**Meet the editor**

Dr Rao, Professor Emeritus, Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, has established a major focus in the area of diet and health. His research has focused on the role of oxidative stress and antioxidant phytochemicals in the causation and prevention of chronic diseases, with particular emphasis on the role of carotenoids and polyphenols. His research

interest also includes the role of prebiotics and probiotics in human health. He has over hundred publications in scientific journals and several books and book chapters. He has a distinguished academic career spanning over 45 years. He is popularly sought by the international media to express his opinions on the subjects of nutrition and health.

Contents

**Preface IX** 

Chapter 3 **Analytical Methods** 

James Hamuel Doughari

Chapter 1 **Phytochemicals: Extraction Methods, Basic Structures and** 

Chapter 2 **Structural Analysis of Flavonoids and Related Compounds – A Review of Spectroscopic Applications 33** 

**for Isolation, Separation and Identification** 

**of Selected Furanocoumarins in Plant Material 57**  Katarzyna Szewczyk and Anna Bogucka - Kocka

**of the Typical Italian Liquor "Limoncello": A Review 93** 

Duduku Krishnaiah, Rajesh Nithyanandam and Rosalam Sarbatly

*anacardioides* **and Their Biological Significance 167** 

Pedro F. Pinheiro and Gonçalo C. Justino

Chapter 4 **Analytical Methods and Phytochemistry** 

Chapter 5 **The Effects of Non-Thermal** 

Chapter 6 **Phytochemical Constituents** 

Mitra Noori

and Olga Martín-Belloso

Chapter 8 **Phytochemicals from** *Beilschmiedia* 

Nkeng-Efouet-Alango Pépin

Marcello Locatelli, Giuseppe Carlucci, Salvatore Genovese and Francesco Epifano

**Technologies on Phytochemicals 107**  Gemma Oms-Oliu, Isabel Odriozola-Serrano

**and Activities of** *Morinda citrifolia* **L. 127** 

Chapter 7 **Flavonoids in some Iranian Angiosperms 151** 

**Mode of Action as Potential Chemotherapeutic Agents 1** 

### Contents

#### **Preface XIII**


Nkeng-Efouet-Alango Pépin


Contents VII

Chapter 21 **Phytochemicals and Their Pharmacological** 

L.G. Rao, N. Kang and A.V. Rao

Chapter 23 **The Pentacyclic Triterpenes , -amyrins:** 

Chapter 25 **The Inhibitory Effect of Natural Stilbenes**

and Arturo Navarro-Ocaña

**Aspects of** *Acanthopanax koreanum* **451** 

Chapter 22 **Polyphenol Antioxidants and Bone Health: A Review 467**

Liliana Hernández Vázquez, Javier Palazon

Chapter 24 **Phytochemical Studies of Fractions and Compounds** 

Olov Sterner and Esther Lilia Peralta Garcìa

**and Their Analogues on Catalytic Activity** 

Renata Mikstacka, Zbigniew Dutkiewicz, Stanisław Sobiak and Wanda Baer-Dubowska

Young Ho Kim, Jeong Ah Kim and Nguyen Xuan Nhiem

**A Review of Sources and Biological Activities 487**

**Present in Vernonanthura Patens with Antifungal Bioactivity and Potential as Antineoplastic 503** Patricia Isabel Manzano Santana, Mario Silva Osorio,

**of Cytochromes P450 Family 1 in Comparison with Other Phenols – Structure and Activity Relationship 519** 


Chapter 21 **Phytochemicals and Their Pharmacological Aspects of** *Acanthopanax koreanum* **451**  Young Ho Kim, Jeong Ah Kim and Nguyen Xuan Nhiem

VI Contents

Chapter 9 **Phenolic Constituents** 

Chapter 11 **The Genus** *Galanthus***:**

Sameh AbouZid

Chapter 13 **Phytocannabinoids 273** 

Chapter 15 **Phytochemistry of some Brazilian** 

**and Antioxidant Properties of some Thai Plants 187** Pitchaon Maisuthisakul

Rodrigo Cassio Sola Veneziani,

Chapter 10 **Lignans: Chemical and Biological Properties 213**  Wilson R. Cunha, Márcio Luis Andrade e Silva,

Sérgio Ricardo Ambrósio and Jairo Kenupp Bastos

Chapter 12 **Silymarin, Natural Flavonolignans from Milk Thistle 255** 

Chapter 14 **Alkaloids and Anthraquinones from Malaysian Flora 287**  Nor Hadiani Ismail, Asmah Alias and Che Puteh Osman

> João X. de Araújo-Júnior, Mariana S.G. de Oliveira, Pedro G.V. Aquino, Magna S. Alexandre-Moreira

Chapter 17 **Phytochemistry, Pharmacology and Agronomy of Medicinal** 

Luzia Kalyne A. M. Leal and Glauce Socorro B. Viana

Maha Aboul Ela, Abdalla El-Lakany and Mohamad Ali Hijazi

**Pharmacology and Biological Properties, a Review 429** Francisco José Queiroz Monte, Telma Leda Gomes de Lemos, Mônica Regina Silva de Araújo and Edilane de Sousa Gomes

**Plants:** *Amburana cearensis***, an Interdisciplinary Study 353** Kirley M. Canuto, Edilberto R. Silveira, Antonio Marcos E. Bezerra,

Cinara V. da Silva, Fernanda M. Borges and Eudes S. Velozo

**A Source of Bioactive Compounds 235**  Strahil Berkov, Carles Codina and Jaume Bastida

Afeef S. Husni and Stephen J. Cutler

**Plants with Aphrodisiac Activity 307**

Chapter 16 **A Phytochemical and Ethnopharmacological Review of the Genus** *Erythrina* **327**

Chapter 18 **General Introduction on Family Asteracea 375**

Chapter 19 **Bioavailability of Phytochemicals 401**  Indah Epriliati and Irine R. Ginjom

Chapter 20 *Ximenia americana***: Chemistry,** 

and Antônio E.G. Sant'Ana


Preface

important field of human nutrition and health.

Since global recognition of the dietary guidelines that include increased consumption of plant-based foods for the prevention of chronic diseases and maintaining good health, there has been a considerable interest in the biologically active compounds that are present in plant foods. These compounds have been referred to, among other terms, as 'Phytochemicals', 'Phytonutrients', 'Nutraceuticals' and 'Functional ingredients". They include a multitude of compounds having different chemical identities, biological activities and mechanisms of action. The scope of 'Phytochemicals' has expanded beyond their initial applications to food to include therapeutics, pharmaceuticals and cosmeceuticals. Although advancements have been made in the field of phytochemicals in the past few decades, more information on the analytical methods of isolation and characterization, their occurrence, biological activity, mechanisms of action and applications to the food and other health industries need to be obtained through systematic scientific investigations. Recognizing this need for more information, a plan to publish a book that brings together up to date information on various aspects of phytochemicals was initiated. This book is a collection of several articles that range in scope from the diversity of their occurrence and chemical characteristics, analytical challenges in their isolation and characterization, and the undertaking of basic and clinical researches to evaluate their biological activities both in animal and human health. The book provides a global perspective related to the phytochemicals present not only in foods but also in medicinal plants. Internationally recognized authors that have expertise in their own respective areas within the phytochemical discipline have contributed to this book. Contents of this important and timely book are useful not only to the researchers but also to health professionals, government regulatory agencies and industrial personnel. It is with this vision that we present this book to our readers and are confident that it will serve as a standard reference book in this

> **Dr. A. V. Rao** Professor Emeritus,

> > Canada

University of Toronto,

Department of Nutritional Sciences, Faculty of Medicine

### Preface

Since global recognition of the dietary guidelines that include increased consumption of plant-based foods for the prevention of chronic diseases and maintaining good health, there has been a considerable interest in the biologically active compounds that are present in plant foods. These compounds have been referred to, among other terms, as 'Phytochemicals', 'Phytonutrients', 'Nutraceuticals' and 'Functional ingredients". They include a multitude of compounds having different chemical identities, biological activities and mechanisms of action. The scope of 'Phytochemicals' has expanded beyond their initial applications to food to include therapeutics, pharmaceuticals and cosmeceuticals. Although advancements have been made in the field of phytochemicals in the past few decades, more information on the analytical methods of isolation and characterization, their occurrence, biological activity, mechanisms of action and applications to the food and other health industries need to be obtained through systematic scientific investigations. Recognizing this need for more information, a plan to publish a book that brings together up to date information on various aspects of phytochemicals was initiated. This book is a collection of several articles that range in scope from the diversity of their occurrence and chemical characteristics, analytical challenges in their isolation and characterization, and the undertaking of basic and clinical researches to evaluate their biological activities both in animal and human health. The book provides a global perspective related to the phytochemicals present not only in foods but also in medicinal plants. Internationally recognized authors that have expertise in their own respective areas within the phytochemical discipline have contributed to this book. Contents of this important and timely book are useful not only to the researchers but also to health professionals, government regulatory agencies and industrial personnel. It is with this vision that we present this book to our readers and are confident that it will serve as a standard reference book in this important field of human nutrition and health.

> **Dr. A. V. Rao**  Professor Emeritus, Department of Nutritional Sciences, Faculty of Medicine University of Toronto, Canada

**1** 

*Nigeria*

James Hamuel Doughari

*Federal University of Technology, Yola*

**Phytochemicals: Extraction Methods,** 

**Potential Chemotherapeutic Agents** 

*Department of Microbiology, School of Pure and Applied Sciences,* 

**Basic Structures and Mode of Action as** 

Medicinal plants have been the mainstay of traditional herbal medicine amongst rural dwellers worldwide since antiquity to date. The therapeutic use of plants certainly goes back to the Sumerian and the Akkadian civilizations in about the third millenium BC. Hippocrates (ca. 460–377 BC), one of the ancient authors who described medicinal natural products of plant and animal origins, listed approximately 400 different plant species for medicinal purposes. Natural products have been an integral part of the ancient traditional medicine systems, e.g. Chinese, Ayurvedic and Egyptian (Sarker & Nahar, 2007). Over the years they have assumed a very central stage in modern civilization as natural source of chemotherapy as well as amongst scientist in search for alternative sources of drugs. About 3.4 billion people in the developing world depend on plant-based traditional medicines. This represents about 88 per cent of the world's inhabitants, who rely mainly on traditional medicine for their primary health care. According to the World Health Organization, a medicinal plant is any plant which, in one or more of its organs, contains substances that can be used for therapeutic purposes, or which are precursors for chemo-pharmaceutical semi synthesis. Such a plant will have its parts including leaves, roots, rhizomes, stems, barks, flowers, fruits, grains or seeds, employed in the control or treatment of a disease condition and therefore contains chemical components that are medically active. These non-nutrient plant chemical compounds or bioactive components are often referred to as phytochemicals ('phyto-' from Greek - *phyto* meaning 'plant') or phytoconstituents and are responsible for protecting the plant against microbial infections or infestations by pests (Abo *et al*., 1991; Liu, 2004; Nweze *et al*., 2004; Doughari *et al*., 2009). The study of natural products on the other hand is called phytochemistry. Phytochemicals have been isolated and characterized from fruits such as grapes and apples, vegetables such as broccoli and onion, spices such as turmeric, beverages such as green tea and red wine, as well as many other sources

The science of application of these indigenous or local medicinal remedies including plants for treatment of diseases is currently called ethno pharmacology but the practice dates back since antiquity. Ethno pharmacology has been the mainstay of traditional medicines the

**1. Introduction** 

(Doughari & Obidah, 2008; Doughari *et al*., 2009).

### **Phytochemicals: Extraction Methods, Basic Structures and Mode of Action as Potential Chemotherapeutic Agents**

James Hamuel Doughari

*Department of Microbiology, School of Pure and Applied Sciences, Federal University of Technology, Yola Nigeria* 

#### **1. Introduction**

Medicinal plants have been the mainstay of traditional herbal medicine amongst rural dwellers worldwide since antiquity to date. The therapeutic use of plants certainly goes back to the Sumerian and the Akkadian civilizations in about the third millenium BC. Hippocrates (ca. 460–377 BC), one of the ancient authors who described medicinal natural products of plant and animal origins, listed approximately 400 different plant species for medicinal purposes. Natural products have been an integral part of the ancient traditional medicine systems, e.g. Chinese, Ayurvedic and Egyptian (Sarker & Nahar, 2007). Over the years they have assumed a very central stage in modern civilization as natural source of chemotherapy as well as amongst scientist in search for alternative sources of drugs. About 3.4 billion people in the developing world depend on plant-based traditional medicines. This represents about 88 per cent of the world's inhabitants, who rely mainly on traditional medicine for their primary health care. According to the World Health Organization, a medicinal plant is any plant which, in one or more of its organs, contains substances that can be used for therapeutic purposes, or which are precursors for chemo-pharmaceutical semi synthesis. Such a plant will have its parts including leaves, roots, rhizomes, stems, barks, flowers, fruits, grains or seeds, employed in the control or treatment of a disease condition and therefore contains chemical components that are medically active. These non-nutrient plant chemical compounds or bioactive components are often referred to as phytochemicals ('phyto-' from Greek - *phyto* meaning 'plant') or phytoconstituents and are responsible for protecting the plant against microbial infections or infestations by pests (Abo *et al*., 1991; Liu, 2004; Nweze *et al*., 2004; Doughari *et al*., 2009). The study of natural products on the other hand is called phytochemistry. Phytochemicals have been isolated and characterized from fruits such as grapes and apples, vegetables such as broccoli and onion, spices such as turmeric, beverages such as green tea and red wine, as well as many other sources (Doughari & Obidah, 2008; Doughari *et al*., 2009).

The science of application of these indigenous or local medicinal remedies including plants for treatment of diseases is currently called ethno pharmacology but the practice dates back since antiquity. Ethno pharmacology has been the mainstay of traditional medicines the

Phytochemicals:

Extraction Methods, Basic Structures and Mode of Action as Potential Chemotherapeutic Agents 3

degree of basicity varies considerably, depending on the structure of the molecule, and presence and location of the functional groups (Sarker & Nahar, 2007). They react with acids to form crystalline salts without the production of water (Firn, 2010). Majority of alkaloids exist in solid such as atropine, some as liquids containing carbon, hydrogen, and nitrogen. Most alkaloids are readily soluble in alcohol and though they are sparingly soluble in water, their salts of are usually soluble. The solutions of alkaloids are intensely bitter. These nitrogenous compounds function in the defence of plants against herbivores and pathogens, and are widely exploited as pharmaceuticals, stimulants, narcotics, and poisons due to their potent biological activities. In nature, the alkaloids exist in large proportions in the seeds

Fig. 1. Basic structures of some pharmacologically important plant derived alkaloids

entire world and currently is being integrated into mainstream medicine. Different catalogues including *De Materia Medica, Historia Plantarum, Species Plantarum* have been variously published in attempt to provide scientific information on the medicinal uses of plants. The types of plants and methods of application vary from locality to locality with 80% of rural dwellers relying on them as means of treating various diseases. For example, the use of bearberry (*Arctostaphylos uva-ursi*) and cranberry juice (*Vaccinium macrocarpon*) to treat urinary tract infections is reported in different manuals of phytotherapy, while species such as lemon balm (*Melissa officinalis*), garlic (*Allium sativum*) and tee tree (*Melaleuca alternifolia*) are described as broad-spectrum antimicrobial agents (Heinrich *et al*., 2004). A single plant may be used for the treatment of various disease conditions depending on the community. Several ailments including fever, asthma, constipation, esophageal cancer and hypertension have been treated with traditional medicinal plants (Cousins & Huffman, 2002; Saganuwan, 2010). The plants are applied in different forms such as poultices, concoctions of different plant mixtures, infusions as teas or tinctures or as component mixtures in porridges and soups administered in different ways including oral, nasal (smoking, snoffing or steaming), topical (lotions, oils or creams), bathing or rectal (enemas). Different plant parts and components (roots, leaves, stem barks, flowers or their combinations, essential oils) have been employed in the treatment of infectious pathologies in the respiratory system, urinary tract, gastrointestinal and biliary systems, as well as on the skin (Rojas *et al*., 2001; R´os & Recio, 2005; Adekunle & Adekunle, 2009).

Medicinal plants are increasingly gaining acceptance even among the literates in urban settlements, probably due to the increasing inefficacy of many modern drugs used for the control of many infections such as typhoid fever, gonorrhoea, and tuberculosis as well as increase in resistance by several bacteria to various antibiotics and the increasing cost of prescription drugs, for the maintenance of personal health (Levy, 1998; Van den Bogaard *et al*., 2000; Smolinski *et al*., 2003). Unfortunately, rapid explosion in human population has made it almost impossible for modern health facilities to meet health demands all over the world, thus putting more demands on the use of natural herbal health remedies. Current problems associated with the use of antibiotics, increased prevalence of multiple-drug resistant (MDR) strains of a number of pathogenic bacteria such as methicillin resistant *Staphylococcus aureus, Helicobacter pylori,* and MDR *Klebsiela pneumonia* has revived the interest in plants with antimicrobial properties (Voravuthikunchai & Kitpipit, 2003). In addition, the increase in cases of opportunistic infections and the advent of Acquired Immune Deficiency Syndrome (AIDS) patients and individuals on immunosuppressive chemotherapy, toxicity of many antifungal and antiviral drugs has imposed pressure on the scientific community and pharmaceutical companies to search alternative and novel drug sources.

#### **2. Classes of phytochemicals**

#### **2.1 Alkaloids**

These are the largest group of secondary chemical constituents made largely of ammonia compounds comprising basically of nitrogen bases synthesized from amino acid building blocks with various radicals replacing one or more of the hydrogen atoms in the peptide ring, most containing oxygen. The compounds have basic properties and are alkaline in reaction, turning red litmus paper blue. In fact, one or more nitrogen atoms that are present in an alkaloid, typically as 1°, 2° or 3° amines, contribute to the basicity of the alkaloid. The

entire world and currently is being integrated into mainstream medicine. Different catalogues including *De Materia Medica, Historia Plantarum, Species Plantarum* have been variously published in attempt to provide scientific information on the medicinal uses of plants. The types of plants and methods of application vary from locality to locality with 80% of rural dwellers relying on them as means of treating various diseases. For example, the use of bearberry (*Arctostaphylos uva-ursi*) and cranberry juice (*Vaccinium macrocarpon*) to treat urinary tract infections is reported in different manuals of phytotherapy, while species such as lemon balm (*Melissa officinalis*), garlic (*Allium sativum*) and tee tree (*Melaleuca alternifolia*) are described as broad-spectrum antimicrobial agents (Heinrich *et al*., 2004). A single plant may be used for the treatment of various disease conditions depending on the community. Several ailments including fever, asthma, constipation, esophageal cancer and hypertension have been treated with traditional medicinal plants (Cousins & Huffman, 2002; Saganuwan, 2010). The plants are applied in different forms such as poultices, concoctions of different plant mixtures, infusions as teas or tinctures or as component mixtures in porridges and soups administered in different ways including oral, nasal (smoking, snoffing or steaming), topical (lotions, oils or creams), bathing or rectal (enemas). Different plant parts and components (roots, leaves, stem barks, flowers or their combinations, essential oils) have been employed in the treatment of infectious pathologies in the respiratory system, urinary tract, gastrointestinal and biliary systems, as well as on

the skin (Rojas *et al*., 2001; R´os & Recio, 2005; Adekunle & Adekunle, 2009).

companies to search alternative and novel drug sources.

**2. Classes of phytochemicals** 

**2.1 Alkaloids** 

Medicinal plants are increasingly gaining acceptance even among the literates in urban settlements, probably due to the increasing inefficacy of many modern drugs used for the control of many infections such as typhoid fever, gonorrhoea, and tuberculosis as well as increase in resistance by several bacteria to various antibiotics and the increasing cost of prescription drugs, for the maintenance of personal health (Levy, 1998; Van den Bogaard *et al*., 2000; Smolinski *et al*., 2003). Unfortunately, rapid explosion in human population has made it almost impossible for modern health facilities to meet health demands all over the world, thus putting more demands on the use of natural herbal health remedies. Current problems associated with the use of antibiotics, increased prevalence of multiple-drug resistant (MDR) strains of a number of pathogenic bacteria such as methicillin resistant *Staphylococcus aureus, Helicobacter pylori,* and MDR *Klebsiela pneumonia* has revived the interest in plants with antimicrobial properties (Voravuthikunchai & Kitpipit, 2003). In addition, the increase in cases of opportunistic infections and the advent of Acquired Immune Deficiency Syndrome (AIDS) patients and individuals on immunosuppressive chemotherapy, toxicity of many antifungal and antiviral drugs has imposed pressure on the scientific community and pharmaceutical

These are the largest group of secondary chemical constituents made largely of ammonia compounds comprising basically of nitrogen bases synthesized from amino acid building blocks with various radicals replacing one or more of the hydrogen atoms in the peptide ring, most containing oxygen. The compounds have basic properties and are alkaline in reaction, turning red litmus paper blue. In fact, one or more nitrogen atoms that are present in an alkaloid, typically as 1°, 2° or 3° amines, contribute to the basicity of the alkaloid. The degree of basicity varies considerably, depending on the structure of the molecule, and presence and location of the functional groups (Sarker & Nahar, 2007). They react with acids to form crystalline salts without the production of water (Firn, 2010). Majority of alkaloids exist in solid such as atropine, some as liquids containing carbon, hydrogen, and nitrogen. Most alkaloids are readily soluble in alcohol and though they are sparingly soluble in water, their salts of are usually soluble. The solutions of alkaloids are intensely bitter. These nitrogenous compounds function in the defence of plants against herbivores and pathogens, and are widely exploited as pharmaceuticals, stimulants, narcotics, and poisons due to their potent biological activities. In nature, the alkaloids exist in large proportions in the seeds

Fig. 1. Basic structures of some pharmacologically important plant derived alkaloids

Phytochemicals:

**2.3 Flavonoids** 

leucoanthocyanidins.

Extraction Methods, Basic Structures and Mode of Action as Potential Chemotherapeutic Agents 5

strophanthidin from *Strophanthus*, digitoxin from *Digitalis*, barbaloin from *Aloes*, salicin from *Salix*, cantharidin from *Cantharides*, and prunasin from *Prunus.* However, the systematic names are invariably coined by replacing the "ose" suffix of the parent sugar with "oside". This group of drugs are usually administered in order to promote appetite and aid digestion. Glycosides are purely bitter principles that are commonly found in plants of the Genitiaceae family and though they are chemically unrelated but possess the common property of an intensely bitter taste. The bitters act on gustatory nerves, which results in increased flow of saliva and gastric juices. Chemically, the bitter principles contain the lactone group that may be diterpene lactones (e.g. *andrographolide*) or triterpenoids (e.g. *amarogentin*). Some of the bitter principles are either used as astringents due to the presence of tannic acid, as antiprotozoan, or to reduce thyroxine and metabolism. Examples include cardiac glycosides (acts on the heart), anthracene glycosides (purgative, and for treatment of skin diseases), chalcone glycoside (anticancer), amarogentin, gentiopicrin, andrographolide, ailanthone and polygalin (Fig. 2). Sarker & Nahar (2007) reported that extracts of plants that contain cyanogenic glycosides are used as flavouring agents in many pharmaceutical preparations. Amygdalin has been used in the treatment of cancer (HCN liberated in stomach kills malignant cells), and also as a cough suppressant in various preparations. Excessive ingestion of cyanogenic glycosides can be fatal. Some foodstuffs containing cyanogenic glycosides can cause poisoning (severe gastric irritations and damage) if not properly handled (Sarker & Nahar, 2007). To test for O-glycosides, the plant samples are boiled with HCl/H2O to hydrolyse the anthraquinone glycosides to respective aglycones, and an aqueous base, e.g. NaOH or NH4OH solution, is added to it. For C-glycosides, the plant samples are hydrolysed using FeCl3/HCl, and and an aqueous base, e.g. NaOH or NH4OH solution, is added to it. In both cases a pink or violet colour in the base layer after

addition of the aqueous base indicates the presence of glycosides in the plant sample.

<sup>α</sup>-Terpineol Cinnamyl acetate Eugenol Taxifolin-7-O- β-glucosid

Fig. 2. Basic structures of some pharmacologically important plant derived glycossides

Flavonoids re important group of polyphenols widely distributed among the plant flora. Stucturally, they are made of more than one benzene ring in its structure (a range of C15 aromatic compounds) and numerous reports support their use as antioxidants or free radical scavengers (Kar, 2007). The compounds are derived from parent compounds known as flavans. Over four thousand flavonoids are known to exist and some of them are pigments in higher plants. Quercetin, kaempferol and quercitrin are common flavonoids present in nearly 70% of plants. Other group of flavonoids include flavones, dihydroflavons, flavans, flavonols, anthocyanidins (Fig. 3), proanthocyanidins, calchones and catechin and

and roots of plants and often in combination with vegetable acids. Alkaloids have pharmacological applications as anesthetics and CNS stimulants (Madziga *et al.,* 2010). More than 12,000-alkaloids are known to exist in about 20% of plant species and only few have been exploited for medicinal purposes. The name alkaloid ends with the suffix –*ine* and plant-derived alkaloids in clinical use include the analgesics morphine and codeine, the muscle relaxant (+)-tubocurarine, the antibiotics sanguinafine and berberine, the anticancer agent vinblastine, the antiarrythmic ajmaline, the pupil dilator atropine, and the sedative scopolamine. Other important alkaloids of plant origin include the addictive stimulants caffeine, nicotine, codeine, atropine, morphine, ergotamine, cocaine, nicotine and ephedrine (Fig. 1). Amino acids act as precursors for biosynthesis of alkaloids with ornithine and lysine commonly used as starting materials. Some screening methods for the detection of alkaloids are summarized in Table 1.


Table 1. Methods for detection of alkaloids

#### **2.2 Glycosides**

Glycosides in general, are defined as the condensation products of sugars (including polysaccharides) with a host of different varieties of organic hydroxy (occasionally thiol) compounds (invariably monohydrate in character), in such a manner that the hemiacetal entity of the carbohydrate must essentially take part in the condensation. Glycosides are colorless, crystalline carbon, hydrogen and oxygen-containing (some contain nitrogen and sulfur) water-soluble phytoconstituents, found in the cell sap. Chemically, glycosides contain a carbohydrate (glucose) and a non-carbohydrate part (aglycone or genin) (Kar, 2007; Firn, 2010). Alcohol, glycerol or phenol represents aglycones. Glycosides are neutral in reaction and can be readily hydrolyzed into its components with ferments or mineral acids. Glycosides are classified on the basis of type of sugar component, chemical nature of aglycone or pharmacological action. The rather older or trivial names of glycosides usually has a suffix 'in' and the names essentially included the source of the glycoside, for instance: strophanthidin from *Strophanthus*, digitoxin from *Digitalis*, barbaloin from *Aloes*, salicin from *Salix*, cantharidin from *Cantharides*, and prunasin from *Prunus.* However, the systematic names are invariably coined by replacing the "ose" suffix of the parent sugar with "oside". This group of drugs are usually administered in order to promote appetite and aid digestion. Glycosides are purely bitter principles that are commonly found in plants of the Genitiaceae family and though they are chemically unrelated but possess the common property of an intensely bitter taste. The bitters act on gustatory nerves, which results in increased flow of saliva and gastric juices. Chemically, the bitter principles contain the lactone group that may be diterpene lactones (e.g. *andrographolide*) or triterpenoids (e.g. *amarogentin*). Some of the bitter principles are either used as astringents due to the presence of tannic acid, as antiprotozoan, or to reduce thyroxine and metabolism. Examples include cardiac glycosides (acts on the heart), anthracene glycosides (purgative, and for treatment of skin diseases), chalcone glycoside (anticancer), amarogentin, gentiopicrin, andrographolide, ailanthone and polygalin (Fig. 2). Sarker & Nahar (2007) reported that extracts of plants that contain cyanogenic glycosides are used as flavouring agents in many pharmaceutical preparations. Amygdalin has been used in the treatment of cancer (HCN liberated in stomach kills malignant cells), and also as a cough suppressant in various preparations. Excessive ingestion of cyanogenic glycosides can be fatal. Some foodstuffs containing cyanogenic glycosides can cause poisoning (severe gastric irritations and damage) if not properly handled (Sarker & Nahar, 2007). To test for O-glycosides, the plant samples are boiled with HCl/H2O to hydrolyse the anthraquinone glycosides to respective aglycones, and an aqueous base, e.g. NaOH or NH4OH solution, is added to it. For C-glycosides, the plant samples are hydrolysed using FeCl3/HCl, and and an aqueous base, e.g. NaOH or NH4OH solution, is added to it. In both cases a pink or violet colour in the base layer after addition of the aqueous base indicates the presence of glycosides in the plant sample.

Fig. 2. Basic structures of some pharmacologically important plant derived glycossides

#### **2.3 Flavonoids**

4 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

and roots of plants and often in combination with vegetable acids. Alkaloids have pharmacological applications as anesthetics and CNS stimulants (Madziga *et al.,* 2010). More than 12,000-alkaloids are known to exist in about 20% of plant species and only few have been exploited for medicinal purposes. The name alkaloid ends with the suffix –*ine* and plant-derived alkaloids in clinical use include the analgesics morphine and codeine, the muscle relaxant (+)-tubocurarine, the antibiotics sanguinafine and berberine, the anticancer agent vinblastine, the antiarrythmic ajmaline, the pupil dilator atropine, and the sedative scopolamine. Other important alkaloids of plant origin include the addictive stimulants caffeine, nicotine, codeine, atropine, morphine, ergotamine, cocaine, nicotine and ephedrine (Fig. 1). Amino acids act as precursors for biosynthesis of alkaloids with ornithine and lysine commonly used as starting materials. Some screening methods for the detection of alkaloids

**Reagent/test Composition of the reagent Result** 

Potassiomercuric iodide solution

Cream precipitate

Yellow precipitate

Precipitation

alkaloids)

pink colour

Reddish-brown precipitate

Orange or reddish-brown precipitate (except with caffeine and a few other

Purine alkaloids produce

A saturated solution of picric acid

Glycosides in general, are defined as the condensation products of sugars (including polysaccharides) with a host of different varieties of organic hydroxy (occasionally thiol) compounds (invariably monohydrate in character), in such a manner that the hemiacetal entity of the carbohydrate must essentially take part in the condensation. Glycosides are colorless, crystalline carbon, hydrogen and oxygen-containing (some contain nitrogen and sulfur) water-soluble phytoconstituents, found in the cell sap. Chemically, glycosides contain a carbohydrate (glucose) and a non-carbohydrate part (aglycone or genin) (Kar, 2007; Firn, 2010). Alcohol, glycerol or phenol represents aglycones. Glycosides are neutral in reaction and can be readily hydrolyzed into its components with ferments or mineral acids. Glycosides are classified on the basis of type of sugar component, chemical nature of aglycone or pharmacological action. The rather older or trivial names of glycosides usually has a suffix 'in' and the names essentially included the source of the glycoside, for instance:

Solution of potassium bismuth iodide potassium chlorate, a drop of hydrochloric acid, evaporated to dryness, and the resulting residue is exposed to ammonia

Iodine in potassium iodide

Tannic acid

vapour

are summarized in Table 1.

Meyer's reagent

Wagner's reagent

Hager's reagent

**2.2 Glycosides** 

Dragendorff's reagent

Murexide test for caffeine

Table 1. Methods for detection of alkaloids

Tannic acid

Flavonoids re important group of polyphenols widely distributed among the plant flora. Stucturally, they are made of more than one benzene ring in its structure (a range of C15 aromatic compounds) and numerous reports support their use as antioxidants or free radical scavengers (Kar, 2007). The compounds are derived from parent compounds known as flavans. Over four thousand flavonoids are known to exist and some of them are pigments in higher plants. Quercetin, kaempferol and quercitrin are common flavonoids present in nearly 70% of plants. Other group of flavonoids include flavones, dihydroflavons, flavans, flavonols, anthocyanidins (Fig. 3), proanthocyanidins, calchones and catechin and leucoanthocyanidins.

Phytochemicals:

Extraction Methods, Basic Structures and Mode of Action as Potential Chemotherapeutic Agents 7

Fig. 4. Basic structures of some pharmacologically important plant derived phenolics

sugar molecule is combined with triterpene or steroid aglycone. There are two major groups of saponins and these include: steroid saponins and triterpene saponins. Saponins are soluble in water and insoluble in ether, and like glycosides on hydrolysis, they give aglycones. Saponins are extremely poisonous, as they cause heamolysis of blood and are known to cause cattle poisoning (Kar, 2007). They possess a bitter and acrid taste, besides causing irritation to mucous membranes. They are mostly amorphous in nature, soluble in alcohol and water, but insoluble in non-polar organic solvents like benzene and n-hexane.

Fig. 3. Basic structures of some pharmacologically important plant derived flavonoids

#### **2.4 Phenolics**

Phenolics, phenols or polyphenolics (or polyphenol extracts) are chemical components that occur ubiquitously as natural colour pigments responsible for the colour of fruits of plants. Phenolics in plants are mostly synthesized from phenylalanine via the action of phenylalanine ammonia lyase (PAL). They are very important to plants and have multiple functions. The most important role may be in plant defence against pathogens and herbivore predators, and thus are applied in the control of human pathogenic infections (Puupponen-Pimiä *et al*., 2008). They are classified into (i) phenolic acids and (ii) flavonoid polyphenolics (flavonones, flavones, xanthones and catechins) and (iii) non-flavonoid polyphenolies. Caffeic acid is regarded as the most common of phenolic compounds distributed in the plant flora followed by chlorogenic acid known to cause allergic dermatitis among humans (Kar, 2007). Phenolics essentially represent a host of natural antioxidants, used as nutraceuticals, and found in apples, green-tea, and red-wine for their enormous ability to combat cancer and are also thought to prevent heart ailments to an appreciable degree and sometimes are anti-inflammatory agents. Other examples include flavones, rutin, naringin , hesperidin and chlorogenic (Fig. 4).

#### **2.5 Saponins**

The term saponin is derived from *Saponaria vaccaria* (*Quillaja saponaria*), a plant, which abounds in saponins and was once used as soap. Saponins therefore possess 'soaplike' behaviour in water, i.e. they produce foam. On hydrolysis, an aglycone is produced, which is called sapogenin. There are two types of sapogenin: steroidal and triterpenoidal. Usually, the sugar is attached at C-3 in saponins, because in most sapogenins there is a hydroxyl group at C-3. *Quillaja saponaria* is known to contain toxic glycosides quillajic acid and the sapogenin senegin. Quillajic acid is strenutatory and senegin is toxic. Senegin is also present in *Polygala senega*. Saponins are regarded as high molecular weight compounds in which, a

Fig. 3. Basic structures of some pharmacologically important plant derived flavonoids

Phenolics, phenols or polyphenolics (or polyphenol extracts) are chemical components that occur ubiquitously as natural colour pigments responsible for the colour of fruits of plants. Phenolics in plants are mostly synthesized from phenylalanine via the action of phenylalanine ammonia lyase (PAL). They are very important to plants and have multiple functions. The most important role may be in plant defence against pathogens and herbivore predators, and thus are applied in the control of human pathogenic infections (Puupponen-Pimiä *et al*., 2008). They are classified into (i) phenolic acids and (ii) flavonoid polyphenolics (flavonones, flavones, xanthones and catechins) and (iii) non-flavonoid polyphenolies. Caffeic acid is regarded as the most common of phenolic compounds distributed in the plant flora followed by chlorogenic acid known to cause allergic dermatitis among humans (Kar, 2007). Phenolics essentially represent a host of natural antioxidants, used as nutraceuticals, and found in apples, green-tea, and red-wine for their enormous ability to combat cancer and are also thought to prevent heart ailments to an appreciable degree and sometimes are anti-inflammatory agents. Other examples include flavones, rutin, naringin ,

The term saponin is derived from *Saponaria vaccaria* (*Quillaja saponaria*), a plant, which abounds in saponins and was once used as soap. Saponins therefore possess 'soaplike' behaviour in water, i.e. they produce foam. On hydrolysis, an aglycone is produced, which is called sapogenin. There are two types of sapogenin: steroidal and triterpenoidal. Usually, the sugar is attached at C-3 in saponins, because in most sapogenins there is a hydroxyl group at C-3. *Quillaja saponaria* is known to contain toxic glycosides quillajic acid and the sapogenin senegin. Quillajic acid is strenutatory and senegin is toxic. Senegin is also present in *Polygala senega*. Saponins are regarded as high molecular weight compounds in which, a

**2.4 Phenolics** 

**2.5 Saponins** 

hesperidin and chlorogenic (Fig. 4).

Fig. 4. Basic structures of some pharmacologically important plant derived phenolics

sugar molecule is combined with triterpene or steroid aglycone. There are two major groups of saponins and these include: steroid saponins and triterpene saponins. Saponins are soluble in water and insoluble in ether, and like glycosides on hydrolysis, they give aglycones. Saponins are extremely poisonous, as they cause heamolysis of blood and are known to cause cattle poisoning (Kar, 2007). They possess a bitter and acrid taste, besides causing irritation to mucous membranes. They are mostly amorphous in nature, soluble in alcohol and water, but insoluble in non-polar organic solvents like benzene and n-hexane.

Phytochemicals:

**2.7 Terpenes** 

Monoterpene Sesquiterpene Diterpene Triterpene Tetraterpene

Polymeric terpenoid

**2.8 Anthraquinones** 

The major groups are shown in Table 2.

**Type of terpenoids Number of carbon** 

<sup>β</sup>-caryophyllene Terpenolen α-Cubebene

**atoms** 

Table 2. Types of terpenoids according to the number of isopropene units

Fig. 6. Basic structures of some pharmacologically important plant derived tarpenes

These are derivatives of phenolic and glycosidic compounds. They are solely derived from anthracene giving variable oxidized derivatives such as anthrones and anthranols (Maurya *et al.,* 2008; Firn, 2010). Other derivatives such as chrysophanol, aloe-emodin, rhein, salinos poramide, luteolin (Fig. 7) and emodin have in common a double hydroxylation at positions C-1 and C-8. To test for free anthraquinones, powdered plant material is mixed with organic solvent and filtered, and an aqueous base, e.g. NaOH or NH4OH solution, is added to it. A

**Number of isoprene units** 

**Example** 

Limonene Artemisinin Forskolin a-amyrin b-carotene Rubber

Extraction Methods, Basic Structures and Mode of Action as Potential Chemotherapeutic Agents 9

Terpenes are among the most widespread and chemically diverse groups of natural products. They are flammable unsaturated hydrocarbons, existing in liquid form commonly found in essential oils, resins or oleoresins (Firn, 2010). Terpenoids includes hydrocarbons of plant origin of general formula (C5H8)n and are classified as mono-, di-, tri- and sesquiterpenoids depending on the number of carbon atoms. Examples of commonly important monterpenes include terpinen-4-ol, thujone, camphor, eugenol and menthol. *Diterpenes (C20)* are classically considered to be resins and taxol, the anticancer agent, is the common example. The t*riterpenes (C30)* include steroids, sterols, and cardiac glycosides with anti-inflammatory, sedative, insecticidal or cytotoxic activity. Common triterpenes: amyrins, ursolic acid and oleanic acid s*esquiterpene (C15*) like monoterpenes, are major components of many essential oils (Martinez *et al.,* 2008). The sesquiterpene acts as irritants when applied externally and when consumed internally their action resembles that of gastrointestinal tract irritant. A number of sesquiterpene lactones have been isolated and broadly they have antimicrobial (particularly antiprotozoal) and neurotoxic action. The sesquiterpene lactone, palasonin, isolated from *Butea monosperma* has anthelmintic activity, inhibits glucose uptake and depletes the glycogen content in *Ascaridia galli* (Fig. 6). Terpenoids are classified according to the number of isoprene units involved in the formation of these compounds.

Saponins are also important therapeutically as they are shown to have hypolipidemic and anticancer activity. Saponins are also necessary for activity of cardiac glycosides. The two major types of steroidal sapogenin are diosgenin and hecogenin. Steroidal saponins are used in the commercial production of sex hormones for clinical use. For example, progesterone is derived from diosgenin. The most abundant starting material for the synthesis of progesterone is diosgenin isolated from *Dioscorea* species, formerly supplied from Mexico, and now from China (Sarker & Nahar, 2007). Other steroidal hormones, e.g. cortisone and hydrocortisone, can be prepared from the starting material hecogenin, which can be isolated from Sisal leaves found extensively in East Africa (Sarker & Nahar, 2007).

#### **2.6 Tannins**

These are widely distributed in plant flora. They are phenolic compounds of high molecular weight. Tannins are soluble in water and alcohol and are found in the root, bark, stem and outer layers of plant tissue. Tannins have a characteristic feature to tan, i.e. to convert things into leather. They are acidic in reaction and the acidic reaction is attributed to the presence of phenolics or carboxylic group (Kar, 2007). They form complexes with proteins, carbohydrates, gelatin and alkaloids. Tannins are divided into hydrolysable tannins and condensed tannins. Hydrolysable tannins, upon hydrolysis, produce gallic acid and ellagic acid and depending on the type of acid produced, the hydrolysable tannins are called gallotannins or egallitannins. On heating, they form pyrogallic acid. Tannins are used as antiseptic and this activity is due to presence of the phenolic group. Common examples of hydrolysable tannins include theaflavins (from tea), daidezein, genistein and glycitein (Fig. 5). Tanninrich medicinal plants are used as healing agents in a number of diseases. In Ayurveda, formulations based on tannin-rich plants have been used for the treatment of diseases like leucorrhoea, rhinnorhoea and diarrhea.

Fig. 5. Basic structures of some pharmacologically important plant derived tannins

#### **2.7 Terpenes**

8 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Saponins are also important therapeutically as they are shown to have hypolipidemic and anticancer activity. Saponins are also necessary for activity of cardiac glycosides. The two major types of steroidal sapogenin are diosgenin and hecogenin. Steroidal saponins are used in the commercial production of sex hormones for clinical use. For example, progesterone is derived from diosgenin. The most abundant starting material for the synthesis of progesterone is diosgenin isolated from *Dioscorea* species, formerly supplied from Mexico, and now from China (Sarker & Nahar, 2007). Other steroidal hormones, e.g. cortisone and hydrocortisone, can be prepared from the starting material hecogenin, which can be isolated

These are widely distributed in plant flora. They are phenolic compounds of high molecular weight. Tannins are soluble in water and alcohol and are found in the root, bark, stem and outer layers of plant tissue. Tannins have a characteristic feature to tan, i.e. to convert things into leather. They are acidic in reaction and the acidic reaction is attributed to the presence of phenolics or carboxylic group (Kar, 2007). They form complexes with proteins, carbohydrates, gelatin and alkaloids. Tannins are divided into hydrolysable tannins and condensed tannins. Hydrolysable tannins, upon hydrolysis, produce gallic acid and ellagic acid and depending on the type of acid produced, the hydrolysable tannins are called gallotannins or egallitannins. On heating, they form pyrogallic acid. Tannins are used as antiseptic and this activity is due to presence of the phenolic group. Common examples of hydrolysable tannins include theaflavins (from tea), daidezein, genistein and glycitein (Fig. 5). Tanninrich medicinal plants are used as healing agents in a number of diseases. In Ayurveda, formulations based on tannin-rich plants have been used for the treatment of diseases like

Fig. 5. Basic structures of some pharmacologically important plant derived tannins

from Sisal leaves found extensively in East Africa (Sarker & Nahar, 2007).

**2.6 Tannins** 

leucorrhoea, rhinnorhoea and diarrhea.

Terpenes are among the most widespread and chemically diverse groups of natural products. They are flammable unsaturated hydrocarbons, existing in liquid form commonly found in essential oils, resins or oleoresins (Firn, 2010). Terpenoids includes hydrocarbons of plant origin of general formula (C5H8)n and are classified as mono-, di-, tri- and sesquiterpenoids depending on the number of carbon atoms. Examples of commonly important monterpenes include terpinen-4-ol, thujone, camphor, eugenol and menthol. *Diterpenes (C20)* are classically considered to be resins and taxol, the anticancer agent, is the common example. The t*riterpenes (C30)* include steroids, sterols, and cardiac glycosides with anti-inflammatory, sedative, insecticidal or cytotoxic activity. Common triterpenes: amyrins, ursolic acid and oleanic acid s*esquiterpene (C15*) like monoterpenes, are major components of many essential oils (Martinez *et al.,* 2008). The sesquiterpene acts as irritants when applied externally and when consumed internally their action resembles that of gastrointestinal tract irritant. A number of sesquiterpene lactones have been isolated and broadly they have antimicrobial (particularly antiprotozoal) and neurotoxic action. The sesquiterpene lactone, palasonin, isolated from *Butea monosperma* has anthelmintic activity, inhibits glucose uptake and depletes the glycogen content in *Ascaridia galli* (Fig. 6). Terpenoids are classified according to the number of isoprene units involved in the formation of these compounds. The major groups are shown in Table 2.

Fig. 6. Basic structures of some pharmacologically important plant derived tarpenes


Table 2. Types of terpenoids according to the number of isopropene units

#### **2.8 Anthraquinones**

These are derivatives of phenolic and glycosidic compounds. They are solely derived from anthracene giving variable oxidized derivatives such as anthrones and anthranols (Maurya *et al.,* 2008; Firn, 2010). Other derivatives such as chrysophanol, aloe-emodin, rhein, salinos poramide, luteolin (Fig. 7) and emodin have in common a double hydroxylation at positions C-1 and C-8. To test for free anthraquinones, powdered plant material is mixed with organic solvent and filtered, and an aqueous base, e.g. NaOH or NH4OH solution, is added to it. A

Phytochemicals:

**2.10 Steroids** 

Extraction Methods, Basic Structures and Mode of Action as Potential Chemotherapeutic Agents 11

handle. Now, the freshly washed lemons are placed in the bowl and rotated repeatedly when the oil glands are punctured (scarified) thereby discharging the oil right into the handle. The liquid, thus collected, is transferred to another vessel, where on keeping the clear oil may be decanted and filtered. For the rasping process, the outer surface of the peel of citrus fruits containing the oil gland is skilfully removed by a grater. The 'raspings' are now placed in horsehair bags and pressed strongly so as to ooze out the oil stored in the oil glands. Initially, the liquid has a turbid appearance but on allowing it to stand the oil separates out which may be decanted and filtered subsequently. The mechanical process involves the use of heavy duty centrifugal devices so as to ease the separation of oil/water emulsions invariably formed and with the advent of modern mechanical devices the oil output has increased impressively. The extraction processes can be carried out with either volatile solvents (e.g hexane, petroleum ether or benzene) resulting into the production of 'floral concretes'- oils with solid consistency and partly soluble in 95% alcohol, or non volatile solvents (tallow, lard or olive oil) which results in the production of perfumes. Examples of volatile oils include amygdaline (volatile oil of bitter almond), sinigrin (volatile oil of black

mustard), and eugenol occurring as gein (volatile oil of *Geum urbanum*) (Fig. 8).

and cevadine (from *Veratrum veride*) are examples of plant steroids (Fig. 9).

**3. Mechanism of action of phytochemicals** 

Fig. 8. Basic structures of some pharmacologically important plant derived essential oils

Plant steroids (or steroid glycosides) also referred to as 'cardiac glycosides' are one of the most naturally occurring plant phytoconstituents that have found therapeutic applications as arrow poisons or cardiac drugs (Firn, 2010). The cardiac glycosides are basically steroids with an inherent ability to afford a very specific and powerful action mainly on the cardiac muscle when administered through injection into man or animal. Steroids (anabolic steroids) have been observed to promote nitrogen retention in osteoporosis and in animals with wasting illness (Maurya *et al.,* 2008; Madziga *et al.,* 2010). Caution should be taken when using steroidal glycosides as small amounts would exhibit the much needed stimulation on a diseased heart, whereas excessive dose may cause even death. Diosgenin

Different mechanisms of action of phytochemicals have been suggested. They may inhibit microorganisms, interfere with some metabolic processes or may modulate gene expression and signal transduction pathways (Kris-Etherton *et al*., 2002; Manson 2003; Surh 2003). Phytochemicals may either be used as chemotherapeutic or chemo preventive agents with chemoprevention referring to the use of agents to inhibit, reverse, or retard tumorigenesis. In this sense chemo preventive phytochemicals are applicable to cancer therapy, since

pink or violet colour in the base layer indicates the presence of anthraquinones in the plant sample (Sarker & Nahar, 2007).

Fig. 7. Basic structures of some pharmacologically important plant derived anthraquinones

#### **2.9 Essential oils**

Essential oils are the odorous and volatile products of various plant and animal species. Essential oils have a tendency evaporate on exposure to air even at ambient conditions and are therefore also referred to as volatile oils or ethereal oils**.** They mostly contribute to the odoriferous constituents or *'essences'* of the aromatic plants that are used abundantly in enhancing the aroma of some spices (Martinez *et al.,* 2008). Essential oils are either secreted either directly by the plant protoplasm or by the hydrolysis of some glycosides and structures such as directly Plant structures associated with the secretion of essential oils include: Glandular hairs (Lamiaceae e.g. *Lavandula angustifolia*), Oil tubes (or vittae) (Apiaceae eg. *Foeniculum vulgare*, and Pimpinella anisum (Aniseed), modified parenchymal cells (Piperaceae e.g. *Piper nigrum -* Black pepper), Schizogenous or lysigenum passages (Rutaceae e.g. *Pinus palustris* - Pine oil. Essential oils have been associated with different plant parts including leaves, stems, flowers, roots or rhizomes. Chemically, a single volatile oil comprises of more than 200 different chemical components, and mostly the trace constituents are solely responsible for attributing its characteristic flavour and odour (Firn, 2010).

Essential oils can be prepared from various plant sources either by direct steam distillation, expression, extraction or by enzymatic hydrolysis. Direct steam distillation involves the boiling of plant part in a distillation flask and passing the generated steam and volatile oil through a water condenser and subsequently collecting the oil in florentine flasks. Depending on the nature of the plant source the distillation process can be either water distillation, water and steam distillation or direct distillation. Expression or extrusion of volatile oils is accomplished by either by sponge method, scarification, rasping or by a mechanical process. In the sponge method, the washed plant part e.g. citrous fruit (*e.g.*, orange, lemon, grape fruit, bergamot) is cut into halves to remove the juice completely, rind turned inside out by hand and squeezed when the secretary glands rupture. The oozed volatile oil is collected by means of the sponge and subsequently squeezed in a vessel. The oil floating on the surface is separated. For the the scarification process the apparatus Ecuelle a Piquer (a large bowl meant for pricking the outer surface of citrus fruits) is used. It is a large funnel made of copper having its inner layer tinned properly. The inner layer has numerous pointed metal needles just long enough to penetrate the epidermis. The lower stem of the apparatus serve two purposes; *first*, as a receiver for the oil; and *secondly*, as a handle. Now, the freshly washed lemons are placed in the bowl and rotated repeatedly when the oil glands are punctured (scarified) thereby discharging the oil right into the handle. The liquid, thus collected, is transferred to another vessel, where on keeping the clear oil may be decanted and filtered. For the rasping process, the outer surface of the peel of citrus fruits containing the oil gland is skilfully removed by a grater. The 'raspings' are now placed in horsehair bags and pressed strongly so as to ooze out the oil stored in the oil glands. Initially, the liquid has a turbid appearance but on allowing it to stand the oil separates out which may be decanted and filtered subsequently. The mechanical process involves the use of heavy duty centrifugal devices so as to ease the separation of oil/water emulsions invariably formed and with the advent of modern mechanical devices the oil output has increased impressively. The extraction processes can be carried out with either volatile solvents (e.g hexane, petroleum ether or benzene) resulting into the production of 'floral concretes'- oils with solid consistency and partly soluble in 95% alcohol, or non volatile solvents (tallow, lard or olive oil) which results in the production of perfumes. Examples of volatile oils include amygdaline (volatile oil of bitter almond), sinigrin (volatile oil of black mustard), and eugenol occurring as gein (volatile oil of *Geum urbanum*) (Fig. 8).

Fig. 8. Basic structures of some pharmacologically important plant derived essential oils

#### **2.10 Steroids**

10 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

pink or violet colour in the base layer indicates the presence of anthraquinones in the plant

Fig. 7. Basic structures of some pharmacologically important plant derived anthraquinones

Essential oils are the odorous and volatile products of various plant and animal species. Essential oils have a tendency evaporate on exposure to air even at ambient conditions and are therefore also referred to as volatile oils or ethereal oils**.** They mostly contribute to the odoriferous constituents or *'essences'* of the aromatic plants that are used abundantly in enhancing the aroma of some spices (Martinez *et al.,* 2008). Essential oils are either secreted either directly by the plant protoplasm or by the hydrolysis of some glycosides and structures such as directly Plant structures associated with the secretion of essential oils include: Glandular hairs (Lamiaceae e.g. *Lavandula angustifolia*), Oil tubes (or vittae) (Apiaceae eg. *Foeniculum vulgare*, and Pimpinella anisum (Aniseed), modified parenchymal cells (Piperaceae e.g. *Piper nigrum -* Black pepper), Schizogenous or lysigenum passages (Rutaceae e.g. *Pinus palustris* - Pine oil. Essential oils have been associated with different plant parts including leaves, stems, flowers, roots or rhizomes. Chemically, a single volatile oil comprises of more than 200 different chemical components, and mostly the trace constituents

are solely responsible for attributing its characteristic flavour and odour (Firn, 2010).

Essential oils can be prepared from various plant sources either by direct steam distillation, expression, extraction or by enzymatic hydrolysis. Direct steam distillation involves the boiling of plant part in a distillation flask and passing the generated steam and volatile oil through a water condenser and subsequently collecting the oil in florentine flasks. Depending on the nature of the plant source the distillation process can be either water distillation, water and steam distillation or direct distillation. Expression or extrusion of volatile oils is accomplished by either by sponge method, scarification, rasping or by a mechanical process. In the sponge method, the washed plant part e.g. citrous fruit (*e.g.*, orange, lemon, grape fruit, bergamot) is cut into halves to remove the juice completely, rind turned inside out by hand and squeezed when the secretary glands rupture. The oozed volatile oil is collected by means of the sponge and subsequently squeezed in a vessel. The oil floating on the surface is separated. For the the scarification process the apparatus Ecuelle a Piquer (a large bowl meant for pricking the outer surface of citrus fruits) is used. It is a large funnel made of copper having its inner layer tinned properly. The inner layer has numerous pointed metal needles just long enough to penetrate the epidermis. The lower stem of the apparatus serve two purposes; *first*, as a receiver for the oil; and *secondly*, as a

Salinos poramide Luteolin

sample (Sarker & Nahar, 2007).

**2.9 Essential oils** 

Plant steroids (or steroid glycosides) also referred to as 'cardiac glycosides' are one of the most naturally occurring plant phytoconstituents that have found therapeutic applications as arrow poisons or cardiac drugs (Firn, 2010). The cardiac glycosides are basically steroids with an inherent ability to afford a very specific and powerful action mainly on the cardiac muscle when administered through injection into man or animal. Steroids (anabolic steroids) have been observed to promote nitrogen retention in osteoporosis and in animals with wasting illness (Maurya *et al.,* 2008; Madziga *et al.,* 2010). Caution should be taken when using steroidal glycosides as small amounts would exhibit the much needed stimulation on a diseased heart, whereas excessive dose may cause even death. Diosgenin and cevadine (from *Veratrum veride*) are examples of plant steroids (Fig. 9).

#### **3. Mechanism of action of phytochemicals**

Different mechanisms of action of phytochemicals have been suggested. They may inhibit microorganisms, interfere with some metabolic processes or may modulate gene expression and signal transduction pathways (Kris-Etherton *et al*., 2002; Manson 2003; Surh 2003). Phytochemicals may either be used as chemotherapeutic or chemo preventive agents with chemoprevention referring to the use of agents to inhibit, reverse, or retard tumorigenesis. In this sense chemo preventive phytochemicals are applicable to cancer therapy, since

Phytochemicals:

mutagenesis.

antioxidants.

**3.2 Anticacinogenesis** 

Evans *et al*., 1997; Diplock *et al*., 1998).

Extraction Methods, Basic Structures and Mode of Action as Potential Chemotherapeutic Agents 13

compensated by making use of natural exogenous antioxidants, such as vitamin C, vitamin E, flavones, β-carotene and natural products in plants (Madsen & Bertelsen, 1995; Rice-

Plants contain a wide variety of free radicals scavenging molecules including phenols, flavonoids, vitamins, terpenoids hat are rich in antioxidant activity (Madsen & Bertelsen, 1995; Cai & Sun, 2003). Many plants, citrus fruits and leafy vegetables are the source of ascorbic acid, vitamin E, caratenoids, flavanols and phenolics which possess the ability to scavenge the free radicals in human body. Significant antioxidant properties have been recorded in phytochemicals that are necessary for the reduction in the occurrence of many diseases (Hertog & Feskens, 1993; Anderson & Teuber, 2001). Many dietary polyphenolic constituents derived from plants are more effective antioxidants *in vitro* than vitamins E or C, and thus might contribute significantly to protective effects *in vivo* (Rice-Evans & Miller, 1997; Jayasri *et al.,* 2009). Methanol extract of *Cinnamon* contains a number of antioxidant compounds which can effectively scavenge reactive oxygen species including superoxide anions and hydroxyl radicals as well as other free radicals *in vitro.* The fruit of *Cinnamon*, an under-utilized and unconventional part of the plant, contains a good amount of phenolic antioxidants to counteract the damaging effects of free radicals and may protect against

Antioxidants are often added to foods to prevent the radical chain reactions of oxidation, and they act by inhibiting the initiation and propagation step leading to the termination of the reaction and delay the oxidation process. Due to safety concerns of synthetic compounds, food industries have focused on finding natural antioxidants to replace synthetic compounds. In addition, there is growing trend in consumer preferences for natural antioxidants, all of which has given more impetus to explore natural sources of

Polyphenols particularly are among the diverse phytochemicals that have the potential in the inhibition of carcinogenesis (Liu, 2004). Phenolics acids usually significantly minimize the formation of the specific cancer-promoting nitrosamines from the dietary nitrites and nitrates. Glucosinolates from various vegetable sources as broccoli, cabbage, cauliflower, and Brussel sprouts exert a substantial protective support against the colon cancer. Regular consumption of Brussel sprouts by human subjects (up to 300 g.day–1) miraculously causes a very fast (say within a span of 3 weeks) an appreciable enhancement in the glutathione-Stransferase, and a subsequent noticeable reduction in the urinary concentration of a specific purine meltabolite that serves as a marker of DNA-degradation in cancer. Isothiocyanates and the indole-3-carbinols do interfere categorically in the metabolism of carcinogens thus causing inhibition of procarcinogen activation, and thereby inducing the 'phase-II' enzymes, namely: NAD(P)H quinone reductase or glutathione S-transferase, that specifically detoxify the selected electrophilic metabolites which are capable of changing the structure of nucleic acids. Sulforaphane (rich in broccoli) has been proved to be an extremely potent phase-2 enzyme inducer. It predominantly causes specific cell-cycle arrest and also the apoptosis of the neoplasm (cancer) cells. Sulforaphane categorically produces d-D-gluconolactone which has been established to be a significant inhibitor of breast cancer. Indole-3-carbinol (most vital and important indole present in broccoli) specifically inhibits the Human Papilloma

Fig. 9. Basic structures of some pharmacologically important plant derived steroids

molecular mechanisms may be common to both chemoprevention and cancer therapy (D'Incalci *et al*., 2005; Sarkar & Li, 2006). Plant extracts and essential oils may exhibit different modes of action against bacterial strains, such as interference with the phospholipids bilayer of the cell membrane which has as a consequence a permeability increase and loss of cellular constituents, damage of the enzymes involved in the production of cellular energy and synthesis of structural components, and destruction or inactivation of genetic material. In general, the mechanism of action is considered to be the disturbance of the cytoplasmic membrane, disrupting the proton motive force, electron flow, active transport, and coagulation of cell contents (Kotzekidou *et al.,* 2008). Some specific modes of actions are discussed below.

#### **3.1 Antioxidants**

Antioxidants protect cells against the damaging effects of reactive oxygen species otherwise called, free radicals such as singlet oxygen, super oxide, peroxyl radicals, hydroxyl radicals and peroxynite which results in oxidative stress leading to cellular damage (Mattson & Cheng, 2006). Natural antioxidants play a key role in health maintenance and prevention of the chronic and degenerative diseases, such as atherosclerosis, cardiac and cerebral ischema, carcinogenesis, neurodegenerative disorders, diabetic pregnancy, rheumatic disorder, DNA damage and ageing (Uddin *et al*., 2008; Jayasri *et al*., 2009). Antioxidants exert their activity by scavenging the 'free-oxygen radicals' thereby giving rise to a fairly 'stable radical'. The free radicals are metastable chemical species, which tend to trap electrons from the molecules in the immediate surroundings. These radicals if not scavenged effectively in time, they may damage crucial bio molecules like lipids, proteins including those present in all membranes, mitochondria and, the DNA resulting in abnormalities leading to disease conditions (Uddin et al. 2008). Thus, free radicals are involved in a number of diseases including: tumour inflammation, hemorrhagic shock, atherosclerosis, diabetes, infertility, gastrointestinal ulcerogenesis, asthma, rheumatoid arthritis, cardiovascular disorders, cystic fibrosis, neurodegenerative diseases (e.g. parkinsonism, Alzheimer's diseases), AIDS and even early senescence (Chen *et al*., 2006; Uddin *et al*., 2008). The human body produces insufficient amount of antioxidants which are essential for preventing oxidative stress. Free radicals generated in the body can be removed by the body's own natural antioxidant defences such as glutathione or catalases (Sen, 1995)**.** Therefore this deficiency had to be compensated by making use of natural exogenous antioxidants, such as vitamin C, vitamin E, flavones, β-carotene and natural products in plants (Madsen & Bertelsen, 1995; Rice-Evans *et al*., 1997; Diplock *et al*., 1998).

Plants contain a wide variety of free radicals scavenging molecules including phenols, flavonoids, vitamins, terpenoids hat are rich in antioxidant activity (Madsen & Bertelsen, 1995; Cai & Sun, 2003). Many plants, citrus fruits and leafy vegetables are the source of ascorbic acid, vitamin E, caratenoids, flavanols and phenolics which possess the ability to scavenge the free radicals in human body. Significant antioxidant properties have been recorded in phytochemicals that are necessary for the reduction in the occurrence of many diseases (Hertog & Feskens, 1993; Anderson & Teuber, 2001). Many dietary polyphenolic constituents derived from plants are more effective antioxidants *in vitro* than vitamins E or C, and thus might contribute significantly to protective effects *in vivo* (Rice-Evans & Miller, 1997; Jayasri *et al.,* 2009). Methanol extract of *Cinnamon* contains a number of antioxidant compounds which can effectively scavenge reactive oxygen species including superoxide anions and hydroxyl radicals as well as other free radicals *in vitro.* The fruit of *Cinnamon*, an under-utilized and unconventional part of the plant, contains a good amount of phenolic antioxidants to counteract the damaging effects of free radicals and may protect against mutagenesis.

Antioxidants are often added to foods to prevent the radical chain reactions of oxidation, and they act by inhibiting the initiation and propagation step leading to the termination of the reaction and delay the oxidation process. Due to safety concerns of synthetic compounds, food industries have focused on finding natural antioxidants to replace synthetic compounds. In addition, there is growing trend in consumer preferences for natural antioxidants, all of which has given more impetus to explore natural sources of antioxidants.

#### **3.2 Anticacinogenesis**

12 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Fig. 9. Basic structures of some pharmacologically important plant derived steroids

actions are discussed below.

**3.1 Antioxidants** 

molecular mechanisms may be common to both chemoprevention and cancer therapy (D'Incalci *et al*., 2005; Sarkar & Li, 2006). Plant extracts and essential oils may exhibit different modes of action against bacterial strains, such as interference with the phospholipids bilayer of the cell membrane which has as a consequence a permeability increase and loss of cellular constituents, damage of the enzymes involved in the production of cellular energy and synthesis of structural components, and destruction or inactivation of genetic material. In general, the mechanism of action is considered to be the disturbance of the cytoplasmic membrane, disrupting the proton motive force, electron flow, active transport, and coagulation of cell contents (Kotzekidou *et al.,* 2008). Some specific modes of

Antioxidants protect cells against the damaging effects of reactive oxygen species otherwise called, free radicals such as singlet oxygen, super oxide, peroxyl radicals, hydroxyl radicals and peroxynite which results in oxidative stress leading to cellular damage (Mattson & Cheng, 2006). Natural antioxidants play a key role in health maintenance and prevention of the chronic and degenerative diseases, such as atherosclerosis, cardiac and cerebral ischema, carcinogenesis, neurodegenerative disorders, diabetic pregnancy, rheumatic disorder, DNA damage and ageing (Uddin *et al*., 2008; Jayasri *et al*., 2009). Antioxidants exert their activity by scavenging the 'free-oxygen radicals' thereby giving rise to a fairly 'stable radical'. The free radicals are metastable chemical species, which tend to trap electrons from the molecules in the immediate surroundings. These radicals if not scavenged effectively in time, they may damage crucial bio molecules like lipids, proteins including those present in all membranes, mitochondria and, the DNA resulting in abnormalities leading to disease conditions (Uddin et al. 2008). Thus, free radicals are involved in a number of diseases including: tumour inflammation, hemorrhagic shock, atherosclerosis, diabetes, infertility, gastrointestinal ulcerogenesis, asthma, rheumatoid arthritis, cardiovascular disorders, cystic fibrosis, neurodegenerative diseases (e.g. parkinsonism, Alzheimer's diseases), AIDS and even early senescence (Chen *et al*., 2006; Uddin *et al*., 2008). The human body produces insufficient amount of antioxidants which are essential for preventing oxidative stress. Free radicals generated in the body can be removed by the body's own natural antioxidant defences such as glutathione or catalases (Sen, 1995)**.** Therefore this deficiency had to be

Polyphenols particularly are among the diverse phytochemicals that have the potential in the inhibition of carcinogenesis (Liu, 2004). Phenolics acids usually significantly minimize the formation of the specific cancer-promoting nitrosamines from the dietary nitrites and nitrates. Glucosinolates from various vegetable sources as broccoli, cabbage, cauliflower, and Brussel sprouts exert a substantial protective support against the colon cancer. Regular consumption of Brussel sprouts by human subjects (up to 300 g.day–1) miraculously causes a very fast (say within a span of 3 weeks) an appreciable enhancement in the glutathione-Stransferase, and a subsequent noticeable reduction in the urinary concentration of a specific purine meltabolite that serves as a marker of DNA-degradation in cancer. Isothiocyanates and the indole-3-carbinols do interfere categorically in the metabolism of carcinogens thus causing inhibition of procarcinogen activation, and thereby inducing the 'phase-II' enzymes, namely: NAD(P)H quinone reductase or glutathione S-transferase, that specifically detoxify the selected electrophilic metabolites which are capable of changing the structure of nucleic acids. Sulforaphane (rich in broccoli) has been proved to be an extremely potent phase-2 enzyme inducer. It predominantly causes specific cell-cycle arrest and also the apoptosis of the neoplasm (cancer) cells. Sulforaphane categorically produces d-D-gluconolactone which has been established to be a significant inhibitor of breast cancer. Indole-3-carbinol (most vital and important indole present in broccoli) specifically inhibits the Human Papilloma

Phytochemicals:

**3.6 Anti-inflammatory** 

**3.7 Multifunctional targets** 

Extraction Methods, Basic Structures and Mode of Action as Potential Chemotherapeutic Agents 15

the amount of TTP (Thrombotic Thrombocytopenic Purpura), IR (Insulin Resistance), and GLUT4 (Glucose Transporter-4) in 3T3-L1 Adipocytes. It was suggested that the mechanism of *Cinnamon*'s insulin-like activity may be in part due to increase in the amounts of TTP, IRβ, and GLUT4 and that *Cinnamon* polyphenols may have additional roles as anti-inflammatory

Essential oil of *C. osmophloeum* twigs has excellent anti- inflammatory activities and cytotoxicity against HepG2 (Human Hepatocellular Liver Carcinoma Cell Line) cells. Previous reports also indicated that the constituents of *C. osmophloeum* twig exhibited excellent anti-inflammatory activities in suppressing nitric oxide production by LPS

Multiple molecular targets of dietary phytochemicals have been identified, from pro- and anti-apoptotic proteins, cell cycle proteins, cell adhesion molecules, protein kinases, transcription factors to metastasis and cell growth pathways (Awad & Bradford, 2005; Aggarwal & Shishodia, 2006; Choi & Friso, 2006). Phytochemicals such as epigallocatechin-3-gallate (EGCG) from green tea, curcumin from turmeric, and resveratrol from red wine tend to aim at a multitude of molecular targets. It is because of these characteristics that definitive mechanisms of action are not available despite decades of research (Francis *et al*., 2002). The multi-target nature of phytochemicals may be beneficial in overcoming cancer drug resistance. This multi-faceted mode of action probably hinders the cancer cell's ability to develop resistance to the phytochemicals. It has also been demonstrated that EGCG has inhibitory effects on the extracellular release of verotoxin (VT) from *E. coli* 0157: H7 (Voravuthikunchai & Kitpipit, 2003). Ethanol pericarp extracts from *Punica granatum* was also reported to inhibited VT production in periplasmic space and cell supernatant. Mechanisms responsible for this are yet to be understood, however the active compounds from the plant are thought to interfere with the transcriptional and translational processes of the bacterial cell (Voravuthikunchai & Kitpipit, 2003). More work is needed to be done in order to establish this assumption. Phytochemicals may also modulate transcription factors (Andreadi *et al*., 2006), redox-sensitive transcription factors (Surh *et al*., 2005), redox signalling, and inflammation. As an example, nitric oxide (NO), a signalling molecule of importance in inflammation, is modulated by plant polyphenols and other botanical extracts (Chan & Fong, 1999; Shanmugam *et al*., 2008). Many phytochemicals have been classified as phytoestrogens, with health-promoting effects resulting in the phytochemicals to be

No single method is sufficient to study the bioactivity of phytochemicals from a given plant. An appropriate assay is required to first screen for the presence of the source material, to purify and subsequently identify the compounds therein. Assay methods vary depending on what bioactivity is targeted and these may include antimicrobial, anti-malarial, anticancer, seed germination, and mammalian toxicity activities. The assay method however

and/or anti-angiogenesis agents (Jakhetia *et al.,* 2010).

marketed as nutraceuticals (Moutsatsou, 2007).

**4. Methods of studying phytochemicals** 

(Lipopolysaccharide)-stimulated macrophages (Jakhetia *et al.,* 2010).

Virus (HPV) that may cause uterine cancer. It blocks the estrogen receptors specifically present in the breast cancer cells as well as down regulates CDK6, and up regulates p21 and p27 in prostate cancer cells. It affords G1 cell-cycle arrest and apoptosis of breast and prostate cancer cells significantly and enhances the p 53 expression in cells treated with benzopyrene. It also depresses Akt, NF-kappaB, MAPK, and Bel-2 signaling pathways to a reasonably good extent. Phytosterols block the development of tumors (neoplasms) in colon, breast, and prostate glands. Although the precise and exact mechanisms whereby the said blockade actually takes place are not yet well understood, yet they seem to change drastically the ensuing cell-membrane transfer in the phenomenon of neoplasm growth and thereby reduce the inflammation significantly.

#### **3.3 Antimicrobial activity**

Phytoconstituents employed by plants to protect them against pathogenic insects, bacteria, fungi or protozoa have found applications in human medicine (Nascimento *et al*., 2000). Some phytochemicals such as phenolic acids act essentially by helping in the reduction of particular adherence of organisms to the cells lining the bladder, and the teeth, which ultimately lowers the incidence of urinary-tract infections (UTI) and the usual dental caries. Plants can also exert either bacteriostatic or bactericidal activity ob microbes. The volatile gas phase of combinations of *Cinnamon* oil and clove oil showed good potential to inhibit growth of spoilage fungi, yeast and bacteria normally found on IMF (Intermediate Moisture Foods) when combined with a modified atmosphere comprising a high concentration of CO2 (40%) and low concentration of O2 (<0.05%) (Jakhetia *et al.,* 2010). *A. flavus,* which is known to produce toxins, was found to be the most resistant microorganism. It is worthy of note that antimicrobial activity results of the same plant part tested most of the time varied from researcher to researcher. This is possible because concentration of plant constituents of the same plant organ can vary from one geographical location to another depending on the age of the plant, differences in topographical factors, the nutrient concentrations of the soil, extraction method as well as method used for antimicrobial study. It is therefore important that scientific protocols be clearly identified and adequately followed and reported.

#### **3.4 Anti-ulcer**

Plants extracts have been reported to inhibit both growth of *H*. *pylori in-vitro* as well as its urease activity (Jakhetia *et al.,* 2010). The efficiency of some extracts in liquid medium and at low pH levels enhances their potency even in the human stomach. Their inhibitory effect on the intestinal and kidney Na+/K+ ATPase activity and on alanine transport in rat jejunum has also been reported (Jakhetia *et al.,* 2010).

#### **3.5 Anti-diabetic**

Cinnamaldehyde, a phytoconstituent extracts have been reported to exhibit significant antihyperglycemic effect resulting in the lowering of both total cholesterol and triglyceride levels and, at the same time, increasing HDL-cholesterol in STZ-induced diabetic rats. This investigation reveals the potential of cinnamaldehyde for use as a natural oral agent, with both hypoglycaemic and hypolipidemic effects. Recent reports indicate that *Cinnamon*  extract and polyphenols with procyanidin type-A polymers exhibit the potential to increase the amount of TTP (Thrombotic Thrombocytopenic Purpura), IR (Insulin Resistance), and GLUT4 (Glucose Transporter-4) in 3T3-L1 Adipocytes. It was suggested that the mechanism of *Cinnamon*'s insulin-like activity may be in part due to increase in the amounts of TTP, IRβ, and GLUT4 and that *Cinnamon* polyphenols may have additional roles as anti-inflammatory and/or anti-angiogenesis agents (Jakhetia *et al.,* 2010).

#### **3.6 Anti-inflammatory**

14 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Virus (HPV) that may cause uterine cancer. It blocks the estrogen receptors specifically present in the breast cancer cells as well as down regulates CDK6, and up regulates p21 and p27 in prostate cancer cells. It affords G1 cell-cycle arrest and apoptosis of breast and prostate cancer cells significantly and enhances the p 53 expression in cells treated with benzopyrene. It also depresses Akt, NF-kappaB, MAPK, and Bel-2 signaling pathways to a reasonably good extent. Phytosterols block the development of tumors (neoplasms) in colon, breast, and prostate glands. Although the precise and exact mechanisms whereby the said blockade actually takes place are not yet well understood, yet they seem to change drastically the ensuing cell-membrane transfer in the phenomenon of neoplasm growth and

Phytoconstituents employed by plants to protect them against pathogenic insects, bacteria, fungi or protozoa have found applications in human medicine (Nascimento *et al*., 2000). Some phytochemicals such as phenolic acids act essentially by helping in the reduction of particular adherence of organisms to the cells lining the bladder, and the teeth, which ultimately lowers the incidence of urinary-tract infections (UTI) and the usual dental caries. Plants can also exert either bacteriostatic or bactericidal activity ob microbes. The volatile gas phase of combinations of *Cinnamon* oil and clove oil showed good potential to inhibit growth of spoilage fungi, yeast and bacteria normally found on IMF (Intermediate Moisture Foods) when combined with a modified atmosphere comprising a high concentration of CO2 (40%) and low concentration of O2 (<0.05%) (Jakhetia *et al.,* 2010). *A. flavus,* which is known to produce toxins, was found to be the most resistant microorganism. It is worthy of note that antimicrobial activity results of the same plant part tested most of the time varied from researcher to researcher. This is possible because concentration of plant constituents of the same plant organ can vary from one geographical location to another depending on the age of the plant, differences in topographical factors, the nutrient concentrations of the soil, extraction method as well as method used for antimicrobial study. It is therefore important

that scientific protocols be clearly identified and adequately followed and reported.

Plants extracts have been reported to inhibit both growth of *H*. *pylori in-vitro* as well as its urease activity (Jakhetia *et al.,* 2010). The efficiency of some extracts in liquid medium and at low pH levels enhances their potency even in the human stomach. Their inhibitory effect on the intestinal and kidney Na+/K+ ATPase activity and on alanine transport in rat jejunum

Cinnamaldehyde, a phytoconstituent extracts have been reported to exhibit significant antihyperglycemic effect resulting in the lowering of both total cholesterol and triglyceride levels and, at the same time, increasing HDL-cholesterol in STZ-induced diabetic rats. This investigation reveals the potential of cinnamaldehyde for use as a natural oral agent, with both hypoglycaemic and hypolipidemic effects. Recent reports indicate that *Cinnamon*  extract and polyphenols with procyanidin type-A polymers exhibit the potential to increase

thereby reduce the inflammation significantly.

has also been reported (Jakhetia *et al.,* 2010).

**3.3 Antimicrobial activity** 

**3.4 Anti-ulcer** 

**3.5 Anti-diabetic** 

Essential oil of *C. osmophloeum* twigs has excellent anti- inflammatory activities and cytotoxicity against HepG2 (Human Hepatocellular Liver Carcinoma Cell Line) cells. Previous reports also indicated that the constituents of *C. osmophloeum* twig exhibited excellent anti-inflammatory activities in suppressing nitric oxide production by LPS (Lipopolysaccharide)-stimulated macrophages (Jakhetia *et al.,* 2010).

#### **3.7 Multifunctional targets**

Multiple molecular targets of dietary phytochemicals have been identified, from pro- and anti-apoptotic proteins, cell cycle proteins, cell adhesion molecules, protein kinases, transcription factors to metastasis and cell growth pathways (Awad & Bradford, 2005; Aggarwal & Shishodia, 2006; Choi & Friso, 2006). Phytochemicals such as epigallocatechin-3-gallate (EGCG) from green tea, curcumin from turmeric, and resveratrol from red wine tend to aim at a multitude of molecular targets. It is because of these characteristics that definitive mechanisms of action are not available despite decades of research (Francis *et al*., 2002). The multi-target nature of phytochemicals may be beneficial in overcoming cancer drug resistance. This multi-faceted mode of action probably hinders the cancer cell's ability to develop resistance to the phytochemicals. It has also been demonstrated that EGCG has inhibitory effects on the extracellular release of verotoxin (VT) from *E. coli* 0157: H7 (Voravuthikunchai & Kitpipit, 2003). Ethanol pericarp extracts from *Punica granatum* was also reported to inhibited VT production in periplasmic space and cell supernatant. Mechanisms responsible for this are yet to be understood, however the active compounds from the plant are thought to interfere with the transcriptional and translational processes of the bacterial cell (Voravuthikunchai & Kitpipit, 2003). More work is needed to be done in order to establish this assumption. Phytochemicals may also modulate transcription factors (Andreadi *et al*., 2006), redox-sensitive transcription factors (Surh *et al*., 2005), redox signalling, and inflammation. As an example, nitric oxide (NO), a signalling molecule of importance in inflammation, is modulated by plant polyphenols and other botanical extracts (Chan & Fong, 1999; Shanmugam *et al*., 2008). Many phytochemicals have been classified as phytoestrogens, with health-promoting effects resulting in the phytochemicals to be marketed as nutraceuticals (Moutsatsou, 2007).

#### **4. Methods of studying phytochemicals**

No single method is sufficient to study the bioactivity of phytochemicals from a given plant. An appropriate assay is required to first screen for the presence of the source material, to purify and subsequently identify the compounds therein. Assay methods vary depending on what bioactivity is targeted and these may include antimicrobial, anti-malarial, anticancer, seed germination, and mammalian toxicity activities. The assay method however

Phytochemicals:

supercritical fluid.

**4.1.3 Microwave-Assisted extraction** 

**4.1.4 Solid phase extraction** 

from various sources, and many more (Patil & Shettigar, 2010).

methods.

2010).

Extraction Methods, Basic Structures and Mode of Action as Potential Chemotherapeutic Agents 17

end product has virtually no solvent residues left in it (CO2 evaporates completely). The downside is that this technology is quite expensive. There are many other gases and liquids that are highly efficient as extraction solvents when put under pressure (Patil & Shettigar,

a. **Coupled SFE-SFC** System in which a sample is extracted with a supercritical fluid which then places the extracted material in the inlet part of a supercritical fluid chromatographic system. The extract is than chromatographed directly using

b. **Coupled SFE-GC and SFE-LC** System in which a sample is extracted using a supercritical fluid which is then depressurized to deposit the extracted material in the inlet part or a column of gas or liquid chromatographic system respectively. SFE is characterized by robustness of sample preparation, reliability, less time consuming, high yield and also has potential for coupling with number of chromatographic

Patil & Shettigar (2010) reported an innovative, microwave-assisted solvent-extraction technology known as Microwave-Assisted Processing (MAP). MAP applications include the extraction of high-value compounds from natural sources including phytonutrients, nutraceutical and functional food ingredients and pharmaceutical actives from biomass. Compared to conventional solvent extraction methods, MAP technology offers some combination of the following advantages: 1. Improved products, increased purity of crude extracts, improved stability of marker compounds, possibility to use less toxic solvents; 2. Reduced processing costs, increased recovery and purity of marker compounds, very fast extraction rates, reduced energy and solvent usage. With microwave-derived extraction as opposed to diffusion, very fast extraction rates and greater solvent flexibility can be achieved. Many variables, including the microwave power and energy density, can be tuned to deliver desired product attributes and optimize process economics. The process can be customized to optimize for commercial/cost reasons and excellent extracts are produced from widely varying substrates. Examples include, but are not limited to, antioxidants from dried herbs, carotenoids from single cells and plant sources, taxanes from taxus biomass, essential fatty acids from microalgae and oilseeds, phytosterols from medicinal plants, polyphenols from green tea, flavor constituents from vanilla and black pepper, essential oils

This involves sorption of solutes from a liquid medium onto a solid adsorbent by the same mechanisms by which molecules are retained on chromatographic stationary phases. These adsorbents, like chromatographic media, come in the form of beads or resins that can be used in column or in batch form. They are often used in the commercially available form of syringes packed with medium (typically a few hundred milligrams to a few grams) through which the sample can be gently forced with the plunger or by vacuum. Solid phase extraction media include reverse phase, normal phase, and ion-exchange media. This is method for sample purification that separates and concentrates the analyte from solution of crude extracts by adsorption onto a disposable solid-phase cartridge. The analyte is

should be as simple, specific, and rapid as possible. An *in vitro* test is more desirable than a bioassay using small laboratory animals, which, in turn, is more desirable than feeding large amounts of valuable and hard to obtain extract to larger domestic or laboratory animals. In addition, *in vivo* tests in mammals are often variable and are highly constrained by ethical considerations of animal welfare. Extraction from the plant is an empirical exercise in which different solvents are utilized under a variety of conditions such as time and temperature of extraction. The success or failure of the extraction process depends on the most appropriate assay. Once extracted from the plant, the bioactive component then has to be separated from the co extractives. Further purification steps may involve simple crystallization of the compound from the crude extract, further solvent partition of the co extractives or chromatographic methods in order to fractionate the compounds based on their acidity, polarity or molecular size. Final purification, to provide compounds of suitable purity for such structural analysis, may be accomplished by appropriate techniques such as recrystallization, sublimation, or distillation.

#### **4.1 Extraction of phytochemicals**

#### **4.1.1 Solvent extraction**

Various solvents have been used to extract different phytoconstituents. The plant parts are dried immediately either in an artificial environment at low temperature (50-60°C) or dried preferably in shade so as to bring down the initial large moisture content to enable its prolonged storage life and . The dried berries are pulverised by mechanical grinders and the oil is removed by solvent extraction. The defatted material is then extracted in a soxhlet apparatus or by soaking in water or alcohol (95% v/v). The resulting alcoholic extract is filtered, concentrated in vacuo or by evaporation, treated with HCl (12N) and refluxed for at least six hours. This can then be concentrated and used to determine the presence of phytoconstituents.

Generally, the saponins do have high molecular weight and hence their isolation in the purest form poses some practical difficulties. The plant parts (tubers, roots, stems, leave etc) are washed sliced and extracted with hot water or ethanol (95% v/v) for several hours. The resulting extract is filtered, concentrated *in vacuo* and the desired constituent is precipitated with ether.

Exhaustive extraction (EE) is usually carried out with different solvents of increasing polarity in order to extract as much as possible the most active components with highest biological activity.

#### **4.1.2 Supercritical fluid extraction (SFE)**

This is the most technologically advanced extraction system (Patil & Shettigar, 2010). Super Critical Fluid Extraction (SFE) involves use of gases, usually CO2, and compressing them into a dense liquid. This liquid is then pumped through a cylinder containing the material to be extracted. From there, the extract-laden liquid is pumped into a separation chamber where the extract is separated from the gas and the gas is recovered for re-use. Solvent properties of CO2 can be manipulated and adjusted by varying the pressure and temperature that one works at. The advantages of SFE are, the versatility it offers in pinpointing the constituents you want to extract from a given material and the fact that your end product has virtually no solvent residues left in it (CO2 evaporates completely). The downside is that this technology is quite expensive. There are many other gases and liquids that are highly efficient as extraction solvents when put under pressure (Patil & Shettigar, 2010).


#### **4.1.3 Microwave-Assisted extraction**

16 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

should be as simple, specific, and rapid as possible. An *in vitro* test is more desirable than a bioassay using small laboratory animals, which, in turn, is more desirable than feeding large amounts of valuable and hard to obtain extract to larger domestic or laboratory animals. In addition, *in vivo* tests in mammals are often variable and are highly constrained by ethical considerations of animal welfare. Extraction from the plant is an empirical exercise in which different solvents are utilized under a variety of conditions such as time and temperature of extraction. The success or failure of the extraction process depends on the most appropriate assay. Once extracted from the plant, the bioactive component then has to be separated from the co extractives. Further purification steps may involve simple crystallization of the compound from the crude extract, further solvent partition of the co extractives or chromatographic methods in order to fractionate the compounds based on their acidity, polarity or molecular size. Final purification, to provide compounds of suitable purity for such structural analysis, may be accomplished by appropriate techniques such as

Various solvents have been used to extract different phytoconstituents. The plant parts are dried immediately either in an artificial environment at low temperature (50-60°C) or dried preferably in shade so as to bring down the initial large moisture content to enable its prolonged storage life and . The dried berries are pulverised by mechanical grinders and the oil is removed by solvent extraction. The defatted material is then extracted in a soxhlet apparatus or by soaking in water or alcohol (95% v/v). The resulting alcoholic extract is filtered, concentrated in vacuo or by evaporation, treated with HCl (12N) and refluxed for at least six hours. This can then be concentrated and used to determine the presence of

Generally, the saponins do have high molecular weight and hence their isolation in the purest form poses some practical difficulties. The plant parts (tubers, roots, stems, leave etc) are washed sliced and extracted with hot water or ethanol (95% v/v) for several hours. The resulting extract is filtered, concentrated *in vacuo* and the desired constituent is precipitated

Exhaustive extraction (EE) is usually carried out with different solvents of increasing polarity in order to extract as much as possible the most active components with highest

This is the most technologically advanced extraction system (Patil & Shettigar, 2010). Super Critical Fluid Extraction (SFE) involves use of gases, usually CO2, and compressing them into a dense liquid. This liquid is then pumped through a cylinder containing the material to be extracted. From there, the extract-laden liquid is pumped into a separation chamber where the extract is separated from the gas and the gas is recovered for re-use. Solvent properties of CO2 can be manipulated and adjusted by varying the pressure and temperature that one works at. The advantages of SFE are, the versatility it offers in pinpointing the constituents you want to extract from a given material and the fact that your

recrystallization, sublimation, or distillation.

**4.1 Extraction of phytochemicals** 

**4.1.1 Solvent extraction** 

phytoconstituents.

biological activity.

**4.1.2 Supercritical fluid extraction (SFE)** 

with ether.

Patil & Shettigar (2010) reported an innovative, microwave-assisted solvent-extraction technology known as Microwave-Assisted Processing (MAP). MAP applications include the extraction of high-value compounds from natural sources including phytonutrients, nutraceutical and functional food ingredients and pharmaceutical actives from biomass. Compared to conventional solvent extraction methods, MAP technology offers some combination of the following advantages: 1. Improved products, increased purity of crude extracts, improved stability of marker compounds, possibility to use less toxic solvents; 2. Reduced processing costs, increased recovery and purity of marker compounds, very fast extraction rates, reduced energy and solvent usage. With microwave-derived extraction as opposed to diffusion, very fast extraction rates and greater solvent flexibility can be achieved. Many variables, including the microwave power and energy density, can be tuned to deliver desired product attributes and optimize process economics. The process can be customized to optimize for commercial/cost reasons and excellent extracts are produced from widely varying substrates. Examples include, but are not limited to, antioxidants from dried herbs, carotenoids from single cells and plant sources, taxanes from taxus biomass, essential fatty acids from microalgae and oilseeds, phytosterols from medicinal plants, polyphenols from green tea, flavor constituents from vanilla and black pepper, essential oils from various sources, and many more (Patil & Shettigar, 2010).

#### **4.1.4 Solid phase extraction**

This involves sorption of solutes from a liquid medium onto a solid adsorbent by the same mechanisms by which molecules are retained on chromatographic stationary phases. These adsorbents, like chromatographic media, come in the form of beads or resins that can be used in column or in batch form. They are often used in the commercially available form of syringes packed with medium (typically a few hundred milligrams to a few grams) through which the sample can be gently forced with the plunger or by vacuum. Solid phase extraction media include reverse phase, normal phase, and ion-exchange media. This is method for sample purification that separates and concentrates the analyte from solution of crude extracts by adsorption onto a disposable solid-phase cartridge. The analyte is

Phytochemicals:

**4.1.6 Advances in chromatographic techniques** 

*a. Preparative high performance liquid chromatography* 

it possible to spend less time on the synthesis conditions. *b. Liquid Chromatography- Mass Spectroscopy (LC-MS)* 

**4.1.6.1 Liquid chromatography** 

Extraction Methods, Basic Structures and Mode of Action as Potential Chemotherapeutic Agents 19

There are basically two types of preparative HPLC. One is low pressure (typically under 5 bar) traditional PLC, based on the use of glass or plastic columns filled with low efficiency packing materials of large particles and large size distribution. A more recent form PLC, Preparative High Performance Liquid Chromatography (Prep.HPLC) has been gaining popularity in pharmaceutical industry. In preparative HPLC (pressure >20 bar), larger stainless steel columns and packing materials (particle size 10-30 μm are needed. The examples of normal phase silica columns are Kromasil 10 μm, Kromasil 16 μm, Chiralcel AS 20 μm whereas for reverse phase are Chromasil C18, Chromasil C8,YMC C18. The aim is to isolate or purify compounds, whereas in analytical work the goal is to get information about the sample. Preparative HPLC is closer to analytical HPLC than traditional PLC, because its higher column efficiencies and faster solvent velocities permit more difficult separation to be conducted more quickly (Oleszek & Marston, 2000; Philipson, 2007). In analytical HPLC, the important parameters are resolution, sensitivity and fast analysis time whereas in preparative HPLC, both the degree of solute purity as well as the amount of compound that can be produced per unit time i.e. throughput or recovery are important. This is very important in pharmaceutical industry of today because new products (Natural, Synthetic) have to be introduced to the market as quickly as possible. Having available such a powerful purification technique makes

In Pharmaceutical industry LC-MS has become method of choice in many stages of drug development. Recent advances includes electro spray, thermo spray, and ion spray ionization techniques which offer unique advantages of high detection sensitivity and specificity, liquid secondary ion mass spectroscopy, later laser mass spectroscopy with 600 MHz offers accurate determination of molecular weight proteins, peptides. Isotopes pattern

The combination of chromatographic separation technique with NMR spectroscopy is one of the most powerful and time saving method for the separation and structural elucidation of unknown compound and mixtures, especially for the structure elucidation of light and oxygen sensitive substances. The online LC-NMR technique allows the continuous registration of time changes as they appear in the chromatographic run automated data acquisition and processing in LC-NMR improves speed and sensitivity of detection (Daffre *et al.,* 2008). The recent introduction of pulsed field gradient technique in high resolution NMR as well as three-dimensional technique improves application in structure elucidation and molecular weight information. These new hyphenated techniques are useful in the areas

of pharmacokinetics, toxicity studies, drug metabolism and drug discovery process.

Coupling capillary column gas chromatographs with Fourier Transform Infrared Spectrometer provides a potent means for separating and identifying the components of different mixtures.

can be detected by this technique (Oleszek & Marston, 2000; Philipson, 2007).

*c. Liquid Chromatography- Nuclear Magnetic Resonance (LC-NMR)* 

*a. Gas Chromatography Fourier Transform Infrared spectrometry* 

**4.1.6.2 Gas chromatography** 

normally retained on the stationary phase, washed and then evaluated with different mobile phase. If an aqueous extract is passed down a column containing reverse phase packing material, everything that is fairly nonpolar will bind, whereas everything polar will pass through (Patil & Shettigar, 2010).

#### **4.1.5 Chromatographic fingerprinting and marker compound analysis**

Chromatographic fingerprint of an Herbal Medicine (HM) is a chromatographic pattern of the extract of some common chemical components of pharmacologically active and or chemical characteristics (Patil & Shettigar, 2010). This chromatographic profile should be featured by the fundamental attributions of "integrity" and "fuzziness" or "sameness" and "differences" so as to chemically represent the HM investigated. It is suggested that with the help of chromatographic fingerprints obtained, the authentication and identification of herbal medicines can be accurately conducted (integrity) even if the amount and/or concentration of the chemically characteristic constituents are not exactly the same for different samples of this HM (hence, "fuzziness") or, the chromatographic fingerprints could demonstrate both the "sameness" and "differences" between various samples successfully. Thus, we should globally consider multiple constituents in the HM extracts, and not individually consider only one and/or two marker components for evaluating the quality of the HM products. However, in any HM and its extract, there are hundreds of unknown components and many of them are in low amount. Moreover, there usually exists variability within the same herbal materials. Hence it is very important to obtain reliable chromatographic fingerprints that represent pharmacologically active and chemically characteristic components of the HM. In the phytochemical evaluation of herbal drugs, TLC is being employed extensively for the following reasons: (1) it enables rapid analysis of herbal extracts with minimum sample clean-up requirement, (2) it provides qualitative and semi quantitative information of the resolved compounds and (3) it enables the quantification of chemical constituents. Fingerprinting using HPLC and GLC is also carried out in specific cases. In TLC fingerprinting, the data that can be recorded using a highperformance TLC (HPTLC) scanner includes the chromatogram, retardation factor (Rf) values, the colour of the separated bands, their absorption spectra, λmax and shoulder inflection/s of all the resolved bands. All of these, together with the profiles on derivatization with different reagents, represent the TLC fingerprint profile of the sample. The information so generated has a potential application in the identification of an authentic drug, in excluding the adulterants and in maintaining the quality and consistency of the drug. HPLC fingerprinting includes recording of the chromatograms, retention time of individual peaks and the absorption spectra (recorded with a photodiode array detector) with different mobile phases. Similarly, GLC is used for generating the fingerprint profiles of volatile oils and fixed oils of herbal drugs. Furthermore, the recent approaches of applying hyphenated chromatography and spectrometry such as High-Performance Liquid Chromatography–Diode Array Detection (HPLC–DAD), Gas Chromatography–Mass Spectroscopy (GC–MS), Capillary Electrophoresis- Diode Array Detection (CE-DAD), High-Performance Liquid Chromatography–Mass Spectroscopy (HPLC–MS) and High-Performance Liquid Chromatography–Nuclear Magnetic Resonance Spectroscopy (HPLC– NMR) could provide the additional spectral information, which will be very helpful for the qualitative analysis and even for the on-line structural elucidation.

#### **4.1.6 Advances in chromatographic techniques**

#### **4.1.6.1 Liquid chromatography**

18 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

normally retained on the stationary phase, washed and then evaluated with different mobile phase. If an aqueous extract is passed down a column containing reverse phase packing material, everything that is fairly nonpolar will bind, whereas everything polar will pass

Chromatographic fingerprint of an Herbal Medicine (HM) is a chromatographic pattern of the extract of some common chemical components of pharmacologically active and or chemical characteristics (Patil & Shettigar, 2010). This chromatographic profile should be featured by the fundamental attributions of "integrity" and "fuzziness" or "sameness" and "differences" so as to chemically represent the HM investigated. It is suggested that with the help of chromatographic fingerprints obtained, the authentication and identification of herbal medicines can be accurately conducted (integrity) even if the amount and/or concentration of the chemically characteristic constituents are not exactly the same for different samples of this HM (hence, "fuzziness") or, the chromatographic fingerprints could demonstrate both the "sameness" and "differences" between various samples successfully. Thus, we should globally consider multiple constituents in the HM extracts, and not individually consider only one and/or two marker components for evaluating the quality of the HM products. However, in any HM and its extract, there are hundreds of unknown components and many of them are in low amount. Moreover, there usually exists variability within the same herbal materials. Hence it is very important to obtain reliable chromatographic fingerprints that represent pharmacologically active and chemically characteristic components of the HM. In the phytochemical evaluation of herbal drugs, TLC is being employed extensively for the following reasons: (1) it enables rapid analysis of herbal extracts with minimum sample clean-up requirement, (2) it provides qualitative and semi quantitative information of the resolved compounds and (3) it enables the quantification of chemical constituents. Fingerprinting using HPLC and GLC is also carried out in specific cases. In TLC fingerprinting, the data that can be recorded using a highperformance TLC (HPTLC) scanner includes the chromatogram, retardation factor (Rf) values, the colour of the separated bands, their absorption spectra, λmax and shoulder inflection/s of all the resolved bands. All of these, together with the profiles on derivatization with different reagents, represent the TLC fingerprint profile of the sample. The information so generated has a potential application in the identification of an authentic drug, in excluding the adulterants and in maintaining the quality and consistency of the drug. HPLC fingerprinting includes recording of the chromatograms, retention time of individual peaks and the absorption spectra (recorded with a photodiode array detector) with different mobile phases. Similarly, GLC is used for generating the fingerprint profiles of volatile oils and fixed oils of herbal drugs. Furthermore, the recent approaches of applying hyphenated chromatography and spectrometry such as High-Performance Liquid Chromatography–Diode Array Detection (HPLC–DAD), Gas Chromatography–Mass Spectroscopy (GC–MS), Capillary Electrophoresis- Diode Array Detection (CE-DAD), High-Performance Liquid Chromatography–Mass Spectroscopy (HPLC–MS) and High-Performance Liquid Chromatography–Nuclear Magnetic Resonance Spectroscopy (HPLC– NMR) could provide the additional spectral information, which will be very helpful for the

**4.1.5 Chromatographic fingerprinting and marker compound analysis** 

qualitative analysis and even for the on-line structural elucidation.

through (Patil & Shettigar, 2010).

#### *a. Preparative high performance liquid chromatography*

There are basically two types of preparative HPLC. One is low pressure (typically under 5 bar) traditional PLC, based on the use of glass or plastic columns filled with low efficiency packing materials of large particles and large size distribution. A more recent form PLC, Preparative High Performance Liquid Chromatography (Prep.HPLC) has been gaining popularity in pharmaceutical industry. In preparative HPLC (pressure >20 bar), larger stainless steel columns and packing materials (particle size 10-30 μm are needed. The examples of normal phase silica columns are Kromasil 10 μm, Kromasil 16 μm, Chiralcel AS 20 μm whereas for reverse phase are Chromasil C18, Chromasil C8,YMC C18. The aim is to isolate or purify compounds, whereas in analytical work the goal is to get information about the sample. Preparative HPLC is closer to analytical HPLC than traditional PLC, because its higher column efficiencies and faster solvent velocities permit more difficult separation to be conducted more quickly (Oleszek & Marston, 2000; Philipson, 2007). In analytical HPLC, the important parameters are resolution, sensitivity and fast analysis time whereas in preparative HPLC, both the degree of solute purity as well as the amount of compound that can be produced per unit time i.e. throughput or recovery are important. This is very important in pharmaceutical industry of today because new products (Natural, Synthetic) have to be introduced to the market as quickly as possible. Having available such a powerful purification technique makes it possible to spend less time on the synthesis conditions.

#### *b. Liquid Chromatography- Mass Spectroscopy (LC-MS)*

In Pharmaceutical industry LC-MS has become method of choice in many stages of drug development. Recent advances includes electro spray, thermo spray, and ion spray ionization techniques which offer unique advantages of high detection sensitivity and specificity, liquid secondary ion mass spectroscopy, later laser mass spectroscopy with 600 MHz offers accurate determination of molecular weight proteins, peptides. Isotopes pattern can be detected by this technique (Oleszek & Marston, 2000; Philipson, 2007).

#### *c. Liquid Chromatography- Nuclear Magnetic Resonance (LC-NMR)*

The combination of chromatographic separation technique with NMR spectroscopy is one of the most powerful and time saving method for the separation and structural elucidation of unknown compound and mixtures, especially for the structure elucidation of light and oxygen sensitive substances. The online LC-NMR technique allows the continuous registration of time changes as they appear in the chromatographic run automated data acquisition and processing in LC-NMR improves speed and sensitivity of detection (Daffre *et al.,* 2008). The recent introduction of pulsed field gradient technique in high resolution NMR as well as three-dimensional technique improves application in structure elucidation and molecular weight information. These new hyphenated techniques are useful in the areas of pharmacokinetics, toxicity studies, drug metabolism and drug discovery process.

#### **4.1.6.2 Gas chromatography**

#### *a. Gas Chromatography Fourier Transform Infrared spectrometry*

Coupling capillary column gas chromatographs with Fourier Transform Infrared Spectrometer provides a potent means for separating and identifying the components of different mixtures.

Phytochemicals:

conditions.

**4.3 Simple assay methods 4.3.1 Antimicrobial assay** 

**4.3.2 Antioxidant assays** 

inhibition and is often calculated using the formula:

Extraction Methods, Basic Structures and Mode of Action as Potential Chemotherapeutic Agents 21

Common methods used in the evaluation of the antibacterial and antifungal activities of plant extracts and essential oils, include the agar diffusion method (paper disc and well), the dilution method (agar and liquid broth) (Yagoub, 2008; Okigbo *et al*., 2009; El-Mahmood, 2009; Aiyegoro *et al*., 2009), and the turbidimetric and impedimetric monitoring of microbial growth (R´os & Recio, 2005). These methods are simple to carry out under laboratory

Most common spectrophotometric assay method applied is the DPPH radical scavenging system in which the hydrogen or electrons donation ability of plant extracts are measured from bleaching of purple methanol solution of 2, 2'-diphenyl-1-picrylhydrazyl (DPPH) free radical (Changwei *et al.,* 2006). This spectrophotometric assay uses the stable radical DPPH as a reagent. DPPH absorbs at 517 nm, and as its concentration is reduced by existence of an antioxidant, the absorption gradually disappears with time. A 2-ml aliquot of a suspension of the ethanol extracts is mixed with 1 ml of 0.5 mM 2,2- diphyenyl-1-picrylhydrazyl (DPPH) solution and 2 ml of 0.1 M sodium acetate buffer (pH 5.5). After shaking, the mixture is incubated at ambient temperature in the dark for 30 minutes, following which the absorbance is measured at 517 nm using a UV-160A spectrometer. A solvent such as ethanol can be used as negative control. Radical scavenging activity is often expressed as percentage

% radical scavenging activity [(A -A )/A ] x control test control 100

Where Acontrol is the absorbance of the control (DPPH solution without test sample) and Atest

Phenolics content and reducing power of extracts is often determined using the Folin-Ciocalteu method. Equal volumes of Folin-Ciocalteu reagent and given quantity (mg) of plant extracts of different concentrations (e.g. 0.4, 0.3, 0.2, 0.1 and 0.05 mg/ml) are often mixed in different sets of test tubes shaken thoroughly, and left to stand for 1 min. Ten percent of NaHCO3 is then added and the mixture once again allowed to stand for 30 minutes, after which the absorbance (725 nm) is measured spectrophotometrically. Gallic acid (0.05-0.5 mg/ml) is often used to produce standard calibration curve and the total phenolic content expressed as mg equivalent of gallic acid (mg GAE) per gram dry weight

of the extract by computing with standard calibration curve (Djeridane *et al.,* 2006).

For determination of reducing power of plant extracts, the ferric reducing/antioxidant power (FRAP) assay method can be applied. The assay is based on the reducing power of a compound (antioxidant). A potential antioxidant reduces the ferric ion (Fe3+) to the ferrous ion (Fe2+); the later forms a blue complex (Fe2+/2, 4, 6, tripyridyl-*s*-triazine (TPTZ)), which increases the absorption at 593 nm. Stronger absorption at this wavelength indicates higher reducing power of the phytochemical, thus higher antioxidant activity. Reaction mixture containing test extract sample at different concentrations (10-100µl) in phosphate buffer (0.2 M, pH 6.6) and equal amounts of 1% (w/v) potassium ferricyanide are incubated at 50ºC for

is the absorbance of the test sample (DPPH solution plus antioxidant).

#### *B. Gas Chromatography-Mass Spectroscopy*

Gas chromatography equipment can be directly interfaced with rapid scan mass spectrometer of various types. The flow rate from capillary column is generally low enough that the column output can be fed directly into ionization chamber of MS. The simplest mass detector in GC is the Ion Trap Detector (ITD). In this instrument, ions are created from the eluted sample by electron impact or chemical ionization and stored in a radio frequency field; the trapped ions are then ejected from the storage area to an electron multiplier detector. The ejection is controlled so that scanning on the basis of mass-to-charge ratio is possible. The ions trap detector is remarkably compact and less expensive than quadrapole instruments.GC-MS instruments have been used for identification of hundreds of components that are present in natural and biological system (Oleszek & Marston, 2000; Philipson, 2007; Daffre *et al.,* 2008).

#### **4.1.6.3 Supercritical Fluid Chromatography (SFC)**

Supercritical fluid chromatography is a hybrid of gas and liquid chromatography that combines some of the best features of each. This technique is an important third kind of column chromatography that is beginning to find use in many industrial, regulatory and academic laboratories. SFC is important because it permits the separation and determination of a group of compounds that are not conveniently handled by either gas or liquid chromatography. These compounds are either non-volatile or thermally labile so that GC procedures are inapplicable or contain no functional group that makes possible detection by the spectroscopic or electrochemical technique employed in LC. SFC has been applied to a wide variety of materials including natural products, drugs, foods and pesticides.

#### **4.2 Other Chromato-Spectrometric studies**

The NMR techniques are employed for establishing connectivities between neighbouring protons and establishing C-H bonds. INEPT is also being used for long range heteronuclear correlations over multiple bondings. The application of Thin Layer chromatography (TLC), High Performance Chromatography (HPLC) and HPLC coupled with Ultra violate (UV) photodiode array detection, Liquid Chromatography-Ultraviolet (LC-UV), Liquid Chromatography-Mass Spectrophotometry (LCMS), electrospray (ES) and Liquid Chromatography-Nuclear Magnetic Resonance (LC-NMR) techniques for the separation and structure determination of antifungal and antibacterial plant compounds is on the increase frequently (Oleszek & Marston, 2000; Bohlin and Bruhn, 1999). Currently available chromatographic and spectroscopic techniques in new drug discovery from natural products Currently, computer modelling has also been introduced in spectrum interpretation and the generation of chemical structures meeting the spectral properties of bioactive compounds obtained from plants (Vlietinck, 2000). The computer systems utilise 1H, 13C, 2D-NMR, IR and MS spectral properties (Philipson, 2007). Libraries of spectra can be searched for comparison with complete or partial chemical structures. Hyphenated chromatographic and spectroscopic techniques are powerful analytical tools that are combined with high throughput biological screening in order to avoid re-isolation of known compounds as well as for structure determination of novel compounds. Hyphenated chromatographic and spectroscopic techniques include LC–UV–MS, LC–UV–NMR, LC–UV– ES–MS and GC–MS (Oleszek & Marston, 2000; Philipson, 2007).

#### **4.3 Simple assay methods**

20 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Gas chromatography equipment can be directly interfaced with rapid scan mass spectrometer of various types. The flow rate from capillary column is generally low enough that the column output can be fed directly into ionization chamber of MS. The simplest mass detector in GC is the Ion Trap Detector (ITD). In this instrument, ions are created from the eluted sample by electron impact or chemical ionization and stored in a radio frequency field; the trapped ions are then ejected from the storage area to an electron multiplier detector. The ejection is controlled so that scanning on the basis of mass-to-charge ratio is possible. The ions trap detector is remarkably compact and less expensive than quadrapole instruments.GC-MS instruments have been used for identification of hundreds of components that are present in natural and biological system (Oleszek & Marston, 2000;

Supercritical fluid chromatography is a hybrid of gas and liquid chromatography that combines some of the best features of each. This technique is an important third kind of column chromatography that is beginning to find use in many industrial, regulatory and academic laboratories. SFC is important because it permits the separation and determination of a group of compounds that are not conveniently handled by either gas or liquid chromatography. These compounds are either non-volatile or thermally labile so that GC procedures are inapplicable or contain no functional group that makes possible detection by the spectroscopic or electrochemical technique employed in LC. SFC has been applied to a

The NMR techniques are employed for establishing connectivities between neighbouring protons and establishing C-H bonds. INEPT is also being used for long range heteronuclear correlations over multiple bondings. The application of Thin Layer chromatography (TLC), High Performance Chromatography (HPLC) and HPLC coupled with Ultra violate (UV) photodiode array detection, Liquid Chromatography-Ultraviolet (LC-UV), Liquid Chromatography-Mass Spectrophotometry (LCMS), electrospray (ES) and Liquid Chromatography-Nuclear Magnetic Resonance (LC-NMR) techniques for the separation and structure determination of antifungal and antibacterial plant compounds is on the increase frequently (Oleszek & Marston, 2000; Bohlin and Bruhn, 1999). Currently available chromatographic and spectroscopic techniques in new drug discovery from natural products Currently, computer modelling has also been introduced in spectrum interpretation and the generation of chemical structures meeting the spectral properties of bioactive compounds obtained from plants (Vlietinck, 2000). The computer systems utilise 1H, 13C, 2D-NMR, IR and MS spectral properties (Philipson, 2007). Libraries of spectra can be searched for comparison with complete or partial chemical structures. Hyphenated chromatographic and spectroscopic techniques are powerful analytical tools that are combined with high throughput biological screening in order to avoid re-isolation of known compounds as well as for structure determination of novel compounds. Hyphenated chromatographic and spectroscopic techniques include LC–UV–MS, LC–UV–NMR, LC–UV–

wide variety of materials including natural products, drugs, foods and pesticides.

*B. Gas Chromatography-Mass Spectroscopy* 

Philipson, 2007; Daffre *et al.,* 2008).

**4.1.6.3 Supercritical Fluid Chromatography (SFC)** 

**4.2 Other Chromato-Spectrometric studies** 

ES–MS and GC–MS (Oleszek & Marston, 2000; Philipson, 2007).

#### **4.3.1 Antimicrobial assay**

Common methods used in the evaluation of the antibacterial and antifungal activities of plant extracts and essential oils, include the agar diffusion method (paper disc and well), the dilution method (agar and liquid broth) (Yagoub, 2008; Okigbo *et al*., 2009; El-Mahmood, 2009; Aiyegoro *et al*., 2009), and the turbidimetric and impedimetric monitoring of microbial growth (R´os & Recio, 2005). These methods are simple to carry out under laboratory conditions.

#### **4.3.2 Antioxidant assays**

Most common spectrophotometric assay method applied is the DPPH radical scavenging system in which the hydrogen or electrons donation ability of plant extracts are measured from bleaching of purple methanol solution of 2, 2'-diphenyl-1-picrylhydrazyl (DPPH) free radical (Changwei *et al.,* 2006). This spectrophotometric assay uses the stable radical DPPH as a reagent. DPPH absorbs at 517 nm, and as its concentration is reduced by existence of an antioxidant, the absorption gradually disappears with time. A 2-ml aliquot of a suspension of the ethanol extracts is mixed with 1 ml of 0.5 mM 2,2- diphyenyl-1-picrylhydrazyl (DPPH) solution and 2 ml of 0.1 M sodium acetate buffer (pH 5.5). After shaking, the mixture is incubated at ambient temperature in the dark for 30 minutes, following which the absorbance is measured at 517 nm using a UV-160A spectrometer. A solvent such as ethanol can be used as negative control. Radical scavenging activity is often expressed as percentage inhibition and is often calculated using the formula:

% radical scavenging activity [(A -A )/A ] x control test control 100

Where Acontrol is the absorbance of the control (DPPH solution without test sample) and Atest is the absorbance of the test sample (DPPH solution plus antioxidant).

Phenolics content and reducing power of extracts is often determined using the Folin-Ciocalteu method. Equal volumes of Folin-Ciocalteu reagent and given quantity (mg) of plant extracts of different concentrations (e.g. 0.4, 0.3, 0.2, 0.1 and 0.05 mg/ml) are often mixed in different sets of test tubes shaken thoroughly, and left to stand for 1 min. Ten percent of NaHCO3 is then added and the mixture once again allowed to stand for 30 minutes, after which the absorbance (725 nm) is measured spectrophotometrically. Gallic acid (0.05-0.5 mg/ml) is often used to produce standard calibration curve and the total phenolic content expressed as mg equivalent of gallic acid (mg GAE) per gram dry weight of the extract by computing with standard calibration curve (Djeridane *et al.,* 2006).

For determination of reducing power of plant extracts, the ferric reducing/antioxidant power (FRAP) assay method can be applied. The assay is based on the reducing power of a compound (antioxidant). A potential antioxidant reduces the ferric ion (Fe3+) to the ferrous ion (Fe2+); the later forms a blue complex (Fe2+/2, 4, 6, tripyridyl-*s*-triazine (TPTZ)), which increases the absorption at 593 nm. Stronger absorption at this wavelength indicates higher reducing power of the phytochemical, thus higher antioxidant activity. Reaction mixture containing test extract sample at different concentrations (10-100µl) in phosphate buffer (0.2 M, pH 6.6) and equal amounts of 1% (w/v) potassium ferricyanide are incubated at 50ºC for

Phytochemicals:

(Phillipson, 2007).

Extraction Methods, Basic Structures and Mode of Action as Potential Chemotherapeutic Agents 23

use with orthodox drugs should be done with extreme caution. Concomitant use of conventional and herbal medicines is reported to lead to clinically relevant herb–drug interactions (Liu et al., 2009). The two may interact either pharmacokinetically or pharmacodynamically resulting into adverse herbal-drug interactions (Izzo, 2005). St John's wort (*Hypericum perforatum*), used for the treatment of mild to moderate depression, interacts with digoxin, HIV inhibitors, theophylline and warfarin. Some medicinal herbs, when ingested, either affect cytochrome P450 isoenzymes by which drugs are metabolised, or, phosphoglycoprotein transporter systems that affect drug distribution and excretion. Concurrent use of some herbal medicines with other medicines may either lower blood plasma concentrations of medicinal drugs, possibly resulting in suboptimal therapeutic amounts, or lead to toxic concentrations in the blood, sometimes with fatal consequences

Despite this observation however, it has been reported that phytochemicals act in synergy with chemotherapeutic drugs in overcoming cancer cell drug resistance and that the application of specific phytochemicals may allow the use of lower concentrations of drugs in

Another advantage with phytochemicals is that, among an estimated 10,000 secondary products (natural pesticides), it has been proposed that human ancestors evolved a generalized defense mechanism against low levels of phytochemicals to enable their consumption of many different plant species containing variable levels of natural pesticides (carcinogens) without subsequent ill health (Liu, 2004). Traces of phytochemicals found in fruits and vegetables may potentiate the immune system and help to protect against cancer (Trewavas and Stewart, 2003). Phytochemicals show biphasic dose responses on mammalian cells. Though at high concentrations they can be toxic, sub-toxic doses may induce adaptive stress response (Ames & Gold, 1991). This includes the activation of signaling pathways that result in increased expression of genes encoding cytoprotective proteins. It is therefore suggested that hormetic mechanisms of action may underlie many of the health benefits of

phytochemicals including their action against cancer drug resistance (Mattson, 2008).

**6. Future prospects of phytochemicals as sources of antimicrobial** 

Molecular mechanisms of herb–drug interaction occur, the most notable is the ATP-binding cassette drug transporters such as P-glycoprotein (You & Moris, 2007) and the drug metabolizing enzymes (known as phase I and phase II enzymes), especially cytochrome

Though there are few disadvantages associated with natural products research. These include difficulties in access and supply, complexities of natural product chemistry and inherent slowness of working with natural products. In addition, there are concerns about intellectual property rights, and the hopes associated with the use of collections of compounds prepared by combinatorial chemistry methods. Despite these limitations, over a 100 natural-product-derived compounds are currently undergoing clinical trials and at least 100 similar projects are in preclinical development (Phillipson, 2007). Among these products the highest number are from plant origin (Table 3). Most are derived from plants and microbial sources. The projects based on natural products are predominantly being studied

cancer treatment with an increased efficacy (Liu, 2004).

P450 3A4 (CYP3A4) (Pal & Mitra, 2006; Meijerman *et al*., 2006).

**chemotherapeutic agents** 

20 minutes and then the reaction terminated by the addition of equal volumes of 10% (w/v) tricarboxyllic acid (TCA) solution and the mixture centrifuged at 3000rpm for 20 minutes. The supernatant is mixed with equal volume of distilled water and 0.1 % (w/v) ferric chloride solution and the absorbance measured at 700 nm. Increased absorbance of the mixture with concentration indicates the reducing power of extract (Jayasri, 2009).

#### **4.3.3 Toxicological studies**

These are often carried out to determine the toxicity of a plant part. Usually animal models such as mice, guinea pigs or rabbits are often employed. In these procedures, the LD50 of the extracts in the experimental animal is often determined via either oral or intradermal administration. The toxic response of experimental animals to the administration of plant alkaloids is usually detected by assay of the serum ALT and AST of the animal as sensitive indicators of hepatocellular damage (Chapatwala *et al*., 1982). Any toxicity usually results in distortion of hepatocytes membrane integrity due to hepatocellular injury and plasma levels rise, as a consequence of high toxin levels present within hepatocytes.

#### **5. Safety concerns for phytochemicals**

Plants are natural reservoir of medicinal agents almost free from the side effects normally caused by synthetic chemicals (Fennel *et al*., 2004). The World Health Organization estimates that herbal medicine is still the main stay of about 75-80% of the world population, mainly in the developing countries for primary health care because of better cultural acceptability, better compatibility with the human body, and lesser side-effects (Kamboj, 2000; Yadav & Dixit, 2008). The over use of synthetic drugs with impurities resulting in higher incidence of adverse drug reactions, has motivated mankind to go back to nature for safer remedies. Due to varied locations where these plants grow, coupled with the problem of different vanacular names, the World Health Organization published standards for herbal safety to minimize adultartion and abuse (WHO, 1999).

A number of modern drugs have been isolated from natural sources and many of these isolations were based on the uses of the agents in traditional medicine (Rizvi *et al*., 2009). Antimicrobial properties of crude extracts prepared from plants have been described and such reports had attracted the attention of scientists worldwide (Falodun *et al*., 2006; El-Mahmood & Amey, 2007; El-Mahmood, 2009). Herbs have been used for food and medicinal purposes for centuries and this knowledge have been passed on from generation to generation (Adedapo *et al*., 2005). This is particularly evident in the rural areas where infectious diseases are endemic and modern health care facilities are few and far thus, compelling the people to nurse their ailments using local herbs. Herbal treatments have been adjudged to be relatively safe (WHO, 1999). For instance, daily oral doses of epigallocatechin-3-gallate (EGCG) for 4 weeks at 800 mg/day in 40 volunteers only caused minor adverse effects (Phillipson, 2007). In a 90-day study of polyphenon E (a formulation of green tea extract with 53% EGCG), the oral no effect level (NOEL) values are 90 mg/kg/day for rats and 600 mg/kg/day for dogs (Boocock *et al*., 2007). For curcumin given to cancer patients at 3600 mg/day for 4 months or 800 mg/day for 3 months, only minor adverse effects are seen. For resveratrol, a single oral dose at 5 g in 10 volunteers only causes minor adverse effects (Boocock *et al*., 2007). Though herbs are relatively safe to use, their combined

20 minutes and then the reaction terminated by the addition of equal volumes of 10% (w/v) tricarboxyllic acid (TCA) solution and the mixture centrifuged at 3000rpm for 20 minutes. The supernatant is mixed with equal volume of distilled water and 0.1 % (w/v) ferric chloride solution and the absorbance measured at 700 nm. Increased absorbance of the

These are often carried out to determine the toxicity of a plant part. Usually animal models such as mice, guinea pigs or rabbits are often employed. In these procedures, the LD50 of the extracts in the experimental animal is often determined via either oral or intradermal administration. The toxic response of experimental animals to the administration of plant alkaloids is usually detected by assay of the serum ALT and AST of the animal as sensitive indicators of hepatocellular damage (Chapatwala *et al*., 1982). Any toxicity usually results in distortion of hepatocytes membrane integrity due to hepatocellular injury and plasma levels

Plants are natural reservoir of medicinal agents almost free from the side effects normally caused by synthetic chemicals (Fennel *et al*., 2004). The World Health Organization estimates that herbal medicine is still the main stay of about 75-80% of the world population, mainly in the developing countries for primary health care because of better cultural acceptability, better compatibility with the human body, and lesser side-effects (Kamboj, 2000; Yadav & Dixit, 2008). The over use of synthetic drugs with impurities resulting in higher incidence of adverse drug reactions, has motivated mankind to go back to nature for safer remedies. Due to varied locations where these plants grow, coupled with the problem of different vanacular names, the World Health Organization published standards for herbal safety to

A number of modern drugs have been isolated from natural sources and many of these isolations were based on the uses of the agents in traditional medicine (Rizvi *et al*., 2009). Antimicrobial properties of crude extracts prepared from plants have been described and such reports had attracted the attention of scientists worldwide (Falodun *et al*., 2006; El-Mahmood & Amey, 2007; El-Mahmood, 2009). Herbs have been used for food and medicinal purposes for centuries and this knowledge have been passed on from generation to generation (Adedapo *et al*., 2005). This is particularly evident in the rural areas where infectious diseases are endemic and modern health care facilities are few and far thus, compelling the people to nurse their ailments using local herbs. Herbal treatments have been adjudged to be relatively safe (WHO, 1999). For instance, daily oral doses of epigallocatechin-3-gallate (EGCG) for 4 weeks at 800 mg/day in 40 volunteers only caused minor adverse effects (Phillipson, 2007). In a 90-day study of polyphenon E (a formulation of green tea extract with 53% EGCG), the oral no effect level (NOEL) values are 90 mg/kg/day for rats and 600 mg/kg/day for dogs (Boocock *et al*., 2007). For curcumin given to cancer patients at 3600 mg/day for 4 months or 800 mg/day for 3 months, only minor adverse effects are seen. For resveratrol, a single oral dose at 5 g in 10 volunteers only causes minor adverse effects (Boocock *et al*., 2007). Though herbs are relatively safe to use, their combined

mixture with concentration indicates the reducing power of extract (Jayasri, 2009).

rise, as a consequence of high toxin levels present within hepatocytes.

**5. Safety concerns for phytochemicals** 

minimize adultartion and abuse (WHO, 1999).

**4.3.3 Toxicological studies** 

use with orthodox drugs should be done with extreme caution. Concomitant use of conventional and herbal medicines is reported to lead to clinically relevant herb–drug interactions (Liu et al., 2009). The two may interact either pharmacokinetically or pharmacodynamically resulting into adverse herbal-drug interactions (Izzo, 2005). St John's wort (*Hypericum perforatum*), used for the treatment of mild to moderate depression, interacts with digoxin, HIV inhibitors, theophylline and warfarin. Some medicinal herbs, when ingested, either affect cytochrome P450 isoenzymes by which drugs are metabolised, or, phosphoglycoprotein transporter systems that affect drug distribution and excretion. Concurrent use of some herbal medicines with other medicines may either lower blood plasma concentrations of medicinal drugs, possibly resulting in suboptimal therapeutic amounts, or lead to toxic concentrations in the blood, sometimes with fatal consequences (Phillipson, 2007).

Despite this observation however, it has been reported that phytochemicals act in synergy with chemotherapeutic drugs in overcoming cancer cell drug resistance and that the application of specific phytochemicals may allow the use of lower concentrations of drugs in cancer treatment with an increased efficacy (Liu, 2004).

Another advantage with phytochemicals is that, among an estimated 10,000 secondary products (natural pesticides), it has been proposed that human ancestors evolved a generalized defense mechanism against low levels of phytochemicals to enable their consumption of many different plant species containing variable levels of natural pesticides (carcinogens) without subsequent ill health (Liu, 2004). Traces of phytochemicals found in fruits and vegetables may potentiate the immune system and help to protect against cancer (Trewavas and Stewart, 2003). Phytochemicals show biphasic dose responses on mammalian cells. Though at high concentrations they can be toxic, sub-toxic doses may induce adaptive stress response (Ames & Gold, 1991). This includes the activation of signaling pathways that result in increased expression of genes encoding cytoprotective proteins. It is therefore suggested that hormetic mechanisms of action may underlie many of the health benefits of phytochemicals including their action against cancer drug resistance (Mattson, 2008).

Molecular mechanisms of herb–drug interaction occur, the most notable is the ATP-binding cassette drug transporters such as P-glycoprotein (You & Moris, 2007) and the drug metabolizing enzymes (known as phase I and phase II enzymes), especially cytochrome P450 3A4 (CYP3A4) (Pal & Mitra, 2006; Meijerman *et al*., 2006).

#### **6. Future prospects of phytochemicals as sources of antimicrobial chemotherapeutic agents**

Though there are few disadvantages associated with natural products research. These include difficulties in access and supply, complexities of natural product chemistry and inherent slowness of working with natural products. In addition, there are concerns about intellectual property rights, and the hopes associated with the use of collections of compounds prepared by combinatorial chemistry methods. Despite these limitations, over a 100 natural-product-derived compounds are currently undergoing clinical trials and at least 100 similar projects are in preclinical development (Phillipson, 2007). Among these products the highest number are from plant origin (Table 3). Most are derived from plants and microbial sources. The projects based on natural products are predominantly being studied

Phytochemicals:

many side effects.

Extraction Methods, Basic Structures and Mode of Action as Potential Chemotherapeutic Agents 25

appropriate pharmacological activity (McGaw *et al*., 2005; Doughari *et al.,* 2009; Okigbo *et al*., 2009). Another approach is the use of 'chemical-genetics profiling' (Doughari *et al.,* 2009). In this method, by building up a database of the effects of a wide range of known compounds, it is possible to interrogate drugs with unknown mechanisms or mixtures of compounds such as natural product mixtures. The technique highlighted unexpected similarities in molecular effects of unrelated drugs (e.g. amiodarone and tamoxifen) and also revealed potential anti-fungal activity of crude extracts. This activity was confirmed by isolation and

Because these compounds are not structurally similar, they would not have been expected to act via the same biological target, thus providing more chances for a very versatile drug component with high efficacy against antibiotic resistant bacteria. It's been reported that despite the popularity of chemical drugs, herbal medicine in Africa and the rest of the world, continued to be practiced due to richness of certain plants in varieties of secondary metabolites such as alkaloids, flavonoids, tannins and terpenoids (Cowan, 1999; Lewis & Ausubel, 2006; Adekunle & Adekunle, 2009). Stapleton *et al.* (2004) reported that aqueous extracts of tea (*Camellia sinensis*) reversed methicillin resistance in methicillin resistant *Staphylococcus aureus* (MRSA) and also to some extent reduced penicillin resistance in betalactamase-producing *Staphylococcus aureus*. Also, Betoni *et al.* (2006) reported synergistic interactions between extracts of guaco (*Mikania glomerata*), guava (*Psidium guajava*), clove (*Syzyguim aromaticum*), garlic (*Allium sativum*) lemon grass (*Cymbopogon citratus*) ginger (*Zingiber officinale*) cargueja (*Baccharis trimera*), and mint (*Mentha pieria*) and some antibiotics against *S. aureus*. However, these are preliminary investigations and more works are needed to actually determine the active ingredients in these plants extracts and this may help in improving management of the different infectious diseases that are developing resistance to commonly used antibiotics and possibly to verocytotoxuic bacteria. Furthermore, toxicological studies can also be carried out to determine the reliance on these herbs without

Researchers have also devised cluster of chemically related scaffolds which are very useful in guiding the synthesis of new compounds. In an attempt to combine the advantages of virtual screening of chemically diverse natural products and their synthetic analogues (scaffolds) with the rapid availability of physical samples for testing, an academic collaboration has established the Drug Discovery Portal (http://www.ddp.strath.ac.uk/). This brings together a wide variety of compounds from academic laboratories in many different institutions in a database that can be used for virtual screening. Academic biology groups can also propose structures as targets for virtual screening with the Portal's database (and with conventional commercially available databases). Access to the Portal is free for academic groups and the continued expansion of the chemical database means that there is a valuable and growing coverage of chemical space through many novel chemical

Despite all of the advances made by the pharmaceutical industry in the development of novel and highly effective medicines for the treatment of a wide range of diseases, there has been a marked increase in the use of herbal medicines even including the more affluent countries of the world. Germany has the largest share of the market in Europe and it was reported that the sales of herbal medicinal products (HMPs) in 1997 were US\$ 1.8 billion (Barnes *et al*., 2007).Numerous scientific medical/pharmaceutical books have been

compounds (Feher & Schmidt, 2003; Galm & Shen, 2007).

testing of defined compounds, stichloroside and theopalauamide (Fig. 10).

for use in cancer or as anti-infectives. There is also, a growing interest in the possibility of developing products that contain mixtures of natural compounds from traditionally used medicines (Charlish, 2008), while, a defined mixture of components extracted from green tea (Veregen TM) has been approved by the US Food and Drug Administration (FDA) and has recently come on the market.


Table 3. Drugs based on natural products at different stages of development

Most of the leads from natural products that are currently in development have come from either plant or microbial sources. Earlier publications have pointed out that relatively little of the world's plant biodiversity has been extensively screened for bioactivity and that very little of the estimated microbial biodiversity has been available for screening (Doughari *et al.,* 2009). Hence, more extensive collections of plants (and microbes) could provide many novel chemicals for use in drug discovery assays. With the growing realization that the chemical diversity of natural products is a better match to that of successful drugs than the diversity of collections of synthetic compounds and with the global emergence of multidrug resistant pathogens (Feher and Schmidt, 2003) the interest in applying natural chemical diversity to drug discovery appears to be increasing once again (Galm & Shen, 2007).

With advances in fractionation techniques to isolate and purify natural products (e.g. counter-current chromatography (Doughari *et al.,* 2009) and in analytical techniques to determine structures (Singh & Barrett, 2006), screening of natural product mixtures is now more compatible with the expected timescale of high-throughput screening campaigns. Singh and Barrett (2006) point out that pure bioactive compound can be isolated from fermentation broths in less than 2 weeks and that the structures of more than 90% of new compounds can be elucidated within 2 weeks. With advances in NMR techniques, complex structures can be solved with much less than 1 mg of compound. It has recently been demonstrated that it is possible to prepare a screening library of highly diverse compounds from plants with the compounds being pre-selected from an analysis of the Dictionary of Natural Products to be drug-like in their physicochemical properties (Oleszek & Marston, 2000; Doughari *et al.,* 2009). It will be interesting to see if such a collection proves to be enriched in bioactive molecules. Several alternative approaches are also being explored in efforts to increase the speed and efficiency with which natural products can be applied to drug discovery. For instance, there is an attraction to screen the mixtures of compounds obtained from extracts of plant material or from microbial broths to select extracts from primary screens that are likely to contain novel compounds with the desired biological activity using the concept of 'differential smart screens'. This approach involves screening extracts of unknown activity against pairs of related receptor sites. By the comparison of the ratios of the binding potencies at the two receptor sites for a known selective ligand and for an extract, it is possible to predict which extract was likely to contain components with the

for use in cancer or as anti-infectives. There is also, a growing interest in the possibility of developing products that contain mixtures of natural compounds from traditionally used medicines (Charlish, 2008), while, a defined mixture of components extracted from green tea (Veregen TM) has been approved by the US Food and Drug Administration (FDA) and has

Development stage Plant Bacterial Fungal Animal Semi-synthetic Total

Most of the leads from natural products that are currently in development have come from either plant or microbial sources. Earlier publications have pointed out that relatively little of the world's plant biodiversity has been extensively screened for bioactivity and that very little of the estimated microbial biodiversity has been available for screening (Doughari *et al.,* 2009). Hence, more extensive collections of plants (and microbes) could provide many novel chemicals for use in drug discovery assays. With the growing realization that the chemical diversity of natural products is a better match to that of successful drugs than the diversity of collections of synthetic compounds and with the global emergence of multidrug resistant pathogens (Feher and Schmidt, 2003) the interest in applying natural chemical

diversity to drug discovery appears to be increasing once again (Galm & Shen, 2007).

With advances in fractionation techniques to isolate and purify natural products (e.g. counter-current chromatography (Doughari *et al.,* 2009) and in analytical techniques to determine structures (Singh & Barrett, 2006), screening of natural product mixtures is now more compatible with the expected timescale of high-throughput screening campaigns. Singh and Barrett (2006) point out that pure bioactive compound can be isolated from fermentation broths in less than 2 weeks and that the structures of more than 90% of new compounds can be elucidated within 2 weeks. With advances in NMR techniques, complex structures can be solved with much less than 1 mg of compound. It has recently been demonstrated that it is possible to prepare a screening library of highly diverse compounds from plants with the compounds being pre-selected from an analysis of the Dictionary of Natural Products to be drug-like in their physicochemical properties (Oleszek & Marston, 2000; Doughari *et al.,* 2009). It will be interesting to see if such a collection proves to be enriched in bioactive molecules. Several alternative approaches are also being explored in efforts to increase the speed and efficiency with which natural products can be applied to drug discovery. For instance, there is an attraction to screen the mixtures of compounds obtained from extracts of plant material or from microbial broths to select extracts from primary screens that are likely to contain novel compounds with the desired biological activity using the concept of 'differential smart screens'. This approach involves screening extracts of unknown activity against pairs of related receptor sites. By the comparison of the ratios of the binding potencies at the two receptor sites for a known selective ligand and for an extract, it is possible to predict which extract was likely to contain components with the

Table 3. Drugs based on natural products at different stages of development

recently come on the market.

Preclinical Phase I Phase II Phase III Pre-registration

Total

appropriate pharmacological activity (McGaw *et al*., 2005; Doughari *et al.,* 2009; Okigbo *et al*., 2009). Another approach is the use of 'chemical-genetics profiling' (Doughari *et al.,* 2009). In this method, by building up a database of the effects of a wide range of known compounds, it is possible to interrogate drugs with unknown mechanisms or mixtures of compounds such as natural product mixtures. The technique highlighted unexpected similarities in molecular effects of unrelated drugs (e.g. amiodarone and tamoxifen) and also revealed potential anti-fungal activity of crude extracts. This activity was confirmed by isolation and testing of defined compounds, stichloroside and theopalauamide (Fig. 10).

Because these compounds are not structurally similar, they would not have been expected to act via the same biological target, thus providing more chances for a very versatile drug component with high efficacy against antibiotic resistant bacteria. It's been reported that despite the popularity of chemical drugs, herbal medicine in Africa and the rest of the world, continued to be practiced due to richness of certain plants in varieties of secondary metabolites such as alkaloids, flavonoids, tannins and terpenoids (Cowan, 1999; Lewis & Ausubel, 2006; Adekunle & Adekunle, 2009). Stapleton *et al.* (2004) reported that aqueous extracts of tea (*Camellia sinensis*) reversed methicillin resistance in methicillin resistant *Staphylococcus aureus* (MRSA) and also to some extent reduced penicillin resistance in betalactamase-producing *Staphylococcus aureus*. Also, Betoni *et al.* (2006) reported synergistic interactions between extracts of guaco (*Mikania glomerata*), guava (*Psidium guajava*), clove (*Syzyguim aromaticum*), garlic (*Allium sativum*) lemon grass (*Cymbopogon citratus*) ginger (*Zingiber officinale*) cargueja (*Baccharis trimera*), and mint (*Mentha pieria*) and some antibiotics against *S. aureus*. However, these are preliminary investigations and more works are needed to actually determine the active ingredients in these plants extracts and this may help in improving management of the different infectious diseases that are developing resistance to commonly used antibiotics and possibly to verocytotoxuic bacteria. Furthermore, toxicological studies can also be carried out to determine the reliance on these herbs without many side effects.

Researchers have also devised cluster of chemically related scaffolds which are very useful in guiding the synthesis of new compounds. In an attempt to combine the advantages of virtual screening of chemically diverse natural products and their synthetic analogues (scaffolds) with the rapid availability of physical samples for testing, an academic collaboration has established the Drug Discovery Portal (http://www.ddp.strath.ac.uk/). This brings together a wide variety of compounds from academic laboratories in many different institutions in a database that can be used for virtual screening. Academic biology groups can also propose structures as targets for virtual screening with the Portal's database (and with conventional commercially available databases). Access to the Portal is free for academic groups and the continued expansion of the chemical database means that there is a valuable and growing coverage of chemical space through many novel chemical compounds (Feher & Schmidt, 2003; Galm & Shen, 2007).

Despite all of the advances made by the pharmaceutical industry in the development of novel and highly effective medicines for the treatment of a wide range of diseases, there has been a marked increase in the use of herbal medicines even including the more affluent countries of the world. Germany has the largest share of the market in Europe and it was reported that the sales of herbal medicinal products (HMPs) in 1997 were US\$ 1.8 billion (Barnes *et al*., 2007).Numerous scientific medical/pharmaceutical books have been

Phytochemicals:

**7. Concluding remarks** 

discovery campaigns.

494-497.

20-24.

**8. References** 

Extraction Methods, Basic Structures and Mode of Action as Potential Chemotherapeutic Agents 27

published in recent years aiming to provide the general public and healthcare professionals with evidence of the benefits and risks of herbal medicines (Barnes *et al*., 2007; Phillipson, 2007). The pharmaceutical industry has met the increased demand for herbal medicines by manufacturing a range of HMPs many of which contain standardized amounts of specific natural products. In the 1950s, it would not have been possible to predict that in 50 years time there would be a thriving industry producing HMPs based on the public demand for herbal medicines. To date European Pharmacopoeia has even published up to 125 monographs on specific medicinal herbs with another 84 currently in preparation (Mijajlovic *et al*., 2006; Phillipson, 2007. The monographs are meant to provide up-to-date knowledge of phytochemistry for defining the chemical profiles of medicinal herbs and an understanding of analytical tests for identification of the herbs and for the quantitative assessment of any known active ingredients (Phillipson, 2007). Several regulatory bodies incuding Traditional Medicines Boards (TMBs, in Nigeria and other African Countries), Medicines and Healthcare products Regulatory Agency (MHRA), Herbal Medicines Advisory Committee (HMAC) (Uk) and American Herbal Products Association (AHPA) and several other pharmacopoeia (British, Chinese, German, Japanese) provide guidelines and advice on the safety, quality and utilization of the plant herbal products in several countries (Yadav & Dixit, 2008). Scientific and Research communities are currently engaged in phytochemical research, and pharmacognosy, phytomedicine or traditional medicine are various disciplines in higher institutions of learning that deals specifically with research in herbal medicines. It is estimated that >5000 individual phytochemicals have been identified in fruits, vegetables, and grains, but a large percentage still remain unknown and need to be identified before we can fully understand the health benefits of phytochemicals (Liu, 2004).

With the increasing interest and so many promising drug candidates in the current development pipeline that are of natural origin, and with the lessening of technical drawbacks associated with natural product research, there are better opportunities to explore the biological activity of previously inaccessible sources of natural products. In addition, the increasing acceptance that the chemical diversity of natural products is well suited to provide the core scaffolds for future drugs, there will be further developments in the use of novel natural products and chemical libraries based on natural products in drug

Abo, K.A.; Ogunleye, V.O. & Ashidi JS (1991). Antimicrobial poteintial of *Spondias mombin,* 

Adedapo, A.A.; Shabi, O.O. & Adedokun OA (2005). Antihelminthic efficacy of the aqueous extract of *Euphorbia hirta* (Linn.) in Nigerian dogs. *Veterinary Archives*. 75(1): 39-47. Adekunle, A.S. & Adekunle, O.C. (2009). Preliminary assessment of antimicrobial properties

*Croton zambesicus* and *Zygotritonia crocea*. *Journal of Pharmacological Research*. 5(13):

of aqueous extract of plants against infectious diseases. *Biology and Medicine.* 1(3):

Fig. 10. Natural products – recently discovered and/or in development. (1) Salinosporamide A; (2) curacin A; (3) dolastatin 10; (4) turbomycin A; (5) cryptophicin; (6) vancomycin; (7) platensimycin; (8) platencin; (9) stichloroside; (10) theopalauamide (Source; Doughari *et al.,*  2009).

published in recent years aiming to provide the general public and healthcare professionals with evidence of the benefits and risks of herbal medicines (Barnes *et al*., 2007; Phillipson, 2007). The pharmaceutical industry has met the increased demand for herbal medicines by manufacturing a range of HMPs many of which contain standardized amounts of specific natural products. In the 1950s, it would not have been possible to predict that in 50 years time there would be a thriving industry producing HMPs based on the public demand for herbal medicines. To date European Pharmacopoeia has even published up to 125 monographs on specific medicinal herbs with another 84 currently in preparation (Mijajlovic *et al*., 2006; Phillipson, 2007. The monographs are meant to provide up-to-date knowledge of phytochemistry for defining the chemical profiles of medicinal herbs and an understanding of analytical tests for identification of the herbs and for the quantitative assessment of any known active ingredients (Phillipson, 2007). Several regulatory bodies incuding Traditional Medicines Boards (TMBs, in Nigeria and other African Countries), Medicines and Healthcare products Regulatory Agency (MHRA), Herbal Medicines Advisory Committee (HMAC) (Uk) and American Herbal Products Association (AHPA) and several other pharmacopoeia (British, Chinese, German, Japanese) provide guidelines and advice on the safety, quality and utilization of the plant herbal products in several countries (Yadav & Dixit, 2008). Scientific and Research communities are currently engaged in phytochemical research, and pharmacognosy, phytomedicine or traditional medicine are various disciplines in higher institutions of learning that deals specifically with research in herbal medicines. It is estimated that >5000 individual phytochemicals have been identified in fruits, vegetables, and grains, but a large percentage still remain unknown and need to be identified before we can fully understand the health benefits of phytochemicals (Liu, 2004).

#### **7. Concluding remarks**

26 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Fig. 10. Natural products – recently discovered and/or in development. (1) Salinosporamide A; (2) curacin A; (3) dolastatin 10; (4) turbomycin A; (5) cryptophicin; (6) vancomycin; (7) platensimycin; (8) platencin; (9) stichloroside; (10) theopalauamide (Source; Doughari *et al.,* 

2009).

With the increasing interest and so many promising drug candidates in the current development pipeline that are of natural origin, and with the lessening of technical drawbacks associated with natural product research, there are better opportunities to explore the biological activity of previously inaccessible sources of natural products. In addition, the increasing acceptance that the chemical diversity of natural products is well suited to provide the core scaffolds for future drugs, there will be further developments in the use of novel natural products and chemical libraries based on natural products in drug discovery campaigns.

#### **8. References**


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African tuberous plant extracts on *Escherichia coli, Staphylococcus aureus* and *Candida* 


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**2** 

*Portugal* 

**Structural Analysis of Flavonoids** 

**of Spectroscopic Applications** 

Pedro F. Pinheiro and Gonçalo C. Justino

**and Related Compounds – A Review** 

*Centro de Química Estrutural, Instituto Superior Técnico, Technical University of Lisbon* 

In 1936 St. Rusznyák and A. Szent-Györgyi described, in a paper in Nature (Rusznyàk & Szent-György, 1936), the relief of certain pathological conditions, characterized by an increased permeability or fragility of the capillary wall, by extracts of Hungarian red pepper containing flavonols, a type of flavonoids, which were then named "vitamin P". The following five decades saw a slow but steady rise in the interest in the group of flavonoids, and their benefits in the treatment of a vast number of diseases and conditions, including pregnancy toxaemia, rheumatic fever, diabetes and cancer. In the late 1980s and throughout the 1990s flavonoids were intensely studied concerning their actions as mutagenic agents and as antioxidants and pro-oxidants as their likely roles in biological systems (for example, Aviram & Fuhrman, 1998; Lambert & Elias, 2010). In the early 90s the antioxidant activity of flavonoids was extensively studied *in vitro*, and it was assumed that such activity would be at the basis of the health promoting benefits of these compounds. However, in the late 90s and early 00s the metabolism of flavonoids was deeply scrutinized, and the results indicated that their antioxidant activity *in vivo* could not account for the overall actions attributed to them (Fraga et al., 2010). The paradigm for flavonoid action changed towards the establishment of flavonoids as inflammation modulators, and more recently their role in neuroprotection, memory and cognition has been under scrutiny (Gomes et al., 2008; Spencer et al., 2009; Spencer, 2010). However, exact mechanisms for many of the actions attributed to flavonoids have not yet been established, but the relationship between their activity and the presence of specific functional groups in the molecules is undeniable. Moreover, each role attributed to flavonoids has been linked to different structural features – for example, while antioxidant activity depends essentially on the number and location of OH groups in the molecules, their antagonist effect towards adenosine receptors depends more on the overall planarity than on the hydroxyl groups; in fact, the latter even appear to

The work developed in this area strongly depends on powerful analytical techniques for quantification and structural identification, as circulating forms are usually found in the low micromolar range, and intracellular levels are even lower. This chapter will briefly review the analytical techniques employed to determine the flavonoids structure from *in vitro* and *in vivo* studies. Albeit it will be focused on the more common classes of flavonoids (flavones,

**1. Introduction** 

be counter-productive (González et al., 2007).


### **Structural Analysis of Flavonoids and Related Compounds – A Review of Spectroscopic Applications**

Pedro F. Pinheiro and Gonçalo C. Justino *Centro de Química Estrutural, Instituto Superior Técnico, Technical University of Lisbon Portugal* 

#### **1. Introduction**

32 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

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Van den Bogaard, A.E. & Stobberingh, E.E. (2000). Epidemiology of resistance to antibiotics:

Vlietinck, A.J. (2000). *The future of phytochemistry in the new millennium*. In: LuijendijkTJC

Voravuthikunchai, S.P. & Kitpipit, L. (2003). Activities of crude extracts of Thai medicinal

Yadav, N.P. & Dixit, V.K. (2008). Recent approaches in herbal drug standardization.

Yagoub, S.O. (2008). Antimicrobial activity of *Tamarindus indica* and *Adansonia digitata* 

You, G.; Morris, M.E. & Editors (2007). *Drug Transporters.* Hoboken: John Wiley & Sons.

Antibacterial activities of *Trema cannabina. Middle*-*East Journal of Scientific Research*.

Links between animals and humans. *International Journal of Antimicrobial*

(Ed.), 2000 years of natural product research - past, present and future.

plants on methicillin- resistant *Staphylococcus aureus. Journal of Clinical Microbiology* 

extracts against *E. coli* isolated from urine and water specimens. *Research Journal of* 

*Current Opinions in Plant Biology*. 6: 185–190.

Phytoconsult, Leiden, pp. 212–215.

W.H.O. (1999). *WHO Monographs on Selected Medicinal Plants*. 1: 1-295.

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3: 105-108.

*Agents*.14:327-335

*and Infection*. 9:236.

*Microbiology*. 3(3): 193-197.

In 1936 St. Rusznyák and A. Szent-Györgyi described, in a paper in Nature (Rusznyàk & Szent-György, 1936), the relief of certain pathological conditions, characterized by an increased permeability or fragility of the capillary wall, by extracts of Hungarian red pepper containing flavonols, a type of flavonoids, which were then named "vitamin P". The following five decades saw a slow but steady rise in the interest in the group of flavonoids, and their benefits in the treatment of a vast number of diseases and conditions, including pregnancy toxaemia, rheumatic fever, diabetes and cancer. In the late 1980s and throughout the 1990s flavonoids were intensely studied concerning their actions as mutagenic agents and as antioxidants and pro-oxidants as their likely roles in biological systems (for example, Aviram & Fuhrman, 1998; Lambert & Elias, 2010). In the early 90s the antioxidant activity of flavonoids was extensively studied *in vitro*, and it was assumed that such activity would be at the basis of the health promoting benefits of these compounds. However, in the late 90s and early 00s the metabolism of flavonoids was deeply scrutinized, and the results indicated that their antioxidant activity *in vivo* could not account for the overall actions attributed to them (Fraga et al., 2010). The paradigm for flavonoid action changed towards the establishment of flavonoids as inflammation modulators, and more recently their role in neuroprotection, memory and cognition has been under scrutiny (Gomes et al., 2008; Spencer et al., 2009; Spencer, 2010). However, exact mechanisms for many of the actions attributed to flavonoids have not yet been established, but the relationship between their activity and the presence of specific functional groups in the molecules is undeniable. Moreover, each role attributed to flavonoids has been linked to different structural features – for example, while antioxidant activity depends essentially on the number and location of OH groups in the molecules, their antagonist effect towards adenosine receptors depends more on the overall planarity than on the hydroxyl groups; in fact, the latter even appear to be counter-productive (González et al., 2007).

The work developed in this area strongly depends on powerful analytical techniques for quantification and structural identification, as circulating forms are usually found in the low micromolar range, and intracellular levels are even lower. This chapter will briefly review the analytical techniques employed to determine the flavonoids structure from *in vitro* and *in vivo* studies. Albeit it will be focused on the more common classes of flavonoids (flavones,

Structural Analysis

the general group of flavonoids.

**3. Mass Spectrometry** 

2010) is highly adequate.

**3.1 Ionization techniques** 

**3.1.1 FAB-MS and LSIMS** 

of Flavonoids and Related Compounds – A Review of Spectroscopic Applications 35

on the basis of the central ring oxidation and on the presence of specific hydroxyl groups. Most common flavonoids are flavones (with a C2-C3 double bond and a C4-oxo function), flavonols (flavones with a 3-OH group) and flavanones (flavone analogues but with a C2-C3 single bond), and abundant isoflavonoids include isoflavones (the analogue of flavones). 4 arylcoumarin (a neoflavonoid with a C3-C4 double bond) and its reduced form, 3,4 dihydro-4-arylcoumarin, are the major neoflavonoids. Other natural compounds, such as chalcones and aurones also possess the C6-C3-C6 backbone, and are henceforth included in

Mass Spectrometry (MS) has proved to be one of the most effective techniques in biomedical research, in special when complex matrixes of biological samples must be analysed. The main advantages of MS are its high sensitivity, which allows analysis of compounds present in the g scale, and high specificity, as it is able to separate molecules of the same molecular weight but different atom composition, and sometimes even to differentiate stereoisomeric compounds. Its easy coupling to separation techniques such as liquid and gas chromatography is also an excellent advantage. A review of separation methods, applied to flavonols, isoflavones and anthocyanidins, has been recently published (Valls et al., 2009). Sample preparation may also be critical, but that varies from sample to sample; nevertheless, some general guidelines have been reviewed (Prasain et al., 2004). For more detail on mass spectrometry equipment and experiments, an excellent book has been published in 2007 (de Hoffmann & Stroobant, 2007). Specifically for electrospray and MALDI mass spectrometry applications in the biology area, the work edited by Cole (Cole,

Gas Chromatography (GC) is one of the key techniques for the separation of organics and, coupled to MS, one of the most common techniques of structural identification. However, flavonoids are largely nonvolatile, and need be derivatized; also, they are usually thermally unstable. Both these characteristics have led to the establishment of Liquid Chromatography, in particular High Performance (HPLC), as the fundamental separation technique for flavonoids. Consequently, LC-MS coupling is routinely used for the overall

Various ionization techniques are available, and each has their own specificities which make them more or less useful depending on the molecules under study and on the aim of such

Fast-Atom Bombardment (FAB) and Liquid Secondary Ion Mass Spectrometry (LSIMS) are ionization techniques used in Secondary Mass Ion Spectrometry (SIMS) in which secondary ions, emitted by sample irradiation with a beam of energetic (primary) ions, are analyzed. Typically, these techniques are able to produce ions from polar compounds with molecular weights up to 10 kDa, but they require the analyte to be dissolved in a matrix, which may lead to the formation of more complex spectra. Also, being soft techniques, ion abundance is

study. The ones most applied to flavonoid research are hereafter presented.

structure elucidation of flavonoids (Fossen & Andersen, 2005).

flavonols, flavanones, catechins, isoflavones and anthocyanidins), it will also address recent developments in minor flavonoid classes. Two fundamental works must be distinguished here. The first is the 1982 book by Markham entitled *Techniques of Flavonoid Identification*, which addresses the state-of-the-art of flavonoid structural identification at the time, and focused largely on UV spectroscopy (Markham, 1982). More recently, in 2005, Markham and Andersen have edited *Flavonoids: Chemistry, Biochemistry and Applications*, which is a reference book for those studying either structure or activity (or both) of flavonoids, containing large tables of collected NMR data and addressing many of the topics under research at that time (Fossen & Andersen, 2005). Hence, this chapter will be mainly focused on developments in the field from 2005 onwards.

#### **2. Overview of flavonoid structural classification**

Flavonoids are polyphenols of plant origin that are among the most important compounds in human diet due to their widespread distribution in foods and beverages. They can occur both in the free form (aglycones) and as glycosides, and differ in their substituents (type, number and position) and in their insaturation. As mentioned, the most common classes are the flavones, flavonols, flavanones, catechins, isoflavones and anthocyanidins, which account for around 80 % of flavonoids. Figure 1 shows the basic structure of the different flavonoid classes addressed in this chapter.

All flavonoids share a basic C6-C3-C6 phenyl-benzopyran backbone. The position of the phenyl ring relative to the benzopyran moiety allows a broad separation of these compounds into flavonoids (2-phenyl-benzopyrans), isoflavonoids (3-phenyl-benzopyrans) and neoflavonoids (4-phenyl-benzopyrans) (Figure 1). Division into further groups is made

Fig. 1. Structure of the structural backbones of the main flavonoid groups (flavan, isoflavan and neoflavan) and of relevant flavonoid classes. Atom numbering and ring nomenclature are also included.

on the basis of the central ring oxidation and on the presence of specific hydroxyl groups. Most common flavonoids are flavones (with a C2-C3 double bond and a C4-oxo function), flavonols (flavones with a 3-OH group) and flavanones (flavone analogues but with a C2-C3 single bond), and abundant isoflavonoids include isoflavones (the analogue of flavones). 4 arylcoumarin (a neoflavonoid with a C3-C4 double bond) and its reduced form, 3,4 dihydro-4-arylcoumarin, are the major neoflavonoids. Other natural compounds, such as chalcones and aurones also possess the C6-C3-C6 backbone, and are henceforth included in the general group of flavonoids.

### **3. Mass Spectrometry**

34 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

flavonols, flavanones, catechins, isoflavones and anthocyanidins), it will also address recent developments in minor flavonoid classes. Two fundamental works must be distinguished here. The first is the 1982 book by Markham entitled *Techniques of Flavonoid Identification*, which addresses the state-of-the-art of flavonoid structural identification at the time, and focused largely on UV spectroscopy (Markham, 1982). More recently, in 2005, Markham and Andersen have edited *Flavonoids: Chemistry, Biochemistry and Applications*, which is a reference book for those studying either structure or activity (or both) of flavonoids, containing large tables of collected NMR data and addressing many of the topics under research at that time (Fossen & Andersen, 2005). Hence, this chapter will be mainly focused

Flavonoids are polyphenols of plant origin that are among the most important compounds in human diet due to their widespread distribution in foods and beverages. They can occur both in the free form (aglycones) and as glycosides, and differ in their substituents (type, number and position) and in their insaturation. As mentioned, the most common classes are the flavones, flavonols, flavanones, catechins, isoflavones and anthocyanidins, which account for around 80 % of flavonoids. Figure 1 shows the basic structure of the different

All flavonoids share a basic C6-C3-C6 phenyl-benzopyran backbone. The position of the phenyl ring relative to the benzopyran moiety allows a broad separation of these compounds into flavonoids (2-phenyl-benzopyrans), isoflavonoids (3-phenyl-benzopyrans) and neoflavonoids (4-phenyl-benzopyrans) (Figure 1). Division into further groups is made

Fig. 1. Structure of the structural backbones of the main flavonoid groups (flavan, isoflavan and neoflavan) and of relevant flavonoid classes. Atom numbering and ring nomenclature

on developments in the field from 2005 onwards.

flavonoid classes addressed in this chapter.

are also included.

**2. Overview of flavonoid structural classification** 

Mass Spectrometry (MS) has proved to be one of the most effective techniques in biomedical research, in special when complex matrixes of biological samples must be analysed. The main advantages of MS are its high sensitivity, which allows analysis of compounds present in the g scale, and high specificity, as it is able to separate molecules of the same molecular weight but different atom composition, and sometimes even to differentiate stereoisomeric compounds. Its easy coupling to separation techniques such as liquid and gas chromatography is also an excellent advantage. A review of separation methods, applied to flavonols, isoflavones and anthocyanidins, has been recently published (Valls et al., 2009). Sample preparation may also be critical, but that varies from sample to sample; nevertheless, some general guidelines have been reviewed (Prasain et al., 2004). For more detail on mass spectrometry equipment and experiments, an excellent book has been published in 2007 (de Hoffmann & Stroobant, 2007). Specifically for electrospray and MALDI mass spectrometry applications in the biology area, the work edited by Cole (Cole, 2010) is highly adequate.

Gas Chromatography (GC) is one of the key techniques for the separation of organics and, coupled to MS, one of the most common techniques of structural identification. However, flavonoids are largely nonvolatile, and need be derivatized; also, they are usually thermally unstable. Both these characteristics have led to the establishment of Liquid Chromatography, in particular High Performance (HPLC), as the fundamental separation technique for flavonoids. Consequently, LC-MS coupling is routinely used for the overall structure elucidation of flavonoids (Fossen & Andersen, 2005).

#### **3.1 Ionization techniques**

Various ionization techniques are available, and each has their own specificities which make them more or less useful depending on the molecules under study and on the aim of such study. The ones most applied to flavonoid research are hereafter presented.

#### **3.1.1 FAB-MS and LSIMS**

Fast-Atom Bombardment (FAB) and Liquid Secondary Ion Mass Spectrometry (LSIMS) are ionization techniques used in Secondary Mass Ion Spectrometry (SIMS) in which secondary ions, emitted by sample irradiation with a beam of energetic (primary) ions, are analyzed. Typically, these techniques are able to produce ions from polar compounds with molecular weights up to 10 kDa, but they require the analyte to be dissolved in a matrix, which may lead to the formation of more complex spectra. Also, being soft techniques, ion abundance is

Structural Analysis

mass analyzers lead to similar fragmentation patterns.

different tasks successively (de Hoffmann & Stroobant, 2007).

**3.3 Tandem mass spectrometry** 

Stroobant, 2007; Abrankó et al., 2011).

**3.3.1 Scan modes**

of Flavonoids and Related Compounds – A Review of Spectroscopic Applications 37

the conclusions also apply to other classes of compounds, such as flavonoids, where different

Tandem mass spectrometry, usually abbreviated MS/MS, or MSn for *nth* order fragmentation, is any method that involves at least two stages of mass analysis, in conjunction with a fragmentation process, either dissociation of reaction, which causes a change in the *m/z* ratio on an ion. Most commonly, a mass analyser is used to isolate a precursor ion, which is then fragment to yield product ions (and, eventually, neutral fragments) that will be detected in the second mass analysis – a typical MS2 experiment. This can, at least conceptually, be expanded with further successive modification and detection steps, giving rise to MS3,… , MSn. However, as only a very small fraction of ions detected in one analyser makes it to the following analyzers, MS3 is usually the highest order achieved. This spatial arrangement of equipment, analyser-modified-analyser, corresponds to tandem MS in space, where ions are treated in different regions of space. Alternatively, tandem MS can be performed in time, with analysers such as ion traps, orbitraps or FT-ICR, where the same analyser performs

*m/z* modification can be achieved by various techniques, but the most common is Collision Induced (or Activated) Dissociation (CID or CAD), where precursor ions undergo collisional activation with neutral atoms or molecules (such as inert gases) in the gas phase. CID is an example of a post-source fragmentation, in which energy is added to the already vibrationally excited ions. An alternative to CID is ECD (Electron Capture Dissociation), in which multiply charged positive ions are submitted to a beam of low energy electrons, producing radical cations. In opposition to post-source fragmentation, in in-source fragmentation, ions already possess sufficient internal energy and fragment spontaneously within the mass spectrometer. Although usually this is an undesired effect, because it leads to lower abundance of precursor ions, it may in some cases become useful (de Hoffmann &

Four general types of tandem MS scans are possible, and all may generate valuable information. A *product ion scan* analyses all the fragment ions resulting from a single selected precursor ion (these are usually called *MS2 spectra*). Conversely, a *precursor ion scan* will identify all the precursors of a selected product ion; a *neutral loss scan* is performed from a selected neutral fragment and will identify the fragmentations leading to the loss of that neutral fragment; these two techniques cannot be performed in time-based analysers (de Hoffmann & Stroobant, 2007). A particular application of neutral loss scans is in the identification of phase II conjugation metabolites, that can be identified by specific neutral losses (Table 1) (Prasain & Barnes, 2007). Neutral loss scans are widely used to detect phase II conjugation metabolites, such as glucuronides (loss of 176 Da) and sulfates (loss of 80 Da), as well as for the detection of glutathione adducts (loss of 129 Da). Scanning for neutral losses of 162 and 132 Da has also been used to separate flavonoids with hexose residues from those with pentose residues, respectively. More selective than these three techniques, *selected reaction monitoring* (SRM) will analyse if a specific product ion comes from the fragmentation of a specific precursor ion; although more sensitive, it is much more specific.

low, and it has been used essentially to identify flavonoid glycosides and for molecular weight determination (Stobiecki, 2000; Prasain et al., 2004).

#### **3.1.2 ESI and APCI**

Electrospray Ionization (ESI) is a technique in which ions are generated by solvent evaporation under a high voltage potential, and can be applied directly, by infusion of the sample with a flow-controlled syringe, or coupled to separation techniques such as LC or capillary electrophoresis. In both cases, a steady liquid stream enters the system, allowing multiple analyses to be performed over a relatively large period of time. ESI interfaces are mostly coupled to quadrupole mass spectrometers; both are simple and robust equipments, able to produce either positive or negative ions, and their main limitation is the relatively limited *m/z* range, usually below 2 kDa. In Atmospheric Pressure Chemical Ionization (APCI) sources ionization occurs via a corona discharge on a heated solvent spray, which produces solvent-derived primary ions that will, in turn, ionize the solute (Prasain et al., 2004; de Hoffmann & Stroobant, 2007). Both ESI and APCI use atmospheric pressure and high collision frequency, and thus generate large amounts of ions; as they involve solvent evaporation, the decomposition of the analytes is reduced, and full scans show limited fragmentation. The main disadvantage of both these techniques is that some HPLC solvents interfere with the ionization process, and thus chromatographic separations need to be specifically designed (Prasain et al*.*, 2004).

#### **3.1.3 MALDI**

Matrix-Assisted Laser Desorption/Ionization (MALDI) is a soft ionization technique in which the analytes are co-crystallized with a matrix; this mixture is deposited on a plate upon which a laser beam is aimed. The laser discharge ultimately leads to analyte ionization and projection from the matrix and onto the analyser. Typical matrixes are derivatives of 4 hydroxycinnamic acid, and also 2,5-dihydroxybenzoic acid (2,5DHB) (de Hoffmann & Stroobant, 2007). Due to the structural similarity between these matrixes and the flavonoids, only recently has MALDI been applied to flavonoid structural elucidation, using a FT-ICR spectrometer, and a significant, although still informative, number of flavonoid-matrix clusters are observable (Madeira & Florêncio, 2009).

#### **3.2 Mass analysis**

Mass analysis is the second step in a MS experiment. Following ion generation, mass analysers measure the mass-to-charge ratio, *m/z*, of the ions, by using a combination of electromagnetic fields. There are many types of mass analysers, as there are of ion sources and detectors. The most common are of the ion type and of the quadrupole type, which analyse *m/z* ratios by the resonance frequency and by the trajectory stability, respectively, and time-of-flight (TOF) analysers, which measure ion velocity (or flight time). More recent resonance frequency analysers, namely Fourier Transform (FT) ion cyclotron resonance (FT-ICR) and FT orbitraps, are now starting to be applied to flavonoids (de Hoffmann & Stroobant, 2007). A recent work has compared the performance of different mass analysers coupled to the same ion source and it was concluded that fragmentation patterns are transferable among different mass analysers, only the relative abundances are changed; although applied to cyano dyes (Volná et al., 2007), the conclusions also apply to other classes of compounds, such as flavonoids, where different mass analyzers lead to similar fragmentation patterns.

#### **3.3 Tandem mass spectrometry**

36 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

low, and it has been used essentially to identify flavonoid glycosides and for molecular

Electrospray Ionization (ESI) is a technique in which ions are generated by solvent evaporation under a high voltage potential, and can be applied directly, by infusion of the sample with a flow-controlled syringe, or coupled to separation techniques such as LC or capillary electrophoresis. In both cases, a steady liquid stream enters the system, allowing multiple analyses to be performed over a relatively large period of time. ESI interfaces are mostly coupled to quadrupole mass spectrometers; both are simple and robust equipments, able to produce either positive or negative ions, and their main limitation is the relatively limited *m/z* range, usually below 2 kDa. In Atmospheric Pressure Chemical Ionization (APCI) sources ionization occurs via a corona discharge on a heated solvent spray, which produces solvent-derived primary ions that will, in turn, ionize the solute (Prasain et al., 2004; de Hoffmann & Stroobant, 2007). Both ESI and APCI use atmospheric pressure and high collision frequency, and thus generate large amounts of ions; as they involve solvent evaporation, the decomposition of the analytes is reduced, and full scans show limited fragmentation. The main disadvantage of both these techniques is that some HPLC solvents interfere with the ionization process, and thus chromatographic separations need to be

Matrix-Assisted Laser Desorption/Ionization (MALDI) is a soft ionization technique in which the analytes are co-crystallized with a matrix; this mixture is deposited on a plate upon which a laser beam is aimed. The laser discharge ultimately leads to analyte ionization and projection from the matrix and onto the analyser. Typical matrixes are derivatives of 4 hydroxycinnamic acid, and also 2,5-dihydroxybenzoic acid (2,5DHB) (de Hoffmann & Stroobant, 2007). Due to the structural similarity between these matrixes and the flavonoids, only recently has MALDI been applied to flavonoid structural elucidation, using a FT-ICR spectrometer, and a significant, although still informative, number of flavonoid-matrix

Mass analysis is the second step in a MS experiment. Following ion generation, mass analysers measure the mass-to-charge ratio, *m/z*, of the ions, by using a combination of electromagnetic fields. There are many types of mass analysers, as there are of ion sources and detectors. The most common are of the ion type and of the quadrupole type, which analyse *m/z* ratios by the resonance frequency and by the trajectory stability, respectively, and time-of-flight (TOF) analysers, which measure ion velocity (or flight time). More recent resonance frequency analysers, namely Fourier Transform (FT) ion cyclotron resonance (FT-ICR) and FT orbitraps, are now starting to be applied to flavonoids (de Hoffmann & Stroobant, 2007). A recent work has compared the performance of different mass analysers coupled to the same ion source and it was concluded that fragmentation patterns are transferable among different mass analysers, only the relative abundances are changed; although applied to cyano dyes (Volná et al., 2007),

weight determination (Stobiecki, 2000; Prasain et al., 2004).

specifically designed (Prasain et al*.*, 2004).

clusters are observable (Madeira & Florêncio, 2009).

**3.1.2 ESI and APCI** 

**3.1.3 MALDI** 

**3.2 Mass analysis** 

Tandem mass spectrometry, usually abbreviated MS/MS, or MSn for *nth* order fragmentation, is any method that involves at least two stages of mass analysis, in conjunction with a fragmentation process, either dissociation of reaction, which causes a change in the *m/z* ratio on an ion. Most commonly, a mass analyser is used to isolate a precursor ion, which is then fragment to yield product ions (and, eventually, neutral fragments) that will be detected in the second mass analysis – a typical MS2 experiment. This can, at least conceptually, be expanded with further successive modification and detection steps, giving rise to MS3,… , MSn. However, as only a very small fraction of ions detected in one analyser makes it to the following analyzers, MS3 is usually the highest order achieved. This spatial arrangement of equipment, analyser-modified-analyser, corresponds to tandem MS in space, where ions are treated in different regions of space. Alternatively, tandem MS can be performed in time, with analysers such as ion traps, orbitraps or FT-ICR, where the same analyser performs different tasks successively (de Hoffmann & Stroobant, 2007).

*m/z* modification can be achieved by various techniques, but the most common is Collision Induced (or Activated) Dissociation (CID or CAD), where precursor ions undergo collisional activation with neutral atoms or molecules (such as inert gases) in the gas phase. CID is an example of a post-source fragmentation, in which energy is added to the already vibrationally excited ions. An alternative to CID is ECD (Electron Capture Dissociation), in which multiply charged positive ions are submitted to a beam of low energy electrons, producing radical cations. In opposition to post-source fragmentation, in in-source fragmentation, ions already possess sufficient internal energy and fragment spontaneously within the mass spectrometer. Although usually this is an undesired effect, because it leads to lower abundance of precursor ions, it may in some cases become useful (de Hoffmann & Stroobant, 2007; Abrankó et al., 2011).

#### **3.3.1 Scan modes**

Four general types of tandem MS scans are possible, and all may generate valuable information. A *product ion scan* analyses all the fragment ions resulting from a single selected precursor ion (these are usually called *MS2 spectra*). Conversely, a *precursor ion scan* will identify all the precursors of a selected product ion; a *neutral loss scan* is performed from a selected neutral fragment and will identify the fragmentations leading to the loss of that neutral fragment; these two techniques cannot be performed in time-based analysers (de Hoffmann & Stroobant, 2007). A particular application of neutral loss scans is in the identification of phase II conjugation metabolites, that can be identified by specific neutral losses (Table 1) (Prasain & Barnes, 2007). Neutral loss scans are widely used to detect phase II conjugation metabolites, such as glucuronides (loss of 176 Da) and sulfates (loss of 80 Da), as well as for the detection of glutathione adducts (loss of 129 Da). Scanning for neutral losses of 162 and 132 Da has also been used to separate flavonoids with hexose residues from those with pentose residues, respectively. More selective than these three techniques, *selected reaction monitoring* (SRM) will analyse if a specific product ion comes from the fragmentation of a specific precursor ion; although more sensitive, it is much more specific.

Structural Analysis

**Fragments - 0,1X**

Hexose 150

Deoxyhexose 134

Pentose 120

Claeys, 2004).

of Flavonoids and Related Compounds – A Review of Spectroscopic Applications 39

Fig. 2. Ion nomenclature used for flavonoid glycosides. Fragmentation of quercetin-7-*O*rutinoside is depicted. Glycoside ions are named according to Domon & Costello, 1988;

**- 0,3X - 1,5X**

90 134 <sup>66</sup>

74 120 <sup>66</sup>

60 104

**- 2,3X – 2H2O (- 3H2O)** 

**- 0,4X** 

(84) 96 162 180

(84) 80 146 164

() 66 132 150

**- 2 H2O - Yi - Xi**

aglycone ions follow the nomenclature of Ma et al., 1997.

**- 0,2X (- H2O) (- 2H2O)** 

> 120 (138) (156)

> 104 (122) (140)

> 90 (108) (126)

Table 2. Characteristic mass losses of flavonoid glycosides and characteristic cross-ring cleavages in the sugar rings of hexoses and pentoses (Vukic & Guttman, 2010; Cuyckens &


Table 1. Mass shift associated with possible metabolic reactions of flavonoids and correspondent detection by neutral loss scanning. Adapted from Prasain & Barnes, 2007.

The following subsections will address the various applications of mass spectrometry to flavonoid structural elucidation, and are organized as a potential guide to explore the structure of novel compounds.

#### **3.4 Flavonoid glycosides – differentiation and characterization**

In plants, flavonoids are often found to be glycosylated; the glycoside residues can be attached to O and C atoms of the flavonoids, giving rise to *O*-glycosides, *C*-glycosides and *O*-*C*-glycosides. These can be differentiated by soft ionization techniques, with low fragmentation energy, usually by FAB-MS, in which glycoside loss from *O-*glycosides undergo heterolysis of their hemi-acetal O-C bonds, gives rise to Yi + ions; at low energy, *C*glycosides only produce [M+H]+ ions, and, at higher energies, intraglycosidic cleaveages rive rise to i,jX fragments and water loss gives rise to characteristic ions (Cuyckens et al., 2000; Li & Claeys, 1994; Vukics & Guttman, 2010). Higher fragmentation energies lead to intraglycosidic cleavage in *O*-glycosides, to the generation of Yi fragments in *C*-glycosides, and to both i,jX and Yi fragments in *O*-*C*-glycosides, all of which correspond to complex, often misleading, mass spectra. (Li & Claeys, 1994). This nomenclature, proposed by Domon & Costello (1988) for the MS study of glycoconjugates, is presented in Figure 2.

The sugar type can be easily determined by the characteristic *m/z* values of the Ai, Bi and Ci fragments arising from hexoses, deoxyhexoses and pentoses, which are not directly observable in the mass spectra but can be computed from the *m/z* differences of the parent ions and corresponding Xi, Yi and Zi fragments (Vukics & Guttman, 2010; Ferreres et al., 2007; Li & Claeys, 1994). The *m/z* values for these fragments are presented in Table 2.

Flavonoid glycosides usually contain one or two glycoside residues, but molecules with more residues have been identified in nature. By definition, diglycosides can have the residues attached at different positions (di-*O*-glycosides and di-*C*,*O*-glycosides) or at the same position (*O*-diglycosides and *C,O*-diglycosides). The differentiation of the different types can be made from the product ions identified in the spectra, particularly from the Zi

**(Neutral loss) Metabolic Reaction Mass change** 

(176) Methylation +14

(162) Hydroxylation +16

(146) Acetylation +42

(132) Carboxylation +44

(80) Decarboxylation -44

(129) Demethylation -14

**(Neutral loss)** 

(–)

(–)

(60)

(44)

(44)

(–)

+ ions; at low energy, *C*-

**Metabolic Reaction Mass change**

Glucuronidation +176

Glycosylation, hexose +162

Glycosylation, deoxyhexose +146

Glycosylation, pentose +132

Sulfation +80

Table 1. Mass shift associated with possible metabolic reactions of flavonoids and correspondent detection by neutral loss scanning. Adapted from Prasain & Barnes, 2007.

**3.4 Flavonoid glycosides – differentiation and characterization** 

undergo heterolysis of their hemi-acetal O-C bonds, gives rise to Yi

& Costello (1988) for the MS study of glycoconjugates, is presented in Figure 2.

The following subsections will address the various applications of mass spectrometry to flavonoid structural elucidation, and are organized as a potential guide to explore the

In plants, flavonoids are often found to be glycosylated; the glycoside residues can be attached to O and C atoms of the flavonoids, giving rise to *O*-glycosides, *C*-glycosides and *O*-*C*-glycosides. These can be differentiated by soft ionization techniques, with low fragmentation energy, usually by FAB-MS, in which glycoside loss from *O-*glycosides

glycosides only produce [M+H]+ ions, and, at higher energies, intraglycosidic cleaveages rive rise to i,jX fragments and water loss gives rise to characteristic ions (Cuyckens et al., 2000; Li & Claeys, 1994; Vukics & Guttman, 2010). Higher fragmentation energies lead to intraglycosidic cleavage in *O*-glycosides, to the generation of Yi fragments in *C*-glycosides, and to both i,jX and Yi fragments in *O*-*C*-glycosides, all of which correspond to complex, often misleading, mass spectra. (Li & Claeys, 1994). This nomenclature, proposed by Domon

The sugar type can be easily determined by the characteristic *m/z* values of the Ai, Bi and Ci fragments arising from hexoses, deoxyhexoses and pentoses, which are not directly observable in the mass spectra but can be computed from the *m/z* differences of the parent ions and corresponding Xi, Yi and Zi fragments (Vukics & Guttman, 2010; Ferreres et al.,

Flavonoid glycosides usually contain one or two glycoside residues, but molecules with more residues have been identified in nature. By definition, diglycosides can have the residues attached at different positions (di-*O*-glycosides and di-*C*,*O*-glycosides) or at the same position (*O*-diglycosides and *C,O*-diglycosides). The differentiation of the different types can be made from the product ions identified in the spectra, particularly from the Zi

2007; Li & Claeys, 1994). The *m/z* values for these fragments are presented in Table 2.

Glutathionylation +129

structure of novel compounds.

Fig. 2. Ion nomenclature used for flavonoid glycosides. Fragmentation of quercetin-7-*O*rutinoside is depicted. Glycoside ions are named according to Domon & Costello, 1988; aglycone ions follow the nomenclature of Ma et al., 1997.


Table 2. Characteristic mass losses of flavonoid glycosides and characteristic cross-ring cleavages in the sugar rings of hexoses and pentoses (Vukic & Guttman, 2010; Cuyckens & Claeys, 2004).

Structural Analysis

of Flavonoids and Related Compounds – A Review of Spectroscopic Applications 41

specific fragmentation patterns. This is especially true in the case of flavan-derived flavonoids, and, to a lesser extent, in the case of some isoflavan-derived flavonoids. For the remaining classes, qualitative MS spectra are still scarce, and, in particular, systematic

The most useful fragmentations of flavonoids, in terms of structural elucidation, are those that involve breaking the C-ring bonds, which are termed retro Diels-Alder (RDA) by analogy with the Diels-Alder cycloaddition. These fragmentations (included in Figure 2) give rise to product ions containing the A or B ring and part of the C ring; for example, the 1,3A+ ion derived from a flavone, formed by a 1/3 fragmentation (cleavage of bonds 1 and 3),

These RDA fragmentations allow the establishment of diagnostic product ions for the various types of flavonoids. These are valid for a large of experimental MS conditions, but it must be kept in mind that the higher fragmentation energies will lead to increased

Acetyl [M+H-acetylhexose]+ (204)

**(mass change, amu)** 

[M±HCO2] (44) [M+H-malonyl]+ (86) [M+H-malonylhexose]+ (248)

[M+H-benzoylhexose]+ (266) [benzoyl]+ *m/z*=105 [benzoylhexose]+ *m/z*=267

[M±H-galloyl] (152)

[M+H-galloylhexose]+ (314) [Gallic acid – H]+ *m/z*=169

[M+H-coumaroylhexose]+ (308)

[M±H-feruloyl] (176) [M±H-feruloylhexose] (338) [feruloyl]+ m/z=177 [ferulic acid]•+ m/z=194 [feruloylhexose]+ m/z=339

[M±H-sinapoyl] (206) [M±H-sinapoylhexose] (368) [sinapoyl]+ m/z=207 [sinapic acid]+ m/z=224 [sinapoylhexose]+ m/z=369

Table 3. Characteristic acyl groups found in acylated glycosyl flavonoids and corresponding characteristic fragmentations. ± stands for either + or –. Adapted from Cuyckens & Claeys,

Coumaroyl [M+H-coumaroyl]+ (146)

(170)

m/z=367

**Acyl group Characteristic fragments** 

[M-H-gallic acid]

[sinapoylhexose]

studies of compounds of the same class are still to be performed.

**3.5.1 Flavones, flavonols, flavanones and flavanonols** 

will contain the whole A ring plus the O1, C4 and O4 atoms.

Malonyl

Benzoyl

Galloyl

Feruloyl

Sinapoyl

2004.

ions – for example, Z1 fragments do not occur in monoglycosides. Also, Yi fragments undergo well-known losses to form radical ions, which can be used to characterize the distribution of sugar residues. It must be noted that in some *O*-glycosides an internal sugar loss may take place, in which the aglycone-bound sugar is released first and, simultaneously, the other residue attaches itself to the aglycone; although this may lead to more complex spectra, this is well described in the literature, and can be overcome by the study of flavonoid-sodium adducts (Vukics & Guttman, 2010; Ma et al., 2000; Brüll et al., 1998; Cuyckens et al., 2001).

Concerning the glycoside sequence of di-, tri- and tetra-glycosides, there is no general procedure to apply for the ordering of the different residues, and it must be done rationally case by base. However, it has been shown that the interglycosidic linkage is easily accessible by MS; although these bonds can be of different types, 12 and 16 bonds are the preferred ones. For these two bond types, the Y0+/Y1+ is always larger for a 12 bond than for a 16 bond – the Y0 + ion is more abundant than the Y1 + ion for glycosides with a 12 bond, and the relative abundance is reversed in the case of a 16 bond. The negative ionisation mode may be particularly important in this case as the Z1 appears to be exclusive of 12 bonded glycosides. In the case of *C*,*O*-diglycosides, it is usually necessary to obtain MS3 spectra to confirm the various interglycosidic linkages, because while 12 glycosides yield, by internal cleavage of the C-glycosyl moiety, 0,2X0 ions, all other possibilities (13, 14 and 16) do not have characteristic fragmentation products (Ma et al., 2000; Cuyckens et al., 2000; Vukics & Guttman, 2010).

In theory, flavonoids could be glycosylated in any position, but *O-*glycosylation occurs mainly at position 7, as in flavones, isoflavones, flavanones and flavonols; *O*-glycosylation in positions 3 and 5 is also frequent, albeit less than in position 7. *C*-glycosyl flavonoids are usually flavones, and the glycosyl moieties are attached at positions 6 or 8; the literature has two reports of 3-*C*-glycosyl-flavones. Positions can be identified with basis of the product ions of the compounds because each glycosylation site appears to yield specific fragmentations. The [Y0-CO] is specific of 7-*O*-monoglycosides, while 3-*O*-monoglycosides are characterized by the [Y0-2H-CO] ion. Also, higher energy fragmentations yield product ions that contain both the glycoside moiety and fragments of the aglycone, particularly Bring derived fragments. In what concerns *C*-glycosides, 6-*C*-glycosides usually undergo more extent fragmentations than 8-*C*-glycosides, and the former are typically associated with a 2,3X+-2H2O fragment and an abundant 0,3X ion, while for the latter no typical product ions have been out forward and the 0,3X ion usually has a low abundance (Li & Claeys, 1994; Vukics & Guttman, 2010; Ferreres et al., 2007; Cuyckens & Claeys, 2005).

Many flavonoid glycosides possess acylated glycosyl moieties. Various acyl groups have been reported in the literature, and they are usually identifiable by characteristic mass losses, which are shown in Table 3. However, the position of glycoside acylation is not easily accessible by mass spectrometry, except for the cases in which a 0,4X fragment is present in the spectra of a hexose-containg flavonoid as it inequivocally establishes that the hexosyl moiety is acylated in position 6 (Cuyckens & Claeys, 2004).

#### **3.5 Aglycones – identification of flavonoid classes**

The identification of the diverse flavonoid classes is often achieved by the MS2 spectra of the various compounds, because, as a general rule, each class of flavonoids is characterized by specific fragmentation patterns. This is especially true in the case of flavan-derived flavonoids, and, to a lesser extent, in the case of some isoflavan-derived flavonoids. For the remaining classes, qualitative MS spectra are still scarce, and, in particular, systematic studies of compounds of the same class are still to be performed.

#### **3.5.1 Flavones, flavonols, flavanones and flavanonols**

40 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

ions – for example, Z1 fragments do not occur in monoglycosides. Also, Yi fragments undergo well-known losses to form radical ions, which can be used to characterize the distribution of sugar residues. It must be noted that in some *O*-glycosides an internal sugar loss may take place, in which the aglycone-bound sugar is released first and, simultaneously, the other residue attaches itself to the aglycone; although this may lead to more complex spectra, this is well described in the literature, and can be overcome by the study of flavonoid-sodium adducts (Vukics & Guttman, 2010; Ma et al., 2000; Brüll et al., 1998; Cuyckens et al., 2001).

Concerning the glycoside sequence of di-, tri- and tetra-glycosides, there is no general procedure to apply for the ordering of the different residues, and it must be done rationally case by base. However, it has been shown that the interglycosidic linkage is easily accessible by MS; although these bonds can be of different types, 12 and 16 bonds are the preferred ones. For these two bond types, the Y0+/Y1+ is always larger for a 12 bond than for a 16 bond – the Y0+ ion is more abundant than the Y1+ ion for glycosides with a 12 bond, and the relative abundance is reversed in the case of a 16 bond. The negative ionisation mode

glycosides. In the case of *C*,*O*-diglycosides, it is usually necessary to obtain MS3 spectra to confirm the various interglycosidic linkages, because while 12 glycosides yield, by internal

not have characteristic fragmentation products (Ma et al., 2000; Cuyckens et al., 2000; Vukics

In theory, flavonoids could be glycosylated in any position, but *O-*glycosylation occurs mainly at position 7, as in flavones, isoflavones, flavanones and flavonols; *O*-glycosylation in positions 3 and 5 is also frequent, albeit less than in position 7. *C*-glycosyl flavonoids are usually flavones, and the glycosyl moieties are attached at positions 6 or 8; the literature has two reports of 3-*C*-glycosyl-flavones. Positions can be identified with basis of the product ions of the compounds because each glycosylation site appears to yield specific

ions that contain both the glycoside moiety and fragments of the aglycone, particularly Bring derived fragments. In what concerns *C*-glycosides, 6-*C*-glycosides usually undergo more extent fragmentations than 8-*C*-glycosides, and the former are typically associated

Many flavonoid glycosides possess acylated glycosyl moieties. Various acyl groups have been reported in the literature, and they are usually identifiable by characteristic mass losses, which are shown in Table 3. However, the position of glycoside acylation is not easily accessible by mass spectrometry, except for the cases in which a 0,4X fragment is present in the spectra of a hexose-containg flavonoid as it inequivocally establishes that the hexosyl

The identification of the diverse flavonoid classes is often achieved by the MS2 spectra of the various compounds, because, as a general rule, each class of flavonoids is characterized by

1994; Vukics & Guttman, 2010; Ferreres et al., 2007; Cuyckens & Claeys, 2005).

appears to be exclusive of 12 bonded

ions, all other possibilities (13, 14 and 16) do

is specific of 7-*O*-monoglycosides, while 3-*O*-monoglycosides

ion. Also, higher energy fragmentations yield product

ion, while for the latter no typical product

ion usually has a low abundance (Li & Claeys,

may be particularly important in this case as the Z1

cleavage of the C-glycosyl moiety, 0,2X0

& Guttman, 2010).

fragmentations. The [Y0-CO]

are characterized by the [Y0-2H-CO]

with a 2,3X+-2H2O fragment and an abundant 0,3X

moiety is acylated in position 6 (Cuyckens & Claeys, 2004).

**3.5 Aglycones – identification of flavonoid classes** 

ions have been out forward and the 0,3X

The most useful fragmentations of flavonoids, in terms of structural elucidation, are those that involve breaking the C-ring bonds, which are termed retro Diels-Alder (RDA) by analogy with the Diels-Alder cycloaddition. These fragmentations (included in Figure 2) give rise to product ions containing the A or B ring and part of the C ring; for example, the 1,3A+ ion derived from a flavone, formed by a 1/3 fragmentation (cleavage of bonds 1 and 3), will contain the whole A ring plus the O1, C4 and O4 atoms.

These RDA fragmentations allow the establishment of diagnostic product ions for the various types of flavonoids. These are valid for a large of experimental MS conditions, but it must be kept in mind that the higher fragmentation energies will lead to increased


Table 3. Characteristic acyl groups found in acylated glycosyl flavonoids and corresponding characteristic fragmentations. ± stands for either + or –. Adapted from Cuyckens & Claeys, 2004.

Structural Analysis

Simons et al., 2011).

**3.5.3 Chalcones** 

**3.5.4 Other classes**

fragmentation of daidzein (Wei et al., 2000).

double neutral loss of CO (Kuhn et al*.*, 2003).

of Flavonoids and Related Compounds – A Review of Spectroscopic Applications 43

published. Besides common neutral losses, isoflavones undergo RDA fragmentation to yield the 0,4B+-H2O and the 1,3A+ ions; a full B ring loss, yielding the [M-*B ring*-CO]+ ion, is also frequently reported. Also characteristic is the loss of a CO group from Ring C oxo group, leading to ring contraction (Madeira et al., 2010; Heinonen et al., 2003; Borges et al., 2001;

In the negative mode, the 1/3 and 0/3 RDA fragmentations are predominant, and the 0/4 fragmentation is also observed sometimes, accompanied by extensive losses of CO, CO2 and C3O2 moieties. C ring expansion is also commonly observed (Vessecchi et al., 2011; Kang et al., 2007; March et al., 2004; Ablajan, 2011). For isoflavones, rare RDA fragmentations have been described in the literature, such as a 2/3 fragmentation that has also been described for various acylated 7,2'-hydroxy-3',4'-dimethoxyisoflavan glycosides (Qi et al., 2008) and a 2/4

The main difference in the fragmentation pathways of flavan- and isoflavan-derived flavonoids appears to be that in the latter the 0/3 RDA fragmentation occurs frequently while it appears not to occur for flavan-compounds; also, the 1/3 pathway appears to be much more important in isoflavan-compounds, in particular in those without a 2-OH group. Differentiation of isomeric aglycones of flavones and isoflavones is also possible based on a

Chalcones exist in nature with a variety of substituents. The MS spectra of these compounds are characterized by substituent loss, fragmentations of the substituents and chalcone fragmentations. Chalcone fragmentations are dominated by cleavage of a single bond, yielding a B ring derived ion with the attached C=O group in the charged form (C≡O+), from which a CO loss yield the free B ring (Nowakowska & Pankiewicz, 2008). Besides that, 2'-OH-chalcones, the most common ones, with an OH group in ring B adjacent to the propenone chain, are known to be converted to the corresponding flavanones by various processes, and that has also been observed to occur in ESI MS; the patter of chalcone

fragmentation will then be the same of flavanone fragmentation (Zhang et al*.*, 2008).

no useful fragmentation pathways have been established.

Pankiewicz, 2008; Zhang et al., 2007; Zhang et al., 2008).

which may lose a further CO group, and the 1,3B+ ion (Kesari et al., 2004).

Many other flavonoid classes exist. Many of those have been isolated and characterized, but

For aurones, a positive mode of two aurone glycosides was identified as main product ions the ones formed by the loss of the glycosyl moiety, followed by a CO loss involving the heterocyclic O atom in the 5-member ring, and, from the [M-glycosyl+H]•+ ion, the 1,3A+ ion,

Coumarins show a wide diversity of substituents, and many even have fused rings attached. This variability does not allow overall fragmentation patterns, but many studies observe that in many cases the heterocyclic ring undergoes a contraction by CO loss (from the C=O group) to yield an ion containing a five membered ring (for example, Nowakowska &

fragmentation and a higher abundance of ions with lower masses, deviating from these diagnostic ions and fragmentation pathways.

The most important RDA fragmentations in flavones and flavonols (3-hydroxyl-flavones) are the 0/2, the 0/4, the 1/3. While the 0/4 pathway appears to be exclusive of flavones, according to a low-energy CID study, where it leads to the 0,4B+ ion, which looses water to form the 0,4B+ – H2O, the 0,2A+ is an exclusive of flavonols; also, the 1,3B+ ion appears to be exclusive of flavones. In common, flavonoids from both classes give rise to the product ions 1,3A+, and 0,2B+ (Cuyckens et al., 2005). This information is essential when analysing, for example, flavonoid metabolites by MS. Quercetin metabolism leads to the formation of, among others, conjugates with sulphate and glucuronic acid, as well as to methoxyquercetin. Considering a full scan MS of quercetin (molecular weight of 302 g/mol) metabolites collected from rat plasma, an ion at *m/z* = 479 corresponds to a protonate quercetin glucoronide; the 1/4 RDA fragmentation produces a 1,4A+ at *m/z* = 303, that bears the mass increase, meaning the glucuronosyl moiety is present in this ion; therefore, it must be located at either position 5 or 7 (Justino et al., 2004). Similar rationales for other ions lead to the identification of the positions of metabolisation in these compounds.

More recently, a mixed ESI-MS and quantum chemical approach has analysed the fragmentation pathways of flavones and flavanols, and has proposed structures for other ions, other than RDA-derived, that are also informative in terms of structural elucidation. In particular, losses of one and two C2H2O moieties from the precursor ion, involving all the rings, and the formation of 1,3A+ – C2H2O ion, are the most useful (Justino et al., 2009). Similar methodology has been applied in other cases (Madeira et al; 2010).

Flavones and flavonols constitute the vast majority of the flavan-based flavonoids studies. Flavanones, which lack the C ring 2-3 double bond, are less abundant than those two and have received less attention. In terms of MS fragmentations, in particular RDA pathways, flavanone itself undergoes cleavages to yield the 1,3A+ and 1,4B+ ions; these same ions are also observed for other flavanones, such as 5,7,4'-tri-hydroxy-flavanone (Nikolic & van Breemen, 2004). Studies on flavanone derivatives have evidenced that the positive mode CID spectra of isoprenylated flavanones is dominated by isoprenyl loss and 1,3A+ ion formation, while the negative mode is dominated by 1,4A–, [1,3A– - isoprenyl] and [1,4A– - isoprenyl] ions. (Zhang et al., 2008) Derived ions, such as [1,3A + H]+ and [1,4B – H2 + H]+, have also been identified. (Zhou et al., 2007). Negative mode ESI has also allowed dividing flavanones into two groups, one with a 2'-OH group, with a spectra dominated by the 1,4A– ion (for 5-OHcontaining molecules) or the 1,4B- (otherwise), and the other with no 2' substituents, for which the spectra were dominated by an intense 1,3A– ion and a low abundant 1,3B– ion. (Zhang et al., 2008) a 0/3 fragmentation, origination a 0,3B- ion, has also been observed (Xu et al*.*, 2009); an equivalent fragmentation has also been observed in the positive mode , to produce a 0,3B+ derived ion (Kéki et al*.*, 2007). Flavanols (3-hydroxy-flavanones) show fragmentation patterns similar to those of flavanones; the main difference is that positive ESI spectra of flavanols are dominated by loss of water and homolytic H loss from the 3 hydroxy group to generate the [M-H]•+ ion (Zhang et al., 2008).

#### **3.5.2 Isoflavones and Isoflavonols**

Unlike flavan derived flavonoids, the fragmentation pathways of isoflavan derived flavonoids have only been explored more recently, but a good set of systematic studies are published. Besides common neutral losses, isoflavones undergo RDA fragmentation to yield the 0,4B+-H2O and the 1,3A+ ions; a full B ring loss, yielding the [M-*B ring*-CO]+ ion, is also frequently reported. Also characteristic is the loss of a CO group from Ring C oxo group, leading to ring contraction (Madeira et al., 2010; Heinonen et al., 2003; Borges et al., 2001; Simons et al., 2011).

In the negative mode, the 1/3 and 0/3 RDA fragmentations are predominant, and the 0/4 fragmentation is also observed sometimes, accompanied by extensive losses of CO, CO2 and C3O2 moieties. C ring expansion is also commonly observed (Vessecchi et al., 2011; Kang et al., 2007; March et al., 2004; Ablajan, 2011). For isoflavones, rare RDA fragmentations have been described in the literature, such as a 2/3 fragmentation that has also been described for various acylated 7,2'-hydroxy-3',4'-dimethoxyisoflavan glycosides (Qi et al., 2008) and a 2/4 fragmentation of daidzein (Wei et al., 2000).

The main difference in the fragmentation pathways of flavan- and isoflavan-derived flavonoids appears to be that in the latter the 0/3 RDA fragmentation occurs frequently while it appears not to occur for flavan-compounds; also, the 1/3 pathway appears to be much more important in isoflavan-compounds, in particular in those without a 2-OH group. Differentiation of isomeric aglycones of flavones and isoflavones is also possible based on a double neutral loss of CO (Kuhn et al*.*, 2003).

#### **3.5.3 Chalcones**

42 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

fragmentation and a higher abundance of ions with lower masses, deviating from these

The most important RDA fragmentations in flavones and flavonols (3-hydroxyl-flavones) are the 0/2, the 0/4, the 1/3. While the 0/4 pathway appears to be exclusive of flavones, according to a low-energy CID study, where it leads to the 0,4B+ ion, which looses water to form the 0,4B+ – H2O, the 0,2A+ is an exclusive of flavonols; also, the 1,3B+ ion appears to be exclusive of flavones. In common, flavonoids from both classes give rise to the product ions 1,3A+, and 0,2B+ (Cuyckens et al., 2005). This information is essential when analysing, for example, flavonoid metabolites by MS. Quercetin metabolism leads to the formation of, among others, conjugates with sulphate and glucuronic acid, as well as to methoxyquercetin. Considering a full scan MS of quercetin (molecular weight of 302 g/mol) metabolites collected from rat plasma, an ion at *m/z* = 479 corresponds to a protonate quercetin glucoronide; the 1/4 RDA fragmentation produces a 1,4A+ at *m/z* = 303, that bears the mass increase, meaning the glucuronosyl moiety is present in this ion; therefore, it must be located at either position 5 or 7 (Justino et al., 2004). Similar rationales for other ions lead

More recently, a mixed ESI-MS and quantum chemical approach has analysed the fragmentation pathways of flavones and flavanols, and has proposed structures for other ions, other than RDA-derived, that are also informative in terms of structural elucidation. In particular, losses of one and two C2H2O moieties from the precursor ion, involving all the rings, and the formation of 1,3A+ – C2H2O ion, are the most useful (Justino et al., 2009).

Flavones and flavonols constitute the vast majority of the flavan-based flavonoids studies. Flavanones, which lack the C ring 2-3 double bond, are less abundant than those two and have received less attention. In terms of MS fragmentations, in particular RDA pathways, flavanone itself undergoes cleavages to yield the 1,3A+ and 1,4B+ ions; these same ions are also observed for other flavanones, such as 5,7,4'-tri-hydroxy-flavanone (Nikolic & van Breemen, 2004). Studies on flavanone derivatives have evidenced that the positive mode CID spectra of isoprenylated flavanones is dominated by isoprenyl loss and 1,3A+ ion formation, while the negative mode is dominated by 1,4A–, [1,3A– - isoprenyl] and [1,4A– - isoprenyl] ions. (Zhang et al., 2008) Derived ions, such as [1,3A + H]+ and [1,4B – H2 + H]+, have also been identified. (Zhou et al., 2007). Negative mode ESI has also allowed dividing flavanones into two groups, one with a 2'-OH group, with a spectra dominated by the 1,4A– ion (for 5-OHcontaining molecules) or the 1,4B- (otherwise), and the other with no 2' substituents, for which the spectra were dominated by an intense 1,3A– ion and a low abundant 1,3B– ion. (Zhang et al., 2008) a 0/3 fragmentation, origination a 0,3B- ion, has also been observed (Xu et al*.*, 2009); an equivalent fragmentation has also been observed in the positive mode , to produce a 0,3B+ derived ion (Kéki et al*.*, 2007). Flavanols (3-hydroxy-flavanones) show fragmentation patterns similar to those of flavanones; the main difference is that positive ESI spectra of flavanols are dominated by loss of water and homolytic H loss from the 3-

Unlike flavan derived flavonoids, the fragmentation pathways of isoflavan derived flavonoids have only been explored more recently, but a good set of systematic studies are

to the identification of the positions of metabolisation in these compounds.

Similar methodology has been applied in other cases (Madeira et al; 2010).

hydroxy group to generate the [M-H]•+ ion (Zhang et al., 2008).

**3.5.2 Isoflavones and Isoflavonols**

diagnostic ions and fragmentation pathways.

Chalcones exist in nature with a variety of substituents. The MS spectra of these compounds are characterized by substituent loss, fragmentations of the substituents and chalcone fragmentations. Chalcone fragmentations are dominated by cleavage of a single bond, yielding a B ring derived ion with the attached C=O group in the charged form (C≡O+), from which a CO loss yield the free B ring (Nowakowska & Pankiewicz, 2008). Besides that, 2'-OH-chalcones, the most common ones, with an OH group in ring B adjacent to the propenone chain, are known to be converted to the corresponding flavanones by various processes, and that has also been observed to occur in ESI MS; the patter of chalcone fragmentation will then be the same of flavanone fragmentation (Zhang et al*.*, 2008).

#### **3.5.4 Other classes**

Many other flavonoid classes exist. Many of those have been isolated and characterized, but no useful fragmentation pathways have been established.

For aurones, a positive mode of two aurone glycosides was identified as main product ions the ones formed by the loss of the glycosyl moiety, followed by a CO loss involving the heterocyclic O atom in the 5-member ring, and, from the [M-glycosyl+H]•+ ion, the 1,3A+ ion, which may lose a further CO group, and the 1,3B+ ion (Kesari et al., 2004).

Coumarins show a wide diversity of substituents, and many even have fused rings attached. This variability does not allow overall fragmentation patterns, but many studies observe that in many cases the heterocyclic ring undergoes a contraction by CO loss (from the C=O group) to yield an ion containing a five membered ring (for example, Nowakowska & Pankiewicz, 2008; Zhang et al., 2007; Zhang et al., 2008).

Structural Analysis

intense spectra (Satterfield & Brodbelt, 2000).

**4. Nuclear magnetic resonance spectroscopy** 

literature (reviewed in Fossen & Andersen, 2005).

compared to 1H (99.9 %) (Claridge, 1999).

**4.2 Homonuclear 2D NMR** 

various groups, requires various 2D experiments.

**H and 13C** 

**4.1 One Dimensional NMR: <sup>1</sup>**

molecule.

of Flavonoids and Related Compounds – A Review of Spectroscopic Applications 45

used to assist the elucidation of flavonoid glucuronides (Davis et al*.*, 2006; Davis & Brodbelt, 2007) and various diglycosides (Pikulski et al., 2007). This is a result of the spectral changes observed when flavonoids are complexed with metals, giving rise to simpler yet more

Nuclear Magnetic Resonance spectroscopy, hereafter simply designated by NMR, is one the most powerful research techniques used to investigate the structure and some properties of molecules. One of the main applications of NMR in flavonoid research is the structural elucidation of novel compounds, for which nothing is known; although NMR traditionally requires large amounts of sample, which is not easy to obtain when analysing novel compounds, the technical developments in the last decade, both in NMR instrumentation, pulse programs and in computing power, have allowed the complete assignment of all proton and carbon signals using amounts in the order of 1 mg (Fossen & Andersen, 2005). The major goal of this section is to summarize the applications of the various NMR experiments to flavonoid research, together with the information (other than atom connectivity) that can be taken from these experiments. Flavonoid NMR data are not presented here, as these are easily accessible, for a vast number of compounds, from the

The two most basic NMR experiments are the 1H and the 13C NMR experiments, which are aimed at the determination of the resonance frequency of each 1H or 13C nucleus in the

1H NMR experiments register the chemical shifts () and spin-spin couplings, the latter described by the coupling constants (*J*). This provides valuable information about the relative number of hydrogens and also their type, by comparison of the recorded chemical shifts with compiled data. This is particularly useful in establishing the aglycone type and the acyl groups attached to it, as well as in identifying the number and the anomeric configuration of the glycoside moieties attached to the aglycone. 13C NMR data is used to complement 1H NMR data, and is particularly useful at establishing the type of groups present in the samples' molecules by comparison with compiled data; however, it must be noted that 13C NMR is much less sensitive due to the abundance of 13C (1.1 %) when

Together, these two 1D experiments are used primarily to identify aglycone types and substituent groups, but a definite structural elucidation, which the accurate location of the

2D NMR experiments generate contour maps that show the correlations between different nuclei in the molecules, and can be either homonuclear or heteronuclear, depending on whether the interacting nuclei are of the same or different elements (Claridge, 1999). COSY (COrrelation SpectroscopY) was one of the first multidimensional systems. COSY

Pterocarpans, isoflavonoid derivatives, show much more complex fragmentation pathways than the above analysed classes of flavonoids; MS studies of different deuterated peterocarpan derivatives, as well as of pterocarpan glycosides, points out that these are dominated by various and successive ring openings and/or contractions (Tóth et al*.,* 2000*;*  Zhang et al., 2007). A rare 2/4 RDA fragmentation, like that of isoflavan-derived flavonoids, has also been observed for pterocarpans in the negative mode (Simons et al., 2011).

Neoflavonoids, in which the B ring is attached in position 4, and include 4-aryl-coumarins, have been poorly studied by MS. The few reports available indicate, however, that neoflavonoid fragmentation is dominated by loss of the CH3 radical in methoxylated compounds and by B ring loss, to yield the [M+H-B ring] fragment (Charles et al., 2005; Hulme et al., 2005; Liu et al., 2005). Figure 3 summarizes the characteristic RDA fragmentation pathways for flavonoid classes for which there is reliable information.

Fig. 3. Diagnostic products of flavones, flavonols, isoflavones, isoflavonols and pterocarpans. ± stands for either + or –. Adapted from Cuyckens et al, 2000; Kuhn et al., 2003; Madeira et al, 2010; Wei et al., 2000; Kang et al., 2007 and Simons et al., 2011.

#### **3.6 Flavonoid-metal complexes**

Flavonoids are good chelating agents towards metal ions and, in the case of iron and copper, the favoured places of chelation are catechol groups, hydroxyl groups adjacent to oxo groups, and 1-oxo-3-hydroxyl-containing moieties (Ren et al*.*, 2008; Fernandez et al., 2002). This ability to chelate metals has been used to enhance the capabilities of MS; it has been used to assist the elucidation of flavonoid glucuronides (Davis et al*.*, 2006; Davis & Brodbelt, 2007) and various diglycosides (Pikulski et al., 2007). This is a result of the spectral changes observed when flavonoids are complexed with metals, giving rise to simpler yet more intense spectra (Satterfield & Brodbelt, 2000).

#### **4. Nuclear magnetic resonance spectroscopy**

44 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Pterocarpans, isoflavonoid derivatives, show much more complex fragmentation pathways than the above analysed classes of flavonoids; MS studies of different deuterated peterocarpan derivatives, as well as of pterocarpan glycosides, points out that these are dominated by various and successive ring openings and/or contractions (Tóth et al*.,* 2000*;*  Zhang et al., 2007). A rare 2/4 RDA fragmentation, like that of isoflavan-derived flavonoids,

Neoflavonoids, in which the B ring is attached in position 4, and include 4-aryl-coumarins, have been poorly studied by MS. The few reports available indicate, however, that neoflavonoid fragmentation is dominated by loss of the CH3 radical in methoxylated compounds and by B ring loss, to yield the [M+H-B ring] fragment (Charles et al., 2005; Hulme et al., 2005; Liu et al., 2005). Figure 3 summarizes the characteristic RDA

has also been observed for pterocarpans in the negative mode (Simons et al., 2011).

fragmentation pathways for flavonoid classes for which there is reliable information.

Fig. 3. Diagnostic products of flavones, flavonols, isoflavones, isoflavonols and

**3.6 Flavonoid-metal complexes** 

pterocarpans. ± stands for either + or –. Adapted from Cuyckens et al, 2000; Kuhn et al., 2003; Madeira et al, 2010; Wei et al., 2000; Kang et al., 2007 and Simons et al., 2011.

Flavonoids are good chelating agents towards metal ions and, in the case of iron and copper, the favoured places of chelation are catechol groups, hydroxyl groups adjacent to oxo groups, and 1-oxo-3-hydroxyl-containing moieties (Ren et al*.*, 2008; Fernandez et al., 2002). This ability to chelate metals has been used to enhance the capabilities of MS; it has been

Nuclear Magnetic Resonance spectroscopy, hereafter simply designated by NMR, is one the most powerful research techniques used to investigate the structure and some properties of molecules. One of the main applications of NMR in flavonoid research is the structural elucidation of novel compounds, for which nothing is known; although NMR traditionally requires large amounts of sample, which is not easy to obtain when analysing novel compounds, the technical developments in the last decade, both in NMR instrumentation, pulse programs and in computing power, have allowed the complete assignment of all proton and carbon signals using amounts in the order of 1 mg (Fossen & Andersen, 2005).

The major goal of this section is to summarize the applications of the various NMR experiments to flavonoid research, together with the information (other than atom connectivity) that can be taken from these experiments. Flavonoid NMR data are not presented here, as these are easily accessible, for a vast number of compounds, from the literature (reviewed in Fossen & Andersen, 2005).

#### **4.1 One Dimensional NMR: <sup>1</sup> H and 13C**

The two most basic NMR experiments are the 1H and the 13C NMR experiments, which are aimed at the determination of the resonance frequency of each 1H or 13C nucleus in the molecule.

1H NMR experiments register the chemical shifts () and spin-spin couplings, the latter described by the coupling constants (*J*). This provides valuable information about the relative number of hydrogens and also their type, by comparison of the recorded chemical shifts with compiled data. This is particularly useful in establishing the aglycone type and the acyl groups attached to it, as well as in identifying the number and the anomeric configuration of the glycoside moieties attached to the aglycone. 13C NMR data is used to complement 1H NMR data, and is particularly useful at establishing the type of groups present in the samples' molecules by comparison with compiled data; however, it must be noted that 13C NMR is much less sensitive due to the abundance of 13C (1.1 %) when compared to 1H (99.9 %) (Claridge, 1999).

Together, these two 1D experiments are used primarily to identify aglycone types and substituent groups, but a definite structural elucidation, which the accurate location of the various groups, requires various 2D experiments.

#### **4.2 Homonuclear 2D NMR**

2D NMR experiments generate contour maps that show the correlations between different nuclei in the molecules, and can be either homonuclear or heteronuclear, depending on whether the interacting nuclei are of the same or different elements (Claridge, 1999). COSY (COrrelation SpectroscopY) was one of the first multidimensional systems. COSY

Structural Analysis

**4.5 Solid state NMR** 

**5. UV-vis spectrophotometry** 

of Flavonoids and Related Compounds – A Review of Spectroscopic Applications 47

Further developments in NMR experiments, using new 2D and 3D techniques, have been developed in recent years, and are starting to be used for flavonoid analysis. In particular, 2D and 3D HSQC-TOCSY experiments are capable of assigning all 13C signals of individual

While the above mentioned 2D techniques are useful to establish the connectivities between atoms through bonds, the Nuclear Overhauser Effect (NOE), which can be summarized as "A change in the intensity of an NMR signal from a nucleus, observed when a neighboring nucleus is saturated", is useful at establishing non-bonded connectivities, or connectivities through space. The crosspeaks in a 1H-1H NOESY (NOE SpectroscopY) spectrum correspond to correlations between protons that are close to each other in space (up to 4 Å) but not necessarily connected through bonds; these correlations may arise from both intramolecular and intermolecular proton interactions, and has been successfully used to establish rotational conformers and restrictions, establish intermolecular associations and even solve proteinligand and DNA-ligand structures. A 2D NOE experiment, ROESY (Rotating Overhauser Effect SpectroscopY), has been used in flavonoid research mainly to establish the stereochemistry of various flavonoids (Claridge, 1999; Fossen & Andersen, 2005). Figure 4

presents sample NMR spectra of anthocyanidine glucosides (Jordheim et al., 2006).

association. (Fossen & Andersen, 2005; Olejniczak & Potrzebowski, 2004)

304 nm, and that of 3-hydroxylated flavonoles is centred around 352 nm.

using post-column UV shift reagents for the flavonoid analysis of crude extracts.

X-ray crystallography depends on the ability to obtain flavonoid crystals, which has only been achieved for a small number of flavonoids; in alternative, the 1H CP-MAS (Cross Polarization Magic Angle Spinning) NMR techniques have been used to elucidate the solid state conformation of flavonoids, either pure or, for example, in tissues, providing enough sample is available (typically in the 10 mg scale). In particular, such an experiment had provided information on the planarity of the flavonoid rings, on the intramolecular H bonding between the 4-oxo group and the 3- and 5-hydroxy groups, and on intermolecular

Ultraviolet and visible spectroscopy was one of the earliest techniques routinely used for flavonoid analysis due to the existence of two characteristic UV/Vis bands in flavonoids, band I in the 300 to 550 nm range, arising from the B ring, and band II in the 240 to 285 nm range, arising from the A ring. For examples, while the band I of flavones and flavonols lies in the 240 – 285 nm range, that of flavanone (no C ring instauration) lies in the 270 – 295 nm range; conversely, the band II of flavones and flavanones (no 3-OH group) lies around 303 –

Shift reagents, such as sodium methoxide and aluminium chrolide, lead to shifts in the maximum wavelength of these bands due to methoxide-induced deprotonation of OH groups or Al3+ complexation by OH groups, were also routinely used to study flavonoid structure. Nowadays these techniques are not routinely used but still continue to be applied in some cases, in particular to HPLC eluates - a hyphenated LC-UV-MS has been developed

glycosides in polyglycosilated flavonoids (Fossen & Andersen, 2005).

**4.4 Connectivity through space – the nuclear overhauser effect** 

crosspeaks are between protons that are coupled to each other, usually two bonds apart (2*J*HH), but sometimes also three and four bonds apart (3*J*HH and 4*J*HH); the intensity of coupling affects the intensity of the peak. DQF-COSY (Double Quantum Filter COSY) is an improvement of the COSY experiment in which non-coupled proton signals, such as those from solvent, are eliminated as they may overlap signals from the analyte (Claridge, 1999). A further improvement is the TOCSY (TOtal Correlation Spectroscopy) experiment, which creates correlations between *all* protons in a given spin system, as long as there are couplings between every intervening protons; this is extremely useful to identify protons on sugar rings – every proton from one sugar ring will have a correlation with all other protons from the same ring but not with those of other rings. Magnetization is transferred over up to 5 or 6 bonds, and is interrupted by small or null 1H-1H couplings and hetero-atoms; also, the number of transfer steps can be adjusted by changing the spin-lock time (Fossen & Andersen, 2005). A good reference for the TOCSY transfer in various sugars is Gheysen's work (Gheysen et al., 2008). Selective 1D TOCSY (also known as HOHAHA, homonuclear Hartman-Hahn) is particularly useful in compounds with more than one sugar moiety, in which overlap occurs; in this experiment, one peak is selected and that magnetization is transferred stepwisely to the protons in the same spin system; instead of crosspeaks, transfer is shown by increased multiplet intensity (Fossen & Andersen, 2005).

#### **4.3 Heteronuclear 2D NMR**

Heteronuclear 2D NMR experiments correlate nuclei of different elements. The most powerful techniques of all are undoubtedly the 2D proton–carbon experiments HMQC/HSQC (Heteronuclear Multiple Quantum Coherence/Heteronuclear Single Quantum Coherence) and HMBC (Heteronuclear Multiple Bond Correlation) as they provide an opportunity to dovetail proton and carbon NMR data directly.

HMQC and HSQC establish one bond correlations between the protons of a molecule and the carbons to which they are attached (1*J*CH). Both these are much more sensitive than the correspondent 1D 13C experiments; while in 1D experiment the low abundance of the isotope leads to a low signal-to-noise ratio, in the heteronuclear 2D experiment the initial magnetization occurs on the highly sensitive 1H nuclei and is then transferred to the 13C atoms that are connected to each proton. A similar NMR experiment is 1H-13C HMBC (Heteronuclear Multiple Bond Correlation), in which long-range interactions (typically 2*J*CH and 3*J*CH) are analyzed; HMBC is usually more sensitive to 3-bond correlations than to 2-bond correlations, but this depends on the overall signal-to-noise ratios and on the adjustable parameters of each experiment. A newer experiment, 2*J*CH,3*J*CH-HBMC, has been designed to differentiate these two types of correlations (Claridge, 1999; Krishnamurthy, 2000; Fossen & Andersen, 2005).

HBMC application to flavonoids usually addresses assignment on nonprotonated C atoms, from both the aglycones and acyl groups. Unlike TOCSY, HMBC transfer is not stopped by heteroatoms, and so it can also be used to determine the linkage points of heteroatomcontaining groups such as sugar residues. HMBC is also useful to distinguish some classes of flavonoids, as flavones from aurones, which have similar 1H and 13C NMR spectra but very different HMBC spectra. Currently, only enhanced variants of the HSQC and HMBC experiments, namely gradient enhanced (*ge*) ones, are used, due to their higher sensitivity and capacity. These have been used to establish strong intramolecular H bonding between the 4-oxo and 5-hydroxy groups in flavonoids (Exarchou et al., 2002; Kozerski et al., 2003).

Further developments in NMR experiments, using new 2D and 3D techniques, have been developed in recent years, and are starting to be used for flavonoid analysis. In particular, 2D and 3D HSQC-TOCSY experiments are capable of assigning all 13C signals of individual glycosides in polyglycosilated flavonoids (Fossen & Andersen, 2005).

#### **4.4 Connectivity through space – the nuclear overhauser effect**

While the above mentioned 2D techniques are useful to establish the connectivities between atoms through bonds, the Nuclear Overhauser Effect (NOE), which can be summarized as "A change in the intensity of an NMR signal from a nucleus, observed when a neighboring nucleus is saturated", is useful at establishing non-bonded connectivities, or connectivities through space. The crosspeaks in a 1H-1H NOESY (NOE SpectroscopY) spectrum correspond to correlations between protons that are close to each other in space (up to 4 Å) but not necessarily connected through bonds; these correlations may arise from both intramolecular and intermolecular proton interactions, and has been successfully used to establish rotational conformers and restrictions, establish intermolecular associations and even solve proteinligand and DNA-ligand structures. A 2D NOE experiment, ROESY (Rotating Overhauser Effect SpectroscopY), has been used in flavonoid research mainly to establish the stereochemistry of various flavonoids (Claridge, 1999; Fossen & Andersen, 2005). Figure 4 presents sample NMR spectra of anthocyanidine glucosides (Jordheim et al., 2006).

#### **4.5 Solid state NMR**

46 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

crosspeaks are between protons that are coupled to each other, usually two bonds apart (2*J*HH), but sometimes also three and four bonds apart (3*J*HH and 4*J*HH); the intensity of coupling affects the intensity of the peak. DQF-COSY (Double Quantum Filter COSY) is an improvement of the COSY experiment in which non-coupled proton signals, such as those from solvent, are eliminated as they may overlap signals from the analyte (Claridge, 1999). A further improvement is the TOCSY (TOtal Correlation Spectroscopy) experiment, which creates correlations between *all* protons in a given spin system, as long as there are couplings between every intervening protons; this is extremely useful to identify protons on sugar rings – every proton from one sugar ring will have a correlation with all other protons from the same ring but not with those of other rings. Magnetization is transferred over up to 5 or 6 bonds, and is interrupted by small or null 1H-1H couplings and hetero-atoms; also, the number of transfer steps can be adjusted by changing the spin-lock time (Fossen & Andersen, 2005). A good reference for the TOCSY transfer in various sugars is Gheysen's work (Gheysen et al., 2008). Selective 1D TOCSY (also known as HOHAHA, homonuclear Hartman-Hahn) is particularly useful in compounds with more than one sugar moiety, in which overlap occurs; in this experiment, one peak is selected and that magnetization is transferred stepwisely to the protons in the same spin system; instead of crosspeaks, transfer

Heteronuclear 2D NMR experiments correlate nuclei of different elements. The most powerful techniques of all are undoubtedly the 2D proton–carbon experiments HMQC/HSQC (Heteronuclear Multiple Quantum Coherence/Heteronuclear Single Quantum Coherence) and HMBC (Heteronuclear Multiple Bond Correlation) as they

HMQC and HSQC establish one bond correlations between the protons of a molecule and the carbons to which they are attached (1*J*CH). Both these are much more sensitive than the correspondent 1D 13C experiments; while in 1D experiment the low abundance of the isotope leads to a low signal-to-noise ratio, in the heteronuclear 2D experiment the initial magnetization occurs on the highly sensitive 1H nuclei and is then transferred to the 13C atoms that are connected to each proton. A similar NMR experiment is 1H-13C HMBC (Heteronuclear Multiple Bond Correlation), in which long-range interactions (typically 2*J*CH and 3*J*CH) are analyzed; HMBC is usually more sensitive to 3-bond correlations than to 2-bond correlations, but this depends on the overall signal-to-noise ratios and on the adjustable parameters of each experiment. A newer experiment, 2*J*CH,3*J*CH-HBMC, has been designed to differentiate these two types of correlations (Claridge, 1999; Krishnamurthy, 2000; Fossen & Andersen, 2005).

HBMC application to flavonoids usually addresses assignment on nonprotonated C atoms, from both the aglycones and acyl groups. Unlike TOCSY, HMBC transfer is not stopped by heteroatoms, and so it can also be used to determine the linkage points of heteroatomcontaining groups such as sugar residues. HMBC is also useful to distinguish some classes of flavonoids, as flavones from aurones, which have similar 1H and 13C NMR spectra but very different HMBC spectra. Currently, only enhanced variants of the HSQC and HMBC experiments, namely gradient enhanced (*ge*) ones, are used, due to their higher sensitivity and capacity. These have been used to establish strong intramolecular H bonding between the 4-oxo and 5-hydroxy groups in flavonoids (Exarchou et al., 2002; Kozerski et al., 2003).

is shown by increased multiplet intensity (Fossen & Andersen, 2005).

provide an opportunity to dovetail proton and carbon NMR data directly.

**4.3 Heteronuclear 2D NMR** 

X-ray crystallography depends on the ability to obtain flavonoid crystals, which has only been achieved for a small number of flavonoids; in alternative, the 1H CP-MAS (Cross Polarization Magic Angle Spinning) NMR techniques have been used to elucidate the solid state conformation of flavonoids, either pure or, for example, in tissues, providing enough sample is available (typically in the 10 mg scale). In particular, such an experiment had provided information on the planarity of the flavonoid rings, on the intramolecular H bonding between the 4-oxo group and the 3- and 5-hydroxy groups, and on intermolecular association. (Fossen & Andersen, 2005; Olejniczak & Potrzebowski, 2004)

#### **5. UV-vis spectrophotometry**

Ultraviolet and visible spectroscopy was one of the earliest techniques routinely used for flavonoid analysis due to the existence of two characteristic UV/Vis bands in flavonoids, band I in the 300 to 550 nm range, arising from the B ring, and band II in the 240 to 285 nm range, arising from the A ring. For examples, while the band I of flavones and flavonols lies in the 240 – 285 nm range, that of flavanone (no C ring instauration) lies in the 270 – 295 nm range; conversely, the band II of flavones and flavanones (no 3-OH group) lies around 303 – 304 nm, and that of 3-hydroxylated flavonoles is centred around 352 nm.

Shift reagents, such as sodium methoxide and aluminium chrolide, lead to shifts in the maximum wavelength of these bands due to methoxide-induced deprotonation of OH groups or Al3+ complexation by OH groups, were also routinely used to study flavonoid structure. Nowadays these techniques are not routinely used but still continue to be applied in some cases, in particular to HPLC eluates - a hyphenated LC-UV-MS has been developed using post-column UV shift reagents for the flavonoid analysis of crude extracts.

Structural Analysis

2000; Moncada et al., 2004).

**6.1 X-ray crystallography** 

(Fossen & Andersen, 2005).

**6.2 Circular dichroism spectroscopy** 

(Wallace & Janes, 2009).

2010).

**6. Other techniques** 

of Flavonoids and Related Compounds – A Review of Spectroscopic Applications 49

UV/Vis spectrophotometry is still widely used to study anthocyanidins, which change their form and colour depending on pH, concentration, metal ions and copigmentation (Giusti & Wrolstad, 2001). This multistate behaviour has been used to derive molecular machines based on flavonoids, particulary flavylium containing ones like anthocyanins (Melo et al.,

MS, NMR and UV/Vis are the most commonly used techniques to elucidate the structure of flavonoids. Three other techniques are also used: X-ray crystallography, although with the potential to solve complete structures, is hampered by the difficulty to obtain good crystals; circular dichroism and vibrational spectroscopies are used to solve specific structural details.

X-ray crystallography is able to detect the arrangement of atoms within a crystal by the atom-induced diffraction of X-rays. Many materials form crystals, such as salts, metals and organic and biological molecules, in particular proteins. Flavonoids, however, only form crystals in sporadic conditions, and the number of reported flavonoid crystals is very low

Nevertheless, traditional X-ray crystallography has been used to identify the intermolecular ππ interactions that guide the stacking of parallel aglycones to form supramolecular layers, and to identify aminoacyl residues involved in the formation of protein-flavonoid complexes, which are critical to the circulation of flavonoids in mammals (Rolo-Naranjo et al., 2010); this is

More recently, X-ray powder diffraction has been used, either alone or in association with solid-state NMR, to obtain structures of flavonoids, in particular of catechins (Harper et al.,

Circular dichroism (CD) is a spectroscopic technique that allows the analysis of the differential absorption of left and right circularly polarized light. The major advantage of CD over optical rotation measurements is that CD absorption is confined to the narrow absorption range of each individual chromophore, and so it can be used to determine the contribution of individual chromophores and to access their possible substitution patterns

 One of the most immediate applications of electronic CD and vibrational CD is the determination of the absolute configuration of quiral flavonoid molecules, such as isoflavan-4-ols (Kim et al., 2010). Slade et al., 2005, have reviewed the CD configuration characterization of most classes of flavonoids, and in particular of flavan-3,4-diols, which bear three quiral centers (Ferreira et al., 2004), and more recently proanthocyanidins analysis

CD is also routinely used to study the interaction of many flavonoids with biomolecules, providing valuable information on biomolecule-drug interaction, such as DNA binding of

has also been reviewed, with a focus on CD results (Hümmer & Schreier, 2008).

one of the strongest applications of X-ray crystallography to the flavonoid area.

Fig. 4. Sample NMR 2D spectra of malvidin 3-*O*-β-glucopyranoside (R=OCH3) (**a**, **b** and **c**) and of petunidin 3-*O*-β-glucopyranoside (R=OH) (**d**) obtained at 600.13 MHz, at about 11 mM at 25 ºC in CD3OD. a) 1H-13C HMBC spectrum; b) 1H-13C HSQC spectrum; c) NOESY spectrum; A negative cross-peak due to NOE correlation between H-4 and H-1'' of the flavylium cation, locating the position of the monosaccharide to the aglycone, is enclosed in a box. Other labelled cross-peaks are positive and are caused by chemical exchange; d) 1H−1H TOCSY NMR spectrum of petunidin 3-O-β-glucopyranoside. f = assignment for the flavylium; a = assignment for the hemiacetal **a** (major); b = assignment for the hemiacetal **b** (minor); \*/s = impurities. Adapted from Jordheim et al., 2006.

UV/Vis spectrophotometry is still widely used to study anthocyanidins, which change their form and colour depending on pH, concentration, metal ions and copigmentation (Giusti & Wrolstad, 2001). This multistate behaviour has been used to derive molecular machines based on flavonoids, particulary flavylium containing ones like anthocyanins (Melo et al., 2000; Moncada et al., 2004).

#### **6. Other techniques**

48 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Fig. 4. Sample NMR 2D spectra of malvidin 3-*O*-β-glucopyranoside (R=OCH3) (**a**, **b** and **c**) and of petunidin 3-*O*-β-glucopyranoside (R=OH) (**d**) obtained at 600.13 MHz, at about 11 mM at 25 ºC in CD3OD. a) 1H-13C HMBC spectrum; b) 1H-13C HSQC spectrum; c) NOESY spectrum; A negative cross-peak due to NOE correlation between H-4 and H-1'' of the flavylium cation, locating the position of the monosaccharide to the aglycone, is enclosed in a box. Other labelled cross-peaks are positive and are caused by chemical exchange; d) 1H−1H TOCSY NMR spectrum of petunidin 3-O-β-glucopyranoside. f = assignment for the

flavylium; a = assignment for the hemiacetal **a** (major); b = assignment for the hemiacetal **b** (minor); \*/s = impurities. Adapted from Jordheim et al., 2006.

MS, NMR and UV/Vis are the most commonly used techniques to elucidate the structure of flavonoids. Three other techniques are also used: X-ray crystallography, although with the potential to solve complete structures, is hampered by the difficulty to obtain good crystals; circular dichroism and vibrational spectroscopies are used to solve specific structural details.

#### **6.1 X-ray crystallography**

X-ray crystallography is able to detect the arrangement of atoms within a crystal by the atom-induced diffraction of X-rays. Many materials form crystals, such as salts, metals and organic and biological molecules, in particular proteins. Flavonoids, however, only form crystals in sporadic conditions, and the number of reported flavonoid crystals is very low (Fossen & Andersen, 2005).

Nevertheless, traditional X-ray crystallography has been used to identify the intermolecular ππ interactions that guide the stacking of parallel aglycones to form supramolecular layers, and to identify aminoacyl residues involved in the formation of protein-flavonoid complexes, which are critical to the circulation of flavonoids in mammals (Rolo-Naranjo et al., 2010); this is one of the strongest applications of X-ray crystallography to the flavonoid area.

More recently, X-ray powder diffraction has been used, either alone or in association with solid-state NMR, to obtain structures of flavonoids, in particular of catechins (Harper et al., 2010).

#### **6.2 Circular dichroism spectroscopy**

Circular dichroism (CD) is a spectroscopic technique that allows the analysis of the differential absorption of left and right circularly polarized light. The major advantage of CD over optical rotation measurements is that CD absorption is confined to the narrow absorption range of each individual chromophore, and so it can be used to determine the contribution of individual chromophores and to access their possible substitution patterns (Wallace & Janes, 2009).

 One of the most immediate applications of electronic CD and vibrational CD is the determination of the absolute configuration of quiral flavonoid molecules, such as isoflavan-4-ols (Kim et al., 2010). Slade et al., 2005, have reviewed the CD configuration characterization of most classes of flavonoids, and in particular of flavan-3,4-diols, which bear three quiral centers (Ferreira et al., 2004), and more recently proanthocyanidins analysis has also been reviewed, with a focus on CD results (Hümmer & Schreier, 2008).

CD is also routinely used to study the interaction of many flavonoids with biomolecules, providing valuable information on biomolecule-drug interaction, such as DNA binding of

Structural Analysis

of Flavonoids and Related Compounds – A Review of Spectroscopic Applications 51

derivatives. While NMR spectroscopy returns information about atoms and bonding between them, MS gives data about molecular and ion/fragment masses, leading to a more complex and laborious data analysing. This problem can be overcome with the construction of structural databases, which allow an easier and quicker data annotation, as well for NMR spectroscopy. NMR has yet a relevant advantage in the biological studies – the ability of studying them in their natural media. Solid-state NMR can be used to observe flavonoid behaviour in tissues, while solution NMR is useful to determine ligand-acceptor interactions though 2D-NMR experiments such as NOESY, being this one of the greatest tools to undergo

It is fair to conclude that although MS methods rarely provide a full molecular determination they are, due to their intrinsic characteristics, the best approach to study flavonoid structures, in complement, when possible, with NMR experiments. For faster cruder screenings, UV absorption data can be used to develop appropriate methods to

Research was partially funded by Fundação para a Ciência e Tecnologia, by the Strategic Programme PEst-OE/QUI/UI0100/2011 and via research grant SFRH/BPD/27563/2006. Critical reviewing of the manuscript by Dr. Alexandra Antunes is gratefully acknowledged.

Ablajan, K. (2011) A study of characteristic fragmentation of isoflavonoids by using negative

Ahmadi, S.M., Dehghan, G., Hosseinpurfeizi, M.A., Ezzati, J., Dolatabadi, N. & Kashanian,

Quercetin–Molybdenum(VI) Complex. *DNA and Cell Biology,* 30, 7, 517-523. Abrankó, L.; García-Reyes, J.F. & Molina-Diáz, A. (2011) In-source fragmentation and accurate

Aviram, M. & Fuhrman, B. (1998) LDL oxidation by arterial wall macrophages depends on

Borges, C.; Martinho, P.; Martins, A.; Rauter, A.P. & Ferreira, M.A. (2001) Structural

Brüll, L.; Huisman, M.; Schols, H.; Voragen, F.; Critchley, G.; Thomas-Oates, J. &

flight mass spectrometry. *Journal of Mass Spectrometry,* 46, 5, 478-488.

antioxidants. *Molecular and Cellular Biochemistry,* 188, 1-2, 149-159.

*Communications in Mass Spectrometry,* 15, 18, 1760-1767.

S. (2011) Preparation, Characterization, and DNA Binding Studies of Water-Soluble

mass analysis of multiclass flavonoid conjugates by electrospray ionization time-of-

the oxidative status in the lipoprotein and in the cells: role of prooxidants vs.

characterisation of flavonoids and flavonoid-O-glycosides extracted from *Genista tenera* by fast-atom bombardment tandem mass spectrometry. *Rapid* 

Haverkamp, J. (1998) Rapid molecular mass and structural determination of plant cell wall-derived oligosaccharides using off-line high-performance anion-exchange chromatography/mass spectrometry. *Journal of Mass Spectrometry,* 33, 8, 713-720. Charles, L.; Laure, F.; Raharivelomanana, P. & Bianchini, J.-P. (2005) Sheath liquid interface

for the coupling of normal-phase liquid chromatography with electrospray mass spectrometry and its application to the analysis of neoflavonoids. *Journal of Mass* 

ion ESI-MSn. *Journal of Mass Spectrometry,* 46, 1, 77-84.

protein activity inhibition that can be in the base of the flavonoid biological activity.

achieve initial flavonoid class identification.

*Spectrometry*, 40, 1, 75-82.

**8. Acknowledgments** 

**9. References** 

quercetin (Ahmadi et al*.*, 2011), binding to serum albumin (di Bari et al., 2009) and hemoglobin (Chauduri et al., 2011), inhibition of β-amyloid toxicity and fibrillogenesis (Thapa et al., 2011). It must be noted that many of the CD studies of protein-flavonoid association studies are usually accompanied by UV-VIS and/or fluorescence studies, such as the probing of kaempferol interaction with human serum albumin (Matei & Hillebrand, 2010).

#### **6.3 Vibrational spectroscopy**

Vibrational spectroscopy, in its infra-red and Raman variants, is a spectroscopic technique that analyses the vibrational modes of molecules and molecular groups, allowing bond characterization, and, by comparison with known tabulated data, identification of functional groups; in the case of flavonoids, vibrational spectroscopy has been systematically used to study hydroxyl and carbonyl groups, but more recent technical developments have allowed its application to a broader set of research goals. Raman spectra are much less complex than the IR spectra of the same molecules, and for that reason Raman spectroscopy has been gradually taking over IR spectroscopy, although it is common to use both techniques as complement of each other. Vibrational spectroscopy is seldom used alone, and most studies are accompanied by other spectroscopic approaches and/or quantum chemical computations (Siebert & Hildebrandt, 2007).

Both these spectroscopies are routinely applied to study the effects of substituents on the geometry of the molecule, in particular of dihedral angles, and also on the analysis of intramolecular (Li et al., 2011; Erdogdu et al., 2010) and intermolecular H bonding, either to other flavonoid moleculer or to solvent molecules. Similarly, metal complexation by flavonoids is also routinely assessed by vibrational spectroscopy (O'Coinceanainn et al., 2004).

#### **7. Conclusion**

The role of flavonoids in biological systems appears yet to be far from definitively determined, involving a large number of research groups all over the world. Interestingly, although many new actions of flavonoids *in vivo* have been put forward, the previously proposed actions are never dismissed, only relegated to secondary ways of flavonoid action, usually considered to be important in pathological conditions (Gomes et al., 2008).

As described above, various physical-chemistry techniques have been used as means of characterization of flavonoids. The great amount of work developed since the 1940s yielded a vast library of structural and spectroscopic information about these compounds, making the identification of new isolated species an easy and quick task. However, some limitations have yet to be overcome. For instance, the maximum molecular size allowed in mass spectroscopy (*ca.* 10 kDa) and in NMR spectroscopy (*ca.* 30 kDa) limits the role of these techniques in the characterization of some more complex molecules and molecular complexes, nevertheless, these two techniques have lead to great breakthrough in terms of structure elucidation that could not be achieved with the classical spectroscopic techniques like UV/Vis and Vibrational (Raman and Infra-Red) spectroscopy or by X-ray diffraction.

It must be noted that, in many cases, information obtained by NMR or MS needs to be correlated with data from other structural analysis techniques, such as CD, in order to confirm some feeble data. Nevertheless, it has been demonstrated that MS and NMR are the most suitable techniques to determine the chemical structure of flavonoids and its derivatives. While NMR spectroscopy returns information about atoms and bonding between them, MS gives data about molecular and ion/fragment masses, leading to a more complex and laborious data analysing. This problem can be overcome with the construction of structural databases, which allow an easier and quicker data annotation, as well for NMR spectroscopy. NMR has yet a relevant advantage in the biological studies – the ability of studying them in their natural media. Solid-state NMR can be used to observe flavonoid behaviour in tissues, while solution NMR is useful to determine ligand-acceptor interactions though 2D-NMR experiments such as NOESY, being this one of the greatest tools to undergo protein activity inhibition that can be in the base of the flavonoid biological activity.

It is fair to conclude that although MS methods rarely provide a full molecular determination they are, due to their intrinsic characteristics, the best approach to study flavonoid structures, in complement, when possible, with NMR experiments. For faster cruder screenings, UV absorption data can be used to develop appropriate methods to achieve initial flavonoid class identification.

#### **8. Acknowledgments**

Research was partially funded by Fundação para a Ciência e Tecnologia, by the Strategic Programme PEst-OE/QUI/UI0100/2011 and via research grant SFRH/BPD/27563/2006. Critical reviewing of the manuscript by Dr. Alexandra Antunes is gratefully acknowledged.

#### **9. References**

50 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

quercetin (Ahmadi et al*.*, 2011), binding to serum albumin (di Bari et al., 2009) and hemoglobin (Chauduri et al., 2011), inhibition of β-amyloid toxicity and fibrillogenesis (Thapa et al., 2011). It must be noted that many of the CD studies of protein-flavonoid association studies are usually accompanied by UV-VIS and/or fluorescence studies, such as the probing of

Vibrational spectroscopy, in its infra-red and Raman variants, is a spectroscopic technique that analyses the vibrational modes of molecules and molecular groups, allowing bond characterization, and, by comparison with known tabulated data, identification of functional groups; in the case of flavonoids, vibrational spectroscopy has been systematically used to study hydroxyl and carbonyl groups, but more recent technical developments have allowed its application to a broader set of research goals. Raman spectra are much less complex than the IR spectra of the same molecules, and for that reason Raman spectroscopy has been gradually taking over IR spectroscopy, although it is common to use both techniques as complement of each other. Vibrational spectroscopy is seldom used alone, and most studies are accompanied by other spectroscopic approaches and/or quantum chemical computations

Both these spectroscopies are routinely applied to study the effects of substituents on the geometry of the molecule, in particular of dihedral angles, and also on the analysis of intramolecular (Li et al., 2011; Erdogdu et al., 2010) and intermolecular H bonding, either to other flavonoid moleculer or to solvent molecules. Similarly, metal complexation by flavonoids is also routinely assessed by vibrational spectroscopy (O'Coinceanainn et al., 2004).

The role of flavonoids in biological systems appears yet to be far from definitively determined, involving a large number of research groups all over the world. Interestingly, although many new actions of flavonoids *in vivo* have been put forward, the previously proposed actions are never dismissed, only relegated to secondary ways of flavonoid action,

As described above, various physical-chemistry techniques have been used as means of characterization of flavonoids. The great amount of work developed since the 1940s yielded a vast library of structural and spectroscopic information about these compounds, making the identification of new isolated species an easy and quick task. However, some limitations have yet to be overcome. For instance, the maximum molecular size allowed in mass spectroscopy (*ca.* 10 kDa) and in NMR spectroscopy (*ca.* 30 kDa) limits the role of these techniques in the characterization of some more complex molecules and molecular complexes, nevertheless, these two techniques have lead to great breakthrough in terms of structure elucidation that could not be achieved with the classical spectroscopic techniques like UV/Vis and Vibrational (Raman and Infra-Red) spectroscopy or by X-ray diffraction. It must be noted that, in many cases, information obtained by NMR or MS needs to be correlated with data from other structural analysis techniques, such as CD, in order to confirm some feeble data. Nevertheless, it has been demonstrated that MS and NMR are the most suitable techniques to determine the chemical structure of flavonoids and its

usually considered to be important in pathological conditions (Gomes et al., 2008).

kaempferol interaction with human serum albumin (Matei & Hillebrand, 2010).

**6.3 Vibrational spectroscopy** 

(Siebert & Hildebrandt, 2007).

**7. Conclusion** 


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**3** 

*Poland* 

**Analytical Methods for Isolation,** 

Katarzyna Szewczyk and Anna Bogucka - Kocka

**Furanocoumarins in Plant Material** 

**Separation and Identification of Selected** 

*Chair and Department of Pharmaceutical Botany, Medical University of Lublin,* 

Coumarins are α–pyrone derivatives synthesized as secondary metabolites in plants. They occur as free compounds or glycosides in plants. They have been isolated from A. Vogel, since 1820, from the tonka beans (*Coumarouna odorata* Aubl. = *Dipteryx odorata* Will.) and they have been synthesized in 1868 from W. H. Perkin, through the famous Perkin reaction

Furanocoumarins are one of the coumarin derivatives. They can be grouped into the linear type, where the furan ring (dihydro) is attached at C(6) and C(7), and the angular type,

The most abundant linear furanocoumarins are psolaren, xanthotoxin, bergapten and isopimpinellin, whereas the angular type is mostly represented by angelicin, sphondin, and pimpinellin. Some structures of furanocoumarins are presented in table 1. As was mentioned for the simple coumarins, numerous minor furanocoumarins have been described in the literature, for example bergamottin (5-geranoxy-psolaren) (Stanley & Vannier, 1967), which has received attention recently as a major grapefruit component interfering with drug metabolism by intestinal CYP3A4 (Bourgaud et al., 2006; Wen et al.,

Linear furanocoumarins (syn. psolarens) are principally distributed in four angiosperm families: Apiaceae (Umbelliferae), Moraceae *(Brosimum, Dorstenia, Fatoua* and *Ficus),*  Rutaceae and Leguminose (restricted to Psoralea and Coronilla generae). The angular (dihydro) furanocoumarins are less widely distributed and primarily confined to the

Moreover, furanocoumarins have been reported from Asteraceae (Compositae), Pittosporaceae, Rosaceae, Solanaceae and Thymelaeaceae (Milesi et al., 2001; Murray et al., 1982). Certain precursors to this group of compounds are found in the Cneoraceae (Murray,

Apiaceae and Leguminosae (Berenbaum et al., 1991; Bourgaud et al., 1995).

**1. Introduction** 

(Dewick, 2009).

2008).

1982).

carrying the substitution at C(7) and C(8).

**2. Distribution of furanocoumarins in plants** 


## **Analytical Methods for Isolation, Separation and Identification of Selected Furanocoumarins in Plant Material**

Katarzyna Szewczyk and Anna Bogucka - Kocka *Chair and Department of Pharmaceutical Botany, Medical University of Lublin, Poland* 

#### **1. Introduction**

56 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

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Spencer, J.P. (2010) The impact of fruit flavonoids on memory and cognition. *British Journal* 

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Volná, K.; Holčapek, M.; Kolářová, L.; Lemr, K.; Čáslavský, J.; Kačer, P.; Poustka, J. &

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Wei, J.; Liu, S.; Fedoreyev, S. A. & Voinov, V. G. (2000) A study of resonance electron

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Zhang, Y.; Zhang, P. & Cheng, Y. (2008) Structural characterization of isoprenylated

Zhou, D.y.; Xu, Q.; Xue, X.-y.; Zhang, F.-f.; Jing, Y. & Liang, X.-m. (2007) Rapid qualitative

studies of flavonoid glycosides. *Phytochemistry,* 54, 3, 237-256.

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trap MS. *Journal of Separation Science,* 30, 6, 858-867.

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flavonoids from Kushen by electrospray ionization multistage tandem mass

and quantitative analyses of flavanone aglycones in *Fructus aurantii* by HPLC ion-

Coumarins are α–pyrone derivatives synthesized as secondary metabolites in plants. They occur as free compounds or glycosides in plants. They have been isolated from A. Vogel, since 1820, from the tonka beans (*Coumarouna odorata* Aubl. = *Dipteryx odorata* Will.) and they have been synthesized in 1868 from W. H. Perkin, through the famous Perkin reaction (Dewick, 2009).

Furanocoumarins are one of the coumarin derivatives. They can be grouped into the linear type, where the furan ring (dihydro) is attached at C(6) and C(7), and the angular type, carrying the substitution at C(7) and C(8).

The most abundant linear furanocoumarins are psolaren, xanthotoxin, bergapten and isopimpinellin, whereas the angular type is mostly represented by angelicin, sphondin, and pimpinellin. Some structures of furanocoumarins are presented in table 1. As was mentioned for the simple coumarins, numerous minor furanocoumarins have been described in the literature, for example bergamottin (5-geranoxy-psolaren) (Stanley & Vannier, 1967), which has received attention recently as a major grapefruit component interfering with drug metabolism by intestinal CYP3A4 (Bourgaud et al., 2006; Wen et al., 2008).

#### **2. Distribution of furanocoumarins in plants**

Linear furanocoumarins (syn. psolarens) are principally distributed in four angiosperm families: Apiaceae (Umbelliferae), Moraceae *(Brosimum, Dorstenia, Fatoua* and *Ficus),*  Rutaceae and Leguminose (restricted to Psoralea and Coronilla generae). The angular (dihydro) furanocoumarins are less widely distributed and primarily confined to the Apiaceae and Leguminosae (Berenbaum et al., 1991; Bourgaud et al., 1995).

Moreover, furanocoumarins have been reported from Asteraceae (Compositae), Pittosporaceae, Rosaceae, Solanaceae and Thymelaeaceae (Milesi et al., 2001; Murray et al., 1982). Certain precursors to this group of compounds are found in the Cneoraceae (Murray, 1982).

Analytical Methods for Isolation,

1986; Stanley-Horn, 1999).

autumn increases (Kohlmünzer, 2010).

**3. Biosynthesis of furanocoumarins** 

1999).

plant parts, followed by leaves of the inner petioles.

Separation and Identification of Selected Furanocoumarins in Plant Material 59

Diawara et al. (1995) examined the relative distribution of furocoumarins in celery (*Apium graveolens* L. var. *dulce* Miller) plant parts and found that leaves of the outer petioles contained significantly higher levels of the three phototoxic constituents than did other

On the other hand, levels of furanocoumarins observed in plants grown in the field are higher than those observed in plants grown in laboratory or greenhouse conditions and may fluctuate over the season (Trumbe et al., 1992; Diawara et al., 1995). In most studies, bergapten has been found to occur in highest concentrations, followed by xanthotoxin, but psoralen is often observed only in trace quantities (Trumbe et al., 1990; Trumbe et al., 1992; Diawara et al., 1993). However, other studies have found that xanthotoxin (Beier et al., 1983) or psoralen (Diawara et al., 1993; Trumble et al., 1990) is most abundant (Stanley-Horn,

Considering the histological location of furanocoumarins in plant tissues, they are arranged differently. For example, celery contains schizogenous canals scattered throughout the pericycle, which are secretory and are thought to extend through the stem and foliage (Maksymowych & Ledbetter, 1986). Furanocoumarins are thought to be restricted to schizogenous canals in seeds of celery (Berenbaum, 1991) and accumulate primarily in petiolar and foliar canals in cow-parsnip, *Heracleum lanatum* Michx. (Apiaceae). However, there is also evidence suggesting that this group of compounds occur in and on the surfaces of tissues as well. A study of several apiaceous and rutaoeous species by Zobel and Brown (1990) revealed that a large proportion of each furocoumarin was located on the leaf surface in most of the plants studied. Furanocoumarins of *Ruta graveolens* L. are present in the epidermal layer of both stems and leaves and in the mesophyll directly below the epidermis, while glands of leaves contain only traces of furanocoumarins. In fact, the cuticular layer contains 15.60% of the psoralens found in leaves (Zobel et al., 1989). The occurrence of bergapten and xanthotoxin in the surface wax of leaves of wild carrot, *Daucus carota* L., a plant containing only trace levels of furanocoumarins has also been reported (Ceska et al.,

The content of coumarins in plants is conditioned by the degree of the development of the plant and its vegetation stage, too. Concentrations of linear furanocoumarins increase dramatically with plant age between 8 and 18 weeks (Reitz et al., 1997) with a subsequent decline in bergapten concentrations in the last six to eight weeks before harvest (Trumble et al., 1992). Significant decreases in levels of furanocoumarins were also observed both in and on senescing leaves of *Ruta graveolens* (Zobel & Brown, 1991). The content of some furanocoumarins in *Apium graveolens* and *Petroselinum sativum* decreases in summer and in

The biosynthesis of linear and angular furanocoumarins is still poorly understood at the molecular level. They are produced *via* the shikimic acid biosynthetic pathway beginning with the conversion of phenyloalanine to trans-cinnamic acid by phenylalanine ammonia lyase. Orto-hydroxylation of trans-cinnamic acid yields 2'-hydroxycinnamic acid, which is converted to its cis form, the precursor to coumarin, in the presence of UV light. Alternatively, trans-cinnamic acid may undergo parahydroxylation to yield p-coumaric


Table 1. The structure of some furanocoumarins

Coumarins are distributed across different parts of the plants, and they have specific histological locations in the tissues. Within the plant they are most abundant in fruits and roots. However, in flowers and leaves they are evident in fewer quantities. In some plant species coumarins were also found in the bark or stems (Głowniak, 1988).

The amount of particular furanocoumarins depends on the enzymes active in plants secondary metabolism. Plants with similar enzyme profiles contain comparable amount of secondary metabolites that are products of chemical reactions induced by these enzymes. Thus, furanocoumarins' content, in different species, varieties and forms may contribute to their better distinction, and better understanding of the taxonomy of genuses within which they are present.

Coumarins are distributed across different parts of the plants, and they have specific histological locations in the tissues. Within the plant they are most abundant in fruits and roots. However, in flowers and leaves they are evident in fewer quantities. In some plant

The amount of particular furanocoumarins depends on the enzymes active in plants secondary metabolism. Plants with similar enzyme profiles contain comparable amount of secondary metabolites that are products of chemical reactions induced by these enzymes. Thus, furanocoumarins' content, in different species, varieties and forms may contribute to their better distinction, and better understanding of the taxonomy of genuses within which

species coumarins were also found in the bark or stems (Głowniak, 1988).

**Name of compound** Structure

Psoralen

Phellopterin

Xanthotoxin

Isopimpinellin

Bergapten

Pimpinellin

Imperatorin

Angelicin

Table 1. The structure of some furanocoumarins

they are present.

Diawara et al. (1995) examined the relative distribution of furocoumarins in celery (*Apium graveolens* L. var. *dulce* Miller) plant parts and found that leaves of the outer petioles contained significantly higher levels of the three phototoxic constituents than did other plant parts, followed by leaves of the inner petioles.

On the other hand, levels of furanocoumarins observed in plants grown in the field are higher than those observed in plants grown in laboratory or greenhouse conditions and may fluctuate over the season (Trumbe et al., 1992; Diawara et al., 1995). In most studies, bergapten has been found to occur in highest concentrations, followed by xanthotoxin, but psoralen is often observed only in trace quantities (Trumbe et al., 1990; Trumbe et al., 1992; Diawara et al., 1993). However, other studies have found that xanthotoxin (Beier et al., 1983) or psoralen (Diawara et al., 1993; Trumble et al., 1990) is most abundant (Stanley-Horn, 1999).

Considering the histological location of furanocoumarins in plant tissues, they are arranged differently. For example, celery contains schizogenous canals scattered throughout the pericycle, which are secretory and are thought to extend through the stem and foliage (Maksymowych & Ledbetter, 1986). Furanocoumarins are thought to be restricted to schizogenous canals in seeds of celery (Berenbaum, 1991) and accumulate primarily in petiolar and foliar canals in cow-parsnip, *Heracleum lanatum* Michx. (Apiaceae). However, there is also evidence suggesting that this group of compounds occur in and on the surfaces of tissues as well. A study of several apiaceous and rutaoeous species by Zobel and Brown (1990) revealed that a large proportion of each furocoumarin was located on the leaf surface in most of the plants studied. Furanocoumarins of *Ruta graveolens* L. are present in the epidermal layer of both stems and leaves and in the mesophyll directly below the epidermis, while glands of leaves contain only traces of furanocoumarins. In fact, the cuticular layer contains 15.60% of the psoralens found in leaves (Zobel et al., 1989). The occurrence of bergapten and xanthotoxin in the surface wax of leaves of wild carrot, *Daucus carota* L., a plant containing only trace levels of furanocoumarins has also been reported (Ceska et al., 1986; Stanley-Horn, 1999).

The content of coumarins in plants is conditioned by the degree of the development of the plant and its vegetation stage, too. Concentrations of linear furanocoumarins increase dramatically with plant age between 8 and 18 weeks (Reitz et al., 1997) with a subsequent decline in bergapten concentrations in the last six to eight weeks before harvest (Trumble et al., 1992). Significant decreases in levels of furanocoumarins were also observed both in and on senescing leaves of *Ruta graveolens* (Zobel & Brown, 1991). The content of some furanocoumarins in *Apium graveolens* and *Petroselinum sativum* decreases in summer and in autumn increases (Kohlmünzer, 2010).

#### **3. Biosynthesis of furanocoumarins**

The biosynthesis of linear and angular furanocoumarins is still poorly understood at the molecular level. They are produced *via* the shikimic acid biosynthetic pathway beginning with the conversion of phenyloalanine to trans-cinnamic acid by phenylalanine ammonia lyase. Orto-hydroxylation of trans-cinnamic acid yields 2'-hydroxycinnamic acid, which is converted to its cis form, the precursor to coumarin, in the presence of UV light. Alternatively, trans-cinnamic acid may undergo parahydroxylation to yield p-coumaric

Analytical Methods for Isolation,

2007).

Separation and Identification of Selected Furanocoumarins in Plant Material 61

A troublesome extension of these effects can arise from the handling of plants which contain significant levels of furocoumarins. *Apium graveolens* (= celery; Umbelliferae/Apiaceae) is normally free of such compounds, but fungal infection with the natural parasite *Sclerotinia sclerotiorum* induces the synthesis of furanocoumarins as a response to the infection. Field workers handling these infected plants may become very sensitive to UV light and suffer from a form of sunburn termed photophytodermatitis. Infected parsley (*Petroselinum crispum*) can give similar effects. Handling of rue (*Ruta graveolens;* Rutaceae) or giant hogweed (*Heracleum mantegazzianum*; Umbelliferae/Apiaceae), which naturally contain significant amounts of psoralen, bergapten, and xanthotoxin, can cause similar unpleasant reactions, or more commonly rapid blistering by direct contact with the sap. The giant hogweed can be particularly dangerous. Individuals vary in their sensitivity towards furanocoumarins, some are unaffected, whilst others tend to become sensitized by an initial exposure and then develop the allergic response on subsequent exposures (Dewick, 2009). Methoxsalen in combination with ultraviolet light is also used for antineoplastic effects and for treating certain skin disorders, including alopecia, cutaneos T-cell lymphoma, excema, lichen planus, mycosis fungoides and psoriaris. A recent report has found that this drug inhibits the enzyme, CYP2A6, which is responsible for the metabolism of nicotine. When 8 methoxypsoralen is taken with oral nicotine, this drug can reduce the number of cigarettes smoked by about one quarter and decrease overall levels of tobacco smoke exposure by

Xanthotoxin is used orally or topically in combination with controlled exposure to long wavelength ultraviolet radiation (UVA) or sunlight to repigment vitiliginous skin in patients with idiopathic vitiligo. Many studies have shown that naturally occurring furocoumarins, e.g. imperatorin and isopimpinellin, inhibit P450-mediated enzyme activities *in vitro*. Imperatorin and isopimpinellin have also the potential chemopreventive effects when administered in the diet. The stimulation of melanogenesis by bergapten is related to increased tyrosinase synthesis. In addition, bergapten stimulated TRP-1 synthesis and induced a dose-dependent decrease of DCT activity without modification of protein expression. Osthole could prevent postmenopausal osteoporosis. It can also delay aging, build up strength, enhance immune function, and adjust sex hormone levels (Chen et al.,

Psoralen and bergapten exert their photosensibilising effects through a covalent interaction with DNA triggered by light of a specific wavelength (320-400 nm). The resulting complex blocks the DNA interaction with transcriptases and polymerases, avoiding cell replication. This mechanism consist of three steps, i.e., (1) drug intercalation between DNA nucleotide bases, (2) drug absorption of a UVA photon and covalent bond formation between the furan ring double bond and a thymine base (T2) of the DNA molecule, (3) absorption of a second photon (UVA) and covalent bonding between the lactone ring double bond and another thymine base (T1), which, in the end, results in a psoralen cross-linked DNA (da Silva et al., 2009; Panno et al., 2010; Cardoso et al., 2002). The same effects have been alternatively utilized for the treatment of human lymphoma and of autoimmune diseases through

Panno et al. (2010) investigated the pro-apoptotic effects induced by high doses of bergapten (methoxypsoralen; 5-MOP), in the absence of UV rays, in human breast cancer cells. The same authors examined the effects of bergapten, alone and in combination with UV light, on

almost half in tobacco dependent individuals (Lehr et al., 2003).

extracorporeal photochemotherapy (Panno et al., 2010).

acid. P-coumaric acid may undergo 2'–hydroxylation followed by conversion by 4 coumarate: CoAligase to 4-coumaryl CoA. This compound is intermediate in the biosynthesis of both flavonoids and phenylpropanoids, including 7-hydroxycoumarin (umbelliferone). Umbelliferone is the precursor to both the angular and linear furanocoumarins. The production of the latter involves prenylation to form marmesin, followed by oxidative loss of the hydroxypropyl group in marmesin by 'psoralensynthase' to yield psoralen (Berenbaum, 1991; Stanley-Horn, 1999). A second cytochrome P-450 dependent monooxygenase enzyme then cleaves off the hydroxyisopropyl fragment (as acetone) from marmesin to give the furocoumarin psoralen. Psoralen can act as a precursor for the further substituted furanocoumarins bergapten, xanthotoxin and isopimpinellin. On the other hand, angular furanocoumarins, such an angelicin can arise by a similar sequence of reactions, but these involve initial dimethylallylation at the alternative position *ortho* to the phenol (Dewick, 2009).

#### **4. Biological activities of furanocoumarins**

Due to their biological activities, furanocoumarins are very interesting compounds and widely investigated. The various biological and pharmacological activities of coumarins, have been known for a long time.

They play the role of phytoalexin in plants (Szakiel, 1991), which can be synthesized as a result of elicitation by microorganisms, insects, fungi as well as abiotic elicitors such as UV radiation, environment pollutants and mechanical breakage. Defensive activity of furanocoumarins consists in their toxicity against phytopathogens (e.g. retardation of DNA synthesis) (Waksmundzka-Hajnos et al., 2004).

Linear furocoumarins can be troublesome to humans since they can cause photosensitization towards UV light, resulting in sunburn or serious blistering. Used medicinally, this effect may be valuable in promoting skin pigmentation and treating psoriasis. Plants containing psoralens have been used internally and externally to promote skin pigmentation and suntanning. Bergamot oil obtained from the peel of *Citrus aurantium* ssp. *bergamia* (Rutaceae) can contain up to 5% bergapten and is frequently used in external suntan preparations. The psoralen absorbs in near UV light and allows this radiation to stimulate formation of melanin pigments (Dewick, 2009).

Methoxsalen (xanthotoxin; 8-methoxypsoralen), a constituent of the fruit of *Ammi majus* (Umbelliferae/Apiaceae), is used medically to facilitate skin repigmentation where severe blemishes exist (vitiligo). An oral dose of methoxsalen is followed by long – wave UV irradiation, though such treatments must be very carefully regulated to minimize the risk of burning, cataract formation, and the possibility of causing skin cancer. The treatment is often referred to as PUVA (psoralen + UV-A). PUVA is also of value in the treatment of psoriasis, a widespread condition characterized by proliferation of skin cells. Similarly, methoxsalen is taken orally, prior to UV treatment. Reaction with psoralens inhibits DNA replication and reduces the rate of cell division. Because of their planar nature, psoralens intercalate into DNA, and this enables a UV – initiated cycloaddition reaction between pyrimidine bases (primarily thymine) in DNA and the furan ring of psoralens. A second cycloaddition can then occur, this time involving the pyrone ring, leading to interstrand cross – linking of the nucleic acid (Dewick, 2009; Żołek et al., 2003).

acid. P-coumaric acid may undergo 2'–hydroxylation followed by conversion by 4 coumarate: CoAligase to 4-coumaryl CoA. This compound is intermediate in the biosynthesis of both flavonoids and phenylpropanoids, including 7-hydroxycoumarin (umbelliferone). Umbelliferone is the precursor to both the angular and linear furanocoumarins. The production of the latter involves prenylation to form marmesin, followed by oxidative loss of the hydroxypropyl group in marmesin by 'psoralensynthase' to yield psoralen (Berenbaum, 1991; Stanley-Horn, 1999). A second cytochrome P-450 dependent monooxygenase enzyme then cleaves off the hydroxyisopropyl fragment (as acetone) from marmesin to give the furocoumarin psoralen. Psoralen can act as a precursor for the further substituted furanocoumarins bergapten, xanthotoxin and isopimpinellin. On the other hand, angular furanocoumarins, such an angelicin can arise by a similar sequence of reactions, but these involve initial dimethylallylation at the alternative position *ortho* to

Due to their biological activities, furanocoumarins are very interesting compounds and widely investigated. The various biological and pharmacological activities of coumarins,

They play the role of phytoalexin in plants (Szakiel, 1991), which can be synthesized as a result of elicitation by microorganisms, insects, fungi as well as abiotic elicitors such as UV radiation, environment pollutants and mechanical breakage. Defensive activity of furanocoumarins consists in their toxicity against phytopathogens (e.g. retardation of DNA

Linear furocoumarins can be troublesome to humans since they can cause photosensitization towards UV light, resulting in sunburn or serious blistering. Used medicinally, this effect may be valuable in promoting skin pigmentation and treating psoriasis. Plants containing psoralens have been used internally and externally to promote skin pigmentation and suntanning. Bergamot oil obtained from the peel of *Citrus aurantium* ssp. *bergamia* (Rutaceae) can contain up to 5% bergapten and is frequently used in external suntan preparations. The psoralen absorbs in near UV light and allows this radiation to

Methoxsalen (xanthotoxin; 8-methoxypsoralen), a constituent of the fruit of *Ammi majus* (Umbelliferae/Apiaceae), is used medically to facilitate skin repigmentation where severe blemishes exist (vitiligo). An oral dose of methoxsalen is followed by long – wave UV irradiation, though such treatments must be very carefully regulated to minimize the risk of burning, cataract formation, and the possibility of causing skin cancer. The treatment is often referred to as PUVA (psoralen + UV-A). PUVA is also of value in the treatment of psoriasis, a widespread condition characterized by proliferation of skin cells. Similarly, methoxsalen is taken orally, prior to UV treatment. Reaction with psoralens inhibits DNA replication and reduces the rate of cell division. Because of their planar nature, psoralens intercalate into DNA, and this enables a UV – initiated cycloaddition reaction between pyrimidine bases (primarily thymine) in DNA and the furan ring of psoralens. A second cycloaddition can then occur, this time involving the pyrone ring, leading to interstrand

the phenol (Dewick, 2009).

have been known for a long time.

**4. Biological activities of furanocoumarins**

synthesis) (Waksmundzka-Hajnos et al., 2004).

stimulate formation of melanin pigments (Dewick, 2009).

cross – linking of the nucleic acid (Dewick, 2009; Żołek et al., 2003).

A troublesome extension of these effects can arise from the handling of plants which contain significant levels of furocoumarins. *Apium graveolens* (= celery; Umbelliferae/Apiaceae) is normally free of such compounds, but fungal infection with the natural parasite *Sclerotinia sclerotiorum* induces the synthesis of furanocoumarins as a response to the infection. Field workers handling these infected plants may become very sensitive to UV light and suffer from a form of sunburn termed photophytodermatitis. Infected parsley (*Petroselinum crispum*) can give similar effects. Handling of rue (*Ruta graveolens;* Rutaceae) or giant hogweed (*Heracleum mantegazzianum*; Umbelliferae/Apiaceae), which naturally contain significant amounts of psoralen, bergapten, and xanthotoxin, can cause similar unpleasant reactions, or more commonly rapid blistering by direct contact with the sap. The giant hogweed can be particularly dangerous. Individuals vary in their sensitivity towards furanocoumarins, some are unaffected, whilst others tend to become sensitized by an initial exposure and then develop the allergic response on subsequent exposures (Dewick, 2009).

Methoxsalen in combination with ultraviolet light is also used for antineoplastic effects and for treating certain skin disorders, including alopecia, cutaneos T-cell lymphoma, excema, lichen planus, mycosis fungoides and psoriaris. A recent report has found that this drug inhibits the enzyme, CYP2A6, which is responsible for the metabolism of nicotine. When 8 methoxypsoralen is taken with oral nicotine, this drug can reduce the number of cigarettes smoked by about one quarter and decrease overall levels of tobacco smoke exposure by almost half in tobacco dependent individuals (Lehr et al., 2003).

Xanthotoxin is used orally or topically in combination with controlled exposure to long wavelength ultraviolet radiation (UVA) or sunlight to repigment vitiliginous skin in patients with idiopathic vitiligo. Many studies have shown that naturally occurring furocoumarins, e.g. imperatorin and isopimpinellin, inhibit P450-mediated enzyme activities *in vitro*. Imperatorin and isopimpinellin have also the potential chemopreventive effects when administered in the diet. The stimulation of melanogenesis by bergapten is related to increased tyrosinase synthesis. In addition, bergapten stimulated TRP-1 synthesis and induced a dose-dependent decrease of DCT activity without modification of protein expression. Osthole could prevent postmenopausal osteoporosis. It can also delay aging, build up strength, enhance immune function, and adjust sex hormone levels (Chen et al., 2007).

Psoralen and bergapten exert their photosensibilising effects through a covalent interaction with DNA triggered by light of a specific wavelength (320-400 nm). The resulting complex blocks the DNA interaction with transcriptases and polymerases, avoiding cell replication. This mechanism consist of three steps, i.e., (1) drug intercalation between DNA nucleotide bases, (2) drug absorption of a UVA photon and covalent bond formation between the furan ring double bond and a thymine base (T2) of the DNA molecule, (3) absorption of a second photon (UVA) and covalent bonding between the lactone ring double bond and another thymine base (T1), which, in the end, results in a psoralen cross-linked DNA (da Silva et al., 2009; Panno et al., 2010; Cardoso et al., 2002). The same effects have been alternatively utilized for the treatment of human lymphoma and of autoimmune diseases through extracorporeal photochemotherapy (Panno et al., 2010).

Panno et al. (2010) investigated the pro-apoptotic effects induced by high doses of bergapten (methoxypsoralen; 5-MOP), in the absence of UV rays, in human breast cancer cells. The same authors examined the effects of bergapten, alone and in combination with UV light, on

Analytical Methods for Isolation,

neurons (Łuszczki et al., 2007).

(i.p.) administration in mice (Łuszczki et al., 2007a).

Separation and Identification of Selected Furanocoumarins in Plant Material 63

Quite recently, it has been documented that imperatorin in a dose-dependent manner increased the threshold for maximal electro-convulsions in mice (Łuszczki et al., 2007a). The time–course and dose–response relationship analyses revealed that the time to peak of the maximum anticonvulsant effect for imperatorin was established at 30 min after its systemie

Recently results indicate that imperatorin administered at subthreshold doses enhanced the anticonvulsant effects of carbamazepine, phenytoin and phenobarbital, but not those of valproate against maximal electroshock-induced seizures in mice. It is important to note that the anti-seizure effects of carbamazepine combined with imperatorin were greater than those observed for the combinations of phenobarbital and phenytoin with imperatorin.

The difference in the anti-seizure effects of carbamazepine and phenytoin or phenobarbital in the maximal electroshock seizure test may be explained through pharmacokinetic interaction between imperatorin and carbamazepine. It was found that imperatorin significantly increased total brain carbamazepine concentrations, having no impact on the total brain phenytoin and phenobarbital concentrations in experimental animals. The selectivity in the increase in total brain carbamazepine concentration one can try to explain through the fact that imperatorin probably enhances the penetration of carbamazepine into the brain by modifying the blood–brain barrier permeability. On the other hand, it may be hypothesized that the selective increase in carbamazepine content in the brain tissue resulted from imperatorin-induced inhibition of multi-drug resistance proteins or Pglycoproteins that normal physiological activity is related to the removal of drugs from the brain tissue. Thus, inhibitors of these proteins may contribute to the accumulation of

Considering molecular mechanisms of the action of conventional antiepileptic drugs and imperatorin, one can ascertain that imperatorin-induced irreversible inactivation of GABAtransaminase and subsequent increases in GABA content in the brain, as well as, the enhanced GABA-mediated inhibitory neurotransmitter action through the interaction of imperatorin with benzodiazepine receptors. This may exhibit complementary potentials to the anticonvulsant activity of carbamazepine, phenytoin and phenobarbital shown in experimental animals testing. Noteworthy, the main anticonvulsant mechanism of the action of carbamazepine and phenytoin is related to the blockade of Na+ channels in certain

It is interesting to note that imperatorin did not potentiate the protective action of valproate against maximal electroshock-induced seizures. This apparent lack of effects of imperatorin on the antiseizure action of valproate, one can try to explain by the fact that valproate possesses a number of variousmechanisms of action that contribute to its anti-seizure

The evaluation of acute adverse effect potentials is exhibited within combinations of imperatorin with conventional antiepileptic drugs revealing that the combinations did not disturb long-term memory, impair motor co-ordination, or change neuromuscular gripstrength in experimental animals. Therefore, the investigated combinations seem to be

It was shown that imperatorin enhances the protective action of carbamazepine, phenytoin and phenobarbital, but not that of valproate against maxima electroshock-induced seizures

antiepileptic drugs in the brain (Brandt et al., 2006; Łuszczki et al., 2007).

activity in both rodents and humans (Łuszczki et al., 2007).

secure and well tolerated by experimental animals (Łuszczki et al., 2007).

the cellular growth of breast tumoral cells. Their study suggested that bergapten alone, or as a photoactivated product, could be used as an active molecule able to counteract effectively the survival and growth of breast hormone-responsive tumors.

Furanocoumarins isolated from fruits of *Heracleum sibiricum* L. inducing apoptosis by forming adducts with DNA. Bogucka – Kocka (2004) reported a visible influence of these compounds on the inhibition of the proliferation and on induction of apoptosis processes in the human HL-60 cell lines. Moreover, compounds isolated from *Angelica dahurica*  (Apiaceae) were examined regarding their cytotoxic activity against L1210, HL-60, K562, and B16F10 tumor cells lines using the MMT cell assay. It was discovered that pangelin and oxypeucedanin hydrate acetonide exhibited the most cytotoxic activity against all selected tumor cell lines (Heinrich et al., 2004). Um and co-authors (2010) were isolated four furonocoumarins (bergapten, isopimpinellin, xanthotoxin and imperatorin) from *Glehnia littoralis* F. Schmidt ex Miquel (Apiaceae), which exhibited dose-dependent inhibitory effectson the cell proliferation. Their study demonstrated that *G. littoralis* has potent inhibitory effect on proliferation of HT-29 human colon cancer cells.

In addition, Oxypeucedanine (= prangolarin), which was isolated from *Prangos, Hippomarathrum, Angelica* and *Ferulago* (genera of Apiaceae) and *Ruta* genus of Rutaceae, has pharmacological and biological activities. It was reported to have antiarrhytmic, channel blocer and antiestrogenic activity. Razavi et al. (2010) studied phytotoxic, antibacterial, antifungal, antioxidant and cytotoxic effects of oxypeucedanin. Their results revealed that this compound exhibits considerable phytotoxic activity and might play an allelopathic role for plants. On the other hand, oxypeucedanin exhibits considerable cytotoxicity against Hela cell line (IC50 value of 314 µg/ml).

The ethanol extract of the *Cnidii* fructus and coumarins separated from it have growthinhibitory effects on the tumor cells (Chen et al., 2007).

One of the major bioactive components of the fruits of *Cnidium monnieri* (L.) Cusson, bergapten, possesses antiinflammatory and analgesic activities. However, imperatorin exhibits strong cytotoxic activity on human leukemia, chemopreventive effects on hepatitis and skin tumor, and antiinflammatory activity (Li & Chen, 2004).

In addition of bergapten, this plant also contained numbers of others coumarins, such as xanthotoxin, isopimpinellin, bergapten, imperatorin and osthole. These constituents regarded for biological activity of this crude drug, which is used for treatment of pain in female genitalia, impotence and supportive (Chen et al., 2007).

Pharmacological studies have indicated that coumarins such as isoimperatorin, notopterol and bergapten possess anti-inflammatory, analgesic, anti-cancer and anti-coagulant activities (Qian et al., 2007).

Imperatorin (8-isopentenyloxypsoralen; 9-(3-methylbut-2-enyloxy)-7H-furo [3,2-g]chromen-7-one) is a bio-active furanocoumarin isolated e.g. from roots of *Angelica dahurica* and fruits of *Angelica archangelica* (Umbelliferae) (Baek et al., 2000). Experimental evidence indicates that imperatorin irreversibly inactivates γ-aminobutyric acid (GABA)-transaminase (the enzyme responsible for the degradation of GABA) and thus, increases the GABA content in the synaptic clefts of neurons and elevates the inhibitory neurotransmitter GABA level in the brain (Choi et al., 2005; Łuszczki et al., 2007).

the cellular growth of breast tumoral cells. Their study suggested that bergapten alone, or as a photoactivated product, could be used as an active molecule able to counteract effectively

Furanocoumarins isolated from fruits of *Heracleum sibiricum* L. inducing apoptosis by forming adducts with DNA. Bogucka – Kocka (2004) reported a visible influence of these compounds on the inhibition of the proliferation and on induction of apoptosis processes in the human HL-60 cell lines. Moreover, compounds isolated from *Angelica dahurica*  (Apiaceae) were examined regarding their cytotoxic activity against L1210, HL-60, K562, and B16F10 tumor cells lines using the MMT cell assay. It was discovered that pangelin and oxypeucedanin hydrate acetonide exhibited the most cytotoxic activity against all selected tumor cell lines (Heinrich et al., 2004). Um and co-authors (2010) were isolated four furonocoumarins (bergapten, isopimpinellin, xanthotoxin and imperatorin) from *Glehnia littoralis* F. Schmidt ex Miquel (Apiaceae), which exhibited dose-dependent inhibitory effectson the cell proliferation. Their study demonstrated that *G. littoralis* has potent

In addition, Oxypeucedanine (= prangolarin), which was isolated from *Prangos, Hippomarathrum, Angelica* and *Ferulago* (genera of Apiaceae) and *Ruta* genus of Rutaceae, has pharmacological and biological activities. It was reported to have antiarrhytmic, channel blocer and antiestrogenic activity. Razavi et al. (2010) studied phytotoxic, antibacterial, antifungal, antioxidant and cytotoxic effects of oxypeucedanin. Their results revealed that this compound exhibits considerable phytotoxic activity and might play an allelopathic role for plants. On the other hand, oxypeucedanin exhibits considerable cytotoxicity against Hela

The ethanol extract of the *Cnidii* fructus and coumarins separated from it have growth-

One of the major bioactive components of the fruits of *Cnidium monnieri* (L.) Cusson, bergapten, possesses antiinflammatory and analgesic activities. However, imperatorin exhibits strong cytotoxic activity on human leukemia, chemopreventive effects on hepatitis

In addition of bergapten, this plant also contained numbers of others coumarins, such as xanthotoxin, isopimpinellin, bergapten, imperatorin and osthole. These constituents regarded for biological activity of this crude drug, which is used for treatment of pain in

Pharmacological studies have indicated that coumarins such as isoimperatorin, notopterol and bergapten possess anti-inflammatory, analgesic, anti-cancer and anti-coagulant

Imperatorin (8-isopentenyloxypsoralen; 9-(3-methylbut-2-enyloxy)-7H-furo [3,2-g]chromen-7-one) is a bio-active furanocoumarin isolated e.g. from roots of *Angelica dahurica* and fruits of *Angelica archangelica* (Umbelliferae) (Baek et al., 2000). Experimental evidence indicates that imperatorin irreversibly inactivates γ-aminobutyric acid (GABA)-transaminase (the enzyme responsible for the degradation of GABA) and thus, increases the GABA content in the synaptic clefts of neurons and elevates the inhibitory neurotransmitter GABA level in

the survival and growth of breast hormone-responsive tumors.

inhibitory effect on proliferation of HT-29 human colon cancer cells.

cell line (IC50 value of 314 µg/ml).

activities (Qian et al., 2007).

inhibitory effects on the tumor cells (Chen et al., 2007).

and skin tumor, and antiinflammatory activity (Li & Chen, 2004).

female genitalia, impotence and supportive (Chen et al., 2007).

the brain (Choi et al., 2005; Łuszczki et al., 2007).

Quite recently, it has been documented that imperatorin in a dose-dependent manner increased the threshold for maximal electro-convulsions in mice (Łuszczki et al., 2007a). The time–course and dose–response relationship analyses revealed that the time to peak of the maximum anticonvulsant effect for imperatorin was established at 30 min after its systemie (i.p.) administration in mice (Łuszczki et al., 2007a).

Recently results indicate that imperatorin administered at subthreshold doses enhanced the anticonvulsant effects of carbamazepine, phenytoin and phenobarbital, but not those of valproate against maximal electroshock-induced seizures in mice. It is important to note that the anti-seizure effects of carbamazepine combined with imperatorin were greater than those observed for the combinations of phenobarbital and phenytoin with imperatorin.

The difference in the anti-seizure effects of carbamazepine and phenytoin or phenobarbital in the maximal electroshock seizure test may be explained through pharmacokinetic interaction between imperatorin and carbamazepine. It was found that imperatorin significantly increased total brain carbamazepine concentrations, having no impact on the total brain phenytoin and phenobarbital concentrations in experimental animals. The selectivity in the increase in total brain carbamazepine concentration one can try to explain through the fact that imperatorin probably enhances the penetration of carbamazepine into the brain by modifying the blood–brain barrier permeability. On the other hand, it may be hypothesized that the selective increase in carbamazepine content in the brain tissue resulted from imperatorin-induced inhibition of multi-drug resistance proteins or Pglycoproteins that normal physiological activity is related to the removal of drugs from the brain tissue. Thus, inhibitors of these proteins may contribute to the accumulation of antiepileptic drugs in the brain (Brandt et al., 2006; Łuszczki et al., 2007).

Considering molecular mechanisms of the action of conventional antiepileptic drugs and imperatorin, one can ascertain that imperatorin-induced irreversible inactivation of GABAtransaminase and subsequent increases in GABA content in the brain, as well as, the enhanced GABA-mediated inhibitory neurotransmitter action through the interaction of imperatorin with benzodiazepine receptors. This may exhibit complementary potentials to the anticonvulsant activity of carbamazepine, phenytoin and phenobarbital shown in experimental animals testing. Noteworthy, the main anticonvulsant mechanism of the action of carbamazepine and phenytoin is related to the blockade of Na+ channels in certain neurons (Łuszczki et al., 2007).

It is interesting to note that imperatorin did not potentiate the protective action of valproate against maximal electroshock-induced seizures. This apparent lack of effects of imperatorin on the antiseizure action of valproate, one can try to explain by the fact that valproate possesses a number of variousmechanisms of action that contribute to its anti-seizure activity in both rodents and humans (Łuszczki et al., 2007).

The evaluation of acute adverse effect potentials is exhibited within combinations of imperatorin with conventional antiepileptic drugs revealing that the combinations did not disturb long-term memory, impair motor co-ordination, or change neuromuscular gripstrength in experimental animals. Therefore, the investigated combinations seem to be secure and well tolerated by experimental animals (Łuszczki et al., 2007).

It was shown that imperatorin enhances the protective action of carbamazepine, phenytoin and phenobarbital, but not that of valproate against maxima electroshock-induced seizures

Analytical Methods for Isolation,

described as follows.

solid extraction (LSE).

Hajnos et al., 2007).

2006).

**5.1 Extraction from plant material** 

Separation and Identification of Selected Furanocoumarins in Plant Material 65

As furanocoumarins have wide applications in biology and have many therapeutic activities, the study of isolation and identification of these compounds is very important. In this part of our work, review of possible methods of isolation of furanocoumarins will be

Coumarins typically appear as colorless or yellow crystalline substances, well soluble in organic solvents (chloroform, diethyl ether, ethyl alcohol), as well as in fats and fatty oils. Coumarin and its derivatives exhibit sublimation on heating to 100C (Lozhkin & Sakanyan,

In this process of quantitative analysis of plant secondary metabolites, preliminary treatment of the plant materials is one of the most time-consuming steps. The first problem is the extraction of the compounds from the plant material – usually performed by liquid –

In research of the content of pharmacologically active compounds in medicinal plants, the routine procedure of extraction from plant tissues is usually applied. The extraction from plant material is frequently carried out by means of "classic" solvent-based procedures, in Soxhlet apparatus, or more simply, in laboratory flask at the temperature of the solvent's boiling under reflux (de Castro & da Silva, 1997; Saim et al., 1997). The imperfection of the time and solvent-consuming methods consists of poor penetration of the tissues by the solvent and also possible destruction of thermolabile compounds. Advantages of conventional extraction methods result from basic, inexpensive and simple equipment to operate. In the Soxhlet extraction, the sample is repeatedly contacted with fresh portions of the solvent in relatively high temperature and with no filtration required after the leaching step (de Castro & da Silva, 1997; de Castro & Garcia-Ayuso, 1998). Recently, modern alternative extraction methods, applied in the environmental analysis and in phytochemistry, are sometimes reported: (1) ultrasonification (USAE) (maceration in ultrasonic bath at various temperatures) (de Castro & Garcia-Ayuso, 1998; Court et al. 1996; Saim et al., 1997); (2) microwave-assisted solvent extraction in closed and open systems (MASE) (de Castro & Garcia-Ayuso, 1998; Saim et al., 1997); (3) accelerated solvent extraction (ASE) (called also PLE, pressurized solvent extraction) (Boselli et al., 2001; de Castro & Garcia-Ayuso, 1998; Ong et al., 2000; Papagiannopoulos et al., 2002; Saim et al., 1997); and (4) supercritical fluid extraction (SFE) (Saim et al., 1997). The above methods give better penetration of solvents into plant tissues or other solid matrices that are rapid and solvent saving. ASE apart from this advantage is dynamic, fast and also enables automatization of extraction and analysis procedures (Waksmundzka-Hajnos et al., 2004; Waksmundzka-

Coumarins are usually isolated from plants by extraction with solvents such as ethanol, methanol, benzene, chloroform, diethyl and petroleum ethers, or their combinations (Lozhkin & Sakanyan, 2006). The most exhaustive extraction of coumarins is achieved with ethanol and its aqueous solutions, either in cold or on heating. The total dense extract obtained after the evaporation of extractant is purified by treatment with chloroform and

diethyl or petroleum ethers (Lozhkin & Sakanyan, 2006).

**5. Analytical methods of furanocoumarins isolation** 

in mice. The lack of any changes in total brain phenytoin and phenobarbital concentrations suggested that the observed interactions of imperatorin with phenytoin and phenobarbital were pharmacodynamic in nature and thus, they deserve more attention from a preclinical viewpoint. If the results from the study of Łuszczki and co-authors (2007) can be extrapolated to clinical settings, a novel therapeutic option in the management of epilepsy may be created for epileptic patients.

Piao et al. (2004) assayed eleven furanocoumarins, isolated from *Angelica dahuricae* to determine its antioxidant activities. 9-hydroxy-4-methoxypsoralen inhibited DPPH formation by 50% at a concentration of 6.1 µg/ml (IC50), and alloisoimperatorin 9.4 µg/ml, thus the other nine furanocoumarins (oxypeucedanin hydrate, byakangelicol, pabulenol, neobyakangelicol, byakangelicin, oxypeucedanin, imperatorin, phellotorin, and isoimperatorin), with an IC50 values higher than 200 µg/ml, showed only a little DPPH radical-scavenging activities.

Tosun et al. (2008) evaluated the anticonvulsant activity of the furanocoumarins among others compounds, obtained from the fruits of *Heracleum crenatifolium*. This activity was estimated against maximal electroshock seizures induced in mice. Among analyzed compounds, bergapten showed significant anticonvulsant activity.

Osthole, a coumarin derivative extracted from many plants, such as *Cnidium monnieri* and *Angelica pubescens*, has been showed to exhibit estrogen-like effects and prevent postmenopausal osteoporosis in overiectomized rats. The latest research suggested that this compound can alleviate hyperglycemia and could be potentially developed into a novel drug for treatment of diabetes mellitus (Liang et al., 2009).

Tang et al. (2008) have reported that imperatorin and bergapten induce osteoblast differentiation and maturation in primary osteoblasts. These compounds increased also BMP-2 (bone morphogenetic protein type 2) expression via p38 and ERK-dependent (extracellular signal-regulated protein) pathways. Long–term administration of imperatorin and bergapten into the tibia of young rats also increased the protein level of BMP-2 and bone volume of secondary spongiosa.

However, the toxic effects of furanocoumarins are also well known. Da Silva et al. (2009) at computational analysis of psoralen, bergapten and their predicted metabolites revealed the presence of six toxicophoric groups related to carcinogenicity, mutagenicity, photoallergenicity, hepatotoxicity and skin sensitization.

Numerous studies have indicated that furanocoumarins are carcinogenic, and their ability to intercalate into DNA in the presence of long wave UV light accounts for their mutagenicity. Linear furocoumarins have been shown to exhibit varying levels of phototoxicity. It must be stated that with isopimpinellin, it results in having the least photosensitizing activity (Lehr et al., 2003).

Moreover, coumarin derivatives in high doses can produce significant side effects. They may induce headaches, nausea, vomiting, sleepiness, and in extreme cases, serious liver damage with potential hemorrhages as a result of hypoprothrominemia (Lozhkin & Sakanyan, 2006).

### **5. Analytical methods of furanocoumarins isolation**

#### **5.1 Extraction from plant material**

64 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

in mice. The lack of any changes in total brain phenytoin and phenobarbital concentrations suggested that the observed interactions of imperatorin with phenytoin and phenobarbital were pharmacodynamic in nature and thus, they deserve more attention from a preclinical viewpoint. If the results from the study of Łuszczki and co-authors (2007) can be extrapolated to clinical settings, a novel therapeutic option in the management of epilepsy

Piao et al. (2004) assayed eleven furanocoumarins, isolated from *Angelica dahuricae* to determine its antioxidant activities. 9-hydroxy-4-methoxypsoralen inhibited DPPH formation by 50% at a concentration of 6.1 µg/ml (IC50), and alloisoimperatorin 9.4 µg/ml, thus the other nine furanocoumarins (oxypeucedanin hydrate, byakangelicol, pabulenol, neobyakangelicol, byakangelicin, oxypeucedanin, imperatorin, phellotorin, and isoimperatorin), with an IC50 values higher than 200 µg/ml, showed only a little DPPH

Tosun et al. (2008) evaluated the anticonvulsant activity of the furanocoumarins among others compounds, obtained from the fruits of *Heracleum crenatifolium*. This activity was estimated against maximal electroshock seizures induced in mice. Among analyzed

Osthole, a coumarin derivative extracted from many plants, such as *Cnidium monnieri* and *Angelica pubescens*, has been showed to exhibit estrogen-like effects and prevent postmenopausal osteoporosis in overiectomized rats. The latest research suggested that this compound can alleviate hyperglycemia and could be potentially developed into a novel

Tang et al. (2008) have reported that imperatorin and bergapten induce osteoblast differentiation and maturation in primary osteoblasts. These compounds increased also BMP-2 (bone morphogenetic protein type 2) expression via p38 and ERK-dependent (extracellular signal-regulated protein) pathways. Long–term administration of imperatorin and bergapten into the tibia of young rats also increased the protein level of BMP-2 and

However, the toxic effects of furanocoumarins are also well known. Da Silva et al. (2009) at computational analysis of psoralen, bergapten and their predicted metabolites revealed the presence of six toxicophoric groups related to carcinogenicity, mutagenicity,

Numerous studies have indicated that furanocoumarins are carcinogenic, and their ability to intercalate into DNA in the presence of long wave UV light accounts for their mutagenicity. Linear furocoumarins have been shown to exhibit varying levels of phototoxicity. It must be stated that with isopimpinellin, it results in having the least photosensitizing activity (Lehr

Moreover, coumarin derivatives in high doses can produce significant side effects. They may induce headaches, nausea, vomiting, sleepiness, and in extreme cases, serious liver damage with potential hemorrhages as a result of hypoprothrominemia (Lozhkin &

compounds, bergapten showed significant anticonvulsant activity.

drug for treatment of diabetes mellitus (Liang et al., 2009).

photoallergenicity, hepatotoxicity and skin sensitization.

bone volume of secondary spongiosa.

et al., 2003).

Sakanyan, 2006).

may be created for epileptic patients.

radical-scavenging activities.

As furanocoumarins have wide applications in biology and have many therapeutic activities, the study of isolation and identification of these compounds is very important. In this part of our work, review of possible methods of isolation of furanocoumarins will be described as follows.

Coumarins typically appear as colorless or yellow crystalline substances, well soluble in organic solvents (chloroform, diethyl ether, ethyl alcohol), as well as in fats and fatty oils. Coumarin and its derivatives exhibit sublimation on heating to 100C (Lozhkin & Sakanyan, 2006).

In this process of quantitative analysis of plant secondary metabolites, preliminary treatment of the plant materials is one of the most time-consuming steps. The first problem is the extraction of the compounds from the plant material – usually performed by liquid – solid extraction (LSE).

In research of the content of pharmacologically active compounds in medicinal plants, the routine procedure of extraction from plant tissues is usually applied. The extraction from plant material is frequently carried out by means of "classic" solvent-based procedures, in Soxhlet apparatus, or more simply, in laboratory flask at the temperature of the solvent's boiling under reflux (de Castro & da Silva, 1997; Saim et al., 1997). The imperfection of the time and solvent-consuming methods consists of poor penetration of the tissues by the solvent and also possible destruction of thermolabile compounds. Advantages of conventional extraction methods result from basic, inexpensive and simple equipment to operate. In the Soxhlet extraction, the sample is repeatedly contacted with fresh portions of the solvent in relatively high temperature and with no filtration required after the leaching step (de Castro & da Silva, 1997; de Castro & Garcia-Ayuso, 1998). Recently, modern alternative extraction methods, applied in the environmental analysis and in phytochemistry, are sometimes reported: (1) ultrasonification (USAE) (maceration in ultrasonic bath at various temperatures) (de Castro & Garcia-Ayuso, 1998; Court et al. 1996; Saim et al., 1997); (2) microwave-assisted solvent extraction in closed and open systems (MASE) (de Castro & Garcia-Ayuso, 1998; Saim et al., 1997); (3) accelerated solvent extraction (ASE) (called also PLE, pressurized solvent extraction) (Boselli et al., 2001; de Castro & Garcia-Ayuso, 1998; Ong et al., 2000; Papagiannopoulos et al., 2002; Saim et al., 1997); and (4) supercritical fluid extraction (SFE) (Saim et al., 1997). The above methods give better penetration of solvents into plant tissues or other solid matrices that are rapid and solvent saving. ASE apart from this advantage is dynamic, fast and also enables automatization of extraction and analysis procedures (Waksmundzka-Hajnos et al., 2004; Waksmundzka-Hajnos et al., 2007).

Coumarins are usually isolated from plants by extraction with solvents such as ethanol, methanol, benzene, chloroform, diethyl and petroleum ethers, or their combinations (Lozhkin & Sakanyan, 2006). The most exhaustive extraction of coumarins is achieved with ethanol and its aqueous solutions, either in cold or on heating. The total dense extract obtained after the evaporation of extractant is purified by treatment with chloroform and diethyl or petroleum ethers (Lozhkin & Sakanyan, 2006).

Analytical Methods for Isolation,

(Martino et al., 2006).

had the best results.

bergapten was extraction with dichloromethane.

efficient solvent (100%) (Yu et al., 2002).

**5.2 Sample purification** 

et al., 2010).

Separation and Identification of Selected Furanocoumarins in Plant Material 67

Soxhlet extraction, ultrasound-assisted extraction and microwaves-assisted extraction in the closed system have been investigated to determine the content of coumarins in flowering tops of *Melilotus officinalis*. Soxhlet extraction was performed in a Soxhlet apparatus equipped with cellulose extraction thimbles. Extraction was performed with ethanol (85C). Ultrasound-assisted extraction was conducted with 50% (v/v) aq. ethanol, in an ultrasonic bath, and MASE with 50% (v/v) aq. ethanol was performed using a closed-vessel system

Soxhlet extraction was used in the isolation of oxypeucedanin from *Prangos uloptera*. Dried and powdered leaves were extracted with n-hexane, dichloromethane and methanol (Razavi

Celeghini et al. (2001) studied the extraction conditions for coumarin analysis in hydroalcoholic extracts of *Mikania glomerata* Spreng leaves. Maceration, maceration under sonication, infusion and supercritical fluid extraction (SFE) were compared. In SFE method, the solvent extraction system was pressurized in the high pressure vessel with the aid of a nitrogen cylinder. Several solvent mixtures were used including CO2:EtOH (95:5), (90:10), (85:15) and CO2:EtOH:H2O (95:2.5:2.5). The experiment was conducted at the same pressure and temperature. The evaluation of these methods showed that maceration under sonication

Kozyra & Głowniak (2006) examined the influence of using solvent in the isolation of furanocoumarins. They carried out extraction techniques with different eluents such nheptane, dichloromethane and methanol. These extractions were performed on a water bath with boiling eluent and on an ultrasonic bath, for 12 and 24 hrs. The more efficient for

In another study, six solvents (n-hexane, chloroform, ethyl acetate, ethanol, acetonitrile and water) were used to extract *Cnidii Fructus* in order to evaluate their efficiency in extracting osthole. A comparative evaluation showed that aqueous alcoholic solvent was the most

The furanocoumarin determination from air-dried plant material was also performed using 75% methanol in an ultrasonic ice–water bath (Yang et al., 2010), with 100% methanol (Cardoso et al., 2000; Ojala, 2000), 70% methanol (Chen & Sheu, 1995), with hot (70C) pure methanol on a water bath (Bartnik & Głowniak, 2007), with pure ethanol in heated reflux (Yu et al., 2002), with 95% ethanol at 80C (Wang et al., 2007; Zheng et al., 2010), with acetone at room temperature (Taniguchi et al., 2011), with ether at 40C (Liu et al., 2004a), with dichloromethane at room temperature (Um et al., 2010), with petroleum ether at room temperature (Tosun et al., 2008), with chloroform in a sonic bath (Cardoso et al., 2000).

The extraction with all solvents was usually done 2–5 times, obtaining solutions that were filtered and evaporated under reduced pressure. Frequently, residuals after methanol/ethanol extractions were suspended in water and portioned a few times with

The next step in sample preparations is the purification of the crude extract. Plant extracts contain much ballast material, both non-polar (chlorophylls, waxes) and polar such as

chloroform or petroleum ether (Wang et al., 2007; Zheng et al., 2010).

Petroleum ether is the extractant usually used in selective extraction of furanocoumarin fraction from plant tissues (Głowniak, 1988), whereas more polar coumarins—hydroxyderivatives are extracted with methanol. Methanol, used after petroleum ether on the same plant material, extracts more hydrophylic coumarins, but also the residual of furanocoumarins.

Historically, exhaustive extraction with different solvents, which can be performed in Soxhlet apparatus, proved to be the most accurate method of isolation of these groups of compound (Głowniak, 1988; Hadacek et al., 1994). The extraction of the same plant material is usually continued with methanol. For example, peucedanin was successfully isolated using this type of extraction with methanol (Lozhkin & Sakanyan, 2006).

Waksmundzka-Hajnos et al. (2004) compared methods of extraction of furanocoumarins. Some of furanocoumarins from *Pastinaca sativa* fruits were extracted using exhaustive extraction with petroleum ether in Soxhlet apparatus, ultrasonification (USAE), accelerated solvent extraction (ASE) and microwave-assisted solvent extraction (MASE).

USAE was performed with petroleum ether in ultrasonic bath at an ambient temperature of 20C or at a temperature of 60C for 30 min three times.

In the ASE method, the plant material was mixed with neutral glass and placed into a stainless steel extraction cell. The application of neutral glass, playing the role of dispersion agent, is recommended to reduce the volume of the solvent used for the extraction (ASE 200, 1995). This extraction was performed with pure methanol or petroleum ether at the same pressure (60 bar).

MASE was also used in the isolation of furocoumarin fractions performing with 80% methanol in a water bath using a two-step extraction with results of 40% generator power during 1 min and by 60% generator power during 30 mins in open and closed systems.

In most cases of the Waksmundzka-Hajnos et al. (2004) experiment, exhaustive extraction in Soxhlet apparatus indicates low yields of furanocoumarins. For example, the use of ultrasonification at 60C gives, in most cases a higher yield than the exhaustive Soxhlet method. In some cases, this method gives the highest yield of extraction (for xanthotoxin and for isopimpinellin) in comparison to all methods used in experimentation. Also, the use of ASE gives, in most cases, higher yields than the Soxhlet extraction (compare yield of extraction of isopimpinellin, bergapten, imperatorin and phellopterin). In case of bergapten, imperatorin, and phellopterin the yield of extraction by ASE was highest in comparison to all extraction methods used in experiments.

Microwave-assisted solvent extraction gives fair extraction yield for more polar furanocoumarins, probably because of the necessary use of more polar extractant (80% MeOH in water). From the gathered data, it is seen that the extraction yield of phellopterin and imperatorin in pressurized MASE is distinctly lower than in open systems. It shows that in a closed system, the extracted compounds were changed by microwaves. Hence, pressurised MASE cannot be recommended as a leaching method of furanocoumarin fraction (Waksmundzka-Hajnos et al., 2004).

These results are similar to those obtained from the same authors in previous investigations, in which they isolated furanocoumarins from *Archangelica officinalis* fruits. This study indicated the highest yield of psoralens by ASE, using methanol or petroleum ether as the extractant. It was also reported that microwave-assisted solvent extraction in the closed system probably causing the change of analytes (Waksmundzka-Hajnos et al., 2004a).

Petroleum ether is the extractant usually used in selective extraction of furanocoumarin fraction from plant tissues (Głowniak, 1988), whereas more polar coumarins—hydroxyderivatives are extracted with methanol. Methanol, used after petroleum ether on the same plant material,

Historically, exhaustive extraction with different solvents, which can be performed in Soxhlet apparatus, proved to be the most accurate method of isolation of these groups of compound (Głowniak, 1988; Hadacek et al., 1994). The extraction of the same plant material is usually continued with methanol. For example, peucedanin was successfully isolated

Waksmundzka-Hajnos et al. (2004) compared methods of extraction of furanocoumarins. Some of furanocoumarins from *Pastinaca sativa* fruits were extracted using exhaustive extraction with petroleum ether in Soxhlet apparatus, ultrasonification (USAE), accelerated

USAE was performed with petroleum ether in ultrasonic bath at an ambient temperature of

In the ASE method, the plant material was mixed with neutral glass and placed into a stainless steel extraction cell. The application of neutral glass, playing the role of dispersion agent, is recommended to reduce the volume of the solvent used for the extraction (ASE 200, 1995). This extraction was performed with pure methanol or petroleum ether at the same

MASE was also used in the isolation of furocoumarin fractions performing with 80% methanol in a water bath using a two-step extraction with results of 40% generator power during 1 min and by 60% generator power during 30 mins in open and closed systems.

In most cases of the Waksmundzka-Hajnos et al. (2004) experiment, exhaustive extraction in Soxhlet apparatus indicates low yields of furanocoumarins. For example, the use of ultrasonification at 60C gives, in most cases a higher yield than the exhaustive Soxhlet method. In some cases, this method gives the highest yield of extraction (for xanthotoxin and for isopimpinellin) in comparison to all methods used in experimentation. Also, the use of ASE gives, in most cases, higher yields than the Soxhlet extraction (compare yield of extraction of isopimpinellin, bergapten, imperatorin and phellopterin). In case of bergapten, imperatorin, and phellopterin the yield of extraction by ASE was highest in comparison to

Microwave-assisted solvent extraction gives fair extraction yield for more polar furanocoumarins, probably because of the necessary use of more polar extractant (80% MeOH in water). From the gathered data, it is seen that the extraction yield of phellopterin and imperatorin in pressurized MASE is distinctly lower than in open systems. It shows that in a closed system, the extracted compounds were changed by microwaves. Hence, pressurised MASE cannot be recommended as a leaching method of furanocoumarin

These results are similar to those obtained from the same authors in previous investigations, in which they isolated furanocoumarins from *Archangelica officinalis* fruits. This study indicated the highest yield of psoralens by ASE, using methanol or petroleum ether as the extractant. It was also reported that microwave-assisted solvent extraction in the closed system probably causing the change of analytes (Waksmundzka-Hajnos et al., 2004a).

extracts more hydrophylic coumarins, but also the residual of furanocoumarins.

using this type of extraction with methanol (Lozhkin & Sakanyan, 2006).

solvent extraction (ASE) and microwave-assisted solvent extraction (MASE).

20C or at a temperature of 60C for 30 min three times.

all extraction methods used in experiments.

fraction (Waksmundzka-Hajnos et al., 2004).

pressure (60 bar).

Soxhlet extraction, ultrasound-assisted extraction and microwaves-assisted extraction in the closed system have been investigated to determine the content of coumarins in flowering tops of *Melilotus officinalis*. Soxhlet extraction was performed in a Soxhlet apparatus equipped with cellulose extraction thimbles. Extraction was performed with ethanol (85C). Ultrasound-assisted extraction was conducted with 50% (v/v) aq. ethanol, in an ultrasonic bath, and MASE with 50% (v/v) aq. ethanol was performed using a closed-vessel system (Martino et al., 2006).

Soxhlet extraction was used in the isolation of oxypeucedanin from *Prangos uloptera*. Dried and powdered leaves were extracted with n-hexane, dichloromethane and methanol (Razavi et al., 2010).

Celeghini et al. (2001) studied the extraction conditions for coumarin analysis in hydroalcoholic extracts of *Mikania glomerata* Spreng leaves. Maceration, maceration under sonication, infusion and supercritical fluid extraction (SFE) were compared. In SFE method, the solvent extraction system was pressurized in the high pressure vessel with the aid of a nitrogen cylinder. Several solvent mixtures were used including CO2:EtOH (95:5), (90:10), (85:15) and CO2:EtOH:H2O (95:2.5:2.5). The experiment was conducted at the same pressure and temperature. The evaluation of these methods showed that maceration under sonication had the best results.

Kozyra & Głowniak (2006) examined the influence of using solvent in the isolation of furanocoumarins. They carried out extraction techniques with different eluents such nheptane, dichloromethane and methanol. These extractions were performed on a water bath with boiling eluent and on an ultrasonic bath, for 12 and 24 hrs. The more efficient for bergapten was extraction with dichloromethane.

In another study, six solvents (n-hexane, chloroform, ethyl acetate, ethanol, acetonitrile and water) were used to extract *Cnidii Fructus* in order to evaluate their efficiency in extracting osthole. A comparative evaluation showed that aqueous alcoholic solvent was the most efficient solvent (100%) (Yu et al., 2002).

The furanocoumarin determination from air-dried plant material was also performed using 75% methanol in an ultrasonic ice–water bath (Yang et al., 2010), with 100% methanol (Cardoso et al., 2000; Ojala, 2000), 70% methanol (Chen & Sheu, 1995), with hot (70C) pure methanol on a water bath (Bartnik & Głowniak, 2007), with pure ethanol in heated reflux (Yu et al., 2002), with 95% ethanol at 80C (Wang et al., 2007; Zheng et al., 2010), with acetone at room temperature (Taniguchi et al., 2011), with ether at 40C (Liu et al., 2004a), with dichloromethane at room temperature (Um et al., 2010), with petroleum ether at room temperature (Tosun et al., 2008), with chloroform in a sonic bath (Cardoso et al., 2000).

The extraction with all solvents was usually done 2–5 times, obtaining solutions that were filtered and evaporated under reduced pressure. Frequently, residuals after methanol/ethanol extractions were suspended in water and portioned a few times with chloroform or petroleum ether (Wang et al., 2007; Zheng et al., 2010).

#### **5.2 Sample purification**

The next step in sample preparations is the purification of the crude extract. Plant extracts contain much ballast material, both non-polar (chlorophylls, waxes) and polar such as

Analytical Methods for Isolation,

(Lozhkin & Sakanyan, 2006).

acetone mixtures (Wang et al., 2007).

oxypeucedanine (Razavi et al., 2010).

2004).

Separation and Identification of Selected Furanocoumarins in Plant Material 69

mixtures or on silica gel column eluted sequentially with hexane-chloroform and chloroform-ethanol systems with increased proportion of a more hydrophilic component

Separation of the psoralens from *Heracleum sibiricum* L. (Apiaceae) fruits was performed by gravitation column chromatography. Glass columns were filled with silica gel (230-400 mesh) and run, under UV-lamp control, in the following eluents: 1) benzene-ethyl acetate; mixtures of increasing polarity (12.5 to 77.5%); 2) benzene-ethyl acetate (17:3); 3) benzenechloroform-ethyl acetate (1:1, v/v, 5%). Chromatographically pure clean compounds, in this

In another investigation, the coumarin mixture from fruits of *Heracleum crenatifolium* was subjected to CC on silica gel and eluted successively with an n-hexane-ethyl acetate solvent system, with increasing polarity (99:1 to 80:20). The collected fractions were applied to preparative-TLC on silica gel plates and pure furanocoumarins were obtained. After chromatography with the use of n-hexane-ethyl acetate (3:1), isobergapten and pimpinellin were obtained. Fractions, which were chromatographed with n-hexane-dichloromethaneethyl acetate (4:4:2) resulted in a production of bergapten, and fractions, after chromatography using toluene-ethyl acetate (9:1), resulted in a production of yielded

On silica-gel column chromatography was also subjected to chloroform residue from roots of *Angelica dahurica*. The furanocoumarins were eluted stepwise with petroleum ether-

Similar techniques were performed for furanocoumarins from the roots *Angelicae dahuricae*. The methylene chloride soluble was chromatographed using column chromatography over silica gel. In this study, a stepwise gradient solution with hexane-ethyl acetate (5:1 to 0:1) was used. Repeated column chromatography of obtained fractions produced an isoimperatorin, imperatorin, oxypeucedanin, phellotorin, byakangelicol, neobyakangelicol, alloisoimperatorin, pabulenol, byakangelicin, and 9-hydroxy-4-methoxypsoralen (Piao et al.,

A vacuum liquid chromatography on silica gel was developed for the isolation of oxypeucedanin from the leaves of *Prangos uloptera.* Hexane extract was subjected, starting with 100% hexane, followed by step gradient of ethyl acetate mixtures (1:99; 5:95; 10:90; 20:80; 40:60; 60:40; 80:20; 100) and finally methanol. The obtained fractions were purified by preparative-TLC on silica gel using (CH3)2CO-CHCl3, 5:95 as the mobile phase to yield

The CC technique was used to separate furanocoumarins from roots of several *Dorstenia* species. The afforded hexane residues were chromatographed on silica gel (230-400 mesh) eluting with hexane-chloroform mixtures (1:1) (gives psoralen) or with hexane-ethyl acetate mixtures of an increasing polarity to give bergapten, 4-[3-(4,5-Dihydro-5.5-dimethyl-4-oxo-2-furanyl)-butoxy]=7H-furo[3,2-g][1]benzopyran-7-one, psoralen and 7-hydroxycoumarin. The chloroform extracts were eluted using hexane-chloroform mixtures to give psoralen, 7 hydroxycoumarin and 4-[3-(4.5-Dihydro-5.5-dimethyl-4-oxo-2-furanyl)-butoxy]=7Hfuro[3.2-g][1]benzopyran-7-one or hexane-ethyl acetate of increasing polarity to give psoralen, psoralen dimer and 7-hydroxycoumarin. The polar fractions from the methanol

study, were crystallized from 96% ethanol (Bogucka-Kocka, 1999).

isopimpinellin, sphondin and byak-angelicol (Tosun et al., 2008).

tannins or sugars. Most often liquid-liquid extraction (LLE) is used, which takes advantages of solubility differences of hydrophobic substances, which have affinity for non-polar solvents, and hydrophobic substances, which have an affinity for aqueous solutions. Although the analyses can be easily obtained by evaporation of the solvent, the method has many disadvantages – for example emulsions can be formed and the process is timeconsuming. Purification can also be achieved by solid-phase-extraction (SPE). This method uses a variety of adsorbents and ion-exchangers and is widely used for a variety of purposes (Fritz & Macha, 2000; Hennion, 1999; Nilsson, 2000; Snyder et al., 1997; Waksmundzka-Hajnos et al., 2007).

The SPE method is very often used in sample pre-treatment for HPLC. This method has been developed for the purification of furanocoumarins from *Peucedanum tautaricum* Bieb. In the first step, aqueous methanol (50%; v/v) solutions of the samples were passed through conditioned microcolumns to adsorb furanocoumarins on the adsorbent bed. The microcolumns were washed with 50% methanol (Zgórka & Głowniak, 1999), and the compounds of interest group were separated from fatty components and chlorophyll by use of SPE microcolumns (LiChrolut RP-18 E; 500 mg, 3 mL). In the next step, the absorbed furanocoumarins were eluted at a flow-rate of 0.5 mL min-1 with 80% methanol into vials previously calibrated with a pipette (Bartnik & Głowniak, 2007).

Sidwa-Gorycka et al. (2003) used SPE for purification furanocoumaric fractions obtained from *Ammi majus* L. and *Ruta graveolens* L. methanolic (30%) extracts. They were loaded into octadecyl-SPE microcolumns activated previously with 100% methanol, followed by the selective elution of compounds. The cartridges were washed with 20 ml of 60% methanol to elute the coumarins. The eluting solvents were passed through the sorbent beds at a flow rate of 0.5 ml min-1.

In addition, the SPE has been developed for purification of furanocoumarin fractions from creams and pomades. The obtained samples were cleaned-up using two methods. Each extracted sample was re-dissolved in chloroform and fractionated on cartridges, which were previously conditioned with chloroform and sequentially eluted with chloroform (first fraction), chloroform:methanol (90:10; v/v) (second fraction, furanocoumarins), chloroform:methanol (1:1; v/v) (third fraction) and methanol (fourth fraction). Next, each sample extracted above was re-dissolved in methanol in a sonic bath and fractionated on cartridges, which were previously conditioned with methanol and sequentially eluted with methanol (first fraction), methanol:chloroform (80:20; v/v) (second fraction, furanocoumarins), methanol:chloroform (1:1; v/v) (third fraction) and chloroform (fourth fraction). All fractions were evaporated to dryness in a stream of nitrogen (Cardoso et al., 2000).

#### **5.3 Chromatographic methods in the analysis of furanocoumarins**

#### **5.3.1 Column Chromatography (CC)**

The good results for purification, separation of the total furanocoumarins and the isolation of individual compounds give column chromatography (CC) a significant advantage of the use of various sorbents and solvent systems.

Furanocoumarins can be fractionated on an aluminum oxide column eluted with petroleum ether, petroleum ether-chloroform (2:1), chloroform, and chloroform-ethanol (9:1; 4:1; 2:1)

tannins or sugars. Most often liquid-liquid extraction (LLE) is used, which takes advantages of solubility differences of hydrophobic substances, which have affinity for non-polar solvents, and hydrophobic substances, which have an affinity for aqueous solutions. Although the analyses can be easily obtained by evaporation of the solvent, the method has many disadvantages – for example emulsions can be formed and the process is timeconsuming. Purification can also be achieved by solid-phase-extraction (SPE). This method uses a variety of adsorbents and ion-exchangers and is widely used for a variety of purposes (Fritz & Macha, 2000; Hennion, 1999; Nilsson, 2000; Snyder et al., 1997; Waksmundzka-

The SPE method is very often used in sample pre-treatment for HPLC. This method has been developed for the purification of furanocoumarins from *Peucedanum tautaricum* Bieb. In the first step, aqueous methanol (50%; v/v) solutions of the samples were passed through conditioned microcolumns to adsorb furanocoumarins on the adsorbent bed. The microcolumns were washed with 50% methanol (Zgórka & Głowniak, 1999), and the compounds of interest group were separated from fatty components and chlorophyll by use of SPE microcolumns (LiChrolut RP-18 E; 500 mg, 3 mL). In the next step, the absorbed furanocoumarins were eluted at a flow-rate of 0.5 mL min-1 with 80% methanol into vials

Sidwa-Gorycka et al. (2003) used SPE for purification furanocoumaric fractions obtained from *Ammi majus* L. and *Ruta graveolens* L. methanolic (30%) extracts. They were loaded into octadecyl-SPE microcolumns activated previously with 100% methanol, followed by the selective elution of compounds. The cartridges were washed with 20 ml of 60% methanol to elute the coumarins. The eluting solvents were passed through the sorbent beds at a flow

In addition, the SPE has been developed for purification of furanocoumarin fractions from creams and pomades. The obtained samples were cleaned-up using two methods. Each extracted sample was re-dissolved in chloroform and fractionated on cartridges, which were previously conditioned with chloroform and sequentially eluted with chloroform (first fraction), chloroform:methanol (90:10; v/v) (second fraction, furanocoumarins), chloroform:methanol (1:1; v/v) (third fraction) and methanol (fourth fraction). Next, each sample extracted above was re-dissolved in methanol in a sonic bath and fractionated on cartridges, which were previously conditioned with methanol and sequentially eluted with methanol (first fraction), methanol:chloroform (80:20; v/v) (second fraction, furanocoumarins), methanol:chloroform (1:1; v/v) (third fraction) and chloroform (fourth fraction). All fractions

The good results for purification, separation of the total furanocoumarins and the isolation of individual compounds give column chromatography (CC) a significant advantage of the

Furanocoumarins can be fractionated on an aluminum oxide column eluted with petroleum ether, petroleum ether-chloroform (2:1), chloroform, and chloroform-ethanol (9:1; 4:1; 2:1)

previously calibrated with a pipette (Bartnik & Głowniak, 2007).

were evaporated to dryness in a stream of nitrogen (Cardoso et al., 2000).

**5.3 Chromatographic methods in the analysis of furanocoumarins** 

**5.3.1 Column Chromatography (CC)** 

use of various sorbents and solvent systems.

Hajnos et al., 2007).

rate of 0.5 ml min-1.

mixtures or on silica gel column eluted sequentially with hexane-chloroform and chloroform-ethanol systems with increased proportion of a more hydrophilic component (Lozhkin & Sakanyan, 2006).

Separation of the psoralens from *Heracleum sibiricum* L. (Apiaceae) fruits was performed by gravitation column chromatography. Glass columns were filled with silica gel (230-400 mesh) and run, under UV-lamp control, in the following eluents: 1) benzene-ethyl acetate; mixtures of increasing polarity (12.5 to 77.5%); 2) benzene-ethyl acetate (17:3); 3) benzenechloroform-ethyl acetate (1:1, v/v, 5%). Chromatographically pure clean compounds, in this study, were crystallized from 96% ethanol (Bogucka-Kocka, 1999).

In another investigation, the coumarin mixture from fruits of *Heracleum crenatifolium* was subjected to CC on silica gel and eluted successively with an n-hexane-ethyl acetate solvent system, with increasing polarity (99:1 to 80:20). The collected fractions were applied to preparative-TLC on silica gel plates and pure furanocoumarins were obtained. After chromatography with the use of n-hexane-ethyl acetate (3:1), isobergapten and pimpinellin were obtained. Fractions, which were chromatographed with n-hexane-dichloromethaneethyl acetate (4:4:2) resulted in a production of bergapten, and fractions, after chromatography using toluene-ethyl acetate (9:1), resulted in a production of yielded isopimpinellin, sphondin and byak-angelicol (Tosun et al., 2008).

On silica-gel column chromatography was also subjected to chloroform residue from roots of *Angelica dahurica*. The furanocoumarins were eluted stepwise with petroleum etheracetone mixtures (Wang et al., 2007).

Similar techniques were performed for furanocoumarins from the roots *Angelicae dahuricae*. The methylene chloride soluble was chromatographed using column chromatography over silica gel. In this study, a stepwise gradient solution with hexane-ethyl acetate (5:1 to 0:1) was used. Repeated column chromatography of obtained fractions produced an isoimperatorin, imperatorin, oxypeucedanin, phellotorin, byakangelicol, neobyakangelicol, alloisoimperatorin, pabulenol, byakangelicin, and 9-hydroxy-4-methoxypsoralen (Piao et al., 2004).

A vacuum liquid chromatography on silica gel was developed for the isolation of oxypeucedanin from the leaves of *Prangos uloptera.* Hexane extract was subjected, starting with 100% hexane, followed by step gradient of ethyl acetate mixtures (1:99; 5:95; 10:90; 20:80; 40:60; 60:40; 80:20; 100) and finally methanol. The obtained fractions were purified by preparative-TLC on silica gel using (CH3)2CO-CHCl3, 5:95 as the mobile phase to yield oxypeucedanine (Razavi et al., 2010).

The CC technique was used to separate furanocoumarins from roots of several *Dorstenia* species. The afforded hexane residues were chromatographed on silica gel (230-400 mesh) eluting with hexane-chloroform mixtures (1:1) (gives psoralen) or with hexane-ethyl acetate mixtures of an increasing polarity to give bergapten, 4-[3-(4,5-Dihydro-5.5-dimethyl-4-oxo-2-furanyl)-butoxy]=7H-furo[3,2-g][1]benzopyran-7-one, psoralen and 7-hydroxycoumarin. The chloroform extracts were eluted using hexane-chloroform mixtures to give psoralen, 7 hydroxycoumarin and 4-[3-(4.5-Dihydro-5.5-dimethyl-4-oxo-2-furanyl)-butoxy]=7Hfuro[3.2-g][1]benzopyran-7-one or hexane-ethyl acetate of increasing polarity to give psoralen, psoralen dimer and 7-hydroxycoumarin. The polar fractions from the methanol

Analytical Methods for Isolation,

for analysis of coumarins.

Sakanyan, 2006).

Eltayeb, 2004).

cases.

examined under UV light at 366 nm.

Separation and Identification of Selected Furanocoumarins in Plant Material 71

methanol-chloroform (20:1:1:1, v/v); 5) chloroform, and 6) ethyl acetate-hexane (25:75, v/v)

The spots of coumarins on thin-layer and paper chromatograms are usually revealed by UV fluorescence at certain characteristic wavelengths, before or after the treatment with an aqueous-ethanol solution of potassium hydroxide or with ammonia vapor, or using some other color reactions. The fluorescent color does not provide accurate identification of the structure of coumarins; nevertheless, sometimes it is possible to determine the type of

Joint TLC – colorimetric methods based on the azo-addition reaction with TLC separation on an aluminum oxide layer eluted in the hexane – benzene – methanol (5:4:1) system were developed for the quantitative determination of peucedanin in *Peucedanum morrissonii*  (Bess.) and for the analysis of beroxan, pastinacin, and psoralen preparations (Lozhkin & Sakanyan, 2006). Colorimetric determination of xanthotoxin, imperatorin, and bergapten in *Ammi majus* (L.) fruits can be performed after TLC separation on silica gel impregnated with formamide and eluted in dibutyl ether. In order to determine psoralen alone and together with bergapten in *Ficus carica* (L.) leaves, the extract was purified from ballast substances and chromatographed in a thin layer of aluminum oxide in diethyl ether (Lozhkin &

Thin layer chromatographic analyses were made by Celeghini and co-authors (2001) on silica gel 60G. As eluent a mixture of toluene:ethyl ether (1:1) saturated with 10% acetic acid was used. The plates were sprayed with an ethanolic solution (5% v/v) of KOH and

In the other investigation, purification of the eluted furanocoumarins from leaves of *Conium maculatum* was also carried out on TLC plates (Si gel 60, f-254). The chromatograms were developed at room temperature using one of the following solvent systems: chloroform:ethyl acetate (2:1), or chloroform, or toluene:ethyl acetate (1:1) (Al-Barwani &

Bogucka-Kocka (1999) analyzed furanocoumarins in fruits of *Heracleum sibiricum* L., after silica gel column chromatography, using 2D-TLC. Two-dimensional thin-layer chromatography was conducted of silica gel plates and run in the following phases: 1) benzene-chloroform-acetonitrile (1:1, v/v, 5%) or 2) benzene-chloroform-ethyl acetate (1:1, v/v, 5%) (first direction); 3) benzene-ethyl acetate (1:1) (second direction). The chromatograms were analyzed using UV light and daylight, after spaying with one of the

Unfortunately, in furocoumarin' group, these substances have comparable polarity and similar chemical structures. As a result, multi-dimensional separations are required in such

Thin-layer chromatography gives the possibility of performing multi-dimensional separation – two-dimensional separation with the use of the same stationary phase, with different mobile phases (Gadzikowska et al., 2005; Härmäla et al., 1990; Waksmundzka-Hajnos et al., 2006), or by using a stationary phase gradient (Glensk et al., 2002; Waksmundzka-Hajnos et al., 2006). In TLC, there are almost no limits as far as mobile phases are concerned, because they can be easily evaporated from the layer after the

derivativers: 1) 0.5% I dissolvent in KI; 2) Dragendorf's reagent or 25% SbCl5 in CCl4.

functional groups (Celeghini et al., 2001; Lozhkin & Sakanyan, 2006).

extract were acetylated using pyridine and Ac2O to give (2'S, 1''S)-2.3-dihydro-2(2-acetoxy-1-hydroxymethylethyl)-7H-furo [3.2-g][1]benzopyran-7-one (Rojas-Lima et al., 1999).

Another useful adsorbent for column chromatography is Florisil (100-200 mesh), which was used to fractionate furanocoumarins obtained from fruits of *Peucedanum alsaticum* L. and *P. cervaria* (L.) Lap. Concentrated petroleum ether extracts were fractionated on this sorbent with a dichloromethane-ethyl acetate (0-50%) gradient, then ethyl acetate and methanol as mobile phases. After CC separation, the fractions richest in coumarins were analyzed by preparative-TLC on silica gel. Separated zones of selected furocoumarins were eluted from the plates (Skalicka-Woźniak et al., 2009).

The Florisil was also used in an investigation performed by Suzuki et al. (1979). Bergamot oil was eluted on this column with methylene chloride and ethyl acetate. The ethyl acetate fractions were re-chromatographed with methylene chloride. The obtained residue was analyzed by preparative-TLC on silica gel using cyclohexane-tetrahydrofuran (1:1) as eluent. The bergapten zone was scraped and eluted with acetone.

Isolation of the furanocoumarins from grapefruit juice was accomplished by preparative thin layer chromatography. The obtained fractions were applied to tapered silica gel GF TLC plates with a fluorescent indicator. Resolution of compounds was accomplished by using solvent systems consisting of hexane:ethyl acetate (3:1 to 2:3; v/v), chloroform, chloroform/methanol (95:5), and benzene: acetone (9:1). The zones containing furanocoumarins were scraped and extracted with acetone (Manthey et al., 2006).

#### **5.3.2 Thin Layer Chromatography (TLC)**

The physicochemical properties of coumarins depend upon their chemical structure, specifically, the presence and position of functional hydroxy or methoxy groups, and methyl or other alkyl chains. As a result of these differences, group separation of the all groups of coumarins does not cause any difficulties (Jerzmanowska, 1967; Waksmundzka-Hajnos et al., 2006). Separation of individual compounds in each group – structural analogs, i.e. closely related compounds – is, however, a difficult task.

Several analytical methods for the quality control of furanocoumarins in plant materials, such as thin layer chromatography (TLC), high performance liquid chromatography (HPLC), high performance liquid chromatography–mass spectrometry (HPLC–MS), high-speed countercurrent chromatography (HSCCC), gas chromatography (GC), gas chromatography–mass spectrometry (GC–MS), capillary electrophoresis (CE) ), and pressurized capillary electrochromatography (pCEC), has been reported (Chen et al., 2009; Wang et et., 2007).

The oldest publications recommended one- or two- thin layer chromatography for separation and identification of furanocoumarins. This method provides a quite rapid separation of components in a sample mixture. Fractions obtained from column chromatography were usually checked with the use of TLC technique.

Several adsorbents have been applied for the chromatographic analysis of furanocoumarins, e.g. silica gel, C18 layers, alumina, poliamide, Florisil, etc. (Cieśla & Waksmundzka-Hajnos, 2009). Analyzed fractions of studied compounds are eluted using several solvent systems. Borkowski (1973) proposed the following eluents: 1) benzene-acetone (90:10, v/v); 2) toluene-acetone (95:5, v/v); 3) benzene-ethyl acetate (9:1, v/v); 4) benzene-ethylic ether-

extract were acetylated using pyridine and Ac2O to give (2'S, 1''S)-2.3-dihydro-2(2-acetoxy-1-hydroxymethylethyl)-7H-furo [3.2-g][1]benzopyran-7-one (Rojas-Lima et al., 1999).

Another useful adsorbent for column chromatography is Florisil (100-200 mesh), which was used to fractionate furanocoumarins obtained from fruits of *Peucedanum alsaticum* L. and *P. cervaria* (L.) Lap. Concentrated petroleum ether extracts were fractionated on this sorbent with a dichloromethane-ethyl acetate (0-50%) gradient, then ethyl acetate and methanol as mobile phases. After CC separation, the fractions richest in coumarins were analyzed by preparative-TLC on silica gel. Separated zones of selected furocoumarins were eluted from

The Florisil was also used in an investigation performed by Suzuki et al. (1979). Bergamot oil was eluted on this column with methylene chloride and ethyl acetate. The ethyl acetate fractions were re-chromatographed with methylene chloride. The obtained residue was analyzed by preparative-TLC on silica gel using cyclohexane-tetrahydrofuran (1:1) as eluent.

Isolation of the furanocoumarins from grapefruit juice was accomplished by preparative thin layer chromatography. The obtained fractions were applied to tapered silica gel GF TLC plates with a fluorescent indicator. Resolution of compounds was accomplished by using solvent systems consisting of hexane:ethyl acetate (3:1 to 2:3; v/v), chloroform, chloroform/methanol (95:5), and benzene: acetone (9:1). The zones containing

The physicochemical properties of coumarins depend upon their chemical structure, specifically, the presence and position of functional hydroxy or methoxy groups, and methyl or other alkyl chains. As a result of these differences, group separation of the all groups of coumarins does not cause any difficulties (Jerzmanowska, 1967; Waksmundzka-Hajnos et al., 2006). Separation of individual compounds in each group – structural analogs, i.e.

Several analytical methods for the quality control of furanocoumarins in plant materials, such as thin layer chromatography (TLC), high performance liquid chromatography (HPLC), high performance liquid chromatography–mass spectrometry (HPLC–MS), high-speed countercurrent chromatography (HSCCC), gas chromatography (GC), gas chromatography–mass spectrometry (GC–MS), capillary electrophoresis (CE) ), and pressurized capillary electrochromatography (pCEC), has been reported (Chen et al., 2009; Wang et et., 2007).

The oldest publications recommended one- or two- thin layer chromatography for separation and identification of furanocoumarins. This method provides a quite rapid separation of components in a sample mixture. Fractions obtained from column

Several adsorbents have been applied for the chromatographic analysis of furanocoumarins, e.g. silica gel, C18 layers, alumina, poliamide, Florisil, etc. (Cieśla & Waksmundzka-Hajnos, 2009). Analyzed fractions of studied compounds are eluted using several solvent systems. Borkowski (1973) proposed the following eluents: 1) benzene-acetone (90:10, v/v); 2) toluene-acetone (95:5, v/v); 3) benzene-ethyl acetate (9:1, v/v); 4) benzene-ethylic ether-

chromatography were usually checked with the use of TLC technique.

furanocoumarins were scraped and extracted with acetone (Manthey et al., 2006).

the plates (Skalicka-Woźniak et al., 2009).

**5.3.2 Thin Layer Chromatography (TLC)** 

The bergapten zone was scraped and eluted with acetone.

closely related compounds – is, however, a difficult task.

methanol-chloroform (20:1:1:1, v/v); 5) chloroform, and 6) ethyl acetate-hexane (25:75, v/v) for analysis of coumarins.

The spots of coumarins on thin-layer and paper chromatograms are usually revealed by UV fluorescence at certain characteristic wavelengths, before or after the treatment with an aqueous-ethanol solution of potassium hydroxide or with ammonia vapor, or using some other color reactions. The fluorescent color does not provide accurate identification of the structure of coumarins; nevertheless, sometimes it is possible to determine the type of functional groups (Celeghini et al., 2001; Lozhkin & Sakanyan, 2006).

Joint TLC – colorimetric methods based on the azo-addition reaction with TLC separation on an aluminum oxide layer eluted in the hexane – benzene – methanol (5:4:1) system were developed for the quantitative determination of peucedanin in *Peucedanum morrissonii*  (Bess.) and for the analysis of beroxan, pastinacin, and psoralen preparations (Lozhkin & Sakanyan, 2006). Colorimetric determination of xanthotoxin, imperatorin, and bergapten in *Ammi majus* (L.) fruits can be performed after TLC separation on silica gel impregnated with formamide and eluted in dibutyl ether. In order to determine psoralen alone and together with bergapten in *Ficus carica* (L.) leaves, the extract was purified from ballast substances and chromatographed in a thin layer of aluminum oxide in diethyl ether (Lozhkin & Sakanyan, 2006).

Thin layer chromatographic analyses were made by Celeghini and co-authors (2001) on silica gel 60G. As eluent a mixture of toluene:ethyl ether (1:1) saturated with 10% acetic acid was used. The plates were sprayed with an ethanolic solution (5% v/v) of KOH and examined under UV light at 366 nm.

In the other investigation, purification of the eluted furanocoumarins from leaves of *Conium maculatum* was also carried out on TLC plates (Si gel 60, f-254). The chromatograms were developed at room temperature using one of the following solvent systems: chloroform:ethyl acetate (2:1), or chloroform, or toluene:ethyl acetate (1:1) (Al-Barwani & Eltayeb, 2004).

Bogucka-Kocka (1999) analyzed furanocoumarins in fruits of *Heracleum sibiricum* L., after silica gel column chromatography, using 2D-TLC. Two-dimensional thin-layer chromatography was conducted of silica gel plates and run in the following phases: 1) benzene-chloroform-acetonitrile (1:1, v/v, 5%) or 2) benzene-chloroform-ethyl acetate (1:1, v/v, 5%) (first direction); 3) benzene-ethyl acetate (1:1) (second direction). The chromatograms were analyzed using UV light and daylight, after spaying with one of the derivativers: 1) 0.5% I dissolvent in KI; 2) Dragendorf's reagent or 25% SbCl5 in CCl4.

Unfortunately, in furocoumarin' group, these substances have comparable polarity and similar chemical structures. As a result, multi-dimensional separations are required in such cases.

Thin-layer chromatography gives the possibility of performing multi-dimensional separation – two-dimensional separation with the use of the same stationary phase, with different mobile phases (Gadzikowska et al., 2005; Härmäla et al., 1990; Waksmundzka-Hajnos et al., 2006), or by using a stationary phase gradient (Glensk et al., 2002; Waksmundzka-Hajnos et al., 2006). In TLC, there are almost no limits as far as mobile phases are concerned, because they can be easily evaporated from the layer after the

Analytical Methods for Isolation,

normal-phase systems.

pairs of orthogonal TLC systems:

dimension, RP-18 with 55% MeOH + H2O.

Separation and Identification of Selected Furanocoumarins in Plant Material 73

from fruits of *H. sibiricum*, where seven compounds were identified in the extract (Cieśla &

The use of graft thin-layer chromatography of coumarins was also reported (Cieśla et al., 2008; Cieśla et al., 2008a; Cieśla et al., 2008b). The authors applied two combinations of adsorbents: silica + RP-18W, and CN-silica + silica gel. In the first stage of this experiment, plates pre-coated with CN-silica were developed in one dimension by unidimensional multiple development. The same mobile phase (35% ethyl acetate in n-heptane) was used, over the same distance, and the same direction of the development. Plates were tripledeveloped with careful drying of the plate after each run. Unidimensional multiple development (UMD) results in increased resolution of neighboring spots (Poole et al., 1989). After chromatography the plates were linearly scanned at 366 nm with slit dimensions 5 mm × 0.2 mm. This chromatographic system was not suitable for separation of structural analogs. Isopimpinellin and byacangelicol are coeluted and phellopterin and bergapten also have very similar retention behavior. The isopimpinellin and byacangelicol molecules have two medium polarity groups in positions 5 and 8, which have similar physicochemical properties. Therefore, it was also easily noticeable that different non-polar substituents did not cause significant difference in retention behavior. Compounds with polar substituents – hydroxyl groups in simple coumarins are more strongly retained on CN-silica layer in

When other systems, for example silica with AcOEt–n-heptane and RP 18W with 55% MeOH in water, were used only partial separation of standards was achieved. This results from the similar structures and physicochemical properties of the compounds. On silica layers only polar aesculetin and umbelliferone are more strongly retained. Phellopterin with a long chain in the 8 position (with a shielding effect on neighboring oxygen) is weakly retained. These differences cause the aforementioned coumarins to be completely separated from other standards. Byacangelicol and umbelliferone, and bergapten, isopimpinellin, and xanthotoxin, with only slight differences in number and position of medium-polarity methoxy groups, are eluted together. More significant resolution of the investigated compounds was obtained on RP-18 plates, eluted with aqueous mobile phases. The differences in number, length, and position of medium-polarity and non-polar substituents cause differences in retention behavior of the analytes. These differences result in good

separation of bergapten, xanthotoxin, and phellopterin by reversed-phase systems.

dimension, SiO2 with 35% AcOEt + n-heptane (three developments);

In the next step Cieśla et al. (2008b) investigated the search for orthogonal systems, which would ensure better separation selectivity for the coumarins, was conducted. To achieve this, graft TLC, with two distinct layers, was applied. The authors experimentally chose two



An application of multiple development technique (UMD) in the first dimension results in partly separated spots, which are transferred to the second layer with methanol. Use of methanol causes narrowing of starting bands, similarly to the effect of a preconcentrating zone. The preconcentration is responsible for symmetric and well separated spots being

Waksmundzka-Hajnos, 2009; Waksmundzka-Hajnos et al., 2006).

development in the first dimension. Both methods, use of the same layer and different mobile phases or two different layers developed with two mobile phases, make use of different selectivity to achieve complete separation in the two-dimensional process. The largest differences are obtained with a normalphase system, with an adsorption mechanism of separation, and a reversed-phase system, with a partition mechanism of separation, are applied for two-dimensional separations (Nyiredy, 2001). Two-dimensional thin-layer chromatography with adsorbent gradient is an effective method for the separation of large group of substances present in natural mixtures, e.g. plant extracts.

Silica gel is the most popular adsorbent, thus it has been widely used in different chromatographic methods. However, in case of two-dimensional separations of coumarins, it has been rarely applied as it is difficult to select solvent systems which are complementary in selectivity. Härmälä et al. (1990) proposed a very interesting method for the separation of 16 coumarins from the genus *Angelica* with the use of silica gel as an adsorbent. The application of two-dimensional over-pressured layer chromatography enabled complete resolution of the analyzed substances. The authors described a very useful procedure of choosing complementary systems that can be applied in the analysis of complex mixtures. It turned out that the systems, I direction – 100% CHCl3 and II direction – AcOEt/*n-*hexane (30:70, v/v) provided excellent separation of all coumarins, although having only the fourth poorest correlation value.

Due to the possibility of the application of normal- and reversed – phase systems, polar bonded phases have been often a choice for two-dimensional separations. In the case of coumarins, the use of diol- and cyanopropyl-silica have been reported.

Waksmundzka-Hajnos et al. (2006) reported the use of diol-silica for the separation of 10 furanocoumarin standards. Firstly, the compounds were chromatographed with the use of 100% diisopropyl ether (double development), then in the perpendicular direction: 10% MeOH/H2O (v/v) containing 1% HCOOH. The use of the first direction eluent caused the separation of analyzed substances into three main groups, which is useful for group separation of natural mixtures of coumarins. Chromatography in reversed-phase system enabled the complete resolution of all tested standards. The disadvantage of the applied reversed-phase system is the fact that it has low efficiency, and most of the substances, especially those containing hydroxyl groups are tailing. Diol-silica is similar in its properties to deactivated silica, thus the application of aqueous eluent may be responsible for tailing, which was only slightly reduced after the addition of formic acid.

Better results were obtained after the application of CN-silica. In this case, coumarin standards were firstly chromatographed with the use of normal-phase, then in reversedphase system. The plate was triple developed in the first direction to improve separation of strongly retained polar coumarins.

The authors also investigated the use of multiphase plates for identification purposes. Coumarins were firstly chromatographed on a RP-18W strip with 55% MeOH/H2O (v/v), and then in a perpendicular direction they were triple-developed with: 35% AcOEt/*n*heptane (v/v). The use of reversed-phase system caused the separation of investigated coumarins into two groups: coumarins containing hydroxyl group, and furanocoumarins. The separation, according to the differences in polarity, is even greater than that observed on diol-silica. This system was then applied for separation of the furanocoumarin fraction

development in the first dimension. Both methods, use of the same layer and different mobile phases or two different layers developed with two mobile phases, make use of different selectivity to achieve complete separation in the two-dimensional process. The largest differences are obtained with a normalphase system, with an adsorption mechanism of separation, and a reversed-phase system, with a partition mechanism of separation, are applied for two-dimensional separations (Nyiredy, 2001). Two-dimensional thin-layer chromatography with adsorbent gradient is an effective method for the separation of large

Silica gel is the most popular adsorbent, thus it has been widely used in different chromatographic methods. However, in case of two-dimensional separations of coumarins, it has been rarely applied as it is difficult to select solvent systems which are complementary in selectivity. Härmälä et al. (1990) proposed a very interesting method for the separation of 16 coumarins from the genus *Angelica* with the use of silica gel as an adsorbent. The application of two-dimensional over-pressured layer chromatography enabled complete resolution of the analyzed substances. The authors described a very useful procedure of choosing complementary systems that can be applied in the analysis of complex mixtures. It turned out that the systems, I direction – 100% CHCl3 and II direction – AcOEt/*n-*hexane (30:70, v/v) provided excellent separation of all coumarins, although having only the fourth

Due to the possibility of the application of normal- and reversed – phase systems, polar bonded phases have been often a choice for two-dimensional separations. In the case of

Waksmundzka-Hajnos et al. (2006) reported the use of diol-silica for the separation of 10 furanocoumarin standards. Firstly, the compounds were chromatographed with the use of 100% diisopropyl ether (double development), then in the perpendicular direction: 10% MeOH/H2O (v/v) containing 1% HCOOH. The use of the first direction eluent caused the separation of analyzed substances into three main groups, which is useful for group separation of natural mixtures of coumarins. Chromatography in reversed-phase system enabled the complete resolution of all tested standards. The disadvantage of the applied reversed-phase system is the fact that it has low efficiency, and most of the substances, especially those containing hydroxyl groups are tailing. Diol-silica is similar in its properties to deactivated silica, thus the application of aqueous eluent may be responsible for tailing,

Better results were obtained after the application of CN-silica. In this case, coumarin standards were firstly chromatographed with the use of normal-phase, then in reversedphase system. The plate was triple developed in the first direction to improve separation of

The authors also investigated the use of multiphase plates for identification purposes. Coumarins were firstly chromatographed on a RP-18W strip with 55% MeOH/H2O (v/v), and then in a perpendicular direction they were triple-developed with: 35% AcOEt/*n*heptane (v/v). The use of reversed-phase system caused the separation of investigated coumarins into two groups: coumarins containing hydroxyl group, and furanocoumarins. The separation, according to the differences in polarity, is even greater than that observed on diol-silica. This system was then applied for separation of the furanocoumarin fraction

group of substances present in natural mixtures, e.g. plant extracts.

coumarins, the use of diol- and cyanopropyl-silica have been reported.

which was only slightly reduced after the addition of formic acid.

poorest correlation value.

strongly retained polar coumarins.

from fruits of *H. sibiricum*, where seven compounds were identified in the extract (Cieśla & Waksmundzka-Hajnos, 2009; Waksmundzka-Hajnos et al., 2006).

The use of graft thin-layer chromatography of coumarins was also reported (Cieśla et al., 2008; Cieśla et al., 2008a; Cieśla et al., 2008b). The authors applied two combinations of adsorbents: silica + RP-18W, and CN-silica + silica gel. In the first stage of this experiment, plates pre-coated with CN-silica were developed in one dimension by unidimensional multiple development. The same mobile phase (35% ethyl acetate in n-heptane) was used, over the same distance, and the same direction of the development. Plates were tripledeveloped with careful drying of the plate after each run. Unidimensional multiple development (UMD) results in increased resolution of neighboring spots (Poole et al., 1989). After chromatography the plates were linearly scanned at 366 nm with slit dimensions 5 mm × 0.2 mm. This chromatographic system was not suitable for separation of structural analogs. Isopimpinellin and byacangelicol are coeluted and phellopterin and bergapten also have very similar retention behavior. The isopimpinellin and byacangelicol molecules have two medium polarity groups in positions 5 and 8, which have similar physicochemical properties. Therefore, it was also easily noticeable that different non-polar substituents did not cause significant difference in retention behavior. Compounds with polar substituents – hydroxyl groups in simple coumarins are more strongly retained on CN-silica layer in normal-phase systems.

When other systems, for example silica with AcOEt–n-heptane and RP 18W with 55% MeOH in water, were used only partial separation of standards was achieved. This results from the similar structures and physicochemical properties of the compounds. On silica layers only polar aesculetin and umbelliferone are more strongly retained. Phellopterin with a long chain in the 8 position (with a shielding effect on neighboring oxygen) is weakly retained. These differences cause the aforementioned coumarins to be completely separated from other standards. Byacangelicol and umbelliferone, and bergapten, isopimpinellin, and xanthotoxin, with only slight differences in number and position of medium-polarity methoxy groups, are eluted together. More significant resolution of the investigated compounds was obtained on RP-18 plates, eluted with aqueous mobile phases. The differences in number, length, and position of medium-polarity and non-polar substituents cause differences in retention behavior of the analytes. These differences result in good separation of bergapten, xanthotoxin, and phellopterin by reversed-phase systems.

In the next step Cieśla et al. (2008b) investigated the search for orthogonal systems, which would ensure better separation selectivity for the coumarins, was conducted. To achieve this, graft TLC, with two distinct layers, was applied. The authors experimentally chose two pairs of orthogonal TLC systems:


An application of multiple development technique (UMD) in the first dimension results in partly separated spots, which are transferred to the second layer with methanol. Use of methanol causes narrowing of starting bands, similarly to the effect of a preconcentrating zone. The preconcentration is responsible for symmetric and well separated spots being

Analytical Methods for Isolation,

this investigation (Giannetti et al., 2010).

in three oral solutions by Pires et al. (2004).

to the initial eluting system (Martino et al., 2006).

(Bartnik & Głowniak, 2007).

quantitatively.

et al., 2004).

2007).

Separation and Identification of Selected Furanocoumarins in Plant Material 75

In most recent applications, reversed-phase HPLC is used to evaluate furanocoumarins

For example, the quantitative analysis of some furanocoumarins from *Pastinaca sativa* fruits was performed by RP-HPLC in system C18/methanol + water in gradient elution. The authors used the following gradient: 0-10 min, 45% MeOH; 10-20 min, 45-55% MeOH; 20-30 min, 55-70% MeOH, and 30-40 min, 70% MeOH in bidistilled water (Waksmundzka-Hajnos

The determination of two furocoumarins (bergapten and bergamottin) in bergamot fruits, was carried out by the HPLC system equipped with a diode array detector. C18 column and the mobile phase consisted of methanol and 5% (v/v) acetic acid aqueous solution in the following gradient: 5-20% (0-13 min), 20-100% (13-25 min), 100-5% (20-30 min), were used in

The optimized HPLC-UV method was used to evaluate the quality of 21 samples of Radix *Angelica dahurica* from different parts of China. Bergapten, imperatorin and cnidilin were separated on C18 column; the mobile phase was 66:34 (v/v) methanol-water (Wang et al.,

The HPLC technique was ensued for analyses of psoralen and bergapten. HPLC separation of the psoralens was performed using a Shimadzu octadecyl Shim-pack CLC-ODS reversedphase column with a small pre-column containing the same packing. Elution was carried with acetonitrile-water 55:45 (v/v) and detections of the peaks were recording at 223 nm (Cardoso et al., 2002). The same conditions were used for determination of furanocoumarins

A rapid and sensitive reversed-phase HPLC method has been used for the determination of furanocoumarins in methanolic extracts of *Peucedanum tautaricum* Bieb. Compounds were separated on stainless-steel column packed with 5µm particle Hypersil ODS C18. The mobile phase was methanol-water gradient used following: 0-5 mins, isocratic elution with 60% (v/v) methanol; 5–20 mins, linear gradient from 60 to 80% methanol; 20 to 30 mins, linear gradient from 80 to 60% methanol; 30–40 mins isocratic elution with 60% methanol. An acetonitrile–water mobile phase gradient was also used (0–8 mins, isocratic elution with 50% acetonitrile; 8–25 mins, linear gradient from 50 to 70% acetonitrile; 25–28 mins, linear gradient from 70 to 50% acetonitrile; 28–40 mins isocratic elution with 50% acetonitrile)

The mobile phase consisted of water with orthophosphoric acid 1:10000 (solvent A), methanol (solvent B) and acetonitrile (solvent C) was used for analysis of coumarins from *Melilotus officinalis* (L.) Pallas. The starting mixture (80% A, 5% B and 15% C) was modified as follows: within 20 mins the mobile phase composition became 65% A, 20% B, 15% C and was kept constant for 10 mins; in the following 10 mins the mixture composition came back

The search for better conditions for application of HPLC has led to development of UPLC (Ultra Performance Liquid Chromatography), a relatively new liquid chromatography technique enabling faster analysis, consumption of less solvent and better sensitivity. The UPLC method enables a reduction of analysis time by up to a factor of nine compared with conventional HPLC without loss of quality of the analytical data generated. Another very

obtained after development of the plate in the second dimension. This makes the densitometric estimation easier.

In the last step of Cieśla and co-authors (2008b) investigations, the separation of furanocoumarin fractions from *Archangelica officinalis, Heracleum sphondylium,* and *Pastinaca sativa* fruits was performed by the use of grafted plates SiO2 with RP-18W and CN with SiO2, with appropriate mobile phases. The identity of the extract components was confirmed by comparing retardation factors and UV spectra with the Rf values and spectra obtained for the standards.

Graft TLC in orthogonal systems characterized by different separation selectivity enables complete separation of structural analogs such as furanocoumarins. The use of two different TLC systems enables complete separation and identification of some furanocoumarins present in extracts obtained from *Archangelica officinalis, Heracleum sphondylium*, and *Pastinaca sativa* fruits (Cieśla et al., 2008b).

The graft-TLC system silica + RP-18W were successfully applied for construction of chromatographic fingerprints of different plants from the *Heracleum* genus.

Two-dimensional chromatography has also been applied for quantitative analysis of furanocoumarins in plant extracts (Cieśla et al., 2008b). In order to obtain reproducible results, all investigated compounds should be completely separated. Graft-TLC with the use of adsorbents silica + RP-18W was proven to be the most suitable for quantitative analysis. Resolution of compounds was insufficient in case of 2D-TLC on one adsorbent (CN-silica), as the standards had to be divided into two separate groups for an accurate estimation of peak surface area.

Quantitative analysis is difficult to perform after two-dimensional chromatographic run, as densitometers are not adjusted to scan two-dimensional chromatograms. This problem may be overcome if small steps between scans are used. In the proposed method, the authors scanned the plate with the slit of a dimension 5 mm×0.2 mm, operated at λ= 366 nm, obtaining 36 tracks that were not overlapping. This wavelength was chosen to get rid of intensive baseline noise, observed at lower wavelengths. Peak areas were measured with the use of the method called "peak approximation" (Cieśla et al., 2008b; Cieśla & Waksmundzka-Hajnos, 2009).

#### **5.3.3 High Performance Liquid Chromatography (HPLC)**

Furanocoumarins are also examined by means of high performance liquid chromatography (HPLC). This technique has shown to be a very efficient system for separation of this group of compounds. HPLC methods have been reported for the determination of psoralens in callus cultures, vitro culture, serum, dermis, plants, citrus essential oils, phytomedicines, but only the most recently published methods has reported assay validation (Cardoso et al., 2000; Dugo et al., 2000; Markowski & Czapińska, 1997; Pires et al., 2004).

Linear furanocoumarins, such as psoralen, bergapten, xanthotoxin, and isopimpinellin isolated from three varieties of *Apium graveolens* were examined by normal-phase HPLC equipped with a variable wavelength detector set at 250 nm. The mobile phase consisted of a mixture of ethyl acetate (0.1%) and formic acid (0.1%) in chloroform (Waksmundzka-Hajnos & Sherma, 2011).

obtained after development of the plate in the second dimension. This makes the

In the last step of Cieśla and co-authors (2008b) investigations, the separation of furanocoumarin fractions from *Archangelica officinalis, Heracleum sphondylium,* and *Pastinaca sativa* fruits was performed by the use of grafted plates SiO2 with RP-18W and CN with SiO2, with appropriate mobile phases. The identity of the extract components was confirmed by comparing retardation factors and UV spectra with the Rf values and spectra obtained for

Graft TLC in orthogonal systems characterized by different separation selectivity enables complete separation of structural analogs such as furanocoumarins. The use of two different TLC systems enables complete separation and identification of some furanocoumarins present in extracts obtained from *Archangelica officinalis, Heracleum sphondylium*, and

The graft-TLC system silica + RP-18W were successfully applied for construction of

Two-dimensional chromatography has also been applied for quantitative analysis of furanocoumarins in plant extracts (Cieśla et al., 2008b). In order to obtain reproducible results, all investigated compounds should be completely separated. Graft-TLC with the use of adsorbents silica + RP-18W was proven to be the most suitable for quantitative analysis. Resolution of compounds was insufficient in case of 2D-TLC on one adsorbent (CN-silica), as the standards had to be divided into two separate groups for an accurate estimation of

Quantitative analysis is difficult to perform after two-dimensional chromatographic run, as densitometers are not adjusted to scan two-dimensional chromatograms. This problem may be overcome if small steps between scans are used. In the proposed method, the authors scanned the plate with the slit of a dimension 5 mm×0.2 mm, operated at λ= 366 nm, obtaining 36 tracks that were not overlapping. This wavelength was chosen to get rid of intensive baseline noise, observed at lower wavelengths. Peak areas were measured with the use of the method called "peak approximation" (Cieśla et al., 2008b; Cieśla &

Furanocoumarins are also examined by means of high performance liquid chromatography (HPLC). This technique has shown to be a very efficient system for separation of this group of compounds. HPLC methods have been reported for the determination of psoralens in callus cultures, vitro culture, serum, dermis, plants, citrus essential oils, phytomedicines, but only the most recently published methods has reported assay validation (Cardoso et al.,

Linear furanocoumarins, such as psoralen, bergapten, xanthotoxin, and isopimpinellin isolated from three varieties of *Apium graveolens* were examined by normal-phase HPLC equipped with a variable wavelength detector set at 250 nm. The mobile phase consisted of a mixture of ethyl acetate (0.1%) and formic acid (0.1%) in chloroform (Waksmundzka-

chromatographic fingerprints of different plants from the *Heracleum* genus.

densitometric estimation easier.

*Pastinaca sativa* fruits (Cieśla et al., 2008b).

the standards.

peak surface area.

Waksmundzka-Hajnos, 2009).

Hajnos & Sherma, 2011).

**5.3.3 High Performance Liquid Chromatography (HPLC)** 

2000; Dugo et al., 2000; Markowski & Czapińska, 1997; Pires et al., 2004).

In most recent applications, reversed-phase HPLC is used to evaluate furanocoumarins quantitatively.

For example, the quantitative analysis of some furanocoumarins from *Pastinaca sativa* fruits was performed by RP-HPLC in system C18/methanol + water in gradient elution. The authors used the following gradient: 0-10 min, 45% MeOH; 10-20 min, 45-55% MeOH; 20-30 min, 55-70% MeOH, and 30-40 min, 70% MeOH in bidistilled water (Waksmundzka-Hajnos et al., 2004).

The determination of two furocoumarins (bergapten and bergamottin) in bergamot fruits, was carried out by the HPLC system equipped with a diode array detector. C18 column and the mobile phase consisted of methanol and 5% (v/v) acetic acid aqueous solution in the following gradient: 5-20% (0-13 min), 20-100% (13-25 min), 100-5% (20-30 min), were used in this investigation (Giannetti et al., 2010).

The optimized HPLC-UV method was used to evaluate the quality of 21 samples of Radix *Angelica dahurica* from different parts of China. Bergapten, imperatorin and cnidilin were separated on C18 column; the mobile phase was 66:34 (v/v) methanol-water (Wang et al., 2007).

The HPLC technique was ensued for analyses of psoralen and bergapten. HPLC separation of the psoralens was performed using a Shimadzu octadecyl Shim-pack CLC-ODS reversedphase column with a small pre-column containing the same packing. Elution was carried with acetonitrile-water 55:45 (v/v) and detections of the peaks were recording at 223 nm (Cardoso et al., 2002). The same conditions were used for determination of furanocoumarins in three oral solutions by Pires et al. (2004).

A rapid and sensitive reversed-phase HPLC method has been used for the determination of furanocoumarins in methanolic extracts of *Peucedanum tautaricum* Bieb. Compounds were separated on stainless-steel column packed with 5µm particle Hypersil ODS C18. The mobile phase was methanol-water gradient used following: 0-5 mins, isocratic elution with 60% (v/v) methanol; 5–20 mins, linear gradient from 60 to 80% methanol; 20 to 30 mins, linear gradient from 80 to 60% methanol; 30–40 mins isocratic elution with 60% methanol. An acetonitrile–water mobile phase gradient was also used (0–8 mins, isocratic elution with 50% acetonitrile; 8–25 mins, linear gradient from 50 to 70% acetonitrile; 25–28 mins, linear gradient from 70 to 50% acetonitrile; 28–40 mins isocratic elution with 50% acetonitrile) (Bartnik & Głowniak, 2007).

The mobile phase consisted of water with orthophosphoric acid 1:10000 (solvent A), methanol (solvent B) and acetonitrile (solvent C) was used for analysis of coumarins from *Melilotus officinalis* (L.) Pallas. The starting mixture (80% A, 5% B and 15% C) was modified as follows: within 20 mins the mobile phase composition became 65% A, 20% B, 15% C and was kept constant for 10 mins; in the following 10 mins the mixture composition came back to the initial eluting system (Martino et al., 2006).

The search for better conditions for application of HPLC has led to development of UPLC (Ultra Performance Liquid Chromatography), a relatively new liquid chromatography technique enabling faster analysis, consumption of less solvent and better sensitivity. The UPLC method enables a reduction of analysis time by up to a factor of nine compared with conventional HPLC without loss of quality of the analytical data generated. Another very

Analytical Methods for Isolation,

polarity used are very important.

(Yang et al., 2010).

Separation and Identification of Selected Furanocoumarins in Plant Material 77

Coumarins can be detected in both positive and negative ion modes. Whereas, the positive ion mode often generates higher yields, the noise level is lower in the negative ion mode, thus improving the quality of the signals. So, preliminary investigations regarding the

The main problem of working with LC-MS of natural products is the choice of the ionisation technique. Particle beam (PB) and thermospray (TSP) interfaces are the most commonly used for natural component analysis. Both of them exhibit many drawbacks, such as the difficulty to optimize ionisation conditions and the lack of sensitivity. Electrospray (ESI) and atmospheric pressure chemical ionisation (APCI) techniques, which operate under atmospheric pressure, seem to be very promising. These ionisations differ in the way they generate ions, but show many similarities: both operate at atmospheric pressure, giving molecular weight information and additional structural information. Many classes of compounds can be analyzed by both APCI and ESI. However, ESI is the technique of choice for polar and higher molecular weight compounds, while APCI is suitable for less polar

A sensitive, specific and rapid LC–MS method has been developed and validated for the simultaneous determination of xanthotoxin (8-methoxypsoralen), psoralen, isoimpinellin (5,8-dimethoxypsoralen) and bergapten (5-methoxypsoralen) in plasma samples from rats after oral administration of *Radix Glehniae* extract using pimpinellin as an internal standard. A chromatographic separation was performed on a C18 column with a mobile phase composed of 1mmol ammonium acetate and methanol (30:70, v/v). The detection was accomplished by multiple-reaction monitoring (MRM), scanning via electrospray ionization (ESI) source operating in the positive ionization mode. The optimized mass transition ionpairs (m/z) for quantitation were 217.1/202.1 for xanthotoxin, 187.1/131.1 for psoralen, 247.1/217.0 for isoimpinellin, 217.1/202.1 for bergapten, and 247.1/231.1 for pimpinellin

A paper by Zheng et al. (2010) reports the quantitation of eleven coumarins including furocoumarins in *Radix Angelicae dahuricae*. By using this HPLC–ESI-MS/MS method, all coumarins were separated and determined within 10 min. These compounds were detected by ESI ionization method and quantified by multiple-reaction monitoring (MRM). The mass spectral conditions were optimized in both positive- and negative-ion modes, and the positive-ion mode was found to be more sensitive. The all coumarins exhibited their quasimolecular ions [M+H]+, [M+Na]+, [M+NH4 ]+, [M+K]+ and fragment ions [M+H−CO]+,

[M+H−C5H9O]+, [M+H−C5H8]+, [M+H−C5H8−CO]+, [M+H−C5H8−CO2]+, [M+H−CH3]+.

Yang et al. (2010a) proposed a practical method for the characterization of coumarins, i.e. linear furanocoumarins, in *Radix Glehniae* by LC–MS. They described in details over 40 derivatives of psoralens. First, 10 coumarin standards were studied, and mass spectrometry fragmentation patterns and elution time rules for the coumarins were found. Then, an extract of *Radix Glehniae* was analyzed by the combination of two scan modes, i.e., multiple ion monitoring-information-dependent acquisition-enhanced product ionmode (MIM-IDA-EPI) and precursor scan information-dependent acquisition-enhanced product ionmode (PREC-IDA-EPI) on a hybrid triple quadrupole-linear ion trap mass spectrometer. This study has demonstrated the unprecedented advantage of the combination of these two scan modes. The MIM-IDA-EPI mode is sensitive, and no pre-acquisition of MS/MS spectra of

compounds and of lower molecular weight than ESI (Dugo et al., 2000).

important advantage is high column efficiency which increases the possibility of compound identification and results in better quantitative analysis. UPLC is more efficient and therefore has greater resolving power than traditional HPLC (Novakova et al., 2006; Skalicka-Woźniak et al., 2009; Wren & Tchelitcheff, 2006).

The quantitative analysis by UPLC was performed for the furocoumarins in *Peucedanum alsaticum* and *P. cervia* (Skalicka-Woźniak et al., 2009). The optimalization of the RP-UPLC separation of the coumarins was achieved by the use of DryLab. The investigation was performed with an Acquity Ultra Performance LC (Waters, Milford, MA, USA) coupled with a DAD detector. Compounds were separated on a stainless-steel column packed with 1.7 µm BEH C18. Two linear mobile phase gradients from 5 to 100% of acetonitrile with gradient times of 10 and 20 min were used. Detection was at 320 nm.

A paper by Desmortreux et al. (2009) reports separation of furocoumarins of essential oils (lemon residue) by supercritical fluid chromatography (SFE). The authors studied many types of stationary phases and the effects of numerous analytical parameters. Amongst the numerous tested columns, good separation of analyzed furanocoumarins was obtained on a pentafluorophenyl (PFP) phase (Discovery HS F5), based on an aromatic ring substituted by five fluorine atoms. The mobile phase used was CO2-EtOH 90:10 (v/v). Amongst the standard compounds, bergapten was well separated being eluted after the other furocoumarins in the lemon residue sample. The results obtained in this study show that SFC is a perfectly suited method to investigate the psoralens in essential oil composition, because of the great number of compounds separated in a reduced analysis time, and with a very short time for re-equilibration of the system at the end of the gradient analysis. Because of the absence of water in the mobile phase in SFC, the stationary phase can establish more varied interactions than in HPLC, making the stationary phase choice highly significant.

#### **5.3.4 Hyphenated HPLC techniques**

A hyphenated, HPLC-TLC procedure for the separation of couamrins, has been proposed by Hawrył et al. (2000). A mixture of 12 coumarins from *Archangelica officinalis* was completely separated as a result of the different selectivities of the two combined chromatographic techniques, RP-HPLC and NP-TLC. Firstly, the analyzed compounds were separated by means of RP-HPLC. The optimal eluent: 60% MeOH in water was chosen with the use of DryLab program. All HPLC fractions were collected, evaporated and finally developed in normal-phase system, on silica gel, with the use of a solvent mixture: 40% AcOEt (v/v) in dichloromethane/heptane (1:1). All fractions were completely separated. The combination of these methods gave successful results, although both methods, if used separately, failed to give good resolution. This procedure may be useful for micropreparative separation of coumarins (Cieśla & Waksmundzka-Hajnos, 2009).

The liquid chromatography coupled with mass spectrometry (LC-MS) technique is becoming increasingly popular, in particular, the introduction of atmospheric pressure chemical ionization (APCI) has dramatically influenced the possibilities for analyzing poorly ionizable compounds. The use of hyphenated techniques such as LC-MS provides great information about the content and nature of constituents of complex natural matrices prior to fractioning and carrying out biological assays. Moreover, MS presents a great advantage not only in its ability to measure accurate ion masses but also in its use in structure elucidation (Chaudhary et al., 1985; Dugo et al., Waksmundzka-Hajnos & Sherma, 2011).

important advantage is high column efficiency which increases the possibility of compound identification and results in better quantitative analysis. UPLC is more efficient and therefore has greater resolving power than traditional HPLC (Novakova et al., 2006;

The quantitative analysis by UPLC was performed for the furocoumarins in *Peucedanum alsaticum* and *P. cervia* (Skalicka-Woźniak et al., 2009). The optimalization of the RP-UPLC separation of the coumarins was achieved by the use of DryLab. The investigation was performed with an Acquity Ultra Performance LC (Waters, Milford, MA, USA) coupled with a DAD detector. Compounds were separated on a stainless-steel column packed with 1.7 µm BEH C18. Two linear mobile phase gradients from 5 to 100% of acetonitrile with

A paper by Desmortreux et al. (2009) reports separation of furocoumarins of essential oils (lemon residue) by supercritical fluid chromatography (SFE). The authors studied many types of stationary phases and the effects of numerous analytical parameters. Amongst the numerous tested columns, good separation of analyzed furanocoumarins was obtained on a pentafluorophenyl (PFP) phase (Discovery HS F5), based on an aromatic ring substituted by five fluorine atoms. The mobile phase used was CO2-EtOH 90:10 (v/v). Amongst the standard compounds, bergapten was well separated being eluted after the other furocoumarins in the lemon residue sample. The results obtained in this study show that SFC is a perfectly suited method to investigate the psoralens in essential oil composition, because of the great number of compounds separated in a reduced analysis time, and with a very short time for re-equilibration of the system at the end of the gradient analysis. Because of the absence of water in the mobile phase in SFC, the stationary phase can establish more varied interactions than in HPLC, making the stationary phase choice highly significant.

A hyphenated, HPLC-TLC procedure for the separation of couamrins, has been proposed by Hawrył et al. (2000). A mixture of 12 coumarins from *Archangelica officinalis* was completely separated as a result of the different selectivities of the two combined chromatographic techniques, RP-HPLC and NP-TLC. Firstly, the analyzed compounds were separated by means of RP-HPLC. The optimal eluent: 60% MeOH in water was chosen with the use of DryLab program. All HPLC fractions were collected, evaporated and finally developed in normal-phase system, on silica gel, with the use of a solvent mixture: 40% AcOEt (v/v) in dichloromethane/heptane (1:1). All fractions were completely separated. The combination of these methods gave successful results, although both methods, if used separately, failed to give good resolution. This procedure may be useful for micropreparative separation of

The liquid chromatography coupled with mass spectrometry (LC-MS) technique is becoming increasingly popular, in particular, the introduction of atmospheric pressure chemical ionization (APCI) has dramatically influenced the possibilities for analyzing poorly ionizable compounds. The use of hyphenated techniques such as LC-MS provides great information about the content and nature of constituents of complex natural matrices prior to fractioning and carrying out biological assays. Moreover, MS presents a great advantage not only in its ability to measure accurate ion masses but also in its use in structure elucidation (Chaudhary et al., 1985; Dugo et al., Waksmundzka-Hajnos & Sherma, 2011).

Skalicka-Woźniak et al., 2009; Wren & Tchelitcheff, 2006).

**5.3.4 Hyphenated HPLC techniques** 

coumarins (Cieśla & Waksmundzka-Hajnos, 2009).

gradient times of 10 and 20 min were used. Detection was at 320 nm.

Coumarins can be detected in both positive and negative ion modes. Whereas, the positive ion mode often generates higher yields, the noise level is lower in the negative ion mode, thus improving the quality of the signals. So, preliminary investigations regarding the polarity used are very important.

The main problem of working with LC-MS of natural products is the choice of the ionisation technique. Particle beam (PB) and thermospray (TSP) interfaces are the most commonly used for natural component analysis. Both of them exhibit many drawbacks, such as the difficulty to optimize ionisation conditions and the lack of sensitivity. Electrospray (ESI) and atmospheric pressure chemical ionisation (APCI) techniques, which operate under atmospheric pressure, seem to be very promising. These ionisations differ in the way they generate ions, but show many similarities: both operate at atmospheric pressure, giving molecular weight information and additional structural information. Many classes of compounds can be analyzed by both APCI and ESI. However, ESI is the technique of choice for polar and higher molecular weight compounds, while APCI is suitable for less polar compounds and of lower molecular weight than ESI (Dugo et al., 2000).

A sensitive, specific and rapid LC–MS method has been developed and validated for the simultaneous determination of xanthotoxin (8-methoxypsoralen), psoralen, isoimpinellin (5,8-dimethoxypsoralen) and bergapten (5-methoxypsoralen) in plasma samples from rats after oral administration of *Radix Glehniae* extract using pimpinellin as an internal standard. A chromatographic separation was performed on a C18 column with a mobile phase composed of 1mmol ammonium acetate and methanol (30:70, v/v). The detection was accomplished by multiple-reaction monitoring (MRM), scanning via electrospray ionization (ESI) source operating in the positive ionization mode. The optimized mass transition ionpairs (m/z) for quantitation were 217.1/202.1 for xanthotoxin, 187.1/131.1 for psoralen, 247.1/217.0 for isoimpinellin, 217.1/202.1 for bergapten, and 247.1/231.1 for pimpinellin (Yang et al., 2010).

A paper by Zheng et al. (2010) reports the quantitation of eleven coumarins including furocoumarins in *Radix Angelicae dahuricae*. By using this HPLC–ESI-MS/MS method, all coumarins were separated and determined within 10 min. These compounds were detected by ESI ionization method and quantified by multiple-reaction monitoring (MRM). The mass spectral conditions were optimized in both positive- and negative-ion modes, and the positive-ion mode was found to be more sensitive. The all coumarins exhibited their quasimolecular ions [M+H]+, [M+Na]+, [M+NH4 ]+, [M+K]+ and fragment ions [M+H−CO]+, [M+H−C5H9O]+, [M+H−C5H8]+, [M+H−C5H8−CO]+, [M+H−C5H8−CO2]+, [M+H−CH3]+.

Yang et al. (2010a) proposed a practical method for the characterization of coumarins, i.e. linear furanocoumarins, in *Radix Glehniae* by LC–MS. They described in details over 40 derivatives of psoralens. First, 10 coumarin standards were studied, and mass spectrometry fragmentation patterns and elution time rules for the coumarins were found. Then, an extract of *Radix Glehniae* was analyzed by the combination of two scan modes, i.e., multiple ion monitoring-information-dependent acquisition-enhanced product ionmode (MIM-IDA-EPI) and precursor scan information-dependent acquisition-enhanced product ionmode (PREC-IDA-EPI) on a hybrid triple quadrupole-linear ion trap mass spectrometer. This study has demonstrated the unprecedented advantage of the combination of these two scan modes. The MIM-IDA-EPI mode is sensitive, and no pre-acquisition of MS/MS spectra of

Analytical Methods for Isolation,

methanol-water (5:7:3, v/v) changed from 0 to 100%.

**5.3.5 Capillary electrophoresis** 

2004b).

2004a).

Separation and Identification of Selected Furanocoumarins in Plant Material 79

hydrodynamic equilibrium was reached, the sample solution containing the crude extract was injected into the separation coil tube through the injection valve. Each peak fraction was collected according to the chromatogram and evaporated under reduced pressure. The results of HSCCC tests indicated that n-hexane-ethyl acetate-methanol-water (5:5:4.5:5.5, v/v) was the best solvent system for the separation of psoralen and isopsoralen (Liu et al.,

In another investigations, the same authors had used the same HSCCC technique to induce preparative isolation and purification of furanocoumarins from *Angelica dahurica* (Fisch. ex Hoffm) Benth, et Hook. f (Liu et al., 2004) and from *Cnidium monnieri* (L.) Cusson (Liu et al., 2004a). The results of the first study (Liu et al., 2004) indicated that the best separation of imperatorin, oxypeucedanin and isoimperatorin was when the lower phase of n-hexanemethanol-water (5:5:5, v/v) and n-hexane-methanol-water (5:7:3, v/v) were used in gradient elution. The following gradient was used: 0-150 min, only the lower phase of n-hexanemethanol-water (5:5:5, v/v); 150-300 min, the volume ratio of the lower phase of n-hexane-

A HSCCC method for separation and purification of psoralens from *C. monnieri* was developed by using with a pair of two-phase solvent system composed of light petroleumethyl acetate-methanol-water at volume ratios of 5:5:5:5, 5:5:6:4 and 5:5:6.5:3.5 (Liu et al.,

In described cases, the crude extracts obtained by HSCCC technique were analyzed by HPLC method. The column used was a reversed-phase symmetry C18 column. The mobile phase was methanol-water (68:32, v/v) (analysis of extract from *A. dahurica*), methanolwater (40:60, v/v) (analysis of extract from *P. corylifolia*), or methanol-acetonitrile-water system in gradient mode as follows: 30:30:40 to 50:30:20 in 30 min (analysis of extract from *C.monnieri*) (Liu et al., 2004, 2004a, 2004b). Bergapten and imperatorin obtained by HSCCC method from *Cnidium monnieri* were also analyzed by high-performance liquid chromatography. The column used was a reversed-phase symmetry C18 column, and the mobile phase adopted was methanol (solvent A) – water (solvent B) in the gradient mode as

follows: 0-5 min, 60% A; 5-14 min, 60-80% A; 14-15 min, 80-60% A (Li & Chen, 2004).

In some cases, capillary electrophoresis was chosen to determine quantities of furanocoumarins. For example Ochocka et al. (1995) used this method for separating psoralens from roots and aerial parts of *Chrysanthemum segetum* L. The analyses were performed with electrophoresis apparatus with UV detection at 280 nm. The best overall separation was obtained on uncoated silica capillary with 7-s pneumatic injection using a buffer solution of 0.2 M boric acid-0.05 M of borax in water (11:9, v/v) (pH 8.5). In another example, micellar electrokinetic capillary chromatography (MEKC) was used in the separation of coumarins contained in *Angelicae Tuhou Radix* (Chen & Sheu, 1995). In this investigation, the electrolyte was buffer solution [20 mM sodium dodecyl sulfate (SDS) – 15 mM sodium borate – 15 mM sodium dihydrogenphosphate (pH 8.26)] – acetonitrile (24:1). The pressurized capillary electrochromatography (pCEC) was utilized for the separation and determination of coumarins in *Fructus cnidii* extracts from 12 different regions (Chen et al., 2009). Capillary electrochromatography (CEC), as a novel microcolumn separation

the parent ion is required due to the same precursor ion and product ion. A PREC-IDA-EPI mode was used to provide information on the parent ions, fragment ions and retention times of specified ions so the molecular weights of unknown coumarins and their glycosides could be identified. The information on the fragment ions from the MIM-IDA-EPI mode could be supplemented, and the retention time could be verified. Therefore, the characterization of trace furanocoumarins has become very easy and accurate by the combined use of the two modes and may play an important role in controlling the quality of medicinal herbs.

A high performance liquid chromatography–diode array detection–electrospray ionization tandem mass spectrometry (HPLC/DAD/ESI-MSn) method was used for the chromatographic fingerprint analysis and characterization of furocoumarins in the roots of *Angelica dahurica* (Kang et al., 2008)*.* The HPLC fingerprint technique has been considered as a useful method in identification and quality evaluation of herbs and their related finished products in recent years, because the HPLC fingerprint could systematically and comprehensively exhibit the types and quantification of the components in the herbal medicines (Drasar & Moravcova, 2004; Kang et al., 2008; Wang et al., 2007). Kang and coauthors (2008) showed that the samples from different batches had similar HPLC fingerprints, and the method could be applied for the quality control of the roots of *Angelica dahurica*. In addition, they identified a total of 20 furocoumarins by HPLC/DAD/ESI-MS*<sup>n</sup>* technique, and their fragmentation patterns in an electrospray ion trap mass spectrometer were also summarized.

Recently, high-speed counter-current chromatography (HSCCC) equipped with a HPLC system for separation and purification of furanocoumarins from crude extracts of plant materials, was also described.

High-speed counter-current chromatography (HSCCC), which was first invented by Y. Ito (1981), is a kind of liquid–liquid partition chromatography. The stationary phase of this method is also a liquid. It is retained in the separation column by centrifugal force. Because no solid support is used in the separation column, HSCCC successfully eliminates irreversible adsorption loss of samples onto the solid support used in conventional chromatographic columns (Ito, 1986). As an advanced separation technique, it offers various advantages including high sample recovery, high-purity of fractions, and high-loading capacity (Ma et al., 1994). In the past 30 years, HSCCC has made great progress in the preparation of various reference standards for pharmacological studies and good manufacturing practice, such as coumarins, alkaloids, flavonoids, hydroxyanthraquiones (Liu et al., 2004b).

Liu and co-authors (2004b) isolated and purified psoralen and isopsoralen from *Psoralea corylifolia* using HSCCC technique. In their investigation, they utilized TBE-300A HSCCC instrument with three multilayer coil separation column connected in series. The two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water was used for HSCCC separation. Each solvent was added to a separatory funnel and roughly equilibrated at room temperature. The upper phase (stationary phase) and the lower phase (mobile phase) of the two-phase solvent system were pumped into the column with the volume ratio of 60:40. When the column was totally filled with the two phases, the lower phase was pumped, and at the same time, the HSCCC apparatus was run at a revolution speed of 900 rmp. After

the parent ion is required due to the same precursor ion and product ion. A PREC-IDA-EPI mode was used to provide information on the parent ions, fragment ions and retention times of specified ions so the molecular weights of unknown coumarins and their glycosides could be identified. The information on the fragment ions from the MIM-IDA-EPI mode could be supplemented, and the retention time could be verified. Therefore, the characterization of trace furanocoumarins has become very easy and accurate by the combined use of the two modes and may play an important role in controlling the quality of

A high performance liquid chromatography–diode array detection–electrospray ionization tandem mass spectrometry (HPLC/DAD/ESI-MSn) method was used for the chromatographic fingerprint analysis and characterization of furocoumarins in the roots of *Angelica dahurica* (Kang et al., 2008)*.* The HPLC fingerprint technique has been considered as a useful method in identification and quality evaluation of herbs and their related finished products in recent years, because the HPLC fingerprint could systematically and comprehensively exhibit the types and quantification of the components in the herbal medicines (Drasar & Moravcova, 2004; Kang et al., 2008; Wang et al., 2007). Kang and coauthors (2008) showed that the samples from different batches had similar HPLC fingerprints, and the method could be applied for the quality control of the roots of *Angelica dahurica*. In addition, they identified a total of 20 furocoumarins by HPLC/DAD/ESI-MS*<sup>n</sup>* technique, and their fragmentation patterns in an electrospray ion trap mass spectrometer

Recently, high-speed counter-current chromatography (HSCCC) equipped with a HPLC system for separation and purification of furanocoumarins from crude extracts of plant

High-speed counter-current chromatography (HSCCC), which was first invented by Y. Ito (1981), is a kind of liquid–liquid partition chromatography. The stationary phase of this method is also a liquid. It is retained in the separation column by centrifugal force. Because no solid support is used in the separation column, HSCCC successfully eliminates irreversible adsorption loss of samples onto the solid support used in conventional chromatographic columns (Ito, 1986). As an advanced separation technique, it offers various advantages including high sample recovery, high-purity of fractions, and high-loading capacity (Ma et al., 1994). In the past 30 years, HSCCC has made great progress in the preparation of various reference standards for pharmacological studies and good manufacturing practice, such as coumarins, alkaloids, flavonoids, hydroxyanthraquiones

Liu and co-authors (2004b) isolated and purified psoralen and isopsoralen from *Psoralea corylifolia* using HSCCC technique. In their investigation, they utilized TBE-300A HSCCC instrument with three multilayer coil separation column connected in series. The two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water was used for HSCCC separation. Each solvent was added to a separatory funnel and roughly equilibrated at room temperature. The upper phase (stationary phase) and the lower phase (mobile phase) of the two-phase solvent system were pumped into the column with the volume ratio of 60:40. When the column was totally filled with the two phases, the lower phase was pumped, and at the same time, the HSCCC apparatus was run at a revolution speed of 900 rmp. After

medicinal herbs.

were also summarized.

(Liu et al., 2004b).

materials, was also described.

hydrodynamic equilibrium was reached, the sample solution containing the crude extract was injected into the separation coil tube through the injection valve. Each peak fraction was collected according to the chromatogram and evaporated under reduced pressure. The results of HSCCC tests indicated that n-hexane-ethyl acetate-methanol-water (5:5:4.5:5.5, v/v) was the best solvent system for the separation of psoralen and isopsoralen (Liu et al., 2004b).

In another investigations, the same authors had used the same HSCCC technique to induce preparative isolation and purification of furanocoumarins from *Angelica dahurica* (Fisch. ex Hoffm) Benth, et Hook. f (Liu et al., 2004) and from *Cnidium monnieri* (L.) Cusson (Liu et al., 2004a). The results of the first study (Liu et al., 2004) indicated that the best separation of imperatorin, oxypeucedanin and isoimperatorin was when the lower phase of n-hexanemethanol-water (5:5:5, v/v) and n-hexane-methanol-water (5:7:3, v/v) were used in gradient elution. The following gradient was used: 0-150 min, only the lower phase of n-hexanemethanol-water (5:5:5, v/v); 150-300 min, the volume ratio of the lower phase of n-hexanemethanol-water (5:7:3, v/v) changed from 0 to 100%.

A HSCCC method for separation and purification of psoralens from *C. monnieri* was developed by using with a pair of two-phase solvent system composed of light petroleumethyl acetate-methanol-water at volume ratios of 5:5:5:5, 5:5:6:4 and 5:5:6.5:3.5 (Liu et al., 2004a).

In described cases, the crude extracts obtained by HSCCC technique were analyzed by HPLC method. The column used was a reversed-phase symmetry C18 column. The mobile phase was methanol-water (68:32, v/v) (analysis of extract from *A. dahurica*), methanolwater (40:60, v/v) (analysis of extract from *P. corylifolia*), or methanol-acetonitrile-water system in gradient mode as follows: 30:30:40 to 50:30:20 in 30 min (analysis of extract from *C.monnieri*) (Liu et al., 2004, 2004a, 2004b). Bergapten and imperatorin obtained by HSCCC method from *Cnidium monnieri* were also analyzed by high-performance liquid chromatography. The column used was a reversed-phase symmetry C18 column, and the mobile phase adopted was methanol (solvent A) – water (solvent B) in the gradient mode as follows: 0-5 min, 60% A; 5-14 min, 60-80% A; 14-15 min, 80-60% A (Li & Chen, 2004).

#### **5.3.5 Capillary electrophoresis**

In some cases, capillary electrophoresis was chosen to determine quantities of furanocoumarins. For example Ochocka et al. (1995) used this method for separating psoralens from roots and aerial parts of *Chrysanthemum segetum* L. The analyses were performed with electrophoresis apparatus with UV detection at 280 nm. The best overall separation was obtained on uncoated silica capillary with 7-s pneumatic injection using a buffer solution of 0.2 M boric acid-0.05 M of borax in water (11:9, v/v) (pH 8.5). In another example, micellar electrokinetic capillary chromatography (MEKC) was used in the separation of coumarins contained in *Angelicae Tuhou Radix* (Chen & Sheu, 1995). In this investigation, the electrolyte was buffer solution [20 mM sodium dodecyl sulfate (SDS) – 15 mM sodium borate – 15 mM sodium dihydrogenphosphate (pH 8.26)] – acetonitrile (24:1).

The pressurized capillary electrochromatography (pCEC) was utilized for the separation and determination of coumarins in *Fructus cnidii* extracts from 12 different regions (Chen et al., 2009). Capillary electrochromatography (CEC), as a novel microcolumn separation

Analytical Methods for Isolation,

furanocoumarins.

**6. Conclusions** 

2007).

**7. References** 

266.

interpretation of NMR spectra.

furanocoumarin compounds analysis.

Separation and Identification of Selected Furanocoumarins in Plant Material 81

spectroscopy and infrared spectroscopy (IR) are used. NMR spectroscopy is an invaluable technique for the structural determination of all furanocoumarins. As well as providing information on the chemical environment of each proton or carbon nucleus in the molecule, the technique can be employed to determine linkages amongst nearby nuclei, often enabling

Dimethyl sulfoxide (DMSO-d6) and methanol (CD3OD) are both suitable solvents for

The reader is referred to Rojas-Lima et al., 1999; Um et al., 2010; Taniguchi et al., 2011, and Tesso et al., 2005 publications, for details of the principles of NMR and general

As furanocoumarins have a lactone structure, they have a wide range of biological activity. Bergapten and the other furanocoumarins are used to treat dermatological diseases (psoriaris, vitiligo). As a result, their photosensitizing properties are playing an important role (Bhatnagar et al., 2007; Trott et al., 2008). Their ability to covalently modify nucleic acids is used in process called "extracorporeal photopheresis" that is medically necessary for either of the following clinical indications: erythrodermic variants of cutaneous T-cell lymphoma (e.g. mycosis fungioides, Sezary's syndrome) or chronic graft-versus-host disease, refractory to standard immunosuppressive therapy (Hotlick et al., 2008; Lee et al.,

The aim of the present chapter was to present an overview of techniques of isolation, separations and identification of furanocoumarins in plant materials. Various analytical approaches exist for detection of coumarins and the analytical techniques should meet the following prerequisites: short time, relatively inexpensive, highly accurate, and precise for a variety of applications. This review may be helpful in the choice of the method of

Ahn, M.J., Lee. M.K., Kim, Y.C., Sung, S.H. (2008) *The simultaneous determination of coumarins* 

Al-Barwani, F.M. & Eltayeb, E.A. (2004) *Antifungal compounds from indused Conium* 

Baek, N.I., Ahn, E.M., Kim, H.Y., Park, Y.D. (2000). *Furanocoumarins from the root of Angelica* 

Baetas, A.C.S., Arruda, M.S.P., Müller, A.H., Arruda, A.C. (1999) *Coumarins and Alkaloids* 

*from the Stems of Metrodorea Flavida*. J Braz Chem Soc. 10(3), 181-183.

*maculatum L. plants*. Biochem Syst Ecol. 32, 1097-1108.

*dahurica.* Arch Pharm Res. 23, 467-470.

*in Angelica gigas root by high performance liquid chromatography-diode array detector coupled with electrospray ionization/mass spectrometry*. J Pharm Biomed Anal. 46, 258-

a complete structure to be assembled (Rice-Evans & Packer, 2003).

technology, couples the high efficiency of capillary electrophoresis with high selectivity of HPLC. The CEC analytes separation is usually achieved in capillaries containing packed stationary phases by an electroosmotic flow (EOF) generated by a high electric field. The experiments were performed in an in-house packed column with a monolithic outlet frit under the optimal conditions: pH 4.0 ammonium acetate buffer at 10 mM containing 50% acetonitrile at −6 kV applied voltage. This analytical method, with use of the novel column, gives good results in the determination of coumarins.

#### **5.3.6 Gas chromatography**

In the recent decade, tasks related to the isolation of furocoumarins and the quality control of related preparations were most frequently solved using GC techniques. Gas chromatography was predominantly used for the identification and quantitative analysis of furocoumarins in preparations and raw plant materials. Investigations of the chromatographic behavior (retention times) of substituted furocoumarins revealed the following general laws: 1) on passage from hydroxy- to methoxycoumarins, the retention time decreases (because of reduced adsorption via hydrogen bonds); 2) furocoumarins with O-alkyl substituents at C5 are eluted after 8-hydroxy isomers; 3) the logarithm of the relative retention time is a linear function of the molecular weight. This GC data can be used for determining the structure and estimating the retention time of analogous coumarins (Lozhkin & Sakanyan, 2006). A number of methods have been described for the analysis of furanocoumarins using capillary gas chromatography (GC) (Beier et al., 1994; Wawrzynowicz & Waksmundzka-Hajnos, 1990).

Gas chromatographic method was used to determine osthole content in *Cnidii Fructus* extract. The analytical conditions are the following: nitrogen as the carrier gas, the flow rate of 40 mL/min; the split ratio of 120:1. The column used was DBTM-5 (30 m × 0.53 mm I.D., 1.5 μm) equipped with a flame ionization detector (FID). The initial oven temperature was programmed to be at 135°C for 12 minutes. The temperature was then raised to 215°C at a rate of 12°C/min for 20 minutes. Caffeine anhydrous was used as the internal standard (Yu et al., 2002).

In another example, GC-FID was used to analyze of psoralen, bergapten, pimpinellin and isopimpinellin present in phytomedicines (creams and pomades) employed in the treatment of vitiligo in Brazil. The GC-FID assay method present here is rapid, sensitive and robust and can be applied to the determination of furanocoumarins in routine analysis of creams, pomades and other lipophilic phytocosmetics. These analyses were performed in a VARIAN 3400 gas chromatograph equipped with a capillary fused silica LM-5 and with a flame ionization detector (FID). H2 was used as carrier gas at a flow rate 0.8 ml min-1 and the injection split ratio was 1:20. The injection temperature was 280°C. Column temperature was programmed from 150 to 240°C with a linear increase of 10°C min-1, then 240–280°C with a linear increase of 5°C min-1 and was then held for 15 mins. The detector temperature was 280°C (Cardoso et al., 2000).

#### **5.4 Structural analysis**

For the structural identification and characterizing of the psoralen compounds, especially if they are novel, instrumental techniques such as nuclear magnetic resonance (NMR) spectroscopy and infrared spectroscopy (IR) are used. NMR spectroscopy is an invaluable technique for the structural determination of all furanocoumarins. As well as providing information on the chemical environment of each proton or carbon nucleus in the molecule, the technique can be employed to determine linkages amongst nearby nuclei, often enabling a complete structure to be assembled (Rice-Evans & Packer, 2003).

Dimethyl sulfoxide (DMSO-d6) and methanol (CD3OD) are both suitable solvents for furanocoumarins.

The reader is referred to Rojas-Lima et al., 1999; Um et al., 2010; Taniguchi et al., 2011, and Tesso et al., 2005 publications, for details of the principles of NMR and general interpretation of NMR spectra.

### **6. Conclusions**

80 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

technology, couples the high efficiency of capillary electrophoresis with high selectivity of HPLC. The CEC analytes separation is usually achieved in capillaries containing packed stationary phases by an electroosmotic flow (EOF) generated by a high electric field. The experiments were performed in an in-house packed column with a monolithic outlet frit under the optimal conditions: pH 4.0 ammonium acetate buffer at 10 mM containing 50% acetonitrile at −6 kV applied voltage. This analytical method, with use of the novel column,

In the recent decade, tasks related to the isolation of furocoumarins and the quality control of related preparations were most frequently solved using GC techniques. Gas chromatography was predominantly used for the identification and quantitative analysis of furocoumarins in preparations and raw plant materials. Investigations of the chromatographic behavior (retention times) of substituted furocoumarins revealed the following general laws: 1) on passage from hydroxy- to methoxycoumarins, the retention time decreases (because of reduced adsorption via hydrogen bonds); 2) furocoumarins with O-alkyl substituents at C5 are eluted after 8-hydroxy isomers; 3) the logarithm of the relative retention time is a linear function of the molecular weight. This GC data can be used for determining the structure and estimating the retention time of analogous coumarins (Lozhkin & Sakanyan, 2006). A number of methods have been described for the analysis of furanocoumarins using capillary gas chromatography (GC) (Beier et al., 1994;

Gas chromatographic method was used to determine osthole content in *Cnidii Fructus* extract. The analytical conditions are the following: nitrogen as the carrier gas, the flow rate of 40 mL/min; the split ratio of 120:1. The column used was DBTM-5 (30 m × 0.53 mm I.D., 1.5 μm) equipped with a flame ionization detector (FID). The initial oven temperature was programmed to be at 135°C for 12 minutes. The temperature was then raised to 215°C at a rate of 12°C/min for 20 minutes. Caffeine anhydrous was used as the internal standard (Yu

In another example, GC-FID was used to analyze of psoralen, bergapten, pimpinellin and isopimpinellin present in phytomedicines (creams and pomades) employed in the treatment of vitiligo in Brazil. The GC-FID assay method present here is rapid, sensitive and robust and can be applied to the determination of furanocoumarins in routine analysis of creams, pomades and other lipophilic phytocosmetics. These analyses were performed in a VARIAN 3400 gas chromatograph equipped with a capillary fused silica LM-5 and with a flame ionization detector (FID). H2 was used as carrier gas at a flow rate 0.8 ml min-1 and the injection split ratio was 1:20. The injection temperature was 280°C. Column temperature was programmed from 150 to 240°C with a linear increase of 10°C min-1, then 240–280°C with a linear increase of 5°C min-1 and was then held for 15 mins. The detector temperature was

For the structural identification and characterizing of the psoralen compounds, especially if they are novel, instrumental techniques such as nuclear magnetic resonance (NMR)

gives good results in the determination of coumarins.

Wawrzynowicz & Waksmundzka-Hajnos, 1990).

**5.3.6 Gas chromatography** 

et al., 2002).

280°C (Cardoso et al., 2000).

**5.4 Structural analysis** 

As furanocoumarins have a lactone structure, they have a wide range of biological activity. Bergapten and the other furanocoumarins are used to treat dermatological diseases (psoriaris, vitiligo). As a result, their photosensitizing properties are playing an important role (Bhatnagar et al., 2007; Trott et al., 2008). Their ability to covalently modify nucleic acids is used in process called "extracorporeal photopheresis" that is medically necessary for either of the following clinical indications: erythrodermic variants of cutaneous T-cell lymphoma (e.g. mycosis fungioides, Sezary's syndrome) or chronic graft-versus-host disease, refractory to standard immunosuppressive therapy (Hotlick et al., 2008; Lee et al., 2007).

The aim of the present chapter was to present an overview of techniques of isolation, separations and identification of furanocoumarins in plant materials. Various analytical approaches exist for detection of coumarins and the analytical techniques should meet the following prerequisites: short time, relatively inexpensive, highly accurate, and precise for a variety of applications. This review may be helpful in the choice of the method of furanocoumarin compounds analysis.

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**4** 

*Italy* 

**Analytical Methods and Phytochemistry of the** 

**Typical Italian Liquor "Limoncello": A Review** 

The analyses of food or food-derived products that naturally occur in plants are essential especially related to food safety, food composition, adulterations, and food quality for Protected Geographical Indication (IGP), Controlled Origin Denomination (DOC) or

For these reasons in this field of research a multidisciplinary approach between analytical chemistry, phytochemistry, organic synthetic chemistry, and biochemistry is strongly

In this contest, the Italian liqueur "Limoncello", obtained by maceration of lemon skin in a

Limoncello is produced in Southern Italy, mainly in Campania region (Gulf of Naples, the Sorrentine Peninsula, the coast of Amalfi, and islands of Procida, Ischia, and Capri) but also

Following an ancient tradition, limoncello is a homemade liqueur obtained from peels of Sorrento lemons. However nowadays, due to the fact that its production on industrial scale is widespread and the demand on the international market is increasing, virtually all kinds of lemons are used to make this liqueur. Its main distinctive feature is a bright yellow colour. Limoncello is served especially after-dinner as a digestive after having been stored in a refrigerator at 4 °C. It is very popular and commonly consumed in all Italy and many areas of the Mediterranean region but it is also becoming famous in other countries like United

Being so popular, limoncello is also an ingredient for the preparation of several cocktails

A typical procedure for the preparation of limoncello include as ingredients 8 Sorrento lemons, 1 litre of water, 1 litre of absolute ethanol and 800 g of sugar. Lemons are then peeled in such a way to get only the flavedo (the very external part of the skin coloured in yellow); the resulting raw vegetable material is then macerated in the dark for 15 days. The

due to its strong lemon flavour without the sourness or bitterness of lemon juice.

Controlled and Guaranteed Origin Denomination (DOCG) designation.

hydroalcoholic solution of saccharose, is an explicative example.

States, Canada, Australia, New Zealand, and northern Europe.

**1. Introduction** 

recommended.

in Sicily and Sardinia.

*University "G. d'Annunzio" of Chieti-Pescara, Faculty of Pharmacy,* 

Marcello Locatelli, Giuseppe Carlucci, Salvatore Genovese and Francesco Epifano

*Dipartimento di Scienze del Farmaco,* 


### **Analytical Methods and Phytochemistry of the Typical Italian Liquor "Limoncello": A Review**

Marcello Locatelli, Giuseppe Carlucci, Salvatore Genovese and Francesco Epifano *University "G. d'Annunzio" of Chieti-Pescara, Faculty of Pharmacy, Dipartimento di Scienze del Farmaco, Italy* 

#### **1. Introduction**

92 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Zobel, A. M. & Brown, S. A. (1990) *Dermatitis-inducing furanocoumarins on leaf surfaces of eight* 

Zobel, A. M., Brown, S. A., Nighswander, J.E. (1991) *Influence of acid and salt sprays on* 

Żołek, T., Paradowska, K., Wawer, I. (2003) *13C CP MAS NMR and GIAO-CHF calculations of* 

ASE 200 Accelerated Solvent Extraction Operator's Manual, Document no. 031149, revision

*furanocoumarin concentrations on the Ruta graveolens leaf surface.* Ann Bot. 67, 213-

*species of rutaceous and umbelliferous plants.* J Chem Ecol. 16, 693-700.

*coumarins.* Solid State Nucl Magn Reson. 23, 77-87.

01, Dionex, Sunyvale, CA, 1995 (Section 3-5).

218.

The analyses of food or food-derived products that naturally occur in plants are essential especially related to food safety, food composition, adulterations, and food quality for Protected Geographical Indication (IGP), Controlled Origin Denomination (DOC) or Controlled and Guaranteed Origin Denomination (DOCG) designation.

For these reasons in this field of research a multidisciplinary approach between analytical chemistry, phytochemistry, organic synthetic chemistry, and biochemistry is strongly recommended.

In this contest, the Italian liqueur "Limoncello", obtained by maceration of lemon skin in a hydroalcoholic solution of saccharose, is an explicative example.

Limoncello is produced in Southern Italy, mainly in Campania region (Gulf of Naples, the Sorrentine Peninsula, the coast of Amalfi, and islands of Procida, Ischia, and Capri) but also in Sicily and Sardinia.

Following an ancient tradition, limoncello is a homemade liqueur obtained from peels of Sorrento lemons. However nowadays, due to the fact that its production on industrial scale is widespread and the demand on the international market is increasing, virtually all kinds of lemons are used to make this liqueur. Its main distinctive feature is a bright yellow colour. Limoncello is served especially after-dinner as a digestive after having been stored in a refrigerator at 4 °C. It is very popular and commonly consumed in all Italy and many areas of the Mediterranean region but it is also becoming famous in other countries like United States, Canada, Australia, New Zealand, and northern Europe.

Being so popular, limoncello is also an ingredient for the preparation of several cocktails due to its strong lemon flavour without the sourness or bitterness of lemon juice.

A typical procedure for the preparation of limoncello include as ingredients 8 Sorrento lemons, 1 litre of water, 1 litre of absolute ethanol and 800 g of sugar. Lemons are then peeled in such a way to get only the flavedo (the very external part of the skin coloured in yellow); the resulting raw vegetable material is then macerated in the dark for 15 days. The

Analytical Methods and Phytochemistry of the Typical Italian Liquor "Limoncello": A Review 95

In this field the best practices and improvements concerns purification and analytes extraction techniques and especially analytical instrument configuration applied to this kind

Sample preparation steps are often sensitive to the matrix and, generally, contributed with approx. at 75% to the final error. This is particular true when multiple steps are involved

The sample preparation has a straight impact on method performances, in particular trueness, precision, limits of quantitation, linearity, reproducibility, and is often the rate-

Sample processing can broadly range in complexity from simple dissolution in an appropriate solvent to a complicated extraction procedure, followed by derivatization and

There are three general extraction methods, solvent extraction (or liquid-liquid extraction,

The use of these techniques, however, often entails the use of precipitation procedures,

Traditional liquid-liquid extraction procedures employ in a serial of extraction of an aqueous sample with an immiscible organic solvent resulting in a large solvent volume that must be dried and then concentrated prior to analysis, bring to an expensive, and in some

For this reason, further extraction methodologies were developed and validated. In particular, in SPE procedures, a solid sorbent material is packed into a cartridge or imbedded in a disk and performs essentially the same function as the organic solvent in

SPE can be applied in several fields, from bio-analytical to environmental analyses, but it requires a sample volume adequately to extract targets analytes and the possibility to

In the early 1990s, the development of a new sampling method, non-destructive, sensitive, reproducible, relatively inexpensive and in particular solventless, allowed the trapping of volatile organic compounds (VOCs) on a silica optical fibre coated with a polymer thin layer

For the analyses of Limoncello the first extraction procedure used to determine the product

In this methodology, extraction organic solvent generally used for this purpose is a hexane/ethyl acetate mixture (Starrantino et al., 1997, Dugo et al., 2000, Versari et al., 2003). In particular the analyses of lemon-derived products were achieved with a preliminary dilution (1:10)-extraction step with hexane and ethyl acetate (75:25, v/v) (Starrantino et al.,

1997) or pure hexane (Mondello et al., 2003) followed by direct analyses of extracts.

LLE), solid phase extraction (SPE), and solid phase micro-extraction (SPME).

liquid-liquid extraction, with a smaller organic solvent volume consumption.

replicate the analysis because SPE is a destructive methodology.

volatile components was Liquid-Liquid Extraction (LLE).

crude separation processes, and subsequent concentration methods.

into the procedure, where the final uncertainty is compounded.

of analyses.

further extraction.

**2.1 Extraction procedures** 

limiting step for several analytical assays.

cases non-reproducible procedure.

followed by their identification by GC.

alcoholic solution from maceration is then added to the solution of sugar in water and the resulting mixture is filtered. The liqueur so obtained is kept in the dark for 40 days after which period is stored in the refrigerator and consumed as described above.

Resulting from an ethnically ancient tradition and being used as a digestive in several part of the world, it is interesting to investigate the chemical composition and medical properties of Limoncello.

The Council Regulation no. 1576/89 lays (Council Regulation, 1989) down a definition and a description of spirit drinks and also dictates the need for a better comprehension of the chemical nature of limoncello. Besides giving a list of rules on alcoholic beverages, the regulation clearly states that even nature-identical flavouring substances and preparations shall not be authorized in liqueurs derived from *Citrus* fruits.

Apart from some investigations on lemon liquors reported in journals specifically linked to beverage industry (Bonomi et al., 2001; Moio et al., 2000; Naviglio et al., 2001, 2003, 2005a, 2005b; Romano et al., 2004), it can be stated that the increasing interest of the market toward limoncellos is not offset by the number of scientific papers.

The effective problem connected to investigation about phytochemicals is the correct and real identification and quantification in natural sources by means of very sensitive, selective, and validated analytical methodologies.

In 1986, the first study describing the chemical composition of the essential oil derived from Sicilian lemons was reported in the literature by Cotroneo and coworkers (Cotroneo et al., 1986). The same group got further insights on the same topic with a series of manuscript published from the beginning of 90s until 2001 (Dugo G. et al., 1993, 1999, 2001; Dugo P. et al., 1998, 2000; Mondello et al., 1999). These Authors used very sensitive and selective analytical methodologies like high-resolution GC coupled with chiral capillary columnsmass spectrometry (HRGC-MS) and bi-dimensional capillary GC (2D-GC). In 2002 they finally summarized in a review article all the works they made on *Citrus* species (Dugo et al., 2002).

Examining the papers by Dugo and coworkers, the need of using advanced instrumental techniques for the analyses of raw sample extracted from Citrus fruits, mainly due to their chemical complexity, can be pointed out.

In the next paragraph, we'll make a survey of the current reported literature data about extraction procedures and instrumental configuration used for the analyses of this Italian liqueur obtained by maceration of lemon skin.

#### **2. Analytical methods**

In the last twenty years a huge progress was made concerning instrumental configuration, sensitivity and selectivity improvement (Locatelli et al., 2011) for analytical methods applied in many fields, especially for food safety.

The main problem consisted in the great complexity of extracted samples and the large number of components. For these reasons the qualitative and quantitative analyses must be very specific and robust enough to isolate, qualificate, and to quantificate the target compound (s).

In this field the best practices and improvements concerns purification and analytes extraction techniques and especially analytical instrument configuration applied to this kind of analyses.

#### **2.1 Extraction procedures**

94 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

alcoholic solution from maceration is then added to the solution of sugar in water and the resulting mixture is filtered. The liqueur so obtained is kept in the dark for 40 days after

Resulting from an ethnically ancient tradition and being used as a digestive in several part of the world, it is interesting to investigate the chemical composition and medical properties

The Council Regulation no. 1576/89 lays (Council Regulation, 1989) down a definition and a description of spirit drinks and also dictates the need for a better comprehension of the chemical nature of limoncello. Besides giving a list of rules on alcoholic beverages, the regulation clearly states that even nature-identical flavouring substances and preparations

Apart from some investigations on lemon liquors reported in journals specifically linked to beverage industry (Bonomi et al., 2001; Moio et al., 2000; Naviglio et al., 2001, 2003, 2005a, 2005b; Romano et al., 2004), it can be stated that the increasing interest of the market toward

The effective problem connected to investigation about phytochemicals is the correct and real identification and quantification in natural sources by means of very sensitive, selective,

In 1986, the first study describing the chemical composition of the essential oil derived from Sicilian lemons was reported in the literature by Cotroneo and coworkers (Cotroneo et al., 1986). The same group got further insights on the same topic with a series of manuscript published from the beginning of 90s until 2001 (Dugo G. et al., 1993, 1999, 2001; Dugo P. et al., 1998, 2000; Mondello et al., 1999). These Authors used very sensitive and selective analytical methodologies like high-resolution GC coupled with chiral capillary columnsmass spectrometry (HRGC-MS) and bi-dimensional capillary GC (2D-GC). In 2002 they finally summarized in a review article all the works they made on *Citrus* species (Dugo et

Examining the papers by Dugo and coworkers, the need of using advanced instrumental techniques for the analyses of raw sample extracted from Citrus fruits, mainly due to their

In the next paragraph, we'll make a survey of the current reported literature data about extraction procedures and instrumental configuration used for the analyses of this Italian

In the last twenty years a huge progress was made concerning instrumental configuration, sensitivity and selectivity improvement (Locatelli et al., 2011) for analytical methods applied

The main problem consisted in the great complexity of extracted samples and the large number of components. For these reasons the qualitative and quantitative analyses must be very specific and robust enough to isolate, qualificate, and to quantificate the target

which period is stored in the refrigerator and consumed as described above.

shall not be authorized in liqueurs derived from *Citrus* fruits.

limoncellos is not offset by the number of scientific papers.

and validated analytical methodologies.

chemical complexity, can be pointed out.

in many fields, especially for food safety.

**2. Analytical methods**

compound (s).

liqueur obtained by maceration of lemon skin.

of Limoncello.

al., 2002).

Sample preparation steps are often sensitive to the matrix and, generally, contributed with approx. at 75% to the final error. This is particular true when multiple steps are involved into the procedure, where the final uncertainty is compounded.

The sample preparation has a straight impact on method performances, in particular trueness, precision, limits of quantitation, linearity, reproducibility, and is often the ratelimiting step for several analytical assays.

Sample processing can broadly range in complexity from simple dissolution in an appropriate solvent to a complicated extraction procedure, followed by derivatization and further extraction.

There are three general extraction methods, solvent extraction (or liquid-liquid extraction, LLE), solid phase extraction (SPE), and solid phase micro-extraction (SPME).

The use of these techniques, however, often entails the use of precipitation procedures, crude separation processes, and subsequent concentration methods.

Traditional liquid-liquid extraction procedures employ in a serial of extraction of an aqueous sample with an immiscible organic solvent resulting in a large solvent volume that must be dried and then concentrated prior to analysis, bring to an expensive, and in some cases non-reproducible procedure.

For this reason, further extraction methodologies were developed and validated. In particular, in SPE procedures, a solid sorbent material is packed into a cartridge or imbedded in a disk and performs essentially the same function as the organic solvent in liquid-liquid extraction, with a smaller organic solvent volume consumption.

SPE can be applied in several fields, from bio-analytical to environmental analyses, but it requires a sample volume adequately to extract targets analytes and the possibility to replicate the analysis because SPE is a destructive methodology.

In the early 1990s, the development of a new sampling method, non-destructive, sensitive, reproducible, relatively inexpensive and in particular solventless, allowed the trapping of volatile organic compounds (VOCs) on a silica optical fibre coated with a polymer thin layer followed by their identification by GC.

For the analyses of Limoncello the first extraction procedure used to determine the product volatile components was Liquid-Liquid Extraction (LLE).

In this methodology, extraction organic solvent generally used for this purpose is a hexane/ethyl acetate mixture (Starrantino et al., 1997, Dugo et al., 2000, Versari et al., 2003).

In particular the analyses of lemon-derived products were achieved with a preliminary dilution (1:10)-extraction step with hexane and ethyl acetate (75:25, v/v) (Starrantino et al., 1997) or pure hexane (Mondello et al., 2003) followed by direct analyses of extracts.

Analytical Methods and Phytochemistry of the Typical Italian Liquor "Limoncello": A Review 97

Due to the complex chemical composition of Limoncello extracts (comprising several classes of volatile compounds, 85-98%), the election chromatographic techniques are certainly gas-

In mono-dimensional GC the stationary phase is bonded and highly cross-linked; silphenylene polymer (polarity similar to 5% phenyl polymethylsiloxane) in programmable thermal analyses, split/splitless injector in splitless mode. This capillary column is used with both FID and MS detector, with the unique difference in helium flow rate (minor in the

With this system is possible the analyses of all volatile components fraction derived from SPME extraction, while for the analyses of enantiomeric compounds chiral capillary column characterized by diethyltertbutylsilyl-β-cyclodextrins as stationary phases are generally

These analyses are carried out separately to obtain a complete chemical composition of volatile components. Recently Mondello and coworkers (Mondello et al., 2006) coupled these two gas-chromatographic separations in an innovative multi-dimensional GC (MDGC) system, to obtain the complete chemical composition on volatile components including

The main goal of this configuration is especially due to the possibility of evaluating the enantiomeric ratios as genuineness markers, especially in complex matrices as Limoncellos. To detect analytes of interest flame ionisation detector (FID) and mass spectrometric

With the first, that is a "universal" gas-chromatographic detector is possible to obtain, in a single run a complete chemical fingerprint of volatile fraction, with identification of various components by retention index (*Ri*). This configuration is very useful if are at disposition data bank with *Ri* or chemical standards. If no pure chemical standards are available, the best choice is gas-chromatography-mass spectrometry interfacing (GC-MS), because is possible to obtain *Ri* of several volatile components and, in addition, the mass-to-charge

Another trend in the last years is the use of MDGC, coupled with mass spectrometry. Gaschromatographic determination has the disadvantage that by this technique it is difficult to

High performance liquid chromatography (HPLC) is generally used for the non-volatile components. Is a well-defined technique, robust and reproducible that generally is coupled

In the literature several paper dealing with the analysis of flavones, coumarins, and furanocoumarins composition of lemon-derived products by HPLC using different

In particular for the analyses of coumarins and furanocoumarins in Limoncellos samples the most widely used column are silica based (generally 300 x 4 mm, 10 μm particle size) to obtain a complete resolution in normal phase mode (Starrantino et al., 1997, Versari et al., 2003), while for the analyses of phenolic compounds Octadecylsilane (ODS) column in

chromatographic (GC) ones.

enantiomeric resolution in a single analysis.

ratio for the correct identification of targeted compounds.

analyse non-volatile components of extracted samples.

with "universal" detector as Diode Array Detector (DAD).

reversed phase mode was used (Versari et al., 2003).

chromatographic column were reported.

detector (MS) are generally chosen.

MS interfacing).

used.

Solid-phase micro extraction (SPME) derives principally from SPE technologies and improves the concept of reducing solvent consumption, economic characteristic, and involved through two different extraction methods: headspace SPME (HS-SPME) and contact SPME.

The headspace SPME is based on the absorption of the analytes on a fibre coating placed in the sample's headspace volume and on the partition of the target analytes between the sampling matrix and the fibre. After exposure the fibre bearing the concentred analytes was retracted, removed from the sample vial and VOCs were thermally desorbed by insertion of the fibre into the injector port of the chromatograph.

Recently in the preparative sample scenario appeared several automated and fully independent instrumentation that allow to process high sample number (high-throughput), and especially with an improvement of efficiency and process reproducibility.

When gas-chromatographic assays were used to quantify diluted Limoncello extracts it was also necessary to decrease the concentration of ethanol that tended to immediately saturate the SPME fibres. The best election, and generally, used methodology for the analysis of volatile components was headspace SPME (HS-SPME) on polydimethylsiloxane (PDMS) as thin layer (TL) due to non-polar characteristic of samples compounds.

This technique can be easily automated (Crupi et al., 2007) to improve analyses number (high throughput assay) and to obtain a better control on overall analysis steps, which bring to higher methodology reproducibility.

#### **2.2 Instrumental analysis**

Mono-dimensional chromatographic processes (1D) are widely applied in the analysis of food and food-derived products.

Although such methods often provide satisfying analytical results, the complexity of several matrices exceeds the resolution capacity of a single dimension separation system.

In the past years several efforts have been dedicated to the combination of independent techniques with the aim of reinforcement resolving power, until the use of multidimensional chromatography (MDC).

A typical comprehensive bi-dimensional chromatographic separation (2D) is achieved, generally, on two distinct columns connected in series with a special transfer system located between them. The type of interface used is connected to the specific methodology. The function of the interface is to cut and then release continuous fractions of the primary column effluent onto a fast separation column. In order to achieve comprehensive analysis and to preserve the 1D separation, the bands injected onto the secondary column must undergo elution before the following re-injection. Secondary retention times must be, at the most, equal or less than the duration of a single modulation period.

Coupled to these chromatographic techniques (both gas chromatography and highperformance liquid chromatography), several detectors are used for a clear and univocal identification and quantification of target analyte(s).

The must used are Flame Ionisation Detector (FID) coupled with GC, Mass Spectrometry Detector (MS) coupled to GC and HPLC, and Tandem Mass Spectrometry (MS/MS) interfaced with HPLC.

Solid-phase micro extraction (SPME) derives principally from SPE technologies and improves the concept of reducing solvent consumption, economic characteristic, and involved through

The headspace SPME is based on the absorption of the analytes on a fibre coating placed in the sample's headspace volume and on the partition of the target analytes between the sampling matrix and the fibre. After exposure the fibre bearing the concentred analytes was retracted, removed from the sample vial and VOCs were thermally desorbed by insertion of

Recently in the preparative sample scenario appeared several automated and fully independent instrumentation that allow to process high sample number (high-throughput),

When gas-chromatographic assays were used to quantify diluted Limoncello extracts it was also necessary to decrease the concentration of ethanol that tended to immediately saturate the SPME fibres. The best election, and generally, used methodology for the analysis of volatile components was headspace SPME (HS-SPME) on polydimethylsiloxane (PDMS) as

This technique can be easily automated (Crupi et al., 2007) to improve analyses number (high throughput assay) and to obtain a better control on overall analysis steps, which bring

Mono-dimensional chromatographic processes (1D) are widely applied in the analysis of

Although such methods often provide satisfying analytical results, the complexity of several

In the past years several efforts have been dedicated to the combination of independent techniques with the aim of reinforcement resolving power, until the use of multi-

A typical comprehensive bi-dimensional chromatographic separation (2D) is achieved, generally, on two distinct columns connected in series with a special transfer system located between them. The type of interface used is connected to the specific methodology. The function of the interface is to cut and then release continuous fractions of the primary column effluent onto a fast separation column. In order to achieve comprehensive analysis and to preserve the 1D separation, the bands injected onto the secondary column must undergo elution before the following re-injection. Secondary retention times must be, at the

Coupled to these chromatographic techniques (both gas chromatography and highperformance liquid chromatography), several detectors are used for a clear and univocal

The must used are Flame Ionisation Detector (FID) coupled with GC, Mass Spectrometry Detector (MS) coupled to GC and HPLC, and Tandem Mass Spectrometry (MS/MS)

matrices exceeds the resolution capacity of a single dimension separation system.

most, equal or less than the duration of a single modulation period.

identification and quantification of target analyte(s).

two different extraction methods: headspace SPME (HS-SPME) and contact SPME.

and especially with an improvement of efficiency and process reproducibility.

thin layer (TL) due to non-polar characteristic of samples compounds.

the fibre into the injector port of the chromatograph.

to higher methodology reproducibility.

**2.2 Instrumental analysis** 

interfaced with HPLC.

food and food-derived products.

dimensional chromatography (MDC).

Due to the complex chemical composition of Limoncello extracts (comprising several classes of volatile compounds, 85-98%), the election chromatographic techniques are certainly gaschromatographic (GC) ones.

In mono-dimensional GC the stationary phase is bonded and highly cross-linked; silphenylene polymer (polarity similar to 5% phenyl polymethylsiloxane) in programmable thermal analyses, split/splitless injector in splitless mode. This capillary column is used with both FID and MS detector, with the unique difference in helium flow rate (minor in the MS interfacing).

With this system is possible the analyses of all volatile components fraction derived from SPME extraction, while for the analyses of enantiomeric compounds chiral capillary column characterized by diethyltertbutylsilyl-β-cyclodextrins as stationary phases are generally used.

These analyses are carried out separately to obtain a complete chemical composition of volatile components. Recently Mondello and coworkers (Mondello et al., 2006) coupled these two gas-chromatographic separations in an innovative multi-dimensional GC (MDGC) system, to obtain the complete chemical composition on volatile components including enantiomeric resolution in a single analysis.

The main goal of this configuration is especially due to the possibility of evaluating the enantiomeric ratios as genuineness markers, especially in complex matrices as Limoncellos.

To detect analytes of interest flame ionisation detector (FID) and mass spectrometric detector (MS) are generally chosen.

With the first, that is a "universal" gas-chromatographic detector is possible to obtain, in a single run a complete chemical fingerprint of volatile fraction, with identification of various components by retention index (*Ri*). This configuration is very useful if are at disposition data bank with *Ri* or chemical standards. If no pure chemical standards are available, the best choice is gas-chromatography-mass spectrometry interfacing (GC-MS), because is possible to obtain *Ri* of several volatile components and, in addition, the mass-to-charge ratio for the correct identification of targeted compounds.

Another trend in the last years is the use of MDGC, coupled with mass spectrometry. Gaschromatographic determination has the disadvantage that by this technique it is difficult to analyse non-volatile components of extracted samples.

High performance liquid chromatography (HPLC) is generally used for the non-volatile components. Is a well-defined technique, robust and reproducible that generally is coupled with "universal" detector as Diode Array Detector (DAD).

In the literature several paper dealing with the analysis of flavones, coumarins, and furanocoumarins composition of lemon-derived products by HPLC using different chromatographic column were reported.

In particular for the analyses of coumarins and furanocoumarins in Limoncellos samples the most widely used column are silica based (generally 300 x 4 mm, 10 μm particle size) to obtain a complete resolution in normal phase mode (Starrantino et al., 1997, Versari et al., 2003), while for the analyses of phenolic compounds Octadecylsilane (ODS) column in reversed phase mode was used (Versari et al., 2003).

Analytical Methods and Phytochemistry of the Typical Italian Liquor "Limoncello": A Review 99


Sabinene 17.570 211.3 - 14.4 9.0 - 20.8 8.8 - - 9.7 9.0 - - 14.9 - - 9.0


yrcene 11.900 18.8 8.9 16.1 9.0 13.0 35.7 12.1 11.0 11.0 9.2 9.1 10.5 10.2 15.0 9.5 9.3 9.1

*p*-Cymene 340 4.5 9.2 84.9 13.3 17.4 11.8 17.2 17.6 28.7 12.7 10.9 14.5 15.3 58.5 13.2 9.5 11.3

Limonene 626.290 837.2 26.5 634.6 43.6 353.3 1671.0 272.1 212.1 288.78 42.6 45.3 179.3 141.4 475.7 50.2 60.6 57.9

Neral 13.520 390.0 30.4 10.8 - 23.6 11.7 13.5 10.2 11.0 10.1 24.8 64.0 26.5 9.9 44.6 26.3 85.0

Geraniol 270 3.2 3.3 4.9 1.9 64.7 10.9 10.9 296.1 106.2 4.6 2.5 6.2 177.5 4.6 21.8 2.8 3.6

Geranial 20.960 581.6 40.2 10.1 - 24.5 17.9 - 9.7 13.7 10.2 32.1 94.0 19.4 11.3 65.5 40.4 133.5

Bergamottin 1.7 4.7 21.5 1.1 0.9 3.8 2.3 19.6 3.7 1.2 1.0 3.6

methoxycoumarine 0.7 1.6 0.8 0.5 0.5 1.3 0.8 7.0 1.2 0.4 0.2 1.2 Citroptene derivate - - 0.1 0.1 - - - 0.3 - - - -

methoxycoumarine - - 0.4 0.2 - - - 1.0 - - - - Citroptene 2.0 - 1.5 1.0 1.3 0.3 1.8 4.0 0.4 1.1 0.4 1.0 Imperatorin 4.4 0.8 - 0.6 - - - 1.6 - - - - Eriocitrin 16.9 - 27.8 21.7 65.9 - - 71.2 - 29.1 - 22.3 Sinapinic acid 0.4 - 0.5 - 1.0 - - Trace - 1.5 - 0.5 Narirutin 5.2 14.5 1.8 0.8 2.2 - - 2.2 - - - 5.9 Naringin - 1.5 - 1.2 Trace - - Trace Trace Trace 1.4 1.3 Hesperidin 16.7 - 25.3 12.0 32.1 - - 42.3 - 6.7 - 7.6 Neohesperidin 0.6 - Trace Trace Trace - - 1.2 - Trace - 0.4 Diosmin 1.9 13.3 7.6 Trace 7.9 - 5.3 - 0.8 4.0 Trace 8.6

Table 2. Identificated and quantificated coumarins, psoralem, and phenolics composition

(mg/L) of several commercial Limoncello samples (Versari et al., 2003).

Table 1. Amount (mg/L) of major representative volatile components in several lemon samples-derivatives in oil, homemade Limoncello, and commercial Limoncello products

**Compound Commercial Limoncello** 

104.670 143.9 9.5 11.9 10.3 47.8 281.9 31.5 30.2 34.0 12.3 10.1 13.8 21.6 25.1 8.9 10.9 15.9

**#01 #02 #03 #04 #05 #06 #07 #08 #09 #10 #11 #12** 

**Commercial Limoncello #01 #02 #03 #04 #05 #06 #07 #08 #09 #10 #11 #12 #13 #14 #15 #16** 

**Compounds** 


(Crupi et al., 2007).

5-geranyloxy-7-

5-isopentenyloxy-7-

**Oil** 

**Home -made Limon -cello**

Ultraviolet-visible (UV/Vis) detection was carried out at 315-330 nm for coumarins and furanocoumarins and at 280 nm for phenolic compounds.

The analyses, due to the samples matrices complexity, are carried out in gradient elution mode. In particular for the analyses of coumarins and furanocoumarins was used a mobile Phase constituted by hexane: ethyl acetate (92:8 or 88:12, v/v) and hexane: ethyl alcohol (90:10, v/v).

Only recently Crupi and coworkers (Crupi et al., 2007) developed a unique reversed phase method for simultaneous determination at 315 nm of coumarins and furanocoumarins using water and acetonitrile as mobile phase in gradient elution mode.

#### **3. Limoncello chemical composition**

It is a matter of fact that the evaluation of the organoleptic properties of limoncello is, although indirectly, connected to the analysis of the essential oil composition. The aroma of the liquor is actually one of the first consumer's perceptions that are crucial in establishing the preference among several products available in the market. For this reason, the analysis of the aromatic fraction of limoncello liquor seems to be an important item in assessing its genuineness and quality, besides ''tracing" the various steps of the preparation procedure.

The current chemical composition of this Italian liqueur is mainly related to the aromatic fraction that was reported by Crupi and coworkers (Crupi et al., 2007) using previously mentioned analytical techniques.

In their comprehensive studies, after SPME extraction of volatile components and chromatographic analyses, both GC and HPLC, they detected several monoterpenes, sesquiterpenes, and oxygenated compounds, as reported in the Table 1.

Versari and coworkers reported similar analyses of several commercial Limoncello samples and chemometric elaboration with Principal Components Analysis (PCA) technique to obtain two main group: the first that showed a composition similar to lemon essential oils (high content of β–pinene, myrcene, *trans*-α-bergamotene, and β-bisabolene, and a low content in neral and geranial) (Versari et al., 2003). The composition of the second group suggested the presence of oxidative phenomena and (or) addition of flavours, in particular the presence of compounds as ethyl acetate, acetaldehyde, 2-methyl-1-propanol and glycerol indicate that fermentation process probably occurred in the sugar syrup during Limoncello dilution step after the extraction process.

The best recurrent molecules of the oil and of Limoncello (both homemade and commercial ones) belonging the volatile compounds family are -pinene, sabinene, -pinene, myrcene, *p*-cymene, limonene, -terpinene, neral, geraniol, and geranial, and as reported in the Table 1. As results of their investigation, Crupi and coworkers (Crupi et al., 2007) underline that most of analysed Limoncello samples were effectively obtained directly from fruits, but also using terpeneless oils and in some cases synthetic products of reconstituted oils.

From data reported in the Tables 1 and 2 is possible to indicate that the commercial #05 and #02 are of high quality, even if commercial #02, due to high level of *p*-cymene can indicate a long storage time.

Ultraviolet-visible (UV/Vis) detection was carried out at 315-330 nm for coumarins and

The analyses, due to the samples matrices complexity, are carried out in gradient elution mode. In particular for the analyses of coumarins and furanocoumarins was used a mobile Phase constituted by hexane: ethyl acetate (92:8 or 88:12, v/v) and hexane: ethyl alcohol

Only recently Crupi and coworkers (Crupi et al., 2007) developed a unique reversed phase method for simultaneous determination at 315 nm of coumarins and furanocoumarins using

It is a matter of fact that the evaluation of the organoleptic properties of limoncello is, although indirectly, connected to the analysis of the essential oil composition. The aroma of the liquor is actually one of the first consumer's perceptions that are crucial in establishing the preference among several products available in the market. For this reason, the analysis of the aromatic fraction of limoncello liquor seems to be an important item in assessing its genuineness and quality, besides ''tracing" the various steps of the preparation procedure. The current chemical composition of this Italian liqueur is mainly related to the aromatic fraction that was reported by Crupi and coworkers (Crupi et al., 2007) using previously

In their comprehensive studies, after SPME extraction of volatile components and chromatographic analyses, both GC and HPLC, they detected several monoterpenes,

Versari and coworkers reported similar analyses of several commercial Limoncello samples and chemometric elaboration with Principal Components Analysis (PCA) technique to obtain two main group: the first that showed a composition similar to lemon essential oils (high content of β–pinene, myrcene, *trans*-α-bergamotene, and β-bisabolene, and a low content in neral and geranial) (Versari et al., 2003). The composition of the second group suggested the presence of oxidative phenomena and (or) addition of flavours, in particular the presence of compounds as ethyl acetate, acetaldehyde, 2-methyl-1-propanol and glycerol indicate that fermentation process probably occurred in the sugar syrup during Limoncello

The best recurrent molecules of the oil and of Limoncello (both homemade and commercial ones) belonging the volatile compounds family are -pinene, sabinene, -pinene, myrcene, *p*-cymene, limonene, -terpinene, neral, geraniol, and geranial, and as reported in the Table 1. As results of their investigation, Crupi and coworkers (Crupi et al., 2007) underline that most of analysed Limoncello samples were effectively obtained directly from fruits, but also

From data reported in the Tables 1 and 2 is possible to indicate that the commercial #05 and #02 are of high quality, even if commercial #02, due to high level of *p*-cymene can indicate a

using terpeneless oils and in some cases synthetic products of reconstituted oils.

sesquiterpenes, and oxygenated compounds, as reported in the Table 1.

furanocoumarins and at 280 nm for phenolic compounds.

water and acetonitrile as mobile phase in gradient elution mode.

**3. Limoncello chemical composition**

mentioned analytical techniques.

dilution step after the extraction process.

long storage time.

(90:10, v/v).


Table 1. Amount (mg/L) of major representative volatile components in several lemon samples-derivatives in oil, homemade Limoncello, and commercial Limoncello products (Crupi et al., 2007).


Table 2. Identificated and quantificated coumarins, psoralem, and phenolics composition (mg/L) of several commercial Limoncello samples (Versari et al., 2003).

Analytical Methods and Phytochemistry of the Typical Italian Liquor "Limoncello": A Review 101

the main present compounds are terpenes, hydrocarbons, esters, aldehydes, and ketones. The key difference between essential oils from different raw plant varieties is especially related to the composition of the volatile fraction (Steuer et al., 2001). *Citrus* essential oil is generally used as aromatising agent and additive for food and food-derived products, beverages, cosmetics and in some pharmaceuticals. In this sample extract is possible to incur into the presence of organic and inorganic contaminants, as well documented by the presence of pesticide residues (Saitta et al., 2000, Dugo et al., 1997; Verzera et al., 2004) and plasticizers (Di Bella et al., 1999, 2000, 2001; Saitta et al., 1997); however, there is a needs of available data regarding the presence of heavy metals in this products. Some reports concerning the microelements composition of *Citrus* peel extracts were published (Gorinstein et al., 2001; Simpkins et al., 2000). Metals, such as iron, copper, zinc, and cobalt, are non-toxic at modest concentrations, while cadmium, lead, mercury, and arsenic are toxic even in very low concentration level and constitute a significant health hazard (Rojas et al.,

Metals levels in *citrus* essential oils mainly depend on the type of soil and treatment, but are also affected by the extraction procedures to manufacture food-derived products, such as scraping or pressing because the fruits inevitably come in contact with metallic

In this field the best instrumental analysis concerns especially the use of electrochemical techniques, such as derivative potentiometric stripping analysis (dPSA) and Atomic Absorption Spectroscopic methods (AAS) in Graphite Furnace Atomic Absorption Spectroscopy Analysis (GFAAS) configuration to determine trace metals concentrations in a variety of matrices, such as alloys, food, biological materials, and environmental

Recently La Pera and coworkers (La Pera et al., 2003) published an interesting work inherent the use of these two techniques for the simultaneous determination of cadmium, copper, lead, and zinc in *Citrus* essential oils with high recoveries values from real

In the Table 3 were reported the mean concentration determined by dPSA and AAS both on

 **Cd Cu Sample Treatment dPSA AAS dPSA AAS**  Lemon Acid extracts 1.57±0.03 1.43±0.03 16.94±0.21 16.00±0.20 Lemon Methanol extracts 1.63±0.03 1.55±0.05 21.65±1.59 18.10±2.65

 **Pb Zn Sample dPSA AAS dPSA AAS**  Lemon Acid extracts 111.24±0.81 103.60±1.10 802.55±2.48 799.60±3.00 Lemon Methanol extracts 113.24±0.72 103.30±7.15 809.62±2.26 788.99±9.10

by dPSA and AAS in Lemon samples (La Pera et al., 2003).

Table 3. Heavy metal mean concentration (ng/g; n=3) and standard deviation (n=3) detected

1999).

surfaces.

samples.

samples extracts.

acid and methanol extracts.

Terpenes are photosensitive compounds; in particular limonene and terpinenes are involved in this irreversible process that negatively affects the organoleptic properties of the beverage. The terpenes fraction and/or the presence of oxidized by-products can predict the origin and the quality of a Limoncello.

Versari and coworkers (Versari et al., 2003) reported that other mainly present compounds, analysed by HPLC-DAD, were phenolics, coumarins, and psoralem derivatives. These compounds are mainly flavones, coumarins, and furanocoumarins, as represented in Figure 1.

Fig. 1. Chemical structures of flavones, coumarins, and furanocoumarins identified and quantified in Limoncello by Versari and co-worker (Versari et al., 2003).

Problems connected to storage time were reported also by Da Costa and Theodore (Da Costa and Theodore, 2010) in a paper that report a complete study of over two-hundred volatile components in Limoncello and their changes that occurred with aging.

**R1 -Geranyloxy chain R2 -H Bergamottin**

Another well-recognized question investigated by Poiana and coworkers (Poiana et al., 2006) regarded the differentiation from sample-to-sample about chemical and physicalchemical parameters of alcoholic extracts of lemons deriving from the Amalfi and Sorrento areas. These discrepancies are probably correlated with the different cultivars and growing conditions.

In particular this research underlined the evolution during the season for some classes of components. The carbonyl-to-oxygenated, alcohols-to-oxygenated and esters-to-oxygenated compounds ratios are indices of flavouring quality. These showed similar trends for both lemon types. In comparison to other lemon productions a higher amount of minor classes of components were also recorded.

#### **4.** *Citrus* **essential oil heavy metal composition – food quality**

*Citrus* essential oil is a very complex mixture of several classes of volatile (85-98%) and nonvolatile (2-15%) compounds, as reported in the previous paragraphs. In this classification,

**O O**

**O**

**R2**

Terpenes are photosensitive compounds; in particular limonene and terpinenes are involved in this irreversible process that negatively affects the organoleptic properties of the beverage. The terpenes fraction and/or the presence of oxidized by-products can predict the

Versari and coworkers (Versari et al., 2003) reported that other mainly present compounds, analysed by HPLC-DAD, were phenolics, coumarins, and psoralem derivatives. These compounds are mainly flavones, coumarins, and furanocoumarins, as represented in Figure 1.

> **R1 -H R2 -Rutinose Eriocitrin R1 -H R2 -Neohesperidose Neoriocitrin R1 -CH3 R2 -Rutinose Hesperidin R1 -CH3 R2 -Neohesperidose Neohesperidin R1 -H R2 -Rutinose Narirutin R1 -H R2 -Neohesperidose Naringin R1 -CH3 R2 -Rutinose Diosmin**

**R1 -OCH3 R2 -OCH3 Citroptene**

**R1 -H R2 -Isopentenyloxy chain Imperatorin R1 -Geranyloxy chain R2 -H Bergamottin**

Fig. 1. Chemical structures of flavones, coumarins, and furanocoumarins identified and

Problems connected to storage time were reported also by Da Costa and Theodore (Da Costa and Theodore, 2010) in a paper that report a complete study of over two-hundred

Another well-recognized question investigated by Poiana and coworkers (Poiana et al., 2006) regarded the differentiation from sample-to-sample about chemical and physicalchemical parameters of alcoholic extracts of lemons deriving from the Amalfi and Sorrento areas. These discrepancies are probably correlated with the different cultivars and growing

In particular this research underlined the evolution during the season for some classes of components. The carbonyl-to-oxygenated, alcohols-to-oxygenated and esters-to-oxygenated compounds ratios are indices of flavouring quality. These showed similar trends for both lemon types. In comparison to other lemon productions a higher amount of minor classes of

*Citrus* essential oil is a very complex mixture of several classes of volatile (85-98%) and nonvolatile (2-15%) compounds, as reported in the previous paragraphs. In this classification,

quantified in Limoncello by Versari and co-worker (Versari et al., 2003).

**4.** *Citrus* **essential oil heavy metal composition – food quality**

volatile components in Limoncello and their changes that occurred with aging.

**R1 -Geranyloxy chain R2 -OCH3 5-geranyloxy-7-methoxycoumarine R1 -Isopentenyloxy chain R2 -OCH3 5-isopentenyloxy-7-methoxycoumarine**

origin and the quality of a Limoncello.

**OH**

**O**

**O**

components were also recorded.

**OR1**

**R2O O**

**R2 O**

**O O**

**R2**

conditions.

**R1**

**R1**

**OH O**

the main present compounds are terpenes, hydrocarbons, esters, aldehydes, and ketones. The key difference between essential oils from different raw plant varieties is especially related to the composition of the volatile fraction (Steuer et al., 2001). *Citrus* essential oil is generally used as aromatising agent and additive for food and food-derived products, beverages, cosmetics and in some pharmaceuticals. In this sample extract is possible to incur into the presence of organic and inorganic contaminants, as well documented by the presence of pesticide residues (Saitta et al., 2000, Dugo et al., 1997; Verzera et al., 2004) and plasticizers (Di Bella et al., 1999, 2000, 2001; Saitta et al., 1997); however, there is a needs of available data regarding the presence of heavy metals in this products. Some reports concerning the microelements composition of *Citrus* peel extracts were published (Gorinstein et al., 2001; Simpkins et al., 2000). Metals, such as iron, copper, zinc, and cobalt, are non-toxic at modest concentrations, while cadmium, lead, mercury, and arsenic are toxic even in very low concentration level and constitute a significant health hazard (Rojas et al., 1999).

Metals levels in *citrus* essential oils mainly depend on the type of soil and treatment, but are also affected by the extraction procedures to manufacture food-derived products, such as scraping or pressing because the fruits inevitably come in contact with metallic surfaces.

In this field the best instrumental analysis concerns especially the use of electrochemical techniques, such as derivative potentiometric stripping analysis (dPSA) and Atomic Absorption Spectroscopic methods (AAS) in Graphite Furnace Atomic Absorption Spectroscopy Analysis (GFAAS) configuration to determine trace metals concentrations in a variety of matrices, such as alloys, food, biological materials, and environmental samples.

Recently La Pera and coworkers (La Pera et al., 2003) published an interesting work inherent the use of these two techniques for the simultaneous determination of cadmium, copper, lead, and zinc in *Citrus* essential oils with high recoveries values from real samples extracts.


In the Table 3 were reported the mean concentration determined by dPSA and AAS both on acid and methanol extracts.

Table 3. Heavy metal mean concentration (ng/g; n=3) and standard deviation (n=3) detected by dPSA and AAS in Lemon samples (La Pera et al., 2003).

Analytical Methods and Phytochemistry of the Typical Italian Liquor "Limoncello": A Review 103

options such as cryo-trapping and to the enhancement of MS and MS/MS detection in both conventional gas and high performance liquid chromatography, and multi-dimensional gas-

Bonomi, M.; Lubian, E.; Puleo, L.; Tateo, F. & Fasan, S. (2001). Criterio di caratterizzazione

Cotroneo, A.; Dugo, G.; Licandro, G.; Ragonese, C. & Di Giacomo, G. (1986). On the

Cotroneo, A.; Verzera, A.; Lamonica, G.; Dugo, G. & Licandro, G. (1986). On the genuineness

European Union, Council Regulation (EEC) No. 1576/89 (29 May 1989). Appellations of

Crupi, M.L.; Costa, R.; Dugo, P.; Dugo, G. & Mondello, L. (2007). A comprehensive

Da Costa, N.C. & Anastasiou, T.J. (2010) Analysis of volatiles in limoncello liqueur and

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chromatography (MDGC) applications.

**6. References** 

0390-0541.

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28491-2, London and New York.

0021-8561.

These results indicates that *Citrus* lemon essential oils contains several heavy metals and that a deep control on food-derived products is required to obviate to any significant health hazard.

In particular, Verzera and coworkers (Verzera et al., 2004) showed that there are great differences between lemon biological oils and traditional ones inherent the organophosphorus pesticide content.

In particular these Authors reports that Parathion methyl, Parathion ethyl, Quinalphos, Methidathion, Clorpyphos methyl, and Azinphos ethyl are the must representative organophoshorus pesticides founded in lemon oils samples, both in biological and traditional agricultural methods.

Traditional oils were found to contain from 3.52 to 3.85 ppm of previously cited compounds, while biological ones were found to contain from 0.17 to 0.74 ppm.

Only in lemon oils obtained from fruits deriving from traditional agricultural methods were found Dicofol as organochlorine pesticides at 1.0 ppm level.

Previously reported values inherent organophoshorus pesticides were obtained by gas chromatography coupled with Flame Photometric Detector (FPD) that is similar to FID in that the sample exits the analytical column into a hydrogen diffusion flame. Where the FID measures ions produced by organic compounds during combustion, the FPD analyzes the spectrum of light emitted by the compounds as they luminesce in the flame.

Organochlorine pesticides were also obtained by gas chromatography coupled with Electron Capture Detector (ECD). The ECD measures electron-capturing compounds (frequently halogenated compounds) by creating an electrical field in which molecules exiting a GC column can be detected by the drop in current in the field.

#### **5. Conclusion**

A deep knowledge of all the chemical aspects of a Limoncello, in this contest, could greatly help with assessing its authenticity and genuineness. The analysis of food products may be directed to the assessment of food quality and authenticity, the control of a technological and production process, the determination of nutritional values, and the detection of molecules and secondary metabolite eventually present in food-derived products with a possible advantageous effect on human health and safety.

Consequently, a main aim in food chemistry regards especially the continuous improvement, development, and in particular, validation of increasingly sensitive and selective analytical techniques.

The availability of hyphenated analytical methods and 2D chromatographic methods capable of revealing the origin, the authenticity and the quality of a Limoncello may encourage the producers to prepare high quality products, appreciated by the consumer not because of the massive advertisement, but for the characteristics of their composition.

Further investigations must be devoted especially to the improvement of the instrumental performance and high-throughputs analyses, to the implementation of supplementary options such as cryo-trapping and to the enhancement of MS and MS/MS detection in both conventional gas and high performance liquid chromatography, and multi-dimensional gaschromatography (MDGC) applications.

#### **6. References**

102 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

These results indicates that *Citrus* lemon essential oils contains several heavy metals and that a deep control on food-derived products is required to obviate to any significant health

In particular, Verzera and coworkers (Verzera et al., 2004) showed that there are great differences between lemon biological oils and traditional ones inherent the

In particular these Authors reports that Parathion methyl, Parathion ethyl, Quinalphos, Methidathion, Clorpyphos methyl, and Azinphos ethyl are the must representative organophoshorus pesticides founded in lemon oils samples, both in biological and

Traditional oils were found to contain from 3.52 to 3.85 ppm of previously cited compounds,

Only in lemon oils obtained from fruits deriving from traditional agricultural methods were

Previously reported values inherent organophoshorus pesticides were obtained by gas chromatography coupled with Flame Photometric Detector (FPD) that is similar to FID in that the sample exits the analytical column into a hydrogen diffusion flame. Where the FID measures ions produced by organic compounds during combustion, the FPD analyzes the

Organochlorine pesticides were also obtained by gas chromatography coupled with Electron Capture Detector (ECD). The ECD measures electron-capturing compounds (frequently halogenated compounds) by creating an electrical field in which molecules

A deep knowledge of all the chemical aspects of a Limoncello, in this contest, could greatly help with assessing its authenticity and genuineness. The analysis of food products may be directed to the assessment of food quality and authenticity, the control of a technological and production process, the determination of nutritional values, and the detection of molecules and secondary metabolite eventually present in food-derived products with a

Consequently, a main aim in food chemistry regards especially the continuous improvement, development, and in particular, validation of increasingly sensitive and

The availability of hyphenated analytical methods and 2D chromatographic methods capable of revealing the origin, the authenticity and the quality of a Limoncello may encourage the producers to prepare high quality products, appreciated by the consumer not because of the massive advertisement, but for the characteristics of their composition.

Further investigations must be devoted especially to the improvement of the instrumental performance and high-throughputs analyses, to the implementation of supplementary

while biological ones were found to contain from 0.17 to 0.74 ppm.

spectrum of light emitted by the compounds as they luminesce in the flame.

exiting a GC column can be detected by the drop in current in the field.

possible advantageous effect on human health and safety.

found Dicofol as organochlorine pesticides at 1.0 ppm level.

hazard.

organophosphorus pesticide content.

traditional agricultural methods.

**5. Conclusion**

selective analytical techniques.


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**5** 

*Spain* 

*University of Lleida,* 

**The Effects of Non-Thermal** 

**Technologies on Phytochemicals** 

Gemma Oms-Oliu, Isabel Odriozola-Serrano and Olga Martín-Belloso

Phytochemicals are non-nutritive plant chemicals that possess protective roles in the human body, against disease. These phytochemicals are considered to be biologically active secondary metabolites that also provide color and flavor, and are commonly referred to as nutraceuticals (Kalt, 2001). There are thousands of known phytochemicals, which have been found to be derived mainly from phenylalanine and tyrosine, and which perform a variety of functions such as pigmentation, antioxidation, protection against UV light, etc. (Shahidi & Naczk, 2004). Evidences of the benefits to human-health associated with the consumption of plant-derived phytochemicals have caused an increase in the demand for fresh-like fruits and vegetables., where are present in different forms as alkaloids (eg., caffeine and threbromine), carotenoids (e.g. lycopene), flavonoids (e.g., flavon-3-ols), isoflavones (e.g. genistein), phenolic acids (e.g., capsaicin, gallic acid and tannic acid), etc., depending on

The health-promoting effects of many phytochemicals are attributed mainly to their antioxidant activity, although there could also be other modes of action. Fruit and vegetables are known to contain significant amounts of phytochemicals with free-radical and nonradical scavenging capacity towards reactive oxygen species. These deleterious substances have been identified as toxic against cell tissues, thus causing oxidative damage to proteins, membrane lipids and DNA, inhibiting enzymatic pathways, and inducing gene mutation. It is believed that these processes are underpinning several chronic human diseases such as diabetes, certain forms of cancer as well as some cardiovascular and

Changes in the concentration of phytochemicals through processing and storage can greatly compromise the quality, and eventually the acceptance of a food. Despite most of these compounds, to some extent, may be affected by abusive temperatures, thermal processing remain the most commonly used technology for inactivating microorganisms and enzymes in processed food. However, during thermal processing, in addition to the inactivation of microorganism, sensory and nutritional compounds of plant-based foods are negative affected. The consumers demand towards products that keep their original nutritious values and, in this context, non-thermal technologies are preservation treatments that are effective at mild temperatures (up to 40 ºC), thereby minimising negative thermal effects on nutritional and quality of products. These non-thermal processes ensure microbial and

degenerative diseases (Eberhardt et al., 2000; Arab & Steck, 2000).

**1. Introduction**

plant species.

Verzera, A.; Trozzi, A.; Dugo, G.; Di Bella, G. & Cotroneo, A. (2004) Biological lemon and sweet orange essential oil composition. *Flavour And Fragrance Journal*, Vol. 19, No. 6 (November 2004), pp. 544–548, ISSN: 1099-1026.

### **The Effects of Non-Thermal Technologies on Phytochemicals**

Gemma Oms-Oliu, Isabel Odriozola-Serrano and Olga Martín-Belloso *University of Lleida, Spain* 

#### **1. Introduction**

106 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Verzera, A.; Trozzi, A.; Dugo, G.; Di Bella, G. & Cotroneo, A. (2004) Biological lemon and

(November 2004), pp. 544–548, ISSN: 1099-1026.

sweet orange essential oil composition. *Flavour And Fragrance Journal*, Vol. 19, No. 6

Phytochemicals are non-nutritive plant chemicals that possess protective roles in the human body, against disease. These phytochemicals are considered to be biologically active secondary metabolites that also provide color and flavor, and are commonly referred to as nutraceuticals (Kalt, 2001). There are thousands of known phytochemicals, which have been found to be derived mainly from phenylalanine and tyrosine, and which perform a variety of functions such as pigmentation, antioxidation, protection against UV light, etc. (Shahidi & Naczk, 2004). Evidences of the benefits to human-health associated with the consumption of plant-derived phytochemicals have caused an increase in the demand for fresh-like fruits and vegetables., where are present in different forms as alkaloids (eg., caffeine and threbromine), carotenoids (e.g. lycopene), flavonoids (e.g., flavon-3-ols), isoflavones (e.g. genistein), phenolic acids (e.g., capsaicin, gallic acid and tannic acid), etc., depending on plant species.

The health-promoting effects of many phytochemicals are attributed mainly to their antioxidant activity, although there could also be other modes of action. Fruit and vegetables are known to contain significant amounts of phytochemicals with free-radical and nonradical scavenging capacity towards reactive oxygen species. These deleterious substances have been identified as toxic against cell tissues, thus causing oxidative damage to proteins, membrane lipids and DNA, inhibiting enzymatic pathways, and inducing gene mutation. It is believed that these processes are underpinning several chronic human diseases such as diabetes, certain forms of cancer as well as some cardiovascular and degenerative diseases (Eberhardt et al., 2000; Arab & Steck, 2000).

Changes in the concentration of phytochemicals through processing and storage can greatly compromise the quality, and eventually the acceptance of a food. Despite most of these compounds, to some extent, may be affected by abusive temperatures, thermal processing remain the most commonly used technology for inactivating microorganisms and enzymes in processed food. However, during thermal processing, in addition to the inactivation of microorganism, sensory and nutritional compounds of plant-based foods are negative affected. The consumers demand towards products that keep their original nutritious values and, in this context, non-thermal technologies are preservation treatments that are effective at mild temperatures (up to 40 ºC), thereby minimising negative thermal effects on nutritional and quality of products. These non-thermal processes ensure microbial and

The Effects of Non-Thermal Technologies on Phytochemicals 109

number of pulses, but also by intrinsic factors of the treated food product such as food composition and the physiological states of microorganisms (Knorr, 2001; Smelt et al., 2002). Effects of pressure and temperature on food constituents are governed by activation volume and activation energy. Differences in sensitivity of reactions towards pressure (activation volume) and temperature (activation energy) lead to the possibility of retaining or even diminishing some desired natural food quality attributes such as vitamins, pigments and flavour or modifying the structure of food system and food functionality, while optimizing the microbial food safety or minimizing the undesired food quality related enzymes (Barbosa-

Exposure to radiation, either ionizing or non ionizing, is being regarded as one of the non thermal methods for food preservation with the best potential, especially for the decontamination of raw material for food production. The ionizing radiation source could be high-energy electrons, X-rays (machine generated), or gamma rays (from Cobalt-60 or cesium- 137), while the non-ionizing radiation is electromagnetic radiation that does not carry enough energy/quanta to ionize atoms or molecules, represented mainly by ultraviolet rays (UV-A, UV-B, and UV-C), visible light, microwaves, and infrared. Decontamination though ionizing radiation consists of the application of doses of 2-7 kGy. It has been proven that radiation can safely and effectively eliminate pathogenic nonsporeforming bacteria in foods (Alothman et al., 2009). However, radiation is not being widely used because of some misconceptions by consumers about its role in causing cancer. Radiation can influence the levels of phytochemicals and the capacity of a specific plant to produce them at different levels. Radiation treatments have been shown to either increase or decrease the antioxidant content of fresh plant produce, which is dependent on the dose delivered (usually low and medium doses have insignificant effects on antioxidants), exposure time, and the raw material used. The enhanced antioxidant capacity/activity of a plant after radiation is mainly attributed either to increased enzyme activity (e.g., phenylalanine ammonia-lyase (PAL) and peroxidase activity) or to the increased

Although ultrasound is unlike to become a commercial technology on its own, it can be applied in combination with other technologies with preservation purposes. Its lethal microbial effects have been related to cavitation, a phenomenon that generates high temperatures and pressures at a microscopic level that are responsible for the formation of highly reactive free radicals and for the mechanical damage of microorganisms (Raso et al., 1998). Ultrasound processing of juices is reported to have minimal effect on the degradation of key quality parameters such as colour and ascorbic acid in orange juice during storage at 10 ºC (Tiwari et al., 2009a). This positive effect of ultrasound is assumed to be due to the

Vitamin C has received much attention when aiming at evaluating the effect of non thermal processing technologies on the phytochemicals. Vitamin C retention in PEF-treated juices depended on processing factors and thus, the lower the electric field strength, the treatment time, the pulse frequency or the pulse width, the higher the vitamin C retention in tomato juices (Odriozola-Serrano et al., 2007). According to these authors, maximal relative vitamin

Cánovas et al., 1997; Messens et al., 1997; Hendrickx et al., 1998).

extractability from the tissues (Alothman et al., 2009).

effective removal of occluded oxygen from the juice (Knorr et al., 2004).

**2.2 Plant-based foods preserved by non-thermal technologies** 

**2.2.1 Tomato** 

enzyme inactivation with reduced effects on nutritional and quality parameters. Nonthermal technologies as, pulsed electric fields (PEF), high pressure (HP), irradiation or ultrasounds have been studied and developed during the last decades with the final aim of implementing them at an industrial level. The impact of non thermal technologies on microorganisms, enzymes and quality-related parameters has been extensively reviewed. However, many efforts in the last years have been made to evaluate the impact of PEF, HP, irradiation and ultrasounds on the stability of phytochemicals. The present review aims at reviewing the effects of non thermal technologies on health-related compounds in plant based products.

#### **2. Non-thermal technologies to preserve phytochemicals**

#### **2.1 Non-thermal processing basics**

Non-thermal technologies may allow obtaining safe and shelf-stable plant-based products with minor changes or increased content in phytochemicals. Most differences between nonthermal and thermal treatments can be explained through the temperatures reached through processing. In general, temperature during processing and storage are important factors affecting the phytochemical content of the processed product. PEF processing involves the application of a high intensity electric field (20–80 kV/cm) in the form of short pulses to a food placed between two electrodes. PEF technology provides fresh-like and safe foods while reducing quality losses that can be triggered after thermal processing (Morris et al., 2007). Application of continuous PEF processing is not suitable for solid food products that do not allow pumping and is restricted to low-conductive food products without air bubbles. Liquid food products are susceptible to be treated by PEF because they are primarily composed by water and nutrients, which are electrical conductors due of the presence of large concentrations of salts and dipolar molecules. Hence, PEF technology has been suggested for the pasteurization of foods such as juices, milk, yogurt, soups, and liquid eggs (Vega-Mercado et al., 1997; Evrendilek et al., 2004; Elez-Martínez et al., 2005; Monfort et al., 2010). In general, a continuous PEF treatment system is composed of treatment chambers, a pulse generator, a fluid-handling system, and monitoring systems (Elez-Martínez et al., 2006). Temperature- and pulse-monitoring systems are used to supervise the process. The effectiveness of PEF technology not only depends on the type of equipment used but also on the treatment parameters and media to be processed (Barbosa-Cánovas et al., 1999). In addition, processing parameters such as electric field strength, total treatment time, pulse shape, pulse frequency, pulse width, polarity and temperature are involved in the degradation or generation of phytochemicals.

HP processing uses water as a medium to transmit pressures from 300 to 700 MPa to foods resulting in a reduction on microbial loads and thus extending shelf life (Patras et al., 2009). Pressure is nearly transmitted uniformly throughout the food, thus leading to very different applications in many different food products. Although pressure results are uniform, the HP technique cannot completely avoid the well-known classical limitation of heat transfer especially during pressure build up and decompression. An increase or a decrease of pressure is associated with a proportional temperature (T) change of the vessel contents, respectively, due to adiabatic heating or cooling temperature gradient. The effectiveness of HP treatment on the overall food quality and safety is not only influenced by extrinsic (process) factors such as treatment time, pressurisation/decompression rate, pressure/ temperature levels and the

enzyme inactivation with reduced effects on nutritional and quality parameters. Nonthermal technologies as, pulsed electric fields (PEF), high pressure (HP), irradiation or ultrasounds have been studied and developed during the last decades with the final aim of implementing them at an industrial level. The impact of non thermal technologies on microorganisms, enzymes and quality-related parameters has been extensively reviewed. However, many efforts in the last years have been made to evaluate the impact of PEF, HP, irradiation and ultrasounds on the stability of phytochemicals. The present review aims at reviewing the effects of non thermal technologies on health-related compounds in plant

Non-thermal technologies may allow obtaining safe and shelf-stable plant-based products with minor changes or increased content in phytochemicals. Most differences between nonthermal and thermal treatments can be explained through the temperatures reached through processing. In general, temperature during processing and storage are important factors affecting the phytochemical content of the processed product. PEF processing involves the application of a high intensity electric field (20–80 kV/cm) in the form of short pulses to a food placed between two electrodes. PEF technology provides fresh-like and safe foods while reducing quality losses that can be triggered after thermal processing (Morris et al., 2007). Application of continuous PEF processing is not suitable for solid food products that do not allow pumping and is restricted to low-conductive food products without air bubbles. Liquid food products are susceptible to be treated by PEF because they are primarily composed by water and nutrients, which are electrical conductors due of the presence of large concentrations of salts and dipolar molecules. Hence, PEF technology has been suggested for the pasteurization of foods such as juices, milk, yogurt, soups, and liquid eggs (Vega-Mercado et al., 1997; Evrendilek et al., 2004; Elez-Martínez et al., 2005; Monfort et al., 2010). In general, a continuous PEF treatment system is composed of treatment chambers, a pulse generator, a fluid-handling system, and monitoring systems (Elez-Martínez et al., 2006). Temperature- and pulse-monitoring systems are used to supervise the process. The effectiveness of PEF technology not only depends on the type of equipment used but also on the treatment parameters and media to be processed (Barbosa-Cánovas et al., 1999). In addition, processing parameters such as electric field strength, total treatment time, pulse shape, pulse frequency, pulse width, polarity and temperature are involved in

HP processing uses water as a medium to transmit pressures from 300 to 700 MPa to foods resulting in a reduction on microbial loads and thus extending shelf life (Patras et al., 2009). Pressure is nearly transmitted uniformly throughout the food, thus leading to very different applications in many different food products. Although pressure results are uniform, the HP technique cannot completely avoid the well-known classical limitation of heat transfer especially during pressure build up and decompression. An increase or a decrease of pressure is associated with a proportional temperature (T) change of the vessel contents, respectively, due to adiabatic heating or cooling temperature gradient. The effectiveness of HP treatment on the overall food quality and safety is not only influenced by extrinsic (process) factors such as treatment time, pressurisation/decompression rate, pressure/ temperature levels and the

**2. Non-thermal technologies to preserve phytochemicals** 

based products.

**2.1 Non-thermal processing basics** 

the degradation or generation of phytochemicals.

number of pulses, but also by intrinsic factors of the treated food product such as food composition and the physiological states of microorganisms (Knorr, 2001; Smelt et al., 2002). Effects of pressure and temperature on food constituents are governed by activation volume and activation energy. Differences in sensitivity of reactions towards pressure (activation volume) and temperature (activation energy) lead to the possibility of retaining or even diminishing some desired natural food quality attributes such as vitamins, pigments and flavour or modifying the structure of food system and food functionality, while optimizing the microbial food safety or minimizing the undesired food quality related enzymes (Barbosa-Cánovas et al., 1997; Messens et al., 1997; Hendrickx et al., 1998).

Exposure to radiation, either ionizing or non ionizing, is being regarded as one of the non thermal methods for food preservation with the best potential, especially for the decontamination of raw material for food production. The ionizing radiation source could be high-energy electrons, X-rays (machine generated), or gamma rays (from Cobalt-60 or cesium- 137), while the non-ionizing radiation is electromagnetic radiation that does not carry enough energy/quanta to ionize atoms or molecules, represented mainly by ultraviolet rays (UV-A, UV-B, and UV-C), visible light, microwaves, and infrared. Decontamination though ionizing radiation consists of the application of doses of 2-7 kGy. It has been proven that radiation can safely and effectively eliminate pathogenic nonsporeforming bacteria in foods (Alothman et al., 2009). However, radiation is not being widely used because of some misconceptions by consumers about its role in causing cancer. Radiation can influence the levels of phytochemicals and the capacity of a specific plant to produce them at different levels. Radiation treatments have been shown to either increase or decrease the antioxidant content of fresh plant produce, which is dependent on the dose delivered (usually low and medium doses have insignificant effects on antioxidants), exposure time, and the raw material used. The enhanced antioxidant capacity/activity of a plant after radiation is mainly attributed either to increased enzyme activity (e.g., phenylalanine ammonia-lyase (PAL) and peroxidase activity) or to the increased extractability from the tissues (Alothman et al., 2009).

Although ultrasound is unlike to become a commercial technology on its own, it can be applied in combination with other technologies with preservation purposes. Its lethal microbial effects have been related to cavitation, a phenomenon that generates high temperatures and pressures at a microscopic level that are responsible for the formation of highly reactive free radicals and for the mechanical damage of microorganisms (Raso et al., 1998). Ultrasound processing of juices is reported to have minimal effect on the degradation of key quality parameters such as colour and ascorbic acid in orange juice during storage at 10 ºC (Tiwari et al., 2009a). This positive effect of ultrasound is assumed to be due to the effective removal of occluded oxygen from the juice (Knorr et al., 2004).

#### **2.2 Plant-based foods preserved by non-thermal technologies**

#### **2.2.1 Tomato**

Vitamin C has received much attention when aiming at evaluating the effect of non thermal processing technologies on the phytochemicals. Vitamin C retention in PEF-treated juices depended on processing factors and thus, the lower the electric field strength, the treatment time, the pulse frequency or the pulse width, the higher the vitamin C retention in tomato juices (Odriozola-Serrano et al., 2007). According to these authors, maximal relative vitamin

The Effects of Non-Thermal Technologies on Phytochemicals 111

processed samples. The apparently inconsistent results may be explained through the

Tomato juices have been found to be a rich source of flavonoids, containing as the main flavonols quercetin and kaempferol, and minor phenolic acids such as ferulic, *p*-coumaric, caffeic acid, etc. PEF processing (35 kV/cm for 1500 µs with 4 µs bipolar pulses at 100 Hz) and thermal treatments (90 ºC 30 s and 90 ºC 60 s) did not affect phenolic content of tomato juices. Both PEF- and heat-treated tomato juices undergo a substantial loss of phenolic acids (chlorogenic and ferulic) and flavonols (quercetin and kaempferol) during 56 days of storage at 4ºC. Caffeic acid content was slightly enhanced over time, regardless the kind of processing, whereas PEF and heat treated tomato juices underwent a substantial depletion of *p*-coumaric acid during storage (Table 1). The increase of caffeic acid in tomato juices after 28 days of storage could be directly associated with residual hydroxylase activities, which convert coumaric acid in caffeic acid. Total phenolics in tomato based beverages and tomato purées appeared to be relatively resistant to the effect of HP (Patras et al., 2009; Barba et al., 2010).The effect of ionizing radiation on the phenolic content of tomatoes has also been studied. The gamma-radiation treatment (2, 4, and 6 kGy) markedly reduced the concentration of the phenolic compounds (*p*-hydroxybenzaldehyde, *p*-coumaric acid, ferulic

Phenolic compound Process Phenolic retention (%)

TT

TT

TT

TT

TT

TT

Chlorogenic HIPEF

Ferulic HIPEF

p-coumaric HIPEF

Caffeic HIPEF

Quercetin HIPEF

Kaempferol HIPEF

HIPEF: High intensity pulsed electric fields treatment at 35 kV/cm for 1000 μs; bipolar 4-μs pulses at

Table 1. Phenolic acid and flavonols retention during storage for 56 days at 4 ºC of tomato juices stabilized by heat or HIPEF treatments. Adapted from Odriozola-Serrano et al. (2009)

Ascorbic acid is a heat-sensitive bioactive compound in the presence of oxygen. Thus, high temperatures during processing can greatly affect the rates of its degradation through an aerobic pathway. Storage conditions such as storage temperature or oxygen concentration may have a significant influence on the rates of vitamin C degradation. Vitamin C is usually degraded by oxidative processes which are stimulated in the presence of light, oxygen, heat

after 56 days of storage at 4 ºC

86 79

67 69

53 53

132 118

> 80 64

> 82 75

combined effect of pressure and temperature.

acid, rutin and naringenin) in tomatoes (Schindler et al., 2005).

Phenolic acids

Flavonols

**2.2.2 Orange** 

100 Hz; TT: thermal treatment at 90 ºC for 60 s

C content (90.2%), was attained with PEF treatments of 1 μs pulse duration applied at 250 Hz in bipolar mode at 35 kV/cm for 1000 μs. Regarding the stability of vitamin C through the storage, the concentration of vitamin C decreased over time in both heat (90ºC for 60s) and PEF-treated (35 kV/cm for 1500 µs with 4 µs bipolar pulses at 100 Hz) tomato juices following an exponential trend. In addition, these works demonstrated vitamin C is better retained in PEF treated juices than in those thermally processed after 56 days at 4 ºC. Oxidation of ascorbic acid occurs mainly during the processing of juices and depends upon many factors such as oxygen presence, heat and light. Most differences between PEF and heat treatments can be explained through the temperatures reached through processing. Ascorbic acid is a heat-sensitive bioactive compound in the presence of oxygen. Thus, high temperatures during processing can greatly affect the rates of its degradation through an aerobic pathway (Odriozola-Serrano et al., 2008a). Studies in tomato juice showed that HP processing (300 and 500 MPa) could not preserve vitamin C and the depletion of vitamin C after HP treatment is dependent mainly on temperature intensity and treating time (Hsu et al., 2008). A long exposure (up to 6 h) to extreme pressure/temperature combinations (e.g., 850 MPa combined with temperatures from 65 to 80 ºC) degraded AA to a large extent (Oey et al., 2008). In tomato puree, a 40% and 30% decrease, respectively, in the content of ascorbic acid (AA) and total AA was observed after HP treatment of 400 MPa/25 ºC/15 min (Sánchez-Moreno et al., 2006).

Processing by using non thermal technologies may be advantageous regarding the amount and stability of carotenoids. For instance, lycopene concentrations in PEF-processed tomato juice have been found to be higher than those found in untreated juices (Odriozola-Serrano et al., 2008b). Consistently, Sánchez-Moreno, et al. (2005a) observed that the content of total carotenoids in a tomato-based cold soup 'gazpacho', increased roughly a 62% after applying bipolar 4-μs pulses of 35 kV/cm for 750 μs at 800 Hz. Odriozola-Serrano et al. (2009) have observed that β-carotene in treated tomato juice undergo a significant increase (31%-38%), whereas γ-carotene content is depleted (3%-6%) after a PEF treatment (35 kV/cm for 1500 µs with 4 µs bipolar pulses at 100 Hz). Authors suggested that a plausible explanation for this fact is that γ-carotene may undergo cyclization to form six membered rings at one end of the molecule, giving β-carotene as a product. During storage, PEF-processed tomato juices better maintained the individual carotenoid content (lycopene, neurosporene and carotene) than thermally-treated and untreated juices for 54 days at 4 ºC (Odriozola-Serrano et al., 2009). Individual carotenoids with antioxidant activity (β-carotene, β-cryptoxanthin, zeaxanthin and lutein) appeared to be resistant to a HP treatment of 400 MPa at 40 °C for 1 min, thus resulting into a better preservation of the antioxidant activity of a tomato-based soup with respect to the thermally pasteurized (Sánchez-Moreno et al., 2005b). It seems that high pressure influences the extraction yield of carotenoids. A significant increase in the measured carotenoid content of pressurized (400 MPa/25 ºC/15 min) compared to either thermal treated or untreated tomato purée was observed by Sánchez-Moreno et al. (2006). Due to this potential, HP technology has also been studied to extract lycopene from tomato paste waste (Jun, 2006). In contrast, other studies have not reported major effects of HP treatments on tomato products. García et al. (2001) treated a tomato homogenate for 5 min with 500 and 800 MPa and did not find any influence on the total lycopene and β-carotene concentration. Barba et al. (2010) reported that total carotenoids are particularly affected by HP, having the unprocessed vegetable beverages made with tomato, green pepper, green celery, onion, carrot, lemon and olive oil, a higher total carotenoids content than HP-

C content (90.2%), was attained with PEF treatments of 1 μs pulse duration applied at 250 Hz in bipolar mode at 35 kV/cm for 1000 μs. Regarding the stability of vitamin C through the storage, the concentration of vitamin C decreased over time in both heat (90ºC for 60s) and PEF-treated (35 kV/cm for 1500 µs with 4 µs bipolar pulses at 100 Hz) tomato juices following an exponential trend. In addition, these works demonstrated vitamin C is better retained in PEF treated juices than in those thermally processed after 56 days at 4 ºC. Oxidation of ascorbic acid occurs mainly during the processing of juices and depends upon many factors such as oxygen presence, heat and light. Most differences between PEF and heat treatments can be explained through the temperatures reached through processing. Ascorbic acid is a heat-sensitive bioactive compound in the presence of oxygen. Thus, high temperatures during processing can greatly affect the rates of its degradation through an aerobic pathway (Odriozola-Serrano et al., 2008a). Studies in tomato juice showed that HP processing (300 and 500 MPa) could not preserve vitamin C and the depletion of vitamin C after HP treatment is dependent mainly on temperature intensity and treating time (Hsu et al., 2008). A long exposure (up to 6 h) to extreme pressure/temperature combinations (e.g., 850 MPa combined with temperatures from 65 to 80 ºC) degraded AA to a large extent (Oey et al., 2008). In tomato puree, a 40% and 30% decrease, respectively, in the content of ascorbic acid (AA) and total AA was observed after HP treatment of 400 MPa/25 ºC/15 min

Processing by using non thermal technologies may be advantageous regarding the amount and stability of carotenoids. For instance, lycopene concentrations in PEF-processed tomato juice have been found to be higher than those found in untreated juices (Odriozola-Serrano et al., 2008b). Consistently, Sánchez-Moreno, et al. (2005a) observed that the content of total carotenoids in a tomato-based cold soup 'gazpacho', increased roughly a 62% after applying bipolar 4-μs pulses of 35 kV/cm for 750 μs at 800 Hz. Odriozola-Serrano et al. (2009) have observed that β-carotene in treated tomato juice undergo a significant increase (31%-38%), whereas γ-carotene content is depleted (3%-6%) after a PEF treatment (35 kV/cm for 1500 µs with 4 µs bipolar pulses at 100 Hz). Authors suggested that a plausible explanation for this fact is that γ-carotene may undergo cyclization to form six membered rings at one end of the molecule, giving β-carotene as a product. During storage, PEF-processed tomato juices better maintained the individual carotenoid content (lycopene, neurosporene and carotene) than thermally-treated and untreated juices for 54 days at 4 ºC (Odriozola-Serrano et al., 2009). Individual carotenoids with antioxidant activity (β-carotene, β-cryptoxanthin, zeaxanthin and lutein) appeared to be resistant to a HP treatment of 400 MPa at 40 °C for 1 min, thus resulting into a better preservation of the antioxidant activity of a tomato-based soup with respect to the thermally pasteurized (Sánchez-Moreno et al., 2005b). It seems that high pressure influences the extraction yield of carotenoids. A significant increase in the measured carotenoid content of pressurized (400 MPa/25 ºC/15 min) compared to either thermal treated or untreated tomato purée was observed by Sánchez-Moreno et al. (2006). Due to this potential, HP technology has also been studied to extract lycopene from tomato paste waste (Jun, 2006). In contrast, other studies have not reported major effects of HP treatments on tomato products. García et al. (2001) treated a tomato homogenate for 5 min with 500 and 800 MPa and did not find any influence on the total lycopene and β-carotene concentration. Barba et al. (2010) reported that total carotenoids are particularly affected by HP, having the unprocessed vegetable beverages made with tomato, green pepper, green celery, onion, carrot, lemon and olive oil, a higher total carotenoids content than HP-

(Sánchez-Moreno et al., 2006).

processed samples. The apparently inconsistent results may be explained through the combined effect of pressure and temperature.

Tomato juices have been found to be a rich source of flavonoids, containing as the main flavonols quercetin and kaempferol, and minor phenolic acids such as ferulic, *p*-coumaric, caffeic acid, etc. PEF processing (35 kV/cm for 1500 µs with 4 µs bipolar pulses at 100 Hz) and thermal treatments (90 ºC 30 s and 90 ºC 60 s) did not affect phenolic content of tomato juices. Both PEF- and heat-treated tomato juices undergo a substantial loss of phenolic acids (chlorogenic and ferulic) and flavonols (quercetin and kaempferol) during 56 days of storage at 4ºC. Caffeic acid content was slightly enhanced over time, regardless the kind of processing, whereas PEF and heat treated tomato juices underwent a substantial depletion of *p*-coumaric acid during storage (Table 1). The increase of caffeic acid in tomato juices after 28 days of storage could be directly associated with residual hydroxylase activities, which convert coumaric acid in caffeic acid. Total phenolics in tomato based beverages and tomato purées appeared to be relatively resistant to the effect of HP (Patras et al., 2009; Barba et al., 2010).The effect of ionizing radiation on the phenolic content of tomatoes has also been studied. The gamma-radiation treatment (2, 4, and 6 kGy) markedly reduced the concentration of the phenolic compounds (*p*-hydroxybenzaldehyde, *p*-coumaric acid, ferulic acid, rutin and naringenin) in tomatoes (Schindler et al., 2005).


HIPEF: High intensity pulsed electric fields treatment at 35 kV/cm for 1000 μs; bipolar 4-μs pulses at 100 Hz; TT: thermal treatment at 90 ºC for 60 s

Table 1. Phenolic acid and flavonols retention during storage for 56 days at 4 ºC of tomato juices stabilized by heat or HIPEF treatments. Adapted from Odriozola-Serrano et al. (2009)

#### **2.2.2 Orange**

Ascorbic acid is a heat-sensitive bioactive compound in the presence of oxygen. Thus, high temperatures during processing can greatly affect the rates of its degradation through an aerobic pathway. Storage conditions such as storage temperature or oxygen concentration may have a significant influence on the rates of vitamin C degradation. Vitamin C is usually degraded by oxidative processes which are stimulated in the presence of light, oxygen, heat

The Effects of Non-Thermal Technologies on Phytochemicals 113

storage at 4 ºC. In consequence, vitamin A values showed an increase above 40% the value of the untreated sample. The inactivation of enzymes that caused losses of carotenoids during storage and the improvement of the extraction caused by HP treatments are the

Flavonoids are the most common and widely distributed group of plant phenolics. Among them, flavones, flavonols, flavanols, flavanones, anthocyanins, and isoflavones are particularly common in fruits. In this way, Sánchez-Moreno et al. (2005a) evaluated the effect of a PEF treatment at 35 kV/cm for 750 µs with 4-µs bipolar pulses at 800 Hz on the flavanone content of orange juice. No changes in the total flavanones were observed, nor in the individual flavanone glycosides and their aglycons hesperetin and naringenin. Recent results show that HP processed orange juice (400 MPa, 40 ºC, 1 min) presented a significant increased on the extractability of each individual flavanone with regard untreated juice and hence on total flavanone content whereas mild pasteurization (70 ºC, 30 s) treatments retained similar levels to those found in untreated juices (Plaza et al., 2010). Regarding the main flavanones identified in orange juice, HP treatments (400 MPa/40 ºC/1 min) increased the content of naringenin by 20% and by 40% the content of hesperetin in comparison with an untreated orange juice (Sánchez-Moreno et al., 2005a). The increase in the extractability of flavanones by Plaza et al. (2010) in HP orange juice happened at beginning due to treatment. Thus, during refrigerated storage at 4 ºC, flavanone content in HP juice decreased around 50% during the first 20 days of storage at 4 ºC. The degradation of phenolic compounds during storage has been mainly related to the residual activity of polyphenol

Different studies have proven the effectiveness of PEF in achieving higher vitamin C content in comparison with heat treatments in berries. Odriozola-Serrano et al. (2008a) reported that vitamin C retention just after treatment in heat-processed (90ºC, 60s) strawberry juice was significantly lower (94%) than that found in a juice treated at 35 kV/cm for 1700 µs in bipolar 4 µs pulses at 100 Hz (98%). Low processing temperatures reached through PEFprocessing (T<40 ºC) would explain the higher retention of vitamin C in HIPEF-treated strawberry juice compared to the thermally processed samples. The concentration of vitamin C in thermally and PEF-processed juices decreased gradually with storage time. Although during 21 days of storage the concentration of vitamin C was similar among processed strawberry juices, beyond this day, juices subjected to thermal treatment at 90 ºC for 60 s exhibited lower vitamin C content compared to PEF-treated juices (Odriozola et al., 2008c). Recommended daily intake (RDI) of vitamin C is currently revised but should be never below 60 mg, as established by the U.S. Food and Drug Administration (FDA, 1999). According to this recommendation, a strawberry juice 250 mL serving size should contain 24 mg/100 mL in order to contribute to the 100% of the RDI. Vitamin C content of juice processed with either PEF or heat at 90 ºC for 30 s felt below the RDI at 35 days of storage. The concentration of vitamin C in juices treated at 90 ºC for 60 s was reduced below 24 mg/mL after 28 days of storage at 4 ºC. As compared to fruit based products, a high residual ascorbic acid concentration after HP treatment is mostly found. In berries, a high retention of AA in strawberry nectar was observed after HP treatment at 500 MPa/room temperature/3 min (Rovere et al., 1996). Changes of vitamin C content in pressure treated

reasons exposed by some authors to explain that results (De Ancos et al., 2002).

oxidase (PPO) and peroxidase (POD) (Odriozola-Serrano et al., 2009).

**2.2.3 Berries** 

peroxides and enzymes (especially ascorbate oxidase and peroxidase). Many authors have reported that vitamin C in different fruit and vegetable products is not significantly affected by HP processing (Sánchez-Moreno et al., 2009). Sánchez-Moreno et al. (2006) reported 91% retention of ascorbic acid in orange juice after HP processing at 400 MPa/40 ºC/1min. In addition, HP orange juices (400 MPa/ 40 ºC/ 1 min) maintained better the vitamin C during more days of refrigerated storage than low pasteurized treated juice (70 ºC/30 s) (Plaza et al., 2006). However, differences in vitamin C pressure stability during storage could be explained by the initial oxygen content and possible endogenous pro-oxidative enzyme activity. The effects of ultrasonication on the vitamin C content of orange juice have also been studied. Degradation of vitamin C in sonicated orange juices was observed and the degradation level depended on the wave amplitude and treatment time (Tiwari et al., 2009a). Increased shelf life based on ascorbic acid retention was found for sonicated orange juice compared to thermal processed samples at 98 ºC for 21 s due to higher processing temperature (Tiwari et al., 2009a).

Some studies have demonstrated that carotenoid content is increased significantly after PEF processing compared to the untreated orange juices. Cortés et al. (2006) observed that the carotenoid concentration in orange juice rose slightly after applying intense PEF treatments of 35 and 40 kV/cm for 30-240 µs. Carotenoid concentration rose as treatment time increased when HIPEF treatments of 25 or 30 kV/cm were applied to orange-carrot juice (Torregrosa et al., 2005). It has been reported that thermal treatment may imply an increase in some individual carotenoids owing to greater stability, enzymatic degradation, and unaccounted losses of moisture, which concentrate the sample (Rodriguez-Amaya, 1997). However, carotenoids are highly unsaturated compounds with an extensive conjugated double-bonds system and they are susceptible to oxidation, isomerisation and other chemical changes during processing and storage. Cortés et al. (2006) reported a significant decrease in total carotenoids of orange juice when applying bipolar treatments of 30 kV/cm for 100 µs. This decrease in provitamin A carotenoids could be correlated with a significant decrease in vitamin A by 7.52% in high-intensity PEF-treated orange juice (30 kV/cm, 100 µs) and by 15.62% in a pasteurized orange juice (90ºC, 20 s). Moreover, PEF processing (35 kV/cm, 750 µs) or thermal treatments (70 ºC, 30 s and 90 ºC, 30 s) did not exert any effect on vitamin A content of an orange juice (Sánchez-Moreno et al., 2005a). Research efforts have been made to obtain fruit and vegetable juices by HP processing without the quality and nutritional damage caused by heat treatments (Sánchez-Moreno et al., 2009). Individual carotenoids with antioxidant activity (β-carotene, β-cryptoxanthin, zeaxanthin and lutein) appeared to be resistant to a HP treatment of 400 MPa at 40 °C for 1 min, thus resulting into a better preservation of the antioxidant activity of orange juices with respect to those thermally pasteurized (Sánchez-Moreno et al. 2005a). Interestingly, an orange juice treated at 350 MPa/30ºC/5 min exhibited a higher carotenoid content (α-carotene, 60%; β-carotene, 50%; α-cryptoxanthin, 63%; β-cryptoxanthin, 42%) than a freshly squeezed juice (De Ancos et al., 2002), which was attributed to the desnaturation of the carotenoid-binding protein induced by pressure. Regarding the stability of carotenoids through storage of HP-pasteurized juices, non significant changes have been reported for at least 10 days of refrigerated storage in an orange juice treated at 100MPa/60ºC/5 min, whereas substantial losses were found at the end of the storage period of samples processed at 350MPa/30ºC/2.5 min or 400MPa/40ºC/1 min (20.56% and 9.16%, respectively). Plaza et al. (2010) reported that HP-treated orange juice showed a higher content in carotenoids than heat pasteurized juice during refrigerated storage at 4 ºC. In consequence, vitamin A values showed an increase above 40% the value of the untreated sample. The inactivation of enzymes that caused losses of carotenoids during storage and the improvement of the extraction caused by HP treatments are the reasons exposed by some authors to explain that results (De Ancos et al., 2002).

Flavonoids are the most common and widely distributed group of plant phenolics. Among them, flavones, flavonols, flavanols, flavanones, anthocyanins, and isoflavones are particularly common in fruits. In this way, Sánchez-Moreno et al. (2005a) evaluated the effect of a PEF treatment at 35 kV/cm for 750 µs with 4-µs bipolar pulses at 800 Hz on the flavanone content of orange juice. No changes in the total flavanones were observed, nor in the individual flavanone glycosides and their aglycons hesperetin and naringenin. Recent results show that HP processed orange juice (400 MPa, 40 ºC, 1 min) presented a significant increased on the extractability of each individual flavanone with regard untreated juice and hence on total flavanone content whereas mild pasteurization (70 ºC, 30 s) treatments retained similar levels to those found in untreated juices (Plaza et al., 2010). Regarding the main flavanones identified in orange juice, HP treatments (400 MPa/40 ºC/1 min) increased the content of naringenin by 20% and by 40% the content of hesperetin in comparison with an untreated orange juice (Sánchez-Moreno et al., 2005a). The increase in the extractability of flavanones by Plaza et al. (2010) in HP orange juice happened at beginning due to treatment. Thus, during refrigerated storage at 4 ºC, flavanone content in HP juice decreased around 50% during the first 20 days of storage at 4 ºC. The degradation of phenolic compounds during storage has been mainly related to the residual activity of polyphenol oxidase (PPO) and peroxidase (POD) (Odriozola-Serrano et al., 2009).

#### **2.2.3 Berries**

112 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

peroxides and enzymes (especially ascorbate oxidase and peroxidase). Many authors have reported that vitamin C in different fruit and vegetable products is not significantly affected by HP processing (Sánchez-Moreno et al., 2009). Sánchez-Moreno et al. (2006) reported 91% retention of ascorbic acid in orange juice after HP processing at 400 MPa/40 ºC/1min. In addition, HP orange juices (400 MPa/ 40 ºC/ 1 min) maintained better the vitamin C during more days of refrigerated storage than low pasteurized treated juice (70 ºC/30 s) (Plaza et al., 2006). However, differences in vitamin C pressure stability during storage could be explained by the initial oxygen content and possible endogenous pro-oxidative enzyme activity. The effects of ultrasonication on the vitamin C content of orange juice have also been studied. Degradation of vitamin C in sonicated orange juices was observed and the degradation level depended on the wave amplitude and treatment time (Tiwari et al., 2009a). Increased shelf life based on ascorbic acid retention was found for sonicated orange juice compared to thermal processed samples at 98 ºC for 21 s due to higher processing

Some studies have demonstrated that carotenoid content is increased significantly after PEF processing compared to the untreated orange juices. Cortés et al. (2006) observed that the carotenoid concentration in orange juice rose slightly after applying intense PEF treatments of 35 and 40 kV/cm for 30-240 µs. Carotenoid concentration rose as treatment time increased when HIPEF treatments of 25 or 30 kV/cm were applied to orange-carrot juice (Torregrosa et al., 2005). It has been reported that thermal treatment may imply an increase in some individual carotenoids owing to greater stability, enzymatic degradation, and unaccounted losses of moisture, which concentrate the sample (Rodriguez-Amaya, 1997). However, carotenoids are highly unsaturated compounds with an extensive conjugated double-bonds system and they are susceptible to oxidation, isomerisation and other chemical changes during processing and storage. Cortés et al. (2006) reported a significant decrease in total carotenoids of orange juice when applying bipolar treatments of 30 kV/cm for 100 µs. This decrease in provitamin A carotenoids could be correlated with a significant decrease in vitamin A by 7.52% in high-intensity PEF-treated orange juice (30 kV/cm, 100 µs) and by 15.62% in a pasteurized orange juice (90ºC, 20 s). Moreover, PEF processing (35 kV/cm, 750 µs) or thermal treatments (70 ºC, 30 s and 90 ºC, 30 s) did not exert any effect on vitamin A content of an orange juice (Sánchez-Moreno et al., 2005a). Research efforts have been made to obtain fruit and vegetable juices by HP processing without the quality and nutritional damage caused by heat treatments (Sánchez-Moreno et al., 2009). Individual carotenoids with antioxidant activity (β-carotene, β-cryptoxanthin, zeaxanthin and lutein) appeared to be resistant to a HP treatment of 400 MPa at 40 °C for 1 min, thus resulting into a better preservation of the antioxidant activity of orange juices with respect to those thermally pasteurized (Sánchez-Moreno et al. 2005a). Interestingly, an orange juice treated at 350 MPa/30ºC/5 min exhibited a higher carotenoid content (α-carotene, 60%; β-carotene, 50%; α-cryptoxanthin, 63%; β-cryptoxanthin, 42%) than a freshly squeezed juice (De Ancos et al., 2002), which was attributed to the desnaturation of the carotenoid-binding protein induced by pressure. Regarding the stability of carotenoids through storage of HP-pasteurized juices, non significant changes have been reported for at least 10 days of refrigerated storage in an orange juice treated at 100MPa/60ºC/5 min, whereas substantial losses were found at the end of the storage period of samples processed at 350MPa/30ºC/2.5 min or 400MPa/40ºC/1 min (20.56% and 9.16%, respectively). Plaza et al. (2010) reported that HP-treated orange juice showed a higher content in carotenoids than heat pasteurized juice during refrigerated

temperature (Tiwari et al., 2009a).

Different studies have proven the effectiveness of PEF in achieving higher vitamin C content in comparison with heat treatments in berries. Odriozola-Serrano et al. (2008a) reported that vitamin C retention just after treatment in heat-processed (90ºC, 60s) strawberry juice was significantly lower (94%) than that found in a juice treated at 35 kV/cm for 1700 µs in bipolar 4 µs pulses at 100 Hz (98%). Low processing temperatures reached through PEFprocessing (T<40 ºC) would explain the higher retention of vitamin C in HIPEF-treated strawberry juice compared to the thermally processed samples. The concentration of vitamin C in thermally and PEF-processed juices decreased gradually with storage time. Although during 21 days of storage the concentration of vitamin C was similar among processed strawberry juices, beyond this day, juices subjected to thermal treatment at 90 ºC for 60 s exhibited lower vitamin C content compared to PEF-treated juices (Odriozola et al., 2008c). Recommended daily intake (RDI) of vitamin C is currently revised but should be never below 60 mg, as established by the U.S. Food and Drug Administration (FDA, 1999). According to this recommendation, a strawberry juice 250 mL serving size should contain 24 mg/100 mL in order to contribute to the 100% of the RDI. Vitamin C content of juice processed with either PEF or heat at 90 ºC for 30 s felt below the RDI at 35 days of storage. The concentration of vitamin C in juices treated at 90 ºC for 60 s was reduced below 24 mg/mL after 28 days of storage at 4 ºC. As compared to fruit based products, a high residual ascorbic acid concentration after HP treatment is mostly found. In berries, a high retention of AA in strawberry nectar was observed after HP treatment at 500 MPa/room temperature/3 min (Rovere et al., 1996). Changes of vitamin C content in pressure treated

The Effects of Non-Thermal Technologies on Phytochemicals 115

Fig. 1. Effect of HIPEF and heat processing on pelargonidin-3-glucoside (a) and cyaniding-3 glucoside of strawberry juices throughout storage at 4 ºC. Strawberry juices: (■) untreated, (□) heat treated at 90 ºC for 30 s, ( ) heat treated at 90 ºC for 60 s and ( ) HIPEF treated at 35 kV/cm for 1700 μs in bipolar 4-μs pulses and 100 Hz. Data shown are mean standard

0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8

**pelargonidin-3-rutinoside** 

**(mg/100mL)**

0 7 14 21 28 35 42 49 56 **Storage time (days)**

were associated to the presence of residual enzyme activities such as β-glucosidase (Aguiló-Aguayo et al., 2008). HP treatment at ambient temperature has been reported to have minimal effects on the anthocyanins content of various food products (Oey et al., 2008). Anthocyanins have been reported to be stable to HP treatments in different products such as strawberry juice (Zabetakis et al., 2000), blackcurrant juice (Kouniaki et al., 2004) and raspberry juice (Suthanthangjai et al., 2004). No significant changes in anthocyanin content of strawberry and blackberry purées after 15 min treatments at 500-600 MPa was also reported by Patras et al. (2009). Similarly, cyanidin-3-glucoside, a predominant anthocyanin in berries, was found to be stable in a model solution at 600 MPa for 30 min at 20 ºC. However, 30 min application of 600 MPa at 70 ºC reduced cyanidin-3-glucoside by 25%, whereas only 5% was lost after 30 min heating at the same temperature and ambient pressure (Corrales et al., 2008). Combined pressure and temperature treatment of blueberry pasteurized juice led to a slightly faster degradation of total anthocyanins during storage compared to heat treatments at ambient pressure (Buckow et al., 2010). Thus, pressure seems to accelerate anthocyanin degradation at elevated temperatures. This could be related to condensation reactions involving covalent association of anthocyanins with other flavanols present in fruit juices. During refrigerated storage, the stability of anthocyanins in HP-treated fruit juices at moderate temperatures has been related to the residual PPO and POD activities, a sufficient activity for rapid oxidation of anthocyanins and other polyphenols in the presence of oxygen. Ultrasonication may be also considered a potential technology for processing of red juices because of its minimal effect on anthocyanins. Tiwari et al. (2009b) reported a slight increase (1-2%) in the pelargonidin- 3-glucoside content of the juice at low acoustic energy density (0,33 W/mL) and treatment time (3 min) which may be due to the extraction of bound anthocyanins from the suspended pulp. Some authors have also studied the effects of non ionizing radiation in phenolic content of berries. UV-C doses at 0.25 and 1.0 kJ/m2 increased anthocyanins concentrations in the fresh strawberries (Baka et al., 1999). Also, UV-C treatment for different durations (1, 5, and 10 min) increased the antioxidant capacity and the concentrations of anthocyanins and phenolic compounds of strawberries (Erkan et al., 2008). Related to ionizing radiation, gamma-radiation (1-10 kGy) led to the degradation of cinnamic, *p*-coumaric, gallic, and hydroxybenzoic acids

deviation. Adapted from Odriozola-Serrano et al. (2008c)

0 7 14 21 28 35 42 49 56 **Storage time (days)**

**Pelargonidin-3-glucoside** 

**(mg/100mL)**

food products during storage have been followed. It is suggested that further vitamin C degradation after HP processing during storage could be reduced by lowering storage temperature, for example, in pressurized (500 MPa/room temperature/3 min) strawberry nectar (Rovere et al., 1996). A kinetic study on degradation of vitamin C in pressure treated strawberry coulis has shown that a pressure treatment neither accelerated nor slowed down the kinetic degradation of ascorbic acid during subsequent storage. Sancho et al. (1999), observed identical kinetics of vitamin C degradation in pressurized (400 MPa/20 ºC/30 min) and untreated coulis during storage at 4 ºC. In general, it can be concluded that ascorbic acid is unstable at high pressure levels combined with high temperatures (above 65 ºC) and the major degradation is caused by oxidation especially during adiabatic heating. Therefore, eliminating the oxygen content in packaging can decrease the ascorbic acid degradation during processing and subsequent storage (Oey et al., 2008). The effects of ultrasonication on the vitamin C content of juices have been also studied. Degradation of vitamin C in sonicated strawberry juices was observed and the degradation level depended on the wave amplitude and treatment time (Tiwari et al, 2009b). Ascorbic acid degradation during sonication may be due to free radical formation and production of oxidative products on the surface of bubbles (Tiwari et al., 2009a). During refrigerated storage, similar vitamin C depletion in strawberry juice was observed in both sonicated and untreated juices (Tiwari et al., 2009b).

Flavonoids are the most common and widely distributed group of plant phenolics. Among them, flavones, flavonols, flavanols, flavanones, anthocyanins, and isoflavones are particularly common in fruits. In strawberry juices, *p*-hydroxybenzoic content was enhanced slightly but significantly after PEF processing (35 kV/cm for 1700 μs in bipolar 4-μs pulses at 100 Hz) compared to the untreated juice, whereas ellagic acid was substantially reduced when the heat treatment was conducted at 90ºC for 60 s. No significant differences in flavonol (kaempferol, quercetin and myricetin) content were obtained between fresh and treated strawberry juices; thus, these phenolic compounds were not affected by processing (Odriozola-Serrano et al., 2008c). It is well known that anthocyanins are unstable pigments and can be decolorized and degraded by many factors such as temperature, pH, oxygen, enzymes, light, the presence of copigments and metallic ions, ascorbic acid, sulphur dioxide and sugars. Numerous studies have evaluated the stability of anthocyanins to different kinds of nonthermal processing. Zhang et al. (2007) observed that after processing a cyanidin-3-glucoside methanolic solution by PEF (300 pulses of 1.2-3 kV/cm, Tª ≤ 47 ºC), the anthocyanin was degraded and the formation of the colorless anthocyanin species, particularly chalcones took place. In real foods such as strawberry juice, it was suggested that the lower the treatment time and the higher the electric field strength, the greater the anthocyanin retention in strawberry juice (Odriozola-Serrrano et al., 2008b). Contrarily, Zhang et al. (2007) reported that the degradation of cyanidin-3-glucoside in blackberry increased as the electric field strength rose. Jin and Zhang (1999) indicated that the losses of anthocyanins in PEF-treated cranberry juice increased during the storage period at 4 ºC. However, the content over time was higher that of thermally-treated samples, which contained the lowest amount of anthocyanins after two weeks of storage. Pelargonidin-3 glucoside and pelargonidine-3-rutinoside content and, in turn, total anthocyanins of strawberry juices, depleted with storage time after PEF (35 kV/cm for 1,700 µs in bipolar 4 µs pulses at 100 Hz) and thermal treatments (90ºC for 60 or 30 s) (Odriozola-Serrano et al., 2008b) (Figure 1). Changes in anthocyanins content throughout storage of PEF-treated juices

food products during storage have been followed. It is suggested that further vitamin C degradation after HP processing during storage could be reduced by lowering storage temperature, for example, in pressurized (500 MPa/room temperature/3 min) strawberry nectar (Rovere et al., 1996). A kinetic study on degradation of vitamin C in pressure treated strawberry coulis has shown that a pressure treatment neither accelerated nor slowed down the kinetic degradation of ascorbic acid during subsequent storage. Sancho et al. (1999), observed identical kinetics of vitamin C degradation in pressurized (400 MPa/20 ºC/30 min) and untreated coulis during storage at 4 ºC. In general, it can be concluded that ascorbic acid is unstable at high pressure levels combined with high temperatures (above 65 ºC) and the major degradation is caused by oxidation especially during adiabatic heating. Therefore, eliminating the oxygen content in packaging can decrease the ascorbic acid degradation during processing and subsequent storage (Oey et al., 2008). The effects of ultrasonication on the vitamin C content of juices have been also studied. Degradation of vitamin C in sonicated strawberry juices was observed and the degradation level depended on the wave amplitude and treatment time (Tiwari et al, 2009b). Ascorbic acid degradation during sonication may be due to free radical formation and production of oxidative products on the surface of bubbles (Tiwari et al., 2009a). During refrigerated storage, similar vitamin C depletion in strawberry juice was observed in both sonicated and untreated juices

Flavonoids are the most common and widely distributed group of plant phenolics. Among them, flavones, flavonols, flavanols, flavanones, anthocyanins, and isoflavones are particularly common in fruits. In strawberry juices, *p*-hydroxybenzoic content was enhanced slightly but significantly after PEF processing (35 kV/cm for 1700 μs in bipolar 4-μs pulses at 100 Hz) compared to the untreated juice, whereas ellagic acid was substantially reduced when the heat treatment was conducted at 90ºC for 60 s. No significant differences in flavonol (kaempferol, quercetin and myricetin) content were obtained between fresh and treated strawberry juices; thus, these phenolic compounds were not affected by processing (Odriozola-Serrano et al., 2008c). It is well known that anthocyanins are unstable pigments and can be decolorized and degraded by many factors such as temperature, pH, oxygen, enzymes, light, the presence of copigments and metallic ions, ascorbic acid, sulphur dioxide and sugars. Numerous studies have evaluated the stability of anthocyanins to different kinds of nonthermal processing. Zhang et al. (2007) observed that after processing a cyanidin-3-glucoside methanolic solution by PEF (300 pulses of 1.2-3 kV/cm, Tª ≤ 47 ºC), the anthocyanin was degraded and the formation of the colorless anthocyanin species, particularly chalcones took place. In real foods such as strawberry juice, it was suggested that the lower the treatment time and the higher the electric field strength, the greater the anthocyanin retention in strawberry juice (Odriozola-Serrrano et al., 2008b). Contrarily, Zhang et al. (2007) reported that the degradation of cyanidin-3-glucoside in blackberry increased as the electric field strength rose. Jin and Zhang (1999) indicated that the losses of anthocyanins in PEF-treated cranberry juice increased during the storage period at 4 ºC. However, the content over time was higher that of thermally-treated samples, which contained the lowest amount of anthocyanins after two weeks of storage. Pelargonidin-3 glucoside and pelargonidine-3-rutinoside content and, in turn, total anthocyanins of strawberry juices, depleted with storage time after PEF (35 kV/cm for 1,700 µs in bipolar 4 µs pulses at 100 Hz) and thermal treatments (90ºC for 60 or 30 s) (Odriozola-Serrano et al., 2008b) (Figure 1). Changes in anthocyanins content throughout storage of PEF-treated juices

(Tiwari et al., 2009b).

Fig. 1. Effect of HIPEF and heat processing on pelargonidin-3-glucoside (a) and cyaniding-3 glucoside of strawberry juices throughout storage at 4 ºC. Strawberry juices: (■) untreated, (□) heat treated at 90 ºC for 30 s, ( ) heat treated at 90 ºC for 60 s and ( ) HIPEF treated at 35 kV/cm for 1700 μs in bipolar 4-μs pulses and 100 Hz. Data shown are mean standard deviation. Adapted from Odriozola-Serrano et al. (2008c)

were associated to the presence of residual enzyme activities such as β-glucosidase (Aguiló-Aguayo et al., 2008). HP treatment at ambient temperature has been reported to have minimal effects on the anthocyanins content of various food products (Oey et al., 2008). Anthocyanins have been reported to be stable to HP treatments in different products such as strawberry juice (Zabetakis et al., 2000), blackcurrant juice (Kouniaki et al., 2004) and raspberry juice (Suthanthangjai et al., 2004). No significant changes in anthocyanin content of strawberry and blackberry purées after 15 min treatments at 500-600 MPa was also reported by Patras et al. (2009). Similarly, cyanidin-3-glucoside, a predominant anthocyanin in berries, was found to be stable in a model solution at 600 MPa for 30 min at 20 ºC. However, 30 min application of 600 MPa at 70 ºC reduced cyanidin-3-glucoside by 25%, whereas only 5% was lost after 30 min heating at the same temperature and ambient pressure (Corrales et al., 2008). Combined pressure and temperature treatment of blueberry pasteurized juice led to a slightly faster degradation of total anthocyanins during storage compared to heat treatments at ambient pressure (Buckow et al., 2010). Thus, pressure seems to accelerate anthocyanin degradation at elevated temperatures. This could be related to condensation reactions involving covalent association of anthocyanins with other flavanols present in fruit juices. During refrigerated storage, the stability of anthocyanins in HP-treated fruit juices at moderate temperatures has been related to the residual PPO and POD activities, a sufficient activity for rapid oxidation of anthocyanins and other polyphenols in the presence of oxygen. Ultrasonication may be also considered a potential technology for processing of red juices because of its minimal effect on anthocyanins. Tiwari et al. (2009b) reported a slight increase (1-2%) in the pelargonidin- 3-glucoside content of the juice at low acoustic energy density (0,33 W/mL) and treatment time (3 min) which may be due to the extraction of bound anthocyanins from the suspended pulp. Some authors have also studied the effects of non ionizing radiation in phenolic content of berries. UV-C doses at 0.25 and 1.0 kJ/m2 increased anthocyanins concentrations in the fresh strawberries (Baka et al., 1999). Also, UV-C treatment for different durations (1, 5, and 10 min) increased the antioxidant capacity and the concentrations of anthocyanins and phenolic compounds of strawberries (Erkan et al., 2008). Related to ionizing radiation, gamma-radiation (1-10 kGy) led to the degradation of cinnamic, *p*-coumaric, gallic, and hydroxybenzoic acids

The Effects of Non-Thermal Technologies on Phytochemicals 117

60 s of treatment. Although the reductions in carotenoids with provitamin A activity are very small after pasteurization, the decreases in the concentrations of lutein (22.8%) and zeaxanthin (22.5%) after pasteurizing are considerable and must be taken into account, because these two xanthophylls play a fundamental part in sight, prevent degenerative eye diseases, and are

Coumaric acid, narirutin and hesperidin were the most abundant phenolic compounds in a blend fruit juice-soymilk beverage. Immediately after PEF (35 kV/cm with 4 µs bipolar pulses at 200 Hz for 800 or 1400 µs) or thermal (90 ºC, 60 s) treatments, hesperidin content of the beverage showed a huge rise, resulting in a significant increase on the total phenolic concentration. In addition, total phenolic concentration seemed to be highly stable during refrigerated storage (Morales et al., 2011). According to these authors, changes observed on the phenolic content of the fruit juice-soymilk beverage after PEF or thermal treatments could be due to some of the followed reasons: (i) biochemical reactions could have occurred during the PEF or heat processing, which led to the formation of new phenolic compounds; (ii) PEF or thermal processing might have caused significant effects on cell membranes or in phenolic complexes with other compounds, releasing some free phenolic acids or flavonoids; (iii) PEF and thermal process may inactivate PPO, preventing further loss of phenolic compounds; and (iv) PEF treatment might have induced favorable conditions to increase PAL activity, resulting in an enhancement of phenolic concentration in the beverage. Levels of total phenolic compounds also increased significantly by HP in processed orange juice–milk; reaching a maximum at 100 MPa/420 s, when there was a significant increase of 22% in comparison with unprocessed samples (Barba et al., 2011). As it was mentioned before, the increase in total phenolic content may be related to an increased extractability of some of the antioxidant

> 1.0 1.2 1.4 1.6 1.8 2.0

**mg Gin/100mL**

Fig. 2. Individual isoflavone profile: a) daidzein, b) genistein, c) daidzin and d) genisitin of untreated (dotted line, ♦), high intensity pulsed electric field (35 kV/cm with 4 μs bipolar pulses at 200 Hz for 800 (continue line, □) or 1400 μs (continue line, )) and thermal (continue line, ○) (90 ºC, 60 s) treated fruit juice-soymilk beverages throughout storage at

**mg Ge/100mL**

0 10 20 30 40 50 60

0 10 20 30 40 50 60

**Storage time (days)**

**c) d)**

**Storage time (days)**

**a) b)**

antioxidant compounds that give quality to food products (Zulueta et al., 2010b).

components following high-pressure processing.

0 10 20 30 40 50 60

0 10 20 30 40 50 60

**Storage time (days)**

**Storage time (days)**

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

4ºC (Morales et al., 2010b)

**mg Din/100mL**

**mg Da/100mL**

(Breitfellner et al., 2002a). The hydroxylation (decomposition) of these phenolic acids has been attributed to the formation of free hydroxyl (OH·) radicals during the treatment. Catechin and kaempferol components also diminished noticeably due to gamma-radiation (1-6 kGy), whereas ellagic acid derivatives and quercetin concentrations were not affected by the treatment in strawberries (Breitfellner et al., 2002b).

#### **2.2.4 Fruit juice-milk beverage**

Fruit juice and milk beverage is a product in which the antioxidant capacity of fruit constituents can be delivered in combination with the health benefits of milk. Morales et al. (2010a) did not found significant differences in vitamin C retention (87–90%) between (35 kV/cm, 4 µs bipolar pulses at 200 Hz for 800 or 1400 µs) and thermally treated (90 ºC, 60 s) blend fruit juice-soymilk beverages. In addition, the vitamin C content of the beverages decreased gradually during storage, regardless of the treatment applied. However, throughout the first 31 days, vitamin C was better maintained in the 800 μs-PEF treated fruit juice-soymilk beverages (46.4%) than in those treated for 1400 μs (22.6%); whereas, those that where thermally treated showed the lowest retention of vitamin C (6.7%) (Morales et al., 2011). These results showed that the shorter the PEF treatment time, the higher the vitamin C retention, as previously found in other studies focused on individual fruit juices treated by PEF. The higher retention of vitamin C of PEF treated fruit juice-soymilk beverages compared to those thermally treated might be due to the lower processing temperatures achieved during PEF treatments (<32 ºC). Currently, few studies have been carried out about the effect of HP processing on phytochemicals of fruit juice–milk beverages. High ascorbic acid retention (91%) in the orange juice-milk beverage after HP (100-400 MPa/120-540 s) treatment was reported by Barba et al. (2011).

Initial degradation of ascorbic acid was less in orange juice-milk beverages treated by PEF (25 kV/cm and 280 µs) than in a heat-treated (90 ºC, 20 s) juice (Zulueta et al., 2010a). During storage, total carotenoid content of untreated and treated blend of fruit juice-soymilk beverage tended to decrease as the storage time increased (Morales et al., 2011). Moreover, thermally treated juices showed higher rate of degradation than those PEF-treated. Although the pathways of carotenoids degradation have not been well established, oxidation is the main cause of carotenoid loss, which is a spontaneous free-radical chain reaction in the presence of oxygen (Sánchez-Moreno et al., 2003). During autooxidation of carotenoids, alkylperoxyl radicals are formed and these radicals attack the double bounds resulting in formation of epoxides (Odriozola-Serrano et al., 2009). Total carotenoid content was significantly enhanced in orange juice-milk beverage treated by HP (100-400 MPa) when treatment time was 420 and 540 s in comparison with the unprocessed samples (Barba et al., 2011). According to these authors, this may be because when pressures of 100 MPa are applied, they are sufficient to cause breakage of the intracellular vacuoles and the cell walls of the plant or they also suggested that an increase in free carotenoids in juices after HP might be because there is probably an alteration in the structure of the proteins that are linked to the carotenoids. With regard to individual carotenoid concentrations, there were no significant differences for any of them (Neoxanthin + 9-cis-violaxanthin, Mutatoxanthin, Lutein, Zeaxanthin, -Cryptoxanthin, -Carotene, Phytoene+phytofluene, -Carotene, - Carotene, 15-cis--Carotene), and only in the case of the electric field strength of 35 kV/cm there was significant increase in -carotene in comparison with the untreated beverage after

(Breitfellner et al., 2002a). The hydroxylation (decomposition) of these phenolic acids has been attributed to the formation of free hydroxyl (OH·) radicals during the treatment. Catechin and kaempferol components also diminished noticeably due to gamma-radiation (1-6 kGy), whereas ellagic acid derivatives and quercetin concentrations were not affected

Fruit juice and milk beverage is a product in which the antioxidant capacity of fruit constituents can be delivered in combination with the health benefits of milk. Morales et al. (2010a) did not found significant differences in vitamin C retention (87–90%) between (35 kV/cm, 4 µs bipolar pulses at 200 Hz for 800 or 1400 µs) and thermally treated (90 ºC, 60 s) blend fruit juice-soymilk beverages. In addition, the vitamin C content of the beverages decreased gradually during storage, regardless of the treatment applied. However, throughout the first 31 days, vitamin C was better maintained in the 800 μs-PEF treated fruit juice-soymilk beverages (46.4%) than in those treated for 1400 μs (22.6%); whereas, those that where thermally treated showed the lowest retention of vitamin C (6.7%) (Morales et al., 2011). These results showed that the shorter the PEF treatment time, the higher the vitamin C retention, as previously found in other studies focused on individual fruit juices treated by PEF. The higher retention of vitamin C of PEF treated fruit juice-soymilk beverages compared to those thermally treated might be due to the lower processing temperatures achieved during PEF treatments (<32 ºC). Currently, few studies have been carried out about the effect of HP processing on phytochemicals of fruit juice–milk beverages. High ascorbic acid retention (91%) in the orange juice-milk beverage after HP

Initial degradation of ascorbic acid was less in orange juice-milk beverages treated by PEF (25 kV/cm and 280 µs) than in a heat-treated (90 ºC, 20 s) juice (Zulueta et al., 2010a). During storage, total carotenoid content of untreated and treated blend of fruit juice-soymilk beverage tended to decrease as the storage time increased (Morales et al., 2011). Moreover, thermally treated juices showed higher rate of degradation than those PEF-treated. Although the pathways of carotenoids degradation have not been well established, oxidation is the main cause of carotenoid loss, which is a spontaneous free-radical chain reaction in the presence of oxygen (Sánchez-Moreno et al., 2003). During autooxidation of carotenoids, alkylperoxyl radicals are formed and these radicals attack the double bounds resulting in formation of epoxides (Odriozola-Serrano et al., 2009). Total carotenoid content was significantly enhanced in orange juice-milk beverage treated by HP (100-400 MPa) when treatment time was 420 and 540 s in comparison with the unprocessed samples (Barba et al., 2011). According to these authors, this may be because when pressures of 100 MPa are applied, they are sufficient to cause breakage of the intracellular vacuoles and the cell walls of the plant or they also suggested that an increase in free carotenoids in juices after HP might be because there is probably an alteration in the structure of the proteins that are linked to the carotenoids. With regard to individual carotenoid concentrations, there were no significant differences for any of them (Neoxanthin + 9-cis-violaxanthin, Mutatoxanthin, Lutein, Zeaxanthin, -Cryptoxanthin, -Carotene, Phytoene+phytofluene, -Carotene, - Carotene, 15-cis--Carotene), and only in the case of the electric field strength of 35 kV/cm there was significant increase in -carotene in comparison with the untreated beverage after

by the treatment in strawberries (Breitfellner et al., 2002b).

(100-400 MPa/120-540 s) treatment was reported by Barba et al. (2011).

**2.2.4 Fruit juice-milk beverage** 

60 s of treatment. Although the reductions in carotenoids with provitamin A activity are very small after pasteurization, the decreases in the concentrations of lutein (22.8%) and zeaxanthin (22.5%) after pasteurizing are considerable and must be taken into account, because these two xanthophylls play a fundamental part in sight, prevent degenerative eye diseases, and are antioxidant compounds that give quality to food products (Zulueta et al., 2010b).

Coumaric acid, narirutin and hesperidin were the most abundant phenolic compounds in a blend fruit juice-soymilk beverage. Immediately after PEF (35 kV/cm with 4 µs bipolar pulses at 200 Hz for 800 or 1400 µs) or thermal (90 ºC, 60 s) treatments, hesperidin content of the beverage showed a huge rise, resulting in a significant increase on the total phenolic concentration. In addition, total phenolic concentration seemed to be highly stable during refrigerated storage (Morales et al., 2011). According to these authors, changes observed on the phenolic content of the fruit juice-soymilk beverage after PEF or thermal treatments could be due to some of the followed reasons: (i) biochemical reactions could have occurred during the PEF or heat processing, which led to the formation of new phenolic compounds; (ii) PEF or thermal processing might have caused significant effects on cell membranes or in phenolic complexes with other compounds, releasing some free phenolic acids or flavonoids; (iii) PEF and thermal process may inactivate PPO, preventing further loss of phenolic compounds; and (iv) PEF treatment might have induced favorable conditions to increase PAL activity, resulting in an enhancement of phenolic concentration in the beverage. Levels of total phenolic compounds also increased significantly by HP in processed orange juice–milk; reaching a maximum at 100 MPa/420 s, when there was a significant increase of 22% in comparison with unprocessed samples (Barba et al., 2011). As it was mentioned before, the increase in total phenolic content may be related to an increased extractability of some of the antioxidant components following high-pressure processing.

Fig. 2. Individual isoflavone profile: a) daidzein, b) genistein, c) daidzin and d) genisitin of untreated (dotted line, ♦), high intensity pulsed electric field (35 kV/cm with 4 μs bipolar pulses at 200 Hz for 800 (continue line, □) or 1400 μs (continue line, )) and thermal (continue line, ○) (90 ºC, 60 s) treated fruit juice-soymilk beverages throughout storage at 4ºC (Morales et al., 2010b)

The Effects of Non-Thermal Technologies on Phytochemicals 119

retention of 86.6–89.0% (Quitao-Teixeira et al., 2009). Watermelon juice was subjected to high-intensity pulsed electric fields (HIPEF). The effects of process parameters including electric field strength (30–35 kV/cm), pulse frequency (50–250 Hz), treatment time (50–2050 s), pulse width (1–7 s) and pulse polarity (monopolar/bipolar) on lycopene, vitamin C and antioxidant capacity were studied using a response surface methodology (Oms-Oliu et al., 2009). Watermelon juices treated at 25 kV/cm for 50 s at 50 Hz using mono- or bipolar 1-s pulses exhibited the highest vitamin C retention (96.4–99.9%). On the other hand, vitamin C loss was higher than 50% when PEF treatment was set up at 35 kV/cm for 2050 (s at 250 Hz applying mono- or bipolar 7-s pulses. Such severe conditions seem to greater affect vitamin C retention in watermelon juice than in other juices such as orange, orange– carrot or strawberry juices, which exhibited retention of vitamin C above 80%, because more acidic conditions are known to stabilise vitamin C (Oms-Oliu et al., 2009). During storage, vitamin C was better retained in the PEF-treated carrot juice than in the thermally processed juices for 56 days at 4 ºC. Differences in vitamin C reduction between PEF and heat treated juices throughout the storage might be due to the activity of enzymes such as ascorbate oxidase. A first-order kinetic model adequately fitted vitamin C depletion (R2 = 0.9680– 0.838; Af = 1.039–1.068) as a function of the storage time (Quitao-Teixeira et al., 2009). Storage conditions such as temperature or the oxygen concentration may also have a significant influence on the rates of vitamin C degradation. Sonication also showed to increase vitamin C content in sonicated (ficar les condicions) guava juice compared to untreated sample, the most likely reason being the elimination of dissolved oxygen that is essential for ascorbic acid degradation during cavitation (Cheng et al., 2007). Research reporting on the impact of ionizing radiation on vitamins of plant-derived products is restricted to the effect of gamma-radiation on vitamin C. In general, most fresh-cut vegetables (iceberg, romaine, green and red leaf lettuce, spinach, tomato, cilantro, parsley, green onion, carrot, broccoli, red cabbage, and celery) can tolerate up to 1 kGy radiation without significant losses in vitamin C content (Fan and Sokorai, 2008). In the case of minimally processed irradiated cucumber and carrot, no significant differences between the vitamin C content of control and treated samples were reported through refrigerated storage (Khamat et al., 2005; Hajare et al., 2006). Minor vitamin C losses were reported for minimally processed refrigerated capsicum after gamma-radiation (1-3 kGy) during storage (Ramamurthy et al., 2004). Vitamin C content of irradiated fresh-cut celery (0.5-1.5 kGy) or lettuce (1 kGy) during refrigerated storage was higher than in non-irradiated products (Lu et al., 2005; Zhang et al., 2006). Regarding fresh-cut fruits, Fan et al. (2006), reported that vitamin C of cantaloupe was not substantially affected by treatment with non ionizing radiation. No differences have been reported between treated and untreated products regarding the stability of vitamin C during storage. On the other hand, light treatments applied as short pulses with a total fluence of 4.8 and 12 J cm-2 maintained amounts of vitamin C similar to those found in untreated fresh-cut mushrooms during 7 days of

refrigerated storage under modified atmosphere packaging (Oms-Oliu et al., 2010).

An increase in lycopene (114%) was observed in watermelon juice treated with 7-s bipolar pulses for 1050 s at 35 kV/cm and frequencies ranging from 200 to 250 Hz (Oms-Oliu et al., 2009). The increase in lycopene has been related to the conversion of some carotenoids to lycopene as a result of an intense PEF treatment. β-Carotene concentration substantially increased in processed carrot juices compared to the untreated juice and thermally treated juices. PEF-treated carrot juice maintained β-carotene content better than heat treatments

During the last years, soy beverages consumption has gradually increased due to their significant concentration of health-promoting compounds, such as isoflavones. PEF seems to be a good technology in order to obtain fruit juice-soymilk beverage with a high content of isoflavones and fresh-like characteristics. In a blend fruit juice-soymilk beverage, PEF treatment (35 kV/cm with 4 μs bipolar pulses at 200 Hz for 800 or 1400 μs) did not caused significant changes on the total isoflavone content and, during the storage period, total isoflavone content tended to increase throughout the time. Genistein, daidzein and daidzin content increased; while genistin showed a slight decrease, irrespective of the treatment applied (Figure 2) (Morales et al., 2010b). These authors suggested that the concentration of some isoflavones in the fruit juice-soymilk beverage might increase during storage from the flavonoids (mainly naringenin from orange) present in the fruits used for the elaboration of the beverage. Nevertheless, there is a need for more in-depth research to provide biochemical evidence of the observed changes.

#### **2.2.5 Broccoli**

Isothiocyanates are organosulfur compounds formed by enzyme-catalysed hydrolysis of glucosinolates, which are largely found in vegetables of the Brassicaceae family. Some isothiocyanates have shown anticarcinogenic potential (Conaway et al., 2002). Knowledge about the impact of non thermal technologies on the stability of these compounds is really scarce. Van Eylen et al. (2007) studied the pressure (600-800 MPa) and temperature (30-60ºC) stabilities of sulforaphane and phenylethyl isothiocyanate in broccoli juice. Authors concluded that isothiocyanates are relatively thermolabile and pressure stable. At the same time, mild pressure treatments where suggested as the most advantageous, because myrosinase activity is stabilized, thus leading to products with increased isothiocyanate content. In a subsequent study, Van Eylen en al. (2008) observed that the composition of glucosinolate hydrolysis products may greatly differ between different HP process conditions. Upon this base, HP treatments can be selected to optimise the health beneficial properties of plant foods.

The non ionizing radiation treatments have been shown to increase the antioxidant capacity of broccoli, which could be useful from the nutritional point of view. UV-C (4-14 kJm-2) treated broccoli florets displayed lower total phenolic and flavonoid content along with higher antioxidant capacity compared to the control samples (Costa et al., 2006). On the other hand, exposure to UV-C (8 kJm-2) increased total phenolic and ascorbic acid contents, as well as the antioxidant capacity of minimally processed broccoli (Lemoine et al., 2007) These authors related, an increment in the activity of PAL after treatment with UV-C to an increase in the content of phenolic compounds in treated samples since PAL is one of the key enzymes in phenolic synthesis.

#### **2.2.6 Others**

As it was mentioned before, PEF processing may allow obtaining juices with higher antioxidant potential and extended shelf-life, thus becoming a feasible alternative to heat processing. PEF processing may help to achieve fresh-like carrot juices with increased amounts of health-related phytochemicals. PEF processing (35 kV/cm for 1500 μs with 6-μs bipolar pulses at 200 Hz) resulted into a carrot juices with significantly greater vitamin C retention of 95.1% than thermal processing (90 ºC, 30 s and 90 ºC, 60 s), which exhibited a

During the last years, soy beverages consumption has gradually increased due to their significant concentration of health-promoting compounds, such as isoflavones. PEF seems to be a good technology in order to obtain fruit juice-soymilk beverage with a high content of isoflavones and fresh-like characteristics. In a blend fruit juice-soymilk beverage, PEF treatment (35 kV/cm with 4 μs bipolar pulses at 200 Hz for 800 or 1400 μs) did not caused significant changes on the total isoflavone content and, during the storage period, total isoflavone content tended to increase throughout the time. Genistein, daidzein and daidzin content increased; while genistin showed a slight decrease, irrespective of the treatment applied (Figure 2) (Morales et al., 2010b). These authors suggested that the concentration of some isoflavones in the fruit juice-soymilk beverage might increase during storage from the flavonoids (mainly naringenin from orange) present in the fruits used for the elaboration of the beverage. Nevertheless, there is a need for more in-depth research to provide

Isothiocyanates are organosulfur compounds formed by enzyme-catalysed hydrolysis of glucosinolates, which are largely found in vegetables of the Brassicaceae family. Some isothiocyanates have shown anticarcinogenic potential (Conaway et al., 2002). Knowledge about the impact of non thermal technologies on the stability of these compounds is really scarce. Van Eylen et al. (2007) studied the pressure (600-800 MPa) and temperature (30-60ºC) stabilities of sulforaphane and phenylethyl isothiocyanate in broccoli juice. Authors concluded that isothiocyanates are relatively thermolabile and pressure stable. At the same time, mild pressure treatments where suggested as the most advantageous, because myrosinase activity is stabilized, thus leading to products with increased isothiocyanate content. In a subsequent study, Van Eylen en al. (2008) observed that the composition of glucosinolate hydrolysis products may greatly differ between different HP process conditions. Upon this base, HP treatments can be selected to optimise the health beneficial

The non ionizing radiation treatments have been shown to increase the antioxidant capacity of broccoli, which could be useful from the nutritional point of view. UV-C (4-14 kJm-2) treated broccoli florets displayed lower total phenolic and flavonoid content along with higher antioxidant capacity compared to the control samples (Costa et al., 2006). On the other hand, exposure to UV-C (8 kJm-2) increased total phenolic and ascorbic acid contents, as well as the antioxidant capacity of minimally processed broccoli (Lemoine et al., 2007) These authors related, an increment in the activity of PAL after treatment with UV-C to an increase in the content of phenolic compounds in treated samples since PAL is one of the

As it was mentioned before, PEF processing may allow obtaining juices with higher antioxidant potential and extended shelf-life, thus becoming a feasible alternative to heat processing. PEF processing may help to achieve fresh-like carrot juices with increased amounts of health-related phytochemicals. PEF processing (35 kV/cm for 1500 μs with 6-μs bipolar pulses at 200 Hz) resulted into a carrot juices with significantly greater vitamin C retention of 95.1% than thermal processing (90 ºC, 30 s and 90 ºC, 60 s), which exhibited a

biochemical evidence of the observed changes.

**2.2.5 Broccoli** 

properties of plant foods.

key enzymes in phenolic synthesis.

**2.2.6 Others**

retention of 86.6–89.0% (Quitao-Teixeira et al., 2009). Watermelon juice was subjected to high-intensity pulsed electric fields (HIPEF). The effects of process parameters including electric field strength (30–35 kV/cm), pulse frequency (50–250 Hz), treatment time (50–2050 s), pulse width (1–7 s) and pulse polarity (monopolar/bipolar) on lycopene, vitamin C and antioxidant capacity were studied using a response surface methodology (Oms-Oliu et al., 2009). Watermelon juices treated at 25 kV/cm for 50 s at 50 Hz using mono- or bipolar 1-s pulses exhibited the highest vitamin C retention (96.4–99.9%). On the other hand, vitamin C loss was higher than 50% when PEF treatment was set up at 35 kV/cm for 2050 (s at 250 Hz applying mono- or bipolar 7-s pulses. Such severe conditions seem to greater affect vitamin C retention in watermelon juice than in other juices such as orange, orange– carrot or strawberry juices, which exhibited retention of vitamin C above 80%, because more acidic conditions are known to stabilise vitamin C (Oms-Oliu et al., 2009). During storage, vitamin C was better retained in the PEF-treated carrot juice than in the thermally processed juices for 56 days at 4 ºC. Differences in vitamin C reduction between PEF and heat treated juices throughout the storage might be due to the activity of enzymes such as ascorbate oxidase. A first-order kinetic model adequately fitted vitamin C depletion (R2 = 0.9680– 0.838; Af = 1.039–1.068) as a function of the storage time (Quitao-Teixeira et al., 2009). Storage conditions such as temperature or the oxygen concentration may also have a significant influence on the rates of vitamin C degradation. Sonication also showed to increase vitamin C content in sonicated (ficar les condicions) guava juice compared to untreated sample, the most likely reason being the elimination of dissolved oxygen that is essential for ascorbic acid degradation during cavitation (Cheng et al., 2007). Research reporting on the impact of ionizing radiation on vitamins of plant-derived products is restricted to the effect of gamma-radiation on vitamin C. In general, most fresh-cut vegetables (iceberg, romaine, green and red leaf lettuce, spinach, tomato, cilantro, parsley, green onion, carrot, broccoli, red cabbage, and celery) can tolerate up to 1 kGy radiation without significant losses in vitamin C content (Fan and Sokorai, 2008). In the case of minimally processed irradiated cucumber and carrot, no significant differences between the vitamin C content of control and treated samples were reported through refrigerated storage (Khamat et al., 2005; Hajare et al., 2006). Minor vitamin C losses were reported for minimally processed refrigerated capsicum after gamma-radiation (1-3 kGy) during storage (Ramamurthy et al., 2004). Vitamin C content of irradiated fresh-cut celery (0.5-1.5 kGy) or lettuce (1 kGy) during refrigerated storage was higher than in non-irradiated products (Lu et al., 2005; Zhang et al., 2006). Regarding fresh-cut fruits, Fan et al. (2006), reported that vitamin C of cantaloupe was not substantially affected by treatment with non ionizing radiation. No differences have been reported between treated and untreated products regarding the stability of vitamin C during storage. On the other hand, light treatments applied as short pulses with a total fluence of 4.8 and 12 J cm-2 maintained amounts of vitamin C similar to those found in untreated fresh-cut mushrooms during 7 days of refrigerated storage under modified atmosphere packaging (Oms-Oliu et al., 2010).

An increase in lycopene (114%) was observed in watermelon juice treated with 7-s bipolar pulses for 1050 s at 35 kV/cm and frequencies ranging from 200 to 250 Hz (Oms-Oliu et al., 2009). The increase in lycopene has been related to the conversion of some carotenoids to lycopene as a result of an intense PEF treatment. β-Carotene concentration substantially increased in processed carrot juices compared to the untreated juice and thermally treated juices. PEF-treated carrot juice maintained β-carotene content better than heat treatments

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during 56 days of storage at 4 ◦C. The major cause of carotenoid losses in vegetable products is the oxidation of the highly unsaturated carotenoid structure. The severity of oxidation depends on the structure of carotenoids and the environmental conditions, and the compounds being formed may vary upon the oxidation process and the carotenoids structure (Quitao-Teixeira et al., 2009). Therefore, the higher depletion of β-carotene throughout the storage in thermally treated carrot juice compared to those PEF-processed might be due to the greater changes in carotenes structure as a consequence of high temperature. In addition, a better retention of carotenoids in HP treated carrot pureés compared to thermally processed samples was observed (Patras et al., 2009). Such effect has been well documented elsewhere and would appear to be related to an increase in extractability of antioxidant components following high pressure treatment rather than an absolute increase.

No significant differences in the amount of total phenolics were observed between untreated and PEF-treated products such as spinach puree (Yin et al., 2007) and carrot juice (Quitão-Teixeira et al., 2009) just after processing. It has been also reported that radiation treatments can generate free radicals, thus leading to an induction of stress responses in plant foods, which in turn may lead to an increase in the antioxidant synthesis. Results by Song et al. (2006) are consistent with this idea. These authors observed that total phenolic content of carrot and kale juices was substantially increased by applying a radiation treatment. However, reductions in the total phenolic content have been reported for treatments of more than 10 kGy in some irradiated products (Villavicencio et al., 2000; Ahn et al., 2005).

#### **3. Conclusion**

Non thermal technologies may allow obtaining safe and shelf-stable plant-based products with minor changes or increased content in health-related phytochemicals. Most differences between non thermal and heat treatments can be explained through the temperatures reached through processing. In general, temperature during processing and storage are important factors affecting the phytochemicals of the processed product. The stability of these compounds through storage is dependent in each case on the residual amounts of enzymes involved in their degradation. In addition, processing parameters are involved in the degradation or generation of bioactive compounds. In-depth research is needed in order to elucidate the mechanisms involved in the destruction or generation of these compounds in a food matrix processed by these novel technologies.

Few studies assessing the impact of non thermal technologies on the bioavailability of bioactive compounds reported an increase of plasma vitamin C and a decrease of the oxidative stress and inflammation biomarkers in healthy humans. Thus, new applications of non thermal processing technologies should be further explored not only to stabilize the content of health-related phytochemicals but also their bioavailability and biological activity in humans.

#### **4. References**

Ahn, H.J.; Kim, J.H.; Kim., J.K.; Kim, D.H.; Yook, H.S.; & Byun, M.W. (2005). Combined effect of irradiation and modified atmosphere packaging on minimally processed Chinese cabbage (Brassica ropa L.). *Food Chemistry*, 89, pp. 589-597.

during 56 days of storage at 4 ◦C. The major cause of carotenoid losses in vegetable products is the oxidation of the highly unsaturated carotenoid structure. The severity of oxidation depends on the structure of carotenoids and the environmental conditions, and the compounds being formed may vary upon the oxidation process and the carotenoids structure (Quitao-Teixeira et al., 2009). Therefore, the higher depletion of β-carotene throughout the storage in thermally treated carrot juice compared to those PEF-processed might be due to the greater changes in carotenes structure as a consequence of high temperature. In addition, a better retention of carotenoids in HP treated carrot pureés compared to thermally processed samples was observed (Patras et al., 2009). Such effect has been well documented elsewhere and would appear to be related to an increase in extractability of antioxidant components following high pressure treatment rather than an

No significant differences in the amount of total phenolics were observed between untreated and PEF-treated products such as spinach puree (Yin et al., 2007) and carrot juice (Quitão-Teixeira et al., 2009) just after processing. It has been also reported that radiation treatments can generate free radicals, thus leading to an induction of stress responses in plant foods, which in turn may lead to an increase in the antioxidant synthesis. Results by Song et al. (2006) are consistent with this idea. These authors observed that total phenolic content of carrot and kale juices was substantially increased by applying a radiation treatment. However, reductions in the total phenolic content have been reported for treatments of more

than 10 kGy in some irradiated products (Villavicencio et al., 2000; Ahn et al., 2005).

in a food matrix processed by these novel technologies.

Non thermal technologies may allow obtaining safe and shelf-stable plant-based products with minor changes or increased content in health-related phytochemicals. Most differences between non thermal and heat treatments can be explained through the temperatures reached through processing. In general, temperature during processing and storage are important factors affecting the phytochemicals of the processed product. The stability of these compounds through storage is dependent in each case on the residual amounts of enzymes involved in their degradation. In addition, processing parameters are involved in the degradation or generation of bioactive compounds. In-depth research is needed in order to elucidate the mechanisms involved in the destruction or generation of these compounds

Few studies assessing the impact of non thermal technologies on the bioavailability of bioactive compounds reported an increase of plasma vitamin C and a decrease of the oxidative stress and inflammation biomarkers in healthy humans. Thus, new applications of non thermal processing technologies should be further explored not only to stabilize the content of health-related phytochemicals but also their bioavailability and biological activity

Ahn, H.J.; Kim, J.H.; Kim., J.K.; Kim, D.H.; Yook, H.S.; & Byun, M.W. (2005). Combined

Chinese cabbage (Brassica ropa L.). *Food Chemistry*, 89, pp. 589-597.

effect of irradiation and modified atmosphere packaging on minimally processed

absolute increase.

**3. Conclusion** 

in humans.

**4. References** 


The Effects of Non-Thermal Technologies on Phytochemicals 123

Khamat, A.S.; Ghadge, N.; Ramamurthy, M.S. Alud, M.D. (2005). Effect of low-dose

Knorr, D. (2001). 'High pressure processing for preservation, modification and

Knorr, D.; Zenker, M.; Heinz, V.; & Lee, D. U. (2004). Applications and potential of

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**6** 

*Malaysia* 

**Phytochemical Constituents** 

*Universiti Malaysia Sabah,* 

 **and Activities of** *Morinda citrifolia* **L.** 

Duduku Krishnaiah\*, Rajesh Nithyanandam and Rosalam Sarbatly

Both artificial and naturally occurring antioxidants have been reported to play major roles in protecting membranes and tissues from free radical and xenobiotic-induced oxidative damage (Burton, 1989; Carini et al., 1990). Most living organisms harbour both enzymatic and non-enzymatic systems that protect them against excessive reactive oxygen species. However, various external factors (smoke, diet, alcohol and some drugs) and aging decrease the efficiency of these protective systems, thereby disrupting the redox equilibrium that is established under healthy conditions. Thus, antioxidants that scavenge reactive oxygen species may be of great value in preventing the onset and propagation of oxidative diseases (Willet, 1994). Recently, more attention has been paid to the role of natural antioxidants, mainly phenolic compounds, which may have higher antioxidant activity than do conventional antioxidants, such as vitamins C, E and β-carotene (Vinson et al., 1995). The anti-oxidative effects of natural phenolic compounds, in pure form or in extracts from various plant sources (vegetables, fruits and medicinal plants), have been studied *in vitro* using a variety of model systems (Meyer et al., 1998; Pietta et al., 1998; Yen & Hsieh, 1998). Therefore, antioxidants, which can neutralize free radicals, may be of central importance in the prevention of carcinogenicity, cardiovascular disease and neurodegenerative changes associated with aging (Halliwell, 1994; Yu, 1994). Epidemiological studies have shown that the consumption of vegetables and fruits can protect humans against oxidative damage by inhibiting and/or quenching free radicals and reactive oxygen species (Ames et al., 1993). Oxidative stress occurs commonly in living organisms, and it is involved in the pathology of cancer, arteriosclerosis, malaria, and rheumatoid arthritis. Moreover, it may play a role in neurodegenerative diseases and ageing processes. It has been demonstrated that many vegetables, fruits, medicinal plants and other foods contain compounds with bioactivity against oxidative stress, and this activity has been attributed to vitamin C, vitamin E, αtocopherol, β-carotene, and polyphenolic compounds (Krishnaiah et al., 2011; Moure et al., 2001). Therefore, research regarding natural antioxidants from foods and plants, particularly

from folk medicinal plants, is receiving increasing attention throughout the world.

The ancestors of the Polynesians are believed to have brought many plants with them, as food and medicine, when they migrated from Southeast Asia 2000 years ago. Of the 12 most

**1. Introduction** 

**2.** *Morinda citrifolia* **L.** 


### **Phytochemical Constituents and Activities of** *Morinda citrifolia* **L.**

Duduku Krishnaiah\*, Rajesh Nithyanandam and Rosalam Sarbatly *Universiti Malaysia Sabah, Malaysia* 

#### **1. Introduction**

126 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

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degradation and its color change of cyanidin-3-glucoside exposed to pulsed electric

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and vitamin A of a pulsed electric field-treated juice-milk beverage and behavior

Both artificial and naturally occurring antioxidants have been reported to play major roles in protecting membranes and tissues from free radical and xenobiotic-induced oxidative damage (Burton, 1989; Carini et al., 1990). Most living organisms harbour both enzymatic and non-enzymatic systems that protect them against excessive reactive oxygen species. However, various external factors (smoke, diet, alcohol and some drugs) and aging decrease the efficiency of these protective systems, thereby disrupting the redox equilibrium that is established under healthy conditions. Thus, antioxidants that scavenge reactive oxygen species may be of great value in preventing the onset and propagation of oxidative diseases (Willet, 1994). Recently, more attention has been paid to the role of natural antioxidants, mainly phenolic compounds, which may have higher antioxidant activity than do conventional antioxidants, such as vitamins C, E and β-carotene (Vinson et al., 1995). The anti-oxidative effects of natural phenolic compounds, in pure form or in extracts from various plant sources (vegetables, fruits and medicinal plants), have been studied *in vitro* using a variety of model systems (Meyer et al., 1998; Pietta et al., 1998; Yen & Hsieh, 1998). Therefore, antioxidants, which can neutralize free radicals, may be of central importance in the prevention of carcinogenicity, cardiovascular disease and neurodegenerative changes associated with aging (Halliwell, 1994; Yu, 1994). Epidemiological studies have shown that the consumption of vegetables and fruits can protect humans against oxidative damage by inhibiting and/or quenching free radicals and reactive oxygen species (Ames et al., 1993).

Oxidative stress occurs commonly in living organisms, and it is involved in the pathology of cancer, arteriosclerosis, malaria, and rheumatoid arthritis. Moreover, it may play a role in neurodegenerative diseases and ageing processes. It has been demonstrated that many vegetables, fruits, medicinal plants and other foods contain compounds with bioactivity against oxidative stress, and this activity has been attributed to vitamin C, vitamin E, αtocopherol, β-carotene, and polyphenolic compounds (Krishnaiah et al., 2011; Moure et al., 2001). Therefore, research regarding natural antioxidants from foods and plants, particularly from folk medicinal plants, is receiving increasing attention throughout the world.

#### **2.** *Morinda citrifolia* **L.**

The ancestors of the Polynesians are believed to have brought many plants with them, as food and medicine, when they migrated from Southeast Asia 2000 years ago. Of the 12 most

Phytochemical Constituents and Activities of *Morinda citrifolia* L. 129

Nevertheless, extraction of anthraquinones directly from plant roots is still more widely conducted and is conventionally performed by solvent extraction. Other techniques, which include super critical carbon dioxide extraction, subcritical water extraction, ultrasonicassisted extraction (UAE), and microwave-assisted extraction (MAE) have also become of

The genus Morinda (*Rubiaceae*), which includes the species *M. Citrifolia* L., is made up of around 80 species. *M. Citrifolia* is a bush or small tree, 3-10 m tall, with abundant broad

The small tubular white flowers are grouped together and inserted on the peduncle. The petioles leave ring-like marks on the stalks and the corolla is greenish-white (Morton, 1992; Elkins, 1998; Dixon et al., 1999; Ross, 2001; Cardon, 2003). The *M. citrifolia* L. fruit (3-10 cm length, 3-6 cm width) is ovular and fleshy with an embossed appearance (Fig. 1). It is slightly wrinkly, semi-translucent, and ranges in colour from green to yellow to almost white at the time of picking. It is covered with small reddish-brown buds containing the seeds. The ripe fruit emits a strong butyric acid-like, rancid smell (Morton, 1992; Dixon et al., 1999). The pulp is juicy and bitter, a light dull yellow or whitish colour, and gelatinous when the fruit is ripe; numerous hard triangular reddish-brown pits are found, each

Moreover, *M. citrifolia* L. leaves are well known for their strong antioxidant activity, and they have been shown to be safe in acute, subacute, and subchronic oral toxicity tests on mice (West et al., 2007). Inspired by ancient Polynesian legends, *M. citrifolia* L. leaves have been developed into therapeutic teas. The leaves are also the source for a variety of other health-promoting commercial products. Commercial *M. citrifolia* L. leaf products have been available in Japan and United States for more than seven years, used mainly for making infusions. However, some manufacturers produce capsules containing powdered *M.* 

containing four seeds (approximately 3.5 mm) (Dittmar, 1993).

interest as alternatives to the conventional methods.

elliptical leaves (5-17 cm length, 10-40 cm width).

**2.1 Plant description** 

Fig. 1. Unripe fruit

common medicinal plants they brought, *Morinda citrifolia* L. was the second most popular plant used in herbal remedies to treat various common diseases and to maintain overall good health. Other names of *Morinda citrifolia* L. include M. bracteata Roxb.; M. litoralis Blanco; Indian mulberry, Bengkudu, Mengkudu (Malay). It has been reported to have a broad range of health benefits for subjects with cancer, infections, arthritis, diabetes, asthma, hypertension and pain. The Polynesians utilised the whole *Morinda citrifolia* L. plant in their medicinal remedies and as a dye for some traditional clothing. The roots, stems, bark, leaves, flowers, and fruits of the *Morinda citrifolia* L. plant are all involved in various combinations in almost 40 known and recorded herbal remedies. Additionally, the roots were used to produce a yellow or red dye for tapa cloth and fala (mats), and the fruit was eaten for health and nutrition. There are numerous Polynesian stories of heroes and heroines that used *Morinda citrifolia* L. to survive famine. *Morinda citrifolia* has a long history of use as a food in tropical regions throughout the world.

It has also been reported to have broad therapeutic effects, including anti-cancer activity, in both humans and laboratory animal models. However, the mechanisms underlying these effects remain unknown. *M. citrifolia* is unique in view of the large number of medicinal claims that have been made for its efficacy and its rapidly evolving commercial success; nevertheless, little is known about its pharmacological potential compared with other popularly used botanicals.

*M. citrifolia* L., a shrub originating in tropical Asia or Polynesia, has been extensively used in folk medicine and as a dye in Asian countries. In the tropics, it seems to have been greatly valued medicinally, and the plant is normally cultivated for its roots, leaves and fruits. *M. citrifolia*, which grows prevalently in tropical regions, has recently gained a great deal of interest from scientists and medical professionals due to its pharmaceutical value. Wang et al. (2002) have published a review of *M. citrifolia* L. research that summarises the therapeutic effects of various compounds in this plant.

The *M. citrifolia* L. plant is a small evergreen tree found growing in open coastal regions at sea level and in forest areas up to approximately 1300 feet above sea level. The plant is often found growing along lava flows. Polynesians are reported to have successfully used *M. citrifolia* L. to treat breast cancer and eye problems. *M. citrifolia* has been tested for a number of biological activities in animal and anti-microbial studies and found that the dried fruit has smooth muscle stimulatory activity and histaminergic effects.

The roots of these plants are reported to be good sources of anthraquinones, which are usually present as aglycones and, to lesser extent, in the form of glycosides (Thomson, 1971; Zenk, El-Shagy & Schulte, 1975). Most parts of the tree have been widely used medicinally to relieve rheumatism and other pains and for their healing effects (Perry & Metzger, 1980).

Traditionally, the roots of *M. citrifolia* L. plants were used by Polynesians to produce yellow or red dye, but more importantly, they are now known to contain medicinally active components, such as anthraquinones, which due to their anti-oxidative activity, possess various therapeutic properties. These properties include anti-bacterial, anti-viral, and anticancer activities, as well as analgesic effects. These factors make the compounds potentially useful in several medical applications. An increasing number of studies are focusing on finding efficient methods for producing and extracting anthraquinones from these plants. Much of the literature also involves producing the compound in root cultures of *M. citrifolia*. Nevertheless, extraction of anthraquinones directly from plant roots is still more widely conducted and is conventionally performed by solvent extraction. Other techniques, which include super critical carbon dioxide extraction, subcritical water extraction, ultrasonicassisted extraction (UAE), and microwave-assisted extraction (MAE) have also become of interest as alternatives to the conventional methods.

#### **2.1 Plant description**

128 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

common medicinal plants they brought, *Morinda citrifolia* L. was the second most popular plant used in herbal remedies to treat various common diseases and to maintain overall good health. Other names of *Morinda citrifolia* L. include M. bracteata Roxb.; M. litoralis Blanco; Indian mulberry, Bengkudu, Mengkudu (Malay). It has been reported to have a broad range of health benefits for subjects with cancer, infections, arthritis, diabetes, asthma, hypertension and pain. The Polynesians utilised the whole *Morinda citrifolia* L. plant in their medicinal remedies and as a dye for some traditional clothing. The roots, stems, bark, leaves, flowers, and fruits of the *Morinda citrifolia* L. plant are all involved in various combinations in almost 40 known and recorded herbal remedies. Additionally, the roots were used to produce a yellow or red dye for tapa cloth and fala (mats), and the fruit was eaten for health and nutrition. There are numerous Polynesian stories of heroes and heroines that used *Morinda citrifolia* L. to survive famine. *Morinda citrifolia* has a long history

It has also been reported to have broad therapeutic effects, including anti-cancer activity, in both humans and laboratory animal models. However, the mechanisms underlying these effects remain unknown. *M. citrifolia* is unique in view of the large number of medicinal claims that have been made for its efficacy and its rapidly evolving commercial success; nevertheless, little is known about its pharmacological potential compared with other

*M. citrifolia* L., a shrub originating in tropical Asia or Polynesia, has been extensively used in folk medicine and as a dye in Asian countries. In the tropics, it seems to have been greatly valued medicinally, and the plant is normally cultivated for its roots, leaves and fruits. *M. citrifolia*, which grows prevalently in tropical regions, has recently gained a great deal of interest from scientists and medical professionals due to its pharmaceutical value. Wang et al. (2002) have published a review of *M. citrifolia* L. research that summarises the

The *M. citrifolia* L. plant is a small evergreen tree found growing in open coastal regions at sea level and in forest areas up to approximately 1300 feet above sea level. The plant is often found growing along lava flows. Polynesians are reported to have successfully used *M. citrifolia* L. to treat breast cancer and eye problems. *M. citrifolia* has been tested for a number of biological activities in animal and anti-microbial studies and found that the dried fruit

The roots of these plants are reported to be good sources of anthraquinones, which are usually present as aglycones and, to lesser extent, in the form of glycosides (Thomson, 1971; Zenk, El-Shagy & Schulte, 1975). Most parts of the tree have been widely used medicinally to relieve rheumatism and other pains and for their healing effects (Perry & Metzger, 1980). Traditionally, the roots of *M. citrifolia* L. plants were used by Polynesians to produce yellow or red dye, but more importantly, they are now known to contain medicinally active components, such as anthraquinones, which due to their anti-oxidative activity, possess various therapeutic properties. These properties include anti-bacterial, anti-viral, and anticancer activities, as well as analgesic effects. These factors make the compounds potentially useful in several medical applications. An increasing number of studies are focusing on finding efficient methods for producing and extracting anthraquinones from these plants. Much of the literature also involves producing the compound in root cultures of *M. citrifolia*.

of use as a food in tropical regions throughout the world.

therapeutic effects of various compounds in this plant.

has smooth muscle stimulatory activity and histaminergic effects.

popularly used botanicals.

The genus Morinda (*Rubiaceae*), which includes the species *M. Citrifolia* L., is made up of around 80 species. *M. Citrifolia* is a bush or small tree, 3-10 m tall, with abundant broad elliptical leaves (5-17 cm length, 10-40 cm width).

#### Fig. 1. Unripe fruit

The small tubular white flowers are grouped together and inserted on the peduncle. The petioles leave ring-like marks on the stalks and the corolla is greenish-white (Morton, 1992; Elkins, 1998; Dixon et al., 1999; Ross, 2001; Cardon, 2003). The *M. citrifolia* L. fruit (3-10 cm length, 3-6 cm width) is ovular and fleshy with an embossed appearance (Fig. 1). It is slightly wrinkly, semi-translucent, and ranges in colour from green to yellow to almost white at the time of picking. It is covered with small reddish-brown buds containing the seeds. The ripe fruit emits a strong butyric acid-like, rancid smell (Morton, 1992; Dixon et al., 1999). The pulp is juicy and bitter, a light dull yellow or whitish colour, and gelatinous when the fruit is ripe; numerous hard triangular reddish-brown pits are found, each containing four seeds (approximately 3.5 mm) (Dittmar, 1993).

Moreover, *M. citrifolia* L. leaves are well known for their strong antioxidant activity, and they have been shown to be safe in acute, subacute, and subchronic oral toxicity tests on mice (West et al., 2007). Inspired by ancient Polynesian legends, *M. citrifolia* L. leaves have been developed into therapeutic teas. The leaves are also the source for a variety of other health-promoting commercial products. Commercial *M. citrifolia* L. leaf products have been available in Japan and United States for more than seven years, used mainly for making infusions. However, some manufacturers produce capsules containing powdered *M.* 

Phytochemical Constituents and Activities of *Morinda citrifolia* L. 131

*M. citrifolia* L. has recently been the object of many claims concerning its nutraceutical properties. Various publications have shown that *M. citrifolia* L. can be used to relieve multiple diseases, and its registered uses span the Pacific, Asia, and Africa. Two clinical studies have reported that relief from arthritis and diabetes are associated with *M. citrifolia* L. consumption (Elkins, 1998; Solomon, 1999). These beneficial effects may derive from certain compounds, such as scopoletin, nitric oxide, alkaloids and/or sterols, and they also may be due to the antioxidant potential of *M. citrifolia* L. As a result of this reputation, consumption of this fruit is currently high not only in the producing countries but also in

In response to this demand, some countries (such as Costa Rica and Cambodia) have increased their cultivation of *M. citrifolia* L. In these countries, the fruit is often commercialised fresh or as juice in both formal and informal markets, but it is also found as pasteurised juice, either in pure form or in combination with other juices (usually grape or blackberry juice). Commercial interest in *M. citrifolia* L. has increased tremendously in recent years, as indicated by the number of patents registered. In the United States alone, 19 patents have been registered by the US patent and Trademark Office since 1976 (USPTO, 2005). *M. citrifolia* L. juice has been accepted by the European Union as a novel food (European Commission, Scientific Committee for Food, 2002). Nevertheless, despite the real market opportunities, there has been little scientific research addressing the actual

Several classes of compounds have been isolated from *M. citrifolia* L., including amino acids, anthraquinones, coumarins, fatty acids, flavonoids, iridoids, lignans and polysaccharides (Chan-Blan-co et al., 2006). Among these, scopoletin, a coumarin derivative, is one of the representative ingredients in *M. citrifolia* L. Its contribution to anti-microbial, antiinflammatory, and antioxidative activities has been well elucidated (Deng et al., 2007). Samoylenko et al. (2006) recommended scopoletin as a constituent marker for *M. citrifolia* L. quality control. This compound has been ubiquitously found in *M. citrifolia* L. collected from Atlantic and Pacific regions and in all examined squeezed fruit juices. Determination of scopoletin in *M. citrifolia* L. might help to control the quality of *M. citrifolia* L. products. However, no reports have evaluated the antioxidative activity of scopoletin and other

Many reports on the antioxidative activity of *M. citrifolia* L. itself have been published. Assays of free-radical-scavenging activity with 1,1-diphenyl-2-picrylhydrazyl (Su et al., 2005; Yang et al., 2007), inhibition of copper-induced low-density lipoprotein oxidation (Kamiya et al., 2004), nitric oxide scavenging activity (Basu & Hazra, 2006) and quenching of H2O2 (Chong et al., 2004; Jeffers et al., 2007) have been performed to evaluate the antioxidative effects of *M. citrifolia* L. and its products. Polyphenols, reducing glycosides (Calzuola et al., 2006), lignin derivatives (Su et al., 2005) and anthraquinones (Chong et al., 2004) have been suggested as sources of antioxidative activity in *M. citrifolia* L. However, there is little available information with which to quantitatively evaluate the antioxidative activity of *M. citrifolia* L ingredients. Recently, a correlation between total phenol and free-

The chemical components of *M. citrifolia* L. have not been well studied, and several anthraquinones and asperuloside are all that have been previously isolated (Levand &

the United States, Japan and Europe.

coumarin derivatives in *M. citrifolia* L.

nutritional and functional properties of *M. citrifolia* L. products.

radical-scavenging activity was reported (r=0.41, Yang et al., 2007).

*citrifolia* L. leaves. The major world-wide source of *M. citrifolia* L. leaves is French Polynesia because leaves from this nation having undergone a safety evaluation (West et al., 2007). Other sources include Panama, Fiji and Hawaii.

Recently, *M. citrifolia* L. fruit juice is in high demand as an alternative medicine due to its potential anti-microbial, anti-cancer, anti-inflammatory, and antioxidant effects (Wang et al., 2002). However, scientific evidence for the benefits of *M. citrifolia* L. fruit juice is still limited. In the past decade, *M. citrifolia* L. fruit juice has emerged on the worldwide market as a safe and popular health product due to its phytochemicals and nutrients. Written documentation of the consumption of this fruit as a food source precedes the twentieth century. Captain James Cook of the British Navy noted in the late 1700s that the fruit was eaten in Tahiti. An 1866 publication in London explained that *M. citrifolia* L. fruit was consumed as a food in the Fiji islands.

Later publications describe the use of this fruit as a food throughout the Pacific islands, Southeast Asia, Australia, and India. In Roratonga, "the fruit was often eaten by the natives". Australian Aborigines were reported to be "very fond" of the fruit. In Samoa, *M. citrifolia* L. fruit was common fare, and in Burma, the fruit was cooked in curries or eaten raw with salt. In 1943, Merrill described *M. citrifolia* L. as an edible plant in a technical manual of edible and poisonous plants of the Pacific islands, in which the leaves and fruits could be used as emergency food. Abbott also reported that *M. citrifolia* L. had been used as a food, drink, medicine, and colourful dye. The medicinal history and accumulated scientific studies have revealed and confirmed the Polynesians' claims regarding the health benefits of *M. citrifolia* L.

*M. citrifolia* L. has identifiable leaves, white tubular flowers, and a distinctive, ovoid, "grenade-like" yellow fruit. The fruit can grow in size up to 12 cm or more and has a lumpy surface covered by polygonal-shaped sections. The seeds, which are triangular and reddish brown, have an air sac contained at one end, making the seeds buoyant. This could explain, in part, the wide distribution of the plant throughout the Polynesian islands. The mature *M. citrifolia* L .fruit has a foul taste and odour. *M. citrifolia* L. is not considered to be at risk in the wild.

The fruit juice of *M. citrifolia* L. is in high demand in alternative medicine for various illnesses, such as arthritis, diabetes, high blood pressure, muscle aches and pains, menstrual difficulties, headaches, heart disease, Acquired Immune Deficiency Syndrome (AIDS), cancer, gastric ulcers, sprains, mental depression, senility, poor digestion, atherosclerosis, blood vessel problems and drug addiction (Kamiya et al., 2004; Wang et al., 2002). Therefore, one of the challenges in recent years has been to process fruit juice so as to make a more modern drug from a traditional product (Chunhieng et al., 2003). A number of *in vitro* biological activities have been reported, such as angiogenesis inhibition, antioxidant activity, inhibition of cycloxygenases-1 and -2, and tyrosine kinase inhibition. However, most of these have only been tested with crude extracts or fractions of *M. citrifolia* L., and the compound(s) responsible for these biological activities have not been fully determined, except for two compounds, neolignan and americanin A, which were identified in an nbutanol-soluble partition of the methanol extract of *M. citrifolia* L. fruits (Su et al., 2005; Zin et al., 2002).

*citrifolia* L. leaves. The major world-wide source of *M. citrifolia* L. leaves is French Polynesia because leaves from this nation having undergone a safety evaluation (West et al., 2007).

Recently, *M. citrifolia* L. fruit juice is in high demand as an alternative medicine due to its potential anti-microbial, anti-cancer, anti-inflammatory, and antioxidant effects (Wang et al., 2002). However, scientific evidence for the benefits of *M. citrifolia* L. fruit juice is still limited. In the past decade, *M. citrifolia* L. fruit juice has emerged on the worldwide market as a safe and popular health product due to its phytochemicals and nutrients. Written documentation of the consumption of this fruit as a food source precedes the twentieth century. Captain James Cook of the British Navy noted in the late 1700s that the fruit was eaten in Tahiti. An 1866 publication in London explained that *M. citrifolia* L. fruit was consumed as a food in the

Later publications describe the use of this fruit as a food throughout the Pacific islands, Southeast Asia, Australia, and India. In Roratonga, "the fruit was often eaten by the natives". Australian Aborigines were reported to be "very fond" of the fruit. In Samoa, *M. citrifolia* L. fruit was common fare, and in Burma, the fruit was cooked in curries or eaten raw with salt. In 1943, Merrill described *M. citrifolia* L. as an edible plant in a technical manual of edible and poisonous plants of the Pacific islands, in which the leaves and fruits could be used as emergency food. Abbott also reported that *M. citrifolia* L. had been used as a food, drink, medicine, and colourful dye. The medicinal history and accumulated scientific studies have revealed and confirmed the Polynesians' claims regarding the health benefits

*M. citrifolia* L. has identifiable leaves, white tubular flowers, and a distinctive, ovoid, "grenade-like" yellow fruit. The fruit can grow in size up to 12 cm or more and has a lumpy surface covered by polygonal-shaped sections. The seeds, which are triangular and reddish brown, have an air sac contained at one end, making the seeds buoyant. This could explain, in part, the wide distribution of the plant throughout the Polynesian islands. The mature *M. citrifolia* L .fruit has a foul taste and odour. *M. citrifolia* L. is not considered to be at risk in the

The fruit juice of *M. citrifolia* L. is in high demand in alternative medicine for various illnesses, such as arthritis, diabetes, high blood pressure, muscle aches and pains, menstrual difficulties, headaches, heart disease, Acquired Immune Deficiency Syndrome (AIDS), cancer, gastric ulcers, sprains, mental depression, senility, poor digestion, atherosclerosis, blood vessel problems and drug addiction (Kamiya et al., 2004; Wang et al., 2002). Therefore, one of the challenges in recent years has been to process fruit juice so as to make a more modern drug from a traditional product (Chunhieng et al., 2003). A number of *in vitro* biological activities have been reported, such as angiogenesis inhibition, antioxidant activity, inhibition of cycloxygenases-1 and -2, and tyrosine kinase inhibition. However, most of these have only been tested with crude extracts or fractions of *M. citrifolia* L., and the compound(s) responsible for these biological activities have not been fully determined, except for two compounds, neolignan and americanin A, which were identified in an nbutanol-soluble partition of the methanol extract of *M. citrifolia* L. fruits (Su et al., 2005; Zin

Other sources include Panama, Fiji and Hawaii.

Fiji islands.

of *M. citrifolia* L.

wild.

et al., 2002).

*M. citrifolia* L. has recently been the object of many claims concerning its nutraceutical properties. Various publications have shown that *M. citrifolia* L. can be used to relieve multiple diseases, and its registered uses span the Pacific, Asia, and Africa. Two clinical studies have reported that relief from arthritis and diabetes are associated with *M. citrifolia* L. consumption (Elkins, 1998; Solomon, 1999). These beneficial effects may derive from certain compounds, such as scopoletin, nitric oxide, alkaloids and/or sterols, and they also may be due to the antioxidant potential of *M. citrifolia* L. As a result of this reputation, consumption of this fruit is currently high not only in the producing countries but also in the United States, Japan and Europe.

In response to this demand, some countries (such as Costa Rica and Cambodia) have increased their cultivation of *M. citrifolia* L. In these countries, the fruit is often commercialised fresh or as juice in both formal and informal markets, but it is also found as pasteurised juice, either in pure form or in combination with other juices (usually grape or blackberry juice). Commercial interest in *M. citrifolia* L. has increased tremendously in recent years, as indicated by the number of patents registered. In the United States alone, 19 patents have been registered by the US patent and Trademark Office since 1976 (USPTO, 2005). *M. citrifolia* L. juice has been accepted by the European Union as a novel food (European Commission, Scientific Committee for Food, 2002). Nevertheless, despite the real market opportunities, there has been little scientific research addressing the actual nutritional and functional properties of *M. citrifolia* L. products.

Several classes of compounds have been isolated from *M. citrifolia* L., including amino acids, anthraquinones, coumarins, fatty acids, flavonoids, iridoids, lignans and polysaccharides (Chan-Blan-co et al., 2006). Among these, scopoletin, a coumarin derivative, is one of the representative ingredients in *M. citrifolia* L. Its contribution to anti-microbial, antiinflammatory, and antioxidative activities has been well elucidated (Deng et al., 2007). Samoylenko et al. (2006) recommended scopoletin as a constituent marker for *M. citrifolia* L. quality control. This compound has been ubiquitously found in *M. citrifolia* L. collected from Atlantic and Pacific regions and in all examined squeezed fruit juices. Determination of scopoletin in *M. citrifolia* L. might help to control the quality of *M. citrifolia* L. products. However, no reports have evaluated the antioxidative activity of scopoletin and other coumarin derivatives in *M. citrifolia* L.

Many reports on the antioxidative activity of *M. citrifolia* L. itself have been published. Assays of free-radical-scavenging activity with 1,1-diphenyl-2-picrylhydrazyl (Su et al., 2005; Yang et al., 2007), inhibition of copper-induced low-density lipoprotein oxidation (Kamiya et al., 2004), nitric oxide scavenging activity (Basu & Hazra, 2006) and quenching of H2O2 (Chong et al., 2004; Jeffers et al., 2007) have been performed to evaluate the antioxidative effects of *M. citrifolia* L. and its products. Polyphenols, reducing glycosides (Calzuola et al., 2006), lignin derivatives (Su et al., 2005) and anthraquinones (Chong et al., 2004) have been suggested as sources of antioxidative activity in *M. citrifolia* L. However, there is little available information with which to quantitatively evaluate the antioxidative activity of *M. citrifolia* L ingredients. Recently, a correlation between total phenol and freeradical-scavenging activity was reported (r=0.41, Yang et al., 2007).

The chemical components of *M. citrifolia* L. have not been well studied, and several anthraquinones and asperuloside are all that have been previously isolated (Levand &

Phytochemical Constituents and Activities of *Morinda citrifolia* L. 133

Table 1. Evolution of fruit skin colour and firmness in the course of ripening (adapted from

Depending on the post-harvest technology programme adopted, the fruits may be harvested at different stages of development and continue to mature. The evolution of the colour and firmness of fruits left to ripen naturally on the tree is reported in Table 1. Nonetheless, most processors buy *M. citrifolia* L. harvested at the "hard white" stage for juice production as the fruits become soft too quickly once this stage is reached (Nelson, 2001, 2003). The change from stage 4 to 5 occurs very quickly (within a few hours), and the pulp practically liquefies

The fruits are individually selected on the tree and harvested by hand. At the "hard white" stage, they are well able to withstand being transported in baskets or containers, and exposure of the fruits to light or high temperatures immediately after harvest does not affect their overall quality. Before processing, fruits are ripened at room temperature for one day or more, depending on the end product (such as tea, juice, pulp, dietetic products) (Nelson,

Approximately 160 phytochemical compounds have already been identified in the *M. citrifolia* L. plant, and the major micronutrients are phenolic compounds, organic acids and alkaloids (Wang & Su, 2001). Of the reported phenolic compounds, the most important are anthraquinones (such as damnacanthal, morindone, morindin.) and also aucubin, asperuloside, and scopoletin (Wang & Su, 2001). The main organic acids are caproic and caprylic acids (Dittmar, 1993), whereas the principal reported alkaloid is xeronine (Heinicke, 1985). Chan-Blanco et al. (2006) reviewed the chemical constituents of different parts of the

However, chemical composition differs significantly according to the part of the plant. The complete physico-chemical composition of the fruit has not yet been reported, and only partial information is available on *M. citrifolia* L. juice. The fruit contains 90% water, and the main components of the dry matter appear to be soluble solids, dietary fibre and proteins (Chunhieng, 2003). The fruit's protein content is surprisingly high, representing 11.3% of the juice dry matter, and the main amino acids are aspartic acid, glutamic acid and isoleucine

According to a book on Malaysian medicinal plants, the chemical constituents of *M. citrifolia* L. are 5,7-Acacetin-7-O-β-D(+)-glycopyranoside, ajmalicine isomers, alizarin, asperuloside, asperulosidic acid, chrysophanol (1,8-dihydroxy-3-methylanthraquinone), damnacanthol, digoxin, 5,6-dihydroxylucidin, 5,6-dihydroxylucidin-3-β-primeveroside, 5,7-dimethylapigenin-4'-O-β-D(+)-galacto pyranoside, lucidin, lucidin-3-β-primeveroside, 2-methyl-3,5,6-trihydroxy

and turns from green to white, as well as develops the characteristic butyric smell.

Chan-Blanco et al., 2006)

**2.3 Chemical composition of** *M. citrifolia* **L.** 

2003).

plant (Table 2).

(Chunhieng, 2003).

Maturity stage Colour Firmness 1 Dark green Very hard 2 Green-yellow Very hard 3 Pale yellow Very hard 4 Pale yellow Fairly hard 5 Translucent-greyish Soft

Larson, 1979; Srivastava & Singh, 1993). For centuries, scientists and medical professionals have been investigating the chemical constituents in all parts of *M. citrifolia* (Noni or Yor), including leaves, fruit, bark and roots. The plants contain several medicinally active components that exhibit various therapeutic effects. These include anti-bacterial, anti-viral and anti-cancer activities as well as analgesic effects. Critical reviews of the therapeutic properties of the plants are given by Chan-Blanco et al. (2006) and Wang et al. (2002). Anthraquinones have been shown to be responsible for the therapeutic properties of *M. citrifolia* L., and among this group of compounds, damnacanthal, which is present mainly in the roots, is of particular interest due to its important anticancer activity (Hiramatsu et al., 1993).

Previous phytochemical studies revealed that *M. citrifolia* L. leaves contain a variety of phytochemical constituents, including terpenoids (Ahmad & Bano, 1980; Saludes et al., 2002; Takashima et al., 2007) phytosterols, fatty acids and their glycosides (Takashima et al., 2007) iridoids and their glycosides (Sang et al., 2001 a,b,c,d; Sang et al., 2003) and flavonol glycosides (Sang et al., 2001a).

Flavonol glycosides appear to predominate in *M. citrifolia* L. leaves; rutin and other flavonol glycosides have previously been identified in raw *M. citrifolia* L. leaves (Sang et al., 2001a). However, the presence of flavonol aglycones in *M. citrifolia* L. leaves has not been previously reported. Flavonoids have been indicated to possess a variety of biological activities (Garcia-Mediavilla et al., 2006; Kampkotter et al., 2007), and they may play an important role in *M. citrifolia* L. leaves. To date, there has been no validated analytical method for determining the flavonol constituents of *M. citrifolia* L. leaves.

#### **2.2 Yield**

*M. citrifolia* L. is a perennial bush, and it is possible to find fruits at different stages of maturity on the same plant at the same time. The species is generally found from sea level to 400 m, although it adapts better to coastal regions (Luberck & Hannes, 2001). Under favourable conditions, the plant bears fruit approximately nine months to one year after planting. At this stage, the fruits can be harvested, but they are generally small, and the yield per tree is low. Some producers choose not to harvest in the first year, and they prune in order to let the bush grow stronger. In Hawaii, *M. citrifolia*  L. fruits are harvested throughout the year, although there are seasonal patterns in flowering and fruit bearing (meteorological factors, fumigation, and irrigation) (Nelson, 2001, 2003).

In Hawaii, *M. citrifolia* L. plots are usually harvested two or three times per month, although fruit production is lower during winter. With a density of 638 plants per hectare; good soil fertility, drainage, and irrigation; appropriate pest, disease and weed control; and an appropriate fertilisation plan, it is possible to obtain yields of 7 tonnes/ha/year after the fifth year (Nelson, 2001, 2003). With a juice extraction rate of approximately 50% (w/w), one hectare can thus yield around 35 tons of juice. However, many factors may affect these yields, and most producers do not obtain such good results because of diseases and/or poor agricultural practices (growing wide plants). In Hawaii, an average annual yield of 50 tonnes/ha is generally attained (Nelson, 2001, 2003).

Larson, 1979; Srivastava & Singh, 1993). For centuries, scientists and medical professionals have been investigating the chemical constituents in all parts of *M. citrifolia* (Noni or Yor), including leaves, fruit, bark and roots. The plants contain several medicinally active components that exhibit various therapeutic effects. These include anti-bacterial, anti-viral and anti-cancer activities as well as analgesic effects. Critical reviews of the therapeutic properties of the plants are given by Chan-Blanco et al. (2006) and Wang et al. (2002). Anthraquinones have been shown to be responsible for the therapeutic properties of *M. citrifolia* L., and among this group of compounds, damnacanthal, which is present mainly in the roots, is of particular interest due to its important anticancer activity (Hiramatsu et al.,

Previous phytochemical studies revealed that *M. citrifolia* L. leaves contain a variety of phytochemical constituents, including terpenoids (Ahmad & Bano, 1980; Saludes et al., 2002; Takashima et al., 2007) phytosterols, fatty acids and their glycosides (Takashima et al., 2007) iridoids and their glycosides (Sang et al., 2001 a,b,c,d; Sang et al., 2003) and flavonol

Flavonol glycosides appear to predominate in *M. citrifolia* L. leaves; rutin and other flavonol glycosides have previously been identified in raw *M. citrifolia* L. leaves (Sang et al., 2001a). However, the presence of flavonol aglycones in *M. citrifolia* L. leaves has not been previously reported. Flavonoids have been indicated to possess a variety of biological activities (Garcia-Mediavilla et al., 2006; Kampkotter et al., 2007), and they may play an important role in *M. citrifolia* L. leaves. To date, there has been no validated analytical method for determining

*M. citrifolia* L. is a perennial bush, and it is possible to find fruits at different stages of maturity on the same plant at the same time. The species is generally found from sea level to 400 m, although it adapts better to coastal regions (Luberck & Hannes, 2001). Under favourable conditions, the plant bears fruit approximately nine months to one year after planting. At this stage, the fruits can be harvested, but they are generally small, and the yield per tree is low. Some producers choose not to harvest in the first year, and they prune in order to let the bush grow stronger. In Hawaii, *M. citrifolia*  L. fruits are harvested throughout the year, although there are seasonal patterns in flowering and fruit bearing (meteorological factors, fumigation, and irrigation) (Nelson,

In Hawaii, *M. citrifolia* L. plots are usually harvested two or three times per month, although fruit production is lower during winter. With a density of 638 plants per hectare; good soil fertility, drainage, and irrigation; appropriate pest, disease and weed control; and an appropriate fertilisation plan, it is possible to obtain yields of 7 tonnes/ha/year after the fifth year (Nelson, 2001, 2003). With a juice extraction rate of approximately 50% (w/w), one hectare can thus yield around 35 tons of juice. However, many factors may affect these yields, and most producers do not obtain such good results because of diseases and/or poor agricultural practices (growing wide plants). In Hawaii, an average annual yield of 50

1993).

**2.2 Yield** 

2001, 2003).

glycosides (Sang et al., 2001a).

the flavonol constituents of *M. citrifolia* L. leaves.

tonnes/ha is generally attained (Nelson, 2001, 2003).


Table 1. Evolution of fruit skin colour and firmness in the course of ripening (adapted from Chan-Blanco et al., 2006)

Depending on the post-harvest technology programme adopted, the fruits may be harvested at different stages of development and continue to mature. The evolution of the colour and firmness of fruits left to ripen naturally on the tree is reported in Table 1. Nonetheless, most processors buy *M. citrifolia* L. harvested at the "hard white" stage for juice production as the fruits become soft too quickly once this stage is reached (Nelson, 2001, 2003). The change from stage 4 to 5 occurs very quickly (within a few hours), and the pulp practically liquefies and turns from green to white, as well as develops the characteristic butyric smell.

The fruits are individually selected on the tree and harvested by hand. At the "hard white" stage, they are well able to withstand being transported in baskets or containers, and exposure of the fruits to light or high temperatures immediately after harvest does not affect their overall quality. Before processing, fruits are ripened at room temperature for one day or more, depending on the end product (such as tea, juice, pulp, dietetic products) (Nelson, 2003).

#### **2.3 Chemical composition of** *M. citrifolia* **L.**

Approximately 160 phytochemical compounds have already been identified in the *M. citrifolia* L. plant, and the major micronutrients are phenolic compounds, organic acids and alkaloids (Wang & Su, 2001). Of the reported phenolic compounds, the most important are anthraquinones (such as damnacanthal, morindone, morindin.) and also aucubin, asperuloside, and scopoletin (Wang & Su, 2001). The main organic acids are caproic and caprylic acids (Dittmar, 1993), whereas the principal reported alkaloid is xeronine (Heinicke, 1985). Chan-Blanco et al. (2006) reviewed the chemical constituents of different parts of the plant (Table 2).

However, chemical composition differs significantly according to the part of the plant. The complete physico-chemical composition of the fruit has not yet been reported, and only partial information is available on *M. citrifolia* L. juice. The fruit contains 90% water, and the main components of the dry matter appear to be soluble solids, dietary fibre and proteins (Chunhieng, 2003). The fruit's protein content is surprisingly high, representing 11.3% of the juice dry matter, and the main amino acids are aspartic acid, glutamic acid and isoleucine (Chunhieng, 2003).

According to a book on Malaysian medicinal plants, the chemical constituents of *M. citrifolia* L. are 5,7-Acacetin-7-O-β-D(+)-glycopyranoside, ajmalicine isomers, alizarin, asperuloside, asperulosidic acid, chrysophanol (1,8-dihydroxy-3-methylanthraquinone), damnacanthol, digoxin, 5,6-dihydroxylucidin, 5,6-dihydroxylucidin-3-β-primeveroside, 5,7-dimethylapigenin-4'-O-β-D(+)-galacto pyranoside, lucidin, lucidin-3-β-primeveroside, 2-methyl-3,5,6-trihydroxy

Phytochemical Constituents and Activities of *Morinda citrifolia* L. 135

Leaves Valine Dittmar (1993), Elkins (1998)

Plant 3-hydroxymorindone Cardon (2003), Inoue et al.

Plant 5,6-dihydroxylucidin Cardon (2003), Inoue et al.

Plant Aucubin Elkins (1998), Wang et al.

Plant Lucidin Cardon (2003), Inoue et al.

Plant Scopoletin Farine et al. (1996), Wang et al.

Leaves β-sitosterol Sang et al. (2002), Chunhieng

Leaves Cysteine Dittmar (1993), Elkins (1998) Leaves Cystine Dittmar (1993), Elkins (1998)

Leaves Glycine Dittmar (1993), Elkins (1998) Leaves Histidine Dittmar (1993), Elkins (1998) Leaves Isoleucine Dittmar (1993), Elkins (1998)

Plant Linoleic acid Wang et al. (2002)

Leaves Arginine Dittmar (1993) Leaves Aspartic acid Dittmar (1993)

Leaves Citrifolinoside B Sang et al. (2002)

Leaves Glutamic acid Dittmar (1993)

(2003), Elkins (1998), Wang et

Cardon (2003), Inoue et al.

Cardon (2003), Inoue et al.

Cardon (2003), Inoue et al.

Cardon (2003), Inoue et al.

al. (2002)

(1981)

(1981)

(1981)

(1981)

(1981)

(1981)

(2002)

(1981)

(2002)

al. (2002)

Sang et al. (2002)

(1981), Ross (2001)

Cardon (2003), Inoue et al.

(2003), Elkins (1998), Wang et

**Source (plant part) Chemical constituent References**  Leaves Tyrosine Dittmar (1993), Elkins (1998) Leaves Ursolic acid Sang et al. (2002), Cardon

trihydroxyanthraquinone

xylopyranosyl-(1-6)-β-D-

xylopyranosyl-(1-6)-β-D-

xylopyranosyl-(1-6)-β-D-

glucopyranoside

glucopyranoside

glucopyranoside

Plant Lucidin 3-O-β-D-xylopyranosyl-(1-6)-β-D-glucopyranoside

Leaves Kaempferol 3-O-α-L-rhamnopyranosyl- (1-6)-β-D-glucopyranoside

Plant 3-hydroxymorindone 6-O-β-D-

Plant 5,6-dihydroxylucidin 3-O-β-D-

trihydroxyanthraquinone 6-O-β-D-

Plant 2-methyl-3,5,6-

Plant 2-methyl-3,5,6-


Fruit β-D-glucopyranose pentaacetate Sang et al. (2002), Elkins (1998)

Fruit Asperulosidic acid Morton (1992), Elkins (1998),

Fruit Asperuloside tetraacetate Wang et al. (1999), Liu et al.

Fruit Caprylic acid Sang et al. (2002), Dittmar

Fruit Ethyl caprylate Solomon (1999), Dittmar

Fruit Octanoic acid Farine et al. (1996), Sang et al.

Leaves Alanine Sang et al. (2002), Srivastava &

Leaves Serine Dittmar (1993), Elkins (1998) Leaves Threonine Dittmar (1993), Elkins (1998) Leaves Tryptophan Dittmar (1993), Elkins (1998)

Sang et al. (2002)

Dittmar (1993)

(2002)

(1979)

(2002)

Su (2001)

(2002)

Wang et al. (1999)

(2001), Cardon (2003)

Sang et al. (2002), Elkins (1998)

Wang et al. (2002), McClatchey

(1993), Elkins (1998), Wang et al. (2002), Levend and Larson

(1993), Cardon (2003), Elkins (1998), Wang et al. (2002), Levand and Larson (1979)

Farine et al. (1996), Sang et al. (2002), Cardon (2003), Wang &

Wang & Su (2001), Wang et al.

Singh (1993), Cardon (2003)

Sang et al. (2002)

**Source (plant part) Chemical constituent References** 

dimethoxyanthraquinone 4-O-β-D-

glucopyranosyl-(1-4)-α-Lrhamnopyranoside

octanoyl-β-D-glucopyranose

Fruit Ascorbic acid Liu et al. (2001)

Fruit Caproic acid Dittmar (1993)

Fruit Ethyl caproate Dittmar (1993) Fruit Hexanoic acid Dittmar (1993)

3)-β-D-galactopyranosyl-(1-6)-β-D-

Fruit Quercetin 3-O-α-L-rhamnopyranosyl-

Heartwood Physcion 8-O-α-L-arabinopyranosyl-(1-

galactopyranoside

Leaves Quercetin 3-O-α-L-rhamnopyranosyl-

(1-6)-β-D-glucopyranoside

(1-6)-β-D-glucopyranoside

Flower 2-methyl-4-hydroxy-5,7-

Flower 5,8-dimethyl-apigenin 4'-O-β-Dgalacatopyranoside

Flower Aracetin 7-O-β-D-glucopyranoside

Fruit 2,6-di-O-(β-D-glucopyranosyl-1-O-

Fruit 6-O-(β-D-glucopyranosyl-1-O-octanoylβ-D-glucopyranose


Phytochemical Constituents and Activities of *Morinda citrifolia* L. 137

anthraquinone, 3-hydroxymorindone, 3-hydroxymorindone-6-β-primereroside, αmethoxyalizarin, 2-methyl-3,5,6-trihydroxyanthraquinone-6-β-primeveroside, monoethoxyrubiadin, morindadiol, morindin, morindone (1,5,6-trihydroxy-2 methylanthraquinone), morindone-6-β-primeveroside, nordamnacanthal, quinoline, rubiadin, rubiadin 1-methyl ether, saronjidiol, ursolic acid, alkaloids, anthraquinones and their glycosides, caproic acid, caprylic acid, fatty acids and alcohols (C5-9), flavone glycosides, flavonoids, glucose (β-D-glucopyranose), indoles, purines, and β-sitosterol.

Minerals account for 8.4% of the dry matter, and these minerals are mainly potassium, sulphur, calcium and phosphorus; traces of selenium have been reported in the juice (Chunhieng, 2003, Table 3). Vitamins have been reported in the fruit, mainly ascorbic acid (24-158 mg/100 g dry matter) (Morton, 1992; Shovie & Whistler, 2001) and provitamin A (Dixon et al., 1999). Phenolic compounds have been found to be the major group of functional micronutrients in *M. citrifolia* L. juice: damnacanthal, scopoletin, morindone, alizarin, aucubin, nordamnacanthal, rubiadin, rubiadin-1-methyl ether and other anthraquinone glycosides have been identified in *M. citrifolia* L. (Morton, 1992; Dittmar, 1993; Dixon et al., 1999; Wang & Su, 2001). Damnacanthal is an anthraquinone that has been characterised and has some important functional properties (mainly anti-carcinogenic) (Solomon, 1999). Scopoletin is a coumarin that was isolated in 1993 at the University of Hawaii and has been found to have analgesic properties as well as a significant ability to control serotonin levels in the body (Levand & Larson, 1979). Other researchers have shown that scopoletin may also have anti-microbial (Duncan et al., 1998) and anti-hypertensive

Multiple Hawaiian teams (Heinicke, 1985; Solomon, 1999) have reported the presence of a novel component, proxeronine, which is an alkaloid that is claimed to combine with human proteins and improve their functionality. The above authors attribute most of the beneficial effects of *M. citrifolia* L. to xeronine. Nonetheless, neither the chemical characterisation of

(2001)a

European

commission (2002)b

this alkaloid nor the method used to assess its levels has been published to date.

pH 3.72 - 3.4-3.6 Dry matter 9.8±0.4% - 10-11% Total soluble solids (°Brix) 8 - -

Protein content 2.5% 0.4 g/100 g 0.2-0.5% Lipids 0.15% 0.3 g/100 g 0.1-0.2% Glucose 11.9±0.2 g/l - 3-4 g/100 g Fructose 8.2±0.2 g/l - 3-4 g/100 g Potassium 3900 mg/l 188 mg/100 g 30-150 mg/100 g Sodium 214 mg/l 21 mg/100 g 15-40 mg/100 g Magnesium 14 mg/l 14.5 mg/100 g 3-12 mg/100 g Calcium 28 mg/l 41.7 mg/100 g 20-25 mg/100 g Vitamin C - 155 mg/100 g 3-25 mg/100 g Table 3. Physico-chemical composition of *M. citrifolia* L. juice (adapted from Chan-Blanco et

Characteristic Chunhieng (2003)a Shovic and Whistler

effects (Solomon, 1999).

al., 2006)


Table 2. Chemical compounds of *M. citrifolia* L. (adapted from Chan-Blanco et al., 2006)

Leaves Leucine Dittmar (1993), Elkins (1998) Leaves Methionine Dittmar (1993), Elkins (1998) Leaves Phenylalanine Dittmar (1993), Elkins (1998) Leaves Proline Dittmar (1993), Elkins (1998)

Morindone Sang et al. (2002), Inoue et al.

Damnacanthal Sang et al. (2002), Cardon

Sang et al. (2002)

(1981), Dittmar (1993), Ross (2001), Cardon (2003), Wang et

Cardon (2003), Solomon (1999)

Inoue et al. (1981), Ross (2001)

Elkins (1998), Morton (1992)

Elkins (1998), Ross (2001),

Ross (2001)

Alizarin Cardon (2003), Dittmar (1993),

Dittmar (1993)

Wang et al. (2002)

al. (2002)

Sang et al. (2002)

(2003)

**Source (plant part) Chemical constituent References** 

(1-2)- α-L-rhamnopyranosyl-(1-6)-β-D-

Leaves Quercetin 3-O-β-D-glucopyranoside Sang et al. (2002)

2)- α-L-rhamnopyranosyl-(1-6)-β-D-

Root 1,3-dihydroxy-6-methyl anthraquinone Morton (1992) Root Morenone 1 Solomon (1999) Root Morenone 2 Solomon (1999) Root Ruberythric acid Cardon (2003)

Root bark Hexose Dittmar (1993) Root bark Morindadiol Dittmar (1993) Root bark Morindanidrine Dittmar (1993)

Root bark Pentose Dittmar (1993) Root bark Physcion Solomon (1999) Root bark Rubiadin monomethyl ether Dittmar (1993)

Seeds Ricinoleic acid Solomon (1999)

Table 2. Chemical compounds of *M. citrifolia* L. (adapted from Chan-Blanco et al., 2006)

Root bark Trioxymethylanthraquinone monoethyl

ether

Root, root bark,

fruit

Root Rubichloric acid Elkins (1998), Morton (1992)

Root Rubiadin Cardon (2003), Elkins (1998),

Root bark Chlororubin Dittmar (1993), Elkins (1998)

Root bark Morindine Cardon (2003), Dittmar (1993),

Root bark Soranjidiol Dittmar (1993), Elkins (1998),

Leaves Kaempferol 3-O-β-D-glucopyranosyl-

galactopyranoside

Leaves Quercetin 3-O-β-D-glucopyranosyl-(1-

galactopyranoside

Root 8-hydroxy-8-methoxy-2-methylanthraquinone

Root, heartwood,

Root, heartwood,

root bark

seeds

anthraquinone, 3-hydroxymorindone, 3-hydroxymorindone-6-β-primereroside, αmethoxyalizarin, 2-methyl-3,5,6-trihydroxyanthraquinone-6-β-primeveroside, monoethoxyrubiadin, morindadiol, morindin, morindone (1,5,6-trihydroxy-2 methylanthraquinone), morindone-6-β-primeveroside, nordamnacanthal, quinoline, rubiadin, rubiadin 1-methyl ether, saronjidiol, ursolic acid, alkaloids, anthraquinones and their glycosides, caproic acid, caprylic acid, fatty acids and alcohols (C5-9), flavone glycosides, flavonoids, glucose (β-D-glucopyranose), indoles, purines, and β-sitosterol.

Minerals account for 8.4% of the dry matter, and these minerals are mainly potassium, sulphur, calcium and phosphorus; traces of selenium have been reported in the juice (Chunhieng, 2003, Table 3). Vitamins have been reported in the fruit, mainly ascorbic acid (24-158 mg/100 g dry matter) (Morton, 1992; Shovie & Whistler, 2001) and provitamin A (Dixon et al., 1999). Phenolic compounds have been found to be the major group of functional micronutrients in *M. citrifolia* L. juice: damnacanthal, scopoletin, morindone, alizarin, aucubin, nordamnacanthal, rubiadin, rubiadin-1-methyl ether and other anthraquinone glycosides have been identified in *M. citrifolia* L. (Morton, 1992; Dittmar, 1993; Dixon et al., 1999; Wang & Su, 2001). Damnacanthal is an anthraquinone that has been characterised and has some important functional properties (mainly anti-carcinogenic) (Solomon, 1999). Scopoletin is a coumarin that was isolated in 1993 at the University of Hawaii and has been found to have analgesic properties as well as a significant ability to control serotonin levels in the body (Levand & Larson, 1979). Other researchers have shown that scopoletin may also have anti-microbial (Duncan et al., 1998) and anti-hypertensive effects (Solomon, 1999).

Multiple Hawaiian teams (Heinicke, 1985; Solomon, 1999) have reported the presence of a novel component, proxeronine, which is an alkaloid that is claimed to combine with human proteins and improve their functionality. The above authors attribute most of the beneficial effects of *M. citrifolia* L. to xeronine. Nonetheless, neither the chemical characterisation of this alkaloid nor the method used to assess its levels has been published to date.


Table 3. Physico-chemical composition of *M. citrifolia* L. juice (adapted from Chan-Blanco et al., 2006)

Phytochemical Constituents and Activities of *Morinda citrifolia* L. 139

transport the fruit by boat from the scattered Pacific islands to processing plants without

It has been reported that *M. citrifolia* L. inhibits the growth of certain bacteria, such as staphylococcus aureus, Pseudomonas aeruginosa, Proteus morgaii, Bacillus subtilis, Escherichia coli, Helicobacter pylori, Salmonella and Shigella (Atkinson, 1956). The same author claims that the anti-microbial effect observed may be due to the presence of phenolic compounds such as acubin, l-asperuloside, alizarin, scopoletin and other anthraquinones. Another study showed that an acetonitrile extract of the dried fruit inhibits the growth of Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, and Streptococcus pyrogene

It has also been found that ethanol and hexane extracts of *M. citrifolia* L. have an antitubercular effect as they inhibit the growth of Mycobacterium tuberculosis by 89-95% (Saludes et al., 2002). The major components identified in the hexane extract are E-phytol, cycloartenol, stigmasterol, β-sitosterol, campesta-5,7,22-trien-3-β-ol, and the ketosteroids

Other studies have reported a significant antimicrobial effect on various strains of Salmonella, Shigella, and E. coli (Bushnell et al., 1950; Dittmar, 1993). Furthermore, they showed that the anti-microbial effect is highly dependent on the stage of ripeness and on

The immunomodulatory properties (the capacity to enhance the host immune system) of *M. citrifolia* L. juice have been studied by a Japanese research team (Hirazumi et al., 1996; Hirazumi & Furusawa, 1999). The ethanol precipitable fraction (ppt) of *M. citrifolia* L. juice, corresponding to a polysaccharide-rich substance composed of glucuronoic acid, galactose, arabinose and rhamnose, has been found to have immunomodulatory and anti-tumour effects against Lewis lung carcinoma (LLC). In cell models, *M. citrifolia* L.-ppt seems to stimulate the production of T-cells, thymocytes and macrophages that produce cytokines,

*M. citrifolia* L.-ppt also appears to stimulate murine effector cells to release several mediators such as cytokines. These mediators slow down the cell cycle in tumours, increase the response of cells to other immunised cells that fight tumour growth and have potent macrophage activator activity suspected of playing a role in the death of tumours (Hirazumi

In the same study, mice were inoculated with LLC, and those ingesting a daily dose of 15 mg of *M. citrifolia* L. juice showed a significant increase (119%) in lifespan. Nine out of 22 mice with terminal cancer survived for more than 50 days. In addition, the ingestion of *M. citrifolia* L.-ppt combined with conventional chemotherapy proved to increase the lifespan of

Another Japanese team studied the influence of damnacanthal, an anthraquinone extracted from a chloroform extract of *M. citrifolia* L. fruits. Surprisingly, the researchers found that damnacanthal induces normal morphology in a particular type of cell found in human neoplasias (K-ras-NKR cells) that multiply uncontrollably and are highly malignant

specific treatment.

(Locher et al., 1995).

**2.4.2 Anti-cancer activity** 

et al., 1996; Hirazumi & Furusawa, 1999).

mice with cancer, (Hirazumi et al., 1994).

(Hiramatsu et al., 1993).

stigmasta-4-en-3-one and stigmasta-4-22-dien-3-one.

processing, being greater when the fruit is ripe and undried.

which are important mediators of tumour cytostasis and cytotoxicity.

Approximately 51 volatile compounds have been identified in the ripe fruit (Sang et al., 2001), including organic acids (mainly octanoic and hexanoic acids), alcohols (3-methyl-3 butene-1-ol), esters (methyl octanoate, methyl decanoate), ketones (2-heptanone) and lactones ((E)-6-dodeceno-γ-lactone) (Farine et al., 1996).

#### **Major components**

A number of major components have been identified in the *M. citrifolia* L. plant, such as scopoletin, octanoic acid, potassium, vitamin C, terpenoids, alkaloids, anthraquinones (such as nordamnacanthal, morindone, rubiadin, rubiadin-1-methyl ether and anthraquinone glycoside), β-sitosterol, carotene, vitamin A, flavones glycosides, linoleic acid, alizarin, amino acids, acubin, L-asperuloside, caproic acid, caprylic acid, ursolic acid, rutin and a putative proxeronine.

A research group led by Chi-Tang Ho at Rutgers University in the United States (US) is searching for new novel compounds in the *M. citrifolia* L. plant. They have successfully identified several new flavonol glycosides, an iridoid glycoside from *M. citrifolia* L. leaves, a trisaccharide fatty acid ester, rutin, and an asperulosidic acid from the fruit. Two novel glycosides and a new unusual iridoid named citrifolinoside have been shown to have an inhibitory effect on AP-1 transactivation and cell transformation in the mouse epidermal JB6 cell line. James Duke listed 23 different phytochemicals found in *M. citrifolia* L. as well as 5 vitamins and 3 minerals in an authoritative handbook of phytochemicals.

#### **Xeronine system**

Retired biochemist Ralph Heinicke states that *M. citrifolia* L. fruit contains a natural precursor of xeronine that he named Proxeronine. Proxeronine is converted to the alkaloid xeronine in the body by an enzyme he named proxeroninase. His hypothesis is that xeronine is able to modify the molecular structure of proteins. Thus, xeronine has a wide range of biological activities. When a protein such as an enzyme, receptor, or signal transducer is not in the appropriate conformation, it will not work properly. Xeronine will interact with the protein and make it fold into its proper conformation. The result is a properly functioning protein. Whenever a problem arises in the cell due to a structural problem with a protein, xeronine's presence would be beneficial. His hypothesis may explain why Tahitian Noni @ juice (TNJ) can help in many health problems in different ways. He has obtained several patents for xeronine. He states that the active ingredient in many of the effective folklore drugs is xeronine. This alkaloid is a critical normal metabolic coregulator. The ailments that he believes are helped by *M. citrifolia* L. include high blood pressure, menstrual cramps, arthritis, gastric ulcers, sprains, injuries, mental depression, senility, poor digestion, drug addiction, and pain. "I have devoted much of my life to the study of this unique substance that I have named 'xeronine'. I am convinced of the tremendous benefits achieved by furnishing the body with a proper supply of this material" (Heinicke, 2001).

#### **2.4 Biological activity of** *M. Citrifolia* **L.**

#### **2.4.1 Anti-microbial effects**

The anti-microbial activity of *M. citrifolia* L. may have been its first observed property; indeed, the fruit contains relatively large amounts of sugars that do not ferment even when fruits are stored in closed containers at ambient temperature. This property is used to transport the fruit by boat from the scattered Pacific islands to processing plants without specific treatment.

It has been reported that *M. citrifolia* L. inhibits the growth of certain bacteria, such as staphylococcus aureus, Pseudomonas aeruginosa, Proteus morgaii, Bacillus subtilis, Escherichia coli, Helicobacter pylori, Salmonella and Shigella (Atkinson, 1956). The same author claims that the anti-microbial effect observed may be due to the presence of phenolic compounds such as acubin, l-asperuloside, alizarin, scopoletin and other anthraquinones. Another study showed that an acetonitrile extract of the dried fruit inhibits the growth of Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, and Streptococcus pyrogene (Locher et al., 1995).

It has also been found that ethanol and hexane extracts of *M. citrifolia* L. have an antitubercular effect as they inhibit the growth of Mycobacterium tuberculosis by 89-95% (Saludes et al., 2002). The major components identified in the hexane extract are E-phytol, cycloartenol, stigmasterol, β-sitosterol, campesta-5,7,22-trien-3-β-ol, and the ketosteroids stigmasta-4-en-3-one and stigmasta-4-22-dien-3-one.

Other studies have reported a significant antimicrobial effect on various strains of Salmonella, Shigella, and E. coli (Bushnell et al., 1950; Dittmar, 1993). Furthermore, they showed that the anti-microbial effect is highly dependent on the stage of ripeness and on processing, being greater when the fruit is ripe and undried.

#### **2.4.2 Anti-cancer activity**

138 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Approximately 51 volatile compounds have been identified in the ripe fruit (Sang et al., 2001), including organic acids (mainly octanoic and hexanoic acids), alcohols (3-methyl-3 butene-1-ol), esters (methyl octanoate, methyl decanoate), ketones (2-heptanone) and

A number of major components have been identified in the *M. citrifolia* L. plant, such as scopoletin, octanoic acid, potassium, vitamin C, terpenoids, alkaloids, anthraquinones (such as nordamnacanthal, morindone, rubiadin, rubiadin-1-methyl ether and anthraquinone glycoside), β-sitosterol, carotene, vitamin A, flavones glycosides, linoleic acid, alizarin, amino acids, acubin, L-asperuloside, caproic acid, caprylic acid, ursolic acid, rutin and a

A research group led by Chi-Tang Ho at Rutgers University in the United States (US) is searching for new novel compounds in the *M. citrifolia* L. plant. They have successfully identified several new flavonol glycosides, an iridoid glycoside from *M. citrifolia* L. leaves, a trisaccharide fatty acid ester, rutin, and an asperulosidic acid from the fruit. Two novel glycosides and a new unusual iridoid named citrifolinoside have been shown to have an inhibitory effect on AP-1 transactivation and cell transformation in the mouse epidermal JB6 cell line. James Duke listed 23 different phytochemicals found in *M. citrifolia* L. as well as 5

Retired biochemist Ralph Heinicke states that *M. citrifolia* L. fruit contains a natural precursor of xeronine that he named Proxeronine. Proxeronine is converted to the alkaloid xeronine in the body by an enzyme he named proxeroninase. His hypothesis is that xeronine is able to modify the molecular structure of proteins. Thus, xeronine has a wide range of biological activities. When a protein such as an enzyme, receptor, or signal transducer is not in the appropriate conformation, it will not work properly. Xeronine will interact with the protein and make it fold into its proper conformation. The result is a properly functioning protein. Whenever a problem arises in the cell due to a structural problem with a protein, xeronine's presence would be beneficial. His hypothesis may explain why Tahitian Noni @ juice (TNJ) can help in many health problems in different ways. He has obtained several patents for xeronine. He states that the active ingredient in many of the effective folklore drugs is xeronine. This alkaloid is a critical normal metabolic coregulator. The ailments that he believes are helped by *M. citrifolia* L. include high blood pressure, menstrual cramps, arthritis, gastric ulcers, sprains, injuries, mental depression, senility, poor digestion, drug addiction, and pain. "I have devoted much of my life to the study of this unique substance that I have named 'xeronine'. I am convinced of the tremendous benefits achieved by

vitamins and 3 minerals in an authoritative handbook of phytochemicals.

furnishing the body with a proper supply of this material" (Heinicke, 2001).

The anti-microbial activity of *M. citrifolia* L. may have been its first observed property; indeed, the fruit contains relatively large amounts of sugars that do not ferment even when fruits are stored in closed containers at ambient temperature. This property is used to

**2.4 Biological activity of** *M. Citrifolia* **L.** 

**2.4.1 Anti-microbial effects** 

lactones ((E)-6-dodeceno-γ-lactone) (Farine et al., 1996).

**Major components** 

putative proxeronine.

**Xeronine system** 

The immunomodulatory properties (the capacity to enhance the host immune system) of *M. citrifolia* L. juice have been studied by a Japanese research team (Hirazumi et al., 1996; Hirazumi & Furusawa, 1999). The ethanol precipitable fraction (ppt) of *M. citrifolia* L. juice, corresponding to a polysaccharide-rich substance composed of glucuronoic acid, galactose, arabinose and rhamnose, has been found to have immunomodulatory and anti-tumour effects against Lewis lung carcinoma (LLC). In cell models, *M. citrifolia* L.-ppt seems to stimulate the production of T-cells, thymocytes and macrophages that produce cytokines, which are important mediators of tumour cytostasis and cytotoxicity.

*M. citrifolia* L.-ppt also appears to stimulate murine effector cells to release several mediators such as cytokines. These mediators slow down the cell cycle in tumours, increase the response of cells to other immunised cells that fight tumour growth and have potent macrophage activator activity suspected of playing a role in the death of tumours (Hirazumi et al., 1996; Hirazumi & Furusawa, 1999).

In the same study, mice were inoculated with LLC, and those ingesting a daily dose of 15 mg of *M. citrifolia* L. juice showed a significant increase (119%) in lifespan. Nine out of 22 mice with terminal cancer survived for more than 50 days. In addition, the ingestion of *M. citrifolia* L.-ppt combined with conventional chemotherapy proved to increase the lifespan of mice with cancer, (Hirazumi et al., 1994).

Another Japanese team studied the influence of damnacanthal, an anthraquinone extracted from a chloroform extract of *M. citrifolia* L. fruits. Surprisingly, the researchers found that damnacanthal induces normal morphology in a particular type of cell found in human neoplasias (K-ras-NKR cells) that multiply uncontrollably and are highly malignant (Hiramatsu et al., 1993).

Phytochemical Constituents and Activities of *Morinda citrifolia* L. 141

protection using *M. citrifolia* L. extract and morphine, respectively), and it also proved to be

Recent research has demonstrated the ability of *M. citrifolia* L. fruit to prevent arteriosclerosis, a disease related to the oxidation of low density lipoproteins (LDLs). Methanol and ethyl acetate extracts showed 88% and 96% inhibition, respectively, of copperinduced LDL oxidation by the thiobarbituric acid relative substance method. This beneficial effect could be due to the presence of lignans, which are phenylpropanoid dimers (Kamiya

Acubin, l-asperuloside, alizarin in *M. citrifolia* L. fruit, and certain other anthraquinone compounds in *M. citrifolia* L. roots, are all proven antibacterial agents. These compounds have been shown to fight infectious bacteria such as Pseudomonas aeruginosa, Proteus morgaii, Staphylococcus aureus, Bacillis subtilis, Escherichia coli, Salmonella, and Shigela. These antibacterial compounds in *M. citrifolia* L. are responsible for the treatment of skin infections, colds, fevers, and other bacteria-caused health problems. Bushnell reported on the antibacterial properties of certain plants found in Hawaii, including *M. citrifolia* L. He further reported that *M. citrifolia* L. was traditionally used to treat broken bones, deep cuts, bruises, sores and wounds. Extracts from the ripe *M. citrifolia* L. fruit exhibit moderate antibacterial properties against Salmonella typhosa. Salmonella montevideo, Salmonella schottmuelleri, Shigella paradys, BH and Shigella paradys, III-Z. Leach demonstrated that acetone extracts obtained from Cycas circinalis, *M. citrifolia*, Bridelia penangiana, Tridax Procumbens, Hibiscus tiliaceus, and Hypericum papuanun show antibacterial activity. The widespread medicinal use of these plants would suggest that they do contain pharmacologically active substances, and alternative methods of extraction and screening should be utilised to find the major bioactive component in the plants for the purpose of new drug development. Locher reported that selected plants including *M. citrifolia* have a history of use in Polynesian traditional medicine for the treatment of infectious diseases. These plants have been investigated for anti-viral, anti-fungal, and anti-bacterial activity *in vitro*. Their study using *in vitro* biological assays confirmed that some of the Hawaiian medicinal

plants in ethnobotanical reports have curative properties against infectious diseases.

Duncan demonstrated that scopoletin, a health promoter in *M. citrifolia* L., inhibits the activity of E. coli, which is associated with recent outbreaks resulting in hundreds of serious infections, even death. *M. citrifolia* L. also helps stomach ulcers by inhibiting H. pylori

Umezawa and coworkers found that a compound isolated from *M. citrifolia* L. roots named 1-methoxy-2-formyl-3-hydroxyanthraquinone suppresses the cytopathic effect of HIV-

non-toxic (Younos et al., 1990).

**2.4.6 Cardiovascular activity** 

**2.5.1 Antibacterial activity** 

**2.5 Biological activities of** *M. citrifolia* **L. products** 

et al., 2004).

bacteria.

**2.5.2 Antiviral activity** 

infected MT-4 cells without inhibiting cell growth.

Another study showed that commercial *M. citrifolia* L. juice (Tahitian Noni juice) prevents the formation of chemical carcinogen-DNA-adducts. In the above study, rats with artificially induced cancer in specific organs were fed for one week with 10% *M. citrifolia* L. juice in their drinking water and rat food (rat chow) ad libitum. They showed reduced DNA-adduct formation depending on sex and organ. The reduction rates were: in female rats, heart 30%, liver 42%, lungs 41% and kidneys 80%; in male rats, heart 60%, liver 70%, lungs 50% and kidneys 90% (Wang & Su, 2001).

#### **2.4.3 Anti-oxidant properties**

The antioxidant properties of ethanol and ethyl acetate extracts of *M. citrifolia* L. fruit have been assessed using the ferric thiocyanate method (FTC) and thiobarbituric acid test (TBA). The authors found that ethyl acetate extract strong inhibited lipid oxidation, comparably to the same weight of pure α-tocopherol and butylated hydroxyl toluene (BHT) (Mohd et al., 2001).

Radical scavenging activity was also measured *in vitro* by the tetrazolium nitroblue (TNB) assay in commercial juice by assessing the capacity of the juice to protect cells and lipids from oxidative alteration promoted by superoxide anion radicals (SARs). The SAR scavenging activity of *M. citrifolia* L. juice was shown to be 2.8 times higher than that of vitamin C, 1.4 times that of pycnogenol (PYC) and almost the same magnitude as that of grape seed powder (Wang & Su, 2001).

#### **2.4.4 Anti-inflammatory activity**

The anti-inflammatory activity of an aqueous extract from *M. citrifolia* L.-juice was observed by inducing a locally acute inflammatory response with the help of a pro-inflammatory agent (bradykinin). It was found that the oral administration of *M. citrifolia* L. juice extract (200 mg) rapidly inhibits the formation of rat paw oedema. This effect may have resulted from interference with the B2 receptor-mediated mechanism by which bradykinin induces rat paw oedema (Mckoy et al., 2002).

Another study showed that commercial *M. citrifolia* L. juice selectively inhibits cyclooxygenase enzymes (COX-1 and COX-2) involved in breast, colon and lung cancer and also has anti-inflammatory activity (Su et al., 2001). The ability of noni juice to inhibit these enzymes was compared to that of traditional commercial non-steroidal inflammatory drugs, such as aspirin, Indomethacin and Celebrex. *M. citrifolia* L. juice showed selective inhibition of COX activity *in vitro* and a strong anti-inflammatory effect comparable to that of Celebrex, and presumably, this juice lacks side effects.

#### **2.4.5 Analgesic activity**

Recent research has examined the analgesic properties of commercial juice in rats. The results showed that rats fed 10% or 20% *M. citrifolia* L. juice had greater pain tolerance (162% or 212%, respectively) compared with the placebo group (Wang et al., 2002). A French research team has also studied the analgesic and sedative effects of *M. citrifolia* L. on mice through the writhing and hotplate tests. *M. citrifolia* L. root extract (1600 mg/kg) showed significant analgesic activity in the animals, similar to the effect of morphine (75% and 81% protection using *M. citrifolia* L. extract and morphine, respectively), and it also proved to be non-toxic (Younos et al., 1990).

#### **2.4.6 Cardiovascular activity**

140 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Another study showed that commercial *M. citrifolia* L. juice (Tahitian Noni juice) prevents the formation of chemical carcinogen-DNA-adducts. In the above study, rats with artificially induced cancer in specific organs were fed for one week with 10% *M. citrifolia* L. juice in their drinking water and rat food (rat chow) ad libitum. They showed reduced DNA-adduct formation depending on sex and organ. The reduction rates were: in female rats, heart 30%, liver 42%, lungs 41% and kidneys 80%; in male rats, heart 60%, liver 70%, lungs 50% and

The antioxidant properties of ethanol and ethyl acetate extracts of *M. citrifolia* L. fruit have been assessed using the ferric thiocyanate method (FTC) and thiobarbituric acid test (TBA). The authors found that ethyl acetate extract strong inhibited lipid oxidation, comparably to the same weight of pure α-tocopherol and butylated hydroxyl toluene (BHT) (Mohd et al.,

Radical scavenging activity was also measured *in vitro* by the tetrazolium nitroblue (TNB) assay in commercial juice by assessing the capacity of the juice to protect cells and lipids from oxidative alteration promoted by superoxide anion radicals (SARs). The SAR scavenging activity of *M. citrifolia* L. juice was shown to be 2.8 times higher than that of vitamin C, 1.4 times that of pycnogenol (PYC) and almost the same magnitude as that of

The anti-inflammatory activity of an aqueous extract from *M. citrifolia* L.-juice was observed by inducing a locally acute inflammatory response with the help of a pro-inflammatory agent (bradykinin). It was found that the oral administration of *M. citrifolia* L. juice extract (200 mg) rapidly inhibits the formation of rat paw oedema. This effect may have resulted from interference with the B2 receptor-mediated mechanism by which bradykinin induces

Another study showed that commercial *M. citrifolia* L. juice selectively inhibits cyclooxygenase enzymes (COX-1 and COX-2) involved in breast, colon and lung cancer and also has anti-inflammatory activity (Su et al., 2001). The ability of noni juice to inhibit these enzymes was compared to that of traditional commercial non-steroidal inflammatory drugs, such as aspirin, Indomethacin and Celebrex. *M. citrifolia* L. juice showed selective inhibition of COX activity *in vitro* and a strong anti-inflammatory effect comparable to that of

Recent research has examined the analgesic properties of commercial juice in rats. The results showed that rats fed 10% or 20% *M. citrifolia* L. juice had greater pain tolerance (162% or 212%, respectively) compared with the placebo group (Wang et al., 2002). A French research team has also studied the analgesic and sedative effects of *M. citrifolia* L. on mice through the writhing and hotplate tests. *M. citrifolia* L. root extract (1600 mg/kg) showed significant analgesic activity in the animals, similar to the effect of morphine (75% and 81%

kidneys 90% (Wang & Su, 2001).

**2.4.3 Anti-oxidant properties** 

grape seed powder (Wang & Su, 2001).

**2.4.4 Anti-inflammatory activity** 

rat paw oedema (Mckoy et al., 2002).

**2.4.5 Analgesic activity** 

Celebrex, and presumably, this juice lacks side effects.

2001).

Recent research has demonstrated the ability of *M. citrifolia* L. fruit to prevent arteriosclerosis, a disease related to the oxidation of low density lipoproteins (LDLs). Methanol and ethyl acetate extracts showed 88% and 96% inhibition, respectively, of copperinduced LDL oxidation by the thiobarbituric acid relative substance method. This beneficial effect could be due to the presence of lignans, which are phenylpropanoid dimers (Kamiya et al., 2004).

#### **2.5 Biological activities of** *M. citrifolia* **L. products**

#### **2.5.1 Antibacterial activity**

Acubin, l-asperuloside, alizarin in *M. citrifolia* L. fruit, and certain other anthraquinone compounds in *M. citrifolia* L. roots, are all proven antibacterial agents. These compounds have been shown to fight infectious bacteria such as Pseudomonas aeruginosa, Proteus morgaii, Staphylococcus aureus, Bacillis subtilis, Escherichia coli, Salmonella, and Shigela. These antibacterial compounds in *M. citrifolia* L. are responsible for the treatment of skin infections, colds, fevers, and other bacteria-caused health problems. Bushnell reported on the antibacterial properties of certain plants found in Hawaii, including *M. citrifolia* L. He further reported that *M. citrifolia* L. was traditionally used to treat broken bones, deep cuts, bruises, sores and wounds. Extracts from the ripe *M. citrifolia* L. fruit exhibit moderate antibacterial properties against Salmonella typhosa. Salmonella montevideo, Salmonella schottmuelleri, Shigella paradys, BH and Shigella paradys, III-Z. Leach demonstrated that acetone extracts obtained from Cycas circinalis, *M. citrifolia*, Bridelia penangiana, Tridax Procumbens, Hibiscus tiliaceus, and Hypericum papuanun show antibacterial activity. The widespread medicinal use of these plants would suggest that they do contain pharmacologically active substances, and alternative methods of extraction and screening should be utilised to find the major bioactive component in the plants for the purpose of new drug development. Locher reported that selected plants including *M. citrifolia* have a history of use in Polynesian traditional medicine for the treatment of infectious diseases. These plants have been investigated for anti-viral, anti-fungal, and anti-bacterial activity *in vitro*. Their study using *in vitro* biological assays confirmed that some of the Hawaiian medicinal plants in ethnobotanical reports have curative properties against infectious diseases.

Duncan demonstrated that scopoletin, a health promoter in *M. citrifolia* L., inhibits the activity of E. coli, which is associated with recent outbreaks resulting in hundreds of serious infections, even death. *M. citrifolia* L. also helps stomach ulcers by inhibiting H. pylori bacteria.

#### **2.5.2 Antiviral activity**

Umezawa and coworkers found that a compound isolated from *M. citrifolia* L. roots named 1-methoxy-2-formyl-3-hydroxyanthraquinone suppresses the cytopathic effect of HIVinfected MT-4 cells without inhibiting cell growth.

Phytochemical Constituents and Activities of *Morinda citrifolia* L. 143

Hiwasa and coworkers demonstrated that damnacanthal, an anthraquinone compound isolated from the *M. citrifolia* L. roots, has potent inhibitory activity towards tyrosine kinases such as Lck, Src, Lyn and EGF receptors. In his research, he examined the effects of damnacanthal on ultraviolet ray-induced apoptosis in ultraviolet-resistant human UVr-1 cells. Consequently, the ultraviolet light induced a concurrent increase in both phosphorylated extracellular signal-regulated kinases and stress-activated protein kinases. After pretreatment with damnacanthal, there was a stimulatory effect on ultraviolet-induced

Dong reported that two glycosides extracted from *M. citrifolia* L.-ppt were effective at inhibiting cell transformation induced by TPA or EGF in the mouse epidermal JB6 cell line. This inhibition was found to be associated with the inhibitory effects of these compounds on AP-1 activity. The compounds also blocked the phosphorylation of c-jun, a substrate of JNKs, suggesting that JNKs are a critical target for the compounds in mediating Ap-1

An ethanol extract of tender *M. citrifolia* L. leaves was found to induce paralysis and death in the human parasitic nematode Ascaris Lumbricoides within a day. A botanist via Morton reported that *M. citrifolia* L. has been used in the Philippines and Hawaii as an effective

Dang Van Ho of Vietnam demonstrated that total extract of *M. citrifolia* L. roots has a hypotensive effect. Moorthy and coworkers found that an ethanol extract of *M. citrifolia* L. roots lowers blood pressure in anesthetised dogs. Youngken's research team determined that a hot water extract of *M. citrifolia* L. roots lowers blood pressure in anesthetised dogs. A

Asahina found that an alcohol extract of *M. citrifolia* L. fruit at various concentrations inhibits the production of tumour necrosis factor-alpha (TNF-α), which is an endogenous tumour promoter. Therefore, the alcohol extract may inhibit the tumour promoting effect of TNF- α. Hirazumi found that *M. citrifolia* L.-ppt contains a polysaccharide-rich substance that inhibits tumour growth. It does not cause significant cytotoxicity in adopted cultures of lung cancer cells, but it can activate peritoneal exudate cells to impart profound toxicity when co-cultured with tumour cells. This suggests the possibility that *M. citrifolia* L.-ppt may suppress tumour growth through activation of the host immune system. *M. citrifolia* L. ppt is also capable of stimulating the release of several mediators from murine effector cells, including TNF- α, interleukin-1 beta (IL-1β), IL-10, IL-12, interferon-gamma (IFN- γ) and nitric oxide (NO). Hokama separated ripe *M. citrifolia* L. fruit juice into 50% aqueous alcohol and precipitate fractions, which was found to stimulate BALB/c thymus cells in (3H)thymidine analysis. It has been suggested that inhibition of Lewis lung tumours in mice,

Hawaiian physician reported that *M. citrifolia* L. fruit juice has a diuretic effect.

in part, may be due to the stimulation of the T-cell immune response.

apoptosis.

insecticide.

activity and cell transformation.

**2.5.5 Antihelmintic activity** 

**2.5.6 Hypotensive activity** 

**2.5.7 Immunological activity** 

#### **2.5.3 Anti-tubercular effects**

At the International Chemical Congress of the Pacific Basin Societies meeting in Honolulu, Saludes and colleagues from the Philippines reported that *M. citrifolia* L. kills Mycobacterium tuberculosis. A concentration of extracts from *M. citrifolia* L. leaves killed 89% of the bacteria in a test tube, almost as effectively as the leading anti-TB drug Rifampicin, which has an inhibitory rate of 97% at the same concentration. Although there have been anecdotal reports of native use of *M. citrifolia* L. in Polynesia as a medicine against tuberculosis, this is the first report demonstrating the antimycobacterial potential of compounds obtained from *M. citrifolia* L. leaves. "I hope that pharmaceutical companies will pay attention to this research and explore the *M. citrifolia* L. plant as a potential source of drugs", said Saludes in Manila.

#### **2.5.4 Antitumor activity**

At the 83rd Annual meeting of the American Association for Cancer Research in 1992, Hirazumi, a researcher at the University of Hawaii, reported that the alcohol-precipitate of *M. citrifolia* L. fruit juice (noni-ppt) has anticancer activity on lung cancer in C57 B1/6 mice. This *M. citrifolia* L.-ppt was shown to significantly prolong (by up to 75%) the life of mice with implanted Lewis lung carcinoma. It was concluded that the *M. citrifolia* L.-ppt seems to suppress tumour growth indirectly by stimulating the immune system. Improved survival time and curative effects occurred when *M. citrifolia* L.-ppt was combined with sub-optimal doses of the standard chemotherapeutic agents, such as adriamycin (Adria), cisplatin (CDDP), 5-fluorouracil (5-FU) and vincristine (VCR), suggesting that *M. citrifolia* L.-ppt has important clinical utility as a supplemental agent in cancer treatment. These results indicate that *M. citrifolia* L.-ppt may enhance the therapeutic effect of anticancer drugs. Therefore, it may be of benefit to cancer patients by enabling them to use lower doses of anti-cancer drugs to achieve the same or even better results.

Dr. Wang and coworkers demonstrated a cytotoxic effect of TNJ on a cultured leukaemia cell line at various concentrations. TNJ showed dose-dependent cytotoxicity on cultured cancer cells by inducing cancer cell necrosis at high doses and apoptosis at lower doses. Synergistic effects of TNJ with known anticancer drugs have been found. At sub-optimal doses, both prednisolone and TNJ can induce apoptosis. When the dose of prednisolone is fixed and the dose of TNJ increases, apoptotic cells significantly increase. Therefore, TNJ is able to enhance the efficacy of anticancer drugs such as prednisolone. When a single dose of Taxol induces a lower percentage of apoptosis in leukaemia cells, TNJ enhances the rate of apoptosis to 100%. These results indicate that TNJ is able to enhance the therapeutic effects of anticancer drugs such as Taxol. These findings regarding the combination of anticancer drugs with TNJ may be significant. This approach may allow lower doses of synthetic anticancer drugs to be used, increase the tolerance of patients to the toxicity of anticancer drugs, and increase immune function, thus creating a new method for treating cancer patients.

In 1993, Hiramatsu and colleagues reported in Cancer Letters the effects of over 500 extracts from tropical plants on K-Ras-NRK cells. Damnacanthal, isolated from *M. citrifolia* L. roots, is an inhibitor of Ras function. The ras oncogene is believed to be associated with signal transduction in several human cancers such as lung, colon, and pancreatic cancer and leukaemia.

Hiwasa and coworkers demonstrated that damnacanthal, an anthraquinone compound isolated from the *M. citrifolia* L. roots, has potent inhibitory activity towards tyrosine kinases such as Lck, Src, Lyn and EGF receptors. In his research, he examined the effects of damnacanthal on ultraviolet ray-induced apoptosis in ultraviolet-resistant human UVr-1 cells. Consequently, the ultraviolet light induced a concurrent increase in both phosphorylated extracellular signal-regulated kinases and stress-activated protein kinases. After pretreatment with damnacanthal, there was a stimulatory effect on ultraviolet-induced apoptosis.

Dong reported that two glycosides extracted from *M. citrifolia* L.-ppt were effective at inhibiting cell transformation induced by TPA or EGF in the mouse epidermal JB6 cell line. This inhibition was found to be associated with the inhibitory effects of these compounds on AP-1 activity. The compounds also blocked the phosphorylation of c-jun, a substrate of JNKs, suggesting that JNKs are a critical target for the compounds in mediating Ap-1 activity and cell transformation.

#### **2.5.5 Antihelmintic activity**

142 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

At the International Chemical Congress of the Pacific Basin Societies meeting in Honolulu, Saludes and colleagues from the Philippines reported that *M. citrifolia* L. kills Mycobacterium tuberculosis. A concentration of extracts from *M. citrifolia* L. leaves killed 89% of the bacteria in a test tube, almost as effectively as the leading anti-TB drug Rifampicin, which has an inhibitory rate of 97% at the same concentration. Although there have been anecdotal reports of native use of *M. citrifolia* L. in Polynesia as a medicine against tuberculosis, this is the first report demonstrating the antimycobacterial potential of compounds obtained from *M. citrifolia* L. leaves. "I hope that pharmaceutical companies will pay attention to this research and explore the *M. citrifolia* L. plant as a potential source of

At the 83rd Annual meeting of the American Association for Cancer Research in 1992, Hirazumi, a researcher at the University of Hawaii, reported that the alcohol-precipitate of *M. citrifolia* L. fruit juice (noni-ppt) has anticancer activity on lung cancer in C57 B1/6 mice. This *M. citrifolia* L.-ppt was shown to significantly prolong (by up to 75%) the life of mice with implanted Lewis lung carcinoma. It was concluded that the *M. citrifolia* L.-ppt seems to suppress tumour growth indirectly by stimulating the immune system. Improved survival time and curative effects occurred when *M. citrifolia* L.-ppt was combined with sub-optimal doses of the standard chemotherapeutic agents, such as adriamycin (Adria), cisplatin (CDDP), 5-fluorouracil (5-FU) and vincristine (VCR), suggesting that *M. citrifolia* L.-ppt has important clinical utility as a supplemental agent in cancer treatment. These results indicate that *M. citrifolia* L.-ppt may enhance the therapeutic effect of anticancer drugs. Therefore, it may be of benefit to cancer patients by enabling them to use lower doses of anti-cancer

Dr. Wang and coworkers demonstrated a cytotoxic effect of TNJ on a cultured leukaemia cell line at various concentrations. TNJ showed dose-dependent cytotoxicity on cultured cancer cells by inducing cancer cell necrosis at high doses and apoptosis at lower doses. Synergistic effects of TNJ with known anticancer drugs have been found. At sub-optimal doses, both prednisolone and TNJ can induce apoptosis. When the dose of prednisolone is fixed and the dose of TNJ increases, apoptotic cells significantly increase. Therefore, TNJ is able to enhance the efficacy of anticancer drugs such as prednisolone. When a single dose of Taxol induces a lower percentage of apoptosis in leukaemia cells, TNJ enhances the rate of apoptosis to 100%. These results indicate that TNJ is able to enhance the therapeutic effects of anticancer drugs such as Taxol. These findings regarding the combination of anticancer drugs with TNJ may be significant. This approach may allow lower doses of synthetic anticancer drugs to be used, increase the tolerance of patients to the toxicity of anticancer drugs, and increase immune function, thus creating a new method for treating cancer patients. In 1993, Hiramatsu and colleagues reported in Cancer Letters the effects of over 500 extracts from tropical plants on K-Ras-NRK cells. Damnacanthal, isolated from *M. citrifolia* L. roots, is an inhibitor of Ras function. The ras oncogene is believed to be associated with signal transduction in several human cancers such as lung, colon, and pancreatic cancer and

**2.5.3 Anti-tubercular effects** 

drugs", said Saludes in Manila.

drugs to achieve the same or even better results.

**2.5.4 Antitumor activity** 

leukaemia.

An ethanol extract of tender *M. citrifolia* L. leaves was found to induce paralysis and death in the human parasitic nematode Ascaris Lumbricoides within a day. A botanist via Morton reported that *M. citrifolia* L. has been used in the Philippines and Hawaii as an effective insecticide.

#### **2.5.6 Hypotensive activity**

Dang Van Ho of Vietnam demonstrated that total extract of *M. citrifolia* L. roots has a hypotensive effect. Moorthy and coworkers found that an ethanol extract of *M. citrifolia* L. roots lowers blood pressure in anesthetised dogs. Youngken's research team determined that a hot water extract of *M. citrifolia* L. roots lowers blood pressure in anesthetised dogs. A Hawaiian physician reported that *M. citrifolia* L. fruit juice has a diuretic effect.

#### **2.5.7 Immunological activity**

Asahina found that an alcohol extract of *M. citrifolia* L. fruit at various concentrations inhibits the production of tumour necrosis factor-alpha (TNF-α), which is an endogenous tumour promoter. Therefore, the alcohol extract may inhibit the tumour promoting effect of TNF- α. Hirazumi found that *M. citrifolia* L.-ppt contains a polysaccharide-rich substance that inhibits tumour growth. It does not cause significant cytotoxicity in adopted cultures of lung cancer cells, but it can activate peritoneal exudate cells to impart profound toxicity when co-cultured with tumour cells. This suggests the possibility that *M. citrifolia* L.-ppt may suppress tumour growth through activation of the host immune system. *M. citrifolia* L. ppt is also capable of stimulating the release of several mediators from murine effector cells, including TNF- α, interleukin-1 beta (IL-1β), IL-10, IL-12, interferon-gamma (IFN- γ) and nitric oxide (NO). Hokama separated ripe *M. citrifolia* L. fruit juice into 50% aqueous alcohol and precipitate fractions, which was found to stimulate BALB/c thymus cells in (3H)thymidine analysis. It has been suggested that inhibition of Lewis lung tumours in mice, in part, may be due to the stimulation of the T-cell immune response.

Phytochemical Constituents and Activities of *Morinda citrifolia* L. 145

changes after DMBA administration. In the acute liver injury model, the histopathological changes in liver tissue and levels of both super-oxide anion free radicals (SAR) and lipid

DMBA DNA-adduct formation was used as a marker to examine whether TNJ is able to prevent carcinogen-induced DNA damage. Most chemical carcinogens need to be activated by endogenous enzymes to be transformed into a form that readily binds to genetic DNA to form DNA-adducts. Carcinogen-DNA adduct formation is an important DNA damage marker that predicts the possibility of cancer development. Most scientists agree that carcinogen-induced DNA adduct formation is an early critical step in the multiple stages of carcinogenesis. Carcinogen-DNA adducts can be repaired by endogenous enzymes. Unrepaired adducts are fixed after one cell cycle. Unrepaired DNA damage is responsible for mutations and subsequent cancer development. Therefore, preventing carcinogen-DNA adduct formation is a key aspect of preventing the initial steps of carcinogenesis. If TNJ can prevent and/or block the formation of carcinogen-induced DNA adducts, it may prevent

In recent years, increasing demand for higher quality and safer foods and medicines, as well as concern for environmental pollution during their commercial production, have triggered stringent regulations on toxin levels in foods and medicines as well as on the discharge of pollutants to the environment. In addition, there has been increasing consumer preference for natural substances. All these factors have provided strong motivation for the development of cost-effective new technologies, such as the eco-friendly extraction of natural substances employing green and safe solvents. In recent years, supercritical fluid extraction (SFE) has emerged as a highly promising environmentally benign technology for the production of natural extracts such as flavours, fragrances, spice oils, and oleoresins; natural anti-oxidants; natural colours; nutraceuticals and biologically active compounds. The state of a substance is called supercritical when both temperature and pressure exceed their critical point values. A supercritical fluid combines two beneficial properties, namely high density (which imparts high solvent power) and high compressibility (which permits high selectivity due to large changes in solvent power in response to small changes in temperature and pressure). In addition, SFE offers very attractive extraction characteristics owing to its favourable

diffusivity, viscosity, surface tension, and other thermo-physical properties.

conventional techniques for the extraction of bioactive ingredients from spices.

Since the 1980s, several potential applications of SFE have been reported. So far, the most popular SF has been carbon dioxide (CO2), owing to its easy availability, low cost, nonflammability, nontoxicity, and its possession of a wide spectrum of solvent properties. Its critical temperature is 31.1 °C and its critical pressure is 73.8 bar. Dense or supercritical carbon dioxide could very well be the most commonly used solvent in this century due to its wide-ranging applications. Its near-ambient critical temperature makes it ideally suitable for processing thermally labile natural substances. It is generally regarded as safe (GRAS), and it yields microbial-inactivated, contaminant-free, tailor-made extracts of superior organoleptic profile and longer shelf life with highly potent active ingredients. The SFE technique ensures high consistency and reliability in the quality and safety of bioactive heatsensitive botanical products as it does not alter the delicate balance of bioactivity of natural molecules. All of these advantages are almost impossible with conventional processes. Therefore, SFE technology using SC-CO2 as the solvent is an ideal alternative to the

hydroperoxide (LPO) after CCl4 administration were the focus.

cancer at the initiation of multiple-stage carcinogenesis.

Wang and coworkers at the University of Illinois college of Medicine observed that the thymus is enlarged in animals treated with TNJ. The wet weight of the thymus was 1.7 times that of control animals on the seventh day after receiving 10% TNJ in drinking water. The thymus is an important immune organ in the body that generates T cells and is involved in the aging process and cellular immune fractions. TNJ may enhance immune function by stimulating thymus growth and thereby exerting anti-aging and anticancer activities and protecting people from other degenerative diseases.

#### **2.5.8 Mental health and improved high frequency hearing**

A small human clinical trial of the effects of TNJ on auditory function and quality of life in patients with decreased bone mineral density and auditory function was conducted at the UIC College of Medicine, Rockford, IL. This study showed that TNJ improves both mental health and high frequency hearing. The results suggest that increased amounts or extended duration of TNJ intake may be required to influence this disorder.

#### **2.6 Study of TNJ for cancer prevention**

"To take medicine only when you are sick is like digging a well only when you are thirsty – is it not already too late?" (Chi Po, c 2500 B.C.). This proverb suggests that prevention is more important than treatment.

Cancer is the second leading cause of death in the US. According to the American Cancer society, 1500 people per day die from cancer in the United States. Fighting against cancer is a great task for scientists engaged in this field. The aetiology of most cases of human cancer remains unknown. Exposure to environmental carcinogens accounts for more than 90% of human cancers. Cigarette smoke is the number one high-risk environmental factor. Although some cancers are preventable, a means to prevent most cancers is not yet known. Seeking a natural way to prevent human cancer is an urgent task for cancer prevention investigators.

Studies of food, diet, and cancer have indicated that lifestyle changes such as eating more fruits and vegetables and quitting smoking will help prevent cancer. "A new plate" for America (75% vegetables, 25% meat) appeared at the 2001 annual conference of the American Institute for Cancer Research. Although TNJ possesses a broad range of therapeutic effects, its ability to prevent cancer remains unclear. Recently, new hypothesis has been investigated: whether or not TNJ can help prevent cancer during the early stages of chemical carcinogenesis.

This hypothesis was examined using two carcinogenic animal models and one human clinical study of a group of current smokers. The animal models included the following: the DMBA-induced mammary gland tumourigenesis model and an acute liver injury model induced by the liver carcinogen carbon tetrachloride (CCl4). These are classical extrinsic carcinogenic models. DMBA-induced DNA adduct formation and histological examination by light and electron microscopy were used as sensitive biomarkers to evaluate the preventive effects of TNJ at the initiation stage of multiple-step carcinogenesis. In the mammary breast carcinogenic model, to monitor the mechanisms of carcinogenesis and DMBA DNA-adduct formation in mammary tissue, the focus was on the pathogenic

Wang and coworkers at the University of Illinois college of Medicine observed that the thymus is enlarged in animals treated with TNJ. The wet weight of the thymus was 1.7 times that of control animals on the seventh day after receiving 10% TNJ in drinking water. The thymus is an important immune organ in the body that generates T cells and is involved in the aging process and cellular immune fractions. TNJ may enhance immune function by stimulating thymus growth and thereby exerting anti-aging and anticancer activities and

A small human clinical trial of the effects of TNJ on auditory function and quality of life in patients with decreased bone mineral density and auditory function was conducted at the UIC College of Medicine, Rockford, IL. This study showed that TNJ improves both mental health and high frequency hearing. The results suggest that increased amounts or extended

"To take medicine only when you are sick is like digging a well only when you are thirsty – is it not already too late?" (Chi Po, c 2500 B.C.). This proverb suggests that prevention is

Cancer is the second leading cause of death in the US. According to the American Cancer society, 1500 people per day die from cancer in the United States. Fighting against cancer is a great task for scientists engaged in this field. The aetiology of most cases of human cancer remains unknown. Exposure to environmental carcinogens accounts for more than 90% of human cancers. Cigarette smoke is the number one high-risk environmental factor. Although some cancers are preventable, a means to prevent most cancers is not yet known. Seeking a natural way to prevent human cancer is an urgent task for cancer prevention

Studies of food, diet, and cancer have indicated that lifestyle changes such as eating more fruits and vegetables and quitting smoking will help prevent cancer. "A new plate" for America (75% vegetables, 25% meat) appeared at the 2001 annual conference of the American Institute for Cancer Research. Although TNJ possesses a broad range of therapeutic effects, its ability to prevent cancer remains unclear. Recently, new hypothesis has been investigated: whether or not TNJ can help prevent cancer during the early stages of

This hypothesis was examined using two carcinogenic animal models and one human clinical study of a group of current smokers. The animal models included the following: the DMBA-induced mammary gland tumourigenesis model and an acute liver injury model induced by the liver carcinogen carbon tetrachloride (CCl4). These are classical extrinsic carcinogenic models. DMBA-induced DNA adduct formation and histological examination by light and electron microscopy were used as sensitive biomarkers to evaluate the preventive effects of TNJ at the initiation stage of multiple-step carcinogenesis. In the mammary breast carcinogenic model, to monitor the mechanisms of carcinogenesis and DMBA DNA-adduct formation in mammary tissue, the focus was on the pathogenic

protecting people from other degenerative diseases.

**2.6 Study of TNJ for cancer prevention** 

more important than treatment.

investigators.

chemical carcinogenesis.

**2.5.8 Mental health and improved high frequency hearing** 

duration of TNJ intake may be required to influence this disorder.

changes after DMBA administration. In the acute liver injury model, the histopathological changes in liver tissue and levels of both super-oxide anion free radicals (SAR) and lipid hydroperoxide (LPO) after CCl4 administration were the focus.

DMBA DNA-adduct formation was used as a marker to examine whether TNJ is able to prevent carcinogen-induced DNA damage. Most chemical carcinogens need to be activated by endogenous enzymes to be transformed into a form that readily binds to genetic DNA to form DNA-adducts. Carcinogen-DNA adduct formation is an important DNA damage marker that predicts the possibility of cancer development. Most scientists agree that carcinogen-induced DNA adduct formation is an early critical step in the multiple stages of carcinogenesis. Carcinogen-DNA adducts can be repaired by endogenous enzymes. Unrepaired adducts are fixed after one cell cycle. Unrepaired DNA damage is responsible for mutations and subsequent cancer development. Therefore, preventing carcinogen-DNA adduct formation is a key aspect of preventing the initial steps of carcinogenesis. If TNJ can prevent and/or block the formation of carcinogen-induced DNA adducts, it may prevent cancer at the initiation of multiple-stage carcinogenesis.

In recent years, increasing demand for higher quality and safer foods and medicines, as well as concern for environmental pollution during their commercial production, have triggered stringent regulations on toxin levels in foods and medicines as well as on the discharge of pollutants to the environment. In addition, there has been increasing consumer preference for natural substances. All these factors have provided strong motivation for the development of cost-effective new technologies, such as the eco-friendly extraction of natural substances employing green and safe solvents. In recent years, supercritical fluid extraction (SFE) has emerged as a highly promising environmentally benign technology for the production of natural extracts such as flavours, fragrances, spice oils, and oleoresins; natural anti-oxidants; natural colours; nutraceuticals and biologically active compounds. The state of a substance is called supercritical when both temperature and pressure exceed their critical point values. A supercritical fluid combines two beneficial properties, namely high density (which imparts high solvent power) and high compressibility (which permits high selectivity due to large changes in solvent power in response to small changes in temperature and pressure). In addition, SFE offers very attractive extraction characteristics owing to its favourable diffusivity, viscosity, surface tension, and other thermo-physical properties.

Since the 1980s, several potential applications of SFE have been reported. So far, the most popular SF has been carbon dioxide (CO2), owing to its easy availability, low cost, nonflammability, nontoxicity, and its possession of a wide spectrum of solvent properties. Its critical temperature is 31.1 °C and its critical pressure is 73.8 bar. Dense or supercritical carbon dioxide could very well be the most commonly used solvent in this century due to its wide-ranging applications. Its near-ambient critical temperature makes it ideally suitable for processing thermally labile natural substances. It is generally regarded as safe (GRAS), and it yields microbial-inactivated, contaminant-free, tailor-made extracts of superior organoleptic profile and longer shelf life with highly potent active ingredients. The SFE technique ensures high consistency and reliability in the quality and safety of bioactive heatsensitive botanical products as it does not alter the delicate balance of bioactivity of natural molecules. All of these advantages are almost impossible with conventional processes. Therefore, SFE technology using SC-CO2 as the solvent is an ideal alternative to the conventional techniques for the extraction of bioactive ingredients from spices.

Phytochemical Constituents and Activities of *Morinda citrifolia* L. 147

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#### **3. Conclusion**

*Morinda citrifolia* L., commonly known as noni, has a long history of widespread use as a food in tropical regions from Indonesia to the Hawaiian Islands, and it is used as an herbal remedy for multiple diseases. Its fruit, leaves, seeds, bark and roots have been traditionally used for the prevention or improvement of various diseases, including arthritis, infections, colds, cancer, and diabetes. It has been found that *Morinda citrifolia L.*, has antioxidant potential equivalent or similar to that of synthetic antioxidants, such as BHT and BHA, which are currently used as food additives. The antioxidants which are found in *Morinda citrifolia L.*, have no side effects, and thus they could replace synthetic antioxidants in the food processing industry and have potential for use in preventive medicine. Thus, this fruit can be used as an antioxidant additive in the food processing industry.

#### **4. Acknowledgements**

The authors wish to acknowledge the financial support of MOSTI Malaysia. This work was carried out under e-science grant number SCF0049-IND-2007.

#### **5. References**


*Morinda citrifolia* L., commonly known as noni, has a long history of widespread use as a food in tropical regions from Indonesia to the Hawaiian Islands, and it is used as an herbal remedy for multiple diseases. Its fruit, leaves, seeds, bark and roots have been traditionally used for the prevention or improvement of various diseases, including arthritis, infections, colds, cancer, and diabetes. It has been found that *Morinda citrifolia L.*, has antioxidant potential equivalent or similar to that of synthetic antioxidants, such as BHT and BHA, which are currently used as food additives. The antioxidants which are found in *Morinda citrifolia L.*, have no side effects, and thus they could replace synthetic antioxidants in the food processing industry and have potential for use in preventive medicine. Thus, this fruit

The authors wish to acknowledge the financial support of MOSTI Malaysia. This work was

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**7** 

*Iran* 

Mitra Noori

**Flavonoids in some Iranian Angiosperms** 

Flavonoids are as one set of the polyphenolic compounds among secondary metabolites in different organs of plants that possess a wide range of biological activities [Parr and Bolwell 2000, Noori 2002, Noori et al 2009]. Their distribution in plants, synthesis and mode of

All flavonoids contain fifteen carbon atoms in their basic nucleus and these are arranged in a C6-C3-C6 configuration, that is, two aromatic rings linked by a three carbon unit which may or may not form a third ring. They are divided into different groups depending on the configuration of the rings and substitutions on these rings of a variety of side-groups which

The flavonoid variants are all related by a common biosynthetic pathway which incorporates precursors from both the "Shikimate" and "Acetate-Malonate" pathways [Hahlbrock and Grisebach 1975; Wong 1976], the first flavonoid produced immediately

The flavonoid initially formed in the biosynthesis is now thought to be the chalcone and all other forms are derived from this by a variety of routes [Hahlbrock 1981] (Scheme 2). More than 4000 varieties of flavonoids have been identified in different higher and lower plant species (De Groot and Rauen 1998). The main flavonoid groups are flavones (e.g. luteolin), flavanone (e.g. naringenin), flavonols (e.g. kaempferol), anthocyanidins (e.g. pelargonidin)

**1. Introduction** 

action have been extensively studied [Shirley 1996].

characterize the individual compounds [Stace 1980] (Scheme 1).

Scheme 1. The basic nucleus of flavonoids (Stace 1980)

following confluence of the two pathways (Scheme 2).

and chalcones (e.g. butein) [Harborne et al 1975].

**1.1 Structure, biosynthesis and variety** 

*Department of Biology, Faculty of Science, Arak University, Arak,* 


#### Mitra Noori

*Department of Biology, Faculty of Science, Arak University, Arak, Iran* 

#### **1. Introduction**

150 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

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Academic Press (Appropriate sections)

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430-434, ISSN 0032-0943

231, ISSN 0308-8146

Flavonoids are as one set of the polyphenolic compounds among secondary metabolites in different organs of plants that possess a wide range of biological activities [Parr and Bolwell 2000, Noori 2002, Noori et al 2009]. Their distribution in plants, synthesis and mode of action have been extensively studied [Shirley 1996].

#### **1.1 Structure, biosynthesis and variety**

All flavonoids contain fifteen carbon atoms in their basic nucleus and these are arranged in a C6-C3-C6 configuration, that is, two aromatic rings linked by a three carbon unit which may or may not form a third ring. They are divided into different groups depending on the configuration of the rings and substitutions on these rings of a variety of side-groups which characterize the individual compounds [Stace 1980] (Scheme 1).

Scheme 1. The basic nucleus of flavonoids (Stace 1980)

The flavonoid variants are all related by a common biosynthetic pathway which incorporates precursors from both the "Shikimate" and "Acetate-Malonate" pathways [Hahlbrock and Grisebach 1975; Wong 1976], the first flavonoid produced immediately following confluence of the two pathways (Scheme 2).

The flavonoid initially formed in the biosynthesis is now thought to be the chalcone and all other forms are derived from this by a variety of routes [Hahlbrock 1981] (Scheme 2). More than 4000 varieties of flavonoids have been identified in different higher and lower plant species (De Groot and Rauen 1998). The main flavonoid groups are flavones (e.g. luteolin), flavanone (e.g. naringenin), flavonols (e.g. kaempferol), anthocyanidins (e.g. pelargonidin) and chalcones (e.g. butein) [Harborne et al 1975].

Flavonoids are found in fruit, vegetables, grains, bark, roots, stems, leaves, flowers, tea and wine [Middleton 1998, Robles et al 2003]. The flavonoid nucleus is normally linked to a sugar moiety to form a water-soluble glycoside. Most flavonoids are stored in the plant cell vacuoles, although they also occur on the surfaces of leaves and stems (Farman 1990). In contrast to earlier studies, all these compounds are no longer judged as waste products, nor as evolutionary remnants without current function, nor as mere metabolic end products that are toxic to the plant and are therefore to be stored away in vacuoles [Parr and Bolwell

Flavonoids possess a wide range of biological activities, medicinal and pharmacological

A large variety of colours such as orange, scarlet, crimson, mauve, violet, blue and purple that we encounter in different part of plants, especially flowers and fruits, are caused by anthocyanins (=anthocyanidin glycosides). Chalcones and some flavones and flavonols also absorb light in the visible region and are associated with bright yellow or cream coloured flowers. Other flavones account for the whiteness in most white flowers, without which they would perhaps appear translucent. Even some of the brown and black pigments found in plants are either due to oxidative products of flavonoids or related phenolic compounds. [Farman 1990]. They are beneficial for the plant itself as physiological active compounds, as stress protecting agents, as attractants or as feeding deterrents, and, in general, by their significant role in plant resistance [Treutter 2006]. Also these compounds serve essential functions in plant reproduction by recruiting pollinators and seed disperses. They are also responsible for the beautiful display of fall color in many plant species, which has recently been suggested to protect leaf cells from photo-oxidative damage, thereby enhancing the

Flavonoids medicinal and pharmacological effects are their contributions to human health which has made them prominent in the past 10 years (Parr and Bolwell 2000). Many flavonoids are active principles of medicinal plants and exhibit pharmacological effects

Flavonoid compounds are taxonomically important. They are popular characters for chemosystematic studies because: the almost universal presence of flavonoids in vascular plants; 2. Their structural diversity; 3. The fact that each species usually contains several flavonoids; 4. The chemical stability of many flavonoids in dried plant material enabling herbarium material to be used; 5. Flavonoid profiles using different chromatographic techniques are easily obtained. 6. Flavonoids are reasonably easy to identify using published

**1.2 Occurance** 

**1.3 Biological activities and their usages** 

**1.3.1 Biological activities** 

[Yilmaz and Toledo 2004].

**1.4 Chemotaxonomy** 

effects [Parr and Bolwell 2000, Noori 2002, Noori et al 2009].

efficiency of nutrient retrieval during senescence [Field et al 2001].

**1.3.2 Medicinal and pharmacological effects** 

2000].

Scheme 2. Showing two biosynthetic pathways of flavonoids (Hahlbrock and Grisebach 1975; Wong 1976).

#### **1.2 Occurance**

152 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Scheme 2. Showing two biosynthetic pathways of flavonoids (Hahlbrock and Grisebach

1975; Wong 1976).

Flavonoids are found in fruit, vegetables, grains, bark, roots, stems, leaves, flowers, tea and wine [Middleton 1998, Robles et al 2003]. The flavonoid nucleus is normally linked to a sugar moiety to form a water-soluble glycoside. Most flavonoids are stored in the plant cell vacuoles, although they also occur on the surfaces of leaves and stems (Farman 1990). In contrast to earlier studies, all these compounds are no longer judged as waste products, nor as evolutionary remnants without current function, nor as mere metabolic end products that are toxic to the plant and are therefore to be stored away in vacuoles [Parr and Bolwell 2000].

#### **1.3 Biological activities and their usages**

Flavonoids possess a wide range of biological activities, medicinal and pharmacological effects [Parr and Bolwell 2000, Noori 2002, Noori et al 2009].

#### **1.3.1 Biological activities**

A large variety of colours such as orange, scarlet, crimson, mauve, violet, blue and purple that we encounter in different part of plants, especially flowers and fruits, are caused by anthocyanins (=anthocyanidin glycosides). Chalcones and some flavones and flavonols also absorb light in the visible region and are associated with bright yellow or cream coloured flowers. Other flavones account for the whiteness in most white flowers, without which they would perhaps appear translucent. Even some of the brown and black pigments found in plants are either due to oxidative products of flavonoids or related phenolic compounds. [Farman 1990]. They are beneficial for the plant itself as physiological active compounds, as stress protecting agents, as attractants or as feeding deterrents, and, in general, by their significant role in plant resistance [Treutter 2006]. Also these compounds serve essential functions in plant reproduction by recruiting pollinators and seed disperses. They are also responsible for the beautiful display of fall color in many plant species, which has recently been suggested to protect leaf cells from photo-oxidative damage, thereby enhancing the efficiency of nutrient retrieval during senescence [Field et al 2001].

#### **1.3.2 Medicinal and pharmacological effects**

Flavonoids medicinal and pharmacological effects are their contributions to human health which has made them prominent in the past 10 years (Parr and Bolwell 2000). Many flavonoids are active principles of medicinal plants and exhibit pharmacological effects [Yilmaz and Toledo 2004].

#### **1.4 Chemotaxonomy**

Flavonoid compounds are taxonomically important. They are popular characters for chemosystematic studies because: the almost universal presence of flavonoids in vascular plants; 2. Their structural diversity; 3. The fact that each species usually contains several flavonoids; 4. The chemical stability of many flavonoids in dried plant material enabling herbarium material to be used; 5. Flavonoid profiles using different chromatographic techniques are easily obtained. 6. Flavonoids are reasonably easy to identify using published

and flavanone types, and they are distinctly different from internal tissue aglycones [Midiwo et al 2007]. Also Hsu (2006) studies revealed that *Polygonum aviculare* L. extract has

Trichopoulou et al (2000) showed that some wild edible species of *Rumex* such as *R. acetosa* L. and *R. japonicas* Houttuyn have a very high flavonol content. Hasan et al (1995) studies showed besides rutin, quercetin 3-rhamnoside and kaempferol 3-rhamnosyl (1 -> 6) galactoside, a new flavonol glycoside, quercetin 3-glucosyl (1 -> 4) galactoside, and 1, 6, 8 trihydroxy-3-methyl anthraquinone (emodin) have been characterized from leaves of *R.* 

Several studies indicated that flavonoids occurred in various species of *Euphorbia*. Nagase reported the isolation of 5, 7, 4'-trihydroxy-flavone-7-glucoside from the leaves and stems of *E. thymifolia* [Nagase et al 1942]. Ten years later, quercetin was isolated from ethyl acetate extract of aqueous solution of hydroalcoholic extract of *E. pilulifera* [Hallett and Parks 1951]. Sotnikova and his coworkers identified 3′, 4′-pentahydroxyflavone 3β-D-galactopyranoside, steppogenin stepposide, isomyricitrin and nineteen other flavonoids in *E. stepposa* [Sotnikova and Litvinenko 1968, Sotnikova et al. 1968]. Muller and Pohl (1970) isolated six new flavonoids all being glycosides of rhamnetin from *E. amygdaloides*. The qualitative composition of flavonoids in alcoholic extract of *E. helioscopia* indicated fifteen substances with flavonoidal nature, by two-dimentional paper chromatography. Acid hydrolysis of *E. helioscopia* alcoholic extract by Volobueva (1970) yielded quercetin and kaempferol. Quercetin-3-xylosidoglucoside has been identified as one of the two flavonoids found in methanolic extract of *E. chamaesyce* and quercetin-3β-D-galactopyranoside gallate has been reported in *E. verrucosa* and *E. platiphyllos* [Singla and Pathak 1990]. Chromatography on cellulose of methanolic extract of *E. lucida* yielded quercetin and its derivatives, viz., isoquercetin, avicularoside, hyperoside and rutoside [Burzanska 1975]. The hypotensive principales of *E. maddeni* were found to be kaempferol-4'-*O*-glucose and hyperin (Sahai et al 1981). In an effort to identify the constituents responsible for the antiviral activity of *E. grantii*. Van Hoof et al. (1984) isolated derivatives of 3-methylquercetin. Polyphenolic components from the aerial parts of *E. soongarica* and *E. alatavica* have been identified as the esters of gallic acid, luteolin-3-rhamnoside and luteoline-3-galactoside [Omurkhamzinova and Erzhanova 1985]. Gautam and Mukhraya (1981) have isolated quercetin-3-O-β-Dglucopyranosyl (1-4)-O-α-L-rhamnopyranoside from the leaves of *E. dracuncoloides*. Kaempferol 3-O-glucoside and quercetin 3-O-glucoside were obtained from *E. larica*, *E. virgata*, *E. chamaesyce* and *E. magalanta*. Also all these taxa, except *E. chamaesyce* contained kaempferol 3-rutinoside and rutin. *E. larica* also yielded 6-methoxyapigenin while *E. virgata* and *E. magalanta* yielded kaempferol. There is an unknown acytylated kaempferol derivative in *E. chamaesyce* (Ulubelen et al 1983). Murillo and Jakupovic (1998) identified myricetin-3 rhamnoside and one flavonoid glycosides in *E. aucherii* which was collected in Iran. Aimova et al (1999) found quercetin 3-(2′′′-Galloylglucosyl) (1→2)-α-L-arabinofuranoside in *E. pachyrrhiza*. Studies of Halaweish et al (2003) are the first report of quercetin-3-O-β glucuronic acid in *E. esula*. They separated and identified kaempferol-3-O-β glucuronic acid and quercetin-3-O-β glucuronic acid from the species. Papp et al (2005) studies showed arial

high phenolics and flavonoid contents.

**1.7 Euphorbiaceae flavonoids** 

*chalepensis*.

UV spectra data and available standards; 7. Flavonoids often show correlations with existing classifications at the family, genus and species level, and support revisions of existing classifications at the family, genus and species level. However, flavonoids rarely provide key characters (the flavonoid may be absent in one or more members of the taxon, and the same flavonoid may occur in an unrelated taxon, e.g. isoflavonoids occur in the Leguminosae and Iridaceae and biflavonyls in the Gymnospermae and some Angiospermae) [Harborne and Turner 1984].

#### **1.5 Flavonoids in Leguminosae**

The Leguminosae is economically the single most important family in the dicotyledonae, and also of major significance in nature The family is especially rich in flavonoids, producing about 28% of all known flavonoids and 95% of all isoflavonoid aglycones (Hegnauer and Grayer-Barkmeijer 1993). The importance of the phenolic constituents in the family has been stressed by Bate-Smith (Bath-Smith 1962). As Gomes et al (1981a) showed in "Advances in Legume Systematics" the Leguminosae are especially well endowed with flavonoid constituents, many of which are only known in these plants. Whithin the Leguminosae, some 850 compounds, including 362 isoflavones, are known [Dewick 1993]. There is a basic structures, Such as genistein (4´, 5, 7-trihydroxyisoflavone), 5-dexy derivatives (some 66% of structures), prenylated derivatives (some 51% of structures) and compounds with extra hydroxylation (e. g. at the 6-, 8- or 2´-positions). Isoflavonoids usually occur in the free state, and are obtained from root, wood, bark or seed rather than leaf or flower [Ingham 1981, 1983]. Flavonoids, as distinct from isoflavonoids and neoflavonoids, are widespread in the *Papilionoidaea* and there is little doubt that they occur not only in the species of the some tribes, but will eventually be found in all tribes [Gomes et al 1981b]. Harborn (1965) obtained quercetagetin from hydrolyzed petal of *Coronilla glauca* L.. He also found halogenin, 3-*O* rutinoside and limocitrin, 3- *O* rutinoside from C. glauca flower (Harborn 1981). Catechin, epigallocatechin, leucodelphinidin and 3, 3´, 4´, 5, 5´, 7 hexahydroxyflavan have been identified from *Alhaji maurorum* Medikus ground parts [Islambeko et al 1982]. Malvidin from hydrolyzed flower, myricitrin from flower and laef of *Cercis siliquastrum* L. have been isolated [Torck et al 1969, Sagareishvili and Ananiya 1990].

#### **1.6 Polygonaceae flavonoids**

Based on Isobe and Noda (1987) flavonoids and flavonol glycosides are of wide-spread occurrence in the genus *Polygonum*. Among them, glycosylation at C-3 of the quercetin nucleus has been found to be the most common trend, and present in all species of this genus [Park, 1987]. While rhamnose, gloucose, arabinose and rhamnosyl-rhamnose are the most common sugars found as aglycones of the flavonol glycosides [Mun and Park 1995], galactosylation is rather uncommon in the genus *Polygonum* or in the family polygonaceae [Collins et al 1975]. Kawasaki et al (1986) isolated thirty-three kinds of flavonoids from Polygonaceae species leaves. Quercetin glycosides were commonly found in the family. In the quercetin glycosides, 3-*O*-rhamnoside was most frequently found, 3-*O*-glucuronide is also distributed widely. Myricetin glycosides were rare. Methylated flavonols were found in some species of the section *Echinocaulon* and *Persicaria* [Kawasaki et al 1986]. The aerial exudate of *Polygonum senegalense* has been reported to contain 12 flavonoids of the chalcone

UV spectra data and available standards; 7. Flavonoids often show correlations with existing classifications at the family, genus and species level, and support revisions of existing classifications at the family, genus and species level. However, flavonoids rarely provide key characters (the flavonoid may be absent in one or more members of the taxon, and the same flavonoid may occur in an unrelated taxon, e.g. isoflavonoids occur in the Leguminosae and Iridaceae and biflavonyls in the Gymnospermae and some

The Leguminosae is economically the single most important family in the dicotyledonae, and also of major significance in nature The family is especially rich in flavonoids, producing about 28% of all known flavonoids and 95% of all isoflavonoid aglycones (Hegnauer and Grayer-Barkmeijer 1993). The importance of the phenolic constituents in the family has been stressed by Bate-Smith (Bath-Smith 1962). As Gomes et al (1981a) showed in "Advances in Legume Systematics" the Leguminosae are especially well endowed with flavonoid constituents, many of which are only known in these plants. Whithin the Leguminosae, some 850 compounds, including 362 isoflavones, are known [Dewick 1993]. There is a basic structures, Such as genistein (4´, 5, 7-trihydroxyisoflavone), 5-dexy derivatives (some 66% of structures), prenylated derivatives (some 51% of structures) and compounds with extra hydroxylation (e. g. at the 6-, 8- or 2´-positions). Isoflavonoids usually occur in the free state, and are obtained from root, wood, bark or seed rather than leaf or flower [Ingham 1981, 1983]. Flavonoids, as distinct from isoflavonoids and neoflavonoids, are widespread in the *Papilionoidaea* and there is little doubt that they occur not only in the species of the some tribes, but will eventually be found in all tribes [Gomes et al 1981b]. Harborn (1965) obtained quercetagetin from hydrolyzed petal of *Coronilla glauca* L.. He also found halogenin, 3-*O* rutinoside and limocitrin, 3- *O* rutinoside from C. glauca flower (Harborn 1981). Catechin, epigallocatechin, leucodelphinidin and 3, 3´, 4´, 5, 5´, 7 hexahydroxyflavan have been identified from *Alhaji maurorum* Medikus ground parts [Islambeko et al 1982]. Malvidin from hydrolyzed flower, myricitrin from flower and laef of *Cercis siliquastrum* L. have been isolated [Torck et al 1969, Sagareishvili and Ananiya 1990].

Based on Isobe and Noda (1987) flavonoids and flavonol glycosides are of wide-spread occurrence in the genus *Polygonum*. Among them, glycosylation at C-3 of the quercetin nucleus has been found to be the most common trend, and present in all species of this genus [Park, 1987]. While rhamnose, gloucose, arabinose and rhamnosyl-rhamnose are the most common sugars found as aglycones of the flavonol glycosides [Mun and Park 1995], galactosylation is rather uncommon in the genus *Polygonum* or in the family polygonaceae [Collins et al 1975]. Kawasaki et al (1986) isolated thirty-three kinds of flavonoids from Polygonaceae species leaves. Quercetin glycosides were commonly found in the family. In the quercetin glycosides, 3-*O*-rhamnoside was most frequently found, 3-*O*-glucuronide is also distributed widely. Myricetin glycosides were rare. Methylated flavonols were found in some species of the section *Echinocaulon* and *Persicaria* [Kawasaki et al 1986]. The aerial exudate of *Polygonum senegalense* has been reported to contain 12 flavonoids of the chalcone

Angiospermae) [Harborne and Turner 1984].

**1.5 Flavonoids in Leguminosae** 

**1.6 Polygonaceae flavonoids** 

and flavanone types, and they are distinctly different from internal tissue aglycones [Midiwo et al 2007]. Also Hsu (2006) studies revealed that *Polygonum aviculare* L. extract has high phenolics and flavonoid contents.

Trichopoulou et al (2000) showed that some wild edible species of *Rumex* such as *R. acetosa* L. and *R. japonicas* Houttuyn have a very high flavonol content. Hasan et al (1995) studies showed besides rutin, quercetin 3-rhamnoside and kaempferol 3-rhamnosyl (1 -> 6) galactoside, a new flavonol glycoside, quercetin 3-glucosyl (1 -> 4) galactoside, and 1, 6, 8 trihydroxy-3-methyl anthraquinone (emodin) have been characterized from leaves of *R. chalepensis*.

#### **1.7 Euphorbiaceae flavonoids**

Several studies indicated that flavonoids occurred in various species of *Euphorbia*. Nagase reported the isolation of 5, 7, 4'-trihydroxy-flavone-7-glucoside from the leaves and stems of *E. thymifolia* [Nagase et al 1942]. Ten years later, quercetin was isolated from ethyl acetate extract of aqueous solution of hydroalcoholic extract of *E. pilulifera* [Hallett and Parks 1951]. Sotnikova and his coworkers identified 3′, 4′-pentahydroxyflavone 3β-D-galactopyranoside, steppogenin stepposide, isomyricitrin and nineteen other flavonoids in *E. stepposa* [Sotnikova and Litvinenko 1968, Sotnikova et al. 1968]. Muller and Pohl (1970) isolated six new flavonoids all being glycosides of rhamnetin from *E. amygdaloides*. The qualitative composition of flavonoids in alcoholic extract of *E. helioscopia* indicated fifteen substances with flavonoidal nature, by two-dimentional paper chromatography. Acid hydrolysis of *E. helioscopia* alcoholic extract by Volobueva (1970) yielded quercetin and kaempferol. Quercetin-3-xylosidoglucoside has been identified as one of the two flavonoids found in methanolic extract of *E. chamaesyce* and quercetin-3β-D-galactopyranoside gallate has been reported in *E. verrucosa* and *E. platiphyllos* [Singla and Pathak 1990]. Chromatography on cellulose of methanolic extract of *E. lucida* yielded quercetin and its derivatives, viz., isoquercetin, avicularoside, hyperoside and rutoside [Burzanska 1975]. The hypotensive principales of *E. maddeni* were found to be kaempferol-4'-*O*-glucose and hyperin (Sahai et al 1981). In an effort to identify the constituents responsible for the antiviral activity of *E. grantii*. Van Hoof et al. (1984) isolated derivatives of 3-methylquercetin. Polyphenolic components from the aerial parts of *E. soongarica* and *E. alatavica* have been identified as the esters of gallic acid, luteolin-3-rhamnoside and luteoline-3-galactoside [Omurkhamzinova and Erzhanova 1985]. Gautam and Mukhraya (1981) have isolated quercetin-3-O-β-Dglucopyranosyl (1-4)-O-α-L-rhamnopyranoside from the leaves of *E. dracuncoloides*. Kaempferol 3-O-glucoside and quercetin 3-O-glucoside were obtained from *E. larica*, *E. virgata*, *E. chamaesyce* and *E. magalanta*. Also all these taxa, except *E. chamaesyce* contained kaempferol 3-rutinoside and rutin. *E. larica* also yielded 6-methoxyapigenin while *E. virgata* and *E. magalanta* yielded kaempferol. There is an unknown acytylated kaempferol derivative in *E. chamaesyce* (Ulubelen et al 1983). Murillo and Jakupovic (1998) identified myricetin-3 rhamnoside and one flavonoid glycosides in *E. aucherii* which was collected in Iran. Aimova et al (1999) found quercetin 3-(2′′′-Galloylglucosyl) (1→2)-α-L-arabinofuranoside in *E. pachyrrhiza*. Studies of Halaweish et al (2003) are the first report of quercetin-3-O-β glucuronic acid in *E. esula*. They separated and identified kaempferol-3-O-β glucuronic acid and quercetin-3-O-β glucuronic acid from the species. Papp et al (2005) studies showed arial

Mature fresh leaves of eight Legumes, seven *Polygonum*, seven *Rumex*, seventeen *Euphorbia*, two *Chrozophora*, four *Reseda* and five *Scirpus* species from different parts of Iran were collected during 2006-2010 as described in Table 1. Plants identified using available references [Rechinger 1964, Mobayen 1979, 1980, Ghahreman 1979-2006]. Specimens of each sample were prepared for reference as herbarium vouchers that were deposited at the Arak University herbarium. Samples were air dried for detection and identification of flavonoids.

For a comparative analysis of the flavonoids, small extracts of all the accessions were prepared by boiling 200 mg of powdered air dried leaf material for 2 min in 5 ml of 70% EtOH. The mixture was cooled and left to extract for 24 h. The extract was then filtered, evaporated to dryness by rotary evaporation at 40, and taken up in 2 ml of 80% MeOH for

For the detection of flavonoids, ca 20 l of each of the small extracts was applied to the corner of a quarter sheet of Whatman No 1 chromatography paper as a concentrated spot (10 applications of 2l). The chromatogram for each sample was developed in BAW (n-BuOH-HOAc-H2O=4:1:5; V/V; upper layer), 1st direction, and HOAc (=15% aqueous acetic acid), 2nd direction, with rutin (= quercetin 3-*O*-rutinoside) as a standard. After development, the chromatograms were viewed in longwave UV light (366 nm) and any dark absorbing and

When sufficient amounts of purified flavonoids had been obtained, as in the cases of the flavonoids from studied samples, they were identified by means of UV spectroscopy using shift reagents to investigate the substitution patterns of the flavonoids [Mabry et al. 1970, Markham 1982] and by acid hydrolysis to identify the aglycone and sugar moieties. Cochromatography with standards was also performed where possible. Flavonoid standards available for comparison during the study obtained commercially from Merck,

A small amount of each purified flavonoid (ca 0.5 mg) was dissolved in 0.5 ml of 80% MeOH in a test tube. To this sample 2 ml of 2M HCl were added and the mixture was heated in a water bath at 100C for 0.5 h. The solution was cooled, 2 ml of EtOAc were added and thoroughly mixed with the aqueous layer using a whirley mixer. The upper EtOAc layer was removed with a pipette, evaporated to dryness, dissolved in 0.5 ml of MeOH and applied as spots on thin layer chromatograms (cellulose). The TLC plates were run in three solvents alongside standards to identify the aglycone moiety [Harborne 1998].

**2. Materiales and methodes** 

**2.2 Extraction of the plant material** 

**2.1 Collection of plant material and praperation** 

analysis by 2-Dimensional Paper Chromatography (2-D PC).

**2.4 Methods of identification of the flavonoids** 

**2.5 Acid hydrolysis and identification of flavonoid aglycones** 

Sigma and Fluka.

**2.3 Flavonoid analysis by 2-Dimensional Paper Chromatography (2-D PC)** 

fluorescent spots were marked. Rf -values in BAW and 15% HOAc were calculated.

parts of *Euphorbia cyparissias* had 2 main flavonoids: kamfpherol-3-glucuronide and quercetin-3-glucuronide. Adedapo et al (2005) showed arial branch extract of *E. hirta* contains kaempferol, quercitol and quercitrin. The phytochemical studies of Falodun et al (2006) on *E. heterophylla* extract revealed the presence of flavonoids in the extract.

Abdel-Sattar (1985) reported existing flavonoids in *Chrozophora*. Then Hashim et al (1990) found kampferol, acacetin, luteolin and apigenin glycosides in *Chrozophora* species. Isorhamnetin and quercetin glycosides were separated from *C. oblique* [Mohamed 2001]. Talischi et al (2005) reported apigenin and quercetin glycosides from metanolic extract of *C. tinctoria* aerial parts. Also Delazar et al (2006) separated 5-flavonoid glycosid from aerial parts metanolic extract of *C. tinctoria*. Shi et al (2006) has reported 7-flavonoid glycoside from *C. sabulosa* species. Vassallo et al (2006) were separated three new flavon glycosids from *C. senegalensis* leaf metabolic extract. Apigenin and loteulin glycosids were reported from leaf aqueous-etanolic extract of *C. brocchiana* species [Hawas 2007].

#### **1.8 Resedaceae flavonoids**

Several studies indicated that flavonoids occurred in various species of *Reseda*. Eight flavone, 15 flavonols and one isoflavone have been reported from the *Reseda. Reseda luteola* contains 40% flavonoids, primarily luteolin, but also luteolin-7-O glucoside and apigenin [Woelfle et al 2009]. Moiteiro et al (2008) found luteolin 4́-O-glucoside in *Reseda luteola* for first time. Berrahal et al (2006) reported five flavonoid glycosides, quercetin-7-*O*-α-L-rhamnosyl-3-*O*-β-Dglucoside, isorhamnetin-3 *O*-β-D-glycosyl-7-*O*-α-L-rhamnoside, kaempferol-7-*O*-α-Lrhamnoside, kaempferol-7-*O*-α-L-rhamnosyl-3-*O*-β-D-glucoside and kaempferol-3, 7-*O*-α-Ldirhamnoside from aerial parts of *R. villosa* for first time. El-Sayad et al (2001) isolated aglycone flavonols, kaempferol and quercetin from the Mediterranean *Reseda* species. Also Yuldashev et al (1996) reported flavonol diglycosides of kaempferol, quercetin and isorhamnetin from four other *Reseda* species. Rzadkowska (1969) isolated four 3-*O*-glycosides from *R. lutea*.

#### **1.9 Cyperaceae flavonoids**

Clifford and Harborne (1969) studies showed identification of the flavonoid pigment aureusidin from *Scirpus nodosus*. Quercetin, kaempferol, apigenin and luteolin were reported from *S. wichurai* [Ahmed et al 1984]. Naser et al (2000) identified lupeol betulin, betulinalaldehyde and apigenin from *Scirpus tuberosus*. Also β-sitosterol, quercetin 3- βglucoside, quercetin 3, 7- β-diglucoside and isorhamnetin 3, 7- β-glucoside were identified from *Scirpus litoralis* using spectroscopic analyses [Naser et al 2000]. Yang et al (2010) used a developed capillary electrophoresis with amperometric detection method for the determination of some phenolic compounds in the rhizome of *Scirpus yagara* Ohwi. Their work determined existing four phenolic compounds: transresveratrol, scirpusin A, scirpusin B, and p-hydroxycinnamic acid in the species rhizome.

#### **1.10 Aim**

The aim of this study was to compare the leaf flavonoids profiles of some Iranian Angiosperm species from Leguminosae, Polygonaceae, Euphorbiaceae, Resedaceae and Cyperaceae.

#### **2. Materiales and methodes**

156 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

parts of *Euphorbia cyparissias* had 2 main flavonoids: kamfpherol-3-glucuronide and quercetin-3-glucuronide. Adedapo et al (2005) showed arial branch extract of *E. hirta* contains kaempferol, quercitol and quercitrin. The phytochemical studies of Falodun et al

Abdel-Sattar (1985) reported existing flavonoids in *Chrozophora*. Then Hashim et al (1990) found kampferol, acacetin, luteolin and apigenin glycosides in *Chrozophora* species. Isorhamnetin and quercetin glycosides were separated from *C. oblique* [Mohamed 2001]. Talischi et al (2005) reported apigenin and quercetin glycosides from metanolic extract of *C. tinctoria* aerial parts. Also Delazar et al (2006) separated 5-flavonoid glycosid from aerial parts metanolic extract of *C. tinctoria*. Shi et al (2006) has reported 7-flavonoid glycoside from *C. sabulosa* species. Vassallo et al (2006) were separated three new flavon glycosids from *C. senegalensis* leaf metabolic extract. Apigenin and loteulin glycosids were reported

Several studies indicated that flavonoids occurred in various species of *Reseda*. Eight flavone, 15 flavonols and one isoflavone have been reported from the *Reseda. Reseda luteola* contains 40% flavonoids, primarily luteolin, but also luteolin-7-O glucoside and apigenin [Woelfle et al 2009]. Moiteiro et al (2008) found luteolin 4́-O-glucoside in *Reseda luteola* for first time. Berrahal et al (2006) reported five flavonoid glycosides, quercetin-7-*O*-α-L-rhamnosyl-3-*O*-β-Dglucoside, isorhamnetin-3 *O*-β-D-glycosyl-7-*O*-α-L-rhamnoside, kaempferol-7-*O*-α-Lrhamnoside, kaempferol-7-*O*-α-L-rhamnosyl-3-*O*-β-D-glucoside and kaempferol-3, 7-*O*-α-Ldirhamnoside from aerial parts of *R. villosa* for first time. El-Sayad et al (2001) isolated aglycone flavonols, kaempferol and quercetin from the Mediterranean *Reseda* species. Also Yuldashev et al (1996) reported flavonol diglycosides of kaempferol, quercetin and isorhamnetin from four other *Reseda* species. Rzadkowska (1969) isolated four 3-*O*-glycosides

Clifford and Harborne (1969) studies showed identification of the flavonoid pigment aureusidin from *Scirpus nodosus*. Quercetin, kaempferol, apigenin and luteolin were reported from *S. wichurai* [Ahmed et al 1984]. Naser et al (2000) identified lupeol betulin, betulinalaldehyde and apigenin from *Scirpus tuberosus*. Also β-sitosterol, quercetin 3- βglucoside, quercetin 3, 7- β-diglucoside and isorhamnetin 3, 7- β-glucoside were identified from *Scirpus litoralis* using spectroscopic analyses [Naser et al 2000]. Yang et al (2010) used a developed capillary electrophoresis with amperometric detection method for the determination of some phenolic compounds in the rhizome of *Scirpus yagara* Ohwi. Their work determined existing four phenolic compounds: transresveratrol, scirpusin A, scirpusin

The aim of this study was to compare the leaf flavonoids profiles of some Iranian Angiosperm species from Leguminosae, Polygonaceae, Euphorbiaceae, Resedaceae and

(2006) on *E. heterophylla* extract revealed the presence of flavonoids in the extract.

from leaf aqueous-etanolic extract of *C. brocchiana* species [Hawas 2007].

**1.8 Resedaceae flavonoids** 

**1.9 Cyperaceae flavonoids** 

B, and p-hydroxycinnamic acid in the species rhizome.

from *R. lutea*.

**1.10 Aim** 

Cyperaceae.

#### **2.1 Collection of plant material and praperation**

Mature fresh leaves of eight Legumes, seven *Polygonum*, seven *Rumex*, seventeen *Euphorbia*, two *Chrozophora*, four *Reseda* and five *Scirpus* species from different parts of Iran were collected during 2006-2010 as described in Table 1. Plants identified using available references [Rechinger 1964, Mobayen 1979, 1980, Ghahreman 1979-2006]. Specimens of each sample were prepared for reference as herbarium vouchers that were deposited at the Arak University herbarium. Samples were air dried for detection and identification of flavonoids.

#### **2.2 Extraction of the plant material**

For a comparative analysis of the flavonoids, small extracts of all the accessions were prepared by boiling 200 mg of powdered air dried leaf material for 2 min in 5 ml of 70% EtOH. The mixture was cooled and left to extract for 24 h. The extract was then filtered, evaporated to dryness by rotary evaporation at 40, and taken up in 2 ml of 80% MeOH for analysis by 2-Dimensional Paper Chromatography (2-D PC).

#### **2.3 Flavonoid analysis by 2-Dimensional Paper Chromatography (2-D PC)**

For the detection of flavonoids, ca 20 l of each of the small extracts was applied to the corner of a quarter sheet of Whatman No 1 chromatography paper as a concentrated spot (10 applications of 2l). The chromatogram for each sample was developed in BAW (n-BuOH-HOAc-H2O=4:1:5; V/V; upper layer), 1st direction, and HOAc (=15% aqueous acetic acid), 2nd direction, with rutin (= quercetin 3-*O*-rutinoside) as a standard. After development, the chromatograms were viewed in longwave UV light (366 nm) and any dark absorbing and fluorescent spots were marked. Rf -values in BAW and 15% HOAc were calculated.

#### **2.4 Methods of identification of the flavonoids**

When sufficient amounts of purified flavonoids had been obtained, as in the cases of the flavonoids from studied samples, they were identified by means of UV spectroscopy using shift reagents to investigate the substitution patterns of the flavonoids [Mabry et al. 1970, Markham 1982] and by acid hydrolysis to identify the aglycone and sugar moieties. Cochromatography with standards was also performed where possible. Flavonoid standards available for comparison during the study obtained commercially from Merck, Sigma and Fluka.

#### **2.5 Acid hydrolysis and identification of flavonoid aglycones**

A small amount of each purified flavonoid (ca 0.5 mg) was dissolved in 0.5 ml of 80% MeOH in a test tube. To this sample 2 ml of 2M HCl were added and the mixture was heated in a water bath at 100C for 0.5 h. The solution was cooled, 2 ml of EtOAc were added and thoroughly mixed with the aqueous layer using a whirley mixer. The upper EtOAc layer was removed with a pipette, evaporated to dryness, dissolved in 0.5 ml of MeOH and applied as spots on thin layer chromatograms (cellulose). The TLC plates were run in three solvents alongside standards to identify the aglycone moiety [Harborne 1998].


*Chrozophora* \*CAM2 **++++** - - -- - **+++** - **++** - CAM22 **++++** - - -- - **++++** - **-** - *Euphorbia* - \*CMK 23 - - ++ - - - +++ - ++ - CMK 65 - - - -- - +++ - +++ - CMK 57 - - - -- - +++ - +++ - CMK 63 - - - -- - - - ++ - CMK 60 - - +++ - - - +++ - + - CMK 59 - - +++ - - - ++ - +++ - CMK 32 - - - -- - +++ - + - CMK 26 - - +++ - - - ++ - ++ - CMK 54 - - - - +++ - ++ - - - CMK 70 - - - -- - +++ - +++ - CMK 69 - - +++ - - - ++ - + - CMK 62 - - ++ - - - + - ++ - CMK 74 - - - - +++ - +++ - - - CMK 16 - - +++ - - - +++ - +++ - CMK 10 - - - -- - + - + - CMK 48 - - ++ - - - +++ - ++ - CMK 34 - - - -- - - + - *Papilionoideae* \*CMJ148 - - ++ - - - -- - ++ - CMJ149 - - ++ +++ +++ ++ - CMF1 - - - - ± - + + ± - CMJ150 - - -- - - - ++ - ++ - CMJ151 - - - -- - - - - - CMJ152 ++ ++ - - - - ++- - CMJ153 ± - - -- - - - - - CMN1 ± - - -- - - - - - *Polygonum* \*CEM1 - - **+** - **+** - + - **+** - CEM2 - - **-** - **-** - + - **-** - CEM3 - - **+** - **+** - + - **-** - CEM4 - - **+** - **-** - + - **-** - CEM5 - - **+** - **-** - + - **+** - CAM6 - - **+** - **+** - + - **+** - CAM7 - - **-** - **+** - + - **-** - *Reseda* \*CMG27 - - + - + - +++ ++ ++ - CMG21 - - +++ + ++ - - - - - CMG22 - - +++ + ++ - - - - - CMG11 - - +++ ++ - - +++ ± ++ *Rumex* + \*CMR2 + - + + - - + + CMR4 - - - ± - ++ ++ + - - CMR6 - - ± ++ - ± ± + + - CMR7 ++ - ± - - ± ± ++ - - CMR9 +- + + - - ++ + + - CMR12 - - + - - ± +++ - - - CMR14 + - - ± ± ± ++ + - - *Scirpus* \*CNM4 ++ + - + - +++ + + + + CMN23 - ± - + ± ± +++ + ++ +++ CMN8 ++ + - + - +++ - +++ + + CMN6 - + - + - ++ - ++ + + CMN18 ++ + - ++ - +++ ++ ++ + ++

Apigenin Chrycin Kaempferol Luteolin Myricetin Naringenin Quercetin Rhamnetin Rutin Vitexin

**Voucher data Identification**

\*C=Collection number

Table 1. Continued

+++ (high concentration of flavonoid)


Table 1. The sampling and also two-dimensional paper and thin layer chromatographically data of 48 studied plant samples from Markazi Province, Iran.

158 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

\*CAM2 *C. tinctoria* 3 3 2 8 CAM22 *C. hierosolymitana* 3 2 3 8

\*CMK 23 *E. bungei* Boiss. 5 2 3 0 CMK 65 *E. chamaesyce* L. 8 4 4 0 CMK 57 *E. cheiradenia* Boiss. et Hohen. 75 2 0 CMK 63 *E. cordifolia* Ell. 9 3 6 0 CMK 60 *E. esula* L. 73 4 0 CMK 59 *E. falcata* L. 7 3 4 0 CMK 32 *E. helioscopia* L. 5 3 2 0 CMK 26 *E. heteradena* Jaub,et Spach. 9 3 6 0 CMK 54 *E. macroclada* Boiss*.* 61 5 0 CMK 70 *E. microsciadeae* Boiss*.* 95 4 0 CMK 69 *E. ozyridiforma* Parsa*.* 7 4 3 0 CMK 62 *E. peplus* L*.* 8 4 4 0 CMK 74 *E. petiolata* Banks et Soland 9 6 3 0 CMK 16 *E. seguieriana* Necker*.* 8 5 3 0 CMK 10 *E. splendida Mobayen.* 73 4 0 CMK 48 *E. szovitsii* Fisch.& Mey. 8 5 3 0 CMK 34 *E. tehranica* Boiss*.* 86 2 0

\*CMJ148 *Alhagi camelorum* Fisch. 2 1 1 0 CMJ149 *Cercis siliquastrum* L. 21 1 0 CMF1 *Coronilla varia* L. 54 1 0 CMJ150 *Glycyrrhiza glabra* L. 2 1 1 0 CMJ151 *Medicago sativa* L. 00 0 0 CMJ152 *Robina peseudo-acacia L.* 4 3 1 0 CMJ153 *Sophora alopecuroides ssp. alopecuroides* 00 0 0 CMN1 *Sophora alopecuroides ssp. tomentosa* 00 0 0

\*CEM1 *P. aviculare* 4 0 4 0 CEM2 *P. convolvolus* 1 0 1 0 CEM3 *P. hyrcanicum* 4 0 4 0 CEM4 *P. patulum* 3 0 3 0 CEM5 *P. alpestre* 3 0 3 0 CAM6 *P. arenastrum* 4 0 4 0 CAM7 *P. persicaria* 3 1 2 0

\*CMG27 *Reseda aucheri* 8 5 3 0 CMG21 *R. buhseana* Mull-Arg. 10 5 4 1 CMG22 *R. bungei* Boiss. 8 7 1 0 CMG11 *R. lutea* L. 7 6 1 0

\*CMR2 *R. chalpensis* 10 4 6 0 CMR4 *R. crispus* 6 2 4 0 CMR6 *R. obtusifolius* 10 4 6 0 CMR7 *R. tuberosus* 10 5 5 0 CMR9 *R .pulcher* 82 6 0 CMR12 *R. acetosella* 82 6 0 CMR14 *R.conglomeratus* 92 6 1

\*CNM4 *S. holoschenus* L. 8 4 1 3 CMN23 *S. lacustris* L. 6 5 0 1 CMN8 *S. littoralis* Kuntze 10 8 1 1 CMN6 *S. maritimus* L. 95 3 1 CMN18 *S. multicaule* 12 5 2 5 Table 1. The sampling and also two-dimensional paper and thin layer chromatographically

data of 48 studied plant samples from Markazi Province, Iran.

total flavonoids Number of flavonoid sulphates

Number of flavone C-and C-/O-glucosides Number of aglycones

Taxon Number of

Voucher data

*Chrozophora*

*Euphorbia*

*Papilionoideae*

*Polygonum*

*Reseda*

*Rumex* 

*Scirpus*


\*C=Collection number


Table 1. Continued

*Euphorbia* species contained flavonoid compounds in their leaves that their flavonoid profiles show a wide variety between the taxa. There are flavonoid sulphate and flavone *C* and *C*-/*O*-glycosides in all species, but *E. bungei*, *E. heteradena* and *E. microsciadea* in addition these two flavonoid types have dihydroflavonol 3-*O*-monoglycosides. *E. cordifoila*, *E. heteradena*, *E. microsciadeae* and *E. petiolata* have the highest number of total flavonoid compounds (9) and *E. bungei* and *E. helioscopia* have the lowest number of flavonoid compounds (2) in their leaves (Table 1). Identification of flavonoids by standards showed all of studied *Euphorbia* species contain rutin with the exception of *E. macroclada* and *E. petiolata*. Also all taxa studied, except 2 species (*E. cordifolia* and *E. tehranica*) have quercetin. Harborne and Baxter (1999) reported that quercetin is widely distributed in various plant families. Kaempferol found in 8 species and myricetin was found just in *E. macroclada* and *E. petiolata* (Table 1). As Volobueva (1970) showed two-dimentional paper chromatography and acid hydrolysis of *E. helioscopia* alcoholic extract yielded quercetin and kaempferol. Also Gautam and Mukhraya (1981) isolated quercetin 3-*O* glucoside and kaempferol 3-*O* glucoside from *E. larica*, *E. virgata*, *E. chamaesyce* and *E. magalanta* and rutin obtained with the exception *E. chamaesyce*. Both quercetin and kaempferol are flavonols. The flavonols may be among the most important flavonoids, they are the most ancient and widespread of the flavonoids, synthesized even in mosses and ferns, and have a wide range of potent physiological activities [Stafford 1991]. Chemical study of 17 *Euphorbia* species using two dimentional paper chromatography (2-DPC) and thin layer chromatography (TLC) showed rutin, quercetin and kaempferol are the most representative compounds for the genus and the presence of myricetin is a taxonomic character for separation of some *Euphorbia* species. It is believed that *Euphorbia* species can be separated from each other according to their

Application of plant flavonoides data can revealed similarity and relationship between plants and inferring phylogeny and used in their taxonomy. 2-dimentional paper chromatography (2-D PC), on leaves of *Alhagi camelorum* Fisch, *Cersis silliquastrum* L., *Coronilla varia* L., *Glycirhiza glarba* L. and *Robnia peseudoacia* from Markazi Province showed all of five named species contain aglycones. *A. camelorum* and *G. glarba* had the most flavonoid variation and concentration having flavon glycoside and two subspecies of *Sophora alopecuroides* and *Medicago sativa* had not or had the least. The most flavonoid

Phytochemical examination of the studied *polygonum* species showed all of *Polygonum* species contain flavon *C*- and *C*-/*O*-glycosides. *P. hyrcanicum* had the most flavonoid variation and concentration and *P. convolvolus* species with having just one flavonoid had

Chemical study of four *Reseda* species using two dimentional paper chromatography (2-DPC) and thin layer chromatography (TLC) showed all studied *Reseda* species contained flavonoid compounds in their leaves and kempferol is the most representative compound for the genus (Table 1). They may be useful taxonomic markers within the genus. Also *Reseda* flavonoids are very important for their toxicity and some different potential clinical applications such as their antiatherosclerotic, antiinflammatory, antitumor, antithrombogenic, antiosteoporotic and antiviral effects [Nijveldt et al 2001]. The presence of quercetin and absence of myrestin in *R. lutea* are taxonomic characters for separation of the species from two other species (*R. buhseana* and *R. bungei*). Among the many functions of flavonoids at the interface between plant and

compounds similarity was between *C. silliquastrum* and *R. peseudoacia* (Table 1).

the least. Flavonoid sulphates was found just in *P. persicaria* species (Table 1).

flavonoid pattern.

### **3. Results**

All studied plant species exceptional two subspecies *Sophora alopecuroides* and *Medicago sativa* contained flavonoid compounds in their leaves. Their flavonoid profiles show a wide variety between the species. Data in Table 1 shows the sampling and also two-dimensional paper and thin layer chromatographical data of 48 studied *plant samples* from Markazi Province, Iran.

### **4. Discussion**

Studies of leaf flavonoids showed some phytochemical characters such as total number of flavonoids, flavonoid group such as aglycone, flavones *C-* and *C-/O* glycoside and flavonoid sulphate and kind of flavonoids such as kaempherol, quercetin, myricetin are valuable for chemotaxonomy and their usage.

Chemical study of two *Chrozophora* species using two dimentional paper chromatography (2-DPC) and thin layer chromatography (TLC) showed both *Chrozophora* species contain flavonoid sulphates, flavone *C* and *C-/O*-glycosides and aglycon. Also all of studied species have apigenin and quercetin while rutin was just found in *C. tinctoria* species that is recorded first time for Markazi Province. All of studied species have flavonoid compounds that have variation in their flavonoid type and number (Table 1).

Phytochemical studies of the Euphorbiaceae have been extremely useful in clarifying systematic relationships within the family (Simpson and Levin 1994). Flavonoids occur widely in plants and are a biologically major and chemically diverse group of secondary metabolites that are popular compounds for chemotaxonomic surveys of plant genera and families [Harborne 1994]. There are some studies in this connection. Mues and Zinsmeister (1988) have discussed about variation of occurrence phenolic compounds in mosses and liverworts. Also they showed there is a clear flavonoid distinction between the subclasses Marchantiidae and Jungermanniidae. Another important chemotaxonomic programme has concerned the ferns and fern allies [Harborne 1986]. The phenolic patterns appear to be more useful for studying relationships within relatively narrow taxonomic limits, e. g. at the species and genus level. Turning to the angiosperms, a chemotaxonomic survey of 255 species of the family Iridaceae has been carried out by Williams et al (1986), who found that flavone *C*-glycosides were present in 66% of the samples [Harborne 1986]. Another family survey has been carried out in the *Polygonaceae*, in which 28 species were analysed for their flavonoid pattern [Harborne 1986]. Studying flavonoid pattern can be used for chemosystematic and lower taxonomic levels. 25 *Avena* species (Poaceae) were investigated for the flavonoid content of leaf tissue [Saleh et al 1988]. Diploid *triticum* species could be divided into two groups depending on the presence or absence of two major di-*C*glycosylflavones (Harborne et al 1986). Flavonoid data of the genus *Vitis* indicate three chemical groups [Moore and Giannasi 1994]. Several studies indicated that flavonoids occurred in various species of *Euphorbia*. They may be useful taxonomic markers within the genus. Also *Euphorbia* flavonoids are very important for their toxicity and some different potential clinical applications such as their antiatherosclerotic, antiinflammatory, antitumor, antithrombogenic, antiosteoporotic and antiviral effects [Nijveldt et al 2001]. Papp et al (2005) showed populations of *Euphorbia cyparissias* can be separated clearly from each other according to their morphology and flavonoid pattern.Our results showed all studied

All studied plant species exceptional two subspecies *Sophora alopecuroides* and *Medicago sativa* contained flavonoid compounds in their leaves. Their flavonoid profiles show a wide variety between the species. Data in Table 1 shows the sampling and also two-dimensional paper and thin layer chromatographical data of 48 studied *plant samples* from Markazi

Studies of leaf flavonoids showed some phytochemical characters such as total number of flavonoids, flavonoid group such as aglycone, flavones *C-* and *C-/O* glycoside and flavonoid sulphate and kind of flavonoids such as kaempherol, quercetin, myricetin are valuable for

Chemical study of two *Chrozophora* species using two dimentional paper chromatography (2-DPC) and thin layer chromatography (TLC) showed both *Chrozophora* species contain flavonoid sulphates, flavone *C* and *C-/O*-glycosides and aglycon. Also all of studied species have apigenin and quercetin while rutin was just found in *C. tinctoria* species that is recorded first time for Markazi Province. All of studied species have flavonoid compounds

Phytochemical studies of the Euphorbiaceae have been extremely useful in clarifying systematic relationships within the family (Simpson and Levin 1994). Flavonoids occur widely in plants and are a biologically major and chemically diverse group of secondary metabolites that are popular compounds for chemotaxonomic surveys of plant genera and families [Harborne 1994]. There are some studies in this connection. Mues and Zinsmeister (1988) have discussed about variation of occurrence phenolic compounds in mosses and liverworts. Also they showed there is a clear flavonoid distinction between the subclasses Marchantiidae and Jungermanniidae. Another important chemotaxonomic programme has concerned the ferns and fern allies [Harborne 1986]. The phenolic patterns appear to be more useful for studying relationships within relatively narrow taxonomic limits, e. g. at the species and genus level. Turning to the angiosperms, a chemotaxonomic survey of 255 species of the family Iridaceae has been carried out by Williams et al (1986), who found that flavone *C*-glycosides were present in 66% of the samples [Harborne 1986]. Another family survey has been carried out in the *Polygonaceae*, in which 28 species were analysed for their flavonoid pattern [Harborne 1986]. Studying flavonoid pattern can be used for chemosystematic and lower taxonomic levels. 25 *Avena* species (Poaceae) were investigated for the flavonoid content of leaf tissue [Saleh et al 1988]. Diploid *triticum* species could be divided into two groups depending on the presence or absence of two major di-*C*glycosylflavones (Harborne et al 1986). Flavonoid data of the genus *Vitis* indicate three chemical groups [Moore and Giannasi 1994]. Several studies indicated that flavonoids occurred in various species of *Euphorbia*. They may be useful taxonomic markers within the genus. Also *Euphorbia* flavonoids are very important for their toxicity and some different potential clinical applications such as their antiatherosclerotic, antiinflammatory, antitumor, antithrombogenic, antiosteoporotic and antiviral effects [Nijveldt et al 2001]. Papp et al (2005) showed populations of *Euphorbia cyparissias* can be separated clearly from each other according to their morphology and flavonoid pattern.Our results showed all studied

that have variation in their flavonoid type and number (Table 1).

**3. Results** 

Province, Iran.

**4. Discussion** 

chemotaxonomy and their usage.

*Euphorbia* species contained flavonoid compounds in their leaves that their flavonoid profiles show a wide variety between the taxa. There are flavonoid sulphate and flavone *C* and *C*-/*O*-glycosides in all species, but *E. bungei*, *E. heteradena* and *E. microsciadea* in addition these two flavonoid types have dihydroflavonol 3-*O*-monoglycosides. *E. cordifoila*, *E. heteradena*, *E. microsciadeae* and *E. petiolata* have the highest number of total flavonoid compounds (9) and *E. bungei* and *E. helioscopia* have the lowest number of flavonoid compounds (2) in their leaves (Table 1). Identification of flavonoids by standards showed all of studied *Euphorbia* species contain rutin with the exception of *E. macroclada* and *E. petiolata*. Also all taxa studied, except 2 species (*E. cordifolia* and *E. tehranica*) have quercetin. Harborne and Baxter (1999) reported that quercetin is widely distributed in various plant families. Kaempferol found in 8 species and myricetin was found just in *E. macroclada* and *E. petiolata* (Table 1). As Volobueva (1970) showed two-dimentional paper chromatography and acid hydrolysis of *E. helioscopia* alcoholic extract yielded quercetin and kaempferol. Also Gautam and Mukhraya (1981) isolated quercetin 3-*O* glucoside and kaempferol 3-*O* glucoside from *E. larica*, *E. virgata*, *E. chamaesyce* and *E. magalanta* and rutin obtained with the exception *E. chamaesyce*. Both quercetin and kaempferol are flavonols. The flavonols may be among the most important flavonoids, they are the most ancient and widespread of the flavonoids, synthesized even in mosses and ferns, and have a wide range of potent physiological activities [Stafford 1991]. Chemical study of 17 *Euphorbia* species using two dimentional paper chromatography (2-DPC) and thin layer chromatography (TLC) showed rutin, quercetin and kaempferol are the most representative compounds for the genus and the presence of myricetin is a taxonomic character for separation of some *Euphorbia* species. It is believed that *Euphorbia* species can be separated from each other according to their flavonoid pattern.

Application of plant flavonoides data can revealed similarity and relationship between plants and inferring phylogeny and used in their taxonomy. 2-dimentional paper chromatography (2-D PC), on leaves of *Alhagi camelorum* Fisch, *Cersis silliquastrum* L., *Coronilla varia* L., *Glycirhiza glarba* L. and *Robnia peseudoacia* from Markazi Province showed all of five named species contain aglycones. *A. camelorum* and *G. glarba* had the most flavonoid variation and concentration having flavon glycoside and two subspecies of *Sophora alopecuroides* and *Medicago sativa* had not or had the least. The most flavonoid compounds similarity was between *C. silliquastrum* and *R. peseudoacia* (Table 1).

Phytochemical examination of the studied *polygonum* species showed all of *Polygonum* species contain flavon *C*- and *C*-/*O*-glycosides. *P. hyrcanicum* had the most flavonoid variation and concentration and *P. convolvolus* species with having just one flavonoid had the least. Flavonoid sulphates was found just in *P. persicaria* species (Table 1).

Chemical study of four *Reseda* species using two dimentional paper chromatography (2-DPC) and thin layer chromatography (TLC) showed all studied *Reseda* species contained flavonoid compounds in their leaves and kempferol is the most representative compound for the genus (Table 1). They may be useful taxonomic markers within the genus. Also *Reseda* flavonoids are very important for their toxicity and some different potential clinical applications such as their antiatherosclerotic, antiinflammatory, antitumor, antithrombogenic, antiosteoporotic and antiviral effects [Nijveldt et al 2001]. The presence of quercetin and absence of myrestin in *R. lutea* are taxonomic characters for separation of the species from two other species (*R. buhseana* and *R. bungei*). Among the many functions of flavonoids at the interface between plant and

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environment, their activity as signals was intensively studied. Flavonoids are also beneficial for the plant itself as physiological active compounds, as stress protecting agents, as attractants or as feeding deterrents, and, in general, by their significant role in plant resistance [Treutter 2006].

Chemical studies of seven *Rumex* species using two dimentional paper chromatography (2- DPC) and thin layer chromatography (TLC) showed all of studied *Rumex* species contain flavonoid compounds with wide variation. *R. chalpensis*, *R. obtosifolius* and *R. tuberusos* species had the most flavonoid number and *R. crispus* species had the least. Identified flavonoid compounds in all of studied species with the exception *R. crispus* (lack flavonoid sulphate) are flavones *C* and *C-/O* glucoside. *R. acetosella* and *R. conglomerates* had aglycon. Rutin and luteolin found in all of studied species exceptional *R. chalpensis*, *R. obtosifolius* and *R. pulcher*. All of studied species showed wide variation in existing and concentration of myricetin, apigenin, narengenin, rhamnetin, quercetin and kaempferol. All of studied species with the exception *R. chalpensis* and *R. tuberusos* had quercetin and also kaempferol found in 3 species (*R. chalpensis*, *R. pulcher* and *R. acetosella*) (Table 1).

Phytochemical studies on five species of *Scirpus* (*S. holoschenus* L., *S. lacustris* L.*, S. littoralis* Kuntze, *S. maritimus* L. and *S. multicaule*) from different parts of Markazi Province, Iran area using two-dimentional paper chromatography (2-DPC) and thin layer chromatography (TLC) showed all of studied taxa contain vitexin, luteolin, rutin and rhamnetin. There were chrysin and naringenin in all of populations with the exception of *S. lucustris* and apigenin was found in 3 species weheras others lack. Quercetin was not found in *S. maritimus* and *S. littoralis* where as three other species had (Table 1).

Our studies showed the most of collected plant species are weed and grow in poor soils and destroyed pasture. Progress continues to be made in understanding the roles of flavonoids in stress protection, as well as in defining the mechanisms that control the amount and varieties of flavonoids that are produced in plants in responses to diverse environmental cuse [Chalker-Scott 1999]. Finally, further work is needed using high performance liquid chromatography with diode array detection, atmospheric pressure chemical ionization liquid chromatography-mass spectroscopy to evaluate all flavonoid profiles in studied and other species.

#### **5. References**


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environment, their activity as signals was intensively studied. Flavonoids are also beneficial for the plant itself as physiological active compounds, as stress protecting agents, as attractants or as feeding deterrents, and, in general, by their significant role in plant resistance [Treutter

Chemical studies of seven *Rumex* species using two dimentional paper chromatography (2- DPC) and thin layer chromatography (TLC) showed all of studied *Rumex* species contain flavonoid compounds with wide variation. *R. chalpensis*, *R. obtosifolius* and *R. tuberusos* species had the most flavonoid number and *R. crispus* species had the least. Identified flavonoid compounds in all of studied species with the exception *R. crispus* (lack flavonoid sulphate) are flavones *C* and *C-/O* glucoside. *R. acetosella* and *R. conglomerates* had aglycon. Rutin and luteolin found in all of studied species exceptional *R. chalpensis*, *R. obtosifolius* and *R. pulcher*. All of studied species showed wide variation in existing and concentration of myricetin, apigenin, narengenin, rhamnetin, quercetin and kaempferol. All of studied species with the exception *R. chalpensis* and *R. tuberusos* had quercetin and also kaempferol

Phytochemical studies on five species of *Scirpus* (*S. holoschenus* L., *S. lacustris* L.*, S. littoralis* Kuntze, *S. maritimus* L. and *S. multicaule*) from different parts of Markazi Province, Iran area using two-dimentional paper chromatography (2-DPC) and thin layer chromatography (TLC) showed all of studied taxa contain vitexin, luteolin, rutin and rhamnetin. There were chrysin and naringenin in all of populations with the exception of *S. lucustris* and apigenin was found in 3 species weheras others lack. Quercetin was not found in *S. maritimus* and *S.* 

Our studies showed the most of collected plant species are weed and grow in poor soils and destroyed pasture. Progress continues to be made in understanding the roles of flavonoids in stress protection, as well as in defining the mechanisms that control the amount and varieties of flavonoids that are produced in plants in responses to diverse environmental cuse [Chalker-Scott 1999]. Finally, further work is needed using high performance liquid chromatography with diode array detection, atmospheric pressure chemical ionization liquid chromatography-mass spectroscopy to evaluate all flavonoid profiles in studied and

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**8** 

**Phytochemicals from** 

Nkeng-Efouet-Alango Pépin

*University of Dschang,* 

*Cameroon* 

*Beilschmiedia anacardioides*

**and Their Biological Significance** 

Medicinal plants provide a vast array of raw materials for primary health care in Africa and other countries of the world. The World Health Organization (W.H.O) estimates that about 80% of Africans living in the continent have resort to traditional medical practitioners and the use of traditional medicine for the treatment of their diverse ailments. This practice has a

It is estimated that less than 10% of the world's genetic resources have been studied seriously as sources of medicines. Yet from this small fraction, humanity has reaped

The search for bioactive plant natural products from higher plants is gathering momentum, as they have potential to provide new lead compounds or to be of use directly. There is an increasing sense of urgency about this search due to the destruction of natural resources. With regards to these plants, it has been estimated that 25-30 million hectares of the world's rainforests are lost each year. The crucial problem already expressed by several scientists then is how to search efficiently and rapidly for bioactive components from the vast number

Six approaches to the selection of plant materials for study exist: the locally random, the taxonomic, the ethnobotanic, the phytochemical, the information based, and serendipity. The ethnomedical approach appears to be the method of choice for natural product chemists working in Africa and other developing countries. In this method only plants used in

 Very little attention has been paid to *Beilshmiedia* species. Previous studies concern trees and herbs of *Beilshmiedia* species, with the aim of cultivating herbs containing the same endiandric acid derivatives as trees. Other studies led to a patent on interesting synthesis of

In our own search for prospective pharmacological products from ethnobotanic data, we have been looking at some traditional medicines whose therapeutic efficiency is scientifically established towards biomedical analyses of patients on treatment in a specialized clinic. We have selected a traditional medicine based on one plant *Beilshmiedia* 

considerable importance within the economic and cultural milieu of Africa.

of unstudied plants. Part of the solution is to narrow down the search-selection.

**1. Introduction** 

enormous benefits.

traditional medicine are collected.

endiandric acid derivatives.


## **Phytochemicals from**  *Beilschmiedia anacardioides* **and Their Biological Significance**

Nkeng-Efouet-Alango Pépin *University of Dschang, Cameroon* 

#### **1. Introduction**

166 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

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*Euphorbia larica, E. virgata, E. chamaesyce* and *E. magalanta*, *Journal of Natural* 

agents, 3-methoxyflavones as potent inhibitors of viral-induced block of cell

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Mediterranean diet. *Food Chemistry*, 70, 319-323.

Medicinal plants provide a vast array of raw materials for primary health care in Africa and other countries of the world. The World Health Organization (W.H.O) estimates that about 80% of Africans living in the continent have resort to traditional medical practitioners and the use of traditional medicine for the treatment of their diverse ailments. This practice has a considerable importance within the economic and cultural milieu of Africa.

It is estimated that less than 10% of the world's genetic resources have been studied seriously as sources of medicines. Yet from this small fraction, humanity has reaped enormous benefits.

The search for bioactive plant natural products from higher plants is gathering momentum, as they have potential to provide new lead compounds or to be of use directly. There is an increasing sense of urgency about this search due to the destruction of natural resources. With regards to these plants, it has been estimated that 25-30 million hectares of the world's rainforests are lost each year. The crucial problem already expressed by several scientists then is how to search efficiently and rapidly for bioactive components from the vast number of unstudied plants. Part of the solution is to narrow down the search-selection.

Six approaches to the selection of plant materials for study exist: the locally random, the taxonomic, the ethnobotanic, the phytochemical, the information based, and serendipity. The ethnomedical approach appears to be the method of choice for natural product chemists working in Africa and other developing countries. In this method only plants used in traditional medicine are collected.

 Very little attention has been paid to *Beilshmiedia* species. Previous studies concern trees and herbs of *Beilshmiedia* species, with the aim of cultivating herbs containing the same endiandric acid derivatives as trees. Other studies led to a patent on interesting synthesis of endiandric acid derivatives.

In our own search for prospective pharmacological products from ethnobotanic data, we have been looking at some traditional medicines whose therapeutic efficiency is scientifically established towards biomedical analyses of patients on treatment in a specialized clinic. We have selected a traditional medicine based on one plant *Beilshmiedia* 

Phytochemicals from *Beilschmiedia anacardioides* and Their Biological Significance 169

The Bamoun are a Bantu people living in the west Region of Cameroon. They number more than half a million. They have a rich cultural Heritage, including famous traditional Healers. Sultan Njoya wrote a book on Bamoun traditional medicine. Important Bamoun towns are Foumban, Foumbot, Koutaba, Massangam, Magba, Malantouen. Among important villages are Mahoua, Manki 1 and Manki 2, where the plant *Beilshmiedia* 

*B. anacardioides* is found in Central Africa, especially in Cameroon, Tchad and Gabon. In Cameroon, this species is found in the Adamaoua and the West Region (Eyog et al., 2006; Fouilloy, 1974). It is synonymous with *B. ngriki* and *B. Jacques-felixii* and it is commonly named *ntseum* (in Bamoun language) in the Noun subdivision of the West region of

*B. anacardioides* stem bark is used in the Noun sub-division of the West Region of Cameroon to treat uterine tumours, rubella, rheumatisms, bacterial and fungal infections (Tchoula, 2001). Seeds are used as spices (Eyog et al., 2006). *B. lancilimba* is used in the same region to cure skin bacterial infections (Tchouala, 2001). *B. manii* is used to treat dysentery and

A review of the literature revealed that no phytochemial studies have been carried out on *Beilshmiedia anacardioides* prior to the initiation of our study. The various phytochemical and pharmacological studies performed and reported in the literature on the beilshmiedia genus

**Structure and name Source and references** 

Wood of *B. madang* (Kitagawa et al., 1993)

*anacardioides* was collected.

are discussed below.

Me

**9**: Dehatrine

**3.1 Generalities on** *Beilschmiedia* **anacardioides** 

Cameroon (Eyog et al., 2006; Fouilloy, 1974; Tchouala, 2001).

**3.2 Uses of** *Beilschmiedia* **species in traditional medicine** 

headache. It is also used as an appetite stimulant (Iwu, 1993).

**4.1 Alkaloids isolated from the** *Beilschmiedia* **genus** 

Very few alkaloids have been isolated from the *Beilschmiedia* genus.

OMe

N N

O

MeO

OMe

H O

OMe

**4. Phytochemistry of plant constituents of** *Beilshmiedia* **species** 

*anacardioides* (Lauraceae), for its proven efficiency on genital infections and rheumatisms through clinical research. No phytochemical studies of *Beilshmiedia anacardioides* are however to our knowledge available in the literature. We propose that phytochemists looking for novel bioactive natural products should investigate the medicinal plants whose therapeutic efficiency has been established through clinical research on African medicine.

The genus *Beilschmiedia* comprises about 200 species widely distributed in the intertropical region (Fouilloy, 1974). *B. anacardioides* stem bark is used in the Western Province of Cameroon to cure uterine tumours (Tchouala, 2001). Some other species of the genus Beilschmiedia are used in traditional medicine in Africa for the treatment of several ailments (Tchouala, 2001; Iwu, 1993). Previous phytochemical investigations of plants of the genus *Beilschmiedia* reported the presence of bio-active lignans (Chen et al., 2006; Chen et al., 2007), flavonoids (Harbone et al., 1969), triterpenoids (Chen et al., 2006); tetracyclic endiandric acid (Bandaranayake et al., 1981; Banfield et al., 1994) and alkaloids (Clezy et al., 1966; Kitagawa et al., 1993; Chouna et al., 2011).

We have initiated a systematic phytochemical investigation of the extracts of *Beilshmiedia anacardioides* as well as the antibacterial activity of the eight new compounds isolated, towards five strains of microbes, namely *Bacillus subtilis*, *Micrococcus luteus*, *Streptococcus faecalis*, *Pseudomonas palida*, and *Escherichia coli*.

The methods used for the isolation of the compounds were mainly column chromatography and preparative TLC. The structures of all compounds were elucidated by means of modern spectroscopic techniques such as 1D-NMR (1H-NMR, 13C-NMR with DEPT experiments), and 2D-NMR (1H-1H-COSY, HMQC,HMBC,NOESY), MS , IR and X-Ray spectroscopies.

The antibacterial activities of the new compounds were examined using the dilution technique with respect to the zone of inhibition (ZI) and minimum inhibitory concentration (MIC).

We report here the results we have so far obtained and published in three renowned scientific journals (Chouna et al.,2009; 2010; 2011).

#### **2. Study of the ethnomedical preparation**

The ethnomedical preparation is a decoction.The decoction is prepared as follows: Boil about 80 g dry stem bark powder in 3 litres of water for 15 minutes. Filter when lukewarm. Drink a glass twice daily for ten days.

A treatment for fibromes could last about two to three months, depending on the patient's age.

#### **3. Study setting**

Cameroon is a bridge between Central Africa and West Africa, humid Africa and dry sahelian Africa, French speaking and English speaking Africa ( French and English are official languages). The country is open to the Gulf of Guinea in his south-west border. Lake Chad is at the extreme –North border. A country of 475.442 square kilometers, Cameroon is borded in the west by Nigeria, on the east by Chad and the Central African Republic, and on the south by Congo, Gabon, and Equatorial Guinea.

The Bamoun are a Bantu people living in the west Region of Cameroon. They number more than half a million. They have a rich cultural Heritage, including famous traditional Healers. Sultan Njoya wrote a book on Bamoun traditional medicine. Important Bamoun towns are Foumban, Foumbot, Koutaba, Massangam, Magba, Malantouen. Among important villages are Mahoua, Manki 1 and Manki 2, where the plant *Beilshmiedia anacardioides* was collected.

#### **3.1 Generalities on** *Beilschmiedia* **anacardioides**

168 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

*anacardioides* (Lauraceae), for its proven efficiency on genital infections and rheumatisms through clinical research. No phytochemical studies of *Beilshmiedia anacardioides* are however to our knowledge available in the literature. We propose that phytochemists looking for novel bioactive natural products should investigate the medicinal plants whose therapeutic

The genus *Beilschmiedia* comprises about 200 species widely distributed in the intertropical region (Fouilloy, 1974). *B. anacardioides* stem bark is used in the Western Province of Cameroon to cure uterine tumours (Tchouala, 2001). Some other species of the genus Beilschmiedia are used in traditional medicine in Africa for the treatment of several ailments (Tchouala, 2001; Iwu, 1993). Previous phytochemical investigations of plants of the genus *Beilschmiedia* reported the presence of bio-active lignans (Chen et al., 2006; Chen et al., 2007), flavonoids (Harbone et al., 1969), triterpenoids (Chen et al., 2006); tetracyclic endiandric acid (Bandaranayake et al., 1981; Banfield et al., 1994) and alkaloids (Clezy et al., 1966; Kitagawa

We have initiated a systematic phytochemical investigation of the extracts of *Beilshmiedia anacardioides* as well as the antibacterial activity of the eight new compounds isolated, towards five strains of microbes, namely *Bacillus subtilis*, *Micrococcus luteus*, *Streptococcus* 

The methods used for the isolation of the compounds were mainly column chromatography and preparative TLC. The structures of all compounds were elucidated by means of modern spectroscopic techniques such as 1D-NMR (1H-NMR, 13C-NMR with DEPT experiments), and 2D-NMR (1H-1H-COSY, HMQC,HMBC,NOESY), MS , IR and X-Ray spectroscopies.

The antibacterial activities of the new compounds were examined using the dilution technique with respect to the zone of inhibition (ZI) and minimum inhibitory concentration

We report here the results we have so far obtained and published in three renowned

The ethnomedical preparation is a decoction.The decoction is prepared as follows: Boil about 80 g dry stem bark powder in 3 litres of water for 15 minutes. Filter when lukewarm.

A treatment for fibromes could last about two to three months, depending on the patient's

Cameroon is a bridge between Central Africa and West Africa, humid Africa and dry sahelian Africa, French speaking and English speaking Africa ( French and English are official languages). The country is open to the Gulf of Guinea in his south-west border. Lake Chad is at the extreme –North border. A country of 475.442 square kilometers, Cameroon is borded in the west by Nigeria, on the east by Chad and the Central African Republic, and on

efficiency has been established through clinical research on African medicine.

et al., 1993; Chouna et al., 2011).

(MIC).

age.

**3. Study setting** 

*faecalis*, *Pseudomonas palida*, and *Escherichia coli*.

scientific journals (Chouna et al.,2009; 2010; 2011).

**2. Study of the ethnomedical preparation** 

the south by Congo, Gabon, and Equatorial Guinea.

Drink a glass twice daily for ten days.

*B. anacardioides* is found in Central Africa, especially in Cameroon, Tchad and Gabon. In Cameroon, this species is found in the Adamaoua and the West Region (Eyog et al., 2006; Fouilloy, 1974). It is synonymous with *B. ngriki* and *B. Jacques-felixii* and it is commonly named *ntseum* (in Bamoun language) in the Noun subdivision of the West region of Cameroon (Eyog et al., 2006; Fouilloy, 1974; Tchouala, 2001).

#### **3.2 Uses of** *Beilschmiedia* **species in traditional medicine**

*B. anacardioides* stem bark is used in the Noun sub-division of the West Region of Cameroon to treat uterine tumours, rubella, rheumatisms, bacterial and fungal infections (Tchoula, 2001). Seeds are used as spices (Eyog et al., 2006). *B. lancilimba* is used in the same region to cure skin bacterial infections (Tchouala, 2001). *B. manii* is used to treat dysentery and headache. It is also used as an appetite stimulant (Iwu, 1993).

### **4. Phytochemistry of plant constituents of** *Beilshmiedia* **species**

A review of the literature revealed that no phytochemial studies have been carried out on *Beilshmiedia anacardioides* prior to the initiation of our study. The various phytochemical and pharmacological studies performed and reported in the literature on the beilshmiedia genus are discussed below.

#### **4.1 Alkaloids isolated from the** *Beilschmiedia* **genus**

Very few alkaloids have been isolated from the *Beilschmiedia* genus.

#### **Structure and name Source and references**

Wood of *B. madang* (Kitagawa et al., 1993)

#### **9**: Dehatrine

NH

Stem bark of *B. elliptica* (Clezy et al., 1966)

Phytochemicals from *Beilschmiedia anacardioides* and Their Biological Significance 171

A bisbenzylisoquinoline alkaloid dehatrine (**9**) isolated from the wood of *B. madang*, exhibited potent inhibitory activity (IC50 value of 0.017 µM) against the proliferation of the malaria pathogen *P. falciparum* (Kitagawa et al., 1993). Paulo and coworkers (1992)

Lignans and neolignans and flavonoids are the main phenolic compounds encountered in

**Structure and name Source and references** 

 Stem of *B*. *tsangii*  (Chen et al., 2006)

Stem of *B*. *tsangii*  (Chen et al., 2006)

Leaves of *B*. *tsangii*  (Chen et al., 2007)

O

OMe

O

OMe

O

**Pharmacological importance of alkaloids isolated from the Beilschmiedia genus** 

demonstrated the antimicrobial properties of laurelliptine (**10**).

O

OMe OMe

O

OMe OMe

O

O

MeO O

OMe OMe

**4.2 Phenolic and phenolic derived compounds** 

**4.2.1 Lignans and neolignans** 

the *Beilschmiedia* genus.

MeO

MeO

MeO

MeO

MeO

**15**: 4α,5α,-Epoxybeilschmin A

**14**: Beilschmin B

**13**: Beilschmin A

**10**: Laurelliptine

MeO

HO

MeO

OH

**11:** (6,7-Diméthoxy-4-methylisoquinolinyl)-(4' methoxyphenyl)-methanone

Leaves of *B. Brevipes*  (Pudjiastuti et al.,2010)

Stem bark of *B. Obscura*  ( Lenta et al.,2011)

#### **12**: Obscurine

Table 1. Structure of some alkaloids isolated from the *Beilschmiedia* genus

#### **Pharmacological importance of alkaloids isolated from the Beilschmiedia genus**

A bisbenzylisoquinoline alkaloid dehatrine (**9**) isolated from the wood of *B. madang*, exhibited potent inhibitory activity (IC50 value of 0.017 µM) against the proliferation of the malaria pathogen *P. falciparum* (Kitagawa et al., 1993). Paulo and coworkers (1992) demonstrated the antimicrobial properties of laurelliptine (**10**).

#### **4.2 Phenolic and phenolic derived compounds**

#### **4.2.1 Lignans and neolignans**

170 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Stem bark of *B. elliptica* (Clezy et al., 1966)

Leaves of *B. Brevipes*  (Pudjiastuti et al.,2010)

Stem bark of *B. Obscura*  ( Lenta et al.,2011)

NH

MeO

HO

MeO

**11:** (6,7-Diméthoxy-4-methylisoquinolinyl)-(4'-

H

1

6

3

H

7

O

H

16

17 18

H N

H

Table 1. Structure of some alkaloids isolated from the *Beilschmiedia* genus

10

19

14 15

12

22 23

20 21

9

methoxyphenyl)-methanone

O

**12**: Obscurine

4

O

**10**: Laurelliptine

OH

Lignans and neolignans and flavonoids are the main phenolic compounds encountered in the *Beilschmiedia* genus.

#### **Structure and name Source and references**

 Stem of *B*. *tsangii*  (Chen et al., 2006)

**13**: Beilschmin A

**14**: Beilschmin B

Leaves of *B*. *tsangii*  (Chen et al., 2007)

Stem of *B*. *tsangii*  (Chen et al., 2006) Phytochemicals from *Beilschmiedia anacardioides* and Their Biological Significance 173

Leaf of *B*. *volckii*  (Banfield et al., 1994)

Stem of *B. zenkeri* (Lenta

et al., 2009)

OH

Table 2. Structure of some lignans and neolignans isolated from *Beilschmiedia* genus

**Pharmacological importance of flavonoids isolated from the Beilschmiedia genus** 

Lenta and coworkers (2009), evaluated the antibacterial activities of the extract and flavonoids isolated from the stem of *B. zenkeri*, in vitro against three strains of microbes*, pseudomonas agarici, Bacillus subtilis,* and *streptococus minor.* Their activities were moderate compare to reference drugs ampicillin and gentamicin. (2S,4R)**-**5,6,7-trimethoxyflavan-4-ol (**22a**) exhibited the best potency against *S. minor* (IC50 of 197.5 µM) (Lenta et al., 2009).

**Structure and name Source and references** 

**Pharmacological importance of lignans and neolignans isolated from the Beilschmiedia** 

Tetrahydrofuran-type lignans beilschmin A (**13**) and B (**14**), dihydrofuran-type lignan beilschmin C (**17**) together with tsangin A (**18**) and B (**19**) were found cytotoxic (IC50 value below 4 *µ*g/mL) in P-388 and/or HT-29 cell lines *in vitro* (Chen et al., 2006). In addition, beilschmin A (**13**) and B (**14)** exhibited potent antitubercular activity (MIC values of 2.5 and 7.5 *µ*g/mL, respectively) against *Mycobacterium tuberculosis* 90-221387 in *vitro* (Chen et al., 2007). A neolignan, magnolol (**20**) displayed wide biological properties, mainly cytotoxic (Li et al., 2007), antidepressant (Li et al., 2007), antimicrobial (Park et al., 2004) and anti-

HO

**4.2.2 Some flavonoids isolated from** *Beilschmiedia* **genus** 

O

O

OH

**21**: 5-Hydroxy-7,8-dimethoxyflavanone

OMe

**20**: Magnolol

inflammatory (Lee et al., 2005).

MeO

**genus** 

**16**: 4α,5α,-Epoxybeilschmin B

Stem of *B*. *tsangii*  (Chen et al., 2006)

**17**: Beilschmin C

Stem of *B*. *tsangii*  (Chen et al., 2006)

**18**: Tsangin A

Stem of *B*. *tsangii* (Chen et al., 2006)

**19**: Tsangin B

Leaf of *B*. *volckii*  (Banfield et al., 1994)

**20**: Magnolol

172 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

OMe

O

O

O

OMe

O

OMe

O

O

Stem of *B*. *tsangii*  (Chen et al., 2006)

Stem of *B*. *tsangii*  (Chen et al., 2006)

Stem of *B*. *tsangii*  (Chen et al., 2006)

Stem of *B*. *tsangii* (Chen et al., 2006)

O

O

MeO OMe

MeO

**16**: 4α,5α,-Epoxybeilschmin B

MeO

MeO

MeO

MeO

MeO

**18**: Tsangin A

**19**: Tsangin B

**17**: Beilschmin C

OMe OMe

O

MeO O

OMe

OMe

O

OMe OMe

O

O

O

O

Table 2. Structure of some lignans and neolignans isolated from *Beilschmiedia* genus

#### **Pharmacological importance of lignans and neolignans isolated from the Beilschmiedia genus**

Tetrahydrofuran-type lignans beilschmin A (**13**) and B (**14**), dihydrofuran-type lignan beilschmin C (**17**) together with tsangin A (**18**) and B (**19**) were found cytotoxic (IC50 value below 4 *µ*g/mL) in P-388 and/or HT-29 cell lines *in vitro* (Chen et al., 2006). In addition, beilschmin A (**13**) and B (**14)** exhibited potent antitubercular activity (MIC values of 2.5 and 7.5 *µ*g/mL, respectively) against *Mycobacterium tuberculosis* 90-221387 in *vitro* (Chen et al., 2007). A neolignan, magnolol (**20**) displayed wide biological properties, mainly cytotoxic (Li et al., 2007), antidepressant (Li et al., 2007), antimicrobial (Park et al., 2004) and antiinflammatory (Lee et al., 2005).

#### **4.2.2 Some flavonoids isolated from** *Beilschmiedia* **genus**

#### **Pharmacological importance of flavonoids isolated from the Beilschmiedia genus**

Lenta and coworkers (2009), evaluated the antibacterial activities of the extract and flavonoids isolated from the stem of *B. zenkeri*, in vitro against three strains of microbes*, pseudomonas agarici, Bacillus subtilis,* and *streptococus minor.* Their activities were moderate compare to reference drugs ampicillin and gentamicin. (2S,4R)**-**5,6,7-trimethoxyflavan-4-ol (**22a**) exhibited the best potency against *S. minor* (IC50 of 197.5 µM) (Lenta et al., 2009).

> **Structure and name Source and references**  O OMe

Stem of *B. zenkeri* (Lenta et al., 2009)

**21**: 5-Hydroxy-7,8-dimethoxyflavanone

Stem of *B. zenkeri* (Lenta et al., 2009)

Phytochemicals from *Beilschmiedia anacardioides* and Their Biological Significance 175

O

O

1' 9'

1' 9'

1' 9'

12' 4'

7'

Table 4. Structure of other phenolic and phenolic derived compounds isolated from the

4' 8'

4' 8'

4' 8'

11'

13'

HO

HO

3

O

1 2

5 4

10

45

**25c**: 3,4-Dehydrooligandrol methyl ether

OH 1

<sup>2</sup> <sup>3</sup> 3'

1'

10

<sup>8</sup> <sup>9</sup>

<sup>8</sup> <sup>9</sup>

O

2

3

3

1

O

1 2

**24b**: 4-hydroxybenzaldehyde

<sup>5</sup> <sup>4</sup> <sup>6</sup>

10

<sup>8</sup> <sup>9</sup>

**24a**: Vanillin

HO

**25a**: Oligandrol

MeO

MeO

MeO

5

*Beilschmiedia* genus

**26**: Farnesylol

6

7

6

7

**25b**: Oligandrol methyl ether

6

7

MeO

**Structure and name Source and references** 

Stem of *B. tsangii*  (Chen et al., 2006)

Stem of *B. tsangii*  (Chen et al., 2006)

Bark of *B. oligandra* (Banfield et al., 1994)

Root of *B. erytrhophloia* (Yang et al., 2008)

Root of *B. erytrhophloia* (Yang et al., 2008)

Root of *B. erytrhophloia* (Yang et al., 2008)

**22a**: R = H: (2S,4R)**-**5,6,7-trimethoxyflavan-4-ol **22b**: R = CH3: (2S,4R)**-**4**,**5,6,7-trimethoxyflavan

**23a**: R = CH3: Beilschmieflavonoid A **23b**: R = H: Beilschmieflavonoid B

Table 3. Structure of flavonoids isolated from the *Beilschmiedia* genus

#### **4.2.3 Other phenolic and phenolic derived compounds from the** *Beilschmiedia* **genus and their pharmacological importance**

Vanillin (**21a**) and 4-hydroxybenzaldehyde (**24b**) were isolated from *Beilschmiedia tsangii*  (Chen et al., 2006). Both compounds were reported to exhibit analgesic, anti-inflammatory and antifungal activities (Lee et al., 2005; Lee et al., 2006; Fitzgerald et al., 2005).

**24a**: Vanillin

174 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

3'

6'

4'

Stem of *B. zenkeri* (Lenta

Stem of *B. zenkeri* (Lenta

et al., 2009)

et al., 2009)

5'

OR

OMe

O

2

1'

2'

3

1

OR

O

O

OMe OMe

O

Table 3. Structure of flavonoids isolated from the *Beilschmiedia* genus

**4.2.3 Other phenolic and phenolic derived compounds from the** *Beilschmiedia* **genus** 

Vanillin (**21a**) and 4-hydroxybenzaldehyde (**24b**) were isolated from *Beilschmiedia tsangii*  (Chen et al., 2006). Both compounds were reported to exhibit analgesic, anti-inflammatory

and antifungal activities (Lee et al., 2005; Lee et al., 2006; Fitzgerald et al., 2005).

**23a**: R = CH3: Beilschmieflavonoid A **23b**: R = H: Beilschmieflavonoid B

**and their pharmacological importance** 

OCH3

**22a**: R = H: (2S,4R)**-**5,6,7-trimethoxyflavan-4-ol **22b**: R = CH3: (2S,4R)**-**4**,**5,6,7-trimethoxyflavan

8

5 4

10

9

H3CO

MeO

MeO

6

7

H3CO

**24b**: 4-hydroxybenzaldehyde

**25a**: Oligandrol

Root of *B. erytrhophloia* (Yang et al., 2008)

**25b**: Oligandrol methyl ether

**25c**: 3,4-Dehydrooligandrol methyl ether

1'

(Yang et al., 2008)

Root of *B. erytrhophloia*

Root of *B. erytrhophloia* (Yang et al., 2008)

**26**: Farnesylol

Table 4. Structure of other phenolic and phenolic derived compounds isolated from the *Beilschmiedia* genus

7'

13'

#### **Structure and name Source and references**

Stem of *B. tsangii*  (Chen et al., 2006)

Stem of *B. tsangii*  (Chen et al., 2006)

(Banfield et al., 1994)

Bark of *B. oligandra*

#### **4.3 Endiandric acids**

Endiandric acids are a rare class of secondary tetracyclic metabolites generally encountered in *Beilschmiedia* and *Endiandra* species of the Lauraceae family. Endiandric acids are products of electrocyclic cyclization of naturally occurring polyketides (Bandanarayake et al., 1980).

#### **4.3.1 Some endiandric acids previously isolated**

#### **Pharmacological importance of the endiandric acids**

Very few pharmacological studies have been done in this class of metabolites. Endiandric acid H (**41**) is used for the manufacture of medication, in particular for the treatment of asthmatic disorders or concomitant inflammatory symptoms of asthma (Eder et al., 2004).

Erytrophloin C (**34**) exhibited antitubercular activity (MIC value of 50 *μ*g/mL) against *Mycobacterium tuberculosis* H37Rv in *vitro* (Yang et al., 2009).

#### **Structure and name Source and references**

Phytochemicals from *Beilschmiedia anacardioides* and Their Biological Significance 177

Leaves of *Endiandra entrorsa*  (Bandaranayake et al., 1982)

Leaves of *Endiandra entrorsa*  (Bandaranayake et al., 1982)

Leaves of *Endiandra entrorsa*  (Banfield et al., 1983)

Root of *B. erytrhophloia* (Yang et al., 2009)

Bark and leaves of *Endiandra jonesii*  (Banfield et al., 1994)

Barks and leaves of *Endiandra jonesii*  (Banfield et al., 1994)

COOH

5"

Ph

O

O

11' 9'

H

**29**: Endiandric acid B

H

5' 7' 9'

2' 1'

3'

1 11'

O

7'

H

H

2

8'

6' 7'

1

H H

**30**: Endiandric acid C

**31**: Endiandric acid D

Me

O O

H

6 7

3

H

**32**: Erytrophloin A

5

4

H

H

12 13

8

H

<sup>11</sup> <sup>1</sup>

1"

3" 5"

H H H

12' 13'

1' 2'

HO <sup>H</sup>

H

9' 10'

5'

4'

3'

HOOC Ph

1'

2'

H

H

9 10

H

H

1" 5"

1' 5' 6' 8'

H

1"

4'

H

<sup>H</sup> <sup>1</sup>

4

10'

11'

Leaves of *Endiandra entrorsa* and *Endiandra oligandra*  (Bandaranayake et al., 1981) Bark of *B. oligandra* (Banfield et al., 1994)

**27**: Endiandric acid A

**28**: Methylenedioxyendiandric acid A

Leaves of *Endiandra entrorsa* (Banfield et al., 1994) and Stem bark of *B*. *manii*  (Mpetga, 2005)

Leaves of *Endiandra entrorsa*  (Bandaranayake et al., 1982) Barks and leaves of *Endiandra jonesii*  (Banfield et al., 1994)

176 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Endiandric acids are a rare class of secondary tetracyclic metabolites generally encountered in *Beilschmiedia* and *Endiandra* species of the Lauraceae family. Endiandric acids are products of electrocyclic cyclization of naturally occurring polyketides (Bandanarayake et al., 1980).

Very few pharmacological studies have been done in this class of metabolites. Endiandric acid H (**41**) is used for the manufacture of medication, in particular for the treatment of asthmatic disorders or concomitant inflammatory symptoms of asthma (Eder et al., 2004). Erytrophloin C (**34**) exhibited antitubercular activity (MIC value of 50 *μ*g/mL) against

COOH

COOH

1

2

9'

10'

11'

1

2

10' 11'

**Structure and name Source and references** 

Leaves of *Endiandra entrorsa* and *Endiandra oligandra*  (Bandaranayake et al., 1981)

Leaves of *Endiandra entrorsa* (Banfield et al., 1994) and Stem bark of *B*. *manii* 

(Mpetga, 2005)

Bark of *B. oligandra* (Banfield et al., 1994)

**4.3 Endiandric acids** 

**4.3.1 Some endiandric acids previously isolated Pharmacological importance of the endiandric acids** 

*Mycobacterium tuberculosis* H37Rv in *vitro* (Yang et al., 2009).

H

5' 7' 9'

H H H

H

1' 2'

H

5" O

7'

2' 1'

3'

H H H

12' 13'

H

12' 13'

H

O

4'

5'

1" 3"

H

**28**: Methylenedioxyendiandric acid A

**27**: Endiandric acid A

3'

1"

5" 3"

Leaves of *Endiandra entrorsa*  (Bandaranayake et al., 1982) Bark and leaves of *Endiandra jonesii*  (Banfield et al., 1994)

Leaves of *Endiandra entrorsa*  (Banfield et al., 1983)

**31**: Endiandric acid D

Root of *B. erytrhophloia* (Yang et al., 2009)

**32**: Erytrophloin A

Root of *B. erytrhophloia* (Yang et al., 2009)

Phytochemicals from *Beilschmiedia anacardioides* and Their Biological Significance 179

6' 8'

10'

10'

10'

6' 7'

10'

8'

9'

O

O

Root of *B. erytrhophloia* (Yang et al., 2009)

Root of *B. erytrhophloia* (Yang et al., 2009)

Root of *B. erytrhophloia* (Yang et al., 2008)

Root of *B. erytrhophloia* (Yang et al., 2008)

1' 9'

4'

3'

O O

H

6 7

3

H

**37**: Erytrophloin D

11

O

7

5

Me

4

H3C

H

**38**: Beilcyclone

HO

H

HO

H

H

**40**: Endiandric acid I

H

H

H

H

12 13

8

H

<sup>11</sup> <sup>1</sup>

1 3

H

1 3

1'

H H

9

H

10

5 4

H

10

1 3

2

H

9

H

4

H H

1'

5

H

2

10

9

H

<sup>2</sup> 1'

H H

5 <sup>4</sup> <sup>6</sup>

H

11

H

11

6 7

O

6 7

**39**: Endiandric acid J

O

H

9 10

H

**33**: Erytrophloin F

Root of *B. erytrhophloia* (Yang et al., 2009)

Root of *B. erytrhophloia* (Yang et al., 2009)

**35**: Erytrophloin E

**34**: Erytrophloin C

H

Root of *B. erytrhophloia* (Yang et al., 2009)

**36**: Erytrophloin B

Root of *B. erytrhophloia* (Yang et al., 2009)

**37**: Erytrophloin D

178 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

11' 9'

6'

1' 5' 6' 8'

6' 8'

10'

4'

3'

4'

3'

13'

HO O

H

6 7

3

H

**33**: Erytrophloin F

O O

H

6 7

3

H

**34**: Erytrophloin C

H

**35**: Erytrophloin E

O O

H

6 7

3

H

**36**: Erytrophloin B

5

Me

4

H

H

12 13

8

H

<sup>11</sup> <sup>1</sup>

H

1'

9 10

H

5

4

HO O

H

6 7

3

5

Me

4

H

H

12 13

H

8

H

H

12 13

8 9 10

H

11 1

H

H

<sup>11</sup> <sup>1</sup>

5

4

H

H

12 13

8

H

<sup>11</sup> <sup>1</sup>

H

H

9 10

H

1' 5'

9 10

H

1' 5' 6' 8'

O

8' 10'

11'

O

Root of *B. erytrhophloia* (Yang et al., 2009)

Root of *B. erytrhophloia* (Yang et al., 2009)

Root of *B. erytrhophloia* (Yang et al., 2009)

Root of *B. erytrhophloia* (Yang et al., 2009)

Root of *B. erytrhophloia* (Yang et al., 2009)

Root of *B. erytrhophloia* (Yang et al., 2008)

Root of *B. erytrhophloia* (Yang et al., 2008)

**40**: Endiandric acid I

*Stem of B. fulva* (Eder et al., 2004)

Phytochemicals from *Beilschmiedia anacardioides* and Their Biological Significance 181

COOH OH

7

**A B**

13

6

1''

3

2

<sup>H</sup> <sup>H</sup>

1''

COOH H

7

**A B**

13

6

H

H

2

<sup>H</sup> <sup>H</sup>

O

4"

2"

1''

4 3

4

H

H

H

HO CH3

12

**<sup>C</sup> <sup>D</sup>**

1

H

H

H

H

H

13' 10' 12'

**C D**

H

O

7"

8'

H

11'

H

O

11

**D**

H

10

H

2' 1'

**<sup>A</sup> <sup>B</sup>**

12 13

1

H H

7

**C**

<sup>H</sup> <sup>H</sup>

H

7'

**A B**

H

H H 2 1

COOH

H

H

9 8

H

H

1'

6

H

8'

CH3

1

12

**<sup>C</sup> <sup>D</sup>**

H

H

6

8'

CH3

8'

6

1'

H

H

H

1'

H

11

H

9

10

11

H

9

10

6

**A**

1''

COOH

H

2

Beilshmiedic acid A ( **1)** Beilshmiedic acid B ( **2)** 

6

1''

3

2

<sup>H</sup> <sup>H</sup>

Beilshmiedic acid C ( **3)** Beilshmiedic acid D ( **4)** 

H

Beilshmiedic acid E ( **5)** Beilshmiedic acid F ( **6)** 

6

1''

3 2

<sup>H</sup> <sup>H</sup>

4

Beilshmiedic acid G ( **7)** Beilshmiedin (**8**)

H

4 3

H

H

H

4

H

1 11

<sup>H</sup> <sup>H</sup>

COOH H

7

**A B**

13

7

13 10 12

H

H

H

HO CH3

12

**C D**

1 11

COOH H

7

**A B**

13

6

1''

2

<sup>H</sup> <sup>H</sup>

COOH

**A B**

H

H

H

H

1

12

**<sup>C</sup> <sup>D</sup>**

H

H

<sup>H</sup> <sup>H</sup>

1'

CH3

H

6"

O

H

4 3 2

H

5

H

H

4' 3'

6'

8'

6

H

H

6

8'

H H 6

1'

O

H

Scheme 1. endiandric acid derivatives from *Beilshmiedia anacardioides.* 

11

H

H

HO CH3

**<sup>C</sup> <sup>D</sup>**

<sup>13</sup> <sup>10</sup> 12

7 9

1

11

H

9

10

H

**<sup>C</sup> <sup>D</sup>**

**B**

H

1'

H H H CH3

H

H

H

6

8'

H

H H

1'

H

9

10

H

6

8'

4

HO

H

**41**: Endiandric acid H

Table 5. Structure of some endiandric acids previously isolated

#### **5. Results of our own studies**

We have initiated a systematic phytochemical investigation of the extracts of *Beilshmiedia anacardioides* and have so far obtained the following results which have led to three publications in renowned scientific journals (Chouna et al.,2009; 2010; 2011).

Air-dried and ground stem bark of *B. anacardioides* was extracted successively at room temperature with MeOH. The methanol extract was re-extracted in turn with CH2Cl2 and EtOAc. These extracts were concentrated to dryness under reduced pressure.

The CH2Cl2 extract was submitted to repeated column chromatography on silica gel, yielding beilschmiedic acids A (**1**), B (**2**) and C (**3**) and the known β-sitosterol (Chouna et al.,2009) .

Further Successive purifications by column chromatography over silica gel and preparative TLC afforded three new endiandric acid derivatives: beilschmiedic acids D(**4**) and E(**5**), and Beilshmiedin (**8**) (Chouna et al.,2010), together with the known compounds bisabolene (Mossa et al 1992; Barrero et al. 1990), and tricosanoic acid (Erdemoglu et al., 2008).

The ethyl acetate soluble part of the MeOH extract of the stem bark of *B. anacardioides* was fractionated by column chromatography over silica gel. Successive purifications by column chromatography and preparative TLC afforded two new endiandric acid derivatives: beilschmiedic acids F(**6**) and G(**7**) Chouna et al.,2011, along with the known constituents beilschmiedic acid A(**1**), beilschmiedic acid C(**3**) [6] and sitosterol-3-*O*-*β*-*D*-glucopyranoside (Chouna et al.,2011).

Beilshmiedic acid A ( **1)** Beilshmiedic acid B ( **2)** 

180 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

*Stem of B. fulva* (Eder et al.,

2004)

COOH

We have initiated a systematic phytochemical investigation of the extracts of *Beilshmiedia anacardioides* and have so far obtained the following results which have led to three

Air-dried and ground stem bark of *B. anacardioides* was extracted successively at room temperature with MeOH. The methanol extract was re-extracted in turn with CH2Cl2 and

The CH2Cl2 extract was submitted to repeated column chromatography on silica gel, yielding beilschmiedic acids A (**1**), B (**2**) and C (**3**) and the known β-sitosterol (Chouna et

Further Successive purifications by column chromatography over silica gel and preparative TLC afforded three new endiandric acid derivatives: beilschmiedic acids D(**4**) and E(**5**), and Beilshmiedin (**8**) (Chouna et al.,2010), together with the known compounds bisabolene (Mossa et al 1992; Barrero et al. 1990), and tricosanoic acid (Erdemoglu et al.,

The ethyl acetate soluble part of the MeOH extract of the stem bark of *B. anacardioides* was fractionated by column chromatography over silica gel. Successive purifications by column chromatography and preparative TLC afforded two new endiandric acid derivatives: beilschmiedic acids F(**6**) and G(**7**) Chouna et al.,2011, along with the known constituents beilschmiedic acid A(**1**), beilschmiedic acid C(**3**) [6] and sitosterol-3-*O*-*β*-*D*-glucopyranoside

1

2

10'

publications in renowned scientific journals (Chouna et al.,2009; 2010; 2011).

EtOAc. These extracts were concentrated to dryness under reduced pressure.

11'

4'

1" 3"

O

H

5" O

5' 7' 9'

12' 13'

2' 1'

3'

H H H

H

Table 5. Structure of some endiandric acids previously isolated

H

**5. Results of our own studies** 

HO

**41**: Endiandric acid H

al.,2009) .

2008).

(Chouna et al.,2011).

Beilshmiedic acid C ( **3)** Beilshmiedic acid D ( **4)** 

7"

Beilshmiedic acid E ( **5)** Beilshmiedic acid F ( **6)** 

Beilshmiedic acid G ( **7)** Beilshmiedin (**8**)

Scheme 1. endiandric acid derivatives from *Beilshmiedia anacardioides.* 

Phytochemicals from *Beilschmiedia anacardioides* and Their Biological Significance 183

Compound tested *B. subtilis M. luteus S. faecalis S. minor S. ferus* 

**1** 181.60 173.60 363.30 n.t. n.t. **2** 11.30 347.20 45.30 n.t. n.t. **3** 5.60 < 0.70 22.70 n.t. n.t.

 381.00 n.t. n.t. 190.50 - 381.00 n.t. n.t. 190.50 - 343.40 n.t. n.t. - - 87.78 10.95 87.78 - - 422.20 n.t. n.t. - -

Ampicillin 89.5 1.95 3.9 1.05 5.25

Table 7. Antibacterial activity ( MIC in µM) of compounds **1-8** against *B. Subtilis*, *M. luteus*, *S.* 

Beilshmiedic acid C (**3)** was more active than Beilshmiedic acid D (**4)** . The enhanced activity may be due to the additional hydroxyl group at C-4 position in Beilshmiedic acid C (**3)** . Beilshmiedic acid B(**2)** which possesses one hydroxyl group more than Beilshmiedic acid A (**1)** and Beilshmiedic acid C (**3)** was less active. Beilshmiedic acid A (**1)** was more active than Beilshmiedic acid C (**3)** . They are epimers at C-4 position; the modification of the

Based on the skeletal features, it is difficult at this stage to define the contribution of the different functional groups with respect to the activity. The mechanism of action of this class of metabolites on these strains is not yet known. Further investigations will help to establish the mode of action of this particular skeleton. These interesting results highlight the potency of this rare class of metabolites that might be investigated for the search of new antibacterial

In our hypothesis we proposed that phytochemists looking for novel bioactive natural products should investigate the medicinal plants whose therapeutic efficiency has been

We suggested that Natural products from *Beilshmiedia anacardioides* may play a role in treating genital infections due to *B*-s*ubtilis* and *M. luteus*, and rheumatisms due *Streptococcus ferus* and *S. minor*. The biological activities of some of the constituents isolated in our studies, Beilshmiedic acid C presented above, more than lend support to this suggestion.

It is certain that as more and more data become available from the phytochemical and biological analysis of the constituents of therapeutic efficient medicinal plants selected after

(-) inactive, n.t. (not tested)

drugs.

**7. Conclusion** 

*faecalis*, *S. minor*, *S. ferus*, *P. palida* and *E. coli*.

configuration at this position influences significantly the activity.

established through clinical research on African medicine.

#### **6. Biological activity and the significance of some compounds**

Our preliminary antibacterial studies on the new endiandric acid derivatives have yielded chemical entities that have been shown to possess significant activities (Chouna et al., 2009) .

#### **Antibacterial assay on some compounds isolated from** *B.anacardioides*

Compounds **1**-**8** were tested in vitro for their antibacterial activity against *Bacillus subtilis*, *Streptococcus ferus*, *Streptococcus minor*, *Micrococcus luteus*, *Escherichia coli*, and *Pseudomonas agarici*, using the dilution technique.

The ZI (Table 1) and MIC (Table 2) obtained for these compounds indicated that they possessed strong to weak antibacterial activity against *gram* positive bacteria.

Beilshmiedic acid C (**3)** demonstrated the best potency against *B. subtilis* and *M. luteus*, compared to the reference drug ampicillin. The MIC values (Table 2) of Beilshmiedic acids B( **2)**, C (**3)** and G(**7)**, against *B. subtilis* and Beilshmiedic acid C (**3)** against *M. luteus* were found to be greater than that of standard drug ampicillin, indicating that this series of compounds might be possible candidates as antibacterial drugs.

None of the tested compounds was active against *Gram* negative *P. palida* and *E. Coli*. Therefore, they might be well tolerated as antibiotics even for long term treatments.


(-) inactive, n.t. (not tested)

Table 6. Antibacterial activity (Zone of inhibition of compounds in mm) of compounds **1-8** (500*µ*g/mL) against *B. Subtilis, M. luteus*, *S. faecalis, S. minor, S. ferus*, *P. palida* and *E. Coli.*


(-) inactive, n.t. (not tested)

182 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Our preliminary antibacterial studies on the new endiandric acid derivatives have yielded chemical entities that have been shown to possess significant activities (Chouna et al., 2009) .

Compounds **1**-**8** were tested in vitro for their antibacterial activity against *Bacillus subtilis*, *Streptococcus ferus*, *Streptococcus minor*, *Micrococcus luteus*, *Escherichia coli*, and *Pseudomonas* 

The ZI (Table 1) and MIC (Table 2) obtained for these compounds indicated that they

Beilshmiedic acid C (**3)** demonstrated the best potency against *B. subtilis* and *M. luteus*, compared to the reference drug ampicillin. The MIC values (Table 2) of Beilshmiedic acids B( **2)**, C (**3)** and G(**7)**, against *B. subtilis* and Beilshmiedic acid C (**3)** against *M. luteus* were found to be greater than that of standard drug ampicillin, indicating that this series of

None of the tested compounds was active against *Gram* negative *P. palida* and *E. Coli*.

Compound tested *B. subtilis M. luteus S. faecalis S. minor S. ferus P. palida E. coli* 

Table 6. Antibacterial activity (Zone of inhibition of compounds in mm) of compounds **1-8** (500*µ*g/mL) against *B. Subtilis, M. luteus*, *S. faecalis, S. minor, S. ferus*, *P. palida* and *E. Coli.*

15 12 14 n.t. n.t. - - 16 15 15 n.t. n.t. - - 13 30 18 n.t. n.t. - - 10 n.t. n.t. 10 - - - 12 n.t. n.t. - 12 - - 10 n.t. n.t. - - - - 20 15 16 - - - - 10 n.t. n.t. - - - - Ampicillin 29 26 25 22 23 - -

Therefore, they might be well tolerated as antibiotics even for long term treatments.

**6. Biological activity and the significance of some compounds** 

**Antibacterial assay on some compounds isolated from** *B.anacardioides* 

possessed strong to weak antibacterial activity against *gram* positive bacteria.

compounds might be possible candidates as antibacterial drugs.

*agarici*, using the dilution technique.

(-) inactive, n.t. (not tested)

Table 7. Antibacterial activity ( MIC in µM) of compounds **1-8** against *B. Subtilis*, *M. luteus*, *S. faecalis*, *S. minor*, *S. ferus*, *P. palida* and *E. coli*.

Beilshmiedic acid C (**3)** was more active than Beilshmiedic acid D (**4)** . The enhanced activity may be due to the additional hydroxyl group at C-4 position in Beilshmiedic acid C (**3)** . Beilshmiedic acid B(**2)** which possesses one hydroxyl group more than Beilshmiedic acid A (**1)** and Beilshmiedic acid C (**3)** was less active. Beilshmiedic acid A (**1)** was more active than Beilshmiedic acid C (**3)** . They are epimers at C-4 position; the modification of the configuration at this position influences significantly the activity.

Based on the skeletal features, it is difficult at this stage to define the contribution of the different functional groups with respect to the activity. The mechanism of action of this class of metabolites on these strains is not yet known. Further investigations will help to establish the mode of action of this particular skeleton. These interesting results highlight the potency of this rare class of metabolites that might be investigated for the search of new antibacterial drugs.

#### **7. Conclusion**

In our hypothesis we proposed that phytochemists looking for novel bioactive natural products should investigate the medicinal plants whose therapeutic efficiency has been established through clinical research on African medicine.

We suggested that Natural products from *Beilshmiedia anacardioides* may play a role in treating genital infections due to *B*-s*ubtilis* and *M. luteus*, and rheumatisms due *Streptococcus ferus* and *S. minor*. The biological activities of some of the constituents isolated in our studies, Beilshmiedic acid C presented above, more than lend support to this suggestion.

It is certain that as more and more data become available from the phytochemical and biological analysis of the constituents of therapeutic efficient medicinal plants selected after

Phytochemicals from *Beilschmiedia anacardioides* and Their Biological Significance 185

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#### **8. Acknowledgments**

Financial support for this survey from the Alango Foundation (Centre de Phytomédecine Africaine ) at Dschang / Cameroon is gratefully acknowledged.

#### **9. References**


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**9. References** 


**9** 

*Thailand* 

**Phenolic Constituents and Antioxidant** 

Thai plants have been used as medicines for many centuries because they contain active phytochemicals including phenolic compounds. These components function as antibiotics, help to make cell walls impermeable to gas and water, act as structural materials to give plants stability and provide protection against ultraviolet (UV) light. Hence, plants in the tropical zone including Thailand contain a high concentration of phenolic compounds

Several parts of edible plants from tropical and subtropical climates are known to contain many phenolic compounds which are receiving increasing interest from consumers for several reasons (Leong & Shui, 2002). Epidemiological studies have suggested that relationships exist between the consumption of phenolic-rich foods or beverages and the prevention of diseases such as cancer, stroke, coronary heart desease and others. This association has been partially explained by the fact that phenolic compounds retard oxidative modification of low density lipoproteins (LDL), which is implicated in the initiation of arteriosclerosis. More recently, alternative mechanisms have been proposed for the role of antioxidants in reducing the incidence of cardiovascular disease, besides that of the simple protection of LDL from reactive oxygen species (ROS)-induced damage. Several phenolic antioxidants significantly affect cellular responses to different stimuli, including cytokines and growth factors. Although many papers have reported studies on the composition and antioxidant activity of phenolic compounds in tropical edible plants (Auddy et al., 2003; Nũnez sellés et al., 2002; Habsah et al., 2000), information about the phenolics and antioxidant potential of Thai edible plant species is limited compared to the broad biodiversity of edible plants grown. Moreover, many studies have been reported in

The aim of this chapter is to provide a critical review of the composition and antioxidant properties of phenolic compounds of some Thai plants. In addition, factors affecting

Plant materials contain many phytochemicals including compounds with antioxidant activity, which are mostly phenolic in structure(Johnson, 2001). Compounds with

formed as secondary metabolites in plants (Shahidi & Naczk, 2003).

the Thai language, and are not available to English speaking scientists.

extraction of these components from Thai plants are reported.

**2. Phenolic compounds of some Thai plants** 

**1. Introduction** 

**Properties of some Thai Plants** 

*University of the Thai Chamber of Commerce,* 

Pitchaon Maisuthisakul


### **Phenolic Constituents and Antioxidant Properties of some Thai Plants**

Pitchaon Maisuthisakul

*University of the Thai Chamber of Commerce, Thailand* 

#### **1. Introduction**

186 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Paulo, M. Q.; Barbosa-Filho, J. M.; Lima, E.O.; Maria R. F.; Barbosa, R. C.; Kaplan, M.A.

Park, J.; Lee, J.; Jung, E.; Park, Y.; Kim, K.; Park, B.; Yung, K.; Park, E.; Kim, J.; Park, D.: In

Tchouala, J.M.: Inventaire ethnobotanique des plantes médicinales du Noun (Ouest-

de maitrise de Biologie Végétale, Université de Dschang. 2001, p. 33. Yang, P.S.; Cheng, M.J.; Chen, I.J.; Chen, I.S.: Two new endiandric analogs, a new

*Propionibacterium sp. Eur. J. Pharmacol*. 2004, 12, 189-195.

*Ethnopharmacol*. 1992, 36, 39-41.

*Helv.Chim. Act*. 2008, 91, 2130-2138.

Antimicrobial activity of benzylisoquinoline alkaloids from *Annona salzmanii. J. of* 

vitro antibacterial and anti-inflammatory effects of honokiol and magnolol against

Cameroun) utilisées pour soigner les infections fongiques et bactériennes. Mémoire

benzopyran and a new benzenoid from the root of *Beilshmiedia erytroploia*.

Thai plants have been used as medicines for many centuries because they contain active phytochemicals including phenolic compounds. These components function as antibiotics, help to make cell walls impermeable to gas and water, act as structural materials to give plants stability and provide protection against ultraviolet (UV) light. Hence, plants in the tropical zone including Thailand contain a high concentration of phenolic compounds formed as secondary metabolites in plants (Shahidi & Naczk, 2003).

Several parts of edible plants from tropical and subtropical climates are known to contain many phenolic compounds which are receiving increasing interest from consumers for several reasons (Leong & Shui, 2002). Epidemiological studies have suggested that relationships exist between the consumption of phenolic-rich foods or beverages and the prevention of diseases such as cancer, stroke, coronary heart desease and others. This association has been partially explained by the fact that phenolic compounds retard oxidative modification of low density lipoproteins (LDL), which is implicated in the initiation of arteriosclerosis. More recently, alternative mechanisms have been proposed for the role of antioxidants in reducing the incidence of cardiovascular disease, besides that of the simple protection of LDL from reactive oxygen species (ROS)-induced damage. Several phenolic antioxidants significantly affect cellular responses to different stimuli, including cytokines and growth factors. Although many papers have reported studies on the composition and antioxidant activity of phenolic compounds in tropical edible plants (Auddy et al., 2003; Nũnez sellés et al., 2002; Habsah et al., 2000), information about the phenolics and antioxidant potential of Thai edible plant species is limited compared to the broad biodiversity of edible plants grown. Moreover, many studies have been reported in the Thai language, and are not available to English speaking scientists.

The aim of this chapter is to provide a critical review of the composition and antioxidant properties of phenolic compounds of some Thai plants. In addition, factors affecting extraction of these components from Thai plants are reported.

#### **2. Phenolic compounds of some Thai plants**

Plant materials contain many phytochemicals including compounds with antioxidant activity, which are mostly phenolic in structure(Johnson, 2001). Compounds with

Phenolic Constituents and Antioxidant Properties of some Thai Plants 189

antioxidant activity are mainly phenolic acids, flavonoids and polyphenols (Dillard & German, 2000). Phenolic acids such as caffeic acid and gallic acid are widely distributed in the plant kingdom. The most widespread and diverse phenolics are the flavonoids which have the same C15 (C6-C3-C6) skeleton and retard oxidationof a variety of easily oxidizable compounds (Zheng & Wang, 2001). Flavonoids include catechins, proanthocyanins, anthocyanidins, flavones, flavonols and their glycosides (Ho, 1992). Flavonoids are ubiquitous in plants, since almost all plant tissues are able to synthesize flavonoids. Among the most widely distributed are the flavonols quercetin and rutin.

Investigations have increased considerably in recent years in order to find natural plant antioxidants to replace synthetic compounds whose use is being restricted due to possible side effects such as carcinogenecity (Zeng & Wang, 2001). Many papers have reported that phenolic plant constituents provide protection against oxidation (Amarowicz et al., 2003; Pokorny, 2001). Phenolic substances inhibit propagation of the oxidation chain reactions due to their resonance stabilized free-radical forms (Lindsay, 1996).

Phenolic compounds possess one or more aromatic rings bearing two or more hydroxyl groups (Ho, 1992). They are closely associated with the sensory and nutritional quality of fresh and processed foods. In general, the leaves, flowers, fruits and other living tissues of the plant contain glycosides, woody tissues contain aglycones, and seeds may contain either (Huang & Ferraro, 1992).

Agricultural based manufacturers in Thailand produce and export many fruit and vegetable products. These Thai plants are widely distributed throughout the tropics particularly in Southeast Asia. Many researchers have shown that several parts of tropical and subtropical plants contain large amounts of natural phenolic phytochemicals, such as flavonoids (Leong & Shui, 2002; Kähkonen et al., 1999; Demo et al., 1998). Hence, there is a potential for Thai plants to be used as sources of phenolic antioxidants and commercial extracts could be prepared from numerous available raw plant materials. Cost, simplicity and safety should be considered in the development of an acceptable extraction procedure (Pokorny & Korczak, 2001).

Due to the diversity and complexity of natural mixtures of phenolic compounds in plant extracts, it is rather difficult to characterize every phenolic compound. Each plant generally contains different mixtures of phenolic compounds. The Folin-Ciocalteu method is a rapid, widely used assay to determine the total concentration of phenolic compounds. It is known that different phenolic compounds vary in their responses in the Folin-Ciocalteu method. Many researchers have reported the total phenolic content of Thai plants as shown in Table 1.

The data clearly indicate that some of these plants are rich in natural phenolic compounds. Many plants with a high phenolic content have an astringent taste (for leaves) or strong colors (for flowers and fruits) due to flavonoid components. Proanthocyanidins contribute astringency to plants and other flavonoids contribute red or violet colors (anthocyanins) or yellow colors (flavonols). Higher total phenolic and flavonoid contents have been found in seeds compared to other tissues. Typically, leaf photosynthesis products including essential nutrients such as sucrose are translocated from leaves to fruits and seeds which are the food storage organs of the plants (Salisbury & Ross, 1992). This leads to a concentration of phenolic compounds in seeds. Thai plant samples (Table 1) may be classified into two groups with high and low contents of polyphenolic phytochemicals, Samples having


antioxidant activity are mainly phenolic acids, flavonoids and polyphenols (Dillard & German, 2000). Phenolic acids such as caffeic acid and gallic acid are widely distributed in the plant kingdom. The most widespread and diverse phenolics are the flavonoids which have the same C15 (C6-C3-C6) skeleton and retard oxidationof a variety of easily oxidizable compounds (Zheng & Wang, 2001). Flavonoids include catechins, proanthocyanins, anthocyanidins, flavones, flavonols and their glycosides (Ho, 1992). Flavonoids are ubiquitous in plants, since almost all plant tissues are able to synthesize flavonoids. Among

Investigations have increased considerably in recent years in order to find natural plant antioxidants to replace synthetic compounds whose use is being restricted due to possible side effects such as carcinogenecity (Zeng & Wang, 2001). Many papers have reported that phenolic plant constituents provide protection against oxidation (Amarowicz et al., 2003; Pokorny, 2001). Phenolic substances inhibit propagation of the oxidation chain reactions due

Phenolic compounds possess one or more aromatic rings bearing two or more hydroxyl groups (Ho, 1992). They are closely associated with the sensory and nutritional quality of fresh and processed foods. In general, the leaves, flowers, fruits and other living tissues of the plant contain glycosides, woody tissues contain aglycones, and seeds may contain either

Agricultural based manufacturers in Thailand produce and export many fruit and vegetable products. These Thai plants are widely distributed throughout the tropics particularly in Southeast Asia. Many researchers have shown that several parts of tropical and subtropical plants contain large amounts of natural phenolic phytochemicals, such as flavonoids (Leong & Shui, 2002; Kähkonen et al., 1999; Demo et al., 1998). Hence, there is a potential for Thai plants to be used as sources of phenolic antioxidants and commercial extracts could be prepared from numerous available raw plant materials. Cost, simplicity and safety should be considered in the development of an acceptable extraction procedure (Pokorny &

Due to the diversity and complexity of natural mixtures of phenolic compounds in plant extracts, it is rather difficult to characterize every phenolic compound. Each plant generally contains different mixtures of phenolic compounds. The Folin-Ciocalteu method is a rapid, widely used assay to determine the total concentration of phenolic compounds. It is known that different phenolic compounds vary in their responses in the Folin-Ciocalteu method. Many researchers have reported the total phenolic content of Thai plants as shown in Table 1. The data clearly indicate that some of these plants are rich in natural phenolic compounds. Many plants with a high phenolic content have an astringent taste (for leaves) or strong colors (for flowers and fruits) due to flavonoid components. Proanthocyanidins contribute astringency to plants and other flavonoids contribute red or violet colors (anthocyanins) or yellow colors (flavonols). Higher total phenolic and flavonoid contents have been found in seeds compared to other tissues. Typically, leaf photosynthesis products including essential nutrients such as sucrose are translocated from leaves to fruits and seeds which are the food storage organs of the plants (Salisbury & Ross, 1992). This leads to a concentration of phenolic compounds in seeds. Thai plant samples (Table 1) may be classified into two groups with high and low contents of polyphenolic phytochemicals, Samples having

the most widely distributed are the flavonols quercetin and rutin.

to their resonance stabilized free-radical forms (Lindsay, 1996).

(Huang & Ferraro, 1992).

Korczak, 2001).


**Source**: Adapted from ¥Maisuthisakul et al. (2008); \*Maisuthisakul et al. (2007a); €Lee & Scagel (2009); Chuenarom et al. (2010);

Chanwitheesuk, Teerawutgulrag & Rakariyatham (2005)

Areekul (2009);


means not reported or not determined.

a The data were calculated as mg% of pyrocatechol equivalent.

Table 1. Ranges of some phenolic constituents in some Thai plants

Phomkaivon &


**Source**: Adapted from ¥Maisuthisakul et al. (2008); \*Maisuthisakul et al. (2007a); €Lee & Scagel (2009); Chuenarom et al. (2010); Phomkaivon & Areekul (2009); Chanwitheesuk, Teerawutgulrag & Rakariyatham (2005) -

means not reported or not determined. a The data were calculated as mg% of pyrocatechol equivalent. Table 1. Ranges of some phenolic constituents in some Thai plants

Phenolic Constituents and Antioxidant Properties of some Thai Plants 193

investigated; Piper betel Linn, *Careya sphaerica*, *Cratoxylum formosum* Dyer., and *Leucaena* 

**Piper betel Linn.;** which is commonly known as Betel leaf, is chewed with Betel nut and lime by some people in Asia. Its Thai name is different in different areas, for instance, Plu-Cheen, Se-ke, Bul-plao-yuan, she-ke (South), Pu (North-West). The leaves are chewed alone or with other plant materials including the areca nut (Areca catechu Linn.) and lime. Many researchers have focused on the red lime betel quid in the past few years. A little information about the Betel leaf was found. The Betel leaf itself has a spicy taste and yields an essential oil widely used as a medicine. In Thailand, it is used to treat bruises, heal urticaria, cure ringworm and joint pain as well as relieving toothache. It is also used in cough and mucus remedies and infusions to cure indigestion, as a topical cure for constipation, as a decongestant and as an aid to lactation. The characteristics and chemical composition of 100 grams of Betel leaves are 44 kcal for energy, 85-90 g of water, 3-3.5 g of protein, 2.3 g of fiber, 0.63-0.89 mg of nicotinic acid, 0.005-0.01 g of vitamin C, 1.9-2.9 mg of Vitamin A, 10-70 μg of thiamine, 1.9-30 μg of riboflavin, 0.1-1.3 g of tannin, 0.05-0.6 g of phosphorus, 1.1-4.6 g of potassium, 0.2-0.5 g of calcium, 0.005-0.007 g of iron, 3.4 μg of

Other biological activities described for the essential oil include antifungal, antiseptic and anthelmintic effects (Evans et al., 1984). It was reported that Betel leaf was rich in carotenes (80 IU/g fresh wt.) and phenolics. Data on the phenolic compounds of this plant have been reported for chavicol (Amonkar, et al., 1986), chavibetol, chavibetol acetate (Rimando, et al., 1986) and eugenol (Nagabhushan, et al., 1989). The major bioactive phenolic compoundsin Thai Betel leaf extracted with ethyl acetate were found to be relatively low in polarity. They are chavicol and two-unknown compounds which show higher polarity than chavicol (Maisuthisakul, 2008). The chemical structures of phenolic compounds found in betel leaf

**MeO**

Chavibetol

**HO**

**OH**

**OMe**

**CH 2 CH CH 2**

**CH 2 CH CH 2**

*leucocephala* de Wit.

iodine (Guha, 2006).

are shown in Fig. 2.

**HO**

**MeO**

**OAc**

Fig. 2. Some phenolic compounds found in *Piper betel* Linn.

**CH 2 CH CH 2**

Eugenol Chavibetol acetate

**CH 2 CH CH 2**

Chavicol

relatively high concentrations of phenolic phytochemicals (more than 100 mg GAE/g dry weight of material) included seeds of *Antidesma velutinum* Tulas, *Cleistocalyx operculatus* var. paniala (Roxb.), *Eugenia siamensis* Craib., bark of *Acacia catechu* (L.F) Wild., kernel of *Areca catechu* Linn. and stem of *Cassia fistula* Linn. Chanwitheesuk et al. (2005) reported the total phenolic content of Thai plants using Folin Denis reagent and pyrocatechol as reference, with data calculated as mg% of pyrocatechol equivalents. For comparison, the total phenolic content of Leucaena was reported as 405 mg% (Chanwitheesuk et al., 2005) and 51.20 mg GAE/g db plant (Maisuthisakul et al., 2008). The data showed that the different reference compound and the units used required a conversion factor of around eight to convert the values (Table 1).

Quantitative determination of individual flavonoid glycosides is difficult because most standards are not commercially available. Hence, the total flavonoid content determined using a colorimetric method (Bonvehí et al., 2001) is commonly used for flavonoid evaluation. Generally, the calculation of the total flavonoid content is quoted in units of mg rutin equivalent of flavonoid compounds in one gram of plant extract based on the dry weight of the original plant sample. It is well known that flavonoids possess antioxidant properties both in vitro and in vivo. The flavonoids contain a number of phenolic hydroxyl groups attached to aromatic ring structures, which confer the antioxidant activity. Flavonoids were found in leaves, seeds and fruits and were good antioxidants (Bonvehí et al., 2001). The data clearly indicate that some plants in Thailand are rich in natural antioxidant flavonoids. Some data in Table 1 were used to find a relationship between total phenolic and flavonoid content. The flavonoid content correlates moderately with the phenolic content as shown in Fig. 1.

Fig. 1. Relationship between total phenolic and total flavonoid content in Thai plant extracts.

Chanwitheesuk et al. (2005) reported the total carotene, total xanthophyll and tannin contents of edible plants grown in Thailand as shown in Table 1. Total carotenes and total xanthophylls were determined according to Helrich (1990). Tannin contents were determined colorimetrically using the Folin-Dennis reagent according to Helrich (1990).

Identification of the phenolic components in Thai plants has not commonly been reported because it is expensive and standards required for identification are often not available. Hence, this chapter will report the phenolic profile of only four of the Thai plants

relatively high concentrations of phenolic phytochemicals (more than 100 mg GAE/g dry weight of material) included seeds of *Antidesma velutinum* Tulas, *Cleistocalyx operculatus* var. paniala (Roxb.), *Eugenia siamensis* Craib., bark of *Acacia catechu* (L.F) Wild., kernel of *Areca catechu* Linn. and stem of *Cassia fistula* Linn. Chanwitheesuk et al. (2005) reported the total phenolic content of Thai plants using Folin Denis reagent and pyrocatechol as reference, with data calculated as mg% of pyrocatechol equivalents. For comparison, the total phenolic content of Leucaena was reported as 405 mg% (Chanwitheesuk et al., 2005) and 51.20 mg GAE/g db plant (Maisuthisakul et al., 2008). The data showed that the different reference compound and the units used required a conversion factor of around eight to convert the

Quantitative determination of individual flavonoid glycosides is difficult because most standards are not commercially available. Hence, the total flavonoid content determined using a colorimetric method (Bonvehí et al., 2001) is commonly used for flavonoid evaluation. Generally, the calculation of the total flavonoid content is quoted in units of mg rutin equivalent of flavonoid compounds in one gram of plant extract based on the dry weight of the original plant sample. It is well known that flavonoids possess antioxidant properties both in vitro and in vivo. The flavonoids contain a number of phenolic hydroxyl groups attached to aromatic ring structures, which confer the antioxidant activity. Flavonoids were found in leaves, seeds and fruits and were good antioxidants (Bonvehí et al., 2001). The data clearly indicate that some plants in Thailand are rich in natural antioxidant flavonoids. Some data in Table 1 were used to find a relationship between total phenolic and flavonoid content. The flavonoid content correlates moderately with the

Fig. 1. Relationship between total phenolic and total flavonoid content in Thai plant extracts.

Chanwitheesuk et al. (2005) reported the total carotene, total xanthophyll and tannin contents of edible plants grown in Thailand as shown in Table 1. Total carotenes and total xanthophylls were determined according to Helrich (1990). Tannin contents were determined colorimetrically using the Folin-Dennis reagent according to Helrich (1990).

Identification of the phenolic components in Thai plants has not commonly been reported because it is expensive and standards required for identification are often not available. Hence, this chapter will report the phenolic profile of only four of the Thai plants

values (Table 1).

phenolic content as shown in Fig. 1.

investigated; Piper betel Linn, *Careya sphaerica*, *Cratoxylum formosum* Dyer., and *Leucaena leucocephala* de Wit.

**Piper betel Linn.;** which is commonly known as Betel leaf, is chewed with Betel nut and lime by some people in Asia. Its Thai name is different in different areas, for instance, Plu-Cheen, Se-ke, Bul-plao-yuan, she-ke (South), Pu (North-West). The leaves are chewed alone or with other plant materials including the areca nut (Areca catechu Linn.) and lime. Many researchers have focused on the red lime betel quid in the past few years. A little information about the Betel leaf was found. The Betel leaf itself has a spicy taste and yields an essential oil widely used as a medicine. In Thailand, it is used to treat bruises, heal urticaria, cure ringworm and joint pain as well as relieving toothache. It is also used in cough and mucus remedies and infusions to cure indigestion, as a topical cure for constipation, as a decongestant and as an aid to lactation. The characteristics and chemical composition of 100 grams of Betel leaves are 44 kcal for energy, 85-90 g of water, 3-3.5 g of protein, 2.3 g of fiber, 0.63-0.89 mg of nicotinic acid, 0.005-0.01 g of vitamin C, 1.9-2.9 mg of Vitamin A, 10-70 μg of thiamine, 1.9-30 μg of riboflavin, 0.1-1.3 g of tannin, 0.05-0.6 g of phosphorus, 1.1-4.6 g of potassium, 0.2-0.5 g of calcium, 0.005-0.007 g of iron, 3.4 μg of iodine (Guha, 2006).

Other biological activities described for the essential oil include antifungal, antiseptic and anthelmintic effects (Evans et al., 1984). It was reported that Betel leaf was rich in carotenes (80 IU/g fresh wt.) and phenolics. Data on the phenolic compounds of this plant have been reported for chavicol (Amonkar, et al., 1986), chavibetol, chavibetol acetate (Rimando, et al., 1986) and eugenol (Nagabhushan, et al., 1989). The major bioactive phenolic compoundsin Thai Betel leaf extracted with ethyl acetate were found to be relatively low in polarity. They are chavicol and two-unknown compounds which show higher polarity than chavicol (Maisuthisakul, 2008). The chemical structures of phenolic compounds found in betel leaf are shown in Fig. 2.

Fig. 2. Some phenolic compounds found in *Piper betel* Linn.

Phenolic Constituents and Antioxidant Properties of some Thai Plants 195

The HPLC chromatogram of the Kradonbok extract is shown in Fig. 4. The ethanol extract was dissolved in methanol and passed through a Sep-Pak C18 cartridge (Waters, Milford, MA.). The C-18 cartridge was first conditioned by suction with 1 column volume of methanol followed by 2 column volumes of a 3% HCl solution (v/v) in HPLC grade water. The cartridge was not allowed to dry out during conditioning. The aqueous extract was then transferred to the cartridge. The cartridge bed was then rinsed with HCl (3%, 5 mL) and airdried under vacuum for ~10 min. Phytochemicals were eluted with HPLC grade methanol. Samples were filtered through a 0.20 mm Millipore filter (type HA) into a 2 mL autosampler

**Source:** Maisuthisakul (2007)

detected at 260, 280 and 320 nm.

Fig. 4. HPLC chromatogram for the extract from *Careya sphaerica* Roxb. (Kradonbok)

**Careya sphaerica Roxb.;** This plant is also known as *Careya arborea* Roxb. This plant is normally consumed fresh and mostly found in the North – East of Thailand. Its name is different in different areas, for instance, Kradon, Phak–Kradon, Kradonbok, Kradonkhon (North – East), Khui (Khanchanaburee), Phuk-Pui (North), Puikradon (South), Pui - khao (Chiangmai). Thai people traditionally eat shoots, young leaves and young flowers of this plant. It tastes a little astringent due to the phenolic compounds present. The harvesting season of Kradonbok is during March to May of each year. Kradonbok trees are planted commercially in Sakolnakhon, Kalasin, Yasothorn, Mahasarakham, and Bureerum which are provinces in the North – East of Thailand. Kradonbok has some health benefits such as the use of Kradonbok leaf for healing a wound and flowers for remedying a cough. The characteristics and chemical composition of 100 grams of Kradonbok leaves are 83 Kcal for energy, 1.9 g of fiber, 13 mg of calcium, 18 mg of phosphorus, 17 mg of iron, 3958 IU of riboflavin, 1.8 mg of niacin and 126 mg of vitamin C (Nutrition division, 1992).

There have been few reports about the phenolic compounds of *Careya sphaerica* Roxb. during the last 30 years. Gupta et al. (1975) reported that the phenolic consituents present in the leaf extracts when extracted with petroleum ether at room temperature were lupeol, hexacosanol (C26H54O), α-spinosterol (C29H48O), teraxerol (C30H50O), β-sitosterol (C29H50O), quercetin (C15H10O7), taraceryl acetate (C32H52O2) and ellagic acid (C14H6O8). Careaborin, βamyrin, careyagenolide, maslinic acid and α-hydroxyursolic acid were also found in the leaves (Das & Mahato, 1982 Das & Mahato, 1982; Talapatra et al., 1981). The chemical structures of some componentsextracted from *Careya sphaerica* Roxb.leaf are shown in Fig. 3.

Fig. 3. Some phytochemicals extracted from *Careya sphaerica* Roxb.

**Careya sphaerica Roxb.;** This plant is also known as *Careya arborea* Roxb. This plant is normally consumed fresh and mostly found in the North – East of Thailand. Its name is different in different areas, for instance, Kradon, Phak–Kradon, Kradonbok, Kradonkhon (North – East), Khui (Khanchanaburee), Phuk-Pui (North), Puikradon (South), Pui - khao (Chiangmai). Thai people traditionally eat shoots, young leaves and young flowers of this plant. It tastes a little astringent due to the phenolic compounds present. The harvesting season of Kradonbok is during March to May of each year. Kradonbok trees are planted commercially in Sakolnakhon, Kalasin, Yasothorn, Mahasarakham, and Bureerum which are provinces in the North – East of Thailand. Kradonbok has some health benefits such as the use of Kradonbok leaf for healing a wound and flowers for remedying a cough. The characteristics and chemical composition of 100 grams of Kradonbok leaves are 83 Kcal for energy, 1.9 g of fiber, 13 mg of calcium, 18 mg of phosphorus, 17 mg of iron, 3958 IU of

There have been few reports about the phenolic compounds of *Careya sphaerica* Roxb. during the last 30 years. Gupta et al. (1975) reported that the phenolic consituents present in the leaf extracts when extracted with petroleum ether at room temperature were lupeol, hexacosanol (C26H54O), α-spinosterol (C29H48O), teraxerol (C30H50O), β-sitosterol (C29H50O), quercetin (C15H10O7), taraceryl acetate (C32H52O2) and ellagic acid (C14H6O8). Careaborin, βamyrin, careyagenolide, maslinic acid and α-hydroxyursolic acid were also found in the leaves (Das & Mahato, 1982 Das & Mahato, 1982; Talapatra et al., 1981). The chemical structures of some componentsextracted from *Careya sphaerica* Roxb.leaf are shown in Fig. 3.

riboflavin, 1.8 mg of niacin and 126 mg of vitamin C (Nutrition division, 1992).

Fig. 3. Some phytochemicals extracted from *Careya sphaerica* Roxb.

The HPLC chromatogram of the Kradonbok extract is shown in Fig. 4. The ethanol extract was dissolved in methanol and passed through a Sep-Pak C18 cartridge (Waters, Milford, MA.). The C-18 cartridge was first conditioned by suction with 1 column volume of methanol followed by 2 column volumes of a 3% HCl solution (v/v) in HPLC grade water. The cartridge was not allowed to dry out during conditioning. The aqueous extract was then transferred to the cartridge. The cartridge bed was then rinsed with HCl (3%, 5 mL) and airdried under vacuum for ~10 min. Phytochemicals were eluted with HPLC grade methanol. Samples were filtered through a 0.20 mm Millipore filter (type HA) into a 2 mL autosampler

**Source:** Maisuthisakul (2007)

Fig. 4. HPLC chromatogram for the extract from *Careya sphaerica* Roxb. (Kradonbok) detected at 260, 280 and 320 nm.

Phenolic Constituents and Antioxidant Properties of some Thai Plants 197

The known antioxidant components 1 (gallic acid) and 7 (ellagic acid) were present at 0.93 % and 2.37 % of the extract. These concentrations were calculated according to the peak areas from the HPLC chromatogram. The thirteen phenolic compounds represented about 56.16%

> O M e

O

O O

H O O

(a) (b)

O H

O M e

Fig. 6. UV spectrum of (a) peak 7 of *Careya sphaerica* Roxb. (Kradonbok) extract, ellagic acid

*Cratoxylum formosum* **Dyer.**; The name of this plant differs in different local areas, for instance, Teawkon (Central), Teawdang (North) and Tao (South). Thai people traditionally eat shoots and young leaves of this plant. It tastes sour and a little astringent due to the phenolic phytochemicals present. The harvesting season of *Cratoxylum formosum* Dyer. is during March to May of each year. Trees of this plant are planted commercially in Sakolnakhon, Kalasin, Yasothorn, Mahasarakham, Bureerum, which are the provinces in North – East Thailand. The chemical composition per 100 grams of Teaw leaves which provides 58 Kcal for energy includes 1.5 g of fiber, 67 mg of calcium, 19 mg of phosphorus, 205 mg of iron, 4500 µg of β-carotene, 750 µg vitamin A as retinol, 10.04 mg of thiamin, 0.67 mg of riboflavin, 3.1 mg of niasin and 58 mg of vitamin

Relatively little work has been done on the phytochemicals in *Cratoxylum* sp. The only studies concerning the phytochemistry of *Cratoxylum* sp. was published by Kitanov & Assenov (1988), and Kumar et al. (2004) who reported that the phenolic compounds in *Cratoxylum pruniflorum* Kurz were quercetin (C15H10O12), hyperoside (C21H20O12), 1,3,6,7 tetrahydroxyxanthone, mangiferin (C19H18O11) and isomangiferin (C19H18O12), (Kitanov & Assenov, 1988). The phenolic constituents in *Cratoxylum neriifolium* Kurz were biflavonol GB-2, pentahydroxyflavanone chromone and stigmasterol (Kumar et al., 2004). The chemical structures of some phytochemicals found in *Cratoxylum* sp. leaf are shown in

of the total phenolic compounds of Kradonbok extract.

Peak 7

Ellagic acid

235 275 300 325 350 375 400

235 250 275 300 325 350 375 400 **Wavelength (nm)** 

**Source:** Maisuthisakul (2007)

0.0

25.0

**Absorption (%)** 

25

60.0

50

and (b) structure of ellagic acid.

C (Nutrition division, 1992).

Fig. 7.

vial for subsequent analysis by HPLC. The solution (10 mL) was injected into the HPLC and analyzed according to the following conditions: column, Synergi Hydro RP column (150 × 4.6 mm id., 4mm, Phenomenex), fitted with a Allsphere ODS-2 guard column (10 × 4.6 mm id., Alltech). The HPLC system was equipped with diode array detector (Dionex PDA 100 photodiode array, USA) controlled by Chromeleon software version 6.60 Build 1428 (Dionex Corporation, Sunnyvale, USA). Chromatograms were recorded at 260, 280 and 320 nm.

Thirteen main peaks were detected in Thai Tummy wood leaf extracted with ethanol and the components corresponding to peaks 1, 2, 3, 4, 5, and 6 eluted at 11.06, 33.91, 41.79, 48.35, 51.91 and 62.36 min in the more hydrophilic region (short retention time). The other main components eluted at 100.19 min (peak 7), 134.71 min (peak 8), 139.27 min (peak 9), 160.93 min (peak 10), 164.63 min (peak 11), 170.44 min (peak 12) and 174.90 min (peak 13) in the more hydrophobic region (long retention time).

The identity and purity of peak 1 in the HPLC chromatogram of the extract of *Careya sphaerica* Roxb. was determined by comparison of the retention time and UV spectrum (Fig. 5) with that of pure gallic acid. The identification of peak 1 as gallic acid was confirmed by co-injection of gallic acid with the plant extract. HPLC-ESI-MS confirmed the identification of this compound (MS [M + H]+ at m/z 171, MS [M + Na]+ at m/z 193 and MS [M+4Na]+ at m/z 262), which is consistent with the molecular weight of 170.12 for gallic acid. Compound 7 was identified by comparison of the retention time and UV spectrum (Fig. 6) with that of pure ellagic acid. The identification of peak 7 as ellagic acid was confirmed by co-injection of ellagic acid with the plant extract. HPLC-ESI-MS confirmed the identification of this compound (MS [M + H]+ at m/z 303, MS [M + Na]+ at m/z 325), confirming the molecular weight of 302.19 for ellagic acid. Talapatra et al. (1981) reported that ellagic acid was isolated from the leaves of *Careya arborea*, which is the synonym of *Careya sphaerica* Roxb.

**Source:** Maisuthisakul (2007)

Fig. 5. UV spectrum of (a) peaks 1 of *Careya sphaerica* Roxb. (Kradonbok) extract, gallic acid and (b) structure of gallic acid.

The known antioxidant components 1 (gallic acid) and 7 (ellagic acid) were present at 0.93 % and 2.37 % of the extract. These concentrations were calculated according to the peak areas from the HPLC chromatogram. The thirteen phenolic compounds represented about 56.16% of the total phenolic compounds of Kradonbok extract.

**Source:** Maisuthisakul (2007)

196 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

vial for subsequent analysis by HPLC. The solution (10 mL) was injected into the HPLC and analyzed according to the following conditions: column, Synergi Hydro RP column (150 × 4.6 mm id., 4mm, Phenomenex), fitted with a Allsphere ODS-2 guard column (10 × 4.6 mm id., Alltech). The HPLC system was equipped with diode array detector (Dionex PDA 100 photodiode array, USA) controlled by Chromeleon software version 6.60 Build 1428 (Dionex Corporation, Sunnyvale, USA). Chromatograms were recorded at 260, 280 and 320 nm.

Thirteen main peaks were detected in Thai Tummy wood leaf extracted with ethanol and the components corresponding to peaks 1, 2, 3, 4, 5, and 6 eluted at 11.06, 33.91, 41.79, 48.35, 51.91 and 62.36 min in the more hydrophilic region (short retention time). The other main components eluted at 100.19 min (peak 7), 134.71 min (peak 8), 139.27 min (peak 9), 160.93 min (peak 10), 164.63 min (peak 11), 170.44 min (peak 12) and 174.90 min (peak 13) in the

The identity and purity of peak 1 in the HPLC chromatogram of the extract of *Careya sphaerica* Roxb. was determined by comparison of the retention time and UV spectrum (Fig. 5) with that of pure gallic acid. The identification of peak 1 as gallic acid was confirmed by co-injection of gallic acid with the plant extract. HPLC-ESI-MS confirmed the identification of this compound (MS [M + H]+ at m/z 171, MS [M + Na]+ at m/z 193 and MS [M+4Na]+ at m/z 262), which is consistent with the molecular weight of 170.12 for gallic acid. Compound 7 was identified by comparison of the retention time and UV spectrum (Fig. 6) with that of pure ellagic acid. The identification of peak 7 as ellagic acid was confirmed by co-injection of ellagic acid with the plant extract. HPLC-ESI-MS confirmed the identification of this compound (MS [M + H]+ at m/z 303, MS [M + Na]+ at m/z 325), confirming the molecular weight of 302.19 for ellagic acid. Talapatra et al. (1981) reported that ellagic acid was isolated

from the leaves of *Careya arborea*, which is the synonym of *Careya sphaerica* Roxb.

(a) (b)

HO

HO

OH

COOH

Gallic acid

Fig. 5. UV spectrum of (a) peaks 1 of *Careya sphaerica* Roxb. (Kradonbok) extract, gallic acid

235 275 300 325 350 375 400 235 250 275 300 325 350 375 400 **Wavelength (nm)** 

more hydrophobic region (long retention time).

**Source:** Maisuthisakul (2007)

60.0

50

0.0

25

25.0

**Absorption (%)** 

and (b) structure of gallic acid.

Peak 1

Fig. 6. UV spectrum of (a) peak 7 of *Careya sphaerica* Roxb. (Kradonbok) extract, ellagic acid and (b) structure of ellagic acid.

*Cratoxylum formosum* **Dyer.**; The name of this plant differs in different local areas, for instance, Teawkon (Central), Teawdang (North) and Tao (South). Thai people traditionally eat shoots and young leaves of this plant. It tastes sour and a little astringent due to the phenolic phytochemicals present. The harvesting season of *Cratoxylum formosum* Dyer. is during March to May of each year. Trees of this plant are planted commercially in Sakolnakhon, Kalasin, Yasothorn, Mahasarakham, Bureerum, which are the provinces in North – East Thailand. The chemical composition per 100 grams of Teaw leaves which provides 58 Kcal for energy includes 1.5 g of fiber, 67 mg of calcium, 19 mg of phosphorus, 205 mg of iron, 4500 µg of β-carotene, 750 µg vitamin A as retinol, 10.04 mg of thiamin, 0.67 mg of riboflavin, 3.1 mg of niasin and 58 mg of vitamin C (Nutrition division, 1992).

Relatively little work has been done on the phytochemicals in *Cratoxylum* sp. The only studies concerning the phytochemistry of *Cratoxylum* sp. was published by Kitanov & Assenov (1988), and Kumar et al. (2004) who reported that the phenolic compounds in *Cratoxylum pruniflorum* Kurz were quercetin (C15H10O12), hyperoside (C21H20O12), 1,3,6,7 tetrahydroxyxanthone, mangiferin (C19H18O11) and isomangiferin (C19H18O12), (Kitanov & Assenov, 1988). The phenolic constituents in *Cratoxylum neriifolium* Kurz were biflavonol GB-2, pentahydroxyflavanone chromone and stigmasterol (Kumar et al., 2004). The chemical structures of some phytochemicals found in *Cratoxylum* sp. leaf are shown in Fig. 7.

Phenolic Constituents and Antioxidant Properties of some Thai Plants 199

Fig. 8. Some phenolic compounds found in *Cratoxylum formosum* in Thailand.

Fig. 9. Some phenolic compounds found in *Leucaena glauca* Benth

Benth leaf are shown in Fig. 9.

Relatively little information on the phenolic constituents of *Leucaena glauca* Benth has been published during the last 30 years. Chen (1979) found foeniculin (C14H18O) and kaempferol-3-xyloside (C20H18O10) in the leaves. Guaijaverin (C20H18O11), juglania (C20H18O10), kaempferol-3-O-β-xyloside and quercitrin (C21H20O11) were also found in the leaves (Morita et al., 1977). The chemical structures of phenolic compounds found in Thai *Leucaena glauca*

Fig. 7. Some phytochemicals found in *Cratoxylum* sp.

With regards to Thai plants, three active phenolic ingredients were chlorogenic acid, dicaffeoylquinic acid and ferulic acid hexose derivative (Maisuthisakul et al., 2007b). Chlorogenic acid was present at 60 % of the extract. Two minor components (dicaffeoylquinic acid and ferulic acid hexose derivative) were present at 7 % and 2 %, and other components that were present at lower concentrations were also detected. Some chemical structures of phenolic compounds found in Thai *Cratoxylum formosum* leaf are shown in Fig. 8.

*Leucaena glauca* **Benth**; the plant is found throughout Thailand in the settled areas at low and medium altitudes. It occurs widely and is abundant. Its name is different in different areas, for instance, Kratin-Thai (Central), Satorban (South), Katong and Kratin. Thai people traditionally eat young leaves and the young pod of this plant. The young leaf is found in all seasons, however, it is most abundant during March to May of each year. Kratin trees are planted commercially in Roi-ed, Amnat Charoen, Pichit, Nakhonsawan, Songkla, Krabi, Pattani and Trang. Kratin leaves contain leucine which can absorb selenium. The charcateristics and chemical composition of 100 grams of Kratin leaves are 62 Kcal for energy, 8.4 g of protein, 3.8 g of crude fiber, 137 mg of calcium, 11 mg of phosphorus, 9.2 g of iron, 7883 IU of total vitamin A, 0.33 mg of thiamin, 0.09 mg riboflavin, 1.7 mg of niacin and 8 mg of vitamin C (Nutrition division, 1992).

With regards to Thai plants, three active phenolic ingredients were chlorogenic acid, dicaffeoylquinic acid and ferulic acid hexose derivative (Maisuthisakul et al., 2007b). Chlorogenic acid was present at 60 % of the extract. Two minor components (dicaffeoylquinic acid and ferulic acid hexose derivative) were present at 7 % and 2 %, and other components that were present at lower concentrations were also detected. Some chemical structures of

*Leucaena glauca* **Benth**; the plant is found throughout Thailand in the settled areas at low and medium altitudes. It occurs widely and is abundant. Its name is different in different areas, for instance, Kratin-Thai (Central), Satorban (South), Katong and Kratin. Thai people traditionally eat young leaves and the young pod of this plant. The young leaf is found in all seasons, however, it is most abundant during March to May of each year. Kratin trees are planted commercially in Roi-ed, Amnat Charoen, Pichit, Nakhonsawan, Songkla, Krabi, Pattani and Trang. Kratin leaves contain leucine which can absorb selenium. The charcateristics and chemical composition of 100 grams of Kratin leaves are 62 Kcal for energy, 8.4 g of protein, 3.8 g of crude fiber, 137 mg of calcium, 11 mg of phosphorus, 9.2 g of iron, 7883 IU of total vitamin A, 0.33 mg of thiamin, 0.09 mg riboflavin, 1.7 mg of niacin and 8 mg of vitamin C (Nutrition

phenolic compounds found in Thai *Cratoxylum formosum* leaf are shown in Fig. 8.

Fig. 7. Some phytochemicals found in *Cratoxylum* sp.

division, 1992).

Stigmasterol

Fig. 8. Some phenolic compounds found in *Cratoxylum formosum* in Thailand.

Relatively little information on the phenolic constituents of *Leucaena glauca* Benth has been published during the last 30 years. Chen (1979) found foeniculin (C14H18O) and kaempferol-3-xyloside (C20H18O10) in the leaves. Guaijaverin (C20H18O11), juglania (C20H18O10), kaempferol-3-O-β-xyloside and quercitrin (C21H20O11) were also found in the leaves (Morita et al., 1977). The chemical structures of phenolic compounds found in Thai *Leucaena glauca* Benth leaf are shown in Fig. 9.

Fig. 9. Some phenolic compounds found in *Leucaena glauca* Benth

Phenolic Constituents and Antioxidant Properties of some Thai Plants 201

Fig. 10. UV spectrum of (a) peaks 1, 2 of *Leucaena glauca* Benth (Kratin) extract, quercetin, (b) structure of quercetin, (c) UV spectrum of peaks 3 of Kratin extract, kaempferol, (d) structure of kaempferol, (e) UV spectrum of peaks 4 of Kratin extract, luteolin and (f)

**Source:** Maisuthisakul (2007)

structure of luteolin.

The phenolic components of Leucaena glauca Benth in the HPLC chromatogram appeared as 4 peaks which were present at 15.28%, 6.50%, 9.71% and 36.91%, respectively, and other components that were present at lower concentrations were also observed. These concentrations were calculated from each area by dividing by the total peak area from the HPLC chromatogram. The four phenolic compounds were about 68.4% of the total phenolic content of Leucaena glauca Benth extract. The compounds corresponding to peaks 1, 2, 3, and 4 had a similar UV spectrum to that of flavonoids such as quercetin, and kaempferol which are flavonols and luteolin which is a flavone (Fig. 10). The identification of each peak was confirmed by using comparisons of their UV spectra and LC-MS in both positive and negative mode in order to obtain more information on the structural features of the conjugated forms of the phenolic compounds.

#### **3. Antioxidant properties of some Thai plants**

In general, the antioxidant activity of plant extracts is associated with specific compounds or classes of compounds, such as flavones, flavonols and proanthocyanidins in plant materials native to the Mediterranean area (Skerget et al. , 2005), carotenoids (Stahl & Sies, 2003) and melatonin (Chen et al., 2003). Most of the antioxidant substances in plants are phenolic compounds. Phenolic compounds serve as oxidation terminators by scavenging radicals to form resonance stabilized radicals (Rice-Evans et al., 1997).

Although the antioxidant capacities are influenced by many factors, which cannot be fully described with a single method, the DPPH radical scavenging activity is the most commonly used method for assessment of antioxidant properties of natural products. The DPPH• assay overcomes the limitations of monitoring the activity of the numerous samples over a specified period of time. It is reproducible and strongly correlated with phenolic compounds (Maisuthisakul et al., 2007; Katalinic et al., 2006; Miliauskas et al., 2004; Matsuda et al., 2001). In addition the radical scavenging method had many benefits compared to the lipid oxidation method (Nuutila et al., 2002). Gallic acid, which has been used as a standard, has been reported to be the most abundant phenolic compound in plants (Witzell et al., 2003., Nuutila et al., 2002). The EC50 is defined as the amount of antioxidant required to cause a 50% reduction in the absorbance of DPPH. These values were changed to antiradical activity defined as 1/EC50, since this parameter increases with antioxidant activity. The antiradical activity of α-tocopherol was 0.67 (Maisuthisakul et al., 2008). Many researchers have reported the total phenolic content of Thai plants as shown in Table 2. The plants which had strong antiradical activity (Table 2) had high total phenolic and flavonoid contents (Table 1).

In Thailand, there are many plants which show antiradical activity higher than αtocopherol such as *Careya sphaerica* Roxb., *Cratoxylum formosum* Dyer., *Erythrina crista* Galli., *Leucaena glauca* Benth, *Momordica charantia* Linn., *Ocimum basilicum* Linn., *Ocimum sanctum* Linn., *Syzygium gratum* (Wight) S.N.Mitra var. gratum, *Allium ascalonicum* Linn., *Azadirachta indeca* A. Juss Var. siamensis valeton, *Cassia siamea* Britt., *Musa spiantum* Linn., *Sesbania grandiflora* Desv. The antiradical activity of Thai fruits and fruit seeds were higher than that of α-tocopherol except for seeds of *Leucaena glauca* Benth (Table 2).

The phenolic components of Leucaena glauca Benth in the HPLC chromatogram appeared as 4 peaks which were present at 15.28%, 6.50%, 9.71% and 36.91%, respectively, and other components that were present at lower concentrations were also observed. These concentrations were calculated from each area by dividing by the total peak area from the HPLC chromatogram. The four phenolic compounds were about 68.4% of the total phenolic content of Leucaena glauca Benth extract. The compounds corresponding to peaks 1, 2, 3, and 4 had a similar UV spectrum to that of flavonoids such as quercetin, and kaempferol which are flavonols and luteolin which is a flavone (Fig. 10). The identification of each peak was confirmed by using comparisons of their UV spectra and LC-MS in both positive and negative mode in order to obtain more information on the structural features of the

In general, the antioxidant activity of plant extracts is associated with specific compounds or classes of compounds, such as flavones, flavonols and proanthocyanidins in plant materials native to the Mediterranean area (Skerget et al. , 2005), carotenoids (Stahl & Sies, 2003) and melatonin (Chen et al., 2003). Most of the antioxidant substances in plants are phenolic compounds. Phenolic compounds serve as oxidation terminators by scavenging radicals to

Although the antioxidant capacities are influenced by many factors, which cannot be fully described with a single method, the DPPH radical scavenging activity is the most commonly used method for assessment of antioxidant properties of natural products. The DPPH• assay overcomes the limitations of monitoring the activity of the numerous samples over a specified period of time. It is reproducible and strongly correlated with phenolic compounds (Maisuthisakul et al., 2007; Katalinic et al., 2006; Miliauskas et al., 2004; Matsuda et al., 2001). In addition the radical scavenging method had many benefits compared to the lipid oxidation method (Nuutila et al., 2002). Gallic acid, which has been used as a standard, has been reported to be the most abundant phenolic compound in plants (Witzell et al., 2003., Nuutila et al., 2002). The EC50 is defined as the amount of antioxidant required to cause a 50% reduction in the absorbance of DPPH. These values were changed to antiradical activity defined as 1/EC50, since this parameter increases with antioxidant activity. The antiradical activity of α-tocopherol was 0.67 (Maisuthisakul et al., 2008). Many researchers have reported the total phenolic content of Thai plants as shown in Table 2. The plants which had strong antiradical activity (Table 2) had high total phenolic and flavonoid

In Thailand, there are many plants which show antiradical activity higher than αtocopherol such as *Careya sphaerica* Roxb., *Cratoxylum formosum* Dyer., *Erythrina crista* Galli., *Leucaena glauca* Benth, *Momordica charantia* Linn., *Ocimum basilicum* Linn., *Ocimum sanctum* Linn., *Syzygium gratum* (Wight) S.N.Mitra var. gratum, *Allium ascalonicum* Linn., *Azadirachta indeca* A. Juss Var. siamensis valeton, *Cassia siamea* Britt., *Musa spiantum* Linn., *Sesbania grandiflora* Desv. The antiradical activity of Thai fruits and fruit seeds were higher than that of α-tocopherol except for seeds of *Leucaena glauca* Benth

conjugated forms of the phenolic compounds.

contents (Table 1).

(Table 2).

**3. Antioxidant properties of some Thai plants** 

form resonance stabilized radicals (Rice-Evans et al., 1997).

**Source:** Maisuthisakul (2007)

Fig. 10. UV spectrum of (a) peaks 1, 2 of *Leucaena glauca* Benth (Kratin) extract, quercetin, (b) structure of quercetin, (c) UV spectrum of peaks 3 of Kratin extract, kaempferol, (d) structure of kaempferol, (e) UV spectrum of peaks 4 of Kratin extract, luteolin and (f) structure of luteolin.

Phenolic Constituents and Antioxidant Properties of some Thai Plants 203

*Scientific name* Local name Plant part Antiradical activity

*Eugenia siamensis* Craib. ¥ Chom-pu-nam Fruit 5.0

meaw

*Momordica charantia* Linn. ¥ Mara-khee-nok Fruit 1.7 *Phyllanthus emblica*¥ Ma-kham-pom Fruit 2.0 *Spondias pinnata* Kurz. ¥ Ma-kok Fruit 1.6

*Antidesma velutinum* Tulas\* Ma-mao Seed 14.28 *Cleistocalyx operculatus* var. paniala (Roxb.)\* Ma-kieng Seed 11.11 *Eugenia siamensis* Craib.\* Chom-pu-nam Seed 6.67 *Leucaena glauca* Benth. \* Kra-tin Seed 0.14 *Nephelium lappaceum* Linn. ¥ Ngo Seed 2.2 *Parkia speciosa* Hassk. ¥ Sa-tor Seed 1.5 *Piper nigrum* Linn. ¥ Prik-Thai-dum Seed 3.0 *Tamarindus indica* Linn¥ Ma-kham Seed 2.0

**Chewing plants** *Acacia catechu* (L.F) Wild.\* See-sead Bark 20.0

Natural antioxidants are available from raw materials of variable composition. Both the content of active substances and the content of various other compounds may vary. The quality of natural extracts and their antioxidative activity depends not only on the quality of the original plant, date and storage, but also the extraction conditions which affect the plant

Sample preparation is required to keep samples for a certain period of time before analysis since even the total phenolic content was shown to decrease during storage compared with the fresh leaves. In addition, the availability of raw materials is usually limited to a

Temperature is the major factor influencing changes in antioxidant activity during storage (Moure et al., 2001). From numerous publications, the preservative processes before storage vary such as drying citrus peel and seed in an oven at 40 ºC and keeping at room temperature (Bocco et al., 1998) or drying Indian Laburnum by air at 25ºC and keeping at room temperature (Siddhuraju et al., 2002) or keeping berries in a still air freezer at -25 to

harvesting season. The preliminary processing of plants is necessary for storage.

*Cassia fistula* Linn.\* Kaen-khun Stem core 6.25 *Piper betel* Linn\* Bai-plu Leaf 3.13 **Source**: Adapted from ¥Maisuthisakul et al. (2008); \*Maisuthisakul et al., 2007a; €Lee & Scagel, 2009; Kerdchoechuen & Laohakunjit, 2010; Ongard & Dara, 2010; Phomkaivon & Areekul (2009)

Kernel

*Euginia malaccenses* Linn. ¥ Chom-pu-ma-

 **Seeds** 


**4.1 Sample preparation for storage** 


*Areca catechu* Linn.\* Mhak Whole fruit

Table 2. Ranges of antiradical activity in some Thai plants

**4. Factors affecting extraction of plant phenolics** 

phenolic compounds extracted (Moure et al., 2001).

(1/ EC50)

2.13 5.56

Fruit 2.2


#### Phenolic Constituents and Antioxidant Properties of some Thai Plants 203

202 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

*Scientific name* Local name Plant part Antiradical activity

Hia tem and leaves 0.19

Sa-dao Flower 1.2

Stem and leaves 1.04

*Allium ascalonicum* Linn. ¥ Hom Flower 2.6

*Aspidistra sutepensis* K. Larsen Nang-laeo Flower 0.06

*Basella alba* Linn. ¥ Plang Leaf 0.7

Nok-Sai

*Bidens pilosa* Linn. Peen-nok-sai Stem and leaves 1.22 *Buddieia asiatica* Lour Ra-cha-wa-di-pa Stem and leaves 0.26 *Careya sphaerica* Roxb. ¥ Kra-don Young leaf and leaf 2.3 *Cassia siamea* Britt. ¥ Kee-lek Flower 2.4 *Centella asiatica* Linn. ¥ Bua-bok Leaf 0.7 *Cratoxylum formosum* Dyer. ¥ Tew Young leaf and leaf 4.4 *Commelina diffusa* Burm.f. Plap Stem and leaves 0.64 *Conyza sumatrensis* (Retz.) Walker Ya-khamai Stem and leaves 0.46 *Cuscuta australis* R. Br. Khruea-kham Stem 1.08 *Diplazium esculentum* (Retz.) Sw. Kut-khao Stem and leaves 0.63 *Dolichandrone serrulata* (DC.) Seem. Khae-pa Flower 0.06 *Embelia ribes* Burm.f. Som-jee Leaves 2.86 *Embelia sessiliflora* Kurtz Som-kui Leaves 5.0 *Erythrina crista* Galli. ¥ Tong-lang Leaf 3.1 *Hydrocharis dubia* (Bl.) Back.\* Tub-tao Bud 0.82 *Lasia spinosa* (Linn.) Thw. ¥ Nham Leaf 0.1 *Leucaena glauca* Benth¥ Kra-tin Young leaf and leaf 1.5 *Limnocharis flava* Buch. ¥ Pai Leaf 0.1 *Melicope pteleifolia* (Champ.ex Benth.) Sa- Riam -Dong Hartley Leaves 0.18 *Micromelum minutum Wight & Arn*  Sa-mui Stem and leaves 0.83 *Momordica charantia* Linn.\* Ma-ra-khee-nok Bud and leaf 1.70 *Musa spiantum* Linn. ¥ Hua-plee Flower 1.8 *Ocimum basilicum* Linn. ¥ Ho-ra-pa Young leaf and leaf 1.80 *Ocimum sanctum* Linn. ¥ Ka-prow Young leaf and leaf 1.8 *Sauropus androgynus* Linn. ¥ Whan-ban Young leaf and leaf 0.7 *Schima wallichii* (DC.) Korth. Talo Leaves 12.5 *Sesbania grandiflora* Desv. ¥ Kae Flower 1.7 *Spondias pinnata* Kurz. ¥ Ma-kok Young leaf and leaf 0.7

Pun-ngu-keaw Leaf

*Tiliacora triandra* (Colebr.) Diels Ya-nang Leaves 0.15 *Vaccinium sprengelii* (G.Don) Sleum**.**  Som-pi Leaves 1.89

*Capsicum frutescus* Linn. ¥ Prik Fruit 1.8

Stem Root Inflorescence

Mek Young leaf and leaf 1.8

 **Herb and vegetable** 

*Artemisia dubia* Wall. ex DC.

valeton¥

(Syn. *A. vulgaris* L. var. *indica* Maxim.)

*Azadirachta indeca* A. Juss Var. siamensis

*Stachytarpheta jamaicensis* (L.) Vahl(*S.indica* 

*Syzygium gratum* (Wight) S.N.Mitra var.

 **Berries and fruits** 

Vahl)

gratum¥

*Bidens bipinnata* , L. Ya-Puen-Laem

(1/ EC50)

0.016 0.018 0.018 0.020


**Source**: Adapted from ¥Maisuthisakul et al. (2008); \*Maisuthisakul et al., 2007a; €Lee & Scagel, 2009; Kerdchoechuen & Laohakunjit, 2010; Ongard & Dara, 2010; Phomkaivon & Areekul (2009) - means not reported or not determined.

Table 2. Ranges of antiradical activity in some Thai plants

#### **4. Factors affecting extraction of plant phenolics**

Natural antioxidants are available from raw materials of variable composition. Both the content of active substances and the content of various other compounds may vary. The quality of natural extracts and their antioxidative activity depends not only on the quality of the original plant, date and storage, but also the extraction conditions which affect the plant phenolic compounds extracted (Moure et al., 2001).

#### **4.1 Sample preparation for storage**

Sample preparation is required to keep samples for a certain period of time before analysis since even the total phenolic content was shown to decrease during storage compared with the fresh leaves. In addition, the availability of raw materials is usually limited to a harvesting season. The preliminary processing of plants is necessary for storage.

Temperature is the major factor influencing changes in antioxidant activity during storage (Moure et al., 2001). From numerous publications, the preservative processes before storage vary such as drying citrus peel and seed in an oven at 40 ºC and keeping at room temperature (Bocco et al., 1998) or drying Indian Laburnum by air at 25ºC and keeping at room temperature (Siddhuraju et al., 2002) or keeping berries in a still air freezer at -25 to -30ºC (Amakura et al., 2000).

Phenolic Constituents and Antioxidant Properties of some Thai Plants 205

**Sovent effect**; solvent extraction is more frequently used for isolation of antioxidants and both extraction yield and antioxidant activity of extracts are strongly dependent on the solvent, due to the different antioxidant potential of compounds with different polarity (Marinova & Yanishlieva, 1997). Apolar solvents are among the most common solvents for removing polyphenols from water. Ethyl acetate and diethyl ether have been used for extraction of low molecular weight phenols from oak wood (Fernández de Simón et al., 1996). Ethanol and water are the most widely employed solvents for reasons of lack of

With regards to Thai plants, Maisuthisakul (2007) reported that the solvent used had significant effects on antioxidant activity, total phenolic content, yield and partition coefficient of Thai betel leaf extract (p < 0.05). The antioxidant activity assessed by DPPH and ABTS radicals was stronger with less polar solvents (Table 3). The results showed that the DPPH activity of the extract obtained with ethyl acetate was significantly higher than that obtained with the other solvents. Total phenolic content confirmed this finding. The extract with a solvent which has higher polarity was found to contain rather small amounts of phenolic compounds. The EC50 and TEAC value of α- tocopherol was also measured and gave values of 14.95±0.23 µg.mL-1 and 2.30±0.03 mmol of Trolox/g sample, respectively. Betel leaf extracted with ethyl acetate (Table 3) had a weak antioxidant activity compared to α- tocopherol. The antioxidant activity and total phenolic contents were significantly different from various solvent extractions. The antioxidant activity values were consistent

The effectiveness of phenolic antioxidants is often dependent on their polarity. Decker (1998) used the term "antioxidant paradox" to describe how polar antioxidants are most effective in bulk lipids while nonpolar antioxidants are most effective in dispersed lipids. The polarity of the Betel leaf extract was assessed by determination of the oil-water partition coefficient by HPLC. The oil-water partition coefficient was calculated by summing the areas of the three phenolic peaks in the HPLC chromatogram. The oil-water partition coefficient of the Betel leaf extract from ethyl acetate was significantly different from those

> TEAC (mmol Trolox/g sample)

> > 2.01 0.06<sup>c</sup> 2.12 0.03bc 2.21 0.04ab 2.34 0.06<sup>a</sup>

Note: ¥ Data followed by different letters within each column are significantly different according to Duncan's multiple range tests at *P* < 0.05. Data were represented as means from three replicate

Table 3. Antioxidant activity and total phenolic content of Betel leaf extracted by different

Total phenolic content (mg GAE/g sample)

> 49.89 ± 0.21d 50.38 ± 0.08c 53.28 ± 0.19b 55.35 ± 0.14a

**4.2 Extraction conditions** 

toxicity and abundance, respectively.

with those of Chen et al. (2001).

extracted with other solvents as shown in Table 4.

(EC50, g.mL-1)

36.65 ± 2.60a 33.85 ± 2.81ab 30.09 ± 1.21b 17.04 ± 0.51c

Solvent used DPPH activity

Methanol Ethanol Acetone Ethyl acetate

measurements.

solvents¥

**Source:** Maisuthisakul (2007)

Sample preparation methods gave significant effects on total phenolic content and antioxidant activity but had no marked effects on yield of the extract of *Careya sphaerica* or Kradonbok (Maisuthisakul & Pongsawatmanit, 2005). Freezing, especially fast freezing, for keeping the leaves until extraction gave a higher total phenolic content compared with those obtained from other drying methods and slow freezing (Fig. 11). The drying methods studied were (1) hot air drying by tray dryer at 40°C for 18 h with air velocity about 0.5 m/s; (2) vacuum drying by vacuum dryer at 40°C, 100 mmHg (EYELA, model VOS-300SD, Japan) for 10 h; (3) air drying at room temperature 25°C for 12 h with air velocity about 3.2 m/s. The rate of freezing affected the total phenolic content obtained because larger ice crystals grew during slower freezing which would damage plant cells and cause a loss of antioxidant activity. Enzymes from plant cells such as lipoxygenase can oxidize polyphenols (Akoh & Min, 1997). The total phenolic content obtained from air drying was higher than those obtained from vacuum drying and hot air drying because some phenolic components may be degraded by higher temperature (Moure et al., 2001). This effect also found in fresh Mulberry leaves, where the amount of flavonoids was higher in air-dried samples than that in oven-dried samples, probably due to decomposition after storage (Zhishen et al., 1999).

Normally, temperature affects the compounds' stability due to chemical and enzymatic degradation. These mechanisms were reported as mainly responsible for a reduction in phenolic content (Larrauri et al., 1997). Maisuthisakul & Pongsawatmanit also reported that the reduction in antioxidant activity was higher than that expected from the reduction in phenolic contents, probably due to the synergistic effect of natural phenolics (Moure et al., 2001). In addition, phenolics can react with other plant components, and prolonged exposure at moderate temperatures can also cause phenolic degradation. Therefore, sample preparation conditions including temperature and time before storage should be controlled.

**Source:** Adapted from Maisuthisakul & Pongsawatmanit (2005)

Fig. 11. Total phenolic content of the dried extracts from *Careya sphaerica* leaves obtained by various sample preparation methods.

#### **4.2 Extraction conditions**

204 Phytochemicals – A Global Perspective of Their Role in Nutrition and Health

Sample preparation methods gave significant effects on total phenolic content and antioxidant activity but had no marked effects on yield of the extract of *Careya sphaerica* or Kradonbok (Maisuthisakul & Pongsawatmanit, 2005). Freezing, especially fast freezing, for keeping the leaves until extraction gave a higher total phenolic content compared with those obtained from other drying methods and slow freezing (Fig. 11). The drying methods studied were (1) hot air drying by tray dryer at 40°C for 18 h with air velocity about 0.5 m/s; (2) vacuum drying by vacuum dryer at 40°C, 100 mmHg (EYELA, model VOS-300SD, Japan) for 10 h; (3) air drying at room temperature 25°C for 12 h with air velocity about 3.2 m/s. The rate of freezing affected the total phenolic content obtained because larger ice crystals grew during slower freezing which would damage plant cells and cause a loss of antioxidant activity. Enzymes from plant cells such as lipoxygenase can oxidize polyphenols (Akoh & Min, 1997). The total phenolic content obtained from air drying was higher than those obtained from vacuum drying and hot air drying because some phenolic components may be degraded by higher temperature (Moure et al., 2001). This effect also found in fresh Mulberry leaves, where the amount of flavonoids was higher in air-dried samples than that in oven-dried samples, probably due to decomposition after storage (Zhishen et al., 1999).

Normally, temperature affects the compounds' stability due to chemical and enzymatic degradation. These mechanisms were reported as mainly responsible for a reduction in phenolic content (Larrauri et al., 1997). Maisuthisakul & Pongsawatmanit also reported that the reduction in antioxidant activity was higher than that expected from the reduction in phenolic contents, probably due to the synergistic effect of natural phenolics (Moure et al., 2001). In addition, phenolics can react with other plant components, and prolonged exposure at moderate temperatures can also cause phenolic degradation. Therefore, sample preparation conditions including temperature and time before storage

Fig. 11. Total phenolic content of the dried extracts from *Careya sphaerica* leaves obtained by

**Storage time (days)** 0 20 40 60 80

**Fast freezing Slow freezing** 

**C-air drying**

**C-vacuum dring**

**C-hot air dying**

**25 o**

**40 o**

**40 o**

should be controlled.

**Source:** Adapted from Maisuthisakul & Pongsawatmanit (2005)

0

5

10

15

20

25

30

**Total phenolics (mg of GAE/g)**

various sample preparation methods.

**Sovent effect**; solvent extraction is more frequently used for isolation of antioxidants and both extraction yield and antioxidant activity of extracts are strongly dependent on the solvent, due to the different antioxidant potential of compounds with different polarity (Marinova & Yanishlieva, 1997). Apolar solvents are among the most common solvents for removing polyphenols from water. Ethyl acetate and diethyl ether have been used for extraction of low molecular weight phenols from oak wood (Fernández de Simón et al., 1996). Ethanol and water are the most widely employed solvents for reasons of lack of toxicity and abundance, respectively.

With regards to Thai plants, Maisuthisakul (2007) reported that the solvent used had significant effects on antioxidant activity, total phenolic content, yield and partition coefficient of Thai betel leaf extract (p < 0.05). The antioxidant activity assessed by DPPH and ABTS radicals was stronger with less polar solvents (Table 3). The results showed that the DPPH activity of the extract obtained with ethyl acetate was significantly higher than that obtained with the other solvents. Total phenolic content confirmed this finding. The extract with a solvent which has higher polarity was found to contain rather small amounts of phenolic compounds. The EC50 and TEAC value of α- tocopherol was also measured and gave values of 14.95±0.23 µg.mL-1 and 2.30±0.03 mmol of Trolox/g sample, respectively. Betel leaf extracted with ethyl acetate (Table 3) had a weak antioxidant activity compared to α- tocopherol. The antioxidant activity and total phenolic contents were significantly different from various solvent extractions. The antioxidant activity values were consistent with those of Chen et al. (2001).

The effectiveness of phenolic antioxidants is often dependent on their polarity. Decker (1998) used the term "antioxidant paradox" to describe how polar antioxidants are most effective in bulk lipids while nonpolar antioxidants are most effective in dispersed lipids. The polarity of the Betel leaf extract was assessed by determination of the oil-water partition coefficient by HPLC. The oil-water partition coefficient was calculated by summing the areas of the three phenolic peaks in the HPLC chromatogram. The oil-water partition coefficient of the Betel leaf extract from ethyl acetate was significantly different from those extracted with other solvents as shown in Table 4.


Note: ¥ Data followed by different letters within each column are significantly different according to Duncan's multiple range tests at *P* < 0.05. Data were represented as means from three replicate measurements.

**Source:** Maisuthisakul (2007)

Table 3. Antioxidant activity and total phenolic content of Betel leaf extracted by different solvents¥

Phenolic Constituents and Antioxidant Properties of some Thai Plants 207

*C. sphaerica C. formosum S. andrugynus*

Fig. 12. Total phenolic contents and antioxidant activity of the extracts from *Careya sphaerica* Roxb., *Cratoxylum formosum* Dyer. and *Sauropus andrugynus* Merr. leaves obtained with

0

70 80 90 100 110 **Ethanol concentration (%)** 

50

100

**Antioxidant activity,** 

**EC50 (mg.L-1)** 

150

**Other factors**; such as extraction time, pH, the particle size of materials, and extraction methods were reported to affect the antioxidant activity and concentrations of phenolic compounds extracted. Sheabar & Neeman (1988) reported the maximum solubility of phenolic compounds from olive rape at pH 4 in the organic phase. The yield of extracted phenolics was correlated with plant cell wall breakdown. Particle size reduction significantly increased the antioxidant activity as a result of both increased extractability and enhanced enzymatic degradation of polysaccharides (Weinberg et al., 1999). Various process conditions (refluxing, shaking and ultrasonic extraction) also affected the

Many plants in Thailand show potential as a source of extracts rich in phenolic constituents and natural antioxidants. Phenolic compounds are the major antioxidants in plants. Moreover, practical aspects relevant to the use of this class of compounds need to be considered including extraction efficiency, availability of sufficient raw material, and toxicity or safety considerations. To utilize these significant sources of natural antioxidants,

This book chapter was supported by a grant from University of the Thai Chamber of Commerce (UTCC). The author also thanks Professor Michael H. Gordon for helpful

Amakura, Y.; Umino, Y.; Tsuji, S. & Tonogai, Y. (2000). Influence of jam processing on the

radical scavenging activity and phenolic content in berries. *Journal of Agricultural* 

concentrations of antioxidants in extracts from balm leaves (Herodež et al., 2003).

further characterization of the phenolic composition is needed.

*and Food Chemistry,* Vol. 48, pp 6292-6297.

**Source**: Adapted from Maisuthisakul (2007)

**Ethanol concentration (%)** 

70 80 90 100 110 **Ethanol concentration (%)**

various ethanol concentrations.

**5. Conclusion** 

**Total phenolic content**

**(mg of GAE/g db) (mg of GAE/g db)** 

**Total phenolic content** 

**6. Acknowledgement** 

suggestions.

**7. References** 


Note: ¥ Data followed by different letters within each column are significantly different according to Duncan's multiple range test at *P* < 0.05. Data were represented as means from three replicate measurements.
