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## **Meet the editors**

Dr. Dragan Cvetković was born on 26 June, 1977, in Leskovac. He finished his elementary and high school education in Lebane, while he completed his studies at the Faculty of Technology in Leskovac in the year 2002 and finished his PhD thesis in the year 2012 at the Faculty of Technology in Leskovac. Dragan Cvetković participated in the realization of numerous projects funded by

the Ministry of Science, Republic of Serbia. He was engaged in the project 'Folding and Stability of Phycobilisome Proteins' at the Institute of Biology and Technology of Saclay, France. He also participated in the realization of the project entitled 'Contribution of Chemical Quenching of Singlet Oxygen to Pro- and Antioxidant Activity of Carotenoids', funded by the Polish Ministry of Science. He was elected as a teaching assistant in the year 2008 for physical chemistry, colloid chemistry and instrumental analysis, and in the year 2012, he was elected as an assistant professor for a physico-chemical group of subjects at the Faculty of Technology in Leskovac.

Dr. Goran Nikolić was born in Knez Selo (Niš, Serbia) on 1 November 1966. He received his PhD degree in Chemical Engineering (2001) from the University of Niš. Currently, he is a full professor at the same university, for pharmaceutical-cosmetic engineering group of subjects at the Faculty of Technology in Leskovac. His research activities are development of pharmaceutical products

and quality control of drugs. His competences are experience in project management and managing academic institutions at different levels. He is a member of several national projects in the technological development area and is a member of numerous TEMPUS Joint European projects of sustainable technologies, environmental application and management courses. He has authored more than 300 scientific papers, numerous technological solutions for pharmaceutical industry, national monographs, international patents, university textbooks and invitation lectures. He is an editor of two international monographs on FTIR spectroscopy (InTech Open Access Publisher).

### Contents

#### **Preface XI**


Hatta


### Preface

Chapter 7 **Carotenoids Regulate Endothelial Functions and Reduce the**

**Risk of Cardiovascular Disease 105**

Chapter 8 **Biotechnological Production of Carotenoids and Their**

F. Kanno, Liliana I. C. Zoz and Júlio C. Carvalho

Chapter 9 **Synthesis of Antioxidant Carotenoids in Microalgae in Response to Physiological Stress 143** Cecilia Faraloni and Giuseppe Torzillo

Chapter 10 **Carotenoid Production by Corynebacterium: The Workhorse of**

**Broad Spectrum of C40 and C50 Carotenoids 159**

Chapter 11 **Carotenoids in Yellow Sweet Potatoes, Pumpkins and Yellow**

Paul Chavarriaga and Meike S. Andersson

**Industrial Amino Acid Production as Host for Production of a**

Nadja A. Henke, Petra Peters-Wendisch and Volker F. Wendisch

Lucia Maria Jaeger de Carvalho, Gisela Maria Dellamora Ortiz, José Luiz Viana de Carvalho, Lara Smirdele and Flavio de Souza Neves

Hernán Ceballos, Fabrice Davrieux, Elise F. Talsma, John Belalcazar,

**Applications in Food and Pharmaceutical Products 125** Ligia A. C. Cardoso, Susan G. Karp, Francielo Vendruscolo, Karen Y.

Kazuo Yamagata

**VI** Contents

**Section 2 Biotechnological Applications 123**

**Sweet Cassava 175**

Chapter 12 **Carotenoids in Cassava Roots 189**

Cardoso

Carotenoids are one of the most widespread pigment groups distributed in nature; more than 700 natural carotenoids have been described so far. This group of pigments is known for versatile roles they play in living organisms; however, their most pivotal function is in‐ volvement in scavenging of reactive oxygen species and photoprotection.

Within the chapters in this book, different activities (Section I) and biotechnological applica‐ tions (Section II) of carotenoids are discussed.

An important photoprotective mechanism, referred to as the xanthophyll cycle, and the mechanisms of less-known types of the mentioned cycle are described; the current knowl‐ edge on the pigments engaged in the xanthophyll cycles operating in various organisms is also summarized. Electronic properties of six carotenoids such as energy in frontier orbitals and the first molecular orbitals to work in the UV-Vis absorption spectroscopy are also ana‐ lyzed. Electronic structure methodologies were used within the frame of density functional theory using different theoretical methods. Results for the main absorption peak are in agreement with experimental results, and possible use in energy generation systems is dis‐ cussed. In addition to theoretical studies, the application of Raman spectroscopy, providing detailed information about the molecular structure of carotenoids, is examined in the light of a powerful method for mapping carotenoid abundance in cells and tissues. In many ap‐ plications, this technique is compatible with living organisms, providing highly specific mo‐ lecular structure information in intact cells and tissues with subcellular spatial resolution.

Antioxidant activities of carotenoids are discussed through several chapters. Reaction of βcarotene with nitrogen dioxide and nitric oxide, both in pure dioxane and dioxane/water solvent, is investigated by EPR and UV-Vis spectroscopy. An appropriate discussion on the importance of carotenoid compounds and their reactions in biological media, as well as the role and the possible reactions of nitroxide intermediates, is evaluated. The biosynthesis of carotenoids as a response on stress factors in order to investigate variables that regulate car‐ otenoid accumulation through manipulation of drought stress, light intensity and nutrient strength is analyzed. Finally, an overview of carotenoid effects in cardiovascular disease risk reduction, their unique biological activities in staving off atherosclerosis and endothelial dysfunction mitigation is given.

The second part of the book deals with biotechnological applications of carotenoids isolated from different sources. Carotenoids as natural pigments with important biological activities, such as antioxidant and provitamin A activity, have a great potential in the food, feed and pharmaceutical industries. They can be either extracted from plants and algae or synthesized by various microorganisms, including bacteria, yeasts, filamentous fungi and microalgae. Mi‐ crobial production, including the ability of microorganisms to use a wide variety of low-cost

substrates, the better control of cultivation and the minimized production time, has a number of advantages in carotenoid production. Under stress conditions such as high light exposure, nutrient starvation, change in oxygen partial pressure and high or low temperatures, microal‐ gal metabolism is altered, and photosynthetic activity may be reduced. In these conditions, photosynthetic electron transport is reduced, and the increase of intracellular reduction level may be associated to the formation of free radicals and species containing singlet oxygen. In order to prevent damage from photo-oxidation, microalgae are able to adopt strategies to con‐ trast these dangerous oxidant molecules. One of the most active mechanisms is to synthesize large amount of carotenoids, which can act as antioxidants. Among microalgae, *Haematococ‐ cus*, *Chlamydomonas*, *Chlorella* and *Dunaliella*, as well as diatoms and dinoflagellates, such as *Phaeodactylum* and *Isochrysis*, are able to synthesize large amount of carotenoids. The main primary carotenoids usually found are neoxanthin, violaxanthin, lutein and b-carotene. To preserve cells from oxidative damage, their production may be increased, while other carote‐ noids may be synthesized de novo. A yellow-pigmented soil bacterium *Corynebacterium gluta‐ micum* is also used as a workhorse of industrial biotechnology for more than 60 years. Carotenoids, the colourful representatives of terpenoids, are high-value compounds whose bio-based productions are on the rise. Since *C. glutamicum* is a natural producer of the rare C50 carotenoid decaprenoxanthin, this organism is well suited to establish terpenoid-overproduc‐ ing platform strains with the help of metabolic engineering strategies. The carotenogenic background of *C. glutamicum* and the metabolic engineering strategies for the generation of carotenoid-overproducing strains are depicted. In the same time, vitamin A deficiency is a preventable tragedy that affects millions of people, particularly in sub-Saharan Africa. During the last two decades, significant efforts have been made to identify sources of germplasm with high provitamin A carotenoids in an attempt to use them in development of cultivars with satisfactory nutritional characteristics and good agronomic performance. The cassava root is especially examined since a large proportion of people in sub-Saharan Africa relies on diets based on cassava as a source of calories. Furthermore, the presence of provitamin A carote‐ noids, mainly the β-carotene in pumpkins, yellow sweet potato and yellow sweet and bitter cassava, is also reported.

> **Prof. Dragan J. Cvetković and Prof. Goran S. Nikolić** University of Niš Faculty of Technology, Leskovac Serbia

**Carotenoids: Characterization and Activity**

substrates, the better control of cultivation and the minimized production time, has a number of advantages in carotenoid production. Under stress conditions such as high light exposure, nutrient starvation, change in oxygen partial pressure and high or low temperatures, microal‐ gal metabolism is altered, and photosynthetic activity may be reduced. In these conditions, photosynthetic electron transport is reduced, and the increase of intracellular reduction level may be associated to the formation of free radicals and species containing singlet oxygen. In order to prevent damage from photo-oxidation, microalgae are able to adopt strategies to con‐ trast these dangerous oxidant molecules. One of the most active mechanisms is to synthesize large amount of carotenoids, which can act as antioxidants. Among microalgae, *Haematococ‐ cus*, *Chlamydomonas*, *Chlorella* and *Dunaliella*, as well as diatoms and dinoflagellates, such as *Phaeodactylum* and *Isochrysis*, are able to synthesize large amount of carotenoids. The main primary carotenoids usually found are neoxanthin, violaxanthin, lutein and b-carotene. To preserve cells from oxidative damage, their production may be increased, while other carote‐ noids may be synthesized de novo. A yellow-pigmented soil bacterium *Corynebacterium gluta‐ micum* is also used as a workhorse of industrial biotechnology for more than 60 years. Carotenoids, the colourful representatives of terpenoids, are high-value compounds whose bio-based productions are on the rise. Since *C. glutamicum* is a natural producer of the rare C50 carotenoid decaprenoxanthin, this organism is well suited to establish terpenoid-overproduc‐ ing platform strains with the help of metabolic engineering strategies. The carotenogenic background of *C. glutamicum* and the metabolic engineering strategies for the generation of carotenoid-overproducing strains are depicted. In the same time, vitamin A deficiency is a preventable tragedy that affects millions of people, particularly in sub-Saharan Africa. During the last two decades, significant efforts have been made to identify sources of germplasm with high provitamin A carotenoids in an attempt to use them in development of cultivars with satisfactory nutritional characteristics and good agronomic performance. The cassava root is especially examined since a large proportion of people in sub-Saharan Africa relies on diets based on cassava as a source of calories. Furthermore, the presence of provitamin A carote‐ noids, mainly the β-carotene in pumpkins, yellow sweet potato and yellow sweet and bitter

**Prof. Dragan J. Cvetković and Prof. Goran S. Nikolić**

Faculty of Technology, Leskovac Serbia

University of Niš

cassava, is also reported.

VIII Preface

## **Characterisation of Carotenoids Involved in the Xanthophyll Cycle**

Paulina Kuczynska, Malgorzata Jemiola-Rzeminska and Kazimierz Strzalka

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67786

#### **Abstract**

Carotenoids are known for versatile roles they play in living organisms; however, their most pivotal function is involvement in scavenging reactive oxygen species (ROS) and photoprotection. In plant kingdom, an important photoprotective mechanism, referred to as the xanthophyll cycle, has been developed by photosynthetic organism to avoid excess light that might lead to photoinhibition and inactivation of photosystems and induce the formation of reactive oxygen species (ROS), resulting in photodamage and long-term changes in the cells caused by oxidative stress. Apart from high-light driven enzymatic conversion of violaxanthin (Viola) to zeaxanthin (Zea) that occurs mostly in higher plants, mosses and lichens, other less known types of the xanthophyll cycle have been hitherto described. The work is aimed at summarising the current knowledge on the pigments engaged in the xanthophyll cycles operating in various organisms.

**Keywords:** carotenoids, chromatography, diadinoxanthin, diatoms, diatoxanthin, *Phaeodactylum tricornutum*, xanthophyll cycle

#### **1. Introduction**

Carotenoids constitute a large group of pigments with over 700 compounds [1]. They comprise of carotenes and their oxygenated derivatives, xanthophylls. Carotenes are polyunsaturated hydrocarbons with 40 carbon atoms, while xanthophylls contain oxygen atoms, most frequently as hydroxyl and epoxide groups, which increase their polarity. Both groups of carotenoids act as accessory light-harvesting pigments or as quenchers of singlet oxygen and chlorophyll triplet states to provide protection against photooxidative damage [2]. The main photoprotective mechanism occurring in photosynthetic organisms is the xanthophyll

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

cycle—a process of enzymatic reactions of epoxidation and de-epoxidation of xanthophylls [3]. These cyclic conversions can proceed between several pigments including violaxanthin (Viola), antheraxanthin (Anth), zeaxanthin (Zea), diadinoxanthin (Diadino), diatoxanthin (Diato), lutein (Lut), lutein-epoxide (LutE) and oxidised but not epoxidised siphonaxanthin (Siph). Therefore, five types of xanthophyll cycles and additional non-specific cycle have been described [4] (**Figure 1**). The common factor in all of them is conversion of epoxidised xanthophylls to their de-epoxidised forms under strong light to dissipate of excess energy and epoxidation of de-epoxidised xanthophylls in low light or dark [5].

**Figure 1.** Xanthophyll cycles in photosynthetic organisms.

### **2. Carotenoids in the xanthophyll cycle**

cycle—a process of enzymatic reactions of epoxidation and de-epoxidation of xanthophylls [3]. These cyclic conversions can proceed between several pigments including violaxanthin (Viola), antheraxanthin (Anth), zeaxanthin (Zea), diadinoxanthin (Diadino), diatoxanthin (Diato), lutein (Lut), lutein-epoxide (LutE) and oxidised but not epoxidised siphonaxanthin (Siph). Therefore, five types of xanthophyll cycles and additional non-specific cycle have been described [4] (**Figure 1**). The common factor in all of them is conversion of epoxidised xanthophylls to their de-epoxidised forms under strong light to dissipate of excess energy and

epoxidation of de-epoxidised xanthophylls in low light or dark [5].

4 Carotenoids

**Figure 1.** Xanthophyll cycles in photosynthetic organisms.

#### **2.1. Violaxanthin, antheraxanthin and zeaxanthin**

Viola, Anth and Zea are engaged in the most common xanthophyll cycle, referred to as the violaxanthin cycle (VAZ cycle), see **Figure 1**. Di-epoxy Viola is de-epoxidised to epoxy-free Zea in two-step reaction catalysed by Viola de-epoxidase (VDE) with mono-epoxy Anth as an intermediate product. In absence of photosynthesis, VDE is localised in the thylakoid lumen as inactive monomer; however, it undergoes dimerization and binds to the membrane in an acidic pH caused by the light-driven transmembrane proton gradient [6–8]. Additionally, ascorbate as a donor of protons and monogalactosyldiacylglycerol (MGDG) as lipid-forming inverted hexagonal structures are essential for Viola de-epoxidation [9, 10]. In reverse reactions of the VAZ cycle, product of de-epoxidation—Zea—is epoxidised by Zea epoxidase (ZEP) to Viola, also via Anth. The reaction is observed in low light and in darkness due to lack of VDE activity in such conditions, but in higher plants, it can also proceed in high light [11, 12]. ZEP, localized in chloroplast stroma, is active in neutral pH and requires nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and molecular oxygen as co-substrates to epoxidise rings in Viola and then in Anth [11, 13].

The VAZ cycle occurs in higher plants, ferns, mosses, lichens and some groups of algae [3, 4]. However, in few species of algae, two specific xanthophyll cycles with Viola, Anth and Zea have been observed (**Figure 1**). In *Mantoniella squamata* (Chlorophyta), Viola is converted mainly to Anth, which is rapidly epoxidised back to Viola, and Zea occurs in low amount in these cells. It is a result of reduced affinity of VDE to Anth [14]. Second modification in the VAZ cycle has been described in two Rhodophyta species *Gracilaria gracilis* and *Gracilaria multipartita* in which Viola does not occur so de-epoxidation and epoxidation proceed only between Anth and Zea [15].

#### **2.2. Diadinoxanthin and diatoxanthin**

In several algal groups including diatoms, phaeophytes, dinophytes and haptophytes, mainly Diadino and Diato are involved in the xanthophyll cycle therefore named diadinoxanthin cycle (DD cycle). In addition, also the VAZ cycle can be observed in these organisms during strong light stress [16, 17], see **Figure 1**. In DD cycle, mono-epoxy Diadino is converted to epoxy-free Diato by a Diadino de-epoxidase (DDE or VDE), and the reverse reaction is catalysed by a Diato epoxidase (DEP or ZEP). Both enzymes have comparable properties and are able to convert Diadino/Diato as well as Viola/Anth/Zea [17]. De-epoxidation occurs in high light, in decreased pH in thylakoid lumen and in the presence of ascorbate, however, at lower concentration than that in plants [18, 19]. Unlike plants, in diatoms, epoxidation does not occur in high light since the proton gradient between thylakoid lumen and chloroplast stroma inhibits this reaction [20, 21]. It was reported that the rates of Diadino de-epoxidation and Diato epoxidation are several times higher than the conversions of Viola and Zea in plants and green algae [20].

#### **2.3. Lutein, lutein epoxide and siphonaxanthin**

Lut is an epoxy-free xanthophyll bound to antenna proteins and is essential for their stability in higher plants, while LutE occurs in significant amount in some species only [22], see **Figure 1**. Although the presence of LutE does not mean its involvement in cyclic conversions with Lut, the fully operative or truncated LutE cycle has been reported in several plant species [4]. In the first case, the initial LutE pool is fully recovered in the dark, which is not observed in truncated cycle. Both reactions of the LutE cycle are catalysed by VDE and ZEP, which are also engaged in the VAZ cycle but their rates are 2- or 3-fold lower, which might be a result of decreased affinity of enzymes to Lut and LutE or stronger binding of these pigments to antenna proteins [4].

Another xanthophyll cycle is operating in green algae *Caulerpa racemosa* in which interconversions between Siph and Lut have been reported [23]. During illumination, Siph is converted to Lut, and the reverse reaction proceeds in low light. Despite the mechanism of the Siph cycle is still unknown, this suggests a photoprotective role [22].

#### **3.** *In vitro* **assays of the xanthophyll cycle**

Studies on de-epoxidation and epoxidation of pigments involved in the xanthophyll cycle, the mechanism and conditions of these reactions, enzyme properties and factors regulating their efficiencies are usually performed *in vivo* by treatment of the organisms studied with stress or genetic modifications. Such experiments allow to observe holistic effects in natural or seminatural conditions but to analyse specific parameters of a single reaction, it is more convenient to perform them in fully controlled system.

Among two reaction types in the xanthophyll cycles, de-epoxidation of Viola has been extensively studied, and *in vitro* assay was developed [24]. The system comprises phosphatidylcholine liposomes with MGDG and Viola suspended in sodium citrate buffer with sodium ascorbate and VDE isolated from wheat. It was concluded that MGDG is an essential component of lipid membrane, which allows to bind VDE to the membrane which is necessary for its activity. Additionally, de-epoxidation of Viola to Anth seemed to be more sensitive to MGDG concentration than the second step of the reaction. Although this assay has been tested with Viola as a substrate, it is highly probable that Diadino can be also used for such assay. Considerable progress in these studies is the use of purified recombinant enzymes preferably from several species which allow for comparative analysis of their properties.

Epoxidation of Zea was investigated in semi-defined system [25]. Thylakoids of *npq1* mutant of *Arabidopsis thaliana* were the source of an active ZEP suspended in Hepes buffer with sorbitol, MgCl2 and ethylenediaminetetraacetic acid (EDTA). Zea was mixed with MGDG and incorporated into thylakoids by sonication, and also sodium ascorbate, FAD and NADPH were added. During 2 hours, the amount of Zea was reduced by 38%. However, isolated thylakoids contain not only an active enzyme but also additional compounds that could play an essential role in Zea epoxidation. Therefore, such semi-defined system may not be applicable to study of purified enzyme activity.

*In vitro* assay of Zea epoxidation has also been reported [26] using recombinant *Capsicum annuum* β-cyclohexenyl epoxidase isolated from *Escherichia coli*. Reaction was carried out in phosphate buffer with FAD, NADPH, Zea, MGDG, digalactosyldiacylglycerol (DGDG), ferredoxin, ferredoxin: NADP+ oxidoreductase and β-cyclohexenyl epoxidase. It has been reported that epoxidase is able to accept NADP only via reduced ferredoxin activity in the presence of NADPH, ferredoxin oxidoreductase and ferredoxin, and in these conditions, a significant conversion of Zea into Viola was observed.

#### **4. Production of the xanthophyll cycle carotenoids**

**2.3. Lutein, lutein epoxide and siphonaxanthin**

6 Carotenoids

is still unknown, this suggests a photoprotective role [22].

**3.** *In vitro* **assays of the xanthophyll cycle**

to perform them in fully controlled system.

to study of purified enzyme activity.

bitol, MgCl2

Lut is an epoxy-free xanthophyll bound to antenna proteins and is essential for their stability in higher plants, while LutE occurs in significant amount in some species only [22], see **Figure 1**. Although the presence of LutE does not mean its involvement in cyclic conversions with Lut, the fully operative or truncated LutE cycle has been reported in several plant species [4]. In the first case, the initial LutE pool is fully recovered in the dark, which is not observed in truncated cycle. Both reactions of the LutE cycle are catalysed by VDE and ZEP, which are also engaged in the VAZ cycle but their rates are 2- or 3-fold lower, which might be a result of decreased affinity of enzymes to Lut and LutE or stronger binding of these pigments to antenna proteins [4]. Another xanthophyll cycle is operating in green algae *Caulerpa racemosa* in which interconversions between Siph and Lut have been reported [23]. During illumination, Siph is converted to Lut, and the reverse reaction proceeds in low light. Despite the mechanism of the Siph cycle

Studies on de-epoxidation and epoxidation of pigments involved in the xanthophyll cycle, the mechanism and conditions of these reactions, enzyme properties and factors regulating their efficiencies are usually performed *in vivo* by treatment of the organisms studied with stress or genetic modifications. Such experiments allow to observe holistic effects in natural or seminatural conditions but to analyse specific parameters of a single reaction, it is more convenient

Among two reaction types in the xanthophyll cycles, de-epoxidation of Viola has been extensively studied, and *in vitro* assay was developed [24]. The system comprises phosphatidylcholine liposomes with MGDG and Viola suspended in sodium citrate buffer with sodium ascorbate and VDE isolated from wheat. It was concluded that MGDG is an essential component of lipid membrane, which allows to bind VDE to the membrane which is necessary for its activity. Additionally, de-epoxidation of Viola to Anth seemed to be more sensitive to MGDG concentration than the second step of the reaction. Although this assay has been tested with Viola as a substrate, it is highly probable that Diadino can be also used for such assay. Considerable progress in these studies is the use of purified recombinant enzymes preferably from several species which allow for comparative analysis of their properties.

Epoxidation of Zea was investigated in semi-defined system [25]. Thylakoids of *npq1* mutant of *Arabidopsis thaliana* were the source of an active ZEP suspended in Hepes buffer with sor-

incorporated into thylakoids by sonication, and also sodium ascorbate, FAD and NADPH were added. During 2 hours, the amount of Zea was reduced by 38%. However, isolated thylakoids contain not only an active enzyme but also additional compounds that could play an essential role in Zea epoxidation. Therefore, such semi-defined system may not be applicable

and ethylenediaminetetraacetic acid (EDTA). Zea was mixed with MGDG and

The production of carotenoids with well-known beneficial effects is of great importance for various industries including food, cosmetology, pharmacy and medicine and can be performed both by extraction from plans, algae, fungi, yeast and bacteria or through chemical synthesis [27, 28]. Both methods have some advantages and disadvantages but the choice is usually dependent on availability of extraction or synthesis procedure [29]. Due to many technologies of chemical synthesis have been developed and the cost of this production often is relatively low, the majority of carotenoids is obtained chemically. However, a consequence of that way is usually the production of stereoisomers mixture with reduced biological activity or even having side effects. Such disadvantages are not the case of natural pigments extraction; however, difficulties with yield and separation efficiency are present. Chemical and physical properties of various carotenoids are similar which results in limited separation capacity.

Most of the xanthophyll cycle carotenoids such as Anth, Zea, Viola, Lut and LutE are commercially available (data are given by international carotenoid society), but Diadino and Diato production has been developed only recently [30]. The procedure consists of total pigments extraction from marine diatom *Phaeodactylum tricornutum* followed by saponification and pigments partitioning and finally purification by open-column chromatography (**Figure 2**).

**Figure 2.** A process of isolation and purification of diadinoxanthin and diatoxanthin illustrating the successive steps comprising: diatoms cultivation under different light conditions, total pigments extraction, carotenoids separation by partitioning and purification of diadinoxanthin and diatoxanthin by open-column chromatography.

#### **4.1. Diatoms as a rich source of natural pigments**

Diatoms are microalgae widely distributed in marine and freshwater environment. They contain two types of photosynthetic pigments which are involved in light harvesting (chlorophyll *a*, chlorophyll *c* and fucoxanthin) and photoprotection (β-carotene, diadinoxanthin, diatoxanthin, zeaxanthin, antheraxanthin and violaxanthin). Three of above-mentioned pigments including Fuco, Diadino and Diato occur only in few algal groups; therefore, they might be considered as diatom-specific carotenoids. The quantitative composition of above pigments is dependent on growth conditions.

Pigment content is regulated mostly by light which increases the level of photoprotective carotenoids which are involved in the xanthophyll cycle [31]. Therefore, light stress can be used to produce the highest possible content of Diadino or Diato in the cells.

#### **4.2. Diatoms cultivation**

Diatoms *P. tricornutum* Bohlin, strain CCAP 1055/1, were cultivated in f/2-Si medium [32] made with seawater supplemented with inorganic nutrients and vitamin mixture. The temperature of 15°C was proved to be an optimal for increased level of both xanthophylls [30]. Cells were grown under white light of the intensity of 100 μmol photons m−2 s−1 in a 16/8 hours day/night photoperiod. Cells used to Diadino purification were collected after dark phase, while in the case of Diato, purification cells were illuminated with white light of the intensity of 1250 μmol photons m−2 s−1 for 2 hours. Several light conditions during diatoms growth have been tested, which resulted in high level of both xanthophylls (even 19% of Diadino or 17% of Diato); however, *cis* isomers of Diadino and Diato and also Zea, Anth and Viola were detected in addition in these conditions [30]. These additional xanthophylls have similar physical and chemical properties, including polarity, which has crucial impact on separation efficiency, and therefore, obtained Diadino and Diato were contaminated by them. Summarising, to obtain pure Diadino and Diato, diatoms should be cultivated under specific light conditions which result in increased biosynthesis of these pigments and simultaneously do not cause induction of the VAZ cycle and *cis* isomers formation. The pigment composition in *P. tricornutum* cultivated in such conditions is given in **Figure 3**, and the levels of Diadino or Diato in these samples were 10 and 6%, respectively.

#### **4.3. Pigment extraction and saponification**

An essential aspect of pigment quantification and its collecting for further purification is a step of extraction which requires selection of an appropriate method for a particular purpose. To analyse the amount of pigments in the cells, it is important to extract each of them with the same yield to avoid disproportion between their content. Since pigment composition of each organism varies and includes pigments with different polarity and hence the solubility in organic compounds, the extraction technique should be selected individually. On the other hand, to obtain a particular pigment, special attention should be paid to the extraction yield and to minimising formation of degradation products. In view of these issues, many methods of pigment extraction have been developed. The most applicable methods are solvents that

**4.1. Diatoms as a rich source of natural pigments**

dependent on growth conditions.

these samples were 10 and 6%, respectively.

**4.3. Pigment extraction and saponification**

**4.2. Diatoms cultivation**

8 Carotenoids

Diatoms are microalgae widely distributed in marine and freshwater environment. They contain two types of photosynthetic pigments which are involved in light harvesting (chlorophyll *a*, chlorophyll *c* and fucoxanthin) and photoprotection (β-carotene, diadinoxanthin, diatoxanthin, zeaxanthin, antheraxanthin and violaxanthin). Three of above-mentioned pigments including Fuco, Diadino and Diato occur only in few algal groups; therefore, they might be considered as diatom-specific carotenoids. The quantitative composition of above pigments is

Pigment content is regulated mostly by light which increases the level of photoprotective carotenoids which are involved in the xanthophyll cycle [31]. Therefore, light stress can be

Diatoms *P. tricornutum* Bohlin, strain CCAP 1055/1, were cultivated in f/2-Si medium [32] made with seawater supplemented with inorganic nutrients and vitamin mixture. The temperature of 15°C was proved to be an optimal for increased level of both xanthophylls [30]. Cells were grown under white light of the intensity of 100 μmol photons m−2 s−1 in a 16/8 hours day/night photoperiod. Cells used to Diadino purification were collected after dark phase, while in the case of Diato, purification cells were illuminated with white light of the intensity of 1250 μmol photons m−2 s−1 for 2 hours. Several light conditions during diatoms growth have been tested, which resulted in high level of both xanthophylls (even 19% of Diadino or 17% of Diato); however, *cis* isomers of Diadino and Diato and also Zea, Anth and Viola were detected in addition in these conditions [30]. These additional xanthophylls have similar physical and chemical properties, including polarity, which has crucial impact on separation efficiency, and therefore, obtained Diadino and Diato were contaminated by them. Summarising, to obtain pure Diadino and Diato, diatoms should be cultivated under specific light conditions which result in increased biosynthesis of these pigments and simultaneously do not cause induction of the VAZ cycle and *cis* isomers formation. The pigment composition in *P. tricornutum* cultivated in such conditions is given in **Figure 3**, and the levels of Diadino or Diato in

An essential aspect of pigment quantification and its collecting for further purification is a step of extraction which requires selection of an appropriate method for a particular purpose. To analyse the amount of pigments in the cells, it is important to extract each of them with the same yield to avoid disproportion between their content. Since pigment composition of each organism varies and includes pigments with different polarity and hence the solubility in organic compounds, the extraction technique should be selected individually. On the other hand, to obtain a particular pigment, special attention should be paid to the extraction yield and to minimising formation of degradation products. In view of these issues, many methods of pigment extraction have been developed. The most applicable methods are solvents that

used to produce the highest possible content of Diadino or Diato in the cells.

**Figure 3.** Pigment composition in *Phaeodactylum tricornutum* cultivated at 15°C under white light of the intensity of 100 μmol photons m−2 s−1 in a 16/8 hours day/night photoperiod. One day old cells were collected after dark phase (dark) or after illumination with white light of the intensity of 1250 μmol photons m−2 s−1 for 2 hours (light) [30].

comprise acetone, methanol and water in varying ratios, while the most common homogenisation techniques include grinding, sonication, heating and shaking [33].

The aim of pigment extraction from *P. tricornutum* in described protocol was to recover the highest amount of Diadino and Diato neglecting the efficiency of other pigments extraction. The composition of solvent mixture and the proportion between the solvent volume and the number of cells were found to be essential for the yield of this step. Pigments were extracted from frozen cells, which were earlier harvested by centrifugation of liquid diatoms culture, using a medium composed of methanol, 0.2-M ammonium acetate and ethyl acetate (81:9:10, v/v/v) in a ratio of 10 mL per 2 × 109 cells [30].

Saponification and liquid-liquid partitioning are widely practised techniques of carotenoids purification, which allows to remove chlorophylls and lipids—compounds abundantly present in pigment extracts. In general, saponification is performed by adding methanol or ethanol and aqueous solution of KOH in concentration of approx. 5–10% into pigment extract and then incubation of the mixture in darkness for several hours. Numerous modifications of this method have been described [34]. Before performing this step, it should be considered whether the carotenoid is stable in alkali because some of them are not, that is, astaxanthin, fucoxanthin and peridinin [35, 36].

After saponification, chlorophylls and carotenoids are separated through the partitioning with mixture of solvents of varied polarity. Lipophilic carotenoids dissolve in the upper phase, which usually contains petroleum, while chlorophylls dissolve in methanol or ethanol in the lower phase. However, the presence of epoxy, hydroxy and other groups in pigments cause decrease in polarity of chlorophylls, while the polarity of some carotenoids is enhanced [34]. Therefore, the composition of mixture for partitioning should be more complex and specific for the extract.

In the case of Diadino and Diato purification, a saponification was performed through addition of ethanol and 60% aqueous solution of KOH (10:1, v/v) and stirring the mixture in darkness overnight in a cold room. To separate phases following solvents were added consecutively: hexane and diethyl ether (1:1, v/v), extraction petroleum and water (4:1:2, v/v/v). Then, to remove alkali, collected carotenoid fraction was washed three times with one volume of distilled water. The efficiency of a single partitioning was more than 90% so repetitions which resulted in increasing of Fuco level were not advisable. After chlorophylls and carotenoids separation, the level of Diadino or Diato in extract increased to approx. 40 or 20%, respectively, and mixtures contained also β,β-Car, cryptoxanthin and fucoxanthin derivatives. This preliminary purification step was essential for further purification by open-column chromatography.

#### **4.4. Diadinoxanthin and diatoxanthin purification**

The final step of Diadino and Diato purification was separation of carotenoids present in the mixture after saponification and partitioning by open-column chromatography. This technique is commonly used in preparative scale and therefore is applicable for industrial use. Development of appropriate chromatographic conditions consists mainly in selection of the solid phase and composition of eluents, but also in the amount of pigment mixture applied onto the column, the volume of eluents, pressure applied to the column.

Since xanthophylls are susceptible to isomerization under acidic conditions, a modified silica gel, which is chemically converted into a basic form, was used. To separate and elute pigments, the mixtures of hexane and acetone were used. Hexane:acetone (90:10, v/v) mixture allows for β,β-Car elution. Second mixture (80:20, v/v) removes cryptoxanthin epoxide from the column. Continued use of this mixture allows to separate and collect Diato. Then, third mixture (70:30, v/v) allows to collect Diadino. To remove Fuco derivatives from the column and to reuse the silica gel, pure acetone was used.

Described procedure allowed to obtain all-*trans* Diadino and all-*trans* Diato of a purity of 99.9%, and the final efficiency was estimated to be 63 and 73% for Diadino and Diato, respectively. Due to the use of popular reagents, simplicity of the procedure, possibility of diatoms cultivation in bioreactors and estimated low costs, the method is widely accessible and might be performed both in analytical and preparative scale.

#### **5. Interaction of the xanthophyll cycle carotenoids with membranes**

It has been in 1974 when the hypothesis was formed postulating that carotenoids present in some prokaryotic membranes play the similar role as cholesterol in animal membranes [37]. Since then, numerous studies have been carried out to get a deep insight into the molecular mechanism of carotenoid-membrane interactions. A large body of research has been performed using liposomes as model systems of biological membranes, mostly due to their simplicity, stability, and well-characterised properties.

#### **5.1. Localisation within model membranes**

In the case of Diadino and Diato purification, a saponification was performed through addition of ethanol and 60% aqueous solution of KOH (10:1, v/v) and stirring the mixture in darkness overnight in a cold room. To separate phases following solvents were added consecutively: hexane and diethyl ether (1:1, v/v), extraction petroleum and water (4:1:2, v/v/v). Then, to remove alkali, collected carotenoid fraction was washed three times with one volume of distilled water. The efficiency of a single partitioning was more than 90% so repetitions which resulted in increasing of Fuco level were not advisable. After chlorophylls and carotenoids separation, the level of Diadino or Diato in extract increased to approx. 40 or 20%, respectively, and mixtures contained also β,β-Car, cryptoxanthin and fucoxanthin derivatives. This preliminary purification step was essential for further purification by open-column

The final step of Diadino and Diato purification was separation of carotenoids present in the mixture after saponification and partitioning by open-column chromatography. This technique is commonly used in preparative scale and therefore is applicable for industrial use. Development of appropriate chromatographic conditions consists mainly in selection of the solid phase and composition of eluents, but also in the amount of pigment mixture applied

Since xanthophylls are susceptible to isomerization under acidic conditions, a modified silica gel, which is chemically converted into a basic form, was used. To separate and elute pigments, the mixtures of hexane and acetone were used. Hexane:acetone (90:10, v/v) mixture allows for β,β-Car elution. Second mixture (80:20, v/v) removes cryptoxanthin epoxide from the column. Continued use of this mixture allows to separate and collect Diato. Then, third mixture (70:30, v/v) allows to collect Diadino. To remove Fuco derivatives from the column

Described procedure allowed to obtain all-*trans* Diadino and all-*trans* Diato of a purity of 99.9%, and the final efficiency was estimated to be 63 and 73% for Diadino and Diato, respectively. Due to the use of popular reagents, simplicity of the procedure, possibility of diatoms cultivation in bioreactors and estimated low costs, the method is widely accessible and might

**5. Interaction of the xanthophyll cycle carotenoids with membranes**

It has been in 1974 when the hypothesis was formed postulating that carotenoids present in some prokaryotic membranes play the similar role as cholesterol in animal membranes [37]. Since then, numerous studies have been carried out to get a deep insight into the molecular mechanism of carotenoid-membrane interactions. A large body of research has been performed using liposomes as model systems of biological membranes, mostly due to their

onto the column, the volume of eluents, pressure applied to the column.

chromatography.

10 Carotenoids

**4.4. Diadinoxanthin and diatoxanthin purification**

and to reuse the silica gel, pure acetone was used.

be performed both in analytical and preparative scale.

simplicity, stability, and well-characterised properties.

The issue of localisation and orientation of carotenoids in the membrane has been addressed mainly by analysing the position of the maxima in the UV-VIS spectral region of these pigments upon their incorporation into lipids matrix as well as by linear dichroism and X-ray diffraction [38]. Generally, xanthophylls are oriented perpendicularly to the membrane surface with their hydrophilic groups anchored in the two opposite polar regions of the lipid bilayer. Accordingly, to minimalise the energy of the system, zeaxanthin molecule with its two hydroxyl groups located at 3 and 3′ position is thought to span the membrane. Alternatively, all polar groups of a xanthophyll molecule can remain with contact with the same polar headgroup region of the membrane. Such orientation has been proposed for xanthophylls in a conformation *cis* [39]. Interestingly, lutein characterised by the rotational freedom around the 6′–7′ single bond is believed to adopt 2 orthogonal orientations: one roughly vertical and the other horizontal to the lipid bilayer [40, 41].

#### **5.2. The influence on the physical-chemical properties of membranes**

The orientation of carotenoids within lipid bilayer enables them to interact with alkyl chains of lipids via van der Waals interactions, which results in modifications of structural and dynamical properties of membranes. Among experimental methods employed to investigate the effect of carotenoids on the properties of biomembranes are electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC) as well as fluorescence and permeability measurements. An excellent overview of this research is presented in [42]. Considering xanthophylls, Viola, Zea and Lut were reported as modulators of membrane fluidity, increasing it in the ordered phase of the phospholipid bilayer and acting conversely in the liquid crystalline phase [43, 44]. Moreover, in the presence of xanthophylls, an increase of the penetration barrier to molecular oxygen into the hydrophobic region of membrane was observed [45]. In general, polar carotenoids were capable of influencing the thermotropic phase behaviour of phospholipid membranes. As a result, main phase transition (Pβ′→Lα) was shifted to lower temperature in a concentration dependent manner accompanying by a decreased cooperativity and the molar heat capacity of the Pβ′→Lα transition [46]. Lutein and zeaxanthin affected alkyl chains of lipid bilayers by restriction of molecular motions of both CH2 and the terminal CH3 groups [47, 48]. Furthermore, xanthophylls, especially Lut, were responsible for an increase in the thickness of lipid membranes composed of lecithins with myristoyl and palmitoyl moieties [41, 49].

Recently, experiments in atomic force microscope (AFM) showed that the presence of Viola or Zea embedded in the liposomes at a concentration up to 1 mol% does not significantly affect the morphology of the vesicles. However, adhesive forces were 10 times higher for dipalmitoylphosphatidylcholine (DPPC) membranes enriched in Zea, than those observed for an untreated system [50].

Finally, it is worth mentioning that unlike the xanthophylls of the VAZ cycle, diatom-specific carotenoids including Diadino and Diato have hitherto been much less studied in terms of the effect they can exert on the structural and dynamic properties of biomembranes. Interestingly, based on measurements carried out most recently in our laboratory by use of DSC technique and fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene in phospholipid liposomes (manuscript in preparation), it seems that Diadino and Diato affect the lipid bilayer much stronger than Viola and Zea.

#### **6. Conclusion**

Along with the characterisation of physiological roles, various carotenoids play being involved in the xanthophyll cycle, *in vitro* assays of both epoxidation and de-epoxidation reactions enable to study the molecular mechanism of the cycle. Moreover, it is interesting to address the question of effect carotenoids have on biological membranes, as it helps to put some light on their antioxidant activity. This in turn seems to play a key role in terms of nutrition and human health. In view of this, the issue of isolation and purification of diatom-specific xanthophylls, yet less described in literature seems to be of great importance. Recently, developed method of all-*trans* diadinoxanthin (Diadino) and all-*trans* diatoxanthin (Diato) purification from *P. tricornutum* comprises four-step procedure and is dedicated to both analytical and preparative scale.

Particular attention is paid to natural carotenoids that apart from the photoprotective function in photosynthetic organism have been recognised as exhibiting beneficial activities for humans and animals and used for commercial and industrial applications. Diatoms seem to be a promising source of unique bioactive compounds, with diadinoxanthin and diatoxanthin as representatives of the xanthophyll cycle pigments. Given that until now neither Diadino nor Diato were commercially available in amounts greater than those used as standards in high-performance liquid chromatography (HPLC), the efficiency of the described purification procedure reaching up to 73% makes the method economically feasible.

#### **Acknowledgements**

The Jagiellonian University is a partner of the Leading National Research Center (KNOW) supported by the Ministry of Science and Higher Education. HPLC analysis was carried out with the equipment purchased, thanks to the financial support of the European Regional Development Fund in the framework of the Polish Innovation Economy Operational Program POIG.02.01.00-12-167/08. A method of diadinoxanthin and diatoxanthin purification has been reported as patent applications P.412178 and P.412177 (Kuczynska & Jemiola-Rzeminska, 2015) and PCT/PL2016/000047 (Kuczynska & Jemiola-Rzeminska, 2016).

#### **Author details**

Paulina Kuczynska1 , Malgorzata Jemiola-Rzeminska1,2 and Kazimierz Strzalka1,2\*

Address all Correspondence to : kazimierzstrzalka@gmail.com

1 Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland

2 Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland

#### **References**

**6. Conclusion**

12 Carotenoids

preparative scale.

**Acknowledgements**

**Author details**

Paulina Kuczynska1

Along with the characterisation of physiological roles, various carotenoids play being involved in the xanthophyll cycle, *in vitro* assays of both epoxidation and de-epoxidation reactions enable to study the molecular mechanism of the cycle. Moreover, it is interesting to address the question of effect carotenoids have on biological membranes, as it helps to put some light on their antioxidant activity. This in turn seems to play a key role in terms of nutrition and human health. In view of this, the issue of isolation and purification of diatom-specific xanthophylls, yet less described in literature seems to be of great importance. Recently, developed method of all-*trans* diadinoxanthin (Diadino) and all-*trans* diatoxanthin (Diato) purification from *P. tricornutum* comprises four-step procedure and is dedicated to both analytical and

Particular attention is paid to natural carotenoids that apart from the photoprotective function in photosynthetic organism have been recognised as exhibiting beneficial activities for humans and animals and used for commercial and industrial applications. Diatoms seem to be a promising source of unique bioactive compounds, with diadinoxanthin and diatoxanthin as representatives of the xanthophyll cycle pigments. Given that until now neither Diadino nor Diato were commercially available in amounts greater than those used as standards in high-performance liquid chromatography (HPLC), the efficiency of the described purification

The Jagiellonian University is a partner of the Leading National Research Center (KNOW) supported by the Ministry of Science and Higher Education. HPLC analysis was carried out with the equipment purchased, thanks to the financial support of the European Regional Development Fund in the framework of the Polish Innovation Economy Operational Program POIG.02.01.00-12-167/08. A method of diadinoxanthin and diatoxanthin purification has been reported as patent applications P.412178 and P.412177 (Kuczynska & Jemiola-Rzeminska,

, Malgorzata Jemiola-Rzeminska1,2 and Kazimierz Strzalka1,2\*

1 Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and

2 Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland

procedure reaching up to 73% makes the method economically feasible.

2015) and PCT/PL2016/000047 (Kuczynska & Jemiola-Rzeminska, 2016).

Address all Correspondence to : kazimierzstrzalka@gmail.com

Biotechnology, Jagiellonian University, Krakow, Poland


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#### **Electronic Structure of Carotenoids in Natural and Artificial Photosynthesis Electronic Structure of Carotenoids in Natural and Artificial Photosynthesis**

Manuel Flores-Hidalgo, Francisco Torres-Rivas, Jesus Monzon-Bensojo, Miguel Escobedo-Bretado, Daniel Glossman‐Mitnik and Diana Barraza‐Jimenez Rivas, Jesus Monzon-Bensojo, Miguel Escobedo-Bretado, Daniel Glossman‐Mitnik and Diana Barraza‐Jimenez

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

Manuel Flores-Hidalgo, Francisco Torres-

http://dx.doi.org/10.5772/67636

#### **Abstract**

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16 Carotenoids

S0005-2736(02)00688-0

s0005-2736(96)00152-6

This chapter is about a theoretical study applied to six carotenoids present in vegeta‐ bles containing carotenes and xanthophylls. Electronic properties are analyzed such as energy in frontier orbitals and the first molecular orbitals to work in the UV‐Vis absorp‐ tion spectroscopy. Electronic structure methodologies were used within the frame of the density functional theory (DFT) using the theoretical methods B3LYP/6‐31G(d)// B3LYP/6‐31G+(d,p) for ground states and B3LYP/6‐31G(d)//CAM‐B3LYP/6‐31G+(d,p) for excited states. Results for the main absorption peak are in agreement with experimental results with a difference between zeaxanthin and violaxanthin results of 0.1 eV, approxi‐ mately. The UV‐Vis absorption spectra obtained for carotenoids are in good agreement with the experimental results. The possible use in energy generation systems is discussed for these systems. Diade chlorophyllide *a*‐zeaxanthin was formed, and calculation results predicted energy transfer for these photosynthetic systems.

**Keywords:** DFT, artificial photosynthesis, carotenoids, xanthophylls, diade chl *a*‐zx

#### **1. Introduction**

Natural photosynthesis requires the participation of chlorophyll *a* and accessory pigments. Carotenoids are the more commonly used accessory pigments. In photosynthesis, plants and organisms convert light energy into chemical energy that can later be released to fuel organ‐ isms' activities; therefore, it is an energy transformation. It is one of the principal processes in

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

nature and it is fundamental for life existence. Solar energy conversion to chemical fuels using green methodologies may be approached with photosynthesis [1] since this natural process is the main user of solar energy in our planet. This natural process uses effectively the largest exploitable renewable energy resource. Solar energy provides our planet with more energy per hour than the total energy consumed by human activities in 1 year. In other words, direct conversion of solar energy into chemical fuels represents an optimal approach to address the globally growing energy demand in a sustainable way [1–2]. Photosynthesis if reproduced may address a lot of our environmental problems derived from energy conversion.

In this way, mimicking photosynthesis has become a subject of great interest in the scientific world, and this global research trend has given origin to a recently created term, artificial photosynthesis [1–3]. This concept refers to a chemical process that replicates the natural pro‐ cess of photosynthesis; it mainly studies the process to convert sunlight, water, and carbon dioxide into carbohydrates and oxygen. This process aims to emulate natural ways by using man‐made devices to convert and store solar energy using chemical fuels as feedstock [3]. To absorb the visible light part of the solar radiation (350–700 nm), green plants use chlorophyll *a* as the main light absorber along with a number of accessory pigments such as xanthophylls, carotenoids, and a modified form of chlorophyll, called chlorophyll *b*. Chlorophyll *a* absorbs in the blue‐violet, orange‐red spectral regions while the accessory pigments cover the inter‐ mediate yellow‐green‐orange part [3–4].

Carotenoids are important in photosynthesis, and with the mimicking of this natural pro‐ cess, they have raised their importance due to the fundamental need for renewable energy sources such as artificial photosynthesis [5]. There are other fields in which carotenoids are important as well, such as food or health. Fruits and vegetables are the principal sources of carotenoids and play an important role in diet due to vitamin A activity [5–6]. In addition to this, carotenoids are also important for antioxidant activity, intercellular communication, and immune system activity [6–8]. Epidemiological studies reported that the consumption of diets rich in carotenoids is associated with a lower incidence of cancer, cardiovascular diseases, age‐related macular degeneration, and cataract formation [9–10]. Deficiency of carotenoids results in clinical signs of conjunctiva and corneal aberrations, including xerophthalmia, night blindness, corneal ulceration, scarring, and resultant irreversible blindness [11].

Carotenoids are classified in carotenes and xanthophylls. Carotenes contain only a parent hydrocarbon chain without any functional group, such as α‐carotene, β‐carotene, and lyco‐ pene. Xanthophylls contain oxygen as the functional group, including lutein and zeaxanthin [12]. In plant tissues, carotenoids are typically located in chromoplasts (specific organelles) inside cells. Substructures composed of lipids, proteins, and carotenoids are being synthesized during chromoplast development, and depending on their morphology, they can be classi‐ fied as crystalline, globular, fibrillar, membranous, or tubular‐type chromoplasts [13–15]. Carotenoids are embedded in a complex structural organization. Carotenes and xanthophylls contain more than seven conjugate bonds that enable visible light absorption and from here, they have the capability to participate in the photosynthesis [6]. For light energy to be trans‐ formed into chemical energy, the electronic structure is fundamental in understanding how natural photosynthesis occurs and how this process can be associated with clean energy generation through artificial photosynthesis. Due to their chemical structure, carotenoids are tetraterpenoids so they have a long chain of conjugated double bonds; for this reason, these micronutrients are highly lipophilic [13, 16–18].

There are different determination methods to find out the basic chemical structure of carot‐ enoids. Their structure is based on eight isoprenoid units with a conjugated double‐bond system, which makes isomeric forms very common [19]. In addition, double bonds in the carbon chain make carotenoids susceptible to reactions, such as oxidation and isomerization (cis‐trans), especially due to light, heat, acids, and oxygen [20]. Cyclization, hydrogenation, dehydrogenation, or additions of lateral groups, among others, are some modifications that lead to an extremely complex variety of compounds with common structures [19].

Moreover, carotenoids can be found in nature both in their free form and also in a more stable, esterified form with fatty acids [21]. The high variability in their chemical structure and their poor stability greatly contribute to the difficulty of carotenoid analysis. Also, there is a lack of commercially available standards and other important reasons [22] that make it difficult to have more analytical methods to identify and to measure carotenoids in real samples [21–23].

Another analytic alternative is related to theoretical methods. There is a wide chart of choices to model and simulate these compounds that range from macro‐, micro‐, and atomic to sub‐ atomic methodologies. In this work, we use density functional theory (DFT) to learn more about carotenoids. We use DFT to model and simulate carotenoids' ground states as well as excited states and analyze their electronic structure. Fundamentally, carotenoids have a strong absorption of visible light in the blue and green region of the solar spectrum. Most carotenoids found in photosynthetic organisms have the characteristic colors yellow, orange, and red. The lowest excited single state in most pigment molecules represents the lowest energy, which optically allows a one‐photon transition from the ground state [13]. This chap‐ ter provides numerical data to parameterize some of the more important properties of carot‐ enoids. It provides a good insight about their important role in both natural and artificial photosynthesis, and since these results relate to its more basic features, it can be useful for other applications as well, such as in the food and health industries.

#### **2. Computational methods and details**

nature and it is fundamental for life existence. Solar energy conversion to chemical fuels using green methodologies may be approached with photosynthesis [1] since this natural process is the main user of solar energy in our planet. This natural process uses effectively the largest exploitable renewable energy resource. Solar energy provides our planet with more energy per hour than the total energy consumed by human activities in 1 year. In other words, direct conversion of solar energy into chemical fuels represents an optimal approach to address the globally growing energy demand in a sustainable way [1–2]. Photosynthesis if reproduced

In this way, mimicking photosynthesis has become a subject of great interest in the scientific world, and this global research trend has given origin to a recently created term, artificial photosynthesis [1–3]. This concept refers to a chemical process that replicates the natural pro‐ cess of photosynthesis; it mainly studies the process to convert sunlight, water, and carbon dioxide into carbohydrates and oxygen. This process aims to emulate natural ways by using man‐made devices to convert and store solar energy using chemical fuels as feedstock [3]. To absorb the visible light part of the solar radiation (350–700 nm), green plants use chlorophyll *a* as the main light absorber along with a number of accessory pigments such as xanthophylls, carotenoids, and a modified form of chlorophyll, called chlorophyll *b*. Chlorophyll *a* absorbs in the blue‐violet, orange‐red spectral regions while the accessory pigments cover the inter‐

Carotenoids are important in photosynthesis, and with the mimicking of this natural pro‐ cess, they have raised their importance due to the fundamental need for renewable energy sources such as artificial photosynthesis [5]. There are other fields in which carotenoids are important as well, such as food or health. Fruits and vegetables are the principal sources of carotenoids and play an important role in diet due to vitamin A activity [5–6]. In addition to this, carotenoids are also important for antioxidant activity, intercellular communication, and immune system activity [6–8]. Epidemiological studies reported that the consumption of diets rich in carotenoids is associated with a lower incidence of cancer, cardiovascular diseases, age‐related macular degeneration, and cataract formation [9–10]. Deficiency of carotenoids results in clinical signs of conjunctiva and corneal aberrations, including xerophthalmia, night

Carotenoids are classified in carotenes and xanthophylls. Carotenes contain only a parent hydrocarbon chain without any functional group, such as α‐carotene, β‐carotene, and lyco‐ pene. Xanthophylls contain oxygen as the functional group, including lutein and zeaxanthin [12]. In plant tissues, carotenoids are typically located in chromoplasts (specific organelles) inside cells. Substructures composed of lipids, proteins, and carotenoids are being synthesized during chromoplast development, and depending on their morphology, they can be classi‐ fied as crystalline, globular, fibrillar, membranous, or tubular‐type chromoplasts [13–15]. Carotenoids are embedded in a complex structural organization. Carotenes and xanthophylls contain more than seven conjugate bonds that enable visible light absorption and from here, they have the capability to participate in the photosynthesis [6]. For light energy to be trans‐ formed into chemical energy, the electronic structure is fundamental in understanding how natural photosynthesis occurs and how this process can be associated with clean energy

blindness, corneal ulceration, scarring, and resultant irreversible blindness [11].

may address a lot of our environmental problems derived from energy conversion.

mediate yellow‐green‐orange part [3–4].

18 Carotenoids

All calculations were carried out employing Gaussian 09 program suite [24]. This chapter was developed with computational calculations employing electronic structure methods using density functional theory (DFT). Then, a vibrational frequencies' calculation was car‐ ried out to corroborate a global minimum. These calculations, geometry optimization, and vibrational frequencies were performed in the gaseous phase, using a methanol‐like solvent. Molecular orbitals for the different carotenoids were obtained with energy calculations using the B3LYP/6‐31G(d)//B3LYP/6‐31+G(d,p) theoretical method [25]. Excited states in the gaseous phase were carried out for all six carotenoid variants within this work and in the solvent phase for xanthophylls molecules. These later calculations allowed us to obtain molecular orbitals and absorption states. The same set of calculations used in carotenoids was applied to chlorophyll *a* in the gaseous phase, with the objective of forming one of the main diades that has been found as a participant in natural photosynthesis. CAM‐B3LYP [26] functional was used in all excited states' calculations using the time‐dependent density functional theory (TDDFT). Molecular orbitals data was processed to obtain orbitals' diagrams and the absorp‐ tion spectra with Chemissan code [27].

#### **3. Results and discussions**

In this section, calculations results are displayed and analyzed. The first set of results contains ground states data; first, the geometric structures are displayed and next the energy results are displayed, including molecular orbitals, energy gap, and relevant chemical properties. In the second set of results are included excited states data with their corresponding molecular orbital diagrams and absorption spectra based on TDDFT calculations.

#### **3.1. Carotenoid structures**

Carotenoids included in this work are displayed in **Figure 1**. Beta‐carotene and lycopene are carotenes with the characteristic of belonging to the hydrocarbons group, which means that their structure includes only carbon and hydrogen atoms. The rest are four carotenoids that belong to the xanthophylls which characterize themselves by containing within their struc‐ ture carbon and hydrogen with oxygen atoms bonded to the six‐carbon ring.

**Figure 1.** Geometry optimization of carotenoid structures: (a) Beta‐carotene, (b) lycopene, (c) lutein, (d) neoxanthin, (e) violaxanthin, (f) zeaxanthin.

#### *3.1.1. Carotenoids ground states*

to chlorophyll *a* in the gaseous phase, with the objective of forming one of the main diades that has been found as a participant in natural photosynthesis. CAM‐B3LYP [26] functional was used in all excited states' calculations using the time‐dependent density functional theory (TDDFT). Molecular orbitals data was processed to obtain orbitals' diagrams and the absorp‐

In this section, calculations results are displayed and analyzed. The first set of results contains ground states data; first, the geometric structures are displayed and next the energy results are displayed, including molecular orbitals, energy gap, and relevant chemical properties. In the second set of results are included excited states data with their corresponding molecular

Carotenoids included in this work are displayed in **Figure 1**. Beta‐carotene and lycopene are carotenes with the characteristic of belonging to the hydrocarbons group, which means that their structure includes only carbon and hydrogen atoms. The rest are four carotenoids that belong to the xanthophylls which characterize themselves by containing within their struc‐

**Figure 1.** Geometry optimization of carotenoid structures: (a) Beta‐carotene, (b) lycopene, (c) lutein, (d) neoxanthin, (e)

orbital diagrams and absorption spectra based on TDDFT calculations.

ture carbon and hydrogen with oxygen atoms bonded to the six‐carbon ring.

tion spectra with Chemissan code [27].

**3. Results and discussions**

20 Carotenoids

**3.1. Carotenoid structures**

violaxanthin, (f) zeaxanthin.

The ground states energy results allow us to obtain data related to electronic conduction capa‐ bilities of the selected molecules in their ground states. For these calculations, the interpreta‐ tion scheme of the difference between the Highest Occupied Molecular Orbital (HOMO) and the Lowest Unoccupied Molecular Orbital (LUMO) was applied, where HOMO and LUMO are frontier orbitals located in the valence band and in the conduction band, respectively. Resulting values for these orbitals relate to ionization energy in the case of HOMO, which means that a lower ionization energy corresponds to a higher HOMO energy. In fact, a lower energy for LUMO is associated with a higher electronic affinity. These effects were explained in more detail in our published work [28] and can be studied by further reading **Table 1** as shown ahead in this section.

Frontier molecular orbitals' values are shown in **Table 1**, and **Figure 2** displays molecular orbitals obtained for the six selected carotenoid structures in their gaseous phase that will be discussed next. The diagram is divided into two sections: the first belongs to carotenes and the second to xanthophylls. The HOMO‐LUMO energy gap is obtained by calculating the difference between frontier orbitals' energy values. Beta‐carotene and lycopene energy gap results have a small difference. Then, it is observed that these compounds present a similar trend in their HOMO orbitals. Based on these results, xanthophylls have a higher ionization energy which derives in the capability present in one of these variants to form a diade inte‐ grated system configured by chlorophyll *a*‐xanthophyll.


\*Absolute values.

**Table 1.** Global chemical reactivity parameters (IP and EA), energy levels (HOMO/LUMO), and energy gap (HOMO‐ LUMO) in the gaseous phase, and methanol as a solvent for all B3LYP/6‐31+G(d,p)‐analyzed carotenoids.

**Figure 2.** Ground states molecular orbitals for selected carotenoid structures. The calculation was carried out in the gaseous phase using DFT with the B3LYP/6‐31+G(d,p) theoretical method.

From xanthophylls displayed in the molecular orbitals diagram, one can see that zeaxanthin has the narrower HOMO‐LUMO energy gap. A narrow HOMO‐LUMO energy difference benefits energy transfer process.

Photosynthesis requires that plants containing chlorophyll *a* capture light to transform it into chemical energy. The role that light plays consists of producing a luminous excitation that impacts an electron, allowing this charged particle to jump from a ground state of inferior energy to an excited state with higher energy and later return to the lower energy state. This process is known as excited states in the electronic structure.

In photosynthesis, carotenoids play the role of the aforementioned accessory pigments. Excited states analysis explains xanthophylls' performance as pigments that are part of the photosynthetic process.

**Figure 3** displays a molecular orbital diagram for excited states corresponding to all mol‐ ecules within our chapter. This diagram, as occurred for ground states, provides information discussed previously in this section.

#### *3.1.2. Carotenoids excited states molecular orbitals*

From xanthophylls displayed in the molecular orbitals diagram, one can see that zeaxanthin has the narrower HOMO‐LUMO energy gap. A narrow HOMO‐LUMO energy difference

**Figure 2.** Ground states molecular orbitals for selected carotenoid structures. The calculation was carried out in the

Photosynthesis requires that plants containing chlorophyll *a* capture light to transform it into chemical energy. The role that light plays consists of producing a luminous excitation that impacts an electron, allowing this charged particle to jump from a ground state of inferior energy to an excited state with higher energy and later return to the lower energy state. This

In photosynthesis, carotenoids play the role of the aforementioned accessory pigments. Excited states analysis explains xanthophylls' performance as pigments that are part of the

**Figure 3** displays a molecular orbital diagram for excited states corresponding to all mol‐ ecules within our chapter. This diagram, as occurred for ground states, provides information

benefits energy transfer process.

22 Carotenoids

photosynthetic process.

discussed previously in this section.

process is known as excited states in the electronic structure.

gaseous phase using DFT with the B3LYP/6‐31+G(d,p) theoretical method.

As occurs in ground states, in excited states, zeaxanthin is the molecule with the narrower energy gap from the xanthophylls‐type carotenoids which means this is the molecule that can be excited more easily. Other data important to consider in excited states analysis is the UV‐Vis absorption spectra. Absorption spectra enable us to identify the wavelength in which a pigment absorbs sunlight and thus locate the reference electromagnetic radiation working range and the visible light required to favor photosynthesis.

Now, depending on the solvent used, sunlight absorption may have a benefit. In general, the absorption extends in larger wavelength when the solvent is employed if compared to the absorption results in the gaseous phase. **Figure 4** displays the diagram of excited states molecular orbitals with the use of solvents, which in this case is methanol. This excited states calculation was carried out only for xanthophylls because zeaxanthin is the accessory pigment used to form the diade with chlorophyll *a*, considering that the latter is the main photosyn‐ thetic pigment. Molecular orbitals shown so far, both in the gaseous phase and in the solvent phase correspond to those involved in the main absorption peak found.

**Figure 4.** Excited states molecular orbitals for selected carotenoid structures. Calculations were carried out in methanol as a solvent with TDDFT using the CAM‐B3LYP/6‐31+G(d,p) theoretical method.

If one observes the molecular orbitals diagram corresponding to the molecules in methanol as a solvent, one can see that zeaxanthin is the molecule with the narrower energy gap as occurred in the gaseous phase calculations. Another important observation is how violaxan‐ thin keeps the same values in both calculations, in the gaseous phase and in the solvent phase.

#### *3.1.3. Carotenoids excited states UV‐Vis spectra*

Absorption spectra for carotenoids‐type beta‐carotene and lycopene for the gaseous phase are shown in **Figure 5**. In this figure, a high coincidence in the absorption maximum peak between beta‐carotene with 472 nm and lycopene with 476 nm is observed. According to these calculations, the maximum absorption peak for beta‐carotene is surpassed by 20 nm. Meanwhile for lycopene, a better approximation with the experimental value of 470 nm is obtained.

**Figure 5.** UV‐Vis absorption spectra for carotenoids‐type beta‐carotene and lycopene. Calculations were carried out with TDDFT using the CAM‐B3LYP/6‐31+G(d,p) theoretical method.

#### *3.1.4. Xanthophylls excited states UV‐Vis spectra*

Absorption spectra for the gaseous phase and with the solvent for xanthophylls‐type carot‐ enoids are displayed in **Figure 6**. According to the figures, one can observe that the varia‐ tion in absorption results in the gaseous phase with respect to the solvent phase is different only by a wavelength displacement. Zeaxanthin is the carotenoid from xanthophylls with an absorption in a longer wavelength, similar to lutein. The difference between them is the absorbance value where zeaxanthin has the bigger absorbance.

#### **3.2. Diades**

If one observes the molecular orbitals diagram corresponding to the molecules in methanol as a solvent, one can see that zeaxanthin is the molecule with the narrower energy gap as occurred in the gaseous phase calculations. Another important observation is how violaxan‐ thin keeps the same values in both calculations, in the gaseous phase and in the solvent phase.

**Figure 4.** Excited states molecular orbitals for selected carotenoid structures. Calculations were carried out in methanol

Absorption spectra for carotenoids‐type beta‐carotene and lycopene for the gaseous phase are shown in **Figure 5**. In this figure, a high coincidence in the absorption maximum peak between beta‐carotene with 472 nm and lycopene with 476 nm is observed. According to these calculations, the maximum absorption peak for beta‐carotene is surpassed by 20 nm. Meanwhile for lycopene, a better approximation with the experimental value of 470 nm is

*3.1.3. Carotenoids excited states UV‐Vis spectra*

as a solvent with TDDFT using the CAM‐B3LYP/6‐31+G(d,p) theoretical method.

obtained.

24 Carotenoids

Diades structures are formed with two molecules, one from the group of the main photo‐ synthetic pigments and the other from the accessory pigments group. For this work, we used chlorophyll *a*‐carotenoid. In the formation of the system, chlorophyll *a*‐zeaxanthin was employed, and the chlorophyllide *a,* due to the phytol, lacks contribution in the photochemi‐ cal activity related to light absorbance in chlorophyll *a* but brings about some benefit with some computational cost reduction. In the next paragraphs, we discuss our results for the selected diade. Our discussion for these systems is a relative view between ground states and excited states that will enable us to understand their electronic structure properties. Our discussion for this system is a relative view between ground states and excited states that will enable us to understand their electronic structure properties.

For the intermolecular energy transfer study, photosynthetic pigments' diade formed by chlo‐ rophyll *a*‐zeaxanthin was modeled and analyzed. For construction of the diade system, we used chlorophyllide *a,* and zeaxanthin was centered, and to build this diade, the individual optimized structures were used. Once the diade was modeled, the system was subject to a geometric optimization using the B3LYP/6‐31G (d) theoretical method in the gaseous phase.

**Figure 6.** UV‐Vis absorption spectra for xanthophylls‐type carotenoid structures. Calculations are in (a) the gaseous phase and (b) by using methanol as a solvent with TDDFT using the CAM‐B3LYP/6‐31+G(d,p) theoretical method.

For the determination of the intermolecular distances between chlorophyllide *a* and zeaxan‐ thin, we performed a series of prior work to determine the best choice with distances between 5.0 and 9 Å (all calculations included vibrations frequencies analysis to make sure that the global minima was reached).

Since electronic properties of selected photosynthetic pigments depend of nature in both cases, the ground and the excited states, we moved forward with molecular orbitals analysis for iso‐ lated molecules and diade systems (in both ground and excited states). TDDFT was used, with Tamm‐Dancoff approximation (TDA) and the CAMB3LYP/6‐31+G(d,p) theoretical method.

After HOMO/LUMO energy analysis, UV‐Vis absorption spectra were obtained for isolated molecules and their corresponding diade systems. The same theoretical level was used with same functional set.

#### *3.2.1. Diade molecular orbitals (HOMO/LUMO energies)*

Results for molecular orbitals and HOMO‐LUMO energies corresponding to chlorophyll *a*, zeaxanthin, and the respective diade formed by them, in their ground and excited states, are shown in **Figure 7(a)** and **(b)**, respectively. This way of organizing the diagrams enables one Electronic Structure of Carotenoids in Natural and Artificial Photosynthesis http://dx.doi.org/10.5772/67636 27

**Figure 7.** Molecular orbitals for chlorophyllide *a*, zeaxanthin, and their corresponding diade in both ground and excited states. Calculations for ground states were obtained using DFT with B3LYP/6‐31+G(d,p) and excited states were obtained using the TDDFT scheme with TDA approximation employing the CAMB3LYP/6‐31+G(d,p) theoretical method. Energies in eV.

For the determination of the intermolecular distances between chlorophyllide *a* and zeaxan‐ thin, we performed a series of prior work to determine the best choice with distances between 5.0 and 9 Å (all calculations included vibrations frequencies analysis to make sure that the

**Figure 6.** UV‐Vis absorption spectra for xanthophylls‐type carotenoid structures. Calculations are in (a) the gaseous phase and (b) by using methanol as a solvent with TDDFT using the CAM‐B3LYP/6‐31+G(d,p) theoretical method.

Since electronic properties of selected photosynthetic pigments depend of nature in both cases, the ground and the excited states, we moved forward with molecular orbitals analysis for iso‐ lated molecules and diade systems (in both ground and excited states). TDDFT was used, with Tamm‐Dancoff approximation (TDA) and the CAMB3LYP/6‐31+G(d,p) theoretical method.

After HOMO/LUMO energy analysis, UV‐Vis absorption spectra were obtained for isolated molecules and their corresponding diade systems. The same theoretical level was used with

Results for molecular orbitals and HOMO‐LUMO energies corresponding to chlorophyll *a*, zeaxanthin, and the respective diade formed by them, in their ground and excited states, are shown in **Figure 7(a)** and **(b)**, respectively. This way of organizing the diagrams enables one

global minima was reached).

26 Carotenoids

same functional set.

*3.2.1. Diade molecular orbitals (HOMO/LUMO energies)*

to compare both, ground and excited states, and obtain an easy way to compare the results. For the specific case of diade formed by chlorophyllide *a* and zeaxanthin in its ground state, when analyzed separately, chlorophyllide *a* presented an energy gap of 2.357 eV. When the diade was analyzed, the energy gap decreased to 1.883 eV. Meanwhile, at the excited state, chlorophyllide *a's* result had 4.048 eV (isolated), and for the diade, it was obtained as 3.756 eV.

**Figure 7** shows an energy gap for chlorophyllide *a* which decreased in both energetic states (ground and excited), presenting a difference between the isolated chlorophyllide *a* and those integrated into a diade system of 0.474 and 0.292 eV, respectively. This indicates in a general way that chlorophyllide *a* has a higher reactivity when it is found in the diade system, and this is independent of the energetic state.

For the diade system, it was found that the HOMO molecular orbital is provided by the cor‐ responding carotenoid zeaxanthin, while the LUMO orbital corresponds to chlorophyllide *a*. **Figure 8** displays both HOMO and LUMO orbitals for diade chlorophyllide *a*/zeaxantina, indi‐ cating in general that carotenoids are responsible for luminous energy absorption, by means of HOMO molecular orbitals, and interact with LUMO from chlorophyllide *a*.

**Figure 8.** Molecular orbitals HOMO and LUMO's graphical representations for the diade systemchlorophyllide *a*/zeaxanthin.

#### *3.2.2. Absorption UV‐vis spectra for chlorophyllide a and chlorophyll a*

From excited states energy calculations, UV‐vis absorption spectra were determined for each of the two selected molecules including integrated systems in the diade. In **Figure 9** are displayed UV‐vis absorption spectra for experimental and theoretical (corresponding to experimentation similar to calculations within this work) frameworks for chlorophyll *a*. Chlorophyllide *a* presents a similar structure than chlorophyll *a*, except that in chlorophyl‐ lide *a,* the phytol lateral chain is removed which does not present any π bonding (conjugate double bonds). Therefore, it does not present photochemical reactivity [29]. In this way, the

**Figure 9.** UV‐Vis absorption spectra for both chlorophyll *a* (experimental data) and for chlorophyllide *a*. Chlorophyll *a* is analyzed in diethylic ether and chlorophyllide *a* is analyzed in the gaseous phase using TDDFT with CAM‐B3LYP/6‐31+G(d,p).

comparison between experimental data for chlorophyll *a* (with the phytol lateral chain) and theoretical data for chlorophyllide *a* (without phytol) is in agreement.

In **Figure 9** are shown experimental values for the more characteristic chlorophyll *a* absorp‐ tion bands [30], which are 670 y 420 nm for Qy and Soret bands, respectively. For chlorophyl‐ lide *a,* the theoretical values found were 568 y 320 nm, respectively.

As can be seen in **Figure 9**, chlorophyllide *a* presents characteristic absorption maximum peaks. These peaks are displaced of approximately 100 nm in chlorophyllide *a* with respect to chlorophyll *a* values. This difference between experimental and theoretical values may be attributed mainly to the solvent effect. As mentioned in this section, to obtain experimental values, the samples used diethylic ether as a solvent while for theoretical values, and calcu‐ lations were made in the gaseous phase. Another contributor is the methodological differ‐ ence, in concrete, in the computational method employed (which relies on approximations). However, characteristic absorption peaks for these molecules were consistent in all cases.

*3.2.2. Absorption UV‐vis spectra for chlorophyllide a and chlorophyll a*

*a*/zeaxanthin.

28 Carotenoids

CAM‐B3LYP/6‐31+G(d,p).

From excited states energy calculations, UV‐vis absorption spectra were determined for each of the two selected molecules including integrated systems in the diade. In **Figure 9** are displayed UV‐vis absorption spectra for experimental and theoretical (corresponding to experimentation similar to calculations within this work) frameworks for chlorophyll *a*. Chlorophyllide *a* presents a similar structure than chlorophyll *a*, except that in chlorophyl‐ lide *a,* the phytol lateral chain is removed which does not present any π bonding (conjugate double bonds). Therefore, it does not present photochemical reactivity [29]. In this way, the

**Figure 9.** UV‐Vis absorption spectra for both chlorophyll *a* (experimental data) and for chlorophyllide *a*. Chlorophyll *a* is analyzed in diethylic ether and chlorophyllide *a* is analyzed in the gaseous phase using TDDFT with

**Figure 8.** Molecular orbitals HOMO and LUMO's graphical representations for the diade systemchlorophyllide

Regarding theoretical UV‐Vis absorption spectra (theoretical), calculations were developed for the two molecules integrated in the diades, and their diagram was based on the excited states' results. In **Figure 10**, chlorophyllide *a* absorption spectra, zeaxanthin, and their correspond‐ ing diades are displayed. Here, one can observe that Qy bands for isolated chlorophyllide *a* and the corresponding band for the diade are practically identical (in both axes). Meanwhile, the Soret band corresponding to the diade presents absorption values slightly over the cor‐ responding values for chlorophyllide *a* alone (the values being the same for the wavelength in both cases). Concretely for zeaxanthin in the diade, this pigment contributes to a slight dis‐ placement in the x‐axis of 468.6 a 470.5 nm with respect to isolated zeaxanthin. Furthermore,

**Figure 10.** UV‐Vis absorption spectra for chlorophyllide *a*, zeaxanthin, and their corresponding diade (chlorophyllide *a*/zeaxanthin). Calculations were carried out under the TDDFT scheme using the CAMB3LYP/6‐31+G(d,p) theoretical method.

isolated zeaxanthin presents in this particular peak an absorbance slightly above with respect to the one measured in its corresponding diade. Then, it may be observed how integrated diade systems can cover a wider absorption spectra than isolated chlorophyll *a*.

#### **4. Conclusions**

Theoretical calculations allowed us to predict UV‐Vis absorption spectra for carotenoid struc‐ tures selected for this work, achieving a good accuracy with experimental results.

Within the selected carotenoids that were analyzed, zeaxanthin is the pigment with better electronic properties to form an integrated system, chlorophyll *a*‐carotenoid.

Excited states molecular orbitals analysis for the diade system formed by chlorophyllide *a*‐zea‐ xanthin allowed one to observe the specific role played by chlorophyll *a* as a photosynthetic pigment by its LUMO contribution to the integrated system on the one hand and on the other hand, the role of zeaxanthin as an accessory pigment with the contribution made to HOMO.

Analysis of pigments related to the natural photosynthetic process allowed us to set a basis on how these pigments can be used in potential artificial photosynthetic applications and to develop new materials for alternative energy applications.

Electronic structure data are fundamental information for any material and may help to develop systems with carotenoids and accessory pigments in other fields such as the health or food industries.

#### **Acknowledgements**

The authors thank the National Council of Science and Technology (CONACYT, for its acro‐ nym in Spanish) for the financial support in the development of this scientific research to the basic science project, No. 158307. FTR and JMB thank CONACYT for a scholarship to support their doctoral studies.

#### **Author details**

Manuel Flores‐Hidalgo1 , Francisco Torres‐Rivas2 , Jesus Monzon‐Bensojo<sup>2</sup> , Miguel Escobedo‐ Bretado1 , Daniel Glossman‐Mitnik3 and Diana Barraza‐Jimenez1,2\*

\*Address all correspondence to: dianabarraza@ujed.mx

1 Department of Chemical Sciences, Juarez University of Durango State, Durango, Mexico

2 Food and Development Research Center, A. C. Delicias Unit, Chih, Mexico

3 Advanced Materials Research Center, Chihuahua, Chih, Mexico

#### **References**

isolated zeaxanthin presents in this particular peak an absorbance slightly above with respect to the one measured in its corresponding diade. Then, it may be observed how integrated

Theoretical calculations allowed us to predict UV‐Vis absorption spectra for carotenoid struc‐

Within the selected carotenoids that were analyzed, zeaxanthin is the pigment with better

Excited states molecular orbitals analysis for the diade system formed by chlorophyllide *a*‐zea‐ xanthin allowed one to observe the specific role played by chlorophyll *a* as a photosynthetic pigment by its LUMO contribution to the integrated system on the one hand and on the other hand, the role of zeaxanthin as an accessory pigment with the contribution made to HOMO. Analysis of pigments related to the natural photosynthetic process allowed us to set a basis on how these pigments can be used in potential artificial photosynthetic applications and to

Electronic structure data are fundamental information for any material and may help to develop systems with carotenoids and accessory pigments in other fields such as the health

The authors thank the National Council of Science and Technology (CONACYT, for its acro‐ nym in Spanish) for the financial support in the development of this scientific research to the basic science project, No. 158307. FTR and JMB thank CONACYT for a scholarship to support

and Diana Barraza‐Jimenez1,2\*

1 Department of Chemical Sciences, Juarez University of Durango State, Durango, Mexico

, Jesus Monzon‐Bensojo<sup>2</sup>

, Miguel Escobedo‐

, Francisco Torres‐Rivas2

2 Food and Development Research Center, A. C. Delicias Unit, Chih, Mexico

3 Advanced Materials Research Center, Chihuahua, Chih, Mexico

diade systems can cover a wider absorption spectra than isolated chlorophyll *a*.

tures selected for this work, achieving a good accuracy with experimental results.

electronic properties to form an integrated system, chlorophyll *a*‐carotenoid.

develop new materials for alternative energy applications.

**4. Conclusions**

30 Carotenoids

or food industries.

**Acknowledgements**

their doctoral studies.

Manuel Flores‐Hidalgo1

, Daniel Glossman‐Mitnik3

\*Address all correspondence to: dianabarraza@ujed.mx

**Author details**

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[30] Strain HH, Thomas MR, Katz JJ. Spectral absorption properties of ordinary and fully deuteriated chlorophylls *a* and *b*. Biochim Biophys Acta 1963; **75**: 306–311. doi:10.1016/0 006‐3002(63)90617‐6

## **Localizing and Quantifying Carotenoids in Intact Cells and Tissues**

Jerilyn A. Timlin, Aaron M. Collins, Thomas A. Beechem, Maria Shumskaya and Eleanore T. Wurtzel

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/68101

#### **Abstract**

Raman spectroscopy provides detailed information about the molecular structure of carot‐ enoids. Advances in detector sensitivity and acquisition speed have driven the expansion of Raman spectroscopy from a bulk analytical tool to a powerful method for mapping carotenoid abundance in cells and tissues. In many applications, the technique is compat‐ ible with living organisms, providing highly specific molecular structure information in intact cells and tissues with subcellular spatial resolution. This leads to spatial‐temporal‐ chemical resolution critical to understanding the complex processes in the life cycle of carotenoids and other biomolecules.

**Keywords:** vibrational spectroscopy, multivariate analysis, confocal Raman microscopy, Raman imaging, photosynthetic organisms, carotenoids, resonance Raman scattering

#### **1. Introduction**

Carotenoids are tetraterpenoids and have various functions in plants and algae. They extend the range of wavelengths for photosynthesis by harnessing solar radiation where chlorophyll pigments do not appreciably absorb and serve as structural elements within the photosyn‐ thetic apparatus [1]. They also function to dissipate excess solar radiation and prevent the formation of harmful singlet oxygen species [2]. Additionally, in specific photosynthetic eukaryotes, carotenoids accumulate in plastid organelles called chromoplasts and give many fruits, flowers, and roots their bright colors [3]. For these reasons, assessing the presence and distribution of carotenoids at the subcellular level provides a keyhole through which to

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

understand a variety of cellular processes. The use and utility of Raman imaging is addressed here toward this end.

The Raman effect involves an incoming photon being scattered inelastically by a polyatomic molecule. This phenomenon requires that energy is exchanged between the photon and mol‐ ecule. Since energy levels in the molecule are discrete, the difference in energy between the incoming and scattered photon corresponds to a molecular transition, most typically a vibra‐ tion [4]. Molecular vibrations are influenced by the composition of atoms that make up the molecule, the types of bonds that connect atoms, and molecular symmetry. Thus, Raman spectroscopy has been exploited as a highly sensitive analytical tool for the determination of the chemical identity of many molecules [5, 6]. The probability of observing the Raman effect is low, however, as the intensity of scattered radiation scales with the fourth power of incident light's frequency. The Raman effect is oftentimes considerably weaker as compared to the intrinsic fluorescence in cells and tissues, complicating and even prohibiting the detection of the Raman scatter in biologically relevant matrices.

However, if the frequency of incident light matches the energy of an electronic transition, an enhancement of the Raman signal may be observed. This is known as resonance Raman scatter‐ ing (RRS), which can enhance the signal by several orders of magnitude over a "normal," non‐ resonance measurement. In fact, the resonance effect can increase the scattering cross section to exceed any off‐resonance Raman scatter and, importantly, even rival intrinsic fluorescence from the species. RRS thus has profound analytical potential and has been comprehensively reviewed elsewhere for the general life science field [7] and specifically for applications in pho‐ tosynthesis [8]. Here, its role in the assessment of carotenoids is examined explicitly.

The optical properties of carotenoids make them especially suitable for RRS. Carotenoids are π‐electron‐conjugated carbon‐chain molecules consisting of alternating C─C single bonds and C═C double bonds. Individual carotenoids can be distinguished by the number of conju‐ gated carbon double bonds, the number of attached methyl side groups, and the number and type of end groups. These properties result in many of the highly abundant carotenoid mol‐ ecules (e.g., β‐carotene, zeaxanthin, lycopene, and lutein) having distinct, yet broad (100 nm) absorption bands in the visible region of the spectrum. The absorption shifts to longer wave‐ lengths as the effective conjugation length of the carotenoid increases. Fortuitously, these visible absorption bands overlap with the common laser wavelengths for Raman excitation. Thus, when excited under these conditions, carotenoids exhibit a very strong RRS response (enhancement factor of about five orders of magnitude relative to non‐resonant Raman spec‐ troscopy) and little to no fluorescence emission. Additionally, variations in absorption of the different carotenoids can be exploited by shifting the excitation wavelength (a technique also known as "tuning") to preferentially excite different carotenoid molecules. Owing to these amenities, RRS therefore enables the detection of carotenoids, even in complex biological sys‐ tems, such as living photosynthetic cells and tissues.

The majority of carotenoids have linear structures resulting in a limited number of Raman‐ observable vibrations that are easily categorized into a distinct vibrational signature. There are three major Raman modes typically leveraged in the analysis of carotenoids [9, 10]. The *ν*<sup>1</sup> band (∼1530 cm−1) arises from the stretching vibrations of the conjugated C═C back‐ bone. This band is sensitive to conjugation length and molecular conformation and therefore is the most diagnostic for carotenoid identity. The *ν*<sup>2</sup> band (∼1160 cm−1) emerges from the stretching of the C─C vibrations coupled to C─CH3 stretches or C─H in‐plane bending. The *ν*3 band (∼1006 cm−1) is attributed to CH<sup>3</sup> in‐plane–rocking modes. Importantly, the vibra‐ tional modes are more or less insensitive to the molecular environment [11] meaning that a carotenoid found within the tissues of a plant may have a very similar spectrum to the same carotenoid dissolved in solvent. This observation is important when assigning carotenoid sig‐ natures *in situ*. **Figure 1** shows the resonance Raman spectra from six common carotenoids produced in photosynthetic organisms. The spectra are plotted in the order of increasing

understand a variety of cellular processes. The use and utility of Raman imaging is addressed

The Raman effect involves an incoming photon being scattered inelastically by a polyatomic molecule. This phenomenon requires that energy is exchanged between the photon and mol‐ ecule. Since energy levels in the molecule are discrete, the difference in energy between the incoming and scattered photon corresponds to a molecular transition, most typically a vibra‐ tion [4]. Molecular vibrations are influenced by the composition of atoms that make up the molecule, the types of bonds that connect atoms, and molecular symmetry. Thus, Raman spectroscopy has been exploited as a highly sensitive analytical tool for the determination of the chemical identity of many molecules [5, 6]. The probability of observing the Raman effect is low, however, as the intensity of scattered radiation scales with the fourth power of incident light's frequency. The Raman effect is oftentimes considerably weaker as compared to the intrinsic fluorescence in cells and tissues, complicating and even prohibiting the detection of

However, if the frequency of incident light matches the energy of an electronic transition, an enhancement of the Raman signal may be observed. This is known as resonance Raman scatter‐ ing (RRS), which can enhance the signal by several orders of magnitude over a "normal," non‐ resonance measurement. In fact, the resonance effect can increase the scattering cross section to exceed any off‐resonance Raman scatter and, importantly, even rival intrinsic fluorescence from the species. RRS thus has profound analytical potential and has been comprehensively reviewed elsewhere for the general life science field [7] and specifically for applications in pho‐

The optical properties of carotenoids make them especially suitable for RRS. Carotenoids are π‐electron‐conjugated carbon‐chain molecules consisting of alternating C─C single bonds and C═C double bonds. Individual carotenoids can be distinguished by the number of conju‐ gated carbon double bonds, the number of attached methyl side groups, and the number and type of end groups. These properties result in many of the highly abundant carotenoid mol‐ ecules (e.g., β‐carotene, zeaxanthin, lycopene, and lutein) having distinct, yet broad (100 nm) absorption bands in the visible region of the spectrum. The absorption shifts to longer wave‐ lengths as the effective conjugation length of the carotenoid increases. Fortuitously, these visible absorption bands overlap with the common laser wavelengths for Raman excitation. Thus, when excited under these conditions, carotenoids exhibit a very strong RRS response (enhancement factor of about five orders of magnitude relative to non‐resonant Raman spec‐ troscopy) and little to no fluorescence emission. Additionally, variations in absorption of the different carotenoids can be exploited by shifting the excitation wavelength (a technique also known as "tuning") to preferentially excite different carotenoid molecules. Owing to these amenities, RRS therefore enables the detection of carotenoids, even in complex biological sys‐

The majority of carotenoids have linear structures resulting in a limited number of Raman‐ observable vibrations that are easily categorized into a distinct vibrational signature. There are three major Raman modes typically leveraged in the analysis of carotenoids [9, 10]. The *ν*<sup>1</sup> band (∼1530 cm−1) arises from the stretching vibrations of the conjugated C═C back‐ bone. This band is sensitive to conjugation length and molecular conformation and therefore

tosynthesis [8]. Here, its role in the assessment of carotenoids is examined explicitly.

here toward this end.

36 Carotenoids

the Raman scatter in biologically relevant matrices.

tems, such as living photosynthetic cells and tissues.

**Figure 1.** Resonance Raman spectra of six common carotenoids produced in photosynthetic organisms. Major Raman active vibrations are labeled. Spectra were obtained from carotenoids in powder form with the exception of lutein which was dissolved in methanol.

*ν*<sup>1</sup> vibration position (ranging from 1516 to 1524 cm−1) and illustrate the ability to distinguish carotenoids by the position of this vibration.

In recent years, advances in detector sensitivity and acquisition speed have driven the expan‐ sion of Raman spectroscopy from a bulk analytical tool to a powerful method for mapping molecular vibrations in cells and tissues by the addition of a spatial dimension. Spatial resolu‐ tion available with Raman microscopy is dependent on the wavelength of the excitation light and in theory is the same as other optical microscopies, such as fluorescence (e.g., ∼250 nm lateral and ∼500 nm axial, for blue/green excitation). The addition of spatial resolution can be accomplished in several different ways: confocal point‐scanning Raman microscopy [12, 13], wide‐field Raman imaging [14], or Raman line scanning [15, 16]. The relative advantages and disadvantages of each of these approaches have been reviewed elsewhere and will not be covered here [17]. In addition to the imaging methodology, spectral information can be obtained from either a single or a small number of bands through the use of discrete band‐ pass filters or tunable filters or in a hyperspectral fashion, where the entire Raman spectrum is dispersed via a prism or grating allowing for the simultaneous acquisition of hundreds of spectral bands. While there is typically a speed advantage associated with the filter‐based acquisition approach, recent commercial systems utilizing electron‐multiplied charge‐cou‐ pled device (EM‐CCD) detectors and cutting‐edge research systems [18] are able to approach confocal fluorescence microscopy speeds and further improvements are expected.

Given the complexity associated with biological matrices such as live cells and tissues, hyper‐ spectral Raman approaches are often advantageous because they can provide detailed spec‐ tral signatures for subsequent analysis of weak or overlapping features and background noise reduction using chemometric algorithms. These approaches are collectively referred in the lit‐ erature as Raman spectroscopic imaging, or hyperspectral Raman microscopy. Additionally, confocal point scanning or pseudo‐confocal line scanning can have advantages due to the inherent rejection of out‐of‐focus signal provided by the pinhole or entrance slit, respectively. A confocal, point‐scanning, hyperspectral Raman microscope was utilized for the work pre‐ sented in this chapter as it adequately rejects the intense pigment signal from outside the focal plane, while providing reasonable acquisition speeds for live cells.

Recent literature shows that Raman spectroscopic imaging is emerging as a key technology for single‐cell analysis, including *in vivo* lipidomics [19], chemical composition of bacterial cells [20], lignin in plant cell walls [21], and metabolism in combination with stable isotope incorpo‐ ration [22–24]. Researchers have also begun capitalizing on the specific benefits of resonance Raman spectroscopic imaging to localize carotenoids in individual cells and tissues. Pudney et al. demonstrated the localization of lutein, beta‐carotene, and lycopene in several tomato varieties [25]. Zheng and coworkers used Raman imaging to investigate biofilm formation in Rhodococcus sp. SD‐74, showing that biofilm production by this organism is correlated with increasing carotenoid concentrations, a finding not possible with traditional dye staining assays or electron microscopy [26]. Kilcrease et al. utilized a novel combination of confocal Raman microscopy with laser scanning confocal and scanning and transmission electron microscopy to demonstrate subcellular accumulation sites of various carotenoids in *Capsicum annuum* L. (Chile pepper) fruit. Their work discovered an unexpected relationship between carotenoid accumulation and chromoplast structure. Collins and coworkers utilized hyperspectral confo‐ cal Raman microscopy to study fundamental carotenoid biogenesis in *Haematococcus pluvialis*

throughout the organism's life cycle [27]. Additionally, although not single cell work, Toomey et al. recently show the utility of hyperspectral confocal Raman microscopy for investigating carotenoid composition in single oil droplets within the avian retina [28].

While Raman spectroscopic imaging has potential for assessing carotenoid distributions in single cells and tissues for many applications in biomedicine and photosynthesis, there are some noteworthy limitations. First, not all carotenoids are enhanced to the same degree and some will not be enhanced at all. This differential enhancement can be advantageous, but it also can pose limitations for certain carotenoids. To some degree, the choice of laser excitation can target additional carotenoids. However, performing multiple Raman microscopy scans at two or more excitation wavelengths is prohibitive for live‐cell dynamics and may require fixed samples depending on the acquisition times. Second, even with high spectral resolu‐ tion instruments, multiple carotenoid species with highly overlapping, similar peaks can be difficult to identify. Additionally, even with resonance enhancement, Raman peaks may still be weak at *in vivo* and *in planta* relevant concentrations and be overwhelmed by background fluorescence depending on the sample matrix.

In addition to the hardware approaches listed above, the first two limitations are actively being addressed with the development of robust multivariate analysis tools [29–31]. For example, multivariate curve resolution (MCR) has been developed by several research groups for the analysis of hyperspectral confocal fluorescence and Raman image data sets [29, 32–34] find‐ ing success in complex multicomponent biological samples. It is therefore used in the analy‐ sis presented in this chapter. Lastly, while recent advances in fluorescence microscopy have extended the spatial resolution beyond the diffraction limit, the spatial resolution of Raman spectroscopic imaging is still in many cases orders of magnitude larger than the biological processes being investigated. Near‐field approaches can provide higher spatial resolution, but have not found wide‐scale success with living cells and tissues [35].

This chapter presents three separate applications of confocal Raman microscopy to assess carotenoid localization and relative abundance within living cells. The green algae *H. pluvialis* is presented to highlight the ability to discern highly similar carotenoid structures in algal cells. Maize (*Zea mays*) protoplasts demonstrate the localization of carotenoids even amidst a high background of chlorophyll pigment. Finally, *Synechocystis* sp. PCC6803, a model cya‐ nobacterium, is presented to illustrate carotenoid localization even in very small microbes. Together, these applications demonstrate the potential of resonance Raman microscopy and imaging for the analysis of carotenoid localization and abundance at the single‐cell and sub‐ cellular levels in photosynthetic organisms.

#### **2. Materials and methods**

*ν*<sup>1</sup> vibration position (ranging from 1516 to 1524 cm−1) and illustrate the ability to distinguish

In recent years, advances in detector sensitivity and acquisition speed have driven the expan‐ sion of Raman spectroscopy from a bulk analytical tool to a powerful method for mapping molecular vibrations in cells and tissues by the addition of a spatial dimension. Spatial resolu‐ tion available with Raman microscopy is dependent on the wavelength of the excitation light and in theory is the same as other optical microscopies, such as fluorescence (e.g., ∼250 nm lateral and ∼500 nm axial, for blue/green excitation). The addition of spatial resolution can be accomplished in several different ways: confocal point‐scanning Raman microscopy [12, 13], wide‐field Raman imaging [14], or Raman line scanning [15, 16]. The relative advantages and disadvantages of each of these approaches have been reviewed elsewhere and will not be covered here [17]. In addition to the imaging methodology, spectral information can be obtained from either a single or a small number of bands through the use of discrete band‐ pass filters or tunable filters or in a hyperspectral fashion, where the entire Raman spectrum is dispersed via a prism or grating allowing for the simultaneous acquisition of hundreds of spectral bands. While there is typically a speed advantage associated with the filter‐based acquisition approach, recent commercial systems utilizing electron‐multiplied charge‐cou‐ pled device (EM‐CCD) detectors and cutting‐edge research systems [18] are able to approach

confocal fluorescence microscopy speeds and further improvements are expected.

plane, while providing reasonable acquisition speeds for live cells.

Given the complexity associated with biological matrices such as live cells and tissues, hyper‐ spectral Raman approaches are often advantageous because they can provide detailed spec‐ tral signatures for subsequent analysis of weak or overlapping features and background noise reduction using chemometric algorithms. These approaches are collectively referred in the lit‐ erature as Raman spectroscopic imaging, or hyperspectral Raman microscopy. Additionally, confocal point scanning or pseudo‐confocal line scanning can have advantages due to the inherent rejection of out‐of‐focus signal provided by the pinhole or entrance slit, respectively. A confocal, point‐scanning, hyperspectral Raman microscope was utilized for the work pre‐ sented in this chapter as it adequately rejects the intense pigment signal from outside the focal

Recent literature shows that Raman spectroscopic imaging is emerging as a key technology for single‐cell analysis, including *in vivo* lipidomics [19], chemical composition of bacterial cells [20], lignin in plant cell walls [21], and metabolism in combination with stable isotope incorpo‐ ration [22–24]. Researchers have also begun capitalizing on the specific benefits of resonance Raman spectroscopic imaging to localize carotenoids in individual cells and tissues. Pudney et al. demonstrated the localization of lutein, beta‐carotene, and lycopene in several tomato varieties [25]. Zheng and coworkers used Raman imaging to investigate biofilm formation in Rhodococcus sp. SD‐74, showing that biofilm production by this organism is correlated with increasing carotenoid concentrations, a finding not possible with traditional dye staining assays or electron microscopy [26]. Kilcrease et al. utilized a novel combination of confocal Raman microscopy with laser scanning confocal and scanning and transmission electron microscopy to demonstrate subcellular accumulation sites of various carotenoids in *Capsicum annuum* L. (Chile pepper) fruit. Their work discovered an unexpected relationship between carotenoid accumulation and chromoplast structure. Collins and coworkers utilized hyperspectral confo‐ cal Raman microscopy to study fundamental carotenoid biogenesis in *Haematococcus pluvialis*

carotenoids by the position of this vibration.

38 Carotenoids

#### **2.1. Cell culture and sample prep**

*H. pluvialis* cells were obtained from the Hu lab cultured as described previously to produce cul‐ tures at various stages of the life cycle (flagellated, nonmotile palmelloid, and aplanospores)[27]. Cells suspended in growth media were directly loaded to a slide and covered with a glass cover‐ slip. In this arrangement, cells remained hydrated during the duration of the imaging.

*Synechocystis* 6803 cells were obtained from the Pakrasi lab and cultured in BG‐11 media (UTEX recipe) in baffled flasks in a temperature‐controlled incubator under continuous light (∼30 µmol m−2 s−1, cool white fluorescent lights) with shaking (60 rpm). Prior to imaging, a 4‐µl sample was directly pipetted onto a BG‐11 agar‐coated slide and covered with a glass cover‐ slip. Cells were immediately imaged.

Etiolated maize protoplasts were isolated from *Z. mays* var. B73 leaves as described previ‐ ously [36]. Released protoplasts were collected, washed, and shipped in a buffer solution overnight for Raman imaging. Upon arrival, protoplasts were concentrated by centrifugation at 500 × g and removal of all but 100 µl of the supernatant. Concentrated sample (25 µl) was loaded into an *in situ* frame (Gene Frame, ThermoFisher) previously adhered to a gridded microscope slide (Lovins Micro‐Slide Field Finder, Electron Microscopy Sciences) and cov‐ ered with a glass coverslip. The gridded slide was used to perform correlated fluorescence microscopy for a separate experiment. Protoplasts were immediately imaged.

#### **2.2. Confocal Raman microscopy**

Raman images were acquired with a WiTec Alpha300R system equipped with a WiTec UHTS spectrometer utilizing a 600‐l/mm grating and an Andor back‐illuminated electron‐multi‐ plying charge‐coupled device (EMCCD). Light was incident at 532 nm and focused using a 50×/0.55 NA objective (*Synechocystis* 6803), 20×/0.45 NA objective (maize protoplasts), and a 100×/0.9 NA objective (*H. pluvialis*). Laser powers were chosen such that the spectral character was nearly invariant with time and did not cause a visible change in the sample during collec‐ tion. In most cases, power incident on the surface was held to less than 1 mW. The acquisition time per spectrum was chosen to provide adequate signal to noise to perform the multivariate analysis and varied for the different samples analyzed ranging from 4 to 10 ms per spectrum. The spectral response was 3 cm−1 per pixel, which through fitting resulted in the ability to specify peak position to an absolute accuracy of ±1 cm−1. All measurements were performed in an unpolarized back‐scattering arrangement. Images were acquired by scanning the sample using a piezo‐stage possessing a lateral resolution of <5 nm. Raman‐scattered light is collected continuously across the sample such that every spectrum is the average convolution of the beam intensity and sample response over the distance defining an individual voxel. Images were collected by acquiring a spectrum every 333 nm (*Synechocystis* 6803 and *H. pluvialis*) or 500 nm (maize protoplasts). Raman shifts were calibrated using the well‐established positions of silicon, 6H‐SiC, graphite, and 4‐acetameniphol.

#### **2.3. Image data analysis**

All spectral image analysis was performed in Matlab 2012 or 2015 (Mathworks) equipped with the statistics and machine learning, signal processing, image processing, and curve fit‐ ting toolboxes leveraging in‐house written software, functions, and scripts. Hyperspectral confocal Raman images were preprocessed to remove cosmic spikes [37]. When applicable, images from the same sample were compiled into a composite image data set. The use of composite images, rather than analyzing every image independently, increases the number of pixels for the MCR analysis and thus serves to improve spectral signature identification by adding additional variance [38]. The spectral region was trimmed to exclude the excitation laser line but still include about 5–10 pixels of the spectrum where the signal was blocked by the Rayleigh filter. This technique assists in the analysis by providing a "zero‐signal region" for assessing baseline contributions and is discussed in detail by Jones et al. [38]. In most cases, an image mask was created that excluded pixels outside the area of the cells from the analysis as they contain predominantly background signal.

Principal components analysis (PCA) was performed on the composite image and the Scree plot was inspected to determine the number of independent components present in the image data set (measured as being before the bend in the elbow of the Scree plot [29]). Multivariate curve resolution was then performed using a constrained alternating least‐ squares algorithm and employing robust constraints for equality (offset/baseline) and non‐ negativity (all true spectral components) to develop a spectral model that described the spectral variance within the data set. A PCA analysis of the residuals was used to confirm the appropriateness of the spectral model for describing the data and identify any unmod‐ eled spectral signatures. The multivariate curve resolution algorithm [39–43] and specific approaches for success with biological images have been described in detail elsewhere [29, 38]. The details of the spectral models developed are presented during the results and dis‐ cussion for each application in this chapter. The MCR‐identified spectra were then used in a classical least‐squares (CLS) analysis to predict the concentrations of each spectral compo‐ nent in each image pixel. Lastly, the resulting concentration maps were exported as 16‐bit grayscale tiffs such that they could be subject to traditional image analysis. Color image overlays and simple image cropping and scaling for visualization purposes was performed in Fiji [44].

#### **3. Results and discussion**

*Synechocystis* 6803 cells were obtained from the Pakrasi lab and cultured in BG‐11 media (UTEX recipe) in baffled flasks in a temperature‐controlled incubator under continuous light (∼30 µmol m−2 s−1, cool white fluorescent lights) with shaking (60 rpm). Prior to imaging, a 4‐µl sample was directly pipetted onto a BG‐11 agar‐coated slide and covered with a glass cover‐

Etiolated maize protoplasts were isolated from *Z. mays* var. B73 leaves as described previ‐ ously [36]. Released protoplasts were collected, washed, and shipped in a buffer solution overnight for Raman imaging. Upon arrival, protoplasts were concentrated by centrifugation at 500 × g and removal of all but 100 µl of the supernatant. Concentrated sample (25 µl) was loaded into an *in situ* frame (Gene Frame, ThermoFisher) previously adhered to a gridded microscope slide (Lovins Micro‐Slide Field Finder, Electron Microscopy Sciences) and cov‐ ered with a glass coverslip. The gridded slide was used to perform correlated fluorescence

Raman images were acquired with a WiTec Alpha300R system equipped with a WiTec UHTS spectrometer utilizing a 600‐l/mm grating and an Andor back‐illuminated electron‐multi‐ plying charge‐coupled device (EMCCD). Light was incident at 532 nm and focused using a 50×/0.55 NA objective (*Synechocystis* 6803), 20×/0.45 NA objective (maize protoplasts), and a 100×/0.9 NA objective (*H. pluvialis*). Laser powers were chosen such that the spectral character was nearly invariant with time and did not cause a visible change in the sample during collec‐ tion. In most cases, power incident on the surface was held to less than 1 mW. The acquisition time per spectrum was chosen to provide adequate signal to noise to perform the multivariate analysis and varied for the different samples analyzed ranging from 4 to 10 ms per spectrum. The spectral response was 3 cm−1 per pixel, which through fitting resulted in the ability to specify peak position to an absolute accuracy of ±1 cm−1. All measurements were performed in an unpolarized back‐scattering arrangement. Images were acquired by scanning the sample using a piezo‐stage possessing a lateral resolution of <5 nm. Raman‐scattered light is collected continuously across the sample such that every spectrum is the average convolution of the beam intensity and sample response over the distance defining an individual voxel. Images were collected by acquiring a spectrum every 333 nm (*Synechocystis* 6803 and *H. pluvialis*) or 500 nm (maize protoplasts). Raman shifts were calibrated using the well‐established positions

All spectral image analysis was performed in Matlab 2012 or 2015 (Mathworks) equipped with the statistics and machine learning, signal processing, image processing, and curve fit‐ ting toolboxes leveraging in‐house written software, functions, and scripts. Hyperspectral confocal Raman images were preprocessed to remove cosmic spikes [37]. When applicable, images from the same sample were compiled into a composite image data set. The use of composite images, rather than analyzing every image independently, increases the number of pixels for the MCR analysis and thus serves to improve spectral signature identification by adding additional variance [38]. The spectral region was trimmed to exclude the excitation

microscopy for a separate experiment. Protoplasts were immediately imaged.

slip. Cells were immediately imaged.

40 Carotenoids

**2.2. Confocal Raman microscopy**

of silicon, 6H‐SiC, graphite, and 4‐acetameniphol.

**2.3. Image data analysis**

#### **3.1. Resolving different carotenoids in living cells:** *H. pluvialis*

*H. pluvialis* is a freshwater green microalga that can synthesize and accumulate astaxanthin, a high‐value nutraceutical, under stress conditions such as nutrient deprivation. Though some aspects of carotenogenesis leading to astaxanthin production are well understood, spatial‐temporal details of key carotenoids and enzymes have yet to be fully elucidated. In previous work, Collins and coworkers used confocal Raman spectroscopic imaging to provide *in vivo* resolution and localization of beta‐carotene and astaxanthin, along with chlorophyll throughout the life cycle of *H. pluvialis* cells [27]. While this work will not be reproduced in detail, here, some key elements will be presented to illustrate the poten‐ tial of the technique for carotenoids localization in living cells. **Figure 2** demonstrates the advantage of performing MCR analysis on the Raman spectroscopic image data as com‐ pared to simplistic band integration. While the image produced by integrating the area under the *ν*<sup>1</sup> carotenoid vibration provides a highly resolved view of carotenoid location within the cell, the image is a superposition of two different carotenoids found in the cell. By contrast, MCR analysis resulted in a four‐component model consisting of two carotenoid spectra (astaxanthin and beta‐carotene), chlorophyll, and an autofluorescence component. (Note: A background component was also part of the model, but is omitted here for simplic‐

ity.) MCR analysis also generates a corresponding concentration map (i.e., image) for each of these species. This results in an exquisite degree of chemical resolution that allows for quantitative assessment via traditional image analysis methods. In **Figure 2D**, the images for astaxanthin, beta‐carotene, and chlorophyll are pseudocolored and overlaid for qualita‐ tive comparison. Importantly, in this example, the MCR analysis reveals that astaxanthin is located only in the cytosol. Beta‐carotene, meanwhile, is both co‐located with astaxanthin in the cytosol, as well as with the chlorophyll in the chloroplast. Together, these Raman images provide spatial‐temporal details of astaxanthin production and accumulation in this organism.

#### **3.2. Carotenoids in high‐chlorophyll backgrounds: maize protoplasts**

Similar to algae, plant carotenoids have roles in light harvesting and protection against light and heat stress. Metabolic engineering of carotenoids in plants has the potential to create variet‐ ies exhibiting increased adaptation to climate change as well as additional nutritional value. To develop plant cultivars with these enhanced properties, it is critical to understand the detailed spatial‐temporal arrangement of both carotenoids and chlorophyll *in situ*. While chlorophyll is straightforward to detect with a standard confocal fluorescence microscope, carotenoids have little to no detectable fluorescence signal and are not amenable to exogenous labeling.

**Figure 2.** Raman‐spectroscopic imaging of *H. pluvialis*: the advantage of MCR analysis. (A) Raw spectra from Raman‐ spectroscopic image of *H. pluvialis*. (B) Integrated Raman image created by traditional integration over the area of the *ν*<sup>1</sup> band of the carotenoid (1480–1530 cm−1) for the image data in A. C. MCR identified spectral component from image data in A. RGB image created by overlaying the independent component maps generated in the MCR analysis. Scale bar *= 10* µ*m*. (note: **Figure 2** is best viewed in color. Please refer to the online version of this chapter for the color version of **Figure 2**).

Confocal Raman spectroscopic imaging is capable of detecting carotenoids without the use of any exogenous labels and does so by detecting the resonance Raman signal of carotenoids. This signal can be observed also within the background of other pigments, such as the chlo‐ rophyll precursor, protochlorophyllide, which is found in cells of etiolated tissue [45] such as plant protoplasts from etiolated maize (**Figure 3**). Plant protoplasts are plant cells that have their cell wall removed through enzyme treatment and thus are an excellent experimental system for plant biologists because they improve the ease of introducing new genes.

ity.) MCR analysis also generates a corresponding concentration map (i.e., image) for each of these species. This results in an exquisite degree of chemical resolution that allows for quantitative assessment via traditional image analysis methods. In **Figure 2D**, the images for astaxanthin, beta‐carotene, and chlorophyll are pseudocolored and overlaid for qualita‐ tive comparison. Importantly, in this example, the MCR analysis reveals that astaxanthin is located only in the cytosol. Beta‐carotene, meanwhile, is both co‐located with astaxanthin in the cytosol, as well as with the chlorophyll in the chloroplast. Together, these Raman images provide spatial‐temporal details of astaxanthin production and accumulation in this

Similar to algae, plant carotenoids have roles in light harvesting and protection against light and heat stress. Metabolic engineering of carotenoids in plants has the potential to create variet‐ ies exhibiting increased adaptation to climate change as well as additional nutritional value. To develop plant cultivars with these enhanced properties, it is critical to understand the detailed spatial‐temporal arrangement of both carotenoids and chlorophyll *in situ*. While chlorophyll is straightforward to detect with a standard confocal fluorescence microscope, carotenoids have

little to no detectable fluorescence signal and are not amenable to exogenous labeling.

**Figure 2.** Raman‐spectroscopic imaging of *H. pluvialis*: the advantage of MCR analysis. (A) Raw spectra from Raman‐ spectroscopic image of *H. pluvialis*. (B) Integrated Raman image created by traditional integration over the area of

 band of the carotenoid (1480–1530 cm−1) for the image data in A. C. MCR identified spectral component from image data in A. RGB image created by overlaying the independent component maps generated in the MCR analysis. Scale bar *= 10* µ*m*. (note: **Figure 2** is best viewed in color. Please refer to the online version of this chapter for the color

**3.2. Carotenoids in high‐chlorophyll backgrounds: maize protoplasts**

organism.

42 Carotenoids

the *ν*<sup>1</sup>

version of **Figure 2**).

**Figure 3** highlights the spatial location and levels of carotenoids and protochlorophyllide in plastids (immature chloroplasts) of etiolated maize protoplasts as observed by confocal Raman microscopy. Previous high‐performance liquid chromatography data have shown that the dominant carotenoid in maize‐etiolated protoplasts is lutein with a minor amount of violaxanthin (data not shown). The peak positions (*ν*<sup>1</sup> = 1525 cm−1 and *ν*<sup>2</sup> = 1157 cm−1) in the isolated Raman spectrum shown in **Figure 3C** are consistent with the purified lutein spectrum in **Figure 1** and by Collins et al. [27]. The slight deviation is likely a result of the violaxanthin contribution as the *ν*<sup>1</sup> vibration of violaxanthin is typically 1528–1530 cm−1 [46]. For compari‐ son, Panels A and B show the correlated bright‐field and confocal fluorescence chlorophyll images, respectively, for the cell shown in **Figure 3D**. Individual component images are shown in grayscale as well as the red‐green merged image (please refer to the online version for color figure). Green corresponds to the carotenoid and red corresponds to the protochloro‐ phyllide pigments in these figures. Localization is confined to the plastids (bright foci ∼1 µm in size within the protoplast). Interestingly, a high degree of variation in carotenoid and chlo‐ rophyll composition within individual plastids is observed. An example of this heterogeneity is indicated by the yellow arrows in the merged image of **Figure 3D**. These arrows highlight plastids that have predominantly carotenoid (green), predominantly protochlorophyllide (red), or roughly equal amounts of both (yellowish‐orange). These differences in relative abundance could arise from differences in the developmental phase or carotenoid abundance. The observed heterogeneities raise important scientific questions about carotenoid biogenesis that Raman‐spectroscopic imaging is well poised to answer in future research.

#### **3.3. Approaching the limits of spatial resolution:** *Synechocystis* **6803**

Cyanobacteria and closely related organisms are thought to be the evolutionary ancestors to chloroplasts found in plants. Cyanobacteria perform photosynthesis to convert light energy to chemical energy and play key roles in the ecology of the earth. Light is harvested by photo‐ synthetic antennae, which are pigment‐protein complexes composed of pigments with vary‐ ing absorption and emission properties in order to "funnel" the energy to the reaction center where it is converted to chemical energy [47]. Carotenoids are integrated into the membrane‐ associated photosynthetic antennae complexes as well as the photosystems of all cyanobac‐ teria and fundamentally coupled to the processes of light harvesting and photosynthesis. Unfortunately, carotenoids in cyanobacteria are often quite difficult to localize *in situ* because of the very small size of many of the cyanobacteria (1.5–4 µm for the most species) and the co‐localization in complexes with highly fluorescent light‐harvesting pigments, like phyco‐ bilins and chlorophylls. Previous work with hyperspectral confocal fluorescence microscopy has demonstrated sensitivity for localization carotenoids via resonance Raman signatures in

**Figure 3.** Confocal Raman‐spectroscopic imaging of carotenoids and protochlorophyllide in etiolated maize protoplasts. (A) Bright‐field image of several protoplasts with red square highlighting single protoplast imaged in B and D. (B) Confocal fluorescence image of cell highlighted in A (chlorophyll emission channel). (C) Spectral components of MCR model from the analysis of confocal Raman image of maize protoplasts. (D) Left panel: protochlorophyllide concentration map. Center panel: carotenoid concentration map. Right panel: Merged red and green color overlay, where the protochlorophyllide image is assigned to the red channel and carotenoid image is assigned to the green channel. Arrows highlight examples of plastid heterogeneity. (E) and (F). Two additional protoplasts. Image panels for E and F are the same as in D. Scale bars = 5 µm. (note: **Figure 3** is best viewed in color. Please refer to the online version of this chapter for the color version of **Figure 3**).

*Synechocystis* 6803 [48]; however, the spectral resolution of such a system is not sufficient to resolve different carotenoids.

*Synechocystis* 6803 has emerged as a model cyanobacterium due to the ease of genetic manip‐ ulation. Although *Synechocystis* 6803 cells are quite small (1.5–2 µm), Raman spectroscopic imaging can provide information about subcellular carotenoid localization. **Figure 4** shows the results of Raman spectroscopic imaging of wild‐type *Synechocystis* 6803 cells. The carot‐ enoid spectrum isolated in **Figure 4A** has *ν*<sup>1</sup> vibration appearing at 1515 cm−1 and *ν*<sup>2</sup> vibration at 1155 cm−1, identifying that carotenoid most likely corresponds to beta‐carotene, one of the major carotenoid species in *Synechocystis* 6803. The phycobilin spectrum peak is centered at ∼3250 cm−1 (equivalent to 643.2 nm) and thus corresponds to phycocyanin, the primary pig‐ ment found in the antenna structure. The fluorescence emission of phycocyanin is ∼645 nm. While subcellular details are somewhat limited given the small number of pixels in each cell, cell‐to‐cell differences in localization patterns are clearly evident. For example, in **Figure 4**, the carotenoids in the dividing cell on the left are concentrated into one small foci on each cell, whereas in the other, two cells have carotenoids localized more uniformly through the thylakoid membranes that house the light‐harvesting complexes, similar to the phycocyanin.

**Figure 4.** Results from confocal Raman‐spectroscopic imaging of wild‐type *Synechocystis* 6803 cells. (A) MCR identified pure component spectra. A fourth component consisting of a tail at ∼3500 cm−1 was isolated as well and corresponds to chlorophyll. It was omitted for simplicity. (B) MCR concentration maps indicating carotenoid (bottom row) and phycobilin (top row) abundance for three different cells. Image intensities have been independently scaled between for maximum visibility; however, all intensity scales are within 20% of each other. A concentration map for the offset component is not shown. Scale bar = 1 µm.

Cell‐to‐cell heterogeneity and population dynamics are important to understand cyanobacte‐ rial adaptation to changes in the abiotic or biotic environment and could be addressed with Raman spectroscopic imaging in the future. It is also important to note that the images in **Figure 4** were collected under slightly less than diffraction‐limited conditions. Improvements in spatial resolution are possible with a more optimal arrangement.

#### **4. Conclusions**

*Synechocystis* 6803 [48]; however, the spectral resolution of such a system is not sufficient to

**Figure 3.** Confocal Raman‐spectroscopic imaging of carotenoids and protochlorophyllide in etiolated maize protoplasts. (A) Bright‐field image of several protoplasts with red square highlighting single protoplast imaged in B and D. (B) Confocal fluorescence image of cell highlighted in A (chlorophyll emission channel). (C) Spectral components of MCR model from the analysis of confocal Raman image of maize protoplasts. (D) Left panel: protochlorophyllide concentration map. Center panel: carotenoid concentration map. Right panel: Merged red and green color overlay, where the protochlorophyllide image is assigned to the red channel and carotenoid image is assigned to the green channel. Arrows highlight examples of plastid heterogeneity. (E) and (F). Two additional protoplasts. Image panels for E and F are the same as in D. Scale bars = 5 µm. (note: **Figure 3** is best viewed in color. Please refer to the online version

*Synechocystis* 6803 has emerged as a model cyanobacterium due to the ease of genetic manip‐ ulation. Although *Synechocystis* 6803 cells are quite small (1.5–2 µm), Raman spectroscopic imaging can provide information about subcellular carotenoid localization. **Figure 4** shows the results of Raman spectroscopic imaging of wild‐type *Synechocystis* 6803 cells. The carot‐

at 1155 cm−1, identifying that carotenoid most likely corresponds to beta‐carotene, one of the major carotenoid species in *Synechocystis* 6803. The phycobilin spectrum peak is centered at ∼3250 cm−1 (equivalent to 643.2 nm) and thus corresponds to phycocyanin, the primary pig‐ ment found in the antenna structure. The fluorescence emission of phycocyanin is ∼645 nm. While subcellular details are somewhat limited given the small number of pixels in each cell, cell‐to‐cell differences in localization patterns are clearly evident. For example, in **Figure 4**, the carotenoids in the dividing cell on the left are concentrated into one small foci on each cell, whereas in the other, two cells have carotenoids localized more uniformly through the thylakoid membranes that house the light‐harvesting complexes, similar to the phycocyanin.

vibration appearing at 1515 cm−1 and *ν*<sup>2</sup>

vibration

resolve different carotenoids.

44 Carotenoids

of this chapter for the color version of **Figure 3**).

enoid spectrum isolated in **Figure 4A** has *ν*<sup>1</sup>

Localizing carotenoids in living cells and tissues is challenging due to the complex biological matrices of the living cells and intense sometimes colocated interfering fluorescence. Raman spectroscopic imaging coupled with multivariate curve resolution analysis provides the nec‐ essary spatial and chemical resolution to identify highly similar carotenoids even in the midst of highly overlapping, strong chlorophyll emission and complex backgrounds. Three exam‐ ples were presented that highlight different advantages of the methodology for investigating photosynthetic organisms. Recent technological advances in detector speed and sensitivity will most likely catalyze future investigations of carotenoid biogenesis in single cells, includ‐ ing population dynamics and response to changing environmental conditions, by facilitating time‐course studies that were previously prohibitive given the long scan times required for Raman spectroscopic imaging. These capabilities are anticipated to impact a variety of research areas including carbon partitioning and utilization, microbial ecology, and crop analytics.

#### **Acknowledgements**

The authors are grateful to the following people for their assistance with the research pre‐ sented in this chapter: Anthony McDonald for assistance with the collection of Raman spec‐ troscopic image data; Meghan Dailey for culturing the *Synechocystis* 6803; Stephen Anthony for the current implementation of the MCR analysis software used to deconvolve spectral components; Howland Jones, Mark Van Benthem, David Melgaard, Mike Keenan, and David Haaland for original development of the MCR algorithm and software; Wim Vermaas, Arizona State University, for the gift of the purified carotenoid standards, Himadri Pakrasi, Washington University, St. Louis, for the gift of *Synechocystis sp*. PCC 6803; Qiang Hu for the gift of the *H. pluvialis.*

This research was primarily supported as part of the Photosynthetic Antenna Research Center (PARC), an Energy Frontier Research Center funded by the U.S. Department of Energy (DOE), Office of Science, Basic Energy Sciences (BES), under Award # DE‐SC 0001035 (Maize proto‐ plasts imaging, Synechocystis imaging, writing), by Sandia National Laboratories' Laboratory Directed Research and Development (LDRD) Program under Award # 141528 (*H. pluvialis* imaging), and by funding from the National Institutes of Health (GM081160) (to ETW) for research on carotenoids in maize protoplasts. Sandia National Laboratories is a multimis‐ sion laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy's National Nuclear Security Administration under contract DE‐AC04‐94AL85000.

#### **Author details**

Jerilyn A. Timlin1 \*, Aaron M. Collins2 , Thomas A. Beechem<sup>1</sup> , Maria Shumskaya3,5 and Eleanore T. Wurtzel3,4


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This research was primarily supported as part of the Photosynthetic Antenna Research Center (PARC), an Energy Frontier Research Center funded by the U.S. Department of Energy (DOE), Office of Science, Basic Energy Sciences (BES), under Award # DE‐SC 0001035 (Maize proto‐ plasts imaging, Synechocystis imaging, writing), by Sandia National Laboratories' Laboratory Directed Research and Development (LDRD) Program under Award # 141528 (*H. pluvialis* imaging), and by funding from the National Institutes of Health (GM081160) (to ETW) for research on carotenoids in maize protoplasts. Sandia National Laboratories is a multimis‐ sion laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy's National Nuclear Security

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## **The Biochemistry and Antioxidant Properties of Carotenoids**

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2013. 336 p.

50 Carotenoids

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67592

#### **Abstract**

Carotenoids are one of the most widespread pigment groups distributed in nature, and more than 700 natural carotenoids have been described so far, and new carotenoids are introduced each year. Carotenoids are derived from 4 terpenes, including totally 40 carbon atoms. Carotenoids are naturally synthesized by cyanobacteria, algae, plants, some fungi, and some bacteria, but not made by mammals. Lately, the beneficial properties of α-carotene, β-carotene, γ-carotene, lycopene, phytoene, phytofluene, lutein, zeaxanthin, β-cryptoxanthin, astaxanthin, and fucoxanthin carotenoids in prevention of various diseases, such as tumor formation, cardiovascular, and vision, have been documented due to their roles as antioxidants, activation in certain gene expression associated with cell-to-cell communication, provitamin A activity, modulation of lipoxygenase activity, and immune response. In this chapter, in addition to biochemical properties of carotenoids, how the structure of these molecules influences the oxidative stress in health and reducing the risk of formation of various diseases will be described.

**Keywords:** carotenoids, antioxidants, biochemistry, health, toxicity

#### **1. Introduction**

Overproduction of reactive oxygen and nitrogen species, such as superoxide and peroxide radicals, plays important roles in the formation of breast, cervical, ovarian, and colorectal cancer in addition to some other malignancies in the cardiovascular system and eye. The antioxidant property of carotenoids may result from its double carbon-carbon bonds interacting with each other via conjugation and causing electrons in the molecule to move freely across these areas of the molecule. Carotenoid intake from food sources reduces the risk of breast, lung, head and neck, cervical, ovarian, colorectal, and prostate cancers and cardiovascular

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

or eye diseases due to their roles as antioxidants. It has been reported that carotenoids can directly interact with some free radical species.

#### **2. The biochemistry and metabolism of main carotenoids**

#### **2.1. Terpenes**

Terpenes are structures derived from isoprene chains. The chemical formula of the isoprene molecule is CH2 ═C(CH3 )─CH═CH2 (2-methyl-1,3-butadiene) [1, 2]. There are two double bonds in the molecule and these bonds are conjugated (**Figure 1**). When 5-carbon containing isoprene molecules are polymerized, compounds called terpenes are formed [3]. This group includes biologically very important molecules. Some among these are lycopene, β-carotene, vitamin A, and squalene [4].

Terpenes are biosynthetically derived from isoprene units, the chemical formula of this unit is C5 H8 , and the basic molecular formulas of terpenes are multiplied, that is, (C5 H8 ) <sup>n</sup> (n is the number of joined isoprene units). The isoprene units can be connected from head to tail to form straight chains or rings. The isoprene unit is a building brick that is widely used in nature [5, 6].

Terpenes can be classified according to the number of isoprene units used, such as hemiterpenes (prenol and isovaleric acid); one isoprene unit (5C), monoterpenes (geraniol and limonene); two isoprene units (10C), sesquiterpenes (farnesol); three isoprene units (15C), diterpenes (quinagolides, sembren, and taxadiene); four isoprene units (20C), sesterterpenes; five isoprene units (25C), triterpenes (squalene); six isoprene units (30C), tetraterpenes (carotenoids); and eight isoprene units (40C), politerpenes, which include great number of isoprene units (natural rubber) [6–9].

Monoterpenes, sesquiterpenes, diterpenes, and sesterterpenes are formed by head-to-tail association of isoprene units, and triterpenes and tetraterpenes (carotenoids) are formed by head-to-head association [9].

The most important group of terpenes is carotenoids with the C40H64 molecular formula which is formed by a tetraterpene containing eight isoprene units [8]. Carotenoids are terpene

**Figure 1.** Chemical formula for isoprene.

group materials which are formed by binding of 5-carbon-containing isoprene molecules, and they have straight chain structure formed by the condensation of isoprene molecules [1]. Carotenoids which colors vary from light yellow to dark red stemming from the double bonds are soluble in organic solvents and oils like other lipids [10].

#### **2.2. Biosynthesis of terpenes and carotenoids**

or eye diseases due to their roles as antioxidants. It has been reported that carotenoids can

Terpenes are structures derived from isoprene chains. The chemical formula of the isoprene

bonds in the molecule and these bonds are conjugated (**Figure 1**). When 5-carbon containing isoprene molecules are polymerized, compounds called terpenes are formed [3]. This group includes biologically very important molecules. Some among these are lycopene, β-carotene,

Terpenes are biosynthetically derived from isoprene units, the chemical formula of this unit

the number of joined isoprene units). The isoprene units can be connected from head to tail to form straight chains or rings. The isoprene unit is a building brick that is widely used in

Terpenes can be classified according to the number of isoprene units used, such as hemiterpenes (prenol and isovaleric acid); one isoprene unit (5C), monoterpenes (geraniol and limonene); two isoprene units (10C), sesquiterpenes (farnesol); three isoprene units (15C), diterpenes (quinagolides, sembren, and taxadiene); four isoprene units (20C), sesterterpenes; five isoprene units (25C), triterpenes (squalene); six isoprene units (30C), tetraterpenes (carotenoids); and eight isoprene units (40C), politerpenes, which include great number of isoprene

Monoterpenes, sesquiterpenes, diterpenes, and sesterterpenes are formed by head-to-tail association of isoprene units, and triterpenes and tetraterpenes (carotenoids) are formed by

The most important group of terpenes is carotenoids with the C40H64 molecular formula which is formed by a tetraterpene containing eight isoprene units [8]. Carotenoids are terpene

, and the basic molecular formulas of terpenes are multiplied, that is, (C5

(2-methyl-1,3-butadiene) [1, 2]. There are two double

H8 ) <sup>n</sup> (n is

**2. The biochemistry and metabolism of main carotenoids**

)─CH═CH2

directly interact with some free radical species.

═C(CH3

**2.1. Terpenes**

52 Carotenoids

molecule is CH2

is C5 H8

nature [5, 6].

vitamin A, and squalene [4].

units (natural rubber) [6–9].

head-to-head association [9].

**Figure 1.** Chemical formula for isoprene.

Carotenoids which are tetraterpenes and biological pigments including eight isoprene units are found in plants and some other photosynthetic microorganisms (naturally occurring cyanobacteria, algae, plants, some fungi, and some bacteria) [11].

Acetyl CoA, which is synthesized directly from free acetate as well as the oxidation of sugars and condensation of pyruvic acid or fatty acids, is used as a precursor in the synthesis of mevalonic acid in addition to synthesis of many natural compounds [9, 12]. First, two acetyl CoA molecules enter the reaction to give acetoacetyl-CoA, and then β-hydroxy-βmethylglutaryl-CoA (HMG-CoA) is obtained via HMG-CoA synthase with another acetyl CoA. The reduction of the ester group in HMG-CoA by NADPH and HMG-CoA reductase (the pathway's rate limiting enzyme) results in mevalonic acid formation. Mevalonic acid later forms mevalonate 5-phosphate using a total of two molecules of ATP by mevalonate kinase, and then mevalonate 5-phosphate forms mevalonate 5-diphosphate by phosphomevalonate kinase (mevalonate phosphate kinase). Isopentenyl diphosphate (IPP) building blocks from decarboxylation of mevalonate diphosphate via mevalonate diphosphate carboxylase produce isoprene chains. IPP:dimethylallyl-PP isomerase converts IPP to dimethylallyl diphosphate (DMAPP) which is an acceptor by successive transfer of isopentenyl residues [9, 13]. After the release of the diphosphate, the hemiterpenes are produced from DMAPP. Upon condensation of DMAPP and IPP via geranyl-PP synthase (dimethylallyl transferase), geranyl-PP forms. Monoterpenes are produced by geranyl-PP, which is the precursor of volatile oils. Likewise, farnesyl-PP is formed by condensation of geranyl-PP with IPP via farnesyl-PP synthase (geranyl transferase). Sesquiterpenes are produced by condensation of farnesyl-PP. In addition, two molecules of farnesyl-PP give rise to triterpenes [13, 14]. Squalene, a triterpenic compound, is the precursor of sterols that participate in membrane structure. Geranylgeranyl-PP is formed from head-to-tail condensation of farnesyl-PP and IPP by geranylgeranyl-PP synthase (farnesyl transferase). Geranylgeranyl-PP also causes the formation of diterpenes (**Figure 2**) [9].

Tetraterpenes (carotenoids) are formed by the condensation of two molecules of geranylgeranyl-PP [9, 15]. The first carotenoid formed by prephytoene diphosphate in this step is phytoene which is found in green plants in association with chlorophyll. This first stable carotenoid synthesis takes place via the phytoene synthase. The second product of carotenoid biosynthesis, phytofluene, forms as a result of desaturation (catalyzed by the enzyme phytoene desaturase), which leads to the formation of saturated double bonds. Phytofluene, after having a series of dehydrogenation reactions, forms a symmetric molecule, lycopene, containing 13 double bonds. The next step, α-carotene, β-carotene, and γ-carotene are produced from lycopene via ring formation in the end groups by lycopene cyclase. α- and β-carotenes are the xanthophylls as a result of hydroxylation by β-carotene hydroxylase (**Figure 2**) [16, 17].

**Figure 2.** Biosynthesis of terpenes and carotenoids.

#### **2.3. Carotenoids**

More than 700 natural carotenoids have been identified, and new carotenoids have been added to this number every year [18–20]. Carotenoids are found in red-, yellow-, and orangecolored fruits and vegetables as well as in all green leafy vegetables [10].

Carotenoids are found in plant tissues as free forms (crystalline or amorphous) are dissolved form in an oily solvent. They may also be esterified with fatty acids or complexed with sugars and proteins [17]. The conjugate double-bond structure found in carotenoids also determines biological functions, such as absorption of light during photosynthesis, energy transfer, and protection from harmful effects of light on the cells during the photosynthesis. The presence of carotenoids also determines the characteristic color of these compounds [21].

Carotenoids are present in large quantities in the Leydig cells, which produce steroid hormones, and in the outer layer of the adrenal gland [22]. Carotenoids are also found in fresh broccoli, vegetables, butter, egg yolks, and animal-based foods [23].

#### **2.4. Classification of carotenoids according to their structures**

According to structures of carotenoids, two classes are distinguished as hydrocarbon carotenoids and xanthophylls (**Figure 3**) [24]. The apolar characteristics of hydrocarbon carotenoids are also called carotenes which include α-carotene, β-carotene, γ-carotene, lycopene, phytoene, and phytofluene. Xanthophylls are more polar and contain oxygen in the form of methoxy, hydroxy, keto, carboxy, and epoxy positions. Examples of xanthophylls include lutein, zeaxanthin, β-cryptoxanthin, astaxanthin, and fucoxanthin [25, 26]. Carotenoids are also grouped as carotenoids with or without ring groups at the ends of the chain [26].

#### *2.4.1. Hydrocarbon carotenoids*

**Figure 2.** Biosynthesis of terpenes and carotenoids.

54 Carotenoids

Carotenoids with hydroaromatic rings are called carotenes [23]. These rings are located at the both ends of the four isoprene molecules. Thus, there are two hydroaromatic rings in each carotene molecule. These hydrocarbons are called ionospheric rings and have three ionone rings. These are α-, β-, and pseudo-ionone rings. α- and β-ionone rings are closed rings and include one double bond. The position of the double bond α is different in the β-ionone rings and pseudo rings. The pseudo-ionone ring includes two double bonds [3].

Carotene is a biochemically synthesized terpene from eight isoprene units [4, 8]. There are three main types, α-carotene, β-carotene, and γ-carotene. The carotenoids are precursors of vitamin A and converted into vitamin A in the body. From α-carotene and γ-carotene, one molecule of vitamin A while from β-carotene two molecules of vitamin A are synthesized [27–29].

α-Carotene molecule has β-ionone ring at one end and α-ionone ring at the other end. There are four molecules of polymerized isoprene molecules between them. The structure of vitamin A is like half of an α-carotene. Vitamin A has a β-ionone ring at one end and two isoprene molecules attached to it [27]. The second most common form of carotene, α-carotene, is found mostly in carrots, sweet potatoes, squash, tomatoes, red peppers [30], and dark green vegetables [26].

**Figure 3.** Classification of carotenoids according to their structures.

β-Carotene is a fat-soluble provitamin. Its active form is vitamin A [31]. The difference of β from α is that it carries the β-ionone ring at both ends. When β-carotene is divided into two molecules, vitamin A is synthesized [29]. It is found in fruits, cereals, vegetables (carrots, green plants, pumpkin, spinach), and oils [32].

γ-Carotene includes β-ionone ring at one end and the pseudo-ionone ring at the other end, and when the molecule is split, one molecule of vitamin A is synthesized [3].

Lycopene is an aliphatic carotenoid [33]. Lycopene is found among tomatoes, watermelons, pink grapefruit, and rosehip. Since lycopene dissolves in the oil, the presence of oils greatly increases its absorption by the digestive system [34].

Phytoene is a 40-carbon intermediate in the biosynthesis of phytoene carotenoids. Phytoene is a symmetric molecule containing three conjugated double bonds [35].

Phytofluene is an orange-colored carotenoid pigment found naturally in tomatoes and other vegetables [36].

#### *2.4.2. Xanthophylls*

β-Carotene is a fat-soluble provitamin. Its active form is vitamin A [31]. The difference of β from α is that it carries the β-ionone ring at both ends. When β-carotene is divided into two molecules, vitamin A is synthesized [29]. It is found in fruits, cereals, vegetables (carrots,

γ-Carotene includes β-ionone ring at one end and the pseudo-ionone ring at the other end,

Lycopene is an aliphatic carotenoid [33]. Lycopene is found among tomatoes, watermelons, pink grapefruit, and rosehip. Since lycopene dissolves in the oil, the presence of oils greatly

Phytoene is a 40-carbon intermediate in the biosynthesis of phytoene carotenoids. Phytoene is

Phytofluene is an orange-colored carotenoid pigment found naturally in tomatoes and other

and when the molecule is split, one molecule of vitamin A is synthesized [3].

a symmetric molecule containing three conjugated double bonds [35].

green plants, pumpkin, spinach), and oils [32].

**Figure 3.** Classification of carotenoids according to their structures.

increases its absorption by the digestive system [34].

vegetables [36].

56 Carotenoids

Xanthophylls contain oxygen atoms and are yellow pigments commonly found in nature [37].

Lutein is a dihydroxy-carotene formed from carotenoids with an alcohol group containing hydroaromatic α structure [38]. Its both ionone rings carry hydroxyl groups [39]. Lutein is a substance that gives color to chicken fat, egg yolk, and chicken feathers [40]. That yellowcolored lutein found in plants is an organic colorant on leaves of green vegetables, such as spinach and black pepper. It is generally found in covalent interactions within fatty acids [20].

The chemical formulas of lutein and zeaxanthin are the same; in other words, they are isomers, but they are not stereoisomers. The difference between the two is the position of a double bond in the end ring [17, 39].

Zeaxanthin is one of the most common carotenoid alcohols found in the nature. It is a pigment that gives its color to maize, saffron, and many other plants. When the zeaxanthin breaks down, picrocrocin, which is responsible for the taste and aroma of the saffron, forms [41].

β-Cryptoxanthin carotenoid has an alcohol group having a hydroaromatic structure including an OH group in one of its ionic rings. Since the other ring is the β-ionone ring, one molecule of vitamin A can be synthesized from it [27].

β-Cryptoxanthin is a natural carotenoid pigment. β-Cryptoxanthin is found in fruits and vegetables, such as mandarin, red pepper, and zucchini, and has important functions for human health. β-Cryptoxanthin is closely related to β-carotene. Only one OH group was added to β-cryptoxanthin. Although β-carotene is present in large quantities in a large number of fruits and vegetables, β-cryptoxanthin is present in small number of food sources but at high concentrations [42].

Astaxanthin is a keto-carotenoid and is a zeaxanthin metabolite, containing both hydroxyl and ketone functional groups [17, 43]. Astaxanthin is found in microalgae, yeast, salmon, trout, shrimp, shellfish, and some birds' feathers [44].

Fucoxantin, found in the chloroplasts of algae and mosses, gives them their brown or olive green color [45].

### **3. Antioxidant functions of carotenoids at molecular level for health and toxicity**

The interest in carotenoids found in plants over the last years is not only due to their A provitamin activity but also due to their reduction of oxidative stress in the organism by capturing oxygen radicals, that is, their antioxidant effects [46]. Free oxygen radicals play an important role in the stress-related tissue damage and pathogenesis of inflammation. The imbalance between protective and damaging mechanisms results in acute inflammation accompanied by neutrophil infiltration [47, 48]. Superoxide radicals formed by neutrophils react with lipids and cause lipid peroxidation [49–51]. Carotenoids can inhibit active radicals by transferring electrons, giving hydrogen atoms to radicals or attaching to radicals [16].

The number of conjugate double bonds in their structure is closely related to the superoxide inhibitory effect of carotenoids [52, 53]. Carotenoids could remove singlet oxygen and peroxyl radicals from reaction medium and also prevent their formation [53–55]. It is stated that carotenoids can inhibit cell renewal and transformation and regulate gene expression that plays a role in the formation of certain types of cancer. On the other hand, in some studies it has been shown that carotenoids could stimulate cancer in some cases. For example, it has been reported that in smokers, synthetic β-carotene does not create protective activity against lung cancer and cardiovascular diseases and even it fastens the progression of an aforementioned diseases [56–58].

The activity of carotenoids as antioxidants also depends on their interaction with other antioxidants, such as vitamins E and C [58]. In addition, some carotenoids and their metabolites activate the nuclear factor-erythroid 2-related factor-2 (Nrf2) transcription factor, which triggers antioxidant gene expression in certain cells and tissues [58, 59]. Thus, a number of chronic diseases characterized by oxidative stress, inflammation, and impaired mitochondrial function have been reported to reduce Nrf2 expression in some animal models. Elevation of Nrf2 has been shown to be effective in the prevention and treatment of many chronic inflammatory diseases, including various cardiovascular, renal, or pulmonary diseases; toxic liver damage; metabolic syndrome; sepsis; autoimmune disorders; inflammatory bowel disease; and HIV infection [58]. Prevention of low-density lipoproteins (LDL) oxidation by carotenoids has been suggested to be the basis of carotenoids' protective activity against coronary vascular disease [26].

The signaling pathways and molecules influenced by carotenoids to prevent various diseases, such as cancer and cardiovascular diseases, involve growth factor signaling members, cell cycle-associated proteins, differentiation-related proteins, retinoid-like receptors, antioxidant response element, nuclear receptors, AP-1 transcriptional complex, the Wnt/β-catenin pathway, angiogenic proteins, and inflammatory cytokines. During the treatment of cardiovascular and eye diseases and cancer, the dose and the exposure time of β-carotene, lycopene, lutein, and zeaxanthin have been reported to be crucial [37].

α-Carotene intake might decrease the development of non-Hodgkin lymphoma [60]. α-Carotene concentration in the blood may be associated with the development of various cancers [30].

β-Carotene, which is accepted as a provitamin of vitamin A (retinol) which is required for the fulfillment of visual functions [61], effects the oxidative damage that formed on cellular lipids, proteins, and DNA as a result of sunlight and causes the formation of erythema, premature aging of the skin, development of photodermatitis, and skin cancer. β-Carotene protects the skin from harmful effects of UV light by its prevention of reactive oxygen species formation and anti-inflammatory properties [62, 63].

Studies have shown that β-carotene's provitaminase activity or antioxidant properties prevent diseases, such as arthrosclerosis, cataracts, multiple sclerosis, and some types of cancers [64]. Moreover in recent studies, β-carotene has been proposed to decrease cell proliferation and induce apoptosis of various cancer cell lines by inhibiting calcium-/calmodulin-dependent protein kinase IV [65]. In another study, β-carotene has been reported to have anticancer stem cell actions on neuroblastoma, and this anticancer action is enhanced by retinoic acid receptor β [66].

The number of conjugate double bonds in their structure is closely related to the superoxide inhibitory effect of carotenoids [52, 53]. Carotenoids could remove singlet oxygen and peroxyl radicals from reaction medium and also prevent their formation [53–55]. It is stated that carotenoids can inhibit cell renewal and transformation and regulate gene expression that plays a role in the formation of certain types of cancer. On the other hand, in some studies it has been shown that carotenoids could stimulate cancer in some cases. For example, it has been reported that in smokers, synthetic β-carotene does not create protective activity against lung cancer and cardiovascular diseases and even it fastens the progression of an aforementioned

The activity of carotenoids as antioxidants also depends on their interaction with other antioxidants, such as vitamins E and C [58]. In addition, some carotenoids and their metabolites activate the nuclear factor-erythroid 2-related factor-2 (Nrf2) transcription factor, which triggers antioxidant gene expression in certain cells and tissues [58, 59]. Thus, a number of chronic diseases characterized by oxidative stress, inflammation, and impaired mitochondrial function have been reported to reduce Nrf2 expression in some animal models. Elevation of Nrf2 has been shown to be effective in the prevention and treatment of many chronic inflammatory diseases, including various cardiovascular, renal, or pulmonary diseases; toxic liver damage; metabolic syndrome; sepsis; autoimmune disorders; inflammatory bowel disease; and HIV infection [58]. Prevention of low-density lipoproteins (LDL) oxidation by carotenoids has been suggested to be the basis of carotenoids' protective activity against coronary

The signaling pathways and molecules influenced by carotenoids to prevent various diseases, such as cancer and cardiovascular diseases, involve growth factor signaling members, cell cycle-associated proteins, differentiation-related proteins, retinoid-like receptors, antioxidant response element, nuclear receptors, AP-1 transcriptional complex, the Wnt/β-catenin pathway, angiogenic proteins, and inflammatory cytokines. During the treatment of cardiovascular and eye diseases and cancer, the dose and the exposure time of β-carotene, lycopene,

α-Carotene intake might decrease the development of non-Hodgkin lymphoma [60]. α-Carotene concentration in the blood may be associated with the development of various

β-Carotene, which is accepted as a provitamin of vitamin A (retinol) which is required for the fulfillment of visual functions [61], effects the oxidative damage that formed on cellular lipids, proteins, and DNA as a result of sunlight and causes the formation of erythema, premature aging of the skin, development of photodermatitis, and skin cancer. β-Carotene protects the skin from harmful effects of UV light by its prevention of reactive oxygen species formation

Studies have shown that β-carotene's provitaminase activity or antioxidant properties prevent diseases, such as arthrosclerosis, cataracts, multiple sclerosis, and some types of cancers [64]. Moreover in recent studies, β-carotene has been proposed to decrease cell proliferation and induce apoptosis of various cancer cell lines by inhibiting calcium-/calmodulin-dependent

lutein, and zeaxanthin have been reported to be crucial [37].

and anti-inflammatory properties [62, 63].

diseases [56–58].

58 Carotenoids

vascular disease [26].

cancers [30].

Lycopene, synthesized by many plants and microorganisms, is an antioxidant that cannot be synthesized by animals and humans [67]. Conjugated dienes are active in creating antioxidant activity [46], and lycopene is reported to have a higher antioxidant capacity than other carotenoids, and in particular among carotenoids, lycopene inhibits the risk of prostate cancer [52, 68].

The use of lycopene can reduce the risk of cardiovascular disease, diabetes, osteoporosis, and prostate, esophagus, colorectal, and mouth cancer risk. Recent studies indicate that lycopene intake has protective functions against cardiovascular diseases by lowering high-density lipoprotein (HDL)-associated inflammation [69]. It was proposed that there is an inverse association between the occurrence of pancreatic cancer and dietary lycopene intake together with vitamin A and β-carotene [70]. β-Carotene 9′,10′-oxygenase which is a key enzyme for the metabolism of lycopene has been proposed to have an important roles to prevent prostate cancer progression by inhibiting NF-κB signaling [71].

In neurodegenerative diseases, lycopene has been reported to increase permeability of bloodbrain barrier, and a significant reduction in lycopene levels in diseases, such as Parkinson's disease and vascular dementia, has been observed. It has also been suggested that lycopene bestow protection against amyotrophic lateral sclerosis (ALS) impairment in humans [67].

The phytoene and phytofluene found in the diet accumulate in the human skin [72]. The dehydration of these carotenoids has a protective effect on the skin due to its UV absorber, antioxidant, and anti-inflammatory properties [73].

Zeaxanthin is one of two carotenoids found in the retina. Zeaxanthin mostly found at the center of the macula and lutein mostly found at the peripheral retina [39, 74]. Lutein and zeaxanthin are responsible for the formation of yellow pigment in the retina. Yellow pigments play an active role in protecting the eye from light and can prevent retinal damage [56].

β-Cryptoxanthin acts as a chemopreventive agent against lung cancer. β-Cryptoxanthin has been reported to decrease lung cancer through downregulating neuronal nicotinic acetylcholine receptor α7/PI3K signaling pathway [75]. In addition to lung cancer, β-cryptoxanthin enhances the action of a chemotherapeutic agent, oxaliplatin, to treat colon cancer [76]. Moreover, high lycopene and β-cryptoxanthin including diet might protect against aggressive prostate cancer [77].

Astaxanthin has an important role in the treatment and prevention of certain diseases by its antitumor properties and protection against free radicals, oxidation of basic polyunsaturated fatty acids, and UV light effect on cell membranes [20, 78]. For example, high concentrations of astaxanthin may suppress mammary carcinoma [79]. In addition, astaxanthin has beneficial health effects against the formation of prostate carcinogenesis and tumor progression by reactivating the expression of Nrf2 and Nrf2-target genes through epigenetic modification and chromatin remodeling [80]. Astaxanthin has been shown to have antitumorigenic and anti-inflammatory effects on human lung cancer cell lines by inhibiting ERK1/2 activity [81].

In vitro studies indicate that fucoxanthin stimulates apoptosis and decreases proliferation and migration in glioma cancer cell lines U87 and U251 through Akt/mTOR and p38 pathway inhibition [82]. Fucoxanthin intake may have health benefits for the treatment of Alzheimer's disease by inhibiting acetylcholinesterase and enhancing brain-derived neurotrophic factor expression [83].

Supplementation of carotenoids by humans may have some beneficial biological actions against the formation of numerous diseases, such as cancer, cardiovascular diseases, or their skin. In addition to nutrient supplements in humans, carotenoids have applications in animal feed. Optimal health promoting actions of carotenoids depend on their proper doses, lengths of treatment, and combinations of carotenoids to maximize their effects. In this book chapter, latest findings on the biochemical and antioxidant activities of main carotenoids and their possible mechanisms of action will be presented.

#### **Author details**

#### Oguz Merhan

Address all correspondence to: oguzmerhan@hotmail.com

Department of Biochemistry, Faculty of Veterinary, Kafkas University, Kars, Turkey

#### **References**


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In vitro studies indicate that fucoxanthin stimulates apoptosis and decreases proliferation and migration in glioma cancer cell lines U87 and U251 through Akt/mTOR and p38 pathway inhibition [82]. Fucoxanthin intake may have health benefits for the treatment of Alzheimer's disease by inhibiting acetylcholinesterase and enhancing brain-derived neurotrophic factor

Supplementation of carotenoids by humans may have some beneficial biological actions against the formation of numerous diseases, such as cancer, cardiovascular diseases, or their skin. In addition to nutrient supplements in humans, carotenoids have applications in animal feed. Optimal health promoting actions of carotenoids depend on their proper doses, lengths of treatment, and combinations of carotenoids to maximize their effects. In this book chapter, latest findings on the biochemical and antioxidant activities of main carotenoids and their

expression [83].

60 Carotenoids

**Author details**

Oguz Merhan

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res. 2013.07.001

ijms17111781

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Address all correspondence to: oguzmerhan@hotmail.com

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## **β‐Carotene and Free Radical Reactions with Nitrogen Oxides**

Sara N. Mendiara and Luis J. Perissinotti

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67683

#### **Abstract**

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The following presentation is based on experimental work we have already developed and published. We investigated the nitrogen oxides in different solvents and analyzed their reaction with β‐carotene. The electron paramagnetic resonance spectroscopy (EPR) and ultraviolet and visible (UV‐vis) spectroscopy were applied to investigate the reaction of β‐carotene with nitrogen dioxide and nitric oxide in both pure dioxane and dioxane/ water solvent. Free radicals were detected and evaluated with the EPR technique, which is highly selective and sensitive. A reaction mechanism was proposed on the basis of the experimental kinetic and EPR results. The validity of the mechanism was checked by applying simulation set up conditions that reproduced the results achieved. The radical intermediates proposed in the reaction: the β‐carotene neutral radicals and the cyclic nitroxide neutral radicals were theoretically studied. For that purpose, the density functional theory (DFT) level was applied, selecting the most suitable method, the unre‐ stricted Becke‐style 3‐parameter with the Lee‐Yang‐Parr correlation functional (UB3LYP) and the 6‐31G(d) basis sets (d orbital functions). We developed an appropriate discussion on the importance of carotenoids compounds and their reactions in biological media. Also, we evaluated the role and the possible reactions of nitroxide intermediates.

**Keywords:** β‐carotene, EPR, neutral radicals, nitrogen dioxide, nitroxides

#### **1. Introduction**

The following presentation is based on experimental work we have already developed and published. We investigated the behavior of nitrogen oxides in different solvents and analyzed the reaction of nitrogen oxides with β‐carotene.

The reaction of free radicals with carotenoids and the properties of the carotenoid‐free radicals formed are of widespread interest because of their potential role in biological systems. We have

carried out our work only with β‐carotene, an unsaturated and extensively conjugated hydro‐ carbon. In vitro studies had shown the potential of carotenoids to act as free‐radical scaven‐ gers. Nitrogen oxides, such as nitric oxide (NO) and the nitrogen dioxide (NO<sup>2</sup> ), constitute a source of free radicals; they are species that have an unpaired electron. It is expected that nitrogen oxides may react in some important way with carotenoids and their radical inter‐ mediates. It is important to note that in our preliminary investigations, when mixtures of NO and NO<sup>2</sup> were added to certain organic purified compounds, compounds of the type of the nitroxides were detected using the technique of EPR [1].

Carotenoids are a family of pigmented compounds that are synthesized by plants and micro‐ organisms but not animals. However, humans and primates accumulate them in several tis‐ sues. Carotenoids are absorbed in the intestinal mucosa like other lipophilic components. β‐carotene molecule is geared in the lipophilic membranes interacting, through van der Waals forces, with the hydrocarbon chains of the lipids [2–5]. Carotenoids can be traced in the cel‐ lular cytoplasm where they can interact with different components, including the nitrogen oxides. It is also important to take into account the metabolism of carotenoids; β‐carotene is particularly hydrophobic and it is reasonable to hypothesize that it would need to be trans‐ formed to more polar compounds in order to be excreted via urine [6]. As well, lutein and β‐carotene were measured in human brain tissue and related to better cognition in octogenar‐ ians. The protective effect may not merely be an antioxidant effect given that α‐tocopherol was less related to cognition than carotenoids [7]. The first report of the presence of β‐carotene in the brain was in 1976, the patient was taking a high‐dose β‐carotene as a treatment and the carotenoid was measured within whole sections of the cerebrum [8].

We understood that the research might be quite profitable. The purpose of our work was to study and to enlighten the following problems:


#### **2. Development: (a), (b), and (c): conclusions and perspective**

#### **2.1. (a) The behavior of the nitrogen oxides NO and NO2 in some solvents**

Luckily, we had started the research developing a detailed study of solutions of nitrogen oxides in several solvents [1]. Those solutions were monitored with the following spectro‐ scopic techniques: ultraviolet and visible (UV‐vis) and electron paramagnetic resonance (EPR). The research allowed us to know what solvents can be used because they do not react with nitrogen oxides. We learned how to develop a careful control of the solvents. The knowl‐ edge acquired in this first work allowed us to follow, understand, and manage to clear up the results later finally achieved from the β‐carotene assays. Therefore, the solvent dioxane and other later used in our research were adequately tested. It was also verified that they did not react with the nitrogen oxides, NO<sup>2</sup> and NO, even after prolonged times of observation in EPR (unlike what happened with hexane) [9].

carried out our work only with β‐carotene, an unsaturated and extensively conjugated hydro‐ carbon. In vitro studies had shown the potential of carotenoids to act as free‐radical scaven‐

a source of free radicals; they are species that have an unpaired electron. It is expected that nitrogen oxides may react in some important way with carotenoids and their radical inter‐ mediates. It is important to note that in our preliminary investigations, when mixtures of NO

Carotenoids are a family of pigmented compounds that are synthesized by plants and micro‐ organisms but not animals. However, humans and primates accumulate them in several tis‐ sues. Carotenoids are absorbed in the intestinal mucosa like other lipophilic components. β‐carotene molecule is geared in the lipophilic membranes interacting, through van der Waals forces, with the hydrocarbon chains of the lipids [2–5]. Carotenoids can be traced in the cel‐ lular cytoplasm where they can interact with different components, including the nitrogen oxides. It is also important to take into account the metabolism of carotenoids; β‐carotene is particularly hydrophobic and it is reasonable to hypothesize that it would need to be trans‐ formed to more polar compounds in order to be excreted via urine [6]. As well, lutein and β‐carotene were measured in human brain tissue and related to better cognition in octogenar‐ ians. The protective effect may not merely be an antioxidant effect given that α‐tocopherol was less related to cognition than carotenoids [7]. The first report of the presence of β‐carotene in the brain was in 1976, the patient was taking a high‐dose β‐carotene as a treatment and the

We understood that the research might be quite profitable. The purpose of our work was to

(c) Theoretical evaluation of the intermediates proposed: the acyclic β‐carotene neutral radi‐ cals, the acyclic nitrous β‐carotene neutral radicals, and the cyclic nitroxide neutral radi‐

Luckily, we had started the research developing a detailed study of solutions of nitrogen oxides in several solvents [1]. Those solutions were monitored with the following spectro‐ scopic techniques: ultraviolet and visible (UV‐vis) and electron paramagnetic resonance (EPR). The research allowed us to know what solvents can be used because they do not react with nitrogen oxides. We learned how to develop a careful control of the solvents. The knowl‐ edge acquired in this first work allowed us to follow, understand, and manage to clear up the

(b) The interaction and reaction among nitrogen oxides and β‐carotene (2009) [9].

**2. Development: (a), (b), and (c): conclusions and perspective**

in some solvents (2001) [1].

 **in some solvents**

were added to certain organic purified compounds, compounds of the type of the

), constitute

gers. Nitrogen oxides, such as nitric oxide (NO) and the nitrogen dioxide (NO<sup>2</sup>

nitroxides were detected using the technique of EPR [1].

carotenoid was measured within whole sections of the cerebrum [8].

study and to enlighten the following problems:

cals (2015) [10].

(a) The behavior of nitrogen oxides: NO and NO<sup>2</sup>

**2.1. (a) The behavior of the nitrogen oxides NO and NO2**

and NO<sup>2</sup>

68 Carotenoids

NO<sup>2</sup> solutions were very carefully followed. In this chapter, we show only highlights from the developed research. Our first manuscript on nitrogen oxides, carried out in 2001, showed experimental and technical research in detail. **Figure 1** displays a set of EPR spectral records of the radical NO<sup>2</sup> at environmental temperature. Solutions of NO<sup>2</sup> were prepared in different

**Figure 1.** EPR spectra of NO<sup>2</sup> , at 290 K, in: (1) gas‐phase; (2) hexane; (3) carbon tetrachloride; and (4) acetone. General instrumental settings: microwave power, 18,000 mW; attenuation, 9 dB; modulator frequency, 100 kHz; modulation amplitude, 2.5 G; time constant or response time, 0.2 s; scan rate, 200 s. The recipient was previously flushed with argon.

media: hexane, benzene, carbon tetrachloride, acetone, and water. You can see that each spectrum do not differ considerably from the others. The signal is not resolved, due to the movement of the free radical and to the collisions that usually take place at the working tem‐ perature. Noticed that our research was always carried out under argon atmosphere [1, 9].

In **Table 1**, we can see the values of g, the spectroscopic factor and of ΔB, the total spectrum line width. Those data were extracted or calculated from **Figure 1**. We can also observe that the val‐ ues in non‐polar media are near the result measured in the gaseous phase. The general instru‐ mental settings were microwave power, 18.000 mW; attenuation, 9 dB; modulator frequency, 100 kHz; modulation amplitude, 2.5 G; time constant or response time, 0.2 s; scan rate, 200 s.

We also prepared NO solutions. The preparation and handling of solutions of NO were per‐ formed under strict inert atmosphere. Even though precautions were employed to exclude oxygen from the system, a fingerprint UV‐vis spectrum assigned to the presence of NO<sup>2</sup> /N<sup>2</sup> O4 / HNO<sup>2</sup> as impurity was sometimes observed. UV‐vis spectra of solutions of NO/NO<sup>2</sup> in different solvents were analyzed. Those Spectra shown splits in some solvents. Some authors had inter‐ preted them as the spectrum of NO [11]. However, it was confirmed that those UV‐vis spectra were obtained in polar solvents and could be attributed to the formation of HNO<sup>2</sup> [1, 12].

Usually, one has to control nitrogen dioxide samples, recording the UV‐vis spectrum and the corresponding EPR spectrum. The EPR spectrum of NO<sup>2</sup> is looked for in the following field interval: 1000–2000 Gauss, as you can observe in **Figure 1**. If we want to search for nitroxides, we must work in the following scan range: 50–200 Gauss. The EPR spectrum of NO was not detected in our field interval work.

When NO was dissolved in hexane, nitroxide intermediates were detected after 20 minutes or more, the radical was persistent. Probably no reaction took place when pure NO was present, but a trace amount of NO<sup>2</sup> initiated the reaction [13, 14].

In 2003, we carried out the measurement of the N<sup>2</sup> O4 dissociation constant (N<sup>2</sup> O4 /2 NO<sup>2</sup> ) in some solvents [15]. The N2 O4 dissociation constant measured in hexane, carbon tetrachloride,


\*Results obtained in the present work. Instrumental settings are shown in **Figure 1**.

a g values were determined with reference to a diphenylpicrylhydrazyl standard, g = 2.0036. The absolute errors are 0.002–0.004.

**Table 1.** EPR spectroscopic data of NO<sup>2</sup> radical at 290 K: spectroscopic factor, g, and total line width, ΔB\* . and chloroform compares approximately with the values calculated by some previous authors [16–18]. The techniques usually applied were colorimetric and spectrophotometric ones. As the absorption along the appropriate wavelength range was small, the errors in mea‐ surements at low concentrations were considerably large. The EPR technique has also quan‐ tification errors; however, the method is more reliable and the radical is directly detected. As far as we were aware, this was the first attempt to measure this equilibrium with the EPR technique.

#### **2.2. (b) The interaction and reaction among nitrogen oxides and β‐carotene: kinetic analysis and the corresponding modeling work**

#### *2.2.1. UV*‐*vis measurements*

media: hexane, benzene, carbon tetrachloride, acetone, and water. You can see that each spectrum do not differ considerably from the others. The signal is not resolved, due to the movement of the free radical and to the collisions that usually take place at the working tem‐ perature. Noticed that our research was always carried out under argon atmosphere [1, 9].

In **Table 1**, we can see the values of g, the spectroscopic factor and of ΔB, the total spectrum line width. Those data were extracted or calculated from **Figure 1**. We can also observe that the val‐ ues in non‐polar media are near the result measured in the gaseous phase. The general instru‐ mental settings were microwave power, 18.000 mW; attenuation, 9 dB; modulator frequency, 100 kHz; modulation amplitude, 2.5 G; time constant or response time, 0.2 s; scan rate, 200 s. We also prepared NO solutions. The preparation and handling of solutions of NO were per‐ formed under strict inert atmosphere. Even though precautions were employed to exclude

oxygen from the system, a fingerprint UV‐vis spectrum assigned to the presence of NO<sup>2</sup>

as impurity was sometimes observed. UV‐vis spectra of solutions of NO/NO<sup>2</sup>

were obtained in polar solvents and could be attributed to the formation of HNO<sup>2</sup>

initiated the reaction [13, 14].

corresponding EPR spectrum. The EPR spectrum of NO<sup>2</sup>

In 2003, we carried out the measurement of the N<sup>2</sup>

O4

**Medium g a ΔB, Gauss** Gas phase 1.999 1190 ± 58 Hexane 1.981 1194 ± 40 Benzene 1.985 563 ± 20 Carbon tetrachloride 1.978 897 ± 34 Acetone 2.185 499 ± 23 Water 2.176 502 ± 20 \*Results obtained in the present work. Instrumental settings are shown in **Figure 1**.

detected in our field interval work.

but a trace amount of NO<sup>2</sup>

some solvents [15]. The N2

**Table 1.** EPR spectroscopic data of NO<sup>2</sup>

solvents were analyzed. Those Spectra shown splits in some solvents. Some authors had inter‐ preted them as the spectrum of NO [11]. However, it was confirmed that those UV‐vis spectra

Usually, one has to control nitrogen dioxide samples, recording the UV‐vis spectrum and the

interval: 1000–2000 Gauss, as you can observe in **Figure 1**. If we want to search for nitroxides, we must work in the following scan range: 50–200 Gauss. The EPR spectrum of NO was not

When NO was dissolved in hexane, nitroxide intermediates were detected after 20 minutes or more, the radical was persistent. Probably no reaction took place when pure NO was present,

g values were determined with reference to a diphenylpicrylhydrazyl standard, g = 2.0036. The absolute errors are

radical at 290 K: spectroscopic factor, g, and total line width, ΔB\*

O4

HNO<sup>2</sup>

70 Carotenoids

a

0.002–0.004.

/N<sup>2</sup> O4 /

in different

[1, 12].

O4 /2 NO<sup>2</sup>

.

) in

is looked for in the following field

dissociation constant (N<sup>2</sup>

dissociation constant measured in hexane, carbon tetrachloride,

β‐Carotene, a non‐polar molecule, is insoluble in polar solvents like water. We desired to investigate the reaction of β‐carotene with the nitrogen oxides generated in situ from aqueous solutions of sodium nitrite and sulfuric acid. In order to achieve our purpose, we prepared solutions of β‐carotene in pure and adequately checked dioxane solvent, under argon atmosphere and constant temperature. The dioxane‐water mixtures remained homogeneous and so the UV‐vis spectroscopy studies could be applied. Other aprotic sol‐ vents easily cause cloudiness when water is added, then UV‐vis measurements cannot be carried out.

#### *2.2.2. EPR measurements*

EPR measurements were carried out with the β‐carotene solution in pure dioxane plus an aliquot of NO and NO<sup>2</sup> in pure dioxane. The solutions in dioxane had low concentration of the persistent radical, besides the triplet‐type signal was recorded during 5 days. In pure dioxane, we only developed a qualitative approach. On the other hand, we could design a quantitative study carried out with β‐carotene solution in pure dioxane plus an aliquot of the dioxane/ water solution with the nitrogen oxides formed in situ from sodium nitrite in an acid medium. Also the formation and decay of the persistent intermediates was monitored and shown in **Figure 2**. Dioxane solvent was tested, and no EPR signal was detected even after prolonged times of observation.

#### *2.2.3. Kinetic measurements*

Kinetic measurements were developed in dioxane/water solvent at 298 K. All the measure‐ ments were successfully simulated with the software for chemical kinetics, following the reac‐ tion path proposed in **Figure 3**. In **Table 2**, each reaction was described and the corresponding kinetic constant assigned. Unfortunately, in our 2009 manuscript, the sixth equation had one mistake, we must write *P*<sup>1</sup> instead of *P*<sup>2</sup> [9]. In the 2015 manuscript, the rate constants *k*<sup>i</sup> (i = 1 to 7) were not written above the arrows [10].

In **Figure 3** and **Table 2**, we observed that whenever both NO and NO<sup>2</sup> were present, abstrac‐ tion took place and nitroxides formed. Furthermore, it has already been shown that NO is less

**Figure 2.** Formation and decay of a persistent type radical generated from a solution of β‐carotene, 1 × 10−2M, and nitrite anion, 1 × 10−1M in dioxane:water (xdioxane = 0.65) and in the presence of an acid medium, pH = 2 at 298 K. The gray curve and symbols represent the values obtained from the kinetic simulation process; the black square symbols represent our experimental values. After 20 days, we were still able to measure a radical concentration of 3.5 × 10−5 M, not shown in the graph. The reactions were carried out under argon atmosphere.

**Figure 3.** General reaction pathway proposed. The figure shows, in first place, the abstraction reactions of allylic hydrogens from β‐carotene by nitrogen dioxide radicals and the formation of the products *P*<sup>1</sup> . *P*<sup>1</sup> represents different possible β‐carotene neutral radicals. *P*<sup>2</sup> represents the nitrous β‐carotene formed with nitric oxide radicals. *P*<sup>3</sup> represents the products formed with nitrogen dioxide radicals, not studied in this work. *P*<sup>4</sup> represents the neutral radicals that are formed after the reaction of *P*<sup>2</sup> with nitrogen dioxide radicals, another abstraction reaction of allylic hydrogens has taken place. Finally, when *P*<sup>4</sup> generates an internal ring, a radical *P*4(nitroxide) comes up.

#### **Kinetic analysis in acid mediuma**


a T = 298K. *k*<sup>1</sup> , *k*−1, and *k*<sup>2</sup> were already known. The rate constants *k*<sup>4</sup> and *k*<sup>5</sup> were considered diffusion controlled. The rate constants *k*<sup>3</sup> , *k*<sup>6</sup> , and *k*<sup>7</sup> were deduced from our modeling work. The kinetic simulation reproduced successfully the experimental kinetics results [9].

*P*1 represents a possible allylic‐type radical. *P*<sup>2</sup> represents the possible isomers molecules of nitrous carotene. *P*<sup>4</sup> represents the possible allylic‐type radicals obtained from *P*<sup>2</sup> . When a radical *P*<sup>4</sup> cycles internally a radical *P*<sup>4</sup> *(nitroxide)* is formed. *P*<sup>3</sup> represents the products formed with nitrogen dioxide, not studied in this work.

**Table 2.** Reaction of β‐carotene and nitrite in dioxane/water solution.

reactive and less efficient in the abstraction of a hydrogen atom than NO<sup>2</sup> [9]. We can compare the following bond dissociation energies (BDE) values at 298 K:

• BDE (H‐NO) = 195.35 ± 0.25 kJ.mol−1

**Figure 2.** Formation and decay of a persistent type radical generated from a solution of β‐carotene, 1 × 10−2M, and nitrite anion, 1 × 10−1M in dioxane:water (xdioxane = 0.65) and in the presence of an acid medium, pH = 2 at 298 K. The gray curve and symbols represent the values obtained from the kinetic simulation process; the black square symbols represent our experimental values. After 20 days, we were still able to measure a radical concentration of 3.5 × 10−5 M, not shown in the

**Figure 3.** General reaction pathway proposed. The figure shows, in first place, the abstraction reactions of allylic

represents the nitrous β‐carotene formed with nitric oxide radicals. *P*<sup>3</sup>

with nitrogen dioxide radicals, another abstraction reaction of allylic hydrogens has taken

. *P*<sup>1</sup>

represents the neutral radicals that are

represents different

represents

hydrogens from β‐carotene by nitrogen dioxide radicals and the formation of the products *P*<sup>1</sup>

generates an internal ring, a radical *P*4(nitroxide) comes up.

the products formed with nitrogen dioxide radicals, not studied in this work. *P*<sup>4</sup>

graph. The reactions were carried out under argon atmosphere.

possible β‐carotene neutral radicals. *P*<sup>2</sup>

formed after the reaction of *P*<sup>2</sup>

place. Finally, when *P*<sup>4</sup>

72 Carotenoids

• BDE (H‐NO<sup>2</sup> or H‐ONO) = 327.6 ± 2.1 kJ.mol−1

We proposed the formation of β‐carotene neutral radicals that in the presence of nitrogen oxides, originated persistent radical intermediates. The reactions were carried out under argon atmosphere because of the reactivity of triplet oxygen; otherwise oxygenated com‐ pounds would be formed with the carotenoid neutral radicals.

We followed and measured the difficult kinetic of the mechanism proposed. The kinetic of the reaction depended on β‐carotene, nitrite, and acid concentrations. We experimentally verified that the β‐carotene followed a first‐order decay and we measured the corresponding pseudo‐ first‐order‐constant [9].

In **Table 2**, a set of reactions represents the mechanism of the reaction. It is assumed that the system always behaves as if it has reached the acid‐base balance.

*P*4 was considered as *P*<sup>4</sup> *(nitroxide)*. We ran a non‐commercial simulation program of chemi‐ cal kinetics. The validity of the proposed mechanism was therefore tested by numerical integration.

The values of *k*<sup>1</sup> *, k*−1*, and k*<sup>2</sup> were extracted from the literature*.* The rate constant *k*<sup>3</sup> was adjusted following the UV‐vis decay of β‐carotene. The rate constants *k*<sup>6</sup> and *k*<sup>7</sup> were involved in the formation of the persistent radicals and were adjusted to the EPR results. The rate con‐ stants *k*<sup>4</sup> and *k*<sup>5</sup> were the recombination reactions between *P*<sup>1</sup> radicals and nitrogen diox‐ ide and nitric oxide radicals. We calculated the following values: *k*<sup>4</sup> = 1.4×1010M−1s−1 and *k*<sup>5</sup> = 1.1×1010M−1s−1, by applying the equations of Smoluchoski, Stokes, and Einstein [19]. The calculations were developed taking into account the viscosity of the mixture dioxane‐water, η25°C = 1.42 × 10−3 Pa.s and the molecular size of the NO, NO<sup>2</sup> and β‐carotene neutral radi‐ cals [20]. Actually, these constants were smaller because the nitrogen oxides reacted at some selected regions of the β‐carotene neutral radicals. It was confirmed that changes of *k*<sup>4</sup> and *k*<sup>5</sup> in the order of 10<sup>7</sup> to 1011 had no significant effect upon the simulation results, which match very well with our experimental results. So the value of 1.0 × 109 M−1s−1 was selected and shown for *k*<sup>4</sup> and *k*<sup>5</sup> in **Table 2**. *P*<sup>3</sup> represents the products formed with nitrogen dioxide, not studied in this work.

We considered that the nitrous acid was proportional to the nitrite anion through the acid‐ base equilibrium [9]. The simulation results at 298 K reproduced quite well the experimental decay of β‐carotene in the range near the half‐life at pH = 5.3. The formation and the decay of the persistent radical intermediate at pH = 2.2 were also very well reproduced. For example, we showed *Case (a)* and *Case (b)* through which we tested the experiments carried out.

*Case (a)* Initial concentrations: β‐carotene, 8.0 × 10−6M; nitrite anion, 9.3 × 10−3M; pH, 5.3

The simulation program showed that the system achieved approximately the following steady state order of concentrations: [NO<sup>2</sup> ] ≅ 10−8 to 10−9M [NO] ≅ 10−6 to 10−7M [*P*<sup>1</sup> ] ≅ 10−10M [*P*2 ] ≅ 10−6M

The pH achieved in the simulation run remained almost constant: 5.30 ± 0.02.

The maximum radical concentration reached was: [*P*<sup>4</sup> + *P*4(nitroxide)] ≅ 10−11M, in agreement with our EPR results.

The persistent radicals were not detected; evidently, our equipment had not enough sensibility.

*Case (b)* Initial concentrations: β‐Carotene, 1.0 × 10−2M; nitrite anion, 1.0 × 10−1M; pH, 2.2.

The system achieved approximately the following steady state concentrations order: [NO<sup>2</sup> ] ≅ 10−7M [NO] ≅ 10−2M [*P*<sup>1</sup> ] ≅ 0M [*P*<sup>2</sup> ] ≅ 10−3M.

The pH achieved in the simulation run remained almost constant: 2.20 ± 0.03.

The maximum radical concentration reached was: [*P*<sup>4</sup> + *P*<sup>4</sup> *(nitroxide)*]=1.5x10−4M.

In agreement with our EPR results, 1.4 × 10−4M, see **Figure 2**.

After 20 days, the kinetics simulation delivered a radical concentration of 3.5 × 10−5M in per‐ fect agreement with our experimental results.

We can observe that the examples have quite different experimental conditions; however, they both work with the same set of rate constants.

In **Figure 2**, the experimental measurement of the formation and decay of the radical interme‐ diates was represented with black square symbols and the results from the kinetic simulation process were represented with gray symbols. **Figure 4** presents the recorded EPR spectrum of the radical intermediates. Although the spectrum may fit with a nitroxide‐type radical show‐ ing a hyperfine coupling constant, aN = 12.7 G, the spectrum exhibits an increment in the cen‐ tral line. This central increment could be attributed to the contribution of a related allylic‐type radical superimposed or to a set of related allylic‐type radicals. However, the kinetic analysis generated poor concentrations for *P*<sup>1</sup> , which represented the allylic radical intermediates. In addition, finally, with the aid of the computational methods, the hypothesis of the formation of cyclic nitroxides was favored [10].

The values of *k*<sup>1</sup>

74 Carotenoids

stants *k*<sup>4</sup> and *k*<sup>5</sup>

in the order of 10<sup>7</sup>

studied in this work.

and *k*<sup>5</sup>

shown for *k*<sup>4</sup>

[*P*2

[NO<sup>2</sup>

] ≅ 10−6M

our EPR results.

*, k*−1*, and k*<sup>2</sup>

following the UV‐vis decay of β‐carotene. The rate constants *k*<sup>6</sup>

η25°C = 1.42 × 10−3 Pa.s and the molecular size of the NO, NO<sup>2</sup>

very well with our experimental results. So the value of 1.0 × 109

in **Table 2**. *P*<sup>3</sup>

steady state order of concentrations: [NO<sup>2</sup>

] ≅ 10−7M [NO] ≅ 10−2M [*P*<sup>1</sup>

The maximum radical concentration reached was: [*P*<sup>4</sup>

The maximum radical concentration reached was: [*P*<sup>4</sup>

fect agreement with our experimental results.

they both work with the same set of rate constants.

In agreement with our EPR results, 1.4 × 10−4M, see **Figure 2**.

ide and nitric oxide radicals. We calculated the following values: *k*<sup>4</sup>

were extracted from the literature*.* The rate constant *k*<sup>3</sup>

to 1011 had no significant effect upon the simulation results, which match

represents the products formed with nitrogen dioxide, not

] ≅ 10−8 to 10−9M [NO] ≅ 10−6 to 10−7M [*P*<sup>1</sup>

+ *P*4(nitroxide)] ≅ 10−11M, in agreement with

*(nitroxide)*]=1.5x10−4M.

formation of the persistent radicals and were adjusted to the EPR results. The rate con‐

= 1.1×1010M−1s−1, by applying the equations of Smoluchoski, Stokes, and Einstein [19]. The calculations were developed taking into account the viscosity of the mixture dioxane‐water,

cals [20]. Actually, these constants were smaller because the nitrogen oxides reacted at some

We considered that the nitrous acid was proportional to the nitrite anion through the acid‐ base equilibrium [9]. The simulation results at 298 K reproduced quite well the experimental decay of β‐carotene in the range near the half‐life at pH = 5.3. The formation and the decay of the persistent radical intermediate at pH = 2.2 were also very well reproduced. For example,

The simulation program showed that the system achieved approximately the following

The persistent radicals were not detected; evidently, our equipment had not enough sensibility.

The system achieved approximately the following steady state concentrations order:

] ≅ 10−3M.

After 20 days, the kinetics simulation delivered a radical concentration of 3.5 × 10−5M in per‐

We can observe that the examples have quite different experimental conditions; however,

+ *P*<sup>4</sup>

*Case (b)* Initial concentrations: β‐Carotene, 1.0 × 10−2M; nitrite anion, 1.0 × 10−1M; pH, 2.2.

we showed *Case (a)* and *Case (b)* through which we tested the experiments carried out. *Case (a)* Initial concentrations: β‐carotene, 8.0 × 10−6M; nitrite anion, 9.3 × 10−3M; pH, 5.3

The pH achieved in the simulation run remained almost constant: 5.30 ± 0.02.

] ≅ 0M [*P*<sup>2</sup>

The pH achieved in the simulation run remained almost constant: 2.20 ± 0.03.

selected regions of the β‐carotene neutral radicals. It was confirmed that changes of *k*<sup>4</sup>

were the recombination reactions between *P*<sup>1</sup>

and *k*<sup>7</sup>

was adjusted

and *k*<sup>5</sup>

] ≅ 10−10M

were involved in the

= 1.4×1010M−1s−1 and *k*<sup>5</sup>

M−1s−1 was selected and

radicals and nitrogen diox‐

and β‐carotene neutral radi‐

#### **2.3. (c) Theoretical evaluation of the intermediates proposed: the acyclic β‐carotene neutral radicals, the acyclic nitrous β‐carotene neutral radicals, and the cyclic nitroxide neutral radicals**

The purpose of the following research was to unravel the structures of the related persistent intermediate or intermediates, we called them *P*<sup>4</sup>  *(nitroxide)* or cyclic nitroxide neutral radical or radicals.

**Figure 4.** The figure shows a first‐derivative X‐band EPR spectrum of the intermediate called *P*4(nitroxide), one or more type of persistent intermediate formed in the reaction of β‐carotene with nitrite anion in acid medium (dioxane/water) at 298 K, under argon atmosphere. Spectrometer settings: microwave frequency, 9.92 GHz; modulation frequency, 100 kHz; microwave power, 11.3 mW; attenuation, 10 dB; field modulation, 0.125 G; receiver gain, 2×10<sup>5</sup> ; time constant, 0.05 s; scan range, 50 G; scan time, 5 s. The peak‐to‐peak value is of 12.7 Gauss and g = 1.994 [9].

On the basis of the kinetic studies, it was reasonable to propose intermediates of the nitroxide type. In a biological medium, one can expect that species react with each other. Or hope that radicals from β‐carotene react with neighboring molecules of nitrous carotenes generating acy‐ clic nitroxides. However, in the dilute solutions of β‐carotene, where the reactions with the nitro‐ gen oxides were carried out, the radicals kept away and intra‐radical reactions would preferably take place. Thus, on that account, we considered it suitable to propose the formation of the rings.

We looked for the help of the computational and theoretical methods in order to study and compare the characteristics of the proposed structures and found out that the density func‐ tional theory (DFT) met the best conditions, given that it included the effects of electron correlation. DFT used very suitable approaches for energy exchange and for the correlation. The main conclusion was that for neutral radicals we could obtain good results with the approximate method Unrestricted Becke‐style 3‐parameter density functional theory using the Lee‐Yang‐Parr correlation functional (UB3LYP), both for the calculation of the energy of the system and for the calculation of the hyperfine coupling constants (hfccs). The selected basis sets, that used *d orbital functions*, allowed a good resolution, with an adequate cost of time, with a personal computer. The software used allowed to obtain the values of energy of each structure, which permitted to evaluate the relative stability of the radicals. Also, the software provided us the isotropic hfccs of each nucleus of the radical intermediates pro‐ posed [10].

Finally, the hfccs of the nuclei of hydrogen (1 H) and nitrogen (14N) obtained with the theo‐ retical methods allowed us to develop the simulation of the spectra that were experimentally recorded, like that shown in **Figure 4**.

Different possible rings with 3, 5, 6, 7, or 8 atoms were built and tested. However, only with some of them the corresponding geometric optimizations were achieved. The radicals with cycles of five atoms were found to be the most favored and those of lower energy.

In the General Reaction Pathway proposed in **Figure 3**, the following intermediates were appreciated:


With the aim of avoiding confusion in the interpretations described, the process of formation of the radical intermediates follows.

• *Allylic‐type radicals, P*<sup>1</sup>

**Figure 5** helps to visualize the possible allylic hydrogens of β‐carotene (the characteristic atomic symbols (C, H) are usually not shown in this type of molecular representation). The set of neutral allylic radicals from β‐carotene is symbolized by *P*<sup>1</sup> in **Figures 3** and **6**.

The molecule of β‐carotene has allylic hydrogens, which are hydrogen atoms of the methyl groups at positions 5 and 5'; 9 and 9'; 13 and 13'. Allylic‐type radicals are obtained by the loss, by abstraction, of those hydrogen atoms:

On the basis of the kinetic studies, it was reasonable to propose intermediates of the nitroxide type. In a biological medium, one can expect that species react with each other. Or hope that radicals from β‐carotene react with neighboring molecules of nitrous carotenes generating acy‐ clic nitroxides. However, in the dilute solutions of β‐carotene, where the reactions with the nitro‐ gen oxides were carried out, the radicals kept away and intra‐radical reactions would preferably take place. Thus, on that account, we considered it suitable to propose the formation of the rings. We looked for the help of the computational and theoretical methods in order to study and compare the characteristics of the proposed structures and found out that the density func‐ tional theory (DFT) met the best conditions, given that it included the effects of electron correlation. DFT used very suitable approaches for energy exchange and for the correlation. The main conclusion was that for neutral radicals we could obtain good results with the approximate method Unrestricted Becke‐style 3‐parameter density functional theory using the Lee‐Yang‐Parr correlation functional (UB3LYP), both for the calculation of the energy of the system and for the calculation of the hyperfine coupling constants (hfccs). The selected basis sets, that used *d orbital functions*, allowed a good resolution, with an adequate cost of time, with a personal computer. The software used allowed to obtain the values of energy of each structure, which permitted to evaluate the relative stability of the radicals. Also, the software provided us the isotropic hfccs of each nucleus of the radical intermediates pro‐

retical methods allowed us to develop the simulation of the spectra that were experimentally

Different possible rings with 3, 5, 6, 7, or 8 atoms were built and tested. However, only with some of them the corresponding geometric optimizations were achieved. The radicals with

In the General Reaction Pathway proposed in **Figure 3**, the following intermediates were

With the aim of avoiding confusion in the interpretations described, the process of formation

**Figure 5** helps to visualize the possible allylic hydrogens of β‐carotene (the characteristic atomic symbols (C, H) are usually not shown in this type of molecular representation). The

The molecule of β‐carotene has allylic hydrogens, which are hydrogen atoms of the methyl groups at positions 5 and 5'; 9 and 9'; 13 and 13'. Allylic‐type radicals are obtained by the loss,

cycles of five atoms were found to be the most favored and those of lower energy.

• *Cyclic nitroxides formed by internal cyclization of the nitrous allylic radicals, P*4(nitroxide)

set of neutral allylic radicals from β‐carotene is symbolized by *P*<sup>1</sup>

H) and nitrogen (14N) obtained with the theo‐

in **Figures 3** and **6**.

posed [10].

76 Carotenoids

appreciated:

• *Allylic‐type radicals, P*<sup>1</sup>

• *Allylic‐type radicals, P*<sup>1</sup>

• *Nitrous allylic radicals, P*<sup>4</sup>

of the radical intermediates follows.

by abstraction, of those hydrogen atoms:

Finally, the hfccs of the nuclei of hydrogen (1

recorded, like that shown in **Figure 4**.

**Figure 5.** The molecule of β‐carotene has allylic hydrogens, that are hydrogen atoms of the methyl groups at positions 5 and 5'; 9 and 9'; 13 and 13'. Vinylic hydrogen atoms are attached to the polyene chain at carbons: 7,8,10,11,12,14,15,15', 14',12',11',10',8', and 7'. Methyl groups with primary hydrogens, but not allylic type are represented with only a line or a bar in positions 1 and 1'.

*P*10(5 or 5'): The allylic‐type radical is formed by the loss of a hydrogen atom of the methyl attached to carbon *5 or 5'.*

*P*10(9 or 9'): The allylic‐type radical is formed by the loss of a hydrogen atom of the methyl attached to carbon *9 or 9'*.

*P*10(13 or 13'): The allylic‐type radical is formed by the loss of a hydrogen atom of the methyl attached to carbon *13 or 13'.*

The molecule of β‐carotene has also methyl groups (represented by a line or a bar) at posi‐ tions 1 and 1'; they are primary hydrogen atoms. Those primary hydrogen atoms are not abstracted, they have higher bond energy than the allylic ones. In **Figure 5**, you can also appreciate the vinylic hydrogen atoms in the polyene chain. Those vinylic hydrogen atoms are attached to carbons: 7,8,10,11,12,14,15,15',14',12',11',10',8', and 7'; they have even higher bond energy than the primary hydrogen atoms. Observe that the radicals formed by the loss of allylic hydrogens are indeed stabilized by resonance.

In **Figure 6**, we followed the formation of the neutral carotenoid allylic radicals described below:

*P*10(5 or 5'): The β‐carotene loses an allylic hydrogen atom from the methyl group attached to position 5 or 5'. The conjugation effect formed a contributing resonance structure, an allylic tertiary radical on carbon 6 (see **Figure 5**), it is *P*11(5 or 5')

*P*12(5 or 5'): It is another contributing resonance structure with the unpaired electron in carbon 13'.

*P*10(5 or 5') *;P*11(5 or 5') and *P*12(5 or 5') are contributing resonance structures of the *same resonance hybrid*.

**Figure 6.** Several radicals of the allylic type are shown, *P*<sup>1</sup> . There are three possibilities of allylic hydrogens: *P*10 (5 or 5'); *P*10 (9 or 9'), and *P*10 (13 or 13'). The second subscript indicates different contributing resonance structures, for example, *P*11 (5 or 5') *and P*12 (5 or 5')*.* The radical formed by the loss of a methyl hydrogen atom in position 9 or 9' is *P*10 (9 or 9'), observe that the electronic movements are shown in three related structures. *P*1 0(13 or 13') is the radical formed by the loss of a methyl hydrogen atom in position 13 or 13'. For this last case, only one possible structure is designed.

In the case of *P*10(9 or 9'), the resonance or electronic movements are also shown. For *P*10(13 or 13')*,* only one structure is designed.

• *Nitrous allylic radicals, P*<sup>4</sup>

**Figure 6.** Several radicals of the allylic type are shown, *P*<sup>1</sup>

78 Carotenoids

. There are three possibilities of allylic hydrogens: *P*10 (5 or 5');

*P*10 (9 or 9'), and *P*10 (13 or 13'). The second subscript indicates different contributing resonance structures, for example, *P*11 (5 or 5') *and P*12 (5 or 5')*.* The radical formed by the loss of a methyl hydrogen atom in position 9 or 9' is *P*10 (9 or 9'), observe that the electronic movements are shown in three related structures. *P*1 0(13 or 13') is the radical formed by the loss of a methyl

hydrogen atom in position 13 or 13'. For this last case, only one possible structure is designed.

The *P*<sup>4</sup> radicals were also investigated with the theoretical methods:


In **Figure 7**, we appreciate that *P*12 (5 or 5') is the intermediate that react with nitric oxide generat‐ ing *P*22, the nitrous allylic compound, see **Figure 3**.

*P*22 loses by abstraction an allylic hydrogen atom from the methyl in position 9' generating *P*42, the corresponding nitrous allylic radical and finally the formation of *P*42 (nitroxide).

(**Figure 6** in our work of 2015 has an error; the nitroxide displayed is not generated from the listed precursors. The precursors designed would actually lead to another nitroxide not shown. The optimization of that nitroxide was completed on the basis of negligible forces. The stationary point was found but the convergence criteria were reached by only three of the required four cases [10]. In the present work, we have the opportunity of showing and solving the mistake. In **Figure 7**, we appreciate the precursors that actually lead to *P*42*(nitroxide).*)

**Figure 7.** Formation of the cyclic *P*42*(nitroxide)* from the allylic precursor. The tertiary allylic radical *P*12 (5 or 5') is a contributing resonance structure of high weight.

In **Figure 8**, the optimized structure of *P*42 (nitroxide) is displayed. Each atom is labeled with a number.

In order to carry out the simulation, three selected nitroxides were chosen, those of lower energy and those that best met the requirements for optimization. We selected two rings of five atoms (*P*41 (nitroxide); *P*42 (nitroxide) ) and one of eight atoms (*P*43 (nitroxide)).

We simulated quite satisfactorily the experimental recorded EPR spectrum in **Figure 4**, by adding the theoretical spectra of *P*42 (nitroxide) from *P*12 (5 or 5') and *P*41 (nitroxide) from *P*11 (5 or 5'), both cyclic nitroxides of five atoms. Also we built another simulation by adding *P*42(nitroxide), *P*41 (nitroxide), and *P*43 (nitroxide) from *P*10 (9 or 9'), a cyclic nitroxide of eight atoms [10].

**Figure 9** shows the theoretical spectra and simulation of the persistent EPR recorded spec‐ trum of **Figure 4**. In part (a), we may appreciate the theoretical EPR spectra of the nitroxides *P*41*(nitroxide), P*42*(nitroxide), and P*43*(nitroxide)*. In order to simulate the experimental recorded spectra, one must add up the spectra of nitroxides, multiplying the values of *P*41*(nitroxide)* by the factor 1 and *P*42*(nitroxide)* by the factor 0.4 and multiply the values of *P*43*(nitroxide)* by the factor 0.2. Part (b) shows the sum that simulates quite well the experimental spectrum shown in **Figure 4**.

Simulations were also carried out considering the contribution of the β‐carotene neutral radi‐ cals or allylic neutral radicals. It was observed that with less than 0.1 order factors, there were no appreciable changes, as is expected due to the result of the kinetic modeling. Although the β‐carotene neutral radicals could be persistent, the nitroxide‐type signal obtained would not be modified.

The theoretical spectra were built from the calculated isotropic hyperfine coupling constants. The hfccs values larger than 0.4 Gauss are shown in **Table 3**.

**Figure 8.** Visualization of the optimized geometric structure of the intermediate radical proposed in **Figure 7**: *P*42*(nitroxide)*.

In **Figure 8**, the optimized structure of *P*42 (nitroxide) is displayed. Each atom is labeled with a

In order to carry out the simulation, three selected nitroxides were chosen, those of lower energy and those that best met the requirements for optimization. We selected two rings of

We simulated quite satisfactorily the experimental recorded EPR spectrum in **Figure 4**, by adding the theoretical spectra of *P*42 (nitroxide) from *P*12 (5 or 5') and *P*41 (nitroxide) from *P*11 (5 or 5'), both cyclic nitroxides of five atoms. Also we built another simulation by adding *P*42(nitroxide), *P*41 (nitroxide),

**Figure 9** shows the theoretical spectra and simulation of the persistent EPR recorded spec‐ trum of **Figure 4**. In part (a), we may appreciate the theoretical EPR spectra of the nitroxides *P*41*(nitroxide), P*42*(nitroxide), and P*43*(nitroxide)*. In order to simulate the experimental recorded spectra, one must add up the spectra of nitroxides, multiplying the values of *P*41*(nitroxide)* by the factor 1 and *P*42*(nitroxide)* by the factor 0.4 and multiply the values of *P*43*(nitroxide)* by the factor 0.2. Part (b) shows the sum that simulates quite well the experimental spectrum shown

Simulations were also carried out considering the contribution of the β‐carotene neutral radi‐ cals or allylic neutral radicals. It was observed that with less than 0.1 order factors, there were no appreciable changes, as is expected due to the result of the kinetic modeling. Although the β‐carotene neutral radicals could be persistent, the nitroxide‐type signal obtained would

The theoretical spectra were built from the calculated isotropic hyperfine coupling constants.

**Figure 8.** Visualization of the optimized geometric structure of the intermediate radical proposed in **Figure 7**:

five atoms (*P*41 (nitroxide); *P*42 (nitroxide) ) and one of eight atoms (*P*43 (nitroxide)).

and *P*43 (nitroxide) from *P*10 (9 or 9'), a cyclic nitroxide of eight atoms [10].

The hfccs values larger than 0.4 Gauss are shown in **Table 3**.

number.

80 Carotenoids

in **Figure 4**.

not be modified.

*P*42*(nitroxide)*.

**Figure 9.** EPR theoretical spectra and simulation. In the part (a) the EPR theoretical spectra of the nitroxides are recorded: *P41(nitroxide) in gray, P*42**(***nitroxide)* in black, and *P43(nitroxide)* in pale gray color. The values of *P41(nitroxide)* are multiplied by a factor of 1, *P*42*(nitroxide)* are multiplied by a factor of 0.4 and the values of *P43(nitroxide)* by a factor of 0.2. The part (b) shows the sum of the spectra in order to simulate the experimental spectrum. The peak‐to‐peak value is 12.1 Gauss, (10 Gauss ≡ 1 mT). Data from **Table 3** are used.


a The hfccs values were obtained by applying the method of calculation B3LYP/6‐31G (d) to the optimized geometries. The hfccs values larger than 0.4 Gauss are shown. Gaussian offers results in Gauss, 10 G ≡ 1 mT.

```
P41(nitroxide) and P42(nitroxide) are rings of five atoms. P43(nitroxide) corresponds to a ring of eight atoms.
```
Each atom is labeled with a number, as in **Figure 8** for *P*42*(nitroxide)*.

**Table 3.** Isotropic hyperfine coupling constants (hfccs), cyclic *P*<sup>4</sup> *(nitroxides)*, (Gauss)<sup>a</sup> .

#### **3. Conclusions and perspective**

We have provided considerable information about the behavior of nitrogen dioxide in some solvents. Also, we followed the action of both NO<sup>2</sup> and NO over some organic compounds. Most relevant was the study of the reaction of both oxides with the β‐carotene molecule.

It is important to highlight that two spectroscopic techniques were used: UV‐vis and EPR. UV‐vis is fundamental for the tracking of reagents, and EPR is essential for the monitoring of the radical intermediates.

Finally, we used the theoretical and computational methods that provided support to the characteristics of the intermediates. Calculations for β‐carotene cyclic nitroxide intermedi‐ ates, β‐carotene allyl radicals, and nitrous β‐carotene radical showed that the combination of the 6‐31G(d,p) basis set and the B3LYP exchange‐correlation functional provided a quite accurate description of the hfccs of the nuclei of hydrogen and nitrogen. The calculation of the hfcc for nitrogen nuclei (14N, nucleus) in the case of the nitroxide persistent radicals was particularly difficult. This problem with the nitroxides was investigated and finally deduced that the choice of the basis sets was very important. Especially the number and nature of *d orbital functions* must be taken into account [10].

β‐Carotene or other carotenoids compounds would be in the lipid and anaerobic part of cells where the investigated reactions could take place. The β‐carotene could be a convenient scav‐ enger, acting as a protective agent in biological media. The cyclic persistent nitroxides may react by disproportionation giving rise to hydroxylated carotenoid compounds and nitrones. Hydroxylated compounds can pass into the lymphatic system and be replaced by new mol‐ ecules of β‐carotene. The nitrone is a new scavenger that will lead to a continuously replace‐ ment process [10]. It is important to remember that the persistent nitroxides are still present in solutions after 20 days with a concentration of 3.5·10−5 M [9].

On the other hand, the nitroxides may react differently. For example:


Moreover, we can see that nitric oxide is generated in different types of cells. Neuronal nitric oxide synthase (nNOS) is constituvely expressed in specific neurons of the brain. In addi‐ tion to brain tissue, nNOS has been identified in adrenal glands, in epithelial cells of various organs. In mammalians, the largest source of nNOS in terms of tissue mass is in the skeletal muscle [23].

It might be expected that nitrogen oxides and non‐polar carotenoids compounds have together an important biological function. Take into account that in carotenoids with a hydroxyl group, the reaction path may be surely different.

The isolation of the cyclic nitroxides described would also be very important.

#### **Author details**

**3. Conclusions and perspective**

the radical intermediates.

36 H −0.42026

a

82 Carotenoids

solvents. Also, we followed the action of both NO<sup>2</sup>

Each atom is labeled with a number, as in **Figure 8** for *P*42*(nitroxide)*.

**Table 3.** Isotropic hyperfine coupling constants (hfccs), cyclic *P*<sup>4</sup>

*orbital functions* must be taken into account [10].

We have provided considerable information about the behavior of nitrogen dioxide in some

The hfccs values were obtained by applying the method of calculation B3LYP/6‐31G (d) to the optimized geometries.

*(nitroxides)*, (Gauss)<sup>a</sup>

.

The hfccs values larger than 0.4 Gauss are shown. Gaussian offers results in Gauss, 10 G ≡ 1 mT. *P*41*(nitroxide)* and *P*42*(nitroxide)* are rings of five atoms. *P*43*(nitroxide)* corresponds to a ring of eight atoms.

It is important to highlight that two spectroscopic techniques were used: UV‐vis and EPR. UV‐vis is fundamental for the tracking of reagents, and EPR is essential for the monitoring of

Finally, we used the theoretical and computational methods that provided support to the characteristics of the intermediates. Calculations for β‐carotene cyclic nitroxide intermedi‐ ates, β‐carotene allyl radicals, and nitrous β‐carotene radical showed that the combination of the 6‐31G(d,p) basis set and the B3LYP exchange‐correlation functional provided a quite accurate description of the hfccs of the nuclei of hydrogen and nitrogen. The calculation of the hfcc for nitrogen nuclei (14N, nucleus) in the case of the nitroxide persistent radicals was particularly difficult. This problem with the nitroxides was investigated and finally deduced that the choice of the basis sets was very important. Especially the number and nature of *d*

β‐Carotene or other carotenoids compounds would be in the lipid and anaerobic part of cells where the investigated reactions could take place. The β‐carotene could be a convenient scav‐ enger, acting as a protective agent in biological media. The cyclic persistent nitroxides may react by disproportionation giving rise to hydroxylated carotenoid compounds and nitrones.

Most relevant was the study of the reaction of both oxides with the β‐carotene molecule.

**Nuclei** *P***41***(nitroxide)* **Nuclei** *P***42***(nitroxide)* **Nuclei** *P***43***(nitroxide)* 3 H 0.73171 49 H 0.42705 28 H 0.94710 10 H 0.91335 51 H 2.99147 31 H 0.45231 14 H 0.56426 53 H −0.53599 53 H 2.00175 18 H 0.57683 55 H −0.55714 54 H 9.42852 25 H −0.53235 57 **N** 10.95771 56 H 19.03785 27 H −0.58125 60 H −0.45979 57 H 2.93589 29 **N** 11.68685 62 H −0.60386 88 **N** 11.40254 33 H −0.57914 64 H 18.74614 92 H 2.15932 34 H −0.47031 96 H 1.59845

and NO over some organic compounds.

Sara N. Mendiara<sup>1</sup> \* and Luis. J Perissinotti1,2

\*Address all correspondence to: mendiara@gmail.com

1 Department of Chemistry, Faculty of Exact and Natural Sciences, National University of Mar del Plata, Mar del Plata, Buenos Aires, Argentina

2 Commission of Scientific Research of the Province of Buenos Aires (CIC), La Plata, Buenos Aires, Argentina

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## **Influence of Environmental Stress toward Carotenogenesis Regulatory Mechanism through** *In Vitro* **Model System**

Rashidi Othman, Norazian Mohd Hassan and Farah Ayuni Mohd Hatta

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67724

#### **Abstract**

Carotenoid biosynthesis is influenced by some aspects and is liable to geometric isomerisation with the existence of oxygen, light, and heat, which affect color degradation and oxidation. The major problems related to carotenoid accumulation inherently originate from pigment instability. This chapter discusses an overview on the influence of stringent control of genetic, developmental, and environmental factors toward carotenoid biogenesis in potato minitubers through the potential model system for rapid initiation, extraction, and analysis of carotenoids. The outcome of this experimental system is a discovery of variables regulating carotenoid accumulation as a result of the environmental change assessment through manipulation of drought stress, light intensity, and nutrient strength on carotenoid accumulation.

**Keywords:** carotenogenesis, environmental stress, *in vitro*, model system, elicitors

#### **1. Introduction**

Considerable research interest has recently focused on the improvement of both transgenic and conventional propagation techniques to enrich total and individual carotenoid composition in potatoes [1–4]. Unfortunately, little information is available on the influence of the environment on the carotenoid content in potatoes, especially growing seasons and locations. Genotype and environment interactions have been reported to account for alteration in free amino acids, protein, and sugar composition [5–13]. In addition, the total glycoalkaloid content of potato tubers was found greatly affected as a result of environmental changes during

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

the growing season [14], even though there are also strong genetic effects [15, 16]. Seasonal differences, growing conditions, locations, genotypes, and postharvest storage conditions are among the factors that can be significantly affecting the quality and nutritional value of potatoes [17–20]. The bioavailability of carotenoids is characterized as an intricate issue and influenced by various factors [21]. In our study of interseasonal and genotype interactions, the data revealed that variations in total carotenoid content and the concentration of individual carotenoid pigments are due to the strong relationship between genotype and growing seasons. This assumption is supported by Chloupek and Hrstkova [22] in their observations of 26 crops over a 43-year period growing seasons; where yield adaptability over time was controlled largely by weather and small variations from year to year in agronomical practices. In other words, major factors influencing yield are location, year, and their interactions. They also observed that yield variation of the 26 crops, including potato, in the Europe was greater than in the USA by nearly two times. In another case, the level of polyphenols in potatoes has been reported to have significant difference with environmental conditions and genetics [22]. A strong relationship and association between growing location of potato, the yellow color intensity in tuber flesh, and its total carotenoid content have also been reported. Report [1] demonstrated that environmental factors may affect on the yellow intensity of tuber flesh. The correlation between genotypes and environment can be indicative of the particular potato cultivar for best adapted to certain locations. For example, in 2004/2005 growing season in New Zealand, Agria was found to have a substantially higher carotenoid content relative to other cultivars with mostly lutein and no zeaxanthin, whereas in 2006/2007 Agria contained all five carotenoids with relatively high concentration of zeaxanthin [23]. A notable difference between the two seasons was the accumulation of zeaxanthin in 2006/2007 and the absence of zeaxanthin in 2004/2005.

pH can affect epoxidation and de-epoxidation reactions in the xanthophyll cycle [24]. Hydroxylation can convert α- and β-carotene to lutein and zeaxanthin, respectively. Violaxanthin is formed from zeaxanthin due to epoxidation and de-epoxidation that can transform violaxanthin back to zeaxanthin. This reaction sequence is reversible and mediated by pH [25, 26]. Epoxidation will occur in darkness or under low light condition and activity is optimal near pH 7.5 [27, 28], whereas de-epoxidation activity is active at pH below 6.5 and optimal at approximately pH 5.2 [24].

Zeaxanthin happens only in trace amounts under physiological conditions *in vivo* or without stress condition [29–31]. Nevertheless, zeaxanthin occurs upon de-epoxidation through the reversible xanthophyll cycle operation due to exposure under irradiance stress or high light condition [32, 33]. Although zeaxanthin accumulates during irradiance stress, that association is normally only transient. Upon recovery under low light or in darkness, zeaxanthin will disappear [33]. In addition, it was revealed from recent *in vitro* studies [34, 35], upon analysis of zeaxanthin accumulating *Arabidopsis thaliana* mutants [36–38] and from the green alga *Scenedesmus obliquus* [39], that zeaxanthin could replace lutein and violaxanthin under irradiance stress.

There are two possibilities to explain the accumulation of zeaxanthin in 2006/2007 season and not previous season:


Overall, this study clearly demonstrated that the total and the individual pigment content of carotenoids in potato tubers were depending on the growing season, subsequently, affect their quality and nutritional content. Thus, along with genotypic factors, environmental factors also take on an important part in regulating the accumulation of individual carotenoids in potato tubers, especially in Agria and Desiree. Between seasons, lutein has been transformed into zeaxanthin in Agria, whereas neoxanthin has been transformed into zeaxanthin in Desiree. These findings evidently suggest that selection of high or low carotenoid tuber levels cannot be established on the basis of a single year's results. Still, valid comparisons can be established between data from different years if the material is stored and developed under similar environmental conditions. This study suggests that environmental factors such as seasonal climatic variation may influence the accumulation of potato tuber carotenoids content and composition. Apparently, further research using potato plant materials produced under different environmental conditions are needed to support this theory.

#### **2. Experimental design**

the growing season [14], even though there are also strong genetic effects [15, 16]. Seasonal differences, growing conditions, locations, genotypes, and postharvest storage conditions are among the factors that can be significantly affecting the quality and nutritional value of potatoes [17–20]. The bioavailability of carotenoids is characterized as an intricate issue and influenced by various factors [21]. In our study of interseasonal and genotype interactions, the data revealed that variations in total carotenoid content and the concentration of individual carotenoid pigments are due to the strong relationship between genotype and growing seasons. This assumption is supported by Chloupek and Hrstkova [22] in their observations of 26 crops over a 43-year period growing seasons; where yield adaptability over time was controlled largely by weather and small variations from year to year in agronomical practices. In other words, major factors influencing yield are location, year, and their interactions. They also observed that yield variation of the 26 crops, including potato, in the Europe was greater than in the USA by nearly two times. In another case, the level of polyphenols in potatoes has been reported to have significant difference with environmental conditions and genetics [22]. A strong relationship and association between growing location of potato, the yellow color intensity in tuber flesh, and its total carotenoid content have also been reported. Report [1] demonstrated that environmental factors may affect on the yellow intensity of tuber flesh. The correlation between genotypes and environment can be indicative of the particular potato cultivar for best adapted to certain locations. For example, in 2004/2005 growing season in New Zealand, Agria was found to have a substantially higher carotenoid content relative to other cultivars with mostly lutein and no zeaxanthin, whereas in 2006/2007 Agria contained all five carotenoids with relatively high concentration of zeaxanthin [23]. A notable difference between the two seasons was the accumulation of zeaxanthin in 2006/2007 and the absence of

pH can affect epoxidation and de-epoxidation reactions in the xanthophyll cycle [24]. Hydroxylation can convert α- and β-carotene to lutein and zeaxanthin, respectively. Violaxanthin is formed from zeaxanthin due to epoxidation and de-epoxidation that can transform violaxanthin back to zeaxanthin. This reaction sequence is reversible and mediated by pH [25, 26]. Epoxidation will occur in darkness or under low light condition and activity is optimal near pH 7.5 [27, 28], whereas de-epoxidation activity is active at pH below 6.5 and optimal at approxi-

Zeaxanthin happens only in trace amounts under physiological conditions *in vivo* or without stress condition [29–31]. Nevertheless, zeaxanthin occurs upon de-epoxidation through the reversible xanthophyll cycle operation due to exposure under irradiance stress or high light condition [32, 33]. Although zeaxanthin accumulates during irradiance stress, that association is normally only transient. Upon recovery under low light or in darkness, zeaxanthin will disappear [33]. In addition, it was revealed from recent *in vitro* studies [34, 35], upon analysis of zeaxanthin accumulating *Arabidopsis thaliana* mutants [36–38] and from the green alga *Scenedesmus obliquus* [39], that zeaxanthin could replace lutein and violaxanthin under

There are two possibilities to explain the accumulation of zeaxanthin in 2006/2007 season and

zeaxanthin in 2004/2005.

88 Carotenoids

mately pH 5.2 [24].

irradiance stress.

not previous season:

#### **2.1. Tissue culture and minituber initiation**

Virus free *in vitro* plants of cultivars Agria and Desiree were supplied by the New Zealand Institute for Crop & Food Research Ltd. These were cultured in a incubation room at 24°C day and night temperature, with a 16-h photoperiod under cool white fluorescent light at 80–85 μmol m−2 s−1. Every 4 weeks, the *in vitro* plants were subcultured as nodal cuttings on potato multiplication medium (PMM) composed of Murashige and Skoog (MS) salts and vitamins [43] added with 30 g/L sucrose, 40 mg/L ascorbic acid, 500 mg/L casein hydrolysate, and 10 g/L agar in accordance with the procedure of Conner et al. [44]. Media was adjusted to pH 5.7 and sterilized by autoclaving (15 min, 121°C) and 50 ml aliquots poured into presterilized 290 ml plastic bottles (80 mm diameter × 60 mm high; Vertex Plastics, Hamilton, New Zealand). For minituber initiation, individual shoots of 3–4 nodes from vigorously growing 4-week-old cultures were transferred into 40 ml of liquid tuber initiation medium (TIM) in 250 ml polycarbonate culture vessels (7 cm diameter × 8 cm high). The TIM contained the same constituents as PMM, except with the addition of 80 g/L sucrose, 5 mg/L benzyladenine, 2.5 mg/L ancymidol, and no agar. Nine shoots were placed upright into each culture vessel and were incubated in darkness at 25°C. Minitubers were classified as such when their diameter exceeded 2 mm and normally grew up to more than 5 mm diameter within 4 weeks.

#### **2.2. Effect of environmental factors on carotenoid biosynthesis**

In three independent experiments, the influence of light, water stress, and nutrient availability on carotenoid biosynthesis were tested in both Agria and Desiree. Minitubers harvested after 4 weeks from two culture vessels were pooled for each of three replicates established under the following conditions:


#### **2.3. Minituber extraction and analysis of carotenoids**

Minitubers were harvested and pooled for each replicated treatment, cut in half, and freezedried as combined skin and flesh samples for 7 days. The samples were then ground into fine powder and kept at −80°C until further analysis.

The extraction procedure followed the methods described in several reports [45–47]. 0.1 g of each powdered sample was rehydrated with distilled water and extracted with a mixture of acetone and methanol (7:3) at room temperature until colorless. The crude extracted was then centrifuged for 5 min at 10,000 g and stored in darkness at 4°C until analysis. The same volume of hexane and distilled water was added to the combined supernatants to extract carotenoids. The mixture was set aside until separation occurred and the upper layer holding the carotenoids was collected. The upper hexane layer was then removed using a gentle stream of oxygen-free nitrogen until the collected carotenoid was dried completely.

The carotenoids HPLC analysis was performed on an Agilent model 1100 series equipped with a binary pump, autosampler injector, micro vacuum degassers, thermostatted column compartment, and a diode array detector [45]. The column used was a Luna C18 end capped 5 μm, 250 × 4.6 mm reverse phase column (Phenomenex Auckland, New Zealand). The solvents used were (A) acetonitrile: water (9:1, v/v) and (B) ethyl acetate. The gradient of solvent used was developed as follows: 0–40% solvent B (0–20 min), 40–60% solvent B (20–25 min), 60–100% solvent B (25–25.1 min), 100% solvent B (25.1–35 min), and 100–0% solvent B (35–35.1 min) at 1.0 ml min−1 flow rate. The column temperature was maintained at 20°C and was allowed to reequilibrate in 100% solvent A for 10 min prior to the next injection. The volume of an injection was 10 μL. Carotenoid standards β-carotene, violaxanthin, lutein, and neoxanthin were isolated from *Eruca sativa* (roquette or rocket salad) by open column chromatography [48], whereas zeaxanthin was obtained commercially from Sigma-Aldrich (Auckland, New Zealand).

#### **3. Results**

Zealand). For minituber initiation, individual shoots of 3–4 nodes from vigorously growing 4-week-old cultures were transferred into 40 ml of liquid tuber initiation medium (TIM) in 250 ml polycarbonate culture vessels (7 cm diameter × 8 cm high). The TIM contained the same constituents as PMM, except with the addition of 80 g/L sucrose, 5 mg/L benzyladenine, 2.5 mg/L ancymidol, and no agar. Nine shoots were placed upright into each culture vessel and were incubated in darkness at 25°C. Minitubers were classified as such when their diameter

In three independent experiments, the influence of light, water stress, and nutrient availability on carotenoid biosynthesis were tested in both Agria and Desiree. Minitubers harvested after 4 weeks from two culture vessels were pooled for each of three replicates established

**1.** Light versus darkness by incubation under cool white fluorescent light (80–85 μmol m−2 s−1, 16 h photoperiod) with dark condition imposed by carefully wrapping the culture vessels

**3.** Incubation in darkness at three concentrations of MS salts (one tenth, half, and full

Minitubers were harvested and pooled for each replicated treatment, cut in half, and freezedried as combined skin and flesh samples for 7 days. The samples were then ground into fine

The extraction procedure followed the methods described in several reports [45–47]. 0.1 g of each powdered sample was rehydrated with distilled water and extracted with a mixture of acetone and methanol (7:3) at room temperature until colorless. The crude extracted was then centrifuged for 5 min at 10,000 g and stored in darkness at 4°C until analysis. The same volume of hexane and distilled water was added to the combined supernatants to extract carotenoids. The mixture was set aside until separation occurred and the upper layer holding the carotenoids was collected. The upper hexane layer was then removed using a gentle stream of

The carotenoids HPLC analysis was performed on an Agilent model 1100 series equipped with a binary pump, autosampler injector, micro vacuum degassers, thermostatted column compartment, and a diode array detector [45]. The column used was a Luna C18 end capped 5 μm, 250 × 4.6 mm reverse phase column (Phenomenex Auckland, New Zealand). The solvents used were (A) acetonitrile: water (9:1, v/v) and (B) ethyl acetate. The gradient of solvent used was developed as follows: 0–40% solvent B (0–20 min), 40–60% solvent B (20–25 min), 60–100% solvent B (25–25.1 min), 100% solvent B (25.1–35 min), and 100–0% solvent B (35–35.1 min) at

oxygen-free nitrogen until the collected carotenoid was dried completely.

**2.** Incubation in darkness with and without 50 mM PEG 4000 to impose water stress.

exceeded 2 mm and normally grew up to more than 5 mm diameter within 4 weeks.

**2.2. Effect of environmental factors on carotenoid biosynthesis**

**2.3. Minituber extraction and analysis of carotenoids**

powder and kept at −80°C until further analysis.

under the following conditions:

in aluminium foil.

strength).

90 Carotenoids

#### **3.1. Effect of light on carotenoid accumulation in potato minitubers**

Statistical analysis demonstrated that there was a highly significant difference (*P* < 0.0001) in carotenoid content in Agria minitubers developing in the dark and light. Agra minitubers accumulated four individual carotenoid compounds (violaxanthin, zeaxanthin, lutein, and β-carotene) when developing in both dark and light. The two predominant carotenoids were violaxanthin and zeaxanthin. Neoxanthin was not detectable in either dark or light treatments. However, development of Agria minitubers in light resulted in an approximate doubling of the total carotenoid content compared minitubers developing in darkness (**Figure 1**). The amount of each individual carotenoid also approximately doubled upon development in light, especially for violaxanthin and zeaxanthin. Analysis of variance comparing Desiree minitubers grown in the dark and light also exhibits highly significant differences (*P* < 0.0001) in carotenoid content. As shown in **Figure 1**, five individual carotenoids (neoxanthin, violaxanthin, zeaxanthin, lutein, and β-carotene) were found in Desiree minitubers grown in darkness, but upon development in light only four (neoxanthin, violaxanthin, lutein, and β-carotene) were detected, with an absence of zeaxanthin. After development in light, total carotenoid content approximately doubled and reflected an increase in neoxanthin and violaxanthin.

#### **3.2. Effect of PEG on carotenoid accumulation in potato minitubers**

Analysis of variance revealed that there was a highly significant difference (*P* < 0.0001) in carotenoid content in response to the water stress treatment during development of Agria minitubers. Agria minitubers developing in the presence of PEG (**Figure 2**) exhibit an increased total carotenoid content. This increase reflected a substantially higher amount of violaxanthin and occurred despite the total absence of zeaxanthin in the presence of PEG. Analysis of variance also indicates highly significant differences (*P* < 0.0001) in carotenoid content for Desiree minitubers developing in the presence of water stress. As shown in **Figure 2**, total carotenoid content increased in minitubers developing in the PEG treatment. This reflected an increase in both neoxanthin and violaxanthin, with traces of lutein being observed in both treatments.

#### **3.3. Effect of nutrient stress on carotenoid accumulation in potato minitubers**

Nutrient stress during Agria minituber development resulted in a highly significant difference (*P* < 0.0001) in carotenoid content. When MS salt strength increased from 0.1× to 0.5×, total carotenoid, violaxanthin, and β-carotene content decreased, accompanied by a slight increase in lutein concentration. However, when MS salt strength increased from 0.5× to 1.0×, total carotenoid, violaxanthin, and β-carotene increased, whereas lutein concentration decreased (**Figure 3**).

Analysis of variance also establishes highly significant differences (*P* < 0.0001) in carotenoid content in Desiree minitubers developing in varying MS salt strengths. As shown in **Figure 3**, when MS salt strength increased from 0.1× to 0.5×, total carotenoid content slightly increased

**Figure 1.** Analysis of carotenoid content (μg/g DW) of Agria and Desiree minitubers in response to light; (A) individual and total carotenoid content (μg/g DW) of Agria minitubers developing in light and dark; (B) individual and total carotenoid content (μg/g DW) of Desiree minitubers developing in light and dark; error bars represent ± SE.

due to minor changes in neoxanthin and lutein. In contrast, upon further increases in MS salt strength, 0.5–1.0×, total carotenoid content and individual carotenoids, especially neoxanthin, violaxanthin, and lutein, decreased. No changes were observed in β-carotene when MS salt strength increased from 0.1× to 0.5× for the development of Desiree minitubers.

concentration. However, when MS salt strength increased from 0.5× to 1.0×, total carotenoid, violaxanthin, and β-carotene increased, whereas lutein concentration decreased (**Figure 3**).

92 Carotenoids

Analysis of variance also establishes highly significant differences (*P* < 0.0001) in carotenoid content in Desiree minitubers developing in varying MS salt strengths. As shown in **Figure 3**, when MS salt strength increased from 0.1× to 0.5×, total carotenoid content slightly increased

**Figure 1.** Analysis of carotenoid content (μg/g DW) of Agria and Desiree minitubers in response to light; (A) individual and total carotenoid content (μg/g DW) of Agria minitubers developing in light and dark; (B) individual and total

carotenoid content (μg/g DW) of Desiree minitubers developing in light and dark; error bars represent ± SE.

**Figure 2.** Analysis of carotenoid content (μg/g DW) of Agria and Desiree minitubers in response to water stress;(A) individual and total carotenoid content (μg/g DW) of Agria upon development with and without PEG treatment; (B) individual and total carotenoid content (μg/g DW) of Desiree upon development with and without PEG treatment; error bars represent ± SE.

**Figure 3.** Analysis of carotenoid content (μg/g DW) of Agria and Desiree minitubers in response to nutrient levels; (A) individual and total carotenoids content (μg/g DW) of Agria upon 0.1×, 0.5× and 1.0× MS salt stress; (B) individual and total carotenoids content (μg/g DW) of Desiree upon 0.1×, 0.5× and 1.0× MS salt stress; error bars represent ± SE.

#### **4. Discussion**

The development of potato minitubers through *in vitro* system has proved to be an effective experimental system for investigating the environmental factors involved in regulating the carotenoid biosynthesis. This potential model system has been used because of several advantages compared to the field cultivated tubers:


Environmental stress is described as external conditions that adversely affect growth, development, or productivity [49]. Plants respond to stress by various means such as transformed gene expression, trigger cellular metabolism, and variations in growth rates and crop yields. There are two categories of stress:

(i) Biotic – caused by other organisms; and

**4. Discussion**

94 Carotenoids

The development of potato minitubers through *in vitro* system has proved to be an effective experimental system for investigating the environmental factors involved in regulating the

**Figure 3.** Analysis of carotenoid content (μg/g DW) of Agria and Desiree minitubers in response to nutrient levels; (A) individual and total carotenoids content (μg/g DW) of Agria upon 0.1×, 0.5× and 1.0× MS salt stress; (B) individual and total carotenoids content (μg/g DW) of Desiree upon 0.1×, 0.5× and 1.0× MS salt stress; error bars represent ± SE.

(ii) Abiotic – resulted from the physical or chemical environment excess or deficiency.

Abiotic or physical and chemical environmental conditions can trigger stress and regulate the carotenoid biosynthesis and of this light, water stress, and nutrient are among the important factors. Species, genotype, and development age are factors influencing the resistance or sensitivity of plants to stress. Three stress resistance mechanisms are as follows:


Plants cultivated in full sunlight exposure usually receive and absorb more light for photosynthesis process. Carotenoids have a significant role to protect photosynthetic organisms against excessive light [50, 51] and these functions have been proven *in vitro* in photosystem II complexes [52]. Light is a major stress factor in plants causing photoinhibition and photooxidation in photosynthetic tissues and inhibit productivity. Light is also one of the main factors regulating carotenoid biosynthesis [53]. Current researches have reported a clear relationship between the dissipation of excess excitation energy and the conversion of zeaxanthin from violaxanthin in the light-harvesting complexes of plants [54–56]. Under these situations, violaxanthin is reversibly de-epoxidized by violaxanthin de-epoxidase to zeaxanthin [57–59]. To put it simply, violaxanthin becomes an efficient accessory pigment in weak light and zeaxanthin becomes an efficient photoprotector in strong light [60]. This association also has been relevant for a varied range of environmental conditions, for example, water stress and high temperature and not just under strong light exposure [61].

This chapter demonstrates that light exposure to Agria and Desiree minitubers leads to the similarity that both total and individual carotenoids were elevated up to two-fold higher on a μg/g DW basis than the total and individual carotenoids produced by dark treatment except for violaxanthin. The findings were consistent with the result reported in the previous studies [62, 63], where high violaxanthin was detected in sun-grown crop plants. However, they are not in agreement with Havaux and Niyogi [60] who found high violaxanthin in the dark and high zeaxanthin in strong light. Lutein and total carotenoid content also high in accordance with their observations and others [64–66]. In this study, lutein and total carotenoid in Agria and Desiree minitubers also increased with light. The changes were due to the stoichiometric and cyclical transformations among violaxanthin, antheraxanthin, and zeaxanthin [67]*.* Light encourages the de-epoxidase reaction and requires acidity for de-epoxidase activity, which can be caused by ATP hydrolysis or supplied by buffer [24, 68, 69]. The de-epoxidase is stereospecific for xanthophylls and as a consequence of that the carotenoid polyene chain must be all-trans. Otherwise, neoxanthin, which is 9-cis, is a passive substrate but becomes active when isomerized to the all-trans form [70].

The phytohormone abscisic acid (ABA) shows a regulatory role in most physiological processes in plants [71]. Various stress conditions, for instances, water, drought, cold, light, and temperature caused an increased amount of ABA. The action of ABA includes alteration of gene expression and analysis of responsive promoters discovered several potential *cis*- and *trans*-acting regulatory elements. In some of the controls in Agria and Desiree minituber experiments, zeaxanthin was detected. The occurrence of zeaxanthin might be in response to the brief exposure of samples to light. Every week, all minituber samples were checked and observed for contamination and size of minitubers. This brief exposure to light might trigger the accumulation of zeaxanthin in some of the minituber control samples.

The presence of zeaxanthin in Agria and Desiree minitubers developing on dark-grown plants could be justified by modification of gene expression in response to stress. Stress recognition may activate signal transduction pathways that transmit information inside the individual cell and through the plant. This may induce gene expression changes that influence growth and development as well as regulate the carotenoid biosynthesis. A stress will trigger and alter cellular metabolism, and as a result zeaxanthin accumulated as a precursor to ABA biosynthesis. Furthermore, plant resistance or sensitivity against stress can be determined by the species, genotype, and development age. In addition, there was a study revealing that different developmental age will accumulate different carotenoid [45]. In their study, 28 day stolons similar to our 4-week minitubers were detected to have highest total carotenoid compared to 80-day developing tubers and 9-month mature tubers. In both cases, zeaxanthin was also detected. Morris et al. [45] also demonstrated comparable results whereby the orange flesh tubers of DB375/1 were detected with high zeaxanthin, while pale yellow Desiree was detected with high violaxanthin. Besides, yellow flesh cultivars were observed to have the capability and ability to generate more carotenoids compared to the white flesh cultivars. Yellow flesh cultivars with high carotenoid content are able to tolerate stress, mainly light with tolerance mechanism [49]. As a result, Desiree minitubers accumulated violaxanthin and neoxanthin when stored in light, while Agria minitubers accumulated zeaxanthin and violaxanthin. In the experiment involving nutrient stress, the higher nutrient concentration given, the higher content of total and individual carotenoids of Agria found. On the other hand, in Desiree, total and individual carotenoids initially increased with increasing nutrient level, but then decreased at higher nutrient levels. The result showed that Desiree minitubers accumulated violaxanthin and neoxanthin, while Agria minitubers accumulated only violaxanthin.

This chapter demonstrates that light exposure to Agria and Desiree minitubers leads to the similarity that both total and individual carotenoids were elevated up to two-fold higher on a μg/g DW basis than the total and individual carotenoids produced by dark treatment except for violaxanthin. The findings were consistent with the result reported in the previous studies [62, 63], where high violaxanthin was detected in sun-grown crop plants. However, they are not in agreement with Havaux and Niyogi [60] who found high violaxanthin in the dark and high zeaxanthin in strong light. Lutein and total carotenoid content also high in accordance with their observations and others [64–66]. In this study, lutein and total carotenoid in Agria and Desiree minitubers also increased with light. The changes were due to the stoichiometric and cyclical transformations among violaxanthin, antheraxanthin, and zeaxanthin [67]*.* Light encourages the de-epoxidase reaction and requires acidity for de-epoxidase activity, which can be caused by ATP hydrolysis or supplied by buffer [24, 68, 69]. The de-epoxidase is stereospecific for xanthophylls and as a consequence of that the carotenoid polyene chain must be all-trans. Otherwise, neoxanthin, which is 9-cis, is a passive substrate but becomes active

The phytohormone abscisic acid (ABA) shows a regulatory role in most physiological processes in plants [71]. Various stress conditions, for instances, water, drought, cold, light, and temperature caused an increased amount of ABA. The action of ABA includes alteration of gene expression and analysis of responsive promoters discovered several potential *cis*- and *trans*-acting regulatory elements. In some of the controls in Agria and Desiree minituber experiments, zeaxanthin was detected. The occurrence of zeaxanthin might be in response to the brief exposure of samples to light. Every week, all minituber samples were checked and observed for contamination and size of minitubers. This brief exposure to light might trigger

The presence of zeaxanthin in Agria and Desiree minitubers developing on dark-grown plants could be justified by modification of gene expression in response to stress. Stress recognition may activate signal transduction pathways that transmit information inside the individual cell and through the plant. This may induce gene expression changes that influence growth and development as well as regulate the carotenoid biosynthesis. A stress will trigger and alter cellular metabolism, and as a result zeaxanthin accumulated as a precursor to ABA biosynthesis. Furthermore, plant resistance or sensitivity against stress can be determined by the species, genotype, and development age. In addition, there was a study revealing that different developmental age will accumulate different carotenoid [45]. In their study, 28 day stolons similar to our 4-week minitubers were detected to have highest total carotenoid compared to 80-day developing tubers and 9-month mature tubers. In both cases, zeaxanthin was also detected. Morris et al. [45] also demonstrated comparable results whereby the orange flesh tubers of DB375/1 were detected with high zeaxanthin, while pale yellow Desiree was detected with high violaxanthin. Besides, yellow flesh cultivars were observed to have the capability and ability to generate more carotenoids compared to the white flesh cultivars. Yellow flesh cultivars with high carotenoid content are able to tolerate stress, mainly light with tolerance mechanism [49]. As a result, Desiree minitubers accumulated violaxanthin and neoxanthin when stored in light, while Agria minitubers accumulated zeaxanthin and violaxanthin. In the experiment involving nutrient stress, the higher nutrient concentration given,

the accumulation of zeaxanthin in some of the minituber control samples.

when isomerized to the all-trans form [70].

96 Carotenoids

Water deficiency is another significant environmental stress, which could influence plant growth and development as it is an essential element to meet basic needs. Drought, hypersaline environments, low temperatures, and transient loss of turgor at midday are among environmental conditions that can cause water deficit [49]. In a water stress condition, ABA will be synthesized by the roots and carried it into the shoots, with ABA being an important mediator to trigger plant responses, especially carotenoid biosynthesis to adverse environmental stimuli [72]. A major increase in the ABA content, particularly, in crops such as winter wheat, potatoes, and alfalfa was detected during hardening and cold acclimation [73–76]. However, the extent of the ABA response influenced by several differences [76], for instance, in winter wheat, a freeze resistant variety of wheat had a greater ABA level than a less resistant variety. Likewise, an increase in the ABA content was also observed in *Solanum commersonii*, but not in *S. tuberosum*, which failed to acclimate at −3°C. Besides, the total and individual carotenoid concentrations in both cultivars increased slightly during the PEG treatments, where the drought stress was simulated (**Figure 2**).

Oxidative cleavage is the first committed reaction for ABA biosynthesis in plant and it has commonly been assumed to be the key regulatory step. Various types of stress could encourage the ABA synthesis; therefore, ABA may be thought as a plant stress hormone [71]. ABA was known as one of the crucial plant growth and development regulator. A significant role for ABA in modulation at the gene level of adaptive responses of plants in adverse environmental conditions was also reported in several previous researches [77–80]. In some other physiological processes, ABA is also involved, for example, in leaf senescence [72], stomatal closure, embryo morphogenesis, development of seeds, and synthesis of storage proteins and lipids [81], germination [82], and defense against pathogens [83]. Nevertheless, ABA plays a role as a mediator in regulating adaptive plant responses to environmental stresses [84]. In certain cases, it has been involved in signal transduction at the single cell level [85]. Therefore, the findings of this study clearly demonstrated that environmental conditions for plant growth and development, such as light, dark, water stress, and nutrient concentration were significantly affecting and stimulating the carotenoid biosynthesis. At the same time, like other environmental stress response, disease or pathogen infection can also lead to oxidative stress responses, which implicate stress response genes [86], as well as storage period, which can cause physical changes such as sprouting and dehydration [87].

The results also suggested that ABA could facilitate a regulatory step for the carotenoid biosynthetic pathway versus environmental stress and during the first committed step in ABA biosynthesis, the epoxidation of zeaxanthin to violaxanthin by ZEP has happened. Zeaxanthin acts as a key element and indicator for the occurrence of environmental stress. In response to environmental stress conditions, violaxanthin and neoxanthin merely accumulated toward producing xanthoxin or precursors of ABA biosynthesis pathway. Predictably, the potato genotypes response to that environmental condition seemed to be highly genotype dependent and time duration exposed to stress. The activity of functional enzymes and candidate enzymes is another factor, which regulates carotenoid biosynthesis that determines the individual carotenoids type and quantity. Since the environmental conditions can influence carotenoid biosynthesis, undoubtedly, the carotenoid type and concentration that accumulates in potato tubers can be induced.

In a nutshell, the differences in carotenoid profile and tuber flesh color from different growing seasons, locations, and cultivars can be explained by the genes regulation, particularly ZEP and VDE, the existence of structure sequestering carotenoids, and the presence of the environmental stress. White flesh cultivars, which have a limited capacity to tolerate excessive light, exhibited an increased susceptibility to photooxidative damage [60]. In contrast, yellow flesh cultivars whose carotenoid content is much higher can specifically tolerate excessive light and also many environmental stress conditions by regulating ZEP and VDE. On top of better yield production, potatoes nutritional value and quality can be enhanced by selecting the appropriate potato cultivars that meet suitable environmental conditions for applicable agronomic practices. In simple words, to increase the accumulation of specific individual carotenoid pigments, implementing the most appropriate environmental factors is required. This invention could be more effective than selecting potato genotypes with higher carotenoid content as parents in a breeding program for the new potato cultivars development with enriched nutrients.

#### **Acknowledgements**

The authors are thankful to the Ministry of Education (MOE) and International Islamic University Malaysia (IIUM) for the Research Initiative Grant Scheme RIGS 16-396-0560.

#### **Author details**

Rashidi Othman<sup>1</sup> \*, Norazian Mohd Hassan<sup>2</sup> and Farah Ayuni Mohd Hatta<sup>1</sup>

\*Address all correspondence to: rashidi@iium.edu.my

1 International Institute for Halal Research and Training (INHART), Herbarium Unit, Department of Landscape Architecture, Kulliyyah of Architecture and Environmental Design, International Islamic University Malaysia, Kuala Lumpur, Malaysia

2 Department of Pharmaceutical Chemistry, Kulliyyah of Pharmacy, International Islamic University Malaysia, Kuala Lumpur, Malaysia

#### **References**

[1] Lu W, Haynes K, Wiley E, Clevidence B. Carotenoid content and colour in diploid potatoes. J. Amer. Soc. Hort. Sci. 2001; 126: 722–726.

[2] Brown CR, Edwards CG, Yang C-P, Dean BB. Orange flesh trait in potato: Inheritance and carotenoid content. J. Amer. Soc. Hort. Sci. 1993; 118:145–150.

time duration exposed to stress. The activity of functional enzymes and candidate enzymes is another factor, which regulates carotenoid biosynthesis that determines the individual carotenoids type and quantity. Since the environmental conditions can influence carotenoid biosynthesis, undoubtedly, the carotenoid type and concentration that accumulates in potato tubers

In a nutshell, the differences in carotenoid profile and tuber flesh color from different growing seasons, locations, and cultivars can be explained by the genes regulation, particularly ZEP and VDE, the existence of structure sequestering carotenoids, and the presence of the environmental stress. White flesh cultivars, which have a limited capacity to tolerate excessive light, exhibited an increased susceptibility to photooxidative damage [60]. In contrast, yellow flesh cultivars whose carotenoid content is much higher can specifically tolerate excessive light and also many environmental stress conditions by regulating ZEP and VDE. On top of better yield production, potatoes nutritional value and quality can be enhanced by selecting the appropriate potato cultivars that meet suitable environmental conditions for applicable agronomic practices. In simple words, to increase the accumulation of specific individual carotenoid pigments, implementing the most appropriate environmental factors is required. This invention could be more effective than selecting potato genotypes with higher carotenoid content as parents in a breeding program for the new potato cultivars development with enriched nutrients.

The authors are thankful to the Ministry of Education (MOE) and International Islamic University Malaysia (IIUM) for the Research Initiative Grant Scheme RIGS 16-396-0560.

1 International Institute for Halal Research and Training (INHART), Herbarium Unit, Department of Landscape Architecture, Kulliyyah of Architecture and Environmental Design,

2 Department of Pharmaceutical Chemistry, Kulliyyah of Pharmacy, International Islamic

[1] Lu W, Haynes K, Wiley E, Clevidence B. Carotenoid content and colour in diploid pota-

and Farah Ayuni Mohd Hatta<sup>1</sup>

\*, Norazian Mohd Hassan<sup>2</sup>

International Islamic University Malaysia, Kuala Lumpur, Malaysia

\*Address all correspondence to: rashidi@iium.edu.my

toes. J. Amer. Soc. Hort. Sci. 2001; 126: 722–726.

University Malaysia, Kuala Lumpur, Malaysia

can be induced.

98 Carotenoids

**Acknowledgements**

**Author details**

Rashidi Othman<sup>1</sup>

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Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67464

#### **Abstract**

Regular consumption of fruits and vegetables can help reduce the risk for cardiovascular disease (CVD) and its associated mortality. A diet rich in fruits and vegetables is thought to have cardioprotective effects, but the specific components of these foods that provide this protection are unclear. Antioxidants such as vitamin C, carotenoids, and polyphenols in fruits and vegetables likely contribute to the reduction in risk of CVD by minimizing cholesterol oxidation in blood vessel walls. Meanwhile, cardioprotective effects afforded by the carotenoids lycopene, α-carotene, β-carotene, β-cryptoxanthin, lutein, and zeaxanthin have been reported in many studies. Carotenoids are naturally occurring fat-soluble pigments that are present at high levels in tomatoes and carrots. Carotenoids play an important role in staving off atherosclerosis via antioxidant activities that reduce lipid peroxidation in low-density lipoproteins. Lycopene reduces endothelin-1 gene expression by suppressing generation of reactive oxygen species and inducing heme oxygenase-1 expression in human endothelial cells. Thus, carotenoids may mitigate endothelial dysfunction by promoting direct antioxidative effects and inducing expression of several genes. Structural and functional differences among carotenoids may explain their unique biologic activities. In this review, the roles of carotenoids in relation to their influence on vascular endothelial functions and cardioprotective effects are discussed.

**Keywords:** carotenoids, cardiovascular disease, endothelial cells

#### **1. Introduction**

Cardiovascular disease (CVD) is a common disease that has high mortality. Many epidemiological studies indicate that a diet rich in fruits and vegetables can have preventive effects for the development of CVD [1, 2]. As such, sufficient consumption of fruits and vegetables is recommended to ensure that vitamins, fiber, potassium, folate, and phenolic molecules are present

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

in proper amounts to yield health benefits [3]. Several of these nutritive components have antioxidant activity and can modify lipoprotein profiles as well as increase insulin sensitivity, and lower blood pressure [4, 5]. Although carotenoids in particular are thought to provide health benefits, several studies suggested that these preventative effects may not be due to β-carotene and vitamin E present in fruits and vegetables [6]. In fact, some reports demonstrated that other carotenoids such as lycopene in tomatoes have preventive effects for CVD [7, 8].

Dietary carotenoids primarily come from fruits and vegetables, as well as plant seeds, roots, leaves, and flowers. Among 12 types of dietary carotenoids, particularly α-carotene, β-carotene, lycopene, lutein, β-cryptoxanthin, and zeaxanthin, can be found in human blood and tissue samples [9, 10], and these molecules have similar chemical constitutions (**Figure 1**) and health benefits [11] (**Table 3**). α-Carotene, β-carotene, γ-carotene, lycopene, and β-cryptoxanthin are all precursors of vitamin A. These carotenoids also have other beneficial effects beyond their antioxidant activity [12, 13].

**Figure 1.** Chemical structures of several carotenoids.

Vascular endothelial cell disorders are a hallmark CVD. Several epidemiologic studies indicate that carotenoids can have a beneficial effect on vascular endothelial cell dysfunction. For example, in experiments using cultured vascular endothelial cells, carotenoids regulated nitric oxide (NO) expression and endothelin-1 (ET-1) production [14]. Moreover, lycopene inhibits expression of lipopolysaccharide (LPS)-enhanced monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), and vascular cell adhesion molecule-1 (VCAM-1) in human endothelial cells [14]. In contrast, lycopene reduced expression of TNF-α–induced intercellular adhesion molecule-1 (ICAM-1) and adhesion of monocyte endothelial cells [15]. In streptozotocin (STZ)-induced diabetic rats, lycopene inhibited endothelial dysfunction [16]. However, the *in vitro* effects of dietary carotenoids do not always translate to an *in vivo* setting. In the present review, we discuss the influence of carotenoids on vascular endothelial functions. Furthermore, we summarize evidence that carotenoids may have a preventive benefit toward CVD.

#### **2. Source and bioactivity of natural carotenoids**

in proper amounts to yield health benefits [3]. Several of these nutritive components have antioxidant activity and can modify lipoprotein profiles as well as increase insulin sensitivity, and lower blood pressure [4, 5]. Although carotenoids in particular are thought to provide health benefits, several studies suggested that these preventative effects may not be due to β-carotene and vitamin E present in fruits and vegetables [6]. In fact, some reports demonstrated that other

Dietary carotenoids primarily come from fruits and vegetables, as well as plant seeds, roots, leaves, and flowers. Among 12 types of dietary carotenoids, particularly α-carotene, β-carotene, lycopene, lutein, β-cryptoxanthin, and zeaxanthin, can be found in human blood and tissue samples [9, 10], and these molecules have similar chemical constitutions (**Figure 1**) and health benefits [11] (**Table 3**). α-Carotene, β-carotene, γ-carotene, lycopene, and β-cryptoxanthin are all precursors of vitamin A. These carotenoids also have other beneficial effects beyond their

Vascular endothelial cell disorders are a hallmark CVD. Several epidemiologic studies indicate that carotenoids can have a beneficial effect on vascular endothelial cell dysfunction. For example, in experiments using cultured vascular endothelial cells, carotenoids regulated nitric oxide (NO) expression and endothelin-1 (ET-1) production [14]. Moreover, lycopene inhibits expression of lipopolysaccharide (LPS)-enhanced monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), and vascular cell adhesion molecule-1 (VCAM-1) in human endothelial cells [14].

carotenoids such as lycopene in tomatoes have preventive effects for CVD [7, 8].

antioxidant activity [12, 13].

106 Carotenoids

**Figure 1.** Chemical structures of several carotenoids.

Carotenoids are found as α-carotene, β-carotene, lycopene, lutein, β-cryptoxanthin, and zeaxanthin. Carotenoids are tetraterpenoids and are synthesized in plants such as vegetables and fruits as well as by other photosynthetic organisms and some nonphotosynthetic bacteria, yeasts, and molds [17]. Carotenoids confer the orange, yellow, and red color of many fruits and vegetables. Carotenoids can be classified as carotenes and xanthophylls according to the chemical structure. Xanthophylls contain oxygen, whereas carotenes are purely hydrocarbons and lack oxygen. The structures of common carotenoids are shown in **Figure 1**. β-Carotene is the most commonly found carotenoid in raw vegetables, canned fruits, and cooked vegetables [13]. Lycopene is present in tomato-based foods, including tomato paste, catsup, and other processed tomato products. Zeaxanthin and lutein are found in cooked kale and spinach and in a number of processed spinach products. Carotenoids can also be found in insects, fish, and crustaceans. The main sources and contents of dietary carotenoids are listed in **Tables 1** and **2** [13, 18]. Carotenoids can be classified into pro-vitamin A and nonpro-vitamin A groups [19]. Daily vitamin A intake is dependent on the pro-vitamin A content of foods. In developing countries, approximately 70% of vitamin A intake is derived from carotenoids found in vegetables and fruits [17]. Pro-vitamin A is converted into vitamin A in the body via mechanisms that are not fully characterized, such that for purposes of bioequivalence, vitamin A levels are quantified according to vitamin A intake. Moreover, conversion efficiencies from carotenoid to vitamin A may influence the biological activity of carotenoids [20].


**Table 1.** Sources of dietary carotenoids.



**Table 2.** Carotenoid contents in foods.

**Carotenoids Food Content (mg/100 g wet wt)a**

Mangos, canned 13.1

Sweet potato, cooked 9.5

Carrots, cooked 8.0

Pumpkin, canned 6.9

Kale, cooked 6.2

Spinach, cooked 5.2

Winter butternut squash 4.6

Swiss chard, raw 3.9

Apricots, raw 2.6

Pepper, red, raw 2.4

Pepper, red, cooked 2.2

Cantaloupe, raw 1.6

Lettuce, romaine, raw 1.3

Tomato paste 1.2

Catsup 17.0

Tomato puree 16.7

Pasta sauce 16.0

Tomato sauce 15.9

Tomato soup 10.9

Tomato, canned, whole 9.7

Tomato juice 9.3

Watermelon, raw 4.9

Tomato, cooked 4.4

Tomato, raw 3.0

β-Carotene Carrots, raw 18.3

108 Carotenoids

Lycopene Tomato paste 29,3

#### **3. Epidemiological studies of carotenoids**

Many epidemiologic studies showed that carotenoids have beneficial effects toward CVD (**Table 3**). A cohort study that included 91,379 men, 129,701 women, and 5007 coronary heart disease events showed that fruits and vegetables intake was associated with decreased levels of coronary heart disease [2]. Meanwhile, another large cohort study indicated that fruits and vegetables intake can reverse coronary heart disease [21]. Many epidemiological studies indicated that higher serum carotenoid levels have beneficial effects on CVD biomarkers. For example, lycopene intake was associated with decreased levels of CVD in a study of 314 CVD patients, 171 CHD patients, and 99 stroke patients [22]. Hazard ratios (HRs) for CVD onset were inversely correlated with lycopene intake. Another study that examined the intake of dietary carotene by 1312 men and 1544 women showed that dietary lutein and zeaxanthin consumption was clearly related to CVD onset, risk ratios, and biomarker levels such as HDL cholesterol [23]. A significant inverse relationship between LDL cholesterol and


**Table 3.** Epidemiological studies of the effect on cardiovascular disease and atherosclerosis with carotenoids.

β-carotene, lutein, and zeaxanthin consumption as well as levels of dietary β-carotene and homocysteine was observed, whereas serum β-carotene affected the relationship between dietary β-carotene intake and C-reactive protein (CRP) levels. Given that hyperlipidemia, serum CRP, and homocysteine are CVD onset risk factors, serum carotenoids may be markers of dietary carotenoid uptake and CVD risk biomarkers. Indeed, a report by Sesso et al. [7] found that higher plasma lycopene levels were associated with decreased risk of CVD in a survey of 39,876 elderly women. In addition, a prospective study indicated that plasma α-carotene, β-carotene, and lycopene levels were associated with the risk of ischemic stroke [24]. A population-based follow-up study in Japan that examined the relationship between CVD and carotene concentration in 3061 subjects showed that higher serum total carotene levels, including α- and β-carotene and lycopene levels were linked with a reduced risk of CVD mortality [8]. Furthermore, report the inverse significant associations between the highest quintiles of the intake of α-carotene and β-carotene and risk of coronary artery disease [25]. In addition, dietary intake of β-carotene was inversely associated with the risk of cerebral infarction [26].

Marine animals produce the carotenoid astaxanthin that is known to have strong antioxidative activity. A study of 24 volunteers that consumed increasing doses of astaxanthin over the course of 14 days showed inhibition of LDL oxidation relative to control subjects that did not consume astaxanthin [27].

In contrast, other reports indicated that fruits and vegetables consumption is not associated with a reduced risk of coronary heart disease [28]. In a study of overweight adults at high risk for CVD, no dose-dependent reduction in CVD risk factors was seen with increased fruits and vegetables intake [29]. These results indicate that there may be some restrictions in the degree of protection afforded by carotenoids [30]. Moreover, a study of healthy adult subjects showed no effects of lutein, lycopene, or β-carotene on biological markers of oxidative stress, including LDL oxidation [31]. In a prospective study, the relationship between plasma lycopene concentration and CVD risk in 499 men showed that higher plasma lutein, zeaxanthin, and retinol levels were associated with a moderate increase in CVD risk, whereas β-cryptoxanthin, α-carotene, and β-carotene were not associated with increased risk of CVD [32]. Likewise, a prospective study involving a population of male physicians in the United States showed that high plasma levels of retinol and carotenoids had no protective effect toward myocardial infarction [33]. Moreover, four extensive, randomized studies revealed no decrease in CVD events by β-carotene treatment [34, 35]. These conflicting results again suggest that the reduction in the risk of CVD associated with fruits and vegetables intake is so far largely confined to observational epidemiology [30].

#### **4. Protective effects of carotenoid-enriched foods**

#### **4.1. Tomato carotenoids**

of coronary heart disease [2]. Meanwhile, another large cohort study indicated that fruits and vegetables intake can reverse coronary heart disease [21]. Many epidemiological studies indicated that higher serum carotenoid levels have beneficial effects on CVD biomarkers. For example, lycopene intake was associated with decreased levels of CVD in a study of 314 CVD patients, 171 CHD patients, and 99 stroke patients [22]. Hazard ratios (HRs) for CVD onset were inversely correlated with lycopene intake. Another study that examined the intake of dietary carotene by 1312 men and 1544 women showed that dietary lutein and zeaxanthin consumption was clearly related to CVD onset, risk ratios, and biomarker levels such as HDL cholesterol [23]. A significant inverse relationship between LDL cholesterol and

> **The number of subjects**

5133 F, M Coronary mortality

**Sex Outcome (main results) Reference** 

(nonsignificant inverse association between dietary intake of carotenoids with provitamin A activity and the risk of coronary mortality in

women)

plaques)

(significantly decreased risk of myocardial infarction in highest -carotene intake quartile)

of β-carotene was inversely associated with the risk of cerebral infarction)

disease (inverse significant associations between the highest quintiles of intake of α-carotene and β-carotene and risk of coronary artery disease)

plaques (those in the highest quintile of carotenoid consumption had a lower prevalence of

12,773 F, M Prevalence of carotid

4802 F, M Myocardial infarction

26,593 M Stroke (dietary intake

73,286 F Coronary artery

**(author, issue year)**

Knekt et al., 1994

Kritchevsky et al., 1998

Klipstein-Grobusch et al., 1999

Hirvonen et al., 2000

Osganian et al., 2003

**Intake from dietary**

110 Carotenoids

Carotenoids with provitamin A activity

Carotenoids with provitamin A activity

β-Carotene The

β-Carotene, lutein plus zeaxanthin, and lycopene

α- and β-Carotene, lutein αplus zeaxanthin, lycopene, and cryptoxanthin

Voutilainen et al. [17].

**Study name**

Finnish Mobile Clinic Study

ARIC study

Rotterdan study

ATBC study

Nurses Health Study

**Nationality of subjects**

**Follow-up, Time**

Finnish Prospective, 14 y

American Cross-

Dutch Prospective, 4 y

Finnish Prospective, 6.1 y

American Prospective, 12 y

**Table 3.** Epidemiological studies of the effect on cardiovascular disease and atherosclerosis with carotenoids.

sectional

Tomato intake has been hypothesized to prevent endothelial dysfunction. However, one study involving 19 postmenopausal women who ingested tomato puree had increased plasma lycopene levels, but no changes in artery dilation, which suggested that lycopene may not have direct effects on endothelial function [36]. On the other hand, another report demonstrated that tomato extract enhanced nitric oxide (NO) production and decreased endothelin release. These effects of tomato extract were related to suppression of inflammatory NF-κB signaling and prevention of adhesion molecule expression in endothelial cells [37], whereas tomato paste supplementation modified endothelial dysfunction and affected oxidation markers in the plasma of healthy human volunteers enrolled in a recent study [38]. Thus, these studies indicated that tomato paste intake can induce beneficial outcomes on endothelial function. The antioxidant properties of lycopene and β-carotene in tomato products may indeed regulate endothelial functions and protect against CVD. In a study that examined pigs with high cholesterol levels, consumption of a tomato-derived lycopene supplement maintained endothelial function of coronary arteries and regulated expression of apolipoprotein A-I and apolipoprotein J [39]. Lycopene supplementation also prevented vasoactive druginduced coronary vasodilation and reduced lipid peroxidation, while enhancing high-density lipoprotein (HDL) levels and endothelial nitric oxide synthase (eNOS) expression. These results demonstrate that lycopene supplementation likely can protect against LDL-enhanced coronary endothelial dysfunction by augmenting endothelial nitric oxide (NO) expression and HDL levels as well as mediating leukocyte adhesion to endothelial cells in response to inflammation.

#### **4.2. Carrot carotenoids**

Carotenoids contained in carrots have beneficial health effects [40]. For example, drinking carrot juice induces antioxidant activity and reduces lipid peroxidation and can decrease levels of CVD risk markers in adults. In addition, carrot juice intake reduces systolic blood pressure [41]. Carrot juice consumption also improved glucose tolerance and hepatic structure and function, which might be associated with the effect of anthocyanins seen in metabolic syndrome [40, 42].

#### **5. Preventive effects of carotenoids on cardiovascular disease associated with endothelial cell and macrophage dysfunction**

Tomato paste supplementation regulated endothelial cell functions and prevented oxidative conditions in 19 healthy subjects [38]. Enhanced reactive oxygen species (ROS) generation is related to a functional inactivation of NO in endothelial cells and can induce CVD. β-Carotene and lycopene-mediated prevention of TNF-α expression was associated with reduced nitrooxidative stress and inflammatory response in endothelial cells [43]. Meanwhile, in human endothelial cells, lycopene prevents endothelin-1 expression by inhibiting ROS generation and inducing heme oxygenase-1 expression (HO-1) [44], while also inhibiting tumor necrosis factor (TNF)-α–induced NF-κB activation, ICAM-1 expression, and monocyte endothelial adhesion [15]. In an *in vivo* study, lycopene inhibited endothelial dysfunction in STZ-enhanced diabetic rats by lowering oxidative stress, which could have implications for the development of treatments to prevent diabetic vascular complications [16]. In addition, astaxanthin inhibits inflammation-induced inducible NO and ROS generation by suppressing NF-κB pathway activity in macrophages [45]. Thus, carotenoids could be effective for treating diseases associated with oxidative stress, such as CVD [46].

plasma lycopene levels, but no changes in artery dilation, which suggested that lycopene may not have direct effects on endothelial function [36]. On the other hand, another report demonstrated that tomato extract enhanced nitric oxide (NO) production and decreased endothelin release. These effects of tomato extract were related to suppression of inflammatory NF-κB signaling and prevention of adhesion molecule expression in endothelial cells [37], whereas tomato paste supplementation modified endothelial dysfunction and affected oxidation markers in the plasma of healthy human volunteers enrolled in a recent study [38]. Thus, these studies indicated that tomato paste intake can induce beneficial outcomes on endothelial function. The antioxidant properties of lycopene and β-carotene in tomato products may indeed regulate endothelial functions and protect against CVD. In a study that examined pigs with high cholesterol levels, consumption of a tomato-derived lycopene supplement maintained endothelial function of coronary arteries and regulated expression of apolipoprotein A-I and apolipoprotein J [39]. Lycopene supplementation also prevented vasoactive druginduced coronary vasodilation and reduced lipid peroxidation, while enhancing high-density lipoprotein (HDL) levels and endothelial nitric oxide synthase (eNOS) expression. These results demonstrate that lycopene supplementation likely can protect against LDL-enhanced coronary endothelial dysfunction by augmenting endothelial nitric oxide (NO) expression and HDL levels as well as mediating leukocyte adhesion to endothelial cells in response to

Carotenoids contained in carrots have beneficial health effects [40]. For example, drinking carrot juice induces antioxidant activity and reduces lipid peroxidation and can decrease levels of CVD risk markers in adults. In addition, carrot juice intake reduces systolic blood pressure [41]. Carrot juice consumption also improved glucose tolerance and hepatic structure and function, which might be associated with the effect of anthocyanins seen in metabolic syn-

**5. Preventive effects of carotenoids on cardiovascular disease associated** 

Tomato paste supplementation regulated endothelial cell functions and prevented oxidative conditions in 19 healthy subjects [38]. Enhanced reactive oxygen species (ROS) generation is related to a functional inactivation of NO in endothelial cells and can induce CVD. β-Carotene and lycopene-mediated prevention of TNF-α expression was associated with reduced nitrooxidative stress and inflammatory response in endothelial cells [43]. Meanwhile, in human endothelial cells, lycopene prevents endothelin-1 expression by inhibiting ROS generation and inducing heme oxygenase-1 expression (HO-1) [44], while also inhibiting tumor necrosis factor (TNF)-α–induced NF-κB activation, ICAM-1 expression, and monocyte endothelial adhesion [15]. In an *in vivo* study, lycopene inhibited endothelial dysfunction in STZ-enhanced diabetic rats by lowering oxidative stress, which could have implications for the development of treatments to prevent diabetic vascular complications [16]. In addition, astaxanthin inhibits inflammation-induced inducible NO and ROS generation by suppressing NF-κB pathway

**with endothelial cell and macrophage dysfunction**

inflammation.

112 Carotenoids

drome [40, 42].

**4.2. Carrot carotenoids**

*In vitr*o studies indicated that endothelial dysfunction induces atherogenic risk [47]. As shown in **Table 4**, carotenoids have a beneficial effect on endothelial cell function. In a study of healthy men, lycopene supplementation was suggested to inhibit oxidative stressmediated decreases in endothelium function [48]. For example, lycopene prevents LPSinduced MCP-1, IL-6, and VCAM-1 expression in human endothelial cells [14]. Similarly, lycopene inhibits activity of an LPS-enhanced proinflammatory cytokine cascade in human endothelial cells through a mechanism that may involve increased expression of Krüppel-like factor 2 (KLF2) and inhibition of toll-like receptor (TLR) 4 function as well as downstream extracellular signal-regulated kinase (ERK) and NF-κB signaling in human endothelial cells [14].

As mentioned above, ET-1 is a strong vasopressor produced by endothelial cells. ET-1 levels may be affected by lycopene and in turn reduce the risk of CVD by modulating the activity of antiinflammatory pathways. Indeed, one report indicated that lycopene prevents cyclic strain-induced endothelin-1 expression by suppressing ROS production in human endothelial cells [44]. Furthermore, β-carotene and lycopene reduced TNF-α–enhanced inflammatory responses by reducing nitro-oxidative stress. These functions decreased interactions of endothelial cells with monocytes [43]. Another report demonstrated that β-carotene and lycopene treatment reduced TNF-α–induced oxidative stress and inflammatory responses to affect interactions between monocytes and human endothelial cells [43]. Furthermore, lycopene reduces C-reactive protein levels in CVD [49]. Meanwhile, paraoxonase-1 (PON1) prevents the oxidation of lipoproteins induced by oxidative stress and may induce metabolism of lipid peroxides [50]. We demonstrated that β-carotene decreases IL-1β–induced downregulation in PON1 expression by activating the CaMKKII signaling pathway in human endothelial cells that may in turn produce antioxidant activity [51]. Similarly, astaxanthin reduces ROS induced-associated dysfunction in human endothelial cells exposed to glucose [52]. Astaxanthin inhibits streptozotocin-induced endothelial dysfunction in diabetes in male rats [53]. Astaxanthin also has antioxidant activity in human endothelial cells that is related to induction of p22phox expression and reduced peroxisome proliferator activated receptor-γ coactivator (PGC-1α) expression [54]. Together these activities of carotenoids may be responsible for their protective effect on CVD risk.

In cultured mouse macrophages, lutein-induced matrix metalloproteinase (MMP)-9 expression and phagocytosis promoted by intracellular ROS and activation of ERK1/2, p38 MAPK, and RAR β [55]. Furthermore, carotenoids induce increases in intracellular glutathione levels by elevating the activity of glutamate–cysteine ligase, the rate limiting enzyme in GSH synthesis [56]. In addition, preventive effects of β-carotene are associated with the β-carotene cleavage enzyme β-carotene 15,15′-monooxygenase (BCMO1) [57]. In the human macrophage cell line THP-1, β-carotene inhibited 7-ketocholesterol (7KC)-induced apoptosis by reducing expression levels of p53, p21, and Bax and inducing expression of AKT, Bcl-2, and Bcl-xL. Concomitantly, 7KC induced ROS generation with enhanced expression of NAD(P)H oxidase (NOX4). However, β-carotene blocked 7KC-induced ROS generation by inhibiting NOX4 [58]. Together these results indicate a possible antiarteriosclerotic action of β-carotene mediated


ACh: acetylcholine; cGMP: cyclic GMP; CVD: cardiovascular disease; IL-1: interleukin-1; ICAM-1: intercellular adhesion molecule-1; LOX-1: lectin-like oxidized low density lipoprotein (LDL) receptor-1; MDA: malondialdehyde; NO: nitric oxide; oxLDL: oxidized low-density lipoprotein; TLR4: Toll-like receptor 4; TNFα: tumor necrosis factor-alpha; VCAM-1: vascular cell adhesion molecule-1.

**Table 4.** Preventive effect of carotenoids on vascular endothelial cells and macrophages.

through 7KC in human macrophages. β-Carotene also prevents expression of inflammatory genes such as inducible NO synthase (iNOS), cyclooxygenase-2 (COX2), TNF-α, and IL-1 in LPS-enhanced macrophages by inhibiting redox-related NF-κB activation [59].

#### **6. Conclusions**

**Carotenoids Preventive effects Mechanism of effects Experiment procedure Reference (author,** 

Induction of the CaMKKII pathway

Prevention of induced, inflammation, decrease of ROS generation, increased NO/cGMP levels and reduces NF-κB– dependent adhesion molecule expression

Block of ROS generation through NAD(P)H oxidase

Low C-reactive protein levels in CVD and health volunteer

Reduce oxidative stress, low C-reactive protein levels and decreased ICAM-1.

Inhibit TLR4 and NF-kappaB signaling

Reduced ROS generation

Inhibition of the ox-LDL/LOX-1-eNOS

ACh: acetylcholine; cGMP: cyclic GMP; CVD: cardiovascular disease; IL-1: interleukin-1; ICAM-1: intercellular adhesion molecule-1; LOX-1: lectin-like oxidized low density lipoprotein (LDL) receptor-1; MDA: malondialdehyde; NO: nitric oxide; oxLDL: oxidized low-density lipoprotein; TLR4: Toll-like receptor 4; TNFα: tumor necrosis factor-alpha; VCAM-

VCAM-1

pathway

pathway

**Table 4.** Preventive effect of carotenoids on vascular endothelial cells and macrophages.

activity

β-Carotene Reverses the IL-1β-

114 Carotenoids

Lycopene Inhibited

induced decrease in paraoxonase-1 expression

Prevent the TNFαinduced decrease nitro-oxidative stress and interaction with monocytes

endothelin-1 expression and induces heme oxygenase-1

Improved endotheliumdependent vasodilatation

function

Reduce proinflammatory cytokine cascade

ACh.

1: vascular cell adhesion molecule-1.

Astaxanthin Protect against

Increase endothelial

glucose fluctuation.

Ameliorative effect on endothelial dysfunction in streptozotocininduced diabetes rats. Reduced serum oxLDL and aortic MDA. Reduced endotheliumdependent vasodilator with

**issue year)**

*In vitro* Yamagata et al., 2012

*In vitro* Di et al., 2012

*In vitro* Sung et al., 2015

*In vivo* Gajendragadkar et

*In vivo* Kim et al., 2011

*In vitro* Wang et al., 2013

*In vitro* Abdelzaher et al., 2016

*In vivo* Zhao et al., 2011

al., 2014

This review examined the protective effects of carotenoids on CVD and the beneficial health effects of dietary carotenoids. Many studies indicated that carotenoids exhibit bioactivity in vascular endothelial cells. Carotenoids have antioxidant activity and appear to support and maintain normal vascular endothelial cell function. Future research may reveal new beneficial effects of carotenoids and help elucidate their preventive mechanisms in CVD.

#### **Abbreviation**


#### **Author details**

#### Kazuo Yamagata

Address all correspondence to: kyamagat@brs.nihon-u.ac.jp

Laboratory of Molecular Health Science of Food, Department of Food Science &; Technology, College of Bioresource Science, Nihon University (NUBS), Fujisawa, Kanagawa, Japan

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**Author details**

116 Carotenoids

Kazuo Yamagata

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## **Biotechnological Applications**
