**Meet the editors**

Associate Professor Dr. Janka Vašková has completed her PhD in Anthropology. She was appointed Associate Professor in Anthropology in 2016, and received the same scientific qualification level, senior researcher, in clinical biochemistry. She is working as a research scientist in the Department of Medical and Clinical Biochemistry, Faculty of Medicine at Pavol Jozef Šafarik University in Kosice.

She is the author of three monographs/book chapters, four textbooks, and more than 280 original scientific and conference papers. Currently, she deals with the detection of pro- and antioxidant properties of natural and synthetic substances, their effect on the antioxidant status of the organism, as well as assessment of treatment success and patient prognosis by detecting the changes in selected antioxidant markers.

Ladislav Vaško, Associate Professor, DVM, PhD, has been working at the Clinic of Internal Diseases since graduation from the University of Veterinary Medicine in Košice. After doctoral studies he taught chemistry at the Department of Biochemistry and Toxicology. He was appointed Associate Professor in Biochemistry by the Ministry of Education of the Slovak Republic, and in 2006

habilitated to Associate Professor in Physiology and Morphology. Since 2008 he has been working at the Faculty of Medicine in Košice. He is the author of three monographs/book chapters, nine textbooks, and more than 420 scientific and conference papers. Currently, he deals with the effects of humic acids on organisms with simultaneous intake of toxic substances.

Contents

**Preface VII**

**Compounds 1**

**Ghats, India 13**

Janka Vašková and Ladislav Vaško

Soundari and Selvam Tamilarasi

and Kimberly S. George Parsons

**Design and Therapeutic Use 43**

Chapter 4 **Indomethacin from Anti-Inflammatory to**

**Anticancer Agent 45** Shaymaa Emam Kassab

**on M624 Human Melanoma Cells 29**

**Section 1 Effects on Biological Systems: In Vitro Testing 11**

Chapter 1 **Introductory Chapter: Unregulated Mitochondrial Production**

Chapter 2 **Determination of In Vitro Cytotoxicity and Anti-Angiogenesis**

Chapter 3 **The Apoptotic Effects of Methylparaben and Ultraviolet B Light**

**Section 2 Structure-activity Studies of Biological Effectiveness in Drug**

Chapter 5 **1,4-Benzodiazepines and New Derivatives: Description,**

**Analysis, and Organic Synthesis 63**

**Isolated from Aegle marmelos around Western**

**of Reactive Oxygen Species in Testing the Biological Activity of**

**for a Bioactive Compound from Aspergillus terreus FC36AY1**

Rebekah S. Wood, Rebecca S. Greenstein, Isabella M. Hildebrandt

Elisabet Batlle, Enric Lizano, Miquel Viñas and Maria Dolors Pujol

Vellingiri Manon Mani, Arockiam Jeyasundar Parimala Gnana

## Contents

#### **Preface XI**


**Section 3 Pharmacokinetic of Drugs, Effect of Compound Interactions on Cytochrome P450 Activity 91**

Preface

The area covered by this book undoubtedly includes a multidisciplinary approach. It com‐ bines and uses the wide range of methods and knowledge from a variety of disciplines in chemistry, pharmacology, and biology to synthesize new or extracted natural substances and their characterization, in terms of bioefficiency in different systems, pharmacokinetics, and pharmacodynamics. Importance is placed on revealing the interactions and effects on organisms. The process is long term, ranging from synthesis to potential testing of substan‐ ces in animal studies, followed by monitoring effects on patients. The purpose is to define molecular targets of the highest efficacy of the prepared drugs, minimizing the undesirable

The introductory chapter deals with the use or monitoring of influence of production of re‐ active oxygen species by mitochondria after administration of newly prepared substances, which were devoted to this research by Prof. Perjési from Pécs (Hungary). Excessive and unmanageable production of reactive oxygen species leads to disruption of mitochondrial functions and induction of apoptosis. The chapter covers the monitoring of the efficacy of substances in biological systems in vitro. To illustrate the effects on cell substructures and energy production, the following chapters deal with the definition of cytotoxic effects of the isolated endophytic fungus *Aspergillus terrus* FC36AY1, as well as methylparaben, a sub‐ stance used in cosmetics and UVB. Subsequently, the chapters deal with the development and effects of indomethacin-derived structures and derivatives derived from benzodiaze‐ pine and point to possible more pronounced and more targeted effects of these substances. Monitoring of the pharmacokinetics of known and used active substances at various concen‐ trations and in combinations with other active substances as demonstrated in the chapter on clavulanic acid inactivating bacterial B-lactamase or novel iodine complex FS-1 as antituber‐ culosis is another phase of research regarding organ distribution and the desired efficacy. Metabolism of ingested drugs and their bioavailability are mediated by cytochrome P450. The efficacy of its enzyme activity is directly related to the distribution and duration of ac‐ tion of these substances in organs. In this respect, it is necessary to know the effect of the activity of these enzymes on commonly ingested fruits and vegetables. The last part is dedi‐ cated to the authorized in vivo study of the endocrine disruption activity of three pesticides in rat embryos. This section also includes a chapter on the positive results of the propriocep‐ tive neuromuscular facilitation method carried out on 15 football players on muscle stem

It is hoped that this book will be of benefit to all the readers for whom it is intended.

**Janka Vašková, Assoc. Prof. Dr. PhD, and Ladislav Vaško, Assoc. Prof. DVM, PhD**

Faculty of Medicine

Pavol Jozef Šafárik University Košice, Slovak Republic

effects. The content of this book is conceived with these intentions.

cells and growth factor stimulation.


## Preface

**Section 3 Pharmacokinetic of Drugs, Effect of Compound Interactions on**

**Lactamase Isolated from Streptomyces clavuligerus and Its**

Rinat Islamov, Bahkytzhan Kerimzhanova and Alexander Ilin

Xóchitl S. Ramírez-Gómez, Sandra N. Jiménez-García, Vicente Beltrán Campos, Esmeralda Rodríguez Miranda, Gabriel Herrera

Chapter 9 **The Pragmatic Strategy to Detect Endocrine-Disrupting Activity**

Shui-Yuan Lu, Pinpin Lin, Wei-Ren Tsai and Chen-Yi Weng

Chapter 8 **Clinical Relevance of Medicinal Plants and Foods of Vegetal Origin on the Activity of Cytochrome P450 117**

Anab Fatima, Mohammad Jiyad Shaikh, Hina Zahid, Ishart Younus,

Chapter 6 **Clinical Pharmacokinetics of Clavulanic Acid, a Novel β-**

**Cytochrome P450 Activity 91**

Sheikh Abdul Khaliq and Farah Khalid

Chapter 7 **New Antituberculosis Drug FS-1 103**

Pérez and Rafael Vargas-Bernal

**of Xenobiotics in Food 137**

**Section 4 Effects on Biological Systems: In Vivo Testing 135**

**Variability 93**

**VI** Contents

The area covered by this book undoubtedly includes a multidisciplinary approach. It com‐ bines and uses the wide range of methods and knowledge from a variety of disciplines in chemistry, pharmacology, and biology to synthesize new or extracted natural substances and their characterization, in terms of bioefficiency in different systems, pharmacokinetics, and pharmacodynamics. Importance is placed on revealing the interactions and effects on organisms. The process is long term, ranging from synthesis to potential testing of substan‐ ces in animal studies, followed by monitoring effects on patients. The purpose is to define molecular targets of the highest efficacy of the prepared drugs, minimizing the undesirable effects. The content of this book is conceived with these intentions.

The introductory chapter deals with the use or monitoring of influence of production of re‐ active oxygen species by mitochondria after administration of newly prepared substances, which were devoted to this research by Prof. Perjési from Pécs (Hungary). Excessive and unmanageable production of reactive oxygen species leads to disruption of mitochondrial functions and induction of apoptosis. The chapter covers the monitoring of the efficacy of substances in biological systems in vitro. To illustrate the effects on cell substructures and energy production, the following chapters deal with the definition of cytotoxic effects of the isolated endophytic fungus *Aspergillus terrus* FC36AY1, as well as methylparaben, a sub‐ stance used in cosmetics and UVB. Subsequently, the chapters deal with the development and effects of indomethacin-derived structures and derivatives derived from benzodiaze‐ pine and point to possible more pronounced and more targeted effects of these substances. Monitoring of the pharmacokinetics of known and used active substances at various concen‐ trations and in combinations with other active substances as demonstrated in the chapter on clavulanic acid inactivating bacterial B-lactamase or novel iodine complex FS-1 as antituber‐ culosis is another phase of research regarding organ distribution and the desired efficacy. Metabolism of ingested drugs and their bioavailability are mediated by cytochrome P450. The efficacy of its enzyme activity is directly related to the distribution and duration of ac‐ tion of these substances in organs. In this respect, it is necessary to know the effect of the activity of these enzymes on commonly ingested fruits and vegetables. The last part is dedi‐ cated to the authorized in vivo study of the endocrine disruption activity of three pesticides in rat embryos. This section also includes a chapter on the positive results of the propriocep‐ tive neuromuscular facilitation method carried out on 15 football players on muscle stem cells and growth factor stimulation.

It is hoped that this book will be of benefit to all the readers for whom it is intended.

#### **Janka Vašková, Assoc. Prof. Dr. PhD, and Ladislav Vaško, Assoc. Prof. DVM, PhD** Faculty of Medicine Pavol Jozef Šafárik University Košice, Slovak Republic

**Chapter 1**

**Provisional chapter**

**Introductory Chapter: Unregulated Mitochondrial**

**Introductory Chapter: Unregulated Mitochondrial** 

**Biological Activity of Compounds**

**Biological Activity of Compounds**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Janka Vašková and Ladislav Vaško

Janka Vašková and Ladislav Vaško

http://dx.doi.org/10.5772/intechopen.82514

**1. Introduction**

compounds in mitochondria.

**Production of Reactive Oxygen Species in Testing the**

**Production of Reactive Oxygen Species in Testing the** 

Medicinal chemistry is an area that creates important links between the function of living organisms and the action of substances, whether natural or synthetic. This includes studies of structure–activity and dose–response associations in cell culture systems, in vitro and subsequent in vivo studies. The treatment of many diseases requires continuous invention, synthesis, characterization, and final testing of new designed compounds. Recently, there is also growing interest in better and more targeted use of the rich spectrum of effective natural substances extracted from plants. Each study thus contributes to the characterization of the effects of substances in order to achieve the least possible side effects in interactions and metabolism but significant expected ones. Also, part of such studies has been carried out at our workplace, which show the necessary and complementary role of identifying the effects and use of these substances. In this way, we introduce a shortened preview of unregulated endogenous production of reactive oxygen and nitrogen species in the biological activity of

Mitochondria are two membrane organelles present in all cells that have a nucleus. They are the energy center of the cells. Their primary role is the production of ATP in oxidative phosphorylation, and the basis of the aerobic oxidation is the citric acid cycle interconnection representing the final metabolic pathway of oxidation of all major nutrients to the respiratory

**2. Reactive oxygen species production in mitochondria**

© 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.

© 2018 The Author(s). Licensee IntechOpen. 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.

DOI: 10.5772/intechopen.82514

#### **Introductory Chapter: Unregulated Mitochondrial Production of Reactive Oxygen Species in Testing the Biological Activity of Compounds Introductory Chapter: Unregulated Mitochondrial Production of Reactive Oxygen Species in Testing the Biological Activity of Compounds**

DOI: 10.5772/intechopen.82514

Janka Vašková and Ladislav Vaško Janka Vašková and Ladislav Vaško

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

http://dx.doi.org/10.5772/intechopen.82514

## **1. Introduction**

Medicinal chemistry is an area that creates important links between the function of living organisms and the action of substances, whether natural or synthetic. This includes studies of structure–activity and dose–response associations in cell culture systems, in vitro and subsequent in vivo studies. The treatment of many diseases requires continuous invention, synthesis, characterization, and final testing of new designed compounds. Recently, there is also growing interest in better and more targeted use of the rich spectrum of effective natural substances extracted from plants. Each study thus contributes to the characterization of the effects of substances in order to achieve the least possible side effects in interactions and metabolism but significant expected ones. Also, part of such studies has been carried out at our workplace, which show the necessary and complementary role of identifying the effects and use of these substances. In this way, we introduce a shortened preview of unregulated endogenous production of reactive oxygen and nitrogen species in the biological activity of compounds in mitochondria.

## **2. Reactive oxygen species production in mitochondria**

Mitochondria are two membrane organelles present in all cells that have a nucleus. They are the energy center of the cells. Their primary role is the production of ATP in oxidative phosphorylation, and the basis of the aerobic oxidation is the citric acid cycle interconnection representing the final metabolic pathway of oxidation of all major nutrients to the respiratory

© 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. © 2018 The Author(s). Licensee IntechOpen. 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.

chain where oxidation of reduced coenzymes results in ATP formation. By the process of oxidative phosphorylation, the mitochondria have an irreplaceable function in the formation of metabolic energy in the form of ATP. The electrodes released in this process from reduced substrates are transferred to O2 via the H+ pumps of the respiratory chain. Pumps (complexes I–IV) form a H+ gradient through the internal mitochondrial membrane, and the electrochemical energy of this gradient is then used to synthesize ATP complex V, ATP synthase [1]. Gradual reduction of O2 occurs through several interstages when reactive oxygen species (ROS) are formed. One-electron reduction of O<sup>2</sup> to superoxide radical (O2 **˙−**) is thermodynamically more advantageous, even for substances with relative oxidation ability, so in the mitochondria, a number of electron donors potentially allow this reaction [2]. However, only a small number of mitochondrial electron transporters with thermodynamic potential to reduce O2 actually act. In most cases, small-molecule electron transporters such as NADH, NADPH, reduced coenzyme (CoQH<sup>2</sup> ), and reduced glutathione (GSH) do not react with O<sup>2</sup> but regenerate it. Instead, O2 **˙−** production takes place on the redox-active prosthetic groups of proteins or electron-binding proteins such as CoQH<sup>2</sup> , which is a kinetic factor that allows or prevents the reduction of O2 molecules and determines the production of O2 **˙−** in the mitochondria [3]. The mechanism of mitochondrial production and release of H2 O2 and O2 **˙−** can be seen as described in more detail in [4]. Overall, in aerobic metabolism, the mitochondrial oxidative phosphorylation system balances the reduction of O2 to H2 O in maximizing ATP synthesis with the simultaneous production of ROS only to the amount required for cell signaling [5].

They catalyze the oxidation of biogenic amines while releasing H2

**˙−** but also H2

**˙−** and H2

considered to be the source of O2

O2

a significant source of ROS [12].

**3. Testing compounds**

that can be oxidized by O2

formation of H2

of the internal mitochondrial membrane, the conversion of dihydroorotate to orotate catalyzes the de novo synthesis of uridine monophosphate dihydroorotate dehydrogenase. It is

further study. Also, part of a glycerophosphate shuttle, the mitochondrial glycerol-3-phosphate dehydrogenase, is present in all cells but with uneven expression and mediating the

the conversion of citrate to isocitrate within the Krebs cycle. Enzyme contains a Fe-S cluster

lipoamide dehydrogenase from the ketoglutarate dehydrogenase complex, located on the

The subunit of the pyruvate dehydrogenase complex, dihydrolipoyl dehydrogenase, is also

Mitochondria and ROS signaling control cell homeostasis by regulating processes of physiological cell death (apoptosis), including autophagy however also that of survival. Damage of mtDNA, protein carbonylation, or lipid peroxidation due to increased ROS production have been documented in many studies, and due to the localization and metabolic role of the organelles, they can lead to an energy disaster of the cell. Therefore, the production of ROS by mitochondria is considered crucial for cell survival or death. Many proteins that mediate apoptosis and autophagy directly affect ROS signaling by translocation into the mitochondria

Synthetic and natural substances can affect the production of ROS; alter the redox state of the cell and, depending on the extent of the oxidative change; affect proliferation; or induce apoptosis. Chalcones are intermediate products of biosynthesis of a wide variety of plant polyphenols, flavonoids. Chalcones, as α, β-unsaturated carbonyl compounds have a wide range of substituents. The cycles are connected by three strongly electrophilic carbons, and the whole system creates a linear or almost planar structure [13]. They also contain a ketoethylene group (–CO–CH=CH–). They have conjugated double bonds and a fully delocalized π-electron system on both benzene rings [14]. Structure–activity studies have shown that the cytotoxicity of chiral analogues is affected by the shape of the molecules [15, 16]. The multimodal pharmacodynamic, structural diversity of synthetic and natural chalcones and the constitutive elements that create optimal toxicity vary for each class of chalcones, and there are no generally valid rules of relationship between structure and activity [17]. Changes in the structure create a high degree of diversity which, as was shown, is useful for the development of new drugs with better efficacy, lower toxicity, and good pharmacological action. Thus, chalcones have become of interest not only in the academic but also in the industrial sphere and are used as intermediates for the preparation of compounds having therapeutic utility. They are currently used in the treatment of viral, cardiovascular diseases, parasitic infections, pain, inflammation, and gastric cancer, as well as additives and cosmetic ingredients [14].

O2

inner side of the membrane turned into the mitochondrial matrix, produces O2

compartment and subsequent modulation of pro- or antioxidant enzymes [9].

O2

O2

http://dx.doi.org/10.5772/intechopen.82514

leading to **˙**OH production [11]. The subunit of

, although its ability to produce O2

[10]. In the mitochondrial matrix, there is localized aconitase catalyzing

Introductory Chapter: Unregulated Mitochondrial Production of Reactive Oxygen Species…

[9]. On the outside

**˙−** requires

3

**˙−** and H2

O2 .

The major part of the mitochondrial ROS production is formed as a by-product of the respiration on the inner side of the inner mitochondrial membrane. Complex IV (cytochrome c oxidase), a terminal component of the electron transport chain, receives four electrons from cytochrome c and reduces one molecule of O2 to two H2 O. The intermediates remain partially reduced until they are completely reduced and are not secreted in measurable amounts [6]. Historically, the O2 **˙−** producing complex III was described as the first site of ROS production—in the Q-cycle [7]. Theoretically, the oxidation of succinate by succinate dehydrogenase (complex II, SDH) leads to significant O<sup>2</sup> **˙−** formation—but so far it has not been measured. Thus, it is not entirely clear whether SDH produces in situ mitochondrial ROS. Nevertheless, the production of ROS complex II is a significant source in many tissues via the reverse electron transport mechanism. This particular phenomenon results from a high membrane potential that thermodynamically favors complex II as a donor for complex I., thanks to which succinate supports production of ROS in complex I. Thanks to which succinate supports production of ROS in complex I [6]. Complex I (NADH dehydrogenase) is the main entry of electrons into the respiratory chain. It is a significant source of ROS, namely, O<sup>2</sup> **˙−** and H2 O2 , although it is very complicated to find out whether it is the major source of ROS in mitochondria in vivo. All evidence of significant ROS production was obtained in in vitro studies [8].

The mitochondria also contain other sources, outside the respiratory chain that highlight ROS production. On the outer mitochondrial membrane, there are cytochrome b5 reductase and monoamine oxidase. Cytochrome b5 reductase is present in all mammalian tissues and is capable of production O2 **˙−** at a very high rate of about 300 mmol.min−<sup>1</sup> .mg of protein−<sup>1</sup> . Monoamine oxidases (MAO-A and MAO-B) are also present in all tissues of mammals. They catalyze the oxidation of biogenic amines while releasing H2 O2 [9]. On the outside of the internal mitochondrial membrane, the conversion of dihydroorotate to orotate catalyzes the de novo synthesis of uridine monophosphate dihydroorotate dehydrogenase. It is considered to be the source of O2 **˙−** and H2 O2 , although its ability to produce O2 **˙−** requires further study. Also, part of a glycerophosphate shuttle, the mitochondrial glycerol-3-phosphate dehydrogenase, is present in all cells but with uneven expression and mediating the formation of H2 O2 [10]. In the mitochondrial matrix, there is localized aconitase catalyzing the conversion of citrate to isocitrate within the Krebs cycle. Enzyme contains a Fe-S cluster that can be oxidized by O2 **˙−** but also H2 O2 leading to **˙**OH production [11]. The subunit of lipoamide dehydrogenase from the ketoglutarate dehydrogenase complex, located on the inner side of the membrane turned into the mitochondrial matrix, produces O2 **˙−** and H2 O2 . The subunit of the pyruvate dehydrogenase complex, dihydrolipoyl dehydrogenase, is also a significant source of ROS [12].

Mitochondria and ROS signaling control cell homeostasis by regulating processes of physiological cell death (apoptosis), including autophagy however also that of survival. Damage of mtDNA, protein carbonylation, or lipid peroxidation due to increased ROS production have been documented in many studies, and due to the localization and metabolic role of the organelles, they can lead to an energy disaster of the cell. Therefore, the production of ROS by mitochondria is considered crucial for cell survival or death. Many proteins that mediate apoptosis and autophagy directly affect ROS signaling by translocation into the mitochondria compartment and subsequent modulation of pro- or antioxidant enzymes [9].

### **3. Testing compounds**

chain where oxidation of reduced coenzymes results in ATP formation. By the process of oxidative phosphorylation, the mitochondria have an irreplaceable function in the formation of metabolic energy in the form of ATP. The electrodes released in this process from

via the H+

electrochemical energy of this gradient is then used to synthesize ATP complex V, ATP syn-

modynamically more advantageous, even for substances with relative oxidation ability, so in the mitochondria, a number of electron donors potentially allow this reaction [2]. However, only a small number of mitochondrial electron transporters with thermodynamic potential

be seen as described in more detail in [4]. Overall, in aerobic metabolism, the mitochondrial

synthesis with the simultaneous production of ROS only to the amount required for cell sig-

The major part of the mitochondrial ROS production is formed as a by-product of the respiration on the inner side of the inner mitochondrial membrane. Complex IV (cytochrome c oxidase), a terminal component of the electron transport chain, receives four electrons from

reduced until they are completely reduced and are not secreted in measurable amounts [6].

tion—in the Q-cycle [7]. Theoretically, the oxidation of succinate by succinate dehydrogenase

Thus, it is not entirely clear whether SDH produces in situ mitochondrial ROS. Nevertheless, the production of ROS complex II is a significant source in many tissues via the reverse electron transport mechanism. This particular phenomenon results from a high membrane potential that thermodynamically favors complex II as a donor for complex I., thanks to which succinate supports production of ROS in complex I. Thanks to which succinate supports production of ROS in complex I [6]. Complex I (NADH dehydrogenase) is the main entry of electrons into

very complicated to find out whether it is the major source of ROS in mitochondria in vivo. All

The mitochondria also contain other sources, outside the respiratory chain that highlight ROS production. On the outer mitochondrial membrane, there are cytochrome b5 reductase and monoamine oxidase. Cytochrome b5 reductase is present in all mammalian tissues and

Monoamine oxidases (MAO-A and MAO-B) are also present in all tissues of mammals.

**˙−** at a very high rate of about 300 mmol.min−<sup>1</sup>

to two H2

**˙−** producing complex III was described as the first site of ROS produc-

actually act. In most cases, small-molecule electron transporters such as NADH,

molecules and determines the production of O2

pumps of the respiratory chain. Pumps

, which is a kinetic factor that allows

O. The intermediates remain partially

O2

**˙−**) is ther-

**˙−** in the mito-

**˙−** can

and O2

O in maximizing ATP

gradient through the internal mitochondrial membrane, and the

occurs through several interstages when reactive oxygen

), and reduced glutathione (GSH) do not react with O<sup>2</sup>

to H2

**˙−** formation—but so far it has not been measured.

**˙−** and H2

O2

, although it is

.mg of protein−<sup>1</sup>

.

**˙−** production takes place on the redox-active prosthetic groups

to superoxide radical (O2

reduced substrates are transferred to O2

species (ROS) are formed. One-electron reduction of O<sup>2</sup>

of proteins or electron-binding proteins such as CoQH<sup>2</sup>

oxidative phosphorylation system balances the reduction of O2

the respiratory chain. It is a significant source of ROS, namely, O<sup>2</sup>

evidence of significant ROS production was obtained in in vitro studies [8].

chondria [3]. The mechanism of mitochondrial production and release of H2

(complexes I–IV) form a H+

to reduce O2

2 Medicinal Chemistry

naling [5].

Historically, the O2

is capable of production O2

thase [1]. Gradual reduction of O2

NADPH, reduced coenzyme (CoQH<sup>2</sup>

cytochrome c and reduces one molecule of O2

(complex II, SDH) leads to significant O<sup>2</sup>

but regenerate it. Instead, O2

or prevents the reduction of O2

Synthetic and natural substances can affect the production of ROS; alter the redox state of the cell and, depending on the extent of the oxidative change; affect proliferation; or induce apoptosis. Chalcones are intermediate products of biosynthesis of a wide variety of plant polyphenols, flavonoids. Chalcones, as α, β-unsaturated carbonyl compounds have a wide range of substituents. The cycles are connected by three strongly electrophilic carbons, and the whole system creates a linear or almost planar structure [13]. They also contain a ketoethylene group (–CO–CH=CH–). They have conjugated double bonds and a fully delocalized π-electron system on both benzene rings [14]. Structure–activity studies have shown that the cytotoxicity of chiral analogues is affected by the shape of the molecules [15, 16]. The multimodal pharmacodynamic, structural diversity of synthetic and natural chalcones and the constitutive elements that create optimal toxicity vary for each class of chalcones, and there are no generally valid rules of relationship between structure and activity [17]. Changes in the structure create a high degree of diversity which, as was shown, is useful for the development of new drugs with better efficacy, lower toxicity, and good pharmacological action. Thus, chalcones have become of interest not only in the academic but also in the industrial sphere and are used as intermediates for the preparation of compounds having therapeutic utility. They are currently used in the treatment of viral, cardiovascular diseases, parasitic infections, pain, inflammation, and gastric cancer, as well as additives and cosmetic ingredients [14]. Some natural but also synthetic chalcones have demonstrated cytotoxic activity against tumor cell cultures by inhibiting cell growth. However, they are also effective as anticancer and chemopreventive agents in vivo [18–20]. The amount of clinically useful antitumor drugs exhibits a genotoxic effect based on their affinity to amino groups of nucleic acid, but chalcones exhibit a pronounced affinity to thiols [15]. These reactions can alter intracellular redox (redox signaling) that can modulate processes such as DNA synthesis, enzyme activation, selective gene expression, and cell cycle regulation [21]. Many of the pharmacological potentials of chalcones are not used yet.

channels, it cannot pass through the inner mitochondrial membrane into the matrix as the anion. Here, the 2-oxoglutarate antiport is applied [28]. By importing GSH, mitochondria lose an important intermediate of the Krebs cycle, which must be replaced by anaplerotic reactions. It is important to note that in experiments, we have been working with isolated mitochondria where transfer of de novo synthesized glutathione was not possible. However, the energy inten-

the membrane space, and the outer mitochondrial membrane reacts with other electron accep-

mechanism requires cyclic oxidation/reduction of cysteine or selenocysteine residues at the catalytic center where GSH is used as a cofactor and GSSG is formed. Reactivation through glutathione reductase (GR) requires a reduction potential of NADPH, whose production also requires energy. The presence of glucose-6-phosphate dehydrogenase (and also isocitrate dehydrogenase), a source of NADPH formation in mitochondria, have been proven [29], but the

production and changes in GSH levels are associated with the affection of apoptosis, cell division, and growth [30]. According to the decrease in mitochondrial membrane potential, they are able to incorporate into the membrane and induce apoptosis [31]. Conversely, the reduction of ROS production in the mitochondria by partial uncoupling of oxidative phosphorylation, such as observed with tetralone analogues, is a protective mechanism and also corresponds to the

As a result of our observations, (E)-2-arylidene-1-tetralone shows antioxidant and (E)-2 arylidene-1-benzosuberone significant pro-oxidant and cytotoxic properties regardless of the character of the substituent. The findings have contributed to the targeted synthesis of derivatives that are expected to enhance the effect due to structural modification. The mentioned cyclic analogues of chalcones served as a structural substrate, with the aromatic ring B being replaced by a ferrocene. In previous studies on various different ferrocene derivatives, some have been shown to exhibit surprisingly high toxicity and antiproliferative activity [32–34]. Among the unique derivatives available for study, the antiproliferative effect of the (E)-3- (ferroceneethylene)-4-chromanone was the most effective [35]. The pronounced effect of cell viability and colony formation of cancer cells in dose-dependent manner has been shown by 1,1′-bis[(1-oxoindane-2-ylidene) methyl] ferrocene. The mechanism by which tested ferrocenyl compounds could demonstrate these remarkable properties is probably based on their activity with RNOS, which subsequently affected the antioxidant mechanisms of mitochondria. In general, the activity of the compounds with respect to **˙**OH and nitric oxide (NO)

The 1,1′-bis[(1-oxoindane-2-ylidene) methyl]ferrocene and 1,1′-bis[(1-oxotetralin-2-ylidene)

activities of superoxide dismutase; additionally, 1,1′-bis[(1-oxoindan-2-ylidene)methyl]

O2

O2

Introductory Chapter: Unregulated Mitochondrial Production of Reactive Oxygen Species…

levels, leading to its inhibition [29]. Affection of ATP

**˙−** and the peroxynitrite anion (ONO2

**−**).

**˙−**, leading to increased

**−**. In addition to preserved

O2

**˙−** produced in the mitochondrial matrix,

http://dx.doi.org/10.5772/intechopen.82514

5


. The local antioxidant capacity of

sity to maintain the redox status was not reduced. O2

peroxidases then determines oxidative damage or H2

enzyme is extremely sensitive to H2

measured values of antioxidant parameters.

was very weak but, however, marked toward O2

methyl] ferrocene were involved in the significant production of O<sup>2</sup>

ferrocene also exhibited the lowest inhibition of NO and ONO2

**4. Conclusion**

tors such as NO but primarily leads to the production of H2

Summarizing the current knowledge of chalcone efficiency and their cyclic analogues ((*E*)-2 arylmethylene-1-indanone, (E)-2-arylmethylene-1-tetralone, and (*E*)-2-arylmethylene-benzosuberone) with several types of substituents, our studies were then primarily focused on monitoring their effects on mitochondria with respect to the production of ROS and the subsequent effects on selected antioxidant markers and ATP production. As the primary organ of xenobiotic metabolism in the body is the liver, studies with 4′-methyl-, methoxy- [16], 4′-hydroxy- [22], and 4′-dimethylamino-cyclic analogues of chalcones [23] were provided on mitochondria isolated from the rat liver. Analogues with methyl substituents showed rather a protective, antioxidant effect. Observed insufficiency in the antioxidant system and the level of reduced glutathione and associated enzymes such as glutathione peroxidase and glutathione reductase were significantly induced by the presence of benzosuberone in all types of substituents. They act as uncouplers of mitochondrial respiration, thus reducing ATP production. 4′-Hydroxy and 4′-dimethylamino analogues of chalcones exhibited similarly toxic effects as (*E*)-2-arylidene-1-indanones. Chalcones with substituents that increase the electron density of the B-ring, such as methoxy, butoxy, or dimethylamino groups, do not exhibit significant reactivity to reactive species [24].

The current level of knowledge makes it possible to use some of these biological properties of chalcone derivatives influenced by the nature of their substitution, such as the ability to inhibit 12-lipoxygenase and cyclooxygenases with 2′-hydroxychalcones, 4′-hydroxychalcones, and 2′,4′-dihydroxychalcones. Selective inhibitory effects on arachidonic-induced platelet aggregation predict them as antithrombotic or anti-inflammatory agents [25]. Under the low pH, the amino group, which are conditions normally found in tumors, is in protonated form increasing β-carbon electrophilicity in enone linkage, thereby increasing its reactivity as nucleophile acceptor in Michael additions [15], for example, thiol groups. Substantial antiproliferative activity was observed for chalcones with substituted amino groups [26]. All benzosuberone cyclic analogues at incubation with mitochondria caused a significant decrease in reduced glutathione (GSH) levels and simultaneous increase in glutathione peroxidase (GPx) activities. Lowering GSH levels most clearly defines the conditions of strong oxidative stress and leads to changes in the redox potential of the cell [27]. Although many antioxidant defense systems exist in the mitochondria, their maintenance is energy demanding. The first condition is a sufficient amount of ATP needed to synthesize low molecular weight antioxidants and molecules that provide uptake of ROS and ROS by-products. Benzosuberone as well as indanone analogues acted in mitochondria as phosphorylation deactivators, thereby reducing ATP production. GSH itself is able to reduce reactive oxygen and nitrogen species (RNOS); however, it is synthesized only in cytosol. Although it easily passes through the outer mitochondrial membrane via porin channels, it cannot pass through the inner mitochondrial membrane into the matrix as the anion. Here, the 2-oxoglutarate antiport is applied [28]. By importing GSH, mitochondria lose an important intermediate of the Krebs cycle, which must be replaced by anaplerotic reactions. It is important to note that in experiments, we have been working with isolated mitochondria where transfer of de novo synthesized glutathione was not possible. However, the energy intensity to maintain the redox status was not reduced. O2 **˙−** produced in the mitochondrial matrix, the membrane space, and the outer mitochondrial membrane reacts with other electron acceptors such as NO but primarily leads to the production of H2 O2 . The local antioxidant capacity of peroxidases then determines oxidative damage or H2 O2 -mediated signaling. The GPx catalytic mechanism requires cyclic oxidation/reduction of cysteine or selenocysteine residues at the catalytic center where GSH is used as a cofactor and GSSG is formed. Reactivation through glutathione reductase (GR) requires a reduction potential of NADPH, whose production also requires energy. The presence of glucose-6-phosphate dehydrogenase (and also isocitrate dehydrogenase), a source of NADPH formation in mitochondria, have been proven [29], but the enzyme is extremely sensitive to H2 O2 levels, leading to its inhibition [29]. Affection of ATP production and changes in GSH levels are associated with the affection of apoptosis, cell division, and growth [30]. According to the decrease in mitochondrial membrane potential, they are able to incorporate into the membrane and induce apoptosis [31]. Conversely, the reduction of ROS production in the mitochondria by partial uncoupling of oxidative phosphorylation, such as observed with tetralone analogues, is a protective mechanism and also corresponds to the measured values of antioxidant parameters.

#### **4. Conclusion**

Some natural but also synthetic chalcones have demonstrated cytotoxic activity against tumor cell cultures by inhibiting cell growth. However, they are also effective as anticancer and chemopreventive agents in vivo [18–20]. The amount of clinically useful antitumor drugs exhibits a genotoxic effect based on their affinity to amino groups of nucleic acid, but chalcones exhibit a pronounced affinity to thiols [15]. These reactions can alter intracellular redox (redox signaling) that can modulate processes such as DNA synthesis, enzyme activation, selective gene expression, and cell cycle regulation [21]. Many of the pharmacological potentials of

Summarizing the current knowledge of chalcone efficiency and their cyclic analogues ((*E*)-2 arylmethylene-1-indanone, (E)-2-arylmethylene-1-tetralone, and (*E*)-2-arylmethylene-benzosuberone) with several types of substituents, our studies were then primarily focused on monitoring their effects on mitochondria with respect to the production of ROS and the subsequent effects on selected antioxidant markers and ATP production. As the primary organ of xenobiotic metabolism in the body is the liver, studies with 4′-methyl-, methoxy- [16], 4′-hydroxy- [22], and 4′-dimethylamino-cyclic analogues of chalcones [23] were provided on mitochondria isolated from the rat liver. Analogues with methyl substituents showed rather a protective, antioxidant effect. Observed insufficiency in the antioxidant system and the level of reduced glutathione and associated enzymes such as glutathione peroxidase and glutathione reductase were significantly induced by the presence of benzosuberone in all types of substituents. They act as uncouplers of mitochondrial respiration, thus reducing ATP production. 4′-Hydroxy and 4′-dimethylamino analogues of chalcones exhibited similarly toxic effects as (*E*)-2-arylidene-1-indanones. Chalcones with substituents that increase the electron density of the B-ring, such as methoxy, butoxy, or dimethylamino groups, do not exhibit significant

The current level of knowledge makes it possible to use some of these biological properties of chalcone derivatives influenced by the nature of their substitution, such as the ability to inhibit 12-lipoxygenase and cyclooxygenases with 2′-hydroxychalcones, 4′-hydroxychalcones, and 2′,4′-dihydroxychalcones. Selective inhibitory effects on arachidonic-induced platelet aggregation predict them as antithrombotic or anti-inflammatory agents [25]. Under the low pH, the amino group, which are conditions normally found in tumors, is in protonated form increasing β-carbon electrophilicity in enone linkage, thereby increasing its reactivity as nucleophile acceptor in Michael additions [15], for example, thiol groups. Substantial antiproliferative activity was observed for chalcones with substituted amino groups [26]. All benzosuberone cyclic analogues at incubation with mitochondria caused a significant decrease in reduced glutathione (GSH) levels and simultaneous increase in glutathione peroxidase (GPx) activities. Lowering GSH levels most clearly defines the conditions of strong oxidative stress and leads to changes in the redox potential of the cell [27]. Although many antioxidant defense systems exist in the mitochondria, their maintenance is energy demanding. The first condition is a sufficient amount of ATP needed to synthesize low molecular weight antioxidants and molecules that provide uptake of ROS and ROS by-products. Benzosuberone as well as indanone analogues acted in mitochondria as phosphorylation deactivators, thereby reducing ATP production. GSH itself is able to reduce reactive oxygen and nitrogen species (RNOS); however, it is synthesized only in cytosol. Although it easily passes through the outer mitochondrial membrane via porin

chalcones are not used yet.

4 Medicinal Chemistry

reactivity to reactive species [24].

As a result of our observations, (E)-2-arylidene-1-tetralone shows antioxidant and (E)-2 arylidene-1-benzosuberone significant pro-oxidant and cytotoxic properties regardless of the character of the substituent. The findings have contributed to the targeted synthesis of derivatives that are expected to enhance the effect due to structural modification. The mentioned cyclic analogues of chalcones served as a structural substrate, with the aromatic ring B being replaced by a ferrocene. In previous studies on various different ferrocene derivatives, some have been shown to exhibit surprisingly high toxicity and antiproliferative activity [32–34]. Among the unique derivatives available for study, the antiproliferative effect of the (E)-3- (ferroceneethylene)-4-chromanone was the most effective [35]. The pronounced effect of cell viability and colony formation of cancer cells in dose-dependent manner has been shown by 1,1′-bis[(1-oxoindane-2-ylidene) methyl] ferrocene. The mechanism by which tested ferrocenyl compounds could demonstrate these remarkable properties is probably based on their activity with RNOS, which subsequently affected the antioxidant mechanisms of mitochondria. In general, the activity of the compounds with respect to **˙**OH and nitric oxide (NO) was very weak but, however, marked toward O2 **˙−** and the peroxynitrite anion (ONO2 **−**). The 1,1′-bis[(1-oxoindane-2-ylidene) methyl]ferrocene and 1,1′-bis[(1-oxotetralin-2-ylidene) methyl] ferrocene were involved in the significant production of O<sup>2</sup> **˙−**, leading to increased activities of superoxide dismutase; additionally, 1,1′-bis[(1-oxoindan-2-ylidene)methyl] ferrocene also exhibited the lowest inhibition of NO and ONO2 **−**. In addition to preserved concentrations of GSH, the mechanism of action, especially in this most effective derivative, is likely to be the modulation of mitochondrial activity through the induction of nitrosative stress.

[8] Kushnareva Y, Murphy AN, Andreyev A. Complex 1-mediated reactive oxygen species generation: Modulation by cytochrome c and NAD(P)+ oxidation-reduction state. The

Introductory Chapter: Unregulated Mitochondrial Production of Reactive Oxygen Species…

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[9] Marchi S, Giorgi C, Suski JM, Agnoletto C, Bononi A, Bonnora M, et al. Mitochondria-Ros crosstalk in the control of cell death and aging. Journal of Signal Transduction. 2012;

[10] Mráček T, Pecinová A, Vrbacký M, Drahota Z, Houstek J. High efficiency of ROS production by glycerolphosphate dehydrogenase in mammalian mitochondria. Archives of

[11] Vásquez-Vivar J, Kalyanaraman B, Kennedy MC. Mitochondrial aconitase is a source of hydroxyl radical. An electron spin resonance investigation. Journal of Biological

[12] Starkov AA, Fiskum G, Chinopoulos C, Lorenzo BJ, Browne SE, Patel MS, et al. Mitochondrial alpha-ketoglutarate dehydrogenase complex generates reactive oxygen species. The Journal of Neuroscience. 2004;**24**(36):7779-7788. DOI: 10.1523/JNEUROSCI.1899-04.2004

[13] Cheng MS, Li RS, Kenyon G. A solid phase synthesis of chalcones by Claisen-Schmidt

[14] Yerragunta V, Kumaraswamy T, Suman D, Anusha V, Patil P, Samhitha T. A review on

[15] Dimmock JR, Zello GA, Oloo EO, Quail JW, Kraatz H-B, Perjési P, et al. Correlations between cytotoxicity and various physicochemical parameters of some 2-arylidenebenzocyclanones determined by X-ray crystallography. Journal of Medicinal Chemistry. 2002;**45**(14):3103-3111.

[16] Perjési P, Das U, De Clercq E, Balzarini J, Kawase M, Sakagami H, et al. Design, synthesis and antiproliferative activity of some 3-benzylidene-2,3-dihydro-1-benzopyran-4-ones which display selective toxicity for malignant cells. European Journal of Medicinal Che-

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[18] Dimmock JR, Elias DW, Beazely MA, Kandepu NM. Bioactivities of chalcones. Current

[19] Middleton E Jr, Kandaswami C, Theoharides TC. The effect of plant flavonoids on mammalian cells: Implications for inflammation, heart disease, and cancer. Pharmacological

[20] Go ML, Wu X, Liu XL. Chalcones: An update on cytotoxic and chemopreventive proper-

Biochemistry and Biophysics. 2009;**481**(1):30-36. DOI: 10.1016/j.abb.2008.10.011

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Chemistry. 2000;**275**(19):14064-14069. DOI: 10.1074/jbc.275.19.14064

condensations. Chinese Chemical Letters. 2000;**11**(10):851-854

chalcones and its importance. PharmaTutor. 2013;**1**(2):54-59

mistry. 2008;**43**(4):839-845. DOI: 10.1016/j.ejmech.2007.06.017

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ties. Current Medicinal Chemistry. 2005;**12**(4):483-499

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DOI: 10.1021/jm010559p

**2012**(329635):1-17. DOI: 10.1155/2012/329635

The demonstration from our workplace suggests a long-term process of characterizing the effects of compounds, which contributes only a small amount to their complex knowledge. That is why authors have been invited to create the content of this book to bring their work and theoretical experiences in this area through their chapters.

## **Author details**

Janka Vašková\* and Ladislav Vaško

\*Address all correspondence to: janka.vaskova@upjs.sk

Faculty of Medicine, Pavol Jozef Šafárik University in Košice, Košice, Slovak Republic

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[8] Kushnareva Y, Murphy AN, Andreyev A. Complex 1-mediated reactive oxygen species generation: Modulation by cytochrome c and NAD(P)+ oxidation-reduction state. The Biochemical Journal. 2002;**368**(Pt 2):545-553. DOI: 10.1042/BJ20021121

concentrations of GSH, the mechanism of action, especially in this most effective derivative, is likely to be the modulation of mitochondrial activity through the induction of nitrosative

The demonstration from our workplace suggests a long-term process of characterizing the effects of compounds, which contributes only a small amount to their complex knowledge. That is why authors have been invited to create the content of this book to bring their work

Faculty of Medicine, Pavol Jozef Šafárik University in Košice, Košice, Slovak Republic

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**Section 1**

**Effects on Biological Systems: In Vitro Testing**

**Effects on Biological Systems: In Vitro Testing**

**Chapter 2**

**Provisional chapter**

**Determination of** *In Vitro* **Cytotoxicity and Anti-**

**Determination of** *In Vitro* **Cytotoxicity and Anti-**

DOI: 10.5772/intechopen.81046

*Aspergillus terreus* **FC36AY1 Isolated from** *Aegle*

*Aspergillus terreus* **FC36AY1 Isolated from** *Aegle* 

© 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,

© 2018 The Author(s). Licensee IntechOpen. 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.

and reproduction in any medium, provided the original work is properly cited.

The biotechnological research mainly emphasis its investigation on searching new natural drugs at economical to human welfare. With this view in mind, this research has focused to develop a prospective bioactive compound isolating from an efficient endophytic fungus isolated from potential medicinal tree *Aegle marmelos.* The endophytic fungus was isolated from the medicinal tree and identified as *Aspergillus terreus* FC36AY1. This fungus produced maximum of crude metabolites and this was produced in Sabouraud's Dextrose Broth. The produced metabolites were extracted using acetone as a sole solvent and it was taken for the assessment of antimicrobial and antioxidant analysis. The crude metabolites exhibited maximum activity at least concentration and further the crude extract were taken for purification processes through chromatographic techniques. Through purification, five different fractions were eluted and those five different fractions were also assessed for antimicrobial and antioxidant analysis. From these analysis results, TA4 was found to be efficient fraction and it was characterized through FT-IR, GC-MS and UV-VIS analysis. The compound was taken for cytotoxicity determination in HT-29 cancer cells and anti-angiogenesis analysis was assessed through HET-CAM testing. The bio-activities study revealed that the compound TA4 has the ability to target

**Angiogenesis for a Bioactive Compound from**

**Angiogenesis for a Bioactive Compound from** 

*marmelos* **around Western Ghats, India**

*marmelos* **around Western Ghats, India**

Arockiam Jeyasundar Parimala Gnana Soundari and

Arockiam Jeyasundar Parimala Gnana Soundari

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.81046

the cancer cells in an efficient manner.

**Keywords:** *Aspergillus* sp., cytotoxicity, metabolites, HPLC

Vellingiri Manon Mani,

and Selvam Tamilarasi

Vellingiri Manon Mani,

Selvam Tamilarasi

**Abstract**

#### **Determination of** *In Vitro* **Cytotoxicity and Anti-Angiogenesis for a Bioactive Compound from** *Aspergillus terreus* **FC36AY1 Isolated from** *Aegle marmelos* **around Western Ghats, India Determination of** *In Vitro* **Cytotoxicity and Anti-Angiogenesis for a Bioactive Compound from**  *Aspergillus terreus* **FC36AY1 Isolated from** *Aegle marmelos* **around Western Ghats, India**

DOI: 10.5772/intechopen.81046

Vellingiri Manon Mani, Arockiam Jeyasundar Parimala Gnana Soundari and Selvam Tamilarasi Vellingiri Manon Mani, Arockiam Jeyasundar Parimala Gnana Soundari and Selvam Tamilarasi

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

http://dx.doi.org/10.5772/intechopen.81046

#### **Abstract**

The biotechnological research mainly emphasis its investigation on searching new natural drugs at economical to human welfare. With this view in mind, this research has focused to develop a prospective bioactive compound isolating from an efficient endophytic fungus isolated from potential medicinal tree *Aegle marmelos.* The endophytic fungus was isolated from the medicinal tree and identified as *Aspergillus terreus* FC36AY1. This fungus produced maximum of crude metabolites and this was produced in Sabouraud's Dextrose Broth. The produced metabolites were extracted using acetone as a sole solvent and it was taken for the assessment of antimicrobial and antioxidant analysis. The crude metabolites exhibited maximum activity at least concentration and further the crude extract were taken for purification processes through chromatographic techniques. Through purification, five different fractions were eluted and those five different fractions were also assessed for antimicrobial and antioxidant analysis. From these analysis results, TA4 was found to be efficient fraction and it was characterized through FT-IR, GC-MS and UV-VIS analysis. The compound was taken for cytotoxicity determination in HT-29 cancer cells and anti-angiogenesis analysis was assessed through HET-CAM testing. The bio-activities study revealed that the compound TA4 has the ability to target the cancer cells in an efficient manner.

**Keywords:** *Aspergillus* sp., cytotoxicity, metabolites, HPLC

© 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. © 2018 The Author(s). Licensee IntechOpen. 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.

## **1. Introduction**

There are numerous natural pigments in the world. They are collected from the sources such as plants, animals and microorganisms. The uses of natural pigments are increasing worldwide. The natural pigments have several affecting factors like temperature, pH, availability and cost. The natural pigments do not cause any serious health effects but it may cause some side effects only for the hypersensitivity persons. The natural pigments are also called as secondary metabolites in which the plants and microbes will produce in enormous by the metabolic production. Each living organism undergoes several pathways for the production of useful secondary metabolites. In this way the best and economical source is microbial metabolites and they have been extensively used in pharmaceutical and medicinal fields to treat various disorders and diseases. With this focus in mind, this research has been focused on the purification of secondary metabolites from an endophytic fungus which would be isolated from an efficient medicinal tree from biodiversified place.

**2. Materials and methods**

*2.2.1. TLC*

**2.1. Isolation and identification of endophytic fungi**

bioactive secondary metabolite through chromatographic techniques.

**2.2. Partial purification of the bioactive secondary metabolite**

taken. The solvent front was marked and R<sup>f</sup>

**2.3. Antioxidant activity**

secondary metabolite.

*2.3.1. DPPH radical scavenging assay*

*2.2.2. High performance liquid chromatography (HPLC)*

flow rate of 1 mL/min and mobile phase of acetonitrile:H<sup>2</sup>

The potential endophytic fungus was isolated from a prospective medicinal plant *Aegle marmelos* from Western Ghats (Nilgiris cluster), Coimbatore. This fungus was found to exhibit highest antagonistic activity when compared to other 37 different endophytic fungi from the medicinal plant. Further the potential fungus was named as FC36AY1 and identified to be *Aspergillus terreus* FC36AY1 with the NCBI accession number KY807648. The FC36AY1 was taken for the production of metabolites in Sabouraud's Dextrose Broth (SDB) medium for 17 days of incubation at stationary phase in normal cycle. The pigmented crude metabolites extract was extracted using acetone as sole solvent and it was concentrated for further use. The crude metabolites extract was assessed for biological determination such as antimicrobial and antioxidant analysis [7]. The fungus FC36AY1 manifested maximum activity at least concentration so this was taken for further purification process. The mass production of pigmented secondary metabolites was carried out in SDB medium and the yield was calculated according to Mani et al. [7]. The crude extract was taken for purification process to elute the

Determination of *In Vitro* Cytotoxicity and Anti-Angiogenesis for a Bioactive Compound…

http://dx.doi.org/10.5772/intechopen.81046

15

The crude extract was subjected to TLC (thin layer chromatography) [8]. About 5 μL of the crude extract was applied 1 cm above from the lower edge of the thin layer chromatography slides and dried. It was immersed to a depth of 1 cm in the solvents. The different solvent system tested for movement of the pigment was chloroform:methanol and petroleum ether:ethyl acetate. The best solvent system for the separation of the components in the crude extract was

The active fraction was further purified through preparative high performance liquid chromatography (HPLC) (Shimadzu-1100 series), manual injector with quaternary pump, photodiode array detector equipped with C18 column (4.6 × 250 mm) with 5 μL of pore size with the

The purified fractions were taken for antioxidant analysis for determining the prospective

The antioxidants present in fungal crude metabolites were aliquot into different concentrations (20–100 μg) to determine extract's ability to scavenge of 2,2-diphenyl-1-picrylhydrazyl

value was calculated.

O (80:20) at 427 nm.

Microbial pigment production is now one of the emerging fields of research to demonstrate its potential for various industrial applications. Among the molecules produced by microorganisms are carotenoids, melanins, flavins, quinines and more specifically monascins, violacein or indigo. Industries are now able to produce some microbial pigments for applications in food, cosmetics and textiles. Naturally, pigment producing microorganisms like fungi, yeast and bacteria are quite common [1]. The pigments producing microorganisms will produce the antibiotic and inhibit the disease causing pathogens. Antibiotics eliminate or prevent the growth and can therefore cure disease caused by bacterial infection. They cannot however treat viral infection such as common cold or nonbacterial inflammation. Among the microbial pigments the fungi placed a promising rank for its largest and efficient production of potential metabolites which could be applicable in different medicinal and pharmaceutical industries. To which, endophytic fungi explored an evidence in the production of medically used metabolites mainly from medicinal plants. An endophyte is an endosymbiont that lives within a plant for at least part of its life cycle without causing apparent disease [2]. Endophytes are ubiquitous and have been found in all species of plants however, most of the endophyte/plant relationships are not well understood [3]. Endophytes are also known to occur within lichens [4] and algae [5]. Many economically important grasses (e.g., *Festuca* sp. and *Lolium* sp.) carry fungal endophytes in genus *Epichloë*, some of which may enhance host growth [6], nutrient acquisition and may improve the plant's ability to tolerate abiotic stresses, such as drought, and enhance resistance to insects, plant pathogens and mammalian herbivores. Nowadays the studies have focused on endophytic fungi isolated from medicinal tree. From ancient time onwards the medicines have been prepared from trees and plants in order to prevent/or cure the diseases. In this regard the current research has been focused on developing the medicine or drugs from the endophytic fungi residing in potential medicinal plants/or trees. These drugs are nothing but the metabolites produced from the metabolic pathways by the organisms. As the endophytes (i) mimics the metabolism of the host plant, (ii) easy for industrial means and (iii) improvement in activity compared to host plant. The drug derived from endophytes explores the nature of the drugs derived from the medicinal plants and/or trees. The research will be discussed on the bioactive secondary metabolites produced by endophytic fungi through different production, and their usage in different medicinal fields as anti-angiogenic product.

## **2. Materials and methods**

**1. Introduction**

14 Medicinal Chemistry

There are numerous natural pigments in the world. They are collected from the sources such as plants, animals and microorganisms. The uses of natural pigments are increasing worldwide. The natural pigments have several affecting factors like temperature, pH, availability and cost. The natural pigments do not cause any serious health effects but it may cause some side effects only for the hypersensitivity persons. The natural pigments are also called as secondary metabolites in which the plants and microbes will produce in enormous by the metabolic production. Each living organism undergoes several pathways for the production of useful secondary metabolites. In this way the best and economical source is microbial metabolites and they have been extensively used in pharmaceutical and medicinal fields to treat various disorders and diseases. With this focus in mind, this research has been focused on the purification of secondary metabolites from an endophytic fungus which would be

Microbial pigment production is now one of the emerging fields of research to demonstrate its potential for various industrial applications. Among the molecules produced by microorganisms are carotenoids, melanins, flavins, quinines and more specifically monascins, violacein or indigo. Industries are now able to produce some microbial pigments for applications in food, cosmetics and textiles. Naturally, pigment producing microorganisms like fungi, yeast and bacteria are quite common [1]. The pigments producing microorganisms will produce the antibiotic and inhibit the disease causing pathogens. Antibiotics eliminate or prevent the growth and can therefore cure disease caused by bacterial infection. They cannot however treat viral infection such as common cold or nonbacterial inflammation. Among the microbial pigments the fungi placed a promising rank for its largest and efficient production of potential metabolites which could be applicable in different medicinal and pharmaceutical industries. To which, endophytic fungi explored an evidence in the production of medically used metabolites mainly from medicinal plants. An endophyte is an endosymbiont that lives within a plant for at least part of its life cycle without causing apparent disease [2]. Endophytes are ubiquitous and have been found in all species of plants however, most of the endophyte/plant relationships are not well understood [3]. Endophytes are also known to occur within lichens [4] and algae [5]. Many economically important grasses (e.g., *Festuca* sp. and *Lolium* sp.) carry fungal endophytes in genus *Epichloë*, some of which may enhance host growth [6], nutrient acquisition and may improve the plant's ability to tolerate abiotic stresses, such as drought, and enhance resistance to insects, plant pathogens and mammalian herbivores. Nowadays the studies have focused on endophytic fungi isolated from medicinal tree. From ancient time onwards the medicines have been prepared from trees and plants in order to prevent/or cure the diseases. In this regard the current research has been focused on developing the medicine or drugs from the endophytic fungi residing in potential medicinal plants/or trees. These drugs are nothing but the metabolites produced from the metabolic pathways by the organisms. As the endophytes (i) mimics the metabolism of the host plant, (ii) easy for industrial means and (iii) improvement in activity compared to host plant. The drug derived from endophytes explores the nature of the drugs derived from the medicinal plants and/or trees. The research will be discussed on the bioactive secondary metabolites produced by endophytic fungi through different production, and their

isolated from an efficient medicinal tree from biodiversified place.

usage in different medicinal fields as anti-angiogenic product.

#### **2.1. Isolation and identification of endophytic fungi**

The potential endophytic fungus was isolated from a prospective medicinal plant *Aegle marmelos* from Western Ghats (Nilgiris cluster), Coimbatore. This fungus was found to exhibit highest antagonistic activity when compared to other 37 different endophytic fungi from the medicinal plant. Further the potential fungus was named as FC36AY1 and identified to be *Aspergillus terreus* FC36AY1 with the NCBI accession number KY807648. The FC36AY1 was taken for the production of metabolites in Sabouraud's Dextrose Broth (SDB) medium for 17 days of incubation at stationary phase in normal cycle. The pigmented crude metabolites extract was extracted using acetone as sole solvent and it was concentrated for further use. The crude metabolites extract was assessed for biological determination such as antimicrobial and antioxidant analysis [7]. The fungus FC36AY1 manifested maximum activity at least concentration so this was taken for further purification process. The mass production of pigmented secondary metabolites was carried out in SDB medium and the yield was calculated according to Mani et al. [7]. The crude extract was taken for purification process to elute the bioactive secondary metabolite through chromatographic techniques.

#### **2.2. Partial purification of the bioactive secondary metabolite**

#### *2.2.1. TLC*

The crude extract was subjected to TLC (thin layer chromatography) [8]. About 5 μL of the crude extract was applied 1 cm above from the lower edge of the thin layer chromatography slides and dried. It was immersed to a depth of 1 cm in the solvents. The different solvent system tested for movement of the pigment was chloroform:methanol and petroleum ether:ethyl acetate. The best solvent system for the separation of the components in the crude extract was taken. The solvent front was marked and R<sup>f</sup> value was calculated.

#### *2.2.2. High performance liquid chromatography (HPLC)*

The active fraction was further purified through preparative high performance liquid chromatography (HPLC) (Shimadzu-1100 series), manual injector with quaternary pump, photodiode array detector equipped with C18 column (4.6 × 250 mm) with 5 μL of pore size with the flow rate of 1 mL/min and mobile phase of acetonitrile:H<sup>2</sup> O (80:20) at 427 nm.

#### **2.3. Antioxidant activity**

The purified fractions were taken for antioxidant analysis for determining the prospective secondary metabolite.

#### *2.3.1. DPPH radical scavenging assay*

The antioxidants present in fungal crude metabolites were aliquot into different concentrations (20–100 μg) to determine extract's ability to scavenge of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals using the method of Mani et al. [7] in triplicates. DPPH solution (1 mM DPPH radical solution in 95% ethanol) was added to the crude metabolites extracts and made upto 1 mL, vortexed well, and then incubated for 30 minutes in dark hood at room temperature. After incubation, the samples were poured into microfuge tubes and centrifuged for 5 min at 13,500 rpm at RT. The absorbance of each sample at λ = 517 nm was measured and 1 mL of 95% EtOH/MeOH was used as a control, and DPPH were used as reference compounds. The antioxidant activity is given as percent (%) DPPH scavenging assay was calculated using the formula: [(control absorbance − extract absorbance)/(control absorbance) × 100]. The fungi exhibiting the maximum antioxidant activity at minimum concentration and antagonistic profile were taken for further identification studies.

*2.3.5. Hydroxyl radical scavenging assay*

*2.3.6. Chemical characterization of bioactive secondary metabolite*

were studied to determine the presence of functional groups.

Abs control] × 100.

**2.4.** *In vitro* **studies**

*2.4.1. MTT assay*

(1 × 10<sup>5</sup>

The scavenging activity for hydroxyl radicals recommended by Yu et al. [12] was followed with minor changes using the fractions. Reaction mixture contained 0.6 mL of 1.0 mM Deoxy ribose, 0.4 mL of 0.2 mM phenyl hydrazine, 0.6 mL of 10 mM phosphate buffer (pH 7.4). It was incubated for 1 h at room temperature. Then 1 mL of 2–8% TCA, 1 mL of 1% TBA and 0.4 mL of compound (at different concentrations) were added and kept in water bath for 20 min. The absorbance of the mixture at 532 nm was measured with a spectrophotometer. From the readings, the hydroxyl radical scavenging activity was calculated as: [(Abs control − Abs sample)/

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The bioactive purified fraction which evinced maximum antioxidant activity at least concentration was dissolved in acetone and it was taken for UV-analysis using UV-Vis scanning spectroscopy for the detection of single peak. Scanning was performed between 200 and 800 nm wavelength. Later the fraction was subjected to structural elucidation of the compound. 1 mg of purified fraction was dried and analyzed for Infrared (IR) spectra using FTIR spectroscopy. The important IR bands of symmetric and asymmetric stretching and stretching frequencies

The gas chromatography-mass spectrometry was done by advanced equipment (Thermo GC—Trace ultra VER: 5.0, Thermo MS DSQ II). The column used in this experiment was DB 5-MS capillary standard non-polar column with the dimension of 30 mts, ID-0.25 mm and the film was 0.25 μm. The carrier gas used was helium, with the flow rate at 1.0 mL/min and the temperature was as oven temperature 70°C which was raised to 260°C at 6°C/min. The sample of 1 μL which was purified from HPLC analysis was taken and injected for the experiment.

The cytotoxic effect of the bioactive compound was studied using cancer cell lines. The HT-29 cell line was obtained from National Centre for Cell Sciences, Pune (NCCS). The cells were maintained in Minimal Essential Medium supplemented with 10% FBS, penicillin (100 U/mL),

/well) were plated in 24-well plates and incubated in 37°C with 5% CO<sup>2</sup>

After the cell reaches the confluence, the various concentrations of the samples were added and incubated for 24 h. After incubation, the sample was removed from the well and washed with phosphate-buffered saline (pH 7.4) or MEM without serum. 100 μL/well (5 mg/mL) of 0.5% 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) were added and incubated for 4 h. After incubation, 1 mL of DMSO was added in all the wells. The absorbance at 570 nm was measured with UV-Vis Spectrophotometer using DMSO as the blank. Measurements were performed and the concentration required to inhibit 50% of cells (IC50) was determined graphically. The % cell viability was calculated using the following formula 1:

at 37°C. Cells

condition.

and streptomycin (100 μg/mL) in a humidified atmosphere of 50 μg/mL CO2

#### *2.3.2. Reducing power assay*

Total reducing power was determined as described by Oyaizu [9] in triplicates [7]. 1 mL of sample solution at different concentrations (20–100 μg) was mixed with 2.5 mL of phosphate buffer (0.2 mol/L, pH 6.6) and 2.5 mL of 1% potassium ferricyanide. The mixture was incubated at 50°C for 20 min, 2.5 mL of 10% trichloroacetic acid (TCA) was added to the mixture and centrifuged at 3000 × *g* for 10 min. The supernatant (5 mL) was mixed with 1 mL of ferric chloride (0.1%), and the absorbance was measured at 700 nm in a spectrophotometer. Increased absorbance of the reaction mixture indicated increased reducing power.

#### *2.3.3. Metal chelating activity*

The metal chelating activity was analyzed by the method of Dinis et al. [10] with slight modification in triplicates [7]. The reaction was performed in HEPES buffer (20 mM) at pH 7.2. Various concentrations (20–100 μg) of samples were mixed with a solution of 12.5 μM ferrous sulfate solution. Addition of 75 μM ferrozine was to initiate the reaction and the mixture was shaken vigorously and incubated for 20 min at room temperature. After incubation the absorbance was measured at 562 nm. Ascorbic acid was used as the reference compound and the percentage chelating capacity was calculated as; % chelating activity = [(A0 − A1 )/A<sup>0</sup> ] × 100 where, A<sup>0</sup> = absorbance of the blank; A1 = absorbance of the sample.

#### *2.3.4. Superoxide anion radical scavenging assay*

Measurement of the superoxide anion radical scavenging capacity of the eluted fractions were essential according to the method described by Liu et al. [11] using a minor modification. The principle of this method is that superoxide radicals are generated in phenazine methosulfate (PMS)-nicotinamide adenine dinucleotide (NADH) systems by oxidation of NADH and reduction of nitroblue tetrazolium (NBT). In this experiment, the superoxide radicals were generated with 3.0 mL of Tris-HCl buffer (16 mM, pH 8.0) containing 1.0 mL of NBT (50 μM) solution, 1.0 mL NADH (78 μM) solution and samples of the compound MM4 (20–100 μg/mL) in methanol. The reaction was initiated by adding 1.0 mL of phenazine methosulfate (PMS) solution (10 μM) to the mixture. The absorbance at 560 nm was measured against a blank. Ascorbic acid was used as a standard. The scavenging activity was calculated by: [(Abs control − Abs sample)/Abs control] × 100.

#### *2.3.5. Hydroxyl radical scavenging assay*

(DPPH) radicals using the method of Mani et al. [7] in triplicates. DPPH solution (1 mM DPPH radical solution in 95% ethanol) was added to the crude metabolites extracts and made upto 1 mL, vortexed well, and then incubated for 30 minutes in dark hood at room temperature. After incubation, the samples were poured into microfuge tubes and centrifuged for 5 min at 13,500 rpm at RT. The absorbance of each sample at λ = 517 nm was measured and 1 mL of 95% EtOH/MeOH was used as a control, and DPPH were used as reference compounds. The antioxidant activity is given as percent (%) DPPH scavenging assay was calculated using the formula: [(control absorbance − extract absorbance)/(control absorbance) × 100]. The fungi exhibiting the maximum antioxidant activity at minimum concentration and antagonistic

Total reducing power was determined as described by Oyaizu [9] in triplicates [7]. 1 mL of sample solution at different concentrations (20–100 μg) was mixed with 2.5 mL of phosphate buffer (0.2 mol/L, pH 6.6) and 2.5 mL of 1% potassium ferricyanide. The mixture was incubated at 50°C for 20 min, 2.5 mL of 10% trichloroacetic acid (TCA) was added to the mixture and centrifuged at 3000 × *g* for 10 min. The supernatant (5 mL) was mixed with 1 mL of ferric chloride (0.1%), and the absorbance was measured at 700 nm in a spectrophotometer.

The metal chelating activity was analyzed by the method of Dinis et al. [10] with slight modification in triplicates [7]. The reaction was performed in HEPES buffer (20 mM) at pH 7.2. Various concentrations (20–100 μg) of samples were mixed with a solution of 12.5 μM ferrous sulfate solution. Addition of 75 μM ferrozine was to initiate the reaction and the mixture was shaken vigorously and incubated for 20 min at room temperature. After incubation the absorbance was measured at 562 nm. Ascorbic acid was used as the reference compound and

Measurement of the superoxide anion radical scavenging capacity of the eluted fractions were essential according to the method described by Liu et al. [11] using a minor modification. The principle of this method is that superoxide radicals are generated in phenazine methosulfate (PMS)-nicotinamide adenine dinucleotide (NADH) systems by oxidation of NADH and reduction of nitroblue tetrazolium (NBT). In this experiment, the superoxide radicals were generated with 3.0 mL of Tris-HCl buffer (16 mM, pH 8.0) containing 1.0 mL of NBT (50 μM) solution, 1.0 mL NADH (78 μM) solution and samples of the compound MM4 (20–100 μg/mL) in methanol. The reaction was initiated by adding 1.0 mL of phenazine methosulfate (PMS) solution (10 μM) to the mixture. The absorbance at 560 nm was measured against a blank. Ascorbic acid was used as a standard. The scavenging activity was calculated by: [(Abs

)/A<sup>0</sup>

] × 100

Increased absorbance of the reaction mixture indicated increased reducing power.

the percentage chelating capacity was calculated as; % chelating activity = [(A0 − A1

where, A<sup>0</sup> = absorbance of the blank; A1 = absorbance of the sample.

*2.3.4. Superoxide anion radical scavenging assay*

control − Abs sample)/Abs control] × 100.

profile were taken for further identification studies.

*2.3.2. Reducing power assay*

16 Medicinal Chemistry

*2.3.3. Metal chelating activity*

The scavenging activity for hydroxyl radicals recommended by Yu et al. [12] was followed with minor changes using the fractions. Reaction mixture contained 0.6 mL of 1.0 mM Deoxy ribose, 0.4 mL of 0.2 mM phenyl hydrazine, 0.6 mL of 10 mM phosphate buffer (pH 7.4). It was incubated for 1 h at room temperature. Then 1 mL of 2–8% TCA, 1 mL of 1% TBA and 0.4 mL of compound (at different concentrations) were added and kept in water bath for 20 min. The absorbance of the mixture at 532 nm was measured with a spectrophotometer. From the readings, the hydroxyl radical scavenging activity was calculated as: [(Abs control − Abs sample)/ Abs control] × 100.

#### *2.3.6. Chemical characterization of bioactive secondary metabolite*

The bioactive purified fraction which evinced maximum antioxidant activity at least concentration was dissolved in acetone and it was taken for UV-analysis using UV-Vis scanning spectroscopy for the detection of single peak. Scanning was performed between 200 and 800 nm wavelength. Later the fraction was subjected to structural elucidation of the compound. 1 mg of purified fraction was dried and analyzed for Infrared (IR) spectra using FTIR spectroscopy. The important IR bands of symmetric and asymmetric stretching and stretching frequencies were studied to determine the presence of functional groups.

The gas chromatography-mass spectrometry was done by advanced equipment (Thermo GC—Trace ultra VER: 5.0, Thermo MS DSQ II). The column used in this experiment was DB 5-MS capillary standard non-polar column with the dimension of 30 mts, ID-0.25 mm and the film was 0.25 μm. The carrier gas used was helium, with the flow rate at 1.0 mL/min and the temperature was as oven temperature 70°C which was raised to 260°C at 6°C/min. The sample of 1 μL which was purified from HPLC analysis was taken and injected for the experiment.

#### **2.4.** *In vitro* **studies**

#### *2.4.1. MTT assay*

The cytotoxic effect of the bioactive compound was studied using cancer cell lines. The HT-29 cell line was obtained from National Centre for Cell Sciences, Pune (NCCS). The cells were maintained in Minimal Essential Medium supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL) in a humidified atmosphere of 50 μg/mL CO2 at 37°C. Cells (1 × 10<sup>5</sup> /well) were plated in 24-well plates and incubated in 37°C with 5% CO<sup>2</sup> condition. After the cell reaches the confluence, the various concentrations of the samples were added and incubated for 24 h. After incubation, the sample was removed from the well and washed with phosphate-buffered saline (pH 7.4) or MEM without serum. 100 μL/well (5 mg/mL) of 0.5% 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) were added and incubated for 4 h. After incubation, 1 mL of DMSO was added in all the wells. The absorbance at 570 nm was measured with UV-Vis Spectrophotometer using DMSO as the blank. Measurements were performed and the concentration required to inhibit 50% of cells (IC50) was determined graphically. The % cell viability was calculated using the following formula 1:

$$\text{\textquotedblleft cell viability = (A}\_{550} \text{ of treated cells/A}\_{550} \text{of control cells} \times 100\text{)}\tag{1}$$

**3. Results**

**3.1. Purification of secondary metabolite**

value was calculated for obtained peaks and tabulated (**Table 1**).

*3.1.2. High performance liquid chromatography (HPLC)*

**3.2. Determination of antioxidant properties**

*3.2.1. DPPH radical scavenging activity*

The concentrated crude metabolite extract was subjected to thin layer chroamtography and the best solvent system which separated maximum compounds as band was chloroform:me thanol:toluene:acetic acid and at 95% about five visible bands got separated (**Figure 1a**)—R<sup>f</sup>

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The crude extract was forced for preparative HPLC analysis about 6 different peaks were obtained at 427 nm (**Figure 1b**) and this range corresponds to the result of UV spectrum analysis. The eluted fractions from HPLC analysis were assessed for antimicrobial profile. Among the five fractions (TA1–TA5), fraction 4 (TA4) was found to contain highest antimicrobial profile on comparing to other fractions (**Table 2**). The fraction 4 explored highest activity against *C. albicans*, *K. pneumoniae*, *E. coli*, *S. epidermidis* and *S. typhi.* From these results it is evident that this particular fraction was able to control the growth of gastro intestinal and skin pathogens.

The bioactive compound of the present study showed a concentration dependent antiradical activity by inhibiting DPPH radical (**Figure 2a**). The decrease in absorbance of the DPPH radical

**Figure 1.** (a) TLC of crude extract and (b) chromatogram showing different peaks in *Aspergillus* sp. extract.

*3.1.1. Thin layer chromatography*

Graphs were plotted using the % of cell viability as Y-axis and concentration of the sample in X-axis. Cell control and sample control was included in each assay to compare the full cell viability in cytotoxicity assessments.

#### **2.5. Determination of HET-CAM test [Hen's egg test on the chorio-allantoic membrane (HET-CAM) of chick eggs]**

In order to understand the inflammatory tissue reactions of metabolite coated materials on the live tissues, the materials were placed on the surface of Chorio-Allantoic membrane (CAM) of embryonated chick eggs. The inflammatory response on CAM was evaluated by direct evaluation method. Freshly laid fertile eggs were collected from the chicken farm and incubated at 36–37°C for 8 days before implanting (implantation day: 9th day) the sample materials such as compound TA4, acetone, positive and negative solutions. During the incubation time, the eggs were turned twice daily. On the day of implantation (9th day after laid), the eggs were candled to determine the position of the air sac and the embryo. A square, with sides approximately 18–20 mm, was marked on the shell where the chorio-allantoic membrane was best developed. Using a dental drill fitted with a straight hand-piece the sides of the marked square were drilled. In one corner of this large triangle a second smaller square was drilled, with sides of approximately 5 mm. A small slit was drilled in the shell over the air sac.

#### *2.5.1. Application of test sample, solvent, positive and negative control on CAM*

Aseptic technique was used for the implantation of the test sample on biomaterial as filter paper discs. For dropping the material onto chorio-allantoic membrane the egg was mounted on a stand, with the drilled area of shell uppermost; a straight Hagedorn's needle was gently inserted under one corner of the smaller square of shell and this square was raised and removed. The shell and shell membrane circumscribed by the larger square were then removed, and the sterile pre-measured size of sample was inserted and carefully lowered on to the exposed membrane. In order to implement the implanted sample TA4, 0.3 mL of the substance (positive and negative control) was applied to the surface of the CAM on separate eggs. 0.1 N NaOH was added on the CAM of separate egg as a positive control and 0.9% NaCl was used as an appropriate negative control. After a 20-s exposure period, the CAM is rinsed with 5 mL of water.

#### *2.5.2. Direct evaluation of CAM time for development of observed endpoints after exposure to the test substance*

A procedure used to evaluate the time for development of endpoints after exposure to the test substance was to continually observe the CAM during the 5-min observation period and record (typically in seconds) the time at which each of the endpoints developed. Therefore, two separate time values (after 2 and 18 h of incubation) were obtained and recorded for each egg (one time value for each endpoint).

## **3. Results**

%cell viability = (A<sup>570</sup> of treated cells/A<sup>570</sup> of control cells × 100) (1)

Graphs were plotted using the % of cell viability as Y-axis and concentration of the sample in X-axis. Cell control and sample control was included in each assay to compare the full cell

In order to understand the inflammatory tissue reactions of metabolite coated materials on the live tissues, the materials were placed on the surface of Chorio-Allantoic membrane (CAM) of embryonated chick eggs. The inflammatory response on CAM was evaluated by direct evaluation method. Freshly laid fertile eggs were collected from the chicken farm and incubated at 36–37°C for 8 days before implanting (implantation day: 9th day) the sample materials such as compound TA4, acetone, positive and negative solutions. During the incubation time, the eggs were turned twice daily. On the day of implantation (9th day after laid), the eggs were candled to determine the position of the air sac and the embryo. A square, with sides approximately 18–20 mm, was marked on the shell where the chorio-allantoic membrane was best developed. Using a dental drill fitted with a straight hand-piece the sides of the marked square were drilled. In one corner of this large triangle a second smaller square was drilled, with sides of approximately 5 mm. A small slit was drilled in the shell over the air sac.

Aseptic technique was used for the implantation of the test sample on biomaterial as filter paper discs. For dropping the material onto chorio-allantoic membrane the egg was mounted on a stand, with the drilled area of shell uppermost; a straight Hagedorn's needle was gently inserted under one corner of the smaller square of shell and this square was raised and removed. The shell and shell membrane circumscribed by the larger square were then removed, and the sterile pre-measured size of sample was inserted and carefully lowered on to the exposed membrane. In order to implement the implanted sample TA4, 0.3 mL of the substance (positive and negative control) was applied to the surface of the CAM on separate eggs. 0.1 N NaOH was added on the CAM of separate egg as a positive control and 0.9% NaCl was used as an appropriate negative control. After a 20-s exposure period, the CAM is rinsed

*2.5.2. Direct evaluation of CAM time for development of observed endpoints after exposure to* 

A procedure used to evaluate the time for development of endpoints after exposure to the test substance was to continually observe the CAM during the 5-min observation period and record (typically in seconds) the time at which each of the endpoints developed. Therefore, two separate time values (after 2 and 18 h of incubation) were obtained and recorded for each

**2.5. Determination of HET-CAM test [Hen's egg test on the chorio-allantoic** 

*2.5.1. Application of test sample, solvent, positive and negative control on CAM*

viability in cytotoxicity assessments.

18 Medicinal Chemistry

**membrane (HET-CAM) of chick eggs]**

with 5 mL of water.

*the test substance*

egg (one time value for each endpoint).

#### **3.1. Purification of secondary metabolite**

#### *3.1.1. Thin layer chromatography*

The concentrated crude metabolite extract was subjected to thin layer chroamtography and the best solvent system which separated maximum compounds as band was chloroform:me thanol:toluene:acetic acid and at 95% about five visible bands got separated (**Figure 1a**)—R<sup>f</sup> value was calculated for obtained peaks and tabulated (**Table 1**).

#### *3.1.2. High performance liquid chromatography (HPLC)*

The crude extract was forced for preparative HPLC analysis about 6 different peaks were obtained at 427 nm (**Figure 1b**) and this range corresponds to the result of UV spectrum analysis. The eluted fractions from HPLC analysis were assessed for antimicrobial profile. Among the five fractions (TA1–TA5), fraction 4 (TA4) was found to contain highest antimicrobial profile on comparing to other fractions (**Table 2**). The fraction 4 explored highest activity against *C. albicans*, *K. pneumoniae*, *E. coli*, *S. epidermidis* and *S. typhi.* From these results it is evident that this particular fraction was able to control the growth of gastro intestinal and skin pathogens.

#### **3.2. Determination of antioxidant properties**

#### *3.2.1. DPPH radical scavenging activity*

The bioactive compound of the present study showed a concentration dependent antiradical activity by inhibiting DPPH radical (**Figure 2a**). The decrease in absorbance of the DPPH radical

**Figure 1.** (a) TLC of crude extract and (b) chromatogram showing different peaks in *Aspergillus* sp. extract.


**Table 1.** R<sup>f</sup> value of TLC for crude extract.


hydrogen atom. In this study, the reductive ability of the compound TA4 had a maximum reductive power and this was observed by increasing OD units (**Figure 2b**). This confirmed the increasing reducing power through increasing OD units. When compared to the standard ascorbic acid, TA4 exerted a similar activity. This activity was concurrent with the investiga-

**Figure 2.** (a) DPPH radical scavenging activity, (b) reductive power ability of purified fraction, (c) superoxide radical

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The superoxide radicals are generated by PMS and it was assessed using NBT. **Figure 2c** explains that the bioactive compound has a maximum scavenging mechanism at minimum concentration. The 50% of inhibition concentration value was found to be 24 μg/mL. The IC 50

Hydroxyl radical scavenging assay showed the ability of the bioactive compound and standard ascorbic acid in inhibiting hydroxyl radical mediated deoxyribose degradation in a Fe3+

was found distinctly increased with increased concentration when compared to the standard. Hydroxyl radicals are the major active oxygen species causing enormous biological damage by lipid peroxidation in cells. The IC50 concentration of purified fraction was found to 75 μg/mL.

The rate of color reduction was measured which was used in the estimation of chelating activity of the coexisting chelator. In the current analysis, the absorbance of Fe2+ ferrozine complex was decreased in a dose dependent manner which can be meant in other way as the activity increased with the increasing concentration from 20 to 100 μg/mL and the IC50 concentration

reaction mixture (**Figure 2d**). The IC50 value of the compound

tion of \*\*\*Liu et al. (2007) with the same concentration of 20–100 μg/mL.

scavenging activity, (d) hydroxyl radical scavenging activity, and (e) metal chelating activity.

concentration of purified fraction was found to 68 μg/mL.

O2

of purified fraction was found to 70 μg/mL (**Figure 2e**).

*3.2.3. Superoxide radical scavenging activity*

*3.2.4. Hydroxyl radical scavenging activity*

EDTA ascorbic acid and H2

*3.2.5. Metal chelating activity*

**Table 2.** Antimicrobial activity for eluted fractions.

caused by antioxidant was due to the scavenging of the radical by hydrogen donation. It is visually noticeable as a color change from purple to yellow. Also, a lower value of IC50 (concentration at which the 50% scavenging activity is obtained) indicates a higher antioxidant activity at lower concentration. The IC50 concentration of purified fraction was found to 56 μg/mL.

#### *3.2.2. Reductive power ability*

The reducing ability of a bioactive compound generally depends on the presence of reductones, which exert the antioxidant activity by breaking the free radical chain by donating a

Determination of *In Vitro* Cytotoxicity and Anti-Angiogenesis for a Bioactive Compound… http://dx.doi.org/10.5772/intechopen.81046 21

**Figure 2.** (a) DPPH radical scavenging activity, (b) reductive power ability of purified fraction, (c) superoxide radical scavenging activity, (d) hydroxyl radical scavenging activity, and (e) metal chelating activity.

hydrogen atom. In this study, the reductive ability of the compound TA4 had a maximum reductive power and this was observed by increasing OD units (**Figure 2b**). This confirmed the increasing reducing power through increasing OD units. When compared to the standard ascorbic acid, TA4 exerted a similar activity. This activity was concurrent with the investigation of \*\*\*Liu et al. (2007) with the same concentration of 20–100 μg/mL.

#### *3.2.3. Superoxide radical scavenging activity*

The superoxide radicals are generated by PMS and it was assessed using NBT. **Figure 2c** explains that the bioactive compound has a maximum scavenging mechanism at minimum concentration. The 50% of inhibition concentration value was found to be 24 μg/mL. The IC 50 concentration of purified fraction was found to 68 μg/mL.

#### *3.2.4. Hydroxyl radical scavenging activity*

Hydroxyl radical scavenging assay showed the ability of the bioactive compound and standard ascorbic acid in inhibiting hydroxyl radical mediated deoxyribose degradation in a Fe3+ EDTA ascorbic acid and H2 O2 reaction mixture (**Figure 2d**). The IC50 value of the compound was found distinctly increased with increased concentration when compared to the standard. Hydroxyl radicals are the major active oxygen species causing enormous biological damage by lipid peroxidation in cells. The IC50 concentration of purified fraction was found to 75 μg/mL.

#### *3.2.5. Metal chelating activity*

caused by antioxidant was due to the scavenging of the radical by hydrogen donation. It is visually noticeable as a color change from purple to yellow. Also, a lower value of IC50 (concentration at which the 50% scavenging activity is obtained) indicates a higher antioxidant activity at

**TA1 TA2 TA3 TA4 TA5**

 **value (%)**

The reducing ability of a bioactive compound generally depends on the presence of reductones, which exert the antioxidant activity by breaking the free radical chain by donating a

lower concentration. The IC50 concentration of purified fraction was found to 56 μg/mL.

**S. no. Pathogens Zone of inhibition (in cm)**

**S. no. Bands R<sup>f</sup>**

value of TLC for crude extract.

1 I 0.046 2 II 1.125 3 III 1.184 4 IV 1.25 5 V 1.8 6 VI 1.956 7 VII 3.461 8 VIII 9.000

 *Staphylococcus aureus* 0.5 0 1.2 1.2 1.2 *Klebsiella pnuemoniae* 0 0 0.9 1.4 0.9 *Staphylococcus epidermis* 0.9 0 0.8 1.6 0 *Pseudomonas aeruginosa* 1.1 0 1.2 1.1 0 *Enterococcus faecalis* 1.2 0 2.1 2.1 0 *Bacillus subtilis* 1 0 0 2.3 1.3 *E. coli* 0 0 0 1.0 1.2 *Proteus mirabilis* 0 0 1.1 0.9 0.9 *Shigella sp* 0 0 0.9 0 0.7 *Salmonella* 0 0 0 0 0.8 *Candida albicans* 0 0 0 2.1 0.9 *Aspergillus terreus* 2.1 0 0 2.3 0

*3.2.2. Reductive power ability*

<0.5 cm: no inhibition activity.

**Table 1.** R<sup>f</sup>

20 Medicinal Chemistry

**Table 2.** Antimicrobial activity for eluted fractions.

The rate of color reduction was measured which was used in the estimation of chelating activity of the coexisting chelator. In the current analysis, the absorbance of Fe2+ ferrozine complex was decreased in a dose dependent manner which can be meant in other way as the activity increased with the increasing concentration from 20 to 100 μg/mL and the IC50 concentration of purified fraction was found to 70 μg/mL (**Figure 2e**).

#### **3.3. Chemical characterization of the secondary metabolite**

UV-Visible spectrum of the purified pink colored compound was recorded using acetone solution in the range of 200–700 nm showed a single peak for the purified fraction (**Figure 3a**). The spectral measurement showed that the compound had registered its absorption band in the region 427 nm. The peak in the wavelength of 427 nm showed that this is the active fraction. IR spectrum has proven to be the most effective way to give the information about the functional groups present in the compound.

**3.4.** *In vitro* **studies**

of cells. So TA4 was taken for further studies.

Control 0.3855

The present investigation has been carried out for a potential bioactive compound TA4. The studies have been performed on HT-29 colon cancer cell lines with the control of triton X 100 (**Table 3** and **Figure 4**). This result was similar to the investigation of Yuvaraj et al. [13] reported the IC50 at nearest concentration. The present study has showed that *A. terrus* FC36AY1 acetonic fractions could extensively inhibited cell proliferation architecture in dose dependent manner. This report demonstrating the *in vitro* anticancer activity of the TA4 from methanolic extract of *A. terrus* FC36AY1 providing a scientific basis for its effects on human health which is similar to Yuvaraj et al. [13], TA4 was found to be inhibited maximum number

Determination of *In Vitro* Cytotoxicity and Anti-Angiogenesis for a Bioactive Compound…

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23

**Sample concentration (μg/mL) Average OD at 540 nm Percentage viability**

6.25 0.3044 78.96239 12.5 0.2941 76.29053 25 0.2427 62.9572 50 0.2077 53.87808 100 0.1545 40.07782

*3.4.1. MTT assay*

**Table 3.** MTT assay.

**Figure 4.** MTT analysis of TA4.

**Figure 3b** depicted the FT-IR report for the purified fraction. In that, the stretching frequencies of the IR spectrum recorded the highest peak at 1711.92 which correspond to C▬O (carbonyl/ ketone) stretching. The region 1360.41 denotes the region ▬C▬H (sp3 configuration and alkane group) and the region 1221.06 records CN stretching (aromatic primary amine group or phenolic group). The region 1426.80 and 1093.19 denotes ▬C▬H (sp3 configuration with vinyl C▬H) and C▬O (alcohol group) respectively. The frequencies from 3600 to 3200 cm−<sup>1</sup> denoted the alcohol group present in the compound and in the same way the frequencies recorded from 3000 to 2850 cm−<sup>1</sup> denoted the alkane stretching.

#### *3.3.1. GC-MS analysis*

The gas chromatography-mass spectrometry (GC-MS) was analyzed for purified fraction of TA4. The chromatogram was obtained with 4 major peaks in TA4 (**Figure 3c** and **d**). Finally from the results of UV-spectra, FT-IR, GC-MS, analysis we conclude the compound of TA4 was found to be octadecenoic acid 4-hydroxy methyl ester C19H38O3.

**Figure 3.** (a) UV-VS of TA4, (b) FT-IR spectra for TA4, (c) GC-MS of TA4, and (d) GC-MS RA peak of TA4.

#### **3.4.** *In vitro* **studies**

#### *3.4.1. MTT assay*

**3.3. Chemical characterization of the secondary metabolite**

functional groups present in the compound.

recorded from 3000 to 2850 cm−<sup>1</sup>

*3.3.1. GC-MS analysis*

22 Medicinal Chemistry

UV-Visible spectrum of the purified pink colored compound was recorded using acetone solution in the range of 200–700 nm showed a single peak for the purified fraction (**Figure 3a**). The spectral measurement showed that the compound had registered its absorption band in the region 427 nm. The peak in the wavelength of 427 nm showed that this is the active fraction. IR spectrum has proven to be the most effective way to give the information about the

**Figure 3b** depicted the FT-IR report for the purified fraction. In that, the stretching frequencies of the IR spectrum recorded the highest peak at 1711.92 which correspond to C▬O (carbonyl/

alkane group) and the region 1221.06 records CN stretching (aromatic primary amine group

vinyl C▬H) and C▬O (alcohol group) respectively. The frequencies from 3600 to 3200 cm−<sup>1</sup> denoted the alcohol group present in the compound and in the same way the frequencies

denoted the alkane stretching.

The gas chromatography-mass spectrometry (GC-MS) was analyzed for purified fraction of TA4. The chromatogram was obtained with 4 major peaks in TA4 (**Figure 3c** and **d**). Finally from the results of UV-spectra, FT-IR, GC-MS, analysis we conclude the compound of TA4

configuration and

configuration with

ketone) stretching. The region 1360.41 denotes the region ▬C▬H (sp3

or phenolic group). The region 1426.80 and 1093.19 denotes ▬C▬H (sp3

was found to be octadecenoic acid 4-hydroxy methyl ester C19H38O3.

**Figure 3.** (a) UV-VS of TA4, (b) FT-IR spectra for TA4, (c) GC-MS of TA4, and (d) GC-MS RA peak of TA4.

The present investigation has been carried out for a potential bioactive compound TA4. The studies have been performed on HT-29 colon cancer cell lines with the control of triton X 100 (**Table 3** and **Figure 4**). This result was similar to the investigation of Yuvaraj et al. [13] reported the IC50 at nearest concentration. The present study has showed that *A. terrus* FC36AY1 acetonic fractions could extensively inhibited cell proliferation architecture in dose dependent manner. This report demonstrating the *in vitro* anticancer activity of the TA4 from methanolic extract of *A. terrus* FC36AY1 providing a scientific basis for its effects on human health which is similar to Yuvaraj et al. [13], TA4 was found to be inhibited maximum number of cells. So TA4 was taken for further studies.


**Table 3.** MTT assay.

**Figure 4.** MTT analysis of TA4.

#### 24 Medicinal Chemistry


**3.5. HET-CAM test on chick embryo**

**4. Discussion**

The CAM assay is a perceptive, easily feasible, and cheap in vivo check for enquiries of the anti-angiogenic promise of individual compounds. The compound TA4 inhibited the angiogenesis at an interval of 2 and 18 h (**Table 4** and **Figure 5**). This evinced that the compound has

Determination of *In Vitro* Cytotoxicity and Anti-Angiogenesis for a Bioactive Compound…

http://dx.doi.org/10.5772/intechopen.81046

25

In our current scenario there are many new and interesting bioactive metabolites applied as antibiotics, antiviral, anticancer and antioxidant compounds, which are of pharmaceutical, industrial and agricultural importance. Those have been investigated, reported and characterized from several fungal endophytes especially from medicinal plants/trees. Endophytic fungi have the ability to pulse up a plethora of secondary metabolites, typically dependent on the stage of development and environmental factors ranging from nutrient concentrations to light and temperature. The biosynthesis of pigmented secondary metabolite(s) is directly related to cultural conditions that

An investigation of Strobel and Daisy [15] suggested the endophytic fungi since such plants may harbor unique and rare endophytes capable of producing important bioactive metabolites with multiple applications. First and foremost pharmaceutical applied drug Taxol, an anticancer drug was derived from *Taxus*, a gymnosperm is an important anticancer plant. Several endophytic fungi isolated from *Taxus* spp., worldwide have been reported to produce important bioactive metabolites [16, 17]. This current investigation is focused to produce the pigmented crude secondary metabolites and purify a bioactive compound through the antioxidant assessment which could be taken for anticancer applications by *in vitro* and *in vivo* studies. The isolated and identified endophytic fungus FC36AY1 was a prospective and potential strain on analyzing antagonistic and preliminary antioxidant analysis. The fungus explored highest activity in both the assessment. The antagonism revealed the extent of this analysis ability increased as the endophytic fungal colonies matured when compared to immature colonies. The significant inhibition in the growth of fungi without direct contact of mycelia suggests that the prevailing antagonisms may be due to the production of inhibitory substances by the fungi or due to the competition for nutrients or both [18]. However, the mechanisms of inhibition in colony growth of the tested fungi were not addressed in our studies. This was similar to the investigation of Tayung et al. [17] investigated an endophytic fungus *Fusarium* sp. with highest antimicrobial and antioxidant analysis isolate from India. The potential strain was taken for the identification which showed up the organism was *Aspergillus terreus* FC36AY1 with the NCBI BankIt ID accession number was KY807648.

The study focused to produce the mass cultivation of pigmented crude secondary metabolites using SDB as production medium on which the strain FC36AY1 produced about 2.31 U/g in 1 L of production medium. The produced crude pigments were extracted using acetone as sole solvent from the fungal mat (biomass). This was similar to the study of Mani et al. [7] which explored that the extraction of pigmented secondary metabolites was done only in mid polar

the good anti-angiogenic potentiality which could be explored as anticancer agents.

include biomass in the production phase and duration of the incubation periods [14].

**Table 4.** Anti-angiogenesis effect of TA4 in HET-CAM test.

#### **3.5. HET-CAM test on chick embryo**

The CAM assay is a perceptive, easily feasible, and cheap in vivo check for enquiries of the anti-angiogenic promise of individual compounds. The compound TA4 inhibited the angiogenesis at an interval of 2 and 18 h (**Table 4** and **Figure 5**). This evinced that the compound has the good anti-angiogenic potentiality which could be explored as anticancer agents.

## **4. Discussion**

**Sample For 2 h For 18 h**

**No. of vessels in treated CAM**

Sample—1–200 μL 12 10 12 08 Sample—1–400 μL 10 05 13 03

18 10 18 05

11 11 11 11

**No. of vessels in untreated CAM**

**No. of vessels in treated CAM**

**No. of vessels in untreated CAM**

**Table 4.** Anti-angiogenesis effect of TA4 in HET-CAM test.

**Figure 5.** HET-CAM test on chick embryo control.

Negative control—acetone

24 Medicinal Chemistry

Positive control—NaOH

> In our current scenario there are many new and interesting bioactive metabolites applied as antibiotics, antiviral, anticancer and antioxidant compounds, which are of pharmaceutical, industrial and agricultural importance. Those have been investigated, reported and characterized from several fungal endophytes especially from medicinal plants/trees. Endophytic fungi have the ability to pulse up a plethora of secondary metabolites, typically dependent on the stage of development and environmental factors ranging from nutrient concentrations to light and temperature. The biosynthesis of pigmented secondary metabolite(s) is directly related to cultural conditions that include biomass in the production phase and duration of the incubation periods [14].

> An investigation of Strobel and Daisy [15] suggested the endophytic fungi since such plants may harbor unique and rare endophytes capable of producing important bioactive metabolites with multiple applications. First and foremost pharmaceutical applied drug Taxol, an anticancer drug was derived from *Taxus*, a gymnosperm is an important anticancer plant. Several endophytic fungi isolated from *Taxus* spp., worldwide have been reported to produce important bioactive metabolites [16, 17]. This current investigation is focused to produce the pigmented crude secondary metabolites and purify a bioactive compound through the antioxidant assessment which could be taken for anticancer applications by *in vitro* and *in vivo* studies. The isolated and identified endophytic fungus FC36AY1 was a prospective and potential strain on analyzing antagonistic and preliminary antioxidant analysis. The fungus explored highest activity in both the assessment. The antagonism revealed the extent of this analysis ability increased as the endophytic fungal colonies matured when compared to immature colonies. The significant inhibition in the growth of fungi without direct contact of mycelia suggests that the prevailing antagonisms may be due to the production of inhibitory substances by the fungi or due to the competition for nutrients or both [18]. However, the mechanisms of inhibition in colony growth of the tested fungi were not addressed in our studies. This was similar to the investigation of Tayung et al. [17] investigated an endophytic fungus *Fusarium* sp. with highest antimicrobial and antioxidant analysis isolate from India. The potential strain was taken for the identification which showed up the organism was *Aspergillus terreus* FC36AY1 with the NCBI BankIt ID accession number was KY807648.

> The study focused to produce the mass cultivation of pigmented crude secondary metabolites using SDB as production medium on which the strain FC36AY1 produced about 2.31 U/g in 1 L of production medium. The produced crude pigments were extracted using acetone as sole solvent from the fungal mat (biomass). This was similar to the study of Mani et al. [7] which explored that the extraction of pigmented secondary metabolites was done only in mid polar

solvents. The crude pigmented metabolites were experimented for the purification process through HPLC analysis on which the crude was separated into 6 different fractions. Those fractions were taken for the assessment of antimicrobial and antioxidant activities. The 4th fraction TA4 was found to scavenge more free radicals at minimum concentration in a dose dependent manner and this was similar to the investigation of Samaga et al. [19]. In living organisms, oxidative stress created by reactive oxygen species (ROS) resulting from metabolism, in the form of superoxide anion (O−<sup>2</sup> ), hydroxyl radical (·OH), hydrogen peroxide (H2 O2 ) and nitric oxide (NO) leads to conditions like cancer, stroke, myocardial infarctions, diabetes, septic and hemorrhagic shock, and neurodegenerative diseases by inducing biomolecular oxidations. Therefore, effective free radical scavenging molecules are needed by food and pharmaceutical industries. The fraction TA4 exhibited effective free radical scavenging activity comparable with that of BHA. The free radical scavenging activity of this particular fraction could be attributed to the presence of phenolic compounds [7]. This fraction was taken for the elucidation purpose through FT-IR and GC-MS analysis, on which TA4 showed similarity with 96–100% for the mass spectra of the compound and FT-IR exhibited the functional groups present in the compound and this was similar to the study of Liu et al. [11]. From this characterization, we found that the compound was octadecenoic acid 4-hydroxy methyl ester C19H38O3. This compound was taken for cytotoxicity analysis on HT-29 colon cancer cell lines, the compound TA4 explored its activity by showing a promising strategy of killing the cancer cells in an efficient manner. This was similar to the study of Devi and Prabakaran [20] in which they had experimented on four different cancer cell lines by using a potential bioactive compound from endophytic fungus.

**Conflict of interest**

**Author details**

Selvam Tamilarasi1

India

**References**

Vellingiri Manon Mani1,2\*†

Coimbatore, Tamil Nadu, India

† Both the authors contributed equally.

University of Arizona; 2002

Mycobiology. 2015;**43**(3):303-310

aheh.19880160622

2009;**3**(9):1105-1115. DOI: 10.1038/ismej.2009.63

plants. Australian Journal of Botany. 2005;**51**:257-266

The authors declare that we do not have any conflict of interest.

\*Address all correspondence to: manonmanisathee12@gmail.com

, Arockiam Jeyasundar Parimala Gnana Soundari2† and

Determination of *In Vitro* Cytotoxicity and Anti-Angiogenesis for a Bioactive Compound…

http://dx.doi.org/10.5772/intechopen.81046

27

1 PG and Research Department of Biotechnology, Hindusthan College of Arts and Science,

2 Department of Microbial Biotechnology, Bharathiar University, Coimbatore, Tamil Nadu,

[2] Puri J. Endophytic fungi for producing bioactive compounds originally from their host

[3] Faeth RJ. Fungal Endophytes Diversity and Functional Roles. Tucson, AZ 85721, USA:

[4] Grube M, Cardinale M, de Castro JV, Müller H, Berg G. Species-specific structural and functional diversity of bacterial communities in lichen symbioses. The ISME Journal.

[5] Flewelling AJ, Ellsworth KT, Sanford J, Forward E, Johnson JA, Gray CA. Macroalgal endophytes from the Atlantic Coast of Canada: A potential source of antibiotic natural products. Microorganisms. 2013;**1**(1):175-187. DOI: 10.3390/microorganisms1010175 [6] Nassar AH. Activity of crude extracts of endophytic fungi isolated from medicinal

[7] Mani VM, Soundari APG, Karthiyaini D, Preethi K. Bioprospecting for endophytic fungi and their metabolites from medicinal tree *Aegle marmelos* in Western Ghats, India.

[8] Wiener B. F. Geiss. Fundamentals of thin layer chromatography planar chromatography. Heidelberg, Dr. A. Hüthig Verlag, 1987, 482 S., 202 Abb., 40 Tab., DM 192,–, ISBN 3-7785-0854-7. Acta Hydrochimica et Hydrobiologica Banner. 1997;**16**:653. DOI: 10.1002/

[1] Dufosse L. Pigments. Microbial Encyclopdic Microbiology. 2009;**4**:457-471

plants. Cancer Chemotherapeutics Pharmacology. 2015;**44**:355-361

The compound was taken for determination of anti-angiogenesis on chick embryo and this HET-CAM test exhibited maximum anti-angiogenesis by inhibiting maximum number of blood vessels. This manifesting that the compound TA4 has the ability to target the cancer cells and destruct by inhibiting the angiogenesis mechanism which is an important thing in oncology. The drug should have the ability to kill the cancer cells and also destruct the angiogenesis mechanism which could lead to metastasis i.e., taking the cancer cells to other parts of the body through the blood vessels and colonize in some particular area of the human body. In this case, we have found a potential bioactive compound from an efficient endophytic fungus possessing the ability to target the angiogenesis and avoid the metastasis. This is the first report that a bioactive compound TA4 from *A. terrus* FC36AY1 possessing the ability of anti-angiogenesis which was identified through HET-CAM analysis. From this investigation, we are concluding the compound TA4 evincing the ability to target the cancer cells and also it possess multi-functionality in medicinal fields.

## **5. Conclusion**

The current scenario of the biotechnological research is searching for a new natural drug which could efficiently target on different diseases. This research mainly focuses on cancer study by isolating and characterizing a bioactive compound which exhibit its cytotoxicity and anti-angiogenesis against cancer cell lines. The compound TA4 manifested its characteristics by exploring its activities in different analysis. Finally, this investigation concludes that the compound TA4 has broad variety of bio-activities which emphasis in pharma and medicinal fields.

## **Conflict of interest**

solvents. The crude pigmented metabolites were experimented for the purification process through HPLC analysis on which the crude was separated into 6 different fractions. Those fractions were taken for the assessment of antimicrobial and antioxidant activities. The 4th fraction TA4 was found to scavenge more free radicals at minimum concentration in a dose dependent manner and this was similar to the investigation of Samaga et al. [19]. In living organisms, oxidative stress created by reactive oxygen species (ROS) resulting from metabolism, in the form

), hydroxyl radical (·OH), hydrogen peroxide (H2

(NO) leads to conditions like cancer, stroke, myocardial infarctions, diabetes, septic and hemorrhagic shock, and neurodegenerative diseases by inducing biomolecular oxidations. Therefore, effective free radical scavenging molecules are needed by food and pharmaceutical industries. The fraction TA4 exhibited effective free radical scavenging activity comparable with that of BHA. The free radical scavenging activity of this particular fraction could be attributed to the presence of phenolic compounds [7]. This fraction was taken for the elucidation purpose through FT-IR and GC-MS analysis, on which TA4 showed similarity with 96–100% for the mass spectra of the compound and FT-IR exhibited the functional groups present in the compound and this was similar to the study of Liu et al. [11]. From this characterization, we found that the compound was octadecenoic acid 4-hydroxy methyl ester C19H38O3. This compound was taken for cytotoxicity analysis on HT-29 colon cancer cell lines, the compound TA4 explored its activity by showing a promising strategy of killing the cancer cells in an efficient manner. This was similar to the study of Devi and Prabakaran [20] in which they had experimented on four different cancer cell lines by using a potential bioactive compound from endophytic fungus.

The compound was taken for determination of anti-angiogenesis on chick embryo and this HET-CAM test exhibited maximum anti-angiogenesis by inhibiting maximum number of blood vessels. This manifesting that the compound TA4 has the ability to target the cancer cells and destruct by inhibiting the angiogenesis mechanism which is an important thing in oncology. The drug should have the ability to kill the cancer cells and also destruct the angiogenesis mechanism which could lead to metastasis i.e., taking the cancer cells to other parts of the body through the blood vessels and colonize in some particular area of the human body. In this case, we have found a potential bioactive compound from an efficient endophytic fungus possessing the ability to target the angiogenesis and avoid the metastasis. This is the first report that a bioactive compound TA4 from *A. terrus* FC36AY1 possessing the ability of anti-angiogenesis which was identified through HET-CAM analysis. From this investigation, we are concluding the compound TA4 evincing the

ability to target the cancer cells and also it possess multi-functionality in medicinal fields.

The current scenario of the biotechnological research is searching for a new natural drug which could efficiently target on different diseases. This research mainly focuses on cancer study by isolating and characterizing a bioactive compound which exhibit its cytotoxicity and anti-angiogenesis against cancer cell lines. The compound TA4 manifested its characteristics by exploring its activities in different analysis. Finally, this investigation concludes that the compound TA4 has broad variety of bio-activities which emphasis in pharma and

O2

) and nitric oxide

of superoxide anion (O−<sup>2</sup>

26 Medicinal Chemistry

**5. Conclusion**

medicinal fields.

The authors declare that we do not have any conflict of interest.

## **Author details**

Vellingiri Manon Mani1,2\*† , Arockiam Jeyasundar Parimala Gnana Soundari2† and Selvam Tamilarasi1

\*Address all correspondence to: manonmanisathee12@gmail.com

1 PG and Research Department of Biotechnology, Hindusthan College of Arts and Science, Coimbatore, Tamil Nadu, India

2 Department of Microbial Biotechnology, Bharathiar University, Coimbatore, Tamil Nadu, India

† Both the authors contributed equally.

## **References**


[9] Oyaizu M. Studies on product of browning reaction prepared from glucoseamine. Japanese Journal of Nutrition. 1986;**44**:307-315

**Chapter 3**

**Provisional chapter**

ultraviolet

UV-B, apoptosis was induced through

**The Apoptotic Effects of Methylparaben and**

**The Apoptotic Effects of Methylparaben and** 

Rebekah S. Wood, Rebecca S. Greenstein,

Rebekah S. Wood, Rebecca S. Greenstein,

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

ment with 5 mM methylparaben and 25 mJ/cm2

**Keywords:** methylparaben, ultraviolet light, apoptosis

can cause damage to cells.

**1. Introduction**

Isabella M. Hildebrandt and Kimberly S. George Parsons

**Abstract**

Isabella M. Hildebrandt and Kimberly S. George Parsons

http://dx.doi.org/10.5772/intechopen.78575

**Ultraviolet B Light on M624 Human Melanoma Cells**

Methylparaben is a commonly used antimicrobial in cosmetics that has been shown to have negative effects on mammalian cells. Human melanoma M624 cells were treated

B (UV-B) light. Cell proliferation assays showed that 5 mM methylparaben was toxic to M624 cells after 24 hours. Apoptotic signaling pathways were analyzed via isolation of separate cellular compartments and protein analysis via western blot. Upon 5 mM methylparaben treatment, PARP I was cleaved indicating apoptosis, which was mediated by the TNF-α receptor activated in the lipid rafts of the M624 cells. Upon 25 mJ/cm2 UV-B radiation, PARP II was activated indicating cellular damage, cytochrome c was released from the mitochondria, and caspase-3 was expressed. Upon combinatory treat-

mitochondrial release of cytochrome c, expression of caspase-3 and cleavage of PARP I, while methylparaben-induced TNF-α receptor activation and UV-B-induced PARP II activation was inhibited., demonstrating that antimicrobial methylparaben in cosmetics

Paraben compounds are antimicrobials used in cosmetics as preservatives due to their broad antimicrobial functions and their ability to meet the criteria for an ideal preservative [1].

with 1 and 5 mM methylparaben in the presence and absence of 25 mJ/cm2

**Ultraviolet B Light on M624 Human Melanoma Cells**

© 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.

© 2018 The Author(s). Licensee IntechOpen. 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.

DOI: 10.5772/intechopen.78575


#### **The Apoptotic Effects of Methylparaben and Ultraviolet B Light on M624 Human Melanoma Cells The Apoptotic Effects of Methylparaben and Ultraviolet B Light on M624 Human Melanoma Cells**

DOI: 10.5772/intechopen.78575

Rebekah S. Wood, Rebecca S. Greenstein, Isabella M. Hildebrandt and Kimberly S. George Parsons Rebekah S. Wood, Rebecca S. Greenstein, Isabella M. Hildebrandt and Kimberly S. George Parsons

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

http://dx.doi.org/10.5772/intechopen.78575

#### **Abstract**

[9] Oyaizu M. Studies on product of browning reaction prepared from glucoseamine.

[10] Dinis TCP, Madeira VMC, Almeida LM. Action of phenolic derivates (acetoaminophen, salycilate and 5-aminosalycilate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Journal of Archives in Biochemistry and Biophysics.

[11] Liu X, Dong M, Chen X, Jiang M, Lv X, Zhou J. Antimicrobial activity of an endophytic *Xylaria* sp. YX-28 and identification of its antimicrobial compound 7-amino-4-methyl-

[12] Yu W, Zhao Y, Shu B. The radical scavenging activities of radix puerariae isoflavonoids:

[13] Yuvaraj N, Kanmani P, Satishkumar R, Paari KA, Pattukuma V, Arul V. Extraction, purification and partial characterization of *Cladophora glomerata* against multidrug resistant human pathogen, *Acinetobacter baumannii* and fish pathogens. World Journal of Fisheries

[14] Pradeep FS, Pradeep BV. Optimization of pigment and biomass production from *Fusarium moniliforme* under submerged fermentation conditions. International Journal

[15] Strobel G, Daisy D. Bioprospecting for microbial endophytes and their natural products. Microbiology and Molecular Biology Reviews. 2003;**67**:491-502. DOI: 10.1128/

[16] Wang HS, Lu Y, Xue Y, Ruan ZY, Jiang RB, Xing XH, et al. Separation, purification and structure identification of purple pigments from *Dunaliella* sp. B2. Journal of Chemistry

[17] Tayung K, Barik BP, Jagadev PN, Mohapatra UB. Phylogenetic investigation of endophytic *Fusarium* strain producing antimicrobial metabolite isolated from Himalayan Yew Bark. Malaysian Journal of Microbiology. 2011;**7**:1-6. DOI: 10.21161/mjm.23810 [18] Powthong P, Thongmee A, Suntornthiticharoen P. Antioxidant and antimicrobial activities of endophytic fungi isolated from *Sesbania grandiflora* (l.) pers. International Journal

[19] Samaga PV, Rai VR, Rai KML. *Bionectria ochroleuca* NOTL33—An endophytic fungus from Nothapodytes foetida producing antimicrobial and free radical scavenging metab-

[20] Devi NN, Prabakaran JJ. Bioactive metabolites from an endophytic fungus Penicillium sp isolated from *Centella asciatica*. Current Research in Environment and Applied

coumarin. Applied Microbiology and Biotechnology. 2008;**78**:241-247

A chemiluminescence study. Food Chemistry. 2004;**86**:525

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olites. Annals of Microbiology. 2013;**13**:661-666

MMBR.67.4.491-502.2003

Japanese Journal of Nutrition. 1986;**44**:307-315

1994;**315**:161-169

28 Medicinal Chemistry

Methylparaben is a commonly used antimicrobial in cosmetics that has been shown to have negative effects on mammalian cells. Human melanoma M624 cells were treated with 1 and 5 mM methylparaben in the presence and absence of 25 mJ/cm2 ultraviolet B (UV-B) light. Cell proliferation assays showed that 5 mM methylparaben was toxic to M624 cells after 24 hours. Apoptotic signaling pathways were analyzed via isolation of separate cellular compartments and protein analysis via western blot. Upon 5 mM methylparaben treatment, PARP I was cleaved indicating apoptosis, which was mediated by the TNF-α receptor activated in the lipid rafts of the M624 cells. Upon 25 mJ/cm2 UV-B radiation, PARP II was activated indicating cellular damage, cytochrome c was released from the mitochondria, and caspase-3 was expressed. Upon combinatory treatment with 5 mM methylparaben and 25 mJ/cm2 UV-B, apoptosis was induced through mitochondrial release of cytochrome c, expression of caspase-3 and cleavage of PARP I, while methylparaben-induced TNF-α receptor activation and UV-B-induced PARP II activation was inhibited., demonstrating that antimicrobial methylparaben in cosmetics can cause damage to cells.

**Keywords:** methylparaben, ultraviolet light, apoptosis

#### **1. Introduction**

Paraben compounds are antimicrobials used in cosmetics as preservatives due to their broad antimicrobial functions and their ability to meet the criteria for an ideal preservative [1].

© 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. © 2018 The Author(s). Licensee IntechOpen. 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.

However, the side effects of paraben use include increased estrogenic activity of receptors, sensitization of broken skin, endocrine disruptive effects and adverse reproductive effects [2]. The paraben group of compounds includes methyl, ethyl, butyl, heptyl, and benzyl parabens. Methylparaben, a methylester of *p*-hydroxybenzoic acid, is the most widely used paraben in topical skin products [3]. Studies have recently shown that small amounts of methylparaben remain unhydrolyzed in the epidermis [4]. Handa et al. has been the first to describe the effects of methylparaben and ultraviolet B (UV-B) radiation as detrimental to human skin HaCaT keratinocytes [3].

apoptotic signaling pathway, and caspase-3 is known to be responsible for the cleavage of PARP I during cell death. The sequence at which caspase-3 cleaves PARP I is well conserved

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One major regulator of apoptosis is the mitochondria through release of apoptotic factors, such as cytochrome c, through permeabilization of the outer mitochondrial membrane. The rapid increase in permeability causes depolarization, uncoupling of oxidative phosphorylation, and mitochondrial swelling [8]. The mitochondrial pathway of apoptosis through the mitochondrial permeability transition is important for the elimination of UV-B-damaged human keratinocytes [15]. Cytochrome c is a soluble protein that participates in the electron transfer between complex III and complex IV of the respiratory chain in a normal functioning cell [16]. However, an apoptotic signal can lead to the release of cytochrome c from the intermembrane space of the mitochondria into the cytosol. Cells undergoing apoptosis may have an elevation of cytochrome c in the cytosol and a corresponding decrease in the mitochondria [17]. Once released, cytochrome c can bind to the adaptor molecule apoptotic protease activating factor-1 (Apaf-1) and can subsequently activate caspase-9 or caspase-8. Finally, caspase-3 can be activated which is the irreversible point in the mitochondrial apoptosis pathway [9].

Another important regulator of apoptotic induction in the cell is contents of the lipid raft domains. Lipid rafts contain plasma membrane proteins compartmentalized into sphingolipid- and cholesterol-rich microdomains and function as platforms for signal transduction [18]. These lipid raft domains are dynamic, float freely within the bilayer and coalesce upon clustering of their components [19]. Sphingolipids and cholesterol are necessary for the assembly of the lipid raft compartments [20]. Lipid rafts and associated proteins are known to be important in the pathogenesis of several diseases, including cancer progression. In the lipid rafts, caveolin-1 functions through direct protein–protein interactions to regulate diverse cellular processes, including raft-mediated endocytosis, vesicular transport, cell migration, and signal transduction [21]. An upregulation of caveolin-1 expression in clinical studies was associated with the occurrence of metastasis [22]. Caveolin-1 is a lipid raft indicator protein because it is a cholesterol binding protein. This is important because the integrity of the lipid raft is dependent on the ability of cholesterol to pack tightly with the saturated sphingolipids [23]. TNF-α binds to the TNF receptor (TNFR) to initiate apoptosis [24]. TNF-α is a pleiotropic cytokine that can signal for proliferation, stress, inflammation, and cell death [8]. The TNF pathway occurs in a number of different cell types [25]. Cytochrome c is located downstream

While Handa et al. demonstrated the apoptotic effects of methylparaben in normal skin cells, this study examines the apoptotic signaling pathways activated by methylparaben in cancerous melanoma cells, as well as the role (or absence of role) of the mitochondria and lipid raft domains in these signaling pathways. In this study, we have analyzed the expression and activation of the nuclear apoptotic indicator PARP I, PARP II, and cleaved PARP proteins, as well as the expression of TNF-*α* receptor in the lipid rafts in UV-B and methylparaben-treated human M624 melanoma cells. In addition, the expression of caspase-3 and the expression and location of cytochrome c were also analyzed under these cellular conditions in order to fully characterize the cell signaling pathway activated by the cosmetic antimicrobial agent

in the tumor necrosis factor *α* (TNF-*α*) apoptotic pathway.

methylparaben.

in distant species, indicating an important role for PARP I cleavage in apoptosis [14].

Handa et al. studied HaCaT keratinocytes treated with UV-B and methylparaben. HaCaT keratinocytes were cultured in methylparaben-containing medium concentrations of 19.7, 1.97, and 0.197 mM (0.3, 0.03, and 0.003%) for 24 hours, exposed to UV-B (15 or 30 mJ/cm2 ) and further cultured for another 24 hours. The use of parabens in cosmetic products is permitted up to 0.8% (w/w), however the concentration in cosmetics is typically less than 0.32% [4]. Cellular viability, cell death, oxidative stress, nitric oxide production, cellular lipid peroxidation, and activation of nuclear factor kappa B and activator protein-1 were studied, demonstrating that methylparaben treatment increases cell death when the cells are exposed to UV-B and that the detrimental potential of methylparaben is dose and time dependent [3].

Ultraviolet B radiation (290–320 nm) is the main cause of tumor initiation and promotion [5]. UV-B is 1000 times more likely than UVA to cause sunburn and is also known to cause melanoma: while some of the harmful UV-B rays are absorbed by the ozone layer, some still penetrate the atmosphere, causing sunburns that lead to skin cancer.

Melanoma is the deadliest of the skin cancers and accounts for approximately three fourths of all skin cancer deaths [6]. Forms of melanoma currently being diagnosed and treated include superficial spreading melanoma (most common), lentigo maligna, lentiginous melanoma and nodular melanoma. The mechanism for UV-B-induced cancer can be attributed to the dysregulation of apoptosis, or programmed cell death. Apoptosis is a fundamental mechanism characterized by cell shrinkage, membrane blebbing, nuclear breakdown, and DNA fragmentation that is needed for embryonic development, tissue homeostasis, immune defense, and elimination of harmful cells [7]. Ultimately, the cell and its contents are broken down to membrane bound fragments that are phagocytosed by adjacent cells [8]. The apoptotic pathway is regulated by death receptor-mediated extrinsic pathways and mitochondria-mediated intrinsic pathways [9]. The dysregulation of apoptosis can result in pathophysiological states and diseases, such as cancer [7]. Dysregulation in the UV-B-induced apoptosis may also have a major impact on photocarcinogenesis [10].

The PARP protein is an indicator of apoptosis. PARP I cleavage is an indication of induced apoptosis, while PARP II expression is an indicator of advanced cellular damage. PARP I and PARP II are activated by DNA interruptions and are involved in cell survival/death, transcription, DNA repair, and cell division [11, 12]. They act as both damage sensors and signal transducers to down-stream effectors [11, 13]. PARP I and PARP II also function to signal the cell to undergo apoptosis when the amount of DNA damage is beyond repair capacity [13]. PARP I will cleave into 89- and 24-kDa fragments that contain the active site and the DNAbinding domain of the enzyme during apoptosis. The caspase cascade is an important apoptotic signaling pathway, and caspase-3 is known to be responsible for the cleavage of PARP I during cell death. The sequence at which caspase-3 cleaves PARP I is well conserved in distant species, indicating an important role for PARP I cleavage in apoptosis [14].

However, the side effects of paraben use include increased estrogenic activity of receptors, sensitization of broken skin, endocrine disruptive effects and adverse reproductive effects [2]. The paraben group of compounds includes methyl, ethyl, butyl, heptyl, and benzyl parabens. Methylparaben, a methylester of *p*-hydroxybenzoic acid, is the most widely used paraben in topical skin products [3]. Studies have recently shown that small amounts of methylparaben remain unhydrolyzed in the epidermis [4]. Handa et al. has been the first to describe the effects of methylparaben and ultraviolet B (UV-B) radiation as detrimental to human skin

Handa et al. studied HaCaT keratinocytes treated with UV-B and methylparaben. HaCaT keratinocytes were cultured in methylparaben-containing medium concentrations of 19.7, 1.97, and 0.197 mM (0.3, 0.03, and 0.003%) for 24 hours, exposed to UV-B (15 or 30 mJ/cm2

and further cultured for another 24 hours. The use of parabens in cosmetic products is permitted up to 0.8% (w/w), however the concentration in cosmetics is typically less than 0.32% [4]. Cellular viability, cell death, oxidative stress, nitric oxide production, cellular lipid peroxidation, and activation of nuclear factor kappa B and activator protein-1 were studied, demonstrating that methylparaben treatment increases cell death when the cells are exposed to UV-B

Ultraviolet B radiation (290–320 nm) is the main cause of tumor initiation and promotion [5]. UV-B is 1000 times more likely than UVA to cause sunburn and is also known to cause melanoma: while some of the harmful UV-B rays are absorbed by the ozone layer, some still penetrate

Melanoma is the deadliest of the skin cancers and accounts for approximately three fourths of all skin cancer deaths [6]. Forms of melanoma currently being diagnosed and treated include superficial spreading melanoma (most common), lentigo maligna, lentiginous melanoma and nodular melanoma. The mechanism for UV-B-induced cancer can be attributed to the dysregulation of apoptosis, or programmed cell death. Apoptosis is a fundamental mechanism characterized by cell shrinkage, membrane blebbing, nuclear breakdown, and DNA fragmentation that is needed for embryonic development, tissue homeostasis, immune defense, and elimination of harmful cells [7]. Ultimately, the cell and its contents are broken down to membrane bound fragments that are phagocytosed by adjacent cells [8]. The apoptotic pathway is regulated by death receptor-mediated extrinsic pathways and mitochondria-mediated intrinsic pathways [9]. The dysregulation of apoptosis can result in pathophysiological states and diseases, such as cancer [7]. Dysregulation in the UV-B-induced apoptosis may also have

The PARP protein is an indicator of apoptosis. PARP I cleavage is an indication of induced apoptosis, while PARP II expression is an indicator of advanced cellular damage. PARP I and PARP II are activated by DNA interruptions and are involved in cell survival/death, transcription, DNA repair, and cell division [11, 12]. They act as both damage sensors and signal transducers to down-stream effectors [11, 13]. PARP I and PARP II also function to signal the cell to undergo apoptosis when the amount of DNA damage is beyond repair capacity [13]. PARP I will cleave into 89- and 24-kDa fragments that contain the active site and the DNAbinding domain of the enzyme during apoptosis. The caspase cascade is an important

and that the detrimental potential of methylparaben is dose and time dependent [3].

the atmosphere, causing sunburns that lead to skin cancer.

a major impact on photocarcinogenesis [10].

)

HaCaT keratinocytes [3].

30 Medicinal Chemistry

One major regulator of apoptosis is the mitochondria through release of apoptotic factors, such as cytochrome c, through permeabilization of the outer mitochondrial membrane. The rapid increase in permeability causes depolarization, uncoupling of oxidative phosphorylation, and mitochondrial swelling [8]. The mitochondrial pathway of apoptosis through the mitochondrial permeability transition is important for the elimination of UV-B-damaged human keratinocytes [15]. Cytochrome c is a soluble protein that participates in the electron transfer between complex III and complex IV of the respiratory chain in a normal functioning cell [16]. However, an apoptotic signal can lead to the release of cytochrome c from the intermembrane space of the mitochondria into the cytosol. Cells undergoing apoptosis may have an elevation of cytochrome c in the cytosol and a corresponding decrease in the mitochondria [17]. Once released, cytochrome c can bind to the adaptor molecule apoptotic protease activating factor-1 (Apaf-1) and can subsequently activate caspase-9 or caspase-8. Finally, caspase-3 can be activated which is the irreversible point in the mitochondrial apoptosis pathway [9].

Another important regulator of apoptotic induction in the cell is contents of the lipid raft domains. Lipid rafts contain plasma membrane proteins compartmentalized into sphingolipid- and cholesterol-rich microdomains and function as platforms for signal transduction [18]. These lipid raft domains are dynamic, float freely within the bilayer and coalesce upon clustering of their components [19]. Sphingolipids and cholesterol are necessary for the assembly of the lipid raft compartments [20]. Lipid rafts and associated proteins are known to be important in the pathogenesis of several diseases, including cancer progression. In the lipid rafts, caveolin-1 functions through direct protein–protein interactions to regulate diverse cellular processes, including raft-mediated endocytosis, vesicular transport, cell migration, and signal transduction [21]. An upregulation of caveolin-1 expression in clinical studies was associated with the occurrence of metastasis [22]. Caveolin-1 is a lipid raft indicator protein because it is a cholesterol binding protein. This is important because the integrity of the lipid raft is dependent on the ability of cholesterol to pack tightly with the saturated sphingolipids [23]. TNF-α binds to the TNF receptor (TNFR) to initiate apoptosis [24]. TNF-α is a pleiotropic cytokine that can signal for proliferation, stress, inflammation, and cell death [8]. The TNF pathway occurs in a number of different cell types [25]. Cytochrome c is located downstream in the tumor necrosis factor *α* (TNF-*α*) apoptotic pathway.

While Handa et al. demonstrated the apoptotic effects of methylparaben in normal skin cells, this study examines the apoptotic signaling pathways activated by methylparaben in cancerous melanoma cells, as well as the role (or absence of role) of the mitochondria and lipid raft domains in these signaling pathways. In this study, we have analyzed the expression and activation of the nuclear apoptotic indicator PARP I, PARP II, and cleaved PARP proteins, as well as the expression of TNF-*α* receptor in the lipid rafts in UV-B and methylparaben-treated human M624 melanoma cells. In addition, the expression of caspase-3 and the expression and location of cytochrome c were also analyzed under these cellular conditions in order to fully characterize the cell signaling pathway activated by the cosmetic antimicrobial agent methylparaben.

## **2. Materials and methods**

#### **2.1. Cell culture and treatment**

Transformed M624 human melanoma cells were obtained from Dr. Shiyong Wu's laboratory (Edison Biotechnology Institute, Ohio University, Ohio, USA) and originally generated by the National Institute of Health (NIH) (USA). Cells were cultured in DMEM supplemented with fetal bovine serum (FBS) (10%) and penicillin/streptomycin (1%). At 80% cell confluency, cells were treated with methylparaben and UV-B. Cells were incubated with 1 or 5 mM methylparaben in media including supplements for 2 hours. The methylparaben media was removed while treating the cells with 25 mJ/cm2 UV-B radiation and replaced after radiation, and then incubated for another 4 hours before cell lysis.

**2.6. Sample preparation**

saline with 0.05% Tween (TBS-T) for 20 minutes.

**2.7. SDS-page**

**2.8. Western blot**

**3. Results**

Samples were prepared by adding protein loading buffer (0.25 M Tris, pH 6.8, 10% sodium dodecyl sulfate, 0.05% bromophenol blue, 50% glycerol, 10 mM *β*-mercaptoethanol). The

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SDS-PAGE was performed using a 15% separating gel and 4% stacking gel to create a gel with 1.0 mm thickness. Protein samples were separated at 200 volts for 45 minutes and then transferred to a nitrocellulose membrane via wet transfer at 100 volts for 35 minutes. After the transfer, the nitrocellulose membrane was blocked using dried non-fat milk in 1× tris buffered

Western blot was performed using *β*-actin primary antibody (122 M4782, Sigma, St. Louis, MO) for 3 hours followed by three rinse wash cycles and then probed with anti-mouse secondary antibody (Santa Cruz Biotechnology, sc-2371) for 1 hour. PARP protein expression and cleavage was detected by probing overnight with PARP primary antibody (MA5–15031, Thermo Fisher Scientific, Waltham, MA) and anti-rabbit secondary antibody (32,260, Thermo Fisher Scientific, Waltham, MA). Caveolin-1 protein expression was detected by probing overnight with caveolin-1 primary antibody (sc-894, Santa Cruz Biotechnology, Dallas, TX) and anti-rabbit secondary antibody. TNF R1 protein expression was detected by probing overnight with TNF R1 primary antibody (sc-7418, Santa Cruz Biotechnology, Dallas, TX) and anti-mouse secondary antibody. Cytochrome c protein expression was detected by probing with cytochrome c HRP-conjugated primary antibody (sc-13,156, Santa Cruz Biotechnology, Dallas, TX) overnight. Caspase-3 protein expression was detected by probing overnight with Caspase-3 primary antibody (sc-7418, Santa Cruz Biotechnology, Dallas, TX) and anti-rabbit secondary antibody. All overnight incubations were performed shaking at 5°C; all other incubations were performed shaking at room temperature. Protein expression was visualized via

The toxicity of methylparaben and UV-B light to human melanoma M624 cells was initially analyzed using a clonogenic cell proliferation assay (**Figure 1**). The effects of methylparaben and UV-B light on apoptotic pathways in M624 cells were then investigated by analyzing expression of PARP and caspase-3 and cleavage of PARP in the whole cell lysate samples, TNF-*α* expression in the lipid rafts, and cytochrome c expression in both mitochondrial and

Human M624 melanoma cells were treated with methylparaben (1 and 5 mM) and 25 mJ/cm2

UV-B were each shown

chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate).

regular lysate samples, via SDS-PAGE and western blot (**Figures 2**–**4**).

UV-B. The 5 mM concentration of methylparaben and the 25 mJ/cm2

samples were vortexed, boiled for 5 minutes, vortexed and then stored at −20°C.

#### **2.2. Cell proliferation assays**

Cells were plated at approximately 25% confluency and then treated with methylparaben and UV-B radiation as described above, except paraben solutions were allowed to incubate for 24 hours and then viable cells were stained using 0.5% crystal violet solution. Results are averaged from three independent trials.

#### **2.3. Protein samples**

*Whole Cell Lysate Preparation* Cells were washed three times with cold, 1X Phosphate Buffered Saline (PBS) (10 mM phosphate). Cells were then placed in a −20°C freezer for at least 2 hours, removed, and washed once more with PBS. TNET lysis buffer (25 mM Tris–HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, and 0.1% Triton X-100) was mixed with protease inhibitors and added to the cells. The solution containing cells was homogenized with 23 gauge needles and syringes and centrifuged at low speed after which supernatant was collected.

#### **2.4. Lipid raft isolation**

Lipid rafts were prepared as previously described [26]. Briefly, 3,3′-dithiobis (sulfosuccinimidyl propionate) (DTSSP) (1.25 m M) was added to each plate of cells after UV-B and methylparaben treatment and incubated for 1 hour at 4°C. The cells were then collected and frozen overnight. Cells were washed and lysed as described above to prepare samples and placed in an iodixanol solution gradient and ultra-centrifuged for 5 hours. Fractions were collected and the lipid rafts were present in fraction two, indicated by the presence of caveolin-1 protein.

#### **2.5. Mitochondrial isolation**

Cells were washed with PBS and suspended in an isotonic buffer (10 mM Hepes, pH 7.4, 0.2 M mannitol, 0.07 M sucrose) supplemented with protease inhibitors. Samples were homogenized and then centrifuged at 900× g for 5 minutes. Then cells were centrifuged at 10,000× g for 30 minutes at 4° C to obtain the heavy membrane pellet. Samples were re-suspended in an SEM buffer (250 mM Sucrose, 1 mM EDTA, 10 mM MOPS-KOH, pH 7.2).

#### **2.6. Sample preparation**

Samples were prepared by adding protein loading buffer (0.25 M Tris, pH 6.8, 10% sodium dodecyl sulfate, 0.05% bromophenol blue, 50% glycerol, 10 mM *β*-mercaptoethanol). The samples were vortexed, boiled for 5 minutes, vortexed and then stored at −20°C.

#### **2.7. SDS-page**

**2. Materials and methods**

32 Medicinal Chemistry

**2.1. Cell culture and treatment**

**2.2. Cell proliferation assays**

**2.3. Protein samples**

**2.4. Lipid raft isolation**

**2.5. Mitochondrial isolation**

for 30 minutes at 4°

averaged from three independent trials.

removed while treating the cells with 25 mJ/cm2

and then incubated for another 4 hours before cell lysis.

Transformed M624 human melanoma cells were obtained from Dr. Shiyong Wu's laboratory (Edison Biotechnology Institute, Ohio University, Ohio, USA) and originally generated by the National Institute of Health (NIH) (USA). Cells were cultured in DMEM supplemented with fetal bovine serum (FBS) (10%) and penicillin/streptomycin (1%). At 80% cell confluency, cells were treated with methylparaben and UV-B. Cells were incubated with 1 or 5 mM methylparaben in media including supplements for 2 hours. The methylparaben media was

Cells were plated at approximately 25% confluency and then treated with methylparaben and UV-B radiation as described above, except paraben solutions were allowed to incubate for 24 hours and then viable cells were stained using 0.5% crystal violet solution. Results are

*Whole Cell Lysate Preparation* Cells were washed three times with cold, 1X Phosphate Buffered Saline (PBS) (10 mM phosphate). Cells were then placed in a −20°C freezer for at least 2 hours, removed, and washed once more with PBS. TNET lysis buffer (25 mM Tris–HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, and 0.1% Triton X-100) was mixed with protease inhibitors and added to the cells. The solution containing cells was homogenized with 23 gauge needles and

Lipid rafts were prepared as previously described [26]. Briefly, 3,3′-dithiobis (sulfosuccinimidyl propionate) (DTSSP) (1.25 m M) was added to each plate of cells after UV-B and methylparaben treatment and incubated for 1 hour at 4°C. The cells were then collected and frozen overnight. Cells were washed and lysed as described above to prepare samples and placed in an iodixanol solution gradient and ultra-centrifuged for 5 hours. Fractions were collected and the lipid rafts were present in fraction two, indicated by the presence of caveolin-1 protein.

Cells were washed with PBS and suspended in an isotonic buffer (10 mM Hepes, pH 7.4, 0.2 M mannitol, 0.07 M sucrose) supplemented with protease inhibitors. Samples were homogenized and then centrifuged at 900× g for 5 minutes. Then cells were centrifuged at 10,000× g

C to obtain the heavy membrane pellet. Samples were re-suspended in an

syringes and centrifuged at low speed after which supernatant was collected.

SEM buffer (250 mM Sucrose, 1 mM EDTA, 10 mM MOPS-KOH, pH 7.2).

UV-B radiation and replaced after radiation,

SDS-PAGE was performed using a 15% separating gel and 4% stacking gel to create a gel with 1.0 mm thickness. Protein samples were separated at 200 volts for 45 minutes and then transferred to a nitrocellulose membrane via wet transfer at 100 volts for 35 minutes. After the transfer, the nitrocellulose membrane was blocked using dried non-fat milk in 1× tris buffered saline with 0.05% Tween (TBS-T) for 20 minutes.

#### **2.8. Western blot**

Western blot was performed using *β*-actin primary antibody (122 M4782, Sigma, St. Louis, MO) for 3 hours followed by three rinse wash cycles and then probed with anti-mouse secondary antibody (Santa Cruz Biotechnology, sc-2371) for 1 hour. PARP protein expression and cleavage was detected by probing overnight with PARP primary antibody (MA5–15031, Thermo Fisher Scientific, Waltham, MA) and anti-rabbit secondary antibody (32,260, Thermo Fisher Scientific, Waltham, MA). Caveolin-1 protein expression was detected by probing overnight with caveolin-1 primary antibody (sc-894, Santa Cruz Biotechnology, Dallas, TX) and anti-rabbit secondary antibody. TNF R1 protein expression was detected by probing overnight with TNF R1 primary antibody (sc-7418, Santa Cruz Biotechnology, Dallas, TX) and anti-mouse secondary antibody. Cytochrome c protein expression was detected by probing with cytochrome c HRP-conjugated primary antibody (sc-13,156, Santa Cruz Biotechnology, Dallas, TX) overnight. Caspase-3 protein expression was detected by probing overnight with Caspase-3 primary antibody (sc-7418, Santa Cruz Biotechnology, Dallas, TX) and anti-rabbit secondary antibody. All overnight incubations were performed shaking at 5°C; all other incubations were performed shaking at room temperature. Protein expression was visualized via chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate).

### **3. Results**

The toxicity of methylparaben and UV-B light to human melanoma M624 cells was initially analyzed using a clonogenic cell proliferation assay (**Figure 1**). The effects of methylparaben and UV-B light on apoptotic pathways in M624 cells were then investigated by analyzing expression of PARP and caspase-3 and cleavage of PARP in the whole cell lysate samples, TNF-*α* expression in the lipid rafts, and cytochrome c expression in both mitochondrial and regular lysate samples, via SDS-PAGE and western blot (**Figures 2**–**4**).

Human M624 melanoma cells were treated with methylparaben (1 and 5 mM) and 25 mJ/cm2 UV-B. The 5 mM concentration of methylparaben and the 25 mJ/cm2 UV-B were each shown

**Figure 1.** Phototoxicity studies: Crystal violet proliferative assay: cells were treated with methylparaben and UV-B radiation and then stained with a 0.5% crystal violet solution after 24 hours.

5 mM methylparaben, UV-B-induced PARP II expression was completely inhibited (lane 6) while UV-B and 1 mM methylparaben partially inhibited the PARP II expression. Overall, methylparaben at 5 mM had an impact on PARP cleavage with and without UV-B and was

**Figure 4.** Western blot of lipid raft protein samples of TNF-*α*. Samples were standardized using caveolin-1 western blot.

**Figure 3.** Western blot of mitochondrial protein samples of cytochrome c. Samples were standardized using β-actin

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Cytochrome c protein expression in the whole cell and mitochondrial lysate samples was analyzed using *β*-actin standardized loading amounts. Cytochrome c expression was reduced in the whole cell lysate samples treated with 5 mM methylparaben and 5 mM methylparaben plus UV-B (**Figure 2**, lanes 3 and 6 versus lane 1) while cytochrome c expression in the mitochondrial lysate 5 mM methylparaben sample was relatively unchanged compared to the expression seen in the control mitochondrial lysate sample (**Figure 3**, lane 3 versus lane 1). UV-B radiation caused a decrease in cytochrome c in the mitochondrial samples treated with

Caspase-3 expression was analyzed in whole cell lysate samples using *β*-actin standardized loading amounts. It was shown that caspase-3 expression was decreased when the M624 human melanoma cells were treated with 5 mM methylparaben, 1 mM methylparaben plus UV-B radiation, and 5 mM methylparaben plus UV-B radiation when compared to the control

able to inhibit UV-B-induced PARP II expression (**Figure 2**).

western blot.

or without methylparaben (**Figure 3**, lanes 4, 5 and 6 versus lane 1).

**Figure 2.** Western blot of whole cell lysate protein samples of PARP I, and cleaved PARP I, PARP II, cytochrome c and caspase 3. Samples were standardized using β-actin western blot.

to be toxic to the M624 human melanoma cells after 24 hours (**Figure 1**). The combination of methylparaben treatment and UV-B radiation showed amplified toxicity, even at the 1 mM paraben concentration level after 24 hours; 1 mM methylparaben alone was nontoxic to the cells (**Figure 1**).

Results showed that 5 mM methylparaben induced PARP I cleavage (lane 3), while 25 mJ/cm2 UV-B induced PARP II expression instead (lane 4). When cells were treated with UV-B and The Apoptotic Effects of Methylparaben and Ultraviolet B Light on M624 Human Melanoma Cells http://dx.doi.org/10.5772/intechopen.78575 35

**Figure 3.** Western blot of mitochondrial protein samples of cytochrome c. Samples were standardized using β-actin western blot.

**Figure 4.** Western blot of lipid raft protein samples of TNF-*α*. Samples were standardized using caveolin-1 western blot.

5 mM methylparaben, UV-B-induced PARP II expression was completely inhibited (lane 6) while UV-B and 1 mM methylparaben partially inhibited the PARP II expression. Overall, methylparaben at 5 mM had an impact on PARP cleavage with and without UV-B and was able to inhibit UV-B-induced PARP II expression (**Figure 2**).

Cytochrome c protein expression in the whole cell and mitochondrial lysate samples was analyzed using *β*-actin standardized loading amounts. Cytochrome c expression was reduced in the whole cell lysate samples treated with 5 mM methylparaben and 5 mM methylparaben plus UV-B (**Figure 2**, lanes 3 and 6 versus lane 1) while cytochrome c expression in the mitochondrial lysate 5 mM methylparaben sample was relatively unchanged compared to the expression seen in the control mitochondrial lysate sample (**Figure 3**, lane 3 versus lane 1). UV-B radiation caused a decrease in cytochrome c in the mitochondrial samples treated with or without methylparaben (**Figure 3**, lanes 4, 5 and 6 versus lane 1).

to be toxic to the M624 human melanoma cells after 24 hours (**Figure 1**). The combination of methylparaben treatment and UV-B radiation showed amplified toxicity, even at the 1 mM paraben concentration level after 24 hours; 1 mM methylparaben alone was nontoxic to the

**Figure 2.** Western blot of whole cell lysate protein samples of PARP I, and cleaved PARP I, PARP II, cytochrome c and

**Figure 1.** Phototoxicity studies: Crystal violet proliferative assay: cells were treated with methylparaben and UV-B

radiation and then stained with a 0.5% crystal violet solution after 24 hours.

caspase 3. Samples were standardized using β-actin western blot.

Results showed that 5 mM methylparaben induced PARP I cleavage (lane 3), while 25 mJ/cm2 UV-B induced PARP II expression instead (lane 4). When cells were treated with UV-B and

cells (**Figure 1**).

34 Medicinal Chemistry

Caspase-3 expression was analyzed in whole cell lysate samples using *β*-actin standardized loading amounts. It was shown that caspase-3 expression was decreased when the M624 human melanoma cells were treated with 5 mM methylparaben, 1 mM methylparaben plus UV-B radiation, and 5 mM methylparaben plus UV-B radiation when compared to the control sample (**Figure 2**, lanes 3, 5 and 6 versus lane 1). On the other hand, caspase-3 expression was greater in the sample treated with UV-B radiation when combined with 5 mM methylparaben treatment compared to 5 mM methylparaben treatment alone (**Figure 2**, lane 6 versus lane 3).

cytochrome c in the whole cell lysate sample upon 5 mM methylparaben treatment indicating no release of cytochrome c from the mitochondria in these cells. On the other hand, UV-B light treatment showed a decreased cytochrome c expression in the mitochondrial isolated sample while the whole cell lysate sample showed an increased or unchanged cytochrome c expression in cells treated with or without methylparaben indicating a UV-B-induced apop-

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37

The same pattern of expression was seen with caspase-3, indicating caspase-3 activation upon release of cytochrome c from the mitochondria in cells treated with UV-B and UV-B with 5 mM methylparaben. However, 5 mM methylparaben treatment alone inhibited caspase-3 expression. These results suggest that both UV-B light and UV-B light radiation in addition to the 5 mM methylparaben treatment results in M624 human melanoma cells taking a different apoptotic pathway in comparison to cell treatment with only 5 mM methylparaben. When human melanoma M624 cells are exposed to UV-B light or both 5 mM methylparaben and UV-B light, apoptosis occurs through the intrinsic pathway as indicated with the release of cytochrome c from the mitochondria and expression of caspase-3. In comparison, treatment with only 5 mM methylparaben does not result in the release of cytochrome c from the mito-

The TNF-*α* receptor expression in the lipid rafts of M624 melanoma cells induced by methylparaben and UV-B radiation was analyzed by western blot analysis of lipid raft samples. Results show that apoptosis was initiated through the TNF-*α* receptor when treated with 5 mM methylparaben, but the involvement of the TNF-α receptor was inhibited in the presence of UV-B light. The integrity of the lipid raft is dependent on the ability of cholesterol to pack tightly with the saturated sphingolipids [23]. Caveolin-1 in the lipid rafts binds to cholesterol, which is necessary for the assembly of the lipid raft components [20]. Caveolin-1 expression was used to standardize the lipid raft samples in order to examine the expression of the TNF-α receptor in the lipid rafts of cells. TNF-*α* commonly initiates apoptosis by binding to the TNF-α receptor [24]. The activation of TNF-α receptor can lead to formation of TNFR1-associated death domain protein (TRADD). TRADD recruits Fas-associated protein with death domain (FADD) and leads to caspase activation and apoptosis [27]. Death domain formation occurs only when TNF-α receptor is translocated into lipid raft domains [19]; therefore, the expression of the receptor in the lipid rafts was analyzed. Results showed expression of TNF-*α* receptor in the lipid rafts of cells treated with 5 mM methylparaben, which was inhibited in the presence of UV-B radiation (**Figure 4**). These results suggest an alternate TNF-*α* apoptotic pathway that does not involve the release of cytochrome c from the mitochondria

TNF-*α* apoptotic pathways exist that do not cause the release of cytochrome c from the mitochondria that should be investigated in future studies. One of these TNF-*α* apoptotic pathway involves inhibition of NF-*κ*B to allow JNK to induce caspase 8-independent cleavage of Bid to produce jBid. jBid then translocates into the mitochondria to induce the release of Smac, but not cytochrome c. [27, 28]. This research and future steps in this project are important to the understanding of the apoptotic pathway in human M624 melanoma cells when treated with

totic release of cytochrome c from the mitochondria.

into the cytosol or expression of caspase-3.

methylparaben and UV-B radiation.

chondria into the cytosol or caspase-3 expression (**Figures 2** and **3**).

After standardizing the lipid raft samples via caveolin-1, TNF-*α* receptor expression in the lipid raft fraction was analyzed. Western blot analysis revealed that the expression of TNF-*α* receptor was increased in the cells treated with 5 mM of methylparaben compared with control cells (**Figure 4**, lane 3 versus lane 1). This methylparaben-induced increase in expression was inhibited in cells treated with UV-B light (**Figure 4**, lane 6 versus lane 3).

## **4. Discussion**

Melanoma is the deadliest of the skin cancers and accounts for approximately three fourths of all skin cancer deaths [6]. Previous research performed by Handa et al. has shown the detrimental effects of methylparaben and UV-B treatment on HaCaT cells [3]. This study is the first to demonstrate the effects of methylparaben and UV-B in M624 human melanoma cells.

Phototoxicity studies performed on M624 human melanoma cells have shown that 5 mM methylparaben is toxic to M624 cells and UV-B exposure increases this toxicity (**Figure 1**). The PARP protein is an indicator of apoptosis: DNA interruptions activate PARP I and PARP II; PARP I cleavage is an indicator that apoptosis has been induced and PARP II expression is an indicator of cellular damage. PARP I and PARP II function to signal the cell to undergo apoptosis when the amount of DNA damage is beyond the repair capacity [13]. Results indicate that apoptosis occurred after 5 mM methylparaben and 5 mM methylparaben plus UV-B treatment due to the increased cleavage of PARP I. PARP II expression was induced after 25 mJ/cm2 UV-B treatment indicated cellular damage. UV-B-induced PARP II expression was partially inhibited in the cells treated with 1 mM methylparaben and completely inhibited in the cells treated with 5 mM methylparaben, indicating that PARP II is not involved in the signaling pathway activated by 5 mM methylparaben in combination with UV-B radiation and that methylparaben-induced apoptosis is concentration-dependent (**Figure 2**).

Since cleavage of PARP I indicates that apoptosis was occurring in cells treated with methylparaben, and activation of PARP II indicates cellular damage was occurring in cells treated with methylparaben and UV-B radiation, the involvement of the mitochondria and the lipid rafts in the signaling pathways was then analyzed.

The apoptotic signal can be propagated through an intrinsic mitochondrial response that causes permeabilization of the outer mitochondrial membrane in order to release cytochrome c into the cytosol [7]. Analysis of cytochrome c in both whole cell and mitochondrial isolated samples enabled determination of the movement of cytochrome c under different treatment conditions. Most notably, there were changes in cytochrome c expression in the cells treated with 5 mM methylparaben compared to the cells treated with UV-B light radiation with or without methylparaben. In the 5 mM methylparaben treated sample, cytochrome c expression remained unchanged in the mitochondrial lysate while there was decreased expression of cytochrome c in the whole cell lysate sample upon 5 mM methylparaben treatment indicating no release of cytochrome c from the mitochondria in these cells. On the other hand, UV-B light treatment showed a decreased cytochrome c expression in the mitochondrial isolated sample while the whole cell lysate sample showed an increased or unchanged cytochrome c expression in cells treated with or without methylparaben indicating a UV-B-induced apoptotic release of cytochrome c from the mitochondria.

sample (**Figure 2**, lanes 3, 5 and 6 versus lane 1). On the other hand, caspase-3 expression was greater in the sample treated with UV-B radiation when combined with 5 mM methylparaben treatment compared to 5 mM methylparaben treatment alone (**Figure 2**, lane 6 versus lane 3). After standardizing the lipid raft samples via caveolin-1, TNF-*α* receptor expression in the lipid raft fraction was analyzed. Western blot analysis revealed that the expression of TNF-*α* receptor was increased in the cells treated with 5 mM of methylparaben compared with control cells (**Figure 4**, lane 3 versus lane 1). This methylparaben-induced increase in expression

Melanoma is the deadliest of the skin cancers and accounts for approximately three fourths of all skin cancer deaths [6]. Previous research performed by Handa et al. has shown the detrimental effects of methylparaben and UV-B treatment on HaCaT cells [3]. This study is the first to demonstrate the effects of methylparaben and UV-B in M624 human melanoma cells. Phototoxicity studies performed on M624 human melanoma cells have shown that 5 mM methylparaben is toxic to M624 cells and UV-B exposure increases this toxicity (**Figure 1**). The PARP protein is an indicator of apoptosis: DNA interruptions activate PARP I and PARP II; PARP I cleavage is an indicator that apoptosis has been induced and PARP II expression is an indicator of cellular damage. PARP I and PARP II function to signal the cell to undergo apoptosis when the amount of DNA damage is beyond the repair capacity [13]. Results indicate that apoptosis occurred after 5 mM methylparaben and 5 mM methylparaben plus UV-B treatment due to the increased cleavage of PARP I. PARP II expression was induced after

UV-B treatment indicated cellular damage. UV-B-induced PARP II expression was

partially inhibited in the cells treated with 1 mM methylparaben and completely inhibited in the cells treated with 5 mM methylparaben, indicating that PARP II is not involved in the signaling pathway activated by 5 mM methylparaben in combination with UV-B radiation

Since cleavage of PARP I indicates that apoptosis was occurring in cells treated with methylparaben, and activation of PARP II indicates cellular damage was occurring in cells treated with methylparaben and UV-B radiation, the involvement of the mitochondria and the lipid

The apoptotic signal can be propagated through an intrinsic mitochondrial response that causes permeabilization of the outer mitochondrial membrane in order to release cytochrome c into the cytosol [7]. Analysis of cytochrome c in both whole cell and mitochondrial isolated samples enabled determination of the movement of cytochrome c under different treatment conditions. Most notably, there were changes in cytochrome c expression in the cells treated with 5 mM methylparaben compared to the cells treated with UV-B light radiation with or without methylparaben. In the 5 mM methylparaben treated sample, cytochrome c expression remained unchanged in the mitochondrial lysate while there was decreased expression of

and that methylparaben-induced apoptosis is concentration-dependent (**Figure 2**).

rafts in the signaling pathways was then analyzed.

was inhibited in cells treated with UV-B light (**Figure 4**, lane 6 versus lane 3).

**4. Discussion**

36 Medicinal Chemistry

25 mJ/cm2

The same pattern of expression was seen with caspase-3, indicating caspase-3 activation upon release of cytochrome c from the mitochondria in cells treated with UV-B and UV-B with 5 mM methylparaben. However, 5 mM methylparaben treatment alone inhibited caspase-3 expression. These results suggest that both UV-B light and UV-B light radiation in addition to the 5 mM methylparaben treatment results in M624 human melanoma cells taking a different apoptotic pathway in comparison to cell treatment with only 5 mM methylparaben. When human melanoma M624 cells are exposed to UV-B light or both 5 mM methylparaben and UV-B light, apoptosis occurs through the intrinsic pathway as indicated with the release of cytochrome c from the mitochondria and expression of caspase-3. In comparison, treatment with only 5 mM methylparaben does not result in the release of cytochrome c from the mitochondria into the cytosol or caspase-3 expression (**Figures 2** and **3**).

The TNF-*α* receptor expression in the lipid rafts of M624 melanoma cells induced by methylparaben and UV-B radiation was analyzed by western blot analysis of lipid raft samples. Results show that apoptosis was initiated through the TNF-*α* receptor when treated with 5 mM methylparaben, but the involvement of the TNF-α receptor was inhibited in the presence of UV-B light. The integrity of the lipid raft is dependent on the ability of cholesterol to pack tightly with the saturated sphingolipids [23]. Caveolin-1 in the lipid rafts binds to cholesterol, which is necessary for the assembly of the lipid raft components [20]. Caveolin-1 expression was used to standardize the lipid raft samples in order to examine the expression of the TNF-α receptor in the lipid rafts of cells. TNF-*α* commonly initiates apoptosis by binding to the TNF-α receptor [24]. The activation of TNF-α receptor can lead to formation of TNFR1-associated death domain protein (TRADD). TRADD recruits Fas-associated protein with death domain (FADD) and leads to caspase activation and apoptosis [27]. Death domain formation occurs only when TNF-α receptor is translocated into lipid raft domains [19]; therefore, the expression of the receptor in the lipid rafts was analyzed. Results showed expression of TNF-*α* receptor in the lipid rafts of cells treated with 5 mM methylparaben, which was inhibited in the presence of UV-B radiation (**Figure 4**). These results suggest an alternate TNF-*α* apoptotic pathway that does not involve the release of cytochrome c from the mitochondria into the cytosol or expression of caspase-3.

TNF-*α* apoptotic pathways exist that do not cause the release of cytochrome c from the mitochondria that should be investigated in future studies. One of these TNF-*α* apoptotic pathway involves inhibition of NF-*κ*B to allow JNK to induce caspase 8-independent cleavage of Bid to produce jBid. jBid then translocates into the mitochondria to induce the release of Smac, but not cytochrome c. [27, 28]. This research and future steps in this project are important to the understanding of the apoptotic pathway in human M624 melanoma cells when treated with methylparaben and UV-B radiation.

## **5. Conclusion**

This study has demonstrated cell damage, cellular apoptosis and cellular damage upon exposure to the cosmetic antimicrobial methylparaben. In summary, results show that upon 5 mM methylparaben treatment, PARP I was cleaved indicating apoptosis, which was mediated by the TNF-α receptor activated in the lipid rafts of the M624 cells (**Figure 5a**). Upon 25 mJ/cm2

UV-B radiation, PARP II was activated indicating cellular damage, cytochrome c was released from the mitochondria, and caspase-3 was expressed in the cell (**Figure 5b**). Upon combi-

The Apoptotic Effects of Methylparaben and Ultraviolet B Light on M624 Human Melanoma Cells

through mitochondrial release of cytochrome c, expression of caspase-3 (and the caspase cascade) and cleavage of PARP I (**Figure 5c**). Differences in signaling pathways treated with 1 versus 5 mM methylparaben suggest concentration-dependent activation of apoptosis in M624 cells. Future studies will further elucidate the details of the signaling pathways through

Thank you to Dr. Shiyong Wu's lab for donation of cells. Thanks to the Departments of Chemistry and Biochemistry and Biology of Marietta College for all funding for this project.

, Isabella M. Hildebrandt<sup>3</sup>

and

UV-B, apoptosis was induced

http://dx.doi.org/10.5772/intechopen.78575

39

natory treatment with 5 mM methylparaben and 25 mJ/cm2

analysis of additional apoptotic proteins and lipids of the cells.

, Rebecca S. Greenstein2

1 Boonshoft School of Medicine, Wright State University, Dayton, Ohio, USA

3 College of Veterinary Medicine, Ohio State University, Columbus, Ohio, USA

[1] Pugazhendhi D, Pope GS, Darbre PD. Oestrogenic activity of p-hydroxybenzoic acid (common metabolite of paraben esters) and methylparaben in human breast cancer cell

[2] Cabaleiro N, Calle I, Bendich C, Lavilla I. An overview of sample preparation for the determination of parabens in cosmetics. Trends in Analytical Chemistry. 2014;**57**:34-46.

lines. Journal of Applied Toxicology. 2005;**25**:301-309. DOI: 10.1002/jat.1066

2 University of Cincinnati College of Medicine, Cincinnati, Ohio, USA

\*

\*Address all correspondence to: ksg001@marietta.edu

**Acknowledgements**

**Conflict of interest**

No conflicts of interest.

**Author details**

Rebekah S. Wood<sup>1</sup>

**References**

Kimberly S. George Parsons4

4 Marietta College, Marietta, Ohio, USA

DOI: 10.1016/j.trac.2014.02.003

**Figure 5.** Apoptotic pathways: (a) apoptotic proteins activated by 5 mM methylparaben; (b) apoptotic proteins activated by 25 mJ/cm2 UV-B; (c) apoptotic proteins activated by combination of 5 mM methylparaben and 25 mJ/cm2 UV-B.

UV-B radiation, PARP II was activated indicating cellular damage, cytochrome c was released from the mitochondria, and caspase-3 was expressed in the cell (**Figure 5b**). Upon combinatory treatment with 5 mM methylparaben and 25 mJ/cm2 UV-B, apoptosis was induced through mitochondrial release of cytochrome c, expression of caspase-3 (and the caspase cascade) and cleavage of PARP I (**Figure 5c**). Differences in signaling pathways treated with 1 versus 5 mM methylparaben suggest concentration-dependent activation of apoptosis in M624 cells. Future studies will further elucidate the details of the signaling pathways through analysis of additional apoptotic proteins and lipids of the cells.

## **Acknowledgements**

**5. Conclusion**

38 Medicinal Chemistry

by 25 mJ/cm2

This study has demonstrated cell damage, cellular apoptosis and cellular damage upon exposure to the cosmetic antimicrobial methylparaben. In summary, results show that upon 5 mM methylparaben treatment, PARP I was cleaved indicating apoptosis, which was mediated by the TNF-α receptor activated in the lipid rafts of the M624 cells (**Figure 5a**). Upon 25 mJ/cm2

**Figure 5.** Apoptotic pathways: (a) apoptotic proteins activated by 5 mM methylparaben; (b) apoptotic proteins activated

UV-B; (c) apoptotic proteins activated by combination of 5 mM methylparaben and 25 mJ/cm2

UV-B.

Thank you to Dr. Shiyong Wu's lab for donation of cells. Thanks to the Departments of Chemistry and Biochemistry and Biology of Marietta College for all funding for this project.

## **Conflict of interest**

No conflicts of interest.

## **Author details**

Rebekah S. Wood<sup>1</sup> , Rebecca S. Greenstein2 , Isabella M. Hildebrandt<sup>3</sup> and Kimberly S. George Parsons4 \*

\*Address all correspondence to: ksg001@marietta.edu

1 Boonshoft School of Medicine, Wright State University, Dayton, Ohio, USA

2 University of Cincinnati College of Medicine, Cincinnati, Ohio, USA

3 College of Veterinary Medicine, Ohio State University, Columbus, Ohio, USA

4 Marietta College, Marietta, Ohio, USA

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The Apoptotic Effects of Methylparaben and Ultraviolet B Light on M624 Human Melanoma Cells

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41

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**Section 2**

**Structure-activity Studies of Biological**

**Use**

**Effectiveness in Drug Design and Therapeutic**
