1

STUDIES ON ANTIOXIDANT AND ANTICANCER PROPERTIES OF BRINJAL (Solanum melongena L.)

GENOTYPES

Dissertation

Submitted to the Punjab Agricultural University in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY in

BIOCHEMISTRY (Minor Subject: Biotechnology)

By

Himanshu Sharma (L-2013-BS-55-D)

Department of Biochemistry College of Basic Sciences and Humanities

PUNJAB AGRICULTURAL UNIVERSITY LUDHIANA-141 004

2017

2

CERTIFICATE I This is to certify that the dissertation entitled, “Studies on antioxidant and

anticancer properties of Brinjal (Solanum melongena L.) genotypes” submitted for the

degree of Ph.D. in the subject of Biochemistry (Minor subject: Biotechnology) of the Punjab

Agricultural University, Ludhiana, is a bonafide research work carried out by Mr. Himanshu

Sharma (L-2013-BS-55-D) under my supervision and that no part of this dissertation has

been submitted for any other degree.

The assistance and help received during the course of investigation have been fully

acknowledged.

______________________ Major Advisor [Dr. (Mrs.) Neena Chawla] Senior Biochemist Department of Vegetable Science Punjab Agricultural University Ludhiana - 141 004

3

CERTIFICATE II This is to certify that the dissertation entitled, “Studies on antioxidant and

anticancer properties of Brinjal (Solanum melongena L.) genotypes” submitted by

Mr. Himanshu Sharma (L-2013-BS-55-D) to the Punjab Agricultural University, Ludhiana,

in partial fulfilment of the requirements for the degree of Ph.D. in the subject of

Biochemistry (Minor subject: Biotechnology) has been approved by student’s Advisory

Committee after an oral examination on the same.

_________________________ _________________________ [Dr. (Mrs.) Neena Chawla] Dr. (Mrs.) Promila Sama Major Advisor External Examiner

Professor of Biochemistry-cum-Head (MLT) BIS Institute of Sciences & Technology Gagra (Moga) -142043

_________________________ (Dr. Bavita Asthir) Head of the Department _________________________ [Dr. (Mrs.) Neelam Grewal] Dean Post-Graduate Studies

4

AcknowledgementAcknowledgementAcknowledgementAcknowledgement

At the very outset and above all I must bow with all humility before the Divine Power who has

bestowed me with the requisite intelligence, health and above all the will to carry out to

consummation the stupendous task throughout my academic and research period. I also thank him

for bringing me in contact with learned team of guides, all of whom have contributed to my

success.

I express my heartful, deep sense of unbound gratitude and indebtness to my esteemed major

advisor Dr. (Mrs.) Neena ChawlaDr. (Mrs.) Neena ChawlaDr. (Mrs.) Neena ChawlaDr. (Mrs.) Neena Chawla, Sr. Biochemist, Department of Vegetable Science, Punjab

Agricultural University, Ludhiana, who nested me by showering moral encouragement,

supervision, meticulous suggestions and constructive criticism during the entire period of my

study, research and the preparation of this dissertation.

I veraciously realize the inadequacy of words at my command to thank Dr. (Mrs.) Babita Asthir Dr. (Mrs.) Babita Asthir Dr. (Mrs.) Babita Asthir Dr. (Mrs.) Babita Asthir

Head & Sr. Biochemist, Department of Biochemistry for their valuable suggestions. I owe my

sincere thanks to the members of my advisory committee viz. Dr. Sucheta SharmaDr. Sucheta SharmaDr. Sucheta SharmaDr. Sucheta Sharma, Professor (Dean

PGS nominee), Department of Biochemistry, Dr. A.S. Dhatt,Dr. A.S. Dhatt,Dr. A.S. Dhatt,Dr. A.S. Dhatt, Sr. Vegetable Breeder cum Head,

Department of Vegetable Science, Dr. (Mrs.) Satvir KaurDr. (Mrs.) Satvir KaurDr. (Mrs.) Satvir KaurDr. (Mrs.) Satvir Kaur, Assistant Biochemist, Department of

Biochemistry, Dr. (Mrs.) Yogesh VikalDr. (Mrs.) Yogesh VikalDr. (Mrs.) Yogesh VikalDr. (Mrs.) Yogesh Vikal, Geneticist, School of Agricultural Biotechnology, for their

able guidance, constructive suggestions and meticulously scrutinizing this dissertation.

Words seem to be inadequate in the available lexicon to avouch the excellent cooperation, guidance

and the provision of requisite infrastructural facilities by Dr. Dipak Deka,Dr. Dipak Deka,Dr. Dipak Deka,Dr. Dipak Deka, Assistant Scientist,

School of Animal Biotechnology, Guru Angad Dev Veterinary and Animal Sciences University,

Ludhiana.

Mere words of acknowledgement can never suffice my feelings of gratitude to my parents for their

devotion, sacrifices, inspiration and moral support to eliminate my problems by vesting infinite

blessings on me which come a long way in perusing my goal unflinchingly. No choice of words will

suffice to adequately register my gratitude to my loving brother Mohit. I express my thanks to my

friends Ravneet, Ashwani, Tilak, Hemant, Manu, Inderpal, Megha and Harjeet for their support,

memorable company, ever willing help and for providing me the moments of refreshment in

between the exhausting hours to shed the workload which kept me energetic during this period and

made my stay at the University memorable.

The help rendered by Ranbir UncleRanbir UncleRanbir UncleRanbir Uncle in laboratory is highly acknowledged. I also appreciate the

excellent facilities and the cooperation of the staffs of library and computer laboratory.

Place: Himanshu SharmaHimanshu SharmaHimanshu SharmaHimanshu Sharma

Date:

5

Title of the Thesis : “Studies on antioxidant and anticancer properties of brinjal (Solanum melongena L.) genotypes”

Name of the Student and Admission No.

: Himanshu Sharma (L-2013-BS-55-D)

Major Subject : Biochemistry

Minor Subject : Biotechnology

Name and Designation of Major Advisor

: Dr. (Mrs.) Neena Chawla Senior Biochemist

Degree to be awarded : Ph.D.

Year of award of degree : 2017

Total Pages in thesis : 131+ Vita

Name of University : Punjab Agricultural University, Ludhiana-141004

Punjab, India

ABSTRACT

The objectives of the present study were to screen brinjal genotypes for phytonutrientsand antioxidant activity & to evaluate cytotoxic and apoptotic effects of brinjal extracts on cancer cell lines. Study included phenolic compounds, pigments, alkaloids and plant acids in brinjal fruit of thirty different genotypes. Genotypic variation was reported on basis of phytonutrients in all brinjal genotypes. Optimization of parameters was done for anthocyanin extraction by varying time (3-12h), temperature (30-60°C), pH (3-8) and solid/solvent ratio. Time of extraction and pH showed significant effect on anthocyanin extraction from brinjal fruit. Optimized conditions for the anthocyanin extraction were: 7.99 h time, 33.63°C temperature, 5.39 pH and solid/liquid ratio 0.32. Anthocyanin content in brinjal varied on basis of color, as purple colored genotypes had higher anthocyanin content. Anthocyanin content in peel was 3 to 4 folds higher as compared to that in whole fruit. Ten brinjal genotypes were selected on the basis of higher phenolic contents. Selected genotypes were analyzed for enzymes (PAL, TAL & ANS) in leaves and brinjal fruit. Study showed positive correlation between the enzymes (PAL, TAL & ANS) and biosynthesis of phenolic compounds. PPO enzyme showed negative correlation with phenolic compounds. Free radical scavenging capacitieswere analyzed by DPPH, ABTS and FRAP assays. Antioxidative potential of brinjal fruits was positively correlated to the total phenol and flavanol contents. Biochemical studies suggested that brinjal is a rich source of phytochemicals and antioxidants. In silico virtual screening showed the binding and behaviour of phytochemicals with cell surface receptors. Brinjal extracts were prepared to study the potential cytotoxic effects on different cancer cell lines. Combination index and dose reduction index were studied and showed synergistic effect of brinjal extract and standard anticancer drugs.

Keywords: Anthocyanins, brinjal, in silico, phytonutrients, optimization.

________________________ _______________________ Signature of Major Advisor Signature of the student

6

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7

CONTENTS

CHAPTER TITLE PAGE NO.

I INTRODUCTION 1-3

II REVIEW OF LITERATURE 4-19

III MATERIAL AND METHODS 20-33

IV RESULTS AND DISCUSSION 34-108

V SUMMARY 109-112

REFERENCES 113-131

VITA

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LIST OF TABLE

Table No.

Particulars Page No.

2.1 Solubility of different compounds in specific type of solvent. 15

2.2 Some conformational search algorithms 17

4.1 Phenolic Compounds present in different brinjal genotypes 35

4.2 Quinones and tannin content in different brinjal genotypes 43

4.3 Effect of different factors on anthocyanin content 50

4.4 ANOVA for Response Surface Quadratic Model 51

4.5 Optimized conditions (selected) for anthocyanin extraction 52

4.6 Variation in Optimized and experimental values for anthocyaninextraction

54

4.7 Anthocyanins concentration in different genotypes of brinjal (Peel and whole fruit)

55

4.8 Leucoanthocyanins content in different genotypes of brinjal 57

4.9 Oxalic acid and Solanin content in different genotypes of brinjal 66

4.10 Correlation among Different variables studied on brinjal genotypes 71

4.11 Phenylalanine ammonia lyase (PAL) activity in leaf at various stages of growth of brinjal

73

4.12 Phenylalanine ammonia lyase (PAL) activity in brinjal fruit at various stages of growth.

74

4.13 Tyrosine ammonia lyase (TAL) activity in leaf at various stages of growth of brinjal genotypes

76

4.14 Tyrosine ammonia lyase (TAL) activity in fruit at various stages of growth of brinjal genotypes.

77

4.15 Anthocyanidin Synthase (ANS) activity in leaves of brinjal at various stages of growth

78

4.16 Anthocyanidin Synthase (ANS) enzymatic activity in fruit at various stages of growth of brinjal.

79

4.17 PPO activity in brinjal genotypes with mono and diphenol substrates. 80

4.18 Peroxidase (POD) activity in brinjal genotypes 82

4.19 Free radical scavenging activity (DPPH, ABTS assays) in brinjal genotypes

83

4.20 Free radical scavenging activity (FRAP assay) in brinjal genotypes 85

4.21 Protein receptor molecules selected for molecular Docking studies 87

4.22 Details of Docking of standard drugs with protein receptors. 90

4.23 Details of Docking of selected ligands with protein receptors. 91-92

4.24 Selected protein-ligand combinations and amino acids involved in their interaction

93

4.25 IC50 values of chemotherapeutic drugs in human cancer cells 104

4.26 Dose effect relationship of S. melongena extracts with drug combinations in cancer cell line

107

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LIST OF FIGURES

Figure

No. Particulars Page

No.

2.1 Different classes of phenolic compounds 6

2.2 Biosynthesis of Chlorogenic acid in eggplant 7

2.3 Important anthocyanidins present in vegetables 10

2.4 Hydroxylation and oxidation reactions catalysed by polyphenol oxidase

13

2.5 Structure of some natural substrates of PPO 14

2.6 Types of virtual screening and steps in molecular docking 16

2.7 Phytochemicals that block or suppress multistage carcinogenesis. 18

4.1 Frequency of brinjal genotypes selected for study 34

4.2 Variation in total phenols, flavonols and ODH content in different brinjal genotypes

36

4.3 Frequency distribution plots for (A) Total phenol & (B) Flavonol content in different brinjal genotypes

37

4.4 One way analysis for (A) Total phenol & (B) Flavonol content on basis of color of brinjal genotypes

38

4.5 Variation in CGA content in different brinjal genotypes 39

4.6 Frequency distribution plots for (A) Orthodihydroxyphenols (ODHs) & (B) Chlorogenic acid (CGA) content in different brinjal genotypes

40

4.7 One way analysis for (A) Orthodihydroxyphenols (ODHs) & (B) Chlorogenic acid (CGA) content on basis of color of brinjal genotypes

41

4.8 Quinone (µg/100g) concentration in different genotypes of brinjal 44

4.9 Tannin (mg/100g) concentration in different genotypes of brinjal 45

4.10 Frequency distribution plots for (A) Quinones & (B) Tannins content in different brinjal genotypes

46

4.11 One way analysis for (A) Quinones & (B) Tannins content on basis of color of brinjal genotypes

47

4.12 Scatterplot matrix showing multivariate analysis of Total phenols (TP), Flavonols, Chlorogenic acid (CGA), Quinones and tannins

49

4.13 Normal residual plot showing normal vs. studentized probability of anthocyanin

52

4.14 Effect of different factors on anthocyanin concentration 53

4.15 Anthocyanins content in peel and whole fruit of different genotypes of brinjal

58

4.16 Leucoanthocyanin content in leaves and flowers of different genotypes of brinjal

59

4.17 Frequency distribution plots for Anthocyanin content in (A) Peel & (B) Whole fruit of different brinjal genotypes

60

4.18 One way analysis for Anthocyanin content in (A) Peel & (B) Whole fruit on basis of color of brinjal genotypes

61

4.19 Frequency distribution plots for Leucoanthocyanin (LAC) content in (A) Leaves (L) & (B) Flowers (F) of different brinjal genotypes

62

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4.20 One way analysis for Leucoanthocyanin (LAC) content in (A) Leaves (L) & (B) Flowers (F) on basis of color of brinjal genotypes

63

4.21 Scatterplot matrix showing multivariate analysis of anthocyanins (Peel & whole fruit) and leucoanthocyanins (Leaf & flower)

64

4.22 Scatterplot matrix showing multivariate analysis of Oxalic acid (OA) with flavonols and anthocyanins (AC)

65

4.23 Scatterplot matrix showing multivariate analysis of Chlorogenic acid (CGA) with anthocyanins (AC) and Leucoanthocyanins (LAC)

65

4.24 Oxalic acid content in brinjal genotypes 67

4.25 Solanin content in brinjal genotypes 68

4.26 Frequency distribution plots for (A) Oxalic acid (OA) & (B) Solanin (SOL) content of different brinjal genotypes

69

4.27 One way analysis for (A) Oxalic acid (OA) & (B) Solanin (SOL) on basis of color of brinjal genotypes

70

4.28 Graphs between some positively correlated variables 72

4.29 Phenylalanine ammonia lyase (PAL) activity in brinjal leaves at 7 days interval

74

4.30 Phenylalanine ammonia lyase (PAL) activity in brinjal fruits at 7 days interval

75

4.31 Tyrosine Ammonia Lyase (TAL) activity in brinjal leaves at 7 days interval

76

4.32 Tyrosine Ammonia Lyase (TAL) activity in brinjal fruits at 7 days interval

77

4.33 Anthocyanidin Synthase (ANS) activity in leaves of brinjal at 7 days interval

78

4.34 Anthocyanidin Synthase (ANS) activity in brinjal fruit at 7 days interval

80

4.35 Polyphenol (PPO) activity in brinjal fruits 81

4.36 Correlation between quinone content and Polyphenol oxidase (PPO) activity

82

4.37 Peroxidase (POD) activity in brinjal genotypes 83

4.38 DPPH and ABTS assisted radical scavenging activity in brinjal genotypes

84

4.39 Correlation of (A) DPPH and (B) ABTS with other phenolic components

85

4.40 FRAP assisted radical scavenging activity in brinjal genotypes 86

4.41 Correlation of FRAP with other phenolic components 86

4.42 3D Structure of different receptor protein complexes used for virtualscreening

88

4.43 3D Structure of different ligands molecules used for virtual screening 89

4.44 3D Structure of standard drugs (A) Doxorubicin (B) Fluorouracil 89

4.45 Secondary structure of AKT1 protein and its interaction withdelphinidine

94

4.46 Interaction of cyanidine with AKT1 protein and Ramachandran plot showinglocation of amino acids involved in interaction.

95

4.47 Secondary structure of CDK2 protein and its interaction with 96

11

delphinidine

4.48 Interaction of Europinidine with CDK2 protein andRamachandran plotshowing amino acids involved ininteraction.

97

4.49 Secondary structure of EGFR protein and its interaction with delphinidine

98

4.50 Secondary structure of ERK2 protein and its interaction with delphinidine

99

4.51 Secondary structure of JAK1 protein and its interaction with delphinidine

100

4.52 Interaction of JAK1 protein with CGA and Ramachandran plot showing amino acids involved in interaction.

101

4.53 Interaction of MAPKK1 protein with europinidine and Ramachandran plot showing amino acids involved in interaction.

101

4.54 Interaction of Rab5a protein with CGA and Ramachandran plot showingamino acids involved in interaction.

102

4.55 Secondary structure of Rab5a protein and its interaction with delphinidine

103

4.56 Effect of varying concentration of (A) Cisplatin (B) Doxorubicin (C) Fluorouracil on different cell lines

105

4.57 Effect of varying concentration extracts on different cell lines 106

4.58 Showing combination index and dose reduction curve for combination one (E+D1)

107

4.59 Dose effect, combination index and dose reduction curve for combination one (E+D1)

108

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ABBREVIATIONS

% = Percentage

µg = Microgram

µl = Microlitre

°C = Centigrade

AOAC = Association of Official Analytical Chemists

conc. = Concentrated

DEAE = Diethyl amino ethyl

DE = Degree of esterification

EDTA = Ethylene diamine tetra-acetate

HM = High methoxyl

h(s) = Hour(s)

kg = Kilogram

LM = Low methoxyl

l = Litre

M = Molar

mg = Milligram

min = Minute

ml = Milliliter

mM = Millimolar

MW = Molecular weight

N = Normal

rpm = Revolutions per minute

SDS = Sodium Dodecyl Sulphate

s = Second

v = Volume

w = Weight

1

CHAPTER I

INTRODUCTION

Brinjal (Solanum melongena L.) is a common vegetable crop and grown in the tropic

and subtropic regions of the world. Brinjal is a perennial crop but grown commercially in

different regions of world as an annual crop. As a warm weather crop, it is grown at large

scale in Indian subcontinent (India, Bangladesh), China, Japan, and Philippines. It is the most

cultivated vegetable crop in Italy, France, Egypt and the United States. Brinjal is a hardy crop

that yields well even under stress conditions, including high temperature and drought.

Eggplant is a native plant of India with a large number of cultivated varieties exhibiting

different color, shape and size. Brinjal is cultivated in temperate, tropical and subtropical

zones. The fruit of brinjal plant is also known as melanzana, aubergine, garden egg, brinjal or

patlican and is a widely used part of plant which is used as a vegetable in cooking. Elongated

ovoid or slender shaped brinjal fruits with purple skin are the most widely cultivated varieties.

Brinjal is considered as one of the top ten vegetables in terms of oxygen radical scavenging

capacity. The major phenolic content present in the fruit part is responsible for the high

oxygen radical scavenging capacity (Cao et al 1996).

A detailed analysis of eggplant extract has been done at the Institute of Biology of

Sauo Paulo State University, Brazil. It was found that brinjal juice could significantly reduce

body weight and level of plasma cholesterol content in hypercholesterolemic rabbits (Jorge et

al 1998) and rats (Silva et al 1999). Hundred grams of eggplant contains energy (24 kcal),

carbohydrates (5.7 g), fat (0.19 g), protein (1.01 g), vitamin B6 (0.084 mg), thiamine (0.039

mg), riboflavin (0.037 mg), niacin (0.649 mg), pantothenic acid (0.281 mg), folate (0.22 mg),

vitamin C (2.2 mg), manganese (0.25 mg), iron (0.24 mg), calcium (9 mg), magnesium (14

mg), potassium (230 mg), phosphorus (25 mg), and zinc (0.16 mg) (Yoon et al 2012). The

fruits of brinjal are excellent remedy for liver problems (Shukla and Naik 1993). The green

brinjal is good for diabetic patients.

Brinjal fruits contain a variety of biologically active compounds viz. phenols,

flavones, flavonoids and phenolic acid. These phytochemicals are reported to have imperative

health promoting benefits. Sudheesh et al (1997) demonstrated hypolipidemic activity due to

eggplant extract in rats, which were fed normal and high fat diets. The skin and pulp of the

eggplant are considered as a hub of phenolic compounds and antioxidant activities (Huang et

al 2004, Kumari et al 2014). Noda et al (2000) reported that the extract from purple brinjal

possesses a high radical scavenging capacity as it can scavenge superoxide radicals and

inhibit the formation of hydroxyl radical by chelation of ferrous ion.

Phenolic compounds are one of the main classes of secondary metabolites. Among

2

these compounds, flavonoids are an important group of plant phenolics. So far, over 8000

varieties of flavonoids have been identified. These perform various roles in plants such as

protection from UV rays, defence against biotic factors as pathogens and pests, pollen

fertility, signalling with microorganisms, pigmentation and regulation of auxin transport

(Winkel-Shirley 2001). Anthocyanins are the most important class of flavonoids.

Anthocyanins are water-soluble natural pigments which give pink, red, magenta, purple and

dark blue colours to flowers and fruits depending on pH. These pigments are synthesized in

the cytosol and are accumulated in the vacuoles of plant cells. These are mainly present in

epidermal cells and also in flesh. The accumulation of anthocyanins varies with plant species,

cultivar, tissue structure, geographical location and cultivation conditions. They are produced

by secondary metabolism of plants (pentose phosphate, shikimate and flavonoid pathways).

Secondary metabolites are generally flavourless and odourless but these contribute to

moderate astringent sensation and taste. Anthocyanins are most widely distributed in all

tissues of higher plants including leaves, stems, roots, flowers and fruits. Vaccinium species

such as blackberry, blueberry, eggplant and black rice are rich in anthocyanins (Chaudhary

and Mukhopadhyay 2012). They are glycosides of polymethoxy and polyhydroxy derivatives

of 2-phenylbenzopyrylium or flavylium salts. The difference among various anthocyanins

relates to the nature and number of sugars, number of hydroxyl groups attached to the

molecule and the position of their attachment. The nature and number of aliphatic or aromatic

acids attached to sugars in the molecule also vary across different anthocyanins.

Condensed tannis (CTs) also known as proanthocyanidins are the polymers of

flavonoids molecules (Salunkhe et al, 1989; Naczk et al, 1999). Tannins are present in plant

seeds and flesh that become brown from colourless on oxidation. Tannins impart astringent

quality to the developing fruit which deter animals from feeding (Feeny 1970, Forkner et al

2004). Lignin, a major constituent of cell wall is polymer of 4-hydroxyphenylpropanoids. It is

also derivatized from the phenylpropanoid pathway (Boerjan et al 2003). Lignins have a role

in plant growth and development. Because the lignin polymers confer strength and rigidity to

the cell wall, these protect plant from various biotic and abiotic stresses. Flavonols are

required for functional pollen in maize and petunia (Deboo et al 1995) and also play an

important role in UV photoprotection (Lois and Buchanan 1994, Ryan et al 2002). It is a rich

source of antioxidants such as phenolic acids and flavonoids (Mennella et al 2010, Stommell

and Whitaker 2003). These compounds are present in the fruit’s flesh as well as skin (Huang

et al 2004; Somawathi et al, 2014) and their content is regulated during fruit ripening

(Mennella et al 2012). Nasunin is a major anthocyanin found in peel of purple brinjal but not

in white and green and has been reported to contain high antioxidant activity (Igarashi et al

1993, Yoshida and Hatano, 2000).

Plant phenolic phytochemicals have several potential health promoting effects

3

(Kinsella et al 1993). These phenolic compounds have an important role to cope with

peroxidation of cell membrane lipids which is stimulated by free radicals, mainly reactive

oxygen species in living systems. Consumption of polyphenols reduces radical-mediated

diseases, mainly carcinogenesis and atherosclerosis (Ames et al 1993, Sawa et al 1999).

Anthocyanins are known to enhance the anti-ulcer activity (Cristoni and Magistretti 1987),

sight acuteness, the maintenance of normal vascular permeability (Lamer-Zarawska and

Oszmianski, 1994), in vitro inhibition of platelet aggregation (Tamas et al 2000), protection

against liver damage (Wang et al 2000, Wang et al 2002), antimutagenic activity (Hope et al

2004), stimulation of insulin in pancreatic cells (Jayaprakasam et al 2005), anti-tumor activity

(Liu et al 2005 ) and may prevent hyperlipidemia (Xia et al 2007) and cardiovascular diseases

(Gracia-Alonso et al 2004). Due to these medicinal properties, Brinjal phenolic extracts can

be used as components of pharmaceutical preparations and functional foods. Due to a variety

of pharmacological activities in mammalian body, the phenolic compound named

anthocyanin is referred to as “Nutraceutical”.

Brinjal has high antioxidant and phytochemical content which provides various

therapeutic potential health effects. These pharmaceutical uses of brinjal have not been

exploited much in India. The present study was carried out with the following objectives:

Objectives:

i. Screening of brinjal genotypes for phytonutrients and antioxidant activity.

ii. Evaluation of cytotoxic and apoptotic effect of brinjal extracts on cancerous cell lines.

4

CHAPTER II

REVIEW OF LITERATURE

Brinjal (Solanum melongena L.) is an economically important vegetable crop that is

usually grown in the tropics, subtropics and Mediterranean parts of the world. It is a

nightshade plant like tomato and potato and belongs to family Solanaceae. It is of South

Asian origin and a native to South India and Sri Lanka (Alam et al, 2006). It is commonly

known as brinjal in South Asia and aubergine in Europe. Brinjal, an inexpensive crop is one

of the major food components of the human diet in various developing countries (Naujeer,

2009). After China, India is the second largest producer of vegetables in the world. The

production of Brinjal is 8.3% of total vegetable produce in India (135.57 lakh tonnes) which

makes 27.2% of total brinjal production of the world (FAOSTAT, 2015). Area covered under

brinjal is about 711.3 thousand hectares in India which is 7.6% of total vegetable crop area.

Brinjal is a rich source of phenols and flavonoids, due to which it ranks amongst top ten

vegetables as far as antioxidant capacity is concerned (Singh et al, 2009, Huang et al, 2004).

A number of health benefits of brinjal are reported, including scavenging of reactive oxygen

species (antioxidant), anti-diabetic properties and its anticancer nature. These properties are

directly attributed to its phenolic and anthocyanin content (Akanitapichat et al, 2010). Brinjal

is a great storehouse of vitamins and minerals (Dias 2012).

Literature related to present study has been reviewed under following sub-headings:

2.1 Brinjal: a rich source of phytochemicals and antioxidants

2.1.1 Phenolic compounds

2.1.2 Pigments of brinjal

2.2 Enzymes

2.2.1 Phenylalanine ammonia lyase (PAL)

2.2.2 Tyrosine ammonia lyase (TAL)

2.2.3 Anthocyanidin synthase (ANS)

2.2.4 Peroxidase (POD)

2.2.5 Polyphenol oxidase (PPO)

2.3 In silico Virtual Screening

2.4 Extraction of phytochemicals

2.5 Role of phytochemicals in disease prevention

2.1 Brinjal: a rich source of phytochemicals and antioxidants

Phytochemicals (Phyto is a Greek word, which means plant) are naturally occurring

chemical compounds of plants with biological activity. They have protective role in various

biotic and abiotic damages and also contribute to the aroma, plant color and flavor (Gibson et

al, 1998). Phytochemicals are also part of human diet and contribute to health promoting

5

effects by regulating enzymes involved in metabolic reactions and cell signalling pathways

(Sato et al, 2011).

Among vegetables, brinjal is having maximum oxygen radical absorbance capacity

(ORAC) (Cao et al, 1996). Thus, it has been introduced in traditional medicine for treatment

of many diseases (Khan, 1979; Kashyap et al, 2003). Brinjal is also titled as brain food due to

presence of anthocyanins as it helps to protect the lipid backbone present in cell membranes

of brain (Spencer et al 2008). Apart from this, brinjal is a rich source of vitamins namely B1,

B2, B5, B6, C, K and minerals like copper, magnesium, potassium and manganese (Alam et

al, 2006, Gajewski et al, 2009)

2.1.1 Phenolic compounds

Phenolic compounds are naturally occurring organic species containing one or more

aromatic rings having bonded hydroxyl groups. Generally natural phenolic compounds exist

in conjugation with carbohydrates (monosaccharides and polysaccharides), linked to one or

more of the phenolic groups (Harbone, 1998). In plants, phenolic compounds produced as

secondary metabolites are involved in different processes, for instance pigmentation,

pollination, growth, lignification (Duthie et al, 2003) and resistance against biotic and abiotic

stresses (Fraga et al, 2010).

In recent years, these compounds are considered critically because of their potential

role as antioxidants. The varieties of fruits and vegetables have high concentration of

polyphenols. (Maksoud et al 2013). Other plant foods like olive, cereals, legumes, chocolate;

and beverages viz. tea, coffee, beer, wine are also considered as good source of polyphenols

(Siemann and Creasy, 1992). Moreover, phenolic compounds are considerably responsible for

the organoleptic properties like astringency and bitterness of fruit juices and fruit. The

interaction of phenolic compounds like Procyanidin with glycoprotein in saliva is mainly

responsible for such characteristics (Dai and Mumper, 2010). A number of different naturally

occurring phenolic compounds are present in brinjal (Middleton and Kandaswami, 1994).

Phenolic compounds of brinjal are also having antibacterial and immune-stimulant properties,

which makes them beneficial for human health (Gajewski et al, 2009).

The main dietary phenolic compounds are classified as phenolic acids, flavonoids and

tannins as shown in fig 2.1. The compounds like hydroxycinnamic and hydroxybenzoic acids

are examples of phenolic acids. Flavonoids, a diverse group of secondary plant metabolites

include flavanols, flavonols, flavones and flavanones (Soleas 1997; Sanders et al, 2000). In

plants, most of the tannins are present in hydrolysable and condensed form (Harborne, 1998;

Mueller-Harvey, 2001).

Phenolic acid generally contributes to the aroma and taste of various plant food

products. Singleton and Nobel (1976) found that chlorogenic acid was responsible for the

astringency and bitterness of plant products. Dadic and Belleau (1973) also related bitterness

to the presence of phenolic compounds.

6

Fig. 2.1: Different classes of phenolic compounds (Harbone, 1998), (Gajewski et al, 2009)

Chlorogenic acid (CGA) is a family of esters formed between certain trans-cinnamic

acids (caffeic, ferulic and p-coumaric acids) and quinic acid (Clifford, 2000). Chlorogenic

acid results from the reaction involving caffeic acid and (-) quinic acid esterification.

Vegetables, fruits, and beverages like coffee are the major dietary sources of CGA

(Azuma et al, 2000). Chen et al (2009b) has estimated daily consumption of CGA in humans.

Coffee is considered as one of the major source of CGA in the human diet thus regular coffee

drinkers may consume up to 0.5 - 1 g of CGA per day. Olthof et al (2001) reported that

vegetables and fruits also make a substantial contribution to CGA intake.

7

Fig. 2.2: Biosynthesis of Chlorogenic acid in eggplant (Niggeweg et al (2004), Mahesh et al (2007), Clé et al (2008), Comino et al (2009), Joët et al (2010), Menin et al (2010)

Whitaker and Stommel (2003) reported that 5-o-caffeoyl-quinic acid (chlorogenic

acid) is the most abundant phenolic acid in different eggplant samples and typically ranges

from 70 to 95% of total phenolics in brinjal fruit flesh. Concellón et al (2012) also found

chlorogenic acid as the most abundant phenolic acid accumulated in the fruit part of eggplant.

The various studies showed influential effect of different stages of fruit development,

(Arivalagan et al, 2012) environmental factors and storage conditions on variable CGA

content among cultivated germplasm of eggplant (Mennella et al 2012).

Among phenolic acids, CGA is well known for having various biological properties

of interest for human health (dos Santos et al, 2006). These include anti-inflammatory, anti-

oxidant and analgesic properties demonstrated both in vitro (Morishita and Ohnishi, 2001)

and in vivo (Sheu et al, 2009), as well as strong anti-microbial activity (Almeida et al, 2006).

Several studies also highlight CGA cardioprotective and neuroprotective effects (Zhao et al,

2012).

Because of inductive effect on apoptosis in human cancer cells like leukemia cells

(Yang et al, 2012) and lung cancer cells (Burgos-Morón et al, 2012), CGA is also recognized

to exert selective anti-cancer properties. Other biological activities of CGA include its anti-

8

obesity effect with improvement of lipid metabolism (Cho et al, 2010), and a delay in

intestinal glucose absorption and inhibition of gluconeogenesis (Ong et al, 2012), which

contributes to an anti-diabetic effect (Coman et al, 2012).

Chlorogenic acid is one of the most abundant polyphenols in the human diet and is

highly bioavailable in nature (dos Santos et al, 2006). This fact, together with its numerous

bioactive properties potentially beneficial for human health (Bernhoft, 2010), encourages the

use of breeding approaches in order to increase its level in food crops (Niggeweg et al, 2004;

Sekara et al, 2007).

Gajewski et al (2009) reported similar concentrations of CGA in the eggplant fruit

skin as well as in the fruit flesh. CGA contributes to 30% to 75% of total phenolics (Luthria,

2012; Mennella et al, 2012). It was found that the content of CGA in eggplant increases after

the thermal treatments normally used for eggplant cooking (Lo Scalzo et al, 2010; Yoshiaki et

al, 2002). Also, it is worth mentioning that, sometimes glycoalkaloids and saponins are

responsible for bitterness present in some cultivars of brinjal (Aubert et al, 1989) instead of

CGA (Nagel et al, 1987).

The main characteristic feature of flavonoids is presence of flavone nucleus C15-(C6-

C3-C6). It is a heterocyclic ring system containing oxygen bonded pyrone and pyran ring

(Harborne, 1988) Flavonoids are generally pale yellowish in colour and sparingly soluble in

water (Maraisi et al, 2006). Flavonoids form O-glycosides with sugars and are present in food

products in this form (Merken and Beecher, 2000). The most common sugar residue is D-

glucose. Other sugar residues like L-arabinose, D-galactose, D-xylose, D-glucuronic acid and

L-rhamnose are also found. The C3 position is most preferable binding site for the sugar

residue than the C7 position (Macheix et al, 1990)

Flavonoids are widely distributed in leaves, fruits and barks of the plants and provide

the colour to the fruits and vegetables. For example, pink to red coloration of fruits and

vegetables are mainly because of anthocyanins. The other groups responsible for yellow or

ivory flavonoid pigments in plants include flavanones, flavones, aurones, flavonols,

isoflavanones biflavonyls and chalcones (Mazza and Miniati, 1994).

2.1.2 Pigments of brinjal

Anthocyanins are responsible for the orange, red, blue and purple colours of many

fruits and vegetables such as apples, berries, beets, brinjals and onions (Dai and Mumper

2010). Anthocyanins are naturally occurring pigments widely distributed in almost all plant

species. These are accumulated in all parts of fruit plants (cherries, raspberries, blackberries,

blueberries, currants, oranges, grapes) and vegetables (tomatoes, red corn, red onion, red

cabbage, red-skinned potatoes, purple sweet potatoes, eggplant, fennel).These are lavishly

found in fruits and flowers in great amounts (Vinson et al, 2012 ).

9

Nasunin, a major component of anthocyanin pigment of eggplant, was isolated from

the eggplant peels, and its antioxidant activity was evaluated (Igarashi et al, 1993; Okmen et

al, 2009. Matsuzoe et al (1999) examined that nasunin effectively scavenges free radicals and

various reactive oxygen species, such as hydroxyl, hydrogen peroxide, and superoxide. Noda

et al (2000) studied the inhibitory role of nasunin in Fenton reaction probably by chelating

ferrous ions. Oancea and Oprean (2011) found variation in anthocyanin content at inter-

species as well as at intra-species level in plants. Other factors like developmental stages,

environmental conditions, agronomic factors (methods of cultivation) and ultimately

processing of plant products have influential effect on amount of anthocyanin in plants.

Some studies showed that nasunin also acts as an angiogenesis (development of new

blood vessels) inhibitor along with antioxidant properties. Thus, this inhibitory role of

nasunin is implicated against several diseases including cancer in the first place. Nasunin is

present in very few foods and brinjal is one of the good natural sources that contain

comparatively higher nasunin. Gallo et al (2014) found significant amount of this

phytonutrient in the peel of brinjal fruit.

Anthocyanins are greatly soluble in water and polar solvents. Anthocyanins coexist in

four different structural forms in aqueous solutions. These forms are specifically varied in

colour at different pH values. Brouillard (1982) identified these forms viz., flavylium cation

(red-orange, predominantly at pH values below 2), quinonoidal base (blue, predominantly at

pH values between 8 and 10), carbinol pseudobase (colorless) and chalcone C (colorless at pH

between 3 and 6). Brinjal peel is a rich source of anthocyanin (delphinidin) (Wu et al, 2006).

Till now a number of anthocyanidins have been identified in different plants (Harborne,

1998). The structures of six (cyanidin, delphinidin, petunidin, peonidin, pelargonidin, and

malvidin) most common anthocyanidins have been given in figure 2.3. These are

ubiquitously spread and are of great importance in human diet (Jaganath and Crozier, 2010).

Recent studies suggest the role of anthocyanins in cancer prevention, the daily intake

of anthocyanins in the diet is suggested to be between 180 and 215 mg, which are higher as

compared to that of other flavonoids quercetin, apigenin and genistein, which is calculated to

be 20–25 mg/day (Wang and Stoner, 2008).

Different factors play an important role in the stability of anthocyanins. A strong

correlation between pH of the medium and stability of anthocyanins was reported. Horbowicz

et al (2008) has reported the colour variation in anthocyanin containing solution from deep

red or orange under low pH conditions due to positive charge present on eight conjugated

double bonds. Castañeda et al (2009) studied the relation of anthocyanins colour stability with

pH variation and found degradation of anthocyanin colour at alkaline pH (>8). The quinoidal

blue species prevailed at pH ranges from 2 to 4; and carbinol pseudobase and chalcone (both

colourless species) found at low acidic pH (5 to 6). Colour stability was found to be decreased

towards neutral pH but some anthocyanins can show a stable behaviour at local maxima

around 8-9 (Cabrita et al, 1999).

10

Fig. 2.3: Important anthocyanidins present in vagetables

Generally anthocyanidin glycosides are 3-monoglycosides and 3, 5-diglycosides.

Glucose is the most common sugar of anthocyanidine, apart from this, arabinose, rhamnose,

xylose, galactose, and rutinose can also occur (Horbowicz et al, 2008). Sugar moiety is

supposed to be activated by acylation by aromatic acids, namely hydroxyl-cinnamic acids

(caffeic, ferulic, p-coumaric or sinapic acids). The acyl moieties are normally linked to the

sugar at C-3 (Jing, 2006; Pereira et al, 2009). Sometimes acylation reaction involved aliphatic

acids like malic, succinic, malonic, acetic acids and oxalic acids (Wu and Prior, 2005).

Matsuzoe et al (1999) examined the profile of anthocyanins in several eggplant cultivars and

found that nasunin represents between 70% -90% of the total anthocyanins in the peel.

2.2 Enzymes

2.2.1 Phenylalanine ammonia lyase (PAL)

Phenylalanine ammonia lyase (PAL) (EC 4.3.1.24) is a key enzyme in the

biosynthesis of phenolics in plants. It catalyses the conversion of phenylalanine to trans-

cinnamate by non-oxidative deamination process (Koukol and Conn 1961). PAL directs the

carbon flow within the shikimate pathway. It transfers the carbon to branches of

phenylpropanoid metabolism (Bhattacharyya and Ward 1988). PAL is an important enzyme

in plants as it is considered to be involved in the first committed step for biosynthesis of

phenolic compounds (Cheng and Breen 1991). Studies reported that PAL activity is present

only in the coloured portions of fruit skin and concluded that the PAL activity was closely

related to formation of pigments (Aoki et al 1970). Jones (1984) reported that there is

11

concomitant increase in PAL activity and flavonoid compounds. Camm and Towers (1973)

reported that the level of PAL activity depends upon the genotypic and environmental

conditions of the plant. PAL is an important regulatory enzyme in the plant’s secondary

metabolism and is regulated by various biotic and abiotic factors such as nutrient levels, light,

fungal infection and wounding (Jones 1984). PAL is considered to be marker of

environmental stress in plant tissues. Dixon and Paiva (1995) found that PAL induces

biosynthesis of polypropanoid in response to biotic and abiotic stresses such as pathogen

attacks, UV radiation, injury and light. Kuhn et al (1984) found that fungal infection causes

increase in the synthesis of PAL which further increases synthesis of phenolic compounds.

Increased expression of PAL also enhances the accumulation of chlorogenic acid which is a

phenylpropanoid (Howles et al 1996), while some flavonoids do not get altered in plants

(Bate et al 1994, Shadle et al 2003).

Yoshioka et al (1996) studied PAL activity in tubers and found that it increases in

response to light and other biotic stresses. Weisshaar and Jenkins (1998) suggested that PAL

can be responsible for many core functions which include mechanical support, pigments

production such as anthocyanins and signalling with factors of nodulation. Sharma et al

(1999) found that exposure to UV-B led to increase in PAL activity in various crops.

Sanchez-Ballesta et al (2000) reported that there is increase in activity of enzyme during

exposure to low temperature. Kumar and Knowles (2003) found that after wounding or

infection of the tissue, the PAL activity rose to levels of 150 times than normal. PAL activity

and phenylpropanoids increased after wounding, during which a suberized wound periderm is

formed. Butelli et al (2008) reported that in transgenic plant Del-Ros1, the increase in PAL

transcript and enzymatic activity seemed to be important to obtain very high levels of

anthocyanins. Zhang et al (2013) suggested that due to close relation between phenolics and

disease resistance, PAL is regarded as one of the important pathogenesis-related proteins.

2.2.2 Tyrosine ammonia lyase (TAL)

Tyrosine ammonia lyase (TAL EC 4.3.1.23) is an enzyme required for deamination of

the amino acid L-Tyrosine to p-coumaric acid (Neish 1961) with the release of ammonia.

Tyrosine ammonia-lyase (TAL) is described as a member of the aromatic amino acid

ammonia-lyase family and is one of the major enzymes involved in phenol biosynthesis

pathway of plants whose activity is analogous to that of PAL. Koukal and Conn (1961)

partially purified TAL and suggested that it is very labile enzyme. The plant might lose TAL

activity as it ages. Neish (1961) found no TAL in 30- to 40-day old alfaalfa plants, although

young plants showed the enzymatic activity. Higuchi (1966) studied enzyme activity in

bamboo in stem elongation zone and concluded that older tissue has low activity. It is

predominately found in Gramineae (Young et al 1966). Shah and Mehta (1978) also reported

the same in dicotyledons. Morrison and Buxton (1993) proposed that products of PAL and

12

TAL are modified through phenylpropanoid metabolism, produce precursors of secondary

metabolites such as lignin, flavonoid pigments and phytoalexins (Tokusoglu et al, 2005).

Rosler et al (1997) proposed that in monocots, the activities of PAL and TAL reside on the

same polypeptides and both have similar catalytic efficiencies. It is found that TAL is

involved in the production of p-coumaric acid as the chromophore of the bacterial

photosensory yellow protein (Kort et al 1998). Kyndt (2002) found that TAL had 150 times

less catalytic efficiency for L-phenylalanine than that for L-tyrosine and suggested that TAL

was mainly involved in the production of p-hydroxycinnamic acid. Nishiyama et al (2010)

studied TAL in Arabidopsis thaliana and found that expression of TAL enhanced the

metabolic flux into the phenylpropanoid pathway which results into accumulation of

phenylpropanoid derivatives such as quercetin glycosides.

2.2.3 Anthocyanidin synthase (ANS)

ANS is an important enzyme of anthocyanin biosynthetic pathway. It catalyzes the

conversion of colourless leucoanthocyanidins to coloured anthocyanidins. Anthocyanidins are

extremely unstable and are converted to anthocyanins. Saito et al (1999) carried out the first

biochemical investigation of ANS by isolating recombinant form of enzyme from Perrilla

frutescens. Anthocyanidin synthase (ANS) belongs to the 2-oxoglutarate iron-dependent

oxygenases (Akhtar and Wright 1991; Feig and Lippard 1994) which use molecular oxygen

and 2-oxoglutarate as co-substrates, Fe2+ as a cofactor and ascorbate as the reducing agent

(Prescott and John 1996, Prescott et al 2002, Schofield and Zhang 2000). Pelletier et al

(1997) and Gollop et al (2001) reported that the ANS expression is strongly associated with

proanthocyanidin and anthocyanin accumulation. Saito et al (1999) suggested that ANS is an

extremely unstable enzyme; this might be because of oxidation. Turnbull et al (2000) found

that ANS requires high concentration of ascorbate and has less than 5% of full activity in the

absence of ascorbate. Nakajima et al (2001) postulated reaction mechanism of ANS that leads

to the formation of anthocyanidin (flavylium ion) under acidic conditions. In this reaction,

ANS catalysed the formation of pseudobase (2-flaven-3, 4-diol) from leucoanthocyanidin.

This is followed by isomerisation reaction that involved shifting of double bonds positioned at

C2-C3 to C3-C4 concomitant with a shift of the group from C-4 to C-2 and removal of OH-

from C-2. It was found that the synthesis of pure leucoanthocyanidin enantiomers is rather

difficult as the flavan-3,4-diols in aqueous solution were unstable. It was also found that

leucoanthocyanidin was a natural precursor of anthocyanidin as ANS is highly substrate

specific. Turnbull et al (2003) studied that ANS forms quercetin as a major product with

cyanidin being a minor product in vitro.

2.2.4 Peroxidase (POD)

Peroxidase is one of the important enzymes of antioxidant defence system. Different

studies showed this heme group containing enzyme to be present in almost all eukaryotic

13

organisms (Huystee and Cairns 1982). Peroxidase is an oxidoreductase which catalyzes a

reaction in which hydrogen peroxide acts as an acceptor and another compound acts as the

donor of hydrogen atoms (Rodrigo et al 1996). Onsa et al (2004) demonstrated oxidising

capacity of peroxidase extracted from plant tissues. It oxidizes a variety of phenolic

compounds, such as catechol, catechin, chlorogenic acid, guaiacol and pyrogallol.

Andrews et al (2000) observed increase in peroxidase activity along with increasing

fruit ripening in tomato. Different studies showed the important role of peroxidase in plant

growth regulation (MuÈsel et al, 1997; Schopfer et al, 2001). A significant correlation has

been found between lignin-like phenolics in the epidermis of the tomato fruit exocarp (Hunt

and Baker, 1980).

A more detailed comparative transcriptomic analysis of functional peroxidase gene

showed differences in plant genotypes with high phenolic content and poor phenolic content

(Andrews et al, 2002). Peroxidase gene has a role in colour and total polyphenols production

was studied by the Quantitative trait loci (QTLs) analysis which showed that peroxidase gene

has role in colouration as well as in production of total polyphenol. Mathé et al (2010)

investigated the segregating populations and suggested that there is good link of colour

expressing genes with the region where the peroxidase gene is present. It is found that

peroxidases are involved in different physiological processes of plant such as suberization,

lignification, networking of cell wall proteins, auxin catabolism, salinity tolerance and

defense against pathogen attack (Valério et al, 2004; Lüthje et al, 2011). Peroxidases are

related to lignin biosynthesis as dimers and these are major products in the initial steps of

ferulic acid polymerization (Ward et al, 2001).

2.2.5 Polyphenol oxidase (PPO)

PPO belongs to copper containing enzyme family. It is broadly distributed in bacteria,

plants and mammals. Polyphenol oxidase catalyzes the reaction involving oxidation of

phenolics to quinones. This produces brown pigments in plant tissues (Mayer, 1987)

Fig. 2.4: Hydroxylation and oxidation reactions catalysed by polyphenol oxidase

PPO activity is associated with the formation of plant pigments, oxygen scavenging

activity (Trebst and Depka, 1995) and defence system against plant pathogens (Mohammadi

and Kazmi, 2002) and insects (Constabel et al 2000). Plant PPOs have wide-range of

14

substrate specificities and thus are able to oxidize a variety of mono, di or polyphenols. (Fig.

2.5) Some of the PPO substrates occur naturally in fruits and vegetables (Podsedek et al

2000). In brinjal, the PPO and phenolics were reported to be present in chloroplasts and

vacuoles respectively. PPO is released by the physical damage to cellular structure (Plazas et

al, 2013). The enzymatic browning is caused due to the oxidation of polyphenols to

corresponding quinones by PPO (Jaime et al, 2007). Further, these quinones are polymerized

with other quinones or phenolics leading to production of brown pigments (Murata et al,

1995)

Fig. 2.5: Structure of some natural substrates of PPO

PPO assay showed that catechol, 4-methylcatechol, catechin, DL-3,4-

dihydroxyphenylalanine (DL-DOPA) and chlorogenic acid can be the substrates for this

enzyme. Ben-Shalom et al (1977) reported a significantly higher PPO activity in fruits than in

leaves. There is negative correlation between the PPO activity and polyphenols found in the

strawberry as studied by Sharma and Singh (2010).

2.3 Extraction of phytochemicals

Plants contain a wide range of metabolites, although metabolites vary from species to

species. All metabolites are categorized in different groups on the basis of their derivatives

such as amino acids, fatty acids, organic acids and carbohydrates (Fiehn et al, 2000).

Moreover metabolites are highly variable because of vast variation and flexibility in their

physicochemical properties. Therefore suitable extraction protocols need to be chosen, as the

extraction conditions differ widely for different types of compounds.

For homogenization, various techniques like fine grinding in liquid nitrogen with a

mortar and pestle, milling in sophisticated vibration mills with cooled holders, homogenization

15

with electric drill connected to a metal pestle (Edlund et al, 1995) and ultra turrax devices (Orth

et al, 1999) are available. Common way to extract metabolites is to shake the homogenized

plant tissue at low/high temperatures in organic solvents, or mixtures of solvents (Fiehn, 2002).

Solvents can be used for extraction according to their polarity and type of metabolite needed to

be extracted as shown in table 2.1.

Table 2.1: Solubility of different compounds in specific type of solvent (Roger, 1999)

S.No. Solvent Type Solvent Name Material extracted/soluble

1 Polar Water, dichloromethane Tannin, carbohydrate, protein, enzyme

2 Semi polar Acetone, alcohol Glycosides, flavonoids

3 Slightly non polar Chloroform Alkaloids

4 Moderate non polar Benzene, toluene Steroids

5 Strongly non polar Cyclohexane, Petroleum ether

Fatty acids, Lipids, oils

Some alternative extraction techniques include sub-critical water extraction (Ozel et

al, 2003), pressurized liquid extraction (Rostagno et al, 2004), microwave-assisted extraction

(Shu et al, 2003) and supercritical fluid extraction (Roger, 1999). Different extraction

techniques, such as Soxhlet, microwave assisted extraction and supercritical fluid extractions

(Lopez-Sebastian et al, 1998) have been used to isolate different oil based compounds from

the plants.

Some analytical methods for extraction of phenolic compounds from plants are

available (Callemien et al, 2004). Extraction is the first basic and most critical step for the

analysis of phenolic compounds. Thus, extraction method depends on the nature of material to

be evaluated, the class of phenolic compound needed to be investigated and the analytical

procedure to be used (Lee and Widmer, 1996; Chemat et al, 2008). The extraction starts with

fine crushing of the sample in order to increase the surface area of the material which allows

for more effective contact of the extraction solvent with the sample (Makkar, 1999).

Crushing, milling, macerating and grinding help in the mixing of sample. Most of the

phenolic compounds occur as glycosides or esters in nature. So to remove the bound phenolic,

the sample preparation includes alkaline, acid and enzymatic hydrolysis. Makkar (1999)

suggested the exclusion of hydrolysis step in case of phenolic derivatives. Multistep

procedures have been developed to study such compounds (Becker et al, 2004).

2.4 In silico Virtual Screening

Nowadays computational biology helps in virtual screening of phytochemicals and

their effectiveness against the signalling processes involved in medical conditions (Lavecchia

and Di Giovanni, 2013). Phytochemicals react with proteins involved in various biological

reactions (Kopeć et al 2013). Proteins are the biological macromolecules which are

16

responsible for various biochemical reactions with their structure, catalyzing reactions and

signalling. Proteins undergo different biochemical reactions to adopt different free-energy

conformations and bringing it to the lowest level (Andricopoulo et al 2009). Various

molecular forces such as Van der Waals, electrostatics, and hydrogen-bonding energies are

responsible for the conformation (Srivastava, 2008).

Molecular docking is a fundamental tool in structural analysis as well as in computer-

assisted drug designing. It is an advanced approach to study the interaction between a protein

and a small molecule at the atomic level. McConkey et al (2002) characterized the behaviour

of molecules present at binding sites of a particular target protein using molecular docking. It

further hepls in elucidation of various fundamental biochemical processes. Molecular docking

is a standard tool in computational biology in combination with virtual screening. Virtual

screening can be of two types viz. structure based or ligand based, as shown in fig. 2.6. After

docking the compound, scoring is done using mathematical models by estimating the ligand-

receptor interaction and binding affinity of the complex (Lyne, 2002). Different protocols for

docking are tested before determining the correct set of parameters which can be used for

docking (Haga et al, 2016, Zhu et al 2013).

Fig. 2.6: Types of virtual screening and steps in molecular docking (Lipinski et al 2001),

(Huang, 2009)

Classification of Docking Programs

The docking process involves the prediction of the binding geometry and interactions

between ligand and receptor. It helps in estimating the binding affinity of the ligand receptor

complex which is also known as scoring. Flexible docking programs can be divided into two

classes based on the conformational searching algorithms applied, including system methods

like incremental construction, conformational search and random methods such as Monte

Carlo genetic algorithms (Kontoyianni et al, 2004).

17

Table 2.2: Some conformational search algorithms

S. No. Systematic Search Random Search

1 e HiTS (Zsoldos et al 2007) Auto Dock (Morris et al 1996)

2 FRED (McGann 2012) Gold (Jones et al 1997)

3 Surflex Dock (Jain, 2003) EA Dock (Grosdidier et al 2007)

4 DOCK (Ewing et al 2001) Ligand Fit (Venkatachalam et al 2003)

5 GLIDE (Friesner et al 2004 ) C Docker (Wu et al 2003)

6 EUDOC (Pang et al 2001) Glam Dock (Tietze and Apostolakis 2007)

7 Flex X (Rarey et al 1996) Mol Dock (Thomsen & Christensen, 2006)

Widely used docking programs, such as DOCK 6 and Flex X (Kramer et al, 1999)

belong to the first class; while GOLD (Verdonk et al, 2003) and AutoDock (Goodsell et al,

1996) belong to the second one. GLIDE (Friesner et al, 2004) program uses a combination of

conformational searching and random algorithms.

2.5 Role of phytochemicals in disease prevention

Phytochemicals show potential for reducing the risk of cancer and cardiovascular

disease in humans (Gibson et al, 1998). In cancer, phytochemicals can detoxify substances

that cause cancer. According to Meagher and Thomson (1999), such compounds prevent the

formation of new capillaries, which is the prime source for tumor growth and metastasis

(Miller, 2002). These compounds can neutralize free radicals, inhibit responsible enzymes

that activate carcinogens or they can activate enzymes that detoxify carcinogens (Gutiérrez et

al, 2003).

Phenolic compounds are considered as ubiquitous group of plant metabolites. These

are present in substantial quantity in vegetables and fruits and essential part of the human

food (Dai and Mumper, 2010). The inverse relationship between fruit and vegetable intake

and the risk of oxidative stress associated diseases such as cardiovascular diseases, cancer or

osteoporosis has been partially ascribed to phenolics (Scalbert et al 2005). Many studies have

established a significant relationship between consumption of polyphenols rich fruits and

vegetables and reduced risk of various chronic diseases like coronary heart diseases (Mazza,

2007), neurodegenerative diseases, and certain forms of cancer. Phenolics have been

considered powerful antioxidants in vitro and proved to be more potent antioxidants than

Vitamin C and E and carotenoids (Rice-Evans et al, 1996; Bouba et al, 2012). Phenolic

compounds are primarily considered as antioxidants. Nanda (2014) revealed that these

compounds play significant role in variety of biological processes specifically in modulation

of carcinogenesis. Researchers have crucially been considering phytochemicals for their

contribution to chemoprevention and cytotoxic activity (Mathai, 2000). It has been studied

18

that the antioxidant properties of phenolic compounds are effective by the different

mechanisms. These are: scavenging reactive oxygen species (ROS), suppressing ROS

formation by inhibiting some enzymes or by chelating metals which are involved in free

radical production and by protecting antioxidant defence (Cotelle 2001).

Flavonoids are one of the foremost factors that determine the antioxidant activity

(Bors and Michel, 2002; Chen et al, 2009a). Being strong electron donors, they can stabilize

the radical form and participates in electron delocalization. Various in vitro and in vivo

systems have been employed to determine the anti-carcinogenic and anticancer potential of

these natural phenolic compounds or extracts (Dai and Mumper, 2010). These compounds can

stop or slow down the growth as well as development of precancerous cells into malignant

cells. Chemopreventive agents can be of two main types: blocking agents and suppressing

agents. Blocking agents prevent such carcinogens from reaching the target sites, by

undergoing activation of metabolism or by subsequent interaction with cellular

macromolecules. On the other hand suppressing agents inhibit the malignant cell formation in

the promotion or the progression stage as shown in Fig 2.7 (Surh, 2003).

Fig. 2.7: Phytochemicals that block or suppress multistage carcinogenesis (Surh, 2003)

Plant phenolics are potent inhibitors of a number of growth factors and such

compounds can cause modulatory effects in cells through selective actions in various

signalling processes. These inhibit EGFR (Epidermal growth factor receptor) action

(Schroeter et al, 2001) and reduce the invasive potential of cancer cells (Korutla et al 1995,

19

Korutla & Kumar, 1994). These compounds act at kinases (protein kinase and lipid kinase

signalling cascades: phosphoinositide 3 –kinase, Akt/PKB (Kong et al, 2000), tyrosine

kinases (Van der et al, 1994), protein kinase C (PKC) and mitogen-activated protein kinases

(Spencer et al, 2003).

Zhang et al (2008) have done a detailed comparative analysis of polyphenols

(quercetin, anthocyanins, kaempferol, esters of coumaric acid and ellagic acid) extracted from

strawberry against different kinds of cancer cell lines viz. human oral (KB, CAL-27), breast

(MCF-7), colon (HT-29, HCT-116), and prostate (LNCaP, DU-145). They observed dose-

dependent manner with different sensitivity between cell lines. Ross et al (2007) assessed that

ellagitannin extract of raspberry has strong antiproliferative activity against human cervical

cancer (Hela) cells. However, in lingon berry, procyanidins are predominant antiproliferative

agents. Furthermore, by comparing the phytochemical diversity of the berry (raspberry,

strawberry, arctic bramble, and cloudberry) extracts with their antiproliferative effectiveness,

McDougall et al (2008) also suggested that the ellagitannins significantly inhibit the growth

of cancer cell lines. Sarkar and Li (2002) observed the potential role of Genistein, isoflavone

extract from Soybean against various cancer cell lines including leukemia, lymphoma,

prostate, breast, lung etc. The growth of HL-60 leukemia cells is significantly inhibited by

flavonoid extract from citrus (Manthey et al 2001). McCann et al (2007) examined the effect

of phenolic compounds extracted from apple on crucial stages of colorectal carcinogenesis.

Different established cell models named as genotoxicity (HT-29), invasion and metastatic

potential (HT-115), and colonic barrier function (CaCo-2) have been utilized. Many studies

indicate the cytotoxic effects of a number of polyphenols also. During last decades, a lot of

research studies have been conducted to discover the potential role of compounds such as

flavonoids, flavones (apigenin, baicalein, luteolin and rutin) and flavanones (hesperidin and

naringin) in treatment of different types of chronic diseases including cancer (Miyahara et al

2000; Gupta et al 2001; Kanno et al 2005). Kuntz et al (1999) observed the cytotoxic effect

of polyphenols on colon cancer cell lines. Zhang et al (2000) assessed the effect of

polyphenols on liver cancer cell lines.

20

CHAPTER III

MATERIALS AND METHODS

The present investigation, “Studies on antioxidant and anticancer properties of brinjal

(Solanum melongena L.) genotypes” was carried out at the Vegetable Research Farm and

Biochemistry Laboratory of Department of Vegetable Science, Punjab Agricultural

University, Ludhiana & molecular diagnostic and vaccinology laboratory of School of

Animal Biotechnology, Guru Angad Dev Veterinary and Animal Sciences University,

Ludhiana. The materials and methods employed in the present study are described under the

following headings:

3.1 Raising of crop and procurement of Samples

3.2 Collection of Samples

3.3 Extraction and estimation of Phenolic Compounds

3.3.1 Estimation of Total Phenols

3.3.2 Estimation of Ortho-dihydroxy phenols

3.3.3 Estimation of Flavonols

3.3.4 Estimation of Quinones

3.3.5 Estimation of Chlorogenic acid

3.3.6 Estimation of Tannins

3.4 Extraction and estimation of pigments

3.4.1 Optimization of anthocyanin extraction using Response Surface Methodology

3.4.2 Extraction and estimation of anthocyanins

3.4.3 Extraction and estimation of leuco-anthocyanins

3.5 Extraction and estimation of oxalic acid (plant acid)

3.6 Extraction and estimation of solanin (Alkaloid)

3.7 Extraction and assay of phenylalanine ammonia lyase

3.8 Extraction and assay of tyrosine ammonia lyase

3.9 Extraction and assay of anthocyanidin synthase

3.10 Extraction and assay of peroxidase

3.11 Extraction and assay of polyphenol oxidase

3.12 Estimation of protein from enzyme extract

3.13 Determination of antioxidant activity

3.14 Preparation of plant extracts

3.15 In silico molecular docking for bioactive compounds against human cancer cell lines

3.16 Evaluation of cytotoxic and apoptotic effects

3.17 Statistical analysis

21

3.1 Raising of crop and procurement of Samples

Crop of brinjal was raised at the Vegetable Research Farm, Department of Vegetable

Science, Punjab Agricultural University, Ludhiana.

3.2 Collection of Samples

The fruit and leaf samples were collected at 7th day after anthesis (DAA), the

subsequent second and third samples were taken at an interval of 7 days (i.e 14 and 21 DAA).

For enzyme assay, various extracts were prepared and the activity of phenylalanine ammonia

lyase, tyrosine ammonia lyase and anthocyanidin synthase was determined in these extracts.

The brinjal fruit samples were harvested at maturity and analyzed for biochemical parameters

such as anthocyanins, total phenols, ortho-dihydroxy phenols and flavonols.

3.3 Extraction and estimation of Phenolic Compounds

Extraction

500 mg dried fruit samples were weighed and refluxed with 80% methanol for 2 h.

The refluxed material was filtered and volume was made 25 ml by washing with 80%

methanol. The extract thus prepared was used for the estimation of phenolic content viz. total

phenols, ortho-dihydroxyphenols and flavonols.

3.3.1 Estimation of total phenols (Swain and Hillis, 1959)

A) Reagents

i) Folin-Ciocalteu reagent: 2N Folin-ciocalteu reagent diluted 1:1 (v/v) with distilled water

ii) Saturated solution of Na2CO3

B) Procedure

Methanolic extract (5 ml) was evaporated to dryness and the residue was dissolved in

6.5 ml of distilled water. To this 0.5 ml of Folin-Ciocalteu’s reagent was added and shaken

thoroughly. After 3 min, 1 ml of saturated solution of Na2CO3 was added and volume was

made 25 ml with distilled water. After keeping samples in the dark for 1 h, the absorbance of

blue colour was read in a spectrophotometer at 760 nm against reagent blank. The

concentration of total phenols was determined from standard curve prepared by using

catechol (20-100µg/ml).

3.3.2 Estimation of ortho-dihydroxy phenols (Nair and Vaidyanathan, 1964)

A) Reagents

i) 10% Trichloroacetic acid (TCA)

ii) 10% Sodium tungstate

iii) 0.5 N Hydrochloric acid (HCl)

iv) 0.5 % Sodium nitrite

v) 0.5 N Sodium hydroxide

B) Procedure

The methanolic extract (5.0 ml) was evaporated to dryness and residue left behind

22

was dissolved in 1.0 ml of distilled water. To this 0.5 ml of 10% TCA, 1.0 ml of 10% sodium

tungstate, 0.5 ml of 0.5 N HCl, and 1.0 ml of freshly prepared 0.5% sodium nitrite were

added. A yellow colour developed. After 5 min 2.0 ml of 0.5 N sodium hydroxide was added.

The light cherry colour developed, whose absorbance was read after 15 min at 540 nm against

reagent blank. The concentration of ortho-dihyroxyphenols was determined from standard

curve prepared by using catechol (10-100µg/ml).

3.3.3 Estimation of Flavonols (Balabaa et al 1974)

A) Reagents

0.01 M methanolic solution of aluminium chloride (AlCl3).

B) Procedure

5.0 ml of methanolic extract was evaporated to dryness. The residue left was

dissolved in 10 ml of 0.1 M methanolic solution of aluminium chloride. Yellow colour

developed, which was read at 420 nm against 0.1 M methanolic solution of AlCl3 as blank.

The concentration of flavonols was determined from standard curve prepared by using rutin

(50-250 µg/ml).

3.3.4 Estimation of Quinones (Mahadevan and Sridhar, 1986)

A) Reagents

i. 0.1M sodium phosphate buffer, pH 6.6

ii. 0.5N Trichloroacetic acid in 60% ethanol

iii. Catechol solution (5×10-3 M)

B) Procedure

1 g of sample was homogenized with chilled phosphate buffer in a pestle mortar.

Supernatant was collected by centrifugation at 2000g for 30 min at 4 °C. Three ml buffer was

taken in a test tube. Then 3 ml catechol solution and 1.5 ml supernatant were added to test

tube. Contents were incubated in water bath at 45 °C for 15 min. 4 ml of TCA reagent was

added to the sample. Mixture was filtered to remove any precipitates. Absorbance was

recorded at 400 nm against reagent blank. The concentration of quinones was determined

from standard curve prepared by using caffeic acid (20-100µg/ml).

3.3.5 Estimation of Chlorogenic acid (Sadasivam and Manickam (1992)

A) Reagents

i. Titanium reagent : 20% TiCl4 in conc.HCl

ii. Standard : 25-200 µg/ml chlorogenic acid in acetone

iii. Acetone

iv. 80% Ethanol

v. 2.5N HCl

B) Procedure

2g of sample was refluxed twice in 5 ml of 80% ethanol (adjusted to pH 4.0 with 2.5

23

N HCl) for 3 min. The precipitate was discarded and 250 ml of extract was collected. 0.5ml

samples were removed and dried in oven at 50°C for 2h. The dried extract was dissolved in

4.75 ml of acetone and then 0.25ml of TiCl4 was added. The cyan colour developed, whose

absorbance was read at 450 nm against the reagent blank. The concentration of chlorogenic

acid was determined from standard curve prepared by using chlorogenic acid (10-100µg/ml).

3.3.6 Estimation of Tannins (Sadasivam and Manickam (1992)

A) Reagents

i. 35% Sodium carbonate solution

ii. Folin-ciocalteu’s reagent

B) Procedure

500mg of sample was taken into a 250ml conical flask and 75ml water was added.

Flask was heated to boil the components for 30 min. After cooling under tap water,

centrifugation at 2000 rpm for 20 min was done. Supernatant was collected. 1ml of this

supernatant extract was taken. After addition of 5ml of Folin-Ciocalteu’s reagent, 10 ml of

sodium carbonate solution was added and final volume was made 100ml using distilled water.

After shaking the absorbance was read at 700nm after 30 min and compared against the

standard tannic acid (0-100µg).

3.4 Extraction and estimation of pigments

3.4.1 Optimization of anthocyanin extraction using Response Surface Methodology (Box

and Behnken (1960)

Experimental design: To carry out the study on optimization of extraction conditions for

anthocyanin, an experiment was planned by chossing the family of variables as suggested by

Box and Behnken (1960). Using Box- Behnken design for one factor, 29 runs were planned

for experiment. The design was taken from response surface analysis and it fulfilled most of

the requirements needed for optimization of the anthocyanin extraction conditions.

Optimization of process parameters: Response surface methodology was applied to study

the experimental results using a commercial statistical package, Design-Expert version 8.0.1.

The same software was used for the generation of response surface plots, superimposition of

3-D plots and optimization of process variables. Plots were generated to study the interaction

between more than one variable at a time. Different interactions for any two independent

variables were studied while holding the value of other variable as constant (as the central

value). Such interactions could give accurate geometrical analysis and representation to

provide useful information about the behaviour of the system within the experimental

design (Cox and Cochran 1964, Montgomery 2004). The optimization of the anthocyanin

extraction process aimed at finding the levels of independent variables viz. extraction

temperature, extraction time, pH and solid to solvent ratio, which could give maximum

possible yield.

24

3.4.2 Extraction and estimation of Anthocyanins (Rabino et al 1977)

Fruit samples were homogenized and anthocyanins extracted with 10 ml of 1% HCl

(w/v) in methanol under optimized extraction conditions. The absorbance of extracts which

were clarified by filtration was measured at 530 nm and 657 nm with a LABINDIA 3000+

UV/VIS Spectrophotometer using 1% HCl as blank. The anthocyanin content of the extracts

was presented as A530-0.33A657. This formula is used to correct for the contribution of

chlorophyll and its degradation products in acid solution to the absorbance of the extracts at

530 nm.

3.4.3 Extraction and estimation of Leuco-anthocyanins (Swain and Hillis, 1959)

A) Regents

i. Methanol or ethanol

ii. Leuco-anthocyanins reagent: Dilute 25 ml of 36% HCl to 500ml with n-butanol

B) Procedure

5g of tissue was ground in methanol. The supernatant was collected after

centrifugation. Methanolic extract (1.0 ml) was taken into a test tube and evaporated to reduce

the volume of the extract to 0.5ml on a hot water bath. To this, 0.5 ml of distilled water and

10 ml of Leuco-anthocyanins reagent were added and mixed thoroughly. Then the test tubes

were placed in water bath at 97°C for 3 min without covering the tubes. Afterwards, the tubes

were covered with glass stoppers and heated continuously. After 40 min, the tubes were

placed under running tap water. The optical density of sample was measured at 550 nm.

3.5 Extraction and estimation of Oxalic acid (Mahadevan, 1969)

A) Reagents

i. 4N H2SO4

ii. 1N NaOH

iii. Diethyl ether

iv. Calcium chloride-acetate buffer: 25 g of anhydrous calcium chloride was dissolved in

500ml of 50% acetic acid. Separately 330g of sodium acetate were dissolved in water

and volume was made upto 550 ml. The two solutions were mixed and pH was

adjusted to 4.5 by using pH meter.

v. 5% acetic acid saturated with calcium oxalate

vi. 4N H2SO4

vii. Standard: 0.02N potassium permanganate solution

B) Procedure

Sample extraction: The tissue was dried to a constant weight in a hot-air oven at 80°C. Then

the tissue was ground to fine powder in a mortar and pestle and dried again and weighed

500mg sample from it. The sample was mixed with 1.5 ml of 4N H2SO4 and it was placed in

beaker over hot plate apparatus and extraction was done with pure diethyl ether for 48h at 50

25

°C. Five ml of 1N NaOH and 7ml of water were added to the extract and mixed thoroughly.

The ether layer was evaporated and the water phase was transferred to centrifuge tube. To

this, 4 ml of calcium-chloride-acetate buffer was added and allowed to stand overnight.

Estimation: The extract was centrifuged for 10min and the supernatant was discarded. The

pellet was washed with 5ml of 5% acetic acid saturated with calcium oxalate and centrifuged.

The residue was dissolved in 5ml of 4N H2SO4 and heated at 80-90°C on a water bath. Then

the filtrate was titrated against standard 0.02N potassium permanganate solution. The amount

of oxalic acid present in the sample was calculated using the relationship:

1ml of 0.02N Potassium permanganate ≡ 1.2653 mg of oxalic acid

3.6 Extraction and estimation of Solanin (Harborne, 1973)

A) Reagents

i. Methanol

ii. Chloroform

iii. 0.8% Sodium Sulphate

iv. 5M Ammonium hydroxide

v. Phosphoric acid

vi. Formaldehyde solution

B) Procedure

5g sample was weighed and crushed with a mixture of methanol:chloroform (2:1).

The suspension was filtered and the residues were rinsed twice with a 25 ml portion of the

same solvent mixture. Then the filtrate was transferred to a 500 ml separatory funnel, 60 ml

of 0.8% Na2SO4 was added, and the mixture was shaken vigorously. The layers were

separated and the lower layer containing chloroform was discarded. The methanolic layer was

used for reextraction with 10 ml of 0.8% Na2SO4, and then lower layer was discarded again.

Then solution was collected into a beaker, and the funnel rinsed with 1% acetic acid. This

solution was concentrated on a steam bath to make volume near to 10 ml. Five ml of the

sample solution was taken into test tube and the pH was adjusted to 9.4 with 5M ammonium

hydroxide. After that the tube was heated in a water bath at 80°C. The precipitates were

washed with a small amount of distilled water. To the precipitates, 5 ml of H3PO4 was added,

followed immediately by 5 drops of formaldehyde solution. Then the absorbance was studied

at 600 nm to estimate the amount of solanin by comparing against the standard solanin (5-

100µg).

3.7 Extraction and assay of Phenylalanine ammonia lyase (PAL) (Hadwiger and

Schwochau, 1971)

Principle

PAL catalyzes the deamination of L-phenylalanine to yield trans-cinnamic acid which

is the first step in the biosynthesis of phenylpropanoids, which are further modified into a

wide variety of phenolic compounds.

26

Extraction

a) Reagents

i) Extraction Buffer: 0.5M Potassium phosphate buffer (pH 8.0)

ii) 0.5g Polyvinylpyrrolidone

b) Extraction

Five hundred milligram of fresh sample was homogenized in a chilled mortar with 5

ml 0.5M potassium phosphate buffer (pH 8.0) in the presence of polyvinylpyrrolidone (PVP).

This and subsequent extractive operations were carried out at 4˚C. The homogenate was

centrifuged for 10 min at 4˚C. The supernatant was used for PAL assay.

A) Assay

a) Reagents

i) Working buffer (0.05 M Boric acid-borax buffer (pH 9.0): 50 ml of 0.05M boric acid

and 59 ml of 0.05M borax crystals solution were mixed and diluted to 200 ml.

ii) 10mM L-phenylalanine: 0.17g of L-phenylalanine was dissolved in 100 ml of

distilled water.

iii) 5N HCl

iv) 0.05M NaOH

v) Diethyl ether

b) Procedure

The assay mixture contained 0.1 ml enzyme extract, 0.5 ml 10mM L-phenylalanine

and 3.0 ml 0.05 M Boric acid-borax buffer (pH 9.0). The reaction mixture was incubated for 1

h at 37˚C. The reaction was then stopped by adding 0.1 ml of 5N HCl. The acidified mixture

was extracted twice with 7.5 ml diethyl ether. The ether extracts were pooled and evaporated

to dryness under a stream of air. The residue was dissolved in 5ml 0.05M NaOH. The

cinnamic acid formed at the end of the reaction was estimated by measuring the absorbance at

290 nm in a LABINDIA 3000+ UV/VIS Spectrophotometer. Standard curve was prepared by

using trans-cinnamic acid in the concentration range 5-35µg/ml.

3.8 Tyrosine ammonia lyase (TAL) (Burell and Rees 1974)

Tyrosine ammonia lyase is an enzyme required for deamination of amino acid L-

tyrosine to p-coumaric acid. It is the one of the major enzymes involved in phenol

biosynthesis pathway of plants.

A) Extraction

a) Reagents

i) Extraction Buffer: 0.5M Potassium phosphate buffer (pH 8.0)

ii)