ISOLATION AND CHARACTERIZATION OF

PHYTONUTRIENTS AND PHENOLIC ANTIOXIDANTS IN

FRUITS OF SELECTED WILD PLANTS FROM SOON VALLEY

A thesis submitted in partial fulfillment for the award of degree of

DOCTOR OF PHILOSOPHY

IN

CHEMISTRY

BY

Naveed Ahmad

2017

Department of Chemistry

University of Sargodha

Sargodha, Pakistan

i

This thesis is dedicated to my loving

parents and beloved wife for their

endless love, kind prayers, support and

encouragement

ii

DECLARATION

It is certified that the research work included in this thesis entitled “Isolation and

Characterization of Phytonutrients and Phenolic Antioxidants in Fruits of Selected Wild

Plants from Soon Valley” is original and nothing has been stolen /copied/ plagiarized from any

other source.

___________

(Supervisor)

Dr. Farooq Anwar

Associate Professor,

Department of Chemistry,

University of Sargodha, Sargodha

___________

(Co-Supervisor)

Dr. Sohail Hameed

NIBGE, Faisalabad

iii

DECLARATION

It is certified that the research work included in this thesis entitled “Isolation and

Characterization of Phytonutrients and Phenolic Antioxidants in Fruits of Selected Wild

Plants from Soon Valley” by Naveed Ahmad is original and nothing has been

stolen/copied/plagiarized from any source.

___________

Naveed Ahmad

Ph.D Scholar

Department of Chemistry,

University of Sargodha, Sargodha

iv

Certificate

It is certified that the research work contained in the thesis entitled “Isolation and

Characterization of Phytonutrients and Phenolic Antioxidants in Fruits of Selected Wild

Plants from Soon Valley” by Mr. Naveed Ahmad is original and has been completed according

to the requirements of Higher Education Commission (HEC) & University of Sargodha, Sargodha,

Pakistan

Signature of Student Signature of Supervisor with stamp

Name:Naveed Ahmad Name:Dr. Farooq Anwar

Signature of Co-Supervisor with stamp

Name:Dr. Sohail Hameed

v

APPROVAL CERTIFICATE

It is certified that research work reported in this thesis entitled “Isolation and Characterization

of Phytonutrients and Phenolic Antioxidants in Fruits of Selected Wild Plants from Soon

Valley” submitted by Naveed Ahmad is accepted in its present form by the Department of

Chemistry, University of Sargodha, Sargodha, Pakistan, as satisfying the partial requirement for

the award of degree of Doctor of Philosophy in Chemistry.

Dr. Farooq Anwar Dr. Sohail Hameed Supervisor Co-Supervisor

Associate Professor NIBGE, Faisalabad

Department of Chemistry

University of Sargodha

Sargodha

Dr. Muhammad Sher Chairman,

Department of Chemistry

University of Sargodha

Sargodha

vi

ACKNOWLEDGEMENTS

In the name of Allah, the most merciful, the beneficent

I am indebted to have an opportunity to admire countless blessings of ALLAH-The Almighty because

the words are bound, knowledge is limited and time of life is too short to express His dignity. It is the one of His

infinite benedictions that He bestowed upon me with the potential and ability to complete the present research

work. Then the trembling lips and wet eyes praise the greatest man of the Universe, the last Messenger of

ALLLAH, HAZRAT MUHAMMAD (PBUH), whom ALLAH has sent as mercy for worlds, the illuminating

torch, the blessing for the literate, illiterate, rich, poor, powerful, weaker, able and disable. Whose life and sayings

are ultimate source of guidance and way of "NIJAT" for the mankind, who enlighten our conscious with the

essence of faith on ALLAH, merging all his kindness and mercy upon us.

I deem it my utmost pleasure to avail an opportunity to express my heartiest gratitude and deep sense

of obligation to my honorable supervisor, Dr. Farooq Anwar, Associate Professor, Department of Chemistry,

University of Sargodha, Sargodha for his able guidance, kind behavior, generous transfer of knowledge, moral

support, constructive criticism and enlightened supervision during the whole study and research period and

completion of this dissertation.

With a proud sense of gratitude, I acknowledge that this dissertation has found its way to significant

completion due to the kind supervision, inspiration, useful suggestions and sympathetic attitude of my Co-

Supervisor, Dr. Sohail Hameed, Deputy Chief Scientist, National Institute for Biotechnology and Genetic

Engineering (NIBGE), Faisalabad as well.

My sincerest thanks are to the Chairman, Dr. Muhammad Sher, Department of Chemistry, University

of Sargodha, Sargodha for his supportive attitude and providing facilities to conduct this research work. I would

like to pay special thanks to Mr. Imran and Mr. Asif(clerical staff)as well as other teaching and non-teaching

staff of the Department of Chemistry, UoS for their kind support and co-operation.

It is an immense pleasure to express my profound gratitude to Dr. Yuegang Zuo, Professor, Department

of Chemistry and Biochemistry, University of Massachusetts, Darmouth, USA for his guidance and providing

research facilities to carry out a part of my research work in his lab during my stay in USA under IRSIP

fellowship. I would like to express my sincere thanks to Higher Education Commission (HEC), Islamabad,

Pakistan for awarding me funds under IRSIP fellowship towards carry out a part of my PhD research at

University of Massachusetts, Dartmouth, USA.

I am highly obliged and have feelings of thanks to Dr Shahid Mansoor (S.I), Director NIBGE, Dr

Qaiser M Khan (DCS/Ex-Head Soil and Environment Biotechnology Division), Dr. Sajjad Mirza (DCS/ Head

Soil and Environment Biotechnology Division) and my group fellows Dr. Fathia Mubeen (PS), Dr. Muhammad

Shahid (Assistant Professor, GCUF), Dr. Tahir Naqqash, Dr. Muhammad Kashif Hanif, Ms. Afshan Majeed

(PhD Scholar), Mr. Tariq M. Shah, Mr. Zahid Iqbal Sajid, Mr. Asghar Ali and Muhammad Ahmad , Mr.

Muhammad Sarwar, Mr.Imran-ul-Haq, Mr. Zakir Hussain for their cooperation and help. I am also very

thankful to my class fellow, Mr. Ali Abbas (PhD Scholar) for his co-operation. I feel pleasure to greatly

acknowledge my loving parents (Nazir Ahmad and Habiban Bibi), Wife (Rozina Naveed) and Kids (Muhammad

vii

Sarosh Naveed and Muhmmad Hasnain Naveed) who always prayed for me and indeed it is due to their kind

support and endless prayers that I have been able to achieve this high goal. I would also like to extend my thanks

to my brothers (M. Mustafa, M. Murtaza and M Nadeeem) and sister (Mrs. Shugufta Hameed) for their

encouragement and support in this regard.

A cordial thank to all who directly or indirectly helped me during my lab. work. May Allah bless all

above mentioned personalities with a long life, happiness and peace (Ameen)!

Naveed Ahmad

viii

Abbreviations

CCE concentrated crude extract mg milligram

PRF phenolic rich fraction min minute

DW dry weight µ micro

mL milliliter λ wavelength

DNA deoxyribonucleic acid m milli

mmol millimol IC inhibition concentration

e.g. exempli gratia m.p. melting point

et al. et alii (and others) MS mass spectrometry

etc. et cetera (and other things) ESI electrospray ionization

pH pondus (power) of hydrogen DPPH 2,2-diphenyl-1-picrylhydrazy

SD standard deviation PAC phenolic antioxidant component

g gram h hour

ppm parts per million % percent

Hz hertz °C degree Celsius

mm millimeter p probability

kg kilogram MIC minimum inhibition concentration

ND not determined BHT butylated hydroxytoluene

TMCS trimethylchlorosilane pKa -logKa (acid dissociation constant)

BSFTA N,O-bis(trimethylsilyl) UV ultraviolet

trifluoroacetamide

ix

Table of contents

Acknowledgement vi

Abbreviations viii

List of Figures xv

List of Tables xvii

Abstract Xx

1. Introduction 1

1.1.Aims and Objectives 9

2. Review of Literature 10

2.1.Plant Bioactives 10

2.2. Phenolics: An Important Class of Bioactives 11

2.2.1 Simple Phenols 15

2.2.2. Phenolic acids 15

2.2.3. Hydroxybenzoic Acids 16

2.2.4. Hydroxycinnamic Acids 16

2.2.5. Flavonoids 17

2.3. Importance of Phenolics 20

2.4. Role of Phenolic Compounds in Food Industry 20

2.5. Medicinal Properties of Plant Phenolics 21

2.6. Oxidation in lipidic compnents and Phenolic Antioxidants 22

2.7. Mechanism of Antioxidant Action of Phenolics 23

2.8. Fruits as a Potential Source of Phenolic Antioxidants with Multiple Medicinal Benefits 24

2.9. Screening of Plants for Antioxidant and Antimicrobial Phenolic Agents 28

2.10. Extraction of Antioxidant Phenolic Components 31

x

2.10.1 Extraction solvent 31

2.10.2. Extraction Techniques 34

2.10.3. Hydrolysis aids 36

2.10.4. Purification and Fractionation of Crude Phenolic Extract 39

2.11. Measurement of Antioxidant Activity 40

2.12. Detection and Identification of Phenolic Compounds 44

2.12.1. Chromatographic Methods for Analysis of Phenolic Compounds 44

2.12.2. Gas Chromatographic-Mass Spectrometry Analysis of Phenolics 47

2.13. Antimicrobial Activities and Biofilm Inhibition Capacity of Plant Extracts 49

2.13.1. Some Synthetic Antimicrobial Agents 50

2.13.1.1. Sulfonamides 50

2.13.1.2. Penicillin 51

2.13.1.3. Gatifloxacin 51

2.13.1.4. Erythromycin 51

2.13.1.5. Tetracyclines 51

2.13.1.6. Streptomycin 52

2.13.1.7. Albicidins 52

2.13.2. Plant Phenolics as Natural Antimicrobial Agents 52

2.13.3. Antibacterial and Antifungal Activities 53

2.14. Minerals Contents of Fruits 56

2.15. Sugars and Organic Acids in Fruits 58

2.15.1. Sugars 59

xi

2.15.2. Organic acids 59

2.15.3. Analysis of Sugars and Organic Acids 60

2.15.3.1. Colorimetric and Spectrophotometric Techniques 60

2.15.3.2. Chromatographic Techniques 61

2.16. Brief Introduction about the Selected Fruits and the Soon Valley 64

2.16.1. Olive 65

2.16.2. Jujube 66

2.16.3. Common fig 67

2.17. Problem Statement: Need for Research Project 68

3. Materials and Methods 69

3.1. Description of the Analytical Instruments used throughout the Research Work 69

3.2. Chemicals and reagents 69

3.3. Sample collection, extraction and fractionation/purification 70

3.4. Assessment of antioxidant activity of crude concentrated extracts (CCE) and phenolic

rich fraction (PRF)

71

3.4.1. Determination of total phenolics (TP) 71

3.4.2. Determination of total flavonoids (TF) 71

3.4.3. Determination of reducing power 72

3.4.4. DPPH radical scavenging assay

3.4.5. Inhibition of linoleic acid peroxidation (%) by CCE/PRF of selected wild fruits

72

73

3.5. Phenolic acid profiling: 73

3.5.1. Preparation of calibration curves using standards 73

3.5.2. Extraction of free phenolics in fruit samples 74

xii

3.5.3. Extraction of conjugated phenolics in fruits using acid hydrolysis (conventional

hydrolysis)

74

3.5.4. Ultrasound-assisted hydrolysis and extraction of conjugated phenolics in the fruits of

selected wild plants:

74

3.5.5. Derivatization of standards and samples 75

3.5.6. GC-MS Instrumentation and Analysis parameters 75

3.5.7. Identification and Quantitation 76

3.6. Antimicrobial activity 76

3.6.1. Microorganisms tested 76

3.6.2. Disc diffusion assay 76

3.6.3. Estimation of minimum inhibitory concentration 77

3.7. Minerals Profiling using ICP-OES 77

3.8. Analysis of Organic Acids 78

3.8.1. HPLC Condition for Organic Acid Analysis 78

3.9. Analysis of Natural Sugars 78

3.9.1. HPLC Condition Used for Sugars Analysis 79

3.10. Gas Chromatographic Fatty Acid Compositional Analysis 79

3.10.1. GC-FID conditions for fatty acid composition 80

3.11. Assessment of Bio-Film Inhibition 80

3.12. Haemolytic activity 81

3.13. Antithrombotic activity 81

3.14. Statistical analysis 82

4. Results and Discussion 83

4.1. Extraction Yield 111

xiii

4.1.1. Yield of crude extracts 111

4.1.2. Yield of phenolic rich fractions 113

4.2. Total Phenolics (TP) and Flavonoids (TF) Content in Crude Extracts 114

4.3. Total Phenolics (TP) and Flavonoids (TF) Content in Phenolic Rich Fractions (PRF) 115

4.4. Reducing Power of crude extracts 117

4.5. Reducing Power of Phenolic Rich Fractions (PRF) 118

4.6. DPPH Free Radical Scavenging Activity of Crude Extracts 118

4.7. DPPH Free Radical Scavenging Activity of Phenolic Rich Fractions (PRF) 120

4.8. Antioxidant Activity of Crude Extracts in Linoleic Acid System 121

4.9. Antioxidant Activity of Phenolic Rich Fractions (PRF) in Linoleic Acid System 122

4.10. Antimicrobial Activity of Crude Extracts 123

4.11. Antimicrobial Activity of Phenolic Rich Fractions (PRF) 126

4.12. Biofilm Inhibition by the Crude Extracts 128

4.13. Biofilm Inhibition by Phenolic Rich Fractions (PRF) 129

4.14. Thrombolytic Activity of the Crude Extracts of Wild Fruits 130

4.15. Thrombolytic Activity of the Phenolic Rich Fractions (PRF) 132

4.16. Hemolytic Activity of Crude Extracts 133

4.17. Hemolytic Activity of Phenolic Rich Fractions (PRF) 134

4.18. Organic Acid Profile of the Fruits of Selected Wild Plants 135

4.19. Minerals Profile in the Fruits of Selected Wild Plants 137

4.20. Fatty Acid Composition of Wild Olive Lipids 139

4.21. Quantification of individual sugars in fruits of selected wild plants 140

4.22. Chromatographic Separation and Identification of Phenolic Compounds 168

xiv

4.23. Quality Control 169

4.24. Effects of Ultrasound on Hydrolysis of Conjugated Phenolic Compounds 172

4.25. Determination of Free and Total Phenolic Compounds in Fruits of Selected Wild Plants 173

Conclusion 175

Future Work 178

References 179

xv

List of Figures

Sr. No. Title Page

Fig.1.1 The basic structural feature of phenolic compounds with an

aromatic ring bearing one or more hydroxyl groups

3

Fig.1.2. Inter-relationships between the primary and secondary

metabolism in plants

5

Fig.2.1 Phenol, the simplest phenolic compound 12

Fig.2.2 The schematic diagram representing the general classification

of plant phenolics

13

Fig.2.3 Some simple phenols 15

Fig.2.4 Structures of coumrain and some p-hydroxycinnamic acids 16

Fig.2.5 Examples of hydroxycinnamic acids 17

Fig.2.6 Basic structure of flavonoids 18

Fig.2.7 Chemical structure of some important flavonoid compounds 19

Fig.2.8 Distribution of electron upon a phenol radical 24

Fig.2.9 Schematic diagram showing the process of extraction and

characterization of phenolics

30

Fig.4.1A A typical GC chromatogram of saturated fatty acid standards 142

Fig.4.1B A typical GC chromatogram of unsaturated fatty acid standards 143

Fig.4.1C A typical GC chromatogram of wild olive oil FAMEs 144

Fig.4.2 Chemical structures of the tested phenolic compounds 145

Fig.4.3a GC-MS chromatogram (Blank) 157

Fig.4.3b GC-MS chromatogram of calibration standards and the

internal standard of phenolics at the concentration of 5 µg/mL

157

Fig.4.3c GC-MS chromatogram of calibration standards and the internal

standard of phenolics at the concentration of 10 µg/mL

158

Fig.4.3d GC-MS chromatogram of calibration standards and the

internal standard of phenolics at the concentration of 15 µg/mL

158

xvi

Fig.4.3.e GC-MS chromatogram of calibration standards and the

internal standard of phenolics at the concentration of 20 µg/mL

159

Fig.4.4a Typical GC–MS chromatogram of free phenolic acids in wild

Olive fruit.

160

Fig.4.4b Typical GC–MS chromatogram of free phenolic acids in wild

jujube fruit

161

Fig.4.4c Typical GC-MS chromatogram of free phenolic acids in

common fig fruit

162

Fig.4.5a Typical GC-MS chromatogram of conjugated phenolics in

Olive fruit using acid hydrolysis.

163

Fig.4.5b Typical GC-MS chromatogram of conjugated phenolics in

Jujube fruit using acid hydrolysis.

164

Fig.4.5c Typical GC-MS chromatogram of conjugated phenolics in

common fig fruit using acid hydrolysis.

165

Fig.4.6a Typical GC-MS chromatogram of conjugated phenolics in

Olive fruit using ultrasonic-assisted hydrolysis.

166

Fig.4.6b Typical GC-MS chromatogram of conjugated phenolics in

jujube fruit using ultrasonic-assisted hydrolysis

167

xvii

List of Tables

Sr. No. Title Page

Table.2.1 Phenolic classes in plant 14

Table.4.1 Crude extract yield (g/100g of dry weight) from fruits of

selected wild plants

83

Table.4.2 Total phenolics content of crude extracts (GAE g/100g of dry

weight) from fruits of selected wild plants

83

Table.4.3 Total flavonoids content of crude extracts (CE g/100g of dry

weight) from fruits of selected wild plants

84

Table.4.4 Reducing power of crude extracts (λ=700nm) from fruits of

selected wild plants

85

Table.4.5 DPPH radical scavenging activity of crude extracts (IC50 value)

from fruits of selected wild plants

86

Table.4.6 Inhibition of linoleic acid peroxidation (%) of crude extracts

from fruits of selected wild plants

87

Table.4.7 Yield of Phenolic Rich Fraction (PRF) (g/100g of CE) from

fruits of selected wild plants

87

Table.4.8 Total phenolic content of PRF(GAE g/100 of dry weight) from

fruits of selected wild plants

88

Table.4.9 Total flavonoids content of PRF (CE g/100 of dry weight) from

fruits of selected wild plants

88

Table.4.10 Reducing power of PRF(absorbance data at 700 nm) from

fruits of selected wild plants

89

Table.4.11 DPPH radical scavenging activity of PRF (IC50 value) from

fruits of selected wild plants

90

Table.4.12 Inhibition of linoleic acid peroxidation (%) of PRF from fruits

of selected wild plants

90

Table.4.13 Organic acids profile of the fruits of selected wild plants

91

xviii

Table.4.14 Biofilm inhibition by crude extract of fruits of selected wild

plants

92

Table.4.15 Biofilm Inhibition by phenolic rich fraction of fruits of selected

wild plants

93

Table.4.16 Hemolytic and thrombolytic activity of fruit extract from

selected wild plants

94

Table.4.17 Hemolytic and thrombolytic activity of phenolic rich fraction

from selected wild fruits

95

Table.4.18 Antibacterial activity of crude extract from fruits of selected

wild plants

96

Table.4.19 Minimum inhibitory concentration (MIC) of crude extract from

fruits of selected wild plants

97

Table.4.20 Antifungal activity of crude extract from fruits of selected wild

plants

98

Table.4.21 Minimum inhibitory concentration (MIC) of crude extract from

fruits of selected wild plants

99

Table.4.22 Antibacterial activity of phenolic rich fraction from fruits of

selected wild plants

100

Table.4.23 Minimum inhibitory concentration (MIC) of phenolic rich

fraction from fruits of selected wild plants

101

Table.4.24 Antifungal activity of phenolic rich fraction from fruits of

selected wild plants

102

Table.4.25 Minimum inhibitory concentration (MIC) of phenolic rich

fraction from fruits of selected wild plants

103

Table.4.26 Elemental composition of fruits from selected wild plants 104

Table.4.27 GC-MS based retention time, characteristic mass ions,

calibration curves and detection limits of silylated derivatives

of standard phenolic compounds

105

xix

Table.4.28 Free (acid hydrolysis) and total phenolic compounds in fruits

of selected wild plants

106

Table.4.29 Free and ultrasonic-assisted hydrolyzed phenolic compounds in

fruits of selected wild plants

107

Table.4.30 Free and total phenolic compounds in fruits of selected wild

plants

108

Table.4.31 Fatty acid (FA) composition of olive oil 109

Table.4.32 Individual sugar content (g/100g DW) in fruits of selected wild

plants

110

xx

ABSTRACT

Plants have been recognized as rich source of functional food ingredients and high-value

bioactives with medicinal benefits. During the last two to three decades an extensive research is

being carried out towards screening of phytochemicals in plants with special emphasis on

qualitative and quantitative analysis of high-value nutrients and natural antioxidants. The fruits of

wildly grown plants, so called wild species, are valued as a potential source of wide array of

nutrients and antioxidant phenolics with multiple biological and medicinal properties. The current

research was mainly aimed to explore the profile of valuable phyto-nutrients and phenolic

antioxidants in the fruits from naturally distributed wild plants such as olive, jujube and common

fig in Pakistan.

Antioxidant bioactive components (phenolic antioxidants) from the shade-dried fruits of

three wild plants were extracted using different extraction solvents such as aqueous methanol,

absolute methanol, aqueous ethanol, absolute ethanol, aqueous acetone and absolute acetone. A

higher extraction yield of extractable antioxidant components were obtained from olive and jujube

fruit using aqueous ethanol while for common fig, aqueous methanol extracted the maximum

amount of these components. Overall, aqueous ethanol derived extracts as well as phenolic rich

fractions (PRF) from wild olive and common fig fruits, were found to contain higher amounts of

total phenolics (TP) and total flavonoids (TF) alongwith superior antioxidant and biological

activities (antimicrobial, antithrombotic, biofilm inhibition and haemolytic) among others.

Nevertheless, in case of common fig fruit, maximum amount of TP, TF and extent of antioxidant

activity were recorded in aqueous methanolic produced extracts/PRF.

Minerals profile of the tested fruits as analyzed using ICP-OES revealed the presence of

twenty five minerals. Potassium (K) was determined to be the major macro element followed by

appreciable amounts of micro essential elements (Ca, Mg, Na, Zn and Fe etc). The composition of

natural sugars and organic acids was determined using HPLC, while fatty acid composition

analysis of olive lipids was carried out using GC-FID. Of the organic acids analyzed, succinic,

gluconic, and acetic acids were identified as the major organic acids in the present analysis of wild

olive, jujube and common fig, respectively, however, a notable but varying amount of acetic, citric,

oxalic, malic and gluconic acids were also detected. Phenolic acids and flavonols in the selected

wild fruits were analyzed by GC-MS-TIC method after derivatization into silyl esters. As far as

xxi

individual phenolics profiling is concerned, trans-cinnamic acid was found to be the the major

phenolic compound in the tested fruits, however, 2,4-dihydrooxybenzoic acid was dominant in the

fruit juice of olive. Epicatechin was noted to be the only flavonol detected in the fruit juice of

olive. Moreover, GC-FID analysis of n-hexane extracted oil/lipids from the wild olive fruit

established the presence of oleic acid as the principal fatty acid along with considerable levels of

linoleic acid and palmitic acid and small amounts of lauric, myristic and arachidic acids were also

detected.

The present results revealed a significant (p

1

CHAPTER 1 INTRODUCTION

Lipid oxidation, due to its deleterious effects, is looked upon as a severe problem both for

the living systems as well as for the food industry. In biological systems, free radicals are mainly

formed due to inequity in the production of reactive species (especially oxygen species) and the

functionality of antioxidant enzymes. Free radicals are chemically unbalanced atoms which can

harm to biomolecules such as DNA, proteins and lipids [1]. In biological systems, ROS and free

radicals generates oxidative stress which may be precursor of different ailments like aging,

inflammation, cancer, cardiovascular disorders [2-3]. On the other hand, in the food industry, lipid

oxidation is considered to be one of the major factors that are responsible for the development of

rancidity and off flavors and thus contributes towards the losses of nutritional and organoleptic

value of the products. Moreover, lipid oxidation mediated primiray and secondary oxidation

products, due to their perceived toxicity, are negatively linked with the incidence of different

diseases such as aging, and cardiovascular disorders [4-5].

The process of lipid oxidation can be controlled through the use of some compounds known

as antioxidants. Antioxidants, being biomolecules, have potential to provide protection to body

against oxidative stress, caused by free radicals [6]. Similarly, antioxidant compounds, due to their

preservative and free radical scavenging properties, play a key role to protect the food stuff from

oxidative deterioration [7].

Human body is blessed by an internal /built-in antioxidant system constituting different

antioxidant enzymes and trace metals such as selenium. This natural defense system helps to

protect the body from oxidative stress caused by ROS. However, this natural defensive system of

the body is affected by the pattern and quality of food intaken, age and the state of the health and

thus usually needs to be strengthen by an external dietary antioxidant supplementation [8]. It is

now widely accepted that the diets rich in antioxidants maintain a balance between ROS and the

defense system components in our body [9].

Antoxidants are the substances that slow dowon and /or retard the process of lipid oxidation

via different mechanisms such as by neutralizing/scavenging free radicals, scavenging molecular

oxygen or via acting as metal chelators [10-12]. A wide range of antioxidants, both natural and

synthetic, have been searched to suppress and slow down the process of lipid oxidation both in the

2

living organisms and the food industry as well as in therapeutic medicine. The organic compounds

(BHA, BHT, PG and TBHQ) are some of the common synthetic antioxidants which are added to

different foods to protect them from oxidation and off-flavor development. However, due to the

perceived toxicity/carcinogenicity, the use of such synthetic antioxidants in the food industry is

restricted [10, 13-14].

Nevertheless, plant based natural antioxidants have gained greater attention of researchers

and food scientists due to their multiple medicinal benefits and free radical scavenging properties

[15-16]. Plant-derived foods such as fruits, vegetables, legumes, whole grains, species and herbs,

etc., are reported to have beneficial effects towards preventing the incidence of some diseases and

thus impart health benefits [17]. The health beneficial effects of such plant foods have been linked

to the occurrence of different secondary metabolites (bioactive phytochemicals) such as

polyphenols, alkaloids, phytosterols and terpenoids which possess wide array of pharmaceutical

and biological properties [17].

In the last few decades, much work is being reported on the investigation and identification

of diverse classes of plant based dietary antioxidants including vitamins (vitamin A, C, E),

caretoinds, tocopherols, phenolics (phenolic acids, flavonoids, tannins, lignans) and allyl sulfides

among others [18-20]. In this regard, a huge number of food and medicinal plants, especially,

fruits, vegetables, oilseeds, whole grains, cereals, spices and herbs have been searched as the

richest sources of natural antioxidants and nutraceutical bioactives [5, 20-22]. Among the

antioxidant phytochemicals, phenolics exhibit remarkable antioxidant and free radical scavenging

activities together with multiple medicinal benefits against oxidative stress related diseases such

as aging, inflammation, certain cancers and cardiovascular disorders [23-25]. Typically, phenolic

compounds have been investigated to act as antioxidant to avoid cardiovascular ailments [26-28],

minimize inflammation [29-31], reduce the happening of cancers [32-34] and diabetes [35-36], as

well as lower mutagenesis in living cells [34, 37].

3

Plants have been reported to produce different kinds of metabolites such as primary

metabolites and secondary metabolites. Primary metabolites are the substances which are

commonly provided to living organisms and are essential to cell maintenance (protein,

carbohydrates, lipids and nucleic acids). While several biosynthetic pathways produces secondary

metabolites such as alkaloids, terpenoids, steroids, pehnolics etc. [38]. Phenolic molecules are

produced in living systems like plants in response to environmental and physiological stresses such

as pathogenic microbes and insect invasion, UV lights and hurting. Interestingly, phenolic

compounds offer protection and defense to plants against aggression of pathogens (insects,

microbes), parasites and other animals as well as impart coloring properties to plant foods [39-41].

Figure 1.1: The basic structural feature of phenolic compounds with an aromatic ring bearing one

or more hydroxyl groups [42].

Phenolic compounds, due to their wide spread distribution in plant foods such as cereals,

olives, legumes, grapes, citrus, soft fruits, chocolates, beverages (black tea, green tea, coffee etc.,),

are a viable dietary source for human nutrition. Phenolic compounds impart sensorial properties

incluing bitterness, aroma, astringency and flavor to plant foods and play a key role in the

physiological and cellular metabolism of plants [28]. Phenolics are recognized as one of the largest

and extensively distributed secondary metabolites in plants with known 8,000 compounds [43]. A

common characteristic of phenolics is that these compounds contain one aromatic ring (at least)

with one or more hydroxyl groups. In the plant kingdom, these substances usually occurr in bound

form with other molecules, often with sugars and proteins [44].

A large diversity has been observed with regard to occurrence, chemical structures,

biological functionalities, polarity and acidity as well as degree of hydroxyl groups and aromatic

rings and concentration levels among phenolics from different plant sources [28]. Plant phenolics

4

comprise simple phenols such as phenolic acids, more complex polyphenols (flavonoids,

hydrolysable and non-hydrolysable tannins) and miscellaneous compounds (coumarines, stilbenes,

lignans) [45]. Phenolic acids belong to main class of phenolic compounds and are mostly present

in bound form as as esters, glycosides or amides, but infrequently exist in free form. Phenolic acids

were foud in two parental assemblies and exist as hydroxycinnamic (HCA) and hydroxybenzoic

acid (HBA) derivatives [28].

Flavonoids are among the most common phenolics, which are widely distributed in plant

tissues. These compounds mainly impart colors such as blue, purple, yellow, orange and red to

different plant parts such as leaves, flowers, roots, fruits and stem. Different types of link between

the glycosyl residue (GR) and the flavonoids, also steer to the different byproducts that impart

coloring and coloring to flowers [47-51].

5

Figure 1.2: Inter-relationships between the primary and secondary metabolism in plants

6

The antioxidant activity of plant phenolics is of great nutritional and therapeutic interest,

since it has been associated with medicinal effects on human health by reducing the prevelance of

several diseases [52]. The antioxidant activity (AA) of phenolics rely on the chemical structure

of these compounds [43]. In several cases, these biomolecules may also be used for healing

applications because of their multiple pharma- and biological attributes such as anticancer, anti-

inflammatory and antmicrobial activities [28]. Moreover, some low molecular phenolics are

utilized in medicine due to their antibacterial potential [51].

Plants have been a source of food, folk medicine and fuel since the history of mankind.

Interestingly, a huge number (ca. 25%) of modern (synthetic) drugs have been isolated or derived

from plant sources [53]. In perspectives of the modern developments of optimal nutrition, which

provoke the uses of functional foods and nutraceuticals for physiological benefits, presently, there

is a renewal of attention in the utilization of these plant stuff as a source of food and medicine [22,

54]. The multiple medicinal and biological properties of plants are attributed to the presence of

different bioactives such as polyphenols, flavonoids, lignins, alkaloids, terpenoids, carotenoids,

phytosterols [55-56]. As an antioxidant bioactives the phenolics compound also help in protecting

the nutritional quality and shelf-life of foods by inhibiting lipid oxidation and minimizing rancidity

and off-odor development [57-58].

Although, many food plants not only give most of the plant based nutrients required for the

care and regulation of body courses, nevertheless, these are also recognized as a potential source

of bioactives and nutraceuticals with medicinal benefits. However, there is greater need to explore

huge reserves of the under-utilized food plants as a feasible source of high-value nutrients and

functional bioactives in order to meet the challenges of food insecurity and civilization diseases

and public health issues [59]. In this regard, the exploration of wild or semi-wild plants as a viable

source of food nutrients and phytomedicine, has to play a crucial role [60-61]. Many under-utilized

wild plants, being a potential source of high-value nutrients and medicinal agents, can play a

significant role towards improving the status of human nutrition and health. Infact, wild plants

have been a source of food and folk medicine for native peoples in different civilizations since the

history of mankind [62].

There is a strong evidence that the healthy diets rich in fruits and vegetables are linked with

the reduced incidence of diseases such as aging, inflammation, cancer and cardiovascular disorders

7

[28, 63]. Fruits are considered as the most important source of high-value components and

bioactives with multiple medicinal and biological activities. The protective role of fruits is mainly

attributed to the presence of a diverse group of secondary metabolites known as phenolics which

have potent antioxidant and radical scavenging activities. Fruit phenolic compounds possesses

medicinal properties on mutagenesis as well as carcinogensis. Most of the fruits have other

antioxidant biomolecules such as carotenoids and vitamins [64].

It has been reported that foods produced from selected wild plant fruits offer medicinal and

health benefits and provide shielding effect in a fight with cancer, stroke and cardiovascular

diseases [65-67]. Concerning the therapeutic attributes, the mostly studied advantage of having

fruits, is their antioxidant effects [68]. Wild fruits have beneficial pharmacological roles because

of the existence number of secondary metabolites like simple phenols, flavonoids and tannins [69-

70].

Pakistan is blessed with a huge reservers of economically and/ or medicinally important

flora for bioprospecting. A large number of native plant species from Pakistani flora, due to

presence of different high-value nutrients and bioactives, are traditionally popular and used by the

local people as a source of food and folk medicine [14]. In this context, three of the wildly grown

fruit plants including Olive, Common Fig and Jujube seem to be promising candidates for

investigation of valuable phytochemicals profile and hence can have considerable potential for

value-addition by their usage for the progress of functional foods and naturaceuticals/therapeutic

agents.

The Olives is a perennial, one of the most widespread and economic fruit trees that wildy

grows and is largely cultivated in moderate climate, especially in Mediterranean regions of the

world for oil production. Olive and its products are valued as a basic ingredient of the healthy diet,

especially in Mediterranean regions, with numerous health benefits [71]. Olives are particularly

rich source of high-value nutrients and phenolic antioxidants [72], incluing some characteristic

compounds such as polyphenols, ligstroside, verbascoside and oleuropein [73].

Jujube (Ziziphus jujuba Mill), which is largely distributed in tropical and subtropical areas

of continent of Asia and America, is a tree belonging to the Rhamnaceae family and contains

approximately 40 species. The fruits of Jujuba tree have been commonly consumed in folk

medicine for the prevention and curing of different diseases [74-75]. The fruits and leaves of Jujuba

8

are a potential source of different antioxidants and fatty acids [76-77]. The fruits are utilized both

as fresh and dried form as well as indulged into processed products (jams, loaf, cakes, jelly, etc.)

due to their intrinsic sugar levels and high amount of vitamin (A, C and B complexes), phosphorus

(P) and calcium (Ca) [74, 78].

Another food plant namely Common Fig (Ficus carica L.), is a small deciduous tree from

the Moraceae family. This is one of the earliest known and important fruit crops which is native

to Iran, Asia Minor and Syria and spread to the Mediterranean regions by humans. Fig fruits have

been comsumed, both in dry and fresh form, since ancient times due to its high nutritious value

[79-80]. The dried Fig fruits are an excellent source of natural sugars and organic acids, minerals,

vitamins and dietary fiber; they are fat and cholesterol-free and have different amino acids [81].

The growing interest in the health benefits of the wild plants prompts the need to investigate

the composition of nutrients and antioxidant properties of under-utilized wild fruits. As such no

earlier reports are available on the analytical characterization of phenolic bioactives and phyto-

nutrients in the fruits from the selected three wild food plants naturalized in Pakistan. The

importance of phenolics as a natural antioxidant and chemo-preventive agent together with the

existing information gap motivated us to investigate the profile of the high-value nutrients and

phenolic bioactives in the fruits of selected wild plants. The present research project, therefore was

mainly framed to systematically characterize high-value phytonutrients (sugars, organic acids,

minerals and lipids) and phenolic antioxidants in the fruits harvested from selected wild plants

(olive, jujube and fig) from Soon valley, Punjab, Pakistan so as to explore their capacity uses as

constituents of functional foods and nutraceuticals.

9

1.1. Aims and Objectives

The present study was designed with the following major aims and objectives:

1. Investigations of valuable phyto-nutrients (sugars, organic acids, minerals and

lipids) and phenolic antioxidant compounds (flavonoids and phenolic acids) in the fruits of

selected wild plants.

2. Evaluation of the efficiency of different solvents for extraction of potent phenolic

antioxidant components (PAC) from the wild fruits.

3. Isolation/fractionation of PAC and assessment of their biological (antioxidant,

antimicrobial, antithrombotic, biofilm inhibition, and haemolytic) activities using different

bioachemical assays.

4. Characterization and quantification of hydrolysable and non-hydrolysable phenolic

compounds in the selected wild fruits using GC-MS.

10

CHAPTER 2 REVIEW OF LITERATURE

This chapter reviews different study aspects including background knowledge, problem

statement, current research trends and analytical methodologies involved in the present research.

Lipid oxidation is a self-sustained process and is considered as one of the most deleterious recation

going on in the living organisms and the food products. It not only spoils the quality and nutritional

value of food, nevertheless, the reactive oxygen species and free radicals generated in this process,

also cause different degenerative diseases [10, 82-85]. Due to safet concerns of synthetic

antioxidants, currently, food and therapeutic medicine industry is keenly interested in the use of

some safer natural antioxidants to enhance the shelf-life of their products [86-87]. Similarly

infectious diseases, due to increasing microbial drug resisitance, are continuingly spearding with

devastating impacts [87]. The alternative solution to these oxidative stress and infectious diseases

rerlated problems is the use of plant-based natural products and phytochemicals/plant bioactive

agents.

2.1. Plant Bioactives

According to ancient Chinese reference “Let food be thy medicine and medicine be thy

food” presently, attention has been revived in using plants for food and medicinal properties [60,

86]. Medicinally, the plants have extensive past of their uses in the traditional medicine systems

of several civilizations across the globe. It is now largely known that plant-derived bioactives show

defensive role in a fight with the prevalence of different ailments such as cancer and

neurodegenerative and cardiovascular problems disorders [87-88]. In this context, the uses of some

plant-based antioxidant nutrients such as polyphenols, vitamin C, phenolic acids and flavonoids in

foods, in defensive and beneficial benefits, is attaining esteem much interst due to their

anticarcinogenic attributes and potential health benefits [89-92].

Plants have been used as a source of food and phyto-medicine since the start of human

evolution; possibly as early as the origin of mankind due to their healing powers [93]. In the

traditional health care system plant-based medicines have important role. These can be used to

provide relief, stop and cure and /or change the physical procedures of the body during diseases.

In fact plants are a sustainable source of both the traditional and modern medicine [94].

11

Plants yield different compounds which are mainly separated into two major groups

(primary and secondary metabolites). The primary metabolites are reported for the production of

elementary building units of plants for their survival [95]. However laters are being used for the

providing protection against different types of pathogenic poisons and abiotic stresses [96].

In primary metabolites, individual sugars impart a vital role in structural and metabolic

pathways in plants on cell level. The presence of individual sugars in fruit part of plant play a

vital role in distribution of seeds as a source. In order to disperse the seeds of different fruits,

various plants contain eatable fruits through which they have spread their seeds along the transfer

of humans, birds and different animals to make a fruitful interdependent relationship. Individual

sugars also play a vital role to be used as a potential source of energy in different metabolic

pathways in animals [97]. Besides the production of basic metabolites, plants were also reported

to generate various compounds (organic) which are believed to be famous as secondary

metabolites. These secondary metabolites play a vital role in making the fruits if different plants

attracting to make it easy for the dispersal of their seeds and provides shelter against different eco-

stresses etc. [98-99]. Significant research was focused for investigating this type of compounds

due to various uses including medicinal, food, fragrances etc. [98-99].

Secondary metabolites of plants such as alkaloids, tannins, steroids, phenolics and

terpenes are valuable phytochemicals and are employed in the preparation of different

phytomedicines and drugs. These natural substances play a key part in defense mechanism of

plant by providing defense against invasion of insects, micro-organisms (MO) and herbivores [28,

89].

2.2. Phenolics: An Important Class of Bioactives

Among the important plant bioactives, phenolic compounds not only impart characteristic

odor and colors to plants, nevertheless, these compounds are used for the seasoning and stabilizing

of different foods due to their preservative properties [28, 89, 96]. Plant phenolics are broadly

dispersed in plant territory and possess one or more aromatic rings equipped with one or more

hydroxyl groups. Phenolics are considered to be one of the widely available metabolite (secondary)

in plants. Currently there are over eight thousands structure of phenlics available in literature which

includes simple phenol and complex structured substances such as falvonoids, phenolic acids.

These are widely available in plant matrices including olive, legumes, chocolate and drinks

12

(Wines,tea,coffee, etc.), and also accountable for organoleptic values of different food stuff [28].

In fact, plant phenolics represent a large group of natural antioxidants and exhibit multiple

biological roles like to scavenge free radical, antiglycemic, anticarcinogenic, to lower

inflammation, bactericidal and fungicidal activities [28, 63, 100-105]. Moreover, these compounds

contribute significantly to the organoleptic properties, astringency, flavor and color of plant foods

as well as play a protective role in several processed food products [28, 89].

Figure 2.1: Phenol, the simplest phenolic compound

Plant phenolics can be grouped into various categories like phenolic acids and different

flavonoids etc. The phenolics mainly present in plants as glycosylated linkage [106-107]. The

individual sugars such ad arabinose,glucose and galctose are dominant to form different phenolic

glycosides and flavonoid glycosides [28, 108].

Phenolic compounds may also exist in conjugated form with different substances including

lipidic, oligosaccharides and aminesetc [108-109]. The different antioxidant/biological properties

depend upon conjugation in aromatic structure. In order to produce different natural phenolics in

plants depends upon varying complex structure, level of conjugation and

hydroxylation/methoxylation [28, 110-111].

Overall, the phenolic entities may be classified as thirteen groups which depends on the

structure of the carbon arrangement in it [106-108] (Table 2.1). The main difference among

different groups is mainly dependent on the frequency of carbon (constitutive)atoms and their

arrangement.

13

Figure 2.2: The schematic diagram representing the general classification of plant phenolics

14

Table 2.1: Phenolic classes in plants [(adapted from Bravo, [108]

15

2.2.1. Simple Phenols

In nature, six carbon atom phenols are relatively scarce; either they are absent or were

present at a very low content [96]. Different examples simple phenolic compounds are presented

in Figure 2.3. Hydroquinone was reported to be produced as a result of removal of carboxyl group

and reduction of p-hydroxy benzoic acid [112]. Arbutin was reported to be exhibited as ß-D-

glucoside of hydroquinone and that is produced a s a result of substitution reaction [112]. The

different phenolic compounds were believed to be produced after removal of carboxyl group,

degrading lignin thermally in different processes and storing [113].

Figure 2.3: Some simple phenols

2.2.2. Phenolic Acids

The phenolics, derived from plant sources, are composed of benzoic (hydroxyl) and

cinnamic (hydroxyl) acid and their derived compounds. The examples of some vital members of

phenolic acids are para-hydroxy benzoic, syringic acid, vanillic acid, and protocatechuic acid

while the derivatives of hydroxycinnamic acids possesses sinapic acid, para-coumaric acid, caffeic

16

acid and ferulic acid. The basic arrangement and degree of formation of hydroxyl and methoxy

groups attached to aromatic structures [28, 107, 114].

2.2.3. Hydroxy Benzoic Acids

The benzoic (hydroxyl) acids are derivatives of benzoic acid [115-116]. Changes in the

hydroxy benzoic acids takes place because of shapes of hydroxylation/methoxylation in aromatic

structures. A large amount of these acids exhibited in the form of glycosidic/esters linkage attached

to aliphatic substances including different organic acids [117]. Hydroxy benzoic acids like gallic

acid, para-hydroxy benzoic acid and vanillic acid are available in most of the plants [106, 115].

Gallic acid is one of the most important phenolic in this group.

2.2.4. Hydroxycinnamic Acids

The plant based phenolics which has Csix to Cthree arrangement, reported to be named as

as phenyl propanoids and mainly comprised of cinnamic (hydroxyl) acid derivatives and

coumarins. The hydroxy cinnamic acids were reported to be in extensively arising phenyl

propanoid compounds and were supposed to be used as precursors of cyclic derivatives.

Figure 2.4. Structures of coumrain and p-hydroxycinnamic acid

Hydroxycinnamic compounds are extensively spread phenolics indifferent plant matrices

[115]. Their free for is vaery scarce and naturally present conjugated form [118], and major

proportion of phenolics including flavonoids [43, 119].

Para-coumaric, caffeiec acids are the examples of osem naturally present compounds. The

phenolic aicds (caffeic and ferulic) were reported to be present in dominant form in different plant

and food matrics [43, 115]. One of widely available phenolic acids, caffeic acid, are mainly spread

17

in the matrices of different plant, fruits, vegetables; most likely, it exists in ester form. Coffee, one

of the richest sources of caffeic acid. While, ferulic acid is mostly abundant in different cereals as

conjugated form in the cell wall [28, 120].

Figure 2.5. Examples of hydroxycinnamic acids

2.2.5. Flavonoids

The secondary metabolites, known as flavonoids, are known to be the most abundant and

important dietary polyphenols distributes in many food and herbal plants [120]. These belong to

the group consists of polyphenols derived from natural sources like plants, majority of them are

plant phenolics [18]. These contain a base structure of Csix-Cthree-Csix that contain at least double

aromaticity in their structure associated with aliphatic structure (consisting three carbon). This

class of flavonoids were reported to be further classified into different subclasses which depends

upon the changing in the substitution upon carbon ring [18,121]. Flavonoids are known to be the

main coloring components and impart color to flowers and fruits in many plants such as apples,

berries, beets and onions etc. [28].

18

Figure 2.6. Basic structure of flavonoids

Flavonol and flavone were reported to be most abundantly occurring in various parts of

plants like fruits and leaves [122]. The members of flavonoids like flavonols were reported to be

exhibit pale yellow color. These compounds are slightly soluble in polar solvents. This type of

compounds was reported to be present in different food matrices as glycoside linkages. The

different residue of sugars was reported to bind at third carbon atom preferably however often at

seventh carbon atom position [123].

Flavonoids were believed to be spread in different plant matrices and credited for sixty

percent share total phenolics (dietary) [116, 124]. So far, the literature witnessed the presences of

over four thousands compounds of flavonoids [111]. A lot of combinations of various kinds of

groups like sugar, O2, CH3, OH were associated with the structures of flavonoids to get different

groups of flavonoids which includes flavones, flavonols.

19

Figure 2.7. Chemical structure of some important flavonoid compounds (Hakkinen, 2000)

20

2.3. Importance of Phenolics

The secondary metabolites, phenolic compounds, have vital role in plants as a defensive

shield. These protect the plant from dangerous UV rays which cause damage them [138-139].

Phenolic compounds, especially flavonoids, has absorbance maximum in the range of 280 to 320

nm); This kind of UV rays are supposed to harm different metabolic pathways and hereditary

materials like nucleic acids in plants [122, 138].

Some phenolic compounds are important due to their antimicrobial role in protecting the

plants from the attack of pathogenic microorganisms [139-140]. Different plant phenolics are

responsible to retard to grow the pathogens which includes bacteria and fungus [141-142]. The

inhibition potential of these phenolics by producing poisonousness to bacterial and fungal strains

by harming cell wall, inhibiting enzyme catalyzed reactions and by stopping the functions of

different proteins [143-144]. These plant phenolics provide shelter by providing restrictions to

feeding insects [145-146]. Aphids were reported to be destroyed by applying different flavonoids

like luteolin by stopping their feeding [147].

2.4. Role of Phenolic Compounds in Food Industry

Of the different viable sources, plant foods such as legumes, fruits,vegetables, chocolates

and drinks such as beer, tea etc are recognized as the richest sources of plant phenolics. Due to

their widespread occurrence in plant organs, phenolic compounds are considered essential part of

the diet. The organoleptic values of the plant derived foods are attributed to plant phenolics. Plant

phenolics are responsible for sensory qualities like color intensity, taste, sweetness and astringency

[106]. The precipitation of proteins (salivary) attributed to the existence of plant phenolics [148].

Among different plant phenolics, tannins were reported to be most abundant towards astringency

however simple phenolics like para-coumaric acid and catechin communicate astringency [149-

150]. The different derivatives of hydroxycinnamic acid were reported to impart bitterness in fruits

like cranberries [151] while catechin and epicatechin were found to impart bitter flavor in wine

[152], tea and cocoa powder [153]. The glycosides of flavanides are mainly responsible to impart

bitterness in different fruits [154].

It was reported that plant phenolics are also responsible for flavor in the food commodity

which are either desired or undesired [43, 116, 125]. The undesirable flavor in the stored fruit

juices are mainly attributed to the presence of free ferulic acid [126]. Moreover, plant phenolics

21

are also responsible to impart different colors in various fruits. The pigments pf anthocyanin are

also reported to impart colors in various vegetables and fruits [106, 127-128].

Besides, their contribution to the sensory related parameters, phenolics have gained

increasing attention of researchers because of their biological and antioxidant capacity making

them a strong candidate to be used in preservation and shelf-life of food commodities [129]. Food

industry is now keenly looking for the use of plant based natural phenolic antioxidants in order to

retard deteriorating of the food stuff [116].

2.5. Medicinal Properties of Plant Phenolics

Recently, much research is being emphasized on medicinal properties of phenolic

compounds which are widely spread in plants. Due to different biological, antioxidant,

antimicrobial activities and shielding effect against various ailments, much interest has been

developed in dietary plant based phenolic compounds [107]. The data obtained from different in-

vitro and in-vivo experiments revealed that plant phenolics provide shielding effect against

various ailments including heart diseases, certain cancer and oxidative stress [130-134].

Furthermore, polyphenols were found to impart a significant character to maintain activity of

various enzymes. Plant pheolics have been documented to take part considerably in various

biological, antioxidant, bactericidal and fungicidal activity and provide shielding cover against

many ailments [130-134].

It is reported that defensive role of plant extracts against many diseases of plant extracts

by providing antioxidant and free radical scavenging potential [28, 63, 135-137]. Due to their

remarkable antioxidant activity, phenolics rich plant extracts were reported to exhibit protection

against certain type of cancer [157], heart disease [158-159], immune related diseases [160],

bacterial, viral and fungal diseases [160-162], neurodegenerative ailments [137, 163].

Phenolics, due to their multiple biological and pharmaceutical activities, were lengthily

explored for medicinal benefits [63, 137]. Oxidation of low density lipoprotein is a key factor

steers plaques formation [164]. Reactive oxygen species, free radicals and some enzyme were

reported to be responsible in LDL oxidation [165-166]. In this regard the positive role of plant

phenolics in different ailment by offering various biological activities including antioxidant and

free radical scavenging effect, is gaining recognition [167-169]. An important class of

polyphenols, flavonoids were reported to impart substantial effect against various ailments

22

including heart diseases, immune disorders by offering biological activities including antioxidant

and free radical scavenging effect in order to provide protection [170-171]. The data obtained from

epidemiological experiments showed that plant phenolic compounds not always provide shielding

effect against cardiovascular disorders [172-173].

Among the phenolic acids, caffeic acid was reported to exert a positive effect in blocking

the synthesis of leukotrienes which is responsible for certain ailments such as immune disorders,

respiratory diseases and certain types of allergy [174]. In order to retard or slow down the process

of carcinogenesis in colon, anti-tumor potential of caffeic acid and its derivative has been

published [175-176]. The different derivatives of caffeic acid have been found to be very operative

in inhibiting human immunodeficiency diseases. Moreover, the protective effect of phenolic acids

and their derivatives to skin from oxidative stress cause by free radicals [177-178]. These phenolic

compounds and their derivatives have been used to lower the infection caused by herpes simplex

virus type I (in vitro) [179].

2.6. Oxidation in Lipidic components and Phenolic Antioxidants

The oxidation in lipidic components is a severe problem both for the living organisms as

well as for the food industry. It is widely accepted that lipid oxidation leads to deterioration of the

quality of food products. In fact, in the food industry, the oxidation reactions are looked upon to

be one of the main factors responsible for the loss of nutrients such as vitamins, aroma, and taste

of the processed foods products [82]. Moreover, the products of lipid oxidation such as conjugated

dienes, trienes and aldehydes and ketones, by interaction with important biomolecules, can upset

cell homeostasis, and act cytotoxically, resulting in different health disorders including tumours

formation, heart failure, cataract development and brain dysfunction [155].

On the other hand, in living organism, lipid oxidation is the main cause of production of

of free radicals and oxygen species which are linked with the incidence of several diseases [10,

82]. The body has a built-in defense mechanism to neutralize reactive oxygen species, while, the

body is in oxidative stress when the amount of free radicals and reactive oxygen species are in

excess. Oxidative stress caused by free radicals were reported to be impart negative role and thus

leads to the incidence of different ailments such as heart diseases, certain type of cancer, immune

disorder and ageing related problems [84-85].

23

The natural defense system of the body can be boosted via dietary intake of some

antioxidant compounds. Adding synthetic antioxidants have been a common practice to enhance

shelf-life of food products in food industry by retarding the lipid oxidation. However, the use if

synthetic antioxidant is restricted in food industry because of the alleged toxicity and safety issues

[82, 156]. On the other hand, by the last few decades, it has been reported that the increased focus

in exploring and investigating the employing natural antioxidants in the nutraceutical industry and

therapeutic medicine due to their safer nature and potential medicinal benefits [10-11, 82].

Plants are well recognized source of natural antioxidant compounds. Among the natural

antioxidant compounds, phenolics have gained special attraction because of their potential

antioxidant and free radical scavenging ability. There is growing demand for phenolic

antioxidants due to their wide spread applications in food, therapeutic and cosmo-nutraceutical

industries [18, 89]. Phenolics, due to possessing multiple medicinal benefits and biological

activities, are considered to be the most important group of natural antioxidant components [28,

89]. These are widely present in almost all food plants, fruits and vegetables, leguminous plants,

cereals and grains, teas, herbs, spices and wines [18, 185].

Several fruits such as berries, olive, grapes, citrus, apple and vegetable have gained special

attention as an impressive source of phenolic antioxidants [73, 182, 186]. In fact, the protective

role and health benefits that various medicinal plants, cereals, fruits and vegetables exhibit against

different diseases is credited to the occurance of phenolic antioxidants in these foods [18, 89,

187]. Especially, several types of vegetables, fresh and dired fruits are reported to possess various

antioxidant nutrients other than the phenolics like vitamin C, vitamin E, carotenoids, however,

antioxidant capacity of these foods is because of phenolics [61, 89, 188]. A important class of

phenolic compounds, flavonoids, are widely distributed in many fruits demonstrated strong

antioxidant activities [18, 61, 89].

2.7. Mechanism of Antioxidant Action of Phenolics

Plant phenolics exerts beneficial effect by imparting antioxidant activity which involve

either (1) to scavenge ROS/radicals, (2) to chelate metals responsible for release of free radicals

or (3) inhibition of enzymes involved in creating ROS and free radicals [157, 180, 189]. In order

to scavenge free radicals, any substance must be in positon to release electron or hydrogen to stop

the free radical to release [181]. The antioxidant potential of a phenolic extract/compound can be

24

evaluated by assessing their potential to scavenge free radical spectrophotometrically. The

potential of phenolic compounds to scavenge free radicals is associated with their capacity to

release electron or hydrogen atom in order stop the production of free radicals. By accepting

hydrogen atom or electron from phenolic antioxidant, the free radicals are converted into non

radical products resulting in the termination of oxidation reaction [181].

A general mechanism of antioxidant action of phenolics is depicted in Figure 2.8. During

the antioxidant action, plant phenolics were reported to be oxidized to release stable prodcuts

(Figure 2.9) [180]. This reaction is can be depicted as under:

ArOH + acid ion• →ArO•+acid

ArOH + OH• → ArO• +H2O

ArOH represents plant phenolics while ArO• represents the radical

Figure 2.8. Distribution of electron upon a phenolic radical (Gordon, 1990).

The oxidation of the reaction stopped due to the stability of the transitionally produced radicals.

The result of this reaction may produce some products by joining reactive oxygen species with

free radicals which are not active and not helpful for further oxidation [181] as follows:

ArO• + ROO• → ArOROO

2.8. Fruits as a Potential Source of Phenolic Antioxidants with

Multiple Medicinal Benefits

Among widespread plant sources, fruits are well known as an impressive source of

phenolic antioxidants [22, 182]. The medical application of fruits and vegetables may be

attributed to occurrence of high-value nutrients and phenolics. There is growing evidence in the

literature that supports the potential medicinal benefits of consumption of fruits due to their ability

and protective role in decreasing the risk of different health disorders [183-184]. Especially, the

25

intake of healthy diet containing vegetables and fruits exerts positively affect the antioxidant

potential and hence protect serum against lipid oxidation [10, 202].

Recently, the use of vegetables and fruits as ingredients of functional food and

nutraceuticals has gained significant consideration due to their nutritional value and source of

phenolic substances. The quality of food is gaining much attention and affected by various

parameters [203]. The healthy diet containing fruits and vegetables contributes many medicals

benefits due to the presence of high-value phytochemicals which can impart antioxidant activity

potential. These high-value phytochemicals may include vitamins, phenolic compounds,

flavonoids and phenolic acids [132, 134]. The antioxidant and antimicrobial and anti-carcinogenic

activity has been cridted to the availability of secondary metabolites like plant phenolics [190,

204].

The enhanced intake of various types of vegetables, fresh and dried form of fruits was

reported to be linked with reduce incidence of many ailments like heart diseases, cancer and other

immune related diseases [134, 205] . Some phytochemicals, especially antioxidant phenolics,

present in the heathy diet of vegetables, fruit juices and fruits were reported to exhibit various

biological and antioxidant activity to maintain the metabolic pathways and to detoxify the toxins

steering to the reduced incidence of many ailments [134]. To scavenge free radicals and ROS and

slowing down the process of oxidation, involved ageing related problems, plant plenolics have a

vital role [192, 206]. Santhakumar et al. [19] reported the positive association between the use of

vegetables, fresh and dried form of fruits and lowered incidence of heart diseases. Likely, Song et

al. [192] published the reduced incidence of many ailments including certain types of cancer with

the healthy food which contain abundance of fruits and vegetables. This type of healthy food can

offer strong serum antioxidant activity and many biological functions.

The use of healthy diet which contain fruits, juices, vegetables and beverages exerts many

health benefits including protection from ageing, immune related disorders, heart diseases. The

health benefits of the utilization of different types of vegetables, fresh and dried form of fuirts has

been credited to the occurrence of potent biologically active components including antioxidants,

individual sugars, dietary fiber, organic acid and water content [207]. Surprisingly, the enhanced

antioxidant activity to retard serum lipid oxidation was principally credited to the availibility of

potent phenolic antioxidants which have been reported in scavenging ROS and free radicals [208-

26

209]. These combined phytochemicals were reported to impart various biological activities

biological including antimicrobial, antioxidant and anti-cancer etc [134].

Various medical applications of the plant-based phenolic antioxidant compounds were

witnessed by different in vitro and in vivo experiments. There are lot of publications available in

the literature [132, 134, 190-191]. Many medical benefits have been reported to exhibit by the

phenolic compounds derived from tea leaves [190]. The data obtained from in vivo experiments

revealed the inverse relation has been established between the lower incidence of certain types of

cancer and healthy diet containing plant-bassed phenolic antioxidant compounds [116]. Due to

their antioxidant activity, consumption of phenolic rich fruits were reported to exerts various

biological functions to suppress heart diseases, immune related problems, certain type of cancer

and enhances antioxidant and antimicrobial activity [192-193].

The strong antioxidant and various biological functions (reduced lipid oxidation, heart

diseases etc) to protect against different ailments were reported to be attributed to the occurrence

of plant phenolics [194]. Lower incidence of certain type of cancer was attributed to the presence

of plant based natural phenolic substances that possesses the potential to neutralize ROS and free

radicals [134, 190]. The data obtained from different in vitro experiments revealed that phenolic

compounds were reported to hinder the growth of pathogens including bacterial and fungal strains

[195-197]. Another phenolic compound, proanthocyanidin, is noted to display anti-cataract

activity in rats [198]. As the cataract appearance is due to damage caused by oxidative stress [199];

in order to suppress the formation of cataract was attributed to antioxidant potential of phenolic

compounds [198]. The enhanced potential to scavenge free radical plays a vital role in antiulcer

activity. Among phenolic compounds, phenolic acids and flavonoids were found to be in

extensively tested compounds [10, 134, 200].

The literature is abundant of report about the extracts, derived from plant matrices, have

strong antioxidant, anti-cancer [134, 190], inhibition of inflammation [160], vasodilatory[193] and

bactericidal and fungicidal activities [196-197]. There has been reports showing higher antioxidant

activity with enhanced intake of plant phenolics [201]. The quantity and types of phenolic

antioxidant compounds in vegetables and fruits was reported to be dependent upon genotypes,

tissue, level of maturity of fruit and environmental conditions [188]. In addition, total phenolic

content was reported to be varied among various types of fruit species and parts of plant [182].

27

Surprisingly the fruits with peel were contained to have enhanced biological active components in

comparison to peeled fruits [15].

Zainudin et al. [12] investigated the variation in bioactive components and antioxidant

activity of fruits of Carambola at dissimilar ripening steps. Bioactives like total phenolics and

flavonoids, beta carotene, γ- and δ-tocopherol were found to be dominant in immature fruit while

those of individual sugars, TCC, α- and β-tocopherol were prominent in the ripe fruit. In another

recent study, aqueous and acidified methanol extracts from C. limonum fruit residues (CLFR)

were assessed for their TPC, antioxidant & antimutagenic capacities. Acidified methanol extracts

of CFLR exhibited higher extraction yield and antioxidant activity [10].

Lamien-Meda et al. [215] determined TPC, TFC and Aantioxidant activities ( AA) of fruit

extracts from wild plants using various in vitro tests like DPPH radical scavenging ability, FRAP

and ABTS methods. The fruit extracts, recovered with acetone, were found to possess significantly

(p

28

radical. The data of the present study was related with standard phenolics such as trolox, BHT,

BHA. The content of phenolic compounds (TPC) and total amount of flavonoids (TF) were

estimated in the investigated samples of extracts. The species such as S. excelsa, V. mrytillus, B.

vulgaris, T. orientalis, P. amphibium, and R. acetosella were found to exhibit maximum

antioxidant capacities [210]. Murillo et al. [211] investigated the antioxidant potential and total

polyphenolic content in the crude methanolic extracts of some cultivated and wild fruits by

estimating free radical hunting potential and Folin-Ciocalteu method, respectively. Among tested

fruits, jujube exhibited highest antioxidant activity and maximum phenolic contents. The

polyphenolic content of fruits were found to be strongly correlated with antioxidant properties.

Seal, [212] evaluated the effect of solvent extraction systems on TPC, flavonoids and

flavonols levels, reducing power and antioxidant capcity (measuring the capacity to scavenge

DPPH free radical) of the crude extracts from selected wild edible plants. The results revealed that

aqueous methanolic extracts had maximum quantity of total phenolic, flavonoid, flavonol and the

highest DPPH radical scavenging ability. Moreover, it was noted that the extracting solvent

significantly influenced the antioxidant activity of these wild edible plants.

The fruit extracts of different wild plants were analyzed for phytonutrients, antioxidants and

mineral composition. A notable (p

29

antitumor, anti-inflammatory, antioxidant, antibacterial and antifungal activities of plant

phenolics, extraction and sample preparation are the key steps. The main objective to prepare the

sample was reported to increase the extraction of targeted compounds by liberating it from the

interaction with different components of matrix used and thus improving the sensitivity and

performance of the method employed [200, 219].

In a schematic process of extraction and characterization of phenolics, collection of plant

samples is followed by employing methods to dry the sample such as drying in air,drying in oven

and drying be freezing the sample. The sample dried were allowed to grinding to get as specific

size in enhance are of surface. In this way, samples of smaller size will have a improved interaction

with the extracting solvent. Usually the samples of vegetables and fruits were allowed to get

homogenize with solvent used by employing electric blender, followed by recovery of biological

active components using suitable extracting solvents to yield solvent extraction which were further

fractioned to isolate and purify the sample. Purified and isolated samples were analyzed employing

state-of-the-art spectroscopic and chromatographic techniques [115]. Figure 2.9 represents a

comprehensive scheme to prepare and characterize the plant phenolics.

30

Plant Material

Pretreatment

Extraction

Purification

Analysis Analysis

Fig 2.9. Schematic diagram showing the process of extraction and characterization of

phenolics

Abbreviations: MAE, microwave-assisted extraction; UAE, u1trasound-assisted extraction; PFE,

pressurized f1uid extraction; PLE, pressurized 1iquid extraction; ASE, accelerated so1vent

extraction; SWE, subcritica1 water extraction; SFE, supercritica1 f1uid extraction; SPE, so1id

phase extraction; CCC, countercurrent chromatography; FD, FolinDenis method (FD), FC, Fo1in-

Cioca1teu method; GC, gas chromatography; LC, Liquid chromatography; FLU, fluorescence;

PDA, photodiode array; EAD, e1ectro-array detection; ECD, e1ectrochemica1 detection; MS,

mass spectrometric; NMR, nuc1ear magnetic res0nance [28].

Air-drying,

Freeze drying,

Milling, Grinding,

Homogenization

Maceration,Soxhlet

MAE, UAE,

PFE (PLE/ASE,

SWE, SFE)

SPE,

Column

Chromatography,

CCC

Spectrophotometric assays, FD,

F-C, etc,

GC/LC/Electromigration with

instrumental analysis (UV/VIS,

FLU, PDA, EAD, Voltammetry,

ECD, MS, NMR

31

2.10. Extraction of Antioxidant Phenolic Components

2.10.1 Extraction solvent

As shown in schematic diagram (Fig 2.9), the recovery of biologically active componnets

form different parts of plant is the key process for the proper use to develop modern medicine, diet

supplement and cosmo-nutraceuticals. Prior extraction process, plant material needs to be

processed and preconditioned by ant drying method, followed by grinding, mixing and allow to

homogenize. The utilization of appropriate extraction solvent and protocol to recover extractable

plant phenolics was reported to be vital step prior to determine antioxidant potential. The polar

extraction solvents were employed to recover extractable phenolic antioxidant compounds which

were reported to concentrate in certain part of plant body [115, 220, 221]. The determination of

chemical characterization of plant phenolics in different extract was reported to be affect due to

type of recoverable compounds, extraction solvent, technique used and moieties [116].

The phenolic antioxidant compounds were reported to be recovered from either processed

or fresh samples depending upon their physical state, nature and ease of method. Complete

extraction and recovery of phenolic compounds, with wide ranging structural variations and

bioactivities, from a specific plant material is a challenging task. The commonly employed

techniques to extract phenolics involve the selection and use of an appropriate extraction solvent.

Various factors were reported to affect recovery of extractable plant based phenolic compounds

such as solvent nature, time, ratio between solvent and sample and temperature.

The optimal extraction of plant based phenolic antioxidant compound was reported to vary

among different samples which depends upon the nature and type of active component. The

assortment of solvent to recover phytochemicals like MeOH, EtOH, aqueous mixtures [207] was

reported to affect extraction of phytochemical. The recovery of biologically active components

requires specific care due to their sensitivity to oxidize or degrade after getting heat from any

surrounding source. Various techniques have been used to recover phytochemicals from plant

matrices using appropriate solvent, type, nature of extractable components [28, 242].

Being easy, quick and with extensive application, solid sample immersed in liquid is one

of the extensively used method to make solvent extracts of the plant sample which contain

bioactive components. The amount of recovery of extractable phenolic antioxidant compound is

dependent on the nature of solvent, type of sample, extraction techniques, time and temperature.

Polar solvents (MeOH, EtOH etc.) with increased polarity were reported to be extensively used to

32

recover phenolic compounds from plant matrices such as root, shoot, leaves and flowers [222-

223]. Polar alcoholic solvents like MeOH and EtOH were reported to recover plant phenolic

antioxidant components successfully from various plant matrix including fruits like

strawberry[224], cherries [225], sweet cherry [226], mulberry [227], plum [228], mangoseed

kernel[229], citrus peel[230], andfruit peels[231].

It is well documented that different types of plant matrices cannot be processed to get

highest amount of phenolic antioxidant compounds using a single extracting solvent. Different

solvents were mixed in proper ration to make a to make effective, efficient combination of

solvents which would be able to recover maximum amount of phenolic antioxidant compound

[232]. Anwar et al. [233] reported the extraction of plant based extractable phenolic antioxidant

components using different plant matrices of rice, citrus peel, by employing polar solvent. Ozgen

et al. [234] extracted highest amount of plant based phenolic antioxidant compounds from plant

matrix of M. Morusnigra employing different solvent of varying polarity. Highest amount

antioxidant components were reported to be extrcated using aqueous mixture with methyl

alcohol from plant matrix of mulberry [ 235] , and strawberry [236]. Escarpa [237] extract

maximum amount of extractable phenolic antioxidant compounds using absolute methyl

alcohol, aqueous methyl alcohol from pears, apples; and beans, pomace and lentil, respectively.

Hakkinen et al. [238] published the method to recover the extractable phenolic antioxidant

components from different plant matrices including fruits employing aqueous methyl alcohol.

Bae and Suh, [239] recovered phenolic components in different fruit matrices employing

aqueous ethyl alcohol.

According to Sun and Ho, [240], methanol was noted to be the most operative extracting

solvent than acetone and hexane to recover plant based biologically active components from plant

matrices of oat bran. Likely, Siddhuraju and Becker, [23] and Chatha et al. [241] extracted

maximum amount of phenolic antioxidant compound from the plant matrix of leaves of M. oleifera

and rice bran employing aqueous methyl alcohol, respectively. Anwar et al. [233] extracted higher

amount of phenolic antioxidant components from various plant matrices including citurs peel,

leaves of guava, bran of rice, bran of wheat employing aqueous methyl alcohol. Sultana et al. [242]

recovered of phenolic antioxidant compound from different parts of various medicinal plant using

aqueous methyl alcohol. Shabir et al. [243] extracted highest amount of phenolic antioxidant

components from diferent part of a plant (D. regia) employing aqueous methyl alcohol.

33

Sajid et al. [195] also reported the recovery of phenolic compound from different parts of

P. pinnata employing aqueous methyl alcohol and ethyl alcohol. Manzoor et al. [15], reported the

maximum yield recovery of antioxidant components from the peel and pulp of pear using aqueous

methanol (80:20 methanol-water v/v). Anwar et al. [5] also reported 80% methanol to be most

appropriate extracting solvent to extract phenolic components from different parts of Ghaneri.

Sultana et al. [244] extracted phenolic compounds from the clove seeds using aqueous methanol.

Similarly, Zainudin et al. [12] published a article to exhibit the optimum recovery of extractable

phenolic compounds from the Carambola fruits using 100% methanol. Anahita et al. [245]

extracted phenolic compounds from the Pomegranate seed and juice using 70% ethanol. Anwar et

al. [196] reported aqueous methanol to be superior and effective solvent to recover antioxidant

extractable components from the leaves of different species of Mulberry.

Critically, the use of conventional organic solvent based liquid-solid extraction

(COSBLSE), although widely acceptable and a convenient approach on practical grounds, has

some limitations with regard to the concerns for end-use extracts/products safety and quality as

well as environmental pollution [28, 246]. Currently, there is increasing interest in functional food

and nutraceutical to develop valuable medicinal products [28]. Thus, in order to meet the

challenges of competitiveness of the globalized market and environment protection, there has been

increasing concern about the applications of eco-friendly and cost-effective green technologies for

bioactives extraction and product development with the main focus on the minimal use of organic

solvents [246-247]. In this regard, ultrasound and microwave-assisted green solvent extractions

involving the use hot water [247], pressurized hot water [248-249], agro-or bio-solvents including

glycerol [250], ethanol [251], ionic liquids [252], hydrotropes [253] and c