� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � Camellia sinensis � � � � � � � � �

THESIS SUBMITTED TO THE ANNAMALAI UNIVERSITY IN PARTIAL FULFILMENT

OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF ! " # $ " % " & ' ( ) * " + " ' ( , ) - . " $ / - ,By

S.R. SENTHILKUMAR, M.Sc., B.Ed., M.Phil., 0 1 2 2 3 4 1 3 5 6 5 7 8 9 8 8 8 :

Under the guidance of

Dr. T. SIVAKUMAR, ; < = > < ? ; < @ A B C < ? @ A < D < ? E F F G F H I J H K L 1 M N F F 1 LDEPARTMENT OF BOTANY

2014

ANNAMALAI UNIVERSITY

DECLARATION

I hereby declare that the thesis entitled “GREEN SYNTHESIS OF

SILVER (Ag) AND ZINC OXIDE (ZnO) NANOPARTICLES AND STUDIES ON

PHYTOCHEMICALS, ANTIOXIDANT AND ANTIMICROBIAL POTENTIALS OF

INDIAN GREEN TEAS (Camellia sinensis (L.) Kuntze)” submitted for the

degree of Doctor of Philosophy in Botany is my original work and the

dissertation has not previously formed the basis for the award of any

degree, diploma, associateship, fellowship or similar other titles. It has not

been submitted to any other University or institution for the award of any

degree or diploma.

Place : O P Q R Q P S T U V W X Y Z [ \ R ]Date : Signature of the Candidate

ANNAMALAI UNIVERSITY

(DST-FIST & UGC-SAP SPONSORED DEPARTMENT) ^ _ ` a ` b c d e f g h e i j k l m n l o k l p q r s l o p q l t l oAssistant Professor Annamalainagar 608 002

Department of Botany Tamil Nadu, INDIA

Mobile : +91 9486420513 E-mail: drtsiva_19@rediffmail.com

This is to certify that the thesis entitled “GREEN SYNTHESIS OF

SILVER (Ag) AND ZINC OXIDE (ZnO) NANOPARTICLES AND STUDIES ON

PHYTOCHEMICALS, ANTIOXIDANT AND ANTIMICROBIAL POTENTIALS OF

INDIAN GREEN TEAS (Camellia sinensis (L.) Kuntze)” is a bonafide record

of research work done by Mr. S.R. SENTHILKUMAR (Roll No. 1219030005)

Research Scholar, Department of Botany, Annamalai University, Annamalainagar,

under my guidance during the period u v w u x u v w y and that this thesis

has not previously formed the basis for the award of any degree, diploma,

associateship, fellowship or other similar title to the candidate.

This is also to certify that the thesis represents the independent work

of the candidate.

Place : Annamalainagar O U Q P W z \ Y Z [ \ R ]Date : Research Supervisor

I feel immense pleasure in expressing my sincere gratitude to my guide

Dr. T. SIVAKUMAR, Assistant Professor, Department of Botany, Annamalai University, for

assigning me this interesting and challenging problem and for his excellent guidance,

sustained encouragement and effective discussions with utmost patience. His motivation

and strict enforcement of time bound schedule of carrying out this work enabled me to

thrive forward and successfully complete this work in time.

I express my special and sincere thanks to Dr. K. ARUMUGAM, Professor and Head,

Department of Botany, Annamalai University for his encouragements, steering advises

and timely helps to achieve this goal without any snag.

It is my duty and pleasure to thank Dr. R. PANNEERSELVAM, Former Professor and

Head, Department of Botany, Former Dean, Faculty of Science, Annamalai University

for providing me this valuable opportunity to do the Ph.D., programme in this esteemed

university and for his fruitful suggestions for performing the work.

I feel happy to express my heartfelt thanks to Dr. R. SRIDHARAN, Associate

Professor and Dr. G.M. ALAGULAKSHMANAN, Assistant Professor, Department of Botany,

Annamalai University for their kind help.

I register my thanks to Dr. V. UDHYA, Professor, Department of Medical Microbiology,

Raja Muthiah Medical College, Annamalai University, for providing facilities to carry out

antimicrobial studies.

From the depth of my heart, I submit my sincere thanks to the Authorities of

Annamalai University for admitting me to do this programme.

Finally, I extend my boundless thanks to M/S. RAJNOOR COMPUTERS, Annamalai Nagar,

for their neat execution of the thesis.

Place : Annamalainagar

Date : (S.R. SENTHILKUMAR)

CHAPTER TITLE PAGE

LIST OF TABLES … i

LIST OF FIGURES … iii

LIST OF PLATES … iv

LIST OF ABBREVIATIONS … v

ABSTRACT … vii

1 INTRODUCTION … 1-21

1.1 Nanoparticles … 1

1.2 Green synthesis of nanoparticles … 2

1.2.1 Merits of green synthesis … 3

1.3 Antimicrobial application of nanoparticles … 5

1.4 Antimicrobial activities … 7

1.4.1 Silver nanoparticles (Ag NPs) … 10

1.4.2 Zinc oxide nanoparticles (ZnO NPs) … 10

1.4.3 Green tea (Camellia sinensisi) … 11

1.5 Free radicals … 12

1.6 Antioxidants … 14

1.7 Antioxidant assays … 19

2 REVIEW OF LITERATURE … 22-44

2.1 Review articles related to the green synthesis of nanoparticles

… 22

2.2 Reviews on the green synthesis and applications of silver nanoparticles (Ag NPs)

… 24

2.3 Reviews on the green synthesis and antimicrobial activities of zinc oxide nanoparticles (ZnO NPs)

… 28

2.4 Reviews on antioxidant activity … 30

2.5 Reviews related to GC-MS analysis of phytochemicals … 34

2.6 Reviews related to FT-IR analysis of phytochemicals … 37

2.7 Reviews related to green tea (Camellia sinensis) … 38

3 MATERIALS AND METHODS … 45-65

3.1 Introduction … 45

3.2 Collection and preparation of plant materials … 45

3.2.1 Greengram (Vigna radiata (L.) Wilczek) … 45

3.2.2 Green tea (Camellia sinensis) … 47

3.3 General laboratory procedures … 49

3.3.1 Cleaning and sterilization of glasswares … 49

3.3.2 Chemicals … 50

CHAPTER TITLE PAGE

3.4 Phytochemical analysis … 50

3.4.1 Preliminary screening tests … 50

3.4.2 GC-MS analysis of phytochemiccals … 53

3.4.3 Folin-Ciacalteu reagent method … 54

3.4.4 Aluminium chloride method … 55

3.5 Green synthesis of nanoparticles … 56

3.5.1 Flowchart for green synthesis … 56

3.5.2 Silver nanoparticles (Ag NPs) … 57

3.5.3 Zinc oxide nanoparticles (ZnO NPs) … 57

3.6 Antimicrobial assays … 58

3.6.1 Pathogenic bacterial species … 58

3.6.2 Pathogenic fungal species … 59

3.6.3 Agar well diffusion method … 59

3.6.4 Measurement of zone of inhibition … 60

3.7 Antioxidant assays … 60

3.7.1 DPPH free radical scavenging assay … 60

3.7.2 FRAP assay … 62

3.8 Characterization techniques … 63

3.8.1 UV-Vis spectroscopic analysis … 63

3.8.2 X-ray diffraction study … 63

3.8.3 FE-SEM analysis … 64

3.8.4 FT-IR spectroscopic analysis … 64

3.9 Statistical analysis … 65

4 RESULTS … 66-145

4.1 Green synthesis of silver nanoparticles … 66

4.1.1 Preliminary screening tests of greengram sprout … 66

4.1.2 GC-MS analysis of phytochemicals in greengram sprout … 66

4.1.3 Visual and UV-Vis monitoring of Ag NPs formation … 74

4.1.4 XRD pattern of Ag NPs … 74

4.1.5 FE-SEM morphology of Ag NPs … 77

4.1.6 FT-IR spectra of greengram sprout and Ag NPs … 77

4.1.7 Antibacterial assays … 81

4.1.8 Antifungal assays … 81

CHAPTER TITLE PAGE

4.2 Green synthesis of zinc oxide nanoparticles … 86

4.2.1 Photographs of green tea plant, dried leaves and synthesized ZnO NPs

… 86

4.2.2 Preliminary screening test of green tea … 86

4.2.3 GC-MS analysis of green tea … 86

4.2.4 UV-Vis spectrum of ZnO nanoparticle … 89

4.2.5 XRD analysis of ZnO NPs … 89

4.2.6 FE-SEM morphology of ZnO NPs … 89

4.2.7 FT-IR spectra of-green tea extract and ZnO NPs … 93

4.2.8 Antibacterial assays … 95

4.2.9 Antifungal assay … 95

4.3 Phytochemical analysis of Indian green teas … 99

4.3.1 GC-MS analysis of green teas … 99

4.3.2 FT-IR analysis of phytochemical green teas … 121

4.4 Antioxidant potentials of Indian green teas … 125

4.4.1 Total phenolic contents-FCR method … 125

4.4.2 Total flavonoids-aluminium chloride method … 127

4.4.3 DPPH-RSA assay … 129

4.4.4 FRAP – assay … 131

4.5 Correlation studies … 134

4.5.1 Correlation analysis on the antioxidant potentials of Indian green teas with their phenolic and flavonoid contents

… 134

4.5.2 Correlation analysis between FT-IR estimates and calorimetric data of the Indian green teas

… 138

4.6 Antimicrobial potential of Indian green teas … 141

5 DISCUSSION … 146-169

5.1 Characterization and antimicrobial activity of Ag NPs … 146

5.1.1 UV-Vis analysis … 146

5.1.2 XRD analysis … 146

5.1.3 FE-SEM analysis … 147

5.1.4 FT-IR studies … 147

5.1.5 Antimicrobial studies … 148

CHAPTER TITLE PAGE

5.2 Characterization and antimicrobial activity of ZnO NPs … 152

5.2.1 UV-Vis spectrum … 152

5.2.2 XRD studies … 152

5.2.3 FE-SEM analysis … 153

5.2.4 FT-IR analysis … 153

5.2.5 Antimicrobial studies … 154

5.3 Preliminary phytochemical tests … 158

5.4 GC-MS analysis of greengram sprout … 159

5.5 GC-MS analysis of green tea samples … 159

5.5.1 Pyrogallol (1,2,3-Benzenetriol) … 160

5.5.2 Quinic acid (1,3,4,5-Tetrahydroxycyclohexanecarbonyl) … 160

5.5.3 Coffeine (Alkaloid) … 161

5.5.4 Phytol (Deterpene) … 161

5.5.5 Theobromine [1H-purine-2,6-dione,3,7-dihydro-3,7-dimethyl]

… 162

5.5.6 Palmitic acid (n-hexadecanoic acid) … 162

5.5.7 Palmitic acid ethyl ester (hexadecanoic acid) … 162

5.5.8 -Linolenic acid (9,12,15-octadecatrienoic acid, (z,z,z)) … 162

5.6 FT-IR analysis of green tea samples … 163

5.7 Antioxidant activities of green tea samples … 164

5.8 Correlation studies … 165

5.8.1 Correlation analysis on the antioxidant potentials of Indian green teas with their phenolic and flavonoid contents

… 165

5.8.2 Correlation analysis between FT-IR estimates and calorimetric data of the Indian green teas

… 166

5.9 Antimicrobial activities of green tea samples … 168

6 SUMMARY … 170-173

REFERENCES … 174-196

LIST OF PUBLICATIONS … 197

i

LIST OF TABLES

Table No.

Captions Page No.

1.1 Properties of natural and synthetic antioxidants 19

4.1 Phytochemicals screening results of greengram sprout 67

4.2 Peak report derived from the chromatogram of the greengram sprout 70

4.3 GC-MS phytochemical analysis of greengram 71

4.4 XRD parameters of Ag NPs 77

4.5 Absorption band assignment for the FT-IR spectrum of greengram sprout sample

80

4.6 Antibacterial of green synthesized Ag NPs at different concentrations against pathogenic bacterial species

83

4.7 Antifungal of green synthesized Ag NPs at different concentrations against pathogenic fungal species

85

4.8 Phytochemicals screening results of green tea extracts 88

4.9 XRD parameters of ZnO nanoparticles 91

4.10 Antibacterial activity of green synthesized ZnO NPs at different concentrations against pathogenic bacterial species of clinical sources

97

4.11 Antifungal activity of green synthesized ZnO NPs at different concentrations against pathogenic bacterial species of clinical sources

99

4.12 Pea report of MOON tea compounds 101

4.13 Peak report of TAN tea compounds 103

4.14 Peak report of GT tea compounds 105

4.15 Peak report of TET tea compounds 107

4.16 Peak report of KOL tea compounds 109

4.17 Peak report of ASS tea compounds 111

ii

Table No.

Captions Page No.

4.18 Major phytochemicals (area percentage >1) of MOON tea 112

4.19 Major phytochemicals (area percentage >1) of TAN tea 113

4.20 Major phytochemicals (area percentage >1) of GT tea 114

4.21 Major phytochemicals (area percentage >1) of TET tea 115

4.22 Major phytochemicals (area percentage >1) of KOL tea 116

4.23 Major phytochemicals (area percentage >1) of ASS tea 117

4.24 Major health beneficial phytochemicals of green tea samples analyzed by GC-MS

118

4.25 Infrared vibrational bands of green tea samples 124

4.26 Optical density values representing total phenolic contents of green teas and garlic acid for various concentrations

125

4.27 Optical density of values representing total flavonoids of the green teas for various concentrations

127

4.28 DPPH radical scavenging activities of the Indian green teas 129

4.29 FRAP assay values of the Indian green teas 131

4.30 DPPH radical scavenging activity (RSA), ferric reducing antioxidant power (FRAP), total phenolic content (TPC) and total flavonoids (TF) of the green tea samples

133

4.31 Pearson’s correlation coefficient (R) matrix analysis between various parameters of green tea sample

134

4.32 FT-IR absorbance (%) of selected vibrational bands of the green tea samples

138

4.33 FT-IR and colorimetric data of the green tea samples 139

4.34 Pearson’s linear correlation coefficient (R) matrix analysis between FT-IR and colorimetric data of green tea samples

140

4.35 Antibacterial activities of the green tea samples 143

4.36 Antifungal activities of the green tea samples 145

iii

LIST OF FIGURES

Figure No.

Captions Page No.

1.1 Scope of antimicrobial applications of nanoparticles 6

1.2 Probable mechanisms of antimicrobial actions 9

1.3 Classification of enzymatic and non-enzymatic antioxidants 17

3.1 Flowchart for green synthesis, characterization and application of nanoparticles

56

4.1 Chromatogram of greengram sprout 69

4.2 UV-Vis absorption spectrum of Ag NPs synthesized using greengram sprout extract

76

4.3 XRD pattern of Ag NPs synthesized using greengram sprout extract 76

4.4 FE-SEM micrograph of green synthesized Ag NPs 78

4.5 FT-IR spectra of (a) greengram sprout extract and (b) synthesized Ag NPs 79

4.6 UV-Vis spectrum of synthesized ZnO NPs 90

4.7 XRD spectra of a) ‘as prepared’ and b) calcined (100 C) ZnO NPs 90

4.8 FE-SEM micrograph of green synthesized ZnO NPs 92

4.9 FT-IR spectra of a) green tea extract and b) synthesized ZnO NPs 94

4.10 Chromatogram of MOON tea 100

4.11 Chromatogram of TAN tea 102

4.12 Chromatogram of GT tea 104

4.13 Chromatogram of TET tea 106

4.14 Chromatogram of KOL tea 108

4.15 Chromatogram of ASS tea 110

4.16 FT-IR spectra of green tea samples: (a) MOON, (b) TAN, (c) GT, (d) TET, (e) KOL and (f) ASS

123

4.17 Dose-response curves of total phenolic contents of green tea samples 126

4.18 Dose-response curves of total flavonoids of green tea samples 128

4.19 Dose-response curves of DPPH radical scavenging activity (%) and the standard ascorbic acid

130

4.20 Dose-response curves of FRAP assay of the green tea samples and the standard ascorbic acid

132

4.21 Correlation between DPPH radical scavenging activity with (a) TPC and (b) TF of the green tea samples

135

4.22 Correlation between FRAP activity with (a) TPC and (b) TF of the green tea samples

136

4.23 Correlation between DPPH and FRAP assays 137

5.1 Simplified diagram representing various modes of action of Ag NPs on bacteria

151

iv

LIST OF PLATES

Plate No.

Captions Page No.

4.1 Phytochemicals screening of test greengram sprout 68

4.2 (a) Greengram plant, (b) seeds, (c) germinated seeds with sprout and (d) various stages of Ag NPs formation

75

4.3 Antibacterial activity of Ag NPs against (a) K. pneumoniae, (b) P. aeruginosa, (c) E. coli and (d) S. aureus at different concentrations. P - Positive control

82

4.4 Antifungal activity of Ag NPs against (a) A. flavus, (b) A. niger, (c) A. fumigatus and (d) M. gypseum at different concentrations. P - Positive control

84

4.5 a) Green tea plant, b) leaves and c) synthesized ZnO NPs 87

4.6 Phytochemicals screening test of green tea extracts 88

4.7 Antibacterial activity of ZnO NPs against a) K. penumoniae, b) P. aeruginosa, c) E. coli and d) S. aureus at different concentrations. P – Positive control; N – Negative control

96

4.8 Antifungal activity of ZnO NPs against a) A. fumigatus, b) Penicillium sp., c) A. flavus and d) A. niger at different concentrations. P – Positive control; N – Negative control

98

4.9 Antibacterial activities of green tea samples 142

4.10 Antifungal activities of green tea samples 144

v

mm - millimetre

nm - nanometre

g - gram

mg - milligram

g - microgram

L - litre

mL - millilitre

L - microlitre

Ag NP - Silver nanoparticles

ZnO NP - Zinc oxide nanoparticles

AAE - Ascorbic acid equivalent

GAE - Gallic acid equivalent

QE - Quercetin equivalent

R - Pearson’s correlation coefficient

FWHM - Full with at half maximum

- Wavelength

D - Size of nanoparticle

FT-IR - Fourier transform-infrared spectroscopy

GC-MS - Gas chromatography mass spectroscopy

FE-SEM - Field emission-scanning electron microscopy

XRD - X-ray diffraction

UV-Vis - UV-visible spectroscopy

vi

TPC - Total phenolic content

TF - Total flavonoids

DPPH - 1,1-Diphenyl-2-picrylhydrazyl

RSA - Radical scavenging activity

FRAP - Ferric reducing antioxidant power

EC - Epicatechin

ECG - Epicatechingallate

EGC - Epigallocatechin

EGCG - Epigallocatechingallate

DNA - Dinucleic acid

RNA - Ribonucleic acid

EFA - Essential fatty acid

ROS - Reactive oxygen species

RNS - Reactive nitrogen species

MOON -

TAN -

GT - Label of commercial green teas TET -

KOL -

ASS -

Abstract

vii

ABSTRACT

The thesis primarily consists of two topics. First one is the

plant extracts mediated green synthesis and characterization of

silver (Ag) and zinc oxide (ZnO) nanoparticles and studies on their

antimicrobial activities against selected pathogenic bacterial and

fungal species. The second one is concerned with the phytochemical

analysis of greengram (Vigna radiata) sprout and commercial Indian

green teas (Camellia sinensis), for the qualitative and quantitative

identification of bioactive components and studies on their antioxidant

and antimicrobial activities.

Chapter 1 is of introductory nature and deals with the

basic concepts involved toward the plant extracts mediated green

synthesis of nanoparticles. The merits of the green synthesis and

biomedical applications of nanoparticles with special reference to

antimicrobial activities are briefly discussed. Fundamental aspects

about free radicals and antioxidants, their origin and classification are

furnished in detail.

A brief, yet comprehensive review of literature pertaining to

the green synthesis of silver and zinc oxide nanoparticles, GC-MS and

FT-IR analysis of phytochemicals, antioxidant and antimicrobial

activities of nanoparticles and plant extracts with special reference to

tea plant (Camellia sinensis) is provided in Chapter 2.

viii

Chapter 3 describes the common laboratory procedures for

collection and preparation of plant materials and their extracts,

various technological aspects of characterization methods, procedural

details of evaluation assays for determining phytochemicals,

antioxidant potentials and antimicrobial activities.

In Chapter 4, the results obtained from various analytical

methods, preliminary tests and evaluation assays are furnished. The

data and informations derived from the results are presented in

the form of Plates, Tables and Figures. The results of correlation

studies carried out to establish the relationships among the estimated

parameters are also provided.

Discussions on the results obtained regarding qualitative

and quantitative estimation of phytochemicals in the studied plant

extracts are provided in Chapter 5. The antimicrobial activities of the

green synthesized nanoparticles on selected pathogenic bacterial and

fungal species, antioxidant potentials of Indian green teas and their

correlations with total phenolic and flavonoid contents are also

discussed.

The conclusions obtained from the analysis of various

parameters of the plant extracts and green synthesized nanoparticles

are summarized in Chapter 6.