Biomedical Potential of Silver and Gold Nanoparticles Synthesized from Daphne Mucronata and Monotheca Buxifolia As

BIOMEDICAL POTENTIAL OF SILVER AND GOLD

NANOPARTICLES SYNTHESIZED FROM DAPHNE

MUCRONATA AND MONOTHECA BUXIFOLIA AS

NEW PRECURSORS

Ph. D Thesis

ASMA SHAH

CENTRE OF BIOTECHNOLOGY AND MICROBIOLOGY UNIVERSITY OF PESHAWAR

Session: 2012-2017 BIOMEDICAL POTENTIAL OF SILVER AND GOLD

NANOPARTICLES SYNTHESIZED FROMDAPHNE

MUCRONATA AND MONOTHECA BUXIFOLIAAS

NEW PRECURSORS

ASMA SHAH

Thesis submitted to University of Peshawar in partial fulfillment for the degree of Doctor of Philosophy in Biotechnology.

CENTRE OF BIOTECHNOLOGY AND MICROBIOLOGY UNIVERSITY OF PESHAWAR

Session: 2012-2017

AUTHOR’S DECLARATION

I, Asma Shah, hereby state that my PhD thesis titled “Biomedical potential of Silver and Gold Nanoparticles Synthesized from Daphne mucronata and Monotheca buxifolia as New Precursors” is my own work and not been submitted previously by me for tacking any degree from UNIVERSITY OF PESHAWAR or anywhere else in the country /world.

At any time if my statement is found to be incorrect, the university has the right to withdraw my PhD Degree.

Name of student: ASMA SHAH

PLAGIARISM UNDERTAKING

I solemnly declared that, research work presented in thesis title “Biomedical potential of Silver and Gold Nanoparticles Synthesized from Daphne mucronata and

Monotheca buxifolia as New Precursors” is solely my research work with no significant contribution from any other person. Small contribution/help where ever taken has been duly acknowledged and that complete thesis has been written by me.

I understand the zero-tolerance policy of the HEC and UNIVERSITY OF

PESHAWAR towards plagiarism. Therefore, I as an author of the above titled thesis declared that no portion of my thesis plagiarized and any material used as reference is properly referred/cited.

I undertake that if I found guilty of any formal plagiarism in the above titled thesis even after reward of Ph. D. Degree, the university reserves the right to withdraw/revoke my degree. The HEC and University has right to publish my name on the HEC/University website on which names of students are placed who submitted plagiarized thesis.

Name: ASMA SHAH

Student/Author signature: ______

In the Name of Allah, Most Gracious, Most Merciful

Dedication

This Dissertation is dedicated to my Family, my

Siblings, my Friends and my Teachers specially Prof.

Dr. Ghosia Lutfullah who supported me throughout

my research work, to my father Mr. Muhammad

Shah Khan, who not only raised and nurtured me but

also taxed himself dearly over the years for my

education and intellectual development.

And

To my mother, whose prayers are the source of

motivation and strength during moments of despair

and discouragement

TABLE OF CONTENTS

LIST OF TABLES ...... I

LIST OF FIGURES ...... VIII

LIST OF ABBREVIATIONS ...... XVI

SUMMARY ...... XX

1.0 INTRODUCTION AND LITERATURE REVIEW ...... 1

1.1 INTRODUCTION ...... 1

1.2 PLANT NATURAL PRODUCTS...... 1

1.2.1 Alkaloids ...... 2

1.2.2 Polyketides ...... 7

1.2.3 Phenylpropanoids ...... 8

1.2.4 Terpenoids ...... 9

1.3 DAPHNE MUCRONATA ...... 10

1.3.1 Phytochemistry ...... 10

1.3.2 Significance in medicine ...... 10

1.4 MONOTHECA BUXIFOLIA ...... 12

1.4.1 Phytochemistry ...... 12

1.4.2 Significance in medicines ...... 13

1.5 PLANT NATURAL PRODUCTS AND NANOTECHNOLOGY ...... 14

1.6 NANOPARTICLES ...... 16

1.6.1 Gold metal uses ...... 17

1.6.2 Silver metal uses ...... 20

1.7 SYNTHESIS OF GOLD AND SILVER NANOPARTICLES ...... 22

1.7.1 Chemical methods ...... 22

1.7.2 Physical methods ...... 23

1.7.3 Biological method ...... 25

1.8 CHARACTERIZATION OF SILVER AND GOLD NANOPARTICLES ...... 26

1.8.1 Transmission electron microscopy (TEM) ...... 27

1.8.2 Scanning electron microscope (SEM) ...... 27

1.8.3 Atomic Force microscopy (AFM) ...... 27

1.8.4 UV-visible spectroscopy ...... 28

1.8.5 X-ray diffractometry (XRD) ...... 29

1.8.6 Fourier transform infrared spectroscopy (FTIR) ...... 30

1.9 BIOLOGICAL EVALUATION OF NANOPARTICLES (GOLD/SILVER) ...... 31

1.9.1 Antimicrobial activity ...... 31

1.9.2 Haemagglutination activity ...... 34

1.9.3 Phytotoxic activity ...... 37

1.9.4 Insecticidal activity ...... 39

1.9.5 Nitric oxide free radical scavenging assay ...... 41

1.9.6 Anti-termite properties ...... 42

1.9.7 Brine shrimp lethality bioassay ...... 44

1.9.8 DNA damaging activity ...... 45

1.9.9 Hemolytic properties...... 45

1.9.10 Acute toxicity ...... 47

1.9.11 Animal studies ...... 48

1.11 OBJECTIVES ...... 50

2.0 METHODOLOGY ...... 51

2.1 COLLECTION OF PLANT MATERIALS ...... 51

2.2 EXTRACTION ...... 52

2.3 PRELIMINARY PHYTOCHEMICAL SCREENING, ELEMENTAL AND NUTRITIONAL ANALYSIS ...... 52

2.3.1. Preliminary phytochemical screening ...... 52

2.3.1.1 Preparation of plant extracts ...... 52

2.3.1.2 Carbohydrates tests ...... 53

2.3.1.2.1 Fehling’s test...... 53

2.3.1.2.2 Benedict’s test ...... 53

2.3.1.2.3 Molisch’s test ...... 53

2.3.1.2.4 Iodine test...... 53

2.3.1.3 Test for phenols and tannins ...... 54

2.3.1.4 Test for flavonoids ...... 54

2.3.1.5. Test for saponins...... 54

2.3.1.6. Tests for glycosides ...... 55

2.3.1.6.1 Liebermann’s test ...... 55

2.3.1.6.2 Salkowski’s test ...... 55

2.3.1.6.3 Keller-kilani test ...... 55

2.3.2 Elemental analysis (atomic absorption spectroscopy) ...... 57

2.3.3 Nutritional analysis ...... 60

2.3.3.1 Ash determination ...... 60

2.3.3.2 Analysis of moisture ...... 61

2.3.3.3 Analysis of protein ...... 62

2.3.3.4 Analysis of fat ...... 64

2.3.3.5 Analysis of crude fiber ...... 65

2.3.3.6 Carbohydrate contents...... 66

2.4 GREEN BIOGENIC SYNTHESIS OF SILVER AND GOLD NANOPARTICLES ...... 67

2.5 CHARACTERIZATION OF SYNTHESIZED SILVER AND GOLD NANOPARTICLES ...... 68

2.5.1 UV-vis spectroscopy ...... 68

2.5.2 Fourier transform infra-red (FTIR) spectroscopy ...... 68

2.5.3 X-ray diffraction (XRD) ...... 68

2.5.4 Energy dispersive X-ray detection (EDX) ...... 69

2.5.5 Scanning electron microscopy (SEM) ...... 69

2.5.6 Transmission electron microscopy (TEM) ...... 69

2.5.7 Thermo gravimetric/ differential thermal analysis (TG-DTA) ...... 70

2.6 BIOLOGICAL EVALUATION OF CRUDE EXTRACTS AND SYNTHESIZED AGNPS AND AUNPS ...... 71

2.6.1 Anti-oxidant activity ...... 71

2.6.2 Anti-bacterial activity ...... 72

2.6.2.1 Determination of percent inhibition ...... 72

2.6.2.2 Determination of minimum inhibitory concentration (MIC50) ...... 72

2.6.2.3 Determination of minimum bactericidal concentration (MBC) ...... 73

2.6.3 Anti-fungal activity ...... 74

2.6.4 Haemagglutination activity ...... 75

2.6.5 Phytotoxic activity ...... 76

2.6.6 Insecticidal activity ...... 77

2.6.7 Anti-termite activity ...... 78

2.6.8 Cytotoxic activity ...... 79

2.7 DNA DAMAGING, HEMOLYTIC AND ANTI-THROMBOLYTIC PROFILE ...... 80

2.7.1 Thrombolytic assay ...... 80

2.7.2 Hemolytic activity ...... 81

2.7.3 Mutagenicity test ...... 82

2.8 ANIMAL STUDIES ...... 85

2.8.1 Acute toxicity assay ...... 86

2.8.2 Anti-analgesic activity ...... 87

2.8.3 Anti-pyretic activity ...... 88

2.8.4 Gastro intestinal track motility (GIT motility) ...... 89

2.8.5 Anti-inflammatory activity ...... 90

2.9 BIOCHEMICAL PARAMETERS DETERMINATION ...... 91

2.9.1 Animal grouping and extract administration ...... 91

2.9.2 Determination of feed and water intake ...... 92

2.9.3 Preparation of serum ...... 92

2.9.4 Determination of biochemical parameters ...... 92

2.10 STATISTICAL ANALYSIS ...... 93

3.0 RESULTS ...... 94

3.1 PRELIMINARY PHYTOCHEMICAL SCREENING ...... 94

3.2 ELEMENTAL ANALYSIS (ATOMIC ABSORPTION SPECTROSCOPY) ...... 96

3.3 NUTRITIONAL ANALYSIS ...... 99

3.4 CHARACTERIZATION OF AGNPS AND AUNPS ...... 101

3.4.1 UV-vis spectroscopy ...... 101

3.4.2 Fourier transform infra-red (FTIR) spectroscopy ...... 104

3.4.3 X-ray diffraction (XRD) ...... 108

3.4.4 Energy dispersive X-ray detection (EDX) ...... 117

3.4.5 Scanning electron microscopy (SEM) ...... 122

3.4.6 Transmission electron microscopy (TEM) ...... 129

3.4.7 Thermo gravimetric-differential thermal analysis (TG-DTA) ...... 137

3.5 BIOASSAY OF PLANT FRACTIONAL CRUDE EXTRACTS AND BIOGENIC SYNTHESIZED AGNPS AND AUNPS ...... 145

3.5.1 Antioxidant activity ...... 145

3.5.2 Anti-bacterial activity ...... 154

3.5.3 Anti-fungal activity ...... 163

3.5.4 Haemagglutination activity ...... 169

3.5.5 Phytotoxic activity ...... 170

3.5.6 Insecticidal activity ...... 177

3.5.7 Anti-termite activity ...... 182

3.5.8. Cytotoxic activity ...... 188

3.6 DNA DAMAGING, HEMOLYTIC AND ANTI-THROMBOLYTIC PROFILE ...... 194

3.6.1 Thrombolytic activity ...... 194

3.6.2 Hemolytic activity ...... 197

3.6.3 Mutagenicity test (Ames assay) ...... 199

3.7 IN-VIVO PHARMACOLOGICAL STUDIES ...... 201

3.7.1 Acute toxicity ...... 201

3.7.2 Anti-analgesic activity ...... 204

3.7.3 Anti-pyretic activity ...... 216

3.7.4 GIT motility test ...... 227

3.7.5 Anti-inflammatory activity ...... 236

3.8 BIOCHEMICAL PARAMETERS ...... 245

4.0 DISCUSSION ...... 264

4.1 DISCUSSION ...... 264

CONCLUSION ...... 282

RECOMMENDATION ...... 284

REFERENCES ...... 285

LIST OF TABLES

Table 2.1 Composition of reagents used in phytochemical analysis56

Table 2.2 Specified conditions applied for detection of various elements59

Table 2.3 Set up of the fluctuation assay84

Table 2.4 Groups received treatment as follows:86

Table 2.5 Groups received the treatment as follows:87

Table 2.6 The groups received treatment as follows:88

Table 2.7 The groups received treatment as follows89

Table 2.8 The groups received treatment as follows:90

Table 3.1 Phytochemical screening of crude methanolic extracts of D.

mucronata(bark, leaves and root) and M. buxifolia (seed, leaves and fruit) ...... 95

Table 3.2 Atomic Absorption spectroscopy of crude aqueous extracts of D. mucronata

and M. buxifolialeaves (Mean ± StdDev mg/L) ...... 98

Table 3.3 Nutritional analysis of D. mucronata and M. buxifolialeaves in percent .. 100

Table 3.4 Crystalline Size Determination of D. mucronataleaves derived AgNPs using

Debye-Scherer’s Equation ...... 109

Table 3.5 Crystalline Size Determination of M. buxifolialeaves derived AuNPsusing

Debye-Scherer’s Equation ...... 113

Table 3.6 Crystalline Size Determination of M. buxifolialeaves derived AgNPs using

Debye-Scherer’s Equation ...... 114

Table 3.7 Antioxidant activity of D. mucronata barkand leaves extracts ...... 146

Table 3.8 Antioxidant activity of D. mucronata root extracts ...... 147

Table 3.9 EC50 values of D. mucronata bark, leaves and roots extracts ...... 147

Table 3.10 Antioxidant activity of M. buxifolia seed and fruit extracts ...... 149

Table 3.11 Antioxidant activity of M. buxifolialeaves extracts ...... 150

Table 3.12 EC50 values of M. buxifolia seed, leaves and fruit extracts ...... 150

Table 3.13 Antioxidant activity of D. mucronataleaves derived AgNPs and M.

buxifolialeaves derived AgNPs and AuNPs (p˂0.0001) ...... 152

Table 3.14 EC50 values of D. mucronataleaves derived AgNPs and M. buxifolialeaves

derived AgNPs and AuNPs ...... 152

Table 3.15 Anti-bacterial Activity of D. mucronata bark, leaves and roots ...... 157

Table 3.16 Anti-bacterial Activity of M. buxifolia seeds, leaves and fruits ...... 159

Table 3.17 Anti-bacterial activity of both plants mediated AgNPs and AuNPs ...... 161

Table 3.18 MIC50 and MBC of both plants mediated AgNPs and AuNPs ...... 161

Table 3.19 Anti-fungal activity of D. mucronata bark, leaves and roots crude

methanolic extract and fractions ...... 165

Table 3.20 Percent Anti-fungal activity of M. buxifolia seeds, leaves and fruits crude

methanolic extract and fractions ...... 166

Table 3.21 Anti-fungal activity of D. mucronataleaves derived AgNPs and M.

buxifolialeaves derived AgNPs and AuNPs ...... 168

Table 3.22 Phytotoxic activity of D. mucronata bark, root and leaves crude

methanolic extracts and fractions ...... 171

Table 3.23 Percent growth inhibition of L. minor by D. mucronata bark, root and

leaves crude extracts and fractions ...... 172

Table 3.24 Phytotoxic activity of M. buxifolia seed, fruit and leaves crude methanolic

extract and its fractions at 20 mg/ml...... 174

Table 3.25 Percent growth inhibition of L. minor by M. buxifolia crude methanolic

extract and its fractions...... 175

Table 3.26 Insecticidal activity of D. mucronata bark, roots and leaves extracts ..... 178

Table 3.27 Insecticidal activity of M. buxifolia seed, fruit and leaves extracts ...... 179

Table 3.28 Anti-termite activity of D. mucronata bark, roots and leaves ...... 183

Table 3.29 Anti-termite activity of M. buxifolia seed, fruit and leaves crude

methanolic extract and its fractions ...... 185

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Table 3.30 Brine shrimp lethality bioassay of D. mucronata bark, root and leaves

crude methanolic extract and fractions and derived NPs...... 189

Table 3.31 Brine shrimp lethality bioassay of M. buxifolia seed, fruit and leaves crude

methanolic extract its fractions and derived NPs ...... 191

Table 3.32 Effect of D. mucronata and M. buxifolialeaves aqueous extracts and their

derived AgNPs and AuNPs on clot lysis ...... 195

Table 3.33 Hemolytic activity of aqueous extracts of D. mucronataleaves and M.

buxifolialeaves and their derived AgNPs and AuNPs ...... 198

Table 3.34 Mutagenic activity of aqueous extracts of D. mucronataleaves and M.

buxifolialeaves and their derived AgNPs and AuNPs ...... 200

Table 3.35 Acute toxicity effect of crude methanolic extracts of D. mucronata bark,

root and leaves and derived AgNPs ...... 202

Table 3.36 Acute toxicity effects of crude methanolic extracts of M. buxifolia seed,

fruit and leaves and its fruit derived AgNPs and AuNPs ...... 203

Table 3.37 Acetic acid induced writhing test (mean± SEM) values of D. mucronata

bark, root and leaves extracts...... 206

Table 3.38 Acetic acid induced writhing test of M. buxifolia leaves, seed and fruit and

leaves derived AgNPs and AuNPs ...... 209

Table 3.39 Antipyretic activity of the plant D. mucronata leaves, bark and root

extracts ...... 217

Table 3.40 Antipyretic activity of the plant M. buxifolia leaves, seed and fruit extracts

...... 217

Table 3.41 Overall effects of 20% Brewer’s yeast induced on body temperature at

various dose of the plant extracts...... 218

Table 3.42 Overall effects of 20% Brewer’s yeast induced on body temperature at

various dose of the plant extracts...... 219

Table 3.43 Anti-pyretic activity of D. mucronata and M. buxifolialeaves derived

AgNPs and AuNPs ...... 224

Table 3.44 Overall effects of 20% Brewer’s yeast induced on body temperature at

various dose of the biogenic nanoparticles ...... 224

Table 3.45 GIT motility assay for D. mucronata bark, root, leaves and M. buxifolia

seed, fruit, leaves (SEM values are in the table as “±”) ...... 228

Table 3.46 GIT motility assay for D. mucronataleaves derived AgNPs and M.

buxifolialeaves derived AuNPs and AgNPs ...... 230

Table 3.47 Anti-inflammatory effects of D. mucronata leaves, bark and root ...... 237

Table 3.48 Anti-inflammatory effects of M. buxifolia leaves, seed and fruit ...... 237

Table 3.49 Anti-inflammatory effects of D. mucronata and M. buxifolia leaves

derived NPs ...... 239

Table 3.50 Effect of administration of methanolic extract of D. mucronataand M.

buxifolia leaves on some hematological parameters of male G. Pigs, (n=6) ...... 248 v

Table 3.51 Effect of administration of methanolic extract of D. mucronataand M.

buxifolialeaves on some hematological parameters of male Rabbits, (n=6) ...... 249

Table 3.52 Effect of administration of methanolic extracts of D. mucronataand M.

buxifolialeaves on the weight of mail G. Pigs and Rabbits, (n=6) ...... 250

Table 3.53 Liver and kidney function parameters of male G. Pigs administered with

methanolic extracts of D. mucronataand M. buxifolia leaves, (n=6) ...... 251

Table 3.54 Liver and kidney function parameters of male Rabbits administered with

methanolic extracts of D. mucronataleaves and M. buxifolia leaves, (n=6) ...... 252

Table 3.55 Feed intake (g) of male G. Pigs administered with methanolic extracts of

D. mucronataand M. buxifolia leaves, (n=6) ...... 253

Table 3.56 Feed intake (g) of male Rabbits administered with methanolic extracts of

D. mucronataand M. buxifolia leaves, (n=6) ...... 253

Table 3.57 Water intake (ml) of male G. Pigs administered with methanolic extracts

of D. mucronataleaves and M. buxifolia leaves, (n=6) ...... 254

Table 3.58 Water intake (ml) of male Rabbits administered with methanolic extracts

of D. mucronataand M. buxifolia leaves, (n=6) ...... 254

Table 3.59 Effect of administration of D. mucronataand M. buxifolialeaves derived

AgNPs and AuNPs on some hematological parameters of male G. Pigs, (n=6) 257

Table 3.60 Effect of administration of D. mucronataleaves and M. buxifolia leaves

derived AgNPs and AuNPs on some hematological parameters of male Rabbits,

(n=6) ...... 258

Table 3.61 Effect of administration of D. mucronataand M. buxifolia leaves derived

AgNPs and AuNPs on the weight of mail G. Pigs and Rabbits, (n=6) ...... 259

Table 3.62 Liver and kidney function parameters of male G. Pigs administered with

D. mucronataand M. buxifolialeaves derived AgNPs and AuNPs, (n=6) ...... 260

Table 3.63 Liver and kidney function parameters of male Rabbits administered with

D. Mucronate and M. buxifolialeaves derived AgNPs and AuNPs, (n=6) ...... 261

Table 3.64 Feed intake (g) of male G. Pigs administered with D. mucronataand M.

buxifolialeaves derived AgNPs and AuNPs, (n=6) ...... 262

Table 3.65 Feed intake (g) of male Rabbits administered with D. mucronataand M.

buxifolialeaves derived AgNPs and AuNPs, (n=6) ...... 262

Table 3.66 Water intake (ml) of male G. Pigs administered with D. mucronataand M.

buxifolialeaves derived AgNPs and AuNPs, (n=6) ...... 263

Table 3.67 Water intake (ml) of male Rabbits administered with D. mucronataand M.

buxifolialeaves derived AgNPs and AuNPs, (n=6) ...... 263

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LIST OF FIGURES

Figure1.1 Structures of theobromine, theophylline and paraxanthine ...... 3

Figure 1.2 Structure of Cocaine ...... 4

Figure 1.3 Structure of Calystegine ...... 5

Figure 1.4 Structure of Benzoxazinone ...... 7

Figure1.5 Pentagonal cross-section, decahedral, cubic,oCtahedral, tetrahedral and

truncated tetrahedral particles, and platelets nanoparticle shapes ...... 18

Figure 3.1 D. mucronataleaves derived AgNPsUV-absorption (λmax) 425 nm 102

Figure 3.2 M. buxifolialeaves derived AgNPs UV-absorption (λmax) 405 nm 102

Figure 3.3 M. buxifolialeaves derived AuNPs UV-absorption (λmax) 540 nm 103

Figure 3.4 FTIR spectra of D. mucronataleaves aqueous extract and D.

mucronataleaves derived AgNPs 105

Figure 3.5 FTIR Spectra of M. buxifolia leaves aqueous extract, M. buxifolialeaves

derived AgNPs and M. buxifolialeaves derived AuNPs 107

Figure 3.6 XRD analysis of D. mucronataleaves dried powder 110

Figure 3.7 XRD analysis of D. mucronataleaves aqueous extract 110

Figure 3.8 XRD analysis of D. mucronataleaves derived AgNPs 111

viii

Figure 3.9 XRD analysis of M. buxifolialeaves dried Powder 115

Figure 3.10 XRD analysis of M. buxifolia leaves aqueous extract 115

Figure 3.11 XRD analysis of M. buxifolialeaves derived AgNPs 116

Figure 3.12 XRD analysis of M. buxifolialeaves derived AuNPs 116

Figure 3.13 EDX Profile (elemental composition) of dried D. mucronata leaves 118

Figure 3.14 EDX Profile (elemental composition) of D. mucronataleaves derived

AgNPs 118

Figure 3.15 EDX Profile (elemental composition) of dried M. buxifolia leaves 120

Figure 3.16 EDX Profile (elemental composition) of M. buxifolialeaves derived

AgNPs 120

Figure 3.17 EDX Profile (elemental composition) of M. buxifolialeaves derived

AuNPs 121

Figure 3.18 SEM image (shape and rough estimation of size) of D. mucronataleaves

derived AgNPs at 0.5 µm 123

Figure 3.19 SEM image (shape and rough estimation of size) of D. mucronataleaves

derived AgNPs at 0.2 µm 123

Figure 3.20 SEM image (shape and rough estimation of size) of D. mucronataleaves

derived AgNPs at 0.1 µm 124

Figure 3.21 SEM image (shape and rough estimation of size) of M. buxifolialeaves

derived AgNPs at 0.5 µm 126

Figure 3.22 SEM image (shape and rough estimation of size) of M. buxifolialeaves

derived AgNPsat 0.2 µm 126

Figure 3.23 SEM image (shape and rough estimation of size) of M. buxifolia leaves

derived AgNPsat 0.1 µm 127

Figure 3.24 SEM image (shape and rough estimation of size) of M. buxifolialeaves

derived AuNPsat 0.5 µm 127

Figure 3.25 SEM image (shape and rough estimation of size) of M. buxifolialeaves

derived AuNPsat 0.2 µm 128

Figure 3.26 SEM image (shape and rough estimation of size) of M. buxifolialeaves

derived AuNPsat 0.1 µm 128

Figure 3.27 TEM image (exact size and shape) of D. mucronataleaves derived AgNPs

at 30 nm 130

Figure 3.28 TEM image (exact size and shape) of D. mucronataleaves derived AgNPs

at 8 nm 130

Figure 3.29 TEM image (exact size and shape) of M. buxifolialeaves derived AgNPs

at 100 nm 132

Figure 3.30 TEM image (exact size and shape) of M. buxifolialeaves derived AgNPsat

30 nm 132

Figure 3.31 TEM image (exact size and shape) of M. buxifolialeaves derived AgNPs

at 8 nm, fringes evident in spherical NPs 133

Figure 3.32 TEM image (exact size and shape) of M. buxifolialeaves derived AuNPsat

500 nm 135

Figure 3.33 TEM image (exact size and shape) of M. buxifolialeaves derived AuNPs

fringes evident in hexagonal NPs 135

Figure 3.34 TEM image (exact size and shape) of M. buxifolialeaves derived AuNPs

at 100 nm, fringes evident in Nano prisms 136

Figure 3.35 TGA profile (weight loss) of dried D. mucronata leaves 138

Figure 3.36 TGA profile (weight loss) of D. mucronataleaves derived AgNPs 138

Figure 3.37 TGA profile (weight loss) of dried M. buxifolia leaves 139

Figure 3.38 TGA profile (weight loss) of M. buxifolialeaves derived AgNPs 139

Figure 3.39 TGA profile (weight loss) of M. buxifolialeaves derived AuNPs 140

Figure 3.40 DTA profile (endothermic and exothermic reactions) of dried D.

mucronata leaves 142

Figure 3.41 DTA profile (endothermic and exothermic reactions) of D.

mucronataleaves derived AgNPs 142

Figure 3.42 DTA (endothermic and exothermic reactions) profile of dried M. buxifolia

leaves 143

Figure 3.43 DTA profile (endothermic and exothermic reactions) of M. buxifolia

leaves derived AgNPs 143

Figure 3.44 DTA profile (endothermic and exothermic reactions) of M.

buxifolialeaves derived AuNPs 144

Figure 3.45 Percent antioxidant activity of D. mucronata and M. buxifolialeaves

extracts and their derived NPs 153

Figure 3.46 Anti-bacterial activity of D. mucronata and M. buxifolialeaves aqueous

extract and derived AgNPs and AuNPs 162

Figure 3.47 Percent growth inhibition of L. minorby D. mucronataand M. buxifolia

leaves extract and derived NPs 176

Figure 3.48 Insecticidal activity of D. mucronataleaves aqueous extract and derived

AgNPs 181

Figure 3.49 Insecticidal activity of M. buxifolialeaves aqueous extract and M.

buxifolialeaves derived AgNPs and AuNPs 181

Figure 3.50 Anti-termites activity of D. mucronataleaves aqueous extract and derived

AgNPs 187

Figure 3.51 Anti-termites activity of M. buxifolialeaves and derived Ag and AuNPs

187

Figure 3.52 Brine shrimp lethality bioassay of D. mucronata and M. buxifolialeaves

aqueous extract and their derived NPs 193

xii

Figure 3.53 Mean effect of D. mucronataand M. buxifolia leaves aqueous extracts and

their derived AgNPs and AuNPs on clot lysis 196

Figure 3.54 Acetic acid induced writhing test values of D. mucronata and M.

buxifolialeaves and their derived NPs 210

Figure 3.55 Acetic acid induced writhing test for D. mucronataleaves methanolic

extract 211

Figure 3.56 Acetic acid induced writhing test for D. mucronata bark methanolic

extract 211

Figure 3.57 Acetic acid induced writhing test for D. mucronata roots methanolic

extract 212

Figure 3.58 Acetic acid induced writhing test for D. mucronataleaves derived AgNPs

212

Figure 3.59 Acetic acid induced writhing test for M. buxifolialeaves crude methanolic

extract 213

Figure 3.60 Acetic acid induced writhing test for M. buxifolia seed crude methanolic

extract 213

Figure 3.61 Acetic acid induced writhing test for M. buxifolia fruit crude methanolic

extract 214

Figure 3.62 Acetic acid induced writhing test for M. buxifolialeaves derived AuNPs

214

xiii

Figure 3.63 Acetic acid induced writhing test for M. buxifolialeaves derived AgNPs

215

Figure 3.64 Anti-pyretic activity of D. mucronataleaves methanolic extract 220

Figure 3.65 Anti-pyretic activity of D. mucronata bark methanolic extract 220

Figure 3.66 Anti-pyretic activity of D. mucronata root methanolic extract 221

Figure 3.67 Anti-pyretic activity of M. buxifolialeaves methanolic extract 221

Figure 3.68 Anti-pyretic activity of M. buxifolia seed methanolic extract 222

Figure 3.69 Anti-pyretic activity of M. buxifolia fruit methanolic extract 222

Figure 3.70 Anti-pyretic activity of D. mucronataleaves derived AgNPs 225

Figure 3.71 Anti-pyretic activity of M. buxifolialeaves derived AuNPs 225

Figure 3.72 Anti-pyretic activity of M. buxifolialeaves derived AgNPs 226

Figure 3.73 GIT motility assay comparison of plants crude extracts and derived NPs

231

Figure 3.74 GIT Motility of D. mucronata bark crude methanolic extract 231

Figure 3.75 GIT Motility of D. mucronataleaves crude methanolic extract 232

Figure 3.76 GIT Motility of D. mucronata root crude methanolic extract 232

Figure 3.77 GIT Motility of M. buxifolia fruit crude methanolic extract 233

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Figure 3.78 GIT Motility of M. buxifolialeaves crude methanolic extract 233

Figure 3.79 GIT Motility of M. buxifolia seed crude methanolic extract 234

Figure 3.80 GIT Motility of D. mucronataleaves derived AgNPs 234

Figure 3.81 GIT Motility of M. buxifolialeaves derived AgNPs 235

Figure 3.82 GIT Motility of M. buxifolialeaves derived AuNPs 235

Figure 3.83 Anti-inflammatory effects of D. mucronata and M. buxifolia leaves and

their derived NPs 240

Figure 3.84 Anti-inflammatory activity of D. mucronataleaves methanolic extract 240

Figure 3.85 Anti-inflammatory activity of D. mucronata bark methanolic extract 241

Figure 3.86 Anti-inflammatory activity of D. mucronata root methanolic extract 241

Figure 3.87 Anti-inflammatory activity of M. buxifolia seed methanolic extract 242

Figure 3.88 Anti-inflammatory activity of M. buxifolialeaves methanolic extract 242

Figure 3.89 Anti-inflammatory activity of M. buxifolia fruit methanolic extract 243

Figure 3.90 Anti-inflammatory activity of D. mucronataleaves derived AgNPs 243

Figure 3.91 Anti-inflammatory activity of M. buxifolialeaves derived AgNPs 244

Figure 3.92 Anti-inflammatory activity of M. buxifolialeaves derived AuNPs 244

LIST OF ABBREVIATIONS

Full name Abbreviation Monotheca buxifolia M. buxifolia Daphne mucronata D. mucronata Nanoparticles NPs Atomic absorption spectroscopy AAS

Silver nitrate AgNO3

Gold chloride AuCl4 Scanning Electron Microscopy SEM Transmission Electron Microscopy TEM Fourier Transform Infra-Red Spectroscopy FTIR X-Ray Diffraction(XRD)X-Ray Diffraction XRD Thermo gravimetric-Differential Thermal Analysis TG-DTA Energy Dispersive X-Ray Detection EDX Silver nanoparticles AgNPs Gold nanoparticles AuNPs Gastro Intestinal Track motility GIT Surface Plasmon Resonance SPR Gold Au Silver Ag Acenatobacter baumanni A. baumanni Morganella morganii M. morganii Escherichia coli E. coli Pseudomonas aeruginosa P. aeruginosa Vancomycin-Resistant Staphylococcus aureus VRSA Proteus vulgaris P. vulgaris Candida albicans C. albicans Aspergillus niger A. niger Fusarium oxysporum F. oxysporum Aspergillus flavus A. flavus Aspergillus parasiticus A. parasiticus Penicillium digitatum P. digitatum Lemna minor L. minor Tribolium castaneum T. castaneum Rhyzopertha dominica R. dominica Callosobruchus analis C. analis Heterotermes indicola H. indicola Artemia salina A. salina Before Christ BC Central Nervous System CNS National Aeronautics and Space Administration NASA Methicillin Resistant Staphylococcus aureus MRSA

Chloroauric acid HAuCl4

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Atomic force microscopy AFM X-ray photoelectron spectroscopy XPS Dynamic light scattering DLS Surface Enhanced Raman spectroscopy SERS Full width at half maximum FWHM Zinc Zn Haemagglutination assay HA Haemagglutination inhibition assay HIA Human immunodeficiency virus type 1 HIV-1 Monkey pox virus MPV Herpes Simplex Virus type 1 HSV-1 Glycoproteins Gp Nano molar nM Aedes aegypti A. Aegypti 2,2-diphenyl-1-picrylhydrazyl DPPH Red blood cell RBC Phosphate buffered saline PBS Bovine serum albumin BSA Venous leg ulcers VLUs Iron Fe Zinc Zn Calcium Ca Cadmium Cd Chromium Cr Nickel Ni Lead Pb Manganese Mn Cobalt Co Copper Cu

Nitric acid HNO3 Hydrogen fluoride HF

Hydrogen per oxide H2O2

Sulphuric acid H2SO4 Hydrochloric acid HCl

Per chloric acid HClO4 Ultra Voilet Visible Spectroscopy UV-Vis Spectroscopy Fourier Transform Infra-Red FTIR X-Ray Diffraction XRD Energy Dispersive X-Ray Detection EDX Scanning Electron Microscopy SEM Thermo gravimetric/ Differential Thermal Analysis TG-DTA

Effective Concentration at 50% EC50

xvii

ACKNOWLEDGEMENT

All praises are meant to Almighty Allah, The Creator, The Guider and The Sustainer,

Who provided the courage, strength and vision to carry out this research work. After that my immense gratitude, heartiest thanks and deep regards to my research supervisor Prof. Dr. Ghosia Lutfullah for her welcoming attitude, clear guidance, positive criticism and supportive approach which helped me to complete my research work with full expression of my capabilities.

Foremost I prompt my heartfeltappreciation to my consultant Dr. Kafeel Ahmad, for endlesssustenance of my PhD writting for his persistence, enthusiasm, eagerness, and enormousunderstanding. His super visionfacilitated me in writing of this thesis. I am thankful to Associate Prof. Dr. Sumera Afzal,Prof. Dr. Bashir Ahmad, Assist. Prof.

Dr. Nafees Bacha, Assist. Prof. Dr. Saeed Khattak, Assist. Prof. Dr. Jamshaid Ahmad,

Assist. Prof. Dr. Irshad ur rehman,Assist. Prof. Dr. Ibrar Khan, Assist. Prof. Dr. Sadiq

Azam, Mr. Muhammad Jawad Khan, Mr. Fida Hassan and Ms. Gul e Sehra Muneeb for their support and helping attitude.

I am also grateful to my research fellows Ms. Jamila Haider, Ms Nazish Khan, Mr.

Faheem ullah Khan, Ms. Saira Jamil, Ms. Zermina Zeband Ms. Sadia Laraib for accompanying me and maintaining comfortable working atmosphere.

I would like to acknowledge the contribution of office and para teaching staff of

Centre of Biotechnology and Microbiology, University of Peshawar for helping me all the way throughout my study.

xviii

My deep regards are forwarded to Dr. Muhammad Saeed, Professor, Department of

Pharmacy, University of Peshawar; Dr. Zafar Iqbal, Professor, Department of

Pharmacy, University of Peshawar; Dr. Muhammad Shahid, Associate Profesor,

Department of Biochemistry, University of Agriculture, Faisalabad, Pakistan; Dr.

Akhtar Nadhman, Institute of Integrative Biosciences, CECOS University, Peshawar,

Mr. Aftab Ahmad, School of Life Sciences and technology, Beijing university of chemical technology PR China, Mian Mohib Jan, Profesor Jehanzeb College

Swat,Muhammad Siddiqui Afridi, PCSIR Labs Peshawar; and all staff at Veterinary

Research Institute Peshawar, Peshawar for providing professional guidance and equipments during my research work.

I am obliged to my friends Nazish, Ms. Faiza, Seema Gul, Maha, Sidra Javed,

Tasmia, Haseena, Nabila Yasmeen, Sumayya, Sadia Khan and Mr. Ali Talha Khalil,

Rizwanullah Shah and Ali Sher for supporting me all the time.

Last but not the least I am extremely thankful to my Father Mr. Muhammad Shah

Khan, my Mother, my uncle Mr. Khalid Khan, my brothers Mr. Javid Shah Khan and

Mr. Junaid Shah Khan and my sisters for lending moral and social support which keep me going and doing well till completion of this thesis.

This thesis is a product of collection of plants, lab work, and synthesis of nanoparticles, experimentation and analysis. Many people have contributed towards completion of this work in one way or the other. Therefore, I acknowledge all those from the core of my heart who played positive role for this achievement.

ASMA SHAH

xix

SUMMARY

Traditional treatment of diseases is primarily based on empirical learning without scientific proofs. Monotheca buxifolia (M. buxifolia) and Daphne mucronata

(D. mucronata) has been used traditionally across the globe in different communities including Pakistan, for the treatment of arthritis, tooth ache, rheumatism, flue like conditions, inflammations, ulcers, urinary track diseases,fevers and vermifuge. The present study was designed for synthesis and characterization of biogenic nanoparticles (NPs) and their biomedical potential comparison to that of plant extracts. From the plant D. mucronata (leaves, bark and roots) and M. buxifolia

(leaves, seeds and fruits) crude methanolic extracts and fractions were obtained.

Phytochemical analysis for the presence of carbohydrates, phenols/tannins, flavonoid, saponins and glycosides was performed. Elemental analysis through atomic absorption spectroscopy (AAS) was performed for determination of various trace and heavy metals. Nutritional analysis of the plants was also determined. Synthesis of NPs included silver nitrate (AgNO3) and gold chloride (AuCl4) that were reduced using aqueous extracts of both plants. The biogenic NPs were then characterized by UV-

Visible spectroscopy, Scanning Electron Microscopy (SEM), Transmission Electron

Microscopy (TEM), Fourier Transform Infra-Red (FTIR) Spectroscopy, X-Ray

Diffraction(XRD), Thermo gravimetric-Differential Thermal Analysis (TG-DTA) and

Energy Dispersive X-Ray Detection (EDX) techniques. The synthesized NPs and methanolic, n-hexane, chloroform, ethyl acetate and aqueous fractions of both plants were tested for biological activities including antioxidant, anti-bacterial, anti-fungal, haemagglutination, phytotoxic, insecticidal, anti-termite and cytotoxic activities. The aqueous extracts and synthesized silver nanoparticles (AgNPs) of both plants and gold

nanoparticles (AuNPs) mediated by M. buxifolialeaves were screened for DNA damage, hemolytic and anti-thrombolytic activities. Crude methanolic extracts and synthesized NPs of both plants were screened for acute toxicity assay, anti-analgesic, anti-pyretic, GIT motility and anti-inflammatory activities. Hematological parameters were also evaluated using male Guinea pigs and Rabbits. All data were then analyzed and interpreted using Microsoft Excel, Origin Pro 8.5 and Graph Pad Prism 6.

Phytochemical screening oftest samplesrevealed the presence of reducing sugars and carbohydrates. Saponins were detected in D. mucronata leaves, roots and

M. buxifolialeaves and fruits. Phenols and tannins were present in all parts except D. mucronata roots. Glycosides were present in D. mucronate bark, leaves and roots and

M. buxifolia leaves. Flavonoids were present in D. mucronata bark and all parts of M. buxifolia. Elemental analysis revealed that D. mucronata contains Fe (0.316), Zn

(0.176), Ca (42.26), Cr (0.001), Ni (0.018), Pb (0.404), Mn (0.285), Co (0.051) and

Cu (0.035). The M. buxifolia contains Fe (0.169), Zn (0.060), Ca (20.24), Cd (0.022),

Pb (0.119), Mn (0.150), Co (0.012) and Cu (0.016). Nutritional analysis determined that D. mucronata contains carbohydrates (61.56), proteins (4.12), fat (2.733), fibers

(23.58), ash (9.94) and moisture (3.85). M. buxifolia contains carbohydrates (56.53), proteins (3.15), fat (0.87), fibers (24.08), ash (10.71) and moisture (2.54%).

Synthesized NPs were first detected by change in color i.e. Brown for AgNPs and cherry red for AuNPs. The UV-Vis spectral patterns of D. mucronataleaves derived AgNPs were observed with corresponding Surface Plasmon Resonance (SPR) peak at 425 nm, whereas M. buxifolia leaves derived AgNPsand AuNPs at 405 and

540 nm peaks. The FTIR results demonstrated that -CH and -OH groups were involved in synthesis of D. mucronataleaves derived AgNPs as reducing agents. The- xxi

OH, -CH and COOH groups were involved in synthesis of M. buxifolialeaves derived

AgNPs. In case of M. buxifolialeaves derived AuNPsthe -OH, -CH and -CO groups were involved in synthesis of AuNPs. Crystal size through XRD analysis observed in

D. mucronataleaves derived AgNPswas 94.779°A whereas M. buxifolialeaves derived

AgNPsand AuNPs was 96.18°A and 109.94°A.

The EDX analysis confirmed D. mucronataleaves derived AgNPs by containing 42.90% silver (Ag), whereas in M. buxifolialeaves derived AgNPs 53.68%

Ag and in AuNPs 15.43% gold (Au) along some other elements in different amounts.

The SEM and TEM analysis revealed AgNPs derived from D. mucronata and M. buxifolialeaves possess well dispersed spherical shape with size range 8-30 nm and 8-

20 nm respectively. Nano prisms, Nano rods and hexagonal shaped structures were observed in case of M. buxifolialeaves derived AuNPs with approximate size of 10-60 nm. The TGA profile of D. mucronataleaves derived AgNPswas observed with weight loss at 320 and 440°C, whereas M. buxifolialeaves derived AgNPs at 320, 500 and 900 °C, and AuNPs at 320, 480 and 906 °C. The DTA graphs showed endothermic and exothermic reactionsoccurrence of both plants at various degrees.

D. mucronate andM. buxifolia extracts and D. mucronataleaves derived

AgNPs (86.4%), M. buxifolialeaves derivedAgNPs (84.58%) and AuNPs (86.31%) showed significant antioxidant activity at 600 μg/ml. Potential anti-bacterial activity of both plants extracts and synthesized NPs was observed against Acenatobacter baumanni, Morganella morganii, Escherichia coli, Pseudomonas aeruginosa,

Vancomycin-Resistant Staphylococcus aureus (VRSA) and Proteus vulgaris. Both plants mediated AgNPs showed moderate activity against Candida albicans and low activity against Aspergillus niger. These samples were inactive against xxii

Fusariumoxysporum, Aspergillus flavus, Aspergillus parasiticus and Penicillium digitatum. The tested samples did not show heameagglutination activity which means phytolectins were absent in them. Highest phytotoxic activity was observed for ethyl acetate extract with 80% inhibitory effect in D. mucronata at 20 mg/ml against Lemna minor. Resultant insecticidal and anti-termite activity of tested samples showed 100% activity against Tribolium castaneum, Rhyzopertha dominica, Callosobruchus analis and Heterotermes indicola. Brine shrimp lethality assay revealed that plants leaves extract derived AgNPs and AuNPs exhibited higher cytotoxic activity than plants extracts alone. Mild thrombolytic activity was observed that ranges from 15.9 to

25.8% clot lysis by the tested samples. Hemolytic and mutagenic activity showed negative results for the tested samples.

All extracts were found safe as no lethality was observed. D. mucronataleaves derived AgNPs showed higher percent of writhihng inhibition than both the plants alone and M. buxifolia leaves derived NPs. Anti-pyretic activity observed in tested samples showed that D. mucronataleaves derived AgNPs exhibited better antipyretic activity as compared to M. buxifolialeaves derived AgNPs and AuNPs at both the doses tested. Gastro intestinal track motility (GIT motility) showed that with increase in sample concentration, decrease in% GIT motility was observed. Both plants mediated AgNPs and AuNPs were observed to possess significant anti-inflammatory properties. Biochemical parameters analyzed fortest samples revealed a slight increase and decrease in all hematological parameters, liver function tests, body weight, feed and water intake which did not affect the normal health.

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Chapter 1 Introduction & Literature Review

1.0 INTRODUCTION AND LITERATURE REVIEW

1.1 Introduction

From many years natural sources like plants, animals, fungi and marine organisms have been used as traditional remedies for treatment of diseases and various ailments. These sources of natural products have been derived from a wide range of living organisms including marine organisms, microorganisms, fungi, plants and animals. More than 200,000 natural products have been recorded from these living organisms [1]. Among these compounds, the plant natural products have been studied the most [2].

The Egypt ancient scripts far back to 2600 before Christ (BC) have record of plant uses as major constituents in treatment of diseases. About 100 BC old documented Chinese and Greek manuscripts dealing with herbal medicines have been found. Basis of modern medicines and pharmaceutical sciences has been developed by the work of famous Avicenna who emphasized on plant derived medicine. Natural products from plants could ultimately replace synthetic and semi-synthetic drugs development [3].

1.2 Plant natural products

Natural products are typically secondary metabolites in contrast to primary metabolites which are essential for primary growth. Secondary metabolites are those metabolites which are responsible for adaptation or survival of living organisms [4].

Over the past half century, there have been advancements in investigating plant derived natural compounds. Till date around 250,000 plant species have been

Chapter 1 Introduction & Literature Review identified, of which so far 15% have been investigated for active compounds whereas only 6% have been screened for their biochemical properties [5]. Thus, still more plants need to be investigated for their important medicinal constituents. The plant natural products comprise various structures that hold up plant physiology in interaction with biotic and abiotic environmental factors [6].

1.2.1 Alkaloids

The word Alkaloid is derived from 'al-qali' referring to plant ashes. Alkaloids are basically heterocyclic nitrogenous compounds synthesized from proteins and amino acids. Other similar compounds such as benzoxazinoids and glucosinolates are considered alkaloids due to their bioactive nature and background study. Currently, alkaloids account for a total of 12,000 compounds, representing one of the largest groups of secondary metabolites.

Purine alkaloids are synthesized from L-glutamine, L-aspartic acid, formate and L-glycine, by pathway similar to cytokinin metabolism. These compounds are produced in diverse plant species including Coffea arabica, Camellia sinensis,

Theobroma cacao, Ilex paraguariensis, Paullinia cupana and Cola nitida. Some of the examples of purine alkaloids are caffeine that exhibit defense mechanism against snails and slugs [7] theobromine, theophylline and paraxanthine [8]. Their structures are shown in Figure1.1. Applications of these alkaloids include their use as cold medicines, analgesics, treatment for asthma and Parkinson's disease [9].

Chapter 1 Introduction & Literature Review

Figure1.1 Structures of theobromine, theophylline and paraxanthine

Tropane alkaloids, which have bicyclic skeleton, are synthesized from ornithine and arginine and mostly contain esters of pseudotropine and alcohols tropine

(3-tropanol). These compounds were first isolated from Solanaceae family. Other families producing tropane alkaloids are Convolvulaceae, Brassicaceae,

Erythroxylaceae, Euphorbiaceae, Rhizophoraceae and Proteaceae [10]. The principle uses of tropane alkaloids are as anticholinergic drugs, antidote and treatment for delirium tremens [11]. Various classes of tropane alkaloids are the hyoscyamine and

Scopolamine that have rule in reducing salivary, gastric, sweat and bronchial secretion, work as antidote and used as pre-medication before surgery.

Chapter 1 Introduction & Literature Review

Cocaine, the highly addictive central stimulant is present only in shrubs native to Andes. Ester of benzoic acid and methylecgonine are constituents of cocaine

(Figure 1.2). Cocaine blocks Na+ channels of mucous membranes and is used as local anesthesia for surgery of ear, eye, throat and nose. Other functions of the direct uptake of cocaine leads to hyperactivity, fatigue, euphoria and hunger suppression [12].

Figure 1.2 Structure of Cocaine

Calystegines containing glycosylated hydroxyl groups on nortropane skeleton have been isolated from species of Convolvulaceae, Solanaceae, Erythroxylaceae,

Brassicaceae and Moraceae [6]. This compound is useful in prevention of type II diabetes and therapy of Morbus Gaucher disease. Structure of Calystegines is shown in Figure 1.3.

Chapter 1 Introduction & Literature Review

Figure 1.3 Structure of Calystegine

Quinolizidine, the bi, tri and tetracyclic compounds are synthesized from lysine thatoccur in Berberidaceae, Ranunculaceae, Fabaceae, Rubiaceae, Solanaceae and Chenopodiacae [13]. Quinolizidine alkaloids like Lupinine and Sparteine have shown antiarrhythmic, hypoglycemic and hypotensive effects and have been used as

Central Nervous System (CNS) depressant. However, some patients were unable to metabolize these compounds leading to intoxication and hence their use in medicine is restricted [14].

Intermediate compound of tyrosine biosynthesis, reticuline can yield different alkaloid groups such as Aporphine, Benzylisoquinoline, Protopine and many more.

Benzylisoquinolines are mainly produced in angiosperms family Berberidaceae,

Papaveraceae, Fumariaceae, Ranunculaceae and Menispermaceae. These compounds have shown antitussive, analgesic, antimicrobial, anti-inflammatory activity as well as muscle relaxant activity [6, 15].

Chapter 1 Introduction & Literature Review

Tyrosine and monoterpene secologanin derived alkaloids called Ipecac also known as terpenoid-isoquinoline alkaloids possess expectorant, amebicidal and emetic properties [16]. Theoccurence of these compounds has been found in the eudicot family’s Alangiaceae and Rubiaceae. The Dover's powder used for treatment of cold and fever was made of opium, potassium sulphate and Ipecacuanha root.

Monoterpene Indole alkaloids are class of alkaloids biosynthesized from secologanin and tryptophan via (S)-strictosidine intermediate. These compounds are produced in plant families Loganiaceae, Apocynaceae, Rubiaceae and Nyssaceae.

Well-known compounds of this group are Reserpine, Ajmalicine and Vinblastine which exhibit various pharmacological activities such as antihypertensive, antiarrhythmic and anticancer activities. These compounds were also used in therapy of Hodgkin's disease and the non-Hodgkin's lymphomas, melanoma and acute leukemia [17].

Benzoxazinones, derived from precursor of tryptophan (indole-3-glycerol phosphate) occur mainly in monocot family Poaceae and in some eudicot family plants Acanthaceae, Plantaginaceae and Ranunculaceae [18]. The constituent of benzoxazinones are cyclic hemiacetal and cyclic lactam/cyclic hydroxamic acid.

Benzoxazinones possess antimicrobial activity (anti-bacterial and anti-fungal), antialgal, insecticidal activity as well as function as allelochemicals. Structure of

Benzoxazinone is shown in Figure 1.4.

Chapter 1 Introduction & Literature Review

Figure 1.4 Structure of Benzoxazinone

Glucosinolates, derived from many amino acids including alanine, leucine, isoleucine, valine, methionine, tyrosine, tryptophan and phenylalanine, are the hydroximinosulfate ester compounds existing with around 120 different structures

[19]. These compounds have been isolated from mostly eudicot families’ species.

These compounds have shown anti-bacterial, anti-fungal, feeding deterrent, nematicidal, insecticidal activities and also possess cancer preventive properties [20].

1.2.2 Polyketides

Polyketides synthesized from 2 carbon units are derived from similar precursors as of fatty acids (acetyl-CoA and malonyl-CoA) but unlike fatty acid these compounds retain the function of oxygen. Polyketides interaction derives many other plant compounds such as phenylpropanoids, naphthoquinones, terpenoid, flavonoids and polyketide alkaloids. Naphthoquinonesoccur in Droseraceae, Plumbaginaceae,

Nepenthaceae and Polygonaceae [21]. These compounds have shown antifeedant activities in insects, as well as antimicrobial properties [22]. Naphthoquinones are synthesized with 6 acetate units whereas the 8 acetate unit compounds that are

Chapter 1 Introduction & Literature Review structurally related to naphthoquinones are called anthraquinones. The anthraquinones exhibiting anti-bacterial and anti-fungal properties have been reported to synthesize in plant family Rhamnaceae, Polygonaceae and Asphodelaceae [23].

1.2.3 Phenylpropanoids

Phenylpropanoids are aromatic compounds derived from shikimate pathway of

L-Phenylalanine and L-tyrosine. This class of compounds is linked with lignans, lignin’s, coumarins and flavonoids with respect to their synthesis or interaction.

Lignans and lignin’s are composed of monolignols including coniferyl alcohol, p- coumaroyl alcohol and sinapyl alcohol which are also referred to as H, G and S (p- hydroxyphenyl, guaiacyl and syringyl), respectively. The monocots and eudicot plant species produce all three monolignols [24]. These compounds exhibit antiviral, anti- bacterial and anti-fungal properties.

Flavonoids constitute one extra aromatic ring to phenylpropanoid and the major compounds are flavanols, flavones and anthocyanidins whichoccur mostly in higher plants [25]. Flavonoids function as radical scavengers, anti-bacterial and anti- fungal, antioxidant and prevention agent for breast cancer [26].

Chapter 1 Introduction & Literature Review

1.2.4 Terpenoids

Terpenoids are derived from 5 carbon isoprene and include about forty thousand different compounds. Classification of terpenoids is based on number of isoprene units such as 1-unit compounds are called hemiterpenes, 2 unit monoterpenes, and 3 units sesquiterpenes and so on [17]. These compounds are widely spread in plant kingdom including gymnosperms, angiosperms, mosses and ferns. Hemiterpenes are known for protection of leaves in dense ozone and reactive oxygen regions [27]. Oleoresin that kills beetles and pathogenic fungi is because of the active site of monoterpenes limonene. It is also considered as anticancer agent [28,

29]. Farnesene, a sesquiterpenes has been reported as insecticidal agent in maize [30].

Carotene is a tetraterpene precursor of vitamin A which functions as light receptor for the human eye [31].

Chapter 1 Introduction & Literature Review

1.3 Daphne mucronata

The D. mucronata is a wild shrub of the family thymeleaceae, which is well known as a medicinal plant in different regions of Asia [32]. Daphne genus belongs to family Thymeleaceae, which consists of 44 genera and approximately 500 herbal species. In china and Africa tropical regions these plants are used in traditional medicines [33].

1.3.1 Phytochemistry

Daphne plants produce a wide range of biologically active natural compounds which include flavonoids [34, 35], lignin [36], triterpenoids, coumarinolignans [36,

37] and coumarins [35].

The plant D. mucronata has not been exploited much for theoccurrence of active ingredients and biological properties. Coumarins and diterpenes have been reported as main constituents of the plant [38]. In traditional Chinese medicine the leaves and roots of Daphne are used as purgative abortifacient and for the treatment of rheumatism, toothache and ulcers. Woody parts of the plant are used for bone diseases as remedy [39]. Skin and cancer type diseases has been treated with it[40]. Studies show that it has also good antimicrobial and cytotoxic activities. The active compounds have been identified and isolated from Daphne genus.

1.3.2 Significance in medicine

Various Daphne species (Thymelaeaceae) have been used to treat cancer type diseases since 200 A. D.[41]. Pharmacological and phytochemical studies have shown their strong anti-cancer potential [32]. D. mucronata is used for skeleton-muscular 10

Chapter 1 Introduction & Literature Review problems while mash dressing of leaves and fruit is used for curing rheumatism [42,

43]. Liniment of the plant is used in treating infectious wounds and smoke of the plant branches have a relieving effect on fatigued muscles. Its cooked leaves and decoction are used for curing women menstruation disorders, infertility, constipation and gynecological problems [44]. The wooden part of the plant is finely ground to make powder which is used to clean and relieve eye pain [45].

Chapter 1 Introduction & Literature Review

1.4 Monotheca buxifolia

M. buxifolia species is found in hilly regions of Afghanistan and Northern areas of Pakistan. M. buxifolia is a small evergreen tree having broad leaves and belongs to the family Sapotaceae. The plant is used as fodder, fuel, roof thatching material, wood lumber and used particularly as hedge around cultivated fields because of its barbed nature. It is a small fruits bearing specie, which is locally called as

Gurgura, available in local markets as dried and fresh foods [46, 47].

1.4.1 Phytochemistry

M. buxifolia have higher economic value for inhabitants of mountains, especially in regions where agronomic cropping is limited [46]. This species exists either as pure stands or in association with Acacia modesta, Ficus palmate, Olea ferruginea, Quercus baloot and Punica granatum. Dalbergia sisso is also associated sporadicallyat some locations with M. buxifolia [46]. M. buxifolialeaves contain bioactive compounds like flavonoids, cardiac glycosides, anthraquinones, terpenoids, tannins, saponins, poly-phenolic compounds and reducing sugars [48].

Recently, two new compounds, buxilide as pyrone and buxifoline-A as alkaloid were isolated from the ethyl acetate fraction of M. buxifolia fruit [49]. Poly- phenolic and flavonoid compounds are reported to have great influence as analgesic and anti-inflammatory properties. The anti-oxidant activity of the plant is evaluated in-vitro and proved to have higher antioxidant properties [50]. Moreover, the fruit also shows inhibitory potential against urease enzyme [49]. Sapotaceae family is widely studied for antipyretic [51], antimicrobial activities [52, 53], antioxidant [50], anti- inflammatory [54, 55], Central nervous system depressant (CNS depressant)[56], anti- 12

Chapter 1 Introduction & Literature Review nociceptive activities [51, 57] andanthelmintic [58]in different in-vivo and in-vitro experimental models.

1.4.2 Significance in medicines

The fruit of M. buxifolia in traditional medicines has medicinally hematinic, vermicidal and purgative properties. They are also used to lower temperature in fevers

[59-61]. The fruit has medicinally digestive and laxative properties, and is also used for treatment of urinary tract diseases [46, 47, 53].

Chapter 1 Introduction & Literature Review

1.5 Plant natural products and nanotechnology

Plant natural products used as pharmaceutical products have comparatively grown in the present era. Around 50% of standardized drugs were reported in period of 1981-2006, that derived from natural products [62]. Nanotechnology is emerging as new science that brings all technological advancements and background science in one field. It is based on knowledge of many sciences like chemistry, biology, material sciences, physics, chemical, electrical and mechanical engineering [63]. As a result,

Bio-Nanotechnology has emerged that utilizes biological materials with chemical procedures and physical techniques to synthesize Nano sized particles. These Nano- scale particles are having specific properties [64]. Nanotechnology is linked to biotechnology by production of nanomaterial using biological methods and eco- friendly techniques [65]. Metallic NPs synthesis can be achieved both by extracellular and intracellular methods [66]. In biological synthesis of NPs using plant is a relatively new technique. For generation of metal NPs the use of plant extracts is economical and it can serve as a commercialized substituent for fabrication of NPs

[67]. In recent years, increased research and applications of Nano science and nanotechnology has been observed. Its applications in medicines can bring advances in diagnostics and treatment of diseases. In medicines the possible applications of nanotechnology include nutraceuticals, production of improved biocompatible materials, in-vitro and in-vivo diagnostics and drug delivery.

Plants contain a range of phytochemicals, primary metabolites and secondary metabolites that are proficient natural resources for generation of metallic NPs [68].

Reduction of metal salts by biological molecules and their mixtures like amino acids, enzymes, polysaccharides, proteins and vitamins impart their role in synthesis of 14

Chapter 1 Introduction & Literature Review metallic NPs [69]. Solvent selection for NPs synthesis is also an important factor.

These properties underline the stability of synthesized nanostructures [70]. The NPs have larger surface area to volume ratio that imparts extraordinary anti-bacterial properties to them. This characteristic of NPs is of prime importance to researchers as a consequence of resistant strains progress and growingresistance of microbesformetal ions and antibiotics [71].

Nanotechnology in short is an emerging field. Due to potential prospects it still has a charm that is not yet completely discovered. Major fields have been revolutionized by nanotechnology including heavy industry, health, consumer goods, eco-friendly energy systems, opto-electronic devices, information technologies and communication systems. It has potential to transform food and agriculture sectors. It possesses extraordinary abilities in weed control strategies and advancedapparatuses for molecular centeredcure of ailments[72]. It is estimated that until next year more than 15% of yields on universal market will possess some form of combined Nano techniques in their preparation process [73]. Improper production of metal NPs has raised some toxicity concerns. Most likely it might have influence on international finances. Therefore, these issues are important to be considered [74].

Chapter 1 Introduction & Literature Review

1.6 Nanoparticles

Nano word comes from Greek language meaning small, and as prefix refers to billionth part 109. So, any particles having size in range of 1 to 100 nm are termed as

NPs [75]. These small size particles possess unique physical and chemical properties as compared to their original intact state of matter because of increase in surface area as well as radical formation. From the time of discovery, these particles have been extensively used in many applied sides of various sciences such as electrochemistry, photochemical chemistry, and biomedical sciences [76]. In recent research NPs have been made functional by linking them to biological molecules like small molecule ligands, proteins andnucleic acids. Currently, cancer nanotechnology is under intense development for its uses in cancer targeted therapy, molecular diagnostics and cancer imaging [77]. Nanotechnology is an interdisciplinary and multidisciplinary science that covers the fields of biomedicines, chemistry, physics, material science, engineering and biology. Nanotechnology uses the novel characteristics of the synthesized nanomaterial that cannot be achieved from its bulk size. Hybrid NPs due to their unique characteristics have been used in therapeutics and imaging applications. These NPs retain features of both organic and inorganic substances, and modify properties of hybrid by combination of functional elements [78].

Both AuNPs and AgNPs are widely used in various fields such as pharmacy, molecular imaging supported by their intrinsic properties of stability,inert nature, non- cytotoxicity, high disparity and biocompatibility. The AgNPs and AuNPs possess many good characteristics like high scale production, stability, controlled activity, lyophilic properties, and powder formation properties without need of organic solvents. Apart from this NPs possess some disadvantages including low loading 16

Chapter 1 Introduction & Literature Review capacity [79, 80]. Current study highlights synthesis and applications of AuNPs and

AgNPs in the field of drug delivery and pharmacy.

1.6.1 Gold metal uses

The Au based medicinal preparations were used in Egypt and Indian ancient cultures, but China was the first to treat disease with them during 2500 BC [81].

Potable Au, the solutions having Au ion or possesses color like Au, were prepared as elixirs. Powdered Au was used to treat furuncle entirely by going deep into wound.

Smallpox toxin and skin ulcers were treated by powdered Au. Mercury was removed from ear and flesh by setting warm Au in it. Sore eyes were treated by touching red hot Au ring to inside of lower and upper eyelids. Heated Au pin was used to relieve pain immediately in serious toothache. Sores of gums and mouth were treated by gargling with cooked gold and water. According to “channels theory” Au powder and

Au foil could be used in medicine because it has no taste and could enter the lungs and heart channels. French physician Forestier in 1929 for the first time used sodium aurothiopropanol sulfonate in treatment of rheumatic arthritis [82]. In ancient times

Au was used as a therapeutic agent by physicians and surgeons in China. Pure Au was used in treatment of skin ulcers, furuncles and smallpox. The Au drugs were used in cure of measles, lungs and joints diseases and in removing mercury from flesh and skin [83]. In recent years AuNPs have gained much attention because they possess applications in materials science owed to distinctive size-dependent properties [84].

British scientist Michael Faraday worked on colloidal solutions of Au and concluded that their purple or ruby colors were due to existence of very small particles of this metal [85, 86]. These particles could be easily detected by their effect in dispersing incident light and were invisible to human eye. Two main research lines were 17

Chapter 1 Introduction & Literature Review historically developed with two different aims. One approach was directed towards the colloidal gold preparation that was used for transparent glass color, resulting in beautiful red and valuable glass [87]. Second approach was towards the field of medicine, to synthesize drinkable Au that was used as powerful drug in treatment of severe diseases. In old Latin texts it was known as aurum potabile [84]. Origin of potabile Au traced back to Middle Ages, it was still a subject of interest in eighteenth century.

The AuNPs generally give wine-red color in solutions, and it has great impact on interactions [88]. The AuNPs size ranges between 1 nm to 8 μm with various shapes that includes spherical, icosahedral, tetrahedral ring, sub- octahedral,oCtahedral, decahedral and Nano rods (Figure1.5).

Figure1.5 Pentagonal cross-section, decahedral, cubic,oCtahedral, tetrahedral and

truncated tetrahedral particles, and platelets nanoparticle shapes

The AuNPs are among the most suitable and efficient nanomaterial in biotechnology and biomedical fields due to their high electric conduction and larger surface area [89]. Beside these, other applications of AuNPs especially in

Chapter 1 Introduction & Literature Review electrochemistry is the determination and identification of pharmaceutical compounds. The Au nanomaterial as surface immobilizer act like conductor, increasing electron transmissionamongst the surface and target analyte. Other properties such as increasing permeability, less toxicity and retention in cell enhance nanomaterial interaction with cellular machinery [90, 91].

The usability of AuNPs in drug delivery systems is due to its good capacity of carrying loaded material to its specific and targeted areas in efficient manner, making it a good vehicle for drug delivery [92]. Generally, AuNPs possessing good physical, chemical and optical properties can be innovatively used to enhance the efficiency of control and transport of pharmaceutical compounds within biological systems [93,

94].

The Au particles synthesized by sunlight radiations were modified by folic acid and then coupled with 6-mercaptopurine. Modified AuNPs were commonly used in radiation practices as radiant enhancer [80]. The AuNPs are most commonly used in field of biochemistry due to their optical properties and compatibility with other elements/compounds resulting in being good therapeutic agents for the transport of drugs within the system covertly [95]. These small particles can also be introduced into tissues and cells along with proteins and other biological molecules in assessing their pathways and molecular characterization [96].

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1.6.2 Silver metal uses

The Ag has been used since the periods of Egyptian and Roman empires in preservation of drinking water from germs. Milk was used to be preserved by putting

Ag coins in it by immigrants to America since 18th century. Since 1884 1% Silver nitrate (AgNO3) solution has been used as eye drops to prevent eye diseases in newborn [97]. The Ag foil has been used in World War I to protect wounds from infection. National Aeronautics and Space Administration (NASA) has employed containers made from Ag to preserve purity of water on a spacecraft for drinking. The

Ag in ionic form possesses antimicrobial potential against a wide range of microorganisms. Due to antimicrobial activity and less toxicity to human cells, Ag is present in many health care products that are commercially available. In the field of wound care, the use of Ag is increasing rapidly, and wound dressings containing Ag are now available in market e.g. Polyurethane foams, gauzes and hydro fiber® dressing [98]. Since overuse of antibiotics, some bacteria have become resistant like

Methicillin Resistant Staphylococcus aureus (MRSA) [99] and VRSA [100]. To resolve this problem Ag has been modified in its chemical and physical properties by increasing its surface to volume ratio. Metallic AgNPs play important role against germs [101]. Germ killing ability is increased by ionic Ag present in synthesized

AgNPs [102].

The potential application of AgNPs involves Nano medicine, drug delivery, cosmetics, electronic and environmental protection applications [103, 104]. The

AgNPs are also famous for their antimicrobial activity and are commonly utilized in treatments of bacterial, viral and fungal infections throughout the world [105]. Being widely accepted against microbes, AgNPs presently are used in many commercial 20

Chapter 1 Introduction & Literature Review products such as colloidal coatings, paints and polymer scaffolds. In textile industry,

AgNPs is used as microbe filtrate [104]. The AgNPs have diverse uses in medical field [106], water and air filtration processes [107], and individual Plasmon optical spectral properties making them much suitable for bio-sensing applications [108].

One of the most important applications of AgNPs is their inhibiting effect on the infectious cells such as targeting HIV-1. Using in-vitro assays AgNPs were evaluated to have viricidal activity at an early stage of viral infection [109]. The Ag-

Hybrid materials of amphiphilic hyper-branched macromolecules coated on AgNPs are synthesized to use in surface anti-bacterial activities [110]. The Ag surface coated

NPs with paint made from plant fatty acid also showed enhanced antimicrobial activities against a wide range of micro biota [111]. Drinking water-related diseases such as diarrhea and cholera can be controlled by enhancing microbial quality of water. The common method of bactericidal activity for filtration purposes is the use of

Ag coated Nano-carbon filters [112]. Bio hazardous aerosols developed in ventilating, heating and air- conditioning systems can be reduced and controlled using Ag-coated carbon filters [113]. Antiseptic activity of Ag has also been reported and several commercial products for wound treatment are available in the market that contains Ag as an antimicrobial agent. Similarly, Ag is also used in wound dressings to treat ulcers and burn wounds, Ag sulfadiazine creams are applied topically [114].

Chapter 1 Introduction & Literature Review

1.7 Synthesis of gold and silver nanoparticles

Nanotechnology is a broad field and bio-nanotechnology is its branch. In general, nanotechnology as a whole is associated with synthesis of NPs [115].

Different methods are used for synthesis of NPs. Preparation of colloidal Au by citrate reduction has been reported [92]. Synthetic AuNPs of different structures as silica-Au

Nano shells, Au Nano rods and hollow AuNPs are prepared by many different methods [116, 117]. Chemical method results in release of toxic chemicals that causes environment pollution. Energy is required in physical methods and they are very expensive. Keeping an eye on these disadvantages significance of green chemistry has been increased. Efforts were made to follow benign and green procedures for fabrication of NPs [118]. With passage of time the green synthesized NPs are aggregated so the factor of size is a littlenegotiated when storage is necessary[119,

120]. Therefore, Nano-biotechnology is well thought-out to be safe and cost- effectiverouteconsumingorganicmeans of NP synthesis as compared to physical and chemical methods [121, 122]. Synthesis is attained by chemical, physical and biological method [123].

1.7.1 Chemical methods

Synthesis of metal NPs can be performed chemically. In chemical procedures usually a liquid media is used that contain reducing substances and mixture of reactant species. Commonly used reducers include potassium bi-tartarate, hydrazine [124], methoxy polyethylene glycol [125] and sodium borohydride [126]. To prevent clump formation of synthesized NPs sometimes stabilizer is used. Stabilizer might be sodium dodecyl benzyl sulfate or polyvinyl pyrrolidone [124]. Chemical methods are

Chapter 1 Introduction & Literature Review inexpensive and can be used for large scale production of metal NPs. As toxic solvents are used in synthesis process, toxic byproducts are formed. Because of these reasons chemical method is not acceptable, unsafe and non-ecological for synthesis of

NPs.

Synthesis of AuNPs has been reported from reduction of Chloroauric acid

(HAuCl4) in chitosan solution [127]. Other preparatory process of AuNPs has been reported using HAuCl4 reduced by aqueous sodium borohydride in presence of dodecane thiol and sodium citrate with heavy water [128, 129]. Kojima et al. revealed synthesis of AuNPs in encapsulated polyethylene glycol dendrimers and formaldehyde was used as reducing agent that absorbs near IR light [130].

For AgNPs synthesis chemical methods are much easier and robust [131], that is based mainly on stabilizing and reducing agent, and Ag precursor. The synthesis processing, shape and size may differ according to the method used, depending on nucleation of Ag, temperature, reducing agents, precursor and pH. The chemical reduction process for synthesis of AgNPs into reduced silver (Ag0) is achieved both by organic and inorganic agents including ascorbate, sodium citrate, sodium borohydride, N, N-dimethylformamide and ethylene glycol copolymers. The cluster formed from these ions ultimately leads to colloidal Ag particles [132].

1.7.2 Physical methods

Physical methods for synthesis of NPs include grinding, pyrolysis and cutting.

Grinding and crushing of large particles are done by using mill. The term attrition is used for such processes. It is a size reducing technique. To obtain oxidized NPs the particles are elutriated. Characteristics of NPs produced are influenced by the nature 23

Chapter 1 Introduction & Literature Review of material used, the grinding time and the surrounding atmosphere. Pyrolysis is a technique that uses an initiator of organic in nature. This early discussed initiator is passed over a slight hole in pressure and burnt. The produced ash is eluted and oxidized NPs are obtained. Investment of large assets is required in these methods while the yield of NPs is quite less. These are expensive methods and due to pressure and temperature requirement physical methods pose major disadvantage for metal

NPs fabrication. Biological methods in comparison are economical and more productive. They might be accomplished under ambient circumstances of pressure and temperature.

The γ-irradiation method for the synthesis of AuNPs with high purity and uniform size of 45 nm is achieved with polysaccharide alginate as stabilizer [133].

The approach of microwave irradiation using citric acid as reducing agent and cetyl trimethyl ammonium bromide as binding agent were used by Arshi et al. For synthesis of AuNPs [134]. Other reducing agents such as tartrate and malate can be used in photochemical bargain for synthesis of AuNPs. The AuNPs were synthesized using photochemical reduction with polymerization reaction (ethylene glycol). Polyvinyl pyrrolidone was used as stabilizing agent [135].

Synthesis of AgNPs is achieved by physical method of condensation and evaporation. Synthesis of AgNPs via physical method requires much greater time and high energy. According to Lee and Kang oleate binding with Ag in thermal decomposition method can lead to synthesis of fine 10 nm AgNPs [136]. Use of ceramic heating process can result in synthesis of mono dispersed uniform sized metal

NPs [137].

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1.7.3 Biological method

Metal NPs have been produced by chemical, physical and biological methods.

These approaches are mostly in developingphase and issuesare practicedin aggregation and stability of NPs, size distribution, morphology and crystal growth control. Plants derived metal NPs were shown to be more stable than those derived from other organisms. Plants and their extracts contain more efficient reducing agents for metal ions as compared to bacteria or fungi. Plant extracts are better option than living plants and plant biomass in industrial and scale up production of well dispersed metal NPs as it is safe and easy green method. Researcher’s attention has been focused on understanding of mechanisms involved in biogenic synthesis of NPs as well as biomolecules characterization and detection involved in metal NPs synthesis.

The plant biomolecules like amino acids, alkaloids, polysaccharides, vitamins, alcoholic compounds and enzymes/proteins are involved in reducing, fabrication and stabilizing metallic NPs. The presence of enzymes, polyphenols and chelating agents in plants has reducing capacity. These plants reducing capacities along with ion reduction potential have critical effects on production of NPs. Investigations are recommended for optimization of reaction conditions and engineering of recombinant organisms for high yield of enzymes, proteins and biomolecules required for biosynthesis and stability of NPs [69].

The biological method also known as green synthesis has shown efficient and eco-friendly reformed process for synthesis of AgNPs and AuNPs. The production of iron oxides and metal NPs produced from inorganic materials in the extracellular or intracellular compartments is well known. This approach is influenced by many factors like enzyme/catalysts in form of stabilizing and reducing agent, temperature, 25

Chapter 1 Introduction & Literature Review pH and type of solvent. Plants as major reservoirs of biological molecules are considered more beneficial over the microbial counterpart.

In synthesizing NPs, three major factors, including reducing agent, precursors and stabilizing agents have equal effects. One of the main and common problems in green synthesis of NPs is agglomeration which can be prevented by using efficient agents and precursors. Biological techniques are referred to as synthesis of NPs with green plants, algae, yeast, fungi and bacteria [138]. The biosynthesis of AgNPs of size less than 15 nm was reported with sphere-shaped and large surface area using a metal reducing agent Shewanella oneidennis, a facultative bacterium capable of reducing heavy metal ions [139]. Fayaz et al. evaluated synthesis of AgNPs from the fungus

Trichoderma viride using AgNO3 as precursor. The resultant AgNPs had size less than 50 nm and was active against gram positive and gram negative bacteria when used in combination with antibiotics [140]. Phyllanthus amarus was used for synthesis of AgNPs at room temperature with size range 10-20 nm [141]. Subbaiah and Savithramma synthesized AgNPs using leaves extracts of Cadaba fruticosa by using AgNO3 as a precursor. The resultant AgNPs exhibited maximum antimicrobial activity against unrelated microbial species [142].

1.8 Characterization of silver and gold nanoparticles

The characterization of NPs nature is essential to determine its efficient correspondent properties. Different method used for characterization of NPs include

TEM,SEM, atomic force microscopy (AFM), X-ray photoelectron spectroscopy

(XPS), UV-visible spectroscopy, XRD, FTIR and dynamic light scattering (DLS).

Chapter 1 Introduction & Literature Review

The first three techniques i.e. TEM, SEM and AFM are used for determining morphological characteristics of NPs including shape and particle size, whereas the later measure three-dimensional properties. Dynamic light scattering technique can also determine size distribution. UV-visible spectroscopy technique measures the

Surface Plasmon Resonance that is used to confirm the NPs formation. X-ray diffraction technique is used for the crystalline structure determination of NPs.

1.8.1 Transmission electron microscopy (TEM)

In this technique NPs are placed on a copper grid coated with carbon. From a drop of NPs TEM micrograph is taken, where the instrument is operated by high voltage up to 200 kV. The copper grid is kept dry and through ultra-thin specimen the photon beam is transmitted penetrating the specimen of interest. This interaction of electrons and specimen is determined as image on imaging device. The various options for illustrating the image are the shape, diameter and surface area detection.

1.8.2 Scanning electron microscope (SEM)

The SEM is used for determining the size and morphological structure of NPs.

Samples are loaded on a stub which is coated with carbon material through pipetting in SEM instrument. The stub is a small cylindrical device about 1mm and made of copper. The range of samples varies according to SEM model apparatus [143].

1.8.3 Atomic Force microscopy (AFM)

The AFM as for achieving atomic resolution a vibrating tip interacts with surface in an aqueous solution or in ultra-high vacuum. The detection procedure of

AFM has been determined in forming small clusters of solid lipid NPs [143]. The 27

Chapter 1 Introduction & Literature Review

AFM using automated analysis software have shown in providing accurate and rapid characterization of NPs [144]. The AuNPs detection with size 1.5 nm show problem in Coulomb blockade effect of AFM, which can be deceased by using AuNPs around

5.4 nm in diameter and above [145].

Surface Enhanced Raman spectroscopy (SERS)

SERS is determined through the equation:

λsp = (λexc + λRs)/2

Where λsp is Surface Plasmon, λexc is the excitation and λRs is the Raman

wavelength, respectively.

The SERS with ability in detecting the Surface Plasmon wavelength also determines the intensity of electromagnetic field within NPs. Raman scattering cross section is observed to be smaller as compared to excitation of molecular fluorescence.

1.8.4 UV-visible spectroscopy

UV-vis spectroscopy is a technique used in detection of wave length (absorbed and scattered light) of a given sample on a photo detector medium. UV-vis spectroscopy is an ideal instrument to detect NPs as the optical properties can be verified according to size, shape, agglomeration state, concentration and surface refractive index. The surface and electromagnetic radiation of NPs are absorbed with oscillations of light incident. The AgNPs usually exhibit Plasmon absorbance within range of λmax 380-480 nm while AuNPs with λmax 480-580 nm.

Chapter 1 Introduction & Literature Review

1.8.5 X-ray diffractometry (XRD)

The XRD is used in detection of aggregate crystallite size of NPs. Philips diffractometers are used for NPs crystalline size determination. The mainnotion of

XRD is detection of crystalline nature of assumed particles in form of crests. These crests are resoluteusing the electron density of particles and complexes in the element cell. The XRD patterns are calculated by Rota flex diffraction meter, to which radiations are provided and a substantial wavelength is determining round 1.54 Å.

Peaks of XRD for detection of width are observed with Scherer’s formula as given below:

D = kλ/βcos θ

Where D is the crystallite size

K is constant = 0.94

β is full width at half maximum (FWHM) which can be determined as

β = FWHM x 2π /360

and cosθ is known for the Bragg angle

The diffraction peaks of XRD pattern are used to determine crystalline structure and size. Like at 111, 200 and 220 diffraction peaks indicated crystal structure for the cubic Ag. Purity and accuracy of NPs structure is determined by the sharpen peaks in given range.

Chapter 1 Introduction & Literature Review

1.8.6 Fourier transform infrared spectroscopy (FTIR)

The FTIR spectroscopy is used for detection of NPs through emission or absorption of infrared spectrum in a given sample of any matter state (solid, liquid and gas). It is used to effectively detect different functional groups and characterize covalent bonding information. The infrared particle emissiongenerallystimulatesparticles of the illuminated material into a higher vibrational state. Light wavelengths captivated by a particular fragment are the energy variancein the middle of at-rest and excited vibrational conditions, and are distinctive of its molecular arrangement.

The FTIR detector measures the intensity of transmitted or reflected light as a function of its wavelength. The FTIR spectra are presented usually, as plots of intensity versus wavenumber (in cm-1). Wavenumber is the reciprocal of the wavelength. Intensity can be plotted as percentage of light absorbance or transmittance at each wavenumber. The AuNPs synthesized using coriander extract as reducing agent was characterized and determined. The FTIR analysis revealed that carbonyl group of proteins was responsible for reduction of AuCl4 [146]. Fayaz et al. reported synthesis of AgNPs using fungus T. Viride. These were characterized by using ultraviolet visible spectra. The identification of functional groups responsible for reduction was determined with FTIR [140].

Chapter 1 Introduction & Literature Review

1.9 Biological evaluation of nanoparticles (gold/silver)

1.9.1 Antimicrobial activity

The NPs play important role in bacterial growth inhibition due to large surface area to volume ratio which makes them highly reactive. Greater antimicrobial effects have been observed in particles with smaller dimensions. Cellular respiration and cell wall permeability is usually disturbed as the synthesized AgNPs get attached to cell wall. Penetration inside the cell may cause damage by interaction with Sulphur and phosphorous containing compounds like protein and DNA. Release of Ag ions from

AgNPs contributes to bactericidal effect [105]. Several reports exploring the antimicrobial characters of AgNPs could be found in the academic literature. The

AuNPs are regarded as one of the most extensively studied NPs in this respect. Most of these bactericidal studies are performed on the gram negative strains of E. coli.

Sondi et al. synthesized AgNPs, and investigated its activity against E. coli strains in liquid systems as well as on agar plates. These NPs exhibited biocidal effects and results were confirmed by SEM and TEM. Cell wall of microbes was found to be adversely effected by “pits” formation upon interaction with AgNPs [147].

Mechanism investigatory studies on bactericidal property of Ag ions were performed.

It was suggested that Ag+ treatment causes the denaturation of protein molecules.

Consequently, reproductive capacities of bacterial DNA were lost and their proliferation rate was found to be ceased in the presence of these ions [148].

Krishnaraj et al. utilized the greener approach for synthesis of AgNPs and suggested that lytic ability of AgNPs is not only ascribed to cell wall damages but also to unrestricted entry potential of Ag+ ions into the cell interiors through ion channels present in bacterial membranes. These ions suppress the effectual expression of 31

Chapter 1 Introduction & Literature Review proteins and enzymes and stop ATP production in cells causing disruption at the spot

[149]. An indirect approach for getting clear picture of mechanism behind working of

AgNPs was presented by Gogoi et al. [150]. In their experiment fluorescent recombinant E. coli was usedas an exemplary system and the changes caused in bacterial colony by introduction of AgNPs in medium were visually observed. Results indicated that with increment in content of AgNPs not only bactericidal effects had enhanced but apparent reduction in size of treated bacteria was also observed as compared to untreated ones. It was proved by spectrophotometric results that AgNPs are not involved in lytic process but Ag+ ions lysed the bacterial cell. Morphological features of AgNPs were evaluated on its antimicrobial capabilities by examining mortality rate of gram-negative E. colibacteria. Results showed that materials with two dimensional triangular contours possess greater destructive capacity than that of other general nanostructures such as circular or rod-shaped NPs [151]. An interesting application of AgNPs was presented by Maneerung et al.[152]. They incorporated

AgNPs over bacterial cellulose. Bacterial cellulose was immersed in AgNO3 solution and then treated with sodium borohydride. In this way the Ag impregnated to bacterial cellulose was reduced to AgNPs on bacterial cellulose. Bacterial cellulose is a wound dressing material; it was then freeze-dried to prevent NPs leakage. These dressings exhibited strong anti-microbial effects against E. coli and S. aureus. Another technology for commercial implementation of AgNPs as bactericidal was presented by Furno et al. [153]. They synthesized polymer based AgNPs glass device for providing medical based antimicrobial applications. The device was tested under various conditions. It was established that lytic capabilities of AgNPs still persists even after several cycles of reusability and washing. Hence, it could be inferred from above considerations that AgNPs possess excellent bactericidal activity. 32

Chapter 1 Introduction & Literature Review

Perni et al. engineered polymeric hybrid assembly containing AuNPs and methylene blue impregnated on siloxane polymeric networks for antimicrobial purposes. Two bacterial strains, S. aureus as well as E. coli, were treated with this polymeric assembly. The bactericidal effects of this assembly became visually apparent after 5 mins exposure. This enhanced activity was prescribed to AuNPs penetrating abilities which could create pits in the bacterium wall. It was further revealed that when irradiated with laser, this bactericidal effect became even more rapid owing to generation of free radicals in medium because of methylene blue interactions with light. The AuNPs can also become more bioactive because of their ability to couple with the incoming radiations due to presence of Nano-level Plasmon on its surface [154]. Hernández-Sierra et al. evaluated comparative bactericidal effect of NPs by relating antimicrobial efficacies of Ag, Au and Zinc (Zn) NPs. Maximum bactericidal effects were shown by AgNPs at lower concentration than Au and Zn

NPs, having important clinical consequences with lower toxicity [155]. Rai et al.

Assembled core-shell-shell antimicrobial prototype system for biomedical applications. The AuNPs were first synthesized in the form of core-shell structure while employing cefaclor (bactericidal drug) as a capping agent. This assembly contains AuNPs in the middle as a core with surrounding cefaclor chains that act as a shell. It was further cemented and stabilized by doing protective capsular coating over it, so that this system could be easily employed in organic mediums without any leakage issues. Highly enhanced bactericidal activity was documented in this case which was not only ascribed to presence of cefaclor in assembly but also to AuNPs.

The AuNPs generate holes in wall of bacteria causing its immediate death owing to leakage of cytoplasmic cell contents [156].

Chapter 1 Introduction & Literature Review

1.9.2 Haemagglutination activity

The noteworthy application of AgNPs lies with its implementation as an inhibitory agent against hazardous pathogens. The AgNPs have gained phenomenal reception as anti-bacterial agents but the potential antiviral capabilities still remain somewhat undeveloped area in comparison. The AuNPs can also be employed as inhibitory agents against dangerous viral pathogens which cause illnesses of worldwide concerns. Haemagglutination assay (HA) is one of the most widely opted methodologies which are utilized for investigation of antiviral capabilities of NPs.

Normally; pathogens cause diseases by interacting with erythrocytes present in human blood and agglutinate them. This renders these erythrocytes useless in terms of bioactivity and causes infections there. If an introduction of any NPs reduces the agglutination capability of pathogens, that NPs is considered to have antiviral potential.

The efficacy of AgNPs was evaluated as antiviral agent for inhibiting the progression of H1N1 influenza-A virus using various testing assays. This particular virus exhibits extensive agglutination capabilities of chicken erythrocytes.

Haemagglutination inhibition assay (HIA) showed that when AgNPs were introduced into biological environment, agglutination property of this influenza virus was either completely eradicated or became drastically reduced with passage of time [157].

Elechiguerra et al. used similar approach against Human immunodeficiency virus type

1 (HIV-1). They also explained details regarding the possible mechanisms behind these antiviral effects of AgNPs. Firstly, they proposed that this capability of AgNPs is strongly dependent upon the size of engineered NPs. It was proved that, in terms of

HIV-1, only the NPs with the size of ≤10nm were capable of producing inhibitory 34

Chapter 1 Introduction & Literature Review effects. Virus lysis was dependent on size of synthesized AgNPs. The AgNPs with size less than 10 nm were able to get attached with gp-120, receptors of virus. Larger

NPs having more diameter than stabilization sites couldn’t fit into these reception sites and hence doesn’t get involved in lytic process of virus. Secondly, they suggested that AgNPs prevent haemagglutination capacities of this virus by physically attaching itself to glycoprotein receptors. As a result, these sites getoCcupied and virus don’t get chance to interact with erythrocytes of humans. Substantially, the pathogenic abilities of virus are lost [158]. The morphological explanation mentioned above was further cemented while studying preliminary effects of AgNPs against monkey pox virus (MPV) strain. Various assays were performed for investigating inhibitory qualities of AgNPs. The AgNPs only with size of 10 nm were effective against MPV infections, investigated by in-vitro procedures. Moreover, it was projected that AgNPs achieve attenuation of reproduction cycles of MPV by causing disruption of virus cellular pathways, which enhances antiviral potential of these

AgNPs [159]. Pinto et al. utilized similar HIA assay for AgNPs mediated inhibitory reactionfor Herpes Simplex Virus type 1 (HSV-1). Instead of using naked AgNPs, they synthesized mercapto capped AgNPs and investigated the effect of capping agents on inhibitory capacities of these NPs. Results showed that control over antiviral ability of AgNPs could be acquired by manipulating the capping agent content for inhibition of HSV-1 infections [160]. The above mentioned facts demonstrated that AgNPs hold phenomenal antiviral potential and attention should be devoted towards making further progress in this field.

Papp i.e. investigated antiviral efficiency of modified AuNPs against influenza virus. Viruses interact with haemecells by utilizing its sialic acid reception sites.

Chapter 1 Introduction & Literature Review

During synthesis of AuNPs glycerol dendritic structure with terminated sialic acid groups were used as capping agents. This provides dual advantages as not only highly dispersed AuNPs with size of 2 and 14nm were effectually synthesized. But also the sialic acid functionalized dendron aided in providing additional interactive chances to

AuNPs for destroying the influenza virus. A multivalent interaction with sialic acid functionalized AuNPs is expected to completely inhibit viral infection, as binding of viral fusion protein haemagglutinin to host cell surface is mediated by sialic acid receptors. The AuNPs with size 14 nm showed high binding affinity towards haemagglutinin on virus surface and less efficiently bind to isolated haemagglutinin

[161]. Spain et al. utilized similar approach and engineered glycol polymer stabilized

AuNPs. Their biological activity with simple agglomeration assay was demonstrated in lectin coated agarose beads. This manufactured assembly was proposed to possess importance in applications like sensors for viruses and toxins due to presence of glycoproteins (gp) in the shell of assembly [162]. Antiviral potential of ligand capped

AuNPs was investigated against HIV virus. The AuNPs having multiple amphiphillic ligands with sulphate ended functionalities were found to be capable of binding/coupling with protective gp capsule of HIV. Even lower concentration i.e.50% of actually required shell formation density of ligand was enough to enhance antiviral capabilities of NPs up to several degrees. These AuNPs of 2 nm size when applied in Nano molar (nM) concentrations successfully inhibited HIV infection

[163]. Hence, nanotechnology provides new frontiers in terms of developing and tailoring Nano assemblies which could effectively replace expensive medicinal compounds generally employed for treatment. Furthermore, the loading concentration or the dosage amount required for full annihilation of virus is also very low i.e. Only in nM which further arms these assemblies from economic point of view. 36

Chapter 1 Introduction & Literature Review

1.9.3 Phytotoxic activity

Barrena et al. reported that investigation of harmful aspects of particular NPs is an equally important work as studying its beneficiary applications [164]. In fact, the toxicity issues associated with NPs should be determined if suggesting any possible biological application of NPs as it can directly/indirectly affect the surrounding ecological parameters as well as the living biota. Since chemical societies have largely become aware of NPs growing commercial implementation in consumer products; the need of performing critical analysis addressing their impacts on environment has also increased. One of the main controversies is that whether eco-toxicological concerns found linked to usage of engineered NPs outweighs their applications/benefits or not.

Therefore, effort has been channeled in recent years for finding cytotoxic potential of

NPs but lot of work still remains to be done in this respect before finalizing status of particular NPs as safe.

Phytotoxic germination tests of AgNPs were carried out by using cucumber and lettuce seeds. The AgNPs showed non-toxic behavior and no apparent effect on sprouting of seeds was observed [164]. Similar studies carried out and it was observed that AgNPs (1-20 nm of size range) caused retardation in shoot growth and seed germination in barely, flax and ryegrass. However, complete inhibition in germination or growth was not achieved. Furthermore, no indication regarding any possible size dependency was observed [165]. Growth retardation capabilities of AgNPs wereinvestigatedfor Lolium multiflorum. Seedlings were badly affected by AgNPs exposure. High concentration of AgNPs showed destructive behavior and root caps breakage as well as epidermis collapsing. Furthermore, retarded development of root hairs, distorted cortical cells and highly swollen vacuoles were also observed. The 37

Chapter 1 Introduction & Literature Review study also showed that small size of AgNPs exhibited higher damaging power [166].

Stampoulis et al. evaluated AgNPs effects on plant biomass and germination of

Cucurbita pepo (Zucchini plant). Seed sprouting was not affected and 75% reduction in biomass was documented after 15 days exposure. Furthermore, exposure also affected transpiration rate. The bioaccumulation capacities of AgNPs showed that shoots of the plant exhibited 4.7 times greater content compared to untreated [167].

Only seed germination assays are not enough for declaring the status that whether

AgNPs are phytotoxic or not, further studies should be carried out.

Barrena et al. Performed a multivariate analysis and evaluated phytotoxic potential of Au, Ag and iron oxide NPs by seed sprouting assays. The AuNPs (10nm size) were used for treating seedlings of lettuce and cucumber plants. Effect of AuNPs exposure in varied concentration on growth of seeds was documented. Surprisingly, the positive effect (i.e. Increment in seedling growth as well as root growth) was observed in case of AuNPs. However, Ag and iron oxide (NPs) showed negative influence on the growth. Mechanism study regarding this positive response of AuNPs was not performed, due to which more research is required in this field for fully understanding the context of this positive influence [164]. Similar results were reported while investigating AuNPs effect on sprouting of lettuce seeds. The effect of

AuNPsexposure to seeds was expressed after 15 days of incubation period. Similar positive influence in the form of shoot and root length increment was observed indicating that AuNPs do influence the growth of plant in some way. Authors further argued that since the effects appeared after 15 days, it might be possible that AuNPs are not directly involved in growth mechanism. Presence of AuNPs might trigger some reaction or change parameters involved in growth of plant. So mechanism

Chapter 1 Introduction & Literature Review behind interaction of AuNPs with plant is probably of indirect nature [168]. Judy et al. carried out widespread ecological study associated with AuNPs and showed that extensive bio magnification of AuNPs can be found in terrestrial environment which is directly influencing overall food chain. Results revealed that influence of AuNPs on plants and ecosystem cannot be determined by only evaluating these soil based assays. Far-reaching modification is required in these general assays for full wide- scaled collaborative research regarding evaluation of cytotoxic and phytotoxic potential of AuNPs [169].

1.9.4 Insecticidal activity

Contamination of water reservoirs or environment as a whole by use of hazardous and toxic chemical substances as insecticide is one of the major issues of twenty first century which has triggered global concerns. Nanotechnology has the potential solution to these problems. Control over the insect mediated illnesses like dengue and malaria by intercepting breeding of insects at larval stage is one of the commonly employed biological control methodology utilized in tropical and sub- tropical portion of world. Application of AuNPs in this respect also presents some interesting findings.

Moorthi et al. Presented excellent case study in this respect. They engineered

AgNPs while utilizing green chemistry approach and using Sargassum muticum, an alga, as reducing and immobilization medium. Consequently, highly active and monodispersed NPs having size of 20 nm were prepared. The insecticidal assay of

AgNPs was carried out which showed engineered sample portrays lethal capabilities towards the pest of Ergolis merione. The hemolymph sketch revealed that AgNPs

Chapter 1 Introduction & Literature Review caused agglutination in peptides of pest. The adverse effects of AgNPs were also confirmed by rise in defense strategies exerted by pest to neutralize AgNPs perturbation. The treated larval fat bodies, lumen and gastric portion of midgut exhibited extensive degradation and segregation because of their interaction with

AgNPs. Denaturation of protective lipid sheets triggered by AgNPs further facilitates entry of AgNPs into interiors of midgut. De-nucleation of numerous cells was also observed along penetration pathway of AgNPs. It was concluded that activity of

AgNPs and toxic characters of utilized algae were the two contributors for insecticidal activity. This cytotoxic potential of AgNPs can also be exploited as a potential inhibitory product against deadly diseases likemalaria, yellow-fever and dengue fever

[170]. Patil et al. synthesized AgNPs using Pergularia daemia and utilized these

AgNPs against larvae of Aedes aegypti and Anopheles stephensi. The AgNPs exhibited excellent larvicidal abilities against both species [171]. Similar results were reported by Sundaravadivelan et al. They also utilized biogenic approach for AgNPs with leaf extract of Pedilanthus tithymaloides for control of larvae of A. Aegypti.

Hence, utilization of these biogenic NPs not only provides edge in terms of safety but also is more efficient than chemical insecticides [172].

Insecticidal efficacy of AuNPs and AgNPs was evaluated against the larvae of

A. Aegypti. The AuNPs of 2-15nm in size were successfully prepared by using this fungus. Results indicated that AuNPs were three times as more effective as compared to AgNPs. This rapid effectiveness of AuNPs was ascribed to the enhanced cuticle penetration ability of NPs as well as fungus surrounding the AuNPs as dissolution of insect’s cuticle. Fungus allows direct exposure of insect’s protoplasmic content to reactive NPs which adversely affects its working and renders them harmless [173].

Chapter 1 Introduction & Literature Review

Murugan et al. Took this approach one step further and not only evaluated the biogenic AuNPs for larvicidal capabilities but also proposed an indirect controlling methodology by boosting up the Mesocyclops aspericornis (a copepod aquatic predator) predation on mosquitoes by introducing AuNPs in medium. The AuNPs proved to be an effective insecticide against A. Stephensi as well as A. Aegypti.

Furthermore, the efficacy of copepod in AuNPs presence increased from 26.8% to

45.6% and 17% to 26.7% for different larvae of A. Stephensi. While it increased from

56% to 77.3% and 26.7% to 51.6% for A. Aegypti larvae. Investigations are still required for understanding the mechanism behind this boost [174]. Lallawmawma et al. Also evaluated AuNPs efficacy against Culex quinquefasciatus and proved that

AuNPs can provide excellent and effective control against vectors of arbovial and filarial diseases. This Nano bio pesticide assembly reduced the lethal period for 50% population of vector up to several times than normally employed pesticides.

Furthermore, the extract utilized for synthesis of AuNPs also contributed in enhancement of insecticidal capabilities of AuNPs. Hence, it could be inferred from the above case studies that AuNPs mediated control over insects could be utilized as a quick and rapid response strategy against various insect-borne disease. These Nano assemblies with larvicidal capabilities provides greener, eco-friendly and safer approach for vector regulation [175].

1.9.5 Nitric oxide free radical scavenging assay

Accumulation of unstable and reactive radicals inside living organisms because of metabolites formation and respiratory activities creates oxidative complications which directly or indirectly lead to numerous illnesses. Materials having potential to quench these unstable radicals by rendering them inactive are 41

Chapter 1 Introduction & Literature Review called anti-oxidants and are of substantial interest in medicine, nutraceuticals and pharmaceuticals production. Nitric oxide radical is itself a free radical and generally the reductive/anti-oxidant capabilities of any material are evaluated by using protocol based on its quenching. This protocol is termed as nitric oxide scavenging assay.

Ramamurthy et al. utilized the assay for evaluation of reductive potential of AgNPs while using Catechin as positive control. High scavenging activity was observed in case of AgNPs. The AgNPs and AuNPs served as strong nitric oxide, superoxide, hydroxyl and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavengers in comparison to their respective metal oxides [176]. Spherical AgNPs wereengineered using fruit extracts of Piper longum as a reducing and fabrication media. The aforementioned in-vivo assays wereutilized for evaluation of antioxidant potential of

AgNPs. Results showed that biogenic stabilized AgNPs have high protective potential against reactivity of nitric oxide in comparison to other opted controls were determined. Chemical components of extract containing aromatic rings also helped in stabilization of free radical and enhancing the quenching ability of system. Moreover, kinetics study further proved that this quenching ability of AgNPs exhibited direct proportionality to concentration of applied AgNPs [177]. Dalbergia spinosa was used for synthesis of nearly circular AgNPs. The extract from leaves of plant provided much strength to AgNPs. Synthesized AgNPs showed much greater quenching abilities than that of control, highlighting the potential application of AgNPs in the field of pharmaceuticals [178].

1.9.6 Anti-termite properties

In past years an increase was observed in development of preservatives for wood. Developing Nano assemblies that could effectively control termites is highly 42

Chapter 1 Introduction & Literature Review desirable. Unfortunately, AgNPs have not yet been fully exploited and only a few studies that cover their potential as anti-termite agent could be found in literature.

Lotfizadeh et al. carried out a study on AgNPs and argued that exceptionally high thermal and mechanical coefficient of AgNPs could be effectively employed in refining wood drying processes [179]. Inhibition capability of AgNPs was demonstrated against various termites through numerous assays. Termites exhibit symbiotic interactions with bacteria and protozoa species in gut of termites and survive there. The AgNPs directly/indirectly effect termite population by not only physically harming but also by destroying bacterial species present inside its gut

[180]. Kartal et al. revealed that AgNPs have very little scope in terms of anti-termite material and termite assays. These contradictory results raise some questions on efficacy of AgNPs as anti-termite material. Hence, more research should be carried out for these activities of AgNPs [181].

Chapter 1 Introduction & Literature Review

1.9.7 Brine shrimp lethality bioassay

Biological activity of organic or inorganic extract NPs could be conveniently determined by simple Brine shrimp lethality assay. Among species of Artemia, 90% studies utilized Artemia salina (brine shrimp) as model organism. Vijayan et al. synthesized AgNPs by using green synthesis approach and utilized seaweed extract as a capping and reducing agent for synthesis of NPs. Results showed that optimal concentration 160 µl/ml of solution causes maximum mortality rate of Artemia. The mortality rate was also found to be increased with increase in concentration of AgNPs

[182]. Kumar et al. Observed 100% mortality rate against brine shrimp at 100 mM of

AgNPs. Sargassum ilicifolium was used as reducing biomaterial for synthesizing

AgNPs. Results showed that biogenic material which is utilized as a capper could interfere with AgNPs toxicity. However, further studies are required to fully understand the role of capping agents in NPs toxic effects [183]. Arulvasu et al. used

Artemia nauplii and revealed toxic effects of AgNPs on hatching capabilities and mortality rate of species. Size of engineered AgNPs was found to be 30-40 nm.

Increase in AgNPs concentration generated apparent increase in lethality rate of shrimp. Furthermore, damage induced by AgNPs also caused aggregation in the

DNA, gut region and in apoptotic cells. Hatching of cysts was reduced by introduction of AgNPs in medium [184]. It was concluded that nanotechnology, although useful for various applications was recommended for extensive refining before being applied at commercial level. These bioassays which deal with toxicology issues of NPs should be made compulsory. Results acquired should be given significant importance as unrestricted or uncontrolled release of these NPs. Benefits

Chapter 1 Introduction & Literature Review or any potential risks associated with NPs should be distinctively defined and technology recommended for improvement accordingly.

1.9.8 DNA damaging activity

Ames test is a bioassay that utilizes various strains of bacteria for evaluating the DNA scavenging/aggregating/mutating capacities of NPs. This test determines the hazardous or cytotoxic status of any compound. This test elaborates the mutagenic activity which is often directly linked with compound’s carcinogenic potential. Li et al. synthesized AgNPs ofsize 5 nm and evaluated their genotoxicity by utilizing Ames test in five different strains of Salmonella. Results indicated that treatment of strains with AgNPs doesn’t cause any potential increment in mutation frequency. However, anti-bacterial effects were apparent in plate. Different strains also showed varied sensitivities to AgNPs [185]. Kim et al. evaluated biocompatible abilities of AgNPs using various in-vivo as well as in-vitro bioassays. The AgNPs of average size of 2-

5nm were pitted against Salmonella typhimurium bacterial strains. Genotoxic effects depend on concentration of injected AgNPs. Furthermore, they also utilized animal models for investigating intravenous toxicity of AgNPs [186].

1.9.9 Hemolytic properties

In-vitrohemolytic potential of human blood was assessed with AgNPs involved in red blood cell (RBC) damaging. The Ag particle powders dispersed in water were characterized with TEM, DLS, SERS and zeta potential measurement.

Results showed size and agglomerationoccurrence on media type. Hemolysis analysis showed that in phosphate buffered saline (PBS) and plasma, AgNPs size was increased. The citrate AgNPs in media was observed to bring changes in size of 45

Chapter 1 Introduction & Literature Review

AgNPs but on hemolysis no significant effect was recorded. It was concluded that greater surface area of particle had increased release of Ag+ which interacted with red blood cells (RBCs) [187]. Anti-fungal effects of AgNPs and their hemolytic effects on erythrocytes were studied. It was concluded that due to membrane disruption by

AgNPs, fungal cells were destructed and low hemolytic potential was observed. They were recommended for further analysis [188]. Liu et al. revealed that novel nano- composite synthesized in N, N-dimethylformamide demonstrated biocidal effect toward Bacillus subtilis, E. coli and C. albicans. The AgNPs biocompatibility in these cells was determined by hemolysis test [189].

Kullar et al. explored Bovine serum albumin (BSA) conjugated AuNPs applications as drug delivery. Unfolding of BSA has been determined with important physiochemical characteristics. Unfolded BSA was determined by UV-vis spectroscopy in different ionic and zwitter ion surfactants. Anionic and zwitter ion surfactants were found to induce unfolding of BSA at high temperature compared to cationic surfactant. Control treatment i.e. cationic surfactant coated AuNPs showed significantly higher hemolytic activity and very low cell viability. Thus for drug release and biomedical applications BSA coated AuNPs were recommended [190].

Corner examined uptake of NPs and their acute toxicity in human leukemia cells. The

18 nm NPs observed for 3 days were found nontoxic to cells. Citrate-capped NPs in cellular uptake were examined by TEM. Study concluded that NPs were not detrimental but rather precursors of such NPs might have harmful effects on cells

[191]. Goodman et al. studied effect of 2 nm core particles on bacterial viability assays. Results showed that cationic particles had moderate toxicity effect, whereas anionic particles were significantly non-toxic. The dye release study showed that lipid

Chapter 1 Introduction & Literature Review vesicles used for nanoparticle translocation were dose dependent [192]. Cho et al. studied in-vivo toxicity of 13 nm coated NPs induced apoptosis and inflammation in liver. The NPs were observed to be accumulated in spleen and liver for 7 days and circulated in blood stream for long time. Presence of these NPs in spleen and liver was detected by TEM [193].

1.9.10 Acute toxicity

Utilization of AgNPs in biological systems with penetrating, absorption, circulating and distribution property with proper approach have been found to have no harmful effects. For application of AgNPs knowledge of biological system is necessary in depth, as AgNPs alone may not damage the system, but ligand binding

AgNPs result in different toxicity patterns. Zhao and Wang investigated acute and chronic toxicities of AgNP in Daphnia magna. Results showed that at 500 µg AgNP media had no effect on Daphnia for 48 hrs, where accumulation of AgNP was

+ observed 22.9 mg in dry weight. However the free Ag from AgNO3 showed 50% lethality after 48 hrs. It was concluded that acute toxicity effect was because of Ag+ not due to AgNPs. The diet borne Daphnia exposure showed that AgNPs significantly inhibit the growth whereas AgNO3 affected reproduction of Daphnia. Exposure of

AgNPs to aquatic organisms was recommended for analysis of chronic toxicity as for release of Ag+ in environment [194]. Asharani et al. studied deleterious effects and distribution pattern in zebrafish (Danio rerio). Hatching, mortality, heart rate and pericardial edema were observed with synthesized AgNPs using starch and bovine serum albumin (BSA) as capping agents. Results showed that hatching period was extended and mortality rate was increased with increase in concentration of AgNPs.

Abnormal twisted notochord, body axes, pericardial edema, cardiac arrhythmia and 47

Chapter 1 Introduction & Literature Review slow blood flow were observed. The TEM and Electron-dispersive x-ray analysis showed distribution of AgNPs in heart, brain blood and yolk of embryo. It was concluded that AgNPs induced embryo toxicity hindering normal growth of zebra fish

[195]. Maneewattanapinyo et al. conducted acute toxicity on eye and oral irritation, corrosion and dermal toxicity of mice exposed to synthesized colloidal AgNPs. The

AgNPs at 5,000 mg/kg for 72 hrs exposure showed no acute toxic and mortality.

There were no significant changes in body weights of treated and untreated mice.

However, in first 24 hrs a transient eye irritation was observed. It was concluded that exposure of colloidal AgNPs was relatively safe and was recommended for short periods of time [196].

1.9.11 Animal studies

Araújoet al. examined effect of AuNPs on anti-tumor, analgesic and anti- inflammatory activities on various tissues [197]. Applications of AuNPs for bio distribution and comprising therapeutic properties i.e. Anti-tumor, analgesic and anti- inflammatory draw the attention to be investigated further in biological system.

Ahmad et al. synthesized AgNPs using plant leaf extract of Rosa damascena and determine their bio reducing properties using AgNO3. Using Westar rat model analgesic and anti-inflammatory study showed that AgNPs had both characters but with slightly low potency compared to standard drugs [198]. Chiguvareet al. synthesized AgNPs using Buchu plant extract and then investigated the analgesic activity of these AgNPs. Synthesized AgNPs have more effective analgesic activity than the plant extract alone and aspirin drug [199]. Wong et al. reviewed that with arrival of Nano science, natural Ag can now be made into nanometer-sized debris.

Along with anti-bacterial activity, AgNPs appear to have anti-inflammatory effects. 48

Chapter 1 Introduction & Literature Review

The supplied findings showed that AgNPs were powerful at decreasing irritation in peritoneal adhesions without considerable toxic effects. AgNPs can be delivered as novel therapeutic drug for prevention of postoperative adhesions [200]. Sibbald et al. examined the recovery of venous leg ulcers (VLUs) often stalled regardless of compression remedy. Extended bacterial burden and continual irritation are two factors which could prevent these continual VLUs from recovery. It was revealed that

Nanocrystalline Ag dressings may additionally reduce bacterial stages, decrease the continual inflammatory response and function as wound healing products. A

Nanocrystalline Ag dressing blended with 4-layer bandaging was observed in having antimicrobial activity and neutrophil irritation [201]. Chen i.e. investigated organ toxicity, distribution and inflammatory cytokines in adipose tissue. Mice were exposed to AuNPs of spherical size 21 nm, which showed significant fat loss as well as anti-inflammatory activity. Thus from experiment it was concluded that AuNPs can be used as a new avenue for developing fat loss potential therapeutic drugs for treatment of such disorders [202].

The aim of the present study was to study the medicinal importance of D. mucronata and M. buxifolia and then establish green synthesis of silver and gold nanoparticles from them. The study of physical and thermal properties of synthesized

NPs and to establish potential biological properties for medicinal applications.

Chapter 1 Introduction & Literature Review

1.11 Objectives

The aims and objectives of the proposed research are to: • Determine the preliminary phytochemical constituents, nutritional and

elemental profile of the selected plants D. mucronata and M. buxifolia.

• Synthesize and characterize the AgNPs and AuNPs.

• Determine different in-vitro and in-vivopharmacological activities for the test

samples of the plants and synthesized AgNPs and AuNPs.

• Determine the DNA damaging profile for selected plants extracts and

synthesized AgNPs and AuNPs.

• Determine the hemolytic and anti-thrombolytic profile for selected plants

extracts and synthesized AgNPs and AuNPs.

Chapter 2 Methodology

2.0 METHODOLOGY

2.1 Collection of plant materials

Plant material(s); D. mucronata (leaves, bark and roots) and M. buxifolia

(leaves, seeds and fruits) were collected from Marghuzar, District Swat and Dir

Khyber Pukhtunkhwa (KPK), Pakistan. Plants were identified by Mr. Ghulam Jelani,

Department of Botany, University of Peshawar, KPK, Pakistan. M. buxifolia was identified as ahuge thorny shrub or short tree. Short thorns, terminal and axillary.

Leafhabitually fascicled or alternate, 2.5-3.5 x 1-1.5 cm. Upper surface glabrous, lower glaucous, apex rounded, base somewhat cuneate, margin recurved, petiole short, 2-3 mm long. Flowers c.5 mm in diameter, pedicel c.1 mm long. Calyx lobes small, c.3 mm long, acute. Corolla lobes glabrous, c.2 x c.1 mm. Stamens borne on the corolla tube and opposite to the lobes, filaments longer than petals, subulate, glabrous, anthers versatile. Ovary hairy; style tapering, elongated. Fruit less than 1 cm in diameter.Dapne mucronata: D. mucronata was identified by the following physical characters. Shrubs up to 2.5 m tall. Fresh branches often tomentose. Leaves scattered or alternate,long 3-5.8 cm, broad 0.4-1 cm, oblong-elliptic to lanceolate, mucronate, less often obtuse, sessile,coriaceous. Flowers subsessile,white, interminal oraxillary clusters.

Chapter 2 Methodology

2.2 Extraction

Plant material were shade dried and then grinded. The dried powder material of selected plants were macerated in methanol two times (x 2) at room temperature for

15 days, throughoccasional shaking and filtered through filter paper. Filtrates were concentrated by rotary evaporator to get crude methanolic extract. Crude extracts were then partitioned with water (500 ml) and distinct solvents chloroform(3x500 ml),n-hexane (3x500 ml), and ethyl acetate (3x500 ml) to obtain fractions of corresponding solvents. Some of crude methanol extract was preserved for biological screening [203].

2.3 Preliminary phytochemical screening, elemental and nutritional analysis

2.3.1. Preliminary phytochemical screening

Qualitative analysis for detection ofsaponins,alkaloids, carbohydrates, phenols,glycosides, reducing sugars,flavonoids and tannins in the plant materials were performed.

2.3.1.1 Preparation of plant extracts

Plant material (3 gm) in powder form was extracted with 30 ml of methanolfor a week in conical flask followed by filteration and placing in small glass vials without lids to facilitate solvent evaporation. Fresh solvent was used three times for extraction.Dried methanol extracts of both plants were further qualitatively screened for phytochemicals [204]. Composition of reagents used for phytochemical tests is given inTable 2.1.

Chapter 2 Methodology

2.3.1.2 Carbohydrates tests

2.3.1.2.1 Fehling’s test

Fehling A and Fehling B reagents were assorted in equivalent volume together. From this assortment, 2 ml was introduced to crude extractand mixture was heated to boiling gently. Reducing sugars were detected bybrick red precipitateappearance at bottom of tube [205].

2.3.1.2.2 Benedict’s test

Benedict’s reagent (2 ml) was mixed with crude extract (5 mg) and boiled for

3-5 mins. Presence of carbohydrates was indicated by formation of a reddish brown precipitate.

2.3.1.2.3 Molisch’s test

Molisch’s reagent (alcoholic α-naphtol) about 2ml was mixed with crude extract (5 mg) and sample was shaken properly. It was tracked by adding 2 ml of concentrated H2SO4 carefully along the side of test tube. Occurrence of carbohydrate was noticed by presence of a violet ring at the interphase.

2.3.1.2.4 Iodine test

Iodine solution (2 ml) was mixed with crude extract (5 ml). Presence of polymeric carbohydrate (starch) was indicated by a dark blue or purple coloration. Iodine test was performed for polysaccharides. Iodine solution was prepared by dissolving 40 gm potassium iodide (KI) in 100 ml distilled water. When the solution came to room temperature 12.7 gm iodine (I2) was added. When iodine was completely dissolved 3 53

Chapter 2 Methodology drops of Hydrochloric acid were added and solution was diluted with distilled water to 1000 ml.

2.3.1.3 Test for phenols and tannins

For phenols and tannins detection 2 ml of 2% FeCl3 solutionwas mixed with 2 ml aqueous solution of crude methanolic extract. Solution when turn to blue-green or black coloration test is positive [206]. The FeCl3 solution was prepared by addition of

Ammonium hydroxide solution that form precipitate. More FeCl3 solution was added that dissolved the precipitate and then diluted HCl was added to prevent its hydrolysis. When clear solution was obtained it was used as neutral FeCl3 solution for phenolic test. By addition of 3.24 gm of FeCl3 to 100 ml distilled water 2% solution was formed. The FeCl3 used was from Merk.

2.3.1.4 Test for flavonoids

Flavonoids presence was determined through alkaline reagent test. Crude extract (5 mg) was mixed with 2 ml of 2% NaOH solution. Adeep yellow color formation that turned colorless on adding few drops of diluted HCl designated the existence of flavonoids [207].

2.3.1.5. Test for saponins

Presence of saponins was detected by foam test or froth formation test. Plant material in powder 5 mg in a conical flask was diluted with 5 ml distilled water and heated at 55 ºC for 30 mins in a water bath. Filter paper was used to filter the suspension into test tube and at room temperature it was cooled for 15 mins. Froth formation was observed by shaking test tube vigorously. Presence of abundant 54

Chapter 2 Methodology saponins was indicated by 2 cm layer foam in tested sample, whereas low amount of saponins was indicated by little foam formation.

2.3.1.6. Tests for glycosides

2.3.1.6.1 Liebermann’s test

Crude extract (5 mg) was assortedto each of 2 ml chloroform and 2 ml acetic acid. Using ice mixture was then cooled. Cautiously, concentrated H2SO4 (2 ml) was added on sides of test tubeas a resulttwo layers are formed, green color in upper layer designatedexistence of sterols and deep red colorformationshowedexistence of triterpenoids. Change in color from violet to blue to green specifiedoccurrence of steroidal nucleus, i.e. Glycone portion of glycoside [208].

2.3.1.6.2 Salkowski’s test

Crude extract (5 mg) was assortedby 2 ml of chloroform. Then 2 ml of concentrated H2SO4 was putcautiously and the sample was graduallyshaken. A reddish brown color showedoccurrence of steroidal ring, i.e. Glycone portion of glycoside.

2.3.1.6.3 Keller-kilani test

Crude extract (5 mg) was assortedby 2 ml of glacial acetic acid comprising 1-2 drops of 2% FeCl3solution. Assortment was then transferred in a new test tube having

2 ml of concentrated H2SO4. The presence of cardiac glycosides was specified by a brown ring at the interphase.

Chapter 2 Methodology

Table 2.1 Composition of reagents used in phytochemical analysis

S. No. Reagent used Composition

100 ml distilled water was used to dissolve Potassium iodide(5.00 g) 1. Mayer’s reagent andMercuric chloride(1.36 g)

1 liter distilled water was used for dissolving Copper (II) sulphate

2. Benedict’s reagent pentahydrate (17.3 g), Anhydrous sodium carbonate (100 g)

andSodium citrate (173 g)

Two solutions were combined to make Fehling’s solution; equal

amount of Fehling's A and Fehling's B

Two drops of dil. Sulphuric acid with 100 ml dist. Water containing

3. Fehling’s solution 7 g Copper (II) sulphate make Fehling's solution A

100 ml dist. water containing colorless clear solution of sodium

hydroxide (12 g) and aqueous Potasium tartrate (35 g) make

Fehling's solution B

Chapter 2 Methodology

2.3.2 Elemental analysis (atomic absorption spectroscopy)

The plants D. mucronata and M. buxifolia leaves in aqueous solution were subjected to Elemental analysis with atomic absorption spectrophotometry for the following trace and heavy metals; Iron (Fe+2), Zinc (Zn+2), Calcium (Ca+2), Cadmium

(Cd+2), Chromium (Cr+2), Nickel (Ni+2), Lead (Pb+2), Manganese (Mn+2), Cobalt

(Co+2) and Copper (Cu+2).

Reagents and equipment’s: Nitric acid (HNO3), Hydrogen fluoride (HF), Double distilled water, Hydrogen per oxide (H2O2), Sulphuric acid (H2SO4), Hydrochloric acid (HCl) and Per chloric acid (HClO4). Merck (Darmstadt, Germany) made reagents were used. The Cd, Mn, Pb, and Co used was sigma made and Zn, Cu and Fe were

Aldrich made. Glassware were carefully washed with water and then rinsed before use.

Sample preparation: Sample was prepared by Wet digestion method. The plant powder (1g) was taken in conical flask and 10 ml concentrated HNO3 (67%) was added to it. The sample was retained overnight (24h) at room temperature. It was tracked by adding 4 ml HClO4 (67%). Each flask contents were heated after 30 mins on hot plate for evaporation, so that 1 ml clear solution may be obtained. It was then cooled and level was raised to 100 ml by adding double distilled water that was filtered with Whatman # 42 filter paper. For each sample, the filtrate workedin place of stock solution. Samples were stored in air tight vessels for use in elements detection process. Plants in triplicate were investigatedvia flame atomic absorption spectrophotometer (Polarized Zeeman Hitachi 2000) and flame photometer (Jenway

Chapter 2 Methodology

PFP7, UK). Appropriate dilutions (5 mg/ml diluted ten times) of stock solutions were made for each metal calibration standard preparation [209].

Procedure: After adjusting the instrument according to conditions specified inTable

2.2for each element corresponding cathode lamp was turned on and warm up for 10 mins. Air acetylene flame was burnt after warming of cathode lamp. For respective element, gadget was regulated and standardized byoperating values of 2.5, 5, and 10 ppm (Table 2.2). Standard solutions of element used for adjustment were made by dilution of stock solution of Cu, Fe, (Aldrich), Zn and Ni (Parkin Elmer) and Pb, Cr,

Co, Mn, (Sigma). To verify standardization, operationalstandardsran by way of unknown. The stock solution trial was sought in flame and respective elementconcentration was considered in ppm by comparisonto standard curve of corresponding metal [210].

Chapter 2 Methodology

Table 2.2 Specified conditions applied for detection of various elements

Wavelength Slit width Acetylene flow Air oxide flow Cathode lamp Elements Flame type (nm) (nm) (L/min) (L/min) current (mA)

Co Air/ acetylene 240.7 0.2 2.0 17 30

Pb Air/ acetylene 283.3 0.7 2.0 17 10

Cu Air/ acetylene 324.8 0.7 2.0 17 15

Zn Air/ acetylene 213.9 0.7 2.0 17 15

Ni Air/ acetylene 232 0.2 2.0 17 25

Mn Air/ acetylene 279.5 0.2 2.0 17 20

Fe Air/ acetylene 248.3 0.2 2.0 17 30

Cr Air/ acetylene 357.9 0.7 2.5 17 25

Cd Air/ acetylene 228.8 0.7 2.0 17 5

Ca Air/ acetylene 422.7 0.7 2.0 17 10

Chapter 2 Methodology

2.3.3 Nutritional analysis

2.3.3.1 Ash determination

Ash contents were determined in D. mucronata and M. buxifolialeaves by powder drugs ash analysis that comprised total ash, acid insoluble ash and water soluble ash. These tests were determined on behalf of crude powder as such as well as exhausted with hexane and ethanol.

Equipment and glassware: Desiccators, oven, Muffle furnace, Benson burner and silica crucible electric balance.

Procedure: Flat bottomed silica crucible was systematically washed and dried in oven at 70oC for 30 mins. Crucible was burnt and tarred then cooled in desiccators and its weight was measured (W1). About 4 gm of test samples were spread on it evenly. The steady raise in temperature to 550oC in muffle furnace burnt the mildly heated loaded crucible on Bunsen burner. Carbon content in test sample was burnt by maintaining temperature for 24 hrs turning it to greyform. The heater was turned off and crucible containing ash was transferred to desiccators where it was cooled and its weight was measured (W2) [211]. Total ash and percent ash values were calculated by the following formula (AOAC, 2000).

Total ash mg/g) = (W2 − W1 of sample) = mg/g

W2 − W1 % Ash = × 100 Weight of sample

Chapter 2 Methodology

2.3.3.2 Analysis of moisture

Equipment and glassware: Petri dish, Electric oven, electric balance and desiccators

Procedure: Weight of clean Petri dish (W1) was measured and about 2 gm of respective plant sample was taken in it. Partially covered Petri dish (with petri dish lid) was placed in oven for 4-6 hrs at temperature of 105oC, till constant weight was obtained. It was then transferred to desiccators for cooling (30 mins), weight of Petri dish was measured again (W2) [212]. Moisture contents in percent were calculated by this formula (AOAC, 2000).

X %Moisture = × 100 Weight of test

Where, X = Sample Weight (nextto heat) = W2 - W1

W2 = Petri dish empty Weight + test (next to heat)

W1 =Petri dish empty Weight

Chapter 2 Methodology

2.3.3.3 Analysis of protein

Macrojeldahl distillation method was used for protein determination.

Reagents: Conc. H2SO4, CuSO4, 4% Boric Acid, 0.1 N standard HCl solution 32%

NaOH and K2SO4

Preparation of Mixed indicator (0.03g of Bromo cresol green + 0.016g of methyl red in 100 ml of alcohol).

Apparatus: Distillation and digestion apparatus, burette, Kjeldahl flask

Procedure: Macro kjeldahl method was used to determine Protein (% Nx6.25) [212].

In digestion flask 0.5 gm of dry ground sample was taken. Digestion assortment

(CuSO4, K2SO4 and FeSO4 in proportion of 5, 94 and 1 correspondingly) and conc.

H2SO4 (25 ml) were put in flask and digested for 6 hrs in digestion flask

(kjeldatherm). Flask was detached, chilled and stuffing were at that timemoved to 250 mlcontainer. PurifiedH2O was poured to mark 50 ml of the solution mentioned. About

10 ml of strong base was putto mark it alkaline. Few drops mixed indicator and 4%

Boric acid solution (50 ml) were moved to distillation flask followed by addition of

60 ml of 32% NaOH solution and 50 ml of water. It was collected for titration in flask after distillation. For titration 0.1 N HCl was taken in burette through content of the flask. The reading was observed and using the following formula, percent protein content was determined (AOAC, 2000).

(V1 − V2) × 14.01 × 0.5 × 100 (%N) = Sample in mg

Where, V1= Sample Titration reading 62

Chapter 2 Methodology

V2= Blank Titration reading

14.01= Nitrogen (N) Atomic weight

The sum of% protein substanceswas calculated by multiplication oftotal nitrogen

detected with 6.25.

% Protein =% Nitrogen × 6.25.

Chapter 2 Methodology

2.3.3.4 Analysis of fat

Chemicals, glassware and equipment: Water bath, Soxhlet extraction apparatus,

Petroleum ether B. P. (40-60oC), H. T (Tecator). Extraction thimbles and heating mantle

Procedure: Crude fat was extracted by Soxhlet apparatus [213]. About 2 gm model was crammed in filter paper made cellulose extraction thimble that was kept in extraction compartment of apparatus. Pre weighed dried and clean round bottom flask

(250 ml) was occupiedby petroleum ether that was coupledwith extraction tube having thimble. For about 5-6 hrs, Soxhlet apparatus was run and using water bath solvent was evaporated from extract in round bottom flask and weight (W2) was taken again.

The subsequent equation was used to estimate fats proportion (AOAC, 2000).

X % fats (ether extract) = × 100 Weight of sample

Where, X = Fats Weight = W2 - W1

W1 = Empty flask Weight

W2 = Empty flask Weight + sample after evaporation of solvent

Chapter 2 Methodology

2.3.3.5 Analysis of crude fiber

Equipment and glassware: Suction pump, Muffle furnace, Crude fiber extraction apparatus (Fiber Tec System M. Tecator), oven.

Reagents: 0.313 NNaOH, 0.255NH2SO4, Petroleum Ether, Ethyl Alcohol Asbestos.

Procedure: In oven 3 gm of respective sample was dried to constant weight. To remove crude fats, 2 gm of this material was extracted with 30 ml Petroleum Ether.

The residue materials via asbestos (0.5g) were transferred to digestion flask. Flask connected to condenser was filled with 200 ml boiled hot 0.255 N H2SO4that was boiled for 30 mins. The substancdesat that momentwere filtered in fluted funnel via linen fabric. To remove acids the filtrate was washed and moved once more to digestion flask with boiling 0.313N NaOH. Then NaOH was added to make it exactly

200 ml. The reflux condenser and flask were coupled and heatedto boiling for 30 mins. Gooch crucible make with asbestos mat was used then for filteration of this boiling material. It was followed by washingthrough boiling water (50 ml) and then

Ethyl Alcohol (15 ml). Substances were moved to crucible and at a temperature of

110oC were driedtill constant in oven hot air (W1). Container was shifted to stifleheater, burnt till gray and then weighted (W2). Equation used to calculate the

Percent crude fibers is given.

W2 − W1 % Crude fibers = × 100 Weight of sample

Where, W2 – W1 = Crude fiber

Chapter 2 Methodology

2.3.3.6 Carbohydrate contents

For Carbohydrate contents calculation the total weights of moisture, crude fibers, proteins, fat, and ash were subtracted from 100 [212].

% Carbohydrates = 100 – (Protein + moisture + crude fiber + ash + fats)

Chapter 2 Methodology

2.4 Green biogenic synthesis of silver and gold nanoparticles

The AgNPs and AuNPs were synthesized through green approach, withaqueous extracts of D. mucronataand M. buxifolia leaves. Dust was removed from both plant materials by filtering it with mesh. Powdered plant material(s) (100 g) wasboiled in 2000 ml of distilled water for 30 mins to get extract. Solution was then filtered. The aqueous extract was centrifuged two times at 10,000 rpm for 20 min at

4°C to eliminate cell debris. Filter paper (0.2 μm pore size) was used to filter the resulting supernatant and filtrate was then used for formation of AgNPs and AuNPs

[214]. The AgNO3 and HAuCl4.3H2O was purchased from Merck.

Reaction: The Ag solution (1mM), AgNO3 (17 mg) was added in distilled water (100 ml) while for Au solution (1mM) preparation, HAuCl4.3H2O (34 mg) was added in distilled water (100 ml). The HAuCl4 solution (1mM) and AgNO3 solution (1mM) was mixed with plant extract in 1:5 ratio respectively. The ruby red color appearance in few mins showed reduction of Au ions to AuNPs by M. buxifolialeaves extract while the brown color appearance showed reduction of Ag ions to AgNPs. The mixtures were then centrifuged at 10,000 rpm for 30 mins at 4 °C, which removed unbound phytochemicals, re-dispersed in distilled water. The reaction mixture was subjected to sonication for 2-3 mins and then dried using lypholizer. The same procedure was repeated for D. mucronata leaves.

Chapter 2 Methodology

2.5 Characterization of synthesized silver and gold nanoparticles

2.5.1 UV-vis spectroscopy

A dual beam UV-1100 Shimadzu spectrophotometer was utilized for study of colloidal Ag solution and Au solution to endorse the existence of AgNPs and AuNPs.

The aqueous plant extracts along liquid AgNPs and AuNPs solutions of D. mucronataand M. buxifolialeaves diluted 10 times were scanned. Aqueous extracts of plants, AgNPs and AuNPs were scanned in wavelength range 200–600 nm. Points of maximum absorbance regions show characteristic peaks that gives information on charge transfer, purity of sample, and analyte [215].

2.5.2 Fourier transform infra-red (FTIR) spectroscopy

The FTIR Prestige- 21, (Shimadzu, Japan) was used to analyze aqueous extracts and derived AgNPs and AuNPs of both plant materials to examine functional groups taking part in NPs synthesis. Spectrometer operating with 4 cm-1 resolution in diffused reflectance mode was utilized for scanning at range 4000 cm-1 to 400 cm-1.

2.5.3 X-ray diffraction (XRD)

Crystalline nature of D. mucronata and M. buxifolialeaves derived AgNPs and

AuNPs was detected by XRD utilising JDX 3532 JEOL, Japan. The diffraction pattern was recorded by placing the sample in sample holder [216]. Instrument was operated at 20 to 40 kV and at a wavelength of 1.5418Ao in θ to 2θ configurations which gave diffracted intensities in 2θ angle range of 10o to 90o. Crystal size of NPs was measured by using Debye Scherer equation as follows:

Chapter 2 Methodology

D = 0.94 λ / β cosθ

Where, β is the full width at half maximum (FWHM),

λ is the wavelength of X-ray used,

D is the measure of size of crystal that is perpendicular to reflection planes,

and θ is angle of diffraction

2.5.4 Energy dispersive X-ray detection (EDX)

The EDX coupled with SEM (JSM-5910 with model INCA- 200- Oxford instruments, UK) was used for elemental analysis of D. mucronataand M. buxifolia leaves derived AgNPs and AuNPs. The NPs were distributed on both verges of adhesive tape that was fixed on a microscopic stump of aluminum [216].

2.5.5 Scanning electron microscopy (SEM)

Analysis of size, shape, surface topology and morphology of AgNPs and

AuNPs derived from D. mucronataand M. buxifolia leaves was performed with SEM

(JSM5910, JEOL, Japan). A copper grid carbon was smearedby thin film of AgNPs and AuNPs assorted with distilled water and dried in hot air for 5 min. Samples were then observed at 30,000, 33,000, 60,000 and 100,000X magnification [216].

2.5.6 Transmission electron microscopy (TEM)

Thin film of sample was set by releasing a minutequantity of sample onto the copper grid coated viacarbon. Sample was air dried by placing it in mercury lamp for

5 mins. Techni-G2 model -300kV was used for TEM detection [216]. 69

Chapter 2 Methodology

2.5.7 Thermo gravimetric/ differential thermal analysis (TG-DTA)

The TG-DTA (Perkin Elmer Diamond Series, USA) was utilised to testD. mucronata and M. buxifolialeaves powdered plant materials and derived AgNPs and

AuNPs to study oxidation reactions, decomposition rates, and physical progressionssuch as vaporization and sublimation [216].

Chapter 2 Methodology

2.6 Biological evaluation of crude extracts and synthesized AgNPs and

AuNPs

The organic crude and aqueous extracts of D. mucronate (bark, leaves and roots) and M. buxifolia (fruits, leaves and seeds) along with derived AgNPs and

AuNPs were estimated to detect their biological importance.

2.6.1 Anti-oxidant activity

From the stock solution (10 mg/10 ml of methanol) various dilutions; 100,

200, 300, 400, 500 and 600 μg/ml, were made to determine the antioxidant activity.

Two mlDPPH solution prepared freshly was assortedby 1ml of respective dilution.

Mixture was incubated in dark room for 10 mins and absorbance was measured at 517 nm by UV–Vis spectrophotometer (Shimadzu UV-1601) by free radical scavenging effect of DPPH [217]. Scavenging capacities of sample and negative control (1 ml methanol and 2ml DPPH) were compared. Scavenging of free radical of respective sample declared in per cent was identified by following equation.

(Ab − As) Percent Inhibition of DPPH activity = × 100 Ab

Where, ‘As’ signifies the absorbance of test model and ‘Ab’ denotes absorbance of blank [217, 218]. A plot of percent scavenging curve with concentration of smaples determined the 50% Effective Concentration (EC50) [219].

Chapter 2 Methodology

2.6.2 Anti-bacterial activity

2.6.2.1 Determination of percent inhibition

Anti-bacterial activity was determined via Agar well diffusion method [220].

Strains used for anti-bacterial studies included Morganella morganii, Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Acinetobacter baumannii (A. baumannii), Vancomycin Resistant

Staphylococcus aureus (VRSA) and Proteus vulgaris (P. vulgaris). A 20 mg/ml stock solution of test samples was prepared in 1% dimethylsulphoxide (DMSO). Turbidity of bacterial inoculum was adjusted according to 0.5% McFarland standard [221]. The culture was inoculated on nutrient agar medium. Ciprofloxacin was used as standard drug for A. baumanni. Amoxicillin was used as standard drug for E. coli, P. aeruginosa, S. aureus and P. vulgaris. Ampicillin was used as standard antibiotic for

VRSA and Rifampicin was used as standard antibiotic disc for M. morganii. The

DMSO (1%) was used as negative control. Wells were made in the agar medium and

100 µl of extract solution was delivered into them. All plates after incubation for 24 hrs at 37ºC, were studied for any zones of growth inhibition, and diameters of these zones were taken in millimetres. Percent inhibition was deliberatedby means of the subsequent formula.

퐼푛ℎ𝑖푏𝑖푡𝑖표푛 푧표푛푒 표푓 푠푎푚푝푙푒 푃푒푟푐푒푛푡 𝑖푛ℎ𝑖푏𝑖푡𝑖표표푛 = × 100 퐼푛ℎ𝑖푏𝑖푡𝑖표푛 푧표푛푒 표푓푠푡푎푛푑푎푟푑

2.6.2.2 Determination of minimum inhibitory concentration (MIC50)

Minimum inhibitory concentrations (MIC50) of test models were deteced for the test bacteria by thetestifiedmethod with minormodifications[222]. 72

Chapter 2 Methodology

Stock solutions (16 mg/ml) were prepared in DMSO. Various dilutions (0.04 mg/ml,

0.08 mg/ml, 0.12 mg/ml…13.96mg/ml and 14 mg/ml) of each extract were made in

DMSO. Bacterial inoculum was added into each tube containing sterile nutrient broth with test samples. Tubes were then incubated at 37°C for 24 hrs. Results were estimated based on visible growth in test tubes in comparison to negative control.

2.6.2.3 Determination of minimum bactericidal concentration (MBC)

The MBC of test samples were detected by a modified method of Spencer and

Spencer [223]. Models were selected from plates with undetectible growth in

MIC50test and sub-cultured on recentlymade nutrient agar plates and incubated at

37°C for 48 hrs. The MBC was analyzed as concentration of model that did not demonstrated any growth.

Chapter 2 Methodology

2.6.3 Anti-fungal activity

Anti-fungal activity of test samples was determined against Candida albicans

(C. albicans), Fusarium oxysporum (F. oxysporum), Aspergillus flavus (A. flavus),

Aspergillus parasiticus (A. parasiticus), Penicillium digitatum (P. digitatum), and

Aspergillus niger (A. niger). Sabouraud Dextrose Agar (SDA) media was made, autoclaved and incubated at 28oC for 24 hrs. Stock solution containing test samples20 mg/ml in Dimethyl sulfoxide (DMSO)was prepared. The SDA when heated till 50ºC,

67μl of test samples from each stock solution were added to make slants. After sterility check, 5-7 days old fungal culture was streaked on solidified medium and incubated at 28±1oC for 7 days. Miconazole and DMSO were taken as positive and negative controls. Results were taken by measuring linear growth on slanted test tubes

[224].

Growth in sample Percent inhibition = 100 − ( × 100) Growth in negative control

Chapter 2 Methodology

2.6.4 Haemagglutination activity

Haemagglutination test of models was carried out for ABO blood groups of human erythrocytes at different dilutions (1:2, 1:4, 1:8 and 1:16) which were prepared in phosphate buffer (pH 7.4) from stock solution (20 mg/ml of DMSO).

Phosphate buffer (pH 7.0) was made by adding 0.453g of KH2PO4 and0.47g of

Na2HPO4, each in 50 ml of distilled water. The dissolvedKH2PO4and Na2HPO4were assorted in ratio of 3:7 (V/V). The test samples stock solution was made as 20 mg of test sample per ml of DMS0. Blood was taken from healthy volunteers and 2%

RBC’s suspension in phosphate buffer was made through centrifugation. From each dilution, 1 ml each of sample and RBC’s suspension was added. The tubes were incubated at 37oC for 30 mins followed by centrifugation. Deposition of rough granules indicated a positive activity while smooth button formation demonstrated negative result. The overall experiment was conducted accordingly to Ahmad et al.

[225].

Chapter 2 Methodology

2.6.5 Phytotoxic activity

The phytotoxic bioassay of test samples was performed againstL. minoraccording to Hussainet al. [226]. From stock solution of sample (20mg/ml of methanol), the phytotoxic behavior was tested. The samples were spread on pre- labeled petri plates and left in open air for drying. Ten fronds of L. minor plant were introducedin every plate and E-media was put subsequently and plates were incubated at room temperature. Number of viable fronds was noted on daily basis up to seven days.

After the fronds damage rate determination in plates the phytotoxic activity was tested at low concentration of samples. From stock solution (5 mg extract dissolved in 5 ml of methanol),10, 100 and 1000 µl were transferred to the sterilized petri plate, that was equal to 10, 100 and 1000 µg/mlcorrespondingly. The test samples were incubated with tenL. minor plants at 28oC in growth chamber for 7 days. Results were recorded on seven day of incubation by damaged plants counting.

Methanol was used as negative control and Paraquat [(C6H7N)2]Cl2at 0.015 μg/ml was used as standard herbicidal agent. Percent regulation was measuredby the succeeding equation.

No. of fronds in test sample Percent Regulation = 100 − ( × 100) No. of fronds in negative control

Criteria for phytotoxic activity: 0-40% inhibition-low activity, 40-60% inhibition-moderate activity, 60-70% inhibition-good activity and 70% and above- significant

Chapter 2 Methodology

2.6.6 Insecticidal activity

The insect species selected were gatherd from Nuclear Institute of Food and

Agriculture (NIFA), Peshawar. Under normal room temperature cultures were kept in lead crystalcontainers with appropriatenutrients and ventilation conditions. R. dominicaand T. castaneumwere reared with yeast and flour in ratio of 1:19 andC. analis was reared with green gram [227, 228].

Insecticidal activity of models was determined by Filter paper protocol forT. castaneum,R. dominicaandC. analisaccording to the reported procedure [229]. Filter paper was cut 9 cm or 90 mm according to size of petri plate and placed in each petri plate. Sample was prepared by dissolving 20 mg of test sample in 1 ml methanolthat was poured into petri plates containing filter paper (Whatman No.1). Methanol was allowed to evaporate and water spray was done on each plate to meet humidity requirement. Anuncontaminated brush was used to move 10 insects to each plate.

Plates were incubated for one day at 27°C with maintenance of humidity. Positive control used was Permethrin (235.9 μg/cm2) while methanol was used as negative control. Results were observed and percent mortality was calculated with Permethrin

(235.9 μg/cm2) as positive control. After incubation period of 24 hrs, percent mortality was deliberatedconferring to the following formula:

No. Of dead insects in test samples Percent mortality = × 100 No. Of dead insects in control

Chapter 2 Methodology

2.6.7 Anti-termite activity

Anti-termite activity was performed according to Salihah et al. [230] against

H. indicola. Filter paper was cut 9 cm or 90 mm according to size of petri plate and placed in each petri plate. Sample was prepared by dissolving 20 mg of test sample in

1 ml methanol that was poured into petri plates containing filter paper (Whatman

No.1). Methanol was allowed to evaporate and water spray was done on each plate to meet humidity requirement. Anuncontaminated brush was utilized to move 10 H. indicola to each plate. Rate of mortality was established for all platesafter 24, 48 and

72 hrs. Termisolve B-PRO served as positive control while Abbott’s formula was used to calculate Percent mortality after three days.

Total number of dead termites after treatment % Mortality x 100 Total number of termites before treatment

Chapter 2 Methodology

2.6.8 Cytotoxic activity

For assessment of cytotoxicity of test samples brine shrimp (A. salina, leach) is an inexpensive, effective and simple test. Eggs were developed in seawater using commercial salt mixture and double distilled water in thin rectangular plastic bowl (22

× 32 cm). Plastic dish was asymmetrically partitioned by a perforated device. About

50 mg of eggs were spread in big partition. Small partition was kept open for light and incubated for two days at 37oC. By means of a pipette nauplii were taken after two days from the partition where light was provided. Cytotoxic activity was tested as reported [231]. Samples for crude extracts, its fractions and derived NPs were tested against A. salina. Test sample (5 mg) for preparation of stock solution was dissolved in 5 ml of dimethylformamide. From stock solution 10, 100 and 1000 µl were shifted to vials (3 vials for each concentration were used for every sample under observation) that was same as 10, 100 and 1000 µg/ml respectively. When solvent was evaporated seawater solution (38 gm/l) was put in each vial. About 5 ml of seawater with 10 shrimps was transferred to each vial. Thus 30 shrimps were tested for each sample and incubated for 24 hrs at room temperature. Number of survived shrimps was calculated next day. Negative control used was 2 ml of dimethylformamide and positive control was Etoposide for cytotoxicity of test samples comparison. Lethality was determined by comparison of control and mean surviving larvae in vials. Median lethal concentration LD50 of test samples was calculated from plot of percentage the shrimps killed by probit analysis Microsoft Excel. Test samples with LD50≤ 1.0 mg/ml are known to exhibit toxic effects.

Chapter 2 Methodology

2.7 DNA damaging, hemolytic and anti-thrombolytic profile

2.7.1 Thrombolytic assay

Reported procedure was followed for determination of% clot lysis [232]. From healthy volunteers whole blood (6 ml) was takendeprived of a history of anticoagulant or oral contraceptive treatment. Six tubes were selectedfor each treatment and test was three times repeated. In pre weighed sterile micro centrifuge tubes blood sample (1 ml) was distributed and for clot formation incubated at 37 ºC for 45 mins. Serum was entirelyremovedafter clot formation deprived ofinterrupting the clot and to determine the clot weight, tubes wereweighed once more (clot weight = weight of tube containing clot – weight of empty tube). Normal solution was made by adding 10 mg of test samples in 10 ml of water. From stock solutions 20, 40, 60, 80 and 100 µlwere added to the Eppendorf tubes containing pre weighed clot. These were equivalent to

20, 40, 60, 80 and 100 µg/ml. Negative and positive controls used were sterile distilled water (100 µl) and streptokinase (100 µl). Tubes were incubatedfor 90 mins at 37 ºC and detected for clot lysis. The fluid attained was detachedprudently and to observe difference in weight the tubes were weighed again after clot disruption.

Before and after clot lysisdifference in weight was taken and articulated as percentage of clot lysis. Significance of test samples% clot lysis in comparison to control was determined by ANOVA followed by Tukey post-test. Data are expressed as mean ± standard deviation. The following formula was used to calculate percent clot lysis.

Weight of released clot % Clot lysis = × 100 Clot weight

Chapter 2 Methodology

2.7.2 Hemolytic activity

Spectrophotometric method reported for hemolytic assay was followed [233].

About 5 ml blood was taken from normal healthy individual and centrifuged for 3 mins at 1500 rpm. Sterile phosphate buffer (pH 7.2) and saline solution was used to wash blood pallet three times. Stock solution was made by dissolving 10 mg of aqueous extracts of D. mucronata and M. buxifolia leaves and their derived AgNPs and AuNPs in 10 ml saline solution. From stock solution 10, 50, 100, 200 and 250 µl was transferred to Eppendorf tubes and their volume was raised to 1 ml of saline solution. This was equivalent to 10, 50, 100, 200 and 250 µg /ml. Normal (0.5%) saline solution was used for re-suspension of pallet. In cell suspension 0.5 ml of test samples (10, 50, 100, 200 and 250 µg/ml) were introducedin saline correspondingly.

The assortment was incubated for 30 mins at 37oC and then centrifuged for 10 mins at

1500 rpm. At 540 nm, absorbance was measured to determine the hemolytic activity.

Triton X-100 was used as positive control and phosphate buffer (saline) as negative control. Procedure followed was according to Helsinki Declaration [234].

Chapter 2 Methodology

2.7.3 Mutagenicity test

Muta-Chromplate kit (purchased from Environmental Biodetection Products

Incorporation (EBPI), Ontario, Canada) based method was used to determine mutagenicity. Fluctuation test was entirely performed in liquid culture and test kit was based on validated Ames bacterial reverse-mutation test. Chemicals used included 2 mg/mlBromo cresol purple, Davis-Mingioli salt (concentrated 5.5 times), 0.1 mg/ml

L-histidine, D-glucose 40% (w/v) and 0.1 mg/ml D-biotin.2-nitro fluorine (30 µg/100

µl), and sodium azide (0.5 µg/100 µl) were used as sterile standard mutagens. These chemicals were purchased from EBPI.

Reagent mixture contained 4.75 ml D-glucose, 21.62 ml Davis-Mingioli salt,

1.19 ml D-biotin, 2.38 mlBromo cresol purple, and 0.06 ml L-histidine aseptically mixed in sterile bottle. Sterile distilled water, sample (plant extract), standard mutagen and reagent mixture were added in bottles by the amount shown inTable 2.3. Two mutant strains (S. typhimurium TA98 and S. typhimurium TA100) were provided by

EBPI. Inoculation of bacteria in nutrient broth was followed by incubation for 18-24 hrs at 37oC. Culture broth (5 µl) was mixed systematically in bottles. Each bottle matters were moved to multichannel reagent boat and 200 µl aliquots of mix were transferred into each well of titration plate. Plate was prevented from evaporation by placing in airtight polyethylene bag and incubated for 4 days at 37oC. Blank plate was examined first andother plates wereobservedonce plates in blank were totally purple in color. This indicated that the assay was not contaminated [235].

Chapter 2 Methodology

Interpretation of results: The standard, background and test plates were counted visually. All turbid,yellow or partial yellow wells were marked positive and wells with purple color were marked as negative. The positive wells number was noted.

Thetest sample and standard mutagen were not contained in background plate. It shows thetest bacteria background or spontaneous mutation. If entire wells of test plate showed purple color, the test sample was taken aslethal to test strain (Table 2.3).

The test samples were considered mutagenic if positive wellsnumberexceededtwofold positive wells in background plate (spontaneous mutation) [236].

Chapter 2 Methodology

Table 2.3 Set up of the fluctuation assay

Volume added (µl)

Treatment Mutagen Reagent Deionized Salmonella Sample Standard Mixture water test strain

Blank -- -- 2.5 17.5 --

Background -- -- 2.5 17.5 0.05

Standard 0.1 -- 2.5 17.4 0.05 mutagen

Test Sample -- 0.005 2.5 17.5 0.05

Chapter 2 Methodology

2.8 Animal studies

Swiss Albino (Balb-C) mice, G. Pigs and Rabbits of definite weight range and either sex were utilized in the experiments. Experiments were performed at Veterinary

Research Institute Peshawar, Khyber Pukhtunkhwa (KPK). For all experimental procedures, animals were kept in group of six per sample. Animals had free access to standard food and water. Temperature at 22±2°C was maintainedthrough adjustable air condition (AC) and 12 hrs light and dark cycle was provided. All investigations were done in agreement with Animals Scientific Procedure Act (1986) UK.

Samples screened: Synthesized AgNPs and AuNPsandCrude methanolic extracts of both plant material(s); D. mucronata (leaves, bark and roots) and M. buxifolia

(leaves, seeds and fruits) were tested qualitatively for effectiveness tovital pathological illnesses as inflammation, fever and pain through in-vivo studies (acute toxicity test, anti-analgesic, anti-pyretic, gastro intestinal track motility, anti- inflammatory tests and biochemical parameters determination).

Statistical analysis

Two way ANOVA tracked by Tukey’s post hoc exploration was utilized for statisticsinvestigation. P< 0.05 was taken significant.

Chapter 2 Methodology

2.8.1 Acute toxicity assay

Acute toxicity assay was performed in accordance to Shilpi et al. By utilizing

Swiss Albino (Balb-C) mice of either sex weighing 30±10 gm [237]. Animals were allocated into twenty seven groups; each containing six animals (n=6). Methanol crude extract of D. mucronata (leaves, bark and roots) and M. buxifolia (leaves, seeds and fruits) were screened at concentration of 200, 400 and 600 mg/kg body weight of mice. D. mucronataleaves derived AgNPs and M. buxifolia leaves derived AgNPs and

AuNPs were screened at 20, 40 and 60 mg/kg body weight of mice. Test dosages were injected via intraperitoneal means (i.p.) and the animals with free access to water and feed were observed for 24 hrs. After 24 hrs, survived and deceased animals number was noted down. The resultsthat were observed was analyzed by ANOVA following Tukey’s post hoc (P< 0.05) analysis.

Table 2.4 Groups received treatment as follows:

Group Sample Dosage Group Sample Dosage mg/kg 1. D. mucronata leaves 200 13. M. buxifolia leaves 200 2. 400 14. 400 3. 600 16. 600 4. D. mucronatabark 200 16. M. buxifolia seed 200 5. 400 17. 400 6. 600 18. 600 7. D. mucronataroot 200 19. M. buxifolia fruit 200 8. 400 20. 400 9. 600 21. 600 10. D. mucronataleaves 20 22. M. buxifolialeaves 20 11. derived AgNPs 40 23. derived AgNPs 40 12. 60 24. 60 25. M. buxifolia leaves 20 26. derived AuNPs 40 27. 60

Chapter 2 Methodology

2.8.2 Anti-analgesic activity

Anti-analgesicactivity was performed via acetic acid induced writhing test withSwiss Albino (Balb-C) mice of either sex weighing 18-22 gm for possible analgesic effect [238]. For experimentations, animals were allocatedrandominto twenty nine groups, each group consisting of six animals (n=6). Before starting the experiment, animals were withdrawn for at least 2 hrs from food. The experimentalmodels, standard (diclofenac sodium) and the saline (negative) control were i.p.introduced, 30 mins earlier toadministration of 1% acetic acid. Writhing activitiesin mice were induced via i.p.administration of 1% acetic acid (10 ml/kg).

Lastly, number of writhes (contraction of abdominal muscles; accompanied by posteriorbody elongation and limbs extension) remainedunder observation for 20 mins; after 5 mins induction of acetic acid.

Table 2.5 Groups received the treatment as follows:

Dosage Group Sample Group Sample Dosage mg/kg 1 normal saline 10 ml/kg 2 Diclofenac Sodium

3 100 15 100 4 D. mucronata leaves 200 16 M. buxifolia leaves 200 5 300 17 300 6 100 18 100 7 D. mucronatabark 200 19 M. buxifolia seed 200 8 300 20 300 9 100 21 100 10 D. mucronataroot 200 22 M. buxifolia fruit 200 11 300 23 300

12 D. mucronataleaves 10 24 M. buxifolialeaves 10 13, 14 derived AgNPs 20 and 30 25, 26 derived AgNPs 20 and 30 M. buxifolia leaves 10, 20 and 27 derived AuNPs 30

Chapter 2 Methodology

2.8.3 Anti-pyretic activity

Antipyretic property of test samples was examined in Swiss albino mice weighing 20–35 gm. By using digital clinical thermometer (Model CA92121, ACON

Laboratories, USA), preliminary rectal temperature of mice was observed. To induce hyperthermia, 1 ml/100 g of 18% yeast suspension (Saccharomyces cerevisiae) was introduced by subcutaneous injection in mice and were kept in their cages. After 18 hrs of yeast injection, rectal temperatures were recorded again and animals with at least 0.5oC or more elevated body temperature were selected for the test. Animals arranged in groups (n = 6) were selected for test and injected as negative control with normal saline (10 ml/kg). Paracetamol (50 mg/kg) was supplemented as positive control or plant extracts (100, 200 mg/kg) or synthesized AgNPs/AuNPs (10, 20 mg/kg). After treatment of each animal rectal temperature was taken at an interval of

60 mins for 3 hrs. Percentage reduction in rectal temperature was calculated from resulting data. Antipyretic effect was well-defined as the potential of test samples to reduce pyrexia induced by yeast [238].

Table 2.6 The groups received treatment as follows:

Dosage Dosage Group Sample Group Sample (mg/kg) (mg/kg)

1 Normal saline 10 ml/kg 2 Paracetamol 50 mg/kg

3 D. mucronata 100 11 100 M. buxifolia leaves 4 leaves 200 12 200 5 100 13 100 D. mucronataroot M. buxifolia seed 6 200 14 200

7, 8 D. mucronatabark 100 and 200 15, 16 M. buxifolia fruit 100 and 200

D. M. buxifolialeaves 9, 10 mucronataleaves 10 and 20 17, 18 10 and 20 derived AgNPs derived AgNPs

Chapter 2 Methodology

2.8.4 Gastro intestinal track motility (GIT motility)

Balb-C mice (25-30 g) with age range 8-12 weeks were designated for this experiment and were allocated into various treatment groups [239]. Treatment1 and 2 were taken as negative and positive control, receiving normal saline (10 ml/kg) and castor oil (0.1 ml/kg). Methanol crude extracts were administered to other groups at a dose of 100, 200 and 300 mg/kg and synthesized NPs at doses of 10, 20 and 30 mg/kg viai.p.means. Next to fifteen mins of the treatments mentioned above, charcoal suspension (aqueous) at a dose of 0.3 ml was introduced to every mouse. Cervical dislocation was followed for animals killing after 30 mins of treatment with charcoal and small intestine was detached via dissection. Charcoal movement relative to total length of small intestine was calculated for percent GIT motility.

Table 2.7 The groups received treatment as follows

Dosage Group Sample Group Sample Dosage mg/kg 1 Normal saline 10 ml/kg 2 Castor oil 0.1 ml/kg 3 100 15 100 D. mucronata M. buxifolia 4 200 16 200 leaves leaves 5 300 17 300 6 100 18 100 D. M. buxifolia 7 mucronatabark 200 19 seed 200 8 300 20 300 9 100 21 100 D. M. buxifolia 10 mucronataroot 200 22 fruit 200 11 300 23 300 12 10 24 10 D. M. 13 mucronataleaves 20 25 buxifolialeaves 20 derived AgNPs derived AgNPs 14 30 26 30 M. buxifolia 10, 20 and 27 leaves derived 30 AuNPs

Chapter 2 Methodology

2.8.5 Anti-inflammatory activity

Swiss Albino (Balb-C) mice weighing 25-30 gm of either sex were used. Anti- inflammatory activity was performed with male rats weighing around 30 gm according to the protocol reported [238]. In acetone, 1 mg of Arachidonic acid (AA) and ethyl phenylpropiolate (EPP) was dissolved and then topically applied to inner and outer surfaces of both ears. Drugs and extracts were dissolved in acetone and topically applied to ear just before the irritants. Thickness was measured of each ear at1 hr, 2 hrs and 3 hrs with help of Vernier caliper after edema induction. Increase in ear thickness was calculated with comparison to control group and% inhibition was calculated as follow.

% Inhibition = A - B A

Where A is negative control, B is ear edema in tested group.

Table 2.8 The groups received treatment as follows:

Dosage Dosage Group Sample Group Sample (mg/kg) (mg/kg) 1 Normal saline 10 ml/kg 2 Phenidone 2 mg/ear

3 D. mucronata 100 11 100 M. buxifolia leaves 4 leaves 200 12 200 5 100 13 100 D. mucronataroot M. buxifolia seed 6 200 14 200

7 D. 100 15 100 M. buxifolia fruit mucronatabark 8 200 16 200 D. 9 10 17 M. buxifolialeaves 10 mucronataleaves derived AgNPs 10 derived AgNPs 20 18 20 19 M. buxifolialeaves 10

20 derived AuNPs 20

Chapter 2 Methodology

2.9 Biochemical parameters determination

Biochemical parameters were determined according to reported procedure

[240]. Male G. Pigs and male Rabbits were purchased and experiments were performed at Veterinary Research Institute Peshawar, Khyber Pukhtunkhwa (KPK).

Animals were kept in well ventilated house with optimum conditions at a temperature of 28oC, photoperiod 12 hrs natural light and 12 hrs dark with 40-45% humidity and free access to water.

Semi Auto Chemistry Analyzer Chem-o-test VET product of Biogen

Technology (BGT) was used for triacylglycerol (mg/dL), calcium (mg/dL), totalbilirubin (mg/dL) and conjugated bilirubin (µmol/L), urea (mg/dL), uric acid

(mg/dL), phosphorus (mg/dL) and Serum Glutamic-Pyruvic Transaminase (U/L). All other reagents used were of analytical grade and were provided by VRI Peshawar

Pakistan.

2.9.1 Animal grouping and extract administration

One hundred and two male G. Pigs and one hundred and two male Rabbits were randomly grouped into seventeen groups (A-Q) of six animals each. Control

Group A was orally administered with distilled water and normal feed daily for 21 days. Animals in rest of groups were treated the same as control except they were treated with same volume containing D. mucronata and M. buxifolialeaves crude methanolic extracts 100, 200 and 300 mg/kg body weight and D. mucronataleaves derived AgNPs andM. buxifolia leaves derived AgNPs and AuNPs 10, 20 and 30 mg/kg body weight.

Chapter 2 Methodology

2.9.2 Determination of feed and water intake

On daily basis water intake and amount of feed were determined. Supplied feed weight and left over by next day were recorded and difference was taken as daily feed intake. Water consumption was determined the same way. Average of water intake and feed was computed twice after eleven days of experimental period.

2.9.3 Preparation of serum

Blood was collected by cardiac puncturethrough sterile syringes under ether anesthesia prior to experiments and after 21 days of sample administration. Blood was taken in EDTA sample tubes for hematological analysis. Some blood was collected in centrifuge tubes and spinned at a speed of 1000 rpm for 15mins, the serum was aspirated carefully with Pasteur pipette in sample bottles that was used for biochemical assays [241].

2.9.4 Determination of biochemical parameters

Automated Hematological Analyzer was used to analyze different hematological parameters; mean platelet volume (MPV), Mean Corpuscular

Hemoglobin Concentration (MCHC), Mean Corpuscular Hemoglobin (MCH), Red

Blood Cells (RBC), Hemoglobin (Hb) and White Blood Cell (WBC). Other aspects tested included calcium, bilirubin (total and conjugated), urea, uric acid, phosphorus,

Serum Glutamic-pyruvic transaminase (SGPT).

Chapter 2 Methodology

2.10 Statistical analysis

Data was analyzed and interpreted by computer software Microsoft excel and

Graphpad Prism 6. Statistically elemental data was analyzed by arithmetic mean and standard deviation.

Chapter 3 Results

3.0 RESULTS

3.1 Preliminary phytochemical screening

Phytochemical characteristics of methanolic crude extracts of D. mucronata bark, leaves and roots and M. buxifolia seeds, leaves and fruits are given in Table 3.1.

Carbohydrates presence through Fehling and Benedict’s test was confirmed in all parts of both plants. Whereas through Molisch’s Test, presence of carbohydrates was only confirmed in D. mucronata bark and M. buxifolia leaves. Detection of carbohydrate through iodine test generated negative results for all plant parts.

Results showed that saponins were not observed in D. mucronata bark and M. buxifolia seed, whereas in other plant material such as D. mucronataleaves and root, and M. buxifolialeaves and fruit presence of saponin was confirmed. Phenols/tannins were detected in all the tested materials of both plants except for root of D. mucronata. Sodium hydroxide test for detection of flavonoids showed that D. mucronataleaves and root had no flavonoid compounds, whereas D. mucronata bark and all parts of M. buxifolia including seeds, leaves and fruits contained flavonoid compounds.

Liebermann’s test for glycosides was negative for both plant materials.

Salkowski test for glycosides showed negative results for D. mucronata bark and positive results for its leaves and root. On the other hand, positive results were confirmed for M. buxifolialeaves and negative for seed and fruit. Keller killani’s test for glycosides showed positive results for all D. mucronata parts. Keller killani’s test for glycosides showed positive results for M. buxifolialeaves while negative for M. buxifolia seeds and fruits. 94

Chapter 3 Results

Table 3.1 Phytochemical screening of crude methanolic extracts of D.

mucronata(bark, leaves and root) and M. buxifolia (seed, leaves and fruit)

Plants names D. mucronata M. buxifolia

Test Bark Leaves Root Seed Leaves Fruit

Fehling’s test (reducing sugars) + + + + + +

Benedict’s test (carbohydrates) + + + + + +

Molisch’s test (carbohydrates) + ‒ ‒ ‒ + ‒

Iodine test(carbohydrates) ‒ ‒ ‒ ‒ ‒ ‒

Froth formation test(Saponins) ‒ + + ‒ + +

Ferric chloride test (Phenols/Tannins) + + ‒ + + +

Sodium hydroxide test (Flavonoids) + ‒ ‒ + + +

Liebermann’s test(Glycosides) ‒ ‒ ‒ ‒ ‒ ‒

Salkowski’s test(Glycosides) ‒ + + ‒ + ‒

Keller-kilani test(Glycosides) + + + ‒ + ‒

Symbols: (‒): absent, (+): present

Chapter 3 Results

3.2 Elemental analysis (atomic absorption spectroscopy)

In present study aqueous extracts of D. mucronata and M. buxifolialeaves were analyzed for elemental composition. Results are given in (Table 3.2). The Ca is important element that play significant role in metabolism. In D. mucronata leaves the amount of Ca calculated was 42.26 ± 0.232 mg/L while in M. buxifolia leaves it was

20.24 ± 0.126 mg/L. The Pb was found to be 0.404 ± 0.0687 mg/L in D. mucronataleaves and 0.119 ± 0.0207 mg/L in M. buxifolia leaves. InD. mucronata leavesMn was observed to be 0.285 ± 0.0048 mg/L while in M. buxifolia leaves it was

0.150 ± 0.0257 mg/L. In D. mucronataleaves Fe was observed to be 0.316 ± 0.0195 mg/L while in M. buxifolia leaves it was 0.169 ± 0.0278 mg/L. The amount of Zn found in D. mucronataleaves was 0.176 ± 0.0022 mg/L while in M. buxifolia leaves it was 0.060 ± 0.0020 mg/L. Amount of Co in D. Mucronta leaves was observed 0.051±

0.0457 mg/L and in M. buxifolialeaves it was 0.012 ± 0.00947 mg/L. Amount of Cu in D. mucronataleaves was observed 0.035 ± 0.0075 mg/L and in M. buxifolialeaves it was observed 0.016 ± 0.0035 mg/L. Amount of Ni in D. mucronataleaves was observed 0.018 ± 0.0213 mg/L while in M. buxifolialeaves it was not detected. The Cr in D. mucronataleaves was observed 0.001 ± 0.0169 mg/L while in M. buxifolialeaves no chromium was detected. The Cd in D. mucronataleaves was not found while in M. buxifolialeaves it was found to be 0.022 ± 0.0164 mg/L.

The Ca was found as dominant element. Amount of Ca in D. mucronataleaves was 42.26 mg/L and in M. buxifolialeaves it was recorded 20.24 mg/L. Next dominant element was Fein D. mucronataleaves that was recorded 0.316 mg/L and in M. buxifolialeaves it was 0.169 mg/L. Least amount of mineral observed was Cr, in D. mucronataleaves whereas Co was the least content in M. buxifolialeaves (0.012 96

Chapter 3 Results mg/L). Similar other minerals detected in D. mucronataleaves were Zn (0.176 mg/L),

Pb (0.404 mg/L), Mn (0.285 mg/L), Co (0.051 mg/L) and Cu (0.035 mg/L). In M. buxifolia leaves, other minerals detected were Zn (0.06 mg/L), Cd (0.022 mg/L), Pb

(0.119 mg/L), Mn (0.15 mg/L) and Cu (0.016 mg/L).

Chapter 3 Results

Table 3.2 Atomic Absorption spectroscopy of crude aqueous extracts of D. mucronata

and M. buxifolialeaves (Mean ± StdDev mg/L)

Analyte D. mucronataleaves M. buxifolia leaves

Fe 0.316 ± 0.0195 0.169 ± 0.0278

Zn 0.176 ± 0.0022 0.060 ± 0.0020

Ca 42.26 ± 0.232 20.24 ± 0.126

Cd 0.00 ± 0.0153 0.022 ± 0.0164

Cr 0.001 ± 0.0169 0.00 ± 0.0111

Ni 0.018 ± 0.0213 0.00 ± 0.0325

Pb 0.404 ± 0.0687 0.119 ± 0.0207

Mn 0.285 ± 0.0048 0.15 ± 0.0257

Co 0.051 ± 0.0457 0.012 ± 0.00947

Cu 0.035 ± 0.0075 0.016 ± 0.0035

Chapter 3 Results

3.3 Nutritional analysis

For determination of nutritional and curative values nutrient and proximate analysis is important. Medicinal plants species are used for curative purposes of many diseases. Nutrional analysis is important to understand nutritional worth of these plants apart from medicinal value. Nutritional compositions of D. mucronataleaves and M. buxifolia leaves are shown in Table 3.3 Nutritional analysis of D. mucronata and M. buxifolialeaves in percent. High amounts of carbohydrates were detected in D. mucronataleaves (61.56%) and M. buxifolialeaves (56.53%). Protein contents found in D. mucronataleaves were 4.12% and in M. buxifolialeaves 3.15%. Fat contents detected in D. mucronataleaves were 2.73% and in M. buxifolia leaves 0.87%. Crude fibers, ash and moisture contents were found to be 23.58%, 9.94% and 3.85% in D. mucronataleaves whereas in M. buxifolialeaves they were recorded to be 24.08%,

10.71% and 2.54%, respectively.

Chapter 3 Results

Table 3.3 Nutritional analysis of D. mucronata and M. buxifolialeaves in percent

Sample Carbohydrates Proteins Fat Fibers Ash Moisture

D. mucronata 61.56±0.48 4.12±0.34 2.733±0.26 23.58±0.40 9.94±0.22 3.85±0.17 leaves

M. buxifolia 56.53±0.31 3.15±0.26 0.87±0.20 24.08±0.80 10.71±0.18 2.54±0.18 leaves

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Chapter 3 Results

3.4 Characterization of AgNPs and AuNPs

Progress of NPs formation was visualized by a color change after few (5-10) mins at room temperature (28oC) for AgNPs and after 30 mins of heating at 70oC for

AuNPs. Appearance of brown color in case of AgNPs and cherry red solution in case of AuNPs indicated the synthesis of respective NPs.

3.4.1 UV-vis spectroscopy

Figure 3.1 represents UV-Vis spectra of D. mucronataleaves aqueous extract and its derived AgNPs. M. buxifolialeaves aqueous extract and its derived biogenic

AgNPs and AuNPs UV-vis spectra are shown in Figure 3.2 and Figure 3.3 respectively. UV-Vis spectral patterns of D. mucronataleaves derived AgNPs was observed with corresponding Surface Plasmon Resonance (SPR) peak at 425. For M. buxifolialeaves derived AgNPs and AuNPs were observed with corresponding SPR peaks at 405 and 540 nm.

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Chapter 3 Results

2 .0 (a ) P la n t e x tr a c t

(b ) (b ) A g N P s

1 .5

a b

r 1 .0

s b

A (a) 0 .5

0 .0 3 0 0 4 0 0 5 0 0 6 0 0 W a v e L e n g th (n m )

Figure 3.1D. mucronataleaves derived AgNPsUV-absorption (λmax) 425 nm

1 .5 (a ) P la n t e x tr a c t (b ) A g N P s

(b ) e

c 1 .0

o s

b 0 .5 A (a)

0 .0 3 0 0 4 0 0 5 0 0 6 0 0 W a v e L e n g th (n m )

Figure 3.2M. buxifolialeaves derived AgNPs UV-absorption (λmax) 405 nm

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Chapter 3 Results

0 .6 (a ) P la n t e x tr a c t

0 .5 (b ) (b ) A u N P s

e c

n 0 .4

r o

s 0 .3

b A

0 .2 (a)

0 .1 4 5 0 5 0 0 5 5 0 6 0 0 6 5 0 7 0 0 W a v e L e n g th (n m )

Figure 3.3M. buxifolialeaves derived AuNPs UV-absorption (λmax) 540 nm

103

Chapter 3 Results

3.4.2 Fourier transform infra-red (FTIR) spectroscopy

D. mucronataleaves aqueous extract FTIR spectra showed a wide peak at

3334.92 cm-1 which confirmed the presence of OH group. Similarly, a sharp peak was observed at 2920.23 cm-1 that corresponds to C-H methylene asymmetric stretching.

Another characteristic peak was observed at 2850.79 cm-1 and 1614.42 cm-1 demonstrating C-H methylene symmetric and aromatic C-C groups (Figure 3.4).

However, in case of D. mucronataleaves derived AgNPs, the peak at 1614 cm-1 was dissolved, while the OH (hydroxyl group) peak at 3334.92 cm-1 and C-H peaks at

2920.23 cm-1 and 2850.79 cm-1 were also dissolved. Hence it is confirmed that reduction of AgNO3 was due to OH group in case of D. mucronataleaves derived

AgNPs. The peaks at 1066.64 and 1041.56 became wide showing a single peak at

1271.09 that corresponds to –C-O groups of polyols like terpenoids, poly saccharides and flavones. Therefore polyols were responsiblefor capping of fabricated AgNPs.

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Chapter 3 Results

Figure 3.4 FTIR spectra of D. mucronataleaves aqueous extract and D.

mucronataleaves derived AgNPs

105

Chapter 3 Results

The M. buxifolia leavesaqueous extractFTIR spectra showed a wide peak at

3361.93 cm-1 which confirmed -OH group. Similarly, sharp peak was observed at

2920.23 cm-1 that corresponds to C-H olefinic stretching. Another defined peak was observed at 2850.79 cm-1 indicating alkyl C-H olefinic group. Peak at 2324.22 cm-1 demonstrated alkyne C-C group and at 1732.08 cm-1 stretched peak demonstrated aldehyde C-O group (Figure 3.5). However, FTIR spectrum of M. buxifolialeaves derived AgNPsshowed that peaks at 2920.23 cm-1, 2850.79 cm-1 and 1732.08 cm-1 were dissolved that mean -OH, -CH and -CHO groups were responsibleforfabrication of these AgNPs. Similarly, in case of M. buxifolialeaves derived AuNPs, peaks at

3361.93 cm-1, 2920.23 cm-1, 2850.79 cm-1 and 1732.08 cm-1 were dissolved, which mean that -OH, -CH and –CO groups were responsibleforthe synthesis of AuNPs

(Figure 3.5).

106

Chapter 3 Results

Figure 3.5 FTIR Spectra of M. buxifolia leaves aqueous extract, M. buxifolialeaves

derived AgNPs and M. buxifolialeaves derived AuNPs

107

Chapter 3 Results

3.4.3 X-ray diffraction (XRD)

The X-Ray Diffraction in existingexperiment was done to detect the crystal dimensions of models. Modelscomprised dried powder form of each plant material, aqueous extracts and respective aqueous extract derived AgNPs and AuNPs of each selected plant material. Succeeding peaks were examined from XRD results obtained.

Grain size was measured by Debye-Scherer’s equation for AgNPs and AuNPs as shown in Table 3.4, Table 3.5 and Table 3.6.

The XRD analysis confirmed that D. mucronatadriedleaves and aqueous extract were not crystalline in nature as evident from the peaks in Figure 3.6 and

Figure 3.7. Crystal size observed in D. mucronataleaves derived AgNPswas 94.779oA

(Table 3.4). Also, in the D. mucronataleaves derived AgNPs, the diffraction peaks were observed at 29.3°, 32.2°, 38.1°, 44.25°, 46.25°, 64.45° and 77.4°; referring to

111, 200, 220 and 310 planes of face-centered cubic (fcc) Ag, respectively (Figure

3.8).

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Chapter 3 Results

Table 3.4 Crystalline Size Determination of D. mucronataleaves derived AgNPs using

Debye-Scherer’s Equation

Kλ = 0.94×1.54 =1.4476oA

S. No. Peak FWHM β = θ = (θ2+θ1) D=Kλ/β. Cosθ

observed 2πFWHM/360 π/360

at 2θ

1. 29.3 0.44691 0.0078 29.32o=0.51 185.58

2. 32.2 0.24462 0.0042 32.2=0.561 344.66

3. 38.1 0.68547 0.0119 38.1=0.664 121.64

4. 44.25 17.43714 0.304 44.2=0.771 4.76

5. 46.25 56.94104 0.9938 46.2=0.806 1.456

6. 64.45 324.6346 5.665 64.55=1.126 0.255

7. 77.4 16.25979 0.2837 77.3=1.34 5.102

Crystallite size was calculated using Debye–Scherrer’s equation [ = Kλ/b(Δθ1/2) cos θ] whereas: D: is the crystallite size. K or Kappa: is a dimensionless shape factor which is equal to 0.9. λ Lambda: is the wavelength of the x-radiation which is equal to 1.5406. b (Δθ1/2) or Beta (β): is the peak’s FWHM (in radians). θ: is the Bragg angle. FWHM: Full width at half maximum.

Kλ= 0.14476 nm; where K is constant= 0.94 and λ is wavelength of X-ray=1.5418

°A.

Average diameter of D. mucronataleaves derived AgNPs = 94.779oA

109

Chapter 3 Results

4 0 0

) s

t 3 0 0

(

y 2 0 0

n e

t 1 0 0

n I

0 2 0 4 0 6 0 8 0 2 

Figure 3.6 XRD analysis of D. mucronataleaves dried powder

5 0 0 )

s 4 0 0

n u

o 3 0 0

(

t i

s 2 0 0

t n

I 1 0 0

0 2 0 4 0 6 0 8 0 2 

Figure 3.7 XRD analysis of D. mucronataleaves aqueous extract

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Chapter 3 Results

3 0 0 (1 1 1 )

)

n u

o 2 0 0

(

t i

s (2 0 0 )

n (2 2 0 )

e 1 0 0 (3 1 0 )

n I

0 2 0 4 0 6 0 8 0 2 

Figure 3.8 XRD analysis of D. mucronataleaves derived AgNPs

111

Chapter 3 Results

It is evident from peaks that M. buxifolia dried leaves and aqueous extract were not crystalline in nature (Figure 3.9 and Figure 3.10). Crystal size observed for

M. buxifolialeaves derived AgNPs was 96.1885°A as shown inTable 3.6. In M. buxifolialeaves derived AgNPs, peaks were observed at 27.8°, 34.25°, 38.1°,

44.25°,64.4° and 77.35°; equivalent to 111, 200, 220 and 310 planes of fcc Ag, correspondingly (Figure 3.11). Crystal size observed in M. buxifolialeaves derived

AuNPs was 109.94°A as shown inTable 3.5. In M. buxifolialeaves derived AuNPs, peaks were observed at 31.65°, 38.2°, 44.35°,45.4°, 64.75° and 77.6°; analogous to

111, 200, 220 and 310 planes of fcc Au, correspondingly (Figure 3.12).

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Chapter 3 Results

Table 3.5 Crystalline size determination of M. buxifolialeaves derived AuNPsusing

Debye-Scherer’s equation

S. No. Peak FWHM β = θ = (θ2+θ1) D=Kλ/β. Cosθ

observed 2πFWHM/360 π/360

at 2θ

1. 31.65 0.26243 0.0045 31.65=0.552 321.68

2. 38.2 0.52224 0.0091 38.15=0.665 159.07

3. 44.35 0.64715 0.011 44.32=0.773 131.6

4. 45.4 30.02194 0.523 45.42=0.79 2.767

5. 64.75 61.22623 1.068 64.57=1.126 1.35

6. 77.6 1.92102 0.0335 77.25=1.34 43.21

Kλ= 0.14476 nm;where K is constant= 0.94 and λ is wavelength of X-ray= 1.5418

°A.

Average diameter of M. buxifolialeaves derived AuNPs = 109.94°A

113

Chapter 3 Results

Table 3.6 Crystalline size determination of M. buxifolialeaves derived AgNPs using

Debye-Scherer’s Equation

S. Peak FWHM β = θ = (θ2+θ1) D=Kλ/β. Cosθ

No. observed 2πFWHM/360 π/360

at 2θ

1. 27.8 0.78141 0.0136 27.75=0.484 106.44

2. 34.25 184.33066 3.217 34.3=0.598 0.449

3. 38.1 0.43987 0.0076 38.1=0.664 190.47

4. 44.25 0.48433 0.0084 44.22=0.771 172.3

5. 64.4 80.36782 1.402 64.45=1.124 1.032

6. 77.35 0.78245 0.0136 77.42=1.35 106.44

Kλ= 0.14476 nm;where K is constant= 0.94 and λ is wavelength of X-ray= 1.5418

°A.

Average diameter of M. buxifolialeaves derived AgNPs= 96.1885 °A

114

Chapter 3 Results

)

s t

n 4 0 0

(

i s

n 2 0 0

n I

2 0 4 0 6 0 8 0 2 

Figure 3.9 XRD analysis of M. buxifolialeaves dried powder

) 3 0 0

c (

2 0 0

t n

I 1 0 0

2 0 4 0 6 0 8 0 2 

Figure 3.10 XRD analysis of M. buxifolia leaves aqueous extract

115

Chapter 3 Results

(1 1 1 )

6 0 0

)

u o

c 4 0 0

(

i s

n (2 0 0 ) e

t 2 0 0

n ( 2 2 0 ) (3 1 0 ) I

0 2 0 4 0 6 0 8 0 2 

Figure 3.11 XRD analysis of M. buxifolialeaves derived AgNPs

5 0 0

(1 1 1 )

) 4 0 0

n u

o 3 0 0

(

t i

s 2 0 0 (2 0 0 )

n e t (2 2 0 ) n (3 1 0 )

I 1 0 0

0 2 0 4 0 6 0 8 0 2 

Figure 3.12 XRD analysis of M. buxifolialeaves derived AuNPs

116

Chapter 3 Results

3.4.4 Energy dispersive X-ray detection (EDX)

Energy dispersive X-ray study was conducted to regulate the type and quantity of elements occurring in test samples and synthesized NPs. Crystalline nature and crystal size of synthesized NPs was determined by XRD analysis, now presence of Ag and Au along with some other elements was confirmed by EDX elemental analysis.

The EDX analysis showed that dried D. mucronataleaves contain carbon (49.77%), oxygen (48.27%), aluminium (0.26%), potassium (0.59%) and Ca (1.11%) as shown in Figure 3.13. The D. mucronataleaves derived AgNPs contained carbon (15.86%), oxygen (30.04%), sodium (0.92%), Mg (0.85%), aluminium (0.34%), silicon (0.25%), sulphur (0.96%), chlorine (1.53%), potassium (1.17%), Ca (1.99%), Ag (42.90) and

Cd (3.14%) (Figure 3.14).

117

Chapter 3 Results

Figure 3.13 EDX profile (elemental composition) of dried D. mucronata leaves

Figure 3.14 EDX profile (elemental composition) of D. mucronataleaves derived

AgNPs

118

Chapter 3 Results

The dried M. buxifolialeaves EDX analysis revealed presence of carbon

(61.15%), oxygen (37.69%), Mg (0.18%), aluminum (0.14%), silicon (0.46%) and Ca

(0.38%); as shown in Figure 3.15. M. buxifolialeaves derived AgNPs revealed the presence of carbon (21.0%), oxygen (22.33%), chlorine (2.98%), Ag (53.68%); as shown in Figure 3.16. M. buxifolialeaves derived AuNPs showed the presence of carbon (58.19%), oxygen (19.20%), sodium (1.89%), Mg (0.16%), aluminum

(0.19%), chlorine (3.92%), potassium (0.22%), Ca (0.23%), copper (0.56%) and Au

(15.43%); as shown in Figure 3.17.

119

Chapter 3 Results

Figure 3.15 EDX profile (elemental composition) of dried M. buxifolia leaves

Figure 3.16 EDX profile (elemental composition) of M. buxifolialeaves derived

AgNPs

120

Chapter 3 Results

Figure 3.17 EDX profile (elemental composition) of M. buxifolialeaves derived

AuNPs

121

Chapter 3 Results

3.4.5 Scanning electron microscopy (SEM)

Size, shape, surface topology and morphology of synthesized NPs were determined by SEM analysis. The SEM image of D. mucronataleaves derived AgNPs showed small sphericalshaped particles ranging in size from 8 nm- 30 nm (Figure

3.18, Figure 3.19 and Figure 3.20). These AgNPs were well dispersed, revealing a significant degree of stabilization by the active constituents (bio-molecules) in aqueous extract of this plant.

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Chapter 3 Results

Figure 3.18 SEM image (shape and rough estimation of size) of D. mucronataleaves

derived AgNPs at 0.5 µm

Figure 3.19 SEM image (shape and rough estimation of size) of D. mucronataleaves

derived AgNPs at 0.2 µm

123

Chapter 3 Results

Figure 3.20 SEM image (shape and rough estimation of size) of D. mucronataleaves

derived AgNPs at 0.1 µm

124

Chapter 3 Results

The SEM analysis of M. buxifolialeaves derived AgNPs indicated the formation particles with small spherical-shape and size range 8 nm – 20 nm (Figure

3.21, Figure 3.22 and Figure 3.23). While that of M. buxifolialeaves derived AuNPs were observed with size range 10 nm – 60 nm andglobularshaped particles (Figure

3.24, Figure 3.25 and Figure 3.26).

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Chapter 3 Results

Figure 3.21 SEM image (shape and rough estimation of size) of M. buxifolialeaves

derived AgNPs at 0.5 µm

Figure 3.22 SEM image (shape and rough estimation of size) of M. buxifolialeaves

derived AgNPsat 0.2 µm

126

Chapter 3 Results

Figure 3.23 SEM image (shape and rough estimation of size) of M. buxifolia leaves

derived AgNPsat 0.1 µm

Figure 3.24 SEM image (shape and rough estimation of size) of M. buxifolialeaves

derived AuNPsat 0.5 µm

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Chapter 3 Results

Figure 3.25 SEM image (shape and rough estimation of size) of M. buxifolialeaves

derived AuNPsat 0.2 µm

Figure 3.26 SEM image (shape and rough estimation of size) of M. buxifolialeaves

derived AuNPsat 0.1 µm

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Chapter 3 Results

3.4.6 Transmission electron microscopy (TEM)

Figure 3.27 and Figure 3.28 show representative TEM images of NPs synthesized using D. mucronataleaves aqueous extract. The TEM analysis clearly revealed formation of Ag nanostructures in addition to spherical NPs. The TEM analysis revealed that major particle size falls in the range of 8-30 nm. The observed

AgNPs were spherical and semi-spherical in shape. Majority of the particles appeared more or less spherical. A close-up of spherical spots in figure 3.28 showed AgNPs synthesized using D. mucronata leaves.

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Chapter 3 Results

Figure 3.27 TEM image (exact size and shape) of D. mucronataleaves derived AgNPs

at 30 nm

Figure 3.28 TEM image (exact size and shape) of D. mucronataleaves derived AgNPs

at 8 nm 130

Chapter 3 Results

Figure 3.29, Figure 3.30 and Figure 3.31 shows representative TEM images of

NPs synthesized using M. buxifolia leaves aqueous extract. The TEM analysis clearly revealed formation of Ag nanostructures in addition to spherical NPs. The TEM analysis revealed that major particle size falls in the range of 8-15 nm. However few particles of 20 nm were also spotted. The observed AgNPs were spherical, semi- spherical and oblong in shape. Majority of particles appear more or less spherical.

Closeup of spherical spots in Figure 3.31 showed AgNPs synthesized using M. buxifolia leaves. Fringes were observed in TEM images of synthesized AgNPs that confirmed its crystalline nature. Fringes are evident in spherical AgNPs as shown in

Figure 3.30 and Figure 3.31.

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Chapter 3 Results

Figure 3.29 TEM image (exact size and shape) of M. buxifolialeaves derived AgNPs

at 100 nm

Figure 3.30 TEM image (exact size and shape) of M. buxifolialeaves derived AgNPsat

30 nm

132

Chapter 3 Results

Figure 3.31 TEM image (exact size and shape) of M. buxifolialeaves derived AgNPs

at 8 nm, fringes evident in spherical NPs

133

Chapter 3 Results

The TEM images i.e.Figure 3.32, Figure 3.33andFigure 3.34 confirmed the formation of gold metal NPs synthesized using M. buxifolia leaves aqueous extracts.

The figure clearly illustrates NPs size range of 10-60 nm. Most of the particles appeared with a pronounced anisotropic morphology, like Nano prisms, hexagonal and Nano-rods; however, few appearedspheroidal. Variability in shape of AuNPs was due to temperature range (50-70oC) provided during synthesis. Optimization of conditions might help to synthesize same shape and size NPs. Crystallinity of the synthesized AgNPs is confirmed by the formation of fringes in TEM micrographs.

Fringes are evident inhexagonal NPs on left of Figure 3.33. They are also evident in

Nano-prisms (Figure 3.34).

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Chapter 3 Results

Figure 3.32 TEM image (exact size and shape) of M. buxifolialeaves derived AuNPsat

500 nm

Figure 3.33 TEM image (exact size and shape) of M. buxifolialeaves derived AuNPs

fringes evident in hexagonal NPs

135

Chapter 3 Results

Figure 3.34 TEM image (exact size and shape) of M. buxifolialeaves derived AuNPs

at 100 nm, fringes evident in Nano prisms

136

Chapter 3 Results

3.4.7 Thermo gravimetric-differential thermal analysis (TG-DTA)

The TGA profile of dried D. mucronataleaves demonstrated the weight loss at

100, 350 and 500°C as shown in Figure 3.35. Weight loss in D. mucronataleaves derived AgNPs was observed at 320 and 440°C (Figure 3.36). Similarly, TGA profile of dried M. buxifolialeaves displayed weight loss at 52, 106, 342 and 508°C as shown in Figure 3.37. For M. buxifolialeaves derived AgNPs, weight loss was observed at

320, 500 and 900 °C (Figure 3.38). The weight loss in M. buxifolialeaves derived

AuNPs was observed at 320, 480 and 906 °C as shown in Figure 3.39.

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Chapter 3 Results

)

(

t 6

d e

t 4

b u

s 2

n U

0 0 2 0 0 4 0 0 6 0 0 o S a m p le te m p e ra tu r e C

Figure 3.35 TGA profile (weight loss) of dried D. mucronata leaves

) 6

(

e w

t b

u 2

n U

0 0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 o S a m p le te m p e ra tu r e C

Figure 3.36 TGA profile (weight loss) of D. mucronataleaves derived AgNPs

138

Chapter 3 Results

) 6

(

e w

t b

u 2

n U

0 0 2 0 0 4 0 0 6 0 0 o S a m p le te m p e ra tu r e C

Figure 3.37 TGA profile (weight loss) of dried M. buxifolia leaves

)

(

i e

w 5

r t

b 4

n U

3 0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 o S a m p le te m p e ra tu r e C

Figure 3.38 TGA profile (weight loss) of M. buxifolialeaves derived AgNPs

139

Chapter 3 Results

)

g (

d 4

r t

b 3

n U 2

0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 o S a m p le te m p e ra tu r e C

Figure 3.39 TGA profile (weight loss) of M. buxifolialeaves derived AuNPs

140

Chapter 3 Results

The DTA graph of dried D. mucronataleaves showed endothermic reactions at

125, 335 and 450°C and exothermic reactions at 72, 230, 395 and 520°C (Figure

3.40). In D. mucronataleaves derived AgNPs DTA graph, endothermic reactions were observed at 250, 260 and 480 °C and exothermic reactions at 240, and 450 °C (Figure

3.41). The DTA graph of dried M. buxifolia leaves showed endothermic reaction at

350, and 480°C and exothermic reactions at 345, 420 and 500°C (Figure 3.42).

Similarly, DTA graph of M. buxifolialeaves derived AgNPs exhibited endothermic reaction at 280 and 450°C and exothermic reaction at 300 and 520°C (Figure 3.43).

The DTA graph of M. buxifolialeaves derived AuNPs presented endothermic reaction at 350 and 450 °C and exothermic reaction at 400 and 520°C (Figure 3.44).

141

Chapter 3 Results V

 8 0

o v

o 6 0

d 4 0

a r

t 2 0

u s

n 0 U

0 2 0 0 4 0 0 6 0 0 o S a m p le te m p e ra tu r e C

Figure 3.40 DTA profile (endothermic and exothermic reactions) of dried D.

mucronata leaves V

 2 0

t l

o 0

i m

-2 0

c a

r -4 0

u s

n -6 0 U

0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 o S a m p le te m p e ra tu r e C

Figure 3.41 DTA profile (endothermic and exothermic reactions) of D.

mucronataleaves derived AgNPs

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Chapter 3 Results

V 6 0



v o

r 4 0

e t

c 2 0

u s

n 0 U

0 2 0 0 4 0 0 6 0 0 o S a m p le te m p e ra tu r e C

Figure 3.42 DTA (endothermic and exothermic reactions) profile of dried M. buxifolia

leaves V

 2 0

l o

v 0

i m

-2 0

a r

t -4 0

u s

n -6 0 U

0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 o S a m p le te m p e ra tu r e C

Figure 3.43 DTA profile (endothermic and exothermic reactions) of M. buxifolia

leaves derived AgNPs

143

Chapter 3 Results

5 0



r c

i 0

b u

s -5 0

n U

0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 o S a m p le te m p e ra tu r e C

Figure 3.44 DTA profile (endothermic and exothermic reactions) of M.

buxifolialeaves derived AuNPs

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Chapter 3 Results

3.5 Bioassay of plant fractional crude extracts and biogenic synthesized

AgNPs and AuNPs

3.5.1 Antioxidant activity

Results of antioxiant activity of D. mucronata extracts are shown inTable 3.7 and Table 3.8. D. mucronata bark crude methanolic extract, n-hexane, chloroform, ethyl acetate and aqueous fractions showed good scavenging activities (81.63, 85.63,

91.81, 77.48 and 87.15%) at 600 μg/ml. Among D. mucronataleaves highest percent antioxidant activity was observed in aqueous fraction that was 91.99%, followed by ethyl acetate fraction with 86.04%, and crude methanolic extract with 82.55%. The n- hexane and chloroform showed good scavenging activities of 76.59 and 79.82% at

600 μg/ml. Crude methanolic extract, n-hexane, chloroform, ethyl acetate and aqueous fractions from D. mucronata roots showed significant scavenging activities at 600 μg/ml of 80.19, 85.54, 81.55, 89.48 and 93.67% respectively. The EC50 of bark leaves and roots are presented inTable 3.9. In case of D. mucronata bark methanolic fraction lowest EC50 value was observed 290.34 µg/ml and highest EC50 value of

367.83 µg/ml was observed for ethyl acetate fraction. In case of D. mucronataleaves extract, the lowest EC50 value of 288.37µg/ml was observed for n-hexane extract and highest EC50 value of 379.39 µg/ml was observed for chloroform. Similar for D. mucronata root extract, the lowest EC50 value of 294.67 µg/ml was calculated for aqueous fraction and highest EC50 value of 401.92 µg/ml was observed for methanolic extract. The EC50 and percent antioxidant activity has an inverse relationship.

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Chapter 3 Results

Table 3.7 Antioxidant activity of D. mucronata barkand leaves extracts

Extract/Fraction Percent(%) scavenging activity (P˂0.005) Plant Concentration part Methanolic n-hexane Chloroform Ethyl acetate Aqueous (µg/ml)

100 23.15±1.24 28.04±1.29 41.6±1.06 33.54±1.54 35.14±1.28

200 38.65±0.68 38.21±1.25 54.55±0.95 39.99±1.04 45.7±1.07

bark 300 53.86±1.19 54.51±0.94 67.28±0.84 49.1±1.25 57±1.32

400 67.57±1.17 67.46±0.91 75.20±1.33 58.55±1.03 67.17±1.23 D. mucronata D. 500 77.23±1.33 80.41±0.99 82.86±1.16 67.15±1.17 77±1.37

600 81.63±1.08 85.63±1.08 91.81±1.12 77.48±1.62 87.15±1.42

100 28.44±1.03 23.51±1.05 25.03±1.07 28.73±1.36 33.06±1.29

200 36.87±1.34 30.91±1.07 33.35±1.20 35.0±1.25 49.88±1.03

leaves 300 49.74±1.36 52.57±0.93 42.08±0.84 49.85±1.0 55.18±1.30

400 63.28±1.29 58.60±1.01 55.11±1.33 61.19±1.16 69.77±0.96

D. mucronata D. 500 69.84±1.29 72.11±1.32 65.0±1.25 74.97±1.16 81.62±1.60

600 82.55±1.04 76.59±1.16 79.82±1.30 86.04±1.28 91.99±1.17

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Chapter 3 Results

Table 3.8 Antioxidant activity of D. mucronata root extracts

Percent(%) scavenging activity (p˂0.005) Extract/Fraction

Concentration Methanolic n-hexane Chloroform Ethyl acetate Aqueous (µg/ml)

100 22.80±1.65 25.10±1.18 24.07±1.30 25.51±1.64 27.54±1.62

200 28.37±1.01 34.14±1.34 33.41±1.57 38.16±1.15 39.39±1.38

300 39.96±1.22 45.23±1.33 45.61±1.45 51.46±1.26 61.80±2.04

400 51.15±1.28 61.33±1.41 57.4±1.37 69.51±1.44 77.28±1.29

500 69.15±1.20 76.32±1.26 69.44±1.55 78.18±1.30 84.1±1.26

600 80.19±1.38 85.54±1.63 81.55±1.54 89.48±1.50 93.67±1.48

Table 3.9 EC50 values of D. mucronata bark, leaves and roots extracts

EC50 value (µg/ml) of methanolic extract and fractions Plant Part Methanolic n-hexane Chloroform Ethyl acetate Aqueous Vitamin C (Standard)

Bark 290.34 317.95 295.48 367.83 340.76 252.21

Leaves 342.50 288.37 379.39 366.45 350.34 252.21

Roots 401.92 362.67 361.07 333.43 294.67 252.21

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Chapter 3 Results

Antioxidant activity of M. buxifolia seed, leaves and fruit is represented inTable 3.10 and Table 3.11. Results showed that in M. buxifolia seed chloroform

(88.53%) and ethyl acetate (85.54%) have highest antioxidant activity compared to standard i.e. Vitamin C (37.11 and 85.36%). Significant antioxidant activities were observed in M. buxifolialeaves crude methanolicextract (82.67%) and ethyl acetate

(83.27%) at 600 μg/ml concentration. Similarly, in M. buxifoliafruit chloroform

(81.12%), ethyl acetate (82.47%) and aqueous extract (85.73%) showed significant scavenging activities, at 600 μg/ml concentration. Antioxidant activity in all others sample tests were determined to be below the standards.

The EC50 values for M. buxifolia crude methanolic extract and its fractions are represented in Table 3.12. The EC50 showed to be high in all the samples compared to standard vitamin C (252.21). The highest EC50 values in M. buxifolia seeds were observed for chloroform extract (381.92µg/ml), and lowest EC50 was calculated for aqueous extract (300µg/ml). Crude methanolic extract ofM. buxifolialeaves had maximum EC50 value (388.70µg/ml), and aqueous extract had lowest EC50 value

(319.94µg/ml). In M. buxifolia fruits, n-hexane showed maximum EC50 value

(401.28µg/ml), and the minimum EC50 was calculated for crude methanolic extract

(310.45µg/ml).

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Chapter 3 Results

Table 3.10 Antioxidant activity of M. buxifolia seed and fruit extracts

Extract/Fractio Percent(%) scavenging activity of M. buxifolia(p˂0.0001)

n Ethyl Concentration Methanolic n-hexane Chloroform Aqueous acetate (µg/ml)

100 30.34±1.41 24.94±1.31 38.46±1.36 34.78±1.51 32.43±1.86

200 41.56±1.68 39.16±1.54 49.36±1.25 41.83±1.32 46.25±1.65

300 56.78±1.32 48.45±1.49 54.32±1.49 53.91±1.53 57.42±1.27 seeds

400 64.15±1.53 57.34±1.29 65.52±1.63 62.84±1.42 63.29±1.49 M. buxifolia M. 500 75.41±1.45 74.15±1.53 79.67±1.68 76.35±1.32 73.41±1.83

600 83.53±1.38 84.32±1.34 88.53±1.76 85.54±1.93 82.41±1.67

100 23.65±1.23 25.35±1.56 27.31±1.42 29.34±1.76 21.34±1.46

200 32.73±1.57 33.64±1.78 36.52±1.12 41.31±1.32 33.67±1.68

300 41.17±1.72 42.71±1.86 42.38±1.44 51.25±1.34 51.35±1.53 fruits

400 52.81±1.91 56.72±1.19 57.37±1.48 59.98±1.32 62.31±1.67 M. buxifolia M. 500 64.42±1.35 64.51±1.22 68.32±1.53 68.31±1.09 77.23±1.51

600 79.34±1.45 77.93±1.36 81.12±1.42 82.47±1.35 85.73±1.74

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Chapter 3 Results

Table 3.11 Antioxidant activity of M. buxifolialeaves extracts

Extract/Fraction Percent(%) scavenging activity (p˂0.003)

Concentration Methanolic n-hexane Chloroform Ethyl acetate Aqueous (µg/ml)

100 31.21±1.36 35.51±1.34 28.19±1.21 31.42±1.63 23.42±1.64

200 40.36±1.23 40.34±1.72 34.13±1.53 40.19±1.24 37.12±1.65

300 56.31±1.53 48.82±1.63 46.43±1.73 53.51±1.42 45.15±1.82

400 62.34±1.87 57.32±1.24 54.63±1.87 60.81±1.23 56.38±1.58

500 71.23±1.51 64.37±1.83 65.21±1.32 71.31±1.92 67.39±1.74

600 82.67±1.61 79.31±1.56 78.27±1.73 83.27±1.62 78.62±1.85

Table 3.12 EC50 values of M. buxifolia seed, leaves and fruit extracts

EC50 value (µg/ml) of methanolic extract and fractions of M. buxifolia Plant Part Methanolic n-hexane Chloroform Ethyl acetate Aqueous Vitamin C (Standard)

Seed 302.10 369.52 381.92 369.99 300.00 252.21

Leaves 388.70 363.74 378.95 353.32 319.94 252.21

Fruit 310.45 401.28 382.93 352.53 352.27 252.21

150

Chapter 3 Results

The synthesized D. mucronataleaves derived AgNPs, M. buxifolialeaves derived AgNPs and M. buxifolialeaves derived AuNPs antioxidant activity is presented in Table 3.13 and EC50 is represented in Table 3.14. Results presented that highest antioxidant activity was detected at highest concentration i.e.600 µg/ml.

Among the derived NPs highest antioxidant activity was observed for D. mucronataleaves derived AgNPs (86.4%), which was followed by M. buxifolia leaves derived AuNPs(86.31%) and M. buxifolialeaves derived AgNPs (84.58%). Highest

EC50 was observed for M. buxifolialeaves derived AgNPs (365.04 µg/ml), followed by M. buxifolialeaves derived AuNPs (349.15 µg/ml) and D. mucronataleaves derived

AgNPs (322.83 µg/ml).Figure 3.45 showed percent antioxidant activity of D. mucronataleaves extract mean, M. buxifolia leaves extract mean, D. mucronataleaves derived AgNPs, M. buxifolialeaves derived AgNPs and M. buxifolialeaves derived

AuNPs.

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Table 3.13 Antioxidant activity of D. mucronataleaves derived AgNPs and M.

buxifolialeaves derived AgNPs and AuNPs (p˂0.0001)

D. mucronataleaves M. buxifolialeaves M. buxifolialeaves Concentration derived AgNPs derived AgNPs derived AuNPs

100 46.18±0.67 38.67±1.29 36.01±0.92

200 53.87±0.81 46.45±0.81 44.98±0.907

300 64.26±0.99 55.6±1.24 55.95±0.75

400 73.15±0.89 66.35±1.09 66.55±0.78

500 81.15±0.903 76.55±1.05 76.69±0.98

600 86.4±0.91 84.58±1.06 86.31±1.02

Table 3.14 EC50 values of D. mucronataleaves derived AgNPs and M. buxifolialeaves

derived AgNPsand AuNPs

D. mucronataleaves M. buxifolialeaves M. buxifolialeaves Sample derived AgNPs derived AgNPs derived AuNPs

EC50 322.83 365.04 349.15

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Chapter 3 Results

Antioxidant activity 100 90 80 70 60 50 40

% % Scavenging 30 20 10 0 100 200 300 400 500 600 Concentration µg/ml

D. mucronata leaves extract mean D. mucronata leaves derived AgNPs M. buxifolia leaves extract mean M. buxifolia leaves derived AgNPs M. buxifolia leaves derived AuNPs

Figure 3.45 Percent antioxidant activity of D. mucronata and M. buxifolialeaves

extracts and their derived NPs

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Chapter 3 Results

3.5.2 Anti-bacterial activity

MIC50 and MBC values for D. mucronata bark, leaves and roots crude methanolic extract and its fractions are given in Table 3.15. Crude methanolic extract of D. mucronata bark showed good anti-bacterial activity against A. baumanni(65.21%), M. morganii(65.21%) and E. coli(62.96%). Moderate anti- bacterial activity was observed against P. aeruginosa(59.25%), S. aureus (55.76%),

VRSA (52%) and P. vulgaris(56%). The n-hexane fraction of D. mucronata bark showed moderate effect against A. baumanni(47.82%), E. coli(48.12%) and P. vulgaris(44%). Low anti-bacterial effect was observed against S. aureus (38.46%), M. morgani (34.78%) and VRSA (36%) while no activity was observed against P. aeruginosa. Chloroform fraction of D. mucronata bark showed moderate effect against M. morganii(43.47%) and VRSA (44%). Low anti-bacterial activity was observed against A. baumani (34.78%), E. coli(25.92), P. aeruginosa(33.33%), S. aureus (23.07%) and P. vulgaris(36%). Ethyl acetate fraction of D. mucronata bark showed moderate anti-bacterial effect against A. baumanii (43.47%), E. coli(44.44%),

P. aeruginosa(48.14%) and P. vulgaris(40%). Low anti-bacterial effect was observed against S. aureus (34.61%) and VRSA (32%). No activity was observed against M. morganii. The aqueous portion of D. mucronata showed moderate anti-bacterial activity against P. aeruginosa (40.74%) and VRSA (40%). Low anti-bacterial activity was observed against A. baumanni (39.13%), E. coli(37.03%), S. aureus (30.76%) and M. morganii(30.43%) while no anti-bacterial activity was observed against P. vulgaris. Highest anti-bacterial activity was observed by methanolic extract of D. mucronata bark with MIC50=2.56 mg/ml and MBC=5.86 mg/ml against A.

154

Chapter 3 Results baumannii. Lowest anti-bacterial activity was observed by D. mucronata bark chloroform extract with MIC50=6.94 mg/ml and MBC=12.88 mg/ml against S. aureus.

Crude methanolic extracts of D. mucronataleaves showed good anti-bacterial activity against A. baumani (78.26%), E. coli(77.78%), P. aeruginosa(74.07%), S. aureus (73.07%), M. morganii (69.56%), VRSA (68%) and P. vulgaris(60%). The n- hexane fraction of D. mucronataleaves showed moderate anti-bacterial effect against

A. baumanni(43.47%), E. coli(51.85%), P. aeruginosa(48.14%), S. aureus (46.15%),

M. morganii(43.70%), VRSA (50%) and P. vulgaris(52%). Chloroform fraction of D. mucronataleaves showed moderate effect against M. morganii(52.17%), VRSA

(44%) and P. vulgaris(40%). Low anti-bacterial activity was observed against A. baumanii (39.13%), E. coli(37.03%), P. aeruginosa (29.62%) and S. aureus

(34.16%). Ethyl acetate fraction of D. mucronataleaves showed moderate anti- bacterial effect against A. baumanii (52.17%), E. coli (55.56%), P. aeruginosa

(51.85%), S. aureus (50%), M. morganii(47.82%), VRSA (52%) and P. vulgaris(56%). Aqueous portion of D. mucronataleaves showed moderate anti- bacterial activity against A. baumanni(43.47%) and P. vulgaris(44%). Low anti- bacterial effect was observed against E. coli(33.33%), P. aeruginosa(38.89%), S. aureus (36.53%), and VRSA (32%) while no anti-bacterial activity was observed against M. morganii. Highest anti-bacterial activity was observed by D. mucronataleaves methanolic extract with MIC50=2.28 mg/ml and MBC=4.2 mg/ml against E. coli. Lowest anti-bacterial activity was observed by D. mucronataleaves with MIC50=6.34 mg/ml and MBC=12.4 mg/ml against VRSA.

Crude methanolic extract of D. mucronata roots exhibited good anti-bacterial effectfor theA. baumanni(86.95%), E. coli(85.18%), and S. aureus (84.61%). 155

Chapter 3 Results

Moderate anti-bacterial activity was observed against P. aeruginosa(62.96%), M. morganii(56.52%), VRSA (60%) and P. vulgaris(68%). The n-hexane fraction of D. mucronata roots showed good anti-bacterial effect against A. baumanni(78.26%).

Moderate anti-bacterial activity was observed against E. coli(48.14%), S. aureus

(57.69%), M. morganii(47.82%), VRSA (40%) and P. vulgaris(48%). No anti- bacterial activity was observed against P. aeruginosa. The chloroform fraction of D. mucronata roots showed good anti-bacterial effect against A. baumanni (60.86%) and

S. aureus (61.53%). Moderate anti-bacterial activity was observed against E. coli(55.55%), M. morganii(43.47%), VRSA (48%) and P. vulgaris(52%) while low anti-bacterial activity was observed against P. aeruginosa(37.03%). Ethyl acetate fraction of D. mucronata roots showed good anti-bacterial effect against A. baumanii

(69.56%), E. coli(62.96%) and S. aureus (69.23%). Moderate anti-bacterial activity was observed against P. aeruginosa(55.55%), M. morganii(52.17%), VRSA (56%) and P. vulgaris(60%). Aqueous portion of D. mucronata roots showed moderate anti- bacterial activity against A. baumanni(52.17%), E. coli(40.74%) and VRSA (44%).

Low anti-bacterial effect was observed against S. aureus (38.46%). However, no anti- bacterial activity was observed against P. aeruginosa, M. morganiiand P. vulgaris.

Highest anti-bacterial activity was observed by D. mucronata roots methanolic extract with MIC50=1.8 mg/ml and MBC=3.32 mg/ml against A. baumannii. Lowest anti- bacterial activity was observed by D. mucronata roots aqueous extract with

MIC50=6.9 mg/ml and MBC=10.66 mg/ml against S. aureus.

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Chapter 3 Results

Table 3.15 Anti-bacterial Activity of D. mucronata bark, leaves and roots

MIC and MBC (mg/ml) of D. mucronata

Test organism Methanolic Extract n-hexane Chloroform Ethyl acetate Aqueous

MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC

A. baumanni 2.56 5.82 5.54 7.78 6.30 12.68 5.46 7.54 6.7 7.82

E. coli 2.96 5.98 5.58 8.18 6.70 13.12 5.3 9.1 6.02 8.9

P. aeruginosa 3.64 6.38 -- -- 6.18 12.12 5.7 7.9 5.5 11.5 bark S. aureus 3.88 6.78 6.06 11.92 6.94 12.88 6.1 11.3 6.22 11.3

M. morganii 2.96 5.02 6.66 11.18 5.98 11.96 -- -- 6.14 11.8 D. mucronata VRSA 5.66 7.14 6.02 12.20 6.02 11.50 6.7 11.8 5.98 9.5

P. vulgaris 5.66 8.74 5.94 12.0 6.46 12.2 5.9 12.2 -- --

A. baumanni 2.4 4.4 5.46 9.38 6.1 10.5 4.32 7.82 5.74 9.14

E. coli 2.28 4.2 4.9 8.5 6.02 10.9 3.8 7.54 6.18 11.38

P. aeruginosa 2.64 4.12 4.4 7.3 6.7 10.9 4.64 7.7 6.42 11.58 leaves S. aureus 2.36 4.4 4.6 7.5 6.18 11.1 4.32 7.74 6.22 9.98

M. morganii 2.76 4.9 5.5 8.7 5.3 11.8 5.26 8.62 -- --

D. mucronata VRSA 2.52 5.02 4.9 8.5 5.82 12.2 4.12 8.94 6.34 12.4

P. vulgaris 3.6 6.1 4.56 7.5 5.94 11.76 4.2 9.1 5.1 7.5

A. baumanni 1.8 3.32 2.76 4.6 3.6 6.42 2.6 4.86 4.78 9.54

E. coli 1.96 3.2 4.7 6.46 4.78 7.14 2.76 5.94 5.98 10.46

P. aeruginosa 2.4 4.44 -- -- 6.18 10.86 4.64 7.26 -- --

roots

S. aureus 1.4 3.56 4.86 7.22 2.8 5.9 2.2 4.34 6.9 10.66

M. morganii 3.28 6.5 5.7 7.78 5.98 8.46 4.56 9.5 -- --

D. mucronata VRSA 3.6 8.42 6.46 9.74 4.28 8.18 4.44 10.3 5.9 9.1

P. vulgaris 2.6 4.36 4.82 9.54 4.32 7.70 3.6 6.42 -- --

MIC = Minimum inhibitory concentration, MBC = Minimum bactericidal concentration

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Chapter 3 Results

The MIC50 and MBC results obtained for M. buxifolia seed, leaves and fruitcrude methanolic extract and its fractions are shown inTable 3.16. Crude methanolic extract of M. buxifolia seed showed highest anti-bacterial effectforM. morganii(82.60% with MIC50=1.6 mg/ml and MBC=2.96 mg/ml) and VRSA (80% with MIC50=1.8 mg/ml and MBC=3.0 mg/ml). The n-hexane fraction of M. buxifolia seeds showed highest anti-bacterial effect forM. morganii(69.56% with MIC50=2.44 mg/ml and MBC=4.2 mg/ml) followed by A. baumanni(65.21% with MIC50=2.6 mg/ml and MBC=3.52 mg/ml), and VRSA (64% with MIC50=2.36 mg/ml and

MBC=4.16 mg/ml). Lowest effect was detectedforP. aeruginosa(51.85% with

MIC50=4.76 mg/ml and MBC=8.28 mg/ml), followed by S. aureus (53.84% with

MIC50=4.36 mg/ml and MBC=7.68 mg/ml), E. coli(55.55% with MIC50=3.2 mg/ml and MBC=7 mg/ml) and P. vulgaris(60% with MIC50=2.88 mg/ml and MBC=4.96 mg/ml). Chloroform, ethyl acetate and aqueous fractions of M. buxifolia seeds showed moderate to least effect against overall bacterial species except for A. baumanii which showed resistance against ethyl acetate and aqueous seed fraction.

Crude methanolic extract of M. buxifolialeaves and fruit anti-bacterial activity was observed similar to fractions of plant seed, where crude methanolic extract showed highest activity against overall bacterial species followed by n-hexane, chloroform and then ethyl acetate and aqueous fractions. Growth ofA. baumannihad been inhibited by M. buxifolia seed ethyl acetate and aqueous fractions. M. buxifolialeaves ethyl acetate fraction has inhibited growth of VRSA and P. vulgaris.

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Chapter 3 Results

Table 3.16 Anti-bacterial Activity of M. buxifolia seeds, leaves and fruits

MIC and MBC (mg/ml) of M. buxifolia

Test organism Methanolic extract n-hexane Chloroform Ethyl acetate Aqueous

MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC

A. baumanni 2.08 3.12 2.6 3.52 3.56 7.12 ------

E. coli 2.96 3.76 3.2 7.0 4.8 7.6 5.96 11.12 5.0 10.6

P. aeruginosa 2.24 3.48 4.76 8.28 5.68 10.84 7.4 13.08 5.88 10.48 seeds S. aureus 2.92 3.84 4.36 7.68 4.28 8.56 7.16 13.12 5.48 10.52

M. morganii 1.6 2.96 2.44 4.2 4.92 9.2 5.8 9.92 4.72 8.56 M. M. buxifolia VRSA 1.8 3.0 2.36 4.16 5.16 10.24 5.32 9.76 4.56 8.68

P. vulgaris 2.84 3.56 2.88 4.96 4.72 8.52 5.96 10.44 5.52 10.76

A. baumanni 2.56 3.92 4.2 9.32 4.24 9.68 5.64 11.2 4.8 9.6

E. coli 2.24 3.56 4.52 9.32 4.52 9.68 5.76 11.12 5.92 10.8

P. aeruginosa 2.32 4.6 4.68 9.32 5.64 10.52 5.84 10.72 6.24 12.28

leaves

S. aureus 2.76 4.48 4.72 9.44 5.36 10.4 6.24 12.0 5.72 10.84

M. morganii 2.12 3.6 3.0 6.12 4.96 10.48 4.52 9.28 5.36 10.92

M. M. buxifolia VRSA 2.2 4.12 3.16 6.28 3.48 7.72 5.48 10.6 -- --

P. vulgaris 2.4 4.4 3.4 6.8 4.68 10.72 6.00 11.6 -- --

A. baumanni 2.52 4.28 2.84 4.64 4.36 9.64 5.76 10.48 4.76 9.68

E. coli 2.44 4.16 4.56 9.32 4.68 9.72 5.6 10.32 5.76 10.72

P. aeruginosa 2.64 4.48 3.72 7.4 4.36 9.4 5.84 10.72 6.72 12.52

fruits

S. aureus 2.72 4.56 4.68 9.36 4.52 9.56 4.68 9.76 6.56 12.44

M. morganii 2.76 4.84 4.44 9.24 2.92 4.6 4.64 9.36 4.32 9.64 M. M. buxifolia VRSA 2.76 4.92 3.88 7.76 4.4 9.2 5.88 10.92 4.68 9.96

P. vulgaris 3.04 6.16 4.48 9.76 4.6 9.8 3.84 7.92 5.2 10.6

MIC = Minimum inhibitory concentration, MBC = Minimum bactericidal concentration

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Chapter 3 Results

Percent anti-bacterial activity of synthesized AgNPs and AuNPs is presented in Figure 3.46 and Table 3.17 and MIC50/MBC are presented in Table 3.18. The D. mucronataleaves derived AgNPs showed significant anti-bacterial activity againstE. coli (81.48% with MIC50=1.96 mg/ml and MBC=2.76 mg/ml). Good anti-bacterial effect was detectedforS. aureus (76.92% with MIC50=2.32 mg/ml and MBC=3.56 mg/ml),P. vulgaris (76%with MIC50=2.4 mg/ml and MBC=3.68 mg/ml),P. aeruginosa (74.07% with MIC50=2.52 mg/ml and MBC=3.8 mg/ml),A. baumanni

(65.21%with MIC50=2.72 mg/ml and MBC=3.8 mg/ml), M. morganii (65.21% with

MIC50=2.76 mg/ml and MBC=3.92 mg/ml) and VRSA (64% with MIC50=2.8 mg/ml and MBC=4.2 mg/ml). The M. buxifolialeaves derived AgNPs showed good anti- bacterial activity against A. baumanni(73.91% with MIC50=2.56 mg/ml and

MBC=3.92 mg/ml),E. coli(74.07% with MIC50=2.24 mg/ml and MBC=3.56 mg/ml),

P. aeruginosa (70.37% with MIC50=2.32 mg/ml and MBC=4.6 mg/ml), S. aureus(69.23% with MIC50=2.76 mg/ml and MBC=4.48 mg/ml), M. morganii

(78.26% with MIC50=2.12 mg/ml and MBC=3.6 mg/ml), VRSA (76% with

MIC50=2.2 mg/ml and MBC=4.12 mg/ml) and P. vulgaris(72% with MIC50=2.4 mg/ml and MBC=4.4 mg/ml). Good anti-bacterial effect was observed by M. buxifolialeaves derived AuNPs against A. baumanni(69.56% with MIC50=2.64 mg/ml and MBC=4.24 mg/ml),S. aureus(65.38% with MIC50=2.88 mg/ml and MBC=4.56 mg/ml), M. morganii (73.91% with MIC50=2.28 mg/ml and MBC=3.76 mg/ml),

VRSA (72% with MIC50=2.36 mg/ml and MBC=4.24 mg/ml) and P. vulgaris(68% with MIC50=2.52 mg/ml and MBC=4.52 mg/ml) and moderate anti-bacterial activity againstE. coli(59.25% with MIC50=2.32 mg/ml and MBC=3.72 mg/ml) and P. aeruginosa (55.55%).

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Table 3.17 Anti-bacterial activity of both plants mediated AgNPs and AuNPs

D. mucronataleaves M. buxifolia leaves M. buxifolia leaves Zone of derived AgNPs derived AgNPs derived AuNPs Name of Inhibition Zone of Zone of Zone of Bacteria (Standard) Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition mm (%) (%) (%) (mm) (mm) (mm)

A. baumanni 23 (Cipro) 15 65.21 17 73.91 16 69.56

E. coli 27 (Amox) 22 81.48 20 74.07 16 59.25

P. aeruginosa 27 (Amox) 20 74.07 19 70.37 15 55.55

S. aureus 26 (Amox) 20 76.92 18 69.23 17 65.38

M. morganii 23 (Rifamp) 15 65.21 18 78.26 17 73.91

VRSA 25 (Amp) 16 64 19 76.00 18 72

P. vulgaris 25 (Amox) 19 76 18 72.00 17 68

Table 3.18 MIC50 and MBC of both plants mediated AgNPs and AuNPs

D. mucronataleaves M. buxifolia leaves M. buxifolia leaves

Name of Bacteria derived AgNPs derived AgNPs derived AuNPs

MIC50 MBC MIC50 MBC MIC50 MBC

A. baumanni 2.72 3.8 2.56 3.92 2.64 4.24

E. coli 1.96 2.76 2.24 3.56 2.32 3.72

P. aeruginosa 2.52 3.8 2.32 4.6 2.52 4.72

S. aureus 2.32 3.56 2.76 4.48 2.88 4.56

M. morganii 2.76 3.92 2.12 3.6 2.28 3.76

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VRSA 2.8 4.2 2.2 4.12 2.36 4.24

P. vulgaris 2.4 3.68 2.4 4.4 2.52 4.52

Anti-bacterial activity 90 80 70 60 50 40 30

% % zone inhibition 20 10 0 A. baumanni E. coli P. S. aureus M. morganii VRSA P. vulgaris aeruginosa

D. mucronata leaves aqueous extract D. mucronata leaves derived AgNPs M. buxifolia leaves aqueous extract M. buxifolia leaves derived AgNPs M. buxifolia leaves derived AuNPs

Figure 3.46 Anti-bacterial activity of D. mucronata and M. buxifolialeaves aqueous

extract and derived AgNPsand AuNPs

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Chapter 3 Results

3.5.3 Anti-fungal activity

Anti-fungal activity was performed against selected fungal strains C. albicans,

F. oxysporum, A. flavus, A. parasiticus, P. digitatum and A. niger. Mainstream of the experimental samples testedin contrast to test organisms exhibited no activity. Results of D. mucronata bark, leaves and roots crude methanolic extracts and its fractions are shown inTable 3.19.

Crude extracts and fractions of D. mucronata bark were found to be inactive against C. albicans. Crude methanolic extract presented low inhibitory effect (25%) forF. oxysporum and rest of the fractions presented no activity against it. The n- hexane showed low inhibition activity (10%) forA. flavus, and fractionsremained were inactive. Chloroform showed low inhibition activity (10%) against A. parasiticus while rests of the fractions were inactive. Crude methanolic extract and its fractions were inactive forP. digitatum. Crude methanolic extract and fractions of D. mucronata bark showed no activity against A. niger. Crude methanolic extract and n- hexane of D. mucronataleaves showed low inhibition activity (20% and 10%) against

C. albicans respectively, while rest of the fractions were inactive. In case of F. oxysporum, crude methanolic extract and rest of the fractions exhibited no inhibition activity. Crude methanolic extract presented low inhibition activity (30%) forA. flavus and rest of the fractions showed no activity. All fractions were inactive forA. parasiticus and P. digitatum. The n-hexane fraction exhibited low inhibition (30%) against A. niger while rests of the fractions were inactive. The D. mucronata roots crude methanolic extract and its fractions revealed no inhibition activity forC. albicans. The n-hexane fraction showed low inhibition activity (10%) forF. oxysporum while rests of the fractions were inactive. Crude methanolic extract and 163

Chapter 3 Results fractions were extract andfractions were inactive forA. flavus, A. parasiticus and P. digitatum. The n-hexane fraction presented low inhibition (10%) forA. niger while rests of the fractions were inactive against it.

Results of M. buxifolia seeds, leaves and fruits crude methanolic extracts and its fractions are displayed in Table 3.20. Crude methanolic extracts and fractions of

M. buxifolia seeds were found to be inactive against C. albicans, F. oxysporum, A. flavus and P. digitatum. Crude methanolic extract and n-hexane revealed low inhibitory activity (15% and 10%) forA. parasiticuswhereas rest of the fractions showed no activity against it. Crude methanolic extract and n-hexane exhibited low anti-fungal activity (10% and 5%) forA. nigerwhereas rests of the fractions were inactive. The n-hexane fraction of M. buxifolialeaves showed low anti-fungal activity

(10%) against C. albicans while crude extracts and rest of the fractions were found to be inactive. Crude methanolic extract showed low inhibition (10%) against F. oxysporum while rests of the fractions were inactive. Crude methanolic extract and n- hexane displayed low inhibition (20% and 10%) against A. parasiticushowever rests of the fractions were inactive. Crude methanolic extract and fractions of M. buxifolialeaves were inactive forA. flavus, P. digitatum and A. niger. Crude methanolic extracts and fractions of M. buxifolia fruits were inactive forC. albicans,

A. flavus, A. parasiticus and A. niger. The n-hexane fraction of M. buxifolia fruits showed low inhibition activity (10%) forF. oxysporumwhereas rests of the fractions were detected to be inactive. Crude methanolic extract and n-hexane showed low inhibition (35% and 15%) against P. digitatum while rests of the fractions were inactive.

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Chapter 3 Results

Table 3.19 Anti-fungal activity of D. mucronata bark, leaves and roots crude

methanolic extract and fractions

Name of fungi C. albicans F. oxysporum A. flavus A. parasiticus P. digitatum A. niger

Negative Control 0 0 0 0 0 0

Positive Control 100 100 100 100 100 100

Crude methanolic 0 25 0 0 0 0

extract bark n-hexane 10 0 10 0 0 0

Chloroform 0 0 0 10 0 0

Ethyl acetate 0 0 0 0 0 0 D. mucronata D.

Aqueous 0 0 0 0 0 0

Crude methanolic 20 0 30 0 0 0

extract leaves

n-hexane 10 0 0 0 0 30

Chloroform 0 0 0 0 0 0

Ethyl acetate 0 0 0 0 0 0 D. mucronata D. Aqueous 0 0 0 0 0 0

Crude methanolic 0 0 0 15 0 10

extract roots n-hexane 0 10 0 0 0 10

Chloroform 0 0 0 0 0 0

Ethyl acetate 0 0 0 0 0 0 D. mucronata D.

Aqueous 0 0 0 0 0 0

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Table 3.20 Percent Anti-fungal activity of M. buxifolia seeds, leaves and fruits crude

methanolic extract and fractions

Name of fungi C. albicans F. oxysporum A. flavus A. parasiticus P. digitatum A. niger

-ve Control 0 0 0 0 0 0

+ve Control 100 100 100 100 100 100

Crude methanolic

0 0 0 15 0 10 seed extract

n-hexane 0 0 0 10 0 5

M.buxifolia Chloroform 0 0 0 0 0 0

Ethyl acetate 0 0 0 0 0 0

Aqueous 0 0 0 0 0 0

Crude methanolic 0 10 0 20 0 0

extract

leaves n-hexane 10 0 0 10 0 0

Chloroform 0 0 0 0 0 0

Ethyl acetate 0 0 0 0 0 0 M.buxifolia

Aqueous 0 0 0 0 0 0

Crude methanolic 0 0 0 0 35 0

extract

fruit n-hexane 0 10 0 0 15 0

Chloroform 0 0 0 0 0 0

M. buxifolia M.buxifolia Ethyl acetate 0 0 0 0 0 0

Aqueous 0 0 0 0 0 0

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Chapter 3 Results

Percent growth inhibition of synthesized AgNPs and AuNPs is represented in

Table 3.21. Results showed that D. mucronataleaves derived AgNPs and M. buxifolialeaves derived AgNPs and AuNPs exhibited anti-fungal activities only against C. albicans and A. niger. Among the synthesized NPs, D. mucronataleaves derived AgNPs had shown highest anti-fungal activity with percent inhibition of

49.5% and M. buxifolialeaves derived AgNPswith percent inhibition of 47% against

C. albicans. D. mucronataleaves derived AgNPs showed 20% inhibition against A. niger, whereas M. buxifolialeaves derived AgNPs showed 15% inhibition.

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Chapter 3 Results

Table 3.21 Anti-fungal activity of D. mucronataleaves derived AgNPs and M.

buxifolialeaves derived AgNPs and AuNPs

Percent inhibition

Name of fungi D. mucronataleaves M. buxifolialeaves derived M. buxifolialeaves derived

derived AgNPs AgNPs AuNPs

C. albicans 49.5 47 5

F. oxysporum - - -

A. flavus - - -

A. parasiticus - - -

P. digitatum - - -

A. niger 20 15 10

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Chapter 3 Results

3.5.4 Haemagglutination activity

In current study the D. mucronata bark, leaves and roots crude methanolic extract and its fractions and leaves derived AgNPs showed no haemagglutination activity. Similarly M. buxifolia seeds, leaves and fruits crude methanolic extract and its fractions and leaves derived AgNPs and AuNPs showed no activity against human erythrocytes.

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Chapter 3 Results

3.5.5 Phytotoxic activity

Various anti-tumorigenic combinations have been reported to hinder the development of L. minor which designates the presence of inhibitors. The presence of natural stimulants is determined by the propagation of fronds. These characteristics undertake need for herbicide which has certain benefit of having growth stimulants.

Variable phytotoxic activities were observed for different parts of D. mucronata

(Table 3.22). Highest phytotoxic activity was observed for D. mucronataleaves ethyl acetate extract (80%), which was followed by D. mucronataleaves chloroform extract

(70%) and D. mucronata root ethyl acetate extract (70%). This range (60-100%) of phytotoxic activity was designated as inhibitors. Least phytotoxicity was observed for

D. mucronata bark and D. mucronata root crude methanolic extract (30%) which was followed by D. mucronata bark aqueous extract (40%) and D. mucronata bark n- hexane, D. mucronata root aqueous and D. mucronataleaves crude methanolic extract

(40%). These low phytotoxic activity extracts were designated as stimulants.

Percent growth inhibition of L. minor by D. mucronata parts and its fractions has been presented in Table 3.23. Results showed that the phytotoxic activity of various fractions increase with increase in concentration against L. minori.e.10 to

1000 μg/ml. Highest phytotoxic activity was observed for D. mucronata root ethyl acetate extract (70%) and D. mucronataleaves ethyl acetate extract (70%). The least phytotoxic activity was observed for D. mucronata bark crude methanolic extract

(30%) followed byD. mucronata bark aqueous and n-hexane extract and D. mucronataroot aqueous extract (40%).

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Chapter 3 Results

Table 3.22 Phytotoxic activity of D. mucronata bark, root and leaves crude

methanolic extracts and fractions

Sample(s) (20 mg/ ml) Total No. Of healthy Percent growth No. Of Inference fronds left inhibition (%) fronds

Crude methanolic 7 30 stimulants

extract bark n-hexane 6 40 stimulus

Chloroform 5 50 stimulus

Ethyl acetate 4 60 inhibitors D. mucronata D.

Aqueous 6 40 stimulants

Crude methanolic 7 30 stimulants

extract root n-hexane 4 60 inhibitors 10 Chloroform 5 50 inhibitors

Ethyl acetate 3 70 inhibitors D. mucronata D.

Aqueous 6 40 stimulants

Crude methanolic 6 40 stimulants

extract leaves

n-hexane 4 60 inhibitors

Chloroform 3 70 inhibitors

Ethyl acetate 2 80 inhibitors D. mucronata D. Aqueous 5 50 inhibitors

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Chapter 3 Results

Table 3.23 Percent growth inhibition of L. minor by D. mucronata bark, root and

leaves crude extracts and fractions

Sample Percent growthinhibition

Conc. Of Sample (μg/ml) 10 100 1000

Standard drug: Paraquat 100 100 100

Crude methanolic extract 10 20 30

n-hexane 20 30 40

bark

Chloroform 30 40 50

Ethyl acetate 20 30 50

D. mucronata D. Aqueous 10 30 40

Crude methanolic extract 20 30 50

n-hexane 20 40 60

root

Chloroform 10 30 50

Ethyl acetate 40 50 70

D. mucronata D. Aqueous 20 30 40

Crude methanolic extract 20 30 50

n-hexane 20 40 50

leaves

Chloroform 40 50 60

Ethyl acetate 30 50 70

D. mucronata D. Aqueous 10 30 50

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Chapter 3 Results

Phytotoxic activity of M. buxifolia parts and its fractions is presented in Table

3.24. In case of M. buxifolia at 20 mg/ml, highest phytotoxic activity was observed in seed aqueous, fruit n-hexane and leaves n-hexane extract (70%), followed by seed n- hexane, fruit aqueous and leaves crude methanolic extract (60%). Depending on these results, the fractions extract were designated as inhibitors. Least phytotoxic activity was observed for M. buxifolia seed ethyl acetate, fruit crude methanolic extract and leaves aqueous extract (30%) followed by seed chloroform, fruit chloroform and leaves ethyl acetate extract (40%). The resulted low toxicity effects were designated as stimulants.

Percent growth inhibition of L. minor by M. buxifolia parts and its fractions is presented in Table 3.25. Phytotoxic activity of M. buxifolia showed similar increased in effect with increase in concentration against L. minori.e.10 to 1000 µg/ml. The highest phytotoxic activity was observed for seeds aqueous extract (70%), leaves n- hexane extract (70%) and fruits n-hexane extract (70%). Least phytotoxic activity was observed for seed ethyl acetate extract (30%), fruit crude methanolic extract (30%) and leaves aqueous extract (30%), followed by seed and fruit chloroform extract

(40%), and leaves ethyl acetate extract (40%) of M. buxifolia.

The D. mucronatabark aqueous extract showed 20% more phytotoxic activity than M. buxifolialeaves aqueous extract. However the derived NPs of these plants extracts had no phytotoxic activity as shown in Figure 3.47.

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Chapter 3 Results

Table 3.24 Phytotoxic activity of M. buxifolia seed, fruit and leaves crude methanolic

extract and its fractions at 20 mg/ml

Total no. No. of healthy Percent growth Sample(s) (20 mg/ ml) of fronds fronds left inhibition (%) Inference

Crude methanolic 5 50 stimulants

extract

seed n-hexane 4 60 Inhibitors

Chloroform 6 40 stimulants

M.buxifolia Ethyl acetate 7 30 stimulants

Aqueous 3 70 Inhibitors

Crude methanolic 7 30 stimulants

extract

fruit n-hexane 3 70 Inhibitors 10 Chloroform 6 40 stimulants

M.buxifolia Ethyl acetate 5 50 stimulants

Aqueous 4 60 Inhibitors

Crude methanolic 4 60 Inhibitors

extract

leaves n-hexane 3 70 Inhibitors

Chloroform 5 50 stimulants

Ethyl acetate 6 40 stimulants M.buxifolia

Aqueous 7 30 stimulants

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Chapter 3 Results

Table 3.25 Percent growth inhibition of L. minor by M. buxifolia crude methanolic

extract and its fractions.

Sample Percent growth inhibition

Conc. of sample (μg/ml) 10 100 1000

Standard drug: Paraquat 100 100 100

Crude methanolic extract 30 40 50

n-hexane 20 40 60

seeds

Chloroform 20 30 40

Ethyl acetate 10 20 30 M.buxifolia Aqueous 50 60 70

Crude methanolic extract 10 20 30

n-hexane 30 50 70

fruits

Chloroform 20 30 40

Ethyl acetate 20 30 50 M.buxifolia Aqueous 30 40 60

Crude methanolic extract 30 40 60

n-hexane 40 50 70

leaves

Chloroform 30 40 50

Ethyl acetate 20 30 40

M.buxifolia Aqueous 10 20 30

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Chapter 3 Results

20 % % growthinhibition

0 D. mucronata D. mucronata M. buxifolia M. buxifolia M. buxifolia leaves aqueous leaves derived leaves aqueous leaves derived leaves derived extract AgNPs extract AgNPs AuNPs

Figure 3.47 Percent growth inhibition of L. minorby D. mucronataand M. buxifolia

leaves extract and derived NPs

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Chapter 3 Results

3.5.6 Insecticidal activity

Insecticidal activity results are shown in Table 3.26. Crude methanolic extract, n-hexane, chloroform and ethyl acetate of D. mucronata bark, root and leaves showed

100% activity against T. castaneum, R. dominica and C. analis. Insecticidal activity of

D. mucronata bark aqueous extract was 70% against T. castaneumand 50% against R. dominica and C. analis. Activity of D. mucronataleaves aqueous extract was 60% against T. castaneum and 60% against R. dominica and C. analis. TheD. mucronataleaves aqueous extract showed 50% insecticidal effect against T. castaneum, 50% against R. dominica and 60% againstC. analis.

The insecticidal effect of crude methanolic extract, n-hexane, chloroform and ethyl acetate fractions of M. buxifolia seed, fruit and leaves was 100% against T. castaneum, R. dominica and C. analis. However aqueous extract of plant seeds showed good insecticidal activity against T. castaneum(80%), R. dominica(60%) and

C. analis(70%). Aqueous extract of M. buxifolia fruits had moderate activity 50% against T. castaneum and C. analis and good activity 70% against R. dominica.

Aqueous extract of M. buxifolialeaves showed good activity against T. castaneum

(60%), R. dominica (70%) and moderate activity 50% against C. analis (Table 3.27).

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Chapter 3 Results

Table 3.26 Insecticidal activity of D. mucronata bark, roots and leaves extracts

Percent mortality (%)

Samples T. castaneum R. dominica C. analis

Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3

Standard positive control 100 - - 100 - - 100 - -

Crude methanolic 100 - - 100 - - 100 - -

extract

bark n-hexane 100 - - 100 - - 100 - -

Chloroform 100 - - 100 - - 100 - -

Ethyl acetate 100 - - 100 - - 100 - - D. mucronata D. Aqueous 20 40 70 30 40 50 20 30 50

Crude methanolic 100 - - 100 - - 100 - -

extract root n-hexane 100 - - 100 - - 100 - -

Chloroform 100 - - 100 - - 100 - -

Ethyl acetate 100 - - 100 - - 100 - - D. mucronata D. Aqueous 20 30 60 20 50 60 20 40 60

Crude methanolic 100 - - 100 - - 100 - -

extract leaves n-hexane 100 - - 100 - - 100 - -

Chloroform 100 - - 100 - - 100 - -

Ethyl acetate 100 - - 100 - - 100 - - D. mucronata D. Aqueous 10 30 50 20 40 50 20 50 60

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Table 3.27 Insecticidal activity of M. buxifolia seed, fruit and leaves extracts

Percent mortality (%)

Samples T. castaneum R. dominica C. analis

Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3

Standard positive control 100 - - 100 - - 100 - -

Crude methanolic 100 - - 100 - - 100 - -

extract

seed n-hexane 100 - - 100 - - 100 - -

Chloroform 100 - - 100 - - 100 - -

M.buxifolia Ethyl acetate 100 - - 100 - - 100 - -

Aqueous 40 50 80 20 50 60 40 50 70

Crude methanolic 100 - - 100 - - 100 - -

extract

fruit n-hexane 100 - - 100 - - 100 - -

Chloroform 100 - - 100 - - 100 - -

M.buxifolia Ethyl acetate 100 - - 100 - - 100 - -

Aqueous 30 40 50 40 60 70 10 30 50

Crude methanolic 100 - - 100 - - 100 - -

extract

leaves n-hexane 100 - - 100 - - 100 - -

Chloroform 100 - - 100 - - 100 - -

Ethyl acetate 100 - - 100 - - 100 - - M.buxifolia

Aqueous 30 40 60 40 50 70 10 30 50

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Chapter 3 Results

D. mucronataleaves derived AgNPs showed 30, 40 and 50% insecticidal activity against T. castaneum, R. dominica and C. analis on first day. On day 3, 100% activity was observed. Insecticidal activity of D. mucronataleaves derived AgNPs showed higher activity as compared to D. mucronataleaves aqueous extract as shown in Figure 3.48.

Similar results were observed for M. buxifolialeaves derived AgNPs and

AuNPs. On first day M. buxifolialeaves derived AgNPs showed 40, 30 and 40% insecticidal activity against T. castaneum, R. dominica and C. analis. The AuNPs synthesized with M. buxifolialeaves extract showed 30, 50 and 40% insecticidal activity on first day against T. castaneum, R. dominica and C. analis. Overall both M. buxifolialeaves derived AgNPs and AuNPs on day three showed 100% insecticidal activity. These results showed that M. buxifolialeaves derived NPs had greater effect on insecticidal activity than M. buxifolialeaves aqueous extract as shown in Figure

3.49.

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Chapter 3 Results

Insecticidal activity 100 90 80 70 60 50 40

% % mortality 30 20 10 0 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 T. castaneum R. dominica C. analis D. mucronata leaves aqueous extract D. mucronata leaves derived AgNPs

Figure 3.48 Insecticidal activity of D. mucronataleaves aqueous extract and derived

AgNPs

Insecticidal activity 100 90 80 70 60 50 40 30 % mortality % 20 10 0 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 T. castaneum R. dominica C. analis M. buxifolia leaves aqueous extract M. buxifolia leaves derived AgNPs M. buxifolia leaves derived AuNPs

Figure 3.49 Insecticidal activity of M. buxifolialeaves aqueous extract and M.

buxifolialeaves derived AgNPs and AuNPs

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Chapter 3 Results

3.5.7 Anti-termite activity

Results for D. mucronata are shown in Table 3.28. D. mucronata bark crude methanolic extract, n-hexane, ethyl acetate and aqueous fractions exhibitedmodest anti-termite activity (50%, 60%, 50% and 60%) respectively. D. mucronata bark chloroform extract showed good anti-termite activity (70%). On the 2nd day, D. mucronata bark chloroform showed significant anti-termite activity (90%), while D. mucronata bark crude methanolic extract (70%), n-hexane (80%), ethyl acetate (80%) and aqueous (80%) fraction had good anti-termite effect. On the 3rd day, 100% mortality was observed.

On first day, D. mucronataleaves crude methanolic extract, n-hexane, ethyl acetate and aqueous fractions showed moderate anti-termite activity (40%, 50%, 60% and 50% respectively). D. mucronata chloroform extract showed good anti-termite activity (70%). On 2nd day, D. mucronataleaves n-hexane showed significant anti- termite activity (90%), while D. mucronata bark crude methanolic extract (40%), chloroform (80%), ethyl acetate (70%) and aqueous(70%) extracts had good anti- termite effect. On the 3rd day, Cent per cent mortality was resulted (100%) in all samples.

On first day, D. mucronata roots crude methanolic extract (40%), n-hexane

(50%), chloroform (60%), ethyl acetate (50%) and aqueous (50%) fractions showed moderate anti-termite activity. On the 2nd day, D. mucronata roots aqueous fraction showed significant anti-termite activity (90%), while D. mucronata bark crude methanolic extract (60%), n-hexane (70%), chloroform (70%) and ethyl acetate (70%) had good anti-termite effect.

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Table 3.28 Anti-termite activity of D. mucronata bark, roots and leaves

No. of No. of termites killed by No. of termites killed No. of termites killed Test sample termites negative control by positive control by the sample

Day 1 2 3 1 2 3 1 2 3

Crude methanolic 0 0 0 7 9 10 5 7 10

extract bark n-hexane 0 0 0 8 9 10 6 8 10

Chloroform 0 0 0 8 9 10 7 9 10

Ethyl acetate 0 0 0 7 9 10 5 8 10 D. mucronata D.

Aqueous 0 0 0 8 9 10 6 8 10

Crude methanolic 0 0 0 6 8 10 4 6 10

extract roots n-hexane 0 0 0 7 8 10 5 7 10 10 Chloroform 0 0 0 8 9 10 6 7 10

Ethyl acetate 0 0 0 8 9 10 5 7 10 D. mucronata D. Aqueous 0 0 0 7 9 10 5 9 10

Crude methanolic 0 0 0 8 9 10 4 6 10

extract leaves

n-hexane 0 0 0 6 8 10 5 9 10

Chloroform 0 0 0 7 8 10 7 8 10

Ethyl acetate 0 0 0 6 8 10 6 7 10 D. mucronata D. Aqueous 0 0 0 8 9 10 5 7 10

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M. buxifolia seed leaves and fruit crude methanolic extract and its fractions anti-termite activity are shown inTable 3.29. On first day, M. buxifolia seeds crude methanolic extract and ethyl acetate fractions showed low anti-termite activity (40%).

M. buxifolia seed n-hexane and chloroform extract showed moderate anti-termite activity (50%). Good anti-termite activity (70%) was observed by aqueous extract. On

2nd day, M. buxifolia seed crude methanolic extract and chloroform fraction showed moderate anti-termite activity (60%), while M. buxifolia seed n-hexane (70%), ethyl acetate (70%) and aqueous extract (80%) had good anti-termite effect. On 3rd day,

100% mortality was observed for all samples.

On first day, M. buxifolialeaves crude methanolic extract, chloroform and aqueous fractions showed moderate anti-termite activity (60%, 50% and 60% respectively). The M. buxifolialeaves n-hexane (40%) and ethyl acetate extract (40%) showed low anti-termite activity. On 2nd day, M. buxifolialeaves crude methanolic extract (70%), chloroform fraction (70%), n-hexane (70%) and aqueous fraction

(80%) showed good anti-termite activity, while M. buxifolialeaves n-hexane (60%) had moderate anti-termite activity. On the 3rd day, 100% mortality was observed in all samples.

On day first, M. buxifolia fruits crude methanolic extract (40%) and chloroform (40%) showed low anti-termite activity. The M. buxifolia fruits n-hexane

(50%), ethyl acetate (60%) and aqueous extract (60%) showed moderate anti-termite activity. On 2nd day, M. buxifolia fruit crude methanolic extract (50%), n-hexane

(60%) and chloroform fraction (60%) showed moderate anti-termite activity. Good anti-termite activity was revealed by ethyl acetate (70%) and aqueous fraction (80%).

On the 3rd day, cent per cent mortality was resulted (100%) in all samples. 184

Chapter 3 Results

Table 3.29 Anti-termite activity of M. buxifolia seed, fruit and leaves crude

methanolic extract and its fractions

No. of No. of termites killed No. of termites killed No. of termites killed Test Sample termites by Negative Control by Positive Control by the Sample

Day 1 2 3 1 2 3 1 2 3

Crude methanolic 0 0 0 6 8 10 4 6 10

extract

seed n-hexane 0 0 0 7 9 10 5 7 10

Chloroform 0 0 0 6 7 10 5 6 10

M.buxifolia Ethyl acetate 0 0 0 6 9 10 4 7 10

Aqueous 0 0 0 8 9 10 7 8 10

Crude methanolic 0 0 0 6 8 10 4 5 10

extract

fruit n-hexane 0 0 0 7 8 10 5 6 10 10 Chloroform 0 0 0 8 9 10 4 6 10

M.buxifolia Ethyl acetate 0 0 0 8 9 10 6 7 10

Aqueous 0 0 0 7 9 10 6 8 10

Crude methanolic 0 0 0 8 9 10 6 7 10

extract

leaves n-hexane 0 0 0 6 8 10 4 6 10

Chloroform 0 0 0 7 8 10 5 7 10

Ethyl acetate 0 0 0 6 8 10 4 7 10 M.buxifolia

Aqueous 0 0 0 8 9 10 6 8 10

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Anti-termite activity of D. mucronataleaves extract and it’s derived AgNPs showed to have 100% activity on day 3. However, D. mucronataleaves derived

AgNPs anti-termite activity was observed to be higher than that of D. mucronataleaves aqueous extract on first and second day as shown in Figure 3.50.

Anti-termite activity was observed to be 100% for M. buxifolialeaves aqueous extract and its derived AgNPs and AuNPs on day 3. On first and second day, the plant fruit extract and its derived NPs had shown similar results as shown in Figure 3.51.

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Chapter 3 Results

100 90 80 70 60 50

40 % % mortality 30 20 10 0 Day 1 Day 2 Day 3

D. mucronata leaves aqueous extract D. mucronata leaves derived AgNPs

Figure 3.50 Anti-termites activity of D. mucronataleaves aqueous extract and derived

AgNPs

100 90 80 70 60 50

40 % % mortality 30 20 10 0 Day 1 Day 2 Day 3

M. buxifolia leaves aqueous extract M. buxifolia leaves derived AgNPs M. buxifolia leaves derived AuNPs

Figure 3.51 Anti-termites activity of M. buxifolialeaves and derived Ag and AuNPs

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Chapter 3 Results

3.5.8. Cytotoxic activity

Cytotoxic activity was tested for D. mucronata bark, roots and leaves methanolic, n-hexane, chloroform, ethyl acetate and aqueous fractions against brine shrimp and results are presented in Table 3.30. D. mucronata bark crude methanolic extract and its fractions showed increase in percent mortality against brine shrimp with increase in concentration. Among D. mucronata bark extracts highest percent mortality was observed by crude methanolic extract (63.33% with LD50=234.212

µg/ml), which was followed by ethyl acetate extract (53.33% with LD50=361.437

µg/ml). Least mortality was observed for D. mucronata bark aqueous extract (40% with LD50=7159.2 µg/ml), followed by D. mucronata bark n-hexane extract (43.33% with LD50=8415.4 µg/ml).

Among D. mucronata roots highest percent mortality was observed by ethyl acetate (93.33% with LD50=8.109 µg/ml)against brine shrimp which was followed by crude methanolic extract (90% with LD50=44.759 µg/ml). Least mortality was observed for D. mucronata roots aqueous extract (76.67% with LD50=32.023 µg/ml), which was followed by n-hexane (83.33% with LD50=27.125 µg/ml) and chloroform extract (86.67% with LD50=13.519 µg/ml). Similar to D. mucronata roots, the D. mucronataleaves ethyl acetate extract was observed to have highest percent mortality against brine shrimp (86.67% with LD50=57.761 µg/ml), which was followed by crude methanolic extract (83.33% with LD50=16.722 µg/ml). Least percent mortality

(73.33% with LD50=90.213 µg/ml) was observed with aqueous D. mucronataleaves extract which was followed by n-hexane extract (76.67% with LD50=27.956

µg/ml)and chloroform extract (80% with LD50=36.348 µg/ml).

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Table 3.30 Brine shrimp lethality bioassay of D. mucronata bark, root and leaves

crude methanolic extract and fractions and derived NPs

Percent mortality LD50 value Fractions 10 µg/ml 100 µg/ml 1000 µg/ml (µg/ml) Control 0 0 0

Etoposide - - - 7.4625

Crude methanolic extract 33.33 36.67 63.33 234.212

n-hexane 26.67 30.00 43.33 8415.4

bark

Chloroform 30.00 40.00 50.00 974.408

Ethyl acetate 33.33 46.67 53.33 361.437

D. mucronata D. Aqueous 23.33 36.67 40.00 7159.2

Crude methanolic extract 36.67 46.67 90.00 44.759

n-hexane 43.33 56.67 83.33 27.125

root

Chloroform 50.00 63.33 86.67 13.519

Ethyl acetate 56.67 70.00 93.33 8.109

D. mucronata D. Aqueous 40.00 60.00 76.67 32.023

Crude methanolic extract 43.33 70.00 83.33 16.722

n-hexane 40.00 63.33 76.67 27.956 leaves Chloroform 33.33 66.67 80.00 36.348

Ethyl acetate 30.00 50.00 86.67 57.761

Aqueous 26.67 53.33 73.33 90.213 D. mucronata D. AgNPs 53.33 63.33 73.33 5.074

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Cytotoxic activity was tested for M. buxifolia seed, fruit and leaves methanolic, n-hexane, chloroform, ethyl acetate and aqueous fractions against brine shrimp and results are presented in Table 3.31. The M. buxifolia seed ethyl acetate extract was observed with highest percent mortality against brine shrimp (83.33% with LD50=24.122 µg/ml) which was followed by M. buxifolia seed crude methanolic extract (76.67% with LD50=35.196 µg/ml). Least percent mortality was observed with

M. buxifolia seed n-hexane extract (63.33% with LD50=64.286 µg/ml) which was followed by M. buxifolia seed chloroform extract (70% with LD50=31.262 µg/ml).

Similar to M. buxifolia seed, highest percent mortality of M. buxifolia fruit was observed with ethyl acetate (60% with LD50=357.7 µg/ml) against brine shrimp which was followed by M. buxifoliafruit crude methanolic extract (56.67% with

LD50=468.04 µg/ml). Least percent mortality was observed by M. buxifolia fruitn- hexane and aqueous extracts (50% with LD50=659.03 µg/ml and LD50=734.6 µg/ml) which was followed by Chloroform (53.33% with LD50=375.3 µg/ml).

Among M. buxifolialeaves extracts highest percent mortality was observed for crude methanolic extract (56.7% with LD50=662.16 µg/ml). Least percent mortality was observed for M. buxifolialeaves aqueous extract (43.33% with LD50=3794.2

µg/ml).

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Table 3.31 Brine shrimp lethality bioassay of M. buxifolia seed, fruit and leaves crude

methanolic extract its fractions and derived NPs

Percent mortality

LD50 value Fractions 1000 10 µg/ml 100 µg/ml µg/ml (µg/ml) Control 0 0 0

Etoposide - - - 7.4625

Crude methanolic extract 36.67 63.33 76.67 35.196

n-hexane 40 53.33 63.33 64.286

seed

Chloroform 43.33 56.67 70 31.262

Ethyl acetate 43.33 60 83.33 24.122 M.buxifolia Aqueous 33.33 50 73.33 75.584

Crude methanolic extract 26.67 36.67 56.67 468.04

n-hexane 23.33 43.33 50 659.03

fruit

Chloroform 30 46.67 53.33 375.3

Ethyl acetate 23.33 36.67 60 357.7 M.buxifolia Aqueous 20 40 50 734.6

Crude methanolic extract 26.67 30 56.7 662.16

n-hexane 33.33 40 53.33 599.63

Chloroform 23.33 30 46.67 2406.054

leaves

Ethyl acetate 30 36.67 50 1257.169

Aqueous 23.33 33.33 43.33 3794.2

M.buxifolia AgNPs 46.67 60 70 17.34

AuNPs 43.33 53.33 66.67 46.1

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Chapter 3 Results

Among the synthesized NPs, cytotoxicity of D. mucronataleaves derived

AgNPswas recorded with higher activity (73.33%with LD50=5.074µg/ml) against brine shrimp in comparison to D. mucronataleaves aqueous extract at concentration of

10, 100 μg/ml and 1000 μg/ml. Among the M. buxifolialeaves derived AgNPs and

AuNPs higher cytotoxicity effect was observed (70 and 66.67%with LD50=17.34 and

LD50=46.1μg/ml) in comparison to M. buxifolia leaves aqueous extract in all the appliedconcentration i.e.10, 100 and 1000 μg/ml as shown in Figure 3.52.

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Chapter 3 Results

% % mortality 30

0 D. mucronata D. mucronata M. buxifolia M. buxifolia M. buxifolia leaves aqueous leaves derived leaves aqueous leaves derived leaves derived extract AgNPs extract AgNPs AuNPs

10 100 1000

Figure 3.52 Brine shrimp lethality bioassay of D. mucronata and M. buxifolialeaves

aqueous extract and their derived NPs

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Chapter 3 Results

3.6 DNA damaging, hemolytic and anti-thrombolyticprofile

3.6.1 Thrombolytic activity

Five concentrations ranging from 20 to 100 µg/ml of D. mucronataleaves aqueous extract, D. mucronataleaves derived AgNPs, M. buxifolialeaves aqueous extract, M. buxifolialeaves derived AgNPs and AuNPs were tested for their thrombolytic potential (Table 3.32). Positive control (streptokinase) showed 68.77% lysis activity and negative control (distilled water) showed negligible lysis activity

(3.91%). Results showed that D. mucronataleaves aqueous extract, D. mucronataleaves derived AgNPs, M. buxifolialeaves aqueous extract, M. buxifolia leaves derived AgNPs and AuNPs possess 15.9%, 25.8%, 19.6%, 23.6% and 24.6% thrombolytic activity respectively at 100 µg/ml.

Results for D. mucronataleaves aqueous extract showed low thrombolytic activity in comparison to M. buxifolia leaves aqueous extract. However, the D. mucronataleaves derived AgNPs showed the highest thrombolytic activity, which was followed by M. buxifolialeaves derived AgNPs and M. buxifolialeaves derived AuNPs as shown in Figure 3.53.

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Table 3.32 Effect of D. mucronata and M. buxifolialeaves aqueous extracts and their

derived AgNPs and AuNPs on clot lysis

% of clot lysis

Sample Sample in each Eppendorf tube(µg/ml)

20 40 60 80 100

D. mucronata leaves 2.8 ± 0.3 4.6 ± 0.2 7.3 ± 0.2 10.4 ± 0.3 15.9 ± 0.3

D. mucronataderived AgNPs 4.3 ± 0.2 10.1 ± 0.3 15.7 ± 0.1 20.9 ± 0.2 25.8 ± 0.3

M. buxifolia leaves 2.8 ± 0.2 7.8 ± 0.2 11.8 ± 0.2 14.8 ± 0.2 19.6 ± 0.2

M. buxifolia derived AgNPs 3.9 ± 0.2 11 ± 0.2 14.8 ± 0.2 19.5 ± 0.2 23.6 ± 0.1

M. buxifolia derived AuNPs 3.5 ± 0.1 9.9 ± 0.3 15.6 ± 0.2 18.8 ± 0.2 24.6 ± 0.1

Streptokinase (100 µL) 68.77± 0.91

Distilled water (100 µL) 3.91± 0.09

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Thrombolytic activity 16 14 12 10 8

6 % % clot lysis 4 2 0 D. mucronata D. mucronata M. buxifolia M. buxifolia M. buxifolia leaves leaves derived leaves leaves derived leaves derived aqueous AgNP’s aqueous AgNP’s AuNP’s extract extract

Figure 3.53 Mean effect of D. mucronataand M. buxifolia leaves aqueous extracts and

their derived AgNPs and AuNPs on clot lysis

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3.6.2 Hemolytic activity

According to reported literature and best of our understanding, no information was existing about harmfulness of D. mucronata, M. buxifolialeaves aqueous extracts and derived AgNPs and AuNPs. Hemolytic potential was detected with spectrophotometric method for human erythrocytes. Negative control (Phosphate buffer) displays no hemolytic effect. Triton X-100 gives maximum human erythrocyteslysis. Hemolytic potential findings are revealed in Table 3.33. Samples tested were nontoxic, as no hemolytic activity was detected; consequently, aqueous extracts of D. mucronata and M. buxifolialeaves and prepared AgNPs and AuNPs can be considered for use in medicinein treatingnumerous diseases.

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Table 3.33 Hemolytic activity of aqueous extracts of D. mucronataleaves and M.

buxifolialeaves and their derived AgNPs and AuNPs

Sample Optical density (p˃0.05)

Concentration µg/ml

10 50 100 200 250

Positive control 0.41±0.02 0.41±0.02 0.41±0.02 0.41±0.02 0.41±0.02

Negative control 1.07±0.02 1.07±0.02 1.07±0.02 1.07±0.02 1.07±0.02

D. mucronataleaves 1.04±0.02 1.05±0.01 1.07±0.006 1.05±0.01 1.04±0.004

aqueous extract

D. mucronataleaves derived 1.02±0.022 1.06±0.008 1.05±0.004 1.02±0.02 0.99±0.02

AgNPs

M. buxifolia leaves aqueous 0.00±0.023 3.03±0.007 3.03±0.007 3.03±0.003 9.09±0.02

extract

M. buxifolialeaves derived 1.51±0.007 3.03±0.008 4.54±0.008 4.54±0.007 10.15±0.02

AgNPs

M. buxifolialeaves derived 0.00±0.01 1.51±0.006 3.03±0.01 4.54±0.004 4.54±0.007

AuNPs

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3.6.3 Mutagenicity test (Ames assay)

Ames bacterial reverse mutation test was accomplishedfor mutagenic activity in liquid culture (fluctuation test)entirely. Mutagenicity was verifiedfor two bacterial strains: S. typhimuriumTA98 and TA100. Blank plate was firstdetected and remaining were observedonce wells in blank plate were entirely colored purple, demonstratingno contamination in experiment. The blank plate was purple with no change in color, demonstrating no contamination. Results of mutagenicity tests are given inTable 3.34.

For TA-98 strain of S. typhimurium background was non-mutagenic and non-toxic having 5/96 positive wells. Standard was highly mutagenic by having 85/96 positive wells. D. mucronataleaves aqueous extract and M. buxifolialeaves aqueous extract

(0/96) were toxic to bacterial strain, while D. mucronataleaves derived AgNPs (10/96 positive wells), M. buxifolialeaves derived AgNPs (1/96 positive well) and M. buxifolialeaves derived AuNPs (6/96 positive wells) were non-mutagenic and nontoxic to bacterial strain. For TA-100 strain of S. typhimuriumbackground was non- mutagenic and non-toxic having 10/96 positive wells. The standard was highly mutagenic having 87/96 positive wells. M. buxifolia leaves aqueous extract and M. buxifolialeaves derived AgNPs (0/96) were toxic to bacterial strain while D. mucronataleaves aqueous extract (5/96positive wells), D. mucronataleaves derived

AgNPs (6/96 positive wells) and M. buxifolialeaves derived AuNPs (1/96 positive well) were non-mutagenic and non-toxic to bacterial strain. No samples exhibited mutagenic potential therefore, they are safe to use for drug development.

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Table 3.34 Mutagenic activity of aqueous extracts of D. mucronataleaves and M.

buxifolialeaves and their derived AgNPs and AuNPs

TA98 TA100

No. of +ve No. of +ve Sample Result wells/total Result wells/total number of number of wells wells

Background 5/96 - 10/96 -

2-nitro fluorine 85/96 Mutagenic - -

NaN3 - - 87/96 mutagenic

D. mucronata extract 0/96 Toxic 5/96 Non-mutagenic

AgNPs 10/96 Non-mutagenic 6/96 Non-mutagenic

M. buxifolia extract 0/96 Toxic 0/96 Toxic

AgNPs 1/96 Non-mutagenic 0/96 Toxic

AuNPs 3/96 Non-mutagenic 1/96 Non-mutagenic

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Chapter 3 Results

3.7 In-vivo pharmacological studies

3.7.1 Acute toxicity

Crude extracts of D. mucronata bark, root, leaves and derived AgNPs and M. buxifolia seed, fruit, leave and derived AuNPs and AgNPs were detected for their in- vivo biological effectiveness (Table 3.35 and Table 3.36). Crude methanolic extracts were tested at 200, 400 and 600 mg/kg body weight. All extracts of D. mucronata including bark, root, leaves and derived AgNPs were found nontoxic at tested doses and no lethality was detected. Similarly M. buxifolia seed, fruit, leaves and derived

AgNPs and AuNPs were found nontoxic at the tested doses and no lethality was detected. The synthesized NPs were screened at test dose of 20, 40 and 60 mg/kg.

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Table 3.35 Acute toxicity effect of crude methanolic extracts of D. mucronata bark,

root and leaves and derived AgNPs

Dose administered Total no. of No. of animals alive Percent death Treatment (mg/kg) animals after 24 h after 24 h (%)

200 6 6 --- D. mucronata bark 400 6 6 --- methanolic extract 600 6 6 ---

200 6 6 --- D. mucronata root 400 6 6 --- methanolic extract 600 6 6 ---

200 6 6 --- D. mucronataleaves 400 6 6 --- methanolic extract 600 6 6 ---

20 6 6 --- D. mucronataleaves 40 6 6 --- derived AgNPs 60 6 6 ---

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Chapter 3 Results

Table 3.36 Acute toxicity effects of crude methanolic extracts of M. buxifolia seed,

fruit and leaves and its fruit derived AgNPs and AuNPs

Dose administered Total no. of No. of animals alive Percent death Treatment (mg/kg) animals after 24 h after 24 h (%)

200 6 6 --- M. buxifolia seed 400 6 6 --- methanolic extract 600 6 6 ---

200 6 6 --- M. buxifolia fruit 400 6 6 --- methanolic extract 600 6 6 ---

200 6 6 --- M. buxifolialeaves 400 6 6 --- methanolic extract 600 6 6 ---

20 6 6 --- M. buxifolialeaves 40 6 6 --- derived AuNPs 60 6 6 ---

20 6 6 --- M. buxifolialeaves 40 6 6 --- derived AgNPs 60 6 6 ---

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3.7.2 Anti-analgesic activity

Crude methanolic extracts of D. mucronata leaves, stem and root and D. mucronataleaves derived AgNPs presentednoteworthyreduction in amount of writhes analyzed by two way ANOVA and Dunnett’s post-hoc exploration at an intermissions of 30, 60 and 90 min. Values were articulated as Mean ± SEM and effects were taken as significant at P<0.05.

Injection of variousdosages (100, 200 and 300 mg/kg) of D. mucronata leaves, bark and roots crude methanolic extracts and (10, 20 and 30 mg/kg) D. mucronataleaves derived AgNPs viai.p.meansexhibitedreduction in mean quantity of writhing in various test groups as revealed in Table 3.37. In saline treated group mean writhing was 74.67±9.47. Percent writhing inhibitory potential resulted by various test doses of crude methanolic extract of D. mucronataleaves was 41.51% (100 mg/kg),

54.17% (200 mg/kg) and 63.84% (300 mg/kg). Percent inhibition effect of writhing produced by different doses of D. mucronata bark crude methanolic extract also increases with increase of concentration that is 44.66% (100 mg/kg) , 54.31% (200 mg/kg) and 55.49% (300 mg/kg ) while, the similarconsequence of dose dependent rise of writing inhibitory potential was showed by D. mucronata roots crude methanolic extract that is 39.52% (100 mg/kg), 54.76% (200 mg/kg) and 59.15% (300 mg/kg). Percent inhibition effect of writhing detected by various doses of D. mucronataleaves derived AgNPs also increases with increase in concentration that is

38.98% (10 mg/kg), 56.39% (20 mg/kg) and 70.08% (30 mg/kg).

Effect produced by D. mucronata leaves, stem and root crude extracts and D. mucronataleaves derived AgNPs was dependenton dosage. Maximum percent

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Chapter 3 Results inhibition resulted by Diclofenac sodium (standard drug) at 20 mg/kg dose was

81.1%, that is more than the maximum dose of D. mucronata crude extract (300 mg/kg) and D. mucronataleaves derived AgNPs (30 mg/kg). Percent reduction in writhing sumby standard and D. mucronata leaves, stem and roots crude methanolic extract and leaves derived AgNPs is represented by Figure 3.55, Figure 3.56, Figure

3.57 and Figure 3.58 respectively. Crude methanolic extract of D. mucronataleaves, stem and root altogether were noteworthy at a dose of 100 mg/kg (***P< 0.001), 200 mg/kg (***P< 0.001) and 300 mg/kg (***P< 0.001).

Diclofenac sodium (20 mg/kg) treated mice exhibited significant inhibition in writhes with a mean value 14.11+0.22. In crude methanolic extract at a test dose of

100 mg/kg, pattern of an inhibition detected was as: D. mucronatabark (41.32+1.38)

>D. mucronataleaves (43.67+0.57) >D. mucronataroots (45.16+1.77). At a test dose of 200 mg/kg, writhing inhibition detected was in the subsequent order: D. mucronataroots (33.78+1.11) >D. mucronatabark (34.11 + 1.22) >D. mucronataleaves (34.22+0.728). Likewise, at a test dose of 300 mg/kg, inhibition detected in writhes was in the subsequent order: D. mucronataroots (23.89+0.91) >D. mucronataleaves (27+2.84) >D. mucronatabark (33.23+1.28).

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Chapter 3 Results

Table 3.37 Acetic acid induced writhing test (mean± SEM) values of D. mucronata

bark, root and leaves extracts

% Inhibition of Samples Dose administered Mean ± SEM values writhing

Normal Saline 10 ml/kg 74.67 ± 9.47 --

100 mg/kg 43.67 ± 0.57 41.51 D. mucronataleaves crude 200 mg/kg 34.22 ± 0.728 54.17 methanolic extract 300 mg/kg 27 ± 2.84 63.84

100 mg/kg 41.32 ± 1.38 44.66 D. mucronatabark crude methanolic 200 mg/kg 34.11 ± 1.22 54.31 extract 300 mg/kg 33.23 ± 1.28 55.49

100 mg/kg 45.16 ± 1.77 39.52 D. mucronataroot Crude methanolic 200 mg/kg 33.78 ± 1.11 54.76 extract 300 mg/kg 23.89 ± 0.91 59.15

D. mucronataleaves derived AgNPs 10 mg/kg 45.56+2.47 38.98

20 mg/kg 32.56+ 2.56 56.39

30 mg/kg 22.34+0.88 70.08

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Chapter 3 Results

Introduction of various doses (100, 200 and 300 mg/kg) of M. buxifolia leaves, seed and fruit crude methanolic extracts and (10, 20 and 30 mg/kg) M. buxifolialeaves derived AuNPs and AgNPs via i.p. meansexhibited, reduction in mean amount of writhing in various test groups as revealed in Table 3.38. Percent writhing inhibitory potentialexhibited by various test doses of crude methanolic extract of M. buxifolialeaves was 38.1% (100 mg/kg), 53.43% (200 mg/kg) and 63.39% (300 mg/kg). Percent inhibitory potential of writhing produced by different doses of M. buxifolia seeds crude methanolic extract also increases with increase of concentration that is 39.58% (100 mg/kg), 51.78% (200 mg/kg) and 63.54% (300 mg/kg) while, the similarconsequence of dose dependent increase of writing inhibitory effect was showed by M. buxifolia fruit crude extract that is 39.88% (100 mg/kg), 53.71% (200 mg/kg) and 69.19% (300 mg/kg). M. buxifolia leaves derived AuNPs percent writhing inhibition produced by different doses correspondingly increases with increase of concentration that is 39.74% (10 mg/kg), 51.19% (20 mg/kg) and 62.05% (30 mg/kg) while, same effect of dose dependent increase of writing inhibitory effect was showed by M. buxifolia leaves derived AgNPs that is 33.80% (10 mg/kg), 40.63% (20 mg/kg) and 55.51% (30 mg/kg).

Effect produced by M. buxifolia leaves, seed and fruit crude methanolic extracts and M. buxifolia leaves derived AuNPs and AgNPs was dose dependent

(Figure 3.59, Figure 3.60, Figure 3.61, Figure 3.62 and Figure 3.63). Crude methanolic extract of M. buxifolia leaves, seed and fruit were shown to be greatly significant at a dose of 100 mg/kg, 200 mg/kg and 300 mg/kg (***P< 0.001).

In crude methanolic extracts at test dose of 100 mg/kg, pattern of inhibition detected was as: M. buxifolia fruit (44.89+2.85) >M. buxifolia seed (45.11+2.7) >M. 207

Chapter 3 Results buxifolia leaves (46.22+4.0). Likewise, at a test dose of 200 mg/kg, inhibition detected in writhes was in the subsequent order: M. buxifolia fruit (34.56+2.73) >M. buxifolia leaves (34.77+3.08) >M. buxifolia seed (36 +3.33). At a test dose of 300 mg/kg, inhibition detected in writhes was in the succeeding order: M. buxifolia fruit

(23+1.26) > M. buxifolia seed (27.22 +3.33) >M. buxifolia leaves (27.33 +3.84).

D. mucronataleaves derived AgNPs showed higher percent of inhibition of writhihng than that of plant leaves extract alone. On the other hand, M. buxifolia leaves derived NPs had been observed with lower inhibition percent than plant leaves extract alone (Figure 3.54). Thus these results indicated that not only NPs availabilitity can bring change in properties of synthesized compounds but also the chemical reactions that take place due to synthesis of NPs can alter the properties of synthesized compoundsone way or the other.

208

Chapter 3 Results

Table 3.38 Acetic acid induced writhing test of M. buxifolia leaves, seed and fruit and

leaves derived AgNPs and AuNPs

Mean + SEM % Inhibition of Samples Dose administered values writhing

Standard (Diclofenac sodium) 20 mg/kg 14.11+0.22 81.1

100 mg/kg 46.22+4.0 38.1

M. buxifolialeaves methanolic extract 200 mg/kg 34.77+3.08 53.43

300 mg/kg 27.33 +3.84 63.39

100 mg/kg 45.11+2.7 39.58

M. buxifolia seed methanolic extract 200 mg/kg 36 +3.33 51.78

300 mg/kg 27.22+2.54 63.54

100 mg/kg 44.89+2.85 39.88

M. buxifolia fruit methanolic extract 200 mg/kg 34.56+2.73 53.71

300 mg/kg 23+1.26 69.19

M. buxifolialeaves derived AuNPs 10 mg/kg 44.99+2.6 39.74

20 mg/kg 36.44+1.49 51.19

30 mg/kg 28.33+1.57 62.05

M. buxifolialeaves derived AgNPs 10 mg/kg 49.43+3.199 33.80

20 mg/kg 44.33+1.38 40.63

30 mg/kg 33.22+1.56 55.51

209

Chapter 3 Results

% % writhing inhibition 20

0 100 200 300 10 20 30

D. mucronata leaves crude methanolicextract M. buxifolia leaves crude methanolic extract D. mucronata leaves derived AgNPs M. buxifolia leaves derived AuNPs M. buxifolia leaves derived AgNPs

Figure 3.54 Acetic acid induced writhing test values of D. mucronata and M.

buxifolialeaves and their derived NPs

For all in-vivo activities the level of significance was shown by:

(*) shows significant activity at (P<0.05).

(**)shows moderately significant activity at (P<0.01).

(***) shows highly significant activity at (P<0.001).

210

Chapter 3 Results

N /S

1 0 0 d

e S t d ( D ic lo f e n a c s o d iu m )

v r

e 1 0 0 m g /k g

b o

2 0 0 m g /k g

* h

* 3 0 0 m g /k g

t *

5 0 *

* N

0 0 0 3 6 9

T r e a tm e n t

Figure 3.55 Acetic acid induced writhing test for D. mucronataleaves methanolic

extract

N /S

1 0 0

d e

v S t d (D ic lo fe n a c s o d iu m )

r e

s 1 0 0 m g /k g

b o

2 0 0 m g /k g

* t

* 3 0 0 m g /k g

i 5 0

* N

0 0 0 3 6 9

T r e a tm e n t

Figure 3.56 Acetic acid induced writhing test for D. mucronata bark methanolic

extract

211

Chapter 3 Results

N /S

1 0 0 S t d (D ic lo fe n a c s o d iu m )

d e

v 1 0 0 m g /k g

r e

s 2 0 0 m g /k g

s 3 0 0 m g /k g

* *

i 5 0

* N

0 0 0 3 6 9

T r e a tm e n t

Figure 3.57 Acetic acid induced writhing test for D. mucronata roots methanolic

extract

N /S

1 0 0 d

e S t d (D ic lo fe n a c s o d iu m )

v r

e 1 0 m g /k g

s b

o 2 0 m g /k g

* *

h 3 0 m g /k g

* *

i 5 0

* N

0 0 0 3 6 9

T r e a tm e n t

Figure 3.58 Acetic acid induced writhing test for D. mucronataleaves derived AgNPs

212

Chapter 3 Results

1 0 0 N /S d

e S t d (D ic lo fe n a c s o d iu m )

v r

e 1 0 0 m g /k g

o *

2 0 0 m g /k g

* *

* 3 0 0 m g /k g t

5 0 *

* N

0 0 0 3 6 9

T r e a tm e n t

Figure 3.59 Acetic acid induced writhing test for M. buxifolialeaves crude methanolic

extract

1 0 0 N /S d

e S t d (D ic lo fe n a c s o d iu m )

r e

s 1 0 0 m g /k g

b o

2 0 0 m g /k g

h *

* 3 0 0 m g /k g t

5 0 *

* *

* * N

0 0 0 3 6 9

T r e a tm e n t

Figure 3.60 Acetic acid induced writhing test for M. buxifolia seed crude methanolic

extract

213

Chapter 3 Results

1 0 0 N /S d

e S t d (D ic lo fe n a c s o d iu m )

r e

s 1 0 0 m g /k g

* 2 0 0 m g /k g

* * t 3 0 0 m g /k g

i 5 0

* *

* N

0 0 0 3 6 9

T r e a tm e n t

Figure 3.61 Acetic acid induced writhing test for M. buxifolia fruit crude methanolic

extract

1 0 0 N /S d

e S t d (D ic lo fe n a c s o d iu m )

r e

s 1 0 m g /k g

b o

2 0 m g /k g

* *

* 3 0 m g /k g

t 5 0

* N

0 0 0 3 6 9

T r e a tm e n t

Figure 3.62 Acetic acid induced writhing test for M. buxifolialeaves derived AuNPs

214

Chapter 3 Results

1 0 0 N /S d

e S t d (D ic lo fe n a c s o d iu m )

v r

e 1 0 m g /k g

* o

2 0 m g /k g

h *

* 3 0 m g /k g

t *

5 0 *

* *

* * N

0 0 0 3 6 9

T r e a tm e n t

Figure 3.63 Acetic acid induced writhing test for M. buxifolialeaves derived AgNPs

215

Chapter 3 Results

3.7.3 Anti-pyretic activity

As shown inFigure 3.64, Figure 3.65, Figure 3.66, Figure 3.67, Figure 3.68 and Figure 3.69, the D. mucronata (leaves, bark and root) and M. buxifolia (leaves, seed, and fruit) crude methanolic extracts were detected to exhibit significant antipyretic activity in contrast to normal saline the negative control at an intervals of 1, 2 and 3 hrs. Results were presented as Mean + SEM and P< 0.05 was taken as significant. Plants extracts possessed significant anti-pyretic activity at doses of 100 mg/kg and 200 mg/kg (***P< 0.001) of body weight. Presence of certain inhibitory compounds was suggested by increased efficacy of test samples against pyrexia.

These compounds actually act as pyrexia inhibitors and prostaglandin- biosynthesismediators, thus result in reduction of temperature [240]. Results obtained were analyzed statistically by one way ANOVA tracked by Dunnet’s test, and values were articulated as Mean + SEM (Table 3.39 and Table 3.40).

Reduction in temperature was observed in Paracetamol (50 mg/kg) with 36.93

+ 0.204 mean value in comparison to normal saline. The plants extracts also presented results in comparison to paracetamol (reference drug). In crude methanolic extracts the test dosage of 100 mg/kg of body weight, temperaturedecrease was detected as follows: D. mucronata bark (37.12 + 0.038) >M. buxifolia fruits (37.21 + 0.116) >D. mucronata roots (37.22 + 0.04) >D. mucronataleaves (37.29 + 0.063) >M. buxifolia seeds (37.29 + 0.143) >M. buxifolialeaves (37.37 + 0.22). While at doses of 200 mg/ kg body weight, the temperature reduction was observed as follows: D. mucronata roots (37.22 + 0.026) >M. buxifolia seeds (37.237 + 0.148) >D. mucronata bark

(37.25 + 0.141) >D. mucronataleaves (37.25 + 0.113) >M. buxifolia fruits (37.31 +

0.084) >M. buxifolialeaves (37.34 + 0.16). 216

Chapter 3 Results

Table 3.39 Antipyretic activity of the plant D. mucronata leaves, bark and root

extracts

Samples Dose administered Mean ± SEM values

Normal saline 10 ml/kg 38.7 + 0

Standard (Paracetamol) 50 mg/kg 36.93 ± 0.204

100 mg/kg 37.29 + 0.063 Leaves 200 mg/kg 37.25 + 0.113

100 mg/kg 37.12 + 0.038 Bark

crude methanolic methanolic crude 200 mg/kg 37.25 + 0.141 extracts 100 mg/kg 37.22 + 0.04 Root

200 mg/kg 37.22 + 0.026 D. mucronata D.

Table 3.40 Antipyretic activity of the plant M. buxifolia leaves, seed and fruit extracts

Samples Dose administered Mean ± SEM values

Normal saline 10 ml/kg 38.7 + 0

Standard (Paracetamol) 50 mg/kg 36.93 ± 0.204

100 mg/kg 37.37 ± 0.22 Leaves 200 mg/kg 37.34 ± 0.16

100 mg/kg 37.29 ± 0.143 Seed

crude methanolic methanolic crude 200 mg/kg 37.237 ± 0.148 extracts 100 mg/kg 37.21 ± 0.116 Fruit

200 mg/kg 37.31 ± 0.084 M. buxifolia M.buxifolia

217

Chapter 3 Results

Table 3.41 Overall effects of 20% Brewer’s yeast induced on body temperature at

various dose of the plant extracts

20% Temp. after Average temp. Dose Normal Brewer’s Brewer’s Dose recorded (°C)

Treatment administered body yeast yeast administered

After After After (mg/kg) temp.(°C) induced induction (mg/kg)

Sample 1 h 2 h 3 h (ml/kg) (°C)

Paracetamol 50 37.12 38.48 50 37.24 37.00 36.54

100 37.90 38.50 100 37.17 37.38 37.32 Leaves 200 37.38 38.43 200 37.48 37.15 37.12

100 36.48 10 37.23 100 37.09 37.08 37.20 Bark

200 36.57 37.45 200 37.01 37.24 37.50

crude methanolic extract methanolic crude

100 36.54 38.42 100 37.15 37.29 37.23 Root

D. mucronata D. 200 36.92 37.67 200 37.17 37.23 37.26

218

Chapter 3 Results

Table 3.42 Overall effects of 20% Brewer’s yeast induced on body temperature at

various dose of the plant extracts

20% Temp. after Average temp. Dose Normal Brewer’s Brewer’s Dose recorded (°C)

Treatment administered body yeast yeast administered

After After After (mg/kg) temp.(°C) induced induction (mg/kg)

Sample 1 h 2 h 3 h (ml/kg) (°C)

Paracetamol 50 37.12 38.48 50 37.24 37.00 36.54

100 36.97 37.31 100 37.74 37.37 36.98

Leaves

200 37.31 38.21 200 37.62 37.31 37.07

100 35.83 10 36.89 100 37.56 37.27 37.06

Seed methanolic extracts methanolic

200 36.21 37.98 200 37.43 37.34 36.94

100 36.64 37.42 100 37.4 37.23 36.99

M.buxifolia Fruit 200 36.23 38.01 200 37.43 37.37 37.14

219

Chapter 3 Results

N /S 4 0

S t d ( P a r a c e ta m o l)

)

C 

( 1 0 0 m g / k g

* *

3 8 *

* *

t *

* 2 0 0 m g / k g

e t

3 6

e R

3 4

r s s h r r 1 h h 2 3

T r e a tm e n t

Figure 3.64 Anti-pyretic activity of D. mucronataleaves methanolic extract

4 0 )

C N /S

 (

S td ( P a r a c e ta m o l)

* *

* * *

3 8 *

u *

* 1 0 0 m g / k g

* *

* r

* 2 0 0 m g / k g

e t

3 6

e R

3 4

r s s h r r 1 h h 2 3

T r e a tm e n t

Figure 3.65 Anti-pyretic activity of D. mucronata bark methanolic extract

220

Chapter 3 Results

4 0

)

C 

( N /S

* *

* S td ( P a r a c e ta m o l)

* *

u *

3 8 *

* *

* a

* 1 0 0 m g / k g

* p

* 2 0 0 m g / k g

e t

3 6

e R

3 4

r s s h r r 1 h h 2 3

T r e a tm e n t

Figure 3.66 Anti-pyretic activity of D. mucronata root methanolic extract

4 0

N /S

) C

 S td ( P a r a c e ta m o l)

(

* *

r 1 0 0 m g / k g

* *

3 8 *

* *

* a

* 2 0 0 m g / k g

e t

3 6

e R

3 4

r s s h r r 1 h h 2 3

T r e a tm e n t

Figure 3.67 Anti-pyretic activity of M. buxifolialeaves methanolic extract

221

Chapter 3 Results

4 0

N /S

) C

 S td ( P a r a c e ta m o l)

(

* *

* 1 0 0 m g / k g

* *

3 8 *

* a

* 2 0 0 m g / k g

e t

3 6

e R

3 4

r s s h r r 1 h h 2 3

T r e a tm e n t

Figure 3.68 Anti-pyretic activity of M. buxifolia seed methanolic extract

4 0

N /S

) C

 S td ( P a r a c e ta m o l)

(

* * *

* *

* 1 0 0 m g / k g

* *

3 8 *

* a

* 2 0 0 m g / k g

e t

3 6

e R

3 4

r s s h r r 1 h h 2 3

T r e a tm e n t

Figure 3.69 Anti-pyretic activity of M. buxifolia fruit methanolic extract

222

Chapter 3 Results

D. mucronataleaves derived AgNPs, M. buxifolialeaves derived AgNPs and

AuNPs were detected to haveconsiderable anti-pyretic propertiesas compared to negative control (Normal saline) after intervals of 1, 2 and 3 hrs (Figure 3.70, Figure

3.72 and Figure 3.71). Values were revealed as Mean ± SEM and P<0.05 was deliberatednoteworthy (Table 3.43). D. mucronataleaves derived AgNPs, M. buxifolialeaves derived AgNPs and AuNPs exhibitednoteworthy anti-pyretic potential at doses of 10 mg/kg and 20 mg/kg (***P<0.001) of body weight.

D. mucronataleaves derived AgNPs exhibitedwell antipyretic activity in comparison to M. buxifolialeaves derived AgNPs and AuNPsat both doses tested. The

D. mucronataleaves derived AgNPs and M. buxifolialeaves derived AgNPs and

AuNPs showed dose dependent reduction in temperature at dose 10 mg/ kg. D. mucronataleaves derived AgNPs (37.14 + 0.006) >M. buxifolialeaves derived

AgNPs(37.23 + 0.0903) >M. buxifolialeaves derived AuNPs (37.56 + 0.22). While at

20 mg/kg reduction in temperature was observed as: D. mucronataleaves derived

AgNPs (37.15 + 0.084) >M. buxifolialeaves derived AgNPs(37.25 + 0.126) >M. buxifolialeaves derived AuNPs (37.27 + 0.109).

223

Chapter 3 Results

Table 3.43 Anti-pyretic activity of D. mucronata and M. buxifolialeaves derived

AgNPs and AuNPs

Samples Dose administered Mean ± SEM values

10 mg/kg 37.14 ± 0.006 D. mucronataleaves derived AgNPs 20 mg/kg 37.15 ± 0.084

10 mg/kg 37.56 ± 0.22 M. buxifolialeaves derived AuNPs 20 mg/kg 37.27 ± 0.109

10 mg/kg 37.23 ± 0.0903 M. buxifolialeaves derived AgNPs 20 mg/kg 37.25 ± 0.126

Table 3.44 Overall effects of 20% Brewer’s yeast induced on body temperature at

various dose of the biogenic nanoparticles

Temp. Average temp. 20% after recorded (°C) Dose Normal Brewer’s Dose Brewer’s Sample Treatment administered body yeast administered yeast After After After (mg/kg) temp.(°C) induced (mg/kg) induction 1 h 2 h 3 h (ml/kg) (°C)

Leaves 10 36.27 37.32 10 37.14 37.15 37.13

derived D. D.

AgNPs 20 36.45 37.57 20 37.18 37.28 36.99 mucronata

10 36.76 37.49 10 37.40 37.19 37.09 Leaves derived 10 20 36.35 38.04 20 37.50 37.16 37.09 AgNPs

10 36.05 37.68 10 37.36 37.33 38.01 Leaves M.buxifolia derived 20 36.42 37.87 20 37.46 37.27 37.08 AuNPs

224

Chapter 3 Results

4 0

) N /S

 (

S td ( P a r a c e ta m o l)

* *

* * *

u 3 8 *

* 1 0 m g / k g

* *

* e

* 2 0 m g / k g

e t

3 6

e R

3 4

r s s h r r 1 h h 2 3

T r e a tm e n t

Figure 3.70 Anti-pyretic activity of D. mucronataleaves derived AgNPs

4 0 N /S

* *

)

* *

* S t d ( P a r a c e ta m o l)

* *



(

* *

* 1 0 m g / k g

* *

* * *

3 8 *

* *

a 2 0 m g / k g

e t

3 6

e R

3 4

r s s h r r 1 h h 2 3

T r e a tm e n t

Figure 3.71 Anti-pyretic activity of M. buxifolialeaves derived AuNPs

225

Chapter 3 Results

4 0

) N /S



(

* S td ( P a r a c e ta m o l)

* *

* * *

u 3 8

* *

* 1 0 m g / k g

* *

* * *

a r

e 2 0 m g / k g

e t

3 6

e R

3 4

r s s h r r 1 h h 2 3

T r e a tm e n t

Figure 3.72 Anti-pyretic activity of M. buxifolialeaves derived AgNPs

226

Chapter 3 Results

3.7.4 GIT motility test

Crude methanolic extracts of D. mucronata leaves, bark, and root were tested for their influence on GIT motility. GIT motilityreduction was detected in a dose dependent mode, and values were expressed as Mean + SEM, as given in Table 3.45.

Outcomes were noteworthy with (P<0.001) as revealed in Figure 3.74, Figure 3.75 andFigure 3.76. GIT motility inpercentdetectedfor group tested with D. mucronata leaves, bark, and root was 46.09%, 44.32% and 46.77% at 100 mg/kg dose respectively. Similar decrease in GIT motility was detected at 200 mg/kg dose ofD. mucronata leaves, bark, and root (38.58%, 33.04%, and 39.73% respectively). At 300 mg/kg dose administration ofD. mucronata leaves, bark, roots 30.13%, 27.81% and

25.65% GIT motility was observed respectively. Percent GIT motility of test group tested by normal saline was 54.62%. It was clear from outcomes that crude methanolic extracts of different parts of D. mucronata reduce GIT motility in dose dependent manner.

Results of M. buxifolia leaves, seed and fruit crude methanolic extracts were highly significant with (P<0.001) (Figure 3.77, Figure 3.78 and Figure 3.79). Percent

GIT motility detected in test group treated with M. buxifolia leaves, seed and fruit crude methanolic extracts were 48.44%, 45.21% and 46.52% at 100 mg/kg dose correspondingly. The same decrease in GIT motility was detected at 200 mg/kg dose of M. buxifolia leaves, seed and fruit (37.52%, 37.15% and 36.20%). At 300 mg/kg dose administration of M. buxifolia leaves, seed and fruit crude methanolic extract was recorded 26.86%, 27.25% and 27.31% correspondingly. Results showed that crude methanolic extracts of different parts of M. buxifolia reduce GIT motility in dose dependent manner.

227

Chapter 3 Results

Table 3.45 GIT motility assay for D. mucronata bark, root, leaves and M. buxifolia

seed, fruit, leaves (SEM values are in the table as “±”)

Mean total Dose mg or Mean charcoal % GIT

Treatment length of ml/kg movement (cm) Motility

Intestine (cm) Sample Normal Saline 10 54.63 ± 1.09 29.84 ± 1.05 54.62

100 55.06 ±1.19 25.38 ± 1.01 46.09

Leaves 200 53.65 ± 1.34 20.70 ± 1.04 38.58

300 52.97 ± 1.51 15.96 ± 1.15 30.13

100 54.48 ±1.15 24.15 ± 1.07 44.32

Bark 200 54.29 ± 1.15 17.94 ± 1.17 33.04

methanolic extracts

300 53.46 ± 1.44 14.87 ± 1.08 27.81

100 54.59 ± 1.25 25.53 ± 1.11 46.77

D. mucronata Root 200 54.60 ± 1.20 21.69 ± 1.09 39.73

300 55.51 ± 1.25 14.24 ± 1.07 25.65

100 52.64 ± 1.52 25.5 ± 1.15 48.44

Leaves 200 54.74 ± 1.61 20.54 ± 1.19 37.52

300 55.80 ± 1.27 14.99 ± 1.27 26.86

100 55.31± 1.16 25.01 ± 1.17 45.21

Seed 200 54.02 ± 1.41 20.07 ± 1.35 37.15 methanolic extract 300 54.92 ± 1.28 14.97 ± 1.22 27.25

100 54.88 ± 1.22 25.53 ± 1.21 46.52

M. M. buxifolia Fruit 200 54.26 ± 1.35 19.64 ± 1.29 36.20

300 54.49 ± 1.11 14.88 ± 1.43 27.31

228

Chapter 3 Results

Percent decreased in GIT motility for D. mucronataleaves derived AgNPs and

M. buxifolialeaves derived AgNPs and AuNPs were detected in a dose dependent mode, and values were expressed as Mean + SEM, results were given in Table 3.46.

Results of D. mucronataleaves derived AgNPs, M. buxifolialeaves derived AgNPs and M. buxifolialeaves derived AuNPs were highly significant with (P<0.001) (Figure

3.80, Figure 3.81 and Figure 3.82). Percent GIT motility observed at 10 mg/kg with

D. mucronataleaves derived AgNPs was 48.71%. Same decreased in GIT motility was observed in leaves derived AgNPs (37.75) at 20 mg/kg. At 30 mg/kg dose administration of D. mucronataleaves derived AgNPs was recorded 26.81% GIT motility.

Percent GIT motility observed in M. buxifolialeaves derived AgNPs and

AuNPs was 43.50% and 49.10% at 10 mg/kg dose correspondingly. SimilarGIT motilityreduction was detected at 20 mg/kg dose (37.88 and 34.35%) and at 30 mg/kg dose (27.45% and 26.57%). Maximum reduction in GIT motility effect was observed in M. buxifolia leaves derived AuNPs at a concentration of 30 mg/kg.

D. mucronataleaves derived AgNPs had shown higher percent GIT motility than D. mucronataleaves methanolic extract. As for M. buxifolia, M. buxifolialeaves derived AgNPs had lower percent GIT motility whereas M. buxifolia leaves derived

AuNPs had similar percent GIT motility as that of M. buxifolia leaves crude methanolic extract (Figure 3.73).

229

Chapter 3 Results

Table 3.46 GIT motility assay for D. mucronataleaves derived AgNPs and M.

buxifolialeaves derived AuNPs and AgNPs

Dose mg or Mean total length Mean charcoal Treatment % GIT motility ml/kg of intestine (cm) movement (cm)

D. 10 53.86 ±1.25 26.24 ± 1.21 48.71

mucronataleaves 20 54.37 ± 1.37 20.53 ± 1.00 37.75

derived AgNPs 30 53.47 ± 1.38 14.34 ± 1.10 26.81

M. buxifolia 10 55.50 ± 1.35 24.14 ± 1.79 43.50

leaves derived 20 54.70 ± 1.34 20.72 ± 1.32 37.88

AgNPs 30 53.98 ± 1.43 14.82 ± 1.45 27.45

M. buxifolia 10 53.52 ± 1.24 26.28 ± 1.35 49.10

leaves derived 20 55.26 ± 1.12 18.98 ± 1.43 34.35

AuNPs 30 55.10 ± 1.17 14.64 ± 1.30 26.57

230

Chapter 3 Results

20 % Motility GIT % 10

0 100 200 300 10 20 30 Concentration (mg/kg)

D. mucronata leaves crude methanolic extract M. buxifolia leaves crude methanolic extract D. mucronata leaves derived AgNPs M. buxifolia leaves derived AgNPs M. buxifolia leaves derived AuNPs

Figure 3.73 GIT motility assay comparison of plants crude extracts and derived NPs

6 0 N /S

* D . m u c r o n a ta b a r k c r u d e e x t .

i l

4 0 *

I G

2 0 %

0 0 0 0 1 0 0 0 1 2 3

T r e a t m e n t (m g /k g )

Figure 3.74 GIT Motility of D. mucronata bark crude methanolic extract

231

Chapter 3 Results

6 0

* N /S

* *

* D . m u c r o n a ta le a v e s c r u d e e x t .

i *

l 4 0

I G

2 0 %

0 0 0 0 1 0 0 0 1 2 3

T r e a t m e n t (m g /k g )

Figure 3.75 GIT Motility of D. mucronataleaves crude methanolic extract

6 0

N /S

* *

* D . m u c r o n a ta r o o t s c r u d e e x t .

i l

i 4 0

I G

2 0 %

0 0 0 0 1 0 0 0 1 2 3

T r e a t m e n t (m g /k g )

Figure 3.76 GIT Motility of D. mucronata root crude methanolic extract

232

Chapter 3 Results

6 0

* t

i N /S *

l 4 0

t o

* M . b u x ifo lia f r u it s c r u d e e x t .

I G

2 0 %

0 0 0 0 1 0 0 0 1 2 3

T r e a t m e n t (m g /k g )

Figure 3.77 GIT Motility of M. buxifolia fruit crude methanolic extract

6 0

N /S

* *

M . b u x ifo lia le a v e s c r u d e e x t .

i *

l 4 0

I G

2 0 %

0 0 0 0 1 0 0 0 1 2 3

T r e a t m e n t (m g /k g )

Figure 3.78 GIT Motility of M. buxifolialeaves crude methanolic extract

233

Chapter 3 Results

6 0

N /S

* *

* M . b u x ifo lia s e e d s c r u d e e x t .

l *

i 4 0

I G

2 0 %

0 0 0 0 1 0 0 0 1 2 3

T r e a t m e n t (m g /k g )

Figure 3.79 GIT Motility of M. buxifolia seed crude methanolic extract

6 0

* N /S

* *

D . m u c r o n a ta

* t

* le a v e s d e r iv e d

i l

4 0 * i

t A g N P s

I G

2 0 %

0 0 0 0 1 1 2 3

T r e a t m e n t (m g /k g )

Figure 3.80 GIT Motility of D. mucronataleaves derived AgNPs

234

Chapter 3 Results

6 0 *

* N /S

t i

* M . b u x ifo lia f r u it

l 4 0 i

t d e r iv e d A g N P 'S

I G

2 0 %

0 0 0 0 1 1 2 3

T r e a t m e n t (m g /k g )

Figure 3.81 GIT Motility of M. buxifolialeaves derived AgNPs

6 0

* *

N /S

i *

l 4 0 M . b u x ifo lia f r u it d e r iv e d

t *

o A u N P 's

I G

2 0 %

0 0 0 0 1 1 2 3

T r e a t m e n t (m g /k g )

Figure 3.82 GIT Motility of M. buxifolialeaves derived AuNPs

235

Chapter 3 Results

3.7.5 Anti-inflammatory activity

As depicted in Table 3.47 and Table 3.48, crude methanolic extracts of both,

D. mucronata (bark, leaves and root) and M. buxifolia (seed, leaves and fruit) were detected to haveconsiderable anti-inflammatory potential in contrast to negative control (Normal saline) group after 1, 2 and 3 hrs intervals. Findings were presented as Mean ± SEM. Crude methanolic extracts of D. mucronata bark, leaves and root presentedconsiderable anti-inflammatory result at 100 mg/kg (***P<0.001) and 200 mg/kg (***P<0.001) as detected by decrease in edema (Figure 3.84, Figure 3.85 and

Figure 3.86). Similarly, crude methanolic extracts of M. buxifolia seed, leaves and fruit were also established to be greatlynoteworthycontrary to inflammation at a concentration of 100 mg/kg (***P<0.001) and 200 mg/kg (***P<0.001), correspondingly (Figure 3.87, Figure 3.88 and Figure 3.89). Standard drug

(phenidone) showed considerable anti-inflammatory activity (88.32 + 0.193) at a dose of 2 mg/ear in comparison to normal saline. Though, the verified samples revealed outcomesin contrast to standard drug. At a dose of 100 mg/kg, the methanolic extract of D. mucronata root (110.94 ± 1.33) showed better results. Among crude methanolic extract the D. mucronataleaves (111.5 + 0.729) showed good activity as compared to

M. buxifolialeaves (121.67 + 0.44), D. mucronata bark (126.2 + 2.93), M. buxifolia seed (135.61 + 1.28) and M. buxifolia fruit (137.97 ± 2.6). At a concentration of 200 mg/kg, anti-inflammatory potential wasdetected in the subsequentdirection: D. mucronata root (101.72 ± 0.907) >D. mucronataleaves (103.67 + 0.528) >D. mucronata bark (116.96 + 2.85) >M. buxifolia seed (122.13 + 3.26) >M. buxifolialeaves (126.48 + 1.4) >M. buxifolia fruit (130.13 ± 3.37).

236

Chapter 3 Results

Table 3.47 Anti-inflammatory effects of D. mucronata leaves, bark and root

Samples Mean + SEM values Dose administered % Inhibition (µm)

Normal saline 10 ml/kg 249.16 + 0.507 --

Standard (Phenidone) 2 mg/ear 88.32 + 0.193 64.36

100 mg/kg 111.5 + 0.729 55.24 Leaves 200 mg/kg 103.67 + 0.528 58.39

100 mg/kg 126.2 + 2.93 49.34

Bark crude methanolic crude

200 mg/kg 116.96 + 2.85 53.05 extracts

100 mg/kg 110.94 ± 1.33 55.47 Root

200 mg/kg 101.72 ± 0.907 59.17 D. mucronata D.

Table 3.48 Anti-inflammatory effects of M. buxifolia leaves, seed and fruit

Samples Dose administered Mean(µm) + SEM values % Inhibition

Normal saline 10 ml/kg 249.16 + 0.507 --

Standard (Phenidone) 2 mg/ear 88.32 + 0.193 64.36

100 mg/kg 121.67 + 0.44 51.16 Leaves 200 mg/kg 126.48 + 1.4 49.23

100 mg/kg 135.61 + 1.28 45.57 Seed

crude methanolic methanolic crude 200 mg/kg 122.13 + 3.26 50.98 extracts

100 mg/kg 137.97 ± 2.6 44.62 Fruit M. buxifolia M.buxifolia 200 mg/kg 130.13 ± 3.37 47.77

237

Chapter 3 Results

As depicted in Table 3.49, D. mucronataleaves derived AgNPs, M. buxifolialeaves derived AgNPs and M. buxifolialeaves derived AuNPs were detected to have significant anti-inflammatory potentialby comparing to negative control

(Normal saline) group afterinterval of 1, 2 and 3 hrs. D. mucronataleaves derived

AgNPs, M. buxifolialeaves derived AgNPs and M. buxifolialeaves derived AuNPs revealed greatly considerableoutcomes at 10 mg/ kg (***P<0.001) and 20 mg/ kg

(***P<0.001) representingeffectiveness of these AgNPs and AuNPs compared to inflammation (Figure 3.90, Figure 3.91 and Figure 3.92).

Among the AgNPs synthesized, M. buxifolialeaves derived AgNPs (118.64 +

2.309) revealed better anti-inflammatory potential as related to D. mucronataleaves derived AgNPs (122.27 ± 1.13) at the test dose of 10 mg/kg. Comparable anti- inflammatory consequence was detected at test dose of 20 mg/kg where M. buxifolialeaves derived AgNPs(111.24 + 2.067) presented better inhibition of inflammation as related to D. mucronataleaves derived AgNPs (111.81 ± 1.44). M. buxifolialeaves derived AuNPs (118.54+ 2.74) showed good anti-inflammatory properties at the test dose of 10 mg/kg. Likewise at a test dose of 20 mg/kg M. buxifolialeaves derived AuNPs (110.24+ 1.24) showed better inhibition of inflammation.

238

Chapter 3 Results

Table 3.49 Anti-inflammatory effects of D. mucronata and M. buxifolia leaves

derived NPs

Samples Dose administered Mean + SEM values (µm) % Inhibition

D. mucronataleaves 10 mg/kg 122.27 ± 1.13 50.92

derived AgNPs 20 mg/kg 111.81 ± 1.44 55.12

M. buxifolialeaves derived 10 mg/kg 118.64 + 2.309 52.38

AgNPs 20 mg/kg 111.24 + 2.067 55.35

118.54+ 2.74 52.42 10 mg/kg M. buxifolialeaves derived

AuNPs 110.24+ 1.24 55.75 20 mg/kg

239

Chapter 3 Results

60 58 56 54 52 50 % % inhibition 48 46 44 D. mucronata M. buxifolia D. mucronata M. buxifolia M. buxifolia leaves crude leaves crude leaves derived leaves derived leaves derived methanolic methanolic AgNPs AgNPs AuNPs extract extract

100 200 10 20

Figure 3.83 Anti-inflammatory effects of D. mucronata and M. buxifolia leaves and

their derived NPs

3 0 0

) N /S m

µ S td (P h e n id o n )

(

s 1 0 0 m g /k g

a 2 0 0

e r

c 2 0 0 m g /k g

* *

* * *

k 1 0 0

a E 0

r s s h r r 1 h h 2 3

T r e a tm e n t

Figure 3.84 Anti-inflammatory activity of D. mucronataleaves methanolic extract

240

Chapter 3 Results

) 3 0 0 m

µ N /S

(

s S td (P h e n id o n ) a

e 1 0 0 m g /k g

r 2 0 0

c 2 0 0 m g /k g

* * *

* *

1 0 0 *

a E 0

r s s h r r 1 h h 2 3

T r e a tm e n t

Figure 3.85 Anti-inflammatory activity of D. mucronata bark methanolic extract

) 3 0 0

µ (

N /S

e s

a S td (P h e n id o n ) e

r 2 0 0 1 0 0 m g /k g c

n 2 0 0 m g /k g

* *

1 0 0 *

a E 0

r s s h r r 1 h h 2 3

T r e a tm e n t

Figure 3.86 Anti-inflammatory activity of D. mucronata root methanolic extract

241

Chapter 3 Results

3 0 0 )

N /S

µ (

S td (P h e n id o n )

e s

a 1 0 0 m g /k g

e 2 0 0

* *

* 2 0 0 m g /k g

* *

1 0 0 *

r a

E 0

r s s h r r 1 h h 2 3

T r e a tm e n t

Figure 3.87 Anti-inflammatory activity of M. buxifolia seed methanolic extract

) 3 0 0

m N /S

µ (

S td (P h e n id o n )

s a

e 1 0 0 m g /k g

r 2 0 0 c

n 2 0 0 m g /k g

* *

1 0 0 *

a E 0

r s s h r r 1 h h 2 3

T r e a tm e n t

Figure 3.88 Anti-inflammatory activity of M. buxifolialeaves methanolic extract

242

Chapter 3 Results

3 0 0

) N /S

µ (

S td (P h e n id o n )

e s

a 1 0 0 m g /k g

e 2 0 0

* *

* 2 0 0 m g /k g

* k

1 0 0 *

a E 0

r s s h r r 1 h h 2 3

T r e a tm e n t

Figure 3.89 Anti-inflammatory activity of M. buxifolia fruit methanolic extract

3 0 0

) N /S

m µ

( S td (P h e n id o n )

e s

a 1 0 m g /k g

e 2 0 0 r

c 2 0 m g /k g

* * *

* *

k 1 0 0

a E 0

r s s h r r 1 h h 2 3

T r e a tm e n t

Figure 3.90 Anti-inflammatory activity of D. mucronataleaves derived AgNPs

243

Chapter 3 Results

3 0 0 )

N /S

µ (

S td (P h e n id o n )

e s

a 1 0 m g /k g

e 2 0 0 r

c 2 0 m g /k g

* *

k *

1 0 0 *

a E 0

r s s h r r 1 h h 2 3

T r e a tm e n t

Figure 3.91 Anti-inflammatory activity of M. buxifolialeaves derived AgNPs

3 0 0 )

N /S

µ (

S td (P h e n id o n )

e s

a 1 0 m g /k g e

r 2 0 0 c

n 2 0 m g /k g

* *

1 0 0 *

a E 0

r s s h r r 1 h h 2 3

T r e a tm e n t

Figure 3.92 Anti-inflammatory activity of M. buxifolialeaves derived AuNPs

244

Chapter 3 Results

3.8 Biochemical parameters

Hematological responses of G. Pigs and Rabbit to 21 days of oral treatment with methanolic extracts of D. mucronataleaves and M. buxifolia leaves

Results showed that D. mucronataleaves and M. buxifolialeaves methanolic extracts hematological analysis (white blood cell, red blood cell, hemoglobin, mean corpuscularhemoglobin, mean corpuscular hemoglobin concentration and mean platelet volume) fell in range of control for male G. Pigs and Rabbits (Table

3.50andTable 3.51). Significant (p<0.01) increase in hematological parameters was observed in case of D. mucronataleaves and M. buxifolialeaves methanolic extracts.

In case of G. Pigs significant (p<0.01) increase in WBC was observed by D. mucronataleaves (15.57 ±0.67) and M. buxifolialeaves (12.17 ±0.04) methanolic extracts from control (10.5±0.20) 1×109/L at 300 mg/kg body weight.

Effect of methanolic extract of D. mucronata and M. buxifolia leaves on body weight in G. Pigs and Rabbits

Also with the effect of plants extracts the body weights were determined stable. A slight increase and decrease was observed in body weights of male G. Pigs and Rabbits in case of crude methanolic extracts of D. mucronataleaves (1056.16

±8.026 gm and 3504.83 ±3.9 gm) and M. buxifolialeaves (1065 ±11.31 gm and 3505.16

±4.49 gm) from control (1094.5±5.48 gm and 3516.16±4.34 gm) as shown in Table 3.52.

Liver and kidney function parameters of male G. Pigs and Rabbits:

Liver and kidney functions had been observed with variance, such that total bilirubin, conjugated bilirubin and triacylglycerol (D. mucronataleaves 0.47 ±0.025 245

Chapter 3 Results mg/dL, 0.1 ±0.009 µmol/L, 135.92 ±0.24 mg/dL and M. buxifolia leaves 0.46 ±0.015 mg/dL, 0.1 ±0.009 µmol/L and 135.89 ±0.23 mg/dL) had been determined to increase from control (0.39±0.02 mg/dL, 0.035±0.007 µmol/L, 132.73±0.38 mg/dL)in G. Pigs whereas the SGPT, phosphorous, calcium, urea and uric acid had decreased compare to the control group of G. Pigs (Table 3.53). In case of male Rabbits, with application of plant extracts in the conjugated bilirubin, calcium and triacylglycerol had been observed with slight increase (D. mucronataleaves 0.28 ±0.01 µmol/L, 9.53 ±0.12 mg/dL and 155.75 ±0.22 mg/dLand M. buxifolialeaves 0.28 ±0.02 µmol/L, 9.49 ±0.12 mg/dL and 155.61±0.23 mg/dL) from control (0.125±0.007 µmol/L, 9.16±0.04 mg/dL and 152.75±0.33 mg/dL) and other factors had resulted in slight decrease (Table

3.54).

Feed intake (gm) of male G. Pigs and Rabbits administered with methanolic extracts of D. mucronataand M. buxifolia leaves

Results of feed intake showed that at higher concentration (300 mg/kg) feed intake had been observed less (D. mucronataleaves 42.16±0.7 gm and 72.2±0.69 gm and M. buxifolialeaves 42.09±0.4 gm and 72.1±0.4 gm) comparative to control group

(43.8±0.42 gm and 73.94±0.39 gm) of animals (G. Pigs and Rabbits) (Table 3.55 and

Table 3.56). On the other hand, at lower concentration i.e.100 and 200 mg/kg, the animals had been observed with higher feed intake as compared to control group of animals.

246

Chapter 3 Results

Water intake (ml) of male G. Pigs and Rabbit administered with methanolic extracts of D. mucronataand M. buxifolia leaves

Water intake was higher with 100 and 200 mg/kg administration of D. mucronataleaves and M. buxifolialeaves methanolic extract, whereas at 300 mg/kg application the water intake was observed lower (D. mucronataleaves 126.49±2.1 ml and 216.62±2.09 ml and M. buxifolialeaves 126.29±1.2 ml and 216.3±1.22 ml) than that of control group (131.5±1.26 ml and 221.8±1.17 ml) of animals (G. Pigs and

Rabbits) (Table 3.57 and Table 3.58).

247

Chapter 3 Results

Table 3.50 Effect of administration of methanolic extract of D. mucronataand M.

buxifolia leaves on some hematological parameters of male G. Pigs, (n=6)

D. mucronataleaves M. buxifolia leaves Parameters Control (mg/kg body weight) (mg/kg body weight)

Conc. 100 200 300 100 200 300

15.14 13.15 15.57 12.11 12.2 12.17 White blood (1×109/L) 10.5±0.20 ±0.70 ±0.61 ±0.67 ±0.66 ±0.07 ±0.04

Red blood cell 5.30 5.18 5.2 5.13 4.7 5.43 4.98±0.16 (1×1012/L) ±0.19 ±0.22 ±0.15 ±0.10 ±0.08 ±0.18

12.23 12.4 12.48 12.3 12.2 12.36 Hemoglobin (g/dL) 11.88±0.19 ±0.08 ±0.14 ±0.15 ±0.14 ±0.15 ±0.14

Mean corpuscular 15.30 15.25 15.9 16.1 16.31 16.32 15.53±0.13 hemoglobin (pg) ±0.11 ±0.07 ±0.17 ±0.22 ±0.19 ±0.22

Mean corpuscular 34.51 34.81 34.48 34.78 35.78 34.81 hemoglobin 33.4±0.11 ±0.27 ±0.18 ±0.17 ±0.18 ±0.18 ±0.14 concentration (g/dL)

Mean platelet volume 14.11 13.97 14.14 14.15 13.96 15.01 13.4±0.15 (fL) ±0.11 ±0.10 ±0.08 ±0.05 ±0.08 ±0.35

248

Chapter 3 Results

Table 3.51 Effect of administration of methanolic extract of D. mucronataand M.

buxifolialeaves on some hematological parameters of male Rabbits, (n=6)

D. mucronataleaves M. buxifolia leaves Parameters Control (mg/kg body weight) (mg/kg body weight)

Conc. 100 200 300 100 200 300

9.43±0.08 9.85 9.58 9.62 9.59 9.66 9.83 White blood (1×109/L) ±0.19 ±0.05 ±0.09 ±0.07 ±0.04 ±0.12

Red blood cell 5.1 5.2 5.13 5.23 5.08 5.24 4.57±0.04 (1×1012/L) ±0.06 ±0.12 ±0.15 ±0.10 ±0.19 ±0.12

14.16 13.96 13.96 14.00 14.13 14.16 Hemoglobin (g/dL) 13.4±0.06 ±0.16 ±0.22 ±0.13 ±0.22 ±0.07 ±0.09

Mean corpuscular 19.41 19.42 19.47 19.42 19.40 19.4± 18.94±0.19 hemoglobin (pg) ±0.08 ±0.05 ±0.06 ±0.09 ±0.07 0.03

Mean corpuscular 35.16 35.06 34.83 35.01 35.05 35.02 hemoglobin 34.51±0.11 ±0.10 ±0.18 ±0.10 ±0.10 ±0.21 ±0.19 concentration (g/dL)

Mean platelet volume 14.25 14.22 14.32 14.18 14.19 14.21 13.735±0.07 (fL) ±0.08 ±0.08 ±0.06 ±0.01 ±0.08 ±0.15

249

Chapter 3 Results

Table 3.52 Effect of administration of methanolic extracts of D. mucronataand M.

buxifolialeaves on the weight of male G. Pigs and Rabbits, (n=6)

D. mucronataleaves (mg/kg M. buxifolia leaves (mg/kg body Parameters body weight) weight) Control Dose 100 200 300 100 200 300

Animals treated Animals administered

Initial body 1054 1055.67 1055 1056.5 1057 1063.83 1087.5±11.08

weight (g) ±10.89 ±7.21 ±7.91 ±10.01 ±14.63 ±11.27

Final body 1056 1057.67 1056.16 1058.33 1057.5 1065 G. Pigs G. 1094.5±5.48 weight (g) ±11.26 ±7.35 ±8.026 ±9.88 ±14.17 ±11.31

Initial body 3513.33 3511 3508.5 3512.67 3509.67 3508.16 3516.5±4.18

weight (g) ±4.24 ±3.83 ±3.79 ±4.57 ±4.22 ±4.44

Final body 3511.83 3508.5 3504.83 3510.16 3507.33 3505.16 Rabbits 3516.16±4.34 weight (g) ±4.28 ±4.12 ±3.9 ±4.84 ±4.43 ±4.49

250

Chapter 3 Results

Table 3.53 Liver and kidney function parameters of male G. Pigs administered with

methanolic extracts of D. mucronataand M. buxifolia leaves, (n=6)

D. mucronataleaves (mg/kg body M. buxifolia leaves (mg/kg Parameters Control weight) body weight)

Dose administered 100 200 300 100 200 300

0.45 0.47±0.0 0.47 0.46 0.48 0.46 Total bilirubin (mg/dL) 0.39±0.02 ±0.023 17 ±0.025 ±0.02 ±0.018 ±0.015

Conjugated bilirubin 0.083 0.1 0.1 0.11 0.1 0.035±0.007 0.1 ±0.01 (µmol/L) ±0.006 ±0.009 ±0.01 ±0.009 ±0.009

Serum glutamic pyruvic 42.11 42.16 42.24 42.43 42.15 42.26 44.07±0.46 transaminase (U/L) ±0.33 ±0.32 ±0.31 ±0.21 ±0.2 ±0.31

4.58 4.57 4.53 4.6 4.5 4.56 Phosphorous (mg/dL) 5.32±0.14 ±0.07 ±0.14 ±0.18 ±0.13 ±0.13 ±0.16

7.47 7.31 7.19 7.32 7.35 7.2 Calcium (mg/dL) 7.74±0.18 ±0.08 ±0.21 ±0.19 ±0.13 ±0.039 ±0.206

19.37 19.35 19.54 19.19 19.58 19.65 Urea (mg/dL) 20.13±0.19 ±0.09 ±0.09 ±0.08 ±0.23 ±0.26 ±0.08

3.59 3.46 3.6 3.58 3.4 Uric acid (mg/dL) 4.3±0.27 3.4 ±0.06 ±0.22 ±0.05 ±0.08 ±0.19 ±0.08

135.77 135.89 135.92 136.02 135.89 135.89 Triacylglycerol (mg/dL) 132.73±0.38 ±0.27 ±0.28 ±0.24 ±0.22 ±0.26 ±0.23

251

Chapter 3 Results

Table 3.54 Liver and kidney function parameters of male Rabbits administered with

methanolic extracts of D. mucronataleaves and M. buxifolia leaves, (n=6)

D. mucronataleaves (mg/kg M. buxifolialeaves (mg/kg Parameters Control body weight) body weight)

Dose administered 100 200 300 100 200 300

0.68 0.681 0.69 0.69 0.67 0.68 Total bilirubin (mg/dL) 0.73±0.006 ±0.018 ±0.021 ±0.014 ±0.014 ±0.015 ±0.015

Conjugated bilirubin 0.23 0.24 0.28 0.27 0.28 0.28 0.125±0.007 (µmol/L) ±0.02 ±0.01 ±0.01 ±0.02 ±0.01 ±0.02

Serum Glutamic Pyruvic 56.36 56.39 56.33 55.73 55.06 55.24 57.82±0.53 transaminase (U/L) ±0.35 ±0.39 ±0.32 ±0.27 ±0.52 ±0.34

5.72 5.63 5.23 5.38 5.4 5.35 Phosphorous (mg/dL) 6.06±0.14 ±0.09 ±0.102 ±0.06 ±0.09 ±0.06 ±0.08

9.64 9.48 9.53 9.56 9.44 9.49 Calcium (mg/dL) 9.16±0.04 ±0.09 ±0.06 ±0.12 ±0.12 ±0.059 ±0.12

16.92 16.58 16.86 17.04 16.6 16.68 Urea (mg/dL) 18.85±0.14 ±0.25 ±0.18 ±0.15 ±0.15 ±0.12 ±0.209

4.19 4.17 4.17 4.16 4.15 4.14 Uric acid (mg/dL) 4.28±0.007 ±0.009 ±0.009 ±0.007 ±0.012 ±0.013 ±0.013

155.79 156.01 155.75 155.76 155.6 155.61± Triacylglycerol (mg/dL) 152.75±0.33 ±0.19 ±0.22 ±0.22 ±0.18 ±0.52 0.23

252

Chapter 3 Results

Table 3.55 Feed intake (g) of male G. Pigs administered with methanolic extracts of

D. mucronataand M. buxifolia leaves, (n=6)

Animal grouping D. mucronata leaves M. buxifolia leaves

Days 1-11 12-21 1-11 12-21

Control 43.3±0.55 43.8±0.42 43.3±0.55 43.8±0.42

100mg/kg body weight 44.3±0.37 44.56±0.38 44.52±0.33 44.86±0.32

200mg/kg body weight 45.2±0.31 45.42±0.32 45.47±0.31 45.74±0.31

300mg/kg body weight 41.86±0.63 42.16±0.7 41.83±0.4 42.09±0.4

Table 3.56 Feed intake (g) of male Rabbits administered with methanolic extracts of

D. mucronataand M. buxifolia leaves, (n=6)

Animal grouping D. mucronataleaves M. buxifolia leaves

Days 1-11 12-21 1-11 12-21

Control 73.3±0.55 73.94±0.39 73.3±0.55 73.94±0.39

100mg/kg body weight 74.3±0.36 74.6±0.39 74.55±0.32 74.9±0.31

200mg/kg body weight 75.28±0.27 75.44±0.31 75.47±0.31 75.73±0.31

300mg/kg body weight 71.9±0.63 72.2±0.69 71.89±0.36 72.1±0.4

253

Chapter 3 Results

Table 3.57 Water intake (ml) of male G. Pigs administered with methanolic extracts

of D. mucronataleaves and M. buxifolia leaves, (n=6)

Animal grouping D. mucronata leaves M. buxifolia leaves

Days 1-11 12-21 1-11 12-21

Control 129.9±1.65 131.5±1.26 129.9±1.65 131.5±1.26

100mg/kg body weight 132.9±1.13 133.68±1.16 133.56±0.99 134.6±0.96

200mg/kg body weight 135.6±0.95 136.27±0.96 136.42±0.93 137.22±0.94

300mg/kg body weight 125.6±1.91 126.49±2.1 125.5±1.21 126.29±1.2

Table 3.58 Water intake (ml) of male Rabbits administered with methanolic extracts

of D. mucronataand M. buxifolia leaves, (n=6)

Animal grouping D. mucronata leaves M. buxifolia leaves

Days 1-11 12-21 1-11 12-21

Control 220.1±1.6 221.8±1.17 220.1±1.6 221.8±1.17

100mg/kg body weight 223.02±1.08 223.8±1.17 223.67±0.97 224.7±0.95

200mg/kg body weight 225.84±0.82 226.32±0.94 226.42±0.93 227.2±0.95

300mg/kg body weight 215.7±1.8 216.62±2.09 215.6±1.1 216.3±1.22

254

Chapter 3 Results

Hematological responses of male G. Pigs and Rabbit to 21 days of oral treatment with D. mucronataand M. buxifolia leaves mediated AgNPs and AuNPs

Biochemical analysis of both plants derived NPs had shown similar results as that of plant extracts alone. Significant increase (p<0.01) was observed in M. buxifolialeaves mediated AuNPs in hematological parameters. While significant

(p<0.01) decrease was observed for both plants mediated AgNPs in male G. Pigs and

Rabbits (Table 3.59 and Table 3.60. Significant (p<0.01) decrease was observed in

WBC by D. mucronataleaves mediated AgNPs 7.91 ±0.21 (1×109/L) and M. buxifolialeaves mediated AgNPs 7.99 ±0.30 (1×109/L) from control 10.5±0.20

(1×109/L) in case of male G. Pigs.

Effect of D. mucronata and M. buxifolia leaves mediated AgNPs and AuNPs on body weight inmale G. Pigsand Rabbit

Slight decrease was observed in weight of male G. Pigs as well as Rabbits with administration of D. mucronataleaves mediated AgNPs (1030.83±2.98 and

3325.33±17.09) and M. buxifolialeaves mediated AgNPs (1022.83±5.92 and

3327.16±16.72) and AuNPs (1051.67±10.51 and 3507.67±3.61) from control

(1094.5±5.48 and 3516.16±4.34). Effect of weight was presented inTable 3.61.

Liver and kidney function parameters of male G. Pigs and Rabbit

The level of total bilirubin, conjugated bilirubin and triacylglycerol were significantly (p<0.01) increased (D. mucronataleaves mediated AgNPs 0.44 ±0.027 mg/dL, 0.11 ±0.009 µmol/L and 135.98 ±0.193 mg/dL and M. buxifolialeaves mediated AgNPs 0.45 ±0.02 mg/dL, 0.098 ±0.009 µmol/L and 135.79 ±0.229 mg/dL

255

Chapter 3 Results and AuNPs 0.44 ±0.019 mg/dL, 0.1 ±0.007 µmol/L and 135.93±0.199 mg/dL) from control (0.39 ±0.02 mg/dL, 0.035 ±0.007 µmol/L and 132.73 ±0.38 mg/dL) in case of male G. Pigs. Liver and kidney function parameters were presented in Table 3.62 and

Table 3.63.

Feed intake (g) of male G. Pigs and Rabbits administered with D. mucronataand

M. buxifolialeaves mediated AgNPs and AuNPs

Significant decrease (p<0.01) was observed in feed intake of male G. Pigs and

Rabbits when administered with D. mucronataleaves mediated AgNPs (42.43±0.49 gm and 72.45±0.49 gm) and M. buxifolialeaves mediated AgNPs (41.74±0.25 gm and

71.95±0.35 gm) and AuNPs (41.9±0.45 gm and 71.95±0.41 gm) at concentration of

30 mg/kg from control (43.8±0.42 gm and 73.94±0.39 gm) as presented in Table 3.64 and Table 3.65.

Water intake (ml) of male G. Pigs and Rabbit administered with D. mucronataand M. buxifolialeaves mediated AgNPs and AuNPs

Same effect was observed for water as that of feed. Water intake was increased in comparison to control at concentration of 10 mg/kg and 20 mg/kg as presented inTable 3.66 and Table 3.67. Significant decrease (p<0.01) was observed in water intake of male G. Pigs and Rabbits when administered with D. mucronataleaves mediated AgNPs (127.31±1.47ml and 217.37±1.4ml) and M. buxifolialeaves mediated

AgNPs (125.24±0.77ml and 215.86±1.05ml) and AuNPs (125.7±1.36ml and

215.87±1.25ml) at concentration of 30 mg/kg from control (131.5±1.26 ml and

221.8±1.17 ml) . Thus these results indicated that synthesized NPs had no additional effects on animals, but rather these effects were dependent on plant extract. 256

Chapter 3 Results

Table 3.59 Effect of administration of D. mucronataand M. buxifolialeaves derived

AgNPs and AuNPs on some hematological parameters of male G. Pigs, (n=6)

D. mucronataleaves M. buxifolialeaves M. buxifolialeaves Parameters Control derived AgNPs derived AgNPs derived AuNPs

Conc. 10 20 30 10 20 30 10 20 30

White blood 8.008 7.84 7.91 7.88 7.7 7.99 10.99 11.1 11.99 10.5±0.20 (1×109/L) ±0.23 ±0.23 ±0.21 ±0.18 ±0.17 ±0.30 ±0.166 ±0.22 ±0.24

Red blood cell 4.78 4.33 4.29 4.33 4.25 4.195 5.44 5.25 5.15 4.98±0.16 (1×1012/L) ±0.08 ±0.11 ±0.06 ±0.10 ±0.05 ±0.03 ±0.087 ±0.08 ±0.09

Hemoglobin 11.16 11.35 11.31 11.39 11.44 11.36 12.205 12.19 12.28 11.88±0.19 (g/dL) ±0.09 ±0.11 ±0.10 ±0.07 ±0.06 ±0.07 ±0.106 ±0.09 ±0.09

Mean 14.46 14.47± 14.23 14.46 14.4±0. 14.29 16.3 16.11 16.16 corpuscular 15.53±0.13 ±0.10 0.055 ±0.06 ±0.07 046 ±0.05 ±0.18 ±0.11 ±0.09 hemoglobin (pg)

Mean

corpuscular 33.07 32.93± 32.85 33.03 32.99 33.1 35.82 36.08 35.59 33.4±0.115 hemoglobin ±0.03 0.107 ±0.12 ±0.14 ±0.22 ±0.16 ±0.17 ±0.15 ±0.09

conc.(g/dL)

Mean platelet 11.81 11.89 11.85 11.71 11.9 12.02 13.72 13.87 14.4± 13.4±0.15 volume (fL) ±0.39 ±0.38 ±0.29 ±0.28 ±0.22 ±0.24 ±0.19 ±0.10 0.15

257

Chapter 3 Results

Table 3.60 Effect of administration of D. mucronataleaves and M. buxifolia leaves

derived AgNPs and AuNPs on some hematological parameters of male Rabbits,

(n=6)

D. mucronataleaves M. buxifolia leaves M. buxifolia leaves Parameters Control derived AgNPs derived AgNPs derived AuNPs

Concentrations 10 20 30 10 20 30 10 20 30

9.43±0.08 White blood 9.016 8.72 8.85 9.106 8.745 8.89 9.24 9.29 9.575

(1×109/L) ±0.149 ±0.25 ±0.14 ±0.20 ±0.21 ±0.14 ±0.06 ±0.07 ±0.07

Red blood cell 4.26 4.39 4.38 4.331 4.32 4.42 5.23 5.24 5.068 4.57±0.05 (1×1012/L) ±0.041 ±0.07 ±0.05 ±0.05 ±0.09 ±0.04 ±0.07 ±0.12 ±0.21

12.41 12.48 12.39 12.35 12.32 12.37 14.00 14.19 14.30 Hemoglobin (g/dL) 13.4±0.06 ±0.153 ±0.09 ±0.09 ±0.08 ±0.76 ±0.08 ±0.11 ±0.15 ±0.22

Mean corpuscular 18.33 18.36 18.57 18.39 18.46 18.46 19.35 19.34 19.47 18.94±0.19 hemoglobin (pg) ±0.065 ±0.08 ±0.11 ±0.04 ±0.12 ±0.04 ±0.08 ±0.08 ±0.06

Mean corpuscular 34.3 34.29 34.22 34.2 34.19 34.23 34.97 35.11 35.88 hemoglobin 34.51±0.11 ±0.075 ±0.07 ±0.03 ±0.15 ±0.05 ±0.07 ±0.09 ±0.14 ±0.15 conc.(g/dL)

Mean platelet 13.52 13.47 13.49 13.33 13.35 14.48 14.43 14.43 14.33 13.73±0.07 volume (fL) ±0.092 ±0.05 ±0.06 ±0.05 ±0.09 ±0.08 ±0.00 ±0.12 ±0.11

258

Chapter 3 Results

Table 3.61 Effect of administration of D. mucronataand M. buxifolia leaves derived

AgNPs and AuNPs on the weight of mail G. Pigs and Rabbits, (n=6)

Animals treated G. Pigs Rabbits

Parameters Initial body Final body Initial body Final body

weight (g) weight (g) weight (g) weight (g) Dose Dose

Control 1087.5±11.08 1094.5±5.48 3516.5±4.18 3516.16±4.34 administered

D. 10 1052 ±10.93 1032.5 ±1.94 3512.67±3.63 3360.33±21.28 mucronata 20 1045.33 ±5.12 1029 ±2.16 3510 ±3.57 3341.16±18.43 derived 30 1044.16±10.00 1030.83±2.98 3509.16±3.29 3325.33±17.09

AgNPs

10 1053 ±9.67 1036.5±1.94 3516.67±3.91 3334.33±17.7 M. buxifolia derived 20 1043 ±5.57 1028.5±6.83 3513.83±3.45 3331.83±17.7

AgNPs 30 1047.5 ±10.12 1022.83±5.92 3510.33±3.63 3327.16±16.72 mg/kg body weight body mg/kg 10 1053.83 ±9.34 1056±8.92 3514.67±4.27 3512.16±4.24 M. buxifolia derived 20 1051±8.8 1061.83±8.89 3512.16±3.64 3509.67±3.71

AuNPs 30 1039.83±6.17 1051.67±10.51 3510.67±3.32 3507.67±3.61

259

Chapter 3 Results

Table 3.62 Liver and kidney function parameters of male G. Pigs administered with

D. mucronataand M. buxifolialeaves derived AgNPs and AuNPs, (n=6)

D. mucronataleaves M. buxifolialeaves M. buxifolialeaves derived

Parameters Control derived AgNPs (mg/kg derived AgNPs (mg/kg AuNPs (mg/kg body

body weight) body weight) weight)

Dose administered 10 20 30 10 20 30 10 20 30

Total bilirubin 0.39 0.428 0.46 0.44 0.47 0.45 0.45 0.48 0.47 0.44

(mg/dL) ±0.02 ±0.023 ±0.029 ±0.027 ±0.012 ±0.014 ±0.02 ±0.02 ±0.01 ±0.019

Conjugated 0.035 0.085 0.095 0.11 0.1 0.11 0.098 0.096 0.098 0.1 bilirubin (µmol/L) ±0.007 ±0.007 ±0.007 ±0.009 ±0.01 ±0.009 ±0.009 ±0.006 ±0.006 ±0.007

Serum Glutamic 44.07 42.33 42.3 41.95 42.37± 42.13 42.25 42.41 42.14 42.4±0. Pyruvic ±0.46 ±0.28 ±0.36 ±0.23 0.37 ±0.318 ±0.217 ±0.28 ±0.28 25 transaminase (U/L)

Phosphorous 5.32 4.66 4.48 4.67 4.74 4.74 4.6 4.58 4.36 4.66±0.

(mg/dL) ±0.14 ±0.16 ±0.04 ±0.15 ±0.15 ±0.159 ±0.07 ±0.07 ±0.38 14

7.74 7.27 7.12 7.02 6.98 7.26 6.97 7.05 6.78 6.98±0. Calcium (mg/dL) ±0.18 ±0.047 ±0.167 ±0.15 ±0.22 ±0.22 ±0.23 ±0.2 ±0.19 21

20.13 19.43 19.46 19.34 19 19.39 19.36 19.6 19.25 19.69± Urea (mg/dL) ±0.19 ±0.23 ±0.18 ±0.13 ±0.21 ±0.26 ±0.308 ±0.178 ±0.171 0.26

4.3 3.59 3.69 3.33 3.53 3.39 3.81 3.45 3.57 3.4±0.1 Uric acid (mg/dL) ±0.27 ±0.26 ±0.13 ±0.08 ±0.23 ±0.09 ±0.2 ±0.12 ±0.16 1

Triacylglycerol 132.73 135.76 135.83 135.98 136.1 135.73 135.79 136.05 135.91 135.93

(mg/dL) ±0.38 ±0.24 ±0.215 ±0.193 ±0.24 ±0.168 ±0.229 ±0.22 ±0.179 ±0.199

260

Chapter 3 Results

Table 3.63 Liver and kidney function parameters of male Rabbits administered with

D. Mucronate and M. buxifolialeaves derived AgNPs and AuNPs, (n=6)

D. mucronataleaves M. buxifolialeaves derived M. buxifolialeaves derived

Parameters Control derived AgNPs (mg/kg AgNPs (mg/kg body AuNPs (mg/kg body

body weight) weight) weight)

Dose administered 10 20 30 10 20 30 10 20 30

Total bilirubin 0.73 0.678 0.68 0.673 0.69 0.69 0.68 0.69 0.67 0.67

(mg/dL) ±0.006 ±0.017 ±0.016 ±0.017 ±0.017 ±0.02 ±0.017 ±0.02 ±0.012 ±0.018

Conjugated 0.125 0.27 0.28 0.28 0.3 0.275 0.278 0.28 0.25 0.27

bilirubin (µmol/L) ±0.007 ±0.019 ±0.027 ±0.02 ±0.02 ±0.019 ±0.024 ±0.02 ±0.02 ±0.02

Serum glutamic 57.82 55.83 54.3 55.45 55.2 55.63 55.08 54.82 55.84 55.07 pyruvic ±0.53 ±0.34 ±0.402 ±0.34 ±0.44 ±0.24 ±0.32 ±0.33 ±0.28 ±0.49 transaminase (U/L)

Phosphorous 6.06 5.27 5.33 5.43 5.64 5.61 5.57 5.53 5.69 5.7

(mg/dL) ±0.14 ±0.07 ±0.05 ±0.08 ±0.12 ±0.08 ±0.14 ±0.15 ±0.07 ±0.09

9.16 9.5 9.4 9.57 9.73 9.57 9.47 9.51 9.62 9.43 Calcium (mg/dL) ±0.04 ±0.14 ±0.102 ±0.102 ±0.06 ±0.078 ±0.08 ±0.08 ±0.08 ±0.08

18.85 16.57 16.6 16.57 16.6 16.68 16.6±0 16.67± 16.65 16.81 Urea (mg/dL) ±0.14 ±0.13 ±0.143 ±0.17 ±0.15 ±0.13 .146 0.116 ±0.16 ±0.214

4.28 4.185 4.17 4.17 4.16 4.14 4.1±0. 4.18±0. 4.16 4.14 Uric acid (mg/dL) ±0.007 ±0.011 ±0.011 ±0.011 ±0.01 ±0.011 022 007 ±0.007 ±0.008

Triacylglycerol 152.75 155.73 155.74 155.72 155.44 155.64 155,.63 155.57 155.51 155.52

(mg/dL) ±0.33 ±0.22 ±0.17 ±0.26 ±0.16 ±0.19 ±0.176 ±0.24 ±0.163 ±0.153

261

Chapter 3 Results

Table 3.64 Feed intake (g) of male G. Pigs administered with D. mucronataand M.

buxifolialeaves derived AgNPs and AuNPs, (n=6)

D. mucronataleaves M. buxifolialeaves derived M. buxifolialeaves derived Animal grouping derived AgNPs AgNPs AuNPs

Days 1-11 12-21 1-11 12-21 1-11 12-21

Control 43.3±0.55 43.8±0.42 43.3±0.55 43.8±0.42 43.3±0.55 43.8±0.42

10mg/kg body weight 44.5±0.61 44.9±0.61 44.5±0.34 44.88±0.34 44.38±0.3 44.62±0.33

20mg/kg body weight 45.3±0.45 45.52±0.43 45.1±0.27 45.47±0.34 44.9±0.26 45.27±0.26

30mg/kg body weight 42.06±0.44 42.43±0.49 41.4±0.26 41.74±0.25 41.45±0.43 41.9±0.45

Table 3.65 Feed intake (g) of male Rabbits administered with D. mucronataand M.

buxifolialeaves derived AgNPs and AuNPs, (n=6)

D. mucronataleaves M. buxifolialeaves M. buxifolialeaves Animal grouping derived AgNPs derived AgNPs derived AuNPs

Days 1-11 12-21 1-11 12-21 1-11 12-21

Control 73.3±0.55 73.94±0.39 73.3±0.55 73.94±0.39 73.3±0.55 73.94±0.39

10mg/kg body weight 74.62±0.61 74.91±0.6 74.63±0.32 74.88±0.34 74.44±0.3 74.66±0.3

20mg/kg body weight 75.36±0.42 75.32±0.36 75.2±0.22 75.48±0.33 74.9±0.23 75.25±0.25

30mg/kg body weight 72.12±0.4 72.45±0.49 71.57±0.24 71.95±0.35 71.69±0.36 71.95±0.41

262

Chapter 3 Results

Table 3.66 Water intake (ml) of male G. Pigs administered with D. mucronataand M.

buxifolialeaves derived AgNPs and AuNPs, (n=6)

D. mucronataleaves derived M. buxifolialeaves derived M. buxifolialeaves derived Animal grouping AgNPs AgNPs AuNPs

Days 1-11 12-21 1-11 12-21 1-11 12-21

Control 129.9±1.65 131.5±1.26 129.9±1.65 131.5±1.26 129.9±1.65 131.5±1.26

10mg/kg body weight 133.68±1.83 134.7±1.83 133.72±1.02 134.6±1.03 133.15±0.92 133.88±1.01

20mg/kg body weight 135.95±1.36 136.56±1.29 135.4±0.82 136.43±1.02 134.7±0.8 135.82±0.78

30mg/kg body weight 126.2±1.34 127.31±1.47 124.45±0.8 125.24±0.77 124.7±1.3 125.7±1.36

Table 3.67 Water intake (ml) of male Rabbits administered with D. mucronataand M.

buxifolialeaves derived AgNPs and AuNPs, (n=6)

Animal grouping D. mucronataleaves derived M. buxifolialeaves derived M. buxifolialeaves derived

AgNPs AgNPs AuNPs

Days 1-11 12-21 1-11 12-21 1-11 12-21

Control 220.1±1.6 221.8±1.17 220.1±1.6 221.8±1.17 220.1±1.6 221.8±1.17

10mg/kg body weight 223.8±1.84 224.78±1.83 223.89±0.96 224.6±1.02 223.32±0.9 224.005±0.9

20mg/kg body weight 226.09±1.26 225.97±1.09 225.6±0.68 226.4±1.006 224.9±0.7 225.76±0.76

30mg/kg body weight 216.38±1.21 217.37±1.4 214.72±0.74 215.86±1.05 215.08±1.08 215.87±1.25

263

Chapter 4 Discussion

4.0 DISCUSSION

4.1 Discussion

D. mucronata belonging to family Thymeleaceae is a famous medicinal plant mostly used in China and Africa tropical regions [32, 33]. Main constituents of plants are lignin, flavonoids, triterpenoids, coumarins and cumarinolignans [37, 39, 242].

Plant has been used for curing many diseases including rheumatism, ulcer, toothache, skin disorder and also in treatment of cancer [38-40, 43, 45].

M. buxifolia is an important medicinal plant found in Iran and northern areas of Pakistan [46, 47]. Phytochemical screening of M. buxifolia stem, bark, leaves and roots extracts shown the occurrence of saponins, steroids, tannins, glycosides, alkaloids and flavonoids [48, 49]. Similar metabolites have been reported for other members of family Sapotaceae. Saponins are bioactive chemical constituents that possess antimicrobial activities. Polyphenolic compounds tannins possess free radical scavenging activity. Some biological activities have been reported to be possessed by phenolic compounds such as anti-carcinogenic activity, anti-inflammatory activity, anti-apoptosis activity, anti-aging activity, antioxidant activity, cardiovascular protections, improvement of endothelial function, cell proliferation activities, inhibition of angiogenesis and anti-atherosclerosis activity [46, 49].

Plants have capability to mount up essential minerals in all parts that are important in human nutrition [243]. The Co, Cd and certain other elements that possess no direct relation to plants composition are gathered in some plants [244].

Trace elements have an important part in hindrance and treatment of several human diseases [209]. Due to environmental reasons some toxic heavy metals are 264

Chapter 4 Discussion accumulated in plants that create serious health hazards [245]. As for conclusions of numerous minerals in both plants, paralleleffects have been stated by Saeed et al. and

Bilal et al. [209, 246]. The Pb and Cd are toxic trace elements which are dispersed in environment most probably due to human activities causing pollution [244, 245].

These are transported to plants through essential metal transporters of plant [247-249].

Uptake of these elements has also been shown to affect the plant physiology [250].

Other elements have roles in plant metabolism e.g. Ca binding effects on structure and stability of human growth hormone and hypoglycemic activity [251, 252]. Among the trace elements correlations exist, suggested by some workers Herber and Stoeppler

[253]. Correlation of essential elementslike Zn, Fe, Mn, Cr and Co in plants was also suggested by Kumar et al. and Lokhande et al. [254, 255]. For example Mn, Cr and

Zn are known as hypoglycemic elements as they play important role in glucose metabolism. It has been reported that Mn, Cr and Zn are essential in maintaining insulin secretion and glucose tolerance.

For all organisms carbohydrates are deliberatedas main source of energy that play nutritional and structural role. High carbohydrate contents were reported by Gulfraz i.e. in Eruca sativaleaves [257]. Aerva javanica and Calotropis procera reported by

Hussain et al. Also has prominent amount of carbohydrates [258]. Present nutritional analysis showed higher carbohydrate contents in D. mucronata and M. buxifolia

265

Chapter 4 Discussion leaves. Because ofquickgrowth in inhabitants and healthinesscomplicationsexploration for good value proteins is growing day by day. It has been reported that if well strategicprocedures are not implemented to confront the circumstances, protein breach in Pakistan would remain to increase [259]. High protein contents were reported by Hussain i.e. in Trianthema portulacastrum and

Spinacea oleracea [260]. Anwar and Rashid reported high protein contents in

Moringa oleifera that supported the present findings [261]. Unnecessary fat ingestionconsequences includeconditions like cancer, aging and atherosclerosis so plant constituentscomprising 1-2% fat are takenbeneficial to human beings [262]. Fat contents detected in D. mucronata and M. buxifolia leaveswere much lower as compared to those of Baseila alba (8.71%), Talinum triangulare (5.90%),

Amaranthus hybridus (4.80%) and Calchorus africanum (4.20%) [263, 264]. These findings suggested that in respect of fat content these plants are safe for nutritional as well as for medicinal purposes. Crude fiber contents found in present study were much higher than those of Acalypha racemosa (7.20%) and Acalypha hispada

(10.25%) [265]. Crude fibers reported by Naseem et al. Were 27.2% in Crotalaria burhia [266]. These plants might be important source of dietary crude fibers, because the detected crude fibers are in reported range. Ash content in high concentration is indication of available mineral stuff in plant materials [262]. Ash contents reported in

T. triangulare (20.05%) are higher than ash contents in present findings [267].

Conversely ash contents inCcimum graticimum (8.00%) and Hibiscus esculentus

(8.00%) are less than ash contents tested in current study [264]. Moisture stated by

Hameed et al. Hussain et al. and Kochhar i.e. in their research are slightvarying with existingresults[258, 259, 268].

266

Chapter 4 Discussion

For green synthesis of AgNPs and AuNPs 140 ml of crude aqueous extract of

D. mucronata and M. buxifolialeaves solutions were added to 700 ml of AgNO3 and

HAuCl4.3H2O solutions respectively. Aqueous plant crude extracts were added to Ag and Au salt solutions in separate flasks, and progress of NPs formation was visualized by color change after 30 mins of heating at 40oC for AgNPs and 70oC for AuNPs.

Brown color was appeared in case of AgNPs while cherry red solution was formed in case of AuNPs. The observed color change in reaction mixtures arises due to SPR phenomena in these NPs. Free electrons in metallic NPs excite upon interaction with light of a specific wavelength and results in such SPR absorption bands. Generally,

AgNPs exhibit SPR peaks at 400-480 nm while AuNPs shows this phenomenon around 480-580 nm. The AgNPs and AuNPs synthesized were determined with unique color. The observed color change in reaction mixtures arises due to SPR phenomena of these NPs [269]. Our findings of UV absorption of synthesized NPs are in consistent with previous studies on biogenic synthesis of AgNPs and AuNPs using plants extracts as a source of reducing and stabilizing moieties of Chandran et al. and

Fayaz et al. [140, 270].

Plants contain severalforms of secondary metabolites such as tannins,steroids, flavonoids,monoterpenes and alkaloids that play a vitalpart in amalgamation of

AuNPs and AgNPs. The biologicaly active metabolites are elaborated in reduction of

Au ions to AuNPs and Ag ions to AgNPs. Bands at 1731 cm-1, 1588 cm-1 and 3320 cm-1 characterize the carbonyl functional group in ketones, aldehyde and carboxylic acids, aromatic C-C skeletal vibrations and OH functional group in alcohols and phenolic compounds respectively. Chandran et al. reported FTIR spectrum of AuNPs synthesized from Aloe vera leaf extract. A. Vera leaf extract FTIR spectrum possessed

267

Chapter 4 Discussion vibrational peaks at 1770 cm-1, 1710 cm-1and 1120 cm-1 that indicated presence of carbonyl and alcoholic groups in components of reaction. The reduction of Au ions was coupled with oxidation of alcoholic components as the correspondent band at

1120 cm-1 had dissolved [270].

Huang et al. reported FTIR analysis that revealed Cinnamomum camphora dried biomass absorbance bands detected in region 1000-1800 cm-1 centered at 1109,

1244, 1317, 1384, 1446, 1517, 1631, and 1726 cm-1. Absorbance bands at 1109, 1631 and 1726 cm-1 were linked with stretch vibration of –C-O, -C=C and RHC=O respectively. The peak 1109 cm-1 contributed by –C-O groups of polyols like terpenoids, poly saccharides and flavones in biomass. After bio reduction, disappearance of band at 1109 cm-1 showed that Ag ions and chloroaurate ions reduction was due to polyols. Alcohol groups were oxidized to carbonyl groups that lead to 1726cm-1. The FTIR spectra obtained for AgNPs and AuNPs exhibitsnumerous absorption peaks that were positioned at 1042, 1077, 1384, 1606,

1622, 1715 and 1762 cm1. Absorption peaks at 1042 and 1077 cm-1 were allocated to

–C-O-C- or –C-O-. The stretching vibration of -C=C- resulted in wide absorption spectra at 1606 and 1622 cm-1. In region 1700-1800cm-1 there were some weak absorption spectra that were related to stretching vibration of –C=O. Absorption at

-1 1384 cm for AgNPs was notably enhanced which showed NO3 existence in residual solution. Water-soluble heterocyclic compounds such as flavones, anthracenes and alkaloids were capping ligands of NPs. Oxygen atoms facilitated stabilization of NPs by adsorption of heterocyclic components on particle surface [67].

Basavaraja et al. reported FTIR spectrum of Fusarium semitectum synthesized

AgNPs bands at 1640 and 1540 cm-1. They were known as amide I and amide II 268

Chapter 4 Discussion thatrise due to carbonyl stretch and –N-H stretch vibrations in amide linkages of proteins. Metal binding ability of carbonyl group of amino acids residue and peptides of proteins enabled the capping of AgNPs preventing agglomeration of NPs and stabilizing in medium [271].

The XRD investigation in current study was done to determine crystal size of samples. Existingresearch findings were in line with findings of Zhan et al. Geeta et al. and Kaur. Zhan i.e. examined the XRD arrangement of Cacumen platycladi leaves extract fabricated AuNPs [272]. Intense peaks were observed at 111, 200, 220 and

311 signifying the features of crystalline Au. Geetha et al. deliberated the XRD profile of Couroupita guianensisflower water extract derived AuNPs. In agreement to the study, representative XRD crests were detected at 111, 200, 220 and 311 indicating fcc configuration of AuNPs [273]. Similarly Kaur presented the XRD profile of

Punica granatum seed fabricated AuNPs and showed peaks at 38.178°, 44.373°,

64.558° and 77.543°. These points were in contract with the fcc indexed by JCPDS,

USA [274].

The EDX analysis confirms presence of elements in test samples. In case of biogenic NPs, presence of Au and Ag was confirmed along with some other elements in different proportion. In present study EDX results of NPs were supported by the findings of Dwivedi and Gopal, Song et al. Huang et al. and Srnova-Sloufova et al. who studied AgNPs and AuNPs synthesized from various plants including

Chenopodium album, Magnolia kobus, Diopyros kaki and C. camphora [67, 275-

277].

269

Chapter 4 Discussion

Plants synthesize a number of active phytochemicals, which can be used to reduce and stabilize different metal ions for their respective NPs. Ankana et al. reported biosynthesis of well dispersed spherical shaped AgNPs using the potential of

Boswellia ovaliafoliolata aqueous extract as a source of both reducing and capping biomolecules [278]. Similarly, studies from other workers illustrate variety of plants that were successfully used in preparing metal NPs of spherical shapes for different biological applications [270, 279].

Absorption spectra provide solid evidence of NPs formation and their growth kinetics, the shape and size of resultant particles were elucidated with help of TEM. In present study the NPs displayed variety of shapes and sizes. Spherical shaped AgNPs were observed by TEM. The TEM analysis revealed the formation of spherical, triangular, Nano-prisms and small amount of hexagonal planner Au nanostructures.

Fringes formation in some hexagonal AuNPs and spherical AgNPs revealed the crystalline nature and purity of synthesized NPs. Relatedresults have been stated by

Chandran et al. using A. Vera leaf extract as reducing agent for biogenic synthesis of

-3 Au nanotriangles and spherical AgNPs from 10 M solution obtained from HAuCl4 and AgNO3. At 0.5mlA. Vera plant extract the AuNano triangles synthesized were observed with larger edge lengths. At 1 mlA. Vera extract the average edge lengths of

Nano triangles synthesized was reduced, so A. Vera extract was increased up to 4ml to obtain Nano triangles sized up to 50nm. The AgNPs analyzed by TEM showed spherical NPs with size 15.2nm [270]. Nicotiana tobaccum leaf extract was used by

Prasad et al. For green synthesis of AgNPs. The TEM analysis revealed monodispersed NPs with size up to 8.41 nm [269]. Huang et al. synthesized AgNPs using 0.1 g biomass of sundried C. camphora leaf that rangedin size from 55-80 nm

270

Chapter 4 Discussion as analyzed by TEM. The NPs obtained from 1.0 gm biomass were found to be polydispersed, some anisotropic structures were observed like Nano triangles and NPs with irregular contours. Morphology of Ag ions reduction by 0.5 gm biomass indicated that sample was composed of largely uniform NPs. The average diameter of these NPs was estimated to be 64.8 nm. The AgNPs from rapid bio reduction were polydispersed whereas those from mild reduction were almost quasi sphere- shaped[67].

Thermal strength was demonstrated for synthesized AgNPs and AuNPs using

TG-DTA. The existing study results were in linkto theoutcomes of Dick et al.

Maensiri et al. and Si and Mandal [280-282]. In temperature range 40-180oC weight loss is attributed to water vaporization and weight loss other than this range suggests the organic matters being decomposed. In diagram indented portion resembles to ignition; that may result due to increased amount of metal.

Antioxidant properties of different Daphne species supporting the present study have been stated previously by Sanda et al. Manojlovic et al. and Sovrlic et al.

[283-285]. Antioxidant potential, total phenolic and total flavonoid contents of M. buxifolia (Falc.) fruit methanolic extract and its n-hexane, ethyl acetate, butanol and aqueous fractions was reported by Jan et al. [50]. The study concluded that fruits of

M. buxifolia comprised antioxidant activity thus preventing free radicals associated with damage. Sharmin et al. Demonstrated antioxidant activity of Picrasma javanica leaf methanolic, n-hexane, chloroform, carbon tetrachloride and aqueous fractions.

Aqueous and chloroform fractions showed highest activity with IC50 value of 18.6

µg/ml and 14.59 µg/mlcorrespondingly[286]. Pandanus foetidus, Ludwigia repens and Nephelium lappaceumleaves methanolic extracts and Adiantum philippensewhole 271

Chapter 4 Discussion plant extract were subjected to antioxidant activity through DPPH free radical assay by Sikder et al. results showed that the highest effect was observed by

Nepheliumlappaceam L. With IC50 3.93 ± 0.25 µg/ml[287]. The present study results of AgNPs and AuNPs antioxidant activity were supported by Negahdary et al. Who concluded that AgNPs antioxidant activity was significantly higher than AuNPs and

Zn NPs [288].

Anti-bacterial activity of a plant extract is considered significant when MIC values are below 2 mg/ml, good when 2 ≤ MIC50 ≤ 3 mg/ml, moderate when 3 ≤

MIC50 ≤ 6 mg/ml and weak when MIC ˃ 6 mg/ml. Consequently, the activity (MIC50 of 1.4 mg/ml) observed for extracts (methanolic) of D. mucronata roots against S. aureuscan be takenimperative. Moderate or weak anti-bacterial activities (2 ≤ MIC ≤

7µg/ml) were observed for the majority of the extracts contrary toE. coli,A. baumani,

S. aureus,P. aeruginosa, VRSA,P. vulgarisandM. morganii strains. The existingresearch provides data on ability of these plants to combatwith pathogenic bacteria. The plant may well be more exploited for isolation of active compounds that may be successively characterized and exploited as preparatory material for development of anti-microbial drug. Leaf extract of Coleus aromaticus was reported to be used as reducing and capping agent in biogenic synthesis of AgNPs. These

AgNPs synthesized were active against gram positive bacteria B. subtilis having 12.33

± 1.203 mm zone of inhibition. Gram negative bacteria Klebsiella planticola was observed to have 11.0 ± 0.335 mm zone of inhibition at 50 µL concentration of

AgNPs. In comparison to leaf extract and chemically synthesized AgNPs, the biosynthesized AgNPs exhibited more zone of inhibition i.e.14.17 ± 0.602 and 12.83

± 0.442 in B. subtilis and K. planticola respectively established by disc diffusion

272

Chapter 4 Discussion method [289]. In current studies, good anti-bacterial activity was observed for various extracts of different plant parts of D. mucronata and M. buxifolia and both plants leaves derived NPs. Differences in anti-bacterial activities were noted for different fractions. Antimicrobial activity of the methanolic extract of Daphne gnidium stem has been reported previously with good activity against B. Lentus and E. coli. A number of metabolites were detected in studied plant extracts that could provide explanation on differences in anti-bacterial activities of different parts. There could be differences in chemical composition and mechanism of action of bioactive constituents present in different plant parts and differences in solubility of these constituents in different solvents [290]. Crude extracts and various fractions and synthesized NPs showed good MIC values.

The present study findings of anti-fungal activities were supported with results of Inamori et al. Javidnia et al. and Hong-xia i.e. inamori et al. concluded that Daphne odora comprising flavonoids i.e. Daphnodorin A, B and C exhibits anti-fungal as well as insecticidal activities [291]. Preliminary bioassays of D. mucronata Royle has reported that plant extracts had no anti-fungal activity against the tested fungal species, similar to present study that showed no significant anti-fungal activity against the tested fungal species [40].

Hemagglutinins that are present in different parts of plants are isolated just as they are obtained from animals and utilized for production of blood typing reagents.

Agglutinins attainedas ofherbalcradles are beneficial and economical owing to its accessibility in hugeextents. On the other hand, lectins have become a well- established means in past few years for understanding diversecharacteristics of cancer and metastasis. Lectins are used for signal transduction across membranes, cell 273

Chapter 4 Discussion adhesion and localization, mutagenic stimulation, tumor cell recognition (surface markers), apoptosis and cytotoxicity, and augmentation of host immune defense

[292].

Various anti-tumorigenic composites have been described to hinder the growth of L. minor which designates the presence of inhibitors. Presence of natural stimulants is determined by propagation of fronds. These characteristics undertake the need for herbicide which has certain benefits of having growth stimulants. The present results findings of phytotoxicity were in line with results of Ladhari et al. who studied phytotoxicity of D. gnidium[293]. Increase of phytotoxicity due to increase in concentration applied may be because of increased allelochemicals [294]. Many reports have shown that phytotoxins responses are species dependent. Broad spectrum of allelopathic plants has suppressed growth of tested species [295, 296]. Plant species with phytotoxicity effect depends not only on biochemical and physiological characteristics but it also involves environmental conditions [297].

In literature many plants extract have shown to comprise such compounds with effective insecticidal activity. Tedeschi et al. reported that Armoracia rusticana and Allium sativum have shown significant insecticidal and anti-fungal activity [298].

Artemisia herbaalba, Eucalyptus camaldulensis and Rosmarinus officinalis ethanol, petroleum ether and aqueous extract have shown significant insecticidal activity against Myzus persicae [299]. As with the present study findings, Enhydra fluctuans,

Clerodendrum viscosum and Andrographis peniculata had shown significant insecticidal activity against adult T. castaneum[300]. Literature reported that

Decalepis hamiltonii root extract have significant insecticidal activity against R. domonica, Sitophilus oryzae, Stigobium pancieum, T. castaneum and Callosobruchus 274

Chapter 4 Discussion chinensis [300]. Zizyphus jujube exhibit insecticidal effect against T. castaneum, R. dominica and C. analis[301]. Biogenic AgNPs and AuNPs have shown significant insecticidal activity [175, 302].

Present study showed that plant D. mucronata and M. buxifolia extracts as well as their synthesized NPs showed significant anti-termite activity. Many plants comprises bioactive compounds with anti-termite properties, such as Sowmya et al.

Determined four Zingiberaceae rhizome plant extracts anti-termite properties, where the Alpinia galangal was highly effective causing 90% mortality in first two hour exposure [303]. Ahmed et al. reported that garlic showed 100% mortality, which was followed by Tobacco extract and least effect was observed of the Neem extract [304].

For prevention and treatment of different diseases including cancer new medicinal drugs are discovered from biogenic compounds of plants [305]. Primary research for revealing and progress of anticancer drugs used is brine shrimp lethality bioassay. Brine shrimp lethality trials were conceded to examineinitial cytotoxic activity of D. mucronata (leaves, bark and roots) and M. buxifolia (leaves, seeds and fruits) crude methanolic, n-hexane, chloroform, ethyl acetate and aqueous extracts.

The present results of the two plant extracts against Brine shrimp results have also been supported by other reports. In cytotoxic activity methanolic extract of Tridax procumbens showed potent effect having LD50 31.68 µg/ml in comparison to positive control (vincristine sulphate) 0.45 µg/ml after 24 hrs of brine shrimp lethality testing

[306]. P. javanica leaf methanolic, n-hexane, chloroform, carbon tetrachloride and aqueous fractions were screened for their brine shrimp lethality. Brine shrimp lethality bioassay was used to determine cytotoxic activity with LD50 1.04 µg/ml by crude extract and LD50 1.28 µg/ml by n-hexane fraction [286]. Significant brine shrimp 275

Chapter 4 Discussion lethality assay was observed by A. philippense and P. foetidus methanolic extracts with LD50 values 0.50 µg/ml and 0.58 µg/ml that were compared to standard anti- neoplastic drug vincristine sulfate with LD50 0.45 µg/ml[287]. Brine shrimp cytotoxic assay observed for Clausena suffruticosa, Leucas aspera, Ageratum conyzoides, Leea indica, Solanum torvum and Senna sophera showed LD50 values 41.16, 181.67,

508.86, 2.65, 478.40 µg/ml[307].

Thrombus in blood vessels causes atherothrombotic diseases like myocardial or cerebral infarction. The already formed clots in blood vessels are used to dissolve by thrombolytic agents however some serious and fatal consequences can be caused.

For treatment of several diseases herbal preparations have been used since ancient times, although in some cases toxicity have been observed. Organic extracts were investigated for thrombolytic properties which possessed minimal or no toxicity

[307]. Two medicinal plants in Bangladesh i.e.T. procumbens and Vernonia cinerea were investigated for their cytotoxic and in-vitro thrombolytic activities. Methanol extract of T. procumbens possessed highest thrombolytic activity than V. cinerea, the clot lysis value determined was 21.15%. Water was used as negative control with clot lysis 2.64% and Standard Streptokinase was used as positive control that demonstrated 66.77% clot lysis of human blood [306]. Withania somnifera extracts were evaluated for thrombolytic activity by means of human erythrocytes and results were linked with streptokinase. The highest thrombolytic activity was shown by methanolic extract of W. somnifera 68.14%, whereas chloroform and ethanolic extracts showed 21.15% and 17.46% moderate thrombolytic effect [308].

Thrombolytic activity reported for P. javanica carbon tetrachloride fraction was

34.16%; standard positive control streptokinase 66.77% and negative control water

276

Chapter 4 Discussion

3.791% lysis of clot [286]. Clot lysis observed for methanolic extract of N. lappaceum and P. foetidus was 17.29% and 17.31%, the standard streptokinase and water demonstrated 66.77% and 3.79% clot lysis as positive and negative control [287].

Thrombolytic activity was investigated for medicinal plants Averrhoa bilimbi

(Oxalidiaceae), Drynaria quercifolia (Polypodiaceae) and C. viscosum (Verbanaceae).

D. quercifolia aqueous and pet ether extracts exhibited 34.38% and 34.27% clot lysis while C. viscosum carbon tetrachloride extracts exhibited 28.64% clot lysis. The A. bilimbi carbon tetrachloride, Pet ether, aqueous and methanolic extracts showed

27.72%, 27.5%, 17.06% and 23.94% clot lysis respectively. Methanolic and carbon tetrachloride extracts of D. quercifolia showed 23.46% and 20.33% clot lysis respectively [309]. Litsea glutinosaleaves hydrocarbon extracts were used to evaluate in-vitro thrombolytic activity. Crude methanolic extract, n-hexane, chloroform and ethyl acetate extracts showed 46.78 ± 0.9%, 32.23 ± 0.26%, 43.13 ± 0.85% and 37.67

± 1.31% clot lysis respectively in comparison to positive control streptokinase at a dose of 30,000 I. U. With 93.35 ± 0.35% disruption [310]. Thrombolytic activity for

C. Suffruticosa, A. Conyzoides, L. Indica, S. Sophera, L. Aspera and S. Torvum showed 48.9%, 18.12%, 39.30%, 31.61%, 37.32% and 31.51% clot lysis respectively.

In comparison to reference drug streptokinase 75.0%L. Indica, L. Aspera and C.

Suffruticosa showed significant p<0.0001 clot lysis [307].

In present study the tested samples showed negative results to hemolytic activity against human erythrocytes. In another study W. somnifera extracts were evaluated for hemolytic activity. By hypotonic solution induced hemolysis methanol extract inhibited 71.90% hemolysis of RBCs at a concentration of 1.0 mg/ml, in comparison to acetyl salicylic acid that was 73.56% at 0.10 mg/ml. The heat induced

277

Chapter 4 Discussion hemolysis established 54.03%, 53.13% and 38.54% inhibition of RBCs by methanol, ethanol and chloroform extracts of W. somnifera[308]. The A. philippense L.

Methanol extract inhibited heat induced and hypotonic solution induced hemolysis of

RBCs by 35.36 ± 1.60% and 53.09 ± 1.01% that were compared with acetyl salicylic acid 42.20% and 71.77% respectively [287].

The environmental mutagens cause damage to DNA that could be chief factor of incapacity in innovativecultures and death. This destruction, accumulatedin one’s lifespan, initiates genetic defects and human cancer, and is quite likely a major contributor to heart diseases and aging as well. Prevention is the possible solution i.e.to identify environmental mutagens and minimize the human revelations. Quick, precise, in-vitro experiments, such as Salmonella/microsome test, play important part in achieving this objective[185]. The present results showed that there were no mutagenic properties observed for derived AgNPs and AuNPs. At cellular level NPs has an effect of oxidative stress. Mitochondrial respiratory chain has been affected by enhanced generation of reactive oxygen species (ROS). Cellular damage lead to DNA damage, ROS generation and activation of signaling causes apoptosis, necrotic death and inflammation [310]. Many of the reports published have declared NPs being non- mutagenic, and with some which have detected mutagenicity of NPs had declared the results being non-significant. Thus it can be concluded that mutagenecity depends on treatment period, concentration and size of NPs [186, 311].

L. Glutinosaleaves hydrocarbon extracts were used to evaluate in-vivo analgesic potential. Analgesic activity observed at a dose of 500 mg/kg crude methanolic extract was 15.54 ± 0.37 sec that significantly differed by P<0.01 and

P<0.001 to that of standard drug ketorolac 16.38 ± 0.27 sec. Crude methanolic extract 278

Chapter 4 Discussion in acetic acid induced writhing test showed significant analgesic effect by P<0.01 and

P<0.001 at doses 250 and 500 mg/kg body weight with 45.98 and 56.32% inhibition respectively while the standard ketorolac showed 64.36% inhibition [312].

Induction of hyperthermia-like condition was employed by Brewer’s yeast induced pyrexia model. The brewer’s yeast pathogenic behavior results in prostaglandins release that is considered important regulators of pyrexia.

Thermoregulatory center in body is affected by these mediators and cause fever [313,

314]. Therefore, antipyretic agents are required to reduce this increase in temperature.

Aspirin among the available antipyretics is being utilized since 19th century, so far the science in arrears relieving temperature was interpretedsolitary a periodback.

Variety of antipyretics control temperature either by inhibiting cyclooxygenase pathway within hypothalamus or decreasing the level of prostaglandin E2 has been revealed [315].

Plant comprising such beneficial compounds can be utilized for miscellaneous diseases such as Enicostemma littorale with potent antidiabetic agent [316].

Depending on dose concentration these compounds application for any organism have to be determined with plant extract/100 gm body weight. Size of NPs is a dependent factor for uptake of these particles in GIT [316]. Furthermore the toxicity study of

NPs of GIT appliance and approach of incidence in GIT and its effectiveness in formerbody part of being has been studied by Arora et al. [317].

Samad et al. described that cyclooxygenase pathway is the bestmutual pathway convoluted in fever, inflammation and pain [318]. Anti-inflammatory drugs are used in order to reduce inflammation. Edema and inflammation is reduced by

279

Chapter 4 Discussion these drugs by inhibiting either cyclooxygenase [319] or lipoxygenase pathways

[320]. Prolonged usage of definite NSAIDs results in disorders associated with digestiveregion such as blood loss, perforations and ulcers [321]. Hence, there rises a necessity for drugs progress of natural derivation with less toxicity and enriched efficacy [322].

Some toxicity concerns were raised for administration of herbal preparations with no standard dosage without adequate scientific studies on safety [323]. In animals toxicity studies are used to measure latenthealthinessthreatsbegan by inherent incompatible plant extracts or chemical composites in humans [324-326]. Levels of biomolecules like histomorphology and normal function of organs, metabolic products and enzymes may be significantly changed by these adverse effects [327].

Hematological parameters analysis is helpful to test the toxic effect of plant extracts and other foreign compounds on blood components of animals. This toxicity test is related to evaluation of risks as for human toxicity, modifications in hematological system is greatly valued once data is translated as of animal studies [328]. Plant extracts or chemical compounds blood relating functions can be explained by this toxicity test [329].

In present study WBC occurrencehad been observed with application of plant extract as well as with plant derived NPs. The doses at which white blood cell (WBC) parameters were significantly increased in case of male G. Pigs and Rabbits of D. mucronata, M. buxifolia leavesand its derived AuNPswhen compared to control suggested that they may help to boost the immune system. The WBC plays vital roles in immune system so increase in its production is not deleterious to body. They are responsible for antibodies production that helps to fight foreign materials and 280

Chapter 4 Discussion infections in body [330]. The synthesized AgNPs doses at which WBC was reduced suggested that from bone marrow the blood parameter entrance is not equal to its removal from circulation or might be because of less production of hematopoietic regulatory elements by bone marrow macrophages and stroma cells [331]. Red blood cells total population is associated with RBC and Hb while red blood cells individually are associated with MCH and MCHC. There was a slight increase and decrease in these parameters that did not affect the total and individual population of red blood cells. Swelling is usually related to body weight [332]. In clinical diagnosis serum enzyme provides valuable tool because it provides information on nature and effect of tissue pathological damage [333]. Reduction in SGPT, phosphorus, calcium, urea and uric acid at 100, 200 and 300 mg/kg body weight suggested inactivation or inhibition of enzyme molecule [334]. This increase and decrease suggests homeostasis of these ions at different doses. Effects of these doses are relatively safe so they can be consumed in routine. Total and conjugated bilirubin level increased in serum indicated the insignificant hemolysis and barrier in bile normal excretion [335-339].

Increased level of triacylglycerol provides information on coronary heart disease association with tendency of animal’s heart to atherosclerosis [340]. Increased level of triacylglycerol may be linked to increased lipolysis; the plasma cholesterol rate at which they are carried to liver will be increased. In everyday life water is essential nutrient to life, for development and growth water is one of important most nutrients.

Feed consumption affected by a factor will influence the water intake as well. Water intake in present study was increased by animals with doses of 100 and 200 mg/kg body weight of plant extracts and 10 and 20 mg/kg body weight of synthesized

AgNPs and AuNPs. Feed consumption in same way was increased by animals that suggest these doses enhanced appetite and sense of taste [341]. 281

Conclusion

CONCLUSION

D. mucronata and M. buxifolia plant have secondary metabolites i.e. carbohydrates,flavonoids, saponins, phenols and tannins, and minerals such as Ca, Cr,

Co, Fe, Zn, Pb and Mn. Synthesis of NPs was detected by UV-Vis spectroscopy,

XRD analysis, FTIR spectroscopy, and TG-DTA profile. The SEM and TEM images of herb derived NP’s exhibited particles with 8 nm- 60 nm range having various structures i.e. spherical, nano prisms, hexagonal and nano-rods. The NPs synthesized in this study are pure and crystalline in nature. Purity is confirmed by fringes formation in TEM analysis. Crude methanolic extract, n-hexane, chloroform, ethyl acetate and aqueous fractions of both plants and their derived NPs showed good scavenging activity at 600 μg/ml. Promising anti-bacterial activity against A. baumanni, M. morganii,P. aeruginosa, S. aureus, VRSA and E. coliwas shown by both plants and derived NPs. Methanolic and n-hexane D. mucronata extract varied in inhibiting growth of F. oxysporum, C. albicans, A. flavus and A. parasiticus, whereas

M. buxifolia extract slightly inhibited growth of C. albicans, F. oxysporum, A. flavus,

A. parasiticus, P. digitatum and A. niger. The NPs possess anti-fungal activities to some extent against C. albicans and A. niger. Existingresearch provides information on capability of these plants to combatcontrary to pathogenic bacteria. Plants may possibly be more exploited for isolation of active compositesthatmay well be successively characterized and exploited as preparatory material for development of anti-microbial drug. D. mucronataleaves 20 mg/ml ethyl acetate extract showed 80% phytotoxic activity which showedexistence of inhibitors for L. minor. Methanolic extract, n-hexane, chloroform and Ethyl acetate extract and their derived NPs of both plants fullysupressed development of insects i.e.T. castaneum, R. dominica and C.

282

Conclusion analis and termites in three days. Plant derived NPs had higher cytotoxic effect than plants extracts alone. The clot lysis of these plants andderived NPs ranged from15.9% to 25.8%. D. mucronata and M. buxifolia leaves aqueous extracts and their synthesized NPs showed no hemolytic activity. Extracts in general were non-lethal and non-mutagenic. They had high potential of writhing reduction, however D. mucronataleaves derived AgNPs showed higher percent of writhihng inhibition than both the plants alone and M. buxifolia leavesderived NPs. D. mucronataleaves derived

AgNPspossessed slightly higher anti-pyretic activity than M. buxifolia leaves derived

NPs. Percent GIT motility was reduced when plants and their derived NPs were applied at 300 mg/kg and 30 mg/kg respectively. Plants derived NPs also possessed significant anti-inflammatory activities. Plant extracts and their derived NPs had no differential biochemical properties than normal range for animals’ i.e.g. Pigs and male

Rabbits. Hence, these plants might be nontoxic as an oral medicine for treating numerous diseases at the doses tested.

Presentresearchlinked the therapeutic capacity of plants with nanotechnology to synthesize biocompatible AgNPs and AuNPs with less toxicity and greater efficiency. Results not only revealed usefulness of the two plants for synthesis of biological AgNPs and AuNPs but also demonstrated their role in pharmaceutical and medical sciences. The synthesizednanoparticles were more promising and should be exploited further through modern techniques and through experimentation in animals and in-vitro models. Optimal dose used was 30 mg/kg, and are useful against infectious (bacterial) diseases.

283

References

RECOMMENDATION

The plants D. mucronata and M. buxifoliaproduce various bioactive compounds that can be used for dietary and therapeutic purposes. The extracts could be further analyzed through advance chromatography techniques (HPLC/MS or

GC/MS) to identify bioactive compounds from the plants. The present study method is recommended for NPs synthesis as it is eco-friendly, rapid, economical and a convenient method. Characterization of NPs accordingly to the present study approach i.e. UV-Vis spectroscopy, FTIR spectroscopy, SEM and TEM images, XRD analysis, EDXand TG-DTA profile displays efficient information regarding NPs. The

NPs fabricated, and extracts of plant should be further observed forfurtherharmful microbes and insects. Crystalline pure NPs can be used in anti-bacterial, anticancer studies, drug delivery, water splitting processes and catalysis. In present study, the

NPs size control and uniqueness of shape is an issue. Condition optimization likepH,Temperature, salt and plant extract concentration is required to obtain monodispersed NPs. Long period treatments should be followed in mutagenicity tests fornon-mutagenic natureconfirmation of NPs. More tests are required to determine interaction,distribution, absorbance,excretion and metabolism of these compounds to utilize them in production of newmedicine. These NPs production on large scale is recommended because purity is not a problem and plants (precursors) utilized occur in large quantity.

284

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