PHYTOCHEMICAL EVALUATION, BIOASSAY SCREENING AND AERIAL PLANT- MEDIATED SILVER NANOPARTICLES SYNTHESIS USING QUERCUS SEMECARPIFOLIA SMITH Ph. D Thesis By: AISHMA KHATTAK CENTRE OF BIOTECHNOLOGY AND MICROBIOLOGY UNVERSITY OF PESHAWAR Session 2013-2018 PHYTOCHEMICAL EVALUATION, BIOASSAY SCREENING AND AERIAL PLANT- MEDIATED SILVER NANOPARTICLES SYNTHESIS USING QUERCUS SEMECARPIFOLIA SMITH AISHMA KHATTAK A thesis submitted to the University of Peshawar in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biotechnology CENTRE OF BIOTECHNOLOGY AND MICROBIOLOGY UNVERSITY OF PESHAWAR Session 2013-2018 In the name of Allah, The Most Gracious, The Most Merciful Dedication I wish to dedicate this work to my parents who taught me to value myself and told me that I was the most precious thing in their life. CONTENTS Tables V Figures VII Schemes IX Acknowledgment X Summary XI C H A PTE R 1 IN TR O D U C TIO N & L ITE R A TU RE R E V IE W 1.1 General Introduction 1 1.2 Fagaceae (Family) 6 1.2.1 Description 6 1.2.2 Distribution 6 1.2.3 Importance 6 1.3 Quercus (Genus) 7 1.3.1 Description 7 1.3.2 Distribution 7 1.3.3 Importance 8 1.4 Quercus semecarpifolia Smith (Plant) 11 1.4.1 Description 11 1.4.2 Distribution 12 1.4.3 Importance 12 1.5 Preliminary Phytochemical Profile of the Genus Quercus 14 1.6 Nanotechnology 26 1.6.1 Background 26 1.6.2 Current Status 27 1.7 Nanobiotechnology 28 1.7.1 Background 28 1.7.2 Current Status 28 1.8 Silver(Ag) 29 1.8.1 History 29 1.8.2 Importance 30 1.9 Different Methods Used for the Synthesis of 32 Nanoparticles (NPs) 1.9.1 Biological Approaches for the Synthesis of Nanoparticles 33 (NPs) 1.9.1.1 Biosynthesis of AgNPs, Using Plant Extracts 33 1.10 Bioinspired Synthesis and Characterization of the AgNPs 35 1.11 Aims and Objectives 39 i C H A PTE R 2 METHODOLOGY 2.1 General Experimental Conditions 40 2.1.1 Drugs and Chemicals used in Different Experiments 40 2.1.2 Physical Constants 40 2.1.3 Spectroscopy 40 2.1.4 Isolation and Purification of the Compounds 41 2.1.4.1 Column Chromatography (CC) 41 2.1.4.2 Thin layer Chromatography (TLC) 41 2.1.5 Spraying Reagents used for Visualization of Spots 41 2.1.5.1 Ceric Sulfate Solution 42 2.1.5.2 Vanillin-Phosphoric acid reagent 42 2.1.5.3 Iodine (I2) Solution 42 2.1.5.4 Dragendorff‘s Reagent 42 2.2 Phytochemical Investigation 43 2.2.1 Collection and Identification of the Plant 43 2.2.2 Extraction Procedure 43 2.2.3 Fractionation of Crude Methanolic Extracts 43 2.2.4 Screening Tests of Crude Extracts for the presence of 45 Different Classes of Compounds 2.2.4.1 Preparation of Plant Extracts 45 2.2.4.2 Preliminary Phytochemical Screening 45 2.2.4.2.1 Alkaloids 45 2.2.4.2.2 Saponins 45 2.2.4.2.3 Flavonoids 45 2.2.4.2.4 Tannins 46 2.2.4.2.5 Glycosides 46 2.2.4.2.6 Terpenoids 47 2.2.4.2.7 Sterols 47 2.2.4.2.8 Phenols 47 2.2.4.2.9 Carbohydrates 47 2.2.4.2.10 Proteins 47 2.2.4.2.11 Anthraquinones 48 2.2.4.2.12 Phlobatannins 48 2.3 Compounds Isolated from Quercus semecarpifolia 50 2.3.1 Characterization of compounds 52 2.3.1.1 Characterization of benzoic acid (1) 52 2.3.1.2 Characterization of p-hydroxybenzoic acid (2) 53 ii 2.3.1.3 Characterization of Bis (2-ethylhexyl) phthalate (3) 54 2.3.1.4 Characterization of β-Sitosterol (4) 55 2.3.1.5 Characterization of Stigmasterol (5) 56 2.4 Green Biogenic Synthesis of Silver Nanoparticles 57 (AgNPs) 2.4.1 Characterization of Synthesized AgNPs 59 2.4.1.1 UV-Vis Spectroscopic Studies 59 2.4.1.2 X-Ray Diffraction (XRD) Dimension 59 2.4.1.3 Scanning Electron Microscopy (SEM) 59 2.4.1.4 Energy Dispersive X-Ray Spectroscopy (EDX) 61 2.4.1.5 Fourier Transform Infra-Red (FTIR) Spectroscopy 61 2.4.1.6 Transmission Electron Microscopy (TEM) 61 2.4.1.7 Thermo gravimetric/Differential Thermal Analysis 61 (TG/DTA) 2.5 Assessment of Pharmacological/Biological Activities 61 (in-vitro) 2.5.1 Antibacterial Activity 61 2.5.2 Determination of Minimum Inhibitory Concentration 63 (MIC50) Values 2.5.3 Antifungal Activity 64 2.5.4 Antioxidant Activity 66 2.5.5 Phytotoxic Activity 67 2.5.6 Cytotoxic Activity 70 2.5.7 Insecticidal Activity 72 2.5.8 Anti-termite Activity 74 2.5.9 Allelopathic Activity 75 2.5.10 Hemagglutination Activity 76 2.6 Assessment of Pharmacological/Biological Activities 77 (in-vivo) 2.6.1 Acute Toxicity Assay 78 2.6.2 Antinoceceptive Assay 79 2.6.2.1 Acetic Acid Induced Writhing Test 79 2.6.2.2 Hot Plate Assay 81 2.6.3 Anti-inflammatory Assay 82 2.6.4 Anti-pyretic Assay 83 2.7 Analysis of Fixed Oils by Gas Chromatography-Mass 84 Spectrometry (GC-MS) C H A PTE R 3 RESULTS & DISCUSSION 3.1 Phytochemical Studies 85 3.1.1 Qualitative Phytochemical Screening 85 3.2 Spectroscopic characterization of isolated compounds 88 iii from Q.semecarpifolia 3.2.1 Structural elucidation of benzoic acid (1) 88 3.2.2 Structural elucidation of p-hydroxy benzoic acid (2) 91 3.2.3 Structural elucidation of Bis (2-ethylhexyl) phthalate (3) 94 3.2.4 Structure Elucidation of β-Sitosterol (4) 97 3.2.5 Structural elucidation of Stigmasterol (5) 100 3.3 Plant mediated Synthesis of AgNPs 103 3.4 Characterization of Silver Nanoparticles (AgNPs) 103 3.4.1 UV-Vis Spectroscopy 103 3.4.2 X-Ray Diffraction Pattern 107 3.4.3 Scanning Electron Microscopy (SEM) 110 3.4.4 Energy Dispersive X-Ray Spectroscopy (EDX) 115 3.4.5 Fourier Transform Infra-Red (FTIR) Spectroscopy 120 3.4.6 Transmission Electron Microscopy (TEM) Studies: 123 3.4.7 Simultaneous Thermogravimetric Analysis/Differential 125 Thermal Analysis (TGA/DTA): 3.5 Assessment of Pharmacological/Biological Activities ( in 129 vitro) 3.5.1 Antibacterial Activity 129 3.5.2 Antifungal Activity 138 3.5.3 Antioxidant Activity 142 3.5.4 Phytotoxic Activity 146 3.5.5 Cytotoxic Activity 150 3.5.6 Insecticidal Activity 154 3.5.7 Antitermite Activity 159 3.5.8 Allelopathic Activity 163 3.5.9 Hemagglutination Activity 166 3.6 Assessment of Pharmacological/Biological Activities ( in 168 vivo) 3.6.1 Acute Toxicity Assay 168 3.6.2 Antinoceceptive Assay 171 3.6.2.1 Acetic Acid Induced Writhing Test 171 3.6.2.2 Hot Plate Assay 176 3.6.3 Anti-inflammatory Assay 181 3.6.4 Anti-pyretic Assay 186 3.7 Chemical Composition of Fixed Oils 190 CONCLUSION 192 REFERENCES 194 iv TABLES Table 1.1 Compounds isolated from natural products since 2000 Table 1.2 Quercus species in Pakistan and their worldwide distribution Table 1.3 Phytochemical constituents from genus Quercus Table 1.4 Green synthesis of AgNPs using various plant extracts Table 2.1 Reagents composition used in phytochemical investigation Table 2.2 Composition of E medium for phytotoxic activity Table 3.1 Tabular representation of phytochemicals present in Q.semecarpifolia Table 3.2 1H-NMR and 13C-NMR spectra of Benzoic acid (1) Table 3.3 1H-NMR and 13C-NMR spectra of P-Hydroxybenzoic acid (2) Table 3.4 1H-NMR and 13C-NMR spectra of Bis (2-ethylhexyl) phthalate (3) Table 3.5 1H-NMR and 13C-NMR spectra of β-Sitosterol (4) Table 3.6 1H-NMR and 13C-NMR spectra of Stigmasterol (5) Table 3.7 Tabular representation of elemental analysis of the Q. semicarpifolia aqueous extract Table 3.8 Tabular representation of elemental analysis of the Q. semicarpifolia derived AgNPs Table 3.9 Tabular representation of antibacterial activity by Quercus semecarpifolia Table 3.10 Tabular representation of MIC50 assay by Quercus semecarpifolia Table 3.11 Tabular representation of antifungal activity by Quercus semecarpifolia Table 3.12 Tabular representation of antioxidant activity by Quercus semecarpifolia Table 3.13 Tabular representation of phytotoxic activity by Quercus semecarpifolia Table 3.14 Percent growth regulation of Lemna minor Table 3.15 Tabular representation of cytotoxic activity by Quercus semecarpifolia Table 3.16 Tabular representation of insecticidal activity by Quercus semecarpifolia Table 3.17 Tabular representation of anti-termite activity by Quercus semecarpifolia v Table 3.18 Tabular representation of allelopathic activity of Quercus semecarpifolia aqueous extract Table 3.19 Tabular representation of hemagglutination activity of Quercus semecarpifolia Table 3.20 Tabular representation of acute toxicity assay of Quercus semecarpifolia Table 3.21 Tabular representation of analgesic assay by acetic acid induced writhing test of Quercus semecarpifolia Table 3.22 Tabular representation of analgesic effect of Quercus semecarpifolia by hot plate assay Table 3.23 Tabular representation of anti-inflammatory assay by Quercus semecarpifolia Table 3.24 Tabular representation of antipyretic assay by Quercus semecarpifolia Table 3.25 Fatty acid composition of fixed oil from Quercus semecarpifolia vi FIGURES Figure 1.1 Morphology of Q. semecarpifolia plant Figure 1.2 Zoom version of leaf of Quercus semecarpifolia plant Figure 3.1 Structure of Benzoic acid (1) Figure 3.2 Structure of P-hydroxy benzoic acid (2) Figure 3.3 Structure of Bis (2-ethylhexyl) phthalate (3) Figure 3.4 Structure of β-Sitosterol (4) Figure 3.5 Structure of Stigmasterol (5) Figure 3.6(a) Plant leaf extract Figure 3.6(b) Synthesized silver nanoparticles Figure 3.7 Graphical representation of absorbance values of Quercus semecarpifolia AgNPs Figure 3.8 Graphical representation of absorbance values of Quercus semecarpifolia aqueous extract Figure 3.9 Graphical representation of XRD values of Quercus semecarpifolia aqueous extract Figure 3.10 Graphical representation of XRD values of Quercus semecarpifolia derived AgNPs Figure 3.11 SEM micrograph of Quercus semecarpifolia derived AgNPs at 10,000X Figure 3.12 SEM micrograph of Quercus semecarpifolia derived AgNPs at 20,000X Figure 3.13 SEM