Phytochemical Evaluation, Bioassay Screening and Aerial Plant

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

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

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

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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

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

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

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 micrograph of Quercus semecarpifolia derived AgNPs at 60,000X Figure 3.14 SEM micrograph of Quercus semecarpifolia derived AgNPs at 30,000X Figure 3.15 SEM micrograph of Quercus semecarpifolia aqueous extract at 10,000X Figure 3.16 SEM micrograph of Quercus semecarpifolia aqueous extract at 20,000X Figure 3.17 SEM micrograph of Quercus semecarpifolia aqueous extract at 30,000X Figure 3.18 SEM micrograph of Quercus semecarpifolia aqueous extract at 60,000X Figure 3.19 Graphical representation of EDX profile of Quercus semecarpifolia aqueous extract

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Figure 3.20 Graphical representation of EDX profile of Quercus semecarpifolia synthesized AgNPs Figure 3.21 Graphical representation of FTIR spectra of Quercus semicarpifolia aqueous extract Figure 3.22 Graphical representation of FTIR spectra of Quercus semicarpifolia derived AgNPs Figure 3.23 TEM micrograph of Quercus semecarpifolia derived AgNPs at 100 nm magnification Figure 3.24 TEM micrograph of Quercus semecarpifolia derived AgNPs at 200 nm magnification Figure 3.25 TGA profile of Quercus semecarpifolia derived AgNPs Figure 3.26 TGA profile of Quercus semecarpifolia aqueous extract Figure 3.27 DTA profile of Quercus semecarpifolia derived AgNPs Figure 3.28 DTA profile of Quercus semecarpifolia aqueous extract Figure 3.29 Graphical representation of antibacterial activity by Quercus semecarpifolia Figure 3.30 Graphical representation of antifungal activity by Quercus semecarpifolia Figure 3.31 Graphical representation of antioxidant activity by Quercus semecarpifolia Figure 3.32 Graphical representation of phytotoxic activity by Quercus semecarpifolia

Figure 3.33 Graphical representation of cytotoxic activity by Quercus semecarpifolia

Figure 3.34 Graphical representation of insecticidal assay by Quercus semecarpifolia against Tribolium castaneum Figure 3.35 Graphical representation of insecticidal activity by Quercus semicarpifolia against Callosobruchus maculates Figure 3.36 Graphical representation of insecticidal assay by Quercus semecarpifolia against Rhyzopertha dominica Figure 3.37 Growth inhibitions of shoot and radical of Quercus semecarpifolia Figure 3.38 Percent germination of seeds by Quercus semicarpifolia Figure 3.39 Percent analgesic activity of Quercus semicarpifolia derived AgNPs in acetic acid induce pain model Figure 3.40 Percent analgesic activity of Quercus semecarpifolia Cr. MeOH Ext in acetic acid induced pain model Figure 3.41 Hot plate assay for Quercus semecarpifolia Cr.MeOH.Ext

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Figure 3.42 Hot plate assay for Quercus semecarpifolia derived AgNPs Figure 3.43 Anti-inflammatory assay of Quercus semecarpifolia Cr.MeOH.Ext Figure 3.44 Anti-inflammatory assay of Quercus semecarpifolia derived AgNPs Figure 3.45 Antipyretic assay of Quercus semecarpifolia Cr.MeOH.Ext Figure 3.46 Antipyretic assay of Quercus semecarpifolia derived AgNPs

SCHEMES Scheme 2.1 Fractionation of crude MeOH extract of Quercus semecarpifolia Scheme 2.2 Flow chart depicting compounds isolated from EtOAc fraction of Quercus semecarpifolia Scheme 2.3 Flowchart depicting steps involved in AgNPs synthesis

ACKNOWLEDGEMENTS

All praises are for Almighty Allah, the most beneficent, the most merciful who bestowed upon me with the sight to observe, the mind to think and the courage to work more and more. Peace and blessing of Allah be upon the Holy Prophet (S.A.W) who exhorted his follower to seek the knowledge from cradle to grave.

It is my privilege and honor to be a student of Prof. Dr. Bashir Ahmad, Centre for Biotechnology and Microbiology (COBAM), University of Peshawar (UOP). I wish to express my deepest gratitude for his expert guidance, appreciation and sincere advice, marvelous and ongoing support during the period of this research work. His endless encouragement and familiar deeds have been the major driving force throughout my research career.

Perhaps I would not be able to present this work in present form withoutco-operation of Higher

Education Commission (HEC) Pakistan for funding methrough Indigenous PhD fellowship programme.

Words fail me to acknowledge the gratitude of my husband (Mohammad Kazim Khattak) and in- laws for accepting and supporting my ambition. Without them I would have never achieved this far. Further, I would like to extend my thanks to my sisters (Zalanda khattak, Haseena khattak) who were always there for me during this entire journey and supported me.

I am thankful to Dr. Javed Khan, PCSIR Laboratory, Peshawar, Dr Abdur Rauf, Assistant Professor/HOD, Department of Chemistry, and University of Swabi. Dr. Sadiq Azam, Assistant professor COBAM, UOP, Yaqoob ur Rehman, PCSIR Laboratories, Peshawar and Mr. Noshad, lab assistant COBAM, UOP for being helpful during entire research period.

Last but not the least I am thankful to Dr. Ibrar khan, Dr. Kashif Bashir, Dr. Rizwan, Kishwar Sultana, all my lab colleagues who succor and guided me during my study at different occasions.

AISHMA KHATTAK

SUMMARY

The current Ph.D. dissertation predominantly highlights the phytochemical screening, biological evaluation, and phytofabrication of silver nanoparticles (AgNPs), using a medicinally significant plant Quercus semecarpifolia Smith, belonging to the Fagaceae family.

In order to check for the presence of important phytochemical constituents, the qualitative phytochemical studies of the plant were also performed. Therefore, the plant was investigated for the different classes of organic compounds, which have shown the occurrence of phenol, glycosidases, alkaloids, saponins, flavonoids, sterols, tannins, and reducing sugars. The plant material was extracted both in the aqueous and methanolic phases. Different solvents such as n- hexane, chloroform (CHCl3), and ethyl acetate (EtOAc) were employed for the fractionation of the crude methanolic extracts (Cr. MeOH Ext). The maximum number of phytochemicals was present in the EtOAc fraction and therefore, this fraction was used for the isolation of compounds by means of column chromatography (CC). Five compounds were isolated from it among which, three were the carboxylic acids; benzoic acid (1), p-hydroxybenzoic acid (2) and

Bis (2-ethylhexyl) phthalate (3) while the remaining two were the phytosterols; stigmasterol (4) and ß-Sitosterol (5).

The present study illustrates an eco-friendly, green strategy for the production of AgNPs, using Q. semecarpifolia aqueous extract. The process is a single step reaction in which, the reduction of the aqueous leaf extract occurs in the presence of a 1 mM silver nitrate (AgNO3) solution. The different phytoconstituents, present in the aqueous extracts of plants play key part in the formation of AgNPs. Characterization of the synthesized AgNPs was done with the different techniques including; UV-Visible Spectroscopy, X-Ray Diffraction (XRD), Scanning

Electron Microscopy (SEM), Energy Dispersive X-Ray Diffraction (XRD), Fourier Transform

Infra-Red (FTIR), Transmission Electron Microscopy (TEM), and Thermo gravimetric/

Differential Thermal Analysis (TG/DTA).

UV-Vis Spectroscopy is an effective technique for confirming the formation of nanoparticles

(NPs). A characteristic absorption band of the synthesized AgNPs was perceived at 430 nm.

XRD pattern is generally used for the determination of crystalline nature of the AgNPs and the average crystal size of the AgNPs was found to be 8.5Å according to Debye-Scherrer‘s equation.

The size and structure of the particles were confirmed by techniques i.e. SEM and TEM. The

TEM investigation showed that the synthesized particles were spherical shaped with size of 20–

50 nm. The EDX study revealed the presence of the element silver (Ag) alongside the elements such as oxygen (O), magnesium (Mg), silicon (Si), potassium (K), carbon (C), sulfur (S), calcium (Ca), and chlorine (Cl). The TG/DTA results attributed thermal stability to the synthesized AgNPs.

The various fractions of the Cr. MeOH. Ext. and the plant-derived AgNPs were checked for different in vivo and in vitro pharmacological activities. The in vitro assessments included checking for antimicrobial, antioxidant, phytotoxic, cytotoxic, insecticidal, antitermite, allelopathic, and haemagglutination activities.

Accordingly, the test samples were checked for possible antibacterial effects against the selected pathogenic strains such as Serratia marcescens, Escherichia coli, Staphylococcus aureus,

Bacillus subtilis, Proteus mirabilis, Klebsiella pneumoniae, Pseudomonas aeruginosa, and

Streptococcus pneumoniae. The results revealed that the extracts and plant-derived AgNPs possessed antibacterial effects. The plant-derived AgNPs showed stupendous antibacterial activities against K. pneumoniae, B. subtilis, and P. mirabilis. Among the extracts, the CHCl3 and EtOAc fractions showed noteworthy antibacterial activities against K. pneumoniae. The

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antifungal activities of the test samples were determined against Aspergillus flavus, Penicillium notatum, Aspergillus niger, Fusarium oxysporum, Trichoderma harzianum, and Candida albicans. Both the aqueous extract-derived AgNPs and crude extracts showed low antifungal activities against the above-mentioned fungal strains. Similarly, the antioxidant activity was also determined by the DPPH assay. At the highest concentration of 300 μg/mL, excellent antioxidant activities were shown by both the synthesized AgNPs and crude extracts.

Simultaneously, the test samples were also investigated for probable phytotoxic, cytotoxic, insecticidal, antitermite, and allelopathic activities. In the assessment of phytotoxic activity, the percentage of inhibition was amplified at the highest concentration of 1000 μg/mL.

The AgNPs, derived from Q. semecarpifolia showed significant phytotoxic activities. In contrast, the crude extracts, as well as the CHCl3 and EtOAc fractions, displayed moderate phytototoxic activities at the highest concentration of 1000 µg/mL Furthermore, insecticidal activities were checked against the selected species of insects such as Tribolium castaneum, Callosobruchus maculatus, and Rhyzopertha dominica. The plant-derived AgNPs showed excellent insecticidal activities against T.castaneum and C. maculatus but did not show any mortality effects against R. dominica. Similarly, all the extracts showed moderate to significant activities against the test species of insects. The extracts and AgNPs were also screened for antitermite activities against the test species Formosan subterranean termite. The results revealed that all the extracts were effective against the selected termite species. Similarly, the plant extracts and synthesized

AgNPs were screened for allelopathic effects. The results showed that the test samples had a momentous amount of biomolecules. Finally, the absence of phytolectins was displayed by negative haemagglutination activities.

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The Cr. MeOH Ext. and plant-derived AgNPs were also screened for in vivo activities such as acute toxicity assay, antinociceptive assay, anti-inflammatory assay, and antipyretic assay in experimental animal models. The Cr. MeOH Ext and synthesized AgNPs did not show any mortality effects within 24 h. However, significant antinoceceptive activities were shown by the test samples. Significant anti-inflammatory and antipyretic activities were also shown by all the test samples.

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INTRODUCATION & LITERATURE REVIEW

1.1 General Introduction

All through history, humans have depended on plants for their essential requirements such as food, clothes, housing, flavors, and fragrances [1, 2]. The natural products, isolated from plants have been a very important source of medicine used by mankind, in order to cure and prevent various diseases [3]. The traditional medicinal system that is basically formed of plants has been used by humans since the prehistoric period, offering new therapies to people till now [4]. The earliest records of plant-based therapy date back to 2900 BC in Egypt and to 2600 BC in

Mesopotamia, where about 1000 plant-derived medicines are described. One of the earliest

Egyptian pharmacological records is the Ebers Papyrus that comprises over 700 different drugs such as pills, infusions, and poultices along with their uses in 800 prescriptions [5, 6].

Mesopotamia, written on clay tablets in the cuneiform script dates back to about 2600 BC and the substances described in it are the oils of Glycyrrhiza glabra, still used in many parts of the world for curing various diseases like cold, cough, and inflammation [7-9].

Ayurveda, the most ancient of all traditional medicine systems is approximately 5000 years old. A detailed description of over 1500 herbs and 10,000 formulations is present in the ancient Ayurveda transcripts [10]. The Chinese traditional medicine system, also in use since ancient times consists mostly of the drugs of plant origin. Nearly 5000 conventional medicines are available in China, representing one-fifth of the whole Chinese medicine market. The Nei

Ching is one of the earliest health science anthologies available from the thirtieth century [11,

12].

The Greek and Roman civilizations have also contributed extensively to the development and implementation of herbal medicines. Diocles of Carystus, the earliest known Greek

1 Chapter 01 Introducation & Literature Review

pharmacopeia was written during the third century BC. Also, Hippocrates, one of the earliest medical practitioners preserved the Greek and Roman homeopathic preparations in his book De herbis et curis. Gallen gave a detailed prescription about western medicine in his book

Therapeutics [13, 14]. Dioscorides (40–90 AD) was another pharmacist who described the different properties and effects of 700 plants in his book De Materia Medica [15]. The

Benedictine monasteries also contributed in preserving the medical knowledge of the Greeks and

Romans in the early middle ages and most of them were translated into the Arabic language [16,

17]. In the early 800 century, Baghdad was the centermost origin of plant herbalism in Arabia.

The two important books, written at that time were the Book of Simples and the Corpus of

Simples, describing the thousands of new herbal plants present in the Arab world [18]. The other primary pharmacopeias of that time comprised the Al-Qanun fi al-tibb (The Law of Medicine), and Kitab al-shifa (The Book of Healing), which are the most famous books in the history of medicine [19].

The rational discovery of drugs from plants with new techniques began in the nineteenth century, in order to isolate active ingredients from plants and extracts. The first pure compound, morphine was isolated from the plant Papaver somniferum in 1816 [4]. The discovery increased the isolation of other natural constituents from plants and many natural constituents such as cocaine, atropine, caffeine, colchicine, nicotine, and capsaicin were isolated at the beginning of the nineteenth century. A large-scale production of antibiotics and analgesics began in the twentieth century and the discovery of natural products flourished when pharmaceutical enterprises refocused on their quest for the new drugs of plant origin. Consequently, the search for pure compounds from plants increased and major advances were seen in drug development.

Today, many of the vital compounds, meant for pharmacotherapy are derived from plants [20].

2 Chapter 01 Introducation & Literature Review

In developing countries, plants are still an important source of medicine and people living in these countries use plant-based medicines for their overall wellness [21]. All over the world, compounds, derived from natural sources are still used in common practice as the sources of medicine for humans. Many of the important techniques used in chemistry have gained interest in recent years for the synthesis of the important plant-derived drugs such as metformin, salbutamol, salmeterol, paclitaxel, vincristine, vinblastine, topotecan, and irinotecan, which are used to cure a wide range of diseases, including asthma, stomach disorders, and the different types of cancer [22]. Some of the plant species, widely used for therapeutic purposes are described in Table 1.1.

The consumption of natural products is increasing day by day as there are little or no side effects of these products. People, living throughout the world are getting interested in these products, which has opened the doors for pharmaceutical industries in unleashing the detailed pharmacognostic properties of plants, which are yet to be checked. Since the year 2000, approximately 119 of the drugs, in addition to the current 37 are reported to be made from natural products and are in the various stages of clinical development [23]. Some of drugs which are derived from natural products are enlisted in Table 1.2.

3 Chapter 01 Introducation & Literature Review

Table 1.1: Compounds isolated from natural products since 2000

Year Generic Name Clinical use 2000 Rivastigmine Nervous disorders NP derived

2001 Galantamine Anti-Parkinson NP

2002 Nitisinone Mental retardation NP derived

2003 Miglustatl Metabolic disorders NP derived

2004 Tiotropium Lungs inflammation NP derived

2004 Apomorphine Anti-Parkinson's NP derived

2006 Varenicline Addiction NP derived

2007 Lisdexamfetamine Nervous disorders NP derived

2008 Methyl-Naltrexone Gastro-intestinal NP derived

2009 Artemether Anti-leshmanial NP derived

2010 Capsaicin Topical pain NP

2010 Cabazitaxel Liver carcinoma NP derived

2011 Vandetanib Cancer NP derived

2012 Ingenol-Mebutate Leukemia NP

2013 Canagliflozin Diabetes NP derived

2013 Trastuzumab Emtansine NP derived Lymphoma 2014 Dapagliflozin Diabetes NP derived

2014 Empagliflozin Diabetes NP derived

2015 Naloxegol Neoplasm NP derived

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Thus, on one hand, pharmacognosy is developing and on the other, nanotechnology has emerged as an interdisciplinary science in this modern world. Nanotechnology is defined as the use or manipulation of matter on a molecular or atomic scale with applications in pharmacology, biomedical sciences, cosmetics, food processing, formulation, optics, electronics, and chemical industries [24-26]. Throughout the world, exploration in this emerging technology is progressing rapidly, taking most of the knowledge from a variety of sciences such as chemistry, physics, biology, electrical sciences, and material sciences. Consequently, bionanotechnology is a specific area of this field that amalgamates the chemical and physical procedures, using biological principles for the phytosynthesis of nanosized particles, having unique characteristics [24].

Metallic NPs can be synthesized by both chemical and physical methods. However, toxic and high-cost reagents are used as the reducing agents in these methods. Recently, the processes for the green fabrication of environment-friendly metal NPs, using algal, fungal, and plant extracts have received much attention [27]. Among these, the most reliable one is a green method, using plant extracts because of the phytochemical constituents present in them. The method is also cost-effective and can serve as an appropriate substitute for the production of important NPs

[28].

In the current study, the phytochemical evaluations, bio prospecting, including biological and/or pharmacological studies and plant-based production of AgNPs are carried out for the selected plant Quercus semecarpifolia, a member of the Fagaceae family.

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1.2 Fagaceae (Family)

1.2.1 Description

Among the flowering plants, Fagaceae is the largest family of monoecious or deciduous evergreen trees and shrubs, comprising eight genera with about 927 species of oaks and beeches.

The leaves are simple or alternate, often lobed, usually with petioles and stipules. The male flowers are either solitary or arranged in groups and are present on either slight catkins or spines, having five stamens, four to six sepals, and no petals. They have three branched styles, six to eight sepals, and three carpels, which are normally united.. The fruit is a nonvalved nut, mostly consisting of a single seed called an acorn [29, 30].

1.2.2 Distribution

The members of the Fagaceae family are extensively distributed and abundantly present in the subtropical areas of the Northern Hemisphere, North America, and central Europe.

Approximately, eight to ten genera and 900 species are present in the Fagaceae family [31 ].

1.2.3 Importance

The numerous species of plants, included in the Fagaceae family have significant uses. The species of the genera Quercus, Castanea, and Fagus are commonly used as timber for furniture, cabinets, and wine barrels. Edible fruits, obtained from the several species of the genus Castanea are used worldwide. Woodchips, obtained from the various species of the genus Fagus are usually used as flavoring agents [32].

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1.3 Quercus (Genus)

1.3.1 Description

Quercus, commonly known as oak is an important genus of the Fagaceae family and includes monoecious, deciduous, evergreen trees, and rarely shrubs. The leaves of many of the oak species are clearly lobed but some species show variations in shape from small to large and pointed. The buds, occurring in clusters are present at the terminal end of each stem. Oaks are considered monoecious plants, having separate male and female flowers on a single tree.

Generally, the male flowers occur in clusters but sometimes they may occur singly in a form called catkin. The perianth sometimes bugles or copular, having three to six nodes and is commonly enclosed by a number of scales. Sometimes, stamens and pistillode are present and usually, the number of stamens is six. The female flowers are small, brownish green, occurring singly or in the form of a spike. The ovary has many styles and three to five locules. The flowers mostly ripen in the sepals, which later mature into the fruit. The fruit is mostly a nut, which is enclosed in a capsule made by hard scales. The seeds are usually present which may be solitary

[33].

1.3.2 Distribution

Oaks (Quercus) are native to Asia, Europe, and America. The highest number of 180 oak species is present in North America. The second largest center for a variety of oaks is China, which contains approximately 100 species. In Pakistan, this genus is mainly distributed in the northern parts, characterized by a total of six species [34]. The list of oak species is given in Table 1.3.

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1.3.3 Importance

In the traditional medicine system, the members of the genus Quercus have noteworthy medicinal status and use. Local people, living in a particular area, use them as antiseptics and hemostatic for treating gastrointestinal tract (GIT) disorders such as diarrhea and hemorrhoids

[35, 36]. Some of the species have antimicrobial, antioxidant, anti-inflammatory, gastroprotective, and cytotoxic activities [37, 38]. Besides, the plant decoctions, added in the ointments, can be employed for the cure of wounds, cuts, and burns [36]. The antibacterial, antioxidant, and gastroprotective effects are exhibited by certain plant species such as Q. ilex, Q. robur, and Q. alba [39-43]. Moreover, certain species of Quercus are used for the treatment of gonorrhea, gastritis, asthma, pyrexia, Parkinson‘s disease, and hepatoprotective diseases [44].

The bark of the oak has much importance and is used extensively in medicine as an antiseptic and an energizer. Moreover, it has been suggested for the patients with hemorrhages and bruises and is also administered in an injectable form to leucorrhea patients. Until now, it is also used as a good substitute for quinine in a recurrent fever that occurs in some parts of the world. The bark of oak is also useful in chronic diarrhea and dysentery and a decoction made from the bark is very useful in the treatment of a sore throat [45]. Previously, the decoctions made from the barks of Q. robur and Q. petraea were identified to have anti-inflammatory, antibacterial, and acerbic activities [46]. Different extracts, prepared from the bark of oak have several medicinal values, which include their use as pacifying agents in inflammation and as healing agents in burns [47].

In their study, Mecune et al. shed light on the importance of the bark of the various species of

Quercus for the treatment of patients with high levels of blood sugar [48]. Another study revealed that Q. rugosa has a restorative action in the abscesses of the gastric tract [49].

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Gallic acid and tannic acid, broadly employed in the preserving and dyeing industries, are generally prepared from the oak galls. Medicinally, they are powerful astringents, having antimicrobial properties and are used internally as a tincture for gonorrhea, diarrhea, and dysentery [50]. Previously, it was reported that the galls of Q. infectoria are a good source for the healing and restoration of uterine elasticity, postpartum. Besides, local people in many parts of the world use them for treating many inflammatory disorders [51].

The fruit (acorn) of oak species is a rich source of energy, containing high amounts of carbohydrates, proteins, amino acids, lipids, and sterols. The oils obtained from the acorns are easily degradable and are a rich source of energy. In addition, some of the biologically active substances of the acorns are utilized in preparing functional foods [52, 53]. The fruits of Q. pubescens can be used for cooking purposes and the powdered form of the fruits is used in making bread [54]. Linoleic acid, isolated from the two species of Quercus (Q. cerris and Q. robur) is primarily used in the treatment of congestive heart diseases [55]. The acorns of the various species of oak are widely used in curing diarrhea, menorrhagia, and stomach ulcers.

Some of the species have organic constituents, which are antidiabetic and are also used as antihyperlipidemic agents [56-58].

In their study, Viegi et al. postulated that a number of oak species can be used in treating skin problems and the complications of the intestinal tract [59]. In another study, Lentz verified that

Q. hondurensis can be used in treating gastric complications [60]. The leaves of Q. virginiana are renowned for displaying antimicrobial properties. Also, the plant can be used for the treatment of gastrointestinal disorders [61, 47].

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Table 1.2: Quercus species in Pakistan and their worldwide distribution

S.no Plant Flowering Distribution

period

1 Quercus dilata March-August Nepal, Kashmir, Afghanistan, India, Pakistan,

America

2 Quercus latifolia Febuarry -June Japan, China, Pakistan, India,

3 Quercus incana April-June Pakistan, Nepal, China, India, America

4 Quercus May-August Afghanistan, Northern parts of Pakistan, China,

semicarpifolia Mexico

5 Quercus baloot April-June Pakistan, China, Afghanistan

6 Quercus robur March-May Europe, N. America, Turkey, Iran, China,

Pakistan.

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1.4 Quercus semecarpifolia (Plant)

1.4.1 Description

Botanical Name: Quercus semicarpifolia Smith

Kingdom: Plantaeae

Order: Fagales

Family: Fagaceae

Genus: Quercus

Species: semicarpifolia

Quercus semecarpifolia is a large, gregarious, evergreen tree that forms a long trunk with a height of 24–30 m and a width of 210 cm. The underdeveloped parts of the larger trees are generally hollow and covered with soft hairs. The barks of the trees are usually dark grey, rough in morphology, and splintered into rectangular scales. The leaves are elliptical or rectangular (5–

12×2.5–7.5 cm), spiny in the young trees and intact in the older trees, coriaceous, stuffy, dark green above and brownish underneath, having six to twelve pairs of lateral nerves, diverging with a rounded base. The new leaves have brown deciduous stipules, are bright green above and light brown beneath. Often, mosses and lichens are present on the branches. The male catkins, measuring 5–10 cm in length are contained in the seeds. The male catkins and the female spikes occur in compact clusters on the new shoots. The perianth is ciliate and the stamens are indefinite. Sometimes, the acorn (fruit) occurs singly or in clusters of three to six on the shoots of the previous year, blackish when ripe, leathery, 2.5 cm in diameter, and tipped with reddish brown scales [62, 63].

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1.4.2 Distribution

Q. semecarpifolia is native to the Himalayas and the nearby mountains of Tibet, Afghanistan,

India, Nepal, and Pakistan. In Pakistan, this perennial tree is found in the northern mountainous ranges, specifically in the Hazara, Azad Kashmir, Chitral, Swat, Dir, and Murree hills [64, 65].

1.4.3 Importance

Many ethno-botanical uses of this medicinal herb are known. Q. semecarpifolia is used for the cure of chronic diarrhea, dysentery, and hemorrhages. The bark or the galls, produced on the trees, are boiled and applied to swollen tissues, bruises, and varicose veins. Usually, the juice, obtained from the bark is used in the treatment of muscular pains. In Pakistan, this plant is used locally as a diuretic and an astringent as well as in gastritis and asthma [66]. The seeds of this tree can be dried and the powdered form is either used as a thickening agent or mixed with cereals for making bread. The bitter tannins, present in the seeds, can be removed by a thorough washing of the seeds with water [67]. The formation of oak galls is an important feature of this tree, which is caused by insect larvae. The galls are rich in tannins and can also be used as coloring agents [68]. The bark of this tree is also a source of tannins, which are mostly used in the tanning industry. The wood, being very hard, is used in construction and is also used as an excellent fuel since it yields good-quality charcoal [69, 47].

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Figure 1.1: Morphology of Q. semecarpifolia plant

Figure 1.2: Zoom version of leaf of Quercus semecarpifolia plant

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1.5 Preliminary Phytochemical Profile of the Genus Quercus

The different classes of natural compounds such as glycosides, terpenoids, flavonoids, sterols, and tannins are present in the different species of the genus Quercus [70, 71]. The phenolic acids, particularly gallic and ellagic acids and their derivatives, are abundant in all the species of

Quercus [72-74]. Previous studies have shown that the oak galls contain a high amount of tannins, gallic acid, syringic acid, ellagic acid, methyl oleate, methyl betulate, flavones, and isocryptomerin [75, 76]. Many reports on the different species of Quercus have explained that the bark of oak has significant medicinal importance. Previously, the compounds, such as proanthocyanidins and condensed tannins, were isolated from the bark of oak [77-79].

A series of flavones were also isolated from the various species of Quercus [80]. Besides, several chemical constituents such as vanillic acid, toluene, egallic acid, tannic acid, monoterpenes, kaempferol, and coumarin were isolated from the species such as Q. macranthera, Q. infectoria,

Q. libani, and Q. aegilop [81]. Using advanced spectroscopic techniques, Rodriguez et al. performed a detailed investigation on the cyclic polyols, present in the different species of oak.

Of the eight cyclitols characterized by the mass spectrometry studies, four were identified as muco, myo, chiro, and scyllo (inositol). The other four cyclitols were identified as deoxy- inositols, which were similar to inositol but presented some characteristic features [82].

Previously, Kuliev et al. isolated more than 20 phytochemical constituents from the bark of Q. robur [83]. A study, carried out on the leaves of the five different species of Quercus revealed that they contain high levels of chlorogenic acids, gentisic acids, and flavonoids [84]. In another study, flavanols were isolated exclusively from the aerial parts of Q. ilex [85]. Cantos et al. reported that several gallic acid derivatives are found in the species, Q. rotundifolia and Q.

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robur. The different classes of phytochemicals were determined and were found to be associated with the antioxidant and antimicrobial activities of the aerial parts of Q. robur [86]. Using silica- gel column chromatography, Yuan et al. evaluated a study on Q. mongolica and isolated six compounds, which were identified as 1-octadecanol, amylin, fridelin, daucosterol, β-sitosterol, and gallic acid [87]. Previously, five new kaempferol derivatives were isolated from the species,

Q. dentate [88] and three phytochemical constituents namely catechin, epicatechin, and tiliroside were isolated from Q. gilva [89].

In addition to the 26 structurally known tannins, five new tannins, comprising proanthocyanidins and phenol glucoside gallates were isolated from the leaves of Q. phillyraeoides [90]. Gul et al. screened EtOAc extracts from the bark of Q. incana for phytochemical investigations and isolated a new compound called quercuschin, along with six other compounds that were identified as quercetin, betulinic acid, methyl gallate, octadecenoic acid, gallic acid, and β- sitosterol glucoside [91].

Previously, several organic compounds such as phenols, phenolic aldehydes, polyphenols, furanic compounds, lactones, and phenyl ketones have been isolated from Q. pyrenaica and Q. petraea [92]. In another study, along with the 14 known glycosides, Romussi et al. isolated a new flavonoid from the leaf extracts of Q. laurifolia and identified it as 1, 2, 3, 6- tetragalloylglucose [93]. Sohretoglu et al. in their study identified three new phytochemical constituents, viz., kermesoside, cocciferoside, and chlorocatechin from the bark of Q. coccifera along with the five known phenolic compounds [94]. In another study, Q. suber was subjected to column chromatography and three new hydrolysable tannins were isolated [95]. Sakar et al. isolated five new compounds from Q. aucheri leaves and structural elucidations through

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advanced spectroscopic techniques identified two of the compounds as flavonol glycosides, while the remaining three were the tannin precursors, viz., gallocatechin, catechin, and epicatechin [96].

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Table 1.3: Phytochemical constituents from genus Quercus

S.no Specie Mol. formula Mol.mass Compound isolated Ref

1 Q. ilex

Q. annulata 1,2,3 benzenetriol [97] C6H3(OH)3 126.11 Q. penduculata

Q. phillyraeoides

Q. cortex

2 Q.cortex

Q. myrsinaefolia C3H8O3 95.094 1,2,3-Propanetriol [97 ]

3 Q.cortex

Q.ilex C10H22 142.286 Decyl hydride [97] 4 Q.cortex

Q. pyrenaica C5H4O3 112.084 Pyromucic acid [97]

5 Q.cortex C15H32 212.421 Pentadecane [97]

6 Q.cortex C4H6O4 118.088 Butanedioic acid [ 97]

7 Q. resinosa C16H12O6 316.256 7-Methoxy kaempferol

Q. glauca [98]

Q.incana C10H8O4 192.168 7-hydroxy-6-methoxy-2H- [97] chromen-2-one

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9 Q. robur C9H12O4 184.191 3,4,5-trimethoxy-phenol [97]

10 Q. bambusifolia Q. phillyraeoides C7H6O3 138.122 4-Carboxyphenol [105] Q. resinosa

Q. glauca

11 Q.cortex 5 (hydroxymethyl)furan-2- [97] carbaldehyde Q.ilex C6H603 126.111 12 Q.cortex [97]

Q. resinosa C15H28O2 240.381 2-Propenoic acid

13 Q. pyrenaica

Q.cortex 4,6-dihydroxy-2-methyl-5- [97] Q.ilex nitropyrimidine C5H5N3O4 171.112

14 Q.cortex [97]

C10H13N5O5 283.244 Guanine riboside

15 Q.cortex C7H13O5 177.176 1,6-anhydro-β-D- [97] glucofuranose

16 Q.coccifera C10H12O3 180.200 Coniferol [99]

17 Q.coccifera C6H12O5 163.156 Proto-quercitol [99]

18 Q.coccifera C6H5ClO2 144.567 8-chlorocatechin [99]

19 Q.coccifera C11H14O5 226.228 Propiosyringone [99]

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20 Q. glauca

Q. petraea C8H8O3 152.149 Vanillic aldehyde [100, Q. pyrenaica 101]

Q. resinosa

Q. salicina

21 Q. glauca lyoniresinol-9-O-β- [99] xylopyranoside

C20H22O8R 390.388 22 Q. salicina

Q. phillyraeoides HOC6H4COOH 138.122 2-Carboxyphenol; [100, Q. resinosa 102]

23 Q. myrsinaefolia

Q. phillyraeoides C7H6O4 154.121 Protocatehuic acid [100, Q. resinosa 102 ]

24 Q. glauca

Q. myrsinaefolia C9H8O3 164.16 4-Hydroxycinnamic [100, Q. phillyraeoides 102 ]

25 Q. phillyraeoides

Q. salicina C7H6O2 154.12 Hydroquinone carboxylic [100] acid

26 Q. sessilis

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Q. phillyraeoides

Q. robur C7H6O5 170.12 3,4,5-Trihydroxybenzoic [100 102 Q. resinosa acid; 86]

Q. salicina

Q. suber

27 Q. acuta

Q. myrsinaefolia C8H8O4 168.148 Homogentisic acid [100] Q. phillyraeoides

Q. salicina

28 Q. myrsinaefolia

Q. phillyraeoides C7H6O4 154.121 4-Hydroxysalicylic acid [100 ] Q. salicina

29 Q. acuta

Q. glauca C16H14O6 610.188 Hesperidin [100]

30 Q. petraea

Q. faginea C9H6O4 178.183 Cichorigenin [101] Q. pyrenaica

Q. robur

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31 Q. acuta

Q. phillyraeoides C9H10O4 182.175 3,4-Dimethoxybenzoic [100] Q. glauca acid;

32 Q. acuta

Q. myrsinaefolia C9H8O4 180.159 3,4-Dihydroxycinnamic [100, Q. phillyraeoides acid 102]

33 Q. alba

Q. faginea C9H10O4 182.175 Syringic aldehyde [101]

34 Q. alba

Q. faginea C11H12O4 208.221 Sinapic aldehyde [101] Q. petraea

35 Q. phillyraeoides

Q. pyrenaica C10H10O4 194.186 Trans-4-Hydroxy-3- [100, Q. robur methoxycinnamic acid 101]

Q. salicina

36 Q. alba

Q. phillyraeoides C16H12O4 268.248 7-Hydroxy-4'- [100] methoxyisoflavone

37 Q. acuta

Q. pyrenaica [100, 102 ]

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Q. phillyraeoides C15H14O6 290.26 Catechuic acid

40 Q. baloot

Q. glauca [100] Q. phillyraeoides C16H14O6 302.282 3',5,7-Trihydroxy-4'- methoxyflavanone

41 Q. petraea

Q. pyrenaica C14H6O8 304.197 Benzoaric acid [101] Q. robur

42 Q. acuta,

Q. glauca, C15H12O5 272.256 5,7-Dihydroxy-2-(4- [100] hydroxyphenyl)chroman

43 Q. acuta C15H10O8 318.237 Cannabiscetin [100] Q. glauca

Q. glauca C15H10O7 302.236 Meletin [100] 44 Q. myrsinaefolia

45 Q. ilex C15H14O7 306.27 Epigallocatechol [103]

46 Q. resinosa

Q. salicina C16H18O9 354.31 3-O-Caffeoylquinic acid [102]

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47 Q. ilex C22H18O10 442.376 442.376 Catechin 3-O- [103] gallate

48 Q. ilex C27H30O16 610.521 Quercetin pentoside [103]

49 Q. ilex [104,105 ] Q. rotundifolia C41H32O26 940.681 Galloyl glucoside

50 Q. ilex C21H20O11 448.38 Kaempferol hexoside [103]

Quercetin-3-O-beta-D- [103] glucoside 51 Q. ilex C21H19O12 436.314

52 Q. acutissima C14H6O8 302.194 Ellagic acid glucoside [104, Q. macrocarpa 105]

4H-chromen-2- phenoxyoxane-2- 53 Q. ilex C21H18O13 478.362 carboxylic acid [103]

54 Q. ilex C22H22O12 479.406 Rhamnetin hexoside [103]

55 Q. macrocarpa C14H10O10 338.224 Hexa hydroxyl diphenoyl- glucoside [104] 56 Q. palustris

Q. rotundifolia C21H22O12 466.395 Digalloyl glucoside [103- 105]

57 Q. acutissima [104, 105] Q. macrocarpa C18H28O9 388.413 Ellagic acid-4-O-beta-D- xylopyranoside

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58 Q. ilex [86]

Q. rotundifolia 940.653 C41H32O26 Pentagalloyl glucoside

59 Q. rotundifolia

Q. suber C34H28O22 788.556 Tetragalloyl glucoside [86]

60 Q. acutissima

Q. macrocarpa C41H62O14 778.933 Dihexahydroxydiphenoyl- [104, glucoside 105]

61 Q. ilex [86, 103] Q. rotundifolia C27H24O18 636.432 Trigalloyl glucoside

Q. ilex [86]

62 Q. rotundifolia C22H22O11 462.407 Tergallagic C-glucoside Q. suber

63 Q. marilandica

Q. muhlenbergii [86] C13H16O10 322.261 Galloyl- hexahydroxydiphenoyl- glucoside

64 Q. ilex C27H30O14 578.543 Vitexin-2''-rhamnoside

Q. rotundifolia [86] Q. suber

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65 Q. marilandica C27H30O16 610.521 Phytomelin [100]

Q. glauca

Q. myrsinaefolia

66 Q. incana [100]

Q. robur C28H34015 610.132 Hesperidin Q. myrsinaefolia

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1.6 Nanotechnology

1.6.1 Background

Nanotechnology has emerged as an interdisciplinary science, having applications in biomedical sciences, pharmacology, food processing, cosmetics formulation, optics, electronics, and chemical industries [106-108]. The research in this emerging technological field is progressing rapidly throughout the world. According to the ‗National Nanotechnology Initiative of the USA‘, it is defined as ―research at the atomic or molecular levels, using a scale of one to 100 nm in any dimension‖. Using this technology, novel structures, and systems, having unique properties and functions due to their small size can be created [109]. According to R.D Booker, the history of nanotechnology is difficult to describe due to two facts:

1. The ambiguity of the term ―nanotechnology‘‘ and

2. The uncertainty of the time, describing the initial stages in the development of

nanotechnology.

The difference between the ancient and current concepts of nanotechnology is the ability to understand and also to gain knowledge about the basic principles of this technology for future developments.

The basic concept of this technology, as put forward by the Father of Nanotechnology—Richard

Feynman in one of his lectures in the year 1959 is that ‗there is a lot of space at the bottom‘, meaning matter is employed at the atomic level [110]. The above concept opens the doors for new thinking and in the year 1974, almost 15 years after Feynman‘s lecture, the word

―nanotechnology‘‘ was coined by Norio Taniguchi [111 ]. In the early eighties, rapid progress in the field of nanotechnology and nanoscience occurred with two major developments—the beginning of the cluster technology and the discovery of the Scanning Tunneling Microscope

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(STM), [112] which led to the invention of the fullerenes in the year 1985 and the fundamental project of carbon nanotubes (CNTs) in the year 1991 [113, 114 ]. Thus, important discoveries were made and further developments in the field of nanotechnology occurred in the eighties and early nineties [115]. In the year 1991, the first nanotechnological platform of the National

Scientific Fund started functioning in the United States of America (USA). Since then, a lot of technical and practical developments in nanotechnology research have taken place worldwide, particularly in countries like Japan, China, Germany, France, and South Korea [116].

1.6.2 Current Status

Currently, nanotechnology is getting a boost in the various fields of science, bringing out the concept that procedures, structures, and systems can be characterized and managed in a detailed manner. Generally, two strategies are used in this technology—a top-down fabrication strategy, where small structures are created from large substrates and a bottom-up fabrication strategy in which, a patching together of systems gives rise to complex ones. Nowadays, nanotechnology has become the basis for noteworthy industrial applications and exponential growth [117]. By the end of the year 2011, about 800 products of nanotechnology were publically available and new ones are striking the market at a very rapid pace. According to a survey conducted by the scientists of the USA and Europe between the years 2000 and 2010, the application of nanomaterials has grown in the many fields of industry, including medicine, food processing, agriculture, and optics [118 ].

Previously, Chunkrekkul et al. stated that multiple nanoproducts are traded worldwide, including garments, microchips, and medical appliances [119]. The various metallic NPs are now employed for their tenacities. Silver (Ag) is broadly used in the packaging of food, clothing, sanitizers, and household usages, while zinc oxide (ZnO) is used in cosmetics, paints, and

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furniture varnishes. Titanium dioxide (TiO2) can be used in cosmetics, surface coatings, and some food products. The allotropes, nanofibers, and nanotubes of carbon (C) are used in some of the other consumer products [120 ].

1.7 Nanobiotechnology

1.7.1 Background

Nanobiotechnology or nanobiology is a term that confers the integration of biological research with the different fields of nanotechnology. Bioinspired nanotechnology that ushered in the early nineties through scientific perceptions and endurance mainly uses biological methods for the development of many useful nanoproducts. The properties and principles, concerned with the materials, are central in nanobiotechnology because these are used to create new technologies

[121]. The properties and uses of the materials, studied in nanobiotechnology include mechanical, electrical, thermal, optical, biological, biosensing, the nanoscience of diseases as well as their applications in computing and agriculture. Using these hybrid principles of nanobiotechnology, scientists across the globe have fabricated many functional nanodevices

[122].

1.7.2 Current Status

Nanobiotechnology is a novel domain that provides many tools and the knowledge for the biological processes and pathways, refabricating them in a form that will be more useful than in the past. At present, a number of therapeutic uses of nanobiotechnology are in wide practice and some of the new therapies are also in clinical trials [123]. Nanospheres, one of the current products of nanotechnology research, are coated with fluorescent polymers and can be used in the diagnosis of pathological metabolites and cancerous tissues [124]. Another product called

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nanotubes can store the biological data of living organisms and this will be helpful in future for the optical computing processes [125]. Moreover, NPs, which have received much attention in all the fields of science, serve as carriers, containing the genes of interest to the target areas, which cannot be easily targeted by the conventional drugs [126]. Another foremost area of study in nanobiotechnology is lipid nanotechnology, where the unique properties of lipids such as self- assembly are subjugated for the construction of nanodevices, having broad-spectrum applications in the fields of medicine and engineering [127]. In addition, the field of nanobiotechnology offers many new tools such as atomic force microscopy (AFM) and optical tweezers for imaging, nanomechanics for computational studies and dual-polarization interferometry (DPI) for assembly analysis [128].

1.8 Silver (Ag)

1.8.1 History

Silver (Ag) is a white, lustrous, transition metal, present either as an unalloyed, elemental form or sometimes as an alloy with other metals. From ancient civilizations, Ag has been earmarked for its speckled exploitation in the trading of costumes, jewels, and tools [129]. In the year 400

BC, Ag was mined and polished from the ores of lead (Pb) in Europe and Sardinia. The

Egyptians are thought to be the pioneers in separating Ag from gold (Au) by heating the latter with metallic salts. During the Greek and Roman civilizations, Ag coins were considered as the source of the economy. In the year 7 BC, Ag was extracted from galena (PbS) by the Greeks.

From the years 300–600 BC, about 30 tons of Ag was extracted from the Ag mines, located in Laurium. The Roman miners supplied Ag bullion mostly from Spain for which their currency remained stable before the discovery of the New World and in the middle of the second century, their estimated Ag stock reached its highest peak to about 10,000 tons [130]. By the end of the

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fifteenth century, Central Europe became the center of Ag production. At that time, Ag was extracted from the mines, located in Asace, Saxony, Hungary, Norway, and Bohemia and most of the Ag could be separated from the rocks simply. Between the years 60–120 AD, technologies, involving the cupellation of Ag and Pb were developed in the Americas [131]. By the end of the eighteenth century, Central America and South America became the foremost fabricators of Ag [132]. In the beginning of the nineteenth century, the primary producers of Ag were North America, particularly Mexico and Canada. During the 1970s, following the discovery of Cu deposits rich in Ag, Poland emerged as an important producer of Ag. Nowadays, the worldwide supply of Ag is from recycling instead of new production [133].

1.8.2 Importance

Until now, Ag, is used for various purposes worldwide has been a ruling aspect of monetary gadgets. Besides its use as a coinage metal, Ag was used in the production of jewelry in the past [134]. Because of its high electrical conductivity, Ag is mainly used in the manufacture of conductors, electrodes, semiconductor devices, circuits, and chemical equipment. Powdered Ag is used in the ceramic industry [135]. In order to work at high temperatures, Ag-plated equipment are made. In oxidation reactions, Ag plays the role of a catalyst [136]. Moreover, Ag, renowned for its novel antimicrobial properties since ancient times, is used as an antiseptic and is also applied in the medical equipment such as cardiac stents, catheters, and nasogastric tubes [137, 138].

Recently, AgNPs have become a very attractive topic as this novelty shrivels the mass popularity of Ag for specific purposes. The AgNPs are of much importance due to their distinctive nature and exclusive characteristics such as catalytic, optical, electrical, and most importantly, antimicrobial properties [139, 140]. All these attributes render them unique among all the

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metallic NPs [141]. Due to these properties; the AgNPs have been successfully implicated in the different fields of biomedicine such as diagnostics, pharmacology, molecular imaging, and drug delivery [142]. Moreover, they are being added to the topical creams, wound dressings, antiseptic sprays, and in the textile industry [143, 144].

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1.9 Different Methods Used for the Synthesis of Nanoparticles (NPs)

The different methods, applied for the production of the NPs, can be generally categorized as chemical, physical, and biological processes [145]. The physical approach for the fabrication of

NPs involves the techniques such as gas condensation, laser ablation, and arc discharge. In the gas condensation technique, the metallic materials are heated by a source of thermal vaporization and a high residual gas pressure causes the formation of the metallic NPs [146].

Apart from the physical methods, NPs can be synthesized by the chemical procedures among which, the most common is the use of reducing agents for the fabrication of NPs. The different reducing agents such as sodium citrate, sodium borohydroxide [147], Tollen‘s reagent [148], and methoxypolyethylene glycol [149] are used for the reduction of metal ions in liquid media. The stabilizing agents that adsorb the NPs onto the surfaces, avoiding agglomeration are also used

[150]. Though the chemical procedures have gained much attention for the large-scale production of NPs, they are unsafe and non-ecofriendly because chemical solvents are used and toxic byproducts is formed.

Therefore, all these limitations demand uncontaminated, environment-friendly, biocompatible, and cost-effective methods for the manufacture of NPs. Thus, the emphasis on the natural processes for the fabrication of NPs comes into play. For instance, a number of biological approaches, involving the use of microorganisms and plants can be exploited for the assembly of the nanoparticles [151].

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1.9.1 Biological Approaches for the Synthesis of Nanoparticles (NPs)

Several natural sources, including plant extracts, bacteria, fungi, algae, and yeasts are used for the synthesis of NPs. Usually, the biological approaches are nontoxic, biocompatible, and use environment-friendly methods for the fabrication of NPs [152]. Usually, two major strategies, viz., bottom-up and top-down, are used for the synthesis of metallic NPs [153]. Generally, in the top-down approach, bulk materials are broken down to NPs via the physical methods, involving grinding and etching techniques, while the chemical and biological procedures are used the bottom-up approach, which is based on the assembly of molecules or atoms into nanostructures

[154]. A green approach of synthesis makes the use of the enormous bioresources such as microorganisms, plant extracts or plant biomass for the synthesis of NPs.

There are many studies on the biological production of NPs, using plants and microbes such as bacteria, fungi, algae, and yeasts. However, the microbe-mediated synthesis of

NPs is not very appropriate for engineering viability due to their preservation and the requirements of highly aseptic conditions [155]. Thus, the plant extracts are possibly useful over microorganisms because they can be easily scaled up, less toxic, and biocompatible. The different parts of the plants, as well as their extracts in different organic solvents, have been used for the synthesis of NPs [156].

1.9.1.1 Biosynthesis of AgNPs, using Plant Extracts

In recent years, the green, biogenic synthesis of NPs has become more attractive as it is an eco- friendly, cost-effective, and single step process that does not require any toxic chemical substance. Previous reports have described that the different parts of the plants, including stems, leaves, barks, roots, flowers, fruits, and seeds can be used to obtain metallic NPs of numerous forms and morphologies [157]. There are many active mediators present in these plant parts,

33 Chapter 01 Introducation & Literature Review

which make the reduction and stabilization processes possible. A number of phytoconstituents such as phenols, flavonoids, alkaloids, polysaccharides, proteins, terpenoids, enzymes, and amino acids affect the reduction and stabilization processes [158]. Flavonoids and phenols are the nontoxic phytochemicals that have unique reducing properties and also help in the stabilization of NPs [159]. Furthermore, free aldehyde and carbonyl groups, present in sugars and proteins, have a potential to bind metal ions, forming NPs and also preventing agglomeration, thereby suggesting that these organic molecules not only have the ability to carry out the formation of NPs but they also stabilize the process of the biosynthesis of AgNPs [160].

Several factors, including plant source, organic compounds present in plant extracts, the concentration of metallic salts, pH, and temperature play important roles in the efficiency of the bioinspired synthesis of AgNPs [161]. The temperature and pH values have great influence on the formation of the AgNPs by plant extracts because the reducing property of phytochemicals changes with the fluctuations in temperature and pH and this may also affect the morphology, size, and production of fabricated AgNPs [162].

34 Chapter 01 Introducation & Literature Review

1.10 Bio inspired Synthesis and Characterization of the AgNPs

In their study, Patil et al. used the leaf extracts of Lantana camara, in order to synthesize AgNPs in an economical and environment-friendly manner. The leaf extracts were characterized by various advanced spectroscopic techniques. All these AgNPs displayed a sharp and well-defined

SPR band at a wavelength of 439 nm. The XRD configurations indicated that these AgNPs have a crystalline morphology. SEM imaging revealed that the synthesized AgNPs have spherical morphology with an average size of 10–50 nm [163].

Abdeen et al. demonstrated Olea europaea mediated synthesis of AgNPs, using a solution of

AgNO3. The aqueous extracts of the plant were treated with a solution of AgNO3 at a normal temperature and the synthesis of AgNPs was indicated by color change. Using the methods like

UV-Vis Spectroscopy, SEM, FTIR, XRD, and atomic absorption spectroscopy (AAS), the synthesized AgNPs were characterized and an SPR band was observed at the absorption maxima of 430 nm. The XRD patterns showed many diffracted intensities, which were recorded between

10–80o at a diffraction angle of 2θ. The FTIR spectra of the biosynthesized AgNPs revealed that hydroxyl (OH) or amine (NH2) groups were the key factors in the formation of AgNPs. The size of the AgNPs ranged from 10–30 nm with a uniform cubical morphology [164].

Anandalakshmi et al. evaluated using different concentrations of the leaf extracts of Vitex negundo for the bioispired synthesis of AgNPs. An SPR peak was observed at the absorption maxima of 423 nm. In the FTIR studies, flavonoids were shown to be the main reducing agents for the synthesis of AgNPs. The synthesized AgNPs have spherical configuration with a size, ranging from 50–70 nm as shown by FM. The EDX spectra showed strong signals of Ag, along with other elements. As studied by the XRD technique, the AgNPs were found to have a crystalline configuration with face-centered cubic geometry. The photoluminescence band of the

35 Chapter 01 Introducation & Literature Review

synthesized AgNPs displayed absorption spectra at wavelengths, ranging from 429–460 nm. The strength of the radiations was related to the different concentrations of the leaf extracts [165].

A simple process, exploiting the aqueous extracts of Cycas circinalis plant was utilized for the rapid production of the AgNPs of different dimensions. The synthesized AgNPs have spherical morphology with average sizes in the range of 15–50 nm [166]. Dwivedi et al. described a facile process for the biosynthesis of AgNPs from an abhorrent weed called Chenopodium album. As inferred from the TEM imaging studies, both AgNPs and AuNPs, having sizes of 10–30 nm with circular morphologies were successfully prepared from the plant extracts [167].

Jyoti et al. reported a green, biogenic approach for the fabrication of AgNPs, using aqueous and methanolic extracts of Tridax procumbens and AgNO3 as a reducing agent. Intense SPR peaks were observed for the AgNPs, derived from the aqueous and methanolic extracts at the wavelengths of 430 nm and 425 nm, respectively. The sizes of AgNPs were in the range of 20–

150 nm, as determined by the Particle Size Analysis and SEM imaging studies [168]. Swamy et al. employed the extracts of Leptadenia reticulata plant as reducing agents for the bioinspired synthesis of AgNPs. The different techniques, used for the characterizations included XRD,

SEM, and TEM. The formed AgNPs were found to be crystalline and mostly oblong in shape with sizes ranging from 50–70 nm [169].

Mohamed et al. successfully synthesized AgNPs, using the leaf extracts of Chenopodium murale and the results showed that the majority of AgNPs had sizes ranging from 30–50 nm. The total contents of phenolics and flavonoids were also determined and were found to be higher in the

AgNPs, compared to the aqueous leaf extracts [170]. Faria et al. did a detailed study, describing the bioinspired synthesis of AgNPs, using the leaf extracts of Cydonia oblonga under optimal conditions by regulating pH, temperature, and the strength of seed extracts. When a solution of

36 Chapter 01 Introducation & Literature Review

AgNO3 was added to the leaf extracts, the color of the solution changed, indicating the synthesis of AgNPs. The above finding was further confirmed by UV-Vis Spectroscopy and peaks were obtained at the absorption maxima of 462 nm, 432 nm, and 421 nm at the temperatures of 600C,

750C, and 950C, respectively. The FTIR analysis showed that the carbonyl groups played a role in binding with the metallic NPs. The XRD studies revealed that the synthesized AgNPs had a crystalline morphology with face-centered cubic structure [171].

In another report, Paramasivam et al. described a rapid process for the biogenic production of the

AgNPs, using Eclipta alba aqueous extracts. The plant extracts and AgNO3 solution were used in the different ratios of 1:3, 1:4, 1:5, 1:7, and 1:9 and peaks were observed at the absorption maxima of 411 nm and 432 nm in the extract ratios of 1:7 and 1:9, respectively. The XRD studies showed that the synthesized AgNPs had a crystalline morphology, while the SEM analysis elaborated the surface morphology and topology of the synthesized AgNPs and the size of the particles mostly ranged from 40–90 nm [172].

37 Chapter 01 Introducation & Literature Review

Table 1.4: Green synthesis of AgNPs using various plant extracts

Plant Plant part Size (nm) Nature Refrence

Averrhoa carambola Leaf 16 Sphere-shaped 173

Carica papaya Leaf 50-250 Circular 174

Cucurbita maxima Petals 19 Crystalline 175

Acorus calamus Rhizome 20 Circular 176

Skimmia laureola Leaf 46 Hexagonal 177

Tephrosia tinctoria Stem 73 Sphere-shaped 178

Clerodendrum serratum Leaf 5-30 Sphere-shaped 179

Plukenetia volubilis Leaf 4-25 Ocular 180

Euphorbia helioscopia Leaf 2-14 Circular 181

Hypnea musciformis Leaf 40-65 Sphere-shaped 182

Annona muricata Leaf 20-53 Sphere-shaped 183

Momordica cymbalaria Fruit 15-50 Sphere-shaped 184

Quercus brantii Leaf 6 Spherical 185

Premna herbacea Leaves 10-30 Spherical 186

Calotropis procera Plant 19-45 Spherical 187

38 Chapter 01 Introducation & Literature Review

1.11 Aim and Objectives

The present study is designed with the following aims and objectives based on the therapeutic and traditional use of Quercus semicarpifolia Smith;

1. The plant Cr. MeOH Ext will be checked for the occurrence of various groups/classes of

secondary metabolites in the selected plant such as flavonoids, phenols, sterols, alkaloids,

terpenoids, saponins, carbohydrates, proteins and glycosides.

2. Both in-vitro (antibacterial, antifungal, antioxidant, phytotoxic, cytotoxic, anti-termite,

insecticidal, hemagglutination and allelopathic) and in-vivo (acute toxicity, analgesic,

anti-inflammatory, antipyretic) biological evaluation will be accomplished in the present

study.

3. Based on the preliminary phytochemical and biological evaluation, the effective fractions

of Q. semecarpifolia plant will be eluted by column chromatography (CC) and flash

column chromatography (FCC) for the isolation of pure compounds and fixed oils.

4. The structure of the compounds will be elucidated by advanced spectroscopic techniques

such as ESI-MS, 1H-NMR, 13C-NMR, COSY, NOSY, UV, HMBC, IR and GC-MS.

5. Q. semecarpifola aqueous extract will be used for the synthesis of AgNPs and

characterization will be done by techniques such as UV-Vis Spectra, TEM, FTIR, EDX,

TG/DTA, XRD and SEM.

39 Chapter 02 Experimental

Experimental

2.1 General Experimental Conditions

All investigations including phytochemical, biological and instrumental analysis were executed at the Centre of Biotechnology and Microbiology (COBAM), University of Peshawar,

Computerized resource lab (CRL), University of Peshawar, Pakistan Council of Scientific and

Industrial Research (PCSIR), Peshawar and International Centre for Chemical and Biological

Studies (ICCBS) Karachi, University of Karachi.

2.1.1 Drugs and Chemicals used in Different Experiments

For the various tests, the drugs and chemicals of analytical and commercial grade, purchased from Merck were used. The different doses of the Cr. MeOH. Ext, fractions, and bioinspired

AgNPs were prepared in distilled water and normal saline was used for the different pharmacological assays. Normal saline and distilled water were used as the controls in the various pharmacological experiments. For the different experiments, the organic solvents such as methanol (CH3OH), acetone [(CH3)2CO], n-hexane, CHCl3, and EtOAc were used.

2.1.2 Physical Constants

Using Buchi SMP-20 apparatus, melting points of the compounds were determined. With the help of digital Polarimeter, optical rotation of the compounds was measured.

2.1.3 Spectroscopy

The UV-Vis spectrum of the compounds was determined using Hitachi-UV-3200

Spectrophotometer in methanol while an infrared (IR) spectrum was done with the help of Jasco-

320-A IR spectrophotometer in CHCl3. For Electron Ionization Mass Spectroscopy (EI-MS),

Thermo Electron Corporation MAT 95XP-Trap instrument was employed using methanol as solvent while Jeol-JMS-HX-110 mass spectrometer was used for the determination of the HR-

40 Chapter 02 Experimental

EIMS and FAB +ive and –ive. 13C‐NMR (Nuclear Magnetic Resonance) spectra was documented on Bruker AMX 300 (75, 100,125 and 150 MHz) mass spectrometer using while

1H-NMR was recorded on Bruker AMX 400 and 500 mass spectrophotometer. Distortion Less

Enhancement by Polarization Transfer (DEPT) was generally used to determine carbon signals

o o (CH, CH2 and CH3) at 90 and 135 . Heteronuclear Multiple Bond Correlation (HMBC) experiment was used for the determination of two and three bond 1H-13C connectivity. Gas

Chromatography (GC) was used for the determination of qualitative data of oils while Gas

Chromatography/Mass Spectrometer (GC-MS) was used for the quantitative analysis of oils.

2.1.4 Isolation and Purification of the Compounds

Pure compounds from the different fractions of Q. semecarpifolia were isolated through various chromatographic techniques.

2.1.4.1 Column Chromatography (CC)

Column chromatography was used for the isolation of pure compounds using silica gel as a stationary phase. Different organic solvents were used as mobile phases in CC.

2.1.4.2 Thin-layer Chromatography (TLC)

For TLC, silica gel cards were utilized. Purification of the compounds was done with the help of the preparative silica gel plates.

2.1.5 Spraying Reagents used for Visualization of Spots

The spraying reagents, used for visualizing the spots on the thin layer chromatography (TLC) plates were ceric sulfate [Ce (SO4)2], Dragendorff‘s reagent, vanillin-phosphoric acid reagent, and iodine (I2) solution. With the help of a spray gun, these reagents were sprayed on the TLC plate and visualized by UV light at the short and long wavelengths of 254 nm and 365 nm, respectively [189].

41 Chapter 02 Experimental

2.1.5.1 Ceric Sulfate Solution

It was prepared by dissolving ceric sulfate in 65% sulfuric acid (H2SO4). The reagent was employed for the detection of different phytochemicals, present in the test samples. Terpenoids, present in the samples, were confirmed by the appearance of a pink color due to heating, whereas a light yellow or black color without heating showed the occurrence of alkaloids in the samples

[189].

2.1.5.2 Vanillin-Phosphoric acid reagent

It was prepared by dissolving 1 g of vanillin in 50% of aqueous phosphoric acid (H3PO4).

Terpenes and steroids were detected by the appearance of a pink color when vanillin solution was sprayed on TLC plates at temperatures ranging from 100–110 °C [189].

2.1.5.3 Iodine (I2) Solution

A solution of I2 was prepared by adding a few crystals of I2 in the TLC tank and heating up to a temperature of 40–50°C. The spots appeared when a TLC plate was placed in the tank [190].

2.1.5 4 Dragendorff’s Reagent

For its preparation, at first, in 20 mL of distilled water 8 g of potassium iodide (KI) was mixed

(solution A). Then, solution B was prepared by dissolving 0.85 g of bismuth nitrate in 20% acetic acid (CH3COOH) and distilled water. The stock solution was then prepared by mixing these two solutions in a ratio of 1:1. After that, 5 mL from the stock solution was taken and diluted with 90 mL of distilled water. Alkaloids were detected by the appearance of a light brown, light pink or black color, upon spraying the reagent, while a light yellow color indicated the presence of steroids and terpenoids in the samples [190].

42 Chapter 02 Experimental

2.2 Phytochemical Investigation

2.2.1 Collection and Identification of the Plant

Plant material of Q. semecarpifolia was collected from the District Mansehra, Khyber

Pakhtunkhwa (KPK), Pakistan in September-October 2015. Plant species was identified by Dr

Habib Ahmad, VC Islamia College University of Peshawar, Pakistan.

2.2.2 Extraction Procedure

The shade-dried plants were pulverized into a powder form and kept at room temperature.

Analytical grade methanol was used twice for soaking the plant materials that were then kept for

15 days at room temperature. Meanwhile, the soaked plant materials were subjected to stirring from time to time. After soaking, the residue was filtered off with the help of a white, colorless, thin cloth and using a rotary evaporator under vacuum, the filtrate were concentrated at a temperature of 40°C, in order to obtain 1200 g of the MeOH Extract.

2.2.3 Fractionation of Crude Methanolic Extracts

The different solvents such as n-hexane, CHCl3, and EtOAc were used for the fractionation of the Cr. MeOH Ext. As depicted in Scheme 1, 1200 g of the Cr. MeOH Ext. of Q. semecarpifolia was mixed in 400 mL of distilled water and portioned thrice with 400 mL each of n-hexane,

CHCl3, and EtOAc, in order to obtain 100 g, 110 g, 150 g, and 210 g of the n-hexane, CHCl3,

EtOAc, and aqueous fractions, respectively. About 100 g of the Cr. MeOH Ext. was reserved for the assessment of various biological evaluations.

43 Chapter 02 Experimental

Crude MeOH extract (1200g)

water water hexane Distilled

1. n-hexane soluble fraction n-hexane insoluble fraction (100g)

CHCl

CHCl3 insoluble fraction 2. CHCl3 soluble fraction

(110g)

EtOAc

EtOAc insoluble fraction

3. EtOAc soluble fraction

(150 g) EtOAc 5. Aqueous fraction

(210 g)

Scheme 2.1 Fractionation of Crude MeOH extract of Q.semecarpifolia

44 Chapter 02 Experimental

2.2.4 Screening Tests of Crude Extracts for the presence of Phytochemicals

2.2.4.1 Preparation of Plant Extracts

100 g of the powdered plant material was soaked in 200 mL MeOH for 32 hours in order to get crude extract. Further, it was filtered and concentrated using rotary evaporator.

2.2.4.2 Preliminary Phytochemical Screening

Using standard protocols, the crude extract was analyzed for the presence of different organic compounds [191-194].

2.2.4.2.1 Alkaloids

To the Cr. MeOH Ext. of the plant materials, 5 mL of HCl was mixed and heated at 40°C in a water bath for 2 min. The solution was then divided into two parts and Mayer‘s reagent and

Wagner‘s reagent were added separately. The formation of white, cream-colored precipitates with Mayer‘s reagent and dark red colored precipitates with Wagner‘s reagent indicated the presence of alkaloids in the samples.

2.2.4.2.2 Saponins

Foam Test: 0.5 g of the powder of the dried plant materials was mixed with 5 mL of distilled water in a test tube and shaken robustly. The formation of froth indicated the presence of saponins in the samples.

2.2.4.2.3 Flavonoids

To 3 mL of the plant extracts, 10 mL of CH3OH was added and heated for some time. The mixture was filtered after cooling and used for the following tests:

Shinoda Test: A small piece of magnesium ribbon (5 g) was added to plant extract (5 ml) followed by the addition of HCL (1-2 drops). The formation of light pink color indicated positive result for the presence of flavonoids.

45 Chapter 02 Experimental

Alkaline Reagent Test: To 1 mL of the plant extracts, 2 mL of sodium hydroxide (NaOH) was added and treated. The appearance of a yellow color indicated the presence of flavonoids in the samples.

2.2.4.2.4 Tannins

Ferric Chloride Test: In a test tube, 2 mL of the plant extracts was mixed with 10 mL of hot, distilled water by stirring. After cooling, the mixture was filtered and 2 mL of ferric chloride

(FeCl3) was added. The turning of the solution to blue-green coloration confirmed the occurrence of tannins in the samples.

Lead Subacetate Test: In a test tube, 2 mL of the plant extracts was mixed with 2–3 drops of lead subacetate. The appearance of cream colored precipitates indicated the presence of tannins in the samples.

2.2.4.2.5 Glycosides

Keller-Killiani Test: Test tubes, containing 5 mL of the plant extracts, 1 mL of concentrated

H2SO4, 2 mL of glacial acetic acid and 5% FeCl3, were mixed. The formation of brown ring between the layers indicated the presence of glycosides in the samples.

Concentrated H2SO4 Test: Test tubes, containing 5 mL of the plant extracts, 0.5 mL of glacial acetic acid, and a drop of 5% FeCl3 were mixed. The formation of reddish brown color on the sides of the test tubes indicated the presence of glycosides in the samples.

46 Chapter 02 Experimental

2.2.4.2.6 Terpenoids

A mixture of 2 mL of the plant extracts and 2 mL of CHCl3 was prepared in a test tube and shaken well, followed by the addition of 3 mL of concentrated H2SO4 to this mixture. The appearance of a brown color indicated the presence of terpenoids in the samples.

2.2.4.2.7 Sterols

Chloroform Test: In a test tube, 2 mL of the plant extracts was mixed with 2 mL of CHCl3. The formation of red color precipitates confirmed the occurrence of sterols in the samples.

2.2.4.2.8 Phenols

Ellagic Acid Test: The methanolic plant extracts were treated with 5% glacial acetic acid and a

5% solution of sodium nitrate (NaNO3). The turning of the solution to brown colored precipitates indicated the presence of phenols in the samples.

2.2.4.2.9 Carbohydrates

Fehling’s Test: Plant extract (2mL) was mixed with Fehling A and Fehling B reagents. Brick red precipitates were formed which confirmed the presence of carbohydrates in the sample.

2.2.4.2.10 Proteins

In a test tube, 2 mL of the plant extracts and 1 mL of a 10% solution of NaOH were taken and heated for 1–2 min. After cooling, 1 mL of a 7% copper sulfate (CuSO4) solution was added to this mixture. The formation of violet or blue color indicated the presence of proteins in the samples.

47 Chapter 02 Experimental

2.2.4.2.11 Anthraquinones

To 2 mL of the plant extracts, 2 mL of dilute HCl was added and warmed for 5 min. After cooling to room temperature, an equal volume (1 mL) of CHCl3 and 10% ammonia (NH3) was added to this mixture and boiled for 15 min in a water bath. The presence of anthraquinones was confirmed by the appearance of red colored precipitates.

2.2.4.2.12 Phlobatannins

2 mL of HCl was added to 2 mL of the plant extract and boiled. The formation of red colored precipitates indicated the occurrence of phlobatannins in the samples.

48 Chapter 02 Experimental

Table 2.1: Reagents composition used in phytochemical investigation

S.no Reagent Used Composition

1. Wagner‘s Reagent In distilled water (5 ml), Iodine (1.27 g) and Potassium

Iodide (2 g) were mixed and made the volume upto

100 ml.

2. Mayer‘s Reagent In distilled water (100 ml), Mercuric chloride (HgCl2)

(1.36 g) and Potassium iodide (KI) (5.00g) was mixed.

3. Fehling‘s solution It is used for the detection of reducing sugars and is

made from two mixtures;

Fehling solution A: In distilled water (500 ml), copper

sulphate (34.66 g) was mixed.

Fehling solution B: In distilled water (500 ml),sodium

hydroxide and Potassium sodium tartarate (173 g)was

mixed.

49 Chapter 02 Experimental

2.3 Compounds Isolated from Quercus semecarpifolia

The EtOAc fraction of Q.semecarpifolia was selected for the isolation of pure compounds through column chromatography (CC) using n-hexane / EtOAc solvent system. Different ratios of the above solvents were used in order to get sub-fractions at different polarities. At first, pure n-hexane (100 %) was added in the column and then with EtOAc, the polarity was amplified gradually i.e 5% (95:05), 10% (90:10), 15% (85:15), 20% (80:20) and so on up to pure 100%

EtOAc. 12 sub-fractions were obtained which were further tested on the TLC plates for good separation using different solvent systems. In order to isolate pure compounds, sub-fraction B, D and F were selected from the sub fractions and eluted with FCC as shown in scheme 2.2.

Using n-hexane: EtOAc (8.0:2.0) solvent system, compound (1) was isolated from the sub- fraction B. Similarly, further 2 sub-fractions D (a) and D (b) were obtained from sub-fraction D with FCC by utilizing CHCl3 and MeOH as a solvent system in increasing order of polarity. The compounds (2) and (3) were isolated from sub-fraction D (a) eluting with solvent system of n- hexane: EtOAc (0.3:9.7) and (0.7: 9.3) respectively. Further two compounds were isolated from the sub-fraction F utilizing n-hexane and EtOAc solvent system. Compound (4) was eluted with the solvent system of n-hexane: EtOAc in a ratio of 6.0: 4.0 while compound (5) was obtained from the same solvent system in a ratio of 5.0: 5.0.

50 Chapter 02 Experimental

Ethyl Acetate Fraction

(80.0g)

)

100% -

hexEtOAc (0 :

n ColumnChromatography

Sub Fraction B Sub Fraction D Sub Fraction F n-hex : EtOAc n-hex : EtOAc n-hex : EtOAc

(8.0 : 2.0) (3.5 : 6.5) (3.0 : 7.0)

FCC

: MeOH :

CHCl

FCC

hex hex EtOAc : - n Sub Fraction D (a) Sub Fraction D (b)

CHCl3 : MeOH CHCl3 : MeOH

(9.5 : 0.5) (8.0 : 2.0) hex hex EtOAc : (7.5 2.5) :

- n

Flash Column Flash Chromatography (FCC)

FCC hex hex EtOAc :

Compound 1 - n

)

5.0 : 5.0) : 5.0

(

hex : EtOAc : hex

(6.0 : 4.0) : (6.0

hex :EtOAc hex

hex : EtOAc : hex

- -

9.7 (0.3: -

(0.7 : 9.3) (0.7:

hex : EtOAc : hex

- n

Compound 2 Compound 3 Compound 4 Compound 5

Scheme 2.2 Flow chart depicting compounds isolated from EtOAc fraction of Q. semecarpifolia

51 Chapter 02 Experimental

2.3.1 Characterization of Compounds

2.3.1.1 Characterization of Benzoic acid (1)

Physical status White crystalline powder

Yield 0.82 g

o M.P 122 C

Molecular formula C7H6O2

Chemical name benzoic acid

HR-ESI-MS (m/z) 122.02

IR (cm-1) 3260 - 2610 (COOH), 1705 (C=C)

Uv λmax (nm) 272 nm

Proton NMR (CDCl3) Table 3.2

13 Carbon-NMR (CDCl3) Table 3.2

52 Chapter 02 Experimental

2.3.1.2 Characterization of p-hydroxybenzoic acid

Physical status White crystalline solid

Yield 0.98 g

M.P 2140C

Molecular formula C7H6O3

Chemical name P-Hydroxybenzoic acid

HR-ESI-MS (m/z) 138.12

IR (cm-1) 3342 (OH ), 1741(C=O)

Uv λmax (nm) 231 nm

Proton -NMR (CDCl3) Table 3.3

13 Carbon-NMR (CDCl3) Table 3.3

53 Chapter 02 Experimental

2.3.1.3 Characterization of Bis (2-ethylhexyl) phthalate (3)

Physical status Colorless Viscous liquid

Yield 1.4 g

o M.P -50 C

Molecular formula C24H39O4

Chemical name Bis (2-ethylhexyl) phthalate

HR-ESI-MS (m/z) 391.2845

IR (cm-1) 1741, 1541

Uv λmax (nm) 227

Proton -NMR (CDCl3) Table 3.4

13Carbon-NMR (CDCl ) Table 3.4 3

54 Chapter 02 Experimental

2.3.1.4 Characterization of β-Sitosterol (4)

Physical status White crystalline needle

Yield 1.41g

M.P 1360C

Molecular formula C29H50O

Chemical name β-Sitosterol

HR-ESI-MS (m/z) 414.7

IR (cm-1) 3473.6 (OH), 2941.7 (CH), 2837.9 (CH)

Uv λmax (nm) 261

Proton-NMR (CDCl3) Table 3.5

13 Carbon-NMR (CDCl3) Table 3.5

55 Chapter 02 Experimental

2.3.1.5 Characterization of Stigmasterol (5)

Physical status White needle crystal

Yield 0.96 g M.P 1650C

Molecular formula C29H48O

Chemical name Stigmasterol

HR-ESI-MS (m/z) 412.4

IR (cm-1) 3461.2, 2945, 1653.74,1453.23,1365,1047.7

Uv λmax (nm) 257

Proton-NMR (CDCl3) Table 3.6

13 Carbon-NMR (CDCl3) Table 3.6

56 Chapter 02 Experimental

2.4 Green Biogenic Synthesis of Silver Nanoparticles (AgNPs)

Using the aqueous leaf extracts of Q. semecarpifolia, the green AgNPs were synthesized. Briefly in 500 mL of distilled water, 25 g of the powdered leaves were mixed and boiled for 30 min, in order to prepare the aqueous leaf extracts. The extracts, thus obtained were filtered by a

Whatman Grade 1 Qualitative Filter Paper, in order to get a vibrant solution. In order to remove the impurities, the resultant extracts were centrifuged. The resultant extracts were further filtered through a 0.2-μm filter paper and used for the subsequent fabrication of the AgNPs. In 90 mL of a 1 mM solution of AgNO3, 10 mL of the aqueous leaf extracts was added, followed by heating the mixture in a water bath with shaking at 75°C for 1 h. The reduction of Ag+ ions to metallic

Ag was confirmed by a change in the color of the solution from light yellow to dark brown. The mixture was again centrifuged at 10,000 rpm for 15 min at a temperature of 4°C, followed by the redispersion of the AgNPs in distilled water. Finally, using a rotary evaporator, the solution was concentrated and spread onto sterile Petri dishes and allowed to dry [195]. The steps, followed for the synthesis of the AgNPs are depicted in Scheme 2.3.

57 Chapter 02 Experimental

aqueous Leaves extracts (10ml)

1m M AgNO3 Solution (90ml)

Incubate in shaking water bath for 1 hour at 75oC

Scheme 2.3: Flowchart depicting steps involved in AgNPs synthesis

58 Chapter 02 Experimental

2.4.1 Characterization of Synthesized AgNPs

2.4.1.1 UV-Vis Spectroscopic Studies

UV-Vis Spectroscopy is a simple and effective technique for confirming the formation of NPs. A double beam Shimadzu UV 1650 PC UV-Vis Spectrophotometer was used for the UV-Vis spectroscopic analysis. Precisely, 4 mL of a diluted AgNPs sample was taken in a cuvette to obtain the UV-Visible spectra in a wavelength range of 300–800 nm [196].

2.4.1.2 X-Ray Diffraction (XRD) Dimensions

The crystallographic assembly of the purified AgNPs was detected by the X-Ray diffraction spectra. A thin layer of the AgNPs was applied on a carbon-coated Cu grid and examined by an

X‘Pert PRO X-Ray diffractometer, operated with Cu (Kα) X-Rays and a current of 30 mA. The scanning was done at a diffraction angle of 2θ, ranging from 10°–80° [197].

2.4.1.3 Scanning Electron Microscopy (SEM)

Using a Hitachi S 4200 FE-SEM apparatus, the SEM investigations were executed. Briefly, a thin layer of the synthesized AgNPs was prepared and loaded onto a Cu grid by dipping a small quantity of the test sample on the lattice. A blotting sheet was used for eliminating the extra solution. Using a mercury lamp, the test sample was dried for 5 min and the images of NPs were observed under an electron microscope at the magnifications of 15,000X, 30,000X, and 60,000X

[198].

59 Chapter 02 Experimental

2.4.1.4 Energy Dispersive X-Ray Spectroscopy (EDX)

A JED–2300 EDX spectrometer was used for confirming the elemental configuration of the synthesized AgNPs. The EDX technique predominantly detected the radiations, released from the samples during the process of bombardment by a stream of electrons [199].

2.4.1.5 Fourier Transform Infra-Red (FTIR) Spectroscopy

For FTIR analysis, the plant derived AgNPs were centrifuged at 10,000 rpm for 10 min to remove unbound phytochemicals present in the solution. The centrifuged and dried powder sample was then analyzed using JASCO 6200 FTIR instrument [200].

2.4.1.6 Transmission Electron Microscopy (TEM)

Using distilled water, AgNPs were applied on a carbon-coated Cu grid and kept for some time to get dry. The TEM analysis predominantly confirmed the size and morphology of the synthesized

AgNPs (JEOL, 2011) [201].

2.4.1.7 Thermo gravimetric/Differential Thermal Analysis (TG/DTA)

Using an STA 7200 Simultaneous Thermogravimetric Analyzer (Hitachi), the TG/DTA studies were performed. Generally, the powdered form of AgNPs was used for the TG/DTA investigations. A total of two glass aluminum containers—one, containing the sample and the other, acting as the reference—were tied together and kept without touching each other and subjected to heating at temperatures ranging from 100–1000 °C, under a persistent stream of nitrogen (N2) gas. During the investigation, the levels of the gas were checked frequently [202].

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2.5 Assessment of Pharmacological/Biological Activities (in vitro)

The crude plant extracts and the bioinspired AgNPs of Q. semecarpifolia were investigated for the various in-vitro biological activities.

2.5.1 Antibacterial Activity

Requirements

Petri dishes (14 cm), sterile cork borers, micropipettes, incubator, Nutrient broth, Nutrient agar, autoclave, spirit lamp, Laminar flow hood (LFH), Dimethyl sulfoxide (DMSO), test samples

(plant Cr. MeOH.Ext, fractions and AgNPs ) and Amoxilin (standard antibiotic).

Test microorganisms

S. aureus, B. subtilis, P. mirabilis, K. pneumoniae, E. coli, P. aeruginosa, S. marcescens, and S. pneumoniae species were used in the current study

Procedure

The antibacterial assays of the crude extracts, fractions, and AgNPs were executed as previously reported [203]. Briefly, the experiments were performed in the following steps:

1. At first, nutrient broth media was autoclaved, transferred to sterile test tubes, and

incubated overnight at a temperature of 37°C.

2. The test tubes containing the media were then inoculated with the test pathogens and

incubated again overnight at 37°C. In the meantime, nutrient agar medium was prepared,

poured into sterile Petri plates, and incubated overnight at 37°C for ensuring aseptic

conditions.

3. On the following day, the nutrient agar plates were inoculated by spreading the 18–24 h

old cultures of respective bacteria over the entire agar surface utilizing sterile cotton swab

under the Laminar Flow Hood (LFH).

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4. After the introduction of the test pathogens to the nutrient agar plates, holes of 6 mm

diameter were punched aseptically, using a sterile cork borer.

5. Furthermore, the stock solutions were prepared by mixing 3 mg of each test sample in

sterile DMSO at a concentration of ˂1%.

6. Using a sterile micropipette, an aliquot of 100 μL of the stock solutions of the plant

extracts and the AgNPs was poured into the respective wells.

7. The control experiments comprising the inoculum without the plant extracts and the

AgNPs were set up.

8. Amoxicillin (positive control) and sterile DMSO served as the negative control. Finally,

the Petri plates were incubated for 24 h at 37 °C, followed by the measurement of the

diameter of the zones of inhibition in millimeter using a ruler.

9. The percentage inhibition (%) was calculated by the formula illustrated below:

Percent Growth Inhibition = ×100

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2.5.2 Minimum Inhibitory Concentration (MIC50)

Usually, the MIC50 values were determined for calculating the minimum concentration of the antimicrobial agents required to kill bacteria during the incubation period of 24 h. The crude plant extracts and the AgNPs were investigated for the possible MIC50 values against the test pathogens, following the procedure of Banso et al., with slight modifications [204].

Test microorganisms

B.subtilis, K. pneumoniae, E.coli, S.aureus, P. aeruginosa, S. marcescens, P. mirabilis and S. pneumoniae

Procedure

1. At first autoclaved nutrient broth (4mL ) was dispensed into test tubes and incubated at

37°C for 24 h.

2. On the following day, the test tubes, containing the broth media were inoculated with the

test pathogens and incubated overnight at 37°C.

3. From the stock solutions, 100, 150, 200, 300, 400, 500, and 600 μL aliquots were

dispensed into the test tubes, containing the test pathogens.

4. Finally, the test tubes were incubated at 37°C for 24 h and the results were calculated on

the basis of the turbidity developed.

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2.5.3 Antifungal Activity

Requirements

Micropipettes, autoclave, test tubes, stirrer, incubator, inoculating loop, Sabouraud Dextrose

Agar (SDA), DMSO, standard drug (Miconazole) and test samples (Cr. Ext, fractions and

AgNPs).

Test fungal species

Penicillium notatum, Fusarium oxysporum, Aspergillus niger, Aspergillus flavus and Triticum harzianum

Procedure

The assays for the antifungal activities were conducted prior to the implementation of the tube dilution method [205].

1. Firstly, autoclaved SDA media was prepared and incubated overnight at 28 ±1°C.

2. On the following day, the aforementioned fungal strains were streaked onto the SDA

plates and incubated at 28 ±1°C for seven days.

3. The stock solutions were prepared by mixing 24 mg of each test sample (crude extracts

and AgNPs) in 1 mL sterile DMSO.

4. To sterile test tubes, 4 mL of the autoclaved SDA medium was transferred.

5. Using a micropipette, 67.6 μL of the stock solution was pipetted into the sterile test tubes.

6. In order to make the slants, the test tubes were kept in a slanting position and allowed to

solidify inside the LFH.

7. In the next step, the test tubes were inoculated with a 7-day old fungal culture, using a

sterile inoculating loop, followed by incubation at a temperature of 28 ±1°C for 7 days.

8. Miconazole (positive control) and DMSO (negative control) were used in the experiment.

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9. The Petri plates were then incubated for 5–7 days and on the seventh day of the

incubation period, the results were noted by measuring the diameter of the colonies in

millimeter.

10. The percentage lethality (%) was calculated by the following formula:

Percent growth Inhibition = 100 ˗ × 100

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2.5.4 Antioxidant Activity

Requirements

DPPH (1, 1-diphenyl -2-8 picrylhydrazyl), methanol, test samples (Cr. MeOH Ext, fractions and

AgNPs), UV-Vis spectrophotometer

Procedure

The antioxidant potential of the test samples was determined as previously reported procedure by

Bashir et al., [195, 206]. The experiments were performed in the following steps:

1. Firstly, 0.01g of the test samples was dissolved in the organic solvent CH3OH for

preparing the stock solutions from which 100, 200, and 300 μg/mL concentrations of the

test samples were set by dilution.

2. In the next step, a 0.001% DPPH solution was prepared in CH3OH and 2 mL of this

solution was dissolved in 1 mL of each concentration of the test samples.

3. A concentration range of ascorbic acid (20–250 μg/mL) (positive control) and a solution

without DPPH was used as negative control.

4. All the samples were then placed in the dark for 30 min, followed by the measurement of

absorbance at 517 nm. The percentage absorbance (%) was then calculated by the

formula given below:

Percent Absorbance = × 100

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2.5.5 Phytotoxic Activity

Requirements

Test samples (Cr. MeOH Ext, fractions and AgNPs), test organism (Lemna minor), Petri plates, micro-pipettes, growth chamber and organic solvent (methanol).

Procedure

Using the protocol for the Lemna minor bioassay, the phytotoxic activities of the test samples were determined as reported earlier [207]. Briefly, the experiments were performed in the following steps:

1. Firstly, E Medium was prepared in distilled water by mixing various constituents,

according to the protocol described in Table 2.2.

2. Subsequently, 20 mg of the test samples, mixed in 1.5 mL of CH3OH served as stock

solutions, which were serially diluted to give the final concentrations of 1000, 100, and

10 µg/mL.

3. All these concentrations of the test samples were transferred to the different Petri plates

were kept until all the excess organic solvents evaporated.

4. In the next step, 20 mL of the E Medium was added to each Petri plate along with 16

healthy Lemna minor plants.

5. Incubation of the samples was done at a temperature of 28 ±1 °C for 7 days in the LFH.

The E Medium without the plant extracts (negative control) and 0.015 µg/mL

concentration of paraquat was used as the positive control in the experiment.

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6. The numbers of fronds were counted on the seventh day and the results were recorded by

the formula illustrated below:

Percent growth regulation = × 100

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Table 2.2: Composition of E-medium for growth of L.minor [208]

S.No Constituents gm/L

1 Magnesium sulphate 0.492

2 Ferric chloride 0.00540

3 Copper sulphate 0.00032

4 Sodium molybdate 0.00017

5 Boric acid 0.00376

6 Potassium di-hydrogen phosphate 0.76

7 Zinc sulphate 0.000034

8 Ethylene diamine tetra-acetic acid 0.01120

9 Calcium nitrate 1.180

10 Magnesium chloride 0.00362

11 Potassium nitrate 1.518

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2.5.6 Cytotoxic Activity

Requirements

Test samples (Cr. MeOH Ext, fractions and AgNPs), Artemia salina (Shrimp eggs), methanol,

DMSO, double distilled water, pasture pipette, plastic dish, sea salt, syringes, glass vials, aluminum foil and magnifying glass.

Procedure

The cytotoxic potential of the test samples was evaluated by the brine shrimp (Artemia salina) lethality assay [209], which was performed in the following steps:

1. Saline water, serving as a good medium for the hatching of the eggs of brine shrimp. It

was prepared by dissolving 38 g of NaCl in 01 L of distilled water.

2. The saline water was then taken in a rectangular tray and the eggs of brine shrimp (100

mg) were spread over it and allowed to incubate for 24 h to allow hatching.

3. The nauplii were collected after the incubation period using a Pasteur pipette.

4. Stock solutions were prepared by mixing 20 mg of each test sample in methanol and

subjected to a serial dilution to get the final concentrations of 1000, 100, and 10 μg/mL

and were transferred to sterile flasks.

5. The flasks were kept inside the LFH for some time to evaporate the excess organic

solvents.

6. Using a Pasteur pipette, a total of 10 nauplii of A. salina, containing 1 mL of saline water

were transferred to each of the flasks and the final volume of each flask was made up to 5

mL with saline water.

7. Incubation of the flasks was done at 28°C for 24 h. The drug, etoposide (positive control)

and CH3OH were used as negative control in the experiment.

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8. Using a magnifying glass, the lethality of the shrimps was estimated and the percentage

lethality (%) was calculated by the following formula:

Percent shrimp lethality = × 100

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2.5.7 Insecticidal Activity

Requirements

Test samples (Cr. MeOH Ext, fractions and AgNPs), methanol, petri dishes, micropipette, glass vials, filter paper, brush and standard drug (Permithrine)

Test Insect Species

Tribolium castaneum, Rhyzopertha Dominica, Callosobruchus maculatus were used as the test insects in the current study.

Procedure

The insecticidal activities of the test samples were determined by applying the direct contact method as reported by Ahmad et al., [210].

1. Firstly, the stock solutions were prepared in 3 mL of methanol by dissolving 200 mg of

the test samples.

2. In the following step, filter papers were placed inside sterile Petri dishes, according to

their size. Furthermore, the stock solutions were poured onto the filter papers, using

autoclaved micropipette tips.

3. In order to evaporate the organic solvents from the test samples completely, the Petri

plates were kept overnight.

4. Using a sterile brush, a total of 16 species of healthy insects were then released into each

Petri dish, including the test samples and controls.

5. Finally, all the Petri plates were incubated at 28 ±1°C and the mortality of the insects was

calculated after an incubation period of 24 h.

6. CH3OH was used as negative control while Permethrin served as the positive controls.

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7. The percentage mortality (%) was computed by the following formula:

Percent insect lethality = × 100

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2.5.8 Anti-termite Activity

Requirements

Termite culture, test samples (Cr. MeOH Ext, fractions and AgNPs), sterile petri plates, blotting paper, brush, and magnifying glass.

Procedure

1. In the termiticide assay, also conducted prior to the contact toxicity bioassay [205], some

blotting papers were placed inside a sterile Petri dish according to its size.

2. The stock solutions were prepared by mixing 2mg of each test sample in 1 mL of

methanol and poured uniformly onto the Petri dishes.

3. The Petri plates were then left for some time to evaporate the excess CH3OH from the

samples.

4. In the next step, a total of 25 termites were released into each Petri plate and incubated at

28°C.

5. Using desiccators, a constant relative humidity was maintained.

6. CH3OH was used as negative control in the experiment while an insecticide (fipronil)

served as positive control

7. The mortality of the termites was recorded on the basis of the dead and living termites for

three consecutive days and the percentage mortality (%) was calculated by the following

formula:

Percent termite mortality = × 100

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2.5.9 Allelopathic Activity

Requirement

Plant aqueous extract, wheat seeds, distilled water, caliper, Petri plates and digital balance

Procedure

Briefly, the experiments were performed in the following steps [211]:

1. At first, wheat seeds were kept in water at a temperature of 30 °C in a growth chamber, in

order to germinate into seedlings with radicles of about 2 mm in size.

2. In the following step, a total of 10 seedlings were transferred to each Petri plate,

containing a 25–100% concentration of the aqueous plant extracts.

3. A Petri plate, containing water served as the negative control. Finally, all the Petri plates

were incubated in a growth chamber at a temperature of 30 °C for 3–5 days.

4. The weight of the seedlings was determined using a digital balance and with the help of a

caliper, the length of the plumules and radicles was determined after the incubation

period.

5. In order to obtain the dry weight of the seedlings, moisture contents from the seedlings

were evaporated by placing them in an oven for some time.

6. Finally, the percentage inhibition (%) of the treated seedlings was compared with the

control.

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2.5.10 Hemagglutination Activity

Requirements

Blood groups, phosphate buffer, Na2HPO4, KH2PO4, incubator, test tubes, and centrifuge.

Procedure

The hemagglutination assay of the crude plant extracts and AgNPs was performed as previously reported reported protocol by Ahmad et al., [212].

1. The hemagglutination activities were assayed against human erythrocytes.

2. At first, fresh blood samples were collected from healthy human subjects, followed by

centrifugation at 3000 rpm, in order to isolate erythrocytes.

3. In the next step, phosphate buffer, having a pH value of 7.2 was used for preparing a 2%

suspension of erythrocytes.

4. A stock solution was prepared by mixing 1 mg of each test sample in 1 mL of the sterile

DMSO, from which, the different serial dilutions of 1:2, 1:4, 1:8, and 1:16 were set in the

phosphate buffer.

5. Subsequently, 1 mL from each of the sample dilutions was mixed with 1 mL of the 2%

erythrocyte suspension and incubated for 30 min.

6. The results were noted by observing the production of granules or smooth buttons at the

bottom of the test tube.

7. A negative result was indicated by the formation of smooth buttons, while the formation

of rough granules at the bottom of the test tube indicated a positive result.

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2.6 Assessment of Pharmacological/Biological Activities (in-vivo)

The plant-derived AgNPs and Cr. MeOH. Ext were used for the screening of various pharmacological activities (in-vivo).

Experimental Animals

Healthy male and female Swiss albino (BALB/c) mice, weighing 20–25 g were used throughout the experiments. The BALB/c mice were obtained from PCSIR, Peshawar, Pakistan. All the animals were kept and maintained in cages and a total of six animals per sample were used for the experiments. All investigational procedures, relating to the animals were in accordance with the guidelines formulated by the Institute of Laboratory Animal Resources Council, Commission on Life Sciences.

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2.6.1 Acute Toxicity Assay

Specifications

Healthy Swiss albino (BALB/c) mice (male or female) weighing 25–30 g were used for the acute toxicity assay. The mice were kept in cages at the animal house facility and maintained at room temperature with relative humidity and a 12:12 h light-dark cycle. Throughout the experiments, water and food were provided to the animals ad libitum.

Procedure

The acute toxicity assay was conducted in accordance with a protocol, previously reported by

Khan et al., with slight modifications [213].

1. At first, the mice were grouped into three comprising of six animals in each group.

2. In the next step, the test samples (Cr. MeOH. Ext) were administered orally at the doses

of 200, 500, 1000, and 1500 mg/kg i.p.

3. The second treatment group received the plant-derived AgNPs at the doses of 10, 40, 50,

and 100 mg/kg.

4. Normal saline (10mL/kg) was orally given to the negative control group.

5. After the administration of the doses, all the treated animals were observed for any signs

of toxicity, initially within 30 min and then for a period of 14 days.

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2.6.2 Antinoceceptive Assay

Altogether, two procedures were performed for determining the possible antinoceceptive effects— the acetic acid-induced writhing test and the hot plate assay.

2.6.2.1 Acetic Acid Induced Writhing Test

Requirements

Test sample (Cr. MeOH. Ext and AgNPs), BALB/c mice of either sex, Aspirin, surgical gloves, vials, masks, glacial acetic acid (Merck, Germany)

Procedure

The writhing test was executed in mice in accordance with previously reported protocols [214].

1. At least 2 h prior to the experiments, all the animals were withdrawn from food.

2. For the experiments, the mice were grouped into four of six animals in each group.

3. Normal saline (10mL/kg) was orally given to the control group.

4. The second treatment group received Aspirin (standard) at a concentration of 10 mg/kg.

5. The test samples (Cr. MeOH. Ext) were administered orally to the third treatment group

at the doses of 50, 100, and 200 mg/kg.

6. The fourth treatment group also received the plant-derived AgNPs at the doses of 10, 40,

50, and 100 mg/kg.

7. Furthermore, all the mice were intraperitoneally injected with 0.06% CH3COOH at a

dose of 10 mL/kg, following 30 min of the oral administration of the test samples.

8. Following injection with CH3COOH, the writhings was observed for a period of 20 min.

9. Finally, the percentage inhibition (%) was calculated by the standard formula given

below:

Percent Inhibition=1- × 100

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Where

We= No of writhes in test sample

Wc= No of writhes in control group

Statistical Analysis

Values are represented as mean ± SEM. The mean values of control groups were compared with the mean value of treated groups using one way ANOVA; P˂ 0.05 was considered significant.

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2.6.2.2 Hot Plate Assay

Requirements

Test sample (Cr. MeOH. Ext, plant-derived AgNPs), Tramadol, normal saline, surgical gloves, syringes, vials, masks and analgesiometer (Harvard apparatus, USA).

Procedure

According to a protocol, previously reported by Hijazi et al., the test samples were assessed for possible analgesic effects and the hot plate assay was performed [214]. The procedure is briefly illustrated below:

1. For this assay, the Swiss albino mice of both sexes were divided into four groups of six

animals each. At least 2 h prior to the experiments, all the animals were withdrawn from

feeding. Normal saline (10mL/kg) was administered to the control group.

2. Tramadol was orally given to the second treatment group at a standard dose of 10 mg/kg.

3. The test samples (Cr. MeOH. Ext) were orally administered to the third treatment group

at the doses of 50, 100, and 200 mg/kg.

4. The fourth treatment group also received the plant-derived AgNPs at the doses of 10, 40,

50, and 100 mg/kg.

5. All the treated mice were then subjected to the preheated plate of an analgesiometer,

maintained at a temperature of 55 ±1 °C.

6. The reaction time in the hot plate assay was observed by monitoring and measuring the

behavior of the animals such as licking the limbs, jumping, and flicking of the hind limb

in s.

7. Finally, the latency period was recorded at the time intervals of 30 min, 60 min, and 90

min.

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2.6.3 Anti-inflammatory Assay

Requirements

Carrageenan (Sigma, Germany), Digital plethysmometer (Ugo Basile, Italy), 1 cc syringes, surgical gloves, masks, vials, normal saline, test sample and aspirin.

Carrageenan-Induced Edema

Procedure

The assay for anti-inflammatory activities was performed according to the procedure described by Winter et al., [215]. The procedure is briefly described below:

1. For this assay, the Swiss albino mice of both sexes were grouped into four of six animals

each.

2. Normal saline (10mL/kg) was orally given to the control group.

3. Aspirin (10mg/kg) was orally given to the second treatment group.

4. The test samples (Cr. MeOH. Ext) were orally administered to the third treatment group

at the doses of 50, 100, and 200 mg/kg.

5. The fourth treatment group also received the plant-derived AgNPs at the doses of 10, 40,

50, and 100 mg/kg.

6. Acute inflammation was produced by a sub plantar injection of 0.1 mL of a 1% solution

of carrageenan to the left hind paw of the mice, following 1 h of the administration of

samples. Carrageenan was also administered to the mice in the positive and negative

control groups, using the same route.

7. Using a plethysmometer, the swelling of the left hind paw, following carrageenan

injection was measured and the results were recorded at the time intervals of 1 h, 2 h, and

3 h.

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2.6.4 Anti-pyretic Assay

Requirements

Digital thermometer (ACON laboratories, USA), Brewer‘s yeast (Merck, Germany), Paracetamol, normal saline, vials, surgical gloves, 1 cc syringes, and test samples.

Procedure

The assay for the antipyretic activities of the test samples (Cr. MeOH. Ext, AgNPs) against yeast-induced pyrexia was performed in the mice, according to a previously described protocol with slight modifications

[216].

1. For this assay, the animals were grouped into four of six animals each.

2. Normal saline (10 mL/kg) was administered to the control group.

3. Paracetamol (50mg/kg) was orally administered to the second treatment group.

4. The test samples (Cr. MeOH. Ext) were orally administered to the third treatment group at the

doses of 50, 100, and 200 mg/kg.

5. The fourth treatment group also received the plant-derived AgNPs at the doses of 10, 40, 50, and

100 mg/kg.

6. The normal body temperature of the animals was measured by thermometer before the induction

of hyperpyrexia.

7. The thermometer probe, maintained for at least 30 s was used to record the body temperature.

Pyrexia was induced in the mice by injecting a 20% suspension of Brewer's yeast in normal saline

at a dose of 10 ml/kg.

8. The changes in the rectal temperature were recorded after 24 h of injection with a suspension of

Brewer‘s yeast and the animals, showing a rise in temperature by at least 1°C were considered for

further investigations.

9. Finally, the results were recorded at the time intervals of 1 h, 2 h, and 3 h.

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2.7 Analysis of Fixed Oils by Gas Chromatography-Mass Spectrometry (GC-MS)

During CC, an ample quantity of fixed oils was obtained from the EtOAc fractions of Q. semecarpifola. The GC-MS technique was employed for the determination of the chemical composition of these crude fixed oils. The Fatty Acid Methyl Esters (FAME) of the fixed oils was prepared in accordance with the methods, reported previously [217]. The procedure is briefly described below:

1. In the first step, 1 g of oil and 5 mL of a 0.5 N KOH) were mixed in a test tube and boiled

in a water bath for 20 min.

2. In the next step, 10 mL of boron trifluoride (BF3) was added to the mixture and heated in

a water bath for 1–2 min.

3. After cooling down to room temperature, 3 mL of a brine solution and 5 mL of n-hexane

were added to this mixture.

4. For the esterification of FAME, the test tube was shaken vigorously and left undisturbed

for some time after which, two layers were formed.

5. The upper layer consisted of the fixed oils from which, 2 mL was transferred to a vial and

analyzed by the GC-MS technique.

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Results & Discussion

3.1 Phytochemical Studies

3.1.1 Qualitative Phytochemical Screening

The different classes of phytoconstituents such as steroids, alkaloids, phenols, flavonoids, tannins, glycosides, carbohydrates, proteins, and oils, produced by the plants are deposited in the different parts such as flowers, leaves, fruits, seeds, roots, and bark [218]. All these secondary metabolites or phytochemicals have beneficial medicinal effects, including defense mechanisms and protection from various diseases [219].

The test samples (Cr. MeOH. Ext) were screened for the presence of various organic constituents and phytochemical analysis revealed that the plant Q. semecarpifolia possesses a high quantity of biologically active elements called polyphenols. The Cr. MeOH. Ext of Q. semecarpifolia not only had a high quantity of flavonoids, phenols, steroids, tannins, glycosides, carbohydrates, alkaloids, and reducing sugars but also had a moderate amount of saponins, alkaloids, and oils.

However, these extracts did not have anthraquinones and phlobatannins, while having a low amount of quinones and proteins. The results are summarized in Table 3.1.

Previous studies have revealed that phenolics, tannins, alkaloids, flavonoids, and saponins have remarkable antimicrobial, antitumor, antidiarrheal, anticancer, antioxidant, and antidiabetic properties [220, 221]. Tannins are important organic constituents that have been used to inhibit microbial infections and they also serve as a natural defense mechanism against pathogens [222]. Previous reports have shown that tannins also have antioxidant, antihemorrhagic, cytotoxic, and antitumor properties. A study carried out with the methanolic fruit extracts of Q. infectoria, obtained positive results for the presence of tannins [223].

Flavonoids, another class of secondary metabolites, are known for their antimicrobial properties

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Phytochemicals Tests End Result Result Screened [224]. Previous findings have revealed that flavonoids inhibit the progression of the pathogenic microbes in numerous in vitro investigations [225]. Alkaloids also have wide-ranging biological activities, including antimalarial, antileishmanial, analgesic, and anticancer activities [226].

Saponins are effective against the bacterial species such as Salmonella typhi, Bacillus subtilis,

Escherichia coli, and Aeromonas hydrophila as well as the fungal pathogens such as Aspergillus flavus, Penicillium notatum, and Candida albicans [227]. Steroids, terpenoids, and glycosides have analgesic as well as hypocholesterolemic properties and also act as antidiabetic agents

[228-230].

Table 3.1: Tabular representation of phytochemicals present in Q.semecarpifolia

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Shinoda test Pink color formation 1 Flavonoids Alkaline Intense yellow color +++ reagent apperance

2 Saponins Foam test Foarm layer which lasts for ++ 10 mints

3 phenols Ellagic acid Formation of brown +++ test precipitates Libermann Red color appeared at the 4 Burchard upper layer of test tube and +++ Steroids Test) yellow color at the lower layer Mayer‘s White creamy colored reagent precipitates formation ++ 5 Alkaloids Wagner‘s Dark reddish precipitates reagent formation

6 Carbohydrates Fehling‘s Brick red precipitates +++ reagent test formation Ferric Green precipitates 7 Tannins chloride test formation +++ Lead sub White creamy precipitates acetate formation Keller-kiliani Appearance of reddish test brown color +++ 8 Glycosides H2SO4 test Brown ring formation at the sides of test tube

9 Terpenoids Copper Bright green color ++ acetate test formation Salkowski‘s Golden yellow color 10 Sterols Test appearance +++ Precipitate Formation of red _ 11 Phlobatannins test precipitates

NaOH test Blue green color formation 12 Quinones +

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3.2 Spectroscopic Characterization of Isolated Compounds from Q.semecarpifolia

3.2.1 Structural Elucidation of Benzoic acid (1)

From the EtOAc fractions of Q. semecarpifolia, the compound 1 was isolated and purified as white crystalline solid. The molecular formula of this compound was found to be C7H6O2 and the approximate molecular mass of this compound was 122.02, as determined by HR ESI-MS.

The infrared (IR) spectroscopy data displayed absorption spectra at 3260 cm–1 (COOH

–1 stretching) and 1750 cm (C=O stretching); UV λmax= 272 nm.

13C NMR) showed signals at δ129.2, δ130.5, δ133.6, δ181.3, and δ128.6 for C–2, 6, C–1,

C–4, C=O, and C–3, 5, respectively. (1H NMR) showed proton signals at δ6.51, δ8.26, δ11.92, and δ7.34 for H–4, H–2, 6, OH, and H–3, 5, respectively.

When compared to previously published literature [231], the spectroscopy data revealed the configuration of compound 1 to be benzoic acid, as depicted in Table 3.2 and structure is presented in Figure 3.1.

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Table 3.2: 1H-NMR and 13C-NMR spectra of Benzoic acid (1)

C.No δC δH ( mult, J, HZ) Types HMBC 1 130.5 C -

2 129.2 8.12 (2H, t, J =8.5 Hz) C C-1,C-3,C-4

3 128.6 7.46 (2H, t J = 8.5 Hz, CH C-4,C-5

4 133.6 7.60 (1H, t, J = 8.5 Hz) C -

5 128.6 7.46 (2H, t J = 8.5 Hz CH C-4,C-6

6 129.2 8.12 (2H, t, J =8.5 Hz, C -

COOH 181.3 11.92 (1H, s, O-H CH C-5,C-6

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Figure 3.1: Structure of Benzoic acid (1)

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3.2.2 Structural Elucidation of p-hydroxy Benzoic acid (2)

From the EtOAc fractions of Q. semecarpifolia, the compound 2 was isolated and purified as white crystalline solid. The molecular formula of this compound was found to be C7H6O3 and

the approximate molecular mass of this compound was 138.01 as determined by HR ESI-MS.

The infrared (IR) spectroscopy data displayed absorption spectra at 3312 cm–1 (O-H) and 1741

–1 cm (C=O); UV λmax= 231 nm.

13C NMR) showed signals at δ130.2, δ131.5, δ124.6, δ181.3, and δ129.6 for C–2, 6, C–1,

C–4, C=O, and C–3, 5, respectively. 1H NMR showed proton signals at δ6.51, δ8.26, δ11.92, and

δ7.34 for H–4, H–2, 6, OH, and H–3, 5, respectively.

When compared to previously published literature [232], the spectroscopy data revealed the configuration of compound 2 to be p-hydroxybenzoic acid, as shown in Table 3.3 and its structure is presented in Figure 3.2.

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Table 3.3: 1H-NMR and 13C-NMR spectra of P-Hydroxybenzoic acid (2)

C.No 13C 1H Types HMBC

- - 1 131.5 C

2 130.3 8.26 ( t, J =7.4 Hz) CH C-1, C-3, C-4

3 129.6 7.34 ( t, J = 7.4 Hz) CH C-4, C-5

4 124.6 6.51 ( t, J = 7.4 Hz) C -

5 129.6 7.34 ( t ,J = 7.4 Hz) CH C-4, C-6

6 130.3 8.26 (t, J= 7.4 Hz) CH C5, C-6

COOH 181.3 11.92 (s, O-H) C -

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Figure 3.2: Structure of P-hydroxy benzoic acid (2)

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3.2.3 Structural Elucidation of Bis (2-ethylhexyl) phthalate (3)

The compound 3 was isolated from the EtOAc fractions of Q. semecarpifolia and purified as colorless viscous liquid. The molecular formula of this compound was found to be C24H39O4. The mass spectrometry data (m/z values) for this compound at 70 eV were as follows: 391, [M + H]+

+ + + + (traces); 279, [M-C8H15] (9%); 167, [M-C16H31] (35%); 149, [C8H5O3] (100%); 57, [C4H9]

(36%); HRMS (APCI) m/z = 391.2, found: 391.2.

The 13C-NMR spectra displayed twenty four carbon signals which mainly include six methylene, six methane, 4 methyl and quaternary carbons. The spectra exhibited signals at 133.2 (C‐1),

128.1 (C‐2), 132.2 (C-3), 132.2 (C-4), 128.1 (C-5), 133.2 (C-6), 168.2 (C-1/), 65.1 (C-2/), 29.2

(C-3/), 24.1 (C-4/), 39.1 (C-5/), 26.1 (C-6/), 22.2 (C-7/),, (C-8/), 22.2 (C-9/), 168.2 (C-1//), 65.1

(C-2//), 29.2 (C-3//), 24.1 (C-4//), 39.1 (C-5//), 26.1 (C-6//), 22.2 (C-7//), (C-8//) and 22.2 (C-9//).

1 The H-NMR data revealed four proton signals at δ7.78–7.54 for the aromatic protons and similar proton signals at δ0.93–4.42 could be assigned to the six methylene, six methane, and four methyl protons. When compared to previously published literature [233], the spectroscopy data revealed the configuration of compound 3 to be Bis (2-ethylhexyl) phthalate as shown in

Table 3.4 and graphically depicted in Figure 3.3.

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Table 3.4: 1H-NMR and 13C-NMR spectra of Bis (2-ethylhexyl) phthalate (3)

Carbon 13C 1H Types HMBC number 1 133.2 ----- C - 2 128.1 7.78, d, J=8.00 CH C-5, C-4, C-3, C-2, C1/ 3 132.2 7.54 dd, J=7.54 CH C-4, C-5 4 132.2 7.54 dd, J=7.54 CH C-5, C-4, C-3, C-2, C1/ 5 128.1 7.78, d, J=8.00 CH C-2, C-3, C-4, C1/ 6 133.2 ----- C - 1/ 168.2 ----- C - / / / / 2 65.1 4.22, m CH2 C-4 , C-3 , C-1 / / / 3 29.2 1.72, m CH2 C-4 , C-5 / / / 4 24.1 1.28, m CH2 C-5 , C-6 / / / 5 39.1 1.29, m CH2 C-6 , C-7 / / / / 6 26.1 1.34, m CH2 C-5 , C-7 , C-8 7/ 28.1 1.30 m CH C-5/, C-6/, C-8/ / / / / 8 22.2 0.93 m CH3 C-6 , C-7 , C-9 / / / 9 22.2 0.93 m CH3 C-7 , C-8 1// 168.2 1.27,m C - // // // // 2 65.1 4.22, m CH2 C-1 , C-3 , C-4 // // // 3 29.2 1.72, m CH2 C-4 , C-5 // // // 4 24.1 1.28, m CH2 C-5 , C-6 // // // 5 39.1 1.29, m CH2 C-6 , C-7 // // // // 6 26.1 1.34, m CH2 C-5 , C-7 , C-8 7// 28.1 1.30 m CH C-5//, C-6//, C-8// // // // // 8 22.2 0.93 m CH3 C-6 , C-7 , C-9 // // // 9 22.2 0.93 m CH3 C-7 , C-8

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Figure 3.3: Structure of Bis (2-ethylhexyl) phthalate (3)

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3.2.4 Structure Elucidation of β-Sitosterol (4)

From the EtOAc fractions of Q. semecarpifolia, the compound 4 was isolated and purified as white, needle-like crystals. The molecular formula of this compound was found to be C29H50O and the approximate molar mass of this compound was 414.7,as determined by HR ESI-MS.

Different ion peaks were observed at the m/z values of 287.1, 194.2, 163.9, 138.9, 123.0, 85.9,

73.0, 46.9, and 32.9. The IR spectroscopy data displayed absorption spectra at 3473.6 cm–1 (O-H

–1 –1 stretching) as well as at 2941.7 cm and 2837.9 cm (C-H stretching); UV λmax = 245, 266, 430 nm.

13 The C‐NMR spectra displayed twenty nine carbon signals which mainly include eleven methane, nine methylene, six methyl and three quaternary carbons. The spectra have shown signals at 34.9 (C‐22), 118.5

(C‐6), 55.8(C‐17), 150.98 (C‐5), 40.8 (C‐20), 78.12 (C‐3), 56.4 (C‐14), 40.8 (C‐20), 51.37 (C‐9), 27.1 (C‐

15), 27.4 (C‐28), 19.1 (C‐11), 40.1(C‐12), 37.8 (C‐4), 39.8 (C‐13), 37.5 (C‐12), 36.23 (C‐1), 36.11 (C‐

10), 35.4 (C‐8), 34.9 (C‐22), 34.2 (C‐7), 36.3 (C‐8), 29.86 (C‐25), 29.71 (C‐16), 30.41 (C‐2), 21.6 (C‐27),

19.32 (C‐19), 17.71 (C‐21), 15.6 (C‐18, 29).

The 1H NMR data showed two proton signals at δ2.41 and δ5.39, which correlated to the H–3

and H–6 of the steroid skeleton, respectively. Furthermore, the 1H NMR spectra showed two signals at δ1.47 and δ1.19 and two tertiary CH3 groups, one at C–18 and the other at C–19 could be assigned to these two signals. Moreover, two signals at δ0.92 and δ0.87 were obtained from the spectra and these could conceivably be assigned to the two CH3 groups at C–26 and C–27, respectively. However at C–21, a triplet proton signal was assighned to methyl group. Further, the intensity of three protons in the triplet signal at δ0.88 contributed to the methyl group at C–

29. When compared to previously published literature [234,235], the spectroscopic data revealed the configuration of the compound 4 to be β-sitosterol, as shown in Table 3.5 and depicted in

Figure 3.4.

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Figure 3.4: Structure of β-Sitosterol (4)

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Carbon 13C 1H Types HMBC number

1 37.18 1.07 d, (J =3.7/5.11) CH2 C

2 30.41 1.27, 1.38, m CH2 -

3 78.12 3.63,m, 3.2,m CH2 -

4 38.9 2.00, t, (J=7); 1.89, (d, J= 3.17) CH2 C-2, C-3, C-5, C-6, C- 10 5 150.98 ---- C - 6 118.5 5.39,m CH C-4, C-8,C-10

7 34.2 1.32,m CH2 - 8 36.3 1.35,m, 1.45, m CH - 9 50.45 0.76,m, 0.89, m CH - 10 37.12 --- C -

11 19.1 1.51,m,1.62,m CH2 -

12 38.6 2.17, d, (J=2.1); 2.22, d, (J=2.1) CH2 - 13 39.8 ---- C - 14 55.4 0.87,m CH -

15 27.1 1.37,m CH2 -

16 29.71 1.27,m CH2 - 17 55.8 1.02, d, (J=7.7, 3.48) CH -

18 15.6 1.47, m CH3 C-12, C-13,C-14, C-17

19 19.32 1.19, m CH3 C-1, C-9,C-10 20 40.8 2.38, m CH -

21 17.71 1.64 (J = 6.5Hz) CH3 C-17, C-20, C-22

22 34.9 4.68,m CH2 -

23 26.12 5.19,m CH2 - 24 45.9 1.12,m CH - 25 29.86 1.35,m CH -

26 25.7 0.92,d, (J = 6.7) CH3 C-24, C-25, C-27

27 21.6 0.87,d,(J = 6.7) CH3 C-24, C-25, C-26

28 27.4 1.11,m CH2 -

29 15.6 0.88, t, (J=7.7) CH3 C-24, C-28

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3.2.5 Structural Elucidation of Stigmasterol (5)

From the EtOAc fractions of Q. semecarpifolia, compound 5 was isolated and purified as a white, amorphous solid, having a melting point of 165 °C. The molecular formula of the compound was found to be C29H48O. The mass spectrometry data (m/z values) for the compound were as follows: 412 [M+], 394, 351, 314, 300, 271, 229, 213, 55; HRMS (APCI) m/z = 412.7.

The IR spectra showed intense broad peaks at 3461.2 cm–1 (O-H stretching), 2847 cm–1 (C-H bending), 1543.74 cm–1 and 1453.23 cm–1 (methylene). The compound also displayed absorption

–1 –1 spectra (stretching) at 1365 cm [CH2 (CH3)2] and 1047.7 cm (cycloalkane). The UV-Vis spectroscopy data showed absorption peaks at 241 nm and 257 nm.

13C NMR showed δC: 48.2 (C-9), 57.7 (C-14), 18.7 (C-19), 129.8 (C-6), 35.4 (C-

20), 38.6 (C-10), 19.4 (C-21), 23.4 (C-15), 74.2 (C-3), 57.2 (C-17), 27.3 (C-16), 35.4 (C-20),

41.9 (C-12), 40.2 (C-13), 27.3 (C-16), 42.6(C-4), 36.9 (C-1), 29.7 (C-8), 138.1 (C-22), 31.6

(C-7), 30.8 (C-25), 24.14(C-28), 19.8 (C-11,26), 20.2 (C-27), 19.4 (C-21), 12.3 (C-18), 12.6

(C-29).

In the 1H NMR spectra of this compound, the H–3 proton appeared as a triplet of a doublet at

δ3.25 (J=4.5). Furthermore, a multiplet was shown by the H–6 proton at δ6.34. At H–22 and H–

23, two olefinic proton signals appeared at δ4.62 (m) and δ4.61 (m), respectively. A total of six methyl proton signals were also displayed at δ1.08, δ1.16, δ1.01, δ0.93, δ0.91, and δ0.87.

When compared to previously published literature [236,237], the spectroscopic data revealed the structure of compound 5 to be stigma sterol, as shown in Table 3.6 and depicted in Figure 3.5.

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Figure 3.5: Structure of Stigmasterol (5)

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Table 3.6: 1H-NMR and 13C-NMR spectra of Stigmasterol (5)

C No 13C 1H Types HMBC

1 36.9 1.09, dd, (J=3.7/5.11) CH2 C-3, C-5,C-19

2 31.5 1.43, d, (J=1.68) CH2 -

3 74.2 3.25( J=4.5) CH2 -

4 42.6 2.00, t, (J=8); 1.89, d, (J= 3.32) CH2 C-2, C-3, C-5, C-6, C-10 5 141.4 - C - 6 129.8 6.34, d, (J=1.34) CH C-4, C-8,C-10

7 31.6 1.34, m CH2 - 8 29.7 1.52, m CH - 9 48.2 0.94, m CH - 10 38.6 - C -

11 19.8 1.39,m, 1.57 m CH2 -

12 41.9 2.29, d, (J=2.8); 2.35, dd, (J=2.1, 2) CH2 - 13 40.2 - C - 14 57.7 0.86, m CH -

15 23.4 1.46, m CH2 -

16 27.3 1.13, m CH2 - 17 57.2 1.09, dd, (J=6.8, 2.48) CH -

18 12.3 1.08,s CH3 C-12, C-13,C-14, C-17

19 18.7 1.16,s CH3 C-1, C-9,C-10 20 35.4 1.24, m CH -

21 19.4 0.91 , d, (J=6) CH3 C-17, C-20, C-22 22 138.1 4.62, dd, (J=14, 7.5) CH - 23 129.5 4.61 , dd, (J=15.1, 8.5) CH - 24 53.7 0.82, m CH - 25 30.8 1.35, m CH -

26 19.8 1.01, d, (J=6.8) CH3 C-24, C-25, C-27

27 20.2 0.93, d, (J=5.58) CH3 C-24, C-25, C-26

28 24.14 1.12, m CH2 -

29 12.6 0.87, t, (J=7.5) CH3 C-24, C-28

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3.3 Plant Mediated Synthesis of AgNPs

According to previous studies it was reported that AgNPs has dark brownish color. The color of

Q. semicarpifolia leaf extract was light yellow before treating with silver nitrate (AgNO3) but when AgNO3 was added, the solution turned to dark brownish (Figure 3.6 ), specifying the production of AgNPs. This is because of reducing power of biomolecules present in the extract.

3.4 Characterization of Silver Nanoparticles (AgNPs)

3.4.1 UV-Vis Spectroscopy

The plant-mediated synthesis of AgNPs is a one-step process in which, the reduction of metallic salts by plant extracts takes place, resulting in the synthesis of AgNPs. The reduction is observed by a change in the color of the solution [238]. Such a distinctive optical property, exhibited by the noble metals is due to the SPR absorption band, caused by the free movement of the electrons of AgNPs, in resonance with the light wave. When the free oscillation of electrons becomes resonant with the frequency of the electromagnetic field, strong absorption occurs and the color of the solution changes [239]. For AgNPs, SPR gives an intense peak at 350–500 nm. However, absorption mainly depends on the size of the NPs and the dielectric medium [240]

Generally, UV-Vis spectral study is used to detect the formation of AgNPs by measuring the

SPR absorption peaks. On addition of the leaf extracts of Q. semecarpifolia to a solution of

AgNO3, the color of the solution usually changes from light yellow to reddish-brown due to the reduction of Ag+ ions to metallic Ag, using the active phytochemicals present in the aqueous leaf extracts.

According to the UV-Vis spectroscopy data, the absorption maxima (λmax) for the plant-derived

AgNPs ranged between 350 nm and 500 nm. However, the aqueous extracts showed irregular absorbance patterns in the same wavelength range. The λmax for Q. semecarpifolia was arrived at

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430 nm, revealing the highest absorbance value of 0.72 (Figure 3.7-3.8). The biogenic AgNPs were stable for a period of about three months.

Previously, it was reported that the SPR absorption band of AgNPs, synthesized from the leaf extracts of Cannonball was in the range of 300–600 nm [241]. Moreover, another report suggested that the leaf extracts of Ceropegia thwaitesii were evaluated by UV-Vis spectroscopy with an SPR absorption band at 430 nm [242]. The SPR absorption band of the plant-derived

AgNPs, synthesized from an extract of Ficus benghalensis was at 410 nm [243].

Mohammad et al. evaluated a study for the biogenic production of AgNPs, using the aqueous leaf extracts of Zizyphus spina. A characteristic and well-defined SPR band was confirmed at 414 nm [244] In addition, another study stated the fabrication of AgNPs, using the plant Paederia foetida as a reducing agent. A change in the colorless solution into a reddish brown colored solution demonstrated the reduction of Ag+ ions to AgNPs. The SPR absorption band for the synthesized AgNPs was distinctive at 430 nm [245].

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Figure 3.6: a) Plant leaf extract (b) Synthesized silver nanoparticles

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Absorbance

0.8

0.72

0.6

0.4 Absorbance

0.2

0 350 370 390 410 430 450 470 490 510 530 550 Wavelength (nm)

Figure 3.7: Graphical representation of absorbance values of Quercus semecarpifolia AgNps

Absorbance

0.8

0.6

0.4 Absorbance

0.2

0 350 370 390 410 430 450 470 490

Wavelength (nm)

Figure 3.8: Graphical representation of absorbance values of Quercus semecarpifolia aqueous extract

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3.4.2. X-Ray Diffraction Pattern

Generally, XRD patterns are used for the confirmation of the crystalline nature of AgNPs. In the current study, the crystal size of the test samples was determined by an investigation, using the

XRD technique. The samples included the aqueous extracts and the corresponding aqueous extract-derived AgNPs of the selected plants. From the XRD results, different peaks were recorded for the samples and according to the Debye-Scherrer equation, the size of the crystals was determined to be 6.41Å and 7.5Å for the aqueous extracts of Q. semecarpifolia and the plant-derived AgNPs, respectively. In the AgNPs derived from Q. semecarpifolia, the intensities of diffraction were recorded approximately at 64.9°, 44.55°, and 38.09° and were assigned to reflections from the 2θ range, corresponding to the (220), (200), and (111) reflection planes of

Ag crystals, respectively. The absence of impure peaks suggested that the test samples were in a purified form. Many irregular d values were observed within the same 2θ range when the findings were compared to those of the aqueous leaf extracts, which confirmed the presence of the face-centered cubic (FCC) structures of Ag [246]. The other peaks observed at 14.35˚ and

16.9˚were also accountable for the fabrication of AgNPs due to the phytochemical constituents present in the plant extracts [247]. The XRD upshots are depicted in Figure (3.9-3.10).

The leaf-derived AgNPs of Malus domestica were investigated for the determination of the size of Ag crystals. A total of four distinct diffraction peaks were displayed by the AgNPs at

38.28°, 41.3°, 63.6°, and 77.5° in the 2θ range. Furthermore, following the Debye-Scherrer equation, the crystal size of the AgNPs was found to be 4.08Å approximately [248]. Huda et al. investigated derived AgNPs for the first time, using the aqueous extracts of Chamaemelum nobile by the XRD technique. The different diffraction peaks were observed at 77.40°, 64.70°,

44.50°, and 37.10° in the 2θ range corresponding to the (311), (220), (200), and (111) reflection

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planes of the FCC structure of Ag crystals. Also, the size of the crystals was determined and found to be in the range of 24 nm [249].

Subha et al. evaluated a study for the determination of the crystal size of the plant-derived

AgNPs, using the Erythrina indica plant aqueous extract. From that study, the structure of

AgNPs was revealed to be FCC. Furthermore, all the noticeable peaks were observed in the 2θ range at 46°, 32°, and 28°, corresponding to the (420), (311), and (220) Bragg‘s reflections. The crystal size was found to be 8 nm, according to the Debye-Scherrer equation [250].

Anandalakshmi et al., synthesized plant derived AgNPs, using the aqueous extracts of Vitex negundo. Successive peaks, observed by the XRD patterns in the 2θ range at 38.11°, 44.29°,

64.45°, and 77.39 °, corresponding to the (111), (200), (220), and (311) reflection planes of Ag crystals were identified in the diffractogram. The crystal size was found to be 8.4 A.The absence of other diffraction peaks indicated that the synthesized AgNPs were pure in nature [251].

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50 47

30 29 Intensity

20 19 18 16 10

0 0 10 20 30 40 50 60 70 80 Angle of diffraction (2Ө)

Figure 3.9: Graphical representation of XRD values of Quercus semecarpifolia aqueous extract

35 38.09 (111) 30 14.35

16.9

20 Intensity 200 64.9 220 15 44.55

0 0 10 20 30 40 50 60 70 80

Angle of diffraction (2Ө)

Figure 3.10: Graphical representation of XRD values of Quercus semecarpifolia derived AgNPs

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3.4.3 Scanning Electron Microscopy (SEM)

Generally, SEM imaging is used for determining the structure and configuration of synthesized

AgNPs. In the current study, the size and conformation of the AgNPs, derived from Q. semecarpifolia, were detected by SEM imaging at 10,000, 20,000, 30,000 and 60,000 X magnifications. The images obtained from SEM imaging clearly indicated a spherical morphology of the AgNPs with an average size of 20–50 nm, distributed well without any aggregation. As reported earlier, different capping and reducing agents, present in the leaf extracts were responsible for the variations in size [252]. A comparative analysis of AgNPs was done with the aqueous extracts of the selected plant, which displayed irregular images. SEM images are depicted in Figure ( 3.11-3.18)

The plant-derived AgNPs, obtained from the aqueous leaf extracts of a total of six species of plants—Rosa rugosa, Lantana camara L., Murraya koenigii, Artemisia nilagirica, Ceropegia thwaitesii, and Olea europaea showed varied conformations, mainly rectangular, spherical, cuboidal, spheroidal, and triangular [253-257]. The SEM images of the plant-derived AgNPs were due to the interactions of hydrogen bonds and the electrostatic interactions among the bioorganic capping molecules, bound to the AgNPs [258].

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Figure 3.11: SEM micrograph of Quercus semecarpifolia derived AgNPs at 10,000X

Figure 3.12: SEM micrograph of Quercus semecarpifolia AgNPs at 20,000X

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Figure 3.13: SEM micrograph of Quercus semecarpifolia derived AgNPs at 60,000X

Figure 3.14: SEM micrograph of Quercus semecarpifolia derived AgNPs at 30,000X

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Figure 3.15: SEM micrograph of Quercus semecarpifolia aqueous extract at 10,000X

Figure 3.16: SEM micrograph of Quercus semecarpifolia aqueous extract at 20,000X

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Figure 3.17: SEM micrograph of Quercus semecarpifolia aqueous extract at 30,000X

Figure 3.18: SEM micrograph of Quercus semecarpifolia aqueous extract at 60,000X

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3.4.4 Energy Dispersive X-Ray Spectroscopy (EDX)

Generally, an EDX analysis is performed for determining the configuration and quantity of an element. In the current investigation, a comparison of the elemental composition of the aqueous leaf extracts was done with that of the biosynthesized AgNPs, using the EDX analysis. The aqueous extracts of Q. semecarpifolia contained 54.09% carbon (C), 3.57% nitrogen (N),

40.58% oxygen (O), 0.23% aluminum (Al), 0.12% silicon (Si), 0.12% potassium (K), 0.35% calcium (Ca), 0.45% copper (Cu), and 0.41% zinc (Zn), as shown in Figure 3.19 and Table 3.7.

The EDX images revealed that the plant-derived AgNPs possessed 34.96% (C), 40.30% (O),

0.93% magnesium (Mg), 0.38% (Si), 0.93% (K), 2.10% sulfur (S), 2.95% chlorine (Cl), 5.15%

Ca, and 11.81% (Ag), as shown in Figure 3.20 and Table 3.8. The biosynthesis of AgNPs was confirmed by the absence of elemental Ag in the aqueous leaf extracts. From the EDX profile, it was clear that the bioinspired AgNPs had a crystalline morphology and the peaks of Ag atoms were located in the range of 2–4 keV.

In their study, Vijayakumar et al. described the plant-mediated fabrication of AgNPs, using the leaf extracts of Artemisia nilagirica. The presence of elemental Ag was confirmed in the range of 2–4 keV from the EDX analysis and also no other impure peaks were detected

[259]. Koyatti et al. made stable AgNPs, using the leaf extracts of Amaranthus viridis and these biogenic AgNPs were characterized with the help of the SEM, EDX, TEM, and XRD techniques.

Notably, the EDX analysis indicated that the percentage of Ag in these biosynthesized AgNPs was 35.48%. The EDX analysis also confirmed that the aggregates, shown in that typical optical absorption peak at approximately 3 keV, were Ag nanocrystals [260].

In another finding, Fatima et al. narrated the plant-derived AgNPs, using the leaf extracts of the plant, Parkia speciosa. The EDX studies confirmed that the synthesized particles were

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nanocrystals and a peak of elemental Ag was obtained at approximately 3 keV. All these previous reports support the findings from the EDX analysis in the current investigation [261].

In a previous study, elemental composition of the plant-derived AgNPs of Origanum vulgare L was also investigated by EDX profile. The results showed that Ag was the main element and it gave sharp band at 3 KeV. Carbon and oxygen were the other two elements which showed signals in the range of 0–0.5 KeV. The presence of these elements clearly manifested that they act as stabilizing agents in the formation of AgNPs [262].Dinesh et al studied the EDX profile of Glycyrrhiza glabra aqueous leaf extract. The results revealed strong signals of Ag in the region of 3 keV, thus confirmed the formation of AgNPs. Optical absorption band of the Ag nanocrystals generally observed in the region of 3keV and this is mainly due to SPR. Thus the analysis revealed that AgNPs were formed due to silver [263].

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Table 3.7: Elemental analysis of the Quercus semecarpifolia aqueous extract

S. No. Element Weight % Atomic %

1 Carbon (C) 54.09 61.39

2 Nitrogen (N) 3.57 3.47

3 Oxygen (O) 40.58 34.58

4 Aluminum (Al) 0.23 0.12

5 Silicon (Si) 0.20 0.10

6 Potassium (S) 0.12 0.04

7 Calcium (Ca) 0.35 0.12

8 Copper (Cu) .45 0.10

9 Zinc (Zn) 0.41 0.09

Total 100 100

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Table 3.8 Elemental analysis of the Quercus semecarpifolia derived AgNPs

S. No. Element Weight % Atomic %

1 Carbon (C) 34.96 49.31

2 Oxygen (O) 40.30 42.68

3 Magnesium (Mg) 0.93 0.65

4 Phosphorus (P) 0.38 0.21

5 Silicon (Si) 0.38 0.23

6 Sulphur (S) 2.10 1.11

7 Chlorine (Cl) 1.03 0.49

8 Potassium (K) 2.95 1.28

9 Calcium (Ca) 5.15 2.18

10 Silver (Ag) 11.81 1.85

Total 100 100

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Figure 3.19: Graphical representation of EDX profile of Quercus semecarpifolia aqueous extract

Figure 3.20: Graphical representation of EDX profile of Quercus semecarpifolia synthesized AgNPs

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3.4.5 Fourier Transform Infra-Red Spectroscopy (FTIR)

The plants consists of various phytochemical constituents such as phenols, flavonoids, tannins, carbohydrates, proteins, and alkaloids which have important role in the fabrication of AgNPs.

Their main role is basically the reduction of Ag ions to AgNPs. Thus this involvement of the secondary metabolites and the functions groups present in these organic compounds is basically determined by FTIR analysis. Both the aqueous extract and plant-derived AgNPs were scanned from 4000 cm-1 to 400 cm-1 [264].

A sharp peak at 3272 cm-1 was observed in Q.semecarpifolia aqueous extract which confirmed the presence of NH group.Two other crests at 3250 and 1716 cm-1 were observed which corresesponded to C-H and COOH groups respectively (Figure 3,21). However, in plant derived

AgNPs, the NH peak intensity increased and it completely disappeared which clealrly confirmed that olefenic and carboxylic groups are involved in the synthesis of AgNPs (Figure (3.22).

In a study, aqueous extract of rosemary (Rosmarinus officinalis) extract was utilized for the synthesis of AgNPs. The synthesized AgNPs were characterized by different techniques. The

FTIR results clearly showed that proteins and polyphenols present in the plant extract play key role in the synthesis AgNPs [265]. Similar findings from previous studies also have shown that the NH3, OH and COOH groups present in the organic compounds play key role in formation of

AgNPs [266].

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Figure 3.21: FTIR analysis of Quercus semicarpifolia aqueous extract

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Figure 3.22: FTIR analysis of Quercus semicarpifolia aqueous extract derived AgNPs

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3.4.6 Transmission Electron Microscopy (TEM) Studies:

The TEM imaging analysis is mostly performed for confirming the size, configuration, and shape of bioinspired AgNPs. The technique provides a detailed two-dimensional conformation and diameter of AgNPs. In this study, the images obtained from the TEM analysis of biosynthesized

AgNPs, using the leaf extracts of Q. semecarpifolia clearly indicated predominantly uniform, spherical morphologies, ranging from 20–50 nm in size, as shown in Figure (3.22-3.23). Thus,

TEM imaging revealed that the dimension and conformation of biosynthesized AgNPs were consistent with the results obtained from SEM imaging. Most of the AgNPs had a spherical morphology with smooth edges and were able to penetrate through the membrane [267, 268].

All these structures were similar to those of the bioinspired AgNPs, synthesized from the leaves of the plants, Q. incana and Q. brantii because the biomolecules present in both the plant species were similar [269]. Priya et al. investigated the leaf extracts of three plants—Azadirachta indica,

Musa balbisiana, and Ocimum tenuiflorum for the fabrication of AgNPs, using a 1 mM aqueous solution of AgNO3. The TEM imaging studies from various plant aqueous extracts showed a number of configurations such as pentagonal, hexagonal, and triangular but the spherical configuration was predominant with a size of up to 80 nm [270].

Krishnadhas et al., developed an environment friendly process for the phytoproduction of plant- derived AgNPs, using the aqueous leaf extracts of the plant, Volkameria inermis. The resulting

NPs were characterized by different techniques among which, TEM imaging clearly indicated the topology and size of the synthesized AgNPs. In fact, most of the synthesized AgNPs, showing a spherical morphology with size of 50 nm were monodispersed without clumping

[271].

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Figure 3.23: TEM micrograph of Quercus semecarpifolia derived AgNPs at 100 nm magnification

Figure 3.24: TEM micrograph of Quercus semecarpifolia derived AgNPs at 200 nm magnification

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3.3.7 Thermogravimetric Analysis/Differential Thermal Analysis (TGA/DTA)

In order to study the thermal features of AgNPs, Thermogravimetric Analysis (TGA) was performed. Generally, TGA is used for investigating the changes in a mass with respect to temperature, which specifies the moisture content, material purity, and heat resistance of AgNPs.

The approach clearly figures out that upon increasing the temperature, loss in the weight of

AgNPs occurs. In this technique, samples were heated at temperatures, ranging from 100–1000

ᴼC for the decomposition of AgNPs. The decomposition of a test sample starts at a temperature of 100 °C and its size decreases slowly when the temperature reaches up to 361.23°C. In this study, the weight of the test sample was 6.963 mg at the start of the process but upon increasing the temperature to 526.81 °C, its weight was reduced to 2.711 mg because moisture was removed from AgNPs, following which no further loss in weight occurred as shown in Figure

3.25. The TGA profiles of the aqueous extracts showed a loss in weight at a temperature of

536.36 °C, as shown in Figure 3.26.

The Differential Thermal Analysis (DTA) is another method of thermal investigation that is used for the analysis of changes in the temperature of a test sample when it is subjected to heating. In the present investigation, the DTA graph of the plant-derived AgNPs showed an exothermic reaction at a temperature of 492.60 °C, as depicted in Figure 3.27. In the aqueous extracts of Q. semecarpifolia, an endothermic reaction was observed at a temperature of 523.67ᴼC, as shown in

Figure 3.28. Thus, it was concluded that the loss in weight was due to the decomposition of organic matter present in the aqueous extracts [272, 273].

Mustafa et al. reported the thermal behavior of the plant-derived AgNPs, synthesized from the leaf extracts O. europaea. A decrease in the mass of AgNPs was witnessed in the

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temperature ranging from 200–600 °C by studying the TGA profiles. The loss in weight of the

AgNPs is due to the loss of bioorganic compounds [274]. Khan et al reported the fabrication of the phytosynthesis of AgNPs, using the leaf extracts Coriandrum sativum and characterized them by different techniques. The TGA profiles of the bioinspired AgNPs revealed that the loss in weight was constant at temperatures, ranging from 200–500 °C and at temperatures below

500 °C, a slight decrease in weight occurred. However, no loss in weight occurred at temperatures, ranging from 550–800 °C. An analysis of the DTA graphs displayed two exothermic peaks of which, one peak ascended in the range of 200–300 °C and the second peak was present in the range of 400–500 °C [275].

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2 UnsubtractedWeight) (g 1

0 100 200 300 400 500 600 700 800 900 1000

Sample temperature ( 0C)

Figure 3.25: TGA profile of Quercus semecarpifolia derived AgNPs

Unsubtracted Weight (g) Weight Unsubtracted 1

-1 100 200 300 400 500 600 700 800 900 1000 Sample Temperature (0C)

Figure 3.26: TGA profile of Quercus semecarpifolia aqueous extract

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

 40 (

20 ovolts

-20

-40

Unsubtracted Micr -60 100 200 300 400 500 600 700 800 900 1000 0 Sample Temperature ( C )

Figure 3.27: DTA profile of Quercus semecarpifolia derived AgNPs

 40

 (

-20

-40 UnsubtractedMicrovolts

-60 100 200 300 400 500 600 700 800 900 0 Sample Temperature( C )

Figure 3.28: DTA profile of Quercus semecarpifolia aqueous extract

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3.5 Biological Evaluation of AgNPs Compared to Crude Plant Extracts (in vitro)

3.5.1 Antibacterial Activity

Throughout the world, plants are a potential source of antimicrobial agents. In developing countries, about 60–80% of the population use plants as medicines against the various pathogenic microorganisms. Plants are a rich source of secondary metabolites, which have been found to possess antimicrobial properties. The toxic effects caused by antibiotics limit the use of these antimicrobial agents. However, the research against the antibiotic-resistant strains of bacteria, using plant-based drugs, is encouraged due to their effectiveness and safety profile

[276, 277].

In the present study, the test samples were checked for potent antibacterial activities against the selected species of bacteria and the results are presented in Figure (3.29) and Table

3.9. The results obtained from the MIC50 are depicted in Table 3.10. The test samples (Cr.

MeOH. Ext) of Q. semecarpifolia exhibited a noteworthy antibacterial activity of 81% with

MIC50 value of 1.2 μg/mL against S. aureus. Moderate antibacterial activities of 68%, 65%,

62%, and 60% with the MIC50 values of 2.1, 2.6, 2.7, and 2.8 μg/mL were seen against B. subtilis, E. coli, K. pneumoniae, and P. mirabilis, respectively. On the other hand, a low antibacterial effect of 44% with an MIC50 value of 3.9 μg/mL was observed against S. marcescens. However, the extracts were unable to show any antibacterial activities against P. aeruginosa and S. pneumoniae.

The n-hexane extracts of Q. semecarpifolia exhibited a significant bactericidal effect of

70% with an MIC50 value of 2.1 μg/mL against E. coli. A good inhibitory effect of 62% with an

MIC50 value of 2.6 μg/mL was also found against P. mirabilis, whereas moderate antibacterial

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activities of 55%, 52%, and 50% with the MIC50 values of 2.9, 3.0, and 3.2 μg/mL were noted against B. subtilis, S. pneumoniae, and S. aureus, respectively. Furthermore, a low antibacterial effect of 37% with MIC50 value of 5.1 μg/mL was observed against S. marcescens. However, the extracts were found to be inactive against K. pneumoniae and P. aeruginosa.

The CHCl3 fractions significantly inhibited the growth of K. pneumoniae by 90% with

MIC50 value of 0.8 μg/mL. Also, good bactericidal effects of 72%, 68%, and 63% with the

MIC50 values of 1.9, 2.1, and 2.6 μg/mL were observed against B. subtilis, P. mirabilis, and S. marcescens, respectively. Furthermore, moderate inhibitory effects of 56%, 56%, and 54% with the MIC50 values of 3.2, 3.1, and 3.8 μg/mL were noted against E. coli, P. aeruginosa, and S. aureus, respectively. However, a low antibacterial activity of 41% with MIC50 value of 4.1

μg/mL was seen against S. pneumoniae.

The EtOAc fractions of Q. semecarpifolia exhibited momentous inhibitory activities of

86% and 84% with the MIC50 values of 0.7 and 0.9 μg/mL against K. pneumoniae and P. mirabilis, respectively. Further, good bactericidal effects of 78%, 76%, and 65% with the MIC50 values of 1.1, 1.3, and 2.2 μg/mL were noted against P. aeruginosa, B. subtilis, and S. aureus, respectively. However, a moderate antibacterial effect of 52% with an MIC50 value of 3.7 μg/mL was observed against E. coli. On the other hand, the EtOAc fractions showed low antibacterial activities of 45% and 44% with the MIC50 values of 4.3 and 4.4 μg/mL against S. pneumoniae and S. marcescens, respectively.

The aqueous extracts of Q. semecarpifolia showed good inhibitory effects of 71%, 63%, and 61% with the MIC50 values of 2.3, 2.6, and 2.7 μg/mL against K. pneumoniae, P. aeruginosa, and E. coli respectively. Besides, moderate antibacterial activities of 57% and 56%

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with the MIC50 values of 3.5 and 3.6 μg/mL were observed against S. pneumoniae and P. mirabilis, respectively. Low antibacterial activities of 48% and 46% with the MIC50 values of 4.4 and 4.8 μg/mL were shown against S. marcescens and S. aureus, respectively. However, no inhibition was observed against B. subtilis.

The AgNPs, derived from the aqueous extracts of Q. semecarpifolia showed remarkable antibacterial effects of 90%, 88%, and 84% with the MIC50 values of 0.5, 0.8, and 1.2 μg/mL against K. pneumoniae, B. subtilis, and P. mirabilis, respectively. Also, significant antibacterial activities of 74%, 72%, and 70% with the MIC50 values of 1.7, 1.9, and 2.1 μg/mL were observed against S. marcescens, S. pneumoniae, and E. coli, respectively. However, the AgNPs showed a good antibacterial activity of 67% with an MIC50 value of 2.3 μg/mL against P. aeruginosa, whereas a moderate antibacterial activity of 54% with an MIC50 value of 2.9 μg/mL was shown by the AgNPs against S. aureus.

Previously, Serit et al. conducted a study on the methanolic extract and the different fractions, including n-hexane, EtOAc, and CHCl3 of Q. acuta. The findings from their study revealed that the extracts displayed good antibacterial activities against the pathogenic species of bacteria. The EtOAc fractions displayed the maximum bactericidal effects. Two compounds that were also isolated by CC showed promising results against the pathogenic species of bacteria

[278].

In their study, Omar et al. screened a total of 14 species of plants, in order to determine their antimicrobial activities against a total of eight species of bacteria. Among the species of plants, the methanolic extract of Q. rubra showed significant antibacterial activities against

MSSA (methicillin-sensitive Staphylococcus aureus), B. subtilis, and Mycobacterium phlei

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[279]. In their study, Sakar et al. described the antibacterial effects of the crude extracts of Q. aucheri against a total of four pathogenic species of bacteria. Among the extracts, the EtOAc fractions exhibited the highest bactericidal effects against S. aureus with the MIC50 value of 2.2

µg/mL [96].

Previously, it was stated that the Cr. MeOH extract of Q. infectoria revealed influential antibacterial activities against Streptococcus mutans. The results revealed that at an extract concentration of 50 μg/mL, strong antibacterial effects were observed [280]. Supayang et al. screened a total of 38 species of medicinal plants, in order to determine their antimicrobial activities against the different strains of E.coli. In that study, about 58 MeOH and aqueous extracts were used and only a total of 14 extracts, belonging to eight plant species, were revealed to have potent bactericidal effects against the strains of E. coli tested. Among the species of plants, the Cr. MeOH. Ext of Q. infectoria showed the maximum inhibition zones against E. coli

[281].

In their study, Berahou et al. evaluated the antibacterial properties of the different bark extracts of Q. ilex. Among the extracts, the EtOAc and butanol (BuOH) fractions significantly inhibited the growth of the seven species of bacteria—P. aeruginosa, S. aureus, Vibrio cholerae, B. subtilis, P. mirabilis, E. coli, and K. pneumoniae, demonstrating that the bark of the plant is a useful bactericidal agent [282]. Hayouni et al. tested the various organic extracts (Cr. MeOH,

EtOAc, BuOH, and n-hexane) of Q. coccifera against the different pathogenic species of bacteria. Stupendous results were shown by the Cr. MeOH and EtOAc extracts against S. aureus and E. coli [283].

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In their study, Khurram et al. highlighted that using the different extracts of Q. baloot, the different species of bacteria such as B. subtilis, Salmonella typhi, Bacillus cereus, P. aeruginosa,

S. aureus, and E. coli were inhibited [284]. The antimicrobial activities of the different fractions of Q. robur were evaluated against the bacterial species of S. aureus, E. coli, K. pneumoniae,

Staphylococcus epidermidis, and B. subtilis and all the fractions were found to show moderate to significant antibacterial activities [285].

Sakar et al. investigated the antimicrobial activities of Q. macranthera, Q. cerris, Q. pubescens, and Q. coccifera by determining the MIC values of the selected plant extracts.

Among the plants tested, the extracts of Q. coccifera displayed phenomenal bactericidal effects against S. aureus, K. pneumoniae, and B. subtilis. However, the extracts of Q. cerris and Q. pubescens were found to be inactive against these pathogenic species of bacteria [286].

Kumar et al. studied the bactericidal effects of the various extracts of Calotropis gigantea against a total of five species of bacteria— B. subtilis, Shigella sonnei, E. coli, Bacillus megaterium, and P. aeruginosa. The MeOH and CHCl3 extracts showed exemplary results at all the concentrations used [287]. Girish et al. reported the antibacterial potential of the MeOH and aqueous extracts of five plants against nine species of bacteria—E. coli, B. megaterium, K. pneumoniae, B. subtilis, P. aeruginosa, S. typhi, S. aureus, S. faecalis, and Yersinia enterocolitica. The results showed spectacular antibacterial effects, observed with the plant extracts. The MeOH extract showed better antibacterial activities, compared to the aqueous extract, indicating that the MeOH Ext may contain some active phytochemicals, which retard the growth of microorganisms [288 ].

Hassan et al. evaluated the antibacterial activities of the methanolic and aqueous extracts of a total of six species of plants against a total of seven species of bacteria. The patterns of

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inhibition showed variations with respect to the solvent, plant extracts, and test organisms used.

The methanolic extract of Sphaeranthus hirtus was found to be the most active among the plant extracts against the bacterial species of P. aeruginosa and E. coli [289].

Mohammad et al. in their study described the antibacterial activities of the plant-derived AgNPs, using the aqueous extracts of the plant, Origanum vulgare. The biogenic AgNPs showed remarkable activities against the various pathogenic species of bacteria [290] Ahmad et al. synthesized AgNPs from the leaf extracts of the plant, Skimmia laureola and checked their antimicrobial activities against the selected pathogenic species of bacteria. Maximum inhibition zone of 17 mm was shown by the bioinspired AgNPs against P. aeruginosa, followed by the inhibition zones of 15 mm against S. aureus and 13 mm against E. coli [291].

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Table 3.9: Tabular representation of antibacterial activity by Quercus semecarpifolia

AgNPs Crude n-hexane CHCl3 EtOAc Aqueous

Extract

Bacteria

10μg/dis of Zone (mm) Inhibition (%) Inhibition of Zone (mm) Inhibition (%) Inhibition of Zone (mm) Inhibition (%) Inhibition of Zone (mm) Inhibition (%) Inhibition Zone (mm) Inhibition (%) Inhibition of Zone (mm) Inhibition (%) Inhibition Zone of inhibition of of inhibition of Zone (amoxicillin) standard

E. coli 23 16 70 15 65 16 70 13 56 12 52 14 61

S. pneumoniae 29 21 72 00 00 15 52 12 41 13 45 17 57

S. aureus 26 14 54 21 81 13 50 14 54 17 65 12 46

P. aeruginosa 27 18 67 00 00 00 00 15 56 21 78 17 63

K. pneumoniae 21 19 90 13 62 00 00 19 90 18 86 15 71

B.subtilis 25 22 88 17 68 12 55 18 72 19 76 00 00

P.mirabulus 25 21 84 15 60 13 62 17 68 21 84 14 56

S.mercescens 27 20 74 12 44 10 37 17 63 12 44 13 48

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Table 3.10: Tabular representation of MIC50 assay by Quercus semecarpifolia

Bacteria Crude extract n- hexane CHCl3 EtOAc Aqueous AgNPs

E. coli 2.6 2.1 3.2 3.7 2.7 2.1

S. pneumoniae --- 3.0 4.1 4.3 3.5 1.9

S. aureus 1.2 3.2 3.8 2.2 4.8 2.9

P. aeruginosa ------3.1 1.1 2.6 2.3

K. pneumoniae 2.7 ---- 0.8 0.6 2.3 0.5

B.subtilis 2.1 2.9 1.9 1.3 --- 0.8

P.mirabulus 2.8 2.6 2.1 0.9 3.6 1.2

S.mercescens 3.9 5.1 2.6 4.4 4.4 1.7

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100

60 Control

50 AgNPs crude extract 40 n-hexane

PercentInhibition(%) 30 CHCl3 20 EtOAc 10 Aqueous 0

Figure 3.29: Graphical representation of antibacterial activity by Quercus semecarpifolia

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3.5.2 Antifungal Activity

The synthesized AgNPs, Cr. MeOH. Ext and fractions were screened for their antifungal activities against six species of fungi—A. flavus, P. notatum, C. albicans, A. niger, F. oxysporum, and T. harzianum. The results are depicted in Table 3.11and Figure 3.34. The evaluations for the antifungal activities, the green synthesized AgNPs, CHCl3 fractions, and aqueous extracts showed low inhibitory activities of 45%, 30%, and 25%, respectively, against

A. flavus. On the other hand, the Cr. MeOH extract, n-hexane, and EtOAc fractions were unable to show any antifungal effects against A. flavus. The AgNPs, derived from Q. semecarpifolia, n- hexane extracts, and EtOAc fractions showed low inhibitory activities of 20%, 25%, and 20%, respectively, against A. niger, while the other fractions did not exhibit inhibitory effects against the species of fungus tested. The test samples were also checked for any fungicidal effects against P. notatum. With the exception of the AgNPs, which showed a low activity of 25%, the remaining extracts were found to be inactive against the species of fungus tested. Similarly, the bioinspired AgNPs, EtOAc fractions, and Cr.MeOH.Ext demonstrated low fungicidal effects of

30%, 25%, and 15%, respectively, against T. harzianum. On the other hand, the remaining fractions did not possess any inhibitory effects against the species of fungus tested. The Cr.

MeOH.Ext and AgNPs of Q. semecarpifolia were found to be inactive against the fungal species of C. albicans and F. oxysporum.

Sakkar et al., studied the fungicidal effects of the methanolic extract of Q. aucheri against the two species of fungi—C. albicans and C. parapsilosis. Among the fractions, EtOAc was the most active with the MIC50 values of 0.9 and 0.6 µg/mL against C. albicans and C. parapsilosis, respectively [286]. Uddin et al., also reported the antifungal effects of the MeOH and aqueous extracts of the galls of Q. infectoria against Candida sp. Both the extracts had inhibitory effects

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against Candida sp. with some strains showing greater inhibition zones, compared to the positive controls [292].

Gulluce et al., in their investigation determined the possible antifungal effects of the

MeOH Ext of the leaves of Q. ilex against a total of five species of fungi and yeast, using the disk diffusion assay [293]. The results indicated that the extracts exhibited noteworthy antifungal effects against C. albicans. Andrensek et al., revealed that the MeOH. Ext of the bark of Q. robur possessed fungicidal effects against A. niger and A. flavus [294].

Bobbarala et al., investigated the methanolic extracts of various plants against the fungal species of A. niger and A. flavus. Among the plants, the extracts of Grewia arborea showed the maximum antifungal activities, while the extracts of Hildegardia populifoli, Emblica officinalis,

Strychnos nuxvomica, Vitex negundo, Hyptis suaveolens, and Moringa heterophylla did not exhibit antifungal effects [295]. In their study, Bashir et al., used the different fractions of the plant, Ferula narthex against a total of five species of fungi—Fusarium solani, Trichophyton longifusus, C. albicans, A. flavus, and Candida glabrata for determining the antifungal activities.

Among the test samples, significant antifungal activities of 40%, 35%, and 30% were shown against Microsporum canis by the methanolic extract, n-hexane, and chloroform fractions, respectively. The remaining fractions were ineffective toward the species of fungus tested [296].

Hussein et al., screened a total of six different plants—Ocimum basilicum, Acacia nilotica, Azadirachta indica, Prosopis juliflora, Crotalaria juncea, and Eucalyptus camaldulensis against the different pathogenic species of fungi. All these plants showed good to moderate antifungal activities against Rhizoctonia solani, F. solani, A. flavus, C. albicans, and A. niger because their mycelial growth was suppressed by the crude extracts of the plants. Among these plants, A. indica and C. juncea showed the most effective results against the fungal species

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of A. flavus, F. solani, and A. niger. The maximum inhibitory effects of 95%, 90%, and 87% against A. flavus were shown by the plants, C. juncea, O. basilicum, and A. indica, respectively

[297].

Kim et al., investigated the fungicidal properties of AgNPs against the various plant pathogens.

The results indicated that AgNPs possessed peculiar antifungal effects at various concentrations and the maximum inhibitions were obtained at the highest concentration [298]. Nasrollahi et al., reported the antifungal activities of AgNPs against the two species of fungi—Saccharomyces cerevisiae and C. albicans. Compared to amphotericin B and fluconazole, which are used as the standard antifungal drugs, the synthesized AgNPs displayed remarkable fungicidal effects against these species of fungi [299].

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Table 3.11: Tabular representation of antifungal activity by Quercus semecarpifolia

Percent growth inhibition (%)

Fungal Standard

species (mg/ml) AgNPs Crude n-hexane CHCl3 EtOAc Aqueous extract

A.flavus 100 45 0 0 30 0 25

A.niger 100 20 0 25 0 20 0

P. notatum 100 25 0 0 0 0 0

F. oxysporum 100 0 0 0 0 0 0

T. harzianum 100 30 15 0 0 25 0

C-albicans 100 0 0 0 0 0 0

A.flavus A.niger P. notatum F. oxysporum T. harzianum C-albicans 100 100

90 80 70 60 50 45 40 30 30 30 25 25 25 25 20 20

20 15 Percent inhibition(%) Percent 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Control AgNPs Crude n-hexane CHCl3 EtOAc Aqueous extract

Figure 3.30: Graphical representation of antifungal activity by Quercus semecarpifolia

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3.5.3 Antioxidant Activity

The antioxidant activities are due to the redox potential of phytochemicals present in the plant extracts, which play a key role in quenching reactive oxygen species (ROS) and free radicals

[300]. Free radicals and ROS, accountable for the progession of diseases, such as neurodegenerative disorders, cardiovascular diseases, cancer, and diabetes, are increasingly becoming important [301, 302].Current research has shown that antioxidants play important part in the prevention and therapy of infections and, therefore, the search for potential antioxidants from natural sources is essential and very important for the discovery of new drugs.

In the present study, the AgNPs derived from Q. semecarpifolia showed an excellent antioxidant activity of 82% at higher concentrations. Similarly, all the extracts showed highest antioxidant activities at 300 μg/mL. The aqueous extracts of Q. semecarpifolia showed a significant antioxidant potential of 71% at the highest concentration of 300 μg/mL. Moreover, at 300

µg/mL, the EtOAc and CHCl3 fractions showed good antioxidant activities of 62% and 61%, respectively. The Cr. MeOH. Ext and n-hexane extracts possessed moderate free radical scavenging activities of 57% and 51%, respectively. Therefore, at the highest concentration of

300 µg/mL, the antioxidant potential of the test samples increased considerably. The results are depicted in Figure 3.31 and summarized in Table 3.12.

The antioxidant effects of the plant extracts of Q. semecarpifolia were similar to the results of previous investigations, indicating the occurrence of polyphenols and tannins as the primary antioxidants in the species, Quercus. Moreover, gallic acid, gallotannins, digallic acid present in the fruits of Q. acutissima, impart high antioxidant activities [303]. Similar results on the degree of antioxidant activities were reported with the bark of Q. robur L., which contained high

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quantities of phenols and flavonoids that conferred high antioxidant activities because of the presence of phenolic groups and hydroxyl groups [304].

Neibgel et al. evaluated the antioxidant activities of the aqueous extracts of Q. brantii, using the

DPPH assay. The upshots displayed that the EC50 value of the aqueous extracts was 31.1 ±0.19

µg/mL [305]. Rakic et al. evaluated a study on antioxidant potential of the MeOH extract of Q. robur and Q. cerris at different test concentrations. The EC50 values for Q. robur and Q. cerris were 8.04 and 8.88 µg/mL, respectively [306]. In their study, Kim et al. isolated a total of five compounds from the EtOAc fractions of the stems of Q. salicina. The antioxidant activities of these compounds were determined and the results proved them to be excellent antioxidants

[307]. Karimi et al. determined the antioxidant activities of Q. persica and the results revealed that the plant had a high amount of phenolic compounds, which were responsible for the high antioxidant potential [308]. In another study, the galls of Q. infectoria were investigated for possible free radical scavenging activities and the results showed that the galls were strong scavengers in eliminating free radicals [309,310].

Kharat et al. reported the antioxidant activities of the plant-derived AgNPs, using the aqueous extracts of the plant, Elephantopus scaber. Using the DPPH assay, the antioxidant activities of the synthesized AgNPs were evaluated and the results indicated that the bioinspired AgNPs possessed strong antioxidant activities and can be used as potential free radical scavengers [311].

In another study, the MeOH Ext and synthesized AgNPs of the plant, Litsea reticulata were investigated for the possible antioxidant activities, using the DPPH assay. Both the extracts and synthesized AgNPs showed remarkable antioxidant potential compared to the standard ascorbic acid. At the highest test concentration of 400 µg/mL, the AgNPs showed stupendous antioxidant

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activity of 88.7%. In fact, the scavenging activities of the plant extracts and AgNPs increased gradually in a dose-dependent manner [312].

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Table 3.12: Graphical representation of antioxidant activity by Quercus semecarpifolia

Percenct absorbance (%)

No of Control Dilutions (µg/ml) (µg/ml) AgNPs Crude n-hexane CHCl3 EtOAc aqueous extract

300 100 82 57 51 61 62 71

200 100 68 49 48 59 54 58

100 100 53 35 23 34 41 43

300 µg/ml 200 µg/ml 100 µg/ml 100

100 90 82 80 68 71 70 61 62 57 59 58 60 53 54 49 51 48 50 41 43 40 35 34 30 23 20 10 0 Control AgNPs crude n-hexane CHCl3 EtOAc Aqueous

extract Percent Antioxidant Activity ) Activity % ( Antioxidant Percent Concentration (µg/ml)

Figure 3.31: Graphical representation of antioxidant activity by Quercus semecarpifolia

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3.5.4 Phytotoxic Activity

In developing countries, weeds are considered an important element for the protection of the environment. The interference of weeds with agricultural crops reduces the agricultural yield, thereby leading to huge economic losses all over the world [313]. However, these chemically synthesized herbicides cause harmful adverse effects, primarily leading to the production of herbicide residues, soil and water pollution, and herbicide-resistant populations of weed.

Currently, more emphasis is laid on the isolation of natural biomolecules from plants, in order to control weeds for crop protection [314].

In our study, the phytotoxic activities of the test samples were checked at the different concentrations of 1000, 100, and 10 µg/mL, in order to observe any damage to the fronds. At the sample concentration of 10 µg/mL, the plant-derived AgNPs displayed minimum inhibitory effects, which amplified when the concentration of the test samples increased by three times to

1000 µg/mL. The results revealed that the AgNPs, derived from Q. semecarpifolia showed a good phytotoxic potential 75% at the concentration of 1000 μg/mL, a moderate phytotoxic activity of 51% at 100 μg/mL, and a low phytotoxic activity of 36% was observed at concentration of 10 μg/mL. In contrast to the AgNPs, the crude extract, chloroform fractions and aqueous extracts showed moderate phytotoxic activities of 61%, 54%, and 54%, respectively, at the highest test sample concentration of 1000 µg/mL. Finally, at all the concentrations of the test samples, the n-hexane and EtOAc fractions were less active. The results are presented in Figure

3.32 and summarized in Table (3.13-3.14).

Previously, the different crude extracts of plants were studied for their inhibitory activities against the other species of plants and the results revealed that all plants have a variable number of phytoconstituents, which can act as a natural defense mechanism [315-317]. In a

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study carried out by Saima et al., the phytotoxic potential of the different extracts of the plant,

Reinwardtia trigyna was investigated and the results revealed that all the extracts exhibited high phytotoxic activities of 60–100% at concentration of 1000 μg/mL, while low phytotoxic effect

30–50% were displayed by the extracts at 10 μg/mL concentration. All these findings are in accordance with the results of our study [318].

In another study, the Cr. MeOH. Ext. of the plant, Heliotropium strigosum demonstrated moderate phytotoxic effects at 1000 µg/mL concentration, but exhibited low phytotoxic activities of 35% and 40% at the test sample concentrations of 100 µg/mL and 10 µg/mL, respectively

[319]. Saeed et al. explored the crude extracts of the plant, Polygonatum verticillatum for the assessment of phytotoxic activities and the results revealed that at 5 µg/mL, 50 µg/mL, and 500

µg/mL test sample concentrations, the crude extracts showed phytotoxic activities of 7.98%,

23.09%, and 48.67%, respectively. The CHCl3 and BuOH fractions showed exemplary results at concentration of 500 µg/mL. The EtOAc fractions and n-hexane extracts also displayed significant inhibitory results at the highest concentration of the test samples [320].

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No. of fronds Plant Concentratio Standard drug * specie n (μg/ml) (μg/ml) AgNPs Crude n-hexane CHCl3 EtOAc Aqueou extract s

1000 4 6 8 6 9 6 0.015 Lemna minor 100 8 10 14 10 10 12

10 10 15 13 13 15 14 Table 3.13: Tabular representation of phytotoxic activity of Quercus semecarpifolia

Percent growth (%) Concentration (μg/ml) AgNPs Crude n-hexane CHCl3 EtOAc Aqueous Standard extract

1000 75 61 46 54 39 54 100

100 51 36 12 36 37 25 100

10 36 05 15 18 05 07 100

Table 3.14: Percent growth regulation of Lemna minor

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1000 µg/ml 100 µg/ml 10 µg/ml 100 100

90 80 75

70 61 60 51 54 54 50 46 3937 40 36 36 36 30 25 18 20 1215 7 Percent Mortality (%) Mortality Percent 10 5 5 0 Control AgNPs Crude n-hexane CHCl3 EtOAc Aqueous extract Concentration( µg/ml )

Figure 3.32: Graphical representation of phytotoxic activity by Quercus semicarpifolia

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3.5.5 Cytotoxic Activity

In the current study, the cytotoxic activities of the test samples were investigated against the brine shrimp, A. salina. The different concentrations of the test samples were used and the variations in lethality were observed. The results revealed that the Cr.MeOH.Ext, n-hexane, and aqueous extracts of Q. semecarpifolia had stupendous cytotoxic effects at the highest concentration of 1000 µg/mL. The LD50 values noted for the Cr. MeOH. Ext was 16.63 µg/mL and that for n-hexane, and aqueous extracts were found to be, 4.56, and 11.53 µg/mL, respectively. At the concentration of 1000 µg/mL, the plant-derived AgNPs, CHCl3, and EtOAc fractions showed significant cytotoxic activities of 77%, 73%, and 67%, respectively. The LD50 values, reported for the AgNPs, CHCl3, and EtOAc fractions, were found to be 0.413 µg/mL,

34.73 µg/mL, and 42.56 µg/mL, respectively. The aqueous and Cr.MeOH.Ext showed remarkable cytotoxic activities of 97% and 83%, respectively at concentration of 100 µg/mL while the n-hexane extracts and green AgNPs revealed significant lethal effects of 77% and 71%, respectively. The CHCl3 and EtOAc fractions exhibited moderate cytotoxic activities of 57% and

53%, respectively. Moderate cytotoxic activities were demonstrated by all the extracts with the exception of the AgNPs which showed a good lethal effect of 60% at concentration of 10 µg/mL.

The results are presented in Figure 3.33 and summarized in Table 3.15.

In their study, Mayilsamy et al. tested the cytotoxic activities of the crude extracts and different fractions such as n-hexane, EtOAc, CHCl3, and MeOH extract of the plant, Parmelia perlata against the nauplii of A. salina. The highest mortality rates of nauplii were recorded with the n- hexane and MeOH extracts at the concentrations of 100 ppm and 200 ppm, respectively. The

EtOAc fractions showed a mortality rate of 92.6% at the higher concentration of 200 ppm. All

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these results are in accordance with the findings of our study because the maximum lethal effects were observed when the concentration of the test samples increased [321].

Ahmad et al. documented the cytotoxic activities of the different extracts of Q. dilata against A. salina, using a lethality assay in which, the maximum cytotoxic effects were observed at the high concentrations of the extracts. The results manifested that 21.42% of the total extracts were found to be highly cytotoxic with a value of LC50≤50 μg/mL, while 57.14% of the total extracts were found to be moderately cytotoxic with a value of LC50≥50 μg/mL. The other extracts were found to be weakly cytotoxic with a value of LC50>200 μg/mL [322]. In another study, Nenad et al. evaluated the cytotoxic effects against the larvae of A. salina, using the different concentrations of Q. robur L., ranging from 10–1000 µg/mL. The results revealed that the increased concentrations of the extracts of Q. robur L. showed significantly higher cytotoxic effects (p<0.001) against the larvae of A. salina. The aqueous extracts showed a significantly lower cytotoxic activity (p<0.05) at 10 µg/mL concentration, compared to that shown at 1000 µg/mL. The cytotoxicity assay revealed moderate toxic effects with the LC50 values of the aqueous extracts of Q. robur L., ranging between 80 and 250 µg/mL [323]. The Cr.MeOH.Ext. and green AgNPs, synthesized from the plant, Bergenia ciliata, were also screened for their cytotoxic activities at the four different test concentrations of 10, 50, 100, and

500 µg/mL. The LD50 value recorded for the Cr.MeOH.Ext was 83.57 µg/mL. The LD50 value of the synthesized AgNPs was found to be lower than the Cr. MeOH.Ext. of B. ciliata, suggesting a higher cytotoxic potential of the AgNPs. Such an enhanced cytotoxicity of the AgNPs revealed the presence of phytoconstituents, which could be the alternative sources of anticancer drugs [324].

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Table 3.15: Tabular representation of cytotoxic activity by Quercus semecarpifolia

Percent inhibition (%)

No of *Control AgNPs Crude n-hexane CHCl3 EtOAc Aqueous

Dilutions (µg/ml) extract extract

(µg/ml)

inhibition

No 0f survivors 0f No % inhibition % survivors of No inhibition % survivors of No inhibition % survivors of No inhibition % survivors of No inhibition % survivors of No %

10 100 12 60 13 57 17 43 16 47 15 50 17 43

100 100 9 71 5 83 7 77 11 57 14 53 1 97

1000 100 7 77 0 100 0 100 8 73 10 67 0 100

Etoposide at concentration of 7.4625 µg/ml was used as standard drug

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1000 μg/ml 100 μg/ml 10 μg/ml

100 100 100 100 97

100 90 83 77 77 80 71 73 67 70 60 57 57 60 53 47 50 50 43 43 40 30 20 10 0 Control AgNPs Crude n-hexane CHCl3 EtOAc Aqueous Percent Shrimps Killed ( % ) % ( Killed Shrimps Percent extract Concentration ( µg/ml )

Figure 3.33: Graphical representation of cytotoxic activity by Quercus semecarpifolia

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3.5.6 Insecticidal Activity

The plant-derived AgNPs, Cr.MeOH.Ext and different fractions of Q. semecarpifolia were studied for their insecticidal activities and the results are presented in Figure (3.34-3.36) and summarized in Table 3.16. Both the green synthesized AgNPs and Cr. MeOH.Ext of Q. semecarpifolia showed mortality rates of 100% against T. castaneum with the exception of the n- hexane extract, which showed a mortality rate of 60%.

Among the test samples, both the CHCl3 and EtOAc fractions showed mortality rates of

100% against C. maculatus, whereas the aqueous extracts exerted a low insecticidal activity of

40%. The AgNPs derived from Q. semecarpifolia showed a significant insecticidal activity of

80%. However, the Cr. MeOH and n-hexane extracts were found to be inactive against C. maculatus. Both the CHCl3 fractions and aqueous extracts showed remarkable results with mortality rates of 100% against R. dominica. Moreover, the Cr. MeOH.Ext exhibited a decreased mortality rate of 80%, while the EtOAc fractions and n-hexane extracts showed lower mortality rates of 40% and 30% respectively against the species of insects tested. However, the AgNPs, derived from the aqueous extracts did not show any mortality effects against R. dominica.

Akhter et al. studied a total of 30 methanolic extracts of the different species of plants against the two species of insects, Sitophilus oryzae and Callosobruchus chinensis, and strong insect repellent activities were shown by the extracts of the four plant species,

Cinnamomum cassia, Illicium verum, Acorus gramineus, and Eugenia caryophyllata. Among the plant species tested, C. cassia and Acorus calamus showed potential insecticidal activities against S. oryzae after an exposure of 24 h, whereas a low inhibitory effect of 80% was shown by the extracts A. gramineus, I. verum, and E. caryophyllata [325]. Ahmad et al., further described

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the efficacy of the extracts of the different species of plants against R. dominica, in order to check their insect repellent activities. Among all the species of plants studied, the extracts of

Piper nigrum showed the maximum mortality rate of 80% against the species of insects tested

[326].

In another study, Rachid et al., examined the insect repellent effects of the methanolic extracts of a total of four plants against the larvae and adults of T. castaneum. The methanolic extract of the plant, Peganum harmala showed significant insect repellent activities against the species of insect tested. However, the extracts of the plants, Aristolochia baetica, A. iva, and

Raphanus raphanistrum showed moderate insecticidal effects [327]. In a previous study, the Cr.

MeOH.Ext of a total of three plants were investigated for the insect repellent activities against T. castaneum and the results revealed that among the three plants, Cinnamomum tamala showed the highest insect repellent activity. Further, the insect repellent activities were increased when the concentrations of the test samples were increased [328].

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Table 3.16: Tabular representation of insecticidal activity of Quercus semecarpifolia

Percent mortality (%)

Insects Test samples introduced Tribolium castaneum Callosobruchus maculates Rhyzopertha dominica

Standard 24h 48h 72h 24h 48h 72h 24h 48h 72h

AgNPs 100 30 80 100 0 40 80 0 0 0

Crude extract 100 25 100 ---- 0 0 0 0 60 80 n-hexane 25 100 0 40 60 0 0 0 0 40 60

CHCl3 100 100 ------85 100 ----- 80 100 ----

EtOAc 100 25 65 100 85 100 ----- 0 0 30

Aqueous 100 55 70 100 0 20 40 60 100 ----

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24 48 72 100 100 100 100 100 100 100 90 80 80

70 70 65 60 60 55 50 40 40 30

30 25 25 Percentmortality(%) 20 10 0 0 Control AgNPs Crude n-hexane CHCl3 EtOAc Aqueous extract

Figure 3.34: Graphical representation of insecticidal assay by Quercus semecarpifolia against Tribolium castaneum

100 100 100 100 90 85 85 80 80 70 24 h 60 48 h 50 40 40 72 h 40 30 20 20 10 Percent mortality (%) mortality Percent 0 0 0 0 0 0 0 0 0 Positive controlAgNPsCrude extractn-hexane CHCl3 EtOAc Aqueous

Figure 3.35: Graphical representation of insecticidal activity by Quercus semicarpifolia against

Callosobruchus maculates

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24 h 48 h 72 h 100 100 100 100

90 80 80 80 70 60 60 60 60 50 40 40 30 30

20 Percent mortality (%) mortality Percent 10 0 0 0 0 0 0 0 0 Positive AgNPs Crude n-hexane CHCl3 EtOAc Aqueous control extract

Figure 3.36: Graphical representation of insecticidal assay by Quercus semecarpifolia against

Rhyzopertha dominica

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3.5.7 Antitermite Activity

Currently, termite menace is a severe issue throughout the world. Normally, these pests cause economic damage to fibers, clothes, wood, paper, mats, and also damage agricultural products.

However, synthetic pesticides cause hazardous effects and therefore, the development of plant- derived pesticides is very important [329]. Thus, in the present investigation, the plant extracts and green AgNPs of Q. semecarpifolia were screened for their antitermite activities.

On the first day, the methanolic extract, n-hexane, and chloroform fractions showed an exemplary antitermite activity of 100%. Furthermore, the EtOAc fractions and green AgNPs exerted significant antitermite activities of 80% and 70%, respectively. However, the aqueous extracts showed a moderate antitermite activity of 60%. On the second day, the EtOAc fractions and derived AgNPs showed significant antitermite activities of 100% and 90%, respectively, while a good antitermite activity of 70% was also observed, following treatment with the aqueous extracts. On the third day, treatments with all the test samples showed ample lethal effects of 100%. The results are depicted in Table 3.17.

Ravi et al., carried out a study, describing the antitermite activities of the various extracts and pure compounds isolated from the plant, Capparis decidua against the termite species,

Odontotermes obesus. Potential antitermite activities were shown by these extracts and compounds and the mortality rates increased with an increase in the dose. Higher mortality rates were observed with treatments by the isolated compounds, followed by the crude extracts [330].

Similarly, in another finding, the aqueous leaf extracts of the plants, Pogostemon parviflorus and

Polygonum hydropiper, were investigated for the antitermite activities against Odontotermes assamensis and the results revealed high mortality rates against the test organism [331-333].

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In another study, the antitermite activities of the four species of plants, Syzygium cumini, Sida acuta, Achyranthes aspera, and Terminalia arjuna were reported against O. obesus. The different extracts of these plants were utilized for the examination of the antitermite effects and the results revealed that the Cr. MeOH.Ext. of the plants exhibited the highest range of phytoconstituents and hence, they were the most effective against termites, compared to the other extracts. Among the plants, the Cr. Mefn. Ext. of T. arjuna showed the highest termiticidal effect of 90%, followed by the Cr.Mefn. Ext. of S. cumini, which showed a termiticidal effect of 70% over a period of 72 h [334].

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Table 3.17: Graphical representation of antitermite activity by Quercus semecarpifolia

Average Average termites termites Test No of No of termites killed by killed by killed *Positive by sample sample Termit Day Negative control Control es 1 0 8 7

2 0 AgNPs 9 9 3 0 10 10 1 0 7 10 Crude 2 0 Extract 9 ------

3 0 10

------

10 1 0 8 10

n-hexane 2 0 9 ----

3 0 10 -----

1 0 8 10 CHCl3 2 0 9 ----

3 0 10 -----

EtOAc 1 0 8 8

2 0 9 10

3 0 10 -----

1 0 8 6 Aqueous 2 0 9 7

3 0 10 10

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3.5.8 Allelopathic Activities

Allelopathy is the capability of a plant to stop the germination of other plants through the production of secondary metabolites also known as allelochemicals that are either present in the flowers, leaves, stems, seeds, and roots of plants as end products or are produced as by-products, which can act as natural herbicides and pesticides and can also have stimulatory effects on the development of seeds and the germination of plants [335].

In the current investigation, the aqueous extracts of Q. semecarpifolia were checked for the presence of allelochemicals. The aqueous extracts of the plant were applied on the seeds of wheat at the different sample concentrations (25, 50, and 100 μg/mL), in order to check their effects on germination, fresh weight, dry weight, radicle length, moisture content, and shoot length. The upshots are presented in Figure (3.37-3.38), and summarized in Table 3.18. At 100

μg/mL sample concentration, compared to the negative control (water), where the rate of seed germination was found to be 100%, the rate of germination of the seeds of wheat was found to be only 35% but it increased to 45% and 70% when the concentrations of the test samples were decreased to 50 and 25 μg/mL, respectively.

In water, the length of the shoot was found to be 120 mm. However, the lengths of the shoot reduced to 90 mm and 70 mm when the concentrations of the test samples were increased to 25 and 50 μg/mL, respectively. The length further reduced to 45 mm when the concentration of the test samples was amplified to 100 μg/mL. The test samples also showed inhibitory effects on the length of the radicle. When the concentration of the test samples was increased from 0 to

100 μg/mL, the length of the radicle was reduced by half, thereby indicating that at 0 μg/mL sample concentration, the length of the radicle was 65.5 mm, while at 100 μg/mL, its length reduced to 33.5 mm. At the test sample concentrations of 25 and 50 μg/mL, the lengths of the

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root were 65 mm and 60 mm, respectively. Thus, the results showed that the lengths of the shoot and root of the seeds of wheat were inhibited considerably by the test samples.

In the experiments, the moisture content, the difference between the fresh weight and dry weight of the seedlings was also calculated at the various concentrations of the test samples. The moisture content of the seedlings was found to be 305 mg when the test samples were not applied, while it reduced to 278 mg and 170 mg when the concentrations of the test samples were increased to 25 and 50 μg/mL, respectively. At 100 μg/mL test sample concentration, the moisture content reduced further to 100 mg.

In their study, Mahmoud et al. demonstrated that the aqueous extracts of the plant,

Cannabis sativa had inhibitory effects on the lengths of the shoot and root of Lactuca sativa at the test sample concentrations of 50, 75, and 100 μg/mL [336]. Anjum et al. executed experiments, in order to check the allelopathic activities of some medicinal plants on the germination and growth of L. sativa. Among the plants, Albizia lebbeck and Broussonetia papyrifera were found to have potential inhibitory effects on the growth of the shoots and hypocotyls of L. sativa [337]. Hussain et al. evaluated the allelopathic effects of the shoots, leaves, and bark of the plant, Q.baloot on Pennisetum americanum, Setaria italica, and L. sativa.

The results indicated that the aqueous extracts reduced the germination as well as the growth of the plumules and radicles of the selected species of plant significantly [338].

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Table 3.18: Graphical representation of allelopathic activity by Quercus semecarpifolia

Extract Percent Shoot Radical Fresh Dry weight Moisture Conc Germination Length length weight (mg) (mg) Content (mm) (mm)

C1 (0%) 100 120 65.5 365 60 305

C2 (25%) 70 90 65 325 47 278

C3 (50 %) 45 70 60 210 40 170

C4 (100%) 35 45 33.5 115 15 100

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Shoot length (mm) Radical length (mm) 120

100 90

80 70 65 65 60 60 45 40 33.5

Percent inhibition(%) Percent 20

0 C1 (0%) control C2 (25%) C3 (50%) C4 (100 %)

Figure 3.37: Growth inhibitions of shoot and radical of Quercus semecarpifolia

100 100

60 45

40 35 %Germination

0 C1 (0%) control C2 (25%) C3 (50%) C4 (100 %)

Figure 3.38: Percent germination of seeds by Quercus semicarpifolia

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3.5.9 Hemagglutination Activity

Plants and other living organisms contain carbohydrate-binding proteins called lectins, which have the unique property of distinguishing between the different carbohydrate moieties.

Numerous studies have been directed on the structural and functional roles of carbohydrates on the basis of the specificity of lectins [339]. Lectins can be used as blood typing agents for the identification of sugar components in normal and cancerous cells and also for the estimation of virus particles [340]. Lectins can be used for recognizing tumour cells, degree of host immune defense, cell localization and adhesion, transduction of signals across membranes, mitogenic stimulation, cytotoxicity and apoptosis [341]. Based on these properties of lectins, an assay for assessing the hemagglutination activities of Q. semecarpifolia was performed against the erythrocytes of human blood. The test samples showed no agglutination of the human erythrocytes, specifying that the plant species lacks phytolectins. The results are presented in

Table 3.19.

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Table 3.19: Tabular representation of hemagglutination activity by Quercus semecarpifolia

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3.6 Assessment of Pharmacological/Biological Activities in vivo

3.6.1 Acute Toxicity Assay

Plants have been used for medicinal purposes for thousand of years and offering unlimited opportunities for the discovery of new drugs. However, these plants were also used by people in the past either as formulations or in the crude form and usually, contradictory results appeared

[342]. Therefore, it must be ensured that plants are safe for human use before using them as medicines. For this purpose, the toxicological investigations are generally performed for a new drug, in order to check its safety and proficiency, thereby shedding light on whether the drug should be approved for human use or not. Generally, animals like guinea pigs, rats, rabbits, and pigs are used under various conditions for the toxicological studies,in accordance with the guidelines formulated by the Organization for Economic Cooperation and Development(OECD)

[343].

In the present investigation, the plant-derived AgNPs and Cr. MeOH. Ext were screened for possible biological activities in vivo. Initially, an acute toxicity assay was performed as it is the first step toward this approach. The Cr. MeOH. Ext of Q. semecarpifolia were orally administered to the four groups of six mice at the different doses of 200, 500, 1000, and 1500 mg/kg and the results showed that the test samples did not show any toxic or lethal effects on the animals. Similarly, the synthesized AgNPs were screened at the doses of 10, 40, 50 and 100 mg/kg. Neither any toxic nor any lethal effects were observed in the four groups of animals throughout the observation period. The results, demonstrating the acute toxicity assay are depicted in Table 3.20.

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Following the OECD Test Guideline 423, Lalitha et al. checked the acute toxic effects of the

EtOAc fractions, aqueous extracts, and Cr. Mefn. Ext. of Eichhornia crassipes in the Swiss albino mice. The test samples were administered orally to the mice at the different doses. All the treated animals were examined for any sighns of toxicity or lethality, initially for a period of 30 min and then for a period of 24h and 48 h. At the highest test sample dose of 2000 mg/Kg, the animals were unable to produce any toxic effects and these findings clearly demonstrated that the highest dose of the test samples was safe for oral administration [344].

In another study, the adverse effects of the methanolic extract of the plant, Alstonia scholaris were determined in the experimental mice, in order to check their levels of safety. The test samples were given to the mice at the different doses. All the treated mice were examined for any changes in behavior, initially for the first few hours and then for a period of 14 days. The mice were also observed for any toxic or lethal effects and the results revealed that the highest test sample dose of 2000 mg/kg was nontoxic to animals [345]. Jhoti et al., investigated the toxic effects of the MeOH extract of Cassia fistula in the mice. In that study, the mice were orally administered with the different doses of the test samples and the results revealed that the highest test sample dose of 5000 mg/kg resulted in neither mortality nor any adverse effects. The control and test groups of mice were observed for any changes in behavior throughout the observation period of 14 days. The results indicated that the Mefn. Ext. of C. fistula showed neither any lethal nor any toxic effects in the mice [346].

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Table 3.20: Tabular representation of acute toxicity assay of Quercus semecarpifolia

Test Samples Extract Total No of No. of animals (%) Mortality Concentration animals Alive After 24 h (ml or mg /kg)

Normal Saline 10 ml 6 6 -

Crude 200 6 6 - methanolic extract 500 6 6 -

1000 6 6 -

1500 6 6 -

10 6 6 -

40 6 6 -

AgNPs 50 6 6 -

100 6 6 -

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3.6.2 Antinoceceptive Effects

3.6.2.1 Acetic Acid Writhing Test

In the current investigation, the test samples (Cr. MeOH. Ext) and AgNPs were screened for any possible analgesic effects and the results revealed that the Cr. MeOH. Ext. and plant-derived

AgNPs of Q. semecarpifolia exhibited a momentous decrease in the mean number of writhes as the concentrations of the test samples were increased. The upshots are presented in Figure (3.43-

3.44) and outlined in Table 3.21.

Compared to the normal saline (control) group, the Cr. MeOH. Ext. and plant-derived

AgNPs of Q. semecarpifolia showed significant analgesic effects in the test group. The Cr.

MeOH. Ext. of Q. semecarpifolia exhibited moderate analgesic effects, while the plant-derived

AgNPs displayed exceedingly significant antinociceptive effects at the test doses. The oral administration of MeOH extract at the doses of 50, 100, and 200 mg/kg of body weight of the animals showed momentous inhibition in the mean number of writhes and the analgesic effects increases three folds at the highest dose of the test samples and maximum inhibition in the number of writhes was achieved. The test sample at doses of 50 and 100 mg/kg displayed inhibitions of 49.83% and 63.67%, respectively in the number of writhes and as the test sample dose was increased to 200 mg/kg, a significant inhibition in the number of writhes of 79.05% in the number of writhes was observed. Aspirin, used as the standard, also showed an inhibition of

85.91%. The plant-derived AgNPs when compared to the MeOH extract, exhibited better efficiency in suppressing the number of writhes. The plant-derived AgNPs at a dose of 10mg/kg, displayed an inhibitory effect 71.58%. However, the inhibitory effects increased to 74.39% and

83.24% as the test doses were increased to 20 and 40 mg/kg, respectively. Results are depicted in

Table 3.21 and Figure (3.39-3.40).

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Banerjee et al., evaluated a study, which described the antinociceptive effects of the methanolic extracts of the plant, Juniperus communis in the animal model of Swiss albino mice. The oral administration of the test samples at the doses of 100 and 200 mg/kg of body weight of the mice displayed dose-dependent suppressions of writhes of 51.47% and 62.34%, respectively compared to the normal saline (control) group . Acetylsalicylic acid, (100mg/kg) used as the standard in the experiments with an inhibitory effect of 74.11% [347]. Mohammad et al. showed the inhibitory effect of the MeOH extract of the aerial parts of the plant, Viola betonicifolia and the results of their experiments displayed that at the highest sample concentration of 300 mg/kg, stupendous inhibition of 78.9% in the number of abdominal constrictions was achieved. Paracetamol, used as the standard drug in the experiments, showed a remarkable suppression of 96.23% in the number of writhes, which was higher than that displayed by the highest dose of the test samples [348].

Using the writhing test, Proma et al., investigated the analgesic effects of the MeOH extract of the plant, Lablab purpureus in the experimental mice. A significant reduction in the writhes was observed by the administration of the test samples at different doses and this reduction was also dose-dependant, indicating that at the highest dose of the test samples, the maximum inhibition was observed. At the doses of 50 and 100 mg/kg, the test samples displayed inhibitory effects of 23.1% and 34.6%, respectively. As the doses of the test samples were increased to 200 and 400 mg/kg, notable reductions in the number of writhes were observed. Aspirin, used as the standard analgesic drug in the experiments, was shown to reduce the number of writhes by 38.5% at the dose of 200 mg/kg [349]. In another study, suppressions in the number of writhes were determined in the experimental mice, using the metanolic extract of the roots of Crotalaria burhia. The results revealed that significant analgesic activities of 47.38%, 38.95%, and

25.5% were witnessed when the extract were orally administered at the doses of 300, 200, and 100 mg/kg, respectively. The extracts also showed a suppression of 56.54% in the number of writhes in a manner, similar to aspirin [350].

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Table 3.21: Tabular representation of analgesic assay by acetic acid induced writing test of Q.semecarpifolia

No. of writhings Percent inhibition Dose/kg Samples (Mean±SEM) (%)

Saline ( Control) 10 ml 76.83

Aspirin 100 mg 10.83±1.45 85.91

10 mg 21.83±1.78 71.58

AgNPs 20 mg 19.67± 1.52 74.39

40 mg 12.17 ±1.90 83.24

50 mg 38.54±1.98 49.83

Crude extract 100 mg 27.91±1.80 63.67

200 mg 16.09±1.65 79.05

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1 0 0 *** A s p irin

n *** o

i 8 0 E x tra c t t

i *** b i 6 0

h ***

t 4 0

e c

r 2 0

e P

g g g g m m m m 0 0 0 0 0 5 0 0 1 1 2

Q .s e m e c a rp ifo lia C r.M e O H E x t.

Figure 3.39: Percent analgesic activity of Quercus semicarpifolia Cr.MeOH.Ext in acetic acid induce pain model

174 Chapter 03 Results & Discussion

) 1 0 0 *** A s p irin

% *** ( 8 0 ***

n *** E x tra c t

i t

i 6 0

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t n

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c r e P 0

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Figure 3.40: Percent analgesic activity of Q. semecarpifolia derived AgNPs in acetic acid

induced pain model

175 Chapter 03 Results & Discussion

3.6.2.2 Hot Plate Assay

In the current investigation, the test samples (Cr. MeOH. Ext and AgNPs) were screened for any possible analgesic effects by the hot plate assay. The AgNPs, derived from Q. semecarpifolia, showed significant (p<0.05) analgesic activities at the test dose of 10 mg/kg but highly significant (p<0.001) results were obtained when the test dose increased to 20 mg/kg. A significant (p<0.05) analgesic effect was observed at the test dose of 40 mg/kg but was lower than that found at the test dose of 20 mg/kg. The test samples produced moderately significant and stable results at the doses of 50 mg/kg (p<0.01) and 100 mg/kg (p<0.01) but highly significant (p<0.001) results were seen at the test sample dose of 200 mg/kg. Results are summarized in Table 3.22 and Figure 3.41-3.42.

Using the hot plate assay, Sarwar et al conducted a study, in order to determine the analgesic property of the MeOH extract of Litsea glutinosa in the male experimental mice. The extract at the test doses of 250 and 500 mg/kg and the standard drug at the dose of 10 mg/kg were orally administered to the mice, 30 min prior to the experiments. The central analgesic effects were determined at different time intervals and the results revealed that at the dose of 500 mg/kg of the MeOH extract, the highest inhibition of pain due to a thermal stimulus (15.65 ±0.37 s) was achieved and that was comparable to the results obtained with the standard drug-treated mice (16.38 ±0.27 s). However, a highly significant (p<0.001) inhibition of pain was obtained when the data was compared with the (standard) and (control) groups of mice [351].

Zeghad et al., examined the antinoceceptive effcts of the crude methanolic extract of

Punica granatum and Vitis vinifera in vivo, using the hot plate assay. Normal saline and acetylsalicylic acid were used as the control and standard in these experiments. The test samples were administered orally at the doses of 1000, 2000, and 3000 mg/kg and compared to the

176 Chapter 03 Results & Discussion

standard drug and significant results were obtained at the test doses of 2000 and 3000 mg/kg

[352]. Malik et al., reported the analgesic effects of the MeOH, CHCl3 and aqueous extracts of

Cousinia stocksii in the Swiss albino mice, using the hot plate assay. The normal saline (control) and aspirin-treated (standard) groups of mice served as group 1 and group 2, respectively, whereas the test dose of 500 mg/kg was orally given to group 3, group 4, and group 5. The results revealed that compared to the standard drug, the MeOH extracts showed a highly analgesic effect of 71.34%, followed by the CHCl3 and aqueous extracts, which also showed analgesic effects of 35.7% and 33.55%, respectively [353].

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Table 3.22: Tabular representation of analgesic effect of Q.semecarpifolia by hot plate assay

Test Treatment Dose Provided Mean± SEM values

Normal 10 ml/kg 8.21±0.961

Standard 30mg 19.39±2.85

10 mg 16.87±0.97

AgNPs 20 mg 17.43 ± 2.32

40 mg 15.51 ± 1.37

100 mg 12.85 ± 2.17

Crude Methanolic extract 200 mg 17.32 ± 0.72

300 mg 14.36 ± 0.78

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** 2 5 S ta n d a rd

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a L

in in in m m m 0 0 0 3 6 9

Q .s e m e c a rp ifo lia C r.M e O H E x t.

Figure 3.41: Hot plate assay for Quercus semicarpifolia Cr.MeOH.Ext

179 Chapter 03 Results & Discussion

2 5 S ta n d a rd

) *** 1 0 m g /k g s *** *** 2 0 * * * n *** i * *** * 2 0 m g /k g

m *** ( 1 5 *

e 4 0 m g /k g

i T

1 0

n e

t 5

a L

in in in m m m 0 0 0 3 6 9

Q .s e m e c a rp ifo lia d e riv e d A g N P s .

Figure 3.42: Hot plate assay for Quercus semicarpifolia derived AgNPs

180 Chapter 03 Results & Discussion

3.6.3 Anti-inflammatory Assay

The carrageenan-induced paw model is a valuable model for acutely investigating the approximate anti-inflammatory potential. In the present study, following the injection of carrageenan, edema was developed in the paws of the animals due to the production of histamine, serotonin, and prostaglandins. Carrageenan is a phlogistic agent, which induces edema in two phases. In the first phase, mediators such as serotonin and histamine are released within 2 h and in the second phase, bradykinins and prostaglandins are responsible for the development of edema after a period of 4 h [354].

In the current study, anti-inflammatory activity of Cr.MeOH.Ext and plant derived AgNPs were determined by carrageenan induced paw edema model. Both the test samples were seen to possesss significant anti-inflammatory properties when compared to negative control group

(Normal saline) after 1, 2, 3 and 4h time intervals. Significant anti-inflammatory effect was observed when treated with 100 mg/kg and 200 mg/kg and this was noted by the reduction in edema as 0.2244±0.01473 and 0.2019±0.01017 respectively. Moreover, Q.semecarpifolia derived AgNPs also showed significant reduction in edema as noted 0.2673±0.01021 and

0.2502±0.02356 at 10mg/kg and at 20 mg/kg respectively. Results are depicted in Table 3.23 and

Figure (3.43-3.44)

Abhishek et al. evaluated the antiinflammatory effects of the MeOH. Ext. of the leaves of the plant, Aerva pseudotomentosa in the experimental mice. The mice were divided into four groups. The first group, receiving normal saline at the dose of 5 mL/kg served as the negative control group. The antiinflammatory drug, indomethacin, administered to the second group at the dose of (10 mL/kg) served as the standard. The test samples, orally administered at the doses of

200 and 400 mg/kg served as the third and fourth groups, respectively. The results showed that at

181 Chapter 03 Results & Discussion

the higher dose of 400 mg/kg, the Cr. MeOH. Ext. exhibited significant antiinflammatory effects on the paw model of edema, while the lower dose of 200 mg/kg was ineffective, following 3 h of the carrageenan administration. Moreover, following 5 h of the dispensation of carrageenan at the dose of 400 mg/kg, the MeOH extract displayed a momentous inhibition (p<0.001) of the mean increase in paw volume. The standard drug, indomethacin also showed noteworthy antiinflammatory effects (p<0.001) [355].

Paliwal et al carried out a study to evaluate the anti-inflammatory properties of the methanolic extracts of Inula cuspidate in mice models. The animals were grouped into four. One group received normal saline, second group received standard indomethacin at a dose of 10 mg/kg and the other two groups received test samples at a dose of 100 and 200 mg/kg respectively. The results indicated that the extracts significantly reduced the paw edema volume in dose-dependant manner. The reduction in the paw volume at a dose of 100 mg/kg and 200 mg/kg was 40.76% and 44.62% respectively at a time interval of 2 h and 3h when compared to normal saline. Significant inhibition of 64.18% in paw edema volume was shown by indomethacin at 10 mg/kg at time interval of 3h [356].

182 Chapter 03 Results & Discussion

Table 3.23: Tabular representation of anti-inflammatory assay of Quercus semecarpifilia

Treatment Dose/kg NPV 1h 2h 3h 4h

Saline (Control) 10ml 0.1308±0.031 0.4451±0.034 0.4462±0.031 0.4626±0.042 0.4676±0.033

Aspirin 10mg 0.1432±0.030 0.3364 ±0.033 0.3551 ±0.041 0.3736±0.046 0.3886 ±0.048

Crude 100mg 0.1409±0.033 0.2351±0.060 0.2405±0.043 0.2452±0.052 0.2590 ±0.039 Methanolic extract 200mg 0.1345±0.028 0.2144±0.042 0.2163 ±0.037 0.2165 ±0.047 0.2273 ±0.036

10mg 0.1709±0.031 0.2885±0.039 0.2891±0.041 0.2917±0.043 0.2963±0.045 AgNPs

20mg 0.1654±0.022 0.2653±0.025 0.2679±0.054 0.2745±0.056 0.2782±0.058

183 Chapter 03 Results & Discussion

)

A s p ir in 1 0 0 m g

(

2 0 0 m g

n 8 0 ** o **

i ** **

t i ** b 6 0

i h

** n

i 4 0 ** t * *

n ** * e 2 0

c *

r e

0 P h h h h 1 2 3 4

Q .s e m e c a rp ifo lia C r.M e O H E x t.

Figure 3.43: Graphical representation of anti-inflammatory activity of Cr.MeOH.Ext of Quercus semicarpifolia

184 Chapter 03 Results & Discussion

A s p ir in 1 0 m g /k g )

2 0 m g /k g

(

8 0 ** ** ** **

n o

i ** t

i 6 0 b

i **

** h

n 4 0 i

* t ** *

n *

e 2 0 *

c r

0 P h h h h 1 2 3 4

Q .s e m e c a rp ifo lia d e riv e d A g N P s

Figure 3.44: Graphical representation of anti-inflammatory activity of Q. semicarpifolia derived

AgNPs

185 Chapter 03 Results & Discussion

3.6.4 Antipyretic Assay

In the current study crude methanolic extract and plant derived AgNPs were checked for possible antipyretic effect in comparison to negative control group (normal saline) after time interval of 1 h, 2h, and 3h. Both the test samples showed significant anti-pyretic potential at all sample doses.The values are expressed as Mean ±SEM and analysed by one way ANNOVA. Pracetamol showed reduction in temperature with a mean value of 36.72± 0.233 as compared to the negative control. Cr.MeOH extract at the dose of 100 and 200 mg/kg showed reduction in temperature as

35.14±0.342 and 34.67± 0.352 respectively. In the same manner, plant derived AgNPs showed remarkable reduction in the rectal temperature at sample dose of 100 mg/kg and 200mg/kg as

35.74±0.089 and 36.47 ±0.192 respectively. Results are shown in Table 3.24 and Figure (3.45-

3.46).

In a study, Padhan et al., reported antipyretic activity of methanolic extract of Capparis zeylanica in experimental mice. Animal used in the study were adult albino rats of either sex weighing 20-25 g.Throughout the experiment, animals were properly fed. The animals were divided into four groups comprising six animals in each group. Normal saline (5mL/kg) was given to negative control group while Paracetamol (standard) was orally administered to second group.The third and fourth group were orally administed with crude methanolic extract at doses of 100mg/kg and 200mg/kg respectively. The results revealed that MeOH extract significantly reduces the temperature in the mice and also the activity was dose-dependent. Remarkable antipyretic activity was observed at a dose of 200mg/kg [357].

186 Chapter 03 Results & Discussion

Table 3.24: Tabular representation of Antipyretic assay of Q. semecarpifolia after 1, 2 and 3 h interval

Treatment Dose/kg Normal After24hr 1h 2h 3h (X) (Y) (T1) (T2) (T3) Saline (Control) 10ml 37.15±0.16 39.72±0.30 39.65± 0.25 39.68± 0.20 39.61± 0.24

Paracetamol 50mg 36.20±0.10 38.58±0.23 37.39±0.10 36.48±0.35 36.29±0.34

Crude 100mg 37.74±0.09 38.35±0.32 34.18±0.22 35.91±0.41 35.34±0.45 MeOH

Extract 200mg 37.51±0.14 37.97±0.40 34.72±0.39 34.51±0.38 34.80±0.43

AgNPs 10 mg 37.38±0.12 37.81±0.27 35.55±0.42 35.82±0.11 35.87±0.13

20 mg 38.52±0.21 38.64±0.42 36.23±0.34 35.11±0.38 36.34±0.02

187 Chapter 03 Results & Discussion

4 0 ** C o n tro l

* )

c P a ra c e ta m o l  ( ** *

e 3 9

r ** * 1 0 0 m g u

r **

a * 2 0 0 m g r **

p 3 8

m e

T **

l a

t 3 7 c

e **

R **

3 6

h h h h 0 1 2 3 Q .s e m e c a rp ifo lia C r.M e O H E x t.

Figure 3.45: Graphical representation of Anti-pyretic assay of Q. semecarpifolia MeOH extract

188 Chapter 03 Results & Discussion

* * 4 0 * * * * C o n tro l * * * * *

) * * * * *

c * * * * P a ra c e ta m o l  * ( * * * * * * * e 3 8

r *

* 1 0m g u

r *

a * 2 0m g r * p * 3 6 * m *

l a

3 4

c e

3 2

h h h h 0 1 2 3

Q .s e m e c a rp ifo lia d e riv e d A g N P s .

Figure 3.46: Graphical representation of Anti-pyretic assay of Q. semicarpifolia derived AgNPs

189 Chapter 03 Results & Discussion

3.7 Chemical composition of fixed oil

In the current investigation, an ample quantity of fixed oils, obtained from the EtOAc extracts of

Q. semecarpifolia was analyzed through the GC-MS technique, in order to determine the chemical composition of the different saturated and unsaturated fatty acids present in the test samples. The results revealed that the fixed oils comprised of both saturated and unsaturated fatty acids, as shown Table (3.25). The most abundant fatty acids were palmitic acid (44.22%), followed by stearic acid (16.90%), oleic acid (13.09%), linoleic acid (2c) (12.52%), myristic acid

(4.90%), linolenic acid (3n3) (3.16%), and capric acid (2.37%). Caprylic acid (0.96%) and hexanoic acid (0.62%) were also present but in lesser quantities.

190 Chapter 03 Results & Discussion

Table 3.25: Fatty acid composition of fixed oil from Q.semecarpifolia

Peak.No Fatty acid Area RT %

1 Hexanoic acid; C6: O 7.51 4.664 0.62

2 Caprylic acid; C8: O 1158 6.639 0.96

3 Capric acid; C10: O 2861 8.476 2.37

5 Lauric acid; C12:0 1523 10.930 1.26

7 Myristic acid; C14:0 5920 14.642 4.90

11 Palmitic acid; C16:0 53444 19.774 44.22

15 Stearic acid; C18:0 20430 27.522 16.90

16 Oleic acid; C18:1c 15825 27.592 13.09

18 Linoleic acid; C18:2c 15127 28.790 12.52

21 Linolenic acid; C18:3n3 3815 30.279 3.16

191 ______Conclusion

Conclusion

Many ethno-botanical uses of Q.semicarpifolia Smith are known. It is used for the cure of chronic diarrhea, dysentery, and hemorrhages. The bark or the galls, produced on the trees, are boiled and applied to swollen tissues, bruises, and varicose veins. Usually, the juice, obtained from the bark is used in the treatment of muscular pains.

From the current findings, it can be concluded that Q.semecarpifolia plant possess cardinal phytochemicals such as flavonoids, sterols, glycosides and phenolic compounds which can be helpful in bioinspired synthesis of green AgNPs. Subsequently, the plant derived AgNPs were characterized via different techniques. The SPR band was obtained at 430 nm. The biofabricated nanocrystals were further analysed as crystalline, circular and monodispersed having diameter of

20-50 nm and thermally stable in nature. These plant-derived AgNPs were assessed biologically in contrast to Cr.MeOH extract and various fractions. From the comparative analysis it was revealed that plant-derived AgNPs possessed excellent bactericidal effect against bacterial species such as K. pneumoniae, B. subtilis, and P. mirabilis. Among the extracts, EtOAc and

CHCl3 have shown significant results while other fractions were good. Against different fungal species, both the plant-derived AgNPs and crude extracts showed low antifungal activities. The crude extracts and synthesized AgNPs showed significant antioxidant activities and at higher concentration, the scavenging ability of the samples increases. This property of the plant can be utilized in food industries in order to boost the shelf life of several food items. Likewise, high phytotoxic, insecticidal and anti-termite activities were recorded at elevated concentration of

192 ______Conclusion

1000 µg/mL which can be helpful in agricultural site for the production of low cost and environment friendly herbicides and pesticides. The plant aqueous extract have also the potency to reduce growth of wheat seeds due to the presence of allelochemicals. Eminent cytotoxic potential was displayed by Cr. MeOH, n-hexane and aqueous extracts at higher concentration of

1000 µg/mL. This explains good anticancer potential by the crude extracts and plant-derived

AgNPs using Q.semecarpifolia plant. Moreover, the in-vivo activities were executed on

Cr.MeOH extract and synthesized AgNPs and the findings revealed that the Q.semecarpifolia plant has good anti-inflammatory, antinoceceptive, and anti-pyretic activities.

Five compounds were isolated from EtOAc fractions. These compounds are (1) benzoic acid, (2) p-hydroxy benzoic acid (3) Bis (2-ethylhexyl) phthalate (4) β-Sitosterol and (5) Stigmasterol.

These compounds can be used in the formulation of new drugs which are economic.

Furthermore, the analysis of fixed oils obtained from n-hexane fraction showed the presence of

FAME (Fatty Acid Methyl Esters ) in the sample.

193 Refrences

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