ph. d thesis by: aishma khattakprr.hec.gov.pk/jspui/bitstream/123456789/10159/1... · 2.1.2...

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.

i

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


1.3.3 Importance 8

1.4 Quercus semecarpifolia Smith (Plant) 11

1.4.1 Description 11


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

Nanoparticles (NPs)

32

1.9.1 Biological Approaches for the Synthesis of Nanoparticles

(NPs)

33

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

ii

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

Different Classes of Compounds

45

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

iii

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

(AgNPs)

57

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

(TG/DTA)

61

2.5 Assessment of Pharmacological/Biological Activities

(in-vitro)

61

2.5.1 Antibacterial Activity 61

2.5.2 Determination of Minimum Inhibitory Concentration

(MIC50) Values

63

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

(in-vivo)

77

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

Spectrometry (GC-MS)

84

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

iv

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

Thermal Analysis (TGA/DTA):

125

3.5 Assessment of Pharmacological/Biological Activities ( in

vitro)

129

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

vivo)

168

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

v

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 13

C-NMR spectra of Benzoic acid (1)


C-NMR spectra of P-Hydroxybenzoic acid (2)


C-NMR spectra of Bis (2-ethylhexyl) phthalate (3)


C-NMR spectra of β-Sitosterol (4)


C-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

vi

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

vii

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


60,000X


30,000X

Figure 3.15 SEM micrograph of Quercus semecarpifolia aqueous extract at

10,000X


20,000X


30,000X


60,000X

Figure 3.19 Graphical representation of EDX profile of Quercus semecarpifolia

aqueous extract

viii

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

ix

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

x

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

xi

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

xii

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

xiii

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.

xiv

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.

Chapter 01 Introducation & Literature Review

1

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


2

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].


3

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.


4

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

Lymphoma

NP derived

2014 Dapagliflozin

Diabetes

NP derived

2014 Empagliflozin

Diabetes

NP derived

2015 Naloxegol

Neoplasm NP derived


5

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.


6

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].

https://en.wikipedia.org/wiki/Beech


7

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.


8

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].


9

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].


10

Table 1.2: Quercus species in Pakistan and their worldwide distribution

S.no Plant Flowering

period

Distribution

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

semicarpifolia

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

Mexico

5 Quercus baloot April-June Pakistan, China, Afghanistan

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

Pakistan.


11

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].


12

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].


13

Figure 1.1: Morphology of Q. semecarpifolia plant

Figure 1.2: Zoom version of leaf of Quercus semecarpifolia plant


14

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.


15

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


16

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].


17

Table 1.3: Phytochemical constituents from genus Quercus

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

1 Q. ilex

Q. annulata

Q. penduculata

Q. phillyraeoides

Q. cortex

C6H3(OH)3

126.11

1,2,3 benzenetriol

[97]

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

Q. glauca

C16H12O6 316.256 7-Methoxy kaempferol

[98]

8

Q.incana

C10H8O4

192.168

7-hydroxy-6-methoxy-2H-

chromen-2-one

[97]


18

9 Q. robur

C9H12O4

184.191

3,4,5-trimethoxy-phenol [97]

10 Q. bambusifolia

Q. phillyraeoides

Q. resinosa

Q. glauca

C7H6O3

138.122

4-Carboxyphenol

[105]

11 Q.cortex

Q.ilex

C6H603

126.111

5 (hydroxymethyl)furan-2-

carbaldehyde

[97]

12 Q.cortex

Q. resinosa

C15H28O2

240.381

2-Propenoic acid

[97]

13 Q. pyrenaica

Q.cortex

Q.ilex

C5H5N3O4

171.112

4,6-dihydroxy-2-methyl-5-

nitropyrimidine

[97]

14 Q.cortex

C10H13N5O5

283.244

Guanine riboside

[97]

15 Q.cortex C7H13O5 177.176 1,6-anhydro-β-D-

glucofuranose

[97]

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]


19

20 Q. glauca

Q. petraea

Q. pyrenaica

Q. resinosa

Q. salicina

C8H8O3

152.149

Vanillic aldehyde

[100,

101]

21 Q. glauca

C20H22O8R

390.388

lyoniresinol-9-O-β-

xylopyranoside

[99]

22 Q. salicina

Q. phillyraeoides

Q. resinosa

HOC6H4COOH

138.122

2-Carboxyphenol;

[100,

102]

23 Q. myrsinaefolia

Q. phillyraeoides

Q. resinosa

C7H6O4

154.121

Protocatehuic acid

[100,

102 ]

24 Q. glauca

Q. myrsinaefolia

Q. phillyraeoides

C9H8O3

164.16

4-Hydroxycinnamic

[100,

102 ]

25 Q. phillyraeoides

Q. salicina

C7H6O2

154.12

Hydroquinone carboxylic

acid

[100]

26 Q. sessilis


20

Q. phillyraeoides

Q. robur

Q. resinosa

Q. salicina

Q. suber

C7H6O5

170.12

3,4,5-Trihydroxybenzoic

acid;

[100 102

86]

27 Q. acuta

Q. myrsinaefolia

Q. phillyraeoides

Q. salicina

C8H8O4

168.148

Homogentisic acid

[100]

28 Q. myrsinaefolia

Q. phillyraeoides

Q. salicina

C7H6O4

154.121

4-Hydroxysalicylic acid

[100 ]

29 Q. acuta

Q. glauca

C16H14O6

610.188

Hesperidin

[100]

30 Q. petraea

Q. faginea

Q. pyrenaica

Q. robur

C9H6O4

178.183

Cichorigenin

[101]


21

31 Q. acuta

Q. phillyraeoides

Q. glauca

C9H10O4

182.175

3,4-Dimethoxybenzoic

acid;

[100]

32 Q. acuta

Q. myrsinaefolia

Q. phillyraeoides

C9H8O4

180.159

3,4-Dihydroxycinnamic

acid

[100,

102]

33 Q. alba

Q. faginea

C9H10O4

182.175

Syringic aldehyde

[101]

34 Q. alba

Q. faginea

Q. petraea

C11H12O4

208.221

Sinapic aldehyde

[101]

35 Q. phillyraeoides

Q. pyrenaica

Q. robur

Q. salicina

C10H10O4

194.186

Trans-4-Hydroxy-3-

methoxycinnamic acid

[100,

101]

36 Q. alba

Q. phillyraeoides

C16H12O4

268.248

7-Hydroxy-4'-

methoxyisoflavone

[100]

37 Q. acuta

Q. pyrenaica

[100,

102 ]


22

Q. phillyraeoides

C15H14O6 290.26 Catechuic acid

40 Q. baloot

Q. glauca

Q. phillyraeoides

C16H14O6

302.282

3',5,7-Trihydroxy-4'-

methoxyflavanone

[100]

41 Q. petraea

Q. pyrenaica

Q. robur

C14H6O8

304.197

Benzoaric acid

[101]

42 Q. acuta,

Q. glauca,

C15H12O5

272.256

5,7-Dihydroxy-2-(4-

hydroxyphenyl)chroman

[100]

43

Q. acuta

Q. glauca

C15H10O8

318.237

Cannabiscetin

[100]

44

Q. glauca

Q. myrsinaefolia

C15H10O7

302.236

Meletin

[100]

45 Q. ilex C15H14O7 306.27 Epigallocatechol [103]

46 Q. resinosa

Q. salicina

C16H18O9

354.31

3-O-Caffeoylquinic acid

[102]


23

47 Q. ilex C22H18O10 442.376 442.376 Catechin 3-O-

gallate

[103]

48 Q. ilex C27H30O16 610.521 Quercetin pentoside [103]

49 Q. ilex

Q. rotundifolia

C41H32O26

940.681

Galloyl glucoside

[104,105

]

50 Q. ilex C21H20O11 448.38 Kaempferol hexoside [103]

51

Q. ilex

C21H19O12

436.314

Quercetin-3-O-beta-D-

glucoside

[103]

52

Q. acutissima

Q. macrocarpa

C14H6O8

302.194

Ellagic acid glucoside

[104,

105]

53

Q. ilex

C21H18O13

478.362

4H-chromen-2-

phenoxyoxane-2-

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

Q. macrocarpa

C18H28O9

388.413

Ellagic acid-4-O-beta-D-

xylopyranoside

[104,

105]


24

58 Q. ilex

Q. rotundifolia

940.653

C41H32O26

Pentagalloyl glucoside

[86]

59 Q. rotundifolia

Q. suber

C34H28O22

788.556

Tetragalloyl glucoside

[86]

60 Q. acutissima

Q. macrocarpa

C41H62O14

778.933

Dihexahydroxydiphenoyl-

glucoside

[104,

105]

61 Q. ilex

Q. rotundifolia

C27H24O18

636.432

Trigalloyl glucoside

[86,

103]

62

Q. ilex

Q. rotundifolia

Q. suber

C22H22O11

462.407

Tergallagic C-glucoside

[86]

63 Q. marilandica

Q. muhlenbergii

C13H16O10

322.261

Galloyl-

hexahydroxydiphenoyl-

glucoside

[86]

64

Q. ilex

Q. rotundifolia

Q. suber

C27H30O14 578.543 Vitexin-2''-rhamnoside

[86]


25

65

Q. marilandica

Q. glauca

Q. myrsinaefolia

C27H30O16 610.521 Phytomelin [100]

66 Q. incana

Q. robur

Q. myrsinaefolia

C28H34015

610.132

Hesperidin

[100]


26

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


27

(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


28

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


29

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

https://en.wikipedia.org/w/index.php?title=Lipid_nanotechnology&action=edit&redlink=1https://en.wikipedia.org/wiki/Laurium


30

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


31

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].


32

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].


33

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,


34

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].


35

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


36

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


37

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 95

0C, 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].


38

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


39

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.

Chapter 02 Experimental

40

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-


41

EIMS and FAB +ive and –ive. 13

C‐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

(CH, CH2 and CH3) at 90o and 135

o. 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].


42

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,

ph. d thesis by: aishma khattakprr.hec.gov.pk/jspui/bitstream/123456789/10159/1... · 2.1.2...

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