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SYNTHESIS, SPECTROSCOPIC ANALYSES AND

BIOLOGICAL EVALUATION OF SILVER AND

GOLD BASED PRO-NANOMEDICINES DERIVED

FROM FLUOROQUINOLONES

Ph.D Thesis

By

SHUJAAT ALI KHAN

INSTITUTE OF CHEMICAL SCIENCES

UNIVERSITY OF PESHAWAR,

PAKISTAN

January 2017

SYNTHESIS, SPECTROSCOPIC ANALYSES AND

BIOLOGICAL EVALUATION OF SILVER AND

GOLD BASED PRO-NANOMEDICINES DERIVED

FROM FLUOROQUINOLONES

By

SHUJAAT ALI KHAN

DISSERTATION

SUBMITTED TO THE UNIVERSITY OF PESHAWAR IN

PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

IN CHEMISTRY


UNIVERSITY OF PESHAWAR,

PAKISTAN

January 2017


UNIVERSITY OF PESHAWAR

PAKISTAN

It is recommended that this dissertation prepared by Mr. Shujaat Ali Khan entitled

“Synthesis, Spectroscopic Analyses and Biological Evaluation of Silver and Gold

Based Pro-nanomedicines derived from Fluoroquinolones” be accepted as

fulfilling this part of the requirements for the degree of “Doctor of Philosophy in

Chemistry”.

________________________ ________________________

SUPERVISOR CO-SUPERVISOR

Meritorious Prof. Dr. Muhammad Nisar Prof. Dr. Ghias Uddin

Institute of Chemical Sciences, Institute of Chemical Sciences,

University of Peshawar University of Peshawar

________________________ ________________________ INTERNAL EVALUATOR/ EXAMINER Meritorious Prof. Dr. Jasmin Shah Prof. Dr. Muhammad Arfan Director,

HEJ, Research Institute of Chemistry Institute of Chemical Sciences,

ICCBS, university of Karachi University of Peshawar

________________________

EXAMINER

Prof. Dr. Hamidullah Shah

Dean, Faculty of Nutrition Sciences,

Agriculture University, Peshawar

________________________

INTERNAL EXAMINER

Prof. Dr. Muhammad Ishaq

Institute of Chemical Sciences,

University of Peshawar

Author’s Declaration

I Mr. Shujaat Ali Khan hereby state that my Ph.D thesis titled “Synthesis,

Spectroscopic Analyses and Biological Evaluation of Silver and Gold Based Pro-

nanomedicines derived from Fluoroquinolones” is my own work and has not been

submitted to previously by me for taking any degree from this University of Peshawar

or anywhere else in the country/world.

At any time if my statement is found to be incorrect even after my graduation the

University has the right to withdraw my Ph.D degree.

________________

Shujaat Ali Khan

Date: / /2018

DEDICATION

TO

MY LOVING PARENTS

Whose encouragement towards knowledge served me

As beacon of light

And to my younger

Brother and Sister

For their sweet feelings who always

Pray for my bright future and success

Table of Contents

S. No. Title Page No.

Acknowledgments...................................................................................................... i

List of Figures ........................................................................................................... iii

List of Tables ............................................................................................................. ix

List of Schemes .......................................................................................................... x

List of Abbreviations ................................................................................................ xi

Summary ................................................................................................................... xiii

Chapter 1 General introduction

1.1 History of quinolones .................................................................................... 1

1.2 Quinolone nucleus......................................................................................... 1

1.3 Classification ................................................................................................. 2

1.3.1 First generation ............................................................................................. 2

1.3.2 Second generation ......................................................................................... 2

1.3.3 Third generation ............................................................................................ 3

1.3.4 Fourth generation .......................................................................................... 4

1.4 Mechanism of action of quinolones and fluoroquinolones ...................... 5

1.5 Medicinal plants ..................................................................................... 6

1.6 Phytochemistry and bioactivities of Rhododendron arboretum ................... 7

1.6.1 Plant introduction .......................................................................................... 7

1.7 Literature review of genus Rhododendron .................................................... 8

1.7.1 Chemical constituents of the genus Rhododendron ................................................. 8

1.7.2 Medicinal and pharmacological properties ....................................................... 9

1.8 Phytochemistry and bioactivities of Eulophia dabia .................................... 9

1.8.1 Plants introduction ........................................................................................ 9

1.9 Literature review of genus Eulophia ....................................................... 10

1.9.1 Chemical constituents of the genus Eulophia ......................................... 10

1.9.2 Medicinal and Pharmacological Properties ............................................. 10

1.10 Phytochemistry and bioactivities of Kigelia pinnata ........................................ 11

1.10.1 Plant introduction .............................................................................. 11

1.11 Literature review of genus Kigelia .................................................................... 11

1.11.1 Plant introduction .............................................................................. 11

1.11.2 Medicinal and pharmacological properties....................................... 12

1.12 Phytochemistry and bioactivities of Desmodium elegans ................................ 12

1.12.1 Plant introduction .................................................................... 12

1.13 Literature review of genus Desmodium ............................................................. 13

1.13.1 Chemical constituents of the genus Desmodium .............................. 13

1.13.2 Medicinal and pharmacological properties....................................... 14

1.14 Biological evaluation ......................................................................................... 14

1.14.1 Urease inhibition activity ................................................................... 14

1.14.2 Leishmanicidal studies ....................................................................... 15

1.14.3 Antioxidant assay ............................................................................... 15

1.14.4 Antibacterial assay ............................................................................. 16

1.14.5 Antifungal activity ............................................................................. 16

Chapter 2 Introduction to Nanotechnology

2.1 Nanotechnology ..................................................................................... 18

2.2 Noble metal NPs .................................................................................... 18

2.3 Synthesis ................................................................................................ 19

2.4 Characterization techniques .................................................................... 22

2.5 Applications ........................................................................................... 22

2.6 Properties ............................................................................................... 25

2.6.1 Optical properties ................................................................................... 25

2.6.2 Biomedical properties ............................................................................ 25

2.6.3 Magnetic properties ................................................................................ 26

2.6.4 Electronic properties .............................................................................. 26

2.6.5 Energy properties ................................................................................... 26

2.7 Aims and objectives of the present research ................................................. 26

2.8 Review on topical advancements in noble metal NPs (28) ........................... 27

2.8.1 Review on Ag NPs ................................................................................. 27

2.8.2 Review on Au NPs ................................................................................. 31

Chapter 3 Results and Discussion

3.1 Results and discussion ............................................................................ 36

3.1.1 Synthesis of Ag-Mox and Au-Mox NPs capped with

moxifloxacin ................................................................................................. 36

3.1.2 Characterization of moxifloxacin-capped Ag/Au NPs ................................. 38

3.1.3 AFM, SEM and EDX spectroscopy analyses ............................................... 40

3.1.4 Stability check of Ag and Au NPs ................................................................ 41

3.2 Synthesis of ciprofloxacin-capped metallic NPs .......................................... 43

3.2.1 FTIR and AFM studies ................................................................................. 45

3.2.2 EDX and SEM studies .................................................................................. 47


3.3 Synthesis of sparfloxacin mediated Ag and Au NPs .................................... 51

3.3.1 UV visible spectroscopic analysis ................................................................. 51

3.3.2 AFM, SEM and EDX analyses ..................................................................... 52

3.3.3 Fourier transform infrared spectroscopy (FTIR) .......................................... 53


3.4 Synthesis of gemifloxacin-capped noble metal NPs ..................................... 59

3.4.1 UV visible spectroscopy for synthesis of Ag/Au NPs .................................. 59

3.4.2 AFM, SEM and EDX spectroscopy analysis ................................................ 60

3.4.3 FTIR analysis ................................................................................................ 62


3.5 Biological evaluation of fluoroquinolones-capped Ag/Au NPs ................... 67

3.5.1 Urease study .................................................................................................. 67

3.5.2 Leishmanicidal activity ................................................................................. 69

3.5.3 Antioxidant activity....................................................................................... 71

3.5.4 Antibacterial activity ..................................................................................... 73

3.5.5 Antifungal activity ........................................................................................ 74

3.6 Green synthesis of noble metal NPs by using flower extract of

Rhododendron arboretum .............................................................................

76

3.6.1 Visual inspection and UV visible spectroscopy analysis .............................. 76

3.6.2 FTIR spectroscopy analysis .......................................................................... 78

3.6.3 Stability of the biosynthesized Ag/Au NPs ................................................... 79

3.6.4 AFM, SEM and EDX analysis ...................................................................... 82

3.7 Green synthesis of metallic NPs by using Kigelia pinnata ........................... 84

3.7.1 UV Visible spectroscopy .............................................................................. 84

3.7.2 FTIR analysis ................................................................................................ 85

3.7.3 Stability check of the synthesized Ag/Au NPs ............................................. 86

3.7.4 AFM and SEM analysis ................................................................................ 88

3.8 Green phytosynthesis of noble metal NPs using Eulophia dabia

extract as reducing and stabilizing agent ......................................................

89

3.8.1 Visual inspection and UV visible spectroscopy analysis .............................. 89

3.8.2 FTIR spectroscopy analysis .......................................................................... 89


3.8.4 AFM and EDX analysis ................................................................................ 91

3.9 Synthesis of metallic NPs by using Desmodium elegans ............................. 93

3.9.1 UV visible spectroscopy ............................................................................... 93

3.9.2 FTIR Analysis ............................................................................................... 95

3.9.3 Stability check of synthesized metallic NPs ................................................. 96

3.9.4 SEM and EDX analysis................................................................................. 98

3.10 Biological evaluation of selected medicinal plants and their

NPs ................................................................................................................

100

3.10.1 Urease inhibition assay ................................................................. 100

3.10.2 Leishmanicidal activity ................................................................. 102

3.10.3 Antioxidant activity ....................................................................... 104

3.10.4 Antibacterial activity ..................................................................... 106

3.10.5 Antifungal activity ........................................................................ 107

Chapter 4 Experimental

4.1 General experimental procedures .................................................................. 109

4.1.1 Collection of fluoroquinolones and plants material ...................................... 109

4.1.2 Preparation of stock solution......................................................................... 110

4.1.3 Synthesis of Ag and Au NPs capped with fluoroquinolones ........................ 110

4.1.4 Green synthesis of metallic NPs by using selected medicinal

plants .............................................................................................................

111

4.2 Biological evaluation .................................................................................... 112

4.2.1 Protocol for urease assay and inhibition ............................................................ 112

4.2.2 Procedure for leishmanicidal activity (in vitro)................................................. 112

4.2.3 Antioxidant activity ............................................................................................ 113

4.2.4 Bacterial strains assortment and preservation ................................................... 114

4.2.5 Antibacterial activity .......................................................................................... 114

4.2.6 Antifungal activity .............................................................................................. 114

Conclusion 116

References 118

List of Publications 137

Biography 138

i

Acknowledgments

In the name of Almighty Allah (the most Beneficent, the most Merciful) who had

given me courage to accomplish this task. It would not have been possible without

His will and support. All respect for His Holy Prophet (SAW), who enabled us to

recognize our creator.

Pursuing a Ph.D. project is both painful and pleasant experience. It is just like

climbing a peak, step by step, accompanied with bitterness, hardships, frustration,

encouragement and trust. When I found myself at the top enjoying the beautiful

scenario, I realized that it was, in fact, teamwork that got me there. Though it will not

be enough to express my appreciation in simple words to all those people who helped

me, I would still like to give my many thanks to all these people.

First of all, I would like to give a bundle of thanks to my honorable supervisor, Dr.

Muhammad Nisar (meritorious professor), who accepted me as his Ph.D. student

without any reluctance. Furthermore, he patiently supervised me and always guided

me in the right way. I have learnt a lot from him, without his help, I could not have

completed my dissertation successfully.

It is difficult to express in words what I feel and what is in my heart for my respected

Co-supervisor Prof. Dr. Ghias Uddin and Assoc. Prof. Dr. Muhammad Raza Shah,

H.E.J. Research Institute of Chemistry, University of Karachi, because the work

presented in this dissertation would have never been completed without their keen

interest, precious attention, and continuous encouragement. By observing their

personality, hard work and devotion, I learnt about the key to success.

My deep appreciation and sincere thanks are due to all my teachers in I.C.S.,

especially Prof. Dr. Jasmin Shah (Director, ICS), Prof. Dr. Yousaf Iqbal, Prof. Dr.

Imtiaz Ahmad, Dr. Adnan Khan, Dr. Rasool Khan and Dr. Salman Zafar for their kind

help whenever I needed.

I would like to express my heartiest gratitude and regards to my mother, father,

younger brother, sister and my late grandfather (May Allah keep his soul in rest and

peace) for their love, prayers and encouragement throughout my studies. I would like

to express my appreciation and thanks to my colleagues Dr. Ajmal Khan, Ayaz Khan,

ii

Qamar Altaf Jaffery, Rahmanullah, Sher Ayaz, Hanif ur Rahman, Muhmmad Atif

Khan and Mansor Khan for providing a pleasant and helping atmosphere during my

studies.

I am thankful to Mr. Masood Jan and Mr. Zulfiqar (Lab Assistants) for their help in

every step during my bench work at the Institute of Chemical Sciences, University of

Peshawar.

I am also thankful to Higher Education Commission (HEC) of Pakistan for providing

necessary grant for access to spectroscopic facilities.

Last but not the least, I would like to thank all those who assisted and guided me in

the completion of this study.

Shujaat Ali Khan

iii

List of Figures

Figure No. Title Page No.

Fig. 1.1 Mechanism of action of fluoroquinolones..................................................... 5

Fig. 1.2 Photograph of Rhododendron arboreum flowers .......................................... 8

Fig. 1.3 Photograph of Eulophia dabia ...................................................................... 10

Fig. 1.4 Photograph of Kigelia pinnata .......................................................................... 11

Fig. 1.5 Photograph of Desmodium elegans .................................................................. 13

Fig. 2.1 Diagram for silver and gold nanomaterials ................................................... 18

Fig. 2.2 Illustrative chemical reduction diagrams for noble metal NPs

synthesis ........................................................................................................

20

Fig. 2.3 Optical inspection of the colloidal solution of noble metal NPs ................... 25

Fig. 3.1 Optical inspection of Ag-Mox NPs ............................................................... 37

Fig. 3.2 Optical inspection of Au-Mox NPs ............................................................... 37

Fig. 3.3 Optimized UV visible spectra of Ag-Mox NPs............................................. 38

Fig. 3.4 Optimized UV visible spectra of Au-Mox NPs............................................. 38

Fig. 3.5 FTIR spectra of Mox, Ag and Au NPs .......................................................... 39

Fig. 3.6 AFM images of Ag NPs ................................................................................ 40

Fig. 3.7 AFM images of Au NPs ................................................................................ 41

Fig. 3.8 SEM image of Ag-Mox ................................................................................. 41

Fig. 3.9 SEM image of Au-Mox ................................................................................. 41

Fig. 3.10 The EDX spectrum for Ag NPs ..................................................................... 41

Fig. 3.11 The EDX spectrum for Au NPs ..................................................................... 41

Fig. 3.12 Effect of brine on Ag-Mox ............................................................................ 42

Fig. 3.13 Effect of brine on Au-Mox ............................................................................ 42

Fig. 3.14 Effect of pH on stability of Ag NPs .............................................................. 43

Fig. 3.15 Effect of pH on stability of Au NPs .............................................................. 43

iv

Fig. 3.16 Optimized UV visible spectra of Ag NPs (Inset: Optical inspection

of Ag NPs) .....................................................................................................

44

Fig. 3.17 Optimized UV visible spectra of Au-Cip NPs (Inset: Au-Cip NPs

color) .............................................................................................................

44

Fig. 3.18 FTIR spectra of Ag-Cip NPs ......................................................................... 46

Fig. 3.19 FTIR spectra of Au-Cip NPs ......................................................................... 46



Fig. 3.22 EDX spectrum of Ag-Cip NPs ...................................................................... 48

Fig. 3.23 EDX spectrum of Au-Cip NPs ...................................................................... 48

Fig. 3.24 SEM image of Ag-Cip NPs ........................................................................... 48

Fig. 3.25 SEM image of Au-Cip NPs ........................................................................... 48

Fig. 3.26 Effect of brine on the stability of Ag NPs ..................................................... 49

Fig. 3.27 Effect of brine on the stability of Au NPs ..................................................... 49

Fig. 3.28 pH effect on the stability of Ag-Cip NPs ...................................................... 50

Fig. 3.29 pH effect on the stability of Au-Cip NPs ..................................................... 50

Fig. 3.30 Temperature effect on the stability of Ag and Au-Cip NPs (Inset:

effect of temperature on Ag and Au-Cip NPs colors ....................................

50

Fig. 3.31 UV visible spectra of Ag NPs (Inset: color of Ag NPs) ................................ 51

Fig. 3.32 UV visible spectra of Au NPs (Inset: color of Au NPs) ................................ 52

Fig. 3.33 AFM image of Ag-Sp NPs ............................................................................ 52

Fig. 3.34 AFM image of Au-Sp NPs ............................................................................ 52

Fig. 3.35 SEM image of Ag NPs .................................................................................. 53

Fig. 3.36 SEM image of Au NPs .................................................................................. 53

Fig. 3.37 EDX spectrum of Ag NPs ............................................................................. 53

Fig. 3.38 EDX Spectrum of Au NPs............................................................................. 53

Fig. 3.39 FTIR spectra of Ag and Au-Sp NPs .............................................................. 54

Fig. 3.40 Effect of brine on the stability of Ag-Sp NPs ............................................... 55

v

Fig. 3.41 Effect of brine on the stability of Au-Sp NPs ............................................... 55

Fig. 3.42 pH effect on the stability of Ag-Sp NPs ........................................................ 56

Fig. 3.43 pH effect on the stability of Au-Sp NPs ........................................................ 56

Fig. 3.44 Heat effect on the stability of Ag-Sp NPs ..................................................... 57

Fig. 3.45 Temperature effect on the stability of Au-Sp NPs ........................................ 57

Fig. 3.46 Reaction time effect on the stability of Ag-Sp NPs ...................................... 58

Fig. 3.47 Reaction time effect on the stability of Au-Sp NPs ...................................... 58

Fig. 3.48 Molecular structure of gemifloxacin ............................................................. 59

Fig. 3.49 Optimized UV visible spectra of Ag NPs (Inset: Color of Ag NPs) ............. 60

Fig. 3.50 Optimized UV visible spectra of Au NPs. (Inset: Colors of Au NPs) .......... 60






Fig. 3.56 EDX spectrum of Au NPs ............................................................................. 62

Fig. 3.57 FTIR spectra of gemifloxacin mesylate and its noble metal (Ag/Au)

NPs ................................................................................................................

62

Fig. 3.58 Temperature effect on the stability of Ag NPs .............................................. 63

Fig. 3.59 Effect of temperature on the stability of Au NPs .......................................... 63

Fig. 3.60 Salt effect on the stability of Ag NPs ............................................................ 64

Fig. 3.61 Effect of salt on the stability of Au NPs ........................................................ 64

Fig. 3.62 Effect of pH on the stability of Ag NPs ........................................................ 65

Fig. 3.63 Effect of pH on the stability of Au NPs (Insets: Effect of pH on the

colors of Ag/Au NPs) ....................................................................................

65

Fig. 3.64 Effect of reaction time on the stability of Ag NPs ........................................ 66

Fig. 3.65 Effect of reaction time on the stability of Au NPs ........................................ 66

vi

Fig. 3.66 Graphical representation of urease activities of fluoroquinolones-

capped noble metal NPs ................................................................................

69

Fig. 3.67 Antioxidant assay of fluoroquinolnes-capped Ag/Au NPs ........................... 71

Fig. 3.68 Antioxidant assay of fluoroquinolnes-capped Ag/Au NPs ........................... 72

Fig. 3.69 Optimized UV visible spectral data of Ag NPs at reaction ratio of

5:1 ..................................................................................................................

77

Fig. 3.70 UV visible spectral data of Au NPs at optimized reaction ratio of

10:1 ................................................................................................................

77

Fig. 3.71 FTIR analysis for RAFE and Ag NPs ........................................................... 78

Fig. 3.72 FTIR spectra of RAFE and Au NPs .............................................................. 79

Fig. 3.73 Effect of salt (NaCl) on stability of Ag NPs ................................................. 79


Fig. 3.75 Effect of salt (NaCl) on stability of Au NPs ................................................. 80


Fig. 3.77 Effect of brine (1 M NaCl) on the color of Ag NPs ...................................... 80

Fig. 3.78 Effect of pH on the color of Ag NPs ............................................................. 81

Fig. 3.79 Effect of salt (1 M NaCl) on the color of Au NPs ......................................... 81

Fig. 3.80 Effect of pH on the color of Au NPs ............................................................. 81

Fig. 3.81 Atomic force microscope spectrum of RAFE stabilized Ag NPs ................. 82

Fig. 3.82 AFM spectrum of RAFE-capped Au NPs ..................................................... 82




Fig. 3.86 EDX spectrum of Au NPs ............................................................................. 84

Fig. 3.87 Optimized UV visible spectra of Ag NPs ..................................................... 85

Fig. 3.88 Optimized UV visible spectra of Au NPs ..................................................... 85

Fig. 3.89 FTIR spectra of K. pinnata and its Ag/ Au NPs ............................................ 86

Fig. 3.90 Effect of salt (NaCl) on stability of Ag NPs ................................................. 86

vii

Fig. 3.91 Salt (NaCl) effect on stability of Au NPs ...................................................... 87







Fig. 3.98 UV visible spectra of Ag-NPs ....................................................................... 89

Fig. 3.99 UV visible spectra of Au NPs ....................................................................... 89

Fig. 3.100 FTIR spectrum of E. dabia ............................................................................ 90

Fig. 3.101 FTIR spectra of Ag and Au NPs ................................................................... 91

Fig. 3.102 Effect of salt (1 M NaCl) on stability of Ag NPs .......................................... 91

Fig. 3.103 Effect of salt (1 M NaCl) on stability of Au NPs .......................................... 91



Fig. 3.106 AFM image of Ag NPs .................................................................................. 92

Fig. 3.107 AFM image of Au NPs .................................................................................. 92

Fig. 3.108 EDX spectrum of Ag-ED .............................................................................. 93

Fig. 3.109 EDX spectrum of Au-ED .............................................................................. 93

Fig. 3.110 UV visible data of Ag NPs reduced with TEA ............................................. 94

Fig. 3.111 Color of the Ag NPs reduced with TEA........................................................ 94

Fig. 3.112 UV visible data of Au NPs reduced with TEA ............................................. 95

Fig. 3.113 Color of Au NPs reduced with TEA ............................................................. 95

Fig. 3.114 FTIR spectra of D. elegans and its Ag/Au NPs reduced with TEA .............. 96

Fig. 3.115 Effect of temperature on the stability of Ag NPs .......................................... 96

Fig. 3.116 Salt effect on the stability of Ag NPs ............................................................ 97

Fig. 3.117 Effect of NaCl salt concentration on the color of Ag NPs ............................ 97

viii

Fig. 3.118 Effect of pH on the stability of Ag NPs ........................................................ 98

Fig. 3.119 Effect of pH on the color of Ag NPs ............................................................. 98



Fig. 3.122 The EDX spectrum for Ag NPs ..................................................................... 100

Fig. 3.123 The EDX spectrum for Au NPs ..................................................................... 100

Fig. 3.124 Urease inhibitory activity of selected medicinal plants and their

metallic NPs ............................................................................................................

102

Fig. 3.125 Antioxidant assay of selected medicinal plants-capped Ag/Au NPs ............ 105

Fig. 3.126 Antioxidant assay of selected medicinal plants-capped Ag/Au NPs ............ 106

ix

List of Tables

Table No. Title Page No.

Table 3.1 Urease enzyme inhibition studies of fluoroquinolones and

fluoroquinolones-capped Ag and Au NPs .....................................................

68

Table 3.2 In vitro efficacy of fluoroquinolones and their metallic NPs against

promastigotes of L. tropica ...........................................................................

70

Table 3.3 Antioxidant activity of fluoroquinolones and fluoroquinolones-

capped Ag/Au NPs ........................................................................................

71

Table 3.4 Antioxidant activity of fluoroquinolones-capped Ag and Au NPs ............... 72

Table 3.5 Antibacterial activities of fluoroquinolones and fluoroquinolones-

capped Ag and Au NPs .................................................................................

74

Table 3.6 Antifungal activities of fluoroquinolones and fluoroquinolones-

capped Ag and Au NPs .................................................................................

75

Table 3.7 Urease enzyme inhibition studies of selected medicinal plants and

plants mediated Ag and Au NPs....................................................................

101

Table 3.8 In vitro efficacy of selected medicinal plants and their metallic NPs

against promastigotes of L. tropica ...............................................................

103

Table 3.9 Antioxidant activity of selected medicinal plants and their Ag/Au

stabilized NPs ................................................................................................

104

Table 3.10 Antioxidant activity of selected medicinal plants Ag/Au stabilized

NPs ................................................................................................................

105

Table 3.11 Antibacterial activities of selected medicinal plants and their Ag/Au

stabilized nanoparticles .................................................................................

107

Table 3.12 Antifungal activities of selected medicinal plants and their stabilized

Ag/Au NPs ....................................................................................................

108

x

List of Schemes

Scheme No. Description Page No.

Scheme 3.1 Capping action of moxifloxacin with noble metals (Ag and Au) ................. 36

Scheme 3.2 Capping action of ciprofloxacin with noble metals (Ag/Au) ........................ 45

Scheme 3.3 Synthesis of Ag and Au NPs stabilized with sparfloxacin ........................... 51

Scheme 3.4 Synthesis of Ag and Au NPs stabilized with gemifloxacin .......................... 59

Scheme 3.5 R. arboreum flower extract (RAFE) reduces and stabilizes

Ag/Au NPs ....................................................................................................

76

Scheme 3.6 E. dabia extract reduces and stabilizes Ag/Au NPs ...................................... 89

xi

List of Abbreviations

S. No. Abbreviations Full name

1. AFM Atomic force microscope

2. Ag NPs Silver nanoparticles

3. Au NPs Gold nanoparticles

4. Cip Ciprofloxacin

5. -COOH Carboxyl group

6. cm-1

Per centimeter

7. DMSO Dimethyl sulfoxide

8. DLS Dynamic light scattering

9. DPPH 2,2-Diphenyl-1-1picryl hydroxyl

10. DE Desmodium elegans

11. EDX Energy dispersive X-ray

12. ED Eulophia dabia

13. FTIR Fourier transform infrared

14. Gm Gemifloxacin

15. h Hour

16. IC50 Inhibition concentration 50 percent

17. KPFE Kigelia pinnata fruit extract

18. M Molar

19. mM Milli molar

20. min Minute

21. mL Milliliter

22. µM Micro molar

23. Mox Moxifloxacin

24. nm Nanometer

xii

25. NPs Nanoparticles

26. r.t Room temperature

27. RAFE Rhododendron arboreum flower extract

28. SPR Surface plasmon resonance

29.

30.

31.

32.

33.

Sp

SEM

TEA

TEM

UV

Sparfloxacin

Scanning electron microscope

Triethylamine

Transmission electron microscope

Ultraviolet

xiii

SUMMARY

The work presented in this dissertation comprises of synthesis, spectroscopic analyses

and biological evaluation of silver and gold based pronanomedicines derived from

fluoroquinolones. Among others, it includes convenient and time saving production of

noble metals (Ag/Au) nanoparticles (NPs) capped with fluoroquinolone antibiotics

(moxifloxacin (Mox), ciprofloxacin (Cip), sparfloxacin (Sp) and gemifloxacin (Gm).

Different reducing agents such as triethylamine, hydroquinone and sodium

borohydride were employed to transform Ag/Au salts into feasible capping agents.

Among them, sodium borohydride relatively gave better results. As for we understand

and based on FTIR data, the NH moiety of fluoroquinolones were mainly responsible

for the capping of Ag/Au nanoparticles.

In order to manifest alternate green method, Ag and Au NPs were also produced by

using selected medicinal plants; Rhododendron arboreum (RA), Kigelia pinnata (KP)

and Eulophia dabia (ED) as reducing and stabilizing agent, while triethylamine was

used to synthesize NPs of the extract of Desmodium elegans (DE).

The structural framework and size morphology of synthesized NPs were

characterized by using advanced analytical techniques such as atomic force

microscope (AFM), UV visible, fourier transform infrared spectroscope (FTIR),

energy dispersive X-ray (EDX) and scanning electron microscope (SEM).

To find alternate to wide spread resistive strains of pathogenic microbes; new

antimicrobial agents are needed to treat the patients infected with such resistive

pathogenic microbes. The locally synthesized pronanomedicines derived of

fluoroquinolones were evaluated for biological properties namely urease inhibition,

xiv

leishmanicidal, antimicrobial and antioxidant activities. Interestingly and as for our

expectations, these NPs enhanced biological and pharmacological activities.

The synthesized pronanomedicines and the capping ligands were independently

screened for jack bean urease enzyme inhibition potential. Mostly, the Ag-Mox NPs

exhibited higher level of enzyme inhibition activity of 93% at 0.2 mg/mL and IC50

value of 0.66 ± 0.042 μg/mL concentration, while the ligand; Mox revealed weak

inhibition with IC50 value of 183.25 ± 2.06 μg/mL. On the other hand, the Au-Mox

NPs remained inactive as compared to the parent ligand (Mox) having IC50 = 183.25 ±

2.06 μg/mL. These results reflect that after conjugation of Mox with Ag, the activity

of moxifloxacin was significantly increased about 250 times. However, the urease

inhibition activity of the Au conjugated counterpart of moxifloxacin decreased

significantly.

The synthesized metallic nano-conjugates (Ag-Cip and Au-Cip NPs) and the parent

ligand, ciprofloxacin were also tested for jack bean urease enzyme inhibition

potential. Ag-Cip pronanomedicine exhibited better urease enzyme inhibition

indicating 96 % at 0.2 mg/ mL (IC50 = 1.181 ± 0.02 μg/mL) concentration. On the

other hand, Au-Cip NPs showed comparatively weaker urease inhibition (90 % at 0.2

mg/mL concentration) with IC50 = 52.55±2.3 μg/mL. As anticipated, the parent ligand

ciprofloxacin revealed weaker inhibition to the values of 75 % at 0.2 mg/mL and IC50

= 82.95 ±1.62 μg/mL concentrations.

Furthermore, leishmanicidal, antimicrobial and antioxidant activities were tested for

both synthesized pronanomedicines and all the parent ligands under discussion but

they revealed good to moderate activities.

The selected plants namely R. arboreum, K. pinnata, E. dabia and D. elegans and

their metallic NPs were also screened for jack beans urease enzyme, leishmanicidal,

xv

antimicrobial and antioxidant activities which exhibited promising activities, while D.

elegans-capped NPs showed moderate activities.

Convincingly, the synthesized pronanomedicines were monodispersed and revealed

stability to some extent by changing pH, concentration of table salt and temperature.

The silver based pronanomedicines were anticipated to be good candidate for urease

inhibition and leishmanicidal potentials.

Chapter-1 Introduction

1

1.1 History of quinolones

The prolific development of the quinolones initiated in 1962, when George Lesher

and coworkers accidentally discover nalidixic acid as a derivative of the synthesis of

the antimalarial drug chloroquine [1]. This discovery led the expansion of quinolone

compounds, particularly the innovative quinolones in medical use at the current time.

Other discoveries followed, but exclusively a few were of substantial importance

because they supplied us with a fuller apprehension of the mechanisms of activity of

the quinolones. The capability to change the quinolone nucleus to enhance

effectiveness and the spectrum of bactericidal activity. Furthermore, the opportunity

to extend the eradication half-life and to improve the pharmacodynamics and

pharmacokinetic properties of quinolones and understanding of the significance of the

structure-activity relationships (SARs) of the quinolones, with respect to their

comparative susceptibilities to the progress of bacterial resistance and their efficacy

for causing adverse effects in treated patient [2].

1.2 Quinolone nucleus

Quinolones (quinolone carboxylic acids or 4-quinolones) consist of 4-oxo-1, 4-

dihydroquinoline structure.

Basic structure of quinolone antibiotics

The R' is piperazine moiety, while the connection contains fluorine (F), it is a

fluoroquinolone.


2

1.3 Classification

The quinolones can be separated into different generations on the basis of bactericidal

spectrum. The first generation members are more confined antibacterial spectrum than

the advanced ones. The only universal standard employed is the grouping of the non-

fluorinated drugs found within the heading of 'first generation'. However, there is no

standard to determine which drug belongs to which generation.

1.3.1 First generation

Nalidixic acid is the first member of this generation. The first generation quinolones

are rarely used today due to their limited antibacterial activities and also associated

with rapid development of bacterial resistance. Other members of this generation are

oxolinic acid, piromedic acid, pipermidic acid, cinoxacin and rosoxacin.

Nalidixic acid

1.3.2 Second generation

This generation comprises of enoxacin, norfloxacin, ofloxacin and ciprofloxacin with

improved action against Gram negative bacteria and longer half-life than the first

generation, having high serum and tissue concentrations. The second generation class

is used for simple and complicated urinary tract infections, gastroenteritis, prostatitis,

nosocomial infections.


3

Ciprofloxacin

1.3.3 Third generation

The members of this generation are active against bacteria (Gram positive and Gram

negative) and similar pharmacokinetics contour as for second generation.

Sparfloxacin, levofloxacin and grepafloxacin are the members of this generation.

They are considered for community acquired pneumonia in hospitalized patients.

Sparfloxacin

Levofloxacin


4

Grepafloxacin

1.3.4 Fourth generation

Fourth generation fluoroquinolones exposed prolonged activity against Gram positive

and Gram negative bacteria as well as active against a typical bacteria and anaerobes

[3]. They performed dual actions; inhibit topoisomerase IV and DNA gyrase, which

slow the progress of resistance. Moxifloxacin, gemifloxacin, sitafloxacin and

prulifloxacin are the members of fourth generation, which are considered in the

treatment of intra-abdominal infections.

Moxifloxacin

Gemifloxacin


5

Sitafloxacin

Prulifloxacin

1.4 Mechanism of action of quinolones and fluoroquinolones

They inhibit two bactericidal key-enzymes, DNA topoisomerase IV and

topoisomerase II (DNA-gyrase), producing fast cell death. topoisomerase IV is

convoluted in the slackening of the supercoiled circular DNA, allowing the split-up of

the inserted daughter chromosomes at the end of bacterial DNA replication, while

DNA gyrase is a topoisomerase II that speeds up the negative supercoiling of the

circular DNA existing in bacteria [4] (Fig. 1.1).

Fig. 1.1. Mechanism of action of fluoroquinolones [4]


6

1.5 Medicinal plants

Medicinal plant is the best source of herbal medicines/drugs. The connection between

humanity and plants is as old as human evolution. In current decades, medicinal

plants are the chief source of medications for the world‟s population. The human has

an expedition for sound health, long life and everlasting beauty. Through such

creative expeditions, more or less founded on his ideas and practical experiences man

empowered to discriminate facts from imaginary fictions. Up to great extent natural

medicines initiated as story, conveyed to the new age group as a traditional

medication and advanced with time.

The medicine‟s history is an explanation of man‟s attempts to share with human

ailment from the crude efforts of preliterate man to the current multifaceted array of

areas in cures. The Chinese, Babylonian, Egyptian and Indian societies, pursued by

Greek, Roman, Arabic and Persian, all established their own representative materia

medica [5, 6]. New medication traces its ancestry to the Greeks. The Greece

medication was brought over by the Romans and then by the Arabs, after its

development with Chinese and Indian medicine, it was followed by Europe. The

Muslim elites familiarized it in India and merged with it the Ayurvedic medicine. This

combination is now termed as Eastern medicine or Unani medicine [7, 8].

The first ideas about the therapeutic purpose of plants are drawn in the Ayurveda

(2500-600 BC) and Rigveda (4500-1600 BC). Charaka scrutinized about 50 groups of

herbs, while Sushruta explained 760 herbs in 37 groups. Buddhist era developed the

importance of medicinal plants and contributed a significant consideration to harvest

these medicinal plants in a systematic way [9].


7

A natural product has distinctive biosynthetic gateways. The comprehensive studies

of the synthetic protocols are managed by expert professionals. Various techniques

have been recognized by the use of isotopically labeled precursors of the biogenetic

substances along with certain biotic combination.

Photosynthesis performs a key role in biogenesis in green plants, photosynthetic

bacteria and algae [10]. The carbon frameworks of biological products are

accumulated by specific arrangements of enzyme catalyzed reactions. These

interconnected metabolic arrangements form the foundation for a biosynthetic

grouping [11].

Primary metabolites like polysaccharides, nucleic acids and proteins are the basis of

all living things. The entire chains of procedure by which plants prepares and blow up

these materials in order to stay alive, set up the primary metabolic process [12].

Secondary metabolites has an important role in the existence of one species over the

other but they are not indispensable for their subsistence; so they are called secondary

metabolites [12]. Materials from secondary metabolism have a tendency to overlap

with the natural compounds of organic chemistry, like pigments, alkaloids, terpenes,

steroids, phenols, oligosaccharides, coumarins and antibiotics.

Secondary metabolites play a critical part in the natural selection of distinct plant

classes during the evolutionary process and in the interface of plants with the

surrounding.

1.6 Phytochemistry and bioactivities of Rhododendron arboreum

1.6.1 Plant introduction

The genus Rhododendron comprises of 1000 species which are distributed all over the

world mostly concentrated in China, Malaysia, Pakistan, India and Nepal [13].


8

Rhododendron arboreum is an evergreen plant with bright red flowers (Fig. 1.2). The name

„Rhododendron‟ is originated from the Greek term „Rhodo‟ means rose and „dendron‟

means tree, arising in the high altitudes from 1500 m to 6000 m in Himalaya and Nilgiri in

South India. It is the national flower of Nepal, locally identified as Lali Guras or „rose tree‟

in English. R. arboreum belongs to family Ericaceae. The blooming season is from March-

April/June-September bears deep red or crimson to pale pinkish flowers. The aesthetic

flowers owe its spiritual importance; it is considered holy and put up in temples for

ornamenting purposes. A stamp was dispensed by the Indian postal department to honor

this flower [14].

Fig. 1.2. Photograph of Rhododendron arboreum flowers

1.7 Literature review of genus Rhododendron

1.7.1 Chemical constituents of the genus Rhododendron

The petroleum ether extract of the bark revealed the presence of triterpenoid taraxerol

(C30H50O) and ursolic acid acetate (C32H50O4), while the ether extract of the bark revealed

the identity of betulinic acid (C30H48O3). The acetone extract of the bark indicated the

leuco-pelargonidin (C15H14O6) [15]. The green foliage are described to consist of glucoside,

ericolin (arbutin) (C12H16O7), ursolic acid (C30H48O4), α-amyrin (C30H50O), epifriedelinol

(C30H52O), campanulin (C26H34ClN3O3S), quercetin (C15H10O7) and hyperoside


9

(C21H20O12) [16]. R. arboreum var. nilagiricum indicated the presence of hyperoside (3-D-

galactoside of quercetin) (C21H20O12), epifriedelinol (C30H52O), a triterpenoid (C30H48O7S)

and ursolic acid (C30H48O4) [17]. From the flower of this species, quercetin-3-rhamnoside

has been isolated [18]. Biologically active phenolic compounds i.e. coumaric acid

(C9H8O3), quercetin (C15H10O7) and rutin (C27H30O16) also have been isolated from the

flowers of R. arboreum [19].

1.7.2 Medicinal and pharmacological properties

Conventionally, dehydrated flowers deep-fried with vegetable oil are observed very much

effective in checking dysentery and squeeze for the cure of psychological disorders.

Flowers have the potential for cholinergic assay and anti-inflammatory. “Ashoka Aristha”

Ayurvedic preparation comprising R. arboreum has oestrogenic, oxytocic, and

prostaglandin synthetase inhibiting activities. The phytochemistry of dried foliage has been

cited in Homeopathic Materia Medica, to be beneficial in rheumatism and gout [20]. The

fresh corolla is acid sweet in taste and is given when fish bones stuck in the gullet [15]. The

flowers are also used for preparing native wine to avoid high altitude illness in the

Darjeeling hills of the eastern Himalayas. The fresh leaves are smeared on the forehead to

get rid of headache [21].

1.8 Phytochemistry and bioactivities of Eulophia dabia


Eulophia dabia belongs to family Orchidaceae. It is the leading family amongst the

monocotyledons comprising 600 to 800 genera. Orchids consist of epiphytic, saprophytic

and terrestrial forms. The Eulophia genus covers permanent terrestrial orchids with fleshy

tubers (Fig. 1.3).

The genus of Eulophia contains around 230 species and distributed worldwide in

tropical as well as temperate climate. In Pakistan it is present in district Swat and


10

Shangla, while in India, this plant is accessible in tropical Himalayas, from Assam

to Nepal and in Deccan from Konkan southwards [22].

Fig. 1.3. Photograph of Eulophia dabia

1.9 Literature review of genus Eulophia

1.9.1 Chemical constituents of the genus Eulophia

Some orchids have been reported to contain alkaloids, triterpenoids, flavonoids

and stilbenoids. Previous studies showed that lupeol and n-hexacosyl alcohol were

isolated from the rhizomes. Various class of bioactive compounds have been

reported from E. dabia plant like phenanthrene derivative; 9,10-dihydro-2,5-

dimethoxyphenanthrene-1,7-diol which exhibited significant on anti proliferative

assay against breast cancer. Furthermore, similar group magnificently isolated and

synthesized; 2,7-dihydroxy-3,4-dimethoxyphenanthrene or nudol [23].


The tubers are reported to be utilized to treat scrofulous glands of the neck,

bronchitis, tumors and blood ailments. In Thailand, it is employed in local

medications for the cure of skin rash. Fresh tubers are utilized for therapeutic

rheumatoid arthritis. In recent times, it is testified to be anthelmintic and


11

demulcent. Tubers also called as „Salep‟ is used as an aphrodisiac drug. Moreover,

E. dabia tubers are employed to cure piles, acidity and stomach illnesses [24].

1.10 Phytochemistry and bioactivities of Kigelia pinnata


Kigelia pinnata belongs to the family of Bignoniaceae and normally due its huge

fruits it is called the sausage tree (Fig. 1.4). This family is spread in 15 genera and

40 species, comprising K. Pinnata which occurs in Southern and Western India,

Pakistan and few species in the Himalayas. It is extensively grown in the tropics but

found lavishly in West Bengal as an ornamental tree. It is also found in south,

central and West Africa. It is a tree growing up to 20 m tall and has dark red florets

in long drooping panicles and gourd like fruits [25].

Fig. 1.4. Photograph of Kigelia pinnata

1.11 Literature review of genus Kigelia

1.11.1 Chemical constituents of the genus Kigelia

Norviburtinal, an iridoid product has been reported from the fruits, stem bark and roots.

A cytotoxicant, lapachol has been isolated from roots and wood of K. pinnata and

communal steroids, such as stigamsterol and β-sitosterol have also been extracted from


12

the root and bark of K. pinnata. γ -Sitosterol was stated to be existing in the pod of K.

pinnata. The flavonol quercitin and four flavonones i.e. luteolin, 6-OH luteolin,

luteolin-7-glucoside and 6-OH luteolin-7-glucoside were isolated from the foliage and

fruit of K. pinnata. Up till now, only a few phytochemical exertions on K. pinnata

flower were reported. Cyanidin glycoside, cyanidin-3-rutinoside and anthoxanthine

constituents were isolated from the flowers of K. pinnata [26].


K. pinnata, as a medicinal plant has a long history, utilized by African and several rural

countries. It is employed as an interesting use on abscesses and wounds, for the cure of

sexually transmitted diseases and skin sicknesses such as psoriasis, boils and acne.

Inside, the plant also utilize as medication for tape worm, ring worm, post partum

hemorrhage, dysentery, diabetes, pneumonia, toothaches and malaria. K. pinnata fruits

are applied as gauze for injuries and sores, hemorrhoids, for rheumatism as a cleansing,

to upsurge milk in lactating mothers and for skin firming possessions. Diverse portions

of the plant, as well as the fruits, are utilized either in a residue form or as ethanolic or

aqueous drinks, which are drunk or smeared to the pretentious body region. The

medicinal assets associated with K. pinnata are owing to the existence of various

subordinate metabolites, comprising iridoids, furonaphthoquinones, naphthoquinones,

meroterpenoids naphthoquinones, coumarin derivatives, lignans, sterols, flavonoids,

furanones, and volatile constituents [27].

1.12 Phytochemistry and bioactivities of Desmodium elegans


Desmodium elegans is the member of genus Desmodium and family Fabaceae with 650

genera and 18000 species, generally comprising of shrubs or herbs (Fig. 1.5). Flowers

are light purple and organized in terminal panicles from August to September. Pod is


13

sessile and spread via seeds from September to December. The Desmodium genus is

scattered in temperate and tropical zones of the biosphere excluding New Zealand and

Europe. The D. elegans plant is mainly distributed in Pakistan, Kashmir, India, Nepal

and Bhutan [28].

Fig. 1.5. Photograph of Desmodium elegans

1.13 Literature review of genus Desmodium

1.13.1 Chemical constituents of the genus Desmodium

Up till now, 40 alkaloids, 13 steroids, 14 terpenoids, 81 flavonoids, 2 glycosides, 8

phenylpropanoids, 10 phenols and numerous volatile compounds have been isolated

from Desmodium species. Phytochemical analysis revealed that alkaloids and

flavonoids are the main metabolites in this genus. Overall 40 alkaloids were extracted

from Desmodium species and identified mostly as amide, indole, pyrrolidine,

phenylethylamine and alkylamine alkaloids. The principal types of flavonoids present

in Desmodium plants were flavanonols, flavones, flavan-3-ols, flavonols, 7, 8-prenyl-

lactone and flavonoids, whereas isoflavonoids contain isoflavones, isoflavanones,

pterocarpans and coumaronochromone. In addition, alkaloids and flavonoids, a variety


14

of terpenoids, phenols, steroids, glycosides, phenylpropanoids and fixed oils have also

been described from the Desmodium species [29].


Usually, Desmodium plants have been employed to therapy numerous illnesses such

as constipation, jaundice, asthma, fever, paralysis, edema, cold, cough and convulsion

[30]. D. elegans has many applications in common medication and many portions of

the plant have been stated to be utilized for various motives, for instance roots were

used as tonic, diuretic and carminative and the pulverized foliage was smeared on

incisions for soothing wounds [31].

1.14 Biological evaluation

Preliminary bioactivity shows an important feat in the drug discovery project. It offers a

platform for bioassays and comforts in the assortment of leads like drugs for evaluation

of pharmacological assessments. Positively, novel or improved therapeutic agents retain

through the preliminary bioactivity with a suitable safety outline. Biological assays are

the best skill of finding valuable and precious substituents present in medicinal plants

and their nanomaterials. Various biological activities have been carried out for noble

metal nanoparticles capped with fluoroquinolones and selected medicinal plants to

explore the hidden potentials. These bioactivities included urease enzyme inhibition,

antimicrobial, antioxidant and leishmanicidal.

1.14.1 Urease inhibition activity

Metal ions exist in the active sites of metal containing proteins i.e. ureases enzyme and

hemocyanin, lactase, ascorbate oxidase and tyrosinase [32]. Urease is a Ni (nickel)

encompassing enzyme and known to speed up the hydrolysis of urea into NH3 and CO2

(urea amidohydrolase). It lets an entity to utilize urea as nitrogen source [33]. Besides,


15

Urease enzyme is one of the highest sources of pathogenesis persuaded by Helicobacter

pylori, consequently allow them to stick at low pH of the gastrointestinal. It acts

significantly in the pathogenesis of intestinal and peptic ulcers [34].

1.14.2 Leishmanicidal studies

Leishmaniasis is the most dreadful of parasitic sicknesses. Except Australia, it exist in

all regions [35]. It is typically zoonotic, but also occurs in an anthroponotic mode of

transmission in some parts of Asia and Europe. WHO reported that almost 350 million

people in the biosphere are at risk of acquiring leishmaniasis [36].

Symptomatologically, leishmaniasis exists in mucocutaneous (MCL), cutaneous (CL),

visceral (VL) and diffuse (DCL) forms [37], [38]. Leishmania tropica and L. major

grounds the cutaneous form of the sickness, which is a stigmatizing and spoiling disease

[39]. If remains untreated, it becomes visceral infection, which is very lethal.

Approximately 500,000 humans losses, have been caused by L. chagasi/infantum in

Latin America and Southern Europe and by L. donovani in Pakistan, India, Middle East

and Africa [40]. In most endemic countries, the systemic pentavalent antimonials still

remain the recommended drug for treatment, but these have disagreeable and severe

side effect i.e. renal, neural, and cardiac toxicity [41]. Handling of VL or CL with

antimonials is even more problematic in human immunodeficiency virus; (HIV)-

infected individuals and is allied with frequent deteriorations because these drugs

require healthy immune system for optimal anti-parasitic activity. Finally, the treatment

of leishmaniasis is still intricate, with a partial therapeutic arsenal, toxic drugs and

resistance cases [42].

1.14.3 Antioxidant assay

Antioxidants can defend cells from the destruction produced by unstable molecules

identified as free radicals. Antioxidants interact with these unstable molecules (free

radicals) and may inhibit the deterioration of cells. Some of the examples of

antioxidants consist of vitamins C, E, A, lycopene, β-carotene and so forth [43]. An


16

antioxidant is an entity capable of slowing or preventing the oxidation of further

substances. Oxidation is a process that transfers electrons from a molecule to an

oxidizing agent. It can originate unstable molecules (free radicals), which produce chain

reactions that deteriorate cells. Antioxidants discontinue these chain reactions by taking

away free radicals and stop further oxidation by being oxidized themselves.

Antioxidants may be reducing agents such as polyphenols, ascorbic acid or thiols. For

several years chemists have acknowledged that free radicals cause oxidation which can

be controlled or stopped by a variety of antioxidants [44].

1.14.4 Antibacterial assay

Universally, the fast spread of infective diseases is due to various causes like failure of

available drug therapies by microbial resistance, poor health care systems and

population growth. In 20th

century, the death rate due to infections has increased [45].

Major worldwide public health problem is due to antimicrobial resistance.

Around one half of all victims are due to infectious diseases in nations of the humid

region. In developed countries, serious infectious casualties due to antimicrobial

resistance also take place where there is best understanding about microbes and their

control [46].

1.14.5 Antifungal activity

The prevalence of infections triggered by fungi also amplified terrifically in the last two

decades and it is expected to continue in future [47]. The increase ratio of fungal

infection is due to the population growth of immune-suppressed persons [48]. Among

the fungi, Candida causes severe fatal fungal infections. It is found on the mucus

membrane. When there is unscrupulous infection in the buccal cavity of the individual,

Candida becomes pathogenic particularly in the subject with immune-deficiencies [49].


17

Failures of available antifungal treatments are due to the adverse drug reactions, fungal

resistance and toxicity. Severe hepatic disorders, gastrointestinal and endochrinologial

are caused by antifungal azoles [50]. The exposure frequency of medicine and

dimensions of fungal population are significant aspects which contribute towards the

fungal resistance. There is key influence by HIV patients in the field of fungal

resistance [51]. Therefore, it is required to search innovative drugs for infections caused

by the resistance strains and better health care.

Chapter-2 Introduction to nanotechnology

18

2.1 Nanotechnology

Nanotech may be defined as the technology manipulating matter with nano scale

extending from 1 to 100 nm in one dimension. Nanotechnology is a broad term

covering all fields of science, such as organic chemistry, molecular biology, surface

science and microfabrication. Nanomaterials reveal different properties based on their

nano size and shape [52].

2.2 Noble metal NPs

Noble metals containing Ag, Au, Pd and Pt have been used for the formation of NPs.

These nanomaterials are extensively used and motivated a lot of attention for

biomedical applications. Furthermore, their importance are steadily growing in the

field of nanotechnology, photochemistry, photographic chemistry, Raman

spectroscopy, physics and biological sciences. The NH moiety is mainly responsible

for the stabilizing of noble metal NPs [53] (Fig. 2.1).

Fig.2.1. Diagram for silver and gold nanomaterials


19

2.3 Synthesis

Synthesis of noble metal NPs has burst in the preceding eras. The best prevalent practices

are chemical reduction, physical techniques and biological processes. The physical

parameters of NPs comprise composition, shape and size. The capability to overcome of

these properties through slight modifications has led to a main effort in research finding

of NPs moreover, amplified the prospective for applications inside the area of catalysis,

microchip technology, diagnostics and therapeutics. The most generalized scheme for the

formation of Ag and Au NPs is chemical reduction method. In this technique the salts of

noble metal is reduced in the presence of a reductant [54, 55]. The initial renowned report

of the solution phase production of Ag and Au was in 1857, when Michael Faraday

reduced HAuCl4 with phosphorous in an aqueous solution [56]. Turkevitch et al., in 1951

developed the citrate reduction method. This synthesis of citrate capped Au nano scale

materials was founded on a single phase reduction of Au chloride by sodium citrate and

formed nanomaterials around 20 nm in size [57]. Frens suggested varying the ratio

between trisodium citrate and tetrachloroaurate and this technique is still hired. Following

this scheme of merely changing reaction parameters for instance, ratios [58], solution pH

[59] and solvent [60] has indorsed for controlling of the noble metal NPs sizes [61]. In the

last few decades, several groups have concentrated on fabricating monodispersed

nanomaterials by finding out possible NPs development tools in imperative to check the

size distribution. Natan et al., was a poineer for the exploration of seed development of

Au NPs employing alterations on the Frens synthesis [62]. Bastus et al., have effectively

prepared uniform citrate capped NPs via kinetically measured seed development [61]

(Fig. 2.2).


20

Fig. 2.2. Illustrative chemical reduction diagrams for noble metal NPs synthesis [61]

Metallic NPs may also be significantly prepared by using other protocols such as UV and

microwave irradiation. For example, well defined Ag NPs were synthesized from a laser

irradiation of AgNO3 and surfactant [63]. In this case, the surfactant actions as the

stabilizing agent, which additional adjust the shape and size of the nanomaterials.

Furthermore, this method is also using for benzophenone [64]. By fluctuating the laser

power and time, the size of the Ag NPs could be organized; at a low power, short

irradiation formed 20 nm nanomaterials, while 5 nm NPs were produced with a higher

ionizing power and longer irradiation times [64]. With the duration of photolysis, particle

dimension can also be controlled [65]. In this event, the substrate starts the reduction of

Ag+ to form Ag

0 upon excitation at 600 nm. Moreover, developing method utilizing more

intensity laser excitation controlled the development rate of the Ag seeds. Microwave

irradiation can also form uniform water soluble Ag NPs (26 nm) [66].


21

The fast nucleation due to microwave radiation is crucial to the even size dispersal of the

NPs. Suzuki suggested a novel method to formulate monodispersed Ag NPs reaching

from 10 to 80 nm [67]. This sophisticated technique uses a blend of laser treatments and

seeding.

Various reductants have been reported for instance, hydroxylamine [62, 68], ascorbic acid

[69, 70] and biogenic approaches which employ an iodide mediated reduction [71]. In the

light of above mentioned synthesis, several efforts have been forwarded to illuminate

biotic processes to synthesize NPs. Plant fabricated noble metal NPs production has

developed impetus due to eco friendliness and ease [72]. Synthesis with plant extracts as

well as iodide mediated reductions of Au salt has been reported. Zingiber officinale plant

can create NPs extending from 5-15 nm in diameter. The plant acts as a reductant as well

as a capping agent and the biological importance are confirmed through physiological

reliability [73]. For exploring the green synthesis, microbes has also developed as a

substitute to chemical reduction. Photosynthetic bacteria [74], prokaryotic bacteria [75-

77], eukaryotic fungus [78, 79] and medicinal plant extracts [80-83] all have been

examined for the reduction of metal ions to yield nanomaterials. Numerous biological

procedures have a slow reaction rate and a wide distribution in particle size [84]. Though,

a current publication by Darroudi investigated the key role of sodium hydroxide as an

accelerator to produce Ag NPs [85].

In summary, optimizing noble metal NPs formation is a productive zone of research.

Monitoring shape, size and distribution is a sophisticated and laborious process. These

reactions are governed by various parameters for example, reaction rate, reactant

concentration, reduction potential, solubility, heat and time. All of the variables are

basically tangled. More study is firm to be in current area for increased tenability.


22

2.4 Characterization techniques

The synthesized NPs are characterized with various advanced techniques to confirm their

morphology and size. These techniques are:

Electron microscopy including, Transmission Electron Microscope (TEM) and

Scanning Electron Microscope (SEM)

Atomic Force Microscopy (AFM)

Fourier Transform Infrared Spectroscopy (FTIR)

X-ray Photoelectron Spectroscopy (XPS)

Dynamic Light Scattering (DLS)

Aerosol Particle Mass Analyzer (APM)

Ultraviolet visible Spectroscopy (UV vis)

Nanoparticle Tracking Analysis (NTA)

Condensation Particle Counter (CPC)

X-ray Diffraction (XRD)

Differential Mobility Analyzer (DMA)

Nanoparticle Surface Area Monitor (NSAM)

2.5 Applications

Noble metal nanotechnology is a flourishing arena with massive prospective for real

world and clinical applications. To understand this potential, it is essential to engineer

and design Ag and Au NPs that can be targeted to tissues of interest, also to create

particular and anticipated results. Particularly NPs with a metallic core due to their

encouraging safety profile in human beings are used in the preclinical analysis for

diagnostic, therapy and imaging. Consequently, the sizes of the NPs employed in the

arena of bio nanotechnology ranges from 2 to 500 nm. The atomic and molecular


23

scales show really new things due to their advanced molecular design and small

configuration can be exactly produced with a high grade of flexibility. This couture is

mainly due to self assembly of the nanomaterials by charge compatibility and non

covalent interfaces. These NPs, have demonstrated to be the most resourceful and

commonly used constituents with wide applications like delivery vectors [86],

imaging [87], inhibitors [88], and sensors [89]. Therefore, these contrived nano-

conjugates assist as exclusive many dimensional frameworks that differ from their

mass complement [90].

The cellular uptake of inorganic NPs is a space of penetrating exploration. However,

Ag, Au and Pt NPs are noble metals; their mechanism of intracellular internalization is

not essentially similar or well understood. Geiser et al., reported blood cells to explain

intracellular uptake of Au NPs [91]. Their findings sustenance a diffusive mechanism of

entry since, Au NPs were originated in the cytosol free from membrane encapsulation.

Comparatively, it has been revealed that uptake of cellular in Au NPs is due to

micropinocytosis [92, 93] which was deep-rooted by other researchers. Similarly,

macrophages simply take on Ag NPs, which were confine to vacuoles [94]. In a

comparable investigation by Yen et al., indicated that Ag/Au NPs were restricted in

cytoplasmic vesicles of the macrophages [95]. On the other hand, the researchers more

ventured that the protein corona development prejudiced cellular uptake of Au NPs as

compared to Ag NPs, hence confusing the internalization process [95, 96].

The role of Ag NPs as bactericidal agents have been well recognized. Antiviral

possessions of Ag NPs biogenically designed are more energetic than chemically

formed Ag NPs [97]. Metallic NPs have also been designated as a possible HIV

defensive tonic [98, 99]. In different investigations, it is confirmed that Ag NPs

prohibited the virus from binding to the host cells in vitro [100, 101]. Furthermore, It was


24

indicated that Ag acts straight on the virus as an antiviral agent by binding to the

glycoprotein [102] and binding in turn stops the CD4 dependent virion binding which

successfully declines HIV-1‟s infection [103]. These noble metal NPs also have been

active virucidal against herpes simplex virus [104], flu [105], and lung syncytial virus

[106].

Angiogenesis plays an important role in numerous ailments like rheumatoid arthritis,

cancer and macular degeneration [107-109]. Normally, angiogenesis is firmly controlled

between various anti-angiogenic and pro-angiogenic growth factors [110]. But the

angiogenic switch is turned on when the balance is disturbed under pathological

conditions. This incident encourages extremely abnormal blood vessels which becomes

hyper permeable to plasma proteins. Several anti-angiogenic mediators are being

currently used in the hospitals, but most of them have been premeditated to only prevent

[110] mediated signaling [111]. Moreover, further reports have shown acute

poisonousness of these conservative materials containing thrombosis, fatal hemorrhage

and hypertension [107, 108].

Some other common therapy for cancer patients is the utilization of radiation. Even

though this technique is operative for preventing the propagation rate of malignant

neoplastic disease cells, it can be aggressive and side effects are legion and normal tissue

is much injured. Noble metal NPs may provide a benefit in this arena by manipulating

their brilliant optical properties, wave length tunability and surface resonance. For

instance, upon X-ray treatment, Au NPs can create cellular apoptosis through the

generation of unstable molecules [112]. This cure method has amplified the killing of

tumor cells without hurting the neighboring normal tissue [113]. X-ray radiation of mice

inoculated with Au NPs at 250 kV produced a fourfold reduction in tumor and improved

endurance of the animals. Similarly, the inherent radioactive properties of Au-198 and

Au-199 NPs marks them the best candidates for radiotherapy [114].


25

2.6 Properties

Metallic NPs show versatile properties which can be employed in various fields of

nanoscience, analytical chemistry, optical activity catalysis, medicines and

electronics. Various aspects are described as follow.

2.6.1 Optical properties

Optical properties of NPs have been widely studied during the last century and metal-

dielectric nanocomposites have found numerous applications in diverse fields of

science and technology [115-118]. These properties of metallic NPs are administered

by surface plasmon resonance (SPR). When a nanomaterial is illuminated by light, the

plasmonic resonance governs the color of colloidal nanoparticle‟s solution. NPs are

quite small to restrain their electrons and yield quantum effects [119]. For instance,

Au NPs seem deep red to black in solution (Fig. 2.3).

Fig. 2.3. Optical inspection of the colloidal solution of noble metal NPs

2.6.2 Biomedical properties

Antimicrobial coated Ag/Au NPs may be applied directly to the wound. The NPs

capped with antibiotics can increase their activity against microorganism suggesting a

new type of drugs and drug delivery to affected areas like cancer and tumor cells.


26

2.6.3 Magnetic properties

Magnetic NPs can be prepared; their magnetic behaviors may be used in diverse

fields, for instance, they may amend the contrast and detail of MRI images. To show

how magnetic NPs morphology and the resultant features are entangled, we can

employ a particular presentation to key out the factors that refrain vital magnetic

features. In bio sensing, for instance, NPs with greater capacity of magnetization are

preferred because they offer higher efficiency and sensitivity [120].

2.6.4 Electronic properties

Nano scale materials are much appropriate for the manufacturing of extraordinary

performance delicate metallic NPs, which are not only distribute ingredients with high

conductivity, but slicker parts for electronic engineering such as laptops and cell

phones etc. Nano electronics may be used to design digital displays which are brighter

in color and cheap [121].

2.6.5 Energy properties

Metallic NPs can be used in energy batteries with long long-term and have more

energy density. They have affinity for storing of efficient fuel cell and hydrogen gas

might be manufactured by expending the property of electro catalytic agent for such

strategies. They make the engine more efficient and economic when metal NPs are

employed as catalysts, [122].

2.7 Aims and objectives of the present research

The main aims and objectives of the present research activity are to:

synthesize objectively Ag and Au NPs of commercially available

fluoroquinolones (FQs) belonging to 2nd

, 3rd

and 4th

generations.


27

develop and employ green phytosynthetic process of preparing noble metal

NPs mediated by selected medicinal plants (R. arboreum, K. pinnata, E. dabia

and D. elegans) as efficient reductants and stabilizers

provide access to eco-friendly synthetic protocol that can ensure high atom

economy and reasonable selectivity for producing NPs of relatively uniform

sizes and shapes.

isolate Ag and Au NPs derived from commercial fluoroquinolones and

designate plant extracts through selective mode of capping approach.

develop nanomaterial based drugs

evaluate locally synthesized pro-nanomedicines for biological activities

namely antimicrobial, enzyme inhibition, leishmanicidal and antioxidant.

2.8 Reviews on topical advancements in noble metal NPs

2.8.1 Review on Ag NPs

Ghosh et al., prepared Ag NPs by a single pot synthesis method of the antimicrobial

drug; ciprofloxacin. Numerous procedures were employed to illustrate the NPs bound

and free states of ciprofloxacin. In drug delivery, the time dependent release of the

prescription molecules from the NPs displays its importance [123].

Rodríguez León et al., described the formation of Ag NPs from AgNO3 solutions of

Rumex hymenosepalus extracts, which is rich in antioxidant molecules and used as

reductant. The nanomaterials were characterized by UV visible spectroscopy and

transmission electron microscopy, as a function of the ratio of Ag ions to reducing agent

molecules. The NPs were in the size of 2 to 40 nm. The HRTEM and FTIR analysis

revealed hexagonal and face-centered cubic crystal configurations were obtained [124].

Abboud et al., reported the application of Allium cepa in the preparation of Ag NPs

under microwave irradiation. The effect of several reaction factors such as microwave


28

irradiation time and microwave irradiation power was analyzed. Furthermore, these

nanomaterials were exhibited potent antibacterial activity against two dissimilar strains

of bacteria Escherichia coli and Staphylococcus aureus [125].

Gopinath et al., synthesized Ag NPs by facile, reliable and green synthetic route using

the Pterocarpus santalinus aqueous extract, which comprises glycosides, glycerides,

steroids, phenols, saponins, triterpenoids, tannins and flavonoids to be accountable for

bio-reduction during the formation of sphere-shaped Ag NPs. FTIR study was

conducted to investigate the molecules involved in the synthesis of Ag NPs and

inveterate the Ag NPs by XRD. The synthesized Ag NPs presented significant

antibacterial assay against Gram positive and Gram negative bacterial strains [126].

Lavanya et al., described the formation of Ag NPs of Paederia foetida L. leaf extract

and showed significant antibacterial activity against various bacterial species [127].

Thirunavoukkarasu et al., employed the formation of Ag NPs by leaf extract of

Desmodium gangeticum. Robust formation of stable NPs was perceived on disclosure

of the aqueous leaf extract with solution of AgNO3. UV visible peak of the aqueous

medium at 450 nm conforming to the plasmon absorbance of Ag NPs. These

biosynthesized NPs were considered to be very toxic against pathogenic bacteria E.

coli, therefore, suggesting implication of the current study in fabrication of

biomedical products [128].

Kumar et al., fabricated Ag NPs using leaf extract of Sacha inchi (SI) as a nontoxic

reductant with particle size from 4 to 25 nm. Infrared measurement was conceded to

assume the possible phytochemicals accountable for capping of Ag NPs [129].

Olenin et al., described the development of Janus nanoparticles in the chemical

modification of the surface of Ag colloids. This is attained via sorption of

nanomaterials on the surface of chemically modified silica grafted with amine [130].


29

Elias et al., reported the base of in situ assortment using a reaction kinetics factor, a

novel and straight forward technique for evaluating the synthesis of catalytically

dynamic Ag-PVP NPs. The catalytic properties with respect to the reduction of five

nitro aromatic compounds having various substituents at the para position were

determined [131].

Singh et al., studied the bactericidal activity of Ag NPs produced from Tinospora

cordifolia stem were analyzed against multidrug resistant strains of Pseudomonas

aeruginosa extracted from burn patients. Ag NPs possessed significant antibacterial

assay, which makes them an intoxicating source of bactericidal agent [132].

Sun et al., developed simple, environmental friendly and cost-effective process to

produce Ag NPs of tea leaf extract. They have also studied the effects of the tea extract

dosage, reaction time and reaction temperature on the formation of Ag NPs. These NPs

showed low antibacterial activity against E. coli [133].

Elsupikhe et al., reported a green sono-chemical process for preparing Ag NPs in

various concentrations of Kappa carrageenan. Ultrasonic irradiation was operated as a

green reducing agent and the Kappa carrageenan was employed as a natural

ecofriendly stabilizer [134].

Padalia et al., synthesized Ag NPs using of Tagetes erecta flower broth as reducing

agent by ecofriendly and a simple route. The Ag ions when uncovered to flower broth,

were reduced and give rise to green synthesis of Ag NPs. The antimicrobial

evaluation of Ag NPs with antibiotics was enhanced than antibiotics alone against

Gram negative bacteria and the tested fungal strains [135].

Ibrahim reported an ecofriendly, cost efficient, rapid and easy protocol for the

production of Ag NPs with banana peel extract (BPE) as a reductant and capping


30

agent. The different factor affecting Ag reduction was investigated. The Ag NPs

showed effective antibacterial activity against representative pathogens of bacteria

and yeast [136].

Yakout and Mostafa established the preparation of Ag NPs by green method. It was

perceived that use of starch makes expedient technique for the formation of Ag NPs.

The synthesized green Ag NPs revealed a potential antibacterial activity [137].

Ahmed and Ikram reported a simple, cost effective bio-reduction on the principle of

“green biosynthesis” of Ag NPs by Terminalia arjuna extract. Biological evaluation

of Ag NPs were also done against Gram negative (E. coli) and Gram positive (S.

aureus) bacteria for their future applications in nanomedicines especially for the

treatment of wounds [138].

Verma et al., studied an ecofriendly, economic, and simple photo catalytic green

method for the facile synthesis of Ag NPs. An aqueous leaf extract of marine fern,

Salvinia molesta, was employed as a bio-reductant and stabilizing agent. The

synthesized Ag NPs were originated to be an active bactericidal mediator against

Gram negative and Gram positive bacteria [139].

Kumar et al., established low cost and an ecofriendly approach for the green creation

of spherical and stable Ag NPs of Erigeron bonariensis extract, which act as reducing

and capping agent. The Ag NPs revealed catalytic activity towards degradation of

Acridine orange without of involvement of any harmful reductant [140].

Govindarajan et al., investigated green synthesis of Ag NPs employing an economy,

Anisomeles indica leaf extract by reduction of Ag ions from AgNO3 solution. The

acute toxicity of synthesized Ag NPs and leaf extract of A. indica was appraised


31

against larvae of the malaria vector Anopheles subpictus, the dengue vector Aedes

albopictus and the Japanese encephalitis vector Culex tritaeniorhynchus. Both Ag NPs

and the A. indica leaf extract exhibited dose dependent larvicidal effect against all tested

mosquito species [141].

Balakumaran et al., reported the synthesis of both Ag and Au NPs using soil fungi. The

Aspergillus terreus has produced extremely stable NPs. The myco derived Ag NPs

presented greater antimicrobial assay as compare to the standard antibiotic,

streptomycin [142].

Saha et al., synthesized the Ag NPs by distinctive grouping of tyrosine and a natural

polymer (starch) using ultrasound assisted green practice. A comprehensive mechanistic

study on the reactive oxygen species (ROS) mediated filaricidal (against Setaria cervi)

and mosquitocidal (against second and fourth instar larvae of Culex quin-quefasciatus)

activities of Ag NPs has been made for the first time [143].

2.8.2 Review on Au NPs

Naveena and Prakash synthesized Au NPs of red marine algae, Gracilaria corticata

aqueous extract as a reductant. The synthesized NPs were assessed against bacterial

pathogens Gram negative Escherichia coli, Enterobacter aerogenes and Gram positive

Staphylococcus aureus, Enterococcus faecalis and also considered for its antioxidant

assay by DPPH free radical scavenging activity. The antibacterial and antioxidant

activities of the Au NPs exhibited significant assays in contrast with the standards [144].

Li et al., has employed carboxylato pillar arene, a new water soluble macrocyclic

synthetic receptor, as a stabilizing ligand for in situ synthesis of Au NPs.

Supramolecular self-assembly of carboxylato pillar arene-modified Au NPs arbitrated


32

by appropriate guest molecules was also examined, signifying that the novel hybrid

entity is convenient for recognition and sensing of the herbicide [145].

Rajeshkumar et al., reported the synthesis of Au NPs by using marine brown algae

Turbinaria conoides. Finally the bactericidal assay of Au NPs was conducted; it

revealed streptococcus having the maximum inhibition and medium range of inhibition

was examined against bacillus subtilis and klebsiella pneumoniae [146].

Joh et al., studied in cell culture tests and in an animal model of glioblastoma multiforme

(GBM) in which radiation therapy (RT) is supplemented by PEGylated-Au NPs. Au NPs

considerably enlarged cellular DNA destruction caused by ionizing radiation in human

GBM-derived cell lines and occasioned in reduced clonogenic survival. Follow up in

vitro experiments deep-rooted that the arrangement of Au NPs and RT resulted in

significantly increased DNA mutilation in brain derived endothelial cells [147].

Orza et al., described that temozolomide loaded Au nanomaterials were effective in

reducing chemo-resistance and abolish 82.7% of cancer stem cells compared with a

42% damage rate of temozolomide alone [148].

Zare et al., utilized amino acids as a reductant and stabilizing Au NPs. The Au NPs

were synthesized with a reduction solution comprising Au ions with Au chloride and

functionalized by phenylalanine, glutamic acid and tryptophan. The therapeutic uses of

proteins and amino acids may be utilized as a useful process owing to the strong

interface of peripheral amine groups with NPs [149].

Wang et al., stated the facile synthesis of Au NPs-alginate composite spheres. The

fabricated spheres are potential for numerous applications, such as bactericide, drug

carriers and micro sensors [150].


33

Pooja et al., revealed the importance of Xanthan gum (XG) in the formation of Au NPs.

XG was used as reductant and capping agent. The improved NPs were also explored as

drug delivery carrier for doxorubicin hydrochloride [151].

Goldstein et al., reported that the charging artifacts associated to non conductive biotic

sample can be magnificently eradicated by employing the uncoated biological specimen

on a conductive substrate. By developing the cells on glass pre coated with a chromium

layer, they observed the uptake of 10 nm Au NPs inside uncoated and unstained

keratinocytes cells and macrophages [152].

Kim et al., described the light-induced synthesis of numerous Au NPs and demonstrated

their considerable use as effective photo thermal heating constituents and practical

heterogeneous catalysts under the treatment of a solar based light after being loaded

onto a paper based substrate [153].

Dharmatti et al., synthesized biogenic Au nano triangles (GNTs) of Azadirachta indica

leaf extract and its application in competent drug delivery of doxorubicin (DOX)

(anticancer drug) [154].

Schroder et al., reported the application of a new catalyst for cycloisomerizations. The

new catalyst scheme comprises Au NPs reinforced on Aluminum-Santa Barbara

amorphous no.15 (Al-SBA15). The catalyst is very selective and ecofriendly.

Cycloisomerization reaction yields excellent important to the synthesis of two new

classes of six and seven membered heterocycles [155].

Ding et al., synthesized Au core-induced poly pyrrole nano hybrids (Au-PPyNHs)

through in situ oxidation polymerization of pyrrole molecules and was immobilized

onto glassy carbon electrode and applied to construct dopamine (DA) sensor. They


34

found that the fabricated sensor with Au-PPyNH-Au nano hybrids is highly specific

probe or sensing DA [156].

Li et al., successfully synthesized Au NPs-decorated poly (o-phenylenediamine)

(PoPD@Au) hollow microspheres. Furthermore, PoPD@Au hollow microspheres were

immobilized onto the surface of a glassy carbon electrode and applied to construct a

sensor [157].

Ding et al., reported the enhancement of innovative protein Au hybrid nanocubes

(PGHNs), which were accumulated by Au nano-clusters, tryptophan and bovine serum

albumin as building blocks. The PGHNs can assist as a novel kind of dual purpose

gadget; a nano-carrier in drug delivery studies and a blue emitting cell marker in bio

imaging exploration [158].

Kidonakis and Stratakis reported the Au-catalyzed hydrosilylation of allenes using

recyclable Au NPs as catalyst. The hydrosilane addition takes place on the more

substituted double bond of terminal allenes in a highly regioselective manner. The

observed regioselectivity/reactivity modes are attributed to steric and electronic factors

[159].

Sathishkumar et al., synthesized Au NPs using the aqueous fruit extract of Couroupita

guianensis Aubl. (CGFE) as a potential bio-reductant. DLS and EDX findings were

confirmed that the produced CG-Au NPs were stable having negative electric charge

without accumulation [160].

Dhamecha et al., summarized a distinctive green process for the preparation of Au

NPs by modest treatment of Au salts with Nepenthes khasiana extract. Fast synthesis


35

of biocompatible Au NPs possessed exclusive physical and chemical characteristics

which function as a benefit for its purpose in numerous biomedical uses [161].

Paul et al., reported a green method for the formation of Ag/Au nanomaterials

employing dried biomass of Parkia roxburghii leaf. The antibacterial assay of the

created NPs was studied on Gram negative bacteria Escherichia coli and Gram

positive bacteria Staphylococcus aureus and both Ag/Au NPs exhibited slightly

higher efficacy on S. aureus than on E. coli [162].

Yan et al., reported a simplistic, green and single pot synthesis of biomolecule capped

Au NPs with higher catalytic potential. Cellulose nanocrystal (CNC)-capped Au NPs

were arranged by warming the aqueous mixture of Au chloride. The resulting CNC-

supported Au NPs displayed catalytic activities for the reduction of 4-nitrophenol by

NaBH4 [163].

Karthik et al., described the green synthesis of Au NPs by leaves extract of Cerasus

serrulata. The prepared Au NPs revealed greater bactericidal assay against Gram

negative (Escherichia coli) than Gram positive (Staphylococcus aureus) bacteria

[164].

Chapter-3 Results and Discussion

36

3.1 Results and Discussion

3.1.1 Synthesis of Ag-Mox and Au-Mox NPs capped with moxifloxacin

Moxifloxacin was employed as a capping agent for the preparation of Ag and Au

NPs. The main objective behind the selection of this drug is reflected due to the

presence of an amino moiety possessing strong ligating potential in the framework.

This functionality may be exploited to inhibit agglomeration hence, stabilize Ag/Au

metals during NPs (Scheme 3.1) formation.

Scheme 3.1. Capping action of moxifloxacin with noble metals (Ag and Au)

Moxifloxacin hydrochloride was first neutralized with equimolar Na2CO3, to

overcome the problem of precipitation of AgCl in the reaction mixture. Ag-Mox and

Au-Mox NPs were produced utilizing NaBH4 as a moderate reductant. The reaction

was stirred vigorously for about 30 minutes at ambient temperature and then 0.2 mL

of 50 mM NaBH4 was added drop wise. After the addition of a reducing agent, the

light yellow solution gradually turned maroon followed by brown and eventually ruby

red, as demonstrated in the digital photographs (Fig. 3.1 and 3.2). The mixture was

stirred robustly for another 30 min.


37

Anticipated variation in UV visible absorption bands was considered as the initial clue

of Ag/Au NPs. The presence of specific peaks in the areas of 400-500 nm and 500-

600 nm ensured the development of Ag and Au NPs correspondingly. The highest

peak for Ag-Mox NPs was recorded for a reaction with the 8:1 (metal: ligand) molar

ratio, while for Au-Mox NPs, the sharp peak was noted at the 1:6 (metal: ligand)

molar ratio as shown in fig. 3.3 and 3.4 respectively. Ag-Mox NPs revealed an

absorption plasmon band in the region of 410 nm and Au-Mox NPs exhibited an

absorption peak at 540 nm, which indicated the formation of Ag and Au NPs.

Fig. 3.1. Optical inspection of Ag-Mox NPs

Fig. 3.2. Optical inspection of Au-Mox NPs


38

300 400 500 600 700

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Ag

Mox

Ag-Mox NPs (8:1)

Ab

sorb

ance

Wavelength (nm)

Fig. 3.3. Optimized UV visible spectra of Ag-Mox NPs

300 400 500 600 700

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Ab

sorb

ance

Wavelength (nm)

Au

Mox

Au-Mox NPs (1:6)

Fig. 3.4. Optimized UV visible spectra of Au-Mox NPs

3.1.2 Characterization of moxifloxacin-capped Ag/Au NPs

The synthesized noble metal NPs (Ag-Mox and Au-Mox NPs) were characterized

through UV visible, AFM, FTIR, SEM and EDX practices. Absorption maxima of

metal NPs were noted as a function of retention time in the range of 300-700 nm

using UV visible spectroscopy. For FTIR measurements, the freeze dried samples

(0.01 g Ag-Mox and Au-Mox NPs) were ground with KBr and transformed into

uniform pellets suitable for FTIR analysis. For AFM analysis, the Ag/Au NPs samples


39

were prepared by dissolving thin films in deionized water and dispersing on a freshly

cleaved sheet of mica. The AFM images were recorded at ambient temperature

followed by repeating the experiment with various concentrations of the samples. The

surface and size of Ag and Au NPs were analyzed by SEM.

In order to ascertain the presence of various functionalities available in the drug

substrate before and after stabilizing of the Ag/Au NPs, the FTIR spectral data were

recorded and interpreted accordingly. For instance, as regards to the substrate drug

(moxifloxacin), the absorption bands of stretching frequencies for aromatic C-H,

secondary N-H, C=O (keto group), O-H besides bending of O-H in the case of COOH

were observed at 2949, 3354, 1708, 2926 and 1457 cm-1

respectively. However, as

anticipated, the N-H stretching band at 3354 cm-1

shifted to 3446 cm-1

and slightly

extended in the case of Ag-Mox NPs and may be attributed to involvement in

conjugate formation. Similarly, as shown in fig. 3.5, the absorption band at 1323 cm-1

is due to C-N stretching and the carbonyl peak of the carboxylic group was seen to

shift from 1708 to 1600 cm-1

in the Ag-Mox conjugate. It was observed that really the

N-H moiety was involved in the capping and stabilizing of metal NPs.

4000 3500 3000 2500 2000 1500 1000 500

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

Tra

ns

mit

tan

ce

(%

)

Wavenumber (cm-1

)

MOX

Ag NPs

Au NPs

Mox

Au NPs

Ag NPs

3354

3446

1708

1600

3446

Fig. 3.5. FTIR spectra of Mox, Ag and Au NPs


40

3.1.3 AFM, SEM and EDX spectroscopy analyses

Structural features of the synthesized Ag and Au NPs were conducted by AFM

analysis (Fig. 3.6 and 3.7). The micrographs visibly show that the Ag NPs have a

sphere-shaped and have the average sizes in the range of 50-60 nm, while the Au NPs

possess somewhat round shape in the range of 50-80 nm. For confirmation of the size

and surface morphology of Ag/Au NPs, the SEM technique was also performed. The

SEM image showed spherical Ag-Mox NPs (50-60 nm) with uniform distribution

similarly for Au-Mox NPs the SEM image results were comparable and viewed the

consequences of AFM that the shape of Au NPs was found slightly spherical in the

range of 50-80 nm as shown in fig. 3.8 and 3.9. Energy-dispersive X-ray spectroscopy

(EDX) (Fig. 3.10 and 3.11) demonstrated the elemental nature of the created Ag/Au

NPs. The EDX analysis displays a sharp signal in the Ag region and endorses the

synthesis of Ag nanomaterials. Metallic Ag nano-crystals normally show an

absorption peak about at 3 keV due to surface plasmon resonance. The peak was

found at the energy of 3 keV for Ag, and some of the weak peaks for Ca, Mg, C, O,

Cl, N and Na were found. For Au NPs, the EDX spectrum also reveals the presence of

peaks characteristic of gold at 2.12 and 9.71 keV and few weak signals for Cl, Na, O,

Mg C and Ca were recorded.

Fig. 3.6. AFM images of Ag NPs


41

Fig. 3.7. AFM images of Au NPs

Fig.3.8. SEM image of Ag-Mox Fig.3.9. SEM image of Au-Mox

Fig. 3.10. The EDX spectrum for Ag NPs Fig. 3.11. The EDX spectrum for Au NPs

3.1.4 Stability check of Ag and Au NPs

The effect of high concentration of brine solution (1 M) on moxifloxacin-capped Au

and Au NPs was also studied. For this purpose, 3 mL of freshly prepared Ag-Mox and

Au-Mox NPs were taken in five separate vials. Then 0.2, 0.4, 0.6, 0.8 and 1mL of 1 M


42

NaCl solution were added to these vials. The resulting solutions were shaken well and

then kept at room temperature for 24 h. UV visible spectra were recorded for Ag-Mox

and Au-Mox NPs. The results showed that higher concentration of brine decreased the

λ max. This rapid decrease in absorbance of Ag/Au NPs containing NaCl may be

attributed to the aggregation effect promoted by Cl-1

ions. From these clarifications it

was concluded that at a higher concentration of sodium chloride, however,

aggregation turned out to be dominant. As for long term stability, Ag-Mox NPs and

Au-Mox NPs are much more stable in neat water than those in brine solution as

shown in fig. 3.12 and 3.13 respectively. In addition, the stability of synthesized Ag-

Mox and Au-Mox NPs against pH variations ranging from 2 to 13 was also examined.

Freshly prepared nano-conjugates of Ag-Mox and Au-Mox (3 mL) were taken in six

separate vials. The pH of Ag-Mox and Au-Mox NPs was measured and found to be

4.7. The pH of Ag and Au NPs in the range 6-13 was monitored by employing 1 M

NaOH solution. Also, the pH of Ag-Mox and Au-Mox NPs ranging from 2 to 3 was

adjusted with 1 M HCl. The UV visible spectra of the resultant solutions were noted

after 24 h. Ag NPs were stable in the pH range of 4-7 and completely unstable in

highly acidic medium at pH 2-3 and basic medium 8-13 (Fig. 3.14), while the Au NPs

showed stability in basic medium (pH = 8-9) and less stable at pH 12-13 (Fig. 3.15).

The synthesized Ag/Au NPs were also found to be stable up to 60 oC.

300 400 500 600 700

0.0

0.3

0.6

0.9

1.2

1.5

1.8

2.1

2.4

Ab

so

rba

nc

e

Wavelength (nm)

AgNPs

0.2mL Brine

0.4mL Brine

0.6mL Brine

0.8mL Brine

1 mL Brine

300 400 500 600 700

0.0

0.5

1.0

1.5

2.0

2.5

Ab

so

rba

nc

e

Wavelength (nm)

AuNPs

0.2 mL Brine

0.4 mL Brine

0.6 mL Brine

0.8 mL Brine

1 mL Brine

Fig. 3.12. Effect of brine on Ag-Mox Fig. 3.13. Effect of brine on Au-Mox


43

300 400 500 600 700

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Ab

so

rba

nc

e

Wavelenth (nm)

pH=2-3

pH=4-5

pH=6-7

pH=8-9

pH=10-11

pH=12-13

300 400 500 600 700

0.0

0.3

0.6

0.9

1.2

1.5

1.8

2.1

2.4

Ab

so

rba

nc

e

Wavelength (nm)

pH=2-3

pH=4-5

pH=6-7

pH=8-9

pH=10-11

pH=12-13

Fig. 3.14. Effect of pH on stability of Ag

NPs

Fig. 3.15. Effect of pH on stability of Au

NPs

3.2 Synthesis of ciprofloxacin-capped metallic NPs

The present study proves the entitled drug as a stabilizing mediator for the preparation

of noble metal (Ag/Au) NPs. The consequence of pH, temperature and salt (1M NaCl)

were considered. This fluoroquinolone drug was coined due to the NH moiety in its

structure which has the competence to cap Ag/Au NPs (Scheme 3.2). The optical

properties of Ag/Au-cip were recorded by UV visible spectroscopy, most commonly

used technique, to determine the preparation and constancy of metal nanomaterials.

To find out the optimized conditions, reactions with various ligand and metal ratios

were stirred; keeping the ligand (ciprofloxacin), as constant and changing the amount

of metal (Ag/Au), i.e. (5:1, 10:1, 15:1, 20:1) and vice versa i.e. (1:2, 1:4, 1:6, 1:8,

1:9). The best optimized absorption peak for Ag-Cip NPs was observed for a reaction

of 10:1 (metal: ligand), while for Au-Cip NPs, the sharp surface plasmon band was

seemed at 1:4 (metal: ligand) as shown in the fig. 3.16 and 3.17 respectively.


44

300 400 500 600 700

0

1

2

3

4

Ab

sorb

an

ce

Wavelength (nm)

Ag(1 mM)

Cip(1 mM)

Ag-Cip(10:1)

Ag-Cip(15:1)

Ag-Cip(20:1)

300 400 500 600 700

-0.5

0.0

0.5

1.0

1.5

2.0

Ab

so

rba

nc

e

Wavelength (nm)

Au(1 mM)

Cip(1 mM)

Au-Cip(1:1)

Au-Cip(1:4)

Au-Cip(1:6)

Au-Cip(1:8)

Au-Cip(1:9)

Au-Cip(5:1)

Au-Cip(10:1)

Fig. 3.17. Optimized UV visible spectra of Au-Cip NPs (Inset: Au-Cip NPs color)

Fig. 3.16. Optimized UV visible spectra of Ag NPs (Inset: Optical inspection of Ag NPs)


45

Scheme 3.2. Capping action of ciprofloxacin with noble metals (Ag/Au)

3.2.1 FTIR and AFM studies

Spectral data were attained from FTIR study for Ag-Cip, Au-Cip NPs and

ciprofloxacin. The C-H, N-H, C=O and OH stretching were perceived as well as OH

twisting of COOH were apparent at 2918, 3372, 1708, 2706 and 1448 cm-1

correspondingly. Furthermore, the band at 3528 cm-1

displays O-H stretch of

carboxylic group in ciprofloxacin. The band at 3372 cm-1

is moved to 3448 cm-1

and


46

broadened for Ag-Cip NPs, while in case of Au-Cip NPs the signal at 3372 cm-1

shifted to 3425 cm-1

.This peak at 3372 cm-1

is due to N-H stretching of the piperazine

group that is considered to be responsible for stabilizing of Ag/Au-Cip NPs. In case of

Ag NPs the FTIR spectral bands designated a carbonyl peak shift from position of

1708 cm-1

to 1628 cm-1

and 1388 cm-1

, whereas, Au NPs indicated a shift from 1708

cm-1

to 1626 cm-1

and 1384 cm-1

(Fig. 3.18 and 3.19).

4000 3500 3000 2500 2000 1500 1000 500

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

Tra

ns

mit

tan

ce

%

Wavenumber (cm-1)

Cip

Ag-Cip NPs

3528

3448

3372

1708

1628

Fig. 3.18. FTIR spectra of Ag-Cip NPs

4000 3500 3000 2500 2000 1500 1000 500

-0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

Tra

ns

mit

tan

ce

(%

)

Wavenumber (cm-1)

Cip

Au-Cip NPs

1626

1708

3372

3425

Fig. 3.19. FTIR spectra of Au-Cip NPs


47

The structural topographies of the ciprofloxacin-stabilized Ag and Au NPs was achieved

by AFM (Fig. 3.20 and 3.21) and specifies that the nano-conjugates of Ag-Cip NPs are

sphere-shaped with size in the range of 40-50 nm, while the Au-Cip NPs also have round

shape with sizes from 60-85 nm (Fig. 3.21).



3.2.2 EDX and SEM studies

Energy-dispersive X-ray spectroscopy (EDX) verified the chemical make-up of metal

NPs by ciprofloxacin as capping and stabilizing agent. The presence of signal in region of

Ag in EDX study verifies the production of Ag NPs. Metallic Ag typically shows a visual

absorption peak at closely 3 keV due to surface plasmon resonance (SPR). Au-Cip NPs

designate the presence of typical peaks of Au at 2.12 and 9.71 keV as shown in fig. 3.22

and 3.23.


48

Fig. 3.22. EDX spectrum of Ag-Cip NPs Fig. 3.23. EDX spectrum of Au-Cip NPs

Scanning electron microscope (SEM) analysis confirmed the metal NPs sizes. The

spherical shapes of the Ag/Au nanomaterials are shown in the SEM images (Fig. 3.24

and 3.25).

Fig. 3.24. SEM image of Ag-Cip NPs Fig. 3.25. SEM image of Au-Cip NPs


Fig. 3.26 and 3.27 demonstrate the salt effect upon SPR peak of Ag and Au NPs.

More concentration of NaCl declines the absorbance maxima. The decrease in

absorbance of Ag/Au NPs having NaCl is credited to the aggregation endorsed by Cl-

1, which reveals that accumulation leads at higher concentrations of brine. The

constancy of Ag and Au NPs was greater in water as compared to brine solution.


49

Ag-Cip pH was 5.49, while Au-Cip pH was 9.56. The pH of Ag/Au-Cip NPs (7-12)

was attuned by 1 M NaOH solution and the pH of these metallic NPs (1-7) was

attained by using 1 M HCl. The UV visible results were recorded after 24 h (Fig. 3.28

and 3.29).

Temperature is one of the imperative ecological aspects that influence chemical

characteristics and stability of Ag/Au NPs. Effect of temperature was also examined

for these metallic NPs with the help of UV visible spectroscopy. Ag-Cip NPs were

stable up to larger variation in temperature as shown in fig. 3.30. Surface plasmon

resonance (SPR) bands up to 100 oC showed a slight decrease in intensity with a blue

shift from original band, which is attributed to the degradation of NPs with decrease

in particle size. Similarly, Au-Cip NPs displayed constancy up to 100 oC. Discovering

the temperature effect on the color of Au-Cip NPs directed that the color of NPs

altered from pinkish-purple to light blue revealed aggregation of Au NPs with rise in

temperature as shown in inset picture in fig. 3.30.

300 400 500 600 700

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

Ab

so

rba

nce

Wavelength (nm)

AgNPs

0.2 mL Brine soln.

0.4 mL "

0.6 mL "

0.8 mL "

1 mL "

300 400 500 600 700

0.0

0.5

1.0

1.5

2.0

Ab

so

rba

nc

e

Wavelength (nm)

AuNPs

0.2 mL NaCl soln.

0.4 mL "

0.6 mL "

0.8 mL "

1 mL "

Fig. 3.26. Effect of brine on the stability

of Ag NPs

Fig. 3.27. Effect of brine on the stability

of Au NPs


50

300 400 500 600 700

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

Ab

so

rba

nc

e

Wavelength (nm)

pure AgNPs

pH=1-2

pH=3-4

pH=5-6

pH=7-8

pH=9-10

pH=11-12

300 400 500 600 700

0.0

0.5

1.0

1.5

2.0

Ab

so

rba

nc

e

Wavelength (nm)

AuNPs

pH=1-2

pH=3-4

pH=5-6

pH=7-8

pH=9-10

pH=11-12

Fig. 3.28. pH effect on the stability of Ag-

Cip NPs

Fig. 3.29. pH effect on the stability of

Au-Cip NPs

400 500 600 700

0.0

0.5

1.0

1.5

2.0

2.5

Ab

so

rba

nc

e

Wavelength (nm)

Ag-cip NPs (reaction at r.t)

30 Co

50 Co

80 Co

100 Co

400 500 600 700

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Ab

so

rba

nc

e

Wavelength (nm)

Au-cip NPs (at r.t)

30 Co

50 Co

80 Co

100 Co

Fig. 3.30. Temperature effect on the stability of Ag and Au-Cip NPs (Inset: effect of

temperature on Ag and Au-Cip NPs colors


51

3.3 Synthesis of sparfloxacin mediated Ag and Au NPs

Sparfloxacin is a fluoroquinolone antibiotic having capability of capping Ag and Au

NPs as shown in scheme 3.3.

Scheme 3.3: Synthesis of Ag and Au NPs stabilized with sparfloxacin

3.3.1 UV visible spectroscopic analysis

Maximum absorption of metallic NPs was noted as a function of retention time in the

range of 300 to 700 nm. Variations in UV visible absorption bands were reflected as

the preliminary clue for the synthesis of Ag/Au NPs. The presence of specific peaks

in the areas of 400-500 nm and 500-600 nm certified the synthesis of Ag and Au NPs

correspondingly. The sharp peak for Ag-Sp NPs was perceived for a 10:1 (metal:

drug) molar ratio (Fig. 3.31), while for Au-Sp NPs, the maximum peak was at 1:2

(metal: drug) molar ratio as shown in the fig. 3.32.

300 400 500 600 700

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Ab

so

rba

nc

e

Wavelength (nm)

Ag

Sp

Ag-sp (10:1)

Fig. 3.31. UV visible spectra of Ag NPs (Inset: color of Ag NPs)


52

300 400 500 600 700

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Ab

so

rba

nc

e

Wavelength (nm)

Au

Sp

Au-sp (1:2)

Fig. 3.32. UV visible spectra of Au NPs (Inset: color of Au NPs)

3.3.2 AFM, SEM and EDX analyses

Surface topology and structural features of the articulated Ag/Au NPs were studied by

AFM analysis (Fig. 3.33 and 3.34). The micrographs obviously show that the

produced Ag-Sp NPs possess sphere-shaped and have the calculated sizes in the range

of 40 to 50 nm, while the Au NPs have slightly spherical shape and have the mean

sizes in the range of 70 to 80 nm. Further confirmation of the sizes and surface

morphologies of these NPs was done by SEM analysis (Fig. 3.35 and 3.36).

Energy-dispersive X-ray spectroscopy (EDX) (Fig. 3.37 and 3.38) illustrated the

chemical nature of synthesized metallic NPs.

Fig. 3.33. AFM image of Ag-Sp NPs Fig. 3.34. AFM image of Au-Sp NPs


53

Fig. 3.35. SEM image of Ag NPs Fig. 3.36. SEM image of Au NPs

Fig. 3.37. EDX spectrum of Ag NPs Fig. 3.38. EDX Spectrum of Au NPs

3.3.3 Fourier transform infrared spectroscopy (FTIR)

In order to find out various functionalities available in the drug substrate

(sparfloxacin) before and after stabilizing the Ag/Au NPs. Sparfloxacin displayed

absorption bands for aromatic C-H stretch (2933 cm-1

), secondary N-H stretch (3374

cm-1

), C=O stretch for ketone (1715 cm-1

), O-H stretch (2681 cm-1

), O-H bend of

COOH (1446 cm-1

) and C-N stretch (1341 cm-1

). The band at 3374 cm-1

is shifted to

3447 cm-1

and somewhat extended in the case of Ag and Au NPs. The band at 3374

cm-1

is due to N-H stretching of the amine moiety, and the broad band at 3510 cm-1

is

due to O-H stretching of carboxylic acid group. In case of Ag-Sp NPs the FTIR

spectra indicated a shift in carbonyl peak of carboxylic group from 1715 to 1609 cm

-1

and C-N stretching is shifted from 1341 to 1385 cm-1

, while in case of Au-Sp NPs a


54

shift from 1715 to 1620 cm

-1 and for C-N stretch the band is shifted from 1341 to

1390 cm-1

were observed (Fig. 3.39). It was revealed by FTIR analysis that amine and

carboxylate moiety can be liable for stabilizing and capping of sparfloxacin mediated

Ag and Au NPs.

Fig. 3.39. FTIR spectra of Ag and Au-Sp NPs


Fig. 3.40 and 3.41 show the results of different concentrations of aqueous solution of

NaCl on the surface plasmon peak of Ag-Sp and Au-Sp NPs respectively. The results

indicated that high concentration of brine solution caused by a decrease in absorbance

maxima. The full width at half maximum is also increased and thus reducing the

stability of metal NPs. This fast reduction in absorbance of Ag/Au nano-conjugates

containing NaCl is attributed to the aggregation effect caused by Cl-1

ions. As far as

long term stability is concerned, Ag and Au NPs are much more stable in neat water

than those in brine solution.


55

300 400 500 600 700

0.0

0.5

1.0

1.5

2.0

2.5

Ab

so

rba

nc

e

Wavelength (nm)

Ag-sp (salt effect)

0.2 mL (Brine)

0.4 mL

0.6 mL

0.8 mL

1 mL

Fig. 3.40. Effect of brine on the stability of Ag-Sp NPs

300 400 500 600 700

0.5

1.0

1.5

2.0

2.5

3.0

Ab

so

rba

nc

e

Wavelength (nm)

AuNPs (Sp)

0.2 mL (Brine)

0.4 mL

0.6 mL

0.8 mL

1 mL

Fig. 3.41. Effect of brine on the stability of Au-Sp NPs

The effect of pH on the stability of Ag and Au-Sp NPs was also studied. pH of 3 mL

solutions of the freshly prepared NPs were found to be 4.49 (Ag-Sp NPs) and 9.66

(Au-Sp NPs). pH of 1-7 was adjusted by using 1M HCl solution, while that of 7-12

was done with 1M NaOH solution. The UV visible bands of the resultant solutions

were verified after 24 hours (Fig. 3.42 and 3.43).


56

300 400 500 600 700

0.0

0.5

1.0

1.5

2.0

B

A

pH=2-3

pH=4-5 (pure Ag-sp)

pH=6-7

pH=8-9

pH=10-11

pH=12-13

Fig. 3.42. pH effect on the stability of Ag-Sp NPs

300 400 500 600 700

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Ab

so

rba

nc

e

Wavelength (nm)

Au NPs (Sp)

pH=1-2

pH=3-4

pH=5-6

pH=7-8

pH=9-10

pH=11-12

Fig. 3.43. pH effect on the stability of Au-Sp NPs

Temperature is an important ecological factor that distresses the permanency and

chemical features of Ag and Au NPs. Ag-Sp NPs were stable at broad range of

temperatures, as shown in fig. 3.44. Surface plasmon resonance bands up to 100 oC

indicated a reduction in strength with a blue shift from the original band, which is

recognized to the dreadful conditions of NPs, with lessening in NPs size.

Furthermore, Au-Sp NPs revealed less stability by increasing heat up to 100 oC (Fig.

3.45).


57

300 400 500 600 700

-0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

Ab

so

rba

nc

e

Wavelength (nm)

30 Co (Ag-sp NPs)

50 Co

80 Co

100 Co

Fig. 3.44. Heat effect on the stability of Ag-Sp NPs

300 400 500 600 700

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Ab

so

rba

nc

e

Wavelength (nm)

Au-sp NPs (at r.t)

30 Co

50 Co

80 Co

100 Co

Fig. 3.45. Temperature effect on the stability of Au-Sp NPs

Time effect on the achievement of reaction was also performed by UV visible

spectroscopy for colloidal suspension of Ag-Sp and Au-Sp NPs to boost the time needed

for the accomplishment of reaction, where the reaction was checked from 0 to 80 min at

10 min time intermission. The absorbance of the subsequent solutions was recorded

spectrophotometrically. The absorption peak intensity amplified swiftly with increase in


58

reaction time from 10 to 30 minutes due to the unceasing development of Ag/Au NPs in

the reaction scheme. Consequently, it was noted that an optimal time is obligatory for the

completion of reaction due to the instability of designed Ag NPs. The optimum time

necessary for the completion of reaction was observed to be 30 minutes for Ag-Sp NPs,

while for Au-Sp NPs was recorded to be 5 minutes (Fig. 3.46 and 3.47).

300 400 500 600 700

-0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

Ab

sorb

ance

Wavelength (nm)

5 min (rxn time of Ag-sp)

10 min

20 min

40 min

1 hr

1 hr & 20 min

over night

Fig. 3.46. Reaction time effect on the stability of Ag-Sp NPs

Fig. 3.47. Reaction time effect on the stability of Au-Sp NPs


59

3.4 Synthesis of gemifloxacin-capped noble metal NPs

Gemifloxacin is a fluoroquinolone antibiotic having the following molecular structure

(Fig. 3.48). Amine moiety is mainly responsible for the capping action of Ag and Au

NPs (Scheme 3.4).

Fig. 3.48. Molecular structure of gemifloxacin

Scheme 3.4. Synthesis of Ag and Au NPs stabilized with gemifloxacin

3.4.1 UV visible spectroscopy for synthesis of Ag/Au NPs

The presence of particular peaks at 410 nm and 550 nm in regions of 400-500 nm and

500-600 nm confirmed the formation of Ag and Au NPs respectively. Using different

molar ratios, only 10:1 and 1:2 (metal: drug) molar ratios presented sharpest surface

plasmon resonance (SPR) bands for Ag NPs and Au NPs respectively (Fig. 3.49 and

3.50). The width and position of SPR band depend mainly on the NPs size and shape;

sharp absorption bands reveal that Ag/Au NPs are spherical in shapes. Ag NPs

exhibiting dark brown, while Au NPs giving pinkish-purple color in aqueous solution

due to excitation of surface plasmon resonance with Ag/Au NPs band in the UV

visible region as shown in the inset picture in fig. 3.49 and 3.50.


60

Fig. 3.49. Optimized UV visible spectra of Ag NPs (Inset: Color of Ag NPs)

Fig. 3.50. Optimized UV visible spectra of Au NPs. (Inset: Colors of Au NPs)

3.4.2 AFM, SEM and EDX spectroscopy analysis

Structural features were determined by using atomic force microscopy and scanning

electron microscopy analysis. AFM micrographs showed that both Ag NPs and Au

NPs are spherical in shapes and have sizes of about 40 nm and 70 nm respectively

(Fig. 3.51 and 3.52).

0

0.5

1

1.5

2

2.5

3

300 400 500 600 700

Ab

sorb

ance

wave length (nm)

Ag

Gm

1:01

5:01

10:01

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

250 350 450 550 650 750

Ab

sorb

ance

Wavelength (nm)

Au

Gm

1:02

1:03

1:04


61



Scanning electron microscopy also has been used to characterize the size, shape and

morphology of Ag/Au NPs. The SEM analysis further confirmed the sizes of Ag and

Au nano-conjugates, synthesized from gemifloxacin mesylate are 40 nm and 70 nm in

sizes and seem to be spherical (Fig. 3.53 and 3.54).



62

Chemical nature of Ag NPs and Au NPs was determined by using EDX. The sharp

signal peak of elemental Ag strongly confirmed the reduction of AgNO3 to Ag NPs.

Metallic Ag nanocrystals revealed distinctive optical absorption peak around at 3 keV

due to SPR as shown in fig. 3.55. EDX spectrum for Au NPs also revealed the peak

for Ag NPs at 2.12 and 9.71 KeV indicating the formation of Au NPs (Fig. 3.56).

Fig. 3.55. EDX spectrum of Ag NPs Fig. 3.56. EDX spectrum of Au NPs

3.4.3 FTIR analysis

FTIR spectra of gemifloxacin mesylate, Ag NPs and Au NPs are showing the bands at

1725 cm-1

(C=O stretching vibrations of carbonyl groups of carboxylic acid), 3410

cm-1

(-NH2 primary amino group), 1475 cm-1

(C=C ring stretching) and 1617cm-1

(aromatic C=C). Spectra for Ag NPs and Au NPs revealed that amino group was

involved in the capping of Ag and Au NPs (Fig. 3.57).

4000 3500 3000 2500 2000 1500 1000 500

0

20

40

60

80

100

120

140

Tra

nsm

itta

nce

(%

)

Wavenumber (cm-1)

Gm

Ag NPs

Au NPs

3410

1725

Fig. 3.57. FTIR spectra of gemifloxacin mesylate and its noble metal (Ag/Au) NPs


63


Temperature effect was investigated for Ag and Au NPs with the help of UV visible

spectroscopy. Ag NPs were stable up to large changes in temperature, SPR bands up

to 100 oC presented a slight decrease in intensity of the SPR band with a blue shift

from original band showing slight degradation of Ag NPs, also color of the heated

solution (up to 100 o

C) was not much different from original solution‟s color (Fig.

3.58), while in case of Au NPs, SPR bands decreased in intensity, broadened and

slight red shift in absorption wavelength was happened (Fig. 3.59).

Fig. 3.58. Temperature effect on the stability of Ag NPs

Fig. 3.59. Effect of temperature on the stability of Au NPs

0

0.5

1

1.5

2

2.5

368 418 468 518 568 618

Ab

sorb

ance

Wavelength (nm)

20ᵒ C

30° C

50° C

80° C

100° C

-0.1

0.1

0.3

0.5

0.7

0.9

1.1

1.3

1.5

420 520 620

Ab

sorb

ance

Wavelength (nm)

20ᵒC

30°C

50°C

80°C

100°C


64

The effect of 1M brine solution was also checked for gemifloxacin mesylate capped

Ag/Au NPs. As the concentration of brine solution increased the intensity of SPR

band gradually decreased. This decrease was less in case of Ag NPs, showing stability

toward brine (Fig. 3.60), while in case of Au NPs with increasing ionic strength of

NaCl solution, a pinkish-purple color of the solution turned to a bluish-purple color

(Fig. 3.61) quickly due to the flocculation of the Au NPs.

Fig. 3.60. Salt effect on the stability of Ag NPs

Fig. 3.61. Effect of salt on the stability of Au NPs

-0.1

0.1

0.3

0.5

0.7

0.9

1.1

1.3

1.5

370 420 470 520 570

Ab

sorb

ance

Wavelength (nm)

0 mL

0.2 mL

0.4 mL

0.6 mL

0.8 mL

1 mL

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

450 550 650 750

Ab

sorb

ance

Wavelength (nm)

0 mL

0.2 mL

0.4 mL

0.6 mL

0.8 mL

1 mL


65

The stability of Ag/Au NPs was checked at all pH values ranging from 1-13. The Ag

NPs and Au NPs have pH ranges 4-5 and 3-4 respectively, which revealed that Ag

NPs were stable over a biological pH (4.2-8.4). But a large change in the pH of the

solution (both highly acidic and highly basic) greatly reduced the stability of the Ag

NPs (Fig. 3.62). While Au NPs were least stable 1-2 pH resulted in red shift in

wavelength but their stability increased as pH medium increased and were more stable

at 11-12 pH, which is translated as blue shift in absorption wavelength (Fig. 3.63).

Fig. 3.62. Effect of pH on the stability of Ag NPs

Fig. 3.63. Effect of pH on the stability of Au NPs (Insets: Effect of pH on the colors

of Ag/Au NPs)

-0.5

0

0.5

1

1.5

2

2.5

360 410 460 510 560 610

Ab

sorb

ance

Wavelength (nm)

4-5pH

2-3pH

6-7pH

8-9pH

10-11pH

12-13pH

0.2

0.4

0.6

0.8

1

1.2

1.4

450 550 650

Ab

sorb

ance

Wavelength (nm)

1-2 pH

3-4pH

5-6 pH

7-8pH

9-10 pH

11-12 pH


66

Effect on reaction time was also studied by UV visible spectroscopy for gemifloxacin

mesylate stabilized Ag and Au NPs. For Ag NPs intensity of absorption band

increased up to 30 minutes, but further increase in stirring time intensity of SPR bands

decreased and was completely vanished when Ag NPs were stirred up to 24 hours

showing the degradation of Ag NPs (Fig. 3.64). In case of Au NPs very fanciful result

was obtained. Once the reaction has been completed within 3 minutes, giving intense

absorption band, no further decrease was observed with increase in reaction time up to

24 hours, showing significant stability of Au NPs (Fig. 3.65).

Fig. 3.64. Effect of reaction time on the stability of Ag NPs

Fig. 3.65. Effect of reaction time on the stability of Au NPs

0

0.5

1

1.5

2

2.5

3

350 400 450 500 550

Ab

sorb

ance

wavelength (nm)

1 min

10 min

20 min

30 min

40 min

1 hr

2 hr

24 hr

0

0.5

1

1.5

2

2.5

3

300 400 500 600 700 800

Ab

sorb

ance

Wavelength (nm)

5 sec

10 min

20 min

30 min

40 min

60 min

80 min

100 min

2 hrs

4 hrs

24 hrs


67

3.5 Biological evaluation of fluoroquinolones-capped Ag/Au NPs

Different biological activities were evaluated to express the hidden potentials of Ag

and Au nano-conjugates. Some activities revealed significant results, while the

outcomes of other activities were moderate. All activities are described in below

section.

3.5.1 Urease study

The NPs along with the capping ligands (fluoroquinolones) were independently screened

for jack bean urease enzymes inhibition potentials. Mostly the Ag nano-conjugates

showed significant urease inhibition activity. The Ag-Mox NPs exhibited significantly

higher levels of enzyme inhibition activity of 93% at 0.2 mg/mL and IC50 value of 0.66 ±

0.042 μg/mL, while the ligand (Moxifloxacin) revealed weak inhibition with IC50 value of

183.25 ± 2.06 μg/mL. The Au-Mox was found inactive as compared to the parent

compound (Mox) having IC50 = 183.25 ± 2.06 μg/mL. The results deduced that after

conjugation of moxifloxacin with Ag, the activity of moxifloxacin was significantly

increased even more than 250 times. Interestingly, after conjugation with Au the activity

of moxifloxacin was significantly decreased, but conjugation of Mox to Ag had a robust

inhibition effect in comparison to Mox (Table 3.1).

The metal nano-conjugates (Ag-Cip and Au-Cip) along with parent drug, ciprofloxacin

(Cip) were also subjected for urease enzymes inhibition studies to search the inhibition

potential. After screening of Cip, Ag-Cip and Au-Cip against urease enzyme, the Ag-

Cip was found significantly active with % inhibition of 96 at 0.2 mg/mL. It revealed

significant activity with IC50 value of 1.181 ± 0.02 μg/mL. The Au-Cip also exhibited

good urease inhibitory activity with IC50 value of 52.55±2.3 μg/mL and % inhibition 90

at 0.2 mg/mL comparatively ciprofloxacin, which presented less activity with %


68

inhibition 75 at 0.2 mg/ mL and IC50 = 82.95±1.62 μg/mL. The results deduced that

after stabilizing ciprofloxacin with Ag and Au, the activity of ciprofloxacin was

significantly enhanced. Ag-Sp (IC50 = 0.615±0.016) and Ag-Gm (IC50 = 0.509±0.019)

NPs were also active against urease enzyme, while Sp, Au-Sp, Gm and Au-Gm were

inactive against urease enzyme as shown in the table 3.1.

Table 3.1: Urease enzyme inhibition studies of fluoroquinolones and

fluoroquinolones-capped Ag and Au NPs

Samples Concentration

(mg/mL)

% Inhibition IC50 ± S.E.M.

(µg/mL)

Mox 0.2 57 183.25 ± 2.06

Ag-Mox 0.2 93 0.66 ± 0.042

Au-Mox 0.2 47.6 NA

Cip 0.2 75 82.95±1.62

Ag-Cip 0.2 96 1.181±0.02

Au-Cip 0.2 90 52.55±2.3

Sp 0.2 21.7 NA

Ag-Sp 0.2 94.2 0.615±0.016

Au-Sp 0.2 25.4 NA

Gm 0.2 25.2 NA

Ag-Gm 0.2 97 0.509±0.019

Au-Gm 0.2 41.6 NA

Thiourea (STD) 0.5 98.2 21 ± 0.11 (µM)

S.E.M = Standard error mean; STD = Standard; NA = Not active


69

Fig. 3.66. Graphical representation of urease activities of fluoroquinolones-capped

noble metal NPs

3.5.2 Leishmanicidal activity

The four FQs (Fluoroquinolones) along with synthesized noble metal NPs were tested

for leishmanicidal activity against promastigotes of leishmania tropica. Ag-Mox,

Cip, Ag-Cip, Au-Cip Sp, Ag-Gm, showed significant leishmanicidal activity with

(IC50 µg/mL) 9.939, 11.32, 11.80, 10.46, 11.12, and 12.12 µg/mL respectively, while

the activity of Mox, Au-Mox, Ag-Sp, Au-Sp, Gm, and Au-Gm were good with (IC50

µg/mL) 10.50, 10.45, 10.69, 8.816, 5.170, and 9.939 µg/mL respectively (Table 3.2).

0

10

20

30

40

50

60

70

80

90

100

% I

nh

ibit

ion


70

Table 3.2. In vitro efficacy of fluoroquinolones and their metallic NPs against

promastigotes of L. tropica

Sample Leishmanicidal activity

(IC50 μg/mL)

Concentrations

(µg/mL)

Number of

Promastigotes (x104)

Mox 10.50 1000 44

500 56

Ag-Mox 9.939 1000 19

500 33

Au-Mox 10.45 1000 39

500 43

Cip 11.32 1000 21

500 32

Ag-Cip 11.80 1000 17

500 20

Au-Cip 10.46 1000 45

500 57

Sp 11.12 1000 31

500 43

Ag-Sp 10.69 1000 40

500 48

Au-Sp 8.816 1000 48

500 72

Gm 5.170 1000 90

500 92

Ag-Gm 12.12 1000 16

500 19

Au-Gm 9.939 1000 50

500 65

Pentamidine

(STD)

2.56 - -


71

3.5.3 Antioxidant activity

The synthesized NPs of Ag and Au stabilized with selected fluoroquinolones were

evaluated for their scavenging activity by 2, 2-diphenyl-1-pecrylhydrazyl (DPPH) free

radical for different concentration (20, 40, 60, 80, 100 µg/mL). In table 3.3 and 3.4

the antioxidant activity of Mox, Cip, Sp and Gm along with their noble metal NPs

were shown. The parent‟s drugs as well as their NPs have not shown good antioxidant

activity (Fig. 3.67 and 3.68).

Table 3.3: Antioxidant activity of fluoroquinolones and fluoroquinolones-capped

Ag/Au NPs

Mox Ag-Mox Au-Mox Cip Ag-Cip Au-Cip STD

Conc.

µg/mL

% DPPH Activity

20 14.59 15.49 14.68 11.98 16.59 12.98 91.59

40 17.90 19.64 16.04 13.39 22.90 17.39 92.09

60 21.27 25.51 20.07 18.54 25.27 20.54 92.59

80 26.59 34.26 27.93 22.43 31.59 26.93 93.35

100 32.90 46.14 31.49 29.59 37.90 35.14 94.47

STD = Standard (Vitamin C)

Fig. 3.67. Antioxidant assay of fluoroquinolones-capped Ag/Au NPs

0

20

40

60

80

100

20 40 60 80 100

Ab

sorb

an

ce (

%)

Concentration µg/mL

Mox Ag-Mox Au-Mox Cip Ag-Cip Au-Cip STD


72

Table 3.4: Antioxidant activity of fluoroquinolones-capped Ag and Au NPs

Sp Ag-Sp Au-Sp Gm Ag-Gm Au-Gm STD

Conc.

µg/mL

% DPPH Activity

20 5.59 11.49 9.68 2.298 10.59 8.98 91.59

40 7.90 16.64 13.04 3.160 16.90 15.39 92.09

60 10.27 23.51 18.07 7.758 21.27 19.51 92.59

80 17.59 28.26 22.93 27.298 33.59 29.93 93.35

100 25.90 35.14 27.49 33.908 45.90 35.14 94.47


Fig. 3.68. Antioxidant assay of fluoroquinolones-capped Ag/Au NPs

0

10

20

30

40

50

60

70

80

90

100

20 40 60 80 100

Ab

sorb

an

ce (

%)


Sp Ag-Sp Au-Sp Gm Ag-Gm Au-Gm STD


73

3.5.4 Antibacterial activity

The fluoroquinolones and their capped noble metal nano-conjugates were tested against

four bacterial strains: Bacillus subtilis, Staphylococcus aureus, Pseudomonas

aeruginosa and Klebsiella pneumonia. The antibacterial activities of these NPs are

summarized in the table 3.5. The Ag-Mox NPs exhibited good antibacterial activity

against B. subtilis, S. aureus, P. aeruginosa and K. pneumonia with the zone of

inhibition ranging from 15±0.73 to 18±0.98 mm at 3 mg/mL, which were comparable

to the parent compound (Mox) with the inhibitory zone ranging from 14±0.92 to

20±0.98 mm. The Au-Mox NPs showed moderate bactericidal activity against B.

subtilis, S. aureus, P. aeruginosa and K. pneumonia with the zone of inhibition ranging

from 13±0.20 to 17±0.90 mm. Streptomycin was used as the standard drug (Table 3.5).

The ciprofloxacin and its stabilized metallic NPs (Ag-Cip and Au-Cip) were also

tested against B. subtilis, S. aureus, P. aeruginosa and K. pneumonia. These metallic

NPs revealed good antibacterial activity against three tests stains (B. subtilis, S.

aureus and K. pneumonia), while Cip and its noble metal NPs (Ag-Cip and Au-Cip)

were inactive against P. aeruginosa. The Ag-Cip showed inhibitory zone from

20±0.94-24±0.98 mm at 3 mg/ mL, whereas in parent drug (Cip) zone of inhibition

was from 24±0.94-28±0.75 mm. The Au-Cip also exhibited good bactericidal assay

against three strains with inhibition from 22±0.94-24±0.94 mm but showed no

activity against P. aeruginosa as shown in table 3.5. The synthesized nanomedicines

of sparfloxacin and gemifloxacin were also examined against these bacterial strains.

They revealed promising bactericidal activity but in all cases the parent drugs

exhibited high antibacterial activity as compared to their capped Ag and Au NPs. The

results are summarized in table 3.5.


74

Table 3.5: Antibacterial activities of fluoroquinolones and fluoroquinolones-capped

Ag and Au NPs

Zone of inhibition (mm)

Bacterial strain Bacillus

subtilis

Staphylococcus

aureus

Pseudomonas

aeruginosa

Klebsiella

pneumonia Sample

Mox 18±0.98 16±0.56 14±0.92 20±0.98

Ag-Mox 18±0.98 15±0.73 16±0.56 18±0.98

Au-Mox 16±0.94 13±0.20 15±0.73 17±0.90

Cip 28±0.75 26±0.92 -- 24±0.94

Ag-Cip 22±0.79 24±0.94 -- 20±0.98

Au-Cip 24±0.94 22±0.79 -- 24±0.94

Sp 26±0.92 26±0.92 -- 30±0.09

Ag-Sp 22±0.79 24±0.94 2±0.02 20±0.98

Au-Sp 22±0.79 24±0.94 2±0.02 26±0.92

Gm 26±0.92 26±0.92 14±0.09 28±0.04

Ag-Gm 24±0.94 22±0.79 16±0.56 26±0.92

Au-Gm 22±0.79 20±0.98 16±0.56 20±0.98

Streptomycin

(STD)

20±0.98 26±0.92 20±0.98 28±0.75

Well size = 6 mm, STD = Standard, -- Not effective, Mean ± S.E.M.


The results of antifungal activity against four selected fungi strains i.e. Fusarium

oxysporum, Aspergillus parasiticus, Aspergillus flavus and Candida albicans of


75

fluoroquinolones and their nano-conjugates have been presented in table 3.6. The

results obtained give us information about the antifungal potential of pure drugs and

their capped NPs. They revealed moderate antifungal activity against A. parasiticus,

A. flavus and C. albicans while the parent drugs and their Ag/Au nano-conjugates

showed low antifungal activity against F. oxysporum. The antifungal activity was not

enhanced after capping with Ag and Au metal (Table 3.6).

Table 3.6: Antifungal activities of fluoroquinolones and fluoroquinolones-capped

Ag and Au NPs

Antifungal activities (% inhibition)

Sample Fusarium

oxysporum

Aspergillus

parasiticus

Aspergillus

flavus

Candida

albicans

Mox 30 63 70 55

Ag-Mox 22 60 68 50

Au-Mox 28 65 60 52

Cip 10 70 80 55

Ag-Cip 10 68 77 52

Au-Cip 12 66 70 50

Sp 22 80 75 45

Ag-Sp 22 73 70 40

Au-Sp 20 70 66 42

Gm 10 63 73 40

Ag-Gm 10 60 70 35

Au-Gm 9 55 65 40

Miconazole

(STD)

100 100 100 100

-- Not effective


76

3.6 Green synthesis of noble metal NPs of flower extract of

Rhododendron arboreum

The green synthesis of Ag/Au NPs involved the mixing of aliquot amounts of (I mM)

AgNO3 and HAuCl4.3H2O with R. arboreum flower extract (RAFE) in deionized

water at room temperature for 25 minutes (Scheme 3.5). The development of NPs

was indicated by an optical color change to brownish-yellow and pinkish-red solution

indicating the formation of Ag and Au NPs respectively. To search out the optimized

conditions for the synthesis, various ratios of metal to extract were employed; keeping

metal (Ag/Au) constant and changing the amount of ligand (RAFE), i.e. (1:1 to 1:20)

and vice versa i.e. (3:1 to 20:1). The ratios which gave best surface plasmon

resonance (SPR) absorption bands were subjected for further studies. The formation

of Ag/Au NPs was further confirmed by using various spectroscopic methods such as

UV visible, AFM, FTIR, EDX and SEM analyses.

Scheme 3.5: R. arboreum flower extract (RAFE) reduces and stabilizes Ag/Au NPs

3.6.1 Visual inspection and UV visible spectroscopy analysis

Color change of the reaction mixture and subsequent UV visible spectra of Ag/Au

NPs are the first indication of biosynthesized Ag/Au NPs. The presence of particular

SPR absorption peaks in the region of 400-500 nm and 500-600 nm deep-rooted the

creation of Ag/Au NPs. The sharp peak for Ag NPs was observed for a reaction of 5:1

(metal: extract), while for Au NPs, the high peak was apparent at 10:1 (metal: extract)

as shown in the fig. 3.69 and fig. 3.70 respectively.


77

300 400 500 600 700

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Abs

orba

nce

Wavelength (nm)

Ag

RAFE

Ag:ligand=1:1

Ag:ligand=1:5

Ag:ligand=1:10

Ag:ligand=3:1

Ag:ligand=5:1

Ag:ligand=7:1

Ag:ligand=10:1

Fig. 3.69. Optimized UV visible spectral data of Ag NPs at reaction ratio of 5:1

300 400 500 600 700

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Ab

so

rba

nce

Wavelength (nm)

Au

RAFE

Au:ligand=1:1

Au:ligand=1:10

Au:ligand=5:1

Au:ligand=10:1

Au:ligand=15:1

Au:ligand=20:1

Fig. 3.70. UV visible spectral data of Au NPs at optimized reaction ratio of 10:1


78

3.6.2 FTIR spectroscopy analysis

The spectra were obtained from FTIR studies at 4000 to 400 cm-1

for RAFE, Ag-

RAFE NPs and Au-RAFE NPs. The FTIR analyses were taken to ascertain the

conceivable biomolecules accountable for capping and stabilization of the Ag/Au NPs

formed by RAFE (Fig. 3.71 and 3.72). The bands from 1790 to 740 cm-1

reflected the

biochemical compositions, mainly the moieties of polyphenols, carbohydrates, lipids

and proteins in RAFE.

4000 3500 3000 2500 2000 1500 1000 500

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

Tra

nsm

ittan

ce (%

)

Wavenumber (cm-1

)

RAFE

Ag NPs

3398

1730

3432 1629

Fig. 3.71. FTIR analysis for RAFE and Ag NPs

The spectral data revealed a broader band for the extract at 3398 cm-1

area which is

due to the occurrence of -OH groups. Likewise, a sharp peak was observed at 1730

cm-1

due the existence of -C=O. In the instance of Ag NPs, a large shift in the

absorbance band with decreased band intensity was observed from 3398 to 3432 cm−1

and 1730 to 1629 cm−1

, suggesting the binding of Ag ions with -OH and -COO-

groups of the RAFE. There was a prominent change in the FTIR spectrum of Ag NPs

as compared to RAFE. The intensity of the peak at 1629 cm-1

has been reduced when

Ag+ is reduced to Ag

0. In case of Au NPs, the band at 3398 cm

-1 is shifted to 3410 cm

-

1 and also a shift in carbonyl peak of C=O from 1730 cm

-1 to 1633 cm

-1 and 1402 cm

-1

was observed (Fig. 3.72).


79

4000 3500 3000 2500 2000 1500 1000 500

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Tra

nsm

itta

nce

(%

)

Wavenumber (cm-1

)

RAFE

Au NPs

3398

34101730

1633

Fig. 3.72. FTIR spectra of RAFE and Au NPs

3.6.3 Stability of the biosynthesized Ag/Au NPs

The capped Ag/Au NPs were found to be stable up to 70 oC. The result of 1 M NaCl

solution was also tested and indicated instability by increasing concentrations. Metal

NPs were stable in the pH range of 4-12, while unstable in the pH range of 1-2 (Fig.

3.73 to 3.76).

300 400 500 600 700

0.00

0.21

0.42

0.63

0.84

1.05

1.26

1.47

Ab

so

rba

nce

Wavelength (nm)

AgNPs

0.2 mL Brine

0.4 mL Brine

0.6 mL Brine

0.8 mL Brine

1 mL Brine

300 400 500 600 700

0.0

0.5

1.0

1.5

2.0

2.5

Ab

so

rba

nce

Wavelength (nm)

AgNPs

pH=2-3

pH=4-5

pH=6-7

pH=8-9

pH=10-11

pH=12-13

Fig. 3.73. Effect of salt (NaCl) on stability

of Ag NPs

Fig. 3.74. Effect of pH on stability of Ag

NPs


80

300 400 500 600 700

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Ab

so

rba

nce

Wavelength (nm)

AuNPs

0.2 mL Brine

0.4 mL Brine

0.6 mL Brine

0.8 mL Brine

1 mL Brine

300 400 500 600 700

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Ab

so

rba

nce

Wavelength (nm)

AuNPs

pH=1-2

pH=3-4

pH=5-6

pH=7-8

pH=9-10

pH=11-12

Fig.3.75. Effect of salt (NaCl) on stability

of Au NPs

Fig.3.76. Effect of pH on stability of Au

NPs

The visual inspection of the color changes in optimized ratio of Ag/Au NPs due to

different concentration of brine solution and pH was also observed and variation or

persistence of the colors as well as UV visible peaks provided evidence about the

stability of respective NPs (Fig. 3.77 to 3.80).

Fig. 3.77. Effect of brine (1 M NaCl) on the color of Ag NPs


81

Fig. 3.78. Effect of pH on the color of Ag NPs

Fig. 3.79. Effect of salt (1 M NaCl) on the color of Au NPs

Fig. 3.80. Effect of pH on the color of Au NPs


82

3.6.4 AFM, SEM and EDX analysis

The nature and size of the Ag/Au NPs were further confirmed by AFM. Most of the

NPs were monodispersed and spherical in shape (Fig. 3.81 and 3.82) and the majority

of the Ag/Au NPs were less than 40 nm in size. Furthermore, the sizes of both Ag/Au

NPs were also confirmed by SEM images (Fig. 3.83 and 3.84).

Fig. 3.81. Atomic force microscope spectrum of RAFE stabilized Ag NPs

Fig. 3.82. AFM spectrum of RAFE-capped Au NPs


83

Fig. 3.83. SEM image of Ag NPs

Fig. 3.84. SEM image of Au NPs

The EDX analysis (Fig. 3.85 and 3.86) further clarified the chemical nature of

biosynthesized Ag/Au NPs with RAFE. The EDX displays strong signal in the Ag

region and confirms the formation of Ag NPs. Metallic Ag NPs normally exhibit

optical absorption peak nearly at 3 keV due to SPR. For Au NPs, the EDX spectrum

revealed the presence of peaks characteristic of Au at 2.12 and 9.71 keV.


84

Fig.3.85. EDX spectrum of Ag NPs

Fig. 3.86. EDX spectrum of Au NPs

3.7 Green synthesis of metallic NPs of Kigelia pinnata extract

K. pinnata fruits were used as both reductant and capping agent for the green

biosynthesis of Ag and Au NPs.

3.7.1 UV Visible spectroscopy

The colorless mixture turned into brownish-yellow and pinkish-red solution after 30

minutes, indicating the biotransformation of ionic Ag and Au reduced to Ag/Au NPs,

as a result of the surface plasmon resonance phenomenon (SPR). Ag NPs showed

absorption peak at 430 nm (Fig. 3.87), while Au NPs exhibited at 560 nm (Fig. 3.88).

The color change ensued as of the active molecules present in the fruits extract that

owing to the excitation of SPR effect. The sharp peak for Ag NPs was observed for a


85

reaction of 10:1 (metal: extract), while for Au NPs, the highest peak was attained at

20:1 (metal: extract) as shown in fig. 3.87 and 3.88 respectively.

Fig. 3.87. Optimized UV visible spectra of Ag NPs

300 400 500 600 700

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Ab

so

rba

nc

e

Wavelength (nm)

Au

Kp

Au:kp(13:1)

Au:kp(15:1)

Au:kp(17:1)

Au:kp(20:1)

Fig. 3.88. Optimized UV visible spectra of Au NPs

3.7.2 FTIR analysis

The FTIR spectra of K. pinnata fruit extract and Ag and Au NPs were shown in fig.

3.89. Changes were observed in the following bands: 3454 cm-1

represent O-H/N-H

stretching, the band shift from 3454-3456 cm-1

implicated that flavonoid (luteolin)

group may be involved in the process of noble metal NPs synthesis. The peak at 1640

cm-1

corresponds to N-H bend it represent primary amine. The peak shift from 1640-

1642 cm-1

indicated the possible involvement of primary amine groups which may be


86

reduced the Ag and Au ions into Ag and Au NPs. A peak at 1018 cm-1

could be

assigned to C-O stretch was suggestive the possible involvement of alcohols,

carboxylic acids, esters, ethers. The K. pinnata plant has a lot of medicinal properties

due to the existence of various secondary metabolites such as naphthoquinones,

flavonoids, irridoids and volatile constituents etc. Since of these secondary

metabolites existing in the plants, they may be involved in the reduction mechanism.

4000 3500 3000 2500 2000 1500 1000 500

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

Tra

ns

mit

tan

ce

(%)

Wavenumber (cm-1)

Kp

Ag NPs

Au NPs

3454

3456

1640

1018

Fig. 3.89. FTIR spectra of K. pinnata and its Ag/ Au NPs

3.7.3 Stability check of the synthesized Ag/Au NPs

The effect of 1 M brine (NaCl) solution was checked and indicated instability by

increasing concentrations. Noble metal NPs were stable in the pH range of 5-12,

while unstable in the pH range of 1-3 as shown in fig. 3.90 to 3.93.

Fig. 3.90. Effect of salt (NaCl) on stability of Ag NPs

300 400 500 600 700

0.0

0.5

1.0

1.5

2.0

2.5

Abs

orba

nce

Wavelength (nm)

AgNPs(kp)

0.2 mL

0.4 mL

0.6 mL

0.8 mL

1 mL


87

Fig. 3.91. Salt (NaCl) effect on stability of Au NPs

Fig. 3.92. Effect of pH on stability of Ag NPs

Fig. 3.93. Effect of pH on stability of Au NPs

300 400 500 600 700

0.0

0.5

1.0

1.5

2.0

2.5

Ab

sorb

ance

Wavelength (nm)

AgNPs (kp)

pH=1-2

pH=3-4

pH=5-6

pH=7-8

pH=9-10

pH=11-12

300 400 500 600 700

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Ab

so

rba

nc

e

Wavelength (nm)

AuNPs(kp)

pH=1-2

pH=3-4

pH=5-6

pH=7-8

pH=9-10

pH=11-12

300 400 500 600 700

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Ab

so

rba

nc

e

Wavelength (nm)

AuNPs(kp)

0.2 mL

0.4 mL

0.6 mL

0.8 mL

1 mL


88

3.7.4 AFM and SEM analysis

The nature and size of the Ag/Au NPs were further confirmed by AFM. Most of the

NPs were monodispersed and spherical in shape (Fig. 3.94 and 3.95) and most of the

Ag/Au NPs were less than 70 nm in size. Furthermore, the sizes of both Ag/Au NPs

were also confirmed by SEM images (Fig. 3.96 and 3.97).





89

3.8 Green phytosynthesis of noble metal NPs of Eulophia dabia

extract as reducing and stabilizing agent

E. dabia plant extract was used as a reductant and capping agent for the facile green

synthesis of Ag/Au NPs (Scheme 3.6).

Scheme 3.6: E. dabia extract reduces and stabilizes Ag/Au NPs

3.8.1 Visual inspection and UV visible spectroscopy analysis

The sharpest peak for Ag NPs was observed for a reaction of 5:1 (metal: extract),

while for Au NPs, the sharp peak was apparent at 1:6 (metal: extract) as shown in fig.

3.98 and 3.99 respectively.

Fig. 3.98. UV visible spectra of Ag-

NPs

Fig. 3.99. UV visible spectra of Au NPs

300 400 500 600 700

0.0

0.5

1.0

1.5

2.0

Ab

so

rba

nc

e

Wavelength (nm)

Ag

Ed

Ag-Ed (5:1)

300 400 500 600 700

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Ab

so

rba

nc

e

Wavelength (nm)

Au-Ed (1:1)

(1:2)

(1:3)

(1:4)

(1:5)

(1:6)


90

3.8.2 FTIR spectroscopy analysis


for ED, Ag and Au

NPs. The FTIR analyses were taken to ascertain the possible biomolecules responsible

for capping and stabilization of the Ag/Au NPs synthesized by ED. The fig. 3.100 and

3.101 show the FTIR spectra of ED and Ag/Au NPs. The bands from 1785 to 740 cm-

1 reflected the biochemical compositions, mainly the moieties of polyphenols,

carbohydrates, lipids and proteins in ED.

The results showed a broader band for the extract at 3396 cm-1

region, which is due to

the presence of -OH groups. Also, a sharp peak was observed at 1731 cm-1

due the

presence of -C=O. In the case of Ag NPs, a large shift in the absorbance band with

decreased band intensity was observed from 3396 to 3433 cm-1

and 1731 to 1629 cm-

1, suggesting the binding of Ag ions with -OH and -COO

- groups of the ED. There

was a noticeable change in the FTIR spectrum of Ag NPs as compared to ED. The

intensity of the peak at 1629 cm-1

has been reduced when Ag+ is reduced to Ag

0. In

case of Au NPs, the band at 3396 cm-1

is shifted to 3409 cm-1

and also a shift in

carbonyl peak of C=O from 1730 cm-1

to 1631 cm-1

and 1402 cm-1

was observed.

Fig. 3.100. FTIR spectrum of E. dabia


91

4000 3500 3000 2500 2000 1500 1000 500

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

Tra

nsm

itta

nce (

%)

Wavenumber (cm-1)

Ag NPs of EDE

Au NPs "

3433

3409 1631

1629

Fig. 3.101. FTIR spectra of Ag and Au NPs


Fig. 3.102 and 3.103 show the effect of various concentrations of NaCl aqueous

solution on surface plasmon peaks of Ag and Au NPs. The results indicate that high

concentration of brine solution resulted in a decrease in absorbance maxima.

The effect of pH on the stability of ED-capped Ag and Au NPs was also studied. The

UV visible spectra of resulting solutions were recorded after 24 hours (Fig. 3.104 and

3.105).

Fig. 3.102. Effect of salt (1 M NaCl) on

stability of Ag NPs

Fig. 3.103. Effect of salt (1 M NaCl) on

stability of Au NPs

300 400 500 600 700

-0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

Ab

so

rba

nc

e

wavelength (nm)

Ag-Ed (5:1)

0.2 mL (Brine)

0.4 mL

0.6 mL

0.8 mL

1 mL

300 400 500 600 700

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Ab

so

rba

nc

e

Wavelength (nm)

AuNPs (Ed)

0.2 mL

0.4 mL

0.6 mL

0.8 mL

1 mL


92

Fig. 3.104. Effect of pH on stability of

Ag NPs

Fig. 3.105. Effect of pH on stability of

Au NPs

3.8.4 AFM and EDX analysis

The nature and size of the Ag/Au NPs were confirmed by AFM. Most of the NPs

were monodispersed and spherical in shape (Fig. 3.106 and 3.107) and the

mainstream of the Ag/Au NPs were less than 75 nm in size.

Fig. 3.106. AFM image of Ag NPs Fig. 3.107. AFM image of Au NPs

Energy dispersive X-ray spectroscopy verified the elemental nature of nano size

particles using E. dabia as reducing and stabilizing agent. The signal in the region of

Ag in EDX study ascertained the synthesis of Ag NPs. Ag metal generally shows

300 400 500 600 700

0.0

0.5

1.0

1.5

2.0

2.5

Ab

so

rba

nc

e

Wavelength (nm)

AgNPs (Ed)

pH=1-2

pH=3-4

pH=5-6

pH=7-8

pH=9-10

pH=11-12

300 400 500 600 700

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

Ab

so

rba

nc

e

Wavelength (nm)

AuNPs (Ed)

pH=1-2

pH=3-4

pH=5-6

pH=7-8

pH=9-10

pH=11-12


93

absorption peak nearly at 3 keV due to SPR. The peak for Ag seemed at 3 keV; for

Au NPs, the EDX band designated the presence of representative peaks of Au at 2.12

and 9.71 keV as shown in fig. 3.108 and 3.109.

Fig. 3.108. EDX spectrum of Ag-ED Fig. 3.109. EDX spectrum of Au-ED

3.9 Synthesis of metallic NPs of Desmodium elegans extract

The Ag/Au and extract solutions with different ratios (1:1, 3:1, 5:1, 7:1, 10:1) were

taken in vials and kept on stirring for 30 minutes and then reducing agent TEA

(triethylamine) (0.1 mL) were added to each vial. The metallic NPs were then isolated

by centrifuge followed by freeze drying for characterization and other analysis.

3.9.1 UV visible spectroscopy

To find out the optimized ratio using TEA as mild reducing agent, reactions with

different ratios of metal to ligand (metal: extract) were carried out. Ag and Au NPs

formation significantly decreased when ligand (Extract) ratio to metal was increased.

This observation was recorded by UV visible spectrophotometer. The ratio, which

contributed the best result in respect of having the sharp absorption peak on UV

visible spectrophotometer was selected for further studies, which was 5:1 (Metal:

ligand) for Ag NPs (Fig. 3.110), while for Au NPs the ratio which provided the best

result in respect of having the highest absorption peak was 5:1(metal: ligand) as

shown in fig. 3.112.


94

0

0.5

1

1.5

2

2.5

3

3.5

350 400 450 500 550 600 650 700

Ag soln

Extractsoln

Fig. 3.110. UV visible data of Ag NPs reduced with TEA

The intensity of Ag NPs color gradually changed for different ratios of metal to ligand

(Ag: extract) as shown in fig. 3.111. The color of the Ag NPs is dependent on the size

of the NPs.

Ag extract 1:1 3:1 5:1 7:1 8:1

Fig. 3.111. Color of the Ag NPs reduced with TEA

Mostly, the Ag NPs have yellowish-brown color, but other color may be possible

depending on the size and compound which is used as capping agent for Ag NPs

formation.

Ab

sorb

ance

Wavelength


95

Fig. 3.112. UV visible data of Au NPs reduced with TEA

The color of Au NPs reduced with TEA was different for various reactions having

different ratios (metal: ligand) as shown in fig. 3.113, which is dependent on their

particle sizes.

Gold extract 3:1 5:1 8:1

Fig. 3.113. Color of Au NPs reduced with TEA

3.9.2 FTIR analysis


for D. elegans, Ag

and Au NPs. The fig. 3.114 shows the FTIR spectra of DE, Ag and Au NPs. The

absorption band at around 1031 cm-1

can be allocated as absorption bands of -C-O-C-

0

0.5

1

1.5

2

2.5

350 450 550 650 750

Extract soln

Gold soln

1:1

5:1

8:1

Wavelength

Gold: ligand

Ab

sorb

ance


96

or -C-O-. The band at around 1653 cm-1

is consigned to the amide I bonds of proteins.

The functional groups and bonds like -C-O-C-, -C-O- and -C=C- derived from

heterocyclic compounds and the amide I bond resulting from the proteins, which are

existent in the extract, responsible for stabilizing of metallic NPs.

Fig. 3.114. FTIR spectra of D. elegans and its Ag/Au NPs reduced with TEA

3.9.3 Stability check of synthesized metallic NPs

The temperature effect was also studied and Ag NPs exhibited heat stability up to 60

oC. The absorption peaks of the Ag NPs at 25

oC and at 60

oC are shown in fig. 3.115.

Fig. 3.115. Effect of temperature on the stability of Ag NPs

0

0.5

1

1.5

2

2.5

300 400 500 600 700

25 ͦC

60 ͦC

Wavelength

Heat effect

Ab

sorb

ance


97

It was found that the Ag NPs indicated instability by increasing concentration (1 M

NaCl) (Fig. 3.116). When the concentration of brine solution increased the intensity

of peak decreased and precipitate formation was observed (Fig. 3.117).

Fig. 3.116. Salt effect on the stability of Ag NPs

Pure AgNPs 0.2ml 0.4ml 0.6ml 0.8ml 1ml

Fig. 3.117. Effect of NaCl salt concentration on the color of Ag NPs

The pH has a pronounced effect on the stability of the Ag NPs. The UV visible data

showed that the Ag NPs were highly stable in the pH range of 7-8 and least stable in

acidic pH ranges 3-4, as shown in fig. 3.118.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

300 400 500 600 700

pure AgNPs

0.2ml

0.8 ml

0.6 ml

0.4 ml

1 ml

Wavelength (nm)

Ab

sorb

ance


98

Wavelength (nm)

Fig. 3.118. Effect of pH on the stability of Ag NPs

The pH has also effect on the color of Ag NPs, as the color is dependent on the size of

NPs; fig. 3.119 shows the effect of pH on the color of Ag NPs.

Fig. 3.119. Effect of pH on the color of Ag NPs

3.9.4 SEM and EDX analysis

Most of the Ag/Au NPs were fairly monodispersed and spherical in shapes with sizes

60-80 nm (Fig. 3.120 and 3.121).

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

350 400 450 500 550 600

pure 11-12

1-2

3-4

5-6

7-8

9-10

Ab

sorb

ance


99

Fig. 3.120. SEM image of Ag NPs

Fig. 3.121. SEM image of Au NPs

The EDX analysis (Fig. 3.122 and 3.123) further clarified the chemical nature of

synthesized Ag/Au NPs with DE. The EDX displays high signal in the Ag region and

confirms the formation of Ag NPs. Metallic Ag NPs normally exhibit optical

absorption peak closely at 3 keV due to SPR. For Au NPs, the EDX spectrum

revealed the presence of peaks characteristic of Au at 2.12 and 9.71 keV.


100

Fig.3.122. The EDX spectrum for Ag NPs

Fig. 3.123. The EDX spectrum for Au NPs

3.10 Biological evaluation of selected medicinal plants and their NPs

Bioactivity of selected medicinal plants and their Ag and Au NPs are described below.

3.10.1 Urease inhibition assay

The selected medicinal plants along with their metallic NPs were screened for jack

beans urease enzymes inhibition assay. All NPs presented good to significant activity

against urease enzyme except DE capped Ag and Au NPs and Au-ED, which were

inactive against urease enzyme i.e., Ag-DE, Au-DE and Au-ED displayed %

inhibition 36.2, 25.4 and 34.6 respectively (Fig. 3.124), while DE displayed %

inhibition 19.3 (Table 3.7).


101

Table 3.7: Urease enzyme inhibition studies of selected medicinal plants and plants

mediated Ag and Au NPs

Samples Concentration

(mg/mL)

% Inhibition IC50 ± S.E.M.

(µg/mL)

RA 0.2 86 170.9±1.2

Ag-RAFE 0.2 96 1.129±0.053

Au-RAFE 0.2 92 55.51±1.03

KP 0.2 50 196.202±1081

Ag-KP 0.2 72 29.76±0.293

Au-KP 0.2 89 2.6±0.25

ED 0.2 38.1 NA

Ag-ED 0.2 97 2.59±0.092

Au-ED 0.2 34.6 NA

DE 0.2 19.3 NA

Ag-DE 0.2 36.2 NA

Au-DE 0.2 25.4 NA

Thiourea (STD) 0.5 98.2 21 ± 0.11 (µM)

S.E.M = Standard error mean; STD = Standard; NA = Not active


102

Fig. 3.124. Urease inhibitory activity of selected medicinal plants and their metallic NPs

3.10.2 Leishmanicidal activity

The antileishmanial activity of selected medicinal plants and their Ag and Au NPs

was also tested against Leishmania tropica as a modal parasite. The efficacy of all the

tested NPs was studied for 96 h. Ag-RAFE (IC50 µg/mL = 9.45) and Ag-ED (IC50

µg/mL = 7.20) showed significant activity, while the other noble metal NPs displayed

good activities as shown in the table 3.8.

0

10

20

30

40

50

60

70

80

90

100

% I

nh

ibit

ion


103

Table 3.8. In vitro efficacy of selected medicinal plants and their metallic NPs

against promastigotes of L. tropica

Sample Leishmanicidal activity

(IC50 μg/mL)

Concentrations

(µg/mL)

Number of

Promastigotes

(x104)

RA 9.896 1000 49

500 63

Ag-RAFE 9.45 1000 41

500 50

Au-RAFE 10.46 1000 45

500 57

KP 9.591 1000 52

500 66

Ag-KP 8.880 1000 64

500 76

Au-KP 6.250 1000 63

500 89

ED 9.894 1000 48

500 61

Ag-ED 7.20 1000 16

500 20

Au-ED 8.87 1000 46

500 67

DE 6.170 1000 72

500 94

Ag-DE 9.42 1000 46

500 53

Au-DE 9.94 1000 51

500 67

Pentamidine

(STD)

2.56 - -


104


The selected medicinal plants and synthesized NPs were screened for free radical

stability properties against ascorbic acid (vitamin C). RAFE and its Ag and Au NPs

revealed significant % DPPH activity with 83.80, 78.22 and 86.28% at 100 µg/mL

respectively, while KP and its noble metal (Ag/Au) NPs displayed low activity of

35.97, 38.90 and 47.14% at 100 µg/mL respectively (Table 3.9).

In table 3.10, ED and its synthesized Ag/Au nanomaterials, (Ag-ED and Au-ED)

exhibited good antioxidant activity with % scavenging 64.770, 60.49 and 78.14% at

100 µg/mL respectively, while DE (24.82%), Ag-DE (22.90%) and Au-DE (39.14%)

showed moderate activity (Fig. 3.125 and 3.126).

Table 3.9: Antioxidant activity of selected medicinal plants and their Ag/Au

stabilized NPs

RAFE Ag-RAFE Au-RAFE KP Ag-KP Au-KP STD

Conc. µg/mL % DPPH Activity

20 52.61 45.49 59.68 18.98 20.59 22.98 91.59

40 55.90 49.64 66.04 20.86 21.90 25.39 92.09

60 62.43 55.51 72.07 21.58 22.27 31.54 92.59

80 70.59 61.26 78.14 23.50 27.59 39.93 93.35

100 83.80 78.22 86.28 35.97 38.90 47.14 94.47



105

Fig. 3.125. Antioxidant assay of selected medicinal plants-capped Ag/Au NPs

Table 3.10: Antioxidant activity of selected medicinal plants Ag/Au stabilized

NPs

ED Ag-ED Au-ED DE Ag-DE Au-DE STD

Conc.

µg/mL

% DPPH Activity

20 27.241 33.681 65.490 10.79 10.59 15.98 91.59

40 29.160 38.040 68.641 20.50 21.90 23.39 92.09

60 39.597 44.072 72.510 20.86 20.27 25.54 92.59

80 47.908 49.93 73.26 22.54 21.59 31.93 93.35

100 64.770 60.49 78.14 24.82 22.90 39.14 94.47


0

10

20

30

40

50

60

70

80

90

100

20 40 60 80 100

Ab

sorb

an

ce (

%)


RAFE

Ag-RAFE

Au-RAFE

KP

Ag-KP

Au-KP

STD


106

Fig. 3.126. Antioxidant assay of selected medicinal plants-capped Ag/Au NPs


Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella

pneumonia were also employed for measuring antibacterial activity of selected

medicinal plants and their NPs. The results of antibacterial activity of selected

medicinal plants and their mediated synthesized Ag and Au nano-conjugates showed

good activity against all the bacterial strain except Pseudomonas aeruginosa. DE, Ag-

DE, and Au-DE showed moderate activity against P. aeruginosa, inhibitory zone

from 16±0.90 to 18±0.75 mm at 3 µg/mL. KP and its metallic NPs were inactive

against S. aureus. The results are summarized in table 3.11.

0

10

20

30

40

50

60

70

80

90

100

20 40 60 80 100

Ab

sorb

an

ce (

%)


ED

Ag-ED

Au-ED

DE

Ag-DE

Au-DE

STD


107

Table 3.11: Antibacterial activities of selected medicinal plants and their Ag/Au

stabilized NPs

Zone of inhibition (mm)

Bacterial strain Bacillus

subtilis

Staphylococcus

aureus

Pseudomonas

aeruginosa

Klebsiella

pneumonia Sample

RAFE 10±0.04 -- -- 10±0.04

Ag-RAFE 12±0.20 2±0.02 -- 14±0.99

Au-RAFE 9.5±0.03 1±0.05 -- 12±0.65

KP 13±0.09 -- -- 12±0.65

Ag-KP 15±0.50 -- -- 14±0.99

Au-KP 13±0.09 -- -- 12±0.20

ED 15.6±0.08 11±0.75 -- 14±0.99

Ag-ED 17±0.73 13±0.09 -- 16±0.90

Au-ED 16±0.90 12±0.20 -- 12±0.20

DE 11±0.75 8±0.09 18±0.75 14±0.99

Ag-DE 14±0.99 10±0.04 18±0.75 15.6±0.08

Au-DE 12±0.20 9.5±0.03 16±0.90 14±0.99

Streptomycin

(STD)

20±0.98 26±0.09 20±0.98 28±0.04

Well size = 6 mm, STD = Standard, -- Not effective, Mean ± S.E.M.


The selected medicinal plants and there metallic nano-conjugates were also evaluated

for antifungal activity against four fungal strains such as F .oxysporum, A. parasiticus,

A. flavus and C. albicans. Among the four medicinal plants, the two medicinal plants

(RAFE and KP) and their noble metal NPs (Ag-RAFE, Au-RAFE, Ag-KP and Au-


108

KP) showed no activity against C. albicans, while ED, DE and their NPs revealed low

activity against this strain. Almost all medicinal plants and their capped NPs

displayed moderate to good antifungal activity against three fungal strains i.e. F.

oxysporum, A. parasiticus, A. flavus as the results summarized in table 3.12.

Table 3.12: Antifungal activities of selected medicinal plants and their stabilized

Ag/Au NPs

Sample Fusarium

oxysporum

Aspergillus

parasiticus

Aspergillus

flavus

Candida

albicans

RAFE 10 85 52 --

Ag-RAFE 12 88 50 --

Au-RAFE 10 80 50 --

KP 80 87 90 --

Ag-KP 82 85 88 --

Au-KP 80 81 84 --

ED 83 65 70 14

Ag-ED 80 65 70 14

Au-ED 81 69 73 16

DE 15 85 65 10

Ag-DE 10 80 60 8

Au-DE 12 80 63 10

Miconazole

(STD)

100 100 100 100

-- Not effective

Chapter-4 Experimental

109

4.1 General experimental procedures

A digital pH meter model 510 (Oakton, Eutech) equipped with a glass working

electrode and a reference Ag/AgCl electrode was hired for pH measurements. UV

visible spectra were recorded using a Shimadzu UV-240, Hitachi U-3200

spectrophotometer with a path length of 1 cm. Fourier transform infrared (FTIR)

spectra were recorded using a Shimadzu IR-460 spectrophotometer. The shape and

size of nanomedicines were examined using atomic force microscope (AFM),

Multimode, Nanoscope IIIa, Veeco, (California, USA) in the tapping mode and

furthermore, confirmed by scanning electron microscope (SEM) with energy

dispersive X-ray (EDX), (JSM 591 JEOL, Japan).

4.1.1 Collection of fluoroquinolones and plants material

Commercial fluoroquinolones along with tablets (moxifloxacin, ciprofloxacin,

sparfloxacin and gemifloxacin) for parallel study were purchased from local

pharmaceutical market Peshawar and selected medicinal plants (Rhododendron

arboreum, Kigelia pinnata, Eulophia dabia and Desmodium elegans) were collected

from designate area of Pakistan. In February 2011, R. arboreum plant was collected

from Seran Valley, Khyber Pakhtunkhwa, Pakistan. A voucher specimen no.

7212/Bot has been deposited in the National Herbarium of Peshawar University,

Pakistan, while the fruits of K. pinnata were collected from University of Sargodha,

Punjab, Pakistan and identified by an experienced taxonomist Dr. Abdur Rashid,

Department of Botany, University of Peshawar, Pakistan. The E. dabia plant material

was collected from Khwazakhela, Swat, Khyber Pakhtunkhwa, Pakistan, during April

2011 and identified by taxonomist Dr. Hassan Sher, University of Swat, Pakistan. D.

elegans plant was collected during the summer season from Gallyat area, Khyber

Pakhtunkhwa, Pakistan, which was identified by Dr. Abdur Rashid, Department of


110

Botany, University of Peshawar, Pakistan. A reference voucher (726/Bot.) was

deposited in the National Herbarium of Peshawar University, Pakistan.

Silver nitrate (AgNO3) was purchased from Sigma-Aldrich, while hydrogen

tetrachloroaurate (III) trihydrate (HAuCl4 3H2O), sodium carbonate (Na2CO3),

sodium chloride (NaCl), sodium hydroxide (NaOH) and hydrochloric acid (HCl) were

purchased from Merck. Sodium borohydride (NaBH4) was obtained from Wako Pure

Chemical Industries Ltd. Deionized water was used throughout the reactions for the

synthesis of Ag and Au NPs.

4.1.2 Preparation of stock solution

Fresh solutions of metal salts (1 mM AgNO3 and 1 mM HAuCl4) and ligand (1 mM

fluoroquinolones) were prepared in deionized water, while crude extracts of selected

medicinal plants (0.5 g) were dissolved in distilled water (100 mL). The stock

solutions were stored in refrigerator for further studies. Fresh stock solutions (1 M) of

NaCl, HCl and NaOH were also prepared for checking salt and pH stability of Ag/Au

NPs.

4.1.3 Synthesis of Ag and Au NPs capped with fluoroquinolones

Fluoroquinolones were synthesized using NaBH4 as a moderate reductant. Reactions

were carried out by mixing different volumes of fluoroquinolones and Ag/Au salts

solutions. The reaction mixture was stirred vigorously for about 30 minutes at

ambient temperature and then 0.2 mL of 50 mM NaBH4 was added drop wise. A

gradual change in the color of reaction mixture was used as a clue to the formation of

the desired products i.e. fluoroquinolones-capped Ag and Au NPs. After the addition

of a reducing agent, the light yellow solution gradually turned maroon followed by

brown and eventually ruby red, depending on the molar ratio of metals to ligands.


111

Optimization of reaction condition was achieved by varying the molar ratio of metals

to ligands (fluoroquinolones), which resulted in the appearance of the highest

absorption peak in the proposed UV visible region. The residual metal salts and drugs

were removed by centrifugation at 10,000 rpm and the supernatants were freeze dried,

which contained fluoroquinolones-capped Ag and Au NPs. The synthesis of Ag and

Au nano-conjugates was further confirmed by using FTIR, AFM, EDX and SEM.

Bioactivity of these Ag and Au NPs having suitable size and shape were performed

and evaluated accordingly.

4.1.4 Green synthesis of metallic NPs by using selected medicinal plants

The selected medicinal plants (R. arboretum, K. pinnata, E. dabia and D. elegans)

were washed with distill water and air dried under shade. The plants materials were

then ground to obtain fine powder. Shade dried samples (20 g) were kept in conical

flasks and 100 mL of methanol/water solution (70:30 v/v) were added to each conical

flask. The flasks were placed for 10 days and the visual appearances of the liquids

were monitored. The extracts were filtered and utilized as reducing and stabilizing

agents in the synthesis of noble metal NPs.

The green synthesis of metal NPs involved the mixing of aliquot amounts of (I mM)

AgNO3 and HAuCl4.3H2O with selected medicinal plants in deionized water at room

temperature for 30 minutes. The formation of NPs was indicated by an optical color

change to brownish-yellow and pinkish-red solutions indicating the formation of Ag

and Au NPs respectively. To search out the optimized conditions for the synthesis,

different ratios of metals to extracts were used; keeping metal (Ag/Au) constant and

varying the amount of ligands (R. arboretum, K. pinnata, E. dabia and D. elegans).

The ratios which contributed best SPR absorption bands were subjected for further

studies. The metallic NPs were then isolated by centrifugation followed by freeze

drying for characterization and other analyses.


112

4.2 Biological evaluation

Fluoroquinolones and selected medicinal plants along with their Ag/Au NPs were

subjected for various biological activities to evaluate and explore their therapeutic and

medicinal importance. These activities included urease enzyme inhibition,

leishmanicidal, antioxidant and antimicrobial.

4.2.1 Protocol for urease assay and inhibition

Reaction mixtures consisting Jack bean (Canavalia ensiformis) urease (25 μL), 100 mM

of urea, 55 μL of buffer at pH 6.8 and 5 μL of diverse concentrations of test samples

(0.5 to 0.00625 mM) were hatched in 96-well plates at temperature of 30°C for 15

minutes. In kinetics studies, different concentrations of test compounds and substrates

were employed. Subsequently, 45 μL phenol reagents (1% w/v phenol and 0.005% w/v

sodium nitroprussside), and 70 μL of alkali (0.5% w/v NaOH and 0.1% w/v NaOCl)

were added to discrete well. Afterward 50 minutes, the cumulative absorbance at 630

nm was recorded in triplicate in a microplate reader (SpectraMax M2, Molecular

Devices, CA, USA). The making of ammonia (NH3) was measured by using

indophenol method with a standard (thiourea) [165]. Eventually the results were

administered by software SoftMax Pro (Molecular Devices, CA, USA), MS-Excel and

Ez-fit programs. For calculating percent inhibition, the following formula was used:

% Inhibition = 100-(OD test /OD control) ×100.

4.2.2 Procedure for leishmanicidal activity (in vitro)

For this bioassay, Leishmania tropica promastigotes were cultured at 22-25°C in

RPMI-1640 (Sigma) as testified earlier [35]. The medium was complemented with 10%

temperature deactivated (56°C for 30 min) fetal bovine serum (FBS). The

promastigotes culture at logarithmic phase was sedimented at 800 rpm, calculated with


113

the help of advanced Neubaver chamber under the microscope and in the same

condition, washed with saline three times. The last density of 2x106 cells/mL was

attained by diluting the parasites with new culture medium. In 96-well micro titer plate,

180 mL of medium was subjected in the main row, while 100 mL of medium was put in

others wells. The substances to be tested were dissolved to an ultimate concentration of

1.0 mg in 0.1 mL of PBS (Phosphate Buffered Saline, pH 7.4 comprising 0.5% CH3OH,

0.5 % DMSO). One row for standard drugs (Pentamidine and ampicilline to a final

concentration of 1.0 mg/mL was added separately as positive control), while one was

used for control (DMSO) which received medium. Dish was incubated at 22-24°C for

72 h. Data are the replicates of three diverse experiments. The 50% inhibitory

concentrations (IC50) were designed by EZ-Fit 5.03 Perrella Scientific Software.


The antioxidant assay was performed by the DPPH radical scavenging method,

according to previous literature reported and standard protocol [166]. The electron

donation abilities of the Ag/Au NPs and standards were evaluated from the varying of

the purple colored methanol solution of 2, 2-diphenyl-1-picrylhydrazyl (DPPH).

Sample solution of 3 mL (drugs/medicinal plants and noble metal NPs) was variegated

with 1 mL of the 1 mM solution of the DPPH solution in methanol, different

concentrations, i.e. (10-100 µg/mL) for precursors and NPs have been organized, while

control has only methanol and DPPH. The mixed solutions have shaken vigorously and

kept for 30 minutes in the dark and then absorbance was recorded at exactly 517 nm.

The decrease of the 2, 2-diphenyl-1-picrylhydrazyl solution absorbance displays an

increase in the free radical scavenging assay. DPPH as percent radical scavenging

activities (% RSA) was determined as below.

% DPPH = (OD control - OD sample) / OD control X 100


114

Where, OD control is the absorbance of the blank sample and OD sample is the

absorbance of samples.

4.2.4 Bacterial strains assortment and preservation

Two selected strains of Gram positive bacteria (Bacillus subtilis and Staphylococcus

aureus) and two of Gram negative bacteria (Pseudomonas aeruginosa and Klebsiella

pneumonia) were obtained from the stock culture of Phytopharmaceutical and

Neutraceutical Research Laboratory (PNRL); Institute of Chemical Sciences,

University of Peshawar, Peshawar, Pakistan and stored in Mueller Hinton agar at low

temperature (4 0C) prior to subculture.


The bactericidal assay was executed by well diffusion process with Mueller Hinton

agar. The culture was developed in triplicates at 37 °C for 72 h. The broth standards

(0.6 mL) of the species were located in a sterilized Petri dish and then 20 mL of the

sterilized molten Mueller Hinton Broth (MHB) was employed. Streptomycin (2

mg/mL) was used as a standard. Inoculation was accomplished for 1 h to make possible

the diffusion of the antibacterial mediator into the medium. Incubation for 24 h at 37 °C

and the breadths of the zone of inhibition of bacterial development was calculated in the

plate in millimeter (mm). The activities were repeated in triplicate.


Agar tube dilution technique was used to carry out the antifungal activity of precursors

and their NPs as reported procedure [167]. Sterile Sabouraud‟s dextrose agar media was

prepared and 5 mL of media was put in each screw capped test tubes and autoclaved at

121 °C for 15 min. Each sample (24 mg) was dissolved in DMSO (1 mL) in effendorfs.

66.6 μl of solution was put in test tubes in triplicates and kept at slanted position then


115

four fungal strains of 4 mm diameter were inoculated. All the test tubes were kept

upright in incubator at 27-29 °C for 3-7 days and were daily checked. On the 7th

day

measurement was taken with scale and percentage growth inhibition was calculated

according to formula;

100*)(

)(100%

mmcontrolingrwothlinear

mmsampletestingrowthlinearInhibition

Conclusion

116

Conclusion

Modern synthesis focuses on the discovery and the formation of selective noble

metal nanomaterials. Employing sodium borohydride (NaBH4) as a mild

reductant, a convenient protocol to produce Ag and Au NPs capped with

fluoroquinolones was developed.

The other reducing agents such as triethylamine and quinol were also tested but

failed to show anticipated results.

The spectroscopic study revealed that the amine moiety and carboxylate group of

the substrate FQs are responsible for promoting capping of these Ag and Au

nano-conjugates.

The energy-dispersive X-ray (EDX) analysis demonstrated the inorganic

composition of the synthesized Ag/Au NPs. The synthesized pro-nanomedicines

of noble metals showed stability to some extent by changing pH, concentration of

table salt and temperature.

Furthermore, the fluoroquinolone-capped noble metal NPs showed inhibition

against the urease enzyme and also exhibited good antimicrobial and

leishmanicidal activity while displayed low activity against % DPPH scavenging.

The selected medicinal plants mediated biosynthesis of Ag/Au NPs approach

is a facile, green and economical process which yields highly stable Ag/Au

NPs. These medicinal plants were used for the first time for the synthesis of

Ag/Au nano-conjugates by bio-reduction of aqueous Ag+/Au

+ ions. The noble

metal NPs of R. arboretum, K. pinnata, and E. dabia were synthesized through

green selective mode of capping approach. They acted as reductants and

efficient stabilizers, while for the synthesis of D. elegans capped metallic NPs,

trimethylamine was used as reducing agent. It was concluded that this plant

has no active phytochemicals to reduce Ag and Au.

Conclusion

117

Stability of these metal capped NPs was ascertained by optimizing the

experimental parameters such as the effect of salt (1M NaCl), pH and

temperature. Both noble metals NPs showed good pH stability.

Preliminary bioassay screening plays a very vital role in the drug development

program. It provides an opportunity for bioactivities and thus helps in the

selection of leads like compounds for secondary screening of detailed

pharmacological evaluations. Certainly, new or modified therapeutic agents

always possess through the preliminary bioassay system with an acceptable

safety profile. Biological assays are the best skill of finding valuable and

precious substituents present in medicinal plants and their nanomaterials.

Various biological activities have been carried out for noble metal NPs

capped with fluoroquinolones and selected medicinal plants to explore the

hidden potentials. These bioactivities included leishmanicidal, antioxidant,

urease enzyme inhibition and antimicrobial. Ag-Mox, Cip, Ag-Cip, Au-Cip

Sp, Ag-Gm, showed significant leishmanicidal activity with (IC50 µg/mL)

9.939, 11.32, 11.80, 10.46, 11.12, and 12.12 µg/mL respectively, while the

activity of Mox, Au-Mox, Ag-Sp, Au-Sp, Gm, and Au-Gm were good with

(IC50 µg/mL) 10.50, 10.45, 10.69, 8.816, 5.170, and 9.939 µg/mL respectively.

Ag-RAFE (IC50 µg/mL = 9.45) and Ag-ED (IC50 µg/mL = 7.20) also showed

potential leishmanicidal activity against Leishmania tropica promastigotes.

Conclusively, different sized Ag and Au NPs with multi-functions were

synthesized using different capping agents.

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List of Publications

137

List of Publications

1. Nisar, M., Khan, S. A., Qayum, M., Khan, A., Farooq, U., Jaafar, H. Z., &

Ali, R. (2016). Robust Synthesis of Ciprofloxacin-Capped Metallic

Nanoparticles and Their Urease Inhibitory Assay. Molecules, 21(4), 411.

2. Nisar, M., Khan, S. A., Shah, M. R., Khan, A., Farooq, U., Uddin, G., &

Ahmad, B. (2015). Moxifloxacin-capped noble metal nanoparticles as

potential urease inhibitors. New Journal of Chemistry, 39(10), 8080-8086.

3. Nisar, M., Khan, S. A., & Ali, I. (2013). GC-MS Analysis and

Pharmacological Potential of Fixed Oil of Eluphia dabia. Middle-East Journal

of Scientific Research, 14(3), 375-380.

Biograpy

138

BIOGRAPY

The author Mr. Shujaat Ali Khan was born on Feb. 20, 1987 in

village Mashkomai, Tehsil Khwazakhela, District Swat and did

his Secondry School Certificate (matric) from Govt. High school

Chamtali, Swat in 2003, subsequently he passed his Higher

Secondary Examination (HSSC) from Govt. Degree College Matta, Swat. He got

admission in M.Sc. (2008-2009) in the field of Organic Chemistry from Institute of

Chemical Sciences, University of Peshawar. Then he completed B.Ed program (2010)

from University of Peshawar. He enrolled in M. Phil leading to Ph.D program at

Institute of Chemical Sciences, University of Peshawar session, 2010-11 under the

supervision of Prof. Dr. Muhammad Nisar. His Ph.D research work was based on

“Synthesis, Spectroscopic Analyses and Biological Evaluation of Silver and Gold

Based Pro-nanomedicines derived from Fluoroquinolones”. During the research

period, the author participated various national and international conferences.

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