Green Synthesis of Multifunctional Metal Oxide ...

Green Synthesis of Multifunctional Metal Oxide Nanoparticles

from Medicinal Plants and their Biological Applications

By

Javed Iqbal

Department of Plant Sciences

Faculty of Biological Sciences

Quaid-i-Azam University

Islamabad, Pakistan

2020

ii

Green Synthesis of Multifunctional Metal Oxide Nanoparticles

from Medicinal Plants and their Biological Applications

A thesis

Submitted to the Department of Plant Sciences in the partial fulfillment of the

requirement for the degree of

Doctor of Philosophy (D. Phil)

in

Plant Sciences

By

Javed Iqbal

Department of Plant Sciences

Faculty of Biological Sciences

Quaid-i-Azam University

Islamabad, Pakistan

2020

iii

SPECIAL THANKS

I pay my special thanks to my supportive Mother (Hussan Bibi),

Father (Abdul Janan), loving, caring brothers, sisters and supportive

family for their countless love, care, encouragement, guidance and

support throughout my life.

iv

ACKNOWLEDGEMENTS

All praise to Allah Almighty, The most beneficent, the most merciful, who gave me

strength and enabled me to undertake and execute this research task. Countless salutations

upon the Holy Prophet Muhammad (SAWW), source of knowledge for enlightening with

the essence of faith in Allah and guiding the mankind, the true path of life.

I would like to extend my deepest appreciation to those people, who helped me in

one way or other in planning and executing this research work and writing up this thesis

manuscript. I am thankful to Prof. Dr. Tariq Mahmood my PhD supervisor and Dean,

Faculty of Biological Sciences for extending the research facilities of the department to

accomplish this work. I feel highly privileged in taking opportunity to express my deep

sense of gratitude to my supervisor for his scholastic guidance and valuable suggestions

throughout the study and presentation of this manuscript. I am thankful to him for his

inspiration, reassurance and counseling from time to time. I would like to thank Dr. Samad

Mumtaz, Chairman Department of Plant Sciences for his kind support. It is inevitable to

thanks my foreign supervisor Prof. Dr. Ali Raza Khademhosseini, for his humble

support, professional guidance and continuous encouragement during and after my

research visit to University of California, Los Angles under International Research

Support Initiative Program (IRSIP), Pakistan. Their effort and opinions are greatly

appreciated. Perhaps, I would not be able to present this work in present form without

cooperation of Higher Education Commission (HEC) Pakistan for funding me through

IRSIP program.

I also want to say a big thank to Prof. Dr. Irshad Hussain and Dr. Akhtar Munir,

Department of Chemistry, Lahore University of Management Sciences and Prof.

Nureddin Ashammakhi, Dr. Mehboobah Mehmoodi, Dr. Muhammad Ali Darabi,

Post doc fellows in UCLA Los Angeles, for their cooperation throughout my research

activities here in Pakistan and in USA. I also acknowledge the humble cooperation of Liaq

Zia, Muhammad Ashraf, Abdul Jamal Nasir, Ali Talha Khalil, Ikram Ullah, Shakeeb

Afridi, Tanveer Khan, Riaz Ahmed, Banzeer Ahsan Abbasi, Abdul Majid and Abdul

Qyyum.

v

It is inevitable to thank all of my senior PhD lab fellows; Dr. Shazia, Dr. Riffat, Dr.

Wasim, Dr. Ejaz, Dr. Fatima, Yasrab Aman, Dr. Irum for their encouragement and

keen interest. I am also thankful to all junior lab fellows; Banzeer, Anber, Muzaffar,

Amir, Wasba, Sana, Saqlain, Farhat, Sheeza, Haleema, Hina. I wish to express sincere

thanks to my senior-juniors colleagues of department for their continued encouragement,

moral support and necessary guidance. Heartiest thanks to my time-tested friends Syed

Afzal Shah, Banzeer Ahsan Abbasi, Maqsood Alam and Riaz Ahmed for their

enjoyable company, care and concern, persistent support and encouragement in my

research and personal problems.

Solemn gratitude to my Father, Mother, Brothers and Sisters who deserve special

mention for their inseparable support and prayers. They have always been my source of

strength and love. It wouldn’t have been this bearable if I didn’t have these in my life.

Thank you for your unconditional support with my studies. Thank you for giving me a

chance to prove and improve myself through all walks of my life. Finally, I would like to

thank everybody who was important to the successful completion of thesis, as well as

expressing my apology that I could not mention personally one by one.

Javed Iqbal

vi

Foreign Examiners

1). Dr. Ata-ur-Rehman

Senior Research associate, E.H Graham center for Agricultural Innovation School of

Biomedical Sciences, Building 288, Charles Sturt University, Wagga Wagga, NSW 2650

Australia

2). Prof. Dr Fatma Zerrin Saltan

Controller of exams, Anadolu University Faculty of Pharmacy Department of

Pharmacognosy 26470, Eskisehir-Turkey

vii

APPROVAL CERTIFICATE

This is to certify that the dissertation entitled “Green Synthesis of Multifunctional

Metal Oxide Nanoparticles from Medicinal Plants and their Biological

Applications” submitted by Javed Iqbal is accepted in its present form by the

Department of Plant Sciences, Quaid-i-Azam University Islamabad, Pakistan, as

satisfying the dissertation requirement for the degree of Ph.D. in Plant Sciences.

Supervisor: ____________________________

Prof. Dr. Tariq Mahmood Department of Plant Sciences,

Quaid-i-Azam University, Islamabad

External Examiner-I: _____ _____________

Dr. Tayyaba Yasmin Tenured Associate Professor,

Department of Biosciences,

COMSATS University, Park Road, Chak Shahzad, Islamabad

External Examiner-II: ____________________________

Prof. Dr. Ghazala Kaukab

University Institute of Biochemistry and Biotechnology PMAS-Arid Agriculture University, Rawalpindi

Chairman: ___________________________

Prof. Dr. Abdul Samad Mumtaz Department of Plant Sciences,

Quaid-i-Azam University, Islamabad

Date: 27th July, 2020

viii

PLAGIARISM UNDERTAKING

I solemnly declare that research work presented in the thesis titled “Green Synthesis of

Multifunctional Metal Oxide Nanoparticles from Medicinal Plants and their

Biological Applications” is solely my research work with no significant contribution from

any other person. Small contribution/help wherever taken has been duly acknowledge and

I agreed that complete thesis has been written by me.

I understand the zero-tolerance policy of the HEC and Quaid-i-Azam University,

Islamabad towards plagiarism. Therefore, I as an author of the above titled thesis declare

that no portion of my thesis has been plagiarized and any material used as reference is

properly referred/cited.

I undertake that if I am found guilty of any formal plagiarism in the above titled thesis even

after award of PhD degree, Quaid-i-Azam University, Islamabad reserves the rights to

withdraw/revoke my PhD degree and that HEC and Quaid-i-Azam University, Islamabad

has the right to publish my name on the HEC/University website on which names of

students are placed who submitted plagiarized thesis.

Student/ Author Signature: _______________

Name: Javed Iqbal

ix

QUAID-I-AZAM UNIVERSITY

DEPARTMENT OF PLANT SCIENCES

No.______________ 28th July 2020

Subject: Author Declaration

I Mr. Javed Iqbal hereby state that my Ph.D. thesis titled “Green Synthesis of

Multifunctional Metal Oxide Nanoparticles from Medicinal Plants and their

Biological Applications” is my own work and has not been submitted previously by me

for taking any degree from Department of Plant Sciences, Quaid-i-Azam University

Islamabad or anywhere else in the country/world.

At any time if my statement found to be incorrect even after my graduate, the

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

Javed Iqbal

Contents

Chapter 1: Introduction 2

1.1 Nanotechnology: a new frontier 3

1.2. Methods of nanoparticles synthesis 4

1.2.1. Synthesis of MNPs by Physical and chemical methods 4

1.2.2. Bio-inspired synthesis of nanoparticles 6

1.3. Multiple factors affecting the Green synthesis of NPs 8

1.3.1. Protocols used for the synthesis 9

1.3.2. Effects of pH 9

1.3.3. Effect of Temperature 9

1.3.4. Effect of Pressure 10

1.3.5. Effect of plant extract concentration 10

1.3.6. Effect of metal ions concentrations 10

1.3.7. Effect of time 11

1.3.8. Preperation cost 11

1.3.9. Effects of Proximity 11

1.4. Different green metal oxides nanoparticles 11

1.4.1. Iron oxide (γ-Fe2O3) 13

1.4.2. Cobalt oxide (Co3O4) 13

1.4.3. Nickel oxide (NiO) 14

1.4.4. Zinc oxide (NiO) 14

1.5. Medicinal plants used for green synthesis of nanoparticles 15

1.5.1. Geranium wallichianum D.Don ex Sweet 16

1.5.2. Rhamnus virgata Roxb. 16

xii

1.5.3. Rhamnella gilgitica Mansf. & Melch. 17

1.6. Scope of study 17

1.7. Aims and objectives 18

Chapter 2: Biogenic synthesis of green and cost effective iron nanoparticles and

evaluation of their potential biomedical properties 19

2.1. Introduction 22

2.2. Material and methods 24

2.2.1. Rhamnella gilgitica leaves extract preparation 24

2.2.2. Biofabrication of the targated IONPs 24

2.2.3 Physico-chemical characterizations of IONPs 24

2.2.4. Preparation of test sample of IONP 26

2.2.5. Biological potentials of IONPs 26

2.2.5.1. Antibacterial activity of IONPs 26

2.2.5.2. Antifungal activity of IONPs 26

2.2.5.3. PK (Protein kinase) inhabitation 26

2.2.5.4. AA (Alpha amylase) inhibition (antidiabetic potential) 27

2.2.5.5. Anticancer potential 27

2.2.5.6. Antileishmanial potential 28

2.2.5.7. Brine shrimp cytotoxicity 28

2.2.5.8. Antioxidant capacities of IONPs 29

2.2.5.9. Biocompatibility of IONPs with human RBCs 29

2.2.5.10. Biocompatibility of IONPs with human Macrophages 30

2.3. Results and Discussion 30

xiii

2.3.1. In vitro Antimicrobial activities 37

2.3.2. Enzymes inhibition potential 41

2.3.3 Antileishmanial potential 43

2.3.4. Brine shrimps cytotoxicity 45

2.3.5. Anticancer activities 45

2.3.6. Biocompatibility assays 45

2.3.7. Antioxidant activities of IONPs 47

Chapter 3: Plant-extract mediated green approach for the synthesis of ZnONPs:

Characterization and evaluation of cytotoxic, antimicrobial and antioxidant

potentials 50


3.2. Experimental methods 55

3.2.1. Green-route synthesis of ZnONPs 55

3.2.2. Characterizations techniques of ZnONPs 56

3.2.3 Test sample preparation of ZnONPs 57

3.2.4. Biological activities of biogenic ZnONPs 57

3.2.4.1. Antibacterial activity of ZnONPs 57

3.2.4.2. Antifungal activity of ZnNPs 57


3.2.4.4. Antileishmanial activity 58

3.2.4.5. Anticancer assay 59

3.2.4.6. Biocompatibility with human RBCs 59

3.2.4.7. Biocompatibility with human macrophages 59

xiv

3.2.4.8. PKs inhibition activity 60

3.2.4.9. Alpha amylase inhibition activity 60

3.2.4.10. Antioxidant capacities 60


3.3.1. Biogenic synthesis of ZnONPs 61

3.3.2. Physical characterization 61

3.3.3 Antimicrobial activities 66

3.3.4. Antileishmanial activity 71

3.3.5. Anticancer activities 71

3.3.6. Brine shrimps lethality assays 73

3.3.7. Biocompatibility test 73

3.3.8. Enzyme inhibition potential 75

3.3.9. Antioxidant activities 76

Chapter 4: Biogenic synthesis of green and cost effective cobalt oxide nanoparticles

using Geranium wallichianum leaves extract and evaluation of in vitro antioxidant,

antimicrobial, cytotoxic, enzyme inhibition properties 78


4.2. Material and methods 83

4.2.1. Sampling of plant and identification 83

4.2.2. Biosynthesis of CoONPs 83

4.2.3 Characterization of CoONPs 83

4.2.4. Biological activities of CoONPs 84

4.2.4.1. Antibacterial activity of CoONPs 84

xv

4.2.4.2. Antifungal activity of CoONPs 84

4.2.4.3. Antileishmanial activity 84


4.2.4.5. Anticancer assay 85

4.2.4.6. Biocompatibility with human RBCs 85

4.2.4.7. Biocompatibility with human macrophages 86



4.2.4.10. Antioxidant capacities 86


4.3.1. Biogenic synthesis of CoONPs 87

4.3.2. Physical characterization 88

4.3.3 Biological activities 91

4.3.3.1. Bactericidal activity 91

4.3.3.2. Antifungal activities 95

4.3.3.3. Brine shrimps lethality assays 97

4.3.3.4. Antileishmanial assay 97

4.3.3.5. Anticancer activity of CoONPs against HepG2 cancer cells 99

4.3.3.6. Biocompatibility assay 102

4.3.3.7. Antioxidant activities 102

4.3.3.8. Enzyme inhibition potential 104

Chapter 5: Green synthesis and characterizations of Nickel oxide nanoparticles using

leaf extract of Rhamnus virgata and their potential biological applications 107

xvi


5.2. Experimental methods 112

5.2.1. Preparation of plant extract 112

5.2.2. Synthesis of nickel oxide nanoparticles 112

5.2.3 Characterizations techniques 113

5.2.4. Cytotoxic assay 113

5.2.4.1. Brine shrimps cytotoxicity 113

5.2.4.2. Antileishmanial activity of NiONPs 115

5.2.4.3. Anticancer activity 115

5.2.5. Antimicrobial activities 115

5.2.6. Enzymes inhibition potentials 116



5.2.7. Biocompatibility assays 117

5.2.7.1. Biocompatibility against human RBCs 117

5.2.4.2. Biocompatibility against human Macrophages 117

5.2.8. Antioxidant assay 117


5.3.1. Physical characterizations of the NiONPs 118

5.3.2. Cytotoxic assay 119

5.3.2.1. Brine shrimps cytotoxicity 119

5.3.2.2. Antileishmanial activity of NiONPs 119

5.3.2.3. Anticancer activity 124

xvii

5.3.3 Antimicrobial activities 124

5.3.3.1. Antibacterial activity 124

5.3.3.2. Antifungal activity of NiONPs 124

5.3.4. Enzyme inhibition activity 125

3.3.5. Biocompatibility activity 130

5.3.6. Antioxidant activity 130

5.4. Discussion 133

Conclusion and future perspectives 137

Conclusions 138

Future perspectives 139

References 141

List of Figures

Chapter 1

Figure 1.1: Different approaches and strategies for the synthesis of nanoparticles 5

Figure 1.2: Biofabrication of NPs using green technology 7

Chapter 2

Figure 2.1: Study plan for biogenic IONPs 25

Figure 2.2: UV analysis of IONPs (a) UV visible spectra (b) Stability of biosynthesized

IONPs 32

Figure 2.3: Various SEM and TEM images of IONPs (a,b,c) SEM images (d) TEM image.

. 33

Figure 2.4: (a) FTIR spectra of IONPs (b) Raman spectra of biosynthesized IONPs 34

Figure 2.5: (a) Elemental composition using EDX; (B) Schematic representation for the

green synthesis of NPs and their effective stabilization 35

Figure 2.6: XRD spectral analysis for biogenic IONPs (a) XRD spectra of Rhamnella

gilgitica based IONPs (b) Size calculation via scherer equation 36

Figure 2.7: (a) Zeta potential measurement of IONPs (b) Particle size measurement 38

Figure 2.8: (a) Antibacterial activity ob biogenic IONPs (b) Antibacterial activity of

biogenic IONPs 42

Figure 2.9: (a) Inhibition potental of green IONPs against protein kinase; (B): Inhibition

potental of green IONPs against alpha amylase 44

Figure 2.10: Cytotoxicity activity of RG-IONPS against leishmania (b) Cytotoxicity

against brineshrimps (c) Anticancer activities of IONPs 46

Figure 2.11: (a) Biocompatability potential against human RBCs and macrophages (b)

Antioxidant potentials of IONPs 49

Chapter 3

Figure 3.1:Study design for the biosynthesis of NPs 56

Figure 3.2: UV and XRD spectral anaysis of ZnONPs (a) UV visible spectra (b) Stability

of biosynthesized ZnONPs (C) XRD analysis of Rhamnus virgata mediated ZnONPs 63

xix

Figure 3.3: Various SEM and TEM images of RV-IONPs (a,b,c): SEM images (b)TEM

image 64

Figure 3.4: Raman spectra of ZnONPs biosynthesized using zinc nitrate hexahydrate as

precursor (b) FTIR spectra of bioinspired zinc oxide nanoparticles 65

Figure 3.5: Elemental composition of green ZnONPs using EDS analysis 67

Figure 3.6: (a) Size distribution of RV-ZnONPs (b) Zeta potential measurement of ZnONPs

. 68

Figure 3.7: (a) Antibacterial activities of biogenic ZnONPs (b) Antifungal potential of

biogenic ZnONPs 70

Figure 3.8: (a) Antileishmanial activity of Rhamnus virgata mediated ZnONPs by biogenic

IONP’s (b) Cytotoxicity against HepG2 cell lines (c) Cytotoxicity against brine shrimps 74

Figure 3.9: (a) Biocompatibility of Rhamnus virgata mediated ZnONPs against human

RBCs and Macrophages (b) Inhibition potential against protein kinase (c) Inhibition

potential against alpha amylase (d) Antioxidant potentoal of Rhamnus virgata mediated

ZnONPs 77

Chapter 4

Figure 4.1: Schematic description of biogenic CoONPs via green route 90

Figure 4.2: UV and XRD spectra of biogenic CoONPs (a) UV visible spectra (b) Stability

of biosynthesized CoONPs (c) XRD spectra of CoONPs (d) Size calculation via schrer

approximation 92

Figure 4.3: SEM images of CoONPs 93

Figure 4.4: (a) EDS analysis of Geranium wallichianum mediated CoONPs (b) Raman

spectra of CoONPs 94

Figure 4.5: (a) FTIR spectra, Zeta potential and size distribution analysis of biogenic

CoONPs (a) FTIR spectra (b) Zeta potential (c) Size distribution of biogenic CoONP 95

Figure 4.6: Antimicrobial activities of CoONPs (a) Antibacterial potentials of CoONPs

with UV illumination(b) Antibacterial potentials of CoONPs without UV illumination (c)

Antifungal potential of CoONPs 98

xx

Figure 4.7: Cytotoxicity assay of biogenic CoONPs (a): Cytotoxicity against brine shrimps

(b) Cytotoxicity against Leishmania tropica (c) Cytotoxicity against HepG2 cancer cell

lines 100

Figure 4.8: Schematic on the cytotoxic properties of bioinspired CoO as reported in

literature 102

Figure 4.9: (a) Biocompatability against human RBCs and Macrophages (b) Different

antioxidant activities (c) Inhibition potential against protein kinase (d) Inhibition potential

against alpha amylase 106

Chapter 5

Figure 5.1: Study plan for biogenic synthesis of NPs 114

Figure 5.2: Spectral analysis of biogenic NiONPs (a) UV visible spectra (b) Stabitity of

biogenic NiONPs (c) FTIR spectra (d) Raman spectrum 120

Figure 5.3: (a,b,c) SEM photograph of green NiONPs (d) TEM of greenly orchestrated

NiONPs 121

Figure 5.4: (a) XRD spectra of NiONPs (b) Size calculation via scherer equation 122

Figure 5.5: DLS, Zeta potential, Size distribution and EDS analysis of green NiONPs (a)

DLS size distribution (b) Zeta potential (c) EDS analysis 123

Figure 5.6: Cytotoxicity assays for NiONPs (a) Cytotoxicity against brine shrimps (b)

Cytotoxicity against Leishmania tropica (c) Cytotoxicity against HepG2 cancer cells 125

Figure 5.7: Diagrammetic representation of cytotoxicity of NiONPs as reported in the

literature 126

Figure 5.8: Antimicrobial potentials of NiONPs (a) Antibacterial potentials of NiONPs

without UV illumination(b) Antibacterial potentials of NiONPs with UV illumination (c)

Antifungal potential of NiONPs 128

Figure 5. 9: Biocompatability, antioxidant and enzyme inhibition assays of NiONPs (a)

Inhibition potential against protein kinase (b) Inhibition potential against alpha amylase (c)

Biocompatability against human RBCs and Macrophages (d) Different antioxidant

activities of NiONPs 131

xxi

Figure 5.10: Schematic representation for the Rhamnus virgata mediated NiONPs via

green route 136

xix

List of Tables

Chapter 2

Table 2.1: Zeta potential measurement of IONPs 39

Chapter 3

Table 3.1: Major XRD values and calculation of crystalline size 72

Chapter 4

Table 4.1: MIC calculations of different microbial strains 98

Table 4.2: IC50 values of different biological assay 103

Chapter 5

Table 5. 1: MIC calculations of different bacterial strains (BS) 127

Table 5. 2: IC50 calculation for lead oxide nanoparticles 129

Table 5. 3: IC50 values calculation for different assays using GraphPad software 132

xx

List of abbreviations

eV electron volt

g gram

µL microliter

µg microgram

mg milligram

mL milliliter

nm nanometer

MIC Minimum inhibitory concentration

MTT Methylethiozoltetrazolium bromide

DPPH Diphenyle dipicrylhydrazyl

DMSO Dimethyle sulfoxide

HBSS Hanks buffer salt solution

PBS Phosphate buffer saline

IC50 Median lethal concentration

pH Hydrogen ion concentration

XRD X-ray diffraction

FTIR Fourier transformed infrared spectroscopy

EDS Energy dispersive spectroscopy

SAED Selected area electron diffraction

HR-SEM High resolution scanning electron microscopy

HR-TEM High resolution transmission electron microscopy

xxiv

UV Ultraviolet

RBC’s Red blood cells

OD Optical density

FWHM Full width half maximum

ATCC American type culture collection

FCBP Fungal culture bank of Pakistan

ROS Reactive oxygen species

Summary

________________________________________________________________________

______________________________________________________________________________________

1

Summary

Green synthesis of nanoparticles using medicinal plants has become a promising substitute

to traditional physical and chemical approaches. In the current study, different metal oxide

nanoparticles (MNPs) were successfully synthesized using one-pot, eco-friendly, green

synthesis method using aqueous leaves extracts of medicinal plants Rhamnus virgata (RV),

Geranium wallichianum (GW), Rhamnella gilgitica (RG). Further, the greenly

orchestrated Fe2O3 (Iron oxide), NiO (Nickel oxide), ZnO (Zinc oxide) and Co3O4 (Cobalt

oxide) nanoparticles were extensively studied using different microscopic and

spectroscopic techniques such as UV-Vis (Ultraviolet-Visible), XRD (X‐ray diffraction),

FT-IR (Fourier‐transform infrared), Raman, DLS (Dynamic light scattering), EDS

(Energy-dispersive X-ray spectroscopy), SEM (Scanning electron microscopy) and TEM

(Transmission electron microscopy). The average sizes of nanoparticles were found to be

20 nm (ZnO), 21 nm (Fe2O3), 24 nm (NiO), and 21 nm (Co3O4). Further, targated NPs were

evaluated for different biological activities such as antibacterial, antifungal, anticancer,

enzymes inhibition, antileishmanial, antioxidants and biocompatibility assays.

Antibacterial potencies were investigated using different pathogenic bacterial strains via

Disc diffusion method revealing significant antibacterial potentials. This method was used

to confirm the antifungal activities using different fungal strains. Significant cytotoxicity

potential was examined using brine shrimps cytotoxicity assay. Dose-dependent

cytotoxicity assays were reported against Leishamnia tropica (Promastigotes and

amastigotes) and HepG2 liver cancer cell line. In addition, the biosafe and nontoxic nature

of targated nanoparticles was confirmed by biocompatibility assays using human RBCs

and macrophages. Significant protein kinase inhibition was observed for RG@Fe2O3

nanoparticles while strong fungicidal and bactericidal activities were reported for

RV@ZnONPs. Strong DPPH while good total antioxidants and total reducing powers were

reported. Our experimental results concluded, that targated MNPs are biocompatible and

nontoxic and can be used in different biomedical applications.

Chapter 1 Introduction

________________________________________________________________________

______________________________________________________________________________________ 2

1. Chapter 1: Introduction


________________________________________________________________________

______________________________________________________________________________________ 3

1.1. Nanotechnology: a new frontier

Nanotechnology is one of the most rapidly evolving area in scientific research that employs

different approaches and technologies for the formation, characterization and applications

of nanomaterials (Rautela et al. 2019). It is a multi-disciplinary science involving

knowledge from biology, chemistry, physics and material science. Nanotechnology deals

with different approaches and strategies to synthesize materials from 1−100 nm, at least in

one dimension and have unique characteristics such as size, shape surface charge and

porosity (Abbasi et al. 2019). The seed of “nanotechnology” was first planted by Richard

Feynman in 1959. The formation of nanomaterials and investigation of their unique

physicochemical features are the examples of rapid growth of the novel frontiers of this

future technology (Singh et al. 2011). The term “nano” is taken from Greek language which

means extremely small or dwarf. Nanoparticles can be synthesized from different precursor

molecules using diverse physical, chemical and biological routes. Nanoparticles have very

small size, high surface energy and high surface area/volume (S/V) ratio which enhance

their catalytic properties and interaction with other molecules thus can be used in imaging,

targeted drug release, diagnosis and in treatment modalities of various diseases (Iqbal et al.

2019). Nanoparticles exhibit chemical and structural diversity based on their composition

and inherent nature. Generally, it may be metallic (monometallic and polymetallic), metal

oxide, polymers, supramolecular assembling and quantum dots. In addition, nanoparticles

can be differentiated on the basis of their shape such as nanowire, nanotube, nanocube,

nanoflower etc while on the basis of their structure, nanoparticles are known as bimetallic,

clusters, core shells etc (Iqbal et al. 2019; Rautela et al. 2019).

The main reasons why metal nanoparticles (MNPs) have gained special attention of the

researchers is because of their unique and fascinating properties including particle size,

shape, crystal structure, surface effect, magnetic, catalytic, optical, chemical and

mechanical characteristics from their bulk counterpart. Size and shape of nanoparticles

below 100 nm has strong effect on the chemical, physical and biological properties of

nanoparticles. Nanotechnology has initiated new frontiers for designing new materials and

evaluating their potencies by modulating particle size, shape and distributions. The

researchers are engaged in the synthesis of different metals and metal oxide nanoparticles

(Chakraborty et al. 2018; Chakraborty et al. 2019). These different MNPs have unique


________________________________________________________________________

______________________________________________________________________________________ 4

structural and functional characteristics depending on their size, shape, nature and synthesis

method used. Metal nanoparticles have been investigated largely due to their biological

activities; antibacterial, antifungal, anticancer, antileishmaniasis, enzyme inhibition

properties (Iqbal et al. 2019; Abbasi et al. 2019). Few years back, NPs were studied only

for their nanoscale properties while now they are extensively studied for commercial

applications (Murray et al. 2000; Paull et al. 2003). Metal nanoparticles have gained the

specific attention of researchers due to their wide range uses in food, cosmetics, electronics,

photonics, agriculture, environmental remediation, catalysis, drug delivery, diagnosis,

imaging and cancer therapy etc (Behravan et al. 2019).

1.2. Methods of nanoparticles synthesis

There are different approaches for the production of NPs such as physical, chemical and

biological. These approaches are mainly divided into “top down” and “bottom up”.

1.2.1. Synthesis of MNPs by Physical and chemical methods

In general, NPs are synthesized either by using “top down” or “bottom up” pathways

(Ahmed et al. 2016). The “bottom up” approach involve the synthesis of nanoparticles from

smaller building blocks; atom by atom, molecule by molecule and cluster by cluster which

ultimately develop into NPs. It is comprising of different chemical and biological methods.

While the “top down” approach is the process of breaking down or slicing bulk material

into small particles by size reduction using different techniques (Shedbalkar et al. 2014).

Detail information about the different kinds of synthesis methods are provided in Figure

1.1. These physical approaches produce heterogeneous nanoparticles with high energy

consumption and temperature (more than 350°C) which makes these types of synthesis

routes more expensive. Another limitation associated with physical approach is the lower

production yield of nano size particles (Shedbalkar et al. 2014). In addition, physical

approaches use complicated equipments, tedious procedures and rigorous experimental

conditions which are big challenges in the way of physical synthesis (Abbasi et al. 2019).

Different nanoparticles have been synthesized and reported using physical methods (Iqbal

et al. 2019). However, chemical processes are the most preferable routes for the

development of nanoparticles as they require less energy during reduction and stabilization


________________________________________________________________________

______________________________________________________________________________________ 5

Figure 1.1. Different approaches and strategies for the synthesis of nanoparticles


________________________________________________________________________

______________________________________________________________________________________ 6

of homogenous particles with high preciseness in size, shape, dimension, composition and

structure (Albanese et al. 2012; Khalil et al. 2019). Chemical methods utilize toxic capping,

reducing and stabilizing agents. However, chemical route is eco-toxic using expensive

metal salts, harsh reducing agents (sodium borohydrides, hydrazine hydrate, sodium

citrate, ascorbate, Gallic acid, tollens, poly (ethylene glycol)-block copolymers, elemental

hydrogen, hydroquinone) and organic solvents. The presence of surfactants comprising

functional groups (thiols, amines etc) interact with particles surface, stabilize their growth

and protect them from sedimentations and agglomerations (Nath and Banerjee. 2013).

These chemicals remain adsorbed on the surface of nanoparticles as a result they may cause

some undesired effects on human life and surrounding environment and may result in

cytotoxicity, carcinogenicity and genotoxicity and can’t be utilized in medical applications

(You et al. 2013; Shah et al. 2015). Therefore, researchers are doing huge efforts to develop

simple, clean, green, ecofriendly, cost-efficient green chemistry methods for fabrication

NPs that significantly modulates the size, shape and surface chemistry of NPs (Chakraborty

et al. 2019).

1.2.2. Bio-inspired synthesis of nanoparticles

Biosynthesis of MNPs can be achieved using several biological sources; medicinal plants,

algae, fungi and bacteria etc (Figure 1.2) (Nasar et al. 2019). In many cases, the reducing

agents and other components present in the cells may directly involve in chemical reduction

of metallic precursor, nucleation, growth and stabilization of nanoparticles, so there is no

need to add reducing, stabilizing and capping agents from outside. These nanoparticles

capped with functional molecules obtained from biological sources can help in stabilization

of NPs and may show enhanced biological activities (Iqbal et al. 2019; Pirtarighat et al.

2019). The development of NPs using several biological routes have specifically attracted

the attention of researchers worldwide as biogenic route is relatively simple, sustainable,

cost effective and provides greater biocompatibility. Thus, there is a growing interest of

scientific community working in the field of nano-biotechnology to identify biological

sources with strong reducing, stabilizing and capping agents in large quantity, because only

then the formation of NPs be truly possible. The major disadvantages related to microbial

sources include aseptic condition, high isolation cost,


________________________________________________________________________

______________________________________________________________________________________ 7

Figure 1.2. Biofabrication of nanoparticles using green nanotechnology

high incubation time, growth in culture media and their pathogenicity in nature (Khalil et

al., 2018; Ahamad et al., 2019).

By contrast, green synthesis of nanomaterials involves the use of medicinal plant extracts

for reduction and stabilization of metal salts and don’t use toxic chemical substances.

Therefore, plant-orchestrated synthesis of MNPs is known as eco-safe using green

chemistry approaches. Furthermore, there is no need to maintain large-scale culture and

the process does not possess any biohazard problems as reported in case of microbial

synthesis.

Nanoparticles synthesized through green nanotechnology is a bio-safe method with

enhanced stability and unique characteristics, eliminating high temperature, pressure and

pH etc (Abbasi et al. 2019). Due to cost-effective, energy-efficient, nontoxic behavior of

functional molecules available in the plant extract (alkaloids, flavonoids, phenol, ketones,

terpenoids, carboxylic acids, aldehyde, amides, flavones, enzymes, vitamins) the green

synthesis of NPs is rapidly gaining attention. These functional molecules play significant

role in reducing and stabilizing metal ions and result in green nanoparticles. Furthermore,

these biomolecules play promising role in modulating structural and functional properties


________________________________________________________________________

----------------------------------------------------------------------------------------------------------------------------- ---------------- 8

of NPs thereby, attracting considerable attention of researchers and stakeholders working

in the field of green nanotechnology (Parandhaman et al. 2019; Yugandhar et al. 2019).

Formation of green MNPs is a simple and rapid process and can be accomplished in salt

solution within short time. There are different factors influencing the green synthesis of

nanoparticles such as nature of plant extracts, concentration of extracts, metal salt, pH and

synthesis protocol used. Large number of research studies have used plant extracts and

their associated phytochemicals to catalyze reduction process and have achieved stable

nanoparticles (Khalil et al. 2018; Maaza et al. 2019). The NPs so fabricated determined

strong antimicrobial, antioxidant and catalytic properties obtained from phytocompounds

adsorbed on their surface. These properties results in wide range applications of NPs in

drug delivery, enzyme, cosmetics, food and pharmaceuticals. Thus, for green synthesis of

MNPs, the 12 basic principles of green chemistry are now becoming a reference guideline

for the researcher, chemical technologist and chemist worldwide to develop less dangerous

chemical products and byproducts (Anastas et al. 1998; Kharissova et al. 2013). Thus,

green technology is an alternate route for the formation of safe, biocompatible and stable

nano size materials (Narayanan et al. 2011). There are three main steps which must be

taken into consideration while synthesizing nanoparticles using biological sources. First,

the solvent medium used should be nontoxic and biocompatible. Second, natural

reductones (reducing agents) should be ecofriendly and third, stabilizing and capping

agents employed should be natural and non-toxic (Mohanpuria et al. 2008; Singh et al.

2011). Green synthesis is generally favoured over traditional methods due to the presence

of numerous components present in biological systems and has been investigated for the

fabrication of bionanomaterials, which is ecofriendly and can be utilized in diverse

biological applications.

1.3. Multiple factors affecting the green synthesis of nanoparticles

There are different physiochemical factors that influences green synthesis of MNPs such

as pH, temperature, pressure, plant extracts concentrations, raw material concentration, size

and the synthesis method (Pennycook et al. 2012; Baker et al. 2013). Several major factors

that influences the green synthesis nanoparticle are discussed below;


________________________________________________________________________

______________________________________________________________________________________ 9

1.3.1. Protocols used for the synthesis

There are various protocols used for the synthesis of MNPs; physical, chemical and

biological. Each synthesis method has both advantages and disadvantages. However,

biological route for designing nanoparticles uses safe and eco-friendly materials employing

the 12 basic principles of green chemistry and is therefore highly acceptable than traditional

physical and chemical approaches (Kharissova et al. 2013; Varahalarao et al. 2014).

1.3.2. Effect of pH

The pH of reaction solution plays very important role in the phytosynthesis of MNPs using

green chemistry approaches. Previous studies have investigated that pH of reaction mixture

effect size, shape and rate of reduction of NPs (Gardea-Torresdey. 1999; Armendariz et al.

2004). Different plant extracts as well as extracts from various parts of the same plant

possess different pH value and it is therefore important to optimize pH of the reaction

mixture to get optimum synthesis of desired NPs. Thus, controlled size and shape of the

nanoparticle can be regulated by changing pH of reaction mixture. Satishkumar et al.

(1990) studied pH effect in wide range from 1–11, while synthesizing AgNPs using

medicinal plant Cinnamomum zeylanicum. At lower pH, larger AgNPs were formed in a

small quantity, while small spherical AgNPs were formed in high concentration at higher

pH. At high pH more bioactive molecules get adsorbed on the surface of metal ions. The

effect of pH on the size and shape of green AgNPs was also studied by Soni and Prakash

(2011).

1.3.3. Effect of temperature

Temperature is also an important factor that powers the fabrication of MNPs. The

development of NPs using physical method involves highest temperature of more than

350°C, whereas chemical synthesis method is performed at < 350°C. Most frequently,

phytosynthesis of NPs is performed at less than 100°C. The temperature of reaction mixture

defines the nature of synthesized NPs (Rai et al., 2006). Iravani and Zolfaghari. (2013)

studied the biogenic nature of AgNPs using bark extracts of Pinus eldarica and observed

that with increasing temperature such as 25°C, 50°C, 100°C and 150°C, there was decrease

in size of AgNPs. Furthermore, Sheny et al. (2011) studied gold and silver nanoparticles

reduced and stabilized with Anacardium occidentale leaves extract ranging from low to

high temperature. At lower temperature, more leaf extract was used in the reduction and


________________________________________________________________________

______________________________________________________________________________________ 10

stabilization of NPs compared to reaction performed at high temperature. It has been

experimentally proved that only 0.6 mL leaves extract was used in greenly orchestrated

NPs performed at 100°C and 2.5 mL leaves extract was used to fabricate NPs at 27°C.

1.3.4. Effect of pressure

Pressure is another important parameter that effects reduction, stabilization and capping of

NPs. The amount of pressure exerted on the reaction medium greatly influences size, shape

and yield of green NPs (Pandey, 2012). The rate of reduction, stabilization and capping of

metal ions using biological sources (plants, algae, fungi etc) has been confirmed to be much

faster at ambient pressure condition (Tran and Le, 2013).

1.3.5. Effect of plant extract concentration

The plant extracts concentration greatly influences the greenly orchestrated nanoparticles

and significantly control the size and shape of MNPs. Dubey et al. (2010) performed the

phytosynthesis of AgNPs and AuNPs using different concentrations (0.5, 1.0, 1.8, 2.8, 3.8,

4.8 mL) of Tanacetum vulgare fruit extracts. The study results concluded that particle size

decreased with increase in extract concentrations in both AgNPs and AuNPs. Similarly,

Dwivedi and Gopal (2010) synthesized green AgNPs and AuNPs using leaves extract of

Chenopodium album and revealed that increase in leaves extract concentrations resulted

decrease in particle size which was further verified by TEM analyses.

1.3.6. Effect of metal ions concentrations

The quantity of metal ions is another important factor that play significant role in

controlling size, shape of greenly orchestrated NPs. Dubey et al. (2010) used tansy fruit

extract to fabricate AgNPs and AuNPs at different ions concentration (1–3 mM). The

results showed that at high metal ion concentrations, size of AgNPs were reported to be

larger. As compare to AgNPs, AuNPs were bigger in size, which were further confirmed

by TEM analysis using high metal ion concentrations. Previously, Dwivedi and Gopal.

(2010) fabricated AgNPs and AuNPs using Chenopodium album leaf extracts and

concluded similar results. The research studies indicated that increasing metal ions

concentrations, size of both AgNPs and AuNPs was found to be increased. In addition,

AgNPs and AuNPs synthesis was observed to be slow at the lower ion concentrations.


________________________________________________________________________

______________________________________________________________________________________ 11

1.3.7. Effect of time

The incubation/reaction time also influences green synthesis of nanoparticles and has a

profound impact on the size, shape, yield and type of nanoparticle fabricated. The time

required for incubation/reaction is highly significant to obtain optimum synthesis of NPs

and plays significant role in the stabilization of nanoparticles (Darroudi et al. 2011). Hence,

properties of green NPs changed with reaction time and are significantly influenced by the

synthesis route etc (Kuchibhatla et al. 2012; Mudunkotuwa et al. 2012). The reaction time

influences synthesis of NPs in different ways such as agglomeration of nanoparticles due

to its storage for long time, they may have shelf life that might influence their diverse

properties (Baer, 2011).

1.3.8. Preparation Cost

The wide range applications of nanomaterials also depend on the synthesis cost associated

with NPs. Hence, synthesis cost is an important parameter that effect synthesis of NPs and

their diverse applications. Even though the formation of nanoparticles using chemical

approach results in higher concentration of NPs within a short period of time, but this

method uses expensive and toxic chemical substances. Therefore, nanoparticles

synthesized through chemical methods may have limited applications in biomedical fields,

whereas synthesis of nanoparticles using green chemistry protocols is cost-effective and

can be utilized in the formation of nanoparticles at large scale (Patra and Baek. 2014).

1.3.9. Effect of proximity

When the individual NPs come in contact or come close to surface of other nearby NPs,

alterations in physical characteristics have been reported in most cases (Baer et al. 2008).

This fascinating and tunable nature of nanoparticle can be employed in designing more

altered NPs with different functionalities. The proximity effect has significant impact on

diverse magnetic and biological properties of metal nanoparticles, influences particle

charging, substrate interactions etc.

1.4. Different green metal oxide nanoparticles

Metal oxide nanoparticles possess a rich compositional chemistry (small size, high S/V

ratio and high surface energy) with size ranging from 1-100 nm. Metal nanoparticles have

been investigated for their wide applications in biomedicine, agriculture, catalysis, optics,

electronics, imaging, sensing and biomedical field as an antimicrobial, antileishmaniasis


________________________________________________________________________

______________________________________________________________________________________ 12

and anticancer agents etc. Different physicochemical properties such as morphology, size,

shape, surface chemistry, crystal structure contributes to the functional properties of MNPs

in different commercial areas (Abbasi et al. 2019; Jolivet et al. 2010). Due to large S/V

ratio, surface Plasmon resonance (SPR), thermal properties, mechanical stability, small

size, unusual catalytic and redox properties, MNPs can be used in various pharmaceutics,

food, electronics and cosmetics industries (Fierascu et al. 2020). The development of

microbial resistance to antibiotics is significantly increasing which is a big impediment.

The available drugs in the market are poor, ineffective and possess side effects which

demands alternative approaches and strategies. Among the various strategies to address

these problems, metal oxide NPs have emerged as potent candidates with lower toxicity,

high biocompability, higher stability and selectivity (Iqbal et al. 2019). Metal NPs such as

silver, gold, zinc, iron etc can be synthesized with desirable shape, size and composite

having remarkable optical, thermal and imaging application in diagnostics and

therapeutics. These nanoparticles can be altered by conjugating biomolecules such as

peptides, antibodies and DNA and drugs to target specific cells (Lee and El-Sayed. 2006;

Jain et al. 2008).

Metal oxides nanoparticles are frequently gaining attention in the treatment of cancer

theranostics, diabetes, neurochemical monitoring and in medical implants. Some metal

oxides have been successfully used for cell separation, labeling and as a gas sensing

nanoprobes. In addition, MNPs have wide range application in MRI as a contrast agent and

carriers in targeted drug delivery (Andreescu et al. 2012). The noble metal nanoparticle can

also be used as a theranostic agents in diagnosis and in the treatment of different diseases

(Chen et al. 2014). Recently, many researchers have developed MNPs based theranostic

methods to cure different diseases (Zhang et al. 2008; Giljohann et al. 2010; Ahamed et al.

2010; Di Pietro et al. 2016). Research studies have also reported that the biosynthesized

silver nanoparticles can be used in different theranostic application against pathogenic

bacteria and cancer cells as an imaging and therapeutic agents (Mukherjee et al. 2014).

Gold nanoparticles have broad range applications in diagnostic tools like computed

tomography, targeted drug delivery and fluorescent imaging etc (Chen et al. 2005; Hu et

al. 2006). Progress has been made to take these metal nanoparticles based imaging and

therapeutic systems to advance level in clinical trials. Food and drug administration


________________________________________________________________________

______________________________________________________________________________________ 13

authority (FDA) has approved few theranostic nanoparticles (Stylianopoulos et al. 2012;

Thakor and Gambhir 2013). The synthesis of nanoparticles with theranostic behavior are

at nascent stage and still need strong efforts and dedication from researchers to perform

pre-clinical and clinical trials. Biodegradable nanoparticles such as gold nanoshell and

silica NPs is gaining special attention to enhance their translational process for the

commercialization of theranostic nanoparticles in biomedical field. A comprehensive

overview about the targated nanoparticles their nature and diverse applications are given

below;

1.4.1. Iron oxide (Fe2O3)

Iron oxide is the most fascinating and ideal material due to its tunable nature such as high

surface area, high biocompatibility, supermagnetism and chemical stability (Boyer et al.

2010; Muthukumar and Matheswaran 2015). Fe2O3 is an n-type semiconductor with band

gap of 2.0 eV (Bakardjieva et al. 2007). Among the different phases of iron oxide NPs, the

widely used and most studied iron oxide nanoparticles are α-Fe2O3 (hematite), Fe3O4

(magnetite) and γ-Fe2O3 (maghemite) (Cornell and Schwertmann 2003; Wu et al. 2015).

These different phases of iron oxides are characterized by their unique catalytic, magnetic

and biochemical properties which make these nanoparticles suitable candidates in diverse

biological applications. The size and shape of Fe2O3 has significantly influenced their

physical, chemical and biological properties. Iron oxide nanoparticles have numerous

applications in the material science, engineering, cosmetics, biomedicine, bioremediation

and other clinical applications (Muthukumar and Matheswaran 2015). Furthermore,

specific properties of IONPs like biocompatibility hold abundant in vivo applications in

tissue repairing, drug delivery, detoxification, immunoassay, MRI contrast enhancement

(Laurent et al. 2008). Fe2O3 are being investigated for significant antimicrobial,

antileishmaniasis potentials (Prucek et al. 2011; Abbasi et al., 2019). Moreover, it has the

power to generate heat in the presence of magnetic field and makes iron nanoparticles a

promising candidate in hyperthermia applications.

1.4.2. Cobalt oxide (Co3O4)

Co3O4 is also one of the most significant multifunctional p-type antiferromagnetic

semiconducting material with spinel crystal structure. The direct optical bandgap of Co3O4

is 1.48 and 2.19 eV and it can be used as photocatalyst to degrade organic pollutant using


________________________________________________________________________

______________________________________________________________________________________ 14

visible light (Gulino et al. 2003; Saeed et al. 2018). Cobalt oxide nanoparticles has gained

the special attention of researchers due to their unique structural, magnetic and catalytic

properties. The Co3O4 have wide range application in diverse areas; energy storage, gas

sensing, electrochromic sensors, heterogeneous catalysis, solar energy absorption, Li-ion

rechargeable batteries, ceramic dyes and pigments etc (Kandalkar et al. 2008; Chen et al.

2007; Lou et al. 2008). The CoONPs have been reported to effectively catalyze the

degradation of water pollutants (Liang and Zhao 2012) and combustion of hydrocarbons

(Wang et al. 2015). CoONPs have been used in diagnosis, biomedicine and radioactive

vectors in the cancer therapeutics. Furthermore, cobalt has potential role in vitamin B12 as

a cofactors and efficiently used in amino acid, arsenic, nitrates, glucose sensing (Cui et al.,

2013, Khan et al. 2015). Current research reports have studied that CoONPs can self-

assemble to form photonic hyper crystals which indicates their possible applications in

chemical and biological sensing systems (Smolyaninova et al. 2014; Bossi et al. 2016).

1.4.3. Nickel oxide (NiO)

Nickel oxide (NiO) is intrinsic p-type semiconductor metal oxide with a wide band gap of

3.6-4.0 eV (Manikandan et al. 2015). It is antiferromagnetic and exhibits exceptionally

high ionization and high isoelectric point. Nickel oxides have gained the special attention

of many researchers due to their chemical stability, electro-catalysis and super conductivity

(Thema et al. 2016). Among the various technological applications of nano-NiO, it can be

used in gas sensing, sensors, battery material, electrochemical storage devices, anodic

electrochromic smart windows and water remediation (Pejova et al. 2000; Bandara and

Yasomanee 2006; Odobel et al. 2010, Abbasi et al. 2019). It is an eco-active material as it

finds applications in the adsorption of harmful dye and inorganic contaminants (Pandian et

al. 2015). Due to its anti-inflammatory nature, NiONPs can be evaluated in diverse

biomedical applications. Recent studies have indicated that it can functions as a strong

antioxidant, anticancerous and antimicrobial agents (Saikia et al. 2010; Ezhilarasi et al.

2016; Khashan et al. 2016; Khashan et al. 2017).

1.4.4. Zinc oxide (ZnO)

Zinc oxide is an n-type semiconductor metal oxide with numerous applications due to its

versatile and multipurpose spintronic, photonic and morphological properties (Ozgur et al.

2005; Kaminsky 2012). ZnO possess a wide band gap of (3.37 eV). Because of its unique


________________________________________________________________________

______________________________________________________________________________________ 15

characteristics, ZnO is widely used in metal-insulator semiconductors diode, miniaturized

semiconductors laser and gas sensors etc (Kalusniak et al. 2009; Ellmer 2012; Gao et al.

2005). ZnO is among the five compounds that fall under the category of “generally

recognized as safe” (Sawai 2003). Due to its biosafe and biocompatible nature, ZnO has

been widely used in cosmetics, sunscreens, lotions, drug delivery and bioimaging (Iqbal et

al. 2019). Numerous research studies have been conducted on ZnONPs and investigated it

as an antimicrobial and anticancer agent (Hassan et al. 2016; Abbasi et al. 2019). Different

research studies have confirmed that ZnONPs is nontoxic and can be employed in different

biological applications (Iqbal et al. 2019).

1.5. Medicinal plants used for green synthesis of nanoparticles

The bioactive functional molecules wisely distributed in medicinal plants play a promising

role in designing different therapeutic drugs. Numerous medicinal plants have been studied

for the isolation of different phytochemicals using diverse biochemical techniques (Butler

2004). The medicinal plants (herbs, shrubs and trees) are being investigated for

identification of therapeutic phytocompounds with diverse biological applications.

Medicinal plant mediated herbal medicines have been utilized for the treatment of different

diseases (Abbasi et al. 2019). The development in biological activities of different plant

extracts and associated phytochemicals have significantly attracted attention of researchers

to use these phytochemicals in diverse applications. Different nanopharmaceutical

companies are engaged in designing Nanotechnological advances to fabricate different

MNPs with significant applications in biomedical fields. There are different physical and

chemical ways to synthesize these different metal nanoparticles. However, chemical routes

are eco-toxic, use costly metal salts, toxic reducing agents and organic solvents which limit

their biomedical applications due to their toxic nature and less biocompatibility. Therefore,

researchers are trying to formulate an eco-friendly, cost-efficient and green chemistry

method for the formation of MNPs (Orive et al. 2004; Yu et al. 2019). Thus, plant extracts

mediated synthesis was explored as an alternative solution. Plant extracts are comprised of

large number of functional molecules; alkaloid, flavonoid, phenols, amides, vitamins,

amino acids and are responsible to reduce and stabilize metal nanoparticles (Gangula et al.

2011; Mukunthan and Balaji. 2012). Numerous research studies have confirmed, that these

bio-inspired NPs are non-toxic, biocompatible, have low


________________________________________________________________________

______________________________________________________________________________________ 16

side effects, promote sustained release and improve bio-activity (Borase et al. 2014). In

our current study, different traditionally important medicinal plants were selected after

extensive ethnobotanical surveys and literature review and were used to synthesize

different eco-friendly and biocompatible nanoparticles. The different medicinal plants used

are as follow;

1.5.1. Geranium wallichianum (G. wallichianum) D. Don ex Sweet

Family: Geraniaceae

Genus: Geranium

Species: wallichianum

Local name: Ratanjot

Plant part used: Leaves

Habitat and distribution

The medicinal plant, G. wallichianum is a flowering herbaceous perennial plant with 40-

60 cm tall. The plant was collected from Nathiagali Khyber Pakhtunkhwa, Pakistan in

flowering stage. The G. wallichianum species are also widely distributed in Afghanistan

and Northwest Himalaya. The plant is known as “Shepherd's needles” in English while

locally it is known as “Ratanjot” in Pahari language”.

Medicinal values

The ethnobotanical surveys and literature review have documented the medicinal values of

G. wallichianum. The medicinal plant is used to treat different diseases such as cancer,

sugar and hemorrhages. Among the numerous plants utilized in the fabrication of green

nanoparticles, G. wallichianum was selected and used due to having numerous functional

molecules such as herniarin, stigmasterol, ursolic acids, herniarin and β-sitosterols which

paly significant role in the reduction and stabilization of metal nanoparticles (Ellis et al.

1994, Abbasi et al., 2019).

1.5.2. Rhamnus virgata (R. virgata) Roxb.

Family: Rhamnaceae

Genus: Rhamnus

Species: virgata

Local name: Cane Buckthorn



________________________________________________________________________

______________________________________________________________________________________ 17

Habitat and distribution

The R. virgata was collected from District Swat, Pakistan. The medicinal plant is a large

shrub/small tree with sharp spines and is widely found in different ecological zones of

Swat, Kashmir (Pakistan), Bhotan, China, India and Burma.

Medicinal values

The ethnobotanical surveys and literature review reported strong medicinal and therapeutic

values for R. virgata. The medicinally active phytocompounds reported are maesopsin,

physcion, herbacetins, rhamnocitrin, emodin, kaempferols and quercetin (Kalidhar and

Sharma 1984). The medicinal values associated are strong antioxidants, bactericidal,

fungicidal, laxative and emetic properties and is used to treat parasitic infection and spleen

affection (Iqbal et al. 2019). Recent research studies have reported significant

antimicrobial, antioxidant, antileishmanial and anticancer activities of R. virgata against

Hep-G2 liver cancer cell lines (Iqbal et al. 2019).

1.5.3. Rhamnella gilgitica (R. gilgitica) Mansf. & Melch.

Family: Rhamnaceae

Genus: Rhamnella

Species: gilgitica

Local name: Tondel


Habitat, distribution and Medicinal values

Rhamnella gilgitica (shrub or small tree) is a highly medicinal plant 5-7 m tall and was

collected from Gilgit, Pakistan (Iqbal et al. 2019). The plant is a rich source of numerous

bioactive compounds; kaempferols, quercetins, naringenins, chrysophanols, gallic acids

and have shown significant antioxidants and anti-inflammation activities. The plant is

widely used to treat rheumatism, swelling and pain etc (Chao et al. 1997; Pan et al. 1997;

Pan et al. 1998; Huang et al. 2016).

1.6. Scope of the study

Generally, plants are comprised of important medicinal compounds and natural healing

agents. Considering the importance of metal oxide nanoparticles, current study was

designed to formulate a simple, rapid, eco-friendly and energy efficient method for the


________________________________________________________________________

______________________________________________________________________________________ 18

biosynthesis of MNPs using aqueous leaves extract of medicinally important and phyto-

chemically rich plants. According to the best of our knowledge and literature review, there

is no data available regarding green synthesis of nanoparticles and their biological

applications using G. wallichianum, R. virgata and R. gilgitica. Therefore, the current study

has been designed to synthesize different MNPs. Furthermore, targeted NPs were

extensively studied for various characterizations and biological applications. The

crystalline nature of greenly orchestrated NPs were studied through XRD (X‐ray

diffraction). FTIR (Fourier‐transform infrared) analyses were performed to study different

functional groups involved in reduction and stabilizations of targated MNPs. EDS (Energy-

dispersive X-ray spectroscopy) were performed to study elemental composition.

Vibrational properties of asynthesized NPs were studied using Raman spectroscopy.

Furthermore, SEM (Scanning electron microscopy) analyses were performed to study the

shape of targeted MNPs. Size, shape and crystalline nature of targated NPs were further

studied via TEM (Transmission electron microscopy). After extensive characterizations,

targated NPs (ZnONPs, IONPs, NiONPs) were studied in detail for diverse biological

activities; bactericidal, fungicidal, biocompatibility, enzyme inhibition, anticancer,

antileishmanial and antioxidant properties.

1.7. Aims and objectives

The aims and objectives of the current thesis are as follow;

To design simple, environment-friendly and cost-efficient green chemistry protocol

for the formation of nanoparticles using medicinal plant extracts.

To investigate the therapeutic potentials of green ZnO, Fe2O3, NiO nanoparticles

for different biological applications.

Specific objectives

Phytosynthesis of ZnO, Fe2O3, NiO nanoparticles using aqueous leaves extracts of

G. wallichianum, R. virgata and R. gilgitica.

Physicochemical characterizations of the targated nanoparticles using UV, XRD,

EDS, FT-IR, Raman, SEM, TEM and DLS.

To investigate the in vitro biocompatibility, antimicrobial, cytotoxic, antioxidant

and enzyme inhibition potentials of targated nanoparticles.

Chapter 2 Green synthesis of iron oxide nanoparticles

______________________________________________________________________________________ 19

Chapter 2: Biogenic synthesis of green and cost effective iron oxide

nanoparticles and evaluation of their potential biomedical

properties


______________________________________________________________________________________ 20

Graphical abstract: Schematic representation of the synthesis, characterization and

biological applications of Rhamnella gilgitica mediated IONPs.


______________________________________________________________________________________ 21

Abstract

This present study for the first time reports a facile and ecofriendly route for the synthesis

of IONPs. The IONPs were synthesized using Rhamnella gilgitica leaves extract. The color

change of reaction mixture from dark brown to reddish black determined the biofabrication

of IONPs which was further confirmed by the presence of an absorbance peak at 341 nm.

The biogenic IONPs were further studied using various microscopic and spectroscopic

techniques such as SEM, TEM, XRD, DLS, FT-IR, EDX and Raman spectroscopy. The

IONPs were further evaluated for anticancer potency against HepG2 cancer cells (IC50:

14.30 µg/mL) with concentrations range of 5.47−700 µg/mL. Dose-dependent cytotoxicity

assays were performed against Leishmanial amastigotes (IC50: 26.91 µg/mL) and

promastigotes (IC50: 9.63 µg/mL). In addition, cytotoxicity assay was evaluated against

brine shrimps (IC50: 34.45 µg/mL). Disc diffusion method showed significant antibacterial

and antifungal activities. Furthermore, significant antioxidants and enzyme inhibition

potentials were revealed. Biocompatibility tests were performed against human RBCs and

macrophages to determine biocompatible nature. Overall, IONPs have played significant

role in multiple biological applications. In future, different in vivo studies can be suggested

on toxicity aspects in different animal models and for once biosafety is maintained, then

these nanoparticles can be utilized in nano-pharma industries for the treatment of different

diseases


______________________________________________________________________________________ 22

2.1. Introduction

Nanotechnology is one of the most significant and rapidly growing area of research that

uses biosynthetic and environment-friendly technology for the synthesis of nanoparticles

(NPs) from 1−100 nm, exhibit small size and high S/V ratio (Abbasi et al. 2019; Iqbal et

al. 2019). The NPs has attracted special attention of scientific community due to their

fascinating properties including physical, mechanical, optical, magnetic and electronic,

sensing and considerably vary in many aspects to their bulk counterparts (Karthik et al.

2018b; Khatami et al. 2018). The NPs have been significantly used in diverse disciplines

including food, cosmetics, medicine, engineering, agriculture and energy (Wang et al.

2014; Khan et al. 2017; Manjunatha et al. 2018). Among the different MNPs, IONPs have

attracted the attention of scientific community due to high solubility, low toxicity, high

chemical stability, high biocompatibility and magnetic behavior (Chakraborty et al. 2018;

Nosrati et al. 2018; Chakraborty et al. 2019). IONPs have extensive applications in

different commercial areas including biomedicine, radiology, sensors, cosmetics,

diagnostics, pathogen, enzymes, antigen detection and vaccines (Ruan et al. 2018; Shah et

al. 2018; Zhao et al. 2018). These different uses of NPs are because of their unique

properties; high stability, high surface area and small band gap (2.1 eV). IONPs exist in

different forms such as magmatic (γ-Fe2O3), hematite (α-Fe2O3), goethite (FeO(OH) and

magnetite (Fe3O4) (Beinlich et al. 2018).

Currently, IONPs have been synthesized through different physical and chemical methods

(Gholoobi et al. 2018; Kastrinaki et al. 2018; Abbasi et al. 2019). These physicochemical

approaches face numerous problems; use expensive metal salts, harsh reducing agents,

organic solvents, costly instruments, need high energy, temperature and pressure (Abbasi

et al. 2019; Iqbal et al. 2019). These physicochemical methods are not only expensive at a

large scale in industries, but these particles cannot be used in medicine due to their toxic

effects on human health, especially in clinical field (Naggar et al. 2018; Iqbal et al.2019).

To overcome these limitations, there is an urgent need to develop sustainable, cleaner and

safer approach as chemical synthesis gives low quantity, requires complex purification

methods and high energy consumption. Currently, great developments have been achieved

in biofabrication of NPs using microorganism and different other biological sources such


______________________________________________________________________________________ 23

as plants, algae, fungi (Khatami et al. 2018). The biosynthesis of NPs is ecofriendly, energy

efficient, nontoxic and don’t need expensive chemicals.

Therefore, synthesis of NPs and other material utilizing only one biological source such as

plants, algae, fungi, bacteria, yeast, actinomycetes and diatoms have specifically attracted

the attention of scientific community worldwide (Marousek and Kwan 2013). This earnest

attention towards green synthesis is because of their eco-friendly nature and significant

biological applications in medical sciences (Jamdagni et al. 2018). The major disadvantage

associated with microbial sources is maintenance of contamination free environment, high

isolation cost, their maintenance in culture media. Therefore, plants (phytofabrication)

promise to be excellent reducing agent in the formation of NPs. The plant extracts are

comprised of a wide range of bioactive compounds such as phenolic, alkaloids, flavonoids,

vitamins, saponins, inositol and terpenes (Iqbal et al. 2017; Abbasi et al. 2018; Iqbal et al.

2018). These plant extracts may function as strong reducing and stabilizing agents, reduces

cost and eliminate the use of toxic chemicals agents (Abbasi et al. 2019b). The IONPs have

been synthesized from large number of medicinal plants (Huang et al. 2014; Naseem et al.

2015; Sharma et al. 2015; Abbasi et al. 2019). In the present study, R. gilgitica leaves

extract was utilized to synthetize IONPs. R. gilgitica is a well-known medicinal plant from

Gilgit, Pakistan (Iqbal et al. 2019) which shows high contents of flavonoid such as

kaempferols, quercetins, naringenins, chrysophanols, 8-sterols, mixed fatty acids and have

shown significant antioxidant and anti-inflammatory potencies. In China, R. gilgitica is

used for the treatment of rheumatism, swelling and pain (Pan et al. 1997; Pan et al. 1998;

Huang et al. 2016). The present study first time reports the green synthesis of IONPs using

R. gilgitica leaf extract without utilizing any surfactant, organic and inorganic solvent from

outside. The detailed reaction conditions and synthesis mechanism have been

comprehensively discussed. According to the best of our literature review, this is the first

report investigating uses of leaves extract of R. gilgitica for IONPs formation. The

biosynthesized IONPs were further characterized through different techniques. In addition,

multiple biological assays have been performed such as antimicrobial, cytotoxicity assays,

biocompatibility assays and enzyme inhibition assays.


______________________________________________________________________________________ 24

2.2. Experimental Methods

2.2.1. Rhamnella gilgitica leaves extract preparation

The medicinal plant R. gilgitica (Rhamnaceae) was collected from Gilgit, Pakistan and was

taxonomically identified. The plant leaves were properly washed using distil water to

remove dust and other particulate material, shade dried for ~15-20 days to remove water

content. The dried leaves were carefully grounded into fine powder and were stored in

airtight zipper in Lab. Furthermore, plant extract was prepared using 20 gram of fresh

leaves powder of R. gilgitica into 200 mL distilled water. The mixture was continuously

stirred and heated at 80°C for ~100 min. The solution obtained was cooled and filtered 3−4

times with the help of Whatman filter papers (0.2 µm) to remove any coarse material and

impurity. The filtered leaves extract was preserved at 4°C for the biosynthesis of

nanoparticles.

2.2.2. Biofabrication of the targated IONPs

The IONPs were synthesized by adding 3 gm of iron (II) acetate into 50 mL of filtered R.

gilgitica leaves extract. The reaction mixture was heated at ~70°C for 2 hr with constant

stirring to ensure maximum reactions. The reaction solution was cooled and pH was

determined as 9.73. The mixture was centrifuged at 3000 rpm for 30 min. The supernatant

was discarded. The IONPs pellet was washed with distil water to remove iron ions and R.

gilgitica leaves extract residues. The resulting powder, considered as IONPs, was dried at

75°C for 2 hr. Later on, IONPs were placed in dark, cool, dry place and were extensively

characterized using different techniques. Figure 2.1 shows study plan of IONPs.

2.2.3. Physico-chemical characterizations of IONPs

The bioinspired IONPs were subjected to various analytical methods. The UV-visible

spectra were observed in the range of 200−800 nm wavelength. FT-IR analysis was

performed between 500–4500 cm-1 to characterize the possible biomolecules responsible

for reduction, stabilizations and capping of IONPs. The FT-IR spectra were recorded using

FT-IR spectrometer (Alpha, Bruker, Germany) in the range 500–4000 cm–1. The spectrum

was analyzed by using Origin Pro 8.5 software. The SEM) (EM (NOVA-FEISEM-450

linked to EDX-detectors) was utilized to image and study size and morphology of IONPs.


______________________________________________________________________________________ 25

In addition, TEM analysis was carried out to exactly calculate size distribution and shape

of R. gilgitica mediated IONPs. The XRD analysis was done for IONPs on X-ray

Figure 2.1: Study plan for biogenic synthesis of iron oxide nanoparticles.

diffractometers armed with Cu radiations source at 45 kV and current voltage of 40 mA

and mean crystalline size was recorded via Scherrer approximation. Raman spectroscopy

analysis was performed to study the vibrational properties of IONPs. The zeta potential,

hydrodynamic size distribution were investigated by DLS using Zeta-sizer equipment

(Malvern Zetasizer Nano). Two hundred microgram of IONPs were suspended in 2 mL of

deionized water (Milli-Q) by bath sonication (Elmasonic E-30 H Germany) for 40 min and

suspension was transferred to a folded capillary cell for zeta potential and conductivity

measurements. The EDX analyses was studied to investigate other elements associated

with IONPs. The EDX values reflect the atomic structure of biogenic NPs and shows

atomic content of the nanoparticles.


______________________________________________________________________________________ 26

2.2.4. Preparation of test sample of IONPs

The biosynthesized IONPs after preparation were tested for their different in vitro

biological activities. A colloidal suspension was made by adding 1 mg IONPs with 1 mL

of DMSO and was properly sonicated for around 30 min. Moreover, the stability of

colloidal suspensions was explored by adding 3 mg IONPs into 3 mL of DMSO. The

slightly turbid colloidal suspensions was obtained and was allow to stand. The UV-spectra

were observed after regular time intervals for 48 hr to check their stability.

2.2.5. Biological activities of IONPs

2.2.5.1. Antibacterial activity of IONPs

Antibacterial activities of IONPs were investigated using different bacterial strains. All

bacterial culture were refreshed and multiplied on nutrient broth media, by spreading on

media plates using cotton swabs. Disc-diffusion method was used to examine antibacterial

activities. The filter disc (6mm) loaded with 10 µL of each IONPs (with dilution

46.875−1500 µg/mL) was dried and kept on bacterial lawns to avoid the spread of test

sample. Ten microliter of oxytetracycline served as positive control. The petri plates were

stored in incubator at 37°C/24 hr and were regularly observed for the appearance of zones

of inhibition and their Minimum inhibitory concentration (MIC) values were recorded.

2.2.5.2. Antifungal activities of IONPs

The antifungal activities of IONPs were investigated against the available fungal strains

using disc-diffusion methods. The different fungal spores were cultured in 100 mL flasks

containing sabouraud dextrose liquid media. The liquid cultures were adjusted to OD of

0.5. The SDA media was poured in sterilized petri plates. To achieve uniform lawns of

fungal strains, 50 μL of broth culture were spread on petri plate having SDA media with

the help of autoclaved cotton swab. Each disc was laden with 10 μL of IONPs. The Amp

B served as positive and DMSO as negative controls. Moreover, fungal plates were

incubated for 24 hr/37°C to observe zones of inhibition after time intervals in this period.

Different concentrations of IONPs were utilized in range of 46.875-1500 µg/mL.

2.2.5.3. PK (Protein kinase) inhibition

Inhibition of PK enzymes is determined one of the popular target for screening chemical

entities for their anticancer potencies. The PK inhibition activity was determined using


______________________________________________________________________________________ 27

different doses of IONPs using Streptomyces 85E strain (Chaudhuri et al. 2017; Abbasi et

al. 2019b). The experiment was carried out under aseptic condition. To achieve this

purpose, even lawns of Streptomyces 85E strains were made utilizing ISP4 media. Briefly,

100 µL of inoculum was introduced from the already standardized culture and was

uniformly spread on the petri plates for making the uniform lawns. 6 mm sterilized filter

disc impregnated with IONPs (10 µL) were cautiously kept on microbial lawns. The

surfactin was served as positive and DMSO as negative controls. The petri plates were

incubated at 30°C/72 hr to target the growth of Streptomyces 85E strains. After 72 hr,

different bald and clear zones appeared around the discs. These zones determine inhibition

of spores and mycelia formation in Streptomyces 85E strain and finally, ZOI were

measured in millimeter.

2.2.5.4. AA (Alpha amylase) inhibition (antidiabetic potential)

The antidiabetic potency of R. gilgitica IONPs was explored to determine AA inhibition

potency (Zak et al. 2011). The reaction mixture was prepared by using 25 μL alpha

amylase, 40 μL starch solution and 15 μL phosphate-buffer saline, 10 μL test sample and

was added stepwise into micro-plate, which was incubated for 30 min at 50°C. The 90 µL

iodine solution and 20 µL (1 M HCL) were added to the mixture after incubating micro-

plate for 20 min. The acarbose was served as positive and DMSO as negative controls. The

micro-plate reader (AMP Platos R II, Microplate) was used to calculate the optical density

at 540 nm.

2.2.5.5. Anticancer potential

The anticancer potency of bioinspired IONPs was determined against HepG2

(Hepatocellular carcinoma) cells using already published protocol (Iqbal et al. 2019). The

cells were cultured in DMEM (Dulbecco's Modified Eagle's medium) media loaded with

10% FBS (Fetal bovine serum), Pen-Strep. The HepG2 cells were grown in 5% CO2

incubator at 37°C. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide)

assay was performed in micro-plate using various doses of IONPs (5.47−700 µg/mL) for

~ 48 hr. To perform this experiment, 20 µL MTT solutions was transferred into each well

of the 96-well microplate and was incubated for 3 hr. The DMEM media was removed and

100 µL of DMSO (Dimethyl sulfoxide) was added in each well followed by incubation for


______________________________________________________________________________________ 28

25 min. The quantity of formazans produced by the living cells was determined by plate

reader at 570 nm and IC50 value was calculated.

2.2.5.6. Antileishmanial potential

The cytotoxicity activity of R. gilgitica mediated iron nanoparticles were explored against

Leishmania. tropica “KWH23 strain” (promastigote and amastigote) using MTT (4,5-

Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cells viability assay (Zak et al.

2011; Abbasi et al. 2019). MI99 media containing 10% FBS was prepared to culture both

types of parasites promastigotes and amastigotes. Different concentrations of IONPs were

tested against L. tropica “KWH23 ranging from 1−200 μg/mL to investigate

antileishmanial activities. The reaction mixture (200 µL) is composed of 100 µL of the

standardized culture, 50 µL fresh media and 50 µL of colloidal nanoparticles suspension

in DMSO. The amphotericin B served as positive and DMSO as negative controls. The 96-

well plate containing test sample of iron nanoparticles was incubated in 5% carbon-dioxide

incubator for 72 hr/24°C. After treatment and incubation with IONPs, readings were

recorded at 540 nm using micro-plate readers. Both living promastigote and amastigote

were counted with the help of an inverted microscope and GraphPad software was used to

record IC50 values.

2.2.5.7. Brine shrimp cytotoxicity (BSC)

The cytotoxic potency of R. gilgitica mediated IONPs was investigated on fresh hatched

shrimp, Artemia salina. Before, activity was performed, A. salina eggs were placed in

incubator in artificial sea water (3.8 g/L) at 30°C for ~24 hr in the presence of light to get

mature nauplii. Ten nauplii were collected through Pasteur pipette and were transferred

into glass vial having sea water and the test sample. Various doses of IONPs ranging from

(1–200 μg/mL) were used to explore cytotoxicity activity. The glass vials having sea water,

vincristine sulphate and mature nauplii were served as positive while vials with sea water,

DMSO and mature nauplii served as negative controls. The vials were kept in incubator

for 24 hr at 30°C and dead shrimps were carefully counted in each vial. The IC50 values

were recorded for IONPs via GraphPad software to determine their cytotoxicity.


______________________________________________________________________________________ 29

2.2.5.8. Antioxidant capacities of IONPs

The spectrophotometric method was employed to evaluate the free radical scavenging

potency of iron oxide nanoparticles. To perform the assay, 2.4 mg DPPH was added into

methanol (25 mL) as a free radicals and was properly vortexed. Later on, different

concentrations of IONPs were utilized in the range of 1−200 µg/mL to explore free radicals

quenching activity. The ascorbic acid (AA) served as positive and DMSO as negative

controls. The 200 µL reaction mixture was comprised of 20 µL of IONPs and 180 µL of

reagent solution and was loaded in 96-well plate and incubated in dark for 1 hr. Readings

were recorded at 517 nm using plate reader and formula below was used to calculate %

scavanging of DPPH.

% DPPH scavanging = 1-[Absorbance of sample

Absorbance of control]×100

In addition, total reducing power (TRP) of test sample was examined using previously

described Potassium-ferricyanide procedure (Bhattacharya et al. 2017; Abbasi et al. 2019).

The AA served as positive while DMSO as negative controls. The microplate reader was

used to calculate the absorbance capacity at 630 nm. Total antioxidants capacity (TAC)

was evaluated utilizing phosphomollybdenum standard method (Khalil et al. 2017). The

absorbance was calculated at 695 nm utilizing Eliza reader. The AA was utilized as positive

while DMSO as negative control to better compare the total antioxidant power of IONPs.

The results were as micrograms equivalent of AA per/mg of the test sample.

2.2.5.9. Biocompatibility of IONPs with human RBCs

The biocompatibility assay was done to confirm the biosafe nature of IONPs with human

RBC. To perform this experiment, hemolytic assay was carried out using fresh human

isolated RBCs (Faria et al. 1997; Abbasi et al. 2019b). In order to perform biocompatibility

assay, 1 mL of freshly obtained human RBC were taken in EDTA tube and was centrifuged

at 13000 rpm for 10 min. Supernatant was thrown away and pellet was washed three times

with PBS. The erythrocytes suspensions was prepared by adding 200 µL erythrocytes into

9.8 mL PBS. The 100 µL of erythrocyte suspension was added to various concentrations

of IONPs, incubated for 1 hr/35°C followed by centrifugation at 10,000 rpm/10 min. The

supernatant was shifted into 96-well plate and hemoglobin release was studied at 540 nm


______________________________________________________________________________________ 30

employing micro plate reader. The Triton X-100 served as positive while DMSO as

negative controls. The results are calculated as % hemolysis produced by different

concentration of IONPs and can be calculated employing the formula below;

% hemolysis = [Sampleabs – Neg. controlabs / Pos. controlabs – Neg. controlabs] × 100

2.2.5.10. Biocompatibility of IONPs with human macrophages

In addition, human macrophages were used to examine the biocompatible nature of IONPs

(Yedurkar et al. 2016). The cells were culture in RPMI media along with 10% FBS, 25

mM Hepes, and antibiotic Pen-Strep. In addition, cells were incubated in 5% CO2

incubator and cultured to a density of 1×105 cells/well. Furthermore, cells were treated with

different concentrations of IONPs (1-200 µg/mL). The % inhibition of IONPs was

calculated against human macrophages using the formula below;

% inhibition =1 – Absorbance of sample

Absorbance of control ×100

2.3. Results and Discussion

The plant mediated synthesis of NPs initiate once precursor salt iron (II) acetate

Fe(C2H3O2)2 was introduced into R. gilgitica leaves extract. The gradual color change of

Fe(C2H3O2)2/R. gilgitica solution at 70°C from dark brown to reddish black determine

the formation of iron oxide nanoparticle. This color change in threaction mixture is due to

surface plasmon vibrations (Ibrahim et al. 2015). The biosynthesis of IONPs were further

evaluated using different analytical techniques. The production of the IONPs in aqueous

environment was further determined by UV-visible spectroscopy. The solution was

scanned by fixing the wavelength scale between 200−800 nm. Maximum absorbance was

observed at 341 nm. This maximum absorbance peak shows synthesis of IONPs, which is

within the range of surface plasmon resonance of IONPs. The reduction in the absorption

peak was noticed after 24 hr, which shows the settlement of IONPs at the bottom of

eppendorf. The Figure 2.2 (a, b) depicts UV-Vis spectra of IONPs recorded from the

colloidal suspension after the experiment performed at room temperature. Figure 2.3 (a, b)

shows the SEM images of R. gilgitica leaves extract stabilized IONPs at different

magnifications, which has confirmed that the biogenic IONPs are spherical in shape. The

fabricated IONPs were further characterized by TEM to determine size and morphology of


______________________________________________________________________________________ 31

the nanoparticles. After TEM analysis, the average particle size was reported to be ~21 nm.

The TEM analysis further determines the synthesis of IONPs which were found to be

spherical in shape (Figure 2.3 (c). FT-IR analysis was used to analyze the functional

molecular vibration and possible biomolecules responsible for synthesis and effective

stabilizations of iron oxide nanoparticles. The Figure 2.4 (a) indicates FT-IR spectra of

biosynthesized IONPs. The bands appearing at 2979 cm−1, 1111 cm−1 and 1064 cm−1 were

assigned to stretching vibration of C−H, C−N, and C−O bond, while the peak appearing at

621 cm−1 shows the presence of Fe−O bond vibrations from maghemite phase Fe2O3.

There was shift in peaks in the synthesized IONPs, which suggests that the functional

groups of leaves extract participated in the formation of IONPs.

The vibrational properties of IONPs were also evaluated by Raman spectroscopy. The

distinguished major modes of Raman spectra were present at 354.28 cm-1 (Eg), 511.36 cm-

-1 (T2g), 718.15 cm-1 (A1g). Our results are in line with previous reports of green IONPs

thereby confirming the biosynthesis of IONPs (Jubb et al. 2010; Yadav et al. 2018). Figure

2.4 (b) shows the Raman peaks of IONPs. The previous research studies have reported that

positions of Raman peaks changes with the synthesis protocol and distribution of vacancies

in the unit cell (Faria et al. 1997). EDX analyses was studied to confirm the elemental

composition of IONPs. The EDX peaks indicated strong signals for both iron and oxygen.

The absence of other element in EDX spectra confirms the purity of biogenic IONPs. The

EDX spectra are indicated in Figure 2.5 (a). The reduction of precursor salt iron (II) acetate

occurred frequently upon the contact of iron (II) acetate with the leaves extract of R.

gilgitica. Several researchers have identified different flavonoid compounds (kaempferol,

quercetin, naringenin, chrysophanol and Gallic acid etc) in the leaves extract of R. gilgitica

(Huang et al. 2016; Iqbal et al. 2019). These compounds present in water extract of R.

gilgitica bind on the surface of metal ions and play significant role in stabilizing NPs.

Figure 2.5 (b) indicates binding of biomolecules with metal ions.

The XRD analysis was done to confirm the crystalline nature of bioinspired IONPs. The

XRD pattern in Figure 2.6 (a, b) indicates crystalline nature of IONPs. The observed Bragg

peaks of R. gilgitica mediated IONPs are in agreement with pure phase maghemite IONPs


______________________________________________________________________________________ 32

Figure 2.2: UV analysis for biogenic IONPs (a) UV visible spectra (b) Stability of

biosynthesized IONPs.


______________________________________________________________________________________ 33

Figure 2.3: Various SEM and TEM images of R. gilgitica mediated IONPs (a, b) SEM

images (c) TEM image.


______________________________________________________________________________________ 34

Figure 2.4: (a) FT-IR spectra of biosynthesized IONPs using iron acetate as precursor (b)

Raman spectra of the IONPs.


______________________________________________________________________________________ 35

Figure 2.5: (a) Elemental composition using EDX (b) Schematic representation of the

possible mechanism for the synthesis of nanoparticles and their effective stabilization


______________________________________________________________________________________ 36

Figure 2.6: XRD spectral analysis for biogenic IONPs (a) XRD spectra of R. gilgitica

mediated IONPs (b) Size calculation via Scherer approximation.


______________________________________________________________________________________ 37

with JCPD card No: 00–025–1402. The XRD distinct diffraction peaks of IONPs

synthesized by magnetic stirring are indexed at 2 theta (2 ) values of 20.456, 25.8, 30.22,

33.14, 34.86, 39.14, 44.22, 54.37, 58.02 and 63.59 representing (105), (203), (206), (216),

(119), (209), (0012), (500), (1115) and (4012) crystallographic planes respectively. The

sharp peaks show high crystalline nature of IONPs. The average size of IONPs was

calculated as ~21 nm using Scherer's equation. No other peaks relating to other impurities

were observed indicating single crystalline phase of IONPs. The XRD analysis of IONPs

is in line with previous reports of IONPs using Sageretia thea (Khalil et al. 2017). The size

distributions and zeta potential of R. gilgitica mediated IONPs were analyzed through DLS

analysis. The DLS analysis results have indicated large particles aggregates of 269 d.nm.

The average zeta potential and PDI of IONPs were -8.7 mV and 0.737 (Figure 2.7 (a, b).

Table 2.1 provides detail regarding zeta potential and PDI of IONPs prepared with

magnetic stirring. Our results corroborate with earlier published reports (Demirezenet al.

2018; Yadav et al. 2018). The DLS is mainly used to determine the size of nanoparticles

in colloidal suspension at nano scale and PDI values suggest moderate polydispersity. The

ζ-potential is based on particles movement under an electric fields. The surface charge and

local environments of the nanoparticles also play promising role in measuring ζ-potentials

(Iqbal et al. 2019).

2.3.1. In vitro antimicrobial activities

The traditional medicines available in pharma-industries play important role in the

treatment of different bacterial diseases. However, there are certain problems related to

these drugs (e.g., antibiotic resistance). Novel research to design new approaches to combat

antibiotic resistance and reduce the risks of development and spread of diseases is being

undertaken by researchers (Bhattacharya et al. 2017; Iqbal et al. 2019). The new

advancements in nanobiotechnology are opening new avenues to develop substances with

special antimicrobial applications (Rizzelo et al. 2013). Figure 2.8 (a) shows inhibition

activities of IONPs against various pathogenic bacterial strains using disc diffusion method

in concentrations range of 46.875−1500/mL. The bacteria used were Bacillus subtilis,

Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella


______________________________________________________________________________________ 38

Figure 2.7: (a) Zeta potential measurement of IONPs (b) Particle size distribution of IONPs


______________________________________________________________________________________ 39

Table 1: Zeta potential measurements of the IONPs

Zeta size distribution, Zeta Potential and PDI

Zeta Size (d.nm) 269

PDI 0.737

Intercept 0.717

Zeta Potential (mV) -8.7

Zeta Deviation (mV) 15.1

Conductivity (mS/cm) 0.0314

Result Quality Good


______________________________________________________________________________________ 40

pneumoniae. The results showed that most bacteria strains were susceptible to IONPs and

have shown strong inhibition capability.

The IONPs were most effective against B. subtilis and E. coli (MIC: 46.875 µg/mL) and

least effective against P. aeruginosa (MIC: 187.5 µg/mL). The oxytetracycline (10 µg)

served as positive control. No single dose was more potent than that of positive control. In

nutshell, significant antibacterial activity has been investigated for bioinspired IONPs. The

antibacterial properties of IONPs have been already well reported in the earlier reports

using different medicinal plants (Safawo et al. 2018; Abbasi et al. 2019b). This strong

antibacterial potency may possibly be due to the active biomolecules attached on the

surface of IONPs. In addition, the current study determined that antibacterial potency

increased with increase in IONPs concentrations. The researchers have explained different

mechanisms for the antibacterial property of IONPs. Among these different mechanisms,

ROS production is most important mechanism that provide antimicrobial activity to

IONPs. Some other factors like membrane injury due to NPs adsorption on the surface may

also cause cell damage. Similarly, surface defects in symmetry of NPs may result in

antibacterial activity of different NPs (Li et al. 2012). Furthermore, we consider the

importance of biomolecules attached on the surface of NPs from leaves extract. Antifungal

potency of IONPs was studied against fungal strains using different concentrations of

IONPs ranging from 46.875−1500 µg/mL. The Amphotericin B served as positive controls.

Various fungal strains studied are Mucor racemosus, Aspergillus flavus, Aspergillus niger,

Candida albicans and Fusarium solani (Figure 2.8 (b). This novel report is first

investigating the antifungal activity of R. gilgitica assisted IONPs. Our data regarding

antifungal activity for IONPs have shown dose-dependent response against different fungal

strains. Among different fungal strains, A. flavus was the least susceptible strain with MIC

value of 187.5 µg/mL while M. racemosus was the most susceptible strain (MIC: 46.875

µg/mL). The biogenic IONPs have shown significant antifungal activity at different

concentrations. However, none of the tested IONPs concentration exhibited % inhibition

greater than the positive control. Among these different strains, Fusarium solani, Mucor

racemosus and A. niger were inhibited at all concentrations.


______________________________________________________________________________________ 41

Lower concentration of test sample (IONPs) was found ineffective against Aspergillus

flavus and Candida albicans. Besides, ROS generation, earlier research studies reported

interference of IONPs with fungal spores/hypea thereby also result in fungal growth arrest.

Earlier reports have shown significant dose-dependent response for IONPs against

different fungal strains (Iqbal et al. 2019) and is consistent with our present study. Different

fungal and bacterial strains and their MIC values are provided in Figure 2.8 (a, b).

2.3.2. Enzyme inhibition potential

Protein kinase (PK) are enzymes plays significant role in phosphorylation of serine-

threonine and tyrosine amino acids also plays an important role as signalling cascade in

metabolism and apoptosis etc. The deregulated phosphorylations can result in genetic

problems and cause cancer progression. So, any product with ability to inhibit PK enzyme

can be of great importance in oncology (Yao et al. 2011). Inhibition of PK enzymes is

determined one of the popular target for screening chemical entities for their anticancer

potencies. The protein kinase phosphorylation play significant in developing hyphae and

mycelia in Streptomyces fungal strains and similar mechanism can be utilized to explore

the PK inhibition activities. The strain used has served to confirm biomedical potency of

compounds to determine PK inhibition activities (Waters et al. 2002). Figure 2.9 (a) shows

significant inhibition of activity of IONPs against PK enzyme. The PK inhibition activities

of IONPs was determined using disc-diffusion method. The PK inhibition activity was

carried out in sterile condition using different doses of IONPs (31.25−1000 µg/mL). The

surfactin was served as positive control. The ZOI (zone of inhibition) was ~35 mm at

concentrations of 1000 μg/mL, which show significant PK inhibition potency for IONPs.

The study shown concentration dependent response for IONPs. Due to PK inhibition

potencies, one can conclude that IONPs are non-toxic, environment-friendly and are of

significant importance in cancer treatment. Our results of IONPs corroborate with earlier

studies where Rhamnus virgata IONPs have shown significant results against PK inhibition

(Abbasi et al. 2019).

Alpha amylase enzyme performs its functions by converting carbohydrate into glucose

(Yao et al. 2011) thus, blocking the function of AA can stop carbohydrate conversion to

glucose which is an important area of diabetic research (Oyedemi et al. 2017). In our


______________________________________________________________________________________ 42

Figure 2.8: (a) Antibacterial activities of biogenic IONPs (b) Antifungal activities of

biogenic IONPs


______________________________________________________________________________________ 43

studies IONPs has been shown to significantly inhibit alpha amylase at the concentration

the range of 31.25−1000 µg/mL. The maximum inhibition recorded was 27% at

concentrations of 1000 μg/mL and the rate of inhibition gradually decreased with the

decrease in the concentration of IONPs. Figure 2.9 (b) indicate AA inhibition potency of

IONPs. Our results substantiate the performance of IONPs in the studies done on Sageretia

thea and Rhamnus virgata (Khalil et al. 2017).

2.3.3. Antileishmanial activity

Leishmaniasis is categorized as a tropical disease caused by Leishmanial parasites (Kaye

et al. 2011). The leishmanial parasites are endemic in around 100 countries, affecting ~1.2

million people annually. About 350 million people across the globe face an imminent threat

from these parasites. The drugs currently available in the market against the disease are

scarce and are believed to often toxic, less effective and costly. The antimonials was

formulated as a strong agent for the treatment of leishmaniasis, but has lost its therapeutic

value as Leishmanial parasites have developed resistance against this drug. In order to

develop alternate routes for its treatment significant research work has been done to design

NPs to treat Leishmania. Various NPs were used to assess their cytotoxic potencies against

Leishmania parasite (Hameed et al. 2019; Iqbal et al. 2019; Iqbal et al. 2019). However,

the biogenic IONPs are rarely studied to determine their cytotoxic activity against L.

tropica. In recent work, antileishmanial activities of green IONPs were investigated against

L. tropica as shown in Figure 2.10 (a). The leishmania parasites were treated with different

doses of IONPs (1−200 μg/mL) for 72 hr and showed concentration-dependent response,

showing increased antileishmanial activity with the increased the increased concentrations

of IONPs. The IONPs showed significant activities against L. tropica promastigotes (IC50:

9.63 µg/mL). Similarly, antileishmanial activity of IONPs was also reported against L.

tropica amastigote (IC50: 26.91 µg/mL). Our results are in line with previous reports of

green IONPs using S. thea (Khalil et al. 2017). The dose-dependent response and lower

IC50 value for IONPs demonstrate that these particles may be used for targated drug

delivery in nano-pharma industries for the treatment of leishmaniasis.


______________________________________________________________________________________ 44

Figure 2.9: (a) Inhibition activities of IONPs against protein kinase (b) Inhibition potential

against alpha amylase.


______________________________________________________________________________________ 45

2.3.4. Brine shrimp cytotoxicity (BSC)

The BSC assay was determined to confirm the cytotoxic potency of IONPs against freshly

hatched A. salina. BSC is the most suitable cytotoxicity screening test to confirm the

biological potencies of compounds (Chanda et al. 2011). The cytotoxicity activity of

IONPs was studied using A. salina. Figure 2.10 (b) indicates % mortality of IONPs at

different concentrations ranging from 1−200 μg/mL. The BSC assay of IONPs has shown

dose-dependent response. Our test sample (IONPs) is considered to be in the category of

general cytotoxicity with IC50 values of 34.45 µg/mL. Our R. gilgitica IONPs results are

consistent with the earlier reports of IONPs using Sageretia thea and Rhamnus virgata

(Khalil et al. 2017; Abbasi et al. 2019).

2.3.5. Anticancer activity

Cancer is a deadly disease and the major reason for the mortality around the globe. The

rate of incidence of cancer is constantly increasing and is predicted to be ~21 million by

2030 (Iqbal et al. 2018; Abbasi et al. 2019; Iqbal et al. 2019). There are different types of

cancer, among these, liver cancer is considered to be the sixth most common and second

deadliest cancer in male, sixth in female and is responsible for around 745,517 deaths

worldwide. There are numerous risk factors involved in hepatocellular cancer such as

alcohol, viral infection and toxins (such as aflatoxin) (Daher et al. 2018). The scientific

community is working hard to overcome this menace by formulating novel drugs having

strong therapeutic values. The anti-hepatocellular carcinoma potency of IONPs was

explored against HepG2 with decreasing concentrations of IONPs. The IC50 value recorded

was 14.30 µg/mL indicating strong potency. Our results are consistent with the earlier

studies of biosynthesized IONPs from Rhamnus virgata using HepG2 cancer cells (Wahab

et al. 2014; Hassan et al. 2017; Abbasi et al. 2019). The results are shown in Figure 2.10

(c).

2.3.6. Biocompatibility assays

The biocompatible and toxicological nature of IONPs was evaluated against human RBC

and macrophages. Biological substances showing hemolysis less than (< 2%) are non-

hemolytic, 2−5% are slightly hemolytic and above 5% are known as hemolytic

(Dobrovolskaia et al. 2008). The hemolysis is estimated by measuring released hemoglobin


______________________________________________________________________________________ 46

Figure 2.10: (a) Cytotoxicity activities of R. gilgitica mediated IONPs against leishmania

(b) Cytotoxicity against brine shrimps (c) Anticancer activities of IONPs.


______________________________________________________________________________________ 47

from the ruptured of red blood cells (RBC) after their treatment with IONPs. Nanoparticles

are considered hemolytic, if they can rupture RBC thereby resulting in the release of

hemoglobin. Therefore, the biocompatibility nature of IONPs was investigated against

human RBCs using hemolytic assay in the range of 1–200 µg/mL. The current research

study has confirmed that the bioinspired IONPs are not hemolytic at lower concentrations

of 2 μg/mL, less hemolytic at 5−40 μg/mL, while hemolytic at >45 μg/mL. The results of

R. gilgitica mediated IONPs were found consistent with the earlier reports of IONPs using

Rhamnus virgata and Sageretia thea (Khalil et al. 2017; Abbasi et al. 2019). The IC50 value

was calculated as >200 μg/mL. Our data confirms that green IONPs are non-hemolytic at

low concentration against RBCs confirming their biocompatibility and nan-hazardous

nature.

The bio-safe nature of IONPs was further confirmed by performing MTT cytotoxic assay

against human macrophages. The research study has concluded dose-dependent inhibition

response. The macrophages response increased after treatment with higher concentration

of IONPs whereas the cytotoxic response decreased at lower concentration The results

indicated that IONPs at 200 μg/mL inhibits the growth of macrophages by ~31%,

confirming the non-toxic behavior of IONPs. Usually macrophages have developed

mechanisms to combat ROS produced from external sources. Thus, ROS at lower quantity

is not toxic to human erythrocytes and macrophages unless its concentration increases

beyond limit (Prach et al. 2013). The IC50 value of IONPs was recorded as 371.3 μg/mL.

The biocompatibility assays results are given in Figure 2.11 (a).

2.3.7. Antioxidant activities of IONPs

Different antioxidant potencies of R. gilgitica IONPs are shown in Figure 2.11 (b). The

antioxidant activity of IONPs and their antioxidant nature was evaluated with

concentrations ranging from 1–200 µg/mL. Maximum values of total antioxidants in terms

of AA equivalent/mg was 52.43% at 200 μg/mL. The TAC has revealed high antioxidant

nature of IONPs towards ROS. Since in this study, R. gilgitica leaf extract was used as

strong reducing and capping agents it was assumed that some flavonoids quench ROS

species which are available on the surface of IONPs. The presence of antioxidant species

available on the surface of IONPs were further investigated using total reducing power.


______________________________________________________________________________________ 48

This mechanism was developed to explore reductones involved in antioxidant activities of

IONPs by providing H-atom that may cause damage to free radical chain (Ul-Haq et al.

2012). The R. gilgitica IONPs showed strong antioxidant activity and was found to be

concentration dependent. The reducing power was decreasing as concentrations was

decreasing. The maximum reducing power (59 %) was recorded at concentration of 200

μg/mL. Strong DPPH radical scavenging potencies of IONPs (78.36 %) was reported at a

concentration of 200 μg/mL. From the results indicated in Figure 2.11 (b), it can be deduced

that some antioxidants compound might be involved in reducing and stabilizing IONPs

using R. gilgitica. Our results of IONPs corroborate earlier reports of Sageretia thea and

Rhamnus virgata mediated IONPs (Khalil et al. 2017; Iqbal et al. 2019; Umar et al. 2019).

The range of variations and disagreement with other research studies might be due to

experimental condition (seasonal variations, humidity and location), route of NPs

synthesis, medicinal plant used and size of NPs (Abbasi et al. 2019).


______________________________________________________________________________________ 49

Figure 2.11: (a) Biocompatibility assay of IONPs against human RBCs and macrophages

(b) Antioxidant potential of IONPs.

Chapter 3 Green synthesis of zinc oxide

nanoparticles

________________________________________________________________________

______________________________________________________________________________________ 50

Chapter 3: Plant-extract mediated green approach for the

synthesis of ZnONPs: Characterization and evaluation of

cytotoxic, antimicrobial and antioxidant potentials

Chapter 3 Green synthesis of zinc oxide nanoparticles

______________________________________________________________________________________ 51

Graphical abstract: Diagrammatic representation of synthesis, characterization and

biological application of R. virgata mediated ZnONPs.


______________________________________________________________________________________ 52

Abstract

Plant-extract-mediated green synthesis of nanoparticles is significantly attracting the

attention of scientific community due to its simple, cost-effective, energy-efficient and

environment-friendly nature. The current study focuses on the biological synthesis of

ZnONPs using aqueous solution of Rhamnus virgata as both reducing and stabilizing

agents. The formation of ZnONPs was confirmed using different microscopic and

spectroscopic techniques. Scherer equation deduced mean crystallite size of ~20 nm for

ZnONPs. Detailed in vitro biological activities revealed significant therapeutic properties.

Strong antibacterial and antifungal activities were reported for ZnONPs. Dose-dependent

cytotoxicity assays revealed against Leishmania tropica promastigotes (IC50: 8.34 µg/mL),

amastigotes (IC50: 13.6 µg/mL), HepG2 cancer cell line (IC50: 19.67 µg/mL), brine shrimps

(IC50: 26.34 µg/mL). Significant antioxidant activities and enzyme inhibition assays were

performed. The biocompatibility assays with RBCs and macrophages confirmed the bio-

safe nature of biogenic ZnONPs. In conclusion, ZnONPs have shown significant bio-

applications and can be recommended for further in vivo studies on toxicity side in animal

models to develop its nano-pharmacological relevance in biomedical applications.


______________________________________________________________________________________ 53

3.1. Introduction

Nanotechnology is one of the most significant and fast growing area in the field of science

and engineering, playing effective role in the improvement of different commercial areas

such as food, cosmetics, agriculture, energy, catalysis, optics, drug delivery and

biomedicine (Matinise et al. 2017; Iqbal et al. 2018). Metal-oxide nanoparticles (MNPs)

have received particular attention due to unique magnetic, optical, chemical, physical,

electronic, mechanical and sensing properties. MNPs varies considerably in many

properties from that of bulk materials such as size, shape, surface effect, electrical and

magnetic properties (Han et al. 2017; Mayedwa et al. 2018). Among the different MNPs,

ZnONPs have attracted extraordinary attention due to its multifunctional and tunable

nature. The intrinsic properties of ZnONPs are highly marked by scientists due to their

broad range of radiation absorption, high biocompatibility, photo-chemical stability, large

electrochemical coupling coefficients. The multi-functionality of ZnONPs is evident from

their uses in cosmetics, biomedicine etc. ZnONPs are represented by 3.37 eV direct band

gap and high excitation energy (60 meV), making it an ideal material to be used in UV

photodetectors, transistor and semiconductor diodes (Kalusniak et al. 2009; Thema et al.

2015). Furthermore, ZnONPs are used in mineral based sunscreens, lotions, ointments, bio-

imaging and drug delivery. ZnONPs have shown potential antimicrobial activity against

bacteria and fungi (Matinise et al. 2017; Mirzaei et al. 2017).

Commonly, ZnONPs are manufactured by different physical and chemical approaches. The

physical route include laser ablation (Gondal et al. 2009), pulse laser deposition

(Kawakami et al. 2003), chemical vapor deposition (Liu et al. 2004) and molecular beam

epitaxy (Zhang et al. 2005). These approaches often need costly instruments, high

temperature, pressure and energy (Guzman et al. 2009). Meanwhile, chemical approaches

include pyrolysis (Saravanakkumar et al. 2018), sol-gel (Barhoum et al. 2017), sono-

chemical etc (Yadav et al. 2008). The chemical approaches generally synthesize ZnONPs

by utilizing various reducing agents, expensive metal salts, toxic solvents and reducing

agents (Tahir et al. 2013). Furthermore, stabilizers are required to avoid particles

aggregation and to make them physiologically compatible (Mourdikoudis et al. 2013). The

physicochemical methods are facing many challenges and has significantly gained the

attention of researchers to discover alternative method which is simple, ecofriendly and


______________________________________________________________________________________ 54

non-hazardous under ambient conditions (Marambio-Jones and Hoek 2010). In this

perspective, green synthesis has gained special attention by replacing chemical products,

developing protocols to reduce or even eliminate materials hazardous to both human health

and environment (Sheldon 2012). Green chemistry has advantages over chemical synthesis

of NPs (Duan and Wang 2015). It fuels reaction without toxic solvent, reducing agents and

stabilizers (Ahmad et al. 2010; Anastas and Eghbali 2010; Mayedwa et al. 2018). Among

the several biological approaches, synthesis of ZnONPs using plant extracts has gained

significant popularity. Plant extract-mediated synthesis can be used as a safe alternative to

physicochemical approaches because of its safe and low cost large scale synthesis of

ZnONPs. The plant extract is comprised of diverse natural phytochemicals including

alkaloids, flavonoids, phenolic, saponins, inositol, resins, terpenes, tannins, amino acids

and vitamins (Iqbal et al. 2017; Abbasi et al. 2018).Theses phytochemicals function as a

strong reducing agent and result in the development of capped NPs. Thus, plant-extract

may serve as a natural reducing, stabilizing and capping agent by eliminating multiple steps

and reducing costs and excess chemical utilization (Rice-evans et al. 1995).

Synthesis of ZnONPs have been achieved using plant extracts of Azadirachta indica,

Sageretia thea, Plectranthus amboinicus, Moringa olifera and Fagonia indica (Bhuyan et

al. 2015; Matinise et al. 2017). Rhamnus virgata, a large shrub/small tree armed with

pointed spines is widely distributed in Pakistan (Swat, Kashmir), China and India. The

ethnobotanical survey and literature review has reported the medicinal values of R. virgata

with strong therapeutic properties. The main phytochemicals found in R. virgata are

physcion, emodin, kaempferol, rhamnocitrin, quercetin, herbacetin and maesopsin

(Kalidhar and Sharma 1984). The medicinal benefits of R. virgata include properties such

as antioxidant, antimicrobial, laxative, emetic and purgative, used in the treatment of

parasitic infections, spleen affection and in leg swelling (Muthee et al. 2011). The

therapeutic values, phytochemistry and biological activities of R. virgata leaves extract

necessitate their use in the fabrication of ZnONPs and may function as an ideal candidate

for the reduction of zinc salt to nano-zinc. The present study shows bioreduction of

precursor salt (Zinc nitrate hexahydrate) using leaves extract of R. virgata. The reaction

conditions under which the synthesis occurs have been discussed comprehensively.

According to literature survey, no earlier research work has been published on ZnONPs


______________________________________________________________________________________ 55

and their biological applications. This study is the first attempt reporting the uses of R.

virgata leaves extracts for the biosynthesis of ZnONPs. Furthermore, the bioinspired

ZnONPs have been extensively characterized and shown diverse biological activities such

as antibacterial, antifungal, biocompatibility assays against RBCs, macrophages,

cytotoxicity assays against brine shrimps, leishmanial parasites and HepG2 cell line have

been investigated.


3.2.1. Green-route synthesis of ZnONPs

The medicinal plant R. virgata (Rhamnaceae) was collected from the mountainous region

of district Swat, Pakistan. After taxonomic identification, the herbarium specimen was

deposited in Plant Biochemistry and Molecular Biology Lab with voucher number

(HPBMBL-18-002). The plant leaves were properly rinsed, shade dried and converted into

fine powder. Extraction of plant material was performed as described previously with

minor modifications (Hameed et al. 2019). Briefly, plant material (30 g of leaves powder)

was added into distilled water (250 mL) and was heated at 80°C on hotplate (DIAB, MS-

H280-Pro) with continuous stirring for 3 hr. The resulting brownish solution was filtered

three times using Whatman filter paper (0.2 µm) to achieve pure aqueous extract. The pH

of the extract was determined at room temperature and was stored at 4°C for future use.

Previously optimized protocol was used to synthesize biogenic ZnONPs following the

principles of green nanochemistry (Thema et al. 2015). The synthesis reaction was carried

out in 250 mL flask. Six grams of precursor salt zinc nitrate hexahydrate (Sigma Aldrich)

was slowly added into 100 mL plant extract followed by pH determination. The reaction

mixture was further heated on a hotplate at 60°C for 2 hrs with continuous stirring to allow

maximum reaction. The reaction mixture was allowed to cool followed by centrifugation

at 10,000 rpm/10 min. The resulting precipitate was washed 3 times with distilled water

and centrifuged at (10,000 rpm/10 min). The pellet was then dried at 100°C in the incubator

(K & K Scientific and Medical equipments, Korea) followed by calcination using open air

furnace (KSL-1100X, MTI Corporation, China). The resultant greyish material (assumed

as ZnONPs) was characterized using different microscopic and spectroscopic

characterization techniques. Figure 3.1 shows general study layout.


______________________________________________________________________________________ 56

Figure 3.1: Study plan for biogenic synthesis of NPs.


______________________________________________________________________________________ 57

3.2.2. Characterization techniques of ZnONPs

Diverse characterization techniques were carried out to study the physicochemical

properties of ZnONPs. The synthesis of ZnONPs was confirmed using UV-4000 UV-Vis

spectrophotometer (Germany) ranging from 200−700 nm wavelength. The shape and

morphology of ZnONPs were studied using SEM and TEM at different magnifications.

The particle size and crystal purity of ZnONPs was determined using XRD. The XRD

analysis was performed for thermally annealed sample on X-ray diffractometer

(PANalytical, Netherland) equipped with a Cu radiation source at 45 kV and 40 mA of

current voltage. Raman spectroscopy analysis was performed to study the vibrational

properties of ZnONPs using LabRam confocal microprobe Raman System (Jobin-Yvon).

The hydrodynamic size distribution and ζ-potential were evaluated by DLS system

(Malvern Zetasizer Nano). In addition, EDS analysis was done to determine the elemental

compositions of ZnONPs. The composition and functional groups involved in the synthesis

of ZnONPs were identified by FTIR (Alpha, Bruker, Germany). The spectral range used

for scanning sample was 500–4500 cm-1.

3.2.3. Test sample preparation of ZnONPs

To study in vitro biological properties of ZnONPs, a colloidal suspension was prepared in

DMSO as 1 mg/mL followed by sonication for 20 min. Furthermore, stability of colloidal

suspension was determined by adding 5 mg ZnONPs/5 mL of DMSO. A slightly turbid

colloidal suspension was achieved that was allowed to stand. UV spectra were monitored

periodically for 24 hr to determine stability of colloidal suspension in DMSO.

3.2.4. Biological activities of biogenic ZnONPs

3.2.4.1. Antibacterial activity of ZnONPs

Bactericidal potencies of ZnONPs were determined using disc-diffusion assay (DDA)

against the available bacterial strains. The frozen cultures were refreshed by inoculating

them on nutrient agar media. Before activity was performed, cultures were transferred into

nutrient broth media and were placed in shaking incubator (Temperature: 37°C, 200 rpm)

for 24 hr. The bacterial cultures were standardized by keeping OD at 0.5. The 6 mm filter

discs impregnated with 10 µL of NPs (1000, 500, 250, 125, 62.5, 31.25 µg/mL) were dried

and carefully placed on bacterial lawns using cotton swabs. 10 µg of oxytetracycline disc

was used as positive control. Bacterial plates were placed in incubator for 24-48 hr at 37°C


______________________________________________________________________________________ 58

with periodic observations of inhibition zones. MIC of NPs were determined with

producing ZOI ≥10 mm in diameter in dilutions ranging from 1000−31.25 µg/mL. MIC is

defined as least dilution of NPs that inhibit bacteria growth.

3.2.4.2. Antifungal assay of ZnONPs

To further investigate antifungal activities of ZnONPs, DDA was performed against

different pathogenic fungal strains. Briefly, fungal spores were cultured in the test tubes

containing Sabouraud Dextrose liquid medium and were placed in shaking incubator for

24 hr at 37°C. The liquid culture was standardized to OD of 0.5. The SDA solid media was

prepared and autoclaved for the growth of fungal strains in sterilized petri plates. Filter

discs added with 10 µL of ZnONPs were placed on media plates. Amphotericin B (10 µg)

discs and DMSO were utilized as positive and negative control. Furthermore, fungal plates

were placed in incubator for 48 hr at 28°C to observe the ZOI and were measured in

millimeters. Different doses of ZnONPs were used in concentration range of 1000−31.25

µg/mL. The MICs values were calculated at lowest concentration of ZnONPs.

3.2.4.3. Brine shrimp lethality assay

Brine shrimp cytotoxicity assay was investigated to determine cytotoxicity activities of

biogenic ZnONPs. To achieve this purpose, A. salina eggs were incubated in sea water (3.8

g/L) for around 24−48 hr in the presence of light at 30°C. Pasteur pipettes were used to

harvest 10 mature nauplii and were transferred into glass vials containing sea water and

ZnONPs. Different concentration (1–200 μg/mL) of ZnONPs were utilized. The vials with

mature nauplii, vincristine sulphate and sea water was considered as positive control, while

vials containing nauplii, sea water and DMSO was utilized as negative control. The dead

shrimps were counted in each glass vial after incubation for 24 hr. The IC50 values was

calculated by GraphPad software.

3.2.4.4. Antileishmanial assay

The cytotoxicity of ZnONPs was investigated against L. tropica “KWH23 strain” (both

promastigotes and amastigotes culture) using MTT assay (Ali et al. 2015). The MI99 media

added with 10% FBS was utilized to culture the Leishmanial parasites. Dilutions of the

final concentration of ZnONPs from 1−200 μg/mL were studied for antileishmanial

property. Amphotericin B was used as positive while DMSO as negative control. The 96

well plate with tested NPs was placed in 5% CO2 incubator for 72 hr at 24°C.


______________________________________________________________________________________ 59

Reading were recorded at 540 nm using spectrophotometer and IC50 was calculated using

GraphPad.

3.2.4.5. Anticancer activity

The cytotoxicity potency of ZnONPs was performed against HepG2 cancer cell line using

optimized protocol (Ali et al. 2015). Cells were cultured in DMEM media added with 10%

FBS and Pen-Strep. The HepG2 cells were cultured in humified 5% CO2 incubator at 37°C.

MTT cytotoxicity assay was demonstrated in the 96-well microplate by incubating at

different test concentration (1000−7.8125 µg/mL) of ZnONPs for 48 hr. The MTT solution

(20 µL) was loaded in each well and was placed in incubator for 3 hr. The DMEM media

was removed and100 µL of DMSO was added followed by further incubation for 20 min.

The formazan quantity formed by survived cells was monitored by plate-reader at a

wavelength of 570 nm. IC50 value was recorded through GraphPad software.

3.2.4.6. Biocompatibility with human RBCs

To determine the bio-safe nature of ZnONPs, hemolytic assay was performed using freshly

isolated RBCs as discussed previously (Malagoli 2007). To reach this purpose, 1 mL fresh

human RBCs were isolated and dispensed in EDTA tube to prevent blood coagulation.

Furthermore, RBCs were centrifuged at 14,000 rpm/10 min. The erythrocyte suspension

was made in a test tube by addition of 200 µL of erythrocyte into 9.8 mL of PBS (pH 7.2).

Erythrocytes suspensions of 100 µL was added with different concentration of NPs and

were placed in incubator at 35°C for 1 hr, followed by centrifugation at 10,000 rpm/10

min. The supernatant was transferred in 96-well-plate and hemoglobin release was

measured at 540 nm utilizing micro-plate reader. Triton X-100 and DMSO were utilized

as positive and negative control. The results were recorded as % hemolysis caused by NPs

dilutions and can be calculated using the formula below;

% hemolysis = [Sampleabs – Neg. controlabs / Pos. controlabs – Neg. controlabs] × 100

3.2.4.7. Biocompatibility with human macrophages

To further investigate the bio-safe nature of ZnONPs, biocompatibility assay was examined

against human macrophages (de Almeida et al. 2000). The isolated cells were cultured in

RPMI media added with FBS (10%), Hepes (25 mM) and antibiotic Pen-Strep.

Furthermore, macrophages were placed in 5% (v/v) CO2 incubator and were grown to a

density of 1 × 105 cells/well. The below formula was used to calculate % inhibition;


______________________________________________________________________________________ 60



3.2.4.8. Protein kinase (PK) inhibition

The PK inhibition assay for green ZnONPs was performed using Streptomyces 85E strain

as discussed earlier (Fatima et al. 2015). The whole experiment was performed under

aseptic conditions. Even lawns of Streptomyces 85E strains were prepared using SP4

minimal media. After that 6 millimeters autoclaved filter discs loaded with 10 µL of

ZnONPs were placed on microbial lawns. Surfactin and DMSO were utilized as positive

and negative control. The incubation period was executed for 72 hr at 30°C to target the

growth of strain. The appearance of clear and bald zones around discs shows the inhibition

of spore and mycelia formation. Finally, ZOI were measured in millimeters.

3.2.4.9. Alpha amylase (AA) inhibition

In vitro AA inhibition activity was performed for biogenic ZnONPs using optimized

protocol (Ali et al. 2017). The reaction mixture was prepared by adding step wise 25 μL of

AA enzyme, 10 μL of test sample, 40 μL of starch solution and 15 μL phosphate-buffered

saline. The reaction mixture with all components was placed in incubator for 30 min at

50°C by adding 20 µL of (1 M HCL) and 90 µL of iodine solution. Acarbose and DH2O

were utilized as positive and negative control respectively.

3.2.4.10. Antioxidant capacities

Spectrophotometric procedure was used to determine the radical scavenging activities of

ZnONPs by adding 2.4 mg of DPPH into 25 mL of methanol as free radical. Various doses

of ZnONPs (1−200 µg/mL) were studied for free radical scavenging properties. AA and

DMSO were utilized as positive and negative control. The 200 µL reaction mixture was

comprised of 180 µL of reagent solution and 20 µL of tested sample. The reaction mixture

was incubated in darkness for 30 min and readings were taken at 517 nm using microplate-

reader to determine % scavanging of DPPH employing the formula below:



Previously described Potassium-ferricyanide procedure was utilized to study total reducing

power of ZnONPs (Baqi et al. 2018). The AA was used as positive control and DMSO was

used as a negative control. The absorbance intensity was recorded at 630 nm using

microplate reader. The reducing power was determined as AA equivalent per milligram.


______________________________________________________________________________________ 61

Total antioxidant capacity was investigated using phosphomollybdenum method (Satpathy

et al. 2018). Absorbance was recorded at 695 nm using microplate reader. Results are

shown as microgram equivalent of AA per/µg of test sample. AA was utilized as positive

and DMSO as negative control.


3.3.1. Biogenic synthesis of ZnONPs

Although, NPs can be fabricated through different routes, however, plant-mediated

synthesis is more advantageous compare to physical and chemical routes. Synthesis of NPs

using physicochemical routes often suffer from many disadvantages because of to high

cost, processing time, energy consumption, labor, eco-toxicity and requiring high

temperature and pressure. Moreover, some noxious chemicals can remain adsorbed on the

surface of NPs which can limit their biomedical uses (Sharma et al. 2018). In this study,

bioinspired ZnONPs were synthesized using the principles of green chemistry. The

utilization of plant extracts to synthesize MNPs, including ZnONPs have several

advantages; being simple, cost-effective and are synthesized in the absence of organic

solvents and hazardous materials (Ahluwalia et al. 2018). Among the numerous medicinal

plants used in the green synthesis of MNPs, R. virgata was selected due to numerous

phytochemicals (physcion, emodin, kaempferol, rhamnocitrin, herbacetin and maesopsin)

(Kalidhar and Sharma 1984, Sharma et al. 2018). These phytochemicals are rich source of

electrons, actively involved in the synthesis of NPs and allow the process of reduction

followed by capping and stabilization. The reduction of Zn+ to Zn0 occurred immediately

after reaction of (Zn(NO3)2.6H2O) with leaves extract of R. virgata (Khalil et al. 2018).

In addition, these phytochemicals act as a capping agents on ZnONPs surface. The

formation of ZnONPs was observed by naked eyes in which the reddish brown solution

gets changed to a reddish black-colored solution.

3.3.2. Physical characterization

Diverse characterization techniques were used to confirm biosynthesis of ZnONPs. The

progress of ZnONPs formation was monitored by color change after salt (Zinc nitrate

hexahydrate) addition at 60°C. The change in solution (reddish black color) indicates the

formation of ZnONPs. To determine stability of ZnONPs, 1 mg/mL solution of NPs was

prepared and sonicated for 25 min. The turbid colloidal suspension was allowed to stand


______________________________________________________________________________________ 62

for 48 hr and SPR was checked with time intervals using spectrophotometer. The UV

spectrum of ZnONPs indicated absorption peaks centered at 400 nm, shows that colloidal

suspension remained stable for 48 hr. The reduction in absorption peak was observed after

72 hr which indicated the settlement of ZnONPs at the bottom of test tube. The Figure 3.2

(a, b) indicates UV-Vis spectra of biogenic ZnONPs.

XRD analysis was performed for the biogenic ZnONPs incubated at 100°C and annealed

as shown in Figure 3.2 (c). The observed Bragg peaks were found in agreement to the

single and pure phase hexagonal zincite with JCPD card No: 00–036–1451. The XRD

spectra indicate various diffraction peaks with 2 theta (2Ø) values of 32.45, 35.14, 36.52,

47.21, 57.52, 63.59, 66.67 and 79.27, which are indexed to (100), (002), (101), (102),

(110), (103), (112) and (202) Bragg’s reflection. Interestingly, average size of ZnONPs

was calculated as ~20 nm using Debye Scherer’s equation (Table 3.1). No other peaks

pertaining to the impurities were found which indicate single phase purity of the ZnONPs.

The XRD pattern for ZnONPs is consistent with some of the earlier reports (Khalil et al.

2018; Zak et al. 2011). Therefore, crystalline ZnONPs were successfully synthesized by

green method using R. virgata. Furthermore, the morphology of ZnONPs was determined

by SEM as shown in Figure 3.3 (a, b, c). In order to further investigate the size and shape

of ZnONPs, TEM analysis was performed. The TEM images showed that most of the

ZnONPs are of hexagonal shapes with average size ranging from 20−30 nm, however, few

ZnONPs with triangle shapes were also observed (Figure 3.3 (d).

The vibrational properties of ZnONPs were determined using Raman and FT-IR analysis.

The distinguished major modes of Raman spectra were centered at 94.867 cm-1 (E2L),

184.78 cm-1 (2TAM), 311.54 cm-1 (2E2M), 402.70 cm-1 (E1TO), 560.27 cm-1 (E2H+E2L).

Our Raman spectra are consistent with the earlier studies of biogenic ZnONPs and

therefore confirm the synthesis of ZnONPs (Khalil et al. 2018). Figure 3.4 (a) indicates the

Raman spectra of ZnONPs. Fourier transformed infrared spectroscopy (FT-IR) analysis

was done as shown in Figure 3.4 (b). The peak located at 3322 cm-1 is ascribed to O−H

stretching. The other peak located at 1557 cm-1 and 1404 cm-1 correspond to C−C and C=C


______________________________________________________________________________________ 63

Figure 3.2: UV and XRD spectra analysis for biogenic ZnONPs (a) UV visible spectra (b)

Stability of biosynthesized ZnONPs (c) XRD spectra of R. virgata mediated ZnONPs.


______________________________________________________________________________________ 64

Figure 3.3: Various SEM and TEM images of R. virgata mediated ZnONPs using zinc

nitrate as a precursor (a,b,c) Their HR-SEM image (d) Their TEM image.


______________________________________________________________________________________ 65

Figure 3.4: (a) Raman Spectra of the ZnONPs biosynthesized using zinc nitrate

hexahydrate as precursor (b) FT-IR spectra of ZnONPs.


______________________________________________________________________________________ 66

stretching. The peak at 1048 cm-1 is characteristic to stretching vibration of symmetric

C−O.

While the high absorption peaks at 468 to 799 cm-1 signify the presence of Zn−O bond,

which demonstrates a high level of crystallinity of ZnONPs. Fourier transformed infrared

spectroscopy (FT-IR) was studied to determine the bioactive molecules involved in the

reduction and stabilization of ZnONPs. The FT-IR absorption of biogenic nanoparticles

was in agreement with the previous studies (Thema et al. 2015). In general, size and

positioning of peaks vary depending on the synthesis protocol and plant used. Elemental

composition of ZnONPs was studied using EDS. The EDS peaks have shown that both

Zinc and oxygen are present in the sample while no other elements have been reported in

the EDS spectrum apart from zinc and oxygen which confirm the single crystalline nature

of ZnONPs. The EDS spectrum is shown in Figure 3.5. The size distribution, PDI and ζ-

potential of ZnONPs incubated at 100°C and calcined at 500°C was measured through DLS

analysis. The data have shown larger particle aggregates of 189.6 nm. The zeta potential

and PDI of ZnONPs was -7.11 mV and 0.801 respectively (Figure 3.6 (a, b). Our results

are in line with the earlier studies performed on ZnONPs using Ixora coccinea (Yedurkar

et al. 2016). DLS is primarily used to confirm NPs size in colloidal suspensions in the range

of nano and submicron. The measurement of zeta potential is based on the movement of

particles under electric field. Moreover, surface charge and local environment of the

particles also play significant role in the measurement of zeta potential (Chaudhuri and

Malodia 2017).

3.3.3. Antimicrobial activities

The traditional medicines often provide potential treatment for bacterial infections but there

are problems associated with antibiotics such as antibiotic resistance. Scientific community

is working hard to develop new strategies to overcome this resistance and minimize the

risks of developing and spread of infectious diseases (Woodford and Livermore 2009;

Hameed et al. 2019). New developments in nanotechnology are providing promising tools

to design new substances with special antimicrobial properties (Rizzello et al. 2013).

Figure 3.7 (a) indicates antibacterial potential of ZnONPs against different bacterial strains

using different concentrations (1000−31.25 µg/mL). The gram positive bacteria strains

utilized are S. aureus and B. subtilis, while gram negative strains are P. aeruginosa, K.


______________________________________________________________________________________ 67

Figure 3.5: Elemental composition of ZnONPs using EDS analysis.


______________________________________________________________________________________ 68

Figure 3.6: (a) Size distribution of R. virgata mediated ZnONPs (b) Zeta potential

measurement of ZnONPs.


______________________________________________________________________________________ 69

pneumoniae and E. coli. Most of the studied bacteria were found susceptible to ZnONPs.

However, bacterial strains that were most susceptible to biogenic ZnONPs were B. subtilis

and S. aureus (MIC: 7.8 µg/mL) while P. aeruginosa was reported the least susceptible

strain with MIC values of 62.5 µg/mL. The 10 µg pure oxytetracycline was used as positive

control. No single concentration was found effective than oxytetracycline. Overall,

significant antibacterial activities have been reported for biogenic ZnONPs which are

consistent with earlier findings (Safawo et al. 2018; Hameed et al. 2019). The reason for

such a strong antibacterial efficacy might be bioactive functional groups adsorbed on the

surface of nanoparticles. In nutshell, our study concluded that antibacterial activities were

increasing with increase in concentration of ZnONPs. Different research studies have

explained antibacterial property of ZnONPs and have concluded that ROS generation is

the primary mechanism that imparts antimicrobial potency to ZnONPs. Besides, ROS

generation, other factors like membrane damage due to adsorption of NPs on surface cause

cell damage. Likewise, surface defect in NPs symmetry account for antibacterial activity

(Li et al. 2012). Furthermore, it is revealed that these bioactive functional groups attached

from leaves extract are helpful in capping and stabilization of ZnONPs as a result they also

play strong role in antibacterial potencies.

Antifungal property of ZnONPs was also performed against different fungal strains using

different concentration (1000−31.25 µg/mL). Amp B was used as a positive control.

Different fungal strains used in the study include M. racemosus, A. flavus, A. niger, C.

albicans and F. solani (Figure 3.7 (b). Significant research has been done on antibacterial

activity of ZnONPs while limited antifungal activities have been investigated for ZnONPs.

Present research study is the first, reporting antifungal activity of R. virgata mediated

ZnONPs. Our results concluded direct relation between concentration and test sample of

ZnONPs. The A. flavus was the least susceptible strain with MIC value of 125 µg/mL while

A. niger was the most susceptible strains with MIC value of 15.625 µg/mL. However, none

of the tested samples give % inhibition greater than Amp B. Among the tested strains, M.

racemosus, A. niger and F. solani were inhibited at all concentrations. In addition to ROS,

the earlier reports suggest that interference of ZnONPs with fungal hypea and fungal spores

lead to inhibition of fungal growth. Significant dose-dependent antifungal activities are

also reported for ZnONPs in the earlier studies (Hameed et al. 2019), which are in


______________________________________________________________________________________ 70

Figure 3.7: (a) Antibacterial activities of biogenic ZnONPs (b) Antifungal activities of

biogenic ZnONPs.


______________________________________________________________________________________ 71

agreement with present study. Furthermore, results concluded concentration-dependent

response towards ZnONPs.

3.3.4. Antileishmanial activity (ALA)

Leishmania is a neglected tropical disease caused by L. tropica (Kaye and Scott 2011). The

parasites are endemic to ~100 countries and its incidence rate is ~1.2 million per annum.

The antileishmanial drugs available in the market are often toxic, poor and are highly

expensive. Thus, scientific community is engaged in developing alternative ways for its

treatment. Henceforth, considerable research work has been done to develop MNPs for

Leishmania treatment. Different MNPs have been utilized in different in vitro studies for

the cytotoxicity assessment against Leishmanial parasites (Hameed et al. 2019; Iqbal et al.

2019). However, biogenic ZnONPs have been poorly investigated for cytotoxicity

activities against L. tropica. In current study, ALA activity of biogenic ZnONPs was done

against L. tropica as shown in Figure 3.8 (a). The Leishmanial parasites were exposed to

ZnONPs with different concentrations (1 to 200 μg/mL) for 72 hr and have shown dose

dependent inhibition. The antileishmanial activity was increasing as concentration of

ZnONPs was increasing. The IC50 values have shown promising antileishmanial property

against L. tropica promastigotes for ZnONPs (IC50: 8.34 µg/mL). Similarly, ALA against

L. tropica amastigotes was also shown by ZnONPs (IC50: 13.6 µg/mL) which are in

agreement with the earlier studies of ZnONPs (Baqi et al. 2018; Khalil et al. 2018).

3.3.5. Anticancer activity

Cancer is a devastating disease and remains a significant reason for mortality in both

developed and developing countries and is continuously increasing projected to be ~21

million by the year 2030 (Iqbal et al. 2018; Iqbal et al. 2019). Among the various types of

cancers, hepatocellular carcinoma is currently the sixth most common cancer and

considered to be the second deadliest cancer in men, sixth for women and account for

745,517 deaths. The risk factors associated with liver cancer are viral infections (HBV,

HCV), heavy alcohol consumption and toxins exposure (aflatoxin) (Daher et al. 2018). To

deal with this deadly disease, scientific community is working hard to design new treatment

materials. The anticancer activity of ZnONPs was investigated against HepG2

(hepatocellular carcinoma) cancer cell line. The HepG2 cancer cells were treated with


______________________________________________________________________________________ 72

Table 3.1: Major XRD values and calculation of crystalline size.

Bragg angle

Bragg

Crystalline Average size S. No Beta (ΔƟ1/2) reflection

(Ɵ) rad Size (nm) (nm)

(hkl)

1 0.283006 0.004258 100 33

2 0.306477 0.006383 002 22

3 0.318514 0.006406 101 22

4 0.411798 0.005908 102 23

19.625

5 0.501672 0.000923 110 15

6 0.554633 0.009594 103 15

7 0.581515 0.01169 112 12

8 0.691381 0.00731 202 15


______________________________________________________________________________________ 73

different concentration of ZnONPs (7.8125-1000µg/mL) for 24 hr which resulted in a dose-

dependent inhibition of cancer cells. Our results have shown strong anticancer activities

for Zinc oxide nanoparticles. A dose-dependent inhibition with ~80% mortality was

obtained with highest inhibition reported at 1000 µg/mL. The IC50 value calculated was

19.67 µg/mL, which confirmed strong therapeutic property of ZnONPs. Our research work

is in match with the earlier research work of biogenic ZnONPs using HepG2 cancer cell

line (Wahab et al. 2014; Hassan et al. 2017). The results are indicated in (Figure 3.8 (b).

3.3.6. Brine shrimps cytotoxicity assay (BSC)

For the assessment of cytotoxicity, BSC was performed. The BSC is the most appropriate

test to determine biological properties of naturally occurring compound (Chanda and

Baravalia 2011). Figure 3.8 (c) shows % mortalities of the ZnONPs at different doses

(200−1 μg/mL). The Cytotoxic potency of ZnONPs was investigated through dose-

dependent manner. The cytotoxic activity was increasing as concentration of ZnONPs was

increasing while their IC50 was recorded as 26.34 µg/mL. These results have confirmed

cytotoxic activity of ZnONPs. Our test sample was found to be in category of general

cytotoxicity with IC50 value of 26.34 µg/mL. Our results are in agreement with earlier

reports of ZnONPs using Silybum marianum (Baqi et al. 2018).

3.3.7. Biocompatibility tests

The biocompatibility and toxicological properties of ZnONPs were assessed against human

RBCs and macrophages. The hemolysis is measured, as hemoglobin is released due to

rupturing of RBCs after treatment with sample. If a given NPs is hemolytic, it will rupture

RBC, which leads to hemoglobin release. To investigate the biocompatible nature of

ZnONPs, haemolytic assay was performed against human RBCs in the range of 200–1

µg/mL. The present results have indicated that biogenic ZnONPs are non-hemolytic at

lower dose (2 μg/mL), slightly hemolytic at 5 to 40 μg/mL, while hemolytic at a

concentrations of >45 μg/mL. These results were found to be consistent with earlier report

(Topwal and Uniyal 2018). The IC50 value of ZnONPs against RBCs was calculated to be

>200 μg/mL. The results of the study concluded that biogenic ZnONPs are non-hemolytic

at lower doses.


______________________________________________________________________________________ 74

Figure 3.8: (a) Antileishmanial activities of R. virgata mediated ZnONPs (b) Cytotoxicity

against HepG2 cell line (c) Cytotoxicity against brine shrimps.


______________________________________________________________________________________ 75

To measure the bio-safe nature of ZnONPs, MTT cytotoxic activity was also investigated

against human macrophages. The macrophages response after treatment with ZnONPs was

dose-dependent, means that at a higher dose cytotoxic effect was increasing while at lower

dose there was less cytotoxicity. The results have shown that ZnONPs at concentration of

200 μg/mL inhibited macrophages growth by ~32% which confirm the non-toxic nature of

ZnONPs. Thus, ROS at lower concentration are nontoxic to RBCs and macrophages unless

the dose is increasing beyond the limit (Prach and Stone 2013). The IC50 value of ZnONPs

was calculated as >200 μg/mL. The results of biocompatibility assays of ZnONPs are

summarized in Figure 3.9 (a).

3.3.8. Enzyme inhibition potencies

Figure 3.9 (b) shows PK inhibition potential of ZnONPs to inhibit PK enzyme. PK are

enzymes responsible to phosphorylate amino acids serine-threonine, tyrosine residues,

regulate different processes; metabolism, proliferation etc. Deregulated phosphorylation

can induce genetic abnormalities leading to progression of cancer. Henceforth, any product

having ability to inhibit PK enzymes can be of significant importance in cancer research

(Yao et al. 2011). PK phosphorylation plays significant role in development of hyphae in

Streptomyces fungal strain and same principle is used to investigate PK inhibition

activities. The strain has been largely used to determine medicinal compounds for

determination of PK inhibition (Waters et al. 2002). Following disc diffusion method, PK

inhibition assay was performed in range of 1000−31.25 µg/mL using surfactin as a positive

control. Zones of inhibition up to 17 mm was measured at 1000 μg/mL for ZnONPs, which

indicates significant PK inhibition activities. A dose dependent response is concluded.

Therefore, strong signal transduction inhibitor is recognized in the form of nanoscaled

ZnONPs that can be further investigated for anti-infective and anticancer properties.

Because of PK inhibition property, one can pre-conclude that biogenic ZnONPs are non-

hazardous, eco-friendly, economical and might play significant role in cancer treatment.

Our results are in line with earlier reports, where Periploca aphylla-ZnONPs shown

significant PK inhibition potential (Abbas et al. 2017). In addition, alpha amylase (AA) inhibition potential was performed ranging from

1000−31.25 µg/mL. The AA is responsible to convert carbohydrates into glucose (Yao et

al. 2011), therefore, blocking the function of AA can prevent level of glucose which is


______________________________________________________________________________________ 76

main area of diabetes research (Oyedemi et al. 2017). Bioinspired ZnONPs were

investigated for their AA inhibition potential. Our results have shown that ZnONPs were

effective by inhibiting AA. Maximum inhibition is reported to be 48% at highest

concentrations (1000 μg/mL). Figure 3.9 (c) shows AA inhibition potential of green

ZnONPs. Our results of ZnONPs are consistent with Sageretia thea and Fagonia indica

mediated ZnONPs (Baqi et al. 2018; Iqbal et al. 2018). 3.3.9. Antioxidant activities

Figure 3.9 (d) shows antioxidant activities (TAC, TRP, DPPH free radical scavenging) of

R. virgata mediated ZnONPs. The antioxidant assays were performed in the concentration

range of 200–1 µg/mL. Maximum value for total antioxidant in terms of AA was found to

be 32.31% for 200 μg/mL for ZnONPs. TAC shows scavenging properties of tested

compounds towards ROS species. Since in the current study, aqueous extract of R. virgata

leaves extract was used as capping, reducing and oxidizing agent. It can be deduced, that

several phenolic compounds scavenge ROS which are also attached to ZnONPs. In order to further verify the presence of antioxidant species attached to ZnONPs, total

reducing power (TRP) was investigated. This procedure was performed to examine the

reductones that are involved in antioxidant potency by contributing H-atoms and may result

in damage of free radical of chain (Ul-Haq et al. 2012). Biogenic ZnONPs have shown

strong antioxidant activity. The reducing power was decreasing as concentration of

ZnONPs was decreasing. Maximum reducing power (38%) was found at concentration of

200 μg/mL. Strong DPPH radical scavenging property (81.36 %) was recorded at 200

μg/mL for ZnONPs. From results shown in Figure 3.8 (d) it can be concluded that several

antioxidant compounds might involve in reduction and stabilization of ZnONPs via

R.virgata leaves extract. Our results are consistent with earlier studies of ZnONPs using

Sageretia thea and Coccinia abyssinica (Khalil et al. 2018; Safawo et al. 2018; Umar et al.

2019). Variation and disagreement in relative to other studies might be due to numerous

significant factors like experimental conditions (seasonal variation, location, humidity),

other factors such as method of NP synthesis, plant used and size of NPs etc (Hameed et

al. 2019).


______________________________________________________________________________________ 77

Figure 3.9: (a) Biocompatibility of R. virgata mediated ZnONPs against human RBCs and

macrophages (b) Inhibition activity against protein kinase (c) Inhibition activity against

alpha amylase (d) Antioxidant activities of R. virgata mediated ZnONPs.

Chapter 4 Green synthesis of cobalt oxide nanoparticles

______________________________________________________________________________________ 78

Chapter 4: Biogenic synthesis of green and cost effective cobalt

oxide nanoparticles using Geranium wallichianum leaves extract

and evaluation of in vitro antioxidant, antimicrobial, cytotoxic,

enzyme inhibition properties


______________________________________________________________________________________ 79

Graphical abstract of greenly synthesized Cobalt oxide nanoparticles, their characterization

and biological applications.


______________________________________________________________________________________ 80

Abstract

In the present study, CoONPs were synthesized using Geranium wallichianum leaves

extract as both reducing and stabilizing agents. The biogenic CoONPs were further studied

for diverse in vitro biological applications (antibacterial, antifungal, enzyme inhibition,

biocompatibility, anticancer and antileishmanial). The biogenic CoONPs were further

studied using various microscopic and spectroscopic techniques including SEM, XRD,

DLS, FT-IR, EDX, Raman spectroscopy. The antibacterial activity was determined using

different bacterial strains. Bacillus subtilis (MIC: 21.875 μg/mL) was found to be most

susceptible strain to CoONPs. CoONPs has shown significant results against HepG2 cancer

cells (IC50: 31.4 μg/mL). Dose-dependent cytotoxicity was performed using Leishmanial

amastigotes (IC50: 9.53 μg/mL) and promastigotes (IC50: 3.12μg/mL). The cytotoxic

potency was investigated using brine shrimps (IC50:18.12 μg/mL). Furthermore, CoONPs

were found biocompatible against human macrophages and erythrocytes (IC50: >200

μg/mL). Moderate antioxidant activities: TAC (61.63%), DPPH (89.56%) and TRP

(56.79%) are reported for CoONPs. In addition, alpha amylase and protein kinase

inhibition assays were studied. Our results concluded that asynthesized CoONPs are non-

toxic and biocompatible and can be used for different biomedical applications.


______________________________________________________________________________________ 81

4.1. Introduction

Nanotechnology is focusing on designing and applications of nanoparticles (NPs) which

has made a revolutionary impact in developing diverse areas such as medicines, food,

agriculture, cosmetics, engineering and biomedical instruments (Iqbal et al. 2019). NPs

possess versatile physicochemical characteristics that are quite different from that of their

bulk counterpart. The material mostly shows unique and promising properties when having

size range of 1−100 nm. These unique properties of NPs are because of their high energy

and frictions of the surface atoms, spatial confinement and reduced imperfection (Bai et al.

2009). NPs are beneficial over bulk material as a result of their surface plasmon light

scattering, surface-enhanced Rayleigh and Raman scattering (Ramanavicius et al. 2005).

NPs size, shape, surface chemistry and monodispersity are highly important to modulate

their properties in diverse nano-applications (Suganthy et al. 2016; Iqbal et al. 2019).

Different synthesis protocols have been developed for the formation of NPs such as

chemical (polymeric and colloidal process), physical (Melt mingling, high energy ball

mining, sputter deposition, ion implantation, laser-vaporization) and biological methods.

The chemically mediated synthesis of NPs can lead to the development of toxic compounds

which may remain attached on NP’s surface and have negative impact on human health in

different biomedical applications (Yu et al. 2013; Jodłowski et al. 2014; Hameed et al.,

2019). Recently, bioinspired NPs from plants, microbes algae and fungi have also been

synthesized and has gained significant popularity because of their physical, electrical,

chemical, optical properties, eco-friendly behaviour, nano-size, low cost, deep penetration

and public acceptance (Hameed et al., 2019; Iqbal et al. 2019). Compare to other biological

sources, the phytochemicals available in the plant extract helps in the reduction and

stabilization of NPs (Islam et al. 2016). Keeping in view the design principles of green

chemistry several scientists have designed different metal and metal oxide NPs for

nanomedicines and targeted delivery agents to treat different diseases (Chikkanna et al.

2018; Galan et al. 2018; Iqbal et al. 2018; Khan et al. 2018; Rajendran et al. 2018).

Cobalt based NPs has considerably gained the attention of scientific community because

of their ecofriendly nature, easy handling, low cost and strong electro potential (Bibi et al.

2017). CoONPs is a versatile class of NPs with numerous applications in lithium-ion


______________________________________________________________________________________ 82

batteries, gas-sensors, supercapacitors, heterogeneous catalysis, magnetic semiconductors

and field emission materials (Hu et al. 2017, Shi et al. 2018). Moreover, CoONPs have also

shown resistance against corrosion and oxidation as a result they can be used in electronic

devices and super conducting materials (Chen et al. 2010). CoONPs have been suggested

as a diagnostic tool and adjuvants in vaccination and radioactive vectors for the treatment

of cancer. In addition, it has played significant role as a cofactor in vitamin B12 and have

been effectively used in sensing of amino acid, nitrates, glucose, arsenic and methanol (Cui

et al.2013; Ovais et al. 2018). It has been estimated that by 2026, the world market of NPs

will reach up to 10,000 tons in terms of volume and its sale will mark US$50 billion

(Gharibshahian et al. 2018).

Numerous physical and chemical procedures have been discovered for the formation of

CoONPs (Jang et al. 2017; Malathy et al. 2017; Kwon et al. 2018; Pineda et al. 2018).

However, these techniques have certain limitations as they use expensive metal salts,

reducing agents, need high temperature and pressure and use sophisticated equipments. To

avoid the issues of energy gaps and environmental toxicities, plant-extracts is the best

possible solution (Matinise et al. 2017). The plant extracts are comprised of a wide range

of green chemicals such as phenolic, alkaloids, saponins, flavonoids, vitamins, inositol and

terpenes (Abbasi et al. 2019a). Synthesis of NPs using plant extracts is a rapid, reliable,

nontoxic, biocompatible, benign, ecofriendly and cheap. The green synthesis of CoONPs

have been successfully revealed (Khalil et al. 2017; Matinise et al. 2018). Therefore,

scientists are focusing on the phyto-synthesis of MNPs. Taking in view of biogenic

synthesis, CoONPs have been successfully fabricated using aqueous leaves extract of G.

wallichianum as a strong reducing and stabilizing agents. The plant is commonly known

as “Shepherd’s needles”/English and “Ratanjot”/Pahari and is reported to be used against

leucorrhoea, rheumatism, gonorrhea, arthritis, liver and cardiovascular problems. In

current study, green synthesis of CoONPs have been reported without utilizing any noxious

chemicals and organic/inorganic solvent. According to the best of our knowledge, CoONPs

using G. wallichianum leaves extract has been reported for the first time. The CoONPs

have been extensively characterized through diverse techniques such as UV, XRD, SEM,

EDS, UV, FT-IR and Raman spectroscopy. Moreover, the in vitro anticancer, antibacterial,


______________________________________________________________________________________ 83

antifungal, antileishmanial, biocompatibility and cytotoxic properties of CoONPs were

investigated.


4.2.1. Sampling of plant and identification

The G. wallichianum was collected from Nathiagali at its flowering stage and was properly

identified. The specimen under voucher number (HPBMBL-18-001) was deposited in in

Plant Biochemistry and Molecular Biology Lab, QAU Pakistan. Fresh plant leaves were

properly excised, washed and dried. Further, aqueous leaf extract was prepared using fine

powder of grounded leaves.

4.2.2. Biosynthesis of CoONPs

The biogenic synthesis of CoONPs was carried out using already optimized protocol (Iqbal

et al. 2019). The leaves extract of G. wallichianum was obtained by adding 20 gm of plant

powder to 300 mL of deionized water. The mixture was heated and stirred on hot plate for

2 hr at 85°C. The homogenate was filtered thrice to achieve pure aqueous extract. To 30

mL of pure plant extract, 1.5 gm cobalt acetate salt was added under continuous stirring

conditions at 60°C for 2 hr followed by cooling and centrifugation at 14,000 rpm/10 min.

The black powder at the bottom of falcon was assumed as CoONPs, were incubated for 3

hrs at 100°C.

4.2.3. Characterization of CoONPs

Different characterization techniques were used to study the crystalline, vibrational and

morphological properties of CoONPs. The formation of CoONPs was determined by UV-

Vis spectrophotometry at wavelength of 300−600 nm. XRD was performed for all annealed

samples to study the crystal purity and corresponding size of CoONPs. The morphology

and size distribution of GW-CoONPs was studied using SEM. Furthermore, elemental

composition was determined by EDS. Moreover, for the confirmation of the structural and

vibrational properties of CoONPs, Raman spectra were recorded over the range from 200

to 800 cm-1. Furthermore, FT-IR (500-4000 cm-1) was used to analyze the presence of

different functional groups involved in the reduction and stabilization of CoONPs. The ζ-

potential and hydrodynamic sizes distributions was studied using DLS analysis.


______________________________________________________________________________________ 84

2.2.4. Biological activities of CoONPs:

4.2.4.2. Antibacterial activities

The bactericidal activity was carried out using agar disc diffusion method for calculating

MIC values (Thatoi. Et al. 2016). Five different bacterial strains: P. aeruginosa, B. subtilis,

K. pneumonia, E. coli and S. aureus were cultured on standardized nutrient agar media to

form uniform lawns (100 μL of standardized suspension having optical density of 0.5 was

used). The 6 mm filter discs laden with 15 μL of CoONPs were subsequently placed on

bacterial lawns. Discs loaded with oxytetracycline (15 μg) were considered as standard.

The ZOI were calculated after incubation for 20 hr at 37°C and their individual MIC values

were calculated across 700 μg/mL to 21.875 μg/mL.

4.2.4.2. Antifungal activities

The fungicidal assay was carried out using disc diffusion method against C. albicans, M.

racemosus, A. niger, F. solani and A. flavus as reported earlier (Bakht et al. 2013). Fungal

nutrient media SDA was prepared and autoclaved. Filter discs loaded with 10 μL CoONPs

were placed on bacterial media plates. The positive control used was Amphotericin B (10

μg) while the negative control was DMSO. Furthermore, plates were incubated at 25°C for

48 hr. The ZOI were measured for different concentrations of CoONPs (700−10.9375

μg/mL) and their MICs values were calculated.

4.2.4.3. Antileishmanial assay (ALA)

The Leishmania tropica “KWH23 strain (both promastigotes and amastigotes) was used to

determine cytotoxicity potencies of CoONPs via MTT cytotoxicity assay (Ovais et al.

2018). The Leishmanial parasites were cultured on MI99 media (GIBCO) added with 10%

FBS. Furthermore, cytotoxicity was evaluated for CoONPs (200 to 1 μg/mL) while using

amphotericin B as positive control. The 96 well plate with CoONPs was incubated at 24°C

for 72 hr. Readings were taken at 540 nm utilizing spectrophotometer and their IC50 value

was calculated using GraphPad software. The % inhibitions was calculated using the

formula below:

% age inhibition = [1 − {absorbance (S)}

{absorbance (C)}]×100


______________________________________________________________________________________ 85

4.2.4.4. Brine shrimp cytotoxicity assay (BSC)

Brine shrimp cytotoxicity assay was performed to determine cytotoxicity activity of

CoONPs as described previously using Artemia salina larvae. The 20 mg eggs of A. salina

were incubated in bi-partitioned tray containing 3.8 g/L sea salt for 24 hr under continuous

light at 30°C (Ali et al. 2017). After 24 hr, 30 nauplii were picked up through micropipette

and transferred into glass-vials having CoONPs and sea water. Various test concentration

of biogenic CoONPs were used. The vials having sea water, nauplii and vincristine sulphate

was taken as positive control. However, vials with sea water, nauplii, and DMSO was taken

as a negative control. Following 24 hr of incubation, % inhibition was measured based on

the number of live and dead shrimps. IC50 value was calculated using GraphPad software.


The cytotoxic potency of bioinspired CoONPs was studied using hepatocellular carcinoma

(HepG2) cancer cell line using previously published protocol (Iqbal et al. 2017). The

DMEM media supplemented with Pen-Strep, streptomycin and FBS (10 %) was used for

the growth of cells. Furthermore, cells were grown at 37°C in 5% humified carbondioxide

incubator. The MTT assay was done in 96-well plate while incubating at various

concentration (3.9−500 μg /mL) of CoONPs for 48 hr. Furthermore, each well was loaded

with 20 μL MTT solution and was incubated for 3 hr. Moreover, 100 μL of DMSO was

used to replace the culture media followed by incubation up to 25 min. The formation of

formazan by living cells was observed via plate reader at 570 nm.

4.2.4.6. Biocompatibility with human RBCs

The hemolytic activity was investigated to check the bio-compatible nature of CoONPs

using previously optimized procedure (Malagoli et al. 2007). To perform hemolytic assay,

2 mL freshly isolated human erythrocytes were centrifuged at 10,000 rpm for 5 min. The

erythrocytes suspension was prepared by mixing phosphate buffer saline and 100 μL of red

cells. To 100 μL red cells suspension various concentrations of bioinspired CoONPs were

added followed by incubation at 35°C for 1 hr. Next centrifugation was carried out at

12,000 rpm for 15 min. The supernatant was added to 96 well plate and hemoglobin release

was observed at the wavelength of 540 nm using Elisa reader. Triton X-100 served as a

control. The values for percentage hemolysis were derived using following formula;


______________________________________________________________________________________ 86

Hemolysis(%) =[AbS − AbNC]

[AbPC − AbPC]× 100

4.2.4.7. Biocompatibility with human macrophages

The biocompatibility assay of CoONPs was also studied using human macrophages (de

Almeida et al. 2000). To achieve this purpose, isolated cells were cultured in RPMI media

supplemented with 10% FBS, HEPES buffer, streptomycin and penicillin. Furthermore,

macrophages were treated with different concentrations of CoONPs and were placed in 5%

CO2 incubator. The % inhibition was recorded using formula below;

% inhibition =1 – Abs(s)

Abs (c) ×100

4.2.4.8. Protein kinase (PK) assay

PK activity of CoONPs was also performed as described earlier (Fatima et al. 2015). The

aseptic conditions were used for performing PK assay. Streptomyces 85-E strain was

culture on ISP4 minimal media to obtain uniform lawns. The 6 mm filter discs loaded with

10 μL of CoONPs were placed on microbial lawns. The incubation was performed for

24−36 hr at 30°C for to achieve fungal growth. The surfactin was used as a standard. The

cleared and bald zones were observed, indicating the inhibition of Streptomyces 85-E

strain.

4.2.4.9. α amylase (AA) inhibition assay

The AA inhibition activity of CoONPs was carried out using already published protocol

(Abbasi et al. 2019). The reaction mixture contained 25 μL α-amylase enzyme, 40 μL

starch solution, 15 μL PBS and 10 μL CoONPs. Reaction mixture containing all required

constituents was incubated at 50°C for 30 min followed by adding HCl (1 M) and iodine

solution (90 μL). Acarbose was used as positive control. Finally percentage inhibition was

calculating using the equation below;

inhibition(%) =[Sample(OD) – NC(OD)]

[Sample(OD) – PC(OD)] ×100

4.2.4.10. Antioxidant capacities

Spectrophotometric procedure was used to determine the radical scavenging potency of

CoONPs by adding 2.4 mg of DPPH into 25 mL of methanol as free radical (Abbasi et al.

2019). Various concentrations of CoONPs (1−200 µg/mL) were studied for free radical


______________________________________________________________________________________ 87

scavenging activities. The ascorbic acid (AA) and DMSO were utilized as positive and

negative control. The 200 µL reaction mixture was comprised of 180 µL of reagent solution

and 20 µL of tested sample. The reaction mixture was incubated in darkness for 30 min

and readings were taken at 517 nm using microplate-reader to determine % scavanging of

DPPH employing the formula below;



Previously described Potassium-ferricyanide procedure was utilized to study total reducing

power of CoONPs (Baqi et al. 2018). The AA was used a positive control and DMSO was

used as a negative control. The absorbance intensity was recorded at 630 nm using

microplate reader. The reducing power was determined as AA equivalent per milligram.

Total antioxidant capacity was investigated using phosphomollybdenum method (Satpathy

et al. 2018). Absorbance was recorded at 695 nm using microplate reader. Results are

shown as microgram equivalent of AA per/µg of test sample. The AA was utilized as

positive and DMSO as negative control.


4.3.1. Biogenic synthesis of CoONPs

The green synthesis of MNPs using plant extracts is a rapidly developing field having

numerous advantages compared to chemical and physical routes. Formation of NPs using

physical and chemical methods is expensive, laborious and eco-toxic (Dauthal and

Mukhopadhyay 2016; Sood and Chopra 2017), whereas, highly stable and biocompatible

NPs can be synthesized using plant extracts due to the presence of different

phytocompounds. The plant mediated synthesis of NPs is sample, green and clean, eco-

friendly and don’t need sophisticated equipments (Sood and Chopra 2017). Previously,

CoONPs have been synthesized but their biological applications (antibacterial, antifungal,

anticancer, antileishmanial, antioxidant, enzyme inhibition) are rarely reported. The

ethnobotanical surveys and literature review have clearly elaborated the medicinal values

of G. wallichianum which is used in the treatment of dysentery, ulceration, leucorrhoea,

diabetes, cancer and hemorrhages (Ismail et al. 2012; Ahmad et al. 2014). The present

study provides perspectives on the green synthesis of highly stable CoONPs from G.

wallichianum. The synthesis of CoONPs without using any external reducing stabilizing


______________________________________________________________________________________ 88

and capping agents demonstrate that G. wallichianum leaves extract contained many

natural phytocompounds (stigmasterol, ursolic acid, β-sitosterol, β-sitosterol galactoside

and herniarin) which are involved in reduction and stabilization of metal ions (Ismail et al.

2009). A detailed mechanism for the green synthesis is shown in Figure 4.1.

Largely, flavanoids and phenolic compounds are involved in the phytosynthesis of NPs,

however, the exact mechanisms of phytosynthesis is still not known. Green synthesis of

NPs is the significant aspect of nanobiotechnology as the stability of NPs play significant

role in modulating their biological potentials (Dauthal and Mukhopadhyay 2016; Otunola

and Afolayan 2018). Despites, colloidal and dispersion and stability behavior, NPs must

also possess low toxicity and high biocompatibility. These properties are highly significant

to conserve the physicochemical behaviour and successful utilization in the physiological

environment. The characterization techniques revealed pure Co3O4 NPs synthesized

through green route using G. wallichianum.

4.3.2. Physical characterizations

Different characterization methods were applied for the confirmation of biosynthesis of

CoONPs. The progress of the synthesis of CoONPs was observed by the darkening of aqua

extract after salt addition at 60°C. The stability of CoONPs was determined sonicating 1

mg CoONPs into 1 mL DMSO for 20 min. In next step, suspension was allowed to settle

down for 48 hr and their SPR was observed after regular intervals via spectrophotometer.

Constant absorptions at 503 nm revealed the stability of suspension up to 24 hr. After 24

hr, the broad absorption peak corresponds to the settlement of CoONPs in the tube. The

results are given in Figure 4.2 (a, b). Furthermore, XRD analysis was performed for

calcined NPs. The XRD spectrum has shown successful synthesis of CoONPs. XRD

spectrum revealed pure crystalline nature of CoONPs with 2Ø values of 20.70, 33.3, 37.22,

45.19, 65.16, which are indexed to (111), (220), (311), (400), (440) Bragg reflection of the

fcc crystalline phase of CoONPs (JCPDS card 00-042-1467). The average size of CoONPs

was calculated as ~ 21 nm. Moreover, from Bragg peaks, it can be inferred that the biogenic

CoONPs were synthesized in their pure phase. Results of the XRD are given in Figure 4.2

(c, d).


______________________________________________________________________________________ 89

Figure 4.1: Schematic representation for the biogenic of CoONPs nanoparticles via green

route.


______________________________________________________________________________________ 90

Figure 4.2: UV and XRD spectra of biogenic CoONPs (a) UV visible spectra (b) Stability

of biosynthesized CoONPs (c) XRD spectra of CoONPs (d) Size calculation via Scherer

approximation.


______________________________________________________________________________________ 91

UV-Vis spectra and XRD studies indicated the synthesis of CoONPs. Bragg peaks for these

NPs were of pure CoONPs in XRD pattern as no peaks were found for other related

compounds. Figure 4.3 (a, b, c) represents the shape and surface topology of CoONPs using

SEM. In SEM images, the shape of NPs appeared to be agglomerated. The EDX analysis

was performed to study the elemental composition of GW- CoONPs. The EDX values

reflect the atomic structure of biogenic NPs and shows the atomic content of nanoparticles.

The EDX spectra revealed the presence of cobalt and oxygen in CoONPs and spectrum is

given in Figure 4.4 (a). The spectral modes of Raman were mainly found at 191, 469, 511,

608 and 675 cm-1. The sharp peak located at 675 cm-1 (A1g) is attributed to CoIIIO6

octahedra. The medium intensity peaks located at 191 (F2g), 469 (Eg), 511 (F2g) and 608

cm-1 (F2g) are attributed to combine vibrations of tetrahedral sites and octahedral oxygen

motions. The Raman spectral modes of our study are in correspondence to the previously

described studies (Farhadi et al. 2016). Raman spectroscopy analysis are illustrated in

Figure 4.4 (b). The vibrational properties of CoONPs were studied using FT-IR analysis.

The FT-IR spectra were in the spectral range of 500-4000 cm−1 as shown in the Figure 4.5

(a). An intense peak at 546 cm-1 attributes to the Co-O stretch in a Co3O4 (Ismail et al.

2012; Sood and Chopra 2017). The peaks at 975 and ~3400 cm-1 signify the availability of

–CH and OH groups which play significant role in both stabilization and reduction of

CoONPs. IR spectra support the role of bioactive functional groups which are involved in

the reduction of metal salt to NPs. The absorption bands in FT-IR were found to have

stretching vibrations of Co−O in Co3O4 which are in correspondence with the previously

published data (Matinise et al. 2018; Pineda et al. 2018). The ζ- potential and

hydrodynamic size distribution of the CoONPs was measured using DLS analysis. The

DLS is mainly used to study the size of NPs in colloidal suspension ranging from nano to

submicron. Our results have revealed larger NPs aggregate of 320 ± 2.33 nm. The ζ-

potential and PDI of CoONPs was -10.5 ± 4 mV and 0.766 respectively Figure 4.5 (b, c)

(Nguyen et al. 2018).

4.3.3. Biological activities

4.3.3.1. Bactericidal activity

Bacterial infection is a public health problem not only in terms of mortality and morbidity

but also in terms of treatment expenditures. The traditional medicines have been used as


______________________________________________________________________________________ 92

Figure 4.3: SEM images of the green CoONPs.


______________________________________________________________________________________ 93

Figure 4.4: (a) EDX analysis of G. wallichianum mediated CoONPs, (b) Raman spectra

of CoONPs.


______________________________________________________________________________________ 94

Figure 4.5: FT-IR spectra, zeta potential and size distribution analysis of biogenic CoONPs

(a) FT-IR spectra (b) Zeta potential (c) Size distribution of biogenic CoONPs.


______________________________________________________________________________________ 95

an effective antibiotics but there are still problems related to them, like acquired resistance

of bacteria against antibiotic etc. Moreover, researchers are trying hard to put forward new

strategies in order to minimize the risk of the development and spread of infectious diseases

(Woodford and Livermore 2009; Iqbal et al. 2017). Developments in nanoscale

technologies will provide new tools to design novel compounds with unusual antimicrobial

activities (Rizzello et al. 2013). Different research studies published on antibacterial

potentials of nanoparticles using biogenic routes and have shown significant results

(Abbasi et al. 2019; Iqbal et al. 2019). The bioinspired CoONPs were tested against

different bacterial strains at various concentrations (700−21.875 μg/mL). The gram

positive bacterial strains include B. subtilis and S. aureus while gram negative strains used

were P. aeruginosa, E. coli and K. pneumonia. It was found that the antibacterial activity

was increasing with increase in NPs concentrations. P. aeruginosa and K. pneumonia were

found to be the least susceptible with MIC value of 175 μg/mL. B. subtilis was found to be

most susceptible with MIC values of 21.875 μg/mL. Positive control, oxytetracycline was

found effective compared to all the tested concentrations. The antibacterial activity against

different test concentrations are shown in Figure 4.6 (a). The results of antibacterial

activities are consistent with earlier studies of Rhamnus virgata mediated nickel oxide

nanoparticle using different strains of bacteria (Abbasi et al. 2019).

4.3.3.2. Antifungal assay of CoONPs

Antifungal activity for CoONPs was performed. Nanoparticles have been frequently

studied for antibacterial activities while inadequate research work has been done on their

antifungal properties. In current study, antifungal assay for G. wallichianum mediated

CoONPs has been reported. The antifungal assay was studied using different fungal strains:

M. racemosus, C. albican, A. Niger, A. flavus and F. solanai for assessing the susceptibility

at various concentrations (700 μg/mL to 10.937 μg/mL) of CoONPs Figure 4.6 (b). In

current studies, direct relationship was found between different concentrations of CoONPs

and fungal growth inhibition. The least susceptible strain was Aspergillus flavus (MIC

value: 175 μg/mL) and the most susceptible strain was M. racemosus (MIC: 10.937

μg/mL).


______________________________________________________________________________________ 96

Figure 4.6: Antimicrobial potentials of CoONPs (a) Antibacterial potential of CoONPs

with UV illumination (b) Antibacterial potential of CoONPs without UV illumination (c)

Antifungal potential of bioinspired CoONPs.


______________________________________________________________________________________ 97

The MICs values for various bacterial and fungal strains are provided in Table 4.1. The

results of antifungal assay are in correspondence with the previous report of R. virgata

mediated NPs using different fungal strains (Iqbal et al. 2019). Recently, some scientists

have published their research studies on antifungal potentials using different fungal strains

via green synthesis route and have shown potential results (Ahmad et al. 2014; Abbasi et

al., 2019).


Brine shrimp cytotoxicity assay is mostly used for the determination of biological potency

of any naturally existing compound (Chanda and Baravalia 2003). The brine shrimps were

treated with different concentrations (200 to 1 μg/mL) of CoONPs to investigate their

cytotoxic potential. Cytotoxicity of the CoONPs was confirmed via dose dependent manner

and IC50 value was calculated as 18.12 μg/mL. Figure 4.7 (a) illustrate percentage mortality

and dose dependent response of CoONPs. Our results are in accordance with previously

published study of CoONPs using Sageretia thea (Khalil et al. 2017). CoONPs revealed

general cytotoxicity with IC50 value of 18.12 μg/mL.

4.3.3.4. Antileishmanial assay (ALA)

Leishmaniasis is a tropical disease caused by L. tropica (Kaye and Scot 2011). The

conventional drugs used against Leishmanial parasites are often least effective, toxic and

are expensive. Different MNPs have shown significant antileishmaniasis activities against

promastigotes and amastigotes using different medicinal plants (Jebali and Kazemi 2013;

Abbasi et al. 2019). In the current study, biogenic CoONPs have shown promising

antileishmanial activity against amastigotes compared to promastigote and resulted in a

dose-dependent inhibition. Figure 4.7 (b) shows antileishmanial activity of GW@CoONPs

against L. tropica (KMH23). Exposure of leishmanial cells to CoONPs at various

concentrations (200−1 μg/mL) for 72 hr has shown dose-dependent response. The IC50

values have revealed good antileishmanial activity against promastigotes for CoONPs

(IC50: 9.53 μg/mL). Likewise, strong antileishmanial potency against L. tropica

amastigotes was also confirmed by CoONPs (IC50: 3.12 μg/mL). A concentration

dependent inhibition was observed with highest inhibition observed at 200 μg/mL. The

concentration dependent and the lower IC50 values (promastigotes IC50: 9.53 and amastig-


______________________________________________________________________________________ 98

Table 4.1: MIC calculations of different microbial strains.

Antimicrobial activities

Antibacterial activity

Bacterial strain MIC (µg/mL)

Gram positive

B. subtilis (ATCC: 6633) 21.875

S. aureus (ATCC: 25923) 87.5

Gram negative

P. aeruginosa (ATCC: 9721) 175

E. coli (ATCC:15224) 43.75

K. pneumonia (ATCC: 4617) 175

Antifungal activity

Fungal strain MIC (µg/mL)

Aspergillus flavus (FCBP: 0064) 175

Aspergillus niger (FCBP: 0918) 21.875

Candida albicans (FCBP: 478) 43.75

Fusarium solani (FCBP: 0291) 21.875

Mucor racemosus (FCBP: 0300) 21.875


______________________________________________________________________________________ 99

otes IC50: 3.12) of the CoONPs revealed that these NPs can be used in antileishmanial drug

delivery. Our results of G. wallichianum mediated CoONPs are in match with the

previously published results of Sageretia thea assisted CoONPs (Khalil et al. 2017). The

antileishmaniasis results have indicated that amastigotes are more susceptible compared to

promastigotes. The application of nano-based formulations have shown significant results

in the treatment of leishmaniasis. These NPs will not only help in the treatment of

leishmania but also in the prevention (vaccines) as well. Up till now, no effective drug or

medication tools have been discovered against leishmaniasis (Gutierrez et al. 2016;

Abamor 2017).

4.3.3.5. Anticancer activity of CoONPs against HepG2

Cancer is a harmful disease which remained major cause of mortality across the globe. The

rate of incidence of cancer is constantly increasing and is predicted to be ~21 million by

2030 (Matinise et al. 2017; Nagajyothi et al. 2017; Ebrahiminezha et al. 2018). Among the

different types of cancers, liver cancer is considered to be the sixth most common and

second deadliest cancer in male and sixth in female and is responsible for around 745,517

deaths. Factors that increases the risk are heavy alcohol consumption, infections by HCV

and HBV and aflatoxins etc. The anticancer activity of CoONPs was investigated using

HepG2 cell line. Cancer cells exposed to CoONPs at different concentration (3.9-500

μg/mL) for 24 hr resulted in a concentration dependent inhibition. The current study has

shown significant anticancer activity for CoONPs with an IC50 value of 31.4 μg/mL. Figure

4.7 (c) showed ~ 70% mortality at 500 μg/mL, however, the anticancer potency decreases

at lower concentrations. The possible schematic description of cytotoxicity is illustrated in

Figure 4.8. The obtained results are in agreement to the already published report (Abbasi

et al. 2019). Previous research studies published on anticancer activities of different MNPs

have shown significant anticancer potentials (Gutierrez et al. 2016; Iqbal et al. 2019). NPs

take advantage of the unique pathophysiological properties of tumor (angiogenesis,

permeable vasculature, poor lymphatic system) which are not found in normal cells/tissues

thus act as a strong candidate for targeted drug delivery in cancer cells. Nanobiotechnology

has played significant role in achieving the milestone for detection and diagnosis of cancer

in earlier stage.


______________________________________________________________________________________ 100

Figure 4.7: Cytotoxicity assays for CoONPs (a) Cytotoxicity against brine shrimps (b)

Cytotoxicity against L. tropica (c) Cytotoxicity against HepG2 cancer cell line.


______________________________________________________________________________________ 101

Figure 4.8: Diagrammatic representation of the cytotoxicity of CoONPs as reported in

literature. (A) Extracellular ROS generation which can easily enter into the cell followed

by their interference with DNA resulted in genotoxicity. (B) CoONPs penetrate into the

cell by receptor mediated endocytosis, followed by their entry into lysosomes, where Co++

dissolution occur. (C) Surface defects in CoONPs can result in breaking cellular

membrane. (D) The generated ions not only interfere with proteins, but also enter into

mitochondria, where they disrupt its function by synthesizing further ROS.


______________________________________________________________________________________ 102

Nanoparticles in combination with other chemotherapeutic drugs have shown synergistic

effect by specifically targeting cancer lesion without harming normal cells (Sood and

Chopra 2017). Furthermore, NPs applications in smart imaging of cancer has also

significantly dropped down the death rate of cancer (Brigger et al. 2012).

4.3.3.1. Biocompatibility Assay

The biocompatibility of green CoONPs and their toxicology were investigated using

human erythrocyte and macrophages. According to American Society, materials having

the % hemolysis of > 5% is known as hemolytic, from 2−5% are slightly hemolytic and <

2% is non hemolytic in nature (Dobrovolskaia et al. 2008). In this regard CoONPs are non-

hemolytic at lower doses (2 μg/mL), slightly hemolytic at 5−40 μg/mL and were found

hemolytic at > 45 μg/mL. The IC50value of CoONPs against RBCs was calculated as > 200

μg/mL. To measure the safe nature of biogenic CoONPs, MTT assay was also

demonstrated against macrophages. The concentration dependent response was found after

treating with CoONPs. The results concluded that CoONPs played a prominent role against

macrophages with ~30% inhibition at 200 μg/mL. The IC50 value of CoONPs was

calculated to be > 200 μg/mL which confirmed the nontoxic nature of bioinspired CoONPs.

The results for biocompatible nature of CoONPs are illustrated in Figure 4.9 (a). There are

some other studies which have focused on biocompatibility aspect of green nanoparticles

and have shown non-toxic effects on macrophages (Abbasi et al. 2019b; Abbasi et al.

2019c). The IC50values for different assays are provided in Table 4.2. From the current

biocompatibility tests, it is confirmed that CoONPs are toxic to macrophages at higher

concentrations so can be used in lower concentration for different biomedical application.

Similar results have been reported for CoONPs in earlier studies (Khalil et al. 2017).

However, present findings are preliminary and further studies are needed with normal

human cells.

4.3.3.2. Antioxidant activities

Different metal NPs have shown significant antioxidant results using green synthesis routes

(Iqbal et al., 2018). Figure 4.9 (b) shows the results of TAC, TRP and DPPH free radical

scavenging activities of CoONPs. Highest values for TAC in terms of ascorbic acid Eq/mg


______________________________________________________________________________________ 103

Table 4.2: IC50 values of different biological assays.

Analysis type IC50 values

Brine shrimps cytotoxicity 18.12 µg/mL

Antileishmanial promastigotes 9.53 µg/mL

Antileishmanial amastigotes 3.12 µg/mL

Alpha amylase inhibition > 200 µg/mL

Human Macrophages > 200 µg/mL

Human RBCs > 200 µg/mL

Anticancer 31.4 µg/mL


______________________________________________________________________________________ 104

was reported as 61.63 % at 200 μg/mL for CoONPs. The TAC showed the scavenging

property of CoONPs toward ROS species. Moreover, in our study, G. wallichianum leaf

extract was used as a reducing and stabilizing agent, it can be presumed that phenolics have

the ability to scavenge ROS, may also attach on the surface of CoONPs. Furthermore, TRP

was carried out for the examination of reductones involved as antioxidants which donate

their H-atoms and result in the termination of free radical chain (Ul-Haq et al. 2012).

Bioinspired CoONPs has shown good antioxidant capacity. The reducing power was

decreased with the decrease in concentration of CoONPs. Maximum values for reducing

power (56.79 %) were recorded at 200 μg/mL. Moderate DPPH activity (89.56 %) was

observed at 200 μg/mL for CoONPs. From figure 4.9 (b) it can be deduced that several

antioxidants present in the G. wallichianum leaf extract play important role in reduction

and stabilization of CoONPs. The present study revealed significant antioxidant activities

which is in correspondence with previous studies (Khalil et al. 2017; Iqbal et al. 2019).

4.3.3.3. Enzyme inhibition potential

The protein kinase enzyme inhibition potency of biogenic NPs have been rarely

investigated. Inhibition of PK enzymes is determined one of the popular target for

screening chemical entities for their anticancer potencies. The PK phosphorylation process

play vital role in hyphal formation in Streptomyces fungal strains and same principle is

applied in determining PK inhibition. Streptomyces 85E strain is usually used to evaluate

medicinal compounds and extracts for determining PK inhibition (Waters et al. 2002).

These enzymes are responsible for phosphorylation of different amino residues and

regulate different cellular processes such as cell division, apoptosis, differentiation and

other metabolic processes. Deregulated phosphorylation can induce genetic anomalies

resulting in cancer. Therefore, any product or chemical entity that has potential to inhibit

PK enzymes has great significance in cancer studies (Guangmin et al. 2011). Figure 4.9 (c)

illustrates the ability of biogenic CoONPs to inhibit protein kinase (PK) enzyme. PK

inhibition assay was performed via disc diffusion method where surfactin was used as

standard. ZOI up to 10.7 mm was recorded at 200 μg/mL for CoONPs indicating good PK

inhibition activity of CoONPs. A concentration dependent response is inferred and

CoONPs was found to be a potential signal transduction inhibitor whose role in anticancer

research is pre-concluded. The results of current studies are in correspondence with the


______________________________________________________________________________________ 105

previously published studies (Khalil et al. 2017). Moreover, for the GW-CoONPs, alpha

amylase (AA) inhibition assay was also investigated. The AA is helpful in the conversion

of carbohydrates into glucose (Yao et al. 2011), hence, glucose level can be prevented by

blocking the function of AA which is the principal area of diabetes research (Zohra et al.

2019; Iqbal et al. 2019). Biogenic CoONPs were studied for their AA inhibitory activity.

The results concluded significant inhibition of AA enzyme by CoONPs. Moderate

inhibition was found i.e. 36 % at the concentration of 200 μg/mL. However, no inhibition

was reported at concentration of 1 μg/mL. Figure 4.9 (d) shows the alpha AA inhibitory

potency of bioinspired CoONPs. The results of current studies are in agreement to the

earlier reports of Sageretia thea mediated CoONPs (Khalil et al. 2017). There are different

other reports available on the green synthesis of biogenic nanoparticles using different

medicinal plants and have shown potential AA and PK activities (Ahmad et al. 2014;

Abbasi et al. 2019; Iqbal et al. 2019)


______________________________________________________________________________________ 106

Figure 4.9: (a) Biocompatibility against human RBCs and macrophages (b) Different

antioxidant activities (c) Inhibition potential against protein kinase and (d) inhibition

potential against alpha amylase.

Chapter 5 Green synthesis of nickle oxide nanoparticles

______________________________________________________________________________________ 107

Chapter 5: Green synthesis and characterizations of Nickel oxide

nanoparticles using leaf extract of Rhamnus virgata and their

potential biological applications


______________________________________________________________________________________ 108

Graphical representation of the synthesis, characterization and bioapplications of R. virgata

based NiONPs


______________________________________________________________________________________ 109

Abstract

In the present report, Nickel oxide nanoparticles (NiONPs) were synthesized using

Rhamnus virgata (Roxb.) (Family: Rhamnaceae) leaves extract as stabilizing, reducing and

chelating agent. The formation, morphology, structure and other physicochemical

properties of NiONPs were studied by Ultra violet spectroscopy, XRD, FT-IR, SEM, EDS,

TEM, Raman spectroscopy and dynamic light scattering DLS. Detailed in vitro biological

activities revealed significant therapeutic properties. The antibacterial efficacy of biogenic

NiONPs was demonstrated against five different gram positive and gram negative bacterial

strains. Klebsiella pneumoniae and Pseudomonas aeruginosa (MIC: 125 µg/mL) were

found to be the least susceptible and Bacillus subtilis (MIC: 31.25 µg/mL) was found to be

the most susceptible strain. Furthermore, antifungal potencies have been reported against

different fungal strains. Biogenic NiONPs were reported to be highly potent against HepG2

cells (IC50: 29.68 µg/mL). Moderate antileishmanial activity against Leishmania tropica

(KMH23) promastigotes (IC50: 10.62 µg/mL) and amastigotes (IC50: 27.58 µg/mL)

cultures are reported. The cytotoxic activity was studied using brine shrimps and their IC50

value was calculated as 43.73 µg/mL. To confirm toxicological assessment, NiONPs were

found compatible towards human RBCs (IC50: > 200 μg/mL) and macrophages (IC50: >

200 μg/mL), deeming particles safe for various applications in nanomedicines.

Furthermore, moderate antioxidant activities: total antioxidant capacity (TAC) (51.43%),

2,2-diphenyl-1-picrylhydrazyl (DPPH) activity (70.36%) and total reducing power (TRP)

(45%) are reported for NiONPs. In addition, protein kinase and alpha amylase inhibition

assays were performed. Our results concluded that R. virgata synthesized NiONPs could

find important biomedical applications with low cytotoxicity to normal cells.


______________________________________________________________________________________ 110

5.1. Introduction

Nanotechnology is one of the most significant and fast growing area in the field of science

and engineering, involving the combination of knowledge from biology, chemistry,

materials science and other related branches. The term “nano” is derived from Greek

language meaning dwarf or extremely small, ranging in dimension from 1−100 nm.

Nanoparticles (NPs) have a very high surface-to-volume ratio because of their very small

size. NPs are also called Nano-crystals. Based on their shape, NPs are known as nanocube,

nanoflower, nanotube and nanowire etc. (Khatami et al. 2018). While based on their

structure, nanoparticles are called as cluster, core shell and bimetallic etc. (Karthik et al.

2018; Khatami et al. 2018). NPs have a wide range of applications in different commercial

areas such as food, cosmetics, agriculture, drug delivery, cancer detection/diagnosis, cancer

therapy and many more (Iqbal et al. 2018). The extent of NPs applications is due to unique

and fascinating properties (magnetic, optical, chemical, electronic, mechanical, sensing

properties). Metallic nanoparticles (MNPs) significantly vary in many properties from that

of their bulk materials such as size, shape, surface effect, electrical and magnetic properties

(Han et al. 2018).

Numerous metals and metal oxide nanoparticles exist in nature such as silver, platinum,

gold, copper, magnesium, cobalt, cesium oxide and zinc oxide etc. (Yang et al. 2018).

Among the different MNPs, NiONPs have attracted the attention of scientific community

due to multifunctional and tunable nature. NiONPs are one of the most engineered NPs

with a wide band gap of (3.7-40 eV) and an intrinsic p-type semiconductor with an average

size of 34 nm. NiONPs possess a wide range of applications in lithium ion batteries,

electrochromic test devices, super-capacitors, smart windows, water remediation through

photocatalysis, electrochemical sensing and catalysis of chemical processes (Sone et al.

2016; Khalil et al. 2018). NiONPs also have applications in the adsorption of toxic

environmental pollutants and dyes (Pandian et al. 2015). Because of their anti-

inflammatory property, NiONPs can also be used in biomedicines (Sudhasree et al., 2014).

The previous research work of NiONPs have revealed cytotoxic effects by releasing ROS

and Ni++ to oxidative damage (Gong et al. 2011).


______________________________________________________________________________________ 111

NiONPs are prepared by various physical and chemical routes (Kundu et al. 2015; Soomro

et al. 2015; Zhang 2015; Thema et al. 2016). However, these physicochemical approaches

have numerous harmful side effects which limit their wide range biological applications

(Thovhogi et al. 2015). Chemical synthesis most frequently generate toxic chemical waste

leading to environmental toxicity and non-biodegradable products while that of physical

methods needs massive energy input (Thema et al. 2015). In contrast, biological method to

synthesize MNPs including NiONPs have been emerged as cost effective option in the field

of “green chemistry” (Duran and Seabra. 2018). Biofabrication of MNPs is considered to

be simple, eco-friendly, economically feasible, easily scaled up and accomplished at room

temperature and pressure in aqueous environment (Duran et al., 2012; Singh et al., 2016).

Biogenic synthesis of MNPs can be achieved using plants, bacteria, fungi and algae (Duran

et al. 2011; Ahmed et al. 2016). Among these, plant-extract mediated synthesis of

nanoparticles have significantly gained the attention due to its simplicity (Song and Kim

2009). The plant extract can act as a strong reducing, stabilizing and capping agent and has

drawn the attention of scientific community due to its simple, fast, cost effective and eco-

friendly nature (Song and kim 2009; Singh et al. 2016). The phytochemicals available in

plant extracts function as a strong reducing agent and result in the development of capped

NPs (Ahmed et al. 2016; Iqbal et al. 2018). Furthermore, the phytocompounds in aqueous

environment replace chemical reagents and organic/inorganic solvents (Duran and Seabra.

2018). Currently, plant extracts of various plants parts have been utilized for the formation

of NPs (Kalidhar and Sharma 1984; Iqbal et al. 2018). Plant broth have medicinal values

due to the presence of different phytocompounds such as alkaloids, flavonoids, terpenoids,

polyphenols, amino acids and vitamins (Song and Kim 2009; Ahmed et al. 2016; Iqbal et

al. 2018).

The objective of this study was to synthesize NiONPs using leaves extract of R. virgata.

The R. virgata is a rich source of alkaloids, flavonoids and anthraquinones compounds and

can be used as a natural source in the formation of biogenic nanoparticles. The plant is

comprised of bioactive molecules; physcion, emodin, kaempferol, rhamnocitrin, quercetin,

herbacetin, maesopsin) which may act as strong reducing and stabilizing agents (Gupta

1968; Kalidhar and Sharma 1985). The ethnobotanical survey and literature review has


______________________________________________________________________________________ 112

reported the medicinal values of R. virgata and have shown strong therapeutic properties

such as antioxidant, antimicrobial, laxative, emetic, purgative and mostly used in the

treatment of parasitic infections, spleen affection and in leg swelling (Topwal and Uniyal

2018; Muthee et al. 2019).

Green synthesis of NiONPs has been achieved in current years and there is a growing

interest in the synthesis of NiONPs for different biological applications from different other

medicinal plants. Plant-mediated synthesis of NiONPs have already been reported for

Moringa oleifera Lam. Agathosma betulina (Thovhogi et al 2015), Callistemon viminalis,

(Sone et al. 2016), Tamarix serotine and Nephelium lappaceum (Yuvakkumar et al. 2014).

In this study, fabrication of NiONPs utilizing aqueous leaves extract of R. virgata without

using any surfactant or organic/inorganic solvents has been reported. Furthermore,

biosynthesized NiONPs were extensively characterized using various analytical

techniques. In addition, diverse in vitro biological activities such as, biocompatibility

assays against RBCs and macrophages, antimicrobial assays, cytotoxicity assays against

brine shrimps, leishmanial parasites and HepG2 cell line were also investigated.


5.2.1. Preparation of plant extract

For the preparation of 30 g of cleaned R. virgata (dried leaves powder) was added in 300

mL of deionized water under magnetic stirrer and was placed on hot plate at 80°C for 2 hr.

The plant extract was cooled and was then three times filtered using Whatman filter paper

(0.2 µm) to achieve pure aqueous extract. The pH of aqueous extract was recorded as 7.3

at room temperature. The plant extract was kept in refrigerator at 4°C for future studies.

5.2.2. Synthesis of nickel oxide nanoparticles

Previously described green synthesis protocol with slight changes was used for the

formation of NiONPs (Khalil et al. 2018). The plant extract was shifted to a reaction flask

and 3 g of NiNO3 salt (Sigma Aldrich) was added to synthesize NiONPs. The resulting

solution (pH. 6.89) was followed by heating at 60°C for 2 hr. The collected precipitate was

allowed to cool, followed by three time washing at 3,000 rpm for 25 min. After washing,

sample was dried at 100°C using incubator (K & K Scientific and Medical equipments,

Korea) for 2 hr followed by calcination in open air furnace (KSL-1100X, MTI Corporation


______________________________________________________________________________________ 113

China). The particles formed were considered as NiONPs and were characterized using

diverse characterization techniques. The study plan is shown in Figure 5.1.

5.2.3. Characterizations techniques

Diverse characterization techniques were applied to study the physical, chemical, optical,

thermal, electrochemical properties of NiONPs. The synthesis of NiONPs was confirmed

using UV-4000 UV-Vis spectrophotometer Germany with in the range of 200−400 nm

wavelength. The composition and the functional groups which are involved in capping and

efficient stabilization of the NiONPs were identified by Infrared spectroscopy. The spectral

range used for scanning sample was 400–4000 cm-1. The FT-IR analysis was performed to

study the different functional molecules involved in reduction and stabilization of NiONPs.

FT-IR spectra were observed using FT-IR spectrometer (Alpha, Bruker, Germany). To

further characterize nanoparticles, Raman spectrum was recorded for NiONPs. Moreover,

size and shape was studied using TEM. Particle size distribution and morphological

features of NiONPs were determined using SEM. The XRD analysis was carried out for

thermally annealed sample on X-ray diffractometer (PANalytical, Netherland) equipped

with a Cu radiation source at 45 kV and 40 mA of current voltage and their corresponding

size was calculated using Scherrer equation. The hydrodynamic size distribution, ζ-

potential and PDI were evaluated using DLS system (Malvern Zetasizer Nano). In addition,

elemental composition was confirmed by EDS analysis.

5.2.4. Cytotoxic assays


To confirm the cytotoxic effect of green NiONPs, BSC assay was performed using A.

salina larvae (Ocean Star, USA). Eggs of A. salina were incubated for 48 hr under light at

30°C in saline water (Sea water: 3.8 g/L). After 24 hr incubation, 20 mature nauplii were

harvested through micropipette and were shifted into glass vials containing SW and

NiONPs. Different doses of NiONPs were used. The vials with mature nauplii, vincristine

sulphate and sea water was considered as positive control while vials containing nauplii,

DMSO and sea water was exploited as negative control. After 24-hour incubation alive

shrimps were counted and % inhibition was measured. Median lethality dose (LD50) was

recorded utilizing GraphPad software.


______________________________________________________________________________________ 114

Figure 5.1: Study plan for the biogenic synthesis of NPs.


______________________________________________________________________________________ 115

5.2.4.2. Antileishmanial assay

The L. tropica “KWH23 strain” (amastigotes and promastigotes cultures) were used to

study the cytotoxic activity of NiONPs using MTT cytotoxic assays (Castro‐Aceituno et

al., 2016). The Leishmanial parasites were culture on MI99 media added with 10% FBS.

Amphotericin B was utilized as control. NiONPs in the range of 1−200 μg/mL was studied

for their cytotoxic activity. The 96 well plate with tested NPs was placed in CO2 incubator

for 72 hr/24°C. Reading were taken at 540 nm using spectrophotometer and IC50 value was

calculated by GraphPad software. The percentage inhibition was measured as;

% age inhibition = [ 1 − (AbS)

(AbC)]×100


Previously optimized protocol (Shah et al. 2018) was used to study in vitro cytotoxicity

potential of NiONPs against HepG2 cancer cell line. Cells were cultured in DMEM media

added with FBS, Pen-strep. In addition, cells were grown in humified 5 % CO2 incubator

at 37°C. The MTT assay was demonstrated in 96-well microplate by incubating at different

test concentration of NiONPs (500−3.9 µg/mL) for 48 hr. 20 µL of MTT solution was

loaded in each well and was placed in incubator for 3 hr. The culture medium was

substituted with 100 µL of DMSO followed by further incubation for 20 min. The formazan

formation by living cells was monitored at wavelength of 570 nm.

5.2.5. Antimicrobial activities

Antimicrobial activity of R. virgata orchestrated NiONPs was evaluated via disc diffusion

method against bacterial strains including B. subtilis, S. aureus, E. coli, P. aeruginosa and

Klebsiella pneumonia. To achieve this purpose, 150 µL of bacterial cultures (broth) were

utilized to grow even bacterial lawn. Discs (6 mm) made of filter papers was loaded with

10 µL of NiONPs were carefully placed up on lawns while 10µg oxytetracycline disc was

utilized as control. After 24 hr incubation at 37°C, zones of inhibition were measured and

their respective MIC values were calculated in the concentrations range of (700−21.875

µg/mL). Furthermore, NiONPs were exposed for about 20 min to UV light illumination

(germicidal-6-Watt UV rod 6GT5) to determine their antibacterial activity.


______________________________________________________________________________________ 116

The fungicidal activities of NiONPs was determined using different fungal strains such as

C. albicans (FCBP 478), M. racemosus (FCBP 0300), A. niger (FCBP 0918), F. solani

(FCBP 0291) and A. flavus (FCBP 0064). Sabouraud dextrose agar media (Oxoid

CMO147) was prepared and autoclaved for the growth of different fungal strains and was

allowed to solidify. Filter discs laden with 10 µL of NiONPs were placed on media plates.

10 µg of amphotericin B was used as control. Furthermore, plates were placed in incubator

overnight at 28°C for 48 hr. The zones of inhibition were measured. Different doses of

NiONPs were used in range of 700−10.9375 µg/mL. MICs values were calculated at lowest

concentration of NiONPs.

5.2.6. Enzyme inhibition potentials

5.2.6.1 Protein kinase (PK) inhibition

The PK inhibitory assay for green NiONPs was demonstrated as discussed earlier (Khalil

et al. 2018). The whole experiment was performed under sterile conditions. Streptomyces

85E strain was cultured on ISP4 minimal media to achieve uniform lawns. After that, 10

µL of NiONPs was added on 6 mm autoclaved filter disc and was placed on microbial

lawns. Surfactin was utilized as control. The incubation period was executed for 24−36 hr

at 30°C to target the growth of strain. The appearance of clear and bald zones around discs

indicates the inhibition of spore and mycelia formation.

5.2.6.2. Alpha amylase (AA) inhibition

Alpha amylase inhibitory potency of NiONPs was performed using previously optimized

protocol (Abbasi et al. 2019). The reaction mixture is comprised of 25 μL of AA enzyme,

NiONPs (10 μL), starch solution and PBS (15 μL). Reaction mixture with all components

was placed in incubator for 30 min at 50°C followed by adding 20 µL of HCL and iodine

solution (90 µL). Acarbose was utilized as positive while deionized water (DW) was

exploited as negative control. Following formula was used to calculate percentage

inhibition;

Inhibition(%) =ODs − ODn

ODb − ODn× 100


______________________________________________________________________________________ 117

5.2.7. Biocompatibility assays

5.2.7.1. Biocompatibility against human RBCs

Haemolytic assay was performed to determine the biocompatible nature of NiONPs with

freshly extracted human RBCs. To achieve this purpose, 1 mL fresh human RBCs was

isolated and centrifuged at 10,000 rpm for 12 min. The erythrocyte suspension was

prepared in a test tube by mixing 200 µL of red cells with 9.8 mL of PBS (pH 7.2).

Erythrocytes suspension of 100 µL was treated with different doses of NiONPs and were

incubated at 35°C for 50 min. Moreover, the sample was centrifuged at 12,000 rpm for 20

min. The supernatant was collected and shifted in a 96-well plate and hemoglobin release

was studied at 540 nm. The DMSO and Triton X-100 were used as negative and positive

control respectively. The results were recorded as % hemolysis caused by NiONPs dilution

which can be calculated using the formula below;

% hemolysis[Absorbance of sample – Absorbance of negative control

Absorbance of positive control − Absorbance of negative control]×100

5.2.7.2. Biocompatibility against macrophages

The biocompatible nature of NiONPs was studied against freshly isolated macrophages.

The cells isolated were grown on RPMI media with addition of 10% FBS, streptomycin,

25 mM HEPES and Penicillin. The macrophages were cultured in 5% CO2 incubator to a

density of 1 × 105 cells/well. Furthermore, cells were treated with different concentrations

of NiONPs. The below formula was utilized to calculate the % inhibition of macrophages;



5.2.8. Antioxidant assays

DPPH activity was performed using different doses of NiONPs (1−200 µg/mL) through

microplate reader to investigate their antiradical potentials (Yao et al. 2011). Ascorbic acid

was exploited as positive and DMSO served as negative control. The readings were taken

at 517 nm using microplate reader to determine the % scavanging of DPPH applying the

formula below;

% scavanging = 1-[AbS

AbC]×100


______________________________________________________________________________________ 118

Moreover, previously described method was used to determine the TRP of green NiONPs

(Khalil et al. 2018). Ascorbic acid (AA) was employed as positive while DMSO as negative

control. Absorbance was taken at 630 nm using microplate reader and results are shown as

ascorbic acid equivalent per/mg of test sample. Furthermore, TAC test was performed

using phosphomollybdenum assay (Khalil et al. 2018). The reaction mixture was prepared

by mixing sulfuric acid, (NH4) 6 Mo7O24 4H2O and potassium diphosphate and was

incubated at 95°C for 90 min. Readings were taken at 695 nm. Results are shown as

microgram equivalent of ascorbic acid per/mg of test sample.

5.3. Results

5.3.1. Physical characterizations of the NiONPs

Different characterization techniques were utilized to confirm the synthesis of NiONPs.

The progress of NiONPs formation was monitored by a color change after adding salt

(Nickel nitrate) at 60°C. The change in solution (black color) demonstrates the formation

of NiONPs. To determine the stability of NiONPs, 1 mg/mL solution of NiONPs was made

and sonicated for 25 min. The turbid suspension was allowed to settle for 48 hr and SPR

was checked from time to time using spectrophotometer. Absorption at 332 nm shows that

colloidal suspension remained stable for 24 hr. The results are given in Figure 5.2a, b.

Figure 5.2c represents the FT-IR spectra of NiONPs. The presence of various chemical

bondings in NiO were determined by FT-IR analysis. The FT-IR spectrum of NiO shows

vibration at ~1030 cm−1, ~ 1380 cm−1, ~1630 cm−1 confirming the adsorbed –OH groups

and vibration at ~3425 cm−1 is for H2O which play important role in both reduction and

stabilization of NiONPs. The bands in the region 800–470 cm−1 give information about

NiO. NiONPs were further confirmed using Raman spectroscopy in the range of 200 cm−1

to 2000 cm−1 as shown in Figure 5.2d. The distinguished major modes of Raman spectra

were centered at approximately (~) 561 cm−1 (1 P), ~1047 cm−1 (2 P) and ~1616 cm−1 (2

M). From the high intensity sharp peak at ~561 cm−1, it can be concluded that NiONPs is

defect rich. The presence less intense yet broader peak at 1616 cm-1 corresponds to decrease

in antiferromagnetic spin correlation among individual Ni++ and is an additional evidence

for nanosized nature of NiONPs (crystal size ~ 24 nm). The morphology of NiONPs was

further observed by taking SEM and TEM images of NPs. Figure 5.3 a,b,c indicates typical


______________________________________________________________________________________ 119

SEM and TEM images. In SEM images shape of nanoparticles appeared to be

agglomerated. TEM images revealed spherical morphology of nanoparticles with mean

size of ~24 nm. Moreover, XRD spectrum has confirmed the synthesis of NiONPs. XRD

spectrum further revealed pure crystalline nature of NiONPs along with 2 theta (2Ø) values

of 27.33, 37.14, 43.22, 62.81, 76.95 and 79.27 which are indexed to (110), (111), (220),

(200), (311), (222) Brag’s reflections of the fcc crystalline phase of NiONPs. The average

size was ~24 nm using Debyes Scherer equation and is in agreement with TEM results. No

Bragg peaks for other related compounds were found confirming the pure crystalline nature

of green NiONPs. Results of the XRD analysis are given in Figure 5.4 a, b.

The ζ- potentials, PDI and hydrodynamic sizes of NiONPs was measured using DLS. The

data have shown larger particle aggregates of 173.7 nm. The zeta potential and PDI of

NiONPs was -13 mV and 1.000 respectively (Figure 5.5 a, b). The DLS is mainly used to

confirm the nanoparticles size in colloidal suspensions in the range of nano and submicron.

The average hydrodynamic particle diameter in water shows particle aggregation. Our

characterizations results are consistent with Sudhasree et al. (2014) for aggregation of NPs

in aqueous medium. Furthermore, elemental composition of NiONPs was studied using

EDS analysis which confirm the presence of both Ni and O in sample. The EDS spectrum

is shown in Figure 5.5c.

5.3.2. Cytotoxic assays

5.3.2.1. Brine shrimp cytotoxicity assay (BSCA)

The cytotoxic potency of green NiONPs was studied using brine shrimps as a model

organism. Figure 5.6a shows the % mortalities at different doses (200 to 1 μg/mL) of

biogenic NiONPs. The cytotoxicity of NiONPs was confirmed through dose dependent

manner and was increasing as concentration of NPs was increasing while their median

lethality dose (IC50) was calculated as 43.73 µg/ml.

5.3.2.2. Antileishmanial activity (ALA)

The ALA of biosynthesized NiONPs was determined against L. tropica (KMH23) as

shown in Figure 5.6 b. Leishmanial parasites were exposed to NiONPs at different

concentrations (1 to 200 μg/mL) for 72 hr and has shown dose dependent inhibition. The

antileishmanial activities was increasing as concentration of the NiONPs was increasing.


______________________________________________________________________________________ 120

Figure 5.2: Spectral analysis for biogenic NiONPs (a) UV visible spectra (b) Stability of

biosynthesized NiONPs (c) FT-IR spectra (d) Raman spectrum.


______________________________________________________________________________________ 121

Figure 5.3: SEM and TEM images of the green NiONPs (a, b, c) SEM (d) TEM.


______________________________________________________________________________________ 122

Figure 5.4: (a) XRD spectra of NiONPs (b) Size calculation via Scherer approximation

analysis.


______________________________________________________________________________________ 123

Figure 5: DLS zeta potential, size distribution and EDS images of the green NiONPs (a)

DLS size distribution (b) Zeta potential (c) EDS analysis


______________________________________________________________________________________ 124

The NiONPs have shown strong antileishmanial activity against L. tropica promastigotes

(IC50: 10.62 µg/mL). Similarly, strong ALA against L. tropica amastigotes was shown by

NiONPs (IC50: 27.58 µg/mL). The dose dependent and lower IC50 values of the NiONPs

showed that these materials can be used for strong antileishmanial drug delivery in the

future medicines.


The anticancer property of NiONPs was performed against HepG2 cell line. Cancer cells

exposed to different concentrations of NiONPs (500 to 3.9 µg/mL) for 24 hr resulted in

dose dependent inhibition of cancer cells. Our results have shown significant anticancer

activity for NiONPs. However, ~84% mortality of cancer cells was observed at 500 µg/mL

and anticancer potential was decreasing as NiONPs concentration was decreasing. The IC50

value calculated was 29.68 µg/mL. The data is given in (Figure 5.6c). The plausible

schematic representation of cytotoxicity is shown in (Figure 5.7).

5.3.3. Antimicrobial assay

5.3.3.1. Antibacterial activity

Newly synthesized green NiONPs were used against five different bacterial strains across

six different doses (1000−31.25 µg/mL) with and without UV illumination. The bacterial

strains (BS) used are shown in Figure 5.8a. Our study reported that antibacterial activity

was increasing with increase in NiONPs concentration. Many strains were found

susceptible. K. pneumonia and P. aeruginosa were found least susceptible with MIC values

of 125 µg/mL. MIC values are given in Table 5.1. The bacterial strain that was most

susceptible to NiONPs was B. subtilis with MIC values of 31.25 µg/mL. 10 µg

oxytetracycline drug was used as positive control. No single concentration was found

effective than oxytetracycline drug. The antibacterial activities using different test

concentration are shown in Figure 5.8a. Figure 5.8b shows increase in the bactericidal

activities of NiONPs after UV exposures.

5.3.3.2. Antifungal activity

Antifungal activity of biogenic NiONPs were performed against different fungal strains in

the concentration range of (1000−15.625 µg/mL). The amp B served as control. The

different fungal strains used in the study are shown in Figure 5.8c. Our results concluded


______________________________________________________________________________________ 125

direct relation between concentration and test sample of NiONPs. M. racemosus (MIC:

31.25 µg/mL) was the most susceptible strain and that of A. flavus (MIC: 125 µg/mL) was

found to be the least susceptible strain. The MIC values of different fungal strains are

provided in Table 5.2.

5.3.4. Enzyme inhibition potential

Figure 5.9a depicts protein kinase enzyme inhibition activity of NiONPs. Protein kinase

inhibitors play significant role in cancer therapeutics (Rahdar et al. 2015). These enzymes

are playing crucial role in the phosphorylation of important amino acids and regulate vital

biological processes such as cell division and apoptosis etc. Deregulated phosphorylation

by PK enzymes can lead to tumor development. Hence, chemicals substances with ability

to inhibit PK enzyme is highly important.

Figure 5.6: Cytotoxicity assays for NiONPs (a) Cytotoxicity against brine shrimps (b)

Cytotoxicity against Leishmania tropica (c) Cytotoxicity against HepG2 cell line.


______________________________________________________________________________________ 126

Figure 5.7: Diagrammatic representation of the cytotoxicity of NiONPs. (a) Extracellular

ROS generation which can easily enter into the cell followed by their interference with

DNA resulted in genotoxicity. (b) NiONPs penetrate into the cell by receptor mediated

endocytosis, followed by their entry into lysosomes, where Co++ dissolution occur. (c)

Surface defects in NiONPs can result in braking cellular membrane. (d) The generated ions

not only interfere with proteins, but also enter into mitochondria, where they disrupt its

function by synthesizing ROS.


______________________________________________________________________________________ 127

Table 5.1: Minimum inhibitory concentration (MIC) calculations of different bacterial

strains.

Without UV illumination With UV illumination Positive

control

Bacterial strain MIC

(µg/mL) Bacterial strain

MIC

(µg/mL)

Gram positive

Oxytetracycline

Bacillus subtilis 15.6 Bacillus subtilis 7.8

Staphylococcus

aureus 62.5

Staphylococcus

aureus 15.6

Gram negative

Pseudomonas

aeruginosa 125

Pseudomonas

aeruginosa 31.5

Escherichia coli 62.5 Escherichia coli 15.6

Klebsiella pneumonia 125 Klebsiella pneumonia 62.5


______________________________________________________________________________________ 128

Figure 5.8: Antimicrobial potentials of NiONPs (a) Antibacterial potential of NiONPs

without UV illumination (b) with UV illumination (c) Antifungal potential of NiONPs.


______________________________________________________________________________________ 129

Table 5.2: Minimum inhibitory concentration (MIC) values of different fungal strains.

Fungal strains MIC (µg/mL) Positive control

Aspergillus niger 31.25

Amphotericin B

Aspergillus flavus 125

Candida albicans 62.5

Candida glabrata 87.5

Fusarium solani 31.25

Mucor racemosus 31.25


______________________________________________________________________________________ 130

In Streptomyces-85E strain, PK play significant role in development of hyphae. Thus, this

strain was used to determine PK inhibitory potential of NiONPs. Following disc-diffusion

method, maximum zone of inhibition (17 mm) was observed at highest concentration (1000

μg/mL), which indicates strong PK inhibition activity of NiONPs. Figure 5.9b shows

inhibition potency of biogenic NiONPs against alpha amylase enzyme. Furthermore,

NiONPs has shown insignificant amylase inhibition (36.1 %). Moderate inhibition was

recorded 36.1 % at 1000 μg/mL. However, no inhibition was observed at the concentration

of 125 μg/mL.

5.3.5. Biocompatibility tests

The biocompatible nature of bioinspired NiONPs and their toxicological properties were

studied against freshly isolated human RBCs and macrophages. Results were examined in

the range of 200−l μg/mL. According to Americans Society (AS), bio-substances showing

%hemolysis of > 5% is known as hemolytic, between 2−5% are slightly hemolytic, while

<2% is non-hemolytic (Iqbal et al. 2018). In this regard, NiONPs are non-hemolytic at

lower concentrations (< 5 μg/mL), while highest hemolysis (28.23 %) was reported at 200

μg/mL. The LD50 (The LD50 (lethality dose) value for NiONPs against RBCs was

calculated to be > 200 μg/mL. To further investigate the biocompatible nature of NiONPs,

MTT assay was performed against human macrophages. The results have shown that

NiONPs are non-toxic towards human macrophages. In this regard, NiONPs are non-toxic

at lower doses of 2 μg/mL, slightly toxic at 5–100 μg/mL, while toxic at 200 μg/mL with

40.2 % mortality. The IC50 value of NiONPs was recoded to be > 200 μg/mL. Generally,

the test samples can be recognized as safe at low concentration. The biocompatibility

results for NiONPs are summarized in Figure 5.9c. The IC50 values for different assays are

given in Table 5.3.

5.3.6. Antioxidant activities

Biogenic NiONPs were found to have strong antioxidant properties. Antioxidant potencies

of NiONPs were studied via DPPH activity, TRP and TAC. The results revealed highest

scavanging (70.36%) at 200 µg/mL, while the activity was decreased with decrease in

concentration. The highest TAC (51.43 %) and reducing powers (45 %) were observed on


______________________________________________________________________________________ 131

the highest concentration (200 µg/mL). In short, good radical scavanging potential,

moderate TAC and TRP potential have been shown by NiONPs (Figure 5.9d).

Figure 5.9: Biocompatibility, antioxidant, enzymes inhibition assays of NiONPs (a)

Inhibition potential against protein kinase and (b) inhibition potential against alpha

amylase, (c) Biocompatibility against human RBCs and macrophages (d) Different

antioxidant activities of NiONPs.


______________________________________________________________________________________ 132

Table 3: IC50 values calculation for different assays using GraphPad software.

S. No Assay name IC50 value

1 Brine shrimp cytotoxicity 43.73

2 Anticancer activity 29.68

3 Antileishmanial (promastigotes) 10.62

4 Antileishmanial (amastigotes) 27.58

5 Alpha amylase > 200

6 Human RBCs > 200

7 Human macrophages > 200


______________________________________________________________________________________ 133

5.4. Discussion

Among the different medicinal plants used in the biofabrication of nanoparticles, R. virgata

was selected due to high content of bioactive compounds (physcion, emodin, kaempferol,

rhamnocitrin, quercetin, herbacetin and maesopsin) which can function as strong reducing,

stabilizing and capping agents in the synthesis of nanoparticles (Gupta 1968). These

phytocompounds are rich source of electrons and are responsible for reduction and

stabilization of NiONPs (Singh et al. 2016). A scheme of green synthesis of NiONPs is

given in Figure 5.10. Mostly phenolic compounds play important role in biogenic synthesis

of NPs, however the clear mechanism and processes by which NPs get synthesized are yet

to discover for scientific community. Nasseri et al. (2016) successfully reported the plant

mediated synthesis of NiONPs using R. virgata leaves extract. Previously, biogenic

NiONPs were synthesized but their extensive biological properties; antibacterial,

antifungal, antioxidants, anticancer, antileishmaniasis, enzyme inhibition, biocompatibility

assays are rarely reported.

Biogenic NiONPs were characterized by different techniques. Bragg peaks for these NPs

are of pure NiONPs in XRD pattern as no other peaks were found for other compounds.

The TEM results are consistent with XRD data. Our results are in accordance to the XRD

pattern of NiONPs using Moringa oleifera (Lam.) (Nasseri et al. 2016). The EDS analysis

has further explored the presence of Ni-O in the sample. The absorption bands in FT-IR

were found having stretching vibrations of Ni_O in NiONPs and are in agreement with

previous results (Yang et al. 2016; Chaudhuri and Malodia 2017). Considering the

difference in Raman shifts (that could be because of stress/strain effect, size, intensity and

relative positions) of 5 peaks, spectra are in harmony with reported values of photon modes.

Overall, Raman scattering support purity of NiONPs and our experimental results are

consistent with earlier report (Yang et al. 2018). DLS is primarily used to confirm NPs size

in colloidal suspensions. However, measurement of zeta potential (ZP) is based on the

movement of particles under an electric field. Moreover, surface charge and local

environment of particles also play significant role in measurement of ZP (Kemary et al.,

2013). The results of present study regarding the hydrodynamic size distribution and zeta


______________________________________________________________________________________ 134

potential are in correspondence with the previous studies conducted on NiONPs (Woodford

and Livermore 2009; Ates et al. 2016).

Traditional medicines (antibiotics) are often used for controlling bacterial infections but

certain problems are associated with them such as antibiotic resistance. However, the

scientific community is engaged in developing new strategies to resolve the issue of

antibiotic resistance (Iqbal et al. 2017). In the current study, NiONPs has shown strong

antibacterial and antifungal potentials. The antibacterial and antifungal potential was

increasing as the concentration of NiONPs was increasing. Generally, there was increase

in the antibacterial potential of NiONPs after UV exposure. The MIC values calculated for

P. aeruginosa was reported as 125 µg/mL while after exposure to UV, the MIC was

significantly reduced (31.5µg/mL). Previously, Khalil et al. (2018) has reported that UV

illumination of Sageretia thea-NiONPs has significantly increased bactericidal potential.

The strong antibacterial potential of NiONPs due to UV exposure might be due to high

production of ROS which is already reported by scientists (Rizzello et al., 2013). The ROS

such as, H2O2 intrude inside bacterial cells and initiate genotoxic effect. In addition

research studies have reported generation of new complexes as a result of irradiations by

UV that enhances antibacterial potential against different pathogenic bacterial strains

(Rizzello et al., 2013). The oxytetracycline and amphotericin B were exploited as positive

controls and have shown greater inhibition zones compared with different doses of

nanoparticles. Recent research study has also indicated that reference drugs were more

effective than NiONPs (Khalil et al., 2018). It is reported that the new advancements in

nanotechnology will provide new opening for novel tools and will develop new therapeutic

substances with strong antimicrobial properties (Siddique et al., 2012). Additionally,

NiONPs have shown strong antitumor potential against HepG2 cell line by inhibiting

cancer cell progression. Our results are in correspondence with previous studies using Hep-

2, MCF-7 and HT-29 cell lines, where NiONPs have shown strong cytotoxicity using MTT

measurement assays (Ezhilarasi et al. 2016; Sood and Chopra 2017). NPs take advantage

of the unique tumor microenvironment (angiogenesis, permeable vasculature, poor

lymphatic system etc) which are not associated with normal cells/tissues thus act as a

potential candidate for targeted drug delivery in tumor cells. NPs in combination with other


______________________________________________________________________________________ 135

anticancer drugs have shown synergistic effect by targeting cancer lesion without

damaging normal cells (Brigger et al. 2012). Furthermore, NPs used in smart imaging has

significantly decreased the death rate of cancer (Chanda and Baravalia 2011).

BSC and ALA activities were performed to investigate the cytotoxic effect of NiONPs.

BSC is the most appropriate test to investigate the biological effect of any naturally

occurring compound (Jebali and Kaemi 2013). The present study concluded dose-

dependent response of NiONPs. Our test sample was found to be in the category of general

cytotoxicity (IC50: 43.73µg/ml) and are consistent with the previous results of Ates et al.

(2016). The antileishmanial drugs conventionally used for leishmaniasis are often toxic,

expensive and less effective against leishmanial parasites. However, NPs have shown

potential antileishmaniasis activity (Gutierrez et al. 2016). In the current study, NiONPs

have shown strong antileishmanial effect against amastigotes compared to promastigote

and have shown dose-dependent inhibition of leishmanial parasites. Antileishmaniasis

results revealed that amastigotes are more vulnerable compared to promastigotes and is in

accordance with previous research reports. The applications of various nano-formulations

have also confirmed potential results in the treatment of leishmaniasis (Gutierrez et al.

2016). Biocompatibility assays revealed that NiONPs are biocompatible and can be used

in different therapeutic treatments. Similar results have been reported for NiONPs in the

earlier studies using Sageretia thea (Khalil et al. 2018). Current research findings are

premature and need further research with normal human cells. Our results have also

revealed strong antioxidant potentials which increases with increase in NiONPs

concentration and are consistent with previous studies (Khalil et al. 2018).


______________________________________________________________________________________ 136

Figure 5. 10: Schematic representation for the biogenic of NiONPs nanoparticles via green

route

Conclusion and future perspectives

______________________________________________________________________________________ 137

Conclusions and Future perspectives

Chapter 6 Conclusion and future perspectives

______________________________________________________________________________________ 138

Conclusion

The conventional methods of developing nanomaterials are costly and produce

toxic products, which limit their uses in different biological applications. So, it is

the need of hour to minimize the toxic effects of these noxious chemicals on

environment, plant and animal life. Hence, an alternative approach is developed

which is eco-friendly and cost-effective for the synthesis of small size stable

nanoparticles using R. gilgitica, R. virgata and G. wallichianum. Herein, different

nanoparticles have been developed such as NiO, ZnO, Co2O3 and Fe2O3 using

aqueous leaves extracts of medicinally important plants.

The biosynthesized MNPs were investigated for multiple biological potentials such

as antimicrobial (using fungal and bacterial strains), enzyme inhibition (alpha

amylase, protein kinase inhibition assays), biocompatibility (using RBCs,

macrophages) and cytotoxicity (brine shrimps, HepG2 cancer cell line, leishmanial

parasites) and have shown significant potentials.

Our greenly orchestrated IONPs results revealed significant biological potentials.

The RG-IONPs have shown strong cytotoxicity assays against HepG2 cell lines

(Hepatic cancer) and L. tropica (promastigotes and amastigotes). Results concluded

that biosynthesized RG-IONPs are nontoxic and biocompatible against human

RBCs and macrophages suggesting their biosafe nature. Furthermore, RG-IONPs

were found to have strong antioxidant and enzymes inhibition potentials.

The RV@ZnONPs showed promising fungicidal, bactericidal and leishmanicidal

potentials and determined dose-dependent response. Moreover, excellent

antioxidants and enhanced biocompatibility potentials make them effective in

different biomedical applications.

The GW-CoONPs determined significant antibacterial and antifungal activities.

GW-CoONPs revealed excellent anticancer, antileishmanial and antioxidant

activities. Moderate enzyme inhibitory potentials were reported. The

biocompatibility assays revealed that GW-CoONPs are bio-safe and biocompatible

in nature and can be used in different biomedical applications.

Bioinspired NiONPs revealed varying degree of bactericidal and fungicidal

potentials. Significant leishmanicidal, moderate enzymes inhibition and antiradical


______________________________________________________________________________________ 139

potentials are reported. Furthermore, phytogenic NiONPs determined biosafe and

biocompatible nature using human RBCs and macrophages. Moreover,

RV−NiONPs revealed strong anticancer potentials against HepG2 cancer cell line.

Overall, strong anticancer activities were reported for RG@IONPs followed by

RV-ZnONPs, RV-NiONPs and GW-CoONPs. Further, significant antileishmanial

activities were reported for GW@CoONPs followed by RV-ZnONPs, RG-IONPs

and RV-NiONPs. In addition, highest protein kinase inhibition potential was

reported for RG−IONPs followed by RV-ZnONPs, RV-NiONPs and GW-

CoONPs. The alpha amylase inhibitory activities are reported for RV−ZnONPs

followed by RV-NiONPs, GW-CoONPs, RG-IONPs. Moreover, RV-ZnONPs

revealed highest fungicidal and bactericidal activities. All the MNPs synthesized

using green methods were found to have good antioxidant potentials and are

biocompatible in nature confirming their biosafe nature.

Future perspectives

Phytosynthesis of nanoparticles using aqueous plant extract is simple, eco-friendly

and cost-efficient approach and could have tremendous potentials in designing

nanomedicines compare to conventional therapies against microbes, leishmaniasis

and cancer etc.

However, medicinal plants extracts contain numerous phytocompounds, so we do

not know the actual phytochemicals responsible in reduction and stabilization. It is

therefore recommended to produce NPs using purified and known compounds from

medicinal plants.

In addition, present study should not be limited to these targeted nanoparticles and

medicinal plants used but should be extended to novel NPs and medicinal plants

having potential biological applications.

Different in vitro biological applications have been investigated for biogenic NPs

and we recommend different in vivo studies using different animal models to further

evaluate their biosafety and biocompatibility.

Significant antimicrobial potentials of prepared NPs have been determined.

Further, research studies should be performed to investigate their antimicrobial


______________________________________________________________________________________ 140

potentials against different other bacterial strains which are potentially resistant to

wide range antibiotics.

References

______________________________________________________________________________________ 141

References

Abamor, E. S. (2017). Antileishmanial activities of caffeic acid phenethyl ester loaded

PLGA nanoparticles against Leishmania infantum promastigotes and amastigotes

in vitro. Asian Pacific journal of tropical medicine, 10(1), 25-34.

Abbas, F., Maqbool, Q., Nazar, M., Jabeen, N., Hussain, S. Z., Anwaar, S., & Iftikhar, S.

(2017). Green synthesised zinc oxide nanostructures through Periploca aphylla

extract shows tremendous antibacterial potential against multidrug resistant

pathogens. IET nanobiotechnology, 11(8), 935-941.

Abbasi, B. A., Iqbal, J., Mahmood, T., Khalil, A. T., Ali, B., Kanwal, S., & Ahmad, R.

(2018). Role of dietary phytochemicals in modulation of miRNA expression:

natural swords combating breast cancer. Asian pacific journal of tropical medicine,

11(9), 501.

Abbasi, B. A., Iqbal, J., Mahmood, T., Khalil, A. T., Ali, B., Kanwal, S., & Ahmad, R.

(2018). Role of dietary phytochemicals in modulation of miRNA expression:

natural swords combating breast cancer. Asian pacific journal of tropical medicine,

11(9), 501.

Abbasi, B. A., Iqbal, J., Mahmood, T., Qyyum, A., & Kanwal, S. (2019). Biofabrication of

iron oxide nanoparticles by leaf extract of Rhamnus virgata: Characterization and

evaluation of cytotoxic, antimicrobial and antioxidant potentials. Applied

Organometallic Chemistry, 33(7), e4947.

Abbasi, B. A., Iqbal, J., Mahmood, T., Ahmad, R., Kanwal, S., & Afridi, S. (2019). Plant-

mediated synthesis of nickel oxide nanoparticles (NiO) via Geranium

wallichianum: characterization and different biological applications. Materials

Research Express, 6(8), 0850a7.

Ahamed, M., AlSalhi, M. S., & Siddiqui, M. K. J. (2010). Silver nanoparticle applications

and human health. Clinica chimica acta, 411(23-24), 1841-1848.

Ahluwalia, V., Elumalai, S., Kumar, V., Kumar, S., & Sangwan, R. S. (2018). Nano silver

particle synthesis using Swertia paniculata herbal extract and its antimicrobial

activity. Microbial pathogenesis, 114, 402-408.

References

______________________________________________________________________________________ 142

Ahmad, N., Sharma, S., Alam, M. K., Singh, V. N., Shamsi, S. F., Mehta, B. R., & Fatma,

A. (2010). Rapid synthesis of silver nanoparticles using dried medicinal plant of

basil. Colloids and Surfaces B: Biointerfaces, 81(1), 81-86.

Ahmad, M., Sultana, S., Fazl-i-Hadi, S., Ben Hadda, T., Rashid, S., Zafar, M., & Yaseen,

G. (2014). An Ethnobotanical study of Medicinal Plants in high mountainous

region of Chail valley (District Swat-Pakistan). Journal of ethnobiology and

ethnomedicine, 10(1), 36.

Ahmad, S., Munir, S., Zeb, N., Ullah, A., Khan, B., Ali, J., & Ali, S. (2019). Green

nanotechnology: a review on green synthesis of silver nanoparticles—an

ecofriendly approach. International journal of nanomedicine, 14, 5087.

Ahmed, S., Ahmad, M., Swami, B. L., & Ikram, S. (2016). A review on plants extract

mediated synthesis of silver nanoparticles for antimicrobial applications: a green

expertise. Journal of advanced research, 7(1), 17-28.

Akbari, B., Tavandashti, M. P., & Zandrahimi, M. (2011). Particle size characterization of

nanoparticles–a practicalapproach. Iranian Journal of Materials Science and

Engineering, 8(2), 48-56.

Albanese, A., Tang, P. S., & Chan, W. C. (2012). The effect of nanoparticle size, shape,

and surface chemistry on biological systems. Annual review of biomedical

engineering, 14, 1-16.

Ali, A., Ambreen, S., Javed, R., Tabassum, S., ul Haq, I., & Zia, M. (2017). ZnO

nanostructure fabrication in different solvents transforms physio-chemical,

biological and photodegradable properties. Materials Science and Engineering: C,

74, 137-145.

Anastas, P. T., & Warner, J. C. (1998) Green Chemistry: Theory and Practice Oxford

University Press New York, NY, USA.

Anastas, P., & Eghbali, N. (2010). Green chemistry: principles and practice. Chemical

Society Reviews, 39(1), 301-312.

Andreescu, S., Ornatska, M., Erlichman, J. S., Estevez, A., & Leiter, J. C. (2012).

Biomedical applications of metal oxide nanoparticles. In Fine particles in medicine

and pharmacy (pp. 57-100). Springer, Boston, MA.

References

______________________________________________________________________________________ 143

Armendariz, V., Herrera, I., Jose-yacaman, M., Troiani, H., Santiago, P., & Gardea-

Torresdey, J. L. (2004). Size controlled gold nanoparticle formation by Avena

sativa biomass: use of plants in nanobiotechnology. Journal of nanoparticle

research, 6(4), 377-382.

Ates, M., Demir, V., Arslan, Z., Camas, M., & Celik, F. (2016). Toxicity of engineered

nickel oxide and cobalt oxide nanoparticles to Artemia salina in seawater. Water,

Air, & Soil Pollution, 227(3), 70.

Baer, D. R., Amonette, J. E., Engelhard, M. H., Gaspar, D. J., Karakoti, A. S., Kuchibhatla,

S., & Seal, S. (2008). Characterization challenges for nanomaterials. Surface and

Interface Analysis: An International Journal devoted to the development and

application of techniques for the analysis of surfaces, interfaces and thin films,

40(3‐4), 529-537.

Baer, D. R. (2011). Surface Characterization of Nanoparticles: critical needs and

significant challenges. Journal of Surface Analysis, 17(3), 163-169.

Baer, D. R., Engelhard, M. H., Johnson, G. E., Laskin, J., Lai, J., Mueller, K., & Elder, A.

(2013). Surface characterization of nanomaterials and nanoparticles: Important

needs and challenging opportunities. Journal of Vacuum Science & Technology A:

Vacuum, Surfaces, and Films, 31(5), 050820.

Bai, X., Gao, Y., Liu, H. G., & Zheng, L. (2009). Synthesis of amphiphilic ionic liquids

terminated gold nanorods and their superior catalytic activity for the reduction of

nitro compounds. The Journal of Physical Chemistry C, 113(41), 17730-17736.

Bakardjieva, S., Stengl, V., Subrt, J., Houskova, V., & Kalenda, P. (2007). Photocatalytic

efficiency of iron oxides: degradation of 4-chlorophenol. Journal of Physics and

Chemistry of solids, 68(5-6), 721-724.

Baker, S., Rakshith, D., Kavitha, K. S., Santosh, P., Kavitha, H. U., Rao, Y., & Satish, S.

(2013). Plants: emerging as nanofactories towards facile route in synthesis of

nanoparticles. BioImpacts: BI, 3(3), 111.

Bakht, J., Islam, A., Ali, H., Tayyab, M., & Shafi, M. (2011). Antimicrobial potentials of

Eclipta alba by disc diffusion method. African journal of Biotechnology, 10(39),

7658-7667.

References

______________________________________________________________________________________ 144

Bandara, J., & Yasomanee, J. P. (2006). P-type oxide semiconductors as hole collectors in

dye-sensitized solid-state solar cells. Semiconductor Science and Technology,

22(2), 20.

Barhoum, A., Van Assche, G., Rahier, H., Fleisch, M., Bals, S., Delplancked, M. P., &

Bahnemann, D. (2017). Sol-gel hot injection synthesis of ZnO nanoparticles into a

porous silica matrix and reaction mechanism. Materials & design, 119, 270-276.

Baqi A, Tareen R B and Mengal A 2018 Determination of antioxidants in two medicinally

important plants, Haloxylon griffithii and Convolvulus leiocalycinus, of

Balochistan. Pure appl. Biol. 7 296-308.

Behravan, M., Panahi, A. H., Naghizadeh, A., Ziaee, M., Mahdavi, R., & Mirzapour, A.

(2019). Facile green synthesis of silver nanoparticles using Berberis vulgaris leaf

and root aqueous extract and its antibacterial activity. International journal of

biological macromolecules, 124, 148-154.

Beinlich, A., Austrheim, H., Mavromatis, V., Grguric, B., Putnis, C. V., & Putnis, A.

(2018). Peridotite weathering is the missing ingredient of Earth’s continental crust

composition. Nature communications, 9(1), 1-12.

Bibi I, Nazar N, Iqbal M, Kamal S, Nawaz H, Nouren S, Safa Y, Jilani K, Sultan M, Ata S

and Rehman F 2017 Green and eco-friendly synthesis of cobalt-oxide nanoparticle:

characterization and photo-catalytic activity Adv. Powder. Technol. 28 2035-2043.

Brigger, I., Dubernet, C., & Couvreur, P. (2012). Nanoparticles in cancer therapy and

diagnosis. Advanced drug delivery reviews, 64, 24-36.

Bhattacharya, S., Nandi, S., & Jelinek, R. (2017). Carbon-dot–hydrogel for enzyme-

mediated bacterial detection. RSC advances, 7(2), 588-594.

Bhuyan, T., Mishra, K., Khanuja, M., Prasad, R., & Varma, A. (2015). Biosynthesis of zinc

oxide nanoparticles from Azadirachta indica for antibacterial and photocatalytic

applications. Materials Science in Semiconductor Processing, 32, 55-61.

Borase, H. P., Salunke, B. K., Salunkhe, R. B., Patil, C. D., Hallsworth, J. E., Kim, B. S.,

& Patil, S. V. (2014). Plant extract: a promising biomatrix for ecofriendly,

controlled synthesis of silver nanoparticles. Applied biochemistry and

biotechnology, 173(1), 1-29.

References

______________________________________________________________________________________ 145

Boyer, C., Whittaker, M. R., Bulmus, V., Liu, J., & Davis, T. P. (2010). The design and

utility of polymer-stabilized iron-oxide nanoparticles for nanomedicine

applications. NPG Asia Materials, 2(1), 23.

Butler, M. S. (2004). The role of natural product chemistry in drug discovery. Journal of

natural products, 67(12), 2141-2153.

Castro-Aceituno, V., Ahn, S., Simu, S. Y., Singh, P., Mathiyalagan, R., Lee, H. A., &

Yang, D. C. (2016). Anticancer activity of silver nanoparticles from Panax ginseng

fresh leaves in human cancer cells. Biomedicine & Pharmacotherapy, 84, 158-165.

Chakraborty, B., Gan-Or, G., Raula, M., Gadot, E., & Weinstock, I. A. (2018). Design of

an inherently-stable water oxidation catalyst. Nature communications, 9(1), 1-8.

Chakraborty, B., Gan‐Or, G., Duan, Y., Raula, M., & Weinstock, I. A. (2019). Visible‐

Light‐Driven Water Oxidation with a Polyoxometalate‐Complexed Hematite Core

of 275 Iron Atoms. Angewandte Chemie International Edition, 58(20), 6584-6589.

Chanda, S., & Baravalia, Y. (2011). Brine shrimp cytotoxicity of Caesalpinia pulcherrima

aerial parts, antimicrobial activity and characterisation of isolated active fractions.

Natural product research, 25(20), 1955-1964.

Chao, R., Zhang, Y., Yang, P., & Liu, W. (1997). Determination of maesopsin in

shengdeng (Rhamnella gilgitica Mansf. et Melch) by HPLC. Zhongguo Zhong yao

za zhi= Zhongguo zhongyao zazhi= China journal of Chinese materia medica,

22(4), 233-4.

Chaudhuri, S. K., & Malodia, L. (2017). Biosynthesis of zinc oxide nanoparticles using

leaf extract of Calotropis gigantea: characterization and its evaluation on tree

seedling growth in nursery stage. Applied Nanoscience, 7(8), 501-512.

Chen, Y., Zhang, Y., & Fu, S. (2007). Synthesis and characterization of Co3O4 hollow

spheres. Materials Letters, 61(3), 701-705.

Chen, J. S., Zhu, T., Hu, Q. H., Gao, J., Su, F., Qiao, S. Z., & Lou, X. W. (2010). Shape-

controlled synthesis of cobalt-based nanocubes, nanodiscs, and nanoflowers and

their comparative lithium-storage properties. ACS applied materials & interfaces,

2(12), 3628-3635.

Chen, F., Ehlerding, E. B., & Cai, W. (2014). Theranostic nanoparticles. Journal of

Nuclear Medicine, 55(12), 1919-1922.

References

______________________________________________________________________________________ 146

Chikkanna, M. M., Neelagund, S., & Hiremath, M. B. (2018). Biological synthesis,

characterization and antibacterial activity of novel copper Oxide nanoparticles

(CuONPs). Journal of Bionanoscience, 12(1), 92-99.

Cornell, R. M., & Schwertmann, U. (2003). The iron oxides: structure, properties,

reactions, occurrences and uses. John Wiley & Sons.

Cui, L., Pu, T., Liu, Y., & He, X. (2013). Layer-by-layer construction of graphene/cobalt

phthalocyanine composite film on activated GCE for application as a nitrite sensor.

Electrochimica acta, 88, 559-564.

Daher, S., Massarwa, M., Benson, A. A., & Khoury, T. (2018). Current and future

treatment of hepatocellular carcinoma: an updated comprehensive review. Journal

of clinical and translational hepatology, 6(1), 69.

Darroudi, M., Ahmad, M. B., Zamiri, R., Zak, A. K., Abdullah, A. H., & Ibrahim, N. A.

(2011). Time-dependent effect in green synthesis of silver nanoparticles.

International journal of nanomedicine, 6, 677.

Dauthal, P., & Mukhopadhyay, M. (2016). Noble metal nanoparticles: plant-mediated

synthesis, mechanistic aspects of synthesis, and applications. Industrial &

Engineering Chemistry Research, 55(36), 9557-9577.

De Almeida, M. C., Silva, A. C., Barral, A., & Barral Netto, M. (2000). A simple method

for human peripheral blood monocyte isolation. Memorias do Instituto Oswaldo

Cruz, 95(2), 221-223.

De Faria, D. L. A., Venâncio Silva, S., & De Oliveira, M. T. (1997). Raman

microspectroscopy of some iron oxides and oxyhydroxides. Journal of Raman

spectroscopy, 28(11), 873-878.

Demirezen, D. A., Yilmaz, Ş., & Yilmaz, D. D. (2018). Green synthesis and

characterization of iron nanoparticles using Aesculus hippocastanum seed extract.

Int. J. Adv. Sci. Eng. Technol., 6, 2321-8991.

Di Pietro, P., Strano, G., Zuccarello, L., & Satriano, C. (2016). Gold and silver

nanoparticles for applications in theranostics. Current topics in medicinal

chemistry, 16(27), 3069-3102.

Duan, H., Wang, D., & Li, Y. (2015). Green chemistry for nanoparticle synthesis. Chemical

Society Reviews, 44(16), 5778-5792.

References

______________________________________________________________________________________ 147

Dubey, S. P., Lahtinen, M., & Sillanpää, M. (2010). Tansy fruit mediated greener synthesis

of silver and gold nanoparticles. Process Biochemistry, 45(7), 1065-1071.

Dobrovolskaia, M. A., Clogston, J. D., Neun, B. W., Hall, J. B., Patri, A. K., & McNeil, S.

E. (2008). Method for analysis of nanoparticle hemolytic properties in vitro. Nano

letters, 8(8), 2180-2187.

Durán, N., Marcato, P. D., Durán, M., Yadav, A., Gade, A., & Rai, M. (2011). Mechanistic

aspects in the biogenic synthesis of extracellular metal nanoparticles by peptides,

bacteria, fungi, and plants. Applied microbiology and biotechnology, 90(5), 1609-

1624.

Durán, N., & Seabra, A. B. (2012). Metallic oxide nanoparticles: state of the art in biogenic

syntheses and their mechanisms. Applied Microbiology and Biotechnology, 95(2),

275-288.

Duran, N., & Seabra, A. B. (2018). Biogenic synthesized Ag/Au nanoparticles: production,

characterization, and applications. Current Nanoscience, 14(2), 82-94.

Dwivedi, A. D., & Gopal, K. (2010). Biosynthesis of silver and gold nanoparticles using

Chenopodium album leaf extract. Colloids and Surfaces A: Physicochemical and

Engineering Aspects, 369(1-3), 27-33.

Ebrahiminezhad, A., Zare-Hoseinabadi, A., Sarmah, A. K., Taghizadeh, S., Ghasemi, Y.,

& Berenjian, A. (2018). Plant-mediated synthesis and applications of iron

nanoparticles. Molecular biotechnology, 60(2), 154-168.

El-Kemary, M., Nagy, N., & El-Mehasseb, I. (2013). Nickel oxide nanoparticles: synthesis

and spectral studies of interactions with glucose. Materials Science in

Semiconductor Processing, 16(6), 1747-1752.

Ellis, S., Taylor, D. M., & Masood, K. R. (1994). Soil formation and erosion in the Murree

Hills, northeast Pakistan. Catena, 22(1), 69-78.

Ellmer, K. (2012). Past achievements and future challenges in the development of optically

transparent electrodes. Nature Photonics, 6(12), 809.

El-Naggar, N. E. A., Hussein, M. H., & El-Sawah, A. A. (2018). Phycobiliprotein-mediated

synthesis of biogenic silver nanoparticles, characterization, in vitro and in vivo

assessment of anticancer activities. Scientific reports, 8(1), 1-20.

References

______________________________________________________________________________________ 148

Ezhilarasi, A. A., Vijaya, J. J., Kaviyarasu, K., Maaza, M., Ayeshamariam, A., & Kennedy,

L. J. (2016). Green synthesis of NiO nanoparticles using Moringa oleifera extract

and their biomedical applications: Cytotoxicity effect of nanoparticles against HT-

29 cancer cells. Journal of Photochemistry and Photobiology B: Biology, 164, 352-

360.

Farhadi, S., Javanmard, M., & Nadri, G. (2016). Characterization of cobalt oxide

nanoparticles prepared by the thermal decomposition. Acta Chimica Slovenica,

63(2), 335-343.

Fatima, H, Khan K, Zia M, Ur-Rehman T, Mirza B and Haq I U 2015 Extraction

optimization of medicinally important metabolites from Datura innoxia Mill. an in

vitro biological and phytochemical investigation BMC. Complement. Altern. Med.

15 376-393.

Gahlawat, G., & Choudhury, A. R. (2019). A review on the biosynthesis of metal and metal

salt nanoparticles by microbes. RSC advances, 9(23), 12944-12967.

Galan, C. R., Silva, M. F., Mantovani, D., Bergamasco, R., & Vieira, M. F. (2018). Green

synthesis of copper oxide nanoparticles impregnated on activated carbon using

Moringa oleifera leaves extract for the removal of nitrates from water. The

Canadian Journal of Chemical Engineering, 96(11), 2378-2386.

Gangula, A., Podila, R., Karanam, L., Janardhana, C., & Rao, A. M. (2011). Catalytic

reduction of 4-nitrophenol using biogenic gold and silver nanoparticles derived

from Breynia rhamnoides. Langmuir, 27(24), 15268-15274.

Gardea-Torresdey, J. L., Tiemann, K. J., Gamez, G., Dokken, K., & Pingitore, N. E. (1999).

Recovery of gold (III) by alfalfa biomass and binding characterization using X-ray

microfluoresence. Advances in Environmental Research, 3(1), 83-93.

Nourbakhsh, M. S., & Mirzaee, O. (2018). Evaluation of the superparamagnetic and

biological properties of microwave assisted synthesized Zn & Cd doped CoFe 2 O

4 nanoparticles via Pechini sol–gel method. Journal of Sol-Gel Science and

Technology, 85(3), 684-692.

Gholoobi, A., Abnous, K., Ramezani, M., Shandiz, F. H., Darroudi, M., Ghayour-

Mobarhan, M., & Meshkat, Z. (2018). Synthesis of γ-Fe 2 O 3 Nanoparticles Capped with

References

______________________________________________________________________________________ 149

Oleic Acid and their Magnetic Characterization. Iranian Journal of Science and

Technology, Transactions A: Science, 42(4), 1889-1893.

Giljohann, D. A., Seferos, D. S., Daniel, W. L., Massich, M. D., Patel, P. C., & Mirkin, C.

A. (2010). Gold nanoparticles for biology and medicine. Angewandte Chemie

International Edition, 49(19), 3280-3294.

Gondal, M. A., Drmosh, Q. A., Yamani, Z. H., & Saleh, T. A. (2009). Synthesis of ZnO2

nanoparticles by laser ablation in liquid and their annealing transformation into

ZnO nanoparticles. Applied surface science, 256(1), 298-304.

Gong, N., Shao, K., Feng, W., Lin, Z., Liang, C., & Sun, Y. (2011). Biotoxicity of nickel

oxide nanoparticles and bio-remediation by microalgae Chlorella vulgaris.

Chemosphere, 83(4), 510-516.

Guangmin, Y., Sebisubi, F. M., Voo, L. Y. C., Ho, C. C., Tan, G. T., & Chang, L. C. (2011).

Citrinin derivatives from the soil filamentous fungus Penicillium sp. H9318.

Journal of the Brazilian Chemical Society, 22(6), 1125-1129.

Gulino, A., Dapporto, P., Rossi, P., & Fragalà, I. (2003). A novel self-generating liquid

MOCVD precursor for Co3O4 thin films. Chemistry of materials, 15(20), 3748-

3752.

Gupta, R. K. (1968). Flora Nainitalensis, Navyug Traders. New Delhi, India, 160.

Gutierrez, V., Seabra, A. B., Reguera, R. M., Khandare, J., & Calderón, M. (2016). New

approaches from nanomedicine for treating leishmaniasis. Chemical Society

Reviews, 45(1), 152-168.

Guzmán, M. G., Dille, J., & Godet, S. (2009). Synthesis of silver nanoparticles by chemical

reduction method and their antibacterial activity. Int J Chem Biomol Eng, 2(3), 104-

111.

Han, Y., Zhang, S., Shen, N., Li, D., & Li, X. (2017). MOF-derived porous NiO

nanoparticle architecture for high performance supercapacitors. Materials Letters,

188, 1-4.

Hassan, H. F. H., Mansour, A. M., Abo‐Youssef, A. M. H., Elsadek, B. E., & Messiha, B.

A. S. (2017). Zinc oxide nanoparticles as a novel anticancer approach; in vitro and

in vivo evidence. Clinical and Experimental Pharmacology and Physiology, 44(2),

235-243.

References

______________________________________________________________________________________ 150

Hameed, S., Khalil, A. T., Ali, M., Numan, M., Khamlich, S., Shinwari, Z. K., & Maaza,

M. (2019). Greener synthesis of ZnO and Ag–ZnO nanoparticles using Silybum

marianum for diverse biomedical applications. Nanomedicine, 14(6), 655-673.

Hameed, S., Iqbal, J., Ali, M., Khalil, A. T., Abbasi, B. A., Numan, M., & Shinwari, Z. K.

(2019). Green synthesis of zinc nanoparticles through plant extracts: establishing a

novel era in cancer theranostics. Materials Research Express, 6(10), 102005.

Han, Y., Zhang, S., Shen, N., Li, D., & Li, X. (2017). MOF-derived porous NiO

nanoparticle architecture for high performance supercapacitors. Materials Letters,

188, 1-4.

Hu, M., Chen, J., Li, Z. Y., Au, L., Hartland, G. V., Li, X., & Xia, Y. (2006). Gold

nanostructures: engineering their plasmonic properties for biomedical applications.

Chemical Society Reviews, 35(11), 1084-1094.

Hu, Y., Yan, C., Chen, D., Lv, C., Jiao, Y., & Chen, G. (2017). One-dimensional Co3O4

nanonet with enhanced rate performance for lithium ion batteries: Carbonyl-β-

cyclodextrin inducing and kinetic analysis. Chemical Engineering Journal, 321,

31-39.

Huang, L., Weng, X., Chen, Z., Megharaj, M., & Naidu, R. (2014). Synthesis of iron-based

nanoparticles using oolong tea extract for the degradation of malachite green.

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 117, 801-

804.

Huang, S., Liu, H. F., Quan, X., Jin, Y., Xuan, G., An, R. B., & Li, B. (2016). Rhamnella

gilgitica attenuates inflammatory responses in LPS-induced murine macrophages

and complete Freund’s adjuvant-induced arthritis rats. The American journal of

Chinese medicine, 44(07), 1379-1392.

Ibrahim, H. M. (2015). Green synthesis and characterization of silver nanoparticles using

banana peel extract and their antimicrobial activity against representative

microorganisms. Journal of Radiation Research and Applied Sciences, 8(3), 265-

275.

Iqbal, J., Abbasi, B. A., Mahmood, T., Kanwal, S., Ali, B., Shah, S. A., & Khalil, A. T.

(2017). Plant-derived anticancer agents: A green anticancer approach. Asian

Pacific Journal of Tropical Biomedicine, 7(12), 1129-1150.

References

______________________________________________________________________________________ 151

Iqbal, J., Abbasi, B. A., Batool, R., Mahmood, T., Ali, B., Khalil, A. T., & Ahmad, R.

(2018). Potential phytocompounds for developing breast cancer therapeutics:

nature’s healing touch. European journal of pharmacology, 827, 125-148.

Iqbal, J., Abbasi, B. A., Khalil, A. T., Ali, B., Mahmood, T., Kanwal, S., & Ali, W. (2018).

Dietary isoflavones, the modulator of breast carcinogenesis: Current landscape and

future perspectives. Asian Pacific Journal of Tropical Medicine, 11(3), 186.

Iqbal, J., Abbasi, B. A., Ahmad, R., Mahmood, T., Kanwal, S., Ali, B., & Badshah, H.

(2018). Ursolic acid a promising candidate in the therapeutics of breast cancer:

Current status and future implications. Biomedicine & Pharmacotherapy, 108, 752-

756.s

Iqbal, J., Abbasi, B. A., Ahmad, R., Mahmood, T., Ali, B., Khalil, A. T., & Munir, A.

(2018). Nanomedicines for developing cancer nanotherapeutics: from benchtop to

bedside and beyond. Applied microbiology and biotechnology, 102(22), 9449-

9470.

Iqbal, J., Abbasi, B. A., Ahmad, R., Batool, R., Mahmood, T., Ali, B., & Bashir, S. (2019).

Potential phytochemicals in the fight against skin cancer: current landscape and

future perspectives. Biomedicine & Pharmacotherapy, 109, 1381-1393.

Iqbal, J., Abbasi, B. A., Mahmood, T., Kanwal, S., Ahmad, R., & Ashraf, M. (2019). Plant-

extract mediated green approach for the synthesis of ZnONPs: Characterization and

evaluation of cytotoxic, antimicrobial and antioxidant potentials. Journal of

Molecular Structure, 1189, 315-327.kin

Iqbal, J., Abbasi, B. A., Mahmood, T., Hameed, S., Munir, A., & Kanwal, S. (2019). Green

synthesis and characterizations of Nickel oxide nanoparticles using leaf extract of

Rhamnus virgata and their potential biological applications. Applied

Organometallic Chemistry, 33(8), e4950.

Iqbal, J., Shinwari, Z. K., & Mahmood, T. (2019). Phylogenetic relationships within the

cosmopolitan family rhamnaceae using atpB gene promoter. Pakistan journal of

Botany, 51(3), 1027-1040.

Iqbal, J., Abbasi, B. A., Ahmad, R., Shahbaz, A., Zahra, S. A., Kanwal, S., & Mahmood,

T. (2020). Biogenic synthesis of green and cost effective iron nanoparticles and

References

______________________________________________________________________________________ 152

evaluation of their potential biomedical properties. Journal of Molecular Structure,

1199, 126979.

Iravani, S., & Zolfaghari, B. (2013). Green synthesis of silver nanoparticles using Pinus

eldarica bark extract. BioMed research international, 2013.

Ismail, M., Ibrar, M., Iqbal, Z., Hussain, J., Hussain, H., Ahmed, M., & Choudhary, M. I.

(2009). Chemical constituents and antioxidant activity of Geranium wallichianum.

Records of Natural Products, 3(4), 193.

Ismail, M., Hussain, J., Khan, A. U., Khan, A. L., Ali, L., Khan, F. U., & Lee, I. J. (2012).

Antibacterial, antifungal, cytotoxic, phytotoxic, insecticidal, and enzyme inhibitory

activities of Geranium wallichianum. Evidence-Based Complementary and

alternative medicine, 2012.

Islam, N. U., Amin, R., Shahid, M., & Amin, M. (2019). Gummy gold and silver

nanoparticles of apricot (Prunus armeniaca) confer high stability and biological

activity. Arabian Journal of Chemistry, 12(8), 3977-3992.

Jain, P. K., Huang, X., El-Sayed, I. H., & El-Sayed, M. A. (2008). Noble metals on the

nanoscale: optical and photothermal properties and some applications in imaging,

sensing, biology, and medicine. Accounts of chemical research, 41(12), 1578-1586.

Jamdagni, P., Rana, J. S., Khatri, P., & Nehra, K. (2018). Comparative account of

antifungal activity of green and chemically synthesized zinc oxide nanoparticles in

combination with agricultural fungicides. International Journal of Nano

Dimension, 9(2), 198-208.

Jang, G. S., Ameen, S., Akhtar, M. S., Kim, E., & Shin, H. S. (2017). Electrochemical

investigations of hydrothermally synthesized porous cobalt oxide (Co3O4)

nanorods: supercapacitor application. ChemistrySelect, 2(28), 8941-8949.

Jebali, A., & Kazemi, B. (2013). Nano-based antileishmanial agents: a toxicological study

on nanoparticles for future treatment of cutaneous leishmaniasis. Toxicology in

vitro, 27(6), 1896-1904.

Jodłowski, P. J., Jędrzejczyk, R. J., Rogulska, A., Wach, A., Kuśtrowski, P., Sitarz, M., &

Łojewska, J. (2014). Spectroscopic characterization of Co3O4 catalyst doped with

CeO2

References

______________________________________________________________________________________ 153

and PdO for methane catalytic combustion. Spectrochimica Acta Part A: Molecular

and Biomolecular Spectroscopy, 131, 696-701.

Jolivet, J. P., Cassaignon, S., Chanéac, C., Chiche, D., Durupthy, O., & Portehault, D.

(2010). Design of metal oxide nanoparticles: Control of size, shape, crystalline

structure and functionalization by aqueous chemistry. Comptes Rendus Chimie,

13(1-2), 40-51.

Jubb, A. M., & Allen, H. C. (2010). Vibrational spectroscopic characterization of hematite,

maghemite, and magnetite thin films produced by vapor deposition. ACS Applied

Materials & Interfaces, 2(10), 2804-2812.

Kalidhar, S. B., & Sharma, P. (1984). Physcion-8-O-gentiobioside from Rhamnus virgata.

Phytochemistry, 23(5), 1196-1197.

Kalidhar, S. B., & Sharma, P. (1985). Chemical-Components of Rhamnus-Virgata. Journal

of the Indian Chemical Society, 62(5), 411-412.

Kalusniak, S., Sadofev, S., Puls, J., & Henneberger, F. (2009). ZnCdO/ZnO–a new

heterosystem for green‐wavelength semiconductor lasing. Laser & Photonics

Reviews, 3(3), 233-242.

Kaminsky, F. (2012). Mineralogy of the lower mantle: A review of ‘super-deep’mineral

inclusions in diamond. Earth-Science Reviews, 110(1-4), 127-147.

Kandalkar, S. G., Gunjakar, J. L., & Lokhande, C. D. (2008). Preparation of cobalt oxide

thin films and its use in supercapacitor application. Applied Surface Science,

254(17), 5540-5544.

Karthik, K., Dhanuskodi, S., Gobinath, C., Prabukumar, S., & Sivaramakrishnan, S.

(2018). Multifunctional properties of microwave assisted CdO–NiO–ZnO mixed

metal oxide nanocomposite: enhanced photocatalytic and antibacterial activities.

Journal of Materials Science: Materials in Electronics, 29(7), 5459-5471.

Kastrinaki, G., Lorentzou, S., Karagiannakis, G., Rattenbury, M., Woodhead, J., &

Konstandopoulos, A. G. (2018). Parametric synthesis study of iron based

nanoparticles via aerosol spray pyrolysis route. Journal of Aerosol Science, 115,

96-107.

References

______________________________________________________________________________________ 154

Kawakami, M., Hartanto, A. B., Nakata, Y., & Okada, T. (2003). Synthesis of ZnO

nanorods by nanoparticle assisted pulsed-laser deposition. Japanese journal of

Applied Physics, 42(1A), L33.

Kaye, P., & Scott, P. (2011). Leishmaniasis: complexity at the host–pathogen interface.

Nature Reviews Microbiology, 9(8), 604-615.

Khalil, A. T., Ovais, M., Ullah, I., Ali, M., Shinwari, Z. K., & Maaza, M. (2017).

Biosynthesis of iron oxide (Fe2O3) nanoparticles via aqueous extracts of Sageretia

thea (Osbeck.) and their pharmacognostic properties. Green Chemistry Letters and

Reviews, 10(4), 186-201.

Khalil, A. T., Ovais, M., Ullah, I., Ali, M., Shinwari, Z. K., Khamlich, S., & Maaza, M.

(2017). Sageretia thea (Osbeck.) mediated synthesis of zinc oxide nanoparticles

and its biological applications. Nanomedicine, 12(15), 1767-1789.

Khalil, A. T., Ovais, M., Ullah, I., Ali, M., Shinwari, Z. K., & Maaza, M. (2017). Physical

properties, biological applications and biocompatibility studies on biosynthesized

single phase cobalt oxide (Co3O4) nanoparticles via Sageretia thea (Osbeck.).

Arabian Journal of Chemistry, 13(1), 606-619.

Khalil, A. T., Ovais, M., Ullah, I., Ali, M., Shinwari, Z. K., Hassan, D., & Maaza, M.

(2018). Sageretia thea (Osbeck.) modulated biosynthesis of NiO nanoparticles and

their in vitro pharmacognostic, antioxidant and cytotoxic potential. Artificial cells,

nanomedicine, and biotechnology, 46(4), 838-852.

Khan, S., Ansari, A. A., Khan, A. A., Ahmad, R., Al-Obaid, O., & Al-Kattan, W. (2015).

In vitro evaluation of anticancer and antibacterial activities of cobalt oxide

nanoparticles. JBIC Journal of Biological Inorganic Chemistry, 20(8), 1319-1326.

Khan, Z. U. H., Khan, A., Chen, Y., ullah Khan, A., Shah, N. S., Muhammad, N., & Wan,

P. (2017). Photo catalytic applications of gold nanoparticles synthesized by green

route and electrochemical degradation of phenolic Azo dyes using AuNPs/GC as

modified paste electrode. Journal of Alloys and Compounds, 725, 869-876.

Khan, S. A., Noreen, F., Kanwal, S., Iqbal, A., & Hussain, G. (2018). Green synthesis of

ZnO and Cu-doped ZnO nanoparticles from leaf extracts of Abutilon indicum,

Clerodendrum infortunatum, Clerodendrum inerme and investigation of their

References

______________________________________________________________________________________ 155

biological and photocatalytic activities. Materials Science and Engineering: C, 82,

46-59.

Kharissova, O. V., Dias, H. R., Kharisov, B. I., Pérez, B. O., & Pérez, V. M. J. (2013). The

greener synthesis of nanoparticles. Trends in biotechnology, 31(4), 240-248.

Khatami, M., Sharifi, I., Nobre, M. A., Zafarnia, N., & Aflatoonian, M. R. (2018). Waste-

grass-mediated green synthesis of silver nanoparticles and evaluation of their

anticancer, antifungal and antibacterial activity. Green Chemistry Letters and

Reviews, 11(2), 125-134.

Khatami, M., Alijani, H. Q., Nejad, M. S., & Varma, R. S. (2018). Core@ shell

nanoparticles:greener synthesis using natural plant products. Applied Sciences,

8(3), 411.

Khatami, M., Alijani, H. Q., Fakheri, B., Mobasseri, M. M., Heydarpour, M., Farahani, Z.

K., & Khan, A. U. (2019). Super-paramagnetic iron oxide nanoparticles (SPIONs):

Greener synthesis using Stevia plant and evaluation of its antioxidant properties.

Journal of cleaner production, 208, 1171-1177.

Kharissova, O. V., Dias, H. R., Kharisov, B. I., Pérez, B. O., & Pérez, V. M. J. (2013). The

greener synthesis of nanoparticles. Trends in biotechnology, 31(4), 240-248.

Khashan, K. S., Sulaiman, G. M., Ameer, A., Kareem, F. A., & Napolitano, G. (2016).

Synthesis, characterization and antibacterial activity of colloidal NiO nanoparticles.

Pakistan journal of pharmaceutical sciences, 29(2).

Khashan, K. S., Sulaiman, G. M., Hamad, A. H., Abdulameer, F. A., & Hadi, A. (2017).

Generation of NiO nanoparticles via pulsed laser ablation in deionised water and

their antibacterial activity. Applied Physics A, 123(3), 190.

Kuchibhatla, S. V., Karakoti, A. S., Baer, D. R., Samudrala, S., Engelhard, M. H.,

Amonette, J. E., & Seal, S. (2012). Influence of aging and environment on

nanoparticle chemistry: implication to confinement effects in nanoceria. The

Journal of Physical Chemistry C, 116(26), 14108-14114.

Kundu, M., & Liu, L. (2015). Binder-free electrodes consisting of porous NiO nanofibers

directly electrospun on nickel foam for high-rate supercapacitors. Materials

Letters, 144, 114-118.

References

______________________________________________________________________________________ 156

Kwon, H. J., Shin, K., Soh, M., Chang, H., Kim, J., Lee, J., & Hyeon, T. (2018). Large‐

scale synthesis and medical applications of uniform‐sized metal oxide

nanoparticles. Advanced Materials, 30(42), 1704290.

Laurent, S., Forge, D., Port, M., Roch, A., Robic, C., Vander Elst, L., & Muller, R. N.

(2008). Magnetic iron oxide nanoparticles: synthesis, stabilization, vectorization,

physicochemical characterizations, and biological applications. Chemical reviews,

108(6), 2064-2110.

Li, Y., Zhang, W., Niu, J., & Chen, Y. (2012). Mechanism of photogenerated reactive

oxygen species and correlation with the antibacterial properties of engineered

metal-oxide nanoparticles. ACS nano, 6(6), 5164-5173.

Liang, X., & Zhao, L. (2012). Room-temperature synthesis of air-stable cobalt

nanoparticles and their highly efficient adsorption ability for Congo red. Rsc

Advances, 2(13), 5485-5487.

Liu, X., Wu, X., Cao, H., & Chang, R. P. (2004). Growth mechanism and properties of

ZnO nanorods synthesized by plasma-enhanced chemical vapor deposition.

Journal of Applied Physics, 95(6), 3141-3147.

Lou, X. W., Deng, D., Lee, J. Y., Feng, J., & Archer, L. A. (2008). Self‐supported

formation of needlelike Co3O4 nanotubes and their application

aslithium‐ion battery electrodes. Advanced Materials, 20(2), 258-262.

Malagoli, D. (2007). A full-length protocol to test hemolytic activity of palytoxin on human

erythrocytes. Invertebrate Survival Journal, 4(2), 92-94.

Malathy, M., Jayasree, R., & Rajavel, R. (2017). Facile Synthesis of CuO Nanoparticles

from Cu (II) Schiff Base Complexes: Characterization, Antibacterial and

Anticancer Activity. Smart Science, 5(2), 100-115.

Manikandan, E., Kennedy, J., Kavitha, G., Kaviyarasu, K., Maaza, M., Panigrahi, B. K., &

Mudali, U. K. (2015). Hybrid nanostructured thin-films by PLD for enhanced field

emission performance for radiation micro-nano dosimetry applications. Journal of

Alloys and Compounds, 647, 141-145.

Manjunatha, M., Kumar, R., Sahoo, B., Damle, R., & Ramesh, K. P. (2018). Determination

of magnetic domain state of carbon coated iron nanoparticles via 57Fe zero-

external-field NMR.

References

______________________________________________________________________________________ 157

Marambio-Jones, C., & Hoek, E. M. (2010). A review of the antibacterial effects of silver

nanomaterials and potential implications for human health and the environment.

Journal of Nanoparticle Research, 12(5), 1531-1551.

Marousek, J., & Kwan, J. T. H. (2013). Use of pressure manifestations following the water

plasma expansion for phytomass disintegration. Water Science and Technology,

67(8), 1695-1700.

Matinise, N., Fuku, X. G., Kaviyarasu, K., Mayedwa, N., & Maaza, M. (2017). ZnO

nanoparticles via Moringa oleifera green synthesis: Physical properties &

mechanism of formation. Applied Surface Science, 406, 339-347.

Matinise, N., Mayedwa, N., Fuku, X. G., Mongwaketsi, N., & Maaza, M. (2018, May).

Green synthesis of cobalt (II, III) oxide nanoparticles using Moringa Oleifera

natural extract as high electrochemical electrode for supercapacitors. In AIP

Conference Proceedings (Vol. 1962, No. 1, p. 040005). AIP Publishing LLC.

Mayedwa, N., Mongwaketsi, N., Khamlich, S., Kaviyarasu, K., Matinise, N., & Maaza, M.

(2018). Green synthesis of nickel oxide, palladium and palladium oxide synthesized

via Aspalathus linearis natural extracts: physical properties & mechanism of

formation. Applied Surface Science, 446, 266-272.

Mirzaei, H., & Darroudi, M. (2017). Zinc oxide nanoparticles: Biological synthesis and

biomedical applications. Ceramics International, 43(1), 907-914.

Mohanpuria, P., Rana, N. K., & Yadav, S. K. (2008). Biosynthesis of nanoparticles:

technological concepts and future applications. Journal of nanoparticle research,

10(3), 507-517.

Mourdikoudis, S., & Liz-Marzán, L. M. (2013). Oleylamine in nanoparticle synthesis.

Chemistry of Materials, 25(9), 1465-1476.

Mudunkotuwa, I. A., Pettibone, J. M., & Grassian, V. H. (2012). Environmental

implications of nanoparticle aging in the processing and fate of copper-based

nanomaterials. Environmental science & technology, 46(13), 7001-7010.

Mukherjee, S., Chowdhury, D., Kotcherlakota, R., & Patra, S. (2014). Potential

theranostics application of bio-synthesized silver nanoparticles (4-in-1 system).

Theranostics, 4(3), 316.

References

______________________________________________________________________________________ 158

Mukunthan, K. S., & Balaji, S. (2012). Cashew apple juice (Anacardium occidentale L.)

speeds up the synthesis of silver nanoparticles. International Journal of Green

Nanotechnology, 4(2), 71-79.

Murray, C. B., Kagan, A. C., & Bawendi, M. G. (2000). Synthesis and characterization of

monodisperse nanocrystals and close-packed nanocrystal assemblies. Annual

review of materials science, 30(1), 545-610.

Muthee, J. K., Gakuya, D. W., Mbaria, J. M., Kareru, P. G., Mulei, C. M., & Njonge, F. K.

(2011). Ethnobotanical study of anthelmintic and other medicinal plants

traditionally used in Loitoktok district of Kenya. Journal of ethnopharmacology,

135(1), 15-21.

Muthukumar, H., & Matheswaran, M. (2015). Amaranthus spinosus leaf extract mediated

FeO nanoparticles: physicochemical traits, photocatalytic and antioxidant activity.

ACS Sustainable Chemistry & Engineering, 3(12), 3149-3156.

Nagajyothi, P. C., Pandurangan, M., Kim, D. H., Sreekanth, T. V. M., & Shim, J. (2017).

Green synthesis of iron oxide nanoparticles and their catalytic and in vitro

anticancer activities. Journal of Cluster Science, 28(1), 245-257.

Narayanan, K. B., & Sakthivel, N. (2011). Green synthesis of biogenic metal nanoparticles

by terrestrial and aquatic phototrophic and heterotrophic eukaryotes and

biocompatible agents. Advances in colloid and interface science, 169(2), 59-79.

Naseem, T., & Farrukh, M. A. (2015). Antibacterial activity of green synthesis of iron

nanoparticles using Lawsonia inermis and Gardenia jasminoides leaves extract.

Journal of Chemistry, 2015.

Nasseri, M. A., Ahrari, F., & Zakerinasab, B. (2016). A green biosynthesis of NiO

nanoparticles using aqueous extract of Tamarix serotina and their characterization

and application. Applied Organometallic Chemistry, 30(12), 978-984.

Nath, D., & Banerjee, P. (2013). Green nanotechnology–a new hope for medical biology.

Environmental toxicology and pharmacology, 36(3), 997-1014.

Nguyen, N. H., Padil, V. V. T., Slaveykova, V. I., Černík, M., & Ševců, A. (2018). Green

synthesis of metal and metal oxide nanoparticles and their effect on the unicellular

alga Chlamydomonas reinhardtii. Nanoscale research letters, 13(1), 1-13.

References

______________________________________________________________________________________ 159

Nasar, MQ., Zohra, T., Khalil, A. T., Saqib, S., Ayaz, M., Ahmad, A., & Shinwari, Z. K.

(2019). Seripheidium quettense mediated green synthesis of biogenic silver

nanoparticles and their theranostic applications. Green Chemistry Letters and

Reviews, 12(3), 310-322.

Nosrati, H., Salehiabar, M., Attari, E., Davaran, S., Danafar, H., & Manjili, H. K. (2018).

Green and one‐pot surface coating of iron oxide magnetic nanoparticles with

natural amino acids and biocompatibility investigation. Applied Organometallic

Chemistry, 32(2), e4069.

Odobel, F., Le Pleux, L., Pellegrin, Y., & Blart, E. (2010). New photovoltaic devices based

on thesensitization of p-type semiconductors: challenges and opportunities.

Accounts of chemical research, 43(8), 1063-1071.

Otunola, G. A., & Afolayan, A. J. (2018). In vitro antibacterial, antioxidant and toxicity

profile of silver nanoparticles green-synthesized and characterized from aqueous

extract of a spice blend formulation. Biotechnology & Biotechnological Equipment,

32(3), 724-733.

Ovais, M., Khalil, A. T., Raza, A., Islam, N. U., Ayaz, M., Saravanan, M., & Shinwari, Z.

K. (2018). Multifunctional theranostic applications of biocompatible green-

synthesized colloidal nanoparticles. Applied microbiology and biotechnology,

102(10), 4393-4408.

Oyedemi, S. O., Oyedemi, B. O., Ijeh, I. I., Ohanyerem, P. E., Coopoosamy, R. M., &

Aiyegoro, O. A. (2017). Alpha-amylase inhibition and antioxidative capacity of

some antidiabetic plants used by the traditional healers in Southeastern Nigeria. The

Scientific World Journal, 2017.

Ozgur, Ü., Alivov, Y. I., Liu, C., Teke, A., Reshchikov, M., Doğan, S., & Morkoç, A. H.

(2005). A comprehensive review of ZnO materials and devices. Journal of applied

physics, 98(4), 11.

Pal, S. L., Jana, U., Manna, P. K., Mohanta, G. P., & Manavalan, R. (2011). Nanoparticle:

An overview of preparation and characterization. Journal of applied

pharmaceutical science, 1(6), 228-234.

References

______________________________________________________________________________________ 160

Pan, Q., Yang, P., Chen, G., & Liu, W. (1998). Studies on the chemical constituents of

traditional Tibetan medicinal herb Shengdeng (Rhamnella gilgitica) (I). Chinese

traditional and herbal drugs, 29(2), 76-79.

Pan, Q., Yang, P., Chen, G., & Liu, W. (1997). Studies on the chemical constituents of

traditional Tibetan medicinal herb shengdeng (Rhamnella gilgitica Mansf. Et

Melch) (Ⅱ). West China Journal of Pharmaceutical Sciences, 12(3), 153-155.

Pandey, B. D. (2012). Synthesis of zinc-based nanomaterials: a biological perspective. IET

nanobiotechnology, 6(4), 144-148.

Pandian, C. J., Palanivel, R., & Dhananasekaran, S. (2015). Green synthesis of nickel

nanoparticles using Ocimum sanctum and their application in dye and pollutant

adsorption. Chinese journal of Chemical engineering, 23(8), 1307-1315.

Parandhaman, T., Dey, M. D., & Das, S. K. (2019). Biofabrication of supported metal

nanoparticles: exploring the bioinspiration strategy to mitigate the environmental

challenges. Green Chemistry, 21(20), 5469-5500.

Patra, J. K., & Baek, K. H. (2014). Green nanobiotechnology: factors affecting synthesis

and characterization techniques. Journal of Nanomaterials, Article ID 417305,

2014.

Paull, R., Wolfe, J., Peter, H., & Sinkula, M. (2003). Investing in nanotechnology. Nature

biotechnology, 21(10), 1144.

Pejova, B., Kocareva, T., Najdoski, M., & Grozdanov, I. (2000). A solution growth route

to nanocrystalline nickel oxide thin films. Applied Surface Science, 165(4), 271-

278.

Pennycook, T. J., McBride, J. R., Rosenthal, S. J., Pennycook, S. J., & Pantelides, S. T.

(2012). Dynamic fluctuations in ultrasmall nanocrystals induce white light

emission. Nano letters, 12(6), 3038-3042.

Pineda, A., Ojeda, M., Romero, A. A., Balu, A. M., & Luque, R. (2018). Mechanochemical

synthesis of supported cobalt oxide nanoparticles on mesoporous materials as

versatile bifunctional catalysts. Microporous and Mesoporous Materials, 272, 129-

136.

References

______________________________________________________________________________________ 161

Pirtarighat, S., Ghannadnia, M., & Baghshahi, S. (2019). Green synthesis of silver

nanoparticles using the plant extract of Salvia spinosa grown in vitro and their

antibacterial activity assessment. Journal of Nanostructure in Chemistry, 9(1), 1-9.

Prach, M., Stone, V., & Proudfoot, L. (2013). Zinc oxide nanoparticles and monocytes:

impact of size, charge and solubility on activation status. Toxicology and applied

pharmacology, 266(1), 19-26.

Prakash, N. S., & Soni, N. (2011). Factors affecting the geometry of silver nanoparticles

synthesis in Chrysosporium tropicum and Fusarium oxysporum. American journal

of nanotechnology, 2(1), 112-121.

Prucek, R., Tuček, J., Kilianová, M., Panáček, A., Kvítek, L., Filip, J., & Zbořil, R. (2011).

The targeted antibacterial and antifungal properties of magnetic nanocomposite of

iron oxide and silver nanoparticles. Biomaterials, 32(21), 4704-4713.

Rai, A., Singh, A., Ahmad, A., & Sastry, M. (2006). Role of halide ions and temperature

on the morphology of biologically synthesized gold nanotriangles. Langmuir,

22(2), 736-741.

Rajendran, A., Siva, E., Dhanraj, C., & Senthilkumar, S. (2018). A green and facile

approach for the synthesis copper oxide nanoparticles using Hibiscus rosa-sinensis

flower extracts and It’s antibacterial activities. J Bioprocess Biotech, 8(3), 324.

Ramanavicius, A., Kausaite, A., & Ramanaviciene, A. (2005). Biofuel cell based on direct

bioelectrocatalysis. Biosensors and Bioelectronics, 20(10), 1962-1967.

Rahdar, A., Aliahmad, M., Azizi, Y., & NanoStruct, J. (2015). 5, 145 (2015) 7. J. Safaei-

Ghomi, S. Zahedi, M. Javid, MA Ghasemzadeh. J. Nanostruct, 5(153), 8.

Rautela, A., Rani, J., & Das, M. D. (2019). Green synthesis of silver nanoparticles from

Tectona grandis seeds extract: characterization and mechanism of antimicrobial

action on different microorganisms. Journal of Analytical Science and Technology,

10(1), 1-10.

Rice-evans, C. A., Miller, N. J., Bolwell, P. G., Bramley, P. M., & Pridham, J. B. (1995).

The relative antioxidant activities of plant-derived polyphenolic flavonoids. Free

radical research, 22(4), 375-383.

Rizzello, L., Cingolani, R., & Pompa, P. P. (2013). Nanotechnology tools for antibacterial

materials. Nanomedicine, 8(5), 807-821.

References

______________________________________________________________________________________ 162

Ruan, H., Wu, X., Yang, C., Li, Z., Xia, Y., Xue, T., & Wu, A. (2018). A supersensitive

CTC analysis system based on triangular silver nanoprisms and SPION with

function of capture, enrichment, detection, and release. ACS Biomaterials Science

& Engineering, 4(3), 1073-1082.

Saeed, M., Muneer, M., Mumtaz, N., Siddique, M., Akram, N., & Hamayun, M. (2018).

Ag-Co3O4: Synthesis, characterization and evaluation of its photo-catalytic

activity towards degradation of rhodamine B dye in aqueous medium. Chinese

journal of chemical engineering, 26(6), 1264-1269.

Safawo, T., Sandeep, B. V., Pola, S., & Tadesse, A. (2018). Synthesis and characterization

of zinc oxide nanoparticles using tuber extract of anchote (Coccinia abyssinica

(Lam.) Cong.) for antimicrobial and antioxidant activity assessment. OpenNano, 3,

56-63.

Saikia, J. P., Paul, S., Konwar, B. K., & Samdarshi, S. K. (2010). Nickel oxide

nanoparticles: a novel antioxidant. Colloids and Surfaces B: Biointerfaces, 78(1),

146-148.

Saravanakkumar, D., Sivaranjani, S., Kaviyarasu, K., Ayeshamariam, A., Ravikumar, B.,

Pandiarajan, S., & Maaza, M. (2018). Synthesis and characterization of ZnO–CuO

nanocomposites powder by modified perfume spray pyrolysis method and its

antimicrobial investigation. Journal of Semiconductors, 39(3), 033001.

Sathishkumar, M., Sneha, K., Won, S. W., Cho, C. W., Kim, S., & Yun, Y. S. (2009).

Cinnamon zeylanicum bark extract and powder mediated green synthesis of nano-

crystalline silver particles and its bactericidal activity. Colloids and Surfaces B:

Biointerfaces, 73(2), 332-338.

Satpathy, S., Patra, A., Ahirwar, B., & Delwar Hussain, M. (2018). Antioxidant and

anticancer activities of green synthesized silver nanoparticles using aqueous extract

of tubers of Pueraria tuberosa. Artificial cells, nanomedicine, and biotechnology,

46(sup3), S71-S85.

Sawai, J. (2003). Quantitative evaluation of antibacterial activities of metallic oxide

powders (ZnO, MgO and CaO) by methods conductimetric assay . Journal of

microbiological, 54(2), 177-182.

References

______________________________________________________________________________________ 163

Shah, M., Fawcett, D., Sharma, S., Tripathy, S. K., & Poinern, G. E. J. (2015). Green

synthesis of metallic nanoparticles via biological entities. Materials, 8(11), 7278-

7308.

Shah, A., & Dobrovolskaia, M. A. (2018). Immunological effects of iron oxide

nanoparticles and iron-based complex drug formulations: therapeutic benefits,

toxicity, mechanistic insights, and translational considerations. Nanomedicine:

Nanotechnology, Biology and Medicine, 14(3), 977-990.

Shah, A., Lutfullah, G., Ahmad, K., Khalil, A. T., & Maaza, M. (2018). Daphne

mucronata-mediated phytosynthesis of silver nanoparticles and their novel

biological applications, compatibility and toxicity studies. Green Chemistry Letters

and Reviews, 11(3), 318-333.

Sharma, J. K., Srivastava, P., Akhtar, M. S., Singh, G., & Ameen, S. (2015). α-Fe 2 O 3

hexagonal cones synthesized from the leaf extract of Azadirachta indica and its

thermal catalytic activity. New Journal of Chemistry, 39(9), 7105-7111.

Sharma, V., Kaushik, S., Pandit, P., Dhull, D., Yadav, J. P., & Kaushik, S. (2019). Green

synthesis of silver nanoparticles from medicinal plants and evaluation of their

antiviral potential against chikungunya virus. Applied microbiology and

biotechnology, 103(2), 881-891.

Shedbalkar, U., Singh, R., Wadhwani, S., Gaidhani, S., & Chopade, B. A. (2014).

Microbial synthesis of gold nanoparticles: current status and future prospects.

Advances in colloid and interface science, 209, 40-48.

Sheldon, R. A. (2012). Fundamentals of green chemistry: efficiency in reaction design.

Chemical Society Reviews, 41(4), 1437-1451.

Sheny, D. S., Mathew, J., & Philip, D. (2011). Phytosynthesis of Au, Ag and Au–Ag

bimetallic nanoparticles using aqueous extract and dried leaf of Anacardium

occidentale. Spectrochimica Acta Part A: Molecular and Biomolecular

Spectroscopy, 79(1), 254-262.

Shi, Y., Pan, X., Li, B., Zhao, M., & Pang, H. (2018). Co3O4 and its composites for high-

performance Li-ion batteries. Chemical Engineering Journal, 343, 427-446.

Siddiqui, M. A., Ahamed, M., Ahmad, J., Khan, M. M., Musarrat, J., Al-Khedhairy, A. A.,

& Alrokayan, S. A. (2012). Nickel oxide nanoparticles induce cytotoxicity,

References

______________________________________________________________________________________ 164

oxidative stress and apoptosis in cultured human cells that is abrogated by the

dietary antioxidant curcumin. Food and chemical toxicology, 50(3-4), 641-647.

Singh, M., Manikandan, S., & Kumaraguru, A. K. (2011). Nanoparticles: a new technology

with wide applications. Research Journal of Nanoscience and Nanotechnology,

1(1), 1-11.

Singh, P., Kim, Y. J., Zhang, D., & Yang, D. C. (2016). Biological synthesis of

nanoparticles from plants and microorganisms. Trends in biotechnology, 34(7),

588-599.

Smolyaninova, V. N., Yost, B., Lahneman, D., Narimanov, E. E., & Smolyaninov, I. I.

(2014). Self-assembled tunable photonic hyper-crystals. Scientific reports, 4, 5706.

Sone, B. T., Fuku, X. G., & Maaza, M. (2016). Physical & electrochemical properties of

green synthesized bunsenite NiO nanoparticles via Callistemon viminalis’ extracts.

Int. J. Electrochem. Sci, 11, 8204-8220.

Song, J. Y., & Kim, B. S. (2009). Rapid biological synthesis of silver nanoparticles using

plant leaf extracts. Bioprocess and biosystems engineering, 32(1), 79.

Sood, R., & Chopra, D. S. (2018). Metal–plant frameworks in nanotechnology: an

overview. Phytomedicine, 50, 148-156.

Soomro, R. A., Ibupoto, Z. H., Abro, M. I., & Willander, M. (2015). Electrochemical

sensing of glucose based on novel hedgehog-like NiO nanostructures. Sensors and

Actuators B: Chemical, 209, 966-974.

Stylianopoulos, T., Wong, C., Bawendi, M. G., Jain, R. K., & Fukumura, D. (2012).

Multistage nanoparticles for improved delivery into tumor tissue. In Methods in

enzymology (Vol. 508, pp. 109-130). Academic Press.

Sudhasree, S., Shakila Banu, A., Brindha, P., & Kurian, G. A. (2014). Synthesis of nickel

nanoparticles by chemical and green route and their comparison in respect to

biological effect and toxicity. Toxicological & Environmental Chemistry, 96(5),

743-754.

Suganthy, N., Ramkumar, V. S., Pugazhendhi, A., Benelli, G., & Archunan, G. (2018).

Biogenic synthesis of gold nanoparticles from Terminalia arjuna bark extract:

assessment of safety aspects and neuroprotective potential via antioxidant,

References

______________________________________________________________________________________ 165

anticholinesterase, and antiamyloidogenic effects. Environmental Science and

Pollution Research, 25(11), 10418-10433.

Tahir, M. N., Natalio, F., Cambaz, M. A., Panthöfer, M., Branscheid, R., Kolb, U., &

Tremel, W. (2013). Controlled synthesis of linear and branched Au@ ZnO hybrid

nanocrystals and their photocatalytic properties. Nanoscale, 5(20), 9944-9949.

Thakor, A. S., & Gambhir, S. S. (2013). Nanooncology: the future of cancer diagnosis and

therapy. CA: a cancer journal for clinicians, 63(6), 395-418.

Thatoi, P., Kerry, R. G., Gouda, S., Das, G., Pramanik, K., Thatoi, H., & Patra, J. K. (2016).

Photo-mediated green synthesis of silver and zinc oxide nanoparticles using

aqueous extracts of two mangrove plant species, Heritiera fomes and Sonneratia

apetala and investigation of their biomedical applications. Journal of

Photochemistry and Photobiology B: Biology, 163, 311-318.

Thema, F. T., Beukes, P., Gurib-Fakim, A., & Maaza, M. (2015). Green synthesis of

Monteponite CdO nanoparticles by Agathosma betulina natural extract. Journal of

Alloys and Compounds, 646, 1043-1048.

Thema, F. T., Manikandan, E., Dhlamini, M. S., & Maaza, M. (2015). Green synthesis of

ZnO nanoparticles via Agathosma betulina natural extract. Materials Letters, 161,

124-127.

Thema, F. T., Manikandan, E., Gurib-Fakim, A., & Maaza, M. (2016). Single phase

Bunsenite NiO nanoparticles green synthesis by Agathosma betulina natural

extract. Journal of alloys and compounds, 657, 655-661.

Thovhogi, N., Diallo, A., Gurib-Fakim, A., & Maaza, M. (2015). Nanoparticles green

synthesis by Hibiscus sabdariffa flower extract: main physical properties. Journal

of Alloys and Compounds, 647, 392-396.

Tran, Q. H., Le, A. T. (2013). Silver nanoparticles: synthesis, properties, toxicology,

applications and perspectives. Advances in Natural Sciences: Nanoscience and

Nanotechnology, 4(3), 033001.

Topwal, M., & Uniyal, S. (2018). Review on Important Ethno-Medicinal Plants of

Uttarakhand. Int. J. Pure App. Biosci, 6, 455-464.

Ul-Haq, I., Ullah, N., Bibi, G., Kanwal, S., Ahmad, M. S., & Mirza, B. (2012). Antioxidant

and cytotoxic activities and phytochemical analysis of Euphorbia wallichii root

References

______________________________________________________________________________________ 166

extract and its fractions. Iranian journal of pharmaceutical research: IJPR, 11(1),

241.

Umar, H., Kavaz, D., & Rizaner, N. (2019). Biosynthesis of zinc oxide nanoparticles using

Albizia lebbeck stem bark, and evaluation of its antimicrobial, antioxidant, and

cytotoxic activities on human breast cancer cell lines. International journal of

nanomedicine, 14, 87.

Varahalarao, V., & DSVGK, K. (2014). Review: Green synthesis of silver and gold

nanoparticles. Middle-East J Sci Res, 19(6), 834-842.

Wahab, R., Siddiqui, M. A., Saquib, Q., Dwivedi, S., Ahmad, J., Musarrat, J., & Shin, H.

S. (2014). ZnO nanoparticles induced oxidative stress and apoptosis in HepG2 and

MCF-7 cancer cells and their antibacterial activity. Colloids and surfaces B:

Biointerfaces, 117, 267-276.

Wang, Z., Fang, C., & Megharaj, M. (2014). Characterization of iron–polyphenol

nanoparticles synthesized by three plant extracts and their fenton oxidation of azo

dye. ACS Sustainable Chemistry & Engineering, 2(4), 1022-1025.

Wang, H., Chen, C., Zhang, Y., Peng, L., Ma, S., Yang, T., & Zhang, J. (2015). In situ

oxidation of carbon-encapsulated cobalt nanocapsules creates highly active cobalt

oxide catalysts for hydrocarbon combustion. Nature communications, 6(1), 1-6.

Waters, B., Saxena, G., Wanggui, Y., Kau, D., Wrigley, S., Stokes, R., & Davies, J. (2002).

Identifying protein kinase inhibitors using an assay based on inhibition of aerial

hyphae formation in Streptomyces. The Journal of antibiotics, 55(4), 407-416.

Wong, T. S., & Schwaneberg, U. (2003). Protein engineering in bioelectrocatalysis.

Current Opinion in Biotechnology, 14(6), 590-596.

Woodford, N., & Livermore, D. M. (2009). Infections caused by Gram-positive bacteria: a

review of the global challenge. Journal of Infection, 59, S4-S16.

Wu, W., Wu, Z., Yu, T., Jiang, C., & Kim, W. S. (2015). Recent progress on magnetic iron

oxide nanoparticles: synthesis, surface functional strategies and biomedical

applications. Science and technology of advanced materials, 16(2), 023501.

Yacamán, M. J., Ascencio, J. A., Liu, H. B., & Gardea-Torresdey, J. (2001). Structure

shape and stability of nanometric sized particles. Journal of Vacuum Science &

References

______________________________________________________________________________________ 167

Technology B: Microelectronics and Nanometer Structures Processing,

Measurement, and Phenomena, 19(4), 1091-1103.

Yadav, R. S., Mishra, P., & Pandey, A. C. (2008). Growth mechanism and optical property

of ZnO nanoparticles synthesized by sonochemical method. Ultrasonics

sonochemistry, 15(5), 863-868.

Yadav, V. K., & Fulekar, M. H. (2018). Biogenic synthesis of maghemite nanoparticles (γ-

Fe2O3) using Tridax leaf extract and its application for removal of fly ash heavy

metals (Pb, Cd). Materials Today: Proceedings, 5(9), 20704-20710.

Yang, Y., Zhang, W., Yang, F., Zhou, B., Zeng, D., Zhang, N., & Zhang, X. (2018). Ru

nanoparticles dispersed on magnetic yolk–shell nanoarchitectures with Fe 3 O 4

core and sulfoacid-containing periodic mesoporous organosilica shell as

bifunctional catalysts for direct conversion of cellulose to isosorbide. Nanoscale,

10(5), 2199-2206.

Yao, G., Sebisubi, F. M., Voo, L. Y. C., Ho, C. C., Tan, G. T., & Chang, L. C. (2011).

Citrinin derivatives from the soil filamentous fungus Penicillium sp. H9318.

Journal of the Brazilian Chemical Society, 22(6), 1125-1129.

Yedurkar, S., Maurya, C., & Mahanwar, P. (2016). Biosynthesis of zinc oxide

nanoparticles using ixora coccinea leaf extract—a green approach. Open Journal

of Synthesis Theory and Applications, 5(1), 1-14.

You, H., Yang, S., Ding, B., & Yang, H. (2013). Synthesis of colloidal metal and metal

alloy nanoparticles for electrochemical energy applications. Chemical Society

Reviews, 42(7), 2880-2904.

Yu, Y., Mendoza‐Garcia, A., Ning, B., and Sun, S. (2013). Cobalt‐substituted magnetite

nanoparticles and their assembly into ferrimagnetic nanoparticle arrays. Advanced

Materials, 25(22), 3090-3094.

Yu, C., Tang, J., Liu, X., Ren, X., Zhen, M., & Wang, L. (2019). Green biosynthesis of

silver nanoparticles using Eriobotrya japonica (Thunb.) leaf extract for reductive

catalysis. Materials, 12(1), 189.

Yugandhar, P., Vasavi, T., Shanmugam, B., Devi, P. U. M., Reddy, K. S., & Savithramma,

N. (2019). Biofabrication, characterization and evaluation of photocatalytic dye

References

______________________________________________________________________________________ 168

degradation efficiency of Syzygium alternifolium leaf extract mediated copper

oxide nanoparticles. Materials Research Express, 6(6), 065034.

Yuvakkumar, R., Suresh, J., Nathanael, A. J., Sundrarajan, M., & Hong, S. I. (2014).

Rambutan (Nephelium lappaceum L.) peel extract assisted biomimetic synthesis of

nickel oxide nanocrystals. Materials Letters, 128, 170-174.

Zak, A. K., Razali, R., Majid, W. A., & Darroudi, M. (2011). Synthesis and

characterization of a narrow size distribution of zinc oxide nanoparticles.

International journal of nanomedicine, 6, 1399.

Zhang, X. H., Liu, Y. C., Wang, X. H., Chen, S. J., Wang, G. R., Zhang, J. Y., & Fan, X.

W. (2005). Structural properties and photoluminescence of ZnO nanowalls

prepared by two-step growth with oxygen-plasma-assisted molecular beam epitaxy.

Journal of Physics: Condensed Matter, 17(19), 3035.

Zhang, W., Qiao, X., & Chen, J. (2007). Synthesis of nanosilver colloidal particles in

water/oil microemulsion. Colloids and Surfaces A: Physicochemical and

Engineering Aspects, 299(1-3), 22-28.

Zhang, Y. (2015). Thermal oxidation fabrication of NiO film for optoelectronic devices.

Applied Surface Science, 344, 33-37.

Zhao, Y., Zhao, X., Cheng, Y., Guo, X., & Yuan, W. (2018). Iron oxide nanoparticles-

based vaccine delivery for cancer treatment. Molecular pharmaceutics, 15(5),

1791-1799.

Zohra, T., Ovais, M., Khalil, A. T., Qasim, M., Ayaz, M., & Shinwari, Z. K. (2019).

Extraction optimization, total phenolic, flavonoid contents, HPLC-DAD analysis

and diverse pharmacological evaluations of Dysphania ambrosioides (L.)

Mosyakin & Clemants. Natural product research, 33(1), 136-142.

Zhao, Y., Zhao, X., Cheng, Y., Guo, X., & Yuan, W. (2018). Iron oxide nanoparticles-

based vaccine delivery for cancer treatment. Molecular pharmaceutics, 15(5),

1791-1799.

Zohra, T., Ovais, M., Khalil, A. T., Qasim, M., Ayaz, M., & Shinwari, Z. K. (2019).

Extraction optimization, total phenolic, flavonoid contents, HPLC-DAD analysis

and diverse pharmacological evaluations of Dysphania ambrosioides (L.)

Mosyakin & Clemants. Natural product research, 33(1), 136-142.

Green Synthesis of Multifunctional Metal Oxide ...

Documents

Transcript of Green Synthesis of Multifunctional Metal Oxide ...