GREEN SYNTHESIS AND BIOLOGICAL INVESTIGATION OF

NANOPARTICLES USING FAGONIA CRETICA PLANT

EXTRACT WITH TRANSITION METALS

Master of Philosophy in Chemistry

MUHAMMAD ASHFAQ
2021-GU-2493

Department of Chemistry
Faculty of Science
Ghazi University
Dera Ghazi Khan, Pakistan.

2024
GREEN SYNTHESIS AND BIOLOGICAL INVESTIGATION OF
NANOPARTICLES USING FAGONIA CRETICA PLANT
EXTRACT WITH TRANSITION METALS

A Thesis Submitted in Partial Fulfillment of the Requirement for the

Degree of

Master of Philosophy in Chemistry

MUHAMMAD ASHFAQ
2021-GU-2493

Department of Chemistry
Faculty of Science
Ghazi University
Dera Ghazi Khan, Pakistan

2024

ii
 STUDENT’S DECLARATION
I, Muhammad Ashfaq hereby state that M. Phil. thesis entitled “Green synthesis
and biological investigation of nanoparticles using Fagonia cretica plant
extract with transition metals” is my own work and has not been submitted
previously for any degree from Ghazi University, Dera Ghazi Khan, or elsewhere in
the country/world. If my statement is found incorrect even after graduation, the
university has the right to take action against me as per approved rules and
regulations of the University.

______________________________
Signature of the candidate

iii
 PLAGIARISM UNDERTAKING
I solemnly declare that the research work presented in the thesis entitled
“Green synthesis and biological investigation of nanoparticles using
Fagonia cretica plant extract with transition metals” is solely my research
work however small contribution wherever taken has been duly acknowledged. I
understand the zero-tolerance policy of the HEC and Ghazi University, Dera Ghazi
Khan towards plagiarism. Therefore, as an Author of this thesis, I declare that no
portion of my thesis has been plagiarized and material used as reference is
properly cited.

I undertake that if I am found guilty of any formal plagiarism in the above- titled
thesis before the submission date of the thesis of M. Phil. degree, the University
reserves the right to take action as per approved rules and regulations of the
University. HEC and the University have the right to publish my name on the
HEC/University Website on which names of students are placed who submitted
plagiarized thesis.

Signature of the candidate

iv
 CERTIFICATE OF APPROVAL
This is to certify that the research work presented in this thesis, entitled “Green
synthesis and biological investigation of nanoparticles using Fagonia
cretica plant extract with transition metals” was conducted by Muhammad
Ashfaq under the supervision of Dr. Maqbool Ahmad. This thesis is submitted to
the Department of Chemistry, Ghazi University, Dera Ghazi Khan in partial
fulfillment of the requirements for the Degree of MPhil.

Name of External Examiner Signature and Date

Supervisor
Dr. Maqbool Ahmad Signature and Date
Assistant Professor
Department of Chemistry

Chairperson
Prof. Dr. Nadeem Akhter Signature and Date
Professor
Department of Chemistry

v
 ACKNOWLEDGMENTS
In the name of ALLAH (SWT), the creator, the most kind, the most generous and
praiseworthy of all devotion. All tributes be to immense ALLAH (SWT) who guides
me when I puzzle in darkness of unawareness and clarify our ways. Uncountable
greetings to the source of knowledge, our Holy Prophet MUHAMMAD (S.A.W) who
is a burn up of leadership and awareness for humanity in every field of life.

It is my pleasure to express my bottomless wisdom of gratefulness to my respected

Supervisor, Dr. Maqbool Ahmad for his constant encouragement, friendly
appreciated suggestions, beneficial criticism and competent supervision at every
during my thesis ended me able to complete this thesis. I cannot be able to thanks
him his supervision. I offer my warm gratitude to my parents and my siblings
specially my mother for her endless motivation, support and last but not Ieast I
greatly appreciate my fellows Muhammad Zahid, Hafiz Touqeer Riaz, Ameer
Hamza, Khadija Tul Kubra, kainat Khizer, Usma Akhtar and specially Ayesha
Azam for their encouragement and support to complete my M.Phil. Degree.

I also express gratitude to all my beloved teachers and professors, who encourage
me to be sensible and experienced. All the practical staff of Ghazi University D.G
Khan for their support and direction in our experimental work.

Signature of the candidate

vi
 ABSTRACT
In this study, cobalt oxide (CoO NPs), nickel oxide (NiO NPs), and nickel-copper
bimetallic nanoparticles (NiO/CuO NPs) were successfully synthesized using
Fagonia cretica plant extract. This extract was then mixed with respective metal
salt solutions to facilitate formation of nanoparticles. Characterization by UV-
Visible spectroscopy, SEM, XRD, and FT-IR has indicated the formation and
targeted nanoparticles. Findings from SEM analysis indicated that the cobalt
nanoparticles were of random shape which includes cubic and hexagonal
configurations with an average size of about 45.45 nm. The XRD spectrum for CoO
indicated different peaks at 31.96° and 42.96° (2θ) which correspond to (220) and
(400) planes in CoO respectively, crystallite size being around 16.40 nm. The FT-
IR analysis of cobalt oxide nanoparticles (CoO NPs) identified peaks at 1401 cm⁻¹
and 1395 cm⁻¹ corresponding to C-H bending and C-O stretching, 2023 cm⁻¹ for
isothiocyanate groups, 1532 cm⁻¹ for N-H bending or C=C stretch in aromatic
rings, 1046 cm⁻¹ for C-O stretching in alcohols or glycosides, and 534 cm⁻¹ for Co-
O bonds. For nickel oxide nanoparticles (NiO NPs), the FT-IR analysis showed
peaks at 2167 cm⁻¹ and 2039 cm⁻¹ indicating isothiocyanate, 1555 cm⁻¹
corresponding to amide or aromatic compounds, and 661 cm⁻¹ confirming the
presence of Ni-O bonds. The FT-IR analysis of NiO/CuO NPs revealed peaks at 1559
cm⁻¹ for amides and aromatic compounds, 1404 cm⁻¹ for phenolics, 1137 cm⁻¹
and 1054 cm⁻¹ for alcohols, and 667 cm⁻¹ confirming Ni-O and Cu-O bonds. UV-
Visible spectroscopy further confirmed the synthesis of CoO NPs, as evidenced by
the strong absorption peak at about 550 nm. Antioxidant activities were evaluated
for nanoparticles through DPPH, HRSC, and LARC assays. CoO NPs showed
moderate antioxidant activity with 66.46% in DPPH test, 50.26% in HRSC test and
low activity of 17.45% in LARC test respectively. On the other hand, different levels
of NiO NPs complex activities were observed which were 29.67% (DPPH), 55.9%
(HRSC) and 18.03% (LARC). NiO/CuO NPs exhibited high antioxidative potentials;
84.1%, 53.33%, and 30.36%, which were comparable to the standard in DPPH
assay HRSC assay and LARC assay respectively. CoO- NPs showed significant
antibacterial activity with inhibition zones of 32 mm for S. aureus (84%) and 30
mm for E. coli (83%) compared to ciprofloxacin.

vii
 TABLE OF CONTENTS
STUDENT’S DECLARATION ....................................................... iii

PLAGIARISM UNDERTAKING ....................................................iv

CERTIFICATE OF APPROVAL ...................................................... v

ACKNOWLEDGMENTS .................................................................. vi

ABSTRACT .................................................................................... vii

LIST OF GRAPHS ........................................................................ xiii

LIST OF FIGURES ....................................................................... xiv

LIST OF TABLES............................................................................xv

1 INTRODUCTION.......................................................................1
1.1 Nanotechnology .............................................................................................................. 1

1.1.1 Applications of Nanotechnology in Chemistry ................................................... 4

1.2 Introduction to Nanoparticles in Chemistry ........................................................ 4

1.2.1 Types of NPs ...................................................................................................................... 6

1.2.2 Bimetallic NPs................................................................................................................... 7

1.3 Applications of NPs in Modern Science ................................................................. 8

1.3.1 Enhanced Reactivity and Catalytic Properties .................................................... 8

1.3.2 Unique Optical Properties ........................................................................................... 8

1.3.3 Magnetic Properties ....................................................................................................... 9

1.3.4 Electrical Conductivity and Quantum Effects ...................................................... 9

1.3.5 Biocompatibility and Medical Applications ......................................................... 9

1.3.6 Environmental Impact and Sustainability ......................................................... 10

1.4 Resources of NPs .......................................................................................................... 10

1.4.1 Natural Sources ............................................................................................................. 10

1.4.2 Engineered Sources ..................................................................................................... 11

viii
1.5 Methods of Preparation of NPs ............................................................................... 11

1.5.1 Top-Down Approaches .............................................................................................. 12

1.5.2 Mechanical Milling ....................................................................................................... 12

1.5.3 Lithography .................................................................................................................... 12

1.5.4 Laser Ablation ............................................................................................................... 12

1.5.5 Bottom-Up Approaches ............................................................................................. 13

1.5.6 Chemical Vapor Deposition ...................................................................................... 13

1.5.7 Sol-Gel Method .............................................................................................................. 13

1.5.8 Microemulsion Method.............................................................................................. 13

1.5.9 Green Synthesis ............................................................................................................ 14

1.6 Applications of Green-Synthesized NPs ............................................................... 15

1.7 Role of Plant Extracts in Nanoparticle Synthesis ............................................. 15

1.8 Historical Background and Evolution ................................................................... 16

1.9 Mechanism of Nanoparticle Formation ............................................................... 16

1.10 Advantages of Using Plant Extracts................................................................... 16

1.11 Plant-Mediated Nanoparticle Synthesis .......................................................... 17

1.12 Plants Selected for Synthesis of Metal NPs .................................................... 17

1.13 Overview of Fagonia cretica in Nanotechnology ......................................... 18

1.14 Habitat and Distribution: ...................................................................................... 19

1.14.1 Phytochemical Composition of Fagonia cretica............................................... 19

1.14.2 Role in Green Synthesis of NPs ............................................................................... 19

1.14.3 Applications of Fagonia cretica-Mediated NPs ................................................ 20

1.14.4 Synthesis of Cobalt NPs via Fagonia cretica ...................................................... 21

1.14.5 Synthesis of Nickel NPs via Fagonia cretica ...................................................... 21

1.14.6 Synthesis of Nickel-Copper Bimetallic NPs ....................................................... 21

1.15 Factors Affecting Yield of NPs ............................................................................. 22

1.15.1 Concentration of Fagonia cretica Extract ........................................................... 22

1.15.2 pH of Reaction ............................................................................................................... 22

ix
 1.15.3 Temperature of the Reaction ................................................................................... 22

1.15.4 Reaction Time ................................................................................................................ 23

1.15.5 Concentration of Metal Salts.................................................................................... 23

1.16 Characterization techniques ................................................................................ 23

1.16.1 Ultraviolet–Visible Spectrum Absorption (UV-Vis) ........................................ 23

1.16.2 Principle and Application of UV in Nanoparticle Characterization ......... 24

1.16.3 Interpretation of UV-Vis Spectra ............................................................................ 24

1.16.4 Significance of UV-Visible in Nanoparticle Synthesis .................................... 25

1.17 X-Ray Diffraction (XRD) ......................................................................................... 25

1.18 Application of XRD in Nanoparticle Characterization ............................... 25

1.19 Typical XRD Peaks and Values ............................................................................ 26

1.19.1 FT-IR Spectroscopy...................................................................................................... 27

1.19.2 Principles of FT-IR in Nanoparticle Characterization ................................... 27

1.19.3 Typical FT-IR Spectrum and Peak Assignments for Nanoparticles ......... 27

1.19.4 FT-IR in the Confirmation of Nanoparticle Formation.................................. 28

1.20 Importance of FT-IR in Nanoparticle Surface Functionalization ........... 28

1.21 Scanning Electron Microscopy (SEM) .............................................................. 29

1.22 Aims and Objectives ................................................................................................ 30

2 REVIEW OF LITRATURE ..................................................... 31

2.1 Green synthesis of nanoparticles ........................................................................... 31

2.2 Green synthesis of Cobalt nanoparticles ............................................................. 32

2.3 Green synthesis of Silver Oxide NPs ...................................................................... 33

2.4 Green synthesis of NiO/CuO NPs............................................................................ 34

3 MATERIALS AND METHODS ............................................. 38

3.1 Preservation and Collection of Fagonia cretica ................................................ 38

3.2 Plants Identification .................................................................................................... 38

3.3 Sample Treatment ........................................................................................................ 38

x
 3.4 Chemicals and apparatus .......................................................................................... 39

3.4.1 Chemicals: ....................................................................................................................... 39

3.4.2 Apparatus: ....................................................................................................................... 39

3.5 Procedure for Preparing Plant Extracts............................................................... 40

3.6 Preparation of Salt Solutions ................................................................................... 40

3.7 Green Synthesis of CoO, NiO, NiO/CuO, Nanoparticles .................................. 41

3.8 Characterization of Nanoparticles ......................................................................... 41

3.8.1 UV-Visible Spectroscopy ............................................................................................ 41

3.8.2 FT-IR Spectroscopy...................................................................................................... 42

3.9 Biological Activities of Nanoparticles ................................................................... 42

3.9.1 Antioxidant Properties of Green Synthesized NPs ......................................... 42

3.9.2 DPPH Radical Scavenging Activity ........................................................................ 42

3.9.3 Linoleic Acid Reducing Capacity (LARC) ............................................................ 43

4 RESULTS AND DISCUSSIONS ............................................. 45

4.1 FT-IR of Cobalt Oxide NPs ......................................................................................... 45

4.2 FT-IR of Nickle Oxide Nanoparticles ..................................................................... 47

4.3 FT-IR of Nickle-Copper Bimetallic nanoparticles............................................. 48

4.4 SEM of Cobalt Oxide NPs ........................................................................................... 49

4.5 XRD of Cobalt Oxide NPs ........................................................................................... 50

4.6 UV-Visible Spectrum of Cobalt Oxide NPs ........................................................... 51

4.7 Antioxidant Activities ................................................................................................. 52

4.7.1 DPPH ................................................................................................................................. 52

4.7.2 HRSC .................................................................................................................................. 53

4.7.3 LARC .................................................................................................................................. 54

4.8 Antibacterial Activity of Cobalt Oxide NPs ......................................................... 55

4.8.1 Protocols for Antibacterial Activity ...................................................................... 55

4.8.2 Results for Antibacterial Activity........................................................................... 55

xi
5 CONCLUSION ......................................................................... 57

6 BIBLIOGRAPHY .................................................................... 58

xii
 LIST OF GRAPHS
Graph 1: FT-IR Spectra of Cobalt Oxide NPs ....................................................................... 46
Graph 2: FT-IR of Nickle Oxide NPs ....................................................................................... 47
Graph 3: FT-IR of Nickle-Copper ............................................................................................. 48
Graph 4: SEM CoO NPs................................................................................................................ 49
Graph 5: XRD of Cobalt Oxide NPs ......................................................................................... 50
Graph 6: UV-Visible Spectrum of CoO NPs .......................................................................... 51
Graph 7: DPPH Antioxidant of NPs ........................................................................................ 52
Graph 8: HRSC Antioxidant of NPs ......................................................................................... 53
Graph 9: LARC Antioxidant of NPs ......................................................................................... 54

xiii
 LIST OF FIGURES
Figure 1: Different method for synthesis of NPs .............................................................. 14
Figure 2: Fagonia cretica plant ................................................................................................ 18
Figure 3: Cobalt chloride salt solution ................................................................................. 41
Figure 4: Nickle Acetate salt solution ................................................................................... 41
Figure 5: E.coli Bacteria.…………………………………………………………………………………...54
Figure 6: Staphylococcus aureus .............................................................................................. 56

xiv
 LIST OF TABLES
Table 1: XRD of Cobalt ................................................................................................................ 50
Table 2: DPPH Antioxidant of NPs.......................................................................................... 52
Table 3: HRSC Antioxidant of NPs .......................................................................................... 53
Table 4: LARC Antioxidant of NPs .......................................................................................... 54
Table 5: Inhibition Zones (mm) of Cobalt Oxide NPs and Ciprofloxacin ................ 56

xv
 Chapter 1
1 INTRODUCTION
"Nanoscience" represents a dynamic and rapidly evolving domain of study
that concentrates on entities whose dimensions lie between the scale of the largest
molecular structures and the smallest configurations achievable through
contemporary photolithography techniques. These entities are generally
characterized by dimensions that span a few nanometers to less than 100
nanometers (Dowling, 2004; Lieber, 2003). Traditionally, within the realm of
chemistry, this dimensional range has been associated with phenomena such as
colloids, micelles, polymeric macromolecules, and phase-separated domains in
block copolymers—essentially, comprising extensive molecular structures or
aggregates of molecules.

However, novel types of nanostructures such as semiconductor quantum dots,

silicon nanorods, and buckytubes have attracted a lot of attention from academics
recently. Nanoscience often studies quantum phenomena and the behavior of
electrons and photons in these small structures within the fields of electrical
engineering and physics. Concurrently, the fields of biology and biochemistry
exhibit a profound engagement with nanostructures, as numerous essential
cellular components—such as DNA, viruses, subcellular organelles, and gap
junctions—can be fundamentally characterized as nanoscale entities (Bamford,
2000; Service, 2002).

1.1 Nanotechnology
Nanotechnology constitutes an interdisciplinary domain that has
revolutionized various scientific domains, including chemistry. It includes the
methodical control and modification of matter at the supramolecular, molecular,
and atomic scales, usually in dimensions between 1 and 100 nanometers (Drexler,
1987). The Remarkable properties of materials at this nanoscale have led to
significant advancements in various areas of chemistry, from catalysis to drug
delivery, making nanotechnology an integral part of modern chemical research.

The concept of nanotechnology, although relatively recent in its formal

definition, has roots that trace back several decades. The first hints of nanoscience

1
can be seen in the work of Richard Feynman, a renowned physicist, who in 1959
delivered a formative lecture, he deliberated upon the feasibility of exerting
control over discrete atoms and molecular entities (Feynman, 2018). Feynman’s
vision laid the groundwork for the development of nanotechnology, inspiring
scientists to explore the potential of constructing materials and devices on an
atomic scale.

Although nanotechnology is often considered an emerging science, its

origins date to the 5th century in China and Egypt (Huang et al., 2003). The
Lycurgus Cup, a notable 4th-century artifact, exemplifies early utilization of nano-
gold and silver particles. Because of these nanoparticles, the cup changes color,
appearing green in reflected light and red in transmitted light (Colomban, 2009).
In his groundbreaking work "Experimental Relations of Aurum and Other Metals
to Light," published in 1857, Michael Faraday provided the first scientific proof of
the special qualities of nanoparticles (Faraday, 1857). Eventually, in 1908, Mie
solved Maxwell's equations, which was essential to comprehending the optical
characteristics of nanoparticles since it explained the scattering and absorption
spectra of particles of any size (Mie, 1976). Conceptual framework for
nanotechnology as we understand it today was significantly advanced in the 1980s
when Eric Drexler proposed a term "nanotechnology." Drexler further elaborated
in his book "Engines of Creation: The Coming Era of Nanotechnology," where he
introduced the concept of using molecular machines to assemble matter from the
bottom up (Kateb & Heiss, 2013).

In recent developments, nanostructured polymer films, particularly

organic light-emitting diodes (OLEDs), have revolutionized the display technology
industry. These materials are used in screens for devices such as televisions,
laptops, and smartphones, offering advantages like lightweight construction, low
power consumption, improved picture density, brighter images, wider viewing
angles, and longer lifespans (Okamoto et al., 2004). Noble metal nanoparticles,
especially gold and silver, are crucial for OLED advancements. Additionally,
nanoparticles are highly efficient catalysts in petroleum processing, where their
low size large surface area and chemical reactivity. For instance, gold NPs have

2
very good catalytic properties even at ambient conditions and are able to catalyze
the oxidation of CO at temperatures as low as -76°C.

It is also crucially needed in the fight against environmental degradation.

Cost-effectiveness, high adsorption capacity, and improved stability of iron oxide
NPs allow their use on an industrial scale for the treatment of wastewater(Hu et
al., 2005). These nanoparticles efficiently eliminate a variety of heavy metal ions,
such as Pb2+, Hg2+, Cd2+, and Cu2+, according to laboratory studies (Ambashta &
Sillanpaa, 2010; Kaegi et al., 2011).

With the advancement of colloidal science in the 1970s, the field of

chemistry started to take shape with regard to the study of nanomaterials. Colloids
were some of the first nanostructures to be examined. They are particles scattered
inside a medium whose size ranges from 1 to 1000 nm (Hunter et al., 2008).
According to Hunter et al., 2008, during this period, a more general appreciation
of the behavior of materials at the nanoscale especially relating to their surface
chemistry and interaction with surrounding molecules started to emanate.

In 1985, Harold Kroto, Robert Curl, and Richard Smalley discovered a very
important advance in nano chemistry-fullerenes (Kroto et al., 1985). The
fullerenes are a class of spherically shaped carbon allotropes, taking into account
a new form of carbon nanomaterial which leads to the synthesis of carbon
nanotubes and graphene. These findings opened new lines of inquiry, especially in
the production of novel nanostructures with distinct chemical, mechanical, and
electrical properties.

Nanotechnology as a field rapidly advanced during the 1990s with unique

techniques for the synthesis and characterization of nanoparticles. The
introduction of methods, for instance, sol-gel processing, chemical vapor
deposition and laser ablation motivated chemists to produce NPs with tailored
control over size, form and composition (Rao et al., 2006). These techniques
motivated creation of materials with tailored properties, which were previously
unattainable through conventional methods.

3
1.1.1 Applications of Nanotechnology in Chemistry
Nanotechnology significantly influences multiple chemistry disciplines. In
catalysis, nanoparticles enhance reaction kinetics and selectivity because of their
greater surface area-to-volume ratio and distinctive surface characteristics
(Astruc, 2008). Nanotechnology has transformed pharmaceutical drug delivery
systems via facilitating creation of nanoparticles that selectively target diseased
cells, thereby reducing side effects and enhancing therapeutic effectiveness
(Farokhzad & Langer, 2009).

Nanotechnology has advanced environmental chemistry, especially in

creating nanomaterials for water treatment and pollution management.
Photocatalytic nanoparticles like titanium dioxide and zinc oxide are utilized to
effectively degrade pollutants, providing a sustainable method for environmental
remediation (Chen & Mao, 2007).

1.2 Introduction to Nanoparticles in Chemistry

Nanoparticles have at least one dimension in nanoscale range, typically
between one and hundred nanometers. Its nanoscale dimension has special
chemical, biological, and physical characteristics set nanoparticles apart from
their larger counterparts. Due to these distinct characteristics, nanoparticles have
become a valuable point in almost every field of science, especially in chemistry,
where their applications range from catalysis and materials science to medicinal
science (Khan et al., 2019).

The concept of nanoparticles is not new; their utilization, albeit

unintentionally, dates back centuries. However, the scientific exploration of
nanoparticles is relatively modern, emerging as a distinct field within the broader
context of nanotechnology in the late 20th century. Nanoparticles have different
materials like metals, semiconductors, polymers, and ceramics. Each exhibiting
different properties which depend upon their composition and size (Freestone et
al., 2007).

One of basic reasons, nanoparticles are so versatile just because of increased

surface area-to-volume ratio they exhibit as their size decreases. This increased
surface area increased the reactivity of nanoparticles, and made them excellent

4
candidates for applications in catalysis and chemical reactions.(Roduner, 2006)
Additionally, quantum effects become significant at the nanoscale, leading to
properties such as quantum confinement in semiconductor nanoparticles, which
can alter the material's optical and electronic behavior (Alivisatos, 1996b).

Nanoparticles also show unique optical properties due to their interaction

with light, which depends upon size. For example, characteristics set nanoparticles
apart from their larger counterparts, due to a process called surface plasmon
resonance, gold nanoparticles can appear red or blue depending on their size
(SPR) (El-Sayed, 2001). This property has been extensively utilized in applications
ranging from medical diagnostics to environmental sensing.

In the field of chemistry, nanoparticles have opened new ways of research

and application. The high reactivity and selectivity of these species has
transformed several areas, including organic synthesis (Daniel & Astruc, 2004). In
addition, smaller nanoparticles sizes endow them to be the transmembrane
carrier for drug delivery systems as well as new medical imaging technology (Peer
et al., 2020).

NPs have been synthesized via many methods widely classified into top
down and bottom up. Top-down processes are those in which the particle forms
starting by massive materials, initially reduced to nanoparticles followed usually
milling or lithographic techniques (Lu et al., 2007). Bottom-up methods require
the assembly of nanoparticles from atoms or molecules, typically by chemical
reactions (Tiwari et al., 2012). The choice of synthesis method depends on the size,
shape and composition that is desired for the nanoparticles.

While research had continued to forge ahead, the capability of

understanding and manipulating nanoparticles has become more refined; design
and preparation of nanocomposites, nano catalysts, and other value-added
materials with improved performance through the incorporation of nanoparticles
became possible (Wang & Song, 2006). The cross-disciplinary nature in the
nanoparticle research area of chemistry has created collaborations across
boundaries into vast areas of scientific disciplines, further extending both the
applications and benefits of these incredible materials.

5
1.2.1 Types of NPs
It is only because of their small size and unique properties that
nanoparticles can also be categorized into different types: composition, structure,
and function. Each type of nanoparticle has a set of characteristics which make it
suitable for specific applications in its relevant field, such as medicine, electronics,
catalysis, and environmental science.

1.2.1.1Metal NPs
Metal NPs are the most studied and used kind of NP. The particles generally
comprise metals such as Au, Pt, and Cu. Generally speaking, they exhibit peculiar
features like high surface area, catalytic activity, and plasmonic behavior. Gold NPs
are one of the most known in biomedical applications because of their excellent
biocompatibility and SPR features, which enable sensitive detection methods and
controlled drug delivery systems (Milan et al., 2022). Because of their well-known
antibacterial properties, the application of silver nanoparticles is becoming very
popular in medical devices, coatings, and water treatment (Tehri et al., 2022).

1.2.1.2 Metal Oxide NPs

Metal oxide NPs consists of particles like zinc oxide, titanium dioxide and
iron oxide, these NPs are known for their photocatalytic, magnetic, and electronic
properties. Among these, the most common applications of Titanium dioxide NPs
can be found in sunscreens and paints, as photocatalysts in environmental
cleaning processes for their abilities to generate reactive oxygen species upon UV
light irradiation (Moma & Baloyi, 2019). Simultaneously, zinc oxide NPs have the
same fields of applications in the development of cosmetics with UV-blocking
properties, and they are used in antimicrobial treatments (Schneider & Lim,
2019). Iron oxide NPs are valuable because of their magnetic properties in MRI
and drug delivery (Laurent et al., 2010).

1.2.1.3 Semiconductor NPs

Semiconductor NPs are also referred to as quantum dots. They comprise
nanocrystals; these are made up of semiconductor materials such as CdSe, CdS,
and ZnS. Quantum confinement effects in these NPs tend to exhibit size-dependent
optical and electronic properties, hence being very valuable in optical and

6
electronic applications like optoelectronics, photovoltaics, and even in biological
imaging (Yu & Wang, 2020). Quantum dots have tunable fluorescence, depending
on a change in NP size, allowing for highly sensitive and selective sensor
construction (Yan et al., 2019).

Carbon-based NPs include carbon nanotubes, graphene, and carbon dots.

Fullerenes are spherical molecules comprising solely carbon atoms, a
representative example being C₆₀. They exhibit specific electronic properties,
which render them important in drug delivery and modern science (Kazemzadeh
& Mozafari, 2019). Graphene is a one-atom-thick sheet composed of carbon atoms
arranged in a hexagonal lattice. It attracted great attention in its single-layer form
due to its exceptional electrical conductivity, transparency, and mechanical
flexibility (Trivedi et al., 2019).

Polymeric NPs are nanoparticles that are made either of natural or artificial
polymers. Their broad fields of application include drug delivery systems, tissue
engineering, and as vectors in gene therapy. In these ways, such NPs can be
engineered to possess specific surface characteristics, size, and even rates of
degradation that allow for regulated drug release and targeted therapy (Beach et
al., 2024). The excellent biocompatibility of polymeric NPs, such as polylactic-co-
glycolic acid and chitosan, has involved them in extensive study for their ability to
encapsulate a wide range of therapeutic agents-from small molecules to proteins
and nucleic acids (Varga et al., 2019).

1.2.2 Bimetallic NPs

Bimetallic NPs consist of two different metals alloyed together. These would
possess improved properties over their monometallic counterparts. Such NPs
could be prepared through various structures such as core-shell, alloyed, and
heterostructures, allowing fine-tuning of their catalytic, optical, and magnetic
properties. (Idris & Roy, 2023). Among them, the synergic effects in bimetallic NPs,
such as Au-Ag, Au-Pt, and NiO/CuO NPs, will hence lead to enhanced catalytic
performance and stability and selectivity in chemical reactions (Scala et al., 2022).
For example, the catalytic behavior of Au-Pt bimetallic NPs has been reported to
exhibit higher catalytic performances in fuel cells compared to pure NPs (Beltran-
Gastelum et al., 2019).

7
 Accordingly, because of their distinctive electrical and surface properties,
nickel-copper bimetallic Oxide nanoparticles are receiving attention with regard
to environmental remediation and catalytic applications (Tang et al., 2023).

1.3 Applications of NPs in Modern Science

NPs have become an important component in many scientific fields due to
the exceptional and often novel properties that arise at nanoscale. Such qualities
differ from complex material qualities, making NPs important in modern science
and technology. Their small size confers value on their unique physical, chemical,
and biological properties, which are important in a number of applications thanks
to quantum effects.

1.3.1 Enhanced Reactivity and Catalytic Properties

One of the most characteristic features of NPs is their increased reactivity,
mainly attributed to their large surface area-to-volume ratio. if particle size
decreases, the proportion of atoms on the surface increases relative to those inside
the particle. It can lead to a greater number of active sites available for chemical
reactions, making NPs highly effective as catalysts. For instance, gold NPs exhibit
remarkable catalytic activity in reactions such as oxidation of carbon monoxide
(CO) and the reduction of nitro compounds (Haruta, 1997).

This catalytic property is leveraged in both industrial processes and

environmental applications, where NPs help in reducing harmful emissions and
transforming pollutants into less toxic substances.

1.3.2 Unique Optical Properties

NPs, particularly metal NPs for instance Ag and Au exhibit unique optical
properties because of their surface SPR. This situation occurs when conduction
electrons on NPs surface oscillate in response to light, causing strong absorption
and scattering of light at specific wavelengths. This is because one can change the
color of NPs by merely varying their sizes, shapes, or surrounding medium. Some
of these properties are in use within applications ranging from biomedical
imaging-surface-enhanced Raman scattering, where the NPs act as contrast
agents-to the development of biosensors, sensitive enough to detect ultra-low
concentrations of biological molecules (Kneipp et al., 2002). Thanks to SPR, hence

8
making NPs relevant tools in diagnostics and therapeutic monitoring (Sharma et
al., 2012).

1.3.3 Magnetic Properties

NPs are made up of magnetic materials such as FeO, which can show
superparamagnetic properties. This is not seen to occur in the bulk material. At
nano, these particles are capable of being magnetized by the external magnetic
field but do not retain any magnetism upon removal of the field. Thus, this
property is very useful in biomedical applications like MRI and drug targeting,
where magnetic NPs may be guided externally, with the help of a magnetic field, to
precise locations inside the body (Pankhurst et al., 2003). In vivo control of the
movement and concentration of NPs revolutionizes methodologies of drug
delivery in enabling the treatment of diseases such as cancer right at where they
emanate from, hence reducing side effects and enhancing the therapeutic outcome
(Laurent et al., 2008).

1.3.4 Electrical Conductivity and Quantum Effects

Quantized behavior in materials due to quantum effects can alter the
electrical conductivity of a material at the nanoscale. Thus, NPs are highly
significant in the development of modern electronics. As a specific example,
semiconductor NPs exhibit a size-dependent electronic property that makes these
NPs useful in many optoelectronic devices, including LEDs and solar cells (Talapin
et al., 2010). Tunability of band gap makes quantum dots versatile in absorbing
and emitting light across different wavelengths, which is why they are extensively
used in next-generation display technologies and photovoltaic devices (Alivisatos,
1996). NPs also highly contribute to the miniaturization of electronic components,
hence opening a whole new world toward the development of more compact and
efficient devices.

1.3.5 Biocompatibility and Medical Applications

NPs are increasingly being integrated into the medical field because of their
biocompatibility and ability to collaborate with biological systems at the
molecular level. For instance, NPs widely facilitate in drug delivery systems. In
which, they can encapsulate therapeutic agents, protect them from degradation,

9
and release them at targeted site in the body (Panyam & Labhasetwar, 2003). This
targeted delivery increases both the power of the drug and decreases systemic
toxicity. NPs are also applied in tissue engineering, gene therapy,, and serve as
vectors for vaccines (Park, 2007).

1.3.6 Environmental Impact and Sustainability

NPs also contribute to sustainable development. Due to their high reactivity
and selectivity, they find applications in various environmental remediation
processes, ranging from the removal of contaminants from water and air to
complicated processes of pollution remediation. For example, titanium dioxide
NPs are among the most popular photocatalysts used for the degradation of
organic pollutants in water, hence finding an essential place in different water
purification systems (Chen & Mao, 2007). The capability of NPs to adsorb heavy
metals and other contaminants from the environment has imposed their value for
mitigation of pollution and restoration of the ecological balance (Rao et al., 2006).

Indeed, the importance of NPs in modern science is undeniable, and they

continue to open new frontiers in many important fields like catalysis, electronics,
medicine, and environmental science. Unusual properties at the nanoscale have
empowered innovations that were unimaginable a few decades ago, marking an
important contribution to the development of technology and sustainability.

1.4 Resources of NPs

NPs can be procured from various resources. Each resource thus bears
unique characteristics and applications. Knowledge of these resources is the key
to the selection of appropriate NPs for intended chemical processes since it
imposes effects on properties such as reactivity, size, shape, and surface area.
Generally speaking, sources of NPs may be grouped into three broad categories:
natural resources, engineered resources, and incidental resources.

1.4.1 Natural Sources

Natural sources are the ones that appear in the environment without any
human intervention. Such NPs appear in a number of natural processes and have
been present in the environment since millennia. The most common are volcanic
ashes, mineral deposits, and biogenic particles produced by microorganisms.

10
 Similarly, mineral deposits are naturally weathered to release NPs in the soil
and water. For example, mineral-based NPs, such as clays, oxides and silicates, play
a vital role in geochemical processes and are thus generally researched for
catalytic properties (Horwell & Baxter, 2006). Microorganisms e.g. bacteria, algae
and fungi are capable of producing NPs through biological processes. For example,
magneto tactic bacteria synthesize magnetite (Fe3O4) NPs within their cells that
they employ for navigation (Hochella Jr et al., 2008).

These biogenic NPs have been harnessed for applications in MRI and
environmental remediation among (Niemeyer, 2001).

1.4.2 Engineered Sources

In general, NPs are prepared and processed either chemically or physically for
definite purposes. These kinds of NPs have widespread use in medicine,
electronics, catalysis, among others.

Most of the common methods of the production of engineered NPs contain

chemical syntheses, including techniques such as the sol-gel process, CVD, and wet
chemical methods. As an example, Au NPs can be synthesized by the Turkevich
method, in which gold salts are reduced in an aqueous solution by citrate to yield
Au colloids with controllable sizes and shapes. (Turkevich et al., 1951). Similarly,
silver NPs are often produced via chemical reduction methods, and Ag ions are
reduced to metallic silver by reducing agents for instance, sodium borohydride or
plant extracts (Iravani, 2011).

Physical methods for nanoparticle synthesis include techniques like laser

ablation, ball milling, and PVD. These methods typically involve breaking down
bulk materials into NPs through mechanical or thermal processes. For example,
laser ablation can be used to generate NPs from solid target material in liquid
medium, offering precise control over particle’s geometry. (Chanana & Liz-Marzan,
2012).

1.5 Methods of Preparation of NPs

Preparation of NPs is an important aspect of nanotechnology, as the method
of preparation can significantly influence size, shape, surface properties, and
ultimately functionality of NPs. With passage of time and advancement in modern

11
science, Different methods have been developed to produce NPs with precise
control over these characteristics. Methods of nanoparticle preparation can be
widely classified into two categories: top down and bottom-up techniques.

1.5.1 Top-Down Approaches

The top-down approach mainly refers to the material distribution of
complex materials into nanoscale particles. In general, these methods represent
physical and mechanical methods of bulk material reduction through different
techniques.

1.5.2 Mechanical Milling

Mechanical milling, or most commonly ball milling, is one of the top-down
production methods for NPs. In this method, bulk materials are placed in a
cylindrical rotating chamber with grinding media. The complex material breaks
down to small particles by the mechanical energy imparted from the rotating
motion. This method has been widely applied in the production of NPs, both
metals and ceramics (Suryanarayana & Al-Aqeeli, 2013).

In this regard, the particles produced by mechanical milling are often further
processed to fit into the size and shape criterion (Ramezanalizadeh et al., 2016).

1.5.3 Lithography
Another top-down approach to the generation of nanostructures and
nanopatterning is lithography, available in two flavors: photolithography and
electron-beam lithography. In this technique, light or electron beams are used to
etch a pattern onto a substrate that may subsequently be used to realize the NPs.
This allows good control over NP size and shape; it is fairly complicated and thus
generally an expensive process, usually used on only small-scale productions
(Vogel et al., 2012).

1.5.4 Laser Ablation

One of the top-down approaches is laser ablation, in which the high-energy
laser beam focused on a bulk material causes its vaporization for the formation of
NPs. The vaporized material condenses and forms NPs within the controlled
environments like gas or liquid medium. Laser ablation technique is known for

12
giving very pure NPs with narrow size distribution. It is also particularly effective
in the synthesis of metal and oxide NPs (Semaltianos, 2010).

1.5.5 Bottom-Up Approaches

The bottom-up techniques are processes in which the assembly takes place
from atoms or molecules to make NPs. The chemical and biological procedures in
many cases offer the possibility of increased control over the chemical
composition, crystallinity, and morphology of the NPs.

1.5.6 Chemical Vapor Deposition

CVD is among the most usable bottom-up processes of the synthesis of NPs.
In the CVD process, volatile precursors are introduced inside a reaction chamber;
here, the precursors undergo a chemical reaction to generate solid NPs. The
conditions in the reaction chamber can be finely adjusted to regulate the size and
control the morphology of the NPs. CVD is mainly used for producing NPs of
carbon, silicon, and a number of other oxides (Manawi et al., 2018).

1.5.7 Sol-Gel Method

The sol-gel technique is a versatile bottom-up method for the synthesis of
nanoparticles, especially oxides. The process consists in hydrolysis and
polycondensation of the solution containing metal precursors into a gel-like
network. After drying the gel and calcination, one obtains NPs. The main
advantage of the method of sol-gel is that by this method of synthesis of NPs, it
allows control over size, shape, and composition at rather low temperatures. It
finds wide applications in the synthesis of nanoparticles of metal oxides such as
Tio₂, Sio₂, and ZnO (Brinker & Scherer, 2013).

1.5.8 Microemulsion Method

In the microemulsion method, the synthesis of NPs was done via a bottom-
up approach in which the thermodynamically stable mixture of oil, water, and
surfactants was used. Within the emulsion microdroplets, the reactants remain
confined, which in turn allows the formation of NPs. Controlling the size of the
microdroplets allows controlling the size of the NPs. This is one of the most
popular techniques for generating metal and metal oxide NPs with uniform
distribution (Zielinska-Jurek et al., 2012).

13
1.5.9 Green Synthesis
Green synthesis is an environment-friendly bottom-up technique,
representing the use of biological systems such as plant extracts, bacteria, and
fungi for the reduction of metal ions and yielding of NPs. It has been gaining
popularity due to its eco-friendly nature that brings out biocompatible NPs.
Phytochemicals present in extracts of plants serve both as reducing and stabilizing
agents, yield the formation of NPs having new properties. It is the suitable process
for the green synthesis of metal and metal oxide NPs, including Co, Ag, Au, and Zn
(Mittal et al., 2013).

Figure 1: Different method for synthesis of NPs

Mechanisms of Green Synthesis

In the green synthesis, the biological material plays an important role for
the reduction of metal ions to form NPs. For example, plant extracts will contain
several kinds of phytochemicals like alkaloids, flavonoids, phenolic acids, and
terpenoids that may play the roles of reducing agents. These phytochemicals
donate electrons to metal ions and reduce them to their zero-valent state and this

14
is followed by nucleation and growth of NPs (Raghunandan et al., 2011; Rai et al.,
2009).

1.5.9.1 Advantages of Green Synthesis

Green synthesis possesses quite a few advantages compared to conventional
methods. First of all, this is an environment-friendly method because toxic
chemicals, which usually are applied in classic nanoparticle synthesis, can be
avoided. This is also a cost-effective process as it generally avails easily available
natural resources like plant extracts. Green synthesis is also a scalable,
straightforward, and therefore suitable method for large-scale production
processes (Ahmed et al., 2016; Kuppusamy et al., 2016). Besides this, NPs
synthesized through green techniques have a greater degree of biocompatibility
and hence are primarily used in biomedical applications for diagnostics, drug
delivery, and imaging (Bhattacharya & Mukherjee, 2008).

1.6 Applications of Green-Synthesized NPs

NPs synthesized through green synthesis have enormous applications in
several sectors due to their unique properties and biocompatibility. In biomedical
fields, these NPs facilitate routes of drug delivery systems where in their smaller
size and surface functionalization potential allow for targeted drug delivery with
minimal side effects (Mody et al., 2010). They are also used in antimicrobial
coatings, cancer therapies, and other imaging modalities. Applications of green-
synthesized NPs also involve environmental remediation, including treatment of
waste water and removal of heavy metals from contaminated environments
(Nasrollahzadeh & Sajjadi, 2021).

However, there are certain disadvantages or challenges regarding green

synthesis. One such disadvantage is the control over size, shape, and distribution
of NPs. It is expected that biological materials, such as plant extracts, may
introduce variability that could result in inconsistency in nanoparticle synthesis
(Gade et al., 2014).

1.7 Role of Plant Extracts in Nanoparticle Synthesis

The synthesis of NPs using plant extracts has recently gained considerable
attention due to their eco-friendly, cost-effective, and sustainable potential. This

15
green synthesis of NPs is in sharp contrast to the conventional physical and
chemical routes, which normally involve the use of toxic chemicals, high energy
consumption, and costly equipment.

1.8 Historical Background and Evolution

The use of plant extracts for the synthesis of NPs emanates from ancient
practices that used plant-based preparations in medicine. However, modern
applications of plant extracts in the synthesis of nanoparticles started to emerge
in the early 2000s, when scientists were still trying to look for less toxic ways of
synthesizing nanoparticles (Iravani, 2011). This method is rich in biodiversity of
plants that can execute a wide variety of secondary metabolites which may
decrease metal ions forming NPs.

1.9 Mechanism of Nanoparticle Formation

In plant extracts, nanoparticle formation is accomplished by the reduction
of metal ions into their relevant NPs by the action of phytochemicals contained
within. Such phytochemicals, which include flavonoids, alkaloids, terpenoids,
phenols, and proteins, generally act as reducing and stabilizing agents (Mittal et
al., 2013). The synthesis of Au and Ag NPs, for instance, follows such mechanisms
where the phytochemicals reduce the metal ions by donating electrons to them
toward their elemental state and cap the NPs to prevent aggregation (Ahmed et al.,
2016).

1.10 Advantages of Using Plant Extracts

The use of plant extract in synthesizing nanoparticles has a number of
advantages since the process is eco-friendly and eliminates hazardous chemicals.
Hence, it is an environmentally benign technique (Kuppusamy et al., 2016).

Besides, NPs synthesized using plant extracts show more biocompatibility,

making them suitable for biomedical applications. (Singh et al., 2016). Another
advantage is scalability: the plant-mediated synthesis method can be easily scaled
up and hence can be industrially viable (Khalil et al., 2014).

16
1.11 Plant-Mediated Nanoparticle Synthesis
In fact, there are various studies that reported successful preparations of
NPs using different plant extracts. For example, synthesis of silver NPs using the
leaf extract of Azadirachta indica (Neem) has been widely reported, reflecting an
efficient role of flavonoids and terpenoids in the reduction process. Similarly,
extracts of Aloe vera have been tried for the synthesis of gold NPs, and reports
show that polysaccharides in Aloe vera play a crucial role in the reduction process
of gold ions to Au NPs (Nallal et al., 2022).

Despite the promising aspects of plant-mediated nanoparticle synthesis,

several challenges are faced. Among them, a composition variability in plant
extracts influences reproducibility and uniformity in the NPs formation (Chauhan
et al., 2021). Also, the extraction process may be complicated and requires
optimization in order to ensure the production of high-quality NPs consistently.

Moving forward, the research has focused on unfolding the precise

mechanism of how a particular phytochemical influences nanoparticle synthesis.
This will be key to keeping protocols and enhancing scalability for this green
synthesis approach (Kharissova et al., 2013).

1.12 Plants Selected for Synthesis of Metal NPs

Medicinal plants are highly reputed for their abundant phytochemical
content that is environmentally friendly. Such natural occurring compounds,
including amino acids, enzymes, polysaccharides, and vitamins, among others,
have been proven to possess substantial medicinal properties. Such biomolecules
have been proved in studies to play a very vital role in the bio reduction and
stabilization of NPs. This stability and metal ion reduction are solely linked to the
presence of polyols like terpenoids, polysaccharides, and flavonoids in plant
extracts.

Phenolic compounds take center stage in such plants, especially due to the
attachment of hydroxyl groups to aromatic hydrocarbon structures, which tends
to make them quite strong antioxidants. Such phenolic compounds could
deactivate metal ions to form chelates that suppress oxidation processes with
great effectiveness. It is thus expected that those plants which contain a high

17
amount of phenolic compounds could act as excellent precursors in NPs synthesis
(Archana et al., 2017; Lata, 2018).

The biomolecules in plants facilitate the synthesis of NPs by containing

elements that promote such synthesis. The biomolecules also work in
coordination with one another to elevate the whole synthesis process. Such
phytochemicals can fabricate NPs of consistent size without the formation of any
unwanted cluster (Lopes & Courrol, 2018).

1.13 Overview of Fagonia cretica in Nanotechnology

Fagonia cretica is an herbaceous plant, which has several medicinal
properties. It is traditionally used in a number of folk medicines for the treatment
of various diseases, including fever, inflammation, and even cancer. Due to its
unique properties, this plant in recent years has gained great interest in the field
of nanotechnology, especially for green synthesis of NPs.

Figure 2: Fagonia cretica plant

Botanical Characteristics:

• Family: Zygophyllaceae

• Genus: Fagonia

18
 • Species: Fagonia cretica

• Common Names: Dhamasa, Fagonbush, Virgin’s Mantle, Rattlesnake, Wild

Rattle.

• Plant Type: Perennial herb

• Growth Habit: The plant typically grows as a small, thorny shrub with
spreading branches.

• Leaves: The leaves are trifoliate (three leaflets), with small, narrow, and
elongated leaflets.

• Flowers: The plant produces small, star-shaped, purple-pink flowers. The

flowers are usually solitary and have five petals.

• Fruits: The fruit is a small, capsule-like structure containing seeds.

1.14 Habitat and Distribution:

Fagonia cretica is native to arid and semi-arid areas across Africa, Asia-this
is more specifically the Indian subcontinent-and the Mediterranean. It usually
grows on stony, sandy soils in deserts or dry places.

1.14.1 Phytochemical Composition of Fagonia cretica

This efficacy of Fagonia cretica in the synthesis of nanoparticles can be
attributed to its rich phytochemical composition. The plant is known to contain
numerous bioactive compounds including flavonoids, saponins, alkaloids, and
tannins (Tiwari & Rana, 2015) These compounds not only contribute to the
therapeutic properties of the plant but also play a crucial role in the reduction and
stabilization of metallic ions during nanoparticle synthesis. More importantly,
flavonoids and saponins from plants may reportedly act as reducing agents to
reduce metal salts into NPs, while tannins and alkaloids cap and stabilize them,
thus avoiding their aggregation (Hossain et al., 2000).

1.14.2 Role in Green Synthesis of NPs

The term green synthesis in nanotechnology refers to the utilization of
materials and methods that do not harm the environment in the production of NPs.
Fagonia cretica is an ideal candidate since it is found in plenty and contains several

19
kinds of phytochemicals that could be used in the synthesis of NPs. Various studies
document that the aqueous extract of Fagonia cretica can accomplish efficient
reduction of Ag, Au, and Zn metal ions into their respective NPs (Iravani, 2011).
This method of synthesis is cost-effective but also ecologically benign because it
avoids the use of hazardous chemicals normally used in conventional methods of
nanoparticle synthesis.

Another great advantage of using Fagonia cretica in the synthesis of

nanotechnology refers to the biocompatibility of the resulting NPs. Several studies
have indicated that plant extract-based synthesis, involving Fagonia cretica,
results in less toxic NPs compared to those synthesized via chemical routes.
(Zulfiqar et al., 2019). This makes them really helpful for biomedical applications-
say, for drug delivery-where the essence of minimum cytotoxicity is highly needed.

1.14.3 Applications of Fagonia cretica-Mediated NPs

The NPs synthesized using Fagonia cretica have always shown encouraging
results for various applications. As an example, the silver NPs synthesized by using
this plant exhibited strong activities of antibacterial and antifungal nature, thus
recommending their use in antimicrobial paints and wound dressings. Similarly,
the gold nanoparticles prepared through Fagonia cretica have also been explored
for their applications in the treatment of cancer because these can absorb light at
a near-infrared region, thus being helpful for photo-thermal therapy (Huang et al.,
2007).

The photocatalytic properties of Fagonia cretica mediated zinc oxide NPs

have also been explored. The NPs have been used in the degradation of organic
pollutants in water, showing promise in environmental remediation applications.
The fact that Fagonia cretica can synthesize various types of metal NPs makes it a
very vital contributor toward the development of green nanotechnology (Kiani et
al., 2022).

While Fagonia cretica shows promising application in nanotechnology,

there is also additional difficulties. The most important one is variation of size and
shape of nanoparticles, which might arise due to different plant extracts at
different places and on different conditions of harvestings of plants.

20
Standardization of the extraction process and identification of key phytochemicals
responsible for nanoparticle synthesis may minimize such variation.

Fagonia cretica has the potential to be integrated into large-scale

production methods for the production of nanoparticles and hence to open ways
to more environmental-friendly and economical nanotechnological applications.
Further studies are required to establish the full capacity of this plant in
synthesizing a wider range of nanoparticles, as well as finding the underlying
mechanism involved in the synthesis process.

1.14.4 Synthesis of Cobalt NPs via Fagonia cretica

Cobalt NPs synthesized using Fagonia cretica have shown promising results
in different applications because of their magnetic and catalytic properties. The
plant’s bioactive compounds act as both reducing and capping agents, converting
cobalt ions into stable cobalt NPs. These NPs exhibit remarkable magnetic
behavior, making them suitable for applications in data storage, MRI, and catalysis
(Akhlaghi et al., 2020). This synthesis process is not only environmentally friendly
but also produces NPs with high purity and stability.

1.14.5 Synthesis of Nickel NPs via Fagonia cretica

Nickel NPs are known for their great catalytic activities and electrical
conducting properties. During the synthesis using Fagonia cretica, phytochemicals
of the plant reduce the nickel ions into NiO NPs. They were further stabilized
through a capping effect of the phytochemical compounds. These NiO NPs have
been applied in hydrogenation reactions and as catalysts in various organic
transformations. The electrical property makes them useful in sensor and battery
developments (Jaji et al., 2020).

1.14.6 Synthesis of Nickel-Copper Bimetallic NPs

Most notably, among all the bimetals, nickel and copper have gained much
attention due to the fact that they exhibit catalytic and antimicrobial activities
higher than that of their monometallic counterparts. In Fagonia cretica-mediated
synthesis of nickel-copper bimetallic NPs, both metals are reduced using the
phytochemicals present in the plant. Applications of such bimetallic NPs indicated
better performances of catalysis, especially in reactions involving hydrogenation

21
of organic compounds and also as an antimicrobial agent. Synergy between nickel
and copper in these NPs has been observed by Ismail et al., which brings out
properties not existing for individual metal nanoparticles (Ismail et al., 2018).

1.15 Factors Affecting Yield of NPs

Different factors may be involved in the green synthesis of NPs using Fagonia
cretica extract. In order to obtain the best yield of NPs, such factors should be very
carefully optimized. Main factors involve plant extract concentration, pH,
temperature, reaction time, and the concentration of salts of metals involved in the
synthesis process.

1.15.1 Concentration of Fagonia cretica Extract

Besides, the extract concentration of Fagonia cretica is another important
factor that has critical relevance with the yield of NPs. Extract Phyto-constituents
play a double role of acting as reducing agents in the conversion of metal ions into
NPs and capping agents themselves. The extract concentration should be such
that the active molecules will be enough in number to reduce the metal ions
effectively and stabilize the formed NPs. It has also been seen from studies that
an increase in the extract concentration generally results in some increase in the
yield of nanoparticles up to a particular value beyond which the nanoparticles
may start to saturate and thereby show no further increase or even begin to
decrease. (Kalimuthu et al., 2008).

1.15.2 pH of Reaction
pH of the reaction medium is another critical factor that affects the size,
shape, and yield of NPs. The pH would influence the ionization state of the various
phytochemicals present in Fagonia cretica extract, thereby altering its reducing
capacity and ability to stabilize the NPs. Therefore, this optimum pH allows a
higher yield of NPs. With a deviation in pH from the optimum, the yield decreases.
For example, smaller-sized particles are usually formed under alkaline conditions,
while larger ones might be obtained under acidic conditions (Singh et al., 2016).

1.15.3 Temperature of the Reaction

During the synthesis of nanoparticles, the temperature is one of the major
parameters able to modify the kinetics of the reaction. Indeed, the speed of

22
reaction usually grows with an increase in temperature; accordingly, NPs are
nucleating and growing more quickly. On the other hand, too high temperatures
can promote NP agglomeration and reduce their yield, together with changing
morphology. The temperature has to be chosen by balancing two opposite needs:
good efficiency in the synthesis process and well-defined NPs (Ahmed et al., 2016).
In some green synthesis using Fagonia cretica, the temperatures are usually
maintained at a moderate scale because if the aim is to retain as much bioactivity
of the extract as possible with adequate yield (Iravani, 2011).

1.15.4 Reaction Time

Reaction time also affects significantly both the yield and quality of NPs. In
general, longer reaction times allow for complete reduction of metal ions and thus
result in a higher yield of NP. Sometimes, however, longer reaction times induce
growth into bigger particles or even form aggregations, which might be
undesirable given the application of the NPs (Sharma et al., 2009). Hence,
optimization of reaction time has to be one of the main keys for balancing the
aspects between yield and characteristics of the particles themselves.

1.15.5 Concentration of Metal Salts

The concentration of metal salts used in the synthesis is another
determinant of the yield of nanoparticles. A higher concentration of metal ions can
lead to a greater number of nucleation events and hence an increase in the number
of NPs. However, if the concentration of metal ions is too high, incomplete
reduction could occur, forming by-products that decrease the overall yield. Hence,
optimal metal ion concentration is a prime requirement for the achievement of
maximum efficiency in nanoparticle synthesis while ensuring quality in the NPs
(Shankar et al., 2004).

1.16 Characterization techniques

1.16.1 UV-Vis Spectrum Absorption
The popular ways to characterize synthetic nanoparticles include UV-
visible spectroscopy. This technique becomes very effective in the case of metal
NPs such as gold, silver, and transition metal NPs. The method is based on the
ultraviolet and visible light absorption of the electromagnetic spectrum, providing

23
important information on electronic transitions occurring inside the NPs and in
their surroundings (Zhang et al., 2015).

1.16.2 Principle and Application of UV in NPs

Characterization
The UV-Visible absorption spectrum of NPs arises because of the collective
oscillation of conduction band electrons in response to the electromagnetic field-
a phenomenon now christened as surface plasmon resonance in metal NPs. It is
also well known that position and intensity of the SPR band are highly sensitive to
the size, shape, and dielectric environment of NPs (Zhang et al., 2015).

As an example, the SPR peak of gold NPs is usually seen at wavelengths in

the range of about 520-540 nm, which is expected to shift depending on the size
and aggregation state. However, SPR peaks commonly fall in the range of 400-450
nm for silver NPs. Specific absorption peaks in the UV-Vis spectrum may also be
seen for transition metal NPs, such as cobalt and nickel, and even for bimetallic
systems like nickel-copper; however, the position could vary with specific
conditions of the particles, such as size and shape, and the synthesis conditions.

1.16.3 Interpretation of UV-Vis Spectra

More precisely, within the context of the UV-Vis spectrum analysis, the average
size-size distribution can be inferred. Generally, the narrower and sharper the SPR
peak, the greater the tendency toward a monodisperse distribution, while a wider
peak suggests a wider size distribution or nanoparticle aggregation.

Normally, the absorption peaks of cobalt NPs are in the UV region, in the range
of 300-350 nm, depending on size and morphology. These peaks correspond to the
electronic transition of cobalt ions and also to the surface plasmon resonance
effect in these particles.

The specific particle size of Nickel NPs may be responsible, together with the
particular synthesis conditions, for its absorption in the UV region especially
around 250-300 nm. In general, the UV-Vis. absorption spectrum for nickel-copper
bimetallic NPs is a combination of the separate characteristics of each metal, and
a broad absorption band due to the plasmon coupling between metals is often

24
observed. The position of the absorption peaks will depend on the ratio of nickel
to copper and also on particle size.

1.16.4 Significance of UV-Visible in Nanoparticle Synthesis

UV-Visible spectroscopy is one of the real-time analysis techniques
indispensables in synthesizing nanoparticles. From the obtained absorption
spectra, reaction time, temperature, and precursor concentration are some
conditions that can be determined to provide the best nanoparticle formation. This
technique would also provide information about time-evolving changes within the
nanoparticle system, such as oxidation or aggregation, which might impact the
stability and functionality of the NPs.

1.17 X-Ray Diffraction (XRD)

Such an analytical method is the very effective X-ray diffraction, which
helps to identify the crystalline structure of the NPs. The basis for this technique
is the principle of constructive interference between monochromatic X-rays and a
crystalline sample. Filtered X-rays are used to produce monochromatic radiation
by a cathode ray tube, which is directed onto the sample. Incident rays, which hit
a crystal lattice, are diffracted into many different and specific directions. These
diffracted beams can be used in the measurement of angles, and their intensity can
provide an image that shows how electrons are distributed within the crystal, thus
showing the arrangement of atoms. The XRD technique relies on the principle
given by Bragg's formula, which expresses the fact that in cases when the path
difference between two waves reflected by successive planes of atoms is an integer
multiple of the wavelength, then diffraction occurs.

1.18 Application of XRD in Nanoparticle Characterization

In context of nanoparticle characterization, XRD is employed to determine
several critical parameters, including:

1. Crystalline Structure: XRD helps in identifying whether the NPs are

amorphous or crystalline. For crystalline nanoparticles, the diffraction
pattern provides information about type of crystal structure (e.g., face-
centered cubic, hexagonal close-packed).

25
 2. Crystallite Size: Crystallite size of NPs can be estimated using Scherrer
equation, which relates the broadening of the diffraction peaks to the
particle size.

3. Phase Identification: If bimetallic NPs (e.g., nickel-copper) are

synthesized, XRD can reveal whether the two metals form a solid solution,
an alloy, or separate phases.

4. Lattice Strain: The analysis of peak broadening also allows for the
estimation of lattice strain, which can be significant in nanoparticles
because of their high surface area and the presence of defects.

1.19 Typical XRD Peaks and Values

For common transition metal NPs such as cobalt Oxide (CoO), nickel Oxide
(NiO), and Nickle-Copper bimetallic oxides, the XRD patterns exhibit
characteristic peaks. For instance:

• Cobalt Oxide Nanoparticles: The XRD pattern typically shows peaks

corresponding to the (100), (002), and (101) planes of hexagonal close-
packed structure. Most intense peak usually occurs around 2θ = 44.2°.

• Nickel Oxide Nanoparticles: For nickel NPs with a face-centered cubic

(fcc) structure, characteristic peaks appear at 2θ values of approximately
44.5°, 51.8°, and 76.4°, corresponding to the (111), (200), and (220) planes,
respectively.

• Nickel-Copper Bimetallic Oxides Nanoparticles: These often show a

mixed phase pattern depending on the synthesis method, with peaks that
may shift slightly due to the formation of an alloy or a solid solution. Peaks
at positions intermediate between the corresponding XRD peaks of pure
nickel and copper can occur.

Hence, XRD provides the necessary information about the structural

properties of NPs, rendering it an indispensable technique within the field of
nanotechnology and materials science.

26
1.19.1 FT-IR Spectroscopy
One of the important widely used analytical techniques in the
characterization of synthesized NPs is FT-IR spectroscopy. By measuring the
absorption of infrared light at various wavelengths, FT-IR provides a close view of
the molecular vibrations and chemical bonds within a sample. This technique is
especially valued in determining functional groups on the surface of the NPs and
confirmation of specific chemical interactions during synthesis.

1.19.2 Principles of FT-IR in Nanoparticle Characterization

It is a principle of FT-IR that molecules will absorb infrared radiation at
frequencies corresponding to the vibrational modes of their bonds. When infrared
radiation falls on NPs, molecules on the surface of NPs start vibrating at frequency
and result in absorption peaks in the spectrum of FT-IR. These give a "fingerprint"
regarding the functional groups present and enable the researcher to identify the
chemistry at the surface of NPs.

1.19.3 Typical FT-IR Spectrum and Peak Assignments for

Nanoparticles
Generally speaking, FT-IR spectra obtained during the synthesis of
nanoparticles have some characteristic absorption bands which usually
correspond to various functional groups present on the surface of the
nanoparticles. For example:

• O-H Stretching

A broad absorption band around 3200-3600 cm⁻¹ usually indicates

hydroxyl (O-H) groups, which are common functional groups on the NPs
synthesized in aqueous environments.

27
 • C-H Stretching

The peaks around 2800-3000 cm⁻¹ refer to the C-H stretching vibrations of
aliphatic hydrocarbons, indicating that organic capping agents were used
during synthesis to help stabilize the NPs.

• N-H Bending

Hence, absorption bands around 1500-1600 cm⁻¹ could be assigned to N-

H bending vibrations, as a signature of amine groups, which are usually
involved in the functionalization of NPs.

1.19.4 FT-IR in the Confirmation of Nanoparticle Formation

Confirmation by FT-IR is essential, especially for the green methods of
synthesis. This might be employed in the confirmation of the reduction and
capping of NPs when plant extracts are to be used for synthesis; for instance, some
functional groups from the phytochemicals of the plant can easily be identified
with FT-IR. Infrared spectroscopy can identify functional groups that would
appear in the spectrum that might show a mechanism of the formation and
stabilization of nanoparticles.

1.20 Importance of FT-IR in Nanoparticle Surface

Functionalization
Among the critical steps for enhancing stability, biocompatibility, and
functionality toward specific applications, surface functionalization of NPs
provides a clear example. FT-IR may become crucial in the verification of the
presence and success of such surface modifications. Functionalization of gold NPs
with thiol groups is one good example; FT-IR is used to detect the S-H stretching
vibration at approximately 2550 cm⁻¹, confirming the attachment of the thiol-
containing molecule onto the surface of the nanoparticles. FT-IR is a traditional,
absolutely indispensable analytical technique in the NP characterization
methodology since it provides detailed information concerning the surface
chemistry of NPs and the functional groups participating in both their synthesis
and stabilization. Since FT-IR can identify a specific molecular vibration, it was

28
considered the proper technique to confirm the formation and functionalization
of NPs, especially in green synthesis methodologies that use natural plant extracts.

1.21 Scanning Electron Microscopy (SEM)

SEM is one of the powerful techniques that have widely been exploited for
characterization purposes in NPs. It yields high-resolution images, which allow
for a detailed study of morphology, size, and structure of NPs. In the synthesis of
nanoparticles, especially those involving biological systems like extracts from
plants, here Fagonia cretica, SEM becomes indispensable to confirm the
formation of NPs and assess uniformity and stability.

Despite the advancements in green nanotechnology, there remains a gap

in developing sustainable methods for synthesizing high-quality bimetallic
nanoparticles with biological applications. This study addresses this gap by
focusing on the use of Fagonia cretica extract for the eco-friendly synthesis of
nanoparticles.

29
1.22 Aims and Objectives
• To develop an eco-friendly and cost-effective green synthesis method for
the preparation of cobalt (CoO), nickel (NiO), and nickel-copper (NiO-
CuO) bimetallic nanoparticles using Fagonia cretica plant extract.
• To Characterize the synthesized nanoparticles using advanced
techniques such as UV-Vis spectroscopy, FT-IR, SEM, and XRD.
• To Investigate the biological properties of the nanoparticles, including
antioxidant and antibacterial activities.
• To Promote sustainability by reducing the use of toxic chemicals and
utilizing natural phytochemicals for nanoparticle production.

30
 Chapter 2

2 REVIEW OF LITRATURE
2.1 Green synthesis of nanoparticles
Recently, green synthesis of NPs has been gaining momentum since it is an
eco-friendly and economical process. Plant extracts, rich in phytochemicals, have
widely been used for the reduction of metal ions into nanoparticles. The approach
is highly regarded as a sustainable alternative to conventional outdated chemical
synthetic methodologies. The use of Fagonia cretica in the synthesis of NPs,
particularly with transition metals like cobalt, is still in the early stages of research.
With its rich phytochemical profile, this plant emerges as a potential candidate for
the development of stable and effective nanoparticles that open new frontiers in
nanotechnology (Reichsollner et al., 2019).

Approaches followed for the synthesis of nanoparticles primarily include

physicochemical and biological methods. Each method has its advantages and
disadvantages based on cost, feasibility in terms of control of particle shape,
production capacity, and scalability of particle size distribution. Of these methods,
plant-mediated biosynthesis represents one of the simplest, least expensive, and
fast methods for nanoparticle production. In contrast to microbial culture, which
is often not easy to maintain owing to the possibility of contamination, plant-
mediated biosynthesis offers an easier and more reliable alternative method of
generating large quantities of nanoparticles without the risk of microbial infection
(Sastry et al., 2004).

The green synthesis using plant extracts, in general, has received significant
attention in recent years. This is because the process is eco-friendly and
economically feasible. More recently, research has emphasized the synthesis of
metallic nanoparticles from plant extracts, highlighting extraordinary properties
combined with a broad spectrum of applications in medicine and agriculture. This
process relies on organic materials from plants, acting as an alternative green
technology in conventional methods for nanoparticle synthesis (Aswini et al.,
2021).

31
 Recent advances in the green synthesis of NPs have pointed out the growing
trend to apply plant extracts as a promising approach for the biological synthesis
of nanoscale metals such as gold, silver, palladium, copper, and iron. The interest
in environmental-friendly methods of synthesis is growing due to serious
environmental and health risks associated with conventional chemical methods.
Green synthesis is preferable due to cost-effectiveness, lower pollution levels, and
heightened safety for both the environment and human health.

Some challenges to this method include variability in the availability of

plants upon which a plant's geographic location and seasonal changes depend.
Other problems also include, but are not limited to, lower purity and yield. Despite
these obstacles, green synthesis offers a very appealing and greener alternative to
more common chemical synthesis, and hopefully, green synthesis will brighten
prospects in the future years (Ying et al., 2022).

Green nanotechnology is a fast-growing area of research wherein biological

production of NPs emphasizes, to a great deal, natural processes of living cells. To
date, it has played an increasingly vital role in industries dealing with
pharmaceuticals, fuel, nuclear power and energy generation, electronics, and
bioengineering. Biotic pathways for nanoparticle synthesis, particularly those
producing particles in the 1 to 100 nm range, are widely practiced and preferred
over other methods due to their safety, environmental friendliness, non-toxicity,
and cost-effectiveness.

Both approaches-top-down and bottom-up are effectively used for

producing metal nanoparticles, which may involve various techniques of
physiochemical and biological nature. Characterization of these NPs is an
important task; their properties are confirmed using an array of sophisticated
instruments. The analytical techniques employed in the characterization include
UV–Vis, FT-IR, SEM, TEM, XRD, AFM, HAADF, and ICP analysis (Kumar & Yadav,
2009).

2.2 Green synthesis of Cobalt nanoparticles

In this regard, Cobalt oxide nanoparticles Co3O4-NPs have been synthesized
through a green synthesis route using leaf extract of Populus ciliata (safaida) along

32
with Co(NO3)2.6H2O salt as the source of cobalt. The as-obtained NPs were
characterized through different techniques, namely FT-IR, XRD, TEM, and SEM.
Further, Co3O4 NPs were scanned for their antibacterial efficacy against both gram-
negative and gram-positive bacteria.

These NPs have demonstrated quite substantial anti-bacterial potentialities

against E. coil, K. pneumonia, B. subtilis, and B. licheniformis. The antibacterial
activities were statistically analyzed by one-way ANOVA and Dunnett's Multiple
Comparison Test. As evident from the antibiogram, the highest mean activity
displayed against B. subtilis with the value 21.8 ± 0.7 and the minimum was against
E. coli 14.0 ± 0.6 (Hafeez et al., 2020).

The significant attention towards Cobalt nanoparticles is due to resourceful

applications in the field of photocatalysis, antimicrobial activity, and
electrochemical sensing. Different synthesis methods are being researched by
physicists and chemists using various approaches. Among these, green synthesis
using plant extracts has come up to be a promising alternative.

The method is cost-effective and biocompatible, has easy scaling up of

production, and eliminates the need for further stabilizing agents. Phytochemicals
from the plant extracts-phenols, terpenoids, alkaloids and tannins, and a number
of vitamins-play the role of agents of stabilization, reduction and capping during
the synthesis of metal NPs. These phytochemicals have been able to successfully
synthesize the cobalt nanoparticles by taking extracts from roots, leaves, and barks
of various plants.

Accordingly, the synthesized process is well-characterized by using various

analytical tools, while advantages of applying Co NPs in photo-catalytic water
remediation, antimicrobial activities, and electrochemical sensing have been
recently realized (Singh, 2022).

2.3 Green synthesis of Silver Oxide NPs

A study conducted for green synthesis of Ag-NPs using the extract of Fagonia
cretica leaves showed that synthesized Ag-NPs were crystalline, clustered, and of
spherical shape, whose sizes range from 20×10-9m to 50×10-9m. Identification of
various functional groups within the Ag-NPs using FT-IR was done. Further, in-

33
vitro studies including α-amylase and α-glucosidase inhibition assays along with
antioxidant tests such as DPPH and ABTS showed significant enzyme inhibitory
and antioxidant activity (Khan et al., 2023).

2.4 Green synthesis of NiO/CuO NPs

A study showed the green synthesis of photocatalysts for mitigating
environmental pollutants. The work focused on synthesizing bimetallic NiO/CuO
NPs using Mangifera indica L. peel extract by a simultaneous reduction process.
HRTEM, XRD, and EDX are some of the advanced characterization techniques
utilized in the study of nanostructure, crystal conformation, and the chemical
composition of synthesized nanoparticles.

Photocatalytic properties of the nanocomposite were also studied through

UV-Vis. spectroscopy, particularly toward the degradation of harmful organic
glyphosate upon UV irradiation. It was found that the glyphosate treated with
NiO/CuO NPs had a substantially higher kinetic removal rate constant compared
to pure CuO and NiO nanoparticles, indicative of better photocatalytic efficiency.

This green synthesis methodology represented an environmentally friendly

and economic route; thus, it is a promising contribution to environmental
protection, mainly with regard to the removal of dangerous pollutants like
glyphosate (BOONSERM et al., 2024).

A study explored the synthesis of ZnO, CuO, and NiO nanoparticles using
Azadirachta indica (neem) leaf extract. The method was characterized as rapid,
eco-friendly, and cost-effective compared to traditional methods. Characterization
techniques included X-ray diffraction (XRD), Fourier-transform infrared
spectroscopy (FT-IR), and scanning electron microscopy (SEM), confirming the
formation of high-purity nanoparticles with distinct morphologies (Yadeta
Gemachu & Lealem Birhanu, 2024).

Research demonstrated the green synthesis of CuO nanoparticles using

Parthenium hysterophorus aqueous extract. The study evaluated the degradation
efficiency of these nanoparticles against rifampicin, achieving a remarkable
98.43% degradation under optimal conditions. This highlights the potential of

34
plant-mediated synthesis methods for environmental applications (Nzilu et al.,
2023).

A comprehensive review discussed the green synthesis methods for CuO

nanoparticles using various plant extracts. It highlighted the antibacterial
properties of these nanoparticles, suggesting their potential applications in
medical fields (Assefa et al., 2024).

The study reported a rapid biosynthesis approach for producing high-

surface-area CuO and NiO nanoparticles, emphasizing their effectiveness as
photocatalysts in environmental remediation processes (Iqbal et al., 2024).

A study utilized Arachis hypogaea (peanut) shell extract for the green
synthesis of NiO nanoparticles. The synthesized NPs exhibited a face-centered
cubic structure with an average size of 20 nm, demonstrating antibacterial activity
against Proteus vulgaris and Escherichia coli. The method was eco-friendly and
suitable for biomedical applications (Saminathan et al., 2024).

A sustainable method was developed using extracts from strawberries

(Fragaria × ananassa), grapes (Vitis vinifera), and grapefruit (Citrus × paradisi).
The synthesized NiO NPs showed distinct optical properties and were effective in
degrading azo dyes, highlighting their potential in environmental remediation
(Nejo et al., 2024).

This study explored the synthesis of silver nanoparticles (Ag NPs)

using Fagonia cretica leaf extract. The synthesized AgNPs were characterized
morphologically, revealing spherical shapes with sizes ranging from 20 to 50 nm.
The study highlighted the potential of these nanoparticles for biomedical
applications, including anti-diabetic properties demonstrated through in vitro and
in vivo tests (Khan et al., 2023).

The extract of Fagonia cretica was shown to serve as an effective stabilizing

agent for various nanoparticles, including AgNPs and potentially NiO and Co NPs.
The study discussed the bioactive compounds present in the extract that
contribute to the reduction and stabilization processes, emphasizing their
therapeutic applications in treating infections and oxidative stress-related
conditions (NAYILA et al., 2024).

35
 Research demonstrated a novel method for synthesizing cobalt oxide (CoO)
nanoparticles using Fagonia cretica extract. The study focused on optimizing
synthesis parameters and characterizing the CoO NPs using techniques like UV-Vis
spectroscopy and XRD, revealing their potential in catalysis and environmental
remediation (Kiani et al., 2022).

This comparative analysis examined various metal oxide nanoparticles

synthesized from Fagonia cretica, including NiO and CuO. The researchers
evaluated the antibacterial properties of these nanoparticles, finding significant
efficacy against common pathogens, which supports their application in medical
fields (SHEIKH et al., 2024).

While primarily focused on ZnO NPs, this study also discussed the potential
for synthesizing NiO and CuO nanoparticles using similar methodologies
with Fagonia cretica. The research emphasized the eco-friendly nature of plant-
mediated synthesis and its advantages over conventional chemical methods (Kiani
et al., 2022).

This study evaluated the antioxidant properties of NiO nanoparticles

synthesized using Fagonia cretica extract. The findings indicated that these
nanoparticles exhibited significant free radical scavenging activity, suggesting
their potential use in health-related applications.

The green synthesis of cobalt nanoparticles via Fagonia cretica extract was
characterized using FTIR and SEM techniques. The study highlighted how
phytochemicals in the extract facilitated the reduction process, leading to stable
nanoparticle formation suitable for various applications.

A review article discussed various green chemistry approaches for

synthesizing metal oxide nanoparticles, including NiO and CuO, using plant
extracts like Fagonia cretica. This comprehensive overview highlighted the
advantages of using plant-based methods over traditional chemical synthesis
(Almutairi et al., 2024).

The study examined the phytochemical constituents of Fagonia cretica that

contribute to nanoparticle synthesis, particularly focusing on their roles as
reducing and stabilizing agents for NiO and CuO NPs. The findings indicated a rich

36
profile of bioactive compounds beneficial for nanoparticle formation (Kiani et al.,
2022).

37
 Chapter 3

3 MATERIALS AND METHODS

3.1 Preservation and Collection of Fagonia cretica
Fagonia cretica plants were collected in September 2023 from the tribal area
of Sakhi Sarwar, Dera Ghazi Khan, Pakistan. After bringing the plant materials to
the laboratory, they were washed with tap water followed by distilled water to
eliminate dust and other impurities. Then, drying in the shade was carried out and
preserved in dry plastic containers for future use.

3.2 Plants Identification

The plants of Fagonia cretica were identified with the help of Dr. Maqbool
Ahmad from Chemistry Department and Dr. Allah Bakhsh Gulshan from Botany
Department, Ghazi University, Dera Ghazi Khan. They supervised the
identification, keeping in view the prime features regarding leaf shape, stem
structure, and flower morphology of the plant. We further verified our findings
from the descriptions available in the relevant botanical literature. This kind of
collaboration helped us to confidently confirm the identity of plant specimens
used in the study.

3.3 Sample Treatment

The collected plants, Fagonia cretica, were shade-dried at room temperature
for a period of two weeks without any kind of contamination. Further, the dried
plant materials were washed in distilled water in order to remove the adhering
dirt particles. Plant materials were subjected to oven-drying at 60°C for 2 days to
become really well-dried. This was an important step in order to remove residual
moisture, which could affect the quality of the work. The dried plant was ground
into fine powder using a clean mortar and pestle. The uniformity of color, texture,
and most especially the particle size of the powdered material was observed. We
carefully sieved the powder in order to have all passages with uniform particle
sizes.

The final powdered plant material was then kept in airtight glass containers
to protect it from moisture and degradation. We stored the containers in a cool,

38
dry place until we were ready to use them. Before each use, the amount needed of
the plant powder was weighed with high accuracy to make sure our every step was
taken precisely (Ahmed et al.)

3.4 Chemicals and apparatus

3.4.1 Chemicals:
1. Cobalt Chloride (CoCl₂)

2. Nickel Acetate (Ni(CH₃COO)₂)

3. Copper Chloride (CuCl₂)

4. Zinc Acetate (Zn(CH₃COO)₂)

5. Fagonia cretica Plant Extract

6. Sodium Hydroxide (NaOH)

7. Distilled Water

8. Deionized Water

9. Ethanol

10. Methanol

11. Dimethyl Sulfoxide (DMSO)

3.4.2 Apparatus:
1. Conical Flasks (50 mL, 250 mL, 500 mL)

2. Measuring Cylinders (50 mL and 100 mL)

3. Reagent Bottles (250 mL)

4. Petri Dishes

5. Test Tubes

6. Spatula

7. Magnetic Stirrer

8. Beakers (250 mL, 500 mL)

9. Blades

10. Mortar and Pestle

11. pH Strips

12. Funnel

39
 13. Filter medium

14. Aluminum Foil

15. Microwave Oven

16. Centrifuge Machine

17. Hot Plate

18. Electric Weight Balance

19. Grinder

20. Single Beam UV-Spectrophotometer

21. Double Beam UV-Spectrophotometer

22. Dryer

3.5 Procedure for Preparing Plant Extracts

The preparation of plant extract began with 50 grams of the dried and
powdered Fagonia cretica samples, which were mixed with 300 mL of deionized
water in a 500 mL conical flask. The mixture was then gently heated on a Magnetic
Hot Plate, where it was stirred at 50 rpm while maintaining a temperature of 50°C
for 20 to 30 minutes. This step was carried out to facilitate the extraction of
bioactive compounds from the plant material.

After heating, the mixture was left undisturbed at room temperature for 3
days. This resting period ensured that the plant material had ample time to
interact with the water, allowing for thorough extraction.

Once the extraction was completed, the mixture was carefully filtered
through Whatman filter paper No. 1 to eliminate any solid particles. The resulting
clear extract was then collected and stored under standard conditions, ready for
use in the subsequent nanoparticle synthesis.

3.6 Preparation of Salt Solutions

The preparation of salt solutions involved careful dissolution of respective
salts in deionized water to achieve the desired concentrations. The solutions were
prepared as follows:

• CoCl2 (0.1 M) soln.

• Nickel acetate (0.01 M) soln.
40
 • Nickel Acetate + Copper chloride (0.1 M) soln.

For each solution, the appropriate amount of salt was accurately weighed
and then gradually dissolved in deionized water. Solutions were mixed thoroughly
to ensure homogeneity. Once prepared, the solutions were stored in clean reagent
bottles and kept ready for use in subsequent experimental procedures.

Figure 4: Nickle Acetate salt solution Figure 3: Cobalt chloride salt solution

3.7 Green Synthesis of CoO, NiO, NiO/CuO, Nanoparticles

Green synthesis of NiO NPs, CoO NPs and NiO/CuO NPs was initiated by mild
mixing of 50 mL plant extract along with 50 mL of respective salt solution in 250
mL conical flask. Then, the soln. was heated cautiously while stirring on the hot
plate for a period of 30 to 60 minutes; the temperature maintained within the
ranges of 70°C to 85°C, with stirring at 120 rpm to ensure homogeneity and
effectiveness during the reaction. Throughout the course of the reaction, it
changed color from lighter into a darker solution as the NPs formed. On the
completion of reaction, the soln. was centrifuged for ten minutes at approximately
7,000 rpm to afford the nanoparticles as pellets.

3.8 Characterization of Nanoparticles

Nanoparticles were thoroughly characterized by using numerous
techniques, including UV spectroscopy, FT-IR spectroscopy, SEM and XRD.

3.8.1 UV-Visible Spectroscopy

The process began by observing the color change when the plant extract
derived from leaves, stems, flowers, and fruits was mixed with a metallic ion

41
solution. This visual change indicated the formation of nanoparticles. To monitor
the progress, small samples were taken at regular intervals, and UV-Visible spectra
of reaction mixtures were recorded. The change in time was followed between the
range of 200-1200 nm by the UV Visible spectrophotometer.

3.8.2 FT-IR Spectroscopy

The FT-IR spectroscopy was done in order to identify the biomolecules
involved in the plant extracts responsible for the synthesis of the nanoparticles.
The spectrum of the nanoparticles was analyzed through Midac MC 2000
Spectrometer in the spectral range of 500-4000 cm⁻¹. This enabled the
identification of important functional groups forming and stabilizing NPs.

3.9 Biological Activities of Nanoparticles

The biological activities of biosynthesized NPs are generally measured with
their antioxidant capabilities. These activities are quantitatively assayed by DPPH
RSA, HRSA, linoleic acid reducing capacity, and ferric reducing power assay.

3.9.1 Antioxidant Properties of Green Synthesized NPs

The antioxidant potential of green-synthesized nanoparticles was screened
for neutralization capability against different kinds of free radicals by using the
four key assays: DPPH RSA, HRSA, linoleic acid reducing capacity, and ferric
reducing power.

3.9.2 DPPH Radical Scavenging Activity

DPPH is a stable free radical discovered by Goldschmidt and Renn in 1922
and used for the determination of antioxidant activity. This method was refined in
later years by Blois using the molecule DPPH, which measures the capability of an
ingredient to donate hydrogen atoms and neutralize the free radicals. The DPPH
molecule has a deep violet color because of an unpaired electron that is
delocalized, while the absorption peak is approximately around 517 nm in ethanol
solution. On introduction to a substance that can donate hydrogen atoms, the
DPPH radical is reduced by accepting electrons or hydrogen atoms, evidenced by
a loss in violet color of the solution.

42
Materials and Equipment for DPPH

To estimate the antioxidant activity of the prepared NPs, the following

materials and equipment were utilized:

• 2,2-Diphenyl-1-picrylhydrazyl
• Methanol
• Ascorbic acid
• Micropipette
• Spectrophotometer
• Test tubes
Experimental Process

First, we prepared 40 µM DPPH solution in methanol (350mL), and then 1

mL of this soln. was combined with 3.0 mL of the synthesized nanoparticles at a
specified concentration. The solution underwent a color change after being left in
the dark at room temperature for 35 minutes, which means reaction occurred. The
absorbance was determined utilizing a UV-visible spectrophotometer at
wavelength of 517 nm, following incubation. Ascorbic acid used as standard
reference against a blank sample. The procedure was done in triplicate to ensure
the correctness of the outcome. A lower absorbance reading indicates higher free
radical scavenging activities. Then, the scavenging ability of the NPs for the DPPH
radicals was calculated by using a certain formula.

DPPH RSC (%) = Abs control –Abs sample/Abs control × 100

3.9.3 Linoleic Acid Reducing Capacity (LARC)

Linoleic Acid Reducing Capacity test helps determine how effective
antioxidants are at preventing fats from breaking down due to free radicals.

3.9.3.1Chemicals and Equipment

We used the following materials for this experiment:

• Sodium phosphate buffer

• Linoleic acid

43
 • FeCl3
• NH4SCN
• Ascorbic acid (AA)
• EtOH
• MeOH
• Distilled water
• Micropipettes
• Spectrophotometer
• Microwave oven
• Test tubes

3.9.3.2 Procedure
Sample mixture was prepared by mixing 2 mL of nanoparticle sample with
2 mL of 2.5% linoleic acid solution in EtOH. Subsequently, 2 mL of 0.05 M sodium
phosphate buffer and 2 mL of distilled water were introduced into mixture. Then,
Combined solution was placed into microwave oven for incubating at 40°C for 24
hrs. After incubation, 1 mL of the reaction mixture was drawn out and mixed with
10 mL of 75% methanol solution. To this solution, 1 mL of 20-mM FeCl3 solution
and 1 mL of 30% NH4SCN Solution was added. The absorbance at 500nm
wavelength of the final solution was measured by using a spectrophotometer. The
control sample was prepared with all the reagents except for the nanoparticle
extract, which was used for comparing the values. Then, we calculated the
antioxidant activities of the extracts by comparing values of the absorbance for
sample and control using a formula designed to specifically measure LARC.

LARC (%) = [Absorbance control –Absorbance sample/Absorbance control] × 100

44
 Chapter 4

4 RESULTS AND DISCUSSIONS

4.1 FT-IR of Cobalt Oxide NPs
FT-IR analysis of Fagonia cretica-mediated cobalt nanoparticles indicated
existence of several functional groups. FT-IR Peaks at 1401 cm⁻¹ and 1395 cm⁻¹
correspond to C-H bending due to methyl or methylene groups, and C-O stretching
owing to carboxylates or phenolics. The peak at 2023 cm⁻¹ represents
isothiocyanate or thiocyanate groups; they are well-documented in secondary
plant metabolites. Peak at 1532 cm⁻¹ is ascribed to either N-H bending as amide II
or to C=C stretching in aromatic rings, thus confirming the presence of proteins or
aromatic substances. Absorption at 1046 cm⁻¹ is because of C-O stretch assigned
to alcohols or glycosides. Finally, band at 816 cm⁻¹ is because of aromatic C-H
bending. Peak at 534 cm⁻¹ is representative of Co-O bonds. so, it is confirming the
synthesis of Co₃O₄ NPs. It should be noted that these functional groups belong to
the plant extract and serve a dual devotion of reduction as well as stabilization of
the nanoparticles, hence determining their bioactivity.

45
 105
Cobalt NPs

104
Transmittance (%T)

103

102

101

100

4000 3500 3000 2500 2000 1500 1000 500

Wavenumbers (cm-1)

Graph 1: FT-IR Spectra of Cobalt Oxide NPs

46
4.2 FT-IR of Nickle Oxide Nanoparticles
FT-IR of the NiO NPs produced by using plant extract shows existence of
some important functional groups like alkyne, isothiocyanate, amide, aromatic
substances, phenolics, and alcohols. Peaks at 2167 cm⁻¹ and 2039 cm⁻¹ point out
the isothiocyanate or alkyne groups, and at 1555 cm⁻¹, it indicates amide or
aromatic compounds. Peak at 661 cm⁻¹ confirms the presence of Ni-O bonds,
which means that nickel oxide nanoparticles have been successfully synthesized.
These functional groups could be intricate in reduction and stabilization of NPs
and might contribute to their biological activity as well.

101.5
Nickle NPs
101.0
Transmittance (%T)

100.5

100.0
1410

99.5
2039

1052
2167

1555

661

99.0

98.5

98.0
4000 3500 3000 2500 2000 1500 1000 500

Wavenumbers (cm-1)

Graph 2: FT-IR of Nickle Oxide NPs

47
4.3 FT-IR of Nickle-Copper Bimetallic nanoparticles
FT-IR analysis shows that NiO/CuO NPs bimetallic nanoparticles contain
functional groups related to amide and aromatic compounds (1559 cm⁻¹),
phenolic (1404 cm⁻¹), alcohol (1137 cm⁻¹ and 1054 cm⁻¹) and metal-oxygen
linkages (667 cm⁻¹). Functional groups of plant extract may provide to the
reduction and stabilization of NPs. Hence improving the properties and
applications.

102 Nickle-Copper
Transmittance (%T)

100

98

1137
1404
1559

1054

667
96

94

92
4000 3500 3000 2500 2000 1500 1000 500

Wavenumbers (cm-1)

Graph 3: FT-IR of Nickle-Copper

48
4.4 SEM of Cobalt Oxide NPs
SEM images of Cobalt oxide nanoparticles, as revealed in the Figure below,
reveal a diverse morphology with irregular, cubic, and hexagonal shapes. These
nanoparticles appear to be agglomerated, forming clusters of various sizes. Upon
closer inspection at higher magnifications, the surfaces of the nanoparticles are
observed to be smooth. However, at even higher magnifications, the images display
larger particles, which are likely the result of aggregation or clustering of smaller
nanoparticles. Average size of the cobalt nanoparticles, revealed from SEM
analysis, is approximately 45.45 nm.

Graph 4: SEM CoO NPs

49
4.5 XRD of Cobalt Oxide NPs
XRD analysis shown successful production of pure crystalline cobalt oxide
nanoparticles. XRD spectrum represented peaks at 31.96 and 42.96 from 20 to 70.
Average size of CoO NPs was computed to be about 16.40 nm.

Table 1: XRD of Cobalt

Plant Extract Sample 2(degree) FWHM Particle Average

size (nm) size (nm)
Fagonia cretica CoO 31.96 0.5031 16.41 16.40

42.96 0.5204 16.40

XRD

30

25

20
Intensity

15

10

0
20 30 40 50 60 70
2 θ (Degree)

Graph 5: XRD of Cobalt Oxide NPs

50
4.6 UV-Visible Spectrum of Cobalt Oxide NPs
UV-Visible spectrum confirmed synthesis of Co NPs. Analysis provided
information on the optical properties of nanoparticles. There was an absorption
peak at about 550 nm, considered the characteristic peak for the SPR of Co NPs.
This peak indicates that stable nanoparticles have been formed since there were
no significant shifts or additional peaks. The UV-Vis spectra, therefore, evidence
that optical properties are one of the main features to detect the presence of Co
NPs synthesized very uniformly with consistent particle size.

UV-Visible Spectrum
3
2.5
2
Co-Nps

1.5
1
0.5
0
-0.5 0 200 400 600 800 1000 1200 1400
Axis Title

Graph 6: UV-Visible Spectrum of CoO NPs

51
4.7 Antioxidant Activities
The antioxidant activities of cobalt nanoparticles were tested in different
ways to see how well they can neutralize free radicals and stop oxidative damage.

4.7.1 DPPH
In the DPPH test, Co-NPs showed a good antioxidant activity by 66.46%,
while Ni-NPs were less active, only by 29.67%. On the contrary, the combination
of NiO/CuO NPs revealed increased activity to 84.1%, which is close to the
standard.

Table 2: DPPH Antioxidant of NPs

Sample Concentration Absorbance Absorbance Absorbance DPPH %

(mg/L) blank Control Sample

Standard 20 0.165 0.975 0.071 92.71%

Cobalt Oxide 20 0.165 0.975 0.327 66.46%

Nickle Oxide 20 0.165 0.975 0.685 29.67%

Nickle+Copper 20 0.165 0.975 0.155 84.10%

DPPH
Percentage Activity

92.71 92.71 92.71

84.1
66.46

29.67

CO-NPS NI-NPS NI+CU-NPS

Concentration

Standard Sample

Graph 7: DPPH Antioxidant of NPs

52
 4.7.2 HRSC
The scavenging activity was 50.26% for CoO NPs, while NiO NPs
demonstrated a little higher activity of 55.9%. For the NiO/CuO NPs, activity was
53.33%. All samples revealed closer activities in comparison to the standard with
65.13%.

Table 3: HRSC Antioxidant of NPs

Sample Concentration Absorbance Absorbance Absorbance HRSC %

(mg/L) blank Control Sample
Standard 20 0.280 0.585 0.102 65.13%

Cobalt Oxide 20 0.280 0.585 0.291 50.26%

Nickle oxide 20 0.280 0.585 0.258 55.90%

Nickle-Copper 20 0.280 0.585 0.273 53.33%

HRSC

65.13 65.13 65.13

Percentage Activity

55.9 53.33
50.26

CO-NPS NI-NPS NI+CU-NPS

Concentration

Standard Sample

Graph 8: HRSC Antioxidant of NPs

53
4.7.3 LARC
The results of the LARC assay revealed that CoO NPs exhibited very low
scavenging activity, with a value of 17.45%, and NiO NPs had comparatively higher
activity of 18.03%. Among these samples, the nickel-copper Bimetallic Oxide NPs
was higher in the LARC assay with an activity of 30.36%, but all samples were
highly lower compared to the standard activity of 84.06%.

Table 4: LARC Antioxidant of NPs

Sample Concentration Absorbance Absorbance Absorbance LARC %

(mg/L) blank Control Sample
Standard 20 0.355 0.527 0.084 84.06%
CoO 20 0.355 0.527 0.435 17.45%
NiO 20 0.355 0.527 0.432 18.03%
NiO/CuO 20 0.355 0.527 0.367 30.36%

LARC

84.06 84.06 84.06

Percentage Activity

30.36
17.45 18.03

CO-NPS NI-NPS NI+CU-NPS

Concentration

Standard Sample

Graph 9: LARC Antioxidant of NPs

54
4.8 Antibacterial Activity of Cobalt Oxide NPs
4.8.1 Protocols for Antibacterial Activity
The antibacterial activity of cobalt nanoparticles was tested using the agar
well diffusion method. Fresh cultures of Staphylococcus aureus and Escherichia
coli were grown overnight and adjusted to a 0.5 McFarland standard to ensure
uniform bacterial concentration. Mueller-Hinton agar plates were prepared, and
the bacterial suspensions were evenly spread over the agar surface.

Wells of approximately 6 mm in diameter were created in the agar, and each

well was filled with 100 µL of cobalt nanoparticle suspension. Ciprofloxacin (50
µg/mL) was used as a positive control. The plates were incubated at 37°C for 24
hours to allow bacterial growth and diffusion of the nanoparticles. After
incubation, the zones of inhibition around each well were measured to determine
the antibacterial activity.

4.8.2 Results for Antibacterial Activity

In this present work, we have studied the antibacterial activity of cobalt
oxide nanoparticles against the pathogens Staphylococcus aureus and Escherichia
coli. Ciprofloxacin was used as the reference antibiotic for comparison.

The zone of inhibition observed against the following was 32 mm for the
cobalt oxide nanoparticles compared with ciprofloxacin, which had a zone of
inhibition of 38 mm. This simply means that cobalt oxide nanoparticles have a
great deal of antibacterial activity, though slightly less than ciprofloxacin, with an
overall effectiveness of roughly 84% when compared to the reference antibiotic.

In the case of Escherichia coli, cobalt oxide nanoparticles exhibited a zone of

inhibition of 30 mm, whereas ciprofloxacin revealed nearly an increase in the
inhibition zone of 36 mm. Here, cobalt oxide nanoparticles almost showed 83% of
the antibacterial effectiveness of ciprofloxacin.

55
Figure 5: E.Coli Figure 6: Staphylococcus Aureus

Table 5: Inhibition Zones (mm) of Cobalt Oxide NPs and Ciprofloxacin

Nanoparticles Escherichia coli Staphylococcus aureus
(Gram-Negative) (Gram-positive)

Zone of inhibition (mm) Zone of inhibition (mm)

Cobalt Oxide NPs

30 32

Ciprofloxacin* 36 38

56
 Chapter 5

5 CONCLUSION
In this study, cobalt oxide, nickel oxide and nickel-copper bimetallic
nanoparticles were produced using Fagonia cretica plant extract. The nanoparticle
formation was confirmed by various assessment techniques including UV-visible
spectroscopy, SEM, XRD and FT-IR. Antioxidant tests performed (DPPH, HRSC, and
LARC) revealed that NiO/CuO NPs exhibited the highest antioxidant activity,
comparable to the standard, while CoO NPs and NiO NPs showed moderate to low
activities, demonstrating the superior antioxidative potential of NiO/CuO NPs.
Cobalt oxide nanoparticles established notable antibacterial activity, showing
efficacy comparable to ciprofloxacin against both Staphylococcus aureus and
Escherichia coli, highlighting their potential as alternative antimicrobial agents.
Therefore, this study indicates how environmentally friendly synthesis is effective
in producing nano-particles that have potential use in biomedicine and the
environment.

57
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