CHAPTER I

INTRODUCTION

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 1.INTRODUCTION
1.1. Green chemistry
1.2. Nano chemistry
1.3. Nanotechnology
1.4. Nanoparticles
1.5. Classification of NPs
1.6. Synthesis of NPs
1.7. Biosynthesis of metal NPs using plant extract
1.8. Application of NPs
1.9. ZnO NPs and its methods of synthesis
2.0. Green synthesis of ZnO NPs
2.1. Applications of ZnO NPs
2.2. Catalytic activity and photocatalytic degradation of
organic dyes
2.2.1. Methyl orange
2.2.2. Rhodamine B
2.3. Plant introduction
2.3.1. Taxonomical classification
2.3.2. Occurrence
2.3.3. Medicinal uses
2.4. Phytochemistry
2.5. Antibacterial activity

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INTRODUCTION

1.1 GREEN CHEMISTRY

Green chemistry may be defined as chemical-assisted pollution-prevention
strategies employed in specific domains such as green analytical chemistry,
ecologically friendly analytical chemistry and clean analytical methodologies. In
particular fields like green analytical chemistry, environmentally friendly analytical
chemistry, and clean analytical techniques, green chemistry can be characterised as
chemically assisted pollution-prevention strategies. Toxic residues, which are
damaging to people and the environment, have significantly decreased as a result of
green chemistry, which use chemical principles to limit or eliminate the usage of
hazardous compounds. It is defined as the lessening of environmental harm brought on
by the manufacture of materials, as well as the minimization and appropriate disposal
of wastes produced during various chemical processes. Because it is biocompatible,
inert, and harmless for the environment, green synthesis is seen as a potential method
for creating nanoparticles [1].

1.2 NANO CHEMISTRY

Over the past ten years, the prefix "nano" has become more and more applicable
to several branches of knowledge. A large population of non-experts is now familiar
with a number of the new nano-containing phrases that are used often in scientific
reports, popular literature, and news articles. These terms include nanoscience,
nanotechnology, and nano chemistry. The universe at the nanoscale is often measured
in nanometres (1nm equals 10-9 nm), and it includes systems that are larger than
molecules but smaller than macroscopic ones (typically > I nm and 100 nm) [2].

Nanotechnology is the use and manipulation of matter at a microscopic scale. It

is the science of the small, the very small. At this scale, atoms and molecules function
differently and provide a wide range of unexpected and fascinating applications.
Studies on nanoscience and nanotechnology have exploded during the past few years
in a variety of product categories. It offers chances for the creation of materials,
especially those for medical uses, while more traditional methods might have their
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limitations. Pharmaceutical nanoparticles are solid, submicron-sized drug carriers with
a diameter of less than 100 nm that may or may not be biodegradable. This
comprehensive study focuses on the pharmacological and physiological implications of
nanoparticles, as well as their classifications, manufacturing methods, characterisation,
and applications [3].

1.3 NANOTECHNOLOGY
Professor Norio gave a clear definition of nanotechnology as "the processing of
separation, consolidation, and deformation of materials by one atom or by one
molecule." Nanotechnology was coined by Taniguchi of Tokyo Science University. He
defined it as the area of research that deals with the manipulation of matter at the atomic
or molecular level. The scientific advancement of the twenty-first century was the
development of nanotechnology. It is a multidisciplinary field that includes the creation,
handling, and application of materials with a scale smaller than 100 nm. It regulates
matter at the molecular level and has just recently entered the world of applications
(Mansoori 2005). Innovative applied and basic horizons in nanotechnology have been
unbolted up by daily amazing growth in a new branch.

Metal nanoparticles (MNPs) are synthesised via nanotechnology and have

various chemical compositions. In the fields of medicine, biology, material science,
physics, chemistry, photography, catalysis, biological labelling, photonics,
optoelectronics, and surface enhanced Raman Scattering, MNPs have been proven to
have a wide range of possible applications (SERS) [1] The biosynthesis of MNPS is a
one-step, cost-effective, environmentally friendly, and straightforward method.

1.4 NANOPARTICLES
Magnetic nanomaterials, semiconductor nanoparticles like titanium dioxide,
copper oxide, and zinc oxide, and noble metal nanoparticles like silver and gold. Nano
zinc, nano aluminium, TiO2, ZnO, and quantum dots are examples of man-made or
created nanoparticles. Nanoparticles are more advantageous for use as sensors because
of their smaller size and ease of deployment in remote areas. Nowadays, using
nanoparticles as an alternative to the present treatment materials for agricultural
remediation is proving successful in terms of effectiveness, cost-effectiveness, and
environmental friendliness [4]. Because they are stable, eco-friendly, and affordable,
biologically created nanomaterials are increasingly being used in agriculture and health.
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They ought to be used in more extensive applications. Biologically produced
nanomaterials is superior than other technologies due to its quick synthesis, simplicity
in controlling toxicity and size, cost-effectiveness, and environmental friendliness.
Agriculture is being protected from bacterial disease and contaminants, such as heavy
metals, by means of nanoscience on a large scale [5].

1.5 CLASSIFICATION OF NANOPARTICLES

NPs can be broadly categorised into a number of groups based on their morphology,
size, and chemical characteristics. The following list includes some of the well-known
classes of NPs based on their physical and chemical properties.

 CARBON-BASED NANOPARTICLES

Two important kinds of carbon-based NPs are fullerenes and carbon nanotubes
(CNTs). Nanomaterials comprised of globular hollow cages, like allotropic forms of
carbon, are found in fullerenes. They've generated significant business interest due to
their electrical conductivity, high strength, structure, electron affinity, and versatility.

 METAL NANOPARTICLES

Only the metals' precursors are used to make metal NPs. These NPs have special
optoelectrical properties because of the well-known LSPR (localised surface plasmon
resonance) features. Cu, Ag, and Au nanoparticles exhibit a wide absorption band in the
visible region of the solar electromagnetic spectrum. Metal NPs are used in numerous
scientific fields because of their superior optical characteristics.

 CERAMICS NANOPARTICLES

Ceramics NPs are inorganic, non-metallic particles that are created by heating and
then cooling substances repeatedly. They come in amorphous, polycrystalline, dense,
porous, hollow, or forms that lack crystalline structure. Because of their utility in
processes including catalysis, photocatalysis, photodegradation of dyes, and imaging
applications, these NPs are hence attention of researchers.

 SEMICONDUCTOR NANOPARTICLES

Semiconductor materials possess properties between metals and nonmetals and

therefore they found various applications in the literature due to this property.

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Semiconductor NPs have broad bandgaps, which led to a considerable change in their
characteristics when the bandgap was tuned. Because of this, they play a crucial role in
photocatalysis, photo optics, and electronic devices.

 POLYMERIC NANOPARTICLES

They are organic-based NPs, and the term polymer nanoparticle (PNP) collective is
used in the literature to refer to them. Typically, they have nanosphere or nano capsular
shapes. PNPs are simple to functionalize and have a wide range of applications in
literature.

 LIPID BASED NANOPARTICLES

Lipid nanoparticles focuses on the designing and synthesis of lipid NPs for various
applications such as drug carriers and delivery and RNA release in cancer therapy.

1.6 SYNTHESIS OF NANOPARTICLES

Nanoparticles are typically synthesized from a top-down or bottom-up
approach. The first one is the top-down approach where particles are produced from the
bulk material, and the second one is the bottom-up approach where nucleation sites are
formed and finally grow into a nano meter-sized particle. The first approach consists of
a set of techniques also known as “physical” synthesis methods. Several physical
methods for top-down synthesis of silver nanostructures including ball milling, laser
ablation, evaporation–condensation, electromagnetic levitation gas condensation
(ELGC), Ultrasonication, lithography, spray pyrolysis , radiolysis , arc discharge , and
photoirradiation have been utilized to synthesize various morphologies of silver
nanostructures with varied size and size distribution. The physical synthesis method is
primarily used for large-scale production in a short amount of time .

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Fig.1. Different methods for the Synthesis of Metal Nanoparticles

The bottom-up approach, however, mostly relies on the use of reducing agents
for the production of silver nanoparticles. This approach is also categorized into two
distinguishable, but not completely disparate, set of methods. The first category consists
of techniques that use chemical reagents to reduce Ag cations into zero-charged Ag
atoms, which then mount on top of the nuclei, serving as templates for crystal growth
into particles at the nanoscale [33,34]. This set of techniques is also known as the
“chemical” synthesis method. Due to the disadvantages of physical and chemical
methods, the focus has been directed towards the use of facile methods and materials
that are less detrimental and more cost-effective. A common subject of interest in this
area has been the selection of naturally occurring processes, widely known as “green
processes”.

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1.7 BIOSYNTHESIS OF METAL NANOPARTICLES
USING PLANT EXTRACT
Generally, the synthesis of different MNPs by physical method are highly
expensive and chemical methods are harmful to the environment. Most of the
physicochemical reactions involving at high temperatures in organic solvent have been
used for the synthesis of these MNPs. Green synthesis is the most environmentally
friendly and economically affordable [6,7] method.

Fig:.2. Different methods of Nanoparticle Synthesis

Researchers are now interested in biological synthesis, which uses natural

reducing, capping, and stabilising agents instead of dangerous, expensive chemicals
and requires a lot of electricity (Fig. 3). NPs are widely used in fields that come into
touch with people (such as medicine and agriculture), therefore it is becoming more
and more important to adopt safe synthesis techniques.

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 Fig.3. Pictorial Representation of the Synthesis of NPs using Plant
Extract

Nanoparticles of plant-related parts such as leaves, stems, flowers, bark, roots, seeds,
and their metabolites have been used for biosynthesis [35] Plants have the ability to
store heavy metals in their various parts. As a result, biosynthesis techniques employing
plant extracts have produced a simple means to stabilize efficient growth, simple, cost-
effective synthesis process, along with traditional preparation methods for the
production of NPs [37].

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1.8 APPLICATION OF NANOPARTICLES
 Nanoparticles have a great variety of applications in several biomedical,
environmental and agricultural sectors.
 Nanoparticles of iron, gold, silver, copper, zinc and titanium are applied as
antimicrobial agents to inhibit the growth of infectious bacteria and fungus and
thus induce mortality.
 Nanoparticles are used as sensors to identify in environmental stages.
 Nanoparticles are to filter out the organic contaminants from several water
reservoirs has been explored.
 They are used to produce scratchproof eyeglasses, crack-resistant paints,
transparent sunscreens etc.,
 Metallic nanoparticles have attracted attention because of their unique
biological and physicochemical properties.
 Nanoparticles have many potential applications in the use of agriculture due to
their antimicrobial activity.
 Nanoparticles play a very important role in the purification of water through
binding with precipitates, debris and heavy metals.

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1.9 INTRODUCTION ABOUT THE ZINC OXIDE NPS
Zinc oxide nanoparticles (ZnO NPs), as one of the most important metal oxide
nanoparticles, are popularly employed in various fields due to their peculiar physical
and chemical properties [8,9]. ZnO NPs are firstly applied in the rubber industry as they
can provide wearproof of the rubber composite, improve performance of high polymer
in their toughness and intensity and antiaging, and other functions [10,11]. In recent
times, the green method for the synthesis of NPs has become an area of great interest
in this direction because the use of conventional chemical methods is very expensive,
and as a reducing agent of chemical compounds or organic solvents use is required
which produces toxicity.

Fig: 4 Zinc Oxide Nanoparticles

Because of the strong UV absorption properties of ZnO, they are mostly used in
personal care products, such as cosmetics and sunscreen [12]. In addition, ZnO NPs
have superior antibacterial, antimicrobial, and also excellent UV-blocking properties.
Therefore, in the textile industry, the finished fabrics by adding ZnO NPs exhibited the
attractive functions of ultraviolet and visible light resistance, antibacteria, and
deodorant [13]. Apart from the applications mentioned above, zinc oxide can also be
used in other branches of industry, including concrete production, photocatalysis,
electronics, electrotechnology industries, and so on [11,14].

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METHODS USED FOR THE SYNTHESIS OF ZnO NPs:
The biological activity of nanoparticles depends on factors including surface
chemistry, size distribution, particle morphology, and particle reactivity in solution.
Hence, the development of nanoparticles with controlled structures that are uniform in
size, morphology, and functionality is essential for various biomedical applications.

The ZnO NPs occurring in a very rich variety of size and shape will provide a wide
range of properties. The methods include the chemical precipitation method, sol-gel
method, solid-state pyrolytic method, solution-free mechanochemical method, and
biosynthesis method.

Fig.5. Experimental Methods for the Synthesis of ZnO NPs

Chemical precipitation

The most popular method for ZnO NPs preparation is chemical precipitation, which
usually involves two reaction reagents: a highly purified zinc forerunner such as zinc
acetate, zinc nitrate or zinc sulphate and a solution of precipitator such as sodium
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hydroxide [15]. Typically, the precipitator is added dropwise to the dissolved zinc
precursor until the pH level reached about 10. Then, completely mix these solutions to
get a white intermediate of zinc hydroxide. Ultimately, the sample of zinc hydroxide
was converted to ZnO after sintering at high temperature. Bisht et al. synthesized ZnO
NPs using the chemical precipitation method [16].

Bettini et al. introduced a simplified precipitation approach in ZnO NPs using

ZnSO4 and NaOH solution with a molar ratio of 1:2, which was carried out under
vigorous stirring for 12 h at room temperature. The obtained white precipitate was
washed several times and separated by centrifugation [17–20]. Finally, the precipitate
(ZnO) was dried in an oven at 100°C for 6 h. The prepared ZnO NPs with a flake-like
structure showed a size distribution about 100 nm. The obtaining of ZnO NPs by the
chemical precipitation method is not only simple and easily controlled but also easy
industrialized.

Sol-gel method
A novel sol-gel synthesis of ZnO NPs was firstly presented by Spanhel and Anderson
[21], which mainly involved three major steps:

(1) Preparation of zinc precursor:

A sample of zinc acetate was dissolved in ethanol, placed into a distillation apparatus,
and then refluxed for few hours at atmospheric pressure. The solution was boiled nearly
at 80°C and stirred to obtain the condensate and hygroscopic reaction mixture.

(2) Preparation of ZnO clusters

The hygroscopic mixture was diluted to ethanolic solution with the addition of
LiOH·H2O powder. The suspension will become transparent with the help of an
ultrasonic bath. This procedure could accelerate the release of OH ions, and the reaction
at low temperature under air conditions could prevent rapid particle growth and get
ZnO sols.

(3) Crystal growth

Crystal growth is a self-induced procedure occurring at room temperature. But the

amount of LiOH which could strongly affect the crystals growth rate, shape, and size
should be well controlled. Rani et al. synthesized ZnO NPs using NaOH instead of

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LiOH and successfully obtained ZnO NPs with the largest crystallite size of 14 nm at 9
pH value [22].

Solid-state pyrolytic method

The solid-state pyrolytic method was first developed by Wang et al. with
advantages of low cost and easy operation for the growth of high-quality ZnO
nanoparticles [23]. The typical synthesis procedure is as follows:

Zinc acetate and sodium bicarbonate are mixed at room temperature. The
mixture is pyrolyzed at the reaction temperature. Zinc acetate is converted into ZnO,
while NaHCO3 is transformed into CH3COONa and can be eventually cleaned out with
deionized water. Subsequently, white ZnO NPs can be obtained via the thermal
decomposition process. The particle sizes can be regulated by selecting different
pyrolytic temperatures. Using this method, Wang et al. obtained ZnO NPs of different
sizes in the range of 8–35 nm.

Solution-free mechanochemical method

The advantages of this method are the low production costs and high homogeneity
of the crystalline structure and morphology [24]. But the morphology of the ZnO NPs
strongly depends on the milling time of the reactant mixture, a longer time of milling
led to a smaller particle size. The obtained ZnO NPs show an average size ranging from
24 to 40 nm. Pardeshi and Patil synthesized ZnO NPs with different morphologies and
crystallite sizes using this method by varying the calcination temperature from 400°C
to 900°C. It was found that zinc oxide calcined from 400°C to 550°C exhibited the
same crystallite growth rate (38–50 nm) [25].

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2.0 GREEN SYNTHESIS OF ZINC OXIDE
NANOPARTICLES
Zinc oxide plays an important role in current industry due to its special
characteristics such as anti-corrosion, anti-bacteria, has low electrons conductivity and
excellent heat resistance. Therefore, the objective of this study is to synthesize zinc
oxide nanostructures with the most practical ways. Zinc acetate dihydrate, zinc sulphate
or zinc nitrate etc., can be used as a precursor and ethanol (CH2COOH) was used as
solvent, Sodium hydroxide and distilled water were used as medium. ZnO
nanoparticles were characterized by using XRD, SEM, and nano-particles analyser.

Fig.6. Green Synthesis of Zinc Oxide NPs

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2.1 APPLICATIONS OF ZINC OXIDE
NANOPARTICLES:
 ZnO has different chemical and physical properties. It can be used in
numerous fields. Zinc oxide is important in a wide range of applications, from
medicine to agriculture, from paints to chemicals and from tires to ceramics.
 Zinc oxide nanoparticles (ZnO NPs) are used in an increasing number of
industrial products such as rubber, paint, coating.

Fig.7. Various applications of Zinc Oxide Nanoparticles

 Due to its outstanding biocompatibility, affordability, and low toxicity over the
past 20 years. ZnO NPs show excellent luminescent properties and have turned
them into one of the main candidates for bioimaging.
 Being nontoxic, ZnO has of late found extensive use as an excellent
photocatalyst for the degradation of a great many emerging organic pollutants.

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2.2 CATALYTIC ACTIVITY AND PHOTOCATALYTIC
DEGRADATION OF ORGANIC DYES:
Catalysts function by reducing the activation energy, which results in a reaction that
can proceed to completion without the need for external energy, a resource that has
recently become very important. To be more effective, the reactions should specifically
take place in environments with lower temperatures and pressures. In addition to
improving efficiency, this also significantly lowers costs. The Nanocatalysts made from
nanoparticles stand out among many other types of catalysts because they excellent
alternatives to conventional Nanocatalysts due to their outstanding surface area.
robustness, and stability.

In comparison to bulk catalysts, nanoscale catalysts have more open active surfaces,
allowing for effective contact between the reactant and the catalysts. The number of
catalytic sites exposed to the reaction was found to directly correlate with the catalytic
rate; a catalytic site with a bigger area for reactions to occur will provide a faster rate
of reaction and, consequently, a higher catalytic activity. Additionally, a bigger surface
area results in a greater number of reactive sites, which aids in the reaction's more
efficient conclusion. The insolubility of the catalysts non the reaction mixture is another
benefit, making it simple to separate and reuse the Nanocatalysts.

Metal-based nanoparticles have recently been regarded as being quite beneficial for a
variety of catalytic applications. Generally speaking, metal nanoparticles have a wide
range of applications, including enhancing the thermal stability of nanofluids and
creating materials with magnetic capabilities. In chemistry and materials science, the
creation of reliable, recyclable, environmentally friendly catalysis is seen as a major
task.

When the catalyst and the reactant are present in the same chemical or physical
structure, the reaction is said to be homogenous. Catalysts are categorized as
heterogeneous in a larger sense. The usage of these homogenous catalysts is common
in a number of industrial processes where the reactants and products are in the same
phase (either gaseous or liquid). Heterogeneous catalysts, on the other hand, are
catalysts that are in a distinct phase from the reactants. An example of this is a solid
catalyst that reacts with either liquid or gases. Compared to homogeneous catalysts,
heterogenous catalysts are thought to be more active, efficient, and selective.
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Metal nanoparticles in particular that have a large surface area and more active sites
encourage quicker reactions and boost product yield. Noble-metal (Au, Pt. Ag,
supported metal nanoparticles) and non-noble-metal (Fe, Cu, Ni, Co, etc.) based
nanoparticles are the two primary classes into which these particles can be generally
separated [28]. Some organic chemicals are persistent, bio accumulative, poisonous,
and produce hazardous by-product’s, their use and manufacturing contribute to
environmental degradation. Due to their catalytic function in the reduction and
degradation of dyes, MNPs have attracted a lot of interest [29]. Methyl orange and
rhodamine B are two cationic dyes that room-temperature green synthetic ZnO-NPs
have a high degradation activity. In this study, we examined the degradability of ZnO
NPs using methyl orange and rhodamine B [30].

2.2.1 METHYL ORANGE

Methyl orange (C14H14N3NaO3S) is an azo dye which comes under acid-base
indicators which exhibit a colour change from red to yellow with increasing basicity.
An azo dye is prepared from benzene, sulfanilic acid, and dimethylaniline. It is used in
titration that gives different colours at different pH values. It is an indicator that tells
whether a substance is acidic or basic. It is abbreviated as MeO. It gives a red colour
with acids and yellow with the basic medium. Despite not having a full spectrum of
colour change, it gives sharp end points. The structure of methyl orange is shown below,

Fig.8. Structure of Methyl Orange

When acidity decreases in a solution, methyl orange moves from red to orange and
finally to yellow, the opposite occurs with the solution when acidity increases. The pH
range of methyl orange is 3.1-4.4 and is mostly used for acidic solutions.

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It is a pH indicator that is used for titration in laboratories and enterprises because it
exhibits clear and noticeable colour fluctuations at various pH levels. solution. At pH
4.3, a transition occurs in Methyl Orange. It shows red colour – Acidic Medium, below
pH 3.1 Orange colour – weak Acid, above 4.4 Yellow Colour – Basic medium, basic
solution.

2.2.2 RHODAMINE B
Rhodamine B is an organic chloride salt with the counterion N-19-(2-
carboxyphenyl)-6--(diethylamino)-31-xanthen-3-ylidene)-N-ethylethanaminium is an
amphoteric dye that is frequently employed as a fluorochrome. It serves as a fluorescent
probe, a histology dye, and a fluorochrome. It is a xanthene colouring and an organic
chloride salt. Additionally, it is a cationic dye. When rhodamine B green solid crystal
is dissolved in water, the dye's colour changes to a bluish-red hue with an absorption
peak at 556 nm. The structure of rhodamine b [31] is seen in Fig.9

Fig.9. Structure of Rhodamine B

Rhodamine B (RhB) is a chemical that is frequently used in industry for things like
printing and dyeing textiles, paper, paints, and leathers, as well as medications and
cosmetics. To create pink fluorescent pigments, rhodamine B is employed. However,
organic dyes can cause irritate to the skin, eyes, and pose major biological and
environmental risks. Thus, removal of dye from water is a difficult task. Conventional
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procedures for removing RB include physical-chemical and biological techniques such
liquid membrane, onto, and adsorption, which are costly and inefficient. Based on
Semiconductor heterogeneous photocatalysis, photocatalysts provide promising
application for the organic dye decomposition with superior activity [32].

Fig.10. Representation of Dye Degradation

Using ZnO NPs

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2.3 PLANT INTRODUCTION:
Pennisetum pedicellatum, known simply as desho or as desho grass, is an indigenous
grass of Ethiopia of the monocot angiosperm plant family Poaceae. It is also known as
annual kyasuwa grass in Nigeria, bare in Mauritania, and deenanath grass in India. It
grows in its native geographic location, naturally spreading across the escarpment of
the Ethiopian highlands. Nigeria grass (Pennisetum pedicellatum Trin) is a many-
branched leafy annual grass up to 1 m high. The culms are erect and branching, and the
leaves are 15-25 cm long and 4-10 mm wide, flat and glabrous. The inflorescence is a
pink to purple, dense flowered, cylindrical panicle. The spikelets are 4 mm long, usually
solitary. It is used as fodder.

In India, it is considered to be a very palatable species to cattle, but its palatability

has been reported as average in the Sahel. It provides high yields of green herbage
ranging from 30 to 109 tonnes per hectare, comparing favourably with Sorghum
bicolour or other Pennisetum species. It responds well to fertilizer and can be combined
with fodder legumes, either in mixtures or in rotation cropping. In short rotation with
maize or groundnuts it yields better than traditional forage grasses, especially with
adequate fertilizer, while the roots and stubbles also contribute to soil fertility.

To obtain the highest yield, Nigeria grass should be cut 4 months after sowing at 8
cm from ground level. Nigeria grass is used in temporary pastures or in cut-and-carry
systems since it provides huge quantities of good quality green forage and can stand
several cuts per year. Nigeria grass is also useful for hay and silage.

21
 Fig:11 The plant of P. Pedicellatum

Pennisetum pedicellatum prefers drier sites, savannas and forest edges. It can infest
croplands, grasslands, and waste places, and can be difficult to eradicate.

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2.3.1 TAXONAMICAL CLASSIFICATION:

Kingdom : Plantae

Clade : Tracheophytes

Class : Liliopsida

Order : Poales

Family : Poaceae

Genus : Pennisetum

Species : P. pedicellatum

Botanical Name : Pennisetum Pedicellatum

Common Name : Deenanath Grass

2.3.2 OCCURANCE:
Nigeria grass is native to West Africa and was first introduced to India from
where it has spread to South East Asia and Northern Australia (Schmelzer, 1996).

Pennisetum pedicellatum prefers drier sites, savannas and forest edges. It can infest
croplands, grasslands, and waste places, and can be difficult to eradicate. It grows in its
native geographic location, naturally spreading across the escarpment of the Ethiopian
highlands. Widely available in this location, it is ideal for livestock feed and can be
sustainably cultivated on small plots of land. Thus, desho is becoming increasingly
utilized, along with various soil and water conservation techniques, as a local method
of improving grazing land management and combating a growing productivity problem
of the local region.

2.3.3 MEDICINAL USES

Pennisetum pedicellatum is an aggressive weed, used for treatment of wounds,
pain related condition, eye and parasitic infections among the traditional healers in
Kebbi State, Nigeria
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2.4 PHYTOCHEMISTY (CHEMICAL CONSTITUENTS)
The phytochemistry of P. Pedicellatum showed a wide range of chemical
compounds including flavones, phenolic compounds etc.. These compounds are
separated from the aerial part of the plant. Physiochemical properties of nanoparticles
and plant extract are important for their behaviour, bio-distribution, safety and efficacy.

2.5 ANTIBACTERIAL ACTIVITY

ZnO NPs can be selected as an antibacterial material because of its superior
properties, such as high specific surface area and high activity to block a wide scope of
pathogenic agents. But recently, the antibacterial activity of ZnO NPs is still scarcely
known. Prior reports had suggested the main antibacterial toxicity mechanisms of ZnO
NPs were based on their ability to induce excess ROS generation, such as superoxide
anion, hydroxyl radicals, and hydrogen peroxide production [10]. The antibacterial
activity may involve the accumulation of ZnO NPs in the outer membrane or cytoplasm
of bacterial cells and trigger Zn2+ release, which would cause bacterial cell membrane
disintegration, membrane protein damage, and genomic instability, resulting in the
death of bacterial cells.

Fig.12. The Antibacterial Reaction of ZnO NPs

To explore the antibacterial action of ZnO NPs, Gram-positive Staphylococcus aureus

(S. aureus) and Gram-negative Escherichia coli (E. coli) are frequently used as

24
prototype bacteria. Investigations are also being conducted on a number of other Gram-
positive and Gram-negative bacteria, including Proteus vulgaris (P. vulgaris), Bacillus
subtilis (B. subtilis), Pseudomonas aeruginosa (P. aeruginosa), and Vibrio cholerae (V.
cholerae). It has been found that ZnO NPs produced using green methods outperformed
chemically produced ZnO NPs and other widely used antimicrobials in their ability to
suppress the growth of bacteria like Staphylococcus aureus and Escherichia coli.

ZnO NPs' bioactivity has increased significantly as a result of their tiny size and
higher surface area to volume ratio. Despite the fact that the mechanism by which ZnO
NPs display antibacterial activity is still being researched, the most recent
investigations into this subject have identified a few likely pathways: ZnO NPs are
discovered to directly interact with cell surfaces, disrupting cell membrane permeability
(a); produce ROS on the ZnO NPs' surface; and (c) The breakdown of cellular zinc ion
homeostasis due to particle dissolution and subsequent release of free Zn2+ ions in the
cytoplasm (and particularly in lower pH cellular organelles like lysosomes) causes an
increase in intracellular ROS levels, which ultimately causes cell necrosis. Additionally,
it's probable that the aforementioned mechanisms combine to produce the antibacterial
properties of ZnO NPs. It was found that bacteria treated with ZnO nanoparticles were
bound to experience cell membrane damage.

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 CHAPTER II

SCOPE OF THE PRESENT

INVESTIGATION

26
SCOPE OF THE PRESENT INVESTIGATION
The field of nanotechnology is one of the most attractive areas of
research in modern materials science. An important branch of the
biosynthesis of nanoparticles was synthesized from plant extract. However,
rapid and green synthesis method using plant extract has developed
enormous interest in zinc oxide nanoparticles due to green chemistry
approach. Moreover, it is simple, cost effective, eco-friendly, easily scaled
up for large scale synthesis. Biological assisted method is named as green
synthesis of zinc oxide nanoparticles and does not release harmful by-
products to the environment; hence the present study is aimed at rapid
green synthesis of zinc oxide nanoparticles.

For the past few years, faster synthesis rates various chemical methods
have been developed for the synthesis of ZnO NPS If biological synthesis
of NPs can compete with chemical methods, there is a need to achieve
faster synthesis rates. This lengthy reaction is one of the major drawbacks
of biological synthesis Biological NP's synthesis would have greater
commercial viability if the NPs could be synthesized more rapidly in the
reaction vessels. The exact mechanism of ZnO NPS synthesis by plant
extract is not fully understood.

The review of the previous literature studies, research works and articles
reveals that there is no report on the green synthesis of any metal
nanoparticles using the fresh leaf extract Pennisetum pedicellatum.

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The objective of the study is,

1. To synthesis zinc oxide nanoparticles by using Pennisetum

pedicellatum plant extract.
2. To determine the phytochemicals, present in the plant extract of P.
Pedicellatum.
3. To characterize the synthesized zinc oxide nanoparticles.
4. Photocatalytic degradation of organic dyes.
 Methyl orange
 Rhodamine B
5. To determine the antimicrobial activity of zinc oxide nanoparticles.

28
CHAPTER III

EXPERIMENTAL
METHODS

29
 EXPERIMENTAL METHODS

3.1. Chemicals Used

3.2. Collection of Sample

3.3. Preparation of Plant Extract

3.4. Working Procedure

3.5. Green Synthesis of ZnO NPs Using P.

Pedicellatum Plant Extract

3.6. Characterization of ZnO NPs

3.7. Antimicrobial Activity

3.8. Phytochemical Screening Tests

3.9 Photocatalytic Degradation of Organic Dyes

30
 EXPERIMENTAL METHODS

3.1 CHEMICALS USED

Zinc Acetate, Sodium Hydroxide, Ethanol, Rhodamine B, Methyl Orange,
Double and Triple distilled water which was used for the entire synthesis.

3.2 COLLECTION OF SAMPLE

The fresh leaves of Pennisetum Pedicellatum was collected from
Manonmaniam Sundaranar University, Abishekapatti, Tirunelveli, Tamil Nadu India
During the month of January 2023. The plant was authenticated by DR. M.
Udhayakumar, Taxonomist, Assistant Professor, Department of Plant Science, M. S.
University. The fresh leaf was thoroughly washed with running tape water followed by
distilled water to remove the dust. It was dried at room temperature. The voucher
specimen was stored in our department.

3.3 PREAPARATION OF PLANT EXTRACT

About 15g of freshly collected leaves of P. pedicellatum was thoroughly
washed several times with running tape water and further the leaves are chopped into
pieces. The extract was prepared by means of SOXHLET EXTRACTION METHOD.
200 ml of deionised water is used as a solvent for extraction. The temperature
maintained in this process is 70-80°C.

31
 Fig.13. Zinc Oxide NPs

3.4 WORKING PROCEDURE

The solid material ie) leaves of P. pedicellatum is placed inside a thimble. The
Soxhlet extractor is placed onto a flask containing the extraction solvent. The Soxhlet
is then equipped with a condenser. The solvent is heated to reflux.

The solvent vapour travels up a distillation arm and floods into the chamber
housing the thimble of the solid. Solid material in chamber slowly fills warm solvent.
Desired compound dissolve in the warm solvent. When the Soxhlet chamber is almost
full, the chamber gets automatically emptied by a siphon side arm, with the solvent
running back down to the distillation flask.

After extraction the solvent is removed, typically by means of a rotary

evaporator, yielding the extracted compound. The solid particles which remain in the
thimble is usually discarded.

32
 Fig.14. Soxhlet Extraction

3.5 GREEN SYNTHESIS OF ZnO NPS USING P.

PEDICELLATUM PLANT EXTRACT:
Zinc acetate dihydrate {Zn (CH3COO)2·2H2O} and Sodium Hydroxide
(NaOH) reagents were used. Briefly, 2.5 ml of plant extract was added to 25 ml 0.5 M
Zinc acetate dihydrate. The pH of the mixture was 6.13, 2 M NaOH was added drop
wise to maintain pH at 8, then the mixture was then stirred and heated at 70 °C for 2
hours for complete reduction and formation of a white precipitate.

The resulting material was then collected via decantation, washed with distilled water
to remove residuals and oven dried at 70 °C for overnight to yield powdered ZnO

33
nanoparticles. The dried sample was stored at room temperature in airtight container
for further characterization and applications.

3.6 CHARACTERIZATION OF ZnO NPS:

The synthesized ZnO NPs from the leaf extract of P. Pedicellatum were
confirmed and characterized using double-beam UV–visible spectrophotometer in the
range of 200–800 nm to observe the characteristic peak confirming ZnO NPs formation.
Fourier transform infrared spectroscopy (FT-IR) in the range of 400–4000 cm−1 is used
for the determination of the functional groups and phytoconstituents contributing to the
reduction and stabilization of the ZnO NPs. Scanning electron microscope, and X-ray
diffractometer (Bruker D8 Discover Diffractometer, USA) were used to analyze the
surface morphology, identify the elemental composition, size, and shape of green
synthesized ZnO NPs.

3.7 ANTIMICROBIAL ACTIVITY:

The antimicrobial activity of green synthesized ZnO NPs, P. Pedicellatum plant
extract was tested against pathogenic microorganisms including Gram positive bacteria,
Staphylococcus aureus (ATCC 6538). The antimicrobial activities of the synthesized
zinc oxide NPs were carried out using the agar well diffusion method. Overnight
cultures of the test organisms (100 µL) were spread on Mueller–Hinton (MH) (Biolab
Merck, Germany) agar plates. Wells with diameters of about 6 mm were created
aseptically using sterile micropipette tips on the agar plates. Twenty microliters of
different synthesized zinc oxide nanoparticle ratios (Zn acetate: urea) (1:1, 1:2, 1:4, 2:1
μg·mL−1) were loaded in the MH agar wells. The positive controls used were a
standard commercial antibiotic, neomycin (30 mg·mL−1), for bacterial isolates and
amphotericin B (30 mg·mL−1) for yeasts, and they were added in one of the wells. The
MH plates were incubated at 37 °C for 24 h. Any resulting clear zone (in mm) (zone of
inhibition) around the wells was measured.

3.8 PHYTOCHEMICAL SCREENING TESTS:

The presence of active phytoconstituents in P. Pedicellatum leaf extract was
analysed by standard qualitative method.

 Test for alkaloids

34
To a few drops of extract add con. Nitric acid and then it is evaporated to dryness. Cool
and then add NH4OH.

Formation of purple colour indicates the presence of alkaloids.

 Test for saponins

To a few drops of alcoholic extract distilled water is added. Shake well.

Formation of foames indicates the presence of saponins.

 Test for carbohydrates

To a few drops of extract add benedict’s reagent and boil over a flame.

Formation of green, brown red indicates the presence of carbohydrates.

 Test for coumaric glycosides

To a few drops of alcoholic extract add NaOH .

Blue or green fluorescence indicates the presence of coumaric glycosides.

 Test for amino acids

To a few drops of extract add ninhydrin reagent. Boil for few minutes.

Formation of blue or green colour indicates the presence of amino acid.

 Test for steroids (SALKOWSKI REACTION)

To a few drops of extract add con. Sulphuric acid. Shaken well and allowed to stand for
sometime.

Formation of red colour at lower layer indicates the presence of steroids.

 Test for terpenoids

To a few drops of extract add chloroform and evaporate to dryness. To the above
mixture add con. Sulphuric acid and heat for 2 minutes.

Formation of greyish colour indicates the presence of terpenoids.

 Test for anthraquinones

To a few drops of extract add benzene.

35
Formation of pink, red or violet colour indicates the presence of anthraquinones.

 Test for glycosides

To a few drops of alcoholic extract add distilled water and aquous NaOH solution

Formation of yellow colour indicates the presence of glycosides.

 Test for coumarin

To a few drops of extract add chloroform and sodium hydroxide solution.

Formation of yellow colour indicates the presence of coumarin.

 Test for quinones

To a few drops of extract add 1 ml con. Sulphuric acid.

Formation of red colour indicates the presence of quinones.

 Test for flavonoid

To a few drops of extract add distilled water. Warm and filter, then add 10% aq. NaOH.

Formation of yellow colour indicates the presence of flavonoids.

 Test for lipids

Place small quantity of extract in between two filter paper.

Formation of oil stain indicates the presence of lipids.

3.9 PHOTOCATALYTIC DEGRADATION OF ORGANIC

DYES
The catalytic dye reduction ability of ZnO-NPs was performed against
rhodamine b and methyl orange under light visible irradiations. About 0.1 g of
rhodamine b and methyl orange is weighed and made up to 100 ml in a SMF using
distilled water separately. After that 3 ml of the above dyes are pipetted out in a test
tube individually in the ratio of 3:1. To this about 1 ml of supernatant liquid of
synthesised ZnO nanoparticle is added. It was kept under light for visible radiations and
then the degradation was observed for a specific interval of time.

36
CHAPTER IV

RESULTS AND
DISCUSSION

37
QUALITATIVE PHYTOCHEMICAL SCREEN ANALYSIS
Phytochemical screening of P. Pedicellatum leaf extract

S.NO PHTOCHEMICALS AQUOUS

EXTRACT

1 alkaloids _
2 saponins ++
3 carbohydrates ++
4 Coumaric glycosides _
5 Amino acids _
6 steroids _
7 terpenoids _
8 Anthro quinone _
9 glycosides _
10 coumarin ++
11 quinones _
12 flavonoid ++
13 Lipids ++

38
CHARACTERIZATION OF ZnO NANOPARTICLES:

4.1. Colour change

The zinc oxide NPs was synthesised from the aquous zinc ions using NaOH and
the leaf extract of P. Pedicellatum act as a reducing and capping agent. The above said
reaction mixture is kept in stirrer at 70° C. The colourless reaction mixture slowly
changes to yellowish. This colour change within 2 hours has indicated the formation of
ZnO NPs. The formation of ZnO NPs were periodically characterised by UV-Vis
spectra, FTIR, SEM, and XRD.

4.2. UV-VISIBLE ANALYSIS

P. Pedicellatum leaf extract mediated green synthesised ZnO NPs was
characterised by UV-Visible spectroscopy. Size and shape of the nanoparticle with high
sensitivity exhibits the absorbance of surface plasma resonance (SPR). The sharp
absorbance at 345 nm has confirmed the formation of ZnO nanoparticles.

Fig.15. UV-Vis spectrum of ZnO Nanoparticles

39
4.3. FOURIERTANSFORM INFRARED

SPECTROSCOPY:
FTIR measurement wee indicated the disappearance and appearance of some
characteristics wave numbers values, decrease and increase of intensities were
identified by the biomass of P. Pedicellatum leaf extract and biosynthesis of ZnO
nanoparticles. The FTIR spectrum of leaf extract and biosynthesized ZnO nanoparticles
showed distinct peak values at 3697.55, 1638.54, 1384.92, 1400.49, 1004.73, 1024.03,
679.84 cm-1.

Fig.16. FT-IR spectrum of plant extract

40
 Fig.17. FT-IR spectrum of ZnO nanoparticles

A broad peak at 3697.55 cm-1 shows O-H stretching vibration due to hydroxyl
group, peak value at 1638.54 indicates C=C ring stretching vibration of aromatics. The
peak value at 1384.92 cm-1 are ascribed to secondary alcohol in-plane bend or vibration,
the peak value at 1004.73 cm-1 is ascribed to the stretching vibration of C-N bond of
the primary amine or to the stretching vibration of the C-O bond of the primary
alcohol[38]. The peak value at 1400.49 may be due to -CH stretching vibration of
dimethyl group, the peak value at 1024.03 cm-1 is ascribed to the stretching vibration
of C-N bond of the primary amine or to the stretching vibration of the C-O bond of the
primary alcohol [39] and the peak value at 679.84 shows either the presence of
hydroxyl or halogen groups impurities.

41
4.4. X- RAY DIFFRACTION SPECTROSCOPY:
The crystalline nature of the synthesised ZnO Nanoparticles were confirmed by
the X-ray diffraction analysis. The XRD pattern of ZnO NPs shows the presence of
sharp frequency of 2 theta values at 32.1, 34.75, 36.58, 47.84, 56.85, 63.11, 68.21. The
peaks corresponding to the 2 theta of the ZnO NPs confirms the crystalline nature of
the sample [40]. A definite line broadening of the XRD peaks indicates that the prepared
material consist of particles in nanoscale range. It also confirms that the synthesized
nano powder was free of impurities as it does not contain any characteristics XRD peaks
other. The XRD pattern in the above figure:17 shows that ZnO NPs are formed in the
reduction of metal ions by the leaf extract of P. Pedicellatum.

Fig.18. XRD spectrum of ZnO Nanoparticles

42
4.6 PHOTOCATALYTIC DEGRADATION OF ORGANIC
DYES

i) Degradation of Rhodamine B
The degradation occurred in rhodamine b absorption spectra corresponding at 551
nm. The degradation activity of ZnO nanoparticles was assessed for RhB dye under
UV- light irradiation at specific conditions. The UV-Visible spectrum of the above dye
was analyzed at different time interval (3 min) in the presence of ZnO – NPs is shown
in fig.19. The complete degradation of RhB dye was absorbed at 18 min with the
surface plasmon resonance band in 551 nm completely disappeared due to the effect of
green synthesized ZnO NPs from plant extract.

Fig.19. UV-Vis spectrum of ZnO NPs against Rhodamine B

dye under visible light irradiation

43
ii)Degradation Of Methyl Orange:
The degradation occurred in methyl orange absorption spectra corresponding
at 464 nm. The degradation activity of ZnO nanoparticles was assessed for methyl
orange under UV- light irradiation at specific conditions. The UV-Visible spectrum of
the above dye was analyzed at different time interval (2 min) in the presence of ZnO –
NPs is shown in fig.20. The complete degradation of methyl orange dye was absorbed
at 10 min with the surface plasmon resonance band in 464 nm completely disappeared
due to the effect of green synthesized ZnO NPs from plant extract

Fig.20. UV-Vis spectrum of ZnO NPs against methyl

Orange dye under visible light irradiation

44
 iii)Degradation of mixed Rhodamine b and methyl Orange:
The degradation occurred in mixed rhodamine b and methyl orange absorption
spectra corresponding at 553 nm. The degradation activity of ZnO nanoparticles was
assessed for mixed RhB and methyl orange dye under UV- light irradiation at specific
conditions. The UV-Visible spectrum of the above dye was analyzed at different time
interval (2 min) in the presence of ZnO – NPs is shown in fig.21. The complete
degradation of mixed RhB and methyl orange dye was absorbed at 6 min with the
surface plasmon resonance band in 553 nm completely disappeared due to the effect of
green synthesized ZnO NPs from plant extract.

Fig.21. UV-Vis spectrum of ZnO NPs against mixed

Rhodamine B and methyl orange dye under visible light
irradiation

45
4.7. ANTI BACTERIAL ACTIVITY:
A qualitative agar well diffusion test was performed to screen ZnO NPs for their
antimicrobial properties against gram positive bacteria (staphylococcus aureus). The
growth inhibiting effect of ZnO NPs was shown by the formation of clear zones on the
Mueller-Hinton Agar Plates. The larger the zone of inhibition, the more sensitive the
test organism is to the nanoparticle, thus indicating the effectiveness of the
nanoparticles.

Fig.22. Antibacterial activity of green synthesized ZnO NPs

The inhibition zones formed around the nanoparticles is shown in fig:22. The zones
of inhibitions around the controls were ≥10 mm. These zones were larger than those
formed around the test organisms, which confirms that the commercial antibiotic
exhibits higher growth inhibitory effect against Gram Positive Bacteria. The bacterium
staphylococcus aureus has the inhibition zone at 100 µL concentration. This is due to
the strong electrostatic interaction between bacterial organisms and nanoparticles,
which leads to oxidise the bacterial cell wall to destruct.

46
 CHAPTER V

CONCLUSION

47
 In conclusion, P. pedicellatum is easily available and one of the most

important medicinal plant species. We have synthesised ZnO NPs from

aquous solution of zinc acetate using reducing and stabilising agents. This

method is an economically efficient eco-friendly and simple process.

Further the synthesis of ZnO NPs is confirmed by the colour, UV-Vis

spectroscopy absorption value at 345 nm. FTIR shows appearance and

disappearance of some functional groups. XRD analysis revealed that the

particle is crystalline. Finally, the antibacterial activity of nanoparticles has

proven the biological significance in environmental application.

48
CHAPTER VI

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