1. The document discusses the synthesis of silver nanoparticles from green spinach leaves using silver nitrate solution. Silver nanoparticles were extracted from the supernatant after centrifuging the solution containing spinach extract and silver nitrate that was incubated and shaken. 2. Further analysis of the silver nanoparticles can be done using techniques like X-ray diffraction, SEM, TEM to determine their crystalline and chemical properties. Silver nanoparticles have a variety of applications due to their exceptional properties like antibacterial activity and high thermal conductivity. 3. The document then discusses the various methods of synthesizing silver nanoparticles, their properties, and applications - particularly in the medical field as antibacterial agents. Both physical and biological synthesis methods are described.

1. 1
Abstract
Nanotechnology refers to the creation and utilization of materials and is
expected to open new avenue to fight and prevent disease.Nanoparticles are
being used in diverse purposes, for medical treatments, using in various
branches of industry production such as solar and oxide fuel batteries for energy
storage, and also used in diverse materials of everyday use such as cosmetics or
cloths catalytic bactericidal, and treatment of some cancers. Due to their
exceptional properties including antibacterial activity, high thermal
conductivity, nanoparticles have attracted considerable attention in recent
years.In this project synthesised silver nanoparticles from the green spinach by
using silver nitrate solution and green spinach leaves are dried under sun light
and extracted. mix the green spinach extract with silver nitrate solution and
mixed well with orbital shaker then incubated the solution and centrifuged .the
supernatant was suspended and pellet was collected and dried under sunlight
then the silver nanoparticles was extracted.Further analysis can be done by
using X-ray diffraction, SEM, TEM, spectroscopic method in course of
determining the crystalline and chemical properties of AgNPs.
Keywords:silver nanoparticles, silver nitrate,incubator , Green spinach ,orbital
shaker and incubator

2. 2
INTRODUCTION
Materials in the nano dimensions (1 - 100 nm) have remarkable difference in
the properties compared to the same material in the bulk. These differences lie
in the physical and structural properties of atoms, molecules, and bulk materials
of the element due to difference in physiochemical properties and surface to
volume ratio. With advancement in nanotechnology, many nanomaterials are
appearing with unique properties, opening spectrum of applications and
research opportunities.
About five thousand years ago, many Greeks, Romans, Persians, and Egyptians
used silver in one form or other to store food products.Use of silver ware during
ancient period by various dynasties was common across the globe utensils for
drinking and eating and storing various drinkable and eatable items due to the
knowledge of antimicrobial action. There are records regarding therapeutic
application of silver in literature as earlier as 300 BC. In the Hindu religion, till
date silver utensils are preferred for the “panchamrit” preparation using curd,
Ocimum sanctum and other ingredients. The therapeutic potentials of various
metals are mentioned in ancient Indian Arecid medicine book medicinal
literature named “Charak Samhita”. Until the discovery of antibiotics by
Alexzander Flemming, silver was commonly used as antimicrobial agent.
In the recent past, silver nano particles (AgNps) have received enormous
attention of the researchers due to their extraordinary defense against wide
range of microorganisms and due to the appearance of drug resistance against
commonly used antibiotics. The exceptional characteristics of Agnosy have
made them applicable in various fields like biomedical , drug delivery , water
treatment, agricultural etc.
AgNps are applied in inks, adhesives, electronic devises, pastes etc. due to high
conductivity. AgNps have been synthesized by physio-chemical techniques
such as chemical reduction , gamma ray radiation , micro emulsion,
electrochemical method, laser ablation, autoclave , microwave, and
photochemical reduction . These methods have effective yield, but they are
associated with the limitations like use of toxic chemicals and high operational
cost and energy needs. Considering the drawbacks of physio-chemical methods,
cost-effective and energy efficient new alternative for AgNP synthesis using
microorganisms, plant extracts and natural polymersas reducing and capping
agents are emerging extremely fast. The association of nanotechnology and -
green chemistry

3. 3
GREEN CHEMISTRY
The primary requirement of green synthesis of Agop’s is silver metal ion
solution and a reducing biological agent. In most of the cases reducing agents or
other constituents present in the cells acts as stabilizing and capping agents, so
there is no need of adding capping and stabilizing agents from outside.
Metal Ion Solution
The Ag+
ions are primary requirement for the synthesis of agapes which can be
obtained from various water-soluble salts of silver. However, the aqueous
AgNO3 solution with Ag+
ion concentration range between 0.1 - 10 mm (most
commonly 1 mm) has been used by most researchers.
Biological Reducing Agents
The reducing agents are widely distributed in the biological systems. The
Agop’s have been synthesized using different organisms belonging to four
kingdoms out of five kingdom of living organisms i.e., Monera (prokaryotic
organisms without true nucleus) Protista (unicellular organisms with true
nucleus), fungi (eukaryotic, saprophyte/parasite), plantae (eukaryotic,
autotrophs) and animalia (eukaryotic, heterotrophs). Data are not available
regarding use of animal materials for the synthesis of Gap’ till date to the best
of our knowledge. Due to this limitation, green synthesis of agapes has been
discussed under headings microorganisms, plants, and bio-poly-mers.Green
syntheses of Agop’s have been performed using plant extracts, microbial cell
biomass or cell free growth medium and biopolymers. The plants used for Agno
synthesis range from algae to angiosperms; however, limited reports are
available for lower plants and the most suitable choice are the angiosperm
plants. Parts like leaf, bark, root, and stem have been used for the Gan synthesis.
The medicinally important plants like Boerhaavia diffusa, Tensor cordifolia,
Aloe vera , Terminalia chebulagic Catharanthus roseus , Ocimum tubiform,
Azadirachta indica, Emblica officinalis , Cocos nucifera, common spices Piper
nigrum , Cinnamon zeylanicum. Some exotic weeds like Parthenium
heterophorias’ growing in uncontrolled manner due to lack of natural enemies
and causing health problems to have also been used for AgNP’s synthesis. The
other group includes alkaloids (Papaver somniferum) and essential oils (Mentha
piperita) producing plants. All the plant extracts played dual role of potential
reducing and stabilizing agents with an exception in few cases where external
chemical agents like sodium-do-decyl sulphate were used for stabilization the
AgNPs. Metabolites, proteins, and chlorophyll present in the plant extracts were
found to be acting as capping agents for synthesized AgNPs.
The preferred solvent for extracting reducing agents from the plant iin most of
the cases however, there are few reports regarding the use of organic solvents
like methanol, ethanol, and ethyl acetate. Some researchers pre-treated the
plants materials in saline or acetone atmospheres before extraction. Overall,
even though the extracting solvents differed, the nanoparticle suspensions have
made in aqueous medium only. Synthesis using plant extracts generate

4. 4
nanoparticles of well-defined shape, structure, and morphology in compared to
those obtained through the utilization of bark, tissue, and whole plant.
The AgNPs synthesis by microbes is strenuous compared to the use of plant
extracts and biopolymers as reducing and capping agents mainly to the
difficulty in growth, culture maintenance, and inoculums size standardization.
Several fungal and bacterial species have been successfully used in the
synthesis. The AgNPs synthesis mainly followed one of the two distinct routes,
one utilizing extracellular materials secreted in the growth medium whereas the
other utilizing microbial cell biomass directly. The microbes synthesize AgNP
intracellularly as well as extracellularly. The Intracellular synthesis of AgNPs
was observed by few researchers.
AgNPs synthesis supports better control on size and shape of AgNPs, due to
easy down streaming and larger adaptability to nano systems. However,
extracellular AgNP synthesis has been widely reported. One of the commonly
used fungal genera for synthesizing AgNPs is Fusarium. No special capping
agent was used in the work of many researchers for stabilizing synthesized
AgNPs, except Perni et al.and Shahverdi etal.who used L-cystine and piperitone
as stabilizing agents, respectively? Among the wide varieties off biopolymers
used for AgNP synthesis, almost all played the dual role of reducing and
stabilizing agents except for using starch as a capping agents

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PROPERTIES OF SILVER NANO PARTICLES
Physical and chemical properties of AgNPs—including surface chemistry, size,
size distribution, shape, particle morphology, particle composition,
coating/capping, agglomeration, dissolution rate, particle reactivity in solution,
efficiency of ion release, cell type, and finally type of reducing agents used for
synthesis—are crucial factors for determination of cytotoxicity For example,
using biological reducing agents such as culture supernatants of
various Bacillus species, AgNPs can be synthesized in various shapes, such as
spherical, rod, octagonal, hexagonal, triangle, flower-like, and so on Previous
studies supported the assertion that smaller size particles could cause more
toxicity than larger, because they have larger surface area Shape is equally
important to the determination of toxicity . For example, in the biomedical field,
various types of nanostructures have been used, including nanocubes,
nanoplates, nanorods, spherical nanoparticles, flower-like, and so on AgNP
toxicity mainly depends on the availability of chemical and or biological
coatings on the nanoparticle surface. AgNP surface charges could determine the
toxicity effect in cells. For instance, the positive surface charge of these NPs
renders them more suitable, allowing them to stay for a long time in blood
stream compared to negatively-charged NPs which is a major route for the
administration of anticancer agents.
Colloidal silver nanoparticles are widely used for its unique properties in
catalysis , chemical sensing, biosensing, photonics, electronics, and
pharmaceuticals. The diversity and importance of these applications has
generated a great deal of interest in developing versatile methods to synthesize
silver nanoparticles with well defined and controlled properties. Several
approaches used to date includes reduction in solutions; chemical and
photochemical reaction in reverse micelles; thermal decomposition of silver
compounds; radiation assisted, electrochemical; and recently, biosynthesis using
living plant system. Some well known examples of biosynthesis of metal
nanoparticles are gold nano-triangles using Lemmon grass and tamarind leaf
extract .
Synthesis of silver nanoparticles using green spinach leaf Recently synthesis of
silver nanoparticles at room temperature from the extract of parthenium
hysterophorous leaves has been reported but not studied in detailed the
photoluminescence property as well as the variation of particle size with the
reaction temperature and time of reaction.
The medical properties of silver have been known for over 2,000 years. Since
the nineteenth century, silver-based compounds have been used in many
antimicrobial applications. Nanoparticles have been known to be used for

6. 6
numerous physical, biological, and pharmaceutical applications. Silver
nanoparticles are being used as antimicrobial agents in many public places such
as railway stations and elevators in China, and they are said to show good
antimicrobial action.
It is a well-known fact that silver ions and silver-based compounds are highly
toxic to microorganisms which include 16 major species of bacteria. This aspect
of silver makes it an excellent choice for multiple roles in the medical field.
Silver is generally used in the nitrate form to induce antimicrobial effect, but
when silver nanoparticles are used, there is a huge increase in the surface area
available for the microbe to be exposed to. Though silver nanoparticles find use
in many antibacterial applications, the action of this metal on microbes is not
fully known. It has been hypothesized that silver nanoparticles can cause cell
lysis or inhibit cell transduction. There are various mechanisms involved in cell
lysis and growth inhibition.
There are many ways depicted in various literatures to synthesize silver
nanoparticles. These include physical, chemical, and biological methods. The
physical and chemical methods are numerous in number, and many of these
methods are expensive or use toxic substances which are major factors that
make them ‘not so favored’ methods of synthesis. An alternate, feasible method
to synthesize silver nanoparticles is to employ biological methods of using
microbes and plants.Silver nanoparticles find use in many fields, and the major
applications include their use as catalysts, as optical sensors of zeptomole
(10−21
) concentration, in textile engineering, in electronics, in optics, and most
importantly in the medical field as a bactericidal and as a therapeutic agent.
Silver ions are used in the formulation of dental resin composites; in coatings of
medical devices; as a bactericidal coating in water filters; as an antimicrobial
agent in air sanitizer sprays, pillows, respirators, socks, wet wipes, detergents,
soaps, shampoos, toothpastes, washing machines, and many other consumer
products; as bone cement; and in many wound dressings to name a few. Though
there are various benefits of silver nanoparticles, there is also the problem of
nanotoxicity of silver. There are various literatures that suggest that the
nanoparticles can cause various environmental and health problems, though
there is a need for more studies to be conducted to conclude that there is a real
problem with silver nanoparticles.

7. 7
This review provides an idea of the antimicrobial properties silver possesses as
a nanoparticle, the various methods employed to synthesize silver nanoparticles,
and an overview of their applications in the medical field and discusses the
toxicity of silver nanoparticles. The focus is on the characteristics of silver
nanoparticles which make them excellent candidates for use in the medical field
besides delving into the unique ability of certain biological systems to
synthesize silver nanoparticles and looks at the chances of these particles to
induce toxicity in humans and the environment.The exact mechanism which
silver nanoparticles employ to cause antimicrobial effect is not clearly known
and is a debated topic. There are however various theories on the action of
silver nanoparticles on microbes to cause the microbicidal effect.
Silver nanoparticles can anchor to the bacterial cell wall and subsequently
penetrate it, thereby causing structural changes in the cell membrane like the
permeability of the cell membrane and death of the cell. There is formation of
‘pits’ on the cell surface, and there is accumulation of the nanoparticles on the
cell surface. The formation of free radicals by the silver nanoparticles may be
another mechanism by which the cells die. There have been electron spin
resonance spectroscopy studies that suggested that there is formation of free
radicals by the silver nanoparticles when in contact with the bacteria, and these
free radicals can damage the cell membrane and make it porous which can
ultimately lead to cell death.
It has also been proposed that there can be release of silver ions by the
nanoparticles, and these ions can interact with the thiol groups of many vital
enzymes and inactivate them. The bacterial cells in contact with silver take in
silver ions, which inhibit several functions in the cell and damage the cells.
Then, there is the generation of reactive oxygen species, which are produced
possibly through the inhibition of a respiratory enzyme by silver ions and attack
the cell itself. Silver is a soft acid, and there is a natural tendency of an acid to
react with a base, in this case, a soft acid to react with a soft base. The cells are
majorly made up of sulfur and phosphorus which are soft bases. The action of
these nanoparticles on the cell can cause the reaction to take place and
subsequently lead to cell death. Another fact is that the DNA has sulfur and
phosphorus as its major components; the nanoparticles can act on these soft
bases and destroy the DNA which would lead to cell death. The interaction of
the silver nanoparticles with the sulfur and phosphorus of the DNA can lead to
problems in the DNA replication of the bacteria and thus terminate the
microbes.

8. 8
It has also been found that the nanoparticles can modulate the signal
transduction in bacteria. It is a well-established fact that phosphorylation of
protein substrates in bacteria influences bacterial signal transduction.
Dephosphorylation is noted only in the tyrosine residues of gram-negative
bacteria. The phosphotyrosine profile of bacterial peptides is altered by the
nanoparticles. It was found that the nanoparticles dephosphorylate the peptide
substrates on tyrosine residues, which leads to signal transduction inhibition and
thus the stoppage of growth. It is however necessary to understand that further
research is required on the topic to thoroughly establish the claims.
Nanoparticles are of great scientific interest as they bridge the gap between bulk
materials and atomic or molecular structures. Among various
nanoparticles, metal nanoparticles are the most promising ones and this is due to
their anti-bacterial properties which, occurs because of the high surface to
volume ratio.
Change in the size or surface of the composition can change the physical and
chemical properties of the nanoparticles. In recent decades, the application of
metal nanoparticles is very common due to their wide applications in various
industries. By reaching nanoparticles size in a certain range (1–100 nm), their
physical, chemical and electrical properties will change. These properties
depend on silver nanoparticles size and characteristics such as melting
temperature, magnetic behavior, redox potential and their color can be
controlled by changing their size and shapes. In recent years silver nanoparticles
have attracted a lot of attentions due to their good conductivity, chemical
stability, use as catalysts and their applications in various industries including
the medical sciences, in order to deal with HIV virus, food industries as anti-
bacterial agents in food packing, anti-bacterial properties and also their unique
electrical and optical qualities.Studying the mechanism of antibacterial
activity of silver ions and silver nanoparticles showed that this property is
related to the morphological and structural changes in the bacterial cell.
Studies have shown that the size, morphology, stability and (chemical and
physical) properties of the metal nanoparticles are influenced strongly by the
experimental conditions, the kinetics of interaction of metal ions with reducing
agents, and adsorption processes of stabilizing agent with metal nanoparticles.
Generally, specific control of the shape, size and distribution of the produced
nanoparticles is achieved by changing the methods of synthesis, reducing and
stabilizing factor.
Incredible properties of nanomaterials strongly depend on size and, shape of
NPs, their interactions with stabilizers and surrounding media and also on their
preparation method. So, controlled synthesis of nanocrystals is a key challenge
to reach their (nanoparticles) better applied characteristics. The optical,

9. 9
electronical, magnetic, and catalytic properties of nanoparticles depend on their
size, shape, and chemical environment. In recent years, new methods have been
proposed to synthesize non-spherical nanoparticles both planar (triangles, 5 or 6
diagonal, round surfaces, etc.) and three dimensional (cubic, pyramid, etc.).
Spherical particles with the minimum surface for a given volume are
thermodynamically more stable and if the reduction of one-capacity silver ions
is performed under controlled thermodynamic conditions, the main product will
be spherical nanoparticles. The shapes of nanoparticles depend on their
interaction with stabilizers and the inductors around them and also their
preparation method. It is also known that reaction rate is influenced by the
shape of synthesized silver nanoparticles. Xu et al. studied the oxidation
of styrene over three shapes (nano cube, semi round and triangular nano plate)
of silver nanoparticles for this purpose. The results of this study showed that the
reaction rate in cubic nanoparticles is 14 times more than
triangular nanoplates and 4 times higher than the semi-spherical nanoparticles.

10. 10
is an important field of modern research dealing with designsynthesis, and
manipulation of particle structures ranging from approximately 1-100 nm.
Nanoparticles (NPs) have wide range of applications in areas such as health
care, cosmetics, food and feed, environmental health, mechanics, optics,
biomedical sciences, chemical industries, electronics, space industries, drug-
gene delivery, energy science, optoelectronics, catalysis, single electron
transistors, light emitters, nonlinear optical devices, and photo-electrochemical
applications.
NANO BIOTECHNOLOGY
Nanobiotechnology is a rapidly growing scientific fieldof producing and
constructing devices. An important area of research in nanobiotechnology is the
synthesis of NPs with different chemical compositions, sizes, and morphologies,
and controlled dispersities. Nanobiotechnology has turned up as an elementary
division of contemporary nanotechnology and untied novel epoch in the fields
of material science receiving global attention due to its ample applications. It is
a multidisciplinary approach resulting from the investigational use of NPs in
biological systems including the disciplines of biology, biochemistry,
chemistry, engineering, physics, and medicine. Moreover, the nanobio-
technology also serves as an imperative technique in the development of clean,
nontoxic, and eco-friendly procedures for the synthesis and congregation of
metal NPs having the intrinsic ability to reduce metals by specific metabolic
pathways.
Nowadays, there is a growing need to develop eco-friendly processes, which do
not use toxic chemicals in the synthesis protocols. Green synthesis approaches
include mixed valence polyoxometalates, polysaccharides, Tollens, biological,
and irradiation method which have advantages over conventional methods
involving chemical agents associated with environmental toxicity. Selection of
solvent medium and selection of eco-friendly nontoxic reducing and stabilizing
agents are the most important issues which must be considered in green
synthesis of NPs.
The Ag NPs were reported to possess anti-bacterial,antiviral(Rogers), anti-
fungal activities. Synthesis of nanoparticles using plants or microorganisms can
potentially eliminate this problem by making the nanoparticles more
biocompatible. Indeed, over the past several years, plants, algae, fungi, bacteria,
and viruses have been used for low-cost, energy-efficient, and non-toxic
production of metallic nanoparticles

11. 11
Aristolochia indica commonly known as Eeswara mooligai, has been used as a
traditional medicine to treat on the sting of scorpion or centipede and on insect
bites. The hexane, ethyl acetate and methanol extracts of Aristolochia indica
were found to possess adulticidal, repellent and larvicidal activities against adult
and early fourth-instar larvae of Culex gelidus and Culex quinquefasciatus and
the bio insecticidal effects of methanol extract of Aristolochia baetica showed
larval growth inhibition against Tribolium castaneum (Jbilou et The recent
reports include the synthesis of nanoparticles using medicinal plants
(Mukunthan and Elumalai This stands as a great application in the field of
nanomedicine. Medicinal property of the extract and nano-silver could play
vital role in treatment of many diseases.
Silver metal has been used widely across the civilizations fordifferent purposes.
Many societies use silver as jewellery, roan-mentation and fine cutlery. Silver
as jewellery, wares and cut-leery was considered to impart health benefits to the
users.
Silver has a long history of anti-microbial use to discourageAntimicrobial
activities of silver nanoparticles synthesized using plant extracts.Biological
entity Test microorganisms Method ReferencesAlternanthera dentate
Escherichia coli, Pseudomonas aeruginosaKlebsiella pneumonia and,
EnterococcusfaecalisBerkadiadiffuse Aeromonas hydrophile,
Pseudomonasfluorescence and Flavobacterium Brachyphyllum
 Tribulus Terrestre’s Streptococcus pyrogens, Pseudomonas
 aeruginosa, Escherichia coli, Bacillus subtilis
 and Staphylococcus aureus
 Kirby-Bauer
 Cocoas nucifera Klebsiella pneumoniae, Bacillus subtilis,
 Pseudomonas aeruginosa and Salmonella
 Paratyphoid Aloe vera E. coli Standard plate count
 Solanumsthorium P. aeruginosa, S. aureus, A. flavus and
 Aspergillus Niger
 Disc diffusion
 Trian ThemaDeandra E. coli and P. aeruginosa Disc diffusion
 ArgemoneMexicana Escherichia coli; Pseudomonas aeruginosa.
 Aspergillus flavus
 Disc diffusion for bacteria and food poisoning for fungi
 Abutilon indicum S. typhi, E. coli, S. aureus and B. substiles
 Cymbopogoncitrates P. aeruginosa, P. mirabilis, E. coli, Shigella
 Flexner, S. someone and Klebsiella pneumonia

12. 12
 Disc diffusion
 Sven Soniahyperabundances A. Niger, Fusarium exospore, Uvularia
 lunate and Rhizopus arrhizal.
 Disc diffusion. Plants extract mediated synthesis of silver nanoparticles.
Phoenicians who used silver as a natural biocide to coat milk bottles. Silver is a
well-known antimicrobial agent against a wide range of over 650
microorganisms from different classes such as gram-negative and gram-positive
bacteria, fungi or viruses.
More recently the metal is finding use in the form of silver nanoparticles. In
ancient Indian medical system (calledAyurveda), silver has been described as
therapeutic agent for many diseases. In 1884, during childbirth it became a
common practice to administer drops of aqueous silver nitrate to new-burn’s
eyes to prevent the transmission of Neisseria gonorrhea from infected mothers.
Out of all the metals with antimicrobial properties, it was found that silver has
the most effective antibacterial action and is least toxic to animal cells. Silver
became commonly used in medical treatments, such as those of wounded
soldiers in World War I, to deter microbial growth .The medical properties of
silver have been known for over 2000 years
Silver is generally used in the nitrate form to induce antimicrobial effect but
when silver nanoparticles are used, there is a huge increase in the surface area
avail-able for the microbes to be exposed to. Silver nanoparticle synthesized
using plant extracts (from different sources) haven used for analyzing their
antimicrobial activities against different microbes
The antimicrobial properties of silver nanoparticles depend on Size and
environmental conditions
Capping agent.
The exact mechanisms of antimicrobial or toxic activity silver nanoparticles are
still in investigation. The positive charge on the Ag ions is suggested
vitalantimicrobial activities. For silver to have any antimicrobial properties, it
must be in its ionized form. In its ionized form, silver is inert but on meeting
moisture it releases silver ions. Ag+ ions are able to form complexes with
nucleic acids and preferentially interact with the nucleosides rather than with
the phosphate groups nucleic acids. Thus, all forms of silver or silver
containing commit observed antimicrobial properties are in one word another

13. 13
sources of silver ions; these silver ions maybe incorporated into the substance
and released slowly wide as with silver sulfadiazine, or the silver ions can come
from ionizing the surface of a solid piece of silver as with silver nanoparticles.
There is some literature showing the electrostatic attraction between positively
charged nanoparticles and negatively charged bacterial cells and they requested
to be most suitable bactericidal agent. These nanoparticles have been shown to
accumulate inside the membrane and can subsequently penetrate into the cells
causing damage to cell wall or cell membranes. It is thought that silver atoms
bind to thiol groups (ASH) of enzymes form-Ing stable Sag bonds with thiol
containing compounds and then it causes the deactivation of enzymes in the
cell membrane that involve in trans membrane energy generation and ion
transport.
It was proposed that Ag(I) ion enters the cell and intercalates between the
purine and pyrimidine base pairs dis-rusting the hydrogen bonding between the
two anti-parallel strands and denaturing the DNA molecule. Bacterial cell lysis
could be one of reason for its antibacterial.
Gram-positive bacteria are less susceptible to Ag+ tangram-negative bacteria.
This is due to; the gram-positive bac-trial cell wall made up of peptidoglycan
molecules and has more peptidoglycan than gram-negative bacteria. As cell
wall of gram positive is thicker, as peptidoglycan is negative charged and silver
ions are positively charged; more silver may get stuck by peptidoglycan in
gram-positive bacteria than gram-negative bacteria. The decreased liability of
gram-positive bacteria can also simply be explained by the fact that the cell wall
of gram-positive bacteria is thicker than that gram-negative bacterium. Other
mechanisms involving inter-action of silver molecules with biological
macromolecules such as enzymes and DNA through an electron-release
mechanism or free radical production have been proposed. Theo cell wall
synthesis as well as protein synthesis shown to be induced by silver
nanoparticles has been suggest by some literatures with the proteomic data
having evidence of accumulation of envelope protein precursor or
destabilization
outer membrane, which finally leads to ATP leaking. Nano silver is a much
effective and a fast-acting fungicide against a broad spectrum of common fungi
including gene Rauch as Aspergillus, Candida and Saccharomyces . The multi-
resistant pathogens due to antigenic shifts and/or drifts are ineffectively
managed with current medications. This resistance to medication by pathogens
has become a stern

14. 14
problem in public health; therefore, there is a strong require-mint to develop
new bactericides and virucides. Silver is having a long history of use as an
antiseptic and disinfectant andcan interact with desulphated bonds of the
glycoprotein/protein contents of microorganisms such as viruses, bac-tera and
fungi. Both silver nanoparticles and silver ions change the three-dimensional
structure of proteins by interfering with disulphate bonds and block the
functional operations of the microorganism .Advancement of this route (green
synthesis) over chemical and physical method is that it is cost effective,
environment friendly, easily scaled up for large scale synthesis and there is no
need to use high energy, pressure, temperature and toxic chemicals. These of
environmentally benign materials like bacteria, fungi, plant extracts and
enzymes for the syntheses of silver nanoparticles offers numerous benefits of
eco-friendly and compatibility for pharmaceutical and other biomedical
applications as they do not use toxic chemicals for the synthesis protocol.
These disadvantages insisted the use of novel and well refined methods that
opened doors to explore benign and green routes for synthesizing nanoparticles.
VITAMINSIN SPINACH:
Spinach is a superior supplier of vitamin K, vitamin A, manganese,
magnesium, folic acid, iron, vitamin C, vitamin B2, and potassium, and it
includes a lot of dietary fiber, vitamin B6, vitamin E, and omega-3 fatty acids,
which are essential for the maintenance, improvement
, and regulation of human tissues.
Raw spinach is 91% water, 4% carbohydrates, 3% protein, and contains
negligible fat. In a 100 g (3.5 oz) serving providing only 23 calories, spinach
has a high nutritional value, especially when fresh, frozen, steamed, or quickly
boiled. It is a rich source (20% or more of the Daily Value, DV) of vitamin A,
vitamin C, vitamin K, magnesium, manganese, iron and folate. Spinach is a
moderate source (10-19% of DV) of the B vitamins, riboflavin and vitamin B6,
vitamin E, calcium, potassium, and dietary fiber. Although spinach is touted as
being high in iron and calcium content, and is often served and consumed in its
raw form, raw spinach contains high levels of oxalates, which block absorption
of calcium and iron in the stomach and small intestine. Spinach cooked in
several changes of water has much lower levels of oxalates and is better
digested and its nutrients absorbed more comp In addition to preventing
absorption and use, high levels of oxalates remove iron from the body

15. 15
A quantity of 100 g of spinach contains over four times the recommended daily
intake of vitamin K. For this reason, individuals taking the anticoagulant
warfarin – which acts by inhibiting vitamin K – are instructed to minimize
consumption of spinach (as well as other dark green leafy vegetables) to avoid
blunting the effect of warfarin.
The larger portion of dietary iron (nonheme) is absorbed slowly in its many
food sources, including spinach. This absorption may vary widely depending on
the presence of binders such as fiber or enhancers, such as vitamin C. Therefore,
the body's absorption of non-heme iron can be improved by consuming foods
that are rich in vitamin C. However, spinach contains high levels of oxalate.
Oxalates bind to iron to form ferrous oxalate and remove iron from the body.
Therefore, a diet high in oxalate (or phosphate or phytate) leads to a decrease in
iron absorption.
Spinach also has a high calcium content. However, the oxalate content in
spinach likewise binds with calcium, decreasing its absorption. By way of
comparison, the body can absorb about half of the calcium present in broccoli,
yet only around 5 percent of the calcium in spinach.
Oxalate is one of a number of factors that can contribute to gout and kidney
stones. Equally or more notable factors contributing to calcium stones are
genetic tendency, high intake of animal protein, excess calcium intake, excess
vitamin D, prolonged immobility, hyperparathyroidism, renal tubular acidosis,
and excess dietary fiber (Williams, 1993).
Oxalic acid also is the same substance that renders spinach its slightly bitter
taste, which some prize and others find disagreeable.
A distinction can be made between older varieties of spinach and more modern
varieties. Older varieties tend to bolt too early in warm conditions. Newer
varieties tend to grow more rapidly but have less of an inclination to run up to
seed. The older varieties have narrower leaves and tend to have a stronger and
more bitter taste. Most newer varieties have broader leaves and round
seeds.here are three basic types of Spinach:
Savoy has dark green, crinkly and curly leaves. It is the type sold in fresh
bunches in most supermarkets. One heirloom variety of savoy is Bloomsdale,
which is somewhat resistant to bolting.

16. 16
REVIEW OF LITEARTURE
Characteristics and antibacterial activity of green synthesized silver Nano
particles using red spinach:
Published online:22 Jan 2019
(Is fatiman & zera Helga vurida irgani aftrid )
The basic principle of the synthesis is the capability of flavonoid and alkanoid
to reduce metal ion precursor such as a silver Nano particles
Green synthesis of AGNPs is inntresting since many studies reported the
characteristics of synthesized nanoparticles as function of kind of plant extract
composition.
Methods of synthesis, green synthesized AGNPs has been reported to have
antibacterial, fungal, anticancer and antioxidant activities.
Synthesis of silver nanoparticles with different shapes:
Published online : 8 December 2019,
The synthesis of silver nanoparticles is very common due to their numerous
applications in various fields.
Silver nanoparticles have unique properties such as: optical and catalytic
properties, which, depend on the size and shape of the produced nanoparticles.
The production of silver nanoparticles with different shapes which have various
uses in different fields such as medicine, are noted by many researchers.
These properties depend on silver nanoparticles size and characteristics such as
melting temperature, magnetic behavior, redox potential and their color can be
controlled by changing their size and shapes (Gurunathan et al., 2009).and their
applications in various industries including the medical sciences, in order to
deal with HIV virus, food industries as anti-bacterial agents infoodpacking
(Ahmad et al., 2003), anti-bacterial properties (Hill, 1939) and also their unique
electrical and optical qualities.

17. 17
BIOLOGICAL SYNTHESIS OF SILVER NANOPARTICLES BY USING
ONION:
Published online:2, April-June 2010
A Saxena, RM Tripathi, RP Singh
For the synthesis of silver nanoparticles we use onion (Allium cepa) as a plant
extract.
For the synthesis of silver nanoparticles, silver nitrate (AgNO3) is used.
Ultraviolet-visible spectrophotometry is performed.
SEM analysis and tem analysis of silver Nano particles.
Silver nanoparticles exhibits strong antibacterial activity due to their well-
developed surface which provides maximum contact with the
environment.Antibacterial effect of silver nanoparticles were studied by using
optical intensity as function of time for 25 hours with different concentration of
silver nanoparticles.
Concentration of silver nanoparticles increases there is reduction in the bacterial
growth curve of E.coli and Salmonella typhimurium.We used onion (Allium
cepa) extract as a reducing and capping agent to minimize the serious
environmental pollution problems.By using onion extract has desired quality
with low cost and convenient methods.These nanoparticles at concentration
50μg/ml were showed complete antibacterial activity against E.coli and
Salmonella typhimurium.
Synthesis of silver nanoparticles mediated by fungi:
Published online :22 October 2019
The use of fungi as reducing and stabilizing agents in the biogenic synthesis of
silver nanoparticles isattractive due to the production of large quantities of
proteins, high yields, easy handling, and low toxicity of the residues.Silver
nanoparticles synthesized using fungi enable the control of pathogens, with low
toxicity and good biocompatibility. These findings open perspectives for future
investigations concerning the use of these nanoparticles as antimicrobials in the
areas of health and agriculture.
Silver nanoparticles synthesized using fungi have various potential applications
in the areas of health, agriculture, and pest control.

18. 18
However, synthesis based on fungi may be advantageous in terms of
production, due to the large quantities of metabolites produced. Another factor
to consider is the capacity of fungi to produce antibiotics that could be
contained in the capping and act in synergy with the nanoparticle core.
BIO TEMPLATES IN THE GREEN SYNTHESIS OF SILVER NANO
PARTICLES:
Published online:28 July 2010
S.PKamala Nalini
“Green” nanoparticle synthesis has been achieved using environmentally
acceptable solvent systems and eco-friendly reducing and capping agents.
Numerous microorganisms and plant extracts have been applied to synthesize
inorganic nanostructures either intracellularly or extracellularly.
The use of nanoparticles derived from noble metals has spread too many areas
including jewellery, medical fields, electronics, water treatment and sport
utilities, thus improving the longevity and comfort in human life.
Green synthesis of silver nanoparticles from Tectona grandis seeds extract:
Published online: 22 January 2019
Akhil Rautela, Jyoti Rani & Mira Debnath (Das)
Green synthesis of silver nanoparticles makes use of plant constituents, like
carbohydrates, fats, enzymes, flavonoids, terpenoids, polyphenols, and
alkaloids, as reducing agents to synthesize silver nanoparticles.
Nanoparticles was confirmed by visual detection in which the colorless solution
gets changed to a brown-colored solution.
Further characterization was done by UV-visible spectroscopy, XRD, FTIR
analysis, SEM/EDS, FESEM, and TEM. Size of silver nanoparticles was found
to be 10–30 nm approximately as determined by transmission electron
microscopy (TEM).
Well diffusion method showed the antimicrobial effect of AgNPs on different
microorganisms with the zone of inhibition of 16 mm for Staphylococcus
aureus, 12 mm for Bacillus cereus, and 17 mm for E. coli when 50 μg of
AgNPs was used.

19. 19
Mode of action of antimicrobial activity of nanoparticles was investigated by
determining leakage of reducing sugars and proteins, suggesting that AgNPs
were able to destroy membrane permeability.
Mechanism of action of antimicrobial activity was found to be the change in
permeability of membrane by detecting the release of reducing sugars and
proteins through leaky membrane, which was detected by DNS and Bradford’s
method, respectively.
The role of tannic acid and sodium citrate in the synthesis of silver Nano
particles:
Published online : 04 August 2017
The significance of a sodium citrate and tannic acid mixture in the synthesis of
spherical silver nanoparticles (AgNPs).
Monodisperse AgNPs were synthesized via reduction of silver nitrate using a
mixture of two chemical agents: sodium citrate and tannic acid.
The oxidation and reduction potentials of silver, tannic acid and sodium citrate
in their mixtures were determined using cyclic voltammetry.
Typical AgNP chemical synthesis uses either tannic acid or citrate as a reducing
and stabilizing agent. Tannic acid contains a central core of glucose that is
linked by ester bonds to polygalloyl ester chains. At its natural pH, tannic acid
is a weak reducing agent (Tian et al. 2007) that is known to hydrolyse under
acidic/basic conditions into glucose and gallic acid units (Bors .2001)
Tannic acid in the solution containing sodium citrate undergoes oxidation the
most readily. Oxidation starts at a potential of 0.195 V. This value is 0.115 V
smaller than in the case of the solution containing tannic acid alone.
The reason for such a change in the potential might be the interaction between
the particles of tannic acid and citrate anions. Probably, these are interactions of
the type of hydrogen bonds between the hydroxyl groups of tannic acid particles
and the oxygen atoms of carbonyl groups of citrates.
The formation of stable citric acid (CA)–TA complexes was also confirmed by
quantum calculations.Green synthesis of silver nanoparticles makes use of plant
constituents, like carbohydrates, fats, enzymes, flavonoids, terpenoids,
polyphenols, and alkaloids, as reducing agents to synthesize silver
nanoparticles.
Nanoparticles was confirmed by visual detection in which the colorless solution
gets changed to a brown-colored solution.

20. 20
Further characterization was done by UV-visible spectroscopy, XRD, FTIR
analysis, SEM/EDS, FESEM, and TEM. Size of silver nanoparticles was found
to be 10–30 nm approximately as determined by transmission electron
microscopy (TEM).
Well diffusion method showed the antimicrobial effect of AgNPs on different
microorganisms with the zone of inhibition of 16 mm for Staphylococcus
aureus, 12 mm for Bacillus cereus, and 17 mm for E. coli when 50 μg of
AgNPs was used.
GREEN SYNTHESIS OF SILVER NANO PARTICLES USING THE
PLANT EXTRACT OF SALVIA SPINOSA
Published online: 04 December 2018
Saba Pirtarighat, Maryam Ghannadnia
The ability of plant extract of Salvia spinosa grown under in vitro condition for
the biosynthesis of silver nanoparticles (Ag-NPs) for the first time. The surface
plasmon resonance found at 450 nm confirmed the formation of Ag NPs.
MoreoverThe extract of various plants grown in farms is used permanently for
the biosynthesis of nanoparticles, but whether the plants grown under in vitro
condition have this ability or not is still under scrutiny.
The main purposes of this work are considering the potential of the plant extract
of S. spinosa grown in vitro in Murashige and Skoog (MS) medium for the
biosynthesis of Ag NPs and investigation of their antibacterial activities against
both Gram-positive and Gram-negative species of bacteria.The antibacterial
activity of the biosynthesized Ag NPs against Gram-positive and -negative
bacteria species was done by disk diffusion method. Experimented bacteria
were Bacillus subtilis (accession number: M59 KP406766), Bacillus
vallismortis (accession number: M92 KP406765) and Escherichia coli (PTCC:
1276).
Moreover, bactericidal activity assessment of the biosynthesized Ag NPs
showed their inhibitory function against both Gram-positive and Gram-negative
bacteria.
In this study, possible functional groups and effective compounds responsible in
reduction of silver ions were assigned.
The speculated mechanism in this work elucidates the involvement of
carboxylic acid functional group presented in carnosic acid and flavonoids in
the bioreduction process and the stabilization of biosynthesized Ag NPs.

21. 21
PARTHENIUM LEAF EXTRACT MEDIATED SYNTHESIS
OF SILVER NANOPARTICLES:
Published online :1, March 2009
(VYOM PARASHAR, RASHMI PARASHARA , BECHAN SHARMA,
AVINASH C. PANDEY)
synthesis of silver nanoparticles using Parthenium leaf extract have been
prepared by bringing fresh leaves of Parthenium hysterophorus
Silver nanoparticles are single crystalline in nature.Eco-friendly nanoparticles in
bactericidal, wound healing and other medical and electronic applications,
makes this method potentially exciting for the large-scale synthesis of other
inorganic materials (nanomaterials). Toxicity studies of Parthenium.
Use of agricultural waste (coconut shell) for the synthesis of silver
nanoparticles:
Published online : November 2018,
SimranSinsinwar
Extract of an agricultural waste, coconut (Cocos nucifera) shell is used to
synthesize AgNPs anther antibacterialeffect wasinvestigated against selected
human pathogens Staphylococcus aureus, Listeria monocytogenes, Escherichia
coli, Salmonella typhimurium. The AgNPs synthesized using coconut shell
extractdegradation might be the possible mechanism of antibacterial action of
CSE-AgNPs. Different concentrations of AgNPs (0.078–2.5 mg/ml) showed
no toxicity against human PBMC cell line. Hence,such highly effective CSE-
AgNPs could be explored as antibacterial agent.
Carnivorous plants used for green synthesis of silver nanoparticles:
Published online: 1, January 2020,
(RafalBanasiuk,DariaSwigon)
The anti-oxidative potential of four carnivorous plants to produce uniform and
biologically active silver nanoparticles.
Since the 12th century carnivorous plants have been used for the treatment of
illnesses such as dry cough, bronchitis, whooping-cough, asthma, urinal tract
infections or even headache.

22. 22
This is due to their secondary metabolite composition that comprises of
flavonoids, naphthoquinones, anthocyanins, phenolic compounds, and organic
acidsCarnivorous plants belong to a group of endangered species however they
can be obtained with a high yield through vegetative reproduction in in vitro
cultures.
This technique allows to increase the propagation rate of valuable, genetically
identical plants from even a single plant. Carnivorous plants were used in the
pre-antibiotic era and now can return in a modern form to combat antibiotic
resistant pathogens.
This is one of the most energy-consuming technological processes that strongly
contributes to the profitability of the reaction.
Lowering the energy consumption can be beneficial concerning both in the
ecological and economical aspects. Bearing in mind silver ion toxicity and
biological variability of plant extracts, it is prudent to use an additional capping
agent to facilitate the reaction process without the need for full extract
standardisation.
Several modifications. Plants: Drosera indica, Drosera binata, Drosera spatulata,
and Dionaea muscipula.
AgNPs synthesized with the use of carnivorous plants extracts were
characterized by using transmission electron microscopy
The chromatographic separation was carried out using Beckmann Gold System
Since the 12th century carnivorous plants have been used for the treatment of
illnesses such as dry cough, bronchitis, whooping-cough, asthma, urinal tract
infections or even headache.
Lemon peels mediated synthesis of silver nanoparticles and its
antidermatophytic activity:
Published online : 24 April 2014
(S.Najimu NishaaO.S.Aysha)
The extract of lemon peel was prepared and mixed with 1 mM AgNO3 solution
.The bioreduction of Ag+ ion in solution.Skin scales were collected from
patients with suspected dermatophytosis and the dermatophytes were isolated
and identified.
The AgNPs produced from lemon peels showed good activity against the
isolated dermatophytes.

23. 23
The present research work emphasizes the use of lemon peels for the effective
synthesize of AgNPs and could be used against the dermatophytes which are
found to develop drug resistant towards broad-spectrum antibiotics.
The biosynthesis of AgNPs using lemon peel extract is very simple and
economic.
The use of environmentally benign and renewable plant material offers
enormous benefits of eco-friendliness.
ARTEMISIACAPILLARIES EXTRACTSAS A GREEN FACTORYFOR
THE SYNTHESISOF SILVER NANOPARTICLESWITH
ANTIBACTERIAL ACTIVITIES:
Published online : 9, September 2012
( Park, Youmie; Noh, Hwa Jung; Han, Lina; Kim, Hyun-Seok; Kim, Yong-Jae;
Choi, Jae Sue; Kim, Chong-Kook; Kim, Yeong Shik; Cho, SeonHo)Green
synthesis of silver nanoparticles that uses extracts from the aerial part of
Artemisia capillaris. Both water and 70% ethanol extracts successfully
generated silver nanoparticles.The formation of silver nanoparticles was
confirmed by surface plasmon resonance bands, Fourier transform-infrared
spectra, high resolution-transmission electron and atomic force microscopic
images.
A remarkable enhancement (approximately 12-fold) was observed against
Pseudomonas aeruginosa, Escherichia coli, Enterobacter cloacae, Klebsiella
oxytoca, and Klebsiella areogenes when compared with the extract alone.Silver
nanoparticles produced by the 70% ethanol extract showed slightly higher
antibacterial activity than those generated with the water extract. The correlation
between total flavonoid content of each extract and the antibacterial activity did
not exert any significant relationships.This report suggests that plant extracts
have the potential to be used as powerful reducing agents forproduction of
biocompatible silver nanoparticles

24. 24
MATERIALSAND METHODS:
Silver nanoparticles were synthesized using the spinach leave extract freshly
collected fromFresh spinach was collected from the local market and The silver
nitrate was used as a precursorand remaining equipment was used in department
of biotechnology Andhra Loyola college Vijayawada before the experiment
the all glass ware was washed well and maintained sterilized conditions before
doing the experiment.
PREPARATION OF GREEN SPINACH EXTRACT:
Collect the freshly collected green spinach leaves and chop in fine pieces and
then wash with ethyl alcohol then wash with water and dry the leaves under
sunlight for two days then leaves become dry now taken the leaves and
weighed it for 4.471 grams make into fine Paste with the help of NaOH and
distilled water and dissolved in 50 ml distilled water.

25. 25
Other side we weighed 0.01-gram of silver nitrate and dissolved in 25 ml of
distilled water and mix well
In another side we weighed 0.01-gram of silver nitrate and dissolved in 25 ml of
distilled water and mix well.
Then we take green spinach solution and kept on a water bath at 100 c and we
mixed the solution on a water bath until obtaining the extractfor 2-3 hours
SYNTHESIS OF SILVER NANO PARTICLES:
Then we take the green spinach extract and mixen with the silver nitrate
solution for well mixing we had kept on orbitalshaker for 1-2 hours it was
mixed well by keeping in orbital shaker.

26. 26
When the green spinach solution and silver nitrate solution mixed well after this
we kept the solution for incubation for 24-48 hours For incubation after
incubation the solution color was changed then done another process.

27. 27
CENTRIFUAGATION:
The solution was taken in centrifuge tubes and kept in centrifuge and then we
centrifuged with 15000 rpmforonehour later repeatedthe centrifugation for fine
Now supernatant and pellet was obtained then we collected the pellet and kept
it for drying under sun for 2-3 days.
After drying 1-2 days the black colored silver particles was obtained then send
for analysis for seeing silver nanoparticles.
Observation :

28. 28
we have observed the black round tiny silver particles after keeping in the sun
light

29. 29
Result and discussion
The silver particles was formed we synthesizedthe silver nano particles from the
green spinach.From this silver particles we can see silver nano particles by
different analysis here the silver nitrate acts as reagent in this experiment but
normally silver nitrate was sterilizing agent in plant biotechnology we used it
has a reagent to obtain the silver particles this obtain silver particles was very
use full to diabetics and cancer patients in their treatment light amount of silver
particles was used to cure the disease and we used green spinach green spinach
was rich in vitamins and it was good for health.
The major advantage of biological methods is the availability of amino acids,
proteins, or secondary metabolites present in the synthesis process, the
elimination of the extra step required for the prevention of particle aggregation,
and the use of biological molecules for the synthesis of AgNPs is eco-friendly
and pollution-free. Biological methods seem to provide controlled particle size
and shape, which is an important factor for various biomedical applications
Using bacterial protein or plant extracts as reducing agents, we can control the
shape, size, and monodispersity of the nanoparticles. The other advantages of
biological methods are the availability of a vast array of biological resources, a
decrea time requirement, high density, stability, and the ready solubility of
prepared nanoparticles in water

30. 30

31. 31
CONCLUSION:
The isolation of silver nano particles was very use full in biology field and
many research s doing under silver nano particles to treat many diseases like
diabetics and cancer etc.and green spinach is contains many vitamins it is good
for healthThis review comprehensively addressed synthesis, characterization,
and bio-applications of silver nanoparticles, with special emphasis on
anticancer activity and its mechanisms and therapeutic approaches for cancer
using AgNPs. Recently, both academic and industrial research has explored the
possibility of using AgNPs as a next-generation anticancer therapeutic agent,
due to the conventional side effects of chemo- and radiation therapy. Although
AgNPs play an important role in clinical research, several factors need to be
considered, including the source of raw materials, the method of production,
stability, bio-distribution, controlled release, accumulation, cell-specific
targeting, and finally toxicological issues to human beings. The development of
AgNPs as anti-angiogenic molecules is one of the most interesting approaches
for cancer treatment and other angiogenesis-related diseases; it can overcome
poor delivery and the problem of drug resistance. Further, it could provide a
new avenue for other angiogenic diseases, such as atherosclerosis, rheumatoid
arthritis, diabetic retinopathy, psoriasis, endometriosis, and adiposity.In
addition, the potential use of AgNPs for cancer diagnosis and treatment is
immense; to address this issue, a variety of modalities have been developed.
Although various methods are available, the synergistic effects of AgNPs and
antibiotics on antibacterial agents or multiple therapeutic agents on anti-
cancer activity/tumor reduction are still obscure. Therefore, more studies are
required to explain the synergistic effect of the two different cytotoxic agents
at a single time point. These kinds of studies could provide understanding,
mechanisms, and efficiency of the synergistic effect of two different agents or
multiple agents; thus, they would help to develop a novel system bearing
multiple components with synergistic effects for the treatment of various
types of cancer.
Although AgNPs have been focused on therapeutic purposes, further research is
inevitable in animal models to confirm the mechanisms and to gain a
comprehensive picture of biocompatibility vs. toxicity of AgNPs.Finally, if we
succeed in all these studies, it would help the researchers of the nanoscience and
nanotechnology community to develop safer, biocompatible, efficient cancer or
anti-angiogenic agents containing AgNPs. Eventually, to ensure the biosafety
ofthe use of AgNPs in humans, studies dealing with biocompatibility of AgNPs

32. 32
and their interaction with cells and tissues are inevitable. Finally, the great
concern is that the developing nanotechnology-based therapy should be better
INSTRUMENTATION
 We used water bath ,orbital shaker ,Laminar air flow cabinet and
incubator for this experiment
LAMINAR AIR FLOW CHAMBER
Laminar flow cabinet or tissue culture hood is a carefully enclosed bench
designed to prevent contamination of semiconductor wafers, biological samples,
or any particle sensitive materials. Air is drawn through a HEPA filter and
blown in a very smooth, laminar flow towards the user. Due to the direction of
air flow, the sample is protected from the user but the user is not protected from
the sample. The cabinet is usually made of stainless steel with no gaps or joints
where spores might collect
Such hoods exist in both horizontal and vertical configurations, and there are
many different types of cabinets with a variety of airflow patterns and
acceptable uses.
Laminar flow cabinets may have a UV-C germicidal lamp to sterilize the
interior and contents before usage to prevent contamination of the experiment.
Germicidal lamps are usually kept on for fifteen minutes to sterilize the interior
before the cabinet is used. The light must be switched off when the cabinet is
being used, to limit exposure to skin and eyes as stray ultraviolet light emissions
can cause cancer and cataracts.

33. 33
WATER BATH
A water bath is laboratory equipment made from a container filled with heated
water. It is used to incubate samples in water at a constant temperature over a
long period of time. Most water baths have a digital or an analogue interface to
allow users to set a desired temperature, but some water baths have their
temperature controlled by a current passing through a reader. Utilisations
include warming of reagents, melting of substrates or incubation of cell
cultures. It is also used to enable certain chemical reactions to occur at high
temperature. Water baths are preferred heat sources for heating flammable
chemicals, as their lack of open flame prevents ignition.[1]
Different types of
water baths are used depending on application. For all water baths, it can be
used up to 99.9 °C.[2][3]
When temperature is above 100 °C, alternative methods
such as oil bath, silicone bath or sand bath may be used.[4]

34. 34
INCUBATOR
An incubator is a device used to grow and maintain microbiological
cultures or cell cultures. The incubator maintains
optimal temperature, humidity and other conditions such as the
CO2 and oxygen content of the atmosphere inside. Incubators are essential for
much experimental work in cell biology, microbiology and molecular
biology and are used to culture both bacterial and eukaryotic cells.
The simplest incubators are insulated boxes with an adjustable heater, typically
going up to 60 to 65 °C (140 to 150 °F), though some can go slightly higher
(generally to no more than 100 °C). The most commonly used temperature both
for bacteria such as the frequently used E. coli as well as for mammalian cells is
approximately 37 °C (99 °F), as these organisms grow well under such
conditions. For other organisms used in biological experiments, such as the
budding yeast Saccharomyces cerevisiae, a growth temperature of 30 °C (86 °F)
is optimal.
More elaborate incubators can also include the ability to lower the temperature
(via refrigeration), or the ability to control humidity or CO2 levels. This is
important in the cultivation of mammalian cells, where the relative humidity is
typically >80% to prevent evaporation and a slightly acidic pH is achieved by
maintaining a CO2 level of 5%.

35. 35
ORBITAL SHAKER
A shaker is a piece of laboratory equipment used to mix, blend, or agitate
substances in a tube or flask by shaking them. It is mainly used in the fields
of chemistry and biology. A shaker contains an oscillating board that is used to
place the flasks, beakers, or test tubes. Although the magnetic stirrer has lately
come to replace the shaker, it is still the preferred choice of equipment when
dealing with large volume substances or when simultaneous agitation is
required.

36. 36
APPLICATIONS OF SILVER NANO PARTICLES:
Anti-Inflammatory Activity of AgNPS:
Inflammation is an early immunological response against foreign particles by
tissue, which is supported by the enhanced production of pro-inflammatory
cytokines, the activation of the immune system, and the release of
prostaglandins and chemotactic substances such as complement factors,
interleukin-1 (IL-1), TNF-α, and TGF-β In order to overcome inflammatory
action, we need to find effective anti-inflammatory agents. Among several anti-
inflammatory agents, AgNPs have recently played an important role in anti-
inflammatory field. AgNPs have been known to be antimicrobial, but the anti-
inflammatory responses of AgNPs are still limited. Bhol and Schechter reported
the anti-inflammatory activity in rat. Rats treated intra-colonically with 4 mg/kg
or orally with 40 mg/kg of nanocrystalline silver (NPI 32101) showed
significantly reduced colonic inflammation. Mice treated with AgNPs showed
rapid healing and improved cosmetic appearance, occurring in a dose-dependent
manner. Furthermore, AgNPs showed significant antimicrobial properties,
reduction in wound inflammation, and modulation of fibrogenic cytokines
Continuing the previous study, Wong et al. investigated to gain further evidence
for the anti-inflammatory properties of AgNPs, in which they used both in vivo
and in vitro models and found that AgNPs are able to down-regulate the
quantities of inflammatory markers, suggesting that Agnosy could suppress
inflammatory events in the early phases of wound healing A porcine contact
dermatitis model showed that treatment with nanosilver significantly increases
apoptosis in the inflammatory cells and decreased the levels of pro-

37. 37
inflammatory cytokines Biologically-synthesized AgNPs can inhibit the
production of cytokines induced by UV-B irradiation in HaCaT cells, and also
reduces the edema and cytokine levels in the paw tissues.
Anti-Angiogenic Activity of AgNPs:
Pathological angiogenesis is a symbol of cancer and various ischemic and
inflammatory diseases There are several research groups interested in
discovering novel pro- and anti-angiogenic molecules to overcome angiogenic-
related diseases. Although there are several synthetic molecules having anti-
angiogenic properties, the discovery of a series of natural pro- and anti-
angiogenic factors suggests that this may provide a more physiological
approach to treat both classes of angiogenesis-dependent diseases in the near
future Recently, several studies provided supporting evidence using both in
vitro and in vivo models showing that AgNPs have both anti-angiogenic and
anti-cancer properties. Herein, we wish to summarize the important contribution
in cancer and other angiogenic related diseases.Kalishwaralal et al.
demonstrated the anti-angiogenic property of biologically synthesized AgNPs
using bovine retinal endothelial cells (BRECs) as a model system, in which they
found the inhibition of proliferation and migration in BRECs after 24 h of
treatment with AgNPs at 500 nM concentration. The mechanisms of inhibition
of vascular endothelial growth factor (VEGF) induced angiogenic process by
the activation of caspase-3 and DNA fragmentation, and AgNPs inhibited the
VEGF-induced PI3K/Akt pathway in BRECs Followed by this study,
Gurunathan et al. provided evidence for the anti-angiogenic property of AgNPs
by using pigment epithelium derived factor (PEDF) as a bench mark, which is
known as a potent anti-angiogenic agent. Using BRECs as an in vitro model
system, they found that AgNPs inhibited VEGF-induced angiogenic assays.
Furthermore, they demonstrated that AgNPs could block the formation of new
blood microvessels by the inactivation of PI3K/Akt. The same group also
demonstrated the anticancer property of AgNPs using various cytotoxicity
assays in Dalton’s lymphoma ascites (DLA) cells, and a tumor mouse model
showed significantly increased survival time in the presence of AgNPs AgNPs
reduced with diaminopyridinyl (DAP)-derivatized heparin (HP) polysaccharides
(DAPHP) inhibited basic fibroblast growth factor (FGF-2)-induced
angiogenesis compared to glucose conjugation Kim et al. developed an anti-
angiogenic Flt1 peptide conjugated to tetra-N-butyl ammonium modified
hyaluronate (HA-TBA), and it was used to encapsulate genistein. Using human
umbilical vein endothelial cells (HUVECs) as in vitro model system, they found
that genistein/Flt1 peptide–HA micelle inhibited the proliferation of HUVECs,
and the same reagents could drastically reduce corneal neovascularization in
silver nitrate-cauterized corneas of Sprague Dawley (SD) rats. Ag2S quantum

38. 38
dots (QDs) used as nanoprobes to monitor lymphatic drainage and vascular
networks. Ag2S-based nanoprobes showed long circulation time and high
stability. In addition, they were able to track angiogenesis mediated by a tiny
tumor (2–3 mm in diameter) in vivo Recently, Achillea biebersteinii flowers
extract-mediated synthesis of AgNPs containing a concentration of 200 μg/mL
showed a 50% reduction in newly-formed vessels shows the inhibitory effect of
AgNPs on VEGF induced angiogenic activity in bovine retinal endothelial cells
(BRECs) and human breast cancer cells MDA-MB 231.
Anticancer Activity of AgNPs
In our lifetime, 1 in 3 people has the possibility to develop cancer Although
many chemotherapeutic agents are currently being used on different types of
cancers, the side effects are enormous, and particularly, administrations of
chemotherapeutic agents by intravenous infusion are a tedious process
Therefore, it is indispensable to develop technologies to avoid systemic side
effects. At this juncture, many researchers are interested in developing
nanomaterials as an alternative tool to create formulations that can target tumor
cells specifically. Several research laboratories have used various cell lines to
address the possibility of finding a new molecule to battle cancer. Here we
summarized the work from various laboratories reporting anticancer activity
using both in vitro and in vivo model systems. Gopinath et al. investigated the
molecular mechanism of AgNPs and found that programmed cell death was
concentration-dependent under conditions. Further, they observed a synergistic
effect on apoptosis using uracil phosphoribosyltransferase (UPRT)-expressing
cells and non-UPRT-expressing cells in the presence of fluorouracil (5-FU). In
these experimental conditions, they observed that AgNPs not only induce
apoptosis but also sensitize cancer cells. The anticancer property of starch-
coated AgNPs was studied in normal human lung fibroblast cells (IMR-90) and
human glioblastoma cells (U251). AgNPs induced alterations in cell
morphology, decreased cell viability and metabolic activity, and increased
oxidative stress leading to mitochondrial damage and increased production of
reactive oxygen species (ROS), ending with DNA damage. Among these two
cell types, U251 cells showed more sensitivity than IMR-90 The same group
also demonstrated that the cellular uptake of AgNPs occurred mainly through
endocytosis. AgNP-treated cells exhibited various abnormalities, including
upregulation of metallothionein, downregulation of major actin binding protein,
filamin, and mitotic arrest The morphology analysis of cancer cells suggests that
biologically synthesized AgNPs could induce cell death very significantly. Jun
et al. elegantly prepared multifunctional silver-embedded magnetic
nanoparticles, in which the first type consist of silver-embedded magnetic NPs
with a magnetic core of average size 18 nm and another type consist of a thick

39. 39
silica shell with silver having an average size of 16 nm; the resulting silica-
encapsulated magnetic NPs (M-SERS dots) produce strong surface-enhanced
Raman scattering (SERS) signals and have magnetic properties, and these two
significant properties were used for targeting breast-cancer cells (SKBR3) and
floating leukemia cells (SP2/O).
The antineoplastic activities of protein-conjugated silver sulfide nano-crystals
are size dependent in human hepatocellular carcinoma Bel-7402 and C6 glioma
cells Instead of giving direct treatment of AgNPs into the cells, some
researchers developed chitosan as a carrier molecule for the delivery of silver to
the cancer cells. For example, Sanpui et al. demonstrated that chitosan-based
nanocarrier (NC) delivery of AgNPs induces apoptosis at very low
concentrations. They then examined cytotoxic efficiency using a battery of
biochemical assays. They found an increased level of intracellular ROSin HT 29
cells. Lower concentrations of nanocarrier with AgNPs showed better inhibitory
results than AgNPs alone. Boca et alreported that chitosan-coated silver
nanotriangles (Chit-AgNTs) show an increased cell mortality rate. In addition,
human embryonic cells (HEK) were able to take up Chit-AgNTs efficiently, and
the cytotoxic effect of various sizes of AgNPs was significant in acute myeloid
leukemia (AML) cells Recently, the anticancer property of bacterial (B-AgNPs)
and fungal extract-produced AgNPs (F-AgNPs) was demonstrated in human
breast cancer MDA-MB-231 cells. Both biologically produced AgNPs exhibited
significant cytotoxicity Among these two AgNPs, fungal extract-derived
AgNPs had a stronger effect than B-AgNPs, which is due to the type of
reducing agents used for the synthesis of AgNPs. Similarly, AgNPs derived
from Escherichia fergusoni showed dose-dependent cytotoxicity against MCF-7
cells Plant extract-mediated synthesis of AgNPs showed more pronounced
toxic effect in human lung carcinoma cells (A549) than non-cancer cells like
human lung cells, indicating that AgNPs could target cell-specific toxicity,
which could be the lower level of pH in the cancer cells Targeted delivery is an
essential process for the treatment of cancer. To address this issue, Locatelli et
al. Developed multifunctional nanocomposites containing polymeric
nanoparticles (PNPs) containing alisertib (Ali) and AgNPs. PNPs conjugated
with a chlorotoxin (Ali@PNPs–Cltx) showed a nonlinear dose–effect
relationship, whereas the toxicity of Ag/Ali@PNPs–Cltx remained stable.
Biologically synthesized AgNPs showed significant toxicity in MCF7 and
T47D cancer cells by higher endocytic activity than MCF10-A normal breast
cell line Banti and Hadjikakou explained the detailed account of anti-
proliferative and anti-tumour activity of silver compounds Biologically
synthesized AgNPs capable of altering cell morphology of cancer cells which is
an early indicator for apoptosis. Apoptosis can be determined by structural
alterations in cells by transmitted light microscopy.

40. 40
Diagnostic, Biosensor, and Gene Therapy Applications of AgNPs
The advancement in medical technologies is increasing. There is much interest
in using nanoparticles to improve or replace today’s therapies. Nanoparticles
have advantages over today’s therapies, because they can be engineered to have
certain properties or to behave in a certain way. Recent developments in
nanotechnology are the use of nanoparticles in the development of new and
effective medical diagnostics and treatments. The ability of AgNPs in cellular
imaging in vivo could be very useful for studying inflammation, tumors,
immune response, and the effects of stem cell therapy, in which contrast agents
were conjugated or encapsulated to nanoparticles through surface modification
and bioconjugation of the nanoparticles. Silver plays an important role in
imaging systems due its stronger and sharper plasmon resonance. AgNPs, due
to their smaller size, are mainly used in diagnostics, therapy, as well as
combined therapy and diagnostic approaches.

41. 41
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