1.

INTRODUCTION

1.1 Overview

The term Nano particle is used to describe the particle with size in the range of 1-
100nm.They tend to react differently than larger particles of the same
composition because of their larger surface area thus allowing them to be used in
novel application moreover they serve as the function building block of
nanotechnology. Now a day there is a wide application of Nano particle in
diverse fields including catalysis, energy, chemistry and medicine. The
nanoparticles (NPs) received a particular attention for their positive impact in
improving many sectors of economy, including consumer products,
pharmaceutics, cosmetics, transportation, energy and agriculture etc., and are
being increasingly produced for a wide range of new applications within industry
are emerging rapidly (Roco, 2003; Novack and Bucheli, 2007). A very interesting
application of NPs in the scope of life sciences is their use as smart delivery
systems. NPs were already postulated in 1906 by the Nobel Prize winner P.
Ehrlich as “magic bullets‟ (Himmelweit, 1960).
Nanotechnology approaches to control disease in human and plants have recently
been increasing greatly and the unique physiochemical properties of Nano-sizes
metal particles make them successful in biology and medicine.Nanotechnology
provides the ability to engineer the properties of materials by controlling their
size, and this has been driven research towards a multitude of potential uses for
nanomaterials. Nanotechnology has been spread to number of areas including
biomedical services, cosmetics, drug gene delivery, environmental health, food,
health care, catalysis, mechanics, non linear optical devices, optics, photo-
electrochemical application, single electron transistors, space industries etc
(Wang, 1991).
Silver nanoparticles are nanoparticles of silver of between 1nm and 100nm in
size. While frequently described as being silver some are composed of a large
percentage of silver oxide due to their large ratio of surface to bulk silver atoms.
Numerous shapes of nanoparticles can be constructed depending on the

1
application at hand. Commonly used are spherical silver nanoparticles but
diamond, octagonal and thin sheets are also popular. Their extremely large
surface area permits the coordination of a vast number of ligands. The properties
of silvernanoparticles application of human treatments are under investigation in
laboratory and animal studies, assessing potential efficacy, toxicity and costs (V
K Sharma, 2009)

Fig 1: silver nanoparticle

Silver nanoparticles can be synthesized using various approaches including

chemical, physical, and biological. Although chemical method of synthesis
requires short period of time for synthesis of large quantity of nanoparticles, this
method requires capping agents for size stabilization of the nanoparticles.
Chemicals used for silver nanoparticles synthesis and stabilization are toxic and
lead to non-ecofriendly by-products. The need for environmental non-toxic
synthetic protocols for Silver nanoparticles synthesis leads to the developing
interest in biological approaches which are free from the use of toxic chemicals
as by-products. Thus, there is an increasing demand for green nanotechnology.

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 Silver nanoparticle
synthesis

Top-down approach Bottom-down approach

Physical method Chemical synthesis Biological synthesis

Plants
Inert gas condensation Electrochemical/electrolysis

Microbes
Mechanical milling Micro-emulsion/colloidal
Enzymes
Laser ablation/ deposition
Polysaccharide & polymer
photochemical

Vapour deposition

Sonochemical

Fig 2: synthesis of silver nanoparticles (Annamalai et al., 2011).

The problem with the chemical and physical methods is that synthesis is
expensive and can also have toxic substances absorbed on to them. Green
synthesis provides advancement of chemical and physical methods because it is
cost effective, environmental friendly and can easily scaled up for large scale
synthesis.
Plants provide a better platform for nanoparticles synthesis as they are free from
toxic chemicals as well as provide natural capping agents. Moreover, use of plant
extracts also reduces the cost of microorganism’s isolation and culture media
enhancing the cost competitive feasibility over nanoparticles synthesis by

3
microorganisms. Plant mediated synthesis is purely a green synthetic route and
are considered better candidates among the different biological entities as they
provide clean, ecofriendly, cost effective, safe, conveniently utilizable and
beneficial way to the synthesis of metal Nps for the large scale production. Many
plants are reported to facilitate the formation of AgNPs and their potential
applications (Chandran et al., 2006; Saxena et al., 2010; Waidha et al., 2015).
The amount of accumulation of NPs varies with reduction potential of ions and
the reducing capacity of plant depends on the presence of various polyphenols
and other heterocyclic compounds (Nair et al., 2010; Vedpriya, 2010).
Plants are known to harbour a big range of metabolites that are most likely to be
responsible in the green synthesis of metal nanoparticles. Biotechnological
approaches, exclusively plant tissue cultures provide a better platform in
producing specific phyto constituents at a rate similar or superior to that of intact
plants. Explants are cultured under appropriate physiological conditions and the
desired product is extracted from the cultured cells/tissue by means of plant tissue
culture. However, the biosynthetic capacity of cultured plant tissue can be
enhanced by regulating environment factors, which are effective for the high
production of NPs.
Recent developments in plant tissue culture techniques in fabrication of NPs have
shown promising results to improve the productivity many folds. As plants
potentially eliminate the environmental issues by making the NPs more
biocompatible, it is necessary to increase the efficiency of the locally available
and unexplored plants resources for the green synthesis of AgNPs and clarify the
possible mechanism involved in synthesis, is still infancy. The silver
nanoparticles have wide spread antimicrobial resistance.

1.2 ALOE VERA

Aloe Vera is an excellent source for production of silver nano particles. Aloe
Vera is a succulent plant species of the genus Aloe. An evergreen perennial. It
originates from the Arabian peninsula but grows wild in tropical climates around
the world and is cultivated for agricultural and medicinal uses. The species is also
used for decorative purposes and grows successfully indoors as a potted plant.

4
Aloe vera is found in many consumer products including beverages, skin lotion,
cosmetics, or ointment for minor burns and sunburns.

Table 1: Scientific classification

Kingdom Plantae
Clade Angiosperms
Clade Monocots
Order Asparagales
Family Asphodelaceae
Genus Aloe
Species A.vera

Fig 3: Aloe vera

Aloe Vera is a stem less or very short-stemmed plant growing to 60-100cm (24-
39 in) tall, spreading by offsets. The leaves are thick and fleshy, green to grey-
green, with some varieties showing white flecks on their upper and lower stem
surfaces. The margin of the leaf is serrated and has small white teeth. Aloe Vera
leaves contain phytochemicals under study for possible bioactivity, such as
acetylated mannans, Polymannans, anthraquinone C-glycosides, anthrones, other
anthraquinones, such as emodin and various lectins.

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1.2.1 Properties of Aloe Vera

Fig 4: Properties of aloe vera

Vitamins: The plant contains many vitamins, including the important antioxidant
vitamins A, C and F. Vitamins B (thiamine), niacin, vitamin B2 (riboflavin),
vitamin B12, choline and folic acid.

Enzymes: Biochemical catalysts, such as amylase and lipase, can aid digestion by
breaking down fats and sugars. Carboxypeptidase, inactivates bradykinins and
produces an anti-inflammatory effect. During the inflammatory process,
bradykinin produces pain associated with vasodilation and, therefore, its
hydrolysis reduces these two components and produces an analgesic effect .

Minerals: Sodium, potassium, calcium, magnesium, manganese, copper, zinc,

chromium and iron are all found in the aloe plant. Magnesium lactate inhibits
histidine decarboxylase and prevents the formation of histamine from the amino

6
acid, histadine. Histamine is released in many allergic reactions and causes
intense itching and pain. The prevention of its formation may explain the
antipuritic effect of aloe vera.

Sugars: Sugars are derived from the mucilage layer of the plant under the rind,
surrounding the inner parenchyma or gel. They form 25 per cent of the solid
fraction and comprise both mono- and polysaccharides. The most important are
the long chain polysaccharides, comprising glucose and mannose, known as the
gluco-mannans. The polysaccharides are absorbed complete and appear in the
blood stream unchanged hence they act as immunomodulators.

Anthraquinones: The bitter aloes consist of free anthraquinones and their

derivatives, Barbaloin, aloe-emodin-9-anthrone, lsobarbaloin ,Anthrone-C-
glycosides and chromones. In large amounts these compounds exert a powerful
purgative effect, but when smaller they appear to aid absorption from the gut, are
potent antimicrobial agents and possess powerful analgesic effects. They also
reduce the formation of melanin and any tendency to hyper-pigmentation. Lignin
with their penetrative ability to carry other active ingredients deep into the skin to
nourish the dermis .
Sterols: These include Campesterol, Sitosterol and Lupeo . Salicylic acid: This is
an aspirin-like compound possessing antiinflammatory and antibacterial
properties. It has a kerolytic effect which helps to debride a wound of necrotic
tissue.

1.3 Wound healing

Our project is focusing on effective synthesis of silver nano particle from Aloe
Vera and its effient application in wound healing.The ultimate goal for wound
healing is a speedy recovery with minimal scarring and maximum
function.Wound healing proceeds through an overlapping pattern of events
including coagulation, inflammation, proliferation & matrix & tissue
remodelling.Further to re- epithelization, wound contraction is another important
process in the early phase of wound healing.

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1.3.1 Wound Healing, Classification, and Cell Signaling

Burning emerges as tissue trauma that is defined by some factors such as heat,
chemicals, electricity, sunlight, and/or nuclear radiation. Most of burning cases
are caused by building fires, touching the boiled water, water steam, liquids, and
flammable gases.
The burning caused by heat and accidents and the like is assumed as the foremost
cause of mortality and disability in the victims. Two million people received
medical treatments caused by burning traumas every year. Most of the primary
treatments including drug topical dosage are employed to prevent against
penetration of infectious substances into the wound. Improvement in methods of
wound therapy and recovery of tissue may enhance the quality of life in the
patients with burning trauma and also it may probably contribute to reducing the
medical costs. Treatment of dermal wounds is a complicated process that is the
result of common performance between the various tissues with several cellular
colonies. The behavior of each type of effective cells has been characterized
during proliferation, migration, matrix building and contraction, phases and
growth factors, and the existing matrix signals in the wound place at present . A
series of usual events may occur to treat the injury after trauma. As a result of
inflammatory response due to trauma, the cells behind the dermis start building
collagen and this continues up to the end of building epithelium.

Following incidence of any type of trauma on skin of other body tissues, a series
of cellular and molecular events occurs. Wound healing includes three phases of
thrombosis and inflammation, proliferation and formation of new tissue, and
tissue retrieval. The paramount cellular signaling events and extracellular matrix
activities in healing process are controlled by various types of growth factors
including fibroblastic growth factors (FGFs), epidermal growth factors (EGFs),
transforming growth factors (TGFs), and insulin-like growth factors (IGFs). The
insulin-like growth factor includes some peptides, which are very similar to
insulin in terms of structural sequence and they are called somatomedin-C due to

8
stimulation for secretion of growth factor hormone. These compounds play their
role through various paths of autocrine, paracrine, and endocrine.
The insulin-like growth factors (IGFs) are secreted by few numbers of dermis and
epidermis cells in normal skin but during dermal trauma they are secreted by
most of epidermal cells including macrophages and platelets. This family of
growth factors stimulates mitogenic fibroblasts and they also intervene in
angiogenesis trend.
Other studies have shown that IGFs along with other factors such as platelet-
derived growth factors (PDGFs) play important role in process of wound healing
so that they increase the thickness of dermis and epidermis. The rate of
expression of IGF genes is low in epidermal basal layer, but this rate is
significantly increased for one to three days after incidence of the wound. It has
been shown in other investigations that abnormal increase in IGF may raise the
expression and production of pro-alpha-I chain gene from collagen type-I and
pro-alpha-I of collagen type-III in wound fibroblasts and as a result it will prepare
the grounds for increase in size of scar (wound trace). (Jun Tian, Kenneth et al.,
2007)

9
 Figure 5: Signaling in wound healing: platelet-derived growth factor (PDGF), vascular
endothelial growth factor (VEGF), and transforming growth factor-beta (TGF-β) .

Principally, the wound is defined as a lesion and rupture on skin surface that is caused by
physical or thermal traumas, which need medical therapy. Improvement and healing of
wound in human or developed animals occur with a completely complex and advanced
mechanism as a result of passing through several phases including inflammation,
proliferation, healing, and reconstruction .Initially, the created gap on place of wound is
closed quickly and epithelium is created again on surface of wound and a new matrix is
substituted promptly with the lost skin. Nonetheless, if the above-said scheduled events
are disrupted for any reason, the healing trend of skin wound encounters problem, and
wound healing speed is affected by them. A lot of factors affect speed and quality of
wound healing trend out of which one can refer to size of wound, blood storage on place,
the existing external objects and microorganisms, age, health status, and nutrition state of
patient

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 2. OBJECTIVE

• To compare the yield and concentration of silver Nano particles, synthesised by

both chemical and biological method

• Physical and molecular characterisation of newly synthesized silver Nano

particles

• To analyse wound healing properties of the selected most efficient silver Nano
particle

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 3. LITERATURE REVIEW

3.1 Shreya et al., (2014): Nanotechnology approaches are used to control disease in
human and plants have recently been increasing greatly and the unique physicochemical
properties of nano-sized metal particles make them successful in biology and medicine.
They tend to react differently than larger particles of the same composition because of
their large surface area, thus allowing them to be used in novel applications. Metal
nanoparticles show large surface to volume ratio and exhibit antimicrobial properties due
to their ability to interact with cellular membranes through disruption of cell wall
structure.

3.2 Patcharaporn Tippayawat et al., ( 2016) : To avoid the chemical toxicity,

biosynthesis (green synthesis) of metal nanoparticles is proposed as a cost-effective and
environmental friendly alternative. Aloe Vera leaf extract is a medicinal agent with
multiple properties including an antibacterial effect. Moreover the constituents of aloe
Vera leaves include lignin, hemicelluloses, and pectin’s which can be used in the
reduction of silver ions to produce as AgNPs with antibacterial activity.

3.3 Ratree Maenthaisong et al., (2003): Aloe Vera has been traditionally used
for burn healing but clinical evidence remains unclear. Based on a met analysis using
duration of wound healing as an outcome measure, the summary weighted mean
difference in healing time of the aloe Vera group was 8.79 days shorter than those in the
control group (P = 0.006). Existing evidence demonstrates that aloe Vera used in a
variety of dosage forms might be effective in shortening the duration of wound healing
in first to second degree burns, and it tended to increase rate of success to healing and
rate of epithelialization. The amount of active ingredient in aloe Vera varies depending
on the age of plants [18], growing and harvesting conditions, parts of plants, and
extraction methods.

3.4 Jun Tian et al., (2007): The ultimate goal for wound healing is a speedy
recovery with minimal scarring and maximal function. Wound healing proceeds through
an overlapping pattern of events including coagulation, inflammation, proliferation, and
matrix and tissue remodeling. Silver has been used for centuries to prevent and treat a
variety of diseases including pleurodesis, cauterisation, and healing of skin wounds. Its
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antibacterial effect may be due to blockage of the respiratory enzyme pathways and
alteration of microbial DNA and the cell wall. In addition to its recognized antibacterial
properties, some authors have reported on the possible pro-healing properties of silver.

3.5 Priya Banerjee et al., (2014):In general, particles with a size less than 100 nm
are referred to as NPs. Entirely novel and enhanced characteristics such as size,
distribution and morphology have been revealed by these particles in comparison to the
larger particles of the mass material that they have been prepared from . NPs of noble
metals like gold, silver and platinum are well recognized to have significant applications
in electronics, magnetic, optoelectronics and information storage. One such important
member of the noble metal NPs are silver NPs (Ag NPs). They are also broadly applied
in shampoos, soaps, detergents, cosmetics, toothpastes and medical and pharmaceutical
products and are hence directly encountered by human systems Earlier, the antifungal
properties of silver and silver nitrate were well incorporated in the field of medical
science. Also, the medicinal importance of innumerable plants and plant parts were
known. But the plant-mediated silver nanoproduct is a relatively newer concept.
Nanobiotechnology and their derived products are unique not only in their treatment
methodology but also due to their uniqueness in particle size, physical, chemical,
biochemical properties and broad range of application as well. . plant-mediated green
biomimetic synthesis of silver nanoparticle is considered a widely acceptable technology
for rapid production of silver nanoparticles for successfully meeting the excessive need
and current market demand and resulting in a reduction in the employment or generation
of hazardous substances to human health and the environment.

3.6 Mahendra Rai et al., (2009): Detection of silver nanoparticles present in the
sample was confirmed by adapting nitrate reductase assay procedure from this literature.

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 4. MATERIALS AND METHODS

AgNPs was extracted from both biological and chemical methods. The yield and
concentration of AgNPs by both extraction were compared to confirm the efficiency of
method of extraction. Comparison of yield and concentration of both chemical and
biological method was done by spectrophotometry.

4.1 Chemical synthesis of AgNPs

The most common approach for synthesis of silver NPs is chemical reduction by
organic and inorganic reducing agents. In general, different reducing agents such as
sodium citrate, ascorbate, sodium borohydride (NaBH4), elemental hydrogen, polyol
process, Tollens reagent..etc.

The composite was prepared by adding 0.5,1and 2Mm concentration of AgNO3

solutions in sterile distilled water.0.7mM Sodium citrate was added to each AgNO3
solution.10mg of Sodium tetra boride was added to each of the concentrations and kept
the composit sample in shaker for overnight incubation in dark.

4.2 Biological synthesis of AgNPs

Synthesis of NPs using plants is very cost effective, and thus can be used as an
economic and valuable alternative for the large-scale production of NPs. In biological
synthesis ,two methods were adopted for the synthesis of AgNPs by using Aloe Vera leaf
extract as a common reducing agent.

In biological synthesis we adopted two methods. In first method 30%Ammonium

solution act as reducing agent along with Aloe Vera leaf extract. This method was not
suitable for the required observation in spectrophotometer. Hence we select an method
by modifying the procedure. In second method only the Aloe Vera extract was used as
reducing agent.

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 4.3 Method 1:

Synthesis of AgNPs using complete Aloe Vera leaf extract or gel extract and
30%ammonium solution as reducing agent.

4.3.1 Preparation of complete Aloe Vera leaf extract:

Fresh mature leaves of Aloe Vera were collected and washed with sterile distilled
water.30g of leaves were weighed and grinded it well. The grinded leaves were mixed
with 100ml distilled water and heated for 12mnt.The extract was filtered through what
man no:1 filter paper which is placed on a conical flask. The filtrate was collected in the
conical flask and stored in refrigerator.

Composite was prepared by adding 5ml of Aloe Vera extract to each concentration
of 5ml AgNO3 was prepared.30% of 2.5 ml ammonium solution was added in to each
concentration. Finally made up to 50ml by using sterile distilled water, allowed to stir
overnight in dark.

4.3.2 Preparation of Aloe Vera gel extract:

Fresh leaves of Aloe Vera were collected. The leaves were washed with sterile
distilled water. The gel was separated from the leaves and 10g were weighed. The gel
was mixed with 100ml distilled water and heated for 12mnt.

Composite was prepared by adding 5ml of Aloe Vera gel extract to each
concentrations of 5ml AgNO3 was prepared.30% of 2.5 ml ammonium solution was
added in to each concentration. Finally made up to 50ml by using sterile distilled water,
allowed to stir overnight in dark.

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4.4 Method 2:

4.4.1 Preparation of complete Aloe Vera leaf extract:

Fresh mature leaves of Aloe Vera were collected and washed with sterile distilled
water.30g of leaves were weighed and grinded it well. The grinded leaves were mixed
with 100ml distilled water and heated for 12mnt.The extract was filtered through what
man no:1 filter paper which is placed on a conical flask. The filtrate was collected in the
conical flask and stored in refrigerator.

Composite was prepared by adding 0.1575g of AgNO3 directly to 10% of 100ml

Aloe Vera extract. Kept that in shaker for overnight incubation under dark at room
temperature.

4.4.2 Preparation of Aloe Vera gel extract:

Fresh leaves of Aloe Vera were collected. The leaves were washed with sterile
distilled water. The gel was separated from the leaves and 10g were weighed. The gel
was mixed with 100ml distilled water and heated for 12mnt.

Composite was prepared by adding 0.1575g of AgNO3 directly to 10% of 100ml

Aloe Vera gel extract. Kept that in shaker for overnight incubation under dark at room
temperature.

4.5 Detection, physical and molecular characterization of AgNPs:

Physical and molecular characterization of AgNPs was done by U-V spectroscopy,

Nitrate reductase assy and TEM analysis.

4.5.1 U-V Spectroscopy:

Ultraviolet–visible spectroscopy or ultraviolet-visible spectrophotometer (UV-

Vis or UV/Vis) refers to absorption spectroscopy or reflectance spectroscopy in
the ultraviolet-visible spectral region. This means it uses light in the visible and adjacent
ranges. The absorption or reflectance in the visible range directly affects the
perceived colour of the chemicals involved. In this region of the electromagnetic

16
spectrum, atoms and molecules undergo electronic transition. Absorption spectroscopy is
complementary to fluorescence spectroscopy, in that florescence deals with transitions
from the excited state to the ground state, while absorption measures transitions from the
ground state to the excited state. The method is most often used in a quantitative way to
determine concentrations of an absorbing species in solution, using the Beer-Lambert
law Ultraviolet light and visible light have just the right energy to cause an electronic
Transition of electron from one filled orbital to another of higher Energy unfilled orbital
When a molecule absorbs light of an appropriate wavelength and an electron is promoted
to a higher energy molecular orbital, the molecule is then in an excited state..

4.5.2 Chemical method

Prepare the sample as per the same procedure in the chemical synthesis. The
solution was kept as blank. Absorbance of incubated sample was taken by using
spectrometer.

4.5.3 Biological method

Prepare the sample by using both Aloe Vera complete extract and Gel extract by
using method 1 and 2 in the biological synthesis. These samples without incubation was
set as blanks for corresponding incubated samples. The absorbance of incubated sample
was taken.

4.5.4 Nitrate reductase assay:

0.1M KNO3 was added 0.1ml of incubated sample and incubated for 24hr.After
incubation 1ml of diazo coupling reagent was added to the sample. Incubated the
solution for 30 mints in dark. After incubation observed the OD at 540 nm.

4.5.5 TEM Analysis:

Transmission electron microscopy (TEM, also sometimes conventional

transmission electron microscopy or CTEM).is a microscopy technique in which a beam
of electron is transmitted through a specimen to form an image. The specimen is most
often an ultra thin section less than 100nm thick or a specimen on a grid. Samples CA
and CB are subjected to Transmission electron microscopy to determine the physical and
molecular characterization of AgNPs synthesized by biological method.

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 Fig 6: Transmitted Beam

For the characterization of AgNPs synthesized by using complete Aloe Vera leaf
extract in method 2 of the biological synthesis sample was subjected to TEM analysis.
Prepared the replicas of the same sample for accurate result. These samples were named
as CA and CB respectively.

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 5. RESULTS AND OBSERVATIONS

The present investigation entitled “optimization and standardization of silver

nanoparticle synthesis for its efficient application in wound healing” was conducted to
compare the chemical and biological methods for the synthesis of silver nanoparticles
and the application of most efficient silver nanoparticles for wound healing. Chemical
synthesis of silver nanoparticle is a very common approach and is confirmed with
different chemical reducing agents such as sodium tetra borate, sodium thiocitrate etc.
there are lot many disadvantages associated with chemical synthesis of silver
nanoparticles. Chemicals used for silver nanoparticles synthesis and stabilization are
toxic and lead to non-eco-friendly by products. The need for environmental non-toxic
synthetic protocols for Silver nanoparticles synthesis leads to the developing interest in
biological approaches which are free from the use of toxic chemicals as by products.
Thus, there is an increasing demand for green nanotechnology. Green synthesis provides
advancement of chemical and physical methods because it is cost effective,
environmental friendly and can easily scale up for large scale synthesis. Plants provide a
better platform for nanoparticles synthesis as they are free from toxic chemicals as well
as provide natural capping age

Different approaches of comparison between chemical and biological method is required

to prove the most efficient method. There are several techniques are available for this
purpose. In this proposed study, techniques used such as UV-Vis spectroscopy analysis
for the characterization of silver nanoparticles (color intensities of reaction mixture was
recorded by using UV-Vis spectrophotometer), nitrate reductase assay for the detection
of silver nanoparticles in the composites and TEM analysis provided further insight in to
the morphology and size details of silver nanoparticles.

5.1 UV-Vis SPECTROPHOTOMETER ANALYSIS:

Reduction of silver ions into silver nanoparticles during exposure to plant extracts was
observed as a result of the colour change. The colour change is due to the Surface
Plasmon Resonance phenomenon. The metal nanoparticles have free electrons, which

19
give the SPR absorption band, due to the combined vibration of electrons of metal
nanoparticles in resonance with light wave.

5.2 RESULT OBTAINED FROM CHEMICAL SYNTHESIS:

Composites prepared by using chemical method are subjected to UV-Vis spectrometer

analysis after 24 hours of incubation at dark. From different literatures (Jun Tian, 2007)
it was found that the silver nanoparticles show SPR peak at around 420 nm. No
composites show SPR at 420nm (figures: 7, 8 & 9). After 4 days of incubation only the
sample with AgNO3 concentration of 1mM gives SPR peak at 420 nm of wave length (
fig: 10). From the result it can be concluded that only the sample with SPR peak at
420nm (fig: 10) contains AgNPs, so this sample (1mM AgNO3 containing sample) can
be used for future studies.

Fig 7: graph obtained from uv-spectroscopy Fig 8: graph obtained from uv-spectroscopy

With sample1 (0.5mM of AgNO3) with sample2 (1mM of AgNO3)

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 After 4 days

Fig 9: graph obtained from uv-spectroscopy Fig 10: Max absorbance with 1mMAgNO3
withsample3 (2mM AgNO3) solution is 0.63 at 400nm

Table 2: Absorbance of sample prepared by chemical synthesis

Sample no: Wavelength nm Absorbance

S1 400 0.020

S2 200 0.481

S3 240 0.118

1After 4 days 400 0.063

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5.3 RESULT OBTAINED FROM GREEN SYNTHESIS:

5.3.1 METHOD 1:

Composites prepared by using biological method 1 are subjected to UV-Vis spectrometer

analysis for the partial conformation of the presence of AgNPs after 24 hours of
incubation. According to the various literatures green synthesized silver nanoparticles
shows SPR at 450-480nm of wavelength. No sample shows SPR at this range of
wavelength after 24 hours of incubation (fig: 11, 12&13). The procedure was repeated
multiple times to obtain the required result. But due to certain changes occurred in
parameters (pH,temp,storage condition etc) of this method required result was doesn’t
obtained.

Fig 11: graph obtained with sample B1 Fig 12: graph obtained with B2

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Fig 13: graph obtained with sample B3 Fig 14: composites prepared after incubation

Table no 3: Absorbance of samples prepared by first biological method

Sample no: Wave length Absorbance

B1 220 0.113

B2 200 0.144

B3 220 0.02

5.3.2 METHOD 2:

To check reducing efficiency of different components of Aloe Vera was determined by

repeating the same methodology for each components to get maximum yield of AgNPs.

5.3.3 Result from complete leaf extract:

After 24 hours of dark incubation three replicas of Composites prepared by using second
biological method is subjected to uv-vis spectrometer analysis for the initial
conformation of presence of AgNPs. From literature of Shreya Medda, (2015) it was
found that green synthesized AgNPs shows SPR peak at 450-480nm. All the three
samples shows SPR peak at 450nm (figures: 15, 16 &17). Result obtained from this

23
method indicates the presence of AgNPs in the composites.so it can be used for further
studies

Fig 15: Graph obtained from sample A Fig 16: Graph obtained from sample B

Shows max absorbance at 450nm Shows max absorbance at 450nm

Fig 17: Graph obtained from sample C Fig 18: colour indication after incubation
Shows max absorbance at 450nm

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 Table 4: Absorbance of sample prepared by second biological method

Complete grinded sample Max absorbance Wave length nm

CA 0.136 450

CB 0.133 450

CC 0.178 450

5.3.4 Result obtained from gel extract:

Based on second biological method replicas of composites prepared by using gel extract
were subjected to UV-Vis spectroscopy after 24 hours of incubation. All the three
replicas shows SPR peak at 460nm.

From the result it can be concluded that these samples may also contain AgNPs.
Compared to complete extract containing samples absorbance was obtained very low in
gel contain samples. Hence the future studies were preceded with composites prepared
by using complete extract.

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Table 5: Comparison of chemical and biological methods by using spectroscopy analysis

Chemical synthesis Biological synthesis

Sample Wavelength Absorbance Sample Wavelength Absorbance

name (nm) name (nm)

S1 400 0.020 CA 450 0.136

S2 200 0.481 CB 450 0.133

S3 240 0.118 CC 450 0.178

S1 (after 4 400 0.063

days)

Both chemical and biological synthesis of silver nanoparticles was carried out. From
table 5 it’s clear that green synthesis is the most efficient method for the synthesis of
silver nanoparticle. Yield of silver nanoparticles from different component was also
confirmed. From that, the complete extract shows maximum yield of production of silver
nanoparticles than other components.

Hence the samples prepared by using complete extract of aloe Vera extract are subjected
to further studies.

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5.4 NITRATE REDUCTASE ASSAY

Fig 19: Flow diagram of nitrate reduction

PRINCIPLE:

Nitrate + ß-NADH Nitrate Reductase> Nitrite + ß-NAD + H2O

Nitrite + Sulfanilamide + NED > Nitrite Color Complex

Abbreviations used:

ß-NADH = ß-Nicotinamide Adenine Dinucleotide, Reduced Form

ß-NAD = ß-Nicotinamide Adenine Dinucleotide, Oxidized Form

NED = N-(1-Naphthyl)ethylenediamineDihydrochloride

5.4.1 Result of Nitrate Reductase Assay:

The change of red color after addition of diazocoupling reagent and incubation for 30
min in dark it was occur only when there is presence of nitrite in reaction. But no such
change was observed in AgNPs synthesized reaction mix. The Nitrate reductase in the
reaction mixture reduced the KNO3 to nitrite.

27
But in AgNPs mix there is no presence of nitrite which means the nitrate reductase
present in the reaction mixture was fully utilized in the conversion of AgNO3 to Ag0
(Ag Nanoparticles). The colour intensity was increased as the amount of nitrate reductase
in the medium.

Two replicas (CA and CB) from the chosen samples for future study were subjected to
nitrate reductase assay. Samples don’t show colour after incubation (fig: 21), so it may
conclude that there is a presence of silver nanoparticles in the sample. According to
Mahendra Rai (2009) absence of colour indicates the presence of silver nanoparticles.

Fig 20: Samples without AgNPs Fig 21: Samples with AgNPs

5.5 TEM ANALYSIS

Physical and molecular characterization of silver nanoparticle is essentially required for

the conformation of presence of silver nanoparticles in the sample. Hence the samples
are subjected to TEM analysis. Transmission electron microscopy (TEM, also sometimes
conventional transmission electron microscopy or CTEM).is a microscopy technique in
which a beam of electron is transmitted through a specimen to form an image. The
specimen is most often an ultra-thin section less than 100nm thick or a specimen on a
grid.

Samples CA and CB are subjected to Transmission electron microscopy to determine the

physical and molecular characterization of green synthesized AgNPs by using complete
extract of Aloe Vera.

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5.5.1 TEM images of Sample CA

29
30
 Fig 22: Set of TEM images obtained from sample CA

Table 6: Range of particles with diameter

Particle no: Size (nm)

1 16.27
2 18.31
3 20.77
4 25.28
5 25.8
6 30.43
7 31.31

Lowest particles obtained from sample CA with diameter is 16.27 and the highest
particle obtained from sample CA with diameter is 31.31. (From table no: 6)

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5.5.2 TEM images of sample CB

32
33
 Fig 23: Set of TEM images obtained from sample CB

Table no 7: Range of particles with diameter

Particle no: Size(nm)

1 22.52
2 25.57
3 29.13
4 37.74
5 48.13

Lowest particle obtained from the sample CB with diameter is 22.52 and the highest
particle obtained from sample CB with diameter is 48.13. (From table no: 7)

Behavior of silver nanoparticles is a function of their size shape and surface reactivity
with the surrounding tissue. According to Shreya Medda (2015), the size range required
for the efficient wound healing property of silver nanoparticles ranges from 1nm to 40nm
in size. Samples CA and CB contains silver nanoparticles with size range required for
efficient wound healing property (From tables: 6 and 7). Hence we concluded that silver
nanoparticles present in our sample (CA and CB) can be used for wound healing.

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 6. CONCLUSION AND RECOMMENDATIONS

In the present study, we focused on green synthesis of silver nanoparticles using aqueous
leaf extract of Aloe Vera. From the comparative analysis of chemical and biological
methods for synthesis of silver nanoparticles, it was found that biological method (green
synthesis) is the most efficient method for the synthesis of silver nanoparticles. Yield of
production of silver nanoparticles from different components of Aloe Vera was also
conducted, from that complete leaf extract gives the maximum yield of production of
silver nanoparticles than other components. The presence of silver nanoparticles in the
prepared samples was confirmed by using nitrate reductase assay. The physical
properties of synthesised AgNPs were characterized by using relevant techniques. The
conducted study shows that the presence of silver nanoparticles with the properties
required for wound healing such as size range, shape and structural configurations. In
future as a part of Invitro analysis, MTT assay can be conducted to analyse the wound
healing efficiency of silver nanoparticles synthesised by this method.

Furthermore the applications of silver nanoparticles are spread in the areas of medical as
well as agricultural fields. It can be incorporated into the antiseptics and it can also be
used for target specific internal wound healing therapies. The data represented in our
study contributed to a novel and unique virgin area of Nano-materials as an alternative
wound healing agent. With the little uncovered mechanism in the current study, there is a
wide scope for the detailed investigation such as Histopathological study for the vibrant
application of green synthesised silver nanoparticles in the area of life science.

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 7. REFERENCE

 Shreya medda ,A Hajra, U Dey ,et at., (2015)Biosynthesis of silver nano particles
from aloe vera leaf extract Applied Nano science.

 S Prathap Chandran et al.,(2006) Synthesis of gold Nano triangles and silver

Nano particles using aloe Vera plant extract.

 R Maenthisong, N Chaiyakunavruv, S Niruntaporn et al.,(2007)The efficacy

of aloe Vera used for burn wound healing.

 Jun Tian, Kenneth K.Y. Wong, Chi-Ming Ho,et al;(2007)Topical delivery of the
silver nano particles promotes wound healing.
Willey Interscience vol:3

 Patcharaporn Tippayawat , N Phromviyo, P Boueroy et al., (2016) Green

synthesis of silver Nanoparticles in aloe Vera plant extract prepared by a
hydrothermal method and their synergistic antibacterial activity.

 Priya Banergee, M Satapathy, A Mukhopahayay et al.,2014 Leaf extract

mediated green synthesis of silver nanoparticles from widely available Indian
plants synthesis, characteristics, antimicrobial property and toxicity analysis.

 Mahendra Rai, Alka yadav et al., (2009): Silvernanoparticles and as a new

generation of antimicrobial

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