Synthesis and Characterization

of Silver Nanoparticles from Leaf

Aqueous Extract of Aloe Vera
and their Anti-Microbial Activity

By,
Sushma.A (160010167)
Manideepthi.B (160010181)
Nanoparticles are particles between 1 and 100 nanometres(nm) in
size with a surrounding interfacial layer. The interfacial layer is an
integral part of nanoscale matter, fundamentally affecting all of its
properties. The interfacial layer typically consists of ions, inorganic and
organic molecules.

PROPERTIES :
• Nanoparticles often possess unexpected physical and optical
properties, as they are small enough to confine their electrons and
produce quantum effects. For example, gold nanoparticles appear
deep-red to black in solution.
• Nanoparticles of yellow gold and grey silicon are red in colour. 
•Nanoparticles have other physical properties that must be measured
for a complete description, such as size, shape, surface properties,
crystallity,and dispersion state.
Green synthesis of silver nano particles:
Green synthesis’ are required to avoid the production of unwanted
or harmful by-products through the build-up of reliable, sustainable, and
eco-friendly synthesis procedures.
Green synthesis of metallic nanoparticles has been adopted to
accommodate various biological materials (e.g., bacteria, fungi, algae, and
plant extracts).

Advantages of green synthesis over chemical synthesis of silver

nano particles:

1.Eco friendly

2.lowcost

3.can be used for large scale synthesis.

4.no need to use high pressure,energy,temperature and toxic

chemicals

5.not required any isolation techniques.

2. LITERATURE SURVEY
The comprehensive survey of literature showed the green process for
silver nanoparticles synthesis.
1. GangaRao Battub, K. Basavaiaha (2018) :
  The analysis in this Ag NPs was synthesized using Aloe Leaf Gel's aqueous
leaf extract using green route process. The phytochemical present in Aloe Leaf
Gel's aqueous leaf extract can act as a reduction and capping agent for
Ag NPs formation. It is found that the rate of formation of Ag NPs depends on
AgNO3 concentration, aqueous leaf extract concentration of Aloe Leaf Gel pH,
temperature and time of reaction. Although many biological green methods for
Ag NPs synthesis have been published.
2. Anand Kumar Keshari (2018):
The AgNPs have special properties of chemical, physical, electrical, mechanical,
and magnetic. Researchers are interested in exploring their nano-medicine
applications such as anti-plasmodial, anti-microbial, controlled drug delivery,
sensing and imaging, anti-pilot, anti-cancer and wound healing, certain special
properties of AgNPs.
3.Alvaro de Jesus Ruiz-Baltazar (2018):
 The present study uses Aloe natalenis leaf extract in aqueous media to report the
green synthesis of silver nanoparticles (AgNP's). The reduction of silver ions was
accomplished by the antioxidant element in the Aloe natalenis.Transmission
electron micrographs showed the semispheric morphology of the synthesized
AgNP and the particle size was less than 45 nm.
4.Simon Yobbany Reyes-Lopez (2017):
 The green synthesis process through Aloe Perfoliata extract offers high
bio-reduction efficiency, because of the notable antioxidant properties. The
silver nanoparticles ' exceptional properties offer many applications in the field
of biomedicine. One of the key strategies of silver nanoparticles is the design
of antibiotics that are clinically effective against bacteria and drug-resistant
Microorganisms

5.Ezhumalai Parthiban (2018):

The synthesis of silver nanoparticles by Aloe Perryi Baker aqueous extract
has produced small-sized nanoparticles in nature with spherical shapes and
crystalline. Effective larvicidal activity has been shown without harming the
non-target aquatic insect and, in addition, green synthesized silver
nanoparticles have shown possible antimicrobial activity with biocompatibility
with red blood cells against O+ve erythrocytes with respect to consideration for
human welfare
OBJECTIVE:

1) To extract phytoconstituents by using different solvent from selected

plant.

2) To develop and establish safe, ecofriendly, cheaper method for the

synthesis of silver nanoparticles.

3) To characterize synthesized silver nanoparticles with various

analytical methods.

4) To evaluate biological activity of the synthesized silver nanoparticles.

5) To study stability of synthesized nanoparticles in different pH range.

MATERIALS:
• Silver Nitrate (AgNO3)
•Distilled water
•What-man filter paper
•Aloe Vera leaves
•Chromic acid
•Volumetric flask
•Petri plates
•Beakers
•Escherichia coli
•Staphylococcus aureus
•Agar Medium
•Alsever solution
Blood
METHODOLOGY to be adopted:

1) Selection of plant and procurement of chemicals

2) Collection of plant

3) Authentication of plant

4) preparation of aloe barba dense leaves extract

5) preparation of silver nano particles(Ag Np) by bio extract

6) Isolation and characterization of nanoparticle

7) Biological application
PROCEDURE:
1.Selection of plants:
Based on the extensive literature survey and review
plant of Momordica dioica, Aloe indica Royle, Aloe perfoliata L. 
is planned as green source for synthesis of silver
nanoparticles.

2. Collection of plant :
The plants will be collected from native place.

3. Authetication of plant:
Plant will be then authenticated.
4. PREPARATION OF ALOE BARBA DENSE LEAVES EXTRACT:

Fresh leaves of Aloe Vera were collected and washed

several time with distilled water in order to remove the dust particle and then
sun dried to remove the remaining moisture. Weighed 10gram dried Aloe Vera
leaves, crumpled and chopped with glass rod in 500ml glass beaker, added
200ml sterilized distilled water in 250 ml beaker, boiled for 40 minutes, until the
color of the aqueous solution changes from watery to light yellow, Cool the
extract at room temperature then filtered through what-man filter paper,
centrifuged at 3000rpm for 30minutes, and separate the supernatant layer by
pipette. Store the Aloe Vera extract at 16-18 °C temperatures and use within
one week.
5.PREPARATION OF SILVER NANO PARTICLES(AG NP) BY BIO EXTRACT

Took 0.17gm of silver nitrate in 1000ml volumetric flask and dissolved

in distilled water and made up the volume to 1000 ml to Prepare 0.001M/1mM
solution of AgNO3. In typical reaction procedure take 50 ml 0.001mm solution
of AgNO3 then added 20 ml Aloe Vera leaves extract drop-wise with continuous
shaking at room temperature until the solution become grey black colour,
indicated the reduction of silver ion by the aqueous plant extract and the
formation of stable silver nanoparticle.

A : AgNO3 solution
B:AgNPs formation

B
A
Ag NPs powder after drying
 6) Isolation and characterization of nanoparticle
Some methods of evaluation of silver nanoparticles:
UV-visible spectral analysis
Anti-microbial activity
Hemolysis
Micro structural analysis

UV-VISIBLE SPECTRAL ANALYSIS

UV-Visible spectral analysis is a simple technique that can be used to detect

the formation of the nanoparticles ' silver synthesis. The UV-visible spectrum of
the solution extract of aqueous silver nanoparticles has been reported. Regular
sampling of aliquots (0.2 ml) of the bio extract, then diluting the samples with 2
ml of deionized water and subsequently measuring UV –Vis spectra of the
resulting diluents, controlled the reduction of Ag ion in bio extract solution. The
spectrum of absorption is plotted against the wavelength range from 300-600
nm in UV visible spectral analysis of the synthesis of a Nanoparticles.
The silver nanoparticle UV-vis spectra in Figure 3(a) were reported using leaf
extract from the reaction mixture and mixed in aqueous solution
of the silver ion complex. The test shows similar activity at 390-410 nm with
total absorption peak. The overall spectrum width and shift in the absorbance
peak indicate that silver nanoparticles require some time to settle, which
means that the reaction takes time to complete, but the size of these particles
increases slightly compared to the initial reaction stage when the extract is
mixed with the salt It is therefore apparent that as the reaction time increases
the concentration and size of silver nanoparticles that can be detected at
different times. The absorption occurs at 420 nm, suggesting the formation
of silver nanoparticles and increasing their wavelength with the reaction time

 
 
 
 
 
 
 
 
 MICROSTRUCTURAL ANALYSIS:
Microstructure is the very small-scale structure of a material,
defined by an optical microscope above 25 magnifications
as the structure of a prepared material surface. A material's
microstructure can affect physical properties such as strength,
durability, ductility, hardness, corrosion resistance, high / low
temperature behavior or wear resistance. The microstructure
was examined under a light microscope and the image was
captured.
 HEMOCOMPATIBILITY TEST
Hemocompatibility experiments are in vitro studies used to determine the
probability of having antagonistic effects on red platelets (hemolysis),
thrombosis, coagulation, platelets and supplement systems. When the blood
interacts with non-hemocompatible material, it triggers the coagulation
enactment and the insusceptible structure within the human body that can
destroy the patient's life, for which it is important to consider completing a
hemocompatibility. The hemolysis test is intended to determine a therapeutic
gadget / material's haemolytic properties. The research is carried out on
restorative gadgets / materials that have immediate or circuitous blood
interaction and meet the needs of blood tests to pick blood cooperative tests. The
hemocompatibility check is necessary for new restorative devices, medications,
blood products and demonstrative operators to be introduced.

Hemolysis = (OD of test sample) – (OD of –ve control)

(OD of +ve control) - (OD of –ve control)
CONDITIONS FOR HEMOCOMPATIBILITY:
•Higher compatibility (<5% hemolysis)
•Hemo compatible ( Within 10% hemolysis)
Non hemo compatible ( >20% hemolysis)
 PROCEDURE FOR HEMOLYSIS TEST:
 
1) Equal ratio of Alsever solution and blood were added
2) The solution was washed with Isosaline or PBS buffer for 3-4times.
3) The solution was centrifuged at 3000 rpm for 10 mins
4) Centrifugation was repeated for 2-3 times.
5)The hypotonic solution and blood were used as negative control and
blood and deionised water were used as positive control.
6)The sample, hypotonic solution and blood were added according to the
given composition: sample-50µl, hypotonic solution-100µl, Blood-5µl. (Make
up to 1ml by adding distilled water).
7)OD values were measured at 560 nm and hemo compatibility was measured
This hemolysis test was done by taking the nanoparticles samples OD at
560nm at varying concentration, then we got the following values:

SAMPLE OD VALUES(at 560nm) HEMOLYSIS

Positive Control 1.68 -

Negative Control 1.36 -

0.2 1.08 0.875

0.4 1.57 0.656

0.6 2.25 2.781

 BIOLOGICAL APPLICATION

Media Preparation
Agar Medium (1 L) The medium was prepared by dissolving the commercially
available Agar Medium (Media) in1000ml of distilled water. The dissolved
medium was autoclaved at 15 lbs pressure at 121°C for 15 minutes.
Inoculation of micro-organism and Preparation of Media plates
Vortex the organism suspension to make sure it is well-mixed. Then, dipped
a sterile wire lope into the suspension, taken out and inoculated in the
sterilized media ensuring that the temperature of media should not exceed
50 °C, shaken well and poured 20ml of this inoculated media in each petri
plate (sterilized at 160 °C for one and half hour), let it to jellify.
Digging of wells and pouring
Dig wells in a petri dish, containing 20ml agar medium that were seeded with
24hr culture of bacterial strains, with a sterile borer, and fill the well with 20 µl
of the nanoparticle were added in each well. The plates were then incubated
at 37 °C for 24 hours
Anti-microbial activity
DISCUSSIONS OF RESULTS
 
The effect of silver ion on microbe can be detected through changes in structure
and morphology, but the mechanism of action is not properly understood. One of
the strongest methods was proposed that when the microbes ‘ DNA molecule is
in a relaxed state, DNA replication can be done effectively. When the microbial
DNA is treated with silver ions, it loses its ability to replicate as it becomes a
condensation and prevents the cell division which contributes to cell death.
Another study explores the direct attachment of heavy metal like silver ions to
the sulfur based thiol group of bacterial membrane proteins that can be bound
to the cell as well as to microorganisms DNA

Compared to other metal ions, the active antimicrobial property of silver ion
derived from Aloe Vera is attributable to its extremely large surface area, small
size and essential oil portion of Aloe Vera leaves that provide antimicrobial
activity for batter attachment and enhancement. Silver ion's antifungal activity
mechanism has not been suggested yet, but silver nanoparticle also shows the
strongest antifungal behavior.
CONCLUSION
 
 We found in our analysis that leaves from the extract of Aloe Vera could be a
good source of silver synthesis of Nano-particles. The type of synthesis used
in our study is called green synthesis and has many advantages as it is an
environmentally friendly, economically viable and efficient method that removes
external stabilizers or reduction agents. Most significantly, the reaction was
easy to handle and is claimed to have advantages over other biological
synthesis. Silver nanoparticles show a dark yellowish brown color in aqueous
solution due to the resonance effects of surface plasmon modification. Both
Ag Nano particles were developed by green synthesis using UV-spectra and
microstructural analysis. In addition to demonstrating the existence of silver
synthesis of Nano particles, the UV-Visible Analysis also reveals the reaction
time on the absorption frequency. As the reaction time decreases, the rate of
absorption increases when Nano-particles increase in size and concentration.
Anti-microbial activity of this silver synthesis of the Nano particles was tested
against Escherichia coli and Staphylococcus aureus, as a strong anti-bacterial
agent showing high efficacy of silver synthesis of the Nano particles.
Hemolysis test showed hemocompatibility and hemocompatibility of silver
nanoparticles.
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