BIOLOGICAL SILVER SYNTHESIS AND CHARACTERIZATION OF …
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BIOLOGICAL SILVER SYNTHESIS AND CHARACTERIZATION OF
SILVER NANOPARTICLES USING CHRYSOPHYLLUM CANITO LEAF
EXTRACT
Antony Prabhu Y.1* and Shamina S.
2
1*MPhil Biochemistry (Research Scholar) RVS College of Arts and Science Sulur.
2Asso. Prof, Dept. of Biochemistry, RVS College of Arts and Science Sulur.
ABSTRACT
The nanoscience theoretical knowledge and applying innovative idea
together in the diverse research field is termed as nanotechnology. It
gives massive effects and unique features in medicinal drug discovery
and exhibits wide applications. In this study was aimed on biological
silver synthesis in various solvents such as ethanol, methanol,
chloroform and petroleum ether. These types of oddity research works
may rapidly produce silver nanoparticles better than other solvents.
Antioxidant DPPH Non-enzymatic assay revealed maximum presence
of free radical scavenging activity in ethanol solvents. Phytochemical
qualitative analysis has been done indiverse solvents. Best nanoparticle
synthesizing solvent analyzed by double beam UV Spectroscopy and
well synthesized silver nanoparticles were characterized Alongwith Scanning electron
microscope (SEM), Energy dispersive x-ray spectroscopy (EDAX), X-ray Dispersive
spectroscopy (XRD) and synthesized silver nanoparticles used against as Klebsiella
pneumonia.
KEYWORDS: Silver nanoparticles, Various solvents, Antioxidant DPPH assay,
Phytochemicals, SEM, EDAX, XRD and Klebsiella pneumonia.
INTRODUCTION
Ultra fine nanoparticles are particles between the size of 1nm to 100nm in nanoscale, fine
nanoparticles in the ranges between 100 nm to 2500 nm and coarse nanoparticles in the
ranges between 2500 nm to 10000 nm (Cristina et al., 2007).
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
SJIF Impact Factor 6.041
Volume 5, Issue 03, 1482-1498. Research Article ISSN 2278 – 4357
*Correspondence for
Author
S. Shamina
Asso. Prof, Dept. of
Biochemistry, RVS
College of Arts and
Science Sulur.
Article Received on
08 Jan 2016,
Revised on 30 Jan 2016,
Accepted on 21 Feb 2016
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Nanoscience theoretical knowledge with applying innovative idea towards in the diverse
research field is termed as nanotechnology. Nanoparticles researches has been being great
and unique due to the extreme surface area, unique features, wide and variety of potential
applications in medicinal fields (Anna et al., 2015) such as immune response stimulation,
inhalable vaccines, acts as an antioxidant, increase bone formation, inhibit brain tumors and
cancer cell detection.
Suspensions of nanoparticles synthesis with characterization and applying in vitro and in vivo
analysis may utterly reveals iron, gold, silver (Bogumiłaet al., 2013), titanium, cerium,
nanodiamonds, nickel, silicate, zinc and palladium nanoparticles entire actions and uses in
medicinal field (James et al., 2012).
An antioxidant is a unique molecule that interacts directly or indirectly the oxidation reaction
producing free radicals and over production of free radical’s chain reactions leads to bring
cell death or cell damage (Sunday et al., 2014).
Actually antioxidants are terminating free radicals chain via making cleavage. The
superoxide dismutase enzymes are directly inhibiting oxidation and non-enzymatic inasmuch
as vitamin and vitamin c antioxidants are indirectly inhibiting oxidation process.
Phytochemicals are plant synthesizing chemical compounds for growth and development and
some of the phytonutrients are being responsible for color and it has much biological
significance (Francis et al., 2015).
Chrysophyllum canito matured and ripe contain 25 % calories and Moisture 33 % (Nwosu et
al., 2013). Medicinal plant contains diverse types of chemical compounds for their growth
development and defense mechanism scientists unraveled the secondary metabolites actions
and function against microorganisms. Plants naturally synthesizing secondary metabolites for
their defense mechanisms (Bhekumthetho et al.,2015).
Plant defense mechanism is potential for recognizing pathogen- associated molecular patterns
entrees and immediately sense and send signals to plant pattern recognition receptors to
instantly activate immune system. Green silver synthesis possible alleyway may use to
increase silver quantity (Raid et al., 2014) and modifying synthesized silver nanoparticles in
forming oral care products will utterly revert the 99.9% of oral clean, invigorate and applying
targeted drug delivery system may eradicate the cancer progression (Royyuru et al., 2013).
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The chemical drugs suppress the microbial growth and micropropagation. When microbes get
tolerated like Escherichia coli and Staphylococcus aureus (Hongxia et al., 2015) may bring
rigorous problems to humankind but synthesized silver coated antibodies and silver used
targeted drug delivery would perfectly destroy the tolerated microbial growth.
Experimental section
Sample collection
The Chrysophyllum canito leaf chosen for silver nanoparticles synthesis and it obtained from
Anaikatti, Coimbatore. 5 grams of leaf in 10ml of pure water were used for extract
preparation. The cooling centrifuge had been used for crude extract.
Silver synthesis
The silver nitrate solution was prepared in various solvents and the 9 ml of 3mM
concentration of silver solution was prepared and 1 ml of plant crude extract slowly added
upon silver solution and abrupt color changes obviously indicates the silver synthesis.
UV- Visible spectroscopy analysis
The reduction of metallic Ag+ ions was monitored by measuring UV- Visible spectrum after
about 16 hours of reaction. The UV beams emitted upon the samples and its absorbed values
were detected by sensors eventually provides the wavelength from 200nm to 800nm in Ultra
visible spectrophotometer (Systronics Double beam spectrophotometer 2202).Synthesized
silver nanoparticles solution was given to ultra-visible spectroscopy analysis.
Free radical scavenging activity on DPPH
The antioxidant activity of the sample was determined in terms of hydrogen donating or
radical scavenging ability, using the stable radical DPPH, according to the method of Blois
(1958). The sample extracts at various concentrations (0.5 – 2.5 µl) was taken and the volume
was adjusted to 50 µl with solvent. 5 ml of 0.1 methanolic solution of DPPH was added and
allowed to stand for 20 min at 27°C. The absorbance of the sample was measured at 517 nm.
Percentage radical scavenging activity of the sample was calculated as follows: % DPPH
radical scavenging activity = (control OD-sample OD / control OD) × 100 The analysis was
performed in triplicate. The sample concentration providing 50% inhibition (IC50) under the
assay condition was calculated from the graph of inhibition percentage against sample
concentration.
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Phytochemical screening
Phytochemical examinations were carried out for all extracts as per the standard methods
(Mirashfaq et al., .2012).
Detection of alkaloids: Hager’s Test
Extracts were dissolved individually in dilute Hydrochloric acid and filtered. Filtrates were
treated with Hager’s reagent (saturated picric acid solution). Presence of alkaloids confirmed
by the formation of yellow colored precipitate.
Detection of carbohydrates: Benedict’s test
Extracts were dissolved individually in 5 ml distilled water and filtered. The filtrates were
used to test for the presence of carbohydrates. Filtrates were treated with Benedict’s reagent
and heated gently. Orange red precipitate indicates the presence of reducing sugars.
Detection of glycosides: Modified Borntrager’s Test
Extracts were hydrolyzed with dil. HCl, and then subjected to test for glycosides. Extracts
were treated with Ferric Chloride solution and immersed in boiling water for about 5 minutes.
The mixture was cooled and extracted with equal volumes of benzene. The benzene layer was
separated and treated with ammonia solution. Formation of rose-pink color in the ammonical
layer indicates the presence of anthranol glycosides.
Detection of saponins:Froth Test
Extracts were diluted with distilled water to 20ml and this was shaken in a graduated cylinder
for 15 minutes. Formation of 1 cm layer of foam indicates the presence of saponins.
Detection of phytosterols:Salkowski’s Test
Extracts were treated with chloroform and filtered. The filtrates were treated with few drops
of Conc. Sulphuric acid, shaken and allowed to stand. Appearance of golden yellow color
indicates the presence of triterpenes.
Detection of phenols: Ferric Chloride Test
Extracts were treated with 3-4 drops of ferric chloride solution. Formation of bluish black
color indicates the presence of phenols.
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Detection of tannins:Gelatin Test
To the extract, 1% gelatin solution containing sodium chloride was added. Formation of
white precipitate indicates the presence of tannins.
Detection of flavonoids:Alkaline Reagent Test
Extracts were treated with few drops of sodium hydroxide solution. Formation of intense
yellow color, which becomes colorless on addition of dilute acid, indicates the presence of
flavonoids.
Detection of proteins: Xanthoproteic Test
The extracts were treated with few drops of conc. Nitric acid. Formation of yellow color
indicates the presence of proteins.
Detection of amino acids: Ninhydrin Test
To the extract, 0.25% w/v ninhydrin reagent was added and boiled for few minutes.
Formation of blue color indicates the presence of amino acid.
Detection of diterpenes:Copper acetate Test
Extracts were dissolved in water and treated with 3-4 drops of copper acetate solution.
Formation of emerald green colour indicates the presence of diterpenes.
Characterization of silver nanoparticles
Scanning electron microscope and Energy-dispersive X-ray spectroscopy
The 3mM concentration of Silver nanoparticles in ethanol solvent was synthesized via green
chemistry oxido-reduction method. These prepared samples poured in Petri dish and kept at
hot air oven for 24 hours until the silver gets settle down. After that synthesized silver and
stored in plastic tubes. These steps recurred many time for samples preparation. In this
process silver purified by acetone. Small quantities of synthesized silver had given for SEM
and EDAX.
X-ray Dispersive spectroscopy
The 3mM concentration of Silver nanoparticles in ethanol solvent was synthesized via green
chemistry oxido-reduction method. These prepared sample poured in Petri dish and kept at
hot air oven for 24 hours until the silver gets settle down. After that dried well prepared
sample and stored in plastic tubes. These steps recurred many time for sample preparation. In
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this process silver purified by acetone. Small quantities of synthesized nanoparticles analyzed
under X-Ray dispersive spectroscopy.
Antibacterial activity
Principle
The disc impregnated with antibiotic of known concentrations can inhibit growth of
microorganisms. This procedure requires the heavy inoculation of an agar plate with the test
organism. Antibiotic impregnated disc is equally spaced in the inoculated agar plate.
Following incubation, the agar plate is examined for zones of inhibition which is an
indicative of microbial activity against the organisms. The effectiveness of antimicrobial
sensitivity testing is based on the size of zone of inhibition.
Materials required
• Pure culture of test organisms
• 5ml of broth suitable for growth of organisms
• Suitable solid media in petridish
• Antibiotic disc
Preparation of the microorganisms
Organisms used in the study Klebsiella pneumonia. Strains were maintained on nutrient agar
slants at 400C. A loop full of bacterial strain was inoculated into 50ml of sterile nutrient broth
in 100ml conical flask. The flasks were incubated on a rotary shaker for 24 hours to activate
the strain. Muller Hinton agar medium was used as bacterial culture medium in the
antibacterial assay.
Preparation of media
Culture medium
Muller Hinton agar (pH 7.3)
Beef infusion : 300.0g
Casein enzyme hydrolysate : 15.50g
Starch : 1.50g
Agar : 17.0g
Distilled water : 1000ml
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Media was sterilized by autoclaving at 1210C for 15 minutes and cooling to 50
0C and
dispensed about 20ml Muller Hinton agar into sterile petri dishes. The plates were allowed to
solidify.
Anti-bacterial assay – disc diffusion method
The extracts obtained were screened for their antibacterial activity in comparison with
standard antibiotic gentamycin 10mg/ml in vitro by disc diffusion method using various
bacterial strains. The paper disc (6mm diameter, whatman no.1 filter paper) containing
various concentrations of samples 5-20ug/ml was chosen and placed aseptically on the agar
surface with the help of a sterile forceps and paper discs were pressed slightly with the
forceps to make complete contact with the surface of the medium. The plates were kept at
room temperature for half an hour and subsequently incubated at 370C and observed for zone
of inhibition after 24 hours. The inhibition zone around each disc was measured in
millimeter. The results were recorded by measuring the zone of growth inhibition
surrounding the disc.
RESULTS
Double beam ultra-visible spectroscopy
The green synthesis is a plant mediated method to rapidly synthesize silver nanoparticles. In
this research, various solvents used such as methanol, ethanol, petroleum ether and
chloroform principally plant chemical compounds interacting with AgNO3 salt to synthesize
silver nanoparticles. UV- visible beams pass through silver synthesized sample and detects
synthesized silver nanoparticles wavelength at 430nm.
Double beam UV spectroscopy obviously confirm synthesis of nanoparticles in ethanol
solvent. In this obtained silver nanoparticles wavelength exactly similar to (Jancyet al.,
2012).
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Fig 1 Silver synthesis in various solvents
Table 1 Describes synthesized silver wavelengths
S.NO Type of Samples Wavelength in nm
1. 3mM Chloroform silver synthesized 689
2. 3Mm ethanol silver synthesized 430
3. 3mM methanol silver synthesized 295
4. 3mM petroleum ether silver synthesized 756
In this analysis, diverse type of solvents used such as ethanol, methanol, chloroform, and
petroleum ether in spite of best synthesized silver nanoparticle wavelength had emerged in
ethanol solvent.
Antioxidant DPPH assay in Chrysophyllum canito leaf
Oxidation of biomolecules can able to cause free radical in the body more oxidation leads to
responsible for various disorders in human like arthritis, inflammation, heart diseases
immune impairment, and cancer. The antioxidant can able to inhibit oxidation and besides
preventing the various disorders in body.
The antioxidants terminate radical’s chain reaction by abolishing intermediates of free
radical, and suppress other oxidation reactions. DPPH is an organic chemical compound 2,2-
diphenyl-1-picrylhydrazyl is composed of stable free radical molecules used to monitor
chemical reactions associating radicals and expose electro paramagnetic resonance signals.
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In this 2,2-diphenyl-1-picrylhydrazyl test obviously exhibits compounds with a stable free
radical. DPPH gives a strong absorption band at 520nm in visible region. When the single
electron becomes paired off in the presence of a free radical scavenger, absorption band
reduces and occurs color changes from deep violet to light yellow.
The degree of reduction in absorbance measurement is implicating radical scavenging
activity in ethanol solvent of Chrysophylum canito and presence of free radical scavenging
activity was found to be IC50 at 0.48 (µl/ml).
Table 2 Antioxidant DPPH Assay in Chrysophylum canito leaf
S.No Sample Concentration
(µl)
Percentage
activity %
IC50
(µl/ml)
1.
CC- Ethanol
0.5 19.11 ± 0.13
0.48 ± 0.002
1.0 45.10 ± 2.29
1.5 65.46 ± 0.23
2.0 87.09 ± 0.16
2.5 93.35 ± 0.2
Values are means three of independent analyses of the extract (+ -) standard deviation (n=3)
DPPH radical scavenging activity in various solvents.
Graph 1 Chrysophyllum canitoleaf DPPH assay
The Chrysophyllum canito leaf ethanol solvent DPPH maximum free radical scavenging
activity (Inhibition Vs. Concentration µl/ml) was found to be IC50 = 0.48 µl.
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Table 3 DPPH assay of antioxidant standard used as Butyl hydroxyl toluene
S.NO Standard Concentration
(µg)
Percentage
activity (%)
IC50
(g/ml)
1. BHT
2 6.46 ± 0.13
4.05±0.025
4 20.26 ± 0.29
6 27.59 ± 0.53
8 39.73 ± 0.17
10 47.13 ± 0.99
Values are means three of independent analyses of the extract (+ -) standard deviation (n=3).
Fig.2 DPPH radical scavenging activity of Chrysophyllum canito leaf ethanol solvents %
of inhibition
Fig. 3 DPPH radical scavenging activity standard antioxidant Butyl hydroxyl toluene
Various Concentration of Butyl hydroxyl toluene used such as 2, 4, 6, 8, and 10 µg and IC50
value found to be 4.05 (g/ml).
Phytochemical in various solvents of Chrysophylum canito leaf
Phytonutrients qualitative assessment was carried out in diverse solvents of well-prepared
Chrysophylum canito leaf sample.
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Table 4 Phytochemical analysis of Chrysophylum canito leaves in various solvents
S.NO Phytochemicals Ethanol Methanol Chloroform Petroleum
Ether
1. Alkaloids - - - +
2. Carbohydrates + + + -
3. Glycosides + - - -
4. Diterpenes + + + +
5. Phenol + + + -
6. Tannins + + + -
7. Saponins + + - -
8. Flavonoids - + - -
9. Quinines + + - -
10. Proteins + - - -
11. Sterols - - + +
12. Amino acids + + - -
Presence of result indicates +ve Positive and absence of result implies – ve negative results
Ethanol extract of Chrysophylum canito leaves absolutely expose presence of phytonutrients
better than methanol, chloroform, and petroleum ether. The interchangeable adding atoms
and breakage emerges good amount of secondary metabolites. Phytochemical analysis of
Chrysophylum canito leaf implies presence of carbohydrates, glycosides, diterpenes, phenols,
tannins saponins, quinines, proteins and amino acids present (+ve Presence). The observed
analysis implies ethanol solventhas highly presence of phytonutrients and it holds good
results with (Chandrashekar et al., 2013).
Scanning Electron Microscope
Best concentrations of synthesized silver nanoparticles only chosen for characterization
research under scanning electron microscope to divulge approximate nanoparticles size. Fig 4
obtained from Jeol - Model JSM-6390 Scanning electron microscope it certainly exhibits
synthesized silver nanoparticles size in 200nm under the magnification of x55,000. The
synthesized silver nanoparticles size was evidently to be 200nm and analyzed silver
nanoparticles approximate size has been similar with (Stacey et al., 2009).
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Fig. 4 SEM Image of 3mM synthesized silver nanoparticles and its approximate size
distributions and silver nanoparticles prepared from 3mM concentrations of AgNO3
Energy Dispersive Spectroscopy Spectrum
Energy-dispersive X-ray spectroscopy reveals strong silver signal EDAX instruments is
capable for all type of element micro analysis and metallic silver synthesized nanocrystals
exhibits optical obsorption peak approximately at 3kev owing to surface plasmon resonance.
Fig 5 obtained from oxford pentafet Energy-dispersive X-ray spectroscopy and retrieved
image indicates synthesized silver nanoparticles containing elements, app concentration,
intensity, weight and atomic ratio. Precisely confirmed silver nanoparticles 0.8349 intensity,
29.51 weight%, atomic ratio% 6.57 and also found appearance of other elements such as
oxygen, chlorine, calcium and potassium.Micro element analysis utterly revealed synthesized
silver nanoparticles and this obtained results similar with (Rebecca et al., 2013).
Fig. 5 Energy dispersive spectroscopy spectrum of synthesized silver nanoparticles
elemental micro analysis prepared from 3mM concentrations of AgNO3
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Table 5 EDAX elemental micro analysis of synthesized silver nanoparticles
Element Intensity App
concentration Weight% Atomic%
O k 0.5212 15.27 56.00 84.05
Cl k 0.8846 3.96 8.33 5.64
K k 1.0972 1.99 3.38 2.07
Ca k 0.8864 1.32 2.78 1.67
Ag l 0.8349 13.23 29.51 6.57
Total 100.00
Figure 6 accurately implies silver nitrate used quantity 300 mg and synthesized silver weight
3050 mg EDAX micro element analysis reports thoroughly gives 29.21% weight of silver and
percentage (29.21%) calculation was applied in synthesized silver quantity (3050mg).
Eventually foundand acknowledged 748.2 mg of pure silver had synthesized via plant
mediated silver synthesis.
Fig. 6 Silver nitrate used quantity, synthesized silver nanoparticles quantity and
puresilver quantity
X-ray Dispersive spectroscopy
The crystalline nature of AgNPs was confirmed from Shimadzu LabX XRD- 6000X ray
Dispersive spectroscopy analysis. A Rietveld refinement of the XRD data of aqueous AgNPs
was carried out. Meticulously obtained X- ray Dispersive spectroscopy result provides
strongest peaks and it has been applied in Scherrer equation. It is the one can calculate
crystallite size from XRD data and obtained strongest peak 2 thetas 32.3154, 46.2892, and
27.9081,Full Width at Half Maximum (FWHM) 0.68980, 0.67460 and 0.66230 and
angstroms 2.76805, 1.95979 and 3.19436were applied in Scherrer equation and eventually
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noticed crystallite size of first peak 22.50nm, Second peak 16.98nm and third peak 26.78nm.
(XRD analyzed peak data values applied in Originlab Pro 9.0 software)
Fig 7. X-ray Dispersive spectroscopy analysis in ethanol 3mM silver nanoparticles
The lattice strain of first peak 0.0104, second peak 0.0069 and third peak 0.0116. Yielded
value of average crystallite size was found to be 22.08 nm and X- ray Dispersive
spectroscopy has been observed result closely similar to (Mohdet al., 2011).
Antimicrobial activity
Antimicrobial activity is an in vitro laboratory method has used to find Chrysophyllum canito
leaf extract and silver nanoparticles capability to control the growth of microorganism. The
plant secondary metabolites are being responsible for bacterial suppression. In this research
Gentamycin standard kept as control, plant extract, silver nitrate solution, 3mM ethanol
synthesized silver nanoparticles activity was evaluated against Klebsiella pneumonia.
Table 6 zone of inhibition in MM scale
Zone of inhibition in MM scale
Gentamycin
Standard used
for control
Chrysophyllum
canito leaf
Silver
nitrate
Synthesized silver zone
of inhibition.
11 1 3 9
The results obtained from the disc diffusion assay and it showed an increasing inhibitory
effect on bacterial growth in various silver synthesized sample. The ethanol silver
synthesized nanoparticles showed good zone of inhibition against Klebsiella pneumonia
microorganism and synthesized silver nanoparticles obtained results similar to (Jayandran et
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al., 2015).Fig. 8 shows zone of inhibition against Klebsiella pneumonia in 3mM of ethanol
silver synthesis sample. The number 1. Gentamycin, 2. Chrysophyllum canito leaf extract, 3.
Silver nitrate solution and 4. ethanol 3mM of silver sample.
Fig.8 Zone of inhibition
CONCLUSION
It has been concluded as Chrysophyllum canitoleaf phytochemical analysis revealed highly
presence of secondary metabolites in ethanol sample and antioxidant DPPH non enzymatic
assay utterly emerged maximum free radical scavenging activity in same solvents.
Exploration of various solvents silver synthesis undoubtedly confirmedbest wavelength in
same ethanol solvent. Scanning electron microscope, Energy dispersive x-ray spectroscopy
and X-ray dispersive spectroscopy had revealed silver nanoparticles size, present elemental
weight, intensity, and silver crystallite size. The synthesized silver nanoparticles used in vitro
study against Klebsiella pneumonia species and maximum zone of inhibition clearly expose
synthesized silver nanoparticles antimicrobial properties. In this adjacent pertaining results
and correlational utterly provide Chrysophyllum canito leaf medicinal functionality, best
silver nanoparticles synthesizing solvent and well-prepared silver nanoparticle suspension
activity against as Klebsiella pneumonia microorganisms.
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