BIOLOGICAL SILVER SYNTHESIS AND CHARACTERIZATION OF …

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Prabhu et al. World Journal of Pharmacy and Pharmaceutical Sciences

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).

http://www.environmental-expert.com/products/brand-jeol


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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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