green route synthesis of silver nano particles … · green route synthesis of silver nano...

DOI: 10.22623/IJAPSA.2017.3007.IDZBN Page 48

GREEN ROUTE SYNTHESIS OF SILVER NANO PARTICLES USING LEAF

EXTRACTS OF MELALEUCALEUCADENDRA

Mita R. Patel1*, Dr. Rasmikant A. Patel

2, Dr. Dharmesh Varade

3 and Dr. Kespi A. Pithawala

4

1Department of Chemistry, Gujarat Arts and Science College, Ahmedabad, Gujarat, India, 380006 2Department of Chemistry, Municipal Arts and Urban Science College, Mehsana, Gujarat, India

3School of Engineering & Applied Sciences, Ahmedabad University, Gujarat, India, 380009

4Department of Biology, Gujarat Arts and Science College, Ahmedabad, Gujarat, India, 380006

*correspondences Author- Mita R. Patel1

Abstract

The ebb and flow explore in nanotechnology is primarily gone for condition benevolent and savvy

techniques for green amalgamation of nanoparticles. Here watery leaf concentrates of Melaleuca

leucadendra which go about as reducing and in addition topping operators were utilized for the

development of silver nanoparticles (SNP) from 0.1 mM AgNO3 arrangement. The SNP were

framed inside hour and the development was steady for a considerable length of time. The

arrangement, qualities, size and compliance of silver nanoparticles framed were completed

utilizing UV - VIS spectroscopy, FTIR, Dynamic Light Scattering (DLS), SEM, TEM and EDX.

Likewise the SNP framed along these lines demonstrated reasonable antimicrobial movement

against Gram positive and Gram negative microorganisms. Additionally SNP showed great

antioxidant (cancer prevention) agent properties. On the premise of result acquired one might say

that the simple generation of silver nanoparticle utilizing green science can be successfully used

in different fields in biomedical-nanotechnology and in addition decreasing the pathogenic

response of microbial flora.

Keywords: Silver nanoparticles, Melaleuca leucadendra, Antibacterial Activity, FTIR, DLS, SEM,

TEM, EDX, antioxidant activity.

I. INTRODUCTION

Nano particles of different metals and metal salts have been shaped utilizing different

synthetic, physical and green synthesis [1-5]. Of the previously mentioned strategies the most simple

and eco-friendly technique is through green union and today it is the most well-known strategy for

nanoparticle combination [6-10]. Of the metals and the salts the most regularly utilized are the

coinage metals as Au, Ag and Cu however Fe, Zn and Pt has likewise been used [11-14]. Ag NPs are

for the most part put to use in pharmaceutical and biomedical items and in this way should be

incorporated by green course utilizing characteristic plant materials which is naturally savvy as well

as expands amalgamation that does not have to utilize high weight, vitality, temperature and harmful

chemicals making it ecofriendly and biocompatible [15]. Bio-motivated union of nanoparticles gives

headway over concoction and physical strategies as it is quick and particular in their objective

towards the applications where they are evaluated for their antimicrobial action [16]. Medicinal

plants are utilized as a part of extensive extents nowadays in view of the enduring change against

maladies after natural treatment [17]. In this exploration silver nano particles have been combined

utilizing the green course by utilizing the fluid leaf concentrates of Melaleuca leucadendra regularly

called paper bark tree [18].

International Journal of Applied and Pure Science and Agriculture (IJAPSA)

Volume 03, Issue 3, [March- 2017] e-ISSN: 2394-5532, p-ISSN: 2394-823X

@IJAPSA-2017, All rights Reserved Page 49

II. MATERIALS AND METHOD

Fresh new leaves of Melaleuca leucadendra were gathered from the tree grownin botanical

gardenof Gujarat College, Ahmedabad [19]. Silver nitrate was bought from Hi-Media and chemicals

for cell reinforcement movement including 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic

corrosive) diammonium salt (ABTS+), 2,2-diphenyl-1-picrylhydrazyl (DPPH), were acquired from

Sigma-Aldrich (St. Louis, MO, USA). BHA, 2-thiobarbituric corrosive (TBARS), trichloroacetic

corrosive (TCA), Ammonium thiocyanate, Nitroblue tetrazolium (NBT), phosphate cradle (pH 7.4)

and nonenzymatic phenazine methosulfate (PMS) were purchased from SRL chemicals, and n-

butanol from Burgoyn, Methanol, ethanol, (CH3)2CO, chloroform, hydrochloric corrosive from

Rankem, Nicotinamide adenine dinucleotide (PMS-NADH) from Spectrochem, Potassium persulfate

from HPLC, Acetic corrosive, vitamin C, vitamin E and BHA from Merk, K3Fe(CN)6, FeCl3 and

FeSO4 from Nice, Bradford Reagent was prepared freshly, All chemicals were of logical review with

98-100% immaculateness measure as analytical reagents and chemical grade. Deionized and twofold

distilled water was utilized wherever required.

2.1 Preparation of Extracts Freshleaves of Melaleucaleucadendra leaves were initially washed with deionized water and

after that with refined water to free them of any earth material these were then subjected to dry in

space for around 5 days andlastly pounded into fine powder utilizing a clean sanitized local

mechanized processor (stainless steel cutting edges).This powder was put away in water/air proof

compartment and was utilized for planning fluid concentrates. These were set up by including 1 g of

leaf powder to 50 ml of twofold distilled water and left overnight. The arrangement accordingly

framed was sifted through Whatmann filter paper No. 1 and the concentrate was utilized for planning

of nanoparticles.

2.2 Synthesis of Ag nanoparticles To 30 ml of 0.1mM AgNO3 10 ml of leaf extract was added and at interval of every 5

minutes colorimetric readings were taken at 410nm to find the presence of formation of nano

particles the color change was an indication of formation of nano particles.These were formed within

15 minutes and these particles were stable for about one month.

2.3 Characterization techniques

The biosynthesized silver nanoparticles were characterized by the following methods:

2.3.1 Visual Observation A change of colour from pale yellow to reddish brown was observed in the solution after

visible irradiation.

2.3.2 UV Spectrophotometric analysis The characterization technique involves ultra-violet and visible spectroscopy. UV-Vis

absorption spectra were measured using Systronic UV-117 spectrometer from 300nm to 700nm

continuously and the leaf powder extract was used as the reference for the baseline correction.

2.3.3 Fourier Transform Infrared Spectroscopy Analysis: FTIR analysis was carried out to determine the functional groups present in leaf extract and

their possible involvement in the synthesis of silver nanoparticles. FTIR analysis were carried using

a FTIR SHIMADZU 8300 instrument with a wavelength range of 4000 to 400 nm where the

samples were incorporated with KBr pellets to acquire the spectra. The results were compared for

shift in functional peaks. A FTIR graph can be useful for preliminary investigation of surface

chemistry of biogenic nanoparticles (i.e. those chemicals that contain carbon). This technique is

widely used for identification of chemical residues such as amine, carbonyl and hydroxyl functional

groups in a molecule [20]. The FTIR analysis was performed with reduced silver nanoparticles. The

synthesized AgNPs sample was mixed with KBr to make a pellet in the ratio of 1:100. The FTIR

instrument with diffuse reflectance mode attachment. All measurements were carried out in the range




of 400-4,000 cm-1 at a resolution of 4 cm-1 [21]. For this fresh sample were sent for FTIR Analysis

at Gujarat Laboratory, Ahmedabad. Samples with total of volume 1-2 ml were given in aqueous form

formed by producing SNPs using the reduction reaction of 9 ml of 0.1 mM Silver Nitrate solution

through 3 ml of plant extract.

2.3.4 Dynamic Light Scattering

These studies were carried out to get to know the particle size distribution in the solution. The

particle size comes out to be 100nm and hence this can be further verified from SEM analysis.

2.3.5 SEM & EDX Analysis The surface morphology of silver nanoparticles was examined using a scanning electron

microscopy (6010 LA, Jeol).The elemental composition of the synthesized silver nanoparticles was

analyzed using Energy Dispersive X-Ray Spectrometer.

2.3.6. TEM Analysis

Since the particle sizes were small TEM studies were carried out to get to know the exact

shape of the particles this was done using transmission electron microscope (JEM1400 Plus, Jeol).

The particle sizes of about 30nm to 60nm can be seen clearly.

2.3.7Determination of antimicrobial activity

A. Microorganisms Human pathogens were obtained from the Department of Microbiology, Gujarat Arts and

Science College, Ahmedabad. The bacterial pathogens namely Staphylococcus aureus, Streptococcus

pneumonia, Escherchia coli, Salmonella typhi, were used to study the antibacterial activity. The

fungal pathogens such as Aspergillus niger, Penicillium sp, Rhizopus stolonifer were used to study

the antifungal activity. The nutrient broth, potato dextrose broth, nutrient agar and potato dextrose

agar were used growing the test bacterial and fungal strains and were maintained on corresponding

agar slants at 4°C.

B. Preparation of inoculums

The bacterial pathogens were inoculated into sterile nutrient broth and incubated at 37°C for

24 hours until the culture attained a turbidity of 0.5 McFarland units. The final inoculum was

standardized to 105 CFU/ml by diluting fresh cultures with sterile distilled water. For fungal

pathogens, inoculums was prepared in potato dextrose broth by choosing five distinct colonies

approximately 1 mm from 24 h old culture grown on Potato dextrose agar (PDA) and incubated at

28±2ºC.

Colonies were suspended in 5 ml of sterile 0.85% saline. The resulting suspension was vortex

and the turbidity was adjusted to yield 2×106 cells/ml (≅0.5 McFarland standards).

C. Antibacterial activity

Antibacterial activity of AgNPs was determined by the agar disc diffusion method [22].

Plates of Nutrient agar were evenly streaked across the complete surface throughout the petri plate so

as to get a loan growth of the inoculums with the help of spread plate technique with a known

volume of 0.01 ml of active young culture with approx. Microbial count as 105 CFU/ml. Sterile filter

paper discs (5 mm diameter) were immersed in the 50 µl of synthesized AgNPs (10, 20, 30, 40, 50

µg/ml) and allowed to dry at room temperature and it was placed over the Nutrient agar plates.

Streptomycin 10 mcg/disc was used as positive control and the disc immersed in distilled water was

used as negative control. The plates were incubated overnight at 37 ºC and the zone of inhibition

around each disc was measured. Experiments were done in triplicate and mean values of zone

diameter were taken.

D. Antifungal activity The growth inhibition of fungal pathogens by AgNPs was determined by the agar disc

diffusion method [23]. Sterile swabs were dipped in the microbial suspensions which contains fungal




strains were uniformly applied to petridishes containing PDA. Sterile filter paper discs (5 mm

diameter) were immersed in the 50 µl of synthesized AgNPs (10, 20, 30, 40, 50 µg/ml) and allowed

to dry at room temperature and it was placed over the petridishes. Nystatin was used as positive

control and plates were incubated at 28 ± 2 ºC for 48-72 h and the zone of inhibition around each

disc was measured.

2.3.5 Determination of antioxidant activity

A. DPPH Radical-Scavenging Activity DPPH radical-scavenging activity was estimated according to the method of Ravikumar et al.

(2008) [24]. Briefly, 1.0 ml of synthesized AgNPs (50 µg/ml) was added to 1.0 ml of 0.16mM DPPH

methanolic solution. Similiarly simple 50 µg/ml plant extract and 0.1 M of AgNO3was also tested for

antioxidant properties. The mixture was vortexed for 1 min and left to stand at room temperature for

30 min in dark, and the absorbance was read at 517 nm. Lower absorbance indicated higher radical-

scavenging activity. The ability to scavenge the DPPH radical was calculated using the following

equation:

Scavenging activity (%) = [(A control – A sample) /A control] × 100

Where, A control is the absorbance of the control (DPPH solution without AgNPs), A sample

is the absorbance of the test sample (DPPH solution plus AgNPs) BHT was used as the standard.

Each samples were performed for triplicate to minimize error.

B. Superoxide Radical Scavenging Activity The superoxide radical scavenging activity was assayed according to the method mentioned

by Nishimiki M. et al. (1972)[25]. Superoxide anions were generated in a phenazine methosulfate-

nicotinamide adenine dinucleotide (PMS-NADH) system through the reaction of PMS-NADH and

oxygen. It was assayed by the reduction of nitroblue tetrazolium (NBT). All the solutions used in this

experiment were prepared in phosphate buffer (pH 7.4). 1ml of NBT (156µM), 1ml of NADH

(468µM) and each 1ml of plant extract (10-50 µg/ml) and each AgNPs (10-50 µg/ml) were mixed.

The reaction was started by adding 1ml of PMS (60µM) and the mixture was incubated at 25°C for

5min followed by measurement of absorbance at 560nm spectrophotometrically. Decreased

absorbance of the reaction mixture indicated increased superoxide anion scavenging activity. The

percentage inhibition was calculated using the formula,

Inhibition (%) = [(A0 – AS) / A0] × 100……………. (Nishimiki M. et al. 1972)

C.Reducing power assay The reducing power of the AgNPs was measured according to the method of Oyaizu, (1986)

[26].Briefly, 500 µl of synthesized AgNPs (100, 200, 300, 400, 500 µg/ml) was mixed with 2.5ml of

phosphate buffer (0.2 M, pH 6.6) and 2.5ml of one percent potassium ferricyanide. Reaction mixture

was incubated at 50°C for 20 min. After incubation, 2.5ml of trichloroaceticacid (10%) was added

and centrifuged at 650×g for 10 min. 2.5ml of upper layer was mixed with 2.5ml of distilled water

and 0.5ml ferric chloride (0.1%). Absorbance of the samples was read at 700 nm. Increased

absorbance of the reaction mixture indicated increased reducing power. BHT was used as the

standard.

D. ABTS+ Scavenging Capacity Assay

The ABTS decolonization assays were carried out involving the generation of ABTS+

chromophore by oxidation of ABTS with potassium persulfate. The ABTS radical cation (ABTS+)

was produced by reacting 7 mM stock solution of ABTS with 2.45 mM potassium persulfate and

allowing the mixture to incubate in the dark condition at room temperature for at least 6 hour before

use. Absorbance at 734 nm was measured after 10 minutes of incubation after mixing different

concentrations of the Melaleuca leucadendra extracted (final concentration as 10, 15, 20 %; v/v)

with 1 ml of ABTS+ solution. The ABTS

+ scavenging capacity of the filtrate was compared with that

of vitamin C (50 µM), vitamin E (50 µM) and BHA (0.1 mg/ml).

International Journal of Applied and Pure Science and Agriculture (IJAPSA) Volume 03, Issue 3, [March

@IJAPSA-2017, All rights Reserved

III. RESULT

A robust practical way for eco

extract of Melaleuca leucadendra (White paper bark tree) as both reducing and capping agent, under

the prescribed condition of room temperature has been adopted without applying any other

precursors, inducers or hazardous chemical additives or rigorous reactions.

The formation of AgNP was predicted usi

confirmed by SEM,Figure-2, and also EDX

characteristics of AgNP was confirmed,

and 3328.189 wave numbers confirming the formation of nano particles of silver.

determined c.a. as 60 nm using DLS,

the shape of the particles was known using transmission

The prepared silver nanoparticles exhibited considerable antioxidant as

activity,Table-1 and 2. The effects were more pronounced on Gram

typhii (MTCC: 733) and Escherichia coli

activity on Gram-positive bacteria S

(MTCC:430). A bactericidal mode of action was observed more for both Gram

negative bacteria by the nanoparticles as compared to Plant extracts. Similarly antifungal activity

also showed more pronouncing activity through nanoparticles formed using extracts of

Melaleucaleucadendra as compared to simple extracts of

property reveals dose dependent relationship

Figure

International Journal of Applied and Pure Science and Agriculture (IJAPSA)Volume 03, Issue 3, [March- 2017] e-ISSN: 2394-5532, p

2017, All rights Reserved

RESULTS AND DISCUSSION

A robust practical way for eco-friendly synthesis of silver nanoparticles using aqueous leaf

(White paper bark tree) as both reducing and capping agent, under


precursors, inducers or hazardous chemical additives or rigorous reactions.

NP was predicted using UV-visible spectroscopy (410 nm),

and also EDX, Figure-3, of AgNP.Also using FT-IR Spectroscopy the

NP was confirmed, Figure-4,FTIR studies show peaks at 1634.550, 2113.342

and 3328.189 wave numbers confirming the formation of nano particles of silver.

DLS,Figure-5, this was further affirmed from the TEM studies also

s known using transmission electron microscopy, Figure

The prepared silver nanoparticles exhibited considerable antioxidant as well as antibacterial

The effects were more pronounced on Gram-negative bacteria

Escherichia coli (MTCC:425). The nanoparticles also showed prominent

positive bacteria Staphylococcus aureus (MTCC:96) and

(MTCC:430). A bactericidal mode of action was observed more for both Gram-positive a



as compared to simple extracts of Melaleucaleucadendra

property reveals dose dependent relationship, Figure-7, 8, 9 & 10.

Figure .1 UV-VIS scan of AgNP

Figure .2 SEM of AgNP

Figure .3EDX of AgNP


5532, p-ISSN: 2394-823X

2

friendly synthesis of silver nanoparticles using aqueous leaf

(White paper bark tree) as both reducing and capping agent, under


nm),Figure-1,further

IR Spectroscopy the

studies show peaks at 1634.550, 2113.342

and 3328.189 wave numbers confirming the formation of nano particles of silver. Size was

this was further affirmed from the TEM studies also

Figure-6.

well as antibacterial

negative bacteria Salmonella

(MTCC:425). The nanoparticles also showed prominent

(MTCC:96) and Bacillus cerus

positive and Gram-



aleucadendra. Antioxidant

International Journal of Applied and Pure Science and Agriculture (IJAPSA) Volume 03, Issue 3, [March

@IJAPSA-2017, All rights Reserved

Table.1.: Anti-Bacterial and Anti

Test Culture Plant

Extract

(50

µg/ml)

AgNPs Concentrations

10

µg/ml

International Journal of Applied and Pure Science and Agriculture (IJAPSA)Volume 03, Issue 3, [March- 2017] e-ISSN: 2394-5532, p

2017, All rights Reserved

Figure .4 FTIR of AgNP

Figure .5 DLS of AgNP

Figure .6 TEM of AgNP

Bacterial and Anti-fungal Activity of formed AgNPs using Melaleuca

AgNPs Concentrations Positive

Control

(Strepto

mycin)

10

µg/ml

20

µg/ml

30

µg/ml

40

µg/ml

50

µg/ml

Antibacterial Property


5532, p-ISSN: 2394-823X

Page 53

extracts

Positive

Control

(Strepto-

mycin)

Negative

Control

(Sterile

D/W)




Staphylococcus aureus 18 16 18 21 17 12 24 5

Bacillus cerus 17 15 14 14 19 11 24 5

Escherchia coli 16 19 21 18 22 24 24 5

Salmonella typhi 15 17 19 22 24 24 24 5

Antifungal Property

Aspergillus niger 22 21 27 24 28 29 32 5

Penicillium sp 24 24 24 23 34 30 32 5

Rhizopus stolonifer 24 26 24 26 30 31 32 5

Table.2. : Activity Index of the AgNPs at various concentrations

Test Culture Plant Extract

(50 µg/ml)

AgNPs Concentrations

10 µg/ml 20 µg/ml 30 µg/ml 40 µg/ml 50 µg/ml

Antibacterial Property

Staphylococcus aureus 0.75 0.66 0.75 0.87 0.70 0.50

Bacillus cerus 0.70 0.625 0.58 0.58 0.79 0.45

Escherchia coli 0.66 0.79 0.87 0.75 0.91 1.00

Salmonella typhi 0.625 0.70 0.79 0.91 1.00 1.00

Antifungal Property

Aspergillus niger 0.68 0.65 0.84 0.75 0.87 0.90

Penicillium sp 0.75 0.75 0.75 0.71 1.06 0.93

Rhizopus stolonifer 0.75 0.81 0.75 0.81 0.93 0.96

Note: Zone of inhibition by streptomycin as a standard drug = 24 mm (Mean Value) and for antifungal Nistatin as a

standard drug = 32 mm (Mean Value)

��(�. �. ) =MeanofZoneofInbitionbyAgNPs

ZoneofInhibitionobtainedforstandardAntibioticDrug

Figure .7 DPPH Radical-Scavenging Activity

Figure .8Superoxide Radical Scavenging Activity UV-VIS scan of AgNP




Figure .9 Reducing power assay

Figure .10 ABTS+ Scavenging Capacity Assay

IV. CONCLUSIONS

We have developed a very simple, efficient, and practical method for the bio-fabrication of

silver nanoparticles using aqueous leaf extract of Melaleuca leucadendra under the normal influence

of light and temperature. The biosynthesis of silver nanoparticles making use of such a traditionally

important medicinal plant without applying any other chemical additives, thus offers a cost-effective

and environmentally benign route for their large-scale commercial production.The AgNPs were

characterized by UV-visible, SEM-EDX and FT-IR spectrum. Biosynthesis of Ag-NPs using green

resources like Melaleucaleucadendra is a better alternative to chemical synthesis, since this green

synthesis is pollutant free and ecofriendly. The biosynthesized AgNPs have shown good antibacterial

efficacy and hence has a potential to be used as antibacterial agent against Gram-negative bacteria

Salmonella typhii (MTCC: 733) and Escherichia coli (MTCC:425) and Gram positive bacteria

Staphylococcus aureus (MTCC:96) and Bacillus cerus (MTCC:430) as well more effectively then

the aqueous extracts of Melaleuca leucadendra. These AgNPs were proved to be powerful weapons

as antibacterial and antioxidant activity.

VII. ACKNOWLEDGEMENT

The authors are thankful to Dr. Edwin A. Pithawala, Asst. Prof. in Microbiology, Khyati

Foundation, Ahmedabad, for his valuable inputs and comments.




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