ANTIMICROBIAL EFFICACY OF SILVER NANOPARTICLES CONJUGATED

WITH Ageratina adenophora LEAF EXTRACT

RAMYA .N1 , MAYURI.P.K1 ,SANTNY.A.S1 , ANGAYARKANNI, T*,

Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam Institute for

Home Science and Higher Education for Women, Coimbatore, Tamil Nadu, India.

Email:angait73@gmail.com

Corresponding author*

Abstract

In the present study, the synthesis of nanoparticles using the methanolic leaf extract of

Ageratina adenophora was done to optimize the method for the synthesis of silver nanoparticles

and to analyse their antimicrobial potential. In this study, the antimicrobial activity of silver

nanoparticles was analysed against the clinical isolates. Among the bacterial strains, Escherichia

coli, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi, Shigella flexneri,

Proteus vulgaris and Klebsiella pneumoniae were used and among the fungal strains Aspergillus

niger, Aspergillus flavus, Aspergillus fumigatus, Candida albicans, Mucor oryzae and Rhizopus

indicus were used. The present study strongly proves the antimicrobial efficacy of silver

nanoparticles synthesised from A. adenophora leaf extract.

Keywords - Ageratina adenophora, silver nanoparticles, antimicrobial activity, Aspergillus

fumigates.

Introduction

Ageratina adenophora (Spreng) (Syn. Eupatorium glandulosum, Eupatorium

adenophorum) is a profusely branching under shrub up to 90-120 cm in height with a few

ascending branches, leaves simple, opposite, subsessile, subentire, lanceolate and glabrous type

which belongs to the family Asteraceae. A. adenophora is a very common weed and invasion of

this weed have replaced the larger part of the vegetation in throughout our country and are

considered as a major threat to native plants and animals. It is reported to possess diverse

medicinal properties and finds use in traditional medicines. The leaves are used as astringent,

thermogenic and stimulant in folklore medicine in India (Vasanthi and Gopalakrishnan, 2013).

A. adenophora leaf juice is used to stop bleeding of cut and wounds, forming clots. Root

juice is used to treat fever. Pure juice of the leaf is used to treat insomnia. A decoction of the

plant has been recommended to treat jaundice and ulcers (Subba and Kandel, 2012).

MATERIALS AND METHODS

COLLECTION OF PLANT SAMPLE

The leaves of the plant sample were collected from in and around The Nilgiri district of Tamil

Nadu. The plant sample is authenticated from Tamil Nadu Agricultural University (TNAU),

Coimbatore, India.

PREPARATION OF METHANOLIC EXTRACT

Fresh leaves of A. adenophora were collected and cleaned to remove adhering dust

particles, washed under running tap water, gently blotted dry between folds of tissue paper. 10g

of leaf sample was weighed, cut into small pieces and added to 100ml of methanol. This was

stored in dark with mild shaking for 24 hrs. The mixture was then filtered through Whatman No.

1 filter paper. The final extract was stored at 4°C for further experiments.

PREPARATION OF SILVER NANOPARTICLES

Silver nanoparticles were prepared from the methanolic extract of A. adenophora leaves.

To 10 ml of the leaf extract 90 ml of 1mM silver nitrate solution was added (Donda et al., 2013).

The extent of nanoparticles synthesis was monitored by measuring the absorbance at 400-600nm.

INCUBATION and HEATING IN WATER BATH

The samples and silver nitrate solution were incubated at room temperature for 72 hrs

(Paulkumar et al., 2014).

The methanolic extract of A. adenophora leaves in the presence of silver nitrate was

heated for various durations (5, 10, 15 and 20 min) in a water bath at a temperature of 60° C

(Gulcin et al., 2011; Mubarakali et al., 2011).

HEATING BY MICROWAVE

The mixture of methanolic extract of leaves with silver nitrate solution was heated in

microwave for various durations namely 10, 20, 30 and 40 seconds (Nooroozi et al., 2012).

EXPOSURE TO SUNLIGHT

The methanolic extracts of A. adenophora with silver nitrate solution were exposed to

sunlight for various durations (5, 10, 15 and 20 min) with silver nitrate solution (Sulaiman et al.,

2013).

SEPARATION OF SILVER NANOPARTICLES

To separate the synthesized silver nanoparticles, samples were centrifuged at 13,000 rpm

for 20 min under refrigeration and washed 3 times with deionized water. A dried powder of the

silver nanoparticles was obtained by freeze drying.

CHARACTERIZATION OF SILVER NANOPARTICLES

The synthesized silver nanoparticles were characterized as per the methods explained below.

UV- VISIBLE SPECTRA

The light nanoparticles like silver (Ag) exhibit unique and tunable optical properties on

account of their Surface Plasmon Resonance (SPR).

SCANNING ELECTRON MICROSCOPY

Silver nanoparticles synthesized were characterized using high resolution scanning

electron microscopy (SEM).

EDAX SPECTRUM MEASUREMENTS

A.adenophora leaf extract– reduced silver solutions were dried, drop coated on to carbon

film, and tested using EDAX analyses.

FTIR (FOURIER – TRANSFORM IR) ANALYSIS

FTIR analysis is done to obtain the infra red spectra of absorption, emission and to ensure the

formation of silver nanoparticles. The advantage of using an FTIR is that it simultaneously

collects spectral data in a wide spectral range.

TEST ORGANISMS

The bacterial and fungal strains used in the present study were obtained from P.S.G Hospitals,

Coimbatore. The bacterial strains used were Escherichia coli, Staphylococcus aureus,

Pseudomonas aeruginosa, Salmonella typhi, Shigella flexneri, Proteus vulgaris and Klebsiella

pneumoniae. The fungal strains used were Asperillus niger, Aspergillus flavus, Aspergillus

fumigatus, Candida albicans, Mucor oryzae and Rhizopus indicus.

PREPARATION OF MEDIUM

The medium was prepared by dissolving 33.9g of the commercially available Muller Hinton

Agar and potato dextrose agar medium (Hi Media) in 1000ml of distilled water. The dissolved

medium was autoclaved at 15 lbs pressure at 121°C for 15 minutes. The autoclaved medium was

mixed well and poured onto petriplates (25- 30ml/plate) .

PREPARATION OF THE TEST CULTURE

Inoculums of the microorganisms were prepared from overnight culture grown in nutrient broth

and the suspension was adjusted with a turbidity equivalent to that of 0.5 MacFarland standards.

SCREENING OF ANTIBACTERIAL ACTIVITY

Petriplates containing 20ml Muller Hinton Agar medium were seeded, with the inoculums

prepared from a broth that has been incubated for 6 hours, when the growth is in logarithmic

phase, 100μl were spread in plates. Wells were cut in the agar and 10μl of the synthesized silver

nanoparticles was added. The plates were incubated at 37°C for 24 hours. The antibacterial

activity was assessed by the diameter of zone of inhibition formed around the wells.

Chloramphenicol was used as standard antibacterial agent.

SCREENING OF ANTIFUNGAL ACTIVITY

Petriplates with 20ml of Potato Dextrose Agar were prepared. A fungal plug was placed in the

center of the plate. Sterile discs impregnated with the Isatin were placed in the plates. Nystatin

was kept as positive control.

RESULT AND DISCUSSION

Visible color change of AgNPs

The silver nanoparticles were synthesized using four different methods, namely heating in

water bath (60°C), microwave heating, room temperature and exposure to sunlight. The synthesis

of nanoparticles was noticed by a change in color and the increase in absorbance. As per the

results of the study, all the four methods, namely heating in water bath (60°C), microwave

heating, room temperature and exposure to sunlight were efficient in the synthesis of AgNPs.

But, there were some difference in the pattern of synthesis of particles between each method.

The silver nanoparticles (AgNPs) synthesized on exposure to sunlight for 20 minutes

showed the maximum yield and efficiency after 24 hrs. From the visual color change we can get

preliminary information regarding the formation of silver nanoparticles. As the silver

nanoparticles are formed, the color of the solution changes from pale green to brown which is an

indication of the presence of silver nanoparticles. The variation of the color was due to the

change in surface plasmon resonance of silver nanoparticles during the formation.

The fresh suspension of E. Chapmaniana was initially yellow in colour and after addition of

AgNO3 and exposure to bright sunlight, the suspension turned reddish brown (Sulaiman et al.,

2013). Khan et al. (2013) reported that the addition of P. glutinosa plant extract into the aqueous

solution of AgNO3, the color of the solution gradually changed from light yellow to brown,

which indicates the formation of Ag NPs.

CHARACTERIZATION OF SILVER NANOPARTICLES

The synthesized silver nanoparticles were characterized as per the methods explained below.

UV-VISIBLE SPECTROSCOPY

Ultraviolet-visible spectrometry was used to examine the size and shape of the nanoparticles

in aqueous suspension. The synthesized silver nanoparticles mediated by A. adenophora leaf

extract were subjected to optical measurements by UV-visible spectroscopy, showed absorbance

peaks within the range 400-450 nm which indicates the presence of silver nanoparticle.

The production of the silver nanoparticles synthesized from the E. chapmaniana leaf extract

was evaluated through spectrophotometry at a wavelength range of 350-700 nm which revealed a

characteristic peak at 413 nm for E. chapmaniana AgNPs, which confirmed the formation of the

silver nanoparticles (Sulaiman et al., 2013). Depending upon the shape and size of the NPs,

AgNPs exhibit absorption under a visible range of 380–450 nm (Kumar et al., 2012).

SCANNING ELECTRON MICROSCOPY (SEM) - EDAX SPECTRA

Scanning Electron Microscopic studies were carried out to study the morphology of the silver

nanoparticles. It is evident that the AgNPs were roughly spherical in shape. The average size was

around 100nm for the AgNPs synthesized from methanolic extract of A. adenophora leaf extract.

EDAX analysis confirmed the presence of the element silver as the major constituent.

The SEM image showing the high density Ag-NPs synthesized by the Gelidiella acerosa

confirmed the development of silver nanostructures. The SEM micrographs of silver nanoparticle

obtained in the filtrate showed that Ag-NPs are spherical in shape, which is well distributed

without aggregation in solution (Vivek et al., 2011).

FOURIER TRANSFORM INFRARED (FTIR) SPECTROSCOPY

The FTIR measurements of biosynthesized silver nanoparticles were carried out to identify

the possible interaction between protein and silver nanoparticles. The FTIR analysis was carried

out to find out the capping material (i.e the possible functional groups involved) present in the

particles which enable the synthesis. The FTIR spectra of leaf extract mediated silver

nanoparticle showed peaks indicating prominent absorption at 2924 cm -1, 2301 cm-1, 1516

cm1,1383 cm-1, 1070cm-1and 678 cm-1.

The result indicates that the carboxyl (-C =O), hydroxyl (-OH), and amine (-NH) groups

are mainly involved in the synthesis of silver nanoparticles. These groups which are commonly

found in the proteins indicate that the presence of proteins as capping agents for silver

nanoparticles increase the stability of the nanoparticle synthesis. Minor peaks indicate that the

formed silver nanoparticles were surrounded by proteins, terpenoids and other secondary

metabolites.

Kora et al. (2012) reported that both hydroxyl and carboxyl groups of gum are involved in

the synthesis of silver nanoparticles. Phenolic compounds and saponins present in plants extract

bind to nanoparticles via either free amine groups or cystein residues in protein (Shankar et al,

2003). The M. umbellatum proteins adsorb as a layer over the green-synthesized silver

nanoparticles, which stabilizes them. Proteins can bind to silver and gold nanoparticles via either

free amine groups or cysteine residues in the phenolic compounds, quinones and saponins from

M. umbellatum, which stabilize the nanoparticles formed through the surface-bound proteins

(Arunachalam et al., 2013).

ANTIBACTERIAL ACTIVITY OF SILVER NANOPARTICLES

The antibacterial activity of the AgNPs was tested at 10µg concentration. The results were

assessed on the basis of zone of inhibition. Based on the optimization of AgNP synthesis, it was

decided to analyze the AgNPs synthesized only by exposure to sunlight for 20 minutes.

Chloramphenicol was used as the standard antibiotic. . The AgNPs was found to be more

effective against Proteus vulgaris, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus

aureus and Salmonella typhi. It was moderately effective against, Klebsiella pneumoniae and

Shigella flexneri.

Augustine et al. (2013) demonstrated that silver nanoparticles synthesized at all the silver

nitrate concentrations displayed antibacterial activity against both E. coli and S. aureus.

Nanoparticles prepared using 1 mM silver nitrate was the most efficient to inhibit both E. coli

and S. aureus. There is a decrease in antibacterial activity as the concentration of silver nitrate

increased.

ANTIFUNGAL ACTIVITY OF SILVER NANOPARTICLES

The results of agar plug method indicated that the antifungal activity of AgNPs showed

100% activity against Aspergillus niger, Aspergillus flavus and Candida albicans. It exhibited

more than 50% inhibition against Rhizopus indicus, Aspergillus fumigatus and Mucor oryzae.

Our results indicated that AgNPs showed better antifungal activity against Candida albicans,

Aspergillus niger and Aspergillus flavus.

Samuel and Guggenbichler, (2004) suggested that the nanoparticles efficiently penetrate

microbial cells, that lower concentrations of nanosized silver particles would be sufficient for

microbial control. This approach could be more efficient than existing treatments, especially for

certain organisms that are less sensitive to antibiotics because of their resistance to cell

penetration.

ANTIBACTERIAL ACTIVITY OF AgNPs BY AGAR WELL

DIFFUSION METHOD

MICROORGANISMS

Diameter of zone of inhibition in mm

AgNPsCONTROL

(Chloramphenicol)

Proteus vulgaris 10 15

Escherichia coli 12 21

Staphylococcus aureus 12 19

Salmonella typhi 11 15

Pseudomonas aeruginosa 6.0 10

Klebsiella pneumonia 5.0 15

Shigella flexneri 3.0 10

ANTIFUNGAL ACTIVITY OF AgNPs BY AGAR PLUG METHOD

MICROORGANISMS

GROWTH INHIBITION

AgNPsCONTROL

(Nystatin)

Aspergillus niger +++ +++

Aspergillus fumigates ++ +++

Aspergillus flavus +++ +++

Rhizopus indicus ++ +++

Mucor oryzae ++ +++

Candida albicans +++ +++

CONCLUSION

The bioactivity of the AgNPs was monitored in terms of antimicrobial activity. The

results obtained from the experiments conducted in this study showed that the synthesized

AgNPs found to have maximum activity against all the pathogenic strains tested to different

extent.

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NAME : Dr.(Mrs) Angayarkanni .T

PHYSICAL ADDRESS : Dr.(Mrs) Angayarkanni .T

Dept. of Biochemistry, Biotechnology & Bioinformatics

Avinashilingam University

103, A K Nagar,

Coimbatore, Tamil Nadu 641011

AFFILIATION INSTITUTION : Avinashilingam University, cbe

CURRENT POSITION : Assistant Professor

e.mail : angait73@gmail.com

MOBILE NO. : 9942972015 , 9751148155, 8148989336