Prof. J.P. Yadav, E.mail: yadav1964@rediffmail.com
Leaf Extract mediated Green synthesis of silver nanoparticles from Phyllanthus amarus: As an antibacterial agent against wide range of microbes Presented By: Prof. J.P. Yadav, E.mail: Department of Genetics, M. D. University, Rohtak , Haryana, India Nanobiotechnology is presently one of the most dynamic and importantdisciplines of research in contemporary material science whereby plants anddifferent plant products are finding an imperative use in the synthesis ofnanoparticles (NPs). The biological method has advancement over chemical and physical method asit is cost effective and ecofriendly (Raut et al., 2009; Talebi et al., 2010). Hence, in recent years, researchers have drawn their attention towardsecofriendly synthesis of nanoparticles and their activity against different kinds ofmicrobes. It is a common pantropical weed.
Phyllanthus amarus is an important plant of Indian Ayurvedic system of medicine, belongs to the family Euphorbiaceae (Figure-1). It is a common pantropical weed. Commonly known as bhumiamalaki/ bahupatra. Figure-1 showing picture of P. amarus growing in the field It is a small herb well known for its medicinal properties and has been used traditionally (Patel et al., 2011). Used for treatment of liver, kidney, bladder problems etc. Different plant parts have various therapeutic activities, such as: leaves as expectorant, diaphoretic and seeds as carminative, laxative, tonic to the liver, diuretic, useful in bronchitis, earache, opthalmia. Synthesis of Silver nanoparticles
Boiled 25 g of plant leaves in 100 mL distilled waterfor 5 min & filter Added 15 mL of this extract with 100 mL of 1 mM silver nitrate and heated to75C Maintain this temp. for the entire reaction (different plant extracts requiredifferent times to complete the reaction) Centrifuge the product. Wash 3 x with distilled water. The colour changes from pale yellow to dark brown (Figure-2).
The AgNPs were synthesized through plant extract by mixing the aqueous plant extracts ofP. amarus with silver nitrate solution (1mM). The colour changes from pale yellow to dark brown (Figure-2). It was due to the reduction of Ag+ which indicates the formation of AgNPs. Figure-2 showing colour change from pale yellow to dark brownindicating the synthesis of AgNPs of P. amarus Characterization of Silver nanoparticles
UV-Vis Spectroscopy Transmission Electron Microscopy (TEM) Energy Dispersive X- Ray Spectroscopy (EDX) Dynamic Light Scattering (DLS) Zeta potential X-Ray Diffraction (XRD) Fourier Transform Infra Red Spectroscopy (FTIR) UV-Vis spectroscopy Synthesis of AgNPs was observed by UV-VIS spectroscope (Shimadzu). The UV-VIS absorption spectra of the AgNPs were monitored in a range of nm. Figure-3 shows the absorption spectra of AgNPs synthesized by P. amarus at nm. Figure-3UV-Vis Spectra of silver nanoparticles of P. amarus Transmission Electron Microscopy (TEM)
TEM confirmed the development of silver nanostructures and gave clear image of silver nanoparticles. The shape of silver nanoparticles was spherical and size was 369 nm synthesized from P. amarus (Figure-4). Figure-4 Image of TEM of silver nanoparticles of P.amarus Energy Dispersive X-Ray (EDX)
EDX performed by energy and intensity distributions of X-ray signals generated by focused electron beam on a specimen. EDX characterization has shown absorption of strong silver signal along with other elements, which may be originate from the biomolecules that are bound to the surface of silver nanoparticles (Figure-5). Figure-5 Image of EDX of silver nanoparticles of P. amarus Dynamic Light Scattering (DLS)
Dynamic light scattering (DLS) is a technique used to determine the size, size distribution profile and poly dispersity index of particles in a colloidal suspension. Figure-6 shows the DLS graph of AgNPs of P. amarus Poly disparity index (PDI) is a measurement for distribution of AgNPs from to 0.5. PDI greater than 0.5 values indicates the aggregation of particles. In this study, PDI was found to be From this, it was clear that AgNPs synthesized fromP. amarus extracts does not aggregate at all. Figure-6 DLS graph of silver nanoparticles of P. amarus Zeta potential Zeta potential measures the potential stability of the particles in the colloidal suspension. Silver nanoparticles generally carry a negative charge. Figure-7 shows the zeta potential graph of AgNPs of P. amarus. The AgNPscarry a charge of mV and were stable at room temperature. Figure-7Zeta potential graph of silver nanoparticles of P. amarus X-Ray Diffraction (XRD)
The particle size and crystalline nature of AgNPs was determined by XRD. The XRD pattern was showing nine strong peaks that corresponds to the planes, which are indexed to the face centered cubic structures of silver nanoparticles. The mean size of silver nanoparticles was calculated using the Debye-Scherrers equation. An average size of the silver nanoparticles synthesized by P. amarus was 15.7 nm with size ranging from 7.43 nm and 26.6 nm XRD diffraction pattern of synthesized AgNPs of P. amarus was shown in Figure-8 (Table-1). Figure-8 XRD of silver nanoparticles of P. amarus
Measurement conditions Qualitative analysis results Figure-8 XRD of silver nanoparticles of P. amarus Table-1 Size of AgNPs of P. amarus by using Debye-Scherrer`s equation
S. No. 2-theta(deg) D (ang.) FWHM(deg) Int. I(cps deg) Int. W(deg) Size(nm) 1 27.797 3.206 0.464 488.7 0.651 18.4 2 32.210 2.776 0.450 0.622 19.1 3 38.094 2.306 0.647 845.25 0.992 13.5 4 44.195 2.047 1.204 224.79 1.535 7.43 5 46.250 1.961 0.576 492.67 0.702 15.6 6 54.750 1.675 0.532 152.66 0.722 17.5 7 57.450 1.602 0.610 148.96 0.825 15.4 8 64.485 1.443 0.367 171.27 0.758 26.6 9 77.068 1.236 1.285 258.17 1.368 8.25 Fourier Transform Infra-Red Spectroscopy (FTIR)
The aim of IR spectroscopic analysis is to determine chemical functional groups in the sample. The amide linkages between amino acid residues in polypeptides and proteins give rise to well known signatures in the infra-red region of the electromagnetic spectrum. Different functional groups absorb characteristic frequencies of IR radiation. Thus, IR spectroscope is an important and popular tool for structural elucidation and compound identification. Our observation confirms the presence of such compounds in the sample which coat covering the silver nanoparticles known as capping agents. FTIR analysis of AgNPs of P. amarus has been shown in Figure-9.
FTIR analysis of AgNPs from P. amarus showed the presence of bands due to: CH stretch(aldehydic) (2,915 and 2,848 cm-1), C-O stretch (1,732 and 1,634cm-1), N-H (1517 cm-1 ) N-O stretch (1462 and 1,377 cm-1) C-O stretch (dialkyl) (1,169 cm-1), C-N stretch (1,037 cm-1), C-H stretch (718 cm-1). This FTIR results gives the evidence of formation and stabilization of silver nanoparticles in the aqueous medium by using biological molecules. Figure-9 FTIR of silver nanoparticles of P. amarus Antimicrobial activity of silver nanoparticles from P. amarus Efficacy of synthesized nanoparticles ) Antimicrobial activity against Reference bacterial & fungal strains ) Antimicrobial activity against Multi Drug Resistant (MDR) strains of Pseudomonasaeruginosa Anti microbial activity of silver nanoparticles
Agar well-diffusion Method (Perez et al., 1990) Resazurin based Micro broth Dilution Method (Sarker et al., 2007) Antimicrobial activity against Reference bacterial & fungal strains
In this study, we compared the antimicrobial properties of synthesized AgNPs (were found to be effective antimicrobial agents) with different plant extracts. Antimicrobial activity of AgNPs was studied against 9 different referenceAmerican Type Cell Culture ( ATCC) bacterial and 2 fungal strains. The effect of different concentration i.e. 100 g/ml of AgNPs, 1mg/ml of plant extracts, against different bacteria and fungus were reported in case of P. amarus (Table-2) (Figure-10). The zone of inhibition was found to be in the range of 121-160.58 mm. Maximum antibacterial activity was shown by AgNP extract with inhibition zone minimum of 160.58 mm (at AgNPs concentration of 100 g/ml) against P. aeruginosa. The plant extracts has not been shown any antibacterial activity against any even at 1 mg/ml. Moreover, AgNPs have shown more antimicrobial activity than that of the standard drug i.e. streptomycin (for bacteria) and ketoconazole (for fungus). Table -2 Results of antibacterial activity of different plant extracts and AgNPs of P. amarus against reference ATCC bacterial and fungal strains S.No. ATCC Reference Strains Zone of Inhibition (in mm) +ve control streptomycin Acetone extract (1mg/ml) Methanol extract Aqueous AgNPs extract (100g/ml) 1 Enterococcus faecalis ATCC 29212 130.39 - 140.13 2 Klebsiella pneumonia ATCC 120.31 151 3 Escherichia coli ATCC 25922 111 130.58 4 Shigella flexneri ATCC 12022 120.58 130.73 5 Salmonella typhi ATCC 13311 130.87 6 Proteus mirabilis ATCC 43071 110.76 131 7 Pseudomonas aeruginosa ATCC 27853 100.13 150.58 8 Staphylococcus aureus ATCC 130.77 130.17 9 Serratia marcescens ATCC 27137 100.87 121 10 Aspergillus niger ATCC 16404 130.51 11 Candida albicans ATCC 10231 120.33 140.58 Figure-10 showing antimicrobial activity of AgNPs of P. amarus
against reference ATCC bacterial & fungal strains Antibacterial Assay against MDR strains of P. aeruginosa
The 10 multidrug resistant strains of P. aeruginosa isolated from burnpatients tested at various concentrations of AgNPs i.e. 12.5, 25, 50, 100 and200 g/ml to determine the antibacterial effect by agar well diffusionmethod. The AgNPs showed antimicrobial activity against all the tested pathogens. The antibacterial activity is concentration dependent as it increased with theconcentration of AgNPs (Figure-11). Figure-11 showing antibacterial activity of AgNPs of P. amarus against
MDR strains of P. aeruginosa from burn patients The zone of inhibition measured in a range of 10 0. 53 to 21 0
The zone of inhibition measured in a range of 10 0.53 to 21 0.11 mm (Figure-12). MDR Strain 1 was found to be most susceptible where zone of inhibition ranged from 13 1 to 21 0.11 mmwith AgNPs concentration of 12.5 to 200 g/ml synthesized from P. amarus. Figure-12 showing antibacterial activity of AgNPs of P
Figure-12 showing antibacterial activity of AgNPs of P. amarus against MDR strain 1,2,3,6,7 and 8 of P. aeruginosa Minimum Inhibitory Concentration (MIC)
The MIC in case of reference ATCC strains was found to be 6.25 to 25 g/ml. Most of the strains have shown MIC at 12.5 g/ml (Figure-13). The MIC of AgNPs from P. amarus against MDR strains of P. aeruginosa was 6.25 to 12.5 gml (Table-3 ). The MIC of AgNPs was found to be in a range from g/ml, almost eight MDR strains have shown the MIC at 6.25 g/ml which was lower than that of the standard antibiotic (10 g). Against reference bacterial & fungal strains
Minimum Inhibitory Concentration (MIC) Reference ATCC bacterial &fungal strains Figure-13 showing Minimum inhibitory concentration (MIC) of AgNPs of P. amarus Against reference bacterial & fungal strains MIC(in g/ml) of AgNPs of P. amarus
Table-3 MIC of silver nanoparticles against MDR strains of P. aeruginosa from burn patients S.No Name of Strains MIC(in g/ml) of AgNPs of P. amarus 1 P. aeruginosa MDR Strain 1 6.25 2 P. aeruginosa MDR Strain 2 3 P. aeruginosa MDR Strain 3 4 P. aeruginosa MDR Strain 4 5 P. aeruginosa MDR Strain 5 6 P. aeruginosa MDR Strain 6 12.5 7 P. aeruginosa MDR Strain 7 8 P. aeruginosa MDR Strain 8 9 P. aeruginosa MDR Strain 9 10 P. aeruginosa MDR Strain 10 Conclusion AgNPs from P. amarus possess good antimicrobial activity, even at very small concentration (in g/ml), which makes them a potent source of antimicrobial agent against reference strains. As infection of P. aeruginosa always remains one of the most challenging concerns in burn units and the synthesized AgNPs are highly effective antibacterial agent against the MDR burn isolates of P. aeruginosa. These synthesized AgNPs exhibitedthe excellent antibacterial agent against wide range of microbes and can provides a potent alternative nanomedicine in the health care system. Also, green synthesis of AgNPs can potentially eliminate the problem of chemical agents that may have adverse affects, thus making nanoparticles more compatible with the ecofriendly approach. Moreover the synthesized AgNPs enhance the therapeutic efficacy and strengthen the medicinal values ofmedicinal plant, P. amarus. References Raut RW, Lakkakula JR, Kolekar NS, Mendhulkar VD, Kashid SB: Phytosynthesis of silver nanoparticle using Gliricidia sepium (Jacq.). Curr Nanosci 2009,5:117122. Talebi S, Ramezani F, Ramezani M: Biosynthesis of metal nanoparticles by micro-organisms. Nanocon Olomouc, Czech Republic, EU 2010, 10:1218. Patel JR, Tripathi P, Sharma V, Chauhan NS, Dixit VK: Phyllanthus amarus: ethnomedicinal uses, phytochemistry and pharmacology: a review.J Ethnopharmacol 2011, 138:286313. Perez C, Pauli M, Bezevque P: An antibiotic assay by agar well diffusion method. Acta Biologiae Med Experimentalis 1990, 15:113115. Sarker SD, Nahar L, Kumarasamy Y: Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals. Methods 2007, 42:321324. THANKS