BIOSYNTHESIS OF SILVER NANOPARTICLES USING MALUS DOMESTICA LEAF EXTRACT AND ITS BIOLOGICAL APPLICATIONS AGAINST HUMAN PATHOGENS .

Shantanu Raikar, 3 Chetan J. D. 3 , Mangesh S. Jadhav, 1 Sameer Kulkarni, 1 P. U. Raikar, 2 U. S. Raikar 1 and Shyam Kumar Vootla 3 . 1. Department of Physics, Karnatak University Dharwad – 580003, Karnataka, India. 2. Visvesvaraya Technological University, Machhe-590018, Belagavi, Karnataka, India . 3. Department of Biotechnology and Microbiology Karnatak University Dharwad – 580003, Karnataka, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 16 August 2018 Final Accepted: 18 September 2018 Published: October 2018

The silver nanoparticles were synthesized by employing a green Malus domestica leaf extract assisted process. The synthesized nanoparticles were analyzed using the instruments FTIR, UV-Vis, PXRD, AFM and SEM with EDS for chemical, structural and morphological studies. It is observed that the phytochemicals present in the leaf extract are responsible for the stable nature of synthesized nanoparticles. The green synthesized silver nanoparticles have shown effective radical scavenging as well as antibacterial activities. From our current findings that the synthesized nanoparticles showing growth inhibitory activities against both gram positive and gram negative pathogenic bacteria, we can say that the synthesized nanoparticles can be used in medicine as the therapeutic agents.

…………………………………………………………………………………………………….... Introduction:-
In recent decades as Feynman once said there is plenty of room at the bottom although he didn"t mean Nanotechnology the area has been very fascinating lots of research work is going to explore possible various applications of nanoparticles (Cao, 2004;Kimling, 2006;Iravani, 2011;). Various approaches have been explored to synthesize nanoparticles broadly classified into bottom up and top down processes (Cao, 2004;Kavitha et al. 2013). The green synthesis of nanoparticles using leaf extract has been attracting scientific community because of environmentally benign, cost effective nature. The constituting of green method for the synthesis of silver nanoparticles is an important aspect of nano research. Syntheses of metal nanoparticles using different methods have been reported so far. Biosynthesis of metal nanoparticles using algae, plant leaves, stem, flowers, and bacteria are some new approach in synthesis (Hutchison, 2008;Links, 2011). This synthesis method is environment friendly, non-toxic, reliable, low cost and synthesized nanoparticles have various applications. The noble metal nanoparticles like Au, Ag, and Pt and their composites have been synthesized for various applications like sensor, solar cell, catalysis and biomedical (Bar et al. 2009;Iravani, 2011). Silver nanoparticles were synthesized using various methods for enormous applications. Among which green synthesized silver nanoparticles play a significant important role in biomedical field due to its unique physiochemical properties like high surface to volume ratio, surface Plasmon resonance in visible region and reactivity towards bacterial cell wall. The silver nanoparticles have shown good antibacterial activity against both gram positive as well as gram negative bacteria like Staphylococcus aureus and Escherichia coli (Tabassum et al. 2015). Numerous works have been published on the anti bacterial activity of Silver nanoparticles.

1398
Owing to the biomedical applications of silver nanoparticles and its composites towards antibacterial, antifungal, anticancer and free radical scavenging activities, here in this communication we report synthesis of bio friendly Silver nanoparticles using simple cost effective Malus domestica leaf extract mediated green process (Huang et al. 2007; Kulkarni et al. 2017;Jadhav et al. 2018). The synthesized nanoparticles are subjected to UV-Vis, FTIR, XRD, AFM and SEM with EDS to study physical, compositional, structural and morphological nature of the nanoparticles. The synthesized silver nanoparticles have shown good antibacterial activity against both gram positive (S. aureus) as well as gram negative (E. coli) bacteria (Liu et al. 2002). The free radical scavenging activity has been evaluated for synthesized silver nanoparticles. Obtained Ag particles are used for further characterization to study surface morphology, particle size, functional group, elemental analysis etc. through different techniques.

Characterization Techniques:-
The surface Plasmon resonance is observed using UV-Vis spectra (UV-Vis spectrophotometer, Ocean Optics, HR4000). Crystalline nature data is gathered using X-ray diffractometer (Rigaku) with Cu Kα = 1.54178 Å over the 2θ angles ranging from -. FTIR-spectra of the silver nanoparticles were recorded in the range of 400-4000 cm -1 using a NICOLET 6700, USA instrument. . The shape, size and elemental analysis of the nanoparticles was investigated using SEM with EDX (JEOL JSM-6360, Mira-3, Tescan, Brno-Czech Republic). The size of the synthesized nanoparticles was studied using atomic force microscopy (AFM) (Cao, 2004;Huang et al. 2007).

Agar well diffusion method:-
The antibacterial activity of the green synthesized silver nanoparticles was determined using the Agar Well diffusion method (Khan et al. 2016;Kulkarni et al. 2017;Jadhav et al. 2018). Lag phase cultures of gram positive bacteria S. aureus and gram negative bacteria E. coli were used as the test microorganisms. 100 mL of the test organisms were swab inoculated onto sterile nutrient agar petriplates for bacteria and 4 wells were bored. Different concentrations (20, 40, 60, 80 and 100 µg/mL in sterile distilled water) of the synthesized silver nanoparticles were added to the labeled wells respectively. These plates were incubated at 37 ᵒC for 24 h for bacteria culture followed by observing and measuring the antibacterial activity of the synthesized silver nanoparticles by measuring the zones of inhibition in millimeters using the scale.

Evaluation of Antioxidant activity of Ag NPs by DPPH radical scavenging activity:-
The antioxidant activity of the synthesized silver nanoparticles was measured on the basis of the free radical scavenging activity by the DPPH (1, 1-diphenyl 2-picrylhydrazyl) method (Mittal et al. 2012;Liaudanskas et al. 2014). The stock solution of DPPH· was prepared by dissolving 3.9432 mg DPPH in 100 mL of methanol (0.1 mM) and stored at 4 ºC until use. 2 mL of DPPH solution was mixed with 1mL of different concentrations (20 -100 μg/mL) of the synthesized silver nanoparticles. Ascorbic acid (100 µg/mL) was used as the reference standard. Mixer of 1 mL distilled water and 2 mL DPPH solution was used as the control. The reaction mixture was mixed 1399 and incubated at room temperature in the dark for 30 min. The absorbance was recorded spectrophotometrically at 517 nm.
The antioxidant activity of the synthesized nanoparticles was estimated based on the percentage of DPPH radical scavenged as the following equation (Tabassum et al. 2015): Scavenging effect %= [control absorbancesample absorbance] x 100 Control absorbance Statistical analysis was carried out using SPSS software, version 20.0. The experiments were carried out in triplicates and the data was expressed as mean ± standard deviation by One-Way ANOVA. Turkey"s multiple comparison test was used to determine significant differences between the Standard and synthesized compounds. Correlation analysis was carried out using Pearson"s correlation analysis using p= 0.01.

Results And Discussion:-
Malus domestica leaf extract assisted green synthesis of Silver nanoparticles is carried out. The synthesized nanoparticles have been characterized in terms of morphology, optical properties, structure, and crystallinity. Furthermore the antibacterial activity was evaluated against gram negative bacteria (E. coli) as well as gram positive bacteria (S. aureus). Free radical scavenging activity of synthesized silver nanoparticles has been carried out. Optical studies:-UV-Vis absorption spectroscopy has been developed as a preliminary tool to study the surface Plasmon resonance of metal nanoparticles. Fig. 2 shows the UV-Vis absorption spectra of the Silver nanoparticles. The synthesized colloidal nanoparticles show a surface plasmon resonance peak at 416 nm indicating the formation of Silver nanoparticles with 10 -15 nm size (Huang et al. 2007;Vigneshwaran et al. 2006).

XRD Analysis:-
An X-ray diffraction analysis was carried out to study the crystallanity and grain size of the synthesized silver nanoparticles. The XRD spectrum of the synthesized silver nanoparticles is shown in figure.3. The XRD pattern shows the synthesized nanoparticles are having nano crystalline structure with Bragg reflections (111) The Debye-Scherrer calculations show that the green synthesized silver nanoparticles have average grain size of 10nm. 1400

Microscopic Analysis:-
The Atomic force microscope (AFM) image (Fig.4) shows the synthesized nanoparticles are spherical in shape with average 15 nm diameter size particles. The fig.5 shows the SEM image showing the well dispersed silver nanoparticles of spherical shape having 10-15 nm size. The elemental analysis of synthesized silver nanoparticles is carried out using EDS fitted with SEM. The elemental analysis shows that indeed the synthesized nanoparticles are having the presence of Ag element indicating the formation of silver nanoparticles.
Radical Scavenging Activity:-Free radical such as hydroxyl ions, are responsible for the faster cell decay of the healthy cells, so we performed free radical scavenging activity of the green synthesized silver nanoparticles taking ascorbic acid as the standard. The table.1 shows the free radical scavenging activity of silver nanoparticles from where we can see that the IC 50 for the silver nanoparticles is 55.73µg/mL where as for ascorbic acid the IC 50 value is 24.73. Although the IC 50 value of green silver nanoparticles is slightly more than that of the ascorbic acid but owing to the green nature of the silver nanoparticles compared to the ascorbic acid the synthesized nanoparticles are very much useful in case of free radical scavenging processes (Tabassum et al. 2015).

Antibacterial Activity:-
The antibacterial activity of synthesized green nanoparticles is evaluated using the S. aureus (gram positive) and E. coli (gram negative) as test samples which are multi drug resistant bacteria. The figure 6 (a & b) shows that the green silver nanoparticles exhibit excellent antibacterial activity against both gram positive (S. aureus) and gram negative bacteria (E. coli). The zone of inhibition is shown in the table. 2 in that we can see that the green synthesized nanoparticles are more active towards S. aureus with an minimum inhibition concentration of about 25 µg/mL while for E. coli the minimum inhibition concentration is about 50µg/mL. Although the action of silver nanoparticles on inhibition of bacteria is largely unknown but some of the researchers predict that the silver nanoparticles produce hydroxyl ions on the cell wall of the bacteria and penetrating through it and causing cell apoptosis leading to the inhibition of the bacteria.

Conclusion:-
We have successfully synthesized silver nanoparticles via Malus domestica leaf extract mediated process. The synthesized nanoparticles are capped by the phytochemicals present in the leaf extract indicated by the FTIR spectrum of the nanoparticles. The UV-Vis spectrum shows the formation of silver nanoparticles with the surface Plasmon resonance peak at 416 nm. The synthesized nanoparticles are having crystalline structure as indicated by the XRD pattern. The analyses indicate the formation of silver nanoparticles with an average size of 15 nm. The synthesized silver nanoparticles have shown excellent free radical activity and antibacterial activity. From our current findings that the synthesized nanoparticles showing growth inhibitory activities against both gram positive and gram negative pathogenic bacteria, we can say that the synthesized nanoparticles can be used in medicine as the therapeutic agents due to its antimicrobial activities against bacteria evaluated in vitro.

Conflict of interest
Authors do not have any conflict of interest related to the manuscript.