INTRODUCTION:

Nanotechnology is a fast growing field of modern scientific research involving in synthesis, design, characterization, production, application and systems by controlling shape and size at the nanometer scale^1and2. Nanotechnology also involves the synthesis of nanoparticles with different sources and techniques ranging from 1 to 100 nm size³. The new branch of nanotechnology is nanobiotechnology that integrates principles of biology with physical, biological and chemical procedures to generate nano-sized particles with specific scientific functions and other medical applications^{4, 5}. Biosynthesis of nanoparticles are both environmentally safe and economically cost effective as using biological agents such as microbes or plant extracts has gained much attention in the area of nanotechnology in last few decades^{6, 7}. Biosynthesis based on green chemistry principles is simple, relatively inexpensive and easily scaled up for larger scale production⁸.

In recent decades nanoparticles are considered as viable alternative therapeutic agents to the conventional chemotherapeutic agents and antibiotics. Moreover it seems to have a high potential to solve the problem of the new emerging infectious diseases caused by of bacterial multidrug resistance⁹. In particular, silver nanoparticles (AgNPs) have attracted much attention in the field of current clinical practices¹⁰. Silver has always been used against various infectious diseases in the past era and it found to use as an antiseptic and antimicrobial against both Gram-positive and Gram-negative bacteria¹¹ due to its low cytotoxicity¹². Mochochoko et al¹³ synthesized metal nanoparticles by using water and starch acting as both reducing agent and stabilizing agents. The fabrication of silver nanoparticles can be performed by using starch as a protective agent and β-d-glucose as a reductant in a mild heating. But in the present studies silver nanoparticles were produced by using DMSO and Milli-Q water separately with starch acting as both reduction and stabilization of silver metal ions in a high (80^oC) temperature. The produced nanoparticles were subjected to perform UV-visible spectroscopy analysis to confirm the nanoparticle. The produced silver nanoparticles were adopted to perform antimicrobial efficacy against selected pathogens.

MATERIALS AND METHODS:

Chemicals

All chemicals, media components of analytical grade and Hi media were procured form Hi media Laboratory Private Limited (Mumbai, India) for the present silver nanoparticle production.

Chemical refereed production of silver nanoparticles (Ag-NPs)

Chemical refereed production of silver nanoparticles (Ag-NPs) have been used by two different solvents viz., DMSO and Millii-Q water using starch as a reducing and stabilizing agent to obtain nanoparticles with potential application. Analytical grade (AG) of starch as a reducing agent and silver nitrate (AgNO₃) as starting material were used for the production of nanoparticle at 80^oC under constant stirring condition. Two sets of production technique have been adopted for silver nanoparticle synthesis. First set, 25 mg of starch was dissolved in 50 ml of DMSO solution in 250ml of Erlenmeyer flask by continuous stirring to dissolve completely. Followed by 17 mg of of silver nitrate was added into the flask. In second set, 25mg of starch dissolved in 50 ml of Milli-Q water in 250ml of Erlenmeyer flask by continuous stirring to dissolve completely. Now 17 mg of silver nitrate was added into each flask separately. Each reaction mixture was continuously heated to 80^oC using heating mantle separately by stirring with clean glass rod until colour change was noticed to yellowish brown. The colour change was confirmed that the production of silver nanoparticles. The produced silver nanoparticles were subjected to performing by the following standard technique of UV-visible spectroscopy analysis.

UV-Vis spectroscopy analysis

UV-Visible spectroscopy analysis was carried by using Systronics type 118 UV-Vis spectrophotometer. The chemical refereed reduction of silver metal ion was examined by measuring the UV-Vis spectrum of the reaction mixture.

Selected pathogens

The produced silver nanoparticles were subjected to perform antimicrobial assay against selected bacterial pathogens viz., E. coli, Pseudomonas aeruginosa, Salmonella paratyphi A, Bacillus subtilis, S. aureus, and yeast C. albicans. The selected pathogens were maintained as auxenic culture in our microbiology laboratory.

Antimicrobial assay

The antimicrobial susceptibility assay of silver nanoparticles was evaluated by standard disc diffusion method against selected pathogens. Different concentrations (10 µl, 20 µl, 30 µl, 40 µl, 50 µl per disc) of silver nanoparticles were impregnated with commercially available sterile empty disc (Hi-media) for antimicrobial assay. Sterile Muller Hinton agar (MHA) plates were prepared and cotton swabbed with overnight broth cultures of each selected pathogens (10⁸ cells) separately. Now silver nanoparticle impregnated disc was placed on the Petri plate at the center to center manner with equal distance aseptically. The disc impregnated with starch aqueous solution (prepared with DMSO and Milli-Q water respectively) was used as a negative control (25µl/disc) to compare the antimicrobial efficacy. Triplicates were maintained for each test pathogens to obtain mean zone of inhibition for each concentration. The zone of inhibition was measured using Vernier Caliper Scale (VCS) after 24 hrs of incubation at 37^oC for bacterial pathogens and 72 hrs at 25^oC for yeast. The zone of inhibition across the discs were measured and recorded in mm in diameter for each tested pathogen.

Statistical analysis

The antimicrobial assay results of silver nanoparticles produced by two different techniques were calculated as mean diameter of zone of inhibition in mm ± standard deviation (mean ± SD).

RESULTS AND DISCUSSION:

Nanobiotechnology is one of the most important emerging disciplines in the field of both nanotechnology and biotechnology. In the present investigation is mooted out to produce chemical mediated silver nanoparticles were produced using DMSO and Milli-Q water as described in the previous section. The reaction mixture containing starch induced the colloidal solution which turned yellowish brown for both the solvent indicating that silver nanoparticles were formed and are displaced figure 1 and 2 respectively. The absorption spectrum of yellowish brown colloidal silver nanoparticles produced by starch as a reducing and stabilizing agent with DMSO and Milli-Q water is portrayed in figure 3and4 respectively. The reaction mixture showed surface plasmon resonance absorption band was observed with a maximum peak of 420 nm. This result clearly shows that the silver nanoparticles were formed over a period of reaction time and are spherical or roughly spherical in shape.

The produced silver nanoparticles were subjected to perform antimicrobial activity against selected pathogens. Antimicrobial activity of starch mediated silver nanoparticle produced by using DMSO and Milli-Q water to assess the efficiency of nanoparticles is portrayed in table 1 and2 respectively. Among the pathogens tested yeast Candida albicans exhibited maximum inhibition zone of 14mm by using silver nanoparticle mediated by starch as a reducing and stabilizing agent by DMSO and Milli-Q water. Among the bacterial pathogens studied both gram positive and gram negative showed susceptibility by the produced silver nanoparticles. None of the activity was reported by silver nanoparticles produced by DMSO and Milli-Q water of least concentration against all the tested pathogens. The results strongly supported that the silver nanoparticle produced by using starch as a reducing and stabilizing agent with Milli-Q water exhibited broad spectrum antimicrobial activity. Chemical mediated starch induced for the production of silver nanoparticle have pharmaceutical importance and could used to treat infectious diseases.

Table 1 Antimicrobial activity of silver nanoparticles produced by starch with DMSO

S.No	Selected pathogens	Zone of inhibition in different concentration of nanoparticles (mm)
S.No	Selected pathogens	10 µl	20 µl	30µl	40 µl	50 µl
1	E.coli	NA	NA	10	11	11
2	Pseudomonas aeruginosa	NA	NA	6	8	9
3	Salmonella paratyphi A	NA	NA	10	11	11
4	Bacillus subtilis	NA	NA	10	10	10
5	Staphylococcus aureus	NA	NA	10	11	12
6	Candida albicans	NA	NA	12	13	14

Values are the average of three replicates; NA – no activity

Table 2 Antimicrobial activity of silver nanoparticles produced by starch with Milli-Q water

S.No	Selected pathogens	Zone of inhibition in different concentration of nanoparticles (mm)
S.No	Selected pathogens	10 µl	20 µl	30µl	40 µl	50 µl
1	E.coli	NA	NA	10	10	12
2	Pseudomonas aeruginosa	NA	NA	8	9	11
3	Salmonella paratyphi A	NA	NA	10	11	12
4	Bacillus subtilis	NA	NA	11	13	14
5	Staphylococcus aureus	NA	NA	8	9	10
6	Candida albicans	NA	NA	13	14	15

Values are the average of three replicates; NA – no activity

The antimicrobial effect of silver nanoparticles depends on their size, shape, and the surface charge of the particles. Silver nanoparticles have the ability to interact physically and chemically with the cell surface of various bacteria by simple adhesion and accumulation. Previous studies have reported that Ag-NPs can damage cell membranes leading to structural changes, which render bacteria more permeable to enter water and other solutes¹⁴. This effect is highly influenced by size, shape and concentration of nanoparticles¹⁵. The accumulation of Ag-NPs on the cell membrane creates small lacuna and loss the integrity of the lipid bi-layer which increases permeability of the cell and finally bacterial death¹⁶. Franci et al¹⁷ reported that, due to the structural difference in the composition of the cell walls of Gram-positive and Gram-negative AgNPs have significantly less effect on the growth of Gram-positive bacteria. Krishnakumar et al¹⁸ reported that chemical mediated synthesis of silver nanoparticles with tri-sodium citrate as a reducing agent showed maximum inhibition activity against Shigella sp. (25mm) by constant heating.The present investigation results revealed that Candida albicans is more susceptible than the bacterial pathogens tested due to their cell wall composition. The cell wall of yeast is composed of chitin cross linkage and its derivatives favours the affinity towards the accumulation of Ag-NPs. It is assumed that the accumulated silver nanoparticle disturb the cell wall biosynthesis of yeast and reach to cell membrane induce minute pores and ultimately affect the original structure of the lipid bilayer. This will increase the cell permeability leads to death of the yeast cell. The broad spectrum of bioactivity of Ag-NPs makes them promising antimicrobial agents not only to fight infections, but also in many other biomedical applications.

CONCLUSION:

Chemical mediated production of silver nanoparticles was performed by the reduction of silver salt by using starch. Ag-NPs were successfully produced under high temperature by continuous stirring. The formation of Ag-NPs reduced by starch was determined by UV–visible spectroscopy where surface plasmon absorption maxima can be observed at 420 nm. The starch mediated silver nanoparticle produced by Milli-Q water at high temperature exhibit highest inhibition activity against Candida albicans. This study clearly demonstrated that starch induced Ag-NPs with Milli-Q water exhibit utmost antimicrobial efficiency towards selected pathogens. This study is suggested that the possible use of silver nanoparicle produced using starch as an alternative safe, non toxic, cost effective antimicrobial agent that can be used as pharmaceutical ingredients for topical nanomedicine and other biomedical applications.

ACKNOWLEDGMENT:

The authors are thankful to the management of Sathyabama University, Faculty of Bio and Chemical Engineering, Department of Biomedical Engineering, Chennai, Tamil Nadu, India for providing all the needed facilities to complete the research successfully.

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Received on 19.03.2016 Modified on 26.04.2016

Research J. Pharm. and Tech. 9(4): April, 2016; Page 440-444

DOI: 10.5958/0974-360X.2016.00081.0