INTRODUCTION:

In recent years nanoparticles of silver have been found to exhibit interesting antibacterial activities. Presently, the investigation of this phenomenon has regained importance due to the increase of bacterial resistance to antibiotics, caused by their overuse. Recently, silver nanoparticles exhibiting antimicrobial activity have been synthesized. Antibacterial activity of the silver-containing materials can be used, for example, in medicine to reduce infections as well as to prevent bacteria colonization on prostheses, catheters, vascular grafts, dental materials, stainless steel materials and human skin. Plant-mediated biological synthesis of nanoparticles is gaining importance due to its simplicity and eco-friendliness.

Although biosynthesis of gold nanoparticles by plants such as alfalfa ^{1, 2}, Aloe vera³, have been reported, the potential of the plants as biological materials for the synthesis of nanoparticles is yet to be fully explored. Plants provide a better platform for nanoparticle synthesis as they are free from toxic chemicals as well as provide natural capping agents. Moreover, use of plant extracts also reduces the cost of microorganism’s isolation and culture media enhancing the cost competitive feasibility over nanoparticles synthesis by microorganisms. In this article, we report rapid biosynthesis of silver nanoparticles using Occimum sanctum leaf extract. In the present research work green synthesis of silver nanoparticles were carried out using the leaf extract of Occimum sanctum plant. The silver nanoparticles were characterized by using UV-visible spectra, FTIR, scanning electron microscopy and XRD.

MATERIALS AND METHODS:

Collection of Plant Materials:

Occimum sanctum leaves are collected from the local garden. Then the leaves are air dried for 10 days, and then kept in the hot air oven at 80°C for 2 hours. The leaves were ground to a fine powder or Take a fresh Occimum sanctum leaves and keep it air dried for 7 days. For further drying keep the collected air dried leaves in the hot air oven at 80°C for 30 minutes. After 30 minutes, make the leaves into a powder form.10 grams of air dried powder is placed in 100 ml. of distilled water in a conical flask, plugged with cotton and then keep it in dark place for 24 hours. After 24 hours it is filtered using a whatmann filter paper and the filtrate is discarded. Collect the filtered plant extract in the conical flask⁴.

Synthesis of Silver Nano Particles from Occimum sanctum

17mg of silver nitrate is added to 100ml of distilled water. Discard the 10 ml from the above solution and it is replaced by adding 10 ml of the plant extract to make up a final solution of 100ml.And it is taken as the control. For reduction process, the final solution is heated for 30 minutes and color change is observed. Then the reduced solution is centrifuged at 180 rpm for 25 mins the collected pellets are stored at -4°C. The supernatant is removed and the sediment is collected. The collected sample is then transferred into the petriplates and kept in the hot air oven for drying. After drying collect the dried sample by scrapping. Reduction of silver ion into silver nanoparticles during exposure to the plant extract could be followed by color change⁵.

Characterization of Silver Nano Particles by Analytical Technique

Characterization in materials science is the external techniques to probe into the internal structure and properties of a material. Characterization is the material testing, or analysis, different forms of microscope. The characterization method used here are: UV-Spectroscopy analysis, Fourier Transform Infrared radiation (FTIR), Field emission Scanning Electron Microscope (FESEM), Energy dispersive X-Ray analysis (EDAX), X-Ray Diffraction (XRD)^4.

RESULTS AND DISCUSSION

Sample-Occimum Sanctum

Figure 1 Fresh and dried leaves used for the synthesis of silver Nanoparticles

Biosynthesis of Silver Nanoparticles:

Synthesis of silver nanoparticles using Occimum sanctum (Fig 2) when leaf extract was subjected to aqueous solution in 1 mMSilvernitrate. A gradual change colour was observed after 12 hours and changed its colour to reddish brown. This change of colour could be due to the formation of silver nanoparticles of various shape and size. The formation of the reduced silver nanoparticles reaction mixture was further characterized by UV visible spectrophotometer analytical technique⁶.

Figure 2 Synthesis of silver Nanoparticles before reduction and after reduction

Characterization of silver nano particles:

UV Spectroscopy analysis:

It is well known fact that silver nano particles exhibit reddish brown colour in aqueous solution due to excitation of surface Plasmon vibrations in silver nanoparticles. Ag²+ ions of silver nitrate are found to be reduced to Ag atoms. It is generally recognized that UV –Vis spectroscopy could be used to examine the size and shape controlled nanoparticles in aqueous suspensions (khadriet al. 2013). Figure show the UV-Vis spectrum recorded from the reaction medium after 72 hrs and gives rise to an absorption band at 420 nm⁷.

Figure 3 - UV Spectroscopy analyses of silver nano particles

FTIR (Fourier transform of Infrared radiations)

To identify the possible biomolecules responsible for the reduction of the Ag+ ions and escaping of the bio reduced silver nanoparticles synthesised using Occimum sanctum extract. The bio reduced silver nitrate solution which is extracted from Occimum sanctum was centrifuged at 10,000 rpm for 15 min and the dried samples were grinded with KBr pellets used for FTIR measurements.

Figure 4 - FTIR Analysis of synthesised Silver Nanoparticles

Table 1 Absorption peaks and corresponding vibrations of FTIR analysis

S.	IR	TYPES OF VIBRATION
No.	SPECTRUM	TYPES OF VIBRATION
1	3432	Hydrogen-bonded O-H stretch
2	2428.7	Hydrogen-bonded O-H stretch
3	2353.2	P-H stretch phosphines
4	2118.4	C-C stretch
5	1617.2	C-C=C symmetric stretch
6	1388.8	CH3 bend of alkynes
7	1248.4	C-O stretch carboxylic acid
8	1156.1	C-o stretch of esters
9	1121.8	C-O stretch of esters
10	670.1	C-N bond of amides

FTIR studies carried out and the representative spectrum of the nanoparticles obtained in the present study is presented in Figure 3. Among them the absorption peak is around 3432, 2428, 2353, 2118, 1617, 1388, 1248, 1156, 1121, 670 cm^-1and the corresponding vibrations are tabulated (Table 1)⁸.

Fe SEM analysis of synthesized silver nanoparticle

Figure 5 - SEM analysis showing particle size

SEM technique was employed to visualize the size and morphology of the particles. The particles obtained are spherical in shape, mono dispersed, uniform size and in the range of 20- 30 nm⁹.

X- Ray Diffraction analysis:

X-ray diffraction is a very important method to characterize the structure of crystalline material and used for the lattice parameters analysis of single crystals, or the phase, texture or even stress analysis of samples. X-ray diffractogram of the silver nanoparticles showed four distinct diffraction peaks at 38.459^o, 44.555^o, 68.705^o, 77.632^oand these 2° values were indexed in the angle values of (111), (200), (220) ,(311) crystalline planes of cubic Ag was observed in (figure 4.2.5a & 4.2.5b)^10.

Biosynthetic method of silver nanoparticles was followed to synthesise the silver nanoparticles. The silver nanoparticles were synthesized using the Ocimum sanctum leaf extract. 17mg of silver nitrate is added to 100ml of distilled water.

Discard the 10 ml from the above solution and it is replaced by adding 10 ml of the plant extract to make up a final solution of 100ml.And it is taken as the control. For reduction process, the final solution is heated for 30 minutes and color change is observed. Then the reduced solution is centrifuged at 180 rpm for 25 minutes the collected pellets are stored at -4°C. The supernatant is removed and the sediment is collected. The collected sample is then transferred into the petriplates and kept in the hotair oven for drying. After drying collect the dried sample by scrapping. Reduction of silver ion into silver nanoparticles during exposure to the plant extract could be followed by color change. Further silver nanoparticles were characterized by UV spectroscopy, FTIR, FESEM - XRD analysis. UV spectroscopy is primary confirmation of the presence of silver nanoparticles.

Figure 6 - X-Ray Diffraction Showing Peaks

The absorbance peaks came at 420-430 nm and for further confirmation FTIR is used to identify the possible biomolecules responsible for the reduction of the Ag+ ions and escaping of the bio reduced silver nanoparticles synthesised using Ocimum sanctum extract. FESEM revealed a uniform alignment of silver nanoparticles having size in the range of 20-30 nm and mono dispersed, spherical in shape; whereas ¹⁰ synthesized silver nanoparticles using Pleurotus florida and obtained particle size in the range of 20 nm ±5 nm. And of poly dispersed in nature¹¹,synthesized silver nanoparticles using pleurotussajorcaju of size range 5-50 nm. But in our studies particle obtained in the size range 20-30 nm. XRD pattern showed a peaks at 38°, 44°, 68°, 77°indicating the crystalline nature of the reduced silver nanoparticles; this was correlating with the work¹².

CONCLUSION:

In our research work silver nanoparticles were synthesised by leaf Ocimum sanctum(Tulsi) from the leaf extract .The synthesised silver nanoparticles was subjected to UV-visible spectrometer to determine the absorption range which was found to be around 420-430 nm and confirmed the presence of silver nanoparticles. Further to confirm the presence of silver nanoparticles the synthesised particles was confirmed by various analytical techniques such as FTIR, XRD,EDAX and FESEM.FTIR study revealed the presence of silver nanoparticles followed by XRD. XRD study indicates the crystalline nature of the reduced of the silver nanoparticles. The size of synthesized silver nanoparticles was determined by FESEM method the size was around 20-30nm. All these studies revealed that the characteristic of synthesized nanoparticles from leaf extract of Ocimum sanctum. Further studies has to be studied to determine the antibacterial activity by disc diffusion method using synthesized green nano particles to prove the efficacy of antimicrobial activity of nanoparticles against human pathogens.

ACKNOWLEDGEMENTS:

The authors are highly indebted to Sathyabama University for providing all the necessary facilities to carry out the research work throughout the study.

REFERENCES:

1. Shetty S, Udupa S, Udupa L, Somayaji N. Wound healing activity of Ocimum sanctum Linn with supportive role of antioxidant enzymes. Indian J. Physiol. Pharmacol., 2006; 50(2): 163-168.

2. Ahmed M, Ahamed RN, Aladakatti RH, Ghosesawar MG. Reversible anti-fertility effect of benzene extract of Ocimum sanctum leaves on sperm parameters and fructose content in rats. J. Basic Clin. Physiol. Pharmacol. 2002; 13(1):51-59.

3. Gupta S, Mediratta PK, Singh S, Sharma KK, Shukla R. Antidiabetic, antihypercholesterolaemic and antioxidant effect of Ocimum sanctum (Linn) seed oil. Indian J. Exp. Biol. 2006; 44(4):300-304

4. Garima Singhal,Riju Bhavesh, Kunal Kasariya, Ashish Ranjan, Sharma Rajendra Pal Singh. Biosynthesis of silver nanoparticles using Occimum sanctum (Tulasi) leaf extract and screening its antimicrobial activity, J Nanopart Res. 2011; 13: 2981–2988.

5. K Mallikarjuna, Narasimha G, Dillip G R, Praveen B, Shredhar B, Sree Lakshmi C, Reddy BVS. Green synthesis of silver nanoparticles using Occimum leaf extract and their Characterization, Digest journal of nanomaterials and biostructures, 2011; 6(1): 181 – 186.

6. Thukkaram Sudhakar, Anima Nanda, Sam George Babu, Sreenivasan Janani, Melvin Danny Evans, Tinku K Markose, Synthesis of silver nanopartciles from edible mushroom and its microbial activity against human pathogens. International Journal of PharmTech Research, 2014; 6(5): 1718-1723.

7. Anima Nanda, Shahnaz Majeed. Improved bactericidal property of silver nanoparticles from Penicillium pinophilum (MTCC 2192) in a combined form with Carbenicillin and Moxifloxacin, International Journal of Pharmacy and Pharmaceutical Sciences, 2014; 6; 609- 612.

8. Shivakumar Singh P, Vidyasagar G.M. Green synthesis, characterization and antimicrobial activity of Silver Nanoparticles by using Sterculi afoetida L. young leaves aqueous extract. International Journal of Green Chemistry and Bioprocess. 2014; 4(1): 1-5.

9. Amin Bhat M, Nayak BK, Anima Nanda. Exploitation of filamentous fungi for biosynthesis of silver nanoparticle and its enhanced antibacterial activity. Int J Pharm Bio Sci, 2015; 6(1): 506 – 515.

10. Ravishankar Bhat, Raghunandan Deshpande, Sharanabasava V, Ganachari, Do Sung Huh, and A. Venkataraman. Photo-Irradiated Biosynthesis of Silver Nanoparticle Using Edible Mushroom Pleurotus florida and Their Antibacterial Activity Studies Bioinorganic Chemistry and Applications, Volume 2011, 7 pages

11. Nithya R, Raghunathan R. Synthesis of silver nanoparticle using Pleurotussa jorcaju and it antimicrobial study Digest Journal of Nanomaterials and Biostructures, 2009; 4(4); 623-629.

12. Anuradha, Abbasi T, Abbasi SA. Green synthesis of gold nanoparticles with aqueous extracts of neem, Research Journal of Biotechnology, 2010; 5(1); 75-79.

Received on 03.03.2016 Modified on 01.04.2016

Research J. Pharm. and Tech. 9(4): April, 2016; Page 397-400

DOI: 10.5958/0974-360X.2016.00072.X