Indian Journal of Medical Research and Pharmaceutical Sciences March 2018;5(3) ISSN: ISSN: DOI: /zenodo Impact Factor: 4.

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1 GREEN SYNTHESIS, CHARACTERIZATION AND ANTIMICROBIAL ACTIVITY OF SILVER NANOPARTICLES FROM Plectranthus amboinicus PLANT EXTRACTS Niveditha.K* and T H Sukirtha *M.Sc Student, Department of Biotechnology, Nehru Arts and Science College, Coimbatore Associate Professor, Department of Microbiology, Nehru Arts and Science College, Coimbatore Keywords: Nano particles, silver nitrate,ftir, SEM. Abstract Nanotechnology is now creating a growing sense of excitement in the life sciences. Nano particles having a size of 1-100nm can be prepared by different chemical, physical and biological approaches. Among these green synthesis method is the most emerging approach of preparation because, this is easier than the other methods, eco friendly and less time consuming. The green synthesis was done by using the aqueous extract of Plectranthus amboinicus leaf and silver nitrate as the precursor for bio reduction. A fixed ratio of plant extract to metal ion was prepared and the color change was observed which proved the formation of Silver nanoparicles. The silver nanoparticles were characterized by FTIR, Raman Plot and SEM analysis. The silver nanoparticles are highly toxic against different multi drug resistant clinical pathogens. The infectious diseases cause serious threat to the people world- wide. At the earlier day antibiotics are used to control these infectious organisms from the environment. The current study was designed to test the antimicrobial potential of green synthesized silver nanoparticles.the advances in nanotechnology has enabled us to utilize particles in the size of the nano scale, has created new therapeutic application in the coming years. Introduction Nanotechnology is emerging as a rapidly growing field with its application in science and technology for the purpose of manufacturing new materials at the nanoscale level [1]. The synthesis of nanoparticles using plants extracts has various human applications. Compare to other biological processes of nanoparticle synthesis using plants have been many advantages because it eliminates the elaborate process of maintaining cell cultures. Plants use can also be suitably scaled up for large-scale synthesis of nanoparticles [2].Because of the pathogenic organism resistance to the already available drugs, there is always need an alternative approach to search for new bioactive compounds [3]. The use of Aloe vera, Azadirachta indica, Medicago sativa, Emblica officinalis has already been reported. The present investigation demonstrated that silver nanoparticles could be prepared using screened plant extract of Plectranthus amboinicus with potent antimicrobial effects having ability to treat infectious diseases. Materials and methods Materials Collection of plant material The plant materials used for this study were Plectranthus amboinicus (also known as Indain borage) collected in the month of february 2015, from Palakkad district.(fig 1) Methodology Paramasivam et al.,2012 [4]. [41]

2 Preparation of leaf extract The plant leaves were washed thoroughly two to three times using tap water followed by distilled water.25g of leaf sample was boiled with 100 ml of double distilled water, at 60 0 C for about 5 minutes (Fig. 2). Precursor The precursor for the synthesis of silver nanoparticles,silver nitrate was obtained from spectrum reagents and chemicals Pvt.Ltd.,Edayar,cochin. (Fig.3). Preparation of 1mm silver nitrate solution 1Mm silver nitate solution was prepared by the concentration of g in 100ml double distilled water. (Fig. 4) Green synthesis of silver nanoparticles from Plectranthus amboinicus In the typical synthesis of silver nanoparticles, 10 ml of the aqueous extract of Plectranthus amboinicus was added to 90 ml of 1Mm (10-3 M) solution of silver nitrate (AgNO 3) in 250 ml conical flask. The reaction was performed at room temperature and was kept over incubator shaker at 120 rpm, for 5 hours. Suitable controls were maintained throughout the conduct of experiments. Recovery of silver nanoparticles by ultra centrifugation For purification of silver nanoparticles formed in the aqueous extract, about 1 Litre of 1Mm silver nitrate solution containing 100 ml of aqueous leaf extract of Plectranthus amboinicus was prepared and incubated over the orbitary shaker at normal room temperature. After the bioreduction, the solution consisting of hydrosols of silver nanoparticles and biomolecules from the aqueous extract of Plectranthus amboinicus, was subjected to centrifugation at 5000 rpm for 10 minutes, washed twice and the pellet was discarded. Later the supernatant was subjected to centrifuge at rpm (75000 g), for 30 minutes. The pellet formed was dissolved in 1.0 ml of deionized water and air dried (Fig- 5). Characterization of green synthesized silver nanoparticles Several techniques have been reported worldwide for the characterization of herbal and medicinal based nanoparticles. Green synthesized silver nanoparticles characterization was done at Department of Nanoscience and Technology, Bharathiar University. Fourier transformation infra-red (ftir) spectroscopy analysis: Fourier transform infrared (FTIR) spectral measurements were carried out to identify the potential biomolecules in Plectranthus amboinicus leaf extract which is responsible for reducing and capping the bioreduced silver nanoparticles were subjected to FTIR analysis by Potassium Bromide pellet method in 1:100 ratios and spectrum was recorded in Nicolet Impact 500 FT-IR spectrophotometer using diffuse reflectance mode. SEM analysis Scanning Electron Microscope (SEM) analysis was done at Nanoscience department of Bharathiar University,Coimbatore. The silver nanoparticles analysis done by preparing thin films on a gold coated copper grid by dropping a very small amount of the sample on a grid. Extra solution was removed using a blotting paper and thin films on the SEM grid were allowed to dry by putting it under a mercury lamp for 5 minutes. Silver Nanoparticles were examined with SEM (JEOL JSM 6390) at an accelerating voltage of 20kV and a working distance of 15mm. Antimicrobial activity of silver nanoparticles synthesized from Plectranthus amboinicus (Ameer azam et al., 2012 [5].) Preparation of silver nanoparticle samples Silver nanoparticles (synthesized at C) were weighed at different concentrations (100,150 and 200µg). [42]

3 Micro organisms used The MTCC strains used for the study were obtained from the stock cultures of different Gram positive bacteria:staphylococcus aureus (MTCC No 96), Bacillus subtilis (MTCC No ),Gram negative bacteria: Pseudomonas auruginosa (MTCC No.424), Proteus vulgaris (MTCC No.1771 ) Fungal strains of Candia albicans (MTCC No.227) were subcultured and maintained in nutrient broth. Screening of antibiotics The screening of antibiotics was done to identify the standard antibiotic against the pathogenic organisms like Gram negative (Proteus vulgaris MTCC No 1771 and Pseudomonas aeruginosa MTCC No 424) and Grampositive (Bacillus subtilis MTCC No10619 and Staphylococcus aureus MTCC No 96) bacterial and fungal strain Candida albicansmtcc No 227. For that; Tetracycline, Erythromycin, Streptomycin and Ampicillin are selected. And that study showed that, Tetracycline is found sensitive against gram positive and gram negative bacteria. For fungus Chloramphenicol showed sensitivity than Amphotericin and thus Tetracyclin is selected against bacteria and Chloramphencol is selected as the standard for fungus in antibiotic sensitivity testing. Antimicrobial assay (kirby bauer method) Silver nanoparticles synthesized using aqueous leaf extrct of Plectranthus amboinicus were tested for its potential antimicrobial activity against a few bacterial and fungal clinical pathogens by agar disc diffusion method(kirby- Bauer method).using a sterile cotton swab, swabbing the cultures in pre-sterilized Muller Hinton agar plates under aseptic conditionds.plates were left standing for 10 minutes to let the cultures get absorbed.then three 8mm wells were punched into the MHA plates for testing the antimicrobial activity of green synthesized silver nanoparticles.20µl of each test sample at different concentrations (100,150,200µg/ml) was poured into each punched 8mm wells and is sealed using 0.8% molten agar. For comparing the activity, standard antibiotic discs (Tetracycline for bacterial test pathogens and Chloramphenicole for fungal test pathogen) are used. The plates were incubated at 37 0 C For hours.after incubation period the zone of inhibition of test pathogens were measured. Result and discussion Green synthesis of silver nanoparticles from plectranthus amboinicus leaf extracts Silver nanoparticles exhibit brownish colour in aqueous solution due excitation of surface plasmonresonance.the results exhibited that the addition of the aqueous leaf extract Plectranthus amboinicus to 1mM silver nitrate led to the appearace of reddish brown colour as aresult of formation of silver nanoparticles.it was due to the reduction of Ag + which indicates the formation of silver nanoparticles. (Fig. 6) Discussion The appearance of a reddish -brown color confirms the existence of silver nanoparticles in the solution.the time duration of change in color was 24 hours.this change in clour of the reaction mixture was taken as a primary evidence for the formation of silver nanoparticles.the time duration of change in color and thickness of the color varies from plant to plant. The reason could be that the quantitative variation in the formation of silver nanoparticles (or) availability of H+ ions to reduce the silver. The aqueous silver ions when exposed to herbal extracts reduced in solution, there by leading to the formation of silver hydrosols.in figure shows the leaf extract of Plectranthus amboinicus and the figure shows the change in colour of the reaction mixture. Plectranthus amboinicus plays a major role in nano medicine and it is seen everywhere in our state. Characterization of green synthesized silver nanoparticles The obtained results of SEM and FTIR studies confirmed the fact that sample powder (Fig- 7) consists of green synthesized silver nanoparticle from the plant Plectranthus amboinicus. Fourier transform infra red (ftir) spectroscopy analysis FTIR spectroscopy analysis was conducted to identify the biomolecules responsible for capping of the bioreduced silver nanoparticles synthesized using plant extract. Fig. 8 shows FTIR spectra of the synthesized silver nanoparticles using Plectranthus amboinicus amboinicus leaves extract. [43]

4 The results of FTIR analysis of this study show different stretches of bonds at different peaks,3350cm -1 N-H stretch, cm -1 -C=C, cm -1 -C=C and cm -1 C=O/-C-N stretch (aliphatic amines )Peak near to cm -1 C-H rock (alkanes),respectively. FTIR spectra of silver nanopartcles exhibited prominent peaks at cm -1, cm -1, and cm -1 The spectra showed sharp and strong absorption band at cm -1 assigned to the stretching vibration of (NH) C=O group.the band cm -1 developed for C-C and C-N stretching vibration respectively SEM ananlysis SEM images show similar appearance for the presence of silver nanoparticles synthesized from Plectranthus amboinicus.the synthesized nanoparticles show below 4µm in size.the SEM image shows cluster of nanoparticles at different sizes. Fig- 9 shows the SEM analysis of silver nanoparticles. The SEM studies confirmed the formation of silver nanoparticles. Antimicrobial activity of silver nanoparticles synthesized from plectranthus amboinicus leaf extracts The antimicrobial activity of green synthesiszed silvernanoparticles was carried out on few clinical pathogens at dufferent concentration. Green synthesized silver nanoparticles showed clear zone of inhibition as indicated in the Table-1 and Table-2 & Plate 1 to 5 respectively. Table-1 zone of inhibition silver nanoparticles against bacterial test pathogens at different concentration (Control antibiotic,tetracycline zone diameter range ) Sensitive: 19mm, Resistant : 14mm, Intermediate :15 to 18mm Table-2: Zone of inhibition silver nanoparticles against fungal test pathogen at different concentrations (Control antibiotic,chloramphenicol zone diameter range) Sensitive: 18mm, Resistant: 12mm, Intermediate:13 to 17mm [44]

5 Discussion Silver nanoparticles synthesized in our study effectively inhibited the growth and multiplication of pathogenic bacteria like Staphylococcus aureus, Pseudomonas aeuroginosa,proteus vulgaris, Bacillus subtilis fungal pathogens candida albicans. The antimicrobial activity was checked for different concentration of silver nanoparticles. The synthesized silver nanoparticle controlling Proteus vulgaris effectively than other bacterial strains. Next to this, it is controlling Pseudomonas aeroginosa and Staphylococcus aureus.but it has lowest activity against Bacillus subtilis compare to other bacterial strains. In fungal pathogens it showed effective activity against Candida albicans. It is found that in the present study,green synthesized silver nanoparticles from Plectranthus amboinicus leaf extracts inhibited the growth and multiplication of selected pathogenic bacterial and fungal strains.on comparison with the bacterial and fungal strains green synthesized silver nanoparticles outperformed in the bactericidal effect. Fig-1 Plectranthus amboinicus Fig-2 Preparation of aqueous extract [45]

6 Fig-3 Precursor-Agno3 Fig-4 AgNO3 solution(1mm) Fig-5 Recovery of silver nanoparticles by centrifugation [46]

7 Fig-6 Synthesis of silver nanoparticles from Plectranthus amboinicus LEAF EXTRACTS Fig-7 Purified silver nanoparticles [47]

8 Fig-8 FTIR transform infra red spectroscopy (FTIR) analysis Fig-9 SEM image showing silver nanoparticles synthesized from Plectranthus amboinicus [48]

9 Plate 1 to 4. Antibacterial activity of silver nanoparticles synthesized from P.amboinicus against Gram positive and Gram negative bacterial strains GRAM POSITIVE BACTERIA Plate-1. Staphylococcus aureus Plate-2. Bacillus subtilis GRAM NEGATIVE BACTERIA Plate-3. Pseudomonas aeruginosa Plate-4. Proteus vulgaris Plate-5. Antifungal activity of silver nanoparticles synthesized from Plectranthus amboinicus against Candida albicans fungal strains. Plate.5.Candida albicans [49]

10 Acknowledgement The authors wish to express their gratitude to the Bharathiar University,Coimbatore. is acknowledged for technical assistance in the use of FTIR & SEM analysis. The authors are also thankful to the Nehru Arts and Science College, T.M.Palayam,Coimbatore- 105 for providing facilities. Summary Nano-biotechnology is an emerging,interdisciplinary area of research with important commercial applications and would be a dominant technology in new world economics. Nanoparticles is being viewed as fundamental building blocks of Nanotechnology. The most effectively studied nanoparticles today are those made from noble metals,in particular silver, gold, platinum and palladium. Among the four, silver nano particles play significant role in the field of medicine. The present study was carried out to find out the antimicrobial activity of green synthesized silver nanoparticles from Plectranthus amboinicus leaf extract. It is found throughout India. Plectranthus amboinicus has been used in traditional medicine to treat fever, cough, sore throats, nasal congestion.it is used by some tribes as remedy for hepatopathy,chronic asthma, bronchitis, renal and vesical calculi.in this study silver nanoparticles were synthesized from Plectranthus amboinicus plant extracts.all around the global, interest for finding bioactive compounds because of the pathogen resistance to available antibiotic drugs.so to treat such infectious diseases the way changed to search on biofriendly based products.the present study maid an attempt by using green synthesized silver nanoparticles with potent antimicrobial effects to treat infectious diseases References 1. Peter Logeswari, Sivagnanam Silambarasan, Jayanthi Abraham, Ecofriendly synthesis of silver nanoparticles from commercially available plant powders and their antibacterial properties in Journal of saudi chemical society,vol.10, pp , February Monali Gabhiye,Aniket,Mahendra Rai and Avinash Ingle, Fungus mediated synthesis of silver nanoparticles and their activity against pathogenic fungi in combination with fluconazole in science direct journal of nanomedicine,pp ,september Praveena Bhatt,Pradeep S.Negi, Antioxidant and antibacterial activities in the leaf extracts of Indian Borage(P.amboinicus) in Food and nutrition sciences,vol.10, pp August T.Parmasivam,W.Femina,P.Dheepan Shankar,Nameirakpam Nirjinta Devi, Antimicrobial efficacy of green synthesized silver nanoparticles from the medicinal plant Plectranthus amboinicus in International journal of pharmaceutical sciences review and research, vol.12,no. 030, February Ameer Azam,Arham S Ahmed,Sami S Habib,Adnan Memic, Antimicrobial activity of metal oxide nanoparticles against gram positive and gram negative bacteria a comparative study in International journal of nanomedicine, Vol.7,pp ,April [50]

11 Author Biblography Niveditha.k M.Phil Research Scholar Department of Biotechnology Sree Narayana Guru College KG Chavadi Coimbatore Dr.T H Sukirtha Associate Professor Department of Microbiology Nehru Arts and Science College T M Palayam Coimbatore [51]