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1 SYNTHESIS OF SILVER NANOPARTICLES FROM THE METHANOLIC EXTRACT OF ACACIA NILOTICA BARK AND EVALUATING IT S ANTIBACTERIAL ACTIVITY USING MULTI DRUG RESISTANT BACTERIA 1 SHINOVA SINGH, 2 MANISHA PRASAD 1 Student of MSc Chemistry, Chemistry Department, Sam Higginbottom Institute of Agriculture Technology & Sciences Allahabad, Uttar Pradesh, India, singh.shinova@gmail.com 2 Assistant Professor, Chemistry Department, Sam Higginbottom Institute of Agriculture Technology & Sciences Allahabad, Uttar Pradesh, India, sharmamanisha1@rediffmail.com ABSTRACT Multi drug resistance has become an emerging and challenging issue for pharmaceutical industry as more and more bacteria are developing drug resistance towards many antibiotics, also over use of these synthetic drugs causes toxicity leading to further detrimental effects on human body. This study focused on the synthesis of silver nanoparticles, which is necessary for safe and effective exploitation of silver nanoparticles in collaboration with plant derived metabolites as a substitute for harmful synthetic drugs. Silver nanoparticles were synthesized using bark of Acacia nilotica, synthesized silver nanoparticles were then subjected to check antibacterial activity against multidrug resistant bacteria like S. aureus and P. aeruginosa. Spectral techniques like Uv Vis, zetasizer and SEM were performed for the characterization of silver nanoparticles and results shows that AgNPs can be used as tool in combating the issue of drug resistance in future, as in comparison to broad spectrum antibiotic silver nanoparticles showed better bacteriostatic effect against bacteria. Keywords: Silver nanoparticles, antibacterial, antibiotic, bacteriostatic. 1. INTRODUCTION Noble metal nanoparticles are gaining lot of significance for the past few years due to their applicability in the field of medicine, biology, material science, physics and chemistry (1). Among the several noble metal nanoparticles, silver metal nanoparticles have attained special focus in view of their distinctive properties, like good electrical conductivity, chemical stability, catalytic and antibacterial activity (2). Along with chemical synthesis, silver nanoparticles have also been synthesized using plants, such as Azadirachta indica(3), Jatropha carcus(4), Cycas (5), Pongamia pinnata (6), Eucalyptus hybrida (7)as reported earlier in these mentioned studies. Thus making different parts of plants as a source of nanoparticles which might be a helpful tool in medicine, biotechnology, healthcare etc sectors. Acacia nilotica belonging to kingdom Plantae, Order Fabales, Genus Acacia and Species nilotica and commonly known as babool in India. This plant contributes a number of groups among which are alkaloids, flavonoids, saponins, tannins etc (8,9,10,11).Due to all this, Acacia nilotica has an inspiring range of medicinal uses with potential anti oxidant activity (12). Antibacterial agents like broad spectrum synthetic drugs have proved advantageous in fighting infectious diseases. Although with their constant use, these antibacterial drugs have resulted in creating resistance in bacteria which has led to further emergence of diseases that were being successfully treatedbefore. For the treatment of drug resistant diseases high doses are often given resulting in further infections and toxicity in patients, thus an early replacement is required for which several classes of antimicrobial nanoparticles and nanosized carriers for antibiotics delivery have proven their effectiveness for treating infectious diseases, including antibioticresistant ones (13). Therefore plants derived AgNPs can be a good substitute for antimicrobial agents and can help in fighting the problem of drug resistance as nanoparticles exhibit completely new or improved properties based on specific characteristics such as size, distribution and morphology. The cell membrane of microorganisms is negatively charged and silver nanoparticles are positively charged and when these positively charged silver nanoparticles accumulate on negatively charged cell membrane, it brings about a substantial conformational change in the membrane and it ultimately loses permeability control which leads to cell death (14). 2. MATERIAL AND METHODS 2.1 COLLECTION OF THE SAMPLE The powdered bark of Acacia nilotica,1 Kg of powdered stem bark of Acacia nilotica was collected from the SHREE Baidyanath, Ayurved Pvt Ltd., Allahabad Preparation of Methanolic Extract from the bark of Acacia Nilotica The plant sample was weighed and extracted in soxhlet extractor with 100 ml of methanol at 64.7 C. The extraction was monitored continuously at each stage on the completion of extraction and was confirmed by colour. The solvent was taken from the thimble and evaporated to check the absence of residue. The extract was filtered through Whatmann no.1 filter paper and evaporated to dryness at 64.7 C Estimation of Total Phenolic Contents The total Phenolic content in bark of Acacia nilotica (Babool) was determined by the method given by International Journal of Science, Engineering and Technology

2 (Singleton et al.,1999 and Makkar et al,1993) by using Gallic acid as standard Synthesis of AgNPs Fifty ml of M aqueous solution of silver nitrate was prepared in a stoppered Erlenmeyer flask and 1 ml of bark extract (0.2 g/ml) was added at room temperature for 24 h in the dark until the brownish colour was developed, after development of the colour the extract was centrifuged at a speed of around 5000 rpm for 15 min, supernatant was discarded and the obtained pellets were further dried Characterization of silver nanoparticle a. UV vis spectra analysis: indicated by an inhibition zone surrounding the well containing the AgNPs.The zone of inhibition was measured and expressed in millimeters. 3. RESULTS 3.1 ESTIMATION OF TOTAL PHENOLIC CONTENT OF THE BARK EXTRACT OF ACACIA NILOTICA USING UV VIS SPECTROSCOPY Quantitative determination of total phenolic content was done using the linear equation based on the calibration curve of gallic acid and which was found to be 720 mg/g gallic acid equivalent Biosynthesis of Silver nanoparticles using A. nilotica bark The reduction of Ag + to Ag 0 was confirmed using UV vis spectrophotometer, this recording for absorption spectra of the synthesized silver nanoparticles were recorded against water, the change in colour seen, ie, from pale yellow to deep brown depending on the extract concentration probably indicating the formation of silver nanoparticles, which is observed due to excitation of surface Plasmon vibration in the silver nanoparticles. It can be seen that the surface plasmon resonance (SPR) of AgNPs is 440. b. Zeta Sizer analysis To confirm that the size of the synthesized silver nanoparticles are in the nanometer range, Malvern Zetasizer Nano instrument was used, size distribution by intensity, size distribution by number and size distribution by volume graphs were recorded at 24 C and using quartz cuvette with water as reference, which gave Z average (d.nm) of the silver nanoparticles. In the current study, 50 ml of M of aqueous solution of silver nitrate was reduced to AgNPs, upon mixing with 0.2g of Acacia nilotica bark extract followed by incubation for 24 h in the dark at room temperature. The color turned from yellow to reddish brown as shown below in fig 1 this change in colour can be attributed to the surface plasmon resonance of deposited AgNPs which may be due to the rich phenolic content as reported above, this higher content of total phenolics content in Acacia nilotica bark extract helps in the bio reduction of Ag + to Ag 0 as these phenolic compounds have electron donating ability. c. SEM analysis: The Morphological evaluation of the samples the suspension containing AgNPs of Acacia nilotica was analyzed by SEM analysis using Scanning Electron Microscope ( EPM Analyser Jeol JXA 8100), by preparing thin films of the sample on a carbon coated copper grid Testing for antibacterial activity of formed silver nanoparticles against multi drug resistant bacteria. To check the antibacterial activity of the obtained AgNPs aginst multi drug resistant bacteria, MDR strains; S.aureus 0066 and P.aeruginosa were used and agar well diffusion method was followed given by Collins et al., 1995.Forthepreparation of nutrient agar plates 15 ml of molten media was poured into sterile petri plates. About ( ) colony forming unit per ml were used. Four well were made using sterile borer into agar plates. 10µL of microbial broth was spread on the surface of nutrient agar plates. 25µl, 50µl, 75µl of AgNPs was poured into three wells of inoculated plates using a micropipette. Amoxicillin which is also a known antibiotic was taken 25 µl as a control and was introduced in one of the four wells of the petriplate. After incubation for 24 hrs at 37 C, the plates were observed. The antibacterial activity present on the plates was Fig 1 Synthesis of silver nanoparticles using bark of Acacia nilotica Characterization of Silver nanoparticles UV spectra Analysis: UV visible spectra of silver nanoparticle recorded for methanolic bark extract of Acacia nilotica plant was 440 nm, the colour of the solutions changed from deep yellow to deep brown depending upon the concentration of the extract showing the probable formation of silver nanoparticle as the color change observed is due to excitation of surface Plasmon vibration in the silver nanoparticles which confirms that the synthesized particles are silver as same readings has been reported in previous literatures. International Journal of Science, Engineering and Technology

3 Fig 2 Uv Vis analysis of synthesized AgNPs using bark extract of Acacia nilotica Estimation of the size of the synthesized Silver Nanoparticles by Zetasizer The synthesized silver nanoparticles were further demonstrated and confirmed by the characteristic peaks observed in Zeta sizer image, which indicate that the average of the diameter was in the range is d.nm. As depicted in the three graphs in fig 3, fig 4 and fig5respectively based on intensity, number and volume distributions, in all the three distributions, similar Z average (d.nm) ie, and polydispersity index to be Fig 4 Depicting Size Distribution by number of the C. A) Fig 3 Depicting Size Distribution by Intensity of the B. Fig 5 Depicting Size Distribution by Volume of the 3.2 SEM analysis: Scanning electron microscope helps in investigatingthe topographical features of the formed silver nanoparticles as shown in Fig6 which confirms the existence of very small and uniformely formed nanoparticles, from the SEM images it can be observed that at some portion larger particles of AgNPs can be seen which might have been formed due to aggregation of nanoparticles, International Journal of Science, Engineering and Technology

4 induced by the evaporation of solvent during sample preparation. Yet even the number of particles participated in aggregation can be seen from the images, shown with arrow. AgNPs generally showed typical optical absorption peak approximately at 25 kv and 15 kv due to surface Plasmon resonance. Also the size estimated to be around 100nm range, was in agreement with the zetasizer scan output result which was d.nm both by number, intensity and volume. Fig 6 depicting SEM images of silver nanoparticles obtained in extract of Acacia nilotica plant Table 1 Antibacterial activity of Silver nanoparticles obtained from methanolic extract of Acacia nilotica against bacteria by agar well diffusion much explored and could be used for the benefit of many sectors like biotechnology, health, cosmetics etc, this field can be exploited especially in adjunction with plant metabolites to combat the concerning issue of drug resistance developing in bacteria against many harmful diseases which were once in a good control of known antibiotics, Ocimum basilicum(l.) aqueous leaf extract which was found to be rich in metabolites like saponins, terpenoids, phenolics, tannins, anthraquinones, anthocyanins helped in synthesizing silver nanoparticles.(18).in the present study silver nanoparticles were synthesized using methanolic bark extract of Acacia nilotica plant, as higher content of total phenolics in plant extract facilitates the reduction of silver ions to nano scale sized silver particles due to the electron donating ability of these phenolic compounds found in plant extract. Moreover, the quinoid compound produced due to the oxidation of the phenol group in phenolics can be adsorbed on the surface of nanoparticles, accounting for their suspension stabilization(19). The color change which appeared due to the surface plasmon resonance of deposited AgNPs may be attributed to this rich phenolic content itself. Further for the confirmation of formed AgNPs spectroscopic techniques like UV Vis, zetasizer and SEM were performed (20, 21). Finally the synthesized AgNPs were tested against multidrug resistant bacteria and these AgNPs showed good antibacterial efficacy against gram positive and gram negative bacteria, thus making it as a potential antimicrobial agents. When silver nanoparticles obtained from aqueous leaf extract of Carica papaya were compared against standard antibiotic using multidrug Resistant bacteria (E. coli, Klebsiella pneumoniae and P. aeruginosa), nanoparticles showing larger radius of zone of inhibition in comparison with antibiotic. (22). From the present study it can be concluded that noble metals like gold and silver, in this case silver can be exploited efficiently and cost effectively and these metallic nanoparticles in collaboration with plant metabolites having good antioxidant content can be used as a successful alternative for synthetic drugs, and can combat the issue of multi drug resistance. ACKNOWLEDGEMENT The authors are grateful to the authorities of SHIATS University. REFERENCES [1] Yokohama, K,; Welchons, D.R., The conjugation of amyloid beta protein on the gold colloidal nanoparticles surfaces Nanotech, 18,2007, Showing antibacterial activity of AgNPs obtained from the methanolic extract of bark of Acacia nilotica against mutidrug resistant a) S.aureus (0066) b)s.aureus (00351) 4. CONCLUSION In this vastness of techniques and knowledge, is emerging a new field which is nanotechnology yet to be [2] Sharma, V,K,; Yngard, R,A,; Lin, Y., Silver nanoparticles Adv. Colloid Interface Sci,145,2009, [3] Shankar,S,S,; Rai, A,; Singh, A,; Ahmed, M,; Sastry, M., Biological synthesis of triangular gold nanoprism Nat Mat, 3,2004, [4] Bar, H,; Bhui,D,H,; Sahoo, P,G,; Sarkar, P,; De,P,S,; Misra, A., Green synthesis of silver nanoparticles using International Journal of Science, Engineering and Technology

5 latex of Jatropha curcas Colloids Surf A Physicochem Eng Asp,339, 2009a, [5] Jha, A,K,; Prasad, K., Green synthesis of silver nanoparticles using Cycas leaf Int JGreen Nanotech Phys Chem,1, 2010, [6] Raut, W,R,; Kolekar, S,N,; Lakkakula, R,J,; Mendhulkar, D,V,; Kashid, B,S., Extracellular synthesis of silver nanoparticles using dried leaves of Pongamia pinnata (L).Nano Micro lett, 2,2010, [7] Dubey, M,; Bhadauria, S,; Kushwaha,B,S., Green synthesis of nano silver particles from the extract of Eucalyptus hybrida" Dig J Nanomater Bios, 5,2009, [8]Mutai, C,; Abatis, D,; Vagias, C,;Moreau, D,; Roussakis, C,; Roussis,V., Cytotoxic lupanetype triterpenoids from Acacia mellifera J Phytochem, 65,2004, [9] Eldeen, I,M,; Elgorashi, E,E,; Staden, J., Antibacterial, anti inflammatory, anti cholinesterase and mutagenic effects of extracts obtained from some trees used in South African traditional medicine J Ethnopharmacol, 102,2005, [10]Chaubal, R,; Mujumdar A M,; Puranik, V,G., Isolation and X ray study of an anti inflammatory active androstene steroid from Acacia nilotica J Arzneim Forsch, 56,2006, [11] Singh, R,; Singh, B,; Singh, S,; Kumar, N,; Kumar, S,; Arora, S., Umbelliferone An antioxidant isolated from Acacia nilotica (L.) Willd. Ex Del J Food Chem, 120,2010, [17] Collins, C,H,; Lynes, P,; Grange, J., Microbiological Methods 7th Ed., Butterwort Heinemann Ltd, Britain, 1995, [18] Jayapriya, E,; Lalitha, P., Synthesis of Silver nanoparticles using Leaf Aqueous Extract of Ocimum basilicum (L.). Int J ChemTech Res,5(6),2013, [19] Wang, W,; Chen, Q,; Jiang, C,; Yang, D,; Liu, X,; Xu, S., One step synthesis of biocompatible gold nanoparticles using gallic acid in the presence of poly (N vinyl 2 pyrrolidone) Colloids Surf A Physicochem Eng Asp, 301,2007, [20] Usha, C,; Rachel, D., Biogenic synthesis of silver nanoparticles by Acacia nilotica and their antibacterial activity Int J. Scientific Res,3(6),2014, [21] Aruna, A,; Nandhini,R,; Karthikeyan,V,; Bose, P., Synthesis and Characterization of Silver Nanoparticles of Insulin Plant (Costus pictus D. Don) Leaves Asian J. Biomed Pharm Sci,4(34),2014,1 6. [22] Upgade, A,; Prabakaran, P., A Novel Combinatorial Herbal Drug Development using Nanotechnology against MDR Bacterial Uropathogens J. Agric. Food. Chem, 7(2),2015, BIOGRAPHIES MSc Student, Department of Chemistry, Sam Higginbottom Institute of Agriculture Technology & Sciences Allahabad, Uttar Pradesh, INDIA. [12] Singh, B,N,; Singh, B,R,; Prakash, D,; Sarma, B,K,; Singh, H,B., Antioxidant and anti quorum sensing activities of green pod of Acacia nilotica J Food Chem Toxicol, 47,2009a, [13] Huh, A,J,; Kwon, Y,J., Nanoantibiotics : a new paradigm for treating infectious diseases using nanomaterials in the antibiotics resistant era J. Controlled Release, 156,2011, Assistant Professor, Department of Chemistry, Sam Higginbottom Institute of Agriculture Technology & Sciences Allahabad, Uttar Pradesh, INDIA. [14] Ramamurthy, C,; Padma, M,; Samadanam, D,; Mareeswaran, R,; Uyavaran, A,; Suresh, K,M,; Premkumar, K,; Thirunavukkarasu,C., The extra cellular synthesis of gold and silver nanoparticles and their free radical scavenging and antibacterial properties Colloids Surf B, 102,2014, [15] Singleton, V,; Orthofer, R,; Lamuela Raventos, R., Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin Ciocalteu reagent. Methods in Enzymology, 299,1999, [16] Makkar, H,P,S,; Blummel, M,; Borowy, N,K,; Becker, K., Gravimetric determination of tannins and their correlations with chemical and protein precipitation methods Journal of Science and Food Agriculture, 61,1993, International Journal of Science, Engineering and Technology