IJPRD, 2015; Vol 6(11):January-2015 (070 – 075) International Standard Serial Number 0974 – 9446 -------------------------------------------------------------------------------------------------------------------------------------------------EVALUATION OF ANTIMICROBIAL AND ANTIOXIDANT PROPERTIES OF THE BIOLOGICALLY SYNTHESIZED GOLD NANOPARTICLES OF OCIMUM PILOSUM WILLD Thomas Bennans1*, D K Sathish2, Lulu Maria Thomas3 1 Department of Botany, FMN College, Kollam, TN, India. Department of Botany, University College, Thiruvananthapuram, TN, India 3 Department of Pharmacology, Sree Ramakrishna Institute of Paramedical Sciences, Coimbatore, TN, India 2 ABSTRACT Plant extracts mediated biosynthesis of nanoparticles has been emerging as the current trend in the drug delivery mechanism of pharmacology. The immense potential of nano biotechnology makes it an intensively researched field in modern medicine. Gold nano particles (AuNPs) offer promising therapeutic possibilities due to its higher biocompatibility, high surface reactivity and inhibitory activity against a variety of cellular disorders. The present study involves the free synthesis of stable gold nanoparticles (AuNPs) using the water extracts of Ocimum pilosum and the evaluation of their anti microbial and anti oxidant activity. Visual observation and ultraviolet–visible spectroscopy and SEM were used to characterize the synthesized AuNPs. The absorption maxima was obtained at 545 nm within a time frame of 7200 seconds beyond which the encapsulation of particles showed a stationary phase. These particles were screened for the free radical scavenging activity and anti microbial activity and the results were compared. The in vitro anti microbial activity was carried out by Agar diffusion method and the free radical scavenging activity was determined by DPPH method. The inherent antimicrobial activity of gold metals along with that of plant extracts has yielded a higher level of antimicrobial as well as anti oxidant activity under experimentation. This new biomimetic approach using traditional medicinal plants to synthesize biocompatible antibacterial and free radical scavenging AuNPs could easily be scaled up for additional biomedical applications. Correspondence Author Thomas Bennans Department of Botany, FMN College, Kollam, TN, India Key words: Ocimum pilosum, Gold nano particles, AuNPs, antimicrobial, antioxidant. 70 International Journal of Pharmaceutical Research & Development ISSN: 0974 – 9446 The herb Ocimum pilosum (Willd.), a INTRODUCTION popular garden and ornamental plant, is a member Over the last few years, researchers of the family Lamiaceae. The plant grows in the have been aiming at identifying and validating subtropical regions and is widely distributed plant-derived substances for the treatment of throughout India. All the aerial parts were used as various diseases. Interestingly it is estimated that a single effective prescription in folk medicine for more than 25% of the modern medicines are treatment of cold associated symptoms. directly or indirectly derived from plants. It is worth mentioning that Indian medicinal plants are considered as a vast source of several MATERIALS AND METHODS pharmacologically principles and compounds that Plant material are commonly used as home remedies against The study involves the green synthesis of gold nano multiple ailments (1). Since early 1990s, the use of particles with extracts of Ocimum pilosum leaves. forest products for medicine has been emerging as Ocimum pilosum was collected locally and a vital income generating resource for the confirmed to their identified taxonomic position. development of various social groups; hence, there The method was carried out in three parts viz. The is an increased attention for their long-term plant extract is allowed to mediate the synthesis of sustainability (2). AuNPs, followed by its confirmation and Research is being carried out all over characterisation. It was screened for antimicrobial the world to develop environmentally safe, non and anti oxidant activity toxic and economically viable plant based products for the remedy of various ailments. In this context, Preparation of plant extract. biosynthesis of nanoparticles mediated by plant The plant was washed in running water and blotted extracts has emerged as a promising area for dry. The leaves were selected harvested and shade developing environmentally safe and target specific dried for four days. Dried leaves were then drug delivery method in pharmacological research. powdered using mortar and pestle. Two grams of Depending on the type of particle, the active fine filtered powder was boiled in 100ml distilled substance can be encapsulated or attached to the water for 5 minutes and cooled to room temperature. The filtrate was decanted and filtered surface. This means that even if they dissolve first through whatmann filter paper (pore size poorly in water, they can be transported in an 0.45µm) and then through 0.22µm filters. The aqueous solution, such as blood, and are better filtrate was stored in sterile vials and kept in protected against degradation by enzymes. Besides ambient temperature. acting as a delivery system, in some cases nanoparticles can act as an active substance. Recent in vitro and in vivo studies have revealed Synthesis of gold nanoparticles Synthesis of gold nano particles was carried out by that silver and gold nanoparticles are endowed reacting plant extracts with appropriate aliquots of with innate antiplatelet properties (3). Auric chloride. 1mM Auric chloride was carefully In the present investigation gold weighed and added to 180 ml of deionised water in nanoparticles (AuNPs) are used for the preparation of plant extract coated nanoparticles. The use of 250ml Erlenmeyer flask in dark followed by the addition of 20ml of plant extract (4). The flask was plant extracts in the synthesis of nanoparticles has then incubated in rotary shaker at 160rpm in the several advantages compared to methods relying dark at room temperature. The reaction was on microorganisms as agents. Hence the study aims allowed to cool at room temperature for the at the evaluation of the antioxidant and formation of gold ions. The filtrate of the plant antimicrobial activities of the leaf extracts of extract acts as reducing or capping agent for the Ocimum pilosum in association with gold AuNPs. Characterisation of the encapsulated gold nanoparticles. nano particles was carried out by UV Visible Available online on www.ijprd.com 71 International Journal of Pharmaceutical Research & Development ISSN: 0974 – 9446 spectroscopy and Transmission electron was 100 µg/ml. From stock solution 0.25µl, 0.5µl, microscopic analysis(5). 0.75µl and 1µl were taken in four test tubes and the final volume of each test tube was made up to UV Visible Spectroscopy 3 µl with methanol. Freshly prepared DPPH The AuNPs were characterised using UV-Visble solution( 0.004% w/v) was added into the test spectrophotometer. The scanning range of the tubes containing extract. After 30 minutes the absorbance was taken at 517nm using a sample was 300-800nm. Base line correction of the spectrophotometer. Ascorbic acid was used as the spectrophotometer was carried out using a blank reference standard and the scavenging activity and reference. The UV- Visible absorption spectra of all the samples (concentration) were recorded along IC50 values were calculated. with the resulting data recorded in graphical format. RESULTS AND DISCUSSION UV Spectral analysis Scanning Electron Microscopy The formation of AuNPs using extracts of Ocimum SEM was adopted to detect the formation of pilosum Willd and could be visually observed due AuNPs. Preparation for SEM was done by the to the red brown coloration of the sample. It was method of Salini and Mohankumar (6). It was done confirmed by the spectroscopic study. The colour by fixing the samples in 2.5% glutaraldehyde in exhibited by the nanoparticles was due to the 0.1M cacodylate buffer with pH 7.4 for 6 hours at coherent excitation of all the free electrons within 1-4˚C. The fixed sample was washed four times the conduction band leading to an in- phase thoroughly in cold 0.1 M cacodylate buffer (pH 7.4) oscillation which is known as the Surface Plasmon for 30 minutes. It was then subjected to osmication resonance. Figure 1 demonstrates the absorption by again fixing it in 2 % osmium tetroxide in 0.05M spectrum of auric chloride and there is no cacodylate buffer pH 7.4 overnight in a characteristic absorption peak in between 500 and refrigerator. The osmicated tissues were 550 nm. In order to show the formation of gold dehydrated in a graded ethanolic series embedded nano particles from auric chloride and Ocimum in synthetic resin and used at 60˚C for 24 hours. A pilosum extract it was essential to find their small quantity was taken to load the carbon coated absorbancy alone. Figure 2 shows the absorption copper grids for analysis. Dark and bright filed spectrum of the. Ocimum pilosum leaf extract images of samples were recorded. which clearly indicates the absence of absorption peak in between 500 -550 nm. Figure 3 reveals the Anti microbial activity formation of extract encapsulated gold nano It was detected by agar well diffusion method. The particles. The absorption maxima was obtained at log phase cultures were spread on nutrient agar 545 nm which indicates the encapsulation of medium plates using a sterile cotton swab in order AuNPs. A time scan of 0 to 24 hours was set to find to get a uniform microbial growth on test plates. out the formation of AuNPs. It was found that at Four wells of uniform size were made with a cork 7200 seconds the formation was stand still which resulted in a sigmoid curve. borer in each plate and 25, 50, 75 and 100µl of the encapsulated gold nano particles were pipetted The scanning electron microscopic study directly into the well. The plates were incubated in The scanning electron microscopic study was done upright positions at 37˚ C for 18 to 48 hours. for detecting the formation of AuNPs. The micrographs obtained demonstrate the Anti oxidant activity appearance and distribution of Au NPs at different It was done using DPPH Free radical scavenging magnifications. Figure 4 depicts the clubbed assay of Lee et al., 2003(7). DPPH solution was electron microscopic view of gold nanaoparticles as prepared in methanol. The concentration of the dark bodies because its diameter was 0.5 mm biosynthesized gold nano particles extract solution Available online on www.ijprd.com 72 International Journal of Pharmaceutical Research & Development whereas in figure 5, comparatively larger particles are seen because the AuNPs shown are of the size of 100 nm and 50nm respectively. 0.50 ISSN: 0974 – 9446 ABS 0.45 0.40 0.35 0.30 0.25 0.20 0.15 Figure 5 0.10 0.05 0.00 300 350 400 450 500 550 600 650 700 750 nm 800 700 750 nm 800 Legends Figure 1: Absorption spectrum of auric chloride between 500 and 550 nm. Figure 2 : Absorption spectrum of Ocimum pilosum between 500 and 550 nm. Figure 3 : Absorption spectrum of Ocimum pilosum extract coated AuNPs at 545 nm Figure 4 : Clubbed electron microscopic view of gold nanaoparticles as dark bodies under SEM. Figure 5 : Enlarged view of gold nanaoparticles under SEM. Figure 1 2.0 ABS 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 300 350 400 450 500 550 600 650 Figure 2 ABS 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 s 0 1000 2000 3000 4000 Figure 3 5000 6000 7000 Anti microbial activity Table 1 demonstrates the result of disc diffusion assay technique and it shows that the leaf extract coated gold nano particles have maximum activity against Staphylococcus aureus followed by Escherichia coli. The anti fungal activity was highest against Candida albicans. All concentrations of AuNPs under study showed anti microbial activity. The antibacterial properties of drugs coated gold nanoparticles were higher when compared with the pure drugs. The small size of gold nanoparticles, large surface area and high penetrating power might be the reason for the enhanced activity and hence such nanoparticles could effectively bind to the substrates on the outer membrane and cell membranes of organisms (8, 9). Figure 4 Available online on www.ijprd.com 73 International Journal of Pharmaceutical Research & Development ISSN: 0974 – 9446 Table 1. Inhibitory effect Ocimum pilosum leaf extract mediated AuNPs against pathogenic organisms Zone of inhibition for micro organisms Sl No 1 2 3 4 Sl. No 1 2 3 4 Concentration of AuNPs (µg/ml) 25 50 75 100 Escherichia coli 6.2±1 8.5±0.59 14±2.08 20 ± 0.25 Staphylococcus aureus 7.30 ± 0.57 10.80 ± 0.85 16.25 ± 0.39 24.00 ± 1.70 Candida albicans 3.9 ± 0.88 9.8 ± 0.65 15.9 ± 0.45 21.9 ± 1.65 Table 2: Antioxidant activity of AuNPs of Ocimum pilosum. Concentration of Optical density Optical density IC50 Ascorbate and AuNPs of ascorbate of AuNPs (µg/ml) 25 0.162 0.102 37% 50 0.650 0.312 52% 75 0.890 0.285 67.9% 100 1.050 0.312 70.28% Antioxidant activity Table 2 explains the nature of decrease of concentration of DPPH radical at different concentrations of the AuNPs. It was found that the significant free radical scavenging activity was noted at 100µl AuNPs. The IC50 inhibition concentration at which there was 50% reduction of free radical resulted at 50µl concentration. The substantial decrease in the concentration of DPPH radical was due to the scavenging ability of gold nanoparticles formed by Ocimum pilosum leaf extract. Similar results were obtained where Gold nanoparticles embedded 3,6-dihydroxyflavone individually showed maximum percent of inhibition (72.04 %) at the 100 lg/ml concentration compared to native 3,6-dihydroxyflavone (64.21 %).The inclusion of gold nanoparticle embedded 3,6dihydroxyflavone with other dietary nutrients lutein and selenium methyl selenocysteine further increased maximum inhibition (87.13 %) at the same concentration (10). CONCLUSION The present investigation demonstrates the encapsulated gold nano particles of Ocimum pilosum leaf extract showed typical features of AuNPs lie in range of 540-550 nm. The absorption maxima λ of the sample extract was obtained at 545nm. The fine structural analysis of the AuNPs under SEM provided sufficient data for its confirmation. Considering the practical relevance of the AuNPs for the welfare of mankind, it could be used as a potent antimicrobial and antioxidant agent (11). 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