3.0. Materials and methods

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1 Materials and methods 3.1. Plant materials and preparation of extracts Salacia oblonga plants were collected from Western Ghats, Karnataka, India. S. oblonga (RRCBI 7881) authentication was done by Dr. Siddamallayya. N, Research Officer, Regional Research Institute, Bangalore, India. The aerial and root parts of the plant were washed thoroughly in tap water and shade dried so as to keep the heat sensitive compounds intact. They were then finely ground to a powder in an electric blender. The solvents used for the extraction procedure in the present study were ethyl acetate, chloroform, hexane, petroleum ether, methanol, ethanol and water. The solvents were used alone and also in combination with HCl, so as to observe the effect of lower ph on extraction of phytochemicals. About 100 g of dried and powdered aerial and root parts were extracted using a soxhlet apparatus. The filtrate was concentrated using a rota - vapour at 45 o C and stored at 4 o C in airtight containers for further use.

2 Antimicrobial activity of Salacia oblonga Test organisms and culture conditions The six Gram negative and five gram positive pathogenic microorganisms were used for the present study. Gram Positive (+ve) organisms included Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Bacillus subtilis & Listeria monocytogenes (MTCC 1143) and gram negative (-ve) organisms were Klebsiella pneumoniae, Enterobacter aerogenes (MTCC 111), Enterobacter cloacae, Pseudomonas aeruginosa, Escherichia coli & Salmonella typhimurium (MTCC 98). The MTCC cultures were obtained from the Microbial Type Culture Collection, IMTECH, Chandigarh, India and other test microorganisms were collected as clinical isolates from Global Hospitals, Hyderabad, India. The bacterial cultures were maintained on Muller Hilton Agar (Himedia, India) as slants (Table 3.1) at 4 0 C with a subculture period of 15 days. Each bacterial strain was revived from these stored slants in Muller Hinton Broth (Himedia, India) and cultured at 37 0 C before the antimicrobial activity was carried out.

3 65 Components gm/l Beef infusion solids 4.0 Starch 1.5 Casein hydrolysate 1.75 Agar 15.0 ph 7 Table 3.1. Composition of Mueller Hinton Medium Antimicrobial assay The antimicrobial activity of plant extracts was investigated by agarwell diffusion method (Perez et al., 1990). The Muller-Hilton agar (MHA) was poured onto the Petri plates with an inoculum size of 10 6 colony forming units (cfu)/ml of bacteria. The wells were made in the MHA plates with the help of a borer, of a diameter of 8 mm. On each Petri plate, four wells were made using a sterile cork borer no. 4. The extracts at concentrations of 250 µg, 500 µg, 750 µg and 1000 µg were checked for antimicrobial activity. A broad spectrum antibiotic Amikacin at a concentration of 50 µg was used as a positive control. Solvent alone & solvent with HCl was used as negative control. The extracts at neutral ph 7.0 and acidic ph 5.5 were checked for activity. The plates after treating with plant extracts were incubated overnight at 37 0 C for allowing bacterial growth. After incubation, the zone of inhibitions (diameter)

4 66 around the wells (including the well diameter) was measured & tabulated for each test microorganism. Experiments were performed in triplicate Minimum inhibitory concentration (MIC) and minimum bactericidal Count (MBC) of Salacia oblonga extracts The minimum inhibitory concentration (MIC) was determined by broth dilution method (Chattopadhyay et al., 1998a). Two fold serial dilutions of the crude extracts, with appropriate antibiotic as control were prepared in Mueller Hinton broth (Chattopadhyay et al., 1998b). A direct suspension of microorganisms was prepared in saline water from a 24 h old suspension in Mueller Hinton broth. The turbidity of the suspension was adjusted to match 0.5 McFarland standard (McFarland, 1907) using a spectrophotometer (Shimadzu UV 2450 UV Vis Spectrophotometer) at 600 nm, which corresponds to 2.4 X 10 8 cfu/ml. McFarland Standard No % Barium chloride (ml) % Sulfuric acid (ml) 9.95 Approx. cell density (1X10 8 CFU/ml) 1.5 % Transmittance 74.3 Absorbance (at 600nm) Table 3.2 : McFarland Standard (0.5%) composition

5 67 For broth dilution tests, 0.1 ml of standardized suspension of bacteria (10 8 cfu/ml) was added to each tube at a final concentration of mg/ml >5.00 mg/ml, and incubated at 37 0 C. The lowest concentration of the tube which did not show any visible growth after the macroscopic evaluation was considered as the MIC. The dilutions, starting from the tube that did not show any visible growth, were streaked onto Mueller Hinton agar plates. These plates were then incubated overnight at 37 0 C and observed for visible growth. The tube containing the lowest concentration of the extract, which when streaked onto the plate, did not show any visible growth after 24 h, was considered as the minimum bactericidal count (MBC). All the assays were performed in triplicates. Statistical analysis Data were calculated as means ± standard deviations in triplicate. The data were compared by least significant difference test using Statistical Analysis System (ver.9.1)

6 Phytochemical analysis of Salacia oblonga Analysis by GC-MS (Gas Chromatography and Mass spectrum) For GC - MS analysis, the samples were injected into a HP - 5MS capillary column (30 m length x 250 µm dia. x 0.25 µm film thickness), Agilent Technologies, USA GCMS model, consisting of 6890 N Gas Chromatograph coupled with 5,973 insert MSD [Mass Selective Detector]. The injector was set at C and the detector at C. The stepped temperature program-was as follows: held at 50 0 C for 2 min, then, from 50 to C at the rate of 10 0 C/min, held for 5 min. The total running time was of 30 min. The GC-MS interface temperature was at C. The injection volume was 1 µl. The solvent delay was 3 min and was injected in a split less mode. The MS scan range was from 35 6,000 Da. by comparing their mass spectra (MS) and retention indices (RI) with Wiley library literature data and spectra database (Adams, 1995; Vaughan and Berhow, 2005).

7 Silica gel chromatography In this chromatography, hexane 200 ml was run on the wet column then, acidic ethyl acetate root extract (100 mg) packed in silica column (mesh 95). This mixture was separated by column chromatography by using a silica gel column. The mobile phase consisted of hexane (95 ml) and ethyl acetate (5 ml). After the mobile phase run was completed to colored impurities were removed and organic compounds were separated into different fractions Analysis by LC-MS (Liquid Chromatography and Mass spectrum) LC-MS analysis was performed on Acquity BEH-C18 column (50 X 2.1 mm) 1.7 µm (Waters Corp. Milford, MA, USA) and chromatography was performed using 0.1% ammonium acetate [(A) : 0.1% HCOOH (Aq)] & 0.1% acetonitrile [(B) : 0.1% HCOOH (ACN)] as the mobile phases. Separation was accomplished by stepwise gradients from T /% B: 0/10, 15/100, 2.9/100, 3/10 and the flow rate provided 0.5 ml/min with diluent : ACN.