The actinomycetes used in this work were isolated from soil samples collected from Vengodu (agricultural field), Thiruvannamalai district, Tamil Nadu, India (Latitude: 12°58′0033″, North; Longitude: 79° 70′5216″, East; Elevation ft/m 228.6/70.0). The actinomycetes isolation was carried out using the plating technique with serial dilution. Aliquots (0.1 ml) of 10−2, 10−3, 10−4, and 10−5 were spread on the starch casein agar (Himedia, Mumbai). To minimize the fungal and bacterial growth, actidione 20 mg/l and nalidixic acid 100 mg/l were added .
The following Gram positive and Gram negative bacteria and fungi were used for the experiment. Gram positive: Staphylococcus aureus MTCC 96, Micrococcus lutues MTCC 106, Bacillus subtilis MTCC 441, Staphylococcus epidermis MTTC 3615, and Methicillin resistance Staphylococcus aureus (MRSA). Gram negative: Klebsiella pneumoniae MTCC 109, Enterobacter aerogens MTCC 111, Vibrio parahaemolyticus MTCC 450, Yersinia enterocolitica MTCC 840, Salmonella typhimurium MTCC 1251, Shigella flexneri MTCC 1457, Proteus vulgaris MTCC 1771, Salmonella typhi-B (SPB). Fungi: Aspergillus flavus (AF), Botrytis cinerea (BC), Candida.krusei (CK), Candida parapsilosis (CP), Malassesia pachydermatis (MP), Scopulariopsis sp. (57), Trichophyton mentagrophytes (66), Trichophyton rubrum (101), Candida albicans (227), Aspergillus niger (1344). The reference bacterial cultures were obtained from the Institute of Microbial Technology (IMTECH), Chandigarh, India-160 036 and all the fungal cultures were obtained from the Department of Microbiology, Christian Medical College, Vellore, Tamil Nadu, India. Bacterial inoculums were prepared by growing cells in Mueller Hinton broth (MHB) (Hi-media) for 24 h at 37°C. The filamentous fungi were grown on Sabouraud dextrose agar (SDA) slants at 28°C for 10 days and the spores were collected using sterile double distilled water and homogenized. Yeast was grown on Sabouraud dextrose broth (SDA) at 28°C for 48 h.
Cross streak method and media Optimization
The antimicrobial activity of actinomycetes isolates was performed by using cross streak method . Antagonism was observed by the inhibition of test organism. Streptomyces lavendulae strain SCA5 was grown on the following media for the production of bioactive compounds in an orbital shaker (150 rpm at 30°C): Antibiotic production media (APM), Fermentation media (FEM), Glucose yeast extract malt media (GLM), M3 media, Modified nutrient glucose media (MNGA), M6 media and Yeast peptone glucose media (YPG). The culture was grown with continuous shaking on a rotary shaker (150 rpm) at 30°C for 10 days. The antimicrobial activity was tested for fermented broth against microbes using .
Cultural and morphological features of SCA5 were characterized by following . Visual observation by light microscopy and Gram-staining were performed for further identification . Biochemical reactions, different temperatures, NaCl concentration, pH level, pigment production and acid or gas production were done following the methods . The total genomic DNA was extracted by using Hipura Streptomyces DNA spin kit-MB 527-20pr from Hi-media, according to the manufacturer’s protocol. The actinomycetes DNA fragments were amplified using Universal primers 16S rRNA and PCR reactions were standardized as follows: initial denaturation at 94°C for 3 min, followed by 35 cycles of 1 min at 94°C, 54°C for 1 min, 72°C for 2 min and a final extension at 72°C for 8–10 min, stop at 4°C for 1 h. The PCR products were stored at 4°C and visualized by electrophoresis. The gel was photographed in gel documentation system. The amplified product was purified and sequenced with two fragments of the 27F (5′AGT TTG ATC CTG GCT CAG 3′) and 1492R (5′ACG GCT ACC TTG TTA CGA CTT 3′) region in both the directions and the sequences obtained were submitted to Genbank. Phylogenetic tree was constructed using the neighbour-joining DNA distance algorithm using software MEGA (version 4.0) .
Cultivation and extraction of antimicrobial metabolites from Streptomyces lavendulae strain SCA5
Well grown slant culture of the Streptomyces lavendulae strain SCA5 was used for the preparation of seed culture. The seed culture was inoculated in 50 ml medium containing the optimized production media and incubated for 10 days in a rotary shaker (150 rpm) at 30°C. The inoculums (10%) were transferred into 150 ml production medium in 250 ml Erlenmeyer flasks and kept for fermentation for ten days. After fermentation, the broth was filtered through blotting paper and the supernatant was separated. The supernatant was extracted twice with ethyl acetate. After separation, the organic phase was dried over Na2SO4 (anhydrous). The extract was then concentrated in a rotary vacuum. The crude extracts were stored at 4°C.
Antibiogram of Streptomyces lavendulae strain SCA5
The antimicrobial activity of the ethyl acetate extract of SCA5 (EA-SCA5) was assayed using the standard Kirby-Bauer disc diffusion method . Petri plates were prepared with 20 ml of sterile Mueller Hinton agar (MHA) (Hi-media, Mumbai). The test cultures were swabbed on the top of the solidified media and allowed to dry for 10 min. The tests were conducted at 2.5 mg/disc concentrations of EA-SCA5. The loaded discs were placed on the surface of the medium and left for 30 min at room temperature for compound diffusion. Negative control was prepared using respective solvent (DMSO). Streptomycin (25 μg/disc) for bacteria and Ketocanozole (30 μg/disc) for fungi was used as positive controls. The plates were incubated over night at 37°C for bacteria and at 28°C for fungi and the zones of inhibition were recorded. Diameters of the zones of inhibition were measured using a zone scale from Hi-media and expressed in millimetres. When the zone of inhibition was 0 to 4 mm it was considered weak activity; when the zone of inhibition was 5 to 10 mm it was considered moderate activity; when the zone of inhibition was 11 to 15 mm it was considered good activity.
Minimal inhibitory concentrations of EA-SCA5
The minimal inhibitory concentrations (MICs) of EA-SCA5, defined as the lowest concentration in the micro titter plate with no growth (i.e., no turbidity) of the inoculated microorganism, was carried out as described by the Clinical and Laboratory Standards Institute (CLSI, 2005) with slight modification . The EA-SCA5 was dissolved in DMSO in a concentration of 1000 μg/ml. The serial two fold dilutions of the EA-SCA5 (1000, 500, 250, 125, 62.5, 31.2, and 15.6 μg/ml) were prepared for MIC tests. MIC tests were carried out in Muller–Hinton Broth for bacteria and Sabouraud dextrose broth for fungi; the organisms were added to 96 well micro titter plate containing 0.1 ml broth. The 3 μl of log phase culture was introduced into respective wells and the final inoculum size was 1×105 cfu/ml. The plates were incubated at 37°C for bacteria and 28°C for fungi. Streptomycin for bacteria and Ketocanozole for fungi were used as positive controls. Negative (water) and solvent controls (DMSO) were also included. 5 μl of the test broth was introduced on plain Mueller Hinton agar for bacteria and Sabouraud dextrose agar plates for fungi to observe the viability of the organism. MIC was determined as the lowest concentration which inhibited complete growth.
DPPH radical scavenging assay
DPPH quenching ability of EA-SCA5 was measured according to . A methanol DPPH solution (0.15%) was mixed with serial dilutions (200–1,000 μg/ml) of EA-SCA5 and after 10 min, the absorbance was read at 515 nm. The radical scavenging activity was expressed as IC50 (μg/ml), (the dose required to cause a 50% inhibition). Vitamin C was used as standard. The ability to scavenge the DPPH radical was calculated by the following formula:
Where A0 is the absorbance of the control at 30 min and A1 is the absorbance of the sample at 30 min. All samples were analyzed in triplicate.
Determination of hydroxyl radical scavenging activity
The hydroxyl radical scavenging assay was performed as described by the method of  with minor changes. All solutions were prepared freshly. Stock solutions of EDTA (1 mM), FeCl3 (10 mM), ascorbic acid (1 mM), H2O2 (10 mM) and deoxyribose (10 mM) were prepared in distilled deionized water. The assay was performed by adding 0.1 ml EDTA, 0.01 ml of FeCl3, 0.1 ml of H2O2, 0.36 ml of deoxyribose, 1.0 ml of extract (200–1,000 μg/ml) each dissolved in distilled water, 0.33 ml of phosphate buffer (50 mM, pH 7.4) and 0.1 ml of ascorbic acid in sequence. The mixture was then incubated at 37°C for 1 h. About 1.0 ml portion of the incubated mixture was mixed with 1.0 ml of (10%) TCA and 1.0 ml of (0.5%) TBA (in 0.025 M NaOH containing 0.025 M NaOH BHA) to develop the pink chromogen and read at 532 nm. The hydroxyl radical scavenging activity of the extract was reported as the percentage of inhibition of deoxyribose degradation and was calculated according to the formula (1).
Nitric oxide radical inhibition assay
Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide; it interacts with oxygen to produce nitrite ions, which can be estimated by the use of Griess Illosvoy reaction . In the present investigation, Griess Illosvoy reagent was modified using naphthylethylenediamine dihydrochloride (0.1% w/v) instead of 1-naphthylamine (5%). The reaction mixture (3 ml) containing sodium nitroprusside (10 mM, 2 ml), phosphate buffer saline (0.5 ml) and EA-SCA5 (200–1000 μg/ml) or standard solution (0.5 ml) was incubated at 25°C for 150 min. After incubation, 0.5 ml of the reaction mixture containing nitrite was pipetted and mixed with 1 ml of sulphanilic acid reagent (0.33% in 20% glacial acetic acid) and allowed to stand for 5 min for completing diazotization. Then, 1 ml of naphthylethylenediamine dihydrochloride (1%) was added, mixed and allowed to stand for 30 min. A pink colored chromophore was formed in diffused light. The absorbance of these solutions was measured at 540 nm against the corresponding blank. Vitamin C was used as standard. The scavenging activity was calculated using the formula (1).
Superoxide scavenging activity
Superoxide scavenging activity of EA-SCA5 was determined by monitoring the competition of those with NBT for the superoxide anion generated by the PMS–NADH system . Superoxide radicals were generated in 1 ml of 20 mM Tris–HCl buffer pH 8.0 containing 0.05 mM nitroblue tetrazolium (NBT), 0.01 mM phenazine methosulphate (PMS), and different concentrations (200–1,000 μg/ml) of EA-SCA5 were pre-incubated for 2 min. The reaction was initiated by the addition of 0.078 mM NADH. Blue chromogen, formed due to NBT reduction, was read at 560 nm. Results were expressed as percentage of inhibition of superoxide radicals.
Male albino Wistar rats bred in the animal house of Entomology Research Institute, weighing 170 ± 5 g were used in the studies. The animals were kept in polypropylene cages, under controlled temperature, humidity and 12/12 light/dark cycles. The animals were fed pellet diet (Pranav Agro Industries Ltd., Maharashtra) and water ad libitum. This study was carried out with prior approval from Institutional Animal Ethical Committee (IAEC-ERI-LC-04/13).
Inhibition of lipid peroxidation in rat liver homogenate
The inhibition effect of EA-SCA5 on lipid peroxidation was determined according to the thiobarbituric acid method . FeCl2–H2O2 was used to induce liver homogenate peroxidation. In this method, 0.2 ml of EA-SCA5 extract (200–1000 μg/ml) was mixed with 1 ml of 1% liver homogenate (each 100 ml homogenate solution contains 1 g rat liver); then 50 μl of FeCl2 (0.5 mM) and H2O2 (0.5 mM) was added. The mixture was incubated at 37°C for 60 min; then 1 ml of trichloroacetic acid (15%) with thiobarbituric acid (0.67%) was added and the mixture was heated in boiling water for 15 min. The absorbance was recorded at 532 nm. Vitamin C was used as positive control. The percentage of inhibition was calculated using the formula (1).
Total phenolic content (TPC)
Total phenolic content of EA-SCA5 was determined according to the Folin-Ciocalteu spectrophotometric method with some modifications . Briefly, 0.1 (200–1,000 μg/ml), EA-SCA5 was mixed with 1.9 ml of distilled water and 1 ml of diluted Folin-Ciocalteu’s phenol reagent and allowed to react for 5 min. Then, 1 ml of 100 g/l Na2CO3 solution was added. After 2 h of reaction at 25°C, the absorbance at 765 nm was determined. The sample was tested in triplicate and a calibration curve with six data points for gallic acid was obtained. The results were compared to gallic acid calibration curve and the total phenolic content was expressed as mg of gallic acid equivalents per gram of extract.
The determination was carried out as described by Oktay, Gu lc_in, and Ku frevioglu . Briefly, different concentrations of EA-SCA5 (200–1000 μg/ml) were mixed with phosphate buffer (2.5 ml, 0.2 mol/l, pH 6.6) and K3Fe (CN)6 (2.5 ml, 1%). The mixtures were incubated for 20 min at 50°C. A portion (2.5 ml) of trichloroacetic acid solution (10%) was added to the mixture, which was then centrifuged at 10 000 g for 10 min. The upper layer of solution (2.5 ml) was mixed with deionized water (2.5 ml) and FeCl3 (0.5 ml, 0.1%), and the absorbance was measured at 700 nm and was compared with standard BHT absorbance.
Cell line maintenance and growth conditions
A549 adenocarcinoma lung cancer cell line was obtained from National Institute of Cell Sciences, Pune and was maintained in complete tissue culture medium DMEM (Dulbecco’s modified eagle’s medium) with 10% Fetal Bovine Serum and 2 mM L-Glutamine, along with antibiotics (about 100 IU/ml of penicillin, 100 μg/ml of streptomycin) with the pH adjusted to 7.2. The cell lines were maintained at 37°C at 5% CO2 in CO2 incubator . Cultures were viewed using an inverted microscope to assess the degree of confluency and the absence of bacterial and fungal contaminants were confirmed.
The cytotoxicity was determined according to the method with some changes . Cells (5000 cells/well) were seeded in 96 well plates containing medium with different concentrations such as 100 μl, 75 μl, 50 μl, 25 μl, 12.5 μl and 6.25 μl. The cells were cultivated at 37°C with 5% CO2 and 95% air in 100% relative humidity. After various durations of cultivation, the solution in the medium was removed. An aliquot of 100 μl of medium containing 1 mg/ml of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) was loaded to the plate. The cells were cultured for 4 h and then the solution in the medium was removed. An aliquot of 100 μl of DMSO was added to the plate, which was shaken until the crystals were dissolved. The cytotoxicity against cancer cells was determined by measuring the absorbance of the converted dye at 570 nm in an ELISA reader. Cytotoxicity of each sample was expressed as IC50 value. The IC50 value is the concentration of test sample that causes 50% inhibition of cell growth, averaged from three replicate experiments.
The TLC profile of the active extract was carried out on Merck silica gel 60F254 pre coated aluminium plates of layer thickness 0.2 mm developing system chloroform : methanol 4:1. The plates were viewed under normal white light and UV light at 366 nm. Derivatization was carried out by 10% alcoholic sulphuric acid reagent, heated at 110°C for 5 minutes and also by exposure to iodine vapours. Phenol was located by 0.1% alcoholic ferric chloride and quinone was located by 0.1% alcoholic NaOH.
The active ethyl acetate crude was subjected to GC-MS analysis on GC-MS- 5975 (AGILENT), column DB 5 ms Agilent, dimension length- 30.0 m, ID- 0.2 mm, flim thickness- 0.25 μm with temperature program- 70–300°C, 10°C/min, injection temperature- 240°C, carrier gas- helium, flowrate- 1.51 ml/min, equipped with GC-MS NIST-II library.
The data for biochemical and physiological parameters were analyzed and expressed as means ± SEM. The IC50 values were calculated from linear regression analysis. Results were processed by computer program, Microsoft Excel (2007).