Chemicals
All reagents used were of analytical grade, and solvents were of spectroscopic grade; methanol, ethanol, dichloromethane, and ethyl acetate HPLC grade (Fischer Chemical, UK). All other media components, analytical grade chemicals, and reagents were obtained from Sigma Chemical Co., USA, and Qualikemes Fine Chem Pvt. Ltd., India.
Sampling
Samples were collected during 2011, between June and December 2011 from soil surroundings of pyramid area. Samples were collected, kept in sterile tubes in refrigerator until processed in laboratory.
Isolation of streptomycetes
Isolation of streptomycetes from soil samples has been done using the serial dilution method of Hayakawa and Nonomura (1987). Suspensions were plated for isolation of streptomycetes. Petri dishes were prepared 1 day before plating and incubated at room temperature overnight to eliminate films of moisture on the agar surface; 0.1 ml inocula of the proper dilution was placed on each plate and spread with a sterile glass rod using starch nitrate agar medium for isolation of streptomycetes. This medium contains (g/l) starch, 20.0; KNO3, 2.0; K2HPO4, 1.0; MgSO4.7H2O, 0.5; NaCl, 0.5; FeSO4.7H2O, 0.01; CaCO3, 3.0; agar, 20.0; and distilled water 1000 ml. The pH was adjusted to 7.2. The inoculated plates were incubated at 28 °C and examined for streptomycete growth after 7–10 days. The isolation of streptomycetes based on their special morphological characteristics (deep sitting colonies, sporulation, characteristic color, etc.) The plates that showed countable single colonies were selected and purified by streak plate technique.
Molecular identification of Streptomyces isolate P4
Streptomyces isolate (P4) was identified according to a molecular biological protocol by DNA isolation, amplification (PCR), and sequencing of the ITS region. The primers 27F (AGAGTTTGA TCM TGG CTC Ag) and 1492R (TAC GGY TAC CTT GTTACG ACT T) were used at PCR. The purification of the PCR products was carried to remove unincorporated PCR primers and dNTPs from PCR products by using Montage PCR Clean up kit (Millipore). Sequencing was performed by using Big Dye terminator cycle sequencing kit (Applied BioSystems, USA). Sequencing products were resolved on an Applied Biosystems model 3730XL automated DNA sequencing system (Applied BioSystems, USA).
Extraction of crude secondary metabolite from actinomycetes
Spore suspensions of the candidate actinomycete strains were inoculated in 1 L Erlenmeyer flasks each containing 250 ml ISP2 broth medium of the following constituents (g/l): yeast extract, 2.0; malt extract, 1.0; and dextrose, 0.5. The flasks were incubated on shaker at 150 rpm rotation at 30 °C for 10 days. The cultures were centrifuged at 4 °C, 5000 rpm for 30 min. The culture supernatant was extracted with 500 ml ethyl acetate and concentrated in rotary vacuum (Augustine et al. 2005).
Determination of antidermatophytic activity of crude extract from actinomycetes
The test fungal cultures Trichophyton mentagrophytes (RCMB 09285), Microsporum canis (RCMB 07321), and Microsporum gypseum (RCMB 07336) obtained from Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University. The antidermatophytic activity of ethyl acetate extract was tested using the agar well diffusion method (Tepe et al., 2004), 100 μl of the actinomycetes broth culture was placed in the wells made with a sterile cork borer on Sabouraud dextrose agar plates (pH 5.6) seeded with the test fungal cultures Trichophyton mentagrophytes (RCMB 09285), Microsporum canis (RCMB 07321), and Microsporum gypseum (RCMB 07336). The plates were incubated at 28 °C and observed for antibiosis after 3–4 days (Augustine et al. 2005). Amphotericin B was used as controls. The Sabouraud dextrose agar (Fluka) medium contains (g/l) peptone extract, 1.0; dextrose, 0.5; and agar, 20.0. The pH was adjusted to 5.6.
Determination of antimicrobial activity
The obtained extract was dissolved in 10% dichloromethane (CH2Cl2) in methanol (MeOH) at a concentration of 1 mg/ml. Aliquots of 50 μl were soaked on filter paper discs (5 mm, Whatman no.1 filter paper) (Bauer et al. 1966) and dried at room temperature under sterilized conditions. The paper discs were placed on agar plates seeded with test microbes and incubated for 24 h at the appropriate temperature of each test organism. Both bacterial and yeast microbes were grown on nutrient agar medium. The fungal strain was on other hand grown on Czapek-Dox agar medium (DSMZ130). The culture of each microorganism was diluted by sterile distilled water to 107to 108 CFU/ml. The inoculated agar plates were first put in the refrigerator for 2 h and then incubated for 24 h (yeast and bacteria) at 37 °C (bacteria) and 48 h (fungi) at 30 °C. After incubation, the diameter of inhibition zones was measured against a wide range of test microorganisms comprising Gram-positive bacteria (Bacillus subtilis ATCC6633 and Staphylococcus aureus ATCC6538-P), Gram-negative bacteria (Escherichia coli ATCC14169 and Pseudomonas areuginosa ATCC27853), yeasts (Candida albicans ATCC10231 and Saccharomyces cerevisiae ATCC9080), and the fungus (Aspergillus niger NRRLA-326). All test microbes were obtained from the culture collection center, Microbial Chemistry Department, National Research Center, Egypt.
Evaluation of the antitumor activity against Ehrlich cell
This test was performed using in vitro assay. Viability of tumor percentages of tumor cells which was measured by modified cytotoxic Trypan blue exclusion technique of Bennett et al. (1976). Female Swiss albino mice were kept under healthy environmental and nutritional conditions for 2 weeks then injected by Ehrlich Ascites carcinoma cells (EACC).
Ehrlich ascites carcinoma cells were isolated from previously transplanted mice after 7 days of transplantation using a sterilized syringe and obtained from Dr. Mohamed El-awady, Microbial Biotechnology Department, National Research Center. The cells were used immediately for viability study and could be diluted by phosphate buffer solution (PBS) if needed. The number of tumor cells/milliliter was calculated by using the appropriate microscope technique used culture medium which prepared using Roswel Park Mark Institute 1640 (RPMI 1640) media supplemented with 10% fetal bovine serum and 10% l-glutamine.
The viability percentages of tumor cells were measured after incubation with the examined sample as well as PBS as control. Two milliliters of Ehrlich cells (1.6 × 106 cells/ml) were added to 1 ml sample. Then, the tubes were incubated under an atmosphere of 5% carbon dioxide at 37 °C for 72 h. The living Ehrlich cells at the end of the 72-h incubation period were determined by a colorimetric assay based on Trypan blue solution prepared by Trypan blue (0.4%) which was dissolved in 100-ml distilled water and then kept in brown closed glass bottle. Ten microliters of trypan blue solution added to 10 μl of sample as well as control with 80 μl PBS solution and were mixed for count living cells. The number of living cells was calculated using hemocytometer slide, survival cells appeared as unstained bodies while non-viable cells were stained blue color. The in vitro results were expressed as the inhibition ratio of tumor cell proliferation calculated as:
$$ \mathrm{The}\ \mathrm{inhibition}\ \mathrm{ratio}\ \mathrm{of}\ \mathrm{tumor}\ \mathrm{cell}\ \mathrm{proliferation}\ \left(\%\right)=\left[\left(A-B\right)/A\right]\times 100 $$
where A and B are the average numbers of viable tumor cells of the control and the samples, respectively.
Scale-up fermentation and extraction
Streptomyces sp. MER4, which provide higher antidermatophyte activity, was used for scale-up fermentation on ISP2 broth medium. One-liter-volume Erlenmeyer flasks each containing 250 ml of ISP2 medium (Pridham et al. 1956-1957) were inoculated with 5 disks of agar from well-grown subculture of this strain. The flasks were incubated at 28 °C on rotary shaker (150 rpm) and harvested after 7 days. After centrifugation, the supernatant of each flask containing extracellular metabolites was extracted by ethyl acetate as mentioned above. In this study, ethyl acetate was selected for further secondary metabolite purification and isolation. Ethyl acetate extracts were concentrated in vacuo tell dryness, yielding 1.9 g of a reddish brown crude extract.
Purification and structure elucidation of bioactive compounds
Sephadex LH-20 column was used for the separation of different extract components. The components were separated and distributed on the stationary phase according to their molecular size. Solvent extraction and partitioning is a method to separate compounds based on their relative solubilities in two different immiscible liquids, usually water and an organic solvent. Characterization and structure elucidate for the purified compound have been established using modern spectroscopic techniques including NMR, 13C, and LCMS.
