Studies On Halotolerant Fungal Strains: Isolation, Culture And Secondary Metabolites

Haltolerant microorganisms have been considered an important source of bioactive leading compounds, due to their unique living environment. A study on two"talented strains"of halotolerant microorganisms was carried out to investigate the potential anti-tumor compounds. Studies include screening for microbial strains with antitumor acitvities, fermentation studies, bioassay-guided fractionation, structural elucidation, and preliminary evaluation for anti-tumor activities of new compounds.Using MTT or SRB methods, one halotolerant"talented strains"were screened out of one hundred and seventy four halotolerant microorganisms isolated from hypersaline samples, on the bioassay of K562 cell lines. Two"talented strains"were chosen based on the result of re-screening combining with HPLC and TLC chemical analysis. After choosing appropriate fermentation condition, these strains were fermented, respectively. The whole broths were extracted with ethyl acetate to give ferment extracts, which were subjected to silica gel column chromatography, Sephadex LH-20 and HPLC by bioassay-guided fractionation, respectively. Seventy six compounds were isolated from the two strains. Forty nine compounds (1-49) were isolated from fungus B-17 (Aspergillus variecolor) and twenty seven compounds (50-76) were isolated from fungus THW-18.By means of modern spectral analysis (UV, IR, NMR, MS, CD, X-Ray) and physico-chemical properties, the structures (see Fig. 1) of seventy six pure compounds were respectively determined. Among them there are twenty five new compounds, including three novel alkaloids, variecolortides A-C (1-3), which share an unprecedented'spiro-anthronopyranoid diketopiperazine'structure and were deduced by single-crystal X-ray analysis; fourteen new enchinulin variecolorins A-N (4-17); two novel quinone type compounds variecolorquinones A-B (30-31), a new butyrolactone type compound halobutyrolactone A (49); three new cerebroside halotolerantcerebroside A-C (50-52), one new steroide (22E,24R)-3β,5α- Dihydroxy- 23-methylergosta-7,22-dien-6-one (56) and one new diketopiperazine type compound haloacetylaranotin A (64).These compounds were evaluated for their cytotoxicities against several cancer cell lines such as HL-60, P388, K562, etc, by the MTT or SRB method. The results indicated that all of the new compounds showed cytotoxicities in certain extent. Among them, variecolortides A-C (1-3) showed week bioactivity against K562 (IC50 = 63, 84 and 91μM respectively), variecolorquinones B have significant cytotoxicities against P388 and HL-60 cells (IC50 = 3.7 and 1.3μM respectively) and variecolorquinones B selectively inhibited the proliferation of A-549 cell (IC50 = 3.0μM). Compounds 4-17, 50-52 and 64 exhibited weak cytotoxicities. In addition, all the compounds were evaluated for their DPPH scavenging activity. The results indicated that compounds 1-14, 17-18, 20, 22, 23, 26, 28, 29, 31-45, 67-69 showed antioxidative activity in certain extent.Summarily, this work obtained one"talented strains"from halotolerant microorganisms and seventy six compounds from two fungal strains. Among them, twenty four compounds were identified new. Studies mentioned above provided novel structures for searching leading antitumor compounds and strain resources of great value for further study and development of halotolerant microorganism.