OSMAC Approach And Application In The Research Of Marine-derived Fungi Spicaria Elegans KLA03

Microbial secondary metabolites have a wide range of biological activities due to their structural diversity and have been proved a major source for drug lead compounds. However, the traditional method of a single culture restricts the metabolic pathways of microorganisms and as a result many metabolites can not be formed. So, the study of using the OSMAC approach on a fungus Spicaria elegans KLA03 which yielded a series of cytochalasins was performed to activate the silent biosynthetic pathway and produce more active cytochalasins analogs. Studies included comparing and optimizing for microbial secondary metabolites with differernt conditions; isolation, purification and structural elucidation of metabolites; evaluation of antitumor activities for pure compounds.Our previous work had reported the isolation of new skeleton cytochalasins from the marine-derived fungus Spicaria elegans KLA03, which showed obviously cytotoxicity and PTK inhibition activity and might be developed as leading compounds for antitumor drug discovery. Our study firstly began with altering culture conditions. By selecting different culture mediums, the secondary metabolites of KLA03 were drastically shifted when the nitrogen source of the initial medium was replaced to NH4Cl from glutamate whether in static or shake culture conditions. Ten compounds(1-10) were identified, among them, there were three new cytochalasins (1-3), and one precursor of trichodermamide B(10), the physico-chemical property and spectral characters of which were firstly reported. Then KLA03 were cultured with shake in the same medium, and three unusual isobenzofuran dimmer (11-13) were isolated, moreover, compound 11 was an exceptional spiro compound.Secondly, the strain KLA03 were cultured in the initial medium made up with distilled water instead of tap water resulted in the isolation of nine cytochalasins (15-23), and five of them were new compounds (15-19); furthermore, these compounds were almost 6,7-deoxidation cytochalasins, which had more similarity with the present study of adding cytochrome P-450 inhibitor in this strain, and the relation-between these two experiments were discussed. Finally, the application of OSMAC method in fungus Spicaria elegans KLA03 was carried out by adding the precursor of L-Trp in order to compare with the present results of adding D-Trp. L-Trp took effects on the growth of KLA03, which resulted in one new cytochalasins (24), one analog of aspochalasin(26), which had not been found in this strain. But L-Trp was not utilized by fungus KLA03 to generate cytochalasins with tryptophan fragment and might be degradated into several Benzene Derivatives from two comounds (29,30).The anti-tumor activities of these compounds are evaluated in vitro by SRB and MTT methods. Among them 4 new cytochalasins (2,16,17,18) with significant cytotoxicities against A-549 cell line, and compound 10 showed a significant cytotoxicity against P388 cell line. For the cytochalsins without the enol ester moiety at C-21, the exocyclic double at C-6 and the C7-OH might be two essential parts for the activity. The further experiments are still in process.In a word, the study of using the OSMAC approach on a fungus Spicaria elegans KLA03 resulted in the isolation of 30 compounds, with 11 new compouds, including nine cytochalasins and two isobenzofuran dimmers, and firtly reported the physico-chemical property and spectral characters of trichodermamide B's precursor (10). The anti-tumor activities of some compounds like cytochalasins or isobenzofuran dimmers were evaluated. The study may not only provide a major source for drug lead compounds, but also supply evidence for the application of OSMAC methods in the research of natural products.