Studies On The Antitumor Constituents And Biotransformation Of Secondary Metabolites Produced By Marine-derived Fungi

Chemical diversity of metabolites from marine fungi made a prominent contribution to the lead discovery of antitumor compounds. A study on fungi from different marine origins was carried out to explore chemical diversity and search for antitumor metabolites. Studies included screening for microbial strains with antitumor activities; fermentation studies; isolation, purification and structural elucidation of metabolites; evaluation of antitumor activities for pure compounds; microbial biotransformation of active compounds.Fifteen antitumor-active fungal strains were screened out of ninety microbial strains isolated from 11 marine animal samples with different origins by new gradated-combinatorial screening method. After choosing appropriate fermentation conditions, two active strains, Rhizopus sp. 2-PDA-61 and Aspergiilus fumigatus WFZ-25 were fermented, respectively. Furthermore, Spicaria elegans KLA03 originated from marine sediment, which mainly produced cytochalasins, was selected for biotransformation study of cytochalasin.The ethyl acetate extracts of 2-PDA-61 and WFZ-25 were, respectively, separated by modern chromatographic methods, and then seventy compounds were isolated and purified from them. Sixty (1-60) of seventy compounds were identified on the basis of spectroscopic methods and their chemical and physical properties, among them, there were twenty-three new compounds and one new natural products (43). Of the new compounds, there were six sterols (1-6), seven furan derivates (12-18), eight fumitremorgin-type alkaloids (30-37), and two spiroγ-lactams (51-52). New compound 1 was one of rare 14βsterols and first reported from microorganism, and new compounds 30-34 were rare spiro-oxindole fumitremorgin-type alkaloids from nature. The known compounds were identified as five sterols (7-11), thirteen fumitremorgin-type alkaloids (38-50), one furan derivate (19), one spiroγ-lactam (53), three diketopiperazine alkaloids derived from phenylalanine and serine (55-57), three quinazoline alkaloids (58-60), two cyclepeptides (20-21), five benzol derivatives (22-25, 54), two nucleosides (26-27), one cerebroside (28), and one glyceride (29), respectively. Moreover, possible biosynthetic pathways of sterols and funitremorgin-type alkaloids were postulated in the paper.The biotransformation study of cytochalasin in fungus Spicaria elegans KLA03 was carried out by adding different amino acids in peter culture medium, the results showed: a. cytochalasins produced by fungus KLA03 were increased with static culture method; b. D-Trp took effects on the growth of KLA03, and ethyl acetate extracts of which gave six cytochalasins, including three new (61-63). All six cytochalasins were characterized by their 6, 7-epoxyl structure, which maybe due to the improved active of cytochrome P450 enzymes; c. D-Trp were degradated by fungus KLA03 and converted into several indole derivates, which was firstly reported in marine-derived fungi.Forty-six compounds were evaluated for their cytotoxicities against several cancer cell lines such as P388, HL-60, A549, BEL-7402, etc, by the MTT or SRB method. The results indicated that ten of them showed significant cytotoxicities against different cancer cell lines and others were weak. Among them, sterol compounds 1, 3-4 showed significant inhibitory activities against P388, HL-60, A549, and BEL-7402 cell lines respectively; cyclepeptide 20-21 exhibited moderate activities against P388 cell lines; fumitremorgin alkaloids 33-35 showed significant inhibitory activities against MOLT-4, HL-60, A549, and BEL-7402 cell lines; cytochalasins 61, 64 exhibited moderate activities against A549 cell lines (IC50 8.2, 3.1μM). Structure and active relations of sterols and fumitremorgin alkaloids suggested thatα,β-unsaturated 7-ketone sterols (1-5, 7) were more active than 5, 8- epidioxysterols (6, 8-10) against cancer cell lines, and 15-OH in sterols 3-4 and 24-OH in compounds 34-35 and 25-hydroperoxyl group in compounds 33 significantly contributed to antitumor activity. Summarily, this work obtained fifteen active fungi from marine animals and seventy-three compounds, including twenty-six new compounds and one new natural product, from three active marine-derived fungi. Furthermore, possible biosynthetic pathways of sterols and funitremorgin-type alkaloids were postulated and forty-six compounds were evaluated for their antitumor activity, ten of which showed significant activity. Moreover, D-Trp was degradated in fungus KLA03 and converted into several indole derivates, which led to six 6, 7-epoxyl cytochalasins. Based on the results of this research, we could do further more.