Studies On Biotransformation Of 2 Steroidal Compounds By Marine Microorganisms

Steroids were a class of compounds which were characterised by a gonane skeleton of four condensed cycloalkane rings.Steroidal compounds were structurally diverse,many of them with a wide range of biological functions and were used as drugs in different medical fields,mainly for anti-inflammatory,anti-allergic,anti-shock,and antitoxicity especially for the treatment of breast and prostate cancer.Epoxy progesterone and phytosterols belonged to steroidal compounds,their conversion products were widely used as chemical synthesis intermediates.In this work,epoxy progesterone and phytosterols were biotransformated by marine microorganism.Biotransformation products were isolated,and identified as well their anti-tumor activity were evaluated.The conclusions were as follow:Firstly,54 strains of microbes were isolated from water of East China Sea by oligotrophic plate with epoxy progesterone,with other 15 marine microorganisms kept in our lab,were used to transform epoxy progesterone.three active strains were found having the activity of biological transformation.By morphology,ITS segment gene blast and phylogenetic analysis,these three marine fungi were determined as:Rhizopus oryzae Zf-5,Penicillium chrysogenum MNP07010101,and Cladosporium sphaerospermum Zf-35.This is the first report of biotransformation of EP with R.oryzae,P.chrysogenum and C.Sphaerospermum by our research group.Secondly,six biotransformed active compounds were identified as:11?-hydroxy-16?,17?-epoxy-pegna-4-ene-3,20-dione?1?,6?,11?-hyd-roxy-16?,17?-epoxy-pegna-4-ene-3,20-dione?2?,1,4-diene-16?,17?-epoxy-pegna-4-ene-3,20-dione?3?,11?-hydroxy-6-methyl-1,4-diene-16?,17?-epoxy-pegna-4-ene-3,20-dione?4?,7?-hydroxy-16?,17?-epoxy-pegna-4-ene-3,20-dione?5?,and 6?-hydroxy-16?,17?-epoxy-pegna-4-ene-3,20-dione?6?.The biological activity showed that compound 4was of certain anti-tumor activity against PC-3 with IC₅₀ of 75.7?M.The anti-tumor effect of other compounds were not strong,but better than epoxy progesterone.Thirdly,we optimized the biotransformation process of Rhizopus oryzae Zf-5 firstly.The result showed that the optimal process was as follow:glucose 30 g/L and peptone 12 g/L with the initial pH 7 of medium,the fermentation temperature was 34?,the fermentation time was 84 h,the concentration of substrate was 3 g/L.Compared to the initial bioconversion rate 37.2%,it was increased by 2.5 times.Finally,23 strains of fungi and bacteria were isolated from sea water of East China Sea by oligotrophic plate with phytosterols,with other 15marine microorganisms kept in our lab.Finally,we select mycobacteria for the further study.By the separation and purification of the biotransformative extract,androstadienedione?7?,androstenedione?8?,20-hydroxypregna-4-en-3-one?9?,20-hydroxymethylpregna-1,4-dien-3-one?10?,20-hydroxymethylpregna-4-dien-3-one?11?were isolated.The active experiment showed that compounds 10 and 11 have strong activity against Hela cell,with IC₅₀ 9.7 and 19.3?M,seperately.Compound 11 was evaluated for the first time for its cytotoxicity against the Hela cancer cell lines.Result showed that compounds 10 and 11couldn't induce apoptosis of cancer cell,as for the real mechanism still need to be further explored.