| Steroidal saponins are important bioactive natural products in plants which are mostly found in the plant families Anemarrhena asphodeloides Bunge,Ophiopogon japonicus,Dioscorea nipponica Makino and Paris polyphylla.Steroidal saponins exhibited a wide range of pharmacological activities such as anti-tumer,anti-platelet,immunomodulatory,anti-inflammatory and myocardial preservation etc.Microbial biotransformation has attracted extensive attention in the field of pharmaceutical research in recent years.It has many advantages compared to the traditional chemical methods,such as mild reaction conditions,environmental friendly,high stereo-and regio-selectivity.In order to obtain derivatives with higher activity and less toxicity,microbial transformation of two steroidal saponins protodioscin and pseudoprotodioscin(PPD)has been studied in this dissertation.The pharmacological activities of the transformed products were also explored.25 strains were screened for the transformation of protodioscin and pseudoprotodioscin,among which,Chaetomium olivaceum CGMCC 3.3604 and Botrytis cinerea CGMCC3.3789 had good abilities to transform protodioscin.Gibberella fujikuroi CGMCC 3.4663 had a good ability to transform pseudoprotodioscin.These three fungi were selected for the scaled-up stage.The crude extracts were purified by a series of modern separation methods:silica gel column chromatography,Sephadex LH-20 column chromatography,semi-PHPLC and so on.A series of metabolites were isolated.The microbial biotransformation of protodioscin by olivaceum CGMCC 3.3604 yielded 4 known compounds including 26-O-?-D-glucopyranosyl-22?-methoxy-(20S,25R)-furost-5-ene-3?,26-diol-3-O-?-L-rhamnopyranosyl-(1?4)-?-D-glucopyranoside(1),26-O-?-D-glucopyranosyl-(20S,25R)-furost-5-ene-3?,22?,26-triol-3-O-?-L-rhamnopyra nosyl-(1?4)-?-D-glucopyranoside(2),prosapogenin B of dioscin(3),dioscin(4).The microbial biotransformation of protodioscin by cinerea CGMCC3.3789 yielded a hydrolysis product prosapogenin B of dioscin(3).The microbial biotransformation of pseudoprotodioscin by fujikuroi CGMCC 3.4663 yielded 12 compounds including 26-0-?-D-glucopyranosyl-22a-methoxy-(20S,25R)-furost-5-ene-3?,26-diol-3-O-?-L-rhamnopyranosyl-(1?4)-?-D-glucopyranoside(1),26-O-?-D-glucopyranosyl-(20S,25R)-furost-5-ene-3?,22?,26-triol-3-O-?-L-rhamnopyranosyl-(1?4)-?-D-glucopyranoside(2),prosapogenin B of dioscin(3),26-O-?-D-glucopyran-osyl-20?-methoxyl-25R-furosta-5,22-diene-3?,26-diol-3-O-?-L-rhamnopyranosyl-(1?4)-?-D-gluc opyranoside(5),26-O-?-D-glucopyranosyl-22?-methoxy-25R-furosta-5-ene-3?,20?,26-triol-3-O-?-L-rhamnopyranosyl-(1?4)-(3-D-glucopyranoside(6),26-O-?-D-glucopyran osyl-25R-furosta-5,22-diene-3?,20?,26-triol-3-O-?-L-26-rhamnopyranosyl-(1?4)-?-D-glucopyranoside(7),16?-(4'R-methyl-5'-ol-pentanoxyl)-pregn-5-ene-3?-ol-20-one-3-O-a-L-rhamnopyranosyl-(1?4)-?-D-glucopyranoside(8),pregnan-5,16-diene-20-one-3?-hydroxy-3-O-?-L-rhamnopyranosyl-(1?4)-?-D-glucopyranoside(9),25R-spirost-5-ene-3?,20-diol-3-O-?-L-rhamnopyranosyl-(1?4)-?-D-glucopyranoside(10),25R-spirost-5,20-diene-3?-ol-3-O-a-L-rhamnopyranosyl-(1?4)-?-D-glucopyranoside(11),25R-spirost-5-ene-21?-methyl-3?-ol-3-O-?-L-rhamnopyranosyl-(1?4)-?-D-glucopyranoside(12),25S-spirost-5-ene-3?,20?-diol-3-O-?-L-rhamnopyranosyl-(1?4)-?-D-glucopyran oside(13).Three(8,11,13)of them were new compounds.All metabolites were hydrolysis products.Compounds 8 and 9 were pregnane glycoside.Microbial transformation of the two steroidal saponins were mainly concentrated in C20(22)positions.Glycosyl hydrolyzation,hydroxylation,methoxylation and oxidation were observed.The possible transformation passway were also proposed.The pharmacological activity of the products were screened.Compounds 3,4,5,8,11,13 exhibited cytotoxic activity against HepG2 cells.Compounds 3,4,10,11,13 exhibited cytotoxicity to Hela cells.Compounds 7 and 9 have a certain inhibitory effect on the expression of inflammatory cytokines in RAW264.7 cells induced by LPS.Further studies show that its inhibition on NO secretion may be achieved by inhibiting the expression of iNOS protein in RAW264.7 cells. |
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