| Steroidal drugs are the second largest category of drugs only lower than antibiotics. Diosgenin, as rich plant resources, which are widely applied to synthesize steroid drugs have been noted. So far, diosgnenin have been provided as main start materials to synthesize more than 300 steroidal drugs. However, in recent years, many biological activities of diosgein have been reported and many pharmacologists have afforded great attention to these bioactive results.In this thesis, based on the principle of green chemistry, microbial transformations of diosgenin were carried out with microorganisms which could introduce active groups in active or non-activated centers of steroids in order to obtain new derivatives of potential biological activities. Thus, the tendency of finding diosgenin derivatives by fungi which possessed the ability to keep the carbon skeleton of diosgenin has been noted. In this study, the main contents and results are concluded as follow:1. Screening experimentsScreening scale experiments showed that both Coriolus versicolor and Cunninghamella echinulata which are screened from 34 microorganisms including saprophytic fungi, plant pathogens and plant endophytes, have abilities to transform diosgenin into its polar derivatives as determined by TLC. Next, preparative scale biotransformations of diosgenin were carried out by C .versicolor and C. echinulata, respectively.2. Bioconversion products derived from biotransformation of diosgenin by C .versicolor and C. echinulataNine bioconversion products were isolated from the liquid broth of C. Versicolor and the phosphate buffer of C. echinulata by multiple column chromatography on CC, ODS, Sephadex LH-20 and preparative HPLC, and next identified by various spectroscopic methods (1D NMR, 2D NMR) in combination with HR-MS, MS and IR. Three new 21-hydroxymethylated metobolites, (25R)-spirost-5-ene-3β,7β,21-triol(3-2), (25R)-spirost-5- ene-3β,7α,15β,21-tetraol(3-4) and (25R)-spirost-5-ene-3β,7β,12β,21-tetraol(3-5) and a new 5-ene-11-one steroid, (25R)-spirost-5-ene-3β,7β-diol-11-one(4-1) were characterized, together with three known derivatives, (25R)-spirost-5-ene-3β,7β-diol(3-1 and 4-3), (25R)-spirost-5- ene-3β,7β,11α-triol(4-2) and (25R)-spirost-5-ene-3β,7β,12β-triol(3-3 and 4-4). The NMR data and HR-MS for the known metabolites, 3-1(4-3) and 4-2 are described here for the first time.3. Preliminary exploration of biotransformation pathway of diosgenin Adding 3-1 to culture of C .versicolor showed the compound 3-1 was absolutely resulted in recovery of start material, suggesting that 3-1 was not precusor which was further hydroxylated to other 7-OH bioproducts. Thus, the five biotransformation compounds were obtained from diosgenin as results of hydroxylation, respectively. The results of adding 4-1, 4-2, 4-3 and 4-4 to the culture of C. echinulata, respectively, showing that 4-1 was transformed to 4-2, 4-4 together with three trace components; 4-3 was metabolized to 4-2 and 4-3 as only two products; 4-2 and 4-4 were not transformed, leaded to obtain a preliminary biotransformation pathway of diosgenin.4. Effects of 3-OH andΔ5 moieties on biotransformation of diosgenin Results of feeding tigogenin, acetyldiosgenin and diosgenone to C .versicolor, respectively, showed that no detectable transformation compound was accumulated, suggesting that 3-OH andΔ5 moieties are necessary to biotransformation of diosgenin. Feeding experiments with tigogenin, the 5, 6-dihydro derivative of diosgenin and acetyldiosgenin, respectively, were carried out to C. echinulata. The experimental results showed tigogenin was not metabolized and acetyldiosgenin could be catalayzed to two polar analogues as determined by TLC. The introduction of a 5α-dihydro derivative suppresses the hydroxylation, suggestingΔ5 moiety is necessary to biotransformation of diosgenin. Whereas, introduction of an acetyl group at C-3 have small influence to metabolize diosgenin.5. Determination of enzyme system catalyzing diosgenin by C .versicolor Feeding experiment with cytochrome P450 monooxygenase inhibitors to C .versicolor showed that the reactions catalzing diosgenin were inhibited. It is proved that the enzymes which catalyzed diosgenin were cytochrome P450 monooxygenase system, and not laccase which widely exist in this genus.6. Cytotoxic activity assay The cytotoxic activities against Human malignant glioma cell line U87 of diosgenin, 3-1, 3-2, 3-3, 3-4 were assessed by the XTT method. The five compounds showed certain cytotoxic activities which exhibited the inhibition rates of 20.41 %, 15.2 %, 28.15 %, 1.78 %, 5.24 %, respectively, at a concentration of 100μM/ml. In a word, microbial transformations of diosgenin by Coriolus versicolor and Cunninghamella echinulata, respectively afford nine diosgenin analogues, which were elucidated as three new 21-hydroxymethylated derivatives 3-2, 3-4, 3-5 and a new 5-ene-11-one steroid 4-1, together with three known metabolites 3-1(4-3), 3-3(4-4), 4-2. The data of NMR and HR-MS for compounds 3-1(4-3) and 4-2 are described here for the first time. In addition, we discuss biotransformation pathway of diosgenin by two fungi, respectively. The effects ofΔ5 and free 3-OH moieties on the biotransformation were also determined in this study.
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