Biotransformation Of Three Kinds Of Bioactive Natural Compounds-bufadienolides, Curcumins And Pseudolaric Acid B

Biotransformation of three kinds of bioactive natural compounds-bufadienolides, curcumins and pseudolaric acid B were carried out. Twenty-nine metabolites were isolated, and twenty-eight of them were identified on the basis of their spectroscopic data. Among them, twelve metabolites were new compounds. In vivo metabolism research of pseudolaric acid B was also carried out.The biotransformation of bufotalin (4), telocinobufagin (6) and gamabufotalin (7) by cell suspension cultures of Saussurea involucrate yielded thirteen metabolites, and twelve of them were identified as 3-epi-bufotalin (11),3-epi-desacetylbufotalin (12), 33-epi-bufotalin 3-O-β-D-glucoside (13),1β-hydroxybufotalin (14), and 5(3-hydroxybufotalin (15); 3-oxo-scillarenin (16),3-oxo-bufalin (17), 3-epi-telocinobufagin (18) and bufalin (19); 3-epi-gamabufotalin (20), 3-oxo-gamabufotalin (21), and 3-oxo-△4-derivative of gamabufotalin (22). Among them,11,12,13,14,20 and 22 were new compounds. The time-course study of the biotransformation of 3 with cultured S. involucrate cells was carried out and a possible biotransformation pathway was proposed.Alternaria alternata AS 3.4578 could metabolize arenobufagin (8) and cinobufotalin (5) actively. Seven products were isolated from the cultures by repeated silica gel column chromatography. On the basis of their spectroscopic data, their structures were identified as 3-oxo-arenobufagin (24),3-oxo-ψ-bufarenogin (25), andψ-bufarenogin (26); 3-oxo-△4-derivative of cinobufotalin (27),3-oxo-cinobufotalin (28),12(3-hydroxy cinobufotalin (29), and desacetylcinobufotalin (30), respectively. Among them,24,25 and 29 were new compounds.The in vitro cytotoxicity of the biotransformed products against HepG2,MCF-7 cell lines were evaluated by MTT method. Most of them showed strong inhibitory activities.After 6 days of incubation, the biotransformation of curcumin I (31) by Rhizopus chinensis IFFI 3043 produced two metabolites. Their structures were elucidated as curcumin 4'-O-β-D-glucoside (32) and hexahydrocurcumin (33) on the basis of 1H NMR,13C NMR, and MS spectroscopic analyses. The biotransformation time-course of curcumin in R. chinensis was studied for 24 h. It showed that curcumin I could be rapidly metabolized after only 8 h incubation. Curcumin 4'-O-β-D-glucoside (2) was the predominant product, and its productivity was 57%, calculated on the basis of peak areas. In order to fully understand the metabolic properties of filamentous fungi on curcuminⅠ, minor products in three most capable fungal strains, including Rhizopus chinensis, Absidia coerulea, and Cunninghamella elegans were analyzed by liquid chromatography coupled with tandem mass spectrometry (LC/MSn). A total of 15 curcuminⅠderivatives (1a-1m,2,3) were identified by analyzing their UV and MS spectral data. Among them, three compounds (1a, 1c, 1g) were reported for the first time. A total of eleven strains of fungi were screened for the biotransformation of curcuminⅡandⅢ, but unfortunately, few strains of fungi could metabolized them, with low productivity. The qualitative analysis of the biotransformation of curcuminⅡby Rhizopus chinensis IFFI 3043 and curcuminⅢby Aspergillus niger AS 3.795 were carried out using LC/MSn, respectively.A preparative scale biotransformation of pseudolaric acid B was conducted with Chaetomium globosum IFFI 02445. After 6 days of incubation of compound 36, seven pure products were isolated from the culture supernatant, and six of them were respectively identified as pseudolaric acid B 18-oyl-L-alanine (37), Pseudolaric acid I 18-oyl-glycine (38), pseudolaric acid B 18-oyl-glycine (39), Pseudolaric acid I (40), pseudolaric acid F (41), and pseudolaric acid B 18-oyl-serine (42) on the basis of their NMR and MS spectroscopy. Among these compounds,38,39,40 and 43 were new ones. The main reaction of this biotransformation is the amino acid conjugation. During our research on its antifungal mechanism, we found that 36 did not kill all of the filamentous fungi, but only inhibited their growth. It revealed that structural modification through biotransformation was a detoxification mechanism for fungi to resist antifungal drugs.The fragmentation behaviors of ten diterpenoids were investigated by LC-MS" in negative ion mode. In vivo metabolism of pseudolaric acid B (36) was carried out to help clarify the in vivo process of 36. Altogether 6 metabolites were reasonably characterized in rat plasma, bile, urine and feces samples by HPLC-MSn method, including the phaseⅠ(demethylation) and phaseⅡ(glucuronidation) metabolites. The phaseⅡmetabolites mainly eliminated via bile. The result that only metabolite PC-6 was eliminated via bile feces revealed the existence of hepato-enteric circulation, which can keep the resident time of the medicine longer.