蟾蜍内酯类3-表异构体的立体特异性葡萄糖醛酸化研究

Abstract

Bufadienolides, a class of natural products with a unique steroidal skeleton were separated and identified from traditional Chinese medicine Chansu, has been reported with Na+-K+-ATPase inhibitory effects and outstanding antitumor activities. In the current study, the glucuronidation of three pairs of bufadienolide epimers including Cinobufagin (CB), deacetylcinobufagin (DCB) and bufalin (BF), as well as their corresponding 3-epimers (ECB, EDCB and EBF), were investigated by using human liver microsomes (HLM). Interestingly, among these investigated compounds, only EDCB and EBF can form corresponding glucuronidated metabolite when the substrate was incubated with HLM in the presence of UDPGA, while no metabolites of CB, DCB, BF or ECB were detected under the same conditions. The EDCB mono-glucuronide and EBF mono-glucuronide were characterized by using LC-MS/MS, and these two glucuronidated metabolites were also confirmed by hydrolysis with β-glucuronidase. Further investigation on EDCB glucuronidation in human liver microsomes was carried out, due to the detection limitation for quantification of EBF glucuronide. A combination of assays with a panel of recombinant human UGT isoform(s) and chemical inhibition study were used to explore the involved isoform(s) in the EDCB glucuronidation, only UGT2B7 exhibited the catalytic activity toward EDCB glucuronidation, while the inhibitory effect of mefanamic acid (a potent inhibitor of UGT2B7) for EDCB glucuronidation in HLM was similar to its in recombinant UGT2B7. These results revealed that human UGT2B7 was the major isoform involved in the glucuronidation of EDCB. The formation of EDCB glucuronide in pooled human liver microsomes can be well fitted to a substrate inhibition model and the kinetic parameters of EDCB glucuronidation were as follows: Km=48.4 ± 12.6 µM, Ksi=342.2 ± 95.3 µM, and Vmax=1.6 ± 0.3 nmol/min/mg of protein. In summary, this study revealed a stereoselective glucuronidation pathway to bufadienolides, and our results indicated that the structure variants of these epimers including the C-16 ester and the configuration of C-3 hydroxyl may influence the glucuronidation of bufadienolides