| Yin-zhi-huang,a heat-clearing and detoxifying multi-herb formula,has significant pharmacogical activities in eliminating jaundice and lowering alanine aminotransferase. It was used widely in clinical practice for the treatment of neonatal jaundice, acute or chronic hepatitis and even other severe hepatitis before. But applications of Yin-zhi-huang injection was limited because of more and more allergic reactions reported in recent years. The chemical composition is often metabolized by drug-metabolizing enzymes. This process will induce or inhibit the enzyme and affect the metabolism of other drugs which called drug-drug interactions. Uridine diphosphate glucuronosyltransferases(UGTs)located in the endoplasmic reticulum is one of the most common phase II drug-metabolizing enzymes in vertebrates. They can catalyze uridine5’-diphosphate glucuronic acid(UDPGA) in combination with a variety of lipophilic substrates and the glucuronidation reaction is an important detoxication way for various xenobiotics and endogenous compounds.The chemical fingerprint profile of Yinzhihuang was previously established in our lab and many kinds of chemical composition were identified and quantified. They are phenylpropanoids, flavonoids and their glycosides, anthraquinones, iridoid glycosides, which may affect the UGT-mediated metabolic reactions. Therefore, the identification of theinteractions of Yin-zhi-huang components and UGTs will be of great importance in studying the safety issues about Chinese traditional medicine Yinzhihuang. In this thesis, we explored the interactions of active ingredients of Yinzhihuang medicine and UGT isoforms using in vitro method. The major contents include:1) Nine probe substrates including estradiol, chenodeoxycholic acid,trifluoperazine, serotonin, propofol, zidovudine, nicotine, S-oxazepam and testosterone were selected and a validated assay was established to evaluate simultaneously the activities of nine UGT isoforms including1A1, 1A3, 1A4, 1A6, 1A9, 2B7, 2B10, 2B15 and 2B17 in human liver microsomes using LC-MS/MS method. Seven kinds of metabolites were analyzed in one injection for chromatographric measurement.Additionally, by comparing the ion signal of six substrates and their corresponding metabolites, we found that quantifying a metabolite with the standard curve of its substrate is not feasible.2) Micosomal incubation conditions including the concentrations of alamethicin, D-saccharic acid 1,4-lactone and magnesium chloride as well as the buffer solution p H were optimized. By using the optimal in vitro incubation system, 11 ingredients in Yinzhihuang injection incluing geniposidic acid, chlorogenic acid, epicatechin, geniposide, luteolin,quercetin, endothelin, wogonoside, scoparone, emodin and rhubarb phenol were preliminary screened for inhibiting the nine UGT isoforms. Significant inhibitions were found by epicatechin, quercetin and wogonoside. Further studies indicated that quercetin and wogonoside showed a non-competitive inhibition on UGT1A4(trifluoperazine glucuronidation) with inhibitiory parameter(Ki) of 11.6 μM, and UGT1A1(β-estradiol glucuronidation) with Ki of 59.8 μM, respectively.3) Yin-zhi-huang herb ingredients containing hydroxyl, carboxyl compounds were incubated with human liver microsomes for screening of the potential substrate of UGTs. We discussed the glucuronidation stereoselectivity of natural products catalyzed by UGT enzyme in human liver. The glucuronidation of luteolin-7-glucoside was first discovered and the contributions of the nine UGT isozymes were compared, in which UGT1A1 was found the most important isozyme in liver microsomes for this glucuronidation reaction. |
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