Akao T, Aoyama M, Akao T, Hattori M, Imai Y, Namba T, Tezuka Y, Kikuchi T, Kobashi K
Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Japan.
Biochem Pharmacol. 1990 Jul 15;40(2):291-6. doi: 10.1016/0006-2952(90)90690-m.
18 beta-Glycyrrhetic acid (GA, an aglycone of glycyrrhizin) is converted to 3-oxo-18 beta-glycyrrhetic acid (3-oxoGA) in the presence of NADP+ by rat liver homogenates, but GA was converted in the presence of NADPH to two other metabolites showing lower Rf values on thin-layer chromatography (TLC) than those of GA and 3-oxoGA by postmitochondrial supernatant of rat liver. The GA-metabolizing activity in the presence of NADPH was localized in microsomes, similar to localization of GA-oxidizing activity to 3-oxoGA. The GA-metabolizing activity required NADPH as a cofactor and O2 for full activity and was inhibited with CO, suggesting the hydroxylation reaction of GA by cytochrome P450. Two metabolites (I and II, lower and higher Rf values on TLC, respectively) were purified on preparative TLC. Mass spectral (MS) analyses of II and methyl ester of acetylated I indicated the formation of monohydroxylated metabolites. On the basis of 3H- and 13C-NMR assignments I and II were identified to be 22 alpha- and 24-hydroxy-18 beta-glycyrrhetic acids, respectively. 3-OxoGA and 3-epi-18 beta-glycyrrhetic acid (3-epiGA) seem to be also hydroxylated at C-22 and C-24. A metabolite of 3-oxoGA showing a lower Rf value was also identified as 22 alpha-hydroxy-3-oxo-18 beta-glycyrrhetic acid by MS and 3H- and 13C-NMR spectral analyses. In 22 alpha-hydroxylation the best substrate was 3-oxoGA, followed by GA and 3-epiGA. On the other hand, for 24-hydroxylation the best substrate was GA, then 3-oxoGA, and 3-epiGA in order. However, 18 alpha-glycyrrhetic acid (18 alpha-GA) was a poor substrate for both 22 alpha- and 24-hydroxylation.
18β-甘草次酸（GA，甘草酸的苷元）在大鼠肝脏匀浆存在NADP⁺的情况下转化为3-氧代-18β-甘草次酸（3-氧代GA），但在大鼠肝脏线粒体后上清液存在NADPH的情况下，GA转化为另外两种代谢产物，它们在薄层色谱（TLC）上的Rf值低于GA和3-氧代GA。在存在NADPH的情况下，GA代谢活性定位于微粒体中，这与GA氧化为3-氧代GA的活性定位相似。GA代谢活性需要NADPH作为辅因子且需要O₂以实现完全活性，并被CO抑制，这表明GA被细胞色素P450进行羟基化反应。两种代谢产物（I和II，在TLC上Rf值分别较低和较高）在制备型TLC上被纯化。对II和乙酰化I的甲酯进行质谱（MS）分析表明形成了单羟基化代谢产物。基于³H-和¹³C-NMR归属，I和II分别被鉴定为22α-和24-羟基-18β-甘草次酸。3-氧代GA和3-表-18β-甘草次酸（3-表GA）似乎在C-22和C-24处也被羟基化。通过MS以及³H-和¹³C-NMR光谱分析，一种Rf值较低的3-氧代GA代谢产物也被鉴定为22α-羟基-3-氧代-18β-甘草次酸。在22α-羟基化反应中，最佳底物是3-氧代GA，其次是GA和3-表GA。另一方面，对于24-羟基化反应，最佳底物依次是GA、3-氧代GA和3-表GA。然而，18α-甘草次酸（18α-GA）对于22α-和24-羟基化反应都是较差的底物。
