基于超高效液相色谱-质谱联用技术的人参皂苷体外Ⅰ相代谢研究

Study on the Ⅰ Phase Metabolism of Ginsenoside in vitro by Ultra Performance Liquid Chromatography Coupled with Mass Spectrometry

口服药物必须经过胃肠道的吸收代谢等过程才能进入体内发挥疗效,但是药物体内代谢的复杂性使其研究相对困难。因此,建立药物的体外代谢模型研究其代谢产物及代谢规律是简单且必要的实验手段。本研究采用离体肠道菌厌氧培养法对人参皂苷提取物进行体外培养,利用超高效液相色谱-质谱联用法(UPLC/MS)检测分析肠内菌代谢产物。通过比较代谢前后的化学指纹图,共检测到25种三醇型、15种二醇型人参皂苷体外肠内菌代谢物和1种齐墩果酸型体外肠内菌代谢物,这些代谢物主要通过去糖基作用产生,同时包括部分氧化还原反应。实验进一步对人参皂苷的主要肠内菌代谢物(包括Rh_1、Rh_2、Compound K、F1)进行体外肝细胞色素P450酶(CYP450)代谢研究,均检测到氧化还原反应代谢产物。结果表明,人参皂苷类成分在肠道菌群作用下主要产生去糖基化产物,而在CYP450作用下主要产生羟基化等氧化还原反应产物。该结果可为人参皂苷类成分的体内代谢研究提供重要参考。

All the drugs taken orally must undergo the process of absorption and metabolism of gastro-intestinal tract. It is difficult to conduct simply operated experiment in vivo considering the complexity of drug metabolism, such as matrix effect, individual difference and so on. Therefore, it is necessary to establish the model of drug metabolism in vitro and to study the metabolites and rules of metabolism. Drug metabolism is usually divided into 2 phases： Ⅰphase and Ⅱ phase metabolism. Ⅰ phase metabolism was taken via functionalization reactions. This study mainly researched I phase metabolism of ginseng. In vitro biotransformation of ginseng extract by intestinal flora and liver cytochrome （CYP450） in turn was performed. Firstly, ginsenoside extract was in vitro cultured with isolated intestinal bacteria under anaerobic conditions, and the incubation samples were analyzed by ultra-performance liquid chromatography tandem mass spectrometry （UPLC/MS）. And then, the chemical fingerprint before and after metabolism were compared to identify the metabolites. 25 panaxatriol-type saponins metabolites, 15 protopanoxadiol-type saponins metabolites and one oleanane-type saponins metabolites were detected from the incubation samples, among of which were biotransformed mainly through deglycosylation and a few through oxidation reduction reaction. The metabolic profile of ginseng saponins biotransformed via intestinal flora was described based on the information of metabolites. The main intestinal bacteria metabolites （including Rh1, Rh2, Compound K, F1） were studied further via rat liver cytochrome P450 enzyme （CYP450） in vitro. The CYP450 was obtained through a series of differential centrifugation steps. The incubation was under 37 ℃ in shaker incubator. All of these compounds were metabolized by redox reaction detected by UPLC/MS. The results showed that ginsenosides mainly produced deglycosylation products under the action of intestinal bacteria, and oxidation reduction reaction products under the action of CYP450. I phase metabolism plays a role in promoting drug absorption and increasing the polarity of the compounds. The results can provide an important reference for in vivo metabolism studies of ginsenosides in further.