School of Pharmacy, Shenyang Pharmaceutical University, No.103 Wenhua Road, Shenhe District, Shenyang, 110016, China.
Hainan Institute for Drug Control, Haikou, 570311, China.
J Ethnopharmacol. 2023 Oct 5;314:116628. doi: 10.1016/j.jep.2023.116628. Epub 2023 May 15.
Alcoholic liver disease (ALD) is the most serious and irreversible liver damage associated with alcohol consumption. Flos Puerariae and Semen Hoveniae are traditional Chinese medicines (TCM) for dispelling the effects of alcohol. Many studies have shown that the combination of two medicinal materials has the enhanced effect of treating ALD.
The aim of this study is to assess the pharmacological effects of Flos Puerariae-Semen Hoveniae medicine pair, to elucidate its action mechanism in the treatment of alcohol-induced BRL-3A cells, and to reveal the active ingredients in the medicine pair that exerted pharmacological effects by spectrum-effect relationship study.
Firstly, MTT assays, ELISA, fluorescence probe analysis, and Western blot were employed to study the underlying mechanisms of the medicine pair in alcohol-induced BRL-3A cells by examining pharmacodynamic indexes and related protein expression. Secondly, HPLC method was established for chemical chromatograms of the medicine pair with different ratios and the sample extracted by different solvents. Then, principal component analysis, pearson bivariate correlation analysis and grey relational analysis were applied for development of the spectrum-effect correlation between pharmacodynamic indexes and HPLC chromatograms. Moreover, prototype components and their metabolites in vivo were identified by the HPLC-MS method.
Flos Puerariae-Semen Hoveniae medicine pair remarkably increased cell viability, decreased the activity of ALT, AST, TC and TG, reduced the generation of TNF-α, IL-1β, IL-6, MDA and ROS, increased the activity of SOD and GSH-Px, reduced protein expression of CYP2E1, compared with alcohol-induced BRL-3A cells. The medicine pair modulated the PI3K/AKT/mTOR signaling pathways by up-regulating the levels of phospho-PI3K, phospho-AKT and phospho-mTOR. Also, the results of the spectrum-effect relationship study showed that P1 (chlorogenic acid), P3 (daidzin), P4 (6″-O-xylosyl-glycitin), P5 (glycitin), P6 (unknown), P7 (unknown), P9 (unknown), P10 (6″-O-xylosyl-tectoridin), P12 (tectoridin) and P23 (unknown) can be considered as the main components of the medicine pair in the treatment of ALD. Furthermore, 6″-O-xylosyl-tectoridin, tectoridin, daidzin, 6″-O-xylosyl-glycitin and glycitin can be absorbed into the blood and showed clear metabolic and excretion behaviors in rats.
In this study, the hepatoprotective effects and the pharmacology mechanism of Flos Puerariae-Semen Hoveniae medicine pair in alcohol-induced BRL-3A cells were initially investigated and revealed. Through the spectrum-effect relationship study, the potential pharmacodynamic constituents such as daidzin, 6″-O-xylosyl-glycitin, 6″-O-xylosyl-tectoridin, glycitin, and tectoridin exert pharmacological effects on alcohol-induced oxidative stress and inflammation by modulating the PI3K/AKT/mTOR signaling pathways. This study provided experimental basis and data support for revealing the pharmacodynamic substance basis and pharmacology mechanism in the treatment of ALD. Moreover, it provides a robust mean of exploring the primary effective components responsible for the bioactivity of complicated TCM.
酒精性肝病（ALD）是与饮酒相关的最严重和不可逆转的肝脏损伤。葛根和鸡矢藤是用于解酒的中药。许多研究表明，两种药材的组合具有增强治疗 ALD 的效果。
本研究旨在评估葛根-鸡矢藤药对的药理作用，阐明其在治疗酒精诱导的 BRL-3A 细胞中的作用机制，并通过谱效关系研究揭示药对发挥药理作用的活性成分。
首先，通过研究药效学指标和相关蛋白表达，采用 MTT 测定、ELISA、荧光探针分析和 Western blot 等方法研究药对在酒精诱导的 BRL-3A 细胞中的潜在机制。其次，建立了不同比例的葛根-鸡矢藤药对和不同溶剂提取样品的 HPLC 化学图谱。然后，应用主成分分析、皮尔逊双变量相关分析和灰色关联分析，建立药效学指标与 HPLC 图谱之间的谱效相关性。此外，采用 HPLC-MS 法鉴定体内原型成分及其代谢物。
与酒精诱导的 BRL-3A 细胞相比，葛根-鸡矢藤药对显著提高了细胞活力，降低了 ALT、AST、TC 和 TG 的活性，减少了 TNF-α、IL-1β、IL-6、MDA 和 ROS 的生成，增加了 SOD 和 GSH-Px 的活性，降低了 CYP2E1 蛋白表达。药对通过上调磷酸化 PI3K、磷酸化 AKT 和磷酸化 mTOR 的水平来调节 PI3K/AKT/mTOR 信号通路。此外，谱效关系研究的结果表明，P1（绿原酸）、P3（大豆苷元）、P4（6′-O-木糖基-甘草苷）、P5（甘草苷）、P6（未知）、P7（未知）、P9（未知）、P10（6′-O-木糖基-金雀异黄素）、P12（金雀异黄素）和 P23（未知）可以被认为是治疗 ALD 的药对的主要成分。此外，6′-O-木糖基-金雀异黄素、金雀异黄素、大豆苷元、6′-O-木糖基-甘草苷和甘草苷可以被吸收到血液中，并在大鼠体内表现出明显的代谢和排泄行为。
本研究初步探讨和揭示了葛根-鸡矢藤药对在酒精诱导的 BRL-3A 细胞中的保肝作用及其药理学机制。通过谱效关系研究，大豆苷元、6′-O-木糖基-甘草苷、6′-O-木糖基-金雀异黄素、甘草苷和金雀异黄素通过调节 PI3K/AKT/mTOR 信号通路，对酒精诱导的氧化应激和炎症发挥药理作用，成为潜在的药效成分。该研究为揭示治疗 ALD 的药效物质基础和药理学机制提供了实验依据和数据支持，为探索中药复杂体系的主要有效成分提供了有力手段。
