Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, China; National and Local Joint Engineering Laboratory for Energy Plant Bio-Oil Production and Application, Chengdu, China.
National and Local Joint Engineering Laboratory for Energy Plant Bio-Oil Production and Application, Chengdu, China.
J Ethnopharmacol. 2021 Feb 10;266:113454. doi: 10.1016/j.jep.2020.113454. Epub 2020 Oct 13.
Adhatoda vasica Nees., which existed in a large; number of Tibetan medicine prescriptions for hepatopathy, used as an adjuvant to treat liver diseases.
HYPOTHESIS/PURPOSE: Oxidative stress is the key player in the development and progression of liver pathogenesis. In recent years, research is increasingly being focused on exploitation of the active components from medicinal plants to combat the liver oxidative injury. In our study, we aimed to screen the active principles from A. vasica and clarify whether they could relieve oxidative damage induced by tert-Butyl hydroperoxide (t-BHP) and its potential mechanism via activating AMPK/p62/Nrf2 pathway.
Ultra performance liquid chromatography (UPLC) was adopted for analysis of chemical composition in the extracts. Furthermore, the antioxidant activity of the fractions was evaluated using DPPH, ABTS and reducing power assay. Along with this, the compounds in this fraction with highest antioxidant activity were analyzed using UPLC-MS. Based on this, the condition for extracting flavonoids of this subfraction was optimized via response surface method. CCK-8 assay was used to detect cell viability. Detection kits were used to measure the activity changes of AST, ALT, LDH and CAT as well as MDA and GSH levels induced by t-BHP. Detection of reactive oxygen species (ROS) production was used DCFH-DA probe. DAPI staining and flow cytometry was used to detect cell apoptosis. In terms of the mechanistic studies, the expression of proteins involved in AMPK/p62/Nrf2 pathway was measured using western blotting.
Eventually, 70% ethanol extract from leaf of A. vasica was chosen due to its highest active components compared with other extracts. Further, ethyl acetate fraction derived from 70% ethanol extract in A. vasica (AVEA) possess highest ability for scavenging DPPH and ABTS free radicals as well as strongest reducing power than other fractions. Chemical composition analysis showed that AVEA contained 17 compounds, including 1 quinazoline alkaloid, 12 flavonoid-C-glycosides and 4 flavonoid-O-glycosides. In addition, the conditions (ratio of solid-liquid 1:14, the concentration of ethanol 73%, and the temperature 65 °C) were selected to enrich the flavonoids in AVEA. Furthermore, AVEA could attenuate t-BHP induced hepatocyte damage via increasing the cell viability, restoring abnormal the activities of AST, ALT, LDH and CAT as well as the levels of MDA and GSH. ROS fluorescence intensity was reduced by AVEA. Meanwhile, it could inhibit the cell apoptosis of BRL 3 A cells, as evidenced by restoration of cell morphology and decreasing the number of apoptotic cells. Further mechanistic studies indicated AVEA could promote p-AMPK expression to further induce autophagy adaptor-p62 protein expression, which could autophagic degradation of Keap1, leading to Nrf2 release and translocation into nucleus to induce antioxidant genes (HO-1, NQO-1, GCLC and GCLM) expression.
In our study, AVEA was first to screen as the active fraction in A. vasica with alkaloids and abundant flavones. Moreover, the fraction potentiates its beneficial aspect by displaying the protective role on relieving t-BHP induced oxidative stress and activating AMPK/p62/Nrf2 pathway. AVEA helps maintain the redox homeostasis of hepatic cells and could be considered as an effective candidate against oxidative stress related liver disorders.
Adhatoda vasica Nees. 存在于大量藏医药处方中，用于辅助治疗肝脏疾病。
假设/目的：氧化应激是肝脏发病机制发展和进展的关键因素。近年来，研究越来越集中于从药用植物中提取活性成分，以对抗肝脏氧化损伤。在我们的研究中，我们旨在从 A. vasica 中筛选活性成分，并通过激活 AMPK/p62/Nrf2 通路，阐明它们是否可以缓解 tert-Butyl hydroperoxide (t-BHP) 诱导的氧化损伤及其潜在机制。
采用超高效液相色谱 (UPLC) 分析提取物的化学成分。此外，还通过 DPPH、ABTS 和还原力测定法评估了各馏分的抗氧化活性。在此基础上，采用 UPLC-MS 分析了该馏分中具有最高抗氧化活性的化合物。基于此，通过响应面法优化了该亚馏分中黄酮类化合物的提取条件。CCK-8 测定法用于检测细胞活力。检测试剂盒用于测量 t-BHP 诱导的 AST、ALT、LDH 和 CAT 活性变化以及 MDA 和 GSH 水平。使用 DCFH-DA 探针检测活性氧 (ROS) 产生。DAPI 染色和流式细胞术用于检测细胞凋亡。在机制研究方面，采用 Western blot 法测定了 AMPK/p62/Nrf2 通路相关蛋白的表达。
最终，由于与其他提取物相比，叶中 70%乙醇提取物具有最高的活性成分，因此选择了该提取物。此外，从 70%乙醇提取物中得到的乙酸乙酯馏分（AVEA）在清除 DPPH 和 ABTS 自由基方面以及在增强还原力方面的能力均强于其他馏分。化学成分分析表明，AVEA 含有 17 种化合物，包括 1 种喹唑啉生物碱、12 种黄酮类-C-糖苷和 4 种黄酮类-O-糖苷。此外，选择（固液比 1:14、乙醇浓度 73%、温度 65°C）的条件来富集 AVEA 中的黄酮类化合物。此外，AVEA 可以通过增加细胞活力、恢复 AST、ALT、LDH 和 CAT 活性以及 MDA 和 GSH 水平来减轻 t-BHP 诱导的肝细胞损伤。AVEA 还可以降低 ROS 荧光强度。同时，它可以抑制 BRL 3A 细胞的细胞凋亡，这可以从细胞形态的恢复和凋亡细胞数量的减少得到证明。进一步的机制研究表明，AVEA 可以促进 p-AMPK 的表达，从而进一步诱导自噬接头蛋白 p62 蛋白的表达，自噬可以降解 Keap1，导致 Nrf2 释放并转位到细胞核，从而诱导抗氧化基因（HO-1、NQO-1、GCLC 和 GCLM）的表达。
在本研究中，首次筛选出 A. vasica 中的活性部分为生物碱和丰富的类黄酮的 AVEA。此外，该馏分通过显示缓解 t-BHP 诱导的氧化应激和激活 AMPK/p62/Nrf2 通路的保护作用来增强其有益方面。AVEA 有助于维持肝细胞的氧化还原稳态，可作为治疗氧化应激相关肝脏疾病的有效候选药物。
