Du Kun-Ze, Cui Yan, Chen Shujing, Yang Rui, Shang Ye, Wang Chenhong, Yan Yiqi, Li Jin, Chang Yan-Xu
State Key Laboratory of Component-based Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, 301617, China; Tianjin Key Laboratory of Phytochemistry and Pharmaceutical Analysis, Tianjin University of Traditional Chinese Medicine, Tianjin, 301617, China.
State Key Laboratory of Component-based Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, 301617, China.
J Ethnopharmacol. 2022 Apr 6;287:114964. doi: 10.1016/j.jep.2021.114964. Epub 2022 Jan 3.
The cardiovascular and cerebrovascular diseases affect human health globally. Naoxintong capsules (NXTs), a famous Chinese Patent Medicine, has been especially applied to treat cerebral infarction and coronary heart disease in clinical practice. The anticoagulant activity of this prescription plays an important role in this course of treatment.
Thrombin and factor Xa (FXa) are two key targets considering the anticoagulant activity. The purpose of this investigation is to screen the quanlity markers as key thrombin and FXa inhibitors for the anticoagulant activity oriented quality control of Chinese patent medicine.
Simple multi-polar solvent extraction processes using various proportions of solvents were conducted and their thrombin/FXa inhibitory activities were evaluated in vitro. Bivariate correlation analysis (BCA), grey correlation analysis (GCA), and orthogonal partial least squares discriminate analysis (OPLS-DA) were adopted for screening the potential active markers related to the anticoagulant activity. The chemical structures of these active compounds were identified by UHPLC-Q-TOF-MS/MS and their thrombin/FXa inhibitory activity was determined. The molecular docking technology was applied to explore the interaction between the compounds and targets. The contribution of these anticoagulant active ingredients in NXT was also investigated. Last but not the least, the contents of these markers in NXT were determined by liquid chromatography-electrospray ionization tandem triple quadrupole mass spectrometry (HPLC-ESI-MS/MS) method.
The results showed that the NXT extract exhibited great activity against thrombin and FXa, especially extracted by 75% methanol (v/v). Six marker compounds with potential anticoagulant activity were screened out. Therein, four of the active compounds owing thrombin inhibitory activity (paeoniflorin, lithospermic acid, salvianolic acid B, Z-ligustilide) and five of the active compounds owing FXa inhibitory activity (3,5-dicaffeoylquinic acid, rosmarinic acid, lithospermic acid, salvianolic acid B and Z-ligustilide). In addition, these active compounds accounted for a large proportion of thrombin/FXa inhibitory activity of NXTs. The binding energy also showed the strong interaction formed by close connection of the compounds to the residues of targets.
The proposed integrated stategy could be an efficient strategy to screen potential thrombin/FXa inhibitors for the bioactivity related quanlity control of Chinese patent medicine.
心血管疾病在全球范围内影响着人类健康。脑心通胶囊（NXT）是一种著名的中成药，在临床实践中特别用于治疗脑梗死和冠心病。该方剂的抗凝活性在治疗过程中起着重要作用。
考虑到抗凝活性，凝血酶和因子Xa（FXa）是两个关键靶点。本研究的目的是筛选质量标志物，作为针对中成药抗凝活性导向质量控制的关键凝血酶和FXa抑制剂。
采用不同比例溶剂进行简单的多极性溶剂萃取工艺，并在体外评估其对凝血酶/FXa的抑制活性。采用双变量相关分析（BCA）、灰色相关分析（GCA）和正交偏最小二乘判别分析（OPLS-DA）筛选与抗凝活性相关的潜在活性标志物。通过UHPLC-Q-TOF-MS/MS鉴定这些活性化合物的化学结构，并测定其对凝血酶/FXa的抑制活性。应用分子对接技术探索化合物与靶点之间的相互作用。还研究了这些抗凝活性成分在脑心通中的贡献。最后，采用液相色谱-电喷雾电离串联三重四极杆质谱法（HPLC-ESI-MS/MS）测定脑心通中这些标志物的含量。
结果表明，脑心通提取物对凝血酶和FXa具有较强的活性，尤其是用75%甲醇（v/v）提取的提取物。筛选出6种具有潜在抗凝活性的标志物化合物。其中，4种活性化合物具有凝血酶抑制活性（芍药苷、紫草酸、丹酚酸B、Z-藁本内酯），5种活性化合物具有FXa抑制活性（3,5-二咖啡酰奎尼酸、迷迭香酸、紫草酸、丹酚酸B和Z-藁本内酯）。此外，这些活性化合物在脑心通对凝血酶/FXa的抑制活性中占很大比例。结合能也表明化合物与靶点残基紧密连接形成了强相互作用。
所提出的综合策略可能是一种有效的策略，用于筛选潜在的凝血酶/FXa抑制剂，以用于中成药生物活性相关的质量控制。
