An integration strategy combined progressive multivariate statistics with anticoagulant activity evaluation for screening anticoagulant quality markers in Chinese patent medicine

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.