Characterization of thrombin/factor Xa inhibitors in Rhizoma Chuanxiong through UPLC-MS-based multivariate statistical a
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Chinese Medicine Open Access
RESEARCH
Characterization of thrombin/factor Xa inhibitors in Rhizoma Chuanxiong through UPLC‑MS‑based multivariate statistical analysis Yi‑Yao Yang1, Zhao‑Yu Wu1, Fang‑Bo Xia2, Hao Zhang1, Xu Wang1, Jian‑Li Gao3, Feng‑Qing Yang1* and Jian‑Bo Wan2,4*
Abstract Background: The dry root and rhizome of Ligusticum chuanxiong Hort., or Chuanxiong, has been used as a bloodactivating and stasis-removing traditional Chinese medicine for 1000 years. Our previous studies have shown the inhibitory activity on platelet and thrombin (THR) of Chuanxiong. THR and factor Xa (FXa) play significant roles in the coagulation cascade and their inhibitors are of valuable in the treatment of thromboembolic diseases. The aim of the present study is to screen THR and FXa inhibitors from Chuanxiong. Methods: Four extracts [ethyl acetate (EA), butanol (BA) and remained extract (RE) from 75% ethanol extract, and water extract (WE)] of Chuanxiong were prepared, and their THR/FXa inhibitory activities were assessed in vitro. Following silica-gel column chromatography (SC), the active EA extract and BA extract was further partitioned, respectively. Their active fractions (EA-SC1 to EA-SC5; BA-SC1 to BA-SC5) were obtained and analyzed by LC–MS. After modeling by the principal component analysis (PCA) and orthogonal partial least squares discriminate analysis (OPLSDA), the specific marker compounds were predicted and identified. Their enzyme inhibitory was assessed in vitro and interactions with THR/FXa were investigated by molecular docking analysis. Results: Chuanxiong EA extract showed strong activity against THR and BA extract was more effective in inhibit‑ ing FXa activity, and their fractions exhibited obvious difference in enzyme inhibitory activity. Furthermore, marker compounds a–h were predicted by PCA and OPLS-DA, and their chemical structures were identified. Among them, senkyunolide A, Z-ligustilide, ferulic acid and senkyunolide I (IC50 was determined as 0.77 mM) with potential THR inhibitory activity, as well as isochlorogenic acid A with FXa inhibitory activity were screened out. It was found that the four components could interact with the active site of THR, and the binding energy was lower than − 5 kcal/mol. Isochlorogenic acid A were bound to the active site of FXa, and the binding energy was − 9.39 kcal/mol. The IC50 was determined as 0.56 mM.
*Correspondence: [email protected]; [email protected] 1 School of Chemistry and Chemical Engineering, Chongqing University, Chongqing 401331, People’s Republic of China 2 State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macao SAR, People’s Republic of China Full list of author information is available at the end of the article © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit t
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