Chinese Medicine
·
2020
·Vol. 15
·期 1
·1-14
·
IF 2.96
Yang Yi-Yao,Wu Zhao-Yu,Xia Fang-Bo,Zhang Hao,Wang Xu,Gao Jian-Li,Yang Feng-Qing,Wan Jian-Bo
Background The dry root and rhizome of Ligusticum chuanxiong Hort., or Chuanxiong, has been used as a blood-activating 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 (OPLS-DA), 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 inhibiting 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.Conclusion sTHR/FXa inhibitory components in different extracts of Chuanxiong were successfully characterized by the method of enzyme inhibition activity assays with ultra performance liquid chromatography-quadrupole time of flight mass spectrometry-based multivariate statistical analysis.
研究领域:
中药学
·
生物化学
·
药理学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Chinese Medicine
·
2020
·Vol. 15
·期 1
·1-13
·
IF 2.96
Yang Yi-Yao,Wu Zhao-Yu,Zhang Hao,Yin Shi-Jun,Xia Fang-Bo,Zhang Qian,Wan Jian-Bo,Gao Jian-Li,Yang Feng-Qing
Background The dry root and rhizome of Salvia miltiorrhiza Bunge, or Danshen, is a well-known traditional Chinese medicine with anticoagulant activity. Taking into account that thrombin (THR) and factor Xa (FXa) play crucial roles in the coagulation cascade, it is reasonable and meaningful to screening THR and/or FXa inhibitors from Danshen. Methods Four extracts [butanol (BA), ethyl acetate (EA) and remained extract (RE) from 75% ethanol extract, and water extract (WE)] of Danshen were prepared, and their THR/FXa inhibitory activities were assessed in vitro. Then, the active EA extract was further separated by silica-gel column chromatography (SC), and its fractions (SC1–SC5) were analyzed by LC–MS. The principal component analysis (PCA) and orthogonal partial least squares discriminate analysis (OPLS-DA) were employed for predicting the specific marker compounds. The chemical structures of targeted compounds were identified by LC–MS/MS and their interactions with THR/FXa were analyzed by the molecular docking analysis. Results Danshen EA extract showed strong activity against THR and FXa, and its fractions (SC1–SC5) exhibited obvious difference in inhibitory activity against these two enzymes. Furthermore, four marker compounds with potential THR/FXa inhibitory activity were screened by PCA and OPLS-DA, and were identified as cryptotanshinone, tanshinone I, dihydrotanshinone I and tanshinone IIA. The molecular docking study showed that all these four tanshinones can interact with some key amino acid residues of the THR/FXa active cavities, such as HIS57 and SER195, which were considered to be promising candidates targeting THR and/or FXa with low binding energy (< − 7 kcal mol −1 ). Conclusions LC–MS combined with multivariate statistical analysis can effectively screen potential THR/FXa inhibitory components in Danshen.
研究领域:
中药学
·
生物化学
·
分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
JOURNAL OF CHROMATOGRAPHY A
·
2020
·
IF 4.049
Zhao-Yu Wu,Hao Zhang,Yi-Yao Yang,Feng-Qing Yang
In this study, an online capillary electrophoresis (CE) based dual-enzyme (thrombin and factor Xa) co-immobilized microreactor (THR-FXa IMER) was constructed for studying enzyme kinetics and screening dual-target inhibitors against THR and FXa with the aid of the polydopamine/graphene oxide (PDA/GO) coating. Based on the developed THR-FXa IMER, the Michaelis-Menten constants ( K m ) of THR and FXa were calculated to be 187.26 and 48.80 μM, respectively. The inhibition constants ( K i ) for two known inhibitors, argatroban and rivaroxaban, on THR and FXa were determined to be 14.73 and 0.41 nM, respectively. In addition, after 30 consecutive runs, the enzymes’ activity was remained 98% of the initial immobilized activity for both THR and FXa, which shows that the constructed IMER has good stability and repeatability. Finally, the developed method was successfully applied to screen dual-target inhibitors against THR and FXa from 30 small molecular compounds. Among them, 10 compounds such as salvianolic acid C and epigallocatechin gallate (EGCG) have dual-enzyme inhibitory activity, and 2 compounds named saikosaponin A and oleuropein have single THR inhibitory activity, 5 compounds such as rosemary acid and salvianolic acid B have single FXa inhibitory activity. Finally, the molecular interactions between enzyme and potential inhibitors were further verified via the molecular docking, and a new compound with a theoretically good coagulation inhibition effect was designed by the scaffold hopping study. In summary, the developed THR-FXa IMER is a reliable method for screening THR and/or FXa inhibitors.
研究领域:
生物化学
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医学
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分析化学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
MOLECULES
·
2021
·Vol. 26
·期 23
·7293
·
IF 4.412
Xu Wang,Dai-Yan Zhang,Shi-Jun Yin,Hui Jiang,Min Lu,Feng-Qing Yang,Yuan-Jia Hu
In this study; a spectrum–effect relationship analysis combined with a high-performance liquid chromatography–mass spectrometry (LC–MS) analysis was established to screen and identify active components that can inhibit thrombin and factor Xa (THR and FXa) in Salviae Miltiorrhizae Radix et Rhizoma–Chuanxiong Rhizoma (Danshen–Chuanxiong) herbal pair. Ten potential active compounds were predicted through a canonical correlation analysis (CCA), and eight of them were tentatively identified through an LC–MS analysis. Furthermore; the enzyme inhibitory activity of six available compounds; chlorogenic acid;Z-ligustilide; caffeic acid; ferulic acid; tanshinone I and tanshinone IIA; were tested to verify the feasibility of the method. Among them; chlorogenic acid was validated to possess a good THR inhibitory activity with IC50of 185.08 µM. Tanshinone I and tanshinone IIA are potential FXa inhibitors with IC50of 112.59 µM and 138.19 µM; respectively. Meanwhile; molecular docking results show that tanshinone I and tanshinone IIA; which both have binding energies of less than −7.0 kcal·mol−1; can interact with FXa by forming H-bonds with residues of SER214; GLY219 and GLN192. In short; the THR and FXa inhibitors in the Danshen–Chuanxiong herbal pair have been successfully characterized through a spectrum–effect relationship analysis and an LC–MS analysis.Keywords:Danshen–Chuanxiong herbal pair;thrombin inhibitors;factor Xa inhibitors;spectrum–effect relationship analysis
研究领域:
中药学
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生物化学
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药物化学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2017
·
IF 4.95
Zhe Xu,Ruonan Liu,Huashi Guan
An accurate, rapid, and cost-effective methodology for enzyme assay is highly demanded to screen the effect of compounds on target at the molecular level. Thrombin (EC 3.4.21.5) and factor Xa (FXa, EC 3.4.21.6) have been identified as the critical targets for the development of potential drugs with anticoagulant activity. In this study, a rapid, sensitive and accurate assay based on UHPLC-MS/MS method has been developed for inhibitor screening against thrombin and factor Xa simultaneously. For thrombin and factor Xa, the Michaelis-Menten constants ( K m ) were calculated to be 6.14 and 57.27 μM, respectively. The inhibition constants ( K i ) for two known inhibitors, argatroban and rivaroxaban , were determined to be 16.23 and 0.41 nM, respectively. The assay was further validated through the determination of a high Z′ factor value of 0.89. Finally, the developed assay was applied to screen a chemical library against two enzymes . Three hit compounds belonging to a class of sulfated polysaccharides were identified and their targets of inhibition action were further evaluated. The results indicated that the dual-target assay by UHPLC-MS/MS analysis could be used as a reliable method for screening anticoagulant agents.
研究领域:
药理学
·
生物化学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
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