An ultrafiltration and high performance liquid chromatography coupled with diode array detector and mass spectrometry approach for screening and characterizing thrombin inhibitors from Rhizoma Chuanxiong

- One in-solution based THR inhibitor screening method was established.
- The reliability of the proposed method were confirmed by positive and negative controls.
- The accumulation mechanism of nonspecific binding components in ultrafiltration tube during ultrafiltration process was discussed.
- Isochlorogenic acid C and senkyunolide I were discovered as new inhibitors targeting THR in Rhizoma Chuanxiong.
- Four other potential active THR inhibitors might have been found based on the principle of structure-activity relationship.

Thrombin (THR) plays a significant role in thromboembolic diseases, direct THR inhibitors are a class of important clinical anticoagulant drugs. This study established a THR in-solution based biospecific extraction combined with ultrafiltration and high performance liquid chromatography coupled with diode array detector and mass spectrometry analysis (TUA) method to screen and identify ligands for THR in Rhizoma Chuanxiong. After evaluating the reliability of the present TUA method using positive (argatroban) and negative (adenosine, tirofiban, ticagrelor) control drugs, this method was successfully applied to detect eight potential active compounds in Rhizoma Chuanxiong. Two new THR-targeted compounds isochlorogenic acid C and senkyunolide I with high THR inhibitory activity (IC50 206.48 and 197.23 μM, respectively) were identified by liquid chromatography/mass spectrometry and enzyme inhibitory activity test finally. They were reported with direct THR inhibition activity for the first time and their ligand-THR interactions were explored by in silico molecular docking research. In addition, based on the TUA screening result, four compounds gained similar structure with the two hit compounds were also investigated as promising candidates targeting THR with high binding energy (>5.0 kcal/mol). These results may prove that the proposed method could effectively screen THR inhibitors in complex mixtures.