Screening Of Thrombin And Matrix Metalloproteinase-2inhibitors In Danggui Sini Decoction Based On UF-UPLC/MS

Objective: The purpose of this study is to establish a method combining ultrafiltration and liquid chromatography-mass spectrometry to screen and identify the bioactive components of potentially targeted thrombin and matrix metalloproteinase-2 in Danggui Sini Decoction,so as to provide scientific basis for elucidating the material basis of Danggui Sini Decoction’s effect of warming meridian,dispersing cold and nourishing blood circulation,and for the rapid development of bioactive components in traditional Chinese medicine Now it is beneficial to explore with screening.Methods: The components of Danggui Sini Decoction were characterized by liquid chromatography-mass spectrometry,and the potential thrombin inhibitors and matrix metalloproteinase-2 inhibitors in Danggui Sini Decoction were analyzed,identified and verified by enzyme activity determination and ultrafiltration centrifugal mass spectrometry.Firstly,the enzyme inhibitory activity of the water extract of Danggui Sini decoction was analyzed and determined.Secondly,the conditions of the screening method were optimized by using UPLC-MS.Then,the components of Danggui Sini Decoction were screened by ultrafiltration after the combination of thrombin and matrix metalloproteinase-2,and the structure was identified by using mass spectrum fragments.Finally,the potential active sites of thrombin and matrix metalloproteinase-2 inhibition were confirmed by molecular docking experiments.Results: In our study,a total of 47 chemical components in Danggui Sini Decoction were identified to provide a material basis for screening potential enzyme inhibitors.The inhibitory activities of Danggui Sini Decoction on thrombin and matrix metalloproteinase-2 were determined by the chromogenic substrate method and the fluorescent substrate method,respectively,and the half-inhibition rates of Danggui Sini Decoction water extract on thrombin were calculated as 0.12 mg/m L and 1.05 mg/m L.A screening method for ultrafiltration affinity antithrombin and matrix metalloproteinase-2 of water extract of Danggui Sini decoction was established.The incubation time,incubation temperature,and elution solvent ratio of the screening conditions were optimized.Finally,the optimal incubation time of thrombin was 30 minutes,the temperature was 30°C,and the elution solvent was100% methanol.Determine the optimal incubation time of matrix metalloproteinase-2 is 30 minutes,the temperature is 37 ℃,and the elution solvent is 75% methanol.Through optimized screening conditions and ultrafiltration centrifugal mass spectrometry technology,16 thrombin inhibitors and 10 matrix metalloproteinase-2 inhibitors were selected from Danggui Sini Decoction.Molecular docking studies the combination of thrombin with potential direct thrombin inhibitors in Danggui Sini Decoction,and the combination of MMP-2 with potential MMP-2 inhibitors.The results showed that hydrogen bonding and hydrophobic interaction played a key role in the process of combining the water extract of Danggui Sini Decoction with the enzyme.Conclusions: 47 chemical components of Danggui Sini Decoction were identified by LC-MS: 8 acids and their glycosides,including 5 phenolic acids,1 acid glycoside,2 organic acids,6 amino acids,2 purine derivatives,10 flavonoids,7 monoterpenoids and 14 triterpenoids.The half inhibitory rate of Danggui Sini Decoction on thrombin was 0.12 mg / ml by chromogenic substrate method.16 potential thrombin inhibitors were screened from Danggui Sini Decoction by optimized screening conditions and ultrafiltration centrifugation mass spectrometry.It was found that hydrogen bond and hydrophobicity played a key role in the binding process of potential thrombin inhibitors and enzymes.The half inhibitory rate of Angelica Sini Decoction on metalloproteinase-2 was 1.05 mg / ml.Ten potential inhibitors of matrix metalloproteinase-2 were screened from Angelica Sini Decoction by optimized screening conditions and ultrafiltration centrifugation mass spectrometry.It was found that hydrogen bond and hydrophobicity played a key role in the binding process of potential inhibitors of matrix metalloproteinase-2 and enzymes.In this paper,the thrombin and matrix metalloproteinase-2 inhibitors in Danggui Sini Decoction were screened by ultrafiltration centrifugal mass spectrometry,which provided an effective research method for the discovery of natural drugs based on the target,provided a scientific basis for the elucidation of the pharmacodynamic material basis of Danggui Sini Decoction,and provided a useful exploration for the rapid discovery and screening of the active components of traditional Chinese medicine.