Study On Anti-thrombin Substance Basis And Rapid Quality Evaluation On Curcumae Rhizoma And Its Vinegar-processed Products

Curcumae rhizome(CR)is the dried rhizome of C.Phaeocaulis Val,C.Kwangsicensis S.G.Lee et C.F.Liang,and C.Wenyujin Y.H Chen et C.Ling.The processing products of Curcumae rhizoma are produced through vinegar which is called vinegar-processed Curcumae rhizoma.In pharmacodynamics,there are some differences between raw Curcumae rhizome and vinegarprocessed Curcumae rhizome.The raw CR is focused on promoting qi and eliminating the accumulation while the vinegar-processed CR on activating blood and removing stasis.Therefore,the two should not be mixed in clinical practice.However,the current methods to distinguish them are limited to traditional manual identification or chromatography-based methods,which are not conducive to large-scale application.There is no definite index to characterize the effect of CR on promoting blood circulation and removing blood stasis.Based on this background,this essay aims to establish a rapid identification strategy for raw and vinegar CR,which combined chemometrics with the rapid and non-invasive electronic nose(E-Nose),near-infrared(NIR),and other methods.Then,aime to thrombin target,the rapid evaluation method of the pharmacodynamic activity of CR was explored,and the pharmacodynamic material basis of CR was explored,which laid the foundation for elucidating the material basis and action mechanism of CR promoting blood circulation and removing stasis.30 batches of CR and vinegar-processed CR were collected,and the qualitative identification model was established based on E-Nose and NIR detector.The data of the E-Nose was analyzed by unsupervised principal component analysis(PCA)and supervised partial least squares(PLS),which identified the data difference and purpose difference factors.To compare the ability to identify data,the BP artifical neural network model(BP-ANN),decision-making tree(DTM),Naive Bayes(NBM),support vector machine(SVM),K Nearest Neighbor(KNN),and Boost were used.It is found that compared with the linear discriminant model,the nonlinear ANN model based on bionics can better learn and make decisions on the information of the bionic E-Nose,and obtain the best qualitative discriminant results.The specific inhibition of thrombin by CR was found.The reality of the inhibitory effect was confirmed by the NIR spectroscopy-effect relationship.The quantitative determination of antithrombin and antioxidant activity of CR extract by desktop NIR and miniaturized handheld NIR(Micro-NIR)were compared.A suitable waveband selection algorithm was used to improve the prediction performance of the model,and the main difference between the two instruments was the detection range of the instruments.After that,a middlelevel data fusion approach was established,for qualitative analysis of raw CR and vinegar-processed CR was carried out by simulating human identification patterns,combined colorimetter,E-Nose,and Micro-NIR--three nonhomologous,rapid,non-invasive portable detection instruments.The significance of non-homologous data fusion was explained by analyzing the discriminant model.In this study,PLS-DA was used to screen the characteristics of the electronic nose with VIP > 1 as the standard.PCA was used to reduce the dimension of Micro-NIR data to balance the dimension difference of multi-source data,and the accuracy of the PLS-DA model was improved.Multi-source data contributed in different directions of the space.In order to further explore the pharmacological basis of anti-thrombin in CR,the studies were as follows.Firstly,the effect of CR on thrombin in HUVECs cells was determined by ELISA,then,an affinity-ultrafiltrationUPLC-Q Exactive Orbitrap/MS(AUF-LC-MS)approach was applied.Agatroban and adenosine were used as positive and negative drugs,respectively,to verify the reliability of the established method.The in vitro activity of the compounds was determined by specific substrate S-2238.The in vivo effect of the active ingredients was determined using zebrafish;Finally,the action mode of drugs in thrombin was studied by molecular docking.C7,8,and 11 were found to bear an in vitro thrombin inhibitory activity,and be able to significantly inhibit the thrombosis in zebrafish models at a safe dose,which is firstly reported.