Studies On Chemical Probe And Zebrafish Model Based Approaches For Screening Bioactive Compounds From Traditional Chinese Medicine

Exploring the bioactive constituents of traditional Chinese medicine(TCM)contributed to clinical efficacy is an important part of the re-exploitation of TCM.The complicated ingredients of TCM make it difficult to identify the bioactive compounds from TCM mixture.In this thesis,we developed three methods for screening of bioactive compounds from TCM in vivo and in vitro respectively based on the zebrafish model,and the molecular probe.The main contents of this dissertation are as follows:1.A method for screening SIRT3 regulator based on molecular probe with aggregation-induced emission(AIE)effect was developed.The probe contains a tetranethylethylene(TPE)as the fluorophore,and the peptide Gln-Lys(Ac)-Arg-Phe-Asn as the substrate of SIRT3.The optimization of condition such as pH,concentration of buffer salt and the probe was performed.And the probe could detect SIRT3 in vitro with satisfied specificity and low limit of detection,and was potential to be applied in the screening of SIRT3 regulator from TCM.2.A method for screening of bioactive compounds from TCM based on arachidonic acid induced zebrafish thrombosis model was developed and was applied in the identification of bioactive compounds and quality assessment of Danhong Injection.The o-dianisidine was used to stain the red blood cells and the image of thrombus was taken under the microscope.And the intensity of heart red blood cells was measured to assess the thrombus severity.The results showed that this model could comparatively accurately access the anti-thrombosis activity of aspirin(as the positive drug).This model was applied to evaluate the anti-thrombosis activity of Danhong Injection.The results indicated that Danhong Injection could attenuate the zebrafish thrombus induced by arachidonic acid in a dose-dependent manner,and the extract of Danshen and Honghua exhibited satisfactory effect.And a series of compounds which possess the anti-thrombosis activity were screened out such as rosmarinic acid and p-coumaric acid.Moreover,two quality-markers(Q-markers),which could reflect the activity variation and could be used in the batch consistency assessment,were identified based on the anti-thrombosis activity and the content analysis of compounds.3.A method for screening of bioactive compounds from TCM based on combined use of chemical probe and transgenic zebrafish model was developed.Phenylhydrazine was used to damage the zebrafish with Green Fluorescence Protein(GFP)-labeled hemoglobin,and the blood stasis and blood deficiency was characterized by the blood flow observed under a fluorescence microscope.The fluorescence chemical probe NP3 was used to specifically detect ONOO-in the zebrafish,and the oxidative stress injury and the inflammation degree was evaluated by the fluorescence intensity of the probe.The intensity of red blood cells labeled by GFP was measured by flow cytometry and was used to evaluate the degree of blood stasis and blood deficiency.The approach was applied to evaluate the efficacy of Danhong Injection,Danshen extract and Honghua extract.This approach is expected to be used for the double indexes screening of bioactive compounds from traditional Chinese medicine.