| The occurrence and development of blood stasis syndrome are closely related to platelet aggregation and coagulation dysfunction.Blood stasis syndrome can be manifested as abnormal platelet aggregation and activation of coagulation.The herbal pair,also known as the ?pair drug?,is a combination of relatively fixed two drugs in clinical medicine,and is a basic form of compatibility in traditional Chinese medicines(TCMs).The TCMs herbal pairs for promoting blood circulation and removing blood stasis are mainly used to promote blood circulation,blood circulation,and dissipation of traces of blood,such as Danshen-Honghua(DH).In this thesis,the spectrum-effect relationship analysis method is used to screen the main active components of DH herbal pair in promoting blood circulation and removing blood stasis,inhibiting tyrosinase and anti-oxidation,and exploring the interaction between the active components.Furthermore a zebrafish thrombus model was established to evaluate the efficacy of the screened active ingredients.The thesis consists of seven main parts(chapters):In the first part,the chemical constituents and pharmacological effects of DH herbal pair are briefly introduced,and the studies of spectrum-effect relationship and its data processing methods,as well as the research status of zebrafish is briefly summarized.Finally,the purpose and research content of this thesis are introduced.In the second part,a spectrum-effect relationship analysis method was established to screen the active components for the blood-activating effect of DH herbal pair.Firstly,reflux extraction method was used to extract DH herbal pairs with different ratios(1:1,2:1,3:1,5:1,1:5 and 1:3).At the same time,blood stasis model was established by ice-water bath method,using the 0.9% physiological saline as a blank control,the in vivo pharmacological effects of different ratios of DH extracts were compared by anticoagulant experiments,and spectrum-effect analysis was performed by transferring DH extracts' chemical fingerprint peak area and pharmacological tests data into SPSS software for canonical correlation analysis(CCA).The results indicated that 15 potential active ingredients were screened out,and the chemical structures of 8 components were identified by LC-MS,of which 5 components were verified for their activity in vitro.The experimental results show that the established spectrum-effect screening method can quickly and effectively screen out the main blood-activating active components of DH herbal pairs,of which caffeic acid,salvianolic acid B,hydroxysafflor yellow A and lithospermic acid were proved to have significant blood-activating effects,which can provide experimental basis for their further study.In the third part,the spectrum-effect relationship analysis method was used to screen the active components of tyrosinase inhibitors in DH herbal pair.Firstly,reflux extraction method was used to extract DH herbal pairs at different ratios(1:1,2:1,3:1,5:1,1:5 and 1:3).At the same time,kojic acid as positive control,the inhibition rate was used to compare the tyrosinase inhibition activity of different ratios DH extracts.Spectrum-effect analysis was performed by transferring DH extracts' chemical fingerprint peak area and inhibition rate data into SPSS software for CCA.And the active site of active ingredients and tyrosinase was analyzed by molecular docking.Results indicated that 16 potential active ingredients were screened out,and the chemical structures of 13 components were identified by LC-MS,of which 5 components were verified for their activity in vitro.Results showed that protocatechuic aldehyde,tanshinone IIA and hydroxysafflor yellow A had significant inhibition effects on tyrosinase,and some of their active sites are the same as the positive drug kojic acid.These experimental results confirm that the spectrum-effect analysis method can effectively screen complex compounds,and the structure characteristics of these active compounds can also provide references for the discovery of other tyrosinase inhibitors.In the fourth part,the spectrum-effect relationship analysis method was used to screen the anti-oxidant active components in DH herbal pair.Firstly,reflux extraction method was used to extract DH herbal pairs at different ratios(1:1,2:1,3:1,5:1,1:5,1:3).At the same time,vitamin C as positive control,the clearance rate of DPPH was used to compare the anti-oxidation activity of different ratios DH extracts.Spectrum-effect analysis was performed by transferring DH extracts' chemical fingerprint peak area and clearance rate of DPPH data into SPSS software for CCA.Results indicated that 18 potential active ingredients were screened out,and the chemical structures of 13 components were identified by LC-MS,of which 8 components were verified for their activity in vitro.Results showed that the danshensu,protocatechuic acid,hydroxysafflor yellow A,caffeic acid,chlorogenic acid,rutin and salvianolic acid A had significant anti-oxidant effects.In the fifth part,the metabolisms of the screened active monomer components in zebrafish were studied.Firstly,the metabolites of tanshinone IIA,danshensu,salvianolic acid B,protocatechuic aldehyde and hydroxysafflor yellow A in zebrafish were identified by LC-MS analysis,and then the metabolic effect(promote or inhibit)of hydroxysafflor yellow A on danshensu,protocatechuic aldehyde,salvianolic acid B and tanshinone IIA in zebrafish was investigated by the content variation of metabolites.Finally,the metabolic characteristics and metabolic mutural effect of these components were summarized.These experimental results can provide reference for the further interaction study of DH herbal pair.In the sixth part,a zebrafish thrombus model was established to evaluate the efficacy of the active monomer component in DH herbal pair.Firstly,the zebrafish thrombus model was established by phenylhydrazine as the inducer.Then,pure water was used as the blank control,aspirin as positive control to evaluate the anti-thrombotic efficacy of protocatechuic aldehyde,danshensu,lithospermic acid,caffeic acid,salvianolic acid B and hydroxysafflor yellow A.Finally,the staining intensity of zebrafish tail vein thrombosis was semi-quantitatively analyzed.Results showed that zebrafish in the blank group had no thrombosis,but had severe thrombosis in the model group.Zebrafish in the positive control group had a small amount of thrombosis,and other active component groups had different degrees anti-thrombotic effects,among which protocatechuic aldehyde,caffeic acid and hydroxysafflor yellow A had the most significant anti-thrombotic effect.These results confirmed that the zebrafish thrombus model by phenylhydrazine induced can be used to screen the antithrombotic drugs.Furthermore,this experiment verified that the protocatechuic aldehyde,danshensu,lithospermic acid,caffeic acid,salvianolic acid B and hydroxysafflor yellow A have good antithrombotic efficacy.The seventh part is the conclusion and prospective of present study.In this paper,the active components in DH herbal pair for promoting blood circulation to remove blood stasis,inhibiting tyrosinase and anti-oxidation were screened by spectrum-effect analysis method.Then,the metabolic effects of hydroxysafflor yellow A on danshensu,protocatechuic aldehyde,salvianolic acid B and tanshinone IIA in zebrafish were investigated,which provided a reference for the study of synergistic mechanism of DH herbal pair.Finally,the zebrafish thrombus model was established to evaluate the efficacy of the active ingredients in DH herbal pair,which can provide experimental evidence for its clinical use. |
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