| Background:Blood stasis syndrome is associated with a variety of diseases,such as hypertension,coronary heart disease,dysmenorrhea,cancer and so on.Salvia miltiorrhiza,called Danshen(DS)in Chinese,is a common bloodactivating and stasis-removing medicine.Its related preparations such as Fufang Danshen Tablets and Fufang Danshen Dropping Pills have better curative effect on promoting blood circulation and removing blood stasis.The DS single-flavored medicinal materials have the characteristics of “multi-component-multi-target-multi-pathway”,and the therapeutic effect and pharmacological mechanism of blood stasis and phlegm have not been further studied.The quality of medicinal materials is the premise of exerting its efficacy.The Chinese Pharmacopoeia(CHP 2015)controls the quality of DS by limiting the content of salvianolic acid B and tanshinone IIA,which fails to reflect the relationship between the quality of DS and its efficacy.Activity-related quality control methods need to be established to ensure their safety and efficacy in clinical applications.Objective:To clarify the efficacy of DS for promoting blood circulation and removing blood stasis,and to explore pharmacological active ingredient of DS aqueous extract and its possible pharmacological mechanism.To obtain the quality markers(Q-marker)related to the effect of DS’s participation in promoting blood circulation and removing blood stasis.And to establish a quality control method system of DS.Methods:1.Study on the pharmacodynamics of DS for promoting blood circulation and removing blood stasisA rat model of acute blood stasis syndrome(ABS)was established by subcutaneous injection of adrenaline and ice water bath.Hemorheology and coagulation function were used as the indexes.The dose and duration of preventive administration of DS were studied.2.Study on the mechanism of DS’s promoting blood circulation and removing blood stasisA discriminant analysis model based on UPLC-Q/TOF-MS for plasma metabolomics was established.Differential metabolites were obtained by Orthogonal partial least squares discriminant analysis(OPLS-DA),and Metabo Analyst was used to obtain the metabolic pathway involved.Metabolomics results were combined with network pharmacology analysis methods.Metabolite-related proteins were obtained using MBrole.A component-target-differential metabolite network was established using Cytoscape to obtain the components related to the effect of promoting blood circulation and removing blood stasis in DS,the target of action and the changes of metabolites caused.And further obtain the mechanism of the effect of DS on promoting blood circulation and removing blood stasis.3.Quality evaluation of DSThe water content and extract content of DS were determined.The HPLC fingerprint of DS aqueous extract was established to obtain the common peak,and the similarity evaluation was carried out.The principal component analysis(PCA)method based on the common peak area was used to identify DS from different areas.Q-marker related to the blood stasis and salvage effect of DS is obtained through the combination of pharmacodynamic substance and chemical substance basis.Quantitative determination of Q-marker was carried out.PCA method based on Q-marker content was used to identify DS from different habitats.Result1.The pharmacodynamic results of DS in the treatment of ABSThe blood viscosity and coagulation function of rats with ABS were improved by DS aqueous extract effectively.The medium dose of DS with 7 and 14 days had better effect of promoting blood circulation and removing blood stasis.2.The pharmacological mechanism result of DS in the treatment of ABS(1)The results of metabolomicsThrough orthogonal partial least-squares discriminant analysis(OPLSDA),18 differential metabolites including 5-thymidine,L-phenylalanine and Lyso PC(14:0)were obtained.Among them,8 differentially metabolites such as 8-hydroxyadenine,selenic phosphate and phenylalanine were increased in ABS rat plasma,while 7 differentially metabolites such as 4-phosphate-l-aspartamyl,5-thymidylic acid,7β,12-α-dihydroxycholic acid were decreased in ABS rat plasma.After preventive administration of water extract of DS,the above 15 different metabolites showed a tendency to be close to the normal group.DS can activate blood circulation and remove blood stasis by interfering with amino acid metabolism,lipid metabolism and nucleotide metabolism in rats with acute blood stasis syndrome.(2)The results of network pharmacologyThrough the establishment of constituent-target-differential metabolite network,it was found that salvianolic acid B and salvianolic acid E acted on mitochondrial 5’(3’)-deoxyribose nucleotide enzyme to cause the metabolic changes of 5-thymidine in ABS rats.9 components,including salvianolic acid K,rosmarinic acid,and tanshinin acted on thymidine kinase to induce the metabolic changes of 5-thymidine in ABS rats.The metabolic changes of L-phenylalanine in ABS rats were induced by the action of protocatechualdehyde on phenylalanine hydroxylase.3.The quality evaluation results of DS(1)The content of moisture and extractThe moisture content of 36 batches of DS was between 8.34 and 10.88%,and extract content distribution between 37.59 and 59.57%.The moisture and extract content of 36 batches of DS in this study meet the requirements.(2)The results of fingerprint researchA total of 15 common peaks were obtained by HPLC fingerprinting of DS aqueous extract.The similarity results showed that DS was similar in different producing areas.From the perspective of the presence or absence of ingredients,there was no significant difference in DS from four producing areas.PCA analysis of the peak areas of 15 common peaks showed that the clustering of DS in four regions was good,indicating that DS from different places of origin had similarities and differences.(3)The quantitative study results of Q-markerThe Q-marker,Danshensu,protocatechuic aldehyde,rosmarinic acid and salvianolic acid B,were obtained for DS related with promoting blood circulation and removing blood stasis through the comprehensive analysis of the chemical substance and pharmacodynamic substance of DS.The PCA results of four Q-markers contents showed that the clustering of DS in four regions was good.This result was consistent with the fingerprint PCA results.Therefore,Q-marker should be as a supplement to the existing quality standard of DS.ConclusionDS has function of promoting blood circulation and removing blood stasis,which is mainly related to amino acid metabolism,lipid metabolism and nucleotide metabolism.Q-marker related to DS promoting blood circulation and removing blood stasis is Danshensu,protocatechuic aldehyde,rosmarinic acid and salvianolic acid B.Its control can be used as a supplement to the quality standard of DS. |
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