Combination Of Composition-Activity Relationship Approach And Metabolomics To Study The Anticoagululant Compositions And Action Mechanism Of Danggui Sini Decoction

Danggui Sini decoction(DSD)is a well-known Traditional Chinese Medicine(TCM)consisting of seven herbs:Angelica sinensis(AS),Cinnamomi cassia(CC),Paeonia lactiiflora(PL),Tetrapanax papyriferus(TP),Asarum heterotropoides(AH),Glycyrrhiza uralensis(GU),Ziziphus jujuba Mill.(ZJ).It was first recorded in Shanghan Lun.Recent pharmacological research showed that DSD has anti-coagulant efficacy.However,the chemical constituents related to the efficacy of DSD are still unknown.This paper studied DSD using HPLC and 1H-NMR by combining composition-activity relationship with metabmonomics strategy.The aims of this paper were to systematically discuss the active composition group relating the efficacy,establish the characteristic fingerprint,study on the action mechanism of DSD anti-coagulant activity,and to offer the reference of technology for related investigation of other TCM formula.The HPLC chromatograms from the DSD,DSD eliminated AS,CC,PL,TP,AH,GU,ZJ and DSD single herb were collected,thrombin time(TT)of mice as index to anti-coagulant efficacy was determinated.The correlation between HPLC spectrum of DSD and its efficacy of prolonging TT in mice was analyzed in composition-activity relationship approach by means of orthogonal partial least squares(OPLS)method.The active compositions contributed significantly to the efficacy was characterized according to the value of variable importance in projection(VIP),17 chromatographic peaks were then assigned to single herbs.among them,5 peaks from Angelica sinensis,5 peaks from Cinnamomum cassia,4 peaks from Glycyrrhiza uralensis,and 4 peaks from Paeonia lactiflora Pall..17 chromatographic peaks significantly contributed to prolong TT were found.Six of them were identified as liquiritin(2),glycyrrhizic acid(3),ferulic acid(7),isoliquiritin(10),paeoniflorin(12)and cinnamic acid(13).The uniform design was utilized to obtain 16 samples of DSD based on original DSD,HPLC chromatograms of 16 samples were analyzed and TT were determinated.As a result,compared to 17 chromatographic peaks area of DSD,16 samples of DSD similarity were calculated by included angle cosine,the similarity index was in a range of 0.65-0.99;for the 17 chromatographic peaks significantly contributed to prolong TT,the number of different peaks(1～17)as the standard of the similarity calculation.DSD similarity(X)of 16 samples and reciprocal of percentage difference(RPD)of TT(Y)were analyzed by Pearson correlation coefficient to investigate the representative of the number of different peaks for efficacy,the correlation coefficient(R)was 0.821 when chromatographic peaks were 17,which shown that high correlation between similarity and RPD.The characteristic fingerprint was established based on the17 chromatographic peaks concerning the efficacy of prolonging TT by 16 samples of DSD.A new approach to explore the active composition groups relating the efficacy was found.The change of endogenous metabolites in the plasma of anticoagulant activity of DSD was analyzed using 1H-NMR coupled with metabonomics.9 potential biomarkers were identified by http://hmdb.ca/data base and references as isobutyrate,carnitine,tyrosine,phenylalanine,glycerol,lysine,isoleucine,histidine and cholesterol.Compared to the control group,isobutyrate,carnitine,phenylalanine,glycerol,histidine and cholesterol of DSD group were found the significant difference(P<0.05).The MetaboAnalyst was applied to study metabolic pathways of potential biomarkers,the result showed that phenylalani ne,histidine,glycerol and cholesterol in the metabolic pathways were involved in the anti-coagulant process of DSD.The study discuss the onset mechanism of anti-coagulant effects that affer the DSD in complex systems of the body from the perspective of systems biology,the scientific ideas were provided for research the action mechanism of DSD anti-coagulant effects.