| Sini decoction(SND) is one of the most famous and important compound in Shanghan Lun,which was composed with Radix aconite lateralis praeparata(RA), Rhizoma zingiberis(RZ) and Radix et rhizoma glycyrrhizae praeparata cum melle(RE). SND is excellent and compact,and modern study has indicated that SND has expressed distinct superiority and effect characteristic in prevention and clinical treatment of heart and cerebrovascular disease.RA is recognized as principal material containing alkaloids constituents.The chemical constituents of RZ are volatile oils and non-volatile constituents including diarylheptanoids and gingerol-related compounds,and the chemical constituents of RE are triterpene glycosides and flavonids.The HPLC-MS fingerprint chromatograms of component medicinal materials and SND were investigated and the HPLC-TOF-MS were used to analysis the chemical constituents of component medicinal materials and SND,and establish HPLC-TOF-MS fingerprint chromatograms to represent the chemical substances base of SND.THE methods of chemometrics were used to evaluate the SND of different ratio and their anti-myocardial ischemia effects.The pharmacodynamic substance base and the possible pharmaceutical effective constituents were confirmed.Combined with the decomposed recipes of SND,the results of chemometrics were validated.1 Study of chemical constituents analysis of SND(1) Study of HPLC-MS fingerprint chromatograms of SNDThe rapid HPLC methods of aconitine,6-gingerol and glycyrrhizic acid were established respectively,and the contents of three chemical constituents in RA,RZ and RE were set as index to investigate and optimize the conditions of preliminary treatment with orthogonal experimental design of solid-liquid proportion,saturate time and ultrasonic time of ultrasound with methanol.The HPLC-MS fingerprint chromatogram of three component medicinal materials were established and methodology investigation was completed,and the fingerprint chromatogram of methanol and boiling water extract were acquired,respectively.On the foundation of fingerprint chromatogram study of three component medicinal materials,the chromatographic conditions of HPLC-MS of SND,including mobile phase system,content of acetic acid and concentration of ammonium acetate were gradually optimized and the best gradient elution of HPLC-MS fingerprint chromatogram was confirmed as acetonitrile-2 mmol/L ammonium acetate solution(0.01%acetic acid) under positive mode with mass spectra range of m/z 100～1200.The yield of volatile oil of RZ was set as index,and the orthogonal design and homogeneous design was used to optimize the conditions of wet distillation.The 17 times water and extract with 8 h was selected as extract condition,and characterized the chemical constituents of volatile oil of RZ.(2) HPLC-TOF-MS analysis of chemical constituents of SNDAccording to the category of chemical constituents of three component medicinal materials including RA,RZ and RE,The chemical constituents database including alkaloids,diarylheptanoids,gingerol-related compounds,triterpene glycosides and flavonids were established.The chemical constituents of methanol and boiling water extracts of component medicinal materials was analyzed with HPLC-TOF-MS,and the known and unknown compounds were matched and marked,the chemical substances base of SND was represented.156 peaks were found from HPLC-TOF-MS total ions chromatogram of SND,in which 49 peaks from RA,40 peaks from RZ,67 peaks from RE.Depending on the accurate mass,abundance of isotopic distribution,elemental compositions and retention time,19,18,13,12 and 23 peaks were identified as known alkaloids,diarylheptanoids, gingerol-related compounds,triterpene glycosides and flavonids respectively,and other peaks were unknown compounds from three component medicinal materials.2 Study of anti-myocardial ischemia effect of SND(1) Study of pharmacodynamic substance base of SNDThe model of mice myocardial ischemia was established by intraperitoneal injection of posterior pituitary,and the activity of SOD in cardiac muscle tissue was selected as index.On the foundation of chemical constituents fingerprint chromatogram of HPLC-TOF-MS,anti-myocardial ischemia effects of SND with different ratios were investigated.The variation of peak area and activity of SOD in cardiac muscle tissue were associated,and the methods of correlation analysis and multiple linear stepwise regression were used to confirm the pharmacodynamic substance base of SND and possible pharmaceutical effective constituents.Meanwhile,the resource of component medicinal materials was identified,and the chemical substances base was upgrade to the pharmacodynamic substance base.With method of correlation analysis,23 compounds peaks were confirmed as the pharmacodynamic substance base of anti-myocardial ischemia effect of SND,11,3 and 2 peaks were identified as known alkaloids,diarylheptanoids and gingerol-related compounds respectively,and 4 peaks of unknown compounds were from RA,other 2 peaks of unknown compounds were from RZ and RE respectively.Furthermore,with the method of multiple linear stepwise regression,2 alkaloids compounds peaks,Senbusine B and Salsolinol were reckoned as possible effective chemical constituents.From the results of the pharmacodynamic substance base and effective chemical constituents,RA can be found with distinguished effect in anti-myocardial ischemia effect of SND.(2) The decomposed recipes study of anti-myocardial ischemia effect of SNDThe activity of SOD in cardiac muscle tissue was selected as index to compare the effect discrepancy of SND,incompleted SND and component medicinal materials.The whole effect of SND was set as reference,and the important effect of RA in anti-myocardial ischemia effect of SND was further identified.Meanwhile,the correctness of pharmacodynamic substance base of SND was confirmed,and the chemometrics method of confirmation of pharmacodynamic substance base of Chinese herb compound was feasible with the mode of combination of HPLC-TOF-MS chemical constituents fingerprint chromatogram and pharmacodynamic action result.
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