Study On The Compatibility Effect And Bio-active Components Of The Herb Pair Danshen And Honghua

This research work was supported by National Natural Science Foundation of China(81573714)-Part experiment content of "Study on bio-active components and synergy of activating blood for the compatibility of Danshen and Honghua" and 333 High-level Talents Training Project Funded by Jiangsu Province(No.BRA2016387).This master thesis is divided into four chapters.The specific research contents and correlated experimental results are as follows.Chapter 1.Literature research(1)A review of activating blood herb pairsThe compatibility rules of herb pairs for activating blood circulation and blood stasis are reviewed in this paper,which lays a theoretical foundation for the research on the compatibility effect and material basic research.(2)A review of the compatibility of Danshen and HonghuaThe applications,chemical compositions,pharmacokinetics,pharmacological effects and systemic biology of the compatibility of Danshen and Honghua were reviewed systematically.Chapter 2.Study on the material foundation of the compatibility of Danshen and Honghua(1)The collection and preparation of Danshen and Honghua sampleThe dry herb pieces of Danshen(5 kg),Honghua(5 kg)and Danshen-Honghua(3:1,5 kg)were extracted with boiling water to obtain Danshen,Honghua and Danshen-Honghua samples.(2)Study on chemical changes of Danshen-Honghua herb pair based on the chemical fuzzy identification strategyA novel and generally applicable approach was established for the interaction analysis of Danshen-Honghua with different proportions when decocting together by using HPLC-PDA and fuzzy chemical identification strategy with poly-proportion design.A simple project was initially developed for the rapid identification and classification of four types of components(salvianolic acids,tanshinones,quinochalcones,flavonoids)in Danshen-Honghua based on the establishment of relevant component data base,recognition of the reference compound peaks,selection of the characteristics of ultraviolet spectrum and formation of group networks.In this study,the accurate structures of the chemical components did not need to be determined,and only the constituents attributed to different groups were further considered for quantitative analysis.A total of 47 constituents(15 salvianolic acids,20 quinochalcones and 12 flavonoids)from Danshen-Honghua were classified into four kinds of chemical groups,and they were quantitatively analyzed furtherly according to semi-symmetric proportion design.The results showed that the range of total optimization relative dissolution rate of four types of components are from 5:1 to 5:2,and the highest proportion of the total relative dissolution are all 5:2.The study provided reference for clinical application of Danshen and Honghua,and for modern research approach of herb pair.(3)Comparative analysis of main bio-active components in the herb pair Danshen-Honghua and its single herbs by UHPLC-TQ/MSA sensitive,reliable and powerful UHPLC-TQ-MS method was developed for simultaneous quantification of 15 main bio-active components including phenolic acids and flavonoids within 13 min for the first time.The proposed method was first reported and validated by good linearity(r2>0.9975),limit of detections(1.12?7.01 ngmL--),limit of quantitations(3.73?23.37 ngmL-1),intra-and inter-day precisions(RSD ? 1.92%,RSD ? 2.45%),stability(RSD 5? 5.63%),repeatability(RSD ? 4.34%),recovery(96.84%?102.12%)and matrix effects(0.92?1.02).The established analytical methodology was successfully applied to comparative analysis of main bio-active components in the herb pair Danshen-Honghua and its single herbs.Compared to the single herb,the content of danshensu,6-hydroxykaempferol-3-O-rutinoside-6-O-glucoside,protocatechualdehyde,caffeic acid,rutin,anhydrosafflor yellow B,kaempferol-3-O-rutinoside and kaempferol were remarkable increases in their herb pair,and the increase rate is 45.24%,14.12%,21.68%,86.00%,31.62%,4.63%,19.73%,56.60%,respectively.The contents,changes of the main components in the herb pair supported the synergistic effects on promoting blood circulation and removing blood stasis.The results could provide a scientific basis and reference for the quality control of Danshen-Honghua herb pair,and the drug interactions based on variation of bio-active components in herb pairs.Chapter 3.Study on the compatibility effect of Danshen and Honghua(1)Study on the compatibility dose-effect relationship of Danshen and Honghua in vitroThe effect of promoting blood circulation to remove blood stasis of the compatibility of Danshen and Honghua was comparativly studied based on the antioxidant(DPPH,ABTS,OH free radical scavenging assay and FRAP method),inhibit platelet aggregation(ADP)and anticoagulant activity(TT,PT,APTT,FIB)evaluation in vitro.The results showed that the IC50 of Danshen,Honghua and Danshen-Honghua on DPPH was 102.00?61.61?60.36 mg L-1,33.82?26.85?23.99 mg L-1 on ABTS respectively.Each dose group had a strong reduction effect on Fe3+ with a certain dose-effect relationship.Compared with the blank group,except the low-dose group(0.031 g mL-1),other groups showed a significant reduction in ADP-induced platelet aggregation and showed a certain dose-effect relationship.The high dose groups(0.250,0.500,1.000 g mL-1)was significantly extended on TT,and the Danshen-Honghua herb pair was superior to that of the single drugs.Danshen-Honghua herb pair had a significant extension effect on PT.Before and after the compatibility,APTT was significantly extended with a certain dose-effect relationship.At the same time,the content of FIB in rabbit plasma was significantly reduced by Danshen-Honghua.Comprehensive analysis found that Danshen-Honghua herb pair and its single drug had significantly anti-oxidative,inhibiting platelet aggregation and anticoagulant effect with a certain dose-effect relationship.And the ability was enhanced after the compatibility of Danshen and Honghua,which showed a certain synergetic effect.(2)Comparative study on the relationship between activating blood circulation dose and effect of Danshen-Honghua herb pair by different animal modelsIn this experiment,acute blood stasis and myocardial ischemia rat model were used to observe the effect of different doses of Danshen-Honghua herb pair on activating blood circulation.The whole blood viscosity(WBV),plasma viscosity(PV),erythrocyte sedimentation rate(ESR),haematocrit(HCT)activated partial thromboplastin time(APTT),the thrombin time(TT),prothrombin time(PT),plasma fibrinogen(FIB)and adenosine diphosphate(ADP)induced platelet aggregation were tested to observe the effects of Danshen-Honghua herb pair with different doses on hemorheology,blood coagulation function and platelet aggregation of actue blood stasis rats.The results suggest that there were significant differences in hemorheology,coagulation function and platelet aggregation indexes in the model group compared with the blank group.Low,medium and high Danshen-Honghua groups could improve blood rheology,dysfunction of blood coagulation and platelet aggregation in acute blood stasis rats with dose-effect relation,and the optimal dosing dose is 7.20 g kg-1.The creatine kinase(CK),lactate dehydrogenase(LDH),aspartate aminotransferase(AST),malondialdehyde(MDA),superoxide dismutase(SOD),endothelin-1(ET-1),nitric oxide(NO),nitric oxide synthase(NOS),prostacyclin(PGI2),interleukin-6(IL-6),tumor necrosis factor-a(TNF-a),thromboxane B2(TXB2),6-Keto prostaglandin(6-Keto-PGFIa),ATPase and ATP content to observe the protective effect of different doses of Danshen-Honghua herb pair(DH-1,3,5,10 times)on acute myocardial ischemia rats.The results showed that different doses of Danshen-Honghua herb pair could protect myocardial enzyme,oxidation level,endothelial function,inflammation,platelet aggregation and energy metabolism disorders of acute myocardial ischemia rats.The order of blood stasis in different dosage groups was DH-5>DH-10>DH-3>DH-1,the optimal drug dosage is 9 g kg-1,and the acute myocardial ischemia model was selected as the late overall animal model.(3)Study on the compatibility effect of Danshen and Honghua with acute myocardial ischemia model ratsOn the basis of the early study on the relationship between activating blood circulation dose and effect of Danshen-Honghua herb pair,the effect of activating blood circulation before and after the compatibility of Danshen and Honghua was comparatively studied on acute myocardial ischemia model rats by observing the cardiac coefficient,cardiac troponin I(cTnI),creatine kinase(CK),aspartate aminotransferase(AST),lactate dehydrogenase(LDH),malondialdehyde(MDA),superoxide dismutase(SOD),endothelin-1(ET-1)and nitric oxide(NO).The result showed that Danshen,Honghua and its herb pair can significantly improve the abnormality of cardiac coefficient,troponin I,myocardial enzyme,ET-1 and NO,adjust the levels of lipid peroxides in mice and the ability of removal of oxygen free radicals partly.The pharmacodynamic data of each drug group were further analyzed by the principal component analysis(PCA),and the relative distance method of each administration group to the blank group was used to compare the effect of promoting circulation and removing stasis.The order was Danshen-Honghua>Honghua>Danshen.Danshen and Honghua had the synergistic effect after the compatibility on acute myocardial ischemia except MDA leval.It may be related to decreasing the content of cardiac troponin I,myocardial enzyme,inhibiting lipid peroxidation and protecting vascular endothelial cell function.Further results indicated that the active ingredient danshensu,protocatechuic aldehyde,caffeic acid and salvianolic acid B may be the substance of the compatibility effect of Danshen ang Honghua after the integration effect with composition content changes,and the specific mechanism needs to be further studied.Chapter 4.Study on activating blood circulation synergistic effect mechanism of the compatibility of Danshen and Honghua(1)Comparative study on pharmacokinetics of Danshen and Honghua in normal and acute myocardial ischemia model ratsBased on the normal and isoprenaline induced acute myocardial ischemia rat model,a method for the determination of 9 bioactive components in the plasma was established to compare and evaluate the changes of pharmacokinetic parameters of the main effect components in Danshen before and after the compatibility,and finally,to reveal the potential effect of activating blood stasis.The result showed that the correlation coefficient(r2)for the 9 analytes were no less than 0.9958.The precision of inter-day and intra-day was less than 11.53%and 12.72%.The relative error of accuracy was in the range of ± 10.88%.The recovery and matrix effects were less than 11.30%and 10.36%respectively.The vadilated method was successfully applied to investigate the pharmacokinetic study after oral dosing of Danshen and Danshen-Honghua(9 g·kg-1)in normal and acute myocardial ischemia rats,respectively.The half-life of the main effect components is shorter,and basically eliminated within 8 h.Compared with the single drug group,the T1/2 of danshensu was significantly extended,and the pharmacokinetic behavior of other main effect components of Danshen were not significantly different.Compared with the normal group,the T1/2 of protocatechualdehyde and caffeic acid,Tmax of danshensu significantly prolonged,and the pharmacokinetic parameter Cmax,AUC0-t and AUC0-? significantly increased of effect components in the model rats.It is suggested that the constituents of the phenolic acid may be closely related to the improvement of myocardial ischemia.(2)Study on the activating blood circulation mechanism of the compatibility of Danshen and Honghua on metabolomics strategyThe mechanism of activating blood stasis of the compatibility of Danshen and Honghua was studied based on metabolomics in isoproterenol-induced acute myocardial ischemia rat model.First,through the detection of serum myocardial enzyme and oxidative level index(cTnI,CK,AST,LDH,MDA,SOD),the effect of Danshen,Honghua and its herb pair on myocardial injury protection of acute myocardial ischemia rats was evaluated.The result showed that the cardiac tropin I,myocardial enzymes and oxidative levels in the model group were significantly changed compared with the normal group(P<0.01),demonstrating the success of acute myocardial ischemic rat model.Compared with model group,cTnI,CK,AST,LDH and MDA level were significantly lower,SOD activity increased significantly in each dosage groups,the difference had statistical significance(P<0.01,0.05).In addition to MDA level,the levels of cTnI,CK,AST,LDH and SOD in Danshen-Honghua group were significantly changed than Danshen,Honghua group(P<0.01,0.05).Danshen,Honghua and its herb pair had protective effects on acute myocardial injury,and the effect of Danshen-Honghua was better than single herbs.Then,12 potential different biomarkers were obtained from the serum of myocardial ischemia rat based on the metabolomics study.Three in the positive ion mode(glucose,LysoPC(18:2),LysoPC(20:3(8Z,11Z,12Z))and ten in the negative ion mode(cholic acid,ursodeoxycholic acid,LysoPC(20:4),LysoPC(22:6),LysoPC(18:2)(as the positive ion mode),LysoPC(16:0),LysoPC(18:0),palmitic acid,docosahexaenoic acid,stearic acid)were constructed correlation path analysis.And it is closely related to carbohydrate metabolism(starch and sucrose metabolism,galactose metabolism)and lipid metabolism(glycerophospholipid metabolism,biosynthesis of unsaturated fatty acids,fatty acid biosynthesis,fatty acid elongation in mitochondria,fatty acid metabolism,primary bile acid biosynthesis)in the acute myocardial ischemia model.Danshen-Honghua and its single herbs can regulate the disturbance of biological markers to normal.The callback trend of glucose,ursodeoxycholic acid,LysoPC(20:4),LysoPC(22:6),LysoPC(18:0),palmitic acid,stearic acid in Danshen-Honghua group was more significant than single herbs(P<0.01,0.05).The overall effect of Danshen-Hoonghua herb pair on the treatment of the biological markers was better than that of the single drug.