| This work was supported by National Natural Science Foundation of China (81274058)-Part experiment content of "Study on dose-effect relationship and synergistic interation of nourishing and activating blood effect for the compatibility of Danggui and Honghua".This dissertation is divided into two major parts. In the first part, this paper reviewed the methods and progress of interaction research of compatibility of traditional Chinese medicine (TCM). There are four chapters in second part, specific research content and correlated conclusion as follows:Chapter 2. The preparation of the samples used for experiment.The dry herb pieces of Danggui (DG) (500 g) and Honghua (HH) (500 g) were extracted with boiling water (1:8) for once,2 h, filtered through gauze. The residue was refluxed with boiling water (1:6) for twice,1.5 h for each time, filtered through gauze, respectively. Then three filtrates were merged and evaporated with rotary evaporation under vacuum at 50℃, thus DG and HH extracts were obtained, respectively. According to the results of data mining through Chinese medicine prescription and the needs of statistical analysis, a certain proportion (4:1,2: 1,3:2,1:1,2:3,1:2,1:4 w/w) of DG and HH, a total 3.5 kg mixed pieces were extracted through the same procedure, and then GH 4:1、GH2:1、GH3:2、GH1:1、GH2:3、GH 1:2、GH 1:4 extract could also be obtained.Chapter 3. The analysis of promoting blood effects and tonifying blood effects of the compatibility between Danggui and Honghua.(1) Comparing tonifying blood effects of Danggui-Honghua with different doses on blood deficiency miceTo observe the differences of tonifying blood effect for the combination of Danggui and Honghua (GH) with different proportions on blood deficiency mice, and choose the proportion of GH which has the optimal tonifying blood effect. The blood deficient mice model was induced by injection of phenylhydrazine and cyclophosphamide. On the basis of multi-attribute comprehensive index method, the optimal dose was determined through-five doses of GH 1:1, which was the highest frequency proportion of GH used in the "Dictionary of Chinese Medicine Prescriptions". According to the optimal dose, the change regulation of tonifying blood effect of GH with different proportions was observed. The Results showed that among five doses (1,3,4, 5 times of clinical dose), the tonifying blood effect of GH was best when the dosing concentration of GH was clinical dose. Among nine proportions (1:0,4:1,2:1,3:2,1:1,2: 3,1:2,1:4 and 0:1), GH 1:1 had the best effect. The results were consistent with regulations of medicine usage that GH 1:1 had the highest frequency used in Herbal Formulae, which could provide scientific basis for more effective application of Danggui and Honghua in modern clinic medicine.(2) Comparing promoting blood effects of Danggui-Honghua with different doses on blood stasis ratsTo observe the differences of the activating blood circulation and dissipating blood stasis effects for the combination of Danggui and Honghua (GH) with different doses on blood stasis rats, and choose the doses of GH which has the optimal the activating blood circulation and dissipating blood stasis effects. The acute blood stasis rat model was induced by subcutaneous injection of adrenaline and ice water bath. On the basis of multi-attribute comprehensive index method, the optimal dose was determined through five doses of GH 1:1, which was the highest frequency proportion of GH used in the "Dictionary of Chinese Medicine Prescriptions". The blood stasis mice were administrated intragastrically with GH (1,2,3,4 and 5 times of clinical dose). The whole blood viscosity (WBV), plasma viscosity (PV), and high shear whole blood relative index (HSWBRI), low shear whole blood relative index (LSWBRI), erythrocyte aggregation index (EAI) were tested to observe the effects of GH on hemorheology of blood stasis rats. The results showed that the hemorheology indexes of model group had significant differences with normal group. Compared with model group, GH (1,2,3,4 and 5 times of clinical dose) could improve all the hemorheology indexes indexes of acute blood stasis rats. Among five doses (1,2,3,4 and 5 times of clinical dose), the promoting blood effects effect of GH was best when the dosing concentration of GH was clinical dose. The results could provide scientific basis for more effective application of Danggui and Honghua in modern clinic medicine.(3) Comparing the promoting blood effects of Danggui-Honghua with different proportions on blood stasis ratsThe combination of Danggui and Honghua (GH) is a popular herb pair commonly used in clinic for the treatment of blood stasis syndrome in China. To evaluate the activating blood circulation and dissipating blood stasis effects of the compatibility between Danggui and Honghua with different proportions on acute blood stasis rats, and optimize the proportion of GH with the best activating blood circulation and dissipating blood stasis effect. Acute blood stasis rat model was induced by subcutaneous injection of adrenaline and ice water bath. The blood stasis mice were administrated intragastrically with GH (1:0,4:1,2:1,3:2,1:1,2:3,1:2, 1:4 and 0:1) extracts. The whole blood viscosity (WBV), plasma viscosity (PV), and high shear whole blood relative index (HSWBRI), low shear whole blood relative index (LSWBRI), erythrocyte aggregation index (EAI) were tested to observe the effects of GH on hemorheology of blood stasis rats. And the maximum aggregation induced by adenosine diphosphate (ADP) was tested to observe the effect of GH on platelet aggregation index of blood stasis rats. In addition, the prothrombin time (PT), thrombin time (TT), activated partial thromboplastin time (APTT), and plasma fibrinogen (FIB) were tested to observe the effects of GH on blood coagulation function of blood stasis rats. Then principal component analysis and multi-attribute comprehensive index methods were both used to comprehensively evaluate the total activating blood circulation and dissipating blood stasis effects of GH. The results showed that the hemorheological indexes and coagulation parameters of model group both had significant differences with normal group. Compared with model group, GH (1:0,4:1,2:1,3:2,1:1,2: 3,1:2,1:4 and 0:1) could improve all the blood hemorheology indexes and regulate part indexes of blood coagulation function and platelet aggregation in acute blood stasis rats. Based on principal component analysis and multi-attribute comprehensive index methods, GH 1:1 and GH 3:2 both had the best effect of blood circulation and dissipating blood stasis effect, and the effect of GH 1:1 was slightly better than GH 3:2. These results suggest that GH could obviously improve the abnormality of hemorheology and blood coagulation function in acute blood stasis rats. The optimized proportion of GH was consistent with regulations of medicine usage that GH 1:1 had the highest frequency used in traditional Chinese formulae. It could provide scientific basis for more effective application of the compatibility between Danggui and Honghua in modern clinic medicine.(4) The antioxidant effect research of Duangui-Honghua in vitroTo observe the differences of the antioxidant effects for the combination of Danggui and Honghua (GH) with different doses and different proportions by the DPPH, ABTS and OH-scavenging and FRAP methods. The results showed that when the combination of Danggui and Honghua (GH) with different doses and same proportion, the high dose groups (100 mg/mL) have strong ability of scavenge DPPH, ABTS and OH·and strong reduction to Fe3-. And the ability of GH to scavenge DPPH, ABTS and Fe3+had a certain concentration-response relationship.(5) The anticoagulation effect research of Duangui-Honghua in vitroIn this experiment, the maximum aggregation induced by adenosine diphosphate (ADP) was tested to observe the effect of GH on platelet aggregation index of blood stasis rats. In addition, the prothrombin time (PT), thrombin time (TT), activated partial thromboplastin time (APTT), and plasma fibrinogen (FIB) were tested to observe the effects of GH on blood coagulation function. The results showed that all the groups except DG could significantly reduce the the maximum aggregation induced by adenosine diphosphate (ADP) compared with control group, and had a certain concentration-response relationship. Compared with control group, all the proportions of Danggui-Honghua in high dose (0.50 g/mL) groups could significantly prolong PT. And all the groups except GH1:2, GH2:3 and GH2:1 had a certain concentration-response relationship. Compared with control group, all the groups could significantly prolong TT had a certain concentration-response relationship. All the dose groups could reduce the content of FIB in plasma in vitro, and the same proportion of Danggui-Honghua in different dose groups presented a certain concentration-response relationship.Chapter 4. The metabolites research of Gui-Hong herb pair and its combination herbs in blood deficiency/blood stasis rats(1) Identification on the metabolites of Gui-Hong herb pair and its combination herbs in blood deficiency ratsThe major metabolites of FA, CA, HSYA and HKG in DG, HH and GH extracts in blood deficiency rats’plasma, urine and bile were studied. Bio-samples were analyzed and detected by ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS). The metabolites of FA, CA, HSYA and HKG in blood deficiency rats plasma, urine and bile were identified after oral administration of DG, HH and GH extracts using MSE and mass defect filter (MDF) techniques. On the basis of the fragment ion and metabolic pattern,37 metabolites including parent, reduction, hydrolysis, methylation hydroxylation, sulfate conjugation, taurine conjugation, glycine conjugation, glucuronic acid conjugation, acetylation and other metabolites of FA, CA, HSYA and HKG were tentatively identified. The major metabolite of phase I and phase II of FA, CA, HSYA and HKG in blood deficient rats plasma, urine and bile from DG, HH and GH extracts can be identified and provided referential information for material foundation of medicinal effectiveness and action mechanism of DG, HH and GH.(2) Identification on the metabolites of Gui-Hong herb pair and its combination herbs in acute blood stasis ratsThe major metabolites of FA, ChA, HSYA and CoA in DG, HH and GH extracts in acute blood stasis rats’plasma, urine and bile were studied. Bio-samples were analyzed and detected by UHPLC-QTOF/MS. The metabolites of FA, ChA, HSYA and CoA in acute blood stasis rats’ plasma, urine and bile were identified after oral administration of DG, HH and GH extracts using MSE and mass defect filter (MDF) techniques. On the basis of the fragment ion and metabolic pattern,44 metabolites including parent, reduction, hydrolysis, methylation hydroxylation, sulfate conjugation, taurine conjugation, glycine conjugation, cysteine conjugation glucuronic acid conjugation, acetylation and other metabolites of FA, ChA, HSYA and CoA were tentatively identified. The major metabolite of phase Ⅰ and phase Ⅱ of FA, ChA, HSYA and CoA in acute blood stasis rats’plasma, urine and bile from DG, HH and GH extracts can be identified and provided referential information for material foundation of medicinal effectiveness and action mechanism of DG, HH and GH.Chapter 5. Comparative metabolomics analysis on invigorating blood circulation and hematinic effects for herb pair Gui-Hong by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry and pattern recognition approach(1) Urine and plasma metabonomics coupled with UHPLC-QTOF/MS and multivariate data analysis on potential biomarkers in anemia and hematinic effects of herb pair Gui-HongRats were divided into 5 groups, and drugs were administered by oral gavage one time each day for continuous 7 days from the experiment began. Urine and plasma were analyzed by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS). Partial least-squares discriminate analysis (PLS-DA) models were built to evaluate the therapeutic effects of GH and its constituent combination. Pearson correlation matrix analysis method was used to discover the correlations between potential biomarkers and biochemical indicators of HAA rats. The Results show that seven potential biomarkers contributing to the separation of model group and control group were tentatively identified. The levels of 1-kynurenine, phenylalanine, nicotinic acid and sphingosine increased significantly (P< 0.05) in HAA rats, while the levels of I-isoleucine,1-tyrosine and serotonin decreased significantly (P< 0.05) in comparison with control rats. Those endogenous metabolites were chiefly involved in phenylalanine, tyrosine and tryptophan biosynthesis, valine, leucine and isoleucine biosynthesis, tryptophan metabolism and tyrosine metabolism. The metabolic deviations could be regulated closer to normal level after DG, HH and GH intervention. In term of hematinic effects, GH was the most effective as shown by the relative distance in PLS-DA score plots and relative intensity of potential biomarkers, reflecting the synergic action between DG and HH. The above results were found to be reasonable in explaining the hematinic effects mechanism of GH. These results suggest that combined with the results of routine blood and the change of urinary metabolic pattern and plasma metabolic pattern show the model group is moving toward the control group and the HAA was being prevented and alleviated. The effect of GH group is more remarkable than DG and HH group. Some potential biomarkers like 1-kynurenine, phenylalanine,1-isoleucine,1-tyrosine, serotonin, nicotinic acid and sphingosine have been found and identified. The work shows that the metabonomics method is a promising tool in the efficacy and mechanism research of traditional Chinese medicines.(2) Comparative metabolomics analysis on invigorating blood circulation for herb pair Gui-Hong by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry and pattern recognition approachThe compatibility of Angelicae Sinensis Radix (Danggui, DG) and Flos Carthami (Honghua, HH), a famous herb pair Gui-Hong (GH), can produce synergistic and promoting blood effects. Although some physiological and pathological function parameters of the acute blood stasis have been investigated, little information about the changes of small metabolites in biofluids has been reported. In present study, global metabolic profiling with ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS) combined with pattern recognition method was performed to discover the underlying blood-activating regulation mechanisms of DG, HH and GH on the acute blood stasis rats induced by subcutaneous injection of adrenaline hydrochloride and ice water bath. The total 14 metabolites (10 in urine and 4 in plasma), up regulated or down regulated (P< 0.05 or 0.01), were identified and contributed to the acute blood stasis progress. These promising identified biomarkers underpin the metabolic pathway including phenylalanine metabolism, sphingolipid metabolism, arachidonic acid metabolism and arginine and proline metabolism are disturbed in the acute blood stasis rats, which identified by using pathway analysis with MetPA. The altered metabolites and hemorheological indexes could be regulated closer to normal level after DG, HH and GH intervention. In term of activate blood circulation function, GH was the most effective as shown by the relative distance in PLS-DA score plots and relative intensity of metabolomics trategy, reflecting the synergic action between Danggui and Honghua. The results demonstrated that biofluids metabolomics was a powerful tool in clinical diagnosis and treatment of syndrome of blood stasis for providing information on changes in metabolites pathways. |
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