| When traditional Chinese medicine (TCM) specialists at ancient and modern times apply herbs for the treatment of some diseases, two certain herbs are frequently used together in many formulae, which are called as herb pair (Yaodui or Duiyao in Chinese). Herb pairs could achieve mutual enhancement, assistance, restraint and suppression, or mutual antagonism. In recent years, its research focus has been gradually transiting from "whther is effective" to "why is effective" and "how to be more effective". As an intermediate point between single herb and many TCM formulae, the research of it is significant to develop the compatibility theory of TCM formulae and create modern TCM new drugs. Therefore, the herb pair Danggui-Chuanxiong (GX) was as a practical example in this thesis, the bio-active components and compatibility action mechanism of which was researched by modern analytical techniques and advanced evaluation approaches. This thesis was supported by the National Natural Science Foundation of China--"Material basis and related mechanism research on the efficacy changes of Xionggui series formulae" and other projects.This dissertation was divided into three chapters, and the major results were summarized as follows:Chapter1. Literature researchIn this section, the bio-active components, compatibility effects, modern research strategies and approaches of herb pairs were systematically reviewed. First, the latest development of bio-active components of herb pairs in recent years were analyzed and summarized from two major aspects, including the quantitative and qualitative changes of herbs compatibility during boiling process in vitro and the action changes of herbs on body or/and the body-related environment. Secondly, the compatibility effects of herb pairs were summed up from whole animals, tissues, cellular and molecular levels; the synergy or attenuation mechanisms of herb pairs were evaluated with monomeric compound-compounds compatibility-single herb-herb pair-formulae as the main line. Then, modern research strategies and methods of herb pairs were summarized. Finally, the thesis thoughts were obtained and reasonable and feasible experimental programmes and technical courses were designed.Chapter2. Correlation analysis between integrative effects and bio-active components of Gui-Xiong herb pair on tonifying and promoting blood(1) Comparative analysis of main aromatic acids and phthalides in Angelicae Sinensis Radix (Danggui), Chuanxiong Rhizoma (Chuanxiong), and Gui-Xiong herb pair by a validated UHPLC-TQ/MSIn this study, an ultra-high performance liquid chromatography coupled with triple quadrupole mass (UHPLC-TQ/MS) method was developed for simultaneous determination of three aromatic acids (chlorogenic acid, caffeic acid, ferulic acid) and six phthalides (senkyunolide I, senkyunolide H, senkyunolide A, butylphthalide, ligustilide and butylidenephthalide) in Angelicae Sinensis Radix (Danggui, DG), Chuanxiong Rhizoma (Chuanxiong, CX), and Gui-Xiong herb pair (GX). Chromatographic separation was carried out on a ACQUITY UHPLC BEH Cl8column with0.1formic acid in water (A) and acetonitrile (B) at a flow rate of0.4mL·min-1, the column temperature was set at30℃; Waters XevoTM TQ worked in multiple reaction monitoring mode. The nine components were simultaneously determined within10min. The proposed method was fully validated in terms of linearity, sensitivity, precision, repeatability as well as recovery. The results showed that there were significant differences in their contents of DG and CX, and there were remarkable differences between the theorized content and observed content in GX. The content of each component in formulae was not just the simple addition among its content in the single herbs. These research results might be helpful to illustrate the drug interactions during decocting process of herb pair according to the quantity changes of these marker compounds, which would lay foundation to further reveal the compatibility rule of the herb pair and other related formulae.(2) Comparative evaluation of the tonifying blood effect of Gui-Xiong herb pair on blood deficiency miceBlood deficiency mice were induced by the combination of acetyl phenylhydrazine (APH) and cyclophosphamide (CTX). White blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), and hematocrit (HCT) of model group had significant difference in comparison with control mice, illustrating the model was successfully reproduced. The tonifying blood effects of Gui-Xiong herb pair (GX) with different ratios and preparations on blood deficiency mice were compared. Under the same ratio, the water and water-ethanol extracts of GX had better tonifying blood effect than its ethanol extracts. When preparation method of GX was same, the tonifying blood effect of Gui-Xiong1.5:1was superior to other ratios.(3) Comparative assessing the effects of Gui-Xing herb pair on hemorheology and cagulation function in acute blood stasis ratsActivating blood circulation and dissipating blood stasis effects of GX (1:1and1.5:1) herb pair and its combination herbs with different preparation methods were evaluated by using multi-attribute comprehensive index method. The results showed that all samples of GX with different ratios or preparation methods could decrease the hemorheological parameters, and improve the cagulation function indexes. Under the same extraction method, the total promoting blood effect of GX1:1ethanol extract was best. And the total promoting blood effect of Chuanxiong (CX) was better than Danggui (DG). Under the same ratio, the total promoting blood effect of GX ethanol extracts was better than its water and water-ethanol extracts. The compatibility of DG and CX could obviously improve the abnormality of hemorheology and blood coagulation function in acute blood stasis rats.(4) Relevance analysis between the integrative efficacy and components of Gui-Xiong herb pair on basis of artificial neural networkThe content of seven major active components (chlorogenic acid ChA, caffeic acid CA, ferulic acid FA, senkyunolide I SI, senkyunolide H SH, ligustilide LIG, and butylidenephthalide BDP) in Gui-Xiong herb pair and the total effects (NTB nourishing and tonifying blood; ADBS activating blood circulation and disspating blood stasis, RMRP regulating menstruation and relieving pain) were comprehensively analyzed by using the artificial neural network, respectively. Aromatic acids was the main effective components for NTB, especially ChA and CA; both aromatic acids and phthalides were the major active components for ADBS, especially ChA, SI and SH; both aromatic acids and phthalides were also the important active constituents for the effect of RMRP, especially LIG, CA, FA, and SI. Both aromatic acids and phthalides contributed to NTB, ADBS and RMRP. Aromatic acids laid the emphasis on NTB and ADBS, while phthalides laid the emphasis on RMRP. The results could provide a new idea for the basic research about the complicated bio-active constituents of the herbs and TCM formulae.Chapter3. The tonifying and promoting blood mechanism of Gui-Xiong herb pair on basis of metabolomics(1) Identification on the metabolites of main aromatic acids from Gui-Xiong herb pair and its combination herbs in blood deficiency rats plasma, urine and bileThe major metabolites of FA, ChA and CA in DG, CX and GX 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, ChA and CA in blood deficiency rats plasma, urine and bile were identified after oral administration of DG, CX and GX extracts using MSE and mass defect filter (MDF) techniques. On the basis of the fragment ion and metabolic pattern,25metabolites including parent, reduction, hydrolysis, methylation hydroxylation, sulfate conjugation, gucuronide conjugation, taurine conjugation, glycine conjugation, acetylation, cysteine conjugation, glutathione conjugation and other metabolites of FA, ChA and CA were tentatively identified. The major metabolite of phase I and phase Ⅱ of FA, ChA and CA in blood deficient rats plasma, urine and bile from DG, CX and GX extracts can be identified and provided referential information for material foundation of medicinal effectiveness and action mechanism of DG, CX and GX.(2) Comparative metabolomics analysis on hematopoietic functions of herb pair Gui-Xiong by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry with pattern recognition approachIn the present study, global metabolic profiling with UHPLC-QTOF/MS combined with pattern recognition methods was performed to discover differentiating metabolites of blood deficiency. In order to study the integrative efficacy and action mechanism of DG, CX and GX, Pearson correlation statistical analysis and MATLAB software were used to correlate the pharmacological effects and endogenous metabolites. As a result, a total of13endogenous metabolites (9upregulated and4downregulated) were identified, and12metabolic pathways contributing to blood deficiency were found. DG, CX and GX had potential pharmacological effects by regulating multiple perturbed pathways close to normal state. The holistically metabolomic results were consistent with the pharmacological tests (peripheral blood parameters); and they had close correlation. DG and CX had synergetic effect because the potential metabolites after GX intervention were closer to the control group than the two single herbs. These results suggested that GX and its single herbs might play a pivotal role in the treatment of blood deficiency syndrome by down-and up-regulating the levels of the metabolites. Overall, this study successfully demonstrated that the power of metabolomics in unraveling hematopoietic effects of GX; these results can provide help for better understanding the mechanisms of blood deficiency; and comprehensive metabolomic study by using UHPLC-QTOF/MS is potentially useful for studying the action mechanisms of the traditional Chinese herb pairs. What is more, an illustrative example was showed that there were close correlation between pharmacological indexes and endogenous metabolites. And the integrative efficacy and action mechanism of herbs or formulae might be demonstrated and solved by such correlation analysis.(3) Metabolomic strategy to study promoting blood action mechanism of Gui-Xiong herb pair in blood stasis ratsActivating blood circulation and dissipating blood stasis effects of GX and its combination herbs were evaluated by testing hemorheological parameters and cagulation function indexes. Three potential biomarkers including lysoPC (15:0), arachidonic acid and linoleic acid in rat plasma were tentatively identified. Compared with control group, arachidonic acid and linoleic acid in blood stasis rats increased significantly; while lysoPC (15:0) decreased significantly. Three potential biomarkers including L-phenylalanine, L-tyrosine, and L-tryptophan in rat urine were tentatively identified. Compared with control group, L-phenylalanine, L-tyrosine in blood stasis rats increased significantly; while L-tryptophan decreased significantly. Plasma and urine markers primarily involved in linoleic acid metabolism, arachidonic acid metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, tryptophan metabolism, and tyrosine metabolism. DG, CX and GX had potential pharmacological effects by regulating multiple perturbed pathways close to normal state. The compatibility of DG and CX had synergetic promoting blood effect because the potential metabolites after GX intervention were closer to the control group than the two single herbs. And the promoting blood effect of CX was slightly better than DG. Three potential biomarkers in plasma had stronger correlation with whole blood viscosity, whole blood relative index, and fibrinogen concentration; while the potential biomarkers in urine had weak correlation with hemorheology and cagulation function indexes. The combination between pharmacology and metabolomics has a bright prospect in the efficacy evaluation and effect mechanism elucidation of the traditional Chinese medicines.(4) Network pharmacology research on major active components of Gui-Xiong herb pairMolecular mechanism of nine major active components (ferulic acid, cholorogenic acid, caffeic acid, ligustilide, senkyunolide H, senkyunolide I, senkyunolide A, butylphthalide, butylidenephthalide) of Gui-Xiong herb pair was analyzed by using network pharmacology method. And the correlations of multi components-multi targets-multi pathways were investigated. Firstly, multi components-protein network was built on basis of PharmMapper database. Secondly, pathways of the proteins were obtained from MAS and KEGG databases. Finally, the components-targets-pathways network of Gui-Xiong herb pair was built by Cytoscape software. The results showed that nine components of Gui-Xiong herb pair involved31target proteins and54pathways. Several pathways involved in nerve-endocrine-immune-blood system. Aromatic acids and phthalides had ten common pathways; additionlly, they had their own emphasis. Results reflected the multi components-multi targets-multi pathways mode of Gui-Xiong herb pair, providing references for the further study of molecular mechanism. |
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