| Chinese herbal medicine processing is an important part of traditional Chinese medicine(TCM).The chinese medicine properties has significant changed after processing.According to the different pathogenesis of TCM syndromes,different kinds of processed products were selected for treatment,which is a characteristic of TCM.However,the theory of Chinese medicine processing lacks modern scientific arguments and became a bottleneck restricting the modernization and internationalization of Chinese medicine industry.Therefore,it is very important to conduct in-depth research on the mechanism of Chinese herbal medicine processing and clarify its mechanism by using modern frontier science and technology.The fresh rhizome of Curcuma wenyujin Y.H.Chen et C.Ling can processed by cutting,steaming and boiling with vinegar to obtain three differdnt kinds of decoction pieces including Pian-JiangHuang(PJH),Sheng-Ezhu(SEZ)and Cu-Ezhu(CEZ),respectively.The three kinds of decoction pieces all have the effect of promoting Qi,breaking blood and relieving pain.However,they have some differences and similarities in clinical application.PJH is good at breaking blood,promoting qi and inducing menstruation to relieve menalgia;SEZ have the effect of promoting qi,breaking blood,removing qi stagnation to relieve pain,which is good at remove blood stasis by breaking blood;CEZ enters the liver meridian and enhances its effect of breaking blood and removing blood stasis.The effects of three kinds of decoction pieces on promoting qi,relieving pain and removing blood stasis were enhanced in turn.At present,the mechanism of effect change of the three kinds of decoction pieces had not been explored.In view of the above problems,this study innovatively introduced the modern frontier scientific research technologies such as chromatographic pharmacodynamics of TCM,metabonomics and network pharmacology into the research field of Chinese herbal medicine processing.Taking pharmacodynamic substances and endogenous metabolites as entry points,the effective substances were screened by spectrum-effect correlation study.Metabonomics was used to evaluate the different effects of three kinds of decoction pieces on endogenous metabolites and metabolic pathways,and network pharmacology was used to comprehensively analyze the correlation between effective substances,endogenous metabolites and metabolic pathways.The research is to clarify the connotation of scientific hypothesis that the three kinds of decoction pieces of Curcuma wenyujin is "through the effective substances to multiple regulation of endogenous metabolites and metabolic pathways,which leads to the difference of the effects of promoting Qi,breaking blood and relieving pain.The project intends to systematically interprete TCM processing theory from multi-ingredients,multi-targets and multi-way,providing scientific basis for the clinical application of Chinese herbal pieces and a demonstration for the modern research of Chinese herbal pieces.The specific research contents are as follows:1.Literature researchThe historical evolution of the three kinds of processed products of Curcuma wenyujin,including all previous dynasties literature,different versions of Chinese Pharmacopoeia,the preparation standards of provinces and cities,the main chemical constituents and pharmacological effects of the three decoctions,the etiology and pathogenesis of blood stasis syndrome,the spectrum and pharmacodynamics study of TCM,metabolomics and other modern frontier scientific research technologies in the field of TCM were reviewed in detail.The above literature research laid the foundation of the research scheme design and smooth development of the study.2.Study on the effect of processing on components of three kinds of decoction pieces of Curcuma wenyujin(1)The results of UPLC multi-component content determination and fingerprint analysis showed that the contents of Curdione and β-elemene decreased in turn in PJH,SEZ and CEZ,while the contents of Curcumol,Germacrone and Furanodiene increased first and then decreased in the three decoction pieces.The results of multivariate statistical analysis of fingerprint showed that there were significant differences in the quality of the three decoction pieces.Except for β-elemene,the chemical structure of other peaks could not be determined.(2)The results of mass spectrometry analysis showed that the relative content of 21 compounds identified in three kinds of decoction pieces were significantly changed.The contents of Curdione and other four compounds in PJH,SEZ and CEZ were decreased in turn.The relative content of curcumenolactones A in SEZ was lower than that of PJH,but higher than in CEZ.We speculate that this may be related to acidic conditions in the processing of CEZ.In addition,the relative content of 11 compounds in SEZ and CEZ was higher than that of PJH,among which Zederone and Curzeone in the CEZ were higher than that of SEZ,Aerugidiol,Furanodiene and Curcumin in the CEZ were lower than that of SEZ.The results of multivariate statistical analysis showed that the four compounds of Curcumenone,Curcumol,Curzerenone and Furanodiene were potential quality markers between PJH and SEZ/CEZ;Three compounds of Curcumenone,Furanodiene and Germacrone are potential quality markers between SEZ and PJH/CEZ;Three compounds of Curcumenone,Curcumol and Curzerenone are potential quality markers between SEZ and PJH/CEZ.Through the above research,among the 21 chemical constituents in the three kinds of decoction pieces of Curcuma wenyujin after processing.The contents of four chemical constituents were the highest in PJH,eight chemical constituents were the highest in SEZ,and two chemical constituents were the highest in CEZ.The changes of the material basis of the three kinds of decoction pieces were preliminarily clarified,which provided the basis for the follow-up mechanism research.3.Evaluation of the effect of different processed products of Curcuma wenyujin on promoting blood circulation and removing blood stasisThe rat model of qi stagnation and blood stasis was established by hypodermic injection adrenaline hydrochloride plus ice water swimming.The rats were treated by intragastric administration with water decoctions of three kinds of processed products of Curcuma wenyujin.The hemorheology,blood coagulation of the four indicators,fibrinolytic factors and inflammatory factors were detected to evaluate the similarities and differences of the effects of three kinds of decoction pieces on promoting blood circulation and removing blood stasis.The results showed that,except for the low dose group of PJH,which was no improvement in blood viscosity and plasma viscosity,the other groups all had statistical significance,and the high dose group of CEZ had the most significant effect;The results of effect of four blood coagulation indexes showed that the low dose group of PJH had no improvement in activated partial thromboplastin time(APTT).Both low and high dose groups of PJH had no improvement on fibrinogen(FIB).The low dose group of CEZ had no improvement on thrombin time(PT)and APTT.The other groups had significant improvement on four indexes of coagulation.The high dose group of CEZ had the most significant effect on the four indexes than the other groups.All groups had significant improvement on Tissue-Type Plasminogen Activator(t-PA).The high-dose and low-dose group of PJH,low-dose group of SEZ and low-dose group of CEZ had no improvement on tissue plasminogen activator Inhibitor(PAI-1).The high-dose group of SEZ could reduce PAI-1.The high dose group of CEZ had the best effect compared with other groups.Except the low-dose group of SEZ and CEZ,the other groups could significantly reduce the levels of interleukin-6(IL-6)and tumor necrosis factor-alpha(TNF-α).Through pharmacodynamic evaluation of three kinds of decoction pieces of Curcuma wenyujin,the results showed that the high and low dose groups of PJH had no regulating effect on FIB and PAI-1,the low dose group of SEZ had no regulating effect on PAI-1,IL-6 and TNF-α,the low dose group of CEZ had no improving effect on TT,APTT,PAI-1 and IL-6,the other groups all had significant improving effect on Hemorheological indexes,and the high-dose group had the best effect.4.Study on spectrum-effect relationship of different processed products of Curcuma wenyujinAfter the blood stasis model rats were treated with different processed products of Curcuma wenyujin,the time-varying curves of blood components and that of pharmacodynamic indexes of each group were analyzed by bivariate correlation analysis.The results showed that the active ingredients related to pharmacodynamics of promoting blood circulation and removing blood stasis in the three kinds of decoction pieces and the process of internal pharmacodynamics were both significant changed.From the aspect of components in plasma,10 components were identified in the plasma of blood-stasis model rats treated with PJH.Among them,Curcumenolactones A was only detected in PJH.The results of bivariate correlation analysis showed that Curcumenolactones A were significantly correlated with APTT and FIB.It was speculated that Curcumenolactones A might play a role of "breaking blood and promoting qi" in the decoction pieces of PJH through liver protection combined with other components in plasma.Ten components were also identified in the plasma samples of blood stasis model rats treated with SEZ,but the types were different with PJH.Curdione and Curcumenone were unique components of SEZ compared with PJH group.Curdione had no significant correlation with pharmacodynamic indexes,but Curcumenone had significant correlation with blood viscosity and APTT.Germacrone,Neocurdione and Curzerenone in SEZ were their specific components in plasma compared with CEZ group.Eight components were identified in plasma samples of blood stasis model rats treated with CEZ.Although the number of components decreased,the results of bivariate correlation analysis showed a higher correlation between components and pharmacodynamics,and the relative content of Zedoalactone B increased significantly,but it has no correlation with pharmacodynamics.The relative content of Zederone also increased slightly,and had significant correlation with whole blood viscosity,PT and FIB.From the time-varying trend of components in plasma,the metabolic process of components in the three kinds of decoction pieces also significantly changed.The plasma concentration of components in PJH was 4 hours,which was 0.5-2 hours in SEZ,and 0.5 hours in CEZ.In addition,the relative content of components in CEZ can last more time in plasma.This may be related to the changes of intestinal environment and blood environment caused by decoction pieces,leading to changes in the metabolic process of components in plasma,thus producing different pharmacodynamic reactions.From the results of bivariate correlation analysis of components and pharmacodynamic indices of three kinds of decoction pieces,although there are more components in the PJH,the correlation between components and pharmacodynamics is worse than compared with SEZ group and CEZ group.The number of components in CEZ was decreased,but there was a very significant correlation with more pharmacodynamic indicators(P<0.01).Based on the above analysis,Curcumenolactones A was the characteristic component in the 10 blood-entry components of PJH,and the five components of Germacrone,Neocurdione,Zedoalactone B,Zedoarofuran and Zederone had higher correlation with pharmacodynamic indexes;among the 10 blood-entry components of SEZ,Germacrone,Curzerenone and Zedoarofuran had higher correlation with pharmacodynamic indexes;Among the eight components of CEZ,five components of Curdione,Furanodiene,Curcumenol,Curcumenone and Aerugidiol were highly correlated with the pharmacodynamic indexes.The correlation and similarities and differences between the components in plasma of the three kinds of decoction pieces and the pharmacodynamics of promoting blood circulation and removing blood stasis were clearly defined,which provided sufficient material basis for the follow-up study of processing mechanism.5.Metabonomics study on different processed products of Curcuma wenyujinThe mechanism of promoting blood circulation and removing blood stasis in different processed products of Curcuma wenyujin was preliminarily studied by metabonomics.The potential biomarkers of Qi stagnation and blood stasis model and the improvement effect of endogenous biomarkers by administration of three kinds of decoction pieces of Curcuma wenyujin were clarified.Compared three groups of decoction pieces of Curcuma wenyujin with the model group,70 biomarkers in plasma,bile and urine samples with significant differences were screened and identified.Metabolic pathways involved in 70 biomarkers included linoleic acid metabolism,α-linolenic acid metabolism,glycerol phospholipid metabolism,sphingolipid metabolism,arachidonic acid metabolism,primary bile acid biosynthesis,vitamin B6 metabolism and so on.Among them,PJH has a regulatory effect on linoleic acid metabolic pathway,but SEZ and CEZ has no effect.The number of endogenous metabolites in primary bile acid biosynthetic pathway regulated by PJH,SEZ and CEZ increases gradually.PJH and CEZ have regulatory effect on vitamin B6 metabolic pathway,while PJH has no effect.CEZ has a regulatory effect on tryptophan metabolism pathway,but PJH and SEZ have no effect.According to the above research,the improvement of Qi stagnation and blood stasis by three different processed products of Curcuma wenyujin was mainly related to the regulation of lipid metabolism.The related metabolic pathways and the types of endogenous metabolites regulated by different decoction pieces are obviously different.PJH specifically regulates linoleic acid metabolic pathway,SEZ has the regulation effect on all related pathways,CEZ specifically regulates tryptophan metabolic pathway.Metabonomics study preliminarily clarified the processing mechanism of different processed products of Curcuma wenyujin on promoting blood circulation and removing blood stasis.6."Component-Target-Pathway" network pharmacology study of different Processed Products of Curcuma wenyujinWith the method of network pharmacology and the related online database,21 chemical constituents identified from three kinds of Processed Products of Curcuma wenyujin and their components in plasma were used to construct "component-target-pathway" network relationship respectively,the similarities and differences of target proteins and related pathways in three kinds of decoction pieces were compared.The results showed that there were 124 protein targets and 176 metabolic pathways predicted by target database for 21 chemical constituents of Curcuma wenyujin,in which five metabolic pathways including linoleic acid metabolism,sphingolipid metabolism,glyceride metabolism,arachidonic acid metabolism and bile secretion were consistent with the results of metabonomics study.The network relationship of "blood-entry components-target-pathway" was constructed.Because of the different blood-entry components,the three Processed Products of Curcuma wenyujin have different effects on different protein targets and pathways in vivo,thus playing a common role in promoting blood circulation and removing blood stasis,but also have their own characteristics.Through the study on the material basis,pharmacodynamic evaluation of promoting blood circulation and removing blood stasis,Spectrum-Effect correlation and a series of effective substances and mechanisms of the above three different processed products of Curcuma wenyujin,the potential quality markers of PJH were four components including Curcumol;The potential quality markers of SEZ were three components including Furanodiene;The potential quality markers of CEZ were three components including Curcumenone.The pharmacological effects of promoting blood circulation and removing blood stasis showed that the high dose group of CEZ had the most significant effect.Spectrum-effect correlation studies showed that Curcumenolactones A was the characteristic component of 10 blood-entry components of PJH,while 5 components of Germacrone,Neocurdione,Zedoalactone B,Zedoarofuran and Zederone were highly correlated with pharmacodynamic indexes.Among the 10 blood-entry components of SEZ,the three components of Germacrone,Curzerenone and Zedoarofuran had higher correlation with pharmacodynamic indexes.Among the eight blood-entry components of CEZ,the five components of Curdione,Furanodiene,Curcumenol,Curcumenone and Aerugidiol had higher correlation with pharmacodynamic indexes.Metabonomics and network pharmacological analysis showed that the improvement of Qi stagnation and blood stasis by different processed products of Curcuma wenyujin was closely related to lipid metabolism,such as linoleic acid metabolism and glycerol phospholipid metabolism.PJH could specifically regulate linoleic acid metabolism pathway.SEZ could regulate all related pathways.CEZ could specifically regulate tryptophan metabolism pathway,which preliminarily elucidated the effect of different products of Curcuma wenyujin on qi stagnation and blood stasis and the processing mechanism of activating blood circulation and removing blood stasis in different processed products of Curcuma wenyujin.It laid a theoretical and material foundation for the follow-up research,in order to reveal the processing mechanism of "three drugs of the same origin" of different processed products of Curcuma wenyujin more systematically and deeply. |
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