| Traditional Chinese Medicine(TCM)property theory has played an important role in the clinical application of traditional Chinese medicine.It expresses the pharmacological properties of Chinese medicine herbs in highly abstract and macro-level representation.Understanding the relationship between TCM property and its efficacy in the perspective of modern science helps to guide the research and development of TCM.In this paper,the efficacy of promoting blood circulation and removing the blood stasis was studied.The most relevant TCM property to this efficacy is pungent and bitter herbs.So the relationship between the efficacy of promoting blood circulation and pungent,together with the relationship between the effects of removing blood stasis and bitter were investigated First,the potential targets associated with the efficacy of promoting blood circulation and removing blood stasis have been identified using literature mining and data analysis techniques.Then,the active ingredients of TCM and their targets have been found using virtual screening based on molecular modeling followed by high-throughput screening on cellular level.Finally,the relationship between the efficacy of promoting blood circulation and pungent,together with the relationship between the effect of removing blood stasis and bitter have been studied in the scales of compounds,constituents and pieces.Around the content and aim of this subject,three parts are organized in this research:1.Identification of the targets associated with the efficacy "Xin-Xing-Ku-Xie"Identification of the targets associated with "Xin-Xing-Ku-Xie",has been divided into two parts.The first part is the identification of taste receptors,which contains the protein structures,distributions and their ligands message of pungent and bitter taste receptors.The second part is the identification of targets associated with the efficacy of promoting blood circulation and removing blood stasis.These targets have been identified by the following steps:Frist,the database of TCM was established including their properties,effects and modern pharmacology.Then,data mining techniques were used to calculate the related pharmacological effects associated with the efficacy of promoting blood circulation and removing blood stasis.DrugBank and other databases have been searched to find out the potential targets relevant to the pharmacological effects.And the potential targets are finally considered to be the targets of "Xin-Xing-Ku-Xie".The results shows that there are 25 receptors belonging to T2Rs family and 6 common receptors named TRPV1,TRPV2,TRPV3,TRPV4,TRPA1 and TRPM8 are related to the flavor of bitter and pungent.And there are 22 targets are associated with the efficacy of promoting blood circulation and removing blood stasis.These 22 targets have various pharmacological effects containing vasodilation(such as Ca2+channel,Protein kinase C,NOS,ACE,AT1,ETa,ETb,ECE-1 and so on.),regulation of blood lipids(such as HMG-CoA reductase,NPC1L1,PPAR-a,LXR,CETP,CYP7A1 and so on.),anti-platelet aggregation(such as TxA2 receptor,PAF receptor,P2Y12 receptor,GP?b/?a receptor and so on.),cardiac stimulant(such as Na+-K+-ATP enzyme,phosphodiesterase ? and so on.),anticoagulant effect(such as Thrombin,PARI receptor and so on.),anti-inflammatory(such as PDE4A,A2B,COX2 and so on)and other pharmacological effects and related targets.This part has established the relationship between the efficacy of promoting blood circulation or removing blood thrombus(Macro-level)and its related pharmacological effects including biological targets(Micro-level).In addition,this study has also found that some key pharmacological effects including improving the components and flow rate of blood,expansion of blood vessels and cardiac function are related to the efficacy of promoting blood circulation or removing the blockage in the blood.This part of research has laid the foundation for further study to identify the material basis of the efficacy "Xin-Xing-Ku-Xie".2.Identification of the material basis associated with the efficacy "Xin-Xing-Ku-Xie".The purpose of this part is to identify the active ingredients of TCM and their targets based on the previous research.In this part,pharmacophore model,molecular docking and homology modeling have been used to perform virtual screening against the database of TCM for active ingredients.Then,high-throughput screening technology on the cellular level was performed to verify the results of virtual screening.In this part,the pharmacophore models of TRPV1 agonists,TRPM8 agonists,T2R1 agonists,T2R10 agonists,T2R14 agonists and T2R46 agonists have been established.Also the pharmacophore models of the targets associated with the effect of improving the components and flow rate of blood have been established,including NOS?ETa?ETb?ECE-1?AT1?AA1R?PAR1?Thrombin?PDE4A?A2B?HMGCoAR?P2Y12?LXR-alpha?CEPT?NPC1L1?Na+-K+-ATPase?PDE3A?PAF receptor?TXA2_synthase and TXA2_PGH2.Homology modeling technology has been used to build the 3D protein structure of AT1 receptor,ETa receptor and ETb receptor.Molecular docking method has been utilized to perform the docking study of AT1,Thrombin,ECE-1,ETa and ETb.The molecular models above are applied to perform virtual screening against TCMD2009 database resulting in a hit list of 15588 compounds.And the database which contain the information of active ingredients,distribution in TCM herbs,pharmacological effects,targets,herbs' property and efficacy have been established.To verify the reliability of the virtual screening,high throughput screening based on the cellular level methods has been applied together to screen a database including 144 Chinese medicine herbs compounds·Taking AT1 receptor as an example,two novel AT1 receptor antagonists have been found based on high-throughput screening and their IC50 values are less than 10uM in further experiments of dose-response curves.At the same time,9 potential AT1 receptor antagonists are hit by virtual screening based on molecular modeling.Fortunately,the 9 hits contain the 2 ATI receptor antagonists that were confirmed in the high-throughput screening.And the identification index which definite multiple relationship between the efficiency of virtual screening and random screening are calculated to be 16.Compared with the high throughput screening,virtual screening has higher efficiency in lower cost and it has a broad application in TCM active ingredients identification.The results of virtual screening shows that there are 60.11 and 62.28 percent of TCM hit by TRPV1 and TRPM8 agonist pharmacophore models are pungent herbs,respectively.And 71.84 percent of TCM compounds hit by T2R1,T2R10,T2R14 and T2R46 agonist pharmacophore models are bitter herbs.These results indicate that TRPV1 and TRPM8 agonist pharmacophore models have enrichment effect of pungent herbs.T2R1,T2R10,T2R14 and T2R46 agonist pharmacophore models have enrichment effect of bitter herbs.The hits by pharmacophore models of the targets associated with the efficacy of"Xin-Xing-Ku-Xie" has pharmacological effects in antiplatelet,antithrombotic,improving hemodynamics,coronary dilation,regulating blood lipids and improving microcirculation.These results help to understand the mechanism of TCM.3.Studying the relationship between flavors and the efficacy "Xin-Xing-Ku-Xie".The part is carried out to study the relationship between flavors and the efficacy related to"Xin-Xing-Ku-Xie" in three different angles.The first angle is the oral taste caused by TCM ingredients.The second angle is the function of pungent and bitter when TCM ingredients acting on targets in vivo.The third angle is the different scales of TCM material basis containing compounds,constituents and pieces.Based on the database of TCM active ingredients and their targets established in previous study,the relationship between flavors and efficacy is studied in this part.The results shows that 1317 TCM compounds can not only activate TRPV1 channel but also act on AT1,HMGCoA,PDE3A and PAFR targets,which trigger a series of pharmacological effects including vasodilation,antihypertensive,anti-atherosclerosis,regulating blood lipids,anti-platelet aggregation and cardiac effect.It reflects the TCM theory of "Pungent can promote blood circulation".On the other hand,1536 TCM compounds can not only activateT2R1,T2R10 T2R14 and T2R46 receptor,but also act on thrombin,AT1,PAFR,TXA2-synthase and other receptors,which trigger a series of pharmacological effects including anticoagulant,vasodilation,anti-atherosclerotic,anti-platelet aggregation and so on.It reflects the TCM theory of "Bitter can remove the blood stasis".The specificity analysis revealed that the pharmacological effects relevant to pungent are regulating blood lipids,enhancing myocardial motion,anti-platelet aggregation and vasodilation.The pharmacological effects relevant to bitter are anticoagulant,anti-inflammatory,anti-bacterial and antitumor effect.It suggests that pungent and bitter flavors are consistent with their TCM effects in the macro trend.The relationship of flavors and effects has been studied in different scales of compounds,constituents and pieces.Taking Salvia miltiorrhiza as an example,the compounds such as Salvianolic acid B and salvianolic acid A can act on thrombin,AT1 and PAFR receptors,which trigger a series of pharmacological effects including anticoagulation,vasodilation,antihypertensive,anti-atherosclerotic and anti-platelet aggregation.In addition,we find that the constituents of Salvia phenolic acids playing important rule in anticoagulant,anti-inflammatory,antioxidant,antithrombotic,protecting endothelial cells and other pharmacological effects using the technology of literature mining.And the piece of Salvia miltiorrhiza is documented to have the efficacy of removing blood stasis and pains,promoting blood circulation and menstruation.It intimates that the property of pungent and bitter having the characteristics of inheritance,additivity and consistency in different scales of compounds,constituents and pieces.The efficacy of TCM is attributed to the chemical constituents and their multi-targets,which is much meaningful to study the relationship between the multiple constituents-targets and their efficacy.In this paper,the target spectrum of TCM constituents has been proposed to study the multi-targets and efficacy.Taking and Carthamus tinctorius as example,the target spectrum of the main constituents from Salvia miltiorrhiza and Carthamus tinctorius L.are constructed and analyzed.The results show that Salvia miltiorrzahiza and Carthamus tinctorius L both have anti-thrombotic effect,but the ways they trigger the pharmacological effect are different.Carthamus tinctorius L focus on the regulation of blood lipids,cardiac and anti-platelet aggregation,while Salvia miltiorrhiza focus on the effects of anti-clotting,anti-bacterial and anti-cancer.It demonstrates that the target spectrum of TCM constituents is a good method to understand the different effects of TCM.In summary,this paper is carried out to study the target identification associated with the efficacy of promoting blood circulation and removing the blood stasis.Then,the active ingredients of TCM and their targets have been identified.Finally,the relationship between flavors property and efficacy have been studied and discussed.The innovative ideas and methods are proposed to uncover mysterious scientific connotation of the relationship between TCM property and its efficacy.It has important significance for the research and development of TCM under the theoretical guidance of TCM property. |
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