| Background:The theory of the properties of traditional Chinese medicines(TCMs)is the core of traditional Chinese medicine theory,which is the indispensable component in the formation and development of TCMs and summarizes the properties and characteristics of the functions of TCMs.Due to the influence of traditional simple cognitive method and thinking concept,the lack of the modern scientific explanation for the essence of the properties of TCMs makes it impossible that TCMs were used under the guidance of the theoretical system and correctly included in the modern drug production management system.Therefore,establishing a characterization system and standardized standards by utilizing modern scientific methods and technologies to reveal the essence of the of properties TCMs is the key scientific issue in the theoretical research of the properties of traditional Chinese medicine.Rhizoma Coptidis and Rhizoma Corydalis,the representative TCMs for cold and warm,have similarities in material basis and differences in cold and hot nature.They are clinically used for the treatment of heatwith-phlegm and cold cold-coagulation-hear-pulse heart pain,which is the manifestation of treating the same disease with different methods in the treatment of cardiovascular diseases.However,the chemical and biological essence which related to the cold and hot properties of TCMs is not yet clear.Objective:To explore the structure-activity-property relationship and mechanism of action of protoberberine alkaloids from Rhizoma Coptidis and Rhizoma Corydalis,and reveal the chemical biological essence of the cold and hot medicinal properties of Rhizoma Coptidis and Rhizoma Corydalis,and provide reference for the understanding of medicinal properties and the medicinal properties of TCMs components from multiple perspectives and levels.Methods:I Evaluation of cold and hot properties of Chinese medicines containing isoquinoline alkaloids.Chinese medicines containing isoquinoline alkaloids are collected,and the correlation between angiogenesis efficacy and cold and hot drug properties is verified.1.Collection and sorting of traditional Chinese medicine containing isoquinoline alkaloids.TCMs who contain isoquinoline alkaloids are collected according to the content determination item in the Chinese Pharmacopoeia 2020.On the other hand,In CNKI,Chinese medicines containing isoquinoline alkaloids are collected with the subject word as "isoquinoline alkaloids"and the discipline setted as TCMs,and the nature,taste,meridian orientation,and main therapeutic effects of they are sorted.2.Evaluation of angiogenic efficacy.The main evaluation indicators of the qCAM model include survival rate and morphological observation of angiogenesis.Morphological observation of angiogenesis includes angiogenesis area,total number of blood vessels,and microvessel count(third+fourth).The area of angiogenesis,the number of microvessels and the total number of blood vessels of 19 Chinese medicines containing isoquinoline alkaloids were detected by qCAM model after 48 h intervention with water extracts of 5 mg·mL-1,10 mg·mL-1 and 20 mg·mL-1,respectively.3.Correlation test between angiogenic efficacy and cold and hot drug properties.The differences in the effects of 19 Chinese medicines on the angiogenesis area,total vessel count,and microvascular count of qCAM are evaluated by LSD-t test.The chi-square test was used to evaluate the differences between drug properties and angiogenesis groups,in order to verify the correlation between the angiogenic efficacy of the qCAM model and the cold and hot drug properties.Ⅱ Evaluation of the cold and hot pharmacological properties of berberine type alkaloids from Rhizoma Coptidis and Rhizoma Corydalis.The components of protoberberine alkaloids in Rhizoma Coptidis and Rhizoma Corydalis are identified,and the cold and hot effects of protoberberine alkaloids are evaluated from the perspectives of angiogenesis and material and energy metabolism.1.Analysis of protoberberine alkaloids in Rhizoma Coptidis and Rhizoma Corydalis.UPLC-Q-Exocative Plus Orbitrap-MS is used to analyse the components of Rhizoma Coptidis and Rhizoma Corydalis.2.Evaluation of the pharmacological properties of protoberberine alkaloids in Rhizoma Coptidis and Rhizoma Corydalis based on angiogenesis.The areas of angiogenesis after 48 hours of protoberberine alkaloids in Rhizoma Coptidis and Rhizoma Corydalis with the concentration of 10 μM、20 μM、40 μM、80 μM based on qCAM are evaluated.3.Validation of the properties of protoberberine alkaloids based on angiogenesis.(1)qPCR experiment.The levels of VEGFR2 mRNA in qCAM are detectied.(2)HUVEC validation.The effects of 1.25 μM、2.5μM、5 μM、10 μM、20 μM、40 μM、80 μM、160μprotoberberine alkaloids treatment for 24 h on HUVEC cell activity.4.Evaluation of the properties of protoberberine alkaloids based on material and energy metabolism.The biological reagent kits are used to detect the material and energy metabolism related indicators(the content of ATP content,the activity of SDH,the activity of NA+-K+ATPase,the activity of Ca2+-Mg2+-ATPase,the content of NO content and the content of PA)of qCAM after administration.Ⅲ Study on the mechanism of the properties of Rhizoma Coptidis and Rhizoma Corydalis.Systems pharmacology is used to reveal the mechanism of action of the main protoberberine alkaloids in Rhizoma Coptidis and Rhizoma Corydalis.1.The mechanisms of of Rhizoma Coptidis and Rhizoma Corydalis on the treatment of cold and heat syndromes based on network pharmacology.Network pharmacology is used to construct the network of "the components of Rhizoma Coptidis-heat syndrome" and "the component of Rhizoma Corydalis-cold syndrome",and the cytoHubba plugin is used to mine key genes(hub genes)based on maximum cluster centrality(MCC)sorting.The GO biological process and KEGG pathways are analyzed through DAVID 2021.2.The metabolic mechanism of cold-related components in Rhizoma Coptidis based on untargeted metabolomics.DESI-MSI untargeted metabolomics is used to analyse the differential metabolites and key metabolic pathways of qCAM after 40 μM oxyberberine treatment for 48 h.Ⅳ Analysis of the "structure-activity-propery" relationship of berberine type alkaloids in Rhizoma Coptidis and Rhizoma Corydalis.1.Analysis of interaction between property-related components and syndrome targets.The combination of protoberberine alkaloids and key cold-and-heat syndrome targets obtained from network pharmacology is analyzed through molecular docking.Docking binding energy,bonding type,bonding number,bonding site,binding amino acid type and other parameters are obtained.2.Calculation of structural parameters of protoberberine alkaloids and analysis of"structure-activity" relationship.Structural parameters of protoberberine alkaloids were calculated by using Gaussian09W to finish the full optimization of geometric configuration and frequency calculation with B3LYP/6-31G,and B3LYP function and 6-31G(d,p)basis set are used to calculate the quantum chemistry structural parameters of protoberberine alkaloids in Rhizoma Coptidis and Rhizoma Corydalis.Further calculations were conducted using the QSAR module in the Hyperchem 7.0 package to obtain physical and chemical property parameters.Results:I There is a significant difference(P<0.05)between cold(cool)and hot(warm)herbs in angiogenetic efficacy among TCMs contained isoquinoline alkaloids,which showed that cold or cool herbs inhibited angiogenesis,while hot or warm herbs promoted angiogenesis in the qCAM model.II The protoberberine alkaloids are the main components shared by Rhizoma Coptidis and Rhizoma Corydalis.Oxyberberine of Rhizoma Coptidis shows the most significant inhibitory effect on angiogenesis,and reduce energy metabolism indicators such as the content of ATP,the activity of SDH activity,the activity of Na+-K+-ATPase,the activity of Ca2+-Mg2+-ATPase,the content of NO,etc.Tetrahydropalmatine of Rhizoma Corydalis shows the most significant promoting effect on angiogenesis,and can upregulate the content of ATP,the activity of SDH activity,the activity of Na+-K+-ATPase,the activity of Ca2+-Mg2+-ATPase,the content of NO and other energy metabolism indicators.Therefore,it is speculated that oxyberberine is the key pharmacological components that contribute significantly to the cold nature of Rhizoma Coptidis,while tetrahydropalmatine is the key pharmacological component that contributes significantly to the warm nature of Rhizoma Corydalis.Ⅲ It was found that Rhizoma Coptidis and Rhizoma Corydalis can jointly act on common targets both related to heat or cold syndromes such as EGFR,VEGFR2,JAK2,ESR1,etc.,thereby regulate biological processes such as cell proliferation and migration,angiogenesis,inflammatory response,hematopoietic function,and cell apoptosis.There are 63 KEGG pathways related to heat syndrome that Rhizoma Coptidis acts on,including 12 specific pathways.On the other hand,85 KEGG pathways related to cold syndrome that Rhizoma Corydalis acts on,including 34 specific pathways.There are 51 pathways that Rhizoma Coptidis and Rhizoma Corydalis both act on.The effect of the cold medicine Rhizoma Coptidis tends to be related to the disease pathway of excessive cell proliferation,especially various pathways related to tumor occurrence and development,as well as the endocrine system.The warm medicine Rhizoma Corydalis mainly acts on thyroid function,sex hormone regulation,tumor growth related pathways,and angiogenesis pathways in the endocrine system.Further it is revealed that the key pharmacological components of Rhizoma Coptidis mainly regulate the metabolic pathways of D-glutamine and D-glutamate,while affecting the metabolism of Darginine and D-ornithine,the biosynthesis of valine,leucine and isoleucine,and the metabolic pathway of arginine biosynthesis based on untargeted metabolomics.Compared with the key pharmacological components of Rhizoma Coptidis affect the metabolism centered on the arginine metabolism,which has been shown in our previous studies.That is,there are significantly different change in metabolism after the action of Rhizoma Coptidis and Rhizoma Corydalis.Ⅳ The binding energy of berberine,oxyberberine and the syndrome target VEGFR2,JAK2,EGFR,which are the key effective components of Rhizoma Coptidis,are lower than the binding energy of tetrahydropalmatine,the key effective component of Rhizoma Corydalis.Physical-chemical property parameters and quantum chemistry descriptors show that the absolute value of the net charge of nitrogen atom of the main components of Rhizoma Corydalis is lower than that of the components of Rhizoma Coptidis,ELUMO and molecular polarizability are generally higher than those of the components of Rhizoma Coptidis.From the perspective of molecular composition,the proportion of oxygen atoms in the molecules of Rhizoma Coptidis is higher than that of Rhizoma Corydalis.These four parameters have potential relevance and regularity with the trend of the components of Rhizoma Coptidis and Rhizoma Corydalis in angiogenesis and material and energy metabolism regulation.Conclusion:There is a significant difference between cold(cool)and hot(warm)herbs in angiogenetic efficacy among TCMs contained isoquinoline alkaloids,which showed that cold or cool herbs inhibited angiogenesis,while hot or warm herbs promoted angiogenesis.Berberine and oxyberberine can significantly inhibit angiogenesis and material and energy metabolism,and they are key pharmacological components associated with the cold property of Rhizoma Coptidis.Tetrahydropalmatine can promote angiogenesis and material and energy metabolism,which is the key pharmacological component associated with the warm nature of Rhizoma Corydalis.Rhizoma Coptidis and Rhizoma Corydalis act on a large amount of identical biological processes,but their metabolic profiles differ.Specifically,the key pharmacological components of Rhizoma Coptidis affect the biosynthesis of various kinds of amino acids,mainly glutamic acid metabolism,while the key pharmacological component of Rhizoma Corydalis mainly affects arginine metabolism.It is speculated that this is because the main alkaloids contained have partially identical components and the same skeleton some components with similar configurations,but different substituents and conjugated system on the same skeleton lead to different action trends and intensities,which lead to differences in the final cold-andhot properties.Among the components whose structures are protoberberine skeletons,those close to the plane,more complete conjugated system,higher absolute value of net charge of nitrogen atom,higher proportion of oxygen atom compounds with lower lowest molecular orbital energy have stronger correlation with cold,and their binding energy with the same key syndrome target is lower than that of the components of warm Chinese medicine,showing higher response,stronger inhibition of angiogenesis and down-regulation of energy metabolism. |
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