Study On The Difference Of Ingredients And The Effect Of Jianchang Gang Of Stewed Polygala Tenuifolia Before And After Processing

[Objective]Qualitative and quantitative analysis of fingerprint spectra of Polygala tenuifolia before and after brewing using HPLC method,to study the effect of brewing method on the changes in chemical components of Polygala tenuifolia,and to explore the differences in changes in its processed components;Using UPLC-MS/MS method,the effects of processed components of Polygala tenuifolia on pharmacokinetics and tissue distribution in rats were studied to analyze the differences in absorption and distribution in vivo before and after processing;Using Text Data Mining Technology and pharmacological experiments to study the effects of stewed Polygala tenuifolia on calming the nerves,in order to reveal the material basis of its pharmacological effects.Correlation analysis is conducted on the differences in composition,absorption,distribution,and pharmacological effects between pre and post processing Polygala tenuifolia,in order to reveal the processing mechanism of"Jianchang gang"Polygala tenuifolia’s calming effect and provide a theoretical basis for its clinical application and promotion.[Methods and Results]1 Study of differences in fingerprint and processed componentsHPLC method was used to establish the fingerprints of polygala tenuifolia before and after stewing with different core-pulling degree and different grades,and cluster analysis was used for classification,in order to explore the difference of the material basis before and after the stewing,the components which had great influence on the stewing of Polygala tenuifolia were screened by orthogonal partial least squares discriminant analysis.The results showed that there was no significant difference in the number of chromatographic peaks in the HPLC fingerprints between Polygala tenuifolia and stewed Polygala tenuifolia,and between Polygala tenuifolia with and without core-drawing.The similarity ranged from 0.909 to 0.995,the results showed that the chemical composition of Polygala tenuifolia and stewed Polygala tenuifoliawas similar,and the chemical composition of Polygala tenuifolia with and without core-drawing was similar.But raw and stewed Polygala tenuifolia can be divided into two categories.Among them,3,6’-disinapoyl sucrose,sibiricose A5,sibiricose A6,polygalactoneⅢ,and peak 13 contribute the most to distinguish the fingerprints of raw and stewed Polygala tenuifolia,and are the index components of the content changes before and after the processing of Polygala tenuifolia.In terms of chemical content,the contents of main components were compared before and after stewing of Polygala tenuifolia with different core-pulling degree and different grades.The results showed that the contents of sibiricose A5,sibiricose A6and 3,6’-disinapoyl sucrose and polygalactoneⅢdecreased,and the average reduction rates of the four components were 49.75%,58.72%,40.09%and 25.62%.Above 4 kinds of components content,the whole non-core-pulling Polygala tenuifolia is higher than core-pulling Polygala tenuifolia,big cylinder Polygala tenuifolia is higher than medium cylinder Polygala tenuifolia,medium cylinder Polygala tenuifolia is higher than small cylinder Polygala tenuifolia.2 Study of differences in blood drug concentration and tissue distributionThe UPLC-MS/MS method was used to determine the concentrations of sibiricose A5,sibiricose A6,Tenuifoliside A,3,6’-disinapoyl sucrose in the plasma and tissues of rats before and after polyglucose fermentation,in order to compare the differences in blood concentration and tissue distribution between rats before and after fermentation.For blood drug concentration,the results showed that the peak concentration C_maxand the area under the concentration-time curve AUC_0-12of the four components of Polygala tenuifolia after stewing increased significantly（P<0.05）,among which the C_maxand AUC_0-12of sibiricose A5,sibiricose A6,3,6’-disinapoyl sucrose increased by 31.10%,57.93%,52.11%and 103.66%,140.89%and 48.31%respectively,and the C_maxand AUC_0-12of Tenuifoliside A increased by 25.42%and35.98%respectively,Although the difference was not statistically significant（P>0.05）,it showed an increasing trend;The apparent distribution volume V_z/Fand clearance rate CL_z/Fof the four oligosaccharide esters in Polygala tenuifolia after stewing were significantly lower than those in Polygala tenuifolia（P<0.05）,V_z/Fwere reduced by 55.38%,64.14%,34.21%and 38.84%,respectively,and CL_z/Fwas reduced by 49.91%,57.62%,30.27%and 32.45%.For tissue distribution.The results showed that the concentrations of sibiricose A5,sibiricose A6,Tenuifoliside A,and 3,6’-disinapoyl sucrose in the six tissues increased after the processing of Polygala tenuifolia.sibiricose A5 and Tenuifoliside A are distributed in heart,liver,lung,kidney,brain and small intestine;sibiricose A6is distributed in the liver,lung,kidney and small intestine,and 3,6’-disinapoyl sucrose is distributed in the liver,kidney and small intestine.These two components are not detected in the heart and brain,and the concentration in the tissue is not high.The concentrations of the four components are the highest in the small intestine as a whole,followed by higher concentrations in the kidney,liver and lung.3 Research on the Mechanism of Anshen ActionThe top 10 core active ingredients selected by Text Data Mining Technology are3,4,5-trimethoxycinnamic acid,Tenuifoliside A,Licoflavonol,Tenuifoliose J,Tenuifoliose A,Tenuifoliose H,Polygalic acid,Glycyrrhizic acid,Tenuifolisose A,and ferulic acid.The top 10 core targets are PRKCA,MAPK8,JUN,AKT1,SRC,HRAS,STAT3,PRKCB,GSK3B and EGFR.Biological pathways mainly include cancer signal pathway,PI3K-Akt signal pathway,MAPK signal pathway,Erb B signal pathway,EGFR tyrosine kinase inhibitor resistance,etc.Molecular docking verification showed that 3,4,5-trimethoxycinnamic acid,polygala tenuifolia A,Licoflavonol,Liquiritigenin and polygala acid had high connectivity with the core target,suggesting that these five components were closely related to the sedative effect of Stewed Polygala tenuifolia.HPLC was used to establish the fingerprints of 11 batches of Stewed Polygala tenuifolia,PCPA was used to replicate the rat insomnia model to evaluate the sedative effect of Stewed Polygala tenuifolia,and the spectral effect relationship was studied by principal component analysis,grey correlation analysis and orthogonal partial least squares regression analysis,so as to explore the material basis of the pharmacodynamics of Stewed Polygala tenuifolia.Results The similarity of fingerprints of 11 batches of Stewed Polygala tenuifolia was more than 0.997,indicating that the chemical components of each batch of Stewed Polygala tenuifolia were similar.The 11 batches of Polygala tenuifolia were divided into 6 categories,and the classification showed that the origin was not used as the quality evaluation index of Stewed Polygala tenuifolia.Stewed Polygala tenuifolia can increase the level of 5-HT,DA,GABA in hypothalamus and PGD2 in serum of insomnia rats,and reduce the level of Glu in hypothalamus and IL-1βin serum Level;Combined with the symptoms after the intervention,it was found that the rats’daytime activities were significantly reduced,they were tired,the circadian rhythm was gradually restored,and their fur was smooth and shiny,indicating that the stewed Polygala tenuifolia had a good calming effect.There are 11 main components that contribute significantly to the tranquilizing effect of stewed Polygala tenuifolia,among which peaks 12（3,6’-disinapoyl sucrosein）,16（Tenuifoliose C）,14（Tenuifoliose A）,4（sibiricose A6）,22,3（sibiricose A5）,31,26 and 20 may be the main components of stewed Polygala tenuifolia tranquilizing effect,and peaks 5 and 24 may play a synergistic role in stewed Polygala tenuifolia tranquilizing effect.In a word,the material basis of calming the nerves of stewed Polygala tenuifolia is the synergy of"chemical composition group".[Conclusion]In the study of differences in fingerprint and processed components,it is shown that the fermentation method has less impact on the chemical properties of Polygala tenuifolia.It has a greater impact on the content of its main active components,such as sibiricose A5,sibiricose A6,3,6’-disinapoyl sucrose,Polygala tenuifoliaⅢ,These components are important indicator components for distinguishing between raw and stewed Polygala tenuifolia.From the results of component content changes,it can be seen that the core is not removed a high level is a high-quality product of Polygala tenuifolia.In the study of differences in blood drug concentration and tissue distribution,the pharmacokinetic and tissue distribution of the main active components of Polygala tenuifolia in rats,including sibiricose A5,sibiricose A6,3,6’-disinapoyl sucrose,and Tenuifoliside A,showed that after brewing,the blood concentration C_maxand area AUC_0-12under the concentration time curve of these index components increased,while the apparent distribution volume V_z/Fand clearance rate CL_z/Fdecreased.They were found in the heart,liver,lungs,kidneys The overall increase in concentration in the six tissues of the brain and small intestine has led to an increase in the blood concentration of effective ingredients,indicating that the decoction of Polygala tenuifolia can enhance its pharmacological effect,which has a theoretical basis.In the study of the mechanism of calming the nerves,stewed Polygala tenuifolia could increase the levels of 5-HT,DA,GABA in hypothalamus and PGD2 in serum of insomnia rats,decrease the levels of Glu in hypothalamus and IL-1βin serum,and improve the insomnia symptoms of rats,make its circadian rhythm gradually return to normal,play a better role in the nerves.The Text Data Mining Technology with spectral effect relationship combined results showed that 3,6’-disinapoyl sucrose,Tenuifoliose C,Tenuifoliose A,sibiricose A6,sibiricose A5,3,4,5-trimethoxycinnamic acid,Tenuifoliside A,Licoflavonol,Liquiritigenin,Polygala acid and other components were its calming effect substances.In summary,compared with raw Polygala tenuifolia,although the content of components in fermented stewed Polygala tenuifolia has decreased,the content of components in the blood and distributed tissues in the body has significantly increased,indicating that fermented stewed Polygala tenuifolia is beneficial for improving its bioavailability.Based on the results of the sedative efficacy test,it was determined that 3,6’-disinapoyl sucrose,Tenuifoliose C,Tenuifoliose A,sibiricose A6,sibiricose A5,3,4,5-trimethoxycinnamic acid and other components are the main active ingredients of stewed Polygala tenuifolia for its sedative effect,and their sedative mechanism was revealed.This study can provide a scientific basis for the rationality of the processing of stewed Polygala tenuifolia,and also provide a possibility for its later clinical promotion and application.