Analysis Of Chemical Constituents Of Wuzhou Liupao Tea And Its Mechanism Of Action On Rats With Damp-Heat Constipation

Objective:Liupao tea is a post-fermented dark tea,one of the varieties of the tea family [Camellia sinensis(L.)O.Kuntze ].Many studies have shown that tea has the effect of clearing away heat and dampness and regulating the stomach.However,there are few reports on the study of Liupao tea on damp-heat constipation.Taking Liupao tea in Wuzhou,Guangxi as the research object,firstly,it was qualitatively analyzed by liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry,and then combined with network pharmacology to predict the potential mechanism of Liupao tea in improving damp-heat constipation.On the basis of the previous two chapters,by constructing a rat model of damp-heat constipation,the pharmacodynamic mechanism of Liupao tea on damp-heat constipation was evaluated from various aspects.16 s r RNA high-throughput sequencing technology was used to explore the relationship between Liupao tea improving damp-heat constipation and intestinal flora,and to preliminarily explore its microbial mechanism.The project research clarifies the mechanism of Liupao tea improving damp-heat constipation from the perspective of chemical composition analysis,network pharmacology prediction,pharmacodynamic mechanism of animal models and intestinal flora,and provides a research basis for the industrial development of Liupao tea.Methods:(1)Chemical composition analysis of Liupao teaThe chemical composition of Wuzhou Liupao tea was qualitatively analyzed by LC-MS and GC-MS,and the chemical components of Wuzhou Liupao tea were identified,which provided the basis for the chemical basis of Liupao tea.(2)A network pharmacological study on Liupao tea intervening in damp-heat constipationA network pharmacology method was used to predict the potential mechanism of Liupao tea in improving damp-heat constipation.Based on literature mining and database search,the active components of Liupao tea were collected,and the components were identified by mass spectrometry to predict the action targets of the common components.The disease-acting targets were mapped to obtain a set of common acting targets,and the potential mechanism of Liupao tea to improve damp-heat constipation was predicted through PPI,GO,KEGG and other signaling pathways.(3)Pharmacodynamic study of Liupao tea on damp-heat constipation ratsTaking male SD rats as the research object,a TCM syndrome model of damp-heat constipation was established by modeling methods of high temperature and high humidity,abnormal hunger and satiety,fat,sweet and thick taste,and fatigue.The rats were randomly divided into normal group,Model group,Liupao tea low-dose group,Liupao tea high-dose group.In the middle and late stages of the experiment,the hair,mental state and water content of feces of the rats in each group were used as the basis for model formation.After modeling,automatic biochemical detector and ELISA method were used to determine the contents of blood biochemical indexes(blood lipid four items alanine aminotransferase,uric acid,D-lactic acid)and four enteric neurotransmitters(nitric oxide,nitric oxide,D-lactic acid)in serum.Vasoactive intestinal peptide,motilin,substance P);HE staining was performed on the colon,stomach,and kidney tissues of rats to observe the histomorphological changes,and on the basis of pathological sections,the colon,stomach,and kidney tissues were analyzed for morphological changes.Three inflammatory factors(TNF-α,IL-6,IL-1β)were analyzed by immunohistochemistry;real-time quantitative PCR was used to detect the expression of AQP3,AQP4 and AQP8 genes in colon,stomach and kidney tissue.To evaluate the pharmacodynamic effect of Liupao tea in improving damp-heat constipation.(4)Study on the intestinal flora of Liupao tea in rats with damp-heat constipationBased on the animal experiments in the previous chapter,with normal rats as controls,16 s r RNA high-throughput sequencing technology was used to sequence the V3～V4 hypervariable regions,and bioinformatics technology was used to analyze the intestinal flora of the damp-heat constipation rat model structure,and preliminary exploration of its microbial mechanism of action.Results:(1)Chemical composition analysis of Liup Liupaoao tea113 compounds were identified by LC-MS and GC-MS techniques.Flavonoids such as quercetin and epigallocatechin,amino acids such as valine and D-proline,stearamide,hexadecamide,etc.were identified through known literature reports,reference substance information and spectral library analysis.amides,etc.According to the preliminary identification of the classification of compounds,it can be seen that the components of Liupao tea are mainly flavonoids,amides and amino acids.(2)A network pharmacological study on Liupao tea intervening in damp-heat constipationDatabase search and mass spectrometry matched 9 common components,and 74 potential targets of Liupao tea to improve damp-heat constipation.The enrichment analysis showed that 22 core targets,including MAPK1,MAPK3,MAPK8,TP53,IL6,and TNF,were mainly involved in signaling pathways such as PI3K-AKT,Human cytomegalovirus infection,and MAPK.The core targets and enrichment results suggest that Liupao tea may directly or indirectly interfere with damp-heat constipation through multiple components acting on multiple pathways to regulate the expression of inflammatory factors and MAPK factors.(3)Pharmacodynamic study of Liupao tea on damp-heat constipation ratsCompared with the normal group,the hair of the rats in the model group gradually turned yellow and fluffy during the modeling process,the food intake and body weight decreased significantly(P < 0.05),the fecal particles became smaller,the texture was dry,and the water content decreased(P < 0.05).P < 0.05),mentally manifested as squinting and curling up,holding a group and arching the back,and when touching the mouse body,it was more common to be nervous and irritable;The secretion of gastrin,D-lactic acid,motilin,substance P,vasoactive intestinal peptide and nitric oxide in serum increased significantly(P <0.05),and blood lipid levels were abnormal;inflammatory factors IL-6,TNF The secretion of-α and IL-1β in the colon,stomach and kidney tissues of the model group rats was significantly increased(P < 0.05).In addition,compared with the normal group,the gene expressions of AQP3 and AQP4 were significantly enhanced(P < 0.01),and the gene expression of AQP8 was significantly decreased(P < 0.01)in the colon and stomach tissues of the model group rats(P < 0.01).,the expression levels of three aquaporins in the model group were significantly decreased(P < 0.05).After Liupao tea intervention,the yellowing state of the hair of the rats was improved,and the phenomenon of squinting and curling up was rare.Substances,vasoactive intestinal peptide and nitric oxide secretion,reduce the expression of inflammatory factors IL-6,TNF-α,IL-1β,improve the inflammatory infiltration state and three water channels in colon,stomach and kidney tissue protein gene expression.(4)Study on the intestinal flora of Liupao tea in rats with damp-heat constipationJudging from the number of OTUs and α diversity in each group,the relative abundance and diversity of the intestinal flora in the damp-heat constipation rat model were the lowest,and the structure of the intestinal flora was destroyed,indicating that the modeling was successful;at the phylum level At the genus level,15 genera with significant differences were found.Combining with the phyla to which the genus belonged at the genus level,it was found that most of them belonged to the phylum Firmicutes and Bacteroidetes.After Liupao tea intervention,the intestinal flora of the rats was relatively rich and diverse compared with the model group,and gradually tended to the normal group.The correlation analysis between each efficacy index and intestinal flora showed significant correlations at both the phylum level and the genus level.Conclusion:In this study,113 compounds were identified by LC-MS and GC-MS,and then predicted by network pharmacology that Liupao tea might interfere with damp-heat constipation by regulating the expression of inflammatory factors and MAPK factors through multiple components acting on multiple pathways.By constructing a rat model of damp-heat constipation,Liupao tea increased the fecal water content of rats after intervention.It is speculated that Liupao tea may reduce the secretion of SP,VIP,MTL and NO in serum and reduce the inflammatory factor IL-6.,TNF-α,IL-1β expression,and regulate the gene expression and intestinal flora structure of three aquaporins in colon,stomach and kidney tissue,so as to comprehensively improve damp-heat constipation.