| ObjectiveIn this study,on the one hand,fingerprint quality,content determination and metabolomics techniques were used to systematically and comprehensively construct the quality evaluation system of Citrus reticulata ’ Chachi ’Pericarpium(CRCP).On the other hand,this study used SD rats as the research object,using metabolomics technology to identify differential metabolites,analyzing metabolic pathways,exploring the mechanism of hypolipidemic effect of CRCP on biological organisms,and elucidating the pharmacodynamic substances of the effect of reducing the blood lipids.This study provides a reference for the quality evaluation of CRCP,and provides a feasible strategy for the identification of Chinese medicinal materials from different varieties and storage years,and lays a foundation for the development of CRCP lipid-lowering health products or medicines.Methods1.This study used HPLC and UPLC Q_TOF/MS technology to determine the multi-index content,fingerprint map and metabolomics analysis of CRCP and Citri Reticulatae Blanco Pericarpium(CRBP),as well as the CRCP within different storage years,in order to evaluate CRCP quality.2.To establish a hyperlipidemic rat model by feeding high-fat and high-cholesterol diet,and using metabolomics combined with UPLC Q-TOF/MS to study the effects of CRCP on biochemical indicators,metabolic profiles and biomarkers in hyperlipidemic rat models.To clarify the hypolipidemic effect and mechanism of CRCP on hyperlipidemic rats.Results1.Evaluation of CRCP quality(1)Determination of content:the content of hesperidin,tangeretin and nobiletin in the CRCP and CRBP are 1.606～5.707%,0.356～1.417%and 0.251～1.144%,respectively;2.606～3.942%,0·052～0.194%and 0.006%～0.046%,the percentage of hesperidin is different between CRBP and CRCP,and the content of tangeretin and nobiletin in the former is significantly higher than that in the latter.The content of tangeretin and nobiletin is not much different among the CRCPs within different storage years,the content of hesperidin has a certain increasing trend during the storage period of 3 to 8 years.It tends to be flat after 8 years and higher in 14 and 25 years.(2)Fingerprint:The HPLC fingerprint of CRCP with 17 common peaks was established that could be discerned for its quality.The common pattern of 32 batches of CRCP was used as the standard fingerprint,the similarity values of 32 batches of CRCP and 10 batches of CRBP were 0.906～0.999,0.326～0.396 respectively.The Cluster analysis and PCA results showed that CRCP and CRBP are distinguished obviously,and peak 4,peak 7,peak 8,and peak 9(hesperidin)have certain differences in the CRBP and CRCP.The chromatographic peaks and peak areas are almost the same among the CRCPs within different storage years,and the similarity values of CRCPs within different storage years are above 0.906.Cluster analysis can clearly distinguish 8 years and below from 8 years and more.(3)Metabolomics analysis of CRCP:92 compounds were identified by UNIFI software form CRCP and CRBP samples.Multivariate statistical analysis showed that CRCP and CRBP showed significant aggregation separation,and 19 potential marker metabolites were identified as potential biomarkers of the CRCP and CRBP.The 15 markers showed a higher level of CRCP than CRBP in the heat map,accounting for most of the potential markers.The results of metabolomics analysis of the CRCP samples with different storage years showed that the samples were clearly separated in 3,5,and 10 years,and the samples in 15,20,and 30 years were mixed together.31 metabolites were identified as potential biomarkers for different storage years.The heat map shows that the metabolite level in 3-10 years is on the rise and peaks in 5-10 years,then gradually decreases and changes little in 15-30 years,consistent with PCA results.2.Study on the efficacy of CRCP in the prevention and treatment of hyperlipidemia(1)Hyperlipidemia induced hyperlipidemia in rats:body weight,TC,TG,LDL levels in the model group were significantly higher than those in the control group,while HDL was significantly decreased,indicating that the hyperlipidemia model was successfully established.Through metabolomics,the model group and the control group were in different spatial colonies in the PCA map,and 30 urine biomarkers and 19 serum biomarkers were identified in the hyperlipidemia rat model.(2)The intervention effect of CRCP on the hyperlipidemia rat model:Compared with the model group,the TG level in the CRCP group was significantly decreased and the HDL-C level was significantly increased.11 chemical components were found in rats with hyperlipidemia after oral administration of CRCP,8 of which were the prototype components of CRCP;3 were the metabolites of CRCP,which were derived from 5-Demethylnobiletin,tangeretin and 5-hydroxy-3,7,3’,4’-tetramethoxyflavone,respectively.The results of metabolomics analysis showed that the CRCP group and the model group were clearly located in different regions in the PCA score plot,and CRCP has a statistically significant pullback effect on 13 biomarkers in model urine and 11 biomarkers in model serum.Among the blood-shifting components,tangeretin and Glucuronic acid metabolite of 5-Demethylnobiletin are highly related to the hyperlipidemia biomarkers.Conclusion1.The quality control mode constructed by fingerprint and chemometrics can effectively distinguish CRCP from CRBP,reflecting the quantitative difference between the varieties.The UPLC-QTOF/MS-based metabolomics method has proven to be an effective method for distinguishing between CRCP and CRBP,as well as the CRCPs within different storage years,and may also provide a viable strategy for the certification of Chinese herbal medicines from different species and storage years.2.CRCP has a preventive and therapeutic effect on the hyperlipidemia rat model,and regulates the blood lipid of the model by recalling the biomarker metabolites of the model;tangeretin and Glucuronic acid metabolite of 5-Demethylnobiletin are the potential pharmacodynamic basis for the prevention and treatment of hyperlipidemia. |
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