| Tibetan Medicine Berberidis Cortex is the dry endothelium of Berberis kansuensis Schneid.and other plants in berberis genus.It is one of the commonly used medicinal herbs for the clinical treatment of diabetes and its complications in Tibetan medicine.Modern studies have found that Berberidis Cortex can effectively reduce the blood sugar level of diabetic mice induced by alloxan,and prevent and treat retinopathy in diabetic mice,which has broad application prospects.Berberidis Cortex is a typical various species Tibetan Medicine with complex sources and a wide variety of species.It has a serious impact on the quality control and market supervision of Berberidis Cortex,and it is also not conducive to ensuring the accuracy of the medication and the efficacy of clinical use.At present,the quality evaluation and control of Berberidis Cortex mainly use one or more components as indicators for thin-layer identification and content determination.However,this "point-to-face" method undoubtedly does not conform to the multi-component and multi-target integrity of medicinal materials.It is necessary to put a more systematic and comprehensive analysis technology into conduct an overall evaluation of the quality of Berberidis Cortex.Therefore,from the overall perspective,combined with fingerprint and UPLC-Q-Exactive Orbitrap MS metabolomics technology,this study researched the metabolites of B.kansuensis Schneid.,B.dictyophylla Franch.,B.vernae Schneid.and B.diaphana Maxim Four kinds of original Berberidis Cortex were analyzed to provide reference for the quality control evaluation and clinical application of Berberidis Cortex.Objectives:1.Establish the HPLC fingerprint of Berberidis Cortex and determine the content of its main effective components,to lay a foundation for the quality control of Berberidis Cortex.2.Based on metabolomics technology,the differences of chemical components between different species Berberidis Cortex were studied,and the different metabolic components between different species Berberidis Cortex were found to evaluate the quality of different species Berberidis Cortex.Methods:1.The fingerprint of Berberidis Cortex was established by HPLC,and the similarity of four kinds of Berberidis Cortex was evaluated by the software of similarity evaluation system of chromatographic fingerprint of traditional Chinese medicine.Combined with principal component analysis(PCA)and partial least squares discriminant analysis(PLS-DA),the differences among the components of four kinds of Berberidis cortex were analyzed systematically.2.The relative correction factor was determined by HPLC.A method for simultaneous determination of six components in Berberidis cortex,such as berberine,jatrorrhizine and Magnolia alkaloid,was established.The consistency between the method and the external standard method was evaluated by paired t test.3.Based on UPLC-Q-Exactive Orbitrap MS metabolomics technology was used to analyze Berberidis cortex.The chemical components of Berberidis cortex were identified by comparison of reference materials and database retrieval.The different metabolic markers of Berberidis cortex from different species were screened by pattern recognition analysis(PCA and PLS-DA)and analysis of variance.The distribution of different components in different species was observed by thermogram analysis.Results:1.Established the HPLC fingerprint of Berberidis Cortex with 13 shared peaks as the characteristic fingerprint information.The shared peaks 2,4,6,11,12,and 13 respectively refer to bufotenidine,ferulic acid 4-O-β-D-glucopyranoside,magnoflorine,jatrorrhizine,palmatine and berberine.The similarity between 21 batches of Berberidis cortex samples and the common pattern graph ranges from 0.777 to 0.988.Except for individual samples less than 0.9,most samples are greater than 0.9.PCA and PLS-DA pattern recognition analysis can distinguish the four species of Berberidis Cortex,indicating that there are certain differences of chemical constituents among different species of Berberidis Cortex.2.Established a quantitative analysis method of multi-components by single-marker for the simultaneous determination of 6 main active ingredients in Berberidis Cortex.Using berberine as an internal reference,the relative correction factors of bufotenidine,ferulic acid 4-O-β-D-glucopyranoside,magnoflorine,jatrorrhizine and palmatine were0.219,0.435,0.531,0.971 and 0.972,respectively.There was no significant difference in the contents of each component between the quantitative analysis method of multicomponents and the external standard method.Assay results showed that the content of magnoline was the highest,followed by the content of berberine and ferulic acid 4-O-β-D-glucopyranoside;the total content of the6 active ingredients in B.vernae was the highest,while the total content of B.dictyophylla is the lowest;and the content of 6 active ingredients in different batches of Berberidis Cortex samples are quite different.3.A total of 47 chemical constituents were identified,including 24 alkaloids,12 organic acids and their derivatives,5 sugars and their derivatives,and 6 other components.By observing the positive and negative total ion current diagrams of different species Berberidis Cortex,it was found that the chemical components of different species Berberidis Cortex were similar overall,and there were differences in the content of the components;through the comparative analysis of different species Berberidis Cortex by pattern recognition analysis,it was found that the PLS-DA threedimensional score diagram showed that the four original Berberidis Cortex were obviously distinguished,indicating that the chemical components of different species Berberidis Cortex were different.Twelve potential components were screened according to the analysis of variance(p<0.05)and VIP>3,including jatrorrhizine,tetrahydropalmatine,Nmethyltetrahydropalmatine,bufotenidine,8-Oxyberberine,palmatine,eleutheroside E,ferulic acid,demethyleneberberine,α,α-trehalose,norisocorydine and ferulic acid 4-O-β-D glucopyranoside.The results of thermogram analysis showed that there were more jatrorrhizine,tetrahydropalmatine,N-methyltetrahydropalmatine and bufotenidine in B.vernae;there were more demethyleneberberine,8-oxyberberine,α,α-trehalose and palmatine in B.kansuensis;there were more eleutheroside E and norisocorydine in B.dictyophylla;there were more norisocorydine and bufotenidine in B.diaphana.Conclusions:1.The established HPLC fingerprint method and the quantitative analysis method of multi-components by single-marker are simple and reliable,which can be used for the quality control and evaluation of Berberidis cortex.2.Metabolomics studies show that,Berberidis Cortex mainly contains alkaloids,amino acids,nucleotides,organic acids,sugars and other metabolites.A total of 47 chemical constituents were identified,including 24 alkaloids,12 organic acids and their derivatives,5 sugars and their derivatives,and 6 other components.3.There are certain differences in the chemical compositions of different species Berberidis Cortex.Based on the perspective of chemical composition,the quality of B.vernae and B.kansuensis is better than that of There are certain differences in the chemical composition of Berberidis Cortex from different species.The quality of Berberidis Cortex presents a trend of B.dictyophylla and B.diaphana. |
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