| Objective:The commercial grades of Notopterygii Rhizoma et Radix were only stipulated in"Standards for Seventy-Six Kinds of Medicinal Products" formulated by the State Administration of Medicine and the Ministry of Health in 1984.On the one hand,the standard used for more than 30 years has not been updated and not able to adapt to market changes.On the other hand,the standard is only based on the external form.Under the support of the national standardization project of Chinese medicine"Standardization of Six Kinds of Chinese Herbal Slices such as Rhei Radix et Rhizoma".this subject uses the idea of spectral efficiency relationship to study the chemical composition,pharmacodynamic activity and identification of active substances based on spectral efficiency relationship of Notopterygii Rhizoma et Radix from different commercial specifications.The study provides the basis for scientifically classifying the commercial grades of Notopterygii Rhizoma et Radix.comprehensively controlling the quality and determining the active substances of the anti-inflammatory activity.Method:1.The market and literature were studied to understand the existing situation of Notopterygii Rhizoma et Radix goods.Notopterygii Rhizoma et Radix goods were divided into three commercial specifications according to the source and appearance:Canqiang,Tiaoqiang and Datouqiang.The contents of chlorogenic acid,nodakenin,ferulic acid.notopterol and isoimperatorin in 30 batches of Notopterygii Rhizoma et Radix were determined by HPLC method.The content of volatile oil was determined by steam distillation.SPSS 19.0 statistical software was used to make significant difference analysis,cluster analysis and principal component analysis.2.Fingerprints of Notopterygii Rhizoma et Radix from three commercial specifications were established by HPLC method.Similarity evaluation system for chromatographic fingerprint of traditional Chinese medicine(Version 2004 A)was used to confirm the common peaks and evaluate the similarity.SPSS 19.0 statistical software was used to make principal component analysis for HPLC fingerprint.3.The chemical components of Notopterygii Rhizoma et Radix from three commercial specifications were identified by UPLC-LTQ/Orbitrap MS method.4.The volatile components of Notopterygii Rhizoma et Radix from three commercial specifications were identified by GC-MS method,and the NIST 2014 database was used for searching and analysis.5.The xylene-induced mouse ear swelling models were used to compare the differences of anti-inflammatory activity in vivo between Notopterygii Rhizoma et Radix from three commercial specifications.Based on the fingerprint analysis,the correlation between characteristic peak area and anti-inflammatory activity of each sample were analyzed by gray correlation analysis method,which was the study of spectral efficiency relationship.The pharmacodynamic components of anti-inflammatory activity were determined by comparing the correlation degree.6.Mouse mononuclear macrophage RAW264.7 induced by LPS was used as the inflammatory model.The changes of cell viability and secretion of inflammation-related factors were detected by CCK8 method,Griess method and ELISA method.In the case,the differences of anti-inflammatory effects in vitro between Notopterygii Rhizoma et Radix from three commercial specifications were studiedResult:1.There were great differences in the content of chlorogenic acid,nodakenin,ferulic acid,notopterol,isoimperatorin and volatile oil between Notopterygii Rhizoma et Radix from three commercial specifications.The sum of the content of notopterol and isoimperatorin and the content of volatile oil in Datouqiang generally did not up to the standard in Chinese Pharmacopoeia.The results of cluster analysis and principal component analysis were completely consistent with the classification of commercial specifications,indicating that the method of content determination combined with statistical analysis was applicable to distinguish commercial specifications of No topterygii Rhizoma et Radix.2.The common modes of HPLC fingerprint for Canqiang,Tiaoqiang and Datouqiang were established separately.22 common peaks were marked in Canqiang,23 common peaks were marked in Tiaoqiang,29 common peaks were marked in Datouqiang.The results of similarity evaluation and principal component analysis showed that there were great differences among Notopterygii Rhizoma et Radix from different commercial specifications in chemical composition and content.The quality of Notopterygii Rh izoma et Radix from the same commercial specification was stable The results of principal component analysis showed that 30 batches of Notopterygii Rhizoma et Radix were divided into three groups,which were consistent with the classification of commercial specifications.10 characteristic components were selected as the determinants.The comprehensive scores indicated that in terms of chemical composition,the quality of Tiaoqiang was the best,followed by Datouqiang,Canqiang the worst.3.Based on the accurate mass measurements,mass fragmentation patterns,retention time and literature reports,a total of 34 compounds including 24 coumarins,7 phenolic acids.,2 flavonoids and 1 polyene acetylene compound were screened and characterized by UPLC-LTQ/Orbitrap MS method.Six different components of Notopterygii Rhizoma et Radix from three commercial specifications were determined,namely decuroside V,trans-p-hydroxycinnamic acid,6’-0-trans feruloyl praline.5’-hydroxylactone,diosmin and phenethyl ferulate.4.A total of 80 compounds were identified among Notopterygii Rhizoma et Radix from three commercial specifications.63 compounds were identified in Canqiang,45 compounds identified in Tiaoqiang and 44 compounds identified in Datouqiang.There were 26 kinds of common compounds.The identified compounds were mainly terpenoids,followed by compounds such as alcohols,aldehydes,and esters.There were great differences among Notopterygii Rhizoma et Radix from different commercial specifications in volatile oil composition and content,but the content of borneol acetate,guaiacol,a-pinene and orthodene was high.5.The experimental results of xylene-induced ear swelling in mice showed that the most significant specification was Canqiang,followed by Datouqiang and Tiaoqiang had no significant difference.This conclusion was consistent with Canqiang was the most expensive and effective in the market.The result of the spectral efficiency relationship study showed that Notopterygii Rhizoma et Radix exerted anti-inflammatory activity,through the interaction of internal active ingredients,although the correlation between 40 characteristic peaks and the anti-inflammatory activity was different.There were 1 7characteristic peaks that were highly correlated with anti-inflammatory activity,among which 3 were known components,namely chlorogenic acid,ferulic acid and isoimperatorin.6.Alcohol extracts of Notopterygii Rhizoma et Radix from three commercial specifications can exert their anti-inflammatory effects in vitro by inhibiting the release of NO,TNF-α and IL-6 in macrophages,the most significant specification was Canqiang,followed by Datouqiang and Tiaoqiang the worst.Conclusion:This study explores the differences of Notopterygii Rhizoma et Radix from three commercial specifications from the two aspects of chemical composition and efficacy,combining fingerprint with pharmacological activity by statistical analysis to study the spectral efficiency relationship and further identify active substances.The study provides the basis for scientifically classifying the commercial grades of Notopterygii Rhizoma et Radix,comprehensively controlling the quality and determining the active substances of the anti-inflammatory activity. |
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