| The Lycii cortex(Digupi)is the dried root bark of Solanaceae plant Lycium chinense Mill.or Lycium barbarum L..Phenolic amides are characteristic components of Digupi,which may be related to anti-inflammatory and antipyretic effects.However,the research on phenolic amides in Digupi is still insufficient.In addition,there were identification and inspection items of Digupi but no provisions for content determination or fingerprints in Chinese Pharmacopoeia(2015).Digupi in the market are producted all over the country,and the wild and cultivated sources are mixed.The quality of Digupi is difficult to guarantee and may affect the clinical efficacy.Therefore,it is of great significance to establish the quality evaluation methods of Digupi to ensure clinical application.In this paper,the qualitative analysis of phenolic amides in Digupi was carried out in order to find out new phenolic amides in Digupi.And the methods of content determination of index components and HPLC fingerprints were established to provide reference for quality evaluation of Digupi.HPLC-LTQ-Orbitrap method was applied to study the chemical constituents of Digupi.A total of 55 compounds were identified,including 39 phenolic amides,5 organic acids,3 cyclic peptides,2 coumarins,2 alkaloids and 4 other compounds.Phenolic amides were consist of 26 polyamine phenolic amides(15 spermines,6 spermidines,5 putrescines)and 13 non polyamine phenolic amides.By studying the fragmentation pathway of reference standards and the identified phenolic amides,and the fragmentation rules were summarized,which laid the foundation for the subsequent research of phenolic amides.An HPLC method was established for determination of three compounds including N-caffeoylputrescine,kukoamine B and chlorogenic acid simultaneously in Digupi.Under the method,the linear range of N-caffeoylputrescine was 0.000366～0.229 mg/ml.The linear range of kukoamine B was 0.006224～3.89 mg/ml.The linear range of chlorogenic acid was 0.00036～0.225 mg/ml.The limits of detection(LOD)and quantification(LOQ)for each analyte were evaluated on the basis of 3(S/N=3)and 10(S/N=10),respectively.The LOD values of the three analytes were 0.06595,1.6805,0.072 μg/ml,the LOQ values of the three analytes were 0.2198,5.0616,0.24μg/ml,respectively.The extraction process of Digupi was optimized by single-factor study,and the best extraction protocol was 60-mesh powder,100-fold amount of 70%methanol,reflux extraction for 60 min.The results show that the method was simple,well repeatable and reproducible.78 batches of Digupi from 33 cities across the country,were analyzed by using the content determination method above,to determine the content of N-caffeoylputrescine,kukoamine B and chlorogenic acid.Then 78 sets of effective data were collected.The content ranges of N-caffeoylputrescine,kukoamine B and chlorogenic acid were 0.037～3.32 mg/g,1.07～57.91 mg/g,0～1.90 mg/g,respectively.The content range of N-caffeoylputrescine plus kukoamine B was 1.22～58.26 mg/g,and the range of total content of the three was 1.38-58.37 mg/g.The content of each component varied greatly.Data processing software SPSS 22 was used to analyze the data.And the minimum control standard of the content of each analyte,the content of N-caffeoylputrescine plus kukoamine B and the total content of the three were set to be 0.05、2.21、0.02、2.29、2.36 mg/g,respectively.The HPLC fingerprint of Digupi produced in different regions was established and 14 common peaks were chosen in the fingerprints.This study validated the precision,stability and reproducibility of fingerprints of Digupi and the results showed that RSD of the relative retention time and the relative peak area of each characteristic peak in method validation was less than 3%.The similarity is above 0.9.The results showed that the overall quality of 10 batches of Digupi were similar.The results of principal component analysis show that the first principal component that has the greatest effect on Digupi might be related to the phenolic amide component in Digupi.And 21 batches of samples were used to test the HPLC fingerprint of Digupi.78 batches of Digupi were tested by the establiehed HPLC reference fingerprint.The results showed that the similarity of most of the samples with the reference fingerprint was above 0.7,and only two batches had the similarity below 0.7.It was considered that there were differences in the overall quality of Digupi on the market.However,the comprehensive quality of most of the Digupi was similar. |
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