The Improvement Of Quality Standards Of Gentianae Macrophyllae Radix

1.Background and purposeGentianae Macrophyllae Radix?GMR?and Gentiana Radix et Rhizoma?GRR?belong to the genus Gentiana,which are similar in chemical composition,but significantly different on clinical effects.In the current 2015 edition of the Chinese Pharmacopoeia,roburic acid,as a characteristic component,can achieve the purpose of distinguishing GMR from GRR,but could not achieve quality transmission and consistency from medicinal materials to preparations for the decoction as an oral preparation.In view of the problems in the quality standards of GMR,this topic will find and discover a new characteristic components from GMR to make up for the shortcomings in the control of the quality of the medicinal herbs in the current standard.In this work,we hope to establish a TLC identification method to achieve the identification not only with GRR,but also the spurious breeds of GMR,and establish a HPLC quantitative method to satisfy the quality evaluation of the medicinal materials.Finally,the work could provide a experimental basis for the improvement of the quality standards of GMR.2.Study methods?1?Selection and separation of characteristic components of GMRBased on the established HPLC fingerprint,the differences of chemical composition in different polar parts of GMR and GRR were compared and analyzed,and the specific component in GMR was locked.The target component was isolated,identified and enriched using chemical separation technology such as macroporous resin,silica gel,gel columns,etc.The specificity of the target 2-methoxyanofinic acid?MAA?obtained was characterized by ultra performance liquid chromatography-mass spectrometry?UPLC/MS?and thin layer chromatography?TLC?.The biochemical self-development method and DPPH reagent were used to investigate the ability of MAA and 2-hydroxyanofinic acid to scavenge oxygen free radicals to evaluate its antioxidant activity.The anti-inflammatory activity of the targets was evaluated according to the release amount of inflammatory factor NO with the macrophage cells induced by lipopolysaccharide?LPS?as the inflammatory model.The primary activity of MAA and 2-hydroxyanofinic acid was screened.?2?Study on quality standards of GMRTaking 2-methoxy tartaric acid as a qualitative indicator,a thin layer chromatographic?TLC?qualitative method of GMR was established with petroleum ether-ethyl acetate-formic acid?25:20:0.3?as developing solvent and 5%vanillin-sulfuric acid ethanol as the chromogenic reagent.The HPLC fingerprint analysis method of GMR was established by using a unitary C₁₈?250*46mm,5?m,100A?column as the column and acetonitrile-0.1%phosphoric acid aqueous solution as the mobile phase.The similarity of the samples was analyzed by the software of"Chinese medicine chromatographic fingerprint similarity evaluation system"?2004 version?.Furthermore,the results of fingerprint were analyzed by partial least squares discrimination analysis?PLS-DA?and hierarchical cluster analysis?HCA?with SIMCA13.0 software.Characteristic peaks of GMR determined by HPLC fingerprints,the analysis method of the feature fingerprint was established.Using MAA as the index,the qualitative identification method of HPLC was established.Taking MAA as a new indicator,the HPLC method for the determination of GMR was established by using a unitary C₁₈?250*46mm,5?m,100A?column and acetonitrile-0.1%phosphoric acid aqueous solution?42:58,v/v?as mobile phase.Using the SPSS software,the correlation between MAA content and pharmacopeia index?gentiopicroside and loganic acid?content was analyzed to explore the rationality of the new index.3.Results?1?Selection and separation of characteristic components of GMRA new characteristic component was found from the decoction of GMR.It was isolated and identified as 2-methoxyanofinic acid?MAA?and the pure product was prepared to about 100 mg.Seven known compounds else were isolated and identified as gentiopicroside,erythrocentaurin,syringaresinol,macrophylloside A,macrophylloside B,anofinic acid and 2-hydroxyanofinic acid.The results of UPLC/MS and TLC analysis showed that MAA was a common component of 4 species of GMR.It had strong specificity and could be detected in the decoction and GMR dispensing granules,which could achieve the quality transmission from medicinal materials to preparations,and could be used for effective identification of GMR with GRR and spurious breeds?Phlomis dentosa Franch.,PDF,Salvia przewalskii Maxim.,SPM,Veratrilla baillonii Franch.,VBF?.The results of antioxidant tests showed that MAA and 2-hydroxyanofinic acid had certain ability to scavenge free radicals,but were weaker than the positive drug,Vc.Anti-inflammatory test results showed that there was a significant difference in NO content between the positive drug group and the model group?P<0.01?,and the positive drug had anti-inflammatory activity.But the sample groups had no significant difference with the model group.?2?Study on quality standards of GMRThe TLC method of GMR was established and had good durability.The corresponding spots of MAA in samples wre clear,the resolution was good,and there was no interference.The HPLC fingerprints of GMR were established.The similarity analysis showed that the similarity of 29 batches of GMR was greater than 0.9,and the interspecies similarity was higher.The results of discriminant analysis?PLS-DA?and cluster analysis?HCA?showed that GMR,GRR and GMR's spurious breeds?Phlomis dentosa Franch.,PDF,Salvia przewalskii Maxim.,SPM,Veratrilla baillonii Franch.,VBF?were aggregated respectively.It indicated that the method could achieve the identification of GMR.The VIP results showed that MAA was the most main contributing component for discrimination.The characteristic peaks of GMR were determined,and the characteristic fingerprint analysis method was established.The precision,repeatability and stability of the method were all good.An HPLC qualitative identification method for MAA in Gentiana macrophylla was established.The HPLC quantitative method of MAA in GMR was established.The method validation was showed that MAA had a good linear relationship at 0.8096 to 202.4?g·m L^-1.The standard curve of MAA was y=49.263x-32.089 with correlation coefficient?r?no less than 0.9999.The average recovery of MAA was 99.56%with RSD 2.29%.The RSDs of the precision,repeatability,stability tests were less than 3%.The results indicated that the HPLC quantitative method established met the analytical experiment requirements.4.ConclusionEight known compounds were isolated from GMR,2-methoxyanofinic acid?MAA?,gentiopicroside,erythrocentaurin,syringaresinol,macrophylloside A,macrophylloside B,anofinic acid and 2-hydroxyanofinic acid,respectively.MAA and its homologs had certain antioxidant and anti-fungal activities.Due to the strong specificity of MAA,in qualitative identification,it is recommended that MAA be used as a substitute for roburic acid or as an indicator components together for TLC identification.The characteristic map or HPLC identification established can be used for the distinguishing of GMR,GRR and GMR's spurious breeds.In terms of quantification,MAA has a good correlation with iridoids,.It is suggested that the two should be used together for the quality evaluation of GMR,so that the quality standards of GMR could be improved from qualitative and quantitative aspects.