| Objective:In order to explore the quality marker（Q-marker）of Codonopsis Radix and evaluate the quality of Codonopsis Radix more objectively,the content of small molecule components in Codonopsis Radix was determined and analyzed by high performance liquid chromatography（HPLC）,and dispersive liquid-phase microextraction-HPLC（DLPME-HPLC）was applied to enrich and determine lobetyolin and atractylenolide III in Codonopsis Radix to find the chemical difference markers of Codonopsis Radix with different origins and different specifications.In this paper,we applied hollow fiber cell fishing with HPLC（HFCF-HPLC）to initially screen the anti-inflammatory active components and explore the potential bioactive markers in Codonopsis Radix.This paper provides a basis for finding the Q-marker of Codonopsis Radix and improving the quality evaluation system of Codonopsis Radix.Methods:1.Based on the previous study of our group,the contents of four index components-syringin,tangshenoside I,lobetyolin,and atractylenolide III-were determined by HPLC in 45 batches of different origins and different specifications of Codonopsis Radix.One-way ANOVA,hierarchical clustering analysis（HCA）,principal component analysis（PCA）and orthogonal partial least squares discriminant analysis（OPLS-DA）were applied to compare and evaluate the quality of different origins and different specifications of Codonopsis Radix.2.DLPME-HPLC was used for the enrichment and determination of lobetyolin and atractylenolide III in Codonopsis Radix.A deep eutectic solvent（DES）prepared with methyltrioctylammonium chloride（TOMAC）and n-butanol was used as the extractant,and the extraction conditions were optimized using a single factor design.The methodology was investigated under the optimal conditions.Content determination and One-way ANOVA were performed on two target analytes in 17 batches of Codonopsis Radix with different origins.The method was compared with published methods to illustrate the feasibility and advantages of DLPME-HPLC in the quality evaluation of Codonopsis Radix.3.Based on the preliminary clarification of the anti-inflammatory activity of the methanol extract of Codonopsis Radix,RAW264.7 cells were grown in the inner wall of hollow fibers as a tool for activity screening and placed in the methanol extract of Codonopsis Radix for preliminary screening and capture of anti-inflammatory active components in Codonopsis Radix.These components were identified by HPLC-ultraviolet（HPLC-UV）,and the potential biomarkers in Codonopsis Radix were preliminarily screened.Results:1.The results of content determination of 45 batches of Codonopsis Radix showed that the content of syringin was 0.0072-0.032 mg/g,the content of tangshenoside I was0.2981-1.5637 mg/g,the content of lobetyolin 0.1287-1.0466 mg/g,and the content of atractylenolide III was 0-0.084 mg/g.Among the different origins of Codonopsis Radix,the content of syringin has no statistical difference in them,the highest content of tangshenoside I was found in C.pilosula（p<0.05）,the higher contentof lobetyolin was found in C.pilosula var.modesta or C.tangshen（p<0.05）,and the highest content of atractylenolide III in C.pilosula（p<0.05）.Among the different specifications of Codonopsis Radix,the highest content of syringin（p<0.05）was in Wen-Dangshen,the highest content of tangshenoside I（p<0.05）was in Lu-Dangshen,the highest content of lobetyolin（p<0.05）was in Dao-Dangshen,and the highest content of atractylenolide III（p<0.05）was in Lu-Dangshen.The results of HCA,PCA and OPLS-DA analyses further verified that there were differences in the samples of Codonopsis Radix between different origins and different specifications,which could be distinguished.2.The optimal experimental conditions for the DES-based DLPME-HPLC were as follows:the extractant was TOMAC-n-butanol,with a molar ratio of 1:4 and a volume of 110μL;the p H of the sample phase solution was 7;the salt concentration was 0%;the dispersant was methanol,with a volume of 300μL;the extraction time was 90 s;the centrifugation speed was 6000 rpm and the centrifugation time was 2 min.The results showed that the limit of detection（LOD）and limit of quantification（LOQ）of lobetyolin were 6×10-4 and 2×10-3μg/m L,and the LOD and LOQ of atractylenolide III were 3×10-3 and 9×10-3μg/m L,respectively,and the average enrichment factors（EF）of the two target analytes were 37 and 154,respectively.The intra-day RSD of lobetyolin and atractylenolideⅢwere 1.5-5.8%and 2.6-5.2%,respectively.The inter-day RSD values were 1.2-6.4%and 3.1-4.7%.The spiked recovery values were in the range of 87.6-114.7%with the RSD of 1.5-4.7%.The results of sample content determination showed that the content of lobetyolin was 0.204-0.927 mg/g,and the content of atractylenolideⅢwas 0-0.1584 mg/g.The highest content of lobetyolin（p<0.05）was in C.pilosula var.modesta,and the highest content of atractylenolideⅢ（p<0.05）was in C.pilosula.Compared to traditional liquid-liquid extraction（LLE）methods,this method has the advantage of lower LOD,LOQ and wider linear range.3.The experimental conditions of HFCF-HPLC were as follows:polypropylene hollow fiber were used as cell growing carrier,and the concentration of the sample solution was 60 mg/m L.Under these conditions,the chemical components captured in order of cell fishing factor（CFF）>1 were syringin,tangshenoside I,lobetyolin,lobetyolinin and atractylenolide III.Conclusion:In this paper,the results of multi-component content determination of syringin,tangshenoside I,lobetyolin,and atractylenolide III in Codonopsis Radix combined with chemometric analysis showed that these four small molecules can be used as chemical markers to distinguish and compare Codonopsis Radix with different origins and different specifications;DLPME-HPLC based on DES enriched and determined lobetyolin and atractylenolide III in Codonopsis Radix,and by comparing the contents of the two components can be compared to distinguish the the three origins of Codonopsis Radix,which also provides a new idea for the analysis and determination of trace components in traditional Chinese medicine.The application of HFCF-HPLC to screen the potential anti-inflammatory active components in Codonopsis Radix provides a simple method to systematically analyze the potential bioactive components in traditional Chinese medicine.The study showed that syringin,tangshenoside I,lobetyolin,lobetyolinin and atractylenolide III could be used as candidate Q-marker in Codonopsis Radix. |
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