| Objective The changes of main chemical components chromatogram of Radix Paeoniae Alba and Atractylodes macrocephala before and after processing and compatibility were studied using high performance liquid.Each group samples common mode fingerprint were drawn and the number of common peaks were determined to develop fingerprint quality control standards for Radix Paeoniae Alba and Atractylodes macrocephala before and after processing and compatibility.Based on "Chinese medicine fingerprint Similarity evaluation system 2004A version",Similarity was evaluated about the changes of main chemical components of Radix Paeoniae Alba and Atractylodes macrocephala.Meanwhile,in order to investigate the differences of raw medicinal herbs of Radix Paeoniae Alba and Atractylodes macrocephala from different areas and different batches,classification statistic was carried on combined with "SPSS Statistics 23.0" system clustering analysis method.In addition,fingerprint common peaks area of raw medicinal herbs of Radix Paeoniae Alba and Atractylodes macrocephala were analyzed using"SPSS Statistics 23.0" to determine the main component of raw medicinal herbs of Radix Paeoniae Alba and Atractylodes macrocephala,respectively.Then 3D spatial distribution curve of raw medicinal herbs of Radix Paeoniae Alba and Atractylodes macrocephala from different areas and different batches was drawn with the corresponding main components as space coordinates.Intuitive image would be seen about the changes of main component of Radix Paeoniae Alba and Atractylodes macrocephala before and after the compatibility and processing.Finally,we used standard comparison method to calibrate fingerprint common peaks of Radix Paeoniae Alba and Atractylodes macrocephala before and after the compatibility and processing,combined the related literatures in recent years.Six kinds of main chemical component(gallic acid,oxypaeoniflorin,peony albiflorin,paeoniflorin,1,2,3,4,6-O-Pentagalloylglucos,benzoyl paeoniflorin)of Radix Paeoniae Alba and four kinds of main chemical component(Atractylenolide Ⅲ,Atractylenolide Ⅰ2 Atractylenolide Ⅱ,Atractylodin)of Atractylodes macrocephala were confirmed.Furthermore,we quantified these main chemical component to investigate the changes of main chemical component content and provide theoretical basis for stating the mechanisms of chemical component changes of Radix Paeoniae Alba and Atractylodes macrocephala before and after the compatibility and processing.Methods This research mainly had the following two parts:1.The establishment of HPLC fingerprint of Radix Paeoniae Alba and Atractylodes macrocephala:Sample preparation-six groups of samples(raw radix paeoniae alba group,processed radix paeoniae alba group,raw atractylodes macrocephala group,processed atractylodes macrocephala group,raw compatibility group,processed compatibility group)were accurately weighed.By eflux extraction with 10 times amount of 70%alcohol in 80℃water bath for 1 h and 70%methanol to complement the weightlessness,the filtrate 1 was collected after filtering.Then the filter residue continued with 10 times amount of 70%alcohol in 80℃ water bath for 1 h and 70%methanol to complement the weightlessness.The filtrate 2 was collected after filtering again.Discarding the residue and merging filtrate 1 and filtrate 2 to dry on a rotating evaporation instrument,and add 70%methanol to constant volume to 25 mL.Six test samples groups were obtained placing in 4℃ for preservation.HPLC analysisall samples were filtered through 0.45 μm filter membrane,and then separated on Dionex ultimate 3000 HPLC-DAD chromatographic system(Dionex,Sunnyvale,CA,USA).The column was Inertsil ODS-SP Extend C18 column(250 mm × 4.6 mm,5 μm).Flow rate was 1.0 mL/min.Detection wavelength was 255 nm.Mobile phase consisted of 0.1%phosphoric acid solution(a)-acetonitrile(b)with gradient elution program as follows:0-20 min,5-10%B;20-35 min,10-15%B;35-50 min,15-18%B;50-65 min,18-20%B;65-70 min,2030%B;70-80 min,30%-50%B;80-120 min,50-65%B;120-130 min,65-5%B.Sample volume was 10 μL.Column temperature was 25℃.The establishment of fingerprint13 batches of HPLC chromatogram of each group were import into "Chinese medicine fingerprint Similarity evaluation system 2004A version" respectively.Then HPLC fingerprint would be obtained and corresponding control fingerprints would be generated.By determinating the number of each sample common peaks and comparing the changes of HPLC fingerprints of Radix Paeoniae Alba and Atractylodes macrocephala before and after the compatibility and processing,quality control standards was developed about HPLC fingerprints of Radix Paeoniae Alba and Atractylodes macrocephala before and after the compatibility and processing.Combined with similarity evaluation,SPSS Statistics 23.0 system cluster analysis and main component analysis,similarity of six groups Radix Paeoniae Alba and Atractylodes macrocephala before and after the compatibility and processing,the differences of raw medicinal herbs of Radix Paeoniae Alba and Atractylodes macrocephala from different areas and different batches,and the main component changes of Radix Paeoniae Alba and Atractylodes macrocephala before and after the compatibility and processing were investigated.2.The determination of the content of main chemical component of Radix Paeoniae Alba and Atractylodes macrocephala before and after compatibility and processing:Chromatographic conditions were the same with HPLC fingerprint analysis conditions.Using standard comparison method,common peaks of Radix Paeoniae Alba and Atractylodes macrocephala were identified in HPLC fingerprint.After standard comparison,the 6 main effective components(Gallic acid,oxypaeoniflorin,peony albiflorin,paeoniflorin,1,2,3,4,6-O-Pentagalloylglucos,benzoyl paeoniflorin)were identified in Radix Paeoniae Alba and the 4 major components(atractylenolide Ⅲ,atractylenolide Ⅰ,atractylenolide Ⅱ,atractylodin)were identified in Atractylodes macrocephala.Through the comparative analysis of the changes of 10 kinds of main chemical components of Radix Paeoniae Alba and Atractylodes macrocephala before and after compatibility processing,Chinese medicine compatibility processing method changing chemical composition mechanism of action were stated to some extent.To provide experimental basis and theoretical basis for further research on the correlation between chemical components and the differences in the effect of the chemical composition and the effect of the compatibility of traditional Chinese medicine.Thus in a certain extent interpretation of Chinese medicine processing and compatibility on the changes of chemical components of mechanism was stated.To further study the correlation between the changes of chemical components and the differences in the effects of different active components on the processing and compatibility of Chinese Medicine,the study provided experimental basis and theoretical basis.Results 1.We established the HPLC fingerprint of the six groups samples of Radix Paeoniae Alba and Atractylodes macrocephala before and after the compatibility and processing,and determined the number of peaks and the similarity of the fingerprints in six groups.2.Raw medicinal herbs of Radix Paeoniae Alba and Atractylodes macrocephala from different areas and different batches were divided into 3 categories by SPSS system cluster analysis.Three main components of Radix Paeoniae Alba and Atractylodes macrocephala were determined by SPSS principal component analysis,respectively.3.The quantitative and qualitative analysis about the main chemical components of Radix Paeoniae Alba and Rhizoma Atractylodis before and after compatibility processing showed that total glucosides of paeony and Atractylodes lactone were increased in varying degrees.Conclusion The established HPLC fingerprint analysis method is simple,efficient and practical,which could fully express chemical composition information of Radix Paeoniae Alba and Atractylodes macrocephala.Comparing the change of HPLC fingerprint of Radix Paeoniae Alba and Atractylodes macrocephala before and after compatibility processing and evaluating the quality Radix Paeoniae Alba and Atractylodes macrocephala before and after compatibility processing in each group,effectively.The research on HPLC quantitative methodology showed that the established HPLC quantitative analysis method is accurate,reliable and simple.Crude Radix Paeoniae Alba and crude Atractylodes macrocephala from different habitats and different batches were reasonably divided into 3 categories and proved of 3 main components through SPSS cluster analysis and main component analysis.The results were reasonable,credible and intuitive. |
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