| ObjectiveTo optimize the preparation technology of Jichuanjian substance benchmark,carry out the overall quality control of Jichuanjian substance benchmark,determine the scientific and reasonable quality standard draft of Jichuanjian substance benchmark,and lay the foundation for the development and research of Jichuanjian substance benchmark.Methods1.To establish a method for the determination of echinacea,mulitinoside,ligustilide,naringin,neohesperidin and isoferulic acid in Jichuanjian substance benchmark.The method was used to determine the content of multiple index components in Jichuanjian substance benchmark,and the weight of the contents of echinacea,mulitinoside,ligustilide,naringin,neohesperidin and isoferulic acid in Jichuanjian substance benchmark was taken as the index to investigate the influencing factors of the preparation of Jichuanjian substance benchmark,such as the soaking time,decocting temperature,decocting time,filtering method and drying method and optimize the preparation of Jichuanjian substance benchmark.The stability of the preparation process of Jichuanjian substance benchmark were verified by the paste yield.On the basis,the contents of multi index components in 15 batches of Jichuanjian substance benchmark were determined,which provided the basis for the quantitative study of Jichuanjian substance benchmark.The transfer rate of multi index components in Jichuanjian substance benchmark was determined,which revealed the relationship of multi index components in Jichuanjian substance benchmark from “pieces-substance benchmark” was revealed.2.To establish a HPLC fingerprint method of Jichuanjian substance benchmark.The fingerprint of 15 batches of Jichuanjian substance benchmark were established by using the method,which were used to assign and identify the peaks.Similarity Evaluation System for Chromatographic Fingerprint of TCM(2004A edition) was used to evaluate the fingerprints.SPSS 22.0 software and SIMCA 14.1 software was used for cluster analysis and discriminant analysis of partial least squares of 15 batches of Jichuanjian substance benchmark samples and the quality of Jichuanjian substance benchmark was evaluated as a whole.3.In order to control the quality of Jichuanjian substance benchmark,it was qualitatively identified by the physical and chemical properties(character,pH value,relative density)and micro identification and thin layer identification of the Jichuanjian substance benchmark were determined.Finally,based on the preparation process,content determination,fingerprint,physical and chemical properties,microscopic identification,thin layer identification of Jichuanjian substance benchmark,the draft quality standard of Jichuanjian substance benchmark was established,which provides the basis for the quality control of Jichuanjian substance benchmark.Results1.The method for the determination of echinacea,mulitinoside,ligustilide,naringin,neohesperidin and isoferulic acid in Jichuanjian substance benchmark was established.Its specificity,linear relationship,precision,repeatability and sample recovery were all good,which indicated that the method could be used for the determination of Jichuanjian substance benchmark.The optimized preparation process of Jichuanjian substance benchmark is as follows: accurately weighing wine washed Cistanche 7.46 g,Angelica 11.19 g,Achyranthes bidentata 7.46 g,Alisma 5.60 g,Fructus aurantii 3.73 g,Cimicifuga 1.87 g,adding water 300 ml,soaking for 60 min,cover with a gentle fire after boiling to 160 ml,filtering with a 200 mesh screen while hot,filter concentrate and freeze dry the filtrate,the freeze-dried powder of Jichuanjian substance benchmark was obtained.The 15 batches of Jichuanjian substance benchmark were prepared by this preparation process,the average paste yield was 21.59% and no discrete data(70%～130% of the average value)were found,which indicated that the preparation process of Jichuanjian substance benchmark were stable.The contents of echinacea,mulitinoside,ligustilide,naringin,neohesperidin and isoferulic acid in 15 batches of Jichuanjian substance benchmark were 4.0916 mg/g～4.6131 mg/g、1.6521 mg/g～1.9105 mg/g、5.0196 mg/g～5.3010 mg/g、4.2742 mg/g～4.5794 mg/g、2.4102 mg/g～2.5725 mg/g、0.1786 mg/g～0.3131 mg/g,which provided the basis for the quantitative study of Jichuan decoction.The average transfer rates of echinoside,mulitinoside,ligustilide,naringin,neohesperidin and isoferulic acid were 71.20%,66.83%,48.33%,69.72%,68.03% and 72.64% respectively in 15 batches of Jichuanjian substance benchmark,which indicated that echinaoside and mulleioside in Cistanche,ligustilide in Angelica,naringin and neohesperidin in Fructus aurantia,and isoferulic acid in Cimicifuga can be stably transferred to Jichuanjian substance benchmark.2.The HPLC fingerprint method of Jichuanjian substance benchmark was established and the RSD of the relative retention time and relative peak area of the common peak was less than 5%,which indicated that the method could be used for the overall evaluation of Jichuanjian substance benchmark quality.The fingerprints of 15 batches of Jichuanjian substance benchmark were established by use the method,the similarity was more than 0.90,and 18 common peaks were demarcated,among which the peak 1 was from Cistanche,Achyranthes bidentata and Fructus aurantii;the peak 2 was from Alisma orientalis;the peak 3 was from cistanche and Fructus aurantii;the peak 4 was from angelica and Fructus aurantii;the peaks 5,9,11,12,13 and 14 were from Fructus aurantii;the peaks 6 and 10 were from Cistanche;the peak 7 were from Angelica and Cimicifuga;the peaks 8,15,16 and 17 were from Fructus aurantii Cohosh;peak 18 from Angelica.Seven common peaks were identified,among which the peak 6 was Echinacea glycoside,the peak 7 was ferulic acid,the peak 8 was isoferulic acid,the peak 10 was Mullein glycoside,the peak 12 was naringin,the peak 14 was neohesperidin and the peak 18 was ligustilide.Cluster analysis and partial least square discriminant analysis were used to classify the 15 batches of Jichuanjian substance benchmark into two groups: S6,S8,S2,S5,S14,S11 and S12 batches into the first group,and S7,S15,S3,S4,S13,S1,S9 and S10 batches into the second group,which may be due to the different origin of each piece in the 15 batches of Jichuanjian substance benchmark.The components that contributed a lot to the overall quality of Jichuanjian substance benchmark were identified as peak 6(Echinacea glycoside),peak 14(neohesperidin),peak 18(ligustilide),peak 12(naringin),peak 7(ferulic acid),peak 8(isoferulic acid),peak 10(Mullein glycoside),peak 9(unknown)and peak 13(unknown)respectively.The results showed that the overall quality of the reference materials of 15 batches of Jichuanjian substance benchmark was stable,and its fingerprint could reflect the overall quality profile of Jichuanjian substance benchmark.3.The physical and chemical properties of Jichuan Decoction reference material were tested.Its properties are dark brown powder,slightly sweet and bitter;pH value is 5.50,slightly acidic;relative density value is 1.015 g/ml,indicating that the physical and chemical properties of Jichuanjian substance benchmark are relatively stable.Microscopical identification was carried out for Jichuanjian substance benchmark.Among them,the thin-walled cells of Angelica sinensis are spindle shaped,with very fine oblique intertexture texture;the thin-walled cells of Fructus aurantii contain calcium oxalate square crystal;the bast fibers of Cimicifuga are in bundles,light yellow green,with narrow or blunt ends,some of which are bifurcated;the wood fibers of Achyranthes bidentata are in bundles,with thin walls and oblique cracks;the thin-walled cells of Alisma orientalis are round,with oval holes The vertical wall of endothelial cells is wavy and curved,thick,with sparse pores.The results of TLC identification of Jichuanjian substance benchmark showed that in the chromatogram of Jichuanjian substance benchmark,there were fluorescent spots of the same color corresponding to the chromatogram of Cistanche deserticola,angelica,Fructus aurantii,Cimicifuga,Achyranthes bidentata,Alisma and Echinacea,naringin.Based on the above research,the quality standard draft of Jichuanjian substance benchmark was established successfully.ConclusionIn this study,the best preparation technology of Jichuanjian substance benchmark was optimized,and the multi index contents of 15 batches of Jichuanjian substance benchmark were determined.The fingerprints similarity of 15 batches of Jichuanjian substance benchmark were all more than 0.90,18 common peaks were demarcated,and 7 peaks were identified.Through chemical pattern recognition,the 15 batches of Jichuanjian substance benchmark were divided into 2 categories,and 9 components which contributed a lot to the overall quality of Jichuanjian substance benchmark were determined.Physical and chemical properties and qualitative identification of Jichuanjian substance benchmark were carried out.Finally,based on the quantitative research,physical and chemical properties determination,qualitative identificationand fingerprint of Jichuanjian substance benchmark,the corresponding quality evaluation indexes and methods are established,and the scientific and reasonable quality standard draft of Jichuanjian substance benchmark is determined. |
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