| Objective: To establish HPLC fingerprints and the UV assay method of soybean isoflavone in Semen Sojae Praeparatum processed by different accessories,prepare the different of component contents and the content of total soybean isoflavone between pre- and post-processing of Semen Sojae Praeparatum and reveal the principle of processing.Besides, establish a method to detect the contents of Daidzin,Genistin, Daidzein and Genistein in Semen Sojae Praeparatum at the same time in order to control the quality of Semen Sojae Praeparatum, and this method provides a scientific basis for the scientific evaluation and utility control of the quality of Semen Sojae Praeparatum of Hebei Province.Methods:1 Establishment of fingerprint:(1)Extraction: An optimal extracting condition was chose by comparing the experimental results. (2) Chromatographic condition: Choose appropriate column and adjust different formulation and proportion of mobile phase and column temperature in order to establish a better fingerprint chromatogram.(3)System suitability test:In this chromatographic condition,calculate the resolution and theoretical plate of genistin peak.(4)In the experiment of stability, transfer the sample solution from Semen Sojae Praeparatum to determine the relevant retention time and peak area at 0, 4, 8, 12, 24 and 48 hours, respectively.(5)In the experiment of precision,take the same test solution,inject to the apparatus for six times,determine the relevant retention time and peak area,respectively.(6)In the experiment of reproducibility, prepare one concentration sample of Semen Sojae Praeparatum, repeat each concentration solution for six times in the same way,determine the relevant retention time and peak area, respectively.(7)Prepare different batches concentration sample of Semen Sojae Praeparatum processed by different accessories and Black Bean,make concentration solutions, inject to HPLC, and get their relevant fingerprint chromatograms.(8)Prepare herba epimedii,southernwood and folia mori,make concentration solutions,inject to HPLC,and get their relevant chromatograms.2 The assay of total isoflavone component:(9)Preparation of standard curve:prepare a series of the reference solutions of Genistein,determine absorbance,then the regression equation was obtained with the content of Genistein as abscissa and the absorbance as ordinate.(2)In the experiment of stability,transfer the sample solution from Semen Sojae Praeparatum to determine the absorbance at 0, 4, 8, 12, 24 and 48 hours, respectively.(3)In the experiment of precision,take the same test solution,inject to the apparatus for six times,determine the absorbance of soybean isoflavone,respectively,then calculate the precision.(4)In the experiment of reproducibility,prepare one concentration sample of Semen Sojae Praeparatum,repeat each concentration solution for six times in the same way,determine the absorbance respectively,then calculate the content and RSD. (5)In the experiment of recovery,transfer 9 shares of Semen Sojae Praeparatum and add the reference solution of Genistein on three levels and every level repeat three times,determine the absorbance respectively,then calculate the content of soybean isoflavone each.(6)Assay:Under above-mentioned conditions, determine the content of soybean isoflavone in different batches of Semen Sojae Praeparatum processed by different accessories and Black Bean,respectively.3 The assay of four soybean isoflavone components:(1) Preparation of standard curve: prepare a series of the reference solutions of Daidzin,Genistin,Daidzein and Genistein, determine peak areas,then the regression equation was respectively obtained with the content of Daidzin,Genistin,Daidzein and Genistein as abscissa and the relevant peak area as ordinate. (2)In the experiment of stability,transfer the sample solution from Semen Sojae Praeparatum to determine the relevant peak area at 0, 4, 8, 12, 24 and 48 hours,respectively.(3)In the experiment of precision,take the same test solution,inject to the apparatus for six times,determine the relevant peak area of Daidzin,Genistin,Daidzein and Genistein, respectively,and then calculate the precision.(4)In the experiment of reproducebility, prepare one concentration sample of Semen Sojae Praeparatum, repeat each concentration solution for six times in the same way,detect the relevant peak area respectively,then calculate the content and RSD.(5)In the experiment of recovery,transfer 9 shares of Semen Sojae Praeparatum and add the reference solutions of Daidzin,Genistin,Daidzein and Genistein on three levels and every level repeat three times,detect the relevant peak area respectively,then calculate the content of Daidzin, Genistin, Daidzein and Genistein each.(6)Determination of the lowest detection limitation:Dilute the reference solution of Daidzin, Genistin,Daidzein and Genistein respectively until the value of S/N was more than or equal to 3.The relevant concentration was thelowest detection limitation.(7)Assay:Under above-mentioned conditions,determine the content of Daidzin,Genistin,Daidzein and Genistein in different batches of Semen Sojae Praeparatum, respectively.Results:1 Establishment of fingerprint(1)Extraction:The method of supersonic wave-extraction with 70% alcohol for 80 minutes was simple, quick and stable. (2) The HPLC system was performed on a HC-C18 analytical column gradient eluted with a mixture consisting of acetonitrile,3% glacial acetic acid at a flow rate of 1.0ml/min.The temperature of column was 25℃. The UV detection wavelength was set at 261 nm.Injection volume was 10μl.(3)System suitability test:Under the above condition,the peak corresponding to Genistin of the test solution was separated well with the resolution of more than 1.0 and about 100000 of theoretical plate.(4)The test solution was stable in 48 hours.Genistin peak as reference peak,the relevant RSD values of relative retention time and relative area were between 0.02%~1.06% and between 0.07%~1.30%,respectively.(5)The precision of sample was good and the RSD values of relative retention time and relative area were between 0.02%~0.53% and between 0.10%~2.02%,respectively.(6)The reproducibility of sample was good and the RSD values of relative retention time and relative area were between 0.01%~0.58% and between 0.1%~2.21%,respectively.(7)Get the relevant fingerprint chroma tograms of different batches of Semen Sojae Praeparatum processed by different accessories and Black Bean of Hebei Province.(8)Data analysis:Use similarity clustering analysis to analyze data,and the results showed that the similarity of samples was between 0.99~1.00.(9)Get the relevant chroma tograms of herba epimedii,southernwood and folia mori under above-mentioned conditions.2 The assay of total isoflavone component:(1)The regression equation of Genistein was:Y=0.1393X+0.0466,r=0.999(n = 5).The linear range was between 1.590~5.566μg.ml-1.(2)The test solution was stable in 48 hours and the RSD value of soybean isoflavone was 1.00%.(3)The precision of sample was good and the RSD value of soybean isoflavone was 0.10%.(4)In the experiment of reproducibility,the RSD values of soybean isoflavone were 0.73%.(5)The average recovery of soybean isoflavone was 99.27% and the RSD was 0.72%.(6)The results showed the contents of soybean isoflavone in Semen Sojae Praeparatum processed by herba epimedii were between 2.6241~2.9907mg/g, and the average was 2.8187mg/g; the contents of soybean isoflavone in Semen Sojae Praeparatum were between 2.1323~2.7705mg/g, and the average was 2.4405mg/g;the contents of soybean isoflavone in Black Bean were between 2.1119~2.8398mg/g and the average was 2.2357 mg/g.3 The assay of four soybean isoflavone components. (1)Daidzin,Genistin,Daidzein and Genistein of there regression equations were: Y=26.059X+10.462 r = 0.9999(n = 6), Y=35.181X-10.905 r = 0.9999(n = 6),Y=89.756X-7.2494 r =0.9999(n=6),Y=90.618X+57.825 r=0.9999(n=6),respectively. Their linear range were between 10.38~103.80μg,2.32~23.2μg, 6.40~64.00μg and2.98~29.82μg,respectively(2)The test solution was stable in 48 hours and the RSD values of Daidzin, Genistin,Daidzein and Genistein were 1.00%,0.43%, 0.46% and 0.42%,respectively.(3)The precision of sample was good and the RSD values of Daidzin,Genistin,Daidzein and Genistein were 1.59%,0.54%,1.24% and 0.28%, respectively.(4) In the experiment of reproducibility,the RSD values of Daidzin, Genistin,Daidzein and Genistein were 2.36%,0.69%,1.51% and 0.45%,respectively.(5)The average recoveries of Daidzin, Genistin,Daidzein and Genistein were 99.27%,98.32%,98.76% and 99.03% and their RSD values were 0.72%,1.32%,1.11% and 0.80% respectively. (6) The lowest detection limitations of Daidzin,Genistin,Daidzein and Genistein were 0.522ng,0.122ng, 0.318ng and 0.152,respectively.(7)The results showed the contents of Daidzin,Genistin,Daidzein and Genistein in Semen Sojae Praeparatum of different sources were between 426.2~ 453.2μg/g,376.1~401.3μg/g,247.6~263.8μg/g,135.4~149.6μg/g,respectively.Conclusion:1 Through the preparation of the influence of different accessories to the processing of Semen Sojae Praeparatum by establishing the HPLC fingerprint chromatogram and the UV assay method of soybean isoflavone in Semen Sojae Praepara tum processed by different accessories,we reveals the principle of processing:Processing is a course of synthetic action with many ingredients,which can reduce the content of glucoside and increase the content of aglycon.2 It is the first time to establish the method to detect the contents of Daidzin,Genistin,Daidzein and Genistein in Semen Sojae Praeparatum at the same time,and the quality of Semen Sojae Praeparatum was evaluated generally.This method is steady,precise and reproductive.Besides,it provides a scientific basis for the scientific evaluation and utility control of the quality of Semen Sojae Praeparatum of Hebei Province.
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