不同辅料对淡豆豉炮制过程及质量影响的研究
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摘要
目的:通过不同辅料炮制的淡豆豉中大豆异黄酮HPLC指纹图谱及含量变化,比较不同辅料对淡豆豉炮制过程的影响,揭示淡豆豉的炮制机理。在此基础上,建立淡豆豉样品中大豆苷、染料木苷、大豆苷元和染料木素多指标成分含量测定的方法,为科学评价与有效控制河北产淡豆豉药材的质量提供科学依据。
方法:
1指纹图谱的建立:(1)提取:比较了不同提取溶剂、不同提取方法及不同提取时间的提取效果,选择提取成分较多,提取率较高的提取条件,并对提取物进行纯化。(2)色谱条件:选用合适的色谱柱及检测波长,调整流动相的组成、配比,调节柱温,使色谱图符合指纹图谱研究的要求。(3)系统适用性试验:在已确定的色谱条件下,考察指纹图谱中染料木苷峰的分离度和理论板数。(4)稳定性试验:取样品溶液分别于0,4,8,12,24,48h进样,记录保留时间和峰面积。(5)精密度试验:取同一份样品溶液,重复进样6次,记录保留时间和峰面积。(6)重复性试验:取同一批淫羊藿炮制淡豆豉药材6份,平行制备样品溶液,进样,记录保留时间和峰面积。(7)指纹图谱的建立:分别取不同批次的不同辅料炮制的淡豆豉和黑豆样品,制备供试品溶液,进行指纹图谱分析。(8)炮制辅料图谱的建立:分别取淫羊藿、青蒿、桑叶药材,制备供试品溶液,进样,得到各药材图谱。
2总异黄酮成分的含量测定:(1)测定波长的确定:取对照品溶液及供试品溶液适量,以相应试剂作空白,照紫外-可见分光光度法,在200~800nm波长范围内进行光谱扫描,考察最大吸收波长。(2)标准曲线的绘制:配制系列浓度的染料木素对照品溶液,分别测定吸收度,以吸收度为纵坐标、质量浓度为横坐标绘制标准曲线。(3)稳定性试验:取样品溶液放置0,4,8,12,24,48h后测定吸收度。(4)精密度试验:取同一份样品溶液,重复6次,测定吸收度。(5)重复性试验:取同一批次淫羊藿炮制淡豆豉药材,平行制备样品溶液6份,分别测定吸收度。(6)回收率试验:取已知总异黄酮含量的药材适量,分别加入适量的染料木素对照品溶液,各平行制备三个不同浓度,每个浓度的样品分别平行3份,测定总异黄酮的含量。(7)样品测定:在上述条件下,测定10批河北产不同辅料炮制淡豆豉及黑豆原料中大豆异黄酮的含量。
3四种大豆异黄酮成分的含量测定:(1)标准曲线的绘制:配制系列浓度的染料木苷、大豆苷、染料木素和大豆苷元对照品溶液,分别进样,测定峰面积;以染料木苷、大豆苷、染料木素和大豆苷元的含量为横坐标,相应的峰面积为纵坐标,分别绘制标准曲线。(2)稳定性试验:取样品溶液分别放置0,4,8,12,24,48h后进样,测定峰面积。(3)精密度试验:取同一份样品溶液,重复进样6次,测定峰面积。(4)重复性试验:取同一批次淡豆豉药材平行制备样品溶液6份,分别进样,测定峰面积。(5)回收率试验:取已知大豆苷、染料木苷、大豆苷元和染料木素含量的淡豆豉药材适量,分别加入适量的大豆苷、染料木苷、大豆苷元和染料木素对照品溶液,各平行制备三个不同浓度,每个浓度的样品分别平行3份,测定大豆苷、染料木苷、大豆苷元和染料木素的含量。(6)最低检测限的确定:将大豆苷、染料木苷、大豆苷元和染料木素对照品溶液逐步稀释,当信噪比S/N≥3时确定为最低检测限。(7)样品测定:在上述色谱条件下,测定10批河北产淡豆豉药材中大豆苷、染料木苷、大豆苷元和染料木素的含量。
结果:
1指纹图谱的建立:(1)提取:以70%乙醇超声提取2h,然后以AB-8大孔吸附树脂进行吸附,70%乙醇进行洗脱为佳,具有稳定和效率高等优点。(2)色谱条件:色谱柱为Agilent HC-C18柱(250×4.6mm,5μm);乙腈-3%冰乙酸为流动相进行梯度洗脱,流速为1.0ml/min,柱温为25℃,检测波长为261nm,进样量10μl。(3)系统适用性试验:在此色谱条件下,染料木苷的保留时间约为21min,按染料木苷峰计算理论板数约为10万,与其它色谱峰的分离度大于1.0。(4)稳定性试验:以染料木苷峰为内参比峰,计算各主要色谱峰相对保留时间及峰面积占总峰面积5%以上峰的相对峰面积值,其RSD分别为0.02%~1.06%和0.07%~1.30%,稳定性良好。(5)精密度试验:各主要色谱峰相对保留时间和峰面积占总峰面积5%以上峰的峰面积比值均无明显变化,其RSD分别为0.01%~0.58%和0.1%~2.21%,精密度良好。(6)重复性试验:各主要色谱峰相对保留时间和峰面积占总峰面积5%以上峰的峰面积比值均无明显变化,其RSD分别为0.02%~0.53%和0.10%~2.02%,重复性良好。(7)指纹图谱的建立:分别得到10批淫羊藿炮制淡豆豉、淡豆豉、黑豆原料的指纹图谱,并生成各药材对照指纹图谱。(8)数据分析:运用相似度对所得数据进行分析,结果相似度均在0.99~1.00之间。(9)炮制辅料图谱的建立:分别得到淫羊藿、青蒿、桑叶药材在已选定色谱条件下的图谱。
2总异黄酮成分的含量测定:(1)测定波长的确定:对照品溶液和供试品溶液均在263nm处有最大吸收,故确定263nm为测定波长。(2)标准曲线的制备:以染料木素吸光度为纵坐标,浓度为横坐标,绘制吸收曲线,经回归处理得染料木素的标准曲线方程:Y=0.1393X+0.0466,相关系数为r=0.999(n=5),通过回归方程可以看出,染料木素对照品浓度在1.590~5.566μg.ml-1范围内,与吸收度呈良好的线性关系。(3)稳定性试验:样品在48h内稳定,总异黄酮含量的RSD为1.00%。(4)精密度试验:样品精密度良好,总异黄酮含量的RSD为0.10%。(5)重复性试验:样品重复性良好,总异黄酮含量的RSD为0.73%。(6)回收率试验:染料木素的平均回收率为100.70%,RSD为1.36%(n=9)。(7)含量测定结果:淫羊藿炮制淡豆豉药材中总异黄酮的含量从2.6241~2.9907mg/g,平均含量为2.8187mg/g;淡豆豉药材中总异黄酮的含量从2.1323~2.7705mg/g,平均含量为2.4405 mg/g;黑豆原料中总异黄酮的含量从2.1119~2.8398mg/g;平均含量为2.2357mg/g。
3四种大豆异黄酮成分的含量测定:(1)标准曲线的绘制:大豆苷、染料木苷、大豆苷元和染料木素分别在10.38~103.80μg、2.32~23.20μg、6.40~64.00μg、2.98~29.82μg范围内,呈良好的线性关系,回归方程分别为Y=26.059X+10.46,Y=35.181X-10.905,Y=89.756X-7.2494,Y=90.618X+57.825,相关系数均为r=0.9999(n=6)。(2)稳定性试验:样品在48h内稳定,大豆苷、染料木苷、大豆苷元和染料木素含量的RSD分别为1.00%、0.43%、0.46%和0.42%。(3)精密度试验:样品的精密度良好,大豆苷、染料木苷、大豆苷元和染料木素含量的RSD分别为1.59%、0.54%、1.24%和0.28%。(4)重复性试验:样品的重复性好,大豆苷、染料木苷、大豆苷元和染料木素含量的RSD分别为2.36%、0.69%、1.51%和0.45%。(5)回收率试验:大豆苷、染料木苷、大豆苷元和染料木素的回收率分别为99.27%、98.32%、98.76%和99.03%,RSD分别为0.72%、1.32%、1.11%和0.80%。(6)大豆苷、染料木苷、大豆苷元和染料木素的最低检测限分别为0.522ng、0.122ng、0.318ng和0.152ng。(7)含量测定结果:河北产淡豆豉药材中大豆苷的含量从426.2~453.2μg/g,染料木苷的含量从376.1~401.3μg/g,大豆苷元的含量从247.6~263.8μg/g ,染料木素的含量从135.4~149.6μg/g不等。
结论:
1建立了不同辅料炮制的淡豆豉中大豆异黄酮HPLC指纹图谱及UV含量测定方法,比较了不同辅料对淡豆豉炮制过程的影响,揭示了淡豆豉的炮制机理:炮制过程是多种成分综合作用的过程,总体上来讲是苷向苷元转化的过程。
2首次建立了淡豆豉样品中大豆苷、染料木苷、大豆苷元和染料木素多指标成分含量测定的HPLC法,方法稳定,重复性和精密度良好,为科学评价与有效控制河北产淡豆豉药材的质量提供了科学依据。
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.
方法:
1指纹图谱的建立:(1)提取:比较了不同提取溶剂、不同提取方法及不同提取时间的提取效果,选择提取成分较多,提取率较高的提取条件,并对提取物进行纯化。(2)色谱条件:选用合适的色谱柱及检测波长,调整流动相的组成、配比,调节柱温,使色谱图符合指纹图谱研究的要求。(3)系统适用性试验:在已确定的色谱条件下,考察指纹图谱中染料木苷峰的分离度和理论板数。(4)稳定性试验:取样品溶液分别于0,4,8,12,24,48h进样,记录保留时间和峰面积。(5)精密度试验:取同一份样品溶液,重复进样6次,记录保留时间和峰面积。(6)重复性试验:取同一批淫羊藿炮制淡豆豉药材6份,平行制备样品溶液,进样,记录保留时间和峰面积。(7)指纹图谱的建立:分别取不同批次的不同辅料炮制的淡豆豉和黑豆样品,制备供试品溶液,进行指纹图谱分析。(8)炮制辅料图谱的建立:分别取淫羊藿、青蒿、桑叶药材,制备供试品溶液,进样,得到各药材图谱。
2总异黄酮成分的含量测定:(1)测定波长的确定:取对照品溶液及供试品溶液适量,以相应试剂作空白,照紫外-可见分光光度法,在200~800nm波长范围内进行光谱扫描,考察最大吸收波长。(2)标准曲线的绘制:配制系列浓度的染料木素对照品溶液,分别测定吸收度,以吸收度为纵坐标、质量浓度为横坐标绘制标准曲线。(3)稳定性试验:取样品溶液放置0,4,8,12,24,48h后测定吸收度。(4)精密度试验:取同一份样品溶液,重复6次,测定吸收度。(5)重复性试验:取同一批次淫羊藿炮制淡豆豉药材,平行制备样品溶液6份,分别测定吸收度。(6)回收率试验:取已知总异黄酮含量的药材适量,分别加入适量的染料木素对照品溶液,各平行制备三个不同浓度,每个浓度的样品分别平行3份,测定总异黄酮的含量。(7)样品测定:在上述条件下,测定10批河北产不同辅料炮制淡豆豉及黑豆原料中大豆异黄酮的含量。
3四种大豆异黄酮成分的含量测定:(1)标准曲线的绘制:配制系列浓度的染料木苷、大豆苷、染料木素和大豆苷元对照品溶液,分别进样,测定峰面积;以染料木苷、大豆苷、染料木素和大豆苷元的含量为横坐标,相应的峰面积为纵坐标,分别绘制标准曲线。(2)稳定性试验:取样品溶液分别放置0,4,8,12,24,48h后进样,测定峰面积。(3)精密度试验:取同一份样品溶液,重复进样6次,测定峰面积。(4)重复性试验:取同一批次淡豆豉药材平行制备样品溶液6份,分别进样,测定峰面积。(5)回收率试验:取已知大豆苷、染料木苷、大豆苷元和染料木素含量的淡豆豉药材适量,分别加入适量的大豆苷、染料木苷、大豆苷元和染料木素对照品溶液,各平行制备三个不同浓度,每个浓度的样品分别平行3份,测定大豆苷、染料木苷、大豆苷元和染料木素的含量。(6)最低检测限的确定:将大豆苷、染料木苷、大豆苷元和染料木素对照品溶液逐步稀释,当信噪比S/N≥3时确定为最低检测限。(7)样品测定:在上述色谱条件下,测定10批河北产淡豆豉药材中大豆苷、染料木苷、大豆苷元和染料木素的含量。
结果:
1指纹图谱的建立:(1)提取:以70%乙醇超声提取2h,然后以AB-8大孔吸附树脂进行吸附,70%乙醇进行洗脱为佳,具有稳定和效率高等优点。(2)色谱条件:色谱柱为Agilent HC-C18柱(250×4.6mm,5μm);乙腈-3%冰乙酸为流动相进行梯度洗脱,流速为1.0ml/min,柱温为25℃,检测波长为261nm,进样量10μl。(3)系统适用性试验:在此色谱条件下,染料木苷的保留时间约为21min,按染料木苷峰计算理论板数约为10万,与其它色谱峰的分离度大于1.0。(4)稳定性试验:以染料木苷峰为内参比峰,计算各主要色谱峰相对保留时间及峰面积占总峰面积5%以上峰的相对峰面积值,其RSD分别为0.02%~1.06%和0.07%~1.30%,稳定性良好。(5)精密度试验:各主要色谱峰相对保留时间和峰面积占总峰面积5%以上峰的峰面积比值均无明显变化,其RSD分别为0.01%~0.58%和0.1%~2.21%,精密度良好。(6)重复性试验:各主要色谱峰相对保留时间和峰面积占总峰面积5%以上峰的峰面积比值均无明显变化,其RSD分别为0.02%~0.53%和0.10%~2.02%,重复性良好。(7)指纹图谱的建立:分别得到10批淫羊藿炮制淡豆豉、淡豆豉、黑豆原料的指纹图谱,并生成各药材对照指纹图谱。(8)数据分析:运用相似度对所得数据进行分析,结果相似度均在0.99~1.00之间。(9)炮制辅料图谱的建立:分别得到淫羊藿、青蒿、桑叶药材在已选定色谱条件下的图谱。
2总异黄酮成分的含量测定:(1)测定波长的确定:对照品溶液和供试品溶液均在263nm处有最大吸收,故确定263nm为测定波长。(2)标准曲线的制备:以染料木素吸光度为纵坐标,浓度为横坐标,绘制吸收曲线,经回归处理得染料木素的标准曲线方程:Y=0.1393X+0.0466,相关系数为r=0.999(n=5),通过回归方程可以看出,染料木素对照品浓度在1.590~5.566μg.ml-1范围内,与吸收度呈良好的线性关系。(3)稳定性试验:样品在48h内稳定,总异黄酮含量的RSD为1.00%。(4)精密度试验:样品精密度良好,总异黄酮含量的RSD为0.10%。(5)重复性试验:样品重复性良好,总异黄酮含量的RSD为0.73%。(6)回收率试验:染料木素的平均回收率为100.70%,RSD为1.36%(n=9)。(7)含量测定结果:淫羊藿炮制淡豆豉药材中总异黄酮的含量从2.6241~2.9907mg/g,平均含量为2.8187mg/g;淡豆豉药材中总异黄酮的含量从2.1323~2.7705mg/g,平均含量为2.4405 mg/g;黑豆原料中总异黄酮的含量从2.1119~2.8398mg/g;平均含量为2.2357mg/g。
3四种大豆异黄酮成分的含量测定:(1)标准曲线的绘制:大豆苷、染料木苷、大豆苷元和染料木素分别在10.38~103.80μg、2.32~23.20μg、6.40~64.00μg、2.98~29.82μg范围内,呈良好的线性关系,回归方程分别为Y=26.059X+10.46,Y=35.181X-10.905,Y=89.756X-7.2494,Y=90.618X+57.825,相关系数均为r=0.9999(n=6)。(2)稳定性试验:样品在48h内稳定,大豆苷、染料木苷、大豆苷元和染料木素含量的RSD分别为1.00%、0.43%、0.46%和0.42%。(3)精密度试验:样品的精密度良好,大豆苷、染料木苷、大豆苷元和染料木素含量的RSD分别为1.59%、0.54%、1.24%和0.28%。(4)重复性试验:样品的重复性好,大豆苷、染料木苷、大豆苷元和染料木素含量的RSD分别为2.36%、0.69%、1.51%和0.45%。(5)回收率试验:大豆苷、染料木苷、大豆苷元和染料木素的回收率分别为99.27%、98.32%、98.76%和99.03%,RSD分别为0.72%、1.32%、1.11%和0.80%。(6)大豆苷、染料木苷、大豆苷元和染料木素的最低检测限分别为0.522ng、0.122ng、0.318ng和0.152ng。(7)含量测定结果:河北产淡豆豉药材中大豆苷的含量从426.2~453.2μg/g,染料木苷的含量从376.1~401.3μg/g,大豆苷元的含量从247.6~263.8μg/g ,染料木素的含量从135.4~149.6μg/g不等。
结论:
1建立了不同辅料炮制的淡豆豉中大豆异黄酮HPLC指纹图谱及UV含量测定方法,比较了不同辅料对淡豆豉炮制过程的影响,揭示了淡豆豉的炮制机理:炮制过程是多种成分综合作用的过程,总体上来讲是苷向苷元转化的过程。
2首次建立了淡豆豉样品中大豆苷、染料木苷、大豆苷元和染料木素多指标成分含量测定的HPLC法,方法稳定,重复性和精密度良好,为科学评价与有效控制河北产淡豆豉药材的质量提供了科学依据。
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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22 张炳文,宋永生,郝征红,等.发酵处理对大豆制品中异黄酮含量与组分影响的研究.食品与发酵工业,2002,28(7):6~9
1 张玉梅,张学斌,高旭年,等.紫外分光光度法测定大豆异黄酮的含量.中国食品科学杂志,2000,12(4):7~9
2 崔力剑,黄芸,杜淑娟,等.紫外分光光度法测定淡豆豉中异黄酮的含量.河北中医药学报,2004,19(3):32
1 Arjm and iBH.Soy isoflavones’osteoprotective role in post menopausal women:mechanism of action.J NutrBiochem, 2002,13(3):130
2 Lee CH.Relative and tioxidant activity of soybean isofla vones and their glycosides.Food Chem,2005,90(4):735
3 Guo JM.Biphasic of daidzein on cell growth of human colon cancer cell.Food Chem Toxicol,2004,42(10):1641
4 张立.RP-HPLC 法测定大豆提取物中大豆苷元、染料木素、大豆苷、染料木苷的含量.中草药,2001,32(2):118
5 褚襄萍,邱明丰,贾伟.高效液相色谱法同时测定豆粕提取液中 4 种成分的含量.中国医院药学杂志,2005,25(7):631~ 632
1 国家药典委员会编.中华人民共和国药典(一部)北京:化学工业出版社,2005:230
2 曲丽萍,宓鹤鸣,范国荣.RP-HPLC制备色谱法分离淡豆中高纯度异黄酮苷元.中药材,2005,28(12):1062
3 李时珍.明,《本草纲目》(金陵版):1344
4 蒋立文,夏波.浏阳豆豉发酵微生物的初步研究.中国酿造,2004,12(11~12):16
5 张建华.曲霉型豆豉发酵机理及其功能性研究.2003,5
6 韩立,高居金.淡豆豉生产工艺改革.中药材,1997.20(8): 394
7 李祥.细菌型豆豉生产的研究.中国调味品1999,10:14~17
8 马盼香,李雅玲,李富华.试论传统中药的发酵炮制.成都医学院学报,2006,12,1(2):125~126
9 王岙,于文海,杨明远,等.高效液相色谱法测定保健食品中大豆异黄酮.中国卫生检验杂志,2006,3(16):322
10 徐尔雅.淡豆豉的原料应使用黑豆.中药材,1994,17 (4):44~45
11 田鹏娜.淡豆豉对早期动脉粥样硬化损伤的保护作用及机制研究:56
12 余爱农,杨春海,谭志斗,等.细菌型豆豉香气成分的研究.食品科学,2002.23(12):98
13 张炳文,宋永生,郝征红,等.发酵处理对豆豉抗氧化活性影响的研究.营养卫生食品科学,2002,23(8):263
14 牛丽颖,刘娇,崔力剑,等.淡豆豉对早期动脉粥样硬化大鼠血管内皮损伤的保护作用.中药药理与临床,2007. 23(5):120
15 王鑫国,葛喜珍,白霞,等.淡豆豉对去卵巢大鼠脂代谢的影响.中药材,2003.26(9):625
16 毛俊琴,李铁军,黄晓瑾.中药淡豆豉防治去卵巢大鼠骨质疏松的实验研究.解放军药学学报,2006.22(2):236
17 葛喜珍,林强,霍清,等.淡豆豉煎剂对高脂血症大鼠主动脉晚期糖化终末产物的抑制作用.中药药理与临床,2006. 22(3,4): 101
18 Lori C,Neil CB,Kenneth DR,et al.Genistein,Daidzein,and their β-glycoside conjugates:antitumor isoflavones in soy bean foods from American and Asian diets.Agric Food Chem,1993,41:1981
19 Steels VE,Pereira MA, Sigman C, et al.Cancer chemoprev ention a-gent development strategies for genistein[J].Nutr, 1995,125(3):713
20 毛俊琴,李铁军,黄晓瑾.中药淡豆豉提取物的体外抗肿瘤作用研究.解放军药学学报,2003.19(6):407
2 王鑫国,葛喜珍,白霞,等.淡豆豉对去卵巢大鼠脂代谢的影响.中药材,2003,26(9):652~654
3 高淑丽,牛丽颍,曹秀莲,等.淡豆豉提取物抗心肌缺血作用的研究.河北医药,2007,29(9):923~924
4 牛丽颖,刘娇,崔力剑,等.淡豆豉对早期动脉粥样硬化大鼠血 管 内 皮 损 伤 的 保 护 作 用 . 中 药 药 理 与 临床,2007,23(5):923~924
5 葛喜珍,林强,霍清,等.淡豆豉煎剂对高脂血症大鼠主动脉晚 期 糖 化 终 末 产 物 的 抑 制 作 用 . 中 药 药 理 与 临床,2006,22(3,4):101
6 谢明杰等.超声波提取大豆异黄酮.大豆科学,2004,23(1): 75
7 鞠兴荣等.大豆异黄酮提取工艺的优化注.中国粮油学报,2001,16(6):17
8 袁龙等.大豆中异黄酮提取条件及其在提取液中的存在形式.徐州师范大学学报(自然科学版),2004,22(3):55
9 胡卫新等.大豆异黄酮提取条件和大豆蛋白质分离工艺研究,大豆科学,2005,24(1):26
10 汪海波.大豆胚芽甲醇提取液中大豆皂甙、大豆异黄酮分离纯化工艺的研究.食品科学,2001,256(4):40
11 Uu QZ.Preparative seperation of isoflavone compoents in soybeans using high-speed counter-current chromatography, ChromatogrA,2001,923(1~2):271
12 Yang FQ.Separation and purification of isoflavones from a crude soybean extract by high-speed counter-current chroma tography.Chromatogram,2001,928(2):163
13 郭文勇,刘彬果,钟蕾,等.大孔树脂吸附层析法提取淡豆豉中总异黄酮的研究.第二军医大学学报,2004,25(9):1033
14 罗少洪等.大豆总异黄酮的提取及含量测定.广东药学,2005,15(4):7
15 冯建光.大孔吸附树脂对大豆异黄酮的吸附与洗脱性能.无锡轻工大学学报,2003,22(1):82
16 白志川.玄参药材HPLC指纹图谱的研究.中药材,2006,29 (12):1295~1298
17 任德权.中药指纹图谱质控技术的意义与作用.中药材, 2001,24(4):2351
18 谢培山.中药质量控制模式的发展趋势.中药新药与临床药理,2001,12(3):1881
19 陈闽军,吴永江,范骁辉,等.色谱指纹图谱分析技术用于鉴别中药川芎产地.中国中药杂志,2003,28(7):6061
20 Katalinic V.High performance liquid chromatographic determination of flavan fingerprints in plant extracts.J Chro matogrA,1997,775:3591
21 周玉新,雷海民,徐永红,等.中药指纹图谱研究技术.北京:化学工业出版社,2002,111~112
22 张炳文,宋永生,郝征红,等.发酵处理对大豆制品中异黄酮含量与组分影响的研究.食品与发酵工业,2002,28(7):6~9
1 张玉梅,张学斌,高旭年,等.紫外分光光度法测定大豆异黄酮的含量.中国食品科学杂志,2000,12(4):7~9
2 崔力剑,黄芸,杜淑娟,等.紫外分光光度法测定淡豆豉中异黄酮的含量.河北中医药学报,2004,19(3):32
1 Arjm and iBH.Soy isoflavones’osteoprotective role in post menopausal women:mechanism of action.J NutrBiochem, 2002,13(3):130
2 Lee CH.Relative and tioxidant activity of soybean isofla vones and their glycosides.Food Chem,2005,90(4):735
3 Guo JM.Biphasic of daidzein on cell growth of human colon cancer cell.Food Chem Toxicol,2004,42(10):1641
4 张立.RP-HPLC 法测定大豆提取物中大豆苷元、染料木素、大豆苷、染料木苷的含量.中草药,2001,32(2):118
5 褚襄萍,邱明丰,贾伟.高效液相色谱法同时测定豆粕提取液中 4 种成分的含量.中国医院药学杂志,2005,25(7):631~ 632
1 国家药典委员会编.中华人民共和国药典(一部)北京:化学工业出版社,2005:230
2 曲丽萍,宓鹤鸣,范国荣.RP-HPLC制备色谱法分离淡豆中高纯度异黄酮苷元.中药材,2005,28(12):1062
3 李时珍.明,《本草纲目》(金陵版):1344
4 蒋立文,夏波.浏阳豆豉发酵微生物的初步研究.中国酿造,2004,12(11~12):16
5 张建华.曲霉型豆豉发酵机理及其功能性研究.2003,5
6 韩立,高居金.淡豆豉生产工艺改革.中药材,1997.20(8): 394
7 李祥.细菌型豆豉生产的研究.中国调味品1999,10:14~17
8 马盼香,李雅玲,李富华.试论传统中药的发酵炮制.成都医学院学报,2006,12,1(2):125~126
9 王岙,于文海,杨明远,等.高效液相色谱法测定保健食品中大豆异黄酮.中国卫生检验杂志,2006,3(16):322
10 徐尔雅.淡豆豉的原料应使用黑豆.中药材,1994,17 (4):44~45
11 田鹏娜.淡豆豉对早期动脉粥样硬化损伤的保护作用及机制研究:56
12 余爱农,杨春海,谭志斗,等.细菌型豆豉香气成分的研究.食品科学,2002.23(12):98
13 张炳文,宋永生,郝征红,等.发酵处理对豆豉抗氧化活性影响的研究.营养卫生食品科学,2002,23(8):263
14 牛丽颖,刘娇,崔力剑,等.淡豆豉对早期动脉粥样硬化大鼠血管内皮损伤的保护作用.中药药理与临床,2007. 23(5):120
15 王鑫国,葛喜珍,白霞,等.淡豆豉对去卵巢大鼠脂代谢的影响.中药材,2003.26(9):625
16 毛俊琴,李铁军,黄晓瑾.中药淡豆豉防治去卵巢大鼠骨质疏松的实验研究.解放军药学学报,2006.22(2):236
17 葛喜珍,林强,霍清,等.淡豆豉煎剂对高脂血症大鼠主动脉晚期糖化终末产物的抑制作用.中药药理与临床,2006. 22(3,4): 101
18 Lori C,Neil CB,Kenneth DR,et al.Genistein,Daidzein,and their β-glycoside conjugates:antitumor isoflavones in soy bean foods from American and Asian diets.Agric Food Chem,1993,41:1981
19 Steels VE,Pereira MA, Sigman C, et al.Cancer chemoprev ention a-gent development strategies for genistein[J].Nutr, 1995,125(3):713
20 毛俊琴,李铁军,黄晓瑾.中药淡豆豉提取物的体外抗肿瘤作用研究.解放军药学学报,2003.19(6):407