豆酱中Daidzein和Genistein提取纯化及功能性研究
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摘要
豆酱是我国传统豆类发酵食品之一，制曲时霉菌分泌的葡萄糖苷酶在发酵过程中降解大豆中异黄酮糖苷键，释放和游离出异黄酮单体成分，如黄豆苷元（Daidzein，De）、染料木黄酮（Gentstein，Ge）和黄豆黄素(（Glycitein，Gle）等。大量的科学研究已经证明，大豆异黄酮（如：染料木黄酮）具有抗肿瘤、防止骨质疏松症、缓解更年期综合症以及防治心血管疾病等多种功效。
本课题以豆酱中游离异黄酮黄豆苷元和染料木黄酮为研究对象，对成品豆酱中的黄豆苷元和染料木黄酮成分进行分离、提取、纯化，并对其抗氧化活性和抗肿瘤活性进行验证。主要研究结果如下：
1、研究了超声波辅助提取法提取豆酱中的黄豆苷元和染料木黄酮的工艺条件，采用响应面法对黄豆苷元和染料木黄酮的超声波提取工艺条件进行优化，最终确定了该提取方法的最佳工艺参数。黄豆苷元的最佳提取工艺参数为：超声波提取时间为62min，超声波提取温度为48℃，乙醇浓度为74%，超声功率为弱，乙醇用量为150mL，豆酱中黄豆苷元的得率为484.610μg/g；染料木黄酮的最佳提取工艺参数为：乙醇浓度79%，提取时间60min，提取温度49℃，提取功率为弱，乙醇用量为150mL，相应提取液中染料木黄酮的得率为100.07μg/g。
2、对黄豆苷元和染料木黄酮的分离纯化进行了研究。结果显示：筛选出适合黄豆苷元和染料木黄酮分离纯化的大孔树脂为AB-8型大孔树脂。上样量3mg，吸附速度为2mL/min，洗脱速度为2mL/min。在此条件下，黄豆苷元的纯度为71.49%，染料木黄酮的纯度为64.78%。
采用SephadexLH-20葡聚糖凝胶分别对黄豆苷元和染料木黄酮进一步纯化，SephadexLH-20葡聚糖凝胶纯化黄豆苷元、染料木黄酮的工艺条件均为：上样量5mg，洗脱速度2mL/min，乙醇浓度70%。利用高效液相色谱对三个洗脱峰进行检测，洗脱峰3为黄豆苷元或者染料木黄酮，黄豆苷元的纯度为94.31%，染料木黄酮的纯度为90.72%。
3、对黄豆苷元和染料木黄酮的体外抗氧化活性和抗肿瘤活性进行研究。结果表明黄豆苷元、染料木黄酮具有较强的还原能力，均低于各自的对照品，黄豆苷元的还原能力低于维生素C，染料木黄酮的还原能力高于维生素C。黄豆苷元、染料木黄酮对DPPH.自由基、羟自由基都有一定的清除能力，且高于维生素C低于相应的标准对照品。染料木黄酮对羟自由基的清除能力高于黄豆苷元。通过黄豆苷元、染料木黄酮对人乳腺癌细胞生长的抑制情况的考察，采用MTT试验测定抑制率。MTT试验结果表明，黄豆苷元和染料木素对人乳腺癌细胞的生长均具有一定的抑制作用，染料木黄酮的抑制作用强于黄豆苷元。
Soy sauce is one of the traditional beans fermented food in China.Mold secreted glucosidasethat hydrolyze Isoflavones glycosidic bonds to Daidzein、Gentstein、Glycitein, et al. during fer--mentation. An explosion of research have been shown that Isoflavones Fight tumour, prevent bo--ne loose, protect cardiovascular and relaxed women's Menopause Syndrome.
The study of Daidzein and Gentstein which extracted, isolated and purificated from Soy sau--ce,with a view toward testing antioxidant activities and anticancer activity. The results showedthat:
1、This paper studied the extraction of Daidzein and Genistein in soybean paste by,ultrasoundmethod.Based on comprehensive consider--ation of extraction rate,economy and simplicity,theultrasonic extraction process of Daidzein and Genistein was optimized by response surfacemethod,the extraction parameters has been determined finally.The optimum extraction conditionsof Daidzein were:extracting time by ultrasonic was61minutes, extraction temperature was48℃,ethanol concentration was74%,the yield rate of Daidzein in soybean paste was484.610μg/g;The optimum extraction conditions of Genistein were:ethanol concentration was78%,extraction temperature was59min, extraction temperature was49℃,extraction power wasweak,the yield rate of Genistein in extracting solution was100.07μg/g.
2、The isolation, purifying of Daidzein and Gentstein were studied.The results showed thatAB-8resin has a good isolation and purifying ability for Daidzein and Gentstein. And the adsor--ption elution conditions were: sample quantity was3mg, adsorption rate was2mL/min, the eluti--on rate was2mL/min.Under this conditions, the purity of Daidzein and Gentstein respectivelywas71.49%,64.78%. The SephadexLH-20dextran gel used in further respectively purification ofDaidzein and Gentstein. And the process conditions were: sample quantity was5mg, the elutionrate was2mL/min, ethanol concentration was70%.Elution peaks were measured by HPLC and thethird elution peak was Daidzein and Gentstein,and the purity of Daidzein and Gentstein respecti--veely was94.31%,90.72%.
3、The anti-oxidation activity and anti tumor activity of Daidzein and Gentstein extract fromSoy sauce were studied. The Daidzein and Gentstein had strong reducing capacity,it was below itsrespectively standard reference substance. The Vc had better reducing capacity than Daidzein andit less than Gentstein. The DPPH free radical and hydroxyl radical scavenging ability of Daidzeinand Gentstein between Vc and respectively standard reference substance,and Vc is less thanstandard reference substance.Using MTT experiment determined the inhibition rate.The experi--mental results show that the Daidzein and Gentstein had inhibition on the growth of Humanbreast cancer cells, Daidzein was not good as Gentstein.
本课题以豆酱中游离异黄酮黄豆苷元和染料木黄酮为研究对象，对成品豆酱中的黄豆苷元和染料木黄酮成分进行分离、提取、纯化，并对其抗氧化活性和抗肿瘤活性进行验证。主要研究结果如下：
1、研究了超声波辅助提取法提取豆酱中的黄豆苷元和染料木黄酮的工艺条件，采用响应面法对黄豆苷元和染料木黄酮的超声波提取工艺条件进行优化，最终确定了该提取方法的最佳工艺参数。黄豆苷元的最佳提取工艺参数为：超声波提取时间为62min，超声波提取温度为48℃，乙醇浓度为74%，超声功率为弱，乙醇用量为150mL，豆酱中黄豆苷元的得率为484.610μg/g；染料木黄酮的最佳提取工艺参数为：乙醇浓度79%，提取时间60min，提取温度49℃，提取功率为弱，乙醇用量为150mL，相应提取液中染料木黄酮的得率为100.07μg/g。
2、对黄豆苷元和染料木黄酮的分离纯化进行了研究。结果显示：筛选出适合黄豆苷元和染料木黄酮分离纯化的大孔树脂为AB-8型大孔树脂。上样量3mg，吸附速度为2mL/min，洗脱速度为2mL/min。在此条件下，黄豆苷元的纯度为71.49%，染料木黄酮的纯度为64.78%。
采用SephadexLH-20葡聚糖凝胶分别对黄豆苷元和染料木黄酮进一步纯化，SephadexLH-20葡聚糖凝胶纯化黄豆苷元、染料木黄酮的工艺条件均为：上样量5mg，洗脱速度2mL/min，乙醇浓度70%。利用高效液相色谱对三个洗脱峰进行检测，洗脱峰3为黄豆苷元或者染料木黄酮，黄豆苷元的纯度为94.31%，染料木黄酮的纯度为90.72%。
3、对黄豆苷元和染料木黄酮的体外抗氧化活性和抗肿瘤活性进行研究。结果表明黄豆苷元、染料木黄酮具有较强的还原能力，均低于各自的对照品，黄豆苷元的还原能力低于维生素C，染料木黄酮的还原能力高于维生素C。黄豆苷元、染料木黄酮对DPPH.自由基、羟自由基都有一定的清除能力，且高于维生素C低于相应的标准对照品。染料木黄酮对羟自由基的清除能力高于黄豆苷元。通过黄豆苷元、染料木黄酮对人乳腺癌细胞生长的抑制情况的考察，采用MTT试验测定抑制率。MTT试验结果表明，黄豆苷元和染料木素对人乳腺癌细胞的生长均具有一定的抑制作用，染料木黄酮的抑制作用强于黄豆苷元。
Soy sauce is one of the traditional beans fermented food in China.Mold secreted glucosidasethat hydrolyze Isoflavones glycosidic bonds to Daidzein、Gentstein、Glycitein, et al. during fer--mentation. An explosion of research have been shown that Isoflavones Fight tumour, prevent bo--ne loose, protect cardiovascular and relaxed women's Menopause Syndrome.
The study of Daidzein and Gentstein which extracted, isolated and purificated from Soy sau--ce,with a view toward testing antioxidant activities and anticancer activity. The results showedthat:
1、This paper studied the extraction of Daidzein and Genistein in soybean paste by,ultrasoundmethod.Based on comprehensive consider--ation of extraction rate,economy and simplicity,theultrasonic extraction process of Daidzein and Genistein was optimized by response surfacemethod,the extraction parameters has been determined finally.The optimum extraction conditionsof Daidzein were:extracting time by ultrasonic was61minutes, extraction temperature was48℃,ethanol concentration was74%,the yield rate of Daidzein in soybean paste was484.610μg/g;The optimum extraction conditions of Genistein were:ethanol concentration was78%,extraction temperature was59min, extraction temperature was49℃,extraction power wasweak,the yield rate of Genistein in extracting solution was100.07μg/g.
2、The isolation, purifying of Daidzein and Gentstein were studied.The results showed thatAB-8resin has a good isolation and purifying ability for Daidzein and Gentstein. And the adsor--ption elution conditions were: sample quantity was3mg, adsorption rate was2mL/min, the eluti--on rate was2mL/min.Under this conditions, the purity of Daidzein and Gentstein respectivelywas71.49%,64.78%. The SephadexLH-20dextran gel used in further respectively purification ofDaidzein and Gentstein. And the process conditions were: sample quantity was5mg, the elutionrate was2mL/min, ethanol concentration was70%.Elution peaks were measured by HPLC and thethird elution peak was Daidzein and Gentstein,and the purity of Daidzein and Gentstein respecti--veely was94.31%,90.72%.
3、The anti-oxidation activity and anti tumor activity of Daidzein and Gentstein extract fromSoy sauce were studied. The Daidzein and Gentstein had strong reducing capacity,it was below itsrespectively standard reference substance. The Vc had better reducing capacity than Daidzein andit less than Gentstein. The DPPH free radical and hydroxyl radical scavenging ability of Daidzeinand Gentstein between Vc and respectively standard reference substance,and Vc is less thanstandard reference substance.Using MTT experiment determined the inhibition rate.The experi--mental results show that the Daidzein and Gentstein had inhibition on the growth of Humanbreast cancer cells, Daidzein was not good as Gentstein.
引文
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