【作者】 颜廷才；

【导师】 孟宪军；

【作者基本信息】 沈阳农业大学 ， 食品科学， 2008， 博士

【摘要】 本论文以辽宁本溪县产刺嫩芽为原料,系统研究了刺嫩芽皂甙的提取纯化方法与条件、刺嫩芽皂甙基本性质、β-葡萄糖苷酶处理对皂甙影响及酶处理后刺嫩芽皂甙生理活性的提高程度,确定出了刺嫩芽皂甙的提取纯化工艺和降低毒性提高活性的方法,扩大了刺嫩芽皂甙的应用范围,提高了刺嫩芽的经济价值。得到主要结论如下:确立了齐墩果酸作为标准品的波长为291nm的皂甙紫外检测方法,证明了刺嫩芽含有丰富的齐墩果酸类皂甙,可以用齐墩果酸含量计算出刺嫩芽皂甙的总量。极差分析提取正交试验因素的影响大小关系顺序为:提取温度＞提取时间＞固液比＞乙醇浓度,最佳条件为浸提温度70℃、浸提时间3h、固液比1:20、乙醇浓度90%,最大提取率为:8.1%。超声波处理正交试验结果表明:影响超声波辅助提取皂甙的因素从大到小排列依次是料液比、温度、超声波处理时间、乙醇浓度,当料液比为1:20,温度为65℃,超声波时间为20min,乙醇浓度为100%,刺嫩芽皂甙提取率最大为8.7%。刺嫩芽粗皂甙经过4次氯仿脱蛋白处理后,脱去了81.9%的蛋白质。经木瓜蛋白酶正交试验极差分析,影响因素从大到小排列顺序依次为:温度、酶量、处理时间、pH值,最佳处理条件为温度50℃、酶用量为2%、酶解2h、pH为6.0,又脱去剩余蛋白的80.2%,蛋白质浓度降到0.4mg/mL。AB-8树脂对刺嫩芽皂甙的吸附量达63.9mg/g,解吸率为96.5%,当解吸液体积为400mL,乙醇浓度为70%,流速为0.6mL/min,pH值为8.0时,AB-8大孔树脂对刺嫩芽皂甙的纯化效果最好。用硅胶柱层析分离刺嫩芽皂甙的不同组分得到6份样品,质量比例为:34.3%、21.1%、16.9%、15.1%、7.9%、4.7%。泡沫试验、Liebermann反应、三氯化锑-氯仿饱和溶液反应、TCL检测试验都证明了刺嫩芽提取物里面含有丰富的皂甙成分。硅胶薄层层析证明刺嫩芽皂甙的单糖可能由鼠李糖、葡萄糖、甘露糖、半乳糖和木糖组成,这为酶水解提高刺嫩芽皂甙活性研究提供了依据。确立了215nm为检测波长,乙腈与水为流动相梯度洗脱的HPLC皂甙组分检测条件,发现样品1、2、3中含有4个单体物质;样品4、5中含有5个单体物;样品6中含有6个单体物质。6个皂甙样品中都含有齐墩果酸,齐墩果酸保留时间为14.7min。确立了HPLC检测齐墩果酸含量的方法条件为波长215nm、乙腈和水等度洗脱,齐墩果酸保留时间为7.0min。6个样品的齐墩果酸含量分别为45.6%、36.1%、31.4%、10.7%、26.6%、25.5%,精总皂甙里的齐墩果酸含量为33.5%。紫外吸收表明,刺嫩芽总皂甙有六个比较明显的吸收峰,其中在299nm处的吸收峰最大,说明总皂甙中含有多个皂甙单体,分子比较大;皂甙样品1到皂甙样品6六个皂甙样品中有多个明显吸收峰,吸收峰逐渐向长波附近集中,说明皂甙单体分子逐渐变大,极性减弱。红外吸收表明:皂甙组分的波形都非常相近,经过基础图谱分析可以发现皂甙中含有脂肪烃、脂肪族伯醇、脂肪族仲醇等官能团,样品1与2还含有脂肪酸官能团,经详细图谱检索,说明皂甙含有带羟基的长碳链、异麦芽糖和β-D-半乳糖。β-葡萄糖苷酶水解正交试验极差分析表明,影响程度依次为酶解温度＞酶用量＞酶解时间,三因素的最佳水平为:酶解温度为40℃,酶用量为3.0mg,酶解时间为120min。经β-葡萄糖苷酶酶解后刺嫩芽皂甙以甲醇为洗脱剂用硅胶柱分离,共收集6份样品,质量比例为:34.8%、24%、11.8%、11.3%、8.1%、10.0%。经进样量10μL;流动相甲醇与水70:30等度洗脱;流速为1.0mL/min,检测波长215nm,柱温为25℃的高效液相色普检测,β-葡萄糖苷酶酶解后样品E1、E2、E3中含有4个分离比较好的基本单体物质,样品E4、E5、E6中含有5个单体物质,样品都含有齐墩果酸,保留时间为5.1min。酶解后刺嫩芽皂甙在紫外检测条件下仍然含有多个吸收峰,但吸收明显向低波长靠近,多数在236nm处的吸收峰最大,说明酶解后皂甙中单体分子明显变小,单体数量减少。红外吸收表明:酶解后皂甙的波形都非常相近,经过基础图谱分析可以判断皂甙中含有脂肪烃、脂肪族伯醇、脂肪族仲醇、脂肪族伯胺、脂肪酸盐等官能团,说明酶水解掉部分单糖,裸露出了羧基与长的碳链。含有带羟基或氨基的长碳链、烯酸结构、异麦芽糖、β-D-苯基葡糖苷、甘露醇、苯甲酸和β-D-半乳糖成分。皂甙溶液在较低浓度时对羟基自由基与氧自由基就有一定的清除作用,自由基的清除效果与皂甙的浓度呈显著正相关性。解酒与防醉试验表明,刺嫩芽皂甙具有抑制乙醇吸收作用,从而能够显著延长酒精耐受时间,降低醉酒维持时间,有解酒防醉保护肝脏效果。体内抗氧化试验表明,刺嫩芽皂甙可以提高小鼠SOD活性,降低小鼠体内组织的MDA含量。刺嫩芽皂甙能够提高小鼠的脾脏指数与胸腺指数,增强小鼠的免疫能力;也可以降低血清中的总脂、总胆固醇,提高血清中高密度脂蛋白。刺嫩芽皂甙只是在一定程度上调解正常小鼠的血糖与胰岛素水平;显著降低高血糖模型小鼠的血糖值,显著提高高血糖模型小鼠的胰岛素水平,对于高血糖具有非常好的治疗作用。经β-葡萄糖苷酶酶解后的皂甙活性更加显著,说明β-葡萄糖苷酶可以提高刺嫩芽皂甙活性。更多还原

【Abstract】 In this study the material is Aralia elata(Miq) Seem planted in Benxi county of Liaoning province.The method and parameters of extracting and purifying saponins from Aralia elata(Miq) Seem,the basic property of saponins,the hydrolyzing influence ofβ-Glucosidase on saponins and the boosting degree of biological activities hydrolyzed byβ-Glucosidase are studied by the numbers in this paper.The technics of extracting and purifying saponins from Aralia elata(Miq) Seem and the method of reducing toxicity and boosting activity are confirmed by this study.The applied extension is expanded and the economic value of saponins from Aralia elata(Miq) Seem is enhanced.The important conclusions gained in this study are as follows:The ultraviolet method of measuring saponins is established,the standard sample is the oleanolic acid,and the metrical wavelength is 291nm.It proves:the Aralia elata(Miq) Seem contains abundant oleanolic acid and homologous saponins,the saponins concentration can be calculated briefly by the oleanolic acid content.The orthogonal test analysed by difference demonstrates:the sequence of the ponderance affecting extracting saponins from high to low is the extracting temperature,the extracting time,the solvent ratio and the concentration of alcohol.The best extracting parameters are as follows:the extracting temperature is 70℃;the extracting time is 3h;the solvent ratio is 1:20;the concentration of alcohol is 90%.At these conditions the maximum extraction rate are 8.1%.The orthogonal test of ultrasonic-assisting treatment demonstrates:the sequence of the ponderance affecting extracting saponin from high to low is the solvent ratio,the extracting temperature,the ultrasonic-assisting time and the concentration of alcohol.The best extracting parameters are as follows:the solvent ratio is 1:20;the extracting temperature is 65℃;the ultrasonic-assisting time is 20min;the concentration of alcohol is 100%.At these conditions the maximum extraction rate are 8.7%.81.9%protein in the crude saponins of Aralia elata(Miq) Seem is removed by treating four times with chloroform.The orthogonal test of removing protein with pawpaw proteinase analysed by difference demonstrates:the sequence of the ponderance affecting doffing protein from high to low is the treating temperature,enzyme quantity,the treating time and the pH value.The best treating parameters are as follows:the treating temperature is 50℃;the enzyme dosage is 2%;the treating time is 2h;the pH value is 6.0.At these conditions 80.2% protein can be removed again,the protein concentration falls to 0.4mg/mL in the saponins solution.The adsorptive capacity of AB-8 resin reachs to 63.9mg/g,and desorption rate is 96.5%.The best purification condition of AB-8 resin is 400mL eluting solution,70%alchohol, 0.6mL/min velocity of flow and pH8.0.Six samples can be separated by eluting through silica gel column,the quantitive proportion is 34.3%,21.1%,16.9%,15.1%,7.9%and 4.7%.Foam test,Liebermann reaction,stibium chloride-saturated chloroform reaction and TCL test prove:there are abundant saponins component in extraction of Aralia elara(Miq) Seem.The monosaccharide are made up of rhamnose,glucose,mannitose,galactose and xylose.It offers foundation for boosting biological activities by hydrolyzing saponins.The examining condition of saponins components by HPLC is established,the inspecting wavelength is 215nm,acetonitrile and water are mobile phase eluting by gradient.There are four monomers in sample 1,2 and 3,five monomers in sample 4 and 5,six monomers in sample 6.The six samples all contain the oleanolic acid which retaining time is 14.7min.The examining method of the oleanolic acid content by HPLC is established,the inspecting wavelength is 215nm,acetonitrile and water are mobile phase.The oleanolic acid retaining time is 7.0min at these conditions.The oleanolic acid content is 45.6%,36.1%,31.4%,10.7%, 26.6%and 25.5%differently in the six samples,and 33.5%in the general saponins.The ultraviolet test indicates:there are six obvious absorbing peaks in the general saponins,the biggest absorbing peak is at 299nm.It shows:the general saponins contain multiplicate monomers,its molecule is big comparatively.All the six saponins samples contain several obvious absorbing peaks,the main absorbing peak closes with long wavelength gradually.It shows:the saponins monomers molecule become bigger,its polarity become lower.The infrared test indicates:all the saponins absorbing waves are similar,they contain functional groups such as aliphatic hydrocarbons,primary aliphatic alcohols and secondary aliphatic alcohols(Aliphatic carboxylic acids in sample 1 and 2) estimated by basic analyzed chart. They may contain long carbon chain with hydroxy,isomaltose and galactose estimated by particular analyzed chart.The orthogonal test ofβ-Glucosidase hydrolyzation analysed by difference indicates: the sequence of the ponderance affecting hydrolyzing saponins from high to low is the hydrolyzing temperature,the enzyme dosage and the hydrolyzing time.The best hydrolyzing parameters are as follows:the hydrolyzing temperature is 40℃;the enzyme dosage is 3.0mg; the hydrolyzing time is 120min.Six samples can be separated by eluting saponins hydrolyzed byβ-Glucosidase through silica gel column with methanol,the quantitive proportion is 34.8%, 24%,11.8%,11.3%,8.1%and 10.0%.The examining condition of hydrolyzed saponins components by HPLC is established,the injecting dosage is 10μL,the mobile phases are methanol and water with 70:30 proportion,the velocity of flow is 1.0mL/min,the inspecting wavelength is 215nm,the column temperature is 25℃.At these inspecting conditions sample E1,E2 and E3 contain four monomers,sample EA,E5and E6 contain five monomers.The six samples all contain the oleanolic acid which retaining time is 5.1min.The ultraviolet test of hydrolyed saponins indicates:All the six hydrolyzed saponins samples contain several obvious absorbing peaks,the main absorbing peak closes with short wavelength gradually,the biggest absorbing peak is at 236nm in most hydrolyzed samples.It shows:the hydrolyed saponins monomers molecule become smaller,its amount decreases.The infrared test indicates:all the hydrolyed saponins absorbing waves are similar,they contain functional groups such as aliphatic hydrocarbons,primary aliphatic alcohols,secondary aliphatic alcohols,aliphatic Primary amines and aliphatic carboxylic acids estimated by basic analyzed chart.They may contain long carbon chain with hydroxy or amino,unsaturated fatty acid, phenyl glucoside,mannitol,benzoic acid,isomaltose and galactose estimated by particular analyzed chart.Low concentration saponins solution can eliminate hydroxy and oxygen free radicel, the effect of eliminating free radicel submits positive pertinence with saponins concentration. The result of dispelling alcohol and defending ebriety test indicates:saponins of Aralia elata (Miq) Seem can restrain alcohol absorbed,extend alcohol bearing time,reduce ebriety maintaining time remarkably,and have a function of dispelling alcohol,defending ebriety and protecting liver.The result of antioxidating effect in vivo test indicates:saponins of Aralia elata(Miq) Seem can boost SOD activity,reduce the MDA concentration of organic tissue. Saponins of Aralia elata(Miq) Seem can increase spleen exponent and thymus exponent, enhance rat immunity;it also debases totall grease and totall cholesterin content,boosts high density lipoprotein content of blood serum.Saponins of Aralia elata(Miq) Seem can mediate blood sugar and insulin level of normal rat at low degree,can debase blood sugar and enhance insulin level of restraining model rat remarkably,can cure high blood sugar.All the activities become prominent after saponins hydrolyzed byβ-Glucosidase,it indicateβ-Glucosidase can enhances biological activities of saponins of Aralia elata(Miq) Seem.更多还原