鹿茸与牛脊髓中糖脂类物质的分离纯化
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摘要
本文主要是以吉林省长春市双阳区的梅花鹿茸经超临界二氧化碳萃取技术提取的脂溶性成分作为研究对象。研究了梅花鹿茸脂溶性物质中的糖脂类物质（脑苷脂、神经节苷脂）的提取、分离及纯化。同时，本文也研究了基于高压脉冲电场辅助溶剂技术下牛脊髓中糖脂类物质和鹿茸渣中卵磷脂的优化提取。内容主要的包括以下几个方面：
（1）采用响应面的方法，对梅花鹿茸脂溶性物质中脑苷脂的提取进行优化。通过对液料比、静置时间和离心时间的单因素试验，建立了一个3水平的优化设计。结果表明，响应面优化设计对梅花鹿茸脂溶性物质中脑苷脂的提取是实际可行的。根据响应面优化和二次回归方程分析得到脑苷脂提取的优化条件为：液料比11，静置时间60min，离心时间8min。在此优化条件下，得到脑苷脂的提取率为2.08%。利用红外光谱的方法分析了石油醚萃余物中所含的官能团，分析结果表明，石油醚萃余物中含有羟基和酰胺基，此二者均为脑苷脂类物质的特征官能团，因此得出结论：利用石油醚从梅花鹿茸脂溶性物质中萃取所得到的萃余物为脑苷脂。DPPH自由基清除试验证明石油醚萃余得到的脑苷脂类物质具有相对较好的抗氧化活性。
（2）对上述的石油醚萃取所得到的粗脑苷脂进行柱分离，采用氯仿和氯仿/甲醇体系作为洗脱剂，对洗脱得到的组分利用薄层层析的方法进行鉴定。红外光谱分析表明，得到的萃取物中含有脑苷脂的特征官能团羟基和酰胺基，可以证明萃取物为脑苷脂。生物活性研究表明，粗脑苷脂和纯脑苷脂均对大肠杆菌具有较强的抑制作用，它们的最小抑菌浓度分别为125μg/mL和27μg/mL，但对葡萄球菌无抑菌作用。
（3）采用响应面的分析方法研究了鹿茸脂溶性物质中神经节苷脂的优化提取。研究了液料比，温度和时间对神经节苷脂提取的影响，并根据单因素试验结果设计了一个3水平的优化试验。通过优化设计试验分析，得到最优的提取条件为：液料比为13，提取温度为30℃，提取时间为4min，得到最大的提取率为1.25%。并结合红外光谱分析了神经节苷脂的结构。
（4）采用响应面的方法，对牛脊髓中糖脂类物质的提取进行优化。通过对电场强度、脉冲数和液料比的单因素考察，建立了一个3水平的优化设计。结果表明，响应面优化设计对牛脊髓中糖脂类物质的提取是实际可行的。根据响应面优化和二次回归方程分析得到脑苷脂提取的优化条件为：电场强度为19.7kV/cm，脉冲数为9.1，液料比为11。在此优化条件下，得到的糖脂类物质的质量为0.318g/5g牛脊髓，与预测值0.319g/5g牛脊髓相符，这足以说明所建立的模型的模拟值是可以采纳的。同时对三种提取方法进行比较得出结论，高压脉冲电场辅助溶剂提取方法对于牛脊髓中糖脂类物质的提取效果更佳。
（5）采用响应面的方法，对鹿茸渣中卵磷脂的提取进行优化。通过对电场强度、脉冲数和提取溶剂含水率进行了单因素考察，建立了一个3水平的优化设计。结果表明，响应面优化设计对鹿茸渣中卵磷脂的提取是实际可行的。根据响应面优化和二次回归方程分析得到鹿茸渣中卵磷脂提取的优化条件为：电场强度20kV/cm，脉冲数为2.48，液料比为8.5。在此优化条件下，得到的糖脂类物质的质量为1.592mg/g鹿茸渣，与预测值1.589mg/g鹿茸渣相符，这足以说明所建立的模型的模拟值是可以采纳的。同时，通过比较可以看出，高压脉冲电场比溶剂直接提取所得到的卵磷脂的量要多，因此得出结论高压脉冲电场辅助溶剂法提取卵磷脂的效果更佳，它不仅能耗少且作用时间短。
Sika deer antler velvet layer is a kind of precious traditional Chinese medicine,which has been widely used for treatment of some diseases. In this paper, sika deerantler velvet which is productive in Shuangyang, China, was choosen as the object ofstudy. We are extrcting the lipid soluble components by using supercritical carbondioxide extraction. Study on the separation and purification identification ofcompound lipids such as cerebroside and ganglioside. At the same time, this paperalso studies the extraction and optimization of glucolipid substance in bovine spinalcord. The research contents are as follows:
(1) Study on the extraction of cerebroside from the lipid soluble fraction of sikadeer antler velvet layer was investigated using response surface methodology (RSM).The quantitative effects of ratio of liquid to raw material, standing time, centrifugationtime were investigated, and a three-level Box-Behnken design which were based onthe results of a single-factor test was employed to optimize the parameters forextraction of cerebroside. The experimental results were fitted with a second-orderpolynomial equation by a multiple regression analysis. By solving the regressionequation and also by analyzing the response surface contour plots, the optimalcerebroside extraction conditions were determined: ratio of liquid to raw material11,standing time60min and centrifugation time8min, and the maximum yield ofcerebroside extraction was2.08%. Residue which was separated from the lipid solublefraction of sika deer antler velvet layer was analysis the functional groups (hydroxyl,amide) by IR. The results show that the residue contains the two functional groupswhich are the characteristics of cerebroside. Thus draw the conclusion: Residue whichwas separated from the lipid soluble fraction of sika deer antler velvet layer wasproved to be cerebroside. Cerebroside has good bioactives of clearing DPPH freeradicals.
(2) An antimicrobial cerebroside was extracted using petroleum ether from thelipid soluble fraction of sika deer antler velvet layer which was extracted bysupercritical CO2extraction technology. The effect of different parameters, such as ratio of liquid to raw material, standing time and centrifugation time on the extractionof crude cerebroside is using single-factor experiment. The crude cerebroside waspurified using silica gel column chromatography, was identified using TLC and wascharacterized by employing IR. The biological activities show cerebroside hasobvious inhibitory activities, and the pure one is rather good than the crude one. Theinhibitory activities of them were125μg/mL and27μg/mL on E.coli, respectively. Buthave no bacteriostasis against staphylococcus.
(3) In this study the extraction of ganglioside from the lipid soluble fraction ofsika deer antler velvet layer was investigated using response surface methodology(RSM). The quantitative effects of ratio of liquid to raw material, temperature, timewere investigated, and a three-level Box-Behnken design which was based on theresults of a single-factor test was employed to optimize the parameters for extractionof ganglioside. The experimental results were fitted with a second-order polynomialequation by a multiple regression analysis. By solving the regression equation andalso by analyzing the response surface contour plots, the optimal gangliosideextraction conditions were determined: ratio of liquid to raw material13, temperature30℃and time4min, and the maximum yield of ganglioside extraction was1.25%.Residue which was separated from the lipid soluble fraction of sika deer antler velvetlayer was proved to be ganglioside by IR and TLC.
(4) In this study the extraction of glycolipids (cerebroside and ganglioside) inbovine spinal cord was investigated using response surface methodology (RSM). Thequantitative effects of field strength, pulse numbers, ratio of liquid to raw material,and a three-level Box-Behnken design which was based on the results of asingle-factor test was employed to optimize the parameters for extraction of glycolipid.By solving the regression equation and also by analyzing the response surface contourplots, the optimal glycolipid extraction conditions were determined: field strength19.7kV/cm, pulse numbers9.1and ratio of liquid to raw material11, and the weightof glycopilid extraction was0.318g per5g bovine spinal cord. And the extraction ofglycolipid by HIPEF is better than other traditional technologies.
(5) In this study the extraction of phosphatidylcholine (PC) in sika deer velvetantler slag was investigated using response surface methodology (RSM). Thequantitative effects of field strength, pulse numbers, moisture concent of extractionsolvent, and a three-level Box-Behnken design which was based on the results of asingle-factor test was employed to optimize the parameters for extraction of PC. By solving the regression equation and also by analyzing the response surface contourplots, the optimal PC extraction conditions were determined: field strength20kV/cm,pulse numbers2.48and moisture concent of extraction solvent8.5, and the weight ofPC extraction was1.592mg per1g sika deer velvet antler slag. And the extraction ofPC by HIPEF is better than other methods, because it’s quick and getting more PC.
（1）采用响应面的方法，对梅花鹿茸脂溶性物质中脑苷脂的提取进行优化。通过对液料比、静置时间和离心时间的单因素试验，建立了一个3水平的优化设计。结果表明，响应面优化设计对梅花鹿茸脂溶性物质中脑苷脂的提取是实际可行的。根据响应面优化和二次回归方程分析得到脑苷脂提取的优化条件为：液料比11，静置时间60min，离心时间8min。在此优化条件下，得到脑苷脂的提取率为2.08%。利用红外光谱的方法分析了石油醚萃余物中所含的官能团，分析结果表明，石油醚萃余物中含有羟基和酰胺基，此二者均为脑苷脂类物质的特征官能团，因此得出结论：利用石油醚从梅花鹿茸脂溶性物质中萃取所得到的萃余物为脑苷脂。DPPH自由基清除试验证明石油醚萃余得到的脑苷脂类物质具有相对较好的抗氧化活性。
（2）对上述的石油醚萃取所得到的粗脑苷脂进行柱分离，采用氯仿和氯仿/甲醇体系作为洗脱剂，对洗脱得到的组分利用薄层层析的方法进行鉴定。红外光谱分析表明，得到的萃取物中含有脑苷脂的特征官能团羟基和酰胺基，可以证明萃取物为脑苷脂。生物活性研究表明，粗脑苷脂和纯脑苷脂均对大肠杆菌具有较强的抑制作用，它们的最小抑菌浓度分别为125μg/mL和27μg/mL，但对葡萄球菌无抑菌作用。
（3）采用响应面的分析方法研究了鹿茸脂溶性物质中神经节苷脂的优化提取。研究了液料比，温度和时间对神经节苷脂提取的影响，并根据单因素试验结果设计了一个3水平的优化试验。通过优化设计试验分析，得到最优的提取条件为：液料比为13，提取温度为30℃，提取时间为4min，得到最大的提取率为1.25%。并结合红外光谱分析了神经节苷脂的结构。
（4）采用响应面的方法，对牛脊髓中糖脂类物质的提取进行优化。通过对电场强度、脉冲数和液料比的单因素考察，建立了一个3水平的优化设计。结果表明，响应面优化设计对牛脊髓中糖脂类物质的提取是实际可行的。根据响应面优化和二次回归方程分析得到脑苷脂提取的优化条件为：电场强度为19.7kV/cm，脉冲数为9.1，液料比为11。在此优化条件下，得到的糖脂类物质的质量为0.318g/5g牛脊髓，与预测值0.319g/5g牛脊髓相符，这足以说明所建立的模型的模拟值是可以采纳的。同时对三种提取方法进行比较得出结论，高压脉冲电场辅助溶剂提取方法对于牛脊髓中糖脂类物质的提取效果更佳。
（5）采用响应面的方法，对鹿茸渣中卵磷脂的提取进行优化。通过对电场强度、脉冲数和提取溶剂含水率进行了单因素考察，建立了一个3水平的优化设计。结果表明，响应面优化设计对鹿茸渣中卵磷脂的提取是实际可行的。根据响应面优化和二次回归方程分析得到鹿茸渣中卵磷脂提取的优化条件为：电场强度20kV/cm，脉冲数为2.48，液料比为8.5。在此优化条件下，得到的糖脂类物质的质量为1.592mg/g鹿茸渣，与预测值1.589mg/g鹿茸渣相符，这足以说明所建立的模型的模拟值是可以采纳的。同时，通过比较可以看出，高压脉冲电场比溶剂直接提取所得到的卵磷脂的量要多，因此得出结论高压脉冲电场辅助溶剂法提取卵磷脂的效果更佳，它不仅能耗少且作用时间短。
Sika deer antler velvet layer is a kind of precious traditional Chinese medicine,which has been widely used for treatment of some diseases. In this paper, sika deerantler velvet which is productive in Shuangyang, China, was choosen as the object ofstudy. We are extrcting the lipid soluble components by using supercritical carbondioxide extraction. Study on the separation and purification identification ofcompound lipids such as cerebroside and ganglioside. At the same time, this paperalso studies the extraction and optimization of glucolipid substance in bovine spinalcord. The research contents are as follows:
(1) Study on the extraction of cerebroside from the lipid soluble fraction of sikadeer antler velvet layer was investigated using response surface methodology (RSM).The quantitative effects of ratio of liquid to raw material, standing time, centrifugationtime were investigated, and a three-level Box-Behnken design which were based onthe results of a single-factor test was employed to optimize the parameters forextraction of cerebroside. The experimental results were fitted with a second-orderpolynomial equation by a multiple regression analysis. By solving the regressionequation and also by analyzing the response surface contour plots, the optimalcerebroside extraction conditions were determined: ratio of liquid to raw material11,standing time60min and centrifugation time8min, and the maximum yield ofcerebroside extraction was2.08%. Residue which was separated from the lipid solublefraction of sika deer antler velvet layer was analysis the functional groups (hydroxyl,amide) by IR. The results show that the residue contains the two functional groupswhich are the characteristics of cerebroside. Thus draw the conclusion: Residue whichwas separated from the lipid soluble fraction of sika deer antler velvet layer wasproved to be cerebroside. Cerebroside has good bioactives of clearing DPPH freeradicals.
(2) An antimicrobial cerebroside was extracted using petroleum ether from thelipid soluble fraction of sika deer antler velvet layer which was extracted bysupercritical CO2extraction technology. The effect of different parameters, such as ratio of liquid to raw material, standing time and centrifugation time on the extractionof crude cerebroside is using single-factor experiment. The crude cerebroside waspurified using silica gel column chromatography, was identified using TLC and wascharacterized by employing IR. The biological activities show cerebroside hasobvious inhibitory activities, and the pure one is rather good than the crude one. Theinhibitory activities of them were125μg/mL and27μg/mL on E.coli, respectively. Buthave no bacteriostasis against staphylococcus.
(3) In this study the extraction of ganglioside from the lipid soluble fraction ofsika deer antler velvet layer was investigated using response surface methodology(RSM). The quantitative effects of ratio of liquid to raw material, temperature, timewere investigated, and a three-level Box-Behnken design which was based on theresults of a single-factor test was employed to optimize the parameters for extractionof ganglioside. The experimental results were fitted with a second-order polynomialequation by a multiple regression analysis. By solving the regression equation andalso by analyzing the response surface contour plots, the optimal gangliosideextraction conditions were determined: ratio of liquid to raw material13, temperature30℃and time4min, and the maximum yield of ganglioside extraction was1.25%.Residue which was separated from the lipid soluble fraction of sika deer antler velvetlayer was proved to be ganglioside by IR and TLC.
(4) In this study the extraction of glycolipids (cerebroside and ganglioside) inbovine spinal cord was investigated using response surface methodology (RSM). Thequantitative effects of field strength, pulse numbers, ratio of liquid to raw material,and a three-level Box-Behnken design which was based on the results of asingle-factor test was employed to optimize the parameters for extraction of glycolipid.By solving the regression equation and also by analyzing the response surface contourplots, the optimal glycolipid extraction conditions were determined: field strength19.7kV/cm, pulse numbers9.1and ratio of liquid to raw material11, and the weightof glycopilid extraction was0.318g per5g bovine spinal cord. And the extraction ofglycolipid by HIPEF is better than other traditional technologies.
(5) In this study the extraction of phosphatidylcholine (PC) in sika deer velvetantler slag was investigated using response surface methodology (RSM). Thequantitative effects of field strength, pulse numbers, moisture concent of extractionsolvent, and a three-level Box-Behnken design which was based on the results of asingle-factor test was employed to optimize the parameters for extraction of PC. By solving the regression equation and also by analyzing the response surface contourplots, the optimal PC extraction conditions were determined: field strength20kV/cm,pulse numbers2.48and moisture concent of extraction solvent8.5, and the weight ofPC extraction was1.592mg per1g sika deer velvet antler slag. And the extraction ofPC by HIPEF is better than other methods, because it’s quick and getting more PC.
引文
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