莲花蜂花粉多糖的提取、分离及生物活性的研究
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摘要
蜂花粉是纯天然的保健品之一,被誉为全能营养库。多糖是蜂花粉重要有效成分之一,具有较好的潜在开发价值。本文对莲花蜂花粉多糖的提取、分离、初步纯化及生物活性进行了研究,结果如下:
1.在超声波破壁的基础上研究了水提法提取莲花蜂花粉多糖的条件,在单因素试验的基础上,根据Box-Behnken中心组合试验设计原理,选取料液比、浸提时间和浸提温度3个因素的3个水平进行响应面分析,建立多糖得率的二次回归方程,得到提取工艺的优化组合条件。试验结果表明:料液比、浸提时间和浸提温度对莲花蜂花粉多糖得率都有显著影响,当提取工艺条件为料液比1:9.4,时间2.4 h,温度81.6℃,浸提2次时,多糖得率达到极大值。此条件下莲花蜂花粉多糖得率预测值为1.2201%,验证值为1.2317%。
2.不同体积乙醇沉淀莲花蜂花粉多糖效果的研究表明,乙醇体积为多糖液体积的3.5倍时,沉淀效果最好。通过对五种不同脱蛋白方法效果的比较,确定胰蛋白酶+Sevage法为莲花蜂花粉多糖提取时脱蛋白的最佳方法,蛋白清除率为82.51%。多糖经Sephadex G-75的洗脱,出现两个洗脱峰,其中分子量较大的含量少于分子量较小的。
3.根据Fenton反应体系,通过对莲花蜂花粉多糖清除羟自由基试验的研究,可知当精制莲花蜂花粉多糖和粗多糖质量浓度同为2.5 mg/mL时,羟自由基的清除率分别达64.58%和56.82%;采用邻苯三酚自氧化测定法,通过对莲花蜂花粉多糖清除超氧阴离子自由基试验的研究,可知当精制多糖和粗多糖质量浓度同为1.3 mg/mL时,超氧阴离子自由基清除率分别达到58.28%和52.52%。表明莲花蜂花粉多糖对羟自由基、超氧阴离子自由基都具有较好的清除作用,且初步纯化后多糖的清除效果比粗多糖好。
4.通过对莲花蜂花粉多糖降血脂作用的研究,得知大鼠灌胃给药低(150 mg/kg.d)、中(300 mg/kg.d)、高(600 mg/kg.d)剂量的莲花蜂花粉多糖,能显著降低试验性高脂血症大鼠血清中的总胆固醇TC含量,但对大鼠血清中甘油三脂TG和高密度脂蛋白HDL-C含量作用不明显。表明莲花蜂花粉多糖有一定的降血脂功效。
Bee pollen is one of the natural health products, and is known as the almighty nutrition. Polysaccharide is one of the most important components in Bee Pollen, and it is valuable to develop. In this paper, extraction, separation, purification and biological activity of Lotus bee pollen polysaccharide are researched, the results are as follows:
1. We used ultrasound-assisted technology in order to extract polysaccharide from Lotus bee pollen. On the basis of single-factors experiments, the Box-Behnken center-united experimental design principles were used to design the experiments and the responsive surfaces analysis of 3 factors and 3 levels was adopted. The quadratic regression equation for rate of polysaccharides was established, and the optimum combination was obtained by responsive surfaces analysis. Experimental results indicated that the ration of solid-liquid,the extraction time and the extraction temperature significantly affected the yield of lotus bee pollen polysaccharide. When the extraction parameters were ration of solid-liquid 1:9.4, 2. 4 hours and 81. 6℃, the ratio of lotus bee pollen polysaccharides was maximum. In the condition, the estimated value was 1. 2201% and verified value was 1. 2317%.
2. The studies on effects of different volume ethanol on precipitating Lotus bee pollen polysaccharide showed that, when the volume ratio of ethanol and polysaccharide liquid was 3.5 : 1, we can obtain the maximum polysaccharide. Effects of five different methods on precipitating protein of polysaccharide liquid were studied. The results showed that, the Trypsin+Sevage removal of protein was an effective method and the removal rate of protein reached 82.51%. We found two polysaccharide peaks though Sephadex G-75 elution, and the content of high molecular weight was less than low molecular weight.
3. Based on Fenton reaction system,the studies on scavenging effects to hydroxyl radicals of Lotus bee pollen polysaccharide showed that, when the concentration of refined and crude Lotus bee pollen polysaccharide was 2.5mg/mL,the rate of scavenging hydroxyl radical respectively reached 64.58% and 56.82%; Using the pyrogallol autoxidation method, the studies on scavenging effects to superoxide radicals of Lotus bee pollen polysaccharide showed that,when the concentration of refined and crude Lotus bee pollen polysaccharide was 1.3mg/mL, the rate of scavenging superoxide radicals respectively reached 58.28% and 52.52%. So we can conclude that both crude and refined Lotus bee pollen polysaccharide were effective in scavenging superoxide radical and hydroxyl radical.The scavenging capacity for superoxide radical and hydroxyl radical of refined Lotus bee pollen polysaccharide was higher than that of crude Lotus bee pollen polysaccharide.
4. The studies on effects of Lotus bee pollen polysaccharide on Hypolipidemic effect of mice showed that, when the Lotus bee pollen polysaccharide was fed to the Hyperlipidemia mice at different dosages which are 150 mg/kg.d, 300 mg/kg.d and 600 mg/kg.d, the effect of Lotus bee pollen polysaccharide on decreasing the content of serum TC in mice was significant,but not on TG and HDL–C. Therefore, the Lotus bee pollen polysaccharide has the Hypolipidemic effect on Hyperlipidemia mice.
1.在超声波破壁的基础上研究了水提法提取莲花蜂花粉多糖的条件,在单因素试验的基础上,根据Box-Behnken中心组合试验设计原理,选取料液比、浸提时间和浸提温度3个因素的3个水平进行响应面分析,建立多糖得率的二次回归方程,得到提取工艺的优化组合条件。试验结果表明:料液比、浸提时间和浸提温度对莲花蜂花粉多糖得率都有显著影响,当提取工艺条件为料液比1:9.4,时间2.4 h,温度81.6℃,浸提2次时,多糖得率达到极大值。此条件下莲花蜂花粉多糖得率预测值为1.2201%,验证值为1.2317%。
2.不同体积乙醇沉淀莲花蜂花粉多糖效果的研究表明,乙醇体积为多糖液体积的3.5倍时,沉淀效果最好。通过对五种不同脱蛋白方法效果的比较,确定胰蛋白酶+Sevage法为莲花蜂花粉多糖提取时脱蛋白的最佳方法,蛋白清除率为82.51%。多糖经Sephadex G-75的洗脱,出现两个洗脱峰,其中分子量较大的含量少于分子量较小的。
3.根据Fenton反应体系,通过对莲花蜂花粉多糖清除羟自由基试验的研究,可知当精制莲花蜂花粉多糖和粗多糖质量浓度同为2.5 mg/mL时,羟自由基的清除率分别达64.58%和56.82%;采用邻苯三酚自氧化测定法,通过对莲花蜂花粉多糖清除超氧阴离子自由基试验的研究,可知当精制多糖和粗多糖质量浓度同为1.3 mg/mL时,超氧阴离子自由基清除率分别达到58.28%和52.52%。表明莲花蜂花粉多糖对羟自由基、超氧阴离子自由基都具有较好的清除作用,且初步纯化后多糖的清除效果比粗多糖好。
4.通过对莲花蜂花粉多糖降血脂作用的研究,得知大鼠灌胃给药低(150 mg/kg.d)、中(300 mg/kg.d)、高(600 mg/kg.d)剂量的莲花蜂花粉多糖,能显著降低试验性高脂血症大鼠血清中的总胆固醇TC含量,但对大鼠血清中甘油三脂TG和高密度脂蛋白HDL-C含量作用不明显。表明莲花蜂花粉多糖有一定的降血脂功效。
Bee pollen is one of the natural health products, and is known as the almighty nutrition. Polysaccharide is one of the most important components in Bee Pollen, and it is valuable to develop. In this paper, extraction, separation, purification and biological activity of Lotus bee pollen polysaccharide are researched, the results are as follows:
1. We used ultrasound-assisted technology in order to extract polysaccharide from Lotus bee pollen. On the basis of single-factors experiments, the Box-Behnken center-united experimental design principles were used to design the experiments and the responsive surfaces analysis of 3 factors and 3 levels was adopted. The quadratic regression equation for rate of polysaccharides was established, and the optimum combination was obtained by responsive surfaces analysis. Experimental results indicated that the ration of solid-liquid,the extraction time and the extraction temperature significantly affected the yield of lotus bee pollen polysaccharide. When the extraction parameters were ration of solid-liquid 1:9.4, 2. 4 hours and 81. 6℃, the ratio of lotus bee pollen polysaccharides was maximum. In the condition, the estimated value was 1. 2201% and verified value was 1. 2317%.
2. The studies on effects of different volume ethanol on precipitating Lotus bee pollen polysaccharide showed that, when the volume ratio of ethanol and polysaccharide liquid was 3.5 : 1, we can obtain the maximum polysaccharide. Effects of five different methods on precipitating protein of polysaccharide liquid were studied. The results showed that, the Trypsin+Sevage removal of protein was an effective method and the removal rate of protein reached 82.51%. We found two polysaccharide peaks though Sephadex G-75 elution, and the content of high molecular weight was less than low molecular weight.
3. Based on Fenton reaction system,the studies on scavenging effects to hydroxyl radicals of Lotus bee pollen polysaccharide showed that, when the concentration of refined and crude Lotus bee pollen polysaccharide was 2.5mg/mL,the rate of scavenging hydroxyl radical respectively reached 64.58% and 56.82%; Using the pyrogallol autoxidation method, the studies on scavenging effects to superoxide radicals of Lotus bee pollen polysaccharide showed that,when the concentration of refined and crude Lotus bee pollen polysaccharide was 1.3mg/mL, the rate of scavenging superoxide radicals respectively reached 58.28% and 52.52%. So we can conclude that both crude and refined Lotus bee pollen polysaccharide were effective in scavenging superoxide radical and hydroxyl radical.The scavenging capacity for superoxide radical and hydroxyl radical of refined Lotus bee pollen polysaccharide was higher than that of crude Lotus bee pollen polysaccharide.
4. The studies on effects of Lotus bee pollen polysaccharide on Hypolipidemic effect of mice showed that, when the Lotus bee pollen polysaccharide was fed to the Hyperlipidemia mice at different dosages which are 150 mg/kg.d, 300 mg/kg.d and 600 mg/kg.d, the effect of Lotus bee pollen polysaccharide on decreasing the content of serum TC in mice was significant,but not on TG and HDL–C. Therefore, the Lotus bee pollen polysaccharide has the Hypolipidemic effect on Hyperlipidemia mice.
引文
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