CN107213185A - 一种具有抗氧化活性的蓝莓叶多酚制备方法 - Google Patents
一种具有抗氧化活性的蓝莓叶多酚制备方法 Download PDFInfo
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Abstract
本发明公开了一种具有抗氧化活性的蓝莓叶多酚制备方法，该方法包括超声波提取，过滤，离心取上层液，减压浓缩，HPD 400型大孔树脂柱层析纯化等步骤；采用ABTS法和DPPH法评价蓝莓叶多酚粗提物和HPD 400型大孔树脂纯化得到的蓝莓叶多酚的自由基清除活性，并用芦丁作为对照。本发明的蓝莓叶多酚制备方法具有提取效率高、重复性好、条件温和、无污染等特点，制备的蓝莓叶多酚具有显著的抗氧化活性，因此在食品工业及医药行业具有广阔的应用前景。
Description
技术领域
本发明属于植物提取技术领域，具体涉及一种从蓝莓叶中提取高纯度多酚的方法
背景技术
蓝莓又名笃斯越橘、蓝浆果等，原产于北美，按照植物学分类，属于杜鹃花科越橘亚科越橘属多年生落叶或常绿灌木。与蓝莓果实相比，蓝莓叶的多酚含量更高。南京地区兔眼蓝莓蓝莓叶中多酚含量高达81.82mg没食子酸当量/g干重。蓝莓叶多酚组成主要有咖啡酰奎尼酸类、儿茶素及其聚合物、槲皮素及其糖苷衍生物、山奈酚及其糖苷衍生物等。蓝莓叶多酚是蓝莓叶中的主要生理活性物质，具有抗氧化、抗肿瘤、抗病毒以及降血压、血脂等功效，在医药和保健食品领域具有巨大的应用价值。
目前，研究者将研究重点集中在蓝莓果实，对蓝莓果实中所含有的多糖、花色苷、黄酮以及挥发性香气成分等的研究已经非常成熟，然而对蓝莓叶多酚的研究相对匮乏。近年来随着我国蓝莓产业发展迅速，蓝莓叶作为蓝莓产业的副产物，其保健功能越来越受到消费者的关注。目前，蓝莓叶保健茶、蓝莓叶蛋白肽营养品等功能性食品均有报道。深入研究蓝莓叶多酚的提取、纯化工艺，对开发蓝莓叶天然药物和保健食品、实现蓝莓叶资源的综合利用具有积极意义。
发明内容
目前，蓝莓叶中多酚提取多采用溶剂提取法，存在着时间长、有机溶剂用量大、提取率低等一系列缺点。本申请采用超声波法辅助有机溶剂溶液提取蓝莓叶中多酚化合物，利用超声波产生高速、强烈的空化效应和搅拌作用破坏植物细胞壁，使溶剂渗透到细胞中，从而缩短提取时间、提高提取效率。本发明对乙醇溶液浓度、料液比以及大孔树脂类型进行了优化或者筛选，取得了较好的提取率与提取效果。实现本发明的具体方案如下：
1)将采摘后的蓝莓叶冲洗、干燥、组织捣碎机粉碎成粉末；
2)将蓝莓叶粉末加入乙醇水溶液中，经超声波辅助提取；
3)将步骤2)中的混合物经8层纱布过滤去除残渣得到多酚粗提液1；
4)将多酚粗体液1离心取上清液得到淡黄色多酚粗提液2；
5)将多酚粗体液2倒入旋蒸瓶中旋蒸，待溶液变为黄褐色浓稠液体后停止旋蒸；
6)将步骤5)中得到的旋蒸液冷冻干燥后得到蓝莓叶多酚粗提物并保存于-20℃冰箱中备用。
7)将步骤6)得到的蓝莓叶多酚粗提物用去离子水配成溶液，并用稀盐酸酸化。
8)将步骤7)得到的溶液吸附到大孔树脂柱上；
9)将吸附多酚后的树脂用去离子水冲洗，再用乙醇进行洗脱，将所得洗脱液旋蒸除去乙醇，冷冻干燥后得到多酚提取粉末。
10)将步骤6)中得到的蓝莓叶多酚粗提物和步骤9)中得到的蓝莓叶多酚分别用95％乙醇配制成梯度浓度溶液，测定其对ABTS自由基和DPPH自由基的清除能力。
制备得到的蓝莓叶多酚呈深黄色粉末状，该样品对ABTS自由基和DPPH自由基均具有较好的清除能力，并呈现出明显的剂量依赖关系。蓝莓叶多酚对ABTS自由基和DPPH自由基的半抑制浓度分别为90.33和7.37μg/mL。
优选的，步骤1)，粉碎成粉末后过40-50目筛；
优选的，步骤2)，乙醇水溶液浓度为60％，料液比为1∶30；
优选的，步骤2)，超声时间为20-25min，温度为30℃，超声波功率为500W，频率40kHz；
优选的，步骤4)，离心力为4000-5000g，离心时间为15-20min；
优选的，步骤7)，溶液浓度为2.5-3mg/mL，酸化后pH为2-3；
优选的，步骤8)，大孔树脂柱填料可以选择为HPD 400型大孔树脂，径高比为1∶8-1∶10；
优选的，步骤8)，以3-4BV(树脂床体积)/h吸附速率吸附在大孔树脂柱上，上样量为2-2.5BV；
优选的，步骤8)，将吸附好的树脂先用4-6BV的去离子水以1-2BV/h的流速洗净树脂，再用2-3BV 40-60％浓度乙醇溶液进行洗脱，洗脱流速为0.5-1BV/h。
附图说明
图1为乙醇浓度对蓝莓叶多酚提取率的影响图；
图2为料液比对蓝莓叶多酚提取率的影响图；
图3为大孔树脂类型对蓝莓叶多酚吸附率(a)与解析率(b)的影响图；
图4为蓝莓叶多酚提取物、蓝莓叶多酚以及芦丁对ABTS自由基清除作用分析图；
图5为蓝莓叶多酚提取物、蓝莓叶多酚以及芦丁对DPPH自由基清除作用分析图；
具体实施方式
为了使本发明的目的、技术方案、积极效果更加清除明白，通过以下实例对本发明进行进一步详细说明。以下对于实施具体方案的描述仅用于解释本发明，并不限定本发明
实施例1：
1)将采摘后的蓝莓叶冲洗、干燥、粉碎成粉末后过40目筛；
2)将蓝莓叶粉末加入60％乙醇水溶液中，料液比为1∶30，经超声波提取20min，温度为30℃，超声波功率为500W，频率40kHz；
3)将步骤2)中的混合物经8层纱布过滤去除残渣得到多酚粗提液1；
4)将多酚粗提液1 5000g条件下离心15min，取上清液得到淡黄色多酚粗体液2；
5)将多酚粗体液2倒入旋蒸瓶中旋蒸，待溶液变为黄褐色浓稠液体后停止旋蒸；
6)将步骤5)中得到的旋蒸液冷冻干燥后得到蓝莓叶多酚粗提物并保存于-20℃冰箱中备用。
7)将步骤6)得到的蓝莓叶多酚粗提物用去离子水配成3mg/mL溶液，并用稀盐酸调至pH 2。
8)以3BV/h速度将步骤7)得到的溶液吸附到径高比1∶8的HPD 400型大孔树脂柱上，上样量为2BV；
9)将吸附多酚后的树脂用4BV去离子水冲洗，流速为1.5BV/h，再用3BV 50％浓度的乙醇水溶液进行洗脱，将所得洗脱液旋蒸除去乙醇，冷冻干燥得到蓝莓叶多酚粉末。
10)将步骤6)中得到的蓝莓叶多酚粗提物和步骤9)中得到的蓝莓叶多酚分别用95％乙醇溶液配制成梯度浓度溶液，测定其对ABTS自由基和DPPH自由基的清除能力。
制备得到的蓝莓叶多酚呈深黄色粉末状，该样品对ABTS自由基和DPPH自由基均具有较好的清除能力，并呈现出明显的剂量依赖关系。蓝莓叶多酚对ABTS自由基和DPPH自由基的半抑制浓度分别为90.33和7.37μg/mL。
ABTS自由基清除率(％)＝[A0-(A1-A2)]/A0×100％；
A0组是水代替样品溶液，A1组是样品实验，A2组是磷酸缓冲液代替ABTS溶液。
DPPH自由基清除率(％)＝[A0-(A1-A2)]/A0×100％；
A0组是水代替样品溶液，A1组是样品实验，A2组是95％乙醇溶液代替DPPH溶液。
以上举例仅为本发明的具体实施例。本发明并不限于以上实施例，从本发明公开内容直接导出或者联想变形所得的制备方法，均应认为是本发明的保护范围。
Claims (2)
1.一种具有抗氧化活性的蓝莓叶多酚制备方法，其特征在于包括以下操作步骤：
A、将采摘后的蓝莓叶冲洗、干燥、组织捣碎机粉碎成粉末；
B、将蓝莓叶粉末加入乙醇水溶液中，经超声波辅助提取；
C、将步骤B中的混合物经8层纱布过滤去除残渣得到多酚粗提液1；
D、将多酚粗体液1离心取上清液得到淡黄色多酚粗提液2；
E、将多酚粗体液2倒入旋蒸瓶中旋蒸，待溶液变为黄褐色浓稠液体后停止旋蒸；
F、将步骤E中得到的旋蒸液冷冻干燥后得到蓝莓叶多酚粗提物并保存于-20℃冰箱中备用。
G、称取一定量的步骤F得到的蓝莓叶多酚粗提物用去离子水配成溶液，并用稀盐酸酸化。
H、将步骤G得到的溶液吸附到大孔树脂柱上；
I、将吸附多酚后的树脂用去离子水冲洗，再用乙醇进行洗脱，将所得洗脱液旋蒸除去乙醇，冷冻干燥后得到多酚提取粉末。
J、将步骤F中得到的蓝莓叶多酚粗提物和步骤I中得到的蓝莓叶多酚分别用95％乙醇配制成梯度浓度溶液，测定其对ABTS自由基和DPPH自由基的清除能力。
2.如权利要求1所述的一种具有抗氧化活性的蓝莓叶多酚制备方法，其特征在于将蓝莓叶粉碎后过40-50目筛，选择提取液乙醇溶液浓度为60％，料液比为1∶30，超声时间为20-25min，超声温度为30℃，超声功率500W、频率40kHz；选择离心力为4000-5000g，离心时间为15-20min；上样溶液浓度为2.5-3mg/mL，酸化后pH为2-3，选择HPD 400大孔树脂为填料、径高比为1∶8-1∶10；以3-4BV/h的吸附速率将蓝莓叶多酚吸附在大孔树脂柱上，上样量为2-2.5BV，将吸附好的树脂先用4-6BV的去离子水以1-2BV/h的流速洗净树脂，再用2-3BV 40-60％浓度乙醇溶液进行洗脱，洗脱流速为0.5-1BV/h。
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