CN113813305A - 一种北五味子果实中活性成分集成提取的方法 - Google Patents
一种北五味子果实中活性成分集成提取的方法 Download PDFInfo
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- CN113813305A CN113813305A CN202111180006.8A CN202111180006A CN113813305A CN 113813305 A CN113813305 A CN 113813305A CN 202111180006 A CN202111180006 A CN 202111180006A CN 113813305 A CN113813305 A CN 113813305A
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- polysaccharide
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- polyphenol
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Abstract
本发明涉及一种从北五味子果实中集成提取多种活性成分的技术,包括下列步骤:以石油醚为溶剂利用索氏提取法对北五味子果实干粉脱脂。脱脂五味子粉末经高压辅助乙醇‑硫酸铵双水相体系一步提取,分离后得到两相:上相是含有大量多酚和少量多糖的乙醇相,下相是富含另一种多糖的无机盐相。其中,上相多酚及多糖的分离于HPD300大孔树脂柱上完成,依次用酸性水和乙醇水溶液洗脱多糖和多酚,两种洗脱液被分别收集、浓缩、冻干后得到上相多糖及多酚。下相多糖通过透析、醇沉、冻干得到。本发明利用高压辅助乙醇‑硫酸铵双水相体系一步投料提取,同时得到五味子多酚和两种不同性质多糖,提高原料利用率,提高多种活性成分提取率,且操作条件温和,经济环保,易于工业化生产使用。
Description
技术领域:
本发明涉及一种天然产物提取分离的方法,具体涉及一种集成提取北五味子果实中多种活性成分的方法。
背景技术:
五味子(Schisandra chinensis Fructus)为五味子属多年生左旋落叶木质藤本植物,由于生长地域不同可以分为北五味子和南五味子。北五味子呈不规则的球形或扁球形,表面呈红色、紫红色或暗红色,果皮皱缩,显油润,果肉柔软。南五味子颗粒较小,表面棕红色或暗棕色,果皮干瘪皱缩,果肉常紧贴在种子上。据中国传统医书中记录,北五味子比南五味子的药理作用更显著,营养价值更优良,应用范围更宽泛。
北五味子一直以干燥成熟果实作为药用,具有收敛固涩,益气生津,补肾宁心等功效。果实是北五味子的药用部位,其内在的化学成分是临床作用的物质基础。多糖是五味子果实中最重要的活性成分之一。五味子多糖在保肝、提高免疫、降血糖、抗肿瘤、抗病毒、抗疲劳、降血脂、抗突变等方面具有一定的功效。同时,五味子果实中含有大量植物多酚,包括酚酸、黄酮、原花青素等,具有优良的抗氧化、降血压、降血脂、免疫调节、抗癌等生理功能。因此,有必要建立一种高效集成提取五味子果实中多糖和多酚的方法,提高原料利用率,为五味子的开发利用及精深加工提供技术支持。
五味子果实中活性成分的提取方法一般包括:热回流提取法、超临界CO2提取法、加压溶剂萃取法、超声波提取法、超高压提取法、微波提取法、离子液体提取法等。一般来说,这些提取方法只能一次投料提取一种活性成分,而其他活性成分常常作为杂质被去除掉,造成资源浪费。双水相提取技术(Aqueous two-phase extraction,ATPE)具有双相萃取能力和选择性,通过使用两相或多相溶剂,使得目标成分可以分别被提取到上相和下相,实现多种活性成分的集成提取。同时,ATPE技术具有高收率、相形成组分的高回收率、相对于纯有机溶剂对环境更友好的特性。高压提取法(High pressure extraction,HPE)是一种利用高压反应釜在较高的压力和温度下进行提取的技术。高压能够驱动溶剂快速渗透到样品内部,而高温有助于减小溶剂黏度,使溶剂和样品基质之间的相互作用加强,因此HPE能够加速提取过程。本发明将高压优势与双水相选择性提取优势相结合,采用高压辅助双水相提取(HP-ATPE)技术,一次投料,一步提取,从北五味子果实中集成提取多种活性成分,提高原料利用率和多种活性成分提取率。
目前,未见将HP-ATPE技术应用于从五味子果实中集成提取多糖和多酚的专利报道。中国专利CN 112057500 A“一种从五味子中提取多糖、挥发油、黄酮和木脂素的方法”,通过连续的四步提取,将多糖、挥发油、黄酮和木脂素从五味子中提取分离,步骤繁杂,试剂繁多,提取周期长。本发明利用HP-ATPE技术一步将多酚和多糖从五味子果实干粉中提取出来,步骤少、周期短、成本低、利于工业化生产。中国专利CN 106967810 A“一种五味子多糖提取物及其制备方法和应用”,中国专利CN 109651522 A“一种五味子多糖的制备方法”,中国专利CN 107163717 X“五味子酸性多糖及其制备方法和应用”中关于五味子多糖的提取均采用水提醇沉的方法,而多酚类物质被当作杂质去除,造成原料利用度低;且后续纯化步骤繁琐,多糖得率不高。而中国专利CN 107652337 B“一种黑米、黑豆、紫薯、玫瑰、牡丹等花色苷提取制备方法”是先使用双水相浸提,将提取获得的有机相再进行高压脉冲电场提取处理;中国专利CN 110483651 A“美洲帘蛤多糖的制备方法”是先高压脉冲电场提取,然后将帘蛤多糖提取液加入离子液体-无机盐双水相体系中分离。这两个发明的目的是提取一种活性物质,且将高压与双水相提取技术割裂成两步进行,不能发挥高压辅助双水相技术集成提取多种活性成分的优势。本发明采用HP-ATPE技术,一步提取和分离五味子多糖和多酚,将高压与双水相高选择性优势结合,促使多酚与多糖在两相中的有效分离,实现北五味子果实中多种活性成分的集成提取,且后续纯化步骤简单。
发明内容
本发明的目的是提供一种能够克服现有技术中的不足,高效地集成提取北五味子果实中多种活性成分的方法,实现五味子生物资源的综合利用。
本发明的目的是这样实现的:
一种集成提取北五味子果实中多种活性成分的方法,以北五味子果实干粉为原料,包括以下步骤:①称取适量过50目筛五味子粉末,进行脱脂,所使用的方法为索氏提取法。所用溶剂为60~90℃沸点的石油醚(原料:石油醚=1:3),置于60℃恒温加热器中回流提取6h。冷却后,将脱脂五味子粉末取出,待残留的石油醚挥发干净后真空保存。②将步骤①得到脱脂五味子干粉按一定液料比,加入到已制备好的乙醇-硫酸铵双水相体系中,在最佳高压辅助条件下进行提取;③将步骤②所得提取液离心分离,分别得到上、下相提取液;④将步骤③中所得上相提取液经无水乙醇洗涤去盐后,减压浓缩,真空冷冻干燥得到上相粗提物;⑤将步骤③所得下相提取液在室温下进行透析,除去无机盐等杂质,然后将透析液减压浓缩,真空冷冻干燥得到下相粗提物;⑥将步骤④所得上相粗提物溶解,上样HPD300大孔树脂柱,使用酸性水溶液洗脱树脂直至洗脱液中检测不到多糖,将洗脱液收集、浓缩、醇沉、冷冻干燥得到上相多糖,而多酚吸附于大孔树脂中;⑦利用酸性乙醇水溶液对步骤⑥中HPD300大孔树脂柱吸附的多酚进行洗脱,洗脱液经收集、浓缩、冷冻干燥得到上相多酚;⑧将步骤⑤所得下相粗提物溶解于去离子水中,乙醇醇沉后,离心收集底部沉淀物,冷冻干燥得到下相多糖。
与现有技术相比,本发明具有以下有益效果:
1.本发明中使用的高压辅助双水相提取技术,结合高压及双水相技术优势,能够一步提取并分离五味子果实多糖和多酚,简化了操作步骤,提高目标物质的提取效率,且操作方便,原料利用率高。
2.本发明采用最常见的乙醇-硫酸铵双水相体系,制备简单,溶剂低毒,环保,且易于连续操作以及工艺放大,具有用于工业化提取及分离五味子果实中功能性天然产物的潜力。
3.本发明优化的HP-ATPE提取条件能够有效加速多糖及多酚从五味子果实细胞中溶出,同时获得最高提取率。
4.本技术一步提取得到的五味子多糖和多酚,可用于相关药品、保健品和食品的开发,显著提高五味子的附加值。
附图说明:
图1:五味子多酚提取物在负离子模式下的基峰色谱图(1:矢车菊素-3-O-葡萄糖苷;2:芦丁;3:金丝桃苷;4:忍冬苦苷;5:紫丁香苷;6:槲皮素;7:山奈酚;8:异鼠李素)
图2:单糖标准品(A)、上相多糖(B)、下相多糖(C)的液相色谱图(GalA:半乳糖醛酸;Rha:鼠李糖;Ara:阿拉伯糖;Man:甘露糖;Glc:葡萄糖;Gal:半乳糖;GlcA:葡萄糖醛酸)
图3:上相多糖(A)和下相多糖(B)分子量测定液相色谱图
具体实施方式
结合具体实施例对本发明进行进一步描述说明,但本发明的保护范围并不仅限于此。
HP-ATPE最佳提取条件的确定:
单因素试验:
液料比的选择:
称取五味子脱脂干粉,分别按不同液料比加入到由28.21%~31.59%乙醇及15.02%~24.13%硫酸铵组成的双水相体系中,在压力为4.5MPa,温度50℃,提取时间15min下进行提取,计算上相多酚和下相多糖的提取率,确定液料比在60:1~90:1mL/g范围内。
提取压力的选择:
称取五味子脱脂干粉,按液料比为60:1~90:1mL/g加入到由28.21%~31.59%乙醇及15.02%~24.13%硫酸铵组成的双水相体系中,在温度50℃,提取时间15min,不同压力下进行提取,计算上相多酚和下相多糖的提取率,确定压力在2.0~6.0MPa范围内。
提取温度的选择:
称取五味子脱脂干粉,按液料比为60:1~90:1mL/g加入到由28.21%~31.59%乙醇及15.02%~24.13%硫酸铵组成的双水相体系中,在压力为2.0~6.0MPa,提取时间15min,不同温度下进行提取,计算上相多酚和下相多糖的提取率,确定提取温度在50~70℃范围内。
提取时间的选择:
称取五味子脱脂干粉,按液料比为60:1~90:1mL/g加入到由28.21%~31.59%乙醇及15.02%~24.13%硫酸铵组成的双水相体系中,在压强为2.0~6.0MPa,温度为50~70℃,不同时间下进行提取,计算上相多酚和下相多糖的提取率,确定提取时间在10~30min范围内。
响应面优化实验:
根据单因素试验结果,选择液料比(X1,60:1~90:1mL/g),压力(X2,2.0~6.0MPa),温度(X3,50~70℃),时间(X4,10~30min)四个因素,以下相多糖提取率(Y1)及上相多酚提取率(Y2),下相DPPH清除率(Y3)及上相DPPH清除率(Y4)为考查指标(设3个重复),进行四因素三水平响应面优化试验。
表1模型1(下相多糖提取率,Y1)方差分析结果:
*p<0.05,**p<0.01,***p<0.001
如表1所示,模型方差分析显示实验结果Y1显著(p<0.01)拟合响应面模型(F=43.31),失拟项不显著(p>0.05)且变异系数C.V.%为3.47%,表明模型能准确预测实验结果。R2为0.9774,表明该模型与实验数据有97.74%的符合度,该方程模型具有高可信度。平方项(X2)均在p<0.001水平上显著。比较F值可知各因素对下相多糖提取率的影响顺序为:温度>料液比>压强>时间。
表2模型2(上相多酚提取率,Y2)方差分析结果
*p<0.05,**p<0.01,***p<0.001
如表2所示,模型方差分析显示实验结果Y2显著(p<0.01)拟合响应面模型(F=13.56),失拟项不显著(p>0.05)且变异系数C.V.为2.48%,表明模型能准确预测实验结果。R2为0.9313,表明该模型与实验数据有93.13%的符合度,该方程模型具有高可信度。X3极显著(p<0.001),说明提取温度对上相多酚提取率的影响最大。比较F值可知因素对上相多酚提取率的影响顺序为:温度>料液比>压强>时间。
表3模型3(下相DPPH清除率,Y3)方差分析结果
*p<0.05,**p<0.01,***p<0.001
如表3所示,模型方差分析显示实验结果Y3显著(p<0.01)拟合响应面模型(F=14.79),失拟项不显著(p>0.05)且变异系数C.V.%为3.15%,表明模型能准确预测实验结果。R2为0.9367,表明该模型与实验数据有93.67%的符合度,该方程模型具有高可信度。X2、X3极显著(p<0.001),说明提取压力和温度对下相DPPH自由基清除活性影响显著。比较F值可知因素对下相DPPH自由基清除活性影响顺序为:温度>压强>时间>料液比。
表四 模型4(上相DPPH清除率,Y4)方差分析结果
*p<0.05,**p<0.01,***p<0.001
如表4所示,模型方差分析显示实验结果Y4显著(p<0.01)拟合响应面模型(F=9.08),失拟项不显著(p>0.05)且变异系数C.V.为3.60%,表明模型能准确预测实验结果。R2为0.9008,表明该模型与实验数据有90.08%的符合度,该方程模型具有高可信度。X3极显著(p<0.001),说明温度对上相DPPH自由基清除活性影响最大。比较F值可知因素对上相DPPH自由基清除活性影响顺序为:温度>时间>料液比>压强。
根据四个响应面拟合模型,预测出HP-ATPE最佳工艺条件为:液料比60:1~90:1mL/g,高压压强2.0~6.0MPa,提取温度50~70℃,提取时间10~30min。此时。四个响应值的预估范围分别为:下相多糖得率15%~20%,上相多酚得率1.5~2.0mg/g,下相提取液DPPH自由基清除率50%~70%,上相提取液DPPH自由基清除率70%~90%。
实施例1
一种北五味子果实中活性成分集成提取的方法,步骤如下:
(1)将经过索氏提取的五味子干粉按液料比70:1mL/g加入到由29.37%乙醇和19.96%硫酸铵组成的双水相体系中,在提取温度为50℃、提取压强为2.5MPa条件下处理15min。离心分离收集上相及下相提取液,并分别量取体积。
(2)取上相提取液,用乙醇洗涤除去硫酸铵,浓缩、冷冻干燥得到上相粗提物。
(3)取下相提取液在室温下进行48h透析处理,除去无机盐及其他小分子杂质,透析液浓缩,冷冻干燥,得到下相粗提物。
(4)将上相粗提物溶解,上样HPD300大孔树脂柱,多酚被大孔树脂吸附于柱内,用酸化水洗脱未被吸附的多糖,洗脱流速为1mL/min,收集洗脱液,浓缩,醇沉、冷冻干燥得到上相多糖。
(5)将大孔树脂内被吸附的多酚用75%含水乙醇洗脱,洗脱流速为0.8mL/min,浓缩,冷冻干燥得到多酚。
(6)将下相粗提物溶解,醇沉,离心收集底部沉淀物,冷冻干燥得到下相多糖。
经测定,上相多酚提取率为1.62±0.05mg/g,上相多糖提取率为5.77±0.09%,下相多糖提取率为15.04±0.12%,多糖总提取率为20.81±0.11%,上相提取液DPPH自由基清除率为84.99±0.46%,下相提取液DPPH自由基清除率为53.74±0.52%。
本方法多糖、多酚提取率及DPPH自由基清除活性测定方法如下:
采用苯酚-硫酸法测定多糖,分别向试管中添加浓度为0.02~0.10mg/mL的葡萄糖标准溶液1.0mL,之后加入去离子水和苯酚溶液(5%,w/v)各1.0mL,充分振荡,再加入浓硫酸5.0mL,混匀。待反应体系降至常温,测定样液在490nm下的吸光度,空白对照以去离子水代替葡萄糖溶液。分别以葡萄糖溶液浓度和对应吸光度为横纵坐标,绘制标准曲线,得到线性回归方程。五味子下相多糖提取率测定:取多糖提取液稀释至适当浓度,与上述步骤相同测定吸光度。根据之前的标准曲线,计算出待测液多糖浓度,然后根据公式(1),计算出提取率。
式中:m-五味子果实的质量,mg;
C-由标准曲线计算所得五味子多糖提取液的浓度,mg/mL;
V-待测溶液体积,mL;
D-稀释倍数。
使用Folin-Ciocalteu方法测定多酚提取率,将0.5mL,0.1~1.0mg/mL没食子酸标准样品溶液分别与1.0mL Folin-Ciocalteu试剂和3.0mL Na2CO3(20%,w/v)溶液混合,然后用去离子水稀释至25.0mL。混合溶液在50℃下加热5min,并在室温下保持30min。于765nm下测量混合物的吸光度。空白对照以去离子水代替没食子酸溶液,分别以没食子酸溶液浓度和对应吸光度为横纵坐标,绘制标准曲线,得到线性回归方程。根据标准曲线,计算出待测液多酚浓度,根据公式(2),计算得出提取率。多酚提取率表示为没食子酸当量(mg)/五味子干粉重(g)。
式中:m-五味子果实的质量,mg;
C-由标准曲线计算所得五味子多酚提取液的浓度,mg/mL;
V-待测溶液体积,mL;
D-稀释倍数。
下相和上相提取液的DPPH自由基清除率测定方法:用移液管分别移取浓度为0.20、0.40、0.60、0.80、1.00、1.20mg/mL五味子下相及上相提取溶液(2.00mL)于试管中,加入DPPH溶液(2.00mL,0.20mmol/L,DPPH溶液用无水乙醇配制),混匀后在25℃下避光30min,在517nm处测定其吸光度。DPPH自由基的清除率计算公式为:
式中:A0-DPPH-乙醇溶液的吸光度;
A1-样品-乙醇溶液的吸光度;
A2-加样品溶液的试验组的吸光度
对比例1不同提取方法提取多糖、多酚的提取率对比
根据本发明确定的乙醇-硫酸铵双水相体系组成,分别采用双水相提取(ATPE)、微波辅助双水相提取(MA-ATPE)和超声辅助双水相提取方法(UA-ATPE)对北五味子果实中多糖与多酚进行集成提取,并与本发明HP-ATPE进行对比。按照实施例1的方法分别测定多酚、多糖提取率。具体操作条件以及多糖、多酚提取率列于表5。
表5五味子多糖及多酚不同提取方法的对比
如表5所示,在相同双水相体系作为提取溶剂的情况下,采用不同辅助方法对于北五味子果实中活性成分的提取率均高于单独双水相提取。微波/超声辅助双水相方法提取得到的五味子多糖、多酚的提取率均小于高压辅助双水相提取法。因此,高压辅助乙醇-硫酸铵双水相提取是一种从北五味子果实中高效集成提取活性成分的技术。
高压辅助乙醇-硫酸铵双水相提取北五味子多酚及两种多糖的组分分析
1.多酚组成分析
使用液相质谱联用技术确定北五味子多酚的主要成分,结果如附图1,表6所示。五味子多酚的主要由芦丁、矢车菊素-3-O-葡萄糖苷、山奈酚、金丝桃苷、槲皮素、忍冬苦苷、紫丁香苷及异鼠李素8个组分组成。
表6五味子多酚提取物的保留时间,质谱数据
2.两种多糖的单糖组成分析
使用高效液相色谱法测定北五味子果实中上、下相多糖的单糖组成,结果如附图2,表7所示。结果表明:本专利制备的北五味子上、下相多糖均由6种单糖组成,包括甘露糖,鼠李糖,半乳糖醛酸,葡萄糖,半乳糖和阿拉伯糖,但是上、下相多糖的单糖组成比例不同。其中,葡萄糖和甘露糖是组成上相多糖的主要单糖,而葡萄糖、半乳糖醛酸是组成下相多糖的主要单糖。
表7标准品及两种多糖中单糖的液相色谱出峰时间
3.两种多糖分子量分析
使用高效液相色谱法,测定葡聚糖标准品的分子量,然后利用GPC软件处理葡聚糖标准品的试验数据,得到回归方程:lgMw=30.2-4.87t+0.354t2-0.00965t3(R2=0.9917)。上、下相多糖的HPLC图,如附图3。上相多糖的保留时间为13.899min,下相多糖的保留时间为10.687min。根据回归方程计算上相多糖Mw为820480Da,下相多糖Mw为6456542Da。
4.测定条件
HPLC-MS/ES测定:
HPLC检测条件:型号为ACQUITYUPLC BEH C18的色谱柱(100mm×2.1mm,1.7μm);柱温40℃;进样量为5μL;流速0.4mL/min;检测时间30min,流动相A为含0.1%甲酸的水溶液,流动相B为纯乙腈;洗脱程序为0-3min,5%-15%B;3-3.5min,15%B;3.5-6min,15%-30%B;6-6.5min,30%B;6.5-12min,30%-70%B;12-12.5min,70%B;12.5-18min,70%-100%B;18-25min,100%B;25-25.5min,100%-5%B;25.5-29min,5%B。
MS检测条件:质谱仪(Triplc TDF R 5600+,SCIEX,USA)配备了ES工系统并由AB分析软件(SCIEX,1.7.1)控制。使用覆盖质量范围从m/z 50到1200的全扫描进行分析。使用氮气作为雾化气体,压力为50psi,并将流速调整为10.0L/min。干燥气体温度为550℃,离子喷雾电压维持在5.5kV。质谱数据在阳离子模式下获取,并使用Master View软件进行分析。
单糖组成测定:
HPLC条件:仪器型号:岛津LC–10aA;色谱柱:Symmetry C18 column,150×4.6mm;检测器:紫外吸收检测器。进样条件:流动相:磷酸盐缓冲溶液和乙腈;柱温:30℃;进样量:10μL;流速:0.6mL/min;洗脱方式:等度洗脱(乙腈:磷酸盐缓冲溶液=17:83,v/v)。
分子量测定:
HPLC条件:仪器型号:Waters2695/2414;色谱柱:Waters Ultrahydrogel Linear,7.8×300mm;检测器:示差折光检测器(WATERS-2414)。
Claims (11)
1.一种北五味子果实中集成提取多种活性成分的方法,所使用的方法为高压辅助乙醇-硫酸铵双水相提取,其特征在于以北五味子果实干粉为原料,包括下列步骤:
①称取适量过50目筛北五味子粉末进行脱脂,所使用的方法为索氏提取法,所用溶剂为60~90℃沸点的石油醚(原料:石油醚=1:3),置于60℃恒温加热器中回流提取6h。冷却后,将脱脂五味子粉末取出,待残留石油醚挥发干净后真空保存;
②将步骤①得到脱脂五味子干粉按一定液料比,加入到已制备好的乙醇-硫酸铵双水相体系中,在最佳高压辅助条件下进行提取;
③将步骤②所得提取液离心分离,分别得到上、下相提取液;
④将步骤③所得上相提取液经无水乙醇洗涤去盐后,减压浓缩,真空冷冻干燥得到上相粗提物;
⑤将步骤③所得下相提取液在室温下进行透析,除去无机盐等小分子杂质,然后将透析液减压浓缩,真空冷冻干燥得到下相粗提物;
⑥将步骤④所得上相粗提物溶解,上样HPD300大孔树脂柱,使用酸性水溶液洗脱树脂直至洗脱液中检测不到多糖,将洗脱液收集、浓缩、醇沉、冷冻干燥得到上相多糖,而多酚吸附于大孔树脂中;
⑦利用酸性乙醇水溶液对步骤⑥中HPD300大孔树脂柱吸附的多酚进行洗脱,洗脱液经收集、浓缩、冷冻干燥得到上相多酚;
⑧将步骤⑤所得下相粗提物溶解于去离子水中,乙醇醇沉后,离心收集底部沉淀物,冷冻干燥得到下相多糖。
2.根据权利要求1所述的方法,其特征在于所述步骤②中乙醇-硫酸铵双水相体系组成为:乙醇质量分数28.21%~31.59%,硫酸铵质量分数15.02%~24.13%。
3.根据权利要求1所述的方法,其特征在于所述步骤②中乙醇-硫酸铵双水相体系与脱脂五味子粉末的液料比为60:1~90:1mL/g。
4.根据权利要求1所述的方法,其特征在于所述步骤②中最佳高压辅助提取条件为:提取压力2.0~6.0MPa,提取温度50~70℃,提取时间10~30min。
5.根据权利要求1所述的方法,其特征在于所述步骤③中离心分离转速为3000~6000r/min,离心时间为5~25min。
6.根据权利要求1所述的方法,其特征在于所述步骤⑤中透析袋截留分子量为2000~6000Da,透析时间为12~48h。
7.根据权利要求1所述的方法,其特征在于所述步骤⑥中在HPD300大孔树脂柱中样品上样量为10~30mL,上样液浓度为10~30mg/mL,pH 1.0~5.0。
8.根据权利要求1所述的方法,其特征在于所述步骤⑥中从HPD300大孔树脂柱中洗脱多糖的洗脱剂为含0.01%~0.10%HCl的酸性水溶液,洗脱流速为0.5~3.0mL/min。
9.根据权利要求1所述的方法,其特征在于所述步骤⑥中醇沉多糖的无水乙醇的用量为多糖浓缩液体积的2~6倍,醇沉时间10~40h。
10.根据权利要求1所述的方法,其特征在于所述步骤⑦中从HPD300大孔树脂柱中洗脱多酚的洗脱剂为质量分数50%~90%的乙醇水溶液,pH 1.0~5.0,洗脱流速为0.5~2.0mL/min。
11.根据权利要求1所述的方法,其特征在于所述步骤⑧中离心分离转速为3000~8000r/min,离心时间为5~30min。
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