CN111487349A - 用于选择性富集纯化皂苷的色谱柱及其使用方法 - Google Patents
用于选择性富集纯化皂苷的色谱柱及其使用方法 Download PDFInfo
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Abstract
本发明公开了一种用于选择性富集纯化皂苷的色谱柱,所述色谱柱的柱管中装填有混合填料,所述混合填料由大孔树脂、弱阴离子交换树脂和弱阳离子交换树脂组成,三者的质量比为(2~5):1:1。本发明还公开了使用所述色谱柱选择性富集纯化皂苷的方法。与单独使用一种色谱柱或者多种色谱柱联用相比,本发明的色谱柱具有更好的分离富集皂苷的能力。
Description
技术领域
本发明涉及天然产物分离纯化技术领域,具体涉及一种用于选择性富集纯化皂苷的色谱柱及其使用方法。
背景技术
皂苷是中草药中广泛存在的一种有效活性成分,具有调节机体免疫功能、抗肿瘤、抗衰老、抗病毒、护肝、抗脂质过氧化及高脂血症、促进细胞增殖、调节中枢神经系统、内分泌和呼吸系统等作用。皂苷存在于各种中药植物中,种类繁多。人参皂苷可影响多种肝炎病毒,通过抑制HAV的活性来影响甲型肝炎病毒,还能抑制乙型肝炎病毒DNA复制、蛋白形成和核转录激活蛋白活性,抑制HCV感染细胞增殖。白头翁皂苷对消化系统肿瘤具有抑制作用,还能强烈抑制结肠癌细胞、胰腺癌细胞、胃癌细胞的生长和增殖,诱导其凋亡,且白头翁皂苷B4具有潜在的逆转肿瘤细胞多药耐药的作用。广泛存在于豆科植物和薯蓣科植物中的薯蓣皂苷元除了具有良好的抗肿瘤,抗炎,降血脂等作用外,在中药治疗阿尔兹海默症中具有积极的影响。柴胡皂苷使大脑海马区ChAT的蛋白表达、AChE活性显著下调,保护海马区神经细胞凋亡,从而起到抗抑郁的作用。中草药等植物中的皂苷含量高,临床上药用价值大,是一种潜力极大的中药活性组分。
虽然皂苷在中草药中含量高,但是如何把中草药中提取的皂苷组分进行快速富集纯化成为目前亟待解决的问题。大孔吸附树脂柱是一种重要的分离纯化方法,具有工艺流程简单、有效组分损失小、易于工业化生产等优点,在中药纯化领域得到了广泛的应用。文献1(海峡药学,2013,25(06):8-9)采用大孔树脂填充的2.0cm×50cm的玻璃柱优化条件来分离纯化地肤子总皂苷,文献2(常熟理工学院学报2007(02):45-48+52)用大孔树脂填充的色谱柱来分离纯化重楼根茎的皂苷。文献3(医药导报,2011,30(04):423-426)先使用大孔吸附树脂富集皂苷,再使用弱碱性阴离子树脂进一步脱色纯化柴胡总皂苷。这些公开的文献涉及到的色谱柱及方法联用一种或两种不同的树脂,但是采用大孔树脂对皂苷的纯化分离效率不高,而联用两种不同的树脂进行纯化的操作步骤复杂,并且并不能有效地实现对立体构型复杂、具有多元糖基、亲水亲脂两性皂苷的大量高纯度制备,限制了其在工业化上的应用。
发明内容
本发明要解决的技术问题是提供一种用于选择性富集纯化皂苷的色谱柱,与单独的一种色谱柱或者多种色谱柱联用相比,本发明的混合填料色谱柱具有更好的分离富集皂苷的能力。
为了解决上述技术问题,本发明提供了如下所述的技术方案:
本发明第一方面提供了一种用于选择性富集纯化皂苷的色谱柱,所述色谱柱的柱管中装填有混合填料,所述混合填料由大孔树脂、弱阴离子交换树脂和弱阳离子交换树脂组成,三者的质量比为(2~5):1:1。
本发明中,所述大孔树脂、弱阴离子交换树脂和弱阳离子交换树脂的尺寸相近。进一步地,所述大孔树脂、弱阴离子交换树脂和弱阳离子交换树脂的粒径均为5~50μm。
进一步地,所述大孔树脂、弱阴离子交换树脂和弱阳离子交换树脂的骨架均独立地选自苯乙烯类、丙烯酸类、酚醛类中的一种或多种。
进一步地,所述大孔树脂、弱阴离子交换树脂和弱阳离子交换树脂的骨架均为丙烯酸类。
进一步地,所述柱管为玻璃管或不锈钢管。
进一步地,所述柱管的内径为20~60cm,长度为90~120cm。
本发明第二方面提供了使用第一方面所述的色谱柱选择性富集纯化皂苷的方法,包括以下步骤:
将皂苷粗品溶解并稀释,得稀释溶液,控制所述稀释溶液流经色谱柱;以及
使用有机溶剂洗脱所述色谱柱,收集洗脱液,蒸发结晶,得到皂苷成品。
进一步地,溶解皂苷粗品的溶剂为甲醇或乙腈。
进一步地,采用水进行稀释,稀释倍数为4~10倍。
进一步地,采用恒流泵控制稀释溶液以200~600ml/min的流速流经色谱柱。
进一步地,洗脱所使用的有机溶剂为甲醇或乙腈,有机溶剂的用量为5~20ml。
与现有技术相比,本发明的有益效果在于:
1.本发明通过在色谱柱中装填了按一定比例混合的大孔树脂、弱阴离子交换树脂、弱阳离子交换树脂,其中大孔树脂和弱阴离子交换树脂能够分别通过物理吸附和离子交换作用于皂苷,同时弱阳离子交换树脂能够通过氢键作用吸附皂苷。与单独的一种色谱柱或者多种色谱柱联用相比,本发明的混合填料色谱柱具有更好的分离富集皂苷的能力。
2.采用本发明的色谱柱纯化皂苷,无需使用两支甚至多支色谱柱,即可达到高度纯化的效果,并且操作简便、成本低廉,可用于工业化生产。
附图说明
附图1是几种皂苷的结构式:(1)地榆Ⅰ(熊果酸型);(2)地榆Ⅰ异构体(齐墩果型);(3)白头翁皂苷B5(齐墩果酸型);(4)白头翁皂苷B5异构体(熊果酸型);(5)科罗索酸葡萄糖苷(熊果酸型);(6)科罗索酸葡萄糖苷异构体(齐墩果酸型);
附图2是地榆Ⅰ皂苷标准品的液相色谱图;
附图3是实施例1中溶剂法提取的地榆Ⅰ皂苷粗品液相色谱图;
附图4是实施例1中地榆Ⅰ皂苷粗品经色谱柱富集纯化后的液相色谱图;
附图5是白头翁皂苷B5标准品的液相色谱图;
附图6是实施例2中溶剂法提取的白头翁皂苷B5粗品液相色谱图;
附图7是实施例2中白头翁皂苷B5粗品经色谱柱富集纯化后的液相色谱图;
附图8是科罗索酸葡萄苷标准品的液相色谱图;
附图9是实施例3中溶剂法提取的科罗索酸葡萄苷粗品液相色谱图;
附图10是实施例3中科罗索酸葡萄苷粗品经色谱柱富集纯化后的液相色谱图。
具体实施方式
下面结合具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
实施例1:纯化地榆Ⅰ皂苷
本实施例提供了一种选择性富集地榆Ⅰ皂苷的方法,包括以下步骤:
(1)自制内径为26cm,长为100cm的玻璃柱管。
(2)选取粒径分别为30μm、20μm、20μm的丙烯酸骨架的大孔树脂、弱阴离子交换树脂、弱阳离子交换树脂,将其活化;然后按照3:1:1的比例将活化后的大孔树脂、弱阴离子交换树脂、弱阳离子交换树脂用水浸泡完全,搅拌30min混合均匀,装填入玻璃柱管内。
(3)取10g通过溶剂法提取得到的地榆Ⅰ皂苷粗品,溶于100ml甲醇,用水稀释10倍至1000mL。
(4)将稀释后的溶液用恒流泵以100ml/min的流速泵入色谱柱内。
(5)用5mL的甲醇洗脱色谱柱,收集洗脱液,蒸发除去甲醇,冷却结晶,得到高纯度地榆Ⅰ皂苷。
参见图2-4,通过和地榆Ⅰ皂苷标准品对比,地榆Ⅰ皂苷粗品HPLC图中地榆Ⅰ的保留时间5.248min。冷却结晶后得到的地榆Ⅰ皂苷质量为4.2g,纯度为98.6%。这说明本发明可以实现对皂苷的选择性富集纯化。自然界天然产物广泛纯在同分异构体,纯化后的地榆Ⅰ皂苷晶体中也会少量存在地榆Ⅰ皂苷的异构体,这并不影响本发明的效果。
实施例2:纯化白头翁皂苷B5
取10g通过溶剂法提取得到的白头翁皂苷粗品,按照实施例1中的方法进行纯化,得到的白头翁皂苷B5结晶质量为4.27g,纯度高达97.84%。
参见图5-7,通过和白头翁皂苷B5标准品对比,白头翁皂苷B5粗品在HPLC图中的保留时间为5.982min。
实施例3:纯化科罗索酸葡萄苷
取10g通过溶剂法提取得到的科罗索酸葡萄苷粗品,按照实施例1中的方法进行纯化,得到的科罗索酸葡萄苷结晶质量为2.93g,纯度高达97.99%。
参见图8-10,通过和科罗索酸葡萄苷标准品对比,科罗索酸葡萄苷粗品在HPLC图中的保留时间为15.943min。
以上实施例表明,本发明的色谱柱,对于地榆Ⅰ皂苷、白头翁皂苷B5以及科罗索酸葡萄苷均具有良好的纯化效果,并且仅需一支色谱柱即可,无需联用两种甚至多种色谱柱,简化了纯化的操作,可用于工业化生产。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例,本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换,均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
Claims (10)
1.一种用于选择性富集纯化皂苷的色谱柱,其特征在于,所述色谱柱的柱管中装填有混合填料,所述混合填料由大孔树脂、弱阴离子交换树脂和弱阳离子交换树脂组成,三者的质量比为(2~5):1:1。
2.根据权利要求1所述的一种用于选择性富集纯化皂苷的色谱柱,其特征在于,所述大孔树脂、弱阴离子交换树脂和弱阳离子交换树脂的粒径均为5~50μm。
3.根据权利要求1所述的一种用于选择性富集纯化皂苷的色谱柱,其特征在于,所述大孔树脂、弱阴离子交换树脂和弱阳离子交换树脂的骨架均独立地选自苯乙烯类、丙烯酸类、酚醛类中的一种或多种。
4.根据权利要求3所述的一种用于选择性富集纯化皂苷的色谱柱,其特征在于,所述大孔树脂、弱阴离子交换树脂和弱阳离子交换树脂的骨架均为丙烯酸类。
5.根据权利要求1所述的一种用于选择性富集纯化皂苷的色谱柱,其特征在于,所述柱管为玻璃管或不锈钢管,所述柱管的内径为20~60cm,长度为90~120cm。
6.使用权利要求1~5任一项所述的色谱柱选择性富集纯化皂苷的方法,其特征在于,包括以下步骤:
将皂苷粗品溶解并稀释,得稀释溶液,控制所述稀释溶液流经色谱柱;以及
使用有机溶剂洗脱所述色谱柱,收集洗脱液,蒸发结晶,得到皂苷成品。
7.根据权利要求6所述的选择性富集纯化皂苷的方法,其特征在于,溶解皂苷粗品的溶剂为甲醇或乙腈。
8.根据权利要求6所述的选择性富集纯化皂苷的方法,其特征在于,稀释倍数为4~10倍。
9.根据权利要求6所述的选择性富集纯化皂苷的方法,其特征在于,采用恒流泵控制稀释溶液以200~600ml/min的流速流经色谱柱。
10.根据权利要求6所述的选择性富集纯化皂苷的方法,其特征在于,洗脱所使用的有机溶剂为甲醇或乙腈,有机溶剂的用量为5~20ml。
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