CN114949043B - 一种紫金龙提取液的脱色与生物碱富集方法 - Google Patents
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Abstract
本发明提供了一种紫金龙提取液的脱色与生物碱富集方法。具体为紫金龙提取液过滤膜,经阴离子交换树脂柱(柱1)富集后得到流穿液,流穿液经过阳离子交换硅胶柱(柱2)纯化后得到富集液,富集液即为紫金龙生物碱类组分。该方法利用阴离子交换树脂串联阳离子交换硅基材料,提取液直接上样,可同时去除提取液中酸性、中性杂质以及大部分色素,获得生物碱类组分。方法中所使用的溶剂对环境友好,整个体系对pH敏感样品友好。并且快速高效,操作简便,脱色除杂率高,生物碱回收率高,利于工业化生产。
Description
技术领域
本发明属于天然产物提取、分离领域,具体涉及一种紫金龙提取液的脱色与生物碱富集方法。
背景技术
紫金龙Dactylicapno sscandens(D.Don)Hutch.系罂粟科紫金龙属植物,为罂粟科植物紫金龙的根。在我国主要分布于四川、云南、广西西部和西藏东南部等地,是我国云南白族、彝族常用于治疗跌打损伤的民族药;药名:紫金龙(《云南中草药》),别名:串枝莲、黑牛膝、川山七、豌豆跌打、大麻药、豌豆七、野豌豆。现代药理学研究证明,紫金龙提取物及所含的生物碱类成分在调节心血管系统、镇痛、抗抑郁等方面有显著活性。与本属相关的各类生物碱活性已有很多相关报道,其中阿朴菲类生物碱具有广泛的生物活性,尤其是对肾上腺素受体(对α1受体及其亚型)的选择性阻断作用、细胞毒性、免疫调节活性及潜在的抗病毒活性等,苯菲啶类生物碱如血根碱,具有显著的抗肿瘤作用。因紫金龙明确的疗效在民间得到了广泛应用,而且已有风湿跌打酊等多个以其为主药的中药品种上市,云南红药、紫楼胃康胶囊等中成药也以其为主要成分。
但是,紫金龙的提取液具有较深的颜色,且含有大量杂质,在紫金龙脂溶性部分中,鉴定的成分中脂肪酸酯含量高达34.43%,甾醇和脂肪酸含量也较高,分别占13.06%和4.55%,另外,还检测出少量烷烃、酚等化合物。有色杂质的存在影响样品性状,非生物碱的存在增加了生物碱富集纯化的难度,阻碍生物碱进一步的药理研究。传统的碱沉法提取以及有机溶剂萃取模式富集生物碱存在污染环境、操作繁琐、对pH敏感生物碱造成不可逆结构破坏等缺点,因此发展可以同时实现紫金龙提取液的脱色和生物碱富集的高效、简单、条件温和且可以大规模生产新工艺意义重大。
发明内容
本发明提供了一种紫金龙提取液的脱色与生物碱富集方法。具体为以紫金龙提取液为原料,通过提取、流穿和富集,从紫金龙提取液中获得生物碱类组分,其中富集液即为紫金龙生物碱类组分。
为实现上述目的,本发明采用的技术方案为:
1)紫金龙提取液的制备:取过筛后紫金龙干燥根粗粉,按1g:5~15mL用50~100%V/V乙醇水溶液超声或回流提取1~3次,每次提取时间为1~5小时,每次提取后均抽滤,合并滤液,过膜,即得提取液;
2)流穿液的制备:将阴离子交换树脂柱(柱1)用3~5倍柱体积50~100%V/V乙醇水溶液平衡后,取步骤(1)所得提取液上样至平衡后的柱1,上样结束后,使用1~10倍柱体积50~100%V/V乙醇水溶液淋洗,然后用5~15倍柱体积1~5%V/V盐酸乙醇水溶液(乙醇水比例为80~100%V/V)洗脱,最后用5~20倍柱体积水溶液冲柱,合并上样流出液与淋洗液即为流穿液;
3)富集液的制备:将阳离子交换硅胶柱(柱2)用2~5倍柱体积50~100%V/V乙醇水溶液平衡后,取步骤(2)所得流穿液上样至平衡后的柱2,上样结束后,使用1~5倍柱体积50~100%V/V乙醇水溶液淋洗,再采用3~8倍柱体积含有20~100mM甲酸铵的50~100%V/V乙醇水溶液洗脱,对收集的洗脱液进行脱盐后即得富集液。
该方法利用阴离子交换树脂串联阳离子交换硅基材料,提取液直接上样,整个过程无需对样品进行浓缩,可同时去除提取液中酸性、中性杂质以及大部分色素,获得生物碱类组分,方法中所使用的溶剂对环境友好,整个体系对pH敏感样品友好。并且快速高效,操作简便,脱色除杂率高,生物碱回收率高,利于工业化生产。
本发明的优点
1.提取液直接上样至阴离子交换树脂柱,得到的流穿液直接上样至阳离子交换硅胶柱,整个过程无需对样品进行浓缩,快速高效,简便可行,节能减排;
2.本发明的整个体系所使用的溶剂绿色环保,条件温和,对pH敏感样品不会造成不可逆改变,并且富集液中不含有非挥发性盐,适合大规模制备的需要;
3.紫金龙提取液经过本发明脱色及富集后,生物碱实现高回收回收率98%,提取液脱色率和除杂率分别高达80%、85%以上,生物碱回收率高,脱色除杂效果尤佳。本发明的阴离子交换树脂柱1可以去除提取液中酸性色素、酸性杂质,且上样量大,阳离子交换硅胶柱能够去除中性杂质以及强极性杂质,对于生物碱类组分能够实现高选择性富集。
该方法利用阴离子交换树脂串联阳离子交换硅基材料,提取液直接上样,可同时去除提取液中酸性、中性杂质以及大部分色素,获得生物碱类组分。方法中所使用的溶剂对环境友好,整个体系对pH敏感样品友好。并且快速高效,操作简便,脱色除杂率高,生物碱回收率高,利于工业化生产。
附图说明
图1为本发明的技术路线;
图2为本发明实施例一紫金龙提取液在阴离子交换树脂柱上制备图;横坐标为体积,单位为mL,纵坐标为紫外响应值,单位为mAU;
图3为本发明实施例一紫金龙流穿液在阳离子交换硅胶柱上制备图;横坐标为时间,单位为min,纵坐标为紫外响应值,单位为AU;
图4为本发明实施例一脱盐过程色谱图;横坐标为时间,单位为min,纵坐标为紫外响应值,单位为AU;
图5为本发明实施例一紫金龙提取液、流穿液、富集液相分析谱图;
图6为本发明实施例一紫金龙提取液与生物碱标准品保留时间与吸收光谱对比分析谱图;本发明实施例一紫金龙提取液与生物碱标准品保留时间与吸收光谱对比分析谱图(a)紫金龙提取液与4个生物碱标准品色谱图的对比;(b)紫金龙提取液中色谱峰1-4吸收光谱;(c)4个生物碱色谱峰吸收光谱
表1为本发明实施例一紫金龙提取液处理前后生物碱回收率、除杂率、脱色率数据。
具体实施方式
下面结合实例,对本发明做进一步说明。实例仅限于说明本发明,而非对本发明的限定。
实施例1:
1)紫金龙提取液的制备:取紫金龙干燥根粉碎成粗粉,过3号筛(50目),取100g通过筛子的粗粉,用1000mL 80%V/V乙醇水溶液,90℃条件下加热回流提取3h,抽滤,滤渣再次用1000mL 80%V/V乙醇水溶液90℃加热回流提取3h,抽滤,共提取2次,合并滤液后过截留分子量12k Da,收集通过滤膜的液体,得紫金龙提取液,提取液固含量15mg/mL;
2)流穿液的制备:使用配备有紫外检测器的中低压制备色谱系统AKTA Pure150制备流穿液,将阴离子交换树脂柱(柱1,填料为PA408,粒度为300~1180μm,填料干重8.19g,内径为16mm,柱长为20cm,柱体积为30mL)用5倍柱体积80%V/V乙醇水溶液平衡后,取5倍柱体积步骤(1)所得提取液上样至平衡后的柱1,得上样流出液,上样结束后,使用5倍柱体积80%V/V乙醇水溶液淋洗,得淋洗液,然后用8倍柱体积2:80:18%V/V/V盐酸-乙醇-水溶液洗脱,最后用14倍柱体积水溶液冲柱,合并上样流出液与淋洗液即得到流穿液;
3)富集液的制备:将高压恒流泵、柱2、2989紫外/可见光检测器依次连接,利用该自组装系统对富集液进行制备。将阳离子交换硅胶柱(柱2,填料为HSCX,粒度为40μm,内径10mm,柱长250mm,柱体积为20mL)用2.5倍柱体积80%V/V乙醇水平衡后,取3倍柱体积步骤(2)所得的流穿液上样至平衡好的柱2,先用2.5倍柱体积80%V/V乙醇水淋洗,再用5倍柱体积含有80mM甲酸铵的60%V/V乙醇水溶液洗脱,收集洗脱液。
洗脱液的脱盐:脱盐实验在Waters Alliance高效液相色谱仪上进行,包括2695四元梯度泵,2989紫外/可见光检测器,自动进样器和柱温箱。将收集的洗脱液浓缩蒸干并以5%V/V甲醇复溶至1mL作为样品,色谱柱为XCharge C18(7μm,10×250mm),流速为3.3mL/min,柱温为30℃,波长为280nm,上样体积100μL,流动相A为添加了0.1%V/V甲酸的甲醇,流动相B为添加了0.1%V/V甲酸的水,流动相梯度条件为:0-12min:5%A,12-12.1min:5%-95%A,12.1-35min:95%A。收集15-20min段馏分,即为富集液;
4)生物碱回收率计算:标准品原阿片碱(PS011355)、海罂粟碱(PS011395)和异紫堇定(PS011328)购于成都普思生物科技股份有限公司,紫堇定(Z12M8S35796)购于上海源叶生物科技有限公司。利用Waters ACQUITY UPLC系统对紫金龙提取液以及单标样品进行分析,所用色谱柱为CSH C18column(2.1mm×50mm,1.7μm),流动相分别为5mM HCOONH4/甲醇(A)和5mM HCOONH4/水(B),流速为0.4mL/min,柱温40℃,,进样体积0.2μL,梯度条件为0-0.15min,12-12%(A);0.15-0.5min,12-22%(A);0.5-1.5min,22-30%(A);30-60%(A),1.5-2.5min;2.5-3min,60-90%(A);3-4min,90%(A)。通过对比生物碱单标样品与紫金龙提取液中色谱峰的保留时间与吸收光谱指认紫金龙涂鸦墙中的生物碱色谱峰。生物碱的回收率分别以流穿液(浓缩定容后)、富集液(浓缩定容后)中主要生物碱色谱峰峰面积之和与对应色谱峰峰面积和的比值计算;
5)除杂率和脱色率计算:除杂率以流穿液、富集液液浓度与提取液浓度的比值计算;脱色率以流穿液、富集液与提取液在356nm下吸光度的比值计算。
实验结果:
1.将150mL提取液上样至平衡后的阴离子交换树脂柱(柱1)上,当紫外检测器检测到有生物碱流出则开始收集,得到上样流出液。上样结束后,使用80%V/V乙醇水溶液对柱1上残留的上样液进行淋洗,同时收集淋洗液。合并上样流出液与淋洗液即得流穿液,流穿液对应附图2中横坐标100~400mL(共300mL);
2.将60mL流穿液上样至平衡后的阳离子交换硅胶柱(柱2)上,上样结束后,使用80%V/V乙醇水溶液对柱2上的非生物碱成分进行淋洗,上样与淋洗阶段对应附图3中15~52min。淋洗结束后,使用80mM甲酸铵的60%V/V乙醇水溶液对生物碱进行洗脱,收集附图3中的53~90min即得洗脱液。洗脱液经减压旋转蒸发仪蒸干后,后复溶至1mL,对200μL复溶后样品进行脱盐处理,收集附图4中的15~20min段馏分得到富集液(~33mL),即紫金龙生物碱组分;
3.将收集的流穿液、富集液浓缩定容至上样液体积(即:取100mL流穿液浓缩定容至50mL,取33mL富集液浓缩定容至6mL),利用相同的方法对上样液、流穿液(浓缩定容后)、富集液(浓缩定容后)进行超高效液相色谱分析,分析结果如附图5所示,利用生物碱主峰(附图6,经过与标准品的保留时间和紫外吸收光谱比对,色谱峰1~4分别为原阿片碱、紫堇定、异紫堇定和海罂粟碱,为紫金龙中主要生物碱成分)峰面积之和计算生物碱回收率,流穿液、富集液的生物碱回收率高达99.49%、98.74%,因此本发明富集得到的紫金龙生物碱类组分回收率高;
4.分别取提取液、流穿液(浓缩定容后)、富集液(浓缩定容后)各取1mL,使用低温冷冻离心机获得各样品的浓度,以流穿液(浓缩定容后)或富集液(浓缩定容后)的浓度与提取液的浓度之比计算除杂率,流穿液(浓缩定容后)、富集液(浓缩定容后)的除杂率分别为46.91%、81.45%;此外,利用紫外分光光度计测量提取液、流穿液(浓缩定容后)、富集液(浓缩定容后)在356nm下的吸光度,以流穿液(浓缩定容后)或富集液(浓缩定容后)的吸光度与上样液的吸光度之比计算脱色率,流穿液(浓缩定容后)、富集液(浓缩定容后)的脱色率分别为77.33%、87.33%;因此本发明可实现对金龙生物提取液进行高效除杂及脱色,得到高纯度紫金龙生物碱组分。流穿液(浓缩定容后)和富集液(浓缩定容后)的生物碱回收率、除杂率和脱色率列于附表1。
表1为本发明实施例一流穿液与富集液的生物碱回收率、除杂率、脱色率数据
样品名称 | 生物碱回收率 | 脱色率 | 除杂率 |
流穿液 | 99.49% | 46.91% | 77.33% |
富集液 | 98.74% | 81.45% | 87.33% |
注:生物碱回收率,方法除杂率和脱色率的计算:生物碱的回收率为处理后与处理前液相色谱分析中提取液中生物碱主峰峰面积和的比值;除杂率为处理后与处理前提取液浓度的比值;脱色率为提取液处理后与处理前的吸光度的比值。
Claims (5)
1.一种紫金龙提取液脱色与生物碱富集方法,其特征在于,包括如下步骤:
(1)紫金龙提取液的制备:取过筛后紫金龙干燥根粗粉,加入乙醇或乙醇水溶液,采用超声提取和/或加热回流法提取1~3次,每次提取时间为1~5小时,每次提取后均过滤,合并滤液,过膜,即得提取液;
(2)流穿液的制备:将阴离子交换树脂柱1用3~5倍柱体积80%-85% V/V乙醇水溶液平衡后,取步骤(1)所得提取液上样至平衡后的柱1,得上样流出液,上样结束后,使用1~10倍柱体积80%-85% V/V乙醇水溶液淋洗,得淋洗液,合并上样流出液与淋洗液即为流穿液;步骤(2)中所用阴离子交换树脂柱1填料为PA408,粒度为300~1300 μm,柱1内径大小为16~50mm,柱长为20~60 cm,上样样品载样量为40 %~100%,所述的载样量=[上样样品固含量/色谱柱填料干重质量]×100%;
(3)富集液的制备:将阳离子交换硅胶柱2用2.5-3倍柱体积80%-85% V/V乙醇水溶液平衡后,取步骤(2)所得流穿液上样至平衡后的柱2,上样结束后,使用2-3倍柱体积80%-85%V/V乙醇水溶液淋洗,再采用3~5倍柱体积含有80-90 mM甲酸铵的60%-70% V/V乙醇水溶液洗脱,收集洗脱液,将洗脱液脱盐后即得富集液;步骤(3)中所用阳离子交换硅胶柱2填料为HSCX,粒度为30~60 μm,柱2内径大小为30~100 mm,柱长为100~250 mm,上样样品载样量为0.5 %~3 %,所述的载样量=[上样样品固含量/色谱柱填料干重质量]×100%。
2.按照权利要求1所述的方法,其特征在于:步骤(1)中所用粗粉的粒度为通过10~100目筛子的粗粉,过膜所用的滤膜的截留分子量10-15 kDa。
3.按照权利要求1所述的方法,其特征在于:步骤(1)中所用乙醇水浓度80%-85% V/V。
4.按照权利要求1所述的方法,其特征在于:步骤(1)中每次提取过程的紫金龙根粉末:乙醇水溶液的料液比为1 g:10-12 mL,2~3次提取过程,是将上一次提取后固液分离的滤渣再进行下一次提取操作。
5.按照权利要求1所述的方法,其特征在于:步骤(2)中淋洗后柱子,然后用6-8倍柱体积2: 80: 18% V/V/V盐酸-乙醇-水溶液洗脱,最后用5~20倍柱体积水溶液冲柱。
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