CN106916859B - 一种从甘草废渣中快速提取甘草素的方法 - Google Patents
一种从甘草废渣中快速提取甘草素的方法 Download PDFInfo
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Abstract
本发明属于中药提取领域,尤其涉及一种从甘草废渣中快速提取甘草素的方法。以碱提酸沉法提取甘草酸后的剩余废渣为原料,超声提取后,降温酶解;酶解液真空抽滤,滤液浓缩至干后,用乙醇溶解,得到浓缩液;将浓缩液注射于CHEETAH中高压快速制备液相色谱柱,分析色谱检测含甘草素浓度最大的馏分及前、后两侧共三个馏分合并,减压浓缩干燥后,得到高纯度甘草素产品。从甘草废渣中快速提取甘草素的方法,该方法操作简单、环保无污染、得到的产品纯度高。
Description
技术领域
本发明属于中药提取领域,尤其涉及一种从甘草废渣中快速提取甘草素的方法。
背景技术
甘草为豆科甘草属植物,其根茎是常用的中草药和经济植物。甘草中含有大量活性成分,具有抗炎、抗变态反应、抗溃疡、抗HIV病毒、抗SARS病毒、抗氧化和抗癌等药理活性;从甘草中提取活性成分甘草酸等萜类产品后,剩余物中主要含有黄酮类化合物,早期作为废渣丢弃。近年来,随着对中药的深入研究,人们发现黄酮类成分也具有重要的药理作用,而甘草黄酮类化合物中主要的活性成分有甘草苷、异甘草苷、甘草素、异甘草素等。
甘草素是甘草苷的苷元,为白色针状晶体,分子式为C15H12O4,分子量256.25。据研究报道,甘草中甘草苷含量约为3.6%,而甘草素的含量仅为0.1%左右,有些甘草甚至检测不出该成分,从甘草中直接分离提取高纯度的甘草素十分困难,并且现有技术中公开的提取方法,均存在提取率较低的问题。例如,中国专利CN102112142A,用于提高甘草或甘草提取物中甘草素含量的提取方法,是先用水或有机溶剂提取,配合酸水解提高甘草素含量,不仅提取效率低,而且产物中甘草素的含量较低;中国专利CN102391232B,从甘草中提取甘草素,使用聚酰胺和大孔树脂的混合柱,在一定程度上提高了甘草素的纯度,但纯化后需经两次8小时的析晶过程,才能得到甘草素产品,该操作过程比较繁琐,对工艺参数要求比较严格,不易控制。
综上所述,研究一种可以高效率提取甘草素的提取方法,对于其药物应用十分有必要。
发明内容
针对上述问题,本发明提供从甘草废渣中快速提取甘草素的方法,该方法操作简单、环保无污染、得到的产品纯度高。
为了实现上述目的,本发明提供的从甘草废渣中快速提取甘草素的方法,包括以下步骤。
步骤1、以碱提酸沉法提取甘草酸后的剩余废渣为原料,加入5-10倍体积的蒸馏水,混合均匀后,90℃条件下超声提取2小时;降温至50-60℃后加入原料重量的2-5%的纤维素酶酶解。
步骤2、将酶解液真空抽滤,取滤渣重复步骤1操作两次,将酶解液真空抽滤;合并三次抽滤得到的滤液,浓缩至干后,用1-2倍体积的95%乙醇溶解两次,得到浓缩液。
步骤3、将浓缩液注射于CHEETAH中高压快速制备液相色谱柱,该制备液相色谱配有二元溶剂混合系统、监测系统和馏分自动收集系统,可在不超过10Mpa压力下操作,洗脱流速设定范围为0-100mL/min;本发明所用色谱柱内填充YWG C18和MCI混合固定相,进样体积为3.5-7mL,设定制备色谱仪的检测波长为276nm,洗脱流速5mL/min,洗脱流动相为30%-70%乙醇溶剂,每20分钟自动收集一个馏分,将通过分析色谱检测含甘草素浓度最大的馏分及前、后两侧共三个馏分合并,减压浓缩在60℃下干燥30min-1h后,得到高纯度甘草素产品。
所述步骤1中酶解得温度为50-60℃,酶解时间为4小时。
所述步骤2中浓缩为真空旋转蒸发浓缩,浓缩温度为60℃。
所述步骤3中色谱柱的规格为40g,结构为上粗下细台柱型结构;所述的YWG C18和MCI混合固定相为两种填料顺序填充至色谱柱,体积比为1:2-4,其中YWG C18填料的颗粒粒径为15 μm,MCI颗粒粒径为10 μm。
所述的顺序填充是指先填充YWG C18固定相,再填充MCI固定相,且两种固定相的体积比为1:2或1:3或1:4。
本发明的有益效果。
本发明从提取甘草酸剩余废渣中提取甘草素,不仅避免了原料资源的浪费,而且甘草总黄酮得到了富集,更有利于利用酶解和色谱分离方法,得到更多具有更高纯度的甘草素产品;本发明使用碱提酸沉法提取甘草酸后的剩余废渣为原料,提高了甘草资源的利用率,碱提酸沉法提取甘草酸及萜类等比较彻底,得到的总黄酮含量更高,也更利于制备甘草素,并且如前所述从甘草中直接分离提取高纯度的甘草素十分困难,如果直接以甘草为原料进行提取,杂质含量过高,色谱分离中容易产生过载效应和谱峰重叠现象,很难得到具有一定质量和较高纯度的甘草素产品;提取和制备过程中使用水-乙醇为溶剂,并且严格控制乙醇的用量,避免了诸如甲醇、三氯甲烷等毒性有机试剂的应用,工艺绿色,无有机溶剂残留,属于环保型提取方法;本发明采用纤维素酶对原料进行酶解,使得甘草总黄酮中的苷类水解生成苷元甘草素,因而甘草素含量大大提高,从原料含量的3%提高到30%,降低了甘草素分离纯化的难度。
本发明在提取过程中在CHEETAH中高压快速纯化制备色谱仪器,采用成熟的工业部件,全自动的软件控制,操作人员只需要输入建立的方法,系统自动实现从溶剂注入到馏分收集,制备过程自动化程度高,直接提高了提取效率。
本发明经快速制备得到的甘草素产品纯度高(90%-100%),最高可制得甘草素纯品。色谱柱采用顺序混合制备填料,相比完全采用C18固定相,生产成本降低了1/3,甘草素的纯度提高了近1.2倍;而相比两种填料完全混合后填充色谱柱再进行分离纯化,甘草素的纯度提高超过1.5倍,同时该工艺简单,可实现大规模生产。
具体实施方式
下面结合具体实施例对本发明作进一步的说明。
实施例1。
取100g提取甘草酸后的剩余废渣原料,加入5倍量蒸馏水,混匀,90℃超声提取2小时,降温至50℃,加入5%重量的纤维素酶酶解,酶解4小时。将酶解液真空抽滤,滤渣重复上述操作两次,合并滤液,在60℃下真空旋转蒸发浓缩至干,用少量95%乙醇溶解。取溶解液4mL注射入CHEETAH制备色谱仪的色谱柱端口,色谱柱内固定相是顺序填充的体积比为1:4的YWG C18和MCI混合填料。设定制备检测波长为276nm,流动相流速5mL/min,用30%乙醇水流动相洗脱,收集目标馏分,在60℃下减压浓缩得纯化后甘草素产品。甘草素纯度为95.4%。
实施例2。
取100g提取甘草酸后的剩余废渣原料,加入6倍量蒸馏水,混匀,90℃超声提取2小时,降温至50℃,加入5%重量的纤维素酶酶解,酶解4小时。将酶解液真空抽滤,滤渣重复上述操作两次,合并滤液,在60℃下真空旋转蒸发浓缩至干,用少量95%乙醇溶解。取溶解液4mL注射入CHEETAH制备色谱仪的色谱柱端口,色谱柱内固定相是顺序填充的体积比为1:4的YWG C18和MCI混合填料。设定制备检测波长为276nm,流动相流速5mL/min,用45%乙醇水流动相洗脱,收集目标馏分,在60℃下减压浓缩得纯化后甘草素产品。甘草素纯度为92.1%。
实施例3。
取100g提取甘草酸后的剩余废渣原料,加入5倍量蒸馏水,混匀,90℃超声提取2小时,降温至50℃,加入5%重量的纤维素酶酶解,酶解4小时。将酶解液真空抽滤,滤渣重复上述操作两次,合并滤液,在60℃下真空旋转蒸发浓缩至干,用少量95%乙醇溶解。取溶解液3.5mL注射入CHEETAH制备色谱仪的色谱柱端口,色谱柱内固定相是顺序填充的体积比为1:2的YWG C18和MCI混合填料。设定制备检测波长为276nm,流动相流速5mL/min,用30%乙醇水流动相洗脱,收集目标馏分,在60℃下减压浓缩得纯化后甘草素产品。甘草素纯度为89.4%。
实施例4。
取100g提取甘草酸后的剩余废渣原料,加入5倍量蒸馏水,混匀,90℃超声提取2小时,降温至50℃,加入5%重量的纤维素酶酶解,酶解4小时。将酶解液真空抽滤,滤渣重复上述操作两次,合并滤液,在60℃下真空旋转蒸发浓缩至干,用少量95%乙醇溶解。取溶解液7mL注射入CHEETAH制备色谱仪的色谱柱端口,色谱柱内固定相是顺序填充的体积比为1:2的YWG C18和MCI混合填料。设定制备检测波长为276nm,流动相流速5mL/min,用45%乙醇水流动相洗脱30min后换70%乙醇水流动相继续洗脱,收集目标馏分,在60℃下减压浓缩得纯化后甘草素产品。甘草素纯度为100%。
对比例。
实施例5。
取100g提取甘草酸后的剩余废渣原料,加入5倍量蒸馏水,混匀,90℃超声提取2小时,降温至50℃,加入5%重量的纤维素酶酶解,酶解4小时。将酶解液真空抽滤,滤渣重复上述操作两次,合并滤液,在60℃下真空旋转蒸发浓缩至干,用少量95%乙醇溶解。取溶解液4mL注射入CHEETAH制备色谱仪的色谱柱端口,色谱柱内固定相是YWG C18填料。设定制备检测波长为276nm,流动相流速5mL/min,用40%乙醇水流动相洗脱,收集目标馏分,在60℃下减压浓缩得纯化后甘草素产品。甘草素纯度为77%。
实施例6。
取100g提取甘草酸后的剩余废渣原料,加入5倍量蒸馏水,混匀,90℃超声提取2小时,降温至50℃,加入5%重量的纤维素酶酶解,酶解4小时。将酶解液真空抽滤,滤渣重复上述操作两次,合并滤液,在60℃下真空旋转蒸发浓缩至干,用少量95%乙醇溶解。取溶解液3mL注射入CHEETAH制备色谱仪的色谱柱端口,色谱柱内固定相是正相硅胶填料(40μm)。设定制备检测波长为276nm,流动相流速3mL/min,用体积比为1:1的正己烷-乙醇流动相洗脱,收集目标馏分,在60℃下减压浓缩得纯化后甘草素产品。甘草素纯度为39%。
实施例7。
取100g提取甘草酸后的剩余废渣原料,加入5倍量蒸馏水,混匀,90℃超声提取2小时,降温至50℃,加入5%重量的纤维素酶酶解,酶解4小时。将酶解液真空抽滤,滤渣重复上述操作两次,合并滤液,在60℃下真空旋转蒸发浓缩至干,用少量95%乙醇溶解。取溶解液3.5mL注射入CHEETAH制备色谱仪的色谱柱端口,色谱柱内固定相是体积比1:2的YWG C18和MCI完全混合后填充而成。设定制备检测波长为276nm,流动相流速5mL/min,用45%乙醇作为流动相洗脱,收集目标馏分,在60℃下减压浓缩得纯化后甘草素产品。甘草素纯度为59%。
由上述7个实施例可以看出:采用正相硅胶填料分离纯化甘草素,效果较差;完全采用YWG C18固定相分离纯化甘草素不仅成本高,且分离纯化效果一般;将YWG C18和MCI两种填料完全混合后用其分离甘草素,效果也较差;而将YWG C18和MCI两种填料按一定次序和比例进行混合,填充于色谱柱中,再进行甘草素的分离纯化则可获得较佳的分离效果,甚至得到100%纯度的甘草素产品。
上述实施例不作为对本发明的限定,凡在本发明的范围内所作的任何修改、等同替换、改进等,均属于本发明的保护范围。
Claims (5)
1.一种从甘草废渣中快速提取甘草素的方法,其特征在于,包括以下步骤:
步骤1、以碱提酸沉法提取甘草酸后的剩余废渣为原料,加入5-10倍体积的蒸馏水,混合均匀后,90℃条件下超声提取2小时;降温至50-60℃后加入原料重量的2-5%的纤维素酶酶解;
步骤2、将酶解液真空抽滤,取滤渣重复步骤1操作两次,将酶解液真空抽滤;合并三次抽滤得到的滤液,浓缩至干后,用1-2倍体积的95%乙醇溶解两次,得到浓缩液;
步骤3、将浓缩液注射于CHEETAH中高压快速制备液相色谱柱,该制备液相色谱配有二元溶剂混合系统、监测系统和馏分自动收集系统,在不超过10Mpa压力下操作,洗脱流速设定范围为0-100mL/min;所用色谱柱内填充YWG C18和MCI混合固定相,所述的YWG C18和MCI混合固定相为两种填料顺序填充至色谱柱,所述的顺序填充是指先填充YWG C18固定相,再填充MCI固定相;进样体积为3.5-7mL,设定制备色谱仪的检测波长为276nm,洗脱流速5mL/min,洗脱流动相为30%-70%乙醇溶剂,每20分钟自动收集一个馏分,将通过分析色谱检测含甘草素浓度最大的馏分及前、后两侧共三个馏分合并,减压浓缩在60℃下干燥30min-1h后,得到高纯度甘草素产品。
2.如权利要求1所述的一种从甘草废渣中快速提取甘草素的方法,其特征在于,所述步骤1中酶解的温度为50-60℃,酶解时间为4小时。
3.如权利要求1所述的一种从甘草废渣中快速提取甘草素的方法,其特征在于,所述步骤2中浓缩为真空旋转蒸发浓缩,浓缩温度为60℃。
4.如权利要求1所述的一种从甘草废渣中快速提取甘草素的方法,其特征在于,所述步骤3中色谱柱的规格为40g,结构为上粗下细台柱型结构;其中YWG C18和MCI两种填料的体积比为1:2-4,其中YWG C18填料的颗粒粒径为15 μm,MCI颗粒粒径为10 μm。
5.如权利要求4所述的一种从甘草废渣中快速提取甘草素的方法,其特征在于,其中YWG C18和 MCI两种填料的体积比为1:2或1:3或1:4。
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