CN107913298B - 一种从人参叶中提取分离人参总黄酮的方法 - Google Patents
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Abstract
本发明涉及一种从人参叶中提取分离人参总黄酮的方法。人参叶提取物用大孔树脂吸附后，使用碱的水溶液洗脱，将洗脱液中和至pH＝7。洗脱液重新用大孔树脂吸附，用高浓度的乙醇水溶液洗脱，将洗脱液减压浓缩、干燥，得到人参总黄酮。
Description
技术领域
本发明属于天然药物分离领域，涉及一种从人参叶中提取分离人参总黄酮的方法。
背景技术
人参叶是人参(Panax ginseng C.A.Mey.)的干燥叶，秋季采收，晾干或烘干；具有补气，益肺，祛暑，生津之功能；用于气虚咳嗽，暑热烦躁，津伤口渴，头目不清，四肢疲乏。
人参叶的主要成分为人参皂苷类成分和人参黄酮类成分等。现有的提取分离方法，一般通过大孔树脂吸附得到人参总皂苷和人参总黄酮的提取物，再通过硅胶柱层析进行分离。分离技术的现状限制了人参黄酮类成分的活性研究。
因此，探寻一种快速从人参叶中提取分离人参黄酮类成分的方法，有利于人参黄酮类成分的活性研究，有利于人参叶活性的研究。
发明内容
本发明的目的是提供一种从人参叶中提取分离人参总黄酮的方法，具体为一种利用大孔树脂快速分离人参总黄酮的方法，以克服现有技术中的不足。
本发明的目的通过以下技术方案实现：
一种从人参叶中提取分离得到人参总黄酮的方法，由产物的分离和分离后检测两部分组成。产物的分离通过大孔树脂进行，所述方法包括以下步骤：人参叶加水煎煮提取，提取液用大孔树脂吸附后，使用碱的水溶液洗脱。将洗脱液中和至pH＝7。再将洗脱液重新用大孔树脂吸附，用高浓度的乙醇水溶液洗脱，将洗脱液减压浓缩、干燥，得到人参总黄酮。
所述碱是氢氧化钠或氢氧化钾。
本发明同时提供了上述方法制备的人参总黄酮以及该人参总黄酮在制备保健食品或药物中的应用和在制备各种人参黄酮类单体中的应用。
本发明的分离方法避免样品损耗，具有分离效果好，有机溶剂用量少，无污染，高效等优点。
附图说明
图1显示人参叶中10种常见人参皂苷的高效液相色谱图。
图2显示从人参叶中提取分离的人参总黄酮的高效液相色谱图。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照制造厂商所建议的条件。除非另外说明，否则所有的百分数、比率、比例或份数按重量计。
除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
实施例1
干燥的人参叶2kg，加水煎煮提取三次，每次加水量分别为30L、24L、20L，煎煮时间分别为2h、1.5h、1h，合并煎煮液，过D101大孔树脂吸附，用0.5％的NaOH水溶液洗脱干净。用盐酸中和洗脱液值pH＝7。洗脱液重新过D101大孔树脂吸附，用85％乙醇水溶液洗脱。将洗脱液减压浓缩、干燥，得到人参总黄酮。
HPLC检测条件如下：
色谱柱:Agilent Eclipse XDB-C18(250×4.6mm,5μm)；流动相：乙腈-水(梯度洗脱0-35min,19:81；36-50min,29:71；51-60min,32:68；61-70min,34:66)；流速：1.2mL/min；柱温：30℃；检测波长:203nm；以下相同。用该HPLC检测条件检测10种常见人参皂苷的高效液相色谱图如图1所示，检测实施例1从人参叶中提取分离的人参总黄酮的高效液相色谱图如图2所示。
实施例2
干燥的人参叶20g，加水煎煮提取三次，每次加水量分别为300mL、240mL、200mL，煎煮时间分别为2h、1.5h、1h，合并煎煮液，过D101大孔树脂吸附，用0.5％的NaOH水溶液洗脱干净。用盐酸中和洗脱液值pH＝7。洗脱液重新过D101大孔树脂吸附，用85％乙醇水溶液洗脱。将洗脱液减压浓缩、干燥，得到人参总黄酮。
对比例1
干燥的人参叶20g，加水煎煮提取三次，每次加水量分别为300mL、240mL、200mL，煎煮时间分别为2h、1.5h、1h，合并煎煮液，减压浓缩、干燥，得到人参总黄酮。
试验例一
6-8周的雌性BALB/c小鼠，体重18-22g，购自长春生物制品研究所有限责任公司。小鼠饲养采取常规分笼方式，饮水和采食自由，温度24±2℃，相对湿度40-80％。实验在吉林大学动物中心的指导下完成。小鼠饲养一周适应环境后进行实验。共36只小鼠，分为4组：对照组，模型组，实施例2组，对比例1组，其中实施例2组和对比例1组按10mg/kg提取物给药。模型组，实施例2组和对比例1组采用腹腔和皮下注射结合的方式在第0、7、14天每只每次注射OVA 0.2mL致敏，空白组注射生理盐水。在第21、22、23天，实施例2组和对比例1组经腹腔注射相应浓度的提取物。注射量为0.2mL。模型组和空白组注射生理盐水。注射1h后，对模型组，实施例2组和对比例1组的小鼠进行滴鼻激发，激发液为50μL OVA，空白组使用生理盐水。于最后一次滴鼻激发24h后，进行小鼠眼球取血，获得血液。室温放置30min后，4℃放置过夜，离心制备血清。使用ELISA试剂盒检测血清中IgE含量。小鼠取血后，用PBS缓冲液对肺组织进行灌洗，收集灌洗液约1mL。离心获得肺泡灌洗液上清液，使用ELISA试剂盒检测上清液中IL-4，IL-5，IL-13含量。血清和肺泡灌洗液中细胞因子结果见表1。
表1不同提取方法的人参总黄酮提取物对IgE,IL-4,IL-5和IL-13的影响(Mean±SE)
#P＜0.05,##P＜0.01，表示相对对照组有显著性差异.*P＜0.05,**P＜0.01，表示相对模型组有显著性差异。
结果显示，相比于模型组和对比例1组，实施例2组显著降低了血清中IgE的含量，并且使IL-4显著降低。说明本申请的方法提取获得的人参总黄酮提取物由于分离效果好而取得了功效更加优异的效果。
以上所述仅为本发明的较佳实施例而已，并非用以限定本发明的实质技术内容范围，本发明的实质技术内容是广义地定义于申请的权利要求范围中，任何他人完成的技术实体或方法，若是与申请的权利要求范围所定义的完全相同，也或是一种等效的变更，均将被视为涵盖于该权利要求范围之中。
Claims (5)
1.一种从人参叶中提取分离人参总黄酮的方法，其特征在于：将人参叶加水煎煮提取，提取液用大孔树脂吸附后，利用碱的水溶液洗脱，将洗脱液中和至pH＝7，再将洗脱液重新用大孔树脂吸附，用浓度为85％的乙醇水溶液洗脱，将洗脱液减压浓缩、干燥，得到人参总黄酮。
2.根据权利要求1所述的方法，其特征在于，所述碱是氢氧化钠或氢氧化钾。
3.根据权利要求1或2所述的方法制备的人参总黄酮。
4.根据权利要求1或2所述的方法制备的人参总黄酮在制备保健食品或药物中的应用。
5.根据权利要求1或2所述的方法制备的人参总黄酮在制备各种人参黄酮类单体中的应用。
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| CN101249125A (zh) * | 2007-12-29 | 2008-08-27 | 重庆希尔安药业有限公司 | 一种中药组合物及其制备方法和医药用途 |
| CN103772337A (zh) * | 2013-12-18 | 2014-05-07 | 闻永举 | 大孔吸附树脂辅助各种苷类水解制备苷元、次生苷的方法 |
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| CN101249125A (zh) * | 2007-12-29 | 2008-08-27 | 重庆希尔安药业有限公司 | 一种中药组合物及其制备方法和医药用途 |
| CN103772337A (zh) * | 2013-12-18 | 2014-05-07 | 闻永举 | 大孔吸附树脂辅助各种苷类水解制备苷元、次生苷的方法 |
Non-Patent Citations (2)
| Title |
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| Preparation and quality assessment of high-purity ginseng total saponins by ion exchange resin combined with macroporous adsorption resin separation;ZHAO Yu-Nan，etal;《Chinese Journal of Natural Medicines 》;20141231;第12卷(第5期);第382−392页 * |
| 吉林省人参产品中总黄酮含量的分析;马莹等;《人参研究》;20151231;第16-17页 * |
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