CN111297887B - 一种白云参保肝活性组分的制备方法及用途 - Google Patents
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Abstract
本发明属于生物技术领域，公开了白云参（Campanumoea javanica BI.）多糖制备、对急性肝损伤的保护作用及其应用。白云参药材用甲醇脱脂后80℃水提，将得到的提取物进行醇沉、除蛋白，最终得到白云参多糖。白云参多糖能够显著改善肝功能指标以及氧化应激指标，具有毒副作用小、药效显著的特点，应用前景广阔。
Description
技术领域
本发明属于生物技术领域，具体涉及白云参多糖的提取方法以及在预防、治疗急性肝损伤疾病中的应用。
背景技术
肝是人体最大的实质性器官，不仅参与糖类、脂肪、蛋白质及维生素等生命物质的代谢，也是人体解毒的重要器官，同时也是有毒物质最易攻击的主要器官之一。虽然目前对于肝脏疾病有效的防治手段及药物治疗的方法很多，但仍然存在很多方面的不足。患者自身免疫***的差异、患者在疾病初期的不重视、依从性不高等问题，使得肝病已成为世界范围内死亡率极高的疾病。
自身免疫性肝炎是临床常见疾病类型，是一种以肝脏急、慢性炎症，以淋巴细胞浸润、肝细胞坏死为主要表现的自身免疫性疾病。随着病程的进展，急、慢性肝损伤若不进行及时有效治疗，则可能发展成为肝衰竭、肝纤维、肝硬化等难治性肝病。现临床主要治疗自身免疫性肝损伤的治疗药物主要有糖皮质激素等药物，具有很大的不良反应。
在寻找低毒且作用显著的保肝药，我们将目光投向了无毒的多糖大分子生物。白云参是一种多糖含量极高的植物，该药材是云南省彝药、苗药常用地方中草药之一。其活性成分白云参多糖具有免疫调节、抗氧化等药理作用。
刀豆蛋白A（Concanavalin A,ConA）是一种强促有丝***的植物凝集素。ConA诱导的急性免疫性肝损伤模型是一种经典的辅助型T细胞介导的肝细胞损伤模型，它能够较好地模拟自身免疫性肝炎的发病机制及病理变化。本研究建立了ConA诱导的小鼠急性免疫性肝损伤模型，以白云参多糖干预后的小鼠肝功能指标、血清细胞因子及氧化应激指标评价白云参多糖的保肝药效。
发明内容
本发明旨在提供一种白云参保肝活性组分的制备方法及应用。
为实现上述发明目的，本发明提供如下技术方案：
白云参多糖在制备预防或治疗肝损伤疾病药物中的应用。
白云参多糖的制备方法为：将龙胆药材粉碎，甲醇回流脱脂后进行80℃水提、80%乙醇醇沉，得到的粗多糖经sevege法除蛋白、弃去沉淀得到精制的白云参多糖。具体步骤为：
1.将白云参药材粉碎、60℃下用甲醇回流脱脂。
2.脱脂后的白云参多糖进行80℃水提，每次3小时，共提取2次，将提取液合并减压浓缩。
3.将浓缩后的提取液以80%乙醇醇沉，离心，弃去上清液。
4.将醇沉后的粗多糖以sevege法除蛋白,离心，弃去沉淀，共5次，直至无沉淀。
5.将除蛋白后的白云参多糖以硫酸苯酚法检测多糖的含量。
白云参多糖通过降低血清的中丙氨酸转氨酶（ALT）值与天冬氨酸转氨酶（AST）值来达到预防或治疗肝损伤疾病的作用。
白云参多糖通过降低血清炎症因子（IL-6）值来达到预防或治疗肝损伤疾病的作用。
白云参多糖通过升高肝脏的中氧化应激指标SOD值和MDA值、降低GSH-PX值来达到预防或治疗肝损伤疾病的作用。
白云参多糖的剂量为每日2-3次，每次0.8-3.6g。
白云参多糖的剂量为每日3次，每次2g。
白云参多糖的制剂，其特征在于所述的白云参多糖加入药学上可接受的辅料制备成片剂、硬胶囊、软胶囊、散剂、丸剂、颗粒剂。
一种药物组合物，白云参多糖作为活性成分或者是药用载体。
为证实上述白云参多糖的保肝活性，本发明采用了ConA诱导小鼠急性免疫性肝损伤模型，评价其对肝损伤的预防和治疗作用，实验结果表明：白云参多糖能够显著降低肝功能指标、血清炎症因子以及改善氧化应激指标。
本发明的优点：
1.本发明以白云参为原料，经本发明的制备方法，可以有效快捷地得到含量较高的多糖成分。
2.经药理活性实验证实了白云参多糖的保肝作用，且多糖具有毒副作用小的特点，可用于保肝护肝保健品及药品的研发。
附图说明
为了使本发明的目的、技术方案及实验结果更加清楚，本发明提供如下附图进行说明：
所有图中NC为正常对照组、MC为模型对照组、PC为联苯双酯阳性对照组、BL为白云参低剂量组400mg/kg、BH为白云参高剂量组1200mg/kg；与NC组相比，“#”表示P＜0.05，“##”表示P＜0.01，“###”表示 P<0.001；与MC组相比，“*”表示 P<0.05，“**”表示P＜0.01，“***”表示 P<0.001；
图1显示了白云参多糖对ConA诱导的小鼠急性免疫性肝损伤模型肝功能指标（a:AST、b:ALT）的干预效果；
图2显示了白云参多糖对ConA诱导的小鼠急性免疫性肝损伤模型血清炎症因子指标（IL-6）的干预效果；
图3显示了白云参多糖对ConA诱导的小鼠急性免疫性肝损伤模型氧化应激指标（a:SOD、b:MDA、c:GSH-PX）的干预效果。
具体实施方式
下面结合实施例对本发明的技术方案作进一步的详细介绍，但不以任何方式对本发明加以限制，基于本发明所作的任何变换，均属于本发明的保护范围。
白云参多糖在制备预防或治疗肝损伤疾病药物中的应用。
白云参多糖的制备方法为：将龙胆药材粉碎，甲醇回流脱脂后进行80℃水提、80%乙醇醇沉，得到的粗多糖经sevege法除蛋白、弃去沉淀得到精制的白云参多糖。具体步骤为：
1.将白云参药材粉碎、60℃下用甲醇回流脱脂。
2.脱脂后的白云参多糖进行80℃水提，每次3小时，共提取2次，将提取液合并减压浓缩。
3.将浓缩后的提取液以80%乙醇醇沉，离心，弃去上清液。
4.将醇沉后的粗多糖以sevege法除蛋白,离心，弃去沉淀，共5次，直至无沉淀。
5.将除蛋白后的白云参多糖以硫酸苯酚法检测多糖的含量。
白云参多糖通过降低血清的中丙氨酸转氨酶（ALT）值与天冬氨酸转氨酶（AST）值来达到预防或治疗肝损伤疾病的作用。
白云参多糖通过降低血清炎症因子（IL-6）值来达到预防或治疗肝损伤疾病的作用。
白云参多糖通过升高肝脏的中氧化应激指标SOD值和MDA值、降低GSH-PX值来达到预防或治疗肝损伤疾病的作用。
白云参多糖的剂量为每日2-3次，每次0.8-3.6g。
白云参多糖的剂量为每日3次，每次2g。
白云参多糖的制剂，其特征在于所述的白云参多糖加入药学上可接受的辅料制备成片剂、硬胶囊、软胶囊、散剂、丸剂、颗粒剂。
一种药物组合物，白云参多糖作为活性成分或者是药用载体。
实施例1
白云参粗多糖的制备：
将白云参药材粉碎后，60℃下用甲醇回流脱脂3次。将脱脂后的白云参药材以80℃进行热水煮提，每次提取3小时，共提取两次，合并提取液，减压浓缩。将浓缩后的浸膏以80%乙醇醇沉，离心，弃去上清液，得到白云参粗多糖。
实施例2
白云参多糖的精制：
将得到的白云参多糖用蒸馏水重溶，以sevege法除蛋白，离心，弃去沉淀，共5次，直至离心再无沉淀。最后将除蛋白后的多糖减压浓缩，冷冻干燥，以硫酸-苯酚法检测多糖的含量。最终得到的白云参多糖含量为96.48%。
实施例3
白云参多糖的保肝药效研究
白云参多糖由实施例2提供。
1.动物分组、造模及给药方法
将ICR小鼠随机分为正常对照组、模型组、阳性组（联苯双酯150mg/kg）、白云参多糖低剂量组（400mg/kg）、白云参多糖高剂量组（1200mg/kg），每组10只。灌胃给药，正常组及模型组给予生理盐水，其它组给予相应剂量药物，1天1次，连续7天。末次给药30min后，模型组及各给药组均静脉注射12mg/kgConA制备急性免疫性肝损伤模型，正常组静脉注射相等体积的生理盐水。禁食不禁水7h后，眼眶采血处死动物，收集肝脏。
2.血清中肝功能指标检测
眼球取血于1.5mL离心管中，常温离心4500r/min离心10min，留取上清液。按丙氨酸转氨酶（ALT）和天冬氨酸转氨酶（AST）试剂盒操作步骤处理，使用酶标仪测定血清中ALT、AST吸光度值（OD值）。通过查找标准曲线，根据OD值计算相应酶活力值。
实验结果见图1，与正常组相比，模型组AST、ALT值均显著升高，与模型组相比，阳性药及不同剂量的给药组AST、ALT均显著降低，且呈现剂量依赖性。结果表明白云参多糖对急性免疫性肝损伤小鼠具有保护作用。
3.血清中炎症因子指标检测
采用ELISA法对血清中IL-6及IFN-γ进行检测。按照IL-6试剂盒操作步骤处理，使用酶标仪测定血清中IL-6吸光度（OD）值，通过查找标准曲线进行计算。
实验结果见图2，与正常组相比模型组IL-6显著升高，与模型组相比，不同剂量给药组显著降低，提示白云参多糖可以减轻肝损伤小鼠的炎症反应。
4.肝脏组织中氧化应激指标检测
将小鼠肝组织进行匀浆，按照肝组织重量：生理盐水=1:9制作10%组织匀浆液待测。按照SOD、MDA、GSH-PX试剂盒操作步骤处理，使用酶标仪测定肝组织中SOD、MDA、GSH-PX的吸光度值，通过相应的公式进行计算。
实验结果见图3，与正常组相比，模型组SOD、GSH-PX显著降低，与模型组相比，各给药组SOD、GSH-PX均显著升高；与正常组相比模型组MDA显著升高，与模型组相比，各给药组均显著降低，结果表明白云参多糖可以改善肝损伤引起的氧化应激反应。
综上可以说明，白云参多糖具有显著的保肝活性，且具有毒副作用小的特点，在保肝类保健品及药品的开发上具有广泛的应用。
在本发明所述的小鼠急性免疫性肝损伤试验中，白云参多糖的剂量为400-1200mg/kg/d。换算为人（以成人体重60kg计算）剂量为2.4-7.2g/d。考虑到人的体重有不同，及各种其他病理情况，推荐剂量为每日2-3次，每次0.8-3.6g。最常用的剂量为每日3次，每次2g。
Claims (1)
1.白云参多糖在制备预防或治疗急性免疫性肝损伤疾病药物中的应用，所述白云参多糖通过降低血清炎症因子IL-6值来达到预防或治疗肝损伤疾病的作用；所述白云参多糖的制备方法为：
1）将白云参药材粉碎、60℃下用甲醇回流脱脂；
2）脱脂后进行80℃水提，每次3小时，共提取2次，将提取液合并减压浓缩；
3）将浓缩后的提取液以80%乙醇醇沉，离心，弃去上清液；
4）将醇沉后的粗多糖以sevege法除蛋白,离心，弃去沉淀，共5次，直至无沉淀。
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| CN101966199A (zh) * | 2010-09-27 | 2011-02-09 | 贵州省中国科学院天然产物化学重点实验室 | 土党参多糖在制备药物和保健食品中的应用 |
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