CN110776581B - 一种八仙花多糖及治疗心肌缺血再灌注损伤的用途 - Google Patents
一种八仙花多糖及治疗心肌缺血再灌注损伤的用途 Download PDFInfo
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Abstract
本发明公开了一种八仙花多糖及治疗心肌缺血再灌注损伤的用途。本发明提供了一种八仙花多糖，通过如下方法制备：收集八仙花Hydrangea macrophylla叶，洗净，阴干，粉碎，用去离子水热回流提取，过滤，滤液减压浓缩，离心收集上清液，加入三倍体积无水乙醇，混匀、静置醇析，收集沉淀，用去离子水复溶，按照同样的方法重复离心、醇沉一次，收集沉淀，无水乙醇、丙酮洗涤，去离子水复溶，Sevag法脱蛋白，冷冻干燥。使用该八仙花多糖预处理可以有效降低大鼠心肌缺血再灌注损伤，其作用机制与降低缺血再灌注大鼠心肌细胞凋亡有关，可以用于制备成拮抗心肌缺血再灌注损伤的药物。
Description
技术领域
本发明属于医药领域，具体涉及一种八仙花多糖及治疗心肌缺血再灌注损伤的用途。
背景技术
心肌缺血再灌注损伤作为一种复杂的生理病理过程，一直是心血管领域的关注焦点。近年文献报道，心肌缺血再灌注损伤发生时采用药物预处理的方式对缺血再灌注的心肌组织具有保护功能，这可能与药物激活机体内源性保护机制或促使内源性保护介质释放有关。
八仙花Hydrangea macrophylla(Thunb.)Ser.是一种生长于我国长江以南地区的落叶灌木，民间常用其全草治疗痢疾、心热惊悸等疾病，现代药理及临床研究表明八仙花叶具有很好的抗疟、抗过敏﹑调血脂、降血糖等作用。
前期研究发现八仙花提取物具有抗心肌缺血再灌注损伤的作用，但是尚不清楚药效物质基础，八仙花中的多糖是否具有抗心肌缺血再灌注损伤作用还不清楚。
发明内容
本发明一是为了提供一种八仙花多糖，二是为了提供该八仙花多糖用于治疗心肌缺血再灌注损伤的用途。
本发明技术方案如下：
一种八仙花多糖，通过如下方法制备：收集八仙花Hydrangea macrophylla叶，洗净，阴干，粉碎，用去离子水热回流提取，过滤，滤液减压浓缩，离心收集上清液，加入三倍体积无水乙醇，混匀、静置醇析，收集沉淀，用去离子水复溶，按照同样的方法重复离心、醇沉一次，收集沉淀，无水乙醇、丙酮洗涤，去离子水复溶，Sevag法脱蛋白，冷冻干燥。
其中，热回流提取3次，每次1.5h。
其中，离心条件为5000r/min转速离心15min。
其中，每次静置醇析12h。
上述八仙花多糖治疗心肌缺血再灌注损伤的用途。
上述八仙花多糖用于制备拮抗心肌缺血再灌注损伤的药物的用途。
具体实施方式中，与假手术组相比，心肌缺血再灌注模型组大鼠心肌损伤标志物CK-MB和LDH的含量显著升高；与模型组相比，八仙花多糖组大鼠心肌损伤标志物CK-MB和LDH的含量显著降低。
具体实施方式中，与假手术组相比，心肌缺血再灌注模型组大鼠心肌梗死率显著升高；与模型组相比，八仙花多糖组大鼠心肌梗死率显著降低。
具体实施方式中，与假手术组相比，心肌缺血再灌注模型组大鼠心肌细胞中的凋亡蛋白Bax、caspase-3表达显著升高；与模型组相比，八仙花多糖组大鼠心肌细胞中的凋亡蛋白Bax、caspase-3表达显著降低。
有益的技术效果：
本发明提供了一种八仙花多糖，使用该八仙花多糖预处理可以有效降低大鼠心肌缺血再灌注损伤，其作用机制与降低缺血再灌注大鼠心肌细胞凋亡有关，可以用于制备成拮抗心肌缺血再灌注损伤的药物。
附图说明
图1为各组大鼠血清中心肌损伤标志物的含量测定结果；
图2为各组大鼠心肌梗死率的测定结果；
图3为各组大鼠心肌细胞中凋亡蛋白表达水平的Westernblot测定结果。
具体实施方式
下面结合附图具体介绍本发明实质性内容，但本领域技术人员应当知道并不以下述内容限定本发明保护范围。
实施例1：提取纯化实验
收集八仙花Hydrangea macrophylla叶，洗净，阴干，粉碎，取粉末1kg，加10L去离子水，热回流提取3次，每次1.5h，合并提取液，过滤，减压浓缩至3L，于5000r/min转速离心15min，收集上清液，加入三倍体积无水乙醇，混匀、静置醇析12h，收集沉淀，用3L去离子水复溶，于5000r/min转速离心15min，收集上清液，按照同样的方法重复醇沉一次，收集沉淀，无水乙醇、丙酮洗涤，去离子水复溶，Sevag法脱蛋白，冷冻干燥。
最终收集得到21.8g多糖，苯酚-硫酸法测定多糖纯度为92.5％。
实施例2：药理活性实验
一、材料和方法
1、动物分组、造模和给药
将30只成年雄性SD大鼠(体质量260～300g)，随机分为假手术组、心肌缺血再灌注模型组和八仙花多糖组，每组10只。使用本领域惯用的大鼠冠状动脉左前降支结扎法制作心肌缺血再灌注模型，具体操作如下：
3％戊巴比妥钠麻醉大鼠，固定，四肢皮下插电极，监测Ⅱ导联心电图，插管，连接动物呼吸机；于大鼠左侧第3、4肋间距离胸骨左缘约0.5cm处作切口，对胸壁进行逐层钝性分离，用手术线结扎冠状动脉左前降支，45min后松开结扎线，再灌注60min，以心电图ST抬高，缺血区心肌呈紫绀为造模成功的标志。假手术组穿线后不结扎，模型组于缺血前30min腹腔注射生理盐水，八仙花多糖组大鼠于缺血前30min腹腔注射八仙花多糖(50mg/kg)。
2、测定心肌损伤标志物
再灌注结束后，腹主动脉取血，室温静置30min，3000rpm离心10min，取血清，分别按照CK-MB检测试剂盒和LDH检测试剂盒操作说明书测定大鼠血清CK-MB和LDH。
3、测定心肌梗死率
各组采血后，取大鼠心脏，冰浴生理盐水洗净，弃除脂肪、血管及心房组织，将心室(自冠状动脉结扎线下方至心尖处)平行切成厚度相同的五片，置37℃环境下加入0.1％NBT染色15min。拍照采集图像，利用ImagePro Plus 7.0软件测算心室总面积和梗死区面积(正常心肌呈现蓝色，梗死心肌呈现灰白色)，按如下公式计算心肌梗死率(％)：
心肌梗死率(％)＝梗死区面积÷心室总面积×100％。
4、Western blot测定心肌细胞中凋亡蛋白表达水平
冰上取各组大鼠取心尖部心肌组织，研磨、裂解液提取细胞总蛋白，BCA法测蛋白浓度。各组取等量总蛋白上样进行SDS-PAGE电泳，转膜、封闭后，加入待测一抗(Bax、caspase-3)和内参抗体(β-actin)，于4℃孵育过夜，次日加入相应的二抗，化学发光法检测目的蛋白表达水平，采用Image J软件对显影的条带进行灰度分析。
4、统计学方法
采用SPSS 19.0分析数据，计量资料以均值±SD表示，组间两两比较用LSD-t检验，以P＜0.05代表差异有统计学意义。
二、实验结果
1、各组大鼠血清中心肌损伤标志物的含量差异
各组大鼠血清中心肌损伤标志物的含量测定结果如表1和图1所示：与假手术组相比，心肌缺血再灌注模型组大鼠心肌损伤标志物CK-MB和LDH的含量显著升高；与模型组相比，八仙花多糖组大鼠心肌损伤标志物CK-MB和LDH的含量显著降低。
表1各组大鼠血清中心肌损伤标志物的含量测定结果
心肌酶CK-MB和LDH正常情况下存在于心肌细胞的胞浆中，当缺血再灌注发生时，心肌受损导致细胞膜的通透性升高，CK-MB和LDH溢出胞外进入血清，因而血清CK-MB和LDH活力能够从侧面反映出心肌细胞的坏死程度及缺血再灌注损伤程度。
2、各组大鼠心肌梗死率的差异
各组大鼠心肌梗死率的测定结果如表2和图2所示：与假手术组相比，心肌缺血再灌注模型组大鼠心肌梗死率显著升高；与模型组相比，八仙花多糖组大鼠心肌梗死率显著降低。
表2各组大鼠心肌梗死率测定结果
3、各组大鼠心肌细胞中凋亡蛋白表达水平的差异
各组大鼠心肌细胞中凋亡蛋白表达水平的Western blot测定结果如图3所示：与假手术组相比，心肌缺血再灌注模型组大鼠心肌细胞中的凋亡蛋白Bax、caspase-3表达显著升高；与模型组相比，八仙花多糖组大鼠心肌细胞中的凋亡蛋白Bax、caspase-3表达显著降低。Bax蛋白是细胞凋亡通路中的促凋亡成员，caspase-3是caspase凋亡信号转导共同通路最关键的效应分子，Bax、caspase-3蛋白表达升高表示细胞凋亡严重。
上述实验结果表明，使用本发明提供的八仙花多糖预处理可以有效降低大鼠心肌缺血再灌注损伤，其作用机制与降低缺血再灌注大鼠心肌细胞凋亡有关，可以用于制备成拮抗心肌缺血再灌注损伤的药物。
上述实施例旨在具体介绍本发明的实质性内容，但本领域技术人员应当知道，不应当将本发明保护范围局限于上述具体实施例。
Claims (2)
1.一种八仙花多糖，通过如下方法制备：收集八仙花Hydrangea macrophylla叶，洗净，阴干，粉碎，取粉末1kg，加10L去离子水，热回流提取3次，每次1.5h，合并提取液，过滤，减压浓缩至3L，于5000r/min转速离心15min，收集上清液，加入三倍体积无水乙醇，混匀、静置醇析12h，收集沉淀，用3L去离子水复溶，于5000r/min转速离心15min，收集上清液，按照同样的方法重复醇沉一次，收集沉淀，无水乙醇、丙酮洗涤，去离子水复溶，Sevag法脱蛋白，冷冻干燥。
2.权利要求1所述八仙花多糖用于制备拮抗心肌缺血再灌注损伤的药物的用途。
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