CN1256349C - 玉竹低聚糖硫酸酯的制备方法和用途 - Google Patents
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Abstract
本发明涉及天然物质的提取及其衍生物的制备和应用。通过以玉竹根状茎干粉为原料，先采用高浓度乙醇回流，过滤，滤渣用一定体积和浓度的乙醇浸提，过滤收集滤液，再用高浓度乙醇沉淀，沉淀用无水乙醇、丙酮、乙醚脱水，烘干即得玉竹低聚糖，再通过氯磺酸-吡啶法进行硫酸酯衍生修饰获得玉竹低聚糖的硫酸酯衍生物，该衍生物可应用于抗病毒药物。
Description
本发明涉及天然物质的提取及其衍生物的制备和应用。
玉竹Polygonatum odoratum(Mill.)Druce是我国的一种传统中药，据《中华本草》记载，其根状茎含有玉竹粘多糖(由果糖、葡萄糖、甘露糖和半乳糖醛酸组成)，玉竹果聚糖(由果糖和葡萄糖组成)。
硫酸酯化多糖(sulfated polysaccharides，SPS)或称多糖硫酸酯(polysaccharidessulfated，PSS)为聚阴离子化合物，是多糖大分子链中单糖分子上的某种羟基被硫酸根所取代而形成的一类化学结构复杂、生物活性多样、构效关系鲜明的多糖衍生物。除已发现的抗凝血作用外，近年来不断发现硫酸酯化多糖具有多种生物活性，如增强机体免疫功能、抗肿瘤、抗病毒等，使硫酸酯化多糖的研究成为多糖研究领域内的一个新热点。
病毒性疾病的危害具有严重性和普遍性，寻找和开发新的、安全有效的抗病毒药物使抗病毒活性成为硫酸酯化多糖生物学活性新近研究的重要方面。自1987年发现硫酸酯化葡聚糖具有抑制艾滋病病毒(Human immunodeficiencyvirus，HIV)活性以来，该类多糖的抗病毒活性方面的研究十分活跃，它有可能成为继病毒逆转录酶活性抑制剂、蛋白酶抑制剂的又一类HIV抑制剂，因此，关于糖类经硫酸酯衍生修饰获得抗病毒活性新型药物的有关研究越来越受到人们的重视。目前德国研制的木聚糖硫酸酯临床试用于艾滋病的治疗，获得了良好的结果；而日本科学家研制的产碱杆菌多糖硫酸酯(curdlan sulfate)在美国开展了I/H期临床测试，最近被认为是最具潜在的和实际的抗逆转录病毒活性的多糖制剂之一。
向道斌等综述硫酸酯化多糖抗艾滋病的作用机理主要有干扰病毒对宿主细胞的吸附、抑制病毒抗原的表达、抑制合胞体的形成、抑制逆转录酶活性，增强免疫功能等。而硫酸酯化多糖对HIV-1囊膜糖蛋白gp120与靶细胞膜上CD4分子之间的相互作用的干扰作用，已经深入到HIVgp120的具体结合部位。根据硫酸乙酰肝素是宿主细胞膜上介导病毒吸附的受体的有关研究，多数学者推测硫酸酯化多糖可能作为硫酸乙酰肝素的结构类似物与病毒囊膜糖蛋白结合从而抑制病毒对靶细胞的吸附。由于文献报道艾滋病病毒HIV、单纯疱疹病毒(Herpessimplex virus，HSV)、人巨细胞病毒(Human cytomega lovirus，HCMV)及蛋白酶抗性蛋白(Proteinase resistant protein，PrP)等，都要与硫酸乙酰肝素作用，这使硫酸酯化多糖极有可能成为作用分子基础清楚的新型广谱抗病毒药物。
本发明目的就是提供一种玉竹低聚糖的提取及其硫酸酯衍生物的制备方法和玉竹低聚糖硫酸酯衍生物在制造抗病毒药物中的应用。
玉竹低聚糖可通过以下方式制备：以玉竹根状茎干粉为原料，先采用干粉3-5倍体积的高浓度(≥70％)乙醇回流，过滤，滤渣用一定体积(玉竹干粉的4-20倍)和浓度(30-60％)的乙醇浸提，过滤收集滤液，再用高浓度乙醇(≥70％)沉淀，沉淀依次用无水乙醇、丙酮和乙醚脱水，烘干即得玉竹低聚糖，再通过氯磺酸-吡啶法进行硫酸酯衍生修饰获得玉竹低聚糖的硫酸酯衍生物，该衍生物可应用于抗病毒药物。
本发明的原料药选用《中国药典》(2000年版)中所述的传统中药玉竹无霉变、色泽淡黄、无杂什的块根；块根切片，干燥，粉碎成颗粒状。
玉竹干粉用高浓度乙醇(70％以上的乙醇)进行回流处理，去除色素、单糖等小分子物质，过滤，滤液可蒸馏回收乙醇，滤渣用适当体积(玉竹干粉的4-20倍)和浓度(30-60％)的乙醇浸提过夜，尼龙布过滤，滤液用2-4倍体积的95％乙醇沉淀，终浓度为73-88％，收集沉淀，上清液可蒸馏回收乙醇，沉淀依次用无水乙醇、丙酮和乙醚脱水，烘干即得玉竹低聚糖。
上述获得的玉竹低聚糖，以果糖为标准品用蒽酮-硫酸法测定糖含量为80-90％；以牛血清蛋白为标准品福林(Folin)-酚法测定蛋白质含量为1-2％；氯化钡-明胶法测定硫含量为0.5-2％；薄层层析法进行单糖组成分析，玉竹低聚糖由果糖、葡萄糖和甘露糖组成；溴化钾(KBr)压片，Nicolet200 SXV FT-IR红外光谱分析，玉竹低聚糖具有糖类的特征峰，在3500-3400、2920、2844、1460-1350、1150-1000cm-1有吸收，在937cm-1和815cm-1附近具有果糖的呋喃环对称伸缩振动和碳-氢(C-H)变角振动的吸收峰，在810cm-1和870cm-1附近有甘露糖的特征吸收峰。委托中国科学院大连化学物理研究所国家色谱中心进行玉竹低聚糖基体辅助激光解吸-飞行时间质谱分析，其分子量主要分布在3000-5000。
以上述制备的玉竹低聚糖为原料，采用氯磺酸-吡啶法制备玉竹低聚糖硫酸酯衍生物。在冰浴和搅拌条件下逐滴将氯磺酸按1∶2-10(体积/体积)比例加入吡啶中，再向该硫酸化试剂中加入溶于甲酰胺中的玉竹低聚糖，甲酰胺与玉竹低聚糖的比例为：10-50∶1(体积/重量)，吡啶与玉竹低聚糖的比例为：5∶1(体积/重量)，冰浴和搅拌条件下反应一定时间后，用反应液2-5倍体积的95％(体积/体积)乙醇沉淀，乙醇终浓度为63-80％(体积/体积)，弃上清液，沉淀用少量蒸馏水溶解，醋酸钠中和至中性，再用2-5倍体积的95％(体积/体积)乙醇沉淀，乙醇终浓度为63-80％(体积/体积)，沉淀依序用无水乙醇、丙酮和乙醚脱水，烘干即得玉竹低聚糖硫酸酯。
上述获得的玉竹低聚糖硫酸酯，以果糖为标准品用蒽酮-硫酸法测定糖含量为30-70％；以牛血清蛋白为标准品Folin-酚法测定蛋白质含量为0.5-1.5％；氯化钡-明胶法测定硫含量增加至3-20％，其硫酸酯取代度为0.17-2.79；KBr压片，Nicolet200 SXV FT-IR红外光谱分析，玉竹低聚糖硫酸酯不仅具有玉竹低聚糖的吸收峰，另外新增加了1250cm-1、810cm-1附近的吸收峰，分别为硫＝氧(S＝O)伸缩振动和碳-氧-硫(C-O-S)伸缩振动，620cm-1附近的尖峰说明硫酸酯以钠盐形式存在。
将以上方法制得的玉竹低聚糖硫酸酯衍生物应用于抗病毒试验，可有效抑制单纯疱疹病毒(HSV)2型引起的非洲绿猴肾细胞(Vero)的细胞病变以及病毒空斑形成，可以开发为治疗疱疹、流感、肝炎及艾滋病的广谱抗病毒药物。
本发明具有以下优点：
1、该发明工艺简单易行，所需原材料价廉易得，用于玉竹低聚糖浸提和沉淀的乙醇可以蒸馏回收，可大大节约成本。
2、获得的衍生物具有抗单纯疱疹病毒2型(Herpes simplex virus type 2，HSV-2)活性，可以开发为抗疱疹药物，用于治疗疱疹病毒性角膜炎、生殖器疱疹以及疱疹病毒引起的其它病毒性疾病，改善病人的生活质量。
3、由于硫酸酯化多糖可能作为硫酸乙酰肝素的结构类似物与病毒囊膜糖蛋白结合从而抑制病毒对靶细胞的吸附，而文献报道艾滋病病毒HIV、单纯疱疹病毒(Herpes simplex virus，HSV)、人巨细胞病毒(Human cytomega lovirus，HCMV)及蛋白酶抗性蛋白(Proteinase resistant protein，PrP)等，都是以硫酸乙酰肝素为入侵细胞的受体，因此获得的衍生物极有可能开发为作用分子基础清楚的新型广谱抗病毒药物，用于疱疹、流感、肝炎及艾滋病的治疗。
实施例1玉竹低聚糖的制备
200g玉竹干粉用4倍体积85％乙醇煮沸回流1小时，再用一定体积的85％乙醇淘洗2-3遍，弃滤液，滤渣用14倍体积50％乙醇浸提过夜。过滤弃滤渣，滤液8500rpm，4℃离心30分钟，取上清液用2倍体积的95％乙醇沉淀过夜。收集沉淀，用无水乙醇、丙酮、乙醚脱水，50℃烘箱烘干后得16.77g玉竹低聚糖。氯化钡-明胶法测定硫含量为1.17％，蒽酮-硫酸法测定糖含量为87.88％，Folin-酚法测定蛋白质含量为1.77％。该制备流程的总收率为8.39％。玉竹低聚糖的红外光谱具有多糖的特征峰，飞行时间质谱显示其主要分子量分布在3000-5000，约由18-31个单糖残基组成，薄层层析分析表明玉竹低聚糖含有果糖、甘露糖和葡萄糖。
实施例2玉竹低聚糖硫酸酯的制备
5mL氯磺酸在冰浴和搅拌条件下逐滴加入10mL吡啶中，2g玉竹低聚糖溶于80mL甲酰胺后加入上述磺酸化试剂中，冰浴及搅拌条件下反应6小时。反应结束后将上述反应液倾入400mL0℃预冷的95％乙醇中，置于4℃冰箱中沉淀过夜。弃上清，沉淀用少量蒸馏水溶解，醋酸钠中和至中性，4倍体积的95％乙醇沉淀。无水乙醇、丙酮、乙醚脱水，50℃烘箱烘干后得1.09g玉竹低聚糖硫酸酯产物。经红外光谱分析，确证有硫酸基团取代了单糖残基的羟基。氯化钡-明胶法测定硫含量为8.47％，推算得知硫酸基团取代度为0.588％，蒽酮-硫酸法测定糖含量为50％，福林(Folin)-酚法测定蛋白质含量为1.12％。该制备流程的总收率为39.78％。
实施例3玉竹低聚糖硫酸酯细胞毒性测定
向96孔细胞板中培养至单层的非洲绿猴肾(Vero)细胞分别加入终浓度为250-4000μg/mL的玉竹低聚糖硫酸酯，置37℃、5％CO2培养箱培养，逐日观察细胞生长状态，72小时后，采用中性红活细胞染色法测定玉竹低聚糖硫酸酯对非洲绿猴肾(Vero)细胞无毒的最高浓度，实验结果表明，在4000μg/mL浓度以下，玉竹低聚糖硫酸酯对Vero细胞无明显的毒害作用。
实施例4玉竹低聚糖硫酸酯的细胞病变抑制实验
非洲绿猴肾(Vero)细胞在24孔细胞板上培养至单层，然后分组，每组4个复孔。细胞对照组：不加药物和病毒；病毒对照组：感染100TCID50(病毒半数感染浓度，Tissue Culture Infecting Dose 50)的单纯疱疹病毒(HSV)2型病毒液，不加药物；实验组：感染病毒同时添加不同终浓度的玉竹低聚糖硫酸酯，在无细胞毒浓度范围内考察玉竹低聚糖硫酸酯对单纯疱疹病毒(HSV)2型引起的非洲绿猴肾(Vero)细胞的细胞病变的抑制作用。置37℃、5％CO2培养箱培养72小时后，中性红染色法测定，结果发现，浓度在125-2000μg/mL的玉竹低聚糖硫酸酯能很好地抑制单纯疱疹病毒(HSV)2型引起的细胞病变(p＜0.05)。
实施例5玉竹低聚糖硫酸酯病毒空斑抑制实验
非洲绿猴肾(Vero)细胞在24孔细胞板上培养至单层分组备用。玉竹低聚糖硫酸酯衍生物用90℃融化、43-45℃保温的底层固体培养基稀释至不同终浓度。将病毒稀释至约30-50PFU/孔(空斑形成单位，Plaque Forming Unit，PFU)，加入含糖底层固体培养基，混匀后立即加入对应孔内。细胞对照组只加底层固体培养基；病毒对照组只加含病毒底层固体培养基；实验组加含病毒和相应终浓度玉竹低聚糖硫酸酯的底层固体培养基。待底层固体培养基凝固后置37℃、5％CO2培养箱倒置培养2天，加入含1∶25000中性红的上层固体培养基，凝固后置37℃、5％CO2培养箱倒置遮光培养1天，观察病毒空斑形成情况。结果发现200μg/mL的玉竹低聚糖硫酸酯能完全抑制单纯疱疹病毒(HSV)2型引起的病毒空斑形成，100、50、25μg/mL的玉竹低聚糖硫酸酯也产生了不同程度的空斑形成抑制作用(p＜0.05)。
Claims (3)
1、一种玉竹低聚糖硫酸酯的制备方法，其特征是：以玉竹根状茎干粉为原料，先采用浓度大于70％的高浓度乙醇回流，过滤，滤渣用体积为玉竹干粉的4-20倍、浓度为30～60％的乙醇浸提，浸提时间为过夜，过滤收集滤液，再用大于70％的高浓度乙醇沉淀，沉淀依次用无水乙醇、丙酮和乙醚脱水，烘干即得玉竹低聚糖，再通过氯磺酸-吡啶法进行硫酸酯衍生修饰获得玉竹低聚糖硫酸酯。
2、根据权利要求1所述的玉竹低聚糖硫酸酯的制备方法，其特征是：所述的氯磺酸-吡啶法进行硫酸酯衍生修饰是在冰浴和搅拌条件下逐滴将氯磺酸按1∶2-10(体积/体积)比例加入吡啶中，甲酰胺按10-50∶1(体积/重量)的比例溶解玉竹低聚糖，将此甲酰胺溶解的玉竹低聚糖缓慢加入吡啶-氯磺酸硫酸化试剂中，吡啶与玉竹低聚糖的比例为5∶1(体积/重量)，继续在冰浴和搅拌条件下反应一定时间后，用该反应液2-5倍体积的95％乙醇沉淀，乙醇终浓度为63-80％，弃上清液，沉淀用少量蒸馏水溶解，醋酸钠中和至中性，再用2-5倍体积的95％乙醇沉淀，乙醇终浓度为63-80％，沉淀依次用无水乙醇、丙酮和乙醚脱水，烘干即得玉竹低聚糖硫酸酯。
3、如权利要求1所述的制备方法制备的玉竹低聚糖硫酸酯的用途，可有效抑制单纯疱疹病毒(HSV)2型引起的非洲绿猴肾细胞(Vero)的细胞病变以及病毒空斑形成，可以用于制备治疗疱疹、流感、肝炎或艾滋病的广谱抗病毒药物。
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