CN108392485B - 硫酸化甘露葡萄糖醛酸寡糖在制备治疗或预防神经退行性疾病药物中的应用 - Google Patents
硫酸化甘露葡萄糖醛酸寡糖在制备治疗或预防神经退行性疾病药物中的应用 Download PDFInfo
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Abstract
本发明涉及硫酸化甘露葡萄糖醛酸寡糖在制备治疗或预防神经退行性疾病药物或保健品中的应用;硫酸化甘露葡萄糖醛酸寡糖具有显著的神经保护作用,可以用于制备预防和治疗神经退行性疾病的药物和保健品。
Description
技术领域
本发明属于生物医药领域,涉及硫酸化甘露葡萄糖醛酸寡糖在预防和治疗神经退行性疾病中的应用。
背景技术
帕金森病(Parkinson’s disease,PD)是一种多发于中老年人、以运动障碍为主要表现的神经系统退行性疾病,PD的发生与人口老龄化有着密切的关系,据估计,到2050年我国PD患者人数将由现在的200多万增加至将近800万。由于帕金森病病程长,致残率高,中晚期病人多,治疗效果差和费用昂贵,因此给社会和家庭带来沉重的负担,是老年医学研究的重要课题。
寡糖具有多种作用,如影响机体肠道生态和生理的作用,抗凝血,抗炎,提高机体免疫,抗老年痴呆等活性。到目前为止,未有关于硫酸化甘露葡萄糖醛酸寡糖在预防和治疗神经退行性疾病药物中的应用。
发明内容
本发明提供一种硫酸化甘露葡萄糖醛酸寡糖在制备预防或治疗神经退行性疾病药物或保健品中的应用。
所述的硫酸化甘露葡糖醛酸寡糖具有如下特征:
(1)组成糖:甘露糖和葡糖糖醛酸或其盐;
(2)2-连接的甘露糖和4-连接的葡萄糖醛酸或其盐交替连接;
(3)甘露糖和葡萄糖醛酸残基上均可能有硫酸化取代。
本发明推荐所述的硫酸化甘露葡糖醛酸寡糖结构式为式(I)所示:
式中,R为SO3Na,SO3H,SO3K或者H的一种或者二种以上,R不全为H;R’为H、Na或K中的一种或二种以上,n为0-8之间的整数(n=0即重复单元不存在,重复单元两侧基团互相连接组成化合物)。
优选地,所述硫酸化甘露葡萄糖醛酸寡糖为下列化合物之一:
进一步,所述硫酸化甘露葡萄糖醛酸寡糖作为治疗或预防神经退行性疾病的药物或保健品,适用的神经退行性疾病主要为帕金森病或阿尔茨海默病。
进一步,所述应用为:将硫酸化甘露葡萄糖醛酸寡糖与药学上可接受的载体和/或赋形剂制成注射剂、口服制剂或局部给药制剂,作为治疗或预防神经退行性疾病的药物或保健品。所述载体包括海藻酸钠微球、脂质体等。
所述赋形剂:如甘露醇、硬脂酸镁、淀粉、环糊精等。
本发明所述硫酸化甘露葡萄糖醛酸寡糖的制备方法是基于参考文献(EuropeanJournal of Medicinal Chemistry,2002,37(10),783-791)的基础上进行修改所得,具体推荐按以下方法制备:
将甘露葡萄糖醛酸寡糖单体在氮气保护条件下溶于DMF(0.05g/ml)中,再加入不同剂量的三氧化硫吡啶盐,室温搅拌24小时。将反应液倒入4倍冰蒸馏水中,中和,SephadexG10脱盐。将洗脱液浓缩,Bio-gel P4分离纯化得到相应硫酸化寡糖。
本发明具有如下优点:
硫酸化甘露葡萄糖醛酸寡糖对帕金森病和阿尔茨海默病具有预防和治疗作用,毒副作用小,安全有效,可以用于制备治疗和预防神经退行性疾病的药物和保健品。
附图说明
图1硫酸化甘露葡萄糖醛酸寡糖GM2S1的ESI-MS图
图2硫酸化甘露葡萄糖醛酸寡糖GM2S2的ESI-MS图
图3硫酸化甘露葡萄糖醛酸寡糖GM4S1的ESI-MS图
图4硫酸化甘露葡萄糖醛酸寡糖GM4S2的ESI-MS图
图5硫酸化甘露葡萄糖醛酸寡糖GM4S3的ESI-MS图
图6硫酸化甘露葡萄糖醛酸寡糖GM6S1的ESI-MS图
图7硫酸化甘露葡萄糖醛酸寡糖GM6S2的ESI-MS图
图8硫酸化甘露葡萄糖醛酸寡糖GM6S3的ESI-MS图
图9硫酸化甘露葡萄糖醛酸寡糖对MPP+损伤PC12细胞的抑制率
具体实施方式
下面用实施例对本发明进行具体说明,不过本发明并不只限于以下实施案例范围。
实施例1甘露葡萄糖醛酸寡糖的制备
将干海带采用30倍质量的蒸馏水100℃下提取3小时,提取液经过过滤,浓缩后,加乙醇至终浓度为75%沉淀,静置12小时后收集沉淀,沉到经真空干燥得到海带多糖。将海带多糖样品溶于质量浓度为4%的硫酸溶液中(料液比为60mg/mL)加热回流5小时,用氢氧化钡中和至PH=6-7,离心,上清液浓缩至原始体积的五分之一,浓缩液上活性炭柱层析,首先用蒸馏水平衡,然后用50%-90%乙醇梯度洗脱,将50%-90%乙醇洗脱液浓缩至原始体积的五分之一,蒸去乙醇,直接上Bio-gel P4柱层析,分离得到五个组分,对上面收集得到的样品进行ESI-MS和HPLC分析。结果进一步确认寡糖的结构。结果显示,G1-G4分别为甘露葡萄糖醛酸八糖,六糖,四糖和二糖。其结构均符合下面所示的结构式:
其中G1为甘露葡萄糖醛酸八糖,式中n=3,R=H或者NH4 +;
G2为甘露葡萄糖醛酸六糖,式中n=2,R=H或者NH4 +;
G3为甘露葡萄糖醛酸四糖,式中n=1,R=H或者NH4 +;
G4为甘露葡萄糖醛酸二糖,式中n=0,R=H或者NH4 +;
实施例2硫酸化甘露葡萄糖醛酸寡糖GM2S1的制备
将实施例1中得到的甘露葡萄糖醛酸寡糖单体G4(甘露葡萄糖醛酸二糖)在氮气保护条件下溶于DMF(0.05g/ml)中,再加入3.30g三氧化硫吡啶盐,室温搅拌24小时。将反应液倒入4倍冰蒸馏水中,中和,Sephadex G10脱盐。将洗脱液浓缩,Bio-gel P4分离纯化得到GM2S1。质谱(如下图1)结果显示GM2S1为GlcAMan(SO3H)3-6。
实施例3硫酸化甘露葡萄糖醛酸寡糖GM2S2的制备
将实施例1中得到的甘露葡萄糖醛酸寡糖单体G4(甘露葡萄糖醛酸二糖)在氮气保护条件下溶于DMF(0.05g/ml)中,再加入1.80g三氧化硫吡啶盐,室温搅拌24小时。将反应液倒入4倍冰蒸馏水中,中和,Sephadex G10脱盐。将洗脱液浓缩,Bio-gel P4分离纯化得到GM2S1。质谱(如下图2)结果显示GM2S1为GlcAMan(SO3H)1-3。
实施例4硫酸化甘露葡萄糖醛酸寡糖GM4S1的制备
将实施例1中得到的甘露葡萄糖醛酸寡糖单体G3(甘露葡萄糖醛酸四糖)在氮气保护条件下溶于DMF(0.05g/ml)中,再加入3.54g三氧化硫吡啶盐,室温搅拌24小时。将反应液倒入4倍冰蒸馏水中,中和,Sephadex G10脱盐。将洗脱液浓缩,Bio-gel P4分离纯化得到GM4S1。质谱(如下图3)结果显示GM4S1为GlcA2Man2(SO3H)8-11。
实施例5硫酸化甘露葡萄糖醛酸寡糖GM4S2的制备
将实施例1中得到的甘露葡萄糖醛酸寡糖单体G3(甘露葡萄糖醛酸四糖)在氮气保护条件下溶于DMF(0.05g/ml)中,再加入2.19g三氧化硫吡啶盐,室温搅拌24小时。将反应液倒入4倍冰蒸馏水中,中和,Sephadex G10脱盐。将洗脱液浓缩,Bio-gel P4分离纯化得到GM4S2。质谱(如下图4)结果显示GM4S2为GlcA2Man2(SO3H)5-9。
实施例6硫酸化甘露葡萄糖醛酸寡糖GM4S3的制备
将实施例1中得到的甘露葡萄糖醛酸寡糖单体G3(甘露葡萄糖醛酸四糖)在氮气保护条件下溶于DMF(0.05g/ml)中,再加入0.93g三氧化硫吡啶盐,室温搅拌24小时。将反应液倒入4倍冰蒸馏水中,中和,Sephadex G10脱盐。将洗脱液浓缩,Bio-gel P4分离纯化得到GM4S3。质谱(如下图5)结果显示GM4S3为GlcA2Man2(SO3H)1-5。
实施例7硫酸化甘露葡萄糖醛酸寡糖GM6S1的制备
将实施例1中得到的甘露葡萄糖醛酸寡糖单体G2(甘露葡萄糖醛酸六糖)在氮气保护条件下溶于DMF(0.05g/ml)中,再加入2.85g三氧化硫吡啶盐,室温搅拌24小时。将反应液倒入4倍冰蒸馏水中,中和,Sephadex G10脱盐。将洗脱液浓缩,Bio-gel P4分离纯化得到GM6S1。质谱(如下图6)结果显示GM6S1为GlcA3Man3(SO3H)8-15。
实施例8硫酸化甘露葡萄糖醛酸寡糖GM6S2的制备
将实施例1中得到的甘露葡萄糖醛酸寡糖单体G2(甘露葡萄糖醛酸六糖)在氮气保护条件下溶于DMF(0.05g/ml)中,再加入2.09g三氧化硫吡啶盐,室温搅拌24小时。将反应液倒入4倍冰蒸馏水中,中和,Sephadex G10脱盐。将洗脱液浓缩,Bio-gel P4分离纯化得到GM6S2。质谱(如下图7)结果显示GM6S2为GlcA3Man3(SO3H)4-10。
实施例9硫酸化甘露葡萄糖醛酸寡糖GM6S3的制备
将实施例1中得到的甘露葡萄糖醛酸寡糖单体G2(甘露葡萄糖醛酸六糖)在氮气保护条件下溶于DMF(0.05g/ml)中,再加入1.17g三氧化硫吡啶盐,室温搅拌24小时。将反应液倒入4倍冰蒸馏水中,中和,Sephadex G10脱盐。将洗脱液浓缩,Bio-gel P4分离纯化得到GM6S3。质谱(如下图8)结果显示GM6S3为GlcA3Man3(SO3H)1-6。
实施例10:硫酸化甘露葡萄糖醛酸寡糖体外给药对神经细胞增殖活性影响
硫酸化甘露葡萄糖醛酸寡糖体外给药对神经细胞增殖活性的影响
MTT比色法是一种检测细胞存活和生长的方法。其检测原理为活细胞线粒体中的琥珀酸脱氢酶能使外源性MTT还原为水不溶性的蓝紫色结晶甲瓒并沉积在细胞中,而死细胞无此功能。二甲基亚砜能溶解细胞中的甲瓒,通过酶标仪测定其在490nm波长处光吸收值,可间接反映活细胞数量。在一定细胞数范围内,MTT结晶形成的量与细胞数成正比。该方法已广泛用于一些生物活性因子的活性检测、大规模的药物筛选等。它的特点是灵敏度高、经济。
实验方法
取对数生长期的PC12细胞,胰蛋白酶消化成单细胞悬液,细胞计数后,接种100μL(1×104个/孔的密度)细胞液于96孔培养板,放入5%CO2、37℃的细胞培养箱中培养24h;向96孔板中加入10μL不同浓度样品或者PBS,继续培养24h;弃去上清液,每孔加入100μL培养基或者含有MPP+的培养基,放入培养箱继续培养24h;每孔加入10μL的MTT溶液(浓度为5mg/mL),放入培养箱中再接着培养4小时,而后倒掉96孔板中的培养上清,每孔加入100μL的二甲基亚砜,摇床上振荡10min混匀,用酶标仪测定490nm处的吸光值。样品对MPP+损伤PC12细胞的抑制率(%)=(样品组OD值-模型组OD值)/(对照组OD值-模型组OD值)×100。由上述实施例2-9制得的硫酸化甘露葡萄糖醛酸寡糖中,只有GM6S1和GM6S3在体外对MPP+诱导的PC12细胞损伤具有一定的保护作用,其中GM6S3具有最好的作用。
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