CN112933105A - 甘露葡萄糖醛酸多(寡)糖及硫酸化衍生物在制备治疗非酒精性脂肪肝药物中的应用 - Google Patents
甘露葡萄糖醛酸多(寡)糖及硫酸化衍生物在制备治疗非酒精性脂肪肝药物中的应用 Download PDFInfo
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Abstract
本发明提供一种甘露葡萄糖醛酸多糖或寡糖及其硫酸化衍生物在制备治疗非酒精性脂肪肝药物中的应用。本发明的甘露葡萄糖醛酸多(寡)糖及其硫酸化衍生物具有极强的抑制脂滴积累,可以用于治疗非酒精性脂肪肝,毒副作用小,安全有效。
Description
技术领域
本发明属于生物医药领域,涉及甘露葡萄糖醛酸多糖或寡糖及其硫酸化衍生物在制备预防或治疗非酒精性脂肪肝的药物中的应用。
背景技术
非酒精性脂肪肝是指由于各种原因引起的肝细胞内脂肪沉积过多所产生的病变,是一种常见的肝脏病理改变。脂肪肝正严重危害人类健康,成为仅次于病毒性肝炎的第二大肝病。其发病率呈逐年增加趋势,且发病年龄日趋年轻化,目前尚无有效根治方法。
越来越多的证据表明,非酒精性脂肪肝的发生发展与肝脏组织中自噬的缺失或抑制密切相关。尽管自噬一般被认为是降解细胞内蛋白质(非选择性)和细胞器(选择性)的手段,目前的研究显示自噬也能选择性的降解脂滴,这一过程被称作脂滴自噬(lipophagy)。肝脏细胞中,大脂滴通过脂质分解作用形成小脂滴,小脂滴则通过溶酶体介导的自噬作用进一步降解。因此,自噬调节药物可能对于非酒精性脂肪肝的早期治疗具有良好的效果。
近年来,糖类药物从其药效和安全性方面考虑,其发展趋势逐渐从对多糖的研究转到对低聚糖和寡糖的研究。寡糖在医疗保健领域具有巨大的需求空间。寡糖类药物结构均一,质量可控,作用机制明确,已经成为糖类药物的主攻方向之一。
甘露葡萄糖醛酸多(寡)糖及其硫酸化衍生物是一类酸性多(寡)糖,研究发现甘露葡萄糖醛酸多(寡)糖及其硫酸化衍生物具有极强的抑制脂滴积累并对细胞的毒负作用很小。
发明内容
为了获得一种具有极强的抑制脂滴积累并对细胞的毒负作用很小的药物,本发明提供一种甘露葡萄糖醛酸多糖或寡糖及其硫酸化衍生物在制备治疗非酒精性脂肪肝药物中的应用。
为了实现上述目的,本发明采用如下技术方案:
一种如式(I)或(II)所示的甘露葡萄糖醛酸多糖或寡糖及其硫酸化衍生物在制备治疗非酒精性脂肪肝药物中的应用,
式(I)中,各个R’各自独立为H、Na或K中的一种或二种以上,各个R各自独立为H或SO3 -,n为0-40之间的整数;
式(II)中,各个R1各自独立为H、Na或K中的一种或二种以上,各个R2各自独立为H或SO3 -,m为0-40之间的整数。
进一步,所述甘露葡萄糖醛酸多糖或寡糖及其硫酸化衍生物为式(I)所示化合物,式(I)中,各个R’皆为H,n为0、1、2、3或40;优选R为的SO3 -个数为0-15,R为SO3 -的个数为0时,意味着R全部为H。
具体推荐以下组合:式(I)中n为0,R为SO3 -的个数为1-6;
n为1,R为SO3 -的个数为0-11,优选0或5-11;
n为2,R为SO3 -的个数为0-15;
n为3,R为SO3 -的个数为0-6。
本发明特别推荐式(I)中n=2,R’皆为H,R为的SO3 -个数为8-15。
进一步,所述治疗非酒精性脂肪肝药物为含有式(I)或(II)所示的甘露葡萄糖醛酸多糖或寡糖或硫酸化衍生物和药学可接受的载体和/或赋形剂的组合物。
具体地,所述药学可接受的载体为海藻酸钠微球、脂质体等,所述赋形剂为甘露醇、硬脂酸镁、淀粉、环糊精等。所述治疗非酒精性脂肪肝药物的剂型为注射剂、口服制剂或局部给药制剂。
与现有技术相比,本发明的有益效果主要体现在:甘露葡萄糖醛酸多(寡)糖及其硫酸化衍生物具有极强的抑制脂滴积累,可以用于治疗非酒精性脂肪肝,毒副作用小,安全有效。
附图说明
图1寡糖的制备过程中酒精洗脱液的凝胶色谱图;
图2甘露葡萄糖醛酸寡糖F1-F4的ESI-MS和HPLC图;a为甘露葡萄糖醛酸二糖(G2);b为甘露葡萄糖醛酸四糖(G4);c为甘露葡萄糖醛酸六糖(G6);d为甘露葡萄糖醛酸八糖(G8)。
图3 Gn的氢谱。
图4 Gn的碳谱。
图5 Gn的二维HMBC谱图。
图6 Gn的二维HSQC谱图。
图7 Gn的二维HHCOSY谱图。
图8 Gn的GPC-HPLC谱图。
图9硫酸化甘露葡萄糖醛酸寡糖G2S1的ESI-MS图
图10硫酸化甘露葡萄糖醛酸寡糖G2S2的ESI-MS图
图11硫酸化甘露葡萄糖醛酸寡糖G4S1的ESI-MS图
图12硫酸化甘露葡萄糖醛酸寡糖G4S2的ESI-MS图
图13硫酸化甘露葡萄糖醛酸寡糖G4S3的ESI-MS图
图14硫酸化甘露葡萄糖醛酸寡糖G6S1的ESI-MS图
图15硫酸化甘露葡萄糖醛酸寡糖G6S2的ESI-MS图
图16硫酸化甘露葡萄糖醛酸寡糖G6S3的ESI-MS图
图17甘露葡萄糖醛酸多(寡)糖及其硫酸化衍生物抑制脂滴积累活性
图18 G6S1抑制脂滴积累活性
图19 G6S1处理后细胞脂肪代谢相关蛋白表达变化
图20 G6S1处理后细胞自噬水平变化
图21 G6S1的作用靶点PPARα及通路
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1kg干海带采用30L蒸馏水100℃下提取3小时,提取液经过过滤,除杂,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10g。将150g海带多糖样品溶于2.5L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,用氢氧化钡固体中和至PH=6-7,离心,取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100mL 50%-90%乙醇梯度洗脱,将50%-90%乙醇洗脱液浓缩至20mL,蒸去乙醇,直接以碳酸氢铵为流动相上Bio-gel P4柱层析,分离得到六个组分(图1所示),对上面收集得到的样品进行ESI-MS和HPLC分析(图2所示)。结果进一步确认寡糖的结构。结果显示,F1-F4分别为甘露葡萄糖醛酸八糖(G8),六糖(G6),四糖(G4)和二糖(G2)。其结构均符合下图所示的结构式:
其中F1为甘露葡萄糖醛酸八糖(G8),式中n1=3,R1为H或者NH4 +;
F2为甘露葡萄糖醛酸六糖(G6),式中n1=2,R1为H或者NH4 +;
F3为甘露葡萄糖醛酸四糖(G4),式中n1=1,R1为H或者NH4 +;
F4为甘露葡萄糖醛酸二糖(G2),式中n1=0,R1为H或者NH4 +;
实施例2甘露葡萄糖醛酸多糖的制备
将实施例1中的得到海带多糖150g溶于2.5L质量浓度为4%的硫酸溶液中(料液比为60mg/mL)加热回流4小时,用固体氢氧化钡中和至PH=6-7,离心,上清液浓缩至100ml,浓缩液上DEAE Sepharose Fast Flow分离,分别采用2L水洗,2L 0.2M氯化钠水溶液和2L 2M氯化钠水溶液洗脱,2M氯化钠水溶液浓缩至100ml,通过500Da透析袋透析,浓缩至50ml,冷冻干燥制备得到甘露葡萄糖醛酸多糖Gn(7.0kDa),将Gn进行1H-NMR,13C-NMR,二维NMR和GPC-HPLC(图3-图8所示)。结果进一步确认甘露葡萄糖醛酸多糖的结构。其结构均符合下图所示的结构式:
其中Gn为甘露葡萄糖醛酸多糖,式中n1≈40,R1为H;
实施例3硫酸化甘露葡萄糖醛酸寡糖G2S1的制备
将实施例1中得到的甘露葡萄糖醛酸寡糖二糖G2(1g)在氮气保护条件下溶于20mLDMF(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,2M氢氧化钠中和,Sephadex G10脱盐。将洗脱液浓缩,以0.2M碳酸氢铵为流动相,Bio-gel P4分离纯化,浓缩,冷冻干燥得到G2S1。质谱(如下图9)结果显示G2S1为GlcAMan(SO3H)3-6。
实施例4硫酸化甘露葡萄糖醛酸寡糖G2S2的制备
将实施例1中得到的甘露葡萄糖醛酸寡糖二糖G2(1g)在氮气保护条件下溶于20mLDMF(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,2M氢氧化钠中和,Sephadex G10脱盐。将洗脱液浓缩,以0.2M碳酸氢铵为流动相,Bio-gel P4分离纯化,浓缩,冷冻干燥得到G2S2。质谱(如下图10)结果显示G2S2为GlcAMan(SO3H)1-3。
实施例5硫酸化甘露葡萄糖醛酸寡糖G4S1的制备
将实施例1中得到的甘露葡萄糖醛酸寡糖四糖G4(1g)在氮气保护条件下溶于20mLDMF(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,2M氢氧化钠中和,Sephadex G10脱盐。将洗脱液浓缩,以0.2M碳酸氢铵为流动相,Bio-gel P4分离纯化,浓缩,冷冻干燥得到G4S1。质谱(如下图11)结果显示G4S1为GlcA2Man2(SO3H)8-11。
实施例6硫酸化甘露葡萄糖醛酸寡糖G4S2的制备
将实施例1中得到的甘露葡萄糖醛酸寡糖四糖G4(1g)在氮气保护条件下溶于20mLDMF(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,2M氢氧化钠中和,Sephadex G10脱盐。将洗脱液浓缩,以0.2M碳酸氢铵为流动相,Bio-gel P4分离纯化,浓缩,冷冻干燥得到G4S2。质谱(如下图12)结果显示G4S2为GlcA2Man2(SO3H)5-9。
实施例7硫酸化甘露葡萄糖醛酸寡糖G4S3的制备
将实施例1中得到的甘露葡萄糖醛酸寡糖四糖G4(1g)在氮气保护条件下溶于20mLDMF(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,2M氢氧化钠中和,Sephadex G10脱盐。将洗脱液浓缩,以0.2M碳酸氢铵为流动相,Bio-gel P4分离纯化,浓缩,冷冻干燥得到G4S3。质谱(如下图13)结果显示G4S3为GlcA2Man2(SO3H)1-5。
实施例8硫酸化甘露葡萄糖醛酸寡糖G6S1的制备
将实施例1中得到的甘露葡萄糖醛酸寡糖六糖G6(1g)在氮气保护条件下溶于20mLDMF(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,2M氢氧化钠中和,Sephadex G10脱盐。将洗脱液浓缩,以0.2M碳酸氢铵为流动相,Bio-gel P4分离纯化,浓缩,冷冻干燥得到G6S1。质谱(如下图14)结果显示G6S1为GlcA3Man3(SO3H)8-15。
实施例9硫酸化甘露葡萄糖醛酸寡糖G6S2的制备
将实施例1中得到的甘露葡萄糖醛酸寡糖六糖G6(1g)在氮气保护条件下溶于20mLDMF(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,2M氢氧化钠中和,Sephadex G10脱盐。将洗脱液浓缩,以0.2M碳酸氢铵为流动相,Bio-gel P4分离纯化,浓缩,冷冻干燥得到G6S2。质谱(如下图15)结果显示G6S2为GlcA3Man3(SO3H)4-10。
实施例10硫酸化甘露葡萄糖醛酸寡糖G6S3的制备
将实施例1中得到的甘露葡萄糖醛酸寡糖六糖G6(1g)在氮气保护条件下溶于20mLDMF(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,2M氢氧化钠中和,Sephadex G10脱盐。将洗脱液浓缩,以0.2M碳酸氢铵为流动相,Bio-gel P4分离纯化,浓缩,冷冻干燥得到G6S3。质谱(如下图16)结果显示G6S3为GlcA3Man3(SO3H)1-6。
实施例11甘露葡萄糖醛酸多(寡)糖及其硫酸化衍生物的抑制脂滴积累活性
利用油酸处理小鼠肝细胞系AML12作为细胞筛选模型,100μg/mL浓度下测定实施例1-10中的甘露葡萄糖醛酸多(寡)糖及其硫酸化衍生物的抑制脂滴积累活性,对照为油酸(OA)。AML12细胞、油酸和甘露葡萄糖醛酸多(寡)糖及其硫酸化衍生物共同孵育6,12或24小时,PBS冲洗细胞。一方面,用异丙醇将油酸洗脱下来,酶标仪测定测定油酸含量;另一方面,用多聚甲醛固定液固定,显微镜观察拍照。结果显示甘露葡萄糖醛酸多(寡)糖及其硫酸化衍生物具有较好的活性(图17)。其中,G6S1在100μg/mL浓度下可以减少32%脂滴数量和37%脂滴体积,表现为最好的抑制脂滴积累活性,且比较其在不同浓度下和处理时间的抑制脂滴积累活性,发现其活性与剂量和时间呈正相关(图18)。并通过WB检测脂肪代谢相关蛋白表达变化(图19),脂滴自噬水平变化(图20)和相关作用靶点及通路(图21)。
以上描述了本发明的具体实施例,然并非用以限定本发明,本领域技术人员对在此公开的实施方案课进行并不偏离本发明范畴和精神的改进和变化。
Claims (6)
2.如权利要求1所述的应用,其特征在于:所述甘露葡萄糖醛酸多糖或寡糖及其硫酸化衍生物为式(I)所示化合物,式(I)中,各个R’皆为H,n为0、1、2、3或40;优选R为的SO3 -个数为0-15,R为SO3 -的个数为0时,意味着R全部为H。
3.如权利要求1所述的应用,其特征在于:式(I)中n=2,R’皆为H,R为的SO3 -个数为8-15。
4.如权利要求1所述的应用,其特征在于:所述治疗非酒精性脂肪肝药物为含有式(I)或(II)所示的甘露葡萄糖醛酸多糖或寡糖或硫酸化衍生物和药学可接受的载体和/或赋形剂的组合物。
5.如权利要求4所述的应用,其特征在于:所述药学可接受的载体为海藻酸钠微球或脂质体,所述赋形剂为甘露醇、硬脂酸镁、淀粉或环糊精。
6.如权利要求4所述的应用,其特征在于:所述治疗非酒精性脂肪肝药物的剂型为注射剂、口服制剂或局部给药制剂。
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