CN113633658A - 硫酸葡半乳寡聚糖在制备抗凝血和/或抗血栓药物中的应用 - Google Patents
硫酸葡半乳寡聚糖在制备抗凝血和/或抗血栓药物中的应用 Download PDFInfo
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- CN113633658A CN113633658A CN202110779428.0A CN202110779428A CN113633658A CN 113633658 A CN113633658 A CN 113633658A CN 202110779428 A CN202110779428 A CN 202110779428A CN 113633658 A CN113633658 A CN 113633658A
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- Prior art keywords
- galacto
- oligosaccharide
- dextran sulfate
- sulfate
- dextran
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Abstract
Description
技术领域
本发明属于抗凝血和抗血栓药物领域,涉及一种硫酸葡半乳寡聚糖在制备抗凝血和/或抗血栓药物中的应用。
背景技术
凝血是指血液由流动的液体状态变成不能流动的凝胶状态的过程,其实质是呈水溶性的纤维蛋白原转变为水中不溶解的固态纤维蛋白的过程,该过程是在内源性或/和外源性凝血途径下产生凝血酶原激活物,在凝血因子的作用下产生凝血酶,最后在凝血酶的作用下使纤维蛋白原转变为纤维蛋白。
血栓是血流在心血管系统血管内面剥落处或修补处的表面所形成的小块。血栓的形成涉及血管系统局部血液凝结,经常导致严重的健康相关疾病,如心脏病发作和中风等。血栓形成的危险因素包括异常的高脂血症、高血糖和血浆纤维蛋白原升高。
肝素是应用最为广泛的抗凝血药物,在临床应用中已有90多年的历史。肝素是一种从猪、羊、牛等动物的肠粘膜或肝中提取的粘多糖,这在一定程度上造成了动物资源的浪费。肝素虽然能够起防治血栓的作用,但也会产生副作用,例如易引起自发性出血,表现为各种粘膜出血、关节出血、伤口出血等,在注射肝素之后,血液中的血小板含量会出现大量减少,长期使用会导致骨矿物质密度的降低,引起骨质疏松症,增加骨折风险。低分子量肝素由肝素通过控制解聚制备得到。相比于普通肝素,低分子量肝素的可利用性更高,半衰期更长,并且成为治疗深静脉血栓的首选抗凝剂。但是,低分子量肝素本质上还是来源于动物组织,而且它的抗凝作用不易逆转,导致了过量用药的出血的风险。超低分子量肝素主要是一类化学合成的类肝素,具有良好的药代动力学控制,没有病毒杂质污染。但是,这类药物化学合成复杂,成本非常昂贵,并且其抗凝作用也不易逆转。因此临床上迫切需要寻找与肝素相比副作用更小的新型抗血栓和抗凝血剂。
发明人的在先研究报道了一种葡半乳寡聚糖及其制备方法,其具有确定的分子量、低粘度和良好水溶性的特性,并指出该葡半乳寡聚糖作为益生元具有显著改善肠道微生物菌群的生物活性(Wang,L.,Cheng,R.,Sun,X.,et al.,Preparation and GutMicrobiota Modulatory Property of the Oligosaccharide Riclinoctaose,J.Agric.Food Chem.2021Mar 31;69(12):3667-3676.)。国际专利申请WO2019090203A1公开了一种具有抗炎活性的硫酸化硫酸乙酰肝素寡糖化合物,但是其并不具有抗凝血活性。上述结果表明寡聚糖硫酸化修饰之后并不必然具有抗凝血、抗血栓活性。
发明内容
本发明的目的在于提供一种硫酸葡半乳寡聚糖在制备抗凝血和/或抗血栓药物中的应用。
本发明的硫酸葡半乳寡聚糖具有良好的抑制凝血因子产生凝血酶的活性,可以作为抗凝血药物或者治疗或预防抗血栓药物。
本发明所述的抗凝血和/或抗血栓药物为常规的抗凝血和/或抗血栓药物,具体可以是治疗急性血栓栓塞性疾病的药物,或者治疗脑梗塞、劲动脉内粥样斑块的形成、肺梗塞、心肌梗死、脾梗塞、肠系膜静脉内血栓的形成、肠系膜动脉栓塞、静脉血栓栓塞性疾病以及下肢动脉闭塞硬化症的药物,或者治疗弥散性血管内凝血药物,或者体外抗凝剂,例如输血、体外循环血液透析、腹膜透析的血样标本等体外实验的抗凝剂。
本发明中,所述的硫酸葡半乳寡聚糖,其结构式如下所示:
其中,R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17、R18、R19、R20、R21、R22、R23和R24独立的为SO3 —或H,但不全为H。
本发明所述的硫酸葡半乳寡聚糖中,平均每个糖残基上的硫酸基个数为0.5~3个。平均每个糖残基上的硫酸基个数越多,对FXIa的抑制率也越高。在本发明具体实施方式中,平均每个糖残基上的硫酸基个数为0.5~2.5个,更优选为2.1~2.5个。
本发明所述的硫酸葡半乳寡聚糖含有8个糖单元,包括7个葡萄糖残基和1个半乳糖残基。第1个葡萄糖残基连接一个丙酮酸基团,第8个糖单元为半乳糖残基,7个葡萄糖残基(Glcp)和1个半乳糖残基(Galp)的具体连接方式如下:
D-Glcp(4,6-pyr)-β-(1→3)-D-Glcp-β-(1→3)-D-Glcp-β-(1→6)-D-Glcp-β-(1→6)-D-Glcp-β-(1→4)-D-Glcp-β-(1→4)-D-Glcp-β-(1→3)-D-Galp。
本发明还提供上述硫酸葡半乳寡聚糖的制备方法,利用硫酸化试剂对葡半乳寡聚糖进行硫酸化制得硫酸葡半乳寡聚糖。
本发明中,通过控制葡半乳寡聚糖与硫酸化试剂的比例、反应温度和反应时间可以控制葡半乳寡聚糖的硫酸化程度,即控制平均每个糖残基上的硫酸基个数,得到不同程度硫酸基取代的葡半乳寡聚糖。
本发明中,所述的硫酸化试剂为本领域常规使用硫酸化试剂,只要是能够对葡半乳寡聚糖进行硫酸化均可。在本发明具体实施方式中,采用的硫酸化试剂为吡啶与氯磺酸的混合物。
本发明具体实施方式中,采用的硫酸葡半乳寡聚糖的制备方法,具体步骤为:以吡啶与氯磺酸的混合物为硫酸化试剂,搅拌下在硫酸化试剂中加入葡半乳寡聚糖的二甲基亚砜(DMSO)溶液,50~120℃油浴中搅拌反应,反应结束后,反应液经后处理和纯化,得到硫酸葡半乳寡聚糖。
作为优选,所述的吡啶与氯磺酸的混合物中,吡啶和氯磺酸的体积比为3:1~8:1。
作为优选,葡半乳寡聚糖的二甲基亚砜溶液中,葡半乳寡聚糖与二甲基亚砜的比例为1:10~20,g:mL。
作为优选,葡半乳寡聚糖与硫酸化试剂的比例为1:20~1:60,g:mL。
作为优选,反应温度为60℃。
作为优选,反应时间为2~5小时。
作为优选,后处理和纯化方法如下:使用15%NaOH溶液将反应液pH调至中性,8000rpm、4℃离心去除不溶性杂质后,收集上清液使用500~1000Da透析袋透析,透析5天后,利用有机超滤膜浓缩脱盐,经过冻干或乙醇沉淀得到硫酸葡半乳寡聚糖。
与现有技术相比,本发明具有以下优点:
本发明首次发现硫酸葡半乳寡聚糖具有明显的体外抗凝血活性,且其能选择性地抑制凝血因子XIa(FXⅠa),出血风险小,适用于制备体外抗凝血药物;同时在小鼠模型中,硫酸葡半乳寡聚糖表现出抑制血栓形成的功能,可用于治疗或预防血栓形成。本发明的硫酸葡半乳寡聚糖安全无毒,药效作用强,在抗凝血和/或抗血栓药物中具有广阔的应用前景。
附图说明
图1为硫酸葡半乳寡聚糖对FXIa的抑制活性结果图,其中包括了葡半乳寡聚糖和硫酸葡半乳寡聚糖对FXIa的抑制活性。
图2为硫酸葡半乳寡聚糖对APTT的作用结果图,其中包括了三种不同浓度的硫酸葡半乳寡聚糖对血浆凝固时间的影响。
图3为硫酸葡半乳寡聚糖抑制FeCl3诱导的动脉血栓形成结果图,其中(a)组为注射生理盐水的小鼠的血栓形成情况,(b)组为注射0.5mg/mL硫酸葡半乳寡聚糖的小鼠的血栓形成情况,(c)组为注射1.0mg/mL硫酸葡半乳寡聚糖的小鼠的血栓形成情况。
图4为硫酸葡半乳寡聚糖对小鼠尾部出血时间的影响结果图,其中包括了两种不同浓度的硫酸葡半乳寡聚糖对小鼠尾尖的出血情况的影响。
具体实施方式
除非另有限定,本发明所用所有技术和科学术语都具有为本发明所属领域普通技术人员所普遍理解的含义。如无特殊说明,下述实施例中采用的试剂或材料可通过商业购买或参考现有方法合成。
本发明中的葡半乳寡聚糖由本领域已知的常规方法制备。具体地,可按照Wang,L.,Cheng,R.,Sun,X.等人,J.Agric.Food Chem.,69,pp.3667-3676(2021)中教导的方法制备。
本发明中,术语“和/或”是指单独或组合的意思。
下面结合实施例和附图对本发明做进一步详述。
实施例1
硫酸葡半乳寡聚糖的制备:
称取1.0g葡半乳寡聚糖原料,转移至锥形瓶中,加入10~20mL DMSO,搅拌溶解,得到葡半乳寡聚糖溶液。将吡啶与氯磺酸以体积比3:1的比例在三颈瓶中混合,在冰水浴中磁力搅拌30min,得到硫酸化试剂。在搅拌下,在30mL硫酸化试剂中逐渐加入上述葡半乳寡聚糖溶液,接通冷凝管并塞好另外两个瓶盖,将三颈瓶置于60℃油浴中搅拌反应3h。自然冷却至室温,反应停止。使用15%NaOH溶液将溶液pH调至中性,8000rpm、4℃离心去除不溶性杂质后,收集上清液使用500~1000Da透析袋透析,透析5天后,利用有机超滤膜浓缩脱盐,经过冻干或乙醇沉淀得到平均每个糖残基上的硫酸基个数为1.65个的硫酸葡半乳寡聚糖。
按上述制备方法,将葡半乳寡聚糖与硫酸化试剂分别以质量体积比1:20、1:30、1:45、1:60进行硫酸化反应,分别得到取代度(即平均每个糖残基上的硫酸基个数)为1.23、1.65、2.10、2.52的硫酸葡半乳寡聚糖。
实施例2
硫酸葡半乳寡聚糖的硫酸化取代度(Ds)的测定。
配制10mg/mL的硫酸葡半乳寡聚糖溶液,取200μL于玻璃瓶中,加入等体积的4MHCl,在100℃下完全酸水解8h,氮气吹干后,用100μL纯水重新溶解。配制0.5%明胶溶液,使用时加入0.5%BaCl2。取100μL硫酸葡半乳寡聚糖溶液和等体积的BaCl2-明胶溶液,在360nm处测定OD值。通过以下公式计算取代度:
硫酸化试剂的使用剂量、反应时间、反应温度等条件都会影响硫酸化的取代程度。硫酸化试剂使用剂量减少,硫酸化取代程度也会降低。
实施例3~6中如无特殊说明,采用的硫酸葡半乳寡聚糖均为平均每个糖残基上的硫酸基个数为1.65个的硫酸葡半乳寡聚糖。
实施例3
硫酸葡半乳寡聚糖对凝血因子FXIa抑制活性测定。
1、实验分组:
实验分为三组:第一组为阴性对照组(TBS-BSA 缓冲液)、葡半乳寡聚糖组和硫酸葡半乳寡聚糖组。
2、抑制活性的测定
将100μL HFXIa(人凝血因子XIa,1nM)与50μL硫酸葡半乳寡聚糖样品(0~500nM)于96孔板中混合均匀,37℃孵育1h,加入50μL生色底物(1mM),在405nm波长下检测1h,每分钟检测一次。葡半乳寡聚糖组为100μLHFXIa+50μL葡半乳寡聚糖样品+50μL底物;阴性对照组为100μLHFXIa+50μL TBS-BSA缓冲液+50μL底物。
3、数据分析
使用软件Graphpad Prism 6.0进行数据处理。以反应时间为横坐标,405nm处的吸光度值为纵坐标进行回归分析。
采用生色底物法检测硫酸葡半乳寡聚糖样品和葡半乳寡聚糖对FXIa的抑制活性。以曲线的斜率为酶促反应的速度V,阴性对照组反应速度V0,样品反应速度Vi,计算抑制率=(V0-Vi)/V0×100%。从图1可以看出,平均每个糖残基上的硫酸基个数为1.65个的硫酸葡半乳寡聚糖对FXIa的抑制率为57.6%,葡半乳寡聚糖对FXIa的抑制率为9.1%,表明硫酸葡半乳寡聚糖对FXIa具有抑制活性,而葡半乳寡聚糖对FXIa无抑制活性。通过控制葡半乳寡聚糖与硫酸化试剂的比例控制硫酸葡半乳寡聚糖的硫酸化程度,表2为不同硫酸化程度的硫酸葡半乳寡聚糖对FXIa的抑制活性,可以看出平均每个糖残基上的硫酸基个数越多,硫酸葡半乳寡聚糖对FXIa的抑制率也越高。
同样,采用生色底物法测定硫酸葡半乳寡聚糖对其他酶的抑制活性,判断硫酸葡半乳寡聚糖对FXIa是否具有选择特异性。结合表1表明,硫酸葡半乳寡聚糖对FXIa具有选择特异性,对其他凝血因子无抑制活性。
表1为硫酸葡半乳寡聚糖对不同凝血因子的抑制作用
表2为不同硫酸化程度的硫酸葡半乳寡聚糖对FXIa抑制率
实施例4
硫酸葡半乳寡聚糖对凝血时间的影响。
1、小鼠模型的建立:
实验动物为雄性C57/BL-6J小鼠,18~20g。在标准实验条件下饲养:12小时光照-12小时黑暗循环,自由摄取水和食物。对小鼠腹腔注射5%水合氯醛进行麻醉,确认完全麻醉且无眼睑反射后尾静脉给药。将小鼠随机分为三组,第一组为阴性对照组,尾静脉注射生理盐水;第二组为低剂量组,尾静脉注射0.25mg/mL硫酸葡半乳寡聚糖;第三组为中等剂量组,尾静脉注射0.5mg/mL硫酸葡半乳寡聚糖;第四组为高剂量组,尾静脉注射1.0mg/mL硫酸葡半乳寡聚糖。从小鼠的腹主动脉收集血液于用抗凝剂湿润的硅烷化管中,37℃保温。
2、ATPP的测定
活化部分凝血活酶时间(ATPP),是在受检血浆中加入活化的部分凝血活酶时间试剂和钙离子后,观察血浆凝固所需要的时间,主要反映内源性凝血系统状况。收集全血后适当稀释,1080rpm离心10min,去掉上层富血小板血浆。3000rpm离心5min,取上层贫血小板血浆(PPP)进行凝血时间测定。ATPP测定按照试剂盒说明书中的操作步骤进行,读取血凝仪上纤维蛋白形成时间。
3、数据分析
使用软件Graphpad Prism 6.0软件进行数据处理。以样品浓度为横坐标,血浆凝固时间倍数为纵坐标绘制APTT折线图。P<0.05为有显著性差异。
将生理盐水组的凝血时间记为1倍,计算不同浓度硫酸葡半乳寡聚糖的APTT相对生理盐水组的倍数。结合图2,可以看出不同浓度的硫酸葡半乳寡聚糖对APTT产生明显影响,因此硫酸葡半乳寡聚糖作用于内源性凝血途径。
实施例5
硫酸葡半乳寡聚糖的体内抗血栓作用。
1、动物模型的建立:
实验动物为雄性C57/BL-6J小鼠,18~20g。在标准实验条件下饲养:12小时光照-12小时黑暗循环,自由摄取水和食物。对小鼠腹腔注射5%水合氯醛进行麻醉,确认完全麻醉且无眼睑反射后尾静脉给药。将小鼠随机分为三组,第一组为阴性对照组,尾静脉注射生理盐水;第二组为低剂量组,尾静脉注射0.5mg/mL硫酸葡半乳寡聚糖;第三组为高剂量组,尾静脉注射1.0mg/mL硫酸葡半乳寡聚糖。尾静脉给药后解剖小鼠颈动脉,将蘸有6.0%三氯化铁溶液的滤纸片(1×2mm)敷在小鼠颈动脉外壁上诱导3min,构建三氯化铁诱导颈动脉血栓形成模型。
2、血管堵塞时间测定
使用激光散斑血流成像仪观察小鼠颈动脉血流和血栓形成情况,并将小鼠颈动脉在moorFLPI-2镜头下观察15min。将结束观察的小鼠颈动脉小心剥离并进行HE染色。
3、数据分析
使用GraphPad Prism 6.0软件制作图表,统计学差异采用单因素方差分析和Dunnett′s多重比较。P<0.05为有显著性差异。
血管堵塞时间是指从移除浸有三氯化铁的滤纸片到血管堵塞的时间。如果在三氯化铁滤纸片移除后15min未观察到血栓形成,则将15min视为血管堵塞时间。结合图3,在阴性对照组(生理盐水组)中,平均血管堵塞时间为4.5±0.23min。样品硫酸葡半乳寡聚糖在0.5、1.0mg/mL的给药剂量下,平均血管堵塞时间分别为5.4±1.37min,15min。在1.0mg/mL的给药剂量下血流保持不变,表明硫酸葡半乳寡聚糖具有明显的抗血栓作用。
实施例6
硫酸葡半乳寡聚糖的出血风险的测定。
1、动物模型的建立:
实验动物为雄性C57/BL-6J小鼠,18~20g。在标准实验条件下饲养:12小时光照-12小时黑暗循环,自由摄取水和食物。对小鼠腹腔注射5%水合氯醛进行麻醉,确认完全麻醉且无眼睑反射后尾静脉给药。将小鼠随机分为三组,第一组为阴性对照组,尾静脉注射生理盐水;第二组为剂计量组,尾静脉注射0.5mg/mL硫酸葡半乳寡聚糖;第三组为高剂量组,尾静脉注射1.0mg/mL硫酸葡半乳寡聚糖。
2、尾尖出血时间测定:
给药10min后将小鼠平稳放置,其尾部置于自然伸直状态,在小鼠尾巴距离尖端3mm处切断,将小鼠尾部浸入装有37℃的生理盐水的硅烷化管中。从切断尾尖开始计时20min,另取秒表记录从切断小鼠尾尖开始后累积的出血时间t,若出血时间t大于20min则记录为20min。
3、数据分析
使用软件Graphpad Prism 6.0软件进行数据处理。以出血时间对样品浓度做点状图。P<0.05为有显著性差异。
出血时间是指毛细血管破损后从出血到自然止血所需的时间。在阴性对照组(生理盐水组)中,累积出血时间为10.35±5.48min。样品硫酸葡半乳寡聚糖在0.5、1.0mg/mL的给药剂量下,累积出血时间分别为11.88±4.44min,11.60±4.13min,在统计学上无显著差异(P>0.05)。结合图4,说明硫酸葡半乳寡聚糖具有显著的抗血栓活性同时无明显出血风险。
Claims (10)
2.根据权利要求1所述的应用,其特征在于,所述的抗血栓药物为治疗急性血栓栓塞性疾病的药物,或者治疗脑梗塞、劲动脉内粥样斑块的形成、肺梗塞、心肌梗死、脾梗塞、肠系膜静脉内血栓的形成、肠系膜动脉栓塞、静脉血栓栓塞性疾病或下肢动脉闭塞硬化症的药物;所述的抗凝血药物为治疗弥散性血管内凝血药物,或者输血、体外循环血液透析或腹膜透析的血样标本的抗凝剂。
3.根据权利要求1所述的应用,其特征在于,所述的硫酸葡半乳寡聚糖中,平均每个糖残基上的硫酸基个数为0.5~3个,优选为平均每个糖残基上的硫酸基个数为0.5~2.5个,更优选为2.1~2.5个。
4.根据权利要求1所述的应用,其特征在于,所述的硫酸葡半乳寡聚糖的制备方法为利用硫酸化试剂对葡半乳寡聚糖进行硫酸化制得硫酸葡半乳寡聚糖。
5.根据权利要求4所述的应用,其特征在于,所述的硫酸化试剂为吡啶与氯磺酸的混合物。
6.根据权利要求4所述的应用,其特征在于,所述的硫酸葡半乳寡聚糖的制备方法的具体步骤为:以吡啶与氯磺酸的混合物为硫酸化试剂,搅拌下在硫酸化试剂中加入葡半乳寡聚糖的二甲基亚砜溶液,50~120℃油浴中搅拌反应,反应结束后,反应液经后处理和纯化,得到硫酸葡半乳寡聚糖。
7.根据权利要求6所述的应用,其特征在于,所述的吡啶与氯磺酸的混合物中,吡啶和氯磺酸的体积比为3:1~8:1,葡半乳寡聚糖的二甲基亚砜溶液中,葡半乳寡聚糖与二甲基亚砜的比例为1:10~20,g:mL。
8.根据权利要求6所述的应用,其特征在于,葡半乳寡聚糖与硫酸化试剂的比例为1:20~1:60,g:mL。
9.根据权利要求6所述的应用,其特征在于,反应温度为60℃,反应时间为2~5小时。
10.根据权利要求6所述的应用,其特征在于,后处理和纯化方法如下:使用15%NaOH溶液将反应液pH调至中性,8000rpm、4℃离心去除不溶性杂质后,收集上清液使用500~1000Da透析袋透析,透析5天后,利用有机超滤膜浓缩脱盐,经过冻干或乙醇沉淀得到硫酸葡半乳寡聚糖。
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