CN117362468A - 一种鱼鳔硫酸软骨素-硫酸皮肤素杂合链及其制备方法与应用 - Google Patents
一种鱼鳔硫酸软骨素-硫酸皮肤素杂合链及其制备方法与应用 Download PDFInfo
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- CN117362468A CN117362468A CN202311468537.6A CN202311468537A CN117362468A CN 117362468 A CN117362468 A CN 117362468A CN 202311468537 A CN202311468537 A CN 202311468537A CN 117362468 A CN117362468 A CN 117362468A
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Abstract
本发明公开了一种鱼鳔硫酸软骨素‑硫酸皮肤素杂合链及其制备方法与应用,涉及医药技术领域。具有抗凝血活性的鱼鳔硫酸软骨素(CS)/硫酸皮肤素(DS)杂合链由艾杜糖醛酸、N‑乙酰氨基半乳糖、葡萄糖醛酸及N‑乙酰氨基葡萄糖四种单糖组成,CS/DS杂合链来源于鱼鳔,易溶于水,具有较强的抗凝血活性,可用于制备预防和/或治疗血栓性心血管疾病的药物。
Description
技术领域
本发明涉及医药技术领域,具体涉及一种鱼鳔硫酸软骨素-硫酸皮肤素杂合链及其制备方法与应用。
背景技术
血栓性心血管疾病严重危害人类健康,是人类致死致残的首位病因。抗凝血抗血栓药物在预防和治疗静脉/动脉血栓等相关疾病发挥重要作用。肝素类药物是临床应用最早及最为广泛的抗凝血抗血栓药物,占有巨大的市场份额,目前全球销售额上百亿美元,其中肝素品种之一赛诺菲的依诺肝素钠克赛在2022年全球化药销售Top100里排名66位。然而,现有临床应用的抗凝药物均具有严重的出血倾向等副作用。如肝素类药物除严重的出血倾向副作用外,还具有引起血小板聚集、过敏反应等副作用,从猪、牛、羊等器官制备时易受病毒等病原体污染等问题。从海洋生物如海藻、海参、鱼鳔等发掘高效低毒的抗凝药物仍是抗凝新药研发的热点。
鱼鳔是鱼类重要器官,在我国食用历史悠久,最早可追溯到北魏《齐民要术》。此外,鱼鳔还具有多种药用价值,《本草纲目》记载:鳔,止折伤血出不止;鳔胶,治妇人难产,破伤风痉、止呕血,散瘀血,消肿毒。《海药本草》、《本草新编》等对其药用价值均有记载。其药用价值也得到了现代中医的认可,具有补精益血、止血、消肿等功效,2004年被国家中医药管理局推荐为重点发展的中药材之一。一般来说,绝大多数动物均含有糖胺聚糖类物质,具有广泛的生理功效和药理活性。然而,鱼鳔来源糖胺聚糖研究较少,仅见广东海洋大学课题组的报道,该课题组从鱼鳔中提取得到糖胺聚糖类物质,发现具有抗血管生成、抗凝血、抗氧化、抗炎、吸湿保湿等活性及性能(屈义等.《食品工业科技》,2017,16:118-125;周斯仪等.《食品与机械》,2018,34(7):151-219;屈义等.《广东海洋大学学报》,2018,38(1):47-53)。该系列研究选取的材料为鲈鱼(Lateolabrax japonicus)或鳙鱼(Aristichthysnobilis),所得糖胺聚糖的结构解析确定为鱼鳔硫酸软骨素A(CSA)(周斯仪等.《食品科学》,2019,40(15):84-91;Chen et al.Food Research International,2022,157:111444)。
本发明人前期研究发现某些物种鱼鳔中糖胺聚糖为CS/DS杂合链,且以DS二糖结构单元为主,与以往报道迥异;该CS/DS杂合链具有强效的抗凝血活性、能够抑制内源性因子X酶(FXase)和作用于肝素辅因子II(HC-II)抑制凝血酶。本发明所述的CS/DS杂合链有望成为新型的FXase和HC-II抑制剂,用于血栓性心血管疾病的治疗中。
发明内容
本发明的目的是提供一种结构新颖的鱼鳔CS/DS杂合链及其制备方法与应用,制备的鱼鳔CS/DS杂合链化合物可作为潜在的FXase和HC-II抑制剂用于制备治疗血栓性心血管疾病的药物。
为实现上述目的,本发明提供以下技术方案:
本发明提供了一种具有抗凝血活性的鱼鳔硫酸软骨素-硫酸皮肤素杂合链,所述鱼鳔硫酸软骨素-硫酸皮肤素杂合链由艾杜糖醛酸、N-乙酰氨基半乳糖、葡萄糖醛酸及N-乙酰氨基葡萄糖四种单糖组成;鱼鳔硫酸软骨素-硫酸皮肤素杂合链的结构(I)如下:
其中,[→4)-C-α-(1→3)-D-β-(1→]m二糖单元占总二糖含量的50%~95%,即主要由硫酸皮肤素(DS)的二糖结构单元组成。另外,含量较高的二糖结构单元[→4)-A-α-(1→3)-E-β-(1→]n或[→4)-I-β-(1→3)-G-β-(1→]r分别占总二糖含量的5%~20%和1%~30%。
进一步地,鱼鳔硫酸软骨素-硫酸皮肤素杂合链是从鱼鳔中提取、分离纯化获得的,以右旋糖酐标定的重均分子量为80~150kDa,糖醛酸含量为20%~40%,硫酸酯基含量为15%~40%,其中蛋白质含量<3%,核酸含量<1%。
本发明还提供了鱼鳔硫酸软骨素-硫酸皮肤素杂合链的制备方法,包括以下步骤:
S1、鱼鳔采用常规的酶解碱解提取:将鱼鳔匀浆液或干粉加入蛋白酶中酶解,酶解的混悬液加入终浓度为0.2~1.0M的无机碱中碱解;
S2、等电点除蛋白、盐析醇沉得到鱼鳔粗多糖提取物:提取物采用阴离子交换柱层析纯化,收集0.5~2.0M NaCl溶液洗脱组分,或采用季铵盐沉淀法沉淀解离,反复醇沉、浓缩、冻干,获得鱼鳔硫酸软骨素-硫酸皮肤素杂合链。
进一步地,所述鱼鳔的来源包括大西洋鳕鱼(Gadus morhua)、斑条魣(Sphyraenajello)。
目前仅在大西洋鳕鱼(Gadus morhua)、斑条魣(Sphyraena jello)来源鱼鳔中发现含有本发明定义的鱼鳔CS/DS杂合链,但全球约有数万种鱼类。尽管本发明人开展鱼鳔CS/DS杂合链分离纯化的鱼鳔物种较少,但含有本发明定义的鱼鳔CS/DS杂合链的鱼鳔来源的鱼类物种并不限于大西洋鳕鱼和斑条魣。本领域技术人员可以理解,对于符合本发明定义的鱼鳔CS/DS杂合链,即使来源于其他物种的鱼,也可用于制备本发明所述结构组成的鱼鳔CS/DS杂合链及其药学上可接受的盐。
进一步地,所述蛋白酶包括木瓜蛋白酶、胰蛋白酶、碱性蛋白酶;所述无机碱包括NaOH、KOH。
本发明还提供了一种治疗和/或预防血栓性心血管疾病的药物,所述药物的有效成分含有上述的鱼鳔硫酸软骨素-硫酸皮肤素杂合链。
进一步地,所述药物剂型为注射用冻干粉针剂或注射用水溶液。
血栓性心血管疾病是人类致死致残的首位病因,造成巨大的社会经济负担。目前临床应用及在研的抗血栓药物包括三大类如溶栓药、抗凝药和抗血小板药,但临床现有抗凝药物均具有严重的出血倾向等副作用。临床上仍迫切需要更加安全有效的抗血栓药物。
为此,本发明所述的鱼鳔CS/DS杂合链具有良好的抗凝血活性,能够强效地抑制内源性FXase及作用于HCII。一般来说,作用于这两个靶点具有低出血倾向的优点。因此,本发明所述的鱼鳔CS/DS杂合链可能具有较低的毒副作用,其具有临床预防和治疗血栓性心血管疾病的应用价值。
本发明提供一种治疗血栓性心血管疾病的药物,含有有效剂量的所述鱼鳔CS/DS杂合链及其药学上可接受的盐,所述血栓性心血管疾病包括但不限于血栓形成性心脑血管疾病、深静脉血栓、肺静脉血栓、周围静脉血栓、周围性动脉血栓等。
本发明采用上述具有抗凝血活性的鱼鳔CS/DS杂合链及其制备方法与应用,具有如下有益技术效果:
1、本发明制备的鱼鳔CS/DS杂合链为结构组成新颖的糖胺聚糖,以右旋糖酐标定的重均分子量为80~150kDa,糖醛酸含量为20%~40%,硫酸酯基含量为15%~30%。由艾杜糖醛酸、N-乙酰氨基半乳糖、葡萄糖醛酸及N-乙酰氨基葡萄糖组成,进一步地,主要由硫酸皮肤素(DS)的二糖结构单元组成。其结构组成迥异于已公开制备及文献报道的鱼鳔多糖(硫酸软骨素A)。
2、本发明中的一种鱼鳔CS/DS杂合链可显著延长APTT,抑制FXase及依赖HC-II抑制凝血酶,为开发为新型抗凝血药物及功能性食品提供更多的选择。
下面通过附图和实施例,对本发明的技术方案做进一步的详细描述。
附图说明
图1为本发明实施例中大西洋鳕鱼、斑条魣鱼鳔CS/DS杂合链的高效液相色谱图;
图2为本发明实施例中大西洋鳕鱼、斑条魣鱼鳔CS/DS杂合链的单糖组成分析色谱图;
图3为本发明实施例中大西洋鳕鱼、斑条魣鱼鳔CS/DS杂合链的红外光谱图;
图4为本发明实施例中大西洋鳕鱼、斑条魣鱼鳔CS/DS杂合链的二糖组成分析图;
图5为本发明实施例中大西洋鳕鱼鱼鳔CS/DS杂合链的1H NMR谱图及归属;
图6为本发明实施例中大西洋鳕鱼鱼鳔CS/DS杂合链的13C NMR谱图及归属;
图7为本发明实施例中大西洋鳕鱼鱼鳔CS/DS杂合链的1H-13C HSQC谱图及归属;
图8为本发明实施例中斑条魣鱼鳔CS/DS杂合链的1H NMR谱图及归属;
图9为本发明实施例中斑条魣鱼鳔CS/DS杂合链的13C NMR和DEPT135谱图及归属;
图10为本发明实施例中斑条魣鱼鳔CS/DS杂合链的1H-13CHSQC-TOCSY谱图及归属;
图11为本发明实施例中斑条魣鱼鳔CS/DS杂合链的1H-1H ROESY谱图及归属。
具体实施方式
以下通过附图和实施例对本发明的技术方案作进一步说明。此处所描述的具体实施例和附图仅仅用于本发明的深入解析,并非用于限定本发明。
除非另外定义,本发明使用的技术术语或者科学术语应当为本发明所属领域内具有一般技能的人士所理解的通常意义。
实施例1:鱼鳔CS/DS杂合链的提取与纯化
提取:将干燥的大西洋鳕鱼、斑条魣鱼鳔粉碎,称取500g鱼鳔干粉置双层玻璃反应釜中,加入10倍(g/mL)量的纯水5L,加入终浓度为1%的木瓜蛋白酶55g,55℃搅拌反应16h。酶解结束后,继续升温至60℃,并缓慢加入6M NaOH至终浓度为0.5M,继续搅拌反应2h。反应结束后冷却至室温,缓慢加入6M HCl调pH至2~3,4℃放置4h,4500rpm离心15min,取上清液用6M NaOH调pH至中性,盐析醇沉。4500rpm离心15min得沉淀,加入一定量纯水溶解沉淀,再次醇沉,重复数次后,浓缩、冻干后得到鱼鳔粗多糖。
分离纯化:取鱼鳔粗多糖5g加纯水60mL溶解,上样于Amberliter FPA98Cl强阴离子交换柱(45mm×500mm)进行分离,分别以纯水、0.5、1.0、2.0、3.0M NaCl梯度洗脱,苯酚硫酸法检测,将收集的1.0M NaCl组分浓缩、反复醇沉、冷冻干燥,即得到纯化的鱼鳔CS/DS杂合链。
结果:大西洋鳕鱼、斑条魣鱼鳔经酶解碱解、去蛋白、醇沉得到粗多糖,以原料干重计,得率分别为0.35%和0.24%。经离子交换柱分离纯化得到的鱼鳔CS/DS杂合链易溶于水,难溶于乙醇、丙醇、乙酸乙酯等有机溶剂。
实施例2:鱼鳔CS/DS杂合链理化性质测定
采用间羟联苯法,以GalA为标准品测定鱼鳔CS/DS杂合链的糖醛酸含量。
采用考马斯亮蓝法,以BSA为标准品测定鱼鳔CS/DS杂合链样品中蛋白质含量。
采用氯化钡明胶法,以K2SO4为标准品测定鱼鳔CS/DS杂合链样品硫酸酯基含量。
采用电导率法测定硫酸羧基摩尔比。
高效液相色谱法测定鱼鳔CS/DS杂合链样品的分子量。仪器为LC-2030C3D HPLC,色谱柱为Shodex OHpak SB-804HQ(7μm,8×300mm),流动相为0.1M NaCl溶液,流速为0.5mL/min,柱温为35℃,进样量为20μL。
采用柱前衍生结合高效液相色谱法测定鱼鳔CS/DS杂合链的单糖组成。仪器为LC-2030C 3D HPLC,色谱柱为Agilent ZORBAX Eclipse Plus C18色谱柱(4.6×250mm,5μm),流动相为乙腈和pH为6.7的0.1M磷酸盐缓冲液以体积比17:83混合,流速为1.0mL/min,检测器为DAD,检测波长为245nm,柱温为30℃。
采用红外光谱法初步分析多糖样品的官能团等。分别取1-2mg的鱼鳔多糖及KBr粉末充分干燥,混合,红外灯下研磨,压片机压成透明薄片,取薄片置于Nicolet iS50 FT-IR红外光谱仪中,在4000~400cm-1范围内进行扫描。
结果:纯化得到的大西洋鳕鱼鱼鳔CS/DS杂合链(GMG-1.0)、斑条魣鱼鳔CS/DS杂合链(SJG-1.0)经HPLC分析均为单一色谱峰,峰值分子量计算分别为123.13kDa和109.32kDa(图1);间羟联苯法测得GMG-1.0和SJG-1.0的糖醛酸含量分别为29.49%和35.04%;氯化钡明胶法测得的GMG-1.0和SJG-1.0硫酸酯基含量分别为23.04%和35.04%;电导率法测得GMG-1.0和SJG-1.0硫酸羧酸摩尔比为1.56和1.16,由此计算的硫酸酯基含量分别为22.4%和28.8%;未检测到蛋白质。
单糖组成测定结果如图2所示。于标准单糖出峰时间比对,大西洋鳕鱼、斑条魣鱼鳔CS/DS杂合链中均含有艾杜糖醛酸(IdoA)、葡萄糖醛酸(GlcA)、N-乙酰氨基半乳糖(GalNAc)、N-乙酰氨基葡萄糖(GlcNAc),根据标准曲线,计算得到其单糖组成摩尔比IdoA:GalNAc:GlcA:GlcNAc分别为36.43:26.28:3.95:1.00和19.69:22.76:12.48:1.00。
大西洋鳕鱼、斑条魣鱼鳔CS/DS杂合链的红外光谱如图3所示,在3435cm-1处出现一强吸收峰,是O-H的伸缩振动;吸收带在2943cm-1是由C-H的伸缩振动引起的吸收峰;1643cm-1及1418cm-1的吸收峰是由C=O不对称伸缩振动及C-H的弯曲振动产生;在1260cm-1及843cm-1附近有明显的吸收峰,为硫酸盐S=O的拉伸振动和C-O-S的弯曲振动。
实施例3:鱼鳔CS/DS杂合链的结构解析
采用酶解及二糖组成分析测定鱼鳔CS/DS杂合链的二糖组成。称取2.5mg鱼鳔CS/DS杂合链样品,加纯水0.25mL,配制成10mg/mL样品溶液,取100μL至2mL离心管中,依次加入800μL三羟甲基氨基甲烷缓冲液及100μL(1mU/μL)软骨素酶ABC,37℃水浴酶解1h。反应结束后,沸水浴5min灭酶活,离心取上清得到酶解的二糖样品,过0.22μm滤膜,HPLC分析。色谱柱:WelchXB-SAX(4.6×250mm,3μm)强阴离子交换柱;流动相:A:2mM磷酸二氢钠(pH 3.0),D:含1.2M高氯酸钠的2mM磷酸二氢钠(pH3.0);柱温:40℃;流速:0.6mL/min;检测波长:232nm。
采用核磁共振仪测定鱼鳔CS/DS杂合链的确切结构。取鱼鳔CS/DS杂合链溶解于D2O,反复冻干3次,将充分干燥的鱼鳔多糖样品溶解于0.5mL D2O中,采用一维NMR波谱(1H和13C)和二维NMR波谱(COSY、TOCSY、ROESY、HSQC、HSQC-TOCSY、HMBC)分析鱼鳔CS/DS杂合链的确切结构如各种糖残基的连接方式、硫酸酯基取代位点等。
结果:鱼鳔GMG-1.0经硫酸软骨素酶ABC酶解后进行分析如图4所示。对比二糖标准品可知,GMG-1.0中主要含ΔDi4S单元,还含有少量的ΔDi4,6diS、ΔDi2,4diS、ΔDi0S单元,此外还有极少量的ΔUA-2S、ΔDi2,6diS及ΔDi2,4,6triS单元。其中,主要的二糖组成为ΔDi4S,约占87.25%(表1)。
鱼鳔SJG-1.0经硫酸软骨素酶ABC酶解后进行二糖组成分析如图4及表1所示。SJG-1.0中主要含ΔDi4S,占78.61%,其次是ΔDi6S(8.96%)、ΔDi0S(7.56%)、ΔDi2,4diS(3.21%),还含极少量的ΔDi4,6diS和ΔDi2,6diS。
表1GMG-1.0及SJG-1.0二糖组成
大西洋鳕鱼、斑条魣鱼鳔CS/DS杂合链GMG-1.0和SJG-1.0的核磁图谱如图5-10所示。1D NMR所有信号可由其2D NMR如1H-1H COSY、TOCSY、ROESY、1H-13C HSQC、HSQC-TOCSY、HMBC等进行归属。归属结果见表2和表3。综合单糖组成分析、二糖组成分析及核磁图谱解析结果可知GMG-1.0和SJG-1.0的化学结构式如下所示。
表2.GMG-1.0核磁化学位移归属结果
注:加粗代表连接位点;下划线代表硫酸酯基连接位点。
表3.SJG-1.0核磁化学位移归属结果
注:加粗代表连接位点;下划线代表硫酸酯基连接位点。
实施例4:鱼鳔多糖的抗凝活性测定方法:
APTT测定:将比色杯37.0℃预热,分别加入待测样品或阴性对照Tris-HCl缓冲液5μL,正常凝血质控血浆45μL,37.0℃下孵育2min;然后加入37.0℃预热的APTT试剂50μL,37.0℃孵育3min,继续向反应液中加入37.0℃预热的0.02M CaCl2 50μL,开始计时,记录血液凝固时间。
PT测定:检测管37.0℃预热后加入取待测样品或Tris-HCl缓冲液5μL,正常凝血质控血浆45μL,37.0℃下孵育2min。PT试剂37.0℃预热,并取100μL加入反应液中,记录凝血时间。
TT测定:比色杯37.0℃预热,依次往预热的比色杯中加入待测样品溶液或Tris-HCl缓冲液10μL,正常凝血质控血浆90μL,37.0℃孵育2min后继续加入37.0℃预热的TT试剂50μL,记录凝血时间。
样品抑制内源性FXase活性检测:
样品抑制FXase的活性采用FVIII:C试剂盒进行测定。取96孔板,依次加入系列梯度浓度的样品溶液30μL、FVIII溶液(2IU/mL)30μL、R2溶液30μL,37.0℃振荡孵育2min;取R1溶液30μL加入至反应液,37.0℃振荡孵育1min;取37.0℃预热的R3溶液30μL加入至反应液,振荡混匀,连续读取405nm处吸光度。Tris-HCl缓冲液作为空白对照孔。
样品HC-II依赖的抗凝血酶活性检测:
取96孔板,加入梯度浓度的多糖样品溶液或阴性对照Tris-HCl缓冲液30μL,然后加入HC-II溶液30μL,酶标仪振荡混匀,并于37.0℃孵育1min;然后加入浓度为20NIH/mL的凝血酶溶液30μL,振荡混匀并于37.0℃孵育1min;最后加入37℃预热的浓度为2.5mg/mL的凝血酶底物溶液30μL,振荡混匀,每隔30s连续读取405nm的吸光度。以吸光度的变化速率(ΔOD405/min)表示凝血酶的活性。
抗凝血活性测定结果:
大西洋鳕鱼、斑条魣鱼鳔CS/DS杂合链均能够显著延长APTT和TT(表4),提示:该两种CS/DS杂合链均具有强效的抗凝血活性。SJG-1.0和GMG-1.0倍增APTT所需的药物浓度分别为0.586和0.226μM。SJG-1.0延长APTT的活性与低分子肝素(0.571μM)相近,而GMG-1.0延长APTT的活性强于低分子肝素。SJG-1.0不具有TT延长活性,而GMG-1.0的TT延长活性也显著低于低分子量肝素,提示CS/DS杂合链对共同凝血途径影响较弱。据以往报道,目前临床应用的抗凝药物均作用于共同凝血途径,影响止血功能,因此均具有严重的出血倾向。CS/DS杂合链对共同凝血途径影响较弱,其出血倾向副作用可能更低。GMG-1.0抑制FXase的EC50值为0.058μM,该活性虽然较低分子量肝素活性弱,但其仍表现出强效的FXase抑制活性。SJG-1.0和GMG-1.0均表现出强效的HC-II依赖的抗FIIa活性,EC50值分别为20.410和6.225nM。据以往研究,依赖HC-II抑制FIIa的抗凝药物具有低出血倾向的优点。因此,SJG-1.0和GMG-1.0有望开发为新型抗凝药物。
表4.大西洋鳕鱼、斑条魣鱼鳔CS/DS杂合链对APTT、TT、内源性FXase、因子Ⅱa的影响
Sample | APTT,μM | TT,μM | anti-tenase,μM | anti-IIa(by HC-II),nM |
LMWH | 0.571±0.003 | 0.658±0.043 | 0.010±0.001 | 4.547±0.245 |
SJG-1.0 | 0.586±0.001 | / | / | 20.410±5.357 |
GMG-1.0 | 0.226±0.016 | 12.035±0.404 | 0.058±0.015 | 6.225±0.657 |
注:“/”代表无活性。
实施例5:鱼鳔CS/DS杂合链钠盐、钾盐、钙盐及其冻干制品的制备:
①鱼鳔CS/DS杂合链钠盐、钾盐、钙盐制备:取SJG-1.0和GMG-1.0样品各5g,去离子水50mL溶解,样品溶液经732氢型强酸型阳离子交换树脂(5.0cm×40cm)交换为氢型,流速1mL/min,收集洗脱液,逐滴加入1M NaOH、KOH或Ca(OH)2至样品溶液pH为7.0。
②鱼鳔CS/DS杂合链冻干制品制备:称取处方量的鱼鳔CS/DS杂合链SJG-1.0和GMG-1.0钠盐样品各10g,加入注射用水200mL溶解,22μm的微孔滤膜抽滤除热源,再采用2mL的西林瓶分装,每瓶0.5mL,半压塞,置于真空冷冻干燥机中设置-40℃,保持3h,然后抽真空至25Pa,保持6h,再升温至10℃,继续抽真空,将真空度降至仪器最低值,保持24h冻干,压塞,轧盖。
因此,本发明采用上述的一种鱼鳔硫酸软骨素-硫酸皮肤素杂合链及其制备方法与应用,制备的鱼鳔CS/DS杂合链化合物可作为潜在的FXase和HC-II抑制剂用于制备治疗血栓性心血管疾病的药物。
最后应说明的是:以上实施例仅用以说明本发明的技术方案而非对其进行限制,尽管参照较佳实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对本发明的技术方案进行修改或者等同替换,而这些修改或者等同替换亦不能使修改后的技术方案脱离本发明技术方案的精神和范围。
Claims (7)
1.一种具有抗凝血活性的鱼鳔硫酸软骨素-硫酸皮肤素杂合链,其特征在于,所述鱼鳔硫酸软骨素-硫酸皮肤素杂合链由艾杜糖醛酸、N-乙酰氨基半乳糖、葡萄糖醛酸及N-乙酰氨基葡萄糖四种单糖组成;鱼鳔硫酸软骨素-硫酸皮肤素杂合链的结构如下:
其中,[→4)-C-α-(1→3)-D-β-(1→]m二糖单元占总二糖含量的50%~95%,[→4)-A-α-(1→3)-E-β-(1→]n二糖单元占总二糖含量的5%~20%,[→4)-I-β-(1→3)-G-β-(1→]r二糖单元占总二糖含量的1%~30%。
2.根据权利要求1所述的鱼鳔硫酸软骨素-硫酸皮肤素杂合链,其特征在于,其是从鱼鳔中提取、分离纯化获得的,以右旋糖酐标定的重均分子量为80~150kDa,糖醛酸含量为20%~40%,硫酸酯基含量为15%~40%,其中蛋白质含量<3%,核酸含量<1%。
3.权利要求1或2所述的鱼鳔硫酸软骨素-硫酸皮肤素杂合链的制备方法,其特征在于,包括以下步骤:
S1、鱼鳔采用酶解碱解提取:将鱼鳔匀浆液或干粉加入蛋白酶中酶解,酶解的混悬液加入终浓度为0.2~1.0M的无机碱中碱解;
S2、等电点除蛋白、盐析醇沉得到鱼鳔粗多糖提取物:提取物采用阴离子交换柱层析纯化,收集0.5~2.0M NaCl溶液洗脱组分,或采用季铵盐沉淀法沉淀解离,反复醇沉、浓缩、冻干,获得鱼鳔硫酸软骨素-硫酸皮肤素杂合链。
4.根据权利要求3所述的制备方法,其特征在于,所述鱼鳔的来源包括大西洋鳕鱼、斑条魣。
5.根据权利要求3所述的制备方法,其特征在于,所述蛋白酶包括木瓜蛋白酶、胰蛋白酶、碱性蛋白酶;所述无机碱包括NaOH、KOH。
6.一种治疗和/或预防血栓性心血管疾病的药物,其特征在于,所述药物的有效成分含有如权利要求1或2所述的鱼鳔硫酸软骨素-硫酸皮肤素杂合链。
7.根据权利要求6所述的药物,其特征在于,所述药物剂型为注射用冻干粉针剂或注射用水溶液。
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