CN112316133A - 注射用银耳孢糖免疫佐剂的制备方法及其应用 - Google Patents
注射用银耳孢糖免疫佐剂的制备方法及其应用 Download PDFInfo
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- CN112316133A CN112316133A CN202011281304.1A CN202011281304A CN112316133A CN 112316133 A CN112316133 A CN 112316133A CN 202011281304 A CN202011281304 A CN 202011281304A CN 112316133 A CN112316133 A CN 112316133A
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- tremella polysaccharide
- injection
- polysaccharide
- vaccine
- tremella
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Abstract
本发明涉及一种注射用银耳孢糖及其提取纯化方法以及应用。所述注射用银耳孢糖单糖组成比例为甘露糖:葡萄糖醛酸:葡萄糖:木糖:岩藻糖=66.2:10.75:1.12:19.20:2.82。所述注射用银耳孢糖的提取纯化方法包括银耳孢糖以水溶解后,通过活性炭柱柱层析,醇沉,盐酸水解,氢氧化钠中和后再次以醇沉,制得注射用银耳孢糖。制备得到的注射用银耳孢糖具有分子量较小、纯度高、水溶性好的特点。本发明的注射用银耳孢糖具有良好的免疫增强活性,可以作为佐剂,明显提高多种疫苗的免疫原型,应用在多种疫苗的制备中。
Description
技术领域
本发明是关于注射用银耳多糖作为免疫佐剂在医药产品中的用途。属于生物药学技术领域。
背景技术
免疫佐剂为与抗原一起或预先注入机体时可有效增强免疫应答的强度或改变免疫应答的类型的非特异性免疫增强剂。常用的佐剂可分为4类:无机佐剂,如氢氧化铝,明矾等;有机佐剂;微生物及其产物如分枝杆菌(结核杆菌、卡介苗)、短小杆菌、百日咳杆菌、内毒素、细菌提取物(胞壁酰二肽)等;合成佐剂,如人工合成的双链多聚核苷酸(双链多聚腺苷酸、尿苷酸)等;近年来,由植物或微生物来源的多糖类成分也成为了研发新型佐剂的成分,如海带多糖。免疫佐剂的生物作用包括:改变了抗原的物理性状,可使抗原物质缓慢地释放,延长了抗原的作用时间;佐剂吸附了抗原后,增加了抗原的表面积,使抗原易于被巨噬细胞吞噬;佐剂能刺激吞噬细胞对抗原的处理;佐剂可促进淋巴细胞之间的接触,增强辅助T细胞的作用;可刺激致敏淋巴细胞的***和浆细胞产生抗体。故免疫佐剂的作用可使无免疫原性物质变成有效的免疫原。对新的免疫佐剂的筛选和开发,越来越引起人们的重视。
银耳(Tremella fueiformis Berk)又名白木耳,为担子菌纲银耳科银耳的干燥子实体,为常用保健药品,具有滋阴润肺的功效,已有几百年的药用历史。其主要化学成分为多糖类化合物。还含有脂类、蛋白类(酶、蛋白质、氨基酸)、无机盐、维生素B族等。银耳多糖主要为酸性杂多糖,是由分子量由几万到几十万的系列酸性杂多糖构成;其组成糖为木糖、葡萄糖、岩藻糖、甘露糖和葡萄糖醛酸;大量的化学和药理实验证明,银耳多糖具有明显的抗辐射,升高白细孢、降血糖、抗溃疡、增强免疫、抗肿瘤、对抗放化疗的毒副作用等活性;急性毒性和长期毒性研究表明,该多糖口服或注射给药均未见明显的毒副作用。银耳多糖易溶于热水,稍溶于低浓度的乙醇,不溶于高浓度的乙醇等有机溶剂,其水溶液成透明粘稠状。银耳多糖在体内的吸收、分布和代谢研究结果表明,其口服吸收利用率极低(>1%),肌肉注射和静脉给药时银耳多糖提高免疫功能、升白、抗癌的作用明显高于口服给药。由于其口服生物利用率低,作用不明显,作为新药研发时,应选择注射给药的途径。但由于银耳多糖分子量大,结构复杂,其水溶液还具有一定的粘稠性,影响它作为注射剂应用,也影响对其质量的评价、控制和大规模生产。因此对其进行降解,制备结构相对简单、分子量小、水溶性好的片段,从中筛选得到作用更强的活性部位,对其开发和应用具有重要的意义。
本发明完成前未发现有关注射用银耳多糖作为免疫佐剂的应用。
发明内容
本发明目的之一在于提供一种免疫佐剂,其是以天然多糖与流感疫苗配合使用,能够增强机体免疫功能。
本发明对银耳孢糖经分离纯化、降解后,制备了分子量较小、纯度高、收率高的注射用银耳孢糖。注射用银耳孢糖在提高机体的免疫功能等方面具有明显的作用;同时由于良好的水溶性,也可满足作为注射方式给药的要求,首次应用于免疫增强剂及疫苗的佐剂具有明显的技术创新。
优选地,所述疫苗为H5N1流感疫苗及狂犬疫苗。
本发明的有益效果在于:本发明以天然银耳多糖作为活性成分,与流感疫苗配合使用,在效果上起协同作用,可增强流感疫苗的活性,获得理想的免疫效果。
具体来说,采用本发明的免疫佐剂的流感疫苗在整个免疫周期内,相比于没有添加免疫佐剂的流感疫苗,其免疫表达的抗体滴度会显著提高。
本发明另一目的,提供注射用银耳孢糖的制备方法:
a.取银耳孢糖,加纯净水,40℃条件下搅拌8小时后,离心,收集离心液,通过颗粒活性碳柱,收集洗脱液,浓缩至每ml含0.3g银耳孢糖,放置至室温,搅拌条件下,缓缓加入1.6倍量95%乙醇,继续搅拌20分钟,离心,收集离心液,搅拌条件下继续加入10倍量95%乙醇,继续搅拌20分钟,离心,收集沉淀,自然干燥,得粗多糖;
b、取粗多糖粉末,加入20倍量盐酸水溶液,在90℃水浴中水解,水解结束取出放置至室温,滴加氢氧化钠水溶液调pH至中性,搅拌条件下,加入0.5倍量95%乙醇,继续搅拌2小时,离心,收集沉淀,加入10倍量注射用水溶解,用微孔滤膜滤过,冷冻干燥,得注射用银耳孢糖。
如上所述注射用银耳孢糖的制备方法,其特征在于,步骤a银耳孢糖与蒸馏水料液比为为1:20g/mL,搅拌时转速为500rpm。
如上所述注射用银耳孢糖的制备方法,其特征在于,步骤b盐酸水溶液浓度为0.1mol/L,水解时间为4-10小时。
为了实现以上发明内容,通过以下技术方案进行:
1、注射用银耳孢糖理化性质的测定
1.1中性糖含量测定---苯酚-硫酸法
取注射用银耳孢糖100mg,加水50ml溶解,取1ml,以中国药典2000版一部附录IX S测定,注射用银耳多糖以甘露糖计,不低于75%。
1.2糖醛酸含量测定---间-羟基联苯法
1.2.1对照品溶液的制备
精密称取105℃干燥至恒重的葡萄糖醛酸对照品5mg,置100ml容量瓶中,加水溶解并稀释至刻度,摇匀,即得(每1ml中含葡萄糖醛酸50μg)。
1.2.2标准曲线的制备
精密吸取对照品溶液0ml、0.1ml、0.2ml、0.3ml、0.4ml,分别加水补充体积至0.4ml混匀,在冰水浴中加入2.4ml 0.0125mol/L四硼酸钠—硫酸溶液(取四硼酸钠0.475克,加浓硫酸100ml溶解),混匀,在沸水浴中加热5分钟,取出并迅速冷却至室温,加入0.15%间羟基联苯40μl,立即混匀,以0管为空白,照分光光度法(中国药典2010年版一部附录V A),在525nm波长处20分钟内测定吸收度,以葡萄糖醛酸μg数对应的吸收度,计算回归方程。
1.2.3测定法
精密称取供试品3350mg,置100ml量瓶中,加水稀释至刻度,摇匀,作为供试品溶液,精密吸取供试品溶液0.2ml,加水补充体积至0.4ml,照标准曲线制备项下自“在冰水浴加入”起,依法操作测定吸收度,由回归方程计算酸性糖含量。
1.2.4糖醛酸含量测定结果
表1标准糖醛酸溶液吸收值
根据以上数据,以糖醛酸含量为横坐标,吸收值为纵坐标,求得回归方程为Y=0.0309X+0.0315,R=0.9985。
根据实验结果,注射用银耳孢糖含酸性糖以葡萄糖醛酸计,不低于10%。
1.3蛋白的测定
取注射用银耳孢糖100mg,加水50ml溶解,取1ml,以中国药典2000版一部附录IX S测定,符合规定。
2、注射用银耳孢糖组成糖分析
2.1标准单糖溶液制备
取Fuc(岩藻糖)、Rha(鼠李糖)、Ara(***糖)、Xyl(木糖)、Man(甘露糖)、Gal(半乳糖)、Glu(葡萄糖)、GalA(半乳糖醛酸)GlcA(葡萄糖醛酸)单糖对照品适量,分别加蒸馏水配成2mmol/L的单糖标准溶液。
2.2注射用银耳孢糖的水解液制备
精确称取注射用银耳孢糖样品20mg于具塞试管中,加入2mol/L三氟乙酸2ml,密封,100℃水解2小时,以氮气吹干。残渣加1ml甲醇,吹干,重复4次,除尽三氟乙酸,待衍生化。
2.3衍生物的制备
取已配制好的2mmol/L标准单糖溶液以及注射用银耳孢糖样品的水溶液各3ml,分别置于不同的具塞试管中,依次加入6ml的0.5mol/L的PMP甲醇溶液和0.3mol/L NaOH溶液,混匀,70℃水浴反应30min,取出并冷却5min,用5mL0.3mol/L HCl中和,加入等体积氯仿萃取,充分震荡,取水层,重复3次。取各PMP衍生化单糖300μL混合,过0.45μm微孔滤膜。
2.4HPLC分析
岛津LC-2010自动进样高效液相色谱仪,ZORBAX EclipseXDB-C18分析柱(Agilent)(Φ4.6mm×150mm);柱温:40℃;流动相:A:磷酸缓冲液(KH2PO4-NaOH,PH=6.8):乙睛(85:15,V/V);B:磷酸缓冲液(KH2PO4-NaOH,PH=6.8):乙睛(60:40,V/V);流速:0.9mL/min;检测波长:250nm。梯度洗脱,开始0~10分钟,流动相B比例由0增加至8%,10~30分钟,流动相B再由8%增加至20%,保持10分钟,结束。
注射用银耳孢糖的PMP衍生化-HPLC法分析结果表明,注射用银耳孢糖主要由甘露糖,葡萄糖醛酸,葡萄糖,木糖,岩藻糖组成;其摩尔比为:66.2:10.75:1.12:19.20:2.82。
2.4分子量分布的测定
注射用银耳孢糖样品,加流动相配成5mg/mL的溶液,按照中国药典进行测定。其分子量分布范围为2000-50000Da,重均分子量为5900Da。
3、注射用银耳孢糖作为佐剂对流感疫苗免疫效果评价
佐剂是疫苗中的免疫增强剂,它与抗原共同被免疫***识别加工,可增强或调解机体对抗原的免疫应答。理想的佐剂以较少的剂量就能高效的刺激机体产生免疫反应,且对机体无毒副作用,适合生产和应用。
3.1实验设计
通过大鼠注射流感疫苗后定期检测血清中产生的抗体滴度,考察25μg流感疫苗(H5N1)(HA)腹腔接种后的免疫原性;通过大鼠注射流感疫苗(HA)加不同的佐剂后,定期检测血清中产生的抗体滴度,考察不同佐剂、不同剂量对流感疫苗产生相应抗体的影响。通过免疫后定期检测实验组和对照组的小鼠体重来考察疫苗腹腔接种后对小鼠安全性的影响。此外,同时免疫不同剂量注射用银耳孢糖佐剂与原有铝佐剂进行比较,考察新型佐剂对大流感疫苗免疫原性影响。
3.2实验方法
3.2.1实验材料
流感疫苗(H5N1)(HA);铝佐剂:吉林省亚泰生物制药有限公司药研中心提供。实验动物:昆明种小鼠(雌性):吉林省亚泰生物制药有限公司动物室提供。注射用银耳孢糖:长春中医药大学研发中心提供。
3.2.2实验分组
表2实验分组
组别 | 分组 | 佐剂剂量 | HA含量 | 免疫数量 |
1 | 银耳孢糖+疫苗 | 500μg/剂 | 25μg/剂 | 10 |
2 | 银耳孢糖+疫苗 | 250μg/剂 | 25μg/剂 | 10 |
3 | 银耳孢糖+疫苗 | 125μg/剂 | 25μg/剂 | 10 |
4 | 银耳孢糖+半剂量疫苗 | 250μg/剂 | 12.5μg/剂 | 10 |
5 | 正常铝佐剂疫苗免疫 | 1000μg/剂 | 25μg/剂 | 10 |
6 | 佐剂空白对照 | 25μg/剂 | 10 |
3.2.3免疫程序
雌性昆明小鼠在第0,14天进行免疫,上述各组以生理盐水稀释后,每只腹腔注射1ml。免疫后的第35天采集静脉血,分离血清,2-8℃保存。
3.2.4检测项目
检测血凝抑制抗体滴度及每周定期监测小鼠体重。
3.3实验结果
3.3.1血凝抑制抗体滴度
表3注射用银耳孢糖血凝抑制抗体滴度实验结果
3.3.2体重
所有小鼠随机分成5组后,记录整个免疫过程中各组小鼠体重变化。各组小鼠体重随着时间均呈增加趋势。流感疫苗和流感疫苗联合佐剂免疫小鼠正常生长状态应不受影响。
3.4结果分析
试验结果表明,使用HA含量为25μg疫苗两次免疫小鼠,取血清检测HA抗体滴度,抗体滴度几何平均值(GMT)为1:40,25μg HA疫苗具有一定的免疫原性。使用1000μg/剂铝佐剂、HA含量25μg疫苗两次免疫小鼠,取血清检测HA抗体滴度,抗体滴度平均值(GMT)为1:171.48,阳转率100%,25μg HA疫苗加铝佐剂具有良好的免疫原性。
不同剂量银耳孢糖为佐剂免疫小鼠,均产生抗体。500μg银耳孢糖+25μgHA疫苗组、250μg银耳孢糖+25μgHA疫苗组、50μg银耳孢糖+25μgHA疫苗组、250μg银耳孢糖+12.5μgHA疫苗组抗体滴度几何平均值(GMT)分别为1:80、1:172.81、1:105.56、1:105.56,均具有明显促进抗体生成作用。其中高剂量、低剂量银耳孢糖组抗体生成的促进作用低于中剂量银耳孢糖组。
250μg银耳孢糖+25μgHA疫苗组抗体滴度与1000μg铝佐剂疫苗抗体滴度值相近。250μg银耳孢糖+25μgHA疫苗组与250μg银耳孢糖+12.5μgHA疫苗组比较,在同剂量银耳孢糖情况下,25μgHA疫苗组明显高于12.5μgHA疫苗组,有HA剂量依赖性。各剂量银耳孢糖佐剂疫苗均具有免疫原性,且250μg银耳孢糖+25μgHA疫苗组免疫原性最佳。各组小鼠体重随着时间均呈增加趋势,与正常对照组无明显区别,疫苗安全性良好。
从以上的药效学实验可知,注射用银耳多糖作为免疫佐剂具有明显促进抗体生成作用。
具体实施方式
本发明是通过如下的实验施例得以实现(证实)。
实例1注射用银耳孢糖的制备方法
取银耳孢糖1000克,加纯净水配制成5%(W/V)的水溶液,低温(40℃)下搅拌(500rpm)8小时后,离心(1600rpm,10分钟)。收集离心液,通过颗粒活性碳柱(80cmx10cm),收集洗脱液。浓缩至3升,放置至室温,搅拌下(500rpm),缓缓加入95%乙醇5升,继续搅拌20分钟,离心(1800rpm,6分钟),收集离心液,搅拌下(500rpm)继续加入95%乙醇30升,搅拌20分钟,离心(2000rpm,10分钟),收集沉淀,自然干燥,得粗多糖约100克。取粗多糖粉末,加入0.1mol/L HCL水溶液2.4升,在90℃水浴中搅拌5小时,取出放置至室温,滴加30%NaOH水溶液调多糖酸水解液pH至中性。搅拌下(500rpm)加入95%乙醇12升,继续搅拌2小时,离心(2500rpm,10分钟),收集沉淀,加入1000ml注射用水溶解,滤过(0.40um滤膜),冷冻干燥,得注射用银耳孢糖50克。
实例2注射用银耳孢糖注射液的制备方法
在无菌条件下,注射用银耳孢糖加入重蒸馏水,制成5%的水溶液,通过0.24μm的微孔滤膜除菌,放置备用。
实例3含注射用银耳孢糖为佐剂的流感疫苗注射液的制备方法
在无菌的条件下,与流感疫苗的浓备液混合,制成注射用银耳孢糖与流感疫苗为10:1的溶液,根据需要分装成不同的计量,制得含注射用银耳孢糖为佐剂的流感疫苗注射液。
实例4含注射用银耳孢糖为佐剂的狂犬疫苗冻干粉的制备方法
在无菌的条件下,与狂犬疫苗的浓备液混合为10:1的溶液,根据需要,分装成不同的剂量,冷冻干燥,制成银耳孢糖为佐剂的狂犬疫苗冻干粉针。
Claims (9)
1.一种注射用银耳孢糖,其特征在于,单糖组成及摩尔比为甘露糖:葡萄糖醛酸:葡萄糖:木糖:岩藻糖=66.2:10.75:1.12:19.20:2.82;其分子量分布范围为2000-50000Da,重均分子量为5900Da。
2.根据权利要求1所述注射用银耳孢糖,其特征在于,含多糖以甘露糖计,不低于75%;含酸性糖以葡萄糖醛酸计,不低于10%。
3.权利要求1所述注射用银耳孢糖的制备方法,其特征在于,包括以下步骤:
a、取银耳孢糖,加纯净水,40℃条件下搅拌8小时后,离心,收集离心液,通过颗粒活性碳柱,收集洗脱液,浓缩至每ml含0.3g银耳孢糖,放置至室温,搅拌条件下,缓缓加入1.6倍量95%乙醇,继续搅拌20分钟,离心,收集离心液,搅拌条件下继续加入10倍量95%乙醇,继续搅拌20分钟,离心,收集沉淀,自然干燥,得粗多糖;
b、取粗多糖粉末,加入20倍量盐酸水溶液,在90℃水浴中水解,水解结束取出放置至室温,滴加氢氧化钠水溶液调pH至中性,搅拌条件下,加入0.5倍量95%乙醇,继续搅拌2小时,离心,收集沉淀,加入10倍量注射用水溶解,用微孔滤膜滤过,冷冻干燥,得注射用银耳孢糖。
4.根据权利要求3所述注射用银耳孢糖的制备方法,其特征在于,步骤a银耳孢糖与蒸馏水料液比为为1:20g/mL,搅拌时转速为500rpm。
5.根据权利要求3所述注射用银耳孢糖的制备方法,其特征在于,步骤b盐酸水溶液浓度为0.1mol/L,水解时间为4-10小时。
6.根据权利要求1所述的注射用银耳孢糖的应用,其特征在于,在制备免疫增强剂的应用。
7.根据权利要求1所述的注射用银耳孢糖的应用,其特征在于,在制备疫苗佐剂中的应用。
8.根据权利要求7所述的疫苗佐剂,其特征在于:所述疫苗为H5N1流感疫苗。
9.根据权利要求7所述的疫苗佐剂,其特征在于:所述疫苗为狂犬疫苗。
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