CN117024626A - 一类新型α-葡聚糖及其用途 - Google Patents
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- CN117024626A CN117024626A CN202311014199.9A CN202311014199A CN117024626A CN 117024626 A CN117024626 A CN 117024626A CN 202311014199 A CN202311014199 A CN 202311014199A CN 117024626 A CN117024626 A CN 117024626A
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Abstract
本发明提供了一类新型α‑葡聚糖及其用途。所述α‑葡聚糖是同源系列多糖,具有相同的α‑1,4‑葡聚糖主链,不同的分支度、葡萄糖和葡萄糖醛酸侧链。葡萄糖醛酸侧链是所述α‑葡聚糖特有的结构片段,也对其生物活性有重要贡献。本发明的α‑葡聚糖来源丰富、结构稳定、纯度高,具有显著的免疫调节和抗肿瘤活性,有望开发为改善或治疗免疫相关机体功能障碍和/或抗肿瘤的药物。
Description
技术领域
本发明属于医药领域,具体涉及一类新型α-葡聚糖及其用途。
背景技术
多糖尤其是葡聚糖是一类典型的免疫反应修饰剂和调节剂,其重复的寡糖结构可以与免疫细胞膜受体多效价结合,激活固有免疫和适应性免疫系统,清除进入机体的异物、杀灭病原菌和肿瘤细胞。
病原菌细胞膜外层含有的β-1,3-葡聚糖,是高度保守的病原体相关分子模式(PAMP),动物体内因缺乏这种结构而在免疫细胞膜上进化出特异性模式识别受体,能够识别PAMP从而激活免疫系统对其进行杀灭和清除。因此,从香菇、茯苓、裂褶菌等得到的β-1,3-葡聚糖具有显著的免疫原性,能上调免疫系统功能,发挥抗肿瘤作用。然而,这类外源性结构的多糖易触发免疫应答,同时也增加了网状内皮系统和生物黏附引起的滞留效应,容易被机体清除,而影响其效用强度和持续时间。
研究表明,一些病原菌的最外层有α-1,3-葡聚糖,因其结构与内源性糖原相似,均属于α-葡聚糖,具有很低的免疫原性,通过包裹PAMP协助病原菌逃避免疫识别,增强致病性。因此,α-葡聚糖一直被认为无生物活性。还有研究表明,一些分枝杆菌中如结核杆菌,包裹细胞壁最外层的多糖是α-1,4-葡聚糖,与糖原的树状高分支结构不同的是,这类多糖结构的支化度低、有较长的线性糖链,多项研究表明该类多糖既能协助结核杆菌逃避免疫识别,也能激活树突状细胞而抑制结核杆菌,这表明该类多糖免疫原性的特异性。据报道,从冬虫夏草、草菇、五味子等得到的α-1,4-葡聚糖具有显著的巨噬细胞激活作用,他们也都有线性糖链,进一步证实α-1,4-葡聚糖具有生物活性,且与其精细结构密切相关。但植物主要含有β-葡聚糖和淀粉;菌菇来源的葡聚糖受培养条件和提取方法影响,其结构如分子量和分支度等差别很大,质量难以控制,这是多糖药物研究与应用亟待解决的问题。
发明内容
本发明的目的在于提供一类来源丰富、结构稳定、易于纯化、生物活性强的α-葡聚糖及其在制备改善或治疗免疫相关机体功能障碍或疾病的制剂中的用途。本发明所涉及的α-葡聚糖是从马粪海胆制备而得,其主链均是由α-葡萄糖通过(1→4)糖苷键聚合而成,在主链葡萄糖的6-位连有葡萄糖侧链和葡萄糖醛酸侧链,尤其是葡萄糖醛酸侧链是其它α-1,4-葡聚糖所不具备的特异性结构。该糖醛酸侧链不仅能增加本发明所涉及α-葡聚糖的水溶性,而且能显著增强其生物活性。
本发明的第一个方面,提供了一类新型α-葡聚糖,所述α-葡聚糖具有式Ⅰ所示的基本结构单元,
其中,n选自2.5、2、1.5、1,即所述α-葡聚糖的主链上每6个、5个、4个或每3个葡萄糖残基上连有1个侧链;R选自葡萄糖、葡萄糖醛酸、甘露糖、核糖、半乳糖残基。
优选地,所述α-葡聚糖的主链由葡萄糖通过α-1,4-糖苷键聚合而成,主链葡萄糖的6-位连有单糖侧链,R选自葡萄糖与葡萄糖醛酸,以及甘露糖、核糖、半乳糖的一种或几种。
优选地,所述α-葡聚糖的分子量为1.913×107~3.094×107Da,按照摩尔比计算,所述多糖结构中葡萄糖的含量为84.6~95.6%,葡萄糖醛酸的含量为4.4~8.0%,甘露糖的含量为0.4~4.6%,核糖的含量为0.1~2.3%,半乳糖的含量为0~1.2%。
更为优选地,所述α-葡聚糖,n选自2.5、2时,R选自葡萄糖和葡萄糖醛酸,按照摩尔比计算,葡萄糖的含量为95.6%,葡萄糖醛酸的含量为4.4%。
更为优选地,所述α-葡聚糖,n选自2、1.5时,R选自葡萄糖、葡萄糖醛酸、甘露糖、核糖,按照摩尔比计算,葡萄糖的含量为85.8~93.0%,葡萄糖醛酸的含量为4.5~8.0%,甘露糖的含量为0.4~4.6%,核糖的含量为0.1~2.1%。
更为优选地,所述α-葡聚糖,n选自1.5、1时,R选自葡萄糖、葡萄糖醛酸、甘露糖、核糖、半乳糖,按照摩尔比计算,葡萄糖的含量为84.6%,葡萄糖醛酸的含量为7.9%,甘露糖的含量为4.0%,核糖的含量为2.3%,半乳糖的含量为1.2%。
本发明的第二个方面,提供本发明第一个方面所述α-葡聚糖的一种或几种组合在制备改善或治疗免疫相关机体功能障碍或疾病的制剂中的用途。
优选地,所述一种或几种组合为任意一种α-葡聚糖单独使用,或任意几种α-葡聚糖组合使用。
优选地,所述免疫相关机体功能障碍为因先天不足、感染、营养不良或药物等引起的免疫功能缺陷、免疫力低下或免疫损伤。
优选地,所述免疫相关疾病为肿瘤。
优选地,所述制剂为功能性食品或药物。
附图说明
图1示出了总多糖的DEAE-52色谱柱洗脱曲线。
图2示出了总多糖的cellufine A-500色谱柱洗脱曲线。
图3示出了本发明的α-葡聚糖的高效凝胶渗透色谱分析图。
图4示出了本发明的α-葡聚糖的单糖组成。ManA是甘露糖醛酸,Man是甘露糖,Rib是核糖,Rha是鼠李糖,GlcA是葡萄糖醛酸,GalA是半乳糖醛酸,GlcNAc是乙酰氨基葡萄糖,Glc是葡萄糖,Gal是半乳糖,Xyl是木糖,Ara是阿拉伯糖,Fuc是岩藻糖。
图5示出了本发明的α-葡聚糖的甲基化分析结果。多糖的全甲基化产物于110℃水解3h。
图6示出了本发明的α-葡聚糖(HPP-6S和HPP-7S)的甲基化分析结果。多糖的全甲基化产物于110℃水解3h。
图7示出了本发明的α-葡聚糖的甲基化分析结果。多糖的全甲基化产物于120℃水解4h。
图8示出了本发明的α-葡聚糖的红外光谱图。
图9示出了本发明的α-葡聚糖的核磁共振氢谱和核磁共振碳谱图。
图10示出了本发明的α-葡聚糖对巨噬细胞生存率的影响。
图11示出了本发明的α-葡聚糖促巨噬细胞释放NO作用。
图12示出了本发明的α-葡聚糖激活巨噬细胞分泌TNF-α和IL-6。
图13示出了本发明的α-葡聚糖提高巨噬细胞吞噬能力。
图14示出了本发明的α-葡聚糖对分离自小鼠的脾淋巴细胞增殖的影响
图15示出了本发明的α-葡聚糖对环磷酰胺致免疫功能低下小鼠的脾脏的保护作用
图16示出了本发明的α-葡聚糖对环磷酰胺致免疫功能低下小鼠的胸腺的保护作用
图17示出了本发明的α-葡聚糖对环磷酰胺致免疫功能低下小鼠的外周血T淋巴细胞亚群CD4+/CD8+的调节作用;A.空白对照组,B.模型对照组,C.多糖低剂量组,D.多糖中剂量组,E.多糖高剂量组。
图18示出了本发明的α-葡聚糖提高环磷酰胺致免疫功能低下小鼠血清中IL-2和TNF-α表达量。
图19示出了本发明的α-葡聚糖提高H22荷瘤小鼠血清中IL-2和TNF-α的表达量。
图20示出了本发明的α-葡聚糖对小鼠体内S180肉瘤的抑制作用(A),小鼠脾脏图(B)和胸腺图(C)。
图21示出了本发明的α-葡聚糖提高荷S180肉瘤小鼠血清中IL-2的表达量。
图22示出了本发明的α-葡聚糖对小鼠体内B16黑色素瘤的抑制作用(A),小鼠脾脏图(B)和胸腺图(C)。
图23示出了本发明的α-葡聚糖提高荷B16黑色素瘤小鼠血清中IL-2和TNF-α的表达量。
具体实施方式
以下通过具体实施例对本发明的α-葡聚糖结构及其用途作进一步的说明,以下实例是用于阐释本发明的技术内容,使本发明的优点更加清晰,而不是对本发明内容的限定。所有基于下文公开内容的实质做出的变化或等同替换,均应属于本发明的保护范围。
实施例1α-葡聚糖的分离
马粪海胆生殖腺经匀浆机粉碎后,用无水乙醇浸泡脱脂,得到脱脂粉末。脱脂粉末用10倍量的水于50℃提取3次,提取液减压浓缩后用木瓜蛋白酶于55℃酶解、然后加热至90℃灭活,离心收集上清液。上清液用sevage试剂(氯仿︰正丁醇=4︰1)萃取脱蛋白。脱蛋白后的多糖水溶液用截留分子量6000Da的透析袋,用流动蒸馏水透析24h。向透析后的多糖水溶液中加入4倍量无水乙醇,离心收取沉淀,干燥后得海胆总多糖粉末,命名为HPP。
HPP用DEAE-52离子交换色谱柱分离,用水和梯度浓度的NaCl溶液洗脱,每10mL收集于1个试管中。如图1所示,用水洗脱得到HPP-1D,用1% NaCl溶液洗脱得到HPP-2D,用5%NaCl溶液洗脱得到HPP-3D。HPP-1D、HPP-2D、HPP-3D分别用Sepharose CL-2B琼脂糖凝胶柱色谱纯化,蒸馏水洗脱得到多糖纯品,减压浓缩、冷冻干燥后于-20℃储存,用于测试其巨噬细胞激活作用。
实施例1的分离结果表明,马粪海胆主要含有HPP-1D,而HPP-2D和HPP-3D含量较低,且HPP-2D和HPP-3D分布在多个试管中,说明HPP-2D和HPP-3D含有多种多糖、纯度较低。前期已分析出HPP-1D的结构,为α-1,4-葡聚糖,主链的每5个葡萄糖的6-位连有一个葡萄糖侧链或葡萄糖醛酸侧链。
实施例2本发明的同源系列α-葡聚糖的制备
本发明进一步优化了离子色谱纯化方法,改用离子交换树脂cellufine A-500做固定相,依次用水、0.017、0.034、0.051、0.068、0.085、0.103和0.171mol/L的NaCl溶液洗脱,分别得到HPP-1S、HPP-2S、HPP-3S、HPP-4S、HPP-5S、HPP-6S、HPP-7S和HPP-8S,分别用Sepharose CL-2B琼脂糖凝胶柱色谱纯化,蒸馏水洗脱得到高纯度多糖。
分离结果如图2所示。下文实施例3的结构表征证实HPP-1S结构与HPP-1D结构相同,且更准确的分析了侧链的分布。本发明优化后的方法将HPP-2D和HPP-3D分成7个高纯度的多糖即HPP-2S、HPP-3S、HPP-4S、HPP-5S、HPP-6S、HPP-7S和HPP-8S。
实施例3同源系列α-葡聚糖的结构表征
(1)测试多糖纯度和分子量
苯酚硫酸法测定HPP-1S、HPP-2S、HPP-3S、HPP-4S、HPP-5S、HPP-6S、HPP-7S和HPP-8S的糖含量分别为99.91%、99.84%、99.43%、98.21%、93.52%、94.08%、93.30%和93.82%。
每一个多糖用Bradford法均未检测到蛋白残留。
用高效凝胶渗透色谱分析,每个多糖均显示单个、对称色谱峰(图3),说明每个多糖分子量分布较窄、均一性良好。
基于HPP-1S(用多角度激光光散射仪确定分子量为2.996×107Da)和标准分子量右旋糖酐(T-20、T-50、T-1000和T-2000)绘制的分子量标准曲线(lgMw=-1.713T+18.38,R2=0.994)计算HPP-2S、HPP-3S、HPP-4S、HPP-5S、HPP-6S、HPP-7S和HPP-8S的分子量分别为2.153×107Da、2.928×107Da、2.311×107Da、1.913×107Da、2.975×107Da、3.094×107Da和2.987×107Da。
(2)分析单糖组成
HPP-1S~HPP-8S(5mg)用三氟乙酸水解,减压彻底蒸干。然后加入0.5mol/L的1-(3'-磺酸苯基)-3-甲基-5-吡唑啉酮(PMP)和0.3mol/L的NaOH,70℃反应1h后,用HCl中和到中性,再用二氯甲烷萃取除去多余的PMP,水层用高效液相色谱分析。检测器波长为245nm;柱温35℃;流动相为磷酸盐缓冲液(pH 6.7)/CH3CN(85:15,V:V)。
如图4所示,HPP-1S显示葡萄糖的信号,HPP-2S、HPP-3S、HPP-4S、HPP-5S、HPP-6S、HPP-7S显示葡萄糖、甘露糖、核糖的信号,HPP-8S显示葡萄糖、甘露糖、核糖和半乳糖的信号。而且通过峰面积可看出,甘露糖和核糖的含量在HPP-2S~HPP-7S中基本呈递增趋势。
根据离子色谱的分离原理,洗脱液盐浓度增加,依次将离子强度增加的多糖洗脱出柱,前期已确证HPP-1S含有糖醛酸,说明HPP-2S~HPP-7S的糖醛酸含量依次递增。液相色谱未显示糖醛酸的信号可能是高温下解离下来的糖醛酸被破坏,这也与前期HPP-1S经完全酸水解用高效阴离子交换色谱仪器分析未检测到糖醛酸信号的结果一致。
(3)甲基化分析
为了进一步分析糖醛酸的准确含量,将多糖HPP-2S~HPP-8S(4mg)用箱守法、以NaOH为催化剂、碘甲烷为甲基化试剂,进行甲基化,全甲基化产物用三氟乙酸于110℃水解3h,再用硼氢化钠还原、醋酐乙酰化得到部分甲基化醛糖醇乙酸酯(PMAA),最后用GC-MS分析。结果如图5所示,GC-MS总离子流图给出4个主要的、与HPP-1S相同的结构片段,分别是α-葡萄糖(1→(a)、α-葡萄糖醛酸(1→(b)、→4)-α-葡萄糖(1→(c);→4,6)-α-葡萄糖(1→(d)。不同点是,除这4个峰,还显示几个非单糖衍生化产物的杂质峰,而且末端糖的总和(片段a和片段b)明显高于分支点(片段d)的量,可能的原因是HPP-2S~HPP-8S的分支度增加,主链水解不够彻底,导致分支点的峰减小、并出现多个杂质峰。但在该条件下,侧链上葡萄糖(片段a)和葡萄糖醛酸(片段b)较完全解离下来,可观察到b/a(t-GlcA/t-Glc)的比例依次增加(HPP-1S的比例是2.5:1),这说明结构中的糖醛酸含量从HPP-1S~HPP-6S依次增加。HPP-7S和HPP-8S的b/a比例降低(图6)可能是实验误差引起的,推测其b/a比例高于HPP-6S的1.6:1。
为了进一步准确分析其分支度,将HPP-1S、HPP-2S、HPP-3S、HPP-4S、HPP-6S和HPP-8S的全甲基化产物用更高的温度(120℃)水解更长时间(4h),再用硼氢化钠还原、醋酐乙酰化得到PMAA,GC-MS分析。结果如图7所示,总离子流图只显示4个主要的峰(a、b、c、d),杂质峰消失。α-葡萄糖醛酸(1→(b)的峰显著降低,可能是因反应升温、加时后,部分糖醛酸被破坏,这与单糖分析结果相符。该条件给出了更准确的主链糖与分支点糖的比例,HPP-1S的c/d(1,4-Glc/1,4,6-Glc)比例为4.5:1(110℃和3h反应条件得到的比例是4:1),说明HPP-1S的主链上每5个或每6个糖上连有侧链。而且从HPP-2S到HPP-8S,分支点糖占比逐渐增加,说明分支度增加,HPP-2S的主链上每5个糖上连有侧链,HPP-3S和HPP-4S的主链上每4个糖上连有侧链,HPP-6S和HPP-8S的主链上每3个或每4个糖上连有侧链。HPP-5S和HPP-7S未分析,分支度应与HPP-6S的相近。另外,HPP-8S的色谱图还显示末端甘露糖和末端半乳糖的信号,说明二者在侧链上。主链分支度以及糖醛酸、甘露糖等单糖含量的变化规律,说明HPP-1S~HPP-8S是同源系列α-葡聚糖,具有相同的主链,结构中含量很少的甘露糖、核糖和半乳糖均为侧链,连在主链的C-6位。
根据单糖PMP衍生化分析结果、甲基化分析结果,按照摩尔比计算本发明所涉及α-葡聚糖中各单糖含量列于表1。HPP-1S含有葡萄糖和葡萄糖醛酸,二者含量分别为95.6%和4.4%。HPP-2S~HPP-7S含有葡萄糖、葡萄糖醛酸、甘露糖、核糖,葡萄糖的含量为85.8~93.0%,葡萄糖醛酸的含量为4.5~8.0%,甘露糖的含量为0.4~4.6%,核糖的含量为0.1~2.1%。HPP-8S含有葡萄糖、葡萄糖醛酸、甘露糖、核糖、半乳糖,葡萄糖的含量为84.6%,葡萄糖醛酸的含量为7.9%,甘露糖的含量为4.0%,核糖的含量为2.3%,半乳糖的含量为1.2%。
表1
需要指出的是,改变离子交换树脂种类(DEAE-52或cellufine A-500),用水洗脱得到的本发明所涉及的α-1,4-葡聚糖的结构相同,是海胆主要含有的多糖,具有来源丰富、结构稳定、纯度高、易溶于水的优点。
需要特别强调的是,实施例1、2和3的结果表明,本发明所涉及α-葡聚糖结构中的糖醛酸、甘露糖等含量在固定范围内变化,其具体的含量与洗脱液盐浓度有关。本发明所示α-葡聚糖的具体结构是应用本发明的NaCl浓度洗脱得到。根据该结果可得出如下结论,若使用其它浓度的NaCl溶液,可得到分支度、糖醛酸、甘露糖含量等在此范围内、具有其他比例的同类多糖,均应在本发明保护范围内。
(4)红外光谱
取1mg多糖加适量溴化钾研磨均匀,压片后用傅里叶红外光谱仪检测。HPP-1S~HPP-8S的红外光谱(图8)显示相同的特征峰。以HPP-2S为例,3397cm-1为羟基的伸缩振动吸收峰,2928cm-1为饱和烷基的伸缩振动吸收峰,1643和1414cm-1是羧基的特征峰,1152、1079和1023cm-1是糖苷键的吸收峰,844cm-1是α-吡喃糖的特征峰,说明是α-葡聚糖。
(5)核磁共振波谱分析
HPP-2S~HPP-8S用重水溶解,测试1H NMR和13C NMR。如图9所示,HPP-2S~HPP-8S显示与HPP-1S基本相同的特征质子和碳信号,进一步证实本发明所涉及α-葡聚糖的同源性。由于甘露糖、核糖和半乳糖的含量太低,未能观察到其明显的核磁信号,但糖醛酸的特征信号明显。以HPP-4S为例,其核磁数据列于表2。
表2
实施例4激活巨噬细胞释放一氧化氮(NO)
选择RAW264.7巨噬细胞为细胞模型。将培养皿内处于对数生长期的细胞吹打下来,调整细胞密度为2×104个/mL,于96孔板中每孔加入100μL细胞悬液,置于37℃、5%CO2细胞培养箱中培养24h。测试海胆总多糖HPP、HPP-1D、HPP-2D、HPP-3D对巨噬细胞的激活作用,给药浓度均为30、150、500μg/mL,培养液和脂多糖(LPS,1μg/mL)分别做空白和阳性对照。给药培养24h后取出96孔板,从每孔吸取100μL的细胞上清液至新的96孔板中,在避光条件下加入100μL的Griess试剂,振摇10min后于540nm测每孔的OD值。结果如表3所示,HPP、HPP-1D、HPP-2D和HPP-3D在30μg/mL时显示显著的促NO释放作用,其激活作用均呈剂量依赖性。
表3
与空白组相比,*P<0.05,**P<0.01。
用同样的方法测试了HPP-1S~HPP-8S激活巨噬细胞释放NO的作用,给药浓度:HPP-1S~HPP-3S为15.6、31.3、62.5、125、250、500μg/mL,HPP-4S~HPP-8S为1.95、3.9、7.8、15.6、31.3、62.5、125、250μg/mL。首先评价了本发明的α-葡聚糖对巨噬细胞生存率的影响,结果如图10所示,本发明的多糖在给药浓度范围内均对巨噬细胞无显著抑制作用,说明本发明的α-葡聚糖具有很好的安全性。本发明的α-葡聚糖均能显著刺激巨噬细胞释放NO,均呈现量效关系,而且能观察到明显的激活能力差异(图11)。例如HPP-1S~HPP-4S,活性强度依次增强,HPP-4S在很低浓度3.9μg/mL时即显示显著的激活作用,在15.6μg/mL时NO释放量达到最大值。它们结构中的甘露糖和核糖含量极低,对活性贡献小,结构主要变化是糖醛酸含量从HPP-1S到HPP-4S逐渐增加,活性强度呈极明显增强,这说明糖醛酸作为结构单元的特异性及其对活性强度的显著贡献。
根据上述分离和生物活性测试结果,HPP是本发明所涉及多糖的混合物,HPP-2D和HPP-3D是HPP-2S~HPP-8S的混合物,这些多糖混合物和纯品均能激活巨噬细胞,说明本发明的系列α-葡聚糖中的一个单独使用或多个组合使用均具有巨噬细胞激活作用。
实施例5促巨噬细胞分泌肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)
TNF-α和IL-6是免疫细胞激活后分泌的炎性细胞因子,能杀灭肿瘤细胞,也能作为特异性抗原激活机体适应性免疫,是免疫功能的重要指标。RAW264.7细胞培养24h后,HPP-1S、HPP-3S和HPP-6S按照实施例4设置给药浓度,分组给药,培养24h后收集细胞上清液至1.5mL离心管中,离心后取上清至新的离心管中。使用ELISA试剂盒,根据说明书分别检测HPP-1S、HPP-3S和HPP-6S给药上清中的TNF-α和IL-6含量。结果如图12所示,HPP-1S分别在31.3和15.6μg/mL时显著促TNF-α和IL-6分泌,HPP-3S分别在1.95和3.9μg/mL、HPP-6S分别在1.95和7.8μg/mL时显著促TNF-α和IL-6分泌。活性强度HPP-6S>HPP-3S>HPP-1S,与促一氧化氮实验观察到的活性强度趋势一致。与对照组的TNF-α分泌量(30.3pg/mL)相比,HPP-1S、HPP-3S和HPP-6S最高浓度组的TNF-α分泌量分别提高15、81和124倍,而IL-6的分泌量分别提高51、173和154倍(对照组IL-6的分泌量是99.0pg/mL)。这些生物活性数据说明本发明的α-葡聚糖的α-1,4-主链、葡萄糖和葡萄糖醛酸侧链、较高的分支度是其发挥强免疫调节活性的结构基础,且糖醛酸含量增加时,其活性显著增强。
实施例6提高巨噬细胞吞噬能力
RAW264.7细胞与HPP-1S(12.5、31.25、62.5、125、250和500μg/mL)共培养24h后取出96孔板并弃去上清,每孔加入中性红溶液(0.1%)100μL,于细胞培养箱内继续培养3h,弃去上清后用PBS清洗后加入裂解液100μL,室温过夜,待细胞裂解后测定其540nm的OD值。结果如图13所示,HPP-1S能显著促进巨噬细胞的吞噬能力,提高巨噬细胞的活性。
实施例7对小鼠脾淋巴细胞的作用
无菌摘取雄性昆明种小鼠脾脏,制备脾淋巴细胞悬液,接种于96孔板中,加入相应浓度的药物100μL,培养48h,加入CCK-8增强液20μL置于培养箱内孵育4h,于450nm处测定其OD值,并计算淋巴细胞的存活率。设置空白组,刀豆蛋白(ConA)对照组(终浓度5μg/mL),ConA(5μg/mL)+HPP-1S(31.25,62.5,125,250,500,1000μg/mL)给药组,HPP-1S给药组(31.25,62.5,125,250,500,1000μg/mL)。
结果如图14所示,加入ConA诱导后,与空白对照组相比,各组T淋巴细胞增殖率提高64%~77%。与ConA模型组相比,ConA+HPP-1S联合给药组对T淋巴细胞增殖率无显著影响。未加入ConA诱导只加入HPP-1S作用时,与空白组相比,HPP-1S在31.25~62.5μg/mL浓度范围内对脾淋巴细胞增殖无明显的影响,在125~1000μg/mL浓度范围内能显著促进其增殖。表明HPP-1S在31.25~1000μg/mL浓度范围内在体外对脾淋巴细胞无毒性,高浓度时还能促进脾淋巴细胞增殖,这说明本发明的α-葡聚糖安全性高,能激活脾淋巴细胞。
实施例8α-葡聚糖对环磷酰胺(CTX)导致的免疫损伤的保护作用
将96只体重为20±2g健康SPF级雄性昆明种小鼠随机分为6组,每组16只,各组内及组间小鼠体重无统计学差异。
分组:1)正常对照组:小鼠1~10天注射生理盐水;2)模型对照组:小鼠1~10天腹腔注射生理盐水,9~10天注射环磷酰胺(40mg/Kg/day);3)HPP-1S低剂量组:1~10天注射HPP-1S(20mg/Kg/day),9~10天注射环磷酰胺(40mg/Kg/day);4)HPP-1S中剂量组:1~10天注射HPP-1S(40mg/Kg/day),9~10天注射环磷酰胺(40mg/Kg/day);5)HPP-1S高剂量组:1~10天注射HPP-1S(80mg/Kg/day),9~10天注射环磷酰胺(40mg/Kg/day);6)HPP-1S高剂量灌胃组:1~10天灌胃HPP-1S(80mg/Kg/day),9~10天注射环磷酰胺(40mg/Kg/day)。
(1)α-葡聚糖对脾脏和胸腺的保护作用
末次给药24h后,小鼠麻醉处死解剖,取其脾脏和胸腺去除结缔和脂肪组织,生理盐水清洗后置于4%多聚甲醛溶液中固定过夜,使用自动脱水机对组织进行脱水,石蜡包埋后进行切片(片厚5μm)。HE染色观察脾脏和胸腺组织的病理学变化。结果如图15所示,正常组小鼠的脾脏被膜较厚,其实质中红髓和白髓界限分明,白髓内淋巴细胞较为密集。与正常组相比,模型组小鼠脾脏实质中白髓的结构散乱且淋巴细胞稀疏,红髓与白髓的界限模糊,被膜变薄。HPP-1S低、中、高剂量组对脾脏实质的组织结构具有明显改善,红髓与白髓的分界逐渐清晰,被膜增厚。图16显示,模型对照组小鼠胸腺的皮质变薄且结构散乱、髓质相对增多,皮质和髓质的界限模糊。HPP-1S给药组能使小鼠胸腺皮质相对增厚,皮髓质分界相对清楚。这表明HPP-1S能显著保护脾脏和胸腺,减弱环磷酰胺引起的免疫器官损伤,具有显著的免疫保护作用。
(2)小鼠单核-巨噬细胞吞噬功能提高
末次给药24h后,尾静脉注射稀释的墨汁,注射完毕后开始计时,在第2min(t1)、10min(t2)时分别从内眼眦静脉丛取血至离心管中,使用移液枪取20μL加入到2mLNa2CO3溶液(0.1%)中混匀,测定其在600nm波长处OD值。处死小鼠后解剖取其脾脏和肝脏,小心去除周围脂肪和结缔组织后称重,按下列公式计算清除率(K)和吞噬指数(α)。
K=(lgOD1-lgOD2)/(t2-t1)
α=体重/(肝重+脾重)×K1/3
其中,OD1为t1时的OD值,OD2为t2的OD值。
结果如表4所示,与空白组相比,模型组的清除率K和吞噬指数α显著降低,这表明环磷酰胺抑制了单核巨噬细胞的吞噬能力。与模型对照组相比,HPP-1S给药组的清除率K和吞噬指数α均显著增加。实验结果表明,HPP-1S能够逆转环磷酰胺引起的小鼠体内巨噬细胞活性降低,重新激活免疫损伤状态下的巨噬细胞功能。
表4
组别 | 剂量(mg/Kg/d) | 清除率K | 吞噬指数α |
正常对照组 | — | 0.017±0.003 | 3.612±0.545 |
模型对照组 | — | 0.002±0.001### | 2.045±0.137### |
HPP-1S低剂量组 | 20 | 0.006±0.003* | 2.829±0.242*** |
HPP-1S中剂量组 | 40 | 0.005±0.002* | 2.749±0.439*** |
HPP-1S高剂量组 | 80 | 0.005±0.001** | 2.851±0.282*** |
数值为平均值±SD(n=10)。与空白组比较,#p<0.05,##p<0.01,###p<0.001;与模型组比较,*p<0.05,**p<0.01,***p<0.001。
(3)血常规指标
末次给药24h后,小鼠通过眼眦取血50μL至EDTA·2K抗凝管中,测定血常规指标。
结果如表5所示,与空白对照组相比,模型对照组的白细胞数量(WBC)、嗜中性粒细胞数量(NE#)和淋巴细胞数量(LY#)显著降低,血小板数量(PLT)也有所减少。与模型组相比,HPP-1S给药组对环磷酰胺所致白细胞数量(WBC)、血小板数量(PLT)数量下降有显著的改善,HPP-1S低剂量组能够增加外周血中嗜中性粒细胞数(NE#),HPP-1S高剂量组能增加淋巴细胞数量(LY#)。高剂量口服给药也能显著提高白细胞和血小板数量,其效果总体上低于腹腔注射给药组。这些实验结果表明本发明的α-葡聚糖对环磷酰胺导致的血液和淋巴系统毒副作用具有良好的治疗效果,以及口服本发明的α-葡聚糖的有效性。
表5
数值为平均值±SD(n=10)。与正常对照组比,#p<0.05,##p<0.01,###p<0.001;与模型对照组比较,*p<0.05,**p<0.01,***p<0.001。
(4)测定小鼠外周血T淋巴细胞CD4 +/CD8 +比值
腹腔注射环磷酰胺造模前和末次给药24h后小鼠眼球取血至2.5mL EDTA·2K抗凝管中,等体积的全血稀释液稀释。经红细胞裂解液于室温条件下裂解后离心,加入PBS洗涤后加100μL PBS重悬,于流式细胞仪分析,测定CD4 +/CD8 +比值。
外周血T淋巴细胞的增殖与分化在机体应对外来刺激并在免疫应答发挥重要作用。机体淋巴细胞功能的高低可间接通过T淋巴细胞亚群水平的高低评价。CD4 +亚群能够调节机体免疫功能,数量降低后会导致机体免疫调节受到抑制;CD8 +亚群具有直接杀伤作用,表达过高时会导致免疫缺陷。CD4 +/CD8 +比值作为判断机体免疫失调的指标,两者的比值增大表示对机体的免疫功能有正向调节作用
结果如图17所示,与空白对照组相比,使用环磷酰胺造模后模型对照组CD4 +/CD8 +比值明显降低。与模型对照组相比,HPP-1S给药组均有所升高,尤其是HPP-1S低剂量组与模型对照组相比CD4 +/CD8 +比值提高了1.4倍,表明HPP-1S能逆转环磷酰胺导致的T淋巴细胞免疫功能损伤。
(5)小鼠血清中细胞因子的测定
末次给药后24h,小鼠麻醉后取血至1.5mL离心管中,静置4h后,于4℃、3000rpm/min离心10min,移去上层血清至新的1.5mL离心管中,-80℃低温保存备用。采用ELISA试剂盒测定其IL-2、TNF-α的含量。
结果如图18所示,环磷酰胺能显著降低小鼠血清中IL-2和TNF-α含量。给予HPP-1S后,对环磷酰胺致IL-2和TNF-α含量降低具有明显的逆转作用,且呈剂量依赖性。HPP-1S低剂量组IL-2和TNF-α含量相比模型组高出60%,HPP-1S高剂量组TNF-α的含量为空白组的86%。结果表明,HPP-1S能够促进免疫因子表达量增加,降低环磷酰胺引起的免疫力降低,增强机体的免疫功能。
实施例4、5、6、7、8的数据表明,本发明的α-葡聚糖对巨噬细胞和小鼠脾淋巴细胞无毒副作用,还具有一定的促进增殖作用,显示高安全性。细胞实验中,能显著激活巨噬细胞,促进吞噬,提高NO、TNF-α和IL-6的表达水平。进一步的动物实验证实本发明的α-葡聚糖在体内的有效性以及口服有效性,能够改善环磷酰胺引起的多种免疫抑制作用,保护免疫器官、减弱损伤,提高白细胞和血小板水平,增强免疫细胞功能与免疫因子的表达,有望开发为改善或治疗免疫相关机体功能障碍的功能性食品或药物。
实施例9α-葡聚糖抑制小鼠体内H22肝癌实体瘤与免疫调节活性
将80只荷瘤小鼠随机分为8组,每组10只,各组内及组间体重与肿瘤体积无统计学差异。生理盐水、HPP-1S和环磷酰胺给药方式均为腹腔注射。
1)模型对照组:1~10天注射生理盐水;
2)阳性对照组:1~10天注射环磷酰胺(20mg/Kg/day);
3)HPP-1S低剂量组:1~10天注射HPP-1S(20mg/Kg/day);
4)HPP-1S中剂量组:1~10天注射HPP-1S(40mg/Kg/day);
5)HPP-1S高剂量组:1~10天注射HPP-1S(80mg/Kg/day);
6)联合低剂量组:1~10天注射HPP-1S(20mg/Kg/day)+环磷酰胺(20mg/Kg/day);
7)联合中剂量组:1~10天注射HPP-1S(40mg/Kg/day)+环磷酰胺(20mg/Kg/day);
8)联合高剂量组:1~10天注射HPP-1S(80mg/Kg/day)+环磷酰胺(20mg/Kg/day)。
末次给药后24h,小鼠称重、麻醉后处死解剖,取其肿瘤并小心去除结缔和脂肪组织后称重,按公式计算肿瘤生长抑制率。公式:肿瘤抑制率(%)=(模型对照组肿瘤质量均值-给药组肿瘤质量均值)/模型对照组肿瘤质量均值×100%。
取其脾脏和胸腺小心去除结缔和脂肪组织后称重,按公式计算脾脏指数和胸腺指数。公式:脾脏(胸腺)指数=脾脏(胸腺)重量/小鼠体重×100%。
结果如表6所示,HPP-1S低、中、高剂量组肿瘤抑制率分别为43.70%、43.75%、52.88%,阳性对照组抑瘤率为70.83%,联合低、中、高剂量组肿瘤抑制率分别为69.41%、67.79%、74.82%。其中HPP-1S高剂量组肿瘤抑制率达52.88%,与环磷酰胺联用时肿瘤抑制率高达74.82%。实验结果表明HPP-1S有显著的抗肿瘤作用,其高剂量与环磷酰胺合用时能一定程度提高肿瘤抑制率。
表6
数值为平均值±SD(n=10)。与正常对照组比,#p<0.05,##p<0.01,###p<0.001;与模型对照组比较,*p<0.05,**p<0.01,***p<0.001。
检测了小鼠血清中IL-2和TNF-α含量。末次给药后24h,小鼠麻醉后取血至1.5mL离心管中,室温静置4h后,于4℃、3000rpm/min离心10min,移取上层血清至1.5mL离心管中。采用ELISA试剂盒测定IL-2和TNF-α的含量。结果如图19所示,与模型组相比,HPP-1S单独给药组TNF-α含量均提高,其中高剂量组显著提升;与阳性对照组相比,联合中、高剂量组TNF-α含量显著提高,且HPP-1S对小鼠体内分泌TNF-α的影响呈剂量依赖性。表明HPP-1S能有效改善机体免疫能力下降所致的IL-2、TNF-α分泌水平降低,增强机体的免疫能力。
实施例10α-葡聚糖抑制小鼠体内S180肉瘤与免疫调节作用
将昆明小鼠接种24h后,随机的分为7个组,分别是正常(未接种S180)、模型、阳性药(CTX,20mg/Kg/d)、HPP-1S低剂量(20mg/Kg/d)、HPP-1S中剂量(40mg/Kg/d)、HPP-1S高剂量(80mg/Kg/d)和联合给药组(CTX 20mg/Kg/d+HPP-1S80 mg/Kg/d)。腹腔注射给药,连续12天。最后麻醉脱臼处死,剥离出肿瘤、脾脏以及胸腺组织,分别称重。
结果如图20和表7所示,本发明的α-葡聚糖能显著抑制S180肉瘤生长,随着多糖剂量升高,抑瘤率显著提升,高剂量80mg/Kg/d时抑制率可达55.49%,略低于阳性药CTX对照组。多糖与CTX联用组的抑制率略高于CTX单用组。多糖对小鼠不同治疗剂量组,其脾指数均大于阳性药组,尤其是中、高剂量治疗组的脾指数可达22.38,远高于环磷酰胺组的4.42。联合用药组脾指数为6.76,也高于CTX组的4.42。实验结果表明,HPP-1S表现出明显的抗肿瘤作用、免疫器官保护作用,有一定的联合增效和明显的减毒作用。
表7
数值为平均值±SD(n=12)。与模型组比较,#p<0.05,##p<0.01,###p<0.001.
通过ELISA法检测了小鼠血清中IL-2的表达量。结果如图21所示,肿瘤模型组和CTX阳性对照组中IL-2的浓度均显著低于正常组,这表明该组实验动物的免疫系统被抑制,致使免疫因子表达水平降低,干扰机体正常杀灭肿瘤细胞的功能。而多糖给药组,以及多糖与CTX合用组,尤其是高剂量组中,IL-2的表达水平明显得到改善,说明HPP-1S能治疗肿瘤及化疗药物引起的机体内免疫因子表达水平降低,能通过免疫调节起到抗肿瘤作用。
实施例11α-葡聚糖抑制小鼠体内B16黑色素瘤与免疫调节作用
小黑鼠接种24h后,随机的分为6组,分为模型、阳性药(CTX,20mg/Kg/d)、HPP-1S低剂量(20mg/Kg/d)、HPP-1S中剂量(40mg/Kg/d)、HPP-1S高剂量(80mg/Kg/d)和联合给药组(CTX 20mg/Kg/d+HPP-1S 80mg/Kg/d),腹腔注射给药,连续12天。最后麻醉脱臼处死,剥离出肿瘤、脾脏以及胸腺组织,分别称重。
结果如图22和表8所示,本发明的α-葡聚糖能显著抑制黑色素瘤的生长,呈一定剂量依赖性,最高剂量时的抑制率可达47.60%。不同多糖给药组小鼠的脾指数均高于阳性药CTX组,且高于空白模型组,高剂量80mg/Kg/d HPP-1S给药组达10.72,远高于CTX阳性对照组的4.3。联合用药组脾指数为10.38,与高剂量给药组效果相当,说明多糖能减弱CTX引起的免疫损伤,保护免疫器官,通过上调免疫功能发挥抗肿瘤作用。
表8
数值为平均值±SD(n=10)。与模型组比较,*p<0.05,**p<0.01。
通过ELISA法检测了小鼠血清中IL-2和TNF-α含量。结果如图23所示,HPP-1S能提高血清中IL-2和TNF-α表达水平。
动物实验结果表明本发明的α-葡聚糖能激活机体免疫系统功能,逆转肿瘤与化疗药对机体的免疫抑制作用,提高免疫系统杀灭肿瘤细胞能力,具有肿瘤免疫治疗作用,可用于研制抗肿瘤药物。
Claims (8)
1.一类新型α-葡聚糖,其特征在于,具有式Ⅰ所示的结构:
其中,n选自2.5、2、1.5、1,R选自葡萄糖、葡萄糖醛酸、甘露糖、核糖、半乳糖残基。
2.如权利要求1所述的α-葡聚糖,其特征在于,所述的式Ⅰ结构中的主链由葡萄糖通过α-1,4-糖苷键聚合而成,其结构中的R是单糖侧链,R选自葡萄糖与葡萄糖醛酸,以及甘露糖、核糖、半乳糖的一种或几种。
3.如权利要求1所述的α-葡聚糖,其特征在于,所述多糖的分子量为1.913×107~3.094×107Da,按照摩尔比计算,所述多糖结构中葡萄糖的含量为84.6~95.6%,葡萄糖醛酸的含量为4.4~8.0%,甘露糖的含量为0.4~4.6%,核糖的含量为0.1~2.3%,半乳糖的含量为0~1.2%。
4.如权利要求1-3中任一所述的α-葡聚糖的一种或几种组合在制备改善或治疗免疫相关机体功能障碍或疾病的制剂中的用途。
5.如权利要求4所述的α-葡聚糖的一种或几种组合在制备改善或治疗免疫相关机体功能障碍或疾病的制剂中的用途,其特征在于,所述一种或几种组合为任意一种α-葡聚糖单独使用,或任意几种α-葡聚糖组合使用。
6.如权利要求4所述的α-葡聚糖的一种或几种组合在制备改善或治疗免疫相关机体功能障碍或疾病的制剂中的用途,其特征在于,所述免疫相关机体功能障碍为因先天不足、感染、营养不良或药物等引起的免疫功能缺陷、免疫力低下或免疫损伤。
7.如权利要求4所述的α-葡聚糖的一种或几种组合在制备改善或治疗免疫相关机体功能障碍或疾病的制剂中的用途,其特征在于,所述免疫相关疾病为肿瘤。
8.如权利要求4所述的α-葡聚糖的一种或几种组合在制备改善或治疗免疫相关机体功能障碍或疾病的制剂中的用途,其特征在于,所述制剂为功能性食品或药物。
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