CN115282159A - 鼠李聚糖硫酸酯在制备预防和/或治疗炎症所致肠黏膜o-糖链结构异常疾病产品中的应用 - Google Patents
鼠李聚糖硫酸酯在制备预防和/或治疗炎症所致肠黏膜o-糖链结构异常疾病产品中的应用 Download PDFInfo
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Abstract
本发明属于海洋药物开发技术领域,具体涉及鼠李聚糖硫酸酯在制备预防和/或治疗炎症所致肠黏膜O‑糖链结构异常疾病产品中的应用。采用饮水添加DSS造模的肠炎肠黏膜损伤小鼠模型,证明了鼠李聚糖硫酸酯能显著改善肠炎所致小鼠肠黏膜损伤、小鼠黏膜O‑糖链种类减少及结构异常,尤其能防治因肠黏膜损伤引起的肠黏膜O‑糖链岩藻糖化、硫酸酯及唾液酸化水平异常。本发明产品的原料来源于海洋绿藻,具有资源丰富、制备工艺简单、产品稳定性好、易于产业化和安全性高等诸多优点,所制备的鼠李聚糖硫酸酯在防治肠炎所致肠黏膜O‑糖链结构异常相关疾病的新药和特医食品的开发领域具有广阔的开发应用前景。
Description
技术领域
本发明属于海洋药物开发技术领域,具体涉及鼠李聚糖硫酸酯在制备预防和/或治疗炎症所致肠黏膜O-糖链结构异常疾病产品中的应用。
背景技术
肠道屏障主要由上皮细胞、免疫细胞、分泌细胞以及其分泌的黏液层组成,而黏液层则主要由杯状细胞分泌的富含O-糖链的糖蛋白组成(Yamada et al.,EBioMedicine,2019,48:513-525)。在正常生理条件下,肠道黏膜可以选择性地吸收水分和营养物质并有效防止病原体、毒素和过敏原从腔内扩散到其他组织、器官和血液中。然而,当肠道发生炎症,肠道黏膜受损时,有害微生物和内毒素可跨越肠黏膜屏障,导致肠源性感染,甚至发展为全身炎症反应,导致器官衰竭、炎症性肠病,脓毒症、急性胰腺炎等疾病(Huo et al.,Journal of Agricultural and Food Chemistry,2020,70:711-735)。肠黏膜损伤的一个重要标志就是肠道黏液层破坏,黏液相关糖蛋白表达异常,肠黏膜表面O-糖链数量/结构等受损或表达异常(Larsson et al.,Inflammatory Bowel Diseases,2011,17(11):2299-2307)。有研究表明肠,黏膜表面O-糖链的唾液酸化对肠屏障具有重要保护作用(Yao etal.,Cell,2022,185:1172-1188)。因此,保护肠黏膜O-糖链正常的结构对于维护人体健康具有重要应用价值。
我国绿藻资源丰富,常见的有石莼属、礁膜属、浒苔属等,绿藻具有重要的营养和药用价值,其富含的鼠李聚糖硫酸酯无毒副作用,是绿藻重要的活性物质之一,受到越来越多的关注。本团队研究表明,虽然海洋多糖的口服生物利用度低,但可以和肠道菌群发生互作,进入肠道病灶并发挥功能(Shang et al.,Carbohydrate Polymers,2018,179:173-185)。在此过程中,鼠李聚糖硫酸酯可以被肠道菌群降解利用,作为益生元调节紊乱的肠道菌群(公开号CN108440681A),促进有益菌的生长和定殖,从而作用于机体健康。此外,有研究表明鼠李聚糖硫酸酯具有抗凝抗栓(公开号CN101328226A)、调节免疫(公开号CN202010378781.3)、杀菌(CN201810007951.X)、防治溃疡性结肠炎(韩秋凤等,海峡预防医学杂志,2010,16(2):58-60)、减轻LPS诱导的肠道炎症(Zhang et al.,Food&Function.2022,13(1):52-63)、保护产肠毒素大肠杆菌对小鼠小肠黏膜屏障的破坏(解影等,中国饲料,2019(23):64-68)等多种生物活性,显示了鼠李聚糖硫酸酯在食品和医药领域广阔的开发前景。由于鼠李聚糖硫酸酯来源丰富,其制备(公开号CN107011456B)和降解(公开号CN112592914A)方法也被广泛研究,保证了产品原料供应。目前鼠李聚糖硫酸酯已被广泛应用于食品、制药、化妆品行业,具有很大开发利用价值。鼠李聚糖硫酸酯由海洋绿藻制备而来,尽管有文献研究表明浒苔(韩秋凤等,海峡预防医学杂志,2010,16(2):58-60)和石莼(Li et al.,Marine Drugs,2020,18(10),499)来源的鼠李聚糖硫酸酯具有治疗和保护小鼠结肠炎的作用,但尚无鼠李聚糖硫酸酯在保护炎症所致肠黏膜O-糖链结构异常中的相关报道。
发明内容
为了克服现有技术中存在的上述缺陷,本发明提供一种鼠李聚糖硫酸酯在制备预防和/或治疗炎症所致肠黏膜O-糖链结构异常疾病产品中的应用,采用饮水添加DSS造模的肠炎肠黏膜损伤小鼠模型,证明了鼠李聚糖硫酸酯能显著改善肠炎所致小鼠肠黏膜损伤、小鼠黏膜O-糖链种类减少及结构异常,尤其能防治因肠黏膜损伤引起的肠黏膜O-糖链岩藻糖化、硫酸酯及唾液酸化水平异常。
为了实现上述发明目的,本发明采用下述技术方案:
鼠李聚糖硫酸酯在制备预防和/或治疗炎症所致肠黏膜O-糖链结构异常疾病产品中的应用。
所述的应用,鼠李聚糖硫酸酯是以海洋绿藻为原料,经脱脂、热水提取,浓缩,氧化脱色,有机溶剂沉淀、干燥制得。
所述的应用,按重量百分比计,鼠李聚糖硫酸酯单糖组成主要含有鼠李糖、葡萄糖醛酸和葡萄糖,鼠李糖含量为20~90wt%,葡萄糖醛酸含量为5~20wt%,葡萄糖含量为1~30wt%。
所述的应用,鼠李聚糖硫酸酯单糖组成中还含有木糖、半乳糖和甘露糖;优选的,按重量百分比计,鼠李聚糖硫酸酯单糖的组成和含量如下:鼠李糖含量为30~70wt%,葡萄糖醛酸含量为10~20wt%,葡萄糖含量为5~25wt%,木糖含量为5~15wt%,半乳糖含量为1~3wt%,甘露糖含量为0.5~5wt%。
所述的应用,硫酸根含量为5~35wt%。
所述的应用,鼠李聚糖硫酸酯的平均分子量为0.5~600kDa。
所述的应用,炎症所致肠黏膜O-糖链结构异常疾病包括器官衰竭、炎症性肠病、脓毒症或急性胰腺炎。
所述的应用,产品包括药物、保健品或特医食品。
所述的应用,产品包括用于保护炎症所致黏膜结构异常的保健品、补充膳食纤维、益生元制品、营养补充剂,或者与益生菌、及/或保护肠黏膜的药物联用,用于保护肠黏膜的药物、功能食品或生物制品。
本发明的设计思想是从与受损肠黏膜愈合密切相关的保护肠黏膜O-糖链的结构角度出发,阐述鼠李聚糖硫酸酯在制备预防和/或治疗炎症所致肠黏膜O-糖链结构异常疾病产品中的应用:
目前,有关炎性肠病的确切病因仍不明确,但大量研究表明遗传因素、免疫因素和环境因素等均与炎性肠病的发病相关,对炎性肠病的治疗主要是基于炎症控制、免疫功能调节等展开。虽然,现有的研究从炎症控制和维持缓解方面表明鼠李聚糖硫酸酯具有保护小鼠结肠炎症的作用。但是,在临床诊疗中,肠黏膜愈合是炎症治疗重要的治疗观察终点,本发明鼠李聚糖硫酸酯应用于制备预防和/或治疗炎症所致肠黏膜O-糖链结构异常疾病产品中,与现有技术中鼠李聚糖硫酸酯相比是首次从与肠黏膜愈合密切相关的保护肠黏膜O-糖链的角度出发,阐述鼠李聚糖硫酸酯在制备预防和/或治疗炎症所致肠黏膜O-糖链结构异常疾病产品中的应用,以达到促进受损黏膜愈合的目的。
与现有技术相比,本发明的优点及有益效果是:
1.本发明采用饮水添加DSS造模的肠炎肠黏膜损伤小鼠模型,实验结果证明所述鼠李聚糖硫酸酯能显著改善肠炎肠黏膜损伤小鼠的肠黏膜杯状细胞减少。
2.本发明采用饮水添加DSS造模的肠炎肠黏膜损伤小鼠模型,实验结果证明所述鼠李聚糖硫酸酯能显著改善肠炎肠黏膜损伤小鼠肠黏膜O-糖链组成比例失调、短链O-糖链含量升高和长链O-糖链含量降低。
3.本发明产品的原料来源于海洋绿藻,具有资源丰富、制备工艺简单、产品稳定性好,易于产业化,安全性高等诸多优点,所制备的鼠李聚糖硫酸酯在防治肠炎所致肠黏膜O-糖链结构异常相关疾病的新药和特医食品的开发领域具有广阔的开发应用前景。
附图说明
图1为鼠李聚糖硫酸酯分子量分布图。
图2为鼠李聚糖硫酸酯的单糖组成分析图谱。
图3为小鼠结肠组织病理学切片染色图。其中,H&E表示苏木精-伊红染色,AB表示阿利新蓝染色;NC为正常未造模Control组小鼠,MD为DSS造模Model组小鼠,EP为鼠李聚糖硫酸酯治疗组小鼠。
图4为小鼠结肠三类O-糖链相对含量分布图。其中,NC为正常未造模Control组小鼠,MD为DSS造模Model组小鼠,EP为鼠李聚糖硫酸酯治疗组小鼠。SA,唾液酸化;NSA,非唾液酸化;F,岩藻糖化;NF,非岩藻糖化;S,硫酸化;NS,非硫酸化;*P<0.05,与Control组小鼠相比;**P<0.01,与Control组小鼠相比;#P<0.05,与Model组小鼠相比;##P<0.01,与Model组小鼠相比。
图5为小鼠结肠10种O-糖链相对含量分布图。其中,NC为正常未造模Control组小鼠,MD为DSS造模Model组小鼠,EP为鼠李聚糖硫酸酯治疗组小鼠。*P<0.05,与Control组小鼠相比;**P<0.01,与Control组小鼠相比;#P<0.05,与Model组小鼠相比;##P<0.01,与Model组小鼠相比。
具体实施方式
在具体实施过程中,以青岛产浒苔所提鼠李聚糖硫酸酯(EP)为例,本发明以青岛产浒苔为原料,经脱脂、热水提取、浓缩、氧化剂脱色、有机溶剂沉淀、干燥获得,其重均分子量范围为0.5kDa~600kDa。本发明的原料为海洋绿藻,具有资源丰富、制备工艺简单、安全性高,易于产业化等优点。
以下,结合具体实施例对本发明的技术方案做进一步详细的说明。
实施例1:鼠李聚糖硫酸酯的制备
将浒苔烘干粉碎至粒度为40至100目,烘干所得的浒苔粉与95wt%乙醇按照1:10质量比例在80℃水浴中搅拌脱脂1~2小时,冷却至室温后,减压过滤收集残渣,重复脱脂2次,残渣烘干备用。将干燥残渣按照1:20质量比例加入蒸馏水,在95℃水浴中搅拌提取1~3小时,冷却至室温后,离心(4500r/min,20min)收集上清液,残渣继续重复提取2次,合并提取液,经旋转蒸发浓缩后,在40~60℃水浴中加入双氧水进行氧化脱色2~5小时,去除残余过氧化氢后,加入5倍质量95wt%纯度的食品级乙醇,离心收集沉淀,经减压干燥后获得鼠李聚糖硫酸酯(EP)。
采用十八角度激光散射仪对鼠李聚糖硫酸酯多糖的分子量进行测定,其分子量为10kDa。采用PMP柱前衍生高效液相色谱法测定浒苔硫酸多糖的单糖组成,该多糖含有Rha、Glc、GlcA,以及Xyl、Gal和Man,其中:鼠李糖含量51.84wt%,葡萄糖含量20.07wt%,葡萄糖醛酸含量19.93wt%,木糖、半乳糖和甘露糖的含量分别为5.86wt%、1.52wt%及0.78wt%。采用离子色谱法测定硫酸根含量为25.97wt%。如图1和图2所示,分别为鼠李聚糖硫酸酯的分子量分布图和单糖组成图。如将浒苔换成石莼或者礁膜,采用类似的制备工艺,可以得到类似结构的鼠李聚糖硫酸酯。
实施例2:鼠李聚糖硫酸酯对黏膜损伤小鼠结肠完整度及杯状细胞的影响
C57/6J小鼠(雄性,6周龄)在SPF级动物房通风的笼子里饲养,所有小鼠被饲养在温度可控(约23℃)的环境下,每天12小时光照,自由饮水和进食。经过2周适应期后,18只小鼠随机平均分为3组:正常未造模Control组(NC,n=6)、DSS造模Model模型组(MD,n=6)、鼠李聚糖硫酸酯治疗组(EP,n=6)。在第一个造模周期中,EP组和MD组小鼠在饮用水中添加1wt%DSS,NC组给与正常饮水,连续7天,随后三组小鼠均给予常规饮用水7天。在第二个周期中,即从第14天到第21天,EP组和MD组小鼠在饮用水中添加2.5wt%DSS,NC组小鼠仍给与正常饮水,第22到24天,三组小鼠均给予常规饮用水3天。在第14~24天,EP组小鼠灌胃给予100mg/kg的EP水溶液,每天1次,NC组和MD组小鼠给予等体积的生理盐水。
第25天处死所有小鼠,解剖小鼠,取部分结肠组织做病理学切片分析,其余部分-80℃冻存。H&E染色(苏木精-伊红染色法)结果如图3所示,从小鼠结肠组织病理学切片染色结果可以看出,NC组小鼠结肠绒毛排布整齐,未见明显炎症细胞浸润和肠黏膜损伤情况,MD组小鼠结肠绒毛明显破坏且有炎症细胞浸润现象结肠黏膜损伤严重,EP组小鼠结肠绒毛排列整齐,未见明显炎症细胞浸润和结肠黏膜损伤情况。AB染色(阿利新蓝染色)结果表明,NC组、EP组小鼠结肠杯状细胞排列规整、丰富,黏膜完整;MD组小鼠结肠杯状细胞严重缺损,黏膜破坏严重。
实施例3:鼠李聚糖硫酸酯对黏膜损伤小鼠结肠O-糖链含量的影响
取实施例2中冻存的小鼠结肠进行O-糖链分析,具体实验步骤如下:首先采用高通量组织均质器对结肠组织进行脱脂和均质。胰蛋白酶对组织中的蛋白进行酶解消化,酶解反应结束后,离心取上清,加入三倍乙醇对其中含O-糖链部分的糖肽进行醇沉,后使用3kDa超滤管对其进行分离得到粗粘蛋白型糖肽。采用还原剂保护的β-消除法制备糖醇形式的O-糖链,用LC-ESI-MS/MS对其进行分析,利用DeconTools去卷积化软件和GlycReSoft 2.0软件对O-糖链进行分析处理,进一步用CarbBank数据库对鉴定的O-糖链进行结构验证。使用Xcalibur对基于各O-糖链的峰面积对其进行相对定量分析,采用SNFG命名法用GlycoWorkbench 2.1绘制O-糖链的结构。研究表明,从结肠黏膜中共鉴定得到36种O-糖链(O-Glycan,OG),将其命名为OG1-OG36,见表1各组样品处理后肠黏膜O-糖链含量(Mean±SD)。
如图4所示,从小鼠结肠三类O-糖链相对含量分布可以看出,对小鼠结肠O-糖链进行唾液酸化、非唾液酸化、岩藻糖化、非岩藻糖化、硫酸化和非硫酸化分类分析发现,相较于NC组小鼠,结肠黏膜损伤MD组小鼠结肠O-糖链中唾液酸化O-糖链丰度显著降低,岩藻糖化和硫酸化O-糖链丰度显著增高,与之相反的MD组小鼠结肠O-糖链中非唾液酸化O-糖链丰度显著上升,非岩藻糖化和非硫酸化O-糖链丰度显著降低。给与EP后可显著改善黏膜损伤引起的小鼠肠道O-糖链相对丰度的失调。EP能显著降低小鼠黏膜损伤引起的岩藻糖化和硫酸化O-糖链丰度的增高,同时对唾液酸化降低的情况也有相应的升高唾液酸化O-糖链相对丰度的趋势。
实施例4:鼠李聚糖硫酸酯对黏膜损伤小鼠结肠O-糖链含量及链长的影响
对实施例3中鉴定得到的36种O-糖链进行相对含量定量分析,共发现10种O-糖链在相对含量丰度上存在显著性差异。如图5所示,从小鼠结肠O-糖链相对含量分布可以看出,其中链长较短的OG5、OG6、OG10、OG12、OG16和OG17糖链在黏膜损伤模型组中丰度显著增高,而链长较长的OG29、OG31和OG33糖链在黏膜损伤模型组中丰度显著降低,这一结果表明黏膜损伤显著降低了黏膜表面O-糖链的链长,而EP能缓解黏膜损伤对黏膜表面O-糖链的链长缩短的影响。在链长较长的OG31糖链中,EP有提高OG31相对丰度的趋势,在链长较短的OG6、OG12、OG16、OG17糖链中,EP能不同程度降低这五种短链O-糖链的丰度。同时对于在模型组中丰度显著降低的OG13糖链,EP能显著逆转其含量减少。
综上,实施例结果表明,本发明的以呼他多糖为代表的鼠李聚糖硫酸酯能显著改善黏膜损伤小鼠结肠黏膜的损伤程度。改善由于黏膜损伤引起的小鼠结肠黏膜O-糖链唾液酸化降低,岩藻糖化和硫酸化丰度显著增高的情况,同时保护由于黏膜损伤引起的小鼠结肠O-糖链链长的缩短。
所述的鼠李聚糖硫酸酯具有保护肠黏膜O-糖链结构异常的功能,可用于防治肠黏膜受损的保健品、补充膳食纤维、益生元制品、营养补充剂,或者与益生菌、及抗肠炎的药物联用,或以鼠李聚糖硫酸酯及其衍生物为母核制备的衍生物用于防治肠黏膜受损的药物、功能食品或生物制品中。本发明产品的原料来源于海洋绿藻中的鼠李聚酸硫酸酯活性成分,该类活性成分除了来源于各种浒苔属外,其他绿藻如石莼属和礁膜属也含有该类结构多糖,凡是其中含有鼠李聚糖硫酸酯结构的绿藻都具有本专利公开的作用。由于该类多糖具有资源丰富、制备工艺简单、产品稳定性好,易于产业化,安全性高等诸多有点,在肠黏膜受损的新药和特医食品的开发领域具有广阔的开发应用前景。
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;尽管参照前述实施例对本发明进行了详细的说明,对于本领域的普通技术人员来说,依然可以对前述实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或替换,并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。
Claims (9)
1.鼠李聚糖硫酸酯在制备预防和/或治疗炎症所致肠黏膜O-糖链结构异常疾病产品中的应用。
2.根据权利要求1所述的应用,其特征在于,鼠李聚糖硫酸酯是以海洋绿藻为原料,经脱脂、热水提取,浓缩,氧化脱色,有机溶剂沉淀、干燥制得。
3.根据权利要求1所述的应用,其特征在于,按重量百分比计,鼠李聚糖硫酸酯单糖组成主要含有鼠李糖、葡萄糖醛酸和葡萄糖,鼠李糖含量为20~90wt%,葡萄糖醛酸含量为5~20wt%,葡萄糖含量为1~30wt%。
4.根据权利要求3所述的应用,其特征在于,鼠李聚糖硫酸酯单糖组成中还含有木糖、半乳糖和甘露糖;优选的,按重量百分比计,鼠李聚糖硫酸酯单糖的组成和含量如下:鼠李糖含量为30~70wt%,葡萄糖醛酸含量为10~20wt%,葡萄糖含量为5~25wt%,木糖含量为5~15wt%,半乳糖含量为1~3wt%,甘露糖含量为0.5~5wt%。
5.根据权利要求3或4所述的应用,其特征在于,硫酸根含量为5~35wt%。
6.根据权利要求3或4所述的应用,其特征在于,鼠李聚糖硫酸酯的平均分子量为0.5~600kDa。
7.根据权利要求1或2所述的应用,其特征在于,炎症所致肠黏膜O-糖链结构异常疾病包括器官衰竭、炎症性肠病、脓毒症或急性胰腺炎。
8.根据权利要求1或2所述的应用,其特征在于,产品包括药物、保健品或特医食品。
9.根据权利要求8所述的应用,其特征在于,产品包括用于保护炎症所致黏膜结构异常的保健品、补充膳食纤维、益生元制品、营养补充剂,或者与益生菌、及/或保护肠黏膜的药物联用,用于保护肠黏膜的药物、功能食品或生物制品。
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