CN111436613B - 坛紫菜多糖在制备修复小肠上皮细胞损伤产品中的应用 - Google Patents
坛紫菜多糖在制备修复小肠上皮细胞损伤产品中的应用 Download PDFInfo
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Abstract
本发明公开了坛紫菜多糖在健胃、预防或修复肠胃损伤中的应用。本发明公开了坛紫菜多糖能够对损伤的IEC‑6细胞起到增殖修复作用,能够有效增强IEC‑6细胞的黏附作用;并对IEC‑6细胞中Cdc‑42,Rac‑1,Par‑3,aPKC,PKCβII,paxillin,FAK和GAPDH等8种蛋白的表达上调。将其作为食品例如米稀的原料时,不仅能提高米稀的营养价值,丰富米稀的口感风味,还具备健胃、预防和修复肠胃损伤的效果。
Description
技术领域
本发明涉及坛紫菜多糖的应用领域,具体涉及坛紫菜多糖在健胃、预防或修复肠胃损伤中的应用。
背景技术
坛紫菜作为我国大规模养殖的重要经济海藻之一,其属红藻门,红毛菜科,紫菜属植物,盛产于广东、福建、浙江等沿海地区。坛紫菜多糖是坛紫菜中的活性成分之一,是一种多组分的水溶性多糖,属于半乳聚硫酸酯,1,3-β-D-半乳糖和1,4-α-L-3,6-内醚半乳糖交替连接的二糖重复单位组成,有时被β-D-半乳糖-6-甲氧基和α-L-半乳糖-6-硫酸基取代。研究表明,坛紫菜多糖具有抗氧化、抗肿瘤、免疫调节及调节肠道菌群等多种生物活性,广泛应用于食品及医药的开发。
胃肠道是不仅是人体的重要消化器官,也是人体最大的免疫系统。发挥着消化和吸收营养物质等功能的同时还防御外界刺激,保护机体免受细菌、毒素、药物等有害物质的侵袭。临床上多种胃肠道疾病的发展均可能会对胃肠黏膜上皮造成破坏,进而可能发展为炎症性肠病等重大的肠胃疾病。因此,对胃肠道上的肠上皮进行加速修复或者保护变得尤为重要。
此前的研究表明,鱿鱼墨多糖在化疗条件下对肠道紧密连接和黏附连接具有保护作用,而坛紫菜多糖(PHP)在胃肠道修复保护中的应用尚无报道。
发明内容
本发明的目的是公开了坛紫菜多糖在健胃、预防或修复肠胃损伤中的应用。
在一些优选的实施情况中,上述肠胃损伤包括胃溃疡、肠胃炎症。
本发明中的坛紫菜多糖可以从红藻门红毛菜科紫菜属坛紫菜中提取纯化得到,该紫菜多糖可有效地对小肠隐窝上皮细胞损伤修复。本发明以坛紫菜为原料经水浸提法提取(也可采用超声微波辅助提取方法、超声提取法、微波提取法、碱提法或酶辅助提取法),通过乙醇沉淀后将获得的多糖沉淀复溶在蒸馏水中经DEAE纤维素纯化,透析冻干后得到的紫菜多糖,有利于对胃肠道细胞的损伤进行修复,对胃肠道起到保护的作用。在一些优选的实施情况中,上述坛紫菜多糖通过以下步骤制得:
步骤一、将坛紫菜粉碎后进行水提取,过滤,向滤液中添加乙醇并在0~10℃的条件下静置12~24h;
步骤二、静置12~24h后过滤,向沉淀中加水,然后冻干得到坛紫菜粗多糖,然后经过纯化,洗脱,透析和冻干后制得所述坛紫菜多糖。该坛紫菜多糖的总糖含量在65.9%以上。
其中,步骤一中,坛紫菜与水的质量比为1:(1~5),水提取的条件包括:温度为80~100℃,搅拌浸提2~4h;优选地,坛紫菜与水的质量比为1:4,水提取的条件包括:温度为90℃,搅拌浸提2h。滤液与乙醇的体积比为1:(1~5),乙醇为95%乙醇;优选地,滤液与乙醇的体积比为1:3。
步骤二中,采用DEAE离子交换树脂对坛紫菜粗多糖进行纯化;采用0.2M氯化钠溶液进行洗脱。
本发明还提供了一种米稀,其原料按照重量份包括:粳米40~60份、燕麦10~20份、山药10~20份、茯苓10~20份、坛紫菜多糖5~10份、花生5~10份、百合5~10份。
由于其原料包括了上述中的坛紫菜多糖,该食品具有健胃、预防和修复肠胃损伤的疗效,上述中的坛紫菜多糖还可以但不限于作为饼干、口服咀嚼片和饮料中的一种的原料。食品为饼干时,将上述的坛紫菜多糖加入到纤麦面团中制成饼干即得。
上述米稀的制备方法包括以下步骤:将各原料粉碎研磨,过筛,得到第一混合物,然后将第一混合物进行糊化,得到糊化产物,将糊化产物蒸制熟化后冷冻干燥,最后粉碎,灭菌包装,即制得上述米稀。
优选地,糊化的条件为:温度为70℃~80℃,时间为20~30min。
优选地,蒸制熟化的条件为:温度为100℃~120℃,时间为20~30min。
优选地,冷冻干燥的时间为6~12h。
本发明的有益效果为:本发明公开了坛紫菜多糖能够对损伤的IEC-6细胞起到增殖修复作用,能够有效增强IEC-6细胞的黏附作用;并对IEC-6细胞中Cdc-42,Rac-1,Par-3,aPKC,PKCβII,paxillin,FAK和GAPDH等8种蛋白的表达上调。将其作为食品例如米稀的原料时,不仅能提高米稀的营养价值,丰富米稀的口感风味,还具备健胃、预防和修复肠胃损伤的效果。
附图说明
图1为检测坛紫菜多糖PHP对损伤的IEC-6细胞增殖作用;
图2为细胞划痕实验检测坛紫菜多糖对IEC-6细胞迁移的影响,在划痕0h,12h,24h时的细胞迁移情况;
图3为荧光染色实验检测坛紫菜多糖PHP对IEC-6细胞的伪足生长状况的影响;
图4为检测坛紫菜多糖PHP对IEC-6细胞的细胞黏附作用;
图5为Western-blot实验,检测坛紫菜多糖PHP对IEC-6细胞蛋白表达的影响。
具体实施方式
以下将结合实施例对本发明的构思及产生的技术效果进行清楚、完整的描述,以充分地理解本发明的目的、方案和效果。需要说明的是,在不冲突的情况下,本申请中的实施例及实施例中的特征可以相互组合。
实施例1:坛紫菜多糖的制备
坛紫菜多糖采用以下方法制备:取干燥坛紫菜作为原料,粉碎,加入原料质量的4倍的蒸馏水进行提取,(温度90℃,搅拌浸提2h),过滤分离滤液,向滤液中加入3倍体积的95%乙醇,静置于4℃冰箱中12~24h,过滤;以蒸馏水溶解沉淀并冻干后获得坛紫菜粗多糖;采用DEAE离子交换树脂对坛紫菜粗多糖进行纯化,以0.2M氯化钠溶液洗脱,收集组分液体,透析,冻干获得纯化坛紫菜多糖PHP,通过以上方法制备的坛紫菜多糖其糖含量为72.4%,蛋白质含量为0.58%,糖醛酸含量为9.37%。
实施例2:坛紫菜多糖对细胞增殖效果
分别用DMEM培养基以25μg/mL,50μg/mL,100μg/mL,200μg/mL,400μg/mL的浓度配制坛紫菜多糖溶液,与IEC-6细胞进行培养24h和48h;分别对不同浓度的坛紫菜多糖溶液培养后的细胞进行计数统计分析,结果如图1所示,坛紫菜多糖表现出对IEC-6有一定的增殖作用,增殖的效果与多糖的浓度以及培养的时间成相关性,在培养24h下,100μg/mL坛紫菜多糖PHP对IEC-6表现出显著的增殖(*p<0.05)。
实施例3:坛紫菜多糖PHP对划痕的修复效果
以6.25×104个细胞每平方厘米的细胞密度将IEC-6细胞接种到六孔培养板中,培养24h,细胞达到90%的融合度后,用200μL的移液吸头对细胞做五处划痕损伤并用PBS缓冲液洗去脱落的细胞,实验组为加入含有100μg/mL的坛紫菜多糖PHP的培养基进行培养,对照组为仅有培养基进行培养,分别在0,12及24小时三个时间点对划痕的愈合情况进行显微镜下检查拍照记录,并对划痕面积进行统计分析。结果如图2所示,划痕经过12h和24h的培养后,对照组和实验组的划痕面积均表现为减小的趋势,而含有100μg/mL的坛紫菜多糖PHP的培养基培养下,实验组的划痕面积较对照组显著减小,特别在24h培养后,实验组中的划痕接近愈合。
实施例4:坛紫菜多糖PHP对细胞运动的影响
按实施例3所述的IEC-6细胞划痕的实验方法建立损伤模型,实验组为含有100μg/mL的坛紫菜多糖PHP的培养基进行培养4小时,对照组为培养基培养4小时,培养后使用5%的BSA溶液对细胞进行封闭30分钟,Actin Green TM 488染色剂对细胞进行孵育,细胞核由DAPI染色;在共聚焦显微镜下对划痕边缘的细胞伪足进行观察,随机选取5个区域对细胞伪足面积进行统计分;结果如图3所示,在划痕边缘我们可以看到,实验组中的细胞伪足生长面积较对照组中的面积显著的扩大(**p<0.01)。
实施例5:坛紫菜多糖PHP对细胞黏附的影响
实验组为100μg/mL的坛紫菜多糖PHP处理的96孔细胞培养板,阳性对照组为40μg/mL的I型胶原蛋白溶液处理的96孔细胞培养板,空白对照为未做任何处理的96孔细胞培养板,分别接种IEC-6细胞,培育4小时后用PBS缓冲液清洗,每孔加入10μL MTT溶液(5mg/mL),继续细胞培养4~6h,吸去培养液加入150μL二甲基亚砜,利用酶标仪在570nm处下进行测定吸光度值。结果如图4所示,与空白对照组相比,坛紫菜多糖PHP处理的培养板中,坛紫菜多糖PHP显著促进了IEC-6细胞的黏附作用(*p<0.05)。
实施例6:坛紫菜多糖PHP对细胞中蛋白的表达影响
按实施例3所述的IEC-6细胞划痕的实验方法建立损伤模型,含有100μg/mL的坛紫菜多糖PHP的培养基进行培养,使用冷的PBS溶液收集细胞,利用Western-blot的方法检测坛紫菜多糖PHP对细胞中的Cdc-42,Rac-1,Par-3,aPKC,PKCβII,paxillin,FAK和GAPDH等8种蛋白的表达。结果如图5所示,PHP对IEC-6中的Cdc-42,Rac-1,paxillin,FAK和PKCβⅡ的表达起到上调作用,结果表明PHP通过增强细胞增殖和迁移而特异性地激活PKCβII并促进粘膜伤口愈合。
实施例7:
一种含有坛紫菜多糖的米稀,由下述步骤制成:
(1)将粳米40~60份、燕麦10~20份、山药10~20份、茯苓10~20份、坛紫菜多糖5~10份、花生5~10份、百合5~10份分别进行浸洗并去除杂质;
(2)将原料分别进行粉碎研磨后,过100目筛;
(3)将各原料过筛后混合得到混合物;
(4)将步骤(3)得到的混合物吸水后在72℃进行糊化,糊化时间30min;
(5)对步骤(4)糊化后的混合物进行蒸制熟化,100℃蒸制20-30min得到混合物;
(6)对步骤(5)的混合物进行冷冻干燥6-12h;
(7)将冷冻干燥后所得到的混合物进行粉碎,粉碎颗粒为均一大小;
(8)灭菌包装,热封避光保存即获得坛紫菜多糖米稀。
Claims (1)
1.坛紫菜多糖在制备修复小肠上皮细胞损伤产品中的应用;
所述坛紫菜多糖通过以下步骤制得:
步骤一、将坛紫菜粉碎后进行水提取,过滤,向滤液中添加乙醇并在0~10℃的条件下静置12~24h;
步骤二、步骤一静置后过滤,向沉淀中加水,然后冻干得到坛紫菜粗多糖,然后经过纯化,洗脱,透析和冻干后制得所述坛紫菜多糖;
步骤一中,坛紫菜与水的质量比为1:(1~5),水提取的条件包括:温度为80~100℃,搅拌浸提2~4h;
步骤一中,滤液与乙醇的体积比为1:(1~5),乙醇为95%乙醇;
步骤二中采用DEAE离子交换树脂对坛紫菜粗多糖进行纯化;采用0.2M氯化钠溶液进行洗脱。
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