CN117503810B - 一种免疫调节组合物及其应用 - Google Patents
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Abstract
本发明涉及一种免疫调节组合物及其应用，该免疫调节组合物包括表没食子儿茶素没食子酸酯、人参粉和聚葡萄糖。本发明还提供了该免疫调节组合物在作为提升人体免疫力、抗炎、抑制M1型巨噬细胞极化和促进M2型巨噬细胞极化产品中的应用。本发明提供的免疫调节组合物中，各成分通过协同作用，可从细胞层面有效抑制巨噬细胞M1极化，促进巨噬细胞M2极化，降低促炎细胞因子分泌水平，抑制炎症反应，实现机体免疫应答能力的快速提升。
Description
技术领域
本发明涉及免疫调节功能性产品，具体涉及一种免疫调节组合物及其应用。
背景技术
随着现代生活节奏的加快，工作压力的增大，作息不规律以及膳食不平衡，加之工业化生产对环境的破坏和恶劣天气如雾霾的产生，人类的免疫系统功能紊乱发生率呈上涨趋势，抵御病原菌的能力也随之下降。这种情况下，人体免疫系统可能出现原发性或继发性功能紊乱，进而导致原发性免疫缺陷病、继发性免疫缺陷病、过敏性疾病、自身免疫性疾病、风湿免疫性疾病，感染性疾病以及肿瘤等疾病的发生。因此，加强免疫力对于预防疾病、应对病毒感染以及促进整体健康具有重要意义。
目前日常使用的免疫调节药物多通过直接调节抗炎因子和促炎因子的表达水平来实现抗炎和免疫调节作用，这类药物需要长期服用且见效时间相对较慢，同时现有的免疫调节药物的组成成分复杂、制作成本较高，难以实现药物的广泛应用和普及使用。
M1型巨噬细胞和M2型巨噬细胞是两种巨噬细胞的亚型，它们在免疫系统中扮演不同的型角色和功能。M1型巨噬细胞通常在免疫因子和细胞活化型免疫反应中发挥作用。M2型巨噬细胞通常在组织修复、免疫炎症和抗炎过程中发挥作用。在巨噬细胞的分化过程中，M1型巨噬细胞和M2型巨噬细胞的极化比例对免疫力的调节具有直接作用。但现有免疫调节组合物中尚未有可以直接从细胞组织层面上影响M1型巨噬细胞和M2型巨噬细胞的极化比例，来实现免疫调节作用的组合物。
发明内容
为了解决现有免疫调节药物存在的上述问题，本发明提供了一种免疫调节组合物及其应用。
为了实现上述目的，本发明采用以下技术方案：
一种免疫调节组合物，包括表没食子儿茶素没食子酸酯（epigallocatechin-3-gallate，EGCG）、人参粉和聚葡萄糖（polydextrose，PDX）；
所述表没食子儿茶素没食子酸酯、所述人参粉和所述聚葡萄糖的质量比为1:(0.62-10):(0.6-46.16)。
优选的，所述表没食子儿茶素没食子酸酯、所述人参粉和所述聚葡萄糖的质量比为1:(0.62-10):(13.3-46.16)。
优选的，所述表没食子儿茶素没食子酸酯、所述人参粉和所述聚葡萄糖的质量比为1:(1.79-10):(13.3-46.16)。
优选的，所述表没食子儿茶素没食子酸酯、所述人参粉和所述聚葡萄糖的质量比为1:1.79:46.16。
优选的，所述人参粉中人参皂苷的质量含量在8%及以上。
优选的，所述聚葡萄糖固形物的纯度为90%及以上。
优选的，所述表没食子儿茶素没食子酸酯的纯度为94%及以上。
本发明还提供了一种保健品，所述保健品包含所述的免疫调节组合物。所述保健品为乳制品。
本发明还提供了所述的免疫调节组合物在制备免疫调节产品中的应用，所述产品选自保健品和药品中的一种。
本发明还提供了所述的免疫调节组合物在制备抗炎产品中的应用，所述产品选自保健品和药品中的一种。
本发明还提供了所述的免疫调节组合物在制备抑制M1型巨噬细胞极化产品中的应用。
本发明还提供了所述的免疫调节组合物在制备促进M2型巨噬细胞极化产品中的应用。
M1型巨噬细胞分泌促炎细胞因子（TNF-α和IL-1β），能促进炎症的发展，加速细胞外基质降解和细胞凋亡，调节并促进Th1型免疫应答。M2型巨噬细胞分泌抗炎细胞因子，促进T细胞的增殖和活化，调节Th2型免疫应答，有助于组织重塑。本发明提供的免疫调节组合物中，通过EGCG、人参粉和PDX三者的协同作用，可从细胞层面有效抑制巨噬细胞M1极化，促进巨噬细胞M2极化，有效降低促炎细胞因子，抑制炎症反应，实现机体免疫应答能力的快速提升。
附图说明
图1为实施例1中不同待测试剂在不同浓度下对巨噬细胞的细胞活力影响曲线图；
图2为实施例2中各成分剂量为表1所示的情况下，EGCG、人参粉和PDX复配组合物，EGCG和人参粉复配组合物以及PDX对M1型巨噬细胞极化的抑制作用柱形图；
图3为实施例2中各成分剂量为表2所示的情况下，EGCG、人参粉和PDX复配组合物，EGCG和人参粉复配组合物以及PDX对M1型巨噬细胞极化的抑制作用柱形图；
图4为实施例2中各成分剂量为表3所示的情况下，EGCG、人参粉和PDX复配组合物，EGCG和人参粉复配组合物以及PDX对M1型巨噬细胞极化的抑制作用柱形图；
图5为实施例2中各成分剂量为表1所示的情况下，EGCG、人参粉和PDX复配组合物，EGCG和人参粉复配组合物以及PDX对M2型巨噬细胞极化的促进作用柱形图；
图6为实施例2中各成分剂量为表2所示的情况下，EGCG、人参粉和PDX复配组合物，EGCG和人参粉复配组合物以及PDX对M2型巨噬细胞极化的促进作用柱形图；
图7为实施例2中各成分剂量为表3所示的情况下，EGCG、人参粉和PDX复配组合物，EGCG和人参粉复配组合物以及PDX对M2型巨噬细胞极化的促进作用柱形图；
图8为对比例中检测的不同PDX浓度下对促炎因子TNF-α分泌水平的影响曲线图；
图9为对比例中检测的不同酵母β-葡聚糖（Yeast beta-glucan, YBG）浓度下对促炎因子TNF-α分泌水平的影响曲线图；
图10为对比例中表4和表5中不同配比组合物对促炎因子TNF-α分泌水平的影响柱形图。
图11为对比例中表6和表7中不同配比组合物对M1型巨噬细胞极化的抑制作用柱形图。
图12为对比例中表6和表7中不同配比组合物对M2型巨噬细胞极化的促进作用柱形图。
具体实施方式
下面结合附图和实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。本申请中未明示来源的成份均可从市售渠道获取。
实施例1
细胞活性检测
1、材料与试剂
RAW264.7巨噬细胞、脂多糖（LPS）、表没食子儿茶素没食子酸酯（EGCG）、聚葡萄糖（PDX）、人参粉、TNF-α ELISA Kit（江苏酶标生物科技有限公司，试剂盒型号MB2868A）、细胞培养基、PBS缓冲液。所述聚葡萄糖固形物的纯度为90%，所述表没食子儿茶素没食子酸酯的纯度为94%，所述人参粉中人参皂苷的质量百分含量为8%。
2、细胞活性
基于CCK-8测定复方组合物干预细胞后细胞增殖能力。接种细胞并设置8个细胞浓度梯度（1μg/mL、5μg/mL、10μg/mL、20μg/mL、40μg/mL、60μg/mL、801μg/mL、100μg/mL），每100μL培养基加10μL的待测试剂（EGCG、人参粉或PDX），添加CCK-8后在培养箱中孵育至OD值约等于1，并计算各待测组的细胞活力。
EGCG、人参粉和PDX在不同浓度下对巨噬细胞存活率的影响如图1所示。
用不同浓度的待测试剂处理细胞后，结果显示人参粉及PDX无明显细胞毒性，且人参粉对细胞增殖有促进作用，EGCG在20μg/mL的浓度下呈现一定的细胞毒性作用。
实施例2
分别用以下三组EGCG、人参粉和PDX复配质量比例及剂量进行后续抗炎功效验证实验。
配比I：待测组合物质量配比为EGCG：人参粉：PDX=1:0.62:0.6；
待测组合物用量如表1所示。
表1 待测组合物用量
配比II：待测组合物质量配比为EGCG：人参粉：PDX=1:1.79:46.16；
待测组合物用量如表2所示。
表2 待测组合物用量
配比III：待测组合物质量配比为EGCG：人参粉：PDX=1:10:13.3；
待测组合物用量如表3所示。
表3待测组合物用量
分别用上述三组待测组合物进行巨噬细胞极化检测。
将对数生长期的RAW264.7细胞，以2×105个/mL的浓度接种于24孔板中，过夜培养后，在37℃，5%CO2条件下培养过夜后，用PBS清洗，每孔加入不含血清的DMEM培养基，终浓度100ng/mL的LPS，20ng/mL的IFN-γ诱导细胞向M1型极化，加入终浓度为20 ng/mL的IL-4诱导细胞向M2极化，同时加入不同配比的组合物（对照组加入等量的PBS），培养24 h后，收集M1型和M2型的细胞进行极化检测。
极化的检测方法:
M1型巨噬细胞极化检测方法：
1.收集细胞到流式管中，用预冷的PBS清洗两遍，去除培养基，每次离心300×g，4℃，5min。
2.孵育CD16/32 4℃封闭10min。
3.预冷的PBS清洗三次，每次离心300×g，4℃，5min。
4.孵育F4/80和CD86抗体，4℃孵育30min。
5.预冷的PBS清洗三次，每次离心300×g，4℃，5min。将细胞重悬于300uL PBS中，过细胞网筛上机检测。
M2型巨噬细胞极化检测方法：
1.收集细胞到流式管中，用预冷的PBS清洗两遍，去除培养基，每次离心300×g，4℃，5min。
2.孵育CD16/32 4℃封闭10min。
3.预冷的PBS清洗三次，每次离心300×g，4℃，5min。
4.孵育F4/80抗体，4℃孵育30min。
5.预冷的PBS清洗三次，每次离心300×g，4℃，5min。
6.将细胞重悬于250uL固定液中，4℃，20min。
7.用破膜液清洗细胞两次，每次离心400×g，4℃，5min。
8.在100uL破膜液中孵育CD206抗体，4℃，30min。
9.预冷的PBS清洗三次，每次离心400×g，4℃，5min。将细胞重悬于300uL PBS中，过细胞网筛上机检测。
不同EGCG、人参粉和PDX复配比例下，对M1型巨噬细胞极化的抑制作用如图2-图4所示。
由图2-图4可知，和对照组相比，EGCG、人参粉和PDX复配物可抑制M1型巨噬细胞极化，呈现抗炎功效。另外，在上述三组不同的复配组合物中，“EGCG+人参粉+PDX”组的M1型巨噬细胞比例均低于“EGCG+人参粉”组和“PDX”组，即复配物对M1型巨噬细胞极化的抑制作用呈现协同性。
不同EGCG、人参粉和PDX复配比例下，对M2型巨噬细胞极化的促进作用如图5-图7所示。
由图5-图7可知，和对照组相比，EGCG、人参粉和PDX复配物可促进M2型巨噬细胞极化，呈现抗炎功效。另外，在上述三组不同的复配组合物中，“EGCG+人参粉+PDX”组的M2型巨噬细胞比例均高于“EGCG+人参粉”组和“PDX”组，即复配物对M2型巨噬细胞极化的促进作用呈现协同性。
对比例
酵母β-葡聚糖（YBG）和PDX一样，是一种益生元，其为酵母细胞中存在的一种活性物质，是由β-1,3键相连的D-葡萄糖组成的线性骨架及通过β-1,6键相连的分支结构构成，因具有多种对健康有益的生物学活性，在食品、医药等行业中被广泛应用。细胞、动物、人群研究均发现酵母β-葡聚糖表现出对免疫细胞、受体及细胞因子等的调节作用。
在此对比例中，以TNF-α作为参考指标，在特定复配比例和剂量下，比较“EGCG+人参粉+PDX”和“EGCG+人参粉+YBG”复配物的抗炎功效。具体方法如下：
确定PDX、YBG的比例。基于前述细胞毒性实验设置干预物浓度梯度进行干预物效应实验，以TNF-α为指标，确定半最大效应浓度（concentration for 50% of maximaleffect，EC50）用于复配物比例确定。经非线性拟合形成效应曲线并计算得到PDX、YBG的EC50，如图8、图9所示，当处于各自的EC50时，YBG EC50=8.3μg/mL，PDX EC50=5.1μg/mL，对TNF-α的抑制效应相当，因此对比实验中设置YBG与PDX的质量比为YBG:PDX=1.63:1。
分别根据以下复配质量比例及剂量进行实验：
1）EGCG：人参粉：PDX=1:0.62:0.6；
各成分剂量如表4所示。
表4各成分剂量
2）EGCG：人参粉：YBG=1:0.62:0.98；
各成分剂量如表5所示。
表5各成分剂量
上述两种复配物降低促炎因子TNF-α分泌水平作用的效果如图10所示。
由图10可知，“EGCG+人参粉+PDX”组对两种细胞因子的降低作用明显优于“EGCG+人参粉+YBG”组，且不同剂量呈现稳定的作用效果，而“EGCG+人参粉+YBG”组的干预作用在不同剂量下呈现不同的作用效果，即“EGCG+人参粉+PDX”复配在抗炎效果上优于“EGCG+人参粉+YBG”复配。
同时，按照如下配比和剂量比较“EGCG+人参粉+PDX”与“EGCG+人参粉+YBG”复配物对M1、M2型巨噬细胞极化的影响。
1）EGCG：人参粉：PDX=1:0.62:0.6；
各成分剂量如表6所示。
表6各成分剂量
2）EGCG：人参粉：YBG=1:0.62:0.98；
各成分剂量如表7所示。
表7各成分剂量
上述两种复配物抑制M1型巨噬细胞极化作用的效果如图11所示，上述两种复配物促进M2型巨噬细胞极化作用的效果如图12所示。由图11可知，“EGCG+人参粉+PDX”组对M1型巨噬细胞极化的抑制作用明显优于“EGCG+人参粉+YBG”组，由图12可知，“EGCG+人参粉+PDX”组对M2型巨噬细胞极化的促进作用也明显优于“EGCG+人参粉+YBG”组，证明“EGCG+人参粉+PDX”复配在抗炎效果上优于“EGCG+人参粉+YBG”复配。
Claims (9)
1.一种免疫调节组合物，其特征在于：由表没食子儿茶素没食子酸酯、人参粉和聚葡萄糖组成；所述表没食子儿茶素没食子酸酯、所述人参粉和所述聚葡萄糖的质量比为 1:(0.62-10): (0.6-46.16)；所述人参粉中人参皂苷的质量百分含量在8%及以上；所述聚葡萄糖固形物的纯度为90%及以上；所述表没食子儿茶素没食子酸酯的纯度为94%及以上。
2.如权利要求1所述的免疫调节组合物，其特征在于：所述表没食子儿茶素没食子酸酯、所述人参粉和所述聚葡萄糖的质量比为1:(0.62-10):(13.3-46.16)。
3.如权利要求1所述的免疫调节组合物，其特征在于：所述表没食子儿茶素没食子酸酯、所述人参粉和所述聚葡萄糖的质量比为1:(1.79-10):(13.3-46.16)。
4.如权利要求1所述的免疫调节组合物，其特征在于：所述表没食子儿茶素没食子酸酯、所述人参粉和所述聚葡萄糖的质量比为1:1.79:46.16。
5.一种保健品，其特征在于，所述保健品包含权利要求1-4任一项所述的免疫调节组合物。
6.如权利要求5所述的保健品，其特征在于，所述保健品为乳制品。
7.权利要求1-4任一项所述的免疫调节组合物在制备免疫调节产品中的应用，所述产品选自保健品和药品中的一种。
8.权利要求1-4任一项所述的免疫调节组合物在制备抗炎产品中的应用，所述产品选自保健品和药品中的一种。
9.权利要求1-5任一项所述的免疫调节组合物在制备抑制M1型巨噬细胞极化产品中的应用，或，制备促进M2型巨噬细胞极化产品中的应用。
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