CN116098925A - 一种壳寡糖-马尾藻提取物反应物的制备方法和应用 - Google Patents
一种壳寡糖-马尾藻提取物反应物的制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种壳寡糖‑马尾藻提取物反应物的制备方法和应用，属于海洋生物医药领域。该制备方法包括水体预处理、原料溶解、添加马尾藻提取物、静止沉降、离心除杂和透析的步骤，包括：蒸馏水超声脱气1h，调整pH值为8～9；依次加入壳寡糖和马尾藻提取物避光和室温条件下缓慢搅拌至充分溶解；于4℃过夜静止；冷冻高速离心，去除沉淀；上清液在去离子水中透析除杂，即得。本发明通过壳聚糖与马尾藻提取物反应得到壳寡糖‑马尾藻提取物反应物，其可以通过抑制角质形成细胞产生PEG2，从而减轻对皮下感受器的刺激达到舒缓功效，具有抗炎与舒缓作用，在医用消炎领域具有潜在应用前景。
Description
技术领域
本发明属于海洋生物医药领域，具体涉及一种壳寡糖-马尾藻提取物反应物的制备方法和应用。
背景技术
前列腺素E2(PGE2)是一种具有广泛生物学效应的脂质，属于前列腺素类化合物的一种，是二十碳饱和脂肪酸形成的花生四烯酸经过一系列酶催化反应形成的产物，主要作用是诱发炎症反应。目前为止，有通过西药和中药降低PGE2的含量以此来达到舒缓消炎作用，其中西药有阿司匹林、双氯芬酸钠、美沙拉嗪、氯丙嗪等，中药有紫杉叶素、西黄丸、程氏蠲痹汤等，但以海洋来源的化学成分对PGE2的研究相对较少。
我国拥有丰富的海洋资源，其中马尾藻有130余种，甲壳类动物资源也极其丰富，甲壳素是蟹、虾、小龙虾等甲壳类动物壳的主要成分，通过脱乙酰得到壳寡糖。其中马尾藻具有预防甲状腺肿大、治疗脚气浮肿和水肿、消炎等功效，壳寡糖具有调节肠道微生态、抗肿瘤、抗感染和降低血清胆固醇的作用，均有抗炎功效，而目前还未见海洋来源的壳寡糖结合马尾藻提取物反应物应用于调控角质形成细胞产生PGE2的相关报道。
发明内容
本发明的主要目的是提供一种壳寡糖-马尾藻提取物反应物(COASEC)的制备方法，所得COASEC具有抗炎与舒缓作用，表现为经过COASEC作用后PGE2含量降低，主要作用机制是COASEC与CB2R受体结合从而介导下游炎症抑制作用，还可以通过调节TRPV1来调节神经反应。
本发明的第二目的是提供上述制备方法得到的壳寡糖-马尾藻提取物反应物在抑制角质形成细胞产生前列腺素E2中的应用。
为实现上述目的，本发明采用如下技术方案：
本发明提供一种壳寡糖-马尾藻提取物反应物的制备方法，包括水体预处理、原料溶解、添加马尾藻提取物、静止沉降、离心除杂、透析的步骤，具体地，包括以下步骤：
步骤1、水体预处理：蒸馏水超声脱气1h，调整蒸馏水的pH值在8～9之间；
步骤2、原料溶解：壳寡糖以40:1(g/L)加入到预处理的蒸馏水中，在避光和室温条件下缓慢搅拌30～45min至壳寡糖充分溶解；
步骤3、添加马尾藻提取物：马尾藻提取物以30:1(g/L)加入到充分溶解的壳寡糖溶液中，在避光和室温条件下缓慢搅拌30～45min至马尾藻提取物充分溶解；
步骤4、静止沉降：步骤3中充分溶解后的混合溶液于4℃保存，过夜静止12h；
步骤5、离心除杂：上述静止沉降的混合溶液通过冷冻高速离心机于4℃和10000rmp离心30min，去除沉淀；
步骤6、透析：离心除杂后的上清液转移入截留分子量为3kDa的透析袋中，在去离子水中透析48h，每12h换一次水以除去色素和小分子杂质，得到壳寡糖-马尾藻提取物反应物溶液。
作为优选，还包括抗氧化贮藏的步骤，具体为：透析后的COASEC溶液中加入10％的甘油、0.1％的苯氧乙醇和1％的EDTA，避光缓慢搅拌30min，搅拌均匀后装入棕色瓶中保存。
本发明还提供一种壳寡糖-马尾藻提取物反应物，通过上述壳寡糖-马尾藻提取物反应物的制备方法得到，其分子量为600～1000Da。
本发明还提供所述壳寡糖-马尾藻提取物反应物在抑制角质形成细胞产生前列腺素E2中的应用。
本发明还提供所述壳寡糖-马尾藻提取物反应物在制备具有抗炎与舒缓作用的药物中的应用。
本发明还提供一种组合物，按体积百分比含量由以下组分组成：10％的甘油、0.1％的苯氧乙醇和1％的EDTA，余量为所述壳寡糖-马尾藻提取物反应物。
本发明还提供所述组合物在制备具有抗炎与舒缓作用的药物中的应用。
与现有技术相比，本发明的有益效果在于：本发明通过壳聚糖与马尾藻提取物反应得到壳寡糖-马尾藻提取物反应物，制备方法简单，步骤易于操作，马尾藻和壳寡糖来源丰富，制备过程中脱气避光处理可以有效减少对有效成分的影响，得到的COASEC作用后PGE2含量降低，可以通过其抑制角质形成细胞产生PEG2，从而减轻对皮下感受器的刺激以达到舒缓作用，具有抗炎与舒缓作用，在医用消炎领域具有潜在应用前景。
附图说明
图1是实施例中壳寡糖-马尾藻提取物反应物进行PGE2指标检测的柱状图。
具体实施方式
下面结合具体实施例对本发明的技术方案做进一步解释说明。
实施例1
本实施例提供一种壳寡糖-马尾藻提取物反应物的制备方法，包括水体预处理、原料溶解、添加马尾藻提取物、静止沉降、离心除杂、透析、抗氧化贮藏的步骤，具体步骤如下：
(1)水体预处理：100L蒸馏水超声脱气1h，调整蒸馏水的pH值在8～9之间。
(2)原料溶解：往经过预处理的100L蒸馏水中加入4kg壳寡糖，室温中缓慢搅拌30min，使壳寡糖充分溶解，搅拌过程保证避光进行。
(3)马尾藻提取物添加：将马尾藻提取物3kg加入到充分溶解的壳寡糖溶液中，室温中避光搅拌30min，搅拌保持均速缓慢进行。
(4)静止沉降：充分溶解的混合溶液于4℃环境中保存，过夜静止12h。
(5)离心除杂：将静止分层后的溶液，取上清液通过冷冻高速离心机，于4℃环境下，转速为10000rmp离心30min，去除沉淀。
(6)透析：将离心后上清液转移入透析袋中(截留分子量为3kDa)，在去离子水中透析48h，每12h换一次水,以除去色素和小分子杂质。
(7)抗氧化贮藏：透析后的COASEC溶液，加入10L甘油、100mL苯氧乙醇等抗氧化剂，在避光环境下缓慢搅拌30min，搅拌均匀后装入棕色瓶中保存。
实施例2
本实施例提供一种壳寡糖-马尾藻提取物反应物的制备方法，包括水体预处理、原料溶解、添加马尾藻提取物、静止沉降、离心除杂、透析、抗氧化贮藏的步骤，具体步骤如下：
(1)水体预处理：125L蒸馏水超声脱气1h，调整蒸馏水的pH值在8～9之间。
(2)原料溶解：经过预处理的125L蒸馏水中加入5kg壳寡糖混合物，室温中缓慢搅拌30min，使壳寡糖混合物充分溶解，搅拌过程保证避光进行。
(3)马尾藻提取物添加：将马尾藻提取物3.75kg加入充分溶解的壳寡糖溶液中，室温中避光搅拌30min，搅拌保持均速缓慢进行。
(4)静止沉降：充分溶解的混合溶液于4℃环境中保存，过夜静止12h。
(5)离心除杂：将静止分层后的溶液，取上清液通过冷冻高速离心机，于4℃环境下，转速为10000rmp离心30min，去除沉淀。
(6)透析：将离心后上清液转移入透析袋中(截留分子量为3kDa)，在去离子水中透析48h，每12h换一次水,以除去色素和小分子杂质。
(7)抗氧化贮藏：透析后的COASEC溶液，加入12.5L甘油、125mL苯氧乙醇等抗氧化剂，在避光环境下缓慢搅拌30min，搅拌均匀后装入棕色瓶中保存。
实施例3
本实施例提供一种壳寡糖-马尾藻提取物反应物的制备方法，包括水体预处理、原料溶解、添加马尾藻提取物、静止沉降、离心除杂、透析、抗氧化贮藏的步骤，具体步骤如下：
(1)水体预处理：175L蒸馏水超声脱气1h，调整蒸馏水的pH值在8～9之间。
(2)原料溶解：经过预处理的175L蒸馏水中加入7kg壳寡糖混合物，室温中缓慢搅拌30min，使壳寡糖混合物充分溶解，搅拌过程保证避光进行。
(3)马尾藻提取物添加：将马尾藻提取物5.25kg加入充分溶解的壳寡糖溶液中，室温中避光搅拌30min，搅拌保持均速缓慢进行。
(4)静止沉降：充分溶解的混合溶液于4℃环境中保存，过夜静止12h。
(5)离心除杂：将静止分层后的溶液，取上清液通过冷冻高速离心机，于4℃环境下，转速为10000rmp，离心30min，去除沉淀。
(6)透析：将离心后上清液转移入透析袋中(截留分子量为3kDa)，在去离子水中透析48h，每12h换一次水,以除去色素和小分子杂质。
(7)抗氧化贮藏：透析后的COASEC溶液，加入17.5L甘油、175mL苯氧乙醇等抗氧化剂，在避光环境下缓慢搅拌30min，搅拌均匀后装入棕色瓶中保存。
按照上述实施例1～3相同的制备方法得到壳寡糖-马尾藻提取物反应物，主要使用壳寡糖和马尾藻提取物作为原料，反应物中含有不同大小的壳寡糖产物以及多种马尾藻的有效成分，以多种壳寡糖与马尾藻提取物中的不同成分为基础进行反应，由不同分子量的壳寡糖与马尾藻提取物结合形成COASEC的标志物，其中标志成分的分子量如表1所示。
表1：壳寡糖-马尾藻提取物反应物中标志成分的分子量
注：COASEC中含有多种分子量的标志物，表中取不同数字为代表。
按照上述实施例1～3相同的制备方法得到壳寡糖-马尾藻提取物反应物，以角质形成细胞为检测模型，测定COASEC对PGE2含量的影响，方法为：以角质形成细胞为检测对象，通过ELISA实验检测角质形成细胞中PGE2含量的变化，分别设置不给予样品添加以及刺激条件的空白对照组(BC)、无样品添加且经UVB(300mJ/cm2)刺激作为阴性对照(NC)、添加0.01％地塞米松且经UVB(300mJ/cm2)刺激作为阳性对照组(PC)、添加0.0781％壳寡糖-马尾藻混合物且经UVB(300mJ/cm2)刺激最为样品组(SC)，检测如表2和图1所示：
NC组与BC组相比，NC组的PGE2含量显著上升，说明本测试刺激条件有效；
PC组与NC组相比，PC(地塞米松)组的PGE2含量显著下降，说明本阳性对照检测有效；
SC组与NC组相比，COASEC的PGE2含量显著下降(P＜0.01)，表明COASEC可以有效抑制角质形成细胞的PGE2生成。
表2：壳寡糖-马尾藻提取物反应物的PGE2指标检测
注：空白对照：BC；阴性对照：NC；阳性对照：PC；样品组：SC；用t-test方法进行统计分析时，NC组与BC组相比，显著性以#表示，P-value＜0.05表示为#，P-value＜0.01表示为##；PC组、SC组与NC组相比，显著性以*表示，P-value＜0.05表示为*，P-value＜0.01表示为**。
以上所述仅为本发明的可选实施例，本领域普通技术人员可以在此基础上进行各种变换或改进，在不脱离本发明总的构思的前提下，这些变换或改进都应当属于本发明要求保护的范围之内。
Claims (7)
1.一种壳寡糖-马尾藻提取物反应物的制备方法，其特征在于，包括水体预处理、原料溶解、添加马尾藻提取物、静止沉降、离心除杂和透析的步骤，包括以下步骤：
步骤1、水体预处理：蒸馏水超声脱气1h，调整蒸馏水的pH值在8～9之间；
步骤2、原料溶解：壳寡糖以40:1(g/L)加入到预处理的蒸馏水中，在避光和室温条件下缓慢搅拌30～45min至壳寡糖充分溶解；
步骤3、添加马尾藻提取物：马尾藻提取物以30:1(g/L)加入到充分溶解的壳寡糖溶液中，在避光和室温条件下缓慢搅拌30～45min至马尾藻提取物充分溶解；
步骤4、静止沉降：步骤3中充分溶解后的混合溶液于4℃过夜静止12h；
步骤5、离心除杂：上述静止沉降后的混合溶液于4℃和10000rmp离心30min，去除沉淀；
步骤6、透析：离心除杂后的上清液转移入截留分子量为3kDa的透析袋中，在去离子水中透析48h，每12h换一次水除去色素和小分子杂质，即得。
2.根据权利要求1所述壳寡糖-马尾藻提取物反应物的制备方法，其特征在于，还包括抗氧化贮藏的步骤，具体为：透析后的壳寡糖-马尾藻提取物反应物溶液中加入10％的甘油、0.1％的苯氧乙醇和1％的EDTA，避光缓慢搅拌30min，搅拌均匀后避光保存。
3.一种壳寡糖-马尾藻提取物反应物，通过权利要求1或2所述壳寡糖-马尾藻提取物反应物的制备方法得到，其分子量为600～1000Da。
4.权利要求3所述壳寡糖-马尾藻提取物反应物在抑制角质形成细胞产生前列腺素E2中的应用。
5.权利要求3所述壳寡糖-马尾藻提取物反应物在制备具有抗炎与舒缓作用的药物中的应用。
6.一种组合物，其特征在于，按体积百分比含量由以下组分组成：10％的甘油、0.1％的苯氧乙醇和1％的EDTA，余量为权利要求3所述的壳寡糖-马尾藻提取物反应物。
7.权利要求6所述组合物在制备具有抗炎与舒缓作用的药物中的应用。
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