WO2019237253A1 - 裙带菜来源的岩藻聚糖及其制备方法和应用 - Google Patents

裙带菜来源的岩藻聚糖及其制备方法和应用 Download PDF

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WO2019237253A1
WO2019237253A1 PCT/CN2018/090869 CN2018090869W WO2019237253A1 WO 2019237253 A1 WO2019237253 A1 WO 2019237253A1 CN 2018090869 W CN2018090869 W CN 2018090869W WO 2019237253 A1 WO2019237253 A1 WO 2019237253A1
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wakame
fucoidan
supernatant
preparation
derived
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French (fr)
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续旭
喻波明
陆隽
毕德成
赖秋娴
韩庆国
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Shenzhen University
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Shenzhen University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof

Definitions

  • the invention belongs to the field of biochemical technology, and particularly relates to fucoidan derived from wakame, and a preparation method and application thereof.
  • Fucoidan derived from brown algae has received continuous attention and extensive research in the fields of medicine and functional food.
  • Fucoidan is a water-soluble sulfated polysaccharide containing fucose as the main component and a small amount of galactose, xylose, and uronic acid. It is mainly found in the cell wall and intercellular space of brown algae. 40% of dry weight of brown algae cells, 16% of dry weight of brown algae.
  • the mammalian immune system consists of innate and acquired immune systems.
  • the innate immune system is mediated by phagocytes such as macrophages and neutrophils, and is an important line of defense against pathogens.
  • activated macrophages play an important role in host defense against infection and tumors by secreting inflammatory mediators such as nitric oxide (NO) and pro-inflammatory cytokines.
  • NO nitric oxide
  • cytokines pro-inflammatory mediators
  • iNOS Inducible nitric oxide synthase catalyzes L-arginine to produce NO is the only way for cells to produce NO. NO participates in non-specific and specific immune responses. It is both a signaling molecule and a cytotoxic molecule.
  • cytokines can affect the proliferation of lymphocytes and promote the release of cytokines to mediate immune effects. It has a strong killing effect on tumor cells and pathogenic microorganisms. In addition, the cytokines tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6) play an important role in enhancing the body's immunity.
  • TNF-a tumor necrosis factor-a
  • IL-6 interleukin-6
  • the purpose of the present invention is to overcome the above-mentioned shortcomings of the prior art, provide a fucoidan derived from wakame, and a preparation method and application thereof. technical problem.
  • One aspect of the present invention provides a method for preparing fucoidan, including the following steps:
  • a precipitating agent is added to the second supernatant to obtain the fucoidan.
  • a wakame-derived fucoidan is obtained, which is obtained by the above-mentioned preparation method of the present invention.
  • the present invention provides an application of fucoidan obtained by the above-mentioned preparation method of the present invention in preparing an immunomodulator.
  • the fucoidan preparation method provided by the present invention uses wakame as a raw material and uses water extraction to obtain a low molecular weight fucoidan from the wakame.
  • high performance gel permeation chromatography HPGPC
  • the molecular weight of fucoidan derived from Wakame was less than 10 kDa.
  • the fucoidan can promote the expression of iNOS, and at the same time can increase the production of TNF-a and IL-6. Therefore, it can prepare an immunomodulator for the development of immunomodulatory drugs or health products.
  • Example 1 is a molecular weight distribution diagram of fucoidan derived from Wakame New Zealand prepared in Example 1 of the present invention
  • FIG. 2 is a cell viability experiment result chart of fucoidan derived from New Zealand wakame in Example 2 of the present invention
  • FIG. 3 is a graph showing the results of increasing the NO secretion of RAW264.7 cells by fucoidan derived from Wakame New Zealand in Example 2 of the present invention
  • Example 4 is a nucleic acid electrophoresis diagram of fucoidan derived from Wakame New Zealand in Example 3 of the present invention, which significantly improves the expression of iNOS gene in RAW264.7 cells;
  • Example 5 is a protein electrophoresis diagram of fucoidan derived from Wakame New Zealand in Example 3 of the present invention, which significantly improves the expression of iNOS protein in RAW264.7 cells;
  • Example 6 is a graph showing the result of increasing fucoidan derived from Wakame New Zealand in Example 4 of the present invention to increase TNF-a secretion by RAW264.7 cells;
  • FIG. 7 is a graph showing the results of fucoidan derived from Wakame New Zealand in Example 4 of the present invention increasing IL-6 secretion by RAW264.7 cells.
  • an embodiment of the present invention provides a method for preparing fucoidan, including the following steps:
  • the fucoidan preparation method provided by the embodiment of the present invention uses wakame as a raw material and uses water extraction to obtain a low-molecular weight fucoidan from wakame.
  • Wakame-derived fucoidan has a molecular weight of less than 10 kDa.
  • the fucoidan can promote the expression of iNOS, and at the same time can increase the production of TNF-a and IL-6. Therefore, it can prepare an immunomodulator for the development of immunomodulatory drugs or health products.
  • step S01 the wakame is New Zealand wakame.
  • the conditions for the heat treatment are: a temperature of 65-75 ° C, and a time of 8-12 hours. Under the conditions of the heating temperature and time, the wakame can be quickly dissolved in water to obtain a more uniform and uniform wakame dissolving solution for subsequent fucoidan extraction. Further, the temperature of the heat treatment is preferably 70 ° C. and the time is 10 hours.
  • the conditions of the first centrifugal treatment are: centrifugal force 180-1900 ⁇ g, centrifugation time 110-130 min, further preferably centrifugal force 18500 ⁇ g, centrifugation time 120 min; the second The conditions of centrifugation are: centrifugal force 180-1900 ⁇ g, centrifugation time 110-130 min, further preferably centrifugal force 18500 ⁇ g, centrifugation time 120 min. Under this centrifugation condition, the target supernatant can be better obtained.
  • the mass ratio of the wakame to the water is 1: 3-4, and within the range of the mass ratio, the wakame is sufficiently dissolved in water to extract fucoidan with a high content.
  • the mass ratio of the wakame to the calcium hydroxide is 1: 0.1-0.2, and the calcium hydroxide can precipitate alginate impurities in the wakame to improve the purity of the fucoidan, and within the range of the mass ratio , Can completely remove alginate impurities.
  • the mass ratio of the wakame to the calming agent is 1: 6-8.
  • fucoidan can be better precipitated.
  • the precipitating agent is ethanol, and the effect of ethanol for fucoidan precipitation is better.
  • step S05 after the step of adding a precipitating agent to the second supernatant liquid, a freeze-drying step is further included, and the freeze-drying conditions are: minus 15 ° C-minus 5 ° C That is, (-15 °C) ⁇ (-5 °C), the time is 22-26 h. Under these freeze-drying conditions, fucoidan having high purity and suitable for long-term storage can be obtained.
  • a wakame-derived fucoidan is obtained, which is obtained by the above-mentioned preparation method of the present invention, and the molecular weight of the fucoidan is less than 10 kDa.
  • the embodiment of the present invention provides an application of fucoidan obtained from the preparation method of the embodiment of the present invention in the preparation of an immunomodulator.
  • the immunomodulator promotes iNOS protein expression or promotes TNF-a and IL-6 secretion.
  • the experimental lipopolysaccharide (LPS) in the examples of the present invention was purchased from Sigma; DMEM medium, penicillin, and streptomycin were purchased from Gibco, USA; fetal bovine serum was purchased from Israeli BI company; TNF-a And IL-6 ELISA kits were purchased from Xinbosheng Biological Company; CCk-8 and cell lysate were purchased from Beyotime Company; total RNA extraction kits were purchased from Shanghai Feijie Biotechnology Company; reverse transcription kits were purchased from Takara, Japan Company; iNOS monoclonal antibody was purchased from CST, Germany; developer and fixing solution were purchased from Themo Fisher Scientific.
  • LPS lipopolysaccharide
  • the crushed New Zealand wakame was placed in 70 ° C hot water and stirred at a constant temperature overnight. After the system was completely cooled to room temperature, the supernatant was centrifuged, and the supernatant was removed by adding calcium hydroxide to the supernatant and centrifuged. Then, ethanol was added to precipitate the fucoidan, followed by freeze-drying to obtain fucoidan powder.
  • the molecular weight of fucoidan derived from Wakame New Zealand was less than 10 kDa by HPGPC (see Figure 1).
  • Example 2 New Zealand wakame-derived fucans increase NO secretion from RAW264.7 cells
  • RAW264.7 cells (1 ⁇ 10 5 cells / well) were cultured in a 96-well plate. After 4-6 h, the supernatant was discarded, and fucoidan derived from New Zealand wakame prepared in Example 1 was added at different concentrations to stimulate. After 24 h, the NO content in the culture medium was detected by the Griess method.
  • the CCK-8 kit was used to examine the toxicity of fucoidan derived from Wakame New Zealand to RAW264.7 cells.
  • the CCK-8 cell viability test kit found that fucoidan derived from Wakame New Zealand at different concentrations was not toxic to RAW264.7 cells (see Figure 2). It was found in concentration gradient experiments that fucoidan derived from New Zealand Wakame can increase NO secretion from RAW264.7 cells, and it increased with the concentration of fucoidan derived from New Zealand Wakame, showing a concentration dependence (see Figure 3 ).
  • RAW264.7 cells (1 ⁇ 10 6 cells / well) were cultured in 6-well plates. After 4-6 hours, the supernatant was discarded, and fucoidan from New Zealand Wakame was added at different concentrations. After stimulating for 12 hours, The total RNA extraction kit extracts total RNA, and then uses the reverse transcription kit to reverse transcribe the total RNA into cDNA, and then amplifies the iNOS gene by a PCR instrument.
  • iNOS primers are: FE 5 '-CAACCAGTATTATGGCTCCT-3', RE 5 '-GTGACAGCCCGGTCTTTCCA-3';
  • the ⁇ -actin primers are: FE 5 '-GGAGAAGATCTGGCACCACACC-3', RE 5 '-CCTGCTTGCTGATCCACATCTGCTGG-3'.
  • RAW264.7 cells (1 ⁇ 10 6 cells / well) were cultured in 6-well plates. After 4-6 h, the supernatant was discarded, and fucoidan derived from Wakame from New Zealand was added at different concentrations. After stimulating for 24 h, The total protein was extracted from the cell lysate, and then the protein expression of iNOS was detected by Western Blot. The results are shown in Fig. 5. Fucosan derived from Wakame New Zealand can increase iNOS protein expression in RAW264.7 cells in a concentration-dependent manner.
  • RAW264.7 cells (1 ⁇ 10 5 cells / well) were cultured in 96-well plates. After 4-6 h, the supernatant was discarded, and fucoidan from New Zealand wakame was added at different concentrations. After 24 h of stimulation, ELISA kit was used to detect the production of TNF-a and IL-6 in the culture medium. The results are shown in Figs. 6 and 7. The fucoidan derived from Wakame New Zealand can increase the secretion of TNF-a and IL-6 by RAW264.7 cells in a concentration-dependent manner.
  • Examples 1-4 illustrate that using New Zealand wakame as a raw material, low-molecular-weight fucoidan (fucoid derived from New Zealand wakame) can be obtained by water extraction, and fucoidan derived from New Zealand wakame can be effective Increased secretion of cytokines by RAW264.7 cells.
  • fucoidan derived from New Zealand wakame has no cytotoxicity to RAW264.7 cells; fucoidan derived from New Zealand wakame can effectively increase NO secretion from RAW264.7 cells, and Its effect is concentration-dependent; fucoidan from New Zealand wakame can effectively increase iNOS gene and protein expression of RAW264.7 cells, and its effect is concentration-dependent; fucoidan from New Zealand wakame can effectively increase RAW264.7 cells secrete TNF-a and IL-6, and their effects are concentration-dependent.

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Abstract

一种裙带菜来源的岩藻聚糖及其制备方法和应用。该制备方法包括如下步骤：提供裙带菜；将所述裙带菜粉碎后溶于水中，进行加热处理，得到裙带菜溶解液；将所述裙带菜溶解液进行第一离心处理，得到第一上清液；将氢氧化钙加入所述第一上清液中，进行第二离心处理，得到第二上清液；将沉淀剂加入所述第二上清液中，得到所述岩藻聚糖。该制备方法提取的岩藻聚糖可以促进诱导型一氧化氮合酶(iNOS)的表达，同时还能提高TNF-a和IL-6的生成，因此，其可以制备免疫调节剂，用于免疫调节药物或保健品用途的开发。

Description

裙带菜来源的岩藻聚糖及其制备方法和应用
本发明属于生物化学技术领域，具体涉及一种裙带菜来源的岩藻聚糖及其制备方法和应用。
来源于褐藻的岩藻聚糖在医药和功能食品领域受到持续关注并且被广泛研究。岩藻聚糖是一种以岩藻糖为主要成分同时还伴随着少量的半乳糖、木糖和糖醛酸的水溶性硫酸多糖，其主要存在于褐藻的细胞壁与细胞间隙中，其约占褐藻细胞干重的40%，褐藻干重的16%。
哺乳动物免疫系统由固有免疫和获得性免疫系统组成。固有免疫系统是由巨噬细胞和中性粒细胞等吞噬细胞所介导的，是防御病原体的重要防线。作为固有免疫和获得性免疫系统的重要组成部分，激活状态的巨噬细胞通过分泌一氧化氮（NO）和促炎细胞因子等炎症介质，在抗感染和肿瘤的宿主防御中扮演着重要的角色。诱导型一氧化氮合酶（iNOS）催化左旋精氨酸产生NO是细胞生成NO的唯一途径。NO参与非特异性与特异性免疫反应，既是信号分子，也是细胞毒性分子，能够影响淋巴细胞的增殖和促进细胞因子的释放从而介导免疫效应，对肿瘤细胞和病原微生物产生很强的杀伤作用。另外，细胞因子肿瘤坏死因子-a（TNF-a）和白介素-6（IL-6）在增强机体免疫力中发挥着重要作用。
发明内容
本发明的目的在于克服现有技术的上述不足，提供一种裙带菜来源的岩藻聚糖及其制备方法和应用，旨在解决现有裙带菜来源的岩藻聚糖提取和医药开发有限的技术问题。
为实现上述发明目的，本发明采用的技术方案如下：
本发明一方面提供一种岩藻聚糖的制备方法，包括如下步骤：
提供裙带菜；
将所述裙带菜粉碎后溶于水中，进行加热处理，得到裙带菜溶解液；
将所述裙带菜溶解液进行第一离心处理，得到第一上清液；
将氢氧化钙加入所述第一上清液中，进行第二离心处理，得到第二上清液；
将沉淀剂加入所述第二上清液中，得到所述岩藻聚糖。
本发明另一方面提供一种裙带菜来源的岩藻聚糖，所述岩藻聚糖由本发明的上述制备方法获得。
最后，本发明提供一种由本发明上述制备方法获得的岩藻聚糖在制备免疫调节剂中的应用。
本发明提供的岩藻聚糖的制备方法，以裙带菜为原材料，采用水提取法从裙带菜中获得一种低分子量岩藻聚糖（low molecular weight fucoidan，裙带菜来源的岩藻聚糖），通过高效凝胶渗透色谱（high performance gel permeation chromatography，HPGPC）检测得知所制备的裙带菜来源的岩藻聚糖分子量小于10 kDa。该岩藻聚糖可以促进iNOS的表达，同时还能提高TNF-a和IL-6的生成，因此，其可以制备免疫调节剂，用于免疫调节药物或保健品用途的开发。
附图说明
图1为本发明实施例1制备的新西兰裙带菜来源的岩藻聚糖分子量的分布图；
图2为本发明实施例2中新西兰裙带菜来源的岩藻聚糖的细胞活力实验结果图；
图3为本发明实施例2中新西兰裙带菜来源的岩藻聚糖提高RAW264.7 细胞分泌NO的结果图；
图4为本发明实施例3中新西兰裙带菜来源的岩藻聚糖提高RAW264.7 细胞的iNOS基因显著表达的核酸电泳图；
图5为本发明实施例3中新西兰裙带菜来源的岩藻聚糖提高RAW264.7 细胞的iNOS蛋白显著表达的蛋白质电泳图；
图6为本发明实施例4中新西兰裙带菜来源的岩藻聚糖提高RAW264.7 细胞分泌TNF-a的结果图；
图7为本发明实施例4中新西兰裙带菜来源的岩藻聚糖提高RAW264.7 细胞分泌IL-6的结果图。
具体实施方式
为了使本发明要解决的技术问题、技术方案及有益效果更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
一方面，本发明实施例提供了一种岩藻聚糖的制备方法，包括如下步骤：
S01：提供裙带菜；
S02：将所述裙带菜粉碎后溶于水中，进行加热处理，得到裙带菜溶解液；
S03：将所述裙带菜溶解液进行第一离心处理，得到第一上清液；
S04：将氢氧化钙加入所述第一上清液中，进行第二离心处理，得到第二上清液；
S05：将沉淀剂加入所述第二上清液中，得到所述岩藻聚糖。
本发明实施例提供的岩藻聚糖的制备方法，以裙带菜为原材料，采用水提取法从裙带菜中获得一种低分子量岩藻聚糖，通过高效凝胶渗透色谱检测得知所制备的裙带菜来源的岩藻聚糖分子量小于10 kDa。该岩藻聚糖可以促进iNOS的表达，同时还能提高TNF-a和IL-6的生成，因此，其可以制备免疫调节剂，用于免疫调节药物或保健品用途的开发。
进一步地，上述步骤S01中，所述裙带菜为新西兰裙带菜。
进一步地，上述步骤S02中，所述加热处理的条件为：温度65-75℃，时间8-12 h。在该加热温度和时间条件下，裙带菜可快速地溶解于水中，得到更加分散均匀的裙带菜溶解液，以备后续提取岩藻聚糖。进一步地，优选加热处理的温度为70℃，时间10 h。
进一步地，上述步骤S03和S04中，所述第一离心处理的条件为：离心力18000-19000×g，离心时间110-130 min，进一步优选离心力18500×g，离心时间120 min；所述第二离心处理的条件为：离心力18000-19000×g，离心时间110-130 min，进一步优选离心力18500×g，离心时间120 min。在该离心条件下，可更好地得到目标上清液。进一步优选地，所述裙带菜与所述水的质量比为1∶3-4，该质量比范围内，裙带菜充分溶解在水中，以高含量地提取岩藻聚糖。所述裙带菜与所述氢氧化钙的质量比为1∶0.1-0.2，氢氧化钙可以沉淀裙带菜中的藻酸盐杂质，以提高岩藻聚糖的纯度，而在该质量比范围内，可将藻酸盐杂质完全去除。
进一步地，上述步骤S05中，所述裙带菜与所述沉定剂的质量比为1∶6-8。该质量比范围内，可更好地沉淀岩藻聚糖。进一步优选地，所述沉淀剂为乙醇，乙醇沉淀岩藻聚糖的效果更佳。
进一步地，上述步骤S05中，在所述将沉淀剂加入所述第二上清液中的步骤之后，还包括冷冻干燥的步骤，所述冷冻干燥的条件为：温度零下15℃-零下5℃，即（-15℃）~（-5℃），时间22-26 h。在该冷冻干燥条件下，可得到高纯度、且适合长期保存的岩藻聚糖。
本发明实施例另一方面提供一种裙带菜来源的岩藻聚糖，所述岩藻聚糖由本发明的上述制备方法获得，且所述岩藻聚糖的分子量小于10 kDa。
最后，本发明实施例提供一种由上述本发明实施例的制备方法获得的岩藻聚糖在制备免疫调节剂中的应用。该免疫调节剂促进iNOS蛋白表达或促进TNF-a和 IL-6分泌。
本发明先后进行过多次试验，现举一部分试验结果作为参考对发明进行进一步详细描述，下面结合具体实施例进行详细说明。
实验原料以及相关试剂：本发明实施例中实验的脂多糖（LPS）购买于Sigma公司；DMEM培养基、青霉素、链霉素购买于美国Gibco公司；胎牛血清购买于以色列BI公司；TNF-a和IL-6 ELISA试剂盒购买于欣博盛生物公司；CCk-8和细胞裂解液购买于Beyotime公司；总RNA提取试剂盒购买于上海飞捷生物技术公司；反转录试剂盒购买于日本Takara公司；iNOS单克隆抗体购买于德国CST公司；显影液、定影液购买于Themo Fisher Scientific公司。
实施例1 新西兰裙带菜来源的岩藻聚糖的制备及分子量确定
将粉碎的新西兰裙带菜放入70℃热水中恒温搅拌过夜，待体系完全冷却至室温离心取上清液，通过在上清液中加入氢氧化钙后离心而去除藻酸盐。然后加入无水乙醇使岩藻聚糖沉淀出来，随后冷冻干燥获得岩藻聚糖粉末。通过 HPGPC测定了新西兰裙带菜来源的岩藻聚糖的分子量小于10 kDa（见图1）。
实施例2 新西兰裙带菜来源的岩藻聚糖提高RAW264.7细胞分泌NO
RAW264.7细胞（1×105个/孔）培养在96孔板中，4-6 h后，弃上清，加入不同浓度的实施例1制备的新西兰裙带菜来源的岩藻聚糖，刺激24 h后，用Griess法检测培养基中的NO含量。并用CCK-8试剂盒检查新西兰裙带菜来源的岩藻聚糖对RAW264.7细胞的毒性。
通过CCK-8细胞活力检测试剂盒发现，不同浓度的新西兰裙带菜来源的岩藻聚糖对RAW264.7细胞没有毒性（见图2）。在浓度梯度实验中发现，新西兰裙带菜来源的岩藻聚糖可以提高RAW264.7细胞分泌NO，并且随着新西兰裙带菜来源的岩藻聚糖浓度增加而增加，呈现浓度依赖性（见图3）。
实施例3 新西兰裙带菜来源的岩藻聚糖提高RAW264.7细胞的iNOS基因及蛋白表达
RAW264.7细胞（1×106个/孔）培养在6孔板中，4-6 h后，弃上清，加入不同浓度的新西兰裙带菜来源的岩藻聚糖，刺激12 h后，用总RNA提取试剂盒提取总RNA，然后用反转录试剂盒将总RNA反转录为cDNA，再通过PCR仪扩增得到iNOS基因。
iNOS引物为：FE 5' -CAACCAGTATTATGGCTCCT-3'，RE 5' -GTGACAGCCCGGTCTTTCCA-3'；
β-actin引物为：FE 5' -GGAGAAGATCTGGCACCACACC-3'，RE 5' -CCTGCTTGCTGATCCACATCTGCTGG-3'。
利用琼脂糖凝胶电泳检测iNOS的基因表达量，结果如图4所示，新西兰裙带菜来源的岩藻聚糖可以提高RAW264.7细胞的iNOS基因表达，并且具有浓度依赖性。
RAW264.7细胞（1×106个/孔）培养在6孔板中，4-6 h后，弃上清，加入不同浓度的新西兰裙带菜来源的岩藻聚糖，刺激24 h后，用细胞裂解液提取总蛋白，然后再利用Western Blot检测iNOS的蛋白表达量。结果如图5所示，新西兰裙带菜来源的岩藻聚糖可以提高RAW264.7细胞iNOS蛋白表达，并且具有浓度依赖性。
实施例4 新西兰裙带菜来源的岩藻聚糖提高RAW264.7细胞分泌细胞因子
RAW264.7细胞（1×105个/孔）培养在96孔板中，4-6 h后，弃上清，加入不同浓度的新西兰裙带菜来源的岩藻聚糖，刺激24 h后，利用ELISA试剂盒检测培养基中TNF-a和IL-6的生成量。结果如图6和图7所示，新西兰裙带菜来源的岩藻聚糖可以提高RAW264.7细胞分泌TNF-a和IL-6，并且具有浓度依赖性。
上述实施例1-4说明：以新西兰裙带菜为原料，通过水提法可获得低分子量岩藻聚糖（新西兰裙带菜来源的岩藻聚糖），新西兰裙带菜来源的岩藻聚糖可以有效提高RAW264.7细胞分泌细胞因子。根据研究结果可得出以下结论：不同浓度的新西兰裙带菜来源的岩藻聚糖对RAW264.7细胞没有细胞毒性；新西兰裙带菜来源的岩藻聚糖可有效提高RAW264.7细胞分泌NO，并且其作用呈现浓度依赖性；新西兰裙带菜来源的岩藻聚糖可有效提高RAW264.7细胞的iNOS基因和蛋白表达，并且其作用具有浓度依赖性；新西兰裙带菜来源的岩藻聚糖可有效提高RAW264.7细胞分泌TNF-a和IL-6，并且其作用具有浓度依赖性。
综上所述，本研究首次发现新西兰裙带菜来源的岩藻聚糖具有提高RAW264.7细胞分泌NO、TNF-a和IL-6的作用，并且不具有细胞毒性，证明新西兰裙带菜来源的岩藻聚糖具有良好的免疫调节活性，将其作为一种新型的增强免疫力的药物和保健品，具有广阔的市场应用前景。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种岩藻聚糖的制备方法，其特征在于，包括如下步骤：
    提供裙带菜；
    将所述裙带菜粉碎后溶于水中，进行加热处理，得到裙带菜溶解液；
    将所述裙带菜溶解液进行第一离心处理，得到第一上清液；
    将氢氧化钙加入所述第一上清液中，进行第二离心处理，得到第二上清液；
    将沉淀剂加入所述第二上清液中，得到所述岩藻聚糖。
  2. 如权利要求1所述的岩藻聚糖的制备方法，其特征在于，所述裙带菜为新西兰裙带菜。
  3. 如权利要求1所述的岩藻聚糖的制备方法，其特征在于，所述加热处理的条件为：温度65-75℃，时间8-12 h。
  4. 如权利要求1所述的岩藻聚糖的制备方法，其特征在于，所述第一离心处理的条件为：离心力18000-19000 ×g，离心时间110-130 min；和/或
    所述第二离心处理的条件为：离心力18000-19000 ×g，离心时间110-130min。
  5. 如权利要求1所述的岩藻聚糖的制备方法，其特征在于，所述裙带菜与所述水的质量比为1∶3-4；和/或
    所述裙带菜与所述氢氧化钙的质量比为1∶0.1-0.2；和/或
    所述裙带菜与所述沉定剂的质量比为3∶6-8。
  6. 如权利要求1所述的岩藻聚糖的制备方法，其特征在于，所述沉淀剂为乙醇。
  7. 如权利要求1-6任一项所述的岩藻聚糖的制备方法，其特征在于，在所述将沉淀剂加入所述第二上清液中的步骤之后，还包括冷冻干燥的步骤，所述冷冻干燥的条件为：温度零下15℃-零下5℃，时间22-26 h。
  8. 一种裙带菜来源的岩藻聚糖，其特征在于，所述岩藻聚糖由权利要求1-7任一项所述的制备方法获得，且所述岩藻聚糖的分子量小于10 kDa。
  9. 一种由权利要求1-7任一项所述的制备方法获得的岩藻聚糖在制备免疫调节剂中的应用。
  10. 如权利要求9所述的应用，其特征在于，所述免疫调节剂促进iNOS蛋白表达或促进TNF-a和 IL-6分泌。
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CN101993501A (zh) * 2009-08-26 2011-03-30 浙江科技学院 一种岩藻聚糖硫酸酯的制备方法

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