WO2020000788A1 - 一种酯化硒多糖及其制备方法和应用 - Google Patents

一种酯化硒多糖及其制备方法和应用 Download PDF

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WO2020000788A1
WO2020000788A1 PCT/CN2018/110494 CN2018110494W WO2020000788A1 WO 2020000788 A1 WO2020000788 A1 WO 2020000788A1 CN 2018110494 W CN2018110494 W CN 2018110494W WO 2020000788 A1 WO2020000788 A1 WO 2020000788A1
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polysaccharide
selenium
esterified
reaction
oxo
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PCT/CN2018/110494
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English (en)
French (fr)
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孙玉敏
刘海全
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爱美中科硒科技（天津）有限公司
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Priority to US17/256,015 priority Critical patent/US11345761B2/en
Priority to EP18925019.4A priority patent/EP3800205B1/en
Publication of WO2020000788A1 publication Critical patent/WO2020000788A1/zh

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    • 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
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
    • 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
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • 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
    • 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
    • A61K31/716—Glucans
    • 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
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00

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  • the invention relates to the technical field of selenium polysaccharide, in particular to an esterified selenium polysaccharide, a preparation method and application thereof.
  • Selenium (Se) is an essential trace element in the human body, and plays an important role in many aspects such as the improvement of life immune function, anti-cancer, anti-oxidation, and prevention of nutritional liver necrosis.
  • selenium is also the active component of glutathione peroxidase. Therefore, the role of selenium on the human body is extremely important.
  • the selenium element is unevenly distributed on the earth, and selenium is lacking in about three-fifths of China, and the selenium content in ordinary foods is extremely low, and the effect of selenium supplementation is generally not achieved.
  • selenium exists mainly in two forms: inorganic selenium and organic selenium.
  • organic selenium is a safer and more active selenium-containing substance, which is more significant than inorganic selenium in stimulating immune response.
  • the main sources of organic selenium are natural selenium-enriched organisms and artificial synthesis.
  • selenium polysaccharides have received great attention.
  • selenium polysaccharide As an organic selenium compound, selenium polysaccharide has the activities of both selenium and polysaccharides, and the selenized polysaccharides are more easily absorbed and utilized by the organism.
  • selenium-rich foods and products have appeared, and selenium polysaccharides are the main constituents.
  • the preparation method of selenium polysaccharide includes natural selenium-containing plant polysaccharide, microbial enrichment and cultivation of selenium polysaccharide, and artificial selenium polysaccharide.
  • the selenium-enriched crops grown in selenium-enriched soil have good product safety, but the selenium content is very low, and the normal metabolic balance of the selenium content necessary for blood cannot be basically achieved.
  • Synthetic selenium polysaccharide is a convenient and controllable method.
  • Patent CN 1560088A discloses a method for preparing selenized glucomannan.
  • the selenium element is oxidized to Se 6+ under the action of an oxidant, and ethanol and hydrochloric acid are added to an aqueous solution of Se 6+ ions to obtain a selenization reaction solution.
  • Patent ZL 88103347 discloses one A method for preparing selenized carrageenan, using selenium powder as a raw material, dissolving it with nitric acid to prepare a selenium solution, and adding a Kappa-carrageenan solution for selenization reaction, but the product obtained by this method has a low selenium content (2500- 15000 ppm), and by-product hydrogen selenide is toxic.
  • Patent ZL 200910162003.4 discloses a method for organically preparing Artemisia selengensis polysaccharide or Pteris polysaccharide, and reacting Pterine polysaccharide or Artemisia polysaccharide with selenyl chloride to obtain selenized Artemisia polysaccharide or Pteridium polysaccharide.
  • the method used in this method is difficult to synthesize and selenium chloride is toxic.
  • the selenium chloride when the selenium chloride is added to the air, the selenium chloride will be oxidized, and the obtained selenium chloride may contain impurities such as selenium chloride. .
  • the polysaccharide selenization modification technology mentioned in the above patents and literatures generally has the disadvantages of complex preparation of selenium-containing intermediates, high toxicity, long reaction time, low selenium content and yield of selenized polysaccharides, many side reactions, and unknown biological effects.
  • the present invention provides an esterified selenium polysaccharide, a preparation method and application thereof.
  • the obtained selenium polysaccharide has a clear binding site, a high selenium content, and has no toxic side effects.
  • the invention provides a method for preparing an esterified selenium polysaccharide, comprising the following steps:
  • the molar ratio of the polysaccharide to the acryloyl chloride is from 1: 1 to 50.
  • the molar ratio of the polysaccharide to the sodium bicarbonate is 1: 5-50.
  • step (2) the reaction is completed 2 to 3 hours after the dropwise addition is stopped.
  • the molar ratio of the acrylated polysaccharide to 3,4-oxo-cycloselenoic acid galactosyl glycoside is 1: 1 to 4.
  • step (3) the displacement reaction is completed within 4 to 5 hours.
  • the ethyl acetate extraction further includes a purification step: washing the extract with saturated brine, drying and evaporating the organic phase, and passing through a column to obtain 3,4-oxo-cycloselenose galactose Propyl glycosides.
  • the polysaccharide is a water-soluble natural polysaccharide containing a primary hydroxyl group at the 6-position.
  • the present invention also includes an esterified selenium polysaccharide prepared by any one of the methods described above, and has a structural formula:
  • n is the number of sugar units of a natural polysaccharide.
  • the organic selenium content of the esterified selenium polysaccharide reaches 10,000 to 100,000 ppm.
  • the invention also includes the application of the above-mentioned esterified selenium polysaccharide in non-therapeutic purposes to improve immunity.
  • the present invention has the following advantages:
  • the present invention prepares an esterified selenium product with controllable selenium content through a substitution reaction between 3,4-oxo-cycloselenose galactose allyl glycoside and an acrylated polysaccharide.
  • the preparation of selenium-containing intermediate 3,4-oxo-cycloselenoic acid galactose allyl glycoside is simple and the yield is greater than 90%; during the preparation process, the selenium-containing intermediate can be purified or used without purification.
  • the selenium intermediate is non-toxic.
  • the replacement reaction conditions are simple, the reaction time is short, and the yield is high.
  • the selenium content of the obtained esterified selenium polysaccharide can reach 10,000 to 100,000 ppm, and the selenium content can be adjusted.
  • the selenium element exists in the form of an organic ester, and a selenium atom is simultaneously combined with two sugar hydroxyl groups to form a selenite unit having a cyclic structure.
  • the organic compound carrier forming the esterified selenium structure is a polysaccharide, which has the potential to increase blood selenium content and improve many immune functions. It can significantly improve the immune ability of the test organism by increasing the blood selenium content.
  • FIG. 1 shows the maximum expression levels of cytokines IL-4 and IFN- ⁇ in Example 4.
  • the invention provides a method for preparing an esterified selenium polysaccharide, comprising the following steps:
  • step (1) and step (2) are not limited in order.
  • the reaction process of esterifying selenium polysaccharide of the present invention is as follows:
  • a substitution reaction occurs at room temperature.
  • D-galactosyl glucoside is preferably dissolved in DMF or DMSO to form a D-galactosyl glucoside solution.
  • the reaction time of the above reaction is preferably 2 to 3 hours.
  • the reaction solution was poured into an aqueous phase and extracted with ethyl acetate to obtain 3,4-oxo-cycloselenoic acid galactose allyl glycoside.
  • a purification step is further included: the extract is washed with saturated brine, the organic phase is dried and evaporated to dryness, and passed through a chromatography column to obtain 3,4-oxo-cycloselenose galactose allyl glycoside.
  • the above purification steps can be performed by conventional methods in the art.
  • the organic phase is preferably dried with anhydrous sodium sulfate.
  • the dried organic phase is preferably evaporated to dryness under reduced pressure on a rotary evaporator.
  • the chromatographic column is preferably a silica gel column to obtain a 3,4-oxo-cycloselenous acid half Pure lactosyl glycoside.
  • the yield can reach more than 90%.
  • acrylic acid polysaccharide is prepared by reacting acrylic acid chloride with polysaccharide.
  • the polysaccharide in the present invention is a water-soluble natural polysaccharide containing a primary hydroxyl group at the 6-position, such as ⁇ -1-3-glucan, lentinan, schizopolysaccharide, small molecular weight carrageenan polysaccharide, and chitooligosaccharide Wait.
  • the polysaccharide is first dissolved in a solvent.
  • the invention does not specifically limit the solvent, and DMF can be used to dissolve the polysaccharide.
  • NaHCO 3 is added to the polysaccharide solution, preferably solid sodium bicarbonate.
  • the molar ratio of the polysaccharide to sodium bicarbonate is preferably 1: 5 to 50, and more preferably 1:10 to 30.
  • the acryloyl chloride is added dropwise to the polysaccharide solution, and the molar ratio of the polysaccharide to the dropwise acryloyl chloride is preferably 1: 1 to 50, more preferably 1:10 to 40, and still more preferably 1:20 to 30.
  • the temperature of the reaction solution is maintained at not more than 40 ° C, and more preferably 15 to 35 ° C.
  • the reaction is preferably completed within 2 to 4 hours after the dropwise addition is stopped.
  • the reaction solution is poured into cold water, and cold ethanol is added to precipitate the acrylated polysaccharide.
  • the amount of cold ethanol is preferably 3 to 7 times the volume of the cold water of the reaction solution.
  • the recovery rate of the synthetic acrylated polysaccharide is greater than 95%.
  • the acrylated polysaccharide in the present invention undergoes a substitution reaction with 3,4-oxo-cycloselenose galactose allyl glycoside under the action of a Ru catalyst to obtain an esterified selenium polysaccharide.
  • the Ru catalyst is not particularly limited in the present invention. In specific embodiments of the present invention, a Grubbs-I generation Ru catalyst is preferred.
  • the above-mentioned substitution reaction in the present invention is preferably performed in a DMF solvent. It is preferable to dissolve the acrylated polysaccharide in DMF, then add 3,4-oxo-cycloselenoic acid galactosyl glycoside, and finally add a catalytic amount of Ru catalyst.
  • the molar ratio of the acrylated polysaccharide to the 3,4-oxo-cycloselenoic acid galactosyl glycoside is preferably 1: 1 to 4, more preferably 1: 2 to 3, and the addition amount of Ru catalyst is preferred. It is 0.1 to 0.5 equivalents.
  • the olefin replacement reaction can be completed within 4 to 5 hours, that is, the organic selenosaccharide unit is bound to the polysaccharide.
  • the reaction solution was dispersed in the aqueous phase, and the product was precipitated with 4 to 5 volumes of ethanol at low temperature. Repeated water-ethanol precipitation can purify the product with a yield of 80-95%.
  • the invention also includes the esterified selenium polysaccharide prepared by the above preparation method, and its structural formula is as follows:
  • n is a sugar unit to be selenized
  • n is the number of sugar units of a natural polysaccharide.
  • the organic selenium content of the esterified selenium polysaccharide prepared by the present invention can reach 10,000 to 100,000 ppm.
  • the esterified selenium polysaccharide of the present invention has the potential to increase blood selenium content and improve many immune functions, and can be used in food, medicine or health products to improve immune function.
  • the polysaccharide is a commercially available water-soluble ⁇ -1-3-glucan as an example, but the scope of protection of the present invention is not limited.
  • B-6 splenocytes were used as experimental models.
  • Carrageenan, chitohexaose, 1,3-glucohexaose, and PMA + innomycin were used as controls.
  • the esterified selenium polysaccharide (2 to 5 micrograms of selenium / mL) was used.
  • the culture medium was incubated for 48 hours, and the expression levels of IL-4 and IFN- ⁇ were detected.
  • the culture medium for culturing B-6 spleen cells is a culture medium for conventionally culturing B-6 spleen cells in the art, which includes essential and non-essential amino acids, vitamins, glucose, hormones, growth factors, trace minerals, and low-concentration fetal bovine serum. (2%) and 5 ml penicillin / streptomycin solution.
  • the medium buffer system is a phosphate buffered saline solution PBS, and the pH value is 7.4 after equilibrating in a cell incubator containing 5% CO 2 .
  • the mouse was used as a model, and the esterified selenium-free polysaccharide was used as a control substance.
  • the esterified selenium polysaccharide prepared by the present invention was continuously fed at a dose of 2 to 5 micrograms of selenium per day for 2 weeks, and then from the third week to the sixth Continuous blood test (this
  • red blood cells increased by 20% and white blood cells increased by 10%.
  • the chick is used as a model, and the esterified selenium-free polysaccharide is used as a control substance. After the chicks are born, the esterified selenium polysaccharide of the present invention is continuously fed at a dose of 5 micrograms of selenium / day. Glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activity tests (using commercially available kits EnzyChrom TM Glutahione Peroxidase Assay Kit and MlBio The mouse superoxide dismutase (SOD) kit is complete).
  • Glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activity tests using commercially available kits EnzyChrom TM Glutahione Peroxidase Assay Kit and MlBio The mouse superoxide dismutase (SOD) kit is complete).

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Abstract

本发明公开了一种酯化硒多糖的制备方法，将O＝SeCl 2滴加到含有三乙胺的D-半乳糖烯丙基苷溶液中室温反应得到3,4-氧-环***半乳糖烯丙基苷；将NaHCO 3混合于多糖溶液中，向所述多糖溶液中滴加丙烯酰氯，滴加时维持反应液温度不超过40℃，得到丙烯酸化多糖；丙烯酸化多糖在Ru催化剂的作用下与3,4-氧-环***半乳糖烯丙基苷发生置换反应，得到酯化硒多糖。本发明制备得到的酯化硒多糖能通过提高血硒含量来显著改善受试生物的免疫能力。

Description

一种酯化硒多糖及其制备方法和应用
本申请要求于2018年06月26日提交中国专利局、申请号为2018106726636，发明名称为“一种酯化硒多糖及其制备方法和应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及硒多糖技术领域，尤其涉及一种酯化硒多糖、其制备方法和应用。
背景技术
硒(Se)是人体必不可少的微量元素，在生命免疫机能的提高、抗癌、抗氧化、防止营养性肝坏死等诸多方面发挥着重要作用。在生物代谢中，硒也是谷胱甘肽过氧化物酶的活性成分，因此，硒对人体的作用极为重要。但是，硒元素在地球分布不均匀，在中国大约有五分之三的地区缺硒，而普通食品中硒含量都是极低的，一般达不到补充硒的效果。在自然界，硒的存在形式主要包括无机硒和有机硒两种。与无机硒相比，有机硒是更安全、活性更高的含硒物质，在激发免疫反应上比无机硒更加显著。有机硒的主要来源是天然富硒生物和人工合成，在众多富含有机硒物质中，硒多糖受到了极大关注。硒多糖作为一种有机硒化合物，拥有硒与多糖二者的活性，且硒化后的多糖更易于被有机体吸收和利用。近几年出现了许多富硒食品和制品，硒多糖是其中的主要成分。硒多糖的制备方法包括天然含硒植物多糖，微生物富集培养代谢硒多糖和人工合成硒多糖。通过富硒土壤种植的作物富硒，产品安全性好，但硒含量很低，基本无法实现血液所必须硒含量的正常代谢平衡。人工合成硒多糖是比较方便且可控的方法。
目前国内外文献和专利主要采用以下三种方法制备硒多糖：温和条件下使用单体硒、***或***钠对多糖进行硒化修饰；利用化学性质活泼，具有酰氯结构的中间体作为硒化试剂进行修饰；将含硒的功能基因接枝到多糖分子上。梁淑轩等采用冰醋酸催化下硒酸钠与枸杞多糖反应；李志洲等利用化学合成法，以猪苓多糖和***钠为原料制备猪苓硒多糖，采用连续或超声波辅助化学合成工艺，缺点是硒化位点不明确，硒化程度不确定，且无法完全去除高分子量多糖中混有的较大量的毒性无机硒成份。专利CN 1560088A公开了一种了硒化葡甘聚糖的制备方法，将硒单质在氧化剂作用下氧化成Se 6+，在Se 6+离子的水溶液中加入乙醇和盐酸，得到硒化反应液，将硒化反应液与葡甘聚糖进行反应，制成硒化葡甘聚糖，所得硒化葡甘聚糖硒含量比较低，且衍生物中硒为 Se 6+；专利ZL 88103347公开了一种硒化卡拉胶的制法，以硒粉为原料，用硝酸将其溶解制备成硒液，加入Kappa-角叉菜胶溶液进行硒化反应，但该方法所得产物硒含量较低(2500-15000ppm)，且副产物硒化氢有毒。专利ZL 200910162003.4公开了一种有机法制备硒化沙蒿多糖或蕨麻多糖的方法，将蕨麻多糖或沙蒿多糖与亚硒酰氯反应得硒化的沙蒿多糖或蕨麻多糖。但该方法使用的亚硒酰氯合成困难且亚硒酰氯具有毒性，反应过程中，加入亚硒酰氯时暴露在空气中，亚硒酰氯会被氧化，得到的亚硒酰氯中可能含有硒酰氯等杂质。
以上专利和文献中提到的多糖硒化修饰技术普遍存在含硒中间体制备复杂且毒性大、反应时间长、硒化多糖硒含量和收率低、副反应多、生物学效应不明等缺点。
发明内容
为了克服现有技术的不足，本发明提供一种酯化硒多糖及其制备方法和应用，得到的酯化硒多糖中硒的结合位点明确，硒含量高，无毒副作用。
本发明的技术方案如下：
本发明提供了一种酯化硒多糖的制备方法，包括以下步骤：
(1)0℃，氮气保护下，将1～5个当量的O＝SeCl 2滴加到含有1～5个当量三乙胺的D-半乳糖烯丙基苷溶液中，室温反应1～4小时后倒入水相，用乙酸乙酯萃取，得到3,4-氧-环***半乳糖烯丙基苷；
(2)将多糖悬浮于溶剂中，加入NaHCO 3混合，向所述多糖溶液中滴加丙烯酰氯，滴加时维持反应液温度不超过40℃，反应完成后，将反应液溶于水中醇沉，得到丙烯酸化多糖；
(3)将所述丙烯酸化多糖溶于溶剂中，在Ru催化剂的作用下与3,4-氧-环***半乳糖烯丙基苷发生置换反应，得到的反应液分散在水相中，醇沉得到酯化硒多糖。
优选的，步骤(2)中，多糖与丙烯酰氯的摩尔比为1：1～50。
优选的，步骤(2)中，多糖与碳酸氢钠的摩尔比为1：5～50。
进一步优选的，步骤(2)中，滴加停止后2～3小时反应完成。
优选的，步骤(3)中，丙烯酸化多糖与3,4-氧-环***半乳糖烯丙基苷的摩尔比为1:1～4。
进一步优选的，步骤(3)中，4～5小时内置换反应完成。
优选的，步骤(1)中，乙酸乙酯萃取后还包括纯化步骤：用饱和食盐水洗涤萃取物，有机相干燥蒸干，过色谱柱得到3,4-氧-环***半乳糖烯丙基苷。
优选的，所述多糖为可溶于水的、含有6-位伯羟基的天然多糖。
本发明还包括上述任意一项所述方法制备得到的酯化硒多糖，其结构式为：
Figure PCTCN2018110494-appb-000001
其中，m为被硒化的糖单元，n为天然多糖的糖单元数。
优选的，所述酯化硒多糖的有机硒含量达1万到10万ppm。
本发明还包括上述酯化硒多糖在非治疗目的提高免疫力中的应用。
现有技术相比，本发明具有以下优点：
本发明通过3,4-氧-环***半乳糖烯丙基苷与丙烯酸酯化的多糖发生置换反应，制备含硒量可控的酯化硒产品。含硒中间体3,4-氧-环***半乳糖烯丙基苷制备简单，产率大于90％；含硒中间体在制备过程中，可纯化也可不经纯化直接使用，得到的含硒中间体无毒性。酯化硒多糖在合成过程中，置换反应条件简单，反应时间短，收率高。得到的酯化硒多糖硒含量可达1万到10万ppm，硒含量可调。
本发明制备得到的酯化硒多糖分子中，硒元素以有机酯的形式存在，硒原子同时与两个糖羟基结合，形成具有环状结构的***酯单元。形成酯化硒结构的有机化合物载体为多糖类物质，具有提高血硒含量，改善诸多免疫功能的潜力，能通过提高血硒含量来显著改善受试生物的免疫能力。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1为实施例4中细胞因子IL-4和IFN-γ的最大表达量。
具体实施方式
下面对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明提供了一种酯化硒多糖的制备方法，包括以下步骤：
(1)0℃，氮气保护下，将1～5个当量的O＝SeCl 2滴加到含有1～5个当量三乙胺的D-半乳糖烯丙基苷溶液中，室温反应1～4小时后倒入水相，用乙酸乙酯萃取，得到3,4-氧-环***半乳糖烯丙基苷；
(2)将多糖悬浮于溶剂中，加入NaHCO 3混合，向所述多糖溶液中滴加丙烯酰氯，滴加时维持反应液温度不超过40℃，反应完成后，将反应液溶于水中醇沉，得到丙烯酸化多糖；
(3)将所述丙烯酸化多糖溶于溶剂中，在Ru催化剂的作用下与3,4-氧-环***半乳糖烯丙基苷发生置换反应，得到的反应液分散在水相中，醇沉得到酯化硒多糖。
其中，步骤(1)和步骤(2)没有顺序限制。
本发明酯化硒多糖的反应过程如下：
Figure PCTCN2018110494-appb-000002
本发明合成3,4-氧-环***半乳糖烯丙基苷的步骤中，0℃，氮气保护下，将O＝SeCl 2滴加到含有三乙胺的D-半乳糖烯丙基苷溶液中，室温下发生取代反应。其中，D-半乳糖烯丙基苷优选溶于DMF或DMSO中形成D-半乳糖烯丙基苷溶液。本发明中，O＝SeCl 2的用量优选为2～4个当量，三乙胺的添加量优选为2～4个当量。本发明优选上述反应的反应时间为2～3小时。反应完成后，将反应液倒入水相，用乙酸乙酯萃取，得到3,4-氧-环***半乳糖烯丙基苷。本发明优选的，乙酸乙酯萃取后还包括纯化步骤：用饱和食盐水洗涤萃取物，有机相干燥蒸干，过色谱柱得到3,4-氧-环***半乳糖烯丙基苷。上述纯化步骤均可采用本领域中的常规操作方法。本发明中优选有机相用无水硫酸钠干燥，干燥后的有机相优选在旋转蒸发仪上减压蒸干，所述色谱柱优选为硅胶柱，得到3,4-氧-环***半乳糖烯丙基苷纯品。本发明合成3,4-氧-环***半乳糖烯丙基苷的步骤中，产率能够达到大于90％。
本发明用丙烯酰氯与多糖反应制备丙烯酸化多糖。本发明所述多糖为可溶于水的、含有6-位伯羟基的天然多糖，如β-1-3-葡聚糖、香菇多糖、裂褶多糖、小分子量的卡拉胶多糖、壳寡糖等。首先将多糖溶于溶剂中。本发明对溶剂没有特殊限定，可以采用DMF溶解多糖。在多糖溶液中加入NaHCO 3，优选为固体碳酸氢钠。所述多糖与碳酸氢钠的摩尔比为优选为1：5～50，更优选为1:10～30。向多糖溶液中滴加丙烯酰氯，优选多糖与滴加的丙烯酰氯的摩尔比为1：1～50，更优选为1:10～40，进一步优选为1:20～30。滴加过程中维持反应液温度不超过40℃，更优选为15～35℃。优选滴加停止后的2～4小时反应完成。将反应液倒入冷水中，添加冷乙醇沉淀出丙烯酸化多糖，优选冷乙醇的添加量为反应液冷水体积的3～7倍。本发明合成丙烯酸化多糖的回收率大于95％。
本发明所述丙烯酸化多糖与在Ru催化剂的作用下与3,4-氧-环***半乳糖烯丙基苷发生置换反应，得到酯化硒多糖。本发明对Ru催化剂没有特殊限定，在本发明具体实施例中，优选为Grubbs-I代Ru催化剂。本发明上述置换反应优选在DMF溶剂中进行，优选将丙烯酸化多糖溶于DMF中，再加入3,4-氧-环***半乳糖烯丙基苷，最后加入催化量的Ru催化剂。本发明中，优选丙烯酸化多糖与3,4-氧-环***半乳糖烯丙基苷的摩尔比为1:1～4，更优选为1:2～3，优选Ru催化剂的添加量为0.1～0.5当量。本发明优选4～5小时内可完成烯烃置换反应，即将该有机硒糖单元结合在多糖上。将反应液分散在水相中，低温下用4～5倍体积的乙醇沉淀出产品。重复水溶-乙醇沉淀可以纯化产品，收率80～95％。
本发明还包括上述制备方法制备得到的酯化硒多糖，其结构式如下：
Figure PCTCN2018110494-appb-000003
其中，m为被硒化的糖单元，n为天然多糖的糖单元数。本发明制备得到的酯化硒多糖有机硒含量可达1万到10万ppm。
本发明上述酯化硒多糖具有提高血硒含量，改善诸多免疫功能的潜力，能够用于食品、药品或保健品中提高免疫功能。
以上所述的具体实施方式，对本发明的目的、技术方案和有益效果进行了进一步详细说明，所应理解的是，以上所述仅为本发明的具体实施方式而已，并不用于限定本发明的保护范围，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
以下提供本发明的实施例，多糖以市售的水溶性β-1-3-葡聚糖为例，但并非是对本发明保护范围的限定。
实施例1
0℃，氮气保护下，将1个当量的O＝SeCl 2滴加到含有4个当量三乙胺的市售D-半乳糖烯丙基苷DMSO溶液中，室温反应4小时后倒入水相，用乙酸乙酯萃取，饱和食盐水洗涤，有机相用无水硫酸钠干燥，旋转蒸发仪上减压蒸干，硅胶柱色谱得到纯品，产率大于90％。
将100mgβ-1-3-葡聚糖悬浮于10毫升无水DMF中，加入5当量固体碳酸氢钠、1当量丙烯酰氯，维持反应液温度不超过40℃共2小时，反应液在搅拌下倒入冷水中，添加4倍体积的冷乙醇沉淀出中间产物，冷冻干燥后备用。上述中间体重新悬浮于干燥的DMF中，加入1当量3,4-氧-环***半乳糖烯丙基苷，然后加入0.1当量的Grubbs-I代Ru催化剂，4小时将该有机硒多糖倒入水相，低温下用4倍体积的乙醇沉淀出产品。ICP MS分析其硒含量为1.8万ppm。
实施例2
0℃，氮气保护下，将5个当量的O＝SeCl 2滴加到含有2个当量三乙胺的D-半乳糖烯丙基苷DMF溶液中，室温反应2小时后倒入水相，用乙酸乙酯萃取，饱和食盐水洗涤，有机相用无水硫酸钠干燥，旋转蒸发仪上减压蒸干，硅胶柱色谱得到纯品，产率大于90％。
将100mgβ-1-3-葡聚糖悬浮于20毫升无水DMF中，加入50当量固体碳酸氢钠、50当量丙烯酰氯，维持反应液温度不超过40℃共4小时，反应液在搅拌下倒入冷水中，添加6倍体积的冷乙醇沉淀出中间产物，冷冻干燥后备用。上述中间体重新悬浮于干燥的DMF中，加入50当量3,4-氧-环***半乳糖烯丙基苷，然后加入 0.5当量的Grubbs-I代Ru催化剂，反应5小时后将该有机硒多糖倒入水相，低温下用5倍体积的乙醇沉淀出产品。ICP MS分析其硒含量为9.6万ppm。
实施例3
0℃，氮气保护下，将3个当量的O＝SeCl 2滴加到含有3个当量三乙胺的D-半乳糖烯丙基苷DMF溶液中，室温反应3小时后倒入水相，用乙酸乙酯萃取，饱和食盐水洗涤，有机相用无水硫酸钠干燥，旋转蒸发仪上减压蒸干，硅胶柱色谱得到纯品，产率大于90％。
将100mgβ-1-3-葡聚糖悬浮于20毫升无水DMF中，加入20当量固体碳酸氢钠、20当量丙烯酰氯，维持反应液温度不超过40℃共4小时，反应液在搅拌下倒入冷水中，添加5倍体积的冷乙醇沉淀出中间产物，冷冻干燥后备用。上述中间体重新悬浮于干燥的DMF中，加入20当量3,4-氧-环***半乳糖烯丙基苷，然后加入0.2当量的Grubbs-I代Ru催化剂，反应4小时后将该有机硒多糖倒入水相，低温下用4倍体积的乙醇沉淀出产品。ICP MS分析其硒含量为4.6万ppm。
实施例4
用B-6脾细胞为实验模型，以卡拉胶、壳六糖、1,3-葡六糖、PMA+innomycin为对照物，在含本发明酯化硒多糖(2至5微克硒/mL)的培养液中孵育48小时，检测其IL-4和IFN-γ的表达量。
其中培养B-6脾细胞的培养液为本领域中常规培养B-6脾细胞的培养液，包含必需和非必需氨基酸、维生素、葡萄糖、激素、生长因子、微量矿物质和低浓度胎牛血清(2％)以及5ml青霉素/链霉素溶液。该培养基缓冲体系为磷酸缓冲盐溶液PBS，在含5％CO 2的细胞培养箱中平衡后pH值为7.4。
细胞收获后，以BD Bioscience的Cytofix/Cytoperm试剂盒染色，用流式细胞仪检测，检测结果见图1。与1，3-葡六糖相比，本发明酯化硒多糖处理的B-6脾细胞IL-4最大表达量可以显著提高60％，IFN-γ最大表达量提高了36％，说明使用本发明的酯化硒多糖可以有效改善体系的免疫能力。
实施例5
以小鼠为模型，以不含酯化硒的多糖为对照物，以2至5微克硒/天的硒剂量连续喂饲本发明制备的酯化硒多糖2周后，于第三周至第六周连续进行血液检测(此
时仍然喂饲酯化硒，方式剂量与前相同)，每周一次，发现血清硒含量最大提高了
30％以上，血红细胞提高了20％，白细胞增加了10％，并于第五周开始上述检测数
据趋于稳定。
表1 饲喂小鼠后不同时间段的血检结果
Figure PCTCN2018110494-appb-000004
实施例6
以雏鸡为模型，以不含酯化硒的多糖为对照物，在雏鸡出生后以5微克硒/天的剂量连续喂饲本发明的酯化硒多糖，并在喂饲后第7天、14天、21天、28天开展谷胱甘肽过氧化物酶(GSH-Px)和超氧化物歧化酶(SOD)的活性检测(使用市售的试剂盒EnzyChrom TMGlutahione Peroxidase Assay Kit和MlBio□小鼠超氧化物歧化酶(SOD)试剂盒完成检测)。结果表明雏鸡GHS-Px活性在第14-21天达到最大值，提高了15％，SOD在第7-14天达到最大值，提高了12％，说明本发明的酯化硒多糖具有较好的抗氧化功能。
表2 饲喂雏鸡不同时间段GSH-Px和SOD的活性
(U/mg) 第1天 第7天 第14天 第21天 第28天
血清GSH-Px 1869 1879 1975 2150 2090
血清SOD 180 191 195 190 187
以上所述的具体实施方式，对本发明的目的、技术方案和有益效果进行了进一步详细说明，所应理解的是，以上所述仅为本发明的具体实施方式而已，并不用于限定本发明的保护范围，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种酯化硒多糖的制备方法，其特征在于，包括以下步骤：
    (1)0℃，氮气保护下，将1～5个当量的O＝SeCl 2滴加到含有1～5个当量三乙胺的D-半乳糖烯丙基苷溶液中，室温反应1～4小时后倒入水相，用乙酸乙酯萃取，得到3,4-氧-环***半乳糖烯丙基苷；
    (2)将多糖悬浮于溶剂中，加入NaHCO 3混合，向所述多糖溶液中滴加丙烯酰氯，滴加时维持反应液温度不超过40℃，反应完成后，将反应液溶于水中醇沉，得到丙烯酸化多糖；
    (3)将所述丙烯酸化多糖溶于溶剂中，在Ru催化剂的作用下与3,4-氧-环***半乳糖烯丙基苷发生置换反应，得到的反应液分散在水相中，醇沉得到酯化硒多糖；
    其中，步骤(1)和步骤(2)没有顺序限制。
  2. 根据权利要求1所述酯化硒多糖的制备方法，其特征在于，步骤(2)中，多糖与碳酸氢钠的摩尔比为1：5～50。
  3. 根据权利要求1～3任意一项所述酯化硒多糖的制备方法，其特征在于，步骤(2)中，滴加停止后2～3小时反应完成。
  4. 根据权利要求1所述酯化硒多糖的制备方法，其特征在于，步骤(3)中，丙烯酸化多糖与3,4-氧-环***半乳糖烯丙基苷的摩尔比为1:1～4。
  5. 根据权利要求1或5所述酯化硒多糖的制备方法，其特征在于，步骤(3)中，4～5小时内置换反应完成。
  6. 根据权利要求1所述酯化硒多糖的制备方法，其特征在于，步骤(1)中，乙酸乙酯萃取后还包括纯化步骤：用饱和食盐水洗涤萃取物，有机相干燥蒸干，过色谱柱得到3,4-氧-环***半乳糖烯丙基苷。
  7. 根据权利要求1所述酯化硒多糖的制备方法，其特征在于，所述多糖为可溶于水的、含有6-位伯羟基的天然多糖。
  8. 权利要求1～8任意一项所述方法制备得到的酯化硒多糖，其结构式为
    Figure PCTCN2018110494-appb-100001
    通式中，m为被硒化的糖单元，n为天然多糖的糖单元数。
  9. 根据权利要求1所述的酯化硒多糖，其特征在于，所述酯化硒多糖的有机硒含量达1万到10万ppm。
  10. 权利要求9所述酯化硒多糖在非治疗目的提高免疫力中的应用。
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