WO2021007858A1 - 键合硒多糖及其制备方法和应用 - Google Patents

键合硒多糖及其制备方法和应用 Download PDF

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WO2021007858A1
WO2021007858A1 PCT/CN2019/096583 CN2019096583W WO2021007858A1 WO 2021007858 A1 WO2021007858 A1 WO 2021007858A1 CN 2019096583 W CN2019096583 W CN 2019096583W WO 2021007858 A1 WO2021007858 A1 WO 2021007858A1
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polysaccharide
selenium
bonded
product
present
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PCT/CN2019/096583
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French (fr)
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程青格
董淑敏
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郑州市御合源生物科技有限公司
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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
    • C08B37/0036—Galactans; Derivatives thereof
    • C08B37/0042—Carragenan or carragen, i.e. D-galactose and 3,6-anhydro-D-galactose, both partially sulfated, e.g. from red algae Chondrus crispus or Gigantia stellata; kappa-Carragenan; iota-Carragenan; lambda-Carragenan; 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants

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  • the invention belongs to the field of biomedicine, and specifically relates to a novel bonded selenium polysaccharide and a preparation method thereof, and its application in immunity.
  • Selenium is a very important trace element to the human body. Selenocysteine is the 21st amino acid. Selenium is an important part of the 25 known selenoprotein structures. Its activity depends on the presence of selenium. For example, research The authors found that selenium is an essential component of glutathione peroxidase in animals. However, there are very limited ways for the human body to supplement selenium, and the commonly used so-called food supplements are far from meeting the body's normal selenium metabolism. For this reason, researchers clearly pointed out that selenium is an essential trace element for the human body in 1975. The Chinese Nutrition Society also listed selenium as one of the daily dietary nutrients in 1988.
  • the invention provides a bonded selenium polysaccharide with a controllable selenium content and a preparation method thereof.
  • the formed bonded selenium polysaccharide has a stable structure, a definite organic selenium content, and has the effect of improving many immune functions.
  • the present invention provides a selenium-bonded polysaccharide.
  • the molecular structure features include: using a polysaccharide substance with a 3,6-endoglycan structure as a carrier, and selenium atoms are connected to the polysaccharide 3,6- through the structure of selenocarboxylate. On the 6 carbon atom of the inner ether structure.
  • the polysaccharide is derived from extracted natural plant polysaccharide, more preferably carrageenan.
  • the selenium-bonded polysaccharide molecule of the present invention has any of the following structures:
  • n is related to the molecular weight of natural polysaccharides and is the number of polysaccharide repeating units.
  • the present invention also includes the method for preparing the above-mentioned bonded selenium polysaccharide.
  • the 6-position carbon atom in the 3,6-internal ether bond of the polysaccharide is catalyzed and oxidized by RuO 2 to convert to 3,6-lactone, and the nucleophilic reaction of the lactone by the negative selenium ion A ring-opening reaction to form an ester occurs, bonding selenium to the carbon atom of the ether structure in the polysaccharide.
  • the preparation method of the bonded selenium polysaccharide of the present invention includes the following steps:
  • Product I is reacted with sodium hypochlorite under the catalysis of RuO 2 to convert the 6-position carbon atom in the 3,6-internal ether bond of polysaccharide into 3,6-lactone to obtain product II;
  • the amount of RuO 2 is 2-10% of the weight of product I, more preferably 5%, and the reaction temperature is preferably -10-10°C, more preferably 0°C.
  • the concentration of the aqueous ammonia solution is 100-300 mM, more preferably 150 mM, and the reaction time is 3-10 min, more preferably 4-6 min.
  • the above-mentioned polysaccharide is a natural plant polysaccharide, which can be any carrageenan, such as k-carrageenan, l-carrageenan or t-carrageenan.
  • the present invention also includes the application of the above-mentioned bonded selenium polysaccharide in improving immune function.
  • the selenium-bonded polysaccharide is used as an additive component to prepare immunity-improving drugs.
  • the bonded selenium polysaccharide of the present invention only contains bonded organic selenium (hereinafter referred to as bonded selenium), and the selenium element in the molecule exists in the form of organic ester in the compound, and does not contain harmful sodium selenite and selenium dioxide And other inorganic substances.
  • the invention makes full use of the unique structure of the polysaccharide lactone, and can provide a bonded selenium product with a certain organic selenium content.
  • the binding site of selenium in the polysaccharide is clear, and the selenium content of the bonded selenium polysaccharide is controllable.
  • the invention utilizes organic chemical reaction technology to successfully bond selenocysteine to polysaccharides with selenoester bonds, realizes the conversion of inorganic selenium to organic selenium, and avoids the potential harm of adding inorganic selenium to human health.
  • Selenium cysteine and natural polysaccharides are bonded together in the form of selenocarboxylate, which has a stable structure, which significantly improves the bioavailability of organic selenium and avoids the existing product "methylselenocysteine" "The risk of DNA methylation also avoids the risk of cardiovascular disease caused by homocysteine produced by the metabolism of selenomethionine.
  • the bonded selenium polysaccharide of the present invention can eliminate free radicals in the body, activate selenoproteins in the body by increasing the blood selenium content, significantly improve the immunity of test organisms, and assist radiotherapy and chemotherapy.
  • Figure 1 shows the cytokine secretion of B-6 splenocytes after incubation in different polysaccharide culture media.
  • the invention provides a bonded selenium polysaccharide with a controllable selenium content.
  • the selenium element in the molecule exists in the form of an organic ester in the compound.
  • the molecular structure features include: a polysaccharide with a 3,6-lactone sugar structure
  • the similar substance is the carrier, and the selenium atom is connected to the 6-position carbon atom of the 3,6-internal ether structure of the polysaccharide through the structure of the selenocarboxylate.
  • the polysaccharide of the present invention is preferably a natural plant polysaccharide, such as carrageenan.
  • the present invention does not limit the type of carrageenan, and all carrageenan polysaccharides with 3,6-internal ether structure can be used in the present invention.
  • k-carrageenan is taken as an example to illustrate the molecular structure, preparation method and biological activity of the bonded selenium polysaccharide. But it is not a limitation of the present invention.
  • the selenium-bonded polysaccharide molecule of the present invention has the following structure:
  • n is related to the molecular weight of natural polysaccharides and is the number of polysaccharide repeating units.
  • the above-mentioned carrageenan-bonded selenium polysaccharide is characterized by: oxidizing the 6-position carbon atom in the 3,6-internal ether bond of carrageenan to convert it to 3,6-lactone, and then open the ring with selenocysteine
  • the esterification reaction prepares a bonded selenium polysaccharide product with a clear structure and a controllable selenium content.
  • the 3,6-lactone sugar is catalyzed and oxidized to 3,6-lactone by RuO 2 and further realized by the nucleophilic reaction of the selenium anion to the lactone.
  • the specific preparation process includes the following steps:
  • the carrageenan polysaccharide is suspended in an organic solvent, and chloroacetyl chloride is added dropwise to react while maintaining the temperature.
  • chloroacetyl chloride is added dropwise to react while maintaining the temperature.
  • triethylamine (Et3N) is added to maintain the pH of the solution.
  • the maintaining temperature is preferably within 0-10°C.
  • the pH of the solution is preferably alkaline.
  • product I is obtained.
  • the present invention preferably dissolves product I in water and precipitates product I in cold ethanol.
  • the obtained product I was re-dissolved in an organic solvent and reacted with sodium hypochlorite (NaClO) under the condition of ruthenium dioxide (RuO 2 ) as a catalyst to obtain product II.
  • the sodium hypochlorite of the present invention is preferably a sodium hypochlorite solution, and more preferably the concentration of the sodium hypochlorite solution is 2-10%, more preferably 5%.
  • the reaction temperature is preferably maintained at -10 to 10°C, more preferably 0°C.
  • the obtained product II is dissolved in an organic solvent and reacted with an aqueous ammonia solution to obtain product III.
  • concentration of the aqueous ammonia solution of the present invention is preferably 100 to 300 mM, more preferably 150 mM.
  • the reaction time is preferably 3 to 10 minutes, more preferably 4 to 6 minutes.
  • the product III of the present invention is preferably precipitated in cold ethanol.
  • selenocystine is preferably dissolved in an aqueous alkali solution, more preferably an aqueous NaOH solution.
  • concentration of the aqueous NaOH solution is preferably 1 to 3M.
  • the preferred amount of selenocystine is 1-1000 g, more preferably 5-100 g; the preferred amount of sodium borohydride is 0.5-76 g, more preferably 1-40 g.
  • the reaction time is preferably 2-12 hours, more preferably 4 hours.
  • the final product bound selenium polysaccharide is precipitated from the cold ethanol solution.
  • the precipitated product can be dissolved in hot water and precipitated with cold ethanol to obtain a qualified product.
  • the organic solvent in the method for preparing the above-mentioned bonded selenium polysaccharide of the present invention is preferably dimethylformamide (DMF) or DMSO.
  • the temperature of cold ethanol used in the present invention is preferably 0-15°C, more preferably 0-5°C.
  • the synthesis reaction path of selenium-bonded polysaccharide is as follows:
  • the bonded selenium polysaccharide prepared by the invention can eliminate free radicals in the body and activate selenoproteins in the body by increasing the blood selenium content.
  • Animal experiments have proved that the selenium-bonded polysaccharide of the present invention can significantly improve the immunity of test organisms, and can be used as an additive component to prepare immunity-improving drugs, and to assist radiotherapy and chemotherapy.
  • k-carrageenan polysaccharide 100g is suspended in 200ml of dimethylformamide (DMF), 50ml of triethylamine (Et 3 N) is added, and 25ml of chloroacetyl chloride is added dropwise while maintaining 0°C. During the reaction Add the amount of Et 3 N to keep the pH of the solution at a basic level. The reaction was completed after 4 hours, the product was dissolved in water, and the product was precipitated in cold ethanol.
  • DMF dimethylformamide
  • Et 3 N triethylamine
  • n is related to the molecular weight of natural polysaccharides and is the number of polysaccharide repeating units.
  • reaction intermediate is dissolved in 50ml DMF, poured into an ammonia solution with a concentration of about 200mM at a volume ratio of 1:1, reacted for 2-4min, and cold ethanol is added to the reaction system until the product precipitates.
  • 25g of selenocystine (commercially available) was dissolved in 20ml of 2M NaOH aqueous solution, and 10g of sodium borohydride was added at 0°C and stirred for 4 hours at low temperature.
  • the precipitate in the reactor I is directly added to the reaction system, and after 5 hours of reaction, it is poured into cold ethanol solution.
  • the precipitated product can be dissolved in hot water and precipitated in cold ethanol to obtain a product.
  • n is related to the molecular weight of natural polysaccharides and is the number of polysaccharide repeating units.
  • t-carrageenan polysaccharide 200g is suspended in 200ml of dimethylformamide (DMF), 100ml of triethylamine (Et 3 N) is added, 40ml of chloroacetyl chloride is added dropwise while maintaining zero degree, and added during the reaction The amount of Et 3 N should keep the pH of the solution at a basic level. After 5 hours, the reaction product was dissolved in water and precipitated in cold ethanol.
  • DMF dimethylformamide
  • Et 3 N triethylamine
  • reaction intermediate is dissolved in 80ml DMF, poured into an ammonia solution with a concentration of about 250mM, and after reacting for 4-6min, cold ethanol is added to the reaction system until the product precipitates.
  • 50g selenocystine commercially available product
  • 50ml 1M NaOH aqueous solution 50ml 1M NaOH aqueous solution
  • 15g sodium borohydride was added at 0°C and stirred for 6 hours at low temperature.
  • the precipitate in the reactor I is directly added to the reaction system, and after reacting for 4 hours, it is poured into cold ethanol solution.
  • the precipitated product can be dissolved in hot water and precipitated in cold ethanol to obtain a product.
  • n is related to the molecular weight of natural polysaccharides and is the number of polysaccharide repeating units.
  • mice were fed with different types of polysaccharides, and their serum selenium content, red blood cell content and white blood cell content were detected.
  • the red blood cells of mice fed with chitosan oligosaccharides increased by 9.2% and white blood cells increased by 10.5%; the red blood cells of mice fed with lentinan increased by 9.4%, and white blood cells increased by 10.8%.
  • the number of red blood cells represents the oxygen carrying capacity
  • the number of white blood cells represents the anti-inflammatory capacity. It shows that the bonded selenium polysaccharide of the present invention can effectively increase the blood selenium content in organisms, and significantly improve the bioavailability of organic selenium. It also improves the oxygen carrying capacity and anti-inflammatory ability to a certain extent, and further improves the body's immunity.
  • the B-6 spleen cells were incubated in standard culture medium containing different types of polysaccharides, and the cells were harvested after the incubation was completed, and the expression of cytokines was detected.
  • the bonded selenium polysaccharide of the present invention contains 5-10 micrograms/ml of selenium (equivalent to 500 micrograms of bonded selenium products), and carrageenan, chitooligosaccharides, lentinan, and yam polysaccharides without a bonded selenium structure are used as controls (2mg/mL each), and set up a blank control group and a conventionally used enoxomycin (Ionomycin, 1ng/mL) positive control group.
  • enoxomycin Ionomycin, 1ng/mL
  • the standard culture medium contains 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 culture fluid buffer system is phosphate buffered saline solution PBS, and the pH value is 7.4 after equilibrating in a cell incubator containing 5% CO 2 .
  • the maximum secretion of IL-4 produced by the selenium-bonded polysaccharide of the present invention is 2.3, 1.9 and 2.7 times that of chitosan, lentinan, and yam polysaccharide respectively; the maximum expression of IFN- ⁇ is respectively 2.1, 1.2 and 3.1 times of chitosan, lentinan and yam polysaccharide.
  • the present invention uses polysaccharides without bound selenium (500 mg/day) as a control, and continuously feeds 5 micrograms of selenium/day with the bound selenium of the present invention after birth, and on the 7th, 14th, and 21st days after feeding , 28 days to detect the activity of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in chicks.
  • GSH-Px glutathione peroxidase
  • SOD superoxide dismutase
  • test method refers to published papers (Optimization of selenylation conditions for lycium barbarum polysaccharide based on antioxidant activity, Carbohydrate Polymers, 2014, 103, 148).
  • the detection method uses a commercially available kit EnzyChrom TM Glutahione Peroxidase Assay Kit and Mouse superoxide dismutase (SOD) kit for detection.

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Abstract

本发明属于生物医药领域，具体涉及一种键合硒多糖，该多糖以具有3,6-内醚糖结构的多糖类物质为载体，硒原子通过硒代羧酸酯的结构连接在多糖3,6-内醚结构的6位碳原子上。本发明通过氧化多糖3,6-内醚键中的6位碳原子，转换其为3,6-内酯，再与硒代半胱氨酸发生开环成酯反应，制备结构明确，含硒量可控的键合硒多糖产品。所制备的键合硒产品能通过提高血硒含量来显著改善受试生物的免疫能力。

Description

键合硒多糖及其制备方法和应用
本申请要求于2019年07月15日提交中国专利局、申请号为2019106356563，发明名称为“键合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
本发明属于生物医药领域，具体涉及一种新的键合硒多糖及其制备方法，以及其在免疫方面的应用。
背景技术
硒是一种对人体非常重要的微量元素，硒代半胱氨酸是第21种氨基酸，硒是已知的25中硒蛋白结构中的重要组成部分，其活性依赖于硒的存在，比如研究者发现硒是动物体内谷胱甘肽过氧化物酶的必需成分。但人体补充硒元素的途径非常有限，常用的所谓食补远远不能满足人体正常的硒代谢水平。为此，研究者于1975年首次明确指出硒是人体必需的微量元素，我国营养学会也于1988年将硒元素列为每日膳食营养元素之一。
近几年出现了人体补充硒元素的产品。一是使用无机亚硒酸钠，但由于缺乏对其生物安全性的可靠评价，使用无机亚硒酸钠存在着食品安全隐患。二是通过富硒土壤种植的作物富硒，产品安全性好，但由于作物自身生长的生理需求所限，作物中的硒含量较低，难以快速实现血液所必须硒含量的正常代谢平衡。三是通过化学方法制备富硒产品，主要是通过亚硒酸钠和浓硝酸对多糖羟基的随机酯化反应来实现，缺点是硒化位点不明确，硒化程度重现性差，多糖中混有的较大量的毒性无机硒成份难以完全去除。
发明内容
本发明提供了一种含硒量可控的键合硒多糖及其制备方法，形成的键合硒多糖结构稳定，有机硒含量确定，具有改善诸多免疫功能的作用。
本发明的技术方案如下：
本发明提供一种键合硒多糖，分子结构特征包括：以具有3,6-内醚糖结构的多糖类物质为载体，硒原子通过硒代羧酸酯的结构连接在多糖3,6-内醚结构的6位碳原子上。
作为优选的，所述多糖来源于提取的天然植物多糖，更优选为卡拉胶。
作为优选的，本发明所述键合硒多糖的分子具有如下任意结构：
Figure PCTCN2019096583-appb-000001
其中，上述通式中，n与天然多糖的分子量相关，是多糖重复单元数。
本发明还包括上述键合硒多糖的制备方法，通过RuO 2催化氧化多糖3,6-内醚键中的6位碳原子转换为3,6-内酯，通过硒负离子对内酯的亲核反应发生开环成酯反应，将硒键合到多糖内醚结构的碳原子上。
作为一种更优选的技术方案，本发明所述键合硒多糖的制备方法，包括以下步骤：
(1)所述多糖与氯乙酰氯发生反应，将多糖中羟基中的氢取代为乙酰氯，得到的产物Ⅰ；
(2)产物Ⅰ在RuO 2催化下与次氯酸钠反应，将多糖3,6-内醚键中的6位碳原子转换为3,6-内酯，得到产物Ⅱ；
(3)产物Ⅱ在氨水溶液中反应，将氧乙酰氯转化为羟基，得到产物Ⅲ；
(4)产物Ⅲ与硒代胱氨酸在硼氢化钠存在下发生开环成酯反应，得到键合硒多糖。
本发明作为优选的，步骤(2)中，所述RuO 2的用量为产物Ⅰ重量的2～10％，更优选为5％，反应温度优选为-10～10℃，更优选为0℃。
本发明作为优选的，步骤(3)中，所述氨水溶液的浓度为100～300mM，更优选为150mM，反应时间为3～10min，更优选为4～6min。
本发明中，上述多糖为天然植物多糖，可以为任意一种卡拉胶，如k-卡拉胶、l-卡拉胶或t-卡拉胶。
本发明还包括上述键合硒多糖在改善免疫功能的应用。
作为优选的，所述键合硒多糖作为添加成分制备改善免疫类的药物。
与现有技术相比，本发明的优点在于：
本发明的键合硒多糖只含有键合了的有机硒(下称键合硒)，分子中的硒元素在化合物中以有机酯的形式存在，不含有害的亚硒酸钠、二氧化硒等无机物。本发明充分利用多糖内酯的独特结构，可以提供有机硒含量确定的键合硒产品，同时，硒在多糖中的结合位点明确，键合硒多糖含硒量可控。
本发明利用有机化学反应技术，将硒半胱氨酸成功地以硒代酯键键合到多糖上，实现了无机硒向有机硒的转化，避免了添加无机硒对人体健康的潜在危害。硒半胱氨酸与天然多糖以硒代羧酸酯的形式键合在一起，结构稳定，显著提高了有机硒的生物可利用度，同时避免了已有产品“甲基硒代半胱氨酸”带来的DNA甲基化风险，也避免了硒代蛋氨酸代谢产生的高半胱氨酸导致的心血管疾病风险。
本发明的键合硒多糖能消除体内自由基，通过提高血硒含量活化体内硒蛋白，显著改善受试生物的免疫能力，辅助放化疗等。
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B-6脾细胞子啊不同多糖培养液中孵育后的细胞因子分泌量。
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。
本发明提供了一种含硒量可控的键合硒多糖，分子中的硒元素在化合物中以有机酯的形式存在，分子结构特征包括：以具有3,6-内醚糖结构的多糖类物质为载体，硒原子通过硒代羧酸酯的结构连接在多糖3,6-内醚结构的6位碳原子上。本发明的多糖优选为天然植物多糖，如卡拉胶。本发明不限定卡拉胶的种类，所有具有3,6-内醚结构的卡拉胶多糖都可用于本发明中。本发明中以k-卡拉胶为例说明键合硒多糖的分子结构、制备方法及生物活性。但并非是对本发明的限制。
以下以k-卡拉胶为例，说明本发明键合硒多糖的分子特性及制备方法。
当多糖为k-卡拉胶时，本发明所述键合硒多糖的分子具有如下结构：
Figure PCTCN2019096583-appb-000002
其中，上述通式中，n与天然多糖的分子量相关，是多糖重复单元数。
上述卡拉胶键合硒多糖制备特征为：通过氧化卡拉胶3,6-内醚键中的6位碳原子，转换其为3,6-内酯，再与硒代半胱氨酸发生开环成酯反应，制备得到结构明确，含硒量可控的键合硒多糖产品。其中，3,6-内醚糖通过RuO 2催化氧化成3,6-内酯，并进一步通过硒负离子对内酯的亲核反应来实现。具体制备过程包括如下步骤：
首先，将卡拉胶多糖悬浮在有机溶剂中，在维持温度的条件下滴加氯乙酰氯进行反应，反应过程中加入三乙胺(Et3N)以保持溶液的pH值。所述维持温度优选为0～10℃以内。所述溶液的pH值优选为碱性。反应完成后得到产物Ⅰ。本发明优选将产物Ⅰ溶于水中，在冷的乙醇中沉淀出产物Ⅰ。
将得到的产物Ⅰ重新溶于有机溶剂中，在二氧化钌(RuO 2)作为催化剂条件下，与次氯酸钠(NaClO)反应得到产物Ⅱ。本发明次氯酸钠优选为次氯酸钠溶液，进一步优选次氯酸钠溶液的浓度为2～10％，更优选为5％。本发明优选反应温度在维持在-10～10℃，更优选为0℃。本发明优选反应完成后先过滤，后再将产物Ⅱ在冷乙醇中沉淀出来。
将得到的产物Ⅱ溶于有机溶剂中，与氨水溶液中反应后得到产物Ⅲ。本发明氨水溶液的浓度优选为100～300mM，更优选为150mM。反应时间优选为3～10min，更进一步优选为4～6min。本发明所述产物Ⅲ优选在冷乙醇中析出。
将得到的产物Ⅲ与硒代胱氨酸在硼氢化钠存在下低温搅拌反应得到终产物键合硒多糖。本发明优选硒代胱氨酸溶于碱水溶液中，更优选为NaOH水溶液，本发明优选NaOH水溶液的浓度为1～3M。本发明优选硒代胱氨酸的用量为1-1000克，更优选为5-100克；优选硼氢化钠的用量为0.5-76克，更优选为1-40克。本发明优选反应时间为2～12小时，更优选为4小时。反应完成后，终产物键合硒多糖从冷乙醇液中沉淀出来。作为优选的，沉淀出的产品可经热水溶解和冷乙醇醇沉得到合格产品。
本发明上述键合硒多糖制备方法中有机溶剂优选为二甲基甲酰胺(DMF)或DMSO。
本发明优选所用的冷乙醇的温度为0～15℃，更优选为0～5℃。
本发明以k-卡拉胶为例，键合硒多糖合成反应路径如下：
Figure PCTCN2019096583-appb-000003
本发明制备得到的键合硒多糖能消除体内自由基，通过提高血硒含量活化体内硒蛋白。经动物实验证明，本发明的键合硒多糖能显著改善受试生物的免疫能力，可作为添加成分制备改善免疫类的药物，辅助放化疗等。
下面将结合本发明的实施例，对本发明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。
实施例1：
k-卡拉胶键合硒多糖的合成
(1)k-卡拉胶多糖100g悬浮在200ml二甲基甲酰胺(DMF)中，加入50ml三乙胺(Et 3N)，在维持0℃的条件下滴加氯乙酰氯25ml，反应过程中加入Et 3N量要保持溶液的pH值处于偏碱性。4小时后完成反应，产物溶于水中，在冷的乙醇中沉淀出产物。
(2)上述粗产物重新溶于50ml DMF中，加入5％重量比的催化剂二氧化钌(RuO 2)，然后在维持0℃的条件下加入30ml浓度为5％的次氯酸钠(NaClO)水溶液，反应5小时后产物先过滤，后再将中间体在低温下的乙醇中沉淀出来。
(3)上述反应中间体溶于50ml DMF中，倾倒入浓度大约为150mM的氨水溶液中，反应4-6min后，向该反应体系加入冷乙醇直至产物析出。在另一反应器中，12g硒 代胱氨酸(有市售产品)溶于20ml 1M的NaOH水溶液中，0℃下加入3.5g硼氢化钠零度低温搅拌4小时。向该反应体系直接加入反应器I中的析出物，反应4小时后，倒入冷乙醇液中，沉淀出的产品可经热水溶解和冷乙醇醇沉得到产品。
得到的键合硒多糖结构如下：
Figure PCTCN2019096583-appb-000004
通式中，n与天然多糖的分子量相关，是多糖重复单元数。
实施例2
loca-卡拉胶键合硒多糖的合成
(1)l-卡拉胶多糖100g悬浮在200ml二甲基甲酰胺(DMF)中，加入50ml三乙胺(Et 3N)，在维持零度的条件下滴加氯乙酰氯20ml，反应过程中加入Et 3N量要保持溶液的pH值处于偏碱性。4小时后完成反应产物溶于水中，在冷的乙醇中沉淀出产物。
(2)上述粗产物重新溶于50ml DMF中，加入7％重量比的催化剂二氧化钌(RuO 2)，然后在维持0℃的条件下加入40ml浓度为3％的次氯酸钠(NaClO)水溶液，反应5小时后产物先过滤，后再将中间体在低温下的乙醇中沉淀出来。
(3)上述反应中间体溶于50ml DMF中，以1:1体积比倾倒入浓度大约为200mM的氨水溶液中，反应2-4min后，向该反应体系加入冷乙醇直至产物析出。在另一反应器中，25g硒代胱氨酸(有市售产品)溶于20ml 2M的NaOH水溶液中，0℃下加入10g硼氢化钠低温搅拌4小时。向该反应体系直接加入反应器I中的析出物，反应5小时后，倒入冷乙醇液中，沉淀出的产品可经热水溶解和冷乙醇醇沉得到产品。
得到的键合硒多糖结构如下：
Figure PCTCN2019096583-appb-000005
通式中，n与天然多糖的分子量相关，是多糖重复单元数。
实施例3
Theta-卡拉胶键合硒多糖的合成
(1)t-卡拉胶多糖200g悬浮在200ml二甲基甲酰胺(DMF)中，加入100ml三乙胺(Et 3N)，在维持零度的条件下滴加氯乙酰氯40ml，反应过程中加入Et 3N量要保持溶液的pH值处于偏碱性。5小时后完成反应产物溶于水中，在冷的乙醇中沉淀出产物。
(2)上述粗产物重新溶于80ml DMF中，加入4％重量比的催化剂二氧化钌(RuO 2)，然后在维持0℃的条件下加入50ml浓度为4％的次氯酸钠(NaClO)水溶液，反应5小时后产物先过滤，后再将中间体在低温下的乙醇中沉淀出来。
(3)上述反应中间体溶于80ml DMF中，倾倒入浓度大约为250mM的氨水溶液中，反应4-6min后，向该反应体系加入冷乙醇直至产物析出。在另一反应器中，50g硒代胱氨酸(有市售产品)溶于50ml 1M的NaOH水溶液中，0℃下加入15g硼氢化钠低温搅拌6小时。向该反应体系直接加入反应器I中的析出物，反应4小时后，倒入冷乙醇液中，沉淀出的产品可经热水溶解和冷乙醇醇沉得到产品。
得到的键合硒多糖结构如下：
Figure PCTCN2019096583-appb-000006
通式中，n与天然多糖的分子量相关，是多糖重复单元数。
实施例4
小鼠模型试验
饲喂小鼠不同种类多糖，检测其血清硒含量、红细胞含量和白细胞含量水平。
以壳寡糖(2mg/天)和香菇多糖(2mg/天)为对照物，以5至10微克硒/天的硒剂量(相当于500微克/天本发明键合硒多糖产品)连续喂饲本发明的键合硒多糖2周后，于第三周至第六周连续进行血液检测(此时仍然喂饲键合硒或普通多糖，方式剂量与前相同)，每周检测一次。
检测结果发现试验小鼠血清硒含量最大提高了140％左右，血红细胞提高了20％，白细胞增加了11.2％，相关检测指标从第五周开始趋于稳定表达。而饲喂壳寡糖的小鼠血红细胞提高了9.2％，白细胞增加了10.5％；饲喂香菇多糖的小鼠血红细胞提高了 9.4％，白细胞增加了10.8％。其中，红细胞数代表载氧能力，白细胞数表示抗炎症的能力。说明本发明的键合硒多糖能有效提高生物体内的血硒含量，显著提高了有机硒的生物可利用度。还在一定程度上提高了载氧能力和抗炎能力，进一步提高机体免疫力。
表1试验小鼠饲喂键合硒多糖后的血检结果
Figure PCTCN2019096583-appb-000007
表2试验小鼠饲喂壳寡糖和香菇多糖后的血检结果
Figure PCTCN2019096583-appb-000008
实施例5
B-6脾细胞试验模型
将B-6脾细胞在含不同种类多糖的标准培养液中进行孵育，孵育完成后收获细胞，检测细胞因子的表达量。
其中，本发明键合硒多糖含硒5-10微克/毫升(折合约500微克键合硒产品)，以不含键合硒结构的卡拉胶、壳寡糖、香菇多糖、山药多糖为对照物(各2mg/mL)，并设置空白对照组和按惯例使用的依诺霉素(Ionomycin，1ng/mL)阳性对照组。
标准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。该培养液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。
将上述多糖及阳性对照加入标准培养液中，孵育B-6脾细胞48小时，细胞收获后，以BD Bioscience的Cytofix/Cytoperm试剂盒染色，用流式细胞仪检测细胞因子的表达量。数据用Treestar Inc.公司的软件Flowjo software处理后，结果如附图1所示。与未键合硒处理的卡拉胶对比，本方面的键合硒多糖免疫指标显著性提高，说明键合硒多糖产生的免疫功能完全是通过键合的有机硒来起作用的。以IL-4为比对指标，本发明键合硒多糖产生的IL-4最大分泌量分别为壳寡糖、香菇多糖、山药多糖的2.3，1.9和2.7倍；IFN-γ最大表达量分别为壳寡糖、香菇多糖、山药多糖的2.1，1.2和3.1倍。说明使用本发明的键合硒多糖可以有效改善体系的免疫能力，效果显著地好于市面上常用的壳寡糖(以低聚壳寡糖为代表)、香菇多糖、山药多糖等保健品。
实施例6
雏鸡模型的抗氧化试验
本发明以不含键合硒的多糖(500mg/天)为对照物，在出生后连续喂饲本发明键合硒5微克硒/天，并在喂饲后第7天、14天、21天、28天检测雏鸡体内的谷胱甘肽过氧化物酶(GSH-Px)和超氧化物歧化酶(SOD)的活性。
试验方法参照已公开发表的论文(Optimization of selenylation conditions for lycium barbarum polysaccharide based on antioxidant activity，Carbohydrate Polymers，2014，103，148)。
检测方法使用市售的试剂盒EnzyChrom TMGlutahione Peroxidase Assay Kit和
Figure PCTCN2019096583-appb-000009
小鼠超氧化物歧化酶(SOD)试剂盒进行检测。
检测结果发现雏鸡体内GHS-Px活性在第14-21天达到最大值，提高了17％，SOD在第7-14天达到最大值，提高了14％。说明本发明的键合硒多糖具有良好的抗氧化功能。
表3饲喂键合硒多糖雏鸡体内GSH-Px和SOD的活性
Figure PCTCN2019096583-appb-000010
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. 一种新型键合硒多糖，其特征在于，以具有3,6-内醚糖结构的多糖类物质为载体，硒原子通过硒代羧酸酯的结构连接在多糖3,6-内醚结构的6位碳原子上。
  2. 根据权利要求1所述的键合硒多糖，其特征在于，所述多糖来源于提取的天然植物多糖。
  3. 根据权利要求1所述的键合硒多糖，其特征在于，所述键合硒多糖的分子结构为以下任意一个：
    Figure PCTCN2019096583-appb-100001
    通式中，n与天然多糖的分子量相关，是多糖重复单元数。
  4. 权利要求1～3任意一项所述键合硒多糖的制备方法，其特征在于，通过RuO 2催化氧化多糖3,6-内醚键中的6位碳原子转换为3,6-内酯，通过硒负离子对内酯的亲核反应发生开环成酯反应，将硒键合到多糖内醚结构的碳原子上。
  5. 根据权利要求4所述键合硒多糖的制备方法，其特征在于，包括以下步骤：
    (1)所述多糖与氯乙酰氯发生反应，将多糖中羟基中的氢取代为乙酰氯，得到的产物Ⅰ；
    (2)产物Ⅰ在RuO 2催化下与次氯酸钠反应，将多糖3,6-内醚键中的6位碳原子转换为3,6-内酯，得到产物Ⅱ；
    (3)产物Ⅱ在氨水溶液中反应，将氧乙酰氯转化为羟基，得到产物Ⅲ；
    (4)产物Ⅲ与硒代胱氨酸在硼氢化钠存在下发生开环成酯反应，得到键合硒多糖。
  6. 根据权利要求5所述键合硒多糖的制备方法，其特征在于，步骤(2)中，所述RuO 2的用量为产物Ⅰ重量的2～10％，反应温度为-10℃～10℃。
  7. 根据权利要求5所述键合硒多糖的制备方法，其特征在于，步骤(3)中，所述氨水溶液的浓度为100～300mM，反应时间为3～10min。
  8. 根据权利要求4～7任意一项所述键合硒多糖的制备方法，其特征在于，所述多糖为卡拉胶。
  9. 权利要求1～3任意一项所述的键合硒多糖或采用权利要求4～8任意一项所述方法制备得到的键合硒多糖在改善免疫功能的应用。
  10. 根据权利要求9所述的应用，其特征在于，所述键合硒多糖作为添加成分制备改善免疫类的药物。
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