WO2018166240A1 - 一种太平洋牡蛎中性多糖及其制备方法和应用 - Google Patents

一种太平洋牡蛎中性多糖及其制备方法和应用 Download PDF

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WO2018166240A1
WO2018166240A1 PCT/CN2017/113256 CN2017113256W WO2018166240A1 WO 2018166240 A1 WO2018166240 A1 WO 2018166240A1 CN 2017113256 W CN2017113256 W CN 2017113256W WO 2018166240 A1 WO2018166240 A1 WO 2018166240A1
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oyster
polysaccharide
neutral
neutral polysaccharide
pacific
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French (fr)
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刘杨
夏李轩
张杰良
肖湘
钟名其
伦镜盛
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汕头大学
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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
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • 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
    • 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
    • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof

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  • the invention relates to the field of deep processing of natural products, in particular to a Pacific oyster neutral polysaccharide and a method and application thereof.
  • Oyster is the world's largest farmed shellfish. It is also an important marine shellfish in China. It is an oyster family and a bivalve mollusc. It is distributed in the temperate and tropical coastal waters. The main species are Omi oysters, Pacific oysters, and Dalian Bay oysters. And dense scale oysters. Pacific oysters have two shells on the left and right, connected by ligaments and the occipital muscles. The left shell is concave, large and thick, and can be used to attach other objects. The surface of the shell has more radiating ribs, which are clearly countable; the right shell is small and flat, and the surface has multiple layers of concentric annular scales without significant radiating ribs.
  • Oyster is a kind of marine economic shellfish with medicinal value.
  • the meat in addition to rich protein, the meat also contains a large amount of polysaccharides with glycogen structure.
  • the content of polysaccharides accounts for 20%-40% of the dry weight of oysters. .
  • the crude oyster polysaccharides are mainly composed of neutral and acidic polysaccharides.
  • the structure of oyster glycogen is divided into primary structure and high-grade structure (including secondary, tertiary, and quaternary structures).
  • the extraction methods adopted for the same raw materials are different, the structure of the obtained polysaccharides is also different, and the polysaccharides of different parts of the same raw materials also have different structures, so the treatment steps are also different. Therefore, it is important to choose the appropriate method, and obtain a higher extraction rate based on the original structure of glycogen as little as possible.
  • the extraction methods of oyster polysaccharides mainly include water extraction method, alkali extraction method and enzymatic extraction method.
  • the water extraction method has the advantages of simple equipment, simple operation, wide application, and the like, but the operation time is long. It is inefficient and requires repeated operations, so energy consumption is high.
  • the process of water extraction is roughly as follows: after the fresh oyster meat is accurately weighed, it is homogenized after washing, and then the triangular flask containing the oyster slurry is placed in a boiling water bath and boiled for 1 hour, cooled and filtered, and the alcohol is concentrated.
  • the alkali extraction method can completely extract the polysaccharide from the tissue, which is based on the animal body, the sugar and the protein combine to form a glycoprotein, and the covalent bond is unstable to the alkali, thereby achieving the purpose of releasing the polysaccharide; generally using NaOH, KOH
  • the extraction rate is high, but the polysaccharide molecule may be degraded, which has a certain influence on the structure and activity of the polysaccharide.
  • the process of alkali extraction is roughly as follows: the oyster meat is homogenized, added with 30% potassium hydroxide, heated at 100 ° C for 1 h, cooled to room temperature and then added 95%.
  • Enzymatic extraction method uses biological enzyme to extract active polysaccharide. Because the specificity and selectivity of the enzyme are strong, this method has the advantages of mild conditions, no damage to polysaccharide activity, high purity of extracted polysaccharide, etc., but it will increase production cost and extract The condition requirements are also higher.
  • the enzyme extracting enzyme is generally pepsin and trypsin, and the enzymatically hydrolyzed crude oyster polysaccharide is further purified by ion exchange chromatography to obtain different components.
  • the crude polysaccharide also contains impurities such as pigments, proteins, and oligosaccharides.
  • Impurities such as pigments, proteins, and oligosaccharides.
  • Decolorization of activated carbon or decolorization of H 2 O 2 is often used, which causes problems such as adsorption and oxidation of neutral polysaccharides.
  • Column chromatography is currently the more common method of removing these impurities.
  • the above methods for extracting and removing impurities have many problems such as many operation steps, low product yield, long operation cycle, introduction of impurities, and reduction in activity.
  • the oyster neutral polysaccharide crude extract is dialyzed to obtain the oyster polysaccharide extract, and the oyster neutral polysaccharide can be obtained after concentration and drying.
  • the ultrasonic microwave extractor in the step (2) has a treatment time of 20-30 minutes and a treatment temperature of 80-90 °C.
  • Another object of the present invention is to provide a Pacific oyster neutral polysaccharide prepared by the above method.
  • Still another object of the present invention is to provide an application of the above-mentioned Pacific oyster neutral polysaccharide in the preparation of an immunologically relevant drug.
  • Still another object of the present invention is to provide a use of a Pacific oyster neutral polysaccharide for the preparation of an antitumor drug.
  • the tumor is liver cancer.
  • the invention adopts ultrasonic microwave extraction, ethanol-(NH4)2SO4 aqueous two-phase system extraction and dialysis to extract oyster neutral polysaccharide, which avoids the high cost and low yield of traditional chromatography technology ( ⁇ 5% oyster neutral polysaccharide / oyster powder), long time and other drawbacks, simple extraction process, low cost, suitable for intermittent and large-scale production and processing of high purity, high yield of oyster neutral polysaccharide products.
  • the oyster polysaccharide extracted by the prior art method is usually a mixture of a neutral polysaccharide and an acidic polysaccharide.
  • the oyster polysaccharide extracted by the method of the present invention is a pure neutral polysaccharide.
  • the present inventors have found that oyster neutral polysaccharide can improve the activity of mouse spleen lymphocyte immune cells better than acidic polysaccharide, and has stronger inhibitory effect on proliferation and apoptosis induction of human hepatoma cell line Hepg-2.
  • the Pacific oyster neutral polysaccharide prepared by the present invention can be stored at 4 ° C for a long time without any decrease or loss of activity.
  • the Pacific oyster neutral polysaccharide prepared by the invention can promote the immunological activity of mouse spleen lymphocytes, and has the effects of inhibiting proliferation and inducing apoptosis of human hepatoma cell line Hepg-2.
  • Fig. 1 is a flow chart showing the process of extracting neutral polysaccharides from Pacific oyster according to the present invention.
  • FIG. 2 is a flow chart of a conventional oyster polysaccharide extraction process.
  • Figure 3 shows the enrichment of crude oyster polysaccharide extract using an ethanol-(NH4)2SO4 aqueous two-phase system.
  • Figure 5 is a finished product of a crude polysaccharide of Pacific oyster extracted by a conventional method in a comparative example.
  • Figure 6 is a chromatogram of the purity of oyster neutral polysaccharide prepared by the method of the present invention as determined by HPLC-evaporation photodetector.
  • Figure 7 (a) (b) are scanning spectra of the oyster polysaccharide crude extract and the oyster neutral polysaccharide finished UV spectrophotometer, respectively.
  • Fig. 8 is a chart showing the determination of the neutral polysaccharide monosaccharide of the oyster prepared by the method of the present invention detected by the HPLC-ELSD instrument, which is in agreement with the glucose standard map.
  • Figure 9 is a traditional oyster neutral polysaccharide (OGN1), the oyster neutral polysaccharide (OGN2) prepared by the method of the present invention, the traditional acidic polysaccharide (OGA1), and the oyster acid in the waste liquid after the aqueous two-phase extraction of the method of the invention
  • OOG1 traditional oyster neutral polysaccharide
  • OAA1 traditional acidic polysaccharide
  • OAA1 traditional acidic polysaccharide
  • oyster acid oyster acid in the waste liquid after the aqueous two-phase extraction of the method of the invention
  • the rate of proliferation of mouse spleen lymphocytes was determined by sugar (OGA2) and the crude polysaccharide extract (OG) in the method of the present invention.
  • Figure 10 is a graph showing the determination of IL-2 release from mouse spleen lymphocytes by the oyster neutral polysaccharide prepared by the method of the present invention.
  • Figure 11 is a graph showing the inhibition rate of oyster neutral polysaccharide prepared by the method of the present invention on human hepatoma cell line Hepg-2.
  • Figure 12 shows the detection of apoptosis of human hepatoma cell line Hepg-2 by Annexin V-FITC double staining method, wherein (a), (b), (c) correspond to the blank group and the positive group (pentafluorouracil 50 ⁇ g/mL).
  • the oyster neutral polysaccharide (200 ⁇ g/mL) prepared by the method of the present invention.
  • the finished product of Pacific oyster neutral polysaccharide is prepared according to the process shown in Figure 1. The specific steps are as follows:
  • the defatted oyster powder obtained in the step (1) is placed in a 1000 mL glass extraction vessel, and 600 mL of deionized water is added and placed in a 500 W ultrasonic microwave extractor for treatment at a temperature of 86 ° C. The time was 26 min and it was cooled after extraction. The extract was centrifuged (4000 rpm, 10 min), and the supernatant was combined and concentrated by rotary evaporation to obtain a crude oyster polysaccharide extract, which was freeze-dried to obtain a yellow crude polysaccharide powder, and the protein and polysaccharide content were determined.
  • the polysaccharides from Pacific oysters were extracted by a conventional process, and the specific process is shown in FIG. 2 .
  • the resulting product was a mixture of a neutral polysaccharide and an acidic polysaccharide, which was a yellow floc (as shown in Figure 4).
  • the content of the polysaccharide and protein in the polysaccharide solution in the different extraction steps was determined by the phenol-sulfuric acid method and the BCA method in Example 1 and Comparative Example 1.
  • Phenol-sulfuric acid method accurately weigh 20 mg of glucose dried to constant weight at 105 ° C, place it in a 500 mL volumetric flask, add water to the mark, shake well, and prepare a glucose standard solution of 40 ⁇ g/mL.
  • a standard curve is drawn on the glucose concentration with the A value as the ordinate, and the standard curve blank is used as a control to dilute a certain number of polysaccharide solutions according to the sample.
  • the absorbance value is used to find the polysaccharide content of the sample from the standard curve. Calculated as follows:
  • Polysaccharide content (%) (C oyster polysaccharide ⁇ D) / W oyster polysaccharide finished product ⁇ 100% (1).
  • C oyster polysaccharide is the test solution polysaccharide concentration
  • D is the dilution factor of the test solution
  • W oyster polysaccharide finished product is the weight of the finished oyster polysaccharide.
  • BCA method 50 parts of reagent A and 1 part of reagent B are mixed uniformly. Accurately absorb 25 ⁇ L of sample solution in the enzyme standard well, add 200 ⁇ L of BCA reagent, shake gently, incubate at 37 ° C for 30 min, cool to room temperature, and use blank as control. The colorimetric curve was obtained at 562 nm on the microplate reader, the bovine serum albumin content was plotted on the abscissa, and the absorbance was plotted on the ordinate. Using the standard curve blank as a control, the protein content of the sample was found from the standard curve based on the absorbance of the sample. Calculated as follows:
  • Protein content (%) (C protein ⁇ D) / W oyster polysaccharide finished product ⁇ 100% (2).
  • protein C is protein concentration for the test solution
  • D is the dilution factor for sample solution
  • W is finished oyster oyster polysaccharide polysaccharide by weight of the finished product.
  • Neutral sugar yield (%) W oyster neutral sugar finished product / W oyster dry powder ⁇ 100% (3).
  • Fig. 7(a) and (b) are scanning spectra of the oyster polysaccharide crude extract and the neutral polysaccharide ultraviolet spectrophotometer respectively.
  • the neutral polysaccharide has no obvious absorption peak at 280 nm, indicating that the protein removal effect is achieved well. .
  • Example 2 HPLC detection of molecular weight and purity of Pacific oyster neutral polysaccharide prepared by the method of the present invention
  • HPLC-ELSD instrument detection glucose standard (500 ⁇ g/mL), oyster neutral polysaccharide hydrolyzate, column Carbohydrate ES 5u (250mm ⁇ 4.6mm); mobile phase: acetonitrile-water (75% -25%) Injection volume: 20 mL, flow rate: 1 mL/min, drift tube temperature: 83 ° C, carrier gas flow rate 2.1 L/min.
  • Fig. 8 is a graph showing the determination of monosaccharides in the neutral polysaccharide of Pacific oyster prepared by the method of the present invention by HPLC-ELSD instrument, which is consistent with the glucose standard map, indicating that the monosaccharide composition is glucose.
  • Example 4 Determination of proliferation of mouse spleen lymphocytes by neutral oyster polysaccharide prepared by the method of the present invention
  • mice were immersed in 75% ethanol for 3 minutes, then transferred to a clean bench.
  • the mice were removed and placed on a sterile plastic table.
  • the left abdomen was facing upwards, the small abdomen was cut open, the spleen was exposed, and the spleen was lifted with tweezers.
  • the underlying connective tissue was isolated by ophthalmology scissors, and the spleen was taken out, placed on a 70 ⁇ m cell strainer, placed in a 50 mL centrifuge tube, and slowly rinsed with an appropriate amount (about 15 mL) of sterile PBS solution (pH 7.2-7.4).
  • the spleen was ground with a grinding rod, and individual cells were washed with PBS into a centrifuge tube, and then the slurry was centrifuged at 1000 rpm for 10 min, and the supernatant was discarded.
  • FIG. 9 is a graph showing the proliferation rate of spleen lymphocytes of oyster polysaccharides in mice.
  • OGN1, OGN2, OGA1, OGA2, OG are traditional oyster neutral polysaccharides, oyster neutral polysaccharides prepared by the method of the present invention, traditional acidic polysaccharides, and double water. Finish of oyster acid polysaccharide in the liquid after phase extraction, and polysaccharide in the method of the invention The crude extract (OG) can be seen that the proliferation rate of mouse spleen lymphocytes is 121.08% when the oyster neutral polysaccharide prepared by the method of the invention is 250 ⁇ g/mL, which can effectively increase the proliferation rate of mouse spleen lymphocytes.
  • Example 5 Effect of Pacific oyster neutral polysaccharide prepared by the method of the present invention on IL-2 release from mouse spleen lymphocytes (MSL)
  • MSL was inoculated to a 96-well plate at a density of 1 ⁇ 10 6 /mL, 100 ⁇ L per well, and then 100 ⁇ L of RPMI-1640 medium containing oyster polysaccharide and Con A was added (the final concentration of Con A was The final concentration of neutral polysaccharide of Pacific oyster prepared by the method of the present invention is 5.0 ⁇ g/mL, and each group is set to a control group (only RPMI-1640 medium is added), and 4 replicate holes are provided for each sample.
  • the cells were cultured in an incubator at 5% CO 2 at 37 ° C for 24 hours, and the culture solution was aspirated, centrifuged at 1500 rpm for 5 min, and the supernatant was collected.
  • Washing Dilute the concentrated washing solution and set aside. Remove the sealing film, discard the liquid, dry it, fill each well with the washing solution, let stand for 30 s and discard. Repeat 5 times and pat dry.
  • Color development add 50 ⁇ L of color developer to each well, add 50 ⁇ L of color developer B, mix well by shaking, and develop color at 37 ° C for 15 min.
  • Termination 50 ⁇ L of stop solution was added to each well.
  • the stimulation index was calculated by the following formula:
  • Stimulus index SI (%) (w experiment - w blank ) / w blank ⁇ 100% (5).
  • the w experiment and the w blank were the concentrations of IL-2 in the spleen lymphocytes of the experimental group and the blank group, respectively.
  • Figure 10 is a graph showing the release of IL-2 from mouse spleen lymphocytes prepared by the method of the present invention. It can be seen that the Pacific oyster neutral polysaccharide 250 ⁇ g/mL prepared by the method of the present invention is used for the mouse spleen. The stimulation index of lymphocyte IL-2 reached 124.81%, which can effectively stimulate the immune activity of mouse spleen lymphocytes.
  • Example 6 Determination of the inhibition rate of Pacific oyster neutral polysaccharide prepared by the method of the present invention on Hepg-2 cells in vitro
  • DMEM medium fetal bovine serum: penicillin/streptomycin in a ratio of 89:10:1). /mL. 100 ⁇ L (4000 cells) per well was added to a 96-well culture plate.
  • Inhibition rate (%) (A control group - A drug group ) / A control group ⁇ 100% (6).
  • Figure 11 is a graph showing the inhibition rate of the Pacific oyster neutral polysaccharide prepared by the method of the present invention on human hepatoma cell Hepg-2, and it can be seen that the human umbilical cancer cell Hepg-2 is obtained by using the method of the present invention, the Pacific oyster neutral polysaccharide 200 ⁇ g/mL.
  • the inhibition rate is 55.81%, which can effectively inhibit the growth of liver cancer cells.
  • the cell culture medium was aspirated, washed once with PBS, and 195 ⁇ L of LAnnexin V-FITC binding solution was added, 5 ⁇ L of Annexin V-FITC was added, and the mixture was gently mixed.
  • Annexin V-FITC was green fluorescence and PI was red fluorescence.
  • Figure 12 shows the detection of apoptosis of human hepatoma cell Hepg-2 cells by Annexin V-FITC double staining method, wherein (a), (b), (c), (d) correspond to the blank group, and the positive group (pentafluorouracil) 50 ⁇ g/mL), oyster neutral polysaccharide (200 ⁇ g/mL), neutral polysaccharide obtained by traditional extraction method (200 ⁇ g/mL), the dark color is the tumor cells with necrosis and advanced apoptosis, and the bright color is the early stage of apoptosis. cell.

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Abstract

本发明公开了一种太平洋牡蛎中性多糖的制备方法，包括以下步骤：(1)将太平洋牡蛎干粉通过丙酮脱脂，干燥后得到脱脂牡蛎粉；(2)将脱脂牡蛎粉溶于水中，用超声微波萃取仪处理，将处理后的溶液离心，上清液蒸发浓缩，得到牡蛎多糖粗提物；(3)将牡蛎多糖粗提物加入乙醇-(NH4)2SO4双水相体系进行萃取分离，萃取后的下相为牡蛎中性多糖粗提液；(4)将牡蛎中性多糖粗提液进行透析，得到牡蛎多糖精提液，浓缩干燥后即可得到牡蛎中性多糖。本发明提取工艺简单、成本低，适于间歇性和规模化生产加工高纯度、高收率、高生物活性的牡蛎中性多糖成品，且所制得的牡蛎中性多糖可长时间贮存、纯度高，能够提高小鼠脾淋巴细胞免疫活性，抑制Hepg-2肝癌细胞活性。

Description

一种太平洋牡蛎中性多糖及其制备方法和应用 技术领域
本发明涉及一种涉及天然产物深加工领域，具体涉及一种太平洋牡蛎中性多糖及其提取方法和应用。
背景技术
牡蛎是世界上第一大养殖贝类，也是我国重要海洋贝类，属牡蛎科，双壳类软体动物，分布于温带和热带各大洋沿岸水域，主要种类有近江牡蛎、太平洋牡蛎、大连湾牡蛎和密鳞牡蛎。太平洋牡蛎具有左右两个贝壳，以韧带和闭壳肌等相连。左壳凹，大而厚，能用来附着他物，壳表面有较多放射肋，清楚可数；右壳较小而平，表面有多层同心环状的鳞片，没有显著的放射肋。
牡蛎是一种具有药用价值的海洋经济贝类动物，根据分析，其肉中除含有丰富的蛋白质外还存在大量糖原结构的多糖，多糖的含量占牡蛎干重的20％-40％左右。牡蛎粗多糖主要有中性和酸性多糖两种。与其他多糖分子一样，牡蛎糖原的结构分为一级结构和高级结构(包括二级、三级、四级结构)。
对于多糖的提取，对于相同原料，采取的提取方法不同，所得到的多糖结构也不同，相同原料的不同部位的多糖也具有不同的结构，因此处理步骤也不尽相同。因此，选择合适的方法至关重要，在尽可能少地破坏糖原原有结构的基础上，得到较高的提取率。
目前，牡蛎多糖的提取方法主要有水提取法、碱提取法及酶解提取方法。
水提取法具有设备及操作简单方便、适用面广等优点，但是操作时间长， 效率低，并且需要反复操作，因此能耗较高。水提取法过程大致为：新鲜牡蛎肉准确称取后，清洗后匀浆，然后将装有牡蛎浆的三角瓶放入沸水浴中煮沸1h，冷却过滤，浓缩醇沉。
碱提取方法能从组织中对多糖进行较完全的提取，这基于动物体内，糖多与蛋白结合形成糖蛋白，其共价键遇碱不稳定，从而达到释放多糖的目的；一般用NaOH、KOH等碱溶液作为提取剂，提取率较高，但多糖分子有可能被降解，对多糖的结构和活性有一定的影响。碱提取法过程大致为：将牡蛎肉匀浆，加入30％氢氧化钾，100℃加热1h，冷却至室温后加入95％
乙醇，过滤得沉淀，即牡蛎粗多糖
酶解提取方法采用生物酶提取活性多糖，因为酶的专一性和选择性较强，故此法具有条件温和，不破坏多糖活性，提取的多糖纯度较高等优点，但会提高生产成本，对提取条件要求也较高。酶提法酶一般为胃蛋白酶和胰蛋白酶，酶解后的牡蛎粗多糖采用离子交换层析进一步纯化，可以得到不同的组分。
粗多糖中还含有色素，蛋白质，低聚糖等杂质。常采用活性炭脱色或者H2O2脱色，这会造成中性多糖被吸附，氧化等问题。柱层析法是目前较普遍的去掉这些杂质的方法。
总之，以上这些提取和去除杂质的方法存在操作步骤多，产品收率低，操作周期长、引入杂质、活性降低等问题。
发明内容
本发明的一个目的在于克服现有技术的缺陷，提供一种新的太平洋牡蛎中性多糖的制备方法，包括以下步骤：
(1)将太平洋牡蛎干粉通过丙酮脱脂，干燥后得到脱脂牡蛎粉；
(2)将脱脂牡蛎粉溶于水中，用超声微波萃取仪处理，将处理后的溶液离心，上清液蒸发浓缩，得到牡蛎多糖粗提物；
(3)将牡蛎多糖粗提物加入乙醇-(NH4)2SO4双水相体系进行萃取分离，萃取后的下相为牡蛎中性多糖粗提液；
(4)将牡蛎中性多糖粗提液进行透析，得到牡蛎多糖精提液，浓缩干燥后即可得到牡蛎中性多糖。
作为对上述技术方案的进一步改进，其中步骤(2)中超声微波萃取仪处理时间为20-30分钟，处理温度为80-90℃。
作为对上述技术方案的进一步改进，其中步骤(3)中乙醇-(NH4)2SO4双水相体系中乙醇、(NH4)2SO4和PBS的重量比是0.5:0.32:1，且双水相体系和牡蛎多糖粗提液的比例为：10：1，粗提液浓度约为9mg/mL。
本发明的另一目的在于提供一种采用上述方法制备的太平洋牡蛎中性多糖。
本发明的再另一目的在于提供一种上述太平洋牡蛎中性多糖在制备免疫相关的药物中的应用。
本发明的再另一目的在于提供一种太平洋牡蛎中性多糖在制备抗肿瘤药物中的应用。优选地，其中所述肿瘤为肝癌。
本发明具有以下优点：
(1)本发明采用超声微波浸提、乙醇-(NH4)2SO4双水相体系萃取以及透析相结合的方式来提取牡蛎中性多糖，避免了传统层析技术的成本高、收率低(<5％牡蛎中性多糖/牡蛎粉)、耗时长等弊端，提取工艺简单、成本低，适于间歇性和规模化生产加工高纯度、高收率的牡蛎中性多糖成品。
(2)现有技术的方法提取的牡蛎多糖通常是中性多糖和酸性多糖的混合 物，而本发明的方法所提取的牡蛎多糖为纯的中性多糖。本发明人研究发现，牡蛎中性多糖相比酸性多糖能够更好地提高小鼠脾淋巴免疫细胞的活性，对人肝癌细胞Hepg-2具有更强的抑制增殖、诱导凋亡的作用。
(3)本发明所制备的太平洋牡蛎中性多糖可以长期在4℃下贮存，活性不会降低或丧失。
(4)本发明所制备的太平洋牡蛎中性多糖能够促进小鼠脾淋巴细胞的免疫活性，对人肝癌细胞Hepg-2具有抑制增殖、诱导凋亡的作用。
附图说明
图1是本发明的太平洋牡蛎中性多糖提取工艺流程图。
图2是传统的牡蛎多糖提取工艺流程图。
图3为采用乙醇-(NH4)2SO4双水相体系对牡蛎多糖粗提液进行富集的结果。
图4为采用本发明的乙醇-(NH4)2SO4双水相体系方法提取的太平洋牡蛎粗多糖的成品。
图5为对比实施例中采用传统方法提取的太平洋牡蛎粗多糖的成品。
图6为采用HPLC-蒸发光检测器测定的本发明方法所制备的牡蛎中性多糖的纯度图谱。
图7(a)(b)分别为牡蛎多糖粗提液和牡蛎中性多糖成品的紫外分光光度计的扫描图谱。
图8为HPLC-ELSD仪检测的本发明方法所制备的牡蛎中性多糖单糖测定图谱，与葡萄糖标准品图谱吻合。
图9为传统牡蛎中性多糖(OGN1)、本发明方法所制备的牡蛎中性多糖(OGN2)、传统酸性多糖(OGA1)、本发明方法双水相萃取后弃液中的牡蛎酸性多 糖(OGA2)、以及本发明方法中多糖粗提物(OG)对小鼠脾淋巴细胞的增殖率测定。
图10为本发明方法所制备的牡蛎中性多糖对小鼠脾淋巴细胞的IL-2释放量的测定。
图11为本发明方法所制备的牡蛎中性多糖对人肝癌细胞Hepg-2的抑制率。
图12为Annexin V-FITC双染法对人肝癌细胞Hepg-2细胞凋亡的检测，其中(a),(b),(c)分别对应的空白组、阳性组(五氟尿嘧啶50μg/mL)、本发明方法所制备的牡蛎中性多糖(200μg/mL)。
具体实施方式
下面将结合具体实施例对本发明进行进一步说明。本发明的实施例意在阐述本发明技术方案，并不对本发明的技术方案构成限制。
实施例1：采用本发明方法制备的太平洋牡蛎中性多糖成品
太平洋牡蛎中性多糖成品按图1所示的工艺进行制备，具体步骤如下：
(1)制备脱脂牡蛎粉：牡蛎干粉经粉碎后,过40目筛,在烘箱中60℃干燥至恒重。称取6g的牡蛎粉末加入60ml的去离子水中，加入3倍体积的丙酮洗涤，4℃下6000rpm离心20min,低温烘干，得到脱脂牡蛎粉。
(2)制备牡蛎多糖粗提液：将步骤(1)所得到的脱脂牡蛎粉放入1000mL的玻璃萃取容器中，加入600mL去离子水并置于500W超声微波萃取仪中处理，温度86℃，时间26min，提取完后冷却。将提取液离心(4000rpm，10min)，合并上清液，旋蒸浓缩，得到牡蛎多糖粗提液，冷冻干燥得到黄色粗多糖粉末，并进行蛋白质和多糖含量的测定。
(3)制备牡蛎中性多糖精提液：乙醇-(NH4)2SO4双水相体系溶液相组成为： 17.7wt％(NH4)2SO4，27.29wt％乙醇，55.01wt％PBS。在10mL离心管中依次加入3.54g 40wt％(NH4)2SO4，0.8g粗多糖溶液(浓度9mg/mL)，1.4768g 0.01M PBS(pH＝7.2)，2.1832g乙醇，混匀30min,静置10min。如图3所示，采用乙醇-(NH4)2SO4双水相体系对牡蛎多糖粗提液进行萃取后，色素杂质等富集在乙醇上相，大分子量的酸性多糖和蛋白质富集在中间层，(NH4)2SO4下相主要富集了牡蛎中性多糖。
弃去上相，取下相制得牡蛎中性多糖粗提液。
(4)制备牡蛎中性多糖成品：取步骤(3)所制备的牡蛎中性多糖精提液置于2000KD的透析袋中用流水透析24h，然后于去离子水透析24h,透析液旋转蒸发浓缩，冷冻干燥制得白色粉末状牡蛎中性多糖成品(OGN2)(如图3所示)。
对比实施例1：采用传统方法制备太平洋牡蛎多糖成品
采用传统工艺提取太平洋牡蛎中的多糖，具体工艺如图2所示。所得到的产物为中性多糖和酸性多糖的混合物，为黄色的絮状物(如图4所示)。
在实施例1和对比实施例1中通过苯酚-硫酸法和BCA法测定不同提取步骤中多糖溶液中的多糖和蛋白质的含量。
苯酚-硫酸法：精密称取105℃下干燥至恒重的葡萄糖20mg，置500mL量瓶中，加水至刻度，摇匀，配得40μg/mL的葡萄糖标准溶液。精密吸取葡萄糖标准溶液0.4、0.6、0.8、1.0、1.2、1.4、1.6、1.8mL,分别置于10mL具塞刻度试管中,各以水补至2.0mL,然后分别加入5％苯酚溶液1mL，摇匀,迅速加入浓硫酸5mL，振摇室温放置5min,放入沸水中15min,取出置冷水中冷却30min,最后加水至刻度，摇匀,以2mL的蒸馏水按同样显色操作作为空白,用紫外分光光度计在490nm处测定吸光度。以A值为纵坐标对葡萄糖浓度绘制标准曲线，以标准曲线空白为对照，稀释一定倍数的多糖溶液，根据样品的 吸光值从标准曲线上查出样品的多糖含量。计算公式如下：
多糖含量(％)＝(C牡蛎多糖〃D)/W牡蛎多糖成品×100％    (1)。
式中C牡蛎多糖为供试液多糖浓度，D为供试液的稀释倍数；W牡蛎多糖成品为牡蛎多糖成品的重量。
BCA法：取50份试剂A与1份试剂B混合均匀，准确吸取25μL样品溶液于酶标孔中，加入BCA试剂200μL，轻摇，于37℃保温30min，冷却至室温后，以空白为对照，在酶标仪上562nm处比色，以牛血清白蛋白含量为横坐标，以吸光值为纵坐标，绘制标准曲线。以标准曲线空白为对照，根据样品的吸光值从标准曲线上查出样品的蛋白质含量。计算公式如下：
蛋白质含量(％)＝(C蛋白质〃D)/W牡蛎多糖成品×100％     (2)。
式中C蛋白质为供试液蛋白质浓度，D为供试液的稀释倍数；W牡蛎多糖成品为牡蛎多糖成品的重量。
同时对不同提取步骤中牡蛎中性多糖的收率进行统计分析，并观察不同提取步骤中牡蛎中性多糖的外观。
通过检测得知，采用本发明方法提取的牡蛎中性多糖的收率相比较传统方法从2.86％提高到11.43％，有显著的提高，计算公式如下：
中性糖收率(％)＝W牡蛎中性糖成品/W牡蛎干粉×100％    (3)。
图7(a),(b)分别为牡蛎多糖粗提液和中性多糖的紫外分光光度计的扫描图谱，中性多糖在280nm处已无明显吸收峰，说明达到很好的去除蛋白的效果。
实施例2：HPLC检测采用本发明方法制备的太平洋牡蛎中性多糖的分子量及纯度
条件：分离柱：TSKgel G4000SW(300mm×7.5mm)；流动相：醋酸铵溶液 (0.05mol/L)；样品浓度：后面详细数据；进样量：20μL；流速：0.5mL/min,漂移管温度：55℃，载气流速：2L/min，撞击器：开。结果如图5所示，为峰宽较窄对称的峰型。
实施例3：检测采用本发明方法制备的太平洋牡蛎中性多糖中单糖组成测定
(1)称取采用本发明方法制备的太平洋牡蛎中性多糖10mg，置于10mL具塞刻度试管中，加1mol/L的H2SO42mL，密封。于100℃恒温水解4h，水解液冷却后用饱和Ba(OH)2中和至中性，3000rpm离心10min除去BaSO4沉淀。将上清液全部转移至10mL量瓶中，用蒸馏水稀释至刻度，溶液经0.45μm微孔滤膜过滤后得到牡蛎中性多糖水解液。
(2)HPLC-ELSD仪检测：葡萄糖标准品(500μg/mL)、牡蛎中性多糖水解液，色谱柱Carbohydrate ES 5u(250mm×4.6mm)；流动相：乙腈-水(75％-25％)，进样量：20mL,流速：1mL/min,漂移管温度：83℃，载气流速2.1L/min。
图8为HPLC-ELSD仪检测的采用本发明方法制备的太平洋牡蛎中性多糖中单糖测定图谱，与葡萄糖标准品图谱吻合，说明单糖组成为葡萄糖。
实施例4：采用本发明方法制备的太平洋牡蛎中性多糖对小鼠脾淋巴细胞的增殖测定
1.小鼠脾淋巴细胞悬液的制备
(1)将小鼠用75％乙醇浸泡消毒3min后移入超净台中，取出小鼠置于无菌的塑料台上，左侧腹部朝上，腹中部剪开小口，暴露脾脏，用镊子提起脾脏，以眼科剪分离下面的结缔组织，取出脾脏，置于70μm的细胞过滤网上，下面放置50mL离心管上，用适量(约15mL)无菌PBS液(pH7.2-7.4)缓慢冲洗， 一边用研磨棒将脾脏磨碎，使单个细胞被PBS冲洗到离心管中，之后将研磨液1000rpm离心10min，弃去上层清液。
(2)沉淀中加入红细胞裂解液6mL,吹打混匀,静止5min,离心10min(1000rpm)，弃去上清液，除去血浆成分、细胞碎片及部分红细胞。加入适量RPMI1640完全培养基(RPMI-1640培养基：胎牛血清：青霉素/链霉素的89:10:1比例配制)悬浮脾细胞，用移液枪吹打均匀，置于5％CO2、37℃的培养箱中培养2h后，将RPMI1640完全培养基悬浮脾细胞更换培养瓶，以去除贴壁生长的杂细胞，之后再置于5％CO2、37℃的培养箱中培养24h。用细胞计数板计数细胞的数目，再加入适量的培养基调整细胞浓度为1×106个/mL。
2.采用MTT法测定小鼠脾淋巴细胞的增殖
(1)将制备好的细胞悬液接种到96孔板上，每孔100μL，再加入含牡蛎多糖，刀豆蛋白A(ConA)、RPMI-1640培养液100μL(Con A的终浓度均为5.0μg/m L)，使采用本发明方法制备的太平洋牡蛎中性多糖的终浓度为10、50、250μg/mL)，每组均设对照组(只加RPMI-1640培养液)，每个样本设4个复孔，置于5％CO2、37℃的培养箱中培养48h。
(2)培养结束后加入MTT溶液20μL/孔，继续培养4h，加入三联液(10g十二烷基硫酸钠，5mL异丁醇，0.1mL 12mol/L HCI溶于蒸馏水，定容至100mL)80μL/孔，于37℃放置过夜后，用酶标仪测波长570nm的吸光度A570nm值为实验结果，以增殖率为指标，判断淋巴细胞转化程度：
Figure PCTCN2017113256-appb-000001
图9为牡蛎多糖对小鼠脾淋巴细胞的增殖率测定，OGN1、OGN2、OGA1、OGA2、OG分别为传统牡蛎中性多糖、本发明方法所制备的牡蛎中性多糖、传统酸性多糖、双水相萃取后弃液中的牡蛎酸性多糖成品、以及本发明方法中多糖 粗提物(OG)，能够看出本发明方法所制备的牡蛎中性多糖250μg/mL时对小鼠脾淋巴细胞的增殖率为121.08％，能够有效的提高小鼠脾淋巴细胞增殖率。
实施例5：采用本发明方法制备的太平洋牡蛎中性多糖对小鼠脾淋巴细胞(MSL)释放IL-2的影响
(1)制备样品：以1×106个/mL的密度接种MSL至96孔板，每孔100μL，再加入含牡蛎多糖，Con A的RPMI-1640培养液100μL(使Con A的终浓度均为5.0μg/mL，采用本发明方法制备的太平洋牡蛎中性多糖的终浓度为250μg/mL)，每组均设对照组(只加RPMI-1640培养液)，每个样本设4个复孔，置于5％CO2、37℃的培养箱中培养24h，吸取培养液，1500rpm离心5min，收集上清液。
(2)绘制标准曲线：在酶标包被板上设标准孔10孔(5个浓度，均设2个复孔)，通过稀释使标准品浓度分别为2400、1600、800、400、200pg/mL。
(3)加样：分别设本底孔(不加样品和酶标试剂)、牡蛎多糖孔、阳性对照孔、空白孔，均分别设3个复孔。在酶标包被板上先加样品稀释液40μL，然后再加待测样品10μL，轻轻晃动摇匀。
(4)温育：用封板膜封板后置37℃温育30min。
(5)洗涤：稀释浓缩洗涤液，备用。揭掉封板膜，弃去液体，甩干，每孔加满洗涤液，静置30s后弃去。重复5次，拍干。
(6)加酶：每孔加50μL酶标试剂，本底孔除外。
(7)温育，洗涤。
(8)显色：每孔加显色剂50μL，再加显色剂B50μL，震荡混匀，37℃避光显色15min。
(9)终止：每孔加终止液50μL。
(10)测定：在加入终止液15min以内测定。以本底孔调零，450nm下测定吸光度。
根据标准曲线得到各实验组对应的IL-2浓度后，采用以下公式计算刺激指数：
刺激指数SI(％)＝(w实验-w空白)/w空白×100％    (5)。
式中w实验、w空白分别为实验组和空白组小鼠脾淋巴细胞IL-2的浓度。
图10为采用本发明方法制备的太平洋牡蛎中性多糖对小鼠脾淋巴细胞的IL-2释放量的测定，能够看出采用本发明方法制备的太平洋牡蛎中性多糖250μg/mL对小鼠脾淋巴细胞IL-2的刺激指数达到124.81％，能够有效的刺激小鼠脾淋巴细胞免疫活性。
实施例6：采用本发明方法制备的太平洋牡蛎中性多糖体外对Hepg-2细胞的抑制率测定
(1)取对数生长期的肿瘤细胞,胰蛋白酶消化后用DMEM培养液(DMEM培养基：胎牛血清：青霉素/链霉素的89:10:1比例配制)调整细胞至4×104/mL。按每孔100μL(4000个细胞)加入96孔培养板中。
(2)4h后加入不同稀释浓度的采用本发明方法制备的太平洋牡蛎中性多糖各100μL,对照组加等体积的DMEM培养液,每个浓度均设4个复孔,置37℃体积分数为5％CO2孵箱中连续培养48h。培养结束前4h各孔加入MTT(5g/L)20μL,放置上述孵箱中继续孵育4h,弃上清,每孔加入二甲基亚砜150μL,充分振荡混匀后于酶标仪570nm处测吸光光度值(A),计算抑制率，计算公式如下：
抑制率(％)＝(A对照组-A药物组)/A对照组×100％    (6)。
图11为采用本发明方法制备的太平洋牡蛎中性多糖对人肝癌细胞Hepg-2的抑制率，能够看出采用本发明方法制备的太平洋牡蛎中性多糖200μg/mL时对人肝癌细胞Hepg-2的抑制率为55.81％，能够有效的抑制肝癌细胞生长的活性。
实施例7：Annexin V-FITC细胞凋亡检测
(1)取对数生长期的肿瘤细胞,胰蛋白酶消化后用DMEM完全培养液调整细胞至4×104/mL。按每孔100μL(4000个细胞)加入96孔培养板中。
(2)4h后加入不同稀释浓度的采用本发明方法制备的太平洋牡蛎中性多糖各100μL,对照组加等体积的DMEM培养液,每个浓度均设4个复孔,置37℃体积分数为5％CO2孵箱中连续培养48h。
(3)在凋亡诱导结束后吸除细胞培养液，加入PBS洗涤一次，加入195μLAnnexin V-FITC结合液，加入5μL Annexin V-FITC，轻轻混匀。
(4)室温(20-25℃)避光孵育10min，使用铝箔进行避光。吸除溶液，加入190μL Annexin V-FITC结合液。
(5)加入10μL碘化丙啶染色液，轻轻混匀，冰浴避光放置。
(6)随即在荧光显微镜下观察，Annexin V-FITC为绿色荧光，PI为红色荧光。
图12为Annexin V-FITC双染法对人肝癌细胞Hepg-2细胞凋亡的检测，其中(a)、(b)、(c)、(d)分别对应的空白组，阳性组(五氟尿嘧啶50μg/mL)，牡蛎中性多糖(200μg/mL)，传统提取方法获得的中性多糖(200μg/mL)，所显示的暗色为坏死和凋亡晚期的肿瘤细胞，亮色为凋亡早期的肿瘤细胞。
虽然，上文中已经用一般性说明、具体实施方式及试验，对本发明作了详 尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。

Claims (7)

  1. 一种太平洋牡蛎中性多糖的制备方法，包括以下步骤：
    (1)将太平洋牡蛎干粉通过丙酮脱脂，干燥后得到脱脂牡蛎粉；
    (2)将脱脂牡蛎粉溶于水中，用超声微波萃取仪处理，将处理后的溶液离心，上清液蒸发浓缩，得到牡蛎多糖粗提物；
    (3)将牡蛎多糖粗提物加入乙醇-(NH4)2SO4双水相体系进行萃取分离，萃取后的下相为牡蛎中性多糖粗提液；
    (4)将牡蛎中性多糖粗提液进行透析，得到牡蛎多糖精提液，浓缩干燥后即可得到牡蛎中性多糖。
  2. 如权利要求1所述的制备方法，其中步骤(2)中超声微波萃取仪处理时间为20-30分钟，处理温度为80-90℃。
  3. 如权利要求1所述的制备方法，其中步骤(3)中乙醇-(NH4)2SO4双水相体系中乙醇、(NH4)2SO4和PBS的重量比是0.5:0.32:1，且双水相体系和牡蛎多糖粗提液的比例为：10：1，粗提液浓度约为9mg/mL。
  4. 一种采用权利要求1-3任一项所述的方法所制备的太平洋牡蛎中性多糖。
  5. 如权利要求4所述的太平洋牡蛎中性多糖在制备免疫相关的药物中的应用。
  6. 如权利要求4所述的太平洋牡蛎中性多糖在制备抗肿瘤药物中的应用。
  7. 如权利要求6的应用，其中所述肿瘤为肝癌。
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