技术领域Technical Field
本发明涉及医药技术领域，具体涉及一种酸性罂粟多糖及其制备方法和应用。The invention relates to the technical field of medicines, and in particular to an acidic poppy polysaccharide and a preparation method and application thereof.
背景技术Background technique
植物多糖是由10个以上同类或不同种类的单糖以糖苷键的方式聚合而成的高分子化合物。其结构复杂多样，分子量从几千至几万不等。研究表明多糖具有抗疲劳、抗缺氧、抗氧化、和调节免疫力等多种生物活性。目前已经上市的多糖产品有香菇多糖、人参多糖和黄芪多糖等用于肿瘤放化疗辅助药物，枸杞多糖功能性食品添加剂和多糖面膜等都取得良好的经济效益，表明多糖类产品的研究与开发具有广泛的应用前景。此外，市场目前用于抗缺氧、抗疲劳的产品主要为红景天制品，近年随着市场需求量的不断扩大和农副产品价格上涨，导致红景天资源已经濒临枯竭，因此迫切需要寻找低毒、高效、资源丰富且价格低廉的抗疲劳、抗缺氧的代用品种，以满足市场需求。Plant polysaccharides are high molecular weight compounds formed by the polymerization of more than 10 monosaccharides of the same or different types in the form of glycosidic bonds. Its structure is complex and diverse, and its molecular weight ranges from several thousand to tens of thousands. Studies have shown that polysaccharides have multiple biological activities such as anti-fatigue, anti-hypoxia, anti-oxidation, and immune regulation. Currently, the polysaccharide products on the market include Lentinan, ginseng polysaccharide and astragalus polysaccharide, which are used as adjuvant drugs for tumor radiotherapy and chemotherapy. Lycium barbarum polysaccharide functional food additives and polysaccharide masks have achieved good economic benefits, indicating that the research and development of polysaccharide products has broad application prospects. In addition, the products currently used in the market for anti-hypoxia and anti-fatigue are mainly Rhodiola rosea products. In recent years, with the continuous expansion of market demand and the rise in prices of agricultural and sideline products, Rhodiola rosea resources are on the verge of exhaustion. Therefore, it is urgent to find low-toxic, high-efficiency, resource-rich and low-priced alternative varieties for anti-fatigue and anti-hypoxia to meet market demand.
罂粟壳为罂粟科植物罂粟(Papaver somniferum L.)的干燥成熟果壳，具有敛肺涩肠、固肾止痛之功效，主治久咳劳嗽、喘息和心腹及筋骨疼痛等。罂粟壳气微、味酸、微寒，为镇痛止咳之良药，因其主要的成分吗啡、那可丁和可待因等生物碱具有成瘾性，被国家列入麻醉精神类药品目录严格管制。罂粟壳中除含有生物碱类成分外，还含有大量的多糖类成分。目前生产上对于罂粟壳的利用主要集中在生物碱类成分，提取后的原料被当成废渣、废料处理，造成极大的资源浪费和环境污染。研究发现罂粟壳废渣中粗多糖的提取率约为10.26％，多糖在抗疲劳制药、食品加工和日化产品等方面的应用，对罂粟壳药渣多糖成分进行深入研究和开发，对充分发挥好、利用好这一植物资源具有重要意义。鉴于此，本发明提供一种酸性罂粟多糖及其制备方法和应用。Poppy shell is the dried mature fruit shell of the poppy (Papaver somniferum L.) of the Papaveraceae family. It has the effects of astringing the lungs and intestines, strengthening the kidneys and relieving pain. It is mainly used to treat chronic cough, fatigue cough, wheezing, and pain in the heart, abdomen, muscles and bones. Poppy shell has a slight smell, sour taste, and is slightly cold. It is a good medicine for analgesia and cough. Because its main ingredients, alkaloids such as morphine, narcotine and codeine, are addictive, it has been included in the list of narcotic and psychotropic drugs by the state and is strictly controlled. In addition to alkaloid components, poppy shell also contains a large amount of polysaccharide components. At present, the utilization of poppy shell in production is mainly concentrated on alkaloid components, and the extracted raw materials are treated as waste residues and waste materials, causing great waste of resources and environmental pollution. Studies have found that the extraction rate of crude polysaccharides in poppy shell waste residues is about 10.26%. The application of polysaccharides in anti-fatigue pharmaceuticals, food processing and daily chemical products, in-depth research and development of the polysaccharide components of poppy shell residues are of great significance to give full play to and make good use of this plant resource. In view of this, the present invention provides an acidic poppy polysaccharide and a preparation method and application thereof.
发明内容Summary of the invention
本发明所要解决的技术问题是提供一种酸性罂粟多糖及其制备方法和应用。目的是提供酸性罂粟多糖及制备方法，酸性罂粟多糖作为抗疲劳、抗缺氧药物中间体、功能性保健食品的原料，或日化产品的活性原料。The technical problem to be solved by the present invention is to provide an acidic poppy polysaccharide and a preparation method and application thereof. The purpose is to provide an acidic poppy polysaccharide and a preparation method thereof, and the acidic poppy polysaccharide is used as an intermediate of anti-fatigue and anti-hypoxia drugs, a raw material of functional health food, or an active raw material of daily chemical products.
本发明解决上述技术问题的技术方案如下：The technical solution of the present invention to solve the above technical problems is as follows:
第一方面，提供一种酸性罂粟多糖，所述酸性罂粟多糖包括甘露糖、核糖、鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖、木糖和阿拉伯糖，所述甘露糖、所述核糖、所述鼠李糖、所述葡萄糖醛酸、所述半乳糖醛酸、所述葡萄糖、所述半乳糖、所述木糖与所述阿拉伯糖的质量比为(1.14～1.24)：(0.66～0.76)：(3.79～3.89)：(1.51～1.61)：(23.26～23.36)：(53.30～53.40)：(5.15～5.25)：(4.77～4.87)：(5.96～6.06)。In a first aspect, an acidic poppy polysaccharide is provided, which includes mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose, and the mass ratio of the mannose, the ribose, the rhamnose, the glucuronic acid, the galacturonic acid, the glucose, the galactose, the xylose and the arabinose is (1.14-1.24): (0.66-0.76): (3.79-3.89): (1.51-1.61): (23.26-23.36): (53.30-53.40): (5.15-5.25): (4.77-4.87): (5.96-6.06).
本发明提供的酸性罂粟多糖，采用示差折光检测器与凝胶渗透色谱联用法测分子量，测定的峰1的数均分子量Mn为488824，重均分子量Mw为539174，分散性指数PDI为1.1030，峰2的数均分子量Mn为48698，重均分子量Mw为52319，分散性指数PDI为1.1744。所述酸性罂粟多糖的红外图谱在3445cm-1左右有吸收峰对应的O–H伸缩振动，2923cm-1和2854cm-1处有吸收峰分别对应C–H的对称和不对称伸缩振动，1740cm-1吸收峰对应糖醛酸C＝O伸缩振动，1427cm-1处对应–CH(–CH2OH)的弯曲振动。The acidic poppy polysaccharide provided by the present invention adopts a differential refractive index detector and a gel permeation chromatography method to measure the molecular weight, and the measured number average molecular weight Mn of peak 1 is 488824, the weight average molecular weight Mw is 539174, and the dispersion index PDI is 1.1030, and the number average molecular weight Mn of peak 2 is 48698, the weight average molecular weight Mw is 52319, and the dispersion index PDI is 1.1744. The infrared spectrum of the acidic poppy polysaccharide has an absorption peak corresponding to O-H stretching vibration at about 3445cm-1 , absorption peaks at 2923cm-1 and 2854cm-1 respectively corresponding to symmetric and asymmetric stretching vibrations of C-H, an absorption peak at 1740cm-1 corresponding to uronic acid C=O stretching vibration, and a peak at 1427cm-1 corresponding to the bending vibration of -CH (-CH2OH ).
本发明得到的酸性罂粟多糖抗疲劳抗缺氧活性显著，优于阳性对照药大株红景天胶囊；所述的从罂粟壳中通过提取、分离纯化得到酸性罂粟多糖，包括但不限于抗氧化、抗疲劳、抗缺氧药物、功能性食品和日化产品，为研制新的药物提供了先导化合物，也为资源的二次开发利用提供了理论依据。The acidic poppy polysaccharide obtained by the invention has significant anti-fatigue and anti-hypoxia activities, which are better than the positive control drug Dazhu Hongjingtian capsule; the acidic poppy polysaccharide obtained from the poppy shell by extraction, separation and purification includes but is not limited to antioxidant, anti-fatigue, anti-hypoxia drugs, functional foods and daily chemical products, provides a lead compound for the development of new drugs, and also provides a theoretical basis for the secondary development and utilization of resources.
进一步，所述甘露糖、所述核糖、所述鼠李糖、所述葡萄糖醛酸、所述半乳糖醛酸、所述葡萄糖、所述半乳糖、所述木糖与所述阿拉伯糖的质量比为1.19：0.71：3.84：1.56：23.31：53.35：5.20：4.82：6.01。Furthermore, the mass ratio of the mannose, the ribose, the rhamnose, the glucuronic acid, the galacturonic acid, the glucose, the galactose, the xylose and the arabinose is 1.19:0.71:3.84:1.56:23.31:53.35:5.20:4.82:6.01.
其中，所述酸性罂粟多糖含量测定方法：采用苯酚-硫酸法测定酸性罂粟多糖的含量，具体的包括如下步骤：Wherein, the method for determining the content of acidic poppy polysaccharide: the content of acidic poppy polysaccharide is determined by phenol-sulfuric acid method, which specifically includes the following steps:
(1)绘制葡萄糖标准曲线：精确称量葡萄糖标准品10.0mg，加蒸馏水溶解，定容至100.0mL，标准品终浓度为0.1mg/mL；分别精确吸取0、0.1、0.2、0.4、0.6和0.8mL葡萄糖母液于试管中；分别补加蒸馏水至1.0mL；各试管中分别加入5％的苯酚溶液1.0mL和浓硫酸2.5mL，立即涡旋混匀，室温反应半小时，以0mL管为对照，测定490nm处的吸光度。以标准葡萄糖溶液浓度(x，μg/mL)为横轴，吸光值(y)为纵轴，拟合标准回归方程。(1) Draw a glucose standard curve: accurately weigh 10.0 mg of glucose standard, dissolve it in distilled water, and dilute to 100.0 mL. The final concentration of the standard is 0.1 mg/mL; accurately pipette 0, 0.1, 0.2, 0.4, 0.6 and 0.8 mL of glucose mother solution into test tubes respectively; add distilled water to 1.0 mL respectively; add 1.0 mL of 5% phenol solution and 2.5 mL of concentrated sulfuric acid to each test tube, immediately vortex mix, react at room temperature for half an hour, and use the 0 mL tube as a control to measure the absorbance at 490 nm. Fit the standard regression equation with the concentration of the standard glucose solution (x, μg/mL) as the horizontal axis and the absorbance value (y) as the vertical axis.
(2)总糖含量测定：蒸馏水配制浓度为100μg/mL的罂粟多糖溶液，吸取1.0mL罂粟多糖溶液，按上述测定步骤测定其490nm吸光值，根据葡萄糖标准曲线计算罂粟多糖样品中多糖含量。(2) Determination of total sugar content: prepare a poppy polysaccharide solution with a concentration of 100 μg/mL with distilled water, take 1.0 mL of the poppy polysaccharide solution, measure its absorbance at 490 nm according to the above determination steps, and calculate the polysaccharide content in the poppy polysaccharide sample based on the glucose standard curve.
第二方面，提供一种酸性罂粟多糖的制备方法，包括如下步骤：In a second aspect, a method for preparing acidic poppy polysaccharide is provided, comprising the following steps:
(1)罂粟总多糖提取：采用提取过生物碱的罂粟壳废渣为原料，采用蒸馏水进行加热回流提取，进行过滤得到提取液，将提取液减压浓缩得到浓缩液，再在搅拌的条件下加入醇溶剂，至所述醇溶剂在所述浓缩液中的终浓度为70％～90％，优选为80％，抽滤(静置12h后，去除滤液)，使用无水乙醇洗涤，随后烘箱后进行烘干，得到罂粟总多糖；(1) Extraction of total opium poppy polysaccharide: using waste residue of poppy shell from which alkaloids have been extracted as raw material, heating and refluxing extraction with distilled water, filtering to obtain an extract, concentrating the extract under reduced pressure to obtain a concentrated solution, adding an alcohol solvent under stirring until the final concentration of the alcohol solvent in the concentrated solution is 70% to 90%, preferably 80%, filtering with suction (standing for 12 hours, removing the filtrate), washing with anhydrous ethanol, and then drying in an oven to obtain total opium poppy polysaccharide;
优选的，采用蒸馏水进行加热回流提取前，可以采用蒸馏水浸泡30min左右；提取液进行减压浓缩至原体积的1/6～1/10；Preferably, before using distilled water for heating and reflux extraction, distilled water can be used for soaking for about 30 minutes; the extract is concentrated under reduced pressure to 1/6 to 1/10 of the original volume;
(2)酸性罂粟多糖分离：将所述罂粟总多糖溶解至蒸馏水中，所述罂粟总多糖与所述蒸馏水的质量比为1：(10～50)，得到罂粟总多糖溶液，依次进行脱除蛋白和色素，后通过离子交换树脂柱进行层析，先使用5～8倍柱体积蒸馏水进行洗脱，收集合并得到洗脱液Ⅰ，继续使用6～10倍柱体积的氯化钠溶液洗脱，收集合并得到洗脱液Ⅱ，将所述洗脱液Ⅱ减压浓缩，将浓缩后的洗脱液Ⅱ进行透析，得到透析产物；其中洗脱液Ⅰ先减压浓缩至原体积的1/6～1/10，再真空干燥/冷冻干燥获得中性罂粟多糖；(2) separation of acidic poppy polysaccharides: dissolving the total poppy polysaccharides in distilled water, wherein the mass ratio of the total poppy polysaccharides to the distilled water is 1:(10-50), to obtain a total poppy polysaccharide solution, removing proteins and pigments in sequence, and then chromatographing through an ion exchange resin column, first eluting with 5-8 column volumes of distilled water, collecting and merging to obtain eluent I, and continuing to elute with 6-10 column volumes of sodium chloride solution, collecting and merging to obtain eluent II, concentrating the eluent II under reduced pressure, and dialyzing the concentrated eluent II to obtain a dialyzed product; wherein the eluent I is first concentrated under reduced pressure to 1/6-1/10 of its original volume, and then vacuum dried/freeze dried to obtain neutral poppy polysaccharides;
(3)酸性罂粟多糖纯化：用葡聚糖凝胶柱层析法对所述透析产物进行纯化，收集组分，进行冷冻干燥，得到酸性罂粟多糖。(3) Purification of acidic poppy polysaccharide: The dialyzed product is purified by dextran gel column chromatography, and the components are collected and freeze-dried to obtain acidic poppy polysaccharide.
进一步，步骤(1)在加热回流提取中，所述提取过生物碱的罂粟壳废渣与所述蒸馏水的质量比为1：(15～45)，优选为1：30，加热回流提取的温度为90～100℃，加热回流提取的时间为1～2h，加热回流提取的次数至少2～3次。Furthermore, in step (1), in the heating reflux extraction, the mass ratio of the poppy shell waste residue from which the alkaloids have been extracted to the distilled water is 1:(15-45), preferably 1:30, the heating reflux extraction temperature is 90-100°C, the heating reflux extraction time is 1-2h, and the heating reflux extraction is performed at least 2-3 times.
进一步，所述醇溶剂为无水乙醇、甲醇、丙醇中的至少一种。Furthermore, the alcohol solvent is at least one of anhydrous ethanol, methanol and propanol.
进一步，步骤(2)脱除蛋白中，采用二氯甲烷和正丁醇混合溶液进行脱除蛋白，所述二氯甲烷和正丁醇混合溶液中二氯甲烷与正丁醇的体积比为4:1，所述二氯甲烷和正丁醇混合溶液与所述罂粟总多糖溶液质量比为1：(1～5)，优选为1:3；Furthermore, in step (2), the protein removal is performed by using a mixed solution of dichloromethane and n-butanol, wherein the volume ratio of dichloromethane to n-butanol in the mixed solution of dichloromethane and n-butanol is 4:1, and the mass ratio of the mixed solution of dichloromethane and n-butanol to the total poppy polysaccharide solution is 1:(1-5), preferably 1:3;
步骤(2)脱除色素中，采用大孔树脂D-101进行吸附脱除色素，所述大孔树脂D-101与所述罂粟总多糖溶液质量比为(2～6)1，优选为4：1；In step (2), in removing the pigment, macroporous resin D-101 is used for adsorption removal of the pigment, and the mass ratio of the macroporous resin D-101 to the total poppy polysaccharide solution is (2-6) 1, preferably 4:1;
步骤(2)中所述离子交换树脂柱为DEAE-阴离子交换柱，所述氯化钠溶液的质量浓度为0.15～0.25mol/L，优选为0.2mol/L；In step (2), the ion exchange resin column is a DEAE-anion exchange column, and the mass concentration of the sodium chloride solution is 0.15-0.25 mol/L, preferably 0.2 mol/L;
步骤(2)中所述透析采用截留分子量3500的透析袋进行，透析的时间为36～60小时，优选的为48小时，每3～54小时换一次蒸馏水，优选的为4小时。The dialysis in step (2) is performed using a dialysis bag with a molecular weight cutoff of 3500. The dialysis time is 36 to 60 hours, preferably 48 hours. The distilled water is replaced every 3 to 54 hours, preferably 4 hours.
进一步，步骤(3)中葡聚糖凝胶柱为Sephadex G-100葡萄糖凝胶层析柱，洗脱液采用超纯水，所述超纯水用量为5～8倍柱体积。Furthermore, in step (3), the dextran gel column is a Sephadex G-100 glucose gel chromatography column, and the eluent is ultrapure water, wherein the amount of ultrapure water is 5 to 8 times the column volume.
第三方面，提供一种酸性罂粟多糖的应用，将所述的酸性罂粟多糖用于抗氧化，和/或抗疲劳，和/或抗缺氧的产品中。In a third aspect, a use of an acidic poppy polysaccharide is provided, wherein the acidic poppy polysaccharide is used in a product for anti-oxidation, and/or anti-fatigue, and/or anti-hypoxia.
所述酸性罂粟多糖对钠石灰诱导缺氧模型小鼠存活时间有明显的改善，对小鼠力竭游泳时间有明显的延长，且优于阳性药物大株红景天胶囊。The acidic poppy polysaccharide significantly improves the survival time of mice in the hypoxia model induced by soda lime, significantly prolongs the exhaustive swimming time of mice, and is superior to the positive drug Dazhu Rhodiola rosea capsule.
本发明所述的从罂粟壳中通过提取、分离纯化得到酸性罂粟多糖，包括但不限于抗氧化、抗疲劳、抗缺氧药物、功能性食品和日化产品，为研制新的药物提供了先导化合物，也为资源的二次开发利用提供了理论依据。The acidic poppy polysaccharide obtained from poppy shell by extraction, separation and purification of the present invention includes but is not limited to antioxidant, anti-fatigue, anti-hypoxia drugs, functional foods and daily chemical products, provides a lead compound for the development of new drugs, and also provides a theoretical basis for the secondary development and utilization of resources.
第四方面，提供一种产品，含有所述的酸性罂粟多糖。In a fourth aspect, a product is provided, comprising the acidic poppy polysaccharide.
进一步，所述产品包括药物、功能性食品或日化产品。Furthermore, the product includes medicine, functional food or daily chemical product.
若产品为药物，还包括药学上可接受的缓冲液、赋形剂或载体。其中术语“药学上可接受的”在本文中被限定为指在合理医学判断的范围内适合用于与受试者的组织相接触而无过度毒性、刺激过敏反应和其他问题并发症，并且与合理的益处/风险比相称的那些化合物、材料、组合物和/或剂型。药物的剂型包括溶液、混悬液、乳剂、浸膏、酏剂、粉剂、颗粒剂、片剂、胶囊。If the product is a drug, it also includes a pharmaceutically acceptable buffer, excipient or carrier. The term "pharmaceutically acceptable" is defined herein as compounds, materials, compositions and/or dosage forms that are suitable for contact with the tissues of a subject without excessive toxicity, irritation, allergic reactions and other complications within the scope of reasonable medical judgment, and are commensurate with a reasonable benefit/risk ratio. The dosage form of the drug includes solutions, suspensions, emulsions, extracts, elixirs, powders, granules, tablets, and capsules.
本发明的有益效果是：本发明以生产提取过生物碱的罂粟壳废渣为原料，采用水提醇沉法获得罂粟总多糖，脱除蛋白、色素后，经过DEAE阴离子交换柱层析及Sephadex G-100葡萄糖凝胶层析柱等纯化方法获得酸性罂粟多糖；并通过小鼠缺氧实验和力竭游泳实验研究酸性罂粟多糖的抗疲劳抗缺氧作用；通过动物试验表明：酸性罂粟多糖能明显延长小鼠力竭游泳时间和常压耐缺氧时间，具有良好的抗疲劳抗缺氧活性，且活性优于阳性对照药大株红景天胶囊。因此，本发明所提供的酸性罂粟多糖具有开发为抗疲劳抗缺氧药物的潜力，同时具有开发为功能性保健食品或饮料、日化产品的优势。The invention has the following beneficial effects: the invention uses the waste residue of poppy shell from which alkaloids have been extracted as raw materials, adopts water extraction and alcohol precipitation method to obtain total poppy polysaccharide, removes protein and pigment, and then obtains acidic poppy polysaccharide through purification methods such as DEAE anion exchange column chromatography and Sephadex G-100 glucose gel chromatography column; and studies the anti-fatigue and anti-hypoxia effects of acidic poppy polysaccharide through mouse hypoxia experiment and exhaustive swimming experiment; animal experiments show that acidic poppy polysaccharide can significantly prolong the exhaustive swimming time and normal pressure hypoxia tolerance time of mice, has good anti-fatigue and anti-hypoxia activity, and has better activity than the positive control drug Dazhu Hongjingtian capsule. Therefore, the acidic poppy polysaccharide provided by the invention has the potential to be developed into anti-fatigue and anti-hypoxia drugs, and also has the advantage of being developed into functional health food or beverage, and daily chemical products.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为本发明酸性罂粟多糖的单糖组成UPLC分析图谱；Fig. 1 is a UPLC analysis spectrum of monosaccharide composition of acidic poppy polysaccharide of the present invention;
图2为本发明葡萄糖标准曲线图；Fig. 2 is a glucose standard curve diagram of the present invention;
图3为本发明酸性罂粟多糖的GPC分析图谱图；Fig. 3 is a GPC analysis spectrum diagram of acidic poppy polysaccharide of the present invention;
图4为本发明酸性罂粟多糖的红外光谱图；Fig. 4 is an infrared spectrum of acidic poppy polysaccharide of the present invention;
图5为本发明酸性罂粟多糖的核磁共振氢谱图；Fig. 5 is a hydrogen nuclear magnetic resonance spectrum of the acidic poppy polysaccharide of the present invention;
图6为本发明酸性罂粟多糖的扫描电子显微镜图；Fig. 6 is a scanning electron microscope image of the acidic poppy polysaccharide of the present invention;
图7为本发明不同浓度的酸性罂粟多糖对ABTS自由基清除率曲线图；FIG7 is a graph showing the scavenging rate of ABTS free radicals by acidic poppy polysaccharides of different concentrations according to the present invention;
图8为本发明不同浓度的酸性罂粟多糖对DPPH自由基清除率曲线图。FIG8 is a graph showing the scavenging rate of DPPH free radicals by acidic poppy polysaccharides of different concentrations according to the present invention.
具体实施方式Detailed ways
以下对本发明的原理和特征进行描述，所举实例只用于解释本发明，并非用于限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购得的常规产品。The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.
下述实施例中采用的仪器、材料和试剂的说明：Description of the instruments, materials and reagents used in the following examples:
仪器：旋转蒸发仪、电子分析天平、离心机、真空冷冻干燥机、多功能微孔板检测仪、涡旋振荡器、凝胶渗透色谱系统(GPC)、UPLC系统、傅里叶变换红外光谱仪、扫描电子显微镜、多功能微孔板检测仪。Instruments: rotary evaporator, electronic analytical balance, centrifuge, vacuum freeze dryer, multifunctional microplate detector, vortex oscillator, gel permeation chromatography system (GPC), UPLC system, Fourier transform infrared spectrometer, scanning electron microscope, multifunctional microplate detector.
试剂：阿拉伯糖、核糖、木糖、葡萄糖、甘露糖、半乳糖、鼠李糖、半乳糖醛酸、葡萄糖醛酸等单糖标准品为色谱纯；葡萄糖标准品、苯酚、硫酸、无水乙醇、三氟乙酸、1-苯基-3-甲基-5-吡唑酮、氢氧化钠、抗化血酸标准品、ABTS、DPPH以及所有其他化学物质至少为分析纯；高效液相色谱的溶剂为色谱纯度。Reagents: Monosaccharide standards such as arabinose, ribose, xylose, glucose, mannose, galactose, rhamnose, galacturonic acid, and glucuronic acid were of chromatographic grade; glucose standards, phenol, sulfuric acid, anhydrous ethanol, trifluoroacetic acid, 1-phenyl-3-methyl-5-pyrazolone, sodium hydroxide, anhydrous hematoxylin and eosin standards, ABTS, DPPH, and all other chemicals were of at least analytical grade; solvents for HPLC were of chromatographic grade.
材料：罂粟壳废渣、D101大孔树脂、DEAE-FF纤维素凝胶、Sephadex G-100葡聚糖凝胶、透析袋、康缘药业大株红景天胶囊、昆明种小鼠(兰州大学医学动物实验中心提供)。Materials: Poppy shell waste, D101 macroporous resin, DEAE-FF cellulose gel, Sephadex G-100 dextran gel, dialysis bag, Kangyuan Pharmaceutical Rhodiola rosea capsule, Kunming mice (provided by the Medical Animal Experiment Center of Lanzhou University).
实施例1：酸性罂粟总多糖的提纯制备方法Example 1: Purification and preparation method of acidic poppy total polysaccharide
本实施例一种酸性罂粟总多糖的提纯制备方法，包括如下步骤：The present embodiment provides a method for purifying and preparing acidic poppy total polysaccharides, comprising the following steps:
(1)罂粟壳总多糖提取(1) Extraction of total polysaccharides from poppy shell
采用提取过生物碱的罂粟壳废渣为原料，取7.7kg，加入46.0L蒸馏水浸泡30min，然后在100℃条件下加热回流提取，共提取3次，第1次提取时间为2h，第2次和第3次提取时间均为1.5h。合并提取液，过滤离心，上清液减压浓缩至原体积的1/6，随后边搅拌边加入约为浓缩液7倍量的无水乙醇至最终浓度为80％，静置24h。抽滤得沉淀，使用无水乙醇洗涤沉淀3次，烘箱60℃烘干，即得罂粟壳总多糖77.0g，罂粟壳总多糖得率约为10.2％。The method adopts the waste residue of poppy shell from which alkaloids have been extracted as raw material, takes 7.7 kg, adds 46.0 L distilled water and soaks for 30 minutes, then heats and refluxes at 100°C for extraction, extracts 3 times in total, the first extraction time is 2 hours, and the second and third extraction times are both 1.5 hours. The extracts are combined, filtered and centrifuged, the supernatant is concentrated under reduced pressure to 1/6 of the original volume, and then anhydrous ethanol of about 7 times the amount of the concentrated solution is added while stirring to a final concentration of 80%, and is left to stand for 24 hours. The precipitate is filtered and washed 3 times with anhydrous ethanol, and dried in an oven at 60°C to obtain 77.0 g of total polysaccharide of poppy shell, and the yield of total polysaccharide of poppy shell is about 10.2%.
其中，多糖得率％＝罂粟壳总多糖质量/罂粟壳废渣质量×100％；Wherein, polysaccharide yield % = total polysaccharide mass of poppy shell/mass of poppy shell waste residue × 100%;
(2)酸性罂粟多糖制备(2) Preparation of acidic poppy polysaccharide
取10.0g罂粟壳总多糖分散至300mL蒸馏水中，加入1/3溶液体积的savage试剂振荡30min，然后在室温条件下，以4200r/min转速离心15min，脱除蛋白3次，并用40g的D101大孔树脂吸附脱色2h后，滤除树脂，旋转蒸发浓缩。Take 10.0g of total polysaccharide from poppy shell and disperse it in 300mL of distilled water. Add 1/3 volume of savage reagent and shake for 30min. Then centrifuge at 4200r/min for 15min at room temperature to remove protein 3 times. After adsorption and decolorization with 40g of D101 macroporous resin for 2h, filter out the resin and concentrate by rotary evaporation.
浓缩后的多糖溶液15.0mL上样至DEAE纤维素凝胶层析柱，柱径高比为1:4，蒸馏水洗脱5倍柱体积(约1000mL)，收集合并洗脱溶液，减压浓缩至原体积1/10，浓缩洗脱液装于截留分子量3500的透析袋中，约1/2体积，用透析夹夹紧透析袋两端，放于蒸馏水中透析48h，每4h换一次水。将透析后的洗脱液真空干燥获得中性罂粟多糖0.95g。15.0 mL of the concentrated polysaccharide solution was loaded onto a DEAE cellulose gel chromatography column with a column diameter-to-height ratio of 1:4, and eluted with distilled water for 5 times the column volume (about 1000 mL). The eluted solution was collected and combined, and concentrated under reduced pressure to 1/10 of the original volume. The concentrated eluate was placed in a dialysis bag with a molecular weight cutoff of 3500, about 1/2 volume, and the two ends of the dialysis bag were clamped with dialysis clips, and dialyzed in distilled water for 48 hours, with water changed every 4 hours. The dialyzed eluate was vacuum dried to obtain 0.95 g of neutral poppy polysaccharide.
继续使用0.2mol/L氯化钠溶液冲洗10倍柱体积(约2000mL)，收集洗脱液，减压浓缩至原体积的1/8。浓缩后的洗脱液装于截留分子量3500的透析袋中，约1/2体积，用透析夹夹紧透析袋两端，放于蒸馏水中透析48h，每4h换一次水。将透析后的洗脱液真空干燥得所述酸性罂粟多糖1.61g。Continue to use 0.2mol/L sodium chloride solution to rinse 10 times column volume (about 2000mL), collect eluent, and concentrate under reduced pressure to 1/8 of the original volume. The concentrated eluent is loaded into a dialysis bag with a molecular weight cutoff of 3500, about 1/2 volume, clamped at both ends of the dialysis bag with a dialysis clip, placed in distilled water for dialysis for 48h, and the water is changed every 4h. The eluent after dialysis is vacuum dried to obtain 1.61g of the acidic poppy polysaccharide.
(3)酸性罂粟多糖纯化：用葡聚糖凝胶柱层析法对所述透析产物进行纯化，收集组分，进行冷冻干燥，得到酸性罂粟多糖。步骤(3)中葡聚糖凝胶柱为Sephadex G-100葡萄糖凝胶层析柱，洗脱液采用超纯水，所述超纯水用量为5～8倍柱体积。(3) Purification of acidic poppy polysaccharide: purify the dialyzed product by dextran gel column chromatography, collect the components, and freeze-dry to obtain acidic poppy polysaccharide. The dextran gel column in step (3) is a Sephadex G-100 glucose gel chromatography column, and ultrapure water is used as the eluent, and the amount of ultrapure water used is 5 to 8 times the column volume.
实施例2：酸性罂粟壳多糖含量测定和理化性质研究Example 2: Determination of content and study of physicochemical properties of acidic poppy chitosan
(1)采用苯酚-硫酸法测定酸性罂粟壳多糖含量(1) Determination of acidic poppy chitosan content by phenol-sulfuric acid method
采用苯酚-硫酸法测定酸性罂粟壳多糖含量，具体过程如下：The phenol-sulfuric acid method was used to determine the content of acidic poppy chitosan. The specific process is as follows:
1)绘制葡萄糖标准曲线：精确称量葡萄糖标准品10.0mg，加纯净水溶解，定容至100.0mL，标准品终浓度为0.1mg/mL；分别精确吸取0、0.1、0.2、0.4、0.6和0.8mL葡萄糖母液于试管中；分别补加纯净水至1.0mL；各试管中分别加入5％的苯酚溶液1.0mL和浓硫酸2.5mL，立即涡旋混匀，室温反应半小时，以0管为对照，测定490nm处的OD值。以标准葡萄糖溶液浓度(x，mg/mL)为横轴，OD490值(y)为纵轴，拟合标准回归方程。标准曲线见图2。1) Draw a glucose standard curve: accurately weigh 10.0 mg of glucose standard, dissolve it in pure water, and dilute to 100.0 mL. The final concentration of the standard is 0.1 mg/mL; accurately pipette 0, 0.1, 0.2, 0.4, 0.6 and 0.8 mL of glucose mother solution into test tubes respectively; add pure water to 1.0 mL respectively; add 1.0 mL of 5% phenol solution and 2.5 mL of concentrated sulfuric acid to each test tube respectively, vortex mix immediately, react at room temperature for half an hour, and use tube 0 as a control to measure the OD value at 490 nm. Fit the standard regression equation with the concentration of standard glucose solution (x, mg/mL) as the horizontal axis and the OD490 value (y) as the vertical axis. The standard curve is shown in Figure 2.
2)总糖含量测定：配制浓度为1.0mg/mL的多糖溶液，吸取1.0mL多糖溶液，按上述测定步骤测定其在490nm处的OD值，根据葡萄糖标准曲线计算罂粟壳多糖样品中多糖含量。2) Determination of total sugar content: prepare a polysaccharide solution with a concentration of 1.0 mg/mL, draw 1.0 mL of the polysaccharide solution, determine its OD value at 490 nm according to the above determination steps, and calculate the polysaccharide content in the poppy shell polysaccharide sample according to the glucose standard curve.
得到罂粟壳总多糖的纯度为16.1％；得到水溶性罂粟壳多糖的纯度为79.2％，酸性罂粟多糖的纯度为83.5％。The purity of the total poppy shell polysaccharide obtained is 16.1%; the purity of the water-soluble poppy shell polysaccharide obtained is 79.2%; and the purity of the acidic poppy polysaccharide is 83.5%.
(2)分子量测定(2) Molecular weight determination
利用凝胶渗透色谱(GPC)系统对酸性罂粟多糖样品的相对分子量大小及分布进行测定分析，结果见图3。第一个组分的重均分子量Mw为539174Da，数均分子量Mn为488824Da，分子量分布宽度(Mw/Mn，即分散性指数PDI)为1.1030；第二个组分的重均分子量Mw为52319Da，数均分子量Mn为48698Da，分子量分布宽度为1.1744，由此可知，该酸性多糖为一种杂多糖。The relative molecular weight and distribution of the acidic poppy polysaccharide samples were measured and analyzed using a gel permeation chromatography (GPC) system, and the results are shown in Figure 3. The weight average molecular weight Mw of the first component is 539174Da, the number average molecular weight Mn is 488824Da, and the molecular weight distribution width (Mw/Mn, i.e., the dispersion index PDI) is 1.1030; the weight average molecular weight Mw of the second component is 52319Da, the number average molecular weight Mn is 48698Da, and the molecular weight distribution width is 1.1744, which shows that the acidic polysaccharide is a heteropolysaccharide.
(3)单糖组成分析(3) Monosaccharide composition analysis
采用1-苯基-3-甲基-5-吡唑酮(PMP)柱前衍生法分析了酸性罂粟多糖的单糖组成。2.0mg酸性多糖用2.0mL的2M三氟乙酸(TFA)在110℃下水解4h得到完全水解产物。然后加5.0mL无水乙醇共浓缩干燥四次，以完全去除残余的TFA。将干燥后的水解物再溶解于0.2mL蒸馏水中，与0.2mL浓度为0.5M的PMP-甲醇溶液和0.2mL浓度为0.3M的氢氧化钠溶液在70℃下反应60min，反应产物用0.2mL浓度为0.5M的盐酸溶液中和，然后用二氯甲烷萃取三次，得到酸性罂粟多糖的PMP衍生物。随后，采用C18色谱柱(5μm,4.6×250mm)和紫外检测器进行UPLC分析。以乙腈和0.1M磷酸盐缓冲液(17:83，v/v，pH 6.7)为流动相，流速为0.8mL/min，柱温30℃，检测波长250nm。单糖标准品包括甘露糖、核糖、鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖、木糖、阿拉伯糖，配制成2.0mg/mL的溶液，用PMP衍生化，继续用二氯甲烷萃取四次，在相同条件下收集水相，利用UPLC分析，根据单糖标准品的保留时间可知单糖组成和含量(表1和图1)。The monosaccharide composition of acidic poppy polysaccharide was analyzed by 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization. 2.0 mg of acidic polysaccharide was hydrolyzed with 2.0 mL of 2 M trifluoroacetic acid (TFA) at 110 ° C for 4 h to obtain a complete hydrolyzate. Then, 5.0 mL of anhydrous ethanol was added and concentrated and dried four times to completely remove the residual TFA. The dried hydrolyzate was redissolved in 0.2 mL of distilled water and reacted with 0.2 mL of 0.5 M PMP-methanol solution and 0.2 mL of 0.3 M sodium hydroxide solution at 70 ° C for 60 min. The reaction product was neutralized with 0.2 mL of 0.5 M hydrochloric acid solution and then extracted with dichloromethane three times to obtain the PMP derivative of acidic poppy polysaccharide. Subsequently, UPLC analysis was performed using a C18 column (5 μm, 4.6 × 250 mm) and a UV detector. Acetonitrile and 0.1M phosphate buffer (17:83, v/v, pH 6.7) were used as the mobile phase, the flow rate was 0.8mL/min, the column temperature was 30°C, and the detection wavelength was 250nm. Monosaccharide standards including mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, and arabinose were prepared into a 2.0mg/mL solution, derivatized with PMP, and then extracted with dichloromethane four times. The aqueous phase was collected under the same conditions and analyzed by UPLC. The monosaccharide composition and content can be known according to the retention time of the monosaccharide standards (Table 1 and Figure 1).
表1酸性罂粟多糖中单糖组成和含量Table 1 Monosaccharide composition and content in acidic poppy polysaccharides
(4)红外光谱分析(4) Infrared spectroscopy analysis
红外光谱分析可以很好的表征多糖中的特征官能团。如图4显示，该酸性罂粟多糖具有多糖特征吸收峰，3445cm-1附近宽而强烈的吸收峰对应O–H伸缩振动，2923cm-1和2854cm-1处的吸收峰分别对应C–H的对称伸缩振动和不对称伸缩振动，1427cm-1处对应–CH(–CH2OH)的弯曲振动。1740cm-1吸收峰对应C＝O伸缩振动，表明糖醛酸的存在，这与单糖组成分析结果相一致。1631cm-1处对应C＝O不对称伸缩振动与–OH的弯曲振动。1248cm-1处弱吸收峰是由于O–H的弯曲振动。1000–1200cm-1处的强吸收峰表明存在C–O键，显示了吡喃糖单元的存在。895cm-1和835cm-1处的弱吸收峰表明同时存在β和α构型的吡喃糖。854cm-1处没有吸收峰，表明不存在呋喃糖。Infrared spectroscopy can well characterize the characteristic functional groups in polysaccharides. As shown in Figure 4, the acidic poppy polysaccharide has characteristic absorption peaks of polysaccharides. The broad and strong absorption peak near 3445cm-1 corresponds to the O–H stretching vibration, the absorption peaks at 2923cm-1 and 2854cm-1 correspond to the symmetric stretching vibration and asymmetric stretching vibration of C–H, respectively, and the absorption peak at 1427cm-1 corresponds to the bending vibration of –CH (–CH2 OH). The absorption peak at 1740cm-1 corresponds to the C＝O stretching vibration, indicating the presence of uronic acid, which is consistent with the results of the monosaccharide composition analysis. The absorption peak at1631cm -1 corresponds to the asymmetric stretching vibration of C＝O and the bending vibration of –OH. The weak absorption peak at 1248cm-1 is due to the bending vibration of O–H. The strong absorption peak at 1000–1200cm-1 indicates the presence of C–O bonds, showing the presence of pyranose units. The weak absorption peaks at 895 cm-1 and 835 cm-1 indicate the presence of both β- and α-configuration pyranose. The absence of an absorption peak at 854 cm-1 indicates the absence of furanose.
(5)核磁共振氢谱分析(5) H NMR spectrum analysis
多糖核磁共振氢谱质子信号重叠较多，但可根据端基质子的化学位移很好的揭示糖苷键。酸性罂粟多糖的1H NMR谱如图5所示，信号峰主要出现在3.0～5.5ppm区域。在端基区出现的化学位移5.0～5.3ppm和4.8～4.9ppm信号峰，表明酸性罂粟多糖中存在α-构型和β-构型糖苷键共存。5.70ppm处有化学位移，表明可能存在糖醛酸。The proton signals of the hydrogen nuclear magnetic resonance spectrum of polysaccharides overlap a lot, but the glycosidic bond can be well revealed based on the chemical shift of the terminal proton. The1H NMR spectrum of acidic poppy polysaccharide is shown in Figure 5, and the signal peaks mainly appear in the 3.0-5.5ppm region. The chemical shift signal peaks of 5.0-5.3ppm and 4.8-4.9ppm appearing in the terminal region indicate that α-configuration and β-configuration glycosidic bonds coexist in acidic poppy polysaccharide. There is a chemical shift at 5.70ppm, indicating the possible presence of uronic acid.
(6)扫描电子显微镜分析(6) Scanning electron microscopy analysis
扫描电子显微镜分析是表征多糖形态结构的有效方法。将酸性多糖样品溶解后于硅片上自然析出，进行扫描电子显微镜分析，结果如图6，为不同放大倍数下的酸性多糖，可见该酸性多糖具有疏松层状多孔的表面形貌。Scanning electron microscopy analysis is an effective method for characterizing the morphological structure of polysaccharides. The acidic polysaccharide sample was dissolved and naturally precipitated on a silicon wafer, and then subjected to scanning electron microscopy analysis. The results are shown in Figure 6, which shows the acidic polysaccharide at different magnifications. It can be seen that the acidic polysaccharide has a loose layered and porous surface morphology.
实施例3：酸性罂粟多糖抗氧化、抗缺氧、抗疲劳作用研究Example 3: Study on the Antioxidant, Anti-hypoxic and Anti-fatigue Effects of Acidic Poppy Polysaccharide
(1)ABTS自由基清除活性试验(1) ABTS free radical scavenging activity test
按体积比1:1，将2.45mmol/L过硫酸钾溶液与7mmol/L ABTS溶液混合，常温下避光反应12h，稀释至吸光度在0.5左右，取1.8mL，分别与0.2mL浓度为0.1、0.2、0.5、1.0、2.0mg/mL的酸性罂粟多糖溶液混合，摇匀后避光反应6min，以抗坏血酸标准品为对照，在波长734nm处测定吸光度变化，结果如图7，所有样品均表现出清除ABTS自由基活性，在实验浓度范围对ABTS的清除率在21％左右，显示酸性多糖具有抗氧化潜力。2.45mmol/L potassium persulfate solution was mixed with 7mmol/L ABTS solution in a volume ratio of 1:1, and reacted in the dark for 12h at room temperature. The solution was diluted to an absorbance of about 0.5, and 1.8mL was taken and mixed with 0.2mL of acidic poppy polysaccharide solutions with concentrations of 0.1, 0.2, 0.5, 1.0, and 2.0mg/mL, respectively. The mixture was shaken and reacted in the dark for 6min. The absorbance change was measured at a wavelength of 734nm with ascorbic acid standard as control. The results are shown in Figure 7. All samples showed ABTS free radical scavenging activity. The scavenging rate of ABTS in the experimental concentration range was about 21%, indicating that acidic polysaccharides have antioxidant potential.
(2)DPPH自由基清除活性试验(2) DPPH free radical scavenging activity test
取0.2mmol/L DPPH乙醇溶液2.0mL，分别与1.0mL浓度为0.1、0.2、0.5、1.0、2.0mg/mL的酸性罂粟多糖溶液混合，摇匀后避光反应30min，以抗坏血酸标准品为对照，在波长517nm处测定吸光度变化，结果如图8，所有样品均表现出清除DPPH自由基活性，在实验浓度范围对DPPH的清除率在31％左右，显示酸性多糖具有抗氧化潜力。2.0 mL of 0.2 mmol/L DPPH ethanol solution was taken and mixed with 1.0 mL of acidic poppy polysaccharide solutions with concentrations of 0.1, 0.2, 0.5, 1.0, and 2.0 mg/mL, respectively. After shaking, the mixture was reacted in the dark for 30 min. The absorbance change was measured at a wavelength of 517 nm using ascorbic acid standard as a control. The results are shown in Figure 8. All samples showed DPPH free radical scavenging activity. The scavenging rate of DPPH within the experimental concentration range was about 31%, indicating that acidic polysaccharides have antioxidant potential.
(3)小鼠常压缺氧实验(3) Normal pressure hypoxia experiment in mice
试验动物：昆明种小鼠30只，体重30～35g，雌雄各半，由兰州大学医学动物实验中心提供。Experimental animals: 30 Kunming mice, weighing 30-35 g, half male and half female, provided by the Medical Animal Experiment Center of Lanzhou University.
实验方法：昆明种小鼠30只，随机分为5组，每组6只，设空白组，阳性对照组(大株红景天胶囊)，本发明罂粟壳总多糖组、中性罂粟多糖组、酸性罂粟多糖组，对照组和各试验组动物饲养环境条件一致。试验组小鼠每天用本发明药物按1.0g/kg计量灌胃给药，阳性对照组(大株红景天胶囊，0.7g/kg)灌胃，每组动物均自由饮水，正常饲料饲养，连续7天。Experimental method: 30 Kunming mice were randomly divided into 5 groups, 6 mice in each group, and a blank group, a positive control group (large plant rhodiola capsule), a poppy shell total polysaccharide group of the present invention, a neutral poppy polysaccharide group, and an acidic poppy polysaccharide group were set. The animal breeding environment conditions of the control group and each test group were consistent. The test group mice were intragastrically administered with the drug of the present invention at a dosage of 1.0 g/kg every day, and the positive control group (large plant rhodiola capsule, 0.7 g/kg) was intragastrically administered. Each group of animals had free drinking water and were fed with normal feed for 7 consecutive days.
常压耐缺氧时间：在第7天小鼠末次灌胃1h后，将5组小鼠，分别置于装有钠石灰15g的500mL密闭广口瓶中，瓶盖涂以凡士林密封，观察记录小鼠停止呼吸时间，计算存活时长。Normal pressure hypoxia tolerance time: 1 hour after the last gavage on the 7th day, the five groups of mice were placed in 500 mL sealed wide-mouth bottles containing 15 g of soda lime. The bottle caps were sealed with vaseline. The time when the mice stopped breathing was observed and recorded, and the survival time was calculated.
统计学分析：所有数据均采用GraphPad Prism5.0统计学软件进行分析，数据以均数±标准差表示，组间比较采用单因素方差分析，P<0.05认为差异性显著，P<0.01认为差异性极显著。Statistical analysis: All data were analyzed using GraphPad Prism 5.0 statistical software. The data were expressed as mean ± standard deviation. One-way analysis of variance was used for comparison among the groups. P < 0.05 was considered to be a significant difference, and P < 0.01 was considered to be an extremely significant difference.
实验结果：与空白组比较，本发明中性罂粟多糖、酸性罂粟多糖能明显延长小鼠常压耐缺氧时间(P<0.01)；与阳性对照组比较，酸性罂粟多糖能延长小鼠常压耐缺氧时间(P<0.05)，结果见表2。Experimental results: Compared with the blank group, the neutral poppy polysaccharide and the acidic poppy polysaccharide of the present invention can significantly prolong the normal pressure hypoxia tolerance time of mice (P<0.01); compared with the positive control group, the acidic poppy polysaccharide can prolong the normal pressure hypoxia tolerance time of mice (P<0.05). The results are shown in Table 2.
表2罂粟多糖对小鼠常压缺氧存活时间的影响(n＝6)Table 2 Effect of poppy polysaccharide on the survival time of mice under normal pressure and hypoxia (n=6)
*P<0.05，**P<0.01与空白组比较；#P<0.05与阳性组比较。*P<0.05, **P<0.01 compared with the blank group;# P<0.05 compared with the positive group.
(4)小鼠力竭游泳实验(4) Exhaustive swimming test in mice
试验动物：昆明种小鼠30只，体重30～35g，雌雄各半，由兰州大学医学动物实验中心提供。Experimental animals: 30 Kunming mice, weighing 30-35 g, half male and half female, provided by the Medical Animal Experiment Center of Lanzhou University.
实验方法：昆明种小鼠30只，随机分为5组，每组6只，设空白组，阳性对照组(大株红景天胶囊)，本发明罂粟壳总多糖组、中性罂粟多糖组、酸性罂粟多糖组，对照组和各试验组动物饲养环境条件一致。试验组小鼠每天用本发明药物按1.0g/kg计量灌胃给药，阳性对照组(大株红景天胶囊，0.7g/kg)灌胃，每组动物均自由饮水，正常饲料饲养，连续7天。Experimental method: 30 Kunming mice were randomly divided into 5 groups, 6 mice in each group, and a blank group, a positive control group (large plant rhodiola capsule), a poppy shell total polysaccharide group of the present invention, a neutral poppy polysaccharide group, and an acidic poppy polysaccharide group were set. The animal breeding environment conditions of the control group and each test group were consistent. The test group mice were intragastrically administered with the drug of the present invention at a dosage of 1.0 g/kg every day, and the positive control group (large plant rhodiola capsule, 0.7 g/kg) was intragastrically administered. Each group of animals had free drinking water and were fed with normal feed for 7 consecutive days.
力竭游泳时间：在第7天小鼠末次灌胃1h后，在每只小鼠尾部系一为体重为其5％的铅丝，放入直径30cm，高25cm，加水至水深20cm，温度25±2℃水中游泳并开始计时，至小鼠头部深入水中10s不再浮起结束计时，记录力竭游泳时间。Exhaustive swimming time: 1 hour after the last gavage of mice on the 7th day, tie a lead wire weighing 5% of its body weight to the tail of each mouse, put it into water with a diameter of 30 cm, a height of 25 cm, and a water depth of 20 cm. The temperature is 25±2℃ and the mouse is allowed to swim. Start timing when the mouse's head is immersed in the water for 10 seconds and no longer floats up. The exhaustive swimming time is recorded.
统计学分析：所有数据均采用GraphPad Prism5.0统计学软件进行分析，数据以均数±标准差表示，组间比较采用单因素方差分析，P<0.05认为差异性显著，P<0.01认为差异性极显著。Statistical analysis: All data were analyzed using GraphPad Prism 5.0 statistical software. The data were expressed as mean ± standard deviation. One-way analysis of variance was used for comparison among the groups. P < 0.05 was considered to be a significant difference, and P < 0.01 was considered to be an extremely significant difference.
实验结果：与空白组比较，本发明中性罂粟多糖、酸性罂粟多糖能明显延长小鼠力竭游泳时间(P<0.01)；与阳性对照组比较，酸性罂粟多糖能延长小鼠力竭游泳时间(P<0.05)，结果见表3。Experimental results: Compared with the blank group, the neutral poppy polysaccharide and the acidic poppy polysaccharide of the present invention can significantly prolong the exhaustive swimming time of mice (P<0.01); compared with the positive control group, the acidic poppy polysaccharide can prolong the exhaustive swimming time of mice (P<0.05). The results are shown in Table 3.
表3罂粟多糖对小鼠力竭游泳时间的影响(n＝6)Table 3 Effects of poppy polysaccharide on exhaustive swimming time of mice (n=6)
*P<0.05，**P<0.01与空白组比较；##P<0.01与阳性组比较。*P<0.05, **P<0.01 compared with the blank group; ##P<0.01 compared with the positive group.
综上可知，本发明从生产提取过生物碱的罂粟壳废渣中获得了一种罂粟酸性多糖；通过动物试验表明：得到的酸性罂粟多糖能明显延长小鼠力竭游泳时间和常压耐缺氧时间，具有良好的抗疲劳抗缺氧活性，且活性优于阳性对照药大株红景天胶囊。因此，本发明所提供的酸性罂粟多糖具有开发为抗疲劳抗缺氧药物的潜力，同时具有开发为功能性保健食品或饮料、日化产品的优势。In summary, the present invention obtains a poppy acid polysaccharide from the waste residue of poppy shell from which alkaloids have been extracted; animal experiments show that the obtained acidic poppy polysaccharide can significantly prolong the exhaustive swimming time and the normal pressure hypoxia tolerance time of mice, has good anti-fatigue and anti-hypoxia activities, and the activity is better than that of the positive control drug Dazhu Hongjingtian capsule. Therefore, the acidic poppy polysaccharide provided by the present invention has the potential to be developed into an anti-fatigue and anti-hypoxia drug, and also has the advantage of being developed into a functional health food or beverage, and a daily chemical product.
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.
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| CN113603801A (en)* | 2021-08-17 | 2021-11-05 | 华侨大学 | Extraction method for obtaining two kinds of rhizoma corydalis Decumbentis polysaccharides, the two kinds of rhizoma corydalis Decumbentis polysaccharides and application thereof |
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| JPH01178501A (en)* | 1988-01-05 | 1989-07-14 | Takayoshi Aoki | Extraction and purification of polysaccharide powder from poppy seed of genus papaver |
| JPH07228601A (en)* | 1994-02-16 | 1995-08-29 | Otsuka Pharmaceut Co Ltd | Water-soluble beta-1,3-glucan derivative and antiviral agent containing the derivative |
| JP4806633B2 (en)* | 2003-06-20 | 2011-11-02 | ルブリゾル アドバンスド マテリアルズ， インコーポレイテッド | Galactomannan hydrocolloid |
| CN100551265C (en)* | 2007-04-16 | 2009-10-21 | 周文志 | Poppy seed oil extract and its production and use |
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| CN113603801A (en)* | 2021-08-17 | 2021-11-05 | 华侨大学 | Extraction method for obtaining two kinds of rhizoma corydalis Decumbentis polysaccharides, the two kinds of rhizoma corydalis Decumbentis polysaccharides and application thereof |
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