WO2022062380A1 - 具有抗氧化功效的西藏灵芝多糖glp-1、制备方法与应用 - Google Patents

具有抗氧化功效的西藏灵芝多糖glp-1、制备方法与应用 Download PDF

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WO2022062380A1
WO2022062380A1 PCT/CN2021/089212 CN2021089212W WO2022062380A1 WO 2022062380 A1 WO2022062380 A1 WO 2022062380A1 CN 2021089212 W CN2021089212 W CN 2021089212W WO 2022062380 A1 WO2022062380 A1 WO 2022062380A1
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ganoderma lucidum
polysaccharide
glp
tibetan
solution
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高雄
吴清平
谢意珍
穆静静
胡惠萍
莫伟鹏
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广东省微生物研究所（广东省微生物分析检测中心）
广东粤微生物科技有限公司
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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
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
    • 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
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    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • 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
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the invention relates to the research field of Ganoderma lucidum polysaccharides, in particular to Vietnamese Ganoderma lucidum polysaccharide GLP-1 with antioxidant effect, a preparation method and application.
  • Ganoderma lucidum Karst also known as Lin Zhongling and Qiongzhen, is the fruiting body of Ganoderma lucidum of Polyporaceae, belonging to Eumycota, Basidiomycotina, Hymenomycetes, Polyporus Order (Aphyllophorales), Ganoderma (Gamodermataceae), Ganoderma (Ganoderma) class.
  • Ganoderma lucidum is known as "Sui Cao", “Xiancao”, “Shenzhi”, “Lingzhi in the forest", etc. It is a medicinal and edible fungus of Ganoderma lucidum.
  • Ganoderma lucidum is a commonly used traditional Chinese medicine in my country.
  • Ganoderma lucidum polysaccharide is the earliest and most in-depth research in the active ingredients of Ganoderma lucidum. There are more than 200 kinds of polysaccharides. Relevant studies have shown that its configuration is similar to DNA and RNA, and it is a three-dimensional monosaccharide chain composed of helical three-dimensional configuration substances; studies have shown that the biological activity of polysaccharides is closely related to its configuration. The higher the length and the frequency of side chains, the stronger the activity, while the polysaccharides linked by ⁇ -type and ⁇ -type glycosidic bonds have different biological activities.
  • Ganoderma lucidum has antioxidant activity. Its active ingredient is Ganoderma lucidum polysaccharide, which can significantly inhibit the proliferation of glomerular mesangial cells induced by high glucose, has a significant scavenging effect on hydroxyl free radicals, and can reduce the effect of free radicals on organelles. It can inhibit the lipid peroxidation induced by different oxidants, thereby inhibiting the process of apoptosis and delaying the aging of the body.
  • the present invention aims to provide the Vietnamese Ganoderma lucidum polysaccharide GLP-1 with antioxidant effect, preparation method and application, taking Cambodia Ganoderma lucidum as the research object, and carrying out separation, purification and biological activity research on the polysaccharide of its molecular weight ⁇ 10kDa, To discover the structure of Vietnamese Ganoderma lucidum polysaccharides ( ⁇ 10kDa), and its edible and medicinal value in antioxidant.
  • the present invention provides the following technical solutions:
  • the Vietnamese Ganoderma lucidum polysaccharide GLP-1 with antioxidant effect has the following sugar chain structure:
  • the polysaccharide content of the Vietnamese Ganoderma lucidum polysaccharide GLP-1 is 73.36%
  • the uronic acid content is 2.27%
  • the weight average molecular weight is 6.31kDa.
  • the protein content of the Vietnamese Ganoderma lucidum polysaccharide GLP-1 is extremely low, and is mainly composed of mannose, glucose, galactose, xylose, arabinose and the like.
  • the Vietnamese Ganoderma lucidum polysaccharide GLP-1 has characteristic absorption peaks of -OH, CH, C-OH, ⁇ -isomeric pyranose and ⁇ -, ⁇ -glycosidic bonds, has no triple helix structure, and has Good thermal stability.
  • the second aspect of the present invention also provides the above-mentioned preparation method of Ganoderma lucidum polysaccharide GLP-1, the preparation method comprises the following steps:
  • step (1) includes: adding 95% ethanol to the Vietnamese Ganoderma lucidum powder, extracting for 2 hours according to a material-to-liquid ratio of 1:20 and an extraction temperature of 75° C., repeating the operation once, and finally drying the Vietnamese Ganoderma lucidum to obtain the Vietnamese Ganoderma lucidum degreased powder ;
  • Step (2) includes: adding water to the defatted powder of Ganoderma lucidum from Cambodia, extracting for 2 hours according to the material-to-liquid ratio of 1:20 and the extraction temperature of 90°C, filtering to obtain an aqueous extract, repeating the operation twice, and then reducing the water extract at a temperature of 60°C. Concentrate under pressure to obtain a concentrate.
  • step (3) includes: adding 4 times the volume of anhydrous ethanol to the concentrated solution, standing at a temperature of 4°C for 16 hours, precipitating the polysaccharide, and then centrifuging to collect the precipitate, the centrifugation speed is 6000rpm, and the centrifugation time is 10min.
  • Step (4) includes: taking 1.5 g of crude polysaccharide, preparing a solution with a concentration of 3 mg/mL, performing ultrafiltration with a 10 kDa ultrafiltration membrane, and after freeze-drying, obtaining Vietnamese Ganoderma lucidum polysaccharide CGLP-1 with a molecular weight of ⁇ 10 kDa.
  • step (5) includes: taking 90 mg of Vietnamese Ganoderma lucidum polysaccharide CGLP-1, preparing a solution with a concentration of 15 mg/mL, separating it through a DEAE cellulose ion exchange column, and sequentially using concentrations of 0, 0.1, 0.2, 0.3, 0.4 , 0.5mol/L NaCl solution elution, elution flow rate is 2mL/min, elution time is 4min/tube, then collect with automatic partial collector, and use phenol-sulfuric acid method to detect the content of polysaccharide;
  • Step (6) includes: the solution eluted from 0.1mol/L NaCl, depressurized and concentrated at 60°C, dialyzed and freeze-dried, take 30 mg of sample, prepare a solution with a concentration of 6 mg/mL, and then pass Sephacryl S- 300 molecular sieves were separated, eluted with first-grade water, the elution flow rate was 1mL/min, and the elution time was 8min/tube, and then collected by an automatic partial collector, and the polysaccharide content was detected by the phenol-sulfuric acid method, and finally a peak appeared
  • the 15-26 tubes were combined, and after concentration, dialysis, and freeze-drying, the Vietnamese Ganoderma lucidum polysaccharide GLP-1 was obtained.
  • the step (5) is to separate the polysaccharide according to the electric charge
  • the step (6) is to separate the polysaccharide according to the molecular weight.
  • separation methods for polysaccharides including the use of ion exchange columns or molecular sieve separation, etc., but the polysaccharides obtained by a single separation method are often not uniform enough.
  • an ultrafiltration step is added before the ion exchange column and the molecular sieve, so that the approximate molecular weight of the polysaccharide can be estimated, which plays a key role in the subsequent selection of molecular sieve fillers.
  • ABTS 2,2'-azido-bis-3-ethylbenzothiazoline-6-sulfonic acid
  • the third aspect of the present invention provides the application of the above-mentioned Vietnamese Ganoderma lucidum polysaccharide GLP-1 in the preparation of antioxidant health care products or medicines.
  • the fourth aspect of the present invention provides that the above-mentioned Vietnamese Ganoderma lucidum polysaccharide GLP-1 can be prepared by enhancing the activity of CAT (catalase, catalase), GSH-Px (glutathione peroxidase, glutathione peroxidase), Increase the ratio of GSH/GSSG (total glutathione/oxidized glutathione, total glutathione/oxidized glutathione) and reduce the level of MDA (malondialdehyde, malondialdehyde) to protect cells from oxidant-induced antioxidant damage Application in health products or medicines.
  • CAT catalase, catalase
  • GSH-Px glutathione peroxidase
  • GSH/GSSG total glutathione/oxidized glutathione, total glutathione/oxidized glutathione
  • MDA malondialdehyde, malondialdehyde
  • the present invention has the following advantages:
  • the present invention separates the Vietnamese Ganoderma lucidum polysaccharide GLP-1 from the Vietnamese Ganoderma lucidum for the first time, and determines its specific active use;
  • the GLP-1 of Ganoderma lucidum polysaccharide was determined by gel permeation chromatography, and its weight average molecular weight was 6.31kDa; by HPLC analysis, GLP-1 of Ganoderma lucidum was mainly composed of mannose, glucose, galactose, xylose, arabinose, etc. composition.
  • GLP-1 of Ganoderma lucidum can improve the cell survival rate of NIH3T3 cells damaged by tBHP (tert-butyl hydroperoxide, tert-butyl hydroperoxide), and reduce the cell LDH (lactate dehydrogenase) , lactate dehydrogenase), enhanced the activity of CAT and GSH-Px, increased the ratio of GSH/GSSG, and decreased the level of MDA, thereby exerting its antioxidant function.
  • Fig. 1 is the DEAE cellulose column chromatography elution diagram of Vietnamese Ganoderma lucidum polysaccharide GLP-1;
  • Fig. 2 is the Sephacryl S-300 molecular sieve elution diagram of Vietnamese Ganoderma lucidum polysaccharide GLP-1;
  • Fig. 3 is the GPC map of Vietnamese Ganoderma lucidum polysaccharide GLP-1;
  • Fig. 4 is the monosaccharide composition diagram of Vietnamese Ganoderma lucidum polysaccharide GLP-1;
  • Fig. 5 is the infrared spectrum of Vietnamese Ganoderma lucidum polysaccharide GLP-1;
  • Fig. 6 is the 13 C NMR spectrum of Vietnamese Ganoderma lucidum polysaccharide GLP-1;
  • Fig. 7 is the 1 H NMR spectrum of Vietnamese Ganoderma lucidum polysaccharide GLP-1;
  • Fig. 8 is the HH-COSY map of Vietnamese Ganoderma lucidum polysaccharide GLP-1;
  • Fig. 9 is the HSQC spectrum of Vietnamese Ganoderma lucidum polysaccharide GLP-1;
  • Fig. 10 is the HMBC map of Vietnamese Ganoderma lucidum polysaccharide GLP-1;
  • Fig. 11 is the Congo red experimental map of Vietnamese Ganoderma lucidum polysaccharide GLP-1;
  • Fig. 12 is the thermogravimetric analysis spectrum of Vietnamese Ganoderma lucidum polysaccharide GLP-1;
  • Figure 13A is a graph showing the scavenging of ABTS free radicals by Vietnamese Ganoderma lucidum polysaccharide GLP-1;
  • Figure 13B is a graph showing the scavenging of hydroxyl radicals by Vietnamese Ganoderma lucidum polysaccharide GLP-1;
  • Figure 13C is a graph showing the scavenging of superoxide anion by GLP-1 of Ganoderma lucidum polysaccharide
  • Figure 13D is a TEAC equivalent map of GLP-1FRAP, a Ganoderma lucidum polysaccharide
  • Figure 13E is a graph of the TEAC equivalent of Vietnamese Ganoderma lucidum polysaccharide GLP-1ORAC;
  • Figure 14 shows the effect of GLP-1 on the viability of tBHP-injured NIH3T3 cells
  • Figure 15 shows the effect of GLP-1 on LDH release from tBHP-injured NIH3T3 cells
  • Figure 16 shows the effect of GLP-1 on ROS in tBHP-injured NIH3T3 cells
  • Figure 17 shows the effect of GLP-1 on MDA levels, GSH/GSSG ratio, CAT and GSH-Px activity in tBHP-injured NIH3T3 cells.
  • the present invention will be described in further detail below with reference to the examples, but the embodiments of the present invention are not limited thereto.
  • the reagents, equipment and methods used in the present invention are conventional commercially available reagents, equipment and methods in the technical field.
  • CGLP-1 Take 90 mg of CGLP-1, prepare a solution with a concentration of 15 mg/mL, separate it through a DEAE cellulose ion-exchange column, and elute with a solution of 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol/L NaCl in sequence.
  • the flow rate is 2mL/min
  • the elution time is 4min/tube
  • the elution curve is shown in Figure 1; after elution, it is collected with an automatic partial collector, and the polysaccharide content is detected by the phenol-sulfuric acid method;
  • the solution eluted by NaCl was concentrated under reduced pressure at 60 °C, and after dialysis and freeze-drying, 30 mg of the sample was taken and prepared into a solution with a concentration of 6 mg/mL.
  • the dewatering flow rate was 1 mL/min, the elution time was 8 min/tube, and the elution curve was shown in Figure 2; after elution, an automatic partial collector was used to collect, and the polysaccharide content was detected by the phenol-sulfuric acid method. 26 tubes were combined, concentrated, dialyzed and freeze-dried to obtain Vietnamese Ganoderma lucidum polysaccharide GLP-1.
  • the average relative molecular weight of GLP-1 was measured by Waters ACQUITY APC equipped with Waters ACQUITY APC AQ 900 and ACQUITY APC AQ 450 columns (2.5 ⁇ m ⁇ 4.6mm ⁇ 150mm), the column temperature was 35°C, and the mobile phase was sodium nitrate ( 100 mM) at a flow rate of 0.4 mL/min, the molecular weight of GLP-1 was estimated from calibration curves obtained from dextran standards of different molecular weights (5.2, 11.6, 23.8, 48.6, 148, 273, 410, 668 kDa), The results are shown in Figure 3.
  • HPLC conditions Agilent XDB-C18 chromatographic column 250mm ⁇ 4.6mm ⁇ 5 ⁇ m; program conditions are: column temperature 30°C, detection wavelength 250nm, flow rate 0.8mL/min, mobile phase is phosphate buffer (0.1M, pH6.5) and Acetonitrile in a ratio of 84:16 (v:v).
  • the standard order (1-10) is: mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, fucose, the results are shown in Figure 4 Show.
  • the characteristic peak at 3392.4 cm -1 is caused by the stretching vibration of the OH group, and the peak at 2925.6 cm -1 is caused by the CH absorption, including the stretching vibration of CH, CH2 and CH3 ;
  • the peaks in the range of 1200-1500 cm- 1 may correspond to the deformation vibration of CH and the bending vibration of C-OH;
  • the characteristic peaks at 1154.7, 1076.5 and 1040.0 cm -1 indicate the presence of the pyran form of the glucosyl residue; in addition,
  • the characteristic absorptions at 835.4 and 900.3 cm -1 indicate the presence of ⁇ - and ⁇ -type glycosidic bonds, respectively.
  • the acetylated product was dissolved in 3 mL of chloroform, transferred to a separatory funnel, a small amount of distilled water was added for sufficient shaking, and then the upper aqueous solution was removed, and the process was repeated 4 times.
  • the chloroform layer was dried with an appropriate amount of anhydrous sodium sulfate, and the volume was fixed to 10 mL, and the Shimadzu GCMS-QP 2010 gas chromatography-mass spectrometer was used to analyze the acetylated product samples;
  • GC-MS conditions RXI-5 SIL MS chromatographic column (30m ⁇ 0.25mm ⁇ 0.25 ⁇ m); temperature programmed conditions: initial temperature 120°C, heating at 4°C/min to 280°C/min; hold for 5min; injection The port temperature was 250°C, the detector temperature was 250°C/min, the carrier gas was helium, and the flow rate was 1 mL/min.
  • GLP-1 NMR analysis of Vietnamese Ganoderma lucidum polysaccharide the results are shown in Figure 6-10. According to the NMR spectrum of Figure 6-10, the chemical shift values of each carbon and hydrogen of each residue are assigned, and the assignment results are shown in Table 2 below. .
  • Thermogravimetric (TG) and differential thermogravimetric (DTG) analyses of GLP-1 were performed on a TGA4000 thermogravimetric analyzer.
  • TG Thermogravimetric
  • TTG differential thermogravimetric
  • TTG thermogravimetric
  • TTG differential thermogravimetric
  • ABTS ⁇ + was used by reacting 7 mM ABTS stock solution with 2.45 mM potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 16 hours.
  • the ABTS ⁇ + solution was diluted with PBS, mixed with an equal amount of ultrapure water at 734 nm, and the absorbance was 0.70 ( ⁇ 0.05).
  • Mix 100 ⁇ L of GLP-1 solution with 100 ⁇ L of ABTS ⁇ + solution place in the dark for 6 min at 30°C, and use a microplate reader to measure at 734 nm, with Vc as a positive control.
  • the results are shown in Fig. 13A, it can be seen that GLP-1 can scavenge ABTS free radicals with an IC 50 of 0.56 mg/mL, but the scavenging ability is weaker than that of Vc.
  • pH 3.6 300mM acetate buffer (containing 1.896g sodium acetate and 16mL acetic acid per liter), 10mM 2,4,6-tris(2-pyridyl)triazine solution (40mM hydrochloric acid solution) Preparation) and 20mM ferric chloride solution in a ratio of 10:1:1 to prepare FRAP working solution, which needs to be preheated at 37°C before the experiment.
  • NIH3T3 cells (1 ⁇ 10 4 cells/well) were inoculated into Corning 96-well plates and cultured in a 37°C, 5% CO 2 incubator for 24 h; the old culture medium was discarded, and different concentrations of GLP-1 (0.5, 1, 2mg/mL) or Trolox (80 ⁇ M), then add tBHP (100 ⁇ M), the blank control and model group were added DMEM medium and tBHP (100 ⁇ M), respectively, and cultured for 24h; take out the 96-well plate, according to Nanjing Jiancheng Bioengineering Institute Collect the cell culture supernatant for the LDH release test according to the instructions provided; discard the old medium, add 200 ⁇ L CCK-8 (200 ⁇ L serum-free medium/5 ⁇ L CCK-8 stock solution, as a control group), at 37 ° C, 5% Incubate in a CO 2 incubator for 2 h, and then measure the absorbance at 450 nm with a microplate reader.
  • GLP-1
  • NIH3T3 cells (5 ⁇ 10 4 cells/well) were inoculated into Corning 24-well plates and cultured in a 37°C, 5% CO 2 incubator for 24 h; the old culture medium was discarded, and different concentrations of GLP-1 (0.5, 1, 2 mg/mL) or Trolox (80 ⁇ M), and then tBHP (100 ⁇ M) was added.
  • the blank control and model groups were added with DMEM medium and tBHP (100 ⁇ M), respectively, and cultured for 6 h; the old culture medium was discarded, and serum-free DMEM medium was added.
  • NIH3T3 cells (6 ⁇ 10 5 cells/well) and inoculate them in 60 mm 2 dishes, and culture them in a 37°C, 5% CO 2 incubator for 24 h; discard the old culture medium and add different concentrations of GLP-1 (0.5, 1, 2 mg/mL) or Trolox (80 ⁇ M), then tBHP (100 ⁇ M) was added, DMEM medium and tBHP (100 ⁇ M) were added to the blank control and model groups, respectively, and cultured for 24 h; the cells were collected, and the cell lysate supernatant was used for the next one step analysis. Protein concentration was quantified with BCA kit.

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Abstract

本发明公开了具有抗氧化功效的西藏灵芝多糖GLP-1、制备方法与应用，所述灵芝多糖GLP-1提取于西藏灵芝粉末，其多糖含量为73.36％，糖醛酸含量为2.27％，重均分子量为6.31kDa，蛋白含量极低。本发明首次从西藏灵芝中分离制备出西藏灵芝多糖GLP-1，GLP-1主要由甘露糖、葡萄糖、半乳糖、木糖、阿拉伯糖等组成，具有-OH、C-H、C-OH、α-异构吡喃糖以及α-，β-糖苷键的特征吸收峰，无三螺旋结构，热稳定性良好；此外，得到的多糖GLP-1具有化学抗氧化活性，且可保护小鼠成纤维细胞NIH3T3细胞免受叔丁基氢过氧化物诱导造成的氧化损伤，在抗氧化活性方面具有很好的食用和药用价值。

Description

具有抗氧化功效的西藏灵芝多糖GLP-1、制备方法与应用 技术领域
本发明涉及灵芝类多糖研究领域，具体涉及具有抗氧化功效的西藏灵芝多糖GLP-1、制备方法与应用。
背景技术
灵芝(Ganoderma lucidum Karst)又称林中灵、琼珍，是多孔菌科真菌灵芝的子实体，隶属于真菌门(Eumycota)、担子菌亚门(Basidiomycotina)、曾菌纲(Hymenomycetes)、多孔菌目(Aphyllophorales)、灵芝科(Gamodermataceae)、灵芝属(Ganoderma)类。灵芝有“瑞草”、“仙草”、“神芝”、“林中灵”等美称，为灵芝属药食两用真菌。灵芝是我国常用传统中药，含有灵芝多糖、三萜和甾醇等多种活性物质，具有补气安神、止咳平喘、延年益寿等功效。在《神农本草》、《本草纲目》等众多古代药学专著中大量记载了灵芝的多种药理作用，因此一直被视为中药的珍品和药膳的瑰宝。
2015年，广东省微生物研究所李泰辉等人在真菌学杂志《Mycos cience》发表文章宣布他们在西藏林芝地区发现了一个灵芝新种-西藏灵芝Ganoderma leucocontextum。自此之后行业迫切希望针对西藏灵芝进行系统研究，寻找其食用及药用价值。
灵芝多糖是在灵芝活性成分中研究最早也是研究最深入的一类成分，目前发现的多糖种类有200多种。有关研究表明其构型与DNA、RNA相似，为一种由3股单糖链组成且呈螺旋状的立体构型物质；研 究表明多糖的生物学活性与其构型有很大关联，主链越长、侧链频率越高，活性越强，而以α-型和β-型糖苷键进行连接的多糖，生物学活性存在差异。
国内外一些研究表明灵芝具有抗氧化活性，其活性成分为灵芝多糖，能够显著抑制高糖诱导的肾小球系膜细胞增殖，对羟基自由基具有明显的清除作用，且能减少自由基对细胞器的损伤，抑制不同氧化剂激发的脂质过氧化作用，从而抑制细胞凋亡过程，延缓机体衰老。
但在现有技术中,对西藏灵芝多糖新成分的研究成果甚少,以及关于西藏灵芝多糖在抗氧化生物活性方面鲜有报道。
发明内容
为解决上述技术问题，本发明旨在提供具有抗氧化功效的西藏灵芝多糖GLP-1、制备方法与应用，以西藏灵芝为研究对象，对其分子量<10kDa的多糖进行分离纯化和生物活性研究，发现西藏灵芝多糖(<10kDa)的结构,以及其在抗氧化方面的食用和药用价值。
为达到上述目的，本发明提供如下技术方案：
具有抗氧化功效的西藏灵芝多糖GLP-1，其糖链的结构如下：
Figure PCTCN2021089212-appb-000001
需要说明的是，所述西藏灵芝多糖GLP-1的多糖含量为73.36％，糖醛酸含量为2.27％，重均分子量为6.31kDa。
需要说明的是，所述西藏灵芝多糖GLP-1的蛋白含量极低，主要由甘露糖、葡萄糖、半乳糖、木糖、阿拉伯糖等组成。
需要说明的是，所述西藏灵芝多糖GLP-1具有-OH、C-H、C-OH、α-异构吡喃糖以及α-，β-糖苷键的特征吸收峰，无三螺旋结构，且有良好的热稳定性。
本发明第二方面还提供如上所述的西藏灵芝多糖GLP-1的制备方法，所述制备方法包括以下步骤：
(1)西藏灵芝粉末经脱脂，得到西藏灵芝脱脂粉末；
(2)往西藏灵芝脱脂粉末中加水，提取多次，得水提液，将水提液减压浓缩，得浓缩液；
(3)浓缩液经沉淀，然后除蛋白，并经透析、冷冻干燥后获得粗多糖；
(4)取粗多糖配制成粗多糖溶液，将粗多糖溶液进行超滤，将超滤物冻干后，得到西藏灵芝多糖CGLP-1；
(5)取西藏灵芝多糖CGLP-1配制成西藏灵芝多糖CGLP-1溶液，西藏灵芝多糖CGLP-1溶液经离子交换柱分离洗脱；
(6)将洗脱后的溶液经降压浓缩、透析、冻干再溶解后，经分子筛分离，再用一级水洗脱，采用苯酚-硫酸法检测多糖含量，将出现峰的15-26管溶液合并，合并后的溶液经浓缩、透析、冷冻干燥后得到西藏灵芝多糖GLP-1。
优选的，步骤(1)包括：向西藏灵芝粉末中加入95％乙醇，按照料液比1:20、提取温度75℃提取2h，重复操作一次，最后将西藏灵芝烘干，得西藏灵芝脱脂粉末；
步骤(2)包括：西藏灵芝脱脂粉末加水，按照料液比1:20、提取温度90℃提取2h，过滤后得水提液，重复操作2次，再将水提液于60℃温度下减压浓缩，得浓缩液。
优选的，步骤(3)包括：向浓缩液中加入4倍体积的无水乙醇，于4℃温度下静置16h，沉淀多糖，然后离心收集沉淀，离心转速为6000rpm，离心时间为10min，将沉淀物溶解于一级水中，得多糖溶液；向多糖溶液中加入1/3倍体积的Sevage试剂，所述Sevage试剂为氯仿:正丁醇＝4:1的溶剂，剧烈震荡30min，于4℃温度下离心，离心转速为6000rpm，离心时间为10min，取上清液，重复操作，直到完全去除蛋白；随后将去除蛋白的多糖溶液采用5000Da透析袋透析72h，之后冷冻干燥，得到粗多糖；
步骤(4)包括：取粗多糖1.5g，配制成浓度为3mg/mL的溶液，使用10kDa超滤膜进行超滤，冻干后，得到分子量<10kDa的西藏灵芝多糖CGLP-1。
优选的，步骤(5)包括：取西藏灵芝多糖CGLP-1 90mg，配制成浓度为15mg/mL的溶液，经DEAE纤维素离子交换柱分离，依次用浓度为0、0.1、0.2、0.3、0.4、0.5mol/L的NaCl溶液洗脱，洗脱流速为2mL/min，洗脱时间为4min/管，之后用全自动部分收集器收集，并采用苯酚-硫酸法检测多糖的含量；
步骤(6)包括：将由0.1mol/L NaCl洗脱下来的溶液，经60℃降压浓缩，透析、冻干后，取30mg样品，配制成浓度为6mg/mL的溶液，再经Sephacryl S-300分子筛分离，用一级水洗脱，洗脱流速为1mL/min，洗脱时间为8min/管，之后采用全自动部分收集器收集，并采用苯酚-硫酸法检测多糖含量，最后将出现峰的15-26管合并，经浓缩、透析、冷冻干燥后得到西藏灵芝多糖GLP-1。
上述方法中，步骤(5)是根据多糖所带电荷进行分离，步骤(6)是根据多糖分子量进行分离。目前多糖的分离方法较多，包括使用离子交换柱或使用分子筛分离等，但单一的分离方法得到的多糖往往不够均一。而本发明的制备方法在离子交换柱和分子筛之前加入了超滤的步骤，这样便能够预估多糖的大概分子量，对后续分子筛填料选择具有关键性作用。
所述西藏灵芝多糖GLP-1在五种化学抗氧化实验:(ABTS(2,2'-联氮-双-3-乙基苯并噻唑啉-6-磺酸；2,2'-azino-bis(3-ethylben zothiazoline-6-sulfonic acid))自由基、羟基自由基、超氧阴离子自由基、FRAP(ferric reducing antioxidant power,亚铁还原能力)、ORAC(oxygen radical antioxidant capacity,氧自由基抗氧化能力))中，表现出抗氧化活性。
本发明第三方面提供如上所述的西藏灵芝多糖GLP-1在制备抗氧化保健品或药物中的应用。
本发明第四方面提供如上所述的西藏灵芝多糖GLP-1在制备可通过增强CAT(catalase,过氧化氢酶)、GSH-Px(glutathione  peroxidase,谷胱甘肽过氧化物酶)的活性，提高GSH/GSSG(total glutathione/oxidized glutathione,总谷胱甘肽/氧化型谷胱甘肽)的比例，及降低MDA(malondialdehyde,丙二醛)的水平来保护细胞免受氧化剂诱导的抗氧化损伤保健品或药物中的应用。
与现有技术相比，本发明具有以下优点：
1、本发明首次从西藏灵芝中分离出西藏灵芝多糖GLP-1，并确定其具体的活性用途；
2、西藏灵芝多糖GLP-1经凝胶渗透色谱仪测定，其重均分子量为6.31kDa；经HPLC分析，西藏灵芝多糖GLP-1主要由甘露糖、葡萄糖、半乳糖、木糖、阿拉伯糖等组成。经刚果红分析，无三螺旋结构；经热重分析，有良好的热稳定性；经红外光谱分析，GLP-1具有-OH、C-H、C-OH、α-异构吡喃糖以及α-，β-糖苷键的特征吸收峰，并结合甲基化和核磁分析，解析了GLP-1的分子结构，确定西藏灵芝多糖GLP-1为新物质；
3、西藏灵芝多糖GLP-1在0-2mg/mL浓度范围内，可有效清除ABTS自由基；在0-3.2mg/mL浓度范围内，可有效清除羟基自由基；在0-1.6mg/mL浓度范围内，可有效清除超氧阴离子自由基；且GLP-1表现出对亚铁离子的还原能力，以及对氧化自由基的吸收能力，可用于发挥其抗氧化功能；
4、西藏灵芝多糖GLP-1在0.5-2mg/mL浓度范围内，可提高NIH3T3细胞经tBHP(tert-butyl hydroperoxide，叔丁基氢过氧化物)损伤后的细胞存活率，降低了细胞LDH(lactate dehydrogenase，乳 酸脱氢酶)的释放量，增强了CAT、GSH-Px的活性，提高了GSH/GSSG的比例，降低了MDA的水平，从而发挥其抗氧化功能。
附图说明
图1是西藏灵芝多糖GLP-1的DEAE纤维素柱层析洗脱图；
图2是西藏灵芝多糖GLP-1的Sephacryl S-300分子筛洗脱图；
图3是西藏灵芝多糖GLP-1的GPC图；
图4是西藏灵芝多糖GLP-1的单糖组成图；
图5是西藏灵芝多糖GLP-1的红外光谱图；
图6是西藏灵芝多糖GLP-1的 13C NMR图谱；
图7是西藏灵芝多糖GLP-1的 1H NMR图谱；
图8是西藏灵芝多糖GLP-1的HH-COSY图谱；
图9是西藏灵芝多糖GLP-1的HSQC图谱；
图10是西藏灵芝多糖GLP-1的HMBC图谱；
图11是西藏灵芝多糖GLP-1的刚果红实验图谱；
图12是西藏灵芝多糖GLP-1的热重分析图谱；
图13A是西藏灵芝多糖GLP-1清除ABTS自由基图；
图13B是西藏灵芝多糖GLP-1清除羟基自由基图；
图13C是西藏灵芝多糖GLP-1清除超氧阴离子图；
图13D是西藏灵芝多糖GLP-1FRAP的TEAC当量图；
图13E是西藏灵芝多糖GLP-1ORAC的TEAC当量图；
图14表示GLP-1对tBHP损伤的NIH3T3细胞存活率的影响；
图15表示GLP-1对tBHP损伤的NIH3T3细胞LDH释放量的影响；
图16表示GLP-1对tBHP损伤的NIH3T3细胞内ROS的影响；
图17表示GLP-1对tBHP损伤的NIH3T3细胞MDA水平、GSH/GSSG比值、CAT和GSH-Px活性的影响。
具体实施方式
下面结合实施例对本发明作进一步详细的描述，但本发明的实施方式不限于此。在未作特别说明的情况下，本发明所采用的试剂、设备和方法均为本技术领域常规市购的试剂、设备和常规使用的方法。
实施例1：
一)西藏灵芝多糖GLP-1的制备
西藏灵芝粉末中加入95％乙醇，按照料液比1:20、提取温度75℃提取2h，重复操作一次，最后将西藏灵芝烘干，得到西藏灵芝脱脂粉末；往西藏灵芝脱脂粉末中加水(西藏灵芝脱脂粉末与水按照料液比1:20)，于90℃温度下提取2h，过滤后得水提液，重复操作2次，再将水提液于60℃温度下减压浓缩，得浓缩液；
向浓缩液中加入4倍体积的无水乙醇，4℃下静置16h，沉淀多糖，然后4℃下离心收集沉淀物，离心转速6000rpm,离心时间10min,再将沉淀物溶解于一级水中，得到多糖溶液；
向多糖溶液中加入1/3倍体积的Sevage试剂，所述Sevage试剂为氯仿:正丁醇＝4:1(体积比)的溶剂，剧烈震荡30min后,于4℃温度下离心，离心转速6000rpm,离心时间10min,取上清液，重复操作，直到完全去除蛋白；随后将去除蛋白的多糖溶液采用5000Da透析袋 进行透析72h；之后冷冻干燥得粗多糖；
取粗多糖1.5g，配制成浓度为3mg/mL的溶液，使用10kDa超滤膜进行超滤，冻干后，得到分子量<10kDa的西藏灵芝多糖CGLP-1；
取CGLP-1 90mg，配制成浓度为15mg/mL的溶液，经DEAE纤维素离子交换柱分离，依次用浓度为0、0.1、0.2、0.3、0.4、0.5mol/L NaCl溶液洗脱，洗脱流速为2mL/min，洗脱时间为4min/管，洗脱曲线如图1所示；洗脱后用全自动部分收集器收集，采用苯酚-硫酸法检测多糖含量；接下来将由0.1mol/L NaCl洗脱下来的溶液在60℃下降压浓缩，透析、冻干后，取30mg样品，配制成浓度为6mg/mL的溶液，经Sephacryl S-300分子筛分离，用一级水洗脱，洗脱流速为1mL/min，洗脱时间为8min/管，洗脱曲线如图2所示；洗脱后用全自动部分收集器收集，采用苯酚-硫酸法检测多糖含量，将出现峰的15-26管合并，经浓缩、透析、冷冻干燥得西藏灵芝多糖GLP-1。
二)西藏灵芝多糖GLP-1的分子量的测定
GLP-1的平均相对分子量通过Waters ACQUITY APC测得，该系统配备Waters ACQUITY APC AQ 900和ACQUITY APC AQ 450柱(2.5μm×4.6mm×150mm)，柱温为35℃，流动相为硝酸钠(100mM)，流速为0.4mL/min，根据从不同分子量(5.2、11.6、23.8、48.6、148、273、410、668kDa)的葡聚糖标准中得到的校准曲线，估算了GLP-1的分子量，结果如图3所示。
三)西藏灵芝多糖GLP-1的单糖组成分析
取2mg多糖，用1mL的2M三氟乙酸于110℃温度下水解6h，水 解后的多糖用0.5M PMP(1-phenyl-3-methyl-5-pyrazolone，1-苯基-3-甲基-5-吡唑酮)进行衍生。分析采用Agilent 1200高效液相色谱系统测定衍生产物样品；
HPLC条件：安捷伦XDB-C18色谱柱250mm×4.6mm×5μm；程序条件为：柱温30℃，检测波长250nm，流速0.8mL/min，流动相为磷酸缓冲液(0.1M，pH6.5)和乙腈，比例为84:16(v:v)。
其中标准品顺序(1-10)依次为：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、岩藻糖，结果如图4所示。
由HPLC结果可知，西藏灵芝多糖GLP-1的单糖组成种类及比例为甘露糖:核糖:鼠李糖:葡萄糖醛酸：葡萄糖:半乳糖:木糖:阿拉伯糖：岩藻糖＝7.02:0.58:0.55:1.10:60.85:12.00:8.58:7.51:1.80。
四)西藏灵芝多糖GLP-1的红外光谱分析
取2mg多糖，加入100mg干燥的溴化钾粉末混匀研磨，倒入压片模具压片30s，置于样品扫描窗口，在4000-400cm -1的范围内进行红外光谱扫描，结果如图5所示，可以看出：出现了多糖的特征吸收峰。在3392.4cm -1处的特征峰是由O-H基团的拉伸振动引起的，在2925.6cm -1处的峰值是由C-H吸收引起的，包括CH、CH 2和CH 3的拉伸振动；在1200-1500cm -1范围内的峰可能对应于C-H的变形振动和C-OH的弯曲振动；在1154.7、1076.5和1040.0cm -1处的特征峰表明存在葡萄糖基残基的吡喃形式；此外，在835.4和900.3cm -1处的特征吸收分别表明存在α-和β-型糖苷键。
五)西藏灵芝多糖GLP-1的甲基化分析
6mg GLP-1置于反应瓶中，加入二甲基亚砜，快速加入氢氧化钠粉末，封闭，在超声作用下溶解，再加入碘甲烷反应。最后加入水到上述混合物中终止甲基化反应。取甲基化后的多糖，用1mL的2M三氟乙酸水解90min，再用旋转蒸发仪蒸干。残基加入2mL双蒸水，60mg硼氢化钠还原8h，加入冰醋酸中和，旋蒸，101℃烘箱烘干，然后加入1mL乙酸酐乙酰化100℃反应1h,冷却，然后加入3mL甲苯，减压浓缩蒸干，重复4-5次，以除去多余的醋酐。将乙酰化后的产物用3mL氯仿溶解后转移至分液漏斗，加入少量蒸馏水充分震荡后，除去上层水溶液，如此重复4次。氯仿层以适量的无水硫酸钠干燥，定容10mL，分析采用Shimadzu GCMS-QP 2010气相色谱-质谱联用仪测定乙酰化产物样品；
GC-MS条件：RXI-5 SIL MS色谱柱(30m×0.25mm×0.25μm)；程序升温条件为：起始温度120℃，以4℃/min升温至280℃/min；保持5min；进样口温度为250℃，检测器温度为250℃/min，载气为氦气，流速为1mL/min。
结果如下表1所示。
表1-西藏灵芝多糖GLP-1的甲基化糖残基GC-MS分析
Figure PCTCN2021089212-appb-000002
Figure PCTCN2021089212-appb-000003
六)西藏灵芝多糖GLP-1的核磁共振分析
将50mg GLP-1溶于0.5mL氘水进行检测。西藏灵芝多糖GLP-1核磁共振分析：结果如图6-10所示，根据图6-10的核磁图谱对各残基的各个碳和氢的化学位移值进行归属，归属结果如下表2所示。
表2-西藏灵芝多糖GLP-1中各糖残基的氢、碳信号归属
Figure PCTCN2021089212-appb-000004
Figure PCTCN2021089212-appb-000005
结合单糖组成、红外光谱、甲基化和核磁共振分析可知GLP-1的糖链结构如下：
Figure PCTCN2021089212-appb-000006
七)西藏灵芝多糖GLP-1的刚果红实验
将0.5mL多糖样品(1mg/mL)与1mL刚果红溶液(80μM)混合。随后，加入0.5mL氢氧化钠溶液，使氢氧化钠的最终浓度为0.05-0.5M，混合溶液避光放置10min，用U-2910分光光度计分析了最大吸收波长(λmax)。结果如图11所示，可以看出：GLP-1无三螺旋结构。
八)西藏灵芝多糖GLP-1的热重分析实验
在TGA4000热重分析仪上进行了GLP-1的热重(TG)和差热重力(DTG)分析。在充满氮气的环境中，在三氧化二铝坩埚上放置7.8mgGLP-1，温度从28℃上升到880℃，升温速率为10℃/min。结果如图12所示，图中实线为热重(TG)曲线,虚线为差热重力(DTG)曲线,可以看出：GLP-1有良好的热稳定性。
实施例2：
一)西藏灵芝多糖GLP-1对ABTS自由基的清除能力实验
ABTS· +是通过将7mM的ABTS储备溶液与2.45mM的过硫酸钾(最终浓度)反应，并使混合物在室温下黑暗中放置16小时后使用。用PBS稀释ABTS· +溶液，在734nm处与等量的超纯水混合，吸光值为0.70(±0.05)。将100μL GLP-1溶液与100μL ABTS· +溶液混合，于30℃温度中避光放置6min，使用酶标仪在734nm处进行测定，以Vc作为阳性对照。结果如图13A所示，可以看出：GLP-1可清除ABTS自由基，其IC 50为0.56mg/mL，但清除能力弱于Vc。
二)西藏灵芝多糖GLP-1对羟基自由基的清除能力实验
将50μL硫酸亚铁(1.5mM)和50μL双氧水(0.01％)与100μ L GLP-1溶液混合，最后加入50μL 1,10-菲啰啉(1.5mM)，于37℃温度中避光放置30min，使用酶标仪在536nm处进行测定，以Vc作为阳性对照。结果如图13B所示，可以看出：GLP-1可清除羟基自由基，其IC 50为1.32mg/mL，但清除能力弱于Vc。
三)西藏灵芝多糖GLP-1对超氧阴离子的清除能力实验
将含有375μM β-烟酰胺腺嘌呤二核苷酸和125μM硝基蓝四氮唑的100μL 0.1M磷酸钠缓冲液(pH＝7.4)与100μL GLP-1溶液混合，之后加入50μL 16.5μM吩嗪硫酸甲酯溶液，于25℃温度中避光放置5min，使用酶标仪在560nm处进行测定，以Vc作为阳性对照。结果如图13C所示，可以看出：GLP-1可清除超氧阴离子自由基，其IC 50为0.76mg/mL，但清除能力弱于Vc。
四)西藏灵芝多糖GLP-1铁还原能力实验
在实验前1h，将pH＝3.6的300mM醋酸盐缓冲液(每升含1.896g醋酸钠和16mL醋酸)、10mM 2,4,6-三(2-吡啶基)三嗪溶液(40mM盐酸溶液配制)和20mM三氯化铁溶液按10:1:1的比例混合，制备FRAP工作液，此工作液在实验前需要放置在37℃中预热。将150μL FRAP工作液与50μL GLP-1溶液或者Trolox(6-羟基-2,5,7,8-四甲基-2-羧酸；6-hydro-xy-2,5,7,8-tetramethyl-2-carboxylic acid)(0-40μM，最终浓度)溶液混合，于37℃温度中避光放置4min，使用酶标仪在593nm处进行测定，结果以μmol/g Trolox等效抗氧化能力(TEAC)表示。结果如图13D所示，可以看出：GLP-1的TEAC值为6.85μmol/g。
五)西藏灵芝多糖GLP-1的氧自由基抗氧化能力实验
用75mM磷酸盐缓冲液(pH＝7.4)配制荧光素储备溶液(1.17mM)，再用磷酸缓冲液稀释10000倍后备用。20μL GLP-1与120μL荧光素溶液混合后，于37℃避光放置15min，加入60μL AAPH(2,2'-偶氮(2-甲基丙胺)二盐酸盐；2,2'-azobis(2-methylpropionamidine)dihydrochloride)溶液(12mM，最终浓度)，立即使用荧光酶标仪在493/515nm进行测定，每分钟记录一次数据，持续60min。结果如图13E所示，可以看出：GLP-1的TEAC值为84.8μmol/g。
六)西藏灵芝多糖GLP-1对tBHP损伤的NIH3T3细胞的保护作用和对LDH释放的影响实验
取NIH3T3细胞(1×10 4个/孔)接种于康宁96孔板，于37℃、5％的CO 2培养箱中培养24h；弃去旧培养液，加入不同浓度的GLP-1(0.5、1、2mg/mL)或者Trolox(80μM)，再加入tBHP(100μM)，空白对照和模型组分别加入DMEM培养基和tBHP(100μM)，培养24h；取出96孔板，根据南京建成生物工程研究所提供的说明书收集细胞培养上清液进行LDH释放试验；弃去旧培养基，加入200μL CCK-8(200μL无血清培养基/5μL CCK-8原液，作为对照组)，于37℃、5％的CO 2培养箱中培养2h，之后用酶标仪测定450nm吸光值，结果如图14、15所示。从图14中可以看出，相比模型组，GLP-1(0.5、1、2mg/mL)增加了细胞存活率，且呈剂量依赖关系，GLP-1浓度为2mg/mL时，细胞存活率为90.5％；从图15中可以看出，经100μM tBHP作用后，LDH释放量显著增加，但GLP-1(0.5、1、2mg/mL)作用后，明显降低 了LDH水平，且呈剂量依赖关系，GLP-1浓度为2mg/mL时，LDH释放量回到正常水平。GLP-1在2mg/mL浓度时，保护效果与Trolox(80μM)相当，说明GLP-1可防止tBHP诱导的NIH3T3细胞氧化损伤。
七)西藏灵芝多糖GLP-1对tBHP损伤的NIH3T3细胞中ROS(活性氧；reactive oxygen species)的影响实验
取NIH3T3细胞(5×10 4个/孔)接种于康宁24孔板，于37℃、5％的CO 2培养箱中培养24h；弃去旧培养液，加入不同浓度的GLP-1(0.5、1、2mg/mL)或者Trolox(80μM)，再加入tBHP(100μM)，空白对照和模型组分别加入DMEM培养基和tBHP(100μM)，培养6h；弃去旧培养液，加入无血清DMEM培养基配制的DCFH-DA(2',7'-二氯荧光黄双乙酸盐；2',7'-dichlorodihydrofluorescein diacetate)(10μM)，于37℃、5％的CO 2培养箱中孵育30min，之后用DPBS清洗三次，使用EVOS FL Auto 2显微镜拍摄荧光图像。用image J软件对每个图像的荧光强度进行量化，结果如图16(A：空白对照组；B：100μM tBHP损伤组；C：100μM tBHP+0.5mg/mL GLP-1；D：100μM tBHP+1mg/mL GLP-1；E：100μM tBHP+2mg/mL GLP-1；F：100μM tBHP+80μM Trolox；G：荧光强度量化结果)所示，可以看出：相比于空白对照组，模型组中ROS显著增加，但GLP-1样品组可显著减少ROS的产生。
八)西藏灵芝多糖GLP-1对tBHP损伤的NIH3T3细胞中MDA，GSH，GSSG，CAT和GSH-Px的影响实验
取NIH3T3细胞(6×10 5个/孔)接种于60mm 2皿中，于37℃、5％的 CO 2培养箱中培养24h；弃去旧培养液，加入不同浓度的GLP-1(0.5、1、2mg/mL)或者Trolox(80μM)，再加入tBHP(100μM)，空白对照和模型组分别加入DMEM培养基和tBHP(100μM)，培养24h；收集细胞，细胞裂解液上清液用于下一步分析。用BCA试剂盒定量测定蛋白浓度。用微量丙二醛和总谷胱甘肽/氧化型谷胱甘肽测定试剂盒测定MDA、GSH、GSSG含量。用CAT和GSH-Px检测试剂盒分析CAT和GSH-Px的活性。结果如图17(A)-(D)所示，可以看出：GLP-1增强了CAT、GSH-Px的活性，提高GSH/GSSG的比例，降低MDA水平。以上结果表明GLP-1可能通过酶的作用机制保护NIH3T3细胞免受氧化损伤。
以上所述，仅为本发明的较佳的具体实施例，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，根据本发明的技术方案及其构思加以等同替换或改变，都应涵盖在本发明的保护范围内。

Claims (10)

  1. 一种具有抗氧化功效的西藏灵芝多糖GLP-1，其特征在于，糖链的结构如下：
    Figure PCTCN2021089212-appb-100001
  2. 根据权利要求1所述的具有抗氧化功效的西藏灵芝多糖GLP-1，其特征在于，多糖含量为73.36％，糖醛酸含量为2.27％，重均分子量为6.31kDa。
  3. 根据权利要求1所述的具有抗氧化功效的西藏灵芝多糖GLP-1，其特征在于，主要由甘露糖、葡萄糖、半乳糖、木糖、阿拉伯糖组成，具有-OH、C-H、C-OH、α-异构吡喃糖以及α-，β-糖苷键的特征吸收峰，无三螺旋结构，热稳定性良好。
  4. 一种如权利要求1～3任一项所述的具有抗氧化功效的西藏灵芝多糖GLP-1的制备方法,其特征在于,包括以下步骤：
    (1)西藏灵芝粉末经脱脂，得到西藏灵芝脱脂粉末；
    (2)往西藏灵芝脱脂粉末中加水，提取多次，得水提液，将水提液减压浓缩，得浓缩液；
    (3)浓缩液经沉淀，然后除蛋白，并经透析、冷冻干燥后获得粗多糖；
    (4)取粗多糖配制成粗多糖溶液，将粗多糖溶液进行超滤，再将 超滤物冻干后，得到西藏灵芝多糖CGLP-1；
    (5)取西藏灵芝多糖CGLP-1配制成西藏灵芝多糖CGLP-1溶液，西藏灵芝多糖CGLP-1溶液经离子交换柱分离洗脱；
    (6)将洗脱后的溶液经降压浓缩、透析、冻干再溶解后，经分子筛分离，再用一级水洗脱，采用苯酚-硫酸法检测多糖含量，将15-26管溶液合并，合并后的溶液经浓缩、透析、冷冻干燥后得到西藏灵芝多糖GLP-1。
  5. 根据权利要求4所述的具有抗氧化功效的西藏灵芝多糖GLP-1的制备方法，其特征在于，步骤(1)包括：向西藏灵芝粉末中加入95％乙醇，按照料液比1:20、提取温度75℃提取2h，重复操作一次，最后将西藏灵芝烘干，得西藏灵芝脱脂粉末；
    步骤(2)包括：西藏灵芝脱脂粉末加水，按照料液比1:20、提取温度90℃提取2h，过滤后得水提液，重复操作2次，再将水提液于60℃温度中减压浓缩，得浓缩液。
  6. 根据权利要求4所述的具有抗氧化功效的西藏灵芝多糖GLP-1的制备方法，其特征在于，步骤(3)包括：向浓缩液中加入4倍体积的无水乙醇，于4℃温度下静置16h，沉淀多糖，然后离心收集沉淀，离心转速为6000rpm，离心时间为10min，再将沉淀物溶解于一级水中，得到多糖溶液；向多糖溶液中加入1/3倍体积的Sevage试剂，剧烈震荡30min，于4℃温度下离心，离心转速为6000rpm，离心时间为10min，取上清液，重复操作，直到完全去除蛋白；随后将去除蛋白的多糖溶液采用5000Da透析袋透析72h，之后冷冻干燥,得 到粗多糖；
    步骤(4)包括：取粗多糖1.5g，配制成浓度为3mg/mL的溶液，使用10kDa超滤膜进行超滤，冻干后，得到分子量<10kDa的西藏灵芝多糖CGLP-1。
  7. 根据权利要求6所述的具有抗氧化功效的西藏灵芝多糖GLP-1的制备方法，其特征在于，所述Sevage试剂为氯仿:正丁醇＝4:1的溶剂。
  8. 根据权利要求4所述的具有抗氧化功效的西藏灵芝多糖GLP-1的制备方法，其特征在于，步骤(5)包括：取西藏灵芝多糖CGLP-1 90mg，配制成浓度为15mg/mL的溶液，经DEAE纤维素离子交换柱分离，依次用浓度为0、0.1、0.2、0.3、0.4、0.5mol/L的NaCl溶液洗脱，洗脱流速为2mL/min，洗脱时间为4min/管，之后用全自动部分收集器收集，并采用苯酚-硫酸法检测多糖的含量；
    步骤(6)包括：将由0.1mol/L NaCl洗脱下来的溶液，经60℃降压浓缩，透析、冻干后，取30mg样品，配制成浓度为6mg/mL的溶液，再经Sephacryl S-300分子筛分离，用一级水洗脱，洗脱流速为1mL/min，洗脱时间为8min/管，之后采用全自动部分收集器收集，并采用苯酚-硫酸法检测多糖含量，最后将15-26管合并，经浓缩、透析、冷冻干燥后得到西藏灵芝多糖GLP-1。
  9. 如权利要求1～3任一项所述的西藏灵芝多糖GLP-1在制备抗氧化保健品或药物中的应用。
  10. 如权利要求1～3任一项所述的西藏灵芝多糖GLP-1在制备 可通过增强过氧化氢酶谷胱甘肽过氧化物酶的活性，提高总谷胱甘肽/氧化型谷胱甘肽的比例，降低丙二醛的水平来保护细胞免受氧化剂诱导的抗氧化损伤保健品或药物中的应用。
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