阅读说明：本技术 一种硫酸化甘露-葡聚糖的增强免疫功能活性的应用 (Application of sulfated mannan-glucan in enhancing immune function activity ) 是由 郭云良 刘英娟 金维华 朱琳 王潇璐 王悦 武筱林 于 2019-11-20 设计创作，主要内容包括：本发明提供了一种来源于海洋生物——罗氏海盘车(starfish Asterias rollestoni)的硫酸化甘露-葡聚糖在增强免疫功能方面的应用。硫酸化甘露-葡聚糖通过酸解罗氏海盘车获得,单糖组成为葡萄糖和甘露糖(单糖比例为1:0.27),含13.85％的硫酸基。该聚糖具有显著激活小鼠腹腔巨噬细胞释放一氧化氮及炎症因子,并加强细胞自噬功能的生物活性。可以用于制备免疫增强剂的药物、保健品以及特殊医学用途配方食品。(The invention provides an application of sulfated mannan-glucan derived from marine organisms, namely starfish Asterias rolestoni, in the aspect of enhancing immune function. The sulfated mannan-glucan was obtained by acid hydrolysis of asterias rollestoni, and the monosaccharide consisted of glucose and mannose (monosaccharide ratio 1:0.27) and contained 13.85% sulfate groups. The glycan has the biological activity of remarkably activating macrophages in abdominal cavities of mice to release nitric oxide and inflammatory factors and strengthening autophagy functions of cells. Can be used for preparing immunopotentiators, health products and foods for special medical purposes.)

1. The application of sulfated mannan-glucan in enhancing the immune function activity is characterized in that the specific preparation process of the sulfated mannan-glucan is carried out as follows:

degreasing dried dry asterias rollestoni by isopropanol and ethanol, drying at 60 ℃, and then leaching for 2 hours by using 0.15mol/L diluted HCl acid at 60 ℃; neutralizing the leaching liquor with 0.1M NaOH, dialyzing in running water for 24h, dialyzing again with double distilled water for 24h, centrifuging, precipitating with ethanol to obtain crude Rogowski starfish polysaccharide, adding the crude polysaccharide into 1% trypsin solution by mass-volume ratio, deproteinizing at 37 ℃, centrifuging, collecting supernatant, lyophilizing, and subjecting the lyophilized sample to anion column chromatography (DEAE-Bio agarose FF (50mm × 40cm)) to obtain a water washing component and a NaCl component, wherein the obtained NaCl component is the sulfated mannan-dextran.

2. The use of a sulfated mannan-glucan as claimed in claim 1, wherein the cut off of the dialysis bag used for dialysis desalting is 1 kD.

3. The use of a sulfated mannan-glucan as claimed in claim 1, wherein the sulfated mannan-glucan has a molecular weight of 151.1 kD.

4. The use of a sulfated mannan-glucan as claimed in claim 1, wherein the sulfated mannan-glucan monosaccharides prepared and extracted by the process are composed of glucose and mannose, the monosaccharide composition is 1:0.27, the total sugar yield is 85.31%, and the sulfate content is 13.85%.

5. The use of the sulfated mannan-glucan according to claim 1, wherein the sulfated mannan-glucan is effective in activating mouse peritoneal macrophages to release nitric oxide and inflammatory factors and enhancing autophagy.

6. Use of a sulfated mannan-glucan as claimed in any one of claims 1 to 4 for enhancing the immune function of a human, wherein the sulfated mannan-glucan is prepared for use in enhancing the immunity of a human.

7. The use of the sulfated mannan-glucan according to any one of claims 1 to 4 for enhancing immune function activity, wherein the sulfated mannan-glucan prepared can be prepared into a drug or health product for enhancing immunity in the form of a tablet, a capsule, an oral liquid, a soft capsule, a powder, a tincture, a powder injection, a water injection, a small infusion solution.

The technical field is as follows:

the invention belongs to the field of biological medicine, and particularly relates to application of sulfated mannan-glucan derived from marine organisms, namely starfisch asterias rolestoni, in the aspect of enhancing immune function.

Background art:

the immunity is a very complex physiological process of an organism to tissue damage and infection caused by pathogenic factors such as pathogens, toxic compounds or radiation, etc., and the organism can identify self and non-self components by means of the function and eliminate antigenic foreign matters or damaged cells and tumor cells, etc. generated by the human body by immune response so as to maintain the physiological balance of the organism. Immune disorders can cause a number of diseases including allergies (allergies, immune complex-type, delayed-type, cytotoxic-type), immunodeficiency (AIDS), etc., and an impaired immune system (e.g., immune cells destroyed by AIDS viruses, resulting in immune deficiency in humans), etc. Therefore, maintaining a dynamic immune system balance is a prerequisite for maintaining the health of the body.

Marine algae, as an important source of unique bioactive substances, has been widely used as a new source of drugs in the medical field. Asteroidea (Asteroidea) belonging to Echinodermata, Asteroidea, is distributed in various seas of the world. Starfish is a traditional folk Chinese medicinal material, is salty in taste and neutral in nature, and is mainly used for treating diseases such as thyromegaly, scrofula, stomachache and pantothenic acid, diarrhea, otitis media, impotence, injury from overexertion pain, rheumatism, lumbocrural pain, epilepsy and the like. In recent years, research on the basis of pharmaceutically effective substances of starfish has been reported, and compounds such as saponins, sterols, glycosides, lipids, nucleosides, proteins, alkaloids, etc. all have certain biological activities, but the research on polysaccharides is relatively small, especially the research on the action mechanism thereof. A few reports show that asterias amurensis polysaccharide has the effects of anticoagulation and nerve cell protection, and other polysaccharides and biological activities thereof are rarely reported. Accordingly, the present invention seeks to devise the use of sulfated mannans to enhance immune function activity, which are effective in enhancing immune function.

The invention content is as follows:

the object of the present invention is to overcome the drawbacks of the prior art and to provide the use of sulfated mannans which are derived from the marine organism asterias rollestoni for their immune function enhancing activity.

In order to achieve the aim, the invention relates to an application specific technical scheme of sulfated mannan-glucan for enhancing the immune function activity, which comprises the following steps:

the specific preparation process of the sulfated mannan-glucan related to the invention is carried out as follows:

degreasing dried dry asterias rollestoni by isopropanol and ethanol, drying at 60 ℃, and then leaching for 2 hours by using 0.15mol/L diluted HCl acid at 60 ℃; neutralizing the leaching liquor by 0.1M NaOH, dialyzing in running water for 24h, dialyzing again by double distilled water for 24h, centrifuging, precipitating with ethanol to obtain crude Roche sea pannier polysaccharide, adding the crude polysaccharide into 1% (M/v) trypsin solution, deproteinizing at 37 ℃, centrifuging, collecting supernatant, lyophilizing, and subjecting the lyophilized sample to anion column chromatography (DEAE-Bio agarose FF (50mm multiplied by 40cm)) to obtain a water washing component and a NaCl component, wherein the obtained NaCl component is the sulfated mannan-glucan of the invention;

further, the cut-off molecular weight of the dialysis bag used for dialysis desalination in the invention is 1 kD;

further, the molecular weight of the sulfated mannan-glucan of the present invention is 151.1 kD.

Through inspection, the sulfated mannose-glucan monosaccharide prepared and extracted by the method in the invention in a grading way consists of glucose and mannose, the monosaccharide composition is 1:0.27, the total sugar yield is 85.31%, and the sulfate radical content is 13.85%.

The prepared sulfated mannan-glucan is verified to detect the activation effect on RAW264.7 cells, and the specific operation steps are carried out according to the following modes:

s1, selecting materials: selecting macrophage RAW264.7 cell, adopting DMEM high-glucose culture solution containing 10% fetal calf serum, 100U/mL penicillin and 100U/mL streptomycin, placing at 37 ℃, and controlling the ratio of carbon dioxide to air in the incubator to be 5: 95;

s2 and Griess reagent method for detecting NO release condition of RAW264.7 cells

Taking RAW264.7 cells in logarithmic growth phase, scraping by a cell scraper, blowing into single cell suspension, centrifuging, and then resuspending and diluting into 1 × 10 by using a DMEM (DMEM) culture medium⁵cells/mL, seeded in 96-well plates at 100. mu.L/well in CO₂Incubation in an incubator; after the cells are attached to the wall, the original culture medium is removed, a fresh preparation culture medium (comprising a culture medium added with sulfated mannan-glucan with different concentrations (0.625 mug/mL-320 mug/mL), a culture medium added with Lipopolysaccharide (LPS) with 1 mug/mL serving as a positive control and a culture medium added with PBS with the same volume) is added), the culture is continued for 24 hours, and 3 compound wells are arranged for each sample; after completion of the culture, 50. mu.L of a freshly prepared Griess (A: B ═ 1:1) mixed reagent was mixed with 50. mu.L of each well of the culture supernatant, and the mixture was incubated at room temperature in the dark for 10min, and then the absorbance value was measured at 550 nm.

At different concentrations of NaNO₂Reacting with Griess (A: B ═ 1:1) mixed reagent, drawing a standard curve, and calculating the NO production amount of the sulfated mannan-dextran after acting on RAW264.7 cells;

after the sulfated mannan-glucan with different concentrations on the surface is tested to act on RAW264.7 cells, the release amount of NO is gradually increased, when the concentration of the sulfated mannan-glucan is 1.25 mug/mL, the release amount of NO is remarkably increased, and when the concentration of the sulfated mannan-glucan reaches 80 mug/mL, the release amount of NO is similar to that of LPS (low-pressure polyethylene) of a positive control group, which indicates that the sulfated mannan can remarkably promote the RAW264.7 cells to release NO, so that the generation of immunity is induced;

s3, Western blot method for detecting expression conditions of RAW264.7 cytokine and autophagy-related protein

Taking RAW264.7 cells in logarithmic growth phase, scraping by a cell scraper, blowing into single cell suspension, centrifuging, and then resuspending and diluting into 1 × 10 by using a DMEM (DMEM) culture medium⁶cells/mL, seeded in 6-well plates, 2mL per well in CO₂Incubation in an incubator; after the cells adhere to the wall, the original culture medium is sucked away and added with fresh culture mediumAdding culture medium of sulfated mannan-dextran (0.625-320 μ g/mL) with different concentrations and PBS with equal volume, continuously culturing for 0h, 1h, 3h, 6h and 9h, setting 3 multiple holes for each sample, collecting cells after culturing, adding cell lysate RIPA containing protease inhibitor, lysing the cells on ice for 30min, centrifuging, and collecting cell lysate;

detecting the protein concentration of cell lysate by using a BCA protein determination kit, adjusting the protein concentration of all sample groups to be the same by using the lysate, adding a sample loading buffer solution containing beta mercaptoethanol, boiling water for 5min, performing denaturation treatment on the protein, performing vertical electrophoretic separation on the mixed solution, setting a constant pressure of 80V for a concentrated gel, setting a voltage of 120V for a separation gel, performing wet-turning on the protein on the gel to a PVDF membrane after electrophoresis is finished, and detecting the expression conditions of cell factors and autophagy-related proteins by using an antigen-antibody principle;

the test shows that the sulfated mannan obviously promotes the release of inflammatory factors in RAW264.7 cells, the sulfated mannan is time-dependent on the expression of IL-6 and IL-1 beta, the expression level of the sulfated mannan is obviously increased after the sulfated mannan is acted on the cells for 3 hours, and the expression level of TNF-alpha reaches the highest after the sulfated mannan is acted on the cells for 3 hours.

Meanwhile, after the sulfated mannan-glucan acts on the cells for 1 hour, the expressions of Beclin1 and LC3 II/I are obviously increased, and the statistical significance is achieved, which indicates that the sulfated mannan-glucan obviously activates autophagy of RAW264.7 cells and promotes immune response of the cells.

The sulfated mannan-glucan prepared by the invention can be used for improving the immunity of human bodies.

The sulfated mannan-glucan prepared by the invention can be prepared into a medicine or health-care product for improving immunity, and is in the form of tablets, capsules, oral liquid, soft capsules, powder, tincture, powder injection, water injection and small infusion.

Compared with the prior art, the invention has the following beneficial effects:

1. the sulfated mannan-glucan has good biocompatibility, can be taken for a long time, and has no obvious toxic or side effect within the range of medicinal dosage;

2. the invention has the advantages of small toxic and side effect, safety, effectiveness and the like, and has wide development and application prospect in the aspect of enhancing immunity;

3. the invention has the advantages of ingenious overall concept, simple preparation process and high preparation efficiency, and the prepared sulfated mannan-glucan has good application environment in the aspect of enhancing the immune function activity and extremely wide market prospect.

Description of the drawings:

FIG. 1 is a schematic diagram of a RAW material for promoting NO release of RAW264.7 cells by sulfated mannan-glucan according to the present invention.

FIG. 2 is a schematic diagram showing the action of sulfated mannan-glucan according to the present invention on RAW264.7 cytokines.

FIG. 3 is a schematic diagram of the principle of the sulfated mannan-glucan of the present invention promoting autophagy in RAW264.7 cells.

The specific implementation mode is as follows:

the present invention will be specifically explained below by way of examples. It is to be understood that these embodiments are illustrative and not restrictive. These examples are not intended to limit the scope of the present invention in any way.