Bamboo fungus mycelium selenium polysaccharide as well as preparation and application thereof
Se-DMP, a selenium polysaccharide of bamboo fungus mycelium, was prepared through deep fermentation and fine separation and purification, which solved the application problems of bamboo fungus mycelium, in food and medicine, and achieved efficient preparation and significant protection of vascular endothelial cells.
Patent Information
- Application Number
- CN202510640369.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-19
- Publication Date
- 2025-07-22
AI Technical Summary
The research and preparation of the selenium polysaccharides of bamboo fungus mycelium in the prior art is not yet mature, and there is a lack of effective methods to achieve its application in food and medicine.
Through deep fermentation of selenium-rich bamboo fungus mycelium, the uniform selenium polysaccharide Se-DMP of bamboo fungus mycelium was prepared by using ultrasonic treatment, hot water extraction, ethanol precipitation, centrifugation, ion exchange chromatography and gel filtration chromatography to prepare a uniform selenium polysaccharide Se-DMP for food and pharmaceutical raw materials.
The prepared bamboo fungus mycelium selenium polysaccharide Se-DMP has high purity and high polysaccharide content, which can promote the repair of vascular endothelial cells damaged by lipopolysaccharide. It is used in anti-artherosclerosis and other cardiovascular and cerebrovascular diseases drugs, and has a significant role in protecting vascular endothelial cells.
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Abstract
Description
Technical Field
[0001] The present invention relates to a method for preparing dictyophora indusiata mycelium selenium polysaccharide, which is used as food raw material, food additive and drug raw material, and belongs to the field of biotechnology. Background Art
[0002] Dictyophora indusiata Dictyophora indusiata (Vent.ex Pers) Fisch)), belonging to Phallales, Phallaceae, Dictyophora, is a rare edible and medicinal fungus parasitic on the roots of dead bamboo. There are 4 edible species, namely Dictyophora indusiata, Dictyophora duplicata, Dictyophora echinovolvata and Dictyophora rubrovalvata. Dictyophora indusiata is delicious, nutritious and has various pharmacological activities, such as antioxidant, anti-inflammatory, immunomodulatory effects, and polysaccharide is its main active ingredient.
[0003] Obtaining the mycelium of edible and medicinal fungi through submerged fermentation and extracting the active ingredients therein is an effective means for the in-depth development of edible and medicinal fungi, and can be industrially produced to achieve industrialization. By adding trace elements to the culture medium, the biosynthesis pathway of fungal polysaccharides can be changed to form new polysaccharides, improve biological activity or increase new uses. At present, the research on dictyophora indusiata mycelium selenium polysaccharide is still blank. The present invention solves the technical problems in the preparation of dictyophora indusiata mycelium selenium polysaccharide by culturing selenium-rich dictyophora indusiata mycelium, and obtains a uniform dictyophora indusiata mycelium selenium polysaccharide, which can be used as food raw material, food additive and drug raw material. Summary of the Invention
[0004] One object of the present invention is to provide a method for separating and purifying a pure product Se-DMP of dictyophora indusiata mycelium selenium polysaccharide from selenium-rich dictyophora indusiata mycelium.
[0005] The dictyophora indusiata mycelium selenium polysaccharide described in the present invention is a polysaccharide containing pyranose glycosidic bond, and is composed of mannose, glucosamine, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose and fucose in a molar ratio of 0.16-0.36:0.014-0.034:0.013-0.033:0.04-0.14:0.04-0.06:0.004-0.024:97.85-99.85:0.32-0.52:0.05-0.15:0.04-0.06:0.05-0.15. The molecular weight of the dictyophora indusiata mycelium selenium polysaccharide Se-DMP is 1.15×10 7 Da.
[0006] As a preferred embodiment, the selenium polysaccharide of Dictyophora indusiata mycelium is a polysaccharide containing pyranoid glycosidic bonds, specifically composed of mannose, glucosamine, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose in a molar ratio of 0.260:0.024:0.023:0.089:0.050:0.014:98.853:0.420:0.108:0.054:0.104. The molecular weight of the selenium polysaccharide Se-DMP of Dictyophora indusiata mycelium is 1.15×10 7 Da.
[0007] The method for obtaining the high-quality selenium polysaccharide Se-DMP of Dictyophora indusiata mycelium is as follows: adding the dried product of selenium-rich Dictyophora indusiata mycelium (organic selenium content 200 - 300 m / kg) to deionized water, performing hot water extraction after ultrasonic treatment, ethanol precipitation, standing overnight at 4 °C, centrifuging to remove the supernatant, and drying to obtain the water extract of selenium-rich Dictyophora indusiata mycelium. Dissolving the water extract of selenium-rich Dictyophora indusiata mycelium in deionized water, adding macroporous resin to remove pigments and proteins, filtering the decolorized solution, adding ethanol for precipitation, washing the precipitate 3 times with ethanol, and vacuum drying the precipitate to obtain the crude selenium polysaccharide of Dictyophora indusiata mycelium of the present invention, the polysaccharide content of which accounts for more than 60% of the total solid weight.
[0008] Dissolving the crude selenium polysaccharide of Dictyophora indusiata mycelium in deionized water, purifying it by ion exchange chromatography with deionized water as the eluent, tracking and detecting by the sulfuric acid - anthrone colorimetric method, collecting the main peak, then performing gel filtration chromatography with deionized water as the eluent, tracking and detecting by the sulfuric acid - anthrone colorimetric method, collecting the main peak, and vacuum drying to obtain the high-quality selenium polysaccharide (Se-DMP) of Dictyophora indusiata mycelium of the present invention, the polysaccharide content of which accounts for more than 95% of the total solid.
[0009] In the said method, the hot water extraction is carried out in a water bath, and the water bath temperature is 50 - 70 °C.
[0010] In the said method, the hot water extraction time is 1 - 6 hours.
[0011] In the said method, the liquid-solid ratio for extraction is 5 - 30 (mL / g).
[0012] In the said method, the macroporous resin is an anion exchange resin.
[0013] In the said method, the decolorization time of the macroporous resin is 0.5 - 4 hours.
[0014] In the above method, the ion exchange resin is diethylaminoethyl cellulose (DEAE-52), or diethylaminoethyl dextran gel (DEAE Sephadex A-25 or DEAE Sephadex A-50), or diethylaminoethyl agarose gel (DEAE Sepharose).
[0015] The second object of the present invention is to provide an application of a uniform selenium polysaccharide from Dictyophora indusiata mycelium in the preparation of drugs for cardiovascular and cerebrovascular diseases including vascular endothelial cells, anti-atherosclerosis, etc. The selenium polysaccharide from Dictyophora indusiata prepared by the above method can promote the repair of lipopolysaccharide (LPS)-damaged vascular endothelial cells. Therefore, it can be applied to the preparation of drugs for protecting vascular endothelial cells, anti-atherosclerosis and other cardiovascular system-related diseases, or inflammation-related diseases. Description of the Drawings
[0016] Figure 1 It is the ultraviolet-visible scanning spectrum of the high-quality Se-DMP of selenium polysaccharide from Dictyophora indusiata mycelium.
[0017] Figure 2 It is the infrared spectrum of the high-quality Se-DMP of selenium polysaccharide from Dictyophora indusiata mycelium.
[0018] Figure 3 It is the analysis chart of the monosaccharide composition of the high-quality Se-DMP of selenium polysaccharide from Dictyophora indusiata mycelium. A is the monosaccharide standard, and B is Se-DMP.
[0019] Figure 4 It is the activity of Se-DMP in promoting the repair of LPS-damaged vascular endothelial cells. Detailed Embodiments
[0020] The present invention will be further described below in conjunction with embodiments. The advantages and features of the present invention will become clearer with the description. However, these examples are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that without departing from the spirit and scope of the present invention, modifications or substitutions to the details and forms of the technical solutions of the present invention are made, but these modifications and substitutions all fall within the protection scope of the present invention. Chemical reagents, chromatography columns, etc. used in the specification and embodiments are all operated under conventional experimental conditions or according to the instructions provided by the suppliers if not otherwise specified.
[0021] Example 1: Preparation method of crude selenium polysaccharide from Dictyophora indusiata mycelium The selenium-enriched Dictyophora indusiata mycelium is obtained by liquid fermentation of our laboratory using shake flasks or fermenters. The mycelium yield is above 1.4 g / 100 mL, and the organic selenium content is greater than 150 mg / kg of the mycelium. It is dried and pulverized. Weigh 100 g of the dry powder, add hot water at 60°C according to a liquid-solid ratio of 10:1 (mL / g), treat it with an ultrasonic power of 50 W for 4 min, and perform the treatment 3 times in total. After cooling to room temperature, centrifuge at 3500 rpm for 15 min, take the supernatant, concentrate it to 1 / 10 of the total volume, slowly add absolute ethanol to the concentrated solution until the final ethanol concentration is 80%, let it stand overnight in a refrigerator at 4°C, and centrifuge at 3500 rpm for 15 min. Discard the supernatant to obtain the water extract of the selenium-enriched Dictyophora indusiata mycelium. Dissolve the water extract in deionized water, add D301G macroporous resin to remove pigments and proteins, filter the decolorized solution, add ethanol for precipitation, and vacuum dry the precipitate to obtain the crude polysaccharide of the Dictyophora indusiata mycelium selenium polysaccharide of the present invention.
[0022] Example 2: Preparation method of homogeneous Dictyophora indusiata mycelium selenium polysaccharide Se-DMP Take 500 mg of the crude polysaccharide of the selenium-enriched Dictyophora indusiata mycelium (with an organic selenium content of 200 - 300 mg / kg) obtained in Example 1, dissolve it thoroughly with deionized water, and load it onto a pre-equilibrated DEAE Sephadex A-25 ion exchange chromatography column with a specification of (2.6×30 cm). The equilibration solution is deionized water. Elute with deionized water at a flow rate of 1.5 mL / min, collect in fractions of 3 min / tube, detect the polysaccharide content in each tube of the eluate by the sulfuric acid-anthrone method, plot the polysaccharide elution curve with the collection tube number as the abscissa and the absorbance value as the ordinate, and combine the same components according to the elution curve. The water-washed fraction of the Dictyophora indusiata mycelium selenium polysaccharide separated by the ion exchange column is loaded onto a Sephacryl S-200 molecular sieve chromatography column and eluted with deionized water. The column specification is (1.0×100 cm), collect in fractions, track and detect by the sulfuric acid-anthrone method, and combine the same components. Freeze-dry to obtain the fine product of the Dictyophora indusiata mycelium selenium polysaccharide (code: Se-DMP). The Dictyophora indusiata mycelium selenium polysaccharide is a polysaccharide containing pyranose glycosidic bonds, and is composed of mannose, glucosamine, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose in a molar ratio of 0.260:0.024:0.023:0.089:0.050:0.014:98.853:0.420:0.108:0.054:0.104. The molecular weight of the Dictyophora indusiata mycelium selenium polysaccharide Se-DMP is 1.15×10 7 Da.
[0023] In Figure 1In this study, by performing a full-wavelength scan of Se-DMP in the ultraviolet-visible light range (200 - 700 nm), it was demonstrated that the purified product of selenium polysaccharide from Dictyophora indusiata mycelium had a high purity and was basically free of proteins, nucleic acids, and other impurities.
[0024] In Figure 2 : Characteristic absorption peaks of polysaccharides appeared in the range of 3000 - 3500 cm -1 . A characteristic peak appeared at 3310.21 cm -1 , which was attributed to the stretching vibration of the O-H bond. Characteristic absorption peaks of polysaccharides also appeared in the range of 2750 - 3000 cm -1 . The absorption peak at 2943.81 cm -1 was caused by the stretching vibration of the C-H bond. A deformation absorption peak of =CH2 appeared at 1397.17 cm -1 for Se-DMP-1. Three characteristic peaks appeared in the range of 1000 - 1200 cm -1 , indicating the presence of pyranoid glycosidic bonds. In addition to the characteristic absorption peaks of polysaccharides, vibration absorption peaks representing O-Se-C and Se=O appeared at 1337.39 cm -1 and 561.66 cm -1 for Se-DMP. The appearance of new characteristic peaks indicated that Se atoms had successfully combined with polysaccharides to form selenium polysaccharides.
[0025] Example 3: Physicochemical Property Determination 1. Determination of Polysaccharide Content Using the sulfuric acid-anthrone method, the total polysaccharide content was determined by spectrophotometry at 620 nm. The content of the crude product of selenium polysaccharide from Dictyophora indusiata mycelium was 62.88% calculated as glucose (C6H 12 O6), and the content of selenium polysaccharide (Se-DMP) from Dictyophora indusiata mycelium was 99.12%.
[0026] 2. Ultraviolet Spectrum Analysis The sample was dissolved in distilled water and subjected to a full-wavelength scan in the ultraviolet range of 200 - 400 nm. As Figure 1 shown, Se-DMP had no absorption at 260 and 280 nm, indicating that it did not contain proteins and nucleic acids.
[0027] 3. Monosaccharide Composition Analysis Weigh an appropriate amount of Se-DMP dry powder of Dictyophora indusiata mycelium polysaccharide into a hydrolysis tube, add 1 mL of 72% sulfuric acid, hydrolyze in a water bath at 30 °C for 1 h, make up the volume to 10 mL, fill with nitrogen, hydrolyze in an oven at 110 °C for 2 h, take it out and cool to room temperature. Take 0.5 mL and put it into a 4 mL centrifuge tube, adjust the pH to neutral, and make up the volume to 1 mL; then add 0.2 mL of 0.3 mol / L NaOH solution and 0.4 mL of PMP methanol solution, fill with nitrogen, hydrolyze in a water bath at 70 °C for 60 min, take it out and cool to room temperature, add 0.2 mL of 0.3 mol / L HCl, add water to make up the volume to 2 mL, add 1.5 mL of chloroform, shake well and let it stand for layering, discard the lower chloroform layer, filter the aqueous layer through a 0.45 μm filter membrane, and determine it by an Agilent 1200 liquid chromatograph. The chromatographic column model is C18 (4.6 mm × 250 mm × 5 μm), and the mobile phase composition: Phase A is 15% acetonitrile (acetonitrile diluted with 0.05 mol / L KH2PO4, pH = 6.8); Phase B is 40% acetonitrile (acetonitrile diluted with 0.05 mol / L KH2PO4, pH = 6.8); elute at a flow rate of 1.0 mL / min under the conditions of column temperature of 25 °C, injection volume of 20 μL, and detection wavelength of 254 nm.
[0028] Figure 3 Among them, the Se-DMP of Dictyophora indusiata mycelium polysaccharide is specifically composed of mannose, glucosamine, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose in a molar ratio of 0.260:0.024:0.023:0.089:0.050:0.014:98.853:0.420:0.108:0.054:0.104.
[0029] 4. Molecular weight determination The gel permeation chromatography (GPC) method was used. A gel permeation chromatograph equipped with a chromatographic column PL aquagel-OH Mixed-H (7.5×300mm, 8 μm) and a differential detector was used to determine the molecular weight of the Se-DMP of Dictyophora indusiata mycelium polysaccharide. Detection conditions: injection volume 50 μL, column temperature 45 °C, gradient elution with 0.1 mol / L sodium nitrate as the mobile phase at 1.0 mL / min. Measure the peak elution times of PEG-PEO series standard products with a number average molecular weight of 585 - 1140000 g / mol and a weight average molecular weight of 660 - 1470000 g / mol at 0.2 - 0.8 mg / mL, prepare a standard curve according to standard substances with different relative molecular weights, and thus calculate the relative molecular weight from the peak elution time. The results show that the molecular weight of the Se-DMP of Dictyophora indusiata mycelium polysaccharide is about 1.15×10 7Da。
[0030] Example 4: Regarding Example 2, the effect of polysaccharide DMP from Dictyophora indusiata mycelium on promoting the repair of LPS-damaged vascular endothelial cells Use a 25 cm 2 culture flask. Inoculate SVEC4-10 cells into RPMI 1640 culture medium (complete medium) containing 10% fetal bovine serum and 1% penicillin / streptomycin, and place them in an incubator at 37°C and 5% CO2 for culture. Take out the cells from the medium and observe the cell growth status under an inverted microscope. Replace the complete culture medium in a timely manner according to the cell growth status and the degree of yellowing of the culture medium color. When changing the medium, aspirate the original culture medium in the culture flask, wash the surface 3 times with PBS, add fresh complete culture medium, and place it in an incubator at 37°C and 5% CO2 for continued culture. When the cell growth reaches a coverage rate of 80%, aspirate the original culture medium, wash 3 times with PBS, add 2 mL of 0.25% trypsin to digest for 5 min. After observing the cells becoming round and detaching under the microscope, quickly transfer them to a laminar flow hood and add 6 mL of complete culture medium to terminate the digestion. Gently pipette the cells to make them completely detached and then aspirate them into a centrifuge tube, centrifuge (1000 r / min, 5 min), discard the supernatant, add fresh complete culture medium to resuspend the cells, passage them at a ratio of 1:5, and take cells in the logarithmic growth phase for experiments.
[0031] When the cells grow to 70%, aspirate the supernatant, add complete medium containing 1.5 μg / mL LPS, and set up a control group (add complete medium without LPS). After culturing for 24 h, divide the damaged cells into a model group, a positive group, and a drug administration group. Aspirate the supernatant from all cells and add different complete culture media. The control group and the model group only contain the solvent, the positive group contains 100 μg / mL of VE, and the drug administration group contains 1.25, 2.5, and 5 μg / mL of the fine product of polysaccharide Se-DMP from Dictyophora indusiata mycelium respectively. In addition, set up a cell-free blank group, with 3 replicate wells in each group. After culturing for 24 h, use a CCK-8 kit to measure the cell viability.
[0032] In Figure 4 : Polysaccharide Se-DMP from Dictyophora indusiata mycelium can significantly improve the activity of LPS-damaged vascular endothelial cells at doses of 1.25, 2.5, and 5 μg / mL, indicating that polysaccharide from Dictyophora indusiata mycelium has the effect of protecting vascular endothelial cells.
Claims
1. Dictyophora indusiata mycelium selenium polysaccharide, characterized in that, The Dictyophora indusiata mycelium selenium polysaccharide is a polysaccharide containing pyranoid glycosidic bonds, characterized in that the polysaccharide containing pyranoid glycosidic bonds is composed of mannose, glucosamine, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose and fucose in a molar ratio of 0.16 - 0.36:0.014 - 0.034:0.013 - 0.033:0.04 - 0.14:0.04 - 0.06:0.004 - 0.024:97.85 - 99.85:0.32 - 0.52:0.05 - 0.15:0.04 - 0.06:0.05 - 0.15, and the molecular weight of the Dictyophora indusiata mycelium selenium polysaccharide Se-DMP is 1.15×10 7 Da.
2. The polysaccharide of dictyophora indusiata mycelium containing selenium according to claim 1, characterized in that, The polysaccharide is a polysaccharide containing pyranoid glycosidic bonds and is composed of mannose, glucosamine, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose in a molar ratio of 0.260:0.024:0.023:0.089:0.050:0.014:98.853:0.420:0.108:0.054:0.
104. The molecular weight of the selenium polysaccharide Se-DMP of Dictyophora indusiata mycelium is 1.15×10 7 Da.
3. The preparation method of dictyophora indusiata mycelium selenium polysaccharide according to any one of claims 1-2, characterized in that, It includes the following steps: Step (1): After the selenium-enriched Dictyophora indusiata mycelium is crushed, hot water at 50 - 70 °C is added according to the liquid-solid ratio of 5 - 20:1 (mL / g), ultrasonic treatment is carried out twice, then water bath extraction is carried out for 2 h, cooled to room temperature and centrifuged, the supernatant is taken, after concentration, anhydrous ethanol is added, and it is left standing overnight in a refrigerator at 2 - 8 °C, centrifuged, and the supernatant is discarded to obtain the precipitate of the water extract of selenium-enriched Dictyophora indusiata mycelium, and dried to obtain the water extract of selenium-enriched Dictyophora indusiata mycelium; Step (2): The dried water extract of selenium-enriched Dictyophora indusiata mycelium is dissolved in water, D301G macroporous resin is added, shaken and centrifuged at room temperature, the supernatant is taken and concentrated, anhydrous ethanol is added, and it is left standing overnight in a refrigerator at 2 - 8 °C, centrifuged, the supernatant is discarded, and the precipitate is dried to obtain the crude product of selenium polysaccharide of Dictyophora indusiata mycelium; Step (3): The crude product of selenium polysaccharide of Dictyophora indusiata mycelium is dissolved in water, loaded onto an anion exchange column, eluted with deionized water, fractionally collected, the polysaccharide content in each tube of the eluate is detected by the sulfuric acid-anthrone method, the main peak is collected, concentrated and then precipitated with ethanol, placed at 2 - 8 °C, centrifuged to remove the supernatant, and vacuum dried; Step (4): The dried sample is dissolved in deionized water, loaded onto a Sephacryl S-200 gel filtration chromatography column, eluted with deionized water, fractionally collected, detected by the sulfuric acid-anthrone method, the main peak is collected, concentrated and then freeze-dried to obtain the fine product of selenium polysaccharide of Dictyophora indusiata mycelium Se-DMP.
4. The preparation method of selenium polysaccharide of Dictyophora indusiata mycelium according to claim 3, characterized in that In step (1), the volume of anhydrous ethanol is 1 - 6 times the solid-liquid volume described in step (1); In step (2), the volume of anhydrous ethanol is 1 - 6 times the volume of the concentrated solution in step (2); In step (3), the volume of anhydrous ethanol is 3 - 5 times the volume of the concentrated solution in step (3); The centrifugation speed in steps (1), (2), and (3) is 3000 - 4000 rpm.
5. The preparation method of dictyophora indusiata mycelium selenium polysaccharide according to claim 3, characterized in that, The anion exchange resin described in step (3) is diethylaminoethyl cellulose DEAE-52, or diethylaminoethyl dextran gel, specifically DEAE Sephadex A-25, or DEAE Sephadex A-50, or diethylaminoethyl agarose gel DEAE Sepharose.
6. The application of the selenium polysaccharide of Dictyophora indusiata mycelium according to any one of claims 1 - 2 in the preparation of a pharmaceutical preparation including vascular endothelial cells, specifically the application as the raw material drug for the preparation of a pharmaceutical preparation including atherosclerosis, or a disease related to inflammation.