Coprinus comatus mycelium polysaccharide as well as preparation method and application thereof
Through deep liquid fermentation and multi-step purification technology, high-purity α-D-grape pyranyl polysaccharides were extracted from the mycelium of chicken thigh mushrooms, solving the problem of insufficient research on polysaccharides and realizing the preparation of drugs that protect vascular endothelial cells and anti-atherosclerosis.
Patent Information
- Application Number
- CN202510639220.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-19
- Publication Date
- 2025-07-11
AI Technical Summary
In the prior art, there are few studies on isolating and purifying polysaccharides from the mycelium of the chicken leg mushroom, and have not been effectively applied to the preparation of drugs that protect vascular endothelial cells and anti-atherosclerosis.
The mycelium of chicken thorn beans was prepared by liquid deep fermentation. Through ultrasonic treatment, centrifugation, anion exchange chromatography and gel filtration chromatography, the mycelium of chicken thorn beans with α-D-grape pyran ring structure was isolated and purified. The specific steps include crushing, hot water treatment, ultrasonication, centrifugation, anhydrous ethanol precipitation, anion exchange column and gel filtration chromatography.
High-purity mycelial polysaccharide of chicken thigh mushrooms was obtained, which can promote the repair of vascular endothelial cells damaged by lipopolysaccharides and is used to prepare anti-artherosclerosis and other cardiovascular and cerebrovascular diseases.
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Abstract
Description
Technical Field
[0001] The present invention relates to a method for preparing polysaccharide from Coprinus comatus mycelium, which is used for preparing drugs for protecting vascular endothelial cells and treating cardiovascular and cerebrovascular diseases such as anti-atherosclerosis. It belongs to the biological field, and the international patent classification number is C08B 37 / 00. Background Art
[0002] Coprinus comatus ( Coprinus comatus ), also known as Coprinus comatus and Coprinus ovatus, is a saprophytic edible and medicinal mushroom growing in soil, belonging to Basidiomycota, Hymenomycetes, Agaricales, Coprinaceae, Coprinus. The fruiting body is medium to large, mostly solitary or clustered, and its appearance is like a chicken leg. It is a rare edible and medicinal mushroom rich in nutrients.
[0003] Coprinus comatus is rich in polysaccharides, which have functions such as antioxidant, immune regulation, hypoglycemic and liver protection. Isolating and purifying polysaccharides with uniform composition is of great significance for studying the functions, structure-activity relationships and modification of polysaccharides. At present, most of the studies on Coprinus comatus polysaccharides are extracted from the fruiting body. Compared with the traditional technology of artificially cultivating the fruiting body of Coprinus comatus on solid substrates, liquid submerged fermentation of Coprinus comatus mycelium not only occupies less land, has a shorter cycle and higher yield, but also can be produced on a large scale to ensure the stability of products. Therefore, obtaining mycelium through submerged fermentation and extracting polysaccharides from it is an effective means to deeply develop the active ingredients of Coprinus comatus. However, there are few studies on isolating, purifying polysaccharides from Coprinus comatus mycelium and analyzing their structures and functions. By solving the technical problems in the preparation of Coprinus comatus mycelium polysaccharides, the present invention obtains a uniform Coprinus comatus mycelium polysaccharide, which can be used for preparing drugs for protecting vascular endothelial cells and anti-atherosclerosis. Summary of the Invention
[0004] One of the purposes of the present invention is to provide a method for isolating and purifying pure Coprinus comatus polysaccharide CMP from Coprinus comatus mycelium.
[0005] A Coprinus comatus mycelium polysaccharide, the Coprinus comatus mycelium polysaccharide is a polysaccharide containing α-D-glucopyranose ring, and is characterized in that the mannuronic acid, mannose, glucose, galactose, arabinose and fucose in the polysaccharide containing α-D-glucopyranose ring are composed in a molar ratio of 0.30 - 0.45:0.02 - 0.03:98.5 - 99.5:0.05 - 0.15:0.05 - 0.15:0.12 - 0.22, and the molecular weight of the Coprinus comatus mycelium polysaccharide CMP is 2.56×10 5 Da.
[0006] As a preferred embodiment, the Coprinus comatus mycelium polysaccharide of the present invention is composed of mannuronic acid, mannose, glucose, galactose, arabinose and fucose in a molar ratio of 0.374:0.022:99.248:0.096:0.087:0.172, and the molecular weight of the Coprinus comatus mycelium polysaccharide CMP is 2.56×10 5 Da.
[0007] The method for obtaining the high-quality Coprinus comatus polysaccharide CMP includes the following steps: (1) After crushing the Coprinus comatus mycelium, add hot water at 80-100 °C, perform ultrasonic treatment, then water bath for 1 h, cool to room temperature and centrifuge. Take the supernatant, concentrate it, add anhydrous ethanol, and let it stand overnight at 2-8 °C. Centrifuge and discard the supernatant. Dissolve the precipitate in water, remove proteins by the Sevag method, take the supernatant and concentrate it, add anhydrous ethanol, let it stand overnight at 2-8 °C, centrifuge and discard the supernatant. Dry the precipitate to obtain the crude Coprinus comatus mycelium polysaccharide; the polysaccharide content accounts for more than 60% of the total solid weight.
[0008] (2) Dissolve the crude Coprinus comatus polysaccharide in water, load it onto an anion exchange column, elute with deionized water, collect fractions separately, and detect the polysaccharide content in each eluate tube by the sulfuric acid-anthrone method. Collect the main peak, concentrate it and precipitate with ethanol, place it at 2-8 °C, centrifuge to remove the supernatant, and freeze-dry it under vacuum; (3) Dissolve the dried sample in deionized water, load it onto a Sephacryl S-200 gel filtration chromatography column, elute with deionized water, collect fractions separately, detect by the sulfuric acid-anthrone method, collect the main peak, concentrate it and freeze-dry it to obtain the high-quality Coprinus comatus mycelium polysaccharide, and its polysaccharide content accounts for more than 95% of the total solid.
[0009] After crushing the Coprinus comatus mycelium, add hot water at 80-100 °C, and the liquid-solid volume-mass ratio is 5-20:1 (mL / g).
[0010] The ultrasonic power is 300-400 W, and the ultrasonic treatment time is 6-14 min.
[0011] The centrifugation speed in steps (1) and (2) is 3000-4000 rpm.
[0012] The anion exchange resin in step (2) 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.
[0013] The second object of the present invention is to provide an application of a uniform polysaccharide from Coprinus comatus mycelium in the preparation of drugs for protecting vascular endothelial cells, anti-atherosclerosis and other cardiovascular and cerebrovascular diseases. The polysaccharide from Coprinus comatus prepared by the above method can promote the repair of vascular endothelial cells damaged by lipopolysaccharide (LPS), so it can be applied to the preparation of drugs for protecting vascular endothelial cells, anti-atherosclerosis and other diseases related to the cardiovascular system. Description of the Drawings
[0014] Figure 1 is the ultraviolet-visible scanning spectrum of the fine product CMP of the polysaccharide from Coprinus comatus mycelium.
[0015] Figure 2 is the infrared spectrum of the fine product CMP of the polysaccharide from Coprinus comatus mycelium.
[0016] Figure 3 is the analysis chart of the monosaccharide composition of the fine product CMP of the polysaccharide from Coprinus comatus mycelium, Figure 4 is the activity of the fine product CMP of the polysaccharide from Coprinus comatus mycelium in promoting the repair of vascular endothelial cells damaged by LPS. Detailed Embodiments
[0017] The present invention will be further described below in conjunction with embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. 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 modifications or substitutions to the details and forms of the technical solutions of the present invention without departing from the spirit and scope of the present invention 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.
[0018] Example 1: Preparation method of the crude product of the polysaccharide from Coprinus comatus mycelium The Coprinus comatus mycelium is obtained by liquid submerged fermentation, with a yield of more than 25 g / L, dried, crushed and sieved. Take 100 g of the dry powder of Coprinus comatus mycelium, add hot water at 90 °C according to the liquid-solid ratio of 10:1 (mL / g), treat it with an ultrasonic power of 350 W for 10 min, and treat it 3 times in total. Then, carry out a water bath at 90 °C for 1 h, cool to room temperature and 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 concentration of ethanol is 80%, let it stand overnight at 4 °C, and centrifuge at 3500 rpm for 15 min. Dissolve the precipitate in deionized water, remove proteins by the Sevag method, add ethanol to the supernatant for precipitation, and vacuum freeze-dry the precipitate to obtain the crude product of the polysaccharide from Coprinus comatus mycelium of the present invention. The polysaccharide content in the crude product of the polysaccharide from Coprinus comatus mycelium accounts for 60%-70%.
[0019] Example 2: Preparation method of homogeneous Coprinus comatus polysaccharide CMP Take 500 mg of the crude Coprinus comatus mycelium polysaccharide, dissolve it thoroughly with deionized water, and load it onto a DEAE Sephadex A-25 ion exchange chromatography column that has been equilibrated. The column specifications are (2.6×30 cm), the equilibration solution is deionized water, and it is eluted with deionized water at a flow rate of 1.5 mL / min. It is fractionally collected at 3 mL / tube. The polysaccharide content in each tube of the eluate is detected by the sulfuric acid-anthrone method. Using the collection tube number as the abscissa and the absorbance value as the ordinate, a polysaccharide elution curve is plotted, and the same components are combined according to the elution curve. The Coprinus comatus polysaccharide water-washed fraction obtained by ion exchange column separation is subjected to Sephacryl S-200 molecular sieve chromatography and eluted with deionized water. The column specifications are (1.0×100 cm), fractionally collected, and tracked and detected by the sulfuric acid-anthrone method, and the same components are combined. Freeze-dry to obtain the high-quality Coprinus comatus polysaccharide (code: CMP). The polysaccharide content in the high-quality Coprinus comatus mycelium polysaccharide accounts for 99.28%.
[0020] In Figure 1, by performing a full-wavelength scan of CMP in the ultraviolet-visible light range (200 - 700 nm), it is proved that the high-quality Coprinus comatus mycelium polysaccharide has a high purity and basically no proteins, nucleic acids, and other impurities.
[0021] In Figure 2 : The relatively broad band that appears in the 3600 - 3200 cm -1 region is caused by the O-H stretching vibration within or between polysaccharide molecules. The absorption peak that appears in the 1400 - 1200 cm -1 region is attributed to the stretching vibration of the C-O-C group, indicating that CMP is a carbohydrate compound. In addition, the absorption peak at 856.67 cm -1 represents that the glycosidic bond type of CMP is an α-type glycosidic bond. The absorption peak at 929.19 cm -1 belongs to the vibration of α-D-glucopyranose; the absorption peaks at 1019.79 cm -1 , 1078.04 cm -1 and 1151.39 cm -1 confirm the existence of the pyranose ring. The absorption peaks at 1368.22 cm -1 , 1368.52 cm -1 are characteristic peaks of the C-H bending vibration of carbohydrates. The weak absorption peaks at 1637.45 cm -1 , 1637.72 cm -1 are the stretching vibrations of C=O.
[0022] Example 3: Effect of Coprinus comatus mycelium polysaccharide CMP on promoting the repair of LPS-damaged vascular endothelial cells for Example 2 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 culture them in an incubator at 37°C and 5% CO2. Take out the cells from the medium, observe the cell growth status under an inverted microscope, and change 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 continue to culture in an incubator at 37°C and 5% CO2. When the cell growth reaches 80% confluence, aspirate the original culture medium, wash 3 times with PBS, add 2 mL of 0.25% trypsin to digest for 5 min. After observing that the cells become round and detached 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, 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 the cells in the logarithmic growth phase for experiments.
[0023] 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 contain only the solvent, the positive group contains 100 μg / mL of VE, and the drug administration groups contain 1.25, 2.5, and 5 μg / mL of the fine product of Coprinus comatus mycelium polysaccharide CMP respectively. Another cell-free blank group is set up, with 3 replicates in each group. After culturing for 24 h, use a CCK-8 kit to measure the cell viability.
[0024] In Figure 4 : Coprinus comatus mycelium polysaccharide CMP can improve the activity of LPS-damaged vascular endothelial cells at doses of 2.5 and 5 μg / mL, indicating that Coprinus comatus mycelium polysaccharide has a protective effect on vascular endothelial cells.
[0025] Example 4: Physicochemical property determination 1. Polysaccharide content determination Adopt the sulfuric acid-anthrone method. At 620 nm, use spectrophotometry to determine the total polysaccharide content. Calculated by glucose (C6H 12 O6), the content of the crude product of Coprinus comatus mycelium polysaccharide is 65.82%, and the content of the fine polysaccharide (CMP) of Coprinus comatus mycelium is 99.28%.
[0026] 2. Ultraviolet spectrum analysis Dissolve the sample in distilled water and perform a full-wavelength ultraviolet scan from 200 to 400 nm. AsFigure 1 As shown, CMP has no absorption at 260 and 280 nm, indicating that it does not contain proteins and nucleic acids.
[0027] 3. Monosaccharide composition analysis Weigh an appropriate amount of Coprinus comatus mycelium polysaccharide CMP dry powder into a hydrolysis tube, add 1 mL of 72% sulfuric acid, incubate at 30 °C for 1 h, dilute to 10 mL, fill with nitrogen, hydrolyze in a 110 °C oven for 2 h, take out and cool to room temperature, take 0.5 mL into a 4 mL centrifuge tube, adjust pH to neutral, dilute 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, incubate at 70 °C for 60 min, take out and cool to room temperature, add 0.2 mL of 0.3 mol / L HCl, dilute to 2 mL with water, add 1.5 mL of chloroform, shake well and let stand for stratification, discard the lower chloroform, filter the water layer through a 0.45 μm filter membrane, and use a 1200 liquid chromatograph for determination. The chromatographic column model is C18 (4.6 mm×250 mm×5 μm), and the mobile phase composition is: A Phase A was 15% acetonitrile (acetonitrile diluted with 0.05 mol / L KH2PO4, pH=6.8); phase B was 40% acetonitrile (acetonitrile diluted with 0.05 mol / LKH2PO4, pH=6.8); elution was performed at a flow rate of 1.0 mL / min under the conditions of column temperature of 25 ℃, injection volume of 20 μL, and detection wavelength of 254 nm.
[0028] Figure 3 In the present invention, the mycelium polysaccharide CMP of Coprinus comatus is specifically composed of mannuronic acid, mannose, glucose, galactose, arabinose and fucose in a molar ratio of 0.374:0.022:99.248:0.096:0.087:0.172.
[0029] 4. Molecular Weight Determination The molecular weight of the polysaccharide from the mycelium of *Coprinus comatus* was determined by gel permeation chromatography (GPC) using a GPC instrument equipped with a PL aquagel-OH Mixed-H column (7.5×300 mm, 8 μm) and a differential detector. Detection conditions: injection volume 50 μL, column temperature 45 °C, gradient elution with 0.1 mol / L sodium nitrate as the mobile phase at a flow rate of 1.0 mL / min. 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 were measured, and a standard curve was prepared based on standard substances with different relative molecular weights, so as to calculate the relative molecular weight from the peak elution time. The results showed that the molecular weight of the polysaccharide from the mycelium of *Coprinus comatus* was approximately 2.56×10 5 Da.
Claims
1. A Coprinus comatus mycelium polysaccharide, the Coprinus comatus mycelium polysaccharide being a polysaccharide containing an α-D-glucopyranose ring, characterized in that, The polysaccharide containing α-D-pyranose is composed of mannuronic acid, mannose, glucose, galactose, arabinose and fucose in a molar ratio of 0.30 - 0.45:0.02 - 0.03:98.5 - 99.5:0.05 - 0.15:0.05 - 0.15:0.12 - 0.
22. The molecular weight of the polysaccharide CMP from Coprinus comatus mycelium is 2.56×10 5 Da.
2. The polysaccharide of Coprinus comatus mycelium according to claim 1, characterized in that, The polysaccharide containing α-D-pyranose is composed of mannuronic acid, mannose, glucose, galactose, arabinose and fucose in a molar ratio of 0.374:0.022:99.248:0.096:0.087:0.
172. The molecular weight of the polysaccharide CMP from Coprinus comatus mycelium is 2.56×10 5 Da.
3. The preparation method of the Coprinus comatus mycelium polysaccharide according to claim 1 or 2, characterized in that, It includes the following steps: (1) After pulverizing Coprinus comatus mycelium, add hot water at 80 - 100 °C, perform ultrasonic treatment and then water bath for 1 h, cool to room temperature and centrifuge, take the supernatant, concentrate it, add absolute ethanol, let it stand overnight at 2 - 8 °C, centrifuge, discard the supernatant, dissolve the precipitate in water, remove protein by Sevag method, take the supernatant and concentrate it, add absolute ethanol, let it stand overnight in a refrigerator at 2 - 8 °C, centrifuge, discard the supernatant, and dry the precipitate to obtain the crude Coprinus comatus mycelium polysaccharide; (2) Dissolve the crude Coprinus comatus mycelium polysaccharide in water, load it onto an anion exchange column, elute with deionized water, collect fractions, detect the polysaccharide content in each tube of the eluate by the sulfuric acid - anthrone method, collect the main peak, concentrate it and precipitate with ethanol, place it at 2 - 8 °C, centrifuge to remove the supernatant, and vacuum freeze - dry; (3) Dissolve the dried sample in deionized water, load it onto a Sephacryl S - 200 gel filtration chromatography column, elute with deionized water, collect fractions, detect by the sulfuric acid - anthrone method, collect the main peak, concentrate it and then freeze - dry to obtain the fine Coprinus comatus mycelium polysaccharide CMP.
4. The preparation method of the polysaccharide of Coprinus comatus mycelium according to claim 3, characterized in that, In step (1), the liquid - solid volume - mass ratio is 5 - 20:1 (mL / g).
5. The preparation method of the polysaccharide of Coprinus comatus mycelium according to claim 3, wherein In step (1), the ultrasonic power is 300 - 400 W and the ultrasonic treatment time is 6 - 14 min.
6. The preparation method of the Coprinus comatus mycelium polysaccharide according to claim 3, wherein In steps (1) and (2), the centrifugation speed is 3000 - 4000 rpm.
7. The preparation method of the collective mycelium polysaccharide according to claim 3, characterized in that, The anion exchange resin described in step (2) 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.
8. Use of the Coprinus comatus mycelium polysaccharide according to claim 1 or 2 in the preparation of a drug for protecting vascular endothelial cells.
9. Use of the Coprinus comatus mycelium polysaccharide according to claim 1 or 2 in the preparation of a drug for treating cardiovascular and cerebrovascular system - related diseases such as anti - atherosclerosis.