Bamboo fungus mycelium polysaccharide as well as preparation method and application thereof

Abstract

The invention relates to a preparation method of dictyophora indusiata mycelium polysaccharide. Belongs to the field of pharmacy, and the international patent classification is C08B 37 / 00. The dictyophora indusiata mycelium polysaccharide fine product is prepared by the following steps: crushing dictyophora indusiata mycelium obtained by deep liquid fermentation, sieving, carrying out ultrasonic-assisted extraction, carrying out ethanol precipitation, staying overnight at 4 DEG C, centrifuging, discarding supernatant, dissolving the precipitate in water, and deproteinizing by a Sevag method. Concentrating the supernatant, precipitating with ethanol, centrifuging to remove the supernatant, and drying to obtain a dictyophora indusiata mycelium polysaccharide crude product; and decoloring the dictyophora indusiata mycelium polysaccharide crude product by H2O2, and purifying by ion exchange chromatography and gel filtration chromatography to obtain the dictyophora indusiata mycelium polysaccharide refined product DMP. The obtained dictyophora indusiata mycelium polysaccharide is applied to preparation of drugs for protecting vascular endothelial cells and resisting cardiovascular and cerebrovascular system related diseases such as atherosclerosis.

Description

Technical Field

[0001] The present invention relates to a method for preparing polysaccharide from Dictyophora indusiata 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 / 60. Background Art

[0002] Dictyophora indusiata Dictyophora indusiata (Vent.ex Pers) Fisch), belonging to Phallales, Phallaceae, Dictyophora, is a rare edible and medicinal fungus parasitizing on the roots of dead bamboos. 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 and immunomodulatory effects.

[0003] Polysaccharide from Dictyophora indusiata is its main medicinal ingredient, which has effects such as antioxidant, anti-inflammatory, immunomodulatory and hypoglycemic. Separating and purifying polysaccharide with uniform composition is of great significance for studying the function, structure-activity relationship and modification of polysaccharide. At present, most of the studies on polysaccharide from Dictyophora indusiata are obtained by extracting from the fruiting body. Compared with the traditional solid matrix artificial cultivation technology of Dictyophora indusiata fruiting body, liquid submerged fermentation of Dictyophora indusiata mycelium not only occupies less land, has a short cycle and high yield, but also can be produced on a large scale to ensure the stability of the product. Moreover, by adding culture medium, trace elements and adjusting fermentation parameters, the biosynthesis pathway of polysaccharide from Dictyophora indusiata mycelium can be changed to obtain new polysaccharides and develop more new uses of polysaccharides. However, there are few studies on separating and purifying polysaccharide from Dictyophora indusiata mycelium and analyzing its structure and function. The present invention solves the technical problems in the preparation of polysaccharide from Dictyophora indusiata mycelium and obtains a uniform polysaccharide from Dictyophora indusiata mycelium, which can be used for preparing drugs for protecting vascular endothelial cells and anti-atherosclerosis. Summary of the Invention

[0004] One object of the present invention is to provide a method for separating and purifying pure Dictyophora polysaccharide (DMP) from Dictyophora indusiata mycelium.

[0005] A polysaccharide from Dictyophora indusiata mycelium. The polysaccharide from Dictyophora indusiata mycelium is a polysaccharide containing pyranose glycosidic bonds. It is characterized in that the polysaccharide containing pyranose glycosidic bonds is composed of mannose, glucosamine, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose in a molar ratio of 0.20 - 0.40:0.02 - 0.04:0.01 - 0.03:0.04 - 0.06:0.05 - 0.07:0.005 - 0.015:98.50 - 99.50:0.30 - 0.50:0.03 - 0.05:0.005 - 0.015:0.07 - 0.09. The molecular weight of the polysaccharide DMP from Dictyophora indusiata mycelium is 1.26×10 7 Da.

[0006] As a preferred embodiment, the polysaccharide from Dictyophora indusiata mycelium of the present invention is composed of mannose, glucosamine, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose in a molar ratio of 0.309:0.030:0.018:0.051:0.063:0.012:98.984:0.404:0.038:0.013:0.079. The molecular weight of the polysaccharide DMP from Dictyophora indusiata mycelium is 1.26×10 7 Da.

[0007] The method for obtaining the refined polysaccharide DMP from Dictyophora indusiata includes the following steps: (1) After crushing the Dictyophora indusiata mycelium, add hot water at 50 - 70°C, perform ultrasonic treatment, then centrifuge at room temperature. Take the supernatant, concentrate it, add absolute ethanol, and let it stand overnight in a refrigerator at 2 - 8°C. Centrifuge, discard the supernatant, dissolve the precipitate in water, remove proteins by the 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 polysaccharide from Dictyophora indusiata mycelium. The polysaccharide content accounts for more than 60% of the total solid weight.

[0008] (2) Dissolve the crude polysaccharide from Dictyophora indusiata in water, add 10 - 30% H2O2 and decolorize at 40 - 60°C for 40 - 60 min. 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, 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 refined polysaccharide from Dictyophora indusiata mycelium. The polysaccharide content accounts for more than 95% of the total solid.

[0009] The Dictyophora indusiata mycelium is crushed and added to hot water at 50 - 70 °C, with a liquid-solid volume-mass ratio of 5 - 20:1 (mL / g).

[0010] The ultrasonic power is 20 - 150 W, and the ultrasonic treatment is carried out for 2 - 20 min.

[0011] The centrifugation speed described in steps (1) and (2) is 3000 - 4000 rpm.

[0012] 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.

[0013] The second object of the present invention is to provide an application of a homogeneous Dictyophora indusiata mycelium polysaccharide in the preparation of drugs for protecting vascular endothelial cells, anti - atherosclerosis and other cardiovascular and cerebrovascular diseases. The Dictyophora indusiata polysaccharide prepared by the above method can promote the repair of vascular endothelial cells damaged by lipopolysaccharide (LPS), and thus 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 refined Dictyophora indusiata mycelium polysaccharide DMP.

[0015] Figure 2 is the infrared spectrum of the refined Dictyophora indusiata mycelium polysaccharide DMP.

[0016] Figure 3 is the monosaccharide composition analysis chart of the refined Dictyophora indusiata mycelium polysaccharide DMP, Figure 4 is the activity of the refined Dictyophora indusiata mycelium polysaccharide DMP in promoting the repair of vascular endothelial cells damaged by LPS. Detailed Embodiments

[0017] The following examples are used to further illustrate the present invention, and the advantages and characteristics 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 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. The chemical reagents, chromatography columns, etc. used in the description and examples 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 crude Dictyophora indusiata polysaccharide The Dictyophora indusiata mycelium is obtained by liquid submerged fermentation, with a yield of over 14 g / L. It is dried, pulverized, and sieved. Take 100 g of the dry powder of Dictyophora indusiata mycelium, add hot water at 60°C according to a liquid-solid ratio of 10:1 (mL / g), and treat it with ultrasonic power of 50 W for 4 min, for a total of 3 times. 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. Dissolve the precipitate in deionized water, remove proteins by the Sevag method, add ethanol to precipitate the supernatant, and vacuum freeze-dry the precipitate to obtain the crude polysaccharide of Dictyophora indusiata of the present invention. The polysaccharide content in the crude polysaccharide of Dictyophora indusiata accounts for 60%-70%.

[0019] Example 2: Preparation method of uniform Dictyophora indusiata polysaccharide DMP Take 500 mg of the crude polysaccharide of Dictyophora indusiata mycelium, fully dissolve it in deionized water, add 20% H2O2 for decolorization at 52°C for 42 min, and then 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 mL / 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 polysaccharide of Dictyophora indusiata 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 refined polysaccharide of Dictyophora indusiata (code: DMP). The polysaccharide content in the refined polysaccharide of Dictyophora indusiata mycelium accounts for 98.5%.

[0020] In Figure 1, by performing a full-wavelength scan of DMP in the ultraviolet-visible light range (200-700 nm), it is proved that the refined polysaccharide of Dictyophora indusiata mycelium has a high purity and basically no proteins, nucleic acids, and other impurities.

[0021] In Figure 2 : DMP shows a characteristic absorption peak at 3317.93 cm -1 due to the stretching vibration of the O-H bond; characteristic absorption peaks of polysaccharides also appear in the range of 2750-3000 cm -1 . The characteristic absorption peak at 2924.04 cm -1 is caused by the stretching vibration of the C-H bond; the characteristic peak at 1373.07 cm -1 is the deformation absorption peak of =CH2, and characteristic peaks also appear in the range of 1000~1200 cm -1Three characteristic peaks appeared within the range, indicating the presence of pyranose glycosidic bonds.

[0022] Example 3: Regarding Example 2, the effect of polysaccharide DMP from Dictyophora indusiata mycelium on promoting the repair of LPS-damaged vascular endothelial cells Using 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, 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 place it in an incubator at 37°C and 5% CO2 for continued culture. When the cell growth reaches 80% coverage, aspirate the original culture medium, wash 3 times with PBS, add 2 mL of 0.25% trypsin and 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.

[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 group contains 1.25, 2.5, and 5 μg / mL of the fine product DMP of polysaccharide from Dictyophora indusiata mycelium respectively. Additionally, set up a cell-free blank group, with 3 replicates in each group. After culturing for 24 h, use a CCK-8 kit to measure cell viability.

[0024] In Figure 4 : Polysaccharide 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.

[0025] Example 4: Physicochemical property determination 1. Determination of polysaccharide content Using the sulfuric acid-anthrone method, at 620 nm, the total polysaccharide content was determined by spectrophotometry, with glucose (C6H 12The crude polysaccharide content of Dictyophora indusiata mycelium was 67.16%, and the content of the purified polysaccharide (DMP) of Dictyophora indusiata mycelium was 98.5%.

[0026] 2. UV Spectral Analysis The sample was dissolved in distilled water and scanned at the full UV wavelength range of 200 - 400 nm. As Figure 1 shown, DMP had no absorption at 260 and 280 nm, indicating that it did not contain proteins and nucleic acids.

[0027] 3. Monosaccharide Composition Analysis An appropriate amount of the dry powder of Dictyophora indusiata mycelium polysaccharide DMP was weighed into a hydrolysis tube, 1 mL of 72% sulfuric acid was added, and it was placed in a water bath at 30 °C for 1 h. Then it was made up to 10 mL, filled with nitrogen, and hydrolyzed in an oven at 110 °C for 2 h. After taking it out and cooling to room temperature, 0.5 mL was taken into a 4 mL centrifuge tube, the pH was adjusted to neutral, and it was made up to 1 mL; then 0.2 mL of 0.3 mol / L NaOH solution and 0.4 mL of PMP methanol solution were added, filled with nitrogen, and placed in a water bath at 70 °C for 60 min. After taking it out and cooling to room temperature, 0.2 mL of 0.3 mol / L HCl was added, made up to 2 mL with water, 1.5 mL of chloroform was added, shaken well and left to stand for layering. The lower chloroform layer was discarded, and the aqueous layer was filtered through a 0.45 μm filter membrane. It was determined using a 1200 liquid chromatograph. The chromatographic column model was C18 (4.6 mm × 250 mm × 5 μm), and the mobile phase composition was as follows: 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 / L KH2PO4, pH = 6.8); The elution was carried out at a flow rate of 1.0 mL / min under the conditions of a column temperature of 25 °C, an injection volume of 20 μL, and a detection wavelength of 254 nm.

[0028] Figure 3 Among them, the Dictyophora indusiata mycelium polysaccharide DMP was specifically composed of mannose, glucosamine, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose in a molar ratio of 0.309:0.030:0.018:0.051:0.063:0.012:98.984:0.404:0.038:0.013:0.079.

[0029] 4. Molecular Weight Determination The molecular weight of polysaccharides from Dictyophora indusiata mycelium was determined by gel permeation chromatography (GPC) using a gel permeation chromatograph 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, and 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 standards with number average molecular weights of 585 - 1140000 g / mol and weight average molecular weights 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 polysaccharides from Dictyophora indusiata mycelium was approximately 1.26×10 7 Da.

Claims

1. A polysaccharide from Dictyophora indusiata mycelium, the polysaccharide from Dictyophora indusiata mycelium being a polysaccharide containing pyranose 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 0.20 - 0.40:0.02 - 0.04:0.01 - 0.03:0.04 - 0.06:0.05 - 0.07:0.005 - 0.015:98.50 - 99.50:0.30 - 0.50:0.03 - 0.05:0.01 - 0.02:0.07 - 0.

09. The molecular weight of the polysaccharide DMP of Dictyophora indusiata mycelium is 1.26×10 7 Da.

2. The polysaccharide of dictyophora indusiata mycelium according to claim 1, wherein, The polysaccharide containing D-glucopyranose ring is composed of mannose, glucosamine, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose and fucose in a molar ratio of 0.309:0.030:0.018:0.051:0.063:0.012:98.984:0.404:0.038:0.013:0.

079. The molecular weight of the polysaccharide DMP of Dictyophora indusiata mycelium is 1.26×10 7 Da.

3. The preparation method of the polysaccharide of dictyophora indusiata mycelium according to claim 1 or 2, characterized in that, It includes the following steps: (1) After crushing the dictyophora indusiata mycelium, add hot water at 50 - 70 °C, perform ultrasonic treatment and then centrifuge at room temperature. Take the supernatant, concentrate it, add absolute ethanol, let it stand overnight in a refrigerator at 2 - 8 °C, centrifuge, discard the supernatant, dissolve the precipitate in water, remove proteins 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 polysaccharide of dictyophora indusiata mycelium; (2) Dissolve the crude polysaccharide of dictyophora indusiata mycelium in water, add 10 - 30% H2O2 and decolorize at 40 - 60 °C for 40 - 60 min, load it onto an anion exchange column, elute with deionized water, collect fractions, detect the polysaccharide content in each tube of the eluate by sulfuric acid - anthrone method, collect the main peak, concentrate and precipitate with ethanol, place it at 2 - 8 °C, centrifuge to remove the supernatant, and freeze - dry 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, detect by sulfuric acid - anthrone method, collect the main peak, concentrate and freeze - dry to obtain the refined polysaccharide DMP of dictyophora indusiata mycelium.

4. The preparation method of dictyophora indusiata mycelium polysaccharide 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 dictyophora indusiata mycelium according to claim 3, characterized in that, In step (1), the ultrasonic power is 20 - 150 W and the ultrasonic treatment time is 2 - 20 min.

6. The preparation method of the dictyophora indusiata mycelium polysaccharide according to claim 3, wherein, In steps (1) and (2), the centrifugation speed is 3000 - 4000 rpm.

7. The preparation method of dictyophora indusiata mycelium polysaccharide according to claim 3, wherein, 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 polysaccharide of dictyophora indusiata mycelium according to claim 1 or 2 in the preparation of a drug for protecting vascular endothelial cells.

9. Use of the polysaccharide of dictyophora indusiata mycelium according to claim 1 or 2 in the preparation of a drug for treating cardiovascular and cerebrovascular system - related diseases such as anti - atherosclerosis.

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