Low-molecular-weight flammulina velutipes foot polysaccharide and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to flammulina velutipes foot polysaccharide with low molecular weight and application thereof. The flammulina velutipes foot waste is used as a raw material, after steam explosion and enzymolysis treatment, ethanol with different volume fractions is adopted for gradient separation, the flammulina velutipes foot polysaccharide with the specific molecular weight is obtained, and after test verification, the obtained flammulina velutipes foot polysaccharide has a good inflammation inhibition effect, and the flammulina velutipes foot polysaccharide can be used for preparing the flammulina velutipes foot polysaccharide. Therefore, the compound can be developed and applied as a natural anti-inflammatory agent.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a low-molecular-weight polysaccharide from the root of Flammulina velutipes and its application in the preparation of anti-inflammatory drugs. Background Art

[0002] Enoki mushroom feet are the main by-product produced during the cultivation and processing of Enoki mushrooms, accounting for about 10%-15% of the total mass of the fruiting body. For a long time, Enoki mushroom feet have been directly discarded or treated as low-value feed / fertilizer due to their hard texture, poor taste and easy carry of impurities, resulting in waste of resources and environmental pressure.

[0003] In fact, the roots of Enoki mushrooms are rich in dietary fiber, protein, trace elements and other bioactive ingredients, have good anti-inflammatory activity, and have high development potential.

[0004] At present, there are also relevant studies on the utilization of active ingredients in the roots of Flammulina velutipes. For example, patent CN117413940A uses the roots of Flammulina velutipes to prepare a nutritious dietary fiber, but the technology does not disclose the molecular weight of the dietary fiber produced. Plant polysaccharides with different molecular weights have significant differences in the types and contents of monosaccharides contained therein. In addition, different types of monosaccharides have different metabolic processes in the body, and their effects on the body are quite different. For example, monosaccharides such as fucose and galactose have good anti-inflammatory effects. However, too high a mannuronic acid content may lead to the production of a large amount of inflammatory mediators, which is not conducive to the relief of inflammation.

[0005] Therefore, it is of great significance to obtain a polysaccharide from the foot of Enoki mushroom with good inflammation inhibition effect by treating and extracting the waste of Enoki mushroom foot. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides a low molecular weight Flammulina velutipes mushroom foot polysaccharide, wherein the molecular weight of the Flammulina velutipes mushroom foot polysaccharide is 67.6 kDa.

[0007] Furthermore, the low molecular weight Flammulina velutipes polysaccharide includes the following monosaccharides, calculated on a molar basis: guluronic acid 0.11%, mannuronic acid 0.2%, mannose 13.89%, ribose 3.19%, rhamnose 0.07%, glucuronic acid 0.43%, galacturonic acid 0.17%, N-acetylglucosamine 0.07%, glucose 63.63%, galactose 7.38%, xylose 8.39%, and fucose 2.46%.

[0008] Furthermore, the preparation method of the low molecular weight Flammulina velutipes polysaccharide is to use the Flammulina velutipes as raw materials, steam explode them, and then use neutral protease to hydrolyze them. The obtained enzymatic hydrolysate is then graded and extracted with anhydrous ethanol with a volume fraction of 20%, 40%, and 80%, respectively. The anhydrous ethanol part with a volume fraction of 80% is collected, which is the low molecular weight Flammulina velutipes polysaccharide.

[0009] In the above-mentioned method for preparing low molecular weight Flammulina velutipes polysaccharide, preferably, the steam explosion conditions are: the steam explosion pressure is 0.8-1.2 MPa, and the pressure holding time is 50-120 s.

[0010] Preferably, before using neutral protease for enzymolysis, the steam-exploded enoki mushroom foot sample is first dried, and then 20 to 40 times the mass of water is added, mixed evenly, and then 0.2% to 0.8% of the mass of the mixed solution is added with neutral protease, and enzymolysis is carried out at a temperature of 40 to 48° C. for 50 to 80 minutes. After the enzymolysis is completed, the temperature is raised to 90 to 100° C. and extracted for 100 to 150 minutes.

[0011] In addition, the use of low molecular weight Enoki mushroom foot polysaccharide prepared by the above method in the preparation of anti-inflammatory products is also the technical content protected by the present invention.

[0012] The beneficial effects of the present invention are:

[0013] The present invention performs steam explosion and enzymatic hydrolysis on the mushroom feet, and then further uses ethanol solutions of different concentrations for graded extraction to obtain mushroom foot polysaccharides with molecular weights of 769.9 kDa, 463.5 kDa, and 67.6 kDa, respectively. Cytotoxicity experiments show that the mushroom foot polysaccharide with a molecular weight of 769.9 kDa exhibits certain cytotoxicity, while the other two mushroom foot polysaccharides with lower molecular weights have no cytotoxicity. In addition, anti-inflammatory activity tests further confirm that the mushroom foot polysaccharide with a molecular weight of 67.6 kDa obtained by the present invention has better anti-inflammatory effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is an infrared spectrum of the Enoki mushroom foot polysaccharide prepared by different methods in Example 3 of the present invention;

[0015] Figure 2 This is a graph showing the effects of Enoki mushroom foot polysaccharides prepared by different methods in Experimental Example 1 of the present invention on the survival rate of RAW 264.7 cells;

[0016] Figure 3 This is a graph showing the effects of Enoki mushroom foot polysaccharides prepared by different methods in Experimental Example 2 of the present invention on the NO production rate of RAW 264.7 cells induced by LPS. DETAILED DESCRIPTION

[0017] In order to enable those skilled in the art to better understand the present invention, the present invention will be further explained in conjunction with specific embodiments.

[0018] Example 1

[0019] A low molecular weight Flammulina velutipes foot polysaccharide, the preparation method is as follows:

[0020] Take the waste of the mushroom feet of Flammulina velutipes, dry them in an oven at 60°C until the moisture content is less than 5%, grind them, and pass them through a 60-mesh sieve to obtain the Flammulina velutipes mushroom foot powder for later use.

[0021] Take the powder of the enoki mushroom feet and perform steam explosion treatment. The steam-exploded material is dried at 60° C., crushed, and passed through a 60-mesh sieve for later use.

[0022] The conditions for steam explosion are as follows: material-cavity ratio 5:8, steam explosion pressure 1.0 MPa, and pressure holding time 100 s.

[0023] The dried powder after steam explosion was dissolved in water at a material-liquid ratio of 1:30, and then 0.5% neutral protease of the total mass of the mixture was added. The mixture was enzymatically hydrolyzed at 45°C for 60 minutes. After the enzymatic hydrolysis, the temperature was raised to 95°C and extraction was continued for 120 minutes. After the extraction, the mixture was cooled to room temperature and centrifuged at 4000 r / min for 10 minutes. After reduced pressure concentration, anhydrous ethanol was added until the ethanol volume fraction reached 20%, and the mixture was precipitated at 4°C overnight. The precipitate was collected by centrifugation to obtain a 20% alcohol-precipitated polysaccharide fraction (SEFVP-20%). A certain amount of anhydrous ethanol was added to the supernatant to make the ethanol volume fraction reach 40%, and the mixture was precipitated overnight under the same conditions to collect a 40% alcohol-precipitated polysaccharide fraction (SEFVP-40%). The above steps were repeated to obtain an 80% alcohol-precipitated polysaccharide fraction (SEFVP-80%).

[0024] After freeze-drying the precipitated components, the yield of each graded component was calculated according to the following formula: yield of polysaccharide at each grade (%) = mass of each graded component / mass of mushroom foot powder after steam explosion × 100%.

[0025] Comparative Example 1

[0026] Different from Example 1, the steam explosion treatment was not performed on the enoki mushroom feet in this comparative example. The specific operation was as follows:

[0027] Take the waste of the mushroom feet of Flammulina velutipes, dry them in an oven at 60°C until the moisture content is less than 5%, grind them, and pass them through a 60-mesh sieve to obtain the Flammulina velutipes mushroom foot powder for later use.

[0028] Water was added to dissolve the mixture at a solid-liquid ratio of 1:30, and then 0.5% neutral protease (based on the total weight of the mixture) was added. Enzymatic hydrolysis was carried out at 45°C for 60 minutes. After the enzymatic hydrolysis, the mixture was heated to 95°C and extracted for 120 minutes. After the extraction, the mixture was cooled to room temperature and centrifuged at 4000 rpm for 10 minutes. After concentration under reduced pressure, 4 volumes of anhydrous ethanol were added. After precipitation at 4°C overnight, the mixture was centrifuged at 4000 rpm for 5 minutes. The precipitate was collected and freeze-dried to obtain crude polysaccharide from the root of Flammulina velutipes, designated FVP. The polysaccharide yield was calculated according to the following formula: Polysaccharide extraction rate (%) = crude polysaccharide mass / mushroom root powder mass × 100%.

[0029] Comparative Example 2

[0030] The difference from Example 1 is that the enzymatic hydrolyzate after enzymatic hydrolysis is not subjected to ethanol fractional extraction. Instead, 4 volumes of anhydrous ethanol are directly added, and the solution is precipitated at 4°C overnight. The solution is then centrifuged at 4000 rpm for 5 minutes, and the precipitate is collected and freeze-dried to obtain crude polysaccharide from the root of Flammulina velutipes, which is named SEFVP.

[0031] Example 2

[0032] The components of the Enoki mushroom foot polysaccharides obtained by the methods of Example 1 and Comparative Examples 1-2 were detected. The chemical composition and monosaccharide composition of the Enoki mushroom foot polysaccharides obtained by different methods are shown in Table 1 and Table 2, respectively.

[0033] Table 1 Chemical composition of polysaccharides from the root of Flammulina velutipes

[0034]

[0035] Table 2 Monosaccharide composition and molecular weight of polysaccharide from the root of Flammulina velutipes

[0036]

[0037]

[0038] The data in Table 1 show that the yields of polysaccharides from the root of Enoki mushroom obtained by extraction with different volume fractions of ethanol vary significantly. This may be because the solubility of polysaccharides in organic solvents is related to their number of carbon atoms, molecular weight and degree of polymerization. Therefore, polysaccharides with different degrees of polymerization can be obtained by using solvents of different concentrations.

[0039] Furthermore, it can be seen from Table 2 that the mushroom foot polysaccharide is composed of more than ten monosaccharides, with a relatively complex composition. The monosaccharide composition of each graded component is also different. Among them, glucose, mannose, galactose, and xylose are the main monosaccharides.

[0040] The monosaccharide composition ratios of SEFVP-20% and SEFVP-80% are similar, with glucose as the main monosaccharide, accounting for more than 63%, followed by galactose or mannose. Therefore, it is speculated that the main components of SEFVP-20% and SEFVP-80% are glucomannogalactan; the main monosaccharides contained in SEFVP-40% are mannose (28.43%), glucose (26.05%), galactose (20.79%), xylose (16.39%), fucose (5.93%), etc., among which the content ratios of mannose, galactose and glucose are comparable.

[0041] By comparing the monosaccharide ratios of the above three polysaccharide components, it can be found that the glucose content of SEFVP-40% is lower than that of the other two components, but the mannose and galactose are higher than those of the other two components. In addition, each component contains a small amount of rhamnose, N-acetylglucosamine, glucuronic acid, galacturonic acid, guluronic acid, and mannuronic acid, and their content accounts for less than 1%. The molecular weights of SEFVP-20%, SEFVP-40%, and SEFVP-80% in the enoki mushroom foot polysaccharide are 769.9kDa, 463.5kDa, and 67.6kDa, respectively.

[0042] Example 3 Infrared Spectrum Analysis of Flammulina velutipes Polysaccharide

[0043] The infrared spectrum analysis results of the polysaccharide of Flammulina velutipes are shown in the attached Figure 1 shown.

[0044] Attachment Figure 1 It shows that the FVP group and the fractionated components are at 3420 cm -1 The strong absorption peak near 2928cm is the OH stretching vibration of hydroxyl group. -1 、2924cm -1 、2925cm -1 The absorption peak at 1400 cm is the CH stretching vibration of the methyl group, indicating the existence of intermolecular hydrogen bonds; -1 The absorption peaks appearing near 1045cm are caused by the symmetrical stretching vibration of C=O of carboxyl group, which indicates that all four polysaccharides contain carboxyl groups; -1 A strong absorption peak appears at 1633 cm, which is mainly due to the COC stretching vibration of the ether bond in the polysaccharide skeleton; -1 The absorption peak near the NH angle vibration peak of the amino group is significantly weaker than that of SEFVP-20%, which indicates that the protein mass fraction of SEFVP-80% is higher than that of SEFVP-20%. This result is consistent with the protein mass fraction detection result. -1 The absorption peak at 570 cm-1 may be the CH angle vibration of the α-end isomer of the pyranose ring.-1 The absorption peaks on the left and right are pyranose ring stretching vibrations, indicating the presence of pyranose in SEFVP-20%, SEFVP-40%, and SEFVP-80%.

[0045] Infrared spectral analysis shows that the polysaccharide from the mushroom foot and its graded components have similar structures and have typical characteristic peaks of sugars, indicating that FVP, SEFVP-20%, SEFVP-40%, and SEFVP-80% are all polysaccharides, and steam explosion treatment has not changed the basic structure of the polysaccharide from the mushroom foot.

[0046] Test Example 1 Cytotoxicity Evaluation of Enoki Mushroom Foot Polysaccharide

[0047] This study used the CCK-8 method to explore the effects of different concentrations of polysaccharide components on the cytotoxicity of RAW264.7 cells. The CCK-8 method uses the dehydrogenase in the cells to reduce the reagent to a yellow formazan product. The darker the color, the more formazan products are generated, and the more living cells there are.

[0048] The results of the effects of different concentrations of Flammulina velutipes polysaccharides on the survival rate of RAW 264.7 cells are shown in the attached Figure 2 shown.

[0049] Attachment Figure 2 The results showed that in the range of 50-400 μg / mL, the cell viability of SEFVP-40% and SEFVP-80% samples was higher than 90%, indicating that the polysaccharide samples had no cytotoxicity within this concentration range.

[0050] However, after administration of different concentrations of SEFVP-20%, cell survival rates were significantly reduced, reaching below 80%, indicating that SEFVP-20% exerted a certain toxic effect on RAW 264.7 cells within this concentration range. This may be due to the large molecular weight and high mannuronic acid content of SEFVP-20%, which is the main stimulator of the production of large amounts of cytokines such as TNF-α and interleukins. The production of large amounts of inflammatory mediators reduces cell survival. Based on these results, SEFVP-40% and SEFVP-80% will be selected as research subjects in subsequent experiments.

[0051] Test Example 2 Determination of Anti-inflammatory Activity of Polysaccharide from Enoki Mushroom Foot

[0052] LPS is a component of the outer cell wall of Gram-negative bacteria. When it acts on the body, it can induce inflammation in cells, causing them to produce NO, leading to increased NO concentration. Excessive NO content in cells can cause cell DNA damage, thereby inducing inflammation and promoting cell death. Therefore, reducing the production of NO in cells is important for inhibiting inflammation.

[0053] The anti-inflammatory activity of polysaccharides from the root of Flammulina velutipes was determined according to the following method:

[0054] 3×10 4 RAW 264.7 cells in logarithmic phase growth were added to 96-well cell culture plates and pre-incubated in a CO2 incubator for a certain period of time. Then, different concentrations of SEFVP, SEFVP-40%, and SEFVP-80% were added for pretreatment for 1 hour. LPS was then added for 24 hours. 100 μL of the supernatant was reacted with 100 μL of Griess reagent for 10 minutes. The absorbance was measured at 540 nm, and the NO content produced by the sample was calculated according to the standard curve. The test results are shown in the attached figure. Figure 3 As shown, Figure 3 Different lowercase letters indicate significant differences (P<0.05).

[0055] Figure 3 The results showed that compared with the blank group, the NO content in cells treated with LPS was significantly increased (P<0.05).

[0056] When treated with SEFVP-40% and SEFVP-80% samples, NO production decreased with increasing sample concentration, indicating that the polysaccharide component has certain anti-inflammatory activity. Specifically, SEFVP-40% at 50 μg / mL and 100 μg / mL did not significantly inhibit LPS-induced NO elevation (P>0.05). However, the inhibitory effects of SEFVP-40% and SEFVP-80% samples increased significantly at 200 μg / mL and 400 μg / mL, with SEFVP-80% having a greater inhibitory effect.

[0057] The present invention uses ethanol of different concentrations to grade and precipitate crude polysaccharides from steam-exploded Flammulina velutipes mushroom feet, obtaining three different polysaccharide graded fractions, namely SEFVP-20%, SEFVP-40%, and SEFVP-80%. By studying the chemical composition, structural characteristics, and functional activity of the graded fractions, it was found that although the fractions have similar functional groups and monosaccharide compositions, there are differences in the proportions of the monosaccharides. Among them, the glucose ratio of SEFVP-40% (26.05%) is significantly lower than that of the other fractions (63.63% to 65.98%).

[0058] In addition, the in vitro anti-inflammatory activity results proved that the polysaccharides from the mushroom feet of Flammulina velutipes have certain anti-inflammatory effects. Among them, SEFVP-40% and SEFVP-80% both had significant inhibitory effects on LPS-induced high NO expression in the range of 200-400 μg / mL, and the inhibitory effect of SEFVP-80% was better. Therefore, SEFVP-80% has the potential to be developed as a natural anti-inflammatory agent. At the same time, isolating SEFVP-80% is also beneficial to avoid the side effects of some high-content monosaccharides in SEFVP-20% on the inhibitory effect of inflammation.

Claims

1. A low molecular weight polysaccharide from the root of Flammulina velutipes, characterized in that: The molecular weight of the low molecular weight Flammulina velutipes polysaccharide is 67.6 kDa.

2. The low molecular weight Flammulina velutipes polysaccharide according to claim 1, characterized in that: The low molecular weight Flammulina velutipes foot polysaccharide includes the following monosaccharides, calculated by the amount of substance: 0.11% guluronic acid, 0.2% mannuronic acid, 13.89% mannose, 3.19% ribose, 0.07% rhamnose, 0.43% glucuronic acid, 0.17% galacturonic acid, 0.07% N-acetylglucosamine, 63.63% glucose, 7.38% galactose, 8.39% xylose, and 2.46% fucose.

3. The method for preparing the low molecular weight Flammulina velutipes polysaccharide according to claim 1, characterized in that: The enoki mushroom base is used as raw material, and after steam explosion, it is enzymatically hydrolyzed by using neutral protease, and then the obtained enzymatic hydrolyzate is graded and extracted using anhydrous ethanol with a volume fraction of 20%, 40% and 80%, respectively. The anhydrous ethanol part with a volume fraction of 80% is collected, which is the low molecular weight enoki mushroom base polysaccharide.

4. The method for preparing low molecular weight Flammulina velutipes polysaccharide according to claim 3, characterized in that: The steam explosion conditions are as follows: the steam explosion pressure is 0.8~1.2 MPa, and the pressure holding time is 50~120 s.

5. The method for preparing low molecular weight Flammulina velutipes polysaccharide according to claim 3, characterized in that: Before using neutral protease for enzymatic hydrolysis, the steam-exploded Flammulina velutipes sample was first dried, then 20 to 40 times the mass of water was added, mixed evenly, and then 0.2% to 0.8% of the mass of the mixed solution was added with neutral protease. The enzymatic hydrolysis was carried out at 40 to 48°C for 50 to 80 min. After the enzymatic hydrolysis was completed, the temperature was raised to 90 to 100°C and extracted for 100 to 150 min.

6. Use of the low molecular weight Flammulina velutipes polysaccharide according to any one of claims 1 to 5 in the preparation of anti-inflammatory products.

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