Tricholoma matsutake polysaccharide and deep eutectic solvent extraction method and application thereof

Abstract

The invention provides tricholoma matsutake polysaccharide and a deep eutectic solvent extraction method and application thereof, and relates to the technical field of plant extraction. A deep eutectic solvent extraction method of tricholoma matsutake polysaccharide comprises the following steps: preparing a deep eutectic solution: uniformly mixing and dissolving a hydrogen donor reagent and a hydrogen acceptor reagent, and adding water to obtain a transparent solution; and polysaccharide extraction: drying, crushing and degreasing tricholoma matsutake, adding the tricholoma matsutake into the deep eutectic solution, heating and extracting to obtain an extracting solution, and purifying, alcohol-precipitating, dialyzing and freeze-drying the extracting solution to obtain the tricholoma matsutake polysaccharide. Compared with a traditional solvent and the prior art, the efficiency of extracting the tricholoma matsutake polysaccharide is obviously higher than that of traditional water solvent extraction, the protein content of the extract is low, the tricholoma matsutake polysaccharide in the extract is more stable than that of the traditional solvent extraction, the antioxidant activity of the polysaccharide is better than that of the traditional method, equipment is simple, production cost is low, continuous operation can be achieved, and the method is suitable for industrial production. And the method has extremely high practicability and operability.

Description

Technical Field

[0001] The present application relates to the field of plant extraction technology, and in particular to a method and application of extracting Tricholoma matsutake polysaccharide and its deep eutectic solvent. Background Art

[0002] Matsutake (Scientific name: Tricholoma matsutake (S.Ito & S.Imai) Singer), also known as pine mushroom, pine mushroom, etc., is a plant of the genus Trichoderma of the family Trichostomaceae. It is an ecto-mycorrhizal fungus of trees such as pine and oak. It is a rare and precious natural medicinal fungus in the world. It is known as the "king of fungi" and is a second-level endangered species in China. Matsutake needs to rely on the roots of pine trees to survive. Species include Tricholoma cylindrica, Tricholoma pseudomatus, Tricholoma chestnut brown, etc. Matsutake is rich in protein, 18 kinds of amino acids, 14 kinds of essential trace elements for the human body, etc.

[0003] For the study of plant polysaccharides, it is crucial to choose a suitable extraction method, because different extraction techniques can significantly affect the yield, physicochemical properties and biological activity of polysaccharides. Traditional polysaccharide extraction methods include hot water reflux method, enzyme-assisted water extraction method, ultrasound-assisted water extraction method and microwave-assisted water extraction method. Given the complexity and diversity of materials, as well as different economic costs and environmental impacts, each method has its own unique advantages and limitations. Therefore, the extraction technology of deep eutectic solvent (DES) provides new possibilities for polysaccharide extraction. DES is a natural green solvent that is biodegradable, recyclable and easy to prepare. It consists of a hydrogen donor (HBD) and a hydrogen acceptor (HBA), of which choline chloride (ChCl) is the most commonly used hydrogen acceptor. At present, DES has been applied to the extraction of various plant polysaccharides, such as kelp, sargassum and lotus leaves. The polysaccharides extracted by deep eutectic have better antioxidant activity than those extracted by traditional methods. However, there are relatively few studies on the extraction of fungal polysaccharides.

[0004] At present, the extraction process of Tricholoma matsutake polysaccharides mainly includes traditional hot water extraction, dilute acid (alkali) extraction, enzymatic extraction, ultrasonic assisted extraction and enzyme method. The extract is precipitated by adding ethanol, and Tricholoma matsutake polysaccharides can be obtained after filtration. In the extraction process, ultrasound assisted extraction and microwave assisted extraction are used to improve the extraction efficiency. However, water extraction, alkali extraction and enzyme extraction often lead to the co-extraction of other components such as proteins and glycosides, which not only increases the difficulty of subsequent separation and purification. Although methods such as ultrasound assisted extraction and microwave assisted extraction can improve the extraction efficiency, they require the use of large equipment, resulting in higher investment and increased costs. These traditional methods generally have problems such as long extraction cycle, low extraction rate, large loss of effective ingredients and high cost. In addition, toxic organic solvents (such as dichloromethane, acetonitrile, methanol, etc.) commonly used in traditional extraction processes pose a threat to the environment and human health. Therefore, it is urgent to develop a new green and environmentally friendly extraction method to improve the efficiency of polysaccharide extraction. Summary of the invention

[0005] The purpose of this application is to provide a convenient and quick method for extracting polysaccharides from Tricholoma matsutake. The method uses a deep eutectic solvent, is green and environmentally friendly, takes a short time, and has cheap reagents, and is suitable for promotion and application.

[0006] Another object of the present application is to provide a pine mushroom polysaccharide obtained by the above extraction method, which has a high yield, low protein content and good antioxidant activity.

[0007] Another object of the present application is to provide a use of Tricholoma matsutake polysaccharide in the preparation of a drug for treating testicular damage caused by diabetes, which provides a new direction for the use of Tricholoma matsutake polysaccharide.

[0008] In order to solve the above technical problems, the technical solution adopted in this application is:

[0009] In one aspect, the present application provides a method for deep eutectic solvent extraction of Tricholoma matsutake polysaccharide, comprising the following steps:

[0010] S1. preparing a deep eutectic solution: mixing and dissolving a hydrogen donor and a hydrogen acceptor reagent, and adding water to obtain a transparent solution;

[0011] S2. Extracting polysaccharides: Dry, crush and defatted the Tricholoma matsutake, and then add it to the above-mentioned deep eutectic solution. After heating and extraction, an extract is obtained. The extract is purified, precipitated with alcohol, dialyzed and freeze-dried to obtain Tricholoma matsutake polysaccharides.

[0012] On the other hand, the present application provides a Tricholoma matsutake polysaccharide prepared by the above extraction method.

[0013] On the other hand, the present application provides a use of Tricholoma matsutake polysaccharide in the preparation of a drug for treating testicular damage caused by diabetes.

[0014] Compared with the prior art, the embodiments of the present application have at least the following advantages or beneficial effects:

[0015] 1. The present application provides a deep eutectic solvent system and a method for extracting polysaccharides from Matsutake. Compared with traditional solvents and prior art, the efficiency of extracting Matsutake polysaccharides by this method is significantly higher than that of traditional water solvent extraction, and the protein content produced in the extraction process is low. The Matsutake polysaccharides in the extract are more stable than those extracted by traditional solvents, and the effect of the polysaccharides in alleviating testicular damage caused by diabetes is better than that of traditional methods. The equipment is simple, the production cost is low, and it can be operated continuously, and it has extremely strong practicality and operability.

[0016] 2. The deep eutectic solvent system provided in the present application mainly uses choline chloride as a hydrogen bond acceptor, and 1,2-propylene glycol, 1,4-butanediol, 1,3-butanediol, ethylene glycol, malonic acid and urea as hydrogen bond donors, and has the following advantages: a. Abundant raw materials and moderate prices. b. Non-toxic and pollution-free. The raw materials of the deep eutectic system of the present application are non-toxic and have good biocompatibility. Several raw materials are easily degradable, non-volatile, and environmentally friendly. In summary, the deep eutectic solvent system provided in the present application has strong practicality. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 The three-dimensional and two-dimensional response surface graphs of the significant interaction terms of Example 6 of the present application;

[0019] Figure 2 This is a comparison chart of cell viability of Tricholoma matsutake polysaccharide extracted with water and Tricholoma matsutake polysaccharide extracted with deep eutectic solvent in the treatment of diabetic testicular damage in Example 7 of the present application; DETAILED DESCRIPTION

[0020] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0021] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to specific embodiments.

[0022] A deep eutectic solvent extraction method for Tricholoma matsutake polysaccharide comprises the following steps:

[0023] S1. preparing a deep eutectic solution: mixing and dissolving a hydrogen donor and a hydrogen acceptor reagent, and adding water to obtain a transparent solution;

[0024] S2. Extracting polysaccharides: Dry, crush and defatted the Tricholoma matsutake, and then add it to the above-mentioned deep eutectic solution. After heating and extraction, an extract is obtained. The extract is purified, precipitated with alcohol, dialyzed and freeze-dried to obtain Tricholoma matsutake polysaccharides.

[0025] In some embodiments of the present application, the molar ratio of the hydrogen donor and the hydrogen acceptor reagent is 1:2; the hydrogen donor is 1,2-propylene glycol, 1,4-butanediol, 1,3-butanediol, ethylene glycol, malonic acid or urea, preferably malonic acid; and the hydrogen acceptor is choline chloride.

[0026] In some embodiments of the present application, the dissolution in the above step S1 is specifically stirred at a temperature of 70-90° C. for 20-50 min.

[0027] In some embodiments of the present application, the water content of the deep eutectic solution in step S1 is 10-50%.

[0028] In some embodiments of the present application, the defatting of the Tricholoma matsutake in the above step S2 is specifically performed by soaking the dried and crushed Tricholoma matsutake powder in ethanol for 20-30 hours.

[0029] In some embodiments of the present application, the solid-liquid ratio of the defatted matsutake mushroom to the deep eutectic solution in the above step S2 is 1:(10-50), preferably 1:30; the heating extraction temperature is 70-100°C, preferably 90°C, and the extraction time is 40-60min.

[0030] In some embodiments of the present application, the purification and alcohol precipitation in the above-mentioned step S2 are specifically as follows: centrifuging the extract at 5000 rpm to collect the centrifuge; adding 4 times the volume of ethanol precipitation to the centrifuge, and then centrifuging at 6000 rpm to collect the precipitate.

[0031] In some embodiments of the present application, the dialysis in the above-mentioned step S2 is specifically to add pure water to the centrifugal precipitate to dissolve and dialyze for 24 hours, and the cut-off flow rate used for the dialysis is 3500Da.

[0032] A pine mushroom polysaccharide is prepared by extraction using the deep eutectic solvent extraction method.

[0033] A use of Tricholoma matsutake polysaccharide in preparing a medicine for treating testicular damage caused by diabetes.

[0034] The features and properties of the present application are further described in detail below in conjunction with the examples. The choline chloride, 1,2-propylene glycol, 1,4-butylene glycol, 1,3-butylene glycol, ethylene glycol, malonic acid and urea used in the present application are all analytically pure.

[0035] Example 1

[0036] This example explores the optimal hydrogen donor (HBD).

[0037] In this embodiment, 6 experimental groups were set up, corresponding to different HBD reagents, as shown in Table 1; HBA (choline chloride) and HBD reagent were added to a flask at a molar ratio of 1:2, stirred at 70°C for 30 minutes, and then water was added to prepare a colorless transparent solution with a water content of 30%, i.e., a deep eutectic solvent system;

[0038] Table 1

[0039] Experimental Group HBD HBA:HBD shape 1 1,2-propylene glycol 1∶2 Colorless and transparent 2 1,4-Butanediol 1∶2 Colorless and transparent 3 1,3-Butanediol 1∶2 Colorless and transparent 4 Ethylene glycol 1∶2 Colorless and transparent 5 Malonic acid 1∶2 Colorless and transparent 6 Urea 1∶2 Colorless and transparent

[0040] The matsutake was dried and crushed, and then soaked in ethanol for 24 hours to obtain defatted matsutake powder; the defatted matsutake powder was added to the deep eutectic solvent according to the solid-liquid ratio (W / V=1:30), and heated and extracted at 90°C for 45 minutes. After the extraction, it was centrifuged at 5000rpm and the centrifuge was collected. The centrifuge was added with 4 times the volume of ethanol to precipitate, and after centrifugation at 6000rpm to collect the precipitate, pure water was added to dissolve and dialyzed for 24 hours, and after dialysis, it was freeze-dried to obtain crude matsutake polysaccharide. In addition, in this embodiment, water was set as the solvent as the control group, and the water extraction group was added with defatted matsutake powder according to the solid-liquid ratio of 1:30, boiled at 100°C for 3 hours and repeated three times, and the concentrated extract was collected, and the subsequent steps were the same. The yield of crude polysaccharide (x, n=3) is shown in Table 2.

[0041] Table 2

[0042]

[0043] As can be seen from Tables 1 and 2, the experimental results show that the extraction rate of traditional water extraction is about 3.0%, while the extraction rate of the deep eutectic solvent system using choline chloride plus malonic acid is better than that of water extraction, and the steps and time required are less than water extraction.

[0044] Example 2

[0045] This example explores the effect of deep eutectic solutions with different water contents on the yield of Tricholoma matsutake polysaccharides.

[0046] Add choline chloride and malonic acid to a beaker at a molar ratio of 1:2, add water according to wt% while heating to form a deep eutectic solution with different water contents. Then add crushed and defatted matsutake powder, heat at 90°C for 45 minutes to extract. After extraction, centrifuge at 5000rpm and collect the centrifuge. Add 4 times the volume of ethanol to the centrifuge to precipitate, centrifuge at 6000rpm to collect the precipitate, add pure water to dissolve and dialyze for 24 hours, and freeze-dry after dialysis to obtain crude matsutake polysaccharide. Each group was repeated three times. The yield of crude matsutake polysaccharide is shown in Table 3.

[0047] Table 3

[0048] Serial number HBD Water content Crude polysaccharide yield (%) 1 Malonic acid 10％ 1.3308±0.2018 2 Malonic acid 20％ 2.0918±0.2064 3 Malonic acid 30％ 4.2792±0.3391 4 Malonic acid 40％ 2.9076±0.2063 5 Malonic acid 50％ 2.8195±0.2086

[0049] As can be seen from Table 3, the experimental results show that the extraction efficiency is higher when the water content of the deep eutectic solution is 30%, so the water content is selected to be 30%.

[0050] Example 3

[0051] This example explores the effect of different extraction times on the yield of Tricholoma matsutake polysaccharides.

[0052] In a deep eutectic solution with a water content of 30% (the molar ratio of choline chloride: malonic acid is 1:2), Tricholoma matsutake powder was added at a solid-liquid ratio of 1:30, and the mixture was heated at 90°C for extraction. After the extraction, the mixture was centrifuged at 5000rpm and the centrifuge was collected. The centrifuge was precipitated by adding 4 times the volume of ethanol, and the precipitate was collected by centrifugation at 6000rpm. Pure water was added to dissolve and dialyzed for 24 hours. After dialysis, the crude Tricholoma matsutake polysaccharide was obtained by freeze-drying. The heating extraction time and the corresponding crude polysaccharide yield (x, n = 3) are shown in Table 4.

[0053] Table 4

[0054] Serial number HBD Extraction time (min) Crude polysaccharide yield (%) 1 Malonic acid 15 1.3563±0.2183 2 Malonic acid 25 2.3564±0.3784 3 Malonic acid 35 3.3999±0.102 4 Malonic acid 45 4.1872±0.199 5 Malonic acid 55 5.6512±0.2385 6 Malonic acid 65 3.502±0.2265

[0055] As can be seen from Table 4, the experimental results show that the extraction efficiency is higher when the extraction time is 55 minutes, so the extraction time is selected at 55 minutes.

[0056] Example 4

[0057] This example explores the effect of different extraction heating temperatures on the yield of Tricholoma matsutake polysaccharides.

[0058] Add matsutake powder to a deep eutectic solution with a water content of 30% (molar ratio of choline chloride: malonic acid is 1:2) at a solid-liquid ratio of 1:30 and heat for extraction. The extraction time is 55 minutes. After extraction, centrifuge at 5000rpm and collect the centrifuge. Add 4 times the volume of ethanol to the centrifuge to precipitate. After centrifugation at 6000rpm to collect the precipitate, add pure water to dissolve and dialyze for 24 hours. After dialysis, freeze-dry to obtain crude matsutake polysaccharide. Heating extraction temperature and crude polysaccharide yield As shown in Table 5.

[0059] Table 5

[0060] Serial number HBD Extraction temperature (℃) Crude polysaccharide yield (%) 1 Malonic acid 60 1.9983±0.0406 2 Malonic acid 70 3.2229±0.2034 3 Malonic acid 80 2.5093±0.2482 4 Malonic acid 90 5.526±0.2694 5 Malonic acid 100 2.9615±0.0873

[0061] As can be seen from Table 5, the experimental results show that when the extraction temperature is 90°C, the extraction efficiency is higher, so the extraction temperature is selected at 90°C.

[0062] Example 5

[0063] This example explores the effect of different liquid-to-solid ratios on the yield of Tricholoma matsutake polysaccharides.

[0064] Add matsutake powder to a deep eutectic solution with a water content of 30% (molar ratio of choline chloride: malonic acid is 1:2) at different material-liquid ratios, heat to 90°C for extraction, and extract for 55 minutes. After extraction, centrifuge at 5000rpm and collect the centrifuge. Add 4 times the volume of ethanol to the centrifuge to precipitate, centrifuge at 6000rpm to collect the precipitate, add pure water to dissolve and dialyze for 24 hours, and freeze-dry after dialysis to obtain crude matsutake polysaccharide. Liquid-to-material ratio and polysaccharide yield As shown in Table 6.

[0065] Table 6

[0066] Serial number HBD Liquid to solid ratio (V / g) Crude polysaccharide yield (%) 1 Malonic acid 10:1 3.0512±0.0573 2 Malonic acid 20:1 3.6781±0.2408 3 Malonic acid 30:1 5.578±0.2124 4 Malonic acid 40:1 4.221±0.1511 5 Malonic acid 50:1 3.544±0.1795

[0067] As can be seen from Table 6, the experimental results show that when the liquid-to-solid ratio is 30:1, the extraction efficiency is higher, so the liquid-to-solid ratio is selected to be 30:1.

[0068] Example 6

[0069] This embodiment responds to 27 groups of experiments in total, with extraction temperature (℃), time (min), liquid-to-solid ratio (V / g) and water content (%) as influencing factors, and polysaccharide extraction rate as the investigation index. The design combinations and their corresponding experimental results are shown in Table 7 (factor levels of response surface experiments).

[0070] Table 7

[0071]

[0072]

[0073] The variance results of the response surface experiment are shown in Table 8:

[0074] Table 8

[0075]

[0076]

[0077] According to Table 8, the P value of the regression equation model is extremely significant, indicating that the model has a good fit and the experimental method is feasible; the P value of the lack of fit term is not significant, indicating that the model error is small; the larger the F value of the factor and the smaller the P value, the greater the influence of the factor on the result, so the order of the influence of each factor on the extraction rate of Tricholoma matsutake polysaccharide is: extraction time> liquid-to-solid ratio> water content> extraction temperature.

[0078] The extraction temperature (A), extraction time (B), liquid-to-solid ratio (C) and water content (D) were used as independent variables to fit the data of the extraction rate Y of Tricholoma matsutake polysaccharide and establish a regression equation:

[0079] Y=5.46663+0.102135*A+0.394502*B+0.193767*C+-0.177117*D+0.04598*AB+-0.16075*AC+0.149175*AD +0.1795*BC+-0.164025*BD+-0.12375*CD+-0.974469*A^2+-1.40289*B^2+-0.688872*C^2+-1.10022*D^2

[0080] The three-dimensional and two-dimensional response surface plots of significant interaction terms are shown in Figure 2. Figure 1 As shown; Figure 1 A: Interaction between temperature and liquid-to-solid ratio B: Interaction between time and liquid-to-solid ratio C: Interaction between time and water content;

[0081] Response surface plots are used to analyze certain response target values ​​and independent variables. Together, they form a three-dimensional space diagram. By analyzing the three-dimensional response surface diagram, the influence of different factors on the response target value can be significantly reflected. In order to obtain the effect of the corresponding interaction factor of each factor in the reaction process. The effectiveness of the influence can be determined and the extreme value can be found through the response value parameters under different conditions. Response surface experimental surface plots and contour lines are as follows Figure 1 As shown, it can be seen that the curves of the interaction terms AC, BC, and BD are steeper and have a greater impact on the results.

[0082] The optimal model obtained by response surface analysis is: extraction temperature 90.2℃, extraction time 56min, liquid-to-solid ratio 31.5V / g, water content 29.6%. After three repeated experiments, the average polysaccharide extraction rate was 5.4516%, which was close to the predicted value of 5.5155%, proving that the extraction process obtained by response surface experiment has high credibility.

[0083] Example 7

[0084] This example explores the therapeutic effect of Tricholoma matsutake polysaccharide on diabetic testicular damage.

[0085] TM4 cells were cultured in DMEM / F12 containing 10% FBS and 1% penicillin-streptomycin at 37°C, 5% CO2 and 95% air. When the cells grew and merged to 80-90%, they were divided into 4 groups: normal group (Control), PA model group (PA400μM), water-extracted polysaccharide group, and deep eutectic solvent-extracted polysaccharide group. Among them, the PA model group and the treatment group were given DMEM / F12 medium containing a final concentration of 400μM palmitic acid and the same concentration of matsutake polysaccharide for 48h. After the intervention, 100μL of the 10% CCK8 solution prepared in advance was added to each well, returned to the incubator, and reacted in the dark for 2h, and the results were detected by an ELISA instrument (absorbance 450nM).

[0086] Figure 2 This is a comparison of cell viability of Tricholoma matsutake polysaccharide extracted from water and Tricholoma matsutake polysaccharide extracted from deep eutectic solvent in the treatment of diabetic testicular damage. (TMP-W: Tricholoma matsutake polysaccharide extracted from water, TMP-DES: Tricholoma matsutake polysaccharide extracted from deep eutectic solvent)

[0087] Depend on Figure 2 The results showed that the deep eutectic extracted pine mushroom polysaccharide was more effective than the water extracted pine mushroom polysaccharide in treating diabetic testicular damage.

[0088] Example 8

[0089] This example explores the protein content comparison between deep eutectic solvent and traditional water-extracted Tricholoma matsutake polysaccharide.

[0090] The same solid-liquid ratio of 1:30 was used to extract polysaccharides from 1 g of Matsutake powder, and then the protein concentration of the extract was detected using an ultra-trace nucleic acid protein analyzer using the protein A280 method. The protein concentration comparison results are shown in Table 9.

[0091] Table 9

[0092] Sample name mg / ml A280 Water extraction 25.59 25.591 Deep eutectic solvent extraction 15.018 15.02

[0093] It can be seen from Table 9 that the protein content produced during the extraction process using the deep eutectic solvent method is lower. At the same time, the equipment indicated the presence of nucleic acid contamination after the water extraction sample was tested, while this was not shown when the deep eutectic solvent extracted sample was tested.

[0094] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present application.

Claims

1. A deep eutectic solvent extraction method for Tricholoma matsutake polysaccharide, characterized in that: The following steps are involved: S1. preparing a deep eutectic solution: mixing and dissolving a hydrogen donor and a hydrogen acceptor reagent, and adding water to obtain a transparent solution; S2. Extracting polysaccharides: Dry, crush and defatted the Tricholoma matsutake, and then add it to the above-mentioned deep eutectic solution. After heating and extraction, an extract is obtained. The extract is purified, precipitated with alcohol, dialyzed and freeze-dried to obtain Tricholoma matsutake polysaccharides.

2. The deep eutectic solvent extraction method of Tricholoma matsutake polysaccharide according to claim 1, characterized in that: The molar ratio of the hydrogen donor and the hydrogen acceptor reagent is 1:2; the hydrogen donor is 1,2-propylene glycol, 1,4-butylene glycol, 1,3-butylene glycol, ethylene glycol, malonic acid or urea, and the hydrogen acceptor is choline chloride.

3. The deep eutectic solvent extraction method of Tricholoma matsutake polysaccharide according to claim 1, characterized in that: The dissolution in step S1 is specifically carried out by stirring at a temperature of 70-90° C. for 20-50 min.

4. The deep eutectic solvent extraction method of Tricholoma matsutake polysaccharide according to claim 1, characterized in that: The water content of the deep eutectic solution in step S1 is 10-50%.

5. The deep eutectic solvent extraction method of Tricholoma matsutake polysaccharide according to claim 1, characterized in that: The defatting of the matsutake in step S2 is specifically to soak the dried and crushed matsutake powder in ethanol for 20-30 hours.

6. The deep eutectic solvent extraction method of Tricholoma matsutake polysaccharide according to claim 1, characterized in that: In the step S2, the material-liquid ratio of the defatted matsutake mushroom to the deep eutectic solution is 1:(10-50), the heating extraction temperature is 70-100° C., and the extraction time is 40-60 min.

7. The deep eutectic solvent extraction method of Tricholoma matsutake polysaccharide according to claim 1, characterized in that: The purification and alcohol precipitation in step S2 are specifically as follows: centrifuging the extract at 5000 rpm to collect the centrifuge; adding 4 times the volume of ethanol precipitation to the centrifuge, and then centrifuging at 6000 rpm to collect the precipitate.

8. The deep eutectic solvent extraction method of Tricholoma matsutake polysaccharide according to claim 7, characterized in that: The dialysis in step S2 is specifically performed by adding pure water to the centrifugal precipitate to dissolve and dialyze for 24 hours, and the cut-off flow rate used for the dialysis is 3500Da.

9. A pine mushroom polysaccharide, characterized in that: The deep eutectic solvent extraction method according to any one of claims 1 to 8 is used for extraction and preparation.

10. Use of the Tricholoma matsutake polysaccharide as claimed in claim 9 in the preparation of a drug for treating testicular damage caused by diabetes.

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