Willow mushroom crude polysaccharide preparation and preparation method thereof

Abstract

The invention relates to the field of traditional Chinese medicine preparations, in particular to a mushroom crude polysaccharide preparation and a preparation method thereof. The preparation raw materials of the coarse polysaccharide preparation comprise, by mass, 1-10 parts of coarse polysaccharide, 0.1-50 parts of natural oil and 10-50 parts of wax, and the preparation method of the coarse polysaccharide comprises the steps that the coarse polysaccharide is extracted through a water extraction method; carrying out alcohol precipitation on the agaric crude polysaccharide; and purifying and drying the crude polysaccharide of the agaricus bisporus. The agaricus bisporus crude polysaccharide preparation prepared by the invention inhibits increase of skin capillary permeability caused by histamine, and the effect of the agaricus bisporus crude polysaccharide preparation is obviously superior to that of an existing product stubborn dermatitis ointment.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine preparations, and in particular to a crude polysaccharide preparation of Agaricus scabra and a preparation method thereof. Background Art

[0002] Wanxuandi ointment (approval number: National Medicine Standard Z12020552) is a traditional topical pharmaceutical preparation, mainly used to treat chronic skin diseases such as psoriasis, tinea versicolor, and psoriasis. It uses agaricus powder and beeswax as the main raw materials, supplemented by vegetable oil to make a semi-solid ointment, which has certain anti-inflammatory, detoxification and antipruritic effects. However, the existing Wanxuandi ointment has many limitations: first, the material basis of its efficacy is unclear, and agaricus is used as medicine in the form of raw powder, making it difficult to determine the specific active ingredients; second, agaricus powder has poor solubility and permeability, and the transdermal absorption effect is poor, which affects the efficacy of the drug; in addition, the ointment is dark in color and greasy in texture, which easily stains clothes, is inconvenient to use, and reduces the patient's medication compliance.

[0003] Skin diseases such as psoriasis, eczema, and psoriasis are common chronic skin diseases in clinical practice. Their pathogenesis is complex and treatment is difficult. Current treatments mainly include topical steroid ointments and immunomodulators, but these drugs often have side effects and are difficult to cure. In recent years, polysaccharides from Agaricus salsa have been shown to inhibit the proliferation of skin keratinocytes and may become an effective ingredient for treating skin diseases such as psoriasis. However, there are relatively few studies on the extraction, purification, and application of polysaccharides in preparations, especially their application in scalp-clearing ointment, which has not been systematically reported.

[0004] With the continuous advancement of pharmaceutical formulation technology, the development of novel formulations has become an important approach to improving drug efficacy and user experience. By optimizing the matrix composition and preparation process, drug stability, release characteristics, and transdermal absorption can be improved. Therefore, by integrating modern formulation technology, we have improved and innovated the traditional Wanxuandi ointment to develop a novel formulation with crude polysaccharide from Agaricus scabra as its primary ingredient, which has important clinical application value and social significance. Summary of the Invention

[0005] In order to solve the above technical problems, the first aspect of the present invention provides a crude polysaccharide preparation of Pholiota adiposa, the raw materials of which, by weight, include: 1-10 parts of crude polysaccharide of Pholiota adiposa, 0.1-50 parts of natural oil, and 10-50 parts of wax.

[0006] As an implementable case, the natural oil includes: almond oil, peanut oil, soybean oil, sunflower oil or lanolin.

[0007] Furthermore, the natural oil is peanut oil.

[0008] As an implementable example, the wax includes beeswax or vaseline.

[0009] Furthermore, the wax is beeswax.

[0010] In the present invention, it is preferred that natural oils and waxes are compounded in the crude polysaccharide of Agaricus fasciatus, and in particular, the natural oils are further limited to peanut oil and the wax is beeswax, which can effectively improve the inhibition of skin vascular permeability after the use of the crude polysaccharide preparation product of Agaricus fasciatus; the inventors speculate that this is mainly because beeswax and peanut oil have good affinity and similar compatibility. Beeswax can form a protective film, which helps to lock in skin moisture, and the fatty acid components in peanut oil are similar to the natural lipids of the skin, which are easily absorbed by the skin and are beneficial to the delivery of drug ingredients to the skin. The combination of beeswax and peanut oil can form a relatively stable dispersion system with the crude polysaccharide of Agaricus fasciatus. After being applied on the skin surface, the oil matrix will gradually release the crude polysaccharide of Agaricus fasciatus, so that it can continuously contact the affected skin and exert an anti-inflammatory effect; in addition, beeswax and peanut oil themselves have certain moisturizing and skin protection effects, which can maintain the normal physiological state of the skin, so that the skin maintains good permeability and absorptive capacity, which is beneficial to the absorption and function of the drug. If the raw material system does not include natural oils, due to the high viscosity of wax and vaseline, their spreadability on the skin is relatively poor, which may cause uneven distribution of Agaricus limonene crude polysaccharides on the skin surface, reducing the effective contact area between the drug and the skin, and thus weakening the drug's regulatory effect on skin vascular permeability.

[0011] Furthermore, when the natural oil is peanut oil and the wax is beeswax, the mass ratio of the natural oil to the wax is (2.5-3.5):1.

[0012] Furthermore, the mass ratio of the natural oil and wax is 3:1.

[0013] The present invention limits the natural oil to peanut oil, and when the wax is beeswax, the mass ratio of the natural oil to the wax is (2.5-3.5): 1, and the viscosity and ductility of the matrix reach a better balance; this is mainly because the wax provides a certain structural support, and the natural oil gives the matrix good fluidity and ductility, so the matrix can be evenly applied on the skin and form a continuous drug film, which is conducive to the full contact between the crude polysaccharide of Agaricus argyi and the skin, so as to better exert its anti-inflammatory effect and inhibit the increase in skin capillary permeability. The appropriate mass ratio helps the drug to be effectively released from the matrix and penetrate into the skin. If the wax ratio is too high, the matrix may be too viscous and the drug release is limited; and when the natural oil ratio is too high, the matrix may be too thin and unable to provide sufficient support and adhesion, resulting in uneven distribution of the drug on the skin surface, affecting the efficacy of the crude polysaccharide preparation of Agaricus argyi.

[0014] As an practicable case, the preparation method of the crude polysaccharide of Agaricus scabra includes:

[0015] Extraction of crude polysaccharides from Agaricus salix using water extraction method;

[0016] Alcohol precipitation of crude polysaccharides from Agaricus salsa;

[0017] Purification and drying of crude polysaccharide from Agaricus salsa.

[0018] Furthermore, the water extraction method for preparing crude polysaccharide from Agaricus scabra includes:

[0019] S1. Grind the medicinal material of Agaricus serrata, place the Agaricus serrata powder in a flask, add water and soak for 1-2 hours to obtain an Agaricus serrata aqueous solution;

[0020] S2, heating the aqueous solution of Agaricus sutchuenensis to reflux for 2-3 hours, filtering it while hot, and collecting the first filter residue and the first filtrate;

[0021] S3, adding water to the first filter residue, heating and reflux for 2-3 hours, filtering while hot, and collecting the second filter residue and the second filtrate;

[0022] S4, adding water to the second filter residue collected in step S3, heating and refluxing for 2-3 hours, filtering while hot, and collecting the third filter residue and the third filtrate;

[0023] S5. Combine the first filtrate, the second filtrate and the third filtrate, concentrate under reduced pressure to a volume of 100-200 mL, and collect the concentrate to obtain crude Agaricus scabra polysaccharide.

[0024] As an implementable case, the volume ratio of the amount of water added in step S1, step S3 and step S4 is (1200-1600): (800-1200): (800-1200).

[0025] Furthermore, the volume ratio of the amount of water added in step S1, step S3 and step S4 is 1500:1000:1000.

[0026] During the extraction process, the amount of solvent used is an important factor affecting the efficiency of extracting crude polysaccharides from Agaricus fasciatus. The present invention stipulates that the crude polysaccharides from Agaricus fasciatus are extracted by adding water and filtering three times, wherein 1500 mL of water is added for the first time, and 1000 mL of water is added for the second and third times, respectively. This ensures that the amount of extraction solvent is sufficient, so that the polysaccharide components in the Agaricus fasciatus medicinal material can be fully dissolved and extracted. The process design of adding water three times can ensure the sufficiency and thoroughness of the extraction process. After the first extraction with 1500 mL of water, some crude polysaccharides from Agaricus fasciatus may still remain in the medicinal residue. By continuing the extraction with 1000 mL of water for the second and third times, the extraction rate of the polysaccharide can be further improved; by extracting multiple times and adjusting the amount of solvent, incomplete extraction due to insufficient solvent can be avoided, thereby ensuring the stability of the extraction rate. Experimental data show that with this combination of water addition volume and three extraction times, the average yield of crude polysaccharides from Agaricus fasciatus is relatively stable, reaching more than 4%, and a high-yield crude polysaccharide extract of Agaricus fasciatus can be stably obtained.

[0027] As an implementable case, the alcohol precipitation of the crude polysaccharide of Agaricus keiskei includes: adding 4-8 times the volume of ethanol to the crude polysaccharide of Agaricus keiskei, shaking it well, and then refrigerating it; centrifuging it at 2000-3000rpm, filtering and collecting the precipitate, adding ethanol to the precipitate, centrifuging it, repeating 2-3 times, collecting and combining the precipitates.

[0028] Furthermore, the volume concentration of the ethanol is 90-98%.

[0029] Furthermore, the volume concentration of the ethanol is 95%.

[0030] The present invention preferably uses 95% ethanol by volume to carry out alcohol precipitation treatment on the crude polysaccharide of Agaricus oleifera, mainly because 95% ethanol by volume can effectively destroy the intermolecular forces such as hydrogen bonds in the cell wall, thereby promoting the dissolution of the crude polysaccharide of Agaricus oleifera, while low-concentration ethanol has strong polarity and weak interaction with polysaccharides, and it is difficult to achieve the same effect; at the same time, high-concentration ethanol can significantly reduce the solubility of polysaccharides, so that they are fully precipitated, while low-concentration ethanol is prone to polysaccharide residues due to insufficient precipitation driving force; in addition, 95% ethanol can more efficiently dissolve low-polarity impurities and optimize separation selectivity by changing the polarity and viscosity of the extract, thereby improving the purity and extraction rate of the crude polysaccharide; its hydrogen bonding effect with the polysaccharide molecules can further promote the extraction efficiency, and the overall performance is far superior to that of low-concentration ethanol.

[0031] As an implementable case, the drying temperature is 35-50°C and the drying time is 24-48h.

[0032] A second aspect of the present invention provides a method for preparing a crude polysaccharide preparation of Agaricus scabra, comprising:

[0033] Mix the wax and natural oil, then heat to 85-100°C. When the wax is completely melted, stop heating and let the temperature drop naturally. When the temperature drops to 70-80°C, add the Agaricus oleifera crude polysaccharide, mix well, and cool to 20-30°C to obtain the Agaricus oleifera crude polysaccharide preparation.

[0034] The present invention stipulates that after the wax is melted, the temperature needs to be lowered before adding the crude polysaccharide of Agaricus oxysporum. This is mainly because the crude polysaccharide of Agaricus oxysporum can undergo structural destruction or reduced activity at high temperatures. Under high temperature conditions, glycosidic bond cleavage and molecular structure denaturation may occur, thereby affecting its biological activity and efficacy in the preparation. The crude polysaccharide with reduced activity cannot effectively inhibit the capillary dilation and increased permeability caused by histamine. If the crude polysaccharide is directly added at high temperature without cooling, it will also cause the crude polysaccharide particles of Agaricus oxysporum to be unevenly dispersed in the matrix. The viscosity of the mixture of beeswax and peanut oil at high temperature is low and the fluidity is strong. After the crude polysaccharide of Agaricus oxysporum is added, it may quickly gather together to form larger clumps, which are difficult to fully mix with the matrix. This will not only affect the uniformity and stability of the preparation, but also affect the distribution and absorption of the drug on the skin, reducing the regulatory effect on the permeability of skin blood vessels.

[0035] As an practicable case, when the natural oil is lanolin and the wax is vaseline, the preparation method of the crude polysaccharide preparation of Agaricus scabra includes:

[0036] Mix vaseline and lanolin, then heat to 70°C. When the wax is completely melted, add Agaricus oleifera crude polysaccharide, mix well, and cool to 20-30°C to obtain Agaricus oleifera crude polysaccharide preparation.

[0037] Beneficial effects

[0038] (1) The present application adopts a specific water extraction method and alcohol precipitation method, using water as the solvent. After three extractions and optimizing the amount of extraction solvent, extraction time and alcohol precipitation solvent, the extraction rate of crude polysaccharides of Agaricus scabra can be stably increased to 4.15%, effectively solving the problem of unstable extraction efficiency in the prior art.

[0039] (2) The crude polysaccharide preparation obtained by optimizing the extraction process and preparation formula in this application is significantly effective in inhibiting the increase in skin capillary permeability caused by histamine, and is superior to the existing stubborn tinea versicolor ointment, indicating that it has stronger pharmacological activity in anti-inflammatory and exudation inhibition.

[0040] (3) This application determines that the optimal mass ratio of wax and natural oil is 1:3, which makes the matrix moderately soft and hard and has good ductility, while maintaining the consistency with the original stubborn tinea versicolor ointment, which is beneficial to the preparation and use of the preparation and improves the drug release and skin absorption effect.

[0041] (4) This application makes the preparation easier to apply and absorb by adjusting the matrix components and proportions, and increasing the amount of crude polysaccharide of Agaricus oleifera, reducing the greasy feeling and pollution to clothes, and improving the convenience of use and patient compliance.

[0042] (5) This application further defines the process steps of cooling the mixture after the natural oil and wax are melted and then adding the crude polysaccharide of Agaricus keiskei. This can ensure the skin vascular permeability of the crude polysaccharide preparation of Agaricus keiskei, and the product has a more excellent practical use effect. DETAILED DESCRIPTION

[0043] Example 1

[0044] In the first aspect of this example, a crude polysaccharide preparation of Agaricus oxysporum is provided, and the raw materials for its preparation are specifically as follows by mass: 1.70 g crude polysaccharide of Agaricus oxysporum, 31.25 g peanut oil, and 10.4 g beeswax.

[0045] The preparation method of the crude polysaccharide of Agaricus scabra is as follows:

[0046] Extraction of crude polysaccharides from Agaricus scabra by water extraction:

[0047] S1. Take 10 g of Agaricus sutchuenensis medicinal material, remove the dust on the surface, grind and sieve through a 100-mesh sieve to obtain Agaricus sutchuenensis fine powder, place it in a 2000 mL round-bottom flask, add 1500 mL of water and soak for 1 hour to obtain an Agaricus sutchuenensis aqueous solution;

[0048] S2, heating the aqueous solution of Agaricus oleifera to reflux at 105° C. for 2 h, filtering it while hot, and collecting the first filter residue and the first filtrate;

[0049] S3. Add 1000 mL of water to the filter residue, heat under reflux at 105°C for 2 h, filter while hot, and collect the second filter residue and the second filtrate;

[0050] S4. Add 1000 mL of water to the filter residue, heat under reflux at 105°C for 2 h, filter while hot, and collect the third filter residue and the third filtrate;

[0051] S5. Combine the first filtrate, the second filtrate and the third filtrate, concentrate under reduced pressure to a volume of 200 mL, and collect the concentrate to obtain crude Agaricus scabra polysaccharide.

[0052] Alcohol precipitation of Agaricus sutchuenensis crude polysaccharide: 1 L of 95% ethanol (volume concentration) was added to the Agaricus sutchuenensis crude polysaccharide, shaken, and refrigerated at 4°C for 24 hours; then centrifuged at 3000 rpm for 10 minutes, filtered to collect the precipitate, and discarded the supernatant; 100 mL of ethanol was added to the precipitate, and centrifuged. Repeat this process three times to collect the Agaricus sutchuenensis crude polysaccharide precipitate;

[0053] Purification and drying of crude polysaccharides of Agaricus oleifera: The crude polysaccharides of Agaricus oleifera were precipitated and purified, and placed in a weighed weighing bottle. The weighing bottle was opened and placed in a vacuum drying oven with a vacuum degree of 0 MPa and a temperature of 40°C. It was dried for 24 hours. The bottle was taken out, stoppered, and weighed to obtain the crude polysaccharide extract of Agaricus oleifera.

[0054] The second aspect of this example provides a method for preparing a crude polysaccharide preparation of Agaricus scabra, specifically:

[0055] Mix beeswax and peanut oil, then heat to 90°C. When the beeswax is completely melted, stop heating and allow to cool naturally. When the temperature drops to 80°C, add Agaricus keiskei crude polysaccharide, mix well, and cool to 25°C to obtain Agaricus keiskei crude polysaccharide preparation.

[0056] Example 2

[0057] In the first aspect of this example, a crude polysaccharide preparation of Agaricus oxysporum is provided, and the raw materials for its preparation are specifically: 1.70g crude polysaccharide of Agaricus oxysporum, 0.867g lanolin, and 40.78g vaseline, calculated by mass.

[0058] The preparation method of the crude polysaccharide of Agaricus scabra is as follows:

[0059] Extraction of crude polysaccharides from Agaricus scabra by water extraction:

[0060] S1. Take 10 g of Agaricus sutchuenensis medicinal material, remove the dust on the surface, grind and sieve through a 100-mesh sieve to obtain Agaricus sutchuenensis fine powder, place it in a 2000 mL round-bottom flask, add 1500 mL of water and soak for 1 hour to obtain an Agaricus sutchuenensis aqueous solution;

[0061] S2, heating the aqueous solution of Agaricus oleifera to reflux at 105° C. for 2 h, filtering it while hot, and collecting the first filter residue and the first filtrate;

[0062] S3. Add 1000 mL of water to the filter residue, heat under reflux at 105°C for 2 h, filter while hot, and collect the second filter residue and the second filtrate;

[0063] S4. Add 1000 mL of water to the filter residue, heat under reflux at 105°C for 2 h, filter while hot, and collect the third filter residue and the third filtrate;

[0064] S5. Combine the first filtrate, the second filtrate and the third filtrate, concentrate under reduced pressure to a volume of 200 mL, and collect the concentrate to obtain crude Agaricus scabra polysaccharide.

[0065] Alcohol precipitation of Agaricus sutchuenensis crude polysaccharide: 1 L of 95% ethanol (volume concentration) was added to the Agaricus sutchuenensis crude polysaccharide, shaken, and refrigerated at 4°C for 24 hours; then centrifuged at 3000 rpm for 10 minutes, filtered to collect the precipitate, and discarded the supernatant; 100 mL of ethanol was added to the precipitate, and centrifuged. Repeat this process three times to collect the Agaricus sutchuenensis crude polysaccharide precipitate;

[0066] Purification and drying of crude polysaccharides of Agaricus oleifera: The crude polysaccharides of Agaricus oleifera were precipitated and purified, and placed in a weighed weighing bottle. The weighing bottle was opened and placed in a vacuum drying oven with a vacuum degree of 0 MPa and a temperature of 40°C. It was dried for 24 hours. The bottle was taken out, stoppered, and weighed to obtain the crude polysaccharide extract of Agaricus oleifera.

[0067] The second aspect of this example provides a method for preparing a crude polysaccharide preparation of Agaricus scabra, specifically:

[0068] Mix vaseline and lanolin, then heat to 70°C. When the vaseline is completely melted, add Agaricus keiskei crude polysaccharide, mix well, and cool to 25°C to obtain Agaricus keiskei crude polysaccharide preparation.

[0069] Example 3

[0070] The specific implementation of this example is the same as that of Example 1, except that the natural oil is almond oil (Prunus armeniaca L.).

[0071] Comparative Example 1

[0072] This example provides a blank ointment base, the raw materials for its preparation are, by mass, 31.25 g peanut oil and 10.4 g beeswax. The blank ointment base is prepared by mixing peanut oil and beeswax, heating to 90°C until the beeswax is completely melted, and then stirring to obtain the blank ointment base.

[0073] Comparative Example 2

[0074] The first aspect of this example provides a stubborn tinea versicolor ointment, the raw materials for its preparation are, by weight, specifically:

[0075] 8.35g willow mushroom powder, 31.25g peanut oil, 10.4g beeswax.

[0076] The preparation method of the willow mushroom fine powder is as follows: dust is removed from the willow mushroom medicinal material, and the willow mushroom fine powder is obtained by grinding and sieving the medicinal material through a 100-mesh sieve.

[0077] The second aspect of this example provides a preparation method of a stubborn tinea versicolor ointment, specifically:

[0078] Mix beeswax and peanut oil, then heat to 90℃. When the beeswax is completely melted, stop heating and let it cool naturally. When it cools to 80℃, add willow mushroom powder, mix well, and cool to 25℃ to obtain the stubborn tinea ointment.

[0079] Comparative Example 3

[0080] In the first aspect of this example, a crude polysaccharide preparation of Agaricus oxysporum is provided, and the raw materials for its preparation are specifically as follows by mass: 1.70 g crude polysaccharide of Agaricus oxysporum, 31.25 g peanut oil, and 10.4 g beeswax.

[0081] The preparation method of the crude polysaccharide of Agaricus scabra is as follows:

[0082] Extraction of crude polysaccharides from Agaricus scabra by water extraction:

[0083] S1. Take 10 g of Agaricus sutchuenensis medicinal material, remove the dust on the surface, grind and sieve through a 100-mesh sieve to obtain Agaricus sutchuenensis fine powder, place it in a 2000 mL round-bottom flask, add 1500 mL of water and soak for 1 hour to obtain an Agaricus sutchuenensis aqueous solution;

[0084] S2, ultrasonically treating the aqueous solution of Agaricus sutchuenensis for 1 h at an ultrasonic power of 250 W and an ultrasonic frequency of 50 kHz; then filtering to collect a first filter residue and a first filtrate;

[0085] S3, add 1000 mL of water to the filter residue, ultrasonicate for 1 h, ultrasonic power 250 W, ultrasonic frequency 50 kHz, filter, and collect the second filter residue and the second filtrate;

[0086] S4, adding 1000 mL of water to the filter residue, ultrasonically treating for 1 h at an ultrasonic power of 250 W and an ultrasonic frequency of 50 kHz, filtering, and collecting the third filter residue and the third filtrate;

[0087] S5. Combine the first filtrate, the second filtrate and the third filtrate, concentrate under reduced pressure to a volume of 200 mL, and collect the concentrate to obtain crude Agaricus scabra polysaccharide.

[0088] Alcohol precipitation of Agaricus sutchuenensis crude polysaccharide: 1 L of 95% ethanol (volume concentration) was added to the Agaricus sutchuenensis crude polysaccharide, shaken, and refrigerated at 4°C for 24 hours; then centrifuged at 3000 rpm for 10 minutes, filtered to collect the precipitate, and discarded the supernatant; 100 mL of ethanol was added to the precipitate, and centrifuged. Repeat this process three times to collect the Agaricus sutchuenensis crude polysaccharide precipitate;

[0089] Purification and drying of crude polysaccharides of Agaricus oleifera: The crude polysaccharides of Agaricus oleifera were precipitated and purified, and placed in a weighed weighing bottle. The weighing bottle was opened and placed in a vacuum drying oven with a vacuum degree of 0 MPa and a temperature of 40°C. It was dried for 24 hours. The bottle was taken out, stoppered, and weighed to obtain the crude polysaccharide extract of Agaricus oleifera.

[0090] The second aspect of this example provides a method for preparing a crude polysaccharide preparation of Agaricus scabra, specifically:

[0091] Mix beeswax and peanut oil, then heat to 90°C. When the beeswax is completely melted, stop heating and allow to cool naturally. When the temperature drops to 80°C, add Agaricus keiskei crude polysaccharide, mix well, and cool to 25°C to obtain Agaricus keiskei crude polysaccharide preparation.

[0092] Comparative Example 4

[0093] The specific implementation of this example is the same as that of Example 1, except that the volume concentration of ethanol is 30%.

[0094] Comparative Example 5

[0095] In the first aspect of this example, a crude polysaccharide preparation of Agaricus oxysporum is provided, and the raw materials for its preparation are specifically as follows by mass: 1.70 g crude polysaccharide of Agaricus oxysporum, 31.25 g peanut oil, and 10.4 g beeswax.

[0096] The preparation method of the crude polysaccharide of Agaricus scabra is as follows:

[0097] Extraction of crude polysaccharides from Agaricus scabra by water extraction:

[0098] S1. Take 10 g of Agaricus sutchuenensis medicinal material, remove the dust on the surface, grind and sieve through a 100-mesh sieve to obtain Agaricus sutchuenensis fine powder, place it in a 2000 mL round-bottom flask, add 1500 mL of water and soak for 1 hour to obtain an Agaricus sutchuenensis aqueous solution;

[0099] S2, heating the aqueous solution of Agaricus oleifera to reflux at 105° C. for 2 h, filtering it while hot, and collecting the first filter residue and the first filtrate;

[0100] S3, add 1000 mL of water to the filter residue, heat under reflux at 105°C for 1 h, filter while hot, and collect the second filter residue and the second filtrate;

[0101] S4. Combine the first filtrate and the second filtrate, concentrate under reduced pressure to a volume of 200 mL, and collect the concentrate to obtain crude Agaricus scabra polysaccharide.

[0102] Alcohol precipitation of Agaricus sutchuenensis crude polysaccharide: 1 L of 95% ethanol (volume concentration) was added to the Agaricus sutchuenensis crude polysaccharide, shaken, and refrigerated at 4°C for 24 hours; then centrifuged at 3000 rpm for 10 minutes, filtered to collect the precipitate, and discarded the supernatant; 100 mL of ethanol was added to the precipitate, and centrifuged. Repeat this process three times to collect the Agaricus sutchuenensis crude polysaccharide precipitate;

[0103] Purification and drying of crude polysaccharides of Agaricus oleifera: The crude polysaccharides of Agaricus oleifera were precipitated and purified, and placed in a weighed weighing bottle. The weighing bottle was opened and placed in a vacuum drying oven with a vacuum degree of 0 MPa and a temperature of 40°C. It was dried for 24 hours. The bottle was taken out, stoppered, and weighed to obtain the crude polysaccharide extract of Agaricus oleifera.

[0104] The second aspect of this example provides a method for preparing a crude polysaccharide preparation of Agaricus scabra, comprising:

[0105] Mix beeswax and peanut oil, then heat to 90°C. When the beeswax is completely melted, stop heating and allow to cool naturally. When the temperature drops to 80°C, add Agaricus keiskei crude polysaccharide, mix well, and cool to 25°C to obtain Agaricus keiskei crude polysaccharide preparation.

[0106] Comparative Example 6

[0107] The specific implementation of this example is the same as that of Example 1, except that 1000 mL of water is added in step S1, 700 mL of water is added in step S3, and 700 mL of water is added in step S4.

[0108] Comparative Example 7

[0109] The specific implementation of this example is the same as that of Example 1, except that the raw materials include 27.8 g of peanut oil and 13.9 g of beeswax.

[0110] Comparative Example 8

[0111] The specific implementation of this example is the same as that of Example 1, except that the raw materials include 33.32 g of peanut oil and 8.33 g of beeswax.

[0112] Comparative Example 9

[0113] The specific implementation of this example is the same as that of Example 1, except that the raw materials for its preparation include, by mass, 1.70 g of Agaricus oleifera crude polysaccharide, 31.25 g of vaseline, and 10.4 g of liquid paraffin.

[0114] Comparative Example 10

[0115] The specific implementation method of this example is the same as that of Example 1, except that the preparation method of the Agaricus limonene crude polysaccharide preparation is as follows: beeswax and peanut oil are mixed, and then heated to 90°C. When the beeswax is completely melted, the Agaricus limonene crude polysaccharide is directly added without waiting for cooling, mixed evenly, and cooled to 25°C to obtain the Agaricus limonene crude polysaccharide preparation.

[0116] In the above Examples 1-3 and Comparative Examples 1-10, the medicinal materials of Agaricus oleifera, peanut oil, and beeswax were provided by Tianjin Darentang Jingwanhong Pharmaceutical Co., Ltd., 95% ethanol was purchased from Tianjin Kangkede Technology Co., Ltd., and water was Milli-Q ultrapure water.

[0117] Performance evaluation

[0118] 1. Stability test

[0119] According to the preparation method of crude polysaccharide of Agaricus oleifera in Example 1, crude polysaccharide of Agaricus oleifera was prepared in three groups, and the raw material each time was 70g of Agaricus oleifera medicinal material. The yield was then calculated according to the formula "yield % = (weight of crude polysaccharide of Agaricus oleifera / weight of fine powder of Agaricus oleifera medicinal material) × 100%", and the average yield was further obtained. The results are shown in Table 1.

[0120] Table 1

[0121]

[0122] From the experimental results in Table 1, it can be seen that the yield of the preparation method of Agaricus oleifera crude polysaccharide provided by the present invention is greater than 4%, and the average yield of three extractions is 4.15% (RSD=0.91%). The three extraction yields are close, indicating that the extraction efficiency of the method is stable and the method is highly feasible.

[0123] 2. Yield test

[0124] Calculate the yields of Examples 1-3 according to the formula "yield % = (weight of crude polysaccharide from willow mushroom / weight of fine powder of willow mushroom medicinal material) × 100%", and the yields of crude polysaccharide from willow mushroom in Comparative Examples 3-10. The experimental results are shown in Table 2 for details.

[0125] Table 2

[0126]

[0127]

[0128] It can be seen from the experimental results in Table 2 that the yields of crude polysaccharide from willow mushroom in Examples 1-3 of the present invention are all greater than 4%, while in Comparative Examples 3-6, due to the influence of preparation parameters in the preparation process, the yields of crude polysaccharide products from willow mushroom are lower than 4%.

[0129] 3. Vascular permeability study

[0130] 3.1 Research purpose: By applying the test products of crude polysaccharide preparations from willow mushroom in Examples 1-3, Comparative Examples 3-10, blank matrix ointment in Comparative Example 1, and Wuanxuedi ointment in Comparative Example 2, evaluate and compare the effects on the skin vascular permeability of mice; among which Comparative Example 1 is the negative control group and Comparative Example 2 is the positive control 1.

[0131] 3.2 Experimental reagents

[0132] The reagents used in the experiment are shown in Table 3 for details.

[0133] Table 3

[0134] name batch number Specification Production Unit Evans Blue E808783 10g / bottle Macklin's reagent Histamine phosphate Q / AKF 78-6-93 5g / bottle Shanghai Livzon Dongfeng Biotechnology Co., Ltd. acetone CAS 67-64-1 500mL / bottle Maoming Runjing Chemical Co., Ltd. Normal saline 23H92K4 10mL / bottle China Otsuka Pharmaceutical Co., Ltd.

[0135] 3.3 Test animals

[0136] Species and strain: CD-1 (ICR) mice;

[0137] Quantity: 30 (15 males and 15 females);

[0138] Source: Beijing Vital River Laboratory Animal Technology Co., Ltd.;

[0139] License number: SCXK (Beijing) 2021-0006;

[0140] Animal certificate number: male: 110011241109870884; female: 110011241109870737.

[0141] 3.4 Animal feeding conditions

[0142] Raised under barrier environment;

[0143] Experimental Animal Use License Number: SYXK(Tianjin)2023-0006;

[0144] Temperature and humidity of the breeding room: temperature: 20-26℃, relative humidity: 40-70%;

[0145] Minimum ventilation rate: ≥15 times / h;

[0146] Lighting time: 12 h light / 12 h dark cycle (lights on at 7:00 am and off at 7:00 pm);

[0147] Cage type: PP polypropylene mouse box (length × width × height: 30 cm × 19 cm × 15 cm);

[0148] Stocking density: 5 per box;

[0149] Cage replacement frequency: Change the mouse box twice a week;

[0150] Animal drinking water: sterile water, drinking water in water bottles, free intake.

[0151] Animal feed: SPF growth and breeding feed for mice and rats, Keao Xieli (Tianjin) Feed Co., Ltd., license number: SCXK (Tianjin) 2020-0004.

[0152] 3.5 Related experimental instruments

[0153] The experimental instruments involved in this experiment are detailed in Table 4.

[0154] Table 4

[0155] Instrument name model Manufacturer Equipment Number electronic balance XS205DU Mettler 1208WL091 microplate reader Varioskan LUX Thermo Fisher Scientific 2107GP029 Hot air circulation oven FD56 BINDER, Germany 1610WD024

[0156] 3.6 Statistical Analysis

[0157] SPSS 26.0 software was used for statistics, and the statistical data were expressed as ± SD.

[0158] 3.7 Test methods and results

[0159] 3.7.1. Effects of test drugs on histamine-induced capillary permeability in mouse skin (Evans blue method).

[0160] 3.7.1.1. Thirty mice were randomly divided into three groups, each consisting of 10 mice, half male and half female. One day before the experiment, the mice were shaved on their backs, covering 20% of their total body surface area.

[0161] 3.7.1.2 On the day of the experiment, the three groups of mice were smeared with the test drug on the shaved area, with a dose of 0.1g test substance / 20g mouse body weight, twice a day for 3 consecutive days.

[0162] 3.7.1.3. One hour after the last administration, clean the drug from the shaved area of the mice and inject each mouse subcutaneously with 100 μL of 10 μg / mL histamine phosphate in saline at the administration site. Immediately after injection, inject 0.5% Evans blue in saline into the tail vein at a concentration of 10 mL / kg, i.e., inject 250 μL intravenously for a 25 g mouse. 15 minutes later, sacrifice the mouse by dislocation.

[0163] 3.7.1.4. Cut a 2 cm × 2 cm area of mouse dorsal skin covering the blue-stained area. After mincing, soak in 2 mL of acetone-saline (7:3) solution. Incubate at 45°C for 96 h until the blue color of the skin disappears completely. Centrifuge at 1500 rpm for 10 min. Take 200 μL of the supernatant and measure the absorbance at 610 nm using a spectrophotometer.

[0164] 3.8 Test results

[0165] After subcutaneous injection of histamine phosphate at the dorsal skin site of mice, Evans blue was injected intravenously, and circular blue staining was observed, indicating the model was established. The absorbance of the positive control group (Comparative Example 2, Wanxuandi Ointment) was lower than that of the negative control group (Comparative Example 1, Blank Matrix), with a significant difference of P < 0.05. The absorbance of the test substance group, Example 1, was lower than that of both the negative control group (Blank Matrix) and the positive control group (Wanxuandi Ointment), with a significant difference of P < 0.01. The results are shown in Table 5.

[0166] Table 5

[0167] Skin vascular permeability (absorbance) Example 1 <![CDATA[0.12±0.02 **，▲▲ ]]> Example 2 0.09±0.01 Example 3 0.10±0.02 Comparative Example 1 0.18±0.03 Comparative Example 2 <![CDATA[0.16±0.02 * ]]> Comparative Example 3 0.11±0.02 Comparative Example 4 0.12±0.03 Comparative Example 5 0.10±0.01 Comparative Example 6 0.09±0.01 Comparative Example 7 0.14±0.03 Comparative Example 8 0.15±0.04 Comparative Example 9 0.15±0.05 Comparative Example 10 0.16±0.02

[0168] Note: 1. * indicates: compared with the negative control group (control group 1), statistically analyzed using SPSS 26.0 software, P < 0.05; ** indicates: compared with the negative control group (control group 1), statistically analyzed using SPSS 26.0 software, P < 0.01;

[0169] 2. ▲▲ indicates: P < 0.01 when the test substance group Example 1 is compared with the positive control group Comparative Example 2, using SPSS 26.0 software for statistical analysis.

[0170] From the experimental results in Table 5, it can be seen that the absorbances of Comparative Examples 1-2 and Comparative Examples 7-10 are relatively high, so it can be seen that the skin vascular permeability of the corresponding Agaricus limonene crude polysaccharide preparations is relatively poor, while the absorbance experimental value in Example 1 is 0.12±0.02, which can effectively inhibit the skin capillary permeability caused by histamine; indicating that in terms of anti-inflammatory and exudation inhibition, the Agaricus limonene crude polysaccharide extract is a clear and effective ingredient.

[0171] 4. Quality standards for crude polysaccharide preparations of Agaricus scabra

[0172] Quality standards were established for homemade crude polysaccharide preparations of Agaricus salix.

[0173] The drafting work is described as follows:

[0174] 4.1. Sample condition: The crude polysaccharide preparation of Agaricus oleifera in Example 1.

[0175] 4.2. Prescription: 65g of crude polysaccharide of Agaricus oxyphylla; 400g of beeswax.

[0176] The crude polysaccharide in the polysaccharide preparation is added at 5 times the amount of polysaccharide contained in the original Wanxuandi ointment. The amount of beeswax added remains unchanged.

[0177] 4.3. Preparation method: Grind the crude polysaccharide of Agaricus oxysporum into fine powder, sieve and set aside. Heat 1200g peanut oil in a pot, add beeswax and melt it. When the temperature drops to 100-80℃, add the crude powder of Agaricus oxysporum, stir evenly and wait for it to cool.

[0178] Refer to the recipe for Wanxuandi Ointment, replacing the fine powder of Agaricus scabra with crude Agaricus scabra polysaccharide, and keeping the amount of vegetable oil unchanged. The text description complies with the relevant requirements of the 2020 edition of the Chinese Pharmacopoeia, Part I.

[0179] 4.4. Properties: This product is a light brown to tan semi-solid ointment.

[0180] It was formulated based on the characteristics of polysaccharide preparations.

[0181] 4.5. Inspection: It should comply with the relevant provisions under ointments (General Rule 0109).

[0182] 4.6. Content Assay: Since the crude polysaccharide from Agaricus oxysporum is used as raw material in the preparation, a total polysaccharide content assay was established to control the preparation quality. The established method can accurately and effectively reflect the intrinsic quality of the finished product.

[0183] 4.7 Instruments and Test Drugs

[0184] Instrument: Hitachi U-3900 UV-visible spectrophotometer; anhydrous ethanol, concentrated sulfuric acid, and phenol were of analytical grade, and water was deionized water; D-anhydrous glucose reference substance was purchased from the China Food and Drug Inspection Institute (for content determination), batch number: 110833-202109, 99.9%.

[0185] 4.8. Selection and determination of measurement conditions

[0186] 4.8.1. Selection of detection wavelength: This experiment uses the phenol-sulfuric acid method to determine the total polysaccharide content, and the detection wavelength of this method is 490nm.

[0187] 4.8.2 Determination method: Accurately measure 2 mL of the test solution and place it in a 10 mL stoppered test tube. Accurately add 1 mL of 6 wt% phenol solution and shake well. Quickly and accurately add 7 mL of concentrated sulfuric acid and immediately shake well. After keeping warm in a 60°C water bath for 30 minutes, immediately cool in an ice bath for 15 minutes. Remove the sample and measure the absorbance at a wavelength of 490 nm using the corresponding reagent as a blank according to the UV-Vis spectrophotometric method (General Method 0401). Read the anhydrous glucose content in the test solution from the standard curve and calculate it.

[0188] 4.9. Investigation of extraction conditions: After searching the literature and the relevant content of the 2020 edition of the Chinese Pharmacopoeia, we designed an investigation on factors such as extraction method, solvent dosage, and extraction time. The results are as follows:

[0189] 4.9.1. Examination of Extraction Methods: Accurately weigh 3g of the same sample and place it in a mortar. Accurately weigh an equal amount of diatomaceous earth and place it in a mortar, then grind until homogenous. Accurately weigh approximately 2g of the above mixture and place it in a stoppered conical flask. Accurately add 50mL of water and weigh the weight. Extract for 40 minutes using water bath reflux and ultrasound (power 250W, frequency 50kHz), respectively. Cool, weigh again, make up the lost volume with water, shake well, and filter. Accurately measure 5mL of the filtrate and place it in a 50mL volumetric flask. Dilute with water and bring the volume to the mark. Shake well to obtain the extract.

[0190] Take the above solution and determine the polysaccharide content according to the method in "4.8.2". The results are shown in Table 6.

[0191] Table 6

[0192] Extraction method Water bath reflux Ultrasound Total polysaccharide content (mg / g) 16.9975 2.6818

[0193] The results showed that water bath reflux extraction could extract a larger amount of polysaccharides, while ultrasound extraction was unable to extract most polysaccharides. This may be because temperature plays a key role in the extraction process. Heating can better dissolve polysaccharides and improve extraction efficiency. Therefore, water bath heating reflux extraction was determined to be the extraction method used for the test sample.

[0194] 4.9.2. Investigation of solvent usage: Take 5g of the same sample (Example 1, Agaricus crude polysaccharide preparation), accurately weigh it, and place it in a mortar. Then take an equal amount of diatomaceous earth, accurately weigh it, place it in a mortar, and grind it evenly. Take approximately 2g of the above mixture, accurately weigh it, place it in a stoppered conical flask, accurately add 25, 50, and 100mL of water, weigh it, and extract it in a water bath under reflux for 40 minutes. Let it cool, weigh it again, make up the lost weight with water, shake it well, and filter it. Accurately measure 10, 5, and 2.5mL of the above filtrate, respectively, place it in a 50mL volumetric flask, dilute it with water and make up to the mark, shake it well, and the solution is ready. Take the above solution and determine the polysaccharide content according to the method under "4.8.2". The results are shown in Table 7.

[0195] Table 7

[0196] Solvent usage (mL) 25 50 100 Total polysaccharide content (mg / g) 14.8028 15.5798 15.2528

[0197] The results showed that the polysaccharide content was highest when the solvent dosage was 50 mL. Further increasing the extraction solvent dosage may cause the content to decrease due to loss and increased error. Therefore, the solvent dosage was determined to be 50 mL.

[0198] 4.9.3. Investigation of sampling time: Take 5 g of the same sample, weigh it accurately, put it in a mortar, then take an equal amount of diatomaceous earth, weigh it accurately, put it in a mortar, and grind it evenly. Take about 2 g of the above mixture, weigh it accurately, put it in a stoppered conical flask, accurately add 50 mL of water, weigh it, and use a water bath to reflux and extract for 20, 40, and 60 minutes respectively. Let it cool, weigh it again, make up the lost weight with water, shake it well, and filter it. Accurately measure 5 mL of the above filtrate, put it in a 50 mL volumetric flask, dilute it with water and make up to the scale, shake it well, and you have it. Take the above solution and determine the polysaccharide content according to the method under "4.8.2". The results are shown in Table 8

[0199] Table 8

[0200] Extraction time (minutes) 20 40 60 Total polysaccharide content (mg / g) 13.8176 14.4776 14.7495

[0201] The results showed that the polysaccharide contents were similar when the extraction time was 40 minutes and 60 minutes (RAD% = 0.9%), so the extraction time was determined to be 40 minutes.

[0202] 4.10 Solution Preparation

[0203] 4.10.1 Preparation of reference substance stock solution: Take 30 mg of D-anhydrous glucose reference substance (purity 99.9%), accurately weigh it, place it in a 50 mL volumetric flask, add water to dissolve it, and dilute it to the scale. Shake well to prepare the reference substance stock solution.

[0204] 4.10.2 Preparation of Test Solution: Accurately weigh 5 g of the same sample (Example 1, Agaricus scabra crude polysaccharide preparation) and place in a mortar. Accurately weigh an equal amount of diatomaceous earth and place in a mortar. Grind thoroughly. Accurately weigh approximately 2 g of the above mixture and place in a stoppered conical flask. Accurately add 50 mL of water and weigh the weight. Heat under reflux for 40 minutes, cool, weigh again, make up the lost volume with water, shake well, and filter. Accurately measure 5 mL of the above filtrate and place in a 50 mL volumetric flask. Dilute with water and bring to volume. Shake well.

[0205] 4.10.3 Preparation of Negative Sample Solution: Prepare all the ingredients according to the prescription, except for the crude polysaccharide from Agaricus serrata, and prepare a preparation. Then, follow the preparation method for the test solution in "4.8.2" to prepare a negative sample solution lacking the crude polysaccharide from Agaricus serrata. The test results indicate that the negative sample has no interference.

[0206] 4.11 Preparation of standard curve

[0207] Accurately measure 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, and 1.2mL of the reference stock solution into 10mL volumetric flasks, dilute to the mark with water, and shake well to prepare a series of standard curve solutions. Accurately measure 2mL of each of the above solutions into 10mL stoppered test tubes, accurately add 1mL of 6% phenol solution, shake well, and quickly and accurately add 7mL of concentrated sulfuric acid. Shake well immediately. Incubate in a 60°C water bath for 30 minutes, then immediately cool in an ice bath for 15 minutes. Remove from heat and measure the absorbance at 490nm using the corresponding reagent as a blank according to UV-Vis spectrophotometry (General Method 0401). Plot the standard curve using absorbance as the ordinate and concentration as the abscissa. Obtain the regression equation: Y = 0.0110X + 0.0117, r = 0.9992. The results showed that D-anhydrous glucose had a good linear relationship in the concentration range of 12.938 μg / mL to 77.628 μg / mL. The results are shown in Table 9.

[0208] Table 9

[0209]

[0210] 4.12 Repeatability test

[0211] Six aliquots of the same sample were prepared according to the test solution preparation method in "4.8.2." The total polysaccharide content was determined according to the method in "6.2.2." The average total polysaccharide content was 18.0493 mg / g, with an RSD of 1.30%. The results are shown in Table 10.

[0212] Table 10

[0213]

[0214] 4.13 Precision test

[0215] Take one aliquot of the test solution after color development in the repeatability test in Section 4.12 and measure the absorbance at 490 nm for six consecutive measurements. The average absorbance was 0.413, with an RSD of 0.10%. The results are shown in Table 11.

[0216] Table 11

[0217]

[0218] 4.14 Stability test

[0219] Take one aliquot of the test solution developed in the repeatability test described in Section 4.12 and measure its absorbance at 490 nm at 0, 20, 40, 60, and 90 minutes. The average absorbance was 0.409, and the RSD was 0.82%. The results indicate that the test solution was stable over the 90-minute measurement period. See Table 12 for the results.

[0220] Table 12

[0221] Time (minutes) 0 20 40 60 90 Absorbance (Abs) 0.408 0.413 0.405 0.406 0.411

[0222] Take D-anhydrous glucose reference substance, accurately weigh it, add water to make a solution containing 0.1517 mg of D-anhydrous glucose per 1 mL, and make a reference solution for sample recovery test. Take 3 g of the same sample, accurately weigh it, and place it in a mortar. Then take an equal amount of diatomaceous earth, accurately weigh it, place it in a mortar, and grind it evenly. Take about 2 g of the above mixture, accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of the reference solution for recovery test, weigh it, heat and reflux to extract for 40 minutes, let it cool, weigh it again, make up the lost weight with water, shake it evenly, and filter it. Accurately measure 5 mL of the above filtrate, place it in a 50 mL volumetric flask, dilute it with water and make up to the scale,

[0223] Shake well to obtain the product. The above solution was then used to determine the polysaccharide content. The average recovery was 97.42% with an RSD of 1.39%. The results are shown in Table 13.

[0224] Table 13

[0225]

[0226] 4.15 Determination of content limits

[0227] According to 50% of the test results, the limit of polysaccharide content in Agaricus sutchuenensis crude polysaccharide should be about 18.0493×50%=9.0mg / g: Therefore, it is tentatively determined that each 1g of this product contains polysaccharide in the form of anhydrous glucose (C6H 12 O6), not less than 9.0 mg.

Claims

1. A crude polysaccharide preparation of Agaricus scabra, characterized in that: The raw materials for preparation include 1-10 parts of crude Agaricus liliiflorus polysaccharide, 0.1-50 parts of natural oil and fat, and 10-50 parts of wax in parts by weight.

2. The crude polysaccharide preparation of Agaricus scabra according to claim 1, characterized in that The natural oil comprises one of almond oil, peanut oil, soybean oil, sunflower oil or lanolin.

3. The crude polysaccharide preparation of Agaricus scabra according to claim 2, characterized in that The wax includes beeswax or vaseline.

4. The crude polysaccharide preparation of Agaricus scabra according to claim 3, characterized in that When the natural oil is peanut oil and the wax is beeswax, the mass ratio of the natural oil to the wax is (2.5-3.5):

1.

5. The crude polysaccharide preparation of Agaricus scabra according to claim 1, characterized in that The preparation method of the crude polysaccharide of Agaricus salsa comprises: The crude polysaccharide from Agaricus salsa was extracted by water extraction, and then precipitated with alcohol, purified and dried.

6. The crude polysaccharide preparation of Agaricus scabra according to claim 5, characterized in that The water extraction method for preparing crude polysaccharide of Agaricus scabra includes: S1. Grind the medicinal material of Agaricus serrata, place the Agaricus serrata powder in a flask, add water and soak for 1-2 hours to obtain an Agaricus serrata aqueous solution; S2, heating the aqueous solution of Agaricus sutchuenensis to reflux for 2-3 hours, filtering it while hot, and collecting the first filter residue and the first filtrate; S3, adding water to the first filter residue, heating and reflux for 2-3 hours, filtering while hot, and collecting the second filter residue and the second filtrate; S4, adding water to the second filter residue collected in step S3, heating and refluxing for 2-3 hours, filtering while hot, and collecting the third filter residue and the third filtrate; S5. Combine the first filtrate, the second filtrate and the third filtrate, concentrate under reduced pressure to a volume of 100-200 mL, and collect the concentrate to obtain crude Agaricus scabra polysaccharide.

7. The crude polysaccharide preparation of Agaricus scabra according to claim 6, characterized in that The volume ratio of the amount of water added in step S1, step S3 and step S4 is (1200-1600): (800-1200): (800-1200).

8. The crude polysaccharide preparation of Agaricus salsa according to claim 5, characterized in that The alcohol precipitation comprises: adding 4-8 times the volume of ethanol to the crude polysaccharide of Agaricus oleifera, shaking, and then refrigerating; centrifuging at 2000-3000 rpm, filtering and collecting the precipitate, adding ethanol to the precipitate, centrifuging, repeating 2-3 times, collecting, and combining the precipitates.

9. The crude polysaccharide preparation of Agaricus scabra according to claim 8, characterized in that The volume concentration of the ethanol is 90-98%.

10. A method for preparing the crude polysaccharide preparation of Agaricus salicifolia according to any one of claims 1 to 9, characterized in that: include: Mix the wax and natural oil, then heat to 85-100°C. When the wax is completely melted, stop heating and cool to 70-80°C. Add the crude polysaccharide of Agaricus oleifera, mix well, and cool to 20-30°C to obtain the crude polysaccharide preparation of Agaricus oleifera.

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