CN110840900A

CN110840900A - Application of ergosterol peroxide in agaricus verticillata to CDC25 phosphoprotease

Info

Publication number: CN110840900A
Application number: CN201911125261.5A
Authority: CN
Inventors: 吴疆; 廖志明; 林鹏程
Original assignee: Qinghai Nationalities University
Current assignee: Qinghai Nationalities University
Priority date: 2019-11-18
Filing date: 2019-11-18
Publication date: 2020-02-28

Abstract

The invention discloses an application of active ingredient ergosterol in a sporotrichum mushroom, which is characterized in that the active ingredients of the sporotrichum mushroom are quickly separated by the separation and extraction method, the separation and extraction process is simple and stable, the sporotrichum mushroom is suitable for industrial continuous production, the product yield is high, the production cost is low, and the active ingredients obtained from the sporotrichum mushroom have the effect of inhibiting the activity of CDC25 phosphoric acid protease, so that the ergosterol peroxide can be applied to diseases related to CDC25 phosphoric acid protease.

Description

Application of ergosterol peroxide in agaricus verticillata to CDC25 phosphoprotease

The technical field is as follows:

the invention relates to the technical field of application of natural products, in particular to a method for extracting peroxyergosterol from natural products and application thereof, and more particularly relates to the application of the peroxyergosterol extracted from agaricus bisporus and the peroxyergosterol thereof in CDC25 phosphoprotease.

Background art:

the Agaricus (Agaricaceae) family fungus has one purpose, many kinds, wide distribution, most edible fungus, and distribution all over the world. The Agaricus family has 28 genera including Agaricus. Common species in the Agaricus genus (Agaricus) of the family Agaricaceae are Agaricus gennadii (Chot. et Boud) P.D.Orton, Agaricus bisporus (Large) Sing., Agaricus bisporus, Agaricus blazei murrill, Agaricus rubellus (Gill.) Sacc., Agaricus blazei murrill, and the like. Currently, the chemical components of the genus and the research on pharmacological activities are mainly focused on two kinds of agaricus bisporus and agaricus blazei.

Chemical composition research of Agaricus (Agaricaceae) of Agaricus in the last century began, and it was found that the chemical compositions of fungi of this genus mainly include volatile oils, Agarbilazeispirol, sterols, A-1 (sodium pyrrolidone carboxylate), flavones, etc. May contain ceramide components, daucosterol, diterpenes, triterpenes, physcion, apigenin-7-4' -dimethyl ether, guaiacyl glycerol, butenolide, etc.

Agaricus is called "Pleurotus Cineraricus" belonging to Agaricales of Agaricus. The obtained product has good flavor and taste. The young plenilla is milk white, and then gradually changes from pink to black brown, and the single plenilla weighs 58-350g and can reach 2-5kg at most. The soil can grow in 20-70cm soil under irrigated sandy land and lake reed, and can be opened under the ground to grow single, scattered or clustered, and mostly grow in the west and southwest regions of Xinjiang. Since the nineties of the last century, active ingredients of agaricus blazei murill of the same genus and various activities thereof have been found. However, the chemical composition and related activity of Agaricus blazei in the same genus are still blank. In order to develop and utilize the resource, systematic research is carried out on the micromolecule chemical components of the sporotrichum mushroom under the subsidization of the natural science fund 2018-ZL-919 in Qinghai province.

Perergosterol is a small molecule secondary metabolite with broader biological activity recently discovered from edible and medicinal fungi. The research reports that the compound has wide pharmacological actions of promoting tumor cell apoptosis, resisting inflammation, resisting oxidation and the like. From the result of a primary antibacterial activity experiment, the ergosterol peroxide has an obvious antibacterial effect, and particularly has an obvious inhibitory activity on staphylococcus aureus and bacillus thuringiensis. Perergosterol is widely present in edible and medicinal fungi, however, the content of the compound is low, and the direct extraction of the ergosterol peroxide from the fungi to obtain enough raw materials is still difficult at present.

CDC25 phosphatase is an important regulator of regulation of normal cell division and cellular response to DNA damage. The studies showed that CDC25C phosphatase subtype exerts a synergistic regulation of the cell cycle. CDC25 phosphatase is overexpressed in many tumors, suggesting that specific CDC25 phosphatase inhibitors may be promising therapeutic agents for cancer.

The progression of the cell division cycle is regulated by activation of a specific set of cell cycle dependent proteases by CDC25 phosphatase. CDC25 protein specifically removes phosphate groups from the amino acid/threonine residues of CDKs.

Expression of CDC25A and CDC25B is upregulated in many tumors, generally the higher the extent of deterioration/the more aggressive and less prognostic, the higher expression of CDC25 in the tumor. The research proves that CDC25 is involved in the G2/M phase blocking process of DNA damage activation, and the result can conclude that CDC25 phosphatase is probably also involved in tumorigenesis, so the cell cycle regulatory factors can be potential targets of cancer treatment. Pharmaceutical companies and research laboratories are striving to develop compounds that inhibit CDC25 activity in order to achieve the goal of inhibiting tumor cell proliferation.

Disclosure of Invention

Aiming at the problems, the invention provides a separation and purification method of active ingredients in the sporotrichum mushrooms and application of extracting the active ingredients, through the separation and extraction method, the components of the sporotrichum mushrooms are quickly separated, the separation and extraction process is simple and stable, the method is suitable for industrial continuous production, the product yield is higher, the production cost is low, and the method can be used for extracting the active ingredients from the sporotrichum mushrooms, and can be used for efficiently and simply separating various active ingredients; the obtained active ingredients, wherein the ergosterol peroxide has the effect of inhibiting the activity of CDC25 phosphoprotease, so the ergosterol peroxide can be applied to diseases related to the inhibition of CDC25 phosphoprotease.

In order to achieve the purpose, the invention adopts the following technical scheme:

slicing fresh and undamaged sporophore of Agaricus campestris, soaking in 90-95% ethanol for one week, filtering to obtain filtrate, recovering solvent under reduced pressure, adding 90-95% ethanol into the residue, ultrasonic extracting for 3-5 times (each time for 1-5 hr), and concentrating under reduced pressure to obtain 90-95% ethanol extract; performing the same operation with 60-65% ethanol, and concentrating under reduced pressure to obtain 60-65% ethanol extract; adding distilled water into the alcohol-extracted agaricus bisporus filter residue, performing ultrasonic extraction for three times, extracting for 1-3h each time, and concentrating under reduced pressure to obtain a water extract.

Wherein the ratio of the sporophore of the Agaricus campestris to the ethanol is 0.5-1:1-5, and the sporophore of the Agaricus campestris is completely soaked in the ethanol.

Wherein the reduced pressure recovery condition is 0.1-0.5MPA and the temperature is 25-40 ℃; the ultrasonic extraction condition is 200-800HZ, the ultrasonic treatment is carried out for 30-45min, and the temperature is 30-50 ℃.

Mixing the two ethanol extracts, kneading and dispersing the mixed extracts in pure water, adding petroleum ether (water to petroleum ether ratio is 1:1-0.5) into a separating funnel, adding the extract dispersion into the separating funnel, extracting for 5 times, mixing the extracts, and concentrating to obtain petroleum ether fraction. Adding ethyl acetate (water to ethyl acetate ratio of 0.5-1:1) into the separating funnel, extracting for 6 times, mixing the extractive solutions, and concentrating to obtain ethyl acetate fraction. Adding n-butanol into the separating funnel again (water to n-butanol ratio is 0.4-1:1), extracting for 5 times, mixing extractive solutions, and concentrating to obtain n-butanol fraction.

Wherein, the extraction conditions are as follows: the extraction pressure is 20MPa to 40MPa, the extraction temperature is 30 ℃ to 50 ℃, and the extraction time is 0.8h to 2.5 h.

Separating the petroleum ether part by medium-pressure silica gel column flash chromatography, carrying out gradient elution by a petroleum ether-ethyl acetate system (100: 0-0: 100), detecting eluent by thin-layer chromatography, merging similar fractions, and recovering a solvent to obtain 9 components A-I. And carrying out medium-pressure silica gel chromatographic separation on the component D to obtain D1 and D2. D1 is recrystallized to obtain the needle compound 3. D2 was subjected to multiple medium pressure silica gel chromatography and Sephadex LH-20 column chromatography to obtain white solid compound 2. And (3) performing combined separation on the component E by multiple medium-pressure silica gel chromatographies and Sephadex LH-20 column chromatographies to obtain a yellowish green oily solid compound 1.

Wherein, the separation conditions of the medium-pressure silica gel column in the petroleum ether part are as follows: the silica gel column is prepared by using 120-type 160 mu m chromatographic silica gel as silica gel, the diameter-height ratio of the silica gel is 1:5-1:10, and the flow rate is 30-90 ml/min.

Wherein, the chromatographic separation conditions of the Sephadex LH-20 column are as follows: using acidified methanol as eluent, with concentration of 20-40% (Ph regulated at 3-4) and flow rate of 0.2-1.0 ml/min.

Subjecting the ethyl acetate fraction obtained above to medium pressure reverse phase C₁₈The column fast chromatographic separation, gradient elution in water-methanol solvent system (5-100%), combination of eluents in the same gradient, and solvent recovery to obtain 6 components I-VI. After the high performance liquid chromatography test, the components I to IV are found to be common components in the n-butanol phase, so that the components are merged to the n-butanol phase. The V component is subjected to medium-pressure reverse phase C₁₈After separation, use C₁₈Semi-preparative column separation) to give compounds 4,5 and 24. Compound 4 is a yellow particulate solid; compound 24 is a yellow particulate solid; compound 5 was a red particulate solid.

Wherein the above-mentioned inverse phase C₁₈The conditions for column chromatography separation were: gradient elution is carried out by a water-methanol solvent system (5-100%); flow rate: 1.0-3.5ml/min, column temperature: 25-30 ℃.

Wherein, the above-mentioned C₁₈The semi-preparative column separation conditions were mobile phase a: 1ml/L phosphoric acid aqueous solution (0.2% triethylamine, pH2.0 adjusted with phosphoric acid), mobile phase B: acetonitrile; filtering the mobile phase A, B, and performing ultrasonic treatment for 10 min; mobile phase ratio: mobile phase A: mobile phase B90: 10; flow rate: 1.0ml/min, column temperature: at 30 ℃.

And adding ethanol into the obtained n-butanol part, precipitating crystals on the container wall during precipitation, repeating the operation for multiple times, and recrystallizing for multiple times to obtain the compound 23. The n-butanol fraction is reversed phase C under medium pressure₁₈Separating, gradient eluting with water-methanol solution (5-100%), merging the eluates, and vacuum recovering solvent to obtain 6 components Z-I-Z-VI. The Z-II component is separated by MCI resin, and is eluted by water-methanol solution (0-100 percent) in a gradient way, and is added with C₁₈Separating and purifying with semi-preparative column to obtain compound 7, compound 8, compound 9 and compound 10. And separating and purifying the Z-IV component by using a C18 semi-preparative column to obtain a compound 6. Pure water wash of Z-I component, i.e. MCI resinRemoving the part, separating and cutting the part by an Agilent Zorbax SB-CN full-preparation column, and isocratically eluting the part by usingA water-methanol solution (5 percent) to obtain 4 components of Z-A-Z-D.

Wherein, the MCI resin separation condition is gradient elution of a water-methanol solvent system (0-100 percent); flow rate: 1.0-3.5ml/min, column temperature: 25-30 ℃.

Wherein, the separation and purification conditions of the C18 semi-preparative column are as follows: mobile phase A: 1ml/L phosphoric acid aqueous solution (0.2% triethylamine, pH2.0 adjusted with phosphoric acid), mobile phase B: acetonitrile; filtering the mobile phase A, B, and performing ultrasonic treatment for 10 min; mobile phase ratio: mobile phase A: mobile phase B90: 10; flow rate: 1.0-3.5ml/min, column temperature: 20-30 ℃.

And separating and purifying the Z-A component by an Agilent Zorbax SB-CN semi-preparative column to obtainA compound 13,A compound 14,A compound 16,A compound 17,A compound 19, compounds 20 and 21. Compounds 14, 16, 17, 20 and 21 were all white particles, compound 13 was a white gum; compound 19 was obtained as a purified crystal, a transparent needle crystal. Separating and purifying the Z-B component by an AgilentZorbax SB-CN semi-preparative column to obtain compounds 11 and 12, wherein the compounds 11 and 12 are white particles. And separating and purifying the Z-C component by an Agilent Zorbax SB-CN semi-preparative column to obtain a compound 18 and a compound 22, wherein the compounds 18 and 22 are white powder. And separating and purifying the Z-D component by an Agilent Zorbax SB-CN semi-preparative column to obtain a compound 15, wherein the compound 15 is white powder.

Wherein, the conditions for separating and purifying the Agilent Zorbax SB-CN semi-preparative column are as follows: mobile phase A: using 10-30mM Na2HPO4-NaH2PO4 buffer solution or K2HPO4-KH2PO4 buffer solution or Tris-HCl buffer solution as eluent A (0.2% triethylamine, pH2.0 adjusted by phosphoric acid), mobile phase B: acetonitrile; filtering the mobile phase A, B, and performing ultrasonic treatment for 10 min; mobile phase ratio: mobile phase A: mobile phase B90: 10; flow rate: 1.0-3.5ml/min, column temperature: 20-30 ℃.

Due to the adoption of the technical scheme, the invention has the beneficial effects that:

1. the method for extracting the active components from the sporotrichum mushrooms has the advantages of rapid separation of the components of the sporotrichum mushrooms, simple and stable separation and extraction process, suitability for industrial continuous production, higher product yield and low production cost, and can efficiently and simply separate various active components.

2. The obtained active component, namely the ergosterol peroxide has the effect of inhibiting the activity of CDC25 phosphoprotease, so that the ergosterol peroxide can be applied to diseases related to the inhibition of CDC25 phosphoprotease.

Description of the drawings:

FIG. 1 is a flow chart of the separation and purification of active ingredients from a Agaricus campestris.

The specific implementation mode is as follows:

the technical solutions in the embodiments of the present invention are clearly and completely described below with reference to specific embodiments. It should be understood that the described embodiments are part of the present invention, and are intended to be illustrative only and not limiting in scope. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.

Example 1 preparation of crude extracts of active ingredients of Agaricus campestris

Collecting fresh and undamaged sporocarp 10kg of sporocarp of the sporocarp. Concentrating under reduced pressure to obtain 95% ethanol extract. The 65% ethanol is used for the same operation, and the 65% ethanol extract is obtained by decompression and concentration. Adding 30L distilled water into alcohol extracted Agaricus campestris residue, and ultrasonic extracting at 1800HZ for three times, each time for 2 hr. Concentrating under reduced pressure to obtain 538g of water extract.

Wherein the reduced pressure recovery condition is 0.25MPA and the temperature is 30 ℃; the ultrasonic extraction conditions are 800HZ, ultrasonic extraction is 45min, and the temperature is 30 ℃.

Mixing the two ethanol extracts to obtain 84.5g, kneading and dispersing 84.5g of the mixed extract in 500ml of pure water, adding petroleum ether (the ratio of water to petroleum ether is 1:1) into a 2000ml separating funnel, adding the extract dispersion into the separating funnel, extracting for 5 times, mixing the extracts, and concentrating to obtain 10.0g of petroleum ether part. Ethyl acetate (water to ethyl acetate ratio 1:1) was added to the above separatory funnel, extraction was performed 6 times, and the extracts were combined and concentrated to obtain 6.0g of ethyl acetate fraction. Adding n-butanol (water to n-butanol ratio of 1:1) into the separating funnel, extracting for 5 times, mixing extractive solutions, and concentrating to obtain n-butanol fraction 60.0 g.

Wherein, the extraction conditions are as follows: the extraction pressure is 40MPa, the extraction temperature is 35 ℃, and the extraction time is 2 h.

The flow chart of the separation and purification process of the active ingredients in the sporotrichum mushroom is shown in figure 1.

EXAMPLE 2 separation and purification of Petroleum Ether phase fraction

10.0g of the petroleum ether fraction obtained in the above example 1 is subjected to medium-pressure silica gel column flash chromatography separation, petroleum ether-ethyl acetate system (100: 0-0: 100) gradient elution, eluent is subjected to thin-layer chromatography detection, similar fractions are combined, and solvent is recovered to obtain 9 components A-I. And carrying out medium-pressure silica gel chromatographic separation on the component D to obtain D1 and D2. D1 was recrystallized to give 20mg of needle compound 3. D2 was subjected to multiple medium pressure silica gel chromatography and Sephadex LH-20 column chromatography to obtain 10mg of white solid compound 2. And (3) performing multiple medium-pressure silica gel chromatography and Sephadex LH-20 column chromatography combined separation on the component E to obtain 5mg of a yellow-green oily solid compound 1.

Wherein, the separation conditions of the medium-pressure silica gel column in the petroleum ether part are as follows: the silica gel diameter-height ratio of the silica gel column is 1:8 by using 120-type 130 μm chromatography silica gel, and the flow rate is 40 ml/min.

Wherein, the chromatographic separation conditions of the Sephadex LH-20 column are as follows: acidified methanol was used as eluent, the concentration was 25% (Ph adjusted at 4), and the flow rate was 0.5 ml/min.

And (3) carrying out physical and chemical property identification on the separated and purified compound components by a conventional physical detection method: the detection method comprises the following steps: EIS-MS, H-NMR, C-NMR. The detection method is the same as that of the pharmacopoeia 2015 edition. The detection results are as follows:

compound 1 dibutyl terephthalate (dibutyl terephthalate)

Yellow solid, EIS-MS (negative) M/z 277[ M-H ]]^-,EIS-MS(positive)m/z:279[M+H]⁺The molecular weight was determined to be 278.¹H-NMR(400MHz,CDCl₃)δ:7.62(4H,ddd,J＝4.9,5.6,3.3Hz,H-2,3,5,6),4.30(4H,t,J＝6.7Hz,H-9.9′),1.77～1.65(4H,m,H-10,10′),1.43(4H,td,J＝14.9,7.4Hz,H-11,11′),0.95(4H,t,J＝7.4Hz.H-12,12′)；¹³C-NMR(100MHz,CDCl₃) Delta.: 132.3(C-1,4),130.9(C-2,6),128.8(C-3,5),167.7(C-7,7 '), 65.6(C-9,9 '), 30.6(C-10,10 '), 19.2(C-11,11 '), 13.7(C-12,12 '). The data above are consistent with a dibutyl terephthalate control, and compound 1 was identified as dibutyl terephthalate (dibutyl terephthalate).

Compound 2 wine yeast sterol (Cerevisterol)

White powder.

(c0.11,CHCl₃)；C₂₈H₄₆O₃,mp＝246-284℃。¹H-NMR(600MHz,DMSO)δ:5.23(1H,dd,J＝15.3,7.3Hz,H-23),5.17(1H,dd,J＝15.3,8.3Hz,H-22),5.08(1H,s,H-7),3.77(1H,m,H-3),3.58(1H,s,H-6),0.99(3H,d,J＝6.6Hz,H-21),0.91(3H,s,H-19),0.88(3H,d,J＝6.8Hz,H-28),0.81(3H,d,J＝7.4Hz,H-27),0.80(3H,d,J＝7.2Hz,H-26),0.54(3H,s,H-18)；¹³C-NMR(150MHz,DMSO)δ:139.7(C-8),135.4(C-22),131.4(C-23),119.4(C-7),74.5(C-5),72.2(C-6),66.0(C-3)55.3(C-17),54.2(C-14),43.0(C-13),42.3(C-9),42.0(C-24),40.2(C-4),40.1(C-20),39.0(C-12),36.7(C-10),32.5(C-2,25),31.2(C-1),27.7(C-16),22.6(C-15),21.3(C-11),21.0(C-21),19.8(C-26),19.5(C-27),17.7(C-19),17.3(C-28),12.1 (C-18). The detection data is basically consistent with the saccharomyces cerevisiae sterol data, so that the compound 2 is identified as saccharomyces cerevisiae sterol.

Compound 3 β -sitosterol (Stigmast-5-en-3-ol)

White needle crystal with melting point of 136-137, C₂₉H₅₀O。EI-MS m/z:414[M]⁺,396,381,329.¹H-NMR(500MHz,CDCl₃)δ:5.29(1H,brd,J＝5.2Hz,H-6),3.46(1H,m,H-3),1.02(3H,s,H-19),0.91(3H,d,J＝8.2Hz,H-21),0.85(3H,d,J＝7.4Hz,H-27),0.82(3H,d,J＝7.4Hz,H-26),0.81(3H,t,J＝8.1Hz,H-29),0.65(3H,s,H-18)；¹³C-NMR(CDCl₃) Δ:36.6(C-1),29.7(C-2),71.8(C-3),46.0(C-4),140.8(C-5),121.7(C-6),31.7(C-7),32.0(C-8),50.3(C-9),36.2(C-10),28.3(C-11),39.9(C-12),42.(C-13),56.9(C-14),26.3(C-15)29.3(C-16),56.2(C-17),11.9(C-18),19.8(C-19),32.0(C-20),19.4(C-21),34.1(C-22)24.3(C-23)37.3(C-24),19.1(C-25),12.0(C-26),23.2(C-27), 28.1 (C-18) 24.3(C-23), 7.21.3 (C-18), 28.3 (C-18) and 36 β,28.3, 36 β, 3652, 23, 21, 18, 28.8, 18, 7, 28, 3, 7, 3.

EXAMPLE 3 separation and purification of Ethyl acetate fraction

6.0g of the ethyl acetate fraction obtained in example 1 above was subjected to medium-pressure reverse phase C₁₈The column fast chromatographic separation, gradient elution in water-methanol solvent system (5-100%), combination of eluents in the same gradient, and solvent recovery to obtain 6 components I-VI. After the high performance liquid chromatography test, the components I to IV are found to be common components in the n-butanol phase, so that the components are merged to the n-butanol phase. The V component is subjected to medium-pressure reverse phase C₁₈After separation, use C₁₈Separating with semi-preparative column to obtain 4mg total compound 4, 4mg total compound 5 and 3mg total compound 24. Compound 4 is a yellow particulate solid; compound (I)24 is a yellow particulate solid; compound 5 was a red particulate solid.

Wherein the above-mentioned inverse phase C₁₈The conditions for column chromatography separation were: gradient elution is carried out by a water-methanol solvent system (5-100%); flow rate: 1.5ml/min, column temperature: at 30 ℃.

compound 41-phenazine formic acid (1-Carboxyphenazine)

Yellow powder, FAB-MS M/z:225[ M +1] +; 1H-NMR (CDCl3):8.93(1H, d, J ═ 7.3Hz),8.50(1H, d, J ═ 8.7Hz),8.31(1H, d, J ═ 9.4Hz),8.23(1H, d, J ═ 8.4Hz),8.02 to 7.93(3H, m); 13C-NMR 165.7(s),143.7(s),143.0(s),139.7(s),139.6(s),136.8(s),134.7(d),132.8(d),131.4(d),129.9(d),129.7(d),127.7(d), 124.9(s). The data above are essentially identical to the data for 1-phenazine carboxylic acid in comparison, and compound 4 was identified as 1-phenazine carboxylic acid (1-phenazine carboxylic acid).

Compound 57-Acetyl-4-methylazlene-1-carboxylic acid

Red powder. HR-ESI-MS 251.0688 (C)₁₄H₁₂O₃),[M+Na]⁺；228.0684).–MS(EI,70eV):m/z(％)＝228(90)[M]+,213(100)[M-Me]+,211(19)[M-OH]+,185(17)[M-MeCO]+,183(6)[M-HCOO]+,¹H-NMR(400MHz,CD₃OD),¹³C-NMR(125MHz,CD₃OD is 169, 13, 149, 7, 129, 64, 128, 63, 115, 29, 57, 60. The above data are substantially identical to those of 7-Acetyl-4-methylazene-1-carboxylic acid, and thus compound 5 was identified as 7-Acetyl-4-methylazene-1-carboxylic acid.

Compound 241, 8-Dihydroxyanthraquinone (1, 8-dihydroxyanthhraquinone)

Yellow crystals FAB-MS M/z 240[ M ] + (100),223[ M-OH ] (9),212[ M-CO ] (24),184(27),155(13),138(20),128 (15); 1H-NMR (CDCl3):12.07(2H, s, OH-1 and 8),7.83(2H, d, J ═ 7.2Hz, H-4 and 5),7.69(2H, t, J ═ 7.2,8.4Hz, H-3 and 6),7.30(2H, d, J ═ 8.4Hz, H-2 and 7); 13C-NMR 193.1(s, C-9),181.5(s, C-10),162.5(s, C-1 and 8),137.3(s, C-3 and 6),133.6(s, C-4a and 5a),124.6(s, C-2 and 7),120.1(s, C-4 and 5),115.9(s, C-1a and 8 a). The data above are essentially consistent with the 1,8-dihydroxyanthraquinone data, so compound 24 was identified as 1, 8-dihydroxyanthraquinone.

Example 4 separation and purification of n-butanol fraction

60.0g of the n-butanol fraction obtained in example 1 was added to ethanol, and during precipitation, crystals were precipitated on the container wall, and the operation was repeated several times to obtain 30mg of compound 23 by repeated recrystallization. The n-butanol fraction is reversed phase C under medium pressure₁₈The separation is carried out by gradient elution with water-methanol solution (5-100%), the eluents are combined according to the same gradient, and the solvent is recovered under reduced pressure to obtain 6 components of Z-I-Z-VI. The Z-II component is separated by MCI resin, and is eluted by water-methanol solution (0-100 percent) in a gradient way, and is added with C₁₈Separating and purifying by semi-preparative column to obtain 4mg of compound 7, 5mg of compound 8, 4mg of compound 9 and 5mg of compound 10. The Z-IV fraction was separated and purified by C18 semi-preparative column to give 5mg of Compound 6. The Z-I component, namely the pure water elution part of the MCI resin, is separated and cut into sections by an Agilent ZorbaxSB-CN full preparative column, and is isocratically eluted byA water-methanol solution (5 percent) to obtain 4 components of Z-A-Z-D.

Wherein the above-mentioned inverse phase C₁₈The conditions for column chromatography separation were: water-methanol solvent system (5% -100%) ladderCarrying out mild elution; flow rate: 1.5ml/min, column temperature: at 30 ℃.

Wherein, the MCI resin separation condition is gradient elution of a water-methanol solvent system (0-100 percent); flow rate: 1.5ml/min, column temperature: at 30 ℃.

Wherein, the separation and purification conditions of the C18 semi-preparative column are as follows: mobile phase A: 1ml/L phosphoric acid aqueous solution (0.2% triethylamine, pH2.0 adjusted with phosphoric acid), mobile phase B: acetonitrile; filtering the mobile phase A, B, and performing ultrasonic treatment for 10 min; mobile phase ratio: mobile phase A: mobile phase B90: 10; flow rate: 1.5ml/min, column temperature: at 30 ℃.

The AA-A fraction was isolated and purified by Agilent Zorbax SB-CN semipreparative column to give 4mg of compound 13, 100mg of compound 14, 5mg of compound 16, 4mg of compound 17, 15mg of compound 19, 4mg of compound 20 and 3mg of compound 21. Compounds 14, 16, 17, 20 and 21 were all white particles, compound 13 was a white gum; compound 19 was obtained as a purified crystal, a transparent needle crystal. The Z-B component was separated and purified by Agilent Zorbax SB-CN semi-preparative column to give 4mg of compound 11, 5mg of compound 12, and the compounds 11, 12 were white particles. And separating and purifying the Z-C component by an Agilent Zorbax SB-CN semi-preparative column to obtain 5mg of compound 18 and 4mg of compound 22, wherein the compounds 18 and 22 are white powder. The Z-D fraction was isolated and purified by Agilent Zorbax SB-CN semipreparative column to give 4mg of Compound 15, Compound 15 being a white powder.

Wherein, the conditions for separating and purifying the Agilent Zorbax SB-CN semi-preparative column are as follows: mobile phase A: at 15mM Na₂HPO₄-NaH₂PO₄Buffer solution or K₂HPO₄-KH₂PO₄Buffer or Tris-HCl buffer as eluent a (0.2% triethylamine, PH2.0 adjusted with phosphoric acid), mobile phase B: acetonitrile; filtering the mobile phase A, B, and performing ultrasonic treatment for 10 min; mobile phase ratio: mobile phase A: mobile phase B90: 10; flow rate: 1.5ml/min, column temperature: at 30 ℃.

compound 6 Ergosterol (Ergosterol)

A white powder; EI-MS M/z 428[ M-H ═ M₂O],410[M-2H₂0],382[M-2H₂O-CO].¹H-NMR(CD3OD,500MHz):5.21(2Hm,H-22,23),5.05(1H,brs,H-7),3.88(2H,m,H-3,6)，1.05(3H,S,H-19),1.03(3H,d,J＝6.7Hz,H-21),0.93(3H,d,J＝6.9Hz,H-28),0.86(3H,d,J＝6.8Hz,H-27),0.84(3H,d,J＝6.8Hz,H-26),0.61(3H,S,H-18)。¹³The C-NMR (DEPT) spectrum gives a signal of 28 carbons, 6 methyl groups, 7 methylene groups, 10 methine groups and 5 quaternary carbons. The four carbon signals are at δ c78.6, 75.8, 71.2, 67.9, indicating that they are linked to oxygen. Four carbon signals at δ c143.3,137.0,133.3,122.0 indicate two double bonds. The above data are essentially consistent with ergosterol data, and compound 6 was identified as ergosterol.

Compound 7 Peroxyaergosterol (peroxy-ergosterol)

White powder.¹H-NMR(500MHz,CDCl₃)δ:6.50(1H,d,J＝8.5Hz,H-7),6.24(1H,d,J＝8.5Hz,H-6),5.22(1H,dd,J＝7.6,15.3Hz,H-23),5.14(1H,dd,J＝8.3,15.5Hz,H-22),3.92(1H,m,H-3),1.25(3H,s,Me-19),1.00(3H,d,J＝6.7,Me-21),0.91(3H,d,J＝6.9Hz,Me-28),0.88(3H,s,Me-18),0.83(3H,d,J＝6.8Hz,Me-26),0.82(3H,d,J＝6.8Hz,Me-27)。¹³C-NMR(125MHz,CDCl₃) 34.7(t, C-1),30.1(t, C-2),66.4(d, C-3),36.9(t, C-4),82.2(s, C-5),135.4(d, C-6),130.7(d, C-7),79.4(s, C-8),51.1(d, C-9),36.9(s, C-10),23.4(t, C-11),39.4(t, C-12),44.6(s, C-13),51.8(d, C-14),20.6(t, C-15),28.6(t, C-16),56.2(d, C-17),12.9(q, C-18),18.2(q, C-19),39.4(d, C-20),20.9(q, C-21),135.2(d, C-22, 22.42, 132.42 (d, C-3), c-24),33.1(d, C-25),20.0(q, C-26),19.6(q, C-27),17.6(q, C-28); the above data are consistent with those of ergosterol peroxide, so Compound 7 was identified as wheat peroxideCanthasterol.

Compound 8 Macrospelide A

White powder, ESI-MS M/z 365.2[ M + Na ]]⁺,341.2[M-H]^-From this, it was concluded that the relative molecular mass was 342, and the molecular formula C was deduced from the combination of the hydrogen spectrum and the carbon spectrum₁₆H₂₂O₈,¹H-NMR(CDCl₃):6.88(1H,dd,J＝15.6,1.5Hz,H-7),6.83(1H,dd,J＝15.6,1.5Hz,H-13),6.02(1H,dd,J＝15.6,1.5Hz,H-12),6.01(1H,dd,J＝15.6,1.5Hz,H-6),5.35(1H,m,H-3),4.93(1H,q,J＝6.3Hz,H-9),4.82(1H,q,J＝6.3Hz,H-15),4.18(1H,m,H-8),4.09(1H,m,H-14),3.57(2H,brs,8-OH,14-OH),2.58(2H,dd,J＝7.2,2.4Hz,2-H2),1.40(3H,d,J＝6.6Hz,9-CH₃),1.33(3H,d,J＝6.3Hz,15-CH3),1.29(3H,d,J＝6.6Hz,3-CH3)；¹³C-NMR(CDCl₃) 170.1(C-1),165.7(C-11),164.9(C-5),146.4(13-CH),145.6(7-CH),122.5(6-CH),122.1(12-CH),74.5(9-CH),74.4(8-CH),73.6(15-CH),72.8(14-CH),67.7(3-CH),40.9(2-CH2),19.6(3-CH3),17.8(9-CH3),17.7(15-CH 3); the above data are consistent with those of Macrosphalide A, and therefore Compound 8 was identified as Macrosphalide A.

Compound 9 adenosine (a-denosine)

White powder, mp.230-232 ℃;¹H-NMR(DMSO-d₆,500MHz)δ:3.58(1H,d,J＝10.8Hz,H-5′),3.67(1H,d,J＝11.4Hz,H-5′),5.88(1H,d,J＝9.6Hz,H-1′),7-33(2H,brs,J＝1.0Hz,NH₂),8.14(1H,s,H-8),8.34(1H,s,H-2)；¹³CNMR(DMSO-d₆125MHz) delta 152.3(C-2),149.0(C-4),119.3(C-5),156.1(C-6),139.9(C-8),87.9(C-1 '), 73.4 (C-2'), 70.6(C-3 '), 85,8 (C-4'), 61.6(C-5 '), 156.1 (C-6'), the spectral data and the physicochemical characteristics of which are consistent with those of adenosine, and thus, the compound 9 was identified as adenosine (a-denosine).

Compound 10 Ethyl p-hydroxybenzoate (4-hydroxy ethylbenzoate)

A colorless oil; it is easily soluble in acetone, chloroform and methanol, and is hardly soluble in water. ESIMS M/z 167[ M + H ]]⁺；¹H-NMR (Me2CO-d,6,500Hz) δ 7.87(2H, d, J ═ 9.0Hz, H-2,6),6.90(2H, d, J ═ 9.0Hz, H-3,5),4.22(2H, q, J ═ 7.0Hz, H2-1 '), 1.31(3H, t, J ═ 7.0Hz, H3-2'). The data above are consistent with those for ethyl paraben, so compound 10 was identified as ethyl paraben.

Compound 12 p-hydroxybenzaldehyde (p-hydroxybenzaldehyde)

A colorless oil; easily soluble in acetone, chloroform and methanol, and hardly soluble in water; ESIMS M/z 123[ M + H ]]⁺；¹H-NMR (Me2CO-d6,500hz,) δ 9.84(1H, s, H-7),7.79(2H, d, J ═ 9.0Hz, H-2,6),7.00(2H, d, J ═ 8.5Hz, H-3, 5). The above data are consistent with those for p-hydroxybenzaldehyde, so compound 12 was identified as p-hydroxybenzaldehyde.

Compound 134-hydroxy-3-methoxy-benzyl alcohol (4-hydroxy-3-methoxy-benzyl alcohol)

A white solid. Is easily soluble in acetone, chloroform and methanol and is insoluble in water; ESIMS M/z 155[ M + H ]]⁺；¹H-NM R(Me2CO-,d,6,500Hz)δ:7.53(1H,s,4-OH),6.84(1H,d,J＝1.5Hz,H-2),6.85(1H,d,J＝8.0Hz,H-5),6.75(1H,dd,J＝1.5,8.0Hz,H-6),4.47(2H,d,J＝6.5Hz,H2-7),4.03(1H,t,J＝6.5Hz,7-OH),3.80(3H,s,-OMe)；¹³C-NMR (Me2CO-, d,6,125Hz) delta 136.1(C-1),112.2(C-2),147.3 (C-or C-4),147.0 (C-or C-4),114.7(C-5),118.5(C-6),64.4(C-7),56.3 (-OMe). The above data are consistent with the data for 4-hydroxy-3-methoxy-benzyl alcohol, so compound 13 was identified as 4-hydroxy-3-methoxy-benzyl alcoholBenzyl alcohol.

Compound 145 α,6 α -Epoxy- (22E,24R) -ergosta-8(14),22-diene-3 β,7 α -diol

A white powder;(c0.13,CHCl₃)；HRESIMS:m/z＝451.3167[M+Na]⁺(calcdfor C₂₈H₄₄NaO₃,451.3183)；¹H-NMR(600MHz,CDCl₃)δ:5.21(2H,m,H-22,23),4.42(1H,d,J＝6.3Hz,H-7),3.91(1H,m,H-3),3.14(1H,d,J＝3.5Hz,H-6),1.02(3H,d,J＝6.7Hz,H-21),0.92(3H,d,J＝6.8Hz,H-28),0.87(6H,s,H-18,19),0.84(3H,d,J＝6.8Hz,H-27),0.82(3H,d,J＝6.8Hz,H-26)；¹³C-NM R(150MHz,CDCl₃) 152.75(C-14),135.41(C-22),132.44(C-23),125.38(C-8),68.87(C-3),67.92(C-5),65.25(C-7),61.49(C-6),57.02(C-17),43.15(C-13),43.01(C-24)39.78(C-20),39.39(C-4),38.92(C-9),36.78(C-12),36.01(C-10),33.27(C-25),32.38(C-1),31.31(C-2),27.31(C-16),25.13(C-15),21.40(C-21),19.82(C-27),19.17(C-26),18.24(C-11),17.77(C-18,28),16.70(C-19), the above data are consistent with that of Epdione 2-24, 24-24, α, 24-24, 24-11, 16.70(C-19, 24-7, 24-24, 8, 24, 8, etc. with the above data for which are consistent with the Epdione, 24.

Compound 15 p-hydroxybenzyl alcohol (p-hydroxybenzyl alcohol)

White needle crystals (methanol); is easily soluble in acetone, chloroform and methanol and is insoluble in water; (-) -ESIMS M/z 283[ M-H ]]^-；¹H-NMR (500MHz, DMSO-d6) δ 9.31(1H, s, OH),7.25(2H, d, J ═ 8.0Hz, H-2,6),6.89(2H, d, J ═ 8.0Hz, H-3,5),4.76(1H, s, OH),4.36(2H, s, H-7). The data above are essentially identical to the data comparison for p-hydroxybenzyl alcohol, so that Compound 15 was identified as p-hydroxybenzyl alcohol。

Compound 16 p-hydroxybenzoic acid (p-hydroxybenzoic acid)

White flaky crystal (acetone), easily soluble in chloroform and methanol, insoluble in water (-) -ESIMS M/z 137[ M-H ]]^-；¹H-NMR(500MHz,Me2CO-d6)δ:7.68(2H,d,J＝9.0Hz,H-2,6),6.79(2H,d,J＝9.0Hz,H-3,5)。¹³C-NMR (125MHz, Me2CO-d6) delta: 168.3(COOH),162.1(C-4),132.1(C-2,6),122.2(C-1),114.9(C-3, 5). The above data are consistent with those for parahydroxybenzoic acid, so compound 16 was identified as parahydroxybenzoic acid.

Compound 17 p-hydroxybenzyl methyl ether (p-hydroxybenzyl methyl ether)

White flaky crystals (acetone), which are easily soluble in chloroform and methanol and insoluble in water. (-) -ESIMS M/z 137[ M-H ]]^-；¹H-NMR (500MHz, DMSO-d6) δ 9.25(1H, OH),7.17(2H, d, J ═ 8.0Hz, H-2,6),6.69(2H, d, J ═ 8.0Hz, H-3,5),4.19(2H, s, H-7),3.18(3H, s, OMe). The above data are consistent with those for p-hydroxybenzyl methyl ether, so compound 17 was identified as p-hydroxybenzyl methyl ether.

Compound 18 p-hydroxybenzylethyl ether (p-hydroxybenzyl ethyl ether)

White flaky crystals (acetone), which are easily soluble in chloroform and methanol and insoluble in water; (+) -ESIMS M/z 153[ M + H ]]⁺；¹H-NMR (300MHz, DMSO-d6) δ 9.29(1H, OH),7.09(2H, d, J ═ 8.4Hz, H-2,6),6.80(2H, d, J ═ 8.4Hz, H-3,5),4.31(2H, s, H-7),3.39(2H, q, J ═ 6.9Hz, OCH2CH3),1.11(3H, t, J ═ 6.9Hz, OCH2CH 3). The above data are consistent with those for p-hydroxybenzylether, so compound 18 was identified as p-hydroxybenzylether.

Compound 19 p-methoxybenzoic acid (p-hydroxybenzoic acid)

White flaky crystals (acetone), which are readily soluble in chloroform and methanol and poorly soluble in water. (-) -ESIMS M/z 151[ M-H ═ M]^-；¹H-NMR (500MHz, DMSO-d6) δ 7.87(2H, d, J ═ 8.5Hz, H-2,6),6.77(2H, d, J ═ 8.5Hz, H-3,5),3.74(3H, s, OMe). The above data are consistent with those for p-methoxybenzoic acid, so compound 19 was identified as p-methoxybenzoic acid.

Compound 208-methoxy-1-naphthol (8-Methoyynapthhalene-1-ol)

White powder, ESI-MS M/z 175[ M + H ]]⁺.¹H-NMR(CDC1₃,500MHz)δ:9.31(1H,s,1-OH),7.41(1H,d,J＝8.3Hz,H-4),7.36～7.28(3H,m,H-3,H-5,H-6)6.87(1H,d,J＝7.5Hz,,H-7),6.78(1H,d,J＝7.7Hz,H-2)4.06(3H,s,8-OCH3)；¹³C-NMR(CDCl₃125MHz) delta 156.2(C-8)154.5(C-1)136.8(C-4a)127.7(C-3)125.6(C-6),121.9(C-5)118.8(C-4)115.1(C-8a)110.4(C-2)103.9(C-7),56.1(8-OCH 3). The data above are essentially consistent with 8-methoxy-1-naphthol, and compound 20 was identified as 8-methoxy-1-naphthol.

Compound 22 peroxyergosterol (gallic acid)

White needle crystal; EI-MS M/z (%). 171[ M + H ] +; 1H-NMR (500MHz, CDCl3) 7.15(2H, s, H-2,6),7.60(2H, s,3,5-OH),7.28(1H, s,4-OH),12.10(1H, s, 1-COOH); 13C-NMR (100MHz, CDCl3) 123.1(s, C-1),114.3(d, C-2,6),146.0(s, C-3,5),139.4(s, C-4),170.6(s, COOH). The data are essentially identical to those of peroxyergosterol, and compound 22 was identified as peroxyergosterol.

Compound 233, 4, 5-trihydroxy-benzaldehyde (3,4, 5-trihydroxybenzadhehyde)

White amorphous powder (methanol). 1H-NMR (CD3OD,500MHz) delta (ppm) 9.55(1H, s, -CHO),7.27(2H, s, H-2, 6); 13C-NMR (CD3OD,100MHz) delta (ppm) 192.8(-CHO),149.9(C-3,5),145.2(C-4),128.0(C-1),108.3(C-2, 6). The above data are consistent with 3,4, 5-trihydroxy-benzaldehyde, so compound 23 was identified as 3,4, 5-trihydroxy-benzaldehyde.

Example 5 Perergosterol inhibits CDC25 phosphatase protease Activity

The experimental method comprises the following steps: test experiment for inhibiting the activity of phosphoprotease CDC25A/CDC 25B:

experimental group reactions were carried out in 18. mu.L systems with compositions comprising 2.7. mu.L of 330. mu. mol/L FDP, 1.8. mu. mol/L100. mu. mol/L peroxyergosterol test substance, 2. mu.L of 0.1. mu. mol/L CDC25A/CDC25B, 11.5. mu.L FDP buffer; control 1 the reaction was carried out in an 18. mu.L system, the composition comprising 2.7. mu.L of 330. mu. mol/L FDP, 2. mu.L of 0.1. mu. mol/L CDC25A/CDC25B, 13.3. mu.L FDP buffer; control 2 reactions were carried out in 18. mu.L system, with the composition comprising 2.7. mu.L of 330. mu. mol/L FDP, 1.8. mu.L of 100. mu. mol/L test, 13.5. mu.L FDP buffer; incubating a system needing adding CDC25A/CDC25B enzyme at 37 ℃ for 60min, adding the enzyme, reacting for 60min under the same condition, and detecting the fluorescence intensity of a reaction product generated by the action of the substrate and the enzyme at 530nm under the action of 485nm excitation light when the reaction is complete. The control group was run under the same conditions without the addition of test substance. The enzyme activity survival efficiency was calculated using the following formula: the calculation formula is as follows:

survival efficiency of enzyme activity ═ assay value ÷ control 1 assay value

Wherein a lower efficiency of enzymatic activity survival indicates a stronger inhibitory effect of the test sample on the histone phosphorylase CDC25A/CDC 25B.

2. Results of the experiment

The primary screening results (see table 1) show that the ethanol extract of the agaricus bisporus shows better activity of inhibiting the phosphoprotease CDC25A/CDC25B, and indicate that the extract of the ergosterol peroxide of the sporophore of the agaricus bisporus can inhibit the activity of CDC25A/CDC 25B.

TABLE 1 results of inhibition of CDC25 phosphoprotease by the P-ergosterol extract of Agaricus campestris

Item	Efficiency of enzyme Activity survival
		CDC25A	38.2
CDC25B	24.1

Example 6 application of Perergosterol to mammary hyperplasia

Experimental materials:

animals: 72 healthy uninoculated adult Vistar female rats with weight of 180-.

Experimental samples and positive control drugs: agaricus campestris Peroxyagoterol extract (high/low concentration); positive control drug: ruan tablet (0.3 g/tablet) is ground into powder and made into water solution, and the water solution is stored at 4 ℃ for standby.

The experimental method comprises the following steps: grouping and administration of drugs

(1) Normal control group: drenching distilled water with the volume of 10 ml/kg/d;

(2) disease model group: injecting estradiol benzoate into the medial muscle of the leg of the rat for 25 days continuously, injecting progesterone for 2mg/kg/d for 5 days continuously, and simultaneously filling distilled water for 10 ml/kg/d;

(3) the module is made in the combination of disease and syndrome: adding a tail clamping mode to irritate the rats on the basis of the group modeling in the step (2), feeding every half an hour every day every other day;

(4) positive drug control group (ruan tablets): 0.16g/kg/d of Ruan tablets are drenched on the basis of the model building in the step (3);

(5) experimental group low dose group: filling 0.08g/kg/d of ergosterol peroxide solution on the basis of the molding in the step (3);

(6) experimental group high dose group: and (3) pouring 0.16g/kg/d of ergosterol peroxide solution on the basis of the mold building.

The experimental steps are as follows:

weighing the above 6 groups of rats before/after experiment, killing the rats by dislocation method after 30 days of molding and drug filling, measuring the diameters of the upper and lower breasts of the right anterior axilla by using a precision vernier caliper, taking the upper and lower breasts of the rats, fixing the rats by using 10% formalin, embedding the rats into paraffin, and observing the proliferation degree of the rats by histology under a light microscope through conventional HE staining; PCNA, bcl-2, VEGF, MVD were determined by immunohistochemical SABC method, which was performed as follows:

(1) slicing, dewaxing conventionally, and dehydrating with gradient alcohol to distilled water; (2) 3% hydrogen peroxide incubation 5Min eliminated endogenous oxidase activity; (3) soaking in distilled water in PBS for 5 min; (4) sections stained with F-VIII PAg were digested with 0.1% pancreatin for 10min at room temperature; (5) carrying out PCNA antigen thermal restoration, namely placing the slices in 0.01mol of citric acid buffer saline with the pH value of 6.0 at 95 ℃ and the temperature of 30Min, cooling and then adding the slices into the buffer solution; (6) washing with PBS for 5min3 times; (7) sealing 5% normal goat serum at room temperature for 10 min; (8) the serum was decanted and primary antibody, FaV III Ag1:100, bcl-21:100, PCNA 1: 50, VEGF1:100, 4 ℃ overnight; (9) washing with PBS for 5min3 times; (10) dripping biotin-labeled secondary antibody at 37 deg.C for 30 min; (11) washing with PBS for 5min3 times; (12) horseradish peroxidase-labeled streptoenzyme ovalbumin, at 37 ℃ for 30 min; (13) washing with PBS for 5min3 times; (14) DAB color development is carried out for 10min at room temperature; (15) washing with tap water; (16) counterstaining, dehydrating, transparent, and sealing.

Thirdly, the method comprises the following steps: results of the experiment

(1) Weight comparison before and after rat experiment: TABLE 2

As can be seen from the above table 2, the weight of the rats of the sick model group 2 and the sick and sick model group 3 is not increased much, the weight of the rats of the normal group 1 and the drug administration groups 4,5 and 6 is increased, and the weight increase degrees of the rats of the normal group and the drug administration groups 4,5 and 6 are not different.

(2) Change in mammary diameter in rats: TABLE 3

As can be seen from table 3 above: the diameter of the mammary gland of the groups 2 and 3 is the largest, the change of the breast before and after the experiment is the largest, and the mammary gland has significant difference compared with other groups; the 3 groups increased more significantly than the 2 groups; 4.5 and 6 groups have resistance to mammary gland enlargement, the resistance effects of the experimental drug high-dose group and the positive drug 4 group are closer, the resistance effect of the low-dose group is good, but the curative effect is poorer than that of the high-dose group and the positive drug.

(3) Histological changes of mammary glands of rats under light mirror

The treatment method is divided into four categories according to the existence and the severity of hyperplasia of mammary glands: no hyperplasia, mild hyperplasia, moderate hyperplasia and severe hyperplasia. No proliferation: the lobules of the mammary gland are not hyperplastic, the number of glands is very small, acinus is not expanded, and the mammary gland is in a stationary state; mild hyperplasia: the individual acini have slight hyperplasia and no expansion, and secretion exists in the acini and ducts; moderate hyperplasia: the mammary lobules are mostly hyperplastic, a few acini have slight dilatation, hyperplasia layers of glandular epithelium are increased, and secretion exists in the acinus and ducts; severe hyperplasia: most of mammary lobules are hyperplastic, most acini are obviously dilated, hyperplasia of glandular epithelium is increased, and more secretion exists in acinus and ducts.

The proliferation degree of each mammary tissue under the light microscope is counted according to the standard as follows: TABLE 4

As can be seen from table 4 above: rats in groups 2 and 3 proliferated significantly, compared to group 1. And the 3 groups have more serious hyperplasia degree than the 2 groups, and the groups are improved after the medicine is taken, wherein the effect of the experimental medicine dosage of 5 groups and 6 groups is slightly worse than that of the positive medicine dosage of 4 groups, but the peroxyergosterol of the experimental medicine groups of 2 groups and 3 groups has the function of inhibiting hyperplasia of mammary glands.

(4) Expression level of PCNA, BCL-2 in mammary tissue of rat: TABLE 5

PCNA and BCL-2 of rats are measured by the immunohistochemical method, and the result judgment standard is as follows: more than 20% of the cells were positive.

Group of	Number of cases of organization	PCNA Positive Rate (%)	BCL-2 Positive Rate (%)
				1	10	8	20
2	10	43	90
				3	10	55	90
4	10	18	40
				5	10	32	65
6	10	26	50

As can be seen from table 5 above: PCNA is expressed in cell nuclei of mammary duct epithelium and glandular epithelium, the expression rates of the model groups 2 and 3 are obviously higher than that of the control group 1, and the combined disease model group of the 3 disease groups is higher than that of the 2 simple disease model groups. Each treatment group has the effect of inhibiting PCNA expression, wherein 6 groups of high-dose groups have obvious effect, the effect is slightly poor compared with 4 groups of positive drug control groups, and compared with 2 groups and 3 groups, the experiment 5 group and 6 groups have the effect of reducing PCNA.

The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. The application of the active ingredient peroxyergosterol in the sporotrichum mushrooms is characterized in that: peroxyaergosterol has application in diseases related to CDC25 phosphatase, and inhibits CDC25 phosphatase activity.

2. The use of peroxyergosterol as an active ingredient in a Agaricus campestris according to claim 1, wherein: the CDC25 phosphoprotease related diseases include cancer, hyperplasia of mammary glands and other proliferative diseases.

3. The use of peroxyergosterol as an active ingredient in a Agaricus campestris according to claim 1, wherein: the extraction method of the peroxyergosterol comprises the following steps: slicing fresh and undamaged sporophore of Agaricus campestris, soaking in 90-95% ethanol for one week, filtering to obtain filtrate, recovering solvent under reduced pressure, adding 90-95% ethanol into the residue, ultrasonic extracting for 3-5 times (each time for 1-5 hr), and concentrating under reduced pressure to obtain 90-95% ethanol extract; performing the same operation with 60-65% ethanol, and concentrating under reduced pressure to obtain 60-65% ethanol extract; adding distilled water into the alcohol-extracted agaricus bisporus filter residue, performing ultrasonic extraction for three times, extracting for 1-3h each time, and concentrating under reduced pressure to obtain a water extract;

mixing the two ethanol extracts, kneading and dispersing the mixed extracts in pure water, adding petroleum ether into a separating funnel, adding the extract dispersion liquid into the separating funnel, extracting for 5 times, mixing the extracts, and concentrating to obtain petroleum ether part; adding ethyl acetate into the separating funnel, extracting for 6 times, combining the extracts, and concentrating to obtain an ethyl acetate part; and adding n-butanol into the separating funnel, extracting for 5 times, mixing the extractive solutions, and concentrating to obtain n-butanol fraction.

4. The use of peroxyergosterol as an active ingredient in a Agaricus campestris according to claim 3, wherein: the ratio of the above sporophore of Agaricus campestris to ethanol is 0.5-1:1-5, so that the sporophore of Agaricus campestris is completely soaked in ethanol.

5. The use of peroxyergosterol as an active ingredient in a Agaricus campestris according to claim 3, wherein: the reduced pressure recovery condition is 0.1-0.5MPA, and the temperature is 25-40 ℃; the ultrasonic extraction condition is 200-800HZ, the ultrasonic treatment is carried out for 30-45min, and the temperature is 30-50 ℃.

6. The use of peroxyergosterol as an active ingredient in a Agaricus campestris according to claim 3, wherein: adding petroleum ether into the separating funnel, wherein the ratio of water to petroleum ether is 1: 1-0.5.

7. The use of peroxyergosterol as an active ingredient in a Agaricus campestris according to claim 3, wherein: adding ethyl acetate into a separating funnel, wherein the ratio of water to ethyl acetate is 0.5-1: 1.

8. The use of peroxyergosterol as an active ingredient in a Agaricus campestris according to claim 3, wherein: adding n-butanol into the separating funnel, wherein the ratio of water to n-butanol is 0.4-1: 1.

9. The use of peroxyergosterol as an active ingredient in a Agaricus campestris according to claim 3, wherein: the extraction conditions of the extractum are as follows: the extraction pressure is 20MPa to 40MPa, the extraction temperature is 30 ℃ to 50 ℃, and the extraction time is 0.8h to 2.5 h.

10. The use of peroxyergosterol as an active ingredient in Agaricus campestris according to claim 4, wherein: the ratio of sporophore of Agaricus campestris to ethanol is 1: 3.