CN110938105A

CN110938105A - Extraction and separation method of active ingredients of agaricus tabularis

Info

Publication number: CN110938105A
Application number: CN201911125258.3A
Authority: CN
Inventors: 林鹏程; 廖志明; 吴疆
Original assignee: Qinghai Nationalities University
Current assignee: Qinghai Nationalities University
Priority date: 2019-11-18
Filing date: 2019-11-18
Publication date: 2020-03-31

Abstract

The invention discloses a method for separating and purifying active ingredients in a sporotrichum mushroom, which is used for quickly separating all the components of the sporotrichum mushroom, has simple and stable separation and extraction process, high product yield and low production cost, is suitable for industrial continuous production, can efficiently and simply separate all the active ingredients, can provide raw materials of various natural products for industrial production and enriches the utilization of natural resources.

Description

Extraction and separation method of active ingredients of agaricus tabularis

The technical field is as follows:

the invention relates to the technical field of separation and purification of natural products, in particular to a method for extracting active ingredients from natural products, and more particularly relates to a method for separating and purifying the active ingredients in sporotrichum mushrooms.

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 Agaricus crocopelus peck, etc. 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.

Essential oils are important constituents of the flavour of fungi of the genus Agaricus. Flavors are classified into volatile flavors and nonvolatile flavors. The volatile flavor is the primary factor whether the food is acceptable for people, and is also the core chemical composition of the spice and the essence. Research shows that the characteristic flavor substances of volatile components in Agaricus bisporus are (E) -Linalool oxide (trans-Linalool oxide), 1-octen-3-ol, 1-octen-3-one, 2-octen-1-ol, 2, 6-dimethylpyrazine, 2-pentylfuran, methylpyrazine, 1-octanol and the like. The Liqin and the like are respectively connected with a mass spectrometer and an smelling instrument through gas chromatography to separate and identify flavor components of the agaricus bisporus, and the results show that main flavor active substances of the agaricus bisporus comprise n-hexanal, C8 unsaturated alcohols, 2, 5-dimethyl pyrazine, ketones, phenylacetaldehyde, 2, 6-dimethyl pyrazine, 2-acetyl thiazole, 3-methyl butyraldehyde and the like. Song Shuzo was analyzed for volatile compounds in Agaricus blazei Murill by GC-MS, and it was found that L-octanol-2-en, Acetie acid, Pyridinium, L, 2-dimethyl, perchlorate, ethyl palmate, Bezaldehyde, Methane, sucfinilbis were contained therein.

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 on the micromolecular chemical components of the agaricus bisporus is developed under the subsidization of the natural science fund 2018-ZL-919 in Qinghai province, and preliminary research on the anti-tumor activity of the ethanol extract of the agaricus bisporus is carried out.

For the research on the active ingredients of the sporotrichum mushrooms and the research on the use value of the sporotrichum mushrooms, the active ingredients in the sporotrichum mushrooms need to be separated, extracted, analyzed and identified so as to determine the active ingredients of the sporotrichum mushrooms and the potential value of the sporotrichum mushrooms. However, since studies on the round-spore mushrooms are still few, the components of the round-spore mushrooms need to be separated and purified.

Disclosure of Invention

Aiming at the problems, the invention provides a method for separating and purifying active ingredients in the sporotrichum mushrooms, and the method for separating and extracting the active ingredients from the sporotrichum mushrooms is used for quickly separating the active ingredients of the sporotrichum mushrooms, has simple and stable separation and extraction process, is suitable for industrial continuous production, has higher product yield and low production cost, and can efficiently and simply separate various active ingredients.

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 (water to n-butanol ratio of 0.4-1:1) into the separating funnel, 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 ℃.

Adding ethanol into the n-butanol part, precipitating to separate out crystal on the container wall, repeating for several timesAnd (4) operating, 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. The Z-I component, namely the pure water elution part of the MCI resin, is separated and cut into sections by an Agilent Zorbax SB-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 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:

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.

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.

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). delta.: 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 high performance liquid chromatography inspection, the components I to IV are found to be in a normal butanol phaseAll the components were combined in 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 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

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. Separating the Z-I component, namely the pure water elution part of MCI resin, by an Agilent ZorbaxSB-CN full-preparation column, cutting into sections, and adding waterMethanol solution (5%) is eluted isocratically 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: 1.5ml/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)δ: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),132.3(d, C-23),42.8(d, 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 peroxyergosterol, and compound 7 was identified as peroxyergosterol.

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-d₆,125MHz)δ: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 thereof are consistent with those of adenosine, so that 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-NMR(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, and compound 13 was identified as 4-hydroxy-3-methoxy-benzyl alcohol.

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

A white powder;

HRESIMS:m/z＝451.3167[M+Na]⁺(calcd forC₂₈H₄₄NaO₃,451.3183)；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-NMR(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 control for p-hydroxybenzyl alcohol, so 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(2HS, 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 Gallic acid (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 gallic acid, and compound 22 was identified as gallic acid.

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.

The method for extracting the active components from the sporotrichum mushrooms has the advantages of 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.

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. A method for separating and purifying active ingredients in sporotrichum mushrooms is characterized by comprising 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; extracting with 60-65% ethanol under reduced pressure to obtain 60-65% ethanol extract, extracting the residue with distilled water under ultrasonic extraction for three times (each time for 1-3 hr), and concentrating under reduced pressure to obtain 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.

2. The method for separating and purifying an active ingredient from a sporotrichum mushroom as set forth in claim 1, 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.

3. The method for separating and purifying an active ingredient from a sporotrichum mushroom as set forth in claim 1, 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 ℃.

4. The method for separating and purifying an active ingredient from a sporotrichum mushroom as set forth in claim 1, wherein: adding petroleum ether into the separating funnel, wherein the ratio of water to petroleum ether is 1: 1-0.5.

5. The method for separating and purifying an active ingredient from a sporotrichum mushroom as set forth in claim 1, wherein: adding ethyl acetate into the separating funnel, wherein the ratio of water to ethyl acetate is 0.5-1: 1.

6. The method for separating and purifying an active ingredient from a sporotrichum mushroom as set forth in claim 1, wherein: adding n-butanol into the separating funnel, wherein the ratio of water to n-butanol is 0.4-1: 1.

7. The method for separating and purifying an active ingredient from a sporotrichum mushroom as set forth in claim 1, 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.

8. The method for separating and purifying an active ingredient from a sporotrichum mushroom as set forth in claim 2, wherein: the ratio of sporophore of Agaricus campestris to ethanol is 1: 3.

9. The method for separating and purifying an active ingredient from a Agaricus campestris according to claim 3, wherein: the reduced pressure recovery condition is 0.5MPA and the temperature is 35 ℃; the ultrasonic extraction condition is 800HZ, ultrasonic is 45min, and the temperature is 30-50 ℃.

10. The method for separating and purifying an active ingredient from a Agaricus campestris according to claim 4, wherein: adding petroleum ether into the separating funnel, wherein the ratio of water to petroleum ether is 1: 0.5.