CN115677467A

CN115677467A - New use of fungus dreg substance

Info

Publication number: CN115677467A
Application number: CN202110854188.6A
Authority: CN
Inventors: 李芒; 李骏涛; 魏蔚
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-07-28
Filing date: 2021-07-28
Publication date: 2023-02-03
Anticipated expiration: 2041-07-28
Also published as: CN115677467B

Abstract

The invention provides application of a fungus residue substance in the aspect of obtaining a compound dankasterone B and application of the fungus residue substance containing the compound dankasterone B in the aspect of preparing an anti-tumor medicament or health-care food, wherein the fungus is a fungus of Flammulina or Collybia. Compared with the biological fermentation method commonly used for obtaining dankasterone B in the prior art, the method realizes the utilization of the fungus dreg waste, namely, the fungus dreg of Flammulina or Collybia (particularly Flammulina velutipes), which is commonly used only as feed fertilizer or is buried and combusted, is effectively utilized with higher added value, so that the comprehensive utilization of resources is improved, and the cost is greatly reduced.

Description

New use of fungus dreg substance

Technical Field

The invention relates to the technical field of mushroom dreg utilization, in particular to a new application of fungus dreg substances of Flammulina or Collybia.

Background

China is the world with the largest edible fungus production, and the total production is steadily increased from 608 ten thousand tons in 2000 to 3596 thousand tons in 2016, which accounts for more than 70% of the total production in the world. Among them, needle mushroom is one of the largest edible fungi in production scale. The flammulina velutipes mushroom dregs (also called mushroom bran) are typical agricultural wastes in China, and the annual average yield is 5214 ten thousand tons (fremingsan, "comprehensive resource utilization of flammulina velutipes and vinegar residue wastes and earthworm cultivation research thereof", major treatise of Jiangsu university, 2019 for 6 months). The mushroom dregs contain amino acid, crude protein, crude cellulose, crude fat, iron, calcium, zinc, magnesium and other nutrients, so that the mushroom dregs have high development and utilization values. However, at present, the flammulina velutipes mushroom dregs are only partially applied to secondary cultivation substrates of edible mushrooms, mushroom bran feeds, fertilizers and polysaccharide extraction. Most of the flammulina velutipes mushroom dregs are buried, discarded or burned, which not only causes serious waste of resources, but also causes the problem of environmental pollution. Therefore, how to realize the resource comprehensive utilization of the flammulina velutipes mushroom dregs becomes one of the current research hotspots.

The compound dankasterone B is a known compound isolated from a fungal strain, gymnacella denkaliensis, such as Amagata and Numata (Amagata T., et al, "Variation in cytological constraints of a span-derived Gymnesella dankaliensis by manipulating the carbon source", J.Nat.Prod.,2007, 70 p1731-1740. According to the results of this study, the compound dankasterone B had antitumor activity and showed significant cytotoxicity (ED) in P388 lymphocytic leukemia test ₅₀ ＝2.8μg mL ^-1 )。

Disclosure of Invention

In the research on the mushroom dregs, the inventor finds that the isolate of the mushroom dregs (Flammulina velutipes, also known as Collybia velutipes) can effectively inhibit the germination of seeds. In the subsequent separation and purification process for the isolate, the inventors also found that the compound dankasterone B contained in the flammulina velutipes mushroom residue isolate; in other words, the inventor firstly finds that the flammulina velutipes dregs contain the compound dankasterone B. Therefore, the novel application of fungus dreg substances (particularly Flammulina velutipes dreg substances) of Flammulina or Collybia fungi, which are related to the compound dankasterone B in the aspects of reasonable utilization of the fungus dreg, acquisition of bioactive substances and the like, is expanded.

Based on the above research results, in a first aspect, the present invention provides the use of a fungal pomace material for obtaining a compound dankasterone B, wherein the fungus is a fungus of the genus Flammulina or the genus Collybia. In a preferred embodiment according to the present invention, the fungal marc material is a flammulina velutipes marc material.

In the invention, flammulina velutipes (also known as Collybia velutipes) and winter mushrooms are also commonly known as broussonetia papyrifera, needle mushrooms, flammulina velutipes, frozen mushrooms and the like. The genus Pleurotus, hypocreaceae, hymenomycetes, agaricales, tricholomataceae, flammulina (Lentinus) or Collybia (Lysimachia) (also of the genus Pleurotus, also known as Agrocybe aegerita). According to the invention, the flammulina velutipes can be a test strain or a cultivated variety. The test strain is usually a pure culture (also referred to as a strain) obtained by tissue isolation, spore culture, or mushroom-wood isolation of a fruiting body of a fungus from different sources or a fruiting body of the same source. Among these many test strains, those which have stability in genetic characteristics and uniformity in biological characteristics and thus can be widely popularized as cultivated strains are called cultivars. In the present invention, a common cultivar strain of Flammulina velutipes (Fr) Sing; or Collybia velutipes (Curt. ExFr.) Quel, with the white variety F.var. Velutipes (Zhao Shuying, "evaluation research on the quality and genetic relationship of Flammulina velutipes (Shuzo, university, shuchio academic university, 2008).

According to the present invention, the structural formula of the compound dankasterone B is shown as follows:

according to the present invention, the fungal pomace material may be a material containing at least fungal pomace of the genus Flammulina or the genus Collybia, or a material containing at least a mixture containing the compound dankasterone B derived from the fungal pomace.

In the present invention, the fungal pomace may be, according to the usual definition in the industry, the culture medium comprising part of the fungal residue remaining after harvesting the fruiting body of a fungus of the genus Flammulina or Collybia. According to the invention, the fungal pomace may also be a culture medium, from which fungal residues (e.g. mycelia) are removed, or a mixture of both culture media, remaining after harvesting of fungal fruiting bodies of the genus Flammulina or the genus Collybia. The harvesting period of the fungus fruiting body can be different according to fungus strains, and the operation can be generally carried out according to industry convention or standard. In one embodiment according to the invention, the harvesting period of the fungal fruiting body is: collected after about 50-60 days from the date of inoculation.

The medium is typically (but not limited to) a solid depending on its physical state. In case growth of fungal fruiting bodies of the genus Flammulina or the genus Collybia can be achieved, the culture medium may also be liquid or semi-solid. The medium may include, in its composition, a medium composition generally used in the industry for producing fungi of the Flammulina or Collybia genus, particularly Flammulina velutipes, and preferably, the medium has at least a starch-containing substance as a carbon source. The starch-containing material may be soluble starch material, or one or more of insoluble starch materials such as corn cob, corn flour, potato, rice bran, brewer's grains, bean dregs, bean cake powder, sorghum flour, etc. In one embodiment according to the present invention, the culture medium comprises corncobs, rice bran, bran and/or cottonseed hulls, bean dregs, seaweed dregs, beet pulp, calcium carbonate, and the like. In a preferred embodiment according to the invention, the culture medium comprises (in mass percent): 40-50% of corncob, 30-40% of rice bran, 2-4% of bran and/or cottonseed hull, 5-6% of bean dregs, 5-6% of seaweed dregs, 4-5% of beet pulp and 2-3% of calcium carbonate.

In the present invention, the mixture containing the compound dankasterone B derived from the fungal pomace is a mixture containing the compound dankasterone B obtained from the fungal pomace of Flammulina or Collybia by a feasible technical means. The skilled person can choose to use this possible technical means as desired. In general, separation, extraction and purification techniques in fermentation processes may be applied to the present invention, for example, in one embodiment according to the present invention, the mixture containing the compound dankasterone B derived from the fungal pomace is an extract extracted by ethyl acetate and then separated by a silica gel column. In another embodiment according to the present invention, the mixture containing the compound dankasterone B derived from the fungal pomace is a mixture which is subjected to secondary extraction and separation by ethyl acetate and silica gel column, and then purified.

According to the invention, the fungal marc material may also comprise harvested fruiting bodies of a fungus of the genus Flammulina or the genus Collybia.

In a second aspect, the present invention provides a use of fungal dreg material comprising compound dankasterone B in the preparation of an antitumor drug or health food, wherein the fungus is a fungus of the genus Flammulina or Collybia. In a preferred embodiment according to the present invention, the fungal marc material is a flammulina velutipes marc material.

Dankasterone B has been reported in the prior art to have antitumor activity (see Amagata, et al, 2007, supra). The method for preparing medicine or health food (such as functional yogurt) from mixture extracted from fungus fruiting body, fermented product or fungus residue has been reported in Chinese applications CN1416349A, CN103284981A and Chinese patent ZL 201510302359.9. The present invention introduces the parts of these prior art documents relating to the methods of applying the extraction mixture to the preparation of a pharmaceutical or health food (e.g. functional yoghurt) into the present specification, and thus are not described herein again.

Through the technical scheme, the invention achieves the following beneficial effects:

compared with the biological fermentation method commonly used for obtaining dankasterone B in the prior art, the method provided by the invention has the advantages that the fungus dreg material of the fungus (especially the Flammulina velutipes) of Flammulina or Collybia is used for obtaining the compound dankasterone B, or is used for preparing antitumor drugs or health-care foods (such as functional yoghourt), so that the fungus dreg waste utilization is realized, namely: so that fungus dregs of Flammulina or Collybia fungi (especially Flammulina velutipes), which are usually used as feed, fertilizer or are burned in landfills, are effectively utilized with higher added value, thereby improving the comprehensive utilization of resources and greatly reducing the cost.

Drawings

FIG. 1A-B shows the results of thin layer chromatography silica gel plate of needle mushroom fungus residue isolate in mobile phase petroleum ether/ethyl acetate ratio of 2: 1; wherein, FIG. 1A shows the results of thin layer chromatography silica gel plates for needle mushroom residue isolates W2, W3 and W4, and FIG. 1B shows the results of thin layer chromatography silica gel plates for needle mushroom residue isolate W4 with high concentration (left band) and low concentration (right band).

FIG. 2A-C shows the results of thin layer chromatography silica gel plate of needle mushroom fungus residue isolate in mobile phase at a ratio of petroleum ether to ethyl acetate of 5: 1; wherein, FIG. 2A is the result of thin layer chromatography silica gel plate with low concentration of flammulina velutipes mushroom residue isolate W2, W3 and W4, and FIG. 2B is the result of thin layer chromatography silica gel plate with high concentration of flammulina velutipes mushroom residue isolate W2, W3 and W4; FIG. 2C shows the results of thin layer chromatography silica gel plate of the needle mushroom residue isolate W4 in the mobile phase of petroleum ether and ethyl acetate at a ratio of 2:1 (with a little ethyl acetate added), the left zone is W4, the right zone is the result of the substances obtained after 21-36 tubes are combined and concentrated after the W4 secondary separation, and the right zone is the sample for structure identification.

FIGS. 3A-C are LCMS liquid chromatogram of a part of sample W4 of flammulina velutipes fungus residue isolate; in which FIG. 3A shows the results at a wavelength of 220nm, FIG. 3B shows the results at a wavelength of 254nm, and FIG. 3C shows the results of mass spectrometry.

FIG. 4 is a mass spectrum of dankasterone B compound obtained in the present invention.

FIGS. 5A-B are LCMS liquid chromatography-mass spectrometry spectra of a needle mushroom fungus residue isolate W4 sample; in which fig. 5A is a whole map, and fig. 5B is a partially enlarged view according to the whole map.

FIG. 6 is a hydrogen spectrum of dankasterone B compound obtained in the present invention.

FIG. 7 is a carbon spectrum diagram of dankasterone B compound obtained in the present invention.

FIG. 8 shows the results of activity tests on Flammulina velutipes (Fr.) Sing residue isolates W2, W3, and W4; wherein the upper left panel is treated with W2, the upper right panel is treated with W3, the lower left panel is treated with W4, and the lower right panel is blank CK.

FIGS. 9A-D are mass spectra of crude samples of other edible fungi residues or crude samples of edible fungi fruiting bodies. Wherein, fig. 9A is oyster mushroom dregs; FIGS. 9B, 9C and 9D show Flammulina velutipes, pleurotus ostreatus and Hypsizygus marmoreus, respectively.

Detailed Description

The present invention will be further described with reference to the following specific embodiments and the accompanying drawings, but the present invention should not be limited by the specific embodiments disclosed below. The methods used in the following examples are conventional methods unless otherwise specified. The conclusions that can be shown by the test data results in the following examples can be reasonably inferred by those skilled in the art based on the common general knowledge in the technical field, and are not limited to the written descriptions disclosed below.

Example 1 isolation and purification of Compound dankasterone B from Flammulina velutipes mushroom residue

The needle mushroom fungus residue is taken from fresh fungus residue waste accumulated in Wuhan Ruyi factory, and is dried for standby. The culture medium contained in the flammulina velutipes mushroom dregs is a culture medium commonly used in the industry, and specifically comprises the following components in percentage by mass: 40-50% of corncobs, 30-40% of rice bran, 2-4% of bran and/or cottonseed hulls, 5-6% of bean dregs, 5-6% of seaweed residues, 4-5% of beet pulp and 2-3% of calcium carbonate. From inoculation to harvest, about 50-60 days.

1.1 one-step separation of Flammulina velutipes dregs

The method for primary separation specifically comprises the following steps:

1) Weighing 1.5 kg of dry mushroom dregs (the water content of fresh mushroom dregs is 55 percent), and extracting with 15L of ethyl acetate to obtain 114.13 g of paste;

2) Taking 42.21 g of paste, and removing acetic acid by using sodium carbonate to obtain 32 g of paste;

3) Mixing 32 g of paste with 32 g (80-100 meshes) of silica gel;

4) Weighing 600 g (200 meshes) of silica gel, washing with a column with the diameter of 8 cm, petroleum ether and ethyl acetate (5: 1), collecting by a 200mL tube, and performing chromatographic separation;

5) After sample loading detection by a thin layer chromatography silica gel plate, combining the tubes according to different polarities of substances, and concentrating under reduced pressure to obtain 4 parts of isolate samples, which are specifically shown in the following table 1:

TABLE 1

Sample name	Corresponding separation tube	Mass after concentration (gram)
			W1	1 to 11 tubes	23
W2	12-20 tubes	2.3
			W3	21-30 tubes	2.6
W4	The remainder was washed with ethyl acetate only	2.22

Among the four isolate samples shown in table 1, isolates W2, W3 and W4 were dissolved with ethyl acetate and detected on a thin layer chromatography silica gel plate. The results are shown in FIGS. 1 and 2, respectively.

FIG. 1 shows the mushroom residue isolates W2, W3 and W4 in mobile phase petroleum ether: ethyl acetate ratio of 2: the results of the thin layer chromatography silica gel plate at 1 (FIG. 1A), wherein the results of the thin layer chromatography silica gel plate at a relatively high concentration (loading amount) (left band of FIG. 1B) and a relatively low concentration (loading amount) (right band of FIG. 1B) of the needle mushroom fungus residue isolate W4 at a mobile phase petroleum ether: ethyl acetate ratio of 2:1 are also shown. FIG. 2 shows the result of thin layer chromatography silica gel plate of needle mushroom fungus residue isolate in mobile phase petroleum ether to ethyl acetate ratio of 5: 1; wherein, FIG. 2A is the result of thin layer chromatography silica gel plate with low concentration of needle mushroom fungus residue isolates W2, W3 and W4, and FIG. 2B is the result of thin layer chromatography silica gel plate with high concentration of needle mushroom fungus residue isolates W2, W3 and W4.

As can be seen from the results of the thin layer chromatography silica gel plate shown in FIGS. 1 and 2, the mixture extracted from the mushroom residues of Flammulina velutipes was well separated according to the polarity difference.

1.2 Secondary separation of Flammulina velutipes mushroom residue isolate W4 separated as described in 1.1

The specific separation method is as follows:

1) Dissolving a W4 sample by using ethyl acetate, and then concentrating under reduced pressure to obtain 1.4 g of a sample;

2) 1.4 grams of the sample was mixed with 1.4 grams of silica gel (200 mesh);

3) Weighing 30 g (20 meshes) of silica gel, and carrying out chromatographic separation by using a small column with the diameter of 3 cm; sequentially carrying out gradient washing on petroleum ether and ethyl acetate according to the ratio of 2:1, 1: 1 and 1: 2, finally washing the rest part with ethyl acetate, and collecting by using a 20mL pipe, wherein the gradient washing is specifically shown in the following table 2:

TABLE 2

Corresponding separation tube	Eluent
			1 to 26 tubes	Petroleum ether ethyl acetate (2: 1)
27-45 pipes	Petroleum ether ethyl acetate (1: 1)
		46-56 tubes	Petroleum ether ethyl acetate (1: 2)
57 pipe	Rinsing with ethyl acetate
		58 tubes	Methanol rinse

According to the results of silica gel plate loading, 21-36 tubes were combined to obtain a purer intermediate fraction, yielding 0.55 g of material, which was used as a sample for structural identification. The results are shown in FIG. 2C for thin layer chromatography silica gel plate. In FIG. 2C, the mobile phase is petroleum ether and ethyl acetate at a ratio of 2:1 (a little acetic acid is added), the left zone is W4, the right zone is 21-36 tubes, and the substances are concentrated after being combined. From the sample loading results, the isolate is single and has no tailing condition.

1.3 LCMS analysis of Flammulina velutipes bacterial residue isolate sample isolated as described in 1.2 above

The specific method comprises the following steps: a small amount of the above-described sample for structural identification was taken, dissolved in MS bottles with DMSO and acetonitrile (chromatographic grade), and subjected to LCMS (SHIMADZU LCMS 2020) analysis. The sample analysis profile is shown in FIG. 3.

From the sample analysis, with MS of 355/875/357/444/331 as target MS (see the peak in the chart of FIG. 4), rt =1.577min-1.646min can be determined as the target peak, and separation and purification can be carried out.

1.4 separating and purifying the needle mushroom fungus dreg isolate separated in the step 1.2

1) The isolated needle mushroom residue obtained by separation in the above 1.2 was dissolved in 3mL of ethyl acetate, and a mixed solvent such as DMSO and acetonitrile (chromatographic grade) was added thereto, followed by filtration through a 0.45 μm filter. Transferring the sample into a 5mL pointed-bottom scale centrifuge tube, wherein the total volume is about 15mL, preparing the sample by using an MS-Trigger preparative high performance liquid chromatograph, and collecting MS containing 355/875/357/444/331 substances as fractions.

The preparation chromatographic conditions are as follows:

phase A: 0.1% trifluoroacetic acid (chromatographic grade) in water; phase B: acetonitrile (chromatographic grade)

A chromatographic column: shim-Pack C18 25mm 10 μm

Flow rate: 25mL/min

The separation gradiometer is as follows:

time (minutes)	A％	B％
			0.01	40	60
12.00	10	90
			12.20	5	95
15.00	0	100
			15.20	40	60

The preparation map is shown in FIG. 5.

Collecting target substance fractions, and according to different MS values and retention times, dividing the fractions into the following 3 components:

component 1: rt =11.810min-12.251min; the MS value is 444/875/449;

and (2) component: rt =12.262min-13.039min; the MS value is 355;

and (3) component: rt =14.824min-15.696min; the MS value is 331/357.

2) Fraction post-treatment: and (3) performing rotary evaporation and freeze drying on the separated fraction of the 3 components respectively.

Wherein, for component 1, after fraction freeze-drying (laboconco freeze-dryer), weighing (XPE 205DR electron analytical balance) was performed to obtain 13.0mg of target, which is compound 1;

for component 2, the fractions were lyophilized and weighed to give 22.0mg of target compound 2;

for fraction 3, the fractions were lyophilized and weighed to give 21.6mg of the target compound, compound 3.

1.5 Structure identification of the Compound isolated and purified as described in 1.4 above

The structures of the

compounds

1, 2 and 3 isolated and purified as described in 1.3 were confirmed by nuclear magnetic resonance 1D/2D NMR. The results show that the

compounds

2 and 3 are glyceryl stearate derivatives with the following structural formulas respectively.

Compound 2:

compound 3:

the compound 1 is a compound dankasterone B, wherein the structural identification result aiming at the compound 1 is as follows:

the structure of the compound was confirmed by 1D/2D NMR, the nuclear hydrogen spectrum (FIG. 6) and the carbon spectrum (FIG. 7), and the nuclear magnetic data are shown in the following Table 3:

table 3: 1D/2D NMR data for Compounds:

note: s-singlet, d-doublet, t-triplet, m-multiplet, ov-overlap.

The structural formula of the compound was confirmed by nuclear magnetic resonance 1D/2D NMR analysis to correspond to the following structural formula (spatial configuration not investigated):

this compound was searched for as a known compound dankasterone B, in agreement with literature reports (see Amagata, et al, 2007, supra).

Example 2 Activity test of needle Mushroom dreg isolate

Activity tests were performed on needle mushroom fungus residue isolates W2, W3, and W4 obtained in the primary isolation in example 1.

The specific method comprises the following steps: dissolving about 0.1g of needle mushroom fungus dreg isolate with 5mL of ethyl acetate respectively, taking 1mL of the ethyl acetate solution, dripping the ethyl acetate solution on dry filter paper (weighed in advance), weighing again to determine the mass of substances on the filter paper after the ethyl acetate is volatilized, putting the filter paper into a culture dish, adding a proper amount of purified water according to the weight difference of the filter paper to prepare a solution with 1000 times concentration (assuming that the isolate is completely dissolved in water), putting 30 white rapeseed seeds into the culture dish, culturing in a constant-temperature incubator at 27 ℃, and observing and recording the germination condition after 5 days. The experiment was set up with filter paper plus purified water as a blank Control (CK) and repeated 3 times per treatment.

The results of the activity assay are shown in FIG. 8. As is clear from FIG. 8, germination was observed in the seeds in the petri dishes under the treatment with the blank reference CK and the needle mushroom residue isolates W2 and W3; in contrast, the seeds treated with the needle mushroom residue isolate W4 (lower left panel of FIG. 8) did not germinate. The results indicate that the flammulina velutipes mushroom residue isolate W4 has activity of inhibiting seed germination.

Comparative example 1 detection of Compound dankasterone B in other edible fungus dregs

In this comparative example 1, it was examined whether the compound dankasterone B was contained in the mushroom dregs of other edible mushrooms such as oyster mushroom by the LCMS method.

The specific method comprises the following steps:

firstly, taking a plurality of oyster mushroom residues (provided by peripheral farmers), drying in the sun, weighing 100g of mushroom residues, extracting with ethyl acetate, and concentrating under reduced pressure to obtain 5g of paste; subsequently, 0.5G of sample is weighed, dissolved in 3mL of ethyl acetate solution and sent to LCMS for analysis, and Agilent liquid chromatography G6500 series and quadrupole-time-of-flight mass spectrometer (Agilent/1260 + 6530) are combined.

Chromatographic conditions and separation gradient tables see example 1.4 above, where the flow rates are changed to: 5mL/min.

The crude analytical mass spectrum is shown in FIG. 9A. As can be seen from FIG. 9A, in other edible fungus residues, the target object MS value is not detected to be 425/449/472/875 in corresponding time periods.

Comparative example 2 detection of Dankasterone B Compound in fruiting body of edible fungus

In this comparative example 2, whether or not the compound dankasterone B is contained in the fruit body of commercially available edible fungi such as needle mushroom, oyster mushroom (Pleurotus ostreatus), and Hypsizygus marmoreus (Peck) h.e. bigelow) was examined by the method as described in comparative example 1.

The results are shown in FIGS. 9B-D. As can be seen from FIGS. 9B-D, in the fruiting bodies of edible fungi such as Flammulina velutipes, pleurotus ostreatus, hypsizygus marmoreus, a characteristic map of dankasterone B was not detected in the corresponding period (MS value: 425/449/472/875).

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent should be subject to the appended claims.

Claims

1. The application of fungus residue substances in obtaining the compound dankasterone B is disclosed, wherein the fungus is a fungus of Flammulina or Collybia.

2. Use of a fungal pomace material according to claim 1 for obtaining a compound dankasterone B, wherein the Flammulina or Collybia fungus is Flammulina velutipes (Flammulina velutipes) or Collybia velutipes (Collybia velutipes).

3. Use of a fungal pomace material according to claim 1 for obtaining a compound dankasterone B, characterized in that the fungal pomace material is a material comprising at least fungal pomace of the genus Flammulina or the genus Collybia.

4. Use of a fungal pomace material according to claim 3 for obtaining a compound dankasterone B, wherein the fungal pomace is a culture medium comprising a part of fungal debris remaining after harvesting fungal fruiting bodies of the genus Flammulina or the genus Collybia and/or a culture medium comprising a part of fungal debris remaining after harvesting fungal fruiting bodies of the genus Flammulina or the genus Collybia from which the fungal debris has been removed.

5. The use of fungal pomace material according to claim 4 for obtaining compound dankasterone B, wherein the harvesting period of the fungal fruiting body is: collected 50-60 days after the date of inoculation.

6. Use of fungal pomace material according to claim 4 for obtaining compound dankasterone B, characterized in that the culture medium is solid or liquid or semi-solid capable of achieving growth of fruiting bodies of fungi of the Flammulina or Collybia genera.

7. The use of fungal pomace material to obtain compound dankasterone B according to claim 4, wherein the culture medium uses at least a starch-containing material as a carbon source.

8. Use of fungal pomace material according to claim 7 for obtaining compound dankasterone B, wherein the starch-containing material is soluble starch material or one or more selected from corncob, corn meal, potato, rice bran, brewer's grains, bean dregs, bean cake meal, sorghum meal.

9. Use of fungal pomace material according to claim 7 for obtaining compound dankasterone B, wherein the culture medium comprises corn cobs, rice bran, wheat bran and/or cottonseed hulls, bean dregs, seaweed dregs, sugar beet pulp and calcium carbonate.

10. The use of the fungal pomace material for obtaining the compound dankasterone B according to claim 9, wherein the culture medium comprises the following components in percentage by mass: 40-50% of corncob, 30-40% of rice bran, 2-4% of bran and/or cottonseed hull, 5-6% of bean pulp, 5-6% of seaweed residue, 4-5% of beet pulp and 2-3% of calcium carbonate.

11. The use of a fungal pomace material according to claim 1 for obtaining a compound dankasterone B, wherein the fungal pomace material is a material comprising at least a mixture comprising the compound dankasterone B derived from the fungal pomace.

12. Use of a fungal pomace material according to claim 3 or 11 for obtaining a compound dankasterone B, wherein the fungal pomace material further comprises harvested fungal fruiting bodies of the genus Flammulina or Collybia.

13. An application of fungus dreg substances containing a compound dankasterone B in preparing antitumor drugs or health-care foods, wherein the fungus is a fungus of Flammulina or Collybia.