CN114230631B

CN114230631B - Method for converting ustilaginoidea virens A by using fungi and application thereof

Info

Publication number: CN114230631B
Application number: CN202111580007.1A
Authority: CN
Inventors: 周立刚; 赖道万; 王明安; 李鹏; 匡宇
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2021-12-22
Filing date: 2021-12-22
Publication date: 2023-10-20
Anticipated expiration: 2041-12-22
Also published as: CN114230631A

Abstract

The application discloses a method for converting ustilaginoidea virens A by using fungi and application thereof. The method comprises the steps of converting ustilaginoidea virens A into ustilaginoidea virens I and J in a buffer solution with the pH value of 7 by utilizing an enzyme system in a cell-free extract of the cladosporium cucumerinum; ustilaginoidea can be converted into ustilaginoidea I, J and M in a buffer with pH value of 9. Experiments show that: the cytotoxic activity of the transformation products, ustilaginoidea I, J and M, is generally reduced compared to ustilaginoidea a. The method for converting the ustilaginoidea virens A by using fungi has simple process and can effectively carry out bioconversion detoxification of the ustilaginoidea virens A.

Description

Method for converting ustilaginoidea virens A by using fungi and application thereof

Technical Field

The application belongs to the technical field of biology, and particularly relates to a method for converting ustilaginoidea virens A by using fungi and application thereof.

Background

False smut (Rice false smut) is a fungal ear disease which seriously damages Rice worldwide at present, and the main symptom is that false smut balls are formed on the Rice ear. The pathogenic rice false smut is Rhizoctonia oryzae, has a morphology of Villosiclava virens (Nakata) Tanaka & Tanaka, and has a morphology of Ustilaginoidea virens (Cooke) tanahashi. The disease not only affects the quality of paddy, causes the yield reduction of paddy, but also generates toxins harmful to human and livestock, and seriously threatens the grain safety and the human health. Three major classes of mycotoxins have been reported in Ustilago oryzae and Ustilago oryzae, namely Ustilocins (Ustilocins), rhizoctons (Ustilaginoids) and Sorbiciloids.

The current mycotoxin detoxification method mainly comprises the following steps: physical detoxification, chemical detoxification and bioconversion detoxification. Besides the photocatalytic degradation of ustilaginoidea A reported by the institute of oil crops of China academy of agricultural science, other reports on detoxification methods of ustilaginoidea A toxins are not yet available, and detoxification of ustilaginoidea A toxins has very important significance for reducing toxin harm and guaranteeing food safety.

Of the three toxins of ustilaginoidea virens, ustilaginoidea is the most toxic one. The ustilaginoidea virens is water-soluble cyclic peptide, has wide toxicity and has toxic action on rice seed germination, seedling and callus growth; can cause the reduction of the growth and reproductive capacity of livestock and poultry and the pathological changes of internal organs; has also inhibiting effect on eukaryotic tubulin assembly and cytoskeletal formation. Based on the wide toxicity of the ustilaginoidea virens, it is important to change the ustilaginoidea virens into a compound with weak toxicity or no toxicity. Of the 5 types of ustilaginoidea that have been elucidated in structure, ustilaginoidea A (Ustiloxin A) is the main toxin present in rice and processed products thereof, and its toxicity is also highest.

Bioconversion (Biotransformation) is the use of organisms and their enzymatic systems to alter the structure of toxins, which tend to weaken or disappear. The biotransformation detoxification method has the advantages of strong specificity, mild reaction conditions and no damage to target products, and is considered as a detoxification method with wide application prospect. Therefore, development of a bioconversion method aiming at the ustorium A, reducing the toxicity of the ustorium A, and has very important significance.

Disclosure of Invention

The application aims to provide a method for converting ustilaginoidea virens A into a low-toxicity or nontoxic product by using fungi.

In order to achieve the above object, the present application provides a bioconversion method of ustorin A.

The bioconversion method of the ustorium A provided by the application comprises the step of treating the ustorium A with a cladosporium cucumerinum cell-free extracting solution; the preparation method of the cell-free extract of the black spore shell comprises the following steps: centrifuging the black spore shell fungus liquid to obtain mycelium sediment, adding buffer solution with pH value of 6-10 into the mycelium sediment, performing ultrasonic crushing and centrifugation, and collecting supernatant to obtain the black spore shell cell-free extract (extract containing crude enzyme).

In the above method, the black spore shell (Petriella setifera) can be any strain of black spore shell. In a specific embodiment of the present application, the cladophora is cladophora Nitaf10.

In the above method, the buffer is added in an amount of 30mL.

In the above method, the centrifugation condition is 4 ℃,12000 Xg, and centrifugation is performed for 20min.

In the method, the ultrasonic crushing conditions are as follows: the ultrasonic oscillation power is 80-100% (such as 100%), the ultrasonic oscillation times are 100-150 times (such as 150 times), each time oscillates for 2 seconds, and the interval is 2 seconds.

In the method, the preparation method of the black spore shell fungus liquid comprises the following steps: activating the cladosporium cucumerinum on a PDA culture medium, inoculating the activated cladosporium cucumerinum into a PDB culture medium, and performing shake culture to obtain cladosporium cucumerinum liquid. Further, the shaking culture is carried out for 3 to 5 days. Still further, the inoculum size of the black spore shell of the seta is 3-4 bacterial cakes with the size of 6mm per 100mL of culture medium.

In the above method, the ustilaginoidea can be a material containing ustilaginoidea A, such as ustilaginoidea A solution or ustilaginoidea A-infected rice pellet or ustilaginoidea A-infected rice and processed products thereof, etc.

Further, the ustilaginoidea are a ustilaginoidea a solution.

Still further, the concentration of the curculin A solution was 500. Mu.g/mL.

In the method, the method for treating the ustilaginoidea virens A by using the ustilaginoidea virens acellular extract is to uniformly mix the ustilaginoidea virens acebeing cladi extract with the ustilaginoidea virens A solution for reaction.

Further, the volume ratio of the cell-free extract of the black spore shell of the setaria to the solution of the curculin A is 4:1.

Further, the reaction conditions were 28℃and 180 rpm for 3 days.

In the above method, the buffer solution with pH value of 6-10 may be phosphate buffer solution with pH value of 6 (concentration of 0.5M), phosphate buffer solution with pH value of 7 (concentration of 0.5M), phosphate buffer solution with pH value of 8 (concentration of 0.5M), carbonate buffer solution with pH value of 9 (concentration of 0.5M) or carbonate buffer solution with pH value of 10 (concentration of 0.5M).

In one embodiment of the present application, when a phosphate buffer solution (concentration of 0.5M) having a pH of 7 is added to the mycelium pellet, the enzyme system in the cell-free extract of black-bristled housing performs a 5 '-site specific transamination reaction on ustorium sativum A shown in formula I, and performs a 6' -site decarboxylation reaction on the same, thereby generating ustorium I shown in formula II and ustorium J shown in formula III, respectively.

In another embodiment of the present application, when a carbonate buffer solution (concentration of 0.5M) having a pH of 9 is added to the mycelium pellet, the enzyme system in the cell-free extract of black spore shell of setaria performs an ammonification reaction and a decarboxylation reaction on ustifolin a shown in formula I to produce ustifolin I shown in formula II and ustifolin J shown in formula III, and performs a hydroxylation reaction on the 13-position aromatic ring on ustifolin a to produce ustifolin M shown in formula IV.

The method further comprises the steps of separating, identifying and determining toxicity of the transformation products of the false brinellin I, the false brinellin J and the false brinellin M.

The separation method comprises the following steps: pure products of the conversion products were obtained by liquid chromatography analysis and reference to the known compound ustilaginoidea a by open column chromatography and semi-preparative liquid chromatography.

The identification method comprises the following steps: determining the molecular weight and molecular formula of the converted product by high resolution mass spectrometry; the structure of the conversion product is determined by one-dimensional and two-dimensional spectra of nuclear magnetic resonance carbon spectrum and hydrogen spectrum.

The toxicity assay includes a cytotoxic activity assay and an inhibitory activity assay for the elongation of the radicle and embryo of germinated seeds.

In order to achieve the above purpose, the application also provides the cell-free extract of the black spore shell.

In order to achieve the aim, the application also provides a novel application of the method or the cell-free extract of the black spore shell.

The application provides the application of the method or the cell-free extract of the black spore shell of the cladosporium cucumerinum in any one of the following 1) to 3):

1) Ustilaginoidea A detoxifies;

2) Degrading the curculin A;

3) Reduce toxicity of the ustilaginoidea virens A.

In order to achieve the above object, the present application finally provides a product for degrading ustilaginoidea a.

The active ingredient of the product for degrading the curculin A provided by the application is the cell-free extract of the black spore shell of the seta.

The application provides a method for converting ustilaginoidea virens A by using fungi, which comprises the steps of converting ustilaginoidea virens A into ustilaginoidea virens I and J in a buffer solution with the pH value of 7 by using an enzyme system in a cladosporium cucumerinum cell-free extracting solution; ustilaginoidea can be converted into ustilaginoidea I, J and M in a buffer with pH value of 9. The cytotoxicity activity detection experiment shows that: the cytotoxic activity of the transformation products, ustilaginoidea I, J and M, is generally reduced compared to ustilaginoidea a. The method for converting the ustilaginoidea virens A by using the fungi has simple process, can effectively carry out bioconversion detoxification of the ustilaginoidea virens A, and has important significance on detoxification of ustilaginoidea virens toxin.

Drawings

FIG. 1 is a full wavelength scan of an HPLC plot of ustilaginoidea M converted to ustilaginoidea M using pH7 buffer and its chromatographic peaks in a cell-free extract of Nitaf10 from Nitaf10. FIG. 1a is a liquid phase diagram of substrate, ustilaginoidea A; FIG. 1b is a liquid phase diagram after reaction in a buffer having a pH of 7, and FIG. 1c is a liquid phase diagram after reaction in a buffer having a pH of 9.

FIG. 2 is a HR-ESI-MS spectrum of ustilaginoidea A.

FIG. 3 shows ustorin A ¹ H NMR spectrum (D) ₂ O,400MHz)。

FIG. 4 shows ustorin A ¹³ C NMR spectrum (D) ₂ O,400MHz)。

FIG. 5 shows the HR-ESI-MS spectrum of ustilaginoidea I.

FIG. 6 is a drawing of ustorium I ¹ H NMR spectrum (D) ₂ O,500MHz)。

FIG. 7 shows ustorin I ¹³ C NMR spectrum (D) ₂ O,125MHz)。

FIG. 8 shows ustorin I ¹ H- ¹ H COSY spectrum (D) ₂ O,500MHz)。

FIG. 9 is a HMBC spectrum (D) ₂ O,500MHz)。

FIG. 10 is a HR-ESI-MS spectrum of ustilaginoidea J.

FIG. 11 shows ustorin J ¹ H NMR spectrum (D) ₂ O,500MHz)。

FIG. 12 is a drawing of ustorium J ¹³ C NMR spectrum (D) ₂ O,125MHz)。

FIG. 13 is a HSQC spectrum (D) ₂ O,500MHz)。

FIG. 14 is a HMBC spectrum (D) ₂ O,500MHz)。

FIG. 15 is a HR-ESI-MS spectrum of ustilaginoidea M.

FIG. 16 shows the effect of ustorium M ¹ H NMR spectrum (D) ₂ O,500MHz)。

FIG. 17 shows the composition of ustorium M ¹ H- ¹ H COSY spectrum (D) ₂ O,500MHz)。

FIG. 18 is HSQC spectrum (D) ₂ O,500MHz)。

FIG. 19 is a HMBC spectrum (D) ₂ O,500MHz)。

FIG. 20 shows the inhibition of rice germination seed radicle and embryo elongation by ustilaginoidea virens A and J and the positive control glyphosate.

Detailed Description

The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The biomaterials involved in the following examples were as follows:

rice seeds: seeds of japonica rice (Oryza sativa var. Japonica) No. 2, liaoning Dan nuo, were purchased from an exemplary breeding farm in the east of Liaoning.

The fungus strain, black spore shell (Petriella setifera) Nitaf10 (GenBank accession number KM 095515): the present inventors isolated this strain from tobacco (Nicotiana tabacum) roots, and specific isolation and identification procedures thereof were described in the literature, "Zhou K, wang W, peng Y, yu R, yue Y, lai D, zhou L.endophytic fungi from Nicotiana tabacum L.and their antibacterial activity.Nature Product Research and Development,2015,27 (11): 1847-1852. "in".

5 human cancer cell lines: human colon cancer cell line (HCT-8), human pancreatic cancer cell line (PANC-1), human gastric cancer cell line (HGC-27), human liver cancer cell line (HepG) and human lung cancer cell line (PC 9) are all purchased from the institute of medicine of China medical science.

The media and solutions involved in the following examples are as follows:

PDA culture medium formula: 200g/L of potato, 20g/L of glucose and 15g/L of agar.

PDB culture medium formula: 200g/L of potato and 20g/L of glucose.

0.5M phosphate buffer formulation at pH 6: na (Na) ₂ HPO ₄ ·2H ₂ O 89g/L，NaH ₂ PO ₄ ·2H ₂ O78 g/L, the pH was adjusted to 6 with pure HCl or 1M NaOH solution.

0.5M phosphate buffer formulation at pH 7: na (Na) ₂ HPO ₄ ·2H ₂ O 89g/L，NaH ₂ PO ₄ ·2H ₂ O78 g/L, the pH was adjusted to 7 with pure HCl or 1M NaOH solution.

0.5M phosphate buffer formulation at pH 8: na (Na) ₂ HPO ₄ ·2H ₂ O 89g/L，NaH ₂ PO ₄ ·2H ₂ O78 g/L, the pH was adjusted to 8 with pure HCl or 1M NaOH solution.

0.5M carbonate buffer formulation at pH 9: na (Na) ₂ CO ₃ 53g/L，NaHCO ₃ 42g/L, the pH was adjusted to 9 with pure HCl or 1M NaOH solution.

0.5M carbonate buffer formulation at pH 10: na (Na) ₂ CO ₃ 53g/L，NaHCO ₃ 42g/L, the pH was adjusted to 10 with pure HCl or 1M NaOH solution.

0.5M acetate buffer formulation at pH 4: 41g/L sodium acetate, 20mL glacial acetic acid, was adjusted to pH4 with pure HCl or 1M NaOH solution.

0.5M acetate buffer formulation at pH 5: 41g/L sodium acetate, 20mL glacial acetic acid, was adjusted to pH5 with pure HCl or 1M NaOH solution.

1M NaOH solution formula: 40g of NaOH was dissolved in 1L of ultrapure water.

The reagents involved in the following examples were as follows:

CuCl ₂ ·2H ₂ o: beijing red star chemical plant.

MgCl ₂ ·6H ₂ O: beijing North chemical fine chemicals Limited company.

CaCl ₂ ·2H ₂ O: national medicine group chemical Co., ltd.

CoCl ₂ ·2H ₂ O: national medicine group chemical Co., ltd.

MnCl ₂ ·4H ₂ O: west Long science Co., ltd.

BaCl ₂ ·2H ₂ O: national medicine group chemical Co., ltd.

ZnCl ₂ : west Long science Co., ltd.

FeCl ₃ ·6H ₂ O: national medicine group chemical Co., ltd.

Potato: purchased from the market.

Glucose: national medicine group chemical Co., ltd.

Agar: alatin Corp.

Na ₂ HPO ₄ ·2H ₂ O: national medicine group chemical Co., ltd.

NaH ₂ PO ₄ ·2H ₂ O: national medicine group chemical Co., ltd.

HCl: national medicine group chemical Co., ltd.

NaOH: west Long science Co., ltd.

Na ₂ CO ₃ : national medicine group chemical Co., ltd.

NaHCO ₃ : national medicine group chemical Co., ltd.

Sodium acetate: west Long science Co., ltd.

Glacial acetic acid: national medicine group chemical Co., ltd.

HPLC analysis conditions for ustilaginoidea A and its conversion products are as follows: the mobile phase was methanol/water with 0.02% trifluoroacetic acid (TFA); elution mode: 0-30 minutes, methanol is linearly increased from 10% to 100%; keeping the column flushed by 100% methanol for 30-40 min; the detection wavelength was 210nm at 40-50 minutes equilibrated with 10% methanol/water.

The ustilaginoidea I, J and M referred to in the following examples are pure transformation products obtained by adding substrate ustilaginoidea A to a cell-free extract of fungi in the laboratory of the present inventors, reacting for a period of time, and isolating. Wherein, the ustilaginoidea virens A is converted into ustilaginoidea virens I and J through ammonia conversion and ammonia conversion decarboxylation reaction in a cell-free extract (pH value is 7) of the cladosporium cucumerinum Nitaf10; ustilaginoidea A is converted into ustilaginoidea M by hydroxylation reaction on aromatic ring in cell-free extract (pH 9) of Nitaf10.

Example 1 conversion of Ustilagin A to Ustilagin I and J by Nitaf10 cell-free extract of Ustilaria sinica in phosphate buffer at pH7

1. Experimental method

1. The preparation method of the cell-free extract (pH value is 7) of the cladosporium cucumerinum Nitaf10 comprises the following steps: inoculating activated black spore shell Nitaf10 on potato-glucose-agar medium (PDA) into a 250mL triangular flask containing 100mL potato-glucose-culture solution (PDB), inoculating 3-4 bacterial cakes with the size of 6mm, shaking and culturing for 3-5 days in a shaking table, loading the shaking fermentation liquor (bacterial liquor) into a 50mL round bottom centrifuge tube, centrifuging at 4 ℃ for 20 minutes at 12000 Xg, removing supernatant, adding 0.5M Phosphate Buffer (PBS) with the pH value of 7 into mycelium sediment, carrying out ice bath on an ultrasonic cleaner (100% power) for about 150 times, oscillating for 2 seconds each time, then carrying out shaking for 4 ℃ for 12000 Xg, centrifuging for 20 minutes, and taking supernatant which is an extract containing intracellular crude enzyme, namely the black spore shell Nitaf10 cell-free extract (pH value is 7).

2. In a penicillin bottle were added 800. Mu.L of the cell-free extract of Nitaf10 of Nitaf, prepared in step 1 (pH 7), and 200. Mu.L of a solution of ustilaginoidea virens A (solvent water) at a concentration of 500. Mu.g/mL, and the reaction was carried out at 28℃for 3 days with the control of adding only the cell-free extract of Nitaf10 of Nitaf and only the solution of ustilaginoidea virens A, at 180 rpm, to obtain a reaction product, and HPLC analysis was carried out.

The preparation of the above-mentioned ustilaginoidea A can be referred to in the literature "Shan T, sun W, liu H, gao S, lu S, wang M, chen Z, wang S, zhou L.determination and analysis of ustiloxins A and B by LC-ESI-MS and HPLC in false smut balls of ce.International Journal of Molecular Sciences,2012,13 (9): 11275-11287. The method in "and" Shan T, sun W, wang X, fu X, sun W, zhou L.purification of ustiloxins A and B from rice false smut balls by macroporous resins. Molecules,2013,18 (7): 8181-8199 ". The method comprises the following specific steps: extracting a certain amount of rice false smut ball with water to obtain water extract, filtering with gauze and filter paper, concentrating the filtrate, separating and purifying the crude extract with macroporous adsorbent resin SP207, reversed phase silica gel column ODS-AQ and dextran gel column G-15, and performing spectral identification on the obtained monomer compound to obtain the final product of ustilaginoidea A. The specific operation is as follows: taking rice false smut (1000 g), airing, crushing, carrying out cold leaching extraction for 7 times according to the proportion of dry weight (g) to water volume (mL) of 1:30 (g/mL), each time for 12 hours, combining 7 times of extracting solutions, and concentrating under reduced pressure to obtain the rice false smut water extract. Re-dissolving the extract with water, filtering with filter paper, purifying the filtrate with macroporous adsorbent resin SP207, eluting with water, eluting with 30% ethanol, mixing 30% ethanol eluates, concentrating, and sequentially passing through ODS-AQ and Sephadex G-15 to obtain 80mg pure ustilaginoidea virens A.

The structural identification method of the curculin A comprises the following steps: taking the separated and purified ustilaginoidea virens A monomer compound, and carrying out spectrum identification on the compound, wherein a high resolution mass spectrum (HR-ESI-MS) spectrogram is shown in figure 2; nuclear magnetic resonance hydrogen spectrum [ ] ¹ H NMR,D ₂ O,400 MHz) spectrum as shown in fig. 3; nuclear magnetic resonance carbon spectrum @ ¹³ C NMR,D ₂ O,100 MHz) spectrum is shown in fig. 4. Thus, it was confirmed that the structure of the obtained ustilaginoidea virens A was shown in formula I and was the same as that of the known ustilaginoidea virens A.

The physicochemical properties and spectrum data of the prepared ustilaginoidea virens A are as follows: pure white powder (MeOH). By high resolution mass spectrometry (HR-ESI-MS, m/z 674.26859[ M+H ]] ⁺ ) The molecular formula is determined as follows: c (C) ₂₈ H ₄₃ N ₅ O ₁₂ S, S. Nuclear magnetic resonance hydrogen spectrum (D) ₂ O,400MHz chemical shifts (delta, ppm) and nuclear magnetic resonance carbon spectra (D) ₂ O,100 MHz) chemical shifts (. Delta., ppm) are shown in Table 1. Physicochemical Properties and spectroscopic data of the above-prepared ustorin A and literature (Koiso Y, li Y, iwasaki S, hanaoka K, kobayashi T, sonoda R, fujita)Y, yaegashi H, sato Z. Ustiloxins, antimitotic cyclic peptides from false smut balls on rice panicles caused by Ustilaginoidea virens. The Journal of Antibiotics 1994,47 (7): 765-773.).

Table 1 Nuclear magnetic resonance carbon Spectrum and Hydrogen Spectrum data for substrate, ustilaginomycin A

2. Experimental results

The reaction product is detected by liquid chromatography to find that: the reaction product of the cell-free extract of Nitaf10 from Nitaf10 (pH 7) with the ustilaginoidea virens A solution showed a peak of the conversion product (FIG. 1 b), whereas none of the control groups detected the peak of the conversion product. And (3) separating and purifying the reaction product by a gel column G-15, wherein pure water is used as a mobile phase, and then semi-preparing methanol water with 20% of liquid phase to prepare the ustilaginoidea virens I and J. The method comprises the steps of firstly converting the ustilaginoidea A into the ustilaginoidea I by ammonia and then decarboxylating the ustilaginoidea I to generate the ustilaginoidea J in a 0.5M phosphate buffer with the pH value of 7 by using a cladosporium cucumerinum Nitaf10 cell-free extract. Illustrating that ustilaginoidea can be converted into ustilaginoidea I and J by using a cell-free extract of Nitaf10 of Nitaf in phosphate buffer with pH of 7.

The structural identification method of the curculin I is as follows: taking the separated and purified ustilaginoidea I monomer compound, and carrying out spectrum identification on the compound, wherein a high resolution mass spectrum (HR-ESI-MS) spectrogram is shown in figure 5; nuclear magnetic resonance hydrogen spectrum [ ] ¹ H NMR,D ₂ O,500 MHz) spectrum as shown in fig. 6; nuclear magnetic resonance carbon spectrum @ ¹³ C NMR,D ₂ O,125 MHz) spectrum as shown in fig. 7; two-dimensional nuclear magnetic resonance spectrogram ¹ H- ¹ H COSY and HMBC are shown in FIGS. 8 and 9, respectively. Comparing with known ustorium A, the amino group at 5' position of ustorium I is changed into carbonyl group by ammonia transfer, and the knot of the obtained ustorium I is determinedThe structure is shown as a formula II.

The physicochemical properties and spectrum data of the prepared ustilaginoidea are as follows: the pure product is light white amorphous solid and is easy to dissolve in water. Obtaining the peak mass-to-charge ratio m/z 671.2289[ M-H ] of the excimer ions by high resolution mass spectrometry (HR-ESI-MS)] ^- ) The molecular formula is determined as follows: c (C) ₂₈ H ₄₀ N ₄ O ₁₃ S, S. Nuclear magnetic resonance hydrogen spectrum (D) ₂ O,500MHz chemical shifts (delta, ppm) and nuclear magnetic resonance carbon spectra (D) ₂ O,125 MHz) chemical shifts (. Delta., ppm) are shown in Table 2. ¹ H NMR(D ₂ O,500MHz)δ(ppm)：8.42(1H,s,H-13),7.56(1H,s,H-16),4.88(1H,d,J _10,9 ＝10.0,H-10),4.81(1H,s,H-3),4.62-4.67(1H,m,H-3’),4.28(1H,d,J _9,10 ＝10.5,H-9),4.09(1H,d,J _6,24 ＝10.0,H-6),3.74(2H,s,H-19),2.98(1H,dd,J _2’,2’ ＝11.0,J _2’,3’ ＝2.5,H-2’),3.08(1H,H-2’),2.70(3H,s,NCH ₃ -9),2.17-2.21(1H,m,H-4’),1.82-1.86(1H,m,H-24),1.70(1H,s,H-21),1.63-1.67(1H,m,H-22),2.15-2.22(1H,m,H-22),1.04(3H,t,J＝1.04,H-23),0.83(3H,d,J _26,24 ＝6.5,H-26),0.73(3H,d,J _25,24 ＝6.5,H-25)； ¹³ C NMR(D ₂ O) δ (ppm): 86.6 (C-2), 59.1 (C-3), 169.9 (C-5), 59.6 (C-6), 166.2 (C-8), 66.0 (C-9), 73.4 (C-10), 127.0 (C-11), 136.6 (C-12), 113.6 (C-13), 151.7 (C-14), 145.5 (C-15), 123.9 (C-16), 43.2 (C-19), 175.6 (C-20), 20.7 (C-21), 31.6 (C-22), 7.1 (C-23), 28.3 (C-24), 17.6 (C-25), 17.8 (C-26), 62.0 (C-2 '), 63.5 (C-3 '), 38.8 (C-4 '), 175.6 (C-5 '), 170.6 (C-6 '), and the above data are compared to the structures of Koiscki Y, li Y, iwasaki S, hanaak, soba shiba, soba, yabana, 35H, 35, 37H.

TABLE 2 Nuclear magnetic resonance carbon Spectroscopy and Hydrogen Spectroscopy data for the transformation product, ustilaginomycin I

The structural identification method of the above-mentioned ustilaginoidea J is as follows: and (3) taking the separated and purified ustilaginoidea J monomer compound and carrying out spectrum identification on the compound. The high resolution mass spectrum (HR-ESI-MS) spectrum is shown in FIG. 10; nuclear magnetic resonance hydrogen spectrum [ ] ¹ H NMR,D ₂ O,500 MHz) spectrum as shown in fig. 11; nuclear magnetic resonance carbon spectrum @ ¹³ C NMR,D ₂ O,125 MHz) spectra are shown in fig. 12, and two-dimensional nuclear magnetic resonance spectra HSQC and HMBC are shown in fig. 13 and 14, respectively. Compared with the known ustilaginoidea A, the amino at the 5 '-position on the ustilaginoidea J is changed into carbonyl through ammonia transfer, then the carboxyl at the 6' -position is changed into hydroxyl through decarboxylation, and the structure of the obtained ustilaginoidea J monomer compound is determined as shown in a formula III.

The physicochemical properties and spectrum data of the prepared ustilaginoidea virens J are as follows: the pure product is light white amorphous solid. By high resolution mass spectrometry (HR-ESI-MS, m/z 643.2339[ M-H ]] ^- ) The molecular formula is determined as follows: c (C) ₂₇ H ₄₀ N ₄ O ₁₂ S, S. Nuclear magnetic resonance hydrogen spectrum (D) ₂ O,500MHz chemical shifts (delta, ppm) and nuclear magnetic resonance carbon spectra (D) ₂ O,125 MHz) chemical shifts (. Delta., ppm) are shown in Table 3. ¹ H NMR(D ₂ O,500MHz)δ(ppm)：7.54(1H,s,H-13),7.01(1H,s,H-16),4.86(1H,d,J _10,9 ＝10,H-10),4.84(1H,s,H-3),4.51-4.56(1H,m,H-3’),4.24(1H,d,J _9,10 ＝10.5,H-9),4.05(1H,d,J _6,24 ＝10.0,H-6),3.93(2H,s,H-19),2.95(1H,dd,J _2’,2’ ＝12.5,J _2’,3’ ＝2.0,H-2’),3.30(1H,dd,J _2’,2’ ＝17.0,J _2’3’ ＝11.0,H-2’),2.71(3H,s,NCH ₃ -9),2.16(1H,ddd,J _4’,3’ ＝8.5,J _4’,4’ ＝22.5,J _4’5’ ＝15.5,H-4’),1.77-1.84(1H,m,H-24),1.67(1H,s,H-21),1.60-1.65(1H,m,H-22),2.13-2.20(1H,m,H-22),1.03(3H,t,H-23),0.81(3H,d,J _26,24 ＝6.5,H-26),0.71(3H,d,J _25,24 ＝6.5,H-25)； ¹³ C NMR(D ₂ O)δ(ppm):86.4(C-2),58.7(C-3),170.6(C-5),59.5(C-6),165.4(C-8),66.0(C-9),73.4(C-10),127.5(C-11),135.5(C-12),113.6(C-13),151.5(C-14),145.3(C-15),124.2(C-16),170.6(C-17),41.1(C-19),172.7(C-20),20.8(C-21),31.3(C-22),7.3(C-23),28.4(C-24),17.7(C-25),17.5(C-26),63.3(C-2’),62.7(C-3’),51.7(C-4’),174.6(C-5’),31.5(NCH ₃ -C). The structure of ustilaginoxin J was determined by comparing the above data with those reported in the literature (Koiso Y, li Y, iwasaki S, hanaoka K, kobayashi T, sonoda R, fujita Y, yaegashi H, sato Z. Ustiloxins, antimitotic cyclic peptides from false smut balls on rice panicles caused by Ustilaginoidea virens. The Journal of Antibiotics 1994,47 (7): 765-773.).

Table 3 Nuclear magnetic resonance carbon Spectrum and Hydrogen Spectrum data of the transformant ustilaginoidea J

Example 2 conversion of Ustilagin A to Ustilagin M by Nitaf10 cell-free extract of Ustilaria brio in carbonate buffer at pH9

1. Experimental method

1. The preparation method of the cell-free extract (pH value is 9) of the black spore shell Nitaf10 comprises the following steps: inoculating activated black spore shell Nitaf10 on potato-glucose-agar medium (PDA) into 250mL triangular flask containing 100mL potato-glucose-culture solution (PDB), inoculating 3-4 bacterial cakes with the size of 6mm, shaking and culturing for 3-5 days in a shaking table, loading the shaking fermentation broth (bacterial liquid) into a 50mL round bottom centrifuge tube, centrifuging at 4 ℃ for 20min at 12000 Xg, removing supernatant, adding 0.5M Carbonate Buffer (CBS) with the pH value of 9 into mycelium sediment, performing ice bath on an ultrasonic cleaner (100% power) for about 150 times, oscillating for 2 seconds each time, and then centrifuging at 4 ℃ for 20min, and taking the supernatant which is an extract containing intracellular crude enzyme, namely the black spore shell Nitaf10 cell-free extract (pH value is 9).

2. In a penicillin bottle were added 800. Mu.L of the cell-free extract of Nitaf10 of Nitaf, prepared in step 1 (pH 9), and 200. Mu.L of a solution of ustilaginoidea virens A (solvent water) at a concentration of 500. Mu.g/mL, and the reaction was carried out at 28℃for 3 days with the control of adding only the cell-free extract of Nitaf10 of Nitaf and only the solution of ustilaginoidea virens A, at 180 rpm, to obtain a reaction product, and HPLC analysis was carried out.

2. Experimental results

The reaction product is detected by liquid chromatography to find that: the reaction product of the cell-free extract of Nitaf10 from Nitaf at pH9 with the solution of ustilaginoidea virens A had other transformation products in addition to the transformation products of ustilaginoidea virens I and J (FIG. 1 c), and no transformation product peak was detected in the control group. And (3) separating and purifying the reaction product by a gel column G-15, wherein pure water is used as a mobile phase, and then preparing the ustilaginoidea virens M by semi-preparation of liquid phase 20% methanol water. The cell-free extract of Nitaf10 of Nitaf is prepared by ammonia transfer and decarboxylation in 0.5M phosphate buffer with pH value of 9 to obtain ustilaginoidea I and ustilaginoidea J, but at the same time, the ustilaginoidea A is subjected to hydroxylation reaction on 13-position aromatic ring to obtain ustilaginoidea M shown in formula IV. Illustrating that ustilaginoidea can be converted into ustilaginoidea I, J and M by using a cell-free extract of Nitaf10 of Nitaf under phosphate buffer pH 9.

The structural identification method of the curcurcin M comprises the following steps: the isolated and purified ustilaginoidea M monomer compound is taken and subjected to spectrum identification, and a high resolution mass spectrum (HR-ESI-MS) spectrum is shown in figure 15; nuclear magnetic resonance hydrogen spectrum [ ] ¹ H NMR,D ₂ O,500 MHz) spectrum as shown in fig. 16; two-dimensional nuclear magnetic resonance spectrogram ¹ H- ¹ H COSY, HSQC and HMBC are shown in FIG. 17, FIG. 18 and FIG. 19, respectively. Comparing with the known ustorium A, hydroxylation on the aromatic ring at 13 position of the ustorium M, and determining the structure of the obtained ustorium M monomer compound as shown in the formula IV.

The physicochemical properties and spectrum data of the prepared ustilaginoidea virens M are as follows: the pure product was a pale white amorphous solid, and was obtained by high resolution mass spectrometry (HR-ESI-MS, m/z 690.2292[ M+H ]] ⁺ ) The molecular formula is determined as follows: c (C) ₂₈ H ₄₃ N ₅ O ₁₃ S, S. Nuclear magnetic resonance hydrogen spectrum (D) ₂ O,500 MHz) chemical shifts (. Delta., ppm) are shown in Table 4. ¹ H NMR(D ₂ O,500MHz)δ(ppm)：7.15(1H,s,H-16),4.96(1H,H-10),4.87(1H,s,H-3),4.50-4.55(1H,m,H-3’),4.32(1H,d,J _9,10 ＝10.5Hz,H-9),4.07(1H,d,J _6,24 ＝11.5Hz,H-6),3.88(1H,dd,J _5’,4’ ＝2Hz,13.5Hz,H-5’),3.63(2H,s,H-19),3.44(2H,H-2’),2.73(3H,s,NCH ₃ -9)，2.05-2.11(1H,m,H-4’),2.69(1H,H-4’),1.84-1.92(1H,m,H-24),1.75(1H,s,H-21),2.04-2.12(1H,m,H-22),1.60-1.67(1H,m,H-22),1.02(3H,t,J＝7.5Hz,H-23),0.84(3H,d,J _26,24 ＝6.5Hz,H-26),0.75(3H,d,J _25,24 =6.5 hz, h-25). The structure of ustilaginoxin M was determined by comparing the above data with those reported in the literature (Koiso Y, li Y, iwasaki S, hanaoka K, kobayashi T, sonoda R, fujita Y, yaegashi H, sato Z. Ustiloxins, antimitotic cyclic peptides from false smut balls on rice panicles caused by Ustilaginoidea virens. The Journal of Antibiotics 1994,47 (7): 765-773.). No relevant hydrogen signal was seen from the nmr hydrogen spectrum (fig. 16), which, in combination with high resolution mass spectrum data, indicated that the 13-hydrogen on the aromatic ring was replaced by a hydroxyl group.

TABLE 4 Nuclear magnetic resonance Hydrogen Spectroscopy data for the transformation product, magnaporthin M

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EXAMPLE 3 dynamic changes in consumption and formation of the transformation products, namely, ustilaginoidea I and J, over time during the bioconversion of ustilaginoidea A in the cell-free extract of Nitaf10 of Nitaf

1. Experimental method

1. As in step 1 of example 1.

2. As in step 2 of example 1.

3. Samples were taken at reaction times 0h, 6h, 12h, 24h, 36h, 48h and 72h, the reaction was stopped with an equal volume of methanol, dried, re-dissolved with 1mL of ultra-pure water, and HPLC analysis was performed.

2. Experimental results

The results are shown in Table 5, which shows that: in the bioconversion process of the bristled black spore shell Nitaf10 cell-free extract on the ustilaginoidea virens A, a conversion product is generated at the 6 th hour, two conversion products appear, the content of the ustilaginoidea virens A gradually decreases along with the extension of the reaction time, the amounts of the conversion products ustilaginoidea virens I and J slowly increase, and the synthesis amounts of the ustilaginoidea virens I and the ustilaginoidea virens J respectively reach the maximum values when the reaction reaches the 48 th hour and the 36 th hour.

TABLE 5 time course of the biotransformation of ustifolia A in the cell-free extract of Nitaf10 from Nitaf10

Note that: the data in the table are mean ± standard deviation (n=3), and each column of different letters indicates a significant difference between the data at p.ltoreq.0.05.

Example 4 Effect of different reaction initiation pH values on the conversion of ustilaginoidea A in cell-free extract of Nitaf10

1. Experimental procedure

1. Buffer solutions with different pH values and concentration of 0.5mol/L are prepared: the pH values are 4 and 5 for acetate buffer solutions, 6, 7 and 8 for phosphate buffer solutions, and 9 and 10 for carbonate buffer solutions.

2. The preparation method of the cell-free extract of the cladosporium cucumerinum Nitaf10 comprises the following steps: inoculating activated black spore shell Nitaf10 on potato-glucose-agar medium (PDA) into 250mL triangular flask containing 100mL potato-glucose-culture solution (PDB), inoculating 3-4 6mm bacterial cakes, shake culturing in shaking table for 3-5 days, loading shake cultured fermentation broth (bacterial liquid) into 50mL round bottom centrifuge tube, centrifuging at 4deg.C for 20min, removing supernatant, adding 30mL buffer solution with different pH value of 0.5mol/L to mycelium sediment, ice-bathing on ultrasonic cleaner (100% power) for 150 times, shaking for 2 seconds each time, and centrifuging at 4deg.C for 20min to obtain black spore shell Nitaf10 cell-free extract with different pH values.

3. In a penicillin bottle, 800. Mu.L of cell-free extract of Nitaf10 of black spore with different pH values and 200. Mu.L of ustilaginoidea virens A solution with the concentration of 500. Mu.g/mL are respectively added, and the mixture is reacted for 3 days at the temperature of 28 ℃ and the rotation speed of 180 r/min. Then, the reaction was stopped by adding an equal volume of methanol, and after drying, re-dissolved with 1mL of ultra-pure water, and HPLC analysis was performed to calculate the conversion rate of ustilaginoidea virens A. The conversion of ustilaginoidea A was calculated as follows: conversion (%) = [ (amount of added ustilaginoidea a-amount of ustilaginoidea a remaining after the reaction)/amount of added ustilaginoidea a ] ×100.

2. Experimental results

The results are shown in Table 6. The results show that: the cell-free extract of Nitaf10 of Neurospora crassa did not undergo conversion reaction at pH values of 4 and 5, but at pH value of 6, the conversion rate was about 50%, and at the initial pH value of the reaction at alkaline conditions, i.e., pH values of 9 and 10, the conversion rate was as high as 70%, and at pH value of 9, the conversion rate was as high as 91%.

TABLE 6 influence of different reaction initiation pH values on the conversion of ustilaginoidea A

pH at the beginning of the reaction	Conversion of Ustilbenin A (%)
		4	0±0d
5	0±0d
		6	49.27±4.25c
7	87.50±1.23a
		8	85.98±0.55a
9	91.44±1.33a
		10	71.25±16.02b

Example 5 Effect of different types of Metal ions on the bioconversion of ustorium Nitaf10 cell-free extract to ustorium oryzae A

1. Experimental method

1. The following different types of metal ion solutions with the concentration of 30mmol/L are prepared:

Cu ²⁺ ion solution: cuCl is added ₂ ·2H ₂ Mixing O with sterile water to obtain a solution;

Mg ²⁺ ion solution: mgCl is added ₂ ·6H ₂ Mixing O with sterile water to obtain a solution;

Ca ²⁺ ion solution: caCl is added with ₂ ·2H ₂ Mixing O with sterile water to obtain a solution;

Co ²⁺ ion solution: coCl is to be processed ₂ ·2H ₂ Mixing O with sterile water to obtain a solution;

Mn ²⁺ ion solution: mnCl is added to ₂ ·4H ₂ Mixing O with sterile water to obtain a solution;

Ba ²⁺ ion solution: baCl is added to ₂ ·2H ₂ Mixing O with sterile water to obtain a solution;

Zn ²⁺ ion solution: znCl ₂ Mixing with sterile water to obtain solution;

Fe ³⁺ ion solution: feCl is added ₃ ·6H ₂ O and sterile water are mixed uniformly to obtain the solution.

2. The preparation method of the cell-free extract of the black spore shell Nitaf10 of the cladophora is the same as that of the step 1 of the example 1.

3. 800 mu L of a bristled black spore shell Nitaf10 cell-free extracting solution, 200 mu L of a ustilaginoidea virens A solution with the concentration of 500 mu g/mL and a solution of different types of metal ions with the volume of 200 mu L and the concentration of 30mmol/L are added into a penicillin bottle, so that the final concentration of the different types of metal ions in the whole reaction system of the penicillin bottle is 5mmol/L, and simultaneously, the equal volume of sterile water is used as a control. The reaction was carried out at 28℃and 180 rpm for 3 days. Then adding an equal volume of methanol to stop the reaction, drying, re-dissolving with 1mL of ultrapure water, performing HPLC analysis, calculating the conversion rate of the ustilaginoidea and researching the influence of different types of metal ions on fungus bioconversion of the ustilaginoidea. The conversion of ustilaginoidea A was calculated as follows: ustilaginoidea conversion (%) = [ (amount of ustilaginoidea a added-amount of ustilaginoidea a remaining after the reaction)/amount of ustilaginoidea a added ] ×100. The relative conversion of ustorin A was calculated as follows: relative conversion (%) = (conversion of ustorin a under different types of metal ion reaction systems/conversion of ustorin a under equal volumes of sterile water) ×100.

2. Experimental results

The results are shown in Table 7. The results show that: in Co ²⁺ In the presence, the relative conversion rate of the bristled black spore shell Nitaf10 to the curculin A is reduced to about 50 percent, and the conversion rate of the bristled black spore shell Nitaf10 to the curculin A is reduced to about 50 percent ²⁺ 、Fe ³⁺ And Zn ²⁺ In the presence, little conversion reaction occurs, and other metal ions have substantially no effect on the conversion reaction.

TABLE 7 influence of different types of metal ions on the relative conversion of ustilaginoidea A

Different types of metal ions	Relative conversion of Ustilagin A (%)
		CK	100±0a
Ba ²⁺	98.52±0.26b
		Ca ²⁺	99.18±0.25ab
Co ²⁺	52.59±1.89c
		Cu ²⁺	0±0d
Fe ³⁺	0±0d
		Mg ²⁺	98.62±0.31b
Mn ²⁺	99.85±1.29ab
		Zn ²⁺	0±0d

Note that: the concentration of each metal ion is 5mmol/L; the data in the table are mean ± standard deviation (n=3), and each column of different letters indicates a significant difference between the data at p.ltoreq.0.05.

Example 6 cytotoxic Activity of Ustilagin A and its transformation products Ustilagin I, J and M

1. Experimental method

Test cells: human colon cancer cell line (HCT-8), human gastric gland cell line (HGC-27), human liver cancer cell line (HepG 2), human pancreatic cancer cell line (PANC-1) and human lung cancer cell line (PC 9).

Test compound: curcin A, curcin I, curcin J, curcin M, and paclitaxel as control compound.

The cytotoxic activity of the test compounds was determined as follows:

the test cells cultured in the flask were eluted with 0.5% trypsin to give a cell density of 1X 10 ⁶ Cell suspension per mL. Setting blank control group and 5 different drug gradients (concentration gradient of ustilaginoidea virens is 0.01. Mu. Mol/L, 0.1. Mu. Mol/L, 1. Mu. Mol/L, 10. Mu. Mol/L, 100. Mu. Mol/L, and concentration gradient range of positive control paclitaxel is 1×10) ^-4 Mu mol/L to 1X 10 ^-2 Mu mol/L) of the above-mentioned substances are respectively added into 96-well culture plates, and each dose is respectively set5 parallel wells were placed and 100. Mu.L of cell suspension was added to each well. Adding medicines according to the pre-prepared medicine gradient in the morning, and culturing in a 37 ℃ incubator for 24 hours. mu.L of thiazole blue (MTT) solution was added to each well and the incubation was continued for 4 hours. The culture was terminated and the in-well culture solution was carefully aspirated. 150 mu L of dimethyl sulfoxide is added into each hole, the mixture is placed on a shaking table to oscillate for 10 minutes at a low speed, so that crystals are fully dissolved, and an enzyme-labeled instrument OD is used ₄₉₀ _nm The absorbance of each well was measured. Setting a zeroing hole and a control hole at the same time, sequentially inputting concentration and survival rate through software Graphpad Prism, and calculating semi-inhibition concentration IC ₅₀ Values.

2. Experimental results

The cytotoxic activity of the ustilaginoidea and its transformation products against 5 human cancer cells is shown in Table 8. The results show that: in human colon cancer cell line (HCT-8), human gastric gland cell line (HGC-27) and human liver cancer cell line (HepG 2), the cytotoxic activity of three transformation products, namely, ustilaginoidea virens I, J and M is smaller than that of original toxin ustilaginoidea virens A; in the human pancreatic cancer cell line (PANC-1) and the human lung cancer cell line (PC 9), the cytotoxic activities of the ustilaginoidea virens I and M are smaller than that of the original toxin ustilaginoidea virens A, and the cytotoxic activity of the ustilaginoidea virens J has no obvious difference with that of the original toxin ustilaginoidea virens A.

Table 8, cytoxic Activity of Ultrafil A and its transformation products against human cancer cell lines

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Example 7 inhibition Activity of Ustilagin A and its transformation product Ustilagin J on elongation of the radicle and embryo of germinated Rice seed

1. Experimental method

1. Preparing a compound solution to be tested: the ustilaginoidea virens A and J are weighed, ultrapure water is added to prepare a 1mg/mL mother solution, and then the mother solution is diluted with the ultrapure water into a series of gradient solutions with the concentration of 0.40mg/mL, 0.20mg/mL, 0.10mg/mL and 0.05mg/mL in sequence. The positive control was glyphosate.

2. Accelerating germination of seeds: washing rice seeds with tap water for 3 times, removing upper layer shrunken particles, washing with sterile water for 3 times, sterilizing with 1% sodium hypochlorite (NaClO) solution for 3 minutes, washing with sterile water for 3 times, and transferring the seeds into a culture dish paved with wet filter paper for accelerating germination at 28 ℃ for 12 hours until most of the seeds are exposed to white.

3. Determination of radicle and embryo elongation inhibiting Activity: two layers of filter paper discs are placed in a 24-hole plate, 5 paddy seeds with uniform particle size and white exposure are added into each hole, then 200 mu L of a series of gradient compound solution to be tested is added into each hole, the holes are sealed, and the culture is carried out for 48 hours under the dark condition at 28 ℃. The experiment was run with both solvent control (sterile water) and positive control (glyphosate). 3 replicates were set for each treatment. The lengths of radicles and germs of the rice seeds of the treatment group and the control group were measured respectively after being placed in a constant temperature incubator at 28 ℃ for 48 hours, and the elongation inhibition (%) of radicles and germs of the rice germinated seeds was calculated. The calculation formula is as follows: the rice germination seed radicle and embryo elongation inhibition (%) = [ (solvent control group length (cm) -compound treatment group length (cm))/solvent control group length (cm) ]x100.

2. Experimental results

The results are shown in Table 9 and FIG. 20. The results show that: the inhibition rate of the radicle and embryo of the rice seeds is reduced along with the reduction of the concentration gradient of the compound to be detected, and the inhibition rate of the radicle and embryo elongation of the ustilaginoidea virens A and the converted product ustilaginoidea virens J of the ustilaginoidea virens A are not obviously different at the same concentration.

Table 9 inhibitory Activity of Ustilagin A and Ustilagin J on elongation of radicle and embryo of Rice seed

Note that: the data in the table are mean ± standard deviation (n=10), and each column of different letters indicates a significant difference between the data at p.ltoreq.0.05.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims

1. A bioconversion method of ustorium A comprises the step of treating ustorium A with a cell-free extract of black spore shell; the preparation method of the cell-free extract of the black spore shell comprises the following steps: centrifuging the cladophora liquid to obtain mycelium sediment, adding buffer solution with pH value of 6-10 into the mycelium sediment, performing ultrasonic crushing, centrifuging, and collecting supernatant to obtain the cladophora cell-free extract;

the cladocera is cladocera Nitaf10;

the centrifugation condition is 4 ℃,12000 Xg, and the centrifugation is carried out for 20 min;

the buffer solution with the pH value of 6-10 is phosphate buffer solution with the pH value of 6, phosphate buffer solution with the pH value of 7, phosphate buffer solution with the pH value of 8, carbonate buffer solution with the pH value of 9 or carbonate buffer solution with the pH value of 10;

the ultrasonic crushing conditions are as follows: the ultrasonic oscillation power is 80-100%, the ultrasonic oscillation times are 100-150, each time oscillates for 2 seconds, and the interval is 2 seconds.

2. The method according to claim 1, characterized in that: the preparation method of the black spore shell fungus liquid comprises the following steps: activating the cladosporium cucumerinum on a PDA culture medium, inoculating the activated cladosporium cucumerinum into a PDB culture medium, and performing shake culture to obtain cladosporium cucumerinum liquid.

3. The method according to claim 1 or 2, characterized in that: when phosphate buffer solution with pH value of 7 is added into the mycelium sediment, the ustilaginoidea virens A is converted into ustilaginoidea virens I shown in the formula II and ustilaginoidea virens J shown in the formula III;

a formula II;

formula III.

4. The method according to claim 1 or 2, characterized in that: when adding carbonate buffer solution with pH value of 9 into the mycelium sediment, the ustilaginoidea virens A is converted into ustilaginoidea virens I shown in the formula II, ustilaginoidea virens J shown in the formula III and ustilaginoidea virens M shown in the formula IV;

a formula II;

formula III;

formula IV.

5. Use of the method of any one of claims 1-4 in any one of the following 1) or 2):

1) Ustilaginoidea A detoxifies;

2) Reducing the toxicity of the ustilaginoidea virens A;

the application is for non-disease diagnosis and therapeutic purposes.