CN114230631A

CN114230631A - Method for converting ustilaginoidin A by using fungi and application thereof

Info

Publication number: CN114230631A
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: 2022-03-25
Anticipated expiration: 2041-12-22
Also published as: CN114230631B

Abstract

The invention discloses a method for converting ustilaginoidea virens A by using fungi and application thereof. The method comprises the steps of converting ustilaginoidin A into ustilaginoidin I and J in a buffer solution with the pH value of 7 by utilizing an enzyme system in a setaria nigrospora cell-free extracting solution; converting ustiloxin A into ustiloxin I, J and M in a buffer with pH 9. Experiments show that: the cytotoxic activity of the transformed products ustilaginoids I, J and M was generally reduced compared to ustilaginoids A. The method for converting the ustilaginoidea virens A by using the fungi has simple process, and can effectively carry out biotransformation and detoxification of the ustilaginoidea virens A.

Description

Method for converting ustilaginoidin A by using fungi and application thereof

Technical Field

The invention 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

Rice false smut (Rice face smut) is a fungal ear disease which seriously harms Rice worldwide at present, and the main symptom is Rice curly bulb formation at the Rice ear. The etiology of Ustilaginoidea virens is Rhizoctonia solani (Nakata) Tanaka & Tanaka, and asexual name Ustilaginoidea virens (Cooke) Takahashi. The disease not only affects the quality of paddy rice and causes the reduction of yield of paddy rice, but also produces toxins harmful to human and livestock, and seriously threatens the grain safety and human health. Three major classes of mycotoxins have been reported in ustilago and ustilaginoidea oryzae, namely Ustilaginoidins (Ustiloxins), Ustilaginoidins (ustilaginoids) and sorbosone (Sorbicillinoids).

The current methods for detoxifying mycotoxins mainly comprise: physical detoxification, chemical detoxification and biotransformation detoxification. Except for the photocatalytic degradation of ustilaginoidea virens A reported by the institute of oil crops of Chinese academy of agricultural sciences, the detoxification method of ustilaginoidea virens toxin has not been reported, and the detoxification of ustilaginoidea virens toxin has very important significance for reducing the harm of the toxin and ensuring the safety of grains and foods.

Among the three types of toxins of ustilaginoidea virens, ustilaginoidea virens is the most toxic. 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; also has inhibitory effect on tubulin assembly and cytoskeleton formation of eukaryotic cells. Based on the wide toxicity of ustilaginoids, it is important to change ustilaginoids into compounds with low or no toxicity. Among 5 kinds of ustilagins whose structures have been elucidated, ustilagin a (ustiloxin a) is the main toxin existing in rice and its processed products, and its toxicity is also highest.

Biotransformation (Biotransformation) involves the use of an organism and its enzyme system to change the structure of a toxin, which often weakens or disappears. The biotransformation detoxification method has the advantages of strong specificity, mild reaction conditions and no damage to target products, and is considered to be a detoxification method with wide application prospect. Therefore, it is very important to develop a biotransformation method for ustilaginoidin A, which can reduce the toxicity of ustilaginoidin A.

Disclosure of Invention

The invention aims to provide a method for converting ustilaginoidin A into a weakly toxic or non-toxic product by using fungi.

In order to achieve the above object, the present invention provides a biotransformation method of ustiloxin A.

The biotransformation method of ustilaginoidin A provided by the invention comprises the steps of treating ustilaginoidin A with a setaria nigrospora cell-free extracting solution; the preparation method of the cladosporium cladosporioides cell-free extracting solution comprises the following steps: and (2) centrifuging the chaetomium setosum bacterial liquid to obtain hypha sediment, then adding a buffer solution with the pH value of 6-10 into the hypha sediment, carrying out ultrasonic crushing and centrifugation, and collecting supernatant, namely the chaetomium setosum cell-free extracting solution (extracting solution containing crude enzyme).

In the above process, the chaetomium setosum (Petriella setifera) may be any one of the chaetomium setosum strains. In a specific embodiment of the present invention, the cladosporium cladosporioides is cladosporium cladosporioides Nitaf 10.

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

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

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

In the method, the preparation method of the chaetomium setosum bacterial liquid comprises the following steps: activating the cladophora on a PDA culture medium, inoculating the activated cladophora into a PDB culture medium, and performing shaking culture to obtain a cladophora liquid. Further, the shaking culture time is 3-5 days. Further, the amount of the cladophora inoculated was 3 to 4 fungus cakes having a size of 6mm per 100mL of the medium.

In the above method, the ustilaginoidin A may be a ustilaginoidin A-containing substance, such as ustilaginoidin A solution, ustilaginoidin A-infected rice, or a processed product thereof.

Further, the ustilaginoidin A is ustilaginoidin A solution.

Further, the concentration of the ustilaginoidin A solution is 500. mu.g/mL.

In the method, the method for treating the ustilaginoidea virens A by using the ustilaginoidea virens cell-free extracting solution comprises the steps of uniformly mixing the ustilaginoidea virens cell-free extracting solution and the ustilaginoidea virens A solution, and reacting.

Further, the volume ratio of the chaetomium nigrosporium cell-free extracting solution to the ustiloxin A solution is 4: 1.

Further, the reaction was carried out at 28 ℃ and 180 rpm for 3 days.

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

In one embodiment of the invention, when phosphate buffer (0.5M) with pH7 is added to the hyphal precipitate, the enzyme system in the cell-free extract of Cladosporium cladosporium performs a 5 '-specific transamination of ustiloxin A of formula I and on the basis thereof a 6' -decarboxylation to yield ustiloxin I of formula II and ustiloxin J of formula III, respectively.

In another embodiment of the present invention, when a carbonate buffer solution (concentration of 0.5M) having a pH of 9 is added to the hyphal precipitate, the enzyme system in the cell-free extract of Cladosporium cladosporium performs a transamination reaction and a decarboxylation reaction on ustiloxin A of formula I to produce ustiloxin I of formula II and ustiloxin J of formula III, and also performs a hydroxylation reaction on the 13-position aromatic ring on ustiloxin A to produce ustiloxin M of formula IV.

The method also comprises the steps of separating, identifying and determining toxicity of the converted products of ustilaginoidea virens I, ustilaginoidea virens J and ustilaginoidea virens M.

The separation method comprises the following steps: the pure product of the conversion product is obtained by liquid chromatography analysis and open column chromatography and semi-preparative liquid chromatography with reference to the known compound ustilaginoidin A.

The method for identification comprises the following steps: determining the molecular weight and molecular formula of the conversion product by high-resolution mass spectrometry; and determining the structure of the transformation product through one-dimensional and two-dimensional spectrums of a nuclear magnetic resonance carbon spectrum and a hydrogen spectrum.

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

In order to achieve the above object, the present invention further provides the above cell-free extract of cladophora.

In order to achieve the above object, the present invention also provides the above method or a novel use of the cell-free extract of Cladosporium cladophora.

The invention provides the method or the application of the cladophora acellular extract in any one of the following 1) to 3):

1) the ustilaginoidin A is detoxified;

2) degrading ustilaginoidin A;

3) reduce the toxicity of ustiloxin A.

In order to achieve the above object, the present invention finally provides a product for degrading ustiloxin A.

The active ingredient of the product for degrading the ustilaginoidea virens A provided by the invention is the above-mentioned cladosporium cladosporioides cell-free extracting solution.

The invention provides a method for converting ustilaginoidin A by using fungi, which is characterized in that ustilaginoidin A is converted into ustilaginoidin I and J in a buffer solution with the pH value of 7 by using an enzyme system in a chaetomium nigrosporium cell-free extracting solution; converting ustiloxin A into ustiloxin I, J and M in a buffer with pH 9. The cytotoxic activity detection experiment shows that: the cytotoxic activity of the transformed products ustilaginoids I, J and M was generally reduced compared to ustilaginoids A. The method for converting ustilaginoidea virens A by using fungi has simple process, can effectively carry out biotransformation and detoxification of ustilaginoidea virens, and has important significance for detoxification of ustilaginoidea virens toxin.

Drawings

FIG. 1 is a full wavelength scan of the HPLC profile and its chromatographic peaks for ustilaginoidin A converted to ustilaginoidin I and J in the cell-free extract of Nitaf10 using a buffer solution of pH7 and ustilaginoidin M using a buffer solution of pH 9. FIG. 1a is a liquid phase diagram of a substrate, ustiloxin A; FIG. 1b is a liquid phase diagram after reaction in a buffer at pH7, and FIG. 1c is a liquid phase diagram after reaction in a buffer at pH 9.

FIG. 2 is a chart of HR-ESI-MS of ustiloxin A.

FIG. 3 shows ustilagin A¹H NMR Spectrum (D)₂O,400MHz)。

FIG. 4 shows ustilagin A¹³C NMR Spectrum (D)₂O,400MHz)。

FIG. 5 is a HR-ESI-MS spectrum of ustiloxin I.

FIG. 6 shows ustilagin I¹H NMR Spectrum (D)₂O,500MHz)。

FIG. 7 shows ustilagin I¹³C NMR Spectrum (D)₂O,125MHz)。

FIG. 8 shows ustilagin I¹H-¹H COSY spectrum (D)₂O,500MHz)。

FIG. 9 shows HMBC spectrum of ustiloxin I (D)₂O,500MHz)。

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

FIG. 11 shows ustilagin J¹H NMR Spectrum (D)₂O,500MHz)。

FIG. 12 shows ustilagin J¹³C NMR Spectrum (D)₂O,125MHz)。

FIG. 13 is an HSQC spectrum (D) of ustiloxin J₂O,500MHz)。

FIG. 14 shows an HMBC spectrum of ustiloxin J (D)₂O,500MHz)。

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

FIG. 16 shows ustiloxin M¹H NMR Spectrum (D)₂O,500MHz)。

FIG. 17 shows ustiloxin M¹H-¹H COSY spectrum (D)₂O,500MHz)。

FIG. 18 is an HSQC spectrum (D) of ustiloxin M₂O,500MHz)。

FIG. 19 shows HMBC spectrum of ustiloxin M (D)₂O,500MHz)。

FIG. 20 shows inhibition of radicle and germ elongation of germinating rice seeds by ustilaginoidins A and J and a positive control glyphosate.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The biomaterials referred to in the following examples are as follows:

rice seeds: seeds of round-grained nonglutinous rice (Oryza sativa var. japonica) No. 2 Liaoning were purchased from the model breeding farm in Dongkong City of Liaoning.

Fungus strain nigrospora cladophora (Petriella setifera) nitif 10(GenBank sequence accession number KM 095515): the present inventors isolated from tobacco (Nicotiana tabacum) roots, and the strain and its specific isolation and identification procedures are described in the literature "Zhou K, Wang W, Peng Y, Yu R, Yue Y, Lai D, Zhou L. "in (1).

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 (PC9) were purchased from the institute of medicine of Chinese academy of medicine science.

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

the PDA culture medium formula comprises: 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)₂HPO₄·2H₂O 89g/L，NaH₂PO₄·2H₂O78 g/L, pH adjusted to 6 with pure HCl or 1M NaOH solution.

0.5M phosphate buffer formulation at pH 7: na (Na)₂HPO₄·2H₂O 89g/L，NaH₂PO₄·2H₂O78 g/L, pH adjusted to 7 with pure HCl or 1M NaOH solution.

0.5M phosphate buffer formulation at pH 8: na (Na)₂HPO₄·2H₂O 89g/L，NaH₂PO₄·2H₂O78 g/L, pH adjusted to 8 with pure HCl or 1M NaOH solution.

0.5M carbonate buffer formulation at pH 9: na (Na)₂CO₃ 53g/L，NaHCO₃42g/L, pH was adjusted to 9 with pure HCl or 1M NaOH solution.

0.5M carbonate buffer formulation at pH 10: na (Na)₂CO₃ 53g/L，NaHCO₃42g/L, using pure HClAlternatively, the pH was adjusted to 10 with 1M NaOH solution.

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

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

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

The reagents involved in the following examples are as follows:

CuCl₂·2H₂o: beijing Hongxing chemical plant.

MgCl₂·6H₂O: beijing Fine Chemicals, Inc.

CaCl₂·2H₂O: chemical agents of the national drug group, ltd.

CoCl₂·2H₂O: chemical agents of the national drug group, ltd.

MnCl₂·4H₂O: west longa science corporation.

BaCl₂·2H₂O: chemical agents of the national drug group, ltd.

ZnCl₂: west longa science corporation.

FeCl₃·6H₂O: chemical agents of the national drug group, ltd.

Potato: purchased from the market.

Glucose: chemical agents of the national drug group, ltd.

Agar: aladdin Co.

Na₂HPO₄·2H₂O: chemical agents of the national drug group, ltd.

NaH₂PO₄·2H₂O: chemical agents of the national drug group, ltd.

HCl: chemical agents of the national drug group, ltd.

NaOH: west longa science corporation.

Na₂CO₃: chemical agents of the national drug group, ltd.

NaHCO₃: chemical agents of the national drug group, ltd.

Sodium acetate: west longa science corporation.

Glacial acetic acid: chemical agents of the national drug group, ltd.

The conditions for HPLC analysis of ustilaginoidin A and its conversion products referred to in the following examples are as follows: the mobile phase was methanol/water with 0.02% trifluoroacetic acid (TFA); elution mode: methanol increased linearly from 10% to 100% for 0-30 min; keeping 100% methanol for washing the column for 30-40 min; the mixture was equilibrated with 10% methanol/water for 40-50 minutes, and the detection wavelength was 210 nm.

The ustilaginoids I, J and M referred to in the following examples are pure transformation products isolated by adding the substrate ustilaginoids A to a fungal cell-free extract in the laboratory of the present inventors after a certain period of reaction. Wherein, the ustilaginoidin A is converted into ustilaginoidin I and J by transamination and transamination decarboxylation in a setaria nigrospora nitida Nitaf10 cell-free extract (pH 7); ustilaginoidin A was converted into ustilaginoidin M by hydroxylation on the aromatic ring in a cell-free extract of Nitaf10 (pH 9).

Example 1 conversion of ustilaginoidin A into ustilaginoidin I and J by cell-free extract from Nitaf10 from Sphaerotheca fuliginosus in phosphate buffer at pH7

First, experiment method

1. The preparation method of the cell-free extract (pH value of 7) of the cladosporium cladosporioides Nitaf10 is as follows: inoculating Nitaf10 activated by Nitaf on potato-glucose-agar medium (PDA) into 250mL triangular flask containing 100mL potato-glucose-culture solution (PDB) with 3-4 fungus cakes of 6mm size, shake culturing in shaking table for 3-5 days, placing the shake cultured broth (bacterial solution) into 50mL round bottom centrifuge tube, centrifuging at 4 deg.C and 12000 Xg for 20min, removing supernatant, then 0.5M Phosphate Buffer Solution (PBS) at pH7 in a volume of about 30mL was added to the hyphal pellet, performing ultrasonic oscillation for about 150 times (2 s each time and 2 s each time) in an ultrasonic cleaner (100% power) in an ice bath, centrifuging at 4 deg.C for 20min at 12000 Xg, collecting supernatant, the supernatant is an extract containing crude enzyme in cells, namely a setaria nigrospora nitiff 10 cell-free extract (pH value is 7).

2. 800. mu.L of the cell-free extract of Cladosporium nigrospora Nitaf10 prepared in step 1 (pH 7) and 200. mu.L of a ustiloxin A solution (solvent water) having a concentration of 500. mu.g/mL were added to a vial, and the reaction was carried out at 28 ℃ and 180 rpm for 3 days by adding only the cell-free extract of Cladosporium nigrospora Nitaf10 and only the ustiloxin A solution as a control, to obtain a reaction product, which was then subjected to HPLC analysis.

The preparation of ustilagin A can be found in the literature "Shann T, Sun W, Liu H, Gao S, Lu S, Wang M, Chen Z, Wang S, Zhou L.determination and analysis of ustilagins A and B by LC-ESI-MS and HPLC in the surface of balls of rice International Journal of Molecular Sciences,2012,13(9): 11275-11287. "and" Shann T, Sun W, Wang X, Fu X, Sun W, Zhou L. purification of ustiloxins A and B from the surface microorganisms by macro bacteria resins residues, molecules,2013,18(7): 8181-. The method comprises the following specific steps: water-cooling and leaching a certain amount of rice false bulbs to obtain a water extract, filtering the water extract by gauze and filter paper, concentrating the filtrate, separating and purifying the crude extract by using macroporous adsorption resin SP207, a reverse phase silica gel column ODS-AQ and a sephadex column G-15, and performing chromatographic identification on the obtained monomer compound to finally obtain the monomeric compound of the ustiloxin A. The specific operation is as follows: collecting rice green ball (1000g), air drying, pulverizing, cold soaking and extracting at a ratio of dry weight (g) to water volume (mL) of 1: 30(g/mL) for 7 times (each time for 12 hr), mixing the 7 extractive solutions, and concentrating under reduced pressure to obtain rice green ball water extract. Re-dissolving the extract with water, filtering with filter paper, passing the filtrate through 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 ustiloxin A.

The structural identification method of the ustilaginoidea virens A comprises the following steps: taking the separated and purified ustilaginoidin A monomer compound, and carrying out spectrum identification on the ustilaginoidin A monomer compound, wherein a high resolution mass spectrum (HR-ESI-MS) spectrogram is shown in figure 2; nuclear magnetic resonance hydrogen spectrum (¹H NMR,D₂O,400MHz) spectrum as shown in fig. 3; nuclear magnetic resonance carbon spectrum (¹³C NMR,D₂O,100MHz) spectrum is shown in fig. 4. Therefore, the structure of the obtained ustilaginoidea virens A is determined to be shown in the formula I, and the structure is the same as that of the known ustilaginoidea virens A.

The physicochemical properties and spectral data of the prepared ustilaginoidin A are as follows: pure product is white powder (MeOH). By high resolution mass spectrometry (HR-ESI-MS, M/z 674.26859[ M + H)]⁺) Determining the molecular formula as follows: c₂₈H₄₃N₅O₁₂And S. Nuclear magnetic resonance hydrogen spectroscopy (D)₂O,400MHz) chemical shifts (delta, ppm) and nuclear magnetic resonance carbon spectrum (D)₂O,100MHz) chemical shifts (δ, ppm) are shown in table 1. The physicochemical properties and spectral data of ustilaginoids A prepared as described above are in agreement with those reported in the literature (Koiso Y, Li Y, Iwasaki S, Hanaoka K, Kobayashi T, Sonoda R, Fujita Y, Yaegashi H, Sato Z. Utilities, antibiotic cyclic peptides from a surface bacteria used by brick of bacteria viruses, the Journal of Antibiotics 1994,47(7): 765-773.).

TABLE 1 NMR carbon and hydrogen spectra data for the substrate ustiloxin A

Second, experimental results

The detection of the reaction product by liquid chromatography shows that: the reaction product of the cell-free extract (pH 7) of Nitaf10 and the ustiloxin A solution showed the peak of the conversion product (FIG. 1b), but was not detected in any of the control groups. Separating and purifying the reaction product by a gel column G-15, taking pure water as a mobile phase, and then preparing the ustilaginoidin I and J by using methanol water with a semi-prepared liquid phase of 20%. Cell-free extract of Nitaf10 from Sphaerotheca fuliginosus was first transaminated to give ustilagin I and then decarboxylated to give ustilagin J in 0.5M phosphate buffer pH 7. Shows that the ustilaginoidin A can be converted into ustilaginoidin I and J by using a cell-free extract of Nitaf10 from Histoplasma cladophora under the condition of a phosphate buffer solution with a pH value of 7.

The structural identification method of the ustilaginoidea virens I comprises the following steps: taking the separated and purified ustilaginoidin I monomer compound, and carrying out spectrum identification on the ustilaginoidin I monomer compound, wherein a high resolution mass spectrum (HR-ESI-MS) spectrogram is shown in figure 5; nuclear magnetic resonance hydrogen spectrum (¹H NMR,D₂O,500MHz) spectrum as shown in fig. 6; nuclear magnetic resonance carbon spectrum (¹³C NMR,D₂O,125MHz) spectrum as shown in fig. 7; two-dimensional nuclear magnetic resonance spectrogram¹H-¹H COSY and HMBC are shown in fig. 8 and 9, respectively. Compared with the known ustilaginoidin A, the 5' -amino of the ustilaginoidin I is converted into carbonyl through transamination, and the structure of the obtained ustilaginoidin I is determined to be shown in the formula II.

The physicochemical properties and spectral data of the prepared ustilaginoidin I 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)]^-) Determining the molecular formula as follows: c₂₈H₄₀N₄O₁₃And S. Nuclear magnetic resonance hydrogen spectroscopy (D)₂O,500MHz) chemical shifts (delta, ppm) and nuclear magnetic resonance carbon spectrum (D)₂O,125MHz) chemical shifts (δ, ppm) are shown in table 2.¹H NMR(D₂O,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,NCH₃-9),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)；¹³C NMR(D₂O) delta (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 Koiso, li Y, Iwasaki S, Hanaoka K, Kobayashi T, Sonoda R, Fujita Y, Yaegashi H, Sato Z. Ustiloxins, antibiotic cyclic peptides from a surface bacteria strains on rice used by rice using a nucleic acids virus, the Journal of Antibiotics 1994,47(7):765-773.) were compared to determine the structure of ustilagin I.

TABLE 2 NMR carbon and hydrogen spectra data for the conversion product ustiloxin I

The structural identification method of the ustilaginoidea virens J comprises the following steps: and taking the separated and purified ustilaginoidea virens J monomer compound, and carrying out spectral identification on the ustilaginoidea virens J monomer compound. The high resolution mass spectrum (HR-ESI-MS) spectrum is shown in FIG. 10; nuclear magnetic resonance hydrogen spectrum (¹H NMR,D₂O,500MHz) spectrum as shown in fig. 11; nuclear magnetic resonance carbon spectrum (¹³C NMR,D₂O,125MHz) spectrum is 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 ustilaginoidin A, the 5 '-amino group on the ustilaginoidin J is converted into carbonyl through transamination, and then the 6' -carboxyl group is converted into hydroxyl through decarboxylation, so that the structure of the obtained ustilaginoidin J monomer compound is determined to be shown in the formula III.

The physicochemical properties and spectral data of the prepared ustilaginoidin 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)]^-) Determining the molecular formula as follows: c₂₇H₄₀N₄O₁₂And S. Nuclear magnetic resonance hydrogen spectroscopy (D)₂O,500MHz) chemical shifts (delta, ppm) and nuclear magnetic resonance carbon spectrum (D)₂O,125MHz) chemical shifts (δ, ppm) are shown in table 3.¹H NMR(D₂O,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,NCH₃-9),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)；¹³C NMR(D₂O)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(NCH₃-C). The above data were compared with data reported in the literature (Koiso Y, Li Y, Iwasaki S, Hanaoka K, Kobayashi T, Sonoda R, Fujita Y, Yaegashi H, Sato Z. Utiloxin, antibiotic cyclic peptides from bacteria on rice plants used by using antibiotic peptides viruses. the Journal of Antibiotics 1994,47(7):765-773.) to determine the structure of ustilagin J.

TABLE 3 NMR carbon and hydrogen spectra data for transformant ustilaginoidin J

Example 2 conversion of ustilaginoidin A to ustilaginoidin M by cell-free extract from Nitaf10 from Sphaerotheca fuliginea in a carbonate buffer at pH9

First, experiment method

1. The preparation method of the cell-free extract (pH value of 9) of the cladosporium cladosporioides Nitaf10 is as follows: inoculating Nitaf10 activated by Nitaf on potato-glucose-agar medium (PDA) into 250mL triangular flask containing 100mL potato-glucose-culture solution (PDB) with 3-4 fungus cakes of 6mm size, shake culturing in shaking table for 3-5 days, placing the shake cultured broth (bacterial solution) into 50mL round bottom centrifuge tube, centrifuging at 4 deg.C and 12000 Xg for 20min, removing supernatant, then 0.5M Carbonate Buffer (CBS) at pH9 was added to the hyphal pellet in a volume of about 30mL, performing ultrasonic oscillation for about 150 times (2 s each time and 2 s each time) in an ultrasonic cleaner (100% power) in an ice bath, centrifuging at 4 deg.C for 20min at 12000 Xg, collecting supernatant, the supernatant is an extract containing crude intracellular enzymes, namely a setaria nigrospora Nitaf10 cell-free extract (pH value of 9).

2. 800. mu.L of the cell-free extract of Cladosporium nigrospora Nitaf10 prepared in step 1 (pH 9) and 200. mu.L of a ustiloxin A solution (solvent water) having a concentration of 500. mu.g/mL were added to a vial, and the reaction was carried out at 28 ℃ and 180 rpm for 3 days by adding only the cell-free extract of Cladosporium nigrospora Nitaf10 and only the ustiloxin A solution as a control, to obtain a reaction product, which was then subjected to HPLC analysis.

Second, experimental results

The detection of the reaction product by liquid chromatography shows that: the reaction product of the cell-free extract of Nitaf10 (pH 9) and the ustiloxin A solution contained other conversion products (FIG. 1c) in addition to the conversion products ustiloxin I and J, whereas no peak was detected in the control group. Separating and purifying the reaction product by a gel column G-15, taking pure water as a mobile phase, and then preparing the ustilaginoidin M by using methanol water with the semi-prepared liquid phase of 20%. The cell-free extract of Nitaf10 of Cladosporium cladophora still mainly transaminates and decarboxylates in 0.5M phosphate buffer solution with pH value of 9 to generate ustilaginoidin I and J, but at the same time, hydroxylation reaction on 13-position aromatic ring is carried out on ustilaginoidin A to generate ustilaginoidin M shown in formula IV. Shows that the ustilaginoidin A can be converted into ustilaginoidin I, J and M by using a cell-free extract of Nitaf10 from Histophora setaria under the condition of a phosphate buffer solution with a pH value of 9.

The structural identification method of the ustilaginoidea virens M comprises the following steps: taking the separated and purified ustilaginoidin M monomer compound, and carrying out spectrum identification on the ustilaginoidin M monomer compound, wherein a high resolution mass spectrum (HR-ESI-MS) spectrogram is shown in FIG. 15; nuclear magnetic resonance hydrogen spectrum (¹H NMR,D₂O,500MHz) spectrum as shown in fig. 16; two-dimensional nuclear magnetic resonance spectrogram¹H-¹H COSY, HSQC and HMBC are shown in fig. 17, 18 and 19, respectively. Compared with the known ustilaginoidin A, the 13-position of the ustilaginoidin M is hydroxylated on an aromatic ring, and the structure of the obtained ustilaginoidin M monomer compound is determined to be shown as a formula IV.

The physicochemical properties and spectral data of the prepared ustilaginoidin M are as follows: the pure product is light white amorphous solid, and is obtained by high resolution mass spectrometry (HR-ESI-MS, M/z 690.2292[ M + H ]]⁺) Determining the molecular formula as follows: c₂₈H₄₃N₅O₁₃And S. Nuclear magnetic resonance hydrogen spectroscopy (D)₂O,500MHz) chemical shifts (δ, ppm) are shown in table 4.¹H NMR(D₂O,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,NCH₃-9)，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_25,246.5Hz, H-25). The above data were compared with data reported in the literature (Koiso Y, Li Y, Iwasaki S, Hanaoka K, Kobayashi T, Sonoda R, Fujita Y, Yaegashi H, Sato Z. Utiloxin, antibiotic cyclic peptides from bacteria on rice plants used by using antibiotic peptides viruses. the Journal of Antibiotics 1994,47(7):765-773.) to determine the structure of ustiloxin M. No relevant hydrogen signal was seen from the nmr hydrogen spectrum (fig. 16), which, in combination with high resolution mass spectral data, indicates that the 13-position hydrogen on the aromatic ring was replaced by a hydroxyl group.

TABLE 4 NMR data on conversion product ustilagin M

Example 3 dynamic variation of the consumption of ustilaginoidin A bioconversion time course and of the production of ustilaginoidin I and J as transformation products in cell-free extract of Nitaf10

First, experiment method

1. The same as 1 in step one of example 1.

2. The same as 2 in step one of example 1.

3. Samples were taken at 0h, 6h, 12h, 24h, 36h, 48h and 72h of the reaction, quenched with an equal volume of methanol, dried and redissolved with 1mL of ultrapure water for HPLC analysis.

Second, experimental results

The results are shown in table 5 and show that: in the biotransformation process of ustilaginoidea brasiliensis Nitaf10 cell-free extract on ustilaginoidin A, the generation of transformation products begins at 6 hours, two transformation products appear, the content of ustilaginoidin A gradually decreases with the increase of reaction time, the amount of the transformation products ustilaginoidin I and J gradually increases, and the synthesis amount of the ustilaginoidin I and the ustilaginoidin J reaches the maximum respectively when the reaction time reaches 48 hours and 36 hours.

TABLE 5 time course of the biotransformation of Aspergillus oryzae A in cell-free extracts of Nitaf10 from Sphaerotheca cladophora

Note: the data in the table are mean ± standard deviation (n ═ 3), and different letters in each column indicate significant differences between the data at p ≦ 0.05.

Example 4 Effect of different initial reaction pH values on the conversion of Aspergillus oryzae A in cell-free extracts of Nitaf10

First, the experimental process

1. Preparing buffer solutions with different pH values and the concentration of 0.5 mol/L: acetate buffer solutions at pH4 and 5, phosphate buffer solutions at pH6, 7 and 8, and carbonate buffer solutions at pH9 and 10.

2. The preparation method of the cladophora Nitaf10 cell-free extract comprises the following steps: inoculating the activated cladophora Nitaf10 on potato-glucose-agar medium (PDA) into a 250mL triangular flask containing 100mL potato-glucose-culture solution (PDB), inoculating 3-4 fungus cakes with the size of 6mm, performing shaking culture in a shaking table for 3-5 days, putting the shaking culture broth (bacterial liquid) into a 50mL round-bottom centrifuge tube, centrifuging for 20 minutes at 4 ℃ and 12000 Xg, removing supernatant, adding 30mL buffer solutions with different pH values and concentration of 0.5mol/L into the hypha sediment, performing ultrasonic shaking for 150 times in an ultrasonic cleaner (100% power), performing shaking for 2 seconds each time and intermittent 2 seconds, then performing centrifugation for 20 minutes at 4 ℃ and 12000 Xg, and taking supernatant to obtain the cell-free extract of the cladophora Nitaf10 with different pH values.

3. 800 mu L of Nitaf10 cell-free extract of Cladosporium cladophora with different pH values and 200 mu L of ustiloxin A solution with the concentration of 500 mu g/mL are respectively added into a penicillin bottle, and the reaction is carried out for 3 days at 28 ℃ and 180 r/min. Then adding equal volume of methanol to stop the reaction, drying, re-dissolving with 1mL of ultrapure water, performing HPLC analysis, and calculating the conversion rate of the ustilaginoidea virens A. The conversion rate of ustilaginoidin A is calculated by the following formula: conversion (%) × 100 [ (amount of added ustiloxin a-amount of remaining ustiloxin a after completion of the reaction)/amount of added ustiloxin a ].

Second, experimental results

The results are shown in Table 6. The results show that: the cell-free extract of Nitaf10, from Sphaeria cladosporium, did not undergo a conversion reaction at pH4 and 5, but at pH6, a conversion of about 50% occurred, which was also as high as 70% when the reaction was initiated at alkaline conditions, i.e. pH9 and 10, and reached a maximum of 91% at pH 9.

TABLE 6 influence of different initial reaction pH values on the conversion of ustilaginoidin A

Different initial pH values of the reaction	Conversion of ustiloxin 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 ustilaginoidin A from cell-free extracts of Nitaf10

First, experiment method

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

Cu²⁺ionic solution: adding CuCl₂·2H₂Mixing O and sterile water uniformly to obtain a solution;

Mg²⁺ionic solution: mixing MgCl₂·6H₂Mixing O and sterile water uniformly to obtain a solution;

Ca²⁺ionic solution: adding CaCl₂·2H₂Mixing O and sterile water uniformly to obtain a solution;

Co²⁺ionic solution: adding CoCl₂·2H₂Mixing O and sterile water uniformly to obtain a solution;

Mn²⁺ionic solution: mixing MnCl₂·4H₂Mixing O and sterile water uniformly to obtain a solution;

Ba²⁺ionic solution: adding BaCl₂·2H₂Mixing O and sterile water uniformly to obtain a solution;

Zn²⁺ionic solution: reacting ZnCl₂Mixing with sterile water to obtain solution;

Fe³⁺ionic solution: FeCl is added₃·6H₂And mixing the O and sterile water uniformly to obtain a solution.

2. The cell-free extract of Nitaf10 was prepared in the same manner as in step 1 of example 1.

3. Adding 800 mu L of cladophora Nitaf10 cell-free extract, 200 mu L of ustiloxin A solution with the concentration of 500 mu g/mL and 200 mu L of different types of metal ion solutions with the concentration of 30mmol/L into a penicillin bottle, so that the final concentrations of the different types of metal ions in the whole reaction system of the penicillin bottle are all 5mmol/L, and simultaneously taking sterile water with the same volume as a control. The reaction was carried out at 28 ℃ and 180 rpm for 3 days. And then adding equal volume of methanol to stop the reaction, drying the reaction product, redissolving the reaction product by using 1mL of ultrapure water, carrying out HPLC analysis, calculating the conversion rate of the ustilaginoidin A, and researching the influence of different types of metal ions on fungal biotransformation of the ustilaginoidin A. The conversion rate of ustilaginoidin A is calculated by the following formula: the ustilaginoidin conversion (%) [ (amount of ustilaginoidin a added-amount of ustilaginoidin a remaining after completion of the reaction)/amount of ustilaginoidin a added ] × 100. The relative conversion rate of ustilaginoidin A is calculated as follows: relative conversion (%) × 100 (conversion of ustiloxin a in different types of metal ion reaction systems/conversion of ustiloxin a under equal volume of sterile water).

Second, experimental results

The results are shown in Table 7. The results show that: in Co²⁺In the presence of the enzyme, the relative conversion rate of Nitaf10 of cladophora to ustilagin A is reduced to about 50%, while in Cu²⁺、Fe³⁺And Zn²⁺In the presence of the metal ion, the conversion reaction hardly occurs, and the other metal ion has substantially no influence on the conversion reaction.

TABLE 7 Effect of different types of metal ions on the relative conversion of ustiloxin A

Different types of metal ions	Relative conversion of ustilagin A (%)
		CK	100±0a
Ba²⁺	98.52±0.26b
		Ca²⁺	99.18±0.25ab
Co²⁺	52.59±1.89c
		Cu ²⁺	0±0d
Fe
	³⁺	0±0d
Mg²⁺			98.62±0.31b
	Mn²⁺	99.85±1.29ab
Zn
	²⁺	0±0d

Note: the concentration of each metal ion is 5 mmol/L; the data in the table are mean ± standard deviation (n ═ 3), and different letters in each column indicate significant differences between the data at p ≦ 0.05.

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

First, experiment method

Test cells: human colon cancer cell line (HCT-8), human gastric gland cell line (HGC-27), human hepatoma cell line (HepG2), human pancreatic cancer cell line (PANC-1) and human lung cancer cell line (PC 9).

Test compounds: ustilaginoidin A, ustilaginoidin I, ustilaginoidin J, ustilaginoidin M, and taxol as a control compound.

The cytotoxic activity of the test compound was determined by the following specific steps:

the test cells cultured in the culture flask were eluted with 0.5% trypsin to give a cell density of 1X 10⁶Cell suspension/mL. Setting blank control group and 5 different drug gradients (the concentration gradients of ustilaginoidin are respectively 0.01 μmol/L, 0.1 μmol/L, 1 μmol/L, 10 μmol/L and 100 μmol/L; the concentration gradient range of positive control taxol is 1 × 10^-4Mu mol/L to 1X 10^-2μ mol/L) into 96-well culture plates, 5 parallel wells per dose, and 100 μ L of cell suspension per well. Adding medicine according to the prepared medicine gradient in the morning of the next day, and culturing in an incubator at 37 ℃ for 24 hours. mu.L of thiazole blue (MTT) solution was added to each well and incubation was continued for 4 hours. The culture was terminated and the culture medium in the wells was carefully aspirated. Adding 150 μ L dimethyl sulfoxide into each well, shaking on a shaker at low speed for 10 min to dissolve the crystals, and measuring with enzyme-labeling instrument OD₄₉₀ _nmThe absorbance of each well was measured. Setting a zero setting hole and a control hole at the same time, sequentially inputting the concentration and the survival rate through the software Graphpad Prism, and calculating the semi-inhibitory concentration IC₅₀The value is obtained.

Second, experimental results

The cytotoxic activity of ustiloxin A and its transformed products against 5 human cancer cells is shown in Table 8. The results show that: in a human colon cancer cell strain (HCT-8), a human gastric gland cell strain (HGC-27) and a human liver cancer cell strain (HepG2), the cytotoxic activity of three transformation products of the ustilaginoidin I, J and M is less than that of the protoxin ustilagin A; in human pancreatic cancer cell lines (PANC-1) and human lung cancer cell lines (PC9), the cytotoxic activity of ustilaginoidea virens I and M is less than that of ustilaginoidea virens A, and the cytotoxic activity of ustilaginoidea virens J has no obvious difference from that of ustilaginoidea virens A.

TABLE 8 cytotoxic Activity of ustiloxin A and its transformation products against human cancer cell lines

Example 7 inhibitory Activity of ustiloxin A and ustiloxin J, a transformation product thereof, on radicle and embryo elongation of germinating seeds of rice

First, experiment method

1. Preparing a solution of a compound to be tested: the ustiloxin A and the ustiloxin J are weighed, ultrapure water is added to prepare 1mg/mL mother liquor, and then the mother liquor is diluted 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 by the ultrapure water. The positive control is glyphosate.

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

3. Determination of radicle and embryo elongation inhibitory Activity: putting two layers of filter paper discs into a 24-pore plate, adding 5 exposed rice seeds with uniform particle size into each pore, adding 200 mu L of series gradient compound solution to be tested into each pore, sealing, and culturing for 48 hours at 28 ℃ in the dark. The experiment was set up with both a solvent control (sterile water) and a positive control (glyphosate). Each process set 3 replicates. After being placed in a constant temperature incubator at 28 ℃ for 48 hours, the lengths of the radicles and the germs of the rice seeds of the treated group and the control group are respectively measured, and the elongation inhibition (%) of the radicles and the germs of the germinated seeds of the rice is calculated. The calculation formula is as follows: the rice germinating seed radicle and germ elongation inhibition (%) was ═ length of solvent control group (cm) -length of compound-treated group (cm))/length of solvent control group (cm) ] × 100.

Second, experimental results

The results are shown in Table 9 and FIG. 20. The results show that: the inhibition rate of the concentration gradient of the compound to be detected on the radicle and the germ of the rice seed is reduced, and the inhibition rates of the ustilaginoidin A and the ustilaginoidin J which is a conversion product of the ustilaginoidin A on the radicle and the germ elongation are not obviously different under the same concentration.

TABLE 9 inhibitory Activity of ustilagin A and ustilagin J on Rice seed radicle and embryo elongation

Note: the data in the table are mean ± standard deviation (n ═ 10), and different letters in each column indicate significant differences between the data at p ≦ 0.05.

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

Claims

1. A biotransformation method of ustilaginoidin A, comprising the steps of treating ustilaginoidin A with a cell-free extract of Chaetomium cladosporium; the preparation method of the cladosporium cladosporioides cell-free extracting solution comprises the following steps: and centrifuging the chaetomium setosum bacterial liquid to obtain hypha sediment, adding a buffer solution with the pH value of 6-10 into the hypha sediment, carrying out ultrasonic crushing, centrifuging, and collecting supernatant, namely the chaetomium setosum cell-free extracting solution.

2. The method of claim 1, wherein: the cladophora is cladophora Nitaf 10.

3. The method according to claim 1 or 2, characterized in that: the centrifugation conditions were 4 ℃ at 12000 Xg for 20 min.

4. A method according to any one of claims 1 to 3, wherein: the conditions of the ultrasonic disruption are as follows: the ultrasonic oscillation power is 80-100%, the ultrasonic oscillation frequency is 100-150, each oscillation is 2 seconds, and the intermittence is 2 seconds.

5. The method according to any one of claims 1 to 4, wherein: the preparation method of the chaetomium setosum bacterial liquid comprises the following steps: activating the cladophora on a PDA culture medium, inoculating the activated cladophora into a PDB culture medium, and performing shaking culture to obtain a cladophora liquid.

6. The method according to any one of claims 1 to 5, wherein: when phosphate buffer solution with the pH value of 7 is added into the hypha precipitate, the ustilaginoidea virens A is converted into ustilaginoidea virens I shown in a formula II and ustilaginoidea virens J shown in a formula III;

7. the method according to any one of claims 1 to 6, wherein: when a carbonate buffer solution with the pH value of 9 is added into the hyphal precipitate, the ustilaginoidea virens A is converted into ustilaginoidea virens I shown in a formula II, ustilaginoidea virens J shown in a formula III and ustilaginoidea virens M shown in a formula IV;

8. the cell-free extract of Cladosporium cladophora according to any one of claims 1 to 7.

9. Use of the method according to any one of claims 1 to 7 or the cell-free extract of cladophora according to claim 8 in any one of the following 1) to 3):

1) the ustilaginoidin A is detoxified;

2) degrading ustilaginoidin A;

3) reduce the toxicity of ustiloxin A.

10. A product for degrading ustilaginoidin A, which comprises the cell-free extract of Cladosporium cladosporioides according to claim 8 as an active ingredient.