CN113637010A

CN113637010A - Hydrogenated xanthone derivative and preparation method and application thereof

Info

Publication number: CN113637010A
Application number: CN202110833249.0A
Authority: CN
Inventors: 吴少华; 鲁祎晗; 刘思思
Original assignee: Yunnan University YNU
Current assignee: Yunnan University YNU
Priority date: 2021-07-22
Filing date: 2021-07-22
Publication date: 2021-11-12
Anticipated expiration: 2041-07-22
Also published as: CN113637010B

Abstract

The invention provides a hydrogenated xanthone derivative, belonging to the technical field of biological medicines. By utilizing a chemical epigenetic modification strategy, a chemical epigenetic modifier sodium butyrate is added in a conventional culture condition, and phosphopsisaxanthones H-L are prepared by a microbial fermentation method. The invention also provides a preparation method and application of the hydrogenated xanthone derivative.

Description

Hydrogenated xanthone derivative and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a hydrogenated xanthone derivative, and a preparation method and application thereof.

Background

Xanthone is an isotridecyl compound, derivatives of which are widely found in nature, including analogs in which many of the aromatic rings are hydrogenated or highly oxidized. The compounds show different biological activities due to different substituents, including body immunity regulation, tumor resistance, antibiosis and anti-inflammation, and the like, and are important molecular structures for developing novel antibiotics. The xanthone compounds from natural sources often have more complex chemical structures and more diversified functional group substitutions, so that the xanthone compounds show more remarkable biological activity, and particularly the xanthone compounds obtained from microbial resources are potential molecules for finding drug lead compounds from natural sources.

Disclosure of Invention

The invention aims to provide a series of hydrogenated xanthone derivatives which can resist phytopathogens, have antioxidant activity and can be used for preparing agricultural antibiotics and antioxidant medicines.

The invention also provides a preparation method and application of the hydrogenated xanthone derivative.

The invention is realized by the following technical scheme:

the invention provides a hydrogenated xanthone derivative, which comprises at least one of phosphops I sxanthones H-L, wherein the structural formulas of the phosphops I sxanthones H-L are respectively shown as (I) to (V):

based on the same inventive concept, the invention provides a preparation method of a hydrogenated xanthone derivative, which comprises the following steps:

fermenting phomopsis in a culture medium added with sodium butyrate, and collecting fermentation liquor and mycelia after fermentation is finished;

extracting the fermentation liquor, leaching the mycelium, combining extracting solutions, and volatilizing a solvent to obtain a fermentation crude extract;

carrying out chromatographic separation on the crude fermentation extract, eluting by adopting a chloroform-methanol system, and collecting crude components Fr.1-Fr.3; carrying out chromatographic separation on the crude component Fr.1, and eluting by adopting a chloroform-methanol system to obtain a compound phosphopsisaxanthone H;

carrying out chromatographic separation on the crude component Fr.2, and eluting with a petroleum ether-ethyl acetate system to obtain a compound phosphopsoxanthene I;

and (3) carrying out chromatographic separation on the crude component Fr.3, and eluting by adopting a methanol-water system to obtain the compounds phosphopsisaxanthone J-L.

Further, the Phomopsis sp.YE3350 is a strain which is preserved in China center for type culture Collection (CCTCC for short) at 8/3 of 2020, and the preservation number is CCTCC NO. M2020394. The preservation address is China, Wuhan and Wuhan university, and the taxonomy is named Phomopsis sp.

Further, the phomopsis is fermented in a culture medium added with sodium butyrate, and fermentation liquor and mycelia are collected after fermentation is finished, and the method specifically comprises the following steps:

fermenting phomopsis in a PDB culture medium added with sodium butyrate, and collecting fermentation liquor and mycelia after fermentation is finished;

the PDB culture medium added with the sodium butyrate is prepared by the following method:

cutting 200-400 parts of peeled potatoes into pieces, adding 1000 parts of water, boiling and filtering to obtain a potato filtrate;

to the potatoAdding 20-50 parts of glucose, 3-5 parts of dipotassium hydrogen phosphate, 3-5 parts of magnesium sulfate and vitamin B into the filtrate₁0.01-0.03 part of PDB culture medium is obtained;

adding sodium butyrate into the PDB culture medium to obtain the PDB culture medium added with the sodium butyrate;

in the PDB culture medium, the concentration of the potatoes is 0.2-0.4 g/mL, and the concentration of the glucose is 0.02-0.05 g/mL; in the PDB culture medium added with the sodium butyrate, the concentration of the sodium butyrate is 200-500 mu M.

Further, the extracting of the fermentation liquor, the leaching of the mycelium, the merging of the extracting solutions, and the volatilizing of the solvent to obtain a fermentation crude extract specifically include:

extracting the fermentation liquor by using ethyl acetate, leaching the mycelium by using methanol, combining extracting solutions, and volatilizing a solvent to obtain a fermentation crude extract.

Further, the crude fermentation extract is subjected to chromatographic separation, an eluent adopts a chloroform-methanol system, and crude components Fr.1-Fr.3 are collected, and the method specifically comprises the following steps:

and (3) carrying out chromatographic separation on the fermented crude extract, carrying out gradient elution on the fermented crude extract by using an eluent in a chloroform-methanol system from the volume ratio of 10:1 to 1:1, collecting a crude component Fr.1 eluted when the volume ratio of chloroform to methanol is 9:1, collecting a crude component Fr.2 eluted when the volume ratio of chloroform to methanol is 8:2, and collecting a crude component Fr.3 eluted when the volume ratio of chloroform to methanol is 7: 3.

Further, the crude fraction fr.1 is chromatographed using a chloroform-methanol system as eluent to obtain the compound phosphopsisaxanthone H, which comprises:

and (2) carrying out chromatographic separation on the crude component Fr.1, carrying out gradient elution on the crude component Fr.1 by using an eluent in a chloroform-methanol system and carrying out volume ratio of chloroform to methanol of 15:1 to 7:3, collecting fractions eluted when the volume ratio of chloroform to methanol is 9:1, and recrystallizing the fractions in a mixed solvent of acetone and methanol of 3:1 to obtain a compound phosphopsoxanthine H.

Further, the crude component fr.2 is chromatographically separated, and an eluent adopts a petroleum ether-ethyl acetate system to obtain a compound phosphopsisaxanthone I, which specifically comprises:

carrying out chromatographic separation on the crude component Fr.2, carrying out gradient elution on the crude component Fr.2 by using an eluent through a petroleum ether-ethyl acetate system and using petroleum ether and ethyl acetate in a volume ratio of 9:1 to 7:3, collecting fractions eluted when the volume ratio of the petroleum ether to the ethyl acetate is 9:1, and recrystallizing the fractions in a mixed solvent of methanol and water in a volume ratio of 8:2 to obtain a compound phosphopsisaxanthone I.

Further, the crude component fr.3 is chromatographically separated, and the eluent adopts a methanol-water system to obtain the compounds phosphopsoxanthine J-L, which specifically include:

carrying out chromatographic separation on the crude component Fr.3, carrying out gradient elution on the crude component Fr.3 by using an eluent in a methanol-water system and using methanol and water in a volume ratio of 1:1 to 9:1, collecting fractions eluted when the volume ratio of the methanol and the water is 6:4, and volatilizing the eluent to obtain a compound phosphopsxanthene J; collecting fractions eluted when the volume ratio of methanol to water is 7:3, and volatilizing an eluent to obtain a compound phosphopsisaxanthone K; collecting fractions eluted when the volume ratio of methanol to water is 8:2, and volatilizing the eluent to obtain the compound phosphopsisaxanthone L.

Based on the same inventive concept, the invention provides the application of the hydrogenated xanthone derivative in preparing agricultural antibiotics and antioxidant drugs.

One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

1. the invention provides five novel hydrogenated xanthone derivatives

phosphopsisaxanthones H-L, five hydrogenated xanthone derivatives have good anti-phytopathogen and anti-oxidation activities, and provide a new choice for developing agricultural antibiotics and anti-oxidation medicaments.

2. The preparation method of the hydrogenated xanthone derivative adopts a chemical epigenetic modification strategy, adds a chemical epigenetic modifier sodium butyrate into a fermentation culture medium of a strain Phomopsis sp.YE3350, and utilizes the chemical epigenetic modifier sodium butyrate to induce and generate five new hydrogenated xanthone derivatives phosphopsisaxanthenes H-L.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.

FIG. 1 shows phosphopsisaxanthone H, a compound of the present invention¹H-NMR spectrum;

FIG. 2 shows phosphopsisaxanthone H of the compound of the present invention¹³C-NMR and DEPT spectra;

FIG. 3 is a drawing showing phosphopsisaxanthone I compound of the present invention¹H-NMR spectrum;

FIG. 4 shows phosphopsisaxanthone I of the present invention¹³C-NMR and DEPT spectra;

FIG. 5 shows phosphopsisaxanthone J, a compound of the present invention¹H-NMR spectrum;

FIG. 6 shows phosphopsisaxanthone J, a compound of the present invention¹³C-NMR and DEPT spectra;

FIG. 7 shows phosphopsisaxanthone K, a compound of the present invention¹H-NMR spectrum;

FIG. 8 shows phosphopsisaxanthone K, a compound of the present invention¹³C-NMR and DEPT spectra;

FIG. 9 shows phosphopsisaxanthone L of the compound of the present invention¹H-NMR spectrum;

FIG. 10 shows phosphopsisaxanthone L of the compound of the present invention¹³C-NMR and DEPT spectra;

FIG. 11 is a crystal structure diagram of the X-ray single crystal diffraction of phosphopsisaxanthone H, a compound of the present invention;

FIG. 12 is a crystal structure diagram showing the X-ray single crystal diffraction of the compound phosphopsisaxanthone I according to the present invention.

FIG. 13 shows the structural formulae of phosphopsisaxanthenes H-L of the hydrogenated xanthone derivatives of the present invention.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.

In order to solve the technical problems, the embodiment of the invention provides the following general ideas:

in recent years, with the rapid development of whole-gene sequencing approaches, it has been found that there are a large number of gene clusters encoding compounds in fungi. It has been shown that under the conditions of routine laboratory culture, biosynthetic gene clusters of fungal genomic sequences encoding secondary metabolites are mostly silent. Expression of these silenced gene clusters in microorganisms by genetic engineering techniques to produce more metabolites has been a hot spot for the study of natural products. Exploring secondary metabolite diversity by modifying the fungal epigenome is currently an effective way to solve the problem. The chemical epigenetic modification method can inhibit enzymes influencing the epigenetic inheritance of the fungus, activate silent biosynthesis genes to induce different secondary metabolites, and greatly increase the diversity of the secondary metabolites of the fungus.

Based on the principle, the invention adopts a chemical epigenetic modification strategy, adds a chemical epigenetic modifier sodium butyrate into a fermentation culture medium of a strain Phomopsis sp.YE3350, and utilizes the chemical epigenetic modifier sodium butyrate to induce and generate five novel hydrogenous xanthone derivatives phosphopsisanxthones H-L, wherein the five hydrogenous xanthone derivatives have good anti-plant pathogenic bacteria and anti-oxidation activity, thereby providing a new choice for developing agricultural antibiotics and anti-oxidation drugs.

The following will describe the hydrogenated xanthone derivatives and the preparation method thereof in detail with reference to examples and experimental data.

Example 1

Isolation of the hydrogenated xanthone derivatives Phomopsis xanthones H-L

(1) Preparing a PDB culture medium: weighing 200g of peeled potato, cutting into small pieces, adding 1000mL of distilled water, boiling, filtering with gauze to obtain potato filtrate, adding 20g of glucose, adjusting pH to natural, and sterilizing at 121 ℃ for 30min to obtain the PDB culture medium for later use. Wherein the concentration of potato in the PDB culture medium is 0.2g/mL, and the concentration of glucose is 0.02 g/mL.

Preparing an improved PDB culture medium: weighing peeled potato 200g, cutting into small pieces, adding 1000mL distilled water, boiling, filtering with gauze to obtain potato filtrate, adding glucose 20g, dipotassium hydrogen phosphate 3g, magnesium sulfate 3g, and vitamin B₁Sterilizing at 121 deg.C for 30min at pH of 10mg to obtain improved PDB culture medium. Wherein the concentration of potato in the modified PDB medium is 0.2g/mL, and the concentration of glucose is 0.02 g/mL.

(2) Inoculating Phomopsis sp.YE3350 strain into PDB culture medium, and shake culturing at 28 + -2 deg.C and 200r/min for 4d to obtain seed solution.

(3) Dissolving a chemical epigenetic modifier sodium butyrate into sterile water, and filtering the solution through a microporous filter membrane with the aperture of 0.22 mu m to obtain a sodium butyrate solution, wherein the concentration of the sodium butyrate in the sodium butyrate solution is 400 mM;

(4) and (3) adding the sodium butyrate solution in the step (3) into an improved PDB fermentation culture medium to obtain an improved PDB fermentation culture medium containing the chemical epigenetic modifier, inoculating the seed solution into the improved PDB culture medium of the fermentation culture medium, and performing shake cultivation at the temperature of 28 +/-2 ℃ and at the speed of 200r/min for 7-9 days to obtain a fermentation product, wherein the concentration of the sodium butyrate in the PDB fermentation culture medium containing the chemical epigenetic modifier is 500 mu M, and the inoculation amount of the seed solution is 10%.

(5) And (4) filtering the fermentation product obtained in the step (4) by using gauze to obtain fermentation liquor and mycelium, extracting the fermentation liquor by using ethyl acetate, extracting the mycelium by using methanol, combining extracting solutions, and concentrating under reduced pressure to obtain a fermentation crude extract.

(6) Subjecting the fermented crude extract to chromatographic separation by using a silica gel column (200-300 meshes), performing gradient elution by using a chloroform-methanol system from a volume ratio of 10:1 to 1:1, collecting a crude component Fr.1 eluted by using the chloroform-methanol system from a volume ratio of 9:1, collecting a crude component Fr.2 eluted by using the chloroform-methanol system from a volume ratio of 8:2, and collecting a crude component Fr.3 eluted by using the chloroform-methanol system from a volume ratio of 7: 3.

The crude component Fr.1 is eluted from a chloroform-methanol system at a volume ratio of 15:1 to 7:3, and a fraction eluted at a chloroform-methanol system volume ratio of 9:1 is collected and recrystallized in a mixed solvent of acetone and methanol at a volume ratio of 3:1 to prepare the compound phosphopsisaxanthone H. And (3) eluting the crude component Fr.2 from the volume ratio of 9:1 to 7:3 by using a petroleum ether-ethyl acetate system, collecting fractions eluted when the volume ratio of the petroleum ether-ethyl acetate system is 9:1, and recrystallizing in a mixed solvent of methanol and water at the volume ratio of 8:2 to prepare the compound phosphopsxanthene I. Performing gradient elution on the crude component Fr.3 from 1:1 to 9:1 in a methanol-water system, and collecting fractions eluted when the methanol-water volume ratio is 6:4 to prepare a compound phosphopsoxanthine J; collecting fractions eluted when the volume ratio of methanol to water is 7:3 to prepare a compound phosphopsisaxanthone K; fractions eluted at a methanol-to-water volume ratio of 8:2 were collected and the compound phosphopsisaxanthone L was prepared.

Example 2

The results of structural identification of the hydrogenated xanthone compounds phosphopsisaxanthone H to L are shown in FIG. 13.

Structural identification of hydrogenated xanthone compound phomopsis xanthone H

The structure of the compound phosphopsisaxanthone H prepared in example 1 was identified by 1D/2D NMR (one-dimensional and two-dimensional nuclear magnetic resonance spectrum) and HRESI-MS (high resolution electrospray ionization mass spectrometry). Phomopsis xanthone H has a molecular formula of C₁₅H₁₆O₈；HRESIMS m/z 309.0965[M+H]⁺The unsaturation degree is 8.

As shown in the figures 1 and 2, the above-mentioned figures,¹the H-NMR spectrum showed a hydroxyl proton (. delta.)_H11.68), one methyl proton (. delta.))_H2.27), one methoxy proton (. delta.))_H4.63), two aromatic protons (δ)_H 6.30；δ_H6.28), two methylene groups (. delta.))_H3.83 and 3.22, δ_H2.49 and 2.99), a vicinal oxymethylene radical (. delta.)_H3.84), one methine group (. delta.) of_H4.94) and one vicinal oxymethylene group (. delta.)_H 4.57)。¹³The C DEPT-NMR spectrum showed 15 carbon signals, respectively two carbonyl carbons (. delta.)_C196.0, 174.5), five quaternary carbons (. delta.)_C161.5, 159.2, 150.4, 105.4, 82.4), four methines (. delta.) (Delta.)_C109.3, 108.9, 87.8, 67.2), a continuous oxymethylene group (. delta.) (_C61.8), two methylene groups (. delta.))_C37.7, 36.9) and one methyl group (. delta.))_C21.4)。¹H-¹The H COSY spectrum shows two sets of meta-aromatic hydrogen protons, indicating the presence of two 1,2,3, 5-tetrasubstituted aromatic rings. CH is observed in its HMBC profile₃The correlation of-11 to C-1, C-2, C-4 indicates the presence of a methyl group at the C-3 position. In HMBC spectra, CH₂12 and C-8a (. delta.)_C 37.7)、C-10a(δ_C82.4) and C-5 (. delta.))_C87.8) there is a point of relevance for determining the linkage of the interconnecting oxymethylene group C-12 to the B ring.¹H-¹The H COSY spectral data show that oxygen methylene proton H-6 is connected with methylene H-7 (delta)_H2.99) and methine H-5 (. delta.))_H4.94), indicating the presence of a gamma-lactone moiety. C-8 (. delta.) can be observed from HMBC mapping_C174.5) and H-5, and H-5 with C-10a and C-12, which also confirms the presence of the gamma-lactone moiety and indicates the attachment of the gamma-lactone moiety to the B ringAnd (6) connecting. NOESY map shows CH₂OH-12 is in the alpha orientation at C-10a and 6-OH is in the beta orientation at C-6. Through trial of various solvent systems and ratios, we finally purified the product by acetone: the system of methanol (3: 1) yields a single crystal. The absolute configuration of the compound was determined by X-ray single crystal diffraction as shown in figure 11. The xanthone compound was searched and named Phomopsis xanthone H. The structural formula is shown as the following formula (I):

structure identification of hydrogenated xanthone compound phomopsis xanthone I

The structure of the compound phosphopsisaxanthone I prepared in example 1 was identified by 1D/2D NMR (one-dimensional and two-dimensional nuclear magnetic resonance spectrum) and HRESI-MS (high resolution electrospray ionization mass spectrometry). Phomopsis xanthone I has a molecular formula of C₁₅H₁₆O₈；HRESIMS m/z 347.0737[M+Na]⁺The unsaturation degree is 8.

As shown in the figures 3 and 4, the figure,¹the H-NMR spectrum showed two aromatic protons (. delta.)_H6.87, 6.66), one active hydroxyl proton (. delta.))_H12.64), one continuous oxymethylene group (. delta.))_H3.98), four hydroxyl protons (δ)_H6.12, 5.35, 4.72, 4.86) and one methyl proton (. delta.) (Δ S)_H 2.40)。¹³The C DEPT-NMR spectrum showed 15 carbon atoms including one carbonyl carbon (. delta.)_C183.6), seven aromatic quaternary carbons (. delta.)_C160.2, 147.9, 108.5, 156.1, 117.0, 165.6, 72.5), one methyl carbon (δ)_C22.3), one methylene group (. delta.) of_C63.8) and five methines (. delta.)_C111.9, 107.6, 69.4, 70.0, 75.0). It is similar to the compound phosphopsoxanthine H, both having two meta aromatic methines, with the methyl group attached to C-3. Combined two-dimensional atlas¹H-¹Results for H COSY, HMQC and HMBC suggest that this compound has the same a and B rings as the compound phosphoissanthone H. 5-OH (. delta.) can be seen in HMBC spectra_H6.12) and C-10a (. delta.)_C 165.6)、C-5(δ_C69.4)、C-6(δ_C70.0) and C-7 (. delta.))_C75.0), the manner of attachment of 5-OH to C-5 is demonstrated. Based on the correlation of 5-OH and 6-H and the correlation of 6-OH and 5-H and H-7 and H-6 observed in the NOESY spectrum, the trans configuration of 5-OH and 6-OH is illustrated, and 7-OH, 6-OH and 8-OH are determined to be alpha orientation, 5-OH and 8-CH₂OH is the beta orientation. Utilizing water: methanol (4:1) system, single crystals of the compound were obtained. As shown in FIG. 12, the absolute configuration of the compound is determined by X-ray, and the compound is searched to be a novel xanthone compound which is named as Phomopsis xanthone I. The structural formula is shown as the following formula (II):

structure identification of hydrogenated xanthone compound phosphopsisaxanthone J

The structure of the compound phosphopsisaxanthone J prepared in example 1 was identified by 1D/2D NMR (one-dimensional and two-dimensional nuclear magnetic resonance spectrum) and HRESI-MS (high resolution electrospray ionization mass spectrometry). Phomopsis xanthone J has a molecular formula of C₁₅H₁₆O₈；HRESIMS m/z 347.0737[M+Na]⁺The unsaturation degree is 8.

HRESI-MS showed molecular ion peak M/z 347.0737[ M + Na ]]⁺. Referring to FIGS. 5 and 6, the data of NMR spectrum and mass spectrum are combined to conclude the molecular formula C₁₅H₁₆O₈. One-dimensional of this compound and the compound phosphopsoxanthene I¹H-NMR and¹³the C-NMR spectra were similar and a combination of their one-and two-dimensional spectra revealed that they had the same planar structure with 8 unsaturations and C-5, C-6, C-7 and C-8 substituted with hydroxyl groups. From the hydrogen spectrum, it can be seen that 6-OH, 7-OH, 12. alpha. -H and 8-OH of the compound are shifted to. delta. toward the low field_H 5.58、δ_H 5.26、δ_H4.10 and delta_H5.00, 12-OH to delta_H4.77, indicating that the compound has a different steric configuration at C-8. NOESY patterns show correlation between H-5, 8-OH and 7-OH to identify compounds8-CH of₂The OH orientation is different from that of phosphopsisaxanthone I, and is alpha orientation. The compound was searched and named as Phomopsis xanthone J. The structural formula is shown as the following formula (III):

structural identification of hydrogenated xanthone compound phomopsis xanthone K

The structure of the compound phosphopsisaxanthone K prepared in example 1 was identified by 1D/2D NMR (one-dimensional and two-dimensional nuclear magnetic resonance spectrum) and HRESI-MS (high resolution electrospray ionization mass spectrometry). Phomopsis xanthone K has a molecular formula of C₁₅H₁₄O₇；HRESIMS m/z 305.0601[M-H]^-The unsaturation degree was 9.

As shown in the figures 7 and 8, the figure,¹H-NMR spectrum data at delta_H11.90 shows a reactive hydroxyl proton and, in addition, a methyl proton (. delta.)_H2.41), one methylene proton (. delta.))_H3.72,3.66), 5 methine protons (. delta.)_H6.97,6.72,4.28,4.26,4.14), and 3 hydroxyl protons (delta)_H6.60,6.03,5.40). The spectrum shows 15 carbon atoms, 1 carbonyl carbon (. delta.)_C184.2), 7 aromatic quaternary carbons (. delta.)_C165.9,159.6,156.4,148.8,115.6,108.4,77.9), one methyl group (. delta.))_C22.3), one methylene group (. delta.) of_C75.2), 5 methine groups (. delta.)_C74.3,72.6,70.2,112.5,108.2). By comparing it with the one-dimensional map of the compound Phomopsis xanthone J, it was found that they are relatively similar, both having two meta aromatic methines, and the methyl group being attached to C-3. Bonding of¹H-¹The results of the H COSY, HMQC and HMBC mapping indicate that the compound phosphopsin K has the same A and B rings as the compound phosphopsin J. Analysis of the structures of ring a and ring B and the degree of unsaturation indicate that the compound should have two rings in addition to two benzene rings and one double bond. At delta_HDouble peaks are shown at both 6.60 and 5.40, indicating that the hydroxyl protons 5-OH and 7-OH are attached to the methine group. From HMBC spectra, it can be seen that 5-OH is in phase with C-5 and C-10aIn the close point, 7-OH has a correlation with C-7 and C-8, and thus the connection mode of 5-OH and 7-OH to ring C is deduced. NOESY map does not show H₂The presence of 12 in association with 7-OH and 5-OH, neither 7-OH nor 5-OH, 8-OH in association with CH₃11 has a correlation, and 5-OH is deduced to be in alpha orientation and 7-OH is deduced to be in beta orientation. The compound is searched and named as Phomopsis xanthone K. The structural formula is shown as the following formula (IV):

structural identification of hydrogenated xanthone compound phosphopsisaxanthone L

The structure of the compound phosphopsisaxanthone L prepared in example 1 was identified by 1D/2D NMR (one-dimensional and two-dimensional nuclear magnetic resonance spectrum) and HRESI-MS (high resolution electrospray ionization mass spectrometry). Phomopsis xanthone L has a molecular formula of C₁₅H₁₄O₆；HRESIMS m/z 313.0683[M+Na]^+-The unsaturation degree was 9.

As shown in the figures 9 and 10, the figure,¹the H-NMR spectrum showed a methyl proton (. delta.)_H2.38), one methylene group (. delta.))_H4.03,3.60), six methines (. delta.)_H 3.66,4.20,4.25,4.63,6.53,6.75)。¹³The C DEPT-NMR spectrum showed 15 carbon atoms including one carbonyl carbon (. delta.)_C181.5), 6 aromatic quaternary carbons (. delta.)_C159.9,147.4,163.8,156.7,115.9,108.1), one methyl group (. delta.))_C21.0), one methylene group (. delta.))_C71.5), six methines (. delta.)_C35.0,69.6,69.9,74.2,111.2,107.3). By comparing the spectra of phosphopsisaxanthone K and the compound phosphopsisaxanthone L, they were found to be similar, the main difference being that the compound phosphopsisaxanthone L is delta_H3.66 has one more peak, and H-7 shifts to delta toward low field_H4.63. The molecular weight of the compound phosphopsisaxanthone K is 16 less, and it is presumed that the substitution of the hydroxyl group adjacent to H-7 is changed to the substitution of hydrogen. The HMQC map shows that the H-8 has correlation points with C-8a, C-6 and C-8,¹H-¹the H COSY map shows that H-8 has correlation points with H-12 alpha and H-7,the connection mode of C-8 and C-12 and C-7 is shown. From the NOESY spectrum, the presence of H-7 associated with H-8 and H-6, the presence of H-8 associated with H-12 α, and the absence of H-5 associated with H-7, were observed, indicating that H-8, H-6, and 5-OH are in the α orientation and 7-OH is in the β orientation. The compound was searched and named Phomopsis xanthone L. The structural formula is shown as the following formula (V):

the five hydrogenated xanthone derivatives are all dissolved in methanol, pyridine, dimethyl sulfoxide and the like, and are not dissolved in petroleum ether and water. Process for preparation of phosphopsisaxanthones H-L¹H NMR and¹³the C NMR data are shown in Table 1.

TABLE 1 preparation of the hydrogenated xanthone derivatives phosphopsisaxanthones H-L¹H NMR and¹³c NMR data

The determination solvent of Phomopsis xanthone H is deuterated acetone; the determination solvent of phosphopsisaxanthone I, J, L is deuterated dimethyl sulfoxide; the assay solvent for phosphopsisaxanthone K was deuterated methanol.

Example 3

Determination of the anti-phytopathogen activity and antioxidant capacity of the hydrogenated xanthone compounds phomopsis xanthones H-L:

(1) and (3) activity determination of anti-phytopathogen:

the 4 plant pathogen indicator bacteria include Botrytis cinerea (Botrytis cinerea), Gibberella cerevisi (Gibberella sautiniii), Fusarium solani (Fusarium solani), and Curvularia lunata (Curvularia lunata).

Well-growing pathogens were inoculated into 250mL Erlenmeyer flasks containing 100mL of liquid medium per flask, under sterile conditions. 1024. mu.g of the compound was weighed, dissolved in DMSO solution, and diluted to 516-2. mu.g/mL on a 96-well plate with sterile water at a double ratio. Add 50. mu.L of bacterial suspension to each well, pipette and mix well. The positive control is nystatin, and 2 groups of pathogenic bacteria are subjected to parallel experiments. Incubate at 28 ℃ for 24-48h, observe the results every 8h and record.

(2) DPPH radical scavenging capacity:

weigh 1mg of sample to be tested, add 2mL of DMSO to dissolve to obtain 0.5mg/mL of sample stock solution. Then, the prepared mother liquor is diluted to 5-50 mu g/mL. 7.886mg of DPPH (1, 1-diphenyl-2-trinitrophenylhydrazine) were weighed out and dissolved in 100mL of DMSO solution to give a 0.2mM DPPH stock solution. 1mL of the prepared DPPH mother solution was added to compound solutions of different concentrations (3mL, 5-50. mu.g/mL) and reacted for 30 minutes in the dark. The reacted sample (200. mu.L) was put in a 96-well plate, and the absorbance at 517nm was measured with a microplate reader. DMSO as negative control, vitamin C (V)_C) And vitamin E (V)_E) Is a positive control.

The measured data are expressed by the formula [ (Ac-As)/Ac]X 100% DPPH radical clearance K (%) of the compounds was calculated, respectively. Ac is the absorbance of the negative control DMSO at 517nm, As is the added compound or the positive control V_CAnd V_EAbsorbance measured at 517 nm. Calculating the IC of the compound from its free clearance data₅₀(median inhibitory concentration) value.

(3)ABTS⁺Radical scavenging ability:

weighing 1mg of sample to be detected, adding 2mL of DMSO, and dissolving to obtain 0.5mg/mL of sample mother solution. Then, the prepared mother liquor is diluted to 5-50 mu g/mL. Weighing 109.736mg ABTS⁺(2, 2-Aza-bis (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt) and 66.2284mg of potassium persulfate were dissolved in 100mL of water and reacted at room temperature in the dark for 12 hours to obtain ABTS⁺The mother liquor was at a concentration of 0.2 mM. Absorb 1mL of configured ABTS⁺The mother liquor was added to a diluted solution of the compound of various concentrations (3mL,5-50g/mL) and reacted for 10 minutes under dark conditions. Taking 200 mu L of reacted sample in a 96-well plate, and labeling the sample with enzymeThe absorption at 734nm was determined by the instrument. DMSO was a negative control in this assay, V_CAnd V_EIs a positive control. The measured data are expressed by the formula [ (Ac-As)/Ac]X 100% DPPH radical clearance K (%) of the compounds was calculated, respectively. Ac is the absorbance of the negative control DMSO at 517nm, As is the added compound or the positive control V_CAnd V_EAbsorbance measured at 734 nm. Calculating the IC of the compound from its free clearance data₅₀The value is obtained.

The Minimum Inhibitory Concentration (MIC) values of the hydrogenated xanthone derivatives phomopissxanthones H-L of the invention to 4 pathogenic indicator bacteria are shown in Table 2.

TABLE 2 inhibitory Activity of Phomopsis xanthones H-L against 4 pathogen indicator bacteria

The hydrogenated xanthone derivatives phomopsis xanthones H-L of the invention are used for DPPH free radical and ABTS⁺Half maximal Inhibitory Concentration (IC) for free radical scavenging₅₀) The values are shown in Table 3.

Table 3 radical scavenging ability of the compounds phosphopsisaxanthones H-L

Experimental results show that the hydrogenated xanthone compounds phosphosixantones H-L have antibacterial activity with different degrees on 4 plant pathogen indicator bacteria, particularly the compound phosphosixanone H has the strongest antibacterial activity, and the compounds phosphosixanone I and phosphosixanone J also have stronger activity on wheat scab. Test Compounds on DPPH free radical and ABTS⁺The free radicals have the scavenging capacity, and the compounds phosphosiloxane K and phosphosiloxane L have the strongest scavenging capacity and show stronger antioxidant activity. Thus, the hydrogenated xanthone compounds phomo according to the inventionThe psisxanthones H-L have potential application in preparing novel agricultural antibiotics and antioxidant drugs.

(1) in the preparation method of the hydrogenated xanthone derivative, during the process of preparing the crude fermentation extract, the fermentation liquor is extracted by ethyl acetate, and the mycelium is leached by methanol, so that the effective components in the fermentation liquor and the mycelium are extracted, and the method has the advantage of separating the effective substances in the fermentation product from the unused culture medium components and water-soluble impurities.

(2) According to the preparation method of the hydrogenated xanthone derivative, the improved PDB culture medium is adopted as the fermentation culture medium of phomopsis, compared with the conventional PDB culture medium, the improved PDB culture medium has the beneficial effects that a fermentation product different from the conventional PDB culture medium can be generated, and the principle is that the synthesis of a microbial fermentation product is influenced by the change of the components of the culture medium.

(3) According to the preparation method of the hydrogenated xanthone derivative, the concentration of the sodium butyrate in the fermentation medium of phomopsis longipes is 200-500 mu M, and the advantage that the concentration range can induce the compound of the invention to generate.

(4) In the preparation method of the hydrogenated xanthone derivative, during the processes of collecting crude components Fr.1-Fr.3 and separating phosphosixantones H-L, different volume ratios of selected eluent systems and eluents are used for obtaining corresponding target fractions or target products, and if other eluent systems or other volume ratios are adopted, the separation process for obtaining the target products phosphosixantones H-L is more complicated, takes longer time or is difficult to obtain than the prior art.

Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims

1. A hydrogenated xanthone derivative characterized by comprising at least one of phosphopsisaxanthones H to L, wherein the formulae of the phosphopsisaxanthones H to L are represented by (I) to (V), respectively:

2. a process for the preparation of a hydrogenated xanthone derivative according to claim 1, which comprises:

carrying out chromatographic separation on the crude fermentation extract, eluting by adopting a chloroform-methanol system, and collecting crude components Fr.1-Fr.3;

carrying out chromatographic separation on the crude component Fr.1, and eluting by adopting a chloroform-methanol system to obtain a compound phosphopsisaxanthone H;

3. The method for producing a hydrogenated xanthone derivative according to claim 2, wherein said Phomopsis sp.YE3350, which has been deposited at the China center for type culture Collection on 8/3/2020 with the deposit number of CCTCC NO. M2020394.

4. The method for producing a hydrogenated xanthone derivative according to claim 2, wherein said phomopsis is fermented in a medium containing sodium butyrate, and the fermentation broth and mycelia are collected after the fermentation is completed, the method comprising:

adding 20-50 parts of glucose, 3-5 parts of dipotassium hydrogen phosphate, 3-5 parts of magnesium sulfate and vitamin B into the potato filtrate₁0.01-0.03 part of PDB culture medium is obtained;

5. The method for preparing a hydrogenated xanthone derivative according to claim 2, wherein the steps of extracting the fermentation broth, leaching the mycelium, combining the extractive solutions, and volatilizing the solvent to obtain a crude fermentation extract comprise:

6. The method for preparing a hydrogenated xanthone derivative according to claim 2, wherein the crude fermentation extract is chromatographically separated, and the eluent is chloroform-methanol system, and the crude fractions fr.1 to fr.3 are collected, specifically comprising:

7. A process for the preparation of a hydrogenated xanthone derivative according to claim 2, wherein the crude fraction fr.1 is chromatographed using a chloroform-methanol system as eluent to obtain the compound phosphopsoxanthine H, comprising in particular:

8. A process for the preparation of a hydrogenated xanthone derivative according to claim 2, wherein said crude fraction fr.2 is chromatographed using an eluent in the petroleum ether-ethyl acetate system to obtain the compound phosphopsin I, comprising:

9. A process for the preparation of a hydrogenated xanthone derivative according to claim 2, wherein said crude fraction fr.3 is chromatographed using an eluent in the methanol-water system to obtain the compounds phosphopsoxanthine J-L, comprising in particular:

10. Use of the hydrogenated xanthone derivative according to claim 1 for the preparation of agricultural antibiotics and anti-oxidant drugs.