CN113527248A

CN113527248A - Xanthone compound and preparation method and application thereof

Info

Publication number: CN113527248A
Application number: CN202110831774.9A
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: 2021-10-22
Anticipated expiration: 2041-07-22
Also published as: CN113527248B

Abstract

The invention provides xanthone compounds, which belong to the technical field of biological medicines, and comprise at least one of phosphops i xanthenes A-G, and the series of xanthone compounds can resist plant pathogenic fungi and have antioxidant activity, can be used for preparing agricultural antibacterial agents and antioxidant drugs, and have higher agricultural and medical commercial values. The invention also provides a preparation method and application of the xanthone compound.

Description

Xanthone compound and preparation method and application thereof

Technical Field

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

Background

Xanthone compounds are an isotridecyl compound. It is an important organic synthesis intermediate, the parent body itself does not exist in the plant body, but the derivatives thereof widely exist in the nature, and the derivatives are collectively called as xanthone compounds and are one of the effective components of medicinal plants. These compounds have different biological activities mainly because of the different groups on their substituents. Due to their diverse and special structures, they are generally capable of exhibiting various biological activities of modulating body immunity, anti-tumor, and anti-inflammatory, and they are also important substances for developing novel antibiotics. At present, the main sources of natural extraction of xanthones are plants and microorganisms. In recent years, microorganisms have been considered as a major source of xanthones having important biological activities in the future, because of the relatively small amount of xanthones found in natural products of plant origin.

Disclosure of Invention

The invention aims to provide xanthone compounds, and the series of xanthone compounds can resist plant pathogenic fungi and have antioxidant activity, can be used for preparing agricultural antibacterial agents and antioxidant drugs, and have higher agricultural and medical commercial values.

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

The invention is realized by the following technical scheme:

the invention provides xanthone compounds, which comprise at least one of phosphosixannes A-G, wherein the structural formulas of the phosphosixannes A-G are respectively shown as (I) to (VII):

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

fermenting phomopsis, 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;

subjecting the crude fraction Fr.1 to chromatographic separation with an eluent comprising petroleum ether-ethyl acetate to obtain the compounds phosphopsisaxanthone A, phosphopsisaxanthone B and phosphopsisaxanthone C;

subjecting the crude fraction Fr.2 to chromatographic separation, eluting with a methanol-water system to obtain phosphopsisaxanthone D and phosphopsisaxanthone E;

the crude fraction Fr.3 is chromatographed using chloroform-acetone as eluent to give the compounds phosphopsisaxanthone F and phosphopsisaxanthone G.

Optionally, the Phomopsis sp.YE3350 is a strain which has been 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.

Optionally, the phomopsis is fermented, and fermentation liquor and mycelia are collected after fermentation is finished, and the method specifically comprises the following steps:

placing phomopsis into a PDB culture medium for fermentation, and collecting fermentation liquor and mycelia after the fermentation is finished, wherein the concentration of potatoes in the PDB culture medium is 0.2-0.4 g/mL, and the concentration of glucose in the PDB culture medium is 0.02-0.05 g/mL;

the PDB culture medium 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;

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.

Optionally, the extracting the fermentation broth, leaching the mycelium, combining the extracting solutions, and volatilizing the solvent to obtain a fermentation crude extract specifically includes:

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.

Optionally, 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, wherein the volume ratio of chloroform to methanol is 95:5 to 1:1, collecting a crude component Fr.1 eluted when the volume ratio of chloroform to methanol is 95:5, collecting a crude component Fr.2 eluted when the volume ratio of chloroform to methanol is 9:1, and collecting a crude component Fr.3 eluted when the volume ratio of chloroform to methanol is 8: 2.

Alternatively, the crude fraction fr.1 is chromatographed using an eluent in the petroleum ether-ethyl acetate system to obtain the compounds phosphosiloxane a, phosphosiloxane B and phosphosiloxane C, including in particular:

carrying out chromatographic separation on the crude component Fr.1, carrying out gradient elution on the crude component Fr.1 by using an eluant through a petroleum ether-ethyl acetate system and using petroleum ether and ethyl acetate in a volume ratio of 95:5 to 6:4, collecting fractions eluted when the volume ratio of the petroleum ether to the ethyl acetate is 9:1, and volatilizing the eluant to obtain a xanthone compound phosphopsisaxanthone A; collecting fractions eluted when the volume ratio of petroleum ether to ethyl acetate is 8:2, and volatilizing an eluent to obtain a xanthone compound phosphopsoxanthene B; collecting fractions eluted when the volume ratio of petroleum ether to ethyl acetate is 7:3, and volatilizing the eluent to obtain the xanthone compound phosphopsoxanthine C.

Alternatively, the crude fraction fr.2 is chromatographed using a methanol-water system as eluent to give the compounds phosphopsin D and phosphopsin E, which specifically include:

carrying out chromatographic separation on the crude component Fr.2, carrying out gradient elution on the crude component Fr.2 by using an eluent in a methanol-water system and using methanol and water in a volume ratio of 1:1 to 4:1, collecting fractions eluted when the volume ratio of methanol and water is 6:4, and volatilizing the eluent to obtain a xanthone compound phosphopsanthone D; collecting fractions eluted when the volume ratio of methanol to water is 7:3, and volatilizing the eluent to obtain the xanthone compound phosphopsoxanthine E.

Alternatively, the crude fraction fr.3 is chromatographed using a chloroform-acetone system as eluent to give the compounds phosphopsin F and phosphopsin G, including:

carrying out chromatographic separation on the crude component Fr.3, carrying out gradient elution on the crude component Fr.3 by using a chloroform-acetone system as an eluent according to the volume ratio of 9:1 to 1:1, collecting fractions eluted when the volume ratio of chloroform to acetone is 8:2, and volatilizing the eluent to obtain a xanthone compound phosphopsisaxanthone F; collecting fractions eluted when the volume ratio of petroleum ether to ethyl acetate is 7:3, and volatilizing the eluent to obtain the xanthone compound phosphopsoxanthine G.

Based on the same inventive concept, the invention also provides the application of the xanthone compound in preparing agricultural antibacterial agents and/or 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 xanthone compounds provide seven novel xanthone compounds A-G, can resist phytopathogens and have antioxidant activity, provide a new choice for developing agricultural antibacterial agents and antioxidant drugs, and have high agricultural and medical commercial values.

2. The xanthone compound is prepared by fermenting the strain Phomopsis sp.YE3350, and the method has the advantages of short period, mild culture conditions, few byproducts, low cost, higher economic value, low cost, simple and convenient operation and easy large-scale production, and provides a new way for obtaining the xanthone compound from natural sources.

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 A, a compound of the present invention¹H-NMR spectrum;

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

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

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

FIG. 5 shows phosphopsisaxanthone C of the compound of the present invention¹H-NMR spectrum;

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

FIG. 7 shows phosphopsisaxanthone D compounds of the present invention¹H-NMR spectrum;

FIG. 8 shows phosphopsisaxanthone D compounds of the present invention¹³C-NMR and DEPT spectra;

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

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

FIG. 11 shows phosphopsisaxanthone F, a compound of the present invention¹H-NMR spectrum;

FIG. 12 shows phosphopsisaxanthone F of the present invention¹³C-NMR and DEPT spectra;

FIG. 13 shows phosphopsisaxanthone G of the present invention¹H-NMR spectrum;

FIG. 14 shows phosphopsisaxanthone G of the present invention¹³C-NMR and DEPT spectra;

FIG. 15 shows the structural formulae of phosphopsisaxanthenes A-G of xanthone compounds according to 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.

The xanthone compounds of the present application will be described in detail with reference to examples and experimental data.

Example 1

Isolation and preparation of xanthone compounds phomopsis xanthones A-G

(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, shake-culturing at 28 + -2 deg.C and 200r/min for 4d to obtain seed solution, inoculating the prepared seed solution into fermentation culture medium modified PDB culture medium according to 10% of inoculum size, shake-culturing at 28 + -2 deg.C and 200r/min for 7-9 d to obtain fermentation product.

(3) Filtering the fermentation product obtained in the step (2) 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.

(4) Performing chromatographic separation on the crude fermentation extract by using a silica gel column (200-300 meshes), performing gradient elution on the crude fermentation extract by using a chloroform-methanol system from a volume ratio of 95:5 to 1:1, collecting a crude component Fr.1 eluted by using the chloroform-methanol system from the volume ratio of 95:5, collecting a crude component Fr.2 eluted by using the chloroform-methanol system from the volume ratio of 9:1, and collecting a crude component Fr.3 eluted by using the chloroform-methanol system from the volume ratio of 8: 2.

Gradient elution is carried out on the crude component Fr.1 from a volume ratio of 95:5 to 6:4 by a petroleum ether-ethyl acetate system, fractions obtained by elution when the volume ratio of petroleum ether to ethyl acetate is 9:1 are collected, and the xanthone compound phosphopsisaxanthone A can be prepared; collecting fractions eluted when the volume ratio of petroleum ether to ethyl acetate is 8:2, and preparing a xanthone compound phosphopsoxanthene B; fractions eluted when the volume ratio of petroleum ether to ethyl acetate is 7:3 are collected, and the xanthone compound phosphopsoxanthine C can be prepared.

Performing gradient elution on the crude component Fr.2 from 1:1 to 4:1 in a methanol-water system, and collecting fractions eluted when the volume ratio of methanol to water is 6:4 to prepare a xanthone compound phosphopsisaxanthone D; fractions eluted at a methanol-water volume ratio of 7:3 are collected to prepare the xanthone compound phosphopsoxanthine E.

Performing gradient elution on the crude component Fr.3 by using a chloroform-acetone system from a volume ratio of 9:1 to 1:1, and collecting fractions eluted when the chloroform-acetone volume ratio is 8:2 to prepare a xanthone compound phosphopsisaxanthone F; fractions eluted when the volume ratio of petroleum ether to ethyl acetate is 7:3 are collected, and the xanthone compound phosphopsoxanthine G can be prepared.

Example 2

The results of structural identification of Phomopsis xanthones A to G are shown in FIG. 15.

Structural identification of xanthone compound phomopsis xanthone A

The structure of the compound phosphopsisaxanthone A 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 A has a molecular formula of C₁₅H₁₂O₅；HRESIMS m/z:271.0607[M-H]^-The unsaturation degree was 10.

As shown in the figures 1 and 2, the above-mentioned figures,¹the H-NMR spectrum showed a hydroxyl proton (. delta.)_H13.00), one methyl proton (. delta.))_H2.37), four methine protons (. delta.))_H6.58,6.71,6.80,7.23), a methylene group (delta) linked to oxygen_H5.07). According to its unsaturation of 10, there should be three benzene rings in addition to one carbonyl group.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. From the HMQC spectrum, a peak at 12-OH (. delta.) can be observed_H5.39) and 1-OH (. delta.))_H12.94), also shows the methyl proton CH₃-11(δ_H2.37) and the oxygen-bound hydroxyl proton CH₂-12(δ_H5.07) correlation with C-11 and C-12, indicating CH₃-11 and CH₂-12 is linked to C-11, C-12, respectively. 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. Furthermore, the oxygen-linked proton CH is observed₂The correlation of-12 with C-7, C-8 and C-8a indicates CH₂The connection mode of OH and C-8. From the HMBC spectrum, delta can be seen_HThe connection of 1-OH and C-1 can be determined from the correlation of the hydroxyl proton at position 12.94 with C-1, C-2, C-9 a. From HMBC and¹H-¹the H COSY map and the characteristics of meta substitution infer the connection of 6-OH and C-6, and further infer the correlation of 12-OH and methylene C-12. Compound 1 is xanthone compound, which is substituted by methyl and hydroxymethyl at C-3 and C-8, respectively, and substituted by hydroxyl at C-1 and C-6, and is searched to be newA compound, designated phosphopsisaxanthone A, having the formula (I):

structural identification of xanthone compound phomopsis xanthone B

The structure of the compound phosphopsisaxanthone B 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 B has the molecular formula of C₁₅H₁₂O₅；HRESIMS m/z 271.0506[M-H]^-The unsaturation degree was 10.

As shown in fig. 3 and 4, compared with the compound phosphopsisaxanthone a,¹the H-NMR spectrum is shown at delta_HThe presence of two low field shifted aromatic proton signals at 7.46 and 7.40, both of which were calculated to have coupling constants of 9.1Hz, indicates the presence of ortho substitution on the benzene ring of the compound. By comparison¹H-¹Comparison of the H COSY, HMBC and HMQC spectra reveals that the compound phosphosiloxane A is identical to the compound phosphosiloxane B in both ring A and ring B and that meta substitution is present. From the HMBC profile H-2 (. delta.) can be observed_H 6.61)，H-4(δ_H6.83) and the methyl carbon C-11 (. delta.))_C22.4) there is a correlation point, indicating the attachment of C-11 to C-3. C-8 (delta)_C 125.7),C-6(δ_C 125.6),C-12(δ_C55.8) displacement to low electric field, C-5 (. delta.)_C 111.8),C-7(δ_C152.7) to a high electric field, indicating that the hydroxyl protons are at the C-7 position and are affected by 7-OH, so that their chemical shifts change. In combination with the above analysis, the compound phosphopsisaxanthone A was determined to be a xanthone. Substituted at C-3 and C-8 with methyl and one hydroxymethyl group, respectively, and at C-1 and C-7 with hydroxy, was searched as a novel compound, named phosphosiloxane B, having the formula shown in the following formula (II):

structural identification of xanthone compound phomopsis xanthone C

The structure of the compound phosphopsisaxanthone C 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). The molecular formula of Phomopsis xanthone C is C₁₅H₁₂O₅；HRESIMS m/z 271.0607[M-H]^-The unsaturation degree was 10.

As shown in FIGS. 5 and 6, the hydrogen spectrum and carbon spectrum of the compound have one more methoxy group (. delta.) than that of the compound phosphopsisaxanthone A_H4.04；δ_C62.8), one methylene group less.¹The H-NMR spectrum is shown at delta_H7.46 and delta_HAt 7.40 there are two low field shifted aromatic proton signals, both of which have a coupling constant of 8.8Hz, indicating ortho-substituted benzene rings. From it¹H-¹A group of methine protons (delta) can be seen in the H COSY spectrum_H 6.69；δ_H6.60), we conclude that there is a meta substitution on the phenyl ring. From the HMBC spectrum, the hydroxyl proton 6-OH (. delta.) can be observed_H5.97) and C-5 (. delta.)_C 118.8)，C-6(δ_C 125.6)，C-7(δ_C152.7) has a correlation, which determines the linkage of the hydroxyl proton 6-OH to C-6. The connection of the A and B rings of the compound phosphopsisaxanthone C was determined in the same manner as the compound phosphopsisaxanthone A. The above analysis was combined to determine that the compound phosphopsisaxanthone C also belongs to xanthones, is substituted at C-3 and C-5 with methyl and methoxy groups, respectively, and at C-1 and C-6 with hydroxy groups, and was searched for a new compound, designated phosphopsisaxanthone C, having the formula shown in the following formula (III):

structural identification of xanthone compound phomopsis xanthone D

Phomopsis xanthone D, the compound prepared in example 1, was subjected to 1D/2D NMR (one-dimensional nuclear magnetic resonance)Spectrum and two-dimensional nuclear magnetic resonance spectrum) and HRESI-MS (high-resolution electrospray ionization mass spectrometry) were used to identify their structures. Phomopsis xanthone D has a molecular formula of C₁₅H₁₂O₄；HRESIMS m/z 257.0806[M-H]^-The unsaturation degree was 10.

Referring to FIGS. 7 and 8, the spectra of this compound show one less hydroxyl proton and 16 less molecular weight than the phosphopsinanthone A compound. Combining the comparison results of hydrogen spectrum and carbon spectrum, the compound phosphopsisaxanthone A is judged to be in delta_H7.65 has more signal peaks and is an aromatic methine. From the HMBC spectra, proton H-6 (. delta.) was observed_H7.65) and C-10a (. delta.))_C157.9) and C-8 (. delta.))_C142.7) has remote correlation.¹H-¹The H COSY spectrum can observe the existence of a 2,3, 4-substituted ring in addition to a meta-substituted ring. From the one-dimensional two-dimensional spectrum of nuclear magnetic resonance, it was confirmed that the A ring and the B ring were linked in the same manner as the compound phosphosiloxane A without substitution of the hydroxyl group at the C-6 position of the C ring. The above analysis was combined to determine that the compound phosphopsisaxanthone D is a xanthone compound in which C-3 and C-8 are substituted with methyl and hydroxymethyl groups, respectively, and which is substituted at C-1 with a hydroxyl group. The compound is named phosphopsisaxanthone D through searching, and the structural formula of the compound is shown as the following formula (IV):

structural identification of xanthone compound phomopsis xanthone E

The structure of the compound phosphopsisaxanthone E 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 E has a molecular formula of C₁₅H₁₂O₄；HRESIMS m/z:311.0529[M-H]^-The unsaturation degree was 10.

As shown in FIGS. 9 and 10, the spectrum data of the compound has one more even oxymethylene group and one less methyl group compared with the compound phosphopsoxanthine A. According to which¹As a result of the H-NMR spectrum display, it can be seen that the difference in the value of delta_H7.50 and delta_HAt 7.41 there are low electric field displaced aromatic proton signals with the same coupling constant of 9.12Hz, and we conclude that ortho substitution may occur on the benzene ring. At delta_H6.95 and delta_HAt 6.72 there is a pair of aromatic proton signals, consisting of¹H-¹The H COSY spectrum confirms that a group of meta-substituted aromatic protons exist on the benzene ring. From HMBC spectra, methine CH can be observed₂-11(δ_H4.58) and C-4 (. delta.))_C 103.9)，C-2(δ_C107.4), and C-3 (. delta.))_C154.0) there is a correlation, confirming CH₂-11 and C-3. Furthermore, the oxygen-linked proton CH is observed₂-12(δ_H5.12) and C-7 (. delta.)_C152.7)，C-8(δ_C127.4) and C-8a (. delta.))_C119.1), which also indicates CH₂The connection mode of OH and C-8. From the HMQC spectrum, 11-OH and CH can be observed₂-11 and 12-OH and CH₂The existence of a point with correlation of-12 determines the connection mode of two continuous oxygen methylene groups. In summary, the compound phosphopsisaxanthone E was determined to be a xanthone. Substituted at C-3 and C-8 with hydroxymethyl and at C-1 with hydroxy respectively, and is searched to be a new compound, namely phosphopsoxanthine E, the structural formula of which is shown as the following formula (V):

structural identification of xanthone compound phomopsis xanthone F

The structure of the compound phosphopsisaxanthone F 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 F has a molecular formula of C₁₅H₁₂O₅；HRESIMS m/z:271.0607[M-H]^-The unsaturation degree was 10.

As shown in FIGS. 11 and 12, the compound has one more hydroxymethyl group (. delta.) than the phosphopsin A compound_H4.69；δ_C62.9) and methine (. delta.) groups_H 7.70；δ_C137.5) with one less methyl group. The formula is the same as that of the compound phosphosiloxane A, indicating that the position of the methylene group attachment has changed. From¹H-¹The H COSY spectrum can observe that a 2,3, 4-substituted ring exists in addition to a group of meta-substituted rings. In a two-dimensional HMBC map, methylene CH is shown₂-11(δ_H4.68) and quaternary carbon C-5 (. delta.)_C 113.2)，C-6(δ_C152.1) and C-7 (. delta.))_C119.6) shows a correlation between methine CH₂The-11 linkage is at the C-6 position. CH (CH)₂-12(δ_H5.44) with C-7, C-8 and C-8a (. delta.)_C115.7) are all present, the CH is determined₂-12 is attached at the position of C-12. Based on the two substituted rings and the above data, it was determined that 12-OH, 11-OH is attached to C-12 and C-11, respectively. The compound phosphopsisaxanthone F, which has a hydroxymethyl substitution at both the C-6 and C-8 positions and a hydroxyl substitution at C-1, was searched for as a novel compound, designated phosphopsisaxanthone F, and its formula is shown in formula (VI):

structural identification of xanthone compound phomopsis xanthone G

The structure of the compound phosphopsisaxanthone G prepared in example 1 was identified by 1D/2D NMR (one-dimensional nuclear magnetic resonance spectrum and two-dimensional nuclear magnetic resonance spectrum). Phomopsis xanthone G has a molecular formula of C₁₅H₁₀O₆(ii) a The unsaturation degree was 11.

Referring to FIGS. 13 and 14, the compound phosphopsisaxanthone G was identified as xanthone by comparing the data of one-dimensional and two-dimensional nuclear magnetic resonance of the compound phosphopsisaxanthone G with that of the compound phosphopsisaxanthone A. Compound phosphopsin G has one more carboxyl group (. delta.) than compound phosphopsin A_C168.7) one less methyl and one less hydroxyl group and 14 more relative molecular mass. From HMBC profile, H-5 (a)δ_H 8.33)，H-7(δ_H9.27) methine proton with carboxyl group C-11 (. delta.))_C168.7) indicating the attachment of C-11 and C-6. Taken together, the compound phosphosiloxane G, which has a hydroxyl substitution at C-1 and a carboxyl and hydroxymethyl substitution at C-6 and C-8, respectively, was searched for a new compound, designated phosphosiloxane G, having the formula (VII):

the seven xanthone compounds described in this example are all white amorphous powders, which are soluble in methanol, pyridine, dimethyl sulfoxide, etc., and insoluble in petroleum ether and water. Process for preparation of phosphopsisaxanthones A-G¹The H NMR data are shown in Table 1,¹³the CNMR data are shown in Table 2.

TABLE 1 preparation of xanthones A-G¹H NMR data

The determination solvent of Phomopsis xanthone A and B, F is deuterated dimethyl sulfoxide; the determination solvent of phosphopsisaxanthone C and D is deuterated chloroform; the solvent for phosphopsisaxanthone E, G assay was deuterated pyridine.

TABLE 2 method for preparation of phosphpsisxanthones A-G¹³C NMR data

Example 3

Determination of anti-phytopathogen activity and anti-oxidation activity of xanthone compounds A to G:

(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). The culture medium is PDB culture medium.

Under sterile conditions, well-growing pathogens were inoculated into 250mL Erlenmeyer flasks containing 100mL of liquid medium per flask. 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, 2 groups of pathogenic bacteria are subjected to parallel experiments, the pathogenic bacteria are cultured for 24-48h at 28 ℃, and the result is observed and recorded once every 8 h.

(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⁺Free radicalClearing capacity:

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. 200. mu.L of the reacted sample was placed in a 96-well plate, and the absorbance at 734nm was measured using a microplate reader. 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 xanthone compounds A to G produced by the endophytic fungi Phomopsis sp.YE3350 of the present invention against 4 plant pathogenic bacteria are shown in Table 3.

TABLE 3 inhibitory Activity of the compounds phomopsis xanthones A-G against 4 plant pathogenic fungi

The xanthone compounds Phomopsis xanthones A-G generated by the endophytic fungi Phomopsis sp.YE3350 of the invention can eliminate DPPH free radicals and ABTS⁺Half maximal Inhibitory Concentration (IC) of free radicals₅₀) As shown in table 4.

TABLE 4 free radical scavenging Activity of the Compounds Phomopsis xanthones A-G

Experimental results show that the xanthone compounds phosphopsisaxanthones A-G of the invention all show antibacterial activities of different degrees to 4 plant pathogen indicator bacteria, wherein the compounds have stronger inhibition effect on wheat scab, and especially the MIC values of the compounds phosphopsisaxanthone B and phosphopsisaxanthone G to wheat scab are only 2 mug/mL. Among the compounds tested, phosphoissanthone A showed the strongest inhibitory effect on Fusarium solani with MIC values of 4. mu.g/mL, phosphoissanthone E and phosphoissanthone G showed the strongest inhibitory activity on Botrytis cinerea with MIC values of 4. mu.g/mL. In the antioxidant activity test, the xanthone compounds phosphopsisaxanthones A to G show different degrees of free radical scavenging capacity, and particularly, the compounds phosphopsisaxanthone D, phosphopsisaxanthone E and phosphopsisaxanthone G have stronger activity, which indicates that the compounds have stronger antioxidant capacity. In conclusion, the xanthone compounds phosphopsisaxanthenes A-G have potential application in preparing novel agricultural antibacterial agents and antioxidant drugs.

(1) in 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 xanthone compound, the improved PDB culture medium is adopted as the fermentation culture medium of phomopsis, compared with the conventional PDB culture medium, the method has the beneficial effect that a fermentation product different from the conventional PDB culture medium can be generated, and the synthesis of the microbial fermentation product can be influenced due to the change of the components of the culture medium.

(3) In the process of collecting crude components Fr.1-Fr.3 and separating phospho-isoxanthones A-G, the method for preparing the xanthone compounds aims to obtain corresponding target fractions or target products by selecting different eluent systems and different volume ratios of the eluents, and if other eluent systems or other volume ratios are adopted, the separation process for obtaining the target products phospho-isoxanthones A-G 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. The xanthone compound is characterized by comprising at least one of phosphopsisaxanthones A to G, wherein the structural formulas of the phosphopsisaxanthones A to G are respectively shown as (I) to (VII):

2. a process for the preparation of xanthone compounds according to claim 1, comprising:

3. The method for preparing xanthone compounds according to claim 2, wherein Phomopsis sp.YE3350 is the strain Phomopsis sp.YE3350 which has been deposited in China center for type culture Collection at 8/3 of 2020 with the deposit number of CCTCC NO. M2020394.

4. The method for preparing xanthone compounds according to claim 2, wherein the phomopsis is fermented, and the fermentation broth and mycelium are collected after the fermentation is finished, specifically comprising:

the PDB culture medium is prepared by the following method:

5. The method for preparing xanthone compounds according to claim 2, wherein the steps of extracting the fermentation liquid, leaching the mycelium, combining the extraction liquid, and volatilizing the solvent to obtain a crude fermentation extract comprise:

6. The method for preparing xanthone compounds according to claim 2, wherein the crude fermentation extract is chromatographically separated, and the eluent is chloroform-methanol system, and crude components Fr.1-Fr.3 are collected, which specifically comprises:

7. The process for the preparation of xanthone compounds according to claim 2, wherein the crude fraction fr.1 is chromatographed using an eluent in the petroleum ether-ethyl acetate system to obtain the compounds phosphosiloxane a, phosphosiloxane B and phosphosiloxane C, which comprises:

8. The process for the preparation of xanthone compounds according to claim 2, wherein the crude fraction fr.2 is chromatographed using a methanol-water system as eluent to obtain the compounds phospho-shixantone D and phospho-shixantone E, which comprises:

9. The process for the preparation of xanthone compounds according to claim 2, wherein the crude fraction fr.3 is chromatographed using chloroform-acetone as eluent to obtain the compounds phospho-siloxane F and phospho-siloxane G, which comprises:

10. Use of the xanthone compound of claim 1 in the preparation of agricultural antibacterial agents and/or antioxidant drugs.