An isoflavone compound, a preparation method and application thereof, a derivative of the isoflavone compound and application thereof, and a pharmaceutical composition

Abstract

The application provides an isoflavone compound and a preparation method and application thereof, a derivative of the isoflavone compound and application thereof, and a pharmaceutical composition, and relates to the technical field of medicines.The isoflavone compound and the derivative thereof provided by the application have a significant inhibitory effect on various crop pathogenic fungi, provide a new idea and technical support for preparing agricultural antibacterial agents, and have important application value.As shown in test results of examples, the minimum inhibitory concentration (MIC) of the isoflavone compound provided by the application on Physalospora piricola, Alternaria alternata and Cercospora arachidicola is 6.25 mu g / mL, 12.5 mu g / mL and 12.5 mu g / mL respectively, and the isoflavone compound provided by the application has obvious inhibitory effects on Physalospora piricola, Alternaria alternata and Cercospora arachidicola.

Description

Technical Field

[0001] This invention relates to the field of medicine, specifically to an isoflavone compound and its preparation method and application, derivatives of the isoflavone compound and their application, and pharmaceutical compositions. Background Technology

[0002] Plant pathogens (also called pathogenic microorganisms) are infectious microorganisms or biological pathogenic vectors that, after invading plants, can cause disease, including bacteria, fungi, and viruses. Fungi are the main plant pathogenic microorganisms, interfering with the normal physiological and biochemical functions of plants, causing plant diseases, and reducing crop yields. *Rhizoctonia solani* (apple leaf mold) is an example. Botryosphaeria berengeriana This fungus primarily infects fruits such as apples and pears, causing them to rot and oozing a brown, sour-smelling, sticky sap. With the increasing planting area of ​​high-quality but susceptible varieties, the large-scale occurrence of rotten fruit has become a significant problem in fruit production. Strawberry black spot disease is caused by *Black Spot Fungus* (*Strawberry Black Spot Fungus*). Colletotrichum acutatum Caused by *S. simmonds*, this disease is widespread in strawberry-growing regions worldwide. Under hot and humid conditions, its incidence increases significantly, typically leading to yield reductions of 25-30%, severely impacting strawberry yield and quality. Strawberry black spot has become the third largest disease hindering the development of China's strawberry industry, after strawberry gray mold and strawberry powdery mildew. Peanut leaf spot is caused by *S. simmonds* fungus (…). Cladosporium personatatum These pathogens, primarily affecting leaves, reduce photosynthetic efficiency, leading to premature leaf drop and thus impacting nutrient accumulation. Peanut leaf spot disease typically causes yield reductions of 5-15%, but in severe cases, reductions can exceed 30%. The damage caused to crops by these three pathogenic fungi results in significant economic losses; therefore, the development of green, low-toxicity, safe, and effective antifungal agents for crop pathogens is urgently needed.

[0003] Fine-leaved rock astragalus ( Corethrodendron scoparium As a leguminous plant, *Astragalus membranaceus* is an excellent sand-fixing plant. Its tender branches and leaves are high-quality fodder, its wood is durable and long-lasting fuel, its flowers are an excellent source of nectar, and its seeds are high-quality concentrated feed and oilseeds. Currently, there are no literature reports on the research of dihydroisoflavone compounds with antifungal activity against crop pathogens extracted from *Astragalus membranaceus*. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide an isoflavone compound, its preparation method and application, derivatives of the isoflavone compound and their applications, and pharmaceutical compositions. The isoflavone compound provided by this invention has high antibacterial activity against pathogenic fungi of crops.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides an isoflavone compound having the structure shown in Formula I: Formula I.

[0006] This invention also provides a method for preparing the isoflavone compounds described in the above technical solution, comprising the following steps: (1) Extracting Astragalus membranaceus from dwarf spp. using a first low-grade alcohol aqueous solution to obtain an alcohol extract; the volume fraction of the low-grade alcohol in the first low-grade alcohol aqueous solution is 80~100%; (2) The alcohol extract is extracted using a mixture of organic extractant and water, and the resulting organic extract is concentrated to obtain an organic extract extract paste; the organic extractant includes ethyl acetate and / or petroleum ether, and the volume ratio of organic extractant to water in the mixture is 1:0.8~1.2. (3) The organic extract is subjected to first silica gel column chromatography to obtain component Fr.8; the elution method of the first silica gel column chromatography is first gradient elution; the eluent used in the first gradient elution includes petroleum ether-ethyl acetate mixed solution and dichloromethane-methanol mixed solution in sequence, wherein the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate mixed solution is 98:2~50:50, and the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixed solution is 100:0~0:100; (4) The component Fr.8 was subjected to a second silica gel column chromatography to obtain component Fr.8.4; the elution method of the second silica gel column chromatography was a second gradient elution, and the eluent used in the second gradient elution included dichloromethane-methanol, wherein the volume ratio of dichloromethane to methanol in the dichloromethane-methanol was 100:0~0:100; (5) The component Fr.8.4 was subjected to third silica gel column chromatography to obtain component Fr.8.4.4; the elution method of the third silica gel column chromatography was third gradient elution, and the eluent used in the third gradient elution included a dichloromethane-methanol mixed solution, wherein the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixed solution was 20:1 to 10:1; (6) The component Fr.8.4.4 is separated by a reverse phase separation column to obtain the isoflavone compound; the eluent used for the reverse phase separation column includes an aqueous solution of a second lower alcohol, wherein the volume fraction of the lower alcohol in the aqueous solution of the second lower alcohol is 80~100%.

[0007] Preferably, the alcohol extraction includes soaking extraction; the temperature of the alcohol extraction is 15~40℃, the number of alcohol extractions is 2~4 times, and the time for each alcohol extraction is 1~5 days; The dry weight of the nephrite astragalus and the volume ratio of the first low-grade alcohol aqueous solution used for a single alcohol extraction are 1 kg: 2~20 L. The lower alcohols in the first and second aqueous solutions of lower alcohols independently include methanol and / or ethanol.

[0008] Preferably, during the first gradient elution process, the volume ratio of petroleum ether to ethyl acetate is 98:2, 95:5, 90:0, 80:20, 70:30, 60:40 and 50:50, respectively, and the volume ratio of dichloromethane to methanol is 100:0, 90:10, 80:20 and 0:100, respectively.

[0009] Preferably, the volume ratio of dichloromethane to methanol during the second gradient elution process is 100:0, 70:30, 50:50, 30:70 and 0:100, respectively.

[0010] Preferably, the volume ratio of dichloromethane to methanol in the third gradient elution process is 20:1 and 10:1, respectively.

[0011] The present invention also provides derivatives of the isoflavone compounds described in the above technical solutions, wherein the derivatives include pharmaceutically acceptable salts.

[0012] The present invention also provides the application of the isoflavone compounds or derivatives of the isoflavone compounds described in the above-mentioned technical solutions in the preparation of pharmaceuticals.

[0013] Preferably, the drug includes an antibacterial agent, which includes an agricultural antibacterial agent.

[0014] The present invention also provides a pharmaceutical composition according to the above-mentioned technical solution, comprising an active component and pharmaceutically acceptable excipients, wherein the active component comprises isoflavone compounds and / or derivatives of isoflavone compounds described in the above-mentioned technical solution.

[0015] The isoflavone compounds and their derivatives with the structure shown in Formula I provided by this invention have significant inhibitory effects on various crop pathogenic fungi, especially on *Rhizoctonia solani*, *Black Spot Fungus*, and *Leuciscus chinensis*. This provides a new approach and technical support for the preparation of agricultural antifungal agents and has significant application value. As shown in the test results of the examples, the minimum inhibitory concentrations (MICs) of the isoflavone compounds provided by this invention against *Rhizoctonia solani*, *Black Spot Fungus*, and *Leuciscus chinensis* are 6.25 μg / mL, 12.5 μg / mL, and 12.5 μg / mL, respectively.

[0016] This invention provides a method for preparing isoflavone compounds using Astragalus membranaceus dentata as the raw material, through alcohol extraction, extraction, silica gel column chromatography, and reversed-phase chromatography. The preparation method provided by this invention is simple, easy to operate, has low production costs, is environmentally friendly, and suitable for industrial production. Attached Figure Description

[0017] Figure 1 The ultraviolet spectrum of the isoflavone compound (GCS114) prepared in Example 1; Figure 2 The infrared spectrum of GCS114 prepared in Example 1; Figure 3 The nuclear magnetic resonance H spectrum (500 MHz, CDCl3) of GCS114 prepared for Example 1. Figure 4 The nuclear magnetic resonance C-spectrum and DEPT spectrum (125 MHz, CDCl3) of GCS114 prepared for Example 1. Figure 5 HMBC spectrum of GCS114 prepared in Example 1 (500 / 125 MHz, CDCl3); Figure 6 The HRESIMS spectrum of GCS114 prepared in Example 1. Detailed Implementation

[0018] This invention provides an isoflavone compound having the structure shown in Formula I: Formula I.

[0019] The isoflavone compounds with the structure shown in Formula I provided by this invention exhibit significant inhibitory effects against various crop pathogenic fungi, particularly against *Rhizoctonia solani*, *Black Spot Fungus*, and *Leuciscus chinensis*. This provides a new approach and technical support for the preparation of agricultural antifungal agents and has significant application value. As shown in the test results of the examples, the minimum inhibitory concentrations (MICs) of the isoflavone compounds provided by this invention against *Rhizoctonia solani*, *Black Spot Fungus*, and *Leuciscus chinensis* are 6.25 μg / mL, 12.5 μg / mL, and 12.5 μg / mL, respectively.

[0020] This invention also provides a method for preparing the isoflavone compounds described in the above technical solution, comprising the following steps: (1) Extracting Astragalus membranaceus from dwarf spp. using a first low-grade alcohol aqueous solution to obtain an alcohol extract; the volume fraction of the low-grade alcohol in the first low-grade alcohol aqueous solution is 80~100%; (2) The alcohol extract is extracted using a mixture of organic extractant and water, and the resulting organic extract is concentrated to obtain an organic extract extract paste; the organic extractant includes ethyl acetate and / or petroleum ether, and the volume ratio of organic extractant to water in the mixture is 1:0.8~1.2. (3) The organic extract is subjected to first silica gel column chromatography to obtain component Fr.8; the elution method of the first silica gel column chromatography is first gradient elution; the eluent used in the first gradient elution includes petroleum ether-ethyl acetate mixed solution and dichloromethane-methanol mixed solution in sequence, wherein the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate mixed solution is 98:2~50:50, and the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixed solution is 100:0~0:100; (4) The component Fr.8 was subjected to a second silica gel column chromatography to obtain component Fr.8.4; the elution method of the second silica gel column chromatography was a second gradient elution, and the eluent used in the second gradient elution included dichloromethane-methanol, wherein the volume ratio of dichloromethane to methanol in the dichloromethane-methanol was 100:0~0:100; (5) The component Fr.8.4 was subjected to third silica gel column chromatography to obtain component Fr.8.4.4; the elution method of the third silica gel column chromatography was third gradient elution, and the eluent used in the third gradient elution included a dichloromethane-methanol mixed solution, wherein the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixed solution was 20:1 to 10:1; (6) The component Fr.8.4.4 is separated by a reverse phase separation column to obtain the isoflavone compound; the eluent used for the reverse phase separation column includes an aqueous solution of a second lower alcohol, wherein the volume fraction of the lower alcohol in the aqueous solution of the second lower alcohol is 80~100%.

[0021] Unless otherwise specified, the materials and equipment used in this invention are all commercially available products in the field.

[0022] This invention utilizes a first low-grade alcohol aqueous solution to extract Astragalus membranaceus from dwarf rock to obtain an alcohol extract; the volume fraction of the low-grade alcohol in the first low-grade alcohol aqueous solution is 80~100%.

[0023] In this invention, the *Astragalus membranaceus* twig is preferably the rhizome of *Astragalus membranaceus* twig, which is preferably dried and cut into segments before use to obtain *Astragalus membranaceus* segments. In this invention, the drying temperature is preferably 25-37℃, and in specific embodiments, it can be 25℃, 28℃, 30℃, 32℃, 35℃, or 37℃. This invention does not have a specific limitation on the drying time; drying to a constant weight is sufficient. In this invention, the length of the *Astragalus membranaceus* segments is preferably 1-2 cm, and in specific embodiments, it can be 1 cm, 1.5 cm, or 2 cm.

[0024] In this invention, the volume fraction of the lower alcohol in the first lower alcohol aqueous solution is 80-100%, and in specific embodiments it can be 80%, 82%, 85%, 88%, 90%, 92%, 95%, 98% or 100%; the lower alcohol preferably includes methanol and / or ethanol.

[0025] In this invention, the preferred ratio of the dry weight of the nephrite astragalus to the volume of the first low-grade alcohol aqueous solution used for a single alcohol extraction is 1 kg: 2~20 L. In specific embodiments, it can be 1 kg: 2 L, 1 kg: 5 L, 1 kg: 8 L, 1 kg: 10 L, 1 kg: 12 L, 1 kg: 15 L, 1 kg: 18 L, or 1 kg: 20 L.

[0026] In this invention, the alcohol extraction preferably includes soaking extraction; the temperature of the alcohol extraction is preferably 15~40℃, and in specific embodiments it can be 15℃, 20℃, 25℃, 30℃, 35℃ or 40℃; the number of alcohol extractions is preferably 2~4 times, more preferably 3 times; the time for a single alcohol extraction is preferably 1~5 days, and in specific embodiments it can be 1 day, 1.5 days, 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days or 5 days.

[0027] Following the alcohol extraction, the present invention preferably further includes: performing solid-liquid separation on the alcohol extraction system obtained from the alcohol extraction, and concentrating the resulting alcohol extract to obtain the alcohol extract. The present invention does not have specific limitations on the concentration; any concentration method well-known to those skilled in the art that can remove the solvent can be used, such as vacuum concentration.

[0028] After obtaining the alcohol extract, the present invention uses a mixed extractant of organic extractant and water to extract the alcohol extract, and concentrates the obtained organic extract phase to obtain an organic extract extract paste. In the present invention, the organic extractant preferably includes ethyl acetate and / or petroleum ether, more preferably ethyl acetate. In the present invention, the volume ratio of organic extractant to water in the mixed extractant is preferably 1:0.8~1.2, and in specific embodiments it can be 1:0.8, 1:0.9, 1:1, 1:1.1 or 1:1.2. The present invention does not have a special limitation on the concentration, and any concentration method well known to those skilled in the art that can remove the solvent can be used, such as vacuum concentration.

[0029] After obtaining the organic extract phase extract, the present invention performs a first silica gel column chromatography on the organic extract phase extract to obtain component Fr.8. In the present invention, the elution method of the first silica gel column chromatography is a first gradient elution; the eluent used in the first gradient elution includes, in sequence, a petroleum ether-ethyl acetate mixed solution and a dichloromethane-methanol mixed solution; the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate mixed solution is 98:2~50:50, and in specific embodiments, the volume ratio can be 98:2, 95:5, 90:0, 80:20, 70:30, 60:40 and 50:50; the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixed solution is 100:0~0:100, and in specific embodiments, the volume ratio can be 100:0, 90:10, 80:20 and 0:100; the particle size of the silica gel used in the first silica gel column chromatography is preferably 200~300 mesh. In this invention, 11 components are obtained during the first silica gel column chromatography process, which are sequentially denoted as component Fr.1, component Fr.2, component Fr.3, component Fr.4, component Fr.5, component Fr.6, component Fr.7, component Fr.8, component Fr.9, component Fr.10 and component Fr.11.

[0030] After obtaining component Fr.8, the present invention performs a second silica gel column chromatography to obtain component Fr.8.4. In the present invention, the elution method of the second silica gel column chromatography is a second gradient elution, and the eluent used in the second gradient elution includes dichloromethane-methanol, wherein the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixture is 100:0 to 0:100. In specific embodiments, the volume ratio can be 100:0, 70:30, 50:50, 30:70, and 0:100, respectively. The preferred particle size of the silica gel used in the second silica gel column chromatography is 200-300 mesh. In the present invention, six components are obtained during the second silica gel column chromatography, which are sequentially designated as component Fr.8.1, component Fr.8.2, component Fr.8.3, component Fr.8.4, component Fr.8.5, and component Fr.8.6.

[0031] After obtaining component Fr.8.4, the present invention performs third silica gel column chromatography on component Fr.8.4.4 to obtain component Fr.8.4.4. In the present invention, the elution method of the third silica gel column chromatography is third gradient elution, and the eluent used in the third gradient elution includes a dichloromethane-methanol mixed solution, wherein the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixed solution is 20:1 to 10:1, and in specific embodiments the volume ratio can be 20:1 and 10:1 respectively; the particle size of the silica gel used in the third silica gel column chromatography is preferably 200-300 mesh. In the present invention, the third silica gel column chromatography yields four components, which are denoted as component Fr.8.4.1, component Fr.8.4.2, component Fr.8.4.3, and component Fr.8.4.4 respectively.

[0032] After obtaining component Fr.8.4.4, the present invention performs reversed-phase chromatography to separate component Fr.8.4.4 to obtain the isoflavone compounds. In the present invention, the eluent used for the reversed-phase chromatography separation comprises an aqueous solution of a second lower alcohol, wherein the volume fraction of the lower alcohol in the second lower alcohol aqueous solution is 80-100%, and in specific embodiments it can be 80%, 82%, 85%, 88%, 90%, 92%, 95%, 98%, or 100%; the lower alcohol preferably includes methanol and / or ethanol. In the present invention, the chromatographic column used for the reversed-phase chromatography separation preferably includes an RP18 column or a 5C column. 18 -AR-II chromatographic column.

[0033] This invention uses Astragalus membranaceus dentata as the raw material, and prepares isoflavone compounds through alcohol extraction, extraction, silica gel column chromatography, and reversed-phase chromatography. The preparation method provided by this invention is simple, easy to operate, has low production cost, is environmentally friendly, and suitable for industrial production.

[0034] The present invention also provides derivatives of the isoflavone compounds described in the above-described technical solutions, wherein the derivatives comprise pharmaceutically acceptable salts. In the present invention, the pharmaceutically acceptable salts preferably comprise organic acid salts or metal salts; the organic acid salts preferably comprise acetate, citrate, or oxalate; the metal salts preferably comprise sodium or potassium salts. In the present invention, the salt-forming site of the isoflavone compounds preferably comprises a phenolic hydroxyl group.

[0035] In this invention, the metal salt is preferably obtained by reacting isoflavone compounds with a base to form a salt; the base preferably includes NaOH or KOH. In this invention, the organic acid salt is preferably obtained by reacting the metal salt with an organic acid through ion exchange or metathesis reaction; the organic acid preferably includes acetic acid, citric acid, or oxalic acid.

[0036] This invention provides the application of the isoflavone compounds or derivatives of the isoflavone compounds described in the above-described technical solutions in the preparation of pharmaceuticals.

[0037] In this invention, the drug preferably includes an antibacterial drug, the antibacterial drug preferably includes an agricultural antibacterial agent, and the antibacterial agent preferably includes an antifungal agent; the fungus preferably includes *Rhizoctonia solani* (a type of fungus). Botryosphaeria berengeriana ), strawberry black spot fungus ( Colletotrichum acutatum Simmonds and peanut leaf spot fungus ( Cladosporium personatatum One or more of the following.

[0038] The present invention also provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises isoflavone compounds described in the above-described technical solutions and / or derivatives of isoflavone compounds described in the above-described technical solutions.

[0039] The present invention does not have any particular limitation on the pharmaceutically acceptable excipients, and any pharmaceutically acceptable excipients well known to those skilled in the art can be used.

[0040] In this invention, the mass content of the active component in the pharmaceutical composition is preferably 1-99%, and in specific embodiments it can be 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%.

[0041] In this invention, when the pharmaceutical composition is an agricultural antibacterial agent, the pharmaceutically acceptable excipient is a pesticide-acceptable adjuvant. In this invention, the formulation of the agricultural antibacterial agent preferably includes wettable powder, water-dispersible granules, suspension concentrate, or emulsifiable concentrate.

[0042] To further illustrate the present invention, the following detailed descriptions, in conjunction with embodiments, illustrate the isoflavone compounds, their preparation methods and applications, derivatives of isoflavone compounds and their applications, and pharmaceutical compositions provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0043] In the following examples, the particle size of the packing material used in the first, second, and third silica gel column chromatography was 200-300 mesh. The reversed-phase column chromatography used was a 5C column. 18 -AR-II chromatographic column (specification: 10 mm ID×250 mm; manufacturer: COSMOSIL).

[0044] Apple ring spot fungus (ATCC 12557), strawberry black spot fungus (ATCC 90170) and peanut leaf spot fungus (ATCC 18592) were purchased from the China Agricultural Microbial Culture Collection Center (ACCC).

[0045] Example 1 Preparation of isoflavone compounds (denoted as GCS114): S1: The rhizomes of Astragalus membranaceus were dried to constant weight at 25℃ and then cut into 1cm segments. 20kg of the obtained Astragalus membranaceus segments were soaked in 95v / v% methanol aqueous solution at 30℃ for extraction. The extraction was repeated 3 times. The amount of methanol aqueous solution used in each soaking extraction was 40L and the soaking extraction time was 3 days. The extracts were combined and concentrated under reduced pressure at 40℃ until the solvent was removed to obtain 1.3kg of alcohol extract. S2: Extract the alcohol extract with a mixed solvent of ethyl acetate and water (volume ratio 1:1), and concentrate the obtained ethyl acetate phase under reduced pressure at 40°C until the solvent is removed to obtain an ethyl acetate extract. S3: The ethyl acetate phase extract was subjected to first silica gel column chromatography. The first silica gel column chromatography was performed by gradient elution with a petroleum ether-ethyl acetate mixed solution and a dichloromethane-methanol mixed solution to obtain 11 fractions, denoted as fractions Fr.1 to Fr.11. The 8th fraction (fraction Fr.8) was collected. The specific gradient elution conditions were as follows: the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate mixed solution was 98:2, 95:5, 90:0, 80:20, 70:30, 60:40 and 50:50, respectively; the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixed solution was 100:0, 90:10, 80:20 and 0:100, respectively. S4: Perform a second silica gel column chromatography on fraction Fr.8; the second silica gel column chromatography uses a dichloromethane-methanol mixed solvent as the eluent for gradient elution to obtain 6 fractions (denoted as fractions Fr.8.1~Fr.8.6), and collect the 4th fraction (fraction Fr.8.4); the volume ratio of dichloromethane to methanol during the gradient elution process is 100:0, 70:30, 50:50, 30:70 and 0:100 respectively.

[0046] S5: Perform third silica gel column chromatography on fraction Fr.8.4; the third silica gel column chromatography is performed by gradient elution using a dichloromethane-methanol mixed solvent with a volume ratio of 20:1 and 10:1 respectively as the eluent to obtain 6 fractions (denoted as fractions Fr.8.4.1~Fr.8.4.6), and collect the fourth fraction (fraction Fr.8.4.4). S6: The fraction Fr.8.4.4 was separated by reversed-phase chromatography using a 90 v / v% methanol aqueous solution as the eluent to obtain isoflavones (6.7 mg).

[0047] Results analysis: The GCS114 prepared in Example 1 was subjected to mass spectrometry, infrared spectroscopy, ultraviolet spectroscopy, and NMR analysis. The results are shown in Table 1 and 2. Figures 1-6 ,in, Figure 1 The image shows the ultraviolet spectrum of GCS114. Figure 2 The infrared spectrum of GCS114 is shown below. Figure 3 The nuclear magnetic resonance H spectrum of GCS114 (500 MHz, CDCl3). Figure 4 The nuclear magnetic resonance C-spectrum and DEPT spectrum of GCS114 (125 MHz, CDCl3, from top to bottom: DEPT-135° spectrum, DEPT-90° spectrum and C-spectrum). Figure 5 The HMBC spectrum of GCS114 (500 / 125 MHz, CDCl3). Figure 6 The HRESIMS spectrum of GCS114 is shown below. The structural characterization results of GCS114 are as follows: Molecular formula: C 21 H 20 O6; ESIMS (pos.): m / z 391 [M + Na] + HRESIMS (pos.): m / z 391.1152 [M + Na] + (391.1152 calcd for C 21 H 20 O6Na); UV (MeOH) λ max 204, 264, 335 nm; IR (KBr) ν max :3436, 2927, 1650, 1621, 1507, 1422, 1364, 1308, 1201, 1167, 1127, 1035 cm -1 Based on the results of mass spectrometry, ultraviolet, infrared, and nuclear magnetic resonance analysis, the compound GCS114 prepared in Example 1 has the following structure: .

[0048] Table 1. Nuclear magnetic resonance spectroscopy data (CDCl3)

[0049] This indicates that chemical shift assignments are interchangeable.

[0050] Test Example 1 To investigate the antifungal effect of GCS114 prepared in the examples, three crop pathogenic fungi, namely, *Rhizoctonia solani*, *Black Spot Fungus*, and *Leuciscus chinensis*, were selected, and the antifungal activity of GCS114 was determined by the two-fold dilution method.

[0051] (1) Activation of pathogenic fungi Preparation of potato dextrose agar (PDA) medium: Take 5g potato extract powder, 15g anhydrous glucose and 25g agar and add them to 1L distilled water. Put them in an autoclave and sterilize at 121℃ for 20min. After sterilization, put the PDA medium into a clean bench, pour it into plates and let it cool for later use.

[0052] The pathogenic fungi preserved in glycerol tubes were poured onto PDA medium and incubated at 28°C for 3-4 days.

[0053] (2) Preparation of seed solution Preparation of potato glucose broth (PDB) medium: Weigh 25g of solid PDB medium and heat it to dissolve in 1000mL of distilled water.

[0054] Sterilization of tools: Place the prepared culture medium and inoculation needle into an autoclave and sterilize at 121°C for 20 minutes. After sterilization, place them on a sterile operating table and sterilize with ultraviolet light for 30 minutes. Let them cool before use.

[0055] The pathogenic fungi in PDA medium were inoculated into PDB medium using an inoculation needle and cultured at 28°C and 150 rpm for 3-4 days.

[0056] (3) Antibacterial activity test Prepare PDB medium: Weigh 25g of PDB medium solid and heat it to dissolve in 1000mL of distilled water.

[0057] Sterilization of tools: Place the prepared culture medium, matching pipette tips, and chamber into an autoclave and sterilize at 121°C for 20 minutes. Sterilize the 96-well plate with ultraviolet light in a clean bench or biosafety cabinet for 30 minutes.

[0058] Preparation of bacterial culture: Take the cultured bacterial culture and mix it with the culture medium at a volume ratio of 1:100.

[0059] Prepare sample solutions. The concentration of the sample stock solution should not be lower than the maximum assay concentration of 100 μg / mL. The working solution concentrations are set as follows: 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.13 μg / mL, 1.56 μg / mL, and 0.78 μg / mL. The specific procedure is as follows: Dissolve the sample to be tested to a stock solution concentration of 4 mg / mL using DMSO (dimethyl sulfoxide) as the solvent. Add 195 μL of bacterial culture to wells A1-A12 of a 96-well plate, and add 100 μL of bacterial culture to each of the remaining wells. Add 5 μL of sample stock solution to wells A1-A12 (at this point, the concentration in wells A1-A12 is 100 μg / mL) and mix well. Then, aspirate 100 μL from wells A1-A12 and add it to wells B2-B12, and mix well. Next, 100 μL of each sample was aspirated from wells B1-B12 and added to wells C1-C12, then mixed thoroughly. This process was repeated until wells H1-H12, at which point 100 μL of each sample was aspirated from H1-H12 and added to the waste solution. Ketoconazole was added to well A1 as a positive control, and DMSO was added to well A12 as a negative control. At this point, the concentrations of the sample solutions in the 96-well plate, from left to right, ranged from 100 to 0.78 μg / mL. The plate was incubated at 28°C for 72 h. The wells of the 96-well plate were numbered A1-A12, B1-B12, C1-C12, D1-D12, E1-E12, F1-F12, G1-G12, and H1-H12. The results are shown in Table 2.

[0060] The test results are meaningful when bacteria grow significantly in the negative control wells (i.e., wells without sample solution).

[0061] Table 2. MIC (Minimum Inhibitory Activity) of GCS114 against Crop Pathogenic Fungi

[0062] As can be seen from Table 2, the compound GCS114 prepared in Example 1 of this invention is effective against *Rhizoctonia solani* (…). Botryosphaeria berengeriana ), strawberry black spot fungus ( Colletotrichum acutatum Simmonds and peanut leaf spot fungus ( Cladosporium personatatum It has inhibitory activity.

[0063] In summary, the isoflavone compound GCS114 provided by this invention has the potential application value of being further developed into an antifungal agent for crop pathogenic fungi.

[0064] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing isoflavone compounds, characterized in that, Includes the following steps: (1) Extracting Astragalus membranaceus from dwarf rock using a first low-grade alcohol aqueous solution to obtain an alcohol extract; the volume fraction of the low-grade alcohol in the first low-grade alcohol aqueous solution is 80-98%; (2) The alcohol extract is extracted using a mixture of organic extractant and water, and the resulting organic extract is concentrated to obtain an organic extract extract paste; the organic extractant is selected from ethyl acetate and / or petroleum ether, and the volume ratio of organic extractant to water in the mixture is 1:0.8~1.2; (3) The organic extract is subjected to first silica gel column chromatography to obtain component Fr.8; the elution method of the first silica gel column chromatography is first gradient elution; the eluent used in the first gradient elution is selected from petroleum ether-ethyl acetate mixed solution and dichloromethane-methanol mixed solution in sequence, wherein the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate mixed solution is 98:2~50:50, and the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixed solution is 100:0~0:100; (4) The component Fr.8 was subjected to a second silica gel column chromatography to obtain component Fr.8.4; the elution method of the second silica gel column chromatography was a second gradient elution, and the eluent used in the second gradient elution was selected from dichloromethane-methanol, wherein the volume ratio of dichloromethane to methanol in the dichloromethane-methanol was 100:0~0:100; (5) The component Fr.8.4 was subjected to third silica gel column chromatography to obtain component Fr.8.4.4; the elution method of the third silica gel column chromatography was third gradient elution, and the eluent used in the third gradient elution was selected from dichloromethane-methanol mixed solution, and the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixed solution was 20:1~10:1; (6) The component Fr.8.4.4 is separated by a reverse phase separation column to obtain isoflavone compounds; the eluent used in the reverse phase separation column is selected from an aqueous solution of a second lower alcohol, wherein the volume fraction of the lower alcohol in the aqueous solution of the second lower alcohol is 80-98%; The lower alcohols in the first and second aqueous solutions of lower alcohols are independently selected from methanol and / or ethanol; The isoflavone compounds have the structure shown in Formula I: Equation I.

2. The preparation method according to claim 1, characterized in that, The alcohol extraction is selected from soaking extraction; the temperature of the alcohol extraction is 15~40℃, the number of alcohol extractions is 2~4 times, and the time of a single alcohol extraction is 1~5 days; The dry weight of the nephrite astragalus and the volume ratio of the first low-grade alcohol aqueous solution used for a single alcohol extraction are 1 kg: 2~20 L.

3. The preparation method according to claim 1, characterized in that, During the first gradient elution process, the volume ratios of dichloromethane and methanol were 100:0, 90:10, 80:20, and 0:100, respectively.

4. The preparation method according to claim 1, characterized in that, During the second gradient elution process, the volume ratios of dichloromethane and methanol were 100:0, 70:30, 50:50, 30:70, and 0:100, respectively.

5. The preparation method according to claim 1, characterized in that, In the third gradient elution process, the volume ratios of dichloromethane and methanol are 20:1 and 10:1, respectively.

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