Application of a chloro-p-benzoquinone compound

Abstract

The present invention relates to microbial metabolites and their application technology in the research field of agricultural fungal control drugs, specifically an application of a chloro-p-benzoquinone compound. The application of a chloro-p-benzoquinone compound as a new control drug for agricultural fungi. The chloro-p-benzoquinone compound is 2-chloro-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione, as shown below #imgabs0# The chloro-p-benzoquinone compound involved in the present invention (as shown in structural formula I) can be used as a new drug ingredient or lead compound for resisting agricultural fungi. The chloro-p-benzoquinone compound involved in the present invention has significant anti-agricultural fungal activity, and its mechanism of action can be further explored in order to develop it into a new pesticide or its lead compound.

Description

Technical Field

[0001] The present invention relates to microbial metabolites and their application in the research field of agricultural fungal control drugs, in particular to the application of chloro-p-benzoquinone compounds. Background Art

[0002] Tomato wilt and wheat head blight are among the most significant diseases that harm crop quality and yield. Currently, chemical pesticides are the primary control method. However, their long-term and extensive use can lead to increased resistance in pathogens and environmental pollution. Microbial secondary metabolites, with their high efficiency, low toxicity, low residue levels, and widespread availability from natural, readily available materials, hold great promise for sustainable agricultural production. 2-chloro-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione, isolated from Xylaria sp., has been shown to exhibit antimalarial activity and cytotoxicity against African green monkey kidney fibroblasts (Vero cells) (J. Nat. Prod., 2007, 70, 1620–1623). However, there are no reports of its inhibitory activity against agricultural pathogens. Summary of the Invention

[0003] The purpose of the present invention is to provide an application of chloro-p-benzoquinone compounds.

[0004] To achieve the above object, the technical solution adopted by the present invention is:

[0005] The invention discloses an application of chloro-p-benzoquinone compounds, and an application of chloro-p-benzoquinone compounds as new control drugs for agricultural fungal diseases.

[0006] The chloro-p-benzoquinone compound is 2-chloro-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione, as shown in Formula I

[0007]

[0008] The chloro-p-benzoquinone compounds are used as novel control drugs for agricultural fungal diseases such as Colletotrichum gloeosporioides, Fusarium wilt, Gibberellic acid, or Fusarium oxysporum.

[0009] The chloro-p-benzoquinone compounds are used as novel control drugs for agricultural fungal diseases such as Colletotrichum gloeosporioides or Fusarium wilt.

[0010] The advantages of the present invention are:

[0011] 1. The chloro-p-benzoquinone compounds (as shown in structural formula I) involved in the present invention can be used as new drug ingredients or lead compounds for resisting agricultural fungal diseases.

[0012] 2. The chloro-p-benzoquinone compounds involved in the present invention can be produced by microbial fermentation, which is relatively controllable, rapid, efficient and environmentally friendly;

[0013] 3. The present invention utilizes microbial fermentation to prepare antifungal compounds without causing pollution to the environment.

[0014] 4. The chloro-p-benzoquinone compounds involved in the present invention have significant anti-agricultural fungal activity, and their mechanism of action can be further explored with the goal of developing them into new pesticides or lead compounds. Experiments have shown that the minimum inhibitory concentrations (MIC values) of the compounds against Colletotrichum gloeosporioides, Fusarium wilt, Gibberellic acid, and Fusarium thaliana are 0.5, 2.0, 2.0, and 4.0 μg / mL, respectively, which are superior to the positive control amphotericin B (MIC values ​​of 2.0, 4.0, 2.0, and 4.0 μg / mL, respectively). In addition, in the mycelial growth inhibition rate assay, the compound showed a significant inhibitory effect on mycelial growth against Fusarium wilt and Gibberellic acid at 1.0 μg / mL, and the inhibition efficiency increased with increasing compound dosage. Therefore, the compounds can be used as new drug ingredients or lead compounds against agricultural fungal diseases. DETAILED DESCRIPTION

[0015] In order to clarify the understanding of the features of the present invention, the present invention is further described below in conjunction with some non-limiting embodiments.

[0016] Example 1:

[0017] The chloro-p-benzoquinone compound is 2-chloro-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione, as shown in Formula I.

[0018]

[0019] The preparation process of the chloro-p-benzoquinone compound 2-chloro-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione can be referred to the description in J. Nat. Prod., 2007, 70, 1620–1623.

[0020] The compound has the following physicochemical and spectroscopic properties:

[0021] Yellow needle-shaped crystals; chemical formula C8H7ClO3; melting point 133–135°C; UV (MeOH) λmax (logε)273(4.16); high-resolution ESI mass spectrum m / z 187.0156[M+H] + (calcd for C8H8ClO3,188.0162). 1 H-NMR and 13 C-NMR is shown in Table 1.

[0022] Table 1. H NMR (500 MHz) and C NMR (125 MHz) data (solvent CDCl3)

[0023]

[0024]

[0025] Example 2: Agricultural fungus inhibitory activity test

[0026] 1. The antifungal activity of the compounds of Formula I against agricultural pathogens was determined using the minimum inhibitory concentration (MIC) method. Five fungal strains of plant pathogens were selected for antifungal activity testing: Ceratobasidium cornigerum, Colletotrichum gloeosporioides, Fusarim graminearum, Fusarium oxysporum, and Fusarium proliferatum.

[0027] 1) Minimum inhibitory concentration activity test:

[0028] The minimum inhibitory concentration (MIC) is the lowest drug concentration that can inhibit microbial growth in vitro. In a 96-well microplate, varying concentrations of drug are added to a suspension of the test bacteria. After incubation and observation, if the indicator bacteria grow in a particular well, the drug concentration in that well is insufficient to inhibit its growth. The liquid in that well becomes turbid, with a significant decrease in light transmittance. Conversely, the liquid in that well becomes clear, with no significant decrease in light transmittance. The lowest sample concentration that completely inhibits the growth of the indicator bacteria in a well is the compound's MIC.

[0029] 2) Preparation of bacterial suspension

[0030] The above-mentioned test fungi were inoculated into Sabouraud medium and cultured at 28°C for 24 hours. Then, 4 mL of sterile 0.85% NaCl solution (containing 0.25% Tween 20) was aspirated to wash the medium and the hyphae were gently scraped off with a glass spatula. An appropriate amount of bacterial suspension was pipetted into a sterile test tube and then adjusted to a McFarland turbidity of 0.5 (equivalent to 1.5×10 8 CFU / mL) and further diluted with 0.85% NaCl solution to 5×10 5 CFU / mL;

[0031] 0.5 McFarland turbidity standard:

[0032] 0.5 mL x 0.048 mol / L BaCl2 (1.175% w / v BaCl2·2H2O) was added to 99.5 mL x 0.18 mol / L (0.36 N) H2SO4 (1% v / v) and stirred continuously to maintain a suspension.

[0033] 3) Sample preparation

[0034] Approximately 1 mg of each test sample (the compound obtained above) and positive control (amphotericin B) was dissolved in approximately 100 μL of DMSO. After thorough mixing, the final concentration was 2560 μg / mL. 50 μL of the sample solution was then pipetted into another centrifuge tube, followed by the addition of 50 μL of DMSO to obtain a sample solution with a concentration reduced by half. Following this method, a total of 11 sets of sample solutions with concentrations reduced by half were obtained (2560, 1280, 640, 320, 160, 80, 40, 20, 10, 5, and 2.5 μg / mL).

[0035] 4) Blank control: Pure DMSO solvent without dissolving the sample to be tested was selected as the blank control.

[0036] 5) MIC determination process

[0037] 5.1) Aseptically add serially diluted sample solutions of varying concentrations to a sterile 96-well plate. Add 5 μL of sample solution to wells 1 through 11. Do not add sample to well 12 as a growth control.

[0038] 5.2) Dilute the indicator bacteria suspension to a McFarland turbidity of 0.5 1000-fold with liquid culture medium. Add 95 μL of the suspension to each well of a 96-well plate, so that the final concentrations in wells 1 to 11 are 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / mL, respectively. Gently shake to mix, seal the 96-well plate, and place it in a 28°C incubator. Incubate for 24 hours before observation.

[0039] 5.3) Measure the absorbance of each well at 600 nm using a microplate reader. The lowest sample concentration that completely inhibits the growth of the indicator bacteria in the well is the MIC of the compound. (Note: The experiment is meaningful only when the indicator bacteria clearly grow in the growth control well. If a single jump well occurs during the experiment, the highest drug concentration that inhibits the growth of the strain should be recorded. If multiple jump wells occur, the result should not be reported and the experiment should be repeated.)

[0040] The experimental results showed that the compound had significant inhibitory activity against Colletotrichum gloeosporioides, Fusarium oxysporum, Fusarium graminearum, and Fusarium proliferatum, with minimum inhibitory concentration (MIC) values ​​of 0.5, 2.0, 2.0, and 4.0 μg / mL, respectively, which were better than the positive control amphotericin B (MIC values ​​of 2.0, 4.0, 2.0, and 4.0 μg / mL, respectively).

[0041] 2. The mycelium growth inhibition rate method was used to evaluate the in vitro anti-tomato wilt fungus F. oxysporum and wheat fusarium graminearum effects of the compound represented by formula I.

[0042] 1) Sample preparation

[0043] Take about 1 mg of the compound represented by Formula I and dissolve it in 1 mL of methanol. After thorough mixing, the final concentration is 1 mg / mL. Pipette 100 μL of the sample solution into another centrifuge tube, and then add 900 μL of methanol to obtain a sample solution with a concentration of 100 μg / mL.

[0044] 2) Blank control: Select pure methanol solvent without dissolving the sample to be tested as the blank control.

[0045] 3) In vitro antifungal activity assay process

[0046] Using aseptic techniques, 200 μL, 500 μL, and 1000 μL of the prepared sample solution were added to 20 mL potato dextrose agar (PDA) plates, achieving final concentrations of 1.0 μg / mL, 2.5 μg / mL, and 5.0 μg / mL, respectively. Fusarium oxysporum and Fusarium graminearum were aseptically inoculated onto the PDA plates. After 48 hours, the mycelial growth inhibition rate was calculated using the following formula:

[0047] Mycelial growth inhibition rate (%) = (B-A) / B×100%

[0048] Where A is the average colony diameter of the added compound, and B is the average colony diameter of the blank control.

[0049] The experimental results showed that after 48 hours, the compound exhibited in vitro inhibition rates of 44.4%, 52.2%, and 85.0% against the tomato wilt pathogen F. oxysporum at 1.0 μg / mL, 2.5 μg / mL, and 5.0 μg / mL, respectively; and 66.8%, 86.4%, and 99.9% against the wheat head blight pathogen F. graminearum, respectively. These experimental results demonstrate that the compounds of the present invention have strong inhibitory effects against agricultural fungi such as Colletotrichum gloeosporioides, Fusarium oxysporum, and Head blight, and can be used to prepare novel anti-agricultural fungal drugs or lead compounds.

Claims

1. A use of a chloro-p-benzoquinone compound in preventing and controlling agricultural fungal diseases, characterized in that: The chloro-p-benzoquinone compound is a compound represented by formula I, Formula I The agricultural pathogenic fungi are Colletotrichum gloeosporioides, Fusarium wilt, Gibberellic acid fusarium, or Fusarium oxysporum.

2. A use of a chloro-p-benzoquinone compound as a drug for preventing and treating agricultural fungal diseases, characterized in that: The chloro-p-benzoquinone compound is a compound represented by formula I, Formula I The agricultural pathogenic fungi are Colletotrichum gloeosporioides, Fusarium wilt, Gibberellic acid fusarium, or Fusarium oxysporum.

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