Preparation method of a guaiane sesquiterpene compound and application thereof in resisting plant pathogenic fungi

Abstract

The application belongs to the field of antibacterial activity research, and discloses a preparation method of guaiane sesquiterpenes and application thereof in resisting plant pathogenic fungi. Firstly, leaf blades of Artemisia stolonifera are dried, extracted, concentrated, and then a crude fraction is obtained through extraction, and then the crude fraction is further purified through normal silica gel, dextran gel and reversed-phase macroporous resin column chromatography, and finally three guaiane sesquiterpenes are obtained through liquid phase preparation. The compounds can effectively inhibit the growth of Sclerotinia sclerotiorum, Cochliobolus sativus and Verticillium dahliae, and the minimum inhibitory concentration (MIC) of moxartenolide is 6.25, 12.5 and 12.5 μg / mL respectively, the minimum inhibitory concentration (MIC) of artemdubolide C is 12.5, 12.5 and 12.5 μg / mL respectively, and the minimum inhibitory concentration (MIC) of artemvulactone F is 12.5, 12.5 and 12.5 μg / mL respectively, and the compounds have great application potential in resisting plant pathogenic fungi.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial activity research technology, specifically relating to a method for preparing guaiacane-type sesquiterpenoid compounds and their application in combating plant pathogenic fungi. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Artemisia stolonifera, a plant belonging to the Artemisia genus of the Asteraceae family, is mainly distributed in Heilongjiang, Jilin, Liaoning, Inner Mongolia, Shandong, and Hubei provinces. It grows mostly in forest edges, sparse forests, roadsides, wastelands, and valleys in low-altitude humid areas. Through systematic herbal research, it is believed that the medicinal material "Nine Ox Grass" recorded in the Song Dynasty's "Illustrated Materia Medica" and the Ming Dynasty's "Compendium of Materia Medica" originated from Artemisia stolonifera, which is one of the important sources of the ancient Chinese medicinal material Artemisia argyi. Currently, 32 chemical components have been identified from Artemisia stolonifera, including flavonoids, flavonoid glycosides, and phenolic acids, but no monomeric compounds have yet been isolated from Artemisia stolonifera.

[0004] Sclerotinia sclerotiorum, the rot of rapeseed, is a broad-spectrum fungal disease caused by *Sclerotinia sclerotiorum*. It is one of the three major diseases affecting rapeseed in my country, severely reducing its yield and quality. Helminthosporium maydis, the causal agent of corn leaf blight, is a disease caused by *Helminthosporium*, and is one of the important diseases in corn-producing areas, causing serious yield losses. Verticillium dahliae Kleb, the causal agent of potato wilt, is a systemic soil-borne disease caused by *Verticillium dahliae*, severely damaging potato yield and quality. Currently, common chemical agents for controlling three plant pathogenic fungi include carbendazim, sclerotinia sclerotinia, imazalil, thiophanate-methyl, and benomyl. These chemical agents are characterized by broad spectrum and high efficiency and are widely used, but they all have certain hazards. For example, carbendazim residues can cause liver disease and chromosomal aberrations and are toxic to mammals; benomyl can irritate the respiratory system and skin, causing allergies, etc. Moreover, because chemical agents are not selective, they may act on beneficial microorganisms and pollute the environment.

[0005] Therefore, in order to effectively prevent and control fungal infections in crops while reducing harm and environmental pollution, the discovery and development of highly effective, low-toxicity, and low-pollution plant-derived pesticides has become one of the key focuses in the field of research on anti-plant pathogenic fungi. Currently, many guaiacolane-type sesquiterpenoids have been isolated, but their main applications are in anti-tumor treatment. No guaiacolane-type sesquiterpenoids with good inhibitory effects against plant pathogenic fungi have yet been isolated from a large number of natural plants. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a method for preparing guaiac sesquiterpenoid compounds and their application in combating plant pathogenic fungi. These guaiac sesquiterpenoid compounds can effectively inhibit the growth of *Sclerotinia sclerotiorum*, *Helminthosporium maydis*, and *Verticillium dahliae Kleb* at low concentrations, demonstrating significant application potential.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a guaiacane-type sesquiterpene compound selected from compounds having the following structural formula:

[0009]

[0010] A second aspect of the present invention provides a method for preparing guaiacane-type sesquiterpenoid compounds, comprising:

[0011] After washing and drying Artemisia scoparia, it was extracted with 60-80% ethanol by heating. The extract was then concentrated to obtain a crude extract.

[0012] The crude extract was dissolved in water and then extracted sequentially with petroleum ether and ethyl acetate. The ethyl acetate extract was collected and concentrated to obtain a crude extract.

[0013] The crude extract was prepared by column chromatography and high-performance liquid chromatography.

[0014] Through experiments, this invention has found that when the target compound is extracted with petroleum ether first, the crude extract can be pre-purified to improve the purity of the target compound in ethyl acetate.

[0015] A third aspect of the present invention provides the application of the above-mentioned guaiacol-type sesquiterpenoid compounds in resisting plant pathogenic fungi, said plant pathogenic fungi including: Sclerotinia sclerotiorum (rapeseed sclerotium), Helminthosporium maydis (corn leaf spot fungus), and Verticillium dahliae Kleb (potato wilt fungus).

[0016] Beneficial effects of the present invention

[0017] (1) The application of the guaiacolane-type sesquiterpenoid compounds Moxartenolide, Artemdubolide C, and Artemvulactone F provided by this invention in the treatment of three plant pathogenic fungi: *Sclerotinia sclerotiorum*, *Sclerotinia sclerotiorum*, and *Verticillium wiltii*. These guaiacolane-type sesquiterpenoid compounds can inhibit the growth of these three plant pathogenic fungi even at low concentrations. Therefore, these guaiacolane-type sesquiterpenoid compounds have great potential for developing plant-derived pesticides for the control of plant pathogenic fungi.

[0018] (2) The preparation method of the compound provided by the present invention is simple to operate, highly controllable, and has good stability. Attached Figure Description

[0019] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. Exemplary embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0020] Figure 1 For compound 1 1 H-NMR spectrum.

[0021] Figure 2 For compound 1 13 C-NMR spectrum.

[0022] Figure 3 For compound 2 1 H-NMR spectrum.

[0023] Figure 4 For compound 2 13 C-NMR spectrum.

[0024] Figure 5 For compound 3 1 H-NMR spectrum.

[0025] Figure 6 For compound 3 13 C-NMR spectrum. Detailed Implementation

[0026] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0027] A guaiacane-type sesquiterpene compound selected from compounds having the following structural formula:

[0028]

[0029] The minimum inhibitory concentrations (MICs) of the compound Moxartenolide against three plant pathogens (Sclerotiniasclerotiorum for rapeseed, Helminthosporium maydis for maize leaf spot, and Verticillium dahliae Kleb for potato) were 6.25, 12.5, and 12.5 μg / mL, respectively; the MICs of artemdubolide C against artemvulactone F ...

[0030] A method for preparing a guaiacane-type sesquiterpene compound includes the following steps:

[0031] After washing and drying Artemisia scoparia, it was extracted with 60-80% ethanol by heating, and the extract was concentrated to obtain crude extract.

[0032] After the crude extract is fully dissolved in water, it is extracted 6-8 times with petroleum ether and ethyl acetate respectively. The ethyl acetate extract is collected and concentrated to obtain crude extract (i.e., the crude extract aqueous solution is first extracted with petroleum ether multiple times to remove small polar components; then the crude extract aqueous solution is extracted with ethyl acetate multiple times and the ethyl acetate extract is collected).

[0033] The target compound was prepared by column chromatography and high performance liquid chromatography (HPLC) of the crude extract. The HPLC used a reversed-phase C18 column, and the elution system was methanol-water or acetonitrile-water, with a flow rate of 2-3 mL / min.

[0034] In some embodiments, the temperature for the heating extraction is 55-65°C, and the heating extraction time is 8-12 hours.

[0035] Preferably, the heating and extraction is performed 2-4 times.

[0036] In some embodiments, the column chromatography includes one or two of normal-phase silica gel column chromatography, reverse-phase D101 type macroporous resin column chromatography, and gel column chromatography.

[0037] Preferably, the elution system for column chromatography is methanol, dichloromethane-methanol, petroleum ether-ethyl acetate, or methanol-water.

[0038] Preferably, the column chromatography method is as follows: First, the ethyl acetate extract is adsorbed onto 80-100 mesh silica gel at 1-1.5 times its mass, mixed, and then dry-loaded onto a normal-phase silica gel column. Then, a dichloromethane-methanol system is used for elution at gradients of 100:0, 100:1, 70:1, 50:1, 30:1, 10:1, and 0:100. The gradient eluates are collected and combined to obtain 6 fractions Fr.1-6.

[0039] Fr.2 was adsorbed with 1 to 1.5 times its mass of D101 macroporous resin, mixed, and then dry-loaded onto a reverse macroporous resin column. Gradient elution was then performed with 20%, 40%, 60%, 80%, and 100% ethanol solutions. The gradient eluents were collected, concentrated, and combined by TLC to obtain five fractions Fr.4-1 to Fr.4-5.

[0040] Fr.4-4 was loaded onto a normal-phase silica gel column using a dry method with 1 to 1.5 times its mass of 80-100 mesh, and then eluted using a gradient elution system of petroleum ether-ethyl acetate at ratios of 20:1, 10:1, 8:1, 5:1, and 1:1. The eluents from each gradient were collected and combined to obtain four fractions, Fr.4-4-1 to Fr.4-4-4. Fr.4-4-3 was loaded onto a gel column and eluted using a methanol or dichloromethane-methanol = 1:1 elution system. The eluents were collected and combined to obtain two subfractions. The first subfraction was purified by HPLC preparative chromatography to obtain compounds 1-3.

[0041] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are explanations of the present invention and not limitations thereof.

[0042] In the following examples, the concentration of ethanol is a volume concentration.

[0043] Example 1: Preparation of the compound

[0044] (1) After washing and drying Artemisia scoparia, it was extracted with 70% ethanol by heating and the extract was concentrated to obtain crude extract;

[0045] (2) After the crude extract is fully dissolved in water, it is extracted 6 times with petroleum ether and ethyl acetate in a 1:1 volume ratio of crude extract aqueous solution to extractant. The ethyl acetate extracts are combined and concentrated to obtain crude extract.

[0046] (3) First, the crude ethyl acetate extract was adsorbed onto 80-100 mesh silica gel with 1.5 times its mass and mixed. Then, it was dry-loaded onto a normal phase silica gel column. Then, a dichloromethane-methanol system was used for elution at gradients of 100:0, 100:1, 70:1, 50:1, 30:1, 10:1, and 0:100. The gradient eluents of each gradient were collected and combined to obtain 6 components Fr.1-6.

[0047] Fr.2 was adsorbed with 1.5 times its mass of D101 macroporous resin, mixed, and then dry-loaded onto a reverse-flow D101 macroporous resin column. Gradient elution was then performed with 20%, 40%, 60%, 80%, and 100% ethanol solutions. The gradient eluents were collected, concentrated, and combined by TLC to obtain five fractions Fr.4-1 to Fr.4-5.

[0048] Fr.4-4 was dry-loaded onto a normal-phase silica column at 1.5 times its mass, and then eluted using a gradient elution system of petroleum ether-ethyl acetate at ratios of 20:1, 10:1, 8:1, 5:1, and 1:1. The eluents from each gradient were collected and combined to obtain four fractions, Fr.4-4-1 to Fr.4-4-4. Fr.4-4-3 was loaded onto a gel column and eluted using a dichloromethane-methanol elution system at a ratio of 1:1. The eluents were collected and combined to obtain two subfractions. The first subfraction was purified by preparative HPLC (acetonitrile:water = 50:50, flow rate = 3 mL / min) to obtain compounds 1-3.

[0049] Example 2: Compound Structure Identification

[0050] After NMR identification of all compounds, the properties and spectral data of the obtained compounds are as follows:

[0051] Compound 1: moxartenolide, a colorless oil with the molecular formula C1. 20 H 22 O5. 1 H NMR (500MHz, CDCl3)δ H :6.21(1H,d,J=2.3Hz,H-3),3.73(1H,t,J=10.2Hz,H-6),3.34(1H,tt,J=10.4,3.1Hz,H-7),5.02 (1H,td,J=10.6,2.2Hz,H-8),2.75(1H,dd,J=10.7,13.4Hz,H-9a),2.52(1H,dd,J=2.3,13.3Hz,H -9b),5.64(1H,d,J=2.9Hz,H-13a),6.20(1H,d,J=2.9Hz,H-13b),2.48(3H,s,H-14),2.34(3H,br s,H-15),6.24(1H,dq,J=7.4,1.6Hz,H-3′),2.04(3H,dq,J=7.3,1.7Hz,H-4′),1.92(3H,s,H-5′); 13 CNMR (126MHz, CDCl3)δ CThe values ​​were: 133.7 (C-1), 195.2 (C-2), 136.2 (C-3), 169.4 (C-4), 51.7 (C-5), 81.6 (C-6), 55.4 (C-7), 68.8 (C-8), 44.6 (C-9), 145.0 (C-10), 136.3 (C-11), 168.6 (C-12), 122.0 (C-13), 21.5 (C-14), 20.1 (C-15), 166.5 (C-1′), 126.9 (C-2′), 141.2 (C-3′), 16.2 (C-4′), 20.7 (C-5′). Based on these data and comparison with literature, compound 1 was identified as moxartenolide.

[0052] Compound 2: artemdubolide C, a colorless oily substance with the molecular formula C. 20 H 24 O5. 1 H NMR (500MHz, CDCl3): δ H 6.20(1H,s,H-3),3.51(1H,brd,J=10.6Hz,H-5),3.73(1H,dd,J=10.2,7.5Hz,H-6),3.28(1H,tt,J=10.3,6.2H z,H-7),4.94(1H,td,J=10.6,2.2Hz,H-8),2.70(1H,dd,J=10.6,13.1Hz,H-9a),2.40(1H,dd,J=3.2,10.3Hz,H -9b),5.67(1H,d,J=2.9Hz,H-13a),6.21(1H,d,J=3.1Hz,H-13b),2.34(3H,s,H-14),2.45(3H,s,H-15),2.42( 1H,m,H-2′),1.75(1H,m,H-3′a),1.51(1H,m,H-3′b),0.96(1H,t,J=7.4Hz,H-4′),1.21(3H,d,J=7.1Hz,H-5′); 13 C NMR (126MHz, CDCl3): δ C133.7 (C-1), 195.1 (C-2), 136.2 (C-3), 169.5 (C-4), 51.6 (C-5), 81.6 (C-6), 55.3 (C-7), 69.0 (C-8), 44.4 (C-9), 144.8 (C-10), 136.3 (C-11), 168.52 (C-12), 121.9 (C-13), 21.4 (C-14), 20.1 (C-15), 175.6 (C-1′), 144.8 (C-2′), 26.5 (C-3′), 12.0 (C-4′), 17.0 (C-5′). Based on the above data and comparison with literature, compound 2 was identified as artemdubolide C.

[0053] Compound 3: artemvulactone F, a colorless oil with the molecular formula C. 20 H 24 O5. 1 H NMR (500MHz, CDCl3): δ C 6.20(1H,m,H-3),3.50(1H,d,J=10.2Hz,H-5),3.71(1H,t,J=10.2Hz,H-6),3.26(1H,tt,J=3.1,1 0.3Hz,H-7),4.92(1H,td,J=10.6,2.1,H-8),2.71(1H,dd,J=13.4,10.7Hz,H-9a),2.47(1H,d,J=2 .2Hz,H-9b),5.65(2H,d,J=3.0Hz,H-13a),6.22(d,J=3.1Hz,H-13b),2.46(s,H-14),2.33(3H,s,H -15),2.27(1H,m,H-2′a),2.30(1H,m,H-2′b),2.16(1H,m,H-3′),1.01(6H,d,J=6.6Hz,H-4′,5′); 13 C NMR (126MHz, CDCl3): δ H133.7 (C-1), 195.2 (C-2), 136.2 (C-3), 169.4 (C-4), 51.7 (C-5), 81.6 (C-6), 55.2 (C-7), 69.2 (C-8), 44.5 (C-9), 144.8 (C-10), 136.2 (C-11), 168.5 (C-12), 122.0 (C-13), 20.1 (C-14), 21.4 (C-15), 172.0 (C-16), 44.5 (C-17), 25.7 (C-18), 22.6 (C-19), 22.5 (C-20). Based on the above data and comparison with literature, compound 3 was identified as artemvulactone F.

[0054] Example 3 Antibacterial Activity Experiment

[0055] Experimental Methods: Compounds 1-3 obtained in Example 2 and ketoconazole were prepared into a 10 mg / mL test solution using DMSO and stored at 4°C. The wells containing the bacterial solution without the test sample served as blanks. The wells containing the bacterial solution and the test sample served as test wells. The wells containing the bacterial solution and DMSO served as negative controls, and the wells containing the bacterial solution and ketoconazole served as positive controls. The MIC of the samples against two plant pathogenic fungi was determined using the two-fold dilution method, with three replicates for each sample concentration.

[0056] Specific operating steps: Using *Sclerotinia sclerotiorum*, *Sclerotinia serratifolia*, and *Verticillium wiltii* as test strains, 35g of PDB (potato dextrose broth) powder (Aoboxing, Beijing) was added to 1000mL with distilled water. The mixture was dispensed into Erlenmeyer flasks, each containing 200mL of culture medium. After autoclaving at 121℃ and cooling, the three plant pathogenic fungi were inoculated into the PDB medium and cultured at 28℃ with shaking at 160r / min for 3 days. One mL of the cultured fungal solution from the Erlenmeyer flask was diluted 100-fold in 100mL of PDB medium. 198μL of the diluted solution was added to the first well of a 96-well plate, and 100μL of the diluted solution was added to the second through eighth wells. Add 2 μL of sample, blank, negative control, and positive control to each well in the first row, repeating this process three times per well. After thorough mixing, transfer 100 μL of the mixture to the second row, mix thoroughly, and then transfer 100 μL of the mixture to the third row. Continue this process until the final sample concentrations in each row are 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.12 μg / mL, 1.56 μg / mL, and 0.78 μg / mL, respectively. Incubate the prepared 96-well plate at 28°C for 24 hours. Once the negative control group is fully colonized with fungi, record the activity results. The concentration of the sample corresponding to the clear well in the last row is defined as the minimum inhibitory concentration (MIC) of the compound.

[0057] The results obtained using the above method are shown in Table 1.

[0058] Table 1 shows the inhibitory activity of compounds 1–3 against plant pathogenic fungi, where MIC represents the minimum inhibitory concentration.

[0059] Table 1

[0060]

[0061] Therefore, compounds 1-3 can inhibit the growth of the three plant pathogenic fungi (Sclerotinia sclerotiorum, Helminthosporium maydis, and Verticillium dahliae Kleb) at low concentrations.

[0062] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of a guaiacane-type sesquiterpene compound in the resistance to plant pathogenic fungi; wherein the plant pathogenic fungi are: *Sclerotinia sclerotiorum*. Sclerotinia sclerotiorum Corn leaf blight fungus Helminthosporium maydis and potato verticillium wilt Verticillium dahliae Kleb; The guaiacolane-type sesquiterpenoids are selected from compounds having the following structural formulas: ； Compound 1 against Sclerotinia brasiliensis Sclerotinia sclerotiorum Corn leaf blight fungus Helminthosporium maydis and potato verticillium wilt Verticillium dahliae The minimum inhibitory concentrations (MICs) of Kleb were 6.25, 12.5, and 12.5 μg / mL, respectively; compound 2 showed inhibitory effects against *Sclerotinia brasiliensis*. Sclerotinia sclerotiorum Corn leaf blight fungus Helminthosporium maydis and potato verticillium wilt Verticillium dahliae The minimum inhibitory concentrations (MICs) of Kleb were 12.5, 12.5, and 12.5 μg / mL, respectively; compound 3 showed inhibitory effects against *Sclerotinia brasiliensis*. Sclerotinia sclerotiorum Corn leaf blight fungus Helminthosporium maydis and potato verticillium wilt Verticillium dahliae The minimum inhibitory concentrations (MICs) of Kleb were 12.5, 12.5, and 12.5 μg / mL, respectively.

2. The application of the guaiacol-type sesquiterpenoid compound as described in claim 1 in its resistance to plant pathogenic fungi, characterized in that, The method for preparing the guaiacol-type sesquiterpene compound includes: After washing and drying Artemisia scoparia, it was extracted with 60-80% ethanol by heating. The extract was then concentrated to obtain the crude extract. The crude extract was dissolved in water and then extracted sequentially with petroleum ether and ethyl acetate. The ethyl acetate extract was collected and concentrated to obtain a crude extract. The crude extract was prepared by column chromatography and high-performance liquid chromatography.

3. The application of the guaiacol-type sesquiterpenoid compound as described in claim 2 in its resistance to plant pathogenic fungi, characterized in that, The heating and extraction temperature is 55-65℃, and the heating and extraction time is 8-12 h.

4. The application of the guaiacol-type sesquiterpenoid compound as described in claim 2 in its resistance to plant pathogenic fungi, characterized in that, The heating and extraction process is repeated 2-4 times.

5. The application of the guaiacol-type sesquiterpenoid compound as described in claim 2 in its resistance to plant pathogenic fungi, characterized in that, The column chromatography employs one or two of the following: normal-phase silica gel column chromatography, reverse-phase D101 type macroporous resin column chromatography, and gel column chromatography.

6. The application of the guaiacol-type sesquiterpenoid compound as described in claim 2 in its resistance to plant pathogenic fungi, characterized in that, The elution system for the column chromatography is methanol, dichloromethane-methanol, petroleum ether-ethyl acetate, or methanol-water.

7. The application of the guaiacol-type sesquiterpenoid compound as described in claim 2 in its resistance to plant pathogenic fungi, characterized in that, The specific steps of the column chromatography method are as follows: First, the ethyl acetate extract is adsorbed onto 80-100 mesh silica gel at 1-1.5 times its mass, mixed, and then dry-loaded onto a normal-phase silica gel column. Then, a dichloromethane-methanol system is used for elution at gradients of 100:0, 100:1, 70:1, 50:1, 30:1, 10:1, and 0:

100. The gradient eluents of each gradient are collected and combined to obtain 6 fractions Fr.1-6. Fr.2 was adsorbed and mixed with 1 to 1.5 times its mass of D101 macroporous resin and then dry-loaded onto a reverse macroporous resin column. Gradient elution was then performed with 20%, 40%, 60%, 80%, and 100% ethanol solutions. The gradient eluents were collected, concentrated, and then combined by TLC to obtain five fractions Fr.4-1 to Fr.4-5. Fr.4-4 was loaded onto a normal-phase silica gel column using a dry method with 1 to 1.5 times its mass of 80-100 mesh, and then eluted using a gradient elution system of petroleum ether-ethyl acetate at ratios of 20:1, 10:1, 8:1, 5:1, and 1:

1. The eluents from each gradient were collected and combined to obtain four fractions, Fr.4-4-1 to Fr.4-4-4. Fr.4-4-3 was loaded onto a gel column and eluted using a methanol or dichloromethane-methanol = 1:1 elution system. The eluents were collected and combined to obtain two subfractions. The first subfraction was purified by HPLC preparative chromatography to obtain compounds 1-3.

8. The application of the guaiacol-type sesquiterpenoid compound as described in claim 2 in its resistance to plant pathogenic fungi, characterized in that, High performance liquid chromatography was performed using a reversed-phase C18 column with methanol-water or acetonitrile-water elution system and a flow rate of 2-3 mL / min.

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