A rosinane-type diterpenoid derivative, its preparation method and application

Abstract

This invention belongs to the field of pharmaceutical preparation technology, and discloses a rosinane-type diterpenoid derivative, its preparation method, and its application. The rosinane-type diterpenoid derivative provided by this invention is a novel type of rosinane-type diterpenoid derivative, which has a good inhibitory effect on the growth of pathogenic plant fungi, can prevent and control plant fungal diseases, and can be used to prepare novel agricultural drugs, thus having good practical applications.

Description

Technical Field

[0001] This invention belongs to the field of medicine and relates to a new method for preparing a rosinane-type diterpenoid derivative and its application in inhibiting pathogenic fungi in plants. Background Technology

[0002] Fungi are among the most important pathogens, causing approximately 80% of infectious plant diseases. Each plant species can be affected by several or even dozens of fungal species, such as wheat rust, rice blast, vegetable soft rot, and apple tree canker. The trunk (vegetative body) of a fungus is a fine, branched, mostly septate filamentous structure called mycelium. It lacks chlorophyll and can only absorb nutrients from the plant or its surrounding environment for growth. After reaching a certain stage of growth, the mycelium begins to develop reproductive structures and forms various asexual spores (sporangiospores, conidia, chlamydospores, etc.). These asexual spores, spread by wind, rain, and insects, can reinfect plants and cause disease. Later in the growing season, many fungi can also form various sexual spores (oospores, ascospores, basidiospores, etc.) through the combination of hermaphroditic spores or organs. Asexual or sexual spores attached to diseased plant debris or seeds can generally become sources of infection for the following year; some fungi overwinter as dormant mycelium or special structures such as sclerotia, stromata, and mycelial cords, thus becoming sources of infection for the following year.

[0003] Previous studies isolated several diterpenoid monomeric compounds from the roots, stems, and leaves of rice infected with pathogens. These studies revealed that some of these compounds significantly inhibited the germination of rice blast fungus spores, while others showed marked inhibitory activity against sheath blight fungus, suppressing spore germination and hyphal elongation. This indicates that rice roots, stems, and leaves contain a large number of active monomeric compounds that inhibit plant fungal pathogens.

[0004] Rice seed husks are the largest byproduct in rice processing, accounting for approximately 20% of the rice grain by weight. Extensive research on the comprehensive utilization of rice seed husks has been conducted both domestically and internationally for a long time, resulting in many potential applications. However, few of these applications can be scaled up for large-scale production and consume large quantities of rice husks; some have limited economic benefits and minimal added value; others face challenges in terms of processing, technology, quality, and environmental pollution. Therefore, many places treat rice seed husks as waste, which is not only a huge waste of resources and causes significant economic losses, but also causes substantial environmental pollution. Therefore, researching and solving the problem of the rational utilization of rice seed husks, turning waste into treasure, is a significant task before us. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a novel rosinane-type diterpenoid derivative and its preparation method, as well as its application in inhibiting pathogenic fungi in plants.

[0006] This invention provides the following technical solutions:

[0007] A rosinane-type diterpenoid derivative or a pharmaceutically acceptable salt thereof, said diterpenoid derivative having the structure of Formula I:

[0008]

[0009] A method for preparing a rosinane-type diterpenoid derivative with the structure shown in Formula I includes the following steps:

[0010]

[0011] 1) Preliminary extraction: The plant was extracted with an organic solvent and then concentrated to dryness under reduced pressure at room temperature to obtain a plant extract. The plant was rice seed hull, preferably the seed hull of rice Oryza sativa L.

[0012] 2) Extraction: Dissolve the plant extract in an aqueous solution to prepare a suspension, and add an equal volume of organic solvent for extraction. Concentrate and dry the organic layer under reduced pressure at room temperature to obtain the plant extract.

[0013] 3) Normal-phase silica gel column chromatography separation: The plant extract obtained in step 2) was added to a chromatographic column packed with reversed-phase silica gel and eluted sequentially with petroleum ether-ethyl acetate solution at a volume ratio of 100:0, 100:1, 100:2, 100:4, 100:10, 100:20, and 0:100. The fraction eluted with petroleum ether-ethyl acetate solution at a volume ratio of 100:10 was collected.

[0014] 4) Reversed-phase silica gel column purification: The fraction obtained in step 3) is added to a chromatographic column packed with reversed-phase silica gel and eluted sequentially with methanol solutions of 40%, 50%, 60%, 70%, 80%, 90%, and 100% (v / v). The fraction eluted with 60% methanol solution is then collected.

[0015] 5) Reversed-phase high-performance liquid chromatography purification: The fraction obtained in step 4) is separated and purified by reversed-phase high-performance liquid chromatography to obtain the rosinane-type diterpene derivative.

[0016] Preferably, in step 1), the organic solvent is 95% ethanol.

[0017] Preferably, in step 2), the organic solvent is ethyl acetate.

[0018] Preferably, in step 5), the mobile phase elution system selected by the high-performance liquid chromatography is a methanol-water system, preferably a methanol-water system with a volume ratio of 48:52.

[0019] The present invention also provides the application of the above-mentioned diterpenoid derivatives or the diterpenoid derivatives prepared by the above-mentioned preparation method in the prevention and control of plant fungal diseases.

[0020] Preferably, the pathogen of the plant fungal disease is one or more of the following: Magnaporthe grisea (rice blast fungus), Rhizoctonia solani (rice sheath blight fungus), Blumeria graminearum (wheat scab fungus), and Fusarium oxysporum (cucumber wilt fungus).

[0021] This invention provides the application of the above-mentioned diterpenoid derivatives or the diterpenoid derivatives prepared by the above-mentioned preparation method in the preparation of drugs that inhibit the growth of plant fungal pathogens, wherein the plant fungal pathogens are one or more of rice blast fungi, rice sheath blight fungi, wheat scab fungi, and cucumber wilt fungi.

[0022] Compared with the prior art, the present invention provides a novel rosinane-type diterpenoid derivative, its preparation method and application. In vitro experiments have confirmed that the rosinane-type diterpenoid derivative provided by the present invention has good inhibitory activity against the growth of plant fungal pathogens, especially against rice blast fungus, rice sheath blight fungus, wheat scab fungus and cucumber wilt fungus. It can be used as an active ingredient in drugs that inhibit plant fungal pathogens and has a wide range of applications. Attached Figure Description

[0023] Figure 1 This is an HPLC chromatogram of the rosinane-type diterpene derivative prepared in Example 1 of the present invention;

[0024] Figure 2 This is a hydrogen spectrum of the compound obtained in Example 1 of the present invention;

[0025] Figure 3 This is a carbon spectrum of the compound obtained in Example 1 of the present invention;

[0026] Figure 4 This is a two-dimensional nuclear magnetic resonance (COSY) image of the compound obtained in Example 1 of the present invention;

[0027] Figure 5 This is a two-dimensional nuclear magnetic resonance (HSQC) image of the compound obtained in Example 1 of the present invention;

[0028] Figure 6 This is a two-dimensional nuclear magnetic resonance (HMBC) image of the compound obtained in Example 1 of the present invention;

[0029] Figure 7 This is a high-resolution mass spectrometry (HRESIMS) data image of the compound obtained in Example 1 of the present invention.

[0030] Example 1: Preparation of a compound with the structural formula I

[0031] This invention uses conventional extraction and purification steps under laboratory culture conditions to prepare the compound of this invention. The plant used is the seed husk of rice (Oryza sativa L.).

[0032] The specific compound's process is as follows:

[0033] 1) Extraction

[0034] Rice seed husks (10 kg) were collected, dried in the shade, pulverized, and extracted with 95% ethanol at room temperature (5 L * 3). The extract was concentrated under reduced pressure to dryness, yielding approximately 187 g of the transformed residue (i.e., rice husk extract).

[0035] 2) Extraction

[0036] The rice husk extract was dissolved in 1.5L of water to prepare a suspension, and an equal volume of ethyl acetate was added for extraction (1.5L*3). The extract was concentrated under reduced pressure to dryness to obtain approximately 83g of rice seed husk ethyl acetate extract.

[0037] 3) Normal-phase silica gel column chromatography separation

[0038] Add the rice seed husk ethyl acetate extract obtained in step 2) to a chromatographic column (200-300 mesh) packed with 500g of normal phase silica gel packing, and perform gradient elution (0%-100%) with a petroleum ether-ethyl acetate system. Collect the eluent fraction and take the fraction that has passed through a petroleum ether-ethyl acetate solution with a volume ratio of 100:10.

[0039] 4) Purification using reversed-phase silica gel column

[0040] Add the fraction obtained in step 3) to a chromatographic column packed with 120g reversed-phase silica gel (120 Å, 30–50 mesh), and elute with a gradient of methanol-water system (40%–100% methanol). Collect the eluent and take the fraction eluted with a methanol solution of 60% by volume.

[0041] 5) Reversed-phase high-performance liquid chromatography purification

[0042] The fraction obtained in step 4) was separated and purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The separation conditions were: Hedera C10 column. 18 A-5μm, 4.6mm ID×250mm (Jiangsu Hanbang Technology), elution system was methanol-water isocratic elution, specific conditions: methanol-water (48:52, V / V), flow rate 3.0mL / min. Detection wavelength was 203nm, column temperature 25℃, injection volume 100μL. Compound with structural formula I was obtained. Its HPLC chromatogram, proton NMR chromatogram, carbon NMR chromatogram, two-dimensional nuclear magnetic resonance (COSY), two-dimensional nuclear magnetic resonance (HSQC), two-dimensional nuclear magnetic resonance (HMBC), and high-resolution mass spectrometry (HRESIMS) chromatograms are shown below. Figure 1-7 As shown.

[0043] Compound I, 3β,20β-lactone-5α-abieta-8,11,13-trien-7-one, is a white amorphous powder; its 1H and 1C NMR spectra are shown in Table 1; high-resolution ESI mass spectrometry data: (+)-HR-ESIMS m / z 313.1850 [M+H] + (calcd for C 20 H 25 O3,313.1804)

[0044] Table 1. Proton and carbon spectral data of compound 1 (deuterated chloroform)

[0045]

[0046]

[0047] The above results indicate that the obtained compound has the correct structure.

[0048] The above results indicate that the structure of the obtained compound is shown in Formula I.

[0049] Example 2: Inhibitory activity of compound I of the present invention against plant fungal pathogens

[0050] (1) Experimental materials

[0051] Instruments and reagents: Microbial incubator; homemade potato culture medium; resazu indicator (purchased from Sigma Reagents). Plant fungal pathogen strains used in the tests: Magnaporthe grisea (rice blast fungus), Rhizoctonia solani (rice sheath blight), Blumeria graminearum (wheat scab), and Fusarium oxysporum (cucumber wilt) were purchased from the Chemical Ecology Laboratory, Department of Ecology, College of Agriculture, South China Agricultural University, China.

[0052] Test sample: Secondary metabolite I of rice husk and pine needles, with a purity of over 90%, diluted after being dissolved in DMSO.

[0053] (2) Experimental methods

[0054] 1) Potato glucose medium (PDA): Weigh 100g of potatoes, wash, peel and cut into small pieces, add 500mL of water and boil for 15min, filter with gauze, make up to 500mL, add 10g of anhydrous glucose and 10-15g of agar, dissolve thoroughly, dispense, sterilize for 30min and take out for use.

[0055] 2) Mix 7 mL of 143 μg / mL resplenium solution with 3 mL of solution containing fungi (10... 8 Mix the culture medium (cfu / mL) thoroughly to achieve a final concentration of 100 μg / mL for *Respora sinensis*. Then, add 100 μL of the mixture of *Respora sinensis* and fungal culture medium to each well in columns 1-10 and column 12, respectively. Simultaneously, add 100 μL of the prepared sample solution to each well using a two-fold dilution method, resulting in final concentrations of the compounds of: 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL, 1.56 μg / mL, 0.78 μg / mL, 0.39 μg / mL, and 0.19 μg / mL. Finally, incubate the plate at 28°C until the culture medium in column 12 changes from blue to pink. MIC values ​​for each compound are calculated, and the results are shown in Table 2.

[0056] Table 2. Results of the test for the inhibitory activity of compound I against plant fungal pathogens (IC50). 50 (μM)

[0057]

[0058] The results show that compound I of the present invention has good inhibitory activity against the growth of plant fungal pathogens and can be used as an active ingredient in drugs against plant fungal pathogens.

[0059] 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 rosinane-type diterpenoid derivative or a pharmaceutically acceptable salt thereof, characterized in that, The rosinane-type diterpenoid derivative is 3β,20β-lactone-5α-abieta-8,11,13-trien-7-one, having the structure of formula I:

2. A method for preparing a rosinane-type diterpenoid derivative with the structure shown in Formula I, characterized in that, Includes the following steps: 1) Preliminary extraction: The plant was extracted with 95% ethanol and then concentrated to dryness under reduced pressure at room temperature to obtain a plant extract. The plant was rice seed hull; the plant was rice Oryza sativa L. seed hull. 2) Extraction: The plant extract was dissolved in an aqueous solution to prepare a suspension. An equal volume of ethyl acetate was added for extraction. The organic layer was concentrated and dried under reduced pressure at room temperature to obtain the plant extract. 3) Normal-phase silica gel column chromatography separation: The plant extract obtained in step 2) was added to a chromatographic column packed with normal-phase silica gel and eluted sequentially with petroleum ether-ethyl acetate solution at a volume ratio of 100:0, 100:1, 100:2, 100:4, 100:10, 100:20, and 0:

100. The fraction eluted with petroleum ether-ethyl acetate solution at a volume ratio of 100:10 was collected. 4) Reversed-phase silica gel column purification: The fraction obtained in step 3) is added to a chromatographic column packed with reversed-phase silica gel and eluted sequentially with methanol solutions of 40%, 50%, 60%, 70%, 80%, 90%, and 100% (v / v). The fraction eluted with 60% methanol solution is then collected. 5) Reversed-phase high-performance liquid chromatography purification: The fraction obtained in step 4) is separated and purified by reversed-phase high-performance liquid chromatography to obtain the rosinane-type diterpenoid derivative.

3. The preparation method according to claim 2, characterized in that, In step 5), the mobile phase elution system selected by the high-performance liquid chromatography is a methanol-water system.

4. The preparation method according to claim 3, characterized in that, In step 5), the volume ratio of methanol to water in the methanol-water system is 48:

52.

5. The application of the rosinane-type diterpenoid derivative of claim 1 or the rosinane-type diterpenoid derivative prepared by any one of claims 2-4 in the prevention and control of plant fungal diseases, wherein the pathogen of the plant fungal disease is one or more of rice sheath blight, wheat scab, and cucumber wilt.

6. The use of the rosinane-type diterpenoid derivative of claim 1 or the rosinane-type diterpenoid derivative prepared by any one of claims 2-4 in the preparation of a drug for inhibiting the growth of plant fungal pathogens, wherein the plant fungal pathogen is one or more of rice sheath blight, wheat scab, and cucumber wilt.

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