A diterpene derivative, and a preparation method and application thereof

Abstract

The application belongs to the technical field of medical intermediates, and discloses a diterpene derivative as well as a preparation method and application thereof. The application utilizes modern separation and purification and structure analysis technologies to analyze secondary metabolites of rice seed hulls in detail, and obtains a novel compound. The compound has good allelopathic activity on plant growth, can be used as an active ingredient of a novel herbicide, and has wide application.

Description

Technical Field

[0001] This invention belongs to the field of pesticide intermediate technology, specifically relating to a new class of diterpenoid derivatives and their preparation methods and applications. Background Technology

[0002] Allelochemicals are non-nutritive substances produced within organisms that can influence the growth, health, behavior, or social dynamics of other plants. Allelochemicals act as the mediators of allelopathic processes and are primarily secondary metabolites of plants. The mediators of plant allelopathic processes are chemical substances, known as "allelochemicals." The chemical substances produced by plants that affect the growth, behavior, and population biology of other organisms include not only those between plants but also those between plants and animals. Moreover, these chemical substances can occur within the plant itself without needing to enter the environment. It has been found that many allelochemicals affect not only plants but also microorganisms, animals, and especially insects.

[0003] Rice is my country's most important food crop, and weeds in paddy fields can reduce rice yields by 40%-60%. The use of chemical herbicides is a common method for weed control in paddy fields. However, the large-scale use of herbicides and the increasing dosage year by year have led to the "3R" problem: increasing weed resistance, pesticide residues, and rampant weed resurgence. Rice seed husks, the outer layer of rice grains, 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 usable pathways. Recent studies have shown that rice seed husks contain a large number of diterpenoid monomeric compounds, which possess certain allelopathic activities. Therefore, using modern separation techniques to deeply extract natural products with enhanced allelopathic activity from rice seed husks and using these as precursors to develop novel herbicides provides a theoretical basis for the further effective utilization of rice seed husks and has good practical application value. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide a diterpenoid derivative or a pharmaceutically formable salt thereof and a method for preparing the same, wherein the diterpenoid derivative can be used as an allelopathic compound or in the preparation of novel herbicides.

[0005] The diterpenoid derivative provided by this invention is a compound with the structural formula of Formula I, and is a novel diterpenoid derivative disclosed for the first time in this invention.

[0006]

[0007] The present invention also provides a method for preparing the above-mentioned diterpenoid derivatives, comprising the following steps:

[0008] 1) Preliminary extraction: The rice seed hulls were extracted with 95% ethanol and then concentrated to dryness under reduced pressure at room temperature to obtain the rice seed hull extract; the rice seed hulls preferably used are those of rice Oryza sativa L.

[0009] 2) Extraction: Add rice seed husk extract to 1.5L aqueous solution to prepare a suspension, and add an equal volume of ethyl acetate for extraction. Concentrate the ethyl acetate layer to dryness under reduced pressure at room temperature to obtain rice seed husk extract.

[0010] 3) Purification by normal-phase silica gel column: The rice seed coat extract obtained in step 2) was added to a chromatographic column packed with normal-phase silica gel. The extract was 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.

[0011] 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 80% methanol solution is then collected.

[0012] 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 diterpene derivative.

[0013] Preferably, in step 1) of the above method, the rice seed coat is the seed coat of rice Oryza sativa L., a grass plant.

[0014] Preferably, the organic solvent in step 1) of the above method is 95% ethanol.

[0015] Preferably, the organic solvent in step 2) of the above method is ethyl acetate.

[0016] Preferably, in step 5), the mobile phase elution system selected by the high-performance liquid chromatography is a methanol-water system.

[0017] Preferably, in step 5), the volume ratio of methanol to water in the methanol-water system is 78:22.

[0018] 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 preparation of herbicides.

[0019] This invention utilizes modern separation and purification techniques to systematically separate secondary metabolites from rice (Oryza sativa L.) seed coats, obtaining a new class of diterpenoid derivatives. In vitro experiments have confirmed that these compounds possess good allelopathic activity and can be used as active ingredients in novel herbicides, with a wide range of applications. Attached Figure Description

[0020] Figure 1 This is a high-performance liquid chromatography chromatogram of the compound obtained in Example 1 of the present invention;

[0021] Figure 2 This is a hydrogen spectrum of the compound obtained in Example 1 of the present invention;

[0022] Figure 3 This is a carbon spectrum of the compound obtained in Example 1 of the present invention;

[0023] Figure 4 This is a two-dimensional nuclear magnetic resonance (COSY) image of the compound obtained in Example 1 of the present invention;

[0024] Figure 5 This is a two-dimensional nuclear magnetic resonance (HSQC) image of the compound obtained in Example 1 of the present invention;

[0025] Figure 6 This is a two-dimensional nuclear magnetic resonance (HMBC) image of the compound obtained in Example 1 of the present invention;

[0026] Figure 7 This is a high-resolution mass spectrometry (HRESIMS) data image of the compound obtained in Example 1 of the present invention.

[0027] Figure 8 This is a graph showing the allelopathic activity screening results of the compounds obtained in Example 1 of this invention. Detailed Implementation

[0028] To further illustrate the present invention, the diterpenoid derivatives and their preparation methods provided by the present invention are described in detail below with reference to embodiments.

[0029] Example 1: Preparation of compound with structural formula i

[0030] 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.).

[0031] The specific compound's process is as follows:

[0032] 1) Extraction

[0033] 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 seed husk extract).

[0034] 2) Extraction

[0035] Rice seed 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 (referred to as rice seed husk extract).

[0036] 3) Normal-phase silica gel column chromatography separation

[0037] 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.

[0038] 4) Purification using reversed-phase silica gel column

[0039] 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 80% by volume.

[0040] 5) Reversed-phase high-performance liquid chromatography purification

[0041] 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 (78:22, V / V), flow rate 3.0mL / min. Detection wavelength was 203nm, column temperature 25℃, injection volume 100μL. The 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.

[0042] Compound I, syn-stemoden-18-oic acid, is a white amorphous powder.

[0043] The proton and carbon NMR spectra of compound I are shown in Table 1.

[0044] High-resolution ESI mass spectrometry data of compound i: (-)-HR-ESIMS m / z 301.2196 [MH] - (Calcd.forC 20 H 29 O2, 301.2168).

[0045] Table 1. Proton and carbon spectral data of compound 1 (deuterated chloroform)

[0046]

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

[0048] Example 2: Allelopathic activity of compound I of the present invention

[0049] (1) Experimental materials

[0050] Instruments and reagents: Constant temperature and humidity incubator. Plant seeds used in the test: Echinochloa crusgalli (L.) Beauv. (barnyard grass seeds), Samolus parviflorus Raf. (Chinese cabbage seeds), Lactuca sativa L. angustana Irish. B. (lettuce seeds), purchased from the Chinese Academy of Agricultural Sciences.

[0051] Test sample: Secondary metabolite I, purity above 90%; dissolved in DMSO for later use.

[0052] (2) Experimental methods

[0053] The selected plant seeds were sterilized with 10% hydrogen peroxide for 5 minutes, then rinsed 5 times with sterile water and drained. The sterilized plant seeds were spread evenly on a moist petri dish (lined with two layers of filter paper), and then the petri dish was placed in a constant temperature and humidity incubator (temperature set to 25℃) until they sprouted white buds.

[0054] Secondary metabolite I was diluted with sterile water to prepare solutions with concentrations of 5, 10, and 20 μg / mL. Two layers of filter paper were placed in each petri dish, and solutions of different concentrations of secondary metabolite I were added to each dish until the filter paper was completely moistened (5 ml). Fifteen seedlings with uniform germination were selected from each petri dish, and the process was repeated three times. The control group consisted of plants cultured in sterile water. After culturing in a light incubator (25℃, 12 h / d), the root length and seedling height were measured. The growth inhibition rate was calculated using the following formula:

[0055] Growth inhibition rate (%) = [(Control growth - Treatment growth) / Control growth] × 100

[0056] The control growth was the growth of plants cultured in sterile water, while the treatment growth was the growth of seeds of each plant after the addition of different concentrations of secondary metabolite I.

[0057] (3) Experimental Results

[0058] The experimental results are shown in Table 2 and Figure 8 As shown.

[0059] Table 2. Screening of allelopathic activity of compound I

[0060]

[0061] The results showed that compound I of the present invention has good allelopathic activity and exhibits a certain inhibitory effect on the growth of barnyard grass seedlings, Chinese cabbage seedlings and lettuce seedlings, and can be used as an active ingredient of novel herbicides.

[0062] 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 producing a diterpene derivative, characterized by, The diterpene derivative has the following structure: ， The preparation method comprises the following steps: 1) preliminary extraction: the rice seed shell is extracted by an organic solvent, and then concentrated to dryness at room temperature under reduced pressure to obtain a rice seed shell extract; 2) extraction: the rice seed shell extract is dissolved in an aqueous solution to prepare a suspension, and an equal volume of an organic solvent is added for extraction, and the organic layer is concentrated to dryness at room temperature under reduced pressure to obtain a rice seed shell extract; 3) normal phase silica gel column chromatography separation: the rice seed shell extract obtained in step 2) is added to a chromatographic column filled with reversed-phase silica gel, and eluted with petroleum ether-ethyl acetate solutions with volume ratios of 100:0, 100:1, 100:2, 100:4, 100:10, 100:20 and 0:100, respectively, and the fraction eluted with the petroleum ether-ethyl acetate solution with a volume ratio of 100:10 is collected; 4) reversed-phase silica gel column purification: the fraction obtained in step 3) is added to a chromatographic column filled with reversed-phase silica gel packing, and eluted with methanol solutions with volume concentrations of 40%, 50%, 60%, 70%, 80%, 90% and 100%, respectively, and the fraction eluted with the methanol solution with a volume concentration of 80% is 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 diterpene derivative; the selected mobile phase elution system of the reversed-phase high performance liquid chromatography is a methanol-water system.

2. The production method according to claim 1, characterized by, The rice seed shell is the seed shell of the plant Oryza sativa L.

3. The preparation method according to claim 1, characterized in that, In step 1), the organic solvent is 95% ethanol.

4. The method of claim 1, wherein, In step 2), the organic solvent is ethyl acetate.

5. The preparation method according to claim 1, characterized in that, In step 5), the volume ratio of methanol to water in the methanol-water system is 78:22.

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