Application of hemi-petane-type diterpenoid compounds in the preparation of pest repellents

Abstract

The present invention discloses the application of a labdane-type diterpenoid compound in the preparation of a pest repellent. The present invention belongs to the field of pest repellents. The present invention reveals that this type of compound has insect antifeedant activity, and the insect antifeedant activity is manifested as having antifeedant activity against Plutella xylostella larvae and Spodoptera litura larvae. The compound of the present invention can be isolated and purified from natural plants, and is low-toxic, residue-free, pollution-free and harmless to various economic crops such as grains, fruits and vegetables.

Description

Technical Field

[0001] The present invention belongs to the field of pest repellents, and particularly relates to the application of labdane-type diterpenoids in the preparation of pest repellents. Background Art

[0002] Disclosing the information of this background art section is only intended to increase some understanding of the overall background of the present invention, and it is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Plutella xylostella and Spodoptera litura are widely distributed worldwide and are the main dominant pest populations in cruciferous vegetable fields. Their massive feeding on cruciferous crops directly affects the growth of the crops, causing serious damage to the crops and thus resulting in huge economic losses. To control these pests, many farmers rely on chemical pesticides for pest control. However, the overuse of chemical pesticides has caused environmental pollution of soil, water bodies and air, while threatening the health of consumers and pesticide applicators and having an adverse impact on beneficial pollinating insects. Based on the various problems caused by chemical pesticides, the development of botanical pesticides has become a research hotspot. Research shows that botanical pesticides have the advantages of being easily degraded, safe to use and not easily generating drug resistance. Therefore, exploring the antifeedant activity of plant secondary metabolites and applying them to the development of botanical pesticides is an important direction to promote the sustainable development of "green agriculture" at present.

[0004] Labdane-type diterpenoids have been found in terrestrial plants, microorganisms, marine organisms and insects so far, and bryophytes are an important source. There are many unique labdane-type diterpenoids in bryophytes. These compounds not only have important significance in the chemotaxonomy of mosses, but also exhibit a wide range of biological activities and play an important ecological role in the interaction relationships between organisms. Gymnomitrion rotundatum is a plant of the family Gymnomitriaceae. The family Gymnomitriaceae is considered an isolated taxonomic unit with many unique characteristics. Many labdane-type diterpenoids found only in Gymnomitrion rotundatum have been extracted at present, and these compounds have biological activities such as anti-inflammatory and allelopathic effects.

[0005] At present, there is no report on the application of related labdane-type diterpenoids in the preparation of pest repellents. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the present invention provides the application of the insect antifeedant activities of labdane-type diterpenoids 1-4. Through the insect antifeedant research with compounds 1-4, it is found that compounds 1-4 have antifeedant activities against Plutella xylostella larvae and Spodoptera litura larvae.

[0007] The technical scheme adopted by the present invention is as follows:

[0008] The present invention provides an application of a labdane - type diterpenoid compound in preparing a pest repellent; the labdane - type diterpenoid compound is one or more of compounds 1 - 4;

[0009] 。

[0010] In one or some embodiments of the present invention, the pests controlled by the pest repellent are Spodoptera litura larvae and / or Plutella xylostella larvae.

[0011] In one or some embodiments of the present invention, the pest repellent is a plant - derived drug.

[0012] Preferably, the plant - derived drug is prepared by the following method: dissolving one or more of compounds 1 - 4 in an aqueous acetone solution, and then preparing the compound into a liquid medicine with a concentration of 10 - 200 μg / mL.

[0013] In the present invention, the preparation or acquisition of compounds 1 - 4 is prior art. As a preference, the examples of the present invention provide a preparation method of one of compounds 1 - 4.

[0014] Compared with the related technologies known to the inventors of the present invention, one of the technical solutions of the present invention has the following beneficial effects:

[0015] The present invention discloses for the first time the application of four labdane - type diterpenoid compounds in pest repellents; labdane - type diterpenoid compounds 1 - 4 have antifeedant activities against various insects, and the antifeedant effects are relatively significant, having good application prospects; compounds 1 - 4 of the present invention can be isolated and extracted from plants, are low - toxic, residue - free and pollution - free to cash crops such as fruits, melons and vegetables, are more beneficial to environmental protection, and can achieve the effect of preventing insect pests. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings forming a part of this specification are used to provide a further understanding of the present invention, and the schematic embodiments and descriptions thereof of the present invention are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0017] Figure 1 : The separation and extraction process of compounds 1 - 4 of the present invention.

[0018] Figure 2 : The non - selective antifeedant broken - line graph of Plutella xylostella.

[0019] Figure 3 : The selective antifeedant broken - line graph of Plutella xylostella.

[0020] Figure 4 : Non-selective antifeedant broken line graph of Spodoptera litura

[0021] Figure 5 : Selective antifeedant broken line graph of Spodoptera litura

[0022] Figure 6 : 24-hour non-selective observation of Plutella xylostella with compound 1 at concentrations of 10, 20, 50, 100, and 200 ug / ml in the control group

[0023] Figure 7 : 24-hour non-selective observation of Plutella xylostella with compound 2 at concentrations of 10, 20, 50, 100, and 200 ug / ml in the control group

[0024] Figure 8 : 24-hour non-selective observation of Plutella xylostella with compound 3 at concentrations of 10, 20, 50, 100, and 200 ug / ml in the control group

[0025] Figure 9 : 24-hour non-selective observation of Plutella xylostella with compound 4 at concentrations of 10, 20, 50, 100, and 200 ug / ml in the control group

[0026] Figure 10 : 24-hour selective observation of Plutella xylostella with compound 1 at concentrations of 10, 20, 50, 100, and 200 ug / ml. The marked area is the control group, and the unmarked area is the drug-administered group.

[0027] Figure 11 : 48-hour selective observation of Plutella xylostella with compound 1 at concentrations of 10, 20, 50, 100, and 200 ug / ml. The marked area is the control group, and the unmarked area is the drug-administered group.

[0028] Figure 12 : 24-hour selective observation of Plutella xylostella with compound 2 at concentrations of 10, 20, 50, 100, and 200 ug / ml. The marked area is the control group, and the unmarked area is the drug-administered group.

[0029] Figure 13 : 48-hour selective observation of Plutella xylostella with compound 2 at concentrations of 10, 20, 50, 100, and 200 ug / ml. The marked area is the control group, and the unmarked area is the drug-administered group.

[0030] Figure 14 : 24-hour selective observation of Plutella xylostella with compound 3 at concentrations of 10, 20, 50, 100, and 200 ug / ml. The marked area is the control group, and the unmarked area is the drug-administered group.

[0031] Figure 15:Selective observation of Plutella xylostella at 48 h with compound 3 at concentrations of 10, 20, 50, 100, and 200 μg / ml. The marked areas are the control groups, and the unmarked areas are the dosing groups.

[0032] Figure 16 :Selective observation of Plutella xylostella at 24 h with compound 4 at concentrations of 10, 20, 50, 100, and 200 μg / ml. The marked areas are the control groups, and the unmarked areas are the dosing groups.

[0033] Figure 17 :Selective observation of Plutella xylostella at 48 h with compound 4 at concentrations of 10, 20, 50, 100, and 200 μg / ml. The marked areas are the control groups, and the unmarked areas are the dosing groups.

[0034] Figure 18 :Selective observation of Spodoptera litura at 24 h with compound 1 at concentrations of 10, 20, 50, 100, and 200 μg / ml. The marked areas are the control groups, and the unmarked areas are the dosing groups.

[0035] Figure 19 :Selective observation of Spodoptera litura at 24 h with compound 2 at concentrations of 10, 20, 50, 100, and 200 μg / ml. The marked areas are the control groups, and the unmarked areas are the dosing groups.

[0036] Figure 20 :Selective observation of Spodoptera litura at 24 h with compound 3 at concentrations of 10, 20, 50, 100, and 200 μg / ml. The marked areas are the control groups, and the unmarked areas are the dosing groups.

[0037] Figure 21 :Selective observation of Spodoptera litura at 24 h with compound 4 at concentrations of 10, 20, 50, 100, and 200 μg / ml. The marked areas are the control groups, and the unmarked areas are the dosing groups.

[0038] Figure 22 :Non - selective observation of Plutella xylostella at 48 h for the control group, compound 1, and compound 3 at a concentration of 100 μg / ml. Detailed implementation mode

[0039] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0040] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they specify the presence of features, steps, operations, and / or combinations thereof.

[0041] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0042] Example 1

[0043] The fresh material Gymnomitrion rotundatum (1.4 kg) was soaked in an ethanol solution with a volume fraction of 95% at room temperature for one week for extraction, and then refluxed three times, each time for 2.5 h. The ethanol extracts were combined and filtered, and concentrated under reduced pressure to obtain an extract, yielding a total extract (22.42 g).

[0044] The obtained extract was separated by a CHP20 / P120 column (brand: MCIGEL TM CHP20 / P120, particle size 75 - 150 μmm, produced by Mitsubishi Chemical Industries Ltd.), and gradient eluted with a MeOH - H 2 O system (methanol / water 3:7 - 1:0, v / v). TLC detection was carried out and fractions were combined to obtain 5 parts A - E. Among them, 3.83 g of part B was passed through a silica gel chromatography column (200 - 300 mesh) and gradient eluted with petroleum ether to acetone (petroleum ether / acetone 200:1 - 0:1) to obtain 7 parts Fr.1 - 7 (Fraction 1 - 7). Fr.4 (300 mg) was obtained with petroleum ether / acetone 20:1, Fr.6 (649 mg) was obtained with petroleum ether / acetone 8:1, and Fr.7 (668 mg) was obtained with petroleum ether / acetone 5:1;

[0045] Compound 1 (32 mg), Compound 2 (29 mg): 300 mg of part Fr.4 was subjected to reverse - phase C18 column chromatography and gradient eluted with water to methanol (water / methanol 6:4 - 0:10, v / v). When the eluent was a water / methanol mixed solution with a volume ratio of 5.5:4.5 - 4:6, , separated by semi - preparative HPLC (Agilent ZORBAX SB - C18 5 m column, 9.4 × 250 mm) (eluent: water / methanol 43:57, v / v) to obtain Compound 1 (1.8 ml / min, t R = 30.5 min), Compound 2 (1.8 ml / min, tR = 23.7 min).

[0046] Compound 3 (37 mg): 649 mg of fraction 6 was chromatographed on a reversed-phase C18 column, eluted with a gradient of water to methanol (water / methanol 6:4 - 0:10, v / v). When the eluent was a water / methanol mixed solution with a volume ratio of 4:6, , separated by semi-preparative HPLC (Agilent ZORBAX SB-C18 5 m column, 9.4 × 250 mm) (eluent: water / acetonitrile 40:60, 1.8 ml / min, t R = 45.9 min) to obtain Compound 3.

[0047] Compound 4 (25 mg): Fraction 7 was chromatographed on a silica gel column with a gradient elution of petroleum ether to acetone (petroleum ether / acetone 10:1 - 0:1, v / v). When the eluent was a petroleum ether / acetone mixed solution with a volume ratio of 3:1 - 2:1, , and then chromatographed on a reversed-phase C18 column with a gradient elution of water to methanol (6:4 - 0:10, v / v). When the eluent was a water / methanol mixed solution with a volume ratio of 1:1, Compound 4 was obtained.

[0048] The names of the labdane-type diterpenoids 1 - 4 obtained in this example are Haplomitrenolide A, Haplomitrenolide B, Haplomitrenolide C, and Hapmnioide B, respectively. The separation and preparation process is shown in Figure 1 .

[0049] Structural identification of 4 labdane-type diterpenoids: Compound 1, Haplomitrenolide A is a colorless crystal, 13 13C NMR (DMSO) δ 196.02, 172.76, 155.62, 144.24, 139.17, 128.74, 126.79, 108.70, 72.50, 69.02, 55.19, 51.38, 42.28, 40.06, 34.40, 33.95, 31.85, 26.90, 22.55, 18.85, 18.26. Consistent with the literature comparison.

[0050] Compound 2, Haplomitrenolide B is a colorless crystal, 1313C NMR (CDCl3) δ 195.84, 171.97, 155.10, 144.08, 139.30, 130.17, 127.07, 108.06, 72.48, 61.08, 50.52, 49.37, 43.22, 34.52, 34.32, 32.92, 28.03, 22.41, 21.98, 19.26. Consistent with the literature comparison.

[0051] Compound 3, Haplomitrenolide C is a colorless crystal, 13 13C NMR (DMSO) δ 194.13, 177.91, 171.90, 159.33, 144.24, 139.56, 127.08, 126.53, 108.82, 71.26, 55.84, 51.94, 49.43, 47.77, 42.65, 37.33, 31.12, 27.14, 21.97, 18.49, 17.92. Consistent with the literature comparison.

[0052] Compound 4, Hapmnioide B is a colorless crystal, 13 13C NMR (DMSO) δ 213.10, 173.26, 163.77, 152.04, 143.74, 140.99, 123.84, 119.08, 109.27, 89.49, 70.91, 51.04, 44.51, 40.97, 35.90, 32.79, 29.44, 23.50, 21.40. Consistent with the literature comparison.

[0053] Example 2 Antifeedant Activity of Compounds 1 - 4 against Insects

[0054] Method: Compounds 1 - 4 were separately dissolved in 1 v / v% aqueous acetone solution, and then the compounds were formulated into a liquid medicine with a final concentration of 200 μg / mL. Fresh flowering Chinese cabbage leaves without using chemical pesticides were made into circular leaf discs with a diameter of 15 mm. Randomly selected leaf discs were immersed in solutions of extracts of Gymnotheca chinensis at different concentrations (10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL) for 30 s and then taken out as the experimental group. Randomly selected leaf discs were immersed in 1% aqueous acetone solution as the control group. After the leaves were naturally dried, they were placed in a petri dish (9 cm in diameter) pre-lined with filter paper for moisturizing.

[0055] For the selectivity experiment, 4 treated leaf discs and 4 control leaf discs were placed in each petri dish, arranged in a cross pattern; for the non-selectivity experiment, 8 leaf discs were placed equidistantly in each petri dish, and another control group had 8 control leaf discs placed in each petri dish. One 3rd instar larva of Plutella xylostella or Spodoptera litura that had been starved for 4 h was introduced into each petri dish, and each concentration treatment was replicated 6 times. The samples were kept in an environment with a relative humidity of 50% - 70% and a temperature of 25 - 28 °C for 24 h and 48 h, and the area of the leaves eaten by the larvae was measured at 24 h and 48 h respectively. The eaten leaves were photographed with a camera, and the Image J 180 software for calculating graphic areas was used to measure the eaten area of the leaves, and the antifeedant rate was calculated.

[0056] The antifeedant activities of Compounds 1 - 4 against 3rd instar larvae of Plutella xylostella and Spodoptera litura were tested using flowering Chinese cabbage leaves, and the antifeedant rates after 24 h and 48 h of treatment were measured. The formula for calculating the antifeedant rate is as follows:

[0057] Non-selective antifeedant rate =

[0058] Selective antifeedant rate =

[0059] The non-selectivity of the 4 labdane-type diterpenoid compounds against Plutella xylostella is shown in Table 1, the selectivity of the 4 labdane-type diterpenoid compounds against Plutella xylostella is shown in Table 2, the non-selectivity of the 4 labdane-type diterpenoid compounds against Spodoptera litura is shown in Table 3, and the selectivity of the 4 labdane-type diterpenoid compounds against Spodoptera litura is shown in Table 4.

[0060] Table 1 Non-selective antifeedant effects of Compounds 1 - 4 against Plutella xylostella

[0061]

[0062] Table 2 Selective antifeedant effects of Compounds 1 - 4 against Plutella xylostella

[0063]

[0064] Table 3 Non-selective antifeedant effects of Compounds 1 - 4 against Spodoptera litura

[0065]

[0066] Table 4 Selective antifeedant effects of Compounds 1 - 4 against Spodoptera litura

[0067]

[0068] The purpose of the non-selective experimental group was to establish a high-stress environment. Leaves with the medicinal solution were placed in all the petri dishes to observe the feeding behavior and more intuitively evaluate the antifeeding efficacy of the test substances. The results are as Figures 6 - 9 shown. Compared with the control group, as the concentration of the compound increased, the area of the leaves rejected by the Plutella xylostella larvae in the treated group continuously increased.Figure 22 The marked diamondback moths are dead. The body segments of the larvae are curled up, forming a C-shaped bend. The body surface is darkened, showing a rigid and curled posture. Some individuals have swollen abdomens and are accompanied by the exudation of light yellow body fluid, all of which are abnormal deaths. The diamondback moths in the control group are bright green and full, with significant motor ability. The diamondback moths in the dosing group are small and move slowly.

[0069] The purpose of the selective experimental group is to simulate the natural environment and utilize the ability of insects to naturally select foods with appropriate nutritional potential and without dangerous compounds, so as to judge whether the test substance can prevent insects from feeding and to understand whether insects regard the substance as unsuitable for feeding. The selective test is closer to the natural state and can reflect the food preferences of insects. The results are as Figures 10 - 21 shown. Compared with the control group, the area of the leaves rejected by diamondback moths and Spodoptera litura in the dosing group is larger.

[0070] Experimental conclusion: In this example, the antifeedant research on Compounds 1-4 was carried out, and it was found that these 4 labdane-type diterpenes have antifeedant activity against diamondback moth larvae, and it was found that these 4 labdane-type diterpenes have antifeedant activity against Spodoptera litura larvae.

[0071] In summary, the above examples provide the antifeedant activity and application of Compounds 1-4. Through the antifeedant research on Compounds 1-4, Compounds 1-4 have antifeedant activity against diamondback moth larvae as shown in Figure 2 、 Figure 3 , and it was found that Compounds 1-4 have antifeedant activity against Spodoptera litura larvae as shown in Figure 4 、 Figure 5 .

[0072] The above examples are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above examples. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A use of a hemipentane-type diterpenoid compound in the preparation of an insect repellent, characterized in that: The hemipentane-type diterpenoid compound is one or more of compounds 1 to 4; The pests controlled by the pest repellent are larvae of Spodoptera litura and / or larvae of Plutella xylostella.

2. The use according to claim 1, characterized in that: The pest repellent is a plant-derived medicine.

3. The use according to claim 2, characterized in that: The plant-derived medicine is prepared by the following method: one or more of compounds 1 to 4 is dissolved in an acetone aqueous solution, and then the compound is configured into a 10 to 200 μg / mL medicinal solution.

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