Application of labdane diterpenoid compound in preparation of pest repellent

Abstract

The invention discloses an application of labdane diterpenoid compounds in preparation of a pest repellent, and belongs to the field of pest repellent. The compound disclosed by the invention has insect antifeedant activity, and the insect antifeedant activity shows that the compound has antifeedant activity on plutella xylostella larvae and prodenia litura larvae. The compound provided by the invention can be separated and purified from natural plants, and has low toxicity, no residue, no pollution and no public nuisance for commercial crops such as various grains, melons and fruits, vegetables and the like.

Description

Technical Field

[0001] The invention belongs to the field of pest repellents, and specifically relates to the application of a hemipentane-type diterpenoid compound in the preparation of pest repellents. Background Art

[0002] The information disclosed in this background technology section is only intended to increase some understanding of the overall background of the invention, and should not be necessarily regarded as an admission or any form of suggestion that the information constitutes the prior art already known to ordinary technicians in this field.

[0003] Diamondback moth and Spodoptera litura are widely distributed around the world and are the main dominant pest populations in cruciferous vegetable fields. Their large-scale feeding on cruciferous crops directly affects the growth of crops, causing serious damage to crops and resulting in huge economic losses. In order to control these pests, many farmers rely on chemical pesticides for pest control and insecticide control. However, the excessive use of chemical pesticides has caused environmental pollution of soil, water and air, while threatening the health of consumers and pesticide applicators, and has adverse effects on pollinating beneficial insects. Based on the various problems of chemical pesticides, the development of botanical pesticides has become a research hotspot. Studies have shown that botanical pesticides have the advantages of easy degradation, safe use, and not easy to develop drug resistance. Therefore, exploring the antifeedant activity of plant secondary metabolites and applying them to the development of botanical pesticides is an important direction for promoting the sustainable development of "green agriculture".

[0004] So far, hemane-type diterpenoids have been found in terrestrial plants, microorganisms, marine organisms and insects, with mosses being an important source. There are many unique hemane-type diterpenoids in mosses, which are not only of great significance in the chemical classification of mosses, but also exhibit a wide range of biological activities and play an important ecological role in the interaction between organisms. Gymnocarpus rotundifolius is a plant of the Gymnocarpaceae family, which is considered an isolated taxonomic unit with many unique characteristics. Many hemane-type diterpenoids found only in Gymnocarpus rotundifolius have been extracted, and these compounds have anti-inflammatory, allelopathic and other biological activities.

[0005] At present, there are no reports on the application of related hemipentane-type diterpenoid compounds in the preparation of pest repellents. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides the application of the insect antifeedant activity of the hemipentane diterpenoid compounds 1 to 4. The insect antifeedant activity of compounds 1 to 4 was studied, and compounds 1 to 4 had antifeedant activity against the larvae of Plutella xylostella, and compounds 1 to 4 had antifeedant activity against the larvae of Spodoptera litura.

[0007] The technical solution adopted by the present invention is as follows: The present invention provides an application of a hesperanoid diterpenoid compound in preparing an insect repellent; the hesperanoid diterpenoid compound is one or more of compounds 1 to 4; .

[0008] In one or some embodiments of the present invention, the pests controlled by the pest repellent are larvae of Spodoptera litura and / or larvae of Plutella xylostella.

[0009] In one or some embodiments of the present invention, the pest repellent is a plant-derived drug.

[0010] Preferably, the plant-derived drug is prepared by the following method: dissolving one or more of compounds 1 to 4 in an acetone aqueous solution, and then preparing the compound into a 10-200 μg / mL drug solution.

[0011] In the present invention, the preparation or acquisition of compounds 1 to 4 is prior art. Preferably, an embodiment of the present invention provides a preparation method of compounds 1 to 4.

[0012] Compared with the related art known to the inventor, one of the technical solutions of the present invention has the following beneficial effects: The present invention discloses for the first time the application of four helianthane-type diterpenoid compounds in pest repellents; helianthane-type diterpenoid compounds 1-4 have antifeedant activity against a variety of insects, with a more significant antifeedant effect and good application prospects; compounds 1-4 of the present invention can be separated and extracted from plants, have low toxicity to economic crops such as fruits and vegetables, have no residue and are pollution-free, are more conducive to environmental protection, and can achieve the effect of preventing insect pests. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute improper limitations on the present invention.

[0014] Figure 1 : The separation and extraction process of compounds 1 to 4 of the present invention.

[0015] Figure 2 : Line chart of non-selective feeding refusal of Plutella xylostella.

[0016] Figure 3 : Line chart of selective feeding refusal of Plutella xylostella.

[0017] Figure 4 : Line graph of non-selective feeding resistance of Spodoptera litura.

[0018] Figure 5 : Line chart of selective feeding resistance of Spodoptera litura.

[0019] Figure 6 : Non-selective observation of the diamondback moth in the control group and compound 1 at concentrations of 10, 20, 50, 100 and 200ug / ml for 24 hours.

[0020] Figure 7 : Non-selective observation of Plutella xylostella in the control group and compound 2 at concentrations of 10, 20, 50, 100 and 200ug / ml for 24 hours.

[0021] Figure 8 : Non-selective observation of the diamondback moth in the control group and compound 3 at concentrations of 10, 20, 50, 100 and 200ug / ml for 24 hours.

[0022] Fig. 9 : Non-selective observation of Plutella xylostella in the control group and compound 4 at concentrations of 10, 20, 50, 100 and 200ug / ml for 24 hours.

[0023] Fig.10 : 24h selective observation of Plutella xylostella with concentrations of 10, 20, 50, 100, and 200ug / ml of compound 1. The marked area is the control group, and the unmarked area is the drug-treated group.

[0024] Fig.11 : 48h selective observation of Plutella xylostella treated with compound 1 at concentrations of 10, 20, 50, 100 and 200ug / ml, the marked area is the control group, and the unmarked area is the drug-treated group.

[0025] Fig.12 : 24h selective observation of Plutella xylostella treated with compound 2 at concentrations of 10, 20, 50, 100, and 200ug / ml, respectively. The marked area is the control group, and the unmarked area is the drug-treated group.

[0026] Fig.13 : 48h selective observation of Plutella xylostella treated with compound 2 at concentrations of 10, 20, 50, 100, and 200ug / ml, respectively. The marked area is the control group, and the unmarked area is the drug-treated group.

[0027] Fig.14 : 24h selective observation of Plutella xylostella treated with compound 3 at concentrations of 10, 20, 50, 100, and 200ug / ml, respectively. The marked area is the control group, and the unmarked area is the drug-treated group.

[0028] Fig.15: 48h selective observation of Plutella xylostella treated with compound 3 at concentrations of 10, 20, 50, 100 and 200ug / ml, the marked area is the control group, and the unmarked area is the drug-treated group.

[0029] Fig.16 : 24h selective observation of Plutella xylostella treated with compound 4 at concentrations of 10, 20, 50, 100, and 200ug / ml, respectively. The marked area is the control group, and the unmarked area is the drug-treated group.

[0030] Fig.17 : 48h selective observation of Plutella xylostella treated with compound 4 at concentrations of 10, 20, 50, 100 and 200ug / ml, the marked area is the control group, and the unmarked area is the drug-treated group.

[0031] Fig.18 : 24h selectivity observation of Spodoptera litura with concentrations of 10, 20, 50, 100 and 200ug / ml of compound 1. The marked area is the control group, and the unmarked area is the drug-treated group.

[0032] Fig.19 : 24h selectivity observation of Spodoptera litura with concentrations of 10, 20, 50, 100 and 200ug / ml of compound 2. The marked area is the control group, and the unmarked area is the drug-treated group.

[0033] Fig. 20 : 24h selectivity observation of Spodoptera litura with compound 3 at concentrations of 10, 20, 50, 100 and 200ug / ml, the marked area is the control group, and the unmarked area is the drug-treated group.

[0034] Fig.21 : 24h selectivity observation of Spodoptera litura with compound 4 at concentrations of 10, 20, 50, 100 and 200ug / ml, the marked part is the control group, and the unmarked part is the drug-treated group.

[0035] Fig. 22 : Non-selective observation of the control group and compounds 1 and 3 at a concentration of 100ug / ml for 48 hours on Plutella xylostella. DETAILED DESCRIPTION

[0036] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0037] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit 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, it indicates the presence of features, steps, operations and / or combinations thereof.

[0038] 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.

[0039] Example 1 Fresh material Gymnocarpus rotundifolia (1.4 kg) was extracted by soaking in 95% ethanol solution at room temperature for one week, and then refluxed for extraction three times, each time for 2.5 hours. The ethanol extracts were combined and filtered, and concentrated under reduced pressure to obtain a total extract (22.42 g).

[0040] The obtained extract was purified by CHP20 / P120 column (brand: MCIGEL TM CHP20 / P120, particle size 75~150μmm, produced by Mitsubishi Chemical Industries, Ltd.), separated by MeOH-H2O system gradient elution (methanol / water 3:7~1:0, v / v), TLC detection and merging, to obtain 5 parts A~E, of which 3.83g of part B was passed through a silica gel chromatography column (200~300 mesh) and gradient eluted from petroleum ether to acetone (petroleum ether / acetone 200:1~0:1) to obtain 7 parts Fr.1~7 (Fraction1~7), petroleum ether / acetone 20:1 to obtain Fr.4 (300mg), petroleum ether / acetone 8:1 to obtain Fr.6 (649mg), petroleum ether / acetone 5:1 to obtain Fr.7 (668mg); Compound 1 (32 mg), Compound 2 (29 mg): 300 mg of Fr.4 was subjected to reverse phase C18 column chromatography, with gradient elution from 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, , The HPLC was performed using semi-preparative HPLC (Agilent ZORBAX SB-C18 5 m column, 9.4 250mm) separation (eluent: water / methanol 43:57, v / v) to obtain compound 1 (1.8ml / min, t R =30.5min), compound 2 (1.8ml / min, t R =23.7min).

[0041] Compound 3 (37 mg): 649 mg of Fr.6 was chromatographed on a reverse phase C18 column, using a gradient elution from water to methanol (water / methanol 6:4 to 0:10, v / v). When the eluent was a water / methanol mixed solution with a volume ratio of 4:6, the , Semi-preparative HPLC (Agilent ZORBAX SB-C18 5 m column, 9.4 250mm) separation (eluent: water / acetonitrile 40:60, 1.8ml / min, t R =45.9min) to obtain compound 3.

[0042] Compound 4 (25 mg): Fr.7 was eluted by gradient elution from petroleum ether to acetone (petroleum ether / acetone 10:1~0:1, v / v) on a silica gel chromatography column. When the eluent was a petroleum ether / acetone mixed solution with a volume ratio of 3:1~2:1, the , The product was then passed through a reverse phase C18 chromatography column and eluted with a gradient from 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.

[0043] The names of the hemipentane diterpenoid compounds 1 to 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 .

[0044] Structural identification of four hemi-helichrysanthene-type diterpenoids: Compound 1, Haplomitrenolide A, is a colorless crystal. 13 C 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.

[0045] Compound 2, Haplomitrenolide B is a colorless crystal. 13C 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.

[0046] Compound 3, Haplomitrenolide C is a colorless crystal. 13 C 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.

[0047] Compound 4, Hapmnioide B is a colorless crystal. 13 C 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.

[0048] Example 2 Antifeedant activity of compounds 1 to 4 against insects Methods: Compounds 1-4 were dissolved in 1v / v% acetone aqueous solution, and then the compounds were prepared into a solution with a final concentration of 200μg / mL. Fresh Chinese cabbage leaves that had not been treated with chemical pesticides were made into round leaf discs with a diameter of 15mm. Leaf discs were randomly selected and placed in solutions of different concentrations (10μg / mL, 20μg / mL, 50μg / mL, 100μg / mL and 200μg / mL) of Gymnocarpus spp. extract and immersed for 30s. The leaves were taken out as the experimental group, and leaf discs were randomly selected and placed in 1% acetone aqueous solution as the control group. After the leaves were naturally dried, they were placed in a culture dish (9cm in diameter) pre-padded with filter paper for moisture retention.

[0049] In the selective experiment, 4 treated leaf discs and 4 control leaf discs were placed in each dish, and they were placed crosswise; in the non-selective experiment, 8 leaf discs were placed in each dish at equal distances, and 8 control leaf discs were placed in each dish of the control group. A 3rd instar larva of Plutella xylostella and Spodoptera litura that had been starved for 4 hours was placed in each dish, and each concentration treatment was repeated 6 times. The relative humidity was 50%-70% and the temperature was 25-28℃ for 24 hours and 48 hours, and the area of ​​leaves eaten by larvae was detected after 24 hours and 48 hours, respectively. The fed leaves were photographed with a camera, and the area of ​​leaves eaten was measured using the graphic area calculation software Image J 180 to calculate the refusal rate.

[0050] The antifeedant activity of compounds 1 to 4 against the third-instar larvae of Plutella xylostella and Spodoptera litura was tested on leaves of Chinese cabbage, and the antifeedant rate was calculated after 24h and 48h of treatment. The antifeedant rate was calculated as follows: Non-selective food refusal rate = Selective food refusal rate = The non-selectivity of the four hexaned terpenoids to Plutella xylostella is shown in Table 1, the selective antifeeding effects of the four hexaned terpenoids to Plutella xylostella are shown in Table 2, the non-selectivity of the four hexaned terpenoids to Spodoptera litura is shown in Table 3, and the selectivity of the four hexaned terpenoids to Spodoptera litura is shown in Table 4.

[0051] Table 1 Non-selective antifeedant effects of compounds 1 to 4 on Plutella xylostella Table 2 Selective antifeedant effects of compounds 1 to 4 on Plutella xylostella Table 3 Non-selective antifeedant effects of compounds 1 to 4 on Spodoptera litura Table 4 Compounds 1 to 4 selectively repellent to Spodoptera litura The purpose of the non-selective experimental group is to establish a high-stress environment, put all the leaves with drug solution in the culture dish, observe the feeding behavior, and more intuitively evaluate the anti-feeding effect of the test object. Figure 6~Figure 9 As shown, compared with the control group, with the increase of compound concentration, the leaf area rejected by the diamondback moth larvae in the treatment group continued to increase. Fig. 22 The marked circle represents the dead diamondback moth. The larvae have curled up segments, forming a C-shaped curve, with darkened body surface and a stiff and curled posture. Some individuals have swollen abdomens and exudate yellowish body fluids, indicating abnormal deaths. The diamondback moths in the control group are emerald green and plump, with significant movement ability; the diamondback moths in the drug-treated group are small and move slowly.

[0052] The purpose of the selective test group is to simulate the natural environment and use the insect's natural ability to select food with suitable nutritional potential and without dangerous compounds to determine whether the test substance can prevent insects from feeding and understand whether the insects regard the substance as unsuitable for feeding. Selective tests are closer to the natural state and can reflect the insect's preference for food. Figure 10~Figure 21 As shown, the leaf area rejected by Plutella xylostella and Spodoptera litura in the treatment group was larger than that in the control group.

[0053] Experimental conclusion: In this example, compounds 1 to 4 were studied for insect antifeedant activity, and it was found that these four hemipentane diterpenes had antifeedant activity against the larvae of Plutella xylostella. It was found that these four hemipentane diterpenes had antifeedant activity against the larvae of Spodoptera litura.

[0054] In summary, the above examples provide the insect antifeedant activity and application of compounds 1 to 4. Insect antifeedant studies were conducted using compounds 1 to 4, and compounds 1 to 4 had antifeedant activity against the larvae of Plutella xylostella. Figure 2 , Figure 3 Compounds 1 to 4 were found to have antifeedant activity against Spodoptera litura larvae. Figure 4 , Figure 5 .

[0055] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are 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; 。 2. The use according to claim 1, characterized in that: The pests controlled by the pest repellent are larvae of Spodoptera litura and / or larvae of Plutella xylostella.

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

4. The use according to claim 3, 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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