Trinitropropionic acid derivative with termite control activity as well as preparation method and application of trinitropropionic acid derivative

Abstract

The invention relates to the technical field of pesticides and chemistry, and particularly discloses a trinitropropionic acid derivative which is derived from a hypoxylon fermentum rice fermentation product and has termite control activity as well as a preparation method and application of the trinitropropionic acid derivative. Experiments prove that under the concentration dosage of 100 [mu] mol / L, the termite antifeedant rate of the trinitropropionic acid derivative is 75.00%, and the death rate of termites can reach 60.98%. The trinitropropionic acid derivative disclosed by the invention is obtained by extracting and purifying fungus fermentation products, the whole production process is free of chemical pollution, green and environment-friendly, and the trinitropropionic acid derivative has potential application in preparation of novel green and environment-friendly pesticides.

Description

Technical Field

[0001] The present invention relates to the field of pesticides and chemical technologies, in particular to a method for preparing a pesticide derived from carbonaceous fungus ( Hypoxylon fragiforme ) Trinitropropionic acid derivatives of rice fermentation products, their preparation method and their application in the preparation of highly effective, stable and environmentally friendly termite control agents. Background Art

[0002] Termites are listed as one of the world's top five pests by the International Center for Insect Physiology and Ecology. At present, chemical synthetic pesticides are mainly used to control termites, which will remain in the soil, water and plants for a long time after use, polluting the environment. Therefore, it is urgent to obtain substances with insecticidal activity from natural organisms to replace chemical synthetic pesticides.

[0003] Carbonaceous Hypoxylon ) Fungi are a class of Ascomycetes that are widely distributed around the world and mainly grow on dead wood or rotten wood. At present, the research on the genus Hypocrea mainly focuses on two directions: first, the disease problem caused by Hypocrea; second, the exploration of metabolites of certain marine fungi in this genus. After literature search, no reports related to the present invention have been found. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention aims to provide a fungus ( Hypoxylon fragiforme ) Trinitropropionic acid derivatives of rice fermentation products, their preparation method and application in termite control.

[0005] The trinitropropionic acid derivatives having termite killing / resistance activity have the following structural formula:

[0006] Molecular formula: C 10 H 16 N2O8.

[0007] The preparation method of the trinitropropionic acid derivatives having termite killing / anti-termite activity comprises the following steps: Hypoxylon fragiforme ) is soaked and extracted with a methanol aqueous solution (preferably with a volume concentration of 90%), the extract is concentrated under reduced pressure and then extracted with ethyl acetate for 3-5 times, the ethyl acetate layers are combined and concentrated under reduced pressure to obtain an extract, the extract is dissolved in methanol and then separated and purified by normal phase column chromatography, reverse phase column chromatography, gel column chromatography and high performance liquid chromatography in sequence to obtain the trinitropropionic acid derivatives.

[0008] The use of the trinitropropionic acid derivatives in the preparation of a preparation or composition with killing / anti-termite activity with the trinitropropionic acid derivatives as the main component also falls within the protection scope of the present invention.

[0009] Another object of the present invention is to provide a method for controlling termites, which comprises applying an effective amount of the above-mentioned trinitropropionic acid derivatives to the termites or their growth environment.

[0010] Another object of the present invention is to provide an application of the trinitropropionic acid derivatives in the preparation of biological pesticides against agricultural and forestry pests.

[0011] The experiments of the present invention prove that the trinitropropionic acid derivatives show obvious lethal and antifeedant effects on termites in the antifeedant and lethal experiments, and have potential use as a preparation of new green and environmentally friendly pesticides.

[0012] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The present invention discloses for the first time the Hypoxylon fragiforme A trinitropropionic acid derivative isolated from rice fermentation has the activity of killing / fighting termites, which may lead to the scientific community's attention to the future of the termite-killing / fighting activity of termites from the genus Hypocrea ( Hypoxylon ) to find active substances with termite control effects; 2. The present invention provides a trinitropropionic acid derivative having killing / resisting termite activity, which has a better environmental protection effect than widely used chemical pesticides, is a green and environmentally friendly insecticide, and can be used to prepare a new type of biological pesticide; 3. The trinitropropionic acid derivatives of the present invention can be obtained by extraction and purification from fungal fermentation products. The entire production process is free of chemical pollution and is green and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the hydrogen spectrum (600 MHz) of the compound prepared in Example 1; Figure 2 The carbon spectrum (150 MHz) and DEPT diagram of the compound prepared in Example 1; Figure 3 This is the HSQC diagram of the compound prepared in Example 1; Figure 4 This is the HMBC diagram of the compound prepared in Example 1; Figure 5 The compound prepared in Example 1 1 H- 1 H COSY diagram; Figure 6 This is the ROESY diagram of the compound prepared in Example 1. DETAILED DESCRIPTION

[0014] In order to make the invention purpose and invention content of this application more clearly presented, the applicant will provide a clear and complete description of the technical solution of the present invention in conjunction with specific embodiments below.

[0015] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0016] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0017] The compounds described in Example 1, claims and the invention content of the specification - trinitropropionic acid derivatives are obtained as follows: Carbonaceous bacteria Hypoxylon fragiforme The strain was purchased from China General Microbial Culture Collection Center, and the strain number is 5.1297. Strain fermentation culture: Carbonaceous bacteria ( H. fragiforme ) was inoculated on the PDA medium, and then placed in a 28°C incubator in dark for 7 days. After the culture was completed, 50 kg of rice was prepared, 100 g of rice and 100 mL of tap water were added to each fermentation bottle, and 500 bottles of rice culture medium were prepared after high-temperature sterilization at 120°C for 30 minutes and then cooled to room temperature. The cultured PDA culture medium was cut into small pieces (1 cm*1 cm in size) on a sterile operating table, and the bacterial blocks were placed in the rice culture medium for fermentation and culture, and placed in a sterile culture room at 28°C, and cultured in dark for 30 days before being recovered. H. fragiforme of rice fermentation products.

[0018] Compound separation and purification process: All the above were immersed in 100L of 90% (v / v) methanol aqueous solution. H. fragiforme The rice fermentation product was extracted for 7 days and centrifuged. The filtrate was concentrated to 5L and extracted repeatedly with ethyl acetate (5L of ethyl acetate was used for each extraction) for 5 times. The ethyl acetate parts were combined and concentrated under reduced pressure to obtain 563g of total extract. The total extract was completely dissolved with reagent (methanol: Shanghai Lingfeng Chemical Reagent Co., Ltd., analytical grade), 570g normal phase silica gel (200-300 mesh) was added to adsorb the sample, and the sample was placed in a fume hood to dry and ground into powder. 200-300 mesh normal phase silica gel (Qingdao Ocean Chemical Co., Ltd., thin layer chromatography silica gel) was used for gradient elution with dichloromethane-methanol (100:1, 50:1, 20:1, 10:1, 5:1, 1:1, 0:1, v / v) as the eluent. Each 500mL of the eluent was concentrated in a 20mL vial, and detected by thin layer chromatography (Yantai Jiangyou Silica Gel Development Co., Ltd., thin layer chromatography silica gel plate) and analyzed by colorimetry with vanillin. The developing solvent was dichloromethane-methanol (10:1, v / v). Similar components were combined and concentrated to obtain 13 components, which were numbered T1 to T13 in the order of elution.

[0019] The T9 component (68 g) obtained by eluting with dichloromethane-methanol (volume ratio of 20:1) was completely dissolved in anhydrous methanol, and 70 g of reverse phase silica gel (Fuji Silysia Chemical Co., Ltd., Japan, Lichroprep The sample was adsorbed on RP-18, 40-70 μm), air-dried in a fume hood, ground into powder, and eluted by MPLC medium pressure chromatography (Biotage, Sweden, C-615 pump manager, C-605 pump module, C-660 fraction collector, RP-18 column) with a methanol-water system (10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 100:0, v / v) for gradient elution. 5.5 L of each ratio was eluted, and one bottle of 350 mL was collected. The samples were detected and combined by thin layer chromatography, developed with chloroform-methanol (20:1, v / v) as the developing solvent, and concentrated sulfuric acid vanillin as the color developer. Five subcomponents were obtained, which were numbered in the order of elution (T9-1~T9-5). The T9-3 component obtained by eluting with methanol-water (volume ratio of 30:70) was subjected to Sephadex LH-20 gel column chromatography and isocratically eluted with anhydrous methanol at a flow rate of 10 mL / min, and one bottle was filled with each 15 mL. The component was detected and analyzed by thin layer chromatography, chloroform-methanol (20:1, v / v) was used as the developing solvent, and concentrated sulfuric acid vanillin was used for color development to obtain 9 components (T9-3-1~T9-3-9). The T9-3-8 component (eluted at 90 to 115 min) was purified by preparative high performance liquid chromatography (chromatographic column: Agilent Zorbax SB-C18 column, specification: 9.4 mm×250 mm; acetonitrile-water gradient elution: 0-35 min, 30% acetonitrile-50% acetonitrile (v / v); flow rate of 4 mL / min; detection wavelength of 210 nm), and then concentrated to prepare the compound of the present invention (light yellow crystals, 8.47 mg, R t = 25.3min).

[0020] Structural identification of the compound: The compound prepared in this example was dissolved in 0.5 mL of deuterated methanol, transferred to an NMR tube using a 200 μL pipette, and the hydrogen spectrum, carbon spectrum, and two-dimensional spectrum (such as Figures 1 - 6 ).

[0021] The NMR data of the obtained compound are shown in Table 1 below: Table 1

[0022] Other physical and chemical data of the compound: Appearance: Pale yellow crystal; specific rotation = -14.00 (c = 0.5, MeOH); UV (MeOH) λmax(log ε ): 210 nm; High resolution mass spectrometry HR-ESI-MS m / z 293.0980 [M + H] + (calcd for C 10 H 17 N2O8:293.0979).

[0023] According to the results of the above tests, the structural formula of the obtained compound is confirmed to be:

[0024] Molecular formula: C 10 H 16 N2O8.

[0025] Embodiment 2, bioassay experiment of the present invention: 100 pieces of 3.5 cm * 1.0 cm filter paper were placed in a culture dish for sterilization and dried after sterilization. After drying, the filter paper was taken out from the sterile operating table, 100 μL of sterilized pure water was added to the filter paper of the control group, and 100 μL of samples of different concentrations were added to the filter paper of the treatment group (the test compound was dissolved in sterilized pure water, and the stock solution concentration was 100 mmol / L; 100 μmol / L of the test compound was the highest concentration, and 2-fold gradient dilution was performed, with 5 concentrations: 100, 50, 25, 12.5, and 6.25 μmol / L, and each concentration was repeated three times). After the filter paper was dried (dried at 50°C for 2 hours), it was weighed, and then the filter paper was placed in a sterilized culture dish (9 cm * 9 cm), moisten the sterile sand and filter paper with a spray bottle (filled with sterile water), and put 50 active termites of the same size in it, put the culture dish in an incubator, and culture it in the dark at room temperature (about 25°C) and humidity of 80%-90%. After 36 hours, count the dead termites in the culture dish, pick up the filter paper in the culture dish with tweezers, rinse the termite feces and sterile sand on the filter paper with a rinse bottle, dry and weigh it, and use the mass loss to represent the degree of termite refusal to feed.

[0026] Calculate the repellency rate: Termite antifeedant rate calculation formula:

[0027] Termite Corrected Mortality Calculation Formula:

[0028] Experimental results: Table 2: The results of the antifeedant and insecticidal effects of the trinitropropionic acid derivatives of the present invention on termites

[0029] The results showed that the trinitropropionic acid derivatives (C 10 H 16 N2O8) has significant insecticidal activity. At a concentration of 100 μmol / L, the trinitropropionic acid derivatives of the present invention have a feeding repellency rate of 75.00% against termites, and the termite mortality rate can reach 60.98%.

Claims

1. A trinitropropionic acid derivative, the chemical formula of which is C 10 H 16 N2O8, the structural formula is as follows: 。 2. Use of the trinitropropionic acid derivatives according to claim 1 in controlling termites.

3. The use according to claim 2, characterized in that: The trinitropropionic acid derivatives according to claim 1 are used to prepare a preparation or composition having activity in preventing and controlling termites.

4. A method for preventing and controlling termites, characterized in that: Apply an effective amount of the trinitropropionic acid derivatives according to claim 1 to termites or their growth environment.

5. A method for preparing the trinitropropionic acid derivatives according to claim 1, characterized in that: The following steps are involved: Carbonaceous spores Hypoxylon fragiforme The rice fermentation product is extracted by soaking with methanol aqueous solution, the extract is concentrated and then extracted with ethyl acetate for 3-5 times, the ethyl acetate layers are combined and concentrated to obtain an extract, the extract is dissolved in methanol and then separated and purified by normal phase column chromatography, reverse phase column chromatography, gel column chromatography and high performance liquid chromatography in sequence to obtain the compound.

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