A trinitropropionic acid derivative with termite control activity and its preparation method and application

Abstract

The present invention relates to the field of pesticides and chemical technologies, and specifically discloses a method for preparing a pesticide derived from a fungus ( Hypoxylon fragiforme Trinitropropionic acid derivatives derived from rice fermentation products with termite control activity, their preparation methods, and uses. Experimental studies have shown that at a concentration of 100 μmol / L, the trinitropropionic acid derivatives of the present invention have a termite antifeedant rate of 75.00% and a termite mortality rate of 60.98%. The trinitropropionic acid derivatives of the present invention are obtained by extraction and purification from fungal fermentation products. The entire production process is chemically pollution-free and environmentally friendly, and they have potential applications in the preparation of new green and environmentally friendly pesticides.

Description

Technical Field

[0001] The present invention relates to the field of pesticides and chemical technology, in particular to a method for preparing a pesticide derived from Hyphaenomyces pygme 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 of Insect Physiology and Ecology. Currently, termite control primarily relies on synthetic chemical pesticides, which can leave long-lasting residues in soil, water, and plants, polluting the environment. Therefore, there is an urgent need to develop insecticides derived from natural organisms to replace these synthetic pesticides.

[0003] Carbonaceous Hypoxylon ) fungi are a globally distributed group of Ascomycetes that primarily grow on dead or decaying wood. Currently, research on the genus Hypocrea focuses on two main areas: first, the diseases caused by Hypocrea; and second, the exploration of the metabolites of certain marine fungi in this genus. A literature search has not yet found any reports related to the present invention. 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 / anti-termite activity have the following structural formula:

[0006]

[0007] Molecular formula: C 10 H 16 N2O8.

[0008] 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 3-5 times with ethyl acetate, 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.

[0009] The use of the trinitropropionic acid derivatives in the preparation of a preparation or composition with termite killing / antifungal activity containing the trinitropropionic acid derivatives as a main component also falls within the scope of protection of the present invention.

[0010] Another object of the present invention is to provide a method for controlling termites, specifically comprising: applying an effective amount of the above-mentioned trinitropropionic acid derivatives to the termites or their growth environment.

[0011] Another object of the present invention is to provide a use of the trinitropropionic acid derivatives in the preparation of biological pesticides against agricultural and forestry pests.

[0012] The experiments of the present invention prove that the trinitropropionic acid derivatives show obvious lethal and antifeedant effects on termites in antifeedant and lethal experiments, and have potential use as a preparation for new green and environmentally friendly pesticides.

[0013] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0014] 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 fungus from the genus Hypocrea ( Hypoxylon ) to find active substances with termite control effects;

[0015] 2. The present invention provides a trinitropropionic acid derivative with termite killing / antifungal activity, which has better environmental protection effects than widely used chemical pesticides and is a green and environmentally friendly insecticide that can be used to prepare new biopesticides;

[0016] 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 chemical-free and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the hydrogen spectrum (600 MHz) of the compound prepared in Example 1;

[0018] Figure 2 The carbon spectrum (150 MHz) and DEPT diagram of the compound prepared in Example 1;

[0019] Figure 3 HSQC chart of the compound prepared in Example 1;

[0020] Figure 4 This is the HMBC diagram of the compound prepared in Example 1;

[0021] Figure 5 The compound prepared in Example 1 1 H- 1 H COSY diagram;

[0022] Figure 6This is the ROESY diagram of the compound prepared in Example 1. DETAILED DESCRIPTION

[0023] In order to make the purpose and content of the invention of this application more clear, the applicant will provide a clear and complete description of the technical solution of the present invention in conjunction with specific embodiments below.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0025] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0026] The compounds described in Example 1, the claims, and the summary of the invention - trinitropropionic acid derivatives - are obtained as follows:

[0027] Carbonaceous bacteria Hypoxylon fragiforme The strain was purchased from China General Microbial Culture Collection Center, the strain number is 5.1297. Strain fermentation culture: Carbonaceous bacteria ( H. fragiforme ) was inoculated on the PDA culture medium, and then placed in a 28°C incubator in the dark for 7 days. After the culture was completed, 50 kg of rice was prepared, and 100 g of rice and 100 mL of tap water were added to each fermentation bottle. After sterilization at 120°C for 30 minutes, the cultured PDA culture medium was cooled to room temperature to prepare 500 bottles of rice culture medium. The cultured PDA culture medium was cut into small pieces (1 cm * 1 cm in size) in a sterile operating table, and the bacterial blocks were placed in the rice culture medium for fermentation culture. The culture was placed in a sterile culture room at 28°C and cultured in the dark for 30 days before being recovered. H. fragiforme of rice fermentation products.

[0028] Compound separation and purification process: Use 100L of 90% (v / v) methanol aqueous solution to soak all the above H. fragiformeThe rice fermentation product was collected for 7 days, centrifuged and filtered, 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 in a reagent (methanol: Shanghai Lingfeng Chemical Reagent Co., Ltd., analytical grade). 570 g of normal-phase silica gel (200–300 mesh) was added for adsorption, and the sample was air-dried in a fume hood and ground into a powder. Thin-layer chromatography (TLC) silica gel was performed on 200–300 mesh normal-phase silica gel (Qingdao Ocean Chemical Co., Ltd.) using a gradient elution system of dichloromethane-methanol (100:1, 50:1, 20:1, 10:1, 5:1, 1:1, and 0:1, v / v). Each 500 mL of the eluate was concentrated into a 20-mL vial. Thin-layer chromatography (TLC silica gel plates, Yantai Jiangyou Silica Gel Development Co., Ltd.) was used for detection and colorimetric analysis using vanillin. The developing solvent was dichloromethane-methanol (10:1, v / v). Similar fractions were combined and concentrated to yield 13 fractions, which were numbered T1 to T13 in order of elution.

[0029] The T9 fraction (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) was added. The sample was adsorbed on RP-18, 40-70 μm, air-dried in a fume hood, ground into powder, and subjected to MPLC medium-pressure chromatography (Biotage, Sweden, C-615 pump manager, C-605 pump module, C-660 fraction collector, RP-18 column). Gradient elution was performed with methanol-water system (10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 100:0, v / v). 5.5 L of each ratio was eluted, and each 350 mL was transferred to a bottle. The samples were detected and combined by thin-layer chromatography. Chloroform-methanol (20:1, v / v) was used as the developing solvent and concentrated sulfuric acid vanillin was used as the color developer. Five subfractions were obtained and numbered (T9-1 to T9-5) according to the order of elution. Fraction T9-3, obtained by elution with methanol-water (volume ratio 30:70), was subjected to isocratic elution with anhydrous methanol on a Sephadex LH-20 gel column at a flow rate of 10 mL / min, with each 15 mL portion transferred to a vial. Thin-layer chromatography (TLC) and colorimetric analysis were performed using chloroform-methanol (20:1, v / v) as the developing solvent and concentrated vanillin sulfate as the colorimetric developer to yield nine fractions (T9-3-1 to T9-3-9). Fraction T9-3-8 (eluted between 90 and 115 min) was purified by preparative HPLC (Agilent Zorbax SB-C18 column, dimensions: 9.4 mm × 250 mm; acetonitrile-water gradient elution: 0-35 min, 30% acetonitrile to 50% acetonitrile (v / v); flow rate 4 mL / min; detection wavelength 210 nm). The product was then concentrated to yield the compound of the present invention (pale yellow crystals, 8.47 mg, R t = 25.3min).

[0030] Structural identification of the compound: The compound prepared in this example was dissolved in 0.5 mL of deuterated methanol and transferred to an NMR tube using a 200 μL pipette. The H spectrum, C spectrum, and two-dimensional spectrum (e.g., 1H spectrum, 2C spectrum, and 2D spectrum) were detected on an NMR spectrometer (Bruker Avance III 600 MHz, Germany). Figures 1 - 6 ).

[0031] The nuclear magnetic resonance data of the obtained compound are shown in Table 1 below:

[0032] Table 1

[0033]

[0034] Other physicochemical data of the compound:

[0035] Appearance: light 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).

[0036] According to the results of the above tests, the structural formula of the obtained compound was confirmed to be:

[0037]

[0038] Molecular formula: C 10 H 16 N2O8.

[0039] Example 2, bioassay experiment of the present invention:

[0040] 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 on a 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 to set 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 placed in a sterile culture dish (9 cm * 9 cm) filled with sterile sand. (cm) Wet sterile sand and filter paper with a spray bottle (filled with sterile water) and place 50 active termites of uniform size in the dish. Place the dish in an incubator and incubate in the dark at room temperature (around 25°C) and 80%-90% humidity. After 36 hours, count the dead termites in the dish, remove the filter paper with tweezers, rinse the filter paper clean of termite feces and sterile sand with a rinse bottle, dry it, and weigh it. The weight loss represents the degree of termite refusal to feed.

[0041] Calculate the repellency rate:

[0042] Termite antifeedant rate calculation formula:

[0043]

[0044] Termite Corrected Mortality Calculation Formula:

[0045]

[0046] Experimental results:

[0047] Table 2: Antifeedant and insecticidal effects of the trinitropropionic acid derivatives of the present invention on termites

[0048]

[0049] 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 75.00% antifeedant rate against termites and a termite mortality rate of up to 60.98%.

Claims

1. A 3-nitropropionic acid derivative, chemical formula C 10 H 16 N2O8, the structural formula is as follows: 。 2. Use of the 3-nitropropionic acid derivatives according to claim 1 in controlling termites.

3. The use according to claim 2, characterized in that The 3-nitropropionic acid derivatives according to claim 1 are used to prepare a preparation or composition having termite control activity.

4. A method for preventing and controlling termites, characterized in that: Apply an effective amount of the 3-nitropropionic acid derivatives according to claim 1 to termites or their growth environment.

5. A method for preparing the 3-nitropropionic acid derivatives according to claim 1, characterized in that: The following steps are involved: Carbonaceous bacteria Hypoxylon fragiforme The rice fermentation product is extracted by soaking with methanol aqueous solution, the extract is concentrated and then extracted with ethyl acetate 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 to obtain the compound; Carbonaceous bacteria Hypoxylon fragiforme The rice fermentation product is prepared by the following method: Hypoxylon fragiforme The culture medium was inoculated on PDA medium and cultured in the dark at 28℃ for 7 days. The PDA medium was cut into small pieces and placed in rice medium for fermentation. The culture medium was cultured in the dark at 28℃ for 30 days. Hypoxylon fragiforme of fermented rice; The rice culture medium is prepared by the following method: adding 100 g of rice and 100 mL of tap water into a fermentation bottle, sterilizing at 120° C. for 30 min, and then cooling at room temperature to obtain the rice culture medium; Carbonaceous bacteria Hypoxylon fragiforme The strain number is 5.1297.

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