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 with termite control activity, their preparation methods, and applications. Experimental studies have demonstrated that at a concentration of 100 μmol / L, the trinitropropionic acid derivatives of the present invention have a termite antifeedant rate of 92.31% and a termite mortality rate of 45.53%. The trinitropropionic acid derivatives of the present invention are obtained through extraction and purification from fungal fermentation. The entire production process is chemically pollution-free and environmentally friendly, demonstrating their potential use as novel green pesticides.

Description

Technical Field

[0001] The present invention relates to the field of pesticides and chemical technology, and in particular to trinitropropionic acid derivatives derived from Hypoxylon fragiforme, a preparation method thereof, and an application of the trinitropropionic acid derivatives in preparing a highly effective, stable and environmentally friendly termite control agent. Background Art

[0002] Termites are listed as one of the world's top five pests by the International Center of Insect Physiology and Ecology. According to statistics, termite damage in my country reaches 80% to 90%, resulting in economic losses of approximately 2 to 2.5 billion yuan annually. The damage caused by termites to cultural relics, historical sites, and reservoir dams is difficult to estimate in economic terms. Currently, chemical synthetic insecticides are primarily used for termite control. These chemicals can remain in soil, water, and plants for long periods of time, polluting the environment. Therefore, there is an urgent need to obtain insecticidal substances from natural organisms to replace these synthetic insecticides.

[0003] Hypoxylon fungi are a globally distributed group of Ascomycetes that primarily grow on dead or decaying wood. Current research on Hypoxylon focuses on two main areas: first, the disease problems caused by Hypoxylon; and second, the exploration of metabolites from certain marine fungi in this genus. A literature search has not yet uncovered any reports relevant 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 trinitropropionic acid derivative derived from the fungus (Hypoxylonfragiforme), a preparation method thereof, and an application thereof in termite control.

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

[0006]

[0007] Molecular formula: C8H 13 NO6.

[0008] The method for preparing the trinitropropionic acid derivatives having termite killing / antifungal activity comprises the following steps: extracting rice fermented by Hypoxylon fragiforme by soaking in a methanol aqueous solution (preferably with a volume concentration of 90%), concentrating the extract under reduced pressure, and then extracting 3-5 times with ethyl acetate, combining the ethyl acetate layers, and concentrating under reduced pressure to obtain an extract, dissolving the extract in methanol, and then separating and purifying it 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 a trinitropropionic acid derivative isolated from Hypoxylon fragiforme, which has termite killing / antifungal activity. In the future, the scientific community will focus on finding active substances with termite control effects from Hypoxylon fungi.

[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. It is a green and environmentally friendly insecticide that can be used to prepare new biological pesticides.

[0016] 3. The trinitropropionic acid derivatives of the present invention can be obtained by extraction and purification from fungal fermentation. 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- 1H COSY diagram;

[0022] Figure 6 This 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] Example 1: The compound described in the claims and the invention summary - trinitropropionic acid derivatives - is obtained as follows:

[0027] Hypoxylon fragiforme was purchased from the China General Microbial Culture Collection Center, with the strain number 5.1297. Strain fermentation culture: H. fragiforme, stored in a 4°C refrigerator, was inoculated onto PDA culture medium and then incubated in a 28°C incubator in the dark for 7 days. After incubation, 50 kg of rice was prepared. 100 g of rice and 100 ml of tap water were added to each fermentation bottle. The culture was sterilized at 120°C for 30 minutes and then cooled to room temperature to prepare 500 bottles of rice culture medium. The incubated PDA culture medium was cut into small pieces (1 cm x 1 cm) in a sterile operating table. The pieces were placed in the rice culture medium for fermentation. The culture was placed in a sterile incubator at 28°C and incubated in the dark for 30 days. The H. fragiforme rice fermentation product was recovered.

[0028] Compound isolation and purification process: All the above H. fragiforme rice fermentation products were soaked in 100 L of 90% (v / v) methanol aqueous solution for 7 days, centrifuged, and the filtrate was concentrated to 5 L. The mixture was extracted repeatedly with ethyl acetate (5 L of ethyl acetate was used for each extraction) for 5 times. The ethyl acetate fractions were combined and concentrated under reduced pressure to obtain 563 g of the total extract. The total extract was completely dissolved with a reagent (methanol: Shanghai Lingfeng Chemical Reagent Co., Ltd., analytical grade), 570 g of normal phase silica gel (200-300 mesh) was added to adsorb the sample, placed in a fume hood to dry and ground into powder, and 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 500 ml of eluate was concentrated into a 20 ml vial and detected by thin layer chromatography (Yantai Jiangyou Silica Gel Development Co., Ltd., thin layer chromatography silica gel plate) and analyzed with vanillin for color development. 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.

[0029] 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 subjected to 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 subfractions were obtained, which were numbered (T9-1 to T9-5) according to the order of elution. The T9-3 component obtained by eluting with methanol-water (volume ratio of 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 one bottle filled with 15 mL. The product was detected by thin layer chromatography and colorimetric analysis using chloroform-methanol (20:1, v / v) as the developing solvent and concentrated sulfuric acid vanillin as the colorimetric developer to obtain 9 components (T9-3-1 to T9-3-9). The T9-3-5 fraction (eluted at 40 to 55 min) was purified by preparative high performance liquid chromatography (chromatographic column: Agilent Zorbax SB-C18 column, size: 9.4 mm × 250 mm; acetonitrile-water gradient elution: 0-35 min, 10% acetonitrile-30% acetonitrile (v / v); 35-40 min, 30% (v / v) acetonitrile; flow rate: 4 mL / min; detection wavelength: 210 nm) and concentrated to obtain the compound of the present invention (yellow oil, 18.68 mg, R t =37.8min).

[0030] Structural identification of the compound: The oily 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). Figure 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: yellow oil; specific rotation UV(MeOH)λmax(log ε ):210nm; High resolution mass spectrometry HR-ESI-MS m / z 242.0634[M+Na] + (calcd.for C8H 13 NO6Na:242.0635).

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

[0037]

[0038] Molecular formula: C8H 13 NO6.

[0039] Example 2: Bioassay of the present invention:

[0040] 100 pieces of 3.5cm*1.0cm 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 5 concentrations were set: 100 μmol / L, 100 μmol / L, and 100 μmol / L was the highest concentration). , 50, 25, 12.5, and 6.25 μmol / L, with each concentration repeated three times). After the filter paper is dried (dried at 50°C for 2 hours), it is weighed and then placed in a sterile Petri dish (9 cm * 9 cm) filled with sterile sand. The sterile sand and filter paper are moistened with a spray bottle (filled with sterile water), and 50 active termites of the same size are placed in it. The Petri dish is placed in an incubator and incubated in the dark at room temperature (about 25°C) and a humidity of 80%-90%. After 36 hours, the dead termites in the Petri dish are counted, the filter paper in the Petri dish is removed with tweezers, and the termite feces and sterile sand on the filter paper are rinsed with a rinse bottle. The filter paper is then dried and weighed, and the mass loss is used to represent the degree of termite refusal.

[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 (C8H 13 NO6) has significant insecticidal activity. At a concentration of 100 μmol / L, the trinitropropionic acid derivatives of the present invention have a feeding refusal rate of 92.31% on termites and a termite mortality rate of 45.53%.

Claims

1. A 3-nitropropionic acid derivative with the chemical formula C8H 13 NO6, 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: The rice fermentation product of Hypoxylon fragiforme is extracted by soaking in a methanol-water 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 3-nitropropionic acid derivative; The rice fermentation product of Hypoxylon fragiforme is prepared by the following method: inoculating Hypoxylon fragiforme stored in a refrigerator at 4°C on a PDA culture medium, and then placing the culture medium in a 28°C incubator in the dark for 7 days; after the culture is completed, preparing 50 kg of rice, adding 100 g of rice and 100 ml of tap water to each fermentation bottle, sterilizing the culture medium at 120°C for 30 minutes, and then cooling the culture medium at room temperature to obtain 500 bottles of rice culture medium; cutting the cultured PDA culture medium into 1 cm*1 cm blocks in a sterile operating table, placing the blocks into the rice culture medium for fermentation, placing the blocks in a sterile culture room at 28°C, and incubating the culture medium in the dark for 30 days to obtain the rice fermentation product of Hypoxylon fragiforme; The strain number of the Hypoxylon fragiforme is 5.1297.

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