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 hypoxylon fermentum 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 can reach 92.31%, and the termite death rate is 45.53%. The trinitropropionic acid derivative disclosed by the invention is obtained by extraction and purification from fungus fermentation, and the whole production process is free of chemical pollution, green and environment-friendly, and has potential application in preparation of novel green and environment-friendly pesticides.

Description

Technical Field

[0001] The invention relates to the field of pesticide and chemical technology, and in particular to trinitropropionic acid derivatives derived from Hypoxylon fragiforme and a preparation method thereof, and application of the trinitropropionic acid derivatives in preparing highly efficient, 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. According to statistics, the damage rate of termites in my country is as high as 80% to 90%, causing economic losses of about 2 to 2.5 billion per year, and the damage caused by termites to cultural relics and reservoir dams is difficult to estimate in terms of economic losses. At present, chemical synthetic insecticides are mainly used to prevent and 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 insecticides.

[0003] Hypoxylon fungi are a class of Ascomycetes fungi widely distributed around the world, mainly growing on dead wood or rotten wood. At present, the research on Hypoxylon mainly focuses on two directions: first, the disease problem caused by Hypoxylon; 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 object of the present invention is to provide a trinitropropionic acid derivative derived from a fungus (Hypoxylonfragiforme) and a preparation method thereof and an application thereof in termite control.

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

[0006]

[0007] Molecular formula: C8H 13 NO6.

[0008] The preparation method of the trinitropropionic acid derivatives with termite killing / resistance activity comprises the following steps: extracting rice fermentation product of Hypoxylon fragiforme by immersing in methanol aqueous solution (preferably with a volume concentration of 90%), extracting the extract by ethyl acetate 3-5 times after concentrating the extract under reduced pressure, combining the ethyl acetate layers, concentrating under reduced pressure to obtain an extract, dissolving the extract in methanol, and then separating and purifying 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 killing / anti-termite activity with the trinitropropionic acid derivatives as the main component also falls within the protection scope of the present invention.

[0010] 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.

[0011] 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.

[0012] 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.

[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 the activity of killing / resisting termites. 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 having termite killing / resistance activity, which has better environmental protection effect than widely used chemical pesticides, is a green and environmentally friendly insecticide, and 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 This is the HSQC diagram 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 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.

[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 of the specification - trinitropropionic acid derivatives is obtained as follows:

[0027] Hypoxylon fragiforme was purchased from the China General Microbiological Culture Collection Administration Center, and the strain number is 5.1297. Strain fermentation culture: The charcoal bacteria (H. fragiforme) stored in a 4°C refrigerator was inoculated on the PDA medium, and then placed in a 28°C incubator for 7 days in the dark. 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 high-temperature sterilization at 120°C for 30 minutes, it was cooled at 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, placed in a sterile culture room at 28°C, and the rice fermentation product of H. fragiforme was recovered after 30 days of dark culture.

[0028] Isolation and purification process of the compound: Use 100L of 90% (v / v) methanol aqueous solution to soak all the above H.fragiforme rice fermentation products for 7d, centrifuge and filter, concentrate the filtrate to 5L, extract repeatedly 5 times with ethyl acetate (5L of ethyl acetate for each extraction), combine the ethyl acetate parts and concentrate under reduced pressure to obtain 563g of 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, 200-300 mesh normal phase silica gel (Qingdao Ocean Chemical Co., Ltd., thin layer chromatography silica gel), dichloromethane-methanol (100:1, 50:1, 20:1, 10:1, 5:1, 1:1, 0:1, v / v) was used as the eluent for gradient elution, and each 500 ml of the eluent was concentrated in 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 by vanillin colorimetry, the developing solvent was dichloromethane-methanol (10:1, v / v), similar components were combined and concentrated to obtain 13 components, and the 13 components 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 eluted by MPLC medium pressure chromatography (Biotage, Sweden, C-615 pump manager, C-605 pump module, C-660 fraction collector, RP-18 chromatographic 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 added. The samples were detected and combined by thin layer chromatography, and chloroform-methanol (20:1, v / v) was used as the developing solvent and concentrated sulfuric acid vanillin was used as the color developer to obtain 5 subcomponents, which were numbered (T9-1 to T9-5) according to the order of elution. Take the T9-3 component obtained by eluting with methanol-water (volume ratio of 30:70), and elute it isocratically with anhydrous methanol through Sephadex LH-20 gel column chromatography at a flow rate of 10 mL / min. One bottle is filled with every 15 mL. Detect and color analyze by thin layer chromatography, chloroform-methanol (20:1, v / v) as the developing solvent, and concentrated sulfuric acid vanillin as the color developer to obtain 9 components (T9-3-1 to T9-3-9). The T9-3-5 component (eluted at 40 to 55 min) was taken and then 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, 10% acetonitrile-30% acetonitrile (v / v); 35-40 min, 30% (v / v) acetonitrile; flow rate of 4 mL / min; detection wavelength of 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, transferred to an NMR tube using a 200 μL pipette, and the hydrogen spectrum, carbon spectrum, and two-dimensional spectrum (such as Figure 1-6 ).

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

[0032] Table 1

[0033]

[0034] Other physical and chemical 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 is confirmed to be:

[0037]

[0038] Molecular formula: C8H 13 NO6.

[0039] Embodiment 2: Bioassay experiment of the present invention:

[0040] 100 pieces of 3.5cm*1.0cm filter paper were placed in a petri 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 for 2-fold gradient dilution: 100 μmol / L and 100 μmol / L respectively). , 50, 25, 12.5, 6.25 μmol / L, each concentration was repeated three times), after the filter paper was dried (dried at 50℃ for 2 hours), it was weighed, and then the filter paper was placed in a sterile culture dish (9cm*9cm) filled with sterile sand, the sterile sand and filter paper were moistened with a spray bottle (filled with sterile water), and 50 active termites of the same size were placed in it, and the culture dish was placed in an incubator, and cultured in the dark at room temperature (about 25℃) and humidity of 80%-90%. After 36 hours, the dead termites in the culture dish were counted, the filter paper in the culture dish was clamped out with tweezers, the termite feces and sterile sand on the filter paper were rinsed with a rinse bottle, and the filter paper was dried and weighed, and the mass loss was used to represent the degree of termite refusal to feed.

[0041] Calculate the repellency rate:

[0042] Termite antifeedant rate calculation formula:

[0043]

[0044] Corrected termite mortality calculation formula:

[0045]

[0046] Experimental results:

[0047] Table 2: The results of the 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, wherein at a concentration of 100 μmol / L, the antifeeding rate of the trinitropropionic acid derivatives of the present invention on termites can reach 92.31%, and the termite mortality rate is 45.53%.

Claims

1. A trinitropropionic acid derivative, the chemical formula of which is C8H 13 NO6, the structural formula is as follows:

2. Use of the trinitropropionic acid derivatives according to claim 1 in preventing and controlling agricultural and forestry pests.

3. Use of the trinitropropionic acid derivatives according to claim 1 in controlling termites.

4. The use according to claim 3, 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.

5. 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.

6. A method for preparing the trinitropropionic acid derivatives according to claim 1, characterized in that: The following steps are involved: The rice fermentation product of Hypoxylon fragiforme is soaked and extracted with a 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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