A trinitropropionic acid derivative derived from a fermentation product of carbonaria, and its preparation method and application

Abstract

The present invention relates to the field of pesticides and chemical technology, and specifically discloses a pesticide derived from Hypoxylon fragiforme The trinitropropionic acid derivatives of the present invention are green and environmentally friendly insect repellents and can be used to prepare new biopesticides.

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 Hypoxylonfragiforme, a preparation method thereof, and application of the trinitropropionic acid derivatives in preparing biological pesticides with anthelmintic activity. Background Art

[0002] The most important part of green and environmentally friendly pesticides is biopesticides. Biopesticides refer to pesticides that use certain naturally occurring compounds through non-traditional toxic mechanisms to interfere with the growth and reproduction of pests and diseases, thereby achieving effective prevention and control of pests and diseases.

[0003] The production process of biopesticides primarily derives from natural materials (such as plants, animals, and microorganisms) and their metabolites. For example, effective microorganisms (bacteria or fungi) are first screened from the natural environment. These microorganisms are then cultured in the laboratory to determine the optimal conditions for producing insect repellent ingredients. After screening and initial optimization, large-scale fermentation production can begin. In fermentation tanks, under well-controlled environmental conditions, these microorganisms multiply in large numbers, producing the desired active ingredients. After fermentation, these active ingredients are extracted and purified before being formulated into suitable pesticide products.

[0004] Hypoxylon fragiforme, a member of the Hypoxylon fungus family, grows on the trunks of Rhizophora mangle trees in central Colombia. A study published in the journal Applied Microbiology and Biotechnology documented its ability to convert lignocellulose into advanced biofuels. Analysis of fermentation in PDA medium revealed that the main products produced were 1,8-cineole, isopentanol, and beta-threne. These volatile compounds can be used as liquid transportation fuels. Currently, there are very few reports on the chemical composition and biological activities of the fungus Hypoxylon fragiforme, with only a few compounds demonstrating effective conversion of cellulose resources into energy. Summary of the Invention

[0005] To address the shortcomings of the prior art, the present invention provides trinitropropionic acid derivatives derived from the fungus Hypoxylon fragiforme, their preparation methods, and their use in the preparation of biopesticides with anthelmintic activity. This application is the first to isolate trinitropropionic acid derivatives with anthelmintic activity from the fermentation products of the fungus Hypoxylon fragiforme.

[0006] The first object of the present invention is to disclose a trinitropropionic acid derivative having anthelmintic activity, the structural formula of which is as follows:

[0007]

[0008] Molecular formula: C8H 15 NO5.

[0009] The present invention also provides a method for preparing the above-mentioned trinitropropionic acid derivatives with anthelmintic activity, comprising the following steps: soaking and extracting rice fermentation product of H. fragiforme with a methanol aqueous solution (preferably with a volume concentration of 90%), concentrating the extract under reduced pressure, and then extracting the extract 3-5 times with ethyl acetate to obtain an extract, dissolving the extract in methanol, and then separating and purifying the extract by normal phase silica gel column chromatography, reverse phase silica gel column chromatography, gel column chromatography, and high performance liquid chromatography in sequence to obtain the trinitropropionic acid derivatives.

[0010] The use of the trinitropropionic acid derivatives in the preparation of preparations and compositions with anthelmintic activity containing the trinitropropionic acid derivatives as main components also falls within the scope of protection of the present invention.

[0011] Another object of the present invention is to provide the use of the above-mentioned trinitropropionic acid derivatives in the preparation of biological pesticides with anthelmintic activity.

[0012] Furthermore, the insect repellent is specifically to repel woodworms.

[0013] The experiments of the present invention prove that the trinitropropionic acid derivatives show obvious repellent effect on woodworms in the experiment of repelling woodworms, and have potential use as preparation of new biological pesticides.

[0014] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0015] 1. The present invention is the first to disclose that trinitropropionic acid derivatives isolated from the fungus H. fragiforme have the activity of repelling wood borers, which may enable the scientific community to focus on searching for substances with anthelmintic activity from Hypoxylon in the future.

[0016] 2. 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 compound 1 prepared in Example 1 in the specific embodiment;

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

[0019] Figure 3 This is the HSQC chart of compound 1 prepared in Example 1 in the specific embodiment;

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

[0021] Figure 5 In the specific embodiment, Example 1 is used to prepare compound 1. 1 H- 1 H COSY diagram;

[0022] Figure 6 This is the ROESY diagram of compound 1 prepared in Example 1 in a specific embodiment;

[0023] Figure 7 The figure shows the repellent effect (12 h) of compound 1 (0.68 mg / ml) on woodworm larvae in Example 2 of the specific embodiment. DETAILED DESCRIPTION

[0024] 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.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0026] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0027] Hypoxylon fragiforme was purchased from the China General Microbial Culture Collection Center, with the strain number being 5.1297. The rice culture medium was formulated as follows: 100 g of rice and 100 mL of water were added to each conical flask, sterilized at 120°C for 30 minutes, and then cooled to room temperature.

[0028] Example 1: The trinitropropionic acid derivatives of Compound 1 described in the claims and the invention description are obtained as follows:

[0029] (1) H. fragiforme stored at 4°C was taken out and inoculated on the surface of a PDA culture medium. The culture medium was then transferred to a constant temperature environment at 28°C in the dark for 7 days and then cut into bacterial blocks (1 cm*1 cm in size). Next, 500 bacterial blocks were evenly placed on 500 bottles of rice culture medium for fermentation. After fermentation in a culture room at 28°C in the dark for 30 days, the rice fermentation product of H. fragiforme was obtained.

[0030] (2) The entire H. fragiforme rice fermentation product obtained in step (1) was soaked in 100 L of a 90 v / v% methanol aqueous solution for 7 days. After centrifugation and filtration, the filtrate was concentrated under reduced pressure to 5 L. The concentrate was then extracted five times with ethyl acetate (5 L of ethyl acetate each time). The collected ethyl acetate layers were combined and concentrated under reduced pressure to obtain 563 g of a total extract.

[0031] (3) The total extract was completely dissolved in anhydrous methanol, 570 g of normal phase silica gel (particle size 200-300 mesh) was added to adsorb the sample, placed in a fume hood to air dry and ground into powder. The sample was separated by normal phase silica gel column chromatography (supplier: Qingdao Ocean Chemical Co., Ltd., thin layer chromatography silica gel with a particle size of 200-300 mesh) and eluted with dichloromethane-methanol (volume ratio of 100:1, 50:1, 20:1, 10:1, 5:1, 1:1, 0:1) as the elution gradient. Every 500 ml of eluate was collected once. Thin layer chromatography (supplied by Yantai Jiangyou Silica Gel Development Co., Ltd., thin layer chromatography silica gel plate) was used for detection and color analysis with vanillin. The developing solvent was dichloromethane-methanol (volume ratio 10:1). The components with similar Rf values ​​were combined and concentrated to obtain 13 components. These components were numbered T1 to T13 in the order of elution.

[0032] (4) 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) was added. The sample was adsorbed on RP-18, 40-70 μm, air-dried in a fume hood, ground into powder, and then subjected to 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) and concentrated sulfuric acid vanillin as the color developer. Five subfractions were obtained and numbered (T9-1 to T9-5) according to the order of elution.

[0033] (5) 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, and one bottle was filled with 15 mL. The product was detected by thin-layer chromatography and color analysis, with chloroform-methanol (20:1, v / v) as the developing solvent and concentrated sulfuric acid vanillin as the color development agent, to obtain 9 components (T9-3-1 to T9-3-9). The eluted T9-3-5 component (40 to 55 min) was taken and then subjected to preparative high performance liquid chromatography (column: Agilent Zorbax SB-C18 column, size: 9.4mm×250mm), acetonitrile-water (0-35min, 10:90-30:70, v / v; 4mL / min, detection wavelength 210nm) gradient elution separation, evaporation concentration, to prepare the trinitropropionic acid derivative compound 1 of the present invention (yellow oil, 20.43mg, R t =34.6min).

[0034] Structural identification of the compound: The oily compound prepared in Example 1 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., 1.5 Å) were detected on an NMR spectrometer (Bruker Avance III 600 MHz, Germany). Figure 1-6 ).

[0035] The nuclear magnetic resonance data of the obtained trinitropropionic acid derivative compound 1 are shown in Table 1:

[0036] Table 1

[0037]

[0038]

[0039] Other physical and chemical data of trinitropropionic acid derivative compound 1:

[0040] Appearance: yellow oil; specific rotation (c=0.5,MeOH); UV(MeOH)λmax(log ε ):210nm; High resolution mass spectrometry HR-ESI-MS m / z 228.0842[M+Na] + (calcd for C8H 15 NO5N a :228.0842).

[0041] According to the results of the above tests, the structural formula of the obtained compound 1 is confirmed to be as follows:

[0042]

[0043] Molecular formula: C8H 15 NO5.

[0044] Example 2: Bioassay of the present invention:

[0045] 1. Test insects: early third-instar larvae of woodworms, provided by Tengfei Insect Industry, Qiannan Prefecture, Guizhou Province.

[0046] 2. Samples and reagents:

[0047] Reagents:

[0048] The solvent was sterilized pure water (pure water was provided by Hangzhou Wahaha Group Co., Ltd., and the vertical pressure steam sterilizer was provided by Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory).

[0049] Sample (Compound C8H prepared in Example 1) 15 NO5):

[0050] The test compound was dissolved in sterilized pure water. The highest concentration of the test compound was 1.36 mg / ml, and the mixture was diluted 2-fold to set 5 concentrations: 1.36, 0.68, 0.34, 0.17, and 0.08 mg / ml.

[0051] 3. Bioassay method:

[0052] The filter paper immersion method was used: a 9cm diameter circular filter paper (Situofan Biotechnology Co., Ltd.) was cut in half and sterilized. After sterilization, it was dried. 400μl of sterilized pure water was added to each of the two halves as a control group and 400μl of different concentrations of sample as a treatment group. After the filter paper was dried, solid glue was applied to the back of the filter paper and affixed to both sides of a 9cm culture dish. Five fireworms of the same size were placed in the culture dish with the filter paper affixed (five at a time, with one concentration repeated three times). The culture was incubated in the dark at room temperature (25°C) and a humidity of 60%-80% for 12 hours. After 12 hours, the number of fireworms on both sides of the filter paper was observed and the repellency rate was calculated.

[0053] 4. Calculate the repellency rate:

[0054] The calculation formula of the repellency rate of the treatment group to woodworm is:

[0055]

[0056] RC 50 It refers to the concentration of the compound when the repellency is 50%, RC 50 The calculation software used was GraphPad Prism 5.

[0057] 5. Experimental results:

[0058] Table 2: Repellent effect of the trinitropropionic acid derivative 1 of the present invention on the early third-instar woodworm larvae

[0059]

[0060] The results showed that the trinitropropionic acid derivative 1(C8H 15 NO5) has significant repellent activity against woodworms, among which the RC 50 It is 0.16mg / ml.

Claims

1. A 3-nitropropionic acid derivative having the following structural formula: ； Molecular formula: C8H 15 NO5.

2. The method for preparing the 3-nitropropionic acid derivatives according to claim 1, comprising the following steps: The rice fermentation product of H. fragiforme is extracted by soaking in a methanol-water solution, the extract is concentrated under reduced pressure and then extracted 3-5 times with ethyl acetate to obtain an extract, the extract is dissolved in methanol, and then separated and purified by normal phase silica gel column chromatography, reverse phase silica gel column chromatography, gel column chromatography and high performance liquid chromatography to obtain the 3-nitropropionic acid derivative; The rice fermentation product of H. fragiforme is prepared by the following method: H. fragiforme stored at 4°C was inoculated on the surface of a PDA culture medium, then transferred to a constant temperature environment at 28°C in the dark for 7 days and cut into 1 cm x 1 cm blocks. Subsequently, 500 blocks were evenly placed on 500 bottles of rice culture medium for fermentation. After fermentation in a dark room at 28°C for 30 days, the H. fragiforme rice fermentation product was obtained. The H. fragiforme strain is numbered 5.1297.

3. Use of the 3-nitropropionic acid derivatives according to claim 1 in the preparation of biological pesticides having the activity of repelling woodworms.

4. Use of the 3-nitropropionic acid derivatives obtained by the preparation method according to claim 2 in the preparation of biological pesticides with woodworm repellent activity.

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