Use of a crude fungal metabolite extract 101H12 in the preparation of a mosquito larvicide

Abstract

This invention relates to the field of biotechnology, specifically to the application of a crude extract of a fungal metabolite, 101H12, in the preparation of mosquito repellents. The LC50 of the crude 101H12 extract against Culex pipiens mosquito larvae is also discussed. 50 The concentration was 31.90 mg / L. The LC50 of component 3 in the crude extract against Culex pipiens larvae was... 50 The concentration was 12.13 mg / L, which is the main component with insecticidal activity. Further isolation and identification yielded two monomeric compounds, vermistatin and dihydrovermistatin. The LC50 of both compounds against Culex pipiens larvae was determined. 50 The concentrations were 28.13 mg / L and 83.87 mg / L, respectively. The crude fungal extract 101H12 and the monomeric compounds vermistatin and dihydrovermistatin of the present invention can be used to prepare insecticides.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of a crude extract of fungal metabolite 101H12 in the preparation of mosquito repellents. Background Technology

[0002] Mosquitoes are important vectors for transmitting many serious human and animal diseases (such as malaria and dengue fever), posing a threat to public health. Vector control is considered an effective way to reduce mosquito-borne diseases. Due to the drawbacks of synthetic insecticides, such as residues and pollution, there is an urgent need to develop new insecticides. Entomopathogenic fungi are widely used in pest control, and their extracts also have the potential to be developed into insecticides or mosquito repellents. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art. The purpose of this invention is to provide an application of a crude extract of fungal metabolites, 101H12, in the preparation of mosquito repellents.

[0004] To achieve the above-mentioned application objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides the application of a crude extract of fungal metabolites 101H12 in the preparation of mosquito repellents; in a second aspect, the present invention provides a mosquito repellent.

[0005] The present invention relates to the application of a crude fungal metabolite extract 101H12 in the preparation of a mosquito repellent, wherein the gene sequence of the crude fungal metabolite extract 101H12 is the nucleotide sequence shown in SEQ ID No. 1.

[0006] Furthermore, the crude extract of fungal metabolites includes the monomeric compounds vermistatin and dihydrovermistatin.

[0007] Furthermore, the structural formula (I) of the vermistatin is shown:

[0008]

[0009] Furthermore, the structural formula (II) of the dihydrovermistatin is shown:

[0010]

[0011] Furthermore, the crude fungal metabolite extract 101H12 was added to the water in which mosquito larvae bred. The LC50 of the crude extract against Culex pipiens quinquefolius larvae was determined. 50 The concentration was 31.90 mg / L.

[0012] Furthermore, component 3 of the crude fungal metabolite extract 10¹H¹⁂ was added to the water in which mosquito larvae breed. The median lethal concentration (LC50) of component 3 for Culex pipiens quinquefolius larvae was determined. 50 It was 12.13 mg / L.

[0013] Furthermore, vermistatin, a monomeric compound of component 3 of the crude fungal metabolite extract 101H12, was added to the water in which mosquito larvae breed. The median lethal concentration (LC50) of vermistatin for Culex pipiens quinquefolius larvae was determined. 50 It was 28.13 mg / L.

[0014] Furthermore, the mosquito mentioned is Culex pipiens quinquefasciatus.

[0015] The present invention provides a mosquito repellent comprising the crude extract 101H12 of the fungal metabolite.

[0016] Furthermore, it also includes the monomeric compounds vermistatin and dihydrovermistatin.

[0017] Beneficial effects: The crude fungal extract 101H12 and the monomeric compounds vermistatin and dihydrovermistatin of the present invention can be used to prepare insecticides.

[0018] Compared with the prior art, the present invention has the following advantages: (1) The present invention uses ethyl acetate extraction to screen out the most effective crude fungal extract 101H12 from the fungal library. Bioassay results show that the crude fungal extract has high toxicity to Culex pipiens quinquefolius larvae, especially its component 3. The results of the present invention show the potential of the crude fungal ethyl acetate extract in mosquito control. (2) After discovering these candidate metabolites, the present invention isolates the active compounds and verifies their larvicidal activity. The results of the present invention show that vermistatin and dihydrovermistatin have the potential as novel insecticides. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This study aims to determine the toxicity of different concentrations of the crude fungal metabolite extract 101H12 to Culex mosquito larvae.

[0021] Figure 2The toxicity of four components of the crude fungal metabolite extract 101H12 of the present invention against Culex pipiens mosquito larvae was determined.

[0022] Figure 3 The toxicity of component 3 of the crude extract 101H12 of fungal metabolites at different concentrations of the present invention to Culex pipiens mosquito larvae was determined.

[0023] Figure 4 The toxicity of the monomeric compounds vermistatin and dihydrovermistatin of this invention to Culex mosquito larvae was determined. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] The first aspect of this application is to provide the use of a crude extract of fungal metabolite 101H12 of the present invention in the preparation of mosquito repellents.

[0026] The present invention relates to the application of a crude fungal metabolite extract 101H12 in the preparation of a mosquito repellent, wherein the gene sequence of the crude fungal metabolite extract 101H12 is the nucleotide sequence shown in SEQ ID No. 1.

[0027] The crude extract of fungal metabolites includes the monomeric compounds vermistatin and dihydrovermistatin.

[0028] The structure of vermistatin is shown in formula (I):

[0029]

[0030] The structural formula (II) of dihydrovermistatin is shown below:

[0031]

[0032] The crude fungal metabolite extract 10¹H¹⁂ was added to the water in which mosquito larvae breed. The LC50 of the crude extract against Culex pipiens quinquefolius larvae was determined. 50 The concentration was 31.90 mg / L.

[0033] Component 3 of the crude fungal metabolite extract 101H12 was added to the water in which mosquito larvae breed. The median lethal concentration (LC50) of component 3 for Culex pipiens quinquefolius larvae was determined. 50It was 12.13 mg / L.

[0034] Vermistatin, a monomeric compound of component 3 of the crude fungal metabolite extract 101H12, was added to the water in which mosquito larvae breed. The median lethal concentration (LC50) of vermistatin in Culex pipiens quinquefolius larvae was determined. 50 It was 28.13 mg / L.

[0035] The mosquito mentioned is Culex pipiens quinquefasciatus.

[0036] A second aspect of this application is to provide a mosquito repellent of the present invention, wherein the mosquito repellent comprises the crude extract 101H12 of the fungal metabolite.

[0037] It also includes the monomeric compounds vermistatin and dihydrovermistatin.

[0038] Example 1

[0039] The crude fungal extract 101H12 was provided by Associate Professor Jun Li, tenured, Florida International University, and purchased from Zhejiang Aolanding Biotechnology Co., Ltd. The purified fungal isolate was cultured in solid-state conditions for 4 weeks at room temperature in a culture medium consisting of Cheerios cereal and a 0.3% sucrose solution supplemented with 0.005% chloramphenicol. The scale-up solid-phase cultures were combined and extracted twice by soaking in ethyl acetate overnight. Mycelia in organic solvents were removed by filtration through Whatmann No. 1 filter paper. Finally, the ethyl acetate extract fraction was dried at room temperature using a rotary vacuum evaporator, and the fungal extract was dissolved in dimethyl sulfoxide (DMSO) to prepare a concentrated fungal secondary metabolite solution for later use.

[0040] Isolation and identification of active ingredients: 15–20 mg of crude extract was dissolved in methanol, centrifuged, and the supernatant was evaporated to dryness under reduced pressure to obtain the extract. 10 mg of the extract was dissolved in 1 mL of 75% methanol / water, filtered, and the supernatant was collected and stored in a DAD vial under refrigeration. The HPLC system was as follows: a semi-preparative column (10 × 250 mm, 5 μm) at a flow rate of 0.8 mL / min. Spectral comparison analysis was performed against previously reported compounds, and the structure of the monomer compound was deduced based on the monomer characteristics and its precipitation solvent.

[0041] Culex quinquefasciatus was provided by the Nanchang Municipal Center for Disease Control and Prevention.

[0042] Larvicidal bioassay: Larvicidal bioassays were conducted according to the standard methods recommended by the World Health Organization using 24-well plates. Each well contained 2 mL of distilled water. The test substance was dissolved in dimethyl sulfoxide (DMSO) and pipetted into the wells to prepare the final target concentration. The test concentrations for the crude extract 101H12 were 10, 20, 40, 60, 80, and 100 mg / L. The test concentrations for the four components of the crude extract 101H12 were 20 mg / L, and the test concentrations for component 3 and its monomeric compounds vermistatin and dihydrovermistatin were 5, 10, 15, 20, and 30 mg / L. Twenty-five fourth-instar larvae were transferred to the wells, and mortality was recorded after 24 hours. Larvae were considered dead if they did not react when touched with a soft plastic dropper. During the bioassay, the larvae were not provided with food. Four biological replicates were performed for each treatment, and three independent bioassays were conducted to determine the toxicity of different substances to mosquito larvae.

[0043] Experimental results showed that after 24 hours of treatment, 100 mg / L 10¹H¹² had a 100% lethality rate against Culex pipiens quinquefolius larvae, with a median lethal concentration (LC50) of 10¹H¹². 50 The concentration was 31.90 mg / L, with a 95% confidence interval of 26.957–37.14 mg / L. Figure 1 Component 3 of the four components in the crude 101H12 compound is highly toxic to Culex pipiens quinquefolius and is its main active mosquito-killing ingredient. Figure 2 The mortality rate of Culex pipiens quinquefolius after 24 hours of treatment with 20 mg / L of component 3 reached 90%, and the mortality rate was 100% at 30 mg / L. The median lethal concentration (LC50) was [not specified]. 50 The concentration was 12.13 mg / L, with a 95% confidence interval of 10.89–13.40 mg / L. Figure 3 Further separation and identification revealed that component 3 comprises the structural formulas of two monomeric compounds, vermistatin and dihydrovermistatin. Figure 4 As shown, bioassay results indicate that vermistatin is significantly more toxic to mosquito larvae than dihydrovermistatin. The median lethal concentration (LC50) of vermistatin in mosquito larvae is... 50 The median lethal concentration (LC50) of dihydrovermistatin in mosquito larvae was 28.13 mg / L. 50 The concentration was 83.87 mg / L, indicating that the crude extract 101H12 and its active ingredients have a good effect on mosquito control.

[0044] The above results indicate that the lethality of the crude extract 101H12 and its active ingredients on Culex pipiens quinquefolius larvae is dose-dependent; the higher the concentration, the higher the mortality rate of Culex pipiens quinquefolius larvae.

[0045] The above results all indicate that the crude fungal extract 101H12, component 3, and vermistatin are all important candidate compounds for future mosquito repellent development.

[0046] The foregoing description illustrates and describes several preferred embodiments of the present invention. However, as mentioned above, it should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations and modifications, and can be altered within the scope of the present invention through the foregoing teachings or related technical or knowledge. Any modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.

Claims

1. The application of a monomeric compound vermistatin and a monomeric compound dihydrovermistatin in the preparation of mosquito repellents, characterized in that: The structural formula of the monomeric compound vermistatin is shown in formula (I): The structural formula of the monomer compound dihydrovermistatin is shown in formula (II):

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