Application of (E)-N'-benzylidenenicotinohydrazide compound

Abstract

The present invention discloses the application of an (E)-N'-benzylidene nicotinohydrazide compound, and the application of the (E)-N'-benzylidene nicotinohydrazide compound in the preparation of a drug for preventing and controlling tobacco mosaic virus. (E)-N'-benzylidene nicotinohydrazide can inhibit the infection of TMV to the host. The (E)-N'-benzylidene nicotinohydrazide pharmaceutical preparation of the present invention has a good effect on preventing and controlling tobacco mosaic virus disease and can be used for preparing a pharmaceutical preparation for preventing and controlling tobacco mosaic virus disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological applications, and further belongs to the technical field of biological pesticides. Specifically, it relates to the application of (E)-N'-benzylidene nicotinohydrazide compounds. Background Art

[0002] Plant virus diseases are known as plant cancers and are the second largest plant diseases after plant fungal diseases. Tobacco mosaic virus (TMV) is the pathogen of tobacco mosaic disease and other diseases. It has a wide host range and can infect more than 800 species in 65 families, including tobacco, pepper, tomato, eggplant, etc. Moreover, it is difficult to cure after infecting plants, easily causing serious economic losses. In China, the agricultural production areas are widely distributed and have a large area, resulting in relatively frequent mutations of TMV, and various symptom types have emerged, making the prevention and control work more difficult. Therefore, there is an urgent need to develop new antiviral preparations.

[0003] Currently, the reported effects of nicotinic acid derivatives are mainly in the treatment of nervous system diseases, in vitro antibacterial activity, and insecticidal activity. (E)-N'-benzylidene nicotinohydrazide is a nicotinic acid derivative designed with nicotine as the parent nucleus. So far, there is no report on the application of (E)-N'-benzylidene nicotinohydrazide compounds as a preparation for controlling tobacco mosaic disease and in the preparation of anti-tobacco mosaic virus drugs. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of (E)-N'-benzylidene nicotinohydrazide compounds.

[0005] The purpose of the present invention is achieved as follows: The application of the (E)-N'-benzylidene nicotinohydrazide compound in the preparation of drugs for preventing and controlling tobacco mosaic virus.

[0006] The (E)-N'-benzylidene nicotinohydrazide described in the present invention is synthesizable.

[0007] The molecular formula of the (E)-N'-benzylidene nicotinohydrazide is C 13 H 11 N 3 O, and the structural formula is:

[0008] 。

[0009] This compound is named: (E)-N'-benzylidene nicotinohydrazide

[0010] It has been experimentally proven in the present invention that (E)-N'-benzylidene nicotinohydrazide has high activity in inhibiting TMV infection. The (E)-N'-benzylidene nicotinohydrazide pharmaceutical preparation of the present invention has a good effect on preventing and controlling tobacco mosaic virus disease and can be used to prepare a pharmaceutical preparation for preventing and controlling tobacco mosaic virus disease.

[0011] When the compound of the present invention is used as a drug, it can be used directly or in the form of a pharmaceutical composition. The pharmaceutical composition contains 0.1-99%, preferably 0.5-90%, of the compound of the present invention, and the rest are pharmaceutically acceptable, inert pharmaceutically acceptable carriers and / or excipients. The said pharmaceutical carrier or excipient is one or more solid, semi-solid and liquid diluents, fillers and pharmaceutical product adjuvants. It is prepared into various dosage forms by methods recognized in the pharmaceutical and food fields, such as liquid preparations (aqueous solutions, emulsions, etc.), solid preparations (tablets, granules, etc.), sprays, aerosols, etc. Description of the Drawings

[0012] Figure 1 Fluorescence signal intensity in leaves at 120 h when a mixture of (E)-N'-benzylidenenicotinohydrazide compounds at different concentrations and Agrobacterium tumefaciens TMV-CP was inoculated on Nicotiana benthamiana;

[0013] Figure 2 Anti-TMV activity of (E)-N'-benzylidenenicotinohydrazide compounds at different concentrations;

[0014] Figure 3 Expression of plant defense-related genes induced by (E)-N'-benzylidenenicotinohydrazide compounds. Detailed Embodiments

[0015] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited in any way. Any transformation or substitution based on the teachings of the present invention falls within the protection scope of the present invention.

[0016] The application of the (E)-N'-benzylidenenicotinohydrazide compound described in the present invention is the application of the (E)-N'-benzylidenenicotinohydrazide compound in the preparation of drugs for preventing and treating tobacco mosaic virus.

[0017] The concentration of the (E)-N'-benzylidenenicotinohydrazide compound solution in the preparation of drugs for preventing and treating tobacco mosaic virus is 25-100 μg / mL.

[0018] The molecular formula of the said (E)-N'-benzylidenenicotinohydrazide compound is C 13 H 11 N 3 O, and the structural formula is as follows:

[0019] .

[0020] The said (E)-N'-benzylidenenicotinohydrazide compound solution is obtained by dissolving the (E)-N'-benzylidenenicotinohydrazide compound in a solvent according to the formulated ratio, stirring well and dissolving it.

[0021] The said solvent is DMSO solution.

[0022] The following is a further description of the present invention with specific implementation cases:

[0023] Example 1

[0024] Preparation of 0.5 mg / mL aqueous solution of (E)-N'-benzylidene nicotinohydrazide:

[0025] Weigh 1 mg of (E)-N'-benzylidene nicotinohydrazide compound and dissolve it in a small amount of DMSO solution. Stir well and mix. After it is fully dissolved, add the remaining distilled water to a final volume of 2 mL to make an aqueous solution, that is, an aqueous solution of 0.5 mg / mL (E)-N'-benzylidene nicotinohydrazide. Dilute it according to the required concentration when using.

[0026] Example 2

[0027] Preparation of 100 μg / mL aqueous solution of (E)-N'-benzylidene nicotinohydrazide:

[0028] Weigh 100 μg of (E)-N'-benzylidene nicotinohydrazide compound and dissolve it in a small amount of DMSO solution. Stir well and mix. After it is fully dissolved, add the remaining distilled water to a final volume of 1 mL to make an aqueous solution, that is, an aqueous solution of 100 μg / mL (E)-N'-benzylidene nicotinohydrazide.

[0029] Example 3

[0030] Preparation of 50 μg / mL aqueous solution of (E)-N'-benzylidene nicotinohydrazide:

[0031] Weigh 50 μg of (E)-N'-benzylidene nicotinohydrazide compound and dissolve it in a small amount of DMSO solution. Stir well and mix. After it is fully dissolved, add the remaining distilled water to a final volume of 1 mL to make an aqueous solution, that is, an aqueous solution of 50 μg / mL (E)-N'-benzylidene nicotinohydrazide.

[0032] Example 4

[0033] Preparation of 1000 μg / mL aqueous solution of (E)-N'-benzylidene nicotinohydrazide:

[0034] Weigh 1000 μg of (E)-N'-benzylidene nicotinohydrazide compound and dissolve it in a small amount of DMSO solution. Stir well and mix. After it is fully dissolved, add the remaining distilled water to a final volume of 1 mL to make an aqueous solution, that is, an aqueous solution of 1000 μg / mL (E)-N'-benzylidene nicotinohydrazide. Dilute it according to the required concentration when using.

[0035] Example 5

[0036] Preparation of 1 g of (E)-N'-benzylidene nicotinohydrazide emulsion:

[0037] Weigh 0.99 g of the (E)-N'-benzylidenenicotinohydrazide compound, add a little DMSO to dissolve it completely; then add 10% sodium dodecylbenzenesulfonate as an emulsifier and mix well to form an emulsion. Dilute the emulsion to the required concentration before use.

[0038] Example 6

[0039] Prepare 1 g of (E)-N'-benzylidenenicotinohydrazide tablets:

[0040] Weigh 0.4 g of the (E)-N'-benzylidenenicotinohydrazide compound, add a little DMSO to dissolve it completely; then add 0.1 g of wheat protein (powder), add water and stir well, then add 40% starch as an excipient, mix well, dry and press into tablets. Dissolve the tablets in water and dilute to the required concentration before use.

[0041] Example 7

[0042] Explore the effect of different concentrations of (E)-N'-benzylidenenicotinohydrazide on TMV based on an infectious clone experiment:

[0043] Use a pipette tip to pick the glycerol bacteria of tobacco mosaic virus infectious clone Agrobacterium (agro-p35s-30B:GFP) stored at -80°C into 2 mL of LB liquid medium containing antibiotics (rifampicin and kanamycin), culture at 28°C and 200 RPM for 24 h. After the first activation is completed, take 100 μL of the infection solution into the LB liquid medium and culture at 28°C and 200 RPM for 24 h to complete the activation. Centrifuge the activated TMV infection solution at 4000 RPM for 2 min to obtain a bacterial precipitate, adjust the OD value of the bacterial precipitate to about 0.8 with the infection buffer, and let it stand at 28°C for 8 hours before use.

[0044] Mix 50 μL of (E)-N'-benzylidenenicotinohydrazide compounds with concentrations of 25 μg / mL, 50 μg / mL, and 100 μg / mL respectively with 50 μL of the bacterial solution as injection agents. After mixing, let it stand at room temperature for 30 min and then inject. Use the mixture of 1% DMSO and the bacterial solution as a control group, and dilute the 8% ningnanmycin aqueous solution 1000 times as a positive control. Select Nicotiana benthamiana with similar growth vigor. For each plant, select leaves at the same level and the same part, and select one injection point on both sides of the leaf near the vein. Use a 1 mL sterile syringe to quantitatively inject from the back of the leaf (100 μL / injection point). Treat 3 plants in each group and repeat three times.

[0045] After 120 h of injection, the treated tobacco leaves were placed in an iBright 1500 instrument to photograph and record the fluorescence signal. Then, based on the iBright Analysis Software, the fluorescence intensity on the leaves was analyzed, and the inhibition rate of the compound was calculated. Inhibition rate (%) = [(fluorescence intensity of the control group - fluorescence intensity of the treatment group) / fluorescence intensity of the control group] × 100%

[0046] Test results: Figure 1 The figure shows the effect of (E)-N'-benzylidene nicotinohydrazide compounds at different concentrations on TMV. It can be seen that when treated with (E)-N'-benzylidene nicotinohydrazide compounds, the fluorescence intensity gradually weakens with the increase in concentration, and is significantly weaker than the 1% DMSO treatment group, and is close to the ningnanmycin group. After statistical analysis (see Figure 2 ), the inhibition rate of the ningnanmycin treatment group was 59.8%, and the inhibition rates of the (E)-N'-benzylidene nicotinohydrazide compound groups at 25 μg / mL, 50 μg / mL, and 100 μg / mL were 47%, 48.8%, and 60.9% respectively. Thus, it can be seen that the ability of (E)-N'-benzylidene nicotinohydrazide compounds to resist TMV gradually increases with the increase in concentration, and 100 μg / mL of (E)-N'-benzylidene nicotinohydrazide is most suitable as a drug preparation for preventing and treating tobacco mosaic virus disease.

[0047] Example 8

[0048] (E)-N'-benzylidene nicotinohydrazide's effect on plant-related defense genes:

[0049] Select healthy K326 tobacco plants with 5 - 6 leaves of similar growth, apply 200 μL of (E)-N'-benzylidene nicotinohydrazide at a concentration of 100 μg / mL, and use the group treated with 1% DMSO as a control. Each group treats 3 tobacco plants. After 12 h, samples are collected and ground in liquid nitrogen in a pre-cooled mortar. After extracting RNA, reverse transcription is carried out, and fluorescence quantitative PCR technology is used for relative quantitative analysis of plant defense-related genes.

[0050] The test results are as shown in Figure 3 . After treatment with 100 μg / mL of (E)-N'-benzylidene nicotinohydrazide, the relative expression levels of 4 plant defense-related genes all increased. Among them, RBOHB, PR5, POR1, and PR1 increased by 7.63, 2.15, 2.52, and 2.11 times respectively. These results indicate that (E)-N'-benzylidene nicotinohydrazide can up-regulate defense-related genes in plants, enhance the disease resistance of plants, and thus inhibit the replication and proliferation of TMV in plants.

Claims

1. An application of a (E)-N'-benzylidene nicotinic acid hydrazide compound, characterized in that: The (E)-N'-benzylidene nicotinic acid hydrazide compound is used in the preparation of drugs for preventing and treating tobacco mosaic virus.

2. The use of the (E)-N'-benzylidene nicotinic acid hydrazide compound according to claim 1, characterized in that: The concentration of the (E)-N'-benzylidene nicotinic acid hydrazide compound solution in the preparation of the drug for preventing and treating tobacco mosaic virus is 25-100 μg / mL; the (E)-N'-benzylidene nicotinic acid hydrazide compound solution is obtained by dissolving the (E)-N'-benzylidene nicotinic acid hydrazide compound of a formula ratio in a solvent and stirring and mixing it thoroughly to dissolve it.

3. The use of the (E)-N'-benzylidene nicotinic acid hydrazide compound according to claim 2, characterized in that: The solvent is DMSO solution.

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