Application of nitrogen-containing heterocyclic compound in prevention and treatment of ampullaria gigas

Abstract

The invention belongs to the technical field of pest control, and discloses application of a nitrogen-containing heterocyclic compound in control of ampullaria gigas. According to the invention, a nitrogen-containing heterocyclic compound with the activity of killing ampullaria gigas is screened by taking ampullaria gigas arginine kinase as an action target, and the nitrogen-containing heterocyclic compound is N-[4-(3, 5-diphenyl-4, 5-dihydro-1H-pyrazole-1-yl) benzyl]-N-(1-hexyl-1H-benzimidazole-2-yl) amine. The nitrogen-containing heterocyclic compound is used as an active component to be prepared into a suspending agent or a granule, the suspending agent or the granule has an excellent killing effect on ampullaria gigas, and compared with an existing arginine kinase inhibitor quercetin, the nitrogen-containing heterocyclic compound is higher in activity, good in selectivity, low in toxicity to vertebrates and huge in application potential.

Description

Technical Field

[0001] The invention belongs to the technical field of pest control, and in particular relates to application of a nitrogen-containing heterocyclic compound in controlling golden apple snails. Background Art

[0002] Pomacea canaliculata is an amphibian freshwater mollusk with a strong ability to adapt to the environment. It can survive in different water environments such as rivers, ponds, and rice fields. In addition to threatening the ecosystem of the invaded area, the Pomacea canaliculata is also an intermediate host of the Guangzhou roundworm (Angiostrongylus cantonensis), which can easily cause disease due to ingestion of unprocessed and cooked Pomacea canaliculata. The impact of Pomacea canaliculata on agriculture is becoming increasingly serious. At present, the management of Pomacea canaliculata is still mainly based on chemical agents. The long-term use of traditional chemical agents not only pollutes the environment, but also causes the continuous increase of drug resistance in Pomacea canaliculata, which in turn promotes the continuous increase of drug use, entering a vicious circle. Therefore, it is particularly important to develop new Pomacea canaliculata killing agents based on the drug's target of action.

[0003] Studies have shown that by inhibiting or interfering with arginine kinase (AK), thereby affecting energy metabolism, the purpose of killing or destroying pests can be achieved. Arginine kinase is a phosphokinase widely distributed in invertebrates, and is directly related to energy transport, muscle contraction, and ATP regeneration in insects. In addition to the above functions, arginine kinase also has the functions of participating in adverse stress, maintaining autoimmunity, and paralyzing host insects. And because arginine kinase only exists in invertebrates, it is used as a target molecule in agricultural pest control. Studies on the lethality of arginine kinase RNA interference have been verified in many insects such as psyllids, houseflies, and red flour beetles.

[0004] Computer aided drug design (CADD) is a drug design method based on computational chemistry. It designs and optimizes lead compounds by computer simulation and prediction of the interaction between small molecule compounds and target receptors. With the continuous progress of computer science and the mutual penetration of quantum mechanics, molecular mechanics, molecular dynamics and pharmaceutical science, CADD technology has developed from basic theoretical research to a practical discipline. In recent years, CADD technology has made certain progress in pesticides, molluscicides, fungicidal compounds, herbicides, etc. For example, Yang et al. used the PcRoo protein of Pomacea canaliculata as a receptor and performed simulation screening in the DrugBank database to obtain two target active compounds, baclofen and acetaminophen, which have the ability to kill mollusks. Chen et al. selected PcAdv and PcnWAS of Pomacea canaliculata as target proteins, and finally obtained a composite potassium alginate with the activity of killing Pomacea canaliculata through simulation screening in the DrugBank database.

[0005] However, arginine kinase is an important enzyme in the body of the golden apple snail, but there are few killing compounds that target the golden apple snail arginine kinase. Therefore, it is necessary to screen out killing compounds that target the golden apple snail arginine kinase in order to further achieve effective control of the golden apple snail. Summary of the invention

[0006] In order to further enrich the existing ingredients for killing Pomacea canaliculata and achieve effective prevention and control of Pomacea canaliculata, the present invention uses CADD technology to screen out a nitrogen-containing heterocyclic compound with activity for killing Pomacea canaliculata. The present invention specifically provides the following technical solutions.

[0007] Firstly, the present invention provides an application of a nitrogen-containing heterocyclic compound in preventing and controlling Pomacea canaliculata, wherein the nitrogen-containing heterocyclic compound is N-[4-(3,5-diphenyl-4,5-dihydro-1H-pyrazol-1-yl)benzyl]-N-(1-hexyl-1H-benzimidazol-2-yl)amine.

[0008] Furthermore, in the application, the nitrogen-containing heterocyclic compound is used as an active ingredient to act on Pomacea canaliculata.

[0009] Furthermore, in the application, the nitrogen-containing heterocyclic compound is prepared into a suspension or granules.

[0010] Furthermore, in the application, the concentration of the nitrogen-containing heterocyclic compound in the suspension is ≥3.8999 mg / L; and the content in the granules is ≥1 wt%.

[0011] Based on the above use, the present invention provides a method for treating Pomacea canaliculata, which comprises: using the above nitrogen-containing heterocyclic compound as an active ingredient to act on Pomacea canaliculata.

[0012] Furthermore, in the method, the nitrogen-containing heterocyclic compound is prepared into a suspension or granules.

[0013] Furthermore, in the method, the concentration of the nitrogen-containing heterocyclic compound in the suspension is ≥3.8999 mg / L; and the content in the granules is ≥1 wt%.

[0014] Compared with the prior art, the invention "application of a nitrogen-containing heterocyclic compound in preventing and treating Pomacea canaliculata" has the following beneficial effects:

[0015] The invention uses the arginine kinase of Pomacea canaliculata as a target and screens out a nitrogen-containing heterocyclic compound with the activity of killing Pomacea canaliculata by using CADD technology. After the nitrogen-containing heterocyclic compound is used to kill the Pomacea canaliculata for 24 hours, 48 ​​hours and 72 hours, the killing rates of the Pomacea canaliculata for the Pomacea canaliculata are 30%, 70% and 80% respectively, the corrected mortality rates are 30.00%, 66.67% and 77.78% respectively, and the LC of 72 hours 50 The value is 3.8999 mg / L, with significant results.

[0016] Compared with the existing arginine kinase inhibitor quercetin, the nitrogen-containing heterocyclic compound provided by the present invention has higher activity and low toxicity to vertebrates, is safe, and has great potential for agricultural application. Specifically, after the nitrogen-containing heterocyclic compound is treated with Pomacea canaliculata for 24h, 48h and 72h, the LC 50 The values ​​were 7.9919 mg / L, 5.3477 mg / L, and 3.8999 mg / L, respectively, which were lower than quercetin's 319.2637 mg / L, 53.6744 mg / L, and 30.4203 mg / L, respectively, and the killing effect was better.

[0017] The present invention has been proved through experiments that the nitrogen-containing heterocyclic compound has a good effect of killing golden apple snails in a field environment. Specifically, the average control effects of 1wt% and 3wt% nitrogen-containing heterocyclic compound granules in 14 days are 87% and 95% respectively, indicating that the field application prospects of the nitrogen-containing heterocyclic compound are good.

[0018] The invention is of great significance for reducing the bite damage of golden apple snails to aquatic crops such as rice, lotus, wild rice stem, taro, water chestnut, water caltrop, gorgon fruit, water spinach, etc., and avoiding the reduction of aquatic crops due to the damage of golden apple snails. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the chemical structural formula of the nitrogen-containing heterocyclic compound. DETAILED DESCRIPTION

[0020] The present invention is explained below in conjunction with the embodiments, and the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0022] In the following embodiments, the nitrogen-containing heterocyclic compound is: N-[4-(3,5-diphenyl-4,5-dihydro-1H-pyrazol-1-yl)benzyl]-N-(1-hexyl-1H-benzimidazol-2-yl)amine, purchased from specs company, with a purity of 99%, and its chemical structure is shown in Figure 1 .

[0023] Example 1

[0024] This example describes the screening of the nitrogen-containing heterocyclic compounds and their activity in killing Pomacea canaliculata snails.

[0025] 1. Test of Pomacea canaliculata

[0026] The experiment was carried out by artificially breeding in the Non-polluting Pesticide Research Laboratory of Sichuan Agricultural University, and individuals of the golden apple snail with a shell diameter of 5±3mm were selected.

[0027] 2. Screening Method

[0028] Computer-aided drug design (CADD) technology was used for design: the tertiary structure of arginine kinase was constructed through the I-TASSER server; the target protein was rigidly docked using Discovery Studio; and the target protein was flexibly docked using Autodock4.0 to simulate and screen out inhibitors of Pomacea canaliculata arginine kinase.

[0029] 3. Test on killing golden apple snails

[0030] The killing effect of the nitrogen-containing heterocyclic compound on Pomacea canaliculata under different treatment time conditions was determined by the immersion killing method. The specific method is as follows:

[0031] Weigh the compound screened in step 2 and prepare it into 5mL of suspension with a concentration of 1g / L. The suspension contains 5mg of the compound, 2% of the volume of the suspension by dimethyl sulfoxide, 1% of the volume of the suspension by Tween 80, and the volume is supplemented with water to 100%. Dilute the suspension to 5mg / L and add it to a twelve-well plate, adding 5mL to each well. Then put the tested Pomacea canaliculata snails into the twelve-well plate, 5 heads in each well, and replace the diluent with clean water as a blank control. The rest of the treatment steps are the same. Repeat each treatment 3 times, place it in an incubator at a temperature of 25°C and a humidity of 80%, seal the orifice with gauze to prevent the Pomacea canaliculata snail from escaping, check the death of the Pomacea canaliculata snails at 24h, 48h and 72h, and calculate the corrected mortality rate.

[0032] Adjusted mortality rate = [(mortality rate of treatment group - mortality rate of control group) / (1 - mortality rate of control group)] × 100%.

[0033] 4. Test results

[0034] The results of the killing effect of the nitrogen-containing heterocyclic compound on Pomacea canaliculata at different times are shown in Table 1.

[0035] Table 1. Corrected mortality of Pomacea canaliculata treated with the nitrogen-containing heterocyclic compounds

[0036] Processing time Treatment group mortality Control group mortality Adjusted mortality rate 24h 30％ 0％ 30.00％ 48h 70％ 10％ 66.67％ 72h 80％ 10％ 77.78％

[0037] As shown in Table 1, under the condition of 25°C, the killing rates of the nitrogen-containing heterocyclic compound on the tested Pomacea canaliculata were 30%, 70%, and 80% respectively after immersion treatment for 24h, 48h, and 72h, and the corrected mortality rates were 30.00%, 66.67%, and 77.78% respectively. This indicates that the nitrogen-containing heterocyclic compound has a significant killing effect on Pomacea canaliculata, and the killing effect of the treatment for 48h and 72h is significantly better than that of the treatment for 24h.

[0038] Example 2

[0039] This example describes the comparison of the effects of the nitrogen-containing heterocyclic compound and quercetin (an existing arginine kinase inhibitor) in killing Pomacea canaliculata.

[0040] 1. The tested Pomacea canaliculata snails were the same as those in Example 1.

[0041] 2. Test methods

[0042] The killing effects of the nitrogen-containing heterocyclic compound and quercetin on Pomacea canaliculata at different concentrations were determined by the immersion killing method. The specific process is as follows:

[0043] The compound screened out in step 2 of Example 1 and quercetin were prepared into 5 mL of suspension with a concentration of 1 g / L. The suspension contained 5 mg of the compound, 2% of the volume of the suspension was dimethyl sulfoxide, 1% of the volume of the suspension was Tween 80, and the volume was supplemented with water to 100%. And diluted to 4 mg / L, 6 mg / L, 8 mg / L, 10 mg / L, and 12 mg / L respectively. The diluents were added to twelve-well plates, and 5 mL was added to each well. Then the tested golden apple snails were placed in twelve-well plates, 5 heads were placed in each well, and clean water was used instead of the diluent as a blank control. The rest of the treatment steps were the same, and each treatment was repeated 3 times. Placed in an incubator at a temperature of 25°C and a humidity of 80%, the orifice was sealed with gauze to prevent the golden apple snail from escaping, and the death of the golden apple snail was checked at 24h, 48h and 72h. Establish a toxicity regression equation and calculate the LC 50 value.

[0044] 3. Test results

[0045] The toxicity regression equation and LC of the nitrogen-containing heterocyclic compounds and quercetin in killing Pomacea canaliculata 50 See Table 2 for values.

[0046] Table 2. Comparison of the effects of the nitrogen-containing heterocyclic compounds and quercetin in killing Pomacea canaliculata

[0047]

[0048]

[0049] As shown in Table 2, the LC values ​​of quercetin on the tested Pomacea canaliculata were 24 h, 48 h and 72 h after treatment. 50 The values ​​were 319.2637 mg / L, 53.6744 mg / L, and 30.4203 mg / L, respectively. The LC values ​​of the nitrogen-containing heterocyclic compounds on the tested Pomacea canaliculata were 50 The values ​​were 7.9919 mg / L, 5.3477 mg / L, and 3.8999 mg / L, respectively, all lower than quercetin. The LC values ​​of nitrogen-containing heterocyclic compounds after 48 h and 72 h treatment were 50 The value was significantly lower than the LC after 24 h treatment. 50 The results show that the nitrogen-containing heterocyclic compound is more active than quercetin and has a better killing effect on Pomacea canaliculata, and the killing effect is significant after 48 hours of treatment.

[0050] Example 3

[0051] This example describes the field test effect of the nitrogen-containing heterocyclic compound in killing Pomacea canaliculata snails.

[0052] 1. Test reagents

[0053] The nitrogen-containing heterocyclic compound is prepared into granules, and the granules have the following components: 1-6 wt% of the nitrogen-containing heterocyclic compound, 1 wt% of rapeseed oil, 2 wt% of sodium carboxymethyl cellulose (binder), 5 wt% of white sugar, 5% of cream flavor, and corn flour to 100 wt%. The granules are prepared and provided by the pollution-free pesticide research laboratory of Sichuan Agricultural University.

[0054] 2. Test methods

[0055] The soil type is clay, the soil fertility is medium, and the cultivation conditions of the plots are consistent. The plot area is 5m 2 100 clumps of rice seedlings were transplanted into each plot, with 5 plants in each clump. Plastic film was used to isolate the surrounding areas and the water depth was maintained at 5±2cm. 7 days after transplanting, 100 golden apple snails of the same size were artificially released into each plot. Granules containing 1wt%, 3wt%, and 6wt% of nitrogen-containing heterocyclic compounds were evenly spread in the water, and the control group was not treated. Each treatment was repeated 3 times. The death of golden apple snails in the plot was investigated 1d, 3d, 7d, and 14d after application, and the corrected mortality rate was calculated.

[0056] Adjusted mortality rate = [(mortality rate of treatment group - mortality rate of control group) / (1 - mortality rate of control group)] × 100%.

[0057] 3. Test results

[0058] As can be seen from Table 3, 1wt% nitrogen-containing heterocyclic compound granules and 3wt% nitrogen-containing heterocyclic compound granules have good field control effects on Pomacea canaliculata, with an average control effect of 87% and 95% for 14 days, respectively. However, when 6wt% nitrogen-containing heterocyclic compound granules were used, the mortality rate of Pomacea canaliculata was reduced, mainly because the Pomacea canaliculata refused to eat.

[0059] Table 3. Field control effect of nitrogen-containing heterocyclic compound granules (%)

[0060]

[0061] In summary, the present invention uses the arginine kinase of Pomacea canaliculata as a target, and screens out a nitrogen-containing heterocyclic compound with the activity of killing Pomacea canaliculata, which is N-[4-(3,5-diphenyl-4,5-dihydro-1H-pyrazol-1-yl)benzyl]-N-(1-hexyl-1H-benzimidazol-2-yl)amine. The nitrogen-containing heterocyclic compound is used as an active ingredient to prepare a suspension or a granule, which has an excellent killing effect on Pomacea canaliculata, and compared with the existing arginine kinase inhibitor quercetin, it has higher activity, better selectivity, and lower toxicity to vertebrates, and has great application potential.

[0062] The embodiments described above are only some embodiments of the present invention, not all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but only represents selected embodiments of the present invention. All other embodiments obtained without creative work and related deductions and substitutions made by ordinary technicians in the field under the conditions of the concept of the present invention belong to the scope of protection of the present invention.

Claims

1. An application of a nitrogen-containing heterocyclic compound in preventing and treating Pomacea canaliculata, characterized in that: The nitrogen-containing heterocyclic compound is N-[4-(3,5-diphenyl-4,5-dihydro-1H-pyrazol-1-yl)benzyl]-N-(1-hexyl-1H-benzimidazol-2-yl)amine.

2. The use according to claim 1, characterized in that: The nitrogen-containing heterocyclic compound is used as an active ingredient to act on Pomacea canaliculata.

3. The use according to claim 2, characterized in that: The nitrogen-containing heterocyclic compound is prepared into a suspension or granules.

4. The use according to claim 3, characterized in that: The concentration of the nitrogen-containing heterocyclic compound in the suspending agent is ≥3.8999 mg / L.

5. The use according to claim 3, characterized in that: The content of the nitrogen-containing heterocyclic compound in the granules is ≥1wt%.

6. A method for controlling golden apple snails, characterized in that: The method comprises: using a nitrogen-containing heterocyclic compound N-[4-(3,5-diphenyl-4,5-dihydro-1H-pyrazol-1-yl)benzyl]-N-(1-hexyl-1H-benzimidazol-2-yl)amine as an active ingredient to act on Pomacea canaliculata.

7. The method for controlling Pomacea canaliculata according to claim 6, characterized in that: The nitrogen-containing heterocyclic compound is prepared into a suspension or granules.

8. The method for controlling Pomacea canaliculata according to claim 7, characterized in that: The concentration of the nitrogen-containing heterocyclic compound in the suspending agent is ≥3.8999 mg / L.

9. The method for controlling Pomacea canaliculata according to claim 7, characterized in that: The content of the nitrogen-containing heterocyclic compound in the granules is ≥1wt%.

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