A nicotinic substance antidote, its preparation method and application

Abstract

The present invention discloses a nicotinic substance antidote, its preparation method and application, in particular to the application of methylene blue or its salt in the preparation of an antidote for alleviating thiamethoxam poisoning. The present invention has verified through experiments that methylene blue or its salt can effectively control, alleviate and treat thiamethoxam poisoning symptoms, has a good detoxification effect, and can prevent thiamethoxam poisoning in advance. Therefore, the methylene blue or its salt provided by the present invention can be used in fields such as medicine, etc., can effectively reduce the death of animals such as birds and insects caused by thiamethoxam poisoning, stabilize the biological chain cycle, and ensure the quality and yield of agricultural products.

Description

Technical Field

[0001] The present invention relates to the technical field of antidotes, and in particular to a nicotine antidote and a preparation method and application thereof. Background Art

[0002] Nicotine, a substance extracted from tobacco leaves and stems, is chemically known as (S)-3-(1-methyl-2-pyrrolidinyl)pyridine. It is a highly effective, low-residue, natural botanical insecticide used in agriculture. Based on its insecticidal properties, research on the structure of nicotine-like substances and their modification through chemical synthesis have led to the development of a series of synthetic nicotine derivatives, such as neonicotinoid insecticides. These compounds have a strong insecticide effect on pests and are widely used in agriculture.

[0003] While nicotine and neonicotinoids exhibit a degree of selective toxicity against pests, they still exhibit some toxicity to non-target organisms (such as mammals). Pesticide residues can form during agricultural production, and even improper use can cause toxicity to animals and plants, particularly herbivores. These can act on nicotinic acetylcholine receptors in the animal's central nervous system, effectively blocking normal neurotransmission and causing poisoning or even death. This can further impact processes such as seed dispersal, transfer, and pollination of crops by birds and insects. Therefore, mitigating the harm nicotine poisoning to plants and animals is urgent. Summary of the Invention

[0004] Based on this, it is necessary to provide a nicotine-based antidote with excellent detoxification effect, a preparation method thereof, and an application thereof.

[0005] In a first aspect, the present invention provides use of methylene blue or a salt thereof in the preparation of an antidote for alleviating thiamethoxam poisoning.

[0006] In some embodiments, the content of methylene blue or its salt in the antidote is 0.01-2 mg / L or 0.01-2 mg / kg.

[0007] In some embodiments, the antidote is in the form of tablets, granules, capsules, solutions, powders, suspensions, emulsions, pastes, or aerosols;

[0008] In some embodiments, the formulation of the antidote includes an oral dosage, an injection, a patch, a liniment, a spray, an inhaler, drops, a lotion, or a suppository.

[0009] In a second aspect, the present invention provides use of a composition in preparing an antidote for alleviating thiamethoxam poisoning, wherein the composition comprises methylene blue or a salt thereof, and an auxiliary material.

[0010] In some embodiments, the content of methylene blue or its salt in the composition is 0.01 - 2 mg / L or 0.01 - 2 mg / kg.

[0011] In some embodiments, the application at least includes one of the following (1) - (3):

[0012] (1) The auxiliary materials further include one or more of natural auxiliary materials, semi - synthetic and synthetic auxiliary materials;

[0013] (2) The forms of the composition include tablets, granules, capsules, solutions, powders, suspensions, emulsions, pastes or aerosols;

[0014] (3) The application routes of the composition include oral agents, injections, patches, liniments, sprays, inhalants, drops, lotions or suppositories.

[0015] In some embodiments, the present invention also provides the application of methylene blue or its salt in the preparation of an acetylcholine receptor inhibitor, and the acetylcholine receptor inhibitor includes inhibiting the binding of nicotine - like substances to acetylcholine receptors; the nicotine - like substance is thiamethoxam.

[0016] In some embodiments, the content of methylene blue or its salt is 0.01 - 2 mg / L or 0.01 - 2 mg / kg

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] The present invention provides the application of methylene blue or its salt in the preparation of an antidote for relieving thiamethoxam poisoning. The phenothiazine group possessed by methylene blue or its salt is one of the effective active groups for relieving thiamethoxam poisoning. It has a good alleviating effect on thiamethoxam poisoning and has effective clinical prevention and treatment effects, providing an effective solution for relieving thiamethoxam poisoning at present and filling the gap in this field. Description of the Drawings

[0019] Figure 1 It is the statistical result of the death situation of honeybee larvae acutely poisoned by thiamethoxam with different antidote contents in Test Example 1. The vertical coordinate in the figure is the mortality rate (%), and the horizontal coordinate is respectively the blank group, the model group and 3 treatment groups with different doses. The a and b in the figure represent the results of significant difference analysis by the Tukey HSD method.

[0020] Figure 2 It is the toxicity experiment of the antidote on honeybee larvae in Experimental Example 2. The vertical coordinate in the figure is the mortality rate (%), and the horizontal coordinate is the blank group, the model group and the treatment group. The a and b in the figure represent the results of significant difference analysis by the Tukey HSD method. Detailed Embodiments

[0021] To facilitate the understanding of the present invention, the following provides preferred embodiments of the present invention for a more comprehensive description of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0022] It should be noted that the experimental methods without specific conditions in the following embodiments of the present invention are generally in accordance with conventional conditions or the conditions recommended by the manufacturer. All kinds of commonly used chemical reagents used in the embodiments are commercially available products.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0024] Since nicotine substances are toxic to both animals and plants to a certain extent, there will be a certain residue on the plant surface or in the animal body, ultimately causing poisoning of beneficial animals and plants and affecting the reproduction and cultivation of animals and plants in the field.

[0025] The present invention provides the use of methylene blue or its salt in the preparation of an antidote for relieving thiamethoxam poisoning.

[0026] In some embodiments, in the antidote, the content range of methylene blue or its salt is 0.01 - 2 mg / L or 0.01 - 2 mg / kg, including but not limited to 0.01, 0.02, 0.05, 0.06, 0.064, 0.07, 0.1, 0.2, 0.3, 0.32, 0.35, 0.38, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 mg / L or any range formed by any two of the foregoing and any value within the range; or, including but not limited to 0.01, 0.02, 0.05, 0.06, 0.064, 0.07, 0.1, 0.2, 0.3, 0.32, 0.35, 0.38, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 mg / kg or any range formed by any two of the foregoing and any value within the range.

[0027] In some embodiments, the form of the antidote includes, but is not limited to, solid state, semi-solid state, liquid state, etc., such as flake, granular, capsule, solution, powder, suspension, emulsion, paste or aerosol, etc.

[0028] In some embodiments, the formulations of the antidote include, but are not limited to, oral preparations, injections, patches, liniments, sprays, inhalants, drops, lotions or suppositories, etc.

[0029] The present invention also provides the application of the composition in the preparation of an antidote for alleviating thiamethoxam poisoning, wherein the composition contains methylene blue or its salt, and excipients.

[0030] In some embodiments, the composition may further contain one or more of natural excipients, semi-synthetic and synthetic excipients, such as one or more of solvents, propellants, solubilizers, suspending agents, emulsifiers, coloring agents, binders, disintegrants, fillers, lubricants, glidants, compression aids, flavoring agents, preservatives, suspending agents, coating agents or fragrances.

[0031] In some embodiments, the excipients in the composition include saccharides, not limited to one or more of sucrose (brown sugar, white sugar, granulated sugar, yellow sugar), glucose, fructose, galactose, lactose, maltose or starch.

[0032] In some embodiments, the excipients in the composition include pollen, not limited to one or more of plant-derived pollen and synthetic pollen.

[0033] In some embodiments, in the composition, the content range of methylene blue or its salt is 0.01 - 2 mg / L or 0.01 - 2 mg / kg, including but not limited to 0.01, 0.02, 0.05, 0.06, 0.064, 0.07, 0.1, 0.2, 0.3, 0.32, 0.35, 0.38, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 mg / L or any value within the range formed by any two of the foregoing; or, including but not limited to 0.01, 0.02, 0.05, 0.06, 0.064, 0.07, 0.1, 0.2, 0.3, 0.32, 0.35, 0.38, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 mg / kg or any value within the range formed by any two of the foregoing.

[0034] In some embodiments, the form of the antidote includes, but is not limited to, solid state, semi-solid state, liquid state, etc., such as flake, granular, capsule, solution, powder, suspension, emulsion, paste or aerosol, etc.

[0035] In some embodiments, the preparations of the antidote include, but are not limited to, oral preparations, injections, patches, liniments, sprays, inhalants, drops, lotions or suppositories, etc.

[0036] In addition, the present invention also provides the application of methylene blue or its salts in fields including, but not limited to, the preparation of drugs, etc.

[0037] In some embodiments, the content range of methylene blue or its salts in the drug is 0.01 - 2 mg / L or 0.01 - 2 mg / kg, including, but not limited to, 0.01, 0.02, 0.05, 0.06, 0.064, 0.07, 0.1, 0.2, 0.3, 0.32, 0.35, 0.38, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 mg / L or the range formed by any two of the foregoing and any value within the range; or, including, but not limited to, 0.01, 0.02, 0.05, 0.06, 0.064, 0.07, 0.1, 0.2, 0.3, 0.32, 0.35, 0.38, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 mg / kg or the range formed by any two of the foregoing and any value within the range.

[0038] In some embodiments, the form of the drug is not limited to liquid or solid.

[0039] Optionally, the form of the drug includes, but is not limited to, solid state, semi-solid state, liquid state, etc., such as flake, granular, capsule, solution, powder, suspension, emulsion, paste or aerosol, etc.

[0040] In some embodiments, the drug may further contain natural excipients, semi-synthetic and / or synthetic excipients, such as one or more of solvent, propellant, solubilizer, suspending agent, emulsifier, colorant, binder, disintegrant, filler, lubricant, glidant, compression aid, flavoring agent, preservative, suspending agent, coating agent and fragrance.

[0041] Residual neonicotinoid insecticides and new neonicotinoid insecticides in crop control operations can be toxic to animals such as birds and insects, especially posing a relatively serious threat to the bee population. They not only remain in the bee brain for a long time, affecting their learning ability and foraging activities, but also reduce the survival ability of bees, shorten their lifespan, affect the pollination of bees and other insects, and thus affect the quality and yield of crops. At the same time, it also affects the beekeeping industry.

[0042] In some embodiments, by mixing methylene blue or its salt with pollen, the detoxification effect of bees during thiamethoxam poisoning can be alleviated.

[0043] In some embodiments, methylene blue or its salt is mixed with pollen and sprayed on the comb or added to the feeding trough in the form of supplementary feeding or incentive feeding, or placed on the frame for bees to ingest.

[0044] The antidote and / or composition containing methylene blue or its salt provided by the present invention has obvious preventive, alleviating or therapeutic effects on bee thiamethoxam poisoning, provides a more effective preventive and therapeutic means for alleviating thiamethoxam poisoning, and provides a scientific basis for further developing antidotes for bee pesticide poisoning.

[0045] The operation of the present invention is simple and the cost is low. It can prevent, control and / or treat thiamethoxam poisoning symptoms and reduce the continuous adverse effects of thiamethoxam poisoning on the bee colony. The present invention is also applicable to raising bee colonies outside the hive, directly providing the antidote for ingestion, thereby alleviating the poisoning symptoms.

[0046] In addition, the present invention also provides the use of methylene blue or its salt in the preparation of an acetylcholine receptor inhibitor, and the acetylcholine receptor inhibitor includes inhibiting the binding of neonicotinoid substances to acetylcholine receptors.

[0047] In some embodiments, the neonicotinoid substance is thiamethoxam.

[0048] In some embodiments, the content range of methylene blue or its salt in the acetylcholine receptor inhibitor is 0.01~2 mg / L or 0.01~2 mg / kg, including but not limited to 0.01, 0.02, 0.05, 0.06, 0.064, 0.07, 0.1, 0.2, 0.3, 0.32, 0.35, 0.38, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 mg / L or any value within the range formed by any two of the foregoing; or, including but not limited to 0.01, 0.02, 0.05, 0.06, 0.064, 0.07, 0.1, 0.2, 0.3, 0.32, 0.35, 0.38, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 mg / kg or any value within the range formed by any two of the foregoing and any value within the range.

[0049] In some embodiments, the acetylcholine receptor inhibitor may further contain natural excipients, semi-synthetic and / or synthetic excipients, such as one or more of solvents, propellants, solubilizers, suspending agents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, glidants, compression aids, flavoring agents, preservatives, suspending agents, coating agents, and fragrances.

[0050] In some embodiments, the excipients in the acetylcholine receptor inhibitor include sugars, not limited to one or more of sucrose (brown sugar, white sugar, granulated sugar, yellow sugar), glucose, fructose, galactose, lactose, maltose, and starch.

[0051] In some embodiments, the form of the acetylcholine receptor inhibitor includes but is not limited to solid state, semi-solid state, liquid state, etc., such as tablets, granules, capsules, solutions, powders, suspensions, emulsions, pastes, or aerosols.

[0052] In some embodiments, the preparations of the acetylcholine receptor inhibitor include but are not limited to oral preparations, injections, patches, liniments, sprays, inhalants, drops, lotions, or suppositories.

[0053] For the experimental parameters not specified in the following specific examples, preferably refer to the guidelines given in this application document, and you can also refer to the experimental manuals in this field or other experimental methods known in this field, or refer to the experimental conditions recommended by the manufacturers.

[0054] The raw materials and reagents involved in the following specific examples can be obtained commercially, or those skilled in the art can prepare them according to known means.

[0055] Example 1: Preparation of a Detoxifying Agent for Nicotine-like Substances

[0056] S1. Dissolve white sugar in water to prepare 50 wt% sugar water;

[0057] S2. Take an appropriate amount of methylene blue (detoxifying agent for nicotine-like substances) and mix it evenly in the above 50 wt% sugar water to prepare a detoxifying agent. The concentration of methylene blue (detoxifying agent for nicotine-like substances) in the detoxifying agent can be any concentration in the range of 0.01 - 2 mg / L.

[0058] In the process of preparing the detoxifying agent in this example, step S1 is not limited to white sugar, and similar technical effects can be achieved by using brown sugar, granulated sugar, and / or yellow sugar, etc.

[0059] Test Example 1: Detoxifying Dose Test of a Detoxifying Agent for Nicotine-like Substances

[0060] In this test example, the detoxifying effect of a nicotine-like substance on bees with the same poisoning dose was tested. The experimental method was as follows: The detoxifying agent was applied into the beehive (spraying the detoxifying agent on the comb or adding it to the feeding trough was optional), and after the bees fed on it, measurements were taken. The specific test steps and results are as follows:

[0061] Test insects: Italian honeybee larvae. Select newly hatched one-day-old larvae from a healthy bee colony, transfer them to a 48-well cell culture plate, add larval feed and culture them until the third day, and pick healthy and highly viable larvae as test samples.

[0062] Test samples: The detoxifying agent prepared in Example 1. The addition amount of methylene blue (purchased from the Macklin brand, purity ≥ 98%) in the detoxifying agent was added accordingly according to the requirements of the test plan. The contents of methylene blue in the detoxifying agent used in this test example were 0.064 mg / L, 0.32 mg / L, and 1.6 mg / L respectively.

[0063] Test steps:

[0064] The cultured test insect larvae were divided into five groups: Control, TMX, TMX+Ad320-1, TMX+Ad320-2, and TMX+Ad320-3, with 3 replicates in each group and 24 in each replicate. On the 3rd day of age, the Control group was fed with normal feed, and the remaining groups were fed with feed containing 100 mg / L thiamethoxam in each well. On the 4th day of age, the Control group was fed with normal feed; the TMX group was fed with feed containing 100 mg / L thiamethoxam in each well; the TMX+Ad320-1 group was fed with feed containing an antidote with 0.064 mg / L methylene blue and feed containing 100 mg / L thiamethoxam in each well; the TMX+Ad320-2 group was fed with feed containing an antidote with 0.32 mg / L methylene blue and feed containing 100 mg / L thiamethoxam in each well; the TMX+Ad320-3 group was fed with feed containing an antidote with 1.6 mg / L methylene blue and feed containing 100 mg / L thiamethoxam in each well.

[0065] Note: Normal larvae can grow healthily until they pupate at 7 days of age, with obvious touch responses, and the larvae's bodies are white and plump. Poisoned larvae will show phenomena such as smaller body size, extended larval stage, weak touch response, and yellowish body color.

[0066] After 24 h, the larval mortality rate (%) of each group was statistically analyzed using formula (I), and the specific results are shown in Table 1 and Appendix Figure 1 ;

[0067] (I)

[0068] Table 1: Detoxification effects of different antidote doses on alleviating thiamethoxam poisoning in bee larvae

[0069]

[0070] From the data in Table 1 and Appendix Figure 1 It can be seen that the average larval mortality rate of the blank control group of bee larvae was only 13.89%±2.78%, and the average larval mortality rate of the model group TMX reached 47.22%±3.68%. The average larval mortality rates of the three treatment groups TMX+Ad320-1, TMX+Ad320-2, and TMX+Ad320-3 were in the range of 13.89%±1.39% to 27.78%±6.05%. Compared with the model group, after the experimental group was fed with the antidote, the average larval mortality rate of bee larvae decreased significantly. Especially when the antidote content in TMX+Ad320-2 was 0.32 mg / L, the average larval mortality rate of bee larvae decreased to basically the same level as the blank group. It can be seen that the antidote prepared by the present invention has a significant effect on alleviating thiamethoxam poisoning in bee larvae and can prevent and / or treat thiamethoxam poisoning in insects.

[0071] Experimental Example 2: Toxicity Test of Ad320

[0072] In this experimental example, the toxicity of Ad320 was tested. The experimental method was basically the same as that in Experimental Example 1. The specific experimental steps are as follows:

[0073] The cultured test insect larvae were divided into three groups: Control, Ad320, and TMX, with 3 replicates in each group and 24 larvae in each replicate.

[0074] On the 3rd day of age, the Control and Ad320 groups were fed normal feed, and the treatment group TMX was fed feed with a thiamethoxam concentration of 80 mg / L. On the 4th day of age, the Control group was fed normal feed, the Ad320 group was fed feed containing an antidote with 0.32 mg / L methylene blue, and the TMX group was fed feed with a thiamethoxam concentration of 80 mg / L. After 24 h, the number of deaths in each group was counted.

[0075] The larval mortality rate (%) of each group was calculated using formula (I). The specific results are shown in Table 2 and Appendix Figure 2 ;

[0076] Table 2: Toxicity Test of Ad320 on Bee Larvae

[0077]

[0078] From the data in Table 2 and Appendix Figure 2 , it can be seen that the average mortality rates of the bee larvae in the blank control group and the Ad320 group are at the same level. It can be seen that the antidote provided by the present invention has no obvious toxicity. Combining the results of Experimental Example 1, it further verifies that the antidote prepared by the present invention has a significant effect on alleviating thiamethoxam poisoning in bee larvae, can prevent and / or treat thiamethoxam poisoning in insects, and is safe, non-toxic and reliable at the same time.

[0079] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope recorded in this specification.

[0080] The above-described embodiments only represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. Use of methylene blue or its salt in the preparation of an antidote for relieving thiamethoxam poisoning.

2. The application according to claim 1, wherein The content of methylene blue or its salt in the antidote is 0.01 - 2 mg / L or 0.01 - 2 mg / kg.

3. The application according to claim 1 or 2, characterized in that, The form of the antidote includes tablets, granules, capsules, solutions, powders, suspensions, emulsions, pastes or aerosols.

4. The application according to claim 1 or 2, characterized in that, The preparations of the antidote include oral preparations, injections, patches, liniments, sprays, inhalants, drops, lotions or suppositories.

5. Use of the composition in the preparation of an antidote for alleviating thiamethoxam poisoning, characterized in that, The composition contains methylene blue or its salt, and excipients.

6. The application according to claim 5, characterized in that, The content of methylene blue or its salt in the composition is 0.01 - 2 mg / L or 0.01 - 2 mg / kg.

7. The application according to claim 5 or 6, characterized in that, It contains at least one of the following (1) - (3): (1) The excipients include one or more of natural excipients, semi-synthetic and synthetic excipients; (2) The form of the composition includes tablets, granules, capsules, solutions, powders, suspensions, emulsions, pastes or aerosols; (3) The application routes of the composition include oral administration, injection, patch, liniment, spray, inhalant, drop, lotion or suppository.

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