A small molecule compound for preventing and treating mycotoxin poisoning and its preparation method and application

Abstract

The present invention relates to a small molecule compound for preventing and treating mycotoxin poisoning, and a preparation method and application thereof. The new functional food feed for preventing and treating mycotoxin poisoning proposed by the present invention reduces the losses caused by mycotoxins in the breeding industry to a certain extent. If it is put into actual production and use in large quantities, it is estimated that the economic benefits saved each year can reach tens of millions of yuan.

Description

Technical Field

[0001] The present invention belongs to the technical field of small molecule compounds for preventing and treating mycotoxin poisoning, and specifically relates to a small molecule compound for preventing and treating mycotoxin poisoning, and a preparation method and application thereof. Background Art

[0002] During the growth and storage of animal feed crops, they are very likely to produce secondary metabolites mycotoxins due to mold contamination. In the normal breeding process, the accidental ingestion of such animal feed plants will cause the toxins to accumulate in the body and eventually cause serious damage to the body of livestock, resulting in economic losses and interruption of normal economic activities. There are many types of mycotoxins, aflatoxin (AFT) is one of the more common and harmful ones, among which aflatoxin B1 (AFB1) is the most toxic and carcinogenic. The growth, reproduction and toxin production of aflatoxin require higher temperatures and humidity. Aflatoxin poisoning is prone to occur in the spring and summer in the south and warm and humid areas. Some crops will be contaminated by aflatoxin before and during harvest. In livestock breeding, there is an urgent need for a product that can be easily used to prevent and control aflatoxin to prevent and control the economic losses caused. Summary of the invention

[0003] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0004] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0005] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a small molecule compound for preventing and treating mycotoxin poisoning.

[0006] In order to solve the above technical problems, the present invention provides the following technical solution: a small molecule compound for preventing and treating mycotoxin poisoning, wherein the small molecule compound is GSK1362.

[0007] Another object of the present invention is to provide a method for preparing a small molecule compound for preventing and treating mycotoxin poisoning.

[0008] To solve the above problems, the present invention provides the following technical solution: a method for preparing a small molecule compound for preventing and treating mycotoxin poisoning, comprising the following steps:

[0009] Dilution: dilute small molecule compounds;

[0010] Oral gavage: The diluted small molecule compound is administered orally for a period of time.

[0011] As a preferred embodiment of the method for preparing the small molecule compound for preventing and treating mycotoxin poisoning of the present invention, the method comprises: the small molecule compound is a diluted solution of GSK1362.

[0012] As a preferred embodiment of the method for preparing the small molecule compound for preventing and treating mycotoxin poisoning of the present invention, the method comprises: the small molecule compound is GSK1362 which is prepared by diluting DMSO with one or more of water, a salt solution of water, and an oil phase.

[0013] As a preferred embodiment of the method for preparing the small molecule compound for preventing and treating mycotoxin poisoning of the present invention, the method comprises: the oil phase is one or two of DMSO and corn oil.

[0014] As a preferred embodiment of the method for preparing the small molecule compound for preventing and treating mycotoxin poisoning of the present invention, the method comprises: extracting 100 μL of the diluted small molecule compound for intragastric administration and injecting it into the back of the mouse tongue using an intragastric needle.

[0015] As a preferred embodiment of the method for preparing the small molecule compound for preventing and treating mycotoxin poisoning of the present invention, the method comprises: intragastric administration, wherein the intragastric administration treatment lasts for 30 days.

[0016] As a preferred embodiment of the method for preparing the small molecule compound for preventing and treating mycotoxin poisoning of the present invention, the method comprises: diluting the concentration of GSK1362 to 50 mg / mL.

[0017] Another object of the present invention is to provide an application of a small molecule compound for preventing and treating mycotoxin poisoning.

[0018] In order to solve the above technical solution, the present invention provides the following technical solution: an application of a small molecule compound for preventing and treating mycotoxin poisoning, which includes: the small molecule compound is GSK1362, and GSK1362 is used for preventing and treating mycotoxin poisoning.

[0019] As a preferred embodiment of the application of the small molecule compound for preventing and treating mycotoxin poisoning of the present invention, it includes: GSK1362 is used to prevent and treat aflatoxin B1 poisoning.

[0020] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0021] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a small molecule compound for preventing and treating mycotoxin poisoning.

[0022] In order to solve the above technical problems, the present invention provides the following technical solutions: a small molecule compound for preventing and treating mycotoxin poisoning: the small molecule compound for preventing and treating mycotoxin poisoning is GSK1362.

[0023] Another object of the present invention is to provide a method for preparing a small molecule compound for preventing and treating mycotoxin poisoning.

[0024] In order to solve the above problems, the present invention provides the following technical solution: a method for preparing a small molecule compound for preventing and treating mycotoxin poisoning, which comprises diluting the small molecule cyclic compound.

[0025] As a preferred embodiment of the method for preparing the small molecule compound for preventing and treating mycotoxin poisoning of the present invention, the method comprises: diluting GSK1362 to obtain a diluted GSK1362 dilution solution.

[0026] As a preferred embodiment of the method for preparing the small molecule compound for preventing and treating mycotoxin poisoning of the present invention, the method comprises: diluting GSK1362 using DMSO through one or more of water, water salt solution, and oil phase.

[0027] As a preferred embodiment of the method for preparing the small molecule compound for preventing and treating mycotoxin poisoning of the present invention, the method comprises: the oil phase is one or two of DMSO and corn oil.

[0028] Another object of the present invention is to provide an application of a small molecule compound for preventing and treating mycotoxin poisoning.

[0029] To solve the above problems, the present invention provides the following technical solution: an application of small molecule compounds for preventing and treating mycotoxin poisoning: the small molecules are mixed and diluted and then gavage is performed.

[0030] As a preferred scheme for the application of the small molecule compound for preventing and treating mycotoxin poisoning described in the present invention, it includes: extracting 100 μL of the diluted small molecule compound during gavage and inserting it into the back of the mouse tongue using a gavage needle, wherein the dilution is to dilute the concentration of the small molecule compound to 50 mg / mL.

[0031] As a preferred solution for the application of the small molecule compound for preventing and treating mycotoxin poisoning according to the present invention, it includes: the small molecule compound is GSK1362, and the GSK1362 is used for preventing and treating mycotoxin poisoning.

[0032] As a preferred embodiment of the application of the small molecule compound for preventing and treating mycotoxin poisoning of the present invention, it includes: the small molecule compound is used to prepare a medicament, a food additive, and a bacterial agent for preventing and treating mycotoxin poisoning.

[0033] As a preferred embodiment of the application of the small molecule compound for preventing and treating mycotoxin poisoning of the present invention, it includes: the small molecule compound is used to prevent and treat aflatoxin B1 poisoning. Beneficial effects of the present invention:

[0034] The new small molecule compound for preventing and treating mycotoxin poisoning proposed in the present invention and the functional food feed prepared subsequently can reduce the damage caused by mycotoxins in the aquaculture industry to a certain extent. If it is put into actual production and use on a large scale, it can directly save the aquaculture industry from losses of tens of millions of yuan each year. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0036] Figure 1 The absorbance at 450 nm under six different treatments was detected by the CCK8 kit prepared in Example 1 to reflect the toxic effect of AFB1 and the preventive and therapeutic effect of GSK1362, wherein the six treatments were a control group, an AFB1 addition group (1 μg / mL), a GSK1362 group (5 μM and 2.5 μM), and a GSK1362 and AFB1 combined treatment group (the addition amounts of GSK1362 were 5 μM and 2.5 μM, respectively, and the addition amount of AFB1 was 1 μg / mL);

[0037] Figure 2 The cells per milliliter were counted for six different treatments. The six treatments were control group, AFB1-added group (1 μg / mL), GSK1362 group (5 μM and 2.5 μM), GSK1362 and AFB1 combined treatment group (GSK1362 added at 5 μM and 2.5 μM, AFB1 added at 1 μg / mL);

[0038] Figure 3 To observe the cell growth of different treatment groups at 24, 48, and 72 h under a microscope, there were 12 treatments, including 12 combinations of GSK1362 added at 0, 1, 2.5, and 5 μM and AFB1 added at 0, 0.75, and 1 μg / mL, to intuitively reflect the protective effect of the additives on cells.

[0039] Figure 4 The cell amount in the toxin-added group, control group, GSK1362-added group, and combined treatment group after 5 days of treatment after a period of time;

[0040] Figure 5The figure is a statistical graph of the body weight of mice under various treatments;

[0041] Figure 6 Statistical graph of food intake of mice under various treatment conditions. DETAILED DESCRIPTION

[0042] In order to make the above-mentioned objects, features and advantages of the present invention more concise and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0043] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0044] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0045] The chemical structure of GSK1362 used in the present invention is shown below:

[0046]

[0047] Example 1

[0048] In this example, a GSK1362 solution was prepared and intragastrically administered:

[0049] Place a sterile EP tube on a balance and weigh it to zero. Add 13.2 μL of GSK1362 solution diluted with DMSO to the EP tube. The concentration of GSK1362 is 50 mg / mL. Mix well, add 300 μL of saline and add corn oil to 600 μL, and mix again. Fix the mouse, extract 100 μL of the mixed solution with a gavage needle, insert the needle into the back of the mouse tongue, perform gavage and continue treatment for 30 days.

[0050] Example 2

[0051] This example simulates the consumption of mycotoxins by animals:

[0052] Place a sterile EP tube on a balance and weigh it to zero. Add 13.2 μL of GSK1362 solution diluted with DMSO to the EP tube. The concentration of GSK1362 is 50 mg / mL. Then add 26.4 μL of AFB1 solution diluted with DMSO. After mixing, add 300 μL of saline. Add corn oil to 600 μL and mix again. Fix the mouse, extract 100 μL of the mixed solution with a gavage needle, insert the needle into the back of the mouse tongue, perform gavage and continue the treatment for 30 days.

[0053] Example 3

[0054] This example verifies the role of GSK1362 in preventing and treating AFB1 toxicity and killing cells:

[0055] Set up three sets of experiments as follows:

[0056] CCK8 cell proliferation toxicity detection, cell counting and microscopic observation were performed respectively, and the cells used were Aml12 (normal mouse liver cells). The cell count plating density during the experiment was 8×10 4 / mL absorbance, the experiment used 96-well plates, and the initial cell density was 5000 cells / well.

[0057] For the first two experiments, the following six treatments were performed: control group, AFB1-added group (1 μg / mL), GSK1362 group (5 μM and 2.5 μM), GSK1362 and AFB1 combined treatment group (GSK1362 added at 5 μM and 2.5 μM, respectively, and AFB1 added at 1 μg / mL). The data obtained are shown in the attached figure. Figures 1 to 4 shown.

[0058] like Figure 1 As shown, GSK1362 in our invention can be spatially embedded with NR1D1, and the binding site of GSK1362 is on the ligand binding domain of NR1D1.

[0059] like Figure 2 and Figure 3 As shown in the figure, only 1 μg / mL of AFB1 showed strong cytotoxicity and strong killing ability. Only adding GSK1362 had no toxic effect on cells, and its absorbance was basically consistent with that of the control group, reflecting the safety and non-toxicity of GSK1362. The results of the combined treatment group showed that GSK1362 significantly inhibited the cytotoxicity of AFB1 and greatly reduced cell death, and the addition of 2.5 and 5 μM had significant effects.

[0060] like Figure 4 As shown in Figure 2, on the fifth day of the experiment, the number of cells in the toxin-added group was only 25×10 4 / mL, which was less than one-fifth of the control group and the GSK1362-added group, while the combined treatment group effectively restored the cell number to 1×10 6 / mL.

[0061] Microscope observation more intuitively reflects the rescue effect of GSK1362. In the experiment, 12 different treatment conditions were set up, with GSK1362 addition of 0, 1, 2.5, 5μM and AFB1 addition of 0, 0.75, 1μg / mL. As can be seen from the first column, cell death increased significantly with the change of time and AFB1 addition. The prevention and treatment effect of adding 1μM GSK1362 was not very obvious, but as the dose gradually increased to 2.5 and 5μM, a certain rescue effect was shown. It can be seen that the cell survival rate at 72h was significantly higher than that of the group treated with toxin alone.

[0062] Example 4

[0063] The corresponding gavage operation was performed according to the treatment in Examples 1 and 2, including the following four gavage treatment methods:

[0064] GSK1362: Place a sterile EP tube on a balance and weigh it to zero. Add 13.2 μL of GSK1362 solution diluted with DMSO to the EP tube. The concentration of GSK1362 is 50 mg / mL. Mix well, add 300 μL of saline and add corn oil to 600 μL, and mix again. Fix the mouse, extract 100 μL of the mixed solution with a gavage needle, insert the needle into the back of the mouse tongue, perform gavage and continue treatment for 28 days.

[0065] AFB1: Place a sterile EP tube on a balance and weigh it to zero. Add 13.2 μL of AFB1 solution diluted with DMSO to the EP tube. The concentration of AFB1 is 2 mg / mL. Mix well, add 300 μL of saline and add corn oil to 600 μL, and mix again. Fix the mouse, extract 100 μL of the mixed solution with a gavage needle, insert the needle into the back of the mouse tongue, perform gavage and continue treatment for 28 days.

[0066] GSK1362+AFB1: Place a sterile EP tube on a balance and weigh it to zero. Add 13.2 μL of GSK1362 solution diluted with DMSO to the EP tube. The concentration of GSK1362 is 50 mg / mL. Then add 26.4 μL of AFB1 solution diluted with DMSO. After mixing, add 300 μL of saline. Add corn oil to 600 μL and mix again. Fix the mouse, extract 100 μL of the mixed solution with a gavage needle, insert the needle into the back of the mouse tongue, perform gavage and continue the treatment for 28 days.

[0067] Control group: During intragastric administration, mice were intragastrically administered with the same volume of the mixture of DMSO and corn oil as described above;

[0068] Depend on Figure 5 , 6 It can be seen that the combined treatment group was fed with the premix of GSK1362 and AFB1 from the eighth day. It can be seen that AFB1 seriously affected the weight and food intake of mice, while GSK1362 itself was harmless to mice. The combined treatment also showed that GSK1362 had a significant preventive effect on the toxic effects of AFB1.

[0069] In summary, the addition of GSK1362 described in the present invention can inhibit the toxicity of aflatoxin B1, and at the same time can effectively weaken the killing effect of the toxin on body cells, thereby maintaining body homeostasis and preventing and treating mycotoxin poisoning.

[0070] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. The use of the small molecule compound GSK1362 in the preparation of a drug for preventing and treating mycotoxin poisoning, characterized in that: The mycotoxin is aflatoxin B1.

Images