Pharmaceutical composition and method for constructing diabetic animal model

Abstract

The invention relates to the technical field of animal model construction, and particularly discloses a pharmaceutical composition and a method for constructing a diabetes animal model. The pharmaceutical composition provided by the invention is prepared from the following components in parts by weight: 350 to 450 parts of metformin, 150 to 250 parts of lycium barbarum polysaccharide, 70 to 140 parts of corn stigma polysaccharide and 8 to 15 parts of dendrobium officinale. The invention also provides a method for constructing a diabetic animal model, which comprises the step of screening animals susceptible to diabetes by adopting the pharmaceutical composition, and the method comprises the following specific steps: administering the pharmaceutical composition to the animals in an oral intragastric administration manner, and screening the animals susceptible to diabetes according to the blood glucose reduction values before and after administration of the animals 40-50 minutes later. According to the method for constructing the diabetic animal model, animals susceptible to diabetes can be screened out, then the diabetic animal model with good consistency and stability can be obtained in a short time, and the model construction success rate can reach 85% or above.

Description

Technical Field

[0001] The present application relates to the technical field of animal model construction, and in particular to a pharmaceutical composition and a method for constructing a diabetic animal model. Background Art

[0002] Diabetes is a chronic metabolic disease, which is mainly manifested by hyperglycemia. Long-term hyperglycemia can lead to a variety of complications, which in turn seriously affect the patient's quality of life. Experimental animals and their disease models are the experimental basis for clinical hypotheses and experimental hypotheses. Only through animal experiments can accurate, reliable, and reproducible experimental results be obtained, so that we can have a deep understanding of the pathogenesis, development process, and treatment methods of diabetes. Therefore, animals play an irreplaceable role in diabetes research, and establishing a reliable diabetic animal model is of great significance for diabetes research.

[0003] At present, common methods for constructing diabetic animal models include diet induction or drug induction. Among them, the diet induction method is to give animals a high-fat and high-sugar diet so that the animals gradually develop into diabetic symptoms; while the chemical induction method is to destroy pancreatic β cells by injecting streptozotocin (STZ) or other chemicals into animals to induce diabetes; however, when the diet induction method is used to construct diabetic animal models, there are differences in the acceptance and response of animals to high-sugar and high-fat diets. For example, some animals have strong metabolic regulation capabilities and high tolerance to high-sugar and high-fat diets, and are not prone to diabetic symptoms, resulting in failure in model construction; in addition, the cycle of simple diet-induced diabetes is long, up to six months or even a year, and the time and feeding costs are high; when constructing diabetic animal models through drug induction, due to the different absorption, metabolism and response of different animals to drugs, the dosage and duration of drug modeling are difficult to control; if the dose is too small, it may not be able to effectively induce the occurrence of diabetes; and if the dose is too large, it may cause serious adverse reactions or even death in animals. In summary, these existing methods are often difficult to accurately control the differences between individual animals in the process of constructing diabetic animal models, resulting in poor consistency and stability of the model.

[0004] Therefore, there is an urgent need to develop a more efficient, stable and controllable method for constructing diabetic animal models to obtain diabetic animal models with good consistency and stability, thereby facilitating large-scale experimental research. Summary of the invention

[0005] In order to overcome the defects of poor consistency and stability of existing diabetic animal models, the present application provides a pharmaceutical composition and a method for constructing a diabetic animal model.

[0006] In a first aspect, the present application provides a pharmaceutical composition, which adopts the following technical solution: A pharmaceutical composition comprises the following components in parts by weight: 350-450 parts of metformin, 150-250 parts of wolfberry polysaccharide, 70-140 parts of corn silk polysaccharide and 8-15 parts of dendrobium officinale.

[0007] The present application provides a pharmaceutical composition, which can control the blood sugar content in animals through the following four aspects: ① acting on the liver, inhibiting gluconeogenesis, and reducing glycogen output; ② acting on peripheral tissues such as muscle and fat, improving muscle glycogen synthesis, reducing free fatty acids, improving insulin sensitivity, and increasing glucose uptake and utilization; ③ acting on the intestine, inhibiting the intestinal wall cells from taking in glucose, and increasing the level of incretin (GLP-1); ④ activating adenosine monophosphate-activated protein kinase (AMPK), and improving the energy metabolism of muscle, fat, and liver. The present application injects the above-mentioned pharmaceutical composition into animals, and determines the animal's body tissue's ability to absorb sugar by detecting the blood sugar content of the animal reduced to, thereby screening out animals susceptible to diabetes.

[0008] In the present application, after the above-mentioned pharmaceutical composition is administered to mice, when the blood glucose content of the animal decreases to ≤4mmol / L, it indicates that the production and output of liver glycogen in the animal has not decreased, the absorption of sugar by peripheral tissues has not increased, and the uptake of glucose by intestinal wall cells has not been inhibited, which further indicates that the animal's body tissues have a weak ability to absorb sugar, indicating that the animal is more susceptible to diabetes. The above-mentioned animal can be used to construct a diabetic animal model with a high success rate.

[0009] Optionally, the pharmaceutical composition comprises the following components in parts by weight: 380-410 parts of metformin, 200-230 parts of wolfberry polysaccharides, 100-120 parts of corn silk polysaccharides and 8-10 parts of Dendrobium officinale.

[0010] In this application, the amount of each component added in the pharmaceutical composition will comprehensively affect the amount of dry glycogen generated in the animal body, the amount of sugar absorbed by peripheral tissues, and the amount of glucose uptake by intestinal wall cells, stimulating and characterizing the body tissue's ability to absorb sugar from multiple aspects, thereby screening out animals that are susceptible to diabetes with good consistency and stability. This application has found through experimental research that by further controlling the amount of each component added in the pharmaceutical composition within the above range, the success rate of constructing a diabetes model in the screened animals susceptible to diabetes can reach more than 95%.

[0011] Optionally, the pharmaceutical composition comprises the following components in parts by weight: 410 parts of metformin, 200 parts of wolfberry polysaccharides, 100 parts of corn silk polysaccharides and 10 parts of Dendrobium officinale.

[0012] In a second aspect, the present application provides a method for constructing a diabetic animal model, comprising using the pharmaceutical composition to screen animals susceptible to diabetes.

[0013] Optionally, the step of screening animals susceptible to diabetes is: administering the drug composition to animals by oral gavage, with a dosage of 600-800 mg / kg, measuring the blood glucose level of the animals 40-50 minutes after administration, and screening animals susceptible to diabetes based on the blood glucose reduction values ​​before and after administration.

[0014] Optionally, the screening criterion is: when the blood sugar reduction value is ≤4mmol / L, the animal is determined to be an animal susceptible to diabetes.

[0015] Optionally, the animal is a mouse.

[0016] Optionally, the method further comprises model construction, the specific steps of which are: feeding animals susceptible to diabetes with diabetic feed for 5-6 consecutive weeks, then injecting STZ for 3 consecutive days at a dose of 80 mg / kg, and then testing the blood glucose level of the animals once a week for 2 consecutive weeks. When the blood glucose levels are maintained above 16.7 mmol / L twice, the diabetic animal model is considered to be successfully constructed.

[0017] In summary, this application has the following beneficial effects: 1. The present application provides a pharmaceutical composition made of metformin, wolfberry polysaccharides, corn silk polysaccharides and Dendrobium officinale. After the above-mentioned pharmaceutical composition is administered to animals, when the blood sugar content of the animals is reduced by ≤4mmol / L, it indicates that the production and output of glycogen in the animal's body are not reduced, the absorption of sugar by peripheral tissues is not increased, and the intestinal wall cells cannot be inhibited from taking in glucose, which further indicates that the animal's body tissues have a weak ability to absorb sugar, indicating that the animal is more susceptible to diabetes. The above-mentioned animals can be used to construct a diabetic animal model with a high success rate.

[0018] 2. The present application further controls the weight of each component in the pharmaceutical composition within the following range: 380-410 parts of metformin, 200-230 parts of wolfberry polysaccharides, 100-120 parts of corn silk polysaccharides and 8-10 parts of Dendrobium officinale. The obtained pharmaceutical composition is more accurate in screening animals susceptible to diabetes, and the success rate of constructing a diabetic animal model from the screened diabetic animals can reach more than 95%. DETAILED DESCRIPTION

[0019] In a first aspect, the present application provides a pharmaceutical composition comprising the following components in parts by weight: 350-450 parts of metformin, 150-250 parts of wolfberry polysaccharides, 70-140 parts of corn silk polysaccharides and 8-15 parts of Dendrobium officinale; further, the pharmaceutical composition comprises the following components in parts by weight: 380-410 parts of metformin, 200-230 parts of wolfberry polysaccharides, 100-120 parts of corn silk polysaccharides and 8-10 parts of Dendrobium officinale.

[0020] The preparation method of the above-mentioned drug composition comprises the following steps: mixing the above-mentioned components evenly to obtain the drug composition.

[0021] In a second aspect, the present application provides a method for constructing a diabetic animal model, comprising the following steps: (1) Screening animals susceptible to diabetes: the pharmaceutical composition is dissolved in water, and then administered to animals by oral gavage at a dosage of 600-800 mg / kg. 40-50 minutes after administration, the blood glucose level of the animals is measured, and animals susceptible to diabetes are screened out based on the blood glucose reduction values ​​before and after administration. The screening standard is: when the blood glucose reduction value is ≤4 mmol / L, the animal is judged to be an animal susceptible to diabetes; (2) Model construction: Animals susceptible to diabetes were fed a diabetic diet for 5-6 weeks, with free access to food. They were then injected with STZ at a dose of 80 mg / kg for 3 consecutive days. The blood glucose levels of the animals were then tested once a week for 2 consecutive weeks. When the blood glucose levels were maintained above 16.7 mmol / L on both occasions, the diabetic animal model was successfully constructed.

[0022] In the examples of the present application, metformin was purchased from Merck Pharmaceuticals (Jiangsu) Co., Ltd.; wolfberry polysaccharides were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; corn silk polysaccharides were purchased from Weikeqi Biotechnology Co., Ltd.; Dendrobium officinale was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; the model of diabetic feed was SFD019, purchased from Spef (Beijing) Biotechnology Co., Ltd.; the model of STZ was S17049, purchased from Shanghai Yuanye Biotechnology Co., Ltd.; the raw materials, reagents, solvents, etc. of the present application can all be purchased from merchants.

[0023] The present application is further described in detail below in combination with Preparation Example 1, Examples, and Testing Experiments.

[0024] Preparation Example 1-12 Preparation Examples 1-12 each provide a pharmaceutical composition.

[0025] The difference between the above preparation examples is that the addition amount of each component in the pharmaceutical composition is shown in Table 1 below.

[0026] The preparation method of the pharmaceutical composition provided in Preparation Example 1-12 is: weigh each component separately and mix them evenly to obtain the pharmaceutical composition.

[0027] Table 1 Addition amount of each component in the pharmaceutical composition provided in Preparation Examples 1-12 Comparative Preparation Example 1 Comparative Preparation Example 1 provides a pharmaceutical composition.

[0028] The difference between the comparative preparation example and preparation example 3 is that the amount of Lycium barbarum polysaccharide added to the pharmaceutical composition is 0.

[0029] Comparative Preparation Example 2 Comparative Preparation Example 2 provides a pharmaceutical composition.

[0030] The difference between the comparative preparation example and preparation example 3 is that the amount of corn silk polysaccharide added in the pharmaceutical composition is 0.

[0031] Comparative Preparation Example 3 Comparative Preparation Example 3 provides a pharmaceutical composition.

[0032] The difference between the comparative preparation example and preparation example 3 is that the amount of Dendrobium officinale added to the pharmaceutical composition is 0. Example

[0033] Screening animals for diabetes The drug compositions provided in Preparation Examples 1-12 and Comparative Preparation Examples 1-3 were used to screen mice to obtain mice susceptible to diabetes. The specific method is: (1) First, 450 6-week-old male C57BL / 6J mice were selected and randomly divided into 15 groups (numbered A1-A12 and B1-B3), with 30 mice in each group (numbered 1-30 within the group); the initial blood glucose level of each mouse was measured; (2) The pharmaceutical composition was dissolved in water and then administered to animals by oral gavage at a dosage of 700 mg / kg. 40 minutes after administration, the blood glucose level of the animals was measured again; wherein, groups 1-12 (groups A1-A12) were administered with the pharmaceutical composition provided in Preparation Example 1-12, and groups 3 (groups B1-B3) were administered with the pharmaceutical composition provided in Comparative Preparation Example 1-3; (3) Calculate the reduction in blood sugar content before and after oral gavage of mice, and the calculation formula is: blood sugar content reduction value = initial blood sugar content of mice - blood sugar content 40 minutes after oral gavage of the drug composition. When the blood sugar content reduction value is ≤4mmol / L, the animal is judged to be an animal susceptible to diabetes, and then mice susceptible to diabetes are screened. The results are shown in Table 2 below.

[0034] Table 2 Results of screening of mice susceptible to diabetes Construction of diabetic mouse model The diabetic model was constructed for the above 450 mice. The specific method is as follows: the mice were fed with diabetic feed for 6 consecutive weeks, and they were allowed to eat freely; then STZ was injected for 3 consecutive days, with an injection volume of 80 mg / kg, and then the blood glucose level of the animals was tested once a week for 2 consecutive weeks. When the blood glucose levels were maintained above 16.7 mmol / L twice, the diabetic mouse model was successfully constructed.

[0035] The data of the construction results of 450 mouse models are shown in Table 3 below. The success rate of model construction for diabetic mice was calculated based on the data, and the calculation method is as follows: Model construction success rate = successfully constructed diabetic mouse models / number of mice susceptible to diabetes × 100%.

[0036] Table 3 Results of diabetic mouse model construction According to the results in Tables 2 and 3, the present application first screened 450 mice using the pharmaceutical compositions provided in Preparation Examples 1-12 and Comparative Preparation Examples 1-3 to obtain mice that were not susceptible to diabetes and mice that were susceptible to diabetes, respectively. The diabetic model was then constructed for the 450 mice, and it was found that the mice that were not susceptible to diabetes failed to successfully construct a diabetic mouse model; the diabetic mouse model was partially or completely successfully constructed for the mice susceptible to diabetes; further comparison found that after the model was constructed for the mice susceptible to diabetes screened by the pharmaceutical compositions provided in Preparation Examples 1-12, the success rate of constructing the diabetic mouse model was 88.46-100% (≥85%); and after the model was constructed for the mice susceptible to diabetes screened by the pharmaceutical compositions provided in Comparative Preparation Examples 1-3, the success rate of constructing the diabetic mouse model was 67.86-79.31% (<85%). Therefore, it is shown that the pharmaceutical composition prepared by metformin, wolfberry polysaccharides, corn silk polysaccharides and Dendrobium officinale in the present application can be used to screen animals susceptible to diabetes, thereby ensuring that a diabetic animal model is obtained in a short time, and the success rate of model construction can reach more than 85%.

[0037] Further comparison revealed that after model construction was performed on the diabetic mice screened out from groups A2, A3, A6, A9, and A12, the success rates of constructing diabetic mouse models were all 96.15-100% (≥95%); and after model construction was performed on the diabetic mice screened out from groups A1, A4-5, A7-8, and A10-11, the success rates of constructing diabetic mouse models were 88.46-92.86% (<95%), indicating that the present application further controls the weight portions of each component in the pharmaceutical composition within the following range: 380-410 parts of metformin, 200-230 parts of wolfberry polysaccharides, 100-120 parts of corn silk polysaccharides, and 8-10 parts of Dendrobium officinale, and the obtained pharmaceutical composition is more accurate in screening animals susceptible to diabetes, and the success rate of constructing diabetic animal models for the screened diabetic animals susceptible to diabetes can reach more than 95%.

[0038] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A pharmaceutical composition, characterized in that The invention comprises the following components in parts by weight: 350-450 parts of metformin, 150-250 parts of wolfberry polysaccharide, 70-140 parts of corn silk polysaccharide and 8-15 parts of dendrobium officinale.

2. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition comprises the following components in parts by weight: 380-410 parts of metformin, 200-230 parts of wolfberry polysaccharide, 100-120 parts of corn silk polysaccharide and 8-10 parts of dendrobium officinale.

3. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition comprises the following components in parts by weight: 410 parts of metformin, 200 parts of wolfberry polysaccharides, 100 parts of corn silk polysaccharides and 10 parts of dendrobium officinale.

4. Use of the pharmaceutical composition according to any one of claims 1 to 3 in constructing a diabetic animal model.

5. A method for constructing a diabetic animal model, characterized in that: The method comprises using the pharmaceutical composition according to any one of claims 1 to 3 to screen animals susceptible to diabetes.

6. The method for constructing a diabetic animal model according to claim 5, characterized in that: The step of screening animals susceptible to diabetes is: administering the drug composition to animals by oral gavage, with a dosage of 600-800 mg / kg, measuring the blood sugar content of the animals 40-50 minutes after administration, and screening animals susceptible to diabetes based on the blood sugar reduction values ​​before and after administration.

7. The method for constructing a diabetic animal model according to claim 6, characterized in that: The screening criteria are: when the blood sugar reduction value is ≤4mmol / L, the animal is judged to be an animal susceptible to diabetes.

8. The method for constructing a diabetic animal model according to claim 5, characterized in that: The method also includes model construction, which specifically comprises the following steps: feeding animals susceptible to diabetes with a diabetic feed for 5-6 consecutive weeks, then injecting STZ for 3 consecutive days at a dose of 80 mg / kg, and then testing the blood glucose level of the animals once a week for 2 consecutive weeks. When the blood glucose levels are maintained above 16.7 mmol / L twice, the construction of the diabetic animal model is considered to be successful.

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