Experimental method for evaluating remission uric acid reduction function and application thereof

Abstract

The invention discloses an experimental method for evaluating a remission uric acid reduction function and application of the experimental method. The method comprises a pre-experiment and a formal experiment, and the formal experiment is as follows: SPF-grade rats are divided into a negative control group, a model group, a positive control group and a test substance group, each group is respectively subjected to intragastric administration with a solvent, a molding agent, a positive drug and a test substance, the test substance is firstly administered during administration each time, and then the molding agent is administered after 2 hours; in 0, 7, 14 and 21 days of the experiment, respectively taking about 0.5 ml of angular blood in rat eyes, and detecting the level of uric acid in serum corresponding to the sampled sample; if the serum uric acid level of the model group rats is obviously higher than that of the negative control group, the rat hyperuricemia model is successfully established; on the basis, the serum uric acid level of a positive control group and any one test substance dosage group is obviously lower than that of a model group, which indicates that the test substance has the function of reducing uric acid in a remission manner. The experimental method established by the invention can provide a scientific reference basis for evaluating the function of relieving blood uric acid increase of the postbiotic related food.

Description

Technical Field

[0001] The present invention relates to the technical field of health food function evaluation, and in particular to an experimental method for evaluating the remission uric acid-lowering function and an application thereof. Background Art

[0002] Hyperuricemia is a metabolic disease of abnormal purine metabolism, and the most direct cause of the disease is gout. Gout is caused by long-term and severe excess of uric acid. Uric acid accumulates in the joints to form urate crystals, which causes increased inflammatory factors in the joints and leads to joint pain. Due to the insignificant clinical characteristics of hyperuricemia, people do not pay much attention to this disease. However, as research has found, in addition to causing gout, hyperuricemia is often complicated by hypertension, non-alcoholic fatty liver disease, cardiovascular disease, and diabetes.

[0003] The traditional dietary pattern in my country is mainly based on the intake of soy products, coarse grains, and fruits and vegetables. With the improvement of living standards and the development of food processing technology, people gradually pursue refined and nutritious foods. The dietary pattern of Chinese residents has gradually changed to a Westernized dietary pattern dominated by refined grains, animal meat, dairy products, sugars, processed foods, etc. The intake of purine-rich seafood, beer and other foods as well as high-sugar and high-fat foods has increased significantly, and the incidence of patients with hyperuricemia has increased year by year. In the past 20 years, the global rate of gout caused by high uric acid has more than doubled. At present, the treatment methods for hyperuricemia mainly include reducing the production of uric acid or increasing the excretion of uric acid. When the uric acid level is high and gout symptoms appear, drug treatment is required. Allopurinol and febuxostat mainly reduce the production of uric acid to inhibit xanthine oxidase, while benzbromarone and probenecid mainly promote uric acid excretion. In clinical practice, these drugs can cause side effects such as allergies, abnormal liver and kidney function, headache, vomiting, and diarrhea. As for the treatment of asymptomatic hyperuricemia and uric acid levels within a controllable range, the main treatments are controlling diet, exercising regularly, quitting drinking, and quitting high-fructose foods. As the number of people with high uric acid levels continues to increase, the demand for uric acid lowering is increasing.

[0004] As people's living standards continue to improve, people's health needs are gradually shifting from passive medical treatment to active health. The public is paying more and more attention to healthy diet, and the health benefits of health foods to the human body are becoming more and more popular. There are 24 functional claims in the current health food function catalog, and there are clear function evaluation methods. Functions other than the 24 functional claims are called new functions of health foods. Since the remission uric acid lowering function is outside the 24 functional claims catalog and is a new function of health foods, there is currently no standard evaluation method.

[0005] In view of the current lack of evaluation methods for new uric acid-lowering functions, the lack of scientific basis for health food companies to apply for new uric acid-lowering functions, and the limited development of the industry, the present invention creates an experimental method for evaluating the remission uric acid-lowering function, and applies it to the new function development of health food postbiotics, clarifies the judgment standards, and establishes a practical and reliable evaluation method, providing a scientific basis for the evaluation of health foods with new remission uric acid-lowering functions. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide an experimental method for evaluating the remission uric acid-lowering function, making it practical and reliable, and providing a scientific basis for the evaluation of health foods with new remission uric acid-lowering functions.

[0007] In order to solve the above technical problems, the present invention provides an experimental method for evaluating the remission uric acid-lowering function, including a formal experiment, wherein the formal experiment is conducted using SD rats of SPF grade and weighing 200-220 g, and the specific method is as follows:

[0008] The rats were divided into negative control group, model group, positive control group and test group, with no less than 9 rats in each group. The rats in the negative control group were orally gavaged with 20 ml / kg·BW 0.5% sodium carboxymethyl cellulose solution, the rats in the model group were orally gavaged with 750 mg / kg·BW potassium oxonate solution and 250 mg / kg·BW ethambutol, with the gavage volume of 5 ml / kg, and the rats in the positive control group were orally gavaged with 750 mg / kg BW potassium oxonate solution and 250mg / kg·BW ethambutol, 30mg / kg·BW positive drug, and the gavage volume of potassium oxonate solution, ethambutol and positive drug were 5ml / kg, 5ml / kg and 10ml / kg respectively. The test group was orally gavaged with 750mg / kg·BW potassium oxonate solution and 250mg / kg·BW ethambutol, as well as a certain dose of the test substance, and the gavage volume of potassium oxonate solution, ethambutol and the test substance were 5ml / kg, 5ml / kg and 10ml / kg respectively. Each group was orally gavaged for 21 consecutive days. The order of gavage every day was first gavage the positive control group, the positive drug and the test substance in the test substance group, and then gavage potassium oxonate solution and ethambutol 2h later.

[0009] About 0.5 ml of blood was collected from the inner canthus of the rats on days 0, 7, 14, and 21 of the experiment, and the uric acid level in the corresponding serum of the sampled samples was tested;

[0010] If the serum uric acid level of the rats in the model group is significantly higher than that of the rats in the negative control group, it indicates that the hyperuric acid model of the rats is successfully established; on this basis, the serum uric acid levels of the positive control group and the test group are significantly lower than those of the model group, indicating that the test substance has a remittent uric acid-lowering function.

[0011] Further improvements include: the step of detecting the urea nitrogen and creatinine levels in the serum corresponding to the sampled samples. If there is no significant difference in the serum urea nitrogen and creatinine levels between the test group and the model group, and the average value of the test group is lower than the average value of the model group, it indicates that the test substance will not cause liver and kidney damage while having the function of alleviating uric acid lowering.

[0012] As a further improvement, the formal experiment also includes removing the liver and kidney of each group of rats and weighing them on the 22nd day of the experiment, measuring the liver and kidney indexes, making pathological slides, observing the changes in organs between the groups, and determining the effects of the test substances on the organs.

[0013] As a further improvement, the test substance group in the formal experiment includes at least three dosage groups with different dosages, and the different dosage groups are set according to the recommended human dosage, and one of the dosage groups is calculated according to 5 times the recommended human dosage.

[0014] As a further improvement, a preliminary experiment is also included before the formal experiment. The preliminary experiment uses SPF grade SD rats weighing 200-220g for the experiment. The specific method is as follows:

[0015] The rats were divided into a negative control group and a model group, with no less than 8 rats in each group. The rats in the negative control group were orally gavaged with 20 ml / kg·BW 0.5% sodium carboxymethyl cellulose solution, and the rats in the model group were orally gavaged with 750 mg / kg·BW potassium oxonate solution and 250 mg / kg·BW ethambutol, with the gavage volume of 5 ml / kg. About 0.5 ml of blood was collected from the inner canthus of the rats on days 0, 7, and 14 of the experiment, and the uric acid level in the corresponding serum of the sampled samples was detected.

[0016] If the serum uric acid level of the rats in the model group is significantly higher than the serum uric acid index of the rats in the negative control group, it indicates that the modeling agent and modeling method of the model group can be used to establish a high uric acid model in rats;

[0017] Based on the successful modeling of the preliminary experiment, the formal experiment was carried out.

[0018] As another improvement of the present invention, the present invention also provides an application of the above-mentioned experimental method for evaluating the remission uric acid-lowering function to evaluate whether the postbiotic health food has the remission uric acid-lowering function.

[0019] After adopting such a design, the present invention has at least the following advantages:

[0020] The experimental method for evaluating the remission uric acid-lowering function of the present invention takes SPF rats as the main experimental subjects, establishes a negative control group, a model group, a positive control group, and a test substance group, and by limiting the administration method and administration sequence of each group, establishes a hyperuric acid model and an intervention mode of the test substance on hyperuric acid, and through the limitation of detection indicators, realizes the evaluation of whether the test substance has the remission uric acid-lowering function and the evaluation of whether it has liver and kidney function damage, providing a scientific reference basis for the evaluation of the new remission uric acid-lowering function of health foods. DETAILED DESCRIPTION

[0021] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of 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.

[0022] In this example, the postbiotic inactivation of Akkermansia muciniphila was used as an example to evaluate its mitigating uric acid-lowering function. The specific method is as follows:

[0023] 1. Experimental Animals

[0024] 70 SPF male SD rats, weighing 200-220 g, were provided by SBEF (Beijing) Biotechnology Co., Ltd., with experimental animal use license number: SYXK (Beijing) 2022-0034.

[0025] Under the conditions of temperature 20-26℃ and relative humidity 40-70%, the purchased animals were adaptively fed for 5 days and then animal quarantine was completed. The experiment was carried out after confirming that the animals were healthy.

[0026] 2. Preliminary Experiment

[0027] In the preliminary experiment, 16 rats were divided into a model group and a negative control group, with 8 rats in each group.

[0028] The experimental animals were given oral gavage according to Table 1.

[0029] Table 1 Oral gavage treatment of pre-experimental animals

[0030]

[0031] About 0.5 ml of blood was collected from the inner canthus of each group of rats on days 0, 7, and 14 of the experiment to detect the uric acid level in the corresponding serum of the sampled samples. The results of the uric acid (UA) level in the corresponding serum of the sampled samples of each group of animals on day 14 are shown in Table 2 below.

[0032] Table 2 Uric acid levels in serum of animals in each group of pre-experimental samples collected on day 14

[0033] Group Uric acid (μmol / L) Negative control group 99.05±4.78 Model Group 155.98±10.99

[0034] As shown in Table 2 above, the UA level in the model group was significantly higher than that in the negative control group (P < 0.05), indicating that the hyperuric acid model was successfully established. The results showed that the modeling agent and modeling method of the model group in the preliminary experiment can be used to establish a hyperuric acid model in rats, and formal experiments can be carried out.

[0035] 3. Formal Experiment

[0036] In the formal experiment, 54 rats were selected and divided into negative control group, model group, positive control group, test substance groups 1, 2, and 3. There were 9 animals in each group, including 2.5 times the dose of the test substance, 5 times the dose of the test substance, and 10 times the dose of the test substance.

[0037] The test sample used in the test group was inactivated Akkermansia muciniphila AKK PROBIO, and the recommended dose for humans was 1×10 11 / day, sample count was 3.1×10 12 / g, and the three dosage groups of the experiment were set up according to 2.5 times, 5 times and 10 times the recommended human dose. The oral doses of the dosage groups were 1.39mg / kg·BW, 2.78mg / kg·BW and 5.56mg / kg·BW, respectively.

[0038] At the same time, the experimental animals were orally gavaged according to Table 3.

[0039] Table 3 Oral gavage treatment of formal experimental animals

[0040]

[0041]

[0042] On days 0, 7, 14, and 21 of the formal experiment, about 0.5 ml of blood was collected from the inner canthus of each group of rats to detect the levels of uric acid (UA), urea nitrogen (BUN), and creatinine (Cr) in the corresponding serum of the sampled samples. At the same time, on the 22nd day of the experiment, the liver and kidney of each group of rats were removed and weighed, the liver and kidney indexes were measured, and pathological sections were prepared to observe the changes in organs between the groups.

[0043] Table 4 shows the levels of uric acid (UA), urea nitrogen (BUN) and creatinine (Cr) in the serum of the animals in each group at 21 days.

[0044] Table 4 Uric acid, urea nitrogen and creatinine levels in serum of the samples collected from animals in each experimental group

[0045] Group Creatinine (μmol / L) Urea nitrogen (mmol / L) Uric acid (μmol / L) Negative control group 32.56±3.93 4.67±0.42 144.00±14.45 Model Group 34.23±4.77 7.04±1.31 210.97±44.98 Positive control group 32.36±2.89 6.35±1.37 178.90±30.68 Test group 1 27.81±3.90 5.51±0.89 149.44±29.70 Test Group 2 30.26±4.09 5.85±0.99 148.19±27.05 Test group 3 24.66±5.91 5.50±1.35 140.78±24.69

[0046] As shown in Table 4 above, the uric acid (UA) in the model group was significantly higher than that in the control group (P < 0.05), indicating that the hyperuric acid model was successfully established. The UA values ​​in the positive control group and each test group were significantly lower than those in the model group (P < 0.05), and the BUN and Cr levels in each test group were not significantly different from those in the model group, and the average values ​​were lower than those in the model group. This result is consistent with the results of pathological section observation. The results show that the postbiotics of inactivated Akkermansia muciniphila have a remission-lowering uric acid effect and do not have the side effect of damaging liver and kidney function.

[0047] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Those skilled in the art may make some simple modifications, equivalent changes or modifications using the technical contents disclosed above, which all fall within the protection scope of the present invention.

Claims

1. An experimental method for evaluating the remission uric acid-lowering function, characterized in that: The formal experiment includes a formal experiment using SPF grade SD rats weighing 200-220g. The specific method is as follows: The rats were divided into negative control group, model group, positive control group and test group, with no less than 9 rats in each group. The rats in the negative control group were orally gavaged with 20 ml / kg·BW 0.5% sodium carboxymethyl cellulose solution, the rats in the model group were orally gavaged with 750 mg / kg·BW potassium oxonate solution and 250 mg / kg·BW ethambutol, with the gavage volume of 5 ml / kg, and the rats in the positive control group were orally gavaged with 750 mg / kg BW potassium oxonate solution and 250mg / kg·BW ethambutol, and 30mg / kg·BW positive drug, and the gavage volume of potassium oxonate solution, ethambutol and positive drug were 5ml / kg, 5ml / kg, 10ml / kg respectively. The test group was orally gavaged with 750mg / kg·BW potassium oxonate solution and 250mg / kg·BW ethambutol, as well as a certain dose of test substance, and the gavage volume of potassium oxonate solution, ethambutol and test substance were 5ml / kg, 5ml / kg, 10ml / kg respectively. Each group was orally gavaged for 21 consecutive days. The gavage order every day was first gavage the positive control group, the positive drug and the test substance in the test substance group, and then gavage potassium oxonate solution and ethambutol 2h later. About 0.5 ml of blood was collected from the inner canthus of the rats on days 0, 7, 14, and 21 of the experiment, and the uric acid level in the corresponding serum of the sampled samples was tested; If the serum uric acid level of the rats in the model group is significantly higher than that of the rats in the negative control group, it indicates that the hyperuric acid model of the rats is successfully established; on this basis, the serum uric acid levels of the positive control group and the test group are significantly lower than those of the model group, indicating that the test substance has a remittent uric acid-lowering function.

2. The experimental method for evaluating the remission uric acid-lowering function according to claim 1, characterized in that: Also includes: The step of detecting the urea nitrogen and creatinine levels in the corresponding serum of the sampled samples. If there is no significant difference in the serum urea nitrogen and creatinine levels between the test group and the model group, and the average value of the test group is lower than the average value of the model group, it indicates that the test substance will not cause liver and kidney damage while having the function of alleviating uric acid lowering.

3. The experimental method for evaluating the remission uric acid-lowering function according to claim 2, characterized in that: The formal experiment also includes removing the liver and kidney of each group of rats and weighing them on the 22nd day of the experiment, measuring the liver and kidney indexes, making pathological sections, observing the changes in organs between groups, and determining the effects of the test substances on the organs.

4. The experimental method for evaluating the remission uric acid-lowering function according to claim 1, characterized in that: The test substance groups in the formal experiment include at least three dosage groups with different dosages, and the different dosage groups are set according to the recommended human dosage, and one of the dosage groups is calculated according to 5 times the recommended human dosage.

5. The experimental method for evaluating the remission uric acid-lowering function according to any one of claims 1 to 4, characterized in that: Before the formal experiment, a preliminary experiment was also included. The preliminary experiment was conducted using SPF grade SD rats weighing 200-220 g. The specific method was as follows: The rats were divided into a negative control group and a model group, with no less than 8 rats in each group. The rats in the negative control group were orally gavaged with 20 ml / kg·BW 0.5% sodium carboxymethyl cellulose solution, and the rats in the model group were orally gavaged with 750 mg / kg·BW potassium oxonate solution and 250 mg / kg·BW ethambutol, with the gavage volume of 5 ml / kg. About 0.5 ml of blood was collected from the inner canthus of the rats on days 0, 7, and 14 of the experiment, and the uric acid level in the corresponding serum of the sampled samples was detected. If the serum uric acid level of the rats in the model group is significantly higher than the serum uric acid index of the rats in the negative control group, it indicates that the modeling agent and modeling method of the model group can be used to establish a high uric acid model in rats; Based on the successful modeling of the preliminary experiment, the formal experiment was carried out.

6. Use of the experimental method for evaluating the palliative uric acid-lowering function according to any one of claims 1 to 5 to evaluate the use of postbiotic health foods with a palliative uric acid-lowering function.

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