CN118794905A - A method for rapidly evaluating the efficacy of inhibiting blood sugar rise using a zebrafish model - Google Patents

Abstract

The invention belongs to the technical field of food detection, and particularly relates to a method for rapidly evaluating an effect of inhibiting sugar increase by using a zebra fish model. The method comprises the following steps: (1) Selecting young zebra fish 5-7 days after fertilization, respectively placing the young zebra fish in culture water, and setting a blank control group, a model control group, a positive control group and a sample group to be tested, wherein maltodextrin is added into the model control group, the positive control group and the sample group to be tested for culture; (2) And (3) determining the glucose content in the zebra fish body, and representing the glucose inhibition effect of the sample to be tested by the relative glucose content. The method has the advantages of short detection time, simple and convenient operation, low detection cost and capability of realizing large-batch operation, and can be used for transverse comparison of the blocking capacity of different raw materials.

Description

A method for rapidly evaluating the efficacy of inhibiting blood sugar rise using a zebrafish model

Technical Field

The invention belongs to the technical field of food detection, and in particular relates to a method for rapidly evaluating the efficacy of inhibiting blood sugar rise by using a zebrafish model.

Background Art

As the living standards of Chinese people improve, the amount of refined carbohydrates consumed in their daily diet is also increasing. Excessive carbohydrate intake is one of the main causes of metabolic diseases such as overweight, obesity and diabetes. Faced with the increasing obesity rate and the increasing incidence of metabolic diseases caused by obesity, controlling the intake of refined carbohydrates is one of the important strategies for treating metabolic diseases such as obesity and diabetes.

In recent years, people's health awareness has been continuously improved. Obese or diabetic patients often use drugs or functional foods to control carbohydrate intake, including choosing to eat low-glycemic index, high-satiety foods instead of traditional high-glycemic index foods, eating white kidney bean extract and other amylase blockers to reduce carbohydrates, and even injecting or taking drugs such as semaglutide to suppress appetite, thereby reducing energy intake. However, as a new drug, semaglutide has the risk of adverse gastrointestinal reactions, hypoglycemia, acute pancreatitis, etc. For most people who need to control carbohydrate intake, eating functional foods that block carbohydrate absorption is a lower-risk and cost-effective way. At present, there are many types of carbohydrate blockers on the market, and the quality is uneven. How consumers choose a product with good carbohydrate absorption blocking is a problem. For functional food manufacturers, the number of raw materials that claim to block carbon water absorption and inhibit food glycemic control is also very large. Therefore, how to select better raw materials from a large number of raw materials for formula design is also a problem that needs to be overcome.

At present, conventional methods for evaluating the ability of food to inhibit blood sugar rise mainly include human blood sugar test ("WS/T652-2019 Food Glycemic Index Determination Method"), oral glucose tolerance test of mice, and in vitro α-amylase or α-glucosidase enzyme activity inhibition evaluation test. Human or animal experiments have problems such as long detection cycle, high cost, and high experimental operation requirements. In vitro enzyme activity tests such as α-amylase and α-glucosidase only detect the ability to inhibit enzyme activity at the in vitro level, and cannot fully reflect the overall evaluation of the ability of carbohydrate absorption blocking products to inhibit blood sugar rise in living organisms.

As a new model organism, zebrafish has a gene homology of up to 87% with human genes and is an important evaluation model for health products or functional foods. Chinese invention patent application CN116508687A discloses a method for constructing a zebrafish model for testing the glycemic index. The zebrafish model was used to test the glycemic index of carbohydrates such as glucose, rice, and buckwheat noodles, but the method did not propose an evaluation method for blocking carbohydrate functional raw materials or foods.

Liu Jun et al. evaluated the hypoglycemic effect of white tea on diabetic zebrafish by using a zebrafish diabetes model induced by glucose and alloxan in “Evaluation of the hypoglycemic effect of white tea based on a zebrafish model” (Modern Food Science and Technology, 2023, 39(3):45-54). This model is also not suitable for evaluating the effect of raw materials or products in blocking carbohydrate absorption.

Chinese invention patent application CN117491323A discloses a method for rapidly evaluating anti-glycation efficacy using a zebrafish model, the method comprising: selecting zebrafish fry 48-72 hours after fertilization, placing them in a zebrafish culture medium containing a glycation inducing agent and a sample to be tested, and culturing them at 25-29°C for 6-28 hours; determining the fluorescence intensity of the advanced glycation end products in the zebrafish culture medium, and characterizing the anti-glycation efficacy of the sample to be tested by relative fluorescence intensity. The glycation inducing agent is one or more of methylglyoxal, glyoxal, 3-deoxyglucosone, glucose, and fructose. However, this invention uses zebrafish to evaluate the glycation effect of anti-glycation raw materials on inhibiting sugar on collagen and elastin in the dermis of the skin, and is not suitable for evaluating the efficacy of some raw materials in blocking the absorption of large molecular carbon water.

Therefore, it is of great significance to develop a rapid and low-cost in vivo method to evaluate the ability to block carbohydrate absorption.

Summary of the invention

In view of the deficiencies in the prior art, the present invention provides a method for quickly evaluating the efficacy of inhibiting blood sugar levels using a zebrafish model, and provides a method for evaluating whether a product blocks carbohydrate absorption and inhibits food blood sugar levels, which is simple and fast to operate, has low detection costs, and can be operated in large quantities.

In order to achieve the above-mentioned purpose of the present invention, the specific technical solution adopted by the present invention is:

A method for rapidly evaluating the efficacy of inhibiting blood sugar rise using a zebrafish model comprises the following steps:

(1) Select zebrafish fry 5-7 days after fertilization and place them in culture water to set up a blank control group, a model control group, a positive control group and a test sample group, wherein maltodextrin is added to the model control group, the positive control group and the test sample group for culture;

(2) Determine the glucose content in zebrafish and use the relative glucose content to characterize the anti-glycemic effect of the test sample.

Preferably, the zebrafish is AB strain zebrafish, and the number of zebrafish fry in each group is 15-30.

Preferably, the mass concentration of maltodextrin in aquaculture water is 4%-8%.

Preferably, the raw materials of the aquaculture water include deionized water, artificial sea salt and sodium bicarbonate.

Further preferably, the mass concentration of the artificial sea salt in the aquaculture water is 350-450 mg/L, and the mass concentration of the sodium bicarbonate is 150-250 mg/L.

Preferably, the culture temperature is 26.5-28.5°C, and the culture time is 23-25h.

Preferably, acarbose is further added to the positive control group in step (1), and a test sample is further added to the test sample group, wherein the test sample is food or its active ingredient.

Preferably, the method for determining the glucose content in zebrafish in step (2) comprises the following steps:

All zebrafish in each group were collected, washed with water and PBS solution, then added with PBS solution, frozen and anesthetized to death, homogenized, centrifuged, and the supernatant was taken. The glucose content of each group was detected by a glucose detection kit, and the absorbance at a wavelength of 500-510nm was detected by an enzyme marker. The test was repeated and the average value was taken.

Preferably, the calculation formula for the relative glucose content in step (2) is:

Relative content of glucose = average absorbance of the sample to be tested/average absorbance of the blank control sample × 100.

Compared with the prior art, the present invention has the following beneficial effects:

(1) Compared with conventional human or animal model detection methods for evaluating the glycemic index of food or evaluating the ability of products to inhibit the glycemic index of food, the method of the present invention has the advantages of short detection time, simple operation, low detection cost, and large-scale operation, and can conduct a horizontal comparison of the ability of different raw materials to block carbohydrate absorption.

(2) Compared with the conventional in vitro enzyme activity method for evaluating products that block carbohydrate absorption, the method of the present invention utilizes a zebrafish model that has a high genetic homology with humans, and can more comprehensively reflect the overall evaluation of the ability of products that block carbohydrate absorption to inhibit blood sugar rise in living organisms in vivo.

(3) Compared with the existing zebrafish high-sugar model using glucose as the main glucose-elevating inducer, the present invention introduces for the first time a high-sugar model using maltodextrin as the glucose-elevating inducer and acarbose as the positive control. The present invention uses the zebrafish model to evaluate the efficacy of products that block carbohydrate absorption, while the existing zebrafish model using glucose as the glucose-elevating inducer cannot evaluate the efficacy of products that block carbohydrate absorption.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a diagram showing the blood sugar elevation effect induced by different concentrations of maltodextrin in Example 1 of the present invention;

FIG2 is a graph showing the effect of acarbose on the blood glucose-raising effect of different inducers in Example 1 of the present invention;

FIG3 is a graph showing the effect of different raw materials on the glycemic control effect of maltodextrin-induced zebrafish in Example 2 of the present invention;

FIG4 is a comparison diagram of the glycemic effects of soluble starch and maltodextrin induced in zebrafish in Comparative Example 1 of the present invention;

FIG5 is a comparison chart of the glucose- and maltodextrin-induced hyperglycemic effects on zebrafish in Comparative Example 2 of the present invention.

DETAILED DESCRIPTION

The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

The fish used in the experiment were wild-type AB strains purchased from Hangzhou Huante Biotechnology Co., Ltd., and the broodstock were 5-6 months old.

Experimental reagents: maltodextrin (purchased from Weifang Shengtai Pharmaceutical Co., Ltd.), acarbose (CAS No.: 56180-94-0, Shanghai Yuanye Biotechnology Co., Ltd.), glucose kit (A154-1-1, Nanjing Jiancheng Bioengineering Institute), phosphate buffer solution (Wuhan Punosai Life Science Technology Co., Ltd.), and purified water.

Experimental equipment and consumables: multifunctional microplate reader (Molecular Devices), high-speed centrifuge (Shanghai Lu Xiangyi Centrifuge Instrument Co., Ltd.), fish tank (purchased from Huante Biotechnology Co., Ltd.), constant temperature incubator (HT-250H-T, Huante Biotechnology Co., Ltd.), 6-well plate (CCP06-006, Zhejiang Belanbo Biotechnology Co., Ltd.).

Example 1

A method for rapidly evaluating the efficacy of inhibiting blood sugar rise using a zebrafish model, the specific steps are as follows:

1) Zebrafish breeding and reproduction

Wild-type AB zebrafish were selected as model animals and cultured in a circulating water system. After sexual maturity, males and females were separated and raised according to a photoperiod of 14h light + 10h dark per day. The culture temperature was 28±0.5℃ and the pH was 7.0±0.5. Artemia was fed twice a day for 15 minutes each time. Zebrafish with good vitality were selected as broodstock. The zebrafish were fertilized in vitro. The fish were placed in the breeding tank at a ratio of female: male = 1:2 and separated by partitions. The partition of the spawning tank was pulled open the next morning. Under the stimulation of light, the male fish could be seen chasing the female fish constantly and starting to lay eggs. The fertilized embryos were collected and placed in a 28℃ constant temperature incubator for incubation until the 6th day, which could be used for experiments. During this period, it was necessary to pay attention to changing fresh breeding water every day.

2) Sample processing and construction of glucose-raising model

Twenty zebrafish at 6 dpf (sixth day after fertilization) were placed in each well of a 6-well plate. The test sample group was added with aquaculture water containing a certain concentration (5 μg/mL-1 mg/mL) of the test sample, wherein the aquaculture water consisted of deionized water, artificial sea salt (400 mg/L) and sodium bicarbonate (200 mg/L); the blank control group and the model control group were added with an equal amount of aquaculture water and placed in a constant temperature incubator at 28°C for 1 hour, and then maltodextrin was added to the model control group and the test sample group and cultured for 24 hours.

3) Determination of glucose content in zebrafish

Collect all zebrafish from each experimental group into a 1.5 mL centrifuge tube, add 1 mL of clean water and wash 3 times, each time for 3 minutes; add 1 mL of PBS solution to wash once, and absorb the residual PBS solution at the bottom of the test tube; add 50 μL of PBS solution to each tube, place it in ice water for freezing anesthesia to death, and then use a manual homogenizer to homogenize the zebrafish, centrifuge at 10,000 rpm for 15 minutes, take the supernatant, use a glucose detection kit to detect the glucose content of each group, and use an enzyme reader to detect the absorbance at a wavelength of 505 nm. Take 2 samples from each group for testing and take the average value; the formula for calculating the relative glucose content in zebrafish is: relative glucose content (relative to blank control group) = (average absorbance of the sample to be tested/average absorbance of the blank control sample) × 100.

The process of exploring, optimizing and verifying the conditions of each step in the detection method is as follows. During the exploration of the following conditions, except for the changes in the mentioned steps, the remaining experimental steps are the same as above.

1. Selection of maltodextrin concentration

Twenty zebrafish at 6 dpf (sixth day after fertilization) were accurately taken into each well of a 6-well plate, and 3 mL of culture water containing 0% (pure culture water), 1%, 2%, 4%, 6%, and 8% (mass percentage) maltodextrin was added respectively. After culturing for 24 hours, the glucose content in the zebrafish was detected. The 0% maltodextrin group was used as the control. The relative glucose content of each group is shown in Table 1 and Figure 1.

Table 1 Glycemic effect induced by different concentrations of maltodextrin

Note: Compared with 0% maltodextrin group, P＜0.01, P＜0.0001.

As can be seen from Table 1 and Figure 1, compared with 0% maltodextrin, 1% and 2% maltodextrin can induce a significant increase in the glucose content in zebrafish (P < 0.01), while 4%, 6%, and 8% maltodextrin can induce a very significant increase in the glucose content in zebrafish (P < 0.0001), so 4% maltodextrin was selected as the subsequent experimental condition.

2. Determination of positive control

Twenty zebrafish at 6 dpf (sixth day after fertilization) were accurately taken into each well of a 6-well plate, and a blank control group (pure culture water), 4% glucose group, 4% glucose + 0.01 mg/mL acarbose group, 4% maltodextrin group, and 4% maltodextrin + 0.01 mg/mL acarbose group were set up respectively. After culturing for 24 hours, the glucose content in the zebrafish was detected. The blank control group was used as the control. The relative glucose content of each group is shown in Table 2 and Figure 2.

Table 2 Effect of acarbose on the glucose-raising effect of different inducers

Note: Compared with 4% glucose, ^ns P＞0.05; compared with 4% maltodextrin, P＜0.001.

As shown in Table 2 and Figure 2, 4% glucose and 4% maltodextrin can both induce an increase in the glucose content in zebrafish. Acarbose can significantly inhibit the blood sugar rise induced by maltodextrin (p < 0.001), but cannot inhibit the blood sugar rise induced by glucose (p > 0.05). This shows that acarbose can inhibit the absorption and utilization of maltodextrin by zebrafish, achieving the effect of blocking sugar, but has no obvious absorption inhibition effect on glucose monosaccharide. Repeated experiments have found that acarbose can stably inhibit the increase in glucose content in zebrafish induced by maltodextrin. Therefore, acarbose was selected as the positive control of this model.

Example 2

This example conducts a study on the efficacy of inhibiting blood sugar rise in some commonly used functional food ingredients that claim to have the ability to inhibit the rise in postprandial blood sugar: EGCG (epigallocatechin gallate), chestnut polyphenols, sugarcane polyphenols and other ingredients.

Twenty zebrafish at 6 dpf (sixth day after fertilization) were accurately taken from each well and placed in a 6-well plate, and blank control group (pure culture water), model control group (4% maltodextrin), positive control group (4% maltodextrin + 0.01 mg/mL acarbose), EGCG group (4% maltodextrin + 0.01 mg/mL EGCG), chestnut polyphenol (4% maltodextrin + 0.01 mg/mL chestnut polyphenol), sugarcane polyphenol (4% maltodextrin + 0.01 mg/mL sugarcane polyphenol) were set up respectively. After culturing for 24 hours, the glucose content in zebrafish was detected, and the relative glucose content of each group was shown in Table 3 and Figure 3, with the blank control group as the control.

Table 3 Effects of different raw materials on the glycemic response of zebrafish induced by maltodextrin

Note: Compared with the model control group, ^ns P＞0.05, P＜0.05, P＜0.01, P＜0.001.

The present invention can be used to detect the ability of products or raw materials to inhibit blood sugar rise. As shown in Table 3 and Figure 3, acarbose, a traditional drug for inhibiting postprandial blood sugar rise, can significantly inhibit the increase of glucose content in zebrafish induced by maltodextrin (P < 0.001). This example also detects the inhibitory effects of chestnut polyphenols, sugarcane polyphenols, and EGCG on blood sugar rise. Among them, chestnut polyphenols (P < 0.05) and EGCG (P < 0.01) can also significantly inhibit the increase of glucose content in zebrafish induced by maltodextrin.

Comparative Example 1

This comparative example uses 4% soluble starch as a control to compare the difference between maltodextrin and soluble starch in inducing glucose elevation in zebrafish.

Twenty zebrafish at 6 dpf (sixth day after fertilization) were accurately taken from each well in a 6-well plate, and a blank control group (pure culture water), a 4% maltodextrin group, and a 4% soluble starch group were set up. After 24 hours of culture, the glucose content in the zebrafish was detected, and the relative glucose content of each group was shown in Table 4 and Figure 4, with the blank control group as the control.

Table 4 Comparison of glycemic effects of soluble starch and maltodextrin in zebrafish

Note: Compared with the blank control group, ^ns P＞0.05, P＜0.001.

As shown in Table 4 and Figure 4, 4% maltodextrin can induce a significant increase in the glucose content in zebrafish (P < 0.001), while 4% soluble starch has no such effect (P > 0.05). It can be seen that soluble starch is not suitable for inducing a high-sugar model in zebrafish.

Comparative Example 2

This comparative example uses 4% glucose as a control and EGCG as a test sample to examine the differences in the effects on the high-sugar model induced by glucose and the high-sugar model induced by maltodextrin.

Twenty zebrafish at 6 dpf (sixth day after fertilization) were accurately taken from each well in a 6-well plate, and a blank control group (pure culture water), 4% glucose group, 4% glucose + 0.01 mg/mL EGCG group, 4% maltodextrin group, and 4% maltodextrin + 0.01 mg/mL EGCG group were set up respectively. After culturing for 24 hours, the glucose content in the zebrafish was detected. The blank control group was used as the control. The relative glucose content of each group is shown in Table 5 and Figure 5.

Table 5 Effect of EGCG on the glucose-raising effect of different inducers

Note: Compared with 4% glucose, ^ns P＞0.05; compared with 4% maltodextrin, P＜0.001.

As shown in Table 5 and Figure 5, EGCG can inhibit the increase of glucose content in zebrafish induced by 4% maltodextrin (P < 0.001), but cannot inhibit the increase of glucose content induced by glucose (P > 0.05). Combined with the above results of acarbose inhibiting blood sugar rise, it can be seen that the high sugar model induced by glucose is not suitable for evaluating the efficacy of some raw materials in blocking the absorption of macromolecular carbon water, while the maltodextrin used in the present invention can be used.

The above detailed description is a specific description of one feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims (9)

1. A method for rapidly evaluating the efficacy of inhibiting blood sugar rise using a zebrafish model, comprising the following steps: (1) Select zebrafish fry 5-7 days after fertilization and place them in culture water to set up a blank control group, a model control group, a positive control group and a test sample group, wherein maltodextrin is added to the model control group, the positive control group and the test sample group for culture; (2) Determine the glucose content in zebrafish and use the relative glucose content to characterize the anti-glycemic effect of the test sample. 2. The method according to claim 1, characterized in that the zebrafish is AB strain zebrafish, and the number of zebrafish fry in each group is 15-30. 3. The method according to claim 1, characterized in that the mass concentration of the maltodextrin in the aquaculture water is 4%-8%. 4. The method according to claim 1 is characterized in that the raw materials of the aquaculture water include deionized water, artificial sea salt and sodium bicarbonate. 5. The method according to claim 4, characterized in that the mass concentration of artificial sea salt in the aquaculture water is 350-450 mg/L, and the mass concentration of sodium bicarbonate is 150-250 mg/L. 6. The method according to claim 1, characterized in that the culture temperature is 26.5-28.5°C and the culture time is 23-25h. 7. The method according to claim 1, characterized in that acarbose is further added to the positive control group in step (1), and a test sample is further added to the test sample group, and the test sample is food or its active ingredient. 8. The method according to claim 1, characterized in that the method of determining the glucose content in zebrafish in step (2) comprises the following steps: All zebrafish in each group were collected, washed with water and PBS solution, then added with PBS solution, frozen and anesthetized to death, homogenized, centrifuged, and the supernatant was taken. The glucose content of each group was detected by a glucose detection kit, and the absorbance at a wavelength of 500-510nm was detected by an enzyme marker. The test was repeated and the average value was taken. 9. The method according to claim 1, characterized in that the calculation formula of the relative glucose content in step (2) is: Relative content of glucose = average absorbance of the sample to be tested/average absorbance of the blank control sample × 100.