A method for rapidly evaluating anti-glycation efficacy using a zebrafish model

Abstract

This invention discloses a method for rapidly evaluating anti-glycation efficacy using a zebrafish model, belonging to the field of food and cosmetic testing technology. The method includes: selecting zebrafish juveniles 48–72 hours after fertilization, placing them in a zebrafish culture medium containing a glycation inducing reagent and the sample to be tested, and culturing them at 25–29°C for 6–28 hours; measuring the fluorescence intensity of advanced glycation end products (AGEs) in the zebrafish culture medium, and characterizing the anti-glycation efficacy of the sample by relative fluorescence intensity. This invention utilizes zebrafish juveniles to construct a glycation model, and evaluates the anti-glycation efficacy of the sample by detecting fluorescent AGEs in the zebrafish culture medium. The method has low detection cost, simple experimental operation, short experimental cycle, and accurate detection results, enabling rapid screening of anti-glycation foods or cosmetics in large quantities.

Description

Technical Field

[0001] This invention relates to the field of food and cosmetic testing technology, specifically to a method for rapidly evaluating anti-glycation efficacy using a zebrafish model. Background Technology

[0002] Glycation is a non-enzymatic, irreversible process that binds sugars or sugar degradation products to proteins, thus damaging them. At the molecular level, reducing sugars undergo non-enzymatic glycation reactions with free amino groups in proteins and other molecules, forming advanced glycation end products (AGEs). The accumulation of AGEs in the body can cause tissue damage, inflammation, oxidative stress, and other adverse reactions.

[0003] With the continuous improvement of people's living standards, the demand for nutrition and health is also constantly increasing, and the development of anti-glycation products has become an important trend in nutrition and health. Therefore, studying the formation mechanism of anti-glycation reactions and its detection methods is of great significance for the development of anti-glycation products.

[0004] Currently, conventional methods for evaluating glycation generally involve in vitro protein glycation experiments or glycation evaluation based on cell experiments. These methods suffer from problems such as long detection times, high requirements for experimental operation, and complex and diverse detection principles. Moreover, these detection methods only detect anti-glycation effects at the in vitro level and cannot comprehensively reflect the overall evaluation of anti-glycation products on living organisms.

[0005] Zebrafish models are important evaluation models for health foods and cosmetics. Zebrafish larvae do not need to eat within 5 days post-fertilization (5 dpf), which is considered acceptable under EU ethics and not within the scope of animal ethics, making it a viable alternative for nutritional and safety evaluations. Compared to using laboratory animals such as mice, using zebrafish larvae within 5 dpf is more in line with animal ethics and has greater significance for animal protection. Zebrafish culture medium is an important medium for fluid exchange between zebrafish and the external environment, and also an important carrier for substance exposure; therefore, the composition of substances in zebrafish culture medium has a certain degree of consistency with corresponding components in the zebrafish's body.

[0006] Existing reports disclose some methods for evaluating anti-glycation using zebrafish models. For example, patent document CN112198143A discloses a method for evaluating the anti-photoaging and anti-glycation effects of cosmetics and health foods. This method uses ultraviolet light to induce a zebrafish glycation model and measures the content of glycated glycoproteins in zebrafish to evaluate the anti-glycation effect. This method requires the use of a glycated hemoglobin ELISA kit, which is costly and requires a high level of operational skill. It is also difficult to control the intensity of ultraviolet irradiation on zebrafish, which is not conducive to large-scale rapid analysis and detection. Another literature reports the use of 5dpf zebrafish as experimental subjects and the reaction at 60℃ (Multi-model evaluation of the anti-glycation effect and active ingredient study of Sophora japonica water extract [J]. Food Industry Technology, 2023, 44(05): 371-379.). However, this method is prone to causing zebrafish death, and the detection results after the reaction at 60℃ are greatly affected by the background. Other methods include PCR, Western blot, flow cytometry, and immunofluorescence to detect the expression level of the corresponding protein receptors. However, these methods have drawbacks such as high technical and cost requirements and low sample detection efficiency.

[0007] Therefore, it is of great significance to develop a method for rapid detection of anti-glycation based on a zebrafish model that is simple to operate, convenient and fast, has animal welfare advantages, and can be used for large-scale operations. Summary of the Invention

[0008] The purpose of this invention is to optimize and simplify current anti-glycation evaluation methods and technologies, and to provide a rapid, objective, accurate, and easy-to-operate method for evaluating anti-glycation, enabling large-sample detection.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] This invention provides a method for rapidly evaluating anti-glycation efficacy using a zebrafish model, comprising the following steps:

[0011] (1) Select zebrafish juveniles 48 to 72 hours after fertilization and place them in zebrafish culture medium containing saccharification induction reagent and the sample to be tested, and culture them at 25 to 29°C for 6 to 28 hours.

[0012] (2) The fluorescence intensity of advanced glycation end products in zebrafish culture medium was determined, and the anti-glycation efficacy of the test sample was characterized by the relative fluorescence intensity.

[0013] This invention constructs a zebrafish glycation model by exposing zebrafish juveniles to a glycation inducing agent. When the glycation inducing agent is absorbed by the zebrafish, it accelerates glycation in vivo. Advanced glycation end products (AGEs) in the zebrafish are excreted from the body into the culture medium. Therefore, by detecting the content of fluorescent AGEs in the culture medium and comparing the changes in fluorescence intensity before and after the treatment, the anti-glycation efficacy of the test sample can be determined.

[0014] This invention uses zebrafish juveniles at 48 hpf to 72 hpf for modeling. At this time, the zebrafish juveniles have not been fed, which can avoid the influence of feeding on the fluorescence detection results.

[0015] Furthermore, the zebrafish used is the AB strain zebrafish.

[0016] Furthermore, 10–40 juvenile fish were placed in each well of the culture plate for treatment. The same number of zebrafish were placed in each experimental group. The study showed that placing 10–40 juvenile fish per well maintained the parallelism of the test results across the experimental groups.

[0017] Preferably, 30 zebrafish juveniles within 72 hours of fertilization are placed in each well and treated to a volume of 3 mL.

[0018] Furthermore, the saccharification inducing agent is one or more of acetone aldehyde (also known as methylglyoxal, MGO), glyoxal, 3-deoxyglucuronide, glucose, and fructose.

[0019] Furthermore, the concentrations of acetone aldehyde, glyoxal, and 3-deoxyglucuronide in zebrafish culture medium were 0.002–0.004 mol / L, while the concentrations of glucose and fructose in zebrafish culture medium were 0.05–0.35 mol / L.

[0020] Preferably, the concentration of acetone aldehyde in the zebrafish culture medium is 0.003 mol / L.

[0021] The zebrafish culture medium comprises: 5 mM sodium chloride, 0.17 mM potassium chloride, 0.33 mM calcium chloride, 0.33 mM magnesium sulfate, with the remainder being water.

[0022] Studies have shown that culture temperature and culture time affect the saccharification level of zebrafish. As the culture temperature and culture time increase, the saccharification level increases, but this can lead to mortality in juvenile fish. Therefore, the culture temperature should be controlled below 30℃, and the treatment time should not exceed 28 hours. Preferably, culture at 28℃ for 24 hours.

[0023] In step (2), the fluorescence intensity of the culture medium is measured using a fluorescence microplate reader with an excitation wavelength of 340 nm and an emission wavelength of 420 nm.

[0024] The formula for calculating the relative fluorescence intensity is:

[0025] Relative fluorescence intensity = F 待测样品组 / F 对照组 F 待测样品组 F represents the fluorescence intensity of the sample group to be tested. 对照组 The fluorescence intensity represents the fluorescence intensity of the model control group.

[0026] Based on the formula for relative fluorescence intensity, the relative fluorescence intensity of the control group is 1.000. Through significance analysis, the anti-glycation efficacy of the test sample groups is compared, and the groups with significant differences are determined to have glycation or anti-glycation effects.

[0027] Furthermore, the sample to be tested is food, pharmaceutical, cosmetic, or its active ingredient. The method provided by this invention can be used for the anti-glycation evaluation of food, pharmaceutical, cosmetic, and other systems or their active ingredients.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) This invention is the first to use the detection of fluorescent AGEs in zebrafish culture medium to evaluate the anti-glycation efficacy of the test sample. The method of constructing zebrafish model does not cause zebrafish death and is more in line with animal welfare and ethical requirements than the detection after zebrafish tissue homogenization.

[0030] (2) Compared with molecular biology detection methods, the method provided by this invention has lower detection costs, simpler experimental operation, and more accurate, efficient and convenient detection results, and can screen anti-glycation foods or cosmetics in large quantities quickly.

[0031] (3) Compared with in vitro glycation reactions, the method provided by the present invention can better reflect in vivo glycation reactions.

[0032] (4) The method provided by the present invention has a short experimental cycle and can quickly complete the efficacy evaluation. Attached Figure Description

[0033] Figure 1 This is a diagram illustrating the mechanism for evaluating the anti-glycation properties of zebrafish. Detailed Implementation

[0034] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0036] The zebrafish used in the following examples are wild-type AB strains, and the broodstock are 3-4 months old.

[0037] Acetone aldehyde (CAS No.: 78-98-8), glyoxal (CAS No.: 107-22-2), 3-deoxyglucuronide (CAS No.: 4084-27-9), glucose (CAS No.: 50-99-7), fructose (CAS No.: 7660-25-5).

[0038] Example 1

[0039] I. This embodiment provides a method for rapidly evaluating anti-glycation efficacy using a zebrafish model. The specific steps are as follows:

[0040] 1) Zebrafish culture and spawning:

[0041] Wild-type AB strain zebrafish were selected as the model organism and cultured in a recirculating aquaculture system. Males and females were separated and fed according to a 14-hour light + 10-hour dark photocycle. The culture temperature was 28±0.5℃, and the pH was 7.0±0.5. They were fed brine shrimp twice daily for 3 minutes each time. Sexually mature zebrafish were selected and placed in the spawning tank one night in advance at a 1:1 male-to-female ratio. Males and females were separated by a partition. The next morning, the lights were turned on, and the partition was removed to allow free mating. Fertilized embryos were collected and cultured in zebrafish culture medium (a solution prepared with deionized water containing 5mM sodium chloride, 0.17mM potassium chloride, 0.33mM calcium chloride, and 0.33mM magnesium sulfate). Incubation was carried out in a 28℃ constant temperature incubator with a 14-hour light + 10-hour dark photocycle, and the culture medium was changed daily. Zebrafish with a growth factor of 72 hpf were collected on the third day.

[0042] 2) Simultaneous exposure to chemical reagents and preparation of the sample to be tested

[0043] Thirty zebrafish at 72 hpf were cultured in 6-well plates. A zebrafish culture medium containing a specific concentration of chemical reagents and a specific concentration of the test sample was added, and the volume was adjusted to 3 mL. The final concentrations of the chemical reagents and test sample in the zebrafish culture medium were the concentrations required for the experiment. After a certain treatment time, the fluorescence intensity in the zebrafish culture medium was measured.

[0044] 3) Measurement of fluorescence intensity and evaluation of anti-glycation

[0045] 200 μL of zebrafish culture medium was added to a black ELISA plate. The fluorescence intensity F of the culture medium was measured using a fluorescence microplate reader with an excitation wavelength of 340 nm and an emission wavelength of 420 nm. Zebrafish culture medium containing chemical reagents was used as a model control group. Anti-glycation was evaluated using relative fluorescence intensity, calculated using the formula: Relative fluorescence intensity = F.待测样品组 / F 对照组 F 待测样品组 F represents the fluorescence intensity of the sample group to be tested. 对照组 The fluorescence intensity represents the fluorescence intensity of the model control group.

[0046] II. Explore, optimize and verify the conditions for each step in the detection method. In the exploration of the following conditions, the contents are the same as above, except that the steps mentioned have changed.

[0047] 1. Selection of MGO concentration

[0048] Thirty zebrafish at 72 hpf were cultured in 6-well plates and added to zebrafish culture medium containing different concentrations of MGO (specific group settings are shown in Table 1), with the volume adjusted to 3 mL. The zebrafish were then cultured in an incubator at 28℃ for 24 hours, and the fluorescence intensity in the zebrafish culture medium was measured. Group F was used as the control, and the relative fluorescence intensity of each group is shown in Table 1.

[0049] Table 1. Selection of different MGO concentrations

[0050]

[0051]

[0052] Different letters indicate significance, and p<0.05 is considered significant.

[0053] As shown in Table 1, 0.002–0.0040 mol / L MGO can increase the content of fluorescent AGEs in zebrafish culture medium. Higher concentrations can cause zebrafish death, while the experimental results are not significant at lower concentrations (less than 0.002 mol / L). This may be because zebrafish can self-regulate their glycosylation state and have a certain tolerance at low concentrations.

[0054] 2. Selection of the number of zebrafish

[0055] Zebrafish ranging from 0 to 60 individuals (72 hpf) were cultured in 6-well plates and added to zebrafish culture medium containing 0.003 mol / L LMGO (specific group settings are shown in Table 2), with the volume adjusted to 3 mL. The zebrafish were then cultured at 28°C for 24 hours, after which the fluorescence intensity in the zebrafish culture medium was measured. Group E served as the control, and the relative fluorescence intensities of each group are shown in Table 2.

[0056] Table 2. Selection of different numbers of zebrafish

[0057] A 1 <![CDATA[0.008±0.005 e ]]> B 5 <![CDATA[0.122±0.024 e ]]> C 10 <![CDATA[0.325±0.047 d <!-- 4 -->]]> D 20 <![CDATA[0.578±0.054 c ]]> E 30 <![CDATA[1.000±0.056 b ]]> F 40 <![CDATA[1.125±0.078 b ]]> G 50 <![CDATA[1.368±0.144 a ]]> H 60 <![CDATA[1.456±0.088 a ]]>

[0058] Different letters indicate significance, and p<0.05 is considered significant.

[0059] Table 2 shows that the content of fluorescent advanced glycation end products (AGEs) increases with the number of zebrafish, indicating an increase in glycation level. Specifically, when the number of zebrafish juveniles is less than 10, the standard deviation is greater than the relative fluorescence intensity, indicating low parallelism and large error in the detection results at this number of juveniles. The results for 30 and 40 zebrafish are not statistically significant, and under conditions with too many zebrafish juveniles (50 or 60), the relative fluorescence intensity does not increase exponentially (more than double). Therefore, 10–40 zebrafish juveniles is a suitable range, with 30 showing the best results.

[0060] 3. Selection of modeling reagents

[0061] Thirty zebrafish at 72 hpf were cultured in 6-well plates and added to zebrafish culture medium containing different modeling reagents (specific group settings are shown in Table 3), with the volume adjusted to 3 mL. The zebrafish were then cultured in an incubator at 28℃ for 24 hours, and the fluorescence intensity in the zebrafish culture medium was measured. Group A was used as the control, and the relative fluorescence intensity of each group is shown in Table 3.

[0062] Table 3. Selection of different modeling reagents

[0063] A 0.003 mol / L MGO (acetone aldehyde) <![CDATA[1.000±0.056 c ]]> B 0.003 mol / L glyoxal <![CDATA[1.348±0.049 b ]]> C 0.003 mol / L 3-deoxyglucuronide <![CDATA[0.869±0.152 d ]]> D 0.05 mol / L glucose <![CDATA[0.267±0.044 e ]]> E 0.05 mol / L fructose <![CDATA[0.305±0.037 e ]]> F 0.35 mol / L glucose <![CDATA[1.532±0.068 a ]]> G 0.35 mol / L fructose <![CDATA[1.502±0.073 a ]]> H No modeling reagent <![CDATA[0.003±0.002 f ]]>

[0064] Different letters indicate significance, and p<0.05 is considered significant.

[0065] As shown in Table 3, 0.003 mol / L of MGO, glyoxal, and 3-deoxyglucuronide can all induce glycosylation in zebrafish, while glucose and fructose require higher concentrations.

[0066] 4. Selection of incubation temperature

[0067] Thirty zebrafish at 72 hpf were cultured in 6-well plates and added to zebrafish culture medium containing 0.003 mol / L LMGO (specific group settings are shown in Table 4), with the volume adjusted to 3 mL. The zebrafish were then cultured in an incubator at 25℃–32℃ for 24 hours. The fluorescence intensity in the zebrafish culture medium was then measured. Group D was used as the control, and the relative fluorescence intensities of each group are shown in Table 4.

[0068] Table 4. Selection of different culture temperatures

[0069] A 25℃ <![CDATA[0.995±0.048 b ]]> B 26℃ <![CDATA[1.056±0.052 b ]]> C 27℃ <![CDATA[1.104±0.064 b ]]> D 28℃ <![CDATA[1.000±0.056 b <!-- 5 -->]]> E 28.5℃ <![CDATA[1.067±0.043 b ]]> F 29℃ <![CDATA[1.230±0.101 a ]]> G 30℃ Some zebrafish died H 31℃ Some zebrafish died I 32℃ Some zebrafish died

[0070] Different letters indicate significance, and p<0.05 is considered significant.

[0071] As shown in Table 4, the higher the culture temperature, the higher the glycosylation level of zebrafish. Zebrafish die at temperatures of 30℃ and above, while the effect is not significant at lower temperatures compared to 28℃. 28℃ is the commonly used temperature for zebrafish culture.

[0072] 5. Selection of training time

[0073] Thirty zebrafish at 72 hpf were cultured in 6-well plates and added to zebrafish culture medium containing 0.003 mol / L LMGO (specific group settings are shown in Table 5), with the volume adjusted to 3 mL. The zebrafish were then cultured at 28℃. After different culture times, the fluorescence intensity in the zebrafish culture medium was measured. Group G was used as the control, and the relative fluorescence intensities of each group are shown in Table 5.

[0074] Table 5. Selection of different incubation times

[0075]

[0076]

[0077] Different letters indicate significance, and p<0.05 is considered significant.

[0078] As shown in Table 5, the glycosylation level of zebrafish increases with increasing culture time, and some zebrafish die after 32 hours or more. Therefore, the treatment time should not exceed 28 hours, and the effect is more significant after 6 hours or more.

[0079] Example 2

[0080] This embodiment studies the anti-glycation evaluation of a common anti-glycation drug, aminoguanidine.

[0081] 1. Thirty zebrafish at 72 hpf were cultured in 6-well plates and added to zebrafish culture medium containing 0.003 mol / L MGO and / or 50 μg / mL aminoguanidine (specific group settings are shown in Table 6), with the volume adjusted to 3 mL. The zebrafish were further cultured in an incubator at 28℃ for 24 hours, and the fluorescence intensity in the zebrafish culture medium was measured. Group B was used as the control, and the relative fluorescence intensity of each group is shown in Table 6.

[0082] Table 6. Zebrafish Saccharification Detection Protocol

[0083] A 0 0 <![CDATA[0.003±0.002 c ]]> B 0.003 0 <![CDATA[1.000±0.056 a ]]> C 0 50 <![CDATA[0.008±0.005 c ]]> D 0.003 50 <![CDATA[0.175±0.022 b ]]>

[0084] Different letters indicate significance, and p<0.05 is considered significant.

[0085] Table 6 shows that 0.003 mol / L MGO can increase the content of fluorescent AGEs in zebrafish culture medium. This is because MGO is absorbed by zebrafish and accelerates saccharification in vivo, leading to the excretion of AGEs from the zebrafish into the culture medium. This model can be used to evaluate the anti-glycation effect of aminoguanidine, and it has a highly significant effect, unaffected by the aminoguanidine substrate.

[0086] 2. Thirty zebrafish at 72 hpf were cultured in 6-well plates and added to zebrafish culture medium containing 0.003 mol / L LMGO and different concentrations of aminoguanidine (specific group settings are shown in Table 7), with the volume adjusted to 3 mL. The zebrafish were further cultured in an incubator at 28℃. After 24 hours of culture, the fluorescence intensity in the zebrafish culture medium was measured. Group A was used as the control, and the relative fluorescence intensities of each group are shown in Table 7.

[0087] Table 7. Selection of different aminoguanidine concentrations

[0088] A 0 μg / mL <![CDATA[1.000±0.056 a ]]> B 5μg / mL <![CDATA[0.857±0.043 b ]]> C 10 μg / mL <![CDATA[0.635±0.048 c ]]> D 20 μg / mL <![CDATA[0.454±0.036 d ]]> E 30μg / mL <![CDATA[0.364±0.032 e ]]> F 50 μg / mL <![CDATA[0.175±0.022 f ]]> G 75μg / mL <![CDATA[0.102±0.036 gh ]]> H 100 μg / mL <![CDATA[0.101±0.032 gh ]]> I 150 μg / mL <![CDATA[0.065±0.042 hi ]]> J 200 μg / mL <![CDATA[0.035±0.016 i ]]>

[0089] Different letters indicate significance, and p<0.05 is considered significant.

[0090] As shown in Table 7, increasing the concentration of aminoguanidine can reduce the glycosylation level in zebrafish and has a good dose-response relationship, proving that this method can be used as an effective method for rapid detection of anti-glycation effect in zebrafish models.

[0091] Comparative Example 1

[0092] This comparative example reference reports that 5dpf zebrafish were used as experimental subjects, and the reaction was carried out at 60℃ to evaluate anti-glycation.

[0093] Wild-type AB strain zebrafish were selected as the model organism and cultured in a recirculating aquaculture system. Males and females were separated and fed according to a 14-hour light + 10-hour dark photocycle. The culture temperature was 28±0.5℃, and the pH was 7.0±0.5. They were fed brine shrimp twice daily for 3 minutes each time. Sexually mature zebrafish were selected and placed in the spawning tank one night in advance at a 1:1 male-to-female ratio. Males and females were separated by a partition. The next morning, the lights were turned on, and the partition was removed to allow free mating. Fertilized embryos were collected and cultured in zebrafish culture medium (a solution prepared with deionized water containing 5mM sodium chloride, 0.17mM potassium chloride, 0.33mM calcium chloride, and 0.33mM magnesium sulfate). Incubation was carried out in a 28℃ constant temperature incubator with a 14-hour light + 10-hour dark photocycle, and the culture medium was changed daily. Zebrafish at 5 days post-fertilization (dpf) were collected on the fifth day.

[0094] Thirty zebrafish at 5 dpf were cultured in 1.5 mL centrifuge tubes and added to zebrafish culture medium containing 0.003 mol / L MgO and / or 50 μg / mL aminoguanidine, bringing the volume to 1 mL. The final concentrations of chemical reagents and test samples in the zebrafish culture medium were the concentrations required for the experiment. After incubation at 60℃ with shaking for 24 h, the supernatant was collected by centrifugation at 6000 rpm, and the fluorescence intensity in the zebrafish culture medium was measured. The average fluorescence intensity of group B was taken as 1.000.

[0095] Table 8. Determination of fluorescence intensity in zebrafish culture medium

[0096] A 0 mol / L 0 μg / mL <![CDATA[0.847±0.196 a ]]> B 0.003 mol / L 0 μg / mL <![CDATA[1.000±0.278 a ]]> C 0 mol / L 50 μg / mL <![CDATA[0.815±0.115 a ]]> D 0.003 mol / L 50 μg / mL <![CDATA[0.924±0.143 a ]]>

[0097] Treating zebrafish at 60℃ leads to their death. Furthermore, as shown in groups A through D, the fluorescence intensity was not significant. This may be because the death of zebrafish causes proteins to dissolve in the water, leading to more severe glycosylation reactions, making the reaction results overly susceptible to background factors. Comparative Example 1 not only caused zebrafish death but also showed poor detection results.

[0098] Comparative Example 2

[0099] This comparative study used adult zebrafish for in vitro evaluation, induced glycosylation reactions with different concentrations of MGO, and intervened with 50 μg / mL aminoguanidine.

[0100] Wild-type AB strain zebrafish were selected as the model organism and cultured in a recirculating aquaculture system, with males and females separated. They were fed a 14-hour light + 10-hour dark photocycle at 28℃ and pH 7.0±0.5. They were fed brine shrimp twice daily for 3 minutes each time. Adult zebrafish were selected and fed at a rate of 5 fish / L. Ten zebrafish were used for evaluation. Since AGEs in water were detected in vitro, still water was used instead of recirculating water. The effect of feed on the detection results was also investigated. Glycosylation of zebrafish was induced according to Table 9, and aminoguanidine was applied. After 24 hours of culture, the fluorescence intensity in the culture medium of each group was measured. Group C served as the control group, with an average fluorescence intensity of 1.000.

[0101] Table 9. Results of different treatment groups in adult zebrafish

[0102] A 0 mol / L 0 μg / mL no <![CDATA[0.038±0.009 f ]]> B 0.003 mol / L 0 μg / mL no <![CDATA[0.571±0.087 e ]]> C 0.003 mol / L 0 μg / mL no <![CDATA[1.000±0.124 de ]]> D 0.005 mol / L 0 μg / mL no <![CDATA[1.527±0.384 d ]]> E 0.008 mol / L 0 μg / mL no <![CDATA[2.960±0.494 b ]]> F 0.01 mol / L 0 μg / mL no <![CDATA[4.322±0.501 a ]]> G 0.003 mol / L 50 μg / mL no <![CDATA[0.878±0.206 de ]]> H 0.008 mol / L 50 μg / mL no <![CDATA[2.060±0.352 c ]]> I 0.003 mol / L 200 μg / mL no <![CDATA[0.678±0.156 e ]]> J 0.008 mol / L 200 μg / mL no <![CDATA[1.312±0.366 d ]]> K 0.003 mol / L 50 μg / mL yes <![CDATA[1.342±0.175 d ]]> L 0.008 mol / L 50 μg / mL yes <![CDATA[3.216±0.413 b ]]>

[0103] Table 9 shows that whether or not feed was added significantly affected the fluorescence intensity detection (groups H and L). This is because the feed contains protein, which reacts with the glycosylation inducing agent, thus affecting the fluorescence detection results. Although the experiment can be conducted without adding feed, it does not have animal welfare implications for zebrafish. Furthermore, the evaluation concentration of aminoguanidine needs to be higher when using adult zebrafish (no significant difference was observed at 50 μg / mL in groups C and G). Adult zebrafish are more cumbersome to handle; using 3 dpf (72 hpf) zebrafish allows for faster and more accurate detection. Additionally, animal welfare considerations are necessary for adult zebrafish, while they are not for zebrafish below 5 dpf.

[0104] Finally, it should be noted that the above examples are merely some specific embodiments and comparative examples of the present invention. Obviously, the present invention is not limited to the above embodiments and comparative examples, and many variations are possible. All variations that can be directly derived or associated with the content disclosed in this invention by those skilled in the art should be considered within the scope of protection of this invention.

Claims

1. A method for rapidly evaluating anti-glycation efficacy using a zebrafish model, characterized in that, Includes the following steps: (1) Select zebrafish juveniles 48-72 hours after fertilization and place them in zebrafish culture medium containing saccharification induction reagent and test sample. Place 10-40 juveniles in each well and culture them at 25-29℃ for 6-28 hours. When the saccharification induction reagent is absorbed by the zebrafish, it accelerates the saccharification process in the body. The late saccharification end products in the zebrafish are excreted from the body to the culture medium. The saccharification inducing agent is one or more of acetone aldehyde, glyoxal, 3-deoxyglucuronide, glucose, and fructose. The concentrations of acetone aldehyde, glyoxal, and 3-deoxyglucuronide in zebrafish culture medium are 0.002~0.004 mol / L, and the concentrations of glucose and fructose in zebrafish culture medium are 0.05~0.35 mol / L. The zebrafish culture medium comprises: 5 mM sodium chloride, 0.17 mM potassium chloride, 0.33 mM calcium chloride, 0.33 mM magnesium sulfate, with the remainder being water; (2) The fluorescence intensity of advanced glycation end products in zebrafish culture medium was determined, and the anti-glycation efficacy of the test sample was characterized by relative fluorescence intensity; the formula for calculating the relative fluorescence intensity is as follows: Relative fluorescence intensity = F 待测样品组 / F 对照组 F 待测样品组 F represents the fluorescence intensity of the sample group to be tested. 对照组 The fluorescence intensity represents the fluorescence intensity of the model control group.

2. The method for rapidly evaluating anti-glycation efficacy using a zebrafish model as described in claim 1, characterized in that, In step (1), the zebrafish used is the AB strain zebrafish.

3. The method for rapidly evaluating anti-glycation efficacy using a zebrafish model as described in claim 1, characterized in that, The concentration of acetone aldehyde in zebrafish culture medium was 0.003 mol / L.

4. The method for rapidly evaluating anti-glycation efficacy using a zebrafish model as described in claim 1, characterized in that, Incubate at 28°C for 24 hours.

5. The method for rapidly evaluating anti-glycation efficacy using a zebrafish model as described in claim 1, characterized in that, The samples to be tested are food, medicine, cosmetics, or their active ingredients.

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