Method for constructing zebrafish mental fatigue model and application thereof

Abstract

The present application relates to the field of mental fatigue model, and aims to solve the problems of long cycle and complex detection method in establishing mental fatigue model, and provides a method for constructing zebrafish mental fatigue model and application thereof.The method for constructing zebrafish mental fatigue model is as follows: 2-5 dpf zebrafish is given 100% white light and dark alternating stroboscopic cycle stimulation for 0-24 h; then cognitive response behavior analysis is carried out on the zebrafish.The present application constructs a zebrafish model of mental (brain) fatigue induced by white light stroboscopic stimulation, and explores a mental (brain) fatigue model induced by physical stimulation to simulate the environment and life and learning pressure in life.The present application also provides application of the zebrafish mental fatigue model constructed by the method, which is used for evaluating the anti-mental fatigue efficacy of drugs, health foods, foods or medical devices; and is used for mental (brain) fatigue cognitive test, memory test, learning test, reaction ability test and motor ability test of zebrafish.

Description

Technical Field

[0001] This invention relates to the field of evaluating the efficacy of mental fatigue, and in particular to a method for constructing a zebrafish mental fatigue model and its application. Background Technology

[0002] In today's society, people of all ages (students, working adults, etc.) constantly bear mental burdens in their daily activities. Mental fatigue (also known as mental exhaustion) is a change in psychobiological state caused by prolonged high-intensity cognitive activity or environmental stimuli. Mental fatigue not only impairs cognitive abilities but also affects various aspects of physical performance. During the maintenance phase of mental fatigue, high-intensity exercise leads to a significant decrease in endurance, intensity, and average speed. Other indicators of endurance performance are also impaired by mental fatigue, such as the intensity of interval running and the distance completed in interval recovery tests.

[0003] However, little is known about the mechanisms underlying the detrimental effects of mental fatigue on endurance performance. Variables traditionally considered limiting to endurance performance, such as heart rate, lactate buildup, and neuromuscular function, are unaffected by mental fatigue. Conversely, the negative impact of mental fatigue on endurance performance is primarily mediated by the increased perceived strength experienced by participants with mental fatigue. Mental fatigue affects not only behavior, movement, and cognitive behavior, but also brain waves, heart rate, and neurotransmitter release. Early detection of mental fatigue can prevent the worsening of symptoms in chronic fatigue syndrome and other diseases. To date, in clinical practice, mental fatigue has been assessed using perceptual strength scales. These questionnaires are not very effective due to patient subjectivity and are highly susceptible to treatment bias.

[0004] Currently reported ingredients that improve mental fatigue mainly include octacosanol, rhodioloside, taurine, caffeine, and lecithin. In addition, many more health products claiming to improve mental fatigue have been reported, including Sanlejiang (a traditional Chinese medicine), Rhodiola rosea oral liquid, Life No. 1, and deep-sea fish oil. Despite the abundance of these products, animal testing for their claimed efficacy is severely lacking. Most products rely on predicting efficacy based on potential ingredients without conducting in vivo studies on these components. Rats, mice, and rabbits are commonly used animal models. A review of recent literature and patents reveals that most animal models of mental fatigue to date use rats and mice. The main physical stimulation methods for model construction are water intrusion, weight-bearing, restraint, or sleep deprivation stress. For example, Rao Ziliang, Feng Xuexuan, Huang Xiaohong, et al. Observation of relevant indicators in rat model of chronic fatigue syndrome [J]. Experimental Animals and Comparative Medicine, 2018, 38(4):6.DOI:10.3969 / j.issn.1674-5817.2018.04.007. However, the diurnal rhythm of rats and mice can lead to differences in modeling time. Moreover, the modeling cycle is long, the animal cost is high, and the detection methods are complex. Therefore, it is necessary to select a suitable animal model to explore the mechanism of action of mental fatigue, construct a model to improve mental fatigue, assist in the efficacy evaluation of drugs, health foods, and foods, and develop drugs, health foods, foods, or medical devices to improve mental fatigue. Summary of the Invention

[0005] To overcome the problems of long development cycles and complex detection methods in establishing mental fatigue models, this invention provides a method for constructing a zebrafish mental fatigue model and its application. It constructs a zebrafish model of mental fatigue induced by white light stroboscopic stimulation, explores the use of physical stimulation to simulate mental fatigue induced by environmental and academic stress in daily life, and provides an effective and rapid detection method for the efficacy of drugs, health foods, and foods in improving mental fatigue.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The method for constructing a zebrafish mental fatigue model used zebrafish with a 2-5 day post-fertilization period (dpf) as subjects; they were given alternating stroboscopic stimulation of 100% white light and darkness, with a light stroboscopic cycle of 0-24 hours; after the treatment, the cognitive response behavior of the zebrafish was analyzed.

[0008] A study involving 20 healthy volunteers, which included continuous performance testing and EEG recording, found that white light increases the power of the alpha wave band in brainwaves. Subjects under white light conditions experienced decreased sustained attention, working memory, and reaction time, along with subjective drowsiness during task performance and other negative psychological effects. Therefore, white light stimulation can induce mental fatigue.

[0009] Zebrafish are vertebrates with up to 87% genetic similarity to humans. The hippocampus in the human brain is closely related to declarative memory; in rodents, the hippocampus is the core region for spatial memory formation, and the hippocampus is a major area involved in memory research. In memory research, classical conditioning (CC) is widely used to create animal models, with the amygdala at its center in mammalian CCs. Based on molecular markers, developmental origin, input / output, and role in regulating behavior, the equivalent of the mammalian cerebral cortex and hippocampus is considered to be located in the dorsal aspect of the zebrafish telencephalon. This region has been directly confirmed by behavioral experiments to be involved in memory and spatial localization. It is believed that the dorsal portion of the ventral Vs region of the zebrafish telencephalon is homologous to the central amygdala. The Proceedings of the National Academy of Sciences (PNAS) imaged the zebrafish cerebral cortex, combining anatomy, gene expression, and behavioral function to verify the hypothetical homology of the mammalian amygdala within the zebrafish cerebral cortex.

[0010] The main structural features of the zebrafish brain system are similar to those of mammals, sharing fundamentally similar characteristics with humans in physiology, brain anatomy, and neurochemistry. While fish brains lack regions resembling the mammalian hippocampus or trisynaptic circuits, the lateral cerebral cortex of bony fish is considered similar to the mammalian hippocampus, capable of acquiring and memorizing spatial information. The medial cerebral cortex functions similarly to the amygdala (center of fear and other emotions) in mammals. From fish to mammals, the role of the cholinergic system in memory has been evolutionarily preserved. Zebrafish larvae not only exhibit long-term memory but also association and social learning, making zebrafish one of the best model organisms for studying the biological mechanisms of learning and memory. Behavioral characteristics comparable to humans, such as motor activity, anxiety-like behavior, learning, memory retention, spatial and object recognition, fear responses, social preferences, and interactions, are all observed in zebrafish.

[0011] Since zebrafish fry within 5 days (5dpf) of fertilization do not need to eat and are not considered animals, the EU considers this ethically acceptable and often uses them as an alternative test for evaluating the efficacy and safety of drugs and health foods.

[0012] As the preferred choice, the zebrafish is the wild-type AB strain zebrafish.

[0013] Ideally, zebrafish should be exposed to alternating stroboscopic stimuli at a temperature of 28-30℃.

[0014] As a preferred method, the alternating strobe stimulation of 100% white light and darkness is specifically as follows: 100% white light for 20 seconds, 0% white light for 5 seconds, 100% white light for 15 seconds, and 0% white light for 3 seconds; the stimulation cycle lasts for 0-24 hours.

[0015] Preferably, zebrafish receiving alternating stroboscopic stimulation are exposed to light intensities of 6000-10000 lux.

[0016] As a preferred method, cognitive response behavior analysis involves placing zebrafish in a behavior analyzer and analyzing the proportion of blue areas within a cross-shaped maze of blue, green, red, and yellow. Zebrafish can distinguish different colors, and studies have shown that juvenile zebrafish selectively prefer blue among the four colors: blue, yellow, red, and green. Evaluating the color preferences of juvenile zebrafish can be used to assess their cognitive abilities.

[0017] The present invention also provides the application of the zebrafish mental fatigue model constructed by the method described above, for evaluating the anti-mental fatigue efficacy of drugs, health foods, foods or medical devices.

[0018] Preferably, zebrafish are administered the drug at a dose of 2-5 days per dpf.

[0019] As a preferred method, the detection indicators used for evaluation include one or more of the following: total movement distance, reaction ability, percentage of blue area, and acetylcholinesterase fluorescence value.

[0020] The present invention also provides the application of the zebrafish mental (brain) fatigue model constructed by the method, for zebrafish mental (brain) fatigue cognitive test (cross maze test), memory test (T-maze test), learning test (conditional position preference test), reaction ability test (light and dark response test) and motor ability test (total movement distance test).

[0021] Therefore, the beneficial effects of the present invention are: (1) There is no report on the model of mental (brain) fatigue induced by white light strobe irradiation in zebrafish; (2) Current models are mostly rats and mice, with long modeling cycles (7 days of adaptation, more than 14 days of modeling, and more than 21 days of drug administration), high animal costs, and complex detection methods. The model of mental (brain) fatigue induced by white light strobe irradiation in zebrafish is fast to model, has high throughput, low cost, and strong predictive ability. Attached Figure Description

[0022] Figure 1 These are images of zebrafish after being irradiated with white light in Examples 1-3; the numbers 1-3 in the images correspond to Examples 1-3 respectively, a represents the normal control group, and b represents the model experimental group.

[0023] Figure 2These are behavioral trajectory diagrams of zebrafish after being irradiated with white light in Examples 1-6; the numbers 1-6 in the diagrams correspond to Examples 1-6 respectively, a represents the normal control group, and b represents the model experimental group.

[0024] Figure 3 The image shows the behavioral trajectory of zebrafish after treatment with Sanlejiang oral solution in Example 1, where a represents the normal control group, b represents the model control group, c represents the 0.122 μL / mL group treated with Sanlejiang oral solution, d represents the 0.244 μL / mL group treated with Sanlejiang oral solution, and e represents the 0.488 μL / mL group treated with Sanlejiang oral solution.

[0025] Figure 4 The image shows the total movement distance and behavioral trajectory of zebrafish after treatment with Sanlejiang oral solution in Example 1. In the image, a is the normal control group, b is the model control group, c is the group treated with 0.122 μL / mL of Sanlejiang oral solution, d is the group treated with 0.244 μL / mL of Sanlejiang oral solution, and e is the group treated with 0.488 μL / mL of Sanlejiang oral solution.

[0026] Figure 5 This refers to the reactivity of zebrafish after treatment with the Triple Slurry in Example 1.

[0027] Figure 6 The values ​​are the fluorescence values ​​of acetylcholinesterase (AChE) in zebrafish after treatment with Sanlejiang oral solution (Example 1). In the figure, compared with the model control group, *p<0.05, **p<0.01.

[0028] Figure 7 This is a behavioral trajectory diagram of the proportion of blue areas in zebrafish after treatment with rice bran fatty alkanols in Example 2; in the figure, a is the normal control group, b is the model control group, c is the rice bran fatty alkanol 25.0 μg / mL group, d is the rice bran fatty alkanol 50.0 μg / mL group, and e is the rice bran fatty alkanol 100 μg / mL group.

[0029] Figure 8 This is a behavioral trajectory diagram of the total movement distance of zebrafish after treatment with rice bran fatty alkanols in Example 2; in the figure, a is the normal control group, b is the model control group, c is the rice bran fatty alkanol 25.0 μg / mL group, d is the rice bran fatty alkanol 50.0 μg / mL group, and e is the rice bran fatty alkanol 100 μg / mL group.

[0030] Figure 9 The fluorescence values ​​of acetylcholinesterase (AChE) in zebrafish after treatment with rice bran fatty alkanols as described in Example 2 are shown in the figure. Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001.

[0031] Figure 10This is a behavioral trajectory diagram of the proportion of blue areas in zebrafish after treatment with total ginsenosides water extract in Example 3; in the figure, a is the normal control group, b is the model control group, c is the group with total ginsenosides water extract of 1.41 μL / mL, d is the group with total ginsenosides water extract of 2.82 μL / mL, and e is the group with total ginsenosides water extract of 5.63 μL / mL.

[0032] Figure 11 This is a behavioral trajectory diagram of the total movement distance of zebrafish after treatment with total ginsenosides in Example 3; in the figure, a is the normal control group, b is the model control group, 1.41 μL / mL of total ginsenosides aqueous extract, d is the 2.82 μL / mL of total ginsenosides aqueous extract, and e is the 5.63 μL / mL of total ginsenosides aqueous extract.

[0033] Figure 12 This refers to the reactivity of zebrafish after treatment with the water extract of total ginseng saponins in Example 3. Detailed Implementation

[0034] The technical solution of the present invention will be further described below through specific embodiments.

[0035] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the art. The methods described in the examples, unless otherwise specified, are conventional methods in the art. Unless otherwise indicated, temperatures are expressed in °C or at ambient temperature, and pressures are at or near atmospheric pressure. Various variations and combinations of reaction conditions (e.g., component concentrations, required solvents, solvent mixtures, temperature, pressure, and other reaction ranges) and conditions that can be used to optimize the purity and yield of the product obtained by the method exist, and optimization of such method conditions will only require reasonable routine experiments.

[0036] Example

[0037] 1. Laboratory animals

[0038] Wild-type AB strain zebrafish.

[0039] 2. Main instruments and reagents

[0040] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); Behavior analyzer (Zebra Lab 3.22.3.31, Viewpoint, France); 6-well plate (Zhejiang Belamber Biotechnology Co., Ltd., China); Cross maze mold (Hangzhou Huante Biotechnology Co., Ltd.).

[0041] The cross-shaped maze mold is described in detail in the applicant's patent CN113951200B. The cross-shaped maze mold is shaped like a cross, as shown below. Figure 2 As shown in Figure 1a, the upper box is the blue area, the right box is the green area, the left box is the red area, and the lower box is the yellow area. All the cross mazes in the attached diagram of the instruction manual have the same color settings.

[0042] The light intensity is 6000-10000 lux, preferably 8000 lux.

[0043] The difference between the normal control group and the experimental group lies in the lighting conditions. The normal control group was in a dark environment without any light.

[0044] Example 1

[0045] A method for constructing a zebrafish mental fatigue model includes the following steps:

[0046] Wild-type AB strain zebrafish, 4 days post-fertilization (4 dpf), were randomly selected and placed in 20 mL beakers, with 30 zebrafish treated in each beaker (experimental group). During the day, they were placed in a bright light location maintained at 28.5℃, and at night, they were placed in a behavior analyzer maintained at 28.5℃. The program was set as follows: 100% white light for 20 s, 0% white light for 5 s, 100% white light for 15 s, 0% white light for 3 s, and strong light stimulation for 48 h. The 30 zebrafish in each experimental group were randomly divided into 6 subgroups (5 zebrafish per subgroup) and placed in the behavior analyzer to analyze their proportion of the blue area in the cross maze. Statistical results are expressed as mean ± SE. p < 0.05 indicated statistical significance.

[0047] Example 2

[0048] A method for constructing a zebrafish mental fatigue model includes the following steps:

[0049] Wild-type AB strain zebrafish, 4 days post-fertilization (4 dpf), were randomly selected and placed in 20 mL beakers, with 30 zebrafish treated in each beaker (experimental group). During the day, they were placed in a bright light location at a temperature maintained at 28.5℃, and at night, they were placed in a behavior analyzer at a temperature maintained at 28.5℃. The program was set as follows: 100% white light for 20 s, 0% white light for 5 s, 100% white light for 15 s, 0% white light for 3 s, and strong light stimulation for 24 h. The 30 zebrafish in each experimental group were randomly divided into 6 subgroups (5 zebrafish per subgroup) and placed in the behavior analyzer to analyze their proportion of the blue area in the cross maze. Statistical results are expressed as mean ± SE. p < 0.05 indicated statistical significance.

[0050] Example 3

[0051] A method for constructing a zebrafish mental fatigue model includes the following steps:

[0052] Wild-type AB strain zebrafish, 4 days post-fertilization (4 dpf), were randomly selected and placed in 3 mL systems in 6-well plates, with 30 zebrafish treated in each well (experimental group). During the day, they were placed in a bright light location maintained at 28.5℃, and at night in a behavior analyzer maintained at 28.5℃. The program was set as follows: 100% white light for 20 s, 0% white light for 5 s, 100% white light for 15 s, 0% white light for 3 s, with strong light stimulation for 24 h. The 30 zebrafish in each experimental group were randomly divided into 6 subgroups (5 zebrafish per subgroup) and placed in the behavior analyzer to analyze their proportion of the blue area in the cross maze. Statistical results are expressed as mean ± SE. p < 0.05 indicated statistical significance.

[0053] Example 4

[0054] A method for constructing a zebrafish mental fatigue model includes the following steps:

[0055] Wild-type AB strain zebrafish, 4 days post-fertilization (4 dpf), were randomly selected and placed in 3 mL systems in 6-well plates, with 30 zebrafish treated in each well (experimental group). The plates were placed in a behavior analyzer maintained at 28.5℃, with the following program: 100% white light for 20 s, 0% white light for 5 s, 100% white light for 15 s, 0% white light for 3 s, and strong light stimulation for 12 h. Each experimental group of 30 zebrafish was randomly divided into 6 subgroups (5 zebrafish per subgroup) and placed in the behavior analyzer to analyze the proportion of blue areas in the cross maze. Statistical results are expressed as mean ± SE. p < 0.05 indicated statistical significance.

[0056] Example 5

[0057] A method for constructing a zebrafish mental fatigue model includes the following steps:

[0058] Wild-type AB strain zebrafish, 4 days post-fertilization (4 dpf), were randomly selected and placed in 3 mL systems in 6-well plates, with 30 zebrafish treated in each well (experimental group). The zebrafish were placed in a behavior analyzer maintained at 28.5℃, with the following program: 100% white light for 20 s, 0% white light for 5 s, 100% white light for 15 s, 0% white light for 3 s, and strong light stimulation for 6 h. The 30 zebrafish in each experimental group were randomly divided into 6 subgroups (5 zebrafish per subgroup) and placed in the behavior analyzer to analyze the proportion of blue areas in the cross maze. Statistical results are expressed as mean ± SE. p < 0.05 indicated statistical significance.

[0059] Example 6

[0060] A method for constructing a zebrafish mental fatigue model includes the following steps:

[0061] Wild-type AB strain zebrafish, 5 days post-fertilization (5 dpf), were randomly selected and placed in 3 mL systems in 6-well plates, with 30 zebrafish treated in each well (experimental group). The plates were placed in a behavior analyzer maintained at 28.5℃, with the following program: 100% white light for 20 s, 0% white light for 5 s, 100% white light for 15 s, 0% white light for 3 s, and strong light stimulation for 6 h. The 30 zebrafish in each experimental group were randomly divided into 6 subgroups (5 zebrafish per subgroup) and placed in the behavior analyzer to analyze the proportion of blue areas in the cross maze. Statistical results are expressed as mean ± SE. p < 0.05 indicated statistical significance.

[0062] Analysis of Results of Examples 1-6

[0063] The results of Examples 1-6 were analyzed, and the results are as follows: Figure 1 , 2 As shown in Table 1.

[0064] Table 1. Effects of white light flickering on zebrafish (mean ± SE)

[0065]

[0066]

[0067] From the experimental results: ① In Example 1, zebrafish were irradiated with white light at 4 dpf for 48 hours, from... Figure 1 1b shows that this causes the zebrafish's body pigment to lighten; Table 1 shows a significant decrease in the proportion of blue, but from... Figure 2 The zebrafish in picture 1b appeared to be in poor condition. Therefore, the duration of white light exposure needs to be shortened.

[0068] ② Example 2: Zebrafish were irradiated with white light stroboscopic light for 24 hours at a 4dpf cadence. Figure 1 2b indicates that this causes the zebrafish's body pigment to lighten; Table 1 shows a significant decrease in the proportion of blue. The mental fatigue model was successfully induced, but from... Figure 2 Model 2b shows significant damage and is unsuitable for further operations. Furthermore, the zebrafish placed in a beaker results in an excessively large system, making it unsuitable for high-throughput studies and the study of trace substances.

[0069] ③ Example 3: Zebrafish were irradiated with white light stroboscopic light for 24 hours at a rate of 4 dpf. Figure 1 Even in a 6-well plate, 3b showed that zebrafish body pigmentation still lightened, and Table 1 showed a significant decrease in the proportion of blue. The mental fatigue model was successfully induced, but... Figure 2 The 3b model shows severe damage and is not suitable for further operations.

[0070] ④ Example 4: Irradiation of zebrafish with 4 dpf of white light for 12 hours resulted in a significant decrease in the blue content of the zebrafish, as shown in Table 1. The mental fatigue model was successfully induced, but from... Figure 2 The 4b model shows severe damage and is not suitable for further operations.

[0071] ⑤ Example 5: Irradiation of zebrafish with 4 dpf of white light for 6 hours resulted in a significant decrease in the blue content of the zebrafish, as shown in Table 1. The mental fatigue model was successfully induced, but from... Figure 2 The 5b result suggests that zebrafish may be unsuitable for subsequent operations due to their small swim bladders and underdeveloped locomotion capabilities. Therefore, it is necessary to update the zebrafish's stage of use.

[0072] ⑥ Example 6: Zebrafish were irradiated with 5 dpf white light for 6 hours. Table 1 shows a significant decrease in the proportion of blue light in the zebrafish. Figure 2 The 6b model of mental fatigue was successfully induced.

[0073] Application examples

[0074] Drug evaluation analysis was performed using the model selected in Example 6 above.

[0075] 1. Laboratory animals

[0076] Wild-type AB strain zebrafish.

[0077] 2. Main instruments and reagents

[0078] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); Multifunctional microplate reader (SPARK, TECAN, Switzerland); Behavioral analyzer (Zebra Lab 3.22.3.31, Viewpoint, France); 6-well plate (Zhejiang Belamb Biotechnology Co., Ltd., China); Cross maze mold (Hangzhou Huante Biotechnology Co., Ltd.); Acetylcholinesterase kit (batch number 3040802, AATBioquest, USA).

[0079] Example 1: Wild-type AB strain zebrafish, 5 days post-fertilization (5 dpf), were randomly selected and placed in 6-well plates (experimental group) with 30 zebrafish per well. Sanlejiang oral solution was administered at concentrations of 0.122, 0.244, and 0.488 μL / mL. A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group (no light), all other groups underwent white light stroboscopic irradiation to establish a zebrafish mental fatigue model. The zebrafish were placed in a behavioral analyzer maintained at 28.5℃. The program was set as follows: 100% white light for 20 s, 0% white light for 5 s, 100% white light for 15 s, 0% white light for 3 s, and strong light stimulation for 6 h. On the day of treatment, the zebrafish were rinsed three times with standard dilution water. Four parallel experiments were conducted.

[0080] Tests were conducted after the experiment.

[0081] Thirty zebrafish in each experimental group were randomly divided into six groups (five zebrafish per group). They were placed in a behavior analyzer to record and analyze the proportion of blue areas in the cross-shaped maze. Results are as follows: Figure 3 As shown in Table 2.

[0082] Ten zebrafish were randomly selected from each experimental group and placed in a behavior analyzer. The total movement distance was recorded and analyzed. The results are as follows: Figure 4 As shown in Table 2.

[0083] Table 2. Percentage of blue area in zebrafish after treatment with Sanlejiang oral liquid (mean±SE)

[0084]

[0085] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001.

[0086] Twelve zebrafish were randomly selected from each experimental group and placed in a behavior analyzer. The analyzer was set to provide 5 minutes of darkness, 5 minutes of light, 5 minutes of darkness, and 5 minutes of light, and the average speed was analyzed every 5 minutes. Results are as follows: Figure 5 As shown in Table 3.

[0087] Table 3. Zebrafish reactivity after treatment with Tripler (mean ± SE)

[0088]

[0089] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001.

[0090] (4) Thirty zebrafish were randomly divided into 10 groups and placed in black 96-well plates. The acetylcholinesterase assay kit was used, and data were collected using a multi-functional microplate reader. The fluorescence value of acetylcholinesterase in zebrafish was analyzed. The statistical analysis results of this index were used to evaluate the enhancing effect of the triamcinolone acetonide on acetylcholinesterase activity.

[0091] Statistical results are expressed as mean ± SE. p < 0.05 indicates a statistically significant difference. Results are shown in Table 4 and... Figure 6 As shown.

[0092] Table 4. AChE fluorescence values ​​(mean ± SE) of zebrafish treated with Sanlejiang oral liquid.

[0093]

[0094] Compared with the model control group, *p<0.05, **p<0.01.

[0095] Analysis of experimental results: In the model control group, the proportion of the blue area and the total movement distance of zebrafish were significantly reduced. Acetylcholinesterase fluorescence assays showed a significant decrease in fluorescence in the model control group, indicating the successful development of the white light strobe irradiation-induced mental fatigue model. After administration of Huamei Pharmaceutical's Sanlejiang oral liquid, the cognitive ability (proportion of the blue area), motor ability (total movement distance), reaction ability (average speed of light and dark response), and acetylcholinesterase activity of zebrafish were all significantly increased, indicating that Huamei Pharmaceutical's Sanlejiang oral liquid has a significant effect in improving mental fatigue.

[0096] Application Example 2: Rice Bran Fatty Alkanols

[0097] Wild-type AB strain zebrafish, 5 days post-fertilization (5 dpf), were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Rice bran fatty alkanols at concentrations of 25.0, 50.0, and 100 μg / mL were dissolved in hot DMSO. A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, all other groups underwent white light stroboscopic irradiation to establish a zebrafish mental fatigue model. All zebrafish were placed in a behavioral analyzer maintained at 28.5℃, with the following program: 100% white light for 20 s, 0% white light for 5 s, 100% white light for 15 s, 0% white light for 3 s, and strong light stimulation for 6 h. On the day of treatment, the zebrafish were rinsed three times with standard dilution water. The experiment was repeated in triplicate.

[0098] Tests were conducted after the experiment.

[0099] Thirty zebrafish in each experimental group were randomly divided into six groups (five zebrafish per group). They were placed in a behavior analyzer to record and analyze their proportion of the blue area in the cross-shaped maze. Results are as follows: Figure 7 As shown in Table 5.

[0100] Ten zebrafish were randomly selected from each experimental group and placed in a behavior analyzer. The total movement distance was recorded and analyzed. The results are as follows: Figure 8 As shown in Table 5.

[0101] Table 5. Percentage of blue area in zebrafish after treatment with fatty alkyl alcohols in rice bran (mean ± SE)

[0102]

[0103] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001.

[0104] (3) Thirty zebrafish were randomly divided into 10 groups and placed in black 96-well plates. The acetylcholinesterase assay kit was used, and data were collected using a multi-functional microplate reader. The fluorescence value of acetylcholinesterase in zebrafish was analyzed. The statistical analysis results of this index were used to evaluate the enhancing effect of rice bran fatty alkanol on acetylcholinesterase activity.

[0105] Statistical results are expressed as mean ± SE. p < 0.05 indicates a statistically significant difference. Results are as follows: Figure 9 As shown in Table 6.

[0106] Table 6. AChE fluorescence values ​​(mean ± SE) of zebrafish after treatment with fatty alkanols from rice bran

[0107]

[0108] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001.

[0109] Analysis of experimental results: In the model control group, the proportion of the blue area and the total movement distance of zebrafish were significantly reduced. Acetylcholinesterase fluorescence assays showed a significant decrease in fluorescence in the model control group, indicating the successful development of the white light strobe irradiation-induced mental fatigue model. After administration of rice bran fatty alkanols, the cognitive ability (proportion of the blue area), motor ability (total movement distance), and activity of zebrafish were significantly increased, indicating that rice bran fatty alkanols have a significant effect in improving mental fatigue.

[0110] Application Example 3: Ginseng Total Saponins Aqueous Extract

[0111] Wild-type AB strain zebrafish, 5 days post-fertilization (5 dpf), were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Water-soluble ginseng total saponins were added at concentrations of 1.41, 2.82, and 5.63 μL / mL. A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, all other groups underwent white light stroboscopic irradiation to establish a zebrafish mental fatigue model. All zebrafish were placed in a behavioral analyzer maintained at 28.5℃. The program was set as follows: 100% white light for 20 s, 0% white light for 5 s, 100% white light for 15 s, 0% white light for 3 s, and strong light stimulation for 6 h. On the day of treatment, the zebrafish were washed three times with standard dilution water. Two parallel experiments were conducted.

[0112] Tests were conducted after the experiment.

[0113] Thirty zebrafish in each experimental group were randomly divided into six groups (five zebrafish per group). They were placed in a behavior analyzer to record and analyze their proportion of the blue area in the cross-shaped maze. Results are as follows: Figure 10 As shown in Table 7.

[0114] Ten zebrafish were randomly selected from each experimental group and placed in a behavior analyzer. The total movement distance was recorded and analyzed. The results are as follows: Figure 11 As shown in Table 7.

[0115] Table 7. Percentage of blue area in zebrafish after treatment with total ginsenosides (mean ± SE)

[0116]

[0117] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001.

[0118] Five zebrafish were randomly selected from each experimental group and placed in a behavior analyzer. The analyzer was set to operate for 5 minutes in darkness, 5 minutes in light, 5 minutes in darkness, and 5 minutes in light, and the average speed was analyzed every 5 minutes. Results are as follows: Figure 12 As shown in Table 8.

[0119] Statistical results are expressed as mean ± SE. p < 0.05 indicates that the difference is statistically significant.

[0120] Table 8. Zebrafish reactivity (mean ± SE) after treatment with total ginsenosides

[0121]

[0122] Compared with the model control group, **p<0.05, **p<0.01, ***p<0.001.

[0123] Analysis of experimental results: In the model control group, the proportion of blue area, total movement distance, and reaction ability of zebrafish were significantly reduced, indicating that the white light strobe irradiation-induced mental fatigue model was successful. After administration of total ginsenosides, the cognitive ability (proportion of blue area), motor ability (total movement distance), and reaction ability (average speed of light and dark reaction) of zebrafish were significantly increased, indicating that total ginsenosides have a significant effect on improving mental fatigue.

[0124] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for constructing a zebrafish mental fatigue model, characterized in that, Zebrafish were used as subjects 2-5 days after fertilization; they were given alternating stroboscopic stimulation of 100% white light and darkness for 6-24 hours; after the treatment, the cognitive response behavior of the zebrafish was analyzed. The 100% white light and darkness alternating strobe stimulation cycle is as follows: 100% white light for 20 seconds, 0% white light for 5 seconds, 100% white light for 15 seconds, 0% white light for 3 seconds; cyclic stimulation for 6 hours.

2. The method for constructing a zebrafish mental fatigue model according to claim 1, characterized in that, The zebrafish is a wild-type AB strain zebrafish.

3. The method for constructing a zebrafish mental fatigue model according to claim 1 or 2, characterized in that, When zebrafish are subjected to alternating stroboscopic stimulation, the temperature is 28-30 ℃.

4. The method for constructing a zebrafish mental fatigue model according to claim 1, characterized in that, The light intensity for zebrafish receiving alternating stroboscopic stimulation is 6000-10000 lux.

5. The method for constructing a zebrafish mental fatigue model according to claim 1, characterized in that, The cognitive response behavior analysis specifically involves placing zebrafish in a behavior analyzer and analyzing the proportion of the blue area in a cross-shaped maze with four colors: blue, green, red, and yellow.

6. The application of the model constructed by the method for constructing a zebrafish mental fatigue model according to any one of claims 1-5, characterized in that, Used to evaluate the anti-mental fatigue efficacy of drugs and foods.

7. The application according to claim 6, characterized in that, The administration period for zebrafish is 2-5 days after fertilization.

8. The application according to claim 6, characterized in that, The evaluation indicators include one or more of the following: total movement distance, reaction ability, percentage of blue area, and acetylcholinesterase fluorescence value.

9. The application of the model constructed by the method for constructing a zebrafish mental fatigue model according to any one of claims 1-5, characterized in that, Used for cognitive tests of mental fatigue, memory tests, learning tests, reaction ability tests, or motor ability tests in zebrafish.

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