Construction method and application of senescence accompanied with cognitive impairment and hypomnesia model

Abstract

The invention discloses a construction method and application of a model for senescence with cognitive impairment and hypomnesia, and belongs to the technical field of medicine, health food and food efficacy evaluation. According to the senescence accompanied cognitive disorder and hypomnesia model construction method, zebra fish D-galactose is combined with physical stimulation induction to successfully establish the senescence accompanied cognitive disorder and hypomnesia model, phenomenon observation is visual, senescence accompanied cognitive disorder and hypomnesia definition is accurate, and the method is suitable for popularization and application. The method can effectively and rapidly evaluate the effects of medicines, health-care foods and foods in improving senescence accompanied by cognitive impairment and hypomnesia, and provides an effective and rapid detection approach for medicines, health-care foods and foods in improving senescence accompanied by cognitive impairment and hypomnesia.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug, health food and food efficacy evaluation, and in particular relates to a method for constructing a model of aging-associated cognitive impairment and memory loss and its application, and evaluates the efficacy of drugs, health food and food in improving aging-associated cognitive impairment and memory loss. Background Art

[0002] Globally, population aging is a significant trend. According to the United Nations, the proportion of the global population aged 65 and over is projected to reach 16% by 2050, compared to approximately 9% in 2020. Aging is a natural physiological process involving the degeneration and functional decline of multiple systems. Aging has significant socioeconomic impacts, including increased healthcare expenditures, changes in the labor market, and pressure on pension systems. Patents filed as of September 2024 reveal over 170 patents related to anti-aging compositions. A growing number of aging-related compositions, foods, supplements, and novel compounds are being developed, some of which are intended to aid in the clinical diagnosis of aging-related diseases. However, these patents merely predict the effects of these compounds on aging and do not involve in vivo studies. Therefore, there is an urgent need to develop models of cognitive impairment and memory loss associated with aging to assist in the efficacy evaluation of drugs, health supplements, and foods.

[0003] There is a severe shortage of products on the market that improve the symptoms of aging with cognitive impairment and memory loss, and the animal experiments they claim to have are very scarce. Most animal experiments use a single chemical stimulus (administering D-galactose) to induce an aging model. The modeling time is as long as 45 days to 3 months, and the observation of the phenomenon is not intuitive. The definitions of aging with cognitive impairment and memory loss are inaccurate. For example, the patent with application number CN202411573587.5 uses mice as experimental subjects and administers D-galactose stimulation. In addition, most products predict their efficacy based on possible ingredients, and no in vivo studies have been conducted on the relevant ingredients. Therefore, it is very necessary to select a fast and suitable animal model to explore the mechanism of action of aging and to develop drugs, health foods, and foods that improve aging with cognitive impairment and memory loss.

[0004] Rats, mice, and rabbits are commonly used as model animals, but the results are not intuitive, and behavioral experiments often place high demands on the animals. End-point studies can lead to animal death when necessary, resulting in unnecessary waste. Therefore, identifying more suitable animal models is urgent. A review of recent literature and patents reveals no reports to date of using zebrafish to establish models of aging-related cognitive impairment and memory loss. Summary of the Invention

[0005] To overcome the problems of the existing technology, such as the lack of intuitive observation and inaccurate definitions of aging-associated cognitive impairment and memory loss, the present invention provides a method for constructing a model of aging-associated cognitive impairment and memory loss. This method can successfully and stably establish a zebrafish model of aging-associated cognitive impairment and memory loss, providing intuitive observation and accurate definitions of aging-associated cognitive impairment and memory loss. Also provided is the application of this method for constructing a model of aging-associated cognitive impairment and memory loss in testing drugs, health foods, and foods that improve aging-associated cognitive impairment and memory loss. The method explores the use of oxidative damage combined with simulation of the phenomenon of rapid aging-associated memory loss and cognitive impairment caused by a large number of external stimuli in daily life, providing an effective and rapid detection method for drugs, health foods, and foods that improve aging-associated cognitive impairment and memory loss.

[0006] To achieve the above object, the present invention adopts the following technical solutions: A method for constructing a model of aging with cognitive impairment and memory loss, wherein the model construction method comprises administering water-soluble D-galactose in combination with physical stimulation of zebrafish to detect indicators of aging with cognitive impairment and memory loss.

[0007] Preferably, the zebrafish is a wild-type AB strain zebrafish.

[0008] As the third most popular model organism, the zebrafish, in addition to having a brain structure similar to that of humans, is also the only vertebrate suitable for high-throughput screening using microplates. Zebrafish embryos develop within 24 hours, making experimental cycles short, efficient, and cost-effective. Therefore, using the zebrafish model will significantly shorten the time from raw material screening to formulation, providing a preemptive opportunity for patent applications. Furthermore, the intuitive and visual presentation of zebrafish experimental results is easily understood by consumers, further empowering product technology in a novel way. The primary structures of the zebrafish nervous system are similar to those of mammals, with essentially identical physiology, brain anatomy, and neurochemistry to those of humans. The zebrafish brain is divided into the telencephalon (containing the olfactory bulbs and cerebral hemispheres); the diencephalon (comprising the preoptic area, anterior tectum, epithalamus, thalamus, hypothalamus, and posterior tubercle); the midbrain (comprising the optic tectum, protuberance, and tegmentum); and the hindbrain (comprising the cerebellum and medulla oblongata). Regulatory neurotransmitter systems (dopamine, norepinephrine, serotonin, histamine, acetylcholine, and orexin / hypothalamocretin) are also present in zebrafish. Based on molecular markers, developmental origins, input / output, and roles in regulating behavior, the equivalent of the mammalian cerebral cortex and hippocampus is believed to be located in the dorsal telencephalon (dT) of the zebrafish. This region has been directly confirmed by behavioral experiments to be involved in memory and spatial orientation. Combining anatomy, gene expression, and behavioral function, it has been demonstrated that the dorsal portion of the ventral Vs region of the zebrafish telencephalon is homologous to the central amygdala, responsible for regulating emotion, learning, and memory. Internationally, no zebrafish models of aging-associated cognitive impairment and memory loss induced by D-gal or D-gal + physical stimulation, nor methods for screening corresponding active ingredients, have been reported.

[0009] Preferably, the zebrafish is a 3-10 dpf zebrafish.

[0010] Preferably, the water-soluble dosage concentration of D-galactose is 12.8-51.2 mg / mL.

[0011] At normal concentrations, D-galactose (D-gal) is metabolized to glucose, but at higher doses, it is converted to aldose and hydroperoxides through the action of galactose oxidase, thereby forming superoxide anions and oxygen-derived free radicals. The free radicals formed by D-gal oxidation suppress the cell's defenses, increase lipid peroxides, and release end products that react with proteins and phospholipids, leading to cell damage and central nervous system damage. At the same time, the excessive production of lipid peroxides and oxygen free radicals accelerates the decomposition of biomolecules, leading to abnormal increases in malondialdehyde concentrations in the brain, which in turn induces abnormal neurotoxicity of advanced glycation end products, leading to behavioral and neurochemical changes. However, single peroxide-induced aging does not conform to the stimulation people receive in the environment. Therefore, the aging model we used, induced by D-galactose combined with physical stimulation (simulating staying up late), is more consistent with the human aging process.

[0012] More preferably, the aqueous D-galactose dosage concentration is 25.6 mg / mL.

[0013] Preferably, the physical stimulation is water rotation stimulation with a rotation speed of 1000 rpm.

[0014] Preferably, the indicators of aging-associated cognitive impairment and memory loss are the proportion of the blue area of ​​the plus maze, the difference in light and dark speed, the total movement distance, SA-β-galactosidase staining, the time spent in the enriched area of ​​the T maze, and the latency to enter the enriched area.

[0015] During the aging process, cognition and memory are closely related psychological processes. They jointly participate in human information processing, knowledge acquisition and application, thinking and judgment, and problem solving. Cognition refers to the process of an individual's perception, understanding, processing, and utilization of information from the external world, while memory is the ability to encode, store, and retrieve this information. The cognitive process depends on memory. Memory is the solidification and accumulation of cognitive results. Cognitive strategies affect memory effects, and memory defects affect cognitive functions. Therefore, cognition and memory are inseparable. Therefore, our model can study the following indicators in order to better simulate a model animal model that conforms to human representation. (1) Cross maze: The four directions of the cross maze are painted with blue, red, yellow, and green colors. Color preference tests can be used as an effective program for memory assessment, cognitive dysfunction, neurodegenerative disease assessment, toxic behavior assessment, etc. Zebrafish prefer short-wavelength (blue) colors, but for zebrafish with cognitive dysfunction, hormones and neurotransmitters in their brains change, causing the zebrafish to lose their preference for blue. The proportion of movement in the blue area is used to evaluate the degree of cognitive impairment in zebrafish. (2) Motor ability test: Motor behavior is the most basic behavioral manifestation of animals. Brain aging is usually manifested as a decline in cognitive ability and reaction speed, and there is an inverse relationship between exercise and the risk of cognitive decline. The total movement distance is used to measure the motor ability of zebrafish. (3) Reaction ability test: Aging behavioral studies have shown that overall cognitive function declines with age, and the key time periods for cognitive decline in different areas are different. Among them, contextual memory and information processing speed can better reflect the structural and functional state of the brain during the aging process and can be used as functional markers for evaluating brain aging. Zebrafish are stimulated with alternating light and dark, and the change in movement speed corresponding to the change in light and dark is used to reflect the reaction ability of zebrafish. (4) Golden indicator of aging: β-galactosidase is an enzyme that catalyzes the hydrolysis of lactose into glucose and galactose. In the study of cell aging, the detection of β-galactosidase activity is often used as an indicator for evaluating cell aging. Specifically, the increase in the activity of a enzyme called SA-β-gal (senescence-associated β-galactosidase) is usually associated with cell aging. SA-β-gal, a specific β-galactosidase, has high activity at pH 6.0. This enzyme activity increases significantly in aging cells and is almost absent in young cells. Increased SA-β-gal activity is considered a sign that cells have entered a state of permanent growth arrest, known as cellular senescence.

[0016] Preferably, the D-galactose water-soluble administration combined with physical stimulation of zebrafish is continued at the stage of 5dpf to 7dpf.

[0017] The above-mentioned method for constructing a model of aging-associated cognitive impairment and memory loss is used in detecting drugs, health foods and foods that improve aging-associated cognitive impairment and memory loss.

[0018] Preferably, the administration period of the medicine, health food and food raw materials is 3 dpf to 8 dpf, and the administration cycle is 0 to 120 h.

[0019] Therefore, the present invention has the following beneficial effects: (1) A model of aging with cognitive impairment and memory loss induced by zebrafish D-galactose combined with physical stimulation has not been reported. (2) The model of aging with cognitive impairment and memory loss induced by zebrafish D-galactose combined with physical stimulation has high throughput, low cost, and strong predictability. (3) The model of aging with cognitive impairment and memory loss induced by zebrafish D-galactose combined with physical stimulation was successfully established. The phenomenon can be observed intuitively, and the definition of aging with cognitive impairment and memory loss is accurate. It can effectively and quickly evaluate the effect of drugs, health foods, and foods on improving aging with cognitive impairment and memory loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a typical image of β-galactosidase staining in zebrafish aging induced by 25.6 mg / mL of D-galactose in Example 1.

[0021] Figure 2 This is a trajectory diagram of the zebrafish aging cognitive experiment induced by 25.6 mg / mL of D-galactose in Example 1.

[0022] Figure 3 This is a typical image of β-galactosidase staining of zebrafish aging induced by physical stimulation (water rotation 1000 rpm) in Example 1.

[0023] Figure 4 This is a trajectory diagram of the zebrafish aging cognitive experiment induced by physical stimulation (water body rotation 1000 rpm) in Example 1.

[0024] Figure 5 This is a typical image of β-galactosidase staining of zebrafish aging induced by 25.6 mg / mL of D-galactose combined with physical stimulation (water rotation 1000 rpm) in Example 1.

[0025] Figure 6 This is a trajectory diagram of the zebrafish aging cognitive experiment induced by 25.6 mg / mL of D-galactose combined with physical stimulation (water rotation 1000 rpm) in Example 1.

[0026] Figure 7 This is a typical image of β-galactosidase staining of zebrafish after treatment with Yangshengtang brand natural vitamin E soft capsules in Example 2.

[0027] Figure 8This is a trajectory diagram of the proportion of blue areas in zebrafish after treatment with Yangshengtang brand natural vitamin E soft capsules in Example 2.

[0028] Figure 9 This is a typical image of β-galactosidase staining of zebrafish after treatment with ginseng stem and leaf total saponin tablets in Example 3.

[0029] Figure 10 This is a trajectory diagram of the proportion of blue areas in zebrafish after treatment with the ginseng stem and leaf total saponin tablets in Example 3.

[0030] Figure 11 This is a trajectory diagram of the total movement distance of zebrafish after treatment with the ginseng stem and leaf total saponin tablets in Example 3.

[0031] Figure 12 This is a bar graph showing the results of improving the reaction ability of zebrafish after treatment with ginseng stem and leaf total saponin tablets in Example 3.

[0032] Figure 13 This is a typical image of β-galactosidase staining of zebrafish after DHA treatment in Example 4.

[0033] Figure 14 This is a trajectory diagram of the blue area percentage of zebrafish after DHA treatment in Example 4.

[0034] Figure 15 This is a statistical graph of the T / S ratio after DHA treatment in Example 4. DETAILED DESCRIPTION

[0035] Example 1 Establishment of a zebrafish model of aging with cognitive impairment and memory loss: 1. Experimental Animals Wild-type AB strain zebrafish.

[0036] 2. Main instruments A dissecting microscope (SZX7, OLYMPUS, Japan); a precision electronic balance (CP214, OHAUS, USA); a nine-link magnetic stirrer (MMS9Pro, JOANLAB, China); and a zebrafish behavior analyzer (Hunter Lab HT-XW-2D-12, Hangzhou Huante Biotechnology Co., Ltd., China) were used.

[0037] 3. Experimental Methods 3.1 First exploration of model conditions Wild-type AB zebrafish, 5 days post-fertilization (dpf), were randomly selected and placed in 100 mL wells. Fifty zebrafish were treated in each well (experimental group). A normal control group and a model control group were established, with each well containing 50 mL. All experimental groups, except the normal control group, were treated with 25.6 mg / mL of D-galactose in water for model establishment. Between 5 and 7 dpf, 1) 10 zebrafish were randomly selected from each group and fixed with tissue fixative. The fish were then stained using a β-galactosidase kit. Data were acquired using NIS-Elements D 3.20 Advanced Image Processing Software, and the average opacity (in pixels) of the β-galactosidase staining in the fixed area of ​​the zebrafish was analyzed. 2) 30 zebrafish were randomly selected from each group and placed in a plus-shaped maze. The fish were recorded using a zebrafish behavioral instrument, and their trajectories were saved. Behavioral data were collected using analysis software, and the percentage of blue areas within the plus-shaped maze was analyzed. Statistical results are expressed as mean ± SE. P < 0.05 indicated statistical significance. The results are shown in Tables 1 and 2. Figure 1 、 Figure 2 shown.

[0038] 3.1.1 Experimental Results Table 1. Results of β-galactosidase staining experiments in zebrafish aging induced by D-galactose 25.6 mg / mL (pixels, mean ± SE, n = 10). Group dose Opacity average (pixels) Normal control group - 0.140±0.002 D-Galactose 25.6 mg / mL 0.148±0.004

[0039] Table 2. Results of the zebrafish aging cognitive experiment induced by D-galactose 25.6 mg / mL (pixels, mean ± SE, n = 6). Group dose The proportion of blue areas in the cross maze Normal control group - 40.7±13.6 D-Galactose 25.6 mg / mL 32.0±8.79

[0040] From Table 1, Table 2, Figure 1 、 Figure 2 As shown, 48 hours after administration of 25.6 mg / mL D-galactose solution to 5dpf zebrafish, there was no significant increase in the average opacity of β-galactosidase staining, indicating that the zebrafish did not show signs of aging under this stimulation condition. Furthermore, the results of the plus-maze experiment showed that the proportion of time spent in the blue area of ​​the plus-maze did not change significantly compared to the normal control group, indicating that stimulating zebrafish under these conditions does not produce changes in the aging phenotype or cognitive impairment.

[0041] 3.2 Second exploration of model conditions Wild-type AB strain zebrafish 5 days post-fertilization (5dpf) were randomly selected and placed in 100mL, and 50 zebrafish were processed in each cup (experimental group). A normal control group and a model control group were set up, and the capacity of each well was 50mL. Except for the normal control group, the other experimental groups were given physical stimulation (water body rotation 1000rpm) for model construction, and after 5dpf to 7dpf, 1) 10 zebrafish were randomly selected from each group and stained with a β-galactosidase kit. The data were collected using NIS-Elements D 3.20 advanced image processing software to analyze the average opacity (pixel) of β-galactosidase staining in the fixed area of ​​the zebrafish; 2) 30 zebrafish were randomly selected from each group and placed in a plus maze. The video was recorded under the zebrafish behavior instrument and the trajectory map was saved. The behavioral data were collected using analysis software to analyze the proportion of the blue area of ​​the zebrafish in the plus maze. The statistical analysis results were expressed as mean±SE, and p<0.05 indicated that the difference was statistically significant. The results are shown in Tables 3 and 4 and Figure 3 、 Figure 4 shown.

[0042] 3.2.1 Experimental Results Table 3. Results of β-galactosidase staining experiments on zebrafish aging induced by physical stimulation (water rotation at 1000 rpm) (pixels, mean ± SE, n = 10). Group parameter Opacity average (pixels) Normal control group - 0.143±0.004 Physical stimulation (water movement) 1000rpm 0.148±0.004

[0043] Table 4. Results of the zebrafish aging cognitive experiment induced by physical stimulation (water rotation 1000 rpm) (pixels, mean ± SE, n = 6). Group parameter The proportion of blue areas in the cross maze Normal control group - 34.9±5.77 Physical stimulation (water movement) 1000rpm 17.0±5.00*

[0044] Compared with the normal control group, *p<0.05.

[0045] From Table 3, Table 4, Figure 3 、 Figure 4 As can be seen, after 48 hours of physical stimulation (water rotation at 1000 rpm) inducing 5dpf zebrafish, there was no significant increase in the average opacity of β-galactosidase staining, indicating that the zebrafish had not yet shown signs of aging under this stimulation condition. However, the results of the plus maze experiment showed that the proportion of time the zebrafish spent in the blue area of ​​the plus maze was significantly reduced compared to the normal control group, indicating that stimulating zebrafish under these conditions can cause cognitive impairment.

[0046] 3.3 The third exploration of model conditions Wild-type AB zebrafish, 5 days post-fertilization (dpf), were randomly selected and placed in 100 mL wells. Fifty zebrafish were treated in each well (experimental group). A normal control group and a model control group were established, each well containing 50 mL. All experimental groups, except the normal control group, were treated with 25.6 mg / mL of D-galactose in water and subjected to physical stimulation (water rotation at 1000 rpm) to establish a model. Between 5 and 7 dpf, 1) 10 zebrafish were randomly selected from each group and stained with a β-galactosidase kit. Data were acquired using NIS-Elements D 3.20 advanced image processing software, and the average opacity (pixel value) of the β-galactosidase staining in fixed areas of the zebrafish was analyzed. 2) 30 zebrafish were randomly selected from each group and placed in a plus-shaped maze. The fish were recorded using a zebrafish behavioral instrument, and their trajectories were saved. Behavioral data were collected using analysis software, and the percentage of blue areas within the plus-shaped maze was analyzed. The statistical results are expressed as mean ± SE, and p < 0.05 indicates that the difference is statistically significant. Figure 5 、 Figure 6 shown.

[0047] Table 5. Results of the β-galactosidase staining experiment in zebrafish aging induced by D-galactose 25.6 mg / mL combined with physical stimulation (water rotation 1000 rpm) (pixels, mean ± SE, n = 10).

[0048] Compared with the normal control group, **p<0.01.

[0049] Table 6. Results of the zebrafish aging cognitive experiment induced by D-galactose 25.6 mg / mL combined with physical stimulation (water rotation 1000 rpm) (pixels, mean ± SE, n = 6).

[0050] Compared with the normal control group, **p<0.01 Table 7. Results of the zebrafish aging memory experiment induced by 25.6 mg / mL of D-galactose combined with physical stimulation (water rotation 1000 rpm) (pixels, mean ± SE, n = 6).

[0051] Compared with the normal control group, *p<0.05, **p<0.01 From Table 5, Table 6, Table 7 and Figure 5 、 Figure 6It can be seen that after 48 hours of induction of 5dpf zebrafish with 25.6mg / mL of D-galactose combined with physical stimulation (water rotation at 1000rpm), a significant increase in the average opacity of β-galactosidase staining was observed, indicating that the zebrafish showed signs of aging under this condition. Observation of the plus maze test results showed that the proportion of time the zebrafish spent in the blue area of ​​the plus maze was significantly reduced compared with the normal control group. At the same time, observation of the T maze test results showed that the zebrafish spent significantly less time in the enriched area of ​​the T maze than the normal control group, and the latency to enter the enriched area was significantly increased, indicating that stimulating zebrafish under these conditions can cause cognitive and memory impairment.

[0052] In summary, D-galactose 25.6 mg / mL combined with physical stimulation (water rotation 1000 rpm) can induce aging-associated cognitive impairment and memory loss in zebrafish.

[0053] Example 2 Application of the zebrafish aging model with cognitive impairment and memory loss in the efficacy verification of Yangshengtang brand natural vitamin E soft capsules: 1. Experimental animals Wild-type AB strain zebrafish.

[0054] 2. Main instruments and reagents A dissecting microscope (SZX7, OLYMPUS, Japan); a precision electronic balance (CP214, OHAUS, USA); a nine-link magnetic stirrer (MMS9Pro, JOANLAB, China); and a zebrafish behavior analyzer (Hunter Lab HT-XW-2D-12, Hangzhou Huante Biotechnology Co., Ltd., China) were used.

[0055] D-galactose (Batch No. J2126173, Shanghai Aladdin Biochemical Technology Co., Ltd., China); Yangshengtang brand natural vitamin E soft capsules (Batch No.: 20240301, Hainan Yangshengtang Health Products Co., Ltd., China).

[0056] 3. Experimental Methods 3.1 Evaluation of the anti-aging efficacy of Yangshengtang natural vitamin E soft capsules Wild-type AB zebrafish, 3 days post-fertilization (dpf), were randomly selected and placed in 100 mL wells (experimental groups), with 50 zebrafish per well. A normal control group and a model control group were established, with each well containing 50 mL. From 3 dpf to 5 dpf, all experimental groups, except the normal and model groups, received preventive administration of Yangshengtang brand natural vitamin E soft capsules at 5.20, 10.4, and 20.8 μg / mL, dissolved in water. Furthermore, from 5 dpf to 7 dpf, all experimental groups, except the normal control group, received 25.6 mg / mL of D-galactose dissolved in water and physical stimulation (water rotation at 1000 rpm) to establish the model. After modeling, 10 zebrafish were randomly selected from each group and stained with a β-galactosidase kit. Data were acquired using NIS-Elements D 3.20 advanced image processing software, and the average opacity (pixel value) of the β-galactosidase staining in fixed regions of the zebrafish was analyzed. The statistical results were expressed as mean ± SE, and p < 0.05 indicated that the difference was statistically significant. Figure 7 shown.

[0057] 3.1.2 Experimental Results Table 8. β-galactosidase staining results of zebrafish after treatment with Yangshengtang brand natural vitamin E soft capsules (pixels, mean ± SE, n = 10).

[0058] Compared with the model control group, *p<0.05.

[0059] From Table 8 and Figure 7 The average opacity of β-galactosidase staining in the model control group was significantly higher than that in the normal control group, indicating that a 48-hour induction of 25.6 mg / mL of D-galactose combined with physical stimulation (water rotation at 1000 rpm) can successfully establish a model of aging with cognitive impairment and memory loss. After administration of Yangshengtang Natural Vitamin E Soft Capsules, the average blue opacity of the zebrafish bodies remained unchanged, indicating that Yangshengtang Natural Vitamin E Soft Capsules do not have significant anti-aging effects.

[0060] 3.2 Evaluation of the cognitive improvement efficacy of Yangshengtang Natural Vitamin E Soft Capsules Wild-type AB zebrafish, 3 days post-fertilization (dpf), were randomly selected and placed in 100 mL of water, with 50 zebrafish per well (experimental group). A normal control group and a model control group were established, with each well containing 50 mL. From 3 dpf to 5 dpf, all experimental groups, except the normal and model controls, received preventive administration of Yangshengtang brand natural vitamin E soft capsules at 5.20, 10.4, and 20.8 μg / mL, dissolved in water. Furthermore, from 5 dpf to 7 dpf, all experimental groups, except the normal control group, received 25.6 mg / mL of D-galactose dissolved in water and physical stimulation (water rotation at 1000 rpm) to establish the model. After modeling, 30 zebrafish were randomly selected from each group and placed in a plus maze. The fish were recorded and their trajectories saved using a zebrafish behavior instrument. Behavioral data were collected using analysis software, and the percentage of blue areas within the plus maze analyzed. The statistical results were expressed as mean ± SE, and p < 0.05 indicated that the difference was statistically significant. Figure 8 shown.

[0061] 3.2.1 Experimental Results Table 2-2. Cognitive ability results of zebrafish after treatment with Yangshengtang brand natural vitamin E soft capsules (pixels, mean±SE, n=10).

[0062] Compared with the model control group, ***p<0.001.

[0063] From Table 9 and Figure 8 The proportion of the blue area in the zebrafish plus maze in the model control group was significantly lower than that in the normal control group, indicating that a 48-hour induction of 25.6 mg / mL of D-galactose combined with physical stimulation (water rotation at 1000 rpm) can successfully establish a model of aging-associated cognitive impairment and memory loss. Administration of 20.8 μg / mL of Yangshengtang Natural Vitamin E Soft Capsules significantly increased the proportion of the blue area in the zebrafish plus maze, demonstrating that 20.8 μg / mL of Yangshengtang Natural Vitamin E Soft Capsules significantly improves cognition.

[0064] Example 3 Application of the zebrafish aging model with cognitive impairment and memory loss in the efficacy verification of ginseng stem and leaf total saponins tablets: 1. Experimental Animals Wild-type AB strain zebrafish.

[0065] 2. Main instruments and reagents A dissecting microscope (SZX7, OLYMPUS, Japan); a precision electronic balance (CP214, OHAUS, USA); a nine-link magnetic stirrer (MMS9Pro, JOANLAB, China); and a zebrafish behavior analyzer (Hunter Lab HT-XW-2D-12, Hangzhou Huante Biotechnology Co., Ltd., China) were used.

[0066] D-galactose (Batch No. J2126173, Shanghai Aladdin Biochemical Technology Co., Ltd., China); Ginseng Stem and Leaf Total Saponins Tablets (Batch No.: 231102, Sichuan Kangfulai Pharmaceutical Group Co., Ltd., China).

[0067] 3. Experimental Methods 3.1 Evaluation of the anti-aging efficacy of ginseng stem and leaf total saponins tablets Wild-type AB zebrafish, 3 days post-fertilization (dpf), were randomly selected and placed in 100 mL of water, with 50 zebrafish per well (experimental group). A normal control group and a model control group were established, with each well containing 50 mL. From 3 dpf to 5 dpf, all experimental groups, except the normal and model controls, received preventive administration of ginseng stem and leaf total saponins tablets at 50, 100, or 200 μg / mL of water-soluble solution. During the period of 5dpf to 7dpf, except for the normal control group, all other experimental groups were given 25.6mg / mL of D-galactose in water and physical stimulation (water body rotation 1000rpm) to construct the model. After the model was completed, 10 zebrafish were randomly selected from each group and stained with a β-galactosidase kit. The data were collected using NIS-Elements D 3.20 advanced image processing software to analyze the average opacity (pixel) of β-galactosidase staining in the fixed area of ​​zebrafish. The statistical analysis results of this indicator were used to evaluate the anti-aging effect of ginseng stem and leaf total saponin tablets. The statistical processing results were expressed as mean±SE, and p<0.05 indicated that the difference was statistically significant. The results are shown in Table 10 and Figure 9 shown.

[0068] 3.1.1 Experimental Results Table 10. Results of β-galactosidase staining in zebrafish after treatment with ginseng stem and leaf total saponins tablets (pixels, mean ± SE, n = 10).

[0069] Compared with the model control group, **p<0.01, ***p<0.001.

[0070] From Table 10 and Figure 9The average opacity of β-galactosidase staining in zebrafish in the model control group was significantly higher than that in the normal control group, indicating that a 48-hour induction of 25.6 mg / mL of D-galactose combined with physical stimulation (water rotation at 1000 rpm) can successfully establish a model of aging with cognitive impairment and memory loss. After administration of 50, 100, and 200 μg / mL of ginseng stem and leaf total saponins tablets, the average blue opacity of the zebrafish body was significantly lower than that in the model control group, indicating that ginseng stem and leaf total saponins tablets have significant anti-aging effects with cognitive impairment and memory loss.

[0071] 3.2 Evaluation of the cognitive improvement efficacy of ginseng stem and leaf total saponins tablets Wild-type AB zebrafish, 3 days post-fertilization (dpf), were randomly selected and placed in 100 mL of water, with 50 zebrafish per well (experimental group). A normal control group and a model control group were established, with each well containing 50 mL. From 3 dpf to 5 dpf, all experimental groups, except the normal and model controls, received a water-soluble dose of 50, 100, or 200 μg / mL of ginseng stem and leaf total saponins tablets as a preventive medication. Furthermore, from 5 dpf to 7 dpf, all experimental groups, except the normal control group, received a water-soluble dose of 25.6 mg / mL of D-galactose and physical stimulation (water rotation at 1000 rpm) to establish a cognitive model. After modeling, 30 zebrafish were randomly selected from each group and placed in a plus-shaped maze. The zebrafish behavior instrument recorded the trajectories and saved the data. Behavioral data were collected using analysis software, and the percentage of blue areas in the plus-shaped maze was analyzed. Statistical analysis of this metric was used to evaluate the cognitive efficacy of ginseng stem and leaf total saponins tablets. The statistical results were expressed as mean ± SE, and p < 0.05 indicated that the difference was statistically significant. Figure 10 shown.

[0072] 3.2.1 Experimental Results Table 11. Cognitive ability results of zebrafish after treatment with ginseng stem and leaf total saponins tablets (mean±SE, n=6).

[0073] Compared with the model control group, *p<0.05, ***p<0.001.

[0074] From Table 11 and Figure 10The study found that the proportion of the blue area in the zebrafish plus maze in the model control group was significantly lower than that in the normal control group, indicating that a 48-hour induction of 25.6 mg / mL of D-galactose combined with physical stimulation (water rotation at 1000 rpm) can successfully establish a model of aging-associated cognitive impairment and memory loss. Administration of 50, 100, and 200 μg / mL of ginseng stem and leaf total saponins significantly increased the proportion of the blue area in the zebrafish plus maze, indicating that 50, 100, and 200 μg / mL of ginseng stem and leaf total saponins have significant anti-cognitive effects on cognitive impairment and memory loss in aging-associated cognitive impairment.

[0075] 3.3 Evaluation of the efficacy of ginseng stem and leaf total saponins tablets in improving exercise performance Wild-type AB zebrafish, 3 days post-fertilization (dpf), were randomly selected and placed in 100 mL of water, with 50 zebrafish per well (experimental group). A normal control group and a model control group were set up, with each well containing 50 mL. From 3 dpf to 5 dpf, all experimental groups, except the normal and model groups, received a water-soluble dose of 50, 100, or 200 μg / mL of ginseng stem and leaf total saponins tablets as a preventive medication. Furthermore, from 5 dpf to 7 dpf, all experimental groups, except the normal control group, received a water-soluble dose of 25.6 mg / mL of D-galactose and physical stimulation (water rotation at 1000 rpm) to establish the model. After modeling, 10 zebrafish were randomly selected from each group and placed in a 96-well plate. The fish were recorded using a zebrafish behavior instrument and their trajectories were saved. Total distance traveled was collected using analysis software, and statistical analysis of this indicator was used to evaluate the efficacy of ginseng stem and leaf total saponins tablets in improving motor function. The statistical results were expressed as mean ± SE, and p < 0.05 indicated that the difference was statistically significant. Figure 11 shown.

[0076] 3.3.1 Experimental Results Table 12. Results of zebrafish motor ability after treatment with ginseng stem and leaf total saponins tablets (mean±SE, n=10).

[0077] Compared with the model control group, *p<0.05, **p<0.01.

[0078] From Table 12 and Figure 11The proportion of the blue area in the zebrafish plus maze in the model control group was significantly lower than that in the normal control group, indicating that a 48-hour induction of 25.6 mg / mL of D-galactose combined with physical stimulation (water rotation at 1000 rpm) can successfully establish a model of aging with cognitive impairment and memory loss. Administration of 100 and 200 μg / mL of ginseng stem and leaf total saponins significantly increased the proportion of the blue area in the zebrafish plus maze, indicating that 100 and 200 μg / mL of ginseng stem and leaf total saponins have significant anti-aging effects on cognitive impairment and memory loss.

[0079] 3.4 Evaluation of the efficacy of ginseng stem and leaf total saponins tablets in improving responsiveness Wild-type AB zebrafish, 3 days post-fertilization (dpf), were randomly selected and placed in 100 mL of water, with 50 zebrafish per well (experimental group). A normal control group and a model control group were set up, with each well containing 50 mL. From 3 dpf to 5 dpf, all experimental groups, except the normal and model groups, received a water-soluble dose of 50, 100, or 200 μg / mL of ginseng stem and leaf total saponins tablets as a preventive medication. Furthermore, from 5 dpf to 7 dpf, all experimental groups, except the normal control group, received a water-soluble dose of 25.6 mg / mL of D-galactose and physical stimulation (water rotation at 1000 rpm) to establish a model. After modeling, 10 zebrafish were randomly selected from each group and placed in a 96-well plate. The fish were recorded using a zebrafish behavior instrument and their trajectory was saved. Analysis software was used to collect data on their responsiveness under light and dark conditions. Statistical analysis of this indicator was used to evaluate the efficacy of ginseng stem and leaf total saponins tablets in improving responsiveness. The statistical results were expressed as mean ± SE, and p < 0.05 indicated that the difference was statistically significant. Figure 12 shown.

[0080] 3.4.1 Experimental Results Table 13. Results of improved reaction ability of zebrafish after treatment with ginseng stem and leaf total saponins tablets (mean±SE, n=10).

[0081] Compared with the model control group, *p<0.05, **p<0.01.

[0082] From Table 13 and Figure 12The researchers found that the proportion of the blue area in the zebrafish plus maze in the model control group was significantly lower than that in the normal control group, indicating that a 48-hour induction of 25.6 mg / mL of D-galactose combined with physical stimulation (water rotation at 1000 rpm) can successfully establish a model of aging-associated cognitive impairment and memory loss. Administration of 200 μg / mL of ginseng stem and leaf total saponins significantly increased the proportion of the blue area in the zebrafish plus maze, demonstrating that 200 μg / mL of ginseng stem and leaf total saponins has significant anti-aging-associated cognitive impairment and memory loss response capabilities.

[0083] Example 4 Application of the zebrafish aging model with cognitive impairment and memory loss in DHA efficacy verification: 1. Experimental Animals Wild-type AB strain zebrafish.

[0084] 2. Main instruments and reagents A dissecting microscope (SZX7, OLYMPUS, Japan); a precision electronic balance (CP214, OHAUS, USA); a nine-link magnetic stirrer (MMS9Pro, JOANLAB, China); and a zebrafish behavior analyzer (Hunter Lab HT-XW-2D-12, Hangzhou Huante Biotechnology Co., Ltd., China) were used.

[0085] D-galactose (Batch No. J2126173, Shanghai Aladdin Biochemical Technology Co., Ltd., China); DHA (Batch No.: C11944178, Shanghai MacLean Biochemical Technology Co., Ltd., China).

[0086] 3. Experimental Methods 3.1 Evaluation of DHA’s anti-aging effects Wild-type AB zebrafish, 3 days post-fertilization (dpf), were randomly selected and placed in 100 mL wells (experimental groups), with 50 zebrafish per well. A normal control group and a model control group were established, with each well containing 50 mL. From 3 dpf to 5 dpf, all experimental groups, except the normal and model controls, received preventive administration of DHA at 0.15625, 0.3125, and 0.625 μL / mL of water-soluble solution. During the period of 5dpf to 7dpf, except for the normal control group, all other experimental groups were given 25.6mg / mL of D-galactose in water and physical stimulation (water body rotation 1000rpm) to build the model. After the model was completed, 10 zebrafish were randomly selected from each group and stained with a β-galactosidase kit. The data were collected using NIS-Elements D 3.20 advanced image processing software to analyze the average opacity (pixel) of β-galactosidase staining in the fixed area of ​​zebrafish. The statistical analysis results of this indicator were used to evaluate the anti-aging effect of DHA. The statistical processing results are expressed as mean±SE, and p<0.05 indicates that the difference is statistically significant. The results are shown in Table 14 and Figure 13 shown.

[0087] 3.1.1 Experimental Results Table 14. Results of β-galactosidase staining in zebrafish after DHA treatment (pixels, mean ± SE, n = 10).

[0088] Compared with the model control group, *p<0.05, ***p<0.001.

[0089] From Table 14 and Figure 13 The results show that the average opacity of zebrafish β-galactosidase staining in the model control group was significantly higher than that in the normal control group, indicating that a 48-hour induction of D-galactose 25.6 mg / mL combined with physical stimulation (water rotation at 1000 rpm) can successfully induce a model of aging with cognitive impairment and memory loss. After administration of DHA 0.15625, 0.3125, and 0.625 μL / mL, the average blue opacity of the zebrafish body was significantly lower than that in the model control group, indicating that DHA 0.15625, 0.3125, and 0.625 μL / mL has significant anti-aging effects on cognitive impairment and memory loss.

[0090] 3.2 Evaluation of DHA’s cognitive improvement effects Wild-type AB zebrafish, 3 days post-fertilization (dpf), were randomly selected and placed in 100 mL of water, with 50 zebrafish per well (experimental group). A normal control group and a model control group were established, with each well containing 50 mL. From 3 dpf to 5 dpf, all experimental groups, except the normal and model controls, received preventive administration of DHA at 0.15625, 0.3125, and 0.625 μL / mL of water-soluble solution. Furthermore, from 5 dpf to 7 dpf, all experimental groups, except the normal control group, received 25.6 mg / mL of water-soluble D-galactose and physical stimulation (water rotation at 1000 rpm) to establish the model. After modeling, 30 zebrafish were randomly selected from each group and placed in a plus maze. The fish were recorded using a zebrafish behavioral instrument and their trajectories were saved. Behavioral data were collected using analysis software, and the percentage of blue areas in the plus maze was analyzed. Statistical analysis of this indicator was used to evaluate the cognitive efficacy of DHA. The statistical results were expressed as mean ± SE, and p < 0.05 indicated that the difference was statistically significant. Figure 14 shown.

[0091] 3.2.1 Experimental Results Table 15. Cognitive ability results of zebrafish after DHA treatment (mean±SE, n=6).

[0092] Compared with the model control group, **p<0.01, ***p<0.001.

[0093] From Table 15 and Figure 14 It can be seen that the proportion of the blue area in the zebrafish plus maze in the model control group was significantly lower than that in the normal control group, indicating that 25.6 mg / mL of D-galactose combined with physical stimulation (water rotation at 1000 rpm) for 48 hours can successfully induce a model of aging with cognitive impairment and memory loss. After administering DHA 0.15625, 0.3125, and 0.625 μL / mL, the proportion of the blue area in the zebrafish plus maze increased significantly, indicating that DHA 0.15625, 0.3125, and 0.625 μL / mL has a significant effect on the cognitive ability of aging with cognitive impairment and memory loss.

[0094] 3.3 Evaluation of the efficacy of DHA in extending telomere length Wild-type AB zebrafish, 3 days post-fertilization (dpf), were randomly selected and placed in 100 mL wells. A normal control group and a model control group were set up, with each well containing 50 mL. From 3 dpf to 5 dpf, all experimental groups, except the normal and model controls, received preventive administration of DHA at 0.15625, 0.3125, and 0.625 μL / mL in water. Furthermore, from 5 dpf to 7 dpf, all experimental groups, except the normal control group, received 25.6 mg / mL of D-galactose in water and physical stimulation (water rotation at 1000 rpm) to establish the model. After modeling, zebrafish samples were collected from each experimental group, and total DNA was extracted using a universal genomic DNA extraction kit. The total DNA concentration and purity were determined using a UV-visible spectrophotometer. Using dio2 (deiodinase, iodothyronine, type II) as a single copy gene, the ratio of telomere (telo) repeat copies (T) to single copy (S) gene products was calculated. Telomere length was expressed as the relative T / S ratio. The statistical analysis results of this indicator were used to evaluate the efficacy of DHA in extending telomere length. The statistical processing results were expressed as mean ± SE, and p < 0.05 indicated that the difference was statistically significant. The results are shown in Table 16 and Figure 15 shown.

[0095] 3.3.1 Experimental Results Table 16. Effect of DHA treatment on T / S ratio (mean ± SE, n = 3).

[0096] Compared with the model control group, *p<0.05.

[0097] From Table 16 and Figure 15 Telomere length is closely related to aging. Telomeres, the protective structures at the ends of chromosomes, gradually shorten with increasing cell division. When telomeres shorten to a certain extent, they can lead to a decline in cellular function, increased genomic instability, and ultimately, biological aging and various age-related diseases. Maintaining or extending telomere length can slow the cellular aging process. Under the conditions of this experiment, DHA has the effect of extending telomere length, as evidenced by a significant increase in the relative T / S ratio.

[0098] Therefore, the method for constructing a model of aging with cognitive impairment and memory loss of the present invention uses D-galactose combined with physical stimulation to induce aging with cognitive impairment and memory loss in zebrafish, and can successfully establish a model of aging with cognitive impairment and memory loss. The phenomenon is intuitively observed, and the definition of aging with cognitive impairment and memory loss is accurate. It can effectively and quickly evaluate the effects of drugs, health foods and foods on improving aging with cognitive impairment and memory loss.

Claims

1. A method for constructing a model of aging with cognitive impairment and memory loss, characterized in that: The model construction method is to administer D-galactose water-soluble solution in combination with physical stimulation of zebrafish to detect indicators of aging-associated cognitive impairment and memory loss.

2. The method for constructing a model of aging with cognitive impairment and memory loss according to claim 1, characterized in that: The zebrafish is a wild-type AB strain zebrafish.

3. The method for constructing a model of aging with cognitive impairment and memory loss according to claim 1 or 2, characterized in that: The zebrafish is 3-10 dpf zebrafish.

4. The method for constructing a model of aging with cognitive impairment and memory loss according to claim 1, characterized in that: The water-soluble administration concentration of D-galactose is 12.8-51.2 mg / mL.

5. The method for constructing a model of aging with cognitive impairment and memory loss according to claim 4, characterized in that: The aqueous D-galactose dosage concentration is 25.6 mg / mL.

6. The method for constructing a model of aging with cognitive impairment and memory loss according to claim 1, characterized in that: The physical stimulation is water rotation stimulation, and the rotation speed is 1000 rpm.

7. The method for constructing a model of aging with cognitive impairment and memory loss according to claim 1, characterized in that: The indicators of aging-associated cognitive impairment and memory loss are the proportion of the blue area in the plus maze, the difference in light and dark speed, the total movement distance, SA-β-galactosidase staining, the time spent in the enriched area of ​​the T maze, and the latency to enter the enriched area.

8. The method for constructing a model of aging with cognitive impairment and memory loss according to claim 1, characterized in that: The D-galactose water-soluble administration combined with physical stimulation of zebrafish is continued at the stage of 5dpf to 7dpf.

9. An application of the method for constructing a model of aging with cognitive impairment and memory loss according to any one of claims 1 to 8, characterized in that: It is used to detect drugs, health foods and food raw materials that improve aging-related cognitive impairment and memory loss.

10. The use of the method for constructing a model of aging with cognitive impairment and memory loss according to claim 9, characterized in that: The drug administration stage of the medicine, health food and food raw materials is 3 dpf to 8 dpf, and the drug administration cycle is 0 to 120 hours.

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