CN112034113A - Method for detecting memory improvement effect of health food - Google Patents
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Abstract
The invention relates to the technical field of health food inspection and evaluation, in particular to a method for detecting memory improvement effect of health food, which comprises the following steps: (1) randomly selecting a plurality of zebra fish, putting an inducer after memory training, and establishing a dysmnesia model control group; (2) after memory training, putting an inducer and a sample to be tested, and establishing a sample group; (3) after memory training, adding an inducer and edaravone, and establishing a positive control group; (4) memory training, establishing a normal control group; (5) and respectively enabling the zebra fish of the memory impairment model control group, the test sample group, the positive control group and the normal control group to enter a T-shaped maze experiment, recording the latency time (T) from the starting point to the eutrophication area of the zebra fish, and calculating the memory improvement effect (%) of the test sample to be tested. The detection method has the advantages of short test period, simple judgment standard of the memory improvement experiment and capability of simultaneously carrying out a plurality of samples.
Description
Technical Field
The invention relates to the technical field of health food inspection and evaluation, in particular to a method for detecting memory improvement effect of health food.
Background
With the improvement of living standard of people, more and more people pay attention to health preservation, health food for enhancing memory is more and more popular with people, and the detection of the memory improvement efficacy of the health food is especially important for ensuring the health of consumers. The technical specification for health food inspection and evaluation (2003) indicates that the method for assisting in improving the memory function comprises animal experiments and human body feeding experiments, wherein the animal experiments comprise a diving platform experiment, a dark method, a shuttle box experiment and a water maze experiment, and any two of the four experiments have positive results, and the repeated positive results are consistent, so that the health food can be judged to assist in improving the memory function.
The above prior art has the following disadvantages: (1) the administration time of the test sample is generally 27-70 days, and the experiment period is too long; (2) the positive result can be judged only if any two of the four experiments are positive after the determination indexes are more, namely, a jump table experiment, a dark method, a shuttle box experiment and a water maze experiment; (3) due to the characteristics of high feeding cost, long development period and the like of the mammals, a plurality of samples cannot be evaluated at the same time, and the experimental efficiency of the mammals is low.
Therefore, the research significance of finding an experimental method which has simple experimental steps, short experimental period, simple judgment standard of the memory improvement experiment and can simultaneously evaluate a plurality of samples is important.
Disclosure of Invention
In order to overcome the technical problems in the prior art, the invention provides the method for detecting the memory improvement effect of the health food, which has the advantages of short test period, simple judgment standard of a memory improvement experiment and capability of simultaneously evaluating a plurality of test articles.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for detecting memory improvement efficacy of health food, which is characterized by comprising the following steps:
(1) randomly selecting a plurality of zebra fish, putting an inducer after memory training, and establishing a dysmnesia model control group;
(2) randomly selecting zebra fishes with the same number as that in the step (1), putting an inducer and a sample to be tested after memory training, and establishing a sample group;
(3) randomly selecting zebra fishes with the same number as that in the step (1), and after memory training, adding an inducer and edaravone to establish a positive control group;
(4) randomly selecting zebra fishes with the same number as that in the step (1), and establishing a normal control group after memory training;
(5) respectively putting the zebra fish of the test group, the memory impairment model control group, the positive control group and the normal control group into a T-maze test, recording the latency time (T) from the starting point to the eutrophication area (EC area) (the selection arm for placing the fish school), and calculating the memory improvement efficacy (%) of the sample to be tested according to the following formula:
the zebra fish is a typical aquatic vertebrate, the brain tissue of the zebra fish is simple, but the brain, the diencephalon, the midbrain, the hindbrain, the rhinestone and the like are distinguished, the zebra fish can be used for behavioral evaluation such as movement, learning, memory and the like, and in addition, the functions of main neurotransmitter systems such as cholinergic, dopamine and norepinephrine have similarity with mammals, so that the zebra fish becomes an important animal model for researching the nerve effect. Due to the characteristics of low feeding cost, small volume, short development period and high single spawning frequency of the zebra fish, a plurality of samples can be evaluated simultaneously. The invention takes zebra fish behavioral index-latency time (T) as a judgment basis, and the zebra fish in a test sample group can be judged to have a memory improvement effect as long as the zebra fish is significantly different from a dysmnesia model control group, and the judgment standard of a memory improvement experiment is simple. Compared with 1/3 time of a mouse experiment (27-70 days), the zebra fish memory improvement experiment period is short.
Preferably, in steps (1) to (3), the inducer is bisphenol AF.
Bisphenol AF, also known as hexafluorobisphenol a, has stronger estrogenic activity than bisphenol a, can induce apoptosis in hippocampal cell lines and primary neuronal cells, can increase the level of intracellular calcium, generate Reactive Oxygen Species (ROS), and up-regulate mitogen-activated protein kinase, interrupt normal physiological functions of microglia, affect nerve signal transduction, and interfere with the expression of transmitter receptors such as 5-HT, DA, GABA, Glu and the like, thereby causing memory dysfunction.
Preferably, in steps (1) to (3), the concentration of the inducer is 1. mu.g/mL.
Preferably, in the steps (1) to (4), the zebra fish is wild-type AB strain zebra fish of 3-6 months old.
Selecting a development stage of 3-6 months old because the zebra fish is in a learning and memory peak period; the wild AB strain zebrafish is the earliest wild strain obtained by the research team of George Streisinger, geneticist of Oregon university, and the strain, genetic background, management level and the like are clear and reliable.
Preferably, in the step (3), the concentration of the edaravone is 8.5 μ g/mL.
Edaravone is a brain protective agent, has effects of scavenging oxygen free radicals, promoting the increase of some nutrient substances in brain, inhibiting neuron death, inhibiting lipid peroxidation, and preventing excessive oxidation and injury of brain cells, vascular endothelial cells and nerve cells. According to the invention, edaravone is added on the basis of a dysmnesia model control group to form a positive control group, and the positive control group is used for judging the detection result of the sample to be detected.
Preferably, in steps (1) to (4), the memory training period is 5 days.
Preferably, in step (5), the criterion for entering the eutrophic area is as follows: zebrafish completely entered and found the fish school to stay for more than 30 s.
Preferably, in the step (5), statistical analysis is carried out by analysis of variance and Dunnett's T-test, p <0.05 shows that the food has significant difference, and the health food to be tested is judged to have memory improvement efficacy.
Preferably, in the step (5), in the T maze experiment, the used T maze comprises two selection arms and a starting arm, a movable partition plate is arranged in the middle, and the starting interval is adjustable; food is put in the selection arm. The selection arm comprises a left arm decorated with red (red more readily attracts zebrafish) and holding the fish school, and a right arm decorated with green.
Therefore, the invention has the following beneficial effects: the invention establishes the model by using the zebra fish with low feeding cost, small volume, short development period and high single spawning frequency, and the detection method has the advantages of short test period, simple memory improvement experiment judgment standard and capability of simultaneously carrying out a plurality of samples.
Drawings
FIG. 1 is a schematic diagram of a T-maze.
In the figure: arm 1 is selected, arm 2 is initiated.
Detailed Description
The technical solution of the present invention is further specifically described below by way of specific examples.
In the present invention, all the equipment and materials are commercially available or commonly used in the art, and the methods in the following examples are conventional in the art unless otherwise specified.
The following examples of the invention employ reagents and instrumentation as follows:
(1) t maze (fig. 1) (custom, hangzhou changqing chemical, China);
(2) a camera (1200TVL, sony);
(3) a high-definition coaxial digital hard disk video recorder (DH-HCVR4104HC-V4, China, Dahua Hangzhou);
(4) precision electronic balances (CP214, OHAUS, China);
(5) bisphenol AF, CAS number: 1478-61-1, manufacturer: aladdin
(6) Edaravone, CAS No.: 89-25-8, manufacturer: aladdin
Experimental animals:
wild type AB strain zebra fish, male and female half, 3-6 months of age, total 960 tails, 10 tails per experimental group.
Detecting items:
evaluation of memory improvement efficacy of 12 samples (a to L) to be tested:
(1) randomly selecting 50 wild AB strain zebra fishes of 3-6 months old in 5L beakers, and dissolving bisphenol AF in water for 1 mu g/mL after memory training for 5 days, and establishing a dysmnesia model control group, wherein 10 zebra fishes are placed in each beaker (namely each concentration group);
(2) randomly selecting 50 wild AB strain zebra fishes of 3-6 months old in 5L beakers, and after memory training for 5 days, dissolving 10 zebra fishes in water and giving bisphenol AF 1 microgram/mL and a sample to be tested to establish a test group;
(3) randomly selecting 50 wild AB strain zebra fishes of 3-6 months old in a 5L beaker, and dissolving bisphenol AF (Achilles bealeis) in water for 1 mu g/mL and 8.5 mu g/mL edaravone after memory training for 5 days to establish a positive control group, wherein 10 zebra fishes are in each beaker (namely each concentration group);
(4) randomly selecting 50 wild AB strain zebra fishes of 3-6 months old in 5L beakers, carrying out memory training for 5 days on 10 zebra fishes in each beaker (namely each concentration group), and establishing a normal control group;
(5) the zebra fish of the test group, the dysmnesia model control group, the positive control group and the normal control group are respectively subjected to a T-maze test, as shown in figure 1: the T maze comprises two selection arms and an initial arm, a movable clapboard is arranged in the middle, and the initial interval is adjustable; food is put in the selection arm. The selection arm comprises a left arm decorated with red (red more readily attracts zebrafish) and holding the fish school, and a right arm decorated with green. Recording the latency time (T) from the starting point to the entrance of the zebra fish into the eutrophication area, and calculating the memory improvement effect (%) of the test sample according to the following formula:
6L of water for fish culture in the T maze in the memory training period is used, one fish is placed at the starting point, the testing time is 6min, and the latency time from the starting point to the EC area of the fish is recorded (only when the fish completely enters and finds the fish group to stay for more than 30s, the fish is considered to really enter the EC area). After the fish found the EC zone, the shrimps were fed in the EC zone. Each fish is tested and placed in a corresponding group of containers. Memory training was performed for 5 days, and each latency was recorded. The inducer exposure period was the same as the memory test period, 5 days of exposure to the virus, and the latency time was recorded.
The evaluation concentration settings of 12 samples (a to L) to be tested are shown in table 1:
TABLE 1.12 evaluation concentrations of samples to be tested (A to L)
The results are shown in tables 2 and 3:
1) summary of death and toxicity phenotype
TABLE 2.12 test samples (A. about.L) for each concentration group, mortality and toxicity phenotype (n. about.10)
2) Results of memory improvement
Table 3.12 results of incubation time of zebrafish after treatment of each concentration group of samples to be tested (a to L) (n is 10)
P <0.05, p <0.01, p <0.001, compared to the dysmnesia model control group
And (4) detection conclusion:
in the dose concentration of the experiment, 100% (12/12) of 12 samples (A-L) have the function of improving memory.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.
Claims (9)
1. A method for detecting memory improvement efficacy of health food, which is characterized by comprising the following steps:
(1) randomly selecting a plurality of zebra fish, putting an inducer after memory training, and establishing a dysmnesia model control group;
(2) randomly selecting zebra fishes with the same number as that in the step (1), putting an inducer and a sample to be tested after memory training, and establishing a sample group;
(3) randomly selecting zebra fishes with the same number as that in the step (1), and after memory training, adding an inducer and edaravone to establish a positive control group;
(4) randomly selecting zebra fishes with the same number as that in the step (1), and establishing a normal control group after memory training;
(5) respectively carrying out T maze experiments on the zebra fishes of a dysmnesia model control group, a sample group, a positive control group and a normal control group, recording the latency time (T) from the starting point to the eutrophication area of the zebra fishes, and calculating the memory improvement efficacy (%) of the sample to be tested according to the following formula:
2. the method for detecting the memory improvement efficacy of health food according to claim 1, wherein in the steps (1) to (3), the inducer is bisphenol AF.
3. The method for detecting the memory improvement efficacy of health food according to claim 1, wherein the concentration of the inducer in the steps (1) to (3) is 1 μ g/mL.
4. The method for detecting the memory improvement effect of the health food according to claim 1, wherein in the steps (1) to (4), the zebra fish is wild-type AB strain zebra fish of 3-6 months old.
5. The method for detecting memory improvement efficacy of health food according to claim 1, wherein in the step (3), the concentration of edaravone is 8.5 μ g/mL.
6. The method for detecting the memory improvement efficacy of health food according to claim 1, wherein the memory training time in steps (1) - (4) is 5 days.
7. The method for detecting memory improvement efficacy of health food according to claim 1, wherein in the step (5), the judgment criteria for entering the eutrophic area are: zebrafish completely entered and found the fish school to stay for more than 30 s.
8. The method for detecting memory improvement efficacy of health food according to claim 1, wherein in the step (5), statistical analysis is performed by analysis of variance and Dunnett's T-test, and p <0.05 shows significant difference, and the health food to be detected is judged to have memory improvement efficacy.
9. The method for testing the memory improvement efficacy of health food according to any one of claims 1 to 8, wherein in the step (5), in the T maze test, the T maze comprises two selection arms and a starting arm, a movable partition is arranged in the middle, and the starting interval is adjustable; food is put in the selection arm; the selection arm comprises a left arm and a right arm, the left arm is decorated with red and is used for placing fish stocks, and the right arm is decorated with green.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113951200A (en) * | 2021-09-15 | 2022-01-21 | 杭州环特生物科技股份有限公司 | Multichannel device for detecting color preference of zebra fish juvenile fish and use method and application thereof |
| CN114667956A (en) * | 2021-12-16 | 2022-06-28 | 杭州环特生物科技股份有限公司 | Method for constructing zebra fish memory evaluation model and application thereof |
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