CN115247211B - Method for screening refreshing drugs and application of asperuloside in refreshing - Google Patents

Method for screening refreshing drugs and application of asperuloside in refreshing Download PDF

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CN115247211B
CN115247211B CN202210986368.4A CN202210986368A CN115247211B CN 115247211 B CN115247211 B CN 115247211B CN 202210986368 A CN202210986368 A CN 202210986368A CN 115247211 B CN115247211 B CN 115247211B
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靳梦
张秀军
任擎宇
王利振
李宁
王荣春
孙晨
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Biology Institute of Shandong Academy of Sciences
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Abstract

The invention discloses an application of refreshing marker genes nr1d4a and nr1d4b in screening of refreshing drugs, relates to the technical field of drug screening, and in particular relates to a method for screening the refreshing drugs based on behavioral indexes and the refreshing marker genes, which comprises the following steps: (1) determining the maximum safe concentration of the caffeine to be screened and the caffeine to be controlled on young zebra fish of 5 days old; (2) screening a refreshing drug based on the behavior of the zebra fish under the light stimulus reaction; (3) detecting the expression level of each group of refreshing marker genes nr1d4a and nr1d4b based on Real-time PCR technology, and screening out refreshing drugs; (4) judgment criteria for a refreshment drug; the screening method has the advantages of short operation flow, easy operation, accurate and efficient screening result, combines the behavioral characterization and the gene expression level, and provides a new method for screening the medicines for refreshing the brain in the future.

Description

Method for screening refreshing drugs and application of asperuloside in refreshing
Technical Field
The invention relates to the technical field of drug screening, in particular to a method for screening a refreshing drug and application of asperuloside in refreshing, wherein the screening is performed based on a behavioral index and a refreshing marker gene.
Background
The existing drug screening method mainly comprises molecular level screening, virtual screening, cell level screening, animal model drug screening and the like, wherein the molecular level screening is effective in drug binding with target proteins, but can not play a role in a cell environment; virtual screening refers to screening out possible effective medicines by simulating the interaction between a known target spot and a medicine structure through a computer, and has low cost and high speed, but the screened medicines are verified by further biological experiments; cell level screening refers to treating target cells with candidate drugs, and then checking the effect of the drugs on the target cells through biochemical experiments, wherein the drugs obtained through screening can effectively act on the target cells, but the experimental process is complex, the requirement on experimental environment is high, and whether the drugs have toxic or side effects on the whole organism cannot be evaluated, so that the use of the drugs is limited; animal model drug screening generally requires building primate models such as monkeys and the like, pigs and mice, but the animal disease model building process is a huge project, and requires a system theory and a complex technical means to obtain effective building, and has strict requirements on animal growth environment.
The zebra fish has the advantages of high homology with human genes, formation of tissues and organs 24 hours after fertilization, short development period, small individuals and the like, and provides possibility for the zebra fish to be used as an animal model for screening active drugs. At present, no scientific, rapid and effective screening method for the refreshing drugs exists.
Disclosure of Invention
In order to solve the problems, the invention aims to provide a method for screening a refreshing drug and application of asperuloside in refreshing, wherein the screening is performed based on a behavioral index and a refreshing marker gene.
The invention aims to achieve the aim, and the aim is achieved by the following technical scheme:
application of refreshing marker genes nr1d4a and nr1d4b in screening of refreshing drugs.
The invention also comprises a method for screening a refreshing drug based on behavioral indexes and the refreshing marker gene according to claim 1, which comprises the following steps:
(1) determining the maximum safe concentration of the caffeine to be screened and the caffeine to be contrast agent on the juvenile zebra fish;
(2) screening a refreshing drug based on the behavior of zebra fish under the light stimulus reaction:
when the experiment is carried out at night 24, transferring the juvenile zebra fish with 5dpf into a 48-hole plate, wherein each hole is used for one fish, dividing the juvenile zebra fish into a control drug group, a blank group and a plurality of experiment groups, wherein the control drug group is added with caffeine with the maximum safe concentration, the blank group is added with 1mL of fresh fish raising water, and each experiment group is added with 1mL of drug to be screened with the maximum safe concentration; three parallel tests are set for each group;
putting the 48 pore plate into a camera bellows of a Zebra zebra fish behavior analyzer, starting to detect the illumination stimulus response of the zebra fish, recording data once every minute for 60 minutes, performing data processing by using Zeblab software, calculating the total swimming distance and swimming speed of the zebra fish, and testing the illumination stimulus response of different groups of zebra fish under the sleeping time;
(3) based on Real-time PCR technology, detecting expression levels of refreshing marker genes nr1d4a and nr1d4b in each group, and screening out refreshing drugs:
designing primer sequences for the refreshing marker genes nr1d4a and nr1d4 b:
nr1d4a-RT-F：AGATATCGCATCAGGGTTCC，
nr1d4a-RT-R：CTCGTTCTTCACGCACATCT，
nr1d4b-RT-F：ATCAGGTCATGCTGCTGAAG，
nr1d4b-RT-R：GCGTTCCTTAGCATTGAACA，
collecting zebra fish subjected to behavioral records of different treatment groups, washing with fish-farming water for two to three times, transferring into a centrifuge tube, centrifuging at high speed, and sucking the upper liquid to leave the tissue of the bottom zebra fish; extracting total RNA of different experimental groups of zebra fish by using an RNA extraction kit, measuring the concentration of RNA by using an ultra-micro spectrophotometer, reversing the RNA into cDNA by using a reverse transcription kit, referring to a real-time fluorescent quantitative PCR specification, adding primers to be detected into a qRT-PCR instrument for amplification by using the cDNA after the reverse rotation of each group as a template, setting three parallel experiments in each group, and detecting the expression level of each group of refreshing marker genes nr1d4a and nr1d4b;
(4) judgment criteria for a refreshing drug:
under the sleeping time, compared with a blank group, the total swimming distance of the refreshing medicine is increased, the swimming speed is increased, and the reaction capability is enhanced; the expression changes of the refreshing marker gene of the refreshing medicine and the caffeine of the contrast medicine are the same, and are all down-regulation changes.
Preferably, (1) the specific steps of determining the maximum safe concentration of the drug to be screened and caffeine of the control drug on the young zebra fish are as follows:
the method comprises the steps of randomly dividing 3-day-old juvenile zebra fish into a blank group, a control drug group and a plurality of experimental groups, wherein the number of the juvenile zebra fish in each group is the same, placing the juvenile zebra fish in 24-hole cell culture plates, putting 8-12 fish in each hole, soaking the 24-hole cell culture plates with drugs to be screened with different gradient concentrations for 3 days, repeating each concentration test for 3 times, recording the death rate of the zebra fish, and determining the maximum safe concentrations of the drugs in the experimental groups and the drugs in the control group through SPSS software calculation.
Preferably, the drug to be screened is cortex et radix Polygalae extract, semen Ziziphi Spinosae extract, eucommiae cortex male flower extract, calendula extract and asperuloside.
The medicine for refreshing and restoring consciousness, namely the asperuloside, is screened by adopting the method.
Compared with the prior art, the invention has the following advantages:
according to the method for screening the refreshing drugs based on the behavioural index and the refreshing marker gene, the nr1d4a and nr1d4b are defined as the refreshing marker genes by adopting a transcriptome method for the first time, in the screening process, the individual groups of zebra fish are firstly screened primarily through behaviours, then upstream and downstream primers are designed for the refreshing marker genes nr1d4a and nr1d4b, RT-qPCR verification is carried out, and as the expression trend of the refreshing marker genes nr1d4a and nr1d4b of the asperuloside group is consistent with that of the control group, the effect of refreshing the drugs to be screened, such that the asperuloside is determined. The method for screening the refreshing drugs based on the behavioural index and the refreshing marker gene has the advantages of short operation flow, easy operation, accurate and efficient screening result, combines behavioural characterization and gene expression level, and provides a new method for screening the refreshing drugs in the future.
The screening method of the invention defines the refreshing effect of the medicine phyllanthin to be screened.
Drawings
FIG. 1 is a schematic diagram of total distance traveled by individual groups of young zebra fish in light stimulus response detection; p < 0.05 vs. blank, < P < 0.01 vs. blank);
FIG. 2 is a schematic diagram showing the speed of movement of young zebra fish in the detection of light stimulus response in a blank group;
FIG. 3 is a schematic diagram showing the speed of swimming of young zebra fish in the light stimulus response test of the control drug group (caffeine group);
FIG. 4 is a schematic diagram showing the speed of swimming of young zebra fish in the detection of the light stimulus response;
FIG. 5 is a graph showing expression levels of the refreshing marker gene nr1d4a for young zebra fish of each group (P < 0.001 vs. blank);
FIG. 6 is a graph showing expression levels of the refreshing marker gene nr1d4b for young zebra fish of each group (< 0.01 vs. blank, < 0.001 vs. blank,);
reference numerals:
1 blank group, 2 control drug group (caffeine group), 3 polygala tenuifolia extract group, 4 spina date seed extract group, 5 eucommia male flower extract group, 6 calendula extract group and 7 asperuloside group.
Detailed Description
The invention aims to provide a method for screening a refreshing drug and application of asperuloside in refreshing, wherein the screening is performed based on a behavioral index and a refreshing marker gene, and the method is realized by the following technical scheme:
application of refreshing marker genes nr1d4a and nr1d4b in screening of refreshing drugs.
The invention also comprises a method for screening a refreshing drug based on behavioral indexes and the refreshing marker gene according to claim 1, which comprises the following steps:
(1) determining the maximum safe concentration of the caffeine to be screened and the caffeine to be contrast agent on the juvenile zebra fish;
(2) screening a refreshing drug based on the behavior of zebra fish under the light stimulus reaction:
when the experiment is carried out at night 24, transferring the juvenile zebra fish with 5dpf into a 48-hole plate, wherein each hole is used for one fish, dividing the juvenile zebra fish into a control drug group, a blank group and a plurality of experiment groups, wherein the control drug group is added with caffeine with the maximum safe concentration, the blank group is added with 1mL of fresh fish raising water, and each experiment group is added with 1mL of drug to be screened with the maximum safe concentration; three parallel tests are set for each group;
putting the 48 pore plate into a camera bellows of a Zebra zebra fish behavior analyzer, starting to detect the illumination stimulus response of the zebra fish, recording data once every minute for 60 minutes, performing data processing by using Zeblab software, calculating the total swimming distance and swimming speed of the zebra fish, and testing the illumination stimulus response of different groups of zebra fish under the sleeping time;
the steps detect the ability of the medicine to promote autonomous activity and excite the central nervous system through behaviours or detect the sleep time of experimental animals so as to measure the refreshing efficacy of the medicine;
(3) based on Real-time PCR technology, detecting expression levels of refreshing marker genes nr1d4a and nr1d4b in each group, and screening out refreshing drugs:
designing primer sequences for the refreshing marker genes nr1d4a and nr1d4 b:
nr1d4a-RT-F：AGATATCGCATCAGGGTTCC，
nr1d4a-RT-R：CTCGTTCTTCACGCACATCT，
nr1d4b-RT-F：ATCAGGTCATGCTGCTGAAG，
nr1d4b-RT-R：GCGTTCCTTAGCATTGAACA，
collecting zebra fish subjected to behavioral records of different treatment groups, washing with fish-farming water for two to three times, transferring into a centrifuge tube, centrifuging at high speed, and sucking the upper liquid to leave the tissue of the bottom zebra fish; extracting total RNA of different experimental groups of zebra fish by using an RNA extraction kit, measuring the concentration of RNA by using an ultra-micro spectrophotometer, reversing the RNA into cDNA by using a reverse transcription kit, referring to a real-time fluorescent quantitative PCR specification, adding primers to be detected into a qRT-PCR instrument for amplification by using the cDNA after the reverse rotation of each group as a template, setting three parallel experiments in each group, and detecting the expression level of each group of refreshing marker genes nr1d4a and nr1d4b;
(4) judgment criteria for a refreshing drug:
under the sleeping time, compared with a blank group, the total swimming distance of the refreshing medicine is increased, the swimming speed is increased, and the reaction capability is enhanced; the expression changes of the refreshing marker gene of the refreshing medicine and the caffeine of the contrast medicine are the same, and are all down-regulation changes.
Preferably, (1) the specific steps of determining the maximum safe concentration of the drug to be screened and caffeine of the control drug on the young zebra fish are as follows:
the method comprises the steps of randomly dividing 3-day-old juvenile zebra fish into a blank group, a control drug group and a plurality of experimental groups, wherein the number of the juvenile zebra fish in each group is the same, placing the juvenile zebra fish in 24-hole cell culture plates, putting 8-12 fish in each hole, soaking the 24-hole cell culture plates with drugs to be screened with different gradient concentrations for 3 days, repeating each concentration test for 3 times, recording the death rate of the zebra fish, and determining the maximum safe concentrations of the drugs in the experimental groups and the drugs in the control group through SPSS software calculation.
Preferably, the method for screening the refreshing drugs based on the behavioral indexes and by using the refreshing marker gene comprises the steps of extracting polygala tenuifolia, extracting semen zizyphi spinosae, extracting eucommia male flower, extracting calendula and extracting phyllanthus niruri.
The medicine for refreshing and restoring consciousness, namely the asperuloside, is screened by adopting the method.
The invention is further described below in connection with specific embodiments.
The qRT-PCR instrument in the invention is a fluorescent real-time quantitative PCR instrument.
The manufacturer and model of the RNA extraction kit are RN2802 of Beijing Aidelai biotechnology Co., ltd; the manufacturer and model of the reverse transcription kit is Takara-RR036A; the qRT-PCR instrument was manufactured and model number Takara-RR091Q.
Example 1
Before drug screening, a zebra fish sample and the drug to be screened need to be prepared.
The preparation method of the zebra fish sample comprises the following specific steps:
healthy mature zebra fish females and males are taken 1:1 on the same day 16: putting the mixture into a spawning jar at about 00 hours, and 8 days after the next day: and (3) extracting a partition plate at 30, collecting fertilized eggs after 2 hours, removing dead embryos, repeatedly flushing culture water for 3 times, transferring the fertilized eggs into culture water containing 2mg/L of methylene blue, and placing the culture water into a 28 ℃ incubator for incubation and culture. After 4 hours, embryos are observed under a microscope, normal-developing embryos are selected for incubation and culture until 5 days after fertilization (day post fertilization, dpf), and dead embryos are removed in time every day.
(II) preparing the drug to be screened
The invention prepares the traditional Chinese medicine raw materials to be screened as follows: polygala tenuifolia, spina date seed, eucommia male flower and calendula; and meanwhile, outsourcing the phyllanthus niruri glycoside.
The extraction of the effective substances is carried out by adopting the following methods:
grinding the traditional Chinese medicine raw materials into powder, adding the powder into a round-bottom flask, adding water which is 3-5 times of the weight of the traditional Chinese medicine raw materials into the round-bottom flask, distilling and purifying for at least 5 hours, cooling, centrifuging, collecting supernatant, carrying out suction filtration on lower liquid to obtain filtrate, and carrying out rotary evaporation on the supernatant and the filtrate to remove water to obtain a traditional Chinese medicine extract;
wherein the effective substances of cortex et radix Polygalae, semen Ziziphi Spinosae, eucommiae cortex male flower and calendula flos after water extraction are cortex et radix Polygalae extract, semen Ziziphi Spinosae extract, eucommiae cortex male flower extract and calendula flos extract respectively.
The traditional Chinese medicine extract adopted in the embodiment of the invention adopts water which is 5 times of the weight of the traditional Chinese medicine raw material powder for distillation purification in the extraction process.
The method for screening the refreshing drugs based on the behavioral indexes and by using the refreshing marker genes comprises the following steps:
(1) determining the maximum safe concentration of the caffeine to be screened and the caffeine to be contrast agent on the juvenile zebra fish;
the method comprises the steps of randomly dividing young zebra fish of 3 days into a blank group, a control drug group (caffeine) and a plurality of experimental groups (polygala tenuifolia extract, spina date seed extract, eucommia male flower extract, calendula officinalis extract and asperuloside), wherein the quantity of each group of fish is the same, placing the young zebra fish in 24-hole cell culture plates, placing 8-12 fish, preferably 10 fish, in each hole, putting drugs to be screened with different gradient concentrations into the 24-hole cell culture plates, soaking for 3 days, repeating each concentration test for 3 times, recording the death rate of the zebra fish, and determining the maximum safe concentrations of the drugs of the experimental groups and the drugs of the control group through SPSS software calculation.
The maximum safe concentrations of polygala tenuifolia extract, spine date seed extract, eucommia male flower extract, calendula extract, asperuloside and caffeine are shown in table 1.
TABLE 1 maximum safe concentration table for each substance
Drug name Maximum safe concentration meter
Polygala tenuifolia extract 209μg/mL
Semen Ziziphi Spinosae extract 30μg/mL
Eucommia ulmoides male flower extract 206μg/mL
Calendula extract 26μg/mL
Radix et rhizoma Rhei Alternantherae glycoside 10μg/mL
Caffeine and its preparation method 200μmol/L
(2) Marker gene for clear refreshing of transcriptome
Transferring the juvenile zebra fish with 5dpf into a 48-hole plate 24 in the evening, setting a blank group and a caffeine group in each hole, adding 1mL fresh fish culture water into the blank group, adding caffeine liquid medicine with the maximum safe concentration into the caffeine group, standing for 60 minutes, washing the zebra fish with the fish culture water for 2-3 times, transferring into a centrifuge tube, and respectively extracting total RNA of the two groups of zebra fish;
transcriptome study is carried out on the extracted total RNA of the two groups of zebra fish, significance analysis is carried out on the gene expression difference of the two groups of zebra fish, information analysis is carried out by using a statistical method, the gene expression difference of the two groups of zebra fish is compared, and specific genes related to conditions are found out. Analysis of transcriptome sequencing results shows that nr1d4a and nr1d4b are marker genes for refreshing and restoring consciousness, and the refreshing and restoring consciousness characterization is defined from the gene level;
(3) screening a refreshing drug based on the behavior of zebra fish under the light stimulus reaction:
experiment at 24 hours, transferring the juvenile zebra fish with 5dpf into a 48-well plate, wherein each well is filled with one fish, and dividing the juvenile zebra fish into a control drug group (caffeine group), a blank group and a plurality of experiment groups (polygala tenuifolia extract group, spine date seed extract group, eucommia male flower extract group, calendula extract group and phyllanthus emblica glycoside group), wherein the control drug group is filled with caffeine with the maximum safe concentration, the blank group is filled with 1mL of fresh fish-culturing water, and each experiment group is filled with 1mL of one drug to be screened with the maximum safe concentration; three parallel tests are set for each group;
putting the 48 pore plate into a camera bellows of a Zebra zebra fish behavior analyzer, starting to detect the illumination stimulus response of the zebra fish, recording data once every minute for 60 minutes, performing data processing by using Zeblab software, calculating the total swimming distance and swimming speed of the zebra fish, and testing the illumination stimulus response of different groups of zebra fish under the sleeping time;
as shown in the schematic diagram of the total distance travelled by the young zebra fish in the light stimulus response detection of each group in fig. 1, the polygala tenuifolia group, the eucommia male flower group and the calendula officinalis group in the drug screening group have certain light stimulus response in the sleeping time compared with the blank group, but the difference from the blank group has no statistical significance. The P value of the caffeine group is 0.002, the P value of the spina date seed extract group is 0.04, the P value of the asperuloside group is 0.008, the P values of the three groups are all smaller than 0.05, the statistical significance is achieved, the P values of the polygala tenuifolia extract group, the eucommia male flower extract group and the calendula officinalis extract group are all larger than 0.05, and the statistical significance is not achieved. Therefore, the wild jujube seed extract and the asperuloside can be successfully screened out to improve the total swimming distance of young zebra fish, so that the method has a refreshing effect, the asperuloside effect is optimal, and only the data of the asperuloside are processed in the next step.
As shown in the schematic diagrams of the results of the swimming speed per minute of the young zebra fish groups in the light stimulation reaction detection in fig. 2-4, compared with the blank group, the swimming speed of the zebra fish groups in the control group and the phyllanthus niruri group is increased, the reaction capacity is enhanced, and the phyllanthus niruri has the effect of refreshing and restoring consciousness.
(4) Based on Real-time PCR technology, detecting expression levels of refreshing marker genes nr1d4a and nr1d4b in each group, and screening out refreshing drugs:
designing primer sequences for the refreshing marker genes nr1d4a and nr1d4 b:
nr1d4a-RT-F：AGATATCGCATCAGGGTTCC，
nr1d4a-RT-R：CTCGTTCTTCACGCACATCT，
nr1d4b-RT-F：ATCAGGTCATGCTGCTGAAG，
nr1d4b-RT-R：GCGTTCCTTAGCATTGAACA，
collecting the blank group, washing the zebra fish subjected to behavioral records by the zebra fish of the control group and the phyllostachys praecox group for two to three times by using fish-farming water, transferring the zebra fish into a centrifuge tube, centrifuging at a high speed, and sucking the upper liquid to leave the tissue of the bottom zebra fish; extracting total RNA of different groups of zebra fish by using an RNA extraction kit, measuring the concentration of the RNA by using an ultra-micro spectrophotometer, reversing the RNA into cDNA by using a reverse transcription kit, referring to a real-time fluorescent quantitative PCR specification, adding primers to be detected into a qRT-PCR instrument for amplification by using the cDNA after the reverse rotation of each group as a template, setting three parallel tests in each group, and detecting the expression level of each group of refreshing marker genes nr1d4a and nr1d4b;
as shown in fig. 5 and 6, compared with the blank group, the gene expression of nr1d4a and nr1d4b of the asperuloside group is down-regulated, and the expression direction is consistent with that of the control group, so that the asperuloside can be used as a refreshing medicine.

Claims (4)

1. The application of the refreshing marker gene in screening the refreshing drugs is characterized in that: the refreshing marker genes are nr1d4a and nr1d4b; firstly, primarily screening zebra fish through behaviours, then designing upstream and downstream primers for refreshing marker genes nr1d4a and nr1d4b, carrying out RT-qPCR verification, and determining whether the medicine to be screened has refreshing effect according to the expression trend of the refreshing marker genes nr1d4a and nr1d4 b.
2. A method for screening a refreshing drug based on behavioral indexes and the refreshing marker gene according to claim 1, which is characterized in that: the method comprises the following steps:
(1) determining the maximum safe concentration of the caffeine to be screened and the caffeine to be contrast agent on the juvenile zebra fish;
(2) screening a refreshing drug based on the behavior of zebra fish under the light stimulus reaction:
when the experiment is carried out at night 24, transferring the juvenile zebra fish with 5dpf into a 48-hole plate, wherein each hole is used for one fish, dividing the juvenile zebra fish into a control drug group, a blank group and a plurality of experiment groups, wherein the control drug group is added with caffeine with the maximum safe concentration, the blank group is added with 1mL of fresh fish raising water, and each experiment group is added with 1mL of drug to be screened with the maximum safe concentration; three parallel tests are set for each group;
putting the 48 pore plate into a camera bellows of a Zebra zebra fish behavior analyzer, starting to detect the illumination stimulus response of the zebra fish, recording data once every minute for 60 minutes, performing data processing by using Zeblab software, calculating the total swimming distance and swimming speed of the zebra fish, and testing the illumination stimulus response of different groups of zebra fish under the sleeping time;
(3) based on Real-time PCR technology, detecting expression levels of refreshing marker genes nr1d4a and nr1d4b in each group, and screening out refreshing drugs:
designing primer sequences for the refreshing marker genes nr1d4a and nr1d4 b:
nr1d4a-RT-F：AGATATCGCATCAGGGTTCC，
nr1d4a-RT-R：CTCGTTCTTCACGCACATCT，
nr1d4b-RT-F：ATCAGGTCATGCTGCTGAAG，
nr1d4b-RT-R：GCGTTCCTTAGCATTGAACA，
collecting zebra fish subjected to behavioral records of different treatment groups, washing with fish-farming water for two to three times, transferring into a centrifuge tube, centrifuging at high speed, and sucking the upper liquid to leave the tissue of the bottom zebra fish; extracting total RNA of different experimental groups of zebra fish by using an RNA extraction kit, measuring the concentration of RNA by using an ultra-micro spectrophotometer, reversing the RNA into cDNA by using a reverse transcription kit, referring to a real-time fluorescent quantitative PCR specification, adding primers to be detected into a qRT-PCR instrument for amplification by using the cDNA after the reverse rotation of each group as a template, setting three parallel experiments in each group, and detecting the expression level of each group of refreshing marker genes nr1d4a and nr1d4b;
(4) judgment criteria for a refreshing drug:
under the sleeping time, compared with a blank group, the total swimming distance of the refreshing medicine is increased, the swimming speed is increased, and the reaction capability is enhanced; the expression changes of the refreshing marker gene of the refreshing medicine and the caffeine of the contrast medicine are the same, and are all down-regulation changes.
3. The method for screening a refreshing drug based on behavioral indexes and using a refreshing marker gene according to claim 2, wherein: (1) the method for determining the maximum safe concentration of the medicine to be screened and the caffeine serving as the contrast agent on the juvenile zebra fish comprises the following specific steps:
the method comprises the steps of randomly dividing 3-day-old juvenile zebra fish into a blank group, a control drug group and a plurality of experimental groups, wherein the number of the juvenile zebra fish in each group is the same, placing the juvenile zebra fish in 24-hole cell culture plates, putting 8-12 fish in each hole, soaking the 24-hole cell culture plates with drugs to be screened with different gradient concentrations for 3 days, repeating each concentration test for 3 times, recording the death rate of the zebra fish, and determining the maximum safe concentrations of the drugs in the experimental groups and the drugs in the control group through SPSS software calculation.
4. The method for screening a refreshing drug based on behavioral indexes and using a refreshing marker gene according to claim 2, wherein: the medicine to be screened is polygala tenuifolia extract, wild jujube seed extract, eucommia male flower extract, calendula extract and asperuloside.
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