Mouse drunkenness degree accurate detection mathematical analysis model construction method and application

Abstract

The invention provides a mouse drunkenness degree accurate detection mathematical analysis model construction method and application. Specifically, the invention provides a method for constructing a drunkenness degree accurate detection mathematical analysis model, and the method comprises the following steps: carrying out wine gavage and ethanol gavage on a mouse, carrying out an open field experiment and a rotating bar experiment, and detecting the concentration of ethanol in mouse blood and the like, so as to obtain each index value, thereby obtaining the drunkenness degree of the mouse. The method disclosed by the invention is simple and convenient to operate and high in credibility and sensitivity, and has important significance in actual production and application of the wine industry.

Description

Technical Field

[0001] The present invention relates to the field of medical diagnosis, and in particular to a method for constructing a mathematical analysis model for accurately detecting the drunkenness degree of mice and its application. Background Art

[0002] According to the latest World Health Organization report on global drinking and health, as the world economy develops, the average per capita alcohol consumption of adults worldwide reached 6.4 liters of pure alcohol by 2016. And according to the model prediction, the per capita alcohol consumption level worldwide will continue to grow, especially in developing countries. With such a high level of alcohol consumption, the health and social security problems caused by drinking are particularly prominent around the world, which has placed a huge burden on global public health.

[0003] Ethanol is a highly polar, fat-soluble substance that can quickly enter the blood circulation in a very short time. It is mainly absorbed in trace amounts in the oral mucosa, a small part in the stomach (about 10%-20%), and most of it in the small intestine. More than 90% of the ethanol in the human body is metabolized in the liver. Under the action of liver alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH), ethanol is decomposed into acetic acid. When ethanol enters the human body, as the blood concentration of ethanol gradually increases, the body begins to relax slowly and enters an excited state; and when its level further increases, with a large amount of ethanol crossing the blood-brain barrier and entering the brain, the body presents symptoms of alcohol intoxication such as ataxia and coma. This symptom will last for several hours until the alcohol in the body is completely metabolized and cleared. When in a drunken state, the human body's cognitive, motor and coordination abilities are greatly reduced, which brings a great burden to human health and social security.

[0004] At present, with the rapid development of the national economy, people are paying more and more attention to health, especially food health. Alcohol is a food. After satisfying the "color" and "aroma" before drinking, and the "taste" and "style" when drinking, people's pursuit of alcohol quality has gradually turned to the comfort of the body after drinking. The comfort of good alcohol after drinking usually means that there is no dry mouth, no head, and no easy drunkenness after drinking, and "not easy to get drunk" occupies an important position among them.

[0005] Mice are the most commonly used animal model in animal experiments. They are easy to raise, have a high reproduction rate, are genetically pure, have small differences between individuals, and are easy to observe the results of parallel experiments. Their metabolic types and physiological and pathological characteristics are similar to those of humans. Most importantly, the ethanol metabolism pattern of mice is very similar to that of humans, making them an ideal animal model for detecting drunkenness.

[0006] Therefore, it is of great significance to establish an effective method and its application to accurately and efficiently evaluate the degree of intoxication of alcohol using mice as model animals. Summary of the invention

[0007] The invention provides an effective method for accurately and efficiently evaluating the drunkenness degree of alcoholic beverages and its application.

[0008] In a first aspect of the present invention, a method for constructing a mathematical analysis model for accurately detecting the degree of drunkenness is provided, comprising the steps of:

[0009] (S1) Provide two groups of mice with the same origin and baseline conditions and raise them under the same environmental conditions;

[0010] (S2) administering alcohol orally to one group of mice, and administering ethanol orally to another group of mice;

[0011] (S3) performing an open field test on the mice in the alcohol gavage group and the mice in the ethanol gavage group, respectively, and recording the probability P1 that the distance moved by the mice in the alcohol gavage group in the open field is smaller than the distance moved by the mice in the ethanol gavage group in the open field;

[0012] (S4) performing a rotating rod test on the mice in the alcohol gavage group and the mice in the ethanol gavage group, respectively, and recording the probability P2 that the rotating rod time of the mice in the alcohol gavage group is shorter than that of the mice in the ethanol gavage group;

[0013] (S5) respectively testing the blood ethanol concentration of the mice in the wine gavage group and the ethanol gavage group, and recording the probability P3 that the blood ethanol concentration of the mice in the wine gavage group is greater than the blood ethanol concentration of the mice in the ethanol gavage group; and

[0014] (S8) Based on each recorded probability, different weights W are assigned to each recorded probability according to the correlation between each detection index and the degree of drunkenness. i ; Establish a mathematical analysis model for accurate detection of drunkenness degree, so as to obtain the drunkenness index (Drunk Index) P:

[0015]

[0016] Among them, step (S3), step (S4), and step (S5) can be performed in any order.

[0017] In another preferred embodiment, the method may further optionally perform the following steps:

[0018] (S6) respectively detecting the blood acetaldehyde concentration of the mice in the alcohol gavage group and the mice in the ethanol gavage group, and recording the probability P4 that the blood acetaldehyde concentration of the mice in the alcohol gavage group is greater than the blood acetaldehyde concentration of the mice in the ethanol gavage group;

[0019] (S7) Detecting cerebellar gamma-aminobutyric acid (GABA) in the mice in the alcohol gavage group and the mice in the ethanol gavage group, respectively, and recording the probability P5 that the cerebellar GABA level in the mice in the alcohol gavage group is greater than the cerebellar GABA level in the mice in the ethanol gavage group.

[0020] In another preferred embodiment, the experiment and detection of the method are carried out after mice are gavaged with ethanol or alcohol for a period of time.

[0021] In another preferred embodiment, the experiment and detection of the method are carried out 1-1.5 hours after the mice are gavaged with ethanol or alcohol.

[0022] In another preferred embodiment, the experiment and detection of the method are carried out 1 hour after the mice are gavaged with ethanol or alcohol.

[0023] In another preferred embodiment, the experiment and detection of the method are carried out 1.5 hours after the mice are gavaged with ethanol or alcohol.

[0024] In another preferred embodiment, the mice administered with wine or ethanol are in a non-comatose drunken state.

[0025] In another preferred embodiment, in the intragastric administration of wine and ethanol, the ethanol content of the wine and ethanol is the same.

[0026] In a second aspect of the present invention, there is provided an assessment system for detecting a degree of drunkenness, the system comprising:

[0027] (M1) Input module: The input module is configured to input open field test data, rotating rod test data and blood alcohol concentration data of the subject to be tested;

[0028] (M2) Evaluation module: The evaluation module is configured to evaluate the open field test data, the rotating rod test data, and the blood ethanol concentration data based on the mathematical analysis model for accurate detection of the degree of drunkenness, so as to obtain an evaluation result of the degree of drunkenness of the subject to be tested; wherein the mathematical analysis model for accurate detection of the degree of drunkenness is a mathematical analysis model for accurate detection of the degree of drunkenness constructed by the construction method as described in the first aspect of the present invention, and the model formula is:

[0029] and

[0030] (M3) Output module: The output module is configured to output the evaluation result.

[0031] In another preferred example, the system further includes: (M4) a control module: the control module is configured to control the operation of each module.

[0032] In another preferred example, the greater the evaluation result value of the evaluation, the higher the degree of drunkenness; conversely, the smaller the evaluation result value of the evaluation, the lower the degree of drunkenness.

[0033] In another preferred embodiment, the test subjects include: ethanol-administered mice and alcohol-administered mice.

[0034] In another preferred embodiment, the alcoholic beverage includes types selected from the following group: white wine, yellow wine, fruit wine, beer, rice wine, medicinal wine, or a combination thereof.

[0035] In another preferred example, the data input by the input module further includes data selected from the following group: blood acetaldehyde concentration, cerebellar GABA level, or a combination thereof.

[0036] In another preferred embodiment, the subject to be tested is in a non-comatose drunken state.

[0037] In a third aspect of the present invention, there is provided an evaluation method, comprising the steps of:

[0038] (a) providing the open field test data, rotarod test data and blood ethanol concentration data of the test subject;

[0039] (b) inputting the open field test data, the rotating rod test data and the blood ethanol concentration data into the mathematical analysis model for accurate detection of drunkenness degree constructed by the construction method described in the first aspect of the present invention, wherein the model formula is:

[0040]

[0041] (c) using the mathematical analysis model for accurate detection of drunkenness degree to evaluate the open field test data, the rotating rod test data and the blood ethanol concentration data, thereby obtaining the drunkenness degree of the subject to be tested; wherein, the larger the evaluation result value of the evaluation is, the higher the drunkenness degree is; conversely, the smaller the evaluation result value is, the lower the drunkenness degree is; and

[0042] (d) Output the evaluation results.

[0043] In another preferred embodiment, the test subjects include: ethanol-administered mice and alcohol-administered mice.

[0044] In another preferred embodiment, the subject to be tested is in a non-comatose drunken state.

[0045] In another preferred embodiment, the evaluation method is non-diagnostic and non-therapeutic.

[0046] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The evaluation indicators of the accurate detection method of mouse drunkenness are shown: (A) 1 hour after the mice were gavaged with ethanol, behavioral tests were performed: open field and rotarod tests, cerebellar tissue GABA measurement, blood ethanol and acetaldehyde concentration measurement, liver ADH and ALDH activity measurement.

[0048] Figure 2 The figure shows that 1 h after intragastric administration of ethanol is the time point for detecting the drunkenness of mice: 0.5, 1, 1.5, and 2 h after intragastric administration of 2.5 g / kg ethanol, the mice's moving distance in the open field (A, B), the time on the rotarod (C), the blood ethanol (D) and acetaldehyde (E) concentrations, the cerebellar GABA content (F), and the liver ADH (G) and ALDH (H) activities were measured. The mice in the normal saline gavage group were taken as 0 h, that is, ethanol had no effect.

[0049] Figure 3 The mathematical analysis model for accurately detecting the drunkenness degree of mice is shown: (A) Taking the ethanol group as the control, the probability that each indicator in the test group is greater than or less than the corresponding indicator is calculated. According to the weight, the weighted sum of each indicator is performed to obtain the final drunkenness index (Drunk Index), and it is stipulated that the larger the index, the higher the degree of drunkenness.

[0050] Figure 4 The figure shows the application of the model to detect the drunkenness of alcohol samples: 1 hour after mice were gavaged with pure alcohol, saline and a certain liquor sample (same dose), the moving distance in the open field (A, B), the time on the rod (C), the blood ethanol (D) concentration and acetaldehyde (E) concentration, the cerebellar GABA level (F) and the drunkenness index (G) of the mice were tested; based on the above indicators, the drunkenness index (Drunk Index) of ethanol, saline and a certain liquor sample was calculated using a mathematical analysis model. DETAILED DESCRIPTION

[0051] After extensive and in-depth research, the inventor unexpectedly discovered a method for evaluating the degree of drunkenness. Specifically, the inventor established a mathematical analysis model for accurately predicting the degree of drunkenness through three evaluation indicators (behavior, neurotransmitters, and biochemistry) after mice were gavaged with white wine. The behavior included test results such as the Open Field Test (OFT) and the Rotarod Test, the neurotransmitters included the cerebellar gamma-aminobutyric acid (GABA) level, and the biochemical indicators included blood ethanol and blood acetaldehyde concentrations. The analysis model of the present invention can better evaluate the degree of drunkenness. On this basis, the present invention was completed.

[0052] Method for establishing mathematical analysis model for accurate detection of drunkenness degree

[0053] The present invention provides a method for constructing a mathematical analysis model for accurately detecting the degree of drunkenness, comprising the following steps:

[0054] (S1) Provide two groups of mice with the same origin and baseline conditions and raise them under the same environmental conditions;

[0055] (S2) administering alcohol orally to one group of mice, and administering ethanol orally to another group of mice;

[0056] (S3) performing an open field test on the mice in the alcohol gavage group and the mice in the ethanol gavage group, respectively, and recording the probability P1 that the distance moved by the mice in the alcohol gavage group in the open field is smaller than the distance moved by the mice in the ethanol gavage group in the open field;

[0057] (S4) performing a rotating rod test on the mice in the alcohol gavage group and the mice in the ethanol gavage group, respectively, and recording the probability P2 that the rotating rod time of the mice in the alcohol gavage group is shorter than that of the mice in the ethanol gavage group;

[0058] (S5) respectively testing the blood ethanol concentration of the mice in the wine gavage group and the ethanol gavage group, and recording the probability P3 that the blood ethanol concentration of the mice in the wine gavage group is greater than the blood ethanol concentration of the mice in the ethanol gavage group; and

[0059] (S8) Based on each recorded probability, different weights W are assigned to each recorded probability according to the correlation between each detection index and the degree of drunkenness. i ; Establish a mathematical analysis model for accurate detection of drunkenness degree, so as to obtain the drunkenness index (Drunk Index) P:

[0060]

[0061] Among them, step (S3), step (S4), and step (S5) can be performed in any order.

[0062] In another preferred embodiment, the method may further optionally perform the following steps:

[0063] (S6) respectively detecting the blood acetaldehyde concentration of the mice in the alcohol gavage group and the mice in the ethanol gavage group, and recording the probability P4 that the blood acetaldehyde concentration of the mice in the alcohol gavage group is greater than the blood acetaldehyde concentration of the mice in the ethanol gavage group;

[0064] (S7) Detecting cerebellar gamma-aminobutyric acid (GABA) in the mice in the alcohol gavage group and the mice in the ethanol gavage group, respectively, and recording the probability P5 that the cerebellar GABA level in the mice in the alcohol gavage group is greater than the cerebellar GABA level in the mice in the ethanol gavage group.

[0065] In another preferred embodiment, the experiment and detection of the method are carried out after mice are gavaged with ethanol or alcohol for a period of time.

[0066] In another preferred embodiment, in the intragastric administration of wine and ethanol, the ethanol content of the wine and ethanol is the same.

[0067] In another preferred embodiment, the mice administered with wine or ethanol are in a non-comatose drunken state.

[0068] Intoxication level assessment system (equipment)

[0069] The present invention provides an evaluation system (device) for detecting the degree of drunkenness, the system comprising:

[0070] (M1) Input module: The input module is configured to input open field test data, rotating rod test data and blood alcohol concentration data of the subject to be tested;

[0071] (M2) Evaluation module: The evaluation module is configured to evaluate the open field test data, the rotating rod test data, and the blood ethanol concentration data based on the mathematical analysis model for accurate detection of the degree of drunkenness, so as to obtain an evaluation result of the degree of drunkenness of the subject to be tested; wherein the mathematical analysis model for accurate detection of the degree of drunkenness is a mathematical analysis model for accurate detection of the degree of drunkenness constructed by the construction method described in the first aspect of the present invention; the model formula is:

[0072] and

[0073] (M3) Output module: The output module is configured to output the evaluation result.

[0074] In another preferred example, the system further includes: (M4) a control module: the control module is configured to control the operation of each module.

[0075] In another preferred example, the greater the evaluation result value of the evaluation, the higher the degree of drunkenness; conversely, the smaller the evaluation result value of the evaluation, the lower the degree of drunkenness.

[0076] In another preferred embodiment, the test subjects include: ethanol-administered mice and alcohol-administered mice.

[0077] In another preferred example, the data input by the input module further includes data selected from the following group: blood acetaldehyde concentration, cerebellar GABA level, or a combination thereof.

[0078] In another preferred embodiment, the subject to be tested is in a non-comatose drunken state.

[0079] The main advantages of the present invention are:

[0080] 1. The present invention establishes for the first time a drunkenness degree evaluation model in a non-comatose drunken state, which is highly similar to the common alcohol consumption and symptoms after drinking by the human body and is more practical.

[0081] 2 The mathematical analysis model for drunkenness detection of the present invention can comprehensively, truly and objectively reflect the overall drunkenness state of mice, and can well distinguish the subtle differences between samples in each group. The model has high credibility and sensitivity.

[0082] 3. The mathematical analysis model for accurately detecting the degree of drunkenness of mice of the present invention is simple and convenient to operate and is less susceptible to interference.

[0083] 4. The present invention has a wide range of application scenarios. In addition to being used for the evaluation of finished wine samples, it can also be used to evaluate wine samples in the production process, such as base wine, blended wine, etc.; it can also be used to study the influence of flavor substances on the degree of drunkenness, so as to improve the quality of wine; it can also evaluate the efficacy of alcohol detoxification drugs, detect the drunkenness degree of mice after drug administration, etc.

[0084] 5. The mathematical analysis model for accurately detecting the degree of drunkenness of mice of the present invention has low actual use cost and meets the needs of large-scale use by enterprises.

[0085] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0086] Materials and methods

[0087] Eight-week-old male C57Bl / 6 mice were purchased from Shanghai Slack Company. They were adapted to the standard SPF environment for one week before the formal experiment and fed with standard food during this period.

[0088] Example 1 Mouse drunkenness experiment

[0089] 1.1 Alcohol gavage in mice

[0090] The ethanol gavage dose for mice was 2.5 g / kg body weight, the ethanol concentration was 31.5% (v / v), and the gavage volume was 10 times the body weight of the mice. A disposable sterile syringe combined with a sterilized No. 8 gavage needle was used for gavage to ensure the gavage effect, no secondary damage to the mice, and no choking of ethanol solution.

[0091] After intragastric administration, the mice were placed in cages to rest and wait for testing. The ambient temperature was controlled at around 25°C to ensure that the mice did not suffer from hypothermia.

[0092] The alcohol solution for intragastric administration of mice was prepared with chromatographic grade anhydrous ethanol (Aladdin, E111993-500ml). The alcohol concentration of the intragastric alcohol sample was measured with an alcohol meter and then diluted to 31.5% (v / v).

[0093] After the experiment, the mice were killed by an overdose of isoflurane, blood was drawn from the apex of the heart, and liver and cerebellum tissues were collected, weighed, and quickly frozen in liquid nitrogen for testing.

[0094] 1.2 Behavioral experiments

[0095] When mice are drunk, their overall activities are inhibited, and their movements, coordination, and muscle strength are significantly reduced. In order to objectively and accurately detect the overall state of mice after drunkenness, two behavioral tests, the open field test and the rotarod test, were used to determine the state.

[0096] The mouse open field test is to place drunken mice in an open field under strong light. Since mice are naturally tactile, they will instinctively move to the edge of the open field. Through the mouse open field test, the mouse's reaction to external stimuli, general activity and exploration ability can be tested. The mouse rotating rod test is to place the mouse on a rotating rod with a certain speed to test the mouse's movement, coordination and muscle strength.

[0097] Combining the two behavioral tests can more comprehensively reflect the overall drunkenness level of mice in a non-comatose drunken state.

[0098] Specifically, before the experiment, mice were intragastrically administered with 2.5 g / kg ethanol, with an ethanol concentration of 31.5% (v / v) and an intragastrically administered volume (μL) of 10 times the weight (g) of the mice.

[0099] After gavage, the mice were returned to their cages to rest and then transferred to the behavioral testing room 30 minutes before the test time.

[0100] Each mouse was subjected to only one behavioral test after oral gavage.

[0101] 1.2.1 Open field test for mice

[0102] The open field video analysis system (SA215, Jiangsu Seons) was used to perform open field tests on mice and analyze video data. Before the experiment, the mice were placed in the open field equipment room to adapt to the environment for half an hour. The total moving distance of the mice in the open field (50 cm × 50 cm) for 20 minutes was recorded and analyzed by video. Four mice could be tested and analyzed at the same time.

[0103] 1.2.2 Mouse Rotarod Test

[0104] The mouse rotarod fatigue test was performed using a mouse rotarod fatigue instrument (Jiangsu Seons, SA102). The rotarod setting parameters were an initial speed of 4 r / min, accelerated to 40 r / min within 5 min, and an acceleration of 7.2 r / min. 2 The mice need to be trained on the rotarod continuously for three days before the formal test, three times a day, with an interval of more than 30 minutes between each training. The mice that have an average time on the rod of more than 200 seconds on the last day can be used for the formal test experiment. Before the experiment, the mice need to adapt to the environment for more than 30 minutes.

[0105] Start the rotating rod, place the mice on the rotating rod one by one at the initial speed, and then start to accelerate. The instrument automatically records the time when the mice fall.

[0106] 1.3 Determination of GABA levels in mouse cerebellum

[0107] 1.3.1 GABA extraction from cerebellum

[0108] About 50 mg of mouse cerebellum tissue was added with 300 μL ultrapure water and then ground into a homogenate. Then 700 μL methanol and acetonitrile (chromatographic grade) solution (1:1) was added, and the mixture was shaken for 30 seconds and then ultrasonicated in an ice bath. Then, it was placed in a -20°C refrigerator for 1 hour, centrifuged at 12000 rpm for 15 minutes at 4°C, and the supernatant was transferred to a new 1.5 mL centrifuge tube. All liquid in the supernatant was evaporated using a vacuum freeze dryer, and 300 μL acetonitrile aqueous solution (1:9) was added to dissolve it, and then it was shaken for 30 seconds and then ultrasonicated in an ice bath. Then, it was centrifuged again at 12000 rpm for 15 minutes at 4°C, the supernatant was collected, and it was diluted 50 times with ultrapure water and then tested on the machine.

[0109] 1.3.2 GABA assay

[0110] GABA was determined by liquid chromatography-mass spectrometry, and the instrument information is as follows: liquid chromatography system: Agilent 1200LC system (binary pump, automatic sampler with temperature control device, column oven, online degasser), mass spectrometry system: SCIEX 4000QTRAPLC / MS / MS systems.

[0111] Chromatographic column: Agilent Zorbox Eclipse Plus C18 4.6*150mm, 5um;

[0112] Mobile phase A: water + 0.1% formic acid + 0.01% heptafluorobutyric acid;

[0113] Mobile phase B: methanol + 0.1% formic acid + 0.01% heptafluorobutyric acid, flow rate: 0.8 ml / min;

[0114] Column temperature: 50°C;

[0115] Gradient elution: 0-3 min: 2% B, 3-5 min: 20% B, 5-5.5 min: 90% B, 5.5-7.5 min: 90% B, 7.5-8 min: 2% B, 8-10 min: 2% B;

[0116] Mass spectrometry parameters: ion source: point spray ion source (EIS), positive ion multiple reflection detection (MRM) mode scanning, capillary voltage (IS) was 5500 V, ion source temperature (TEM) was 600 °C, curtain gas (CUR) was 15 psi, collision gas (CAD) was 8 psi, nebulizer gas (GS1) was 60 psi, auxiliary gas (GS2) was 50 psi, declustering voltage (DP) was 43 V, inlet voltage was 10 V, collision cell exit voltage (CXP) was 7 V, and collision voltage (CE) was 15 V;

[0117] Quantitative ion pair: m / z 104.1→87.1, retention time: 3.5min.

[0118] 1.4 Determination of biochemical indicators in mice

[0119] 1.4.1 Determination of blood ethanol and acetaldehyde concentrations

[0120] Headspace gas chromatography (Agilent 7890B GC with FID, Agilent 7697A headspace sampler) was used to determine the ethanol and acetaldehyde content in the blood. 0.4 mL of blood was taken, 0.1 mL of tert-butyl alcohol solution was added as an internal standard, and 0.5 mL of ultrapure water was added to a 20 mL headspace bottle, which was sealed with a crimp clamp.

[0121] The chromatographic column was Agilent J&W DB-BAC1 UI, 30m×0.32mm, 1.8m, column temperature 40℃, lasting 5min. The carrier gas was nitrogen, constant flow, 6mL / min. The injection port temperature was 210℃, split mode, split ratio 1:1. FID conditions: temperature was 250℃, hydrogen flow rate was 30mL / min, air flow rate was 350mL / min, and tail blowing was 35mL / min. HS conditions: column oven 70℃ (equilibration 15min), quantitative loop 80℃, transfer line 90℃. The retention times of acetaldehyde, acetaldehyde, and tert-butyl alcohol were 1.2, 1.5, and 2.0min, respectively.

[0122] 1.4.2 Determination of liver ADH and ALDH activity

[0123] The enzyme activities were determined using ADH (Nanjing Jiancheng, A083-2-1) and ALDH (Solarbio, A083-2-1) activity assay kits, respectively, and the relevant steps were based on their instructions.

[0124] 1.5 Experimental Results

[0125] 1.5.1 1 hour after ethanol gavage is the time point for detecting the degree of intoxication of mice

[0126] When an individual is in a drunken state, his or her cognitive, motor and coordination functions may become impaired or the individual may become completely unconscious.

[0127] If the degree of intoxication is mild, the effect of ethanol on mice cannot be fully reflected; if the mice are completely intoxicated and unconscious, the differences between the alcohol samples and the effect of ethanol on the motor ability of mice may not be reflected. Therefore, finding a suitable degree of intoxication for mice is crucial for the evaluation of intoxication.

[0128] Based on this, mice were given 2.5 g / kg ethanol by gavage, and the changes of various indicators at each time point were monitored for a continuous period of 2 hours. The results are as follows Figure 2 In behavioral tests, the movement and coordination abilities of mice first gradually decreased and then recovered with the passage of time after ethanol gavage ( Figure 2 A, B and C), and the lowest at 1 hour. The concentrations of ethanol and acetaldehyde in the blood and the level of GABA in the cerebellum of mice showed a trend opposite to that of motor ability, increasing first and then decreasing ( Figure 2 D, E and F), with the highest level at 1 h.

[0129] Mouse liver ADH and ALDH showed no significant changes in activity in the presence or absence of metabolic substrates, so they may not be suitable for inclusion in the evaluation of mouse intoxication.

[0130] Based on the changes in the above indicators, it was found that the movement and coordination abilities of mice were lowest 1 hour after ethanol gavage, and the blood ethanol, acetaldehyde and cerebellar GABA levels reached peak values. Therefore, this time point is more suitable as a time point for detecting the degree of drunkenness.

[0131] Example 2 Mathematical analysis model for detecting the degree of drunkenness in mice

[0132] 2.1 Construction of mathematical analysis model for drunkenness detection

[0133] In order to better reflect and describe the degree of drunkenness, based on the above evaluation indicators, this patent establishes a drunkenness index (Drunk Index) mathematical model that can integrate the characteristics of the above indicators. Specifically, the pure ethanol group is used as the control group, and the various wine sample groups are used as the test groups. The probability (P1-P5) that the control group of the above indicators is greater than or less than the test group is calculated, and then these indicators are assigned corresponding probabilities with certain weights (W1-W5). Finally, the probability of each indicator is weighted and summed to obtain the final probability, which is defined as Drunk Index (0, 1). It is stipulated that the larger the probability, the greater the degree of drunkenness.

[0134] The specific analysis model is as follows Figure 3as shown

[0135] Taking the pure ethanol group as the control, compare the various indicators of the test group with those of the control group, calculate the probabilities of each indicator, and then perform weighted summation after assigning certain weights to different probabilities.

[0136] Specifically, define the total moving distance in the open field of the mice as O, and the control group and the test group are O0 and O1 respectively, where o 01 -o 0n and o 11 -o 1n represent the total moving distances of different mice in the open field in the control group and the test group respectively. Similarly, define the time on the rod in the rotarod test of the mice, blood ethanol concentration, blood acetaldehyde concentration, and cerebellar GABA level as R, B, C, and G respectively.

[0137] Next, compare the above two groups of each indicator, define p1 = P(O0>O1), p2 = P(R0>R1), p3 = P(B0<B1), p4 = P(C0>C1), p5 = P(G0<G1), and at the same time define the weights of p1 - p5 as w1 = w2 = w3 = 0.25, w4 = w5 = 0.125, the specific ratio of w1 - w5 is 2:2:2:1:1, and the sum of w1 - w5 is 1.

[0138] Finally, perform weighted summation on p1 - p5 according to the weights w1 - w5, that is Define p as the Drunk Index, and its value is between 0 and 1. The p value of the control group is about 0.5, and the smaller the p value of the test group, the lower its degree of drunkenness.

[0139] The above calculation process can be quickly implemented through R language.

[0140] 2.2 Detection of mouse drunkenness degree and application of mathematical analysis model

[0141] 2.2.1 Experimental method

[0142] Apply the model of this patent using physiological saline (positive control) and white liquor samples with the same dose of ethanol to verify the sensitivity and reliability of the model.

[0143] Taking the pure alcohol group as the control, gavage the above three groups of samples to the mice, and measure the relevant indicators 1 h after gavage.

[0144] 2.2.2 Experimental results

[0145] The results are as Figure 4In terms of the motor ability of mice, their motor ability was significantly reduced after being gavaged with alcohol; however, the motor phenotype of the alcohol-like mice gavaged with the same dose of ethanol was better than that of the ethanol control group, and their moving distance in the open field and the time they stayed on the rotating rod were significantly higher than those of the ethanol control group ( Figure 4 A, B and C). Similarly, the blood ethanol concentration and cerebellar GABA level of mice in the liquor-like group were significantly lower than those in the ethanol control group ( Figure 4 D and F), indicating that they may have a faster rate of ethanol metabolism, while the level of acetaldehyde in the blood did not change significantly ( Figure 4 E).

[0146] After determining the model detection index of this patent, the mathematical analysis model is used to quickly calculate the probability value (P1-P5) corresponding to each group of indicators and the final drunkenness index: Drunk Index ( Figure 4 G), as shown in Table 1.

[0147] Table 1

[0148] EtOH Normal saline Liquor <![CDATA[p1]]> 0.45 0.01 0.12 <![CDATA[p2]]> 0.43 0 0 <![CDATA[p3]]> 0.46 0 0.09 <![CDATA[p4]]> 0.42 0 0.63 <![CDATA[p5]]> 0.39 0 0 Intoxication Index 0.44 0.00 0.13

[0149] The Drunk Index of the saline group was close to 0, indicating that the drunkenness detection and mathematical analysis model based on this patent can well distinguish whether the mice have drunk alcohol, reflecting that the model of this patent has high reliability; and the Drunk Index of a certain liquor sample was 0.13, which was lower than 0.44 of the ethanol group, indicating that the mathematical analysis model of this patent can better integrate and quantify the data characteristics of each detection index, reflecting the drunkenness degree of the liquor sample.

[0150] 2.2.3 Comparative experiments and results

[0151] Given that the weights of the indicators are equal and their sum is 1, if only the open field test P1, only the rotarod test P2, only the two behavioral tests P1 and P2, or only two behavioral tests combined with blood biochemical indicators P1-P4 are used, the final calculated Drunk Index is 0.12, 0, 0.06, and 0.21, respectively.

[0152] The comparative examples given above cannot fully reflect the degree of intoxication of mice and are too one-sided. After large-scale application, they cannot effectively distinguish the differences between samples and treatments, which will lead to large errors in actual application.

[0153] In summary, the accurate detection method and mathematical analysis model of drunkenness degree described in this patent have high reliability and sensitivity, and can be used for accurate detection of drunkenness degree in the actual wine industry.

[0154] discuss

[0155] After alcohol intake, the body's reaction gradually changes from initial excitement, ataxia, sedation to the final coma and drunken state as the blood ethanol concentration increases.

[0156] The existing reports on the evaluation of mouse drunkenness have all focused on the comatose drunken state of mice after oral administration of excessive ethanol (>3.5g / kg), and the evaluation indicators used are the time when the righting reflex (an animal instinctive reflex) disappears or recovers during coma and drunkenness, or the standing time, etc. This results in the effect of various alcohol samples or drug treatments being partially masked under the influence of excessive ethanol, and the sensitivity of the model itself will decrease.

[0157] Secondly, in daily drinking, people emphasize rational drinking and do not encourage excessive drinking. What happens more often is drunkenness in a non-comatose state.

[0158] Therefore, a mouse non-comatose drunkenness evaluation and analysis model is urgently needed to fill the gap and accurately detect the drunkenness of alcohol samples or drug treatments. According to the law of ethanol metabolism, the drunkenness of the non-comatose state can also represent the drunkenness of the coma state after the ethanol dose is increased.

[0159] The present invention uses ethanol drunkenness-related indicators and finds that mice have the most severe drunkenness 1 hour after intragastric administration of 2.5g / kg alcohol, which is an ideal drunkenness detection point. A mathematical analysis method that integrates various indicators is established to quantitatively calculate the drunkenness of ethanol. The model is used to detect the drunkenness of physiological saline and a certain liquor sample, and the reliability and sensitivity of the model are further tested. Therefore, this model can be used for the accurate detection of ethanol drunkenness in mice.

[0160] The invention discloses a method and a mathematical analysis model for accurately detecting the drunkenness degree of mice based on multiple indicators of behavior, nerves and biochemistry, which can accurately detect the drunkenness degree of mice in a comprehensive, objective and quantitative manner and has high credibility and sensitivity.

[0161] The present invention not only has a wide range of applications, but also has great application potential. First, it can be used to accurately detect the degree of drunkenness of conventional wine samples; second, this patent can be used to improve or develop new brewing processes, such as evaluating the impact of various flavor substances on the degree of drunkenness, so as to target the flavor substances to improve the process; in addition, it can also be used to evaluate the efficacy of various types of alcohol relieving drugs and test their actual efficacy in reducing the degree of ethanol drunkenness. Finally, through large-scale experiments, relevant industry standards can be formulated, and the degree of drunkenness can be included in the wine quality evaluation standards, which will promote the development of the entire wine industry, provide consumers with high-quality wine, and actively promote the internationalization of my country's liquor.

[0162] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for constructing a mathematical analysis model for accurately detecting the degree of drunkenness, characterized in that: Includes steps: (S1) Provide two groups of mice with the same origin and baseline conditions and raise them under the same environmental conditions; (S2) administering alcohol orally to one group of mice, and administering ethanol orally to another group of mice; (S3) performing an open field test on the mice in the alcohol gavage group and the mice in the ethanol gavage group, respectively, and recording the probability P1 that the distance moved by the mice in the alcohol gavage group in the open field is smaller than the distance moved by the mice in the ethanol gavage group in the open field; (S4) performing a rotating rod test on the mice in the alcohol gavage group and the mice in the ethanol gavage group, respectively, and recording the probability P2 that the rotating rod time of the mice in the alcohol gavage group is shorter than that of the mice in the ethanol gavage group; (S5) respectively testing the blood ethanol concentration of the mice in the wine gavage group and the ethanol gavage group, and recording the probability P3 that the blood ethanol concentration of the mice in the wine gavage group is greater than the blood ethanol concentration of the mice in the ethanol gavage group; and (S8) Based on each recorded probability, different weights W are assigned to each recorded probability according to the correlation between each detection index and the degree of drunkenness. i ; Establish a mathematical analysis model for accurate detection of drunkenness degree, so as to obtain the drunkenness index (Drunk Index) P: Among them, step (S3), step (S4), and step (S5) can be performed in any order.

2. The method according to claim 1, characterized in that The method may also optionally include the following steps: (S6) respectively detecting the blood acetaldehyde concentration of the mice in the alcohol gavage group and the mice in the ethanol gavage group, and recording the probability P4 that the blood acetaldehyde concentration of the mice in the alcohol gavage group is greater than the blood acetaldehyde concentration of the mice in the ethanol gavage group; (S7) Detecting cerebellar gamma-aminobutyric acid (GABA) in the mice in the alcohol gavage group and the mice in the ethanol gavage group, respectively, and recording the probability P5 that the cerebellar GABA level in the mice in the alcohol gavage group is greater than the cerebellar GABA level in the mice in the ethanol gavage group.

3. The method according to claim 1, characterized in that The experiments and detections of the method are carried out 1-1.5 hours after mice are intragastrically administered with ethanol or alcohol.

4. The method according to claim 1, characterized in that The mice administered with alcohol and the mice administered with ethanol were in a non-comatose drunken state.

5. The method according to claim 1, characterized in that In the intragastric administration of the wine and ethanol, the ethanol content of the wine and ethanol is the same.

6. An evaluation system for detecting the degree of drunkenness, characterized in that The system comprises: (M1) Input module: The input module is configured to input open field test data, rotating rod test data and blood alcohol concentration data of the subject to be tested; (M2) Evaluation module: The evaluation module is configured to evaluate the open field test data, the rotating rod test data, and the blood ethanol concentration data based on the mathematical analysis model for accurate detection of the degree of drunkenness, so as to obtain an evaluation result of the degree of drunkenness of the subject to be tested; wherein the mathematical analysis model for accurate detection of the degree of drunkenness is a mathematical analysis model for accurate detection of the degree of drunkenness constructed by the construction method according to claim 1, and the model formula is: and (M3) Output module: The output module is configured to output the evaluation result.

7. The system according to claim 6, characterized in that The greater the evaluation result value of the evaluation, the higher the degree of drunkenness; conversely, the smaller the evaluation result value of the evaluation, the lower the degree of drunkenness.

8. The system according to claim 6, characterized in that The alcoholic beverages include those selected from the group consisting of: white wine, yellow wine, fruit wine, beer, rice wine, medicinal wine, or a combination thereof.

9. The system according to claim 6, characterized in that The data input by the input module also includes data selected from the following group: blood acetaldehyde concentration, cerebellar GABA level, or a combination thereof.

10. An evaluation method, characterized in that: Includes steps: (a) providing the open field test data, rotarod test data and blood ethanol concentration data of the test subject; (b) inputting the open field test data, the rotating rod test data and the blood ethanol concentration data into the mathematical analysis model for accurate detection of drunkenness degree constructed by the construction method according to claim 1, wherein the model formula is: (c) using the mathematical analysis model for accurate detection of drunkenness degree to evaluate the open field test data, the rotating rod test data and the blood ethanol concentration data, thereby obtaining the drunkenness degree of the subject to be tested; wherein, the larger the evaluation result value of the evaluation is, the higher the drunkenness degree is; conversely, the smaller the evaluation result value is, the lower the drunkenness degree is; and (d) Output the evaluation results.

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