A method for constructing a drunken animal model and application thereof in drunkenness evaluation

Abstract

The application discloses a drunk animal model construction method and application thereof in drunk evaluation, and belongs to the technical field of model construction. The technical problems to be solved are that a traditional Kunming mouse drunk model is genetically hybrid, modeling is uneven, data repeatability is poor, drug efficacy verification is inaccurate, and there is no systematic drunk evaluation system. The technical solution points comprise providing a drunk animal model construction method for light and severe drunk animals and a light and severe drunk evaluation method. The drunk animal model construction method based on ICR mice is provided, the drunk model is reproducible, the evaluation indexes are quantifiable, and the drug screening is accurate, and the light and severe drunk evaluation method is provided.

Description

Technical Field

[0001] This invention belongs to the field of animal model construction technology, specifically relating to a method for constructing an intoxicated animal model and its application in intoxication assessment. Background Technology

[0002] Alcohol, a beverage ingredient widely present in daily life, can have adverse effects on various bodily organs, such as the heart, brain, liver, and kidneys, if consumed in large quantities. Long-term alcohol consumption may lead to specific behavioral and psychological changes, resulting in functional and structural abnormalities of the nervous, circulatory, or immune systems.

[0003] In clinical practice, most people experience mild intoxication after drinking alcohol. However, current research faces two major problems: First, existing alcohol poisoning modeling studies mostly focus on severe intoxication models, which differ significantly from the mild intoxication observed in most people in real life. This leads to insufficient suitability of selected drugs in practical applications, making it difficult to accurately reflect clinical efficacy. Second, existing models have extremely poor statistical performance. Traditional studies often use Kunming mice combined with Red Star Erguotou (a type of Chinese liquor) for modeling. Kunming mice, as a closed population, have high genetic heterozygosity and significant differences in genetic background among different populations. Red Star Erguotou, however, has a complex composition containing various trace impurities, which can easily produce synergistic toxicity with ethanol. This results in significant differences in alcohol tolerance and the activity of key metabolic enzymes among individual mice, leading to drastic fluctuations in intoxication status and core evaluation indicators after modeling. The reproducibility and reliability of experimental data are insufficient, failing to meet the precision requirements for screening hangover remedies. Furthermore, the time to intoxication is highly correlated with the recovery time after severe intoxication. Therefore, modeling groups need to be selected according to the time of intoxication to further improve the scientific validity of the model and the reliability of experimental data, providing precise support for the screening of hangover remedies.

[0004] Relevant patent documents retrieved: This document, published in China (CN120305376A) on July 15, 2025, discloses a method for constructing an acute intoxication model. The method involves daily gavage administration of 56% baijiu (a certain brand of 56° baijiu, 1 mL / 100 g / day) to establish the acute intoxication model. Using this baijiu as an alcohol inducer is problematic. This reagent has a complex composition, containing not only the core component ethanol but also trace impurities such as methanol, fusel oils, and esters. These substances easily produce synergistic toxic effects with ethanol, leading to significant differences in alcohol tolerance and the activity of key alcohol metabolism enzymes (ADH, ALDH) among individuals. After modeling, the intoxication state (e.g., the time to disappearance of the righting reflex, peak blood alcohol concentration, and metabolic curve) exhibits significant heterogeneity, with core evaluation indicators fluctuating drastically. The repeatability and reliability of the experimental data are insufficient, making it difficult to accurately support the efficient screening and objective verification of the efficacy of hangover remedies.

[0005] Relevant non-patent literature retrieved: The journal or book title is "Modern Journal of Integrated Traditional and Western Medicine", the article title is "Experimental Study on the Effect of Detoxifying and Refreshing Formula on Alcohol Poisoning Mouse Model", volume number 10, publication date 2001. This article discloses a method for constructing an alcohol poisoning model. The method uses male Kunming mice and baijiu (Chinese liquor) with an alcohol content of 46% (V / V) produced by a certain winery. The model construction method is as follows: 0.3 mL of liquor per mouse per day is administered by gavage, followed by 0.3 mL of water per mouse half an hour later.

[0006] Using Kunming mice to establish an alcohol intoxication model has significant limitations. As a closed colony, Kunming mice have high genetic heterozygosity and rich gene diversity, and their genetic background varies significantly across different geographical populations. In establishing a severe alcohol intoxication model using Kunming mice, the drawbacks of individual tolerance differences are further amplified under high-dose alcohol induction: some mice are prone to acute alcohol poisoning, acute liver and kidney damage, or even sudden death, resulting in a high mortality rate. Furthermore, the pathological state of the severe model differs significantly from the mild alcohol intoxication scenarios experienced by most people in clinical practice, leading to insufficient suitability of screened drugs for practical applications and making it difficult to accurately reflect clinical efficacy.

[0007] The prior art represented by the aforementioned documents has at least the following unresolved technical problems and defects: Traditional hangover models generally have limitations such as limited clinical translational value, significant individual differences, poor controllability and repeatability of the modeling process, and large fluctuations in alcohol metabolism-related indicators. These shortcomings make it difficult to achieve standardization and precision in the screening of hangover remedies, which in turn leads to the problem of effective hangover remedies being missed and ineffective drugs being misjudged. Summary of the Invention

[0008] The purpose of this invention is to provide: A method for constructing an animal model of intoxication and its application in intoxication evaluation, along with related technologies, are proposed to address technical problems in the traditional Kunming mouse intoxication model, such as genetic heterozygosity, uneven modeling, poor data repeatability, inaccurate drug efficacy verification, and the lack of a systematic intoxication evaluation system.

[0009] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0010] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0011] The definitions of standard chemical terms can be found in the references "Experimental Pharmacology" (2nd Edition), edited by Du Guanhua, Higher Education Press; and "Application of Animal Models in Medical Research", edited by Li Houda, People's Medical Publishing House.

[0012] Unless otherwise stated, conventional methods within the scope of the art, such as intravenous injection, intraperitoneal injection, or gavage, shall be used.

[0013] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0014] The term "naloxone hydrochloride" as used in this article refers to a synthetic opioid receptor-specific antagonist, used as an adjunct treatment for acute alcohol poisoning. It can stimulate the central nervous system, shorten the duration of intoxication and coma, and alleviate symptoms of intoxication such as unsteady gait, drowsiness, and confusion. It is especially suitable for acute alcohol poisoning without serious changes in vital signs.

[0015] The term "metadone" as used in this article refers to a drug used to treat alcoholic liver disease and relieve symptoms of acute alcohol poisoning. It can promote alcohol metabolism, reduce the damage of alcohol and its metabolites to the liver, and improve alcohol-induced neurological and psychological abnormalities.

[0016] In a first aspect, the present invention provides: A method for constructing a mild intoxication animal model includes the following steps: S1. Pre-treat ICR mice by fasting; S2. Administer 38%-50% (v / v) ethanol solution via gavage at a rate of 5-15 mL / kg.

[0017] The technical features include: ICR mice, fasting pretreatment, ethanol solution, and the volume of ethanol solution administered via gavage.

[0018] Furthermore, the ICR mice mentioned in step S1 are SPF-grade ICR mice that are 6-8 weeks old and weigh 27-30 g.

[0019] Furthermore, the ICR mice mentioned in step S1 can be male mice, female mice, or a mixture of male and female mice.

[0020] Furthermore, the ICR mice described in step S1 were acclimatized for 3-5 days before model construction.

[0021] Furthermore, the conditions for adaptive feeding are a temperature of 22±2℃, a humidity of 50±5%, a 12-hour light-dark cycle, and free access to standard feed and sterile water during the adaptive feeding period.

[0022] Furthermore, in step S1, the fasting pretreatment is fasting for 16-18 hours but not water, to avoid individual differences due to dietary differences; preferably, in step S1, the fasting pretreatment is fasting for 16 hours but not water.

[0023] Furthermore, in step S2, the volume concentration of ethanol can be selected as 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, or any two points or a range between two points.

[0024] Furthermore, in step S2, the volume of ethanol administered via gavage can be 5 mL / kg, 6 mL / kg, 7 mL / kg, 8 mL / kg, 9 mL / kg, 10 mL / kg, 11 mL / kg, 12 mL / kg, 13 mL / kg, 14 mL / kg, 15 mL / kg, or any two values ​​or a range between two values.

[0025] Preferably, the concentration of ethanol in step S2 is 38% (v / v).

[0026] Preferably, the volume of ethanol administered via gavage in step S2 is 8 mL / kg or 10 mL / kg.

[0027] Furthermore, the rate of gavage administration in step S2 is 0.1 mL / s.

[0028] The method for determining whether a mild intoxication animal model has been successfully established is as follows: after gavage, the righting reflex of the mice is still present, and the total distance and average speed of open field movement are significantly increased, which can be maintained for 2 hours or more after modeling, indicating that the mild intoxication model has been successfully established.

[0029] In this invention, the behavioral indicators include at least one of the following: rotarod test dwell time, open field test total activity distance / central area dwell time, and hot plate test pain threshold (hot plate temperature 52±0.5℃).

[0030] Secondly, the present invention provides a method for constructing a severely intoxicated animal model, comprising the following steps: S1. Pre-treat ICR mice by fasting; S2. Administer 40-60% (v / v) ethanol solution via gavage at a rate of 17-20 mL / kg.

[0031] Furthermore, the ICR mice mentioned in step S1 are SPF-grade ICR mice that are 6-8 weeks old and weigh 27-30 g.

[0032] Furthermore, the ICR mice mentioned in step S1 can be male mice, female mice, or a mixture of male and female mice.

[0033] Furthermore, the ICR mice described in step S1 were acclimatized for 3-5 days before model construction.

[0034] Furthermore, the conditions for adaptive feeding are a temperature of 22±2℃, a humidity of 50±5%, a 12-hour light-dark cycle, and free access to standard feed and sterile water during the adaptive feeding period.

[0035] Furthermore, in step S1, the fasting pretreatment is fasting but not watering for 16-18 hours to avoid individual differences due to dietary differences; preferably, in step (1), the fasting pretreatment is fasting but not watering for 16 hours.

[0036] Furthermore, in step S2, the volume concentration of ethanol can be selected as 40%, 45%, 50%, 55%, 60%, or any two points or a range between two points.

[0037] Further, the intragastric volume of ethanol in step S2 is 17 mL / kg, 17.1 mL / kg, 17.2 mL / kg, 17.3 mL / kg, 17.4 mL / kg, 17.5 mL / kg, 17.6 mL / kg, 17.7 mL / kg, 17.8 mL / kg, 17.9 mL / kg, 18.0 mL / kg, 18.1 mL / kg, 18.2 mL / kg, 18.3 mL / kg, 18.4 mL / kg, 18.5 mL / kg, 18.6 mL / kg, 18.7 mL / kg, 18.8 mL / kg, 18.9 mL / kg, 19.0 mL / kg, 19.1 mL / kg, 19.2 mL / kg, 19.3 mL / kg, 19.4 mL / kg, 19.5 mL / kg, 19.6 mL / kg, 19.7 mL / kg, 19.8 The values ​​can be selected as mL / kg, 19.9 mL / kg, 20.0 mL / kg, or any two points in between or a range between two points.

[0038] Preferably, the volume of ethanol administered via gavage in step S2 is 17 mL / kg.

[0039] Furthermore, the rate of gavage administration in step S2 is 0.1 mL / s.

[0040] Furthermore, in step S2, the ICR mice were grouped according to the time of intoxication after being administered 50% alcohol by gavage, in order to avoid individual differences in the degree of intoxication interfering with the evaluation of drug efficacy.

[0041] Furthermore, the duration of intoxication is divided into four time periods: <30 min, 30-60 min, 60-90 min, and ≥90 min.

[0042] One method to determine whether a severe alcohol intoxication animal model has been successfully established is: the disappearance of the righting reflex in mice after gavage administration indicates that the severe alcohol intoxication model has been successfully established.

[0043] According to some embodiments of the present invention, the operation of the righting reflex is as follows: the mouse is placed on a mat with its abdomen facing upward and the timing is started. If the mouse can maintain the abdomen-upward position for 30 seconds, the righting reflex is considered to have disappeared. The time from the time of gavage administration to the disappearance of the righting reflex is observed and recorded.

[0044] Thirdly, the present invention provides the application of the above-mentioned mild or severe intoxication animal models in the evaluation of hangover relief products.

[0045] Specifically, the hangover remedies include, but are not limited to, naloxone hydrochloride and metadoxine.

[0046] Fourthly, the present invention provides a method for evaluating mild intoxication, comprising the following steps: S1. Pre-treat ICR mice by fasting; S2. Administer 38% (v / v) ethanol solution via gavage at a rate of 8-10 mL / kg; S3. After gavage, the behavioral indicators of the mice were tested, including: righting reflex, total distance of open field movement, average speed of open field movement, and dwell time in the rotundus test. S4. After gavage, the righting reflex of the mice was still present, and the total distance and average speed of open field movement were significantly increased. The dwell time in the rotundus test was significantly reduced and could be maintained for 2 hours or more after modeling, indicating mild intoxication.

[0047] Furthermore, the ICR mice mentioned in step S1 are SPF-grade ICR mice that are 6-8 weeks old and weigh 27-30 g.

[0048] Furthermore, the ICR mice mentioned in step S1 can be male mice, female mice, or a mixture of male and female mice.

[0049] Furthermore, the ICR mice described in step S1 were acclimatized for 3-5 days before model construction.

[0050] Furthermore, the conditions for adaptive feeding are a temperature of 22±2℃, a humidity of 50±5%, a 12-hour light-dark cycle, and free access to standard feed and sterile water during the adaptive feeding period.

[0051] Furthermore, in step S1, the fasting pretreatment is 16-18 hours of fasting but not water restriction to avoid inaccurate data analysis due to dietary differences; preferably, in step S1, the fasting pretreatment is 16 hours of fasting but not water restriction.

[0052] Furthermore, in step S2, the volume concentration of ethanol can be selected as 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, or any two points or a range between two points.

[0053] Furthermore, in step S2, the volume of ethanol administered via gavage can be 5 mL / kg, 6 mL / kg, 7 mL / kg, 8 mL / kg, 9 mL / kg, 10 mL / kg, 11 mL / kg, 12 mL / kg, 13 mL / kg, 14 mL / kg, 15 mL / kg, or any two values ​​or a range between two values.

[0054] Preferably, the concentration of ethanol in step S2 is 38% (v / v).

[0055] Preferably, the volume of ethanol administered via gavage in step S2 is 8 mL / kg or 10 mL / kg.

[0056] Furthermore, the rate of gavage administration in step S2 is 0.1 mL / s.

[0057] Furthermore, in step S2, the ICR mice were grouped according to the time of intoxication after being administered 38% alcohol by gavage, in order to avoid individual differences in the degree of intoxication interfering with the evaluation of drug efficacy.

[0058] According to some embodiments of the present invention, the open field test method is as follows: the open field test uses an open field box with dimensions of 40×40×30 cm, and records the mouse's activity trajectory within 3 minutes through a video tracking system to quantify the total activity distance and the proportion of time spent in the central area.

[0059] In this invention, the equipment parameters for the rotarod experiment are as follows: the rotation speed is gradually increased from 1 rpm to 15 rpm within 2 minutes (adjusted according to the experiment), each mouse is observed for 2 minutes, and the time the mouse moves on the roller without falling is recorded. If the mouse does not fall off within 2 minutes, it is recorded as 2 minutes.

[0060] Fifthly, the present invention provides a method for evaluating severe intoxication, comprising the following steps: S1. Pre-treat ICR mice by fasting; S2. Administer 50% (v / v) ethanol solution via gavage at a rate of 17-20 mL / kg; S3. Detect the righting reflex in mice. If the righting reflex disappears, it indicates severe intoxication.

[0061] Furthermore, the ICR mice mentioned in step S1 are SPF-grade ICR mice that are 6-8 weeks old and weigh 27-30 g.

[0062] Furthermore, the ICR mice mentioned in step S1 can be male mice, female mice, or a mixture of male and female mice.

[0063] Furthermore, the ICR mice described in step S1 were acclimatized for 3-5 days before model construction.

[0064] Furthermore, the conditions for adaptive feeding are a temperature of 22±2℃, a humidity of 50±5%, a 12-hour light-dark cycle, and free access to standard feed and sterile water during the adaptive feeding period.

[0065] Furthermore, in step S1, the fasting pretreatment is fasting for 16-18 hours but not water, to avoid inaccurate data analysis due to dietary differences; preferably, in step S1, the fasting pretreatment is fasting for 16 hours but not water.

[0066] Furthermore, in step S2, the volume concentration of ethanol can be selected as 40%, 45%, 50%, 55%, 60%, or any two points or a range between two points.

[0067] Further, the intragastric volume of ethanol in step S2 is 17 mL / kg, 17.1 mL / kg, 17.2 mL / kg, 17.3 mL / kg, 17.4 mL / kg, 17.5 mL / kg, 17.6 mL / kg, 17.7 mL / kg, 17.8 mL / kg, 17.9 mL / kg, 18.0 mL / kg, 18.1 mL / kg, 18.2 mL / kg, 18.3 mL / kg, 18.4 mL / kg, 18.5 mL / kg, 18.6 mL / kg, 18.7 mL / kg, 18.8 mL / kg, 18.9 mL / kg, 19.0 mL / kg, 19.1 mL / kg, 19.2 mL / kg, 19.3 mL / kg, 19.4 mL / kg, 19.5 mL / kg, 19.6 mL / kg, 19.7 mL / kg, 19.8 The values ​​can be selected as mL / kg, 19.9 mL / kg, 20.0 mL / kg, or any two points in between or a range between two points.

[0068] Preferably, the volume of ethanol administered via gavage in step S2 is 17 mL / kg.

[0069] Furthermore, the rate of gavage administration in step S2 is 0.1 mL / s.

[0070] Furthermore, in step S2, the ICR mice were grouped according to the time of intoxication after being administered 50% alcohol by gavage, in order to avoid individual differences in the degree of intoxication interfering with the evaluation of drug efficacy.

[0071] Furthermore, the duration of intoxication is divided into four time periods: <30 min, 30-60 min, 60-90 min, and ≥90 min.

[0072] According to some embodiments of the present invention, the operation of the righting reflex is as follows: after placing the mouse on a mat with its abdomen facing upward, start timing. If the mouse can maintain the abdomen-upward position for 30 seconds, the righting reflex is considered to have disappeared. Observe and record the time from the time of gavage administration to the disappearance of the righting reflex, and the time from the disappearance of the righting reflex to the recovery of the righting reflex.

[0073] The present invention has at least the following beneficial effects: (1) This invention completely solves the core pain points of traditional Kunming mouse model, such as large individual differences, low modeling success rate and drastic data fluctuation. The modeling success rate is ≥95%, the coefficient of variation of core indicators (righting reflex disappearance time, total distance of open field movement) is ≤10%, and the consistency of intra-batch / inter-batch experiments is improved by more than 60%.

[0074] SPF-grade ICR mice with strong genetic uniformity and regular alcohol response were selected, and the selection criteria of 6-8 weeks of age and 27-30 g were clearly defined to reduce the interference of individual genetic differences from the source. The modeling success criteria of "time from the disappearance of the righting reflex to the recovery of the righting reflex + open field test" were established to unify the qualified threshold for modeling and remove abnormal individuals.

[0075] (2) Simple random grouping, weight stratified grouping, etc., due to the uneven degree of intoxication within the group, it is difficult to accurately quantify the core effects of the drug on "shortening the duration of intoxication" and "prolonging the intoxication latency period". There may be situations where "effective drugs are not statistically significant due to differences within the group" or "ineffective drugs are mistakenly judged as effective due to the slight intoxication of individual mice".

[0076] This invention groups mice according to the duration of intoxication, ensuring a uniform state of intoxication within each group. The effect of the drug on the duration of intoxication can be directly demonstrated through inter-group data comparison, providing a clearer comparison of differences and higher statistical power. p The value is more likely to reach <0.05), which can accurately distinguish whether a drug is "effective / ineffective" or "highly effective / lowly effective".

[0077] (3) The mortality rate of the model is ≤5%, which is much lower than that of the traditional Kunming mouse model (10%-15%). The experimental process is safer, the animal welfare is better, and the data dispersion is small, resulting in higher statistical power. The experimental environment is strictly controlled (temperature 22±2℃, humidity 50±5%, 12 h light-dark cycle) to avoid experimental deviations caused by environmental stress; clear sample processing standards are established to ensure the stability of the test results.

[0078] (4) This invention provides an evaluation method for mild and severe intoxication models, which determines the state of intoxication by detecting behavioral indicators. The behavioral indicators for mild intoxication include righting reflex, total distance of open field movement, average speed of open field movement, and dwell time in rotundus test. The behavioral indicator for severe intoxication is the sleep time from the disappearance of righting reflex to the recovery of righting reflex. Attached Figure Description

[0079] Figure 1 shows the sleep time of male Kunming mice with severe alcohol intoxication at different intoxication times in Comparative Example 1.

[0080] Figure 2 shows the sleep time of female Kunming mice with severe alcohol intoxication at different intoxication times in Comparative Example 2.

[0081] Figure 3 shows the sleep time of the Kunming mouse model of severe alcohol intoxication after administration of a positive wake-up drug in Comparative Example 3.

[0082] Figure 4 shows the sleep time of male Kunming mice with severe alcohol intoxication in Comparative Example 4 after administration of positive alcohol metabolism-promoting drugs and positive wakefulness-promoting drugs.

[0083] Figure 5 shows the sleep time of female Kunming mice with severe alcohol intoxication in Comparative Example 5 after administration of positive alcohol metabolism-promoting drugs and positive wakefulness-promoting drugs.

[0084] Figure 6 shows the intoxication rate and mortality rate of the ICR mouse severe intoxication model under different alcohol dosages in Example 1.

[0085] Figure 7 shows the sleep time of ICR mice with severe alcohol intoxication after being given different doses of positive wakefulness-inducing drugs in Example 1.

[0086] Figure 8 shows the total distance traveled by ICR mice with mild alcohol intoxication under different alcohol dosages in Example 3. Detailed Implementation

[0087] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0088] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0089] Data analysis and statistical analysis were performed using professional data processing software, and significance analysis was conducted using one-way ANOVA. p <0.05 indicates a significant difference.

[0090] Comparative Example 1 (1) Construction of a severe alcohol intoxication model in male Kunming mice Male Kunming mice were fasted for 16 hours but allowed free access to water. Experiments were conducted when their body weight reached 22 ± 2 g. The mice were given 18 mL / kg of 50% (v / v) ethanol solution. The proportion of mice that lost their righting reflex after alcohol intoxication, the time from the loss of the righting reflex to its recovery (sleep time), and the mortality rate of severely intoxicated mice were recorded.

[0091] (2) Grouping of male Kunming mouse severe illness model Male Kunming mice were grouped according to the time of intoxication, defined as the time from the start of alcohol administration until the mice lost their righting reflex. They were divided into four groups: <30 min, 30-60 min, 60-90 min, and ≥90 min. Figure 1 As shown, the longer the intoxication time in mice, the shorter the sleep time, and there were significant differences in sleep time among the four groups of mice. The intoxication time of the samples was mainly distributed between 30 and 90 minutes. The faster the intoxication, the higher the mortality rate of the mice. The specific results are shown in Table 1.

[0092] Table 1. Mortality and sleep duration of Kunming mice at different intoxication times.

[0093] Comparative Example 2 (1) Construction of a severe alcohol intoxication model in female Kunming mice Female Kunming mice were fasted for 16 hours but allowed free access to water. Experiments were conducted when their body weight reached 22 ± 2 g. A severe alcohol intoxication model was induced in the Kunming mice by administering 18 mL / kg of 50% (v / v) ethanol solution. The proportion of mice that lost their righting reflex after intoxication, the time from the loss of the righting reflex to its recovery, and the mortality rate of severely intoxicated mice were recorded.

[0094] (2) Grouping of female Kunming mice with severe illness Female Kunming mice were grouped according to the time of intoxication, defined as the time from the start of alcohol administration until the mice lost their righting reflex. They were divided into four groups: <30 min, 30-60 min, 60-90 min, and ≥90 min. Figure 2 As shown, the longer the intoxication time in mice, the shorter the sleep time, and there were significant differences in sleep time among the four groups of mice. The intoxication time of the samples was mainly distributed between 30-90 minutes. The faster the intoxication, the higher the mortality rate of the mice, which is consistent with the results of male mice. The specific results are shown in Table 1.

[0095] Comparative Example 3: Efficacy Evaluation Based on the Kunming Mouse Model of Severe Intoxication A severe alcohol intoxication model was constructed in male Kunming mice using the same method as in Comparative Example 1. Mice were randomly divided into two groups based on the duration of intoxication: the control group received intravenous saline, while the naloxone hydrochloride group received intravenous naloxone hydrochloride at 2 mg / kg. The time from the disappearance to the recovery of the righting reflex after intoxication, as well as the mortality rate of severely intoxicated mice, were recorded. This experiment was repeated five times at different time points; the specific results are shown in Figure 3.

[0096] Five repeated experiments at different time points yielded varying results. There was no significant difference in sleep duration between the control group and the two groups of mice receiving intravenous naloxone hydrochloride (2 mg / kg). Naloxone hydrochloride, a positive wake-up-inducing drug, failed to show effectiveness in the Kunming mouse model in any of the five experiments. Therefore, a large sample size is needed to observe statistically significant differences between groups when studying the sobering effect of drugs using Kunming mice.

[0097] Comparative Example 4: Construction of a severe alcohol intoxication model in male Kunming mice and evaluation of the efficacy of antidote. Male Kunming mice were fasted for 18 hours prior to modeling, but allowed free access to water (14:30-8:30). Before modeling, body weight was measured sequentially, and the amount of alcohol administered was calculated based on body weight. Mice were given 18 mL / kg of 50% (v / v) ethanol. Mice were then alternately divided into two groups based on body weight: Group 1 was the blank control group: 1 minute after intoxication, mice received a tail vein injection of metadoxine solution at 0.316 g / kg (3.16 mL / kg); Group 2 was the intraperitoneal injection group: 1 minute after intoxication, mice received a tail vein injection of metadoxine solution at 0.316 g / kg (3.16 mL / kg) and intraperitoneal administration of naloxone hydrochloride at 2 mg / kg (5 mL / kg naloxone hydrochloride intraperitoneal solution). The success of the mouse intoxication model was tested using a righting test (righting test: mice were placed back-down; if they did not turn over within 30 seconds, they were considered intoxicated). The mice's condition was observed in real-time, and the intoxication time and sleep time for each group were recorded.

[0098] The results of Comparative Example 4 are shown in Figure 4. The within-group standard deviation (SD) was generally high in all groups, indicating significant differences in physiological or biochemical indicators among individuals within each group. When comparing between groups, a t-test was used for statistical analysis. The results showed that even after treatment, the differences in indicators between groups did not reach statistical significance. This phenomenon may be related to the limited sample size or the high heterogeneity in the drug response of individual Kunming mice, causing the differences between groups to be masked by large within-group variations.

[0099] Comparative Example 5: Construction of a severe alcohol intoxication model in female Kunming mice and evaluation of the efficacy of antidote. Female Kunming mice were fasted for 18 hours prior to modeling (14:30-8:30). Before modeling, body weight was measured sequentially, and the alcohol dosage was calculated based on body weight. Mice were administered 18 mL / kg of 50% (v / v) ethanol. Mice were then alternately divided into two groups based on body weight: Group 1 (blank control): 1 minute after intoxication, mice received a tail vein injection of metadoxine solution at 0.316 g / kg (3.16 mL / kg); Group 2 (intraperitoneal injection): 1 minute after intoxication, mice received a tail vein injection of metadoxine solution at 0.316 g / kg (3.16 mL / kg) and intraperitoneal administration of naloxone hydrochloride at 2 mg / kg (5 mL / kg naloxone hydrochloride intraperitoneal solution). The success of the mouse intoxication model was tested using a righting test. Mice were observed in real-time, and the intoxication and sleep durations of each group were recorded.

[0100] The results of Comparative Study 5 are shown in Figure 5. The within-group standard deviation (SD) was generally high in all groups, indicating significant differences in physiological or biochemical indicators among individuals within each group. When comparing between groups, a t-test was used for statistical analysis. The results showed that even after treatment, the differences in indicators between groups did not reach statistical significance. This phenomenon may be related to the limited sample size or the high heterogeneity in the drug response of individual Kunming mice, causing the differences between groups to be masked by large within-group variations.

[0101] Example 1 (1) Construction of ICR mouse model of severe alcohol intoxication ICR mice were randomly grouped according to body weight. The animals were fasted for about 16 hours but allowed free access to water. Different volumes of 50% (v / v) ethanol solution were administered to the mice via gavage to induce a mild alcohol intoxication model. The proportion of mice that lost their righting reflex after intoxication, the time from the loss of the righting reflex to its recovery, and the mortality rate of mice with severe alcohol intoxication were recorded to determine the optimal modeling dose for severe alcohol intoxication in mice.

[0102] As shown in Figure 6, the intoxication rate and mortality rate of mice increased with increasing alcohol dosage. When the alcohol dosage was higher than 22 mL / kg, the mortality rate of mice reached over 80%; when the alcohol dosage was higher than 20 mL / kg, the intoxication rate was close to 100%. When the alcohol dosage was 17 mL / kg, the intoxication rate of mice was higher than 80%, and the mortality rate was lower than 10%. Considering all factors, 17 mL / kg was selected as the modeling standard for subsequent pharmacodynamic evaluation.

[0103] (2) Verification of the awakening effect of naloxone hydrochloride on a mouse model of severe alcohol intoxication ICR mice were randomly divided into groups according to body weight. The animals were fasted for approximately 16 hours but allowed free access to water. A mild alcohol intoxication model was induced by gavage with 50% ethanol at a volume of 17 mL / kg. The normal control group received the same volume of ultrapure water. As shown in Figure 7, with increasing naloxone dosage, the sleep time of mice after alcohol intoxication was significantly shortened, indicating that naloxone hydrochloride has a significant ameliorative effect on severe alcohol intoxication.

[0104] Example 2: Construction of a mild ICR mouse model of alcohol intoxication (1) Experimental grouping Animals were adapted for 6 days, and when their weight was approximately 30 g, they were randomly divided into groups according to their weight. The experiment was divided into two parts: In the first part, the animals were divided into normal control group 1, model control group 1, model control group 2, model control group 3, model control group 4, and model control group 5, with model establishment volumes of 5, 8, 10, 12, and 15 mL / kg, respectively, with 3 animals in each group, to explore the volume of ethanol solution required for model establishment by gavage; In the second part, there were normal control group 2, model control group 6 (8 mL / kg), and positive control group (2 mg / kg, administered immediately after model establishment), with 3 animals in each group.

[0105] (2) Modeling method Animals were fasted for about 16 hours but allowed free access to water. A mild alcohol intoxication model was induced by gavage with 38% ethanol. The normal control group was given 15 mL / kg of ultrapure water. The model control groups 1, 2, 3, 4, and 5 were given 5, 8, 10, 12, and 15 mL / kg of 38% ethanol, respectively. The gavage volume of ethanol solution was 8 mL / kg for the model establishment confirmation test.

[0106] (3) Dosage setting and administration Normal control group 2, model control group 6, and positive control group were all administered the drug via tail vein injection immediately after model establishment. The positive control group received 8 mL / kg of the drug, while normal control group 2 and model control group 6 received the same volume of solvent. See Table 2.

[0107] Table 2 Dosage settings and administration methods ( n =3)

[0108] Note: n This represents the number of animals.

[0109] (4) Detection indicators Weight: before grouping and before modeling.

[0110] Righting reflex: At 5, 10, and 30 minutes after modeling, place the mouse on a mat with its abdomen facing up and start timing. If the mouse can maintain the abdomen-up position for 30 seconds, the righting reflex is considered to have disappeared. Observe and record the righting time of the mouse.

[0111] Rotary bar test: Used to test the motor coordination, strength, and balance of mice. Tests are performed at 5, 10, and 30 minutes after model establishment. Mice are placed on a rotundus, and after setting the parameters, the rotundus begins to rotate. The rotation speed is gradually increased from 1 rpm to 15 rpm over 2 minutes. Each mouse is observed for 2 minutes, and the time it takes for the mouse to remain on the rotundus without falling is recorded. A mouse that remains on the rotundus for more than 2 minutes is counted as 2 minutes.

[0112] (5) Data processing Quantitative data are expressed as mean ± standard error (Mean ± SEM) and analyzed using SPSS 25.0 software. Independent t-tests were performed between the normal control group and the model control group, and between the model control group and the drug-treated group. The significance level was set at α = 0.05. p A value ≤0.05 indicates a statistically significant difference.

[0113] Results of Example 2 In the normal control group and the model control group, the righting reflex was present in mice at concentrations of 5, 8, 10, and 12 mL / kg, with a righting time of 1 second. In the model control group at 15 mL / kg, the righting reflex was present in 2 mice and absent in 1 mouse. Since the righting reflex time was relatively similar across groups and there was no significant difference, it was not statistically analyzed.

[0114] Compared with the normal control group 1, the model control group with 10 mL / kg for 5 min of modeling time was significantly reduced ( p ≤0.05); Model control group 12 mL / kg, modeling time 5, 10 min significantly reduced ( p ≤0.05); Model control group 15 mL / kg, modeling time significantly reduced at 5, 10, and 30 min ( p ≤0.05); In the model control group, 8 mL / kg showed a significant decreasing trend in the time to modeling at 5 and 10 min, but there was no significant difference. p >0.05). Since mice in the model control group (10, 12, and 15 mL / kg) immediately dropped off the rotarod when placed in the rotarod, and no statistically significant difference was observed at 8 mL / kg (which is likely related to the small number of animals), the model control group (8 mL / kg) was selected for the drug administration validation experiment. See Table 3 for details.

[0115] Table 3. Effects of different volumes of ethanol solution on the rotarod test in ICR mice. (Mean±SEM, n =3)

[0116] Note: Compared with the normal control group, * p≤ 0.05. n This represents the number of animals.

[0117] Compared with the normal control group 2, the model control group showed a decreasing trend in the time taken for 5, 10, and 30 min at 8 mL / kg for modeling, but the difference was not statistically significant. p >0.05). Compared with the model control group 6, naloxone hydrochloride 2 mg / kg (administered immediately after modeling) showed a trend of increasing rod time at 5, 10, and 30 min, but the differences were not statistically significant. p >0.05); Naloxone hydrochloride 2 mg / kg (administered 10 min before modeling) showed no significant change at 5, 10, and 30 min of modeling time. p >0.05). See Table 4 for details.

[0118] Table 4. Effects of naloxone hydrochloride on rotarod test in ICR mice (Mean ± SEM). n =3)

[0119] Note: n This represents the number of animals.

[0120] Example 3: Construction of a mild ICR mouse model of alcohol intoxication (1) Experimental grouping First batch: When the animals had adapted for 3 days and their weight was about 30 g, they were randomly divided into groups according to their weight. The experiment was divided into two parts: In the first part, the animals were divided into normal control group 1, model control group 1 and 2, with model establishment volumes of 8 and 10 mL / kg, respectively, with 3 animals in each group, to explore the volume of ethanol required for model establishment by gavage; In the second part, there were normal control group 2, model control group 3 (10 mL / kg), positive control low-dose group (2 mg / kg), and positive control high-dose group (4 mg / kg), with 3 animals in each group.

[0121] The second batch: After 3 days of acclimatization and when the animals weighed approximately 30 g, they were randomly grouped according to their weight. The experiment was divided into two parts: In the first part, the animals were divided into a normal control group (3), a model control group (4, 5, 6, 7, 8), and a model control group (8). The modeling volumes were 5, 7.5, 10, 12.5, and 15 mL / kg, respectively, with 3 animals in each group. This was used to explore the volume of ethanol required for modeling by gavage.

[0122] (2) Modeling method Animals were fasted but allowed free access to water for approximately 16 hours. A mild alcohol intoxication model was induced by gavage with 38% ethanol. The normal control group was given 15 mL / kg of ultrapure water. The model control group (numbers 1-8) was given 8, 10, 10, 5, 7.5, 10, 12.5, and 15 mL / kg of 38% ethanol, respectively. For the model establishment confirmation test, the ethanol solution volume was 10 mL / kg, and other modeling conditions were the same as in the previous test.

[0123] (3) Dosage setting and administration The dosage of naloxone hydrochloride in mice was set at 2 and 4 mg / kg.

[0124] Normal control group 2, model control group 3, low-dose positive control group, and high-dose positive control group were all administered the drug via tail vein injection immediately after model establishment. The positive control group received 8 mL / kg of the drug, while normal control group 2 and model control group 3 received the same volume of solvent. See Table 5.

[0125] Table 5 Dosage settings and administration methods ( n =3)

[0126] Note: n This represents the number of animals.

[0127] (4) Detection indicators Weight: before grouping and before modeling.

[0128] Righting reflex: At 5, 10, and 30 minutes after modeling, place the mouse on a mat with its abdomen facing up and start timing. If the mouse can maintain the abdomen-up position for 30 seconds, the righting reflex is considered to have disappeared. Observe and record the righting time of the mouse.

[0129] Autonomous Activity: At 5, 10, 30 min, 1 h, 2 h, 3 h, and 4 h after modeling, the open field test protocol needs to be prepared on the video tracking system before the experiment begins. The mice are gently placed in the center of the test chamber. After the operator leaves the camera's field of vision, the video tracking system is used to observe each mouse for 3 minutes. The total distance, total time, and average speed of movement within 3 minutes are recorded using VisuTrack animal behavior analysis software. Before each mouse is placed in the test chamber, the device is sprayed with 75% alcohol to remove odor, and the alcohol is allowed to dry before the next mouse is placed in. For mice with a modeling volume of 8 mL / kg and 10 mL / kg, behavioral records are taken at 5, 10, 30 min, 1 h, and 3 h after modeling.

[0130] (5) Data processing Quantitative data are expressed as mean ± standard error (Mean ± SEM) and statistically analyzed using SPSS 25.0 software. Independent t-tests were performed between the normal control group and the model control group, and between the model control group and the drug-treated group.

[0131] The significance level α = 0.05. p ≤0.05 indicates a statistically significant difference.

[0132] Example 3 Results Compared with the normal control group 1, the total distance and average speed of the model control group at 8 mL / kg for 5, 10, 30 min and 1 h after modeling showed an increasing trend, but there was no significant difference. p >0.05); In the model control group, 10 mL / kg significantly increased the total distance of movement at 5, 30 min and 3 h after modeling ( p ≤0.05, p ≤0.01), the average speed after 3 hours of modeling increased significantly ( p ≤0.05), see Table 6 for details.

[0133] Table 6. Effects of different ethanol volumes on open field test in ICR mice (Mean ± SEM). n =3)

[0134] Note: Compared with the normal control group, * p≤ 0.05, ** p≤ 0.01. n This represents the number of animals.

[0135] Compared with the normal control group 3, the total distance of the model control group with 5 mL / kg for 5 min and 2 h of modeling was significantly increased (p ≤0.05), the average speed increased significantly at 5 and 30 min during modeling ( p ≤0.05); In the model control group, 7.5 mL / kg significantly increased the total distance and average speed at 5 and 10 min after modeling ( p ≤0.05, p ≤0.01), the total distance of movement increased significantly after 4 hours of modeling ( p ≤0.05); In the model control group (10 mL / kg), the total distance and average speed of movement were significantly increased at 10, 30 min, 1 h, and 2 h after modeling ( p ≤0.05, p ≤0.01), the average speed at 5 min and 4 h of modeling significantly increased ( p ≤0.05); In the model control group (12.5 mL / kg), the total distance and average speed of movement were significantly increased at 10, 30 min, 2 h, and 4 h after modeling ( p ≤0.05, p ≤0.01), the average speed over 5 minutes and the total distance traveled over 1 hour significantly increased during modeling. p ≤0.05); In the model control group, 15 mL / kg was used for 5 min of modeling, resulting in a significant increase in total distance and average speed. p ≤0.05), the total distance of movement in 10 min and the average speed in 4 h increased significantly ( p (≤0.05), see Tables 7 and 8 for details.

[0136] Based on the results in Figures 8 and Tables 6, 7, and 8, the mouse model was maintained for a longer period after gavage administration of 10 mL / kg ethanol solution, lasting up to 2 hours post-modeling, and the results from two administrations were largely consistent, indicating good reproducibility. Decreasing the volume of ethanol solution administered via gavage reduced the degree of intoxication in the mice, while increasing the volume resulted in decreased activity, possibly indicating severe intoxication. Therefore, a gavage administration of 10 mL / kg ethanol solution was chosen for the drug administration validation experiment.

[0137] Compared with the normal control group 2, the model control group with 10 mL / kg significantly increased the total distance and average speed of movement after 10 min of modeling ( p ≤0.01), the average speed increased significantly after 5 minutes of modeling ( p ≤0.01), the total distance and average speed of the modeling process after 30 min and 1 h showed an increasing trend, but there was no significant difference. p>0.05). Compared with the model control group (10 mL / kg), the total distance and average speed of the positive control drug (4 mg / kg) showed a decreasing trend from 5 min to 1 h after administration, but there was no significant difference. p >0.05), but better than the positive control drug 2 mg / kg. See Table 9 for details.

[0138] Table 7. Effects of different ethanol volumes on open field test in ICR mice (Mean ± SEM). n =3)

[0139] Note: Compared with the normal control group, * p≤ 0.05, ** p≤ 0.01. n This represents the number of animals.

[0140] Table 8. Effects of different ethanol volumes on open field test in ICR mice (Mean ± SEM). n =3)

[0141] Note: Compared with the normal control group, * p≤ 0.05, ** p≤ 0.01. n This represents the number of animals.

[0142] Table 9. Effects of naloxone hydrochloride on open field test in ICR mice (Mean ± SEM). n =3)

[0143] Note: Compared with the normal control group, ** p≤ 0.01. n This represents the number of animals.

[0144] Table 10. Effect of naloxone hydrochloride on open field test in ICR mice during model validation (Mean ± SEM). n =10)

[0145] Note: Compared with the normal control group, ** p≤ 0.01; compared with the model control group, # p≤ 0.05, ## p≤ 0.01.n This represents the number of animals. b Animal No. 9 in this group has abnormal data and was not included in the statistics.

[0146] Example 4: Evaluation of the efficacy of hangover remedies based on an ICR mouse model of mild alcohol intoxication (1) Experimental grouping Animals were acclimatized for 3 days and their weight was about 30 g. They were then randomly divided into three groups according to their weight: a normal control group, a model control group (10 mL / kg), and a positive control group (4 mg / kg), with 10 animals in each group.

[0147] (2) Modeling method Animals were fasted but allowed free access to water for approximately 16 hours. A mild alcohol intoxication model was induced by gavage with 38% ethanol at a volume of 10 mL / kg. The normal control group was given the same volume of ultrapure water.

[0148] (3) Dosage setting and administration Based on the results of the model pre-experiment 2, the positive control drug of 4 mg / kg can reduce the total distance and average speed of mice in the open field test. Therefore, the positive control drug in this experiment was set at 4 mg / kg.

[0149] The normal control group, model control group, and positive control group were all administered the drug via tail vein injection immediately after modeling. The positive control drug was administered at a volume of 8 mL / kg, while the normal control group and model control group were given the same volume of solvent.

[0150] Table 11 Dosage settings and administration methods ( n =10)

[0151] Note: n This represents the number of animals.

[0152] (4) Detection indicators Weight: before grouping and before modeling.

[0153] Righting reflex: At 5, 10, and 30 minutes after modeling, place the mouse on a mat with its abdomen facing up and start timing. If the mouse can maintain the abdomen-up position for 30 seconds, the righting reflex is considered to have disappeared. Observe and record the righting time of the mouse.

[0154] Autonomous Activity Test: At 5, 10, 30 min, 1 h, 2 h, 3 h, and 4 h after modeling, the open field test protocol needs to be prepared on the video tracking system before the test begins. The mice are gently placed in the center of the test chamber. After the operator leaves the camera's field of vision, the video tracking system is used to observe each mouse for 3 minutes. VisuTrack animal behavior analysis software is used to record the total distance, total time, and average speed of the mice within 3 minutes. Before each mouse is placed in the test chamber, the device is sprayed with 75% alcohol to remove odor, and the alcohol is allowed to dry before the next mouse is placed in the chamber.

[0155] (5) Data processing Quantitative data are expressed as mean ± standard error (Mean ± SEM) and statistically analyzed using SPSS 25.0 software. Independent t-tests were performed between the normal control group and the model control group, and between the model control group and the drug-treated group.

[0156] The significance level α = 0.05. p ≤0.05 indicates a statistically significant difference.

[0157] Example 4 Results Compared with the normal control group, the model control group with 10 mL / kg of oxygen saturated for 5 min to 1 h showed a significant increase in total distance and average speed. p ≤0.01). Compared with the model control group (10 mL / kg), the total distance and average speed of the positive control drug (4 mg / kg) were significantly reduced after 30 min of administration ( ). p ≤0.05, p ≤0.01), the average speed after 5 min and 1 h of modeling was significantly reduced ( p ≤0.05). See Table 10 for details.

[0158] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for constructing a mild intoxication animal model, characterized in that, Includes the following steps: S1. Pre-treat ICR mice by fasting; S2. Administer 38-50% (v / v) ethanol solution via gavage at a rate of 5-15 mL / kg.

2. The construction method according to claim 1, characterized in that, In step S2, the volume concentration of ethanol is 38% (v / v).

3. A method for constructing a severe alcohol intoxication animal model, characterized in that, Includes the following steps: S1. Pre-treat ICR mice by fasting; S2. Administer 40-60% (v / v) ethanol solution via gavage at a rate of 17-20 mL / kg.

4. The construction method according to claim 3, characterized in that, In step S2, the volume concentration of ethanol is 50% (v / v).

5. The construction method according to claim 3, characterized in that, In step S2, the volume of ethanol administered via gavage is 17 mL / kg.

6. The application of the mild intoxication animal model according to any one of claims 1-2 or the severe intoxication animal model according to any one of claims 3-5 in the evaluation of hangover relief products.

7. A method for evaluating mild intoxication, characterized in that, Includes the following steps: S1. Pre-treat ICR mice by fasting; S2. Administer 38% (v / v) ethanol solution via gavage at a rate of 8-10 mL / kg; S3. After gavage, the behavioral indicators of the mice were tested, including: righting reflex, total distance of open field movement, average speed of open field movement, and dwell time in the rotundus test. S4. After gavage, the righting reflex of the mice was still present, and the total distance and average speed of open field movement were significantly increased. The dwell time in the rotundus test was significantly reduced and could be maintained for 2 hours or more after modeling, indicating mild intoxication.

8. The evaluation method according to claim 7, characterized in that, In step S2, the gavage rate is 0.1 mL / s.

9. A method for evaluating severe intoxication, characterized in that, Includes the following steps: S1. Pre-treat ICR mice by fasting; S2. Administer 50% (v / v) ethanol solution via gavage at a rate of 17-20 mL / kg; S3. Detect the righting reflex of the mouse. If the righting reflex disappears, it indicates severe intoxication. Record the time from when the righting reflex disappears after the mouse is given by gavage, which is the time of intoxication.

10. The evaluation method according to claim 9, characterized in that, In step S2, after the ICR mice were administered 50% (v / v) ethanol by gavage, they were grouped according to the time of intoxication. The time of intoxication was divided into four time periods: <30 min, 30-60 min, 60-90 min and ≥90 min.

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