Application of methylophiopogonanone B in preparation of medicine for preventing and treating acute alcoholism

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of methylophiopogonanone B in preparation of a medicine for preventing and treating acute alcoholism. The methylophiopogon flavanone B can relieve clinical symptoms or physiological abnormalities of acute alcoholism, shorten sobering time and sleep time of experimental animals suffering from acute alcoholism, reduce liver indexes, maintain normal morphological structures of liver tissues and cells, reduce levels of glutamic-pyruvic transaminase and glutamic oxalacetic transaminase in serum and improve the curative effect of acute alcoholism. The composition has the effects of reducing serum total cholesterol and triglyceride levels, increasing the activity of superoxide dismutase and glutathione peroxidase, increasing the content of reduced glutathione, reducing the content of malondialdehyde, improving the functions of organs such as liver, kidney, stomach and the like of acute alcoholism animals, regulating proteome expression of acute alcoholism mice, and treating acute alcoholism. The composition has the effect of preventing and treating acute alcoholism.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of methylophiopogonone B in the preparation of drugs for preventing and treating acute alcohol poisoning. Background Art

[0002] Ethanol is the main component of alcoholic beverages. After entering the human body, ethanol is mainly metabolized through the liver, skin and respiratory system, and the liver is the main metabolic site. The ethanol dehydrogenase oxidation system, catalase oxidation system and microsomal oxidation system in the liver, mainly cytochrome P450 2E1 and other substances, are involved in ethanol metabolism. In the ethanol dehydrogenase oxidation system, ethanol is oxidized by ethanol dehydrogenase to form acetaldehyde, and acetaldehyde is oxidized by acetaldehyde dehydrogenase to acetic acid, which can then enter the tricarboxylic acid cycle and be decomposed into water and CO2 and excreted from the body. Acute alcohol poisoning is an acute disease that causes serious damage to multiple organs such as the liver, stomach and brain of drinkers or even leads to death after one-time or short-term excessive consumption of alcoholic beverages. There is a positive linear relationship between the amount of alcohol consumed and the death risk of various disease factors. An increase in the weekly alcohol consumption will lead to an increase in the all-cause death risk of men, and the death risks caused by alcoholic liver disease or cirrhosis, cardiovascular diseases, cancers and other reasons will all increase. See the study by Iona et al., Alcohol intake and cause-specific mortality: conventional and genetic evidence in a prospective cohort study of 512000 adults in China [J]. The Lancet Public Health, 2023. Alcohol poisoning is highly harmful to human health and may be fatal in severe cases. Clinically, western medicines such as naloxone and alixin A are used to treat acute alcohol poisoning, but there are side effects or limitations such as arrhythmia, increased blood pressure, nausea and vomiting, or complex administration methods. See the study by Meng Qinglin et al., Current situation and prospect of naloxone in the treatment of cerebral infarction [J]. Chinese Journal of Critical Care Medicine, 2000; the study by Gao Wanlu et al., New progress in drug treatment of acute alcohol poisoning [J]. Pharmaceutical and Clinical Research, 2015. It can be seen that the harm of alcohol to the human body is self-evident, and western medicines have certain limitations or side effects in the clinical application of treating alcohol poisoning. Therefore, it has certain practical significance to give play to the advantages of traditional Chinese medicines in the prevention and treatment of alcohol poisoning and to explore active ingredients of traditional Chinese medicines or natural drugs.

[0003] In the prior art, Patent CN113521195B discloses a medicine-food homologous composition for alcohol use disorder, which is composed of Dendrobium officinale, Polygonatum sibiricum, and Pueraria lobata, and has a certain effect on improving alcohol use disorder in mice; Patent CN104286833B discloses a composition for relieving alcohol and anti-fatigue, which is composed of Astragalus membranaceus, Polygonatum sibiricum, Glycyrrhiza uralensis, Ziziphus jujuba, guarana, silkworm pupa powder, honey, and granulated sugar, and has the effects of relieving alcohol and anti-fatigue; Patent CN103705731B discloses a traditional Chinese medicine composition for treating chronic alcoholic liver disease, which is composed of Alisma orientale, Stephania tetrandra, Artemisia capillaris, Polygonum multiflorum, Ligusticum wallichii, Angelica sinensis, Citrus aurantium, Galla chinensis, Bupleurum chinense, Polygonatum sibiricum, Scutellaria baicalensis, Lycium barbarum, Poria cocos, Glycyrrhiza uralensis, etc., and can be used to treat chronic alcoholic liver disease. However, most of the existing technologies mainly prevent and treat chemical liver injury and alcoholic liver injury, and rarely involve lesions or abnormalities at different levels such as tissues and organs like the kidney, stomach, brain, behavioral actions like ataxia, conscious nerves like coma, metabolic stress like glucose metabolism, oxidative stress, and molecular cells like proteome. In fact, acute alcohol poisoning is a comprehensive disease. In addition to liver injury, it often involves damage to tissues and organs such as the stomach, kidney, and brain. In addition, there are usually typical symptoms or abnormalities at the levels of behavioral actions, conscious nerves, metabolic stress, and molecular cells.

[0004] It can be seen that most of the existing technologies mainly use the traditional Chinese medicine Polygonatum sibiricum directly in combination with compound combinations of other traditional Chinese medicines, or use Polygonatum sibiricum water extract, Polygonatum sibiricum polysaccharide, etc. to treat or prevent alcoholic diseases. Further research is needed on whether specific monomeric compounds have applications in preventing and treating acute alcohol poisoning. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides the application of methylophiopogonone B in the preparation of drugs for preventing and treating acute alcohol poisoning.

[0006] The technical solution of the present invention is as follows.

[0007] The application of methylophiopogonone B in the preparation of drugs for preventing and treating acute alcohol poisoning, wherein the methylophiopogonone B is methylophiopogonone B extracted from Polygonatum sibiricum. Among them, methylophiopogonone B is used in the form of a pharmaceutical preparation for preparing drugs for preventing and treating acute alcohol poisoning. The pharmaceutical preparation includes methylophiopogonone B as the sole active ingredient and a pharmaceutically acceptable carrier. The pharmaceutical preparation is a dripping pill, a granule, or a functional food.

[0008] The present invention discovers that methylophiopogonone B can alleviate the clinical symptoms or physiological abnormalities of acute alcohol poisoning. This is mainly because methylophiopogonone B has molecular docking affinity activities for alcohol dehydrogenase, aldehyde dehydrogenase, catalase, cytochrome P450 2E1, etc., and can shorten the sobering-up time and sleep time after acute alcohol poisoning, thereby reducing the liver index, maintaining the normal morphological structure of liver tissues and cells, reducing the levels of serum alanine aminotransferase and aspartate aminotransferase, reducing the levels of serum total cholesterol and triglycerides, increasing the activities of superoxide dismutase and glutathione peroxidase, increasing the content of reduced glutathione, reducing the content of malondialdehyde, improving the functions of organs such as the liver, kidney, and stomach in animals with acute alcohol poisoning, regulating the proteome expression after acute alcohol poisoning, and having the effect of preventing and treating acute alcohol poisoning.

[0009] In another preferred embodiment, the drug is a drug for shortening the sleep time and sobering-up time after acute alcohol poisoning.

[0010] In another preferred embodiment, the drug is a drug for reducing the contents of serum alanine aminotransferase and aspartate aminotransferase after acute alcohol poisoning.

[0011] In another preferred embodiment, the drug is a drug for reducing the liver index after acute alcohol poisoning.

[0012] In another preferred embodiment, the drug is a drug for repairing the abnormal morphological structure of liver tissues and cells after acute alcohol poisoning.

[0013] In another preferred embodiment, the drug is a drug for reducing the levels of serum urea nitrogen and creatinine after acute alcohol poisoning.

[0014] In another preferred embodiment, the drug is a drug for reducing the levels of total cholesterol and triglycerides in serum after acute alcohol poisoning.

[0015] In another preferred embodiment, the drug is a drug for increasing the activities of superoxide dismutase and glutathione peroxidase, reducing the content of malondialdehyde, and increasing the content of reduced glutathione after acute alcohol poisoning.

[0016] In another preferred embodiment, the drug is a drug for improving the functions of the liver, kidney, and stomach organs after acute alcohol poisoning.

[0017] In another preferred embodiment, the drug is a drug for regulating the proteome expression after acute alcohol poisoning.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] In the present invention, methylophiopogonone B can alleviate the clinical symptoms or physiological abnormalities of acute alcoholism. The chemical structure of the monomer compound methylophiopogonone B is clear, and the plant source of methylophiopogonone B is Polygonatum sibiricum, which belongs to the items that are both food and medicine, with high safety, and can be used to make medicines, health foods or ordinary foods, providing a guarantee for the application of methylophiopogonone B in the prevention and treatment of acute alcoholism.

[0020] The present invention discovers that methylophiopogonone B has molecular docking affinity activities for alcohol dehydrogenase, aldehyde dehydrogenase, catalase, cytochrome P450 2E1, etc., and can shorten the sobering time and sleeping time after acute alcoholism, thereby reducing the liver index, maintaining the normal morphological structure of liver tissues and cells, reducing the levels of serum alanine aminotransferase and aspartate aminotransferase, reducing the levels of serum total cholesterol and triglyceride, increasing the activities of superoxide dismutase and glutathione peroxidase, enhancing the content of reduced glutathione, reducing the content of malondialdehyde, improving the functions of organs such as liver, kidney, and stomach in animals with acute alcoholism, regulating the proteome expression after acute alcoholism, and having the effect of preventing and treating acute alcoholism. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the chemical structure diagram of methylophiopogonone B.

[0022] Figure 2 It is the molecular docking energy heat map. Among them, MO-B is methylophiopogonone B, Saponarin is saponarin, Vitexin is vitexin, and A, B, and C all represent the results of the molecular docking energy ranking.

[0023] Figure 3 It is the schematic diagram of the binding mode of methylophiopogonone B and alcohol dehydrogenase.

[0024] Figure 4 It is the schematic diagram of the binding mode of methylophiopogonone B and aldehyde dehydrogenase.

[0025] Figure 5 It is the schematic diagram of the binding mode of methylophiopogonone B and catalase.

[0026] Figure 6 It is the schematic diagram of the binding mode of methylophiopogonone B and cytochrome P450 2E1 enzyme.

[0027] Figure 7 It is the diagram of the effect of methylophiopogonone B intervention on the drunken behavior of mice.

[0028] Figure 8Effect diagram of methylophiopogonone B intervention on serum AST and ALT levels in mice; among them, A is the effect diagram of ALT level; B is the effect diagram of AST level. Different letters in the figure indicate significant differences between treatment groups (P < 0.05).

[0029] Figure 9 Effect diagram of methylophiopogonone B intervention on liver index in mice.

[0030] Figure 10 Effect diagram of methylophiopogonone B intervention on liver tissue pathological sections of mice with acute alcohol poisoning; among them, A is the NC control group; B is the MC model group; C is the low-dose group of methylophiopogonone B (MO-B low); D is the high-dose group of methylophiopogonone B (MO-B high). The magnification of the picture is 200 times.

[0031] Figure 11 Effect diagram of methylophiopogonone B intervention on kidneys of mice with acute alcohol poisoning; among them, A is the content diagram of serum urea nitrogen (BUN); B is the content diagram of serum creatinine (CRE). Different letters in the figure indicate significant differences between treatment groups (P < 0.05).

[0032] Figure 12 Effect diagram of methylophiopogonone B intervention on serum TC and TG contents of mice with acute alcohol poisoning; among them, A is the content diagram of serum TC; B is the content diagram of serum TG. Different letters in the figure indicate significant differences between treatment groups (P < 0.05).

[0033] Figure 13 Effect diagram of methylophiopogonone B intervention on antioxidant capacity of mice with acute alcohol overdose; among them, A is SOD activity; B is GSH-Px activity; C is GSH content; D is MDA content. Different letters in the figure indicate significant differences between treatment groups (P < 0.05). Detailed implementation manners

[0034] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0035] Unless otherwise specified, the experimental methods described in the following embodiments are all conventional methods; unless otherwise specified, the reagents and materials can all be purchased in the market.

[0036] Polygonatum sibiricum Redoute is the dried rhizome of the plants of the genus Polygonatum in the family Liliaceae, and it is a unique Chinese medicinal material with both medicinal and edible properties in China. "Japanese Pharmacopoeia" records that it can "tonify five kinds of overstrains and seven kinds of impairments, strengthen muscles and bones, stop hunger, withstand cold and heat, benefit the spleen and stomach, moisten the heart and lungs, and eating it can maintain a youthful appearance"; "Compendium of Materia Medica of Yunnan" records that it "tonifies the middle qi, dispels wind-dampness, and tranquilizes the five zang-organs. Taking it for a long time can lighten the body and prolong life. It treats five kinds of overstrains and seven kinds of impairments, strengthens muscles and bones, withstands cold and heat, benefits the spleen and stomach, and opens the heart and lungs". Methylophiopogonanone B, with the molecular formula C 19 H 20 O5, and its structural formula is shown in Figure 1 , is one of the flavonoid substances in Polygonatum sibiricum Redoute. Most of the existing technologies mainly use the compound combination of the Chinese medicinal material Polygonatum sibiricum Redoute and other various Chinese medicinal materials or the water extract and polysaccharide of Polygonatum sibiricum Redoute to treat or prevent alcoholic diseases, and there is no report on the prevention and treatment of acute alcoholism using the monomer compound of methylophiopogonanone B.

[0037] The embodiment of the present invention provides the application of methylophiopogonanone B in the preparation of drugs for relieving alcoholism, and the application of methylophiopogonanone B in the prevention and treatment of acute alcoholism, that is, it can relieve the clinical symptoms or physiological abnormalities of acute alcoholism. Specifically, methylophiopogonanone B has molecular docking affinity activities for alcohol dehydrogenase, aldehyde dehydrogenase, catalase, cytochrome P4502E1, etc., can shorten the sobering-up time and sleeping time of experimental animals with acute alcoholism, reduce the liver index, maintain the normal morphological structure of liver tissues and cells, reduce the levels of serum alanine aminotransferase and aspartate aminotransferase, reduce the levels of serum total cholesterol and triglyceride, increase the activities of superoxide dismutase and glutathione peroxidase, increase the content of reduced glutathione, reduce the content of malondialdehyde, improve the functions of organs such as the liver, kidney, and stomach of animals with acute alcoholism, regulate the protein group expression of mice with acute alcoholism, and has the effect of preventing and treating acute alcoholism.

[0038] The clinical manifestations of acute alcoholism in the embodiment of the present invention are: (1) After alcoholism, the sobering-up time, sleeping time, and the extension of the sobering-up time, disappearance of the righting reflex, etc.; (2) Damage to the nervous system, coma or lethargy, etc.; (3) Damage to the functions of organs such as the liver, stomach, kidney, and brain; (4) Appearance of oxidative stress state, etc.; (5) Abnormal protein expression.

[0039] The following specifically describes the application of methylophiopogonanone B in the preparation of drugs for preventing and treating acute alcoholism.

[0040] Methylophiopogonanone B in the following examples was purchased from Shanghai Yuanye Bio-Technology Co., Ltd., polyethylene glycol was purchased from Sigma Company in the United States, maltodextrin was purchased from Sinopharm Chemical Reagent Co., Ltd., arabinose was purchased from Sigma Company in the United States, and citric acid was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0041] Example 1

[0042] Methylophiopogonone B dropping pills, and the specific preparation process of the Methylophiopogonone B dropping pills is as follows:

[0043] Weigh Methylophiopogonone B and polyethylene glycol according to the mass ratio of 1:5, heat them to the molten state at 80 °C, and stir for 15 min at 65 rpm to obtain a mixed liquid medicine. Adjust the dropping distance to 5 cm, and drop the mixed liquid medicine into the liquid paraffin condensate at a speed of 45 drops per minute to prepare Methylophiopogonone B dropping pills.

[0044] Example 2

[0045] Methylophiopogonone B granules, and the specific preparation process of the Methylophiopogonone B granules is as follows:

[0046] Weigh the solid materials of Methylophiopogonone B, maltodextrin, arabinose and citric acid according to the mass ratio of 1:86:10:3, dissolve them evenly in water according to the solid-liquid ratio of 1 kg:5 L, and then spray dry with an inlet air temperature of 170 °C, an outlet air temperature of 85 °C, a sample injection flow rate of 2 mL / min, an air flow rate of 500 L / h, and pass through an 80-mesh sieve to prepare Methylophiopogonone B granules.

[0047] The following further elaborates on the prevention and treatment effects of Methylophiopogonone B of the present invention on acute alcohol poisoning through test examples:

[0048] Test Example 1: Molecular docking study of Methylophiopogonone B

[0049] Experimental method

[0050] 1) Receptor protein

[0051] Alcohol dehydrogenase (PDB ID: 1u3t, abbreviated as ADH), aldehyde dehydrogenase (PDB ID: 5tei, abbreviated as ALDH), catalase (PDB ID: 1qqw, abbreviated as CAT), cytochrome P450 2E1 (PDB ID: 3lc4, abbreviated as CYP2E1), and use PyMOL to remove the water molecules and ligands of the protein.

[0052] 2) Small molecule ligand

[0053] Methylophiopogonone B (CAS No.: 74805-91-7, abbreviated as MO-B), vitexin (CAS No.: 3681-93-4, abbreviated as Vitexin), saponarin (CAS No.: 20310-89-8, abbreviated as Saponarin).

[0054] 3) Molecular docking method: Use AutoDock 1.5.6 software to process and dock small molecule ligands with receptor proteins, and use PyMOL software for visual analysis.

[0055] Experimental results

[0056] Molecular docking studies of small molecule ligands with receptor proteins found that the docking energies of methylophiopogonin B were as follows: the docking energy with ADH was -5.01 kcal / mol, the docking energy with ALDH was -5.27 kcal / mol, the docking energy with CAT was -4.15 kcal / mol, and the docking energy with CYP2E1 was 3.44 kcal / mol. Figure 2 For the energy analysis of molecular docking of three compounds, methylophiopogonin B, vitexin, and saponarin, with ADH, ALDH, CAT, and CYP2E1, it can be seen that the order of docking energies is: methylophiopogonin B < vitexin < saponarin. Thus, it can be known that the molecular docking effect of methylophiopogonin B with the four proteins is the best.

[0057] From Figures 3 to 6 it can be seen that methylophiopogonin B can bind to all four proteins, and the binding sites include: the binding sites with ADH are VAL-6 and LYS-8, as Figure 3 shown; the binding site with ALDH is ASP-392, as Figure 4 shown; the binding sites with CAT are GLU-139, ARG-380, ARG-382, and GLN-415, as Figure 5 shown; the binding site with CYP2E1 is LYS-486, as Figure 6 shown. Alcohol dehydrogenase, aldehyde dehydrogenase, catalase, and cytochrome P450 2E1 are closely related to alcohol metabolism. The results of molecular docking studies suggest that methylophiopogonin B may play an important role in alcohol catabolism.

[0058] Test Example 2: Effects of methylophiopogonin B on the righting reflex and behavior of alcohol-intoxicated mice

[0059] 1) Experimental materials

[0060] SPF-grade Kunming mice, male, 6 weeks old, 100 mice, weighing 18 - 22 g. The experimental animals were purchased from Henan SBKS Biotechnology Co., Ltd. and were raised in the Animal Center of Shaanxi Normal University, and passed the experimental animal ethics review.

[0061] 2) Experimental reagents

[0062] Mainly including: methylophiopogonin B, silymarin capsules (product batch number: B2002580, manufacturer: MADAUS GmbH), 52° Hongxing Erguotou liquor.

[0063] 3) Experimental methods

[0064] Establishment of acute alcoholism model: Determine the amount of alcohol administered to mice through preliminary experiments. Randomly select 20 mice and divide them into 4 groups, with 5 mice in each group. Administer white liquor at doses of 0.12 mL / 10 g, 0.14 mL / 10 g, 0.16 mL / 10 g, and 0.18 mL / 10 g respectively by gavage. According to the animal experiment protocol, whether the mouse shows a righting reflex and maintains the righting reflex state for more than 30 s is used as the main criterion for judging drunkenness.

[0065] Grouping and treatment of animal experiments: The experimental animals are fed with ordinary animal feed. After all mice are adaptively fed for a period of time, the experiment begins.

[0066] During the experiment, the mice are randomly divided into 5 groups: The blank control group is denoted as the NC group, and normal saline is administered by gavage; the model group is denoted as the MC group; the silymarin group is denoted as the SC group, and silymarin at a dose of 50 mg / kg is administered by gavage; the low-dose methylophiopogonin B group is denoted as the MO-B low-dose group, and methylophiopogonin B at a dose of 10 mg / kg is administered by gavage; the high-dose methylophiopogonin B group is denoted as the MO-B high-dose group, and methylophiopogonin B at a dose of 20 mg / kg is administered by gavage. After the treatment, the mice are sacrificed, and the corresponding materials are collected for subsequent research.

[0067] Observation of drunken behavior in mice: Observe and record the mental state, behavioral responses, death conditions, etc. of mice in each group, and statistically analyze indicators such as the time of giving alcohol, the time of disappearance of the righting reflex, and the time of recovery of the righting reflex. The calculation methods for drunkenness time, etc. are as follows:

[0068] Drunkenness time = Time of disappearance of the righting reflex - Time of giving alcohol;

[0069] Sleeping time = Time of recovery of the righting reflex - Time of giving alcohol;

[0070] Sobering-up time = Time of recovery of the righting reflex - Time of disappearance of the righting reflex.

[0071] 4) Experimental results

[0072] Through preliminary experiments, it was found that when the gavage volume of white liquor was 0.16 ml / 10 g, the proportion of mice showing the righting reflex was the highest. When the gavage volume was too small, it was difficult for mice to show the righting reflex. When the gavage volume was too large, mice were prone to death. After comprehensive judgment, 0.16 ml / 10 g was selected as the alcohol gavage dose for mice in the acute alcohol poisoning model. After a single gavage of alcohol, no death occurred in each group of mice, and the appearance rate of the righting reflex was above 90%. A small number of mice may not show the righting reflex due to individual differences in alcohol tolerance, but all mice showed an excited state after gavage of alcohol, with increased activity, uncoordinated movements, irritability. After a period of time, their actions gradually became sluggish, their hair color was dull and not smooth, their muscles were relaxed, most showed the righting reflex phenomenon, and the degree of lethargy increased. Among them, the symptoms of the mice in the model group were the most obvious. The conditions of each group of mice with drug intervention improved to varying degrees compared with the model group. From Figure 7 It can be seen that the intervention of methylophiopogonone B can shorten the sleep time and hangover time of mice, indicating that it has a certain degree of alleviating effect on the drunken symptoms of alcohol-poisoned mice.

[0073] Experimental Example 3: Effect of Methylophiopogonone B on the Liver of Mice with Acute Alcohol Poisoning

[0074] 1) Experimental Materials, Reagents and Instruments

[0075] For experimental animals and their treatment, see Experimental Example 2. Kits for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were both purchased from Nanjing Jiancheng Bioengineering Institute. The instruments mainly included a full-wavelength microplate reader, a high-speed refrigerated centrifuge and an ultra-low temperature refrigerator.

[0076] 2) Experimental Materials and Methods

[0077] Liver function analysis: Take the blood of mice and process it according to the kit instructions to detect the levels of serum ALT and AST in mice.

[0078] Liver index analysis: Weigh and record the body weight of mice before euthanasia; after euthanasia, dissect to take out their livers, wash the blood with physiological saline, dry the water with filter paper and then weigh and record. The obtained results were used for the calculation of the liver index, and the calculation formula was:

[0079] Liver index (%) = liver weight / body weight × 100%.

[0080] 3) Liver pathological section study: Take out the liver tissue of mice, rinse it with physiological saline to remove the blood, then fix it with 4% paraformaldehyde solution, dehydrate it with ethanol, and then carry out xylene transparency, wax infiltration, embedding, sectioning and HE staining in sequence. The processed organ tissues were observed and photographed with a light microscope.

[0081] 4) Experimental Results

[0082] Effect of Methylophiopogonone B Intervention on Serum Transaminase Activity in Mice

[0083] Serum transaminases mainly include aspartate transaminase and alanine transaminase, which are important indicators for evaluating liver function. Under normal conditions of the body, the activities of AST and ALT in serum are relatively low. When the liver is damaged, AST and ALT will enter the blood, leading to an increase in the activity of serum transaminases. Therefore, the activity of transaminases in serum can be used to evaluate the degree of liver damage. As Figure 8 can be seen, the serum AST level in the MC group was significantly higher than that in the blank NC group (P < 0.05), and the serum ALT level in the MC group was also significantly higher than that in the NC group (P < 0.05), indicating that alcohol had a serious adverse effect on the liver function of mice. After the intervention of methylophiopogonone B, the AST and ALT levels in the serum of alcohol-intoxicated mice decreased compared with those in the MC group, and the degree of decrease in the serum AST and ALT levels in the high-dose methylophiopogonone group exceeded that in the silymarin group, indicating that methylophiopogonone B could protect the liver of acute alcohol-intoxicated mice and weaken the liver damage caused by alcohol intoxication.

[0084] Effect of Methylophiopogonone B Intervention on Liver Index in Mice

[0085] Under normal conditions, the ratio between the liver and body weight of mice is relatively stable. When the liver is damaged, the liver index often increases accordingly. From Figure 9 can be seen, the liver index of mice in the model group was extremely significantly higher than that in the blank control group (P < 0.05), indicating that alcohol caused liver damage in mice. After intragastric administration of methylophiopogonone B, the liver index of acute alcohol-intoxicated mice decreased to a certain extent compared with that in the MC group, indicating that MO-B treatment had a certain protective effect on the liver of acute alcohol-intoxicated mice.

[0086] Effect of Methylophiopogonone B on Liver Pathological Sections of Mice

[0087] The liver is the main metabolic organ in vertebrates and also the main metabolic site of alcohol. The physiological state of the liver has a great impact on the metabolic activities of the body. As Figure 10It can be seen that the hepatocytes of normal mice are radially arranged around the central vein to form hepatic plates. Hepatic sinusoids are formed between adjacent hepatic plates. The sinusoids communicate with each other through the pores on the hepatic plates to form a reticular duct. In sections, the cross-section of the hepatic plate is cord-like and is called hepatic cord. Hepatic macrophages, lymphocytes, etc. are present in the hepatic sinusoids; they and some other units together form hepatic lobules. It can be seen from section A that the hepatic lobule structure of the liver in the NC group is complete, the chromatin staining of the liver nucleus is clear, the morphology and size are relatively regular, and there is no obvious lipid accumulation. There are obvious differences between the liver sections of the MC group and the NC group. The hepatocytes are not radially arranged along the central vein. The volume of the hepatocytes is small, some hepatocytes are necrotic, the hepatic sinusoid tissue is damaged, and there are many small lipid vacuoles, indicating liver damage. In the low-dose methylophiopogonanone B treatment group, the volume of the hepatocytes is large and they are radially arranged around the central vein, but some lipid vacuoles can still be seen; in the high-dose methylophiopogonanone B treatment group, except that the hepatocytes are smaller, the arrangement of the hepatocytes, the hepatic sinusoid tissue, etc. are all close to the NC group, and there are no obvious lipid vacuoles. It can be seen that alcohol causes damage to the liver tissue of mice, affects the arrangement of hepatocytes, etc., and hinders the normal physiological function of hepatic lobules. After intervention with different doses of methylophiopogonanone B, the liver tissue and cell problems of mice have improved to varying degrees, indicating that methylophiopogonanone B can repair the abnormal morphological structure of the liver tissue and cells of animals with acute alcohol poisoning and has a relieving effect on liver damage caused by alcohol poisoning in mice.

[0088] Experimental Example 4: Effect of Methylophiopogonanone B on the Kidneys of Mice with Acute Alcohol Poisoning

[0089] 1) Experimental Materials and Reagents

[0090] The experimental animals and their treatments are as described in Experimental Example 2. The urea nitrogen (BUN) kit and creatinine (CRE) kit were both purchased from Nanjing Jiancheng Bioengineering Institute. The instruments mainly include a full-wavelength microplate reader, a high-speed refrigerated centrifuge, and an ultra-low temperature freezer.

[0091] 2) Experimental Method

[0092] Take the serum of mice with acute alcohol poisoning and measure the contents of serum urea nitrogen and creatinine according to the requirements of the kit.

[0093] 3) Experimental Results

[0094] Creatinine is a small molecule formed in muscle, is rarely absorbed in the renal tubules, can be filtered by the glomerulus, and serum CRE is an important indicator reflecting glomerular filtration function. Its concentration change is one of the main indicators for clinically detecting renal function; BUN is a metabolite of protein in the body and is also an important marker for evaluating the degree of kidney injury. From Figure 11It can be seen that compared with the NC group, the contents of serum creatinine and urea nitrogen in the MC group of mice increased significantly, indicating that alcohol caused certain damage to the kidneys of mice. After the intervention of methylophiopogonin B, both CRE and BUN decreased significantly, and were similar to those in the silymarin group, indicating that the intervention of methylophiopogonin B could alleviate kidney injury caused by alcoholism.

[0095] Experimental Example 5: Effect of Methylophiopogonin B on Blood Lipids in Mice with Acute Alcoholism

[0096] 1) Experimental Materials and Reagents

[0097] For experimental animals and their treatment, see Experimental Example 2. Kits for triglyceride (TG) and total cholesterol (TC) were both purchased from Nanjing Jiancheng Bioengineering Institute. The instruments mainly included a full-wavelength microplate reader, a high-speed refrigerated centrifuge, and an ultra-low temperature refrigerator.

[0098] 2) Experimental Method

[0099] Take the serum of mice with acute alcoholism and measure the contents of serum triglyceride and total cholesterol according to the requirements of the kit.

[0100] 3) Experimental Results

[0101] The contents of triglyceride and total cholesterol in serum are important indicators reflecting the body's blood lipid metabolism. When the liver is damaged, it may cause lipid metabolism disorders. Figure 12 It can be seen that the TG content in the MC group was significantly higher than that in the blank control group (P < 0.05), and the TC content was significantly higher than that in the NC group (P < 0.05), indicating that obvious blood lipid metabolism disorders occurred in mice under the action of alcohol; compared with the MC group, the TG and TC contents in both the low-dose MO-B group and the high-dose MO-B group were significantly decreased (P < 0.05). It can be inferred that methylophiopogonin B can regulate the liver lipid metabolism of mice with acute alcoholism and alleviate the blood lipid abnormalities caused by alcoholism.

[0102] Experimental Example 6: Effect of Methylophiopogonin B on Antioxidant Capacity in Mice with Acute Alcoholism

[0103] 1) Experimental Materials

[0104] For experimental animals and their treatment, see Experimental Example 2. Kits for superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), reduced glutathione (GSH), and malondialdehyde (MDA) were all purchased from Nanjing Jiancheng Bioengineering Institute. The instruments mainly included a grinder, a full-wavelength microplate reader, a high-speed refrigerated centrifuge, and an ultra-low temperature refrigerator.

[0105] 2) Experimental Method

[0106] After sacrificing the mice, the livers were dissected. According to the kit instructions, physiological saline and small steel beads for grinding were added, and the mixture was ground evenly with a grinder. After centrifugation to remove the precipitate, the supernatant was used for the detection of antioxidant indexes.

[0107] 3) Experimental results

[0108] The main metabolic site of alcohol in the body is the liver, and acute alcohol poisoning is closely related to the oxidative stress state of the liver. The imbalance between free radicals induced by oxidative stress and the antioxidant defense system can cause tissue and organ damage, which plays an important role in the pathogenesis of acute alcohol poisoning. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and reduced glutathione (GSH) together constitute the body's antioxidant defense system. The content of malondialdehyde (MDA) is one of the commonly used indexes to evaluate the level of oxidative stress. Figure 13 It can be seen that compared with the NC group, the MDA content in the livers of mice in the MC group was significantly increased (P < 0.05), the SOD activity was significantly decreased (P < 0.05), the GSH-Px activity was significantly decreased (P < 0.05), and the GSH content was significantly decreased (P < 0.05). This indicates that the livers of alcohol-intoxicated mice are in a relatively severe oxidative stress state, the redox balance of the mouse liver is disrupted, and the liver function may be affected to a certain extent. After the intervention of methylophiopogonin B, compared with the MC group, the SOD activity was significantly increased (P < 0.05), the GSH-Px activity was also significantly increased (P < 0.05), the MDA content decreased, and the GSH content increased, showing an obvious dose-dependent relationship. This indicates that the activity of redox enzymes in the mouse liver in vivo was enhanced after the intervention of methylophiopogonin B, and the imbalance state of redox was alleviated to a certain extent. It shows that methylophiopogonin B can improve the oxidative stress state of the livers of mice with acute alcohol poisoning.

[0109] Experimental Example 7: Proteomic study on the prevention and treatment of acute alcohol poisoning in mice by methylophiopogonin B

[0110] 1) Experimental materials

[0111] The treatment method for mice with acute alcohol poisoning was the same as above. After the mouse livers were taken out of the body, they were quickly frozen with liquid nitrogen and then stored temporarily in a -80°C refrigerator for later use.

[0112] 2) Experimental methods

[0113] Protein extraction: An appropriate amount of liver tissue stored at -80°C was taken, lysis buffer was added, and after grinding at low temperature, the tissue homogenate was transferred to an EP tube. The sample was sonicated in an ice-water bath with a cell disruptor for 20 min, and the lysed sample was centrifuged at 12,000 rpm and 4°C for 20 min to collect the supernatant.

[0114] Protein quantification: The concentration of proteins in liver tissue samples was determined according to the instructions of the BCA quantification kit.

[0115] Protein enzymolysis: According to the experimental operation procedures, protein samples were taken and enzymolyzed with proteases.

[0116] Polypeptide desalting: The enzymolyzed samples were desalted using a desalting column.

[0117] Electrophoresis: SDS-PAGE gel electrophoresis combined with Coomassie brilliant blue staining was used to analyze the quality of protein samples.

[0118] Mass spectrometry detection and analysis of proteins: After separation by the nano-UPLC liquid phase system, the total peptide samples were used in conjunction with a mass spectrometer for data acquisition. The scanning range of the mass spectrometer was 100 - 1700 m / z. The original mass spectrometry data files were searched against a database using software to obtain data on relevant peptide segments and proteins, analyze the proteomic changes in the NC group, MC group, and high-dose MO-B group, screen for differentially expressed proteins, and perform bioinformatics analysis.

[0119] 3) Experimental results

[0120] A total of 37,132 polypeptides were obtained, and 4,323 proteins were identified, including 186 differentially expressed proteins in the MC group and the high-dose MO-B group. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses found that the differentially expressed proteins were involved in biological processes or functional localizations such as the maintenance of gastrointestinal epithelial cells, nervous system development, alcohol metabolism, and oxidative stress. Acute alcohol intoxication often affects the physiological activities of multiple organs, including the liver, stomach, and brain. Methylophiopogonanone B may affect the physiological activities and functions of the liver, stomach, and brain organs by regulating the expression of related proteins, thereby playing a role in preventing and treating acute alcohol intoxication.

[0121] In summary, methylophiopogonanone B has molecular docking affinity for ADH, ALDH, CAT, and CYP2E1, can shorten the sobering-up time and sleep time of experimental animals with acute alcohol intoxication, reduce the liver index, maintain the normal morphological structure of liver tissues and cells, lower the levels of serum alanine aminotransferase and aspartate aminotransferase, reduce the levels of serum total cholesterol and triglycerides, increase the activities of superoxide dismutase and glutathione peroxidase, increase the content of reduced glutathione, reduce the content of malondialdehyde, improve the functions of organs such as the liver, kidney, and stomach in animals with acute alcohol intoxication, regulate the protein expression in mice with acute alcohol intoxication, and has the effect of preventing and treating acute alcohol intoxication.

[0122] The above content is only to illustrate the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. Any modifications made on the basis of the technical solution according to the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. The use of methyl ophiopogon japonicus flavanone B in the preparation of a drug for the prevention and treatment of acute alcohol poisoning, characterized in that: The methyl ophiopogon japonicus flavanone B is methyl ophiopogon japonicus flavanone B extracted from polygonatum sibiricum.

2. The use according to claim 1, characterized in that: The medicine is a medicine for shortening the sleeping time and sobering time after acute alcohol poisoning.

3. The use according to claim 1, characterized in that: The medicine is a medicine for reducing the levels of serum alanine aminotransferase and aspartate aminotransferase after acute alcohol poisoning.

4. The use according to claim 1, characterized in that: The medicine is a medicine for reducing liver index after acute alcohol poisoning.

5. The use according to claim 1, characterized in that: The medicine is a medicine for repairing the abnormal morphological structure of liver tissue and cells after acute alcohol poisoning.

6. The use according to claim 1, characterized in that: The drug is a drug for reducing serum urea nitrogen and creatinine levels after acute alcohol intoxication.

7. The use according to claim 1, characterized in that: The medicine is a medicine for reducing the total cholesterol and triglyceride content in serum after acute alcohol poisoning.

8. The use according to claim 1, characterized in that: The drug is a drug for increasing the activities of superoxide dismutase and glutathione peroxidase after acute alcohol poisoning, and for reducing the content of malondialdehyde and increasing the content of reduced glutathione.

9. The use according to claim 1, characterized in that: The medicine is a medicine for improving the liver, kidney and stomach organ functions after acute alcohol poisoning.

10. The use according to claim 1, characterized in that: The drug is a drug for regulating the expression of proteome after acute alcohol intoxication.

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