CN113456660A

CN113456660A - Application of epimedin C in preventing and treating acute alcoholism

Info

Publication number: CN113456660A
Application number: CN202110882107.3A
Authority: CN
Inventors: 张华峰; 张梦园; 杨晓华; 梁巧
Original assignee: Xian Jiaotong University; Shaanxi Normal University
Current assignee: Xian Jiaotong University; Shaanxi Normal University
Priority date: 2021-08-02
Filing date: 2021-08-02
Publication date: 2021-10-01
Anticipated expiration: 2041-08-02
Also published as: CN113456660B

Abstract

The invention discloses an application of epimedin C in preventing and treating acute alcoholism, and belongs to the technical field of biological medicines. The invention discloses the epimedin C which can shorten the sobering-up time and the somnolence time of an acute alcoholism model animal, prolong the intoxication latency, reduce the levels of serum total bilirubin, total cholesterol and triglyceride, reduce the activity levels of serum glutamic-pyruvic transaminase and glutamic-oxaloacetic transaminase, increase the content of reduced glutathione, reduce the content of malondialdehyde, maintain the normal shapes and structures of liver tissues and cells, regulate the proteome expression of the acute alcoholism animal, repair the damage of a nervous system, improve the oxidative stress state of an organism and improve the glycometabolism and lipid metabolism for the first time, so the epimedin C can be used for preparing the acute alcoholism prevention and treatment medicine. The medicine for preventing and treating acute alcoholism prepared from the epimedin C is used in the form of medicinal preparations such as aerosol and the like. The invention is beneficial to promoting the research and development process of the medicine for preventing and treating acute alcoholism, and also provides an idea for developing and utilizing epimedium herb resources.

Description

Application of epimedin C in preventing and treating acute alcoholism

Technical Field

The invention belongs to the technical field of biological medicines, relates to a new effect of epimedin C in preventing and treating acute alcoholism, and particularly relates to an application of epimedin C in preparing a medicine for preventing and treating acute alcoholism.

Background

Excessive drinking wine (including various fermented wines such as white wine, beer, wine, yellow wine and the like, distilled wine and compound wine) or alcohol (ethanol) is taken by a human body at one time, acute alcoholism can be caused, and abnormal or pathological changes of different levels such as behavioral movement (such as ataxia), conscious nerves (such as coma), metabolic stress (such as glucose metabolism and oxidative stress), tissue organs (such as stomach, esophagus, liver, pancreas and brain injury), molecular cells (such as apoptosis) and the like can be caused, and the life can be threatened when the abnormal or pathological changes are serious. Acetaldehyde produced by Alcohol metabolism can lead to stem cell mutation and chromosomal damage (see, for example, ethanol and endogenous aldehydes large chromosomes and mutant stem cells [ J ]. Nature, 2018, studied by Garacoechea et al). Alcoholism is a serious and possibly fatal health hazard to human health, and has become a frequent medical problem in recent years. The western medicines such as naloxone and orexin A are clinically used for treating acute alcoholism, but side effects or limitations such as arrhythmia, increased blood pressure, nausea and vomiting or complex administration mode exist (see the current situation and prospect of naloxone for treating cerebral infarction researched by mengqinglin [ J ]. Chinese emergency medicine 2000; new progress of acute alcoholism medicine treatment researched by Gaowanlu [ J ]. pharmacy and clinical research 2015). The traditional Chinese medicine has unique advantages in the aspect of preventing and treating alcoholism, and has important practical significance in screening active monomers for preventing and treating acute alcoholism from the traditional Chinese medicine.

Herba Epimedii is a Chinese herbal medicine with a long application history in China, contains various plant chemical components such as polysaccharide, flavonoid, alkaloid, lignan, etc., has the functions of strengthening will, invigorating kidney yang, strengthening tendons and bones, dispelling pathogenic wind and removing dampness, has high safety, and is an approved medicinal and edible plant (participating in Zhangfeng research, etc. related to medicinal and edible homology)Term problem and countermeasure [ J]Chinese scientific terminology, 2019). Epimedin C (epimedidin C) is a flavonoid compound monomer separated from herba Epimedii, and has molecular formula of C₃₉H₅₀O₁₉. The applicant reports the effect of epimedin C in preventing and treating osteoporosis, cancer, hypoimmunity and other diseases in early team (see the research on the bioactive components of epimedium and development strategy thereof in Zhang Huafeng and the like [ J)]Chinese herbal medicine, 2010), but no application report of epimedin C in preventing and treating acute alcoholism has been found so far.

In the prior art, patent CN106924686A discloses a health product with liver protecting and alcoholism relieving functions, which is prepared from raw materials of radix bupleuri, radix puerariae, gardenia jasminoides, ginseng, radix scutellariae, Chinese date, medlar, herba epimedii, platycodon grandiflorum, medicated leaven, bamboo shavings, fructus viticis, reed rhizome and the like. Patent CN106924602A discloses a liver-protecting and hangover-alleviating traditional Chinese medicine preparation, which is prepared from corn stigma, bamboo leaves, rhizoma anemarrhenae, fructus schizandrae, selenium malt, dogwood, medlar, herba epimedii, poria, cassia twig, pomelo peel, duckweed, selfheal, ground beetle and other raw materials. Patent CN109793235A discloses an oral liquid with auxiliary protection effect on chemical liver injury and a preparation method thereof, wherein the raw materials comprise American ginseng extract, ganoderma lucidum polysaccharide, ganoderma lucidum, hepialus armoricanus, rose powder, kudzu root extract, hovenia dulcis thunb extract, L-cysteine, taurine, vitamins, sweetening agent, honey, blueberry, preservative, cynomorium songaricum extract, cistanche deserticola extract, lycium ruthenicum extract, maca extract, eurycoma longifolia extract, pilose antler extract and epimedium extract. Patent CN104274582A discloses a liver-protecting pharmaceutical composition and its preparation method, wherein the raw materials of the medicinal composition include fructus ligustri lucidi, fructus schizandrae, herba epimedii, rhizoma polygoni cuspidati, placenta hominis, acanthopanax, radix astragali, cortex moutan, radix isatidis, radix scutellariae, herba taxilli and radix bupleuri, and the composition has the effect of inhibiting hepatitis b virus. The study on the extraction and purification of epimedium leaf polysaccharide and the effect of the epimedium leaf polysaccharide on preventing alcoholic liver injury prepares epimedium polysaccharide by an ultrasonic water extraction method, an ion exchange method and the like, and then the obtained polysaccharide is used for preventing alcoholic liver injury. Patent CN101574360A discloses the application of epimedin in preparing medicines for treating diseases requiring estrogen supplementation. Patent CN111494360B discloses the use of epimedin C in the treatment of diabetic liver injury.

In general, the prior art has various features, and the characteristics or limitations thereof are as follows:

(1) the prior art mainly adopts the compound combination of epimedium original medicinal material and other various Chinese medicinal materials, the single epimedium medicinal material or the extract thereof is rarely used, and the report of preventing and treating acute alcoholism by using epimedium flavonoid compound monomer epimedin C alone is not shown.

(2) In the prior art, epimedium medicinal material or compound composition of a plurality of medicinal materials is mainly used for preventing and treating chemical liver injury, alcoholic liver injury or hepatitis B, only liver injury is involved, and behavioral actions (such as ataxia), conscious nerves (such as coma), metabolic stress (such as glycometabolism and oxidative stress), abnormal or pathological changes of different levels of tissues and organs (such as stomach, esophagus, pancreas and brain injury), molecular cells (such as apoptosis) and the like are rarely involved. That is, in the prior art, reports of using epimedium medicinal materials, extracts and monomeric compounds to prevent and treat acute alcoholism are not found. Acute alcoholism is a complex disorder involving not only liver damage but also damage to tissues and organs such as stomach, esophagus, pancreas, brain, etc., and also has typical clinical symptoms in behavioral activities, conscious nerves, metabolic stress, and molecular cell level.

(3) At present, the application of the epimedin C medicine is mainly concentrated in the fields of osteoporosis, cancer, hypoimmunity, climacteric syndrome, diabetic liver injury and the like, and no report on the application aspect of preventing and treating acute alcoholism of the epimedin C is found.

(4) Many of the prior art lacks experimental basis, and the mechanism of action, particularly the molecular mechanism (such as proteomics analysis) is unclear, which affects the credibility and practicability of the technology.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide an application of epimedin C in preventing and treating acute alcoholism, and an application of epimedin C in preparing a medicine for preventing/treating acute alcoholism.

In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:

the invention provides application of epimedin C in preparing a medicine for preventing/treating acute alcoholism.

Preferably, the drug is a drug having molecular docking affinity activity for acetaldehyde dehydrogenase protein.

Preferably, the medicine is capable of shortening the sobering-up time and the somnolence time of the acute alcoholism animal and prolonging the drunkenness latent period.

Preferably, the medicament is a medicament for reducing the liver index of an acute alcoholism animal.

Preferably, the medicament is a medicament for reducing the levels of serum total bilirubin, total cholesterol and triglyceride levels in an acute alcoholism animal.

Preferably, the drug is a drug for improving the content of reduced glutathione in acute alcoholism animals.

Preferably, the medicine is used for maintaining the normal shape and structure of liver tissues and cells of the acute alcoholism animal.

Preferably, the drug is a drug for improving the sugar metabolism of acute alcoholism animals.

Preferably, the medicament is a medicament for regulating the expression of acute alcoholism animal proteome.

Preferably, the medicament is an aerosol prepared from the epimedin C and pharmaceutically acceptable auxiliary materials.

The invention discloses an application of epimedin C in preventing and treating acute alcoholism.

The monomeric compound epimedin C for preventing and treating acute alcoholism is a flavonoid compound with a molecular formula of C₃₉H₅₀O₁₉. In nature, epimedin C is widely distributed in epimedium plants such as wushanense epimedium, epimedium heartleaf, epimedium dauricum, epimedium sagittatum, epimedium koreanum and epimedium koreanum. The epimedin C can be prepared from epimedium plants by using common extraction and refining technology, and the production of the epimedin C has already realized industrialization and commercialization.

The clinical manifestations of acute alcoholism related to the invention are as follows: (1) behavior action level: the righting reflex disappears and sleepiness is caused; (2) level of consciousness and nerves: nervous system damage, coma (lethargy); (3) tissue and organ level: impaired liver function, abnormal liver tissue, cellular morphology and structure, etc.; (4) level of metabolic stress: disorders of carbohydrate metabolism, lipid metabolism, exacerbation of oxidative stress conditions, and the like; (5) molecular cell level: protein expression is abnormal, and proteomics analysis shows that a large number of proteins are differentially expressed.

The invention provides application of epimedin C in preventing and treating acute alcoholism, namely the epimedin C can relieve clinical manifestations of acute alcoholism. Specifically, epimedin C has molecular docking affinity activity on acetaldehyde dehydrogenase protein, can shorten the sobering-up time and the somnolence time of acute alcoholism animals, prolong the intoxication latency, reduce the liver index, reduce the levels of serum total bilirubin, total cholesterol and triglyceride, reduce the levels of serum glutamic pyruvic transaminase and glutamic oxaloacetic transaminase, improve the activities of superoxide dismutase and glutathione peroxidase, increase the content of reduced glutathione, reduce the content of malondialdehyde, maintain the normal forms and structures of liver tissues and cells, regulate the proteome expression of the acute alcoholism animals, repair the damage of nervous systems, improve the oxidative stress state of organisms, relieve the sugar metabolism and lipid metabolism disorder, and has the effect of preventing and treating acute alcoholism.

The epimedin C is used for preparing the medicine for preventing and treating acute alcoholism in the form of a medicinal preparation. The medicinal preparation comprises epimedin C as an active ingredient and a pharmaceutically acceptable carrier (such as a propellant suitable for respiratory tract administration). The medicinal preparation can be aerosol, nano granules and the like, and can also be used for preparing functional food.

The epimedin C medicinal preparation can be prepared according to various conventional preparation processes.

Compared with the prior art, the invention has the following beneficial technical effects:

(1) the medicine has obvious effect of preventing and treating acute alcoholism, and can effectively relieve clinical manifestations of acute alcoholism. The epimedin C can reduce the harm of acute alcoholism in different levels of behavioral action, conscious nerves, metabolic stress, tissues, organs, molecular cells and the like, can shorten the sobering-up time and the somnolence time of an acute alcoholism model animal, prolong the intoxication latency, protect the liver and the nervous system, regulate the expression of proteomes, improve the oxidative stress state of an organism, and relieve the disorder of carbohydrate metabolism and lipid metabolism. The current special drugs for preventing and treating acute alcoholism are scarce, and the invention advances the research and development process of the drugs for preventing and treating acute alcoholism.

(2) The monomeric compound epimedin C for preventing and treating acute alcoholism, which is disclosed by the invention, has a clear chemical structure, is easy to analyze and detect, is easy to obtain raw materials, and has higher application feasibility in the practice of preventing and treating acute alcoholism. The epimedin C is ubiquitous in epimedium plants, can be prepared from epimedium by using a conventional method, and can also be directly purchased from a commercial pure product of the epimedin C on the market. Epimedium herb is widely distributed in provinces of Shaanxi, Hubei, Sichuan, Guizhou, Jilin and the like in China, and artificial planting is started, so that the medicinal material source of epimedin C is ensured. Many research reports on qualitative and quantitative analysis of epimedin C are reported, so that the epimedin C is easy to analyze, detect and control quality and is suitable for clinical application of acute alcoholism.

(3) The epimedin C and the source medicinal material epimedium have higher safety. A large number of researches show that the epimedium is nontoxic to human bodies (see mutational observation [ J ] of epimedium in researches such as Liujia and the like, parasitic diseases and infectious diseases, 2007; long-term toxicity research [ J ] of epimedium total flavonoids in researches such as plum blossom and the like, China journal of Experimental prescriptions, 2008), is a medicine-food homologous plant which is licensed by the national ministry of health, can be used as a medicine and food material, and can also be used as a health-care food raw material. The related research of the invention does not find that the epimedin C has hepatotoxicity and kidney toxicity. The method provides an important guarantee for the application of the epimedin C in the prevention and treatment practice of acute alcoholism.

Drawings

FIG. 1 is a three-dimensional analysis chart of the docking affinity activity of epimedin C and acetaldehyde dehydrogenase molecules;

FIG. 2 is a graph of the effect of epimedin C on the liver index of mice with acute alcoholism;

FIG. 3 shows the effect of epimedin C on the activity of serum ALT and AST of mice with acute alcoholism. Wherein, A is ALT, B is AST;

FIG. 4 is a graph showing the effect of epimedin C on serum bilirubin levels in acute alcoholism mice;

FIG. 5 shows the effect of epimedin C on the content of TC and TG in serum of mice with acute alcoholism. Wherein A is TG and B is TC;

FIG. 6 shows the effect of epimedin C on the antioxidant stress capacity of acute alcoholism mice. Wherein, A is GSH, B is GSH-Px, C is MDA, and D is SOD;

FIG. 7 is a liver tissue section of an acute alcoholism mouse. Wherein, A is MC group, B is EC20 group, C is EC50 group, and D is NC group;

FIG. 8 is the identification of acute alcoholism mouse proteome by mass spectrometry. Wherein, A is EC20 group, and B is MC group.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The invention discloses an application of epimedin C in preventing and treating acute alcoholism, wherein the compound epimedin C for preventing and treating acute alcoholism is a flavonoid compound monomer distributed in epimedium plants, and the molecular formula is C₃₉H₅₀O₁₉. The epimedin C standard (CAS number: 110642-44-9; batch number: 111780-201905) used in the examples of the invention is purchased from China food and drug testing research institute.

The present invention is described in further detail below with reference to examples:

example 1: preparation of epimedin C aerosol

The effective components are as follows: epimedin C;

main auxiliary materials: film forming agent (hydroxypropyl methylcellulose), liquid paraffin, propellant (propane and butane), chitosan;

the preparation process comprises the following steps: collecting epimedin C (molecular formula: C)₃₉H₅₀O₁₉(ii) a CAS number: 110642-44-9; batch number: 111780-201905; provided by China institute for food and drug testing) and (65 +/-3)% ethanol solution according to the solid-to-liquid ratio of 1: 3-1: 4, placing for 23-25 hours in a closed state, concentrating by using a rotary evaporator, filtering the concentrated solution by using a microporous filter membrane (0.45 mu m), taking the filtrate, adding (2.0 +/-0.1)% chitosan solution and a proper amount of liquid paraffin (pre-testing). And canning the mixed solution and the propellant according to the ratio of 1: 8-1: 10 to obtain the epimedin C aerosol.

Example 2: preparation of epimedin C nano granules

The effective components are as follows: epimedin C;

main auxiliary materials: ethyl cellulose;

the preparation process comprises the following steps: dissolving ethyl cellulose in ethanol to obtain (2.0 +/-0.1)% ethyl cellulose solution. Adding a proper amount of epimedin C (the addition amount is tested in advance), dispersing for 2-4 minutes by using a high-speed disperser, and homogenizing for 1 time under the pressure of 50MPa, 70MPa and 90MPa respectively to prepare an organic phase solution. Preparing an emulsion from an organic phase solution, concentrating the emulsion at the temperature of (40 +/-5) DEG C by using a rotary evaporator, removing ethanol, then carrying out vacuum freeze drying, vacuumizing the dispersed suspension, filling nitrogen to atmospheric pressure, precooling to the temperature of (8 +/-1) DEG C, vacuumizing again after 1 hour, then cooling to enable the solution to be cold and formed into ice-shaped solid, introducing nitrogen after 3-5 hours, and carrying out heat preservation for 5-7 hours. Finally, the epimedin C nano granules are prepared.

The prevention and treatment effects of epimedin C according to the invention on acute alcoholism are further illustrated by the following test examples:

molecular docking of epimedin C and acetaldehyde dehydrogenase (ALDH)

1 method of experiment

1.1 receptor protein treatment method: acetaldehyde dehydrogenase (PDB ID: 1O04) is selected as a receptor Protein, the Protein crystal structure is obtained from a Protein Data Bank database, after hydrogenation, preparation and force field giving, the position of a primary ligand (epimedin C monomer compound) is defined as an active site, and the radius of an active pocket is defined as 9.627148 according to the size of the primary ligand.

1.2 ligand small molecule processing method: 4 flavonoid monomers of epimedin C, icariin, puerarin and daidzin are respectively used as ligand molecules, the stereoscopic structure of the compound is generated by using Discovery Studio software, and the compound is stored in an SD format for later use after hydrogenation and force field giving.

1.3 molecular docking method: a CDocker module of a Discovery Studio platform is adopted to carry out molecular docking on 4 flavonoid compound monomers of epimedin C, icariin, puerarin and daidzin and acetaldehyde dehydrogenase respectively, and the molecular docking is scored according to the docking site number and the docking affinity.

2 results and analysis of the experiments

As can be seen from Table 1, 4 flavonoid monomers of epimedin C, icariin, puerarin and daidzin and acetaldehyde dehydrogenase all have certain affinity activity, and the docking effect is from strong to weak: epimedin C, icariin, puerarin and daidzin. The docking mode of epimedin C with acetaldehyde dehydrogenase is shown in figure 1. The epimedin C can form intermolecular hydrogen bonds with amino acid residues LYS127 and GLN289 of the zymoprotein, forms hydrophobic interaction with amino acid residues VAL120, ASP123, MET124, LYS127, PHE292 and VAL458, and has stable combination of the ligand and the protein and better scoring of interaction energy (Table 1). After entering human body, alcohol is firstly oxidized into acetaldehyde under the action of alcohol dehydrogenase, and acetaldehyde is further oxidized into acetic acid under the action of acetaldehyde dehydrogenase, which is the most important way for alcohol metabolism. Acetic acid is nontoxic to human bodies, but acetaldehyde can cause chromosome damage and stem cell mutation, so that the improvement of the activity of acetaldehyde dehydrogenase is particularly important for preventing and treating acute alcoholism. The results of molecular docking experiments suggest that epimedin C has good potential application in preventing and treating acute alcoholism.

TABLE 1 molecular docking experiment results of monomeric compounds and acetaldehyde dehydrogenase proteins

Second, the influence of epimedin C on the liver index of mice with acute alcoholism

1 materials of the experiment

1.1 Experimental animals: male mice of Kunming species 56, 4-5 weeks old, weighing 18-22g, were provided by the laboratory animal center, Sigan university of transportation, and were ethically reviewed.

1.2 reagents and instruments: the chemical reagents include epimedin C standard (batch No. 111780) -201905; provided by China food and drug testing research institute), silymarin capsule (batch No. B0902641; provided by Germany MADAUS company), 52-degree white spirit (food grade) and other conventional chemical reagents; the kit mainly comprises a glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminase (AST), Triglyceride (TC), total cholesterol (TG), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), reduced Glutathione (GSH) and Malondialdehyde (MDA) kit; the instrument mainly comprises a full-wavelength microplate reader, a high performance liquid chromatograph, a freeze dryer, an ultraviolet visible spectrophotometer, a high-speed refrigerated centrifuge and an ultra-low temperature refrigerator.

2 method of experiment

2.1 preparation and grouping treatment method of mouse model: mice were raised adaptively for 1 week using a common feed for experimental animals, weighed one by one, and randomly divided into 5 groups: normal group (NC group), model group (MC group), silymarin group (PC group) (positive control), low dose epimedin C group (EC20 group) and high dose epimedin C group (EC50 group). Wherein, the dosages of the low and high dosages of the epimedin C group are respectively 20 and 50mg/kg · bw. Except the normal group and the model group, the other groups are continuously administered with the corresponding drugs by intragastric administration for 30 days every day, and after the last intragastric administration for 12 hours, the mice are orally administered with 52-degree white spirit (12ml/kg · bw) once to induce the acute alcoholism model.

2.2 liver index measuring and calculating method: taking mouse liver, and calculating liver index according to the following formula: liver index is liver mass/body weight 100%.

2.3 statistical treatment methods: SPSS 13.0 software is used for statistical analysis of experimental data in and among groups, experimental results are expressed by mean (mean), One-Way ANOVA analysis method is adopted for comparison among groups, P < 0.05 shows significant difference, P < 0.01 shows significant difference, and P > 0.05 shows insignificant difference.

3 results and analysis of the experiments

The liver index is the ratio between the wet mass of the liver and the body mass. In general, when the liver is damaged, the liver index increases. As can be seen from Table 2 and FIG. 2, the liver index of the mice in the MC group was significantly higher than that in the NC group (P < 0.01), indicating that white spirit caused liver injury in the mice. After the epimedin C is perfused into the stomach, the liver index of the acute alcoholism mouse is reduced to a certain degree relative to the MC group, which shows that the epimedin C has a certain protection effect on the liver of the acute alcoholism mouse.

Table 2 effect of epimedin C on mouse liver index

Group of	Liver index(％)
		PC	3.83^b
EC20	4.17^ab
		EC50	4.30^a
NC	3.81^b
		MC	4.44^a

Note: the different letter superscripts to the right of the numbers in the same column indicate significant differences.

Influence of epimedin C on behavior of acute alcoholism mouse

1 Experimental materials and methods

The experimental materials and the mouse model were prepared and processed in groups as described above. Observing and recording the mental state, behavior, death condition and the like of the mouse, measuring and recording the activity amount, the food consumption and the like of the mouse, and measuring and recording the indexes of the mouse, such as turning positive reflection performance, sobering time, somnolence time, intoxication latency and the like.

2 results and analysis of the experiments

After the gavage of the stomach, all the mice in each group did not die, and the mice in the model group showed the symptoms of disappearance of righting reflex and drunkenness such as slow action, little sleepiness, obvious reduction of activity and the like. Compared with the model group, the mice in the administration group have shortened sobering-up time (the time for the mice to recover the righting reflex), prolonged intoxication latency and shortened lethargy time.

Influence of epimedin C on liver function of acute alcoholism mouse

1 Experimental materials and methods

The activity of glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase in serum of a mouse is measured by taking the serum of the mouse according to the method of the kit specification. The rest of the experimental materials and experimental methods were as described above.

2 results and analysis of the experiments

ALT and AST are 2 important indicators for evaluating liver function. As can be seen from FIG. 3, the ALT activity level of the MC group is significantly higher than that of the NC group (P < 0.01), and the AST activity of the MC group is also higher than that of the NC group (P > 0.05), indicating that alcohol has a serious adverse effect on the liver function of the mice. After the epimedin C is treated, the ALT and AST vitality of the mice suffering from acute alcoholism is reduced relative to that of the MC group, and the AST and ALT vitality of the mice in the EC20 group is reduced to a degree higher than that of the mice in the PC group, which indicates that the epimedin C can protect the liver function of the mice suffering from acute alcoholism.

Influence of epimedin C on serum bilirubin of acute alcoholism mouse

1 Experimental materials and methods

The serum of the mouse is taken, and the content of the total bilirubin in the serum is determined according to the method of the kit instruction. The rest of the experimental materials and experimental methods were as described above.

2 results and analysis of the experiments

The liver performs the functions of uptake, binding and excretion in bilirubin metabolism, and disorders of any one of these functions can cause jaundice. The content of serum Total Bilirubin (TBiL) can reflect jaundice condition and further indicate liver health condition. As can be seen from FIG. 4, the total bilirubin content of the MC group is significantly higher than that of the NC group (P is less than 0.01), which indicates that alcohol has serious negative effects on liver bilirubin metabolism and increases jaundice risk; the EC20 and EC50 groups effectively reduce the serum total bilirubin content relative to the MC group and show a dose-dependent relationship, which indicates that the epimedin C can improve the liver bilirubin metabolism of acute alcoholism mice and prevent jaundice.

Effects of epimedin C on serum TC and TG of acute alcoholism mouse

1 Experimental materials and methods

And (3) taking mouse serum, and measuring the content of TC and TG in the serum according to a kit instruction method. The rest of the experimental materials and experimental methods were as described above.

2 results and analysis of the experiments

Dyslipidemia is one of the most common reactions of excessive alcohol consumption. The major effects of alcohol metabolism on blood lipid metabolism are hypertriglyceridemia and hypercholesterolemia, with triglyceride and cholesterol levels in serum closely correlated with liver health. As can be seen from FIG. 5, the TG content in the MC group is significantly higher than that in the NC group (P < 0.01), and the TC content is significantly higher than that in the NC group (P < 0.05), which indicates that the mice have obvious dyslipidemia under the action of alcohol; compared with the MC group, the TG content of the EC20 and the EC50 group is obviously reduced (P is less than 0.05), and the TC content is slightly reduced (P is more than 0.05). Therefore, the epimedin C can regulate the liver lipid metabolism of the acute alcoholism mouse and relieve the dyslipidemia caused by the alcohol.

Influence of seven and epimedin C on liver oxidation resistance of acute alcoholism mice

1 Experimental materials and methods

The mouse liver is taken, and the activity of liver SOD and GSH-Px and the content of GSH and MDA are determined according to the method of the kit instruction. The rest of the experimental materials and experimental methods were as described above.

2 results and analysis of the experiments

Acute alcoholism is closely related to the oxidative stress state of the liver. Oxidative stress induces an imbalance between the production of free radicals and the antioxidant defense system, which can cause tissue and organ damage and play an important role in the pathogenesis of acute alcoholism. Superoxide dismutase, glutathione peroxidase, reduced glutathione and the like form an important antioxidant defense system of an organism. MDA is also a common indicator for assessing oxidative stress levels. As can be seen from FIG. 6, compared with the NC group, the liver of the MC group mice has significantly increased MDA content (P < 0.05), significantly decreased SOD activity (P < 0.05), significantly decreased GSH-Px activity (P < 0.01), and significantly decreased GSH content (P < 0.05), indicating that the liver of the acute alcoholism mice is in a more severe oxidative stress state; after the epimedin C stem prognosis, compared with an MC group, the activity of SOD is obviously improved (P is less than 0.05), the activity of GSH-Px is extremely obviously improved (P is less than 0.01), the content of MDA is reduced, and the content of GSH is increased, wherein the content of GSH in the EC50 group is obviously higher than that in the MC group (P is less than 0.05); the GSH content of the EC50 group was also significantly higher than that of the EC20 group (P < 0.05), exhibiting a clear dose-dependent relationship. The above results indicate that epimedin C can improve the oxidative stress state of the liver of acute alcoholism mice.

Liver pathological section analysis of mouse with acute alcoholism intervened by epimedin C

1 Experimental materials and methods

Taking mouse liver, fixing, performing gradient dehydration with ethanol solutions of different concentrations, sequentially performing xylene transparence, wax immersion, embedding, slicing, hematoxylin-eosin staining (HE staining), and finally observing and photographing under a 400-fold optical lens. The rest of the experimental materials and experimental methods were as described above.

2 results and analysis of the experiments

Fig. 7 is a representative photograph of pathological sections of liver of mice with acute alcoholism in which epimedin C intervenes. It can be seen that the liver tissue of the NC group is the normal liver structure of the mice, the liver cells are arranged regularly, the liver cords are arranged in a radial shape by taking the central vein as the center, and the shape and the structure of the liver cells are normal. The MC group exposed by acute alcohol has the pathological changes that the arrangement of liver cells is disordered, liver cords and liver cell structures are damaged, a large number of liver cells are subjected to steatosis, the cavitation similar to emulsion drops is more, and partial liver cells are subjected to necrosis and the like. Compared with the MC group, the mice with the prognosis of the epimedin C stem have less vacuole deformation of liver tissues, the arrangement of liver cords and liver sinuses tends to be regular, the pathological damage is obviously relieved, the local part of the EC20 group still has degenerated liver cells, and the prevention and treatment effect of the EC50 group is better. It can be seen that the epimedin C can block the pathological change process of the liver tissue of the acute alcoholism mouse and has better protective effect on the liver tissue and cells.

Influence of epimedin C on acute alcoholism mouse proteomics

1 Experimental materials and methods

1.1 extraction and quantification of proteins required for proteomics studies: the tissue of NC group and EC20 group mice was taken on dry ice, RIPA working solution (50mM Tris, 150mM NaCl, 1% Trition X-100, 1% sodium deoxycholate, 0.1% SDS and sodium vanadate) was added, steel balls were added, homogenization was carried out, ultrasonication and centrifugation were carried out to take out supernatant. Protein content was determined using BCA protein quantification kit.

1.2 acetone precipitation, redissolution, reduction and alkylation of proteins: the protein sample is diluted and then added with precooled acetone (-20 ℃), mixed evenly and precipitated overnight. Centrifuging to remove supernatant, adding a protein complex solution into the precipitate, dissolving the precipitate, adding DTT (diethylenetriamine pentaacetic acid) for shake incubation to reduce disulfide bonds, and adding IAA (IAA) for alkylation reaction.

1.3 enzymolysis of protein, peptide fragment impurity removal and desalination: mixing Trypsin and a sample sufficiently and uniformly according to a certain proportion, incubating for 12 hours, adding TFA, mixing uniformly, centrifuging at a high speed and taking supernatant. TFA was added and the precipitated polypeptide was extracted by centrifugation. Activating C18 column, adding sample, collecting effluent, washing, eluting, desalting, and vacuum drying.

1.4 detection by liquid chromatography-mass spectrometry: and (3) sampling total peptides, separating by using ultra-high performance liquid chromatography, and then collecting data by using a mass spectrometer equipped with a sodium-ion source. The chromatographic separation adopts a C18 reversed phase chromatographic column, and the mobile phase adopts an acetonitrile-water-formic acid system and is eluted in a gradient way. Mass spectrometry uses a Data Dependent Acquisition (DDA) mode for a total analysis time of 2 hours, with a positive ion detection mode. The range of the primary scanning (Full scan) is 350-1600m/z, AGC is 3E6, the maximum ion injection time is 50ms, and the ions with the highest intensity in the primary scanning are screened by a quadrupole rod and then subjected to fragment ion scanning after being cracked by HCD.

1.5 qualitative, quantitative and bioinformatic analysis of proteins: the mass spectrum raw file was searched using the label-free qualitative and quantitative software MaxQuant No.1.5.2.8 to obtain the corresponding peptides and proteins. Primary mass spectrum accuracy: 4.5ppm, second order mass spectral accuracy: 20ppm, maximum number of missed cuts allowed during enzymatic hydrolysis: 2, database: Uniprot-Swissprot; the quantitative calculation method comprises the following steps: absolute quantification based on iBAQ intensity. Protein samples of mice in the MC group and the EC20 group are analyzed by using a Uniprot-Swissprot database, differential expression proteins between mice in acute alcoholism and mice in a low-dose epimedin C group are screened out, and other bioinformatics analysis is carried out.

2 results and analysis of the experiments

2.1 Mass Spectrometry identification and screening of differentially expressed proteins

The mass spectrometry system of the research is stable and reliable, and the obtained original data is analyzed by using a Proteome scanner software (PD) and a built-in sequence HT search engine to obtain qualitative and quantitative results (figure 8). After the data filtering treatment, 3 thousands of polypeptides and proteins were obtained. In the epimedin group C versus model group alignment (EC20 vs MC), there were 256 differentially expressed proteins, including 91 upregulated proteins and 165 downregulated proteins. These differentially expressed proteins may be closely related to the mechanism of development of epimedin C against acute alcoholism.

2.2 functional analysis of differentially expressed proteins

In order to further clarify the role of DEPs in the alcoholism process, DEPs were functionally clustered, and as a result, they were found to be involved in the development of the nervous system, oxidative stress, calcium ion binding, lipid metabolism (including fatty acid β -oxidation and arachidonic acid metabolism), vitamin metabolism (mainly water-soluble vitamin metabolism), sugar metabolism, purine metabolism, and the like. Among them, DPYLS2 plays an important role in synaptic signaling through interaction with calcium channels, and may be associated with various neurological diseases, such as neuronal ceroid lipofuscinosis and alzheimer's disease; the protein encoded by SERPINA3 belongs to a member of serpin protein family, and the variation thereof can be related to Alzheimer disease and Parkinson disease; CAT encodes catalase, which is a key enzyme for the body to resist oxidative stress and mainly has the function of converting active oxygen hydrogen peroxide into water and oxygen to prevent cells from being damaged by related free radicals; LMNB2 is involved in lipid metabolism and may have close relationship with epilepsy, lipodystrophy, etc.; SHC1 is also involved in lipid metabolism and may be related to sclerosing liver cancer and other diseases; ACACB is involved in fatty acid oxidation process, possibly associated with biotin deficiency or fatty liver; PFKL is the rate-limiting enzyme of glycolysis, catalyzing the irreversible reaction of fructose-6-phosphate to fructose-1, 6-bisphosphate; TIGAR may be involved in metabolic processes of fructose, mannose, etc.; HMGA1 may be associated with insulin resistance or type 2 diabetes, among others. Through the analysis of the function of the differentially expressed protein, the acute alcoholism can cause the abnormal expression of various proteins, and the negative effects are generated on the nervous system, the digestive system, the metabolism (such as sugar metabolism and fat metabolism), the oxidative stress and the like, so that the organism is in an oxidative stress state, various metabolic processes of the organism are interfered, and the nerve damage of different degrees is caused. Epimedin C may have some regulatory effect on these acute alcoholism symptoms.

2.3 differential protein interaction analysis

Proteins typically function themselves by interacting with other proteins. Network interaction analysis of differentially expressed proteins shows that major node proteins of EC20 vs MC include CAT, ADSL, PSMA7, PSMB3, NUDC, MRPL41, RPL37, ATP5ME, MRPS7, RPL18A, MIA3, ACLY, SRSF5, RPL36A and the like. Among them, NUDC may play an important role in neurogenesis and neuronal migration, and it is presumed that alcoholism may cause brain nerve damage; alcoholic neuropathy is closely related to MIA3, and long-term or heavy drinking is presumed to cause damage to the central nervous system, produce mental symptoms and are often accompanied by severe pathological changes of various internal organs; PSMB3 may be associated with certain neurological disorders, typically epilepsy and the like; ADSL may be closely related to nervous system disorders such as epilepsy and slow response; the occurrence of epilepsy may also be associated with ATP5ME, and may involve purine nucleotide biosynthesis, ATP synthesis, etc.; physiological and biochemical processes associated with ACLY may include lipid metabolism and acetylcholine biosynthesis; CAT is closely related to the process of resisting oxidative stress of the organism, and the influence of alcohol on the expression of catalase of the organism is inferred to be large, and the occurrence of acute alcoholism is probably closely related to the expression change of CAT; PSMA7 may be associated with hypoxia or hepatitis, etc.; MRPL41 is a component of the large subunit of mitochondrial ribosome and is involved in apoptosis and cell cycle regulation; RPL37 is closely related to mRNA translation and RNA synthesis; SRSF5 related protein is a member of pre-mRNA splicing factor family rich in serine/arginine and is important for mRNA splicing; RPL36A belongs to the ribosomal protein family and may be involved in transcription and replication of mRNA. It is speculated that acute alcoholism may be associated with apoptosis, transcriptional translation, oxidative stress, nerve damage, and metabolic disorders (e.g., glucose metabolism disorders). Epimedin C may play a role in preventing and treating acute alcoholism by regulating the expression of these DEPs.

In conclusion, the epimedin C has molecular docking affinity activity on acetaldehyde dehydrogenase protein, can shorten the sobering-up time and the sleepiness time of an acute alcoholism experimental animal, prolong the intoxication latency, reduce the liver index, reduce the levels of serum total bilirubin, total cholesterol and triglyceride, reduce the levels of serum glutamic pyruvic transaminase and glutamic oxaloacetic transaminase activities, improve the activities of superoxide dismutase and glutathione peroxidase, increase the content of reduced glutathione, reduce the content of malondialdehyde, maintain the normal forms and structures of liver tissues and cells, regulate the expression of acute alcoholism mouse proteome, repair the damage of a nervous system, improve the oxidative stress state of an organism, relieve the sugar metabolism and lipid metabolism disorder, and has the effect of preventing and treating acute alcoholism.

The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Claims

1. Application of epimedin C in preparing medicine for preventing/treating acute alcoholism.

2. The use of claim 1, wherein the agent is an agent having molecular docking affinity activity for acetaldehyde dehydrogenase protein.

3. The use of claim 1, wherein the medicament is a medicament capable of shortening the sobering-up time and the somnolence time of an acute alcoholism animal and prolonging the intoxication latency.

4. The use of claim 1, wherein the medicament is a medicament for reducing the liver index of an acute alcoholism animal.

5. The use of claim 1, wherein the medicament is a medicament for lowering the levels of serum total bilirubin, total cholesterol and triglyceride levels in an acute alcoholism animal.

6. The use of claim 1, wherein the medicament is a medicament for increasing the reduced glutathione content of an acute alcoholism animal.

7. The use of claim 1, wherein the medicament is a medicament for maintaining normal morphology and structure of liver tissue, cells in an acute alcoholism animal.

8. The use of claim 1, wherein the medicament is a medicament for improving glucose metabolism in an acute alcoholism animal.

9. The use of claim 1, wherein the medicament is a medicament for modulating the expression of acute alcoholism animal proteome.

10. A medicine for preventing/treating acute alcoholism is characterized in that the medicine is an aerosol prepared from epimedin C and pharmaceutically acceptable auxiliary materials.