CN114246941A

CN114246941A - Composition with effects of preventing hangover, relieving alcoholism and protecting liver and application thereof

Info

Publication number: CN114246941A
Application number: CN202111211641.8A
Authority: CN
Inventors: 周飞; 张卫东; 易龙; 郜双林; 彭千真
Original assignee: Guangdong Haobang Medicine Health Co ltd
Current assignee: Guangdong Haobang Medicine Health Co ltd
Priority date: 2021-10-18
Filing date: 2021-10-18
Publication date: 2022-03-29

Abstract

The invention relates to a composition for preventing hangover, relieving alcoholism and protecting liver and application thereof, wherein the composition comprises NAD + replenisher, coenzyme Q10, superoxide dismutase and acetylcysteine. The NAD + supplements include NADH, NMNH and/or NRH. The composition for preventing hangover, relieving alcoholism and protecting liver and a pharmaceutically acceptable carrier are used for preparing a pharmaceutical preparation for preventing hangover, relieving alcoholism and protecting liver, and the preparation comprises capsules, tablets, pills, oral liquid, ointments, gels or aerosols. The experiments showed that each composition increased NAD + levels in various mammalian cell lines and in various tissues in vivo; and the effects of each composition on ethanol and acetaldehyde levels in blood, NAD +/NADH ratio in liver, mouse plasma AST and ALT levels, malondialdehyde and superoxide dismutase levels in liver, loss of mouse righting reflex (LORR) all indicate that the composition has greater hangover prevention and anti-alcohol hepatoprotective effects compared to NADH.

Description

Composition with effects of preventing hangover, relieving alcoholism and protecting liver and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a composition with functions of preventing hangover, dispelling the effects of alcohol and protecting liver and application thereof.

Background

The discovery and brewing of wine by human beings has been a history of thousands of years, and the benefits of drinking and the damage to the human body are also important problems in healthy life. Small amount of drinking can accelerate blood circulation and eliminate fatigue, but excessive drinking can cause certain harm to human body due to alcohol and metabolites thereof; excessive drinking can cause damage to the human body from the liver, and over-dependence of alcohol addicts on drinking can cause irreparable damage to the human body. When excessive drinking occurs, proper measures can protect the human body.

The liver is the central hub of carbohydrate metabolism, lipid metabolism and protein metabolism of human body and has important functions of expelling toxin, detoxifying, secreting bile and the like. After drinking, alcohol is absorbed mainly by the stomach (20%) and duodenum (80%), and about 90% of alcohol is metabolized in the liver. Alcohol has cytotoxicity, and can over-oxidize lipid on the surface of hepatocyte membrane, destroy hepatocyte membrane, further develop and destroy microtubule and mitochondria in hepatocyte, and cause intracellular metabolic disorder, and result in generation of cytotoxic metabolite, thereby causing hepatocyte swelling and necrosis.

Along with the increasing attention of people to the health problems, various anti-alcoholism preparations appear, and the existing anti-alcoholism preparation is roughly divided into three categories, namely a traditional Chinese medicine preparation, and the anti-alcoholism preparation is prepared from traditional Chinese medicine components for relieving alcoholism, strengthening the spleen and stomach, soothing the liver and benefiting the gallbladder by taking the traditional Chinese medicine theory as guidance. The advantages are that besides sobering up, most of them have liver-protecting health-care function, but the disadvantages are slow drug effect, thick and black liquor, large bitter taste and bad palatability. The other is some biochemical preparations developed based on the modern drug metabolism theory and with the main aim of enhancing the metabolism speed of ethanol in vivo. Such as RU21 and naloxone, has the advantages of convenient use, rapid promotion of alcohol decomposition and refreshing after intoxication, and has the defects of only having the function of relieving alcoholism, no viscera organ injury repair function and high price. The third category is enzyme preparations mainly comprising alcohol dehydrogenase and acetaldehyde dehydrogenase, and microorganisms or microorganism fermentation liquids having an anti-hangover effect, which have recently been developed. The advantages are safety, no toxic side effect, and the disadvantage is that enzymes and microorganisms can only play a role under the specific temperature and dissolved oxygen condition, and the high acid environment of gastric juice and the anaerobic environment of intestinal tract of human body often limit the efficacy of the sobering-up agent. The three main types of anti-inebriation products have different efficacies and have own advantages and defects.

In the prior art, the invention with the publication number of CN106492110B discloses an anti-inebriation composition, an anti-inebriation and liver-protecting preparation containing the same and an invention patent application of the application thereof, and discloses an anti-inebriation composition, an anti-inebriation and liver-protecting preparation containing the same and an application thereof, wherein the anti-inebriation composition comprises the following components: maca extract, kudzu root extract, turmeric extract and hovenia dulcis thunb extract. The hangover alleviating composition disclosed by the invention is prepared by selecting specific raw materials and scientifically and reasonably compounding, can promote rapid absorption and decomposition of alcohol in a body, is beneficial to rapid hangover alleviation of an alcohol taker, prevents hangover and has a good liver protection effect. The anti-alcohol composition can be prepared into an anti-alcohol and liver-protecting preparation or applied to liver-protecting functional food; the technical scheme of the invention adopts effective plant extracts to compound to form the composition for anti-inebriation, and the inventor considers that the anti-inebriation composition can be used for preparing products with various taking modes, such as capsules, oral liquid, tablets, pills and the like.

The patent application of the invention discloses a prescription of an anti-alcoholism oral medicine and a preparation method thereof, and discloses the anti-alcoholism oral medicine which is formed by matching anti-alcoholism oral liquid and an anti-alcoholism safety seed, wherein the components of the anti-alcoholism oral liquid comprise 13 Chinese herbal medicines such as kudzu root, red bean flower, red ginseng and the like; the components of the sobering-up safety seed are 16 Chinese herbal medicines of kudzuvine root, small bean flower, kudzuvine flower and the like. The inventor thinks that the anti-inebriation oral medicine can rapidly stimulate the secretion of alpha-2 aldehyde dehydrogenase and beta-2 aldehyde dehydrogenase in vivo, relieve alcoholism and play the efficacy of drinking too much and getting awake after intoxication.

The invention discloses a preparation method of a fermented traditional Chinese medicine beverage with a hangover alleviating function, which comprises the following steps: (a) preparing seed bacteria liquid for traditional Chinese medicine fermentation, red date pulp and fragrant rice fermentation liquid; (b) weighing the traditional Chinese medicine formula of the anti-alcoholism agent; (c) the preparation method provided by the invention adopts a traditional Chinese medicine fermentation mode to obtain the traditional Chinese medicine fermented beverage for relieving the drunkenness state.

Application publication No. CN1706479 discloses an invention patent application entitled method and composition for accelerating alcohol metabolism selected from NAD, NADH oxidation co-substrates, precursors thereof, and combinations thereof, in order to promote the regeneration of NAD that catalyzes alcohol metabolism, thereby reducing intoxication and preventing hangover by administering to a user a composition containing an extract in an amount effective to reduce alcohol poisoning. The object of the invention is to accelerate alcohol metabolism and maintain a healthy redox state in order to prevent/reduce alcohol intoxication, drunkenness and hangover.

Many compositions have been developed to alleviate the damage caused by drinking, but with limited success. For example, U.S. Pat. No. 4,450,153 (to Hopkins) proposes methods and compositions suitable for reducing ethanol in human blood to reduce the effects of ethanol consumption. Hopkins proposes reducing the alcohol content in human blood by administering alcohol oxidase. U.S. Pat. No. 5,759,539 to Whitmire describes a method and formulation for accelerated removal of ethanol from the body. The formulation combines an enzyme that oxidizes ethanol to acetate, an enzyme that regenerates NADH (reduced form of nicotinamide adenine dinucleotide) to NAD (nicotinamide adenine dinucleotide), a substrate to limit the rate of enzyme required, a buffer to protect the enzymes from pH changes (e.g., low pH in the stomach), a gastric acid scavenger to inhibit gastric acid synthesis, protease inhibitors and other agents to protect the active enzyme from proteolysis, carbohydrates to protect the labile enzyme from cholate inactivation.

U.S. Pat. No. 6,284,244 to Owades proposes a method of reducing blood ethanol levels by orally administering an active dry yeast containing an alcohol dehydrogenase either before or while drinking to oxidize a portion of the ethanol while it remains in the stomach. The alcohol dehydrogenase may be administered as a purified enzyme or, more conveniently, as a natural source for consumption of the enzyme, such as active dry baker's yeast, brewer's yeast. According to Owades 'method, active dry baker's yeast (the most readily available yeast) or brewer's yeast, brewer's yeast are consumed just before or simultaneously with alcohol consumption, and a portion of them are oxidized while ethanol remains in the stomach, which can reduce the peak level of blood ethanol and also reduce the area under the curve of blood ethanol level versus time. However, the effect of alcohol dehydrogenase on ethanol can only occur in the stomach, so the source of alcohol dehydrogenase must be taken while the ethanol remains in the stomach. Once ethanol leaves the stomach and enters the bloodstream, it cannot function because the enzymes are destroyed by the acidity and proteolysis in the stomach.

U.S. Pat. No. 4,877,601 (to Wren) provides a composition containing a physiologically inert hydrophobic molecular sieve material, particularly a crystalline zeolite, and a method for producing it in an edible form. The hydrophobic molecular sieve material has a pore size and can absorb ethanol but not other organic substances in blood or small intestine, and according to the Wren method, people take the molecular sieve, especially crystalline zeolite, to reduce the ethanol content in vivo. The zeolite is prepared in the form of: it is suitable for administration by dispersion in an edible or physiologically acceptable base, in particular in unit dosage form relative to the amount of ethanol to be absorbed.

U.S. patent application No. 20020006910 (by the applicant Miamikov and Kashlinsky) describes the use of a composition consisting of a sugar, L-glutamic acid, succinic acid, fumaric acid, ascorbic acid and aspartic acid to reduce intoxication, eliminate alcoholism and prevent hangover. Other methods and compositions for reducing the side effects of alcohol consumption include oral administration of iboinine and its salts to reduce ethanol dependence of U.S. patent 4857523 (issued to Lotsof), compositions of fructose and aqueous extracts of kudzu root (pueria) flowers, mung bean (phaseoliradiati) seeds, and pinellia tuber (pinellia) of U.S. patent 5324516 (issued to Pek, etc.) to reduce blood ethanol concentration, and the use of ephedrine (in the form of a powder sealed in a capsule) in combination with charcoal, vitamin B6 of U.S. patent 6485758 (issued to Mirza, etc.) to treat hangover and reduce alcoholic symptoms.

International patent publication (Mizumoto et al) discloses a composition of fermented lemon syrup and plant worm extract that promotes ethanol metabolism, thereby reducing nausea and vomiting from drinking and hangover. Also, European patent 1066835 (to Kim) proposes the use of extracts of the leaves, stems, and fruits of pepino to lower blood alcohol concentrations and reduce hangover.

NADH (Nicotinamide adenedisulotide) is a solid substance in the form of white powder; the molecular formula is as follows: C21H27N7O14P 2; molecular weight: 663.43, respectively; melting point: 160 ℃. NADH is called reduced nicotinamide adenine dinucleotide, reduced coenzyme 1 for short, also called mitochondria; the preparation method mainly comprises an extraction method, a fermentation method, a biosynthesis method and an organic matter synthesis method.

NADH, as a coenzyme for dehydrogenases, participates in several hundred redox reactions in cells. NADH produces ATP, an energy molecule, for cells, and if oxygen is sufficient, biological hydrogen also reacts with oxygen to produce a large amount of energy and water. NADH is oxidized and then converted into NAD +, which provides a key molecule NAD + for the anti-aging of cells.

NADH is the highest ranking coenzyme in humans and is a promoter of many biological reactions. NADH is essential for cell development and energy production: the production of energy from food is of vital importance and is the main carrier of electrons in the energy production process of cells. NADH is also an important antioxidant. Indeed, scientists have accepted that NADH is the most powerful antioxidant that protects cells from harmful substances.

NADH animal toxicity tests were carried out in rats and dogs, and NADH showed no toxicity or side effects even at high concentrations. NADH is approved as a nutraceutical on the world's largest, most complete drug and drug target resource pool drug bank. As a dietary supplement [2], NADH has been marketed in Europe and America for more than 20 years, and Adverse events caused by oral administration of NADH have never been reported [3] based on data carried by FDA Adverse Event Reporting System (FDA Adverse Event Reporting System) and CFSAN Adverse Event Reporting System (CAERS Adverse Event Reporting System).

However, the chemical properties of NADH are extremely reactive and NADH is easily inactivated by oxygen, moisture, light and acidity. The food source contains almost no NADH in vegetables and little NADH in meat, and the NADH in food has a great deal of denaturation problem in the processing process.

Nevertheless, all existing methods and compositions for reducing the health risks of alcohol consumption and hangover have limited effectiveness. Thus, there remains a need for effective compositions and methods for reducing the health risks of hangover.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a composition with the functions of preventing hangover, relieving alcoholism and protecting liver; the invention also aims to provide an application mode of the composition for preventing hangover, relieving alcoholism and protecting liver.

Prior art studies have found that the physiological functions of NADH include improving energy levels, protecting cells and promoting neurotransmitter production; NADH not only acts as an important coenzyme in aerobic respiration, but also carries a large amount of energy. Studies have demonstrated that extracellular NADH use promotes intracellular ATP levels, indicating that NADH can penetrate the cell membrane and elevate intracellular energy levels. Macroscopically, exogenous supply of NADH helps to restore physical strength and enhance appetite. And the increase of energy level of brain by NADH also helps to improve mental state and sleep quality. It has been reported that NADH is applied to the fields of improving chronic fatigue syndrome, increasing exercise endurance, and the like; NADH is a strong antioxidant naturally present in cells. NADH can react with free radicals to inhibit lipid peroxidation and protect mitochondrial membrane and mitochondrial function. Researches show that NADH can reduce oxidative stress of cells caused by various factors such as radiation, medicaments, toxic substances, strenuous exercise, ischemia and the like, thereby protecting vascular endothelial cells, liver cells, cardiac muscle cells, fibroblasts, neurons and the like. Therefore, the NADH for injection or oral administration is clinically applied to the fields of improving cardiovascular and cerebrovascular diseases, assisting cancer radiotherapy and chemotherapy and the like. Topical NADH has been shown to be effective in treating rosacea and contact dermatitis; studies have shown that NADH significantly promotes the production of the neurotransmitter dopamine, a chemical signal essential to short-term memory, involuntary movements, muscle tone and spontaneous physical responses. It also mediates the release of growth hormone and determines muscle movement. Without enough dopamine, the muscles become stiff. For example, some causes of Parkinson's disease are caused by disturbances in dopamine synthesis in brain cells. Preliminary clinical experimental data indicate that NADH contributes to the amelioration of symptoms of parkinson's disease. NADH can also promote the biosynthesis of norepinephrine and serotonin, and shows good application potential for relieving depression and senile dementia. NADH promotes the synthesis of the neurotransmitters dopamine, serotonin and norepinephrine. Dopamine is a class of neurotransmitters that causes people to feel happy or excited. When the concentration of dopamine in brain is reduced, central excitation is weakened, the reactivity of human is reduced, the muscle control capability is reduced, and the emotion is lowered. Dopamine deficiency in specific areas of the brain leads to parkinson's disease. Parkinson's disease is improved clinically, mainly by restoring dopamine levels. The clinical application of NADH starts with the assistance of parkinson's disease treatment, and thousands of human clinical trials have found that NADH improves the performance deficiencies of parkinson's patients, probably because NADH increases the activity of tyrosine hydroxylase in cells and stimulates dopamine biosynthesis.

Serotonin, also known as 5-hydroxytryptamine (5-HT), is a neurotransmitter in the brain that delivers pleasure and mood stabilization. Norepinephrine has the effects of stimulating the heart and improving blood supply to vital organs (brain) of the body.

Dopamine, serotonin and norepinephrine in the brain of a depression patient are all in a low concentration state, and clinically restoring their concentrations helps to improve the low-falling state of the mood of the person. NADH is used as a novel biological antidepressant, and more than 90 percent of depression patients reflect good clinical effect.

From a chemical point of view, the physiological function of NADH, is related to the following reactions:

wherein NADH and NAD + are a pair of redox pairs in the cell, NADH is a reduced form of coenzyme 1NAD, and NAD + is an oxidized form thereof. In the redox reaction, NADH is used as a donor of hydrogen and electrons, and NAD + is used as an acceptor of hydrogen and electrons, and is involved in physiological processes such as respiration, photosynthesis, alcohol metabolism and the like. They are used as coenzymes for many redox reactions in organisms to participate in life activities and mutually transform.

Also, under anaerobic conditions, ATP produced by glucose metabolism is very small. Under aerobic conditions, NADH or FADH2, produced by glycolysis and the Krebs cycle, can produce large amounts of ATP via oxidative phosphate reactions. The amount of NADH is directly related to the amount of ATP produced, the more NADH that each cell contains, the more energy that is produced. The more energy-requiring organ, the higher the amount of NADH it contains (or requires).

Normally, NADH in human tissue is mainly distributed in heart, muscle, brain, liver and erythrocytes; wherein the NADH content in the heart is the highest, about 90 mg/kg; the NADH content in the brain is about 40 mg/kg. As can be seen, NADH plays an essential role in the physiological metabolism of the human body.

Alcohol metabolism is divided into two steps, first conversion to toxic acetaldehyde and further decomposition to harmless acetic acid, each step requiring catalysis by the oxidized form of NADH.

However, the inventors of the present application, surprisingly found in their studies that NADH, in combination with coenzyme Q10, superoxide dismutase and acetylcysteine, works better in alleviating hangover than NADH; the liver protection function is also achieved objectively; the wine intoxication phenomenon caused by excessive drinking has good relieving effect; and similarly, if the composition with effective dose is deterred in advance, the metabolism of alcohol in a human body can be promoted, the phenomenon of drunkenness is avoided, the sobering can be more quickly performed, and the harm to the human body is avoided.

NADH is oxidized and then converted into NAD +, which provides a key molecule NAD + for the anti-aging of cells. Further studies have found that other NAD + supplements, such as NMNH, NRH, as a substitute for NADH, in combination with coenzyme Q10, superoxide dismutase and acetylcysteine, have similar effects in alleviating hangover and preventing intoxication.

The composition with the functions of preventing hangover, relieving alcoholism and protecting liver is realized by the following technical scheme.

Wherein the composition comprises NAD + supplement, coenzyme Q10, superoxide dismutase and acetyl cysteine.

Further, in the technical scheme of the invention, the NAD + supplement represents NADH, NMNH and/or NRH;

wherein, NADH, NMNH and NRH are respectively: reduced Nicotinamide Adenine Dinucleotide (NADH), reduced Nicotinamide Mononucleotide (NMNH), reduced Nicotinamide Ribose (NRH);

furthermore, in the composition, the weight part ratio of the NAD + replenisher to the coenzyme Q10 to the superoxide dismutase to the acetylcysteine is 0.8-1.2:3.2-4.8: 10.0-15;

further, the composition of the invention preferably comprises the components of NAD + supplement, coenzyme Q10, superoxide dismutase and acetylcysteine, wherein the weight ratio of the components is 0.95-1.05:3.80-4.20:11.875-13.125.

More preferably, the composition of the invention preferably has a weight ratio of the components NAD + supplement, coenzyme Q10, superoxide dismutase and acetylcysteine of 1:4: 12.5;

the composition can be used for preparing pharmaceutical preparations for preventing hangover, relieving alcoholism and protecting liver, wherein the preparations comprise capsules, tablets, pills, oral liquid, ointments, granules, gels or aerosols. Among them, capsules and tablets are preferable. Specifically, the composition of the invention is used as an active ingredient, is matched with medically necessary auxiliary agents to prepare a mixed preparation which is filled into capsules, or is prepared into tablets through a tabletting process.

The composition tabletting preparation is prepared by the following steps:

1. pretreatment of raw and auxiliary materials

1.1, sterilizing the workshop environment, starting a dehumidifier and controlling the humidity to be lower than 45%;

1.2, screening the raw and auxiliary materials by a 40-mesh screen, and removing non-uniform agglomerated raw materials;

1.3 weighing and labeling each required material for further use;

2. mixing the raw materials and auxiliary materials

2.1, cleaning and disinfecting the environment and required equipment and instruments, starting a dehumidifier, and controlling the humidity to be lower than 45%;

2.2 weighing NADH, superoxide dismutase and coenzyme Q10, putting into a two-layer sealed bag, manually mixing for about 15min, or putting into a mixing hopper, setting the rotating speed at 15rpm, and mixing for 15 min;

2.3 weighing acetylcysteine, L-theanine and premixed 2.2 materials, putting into a mixing hopper, setting the rotating speed at 15rpm, and mixing for 15 min;

2.4 weighing the buffer vitamin C and the premixed 2.3 materials, putting into a mixing hopper, setting the rotating speed at 15rpm, and mixing for 5 min;

2.5 after the mixing is finished, sealing and temporarily storing the materials, sticking labels, and recording the total weight of the mixed materials;

3. tabletting

3.1 cleaning and disinfecting the equipment environment. Starting a dehumidifier, and controlling the humidity to be below 45%;

3.2 detecting the tablet weight every 30min, and controlling the tablet weight at the discharge port to be 530 +/-25 mg;

3.3, detecting the hardness once every 30min, and controlling the hardness of the discharge hole to be 70-110N;

3.4 recording the total weight of the tablet cores after tabletting, and calculating the tabletting yield;

4. coating film

4.1 weighing the required coating raw materials, adding 8% (w/w) ethanol (95%) according to the weight part ratio, starting magnetic stirring or mechanical stirring, paying attention to explosion-proof safety, and stirring at a constant speed.

4.2 before use, the desired coating solution is filtered through a 100 mesh screen and the filtered solution is subjected to a coating operation.

Further, preferred compositions of the present invention are tabletted preparations or capsule preparations, and the material content and daily dose of each tablet are shown in table 1:

TABLE 1

Superoxide dismutase (SOD); acetylcysteine (NAC);

beta-nicotinamide adenine dinucleotide reduced (NADH)

NAD + supplements: NADH, NRH and/or NMNH

Wherein, the 7 components in the table 1 account for 21.328% (w/w) of the weight of each tablet or capsule preparation, namely the weight of each tablet or capsule preparation is 2.485 g; the balance is a pharmaceutically acceptable carrier.

When the composition of the present invention is used as a pharmaceutical ingredient, the composition may further contain a pharmaceutically acceptable carrier. As used herein, a "pharmaceutically acceptable carrier" should be compatible with, i.e., capable of being blended with, the components of the compositions of the present invention without substantially diminishing the effectiveness of the pharmaceutical composition in the treatment and/or prevention of helicobacter pylori, as is often the case. Specific examples of some substances that may serve as pharmaceutically acceptable carriers or components thereof are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and methyl cellulose; powdered gum tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyhydric alcohols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting agents, stabilizers; an antioxidant; a preservative; pyrogen-free water; isotonic saline solution; and phosphate buffer, etc., wherein the preferred carrier is physiological saline, or phosphate buffered saline.

In order to make the purpose and technical solution of the embodiments of the present invention clearer, the technical solution of the embodiments of the present invention will be clearly and completely described below with reference to the implementation examples of the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be made by a person skilled in the art without inventive effort based on the described embodiments of the invention, fall within the scope of protection of the invention.

Drawings

FIG. 1NADH, NRH, NMNH and their corresponding compositions increase NAD + in various mammalian cell lines

Horizontal;

FIG. 2 is a dose response of NADH and NADH compositions in AML12 cells;

FIG. 3 shows that the NADH, NRH, NMNH compositions have no significant apoptosis-triggering effect;

FIG. 4 shows that the NADH, NRH, NMNH compositions protect cells from genotoxicity;

FIG. 5 is a graph showing that each of the compositions NADH, NRH, NMNH enhances NAD + levels in various tissues in vivo;

FIG. 6 is a graph of the effect of NADH, NRH, NMNH compositions on the levels of ethanol and acetaldehyde in the blood and the NAD +/NADH ratio in the liver of ethanol-loaded mice;

FIG. 7 is a graph of the effect of NADH, NRH, NMNH compositions on plasma AST and ALT levels in ethanol-loaded mice;

FIG. 8 is a graph of the combination of NADH, NRH, NMNH Malondialdehyde (MDA) in the liver of ethanol-loaded mice

And the effects of superoxide dismutase (SOD) levels;

FIG. 9 is a graph of the liver Triglyceride (TG) of NADH, NRH, NMNH combinations versus ethanol-loaded mice in the liver

And the effect of VLDL levels;

FIG. 10 is a graph of the effect of NADH, NRH, NMNH combinations on loss of the ethanol loaded mouse righting reflex (LORR).

Detailed description of the preferred embodiments

The technical solution of the present invention is further illustrated by the following specific examples. In the following embodiments, the raw materials are all commercial products purchased from the relevant manufacturing enterprises or commercial departments, unless otherwise specified.

Examples 1 to 5

The raw materials were weighed in an amount of 1000 times according to the following table 2, and then, according to the manufacturing procedure of tablets, tablets were prepared.

TABLE 2

NAD + supplements: NADH, NRH and/or NMNH

The 7 components in table 2 account for 21.328% (w/w) of the weight of each tabletted preparation or each capsule preparation, i.e. the weight of each tabletted preparation or each capsule preparation is 2.485 g; the balance is pharmaceutically acceptable carrier.

The preparation method comprises the following steps:

1. pretreatment of raw and auxiliary materials

1.3 weighing and labeling each required material for further use;

2. mixing the raw materials and auxiliary materials

3. tabletting

3.2 detecting the tablet weight every 30min, and controlling the tablet weight at the discharge port to be 450 +/-25 mg;

4. coating film

Control test

In the following tests, the materials and methods used were as follows:

LN229, HEK293, Neuro2a and C2C12 cell lines were purchased from ATCC cell bank, from central office, Beijing, and were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% inactivated Fetal Bovine Serum (FBS), 100U/ml penicillin and streptomycin. MIN6 cell line was purchased from ATCC cell bank, agency of central office of Beijing, and cultured in DMEM containing 15% inactivated fetal bovine serum, 100U/ml penicillin and 100. mu.g/ml streptomycin. AML12 cell line was purchased from ATCC cell bank, agency of Central offices in Beijing, cultured in a 1:1 volume ratio of high glucose DMEM to Ham's F-12 (containing L-glutamine) mixed medium supplemented with 10% inactivated FBS, 15mM HEPES (hydroxyethylpiperazineethiosulfonic acid), 40ng/ml dexamethasone, 0.005mg/ml human recombinant insulin, 5ng/ml sodium selenite and 0.005mg/ml transferrin (Aldrich). Cells were cultured in a humidified incubator supplied with 5% CO2 and 95% air at 37 ℃.

Cellular NAD + assay

For NAD + assay, each cell line was seeded in 6-well plates, and when the confluence of the cells reached about 80%, the cells were incubated with the desired concentrations of NADH, NRH, NMNH monomers and their corresponding compositions containing the same monomer concentrations for 3 hours of incubation treatment unless otherwise specified, and after incubation was completed, the cells were collected by trypsinization and counted by hemocytometer and trypan blue. The collected cells were centrifuged at 3000 Xg for 3 minutes. After the residual medium was decanted, the cells were lysed with 7% perchloric acid to preserve NAD +, and then neutralized with NaOH (2M) and K2HPO4(500 mM). The level of NAD + in the cells is determined according to published methods.

Apoptosis assay

To test whether each of the compositions NADH, NRH, NMNH was cytotoxic, the C2C12, Neuro2a and AML12 cell lines were incubated with each of the compositions NADH, NRH, NMNH (1mM NADH, NRH, NMNH, respectively) for 24 hours, and then separated with trypsin for cell counting. Cells were stained with trypan blue and then counted with a hemocytometer. To test whether each composition triggered apoptosis to AML12 cell line, PBS, NADH, NRH, NMNH each composition (1mM NADH, NRH, NMNH, respectively) was added to 6-well plates and treated for 24 hours. Cells were then stained using Annexin V/PI staining kit and caspase 3/7/SYTOX dead cell staining kit (Life Technologies) and counted using a BD Fascelesta count cytoTM flow cytometer. Quadrants of apoptotic, necrotic and viable cells were identified and quantified according to the manufacturer's instructions.

Effect of NADH, NRH, NMNH compositions on NAD + in vivo

10 male C57BL/6J mice (Jackson laboratories) aged 8 weeks were housed in polycarbonate cages and were subjected to a 12 hour light/dark cycle with free access to food and water. Then dividing the mice into two groups randomly; the treatment group was injected intraperitoneally with 1000mg/kg NADH composition dissolved in PBS, and the control group was injected with PBS alone. After 4 hours, blood samples were collected by cardiac puncture and mice were sacrificed. Adipose tissues of liver, kidney, brain, muscle and epididymis were collected, immediately frozen in liquid nitrogen, and then stored at-80 ℃ until NAD + analysis. To compare the effect of smaller doses of each of the NADH, NRH, NMNH compositions, we also performed similar experiments: each of NADH, NRH, and NMNH compositions (containing 250mg/kg of NADH, NRH, and NMNH, respectively) was injected intraperitoneally. After 4 hours, mice were sacrificed and tissue samples were collected. All animal studies were performed according to the guidelines for care and use of experimental animals. For the NAD + assay, approximately 100mg of frozen tissue was pulverized in liquid nitrogen and sonicated in 7% perchloric acid, then the solution was neutralized and the NAD + assay performed as described above.

Animal(s) production

C57BL/6J male mice (Jackson laboratory) (40 days old) were used for this study. They were kept under standard laboratory conditions at 22. + -. 1 ℃ with a dark/light cycle of 12/12h and a relative humidity of 55. + -. 5%. All animals had free access to food and water except for 2 hours of fasting prior to dosing.

Mice were randomly divided into normal (untreated), control and treated groups of 8 mice each. Mice in the treatment group received each of the NADH, NRH, NMNH compositions (500 mg/kg NADH, NRH, NMNH respectively) or vehicle intragastrically 15 minutes prior to a single intake of 8ml/kg ethanol (52% w/v in tap water). This is the most commonly used animal alcohol intake to simulate human alcoholism, being 5-6g/kg body weight, corresponding to a 75 kg human consumption of 0.75L whisky (40% v/v) [15 ]. The control group was given the same ethanol solution and corresponding distilled water, while the normal group was given only the corresponding distilled water. At various time points of the experiment, mice were anesthetized by abdominal cavity injection of 10% chloral hydrate (350mg/kg body weight). Blood and liver tissue were extracted from each animal. Liver tissue was immediately frozen for the following bioanalysis.

Determination of ethanol and acetaldehyde concentration in blood

An eye blood sample (0.3mL) was taken and placed in an 8mL headspace vial containing 1.2mL of 0.6M perchloric acid, 0.5mL of trichloroacetic acid (10%) and 0.3mL of internal standard (160mg/L t-butanol) and the concentrations of ethanol and acetaldehyde were determined by headspace chromatography. Ethanol and acetaldehyde were quantified by gas chromatography as described in Isse et al.

Determination of liver NAD +/NADH ratio

The NAD +/NADH ratio was measured using a NAD +/NADH quantitative colorimetric kit (ABIN411692) according to the manufacturer's instructions. Fresh tissue lysates were prepared from snap-frozen liver samples stored at 80 deg.C, 20mg of liver homogenized with NAD +/NADH extraction buffer (400. mu.L). To rapidly remove enzymes that may consume NADH, the sample was filtered through a 10kD molecular weight cut-off filter prior to detection. To detect NADH, the extracted sample was heated to 60 ℃ for 30 minutes in a heating block to decompose NAD. Concentration was calculated using NADH standard curve.

Determination of plasma AST and ALT levels

To evaluate the prophylactic effect of each of the NADH, NRH, NMNH compositions, male C57BL/6J mice were fasted for 12 hours and either NADH, NRH, NMNH compositions (containing 500mg/kg NADH, NRH, NMNH, respectively) or vehicle were gavaged 15 minutes prior to the intake of 8ml/kg ethanol (40% w/v in tap water). After the alcohol is treated for 6 hours, a blood sample is taken to detect biochemical indexes. To evaluate the therapeutic effect of each of the compositions NADH, NRH, NMNH, 40% ethanol was administered every 24h in addition to the control group at an equal amount of physiological saline, resulting in acute liver injury for 3 consecutive days. After three consecutive days of alcohol treatment, the mice were administered with each of the compositions NADH, NRH, NMNH (500 mg/kg NADH, NRH, NMNH, respectively) daily for three consecutive days, and blood samples were taken for biochemical tests. Blood samples were collected in anticoagulant tubes and centrifuged at 1500rpm for 10 minutes to obtain plasma. Plasma AST and ALT activities were determined using a commercial kit (Sigma-Aldrich) according to the manufacturer's instructions.

Determination of liver SOD, MDA and TG levels

Liver samples were prepared by homogenization in cold isotonic saline. The homogenate (10%, w/v) was centrifuged at 4500g for 10 min and the supernatant used for biochemical analysis. MDA, SOD and TG levels were determined using commercial kits (Abcam, # ab 118970; Thermofisiher, # EIASODC and Abcam, ab65336) according to the manufacturer's instructions. The total protein was determined by BCA protein assay kit (Abcam, # ab102536) for each set of samples according to the manufacturer's instructions and the results were normalized to total protein concentration as an internal reference (comparing MDA, SOD and TG levels for different samples at the same total protein concentration).

Very Low Density Lipoprotein (VLDL) levels in the liver of experimental mice

Portions of liver tissue were fixed for histopathological evaluation according to standard procedures. The other part of the liver was stored at-80 ℃. Frozen sections 10mm thick were stained with oil red O to detect liver steatosis. The stained sections were observed with a 400-fold optical microscope. Serum samples were examined for VLDL levels using a Bio-Tek synergy2 multi-scan spectrum (Botten Instruments Co.).

Determination of loss of righting (forward) reflection

According to Ozburn et al, the tolerance of mice to alcohol-induced hypnosis was evaluated by the loss of righting reflex (LORR) test [18 ]. LORR is defined as the phenomenon that mice cannot correct themselves 3 times within 30 seconds after drinking alcohol. The LORR delay time is defined as the time from drinking to LORR occurrence, and the LORR duration is defined as the time from occurrence to LORR recovery.

Statistical analysis

Differences between the treated and control groups were analyzed by independent t-test using SPSS 20.0 statistical software. All results are expressed as mean ± standard deviation. Differences with P <0.05 were statistically significant.

Control test and results

NADH, NRH, NMNH and corresponding compositions increase NAD + levels in various mammalian cell lines

To assess whether NADH, NRH, NMNH and their corresponding compositions are taken up by cells and alter intracellular NAD + levels, some mammalian cell lines, such as proinsulin cells (MIN6), neuron-like cells (LN229), muscle-like cells (C2C12), normal liver cells (AML12) and HEK293 cells, were treated with vehicle (control) for 3 h.

The former varied the NAD + concentration in the cells 2.7-11 times the latter after 3h of treatment with each of the compositions NADH, NRH, NMNH (FIG. 1). It is evident that the NADH composition has a greater effect on the promotion of intracellular NAD + than NADH. NADH compositions induced NAD + levels much higher than NADH induced levels in all cell lines (fig. 1). These data indicate that NADH compositions stimulate NAD + increase in cultured mammalian cells more effectively than NADH monomers, NRH, NMNH monomers, and combinations thereof.

FIG. 1NADH, NRH, NMNH and their corresponding compositions increase NAD + levels in various mammalian cell lines.

Referring to FIG. 1(A) (B) (C) (D) (E), LN229, HEK293, MIN6, C2C12, and AML12 cell lines were treated with 1mm NADH, NRH, NMNH monomers and their corresponding compositions containing the same monomer concentration for 3 hours, and the change in NAD + in each cell line was detected.

Note: comparison of cell lines from each group with control group (. about.P)<0.01,

n ═ 3), each monomer was compared with its corresponding composition (# P)<0.05,

n＝3)。

Dose response of NADH and NADH compositions in AML12 cells

These cells were treated with increasing concentrations of NADH composition for 3 hours. At the lowest NADH composition concentration of 50. mu.M, the NAD + concentration was about 4 times that of the corresponding control group. Saturation was achieved at 500. mu.M and the concentration was 10-fold higher than that of the untreated control (FIG. 2A). Time course studies of the effect of NADH compositions were also conducted. AML12 cells were incubated with NADH composition for various times (0-12 hours) and then harvested and NAD + levels were evaluated. We demonstrated that the NAD + concentration increase effect occurred at the earliest 0.5 hours and that the full effect occurred after 3 hours (fig. 2B). Notably, NAD + remained at a high concentration after 12 hours of treatment with the NADH composition. Finally, the NADH and NADH combination can significantly increase the NAD +/NADH ratio of hepatocytes, with the effect of the NADH combination being far superior to NADH (fig. 2C).

FIG. 2 dose response of NADH and NADH compositions in AML12 cells, NAD + levels per mg protein were measured in cell lines after 3 hours of treatment of AML12 cells with 50, 100, 250, 500, 750 and 1000. mu.M NADH and NADH compositions. Note: comparison with control group (. about.P)<0.05,

n-3), NADH is compared to NADH composition (# P)<0.05,

n-3). (B) NADH and NADH combinations enhance NAD + levels in AML12 cells in a time-dependent manner. The level of NAD + contained per mg protein in the cell line was measured after incubating AML12 cells with 1mM NADH and NADH combination for 0.5, 1, 3, 6 and 12 h. Note: comparison with control group (. about.P)<0.05,

n-3), NADH is compared to NADH composition (# P)<0.05,

n-3). (C) The NAD +/NADH ratio was measured in AML12 cells treated with 1mM NADH, NRH, NMNH monomer and their corresponding compositions containing the same monomer concentration for 6 h. Note: each one ofGroup cell lines compared to control group (. about.P)<0.01,*P<0.05,

n＝3)。

3. Low toxicity and rescue effects of genotoxicity

The dramatic increase in NAD + concentration has made us suspected of being potentially toxic to cells. After 24 hours of exposure to the NADH composition (1mm NADH), we did not observe any significant loss of cultured cells (fig. 3A). For example, the cell counts of C2C12, Neuro2a, and AML12 cells were similar in the control (NADH) and NADH composition treated cells. Trypan blue positive cells were similar in the three cases, less than 5%, and there was no significant difference in apoptosis markers. Annexin V or caspase 3/7 did not significantly change in cells treated with the combination of NADH and NADH compared to the control group (fig. 3B and 3C). The results show that the cells are well tolerated at millimolar concentrations of NADH and NADH composition, and that high NAD + levels are well tolerated without significant toxic effects for at least 24 hours.

FIG. 3 the effect of NADH, NRH, NMNH on the triggering of apoptosis is not evident.

(A) The C2C12, Neuro2a and AML12 cell lines were treated with each combination of NADH, NRH, NMNH (1mM NADH, NRH, NMNH, respectively) for 24 hours and then the total number of cells was measured. Note: comparison with control group (P)>0.05,

n-3). (B) The AML12 cell line was stained for annexin V (apoptosis marker) and the cells were counted by flow cytometry. Note: the three cell types were compared between different experimental groups (P)>0.05,

n-3). (B) The AML12 cell line was subjected to caspase 3/7 (apoptosis marker)Twigs) and cells were counted using a flow cytometer. Note: comparison of the three cell types between different experimental groups (P)>0.05,

n＝3)。

Whether NADH compositions can improve cell viability under stress conditions with NAD + depletion

To investigate whether NADH compositions could improve cell viability under stress conditions with NAD + depletion. Thus, we tested genotoxicity that can lead to DNA damage, activate poly (ADP-ribose) polymerase and induce NAD + depletion. Strong genotoxicity can lead to severe depletion of NAD + by the cell, leading to cell death. Therefore, we treated AML12 cells with Hydrogen Peroxide (HP) for 6 hours, then counted the cells and detected NAD + in samples co-treated with NADH composition (containing NADH 1mM) or its carrier. It was demonstrated that cell viability increased from 12% to nearly 72% under treatment with the HP + NADH composition (FIG. 4A). Furthermore, in the cells treated with the HP + NADH composition, NAD + levels remained high, whereas in the HP cells, NAD + concentrations were much lower than the control values (fig. 4A). We obtained similar results in INS1 cells that contained insulin and were sensitive to HP (data not shown). Then, we compared the effect of NADH composition and NADH on AML12 cells at lower concentrations (250. mu.M), and found that NADH composition not only protected NAD + levels, but also protected cell survival. However, although NADH treatment was also very effective, it was much less effective in maintaining NAD + content and cell viability (fig. 4B).

FIG. 4NADH, NRH, NMNH compositions protect cells from genotoxicity.

(A) (B) AML12 cell lines cell viability and intracellular NAD + levels were measured after 3 hours of treatment with 500 μ M HP in the presence or absence of a NADH composition (containing 1mM NADH). Note: comparison with control group (. about.P)<0.05,

n-3), compared to HP group (# P)<0.05,

n-3). (C) (D) AML12 cell lines cell survival and NAD + levels in cells were measured after 3 hours of treatment with 500. mu.M HP in the presence or absence of each of the compositions NADH, NRH, NMNH (500. mu.M NADH, NRH, NMNH respectively). Note: comparison with control group (. about.P)<0.05,

n-3), compared to HP group (# P)<0.05,

n-3) compared to the HP + NMNH composition or HP + NRH composition ($ P)<0.05,

n＝3)。

5. NAD + levels in various tissues in vivo

To evaluate the biological effect of each of the NADH, NRH, NMNH compositions on mice, we injected each of the NADH, NRH, NMNH compositions (containing 1000mg/kg NADH) intraperitoneally into C57BL/6J mice and measured the NAD + content in the tissues after 4 hours. As shown in fig. 5, NAD + levels in blood, brain, fat and kidney increased several-fold, with a maximum increase in liver NAD + concentration of more than 7-fold within 4 hours (fig. 5A). NADH, NRH, NMNH each composition had the least NAD + promoting effect on skeletal muscle. The results show that each of the NADH, NRH, NMNH compositions is an effective NAD + concentration enhancer. After a single administration, NAD + concentrations in many animal tissues and organs are elevated to varying degrees.

To assess whether each of the NADH, NRH, NMNH compositions was more effective in vivo than the other NAD + precursors, each of the NADH, NRH, NMNH compositions (containing 250mg/kg), NADH (250mg/kg) or vehicle was intraperitoneally injected alone and the NAD + content in kidney and liver was assessed after 4 hours (FIG. 5B). The results show that the NADH, NRH and NMNH compositions and NADH can obviously increase the NAD + content in the kidney, wherein the effect of the NADH, NRH and NMNH compositions is strongest, and reaches 350 percent, and the NADH content is 260 percent respectively. In the liver, each combination of NADH, NRH, NMNH increased NAD concentration by 680% while NADH provided 500% (fig. 5B). Thus, at the same dose, each composition of NADH, NRH, NMNH provided a greater increase in NAD + than NADH, while in the liver it provided a rather significant increase in NAD + at low doses, consistent with an improvement in pharmacological efficacy compared to other NAD + precursors. These findings further confirm the results observed in cell culture studies.

FIG. 5NADH, NRH, NMNH compositions enhance NAD + levels in tissues in vivo.

After male C57BL/6J mice were injected intraperitoneally with 1000mg/kg NADH composition for 4h, the level of NAD + in the adipose tissues of blood, liver, kidney, brain and epididymis was significantly higher than that in the control group. Note: comparison with control group (. about.P)<0.05,

n-3). (B) Male C57BL/6J mice were euthanized after intraperitoneal injection of each composition NADH, NRH, NMNH (250mg/kg NADH, NRH, NMNH, respectively) for 4 h. NAD + levels in liver and kidney tissues were compared for each group. Note: comparison with control group (. about.P)<0.05,

n-3), in the same tissue, compared with NMNH composition group (# P)<0.05,

n＝3)。

Effect of NADH, NRH, NMNH compositions on the levels of ethanol and acetaldehyde in blood and the NAD +/NADH ratio in liver in ethanol-loaded mice

As shown in fig. 6A, when NADH, NRH, NMNH compositions and NADH were orally administered 30 minutes before ethanol administration, they could significantly reduce ethanol in blood during the experiment, and the NADH, NRH, NMNH compositions acted faster and stronger than NADH, beginning to act within 15 minutes. The NADH, NRH, NMNH compositions and NADH treatment significantly increased blood acetaldehyde levels in mice given alcohol within two hours, meaning that they increased the first-pass rate of alcohol metabolism (fig. 6B). In other words, ethanol is rapidly metabolized to acetaldehyde, which is slowly metabolized to acetic acid. It is noteworthy that after two hours, they significantly reduced acetaldehyde in the blood, and the effect of each combination of NADH, NRH, NMNH was more significant than NADH. These results all indicate that NADH, NRH, NMNH and NADH promote alcohol metabolism. To further elucidate the respective compositions NADH, NRH, NMNH and the anti-alcohol function of NADH, we examined the ratio NAD +/NADH in the liver of mice. As shown in FIG. 6C, the NAD +/NADH redox ratio in hepatocytes decreased due to alcohol metabolism, and the decrease was significantly suppressed by the NADH, NRH, NMNH compositions or NADH administration. Thus, administering NADH, NRH, NMNH combinations or NADH to mice can physiologically alter the liver redox fraction, NAD +/NADH, allowing the dehydrogenase reaction to progress further towards ethanol oxidation.

FIG. 6 Effect of NADH, NRH, NMNH compositions on the levels of ethanol and acetaldehyde in blood and the NAD +/NADH ratio in liver in ethanol-loaded mice.

(A) (B) Male C57BL/6J mice were fasted for 12 hours and gavaged with either NADH, NRH, NMNH compositions (500 mg/kg NADH, NRH, NMNH respectively) or vehicle 15 minutes prior to the intake of 8ml/kg ethanol (40% w/v in tap water). Blood samples were taken from the infraorbital venous plexus at different time points after ethanol administration and the levels of ethanol and acetaldehyde in the blood were determined, each group containing 6-8 mice. (C) In another set of experiments, mice were sacrificed after different time of ethanol treatment, liver tissue was extracted and used to measure the NAD +/NADH ratio (n ═ 6) therein.

Reduction of acute alcoholic liver toxicity by NADH, NRH, NMNH compositions and NADH

Liver marker enzymes in plasma, such as aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT), are known as sensitive biochemical markers of early liver injury. In this study, we further observed the effect of NADH, NRH, NMNH combinations and NADH on the prevention and treatment of acute alcoholic liver injury. As shown in fig. 5, acute alcohol treatment significantly increased plasma AST and ALT levels. Compared with the model group, the intake of NADH, NRH, NMNH compositions and NADH in advance can significantly reduce plasma AST and ALT levels, indicating that NADH, NRH, NMNH compositions and NADH may protect liver tissues from acute alcoholism. Furthermore, in another set of experiments, we treated mice with alcohol for three days, followed by treatment with each of the compositions NADH, NRH, NMNH and NADH. The NADH, NRH, NMNH compositions and NADH still significantly reduced AST and ALT levels, indicating that they had some therapeutic effect on alcoholic hepatotoxicity, although these effects were not as strong as prophylactic (fig. 7A and 7B).

FIG. 7 Effect of NADH, NRH, NMNH compositions on plasma AST and ALT levels in ethanol-loaded mice.

(A) To evaluate the preventive effect of each of the NADH, NRH, NMNH compositions, male C57BL/6J mice were fasted for 12h and gavaged with either NADH, NRH, NMNH compositions (500 mg/kg NADH, NRH, NMNH respectively) or vehicle 15 minutes prior to the intake of 8ml/kg ethanol (40% w/v in tap water). After the alcohol treatment for 6h, a blood sample is taken to detect biochemical indexes. Note: comparison with control group (. about.P)<0.05,

n ═ 3), compared with ethanol administration group (# P)<0.05,

n-3). (B) To evaluate the therapeutic effect of each of the compositions NADH, NRH, NMNH, 40% ethanol was administered every 24h in addition to the control group at an equal amount of physiological saline, resulting in acute liver injury for 3 consecutive days. After three consecutive days of alcohol treatment, the mice were administered the respective compositions NADH, NRH, NMNH (500 mg/kg NADH, NRH, NMNH, respectively) daily for three consecutive days, and blood samples were taken for biochemical tests. Note: comparison with control group (. about.P)<0.05,

n ═ 3), compared with ethanol administration group (# P)<0.05,

n＝3)。

Protective Effect (prevention) of NADH, NRH, NMNH compositions and NADH against oxidative damage caused by acute alcohol intake

Oxidative stress plays a pathogenic role in many liver diseases such as hepatitis, NASH, fibrosis, cirrhosis and liver cancer. Thus, monitoring endogenous/exogenous antioxidants and enzymes involved in free radical control can make a necessary contribution to the development and progression of the disease, and can also be considered as good adjuvants for antioxidant therapy, in order to assess the protective effect of NADH, NRH, NMNH compositions and NADH on oxidative damage induced by acute alcohol intake in vivo, biochemical parameters Malondialdehyde (MDA) (oxidative damage index) and superoxide dismutase (SOD) (antioxidant index) in liver tissue were examined. As shown in fig. 8, the SOD activity was decreased and MDA level was significantly increased in the model group, almost twice as much as that of the control group. The pre-administration of NADH, NRH and NMNH compositions and NADH can obviously reduce the MDA content of the liver of an alcohol-loaded mouse and improve the SOD activity. Taken together, these results suggest that the NADH, NRH, NMNH compositions and NADH may protect the liver from high oxidative stress caused by acute alcohol intake.

FIG. 8 Effect of NADH, NRH, NMNH compositions on Malondialdehyde (MDA) and superoxide dismutase (SOD) levels in the liver of ethanol-loaded mice.

(B) Male C57BL/6J mice were fasted for 12h and gavaged 30 minutes prior to ingestion of 8ml/kg ethanol (40% w/v in tap water) with each of the NADH, NRH, NMNH compositions (500 mg/kg NADH, NRH, NMNH respectively) or vehicle. After 6h of alcohol treatment, liver tissue was taken for measurement of Malondialdehyde (MDA) and superoxide dismutase (SOD). Note: comparison with control group (. about.P)<0.05,

n ═ 3), compared with ethanol administration group (# P)<0.05,

n＝3)。

Effect of NADH, NRH, NMNH compositions and NADH on liver histopathology and lipid metabolism

The hepatic lipid metabolism index, hepatic tissue Triglyceride (TG) level and Very Low Density Lipoprotein (VLDL) activity in serum were measured to evaluate alcoholic liver injury. As shown in FIG. 9A, the liver TG and serum VLDL activities of the alcohol-induced mice were increased 1.5-fold and 1.3-fold, respectively, as compared with the normal control group. Compared with the model control group, the NADH, NRH, NMNH compositions and NADH prevention reduce liver index, TG and VLDL levels of the alcohol-loaded mice, and the NADH, NRH, NMNH compositions and NADH can relieve lipid metabolism abnormality caused by acute alcoholic liver injury.

FIG. 9 Effect of NADH, NRH, NMNH compositions on hepatic Triglyceride (TG) and VLDL levels in the liver of ethanol-loaded mice.

(A) (B) Male C57BL/6J mice were fasted for 12h and gavaged 30 minutes prior to ingestion of 8ml/kg ethanol (40% w/v in tap water) with each of the NADH, NRH, NMNH compositions (500 mg/kg NADH, NRH, NMNH respectively) or vehicle. Liver tissue was taken for determination of hepatic Triglyceride (TG) and VLDL levels after 6h of alcohol treatment. Note: comparison with control group (. about.P)<0.05,

n ═ 3), compared with ethanol administration group (# P)<0.05,

n＝3)。

Effect of NADH, NRH, NMNH compositions and NADH on acute alcohol exposure tolerance

Alcohol acts on the central nervous system, causing various behavioral problems, such as loss of righting reflex (LORR). As shown in fig. 10A and fig. 10B, acute alcohol intake induced LORR (100% LORR rate) in all mice within an average of 14.76 minutes (latency) in the model group. Meanwhile, the average length of the LORR is about 798.68 minutes. Administration of each of the NADH, NRH, NMNH compositions and NADH significantly increased tolerance to acute alcohol exposure in mice, manifested by decreased LORR rates and durations and increased latency. It is noted that less than half of the mice in each of the NADH, NRH, NMNH composition groups had positive reflex after drinking, the LORR duration was significantly shortened to 242.00 minutes, almost one third of the model group, and the mean latency increased to 58.52 minutes, which is 4 times that of the model group.

FIG. 10 Effect of NADH, NRH, NMNH compositions on loss of the ethanol loaded mouse righting reflex (LORR).

(A) Male C57BL/6J mice were fasted for 12h and gavaged 30 minutes prior to ingestion of 8ml/kg ethanol (40% w/v in tap water) with each of the NADH, NRH, NMNH compositions (500 mg/kg NADH, NRH, NMNH respectively) or vehicle. After 6h of alcohol treatment, the rate of loss of mouse righting reflex (LORR) was observed and recorded. Note: comparison with control group (. about.P)<0.05,

n-3). (B) Male C57BL/6J mice were fasted for 12h and gavaged 30 minutes prior to ingestion of 8ml/kg ethanol (40% w/v in tap water) with each of the NADH, NRH, NMNH compositions (500 mg/kg NADH, NRH, NMNH respectively) or vehicle. After 6h of alcohol treatment, the delay time and duration of LORR (LORR delay time is defined as the time from drinking to LORR occurrence and LORR duration is defined as the time from LORR occurrence to LORR recovery.) were observed and recorded. Note: comparison with control group (. about.P)<0.05,

n＝3)。

The above description further describes a specific embodiment of the present invention with reference to specific examples, which are intended for the detailed description of the present invention and are not intended to limit the present invention. The above-mentioned embodiments are merely descriptions of the preferred embodiments of the present invention, and do not limit the technical concept and the protection scope of the present invention, and those skilled in the art should make various modifications and improvements to the technical concept without departing from the design concept of the present invention.

Claims

1. A composition for preventing hangover, relieving hangover and protecting liver is characterized by comprising NAD + replenisher, coenzyme Q10, superoxide dismutase and acetylcysteine.

2. The composition for preventing hangover, alleviating hangover and protecting liver as claimed in claim 1, wherein the weight ratio of NAD + supplement, coenzyme Q10, superoxide dismutase and acetylcysteine in the composition is 0.8-1.2:3.2-4.8:10.0-15: 4.3-4.7.

3. The composition for preventing hangover, alleviating hangover and protecting liver as claimed in claim 2, wherein the components of the composition, NAD + supplement, coenzyme Q10, superoxide dismutase and acetylcysteine, are 0.95-1.05:3.80-4.20:11.875-13.125:4.4-4.6 by weight.

4. The composition for preventing hangover and protecting liver as claimed in claim 3, wherein the composition comprises NAD + supplement, coenzyme Q10, superoxide dismutase and acetylcysteine at a weight ratio of 1:4: 12.5: 4.5.

5. the composition for preventing hangover and protecting liver as claimed in any one of claims 1 to 4, wherein the NAD + supplements comprise NADH, NMNH and/or NRH.

6. The composition for preventing hangover and protecting liver as claimed in claim 5, wherein the composition is combined with a pharmaceutically acceptable carrier or its components to prepare a pharmaceutical preparation for preventing hangover and protecting liver, and the preparation comprises capsules, tablets, pills, oral liquids, ointments, granules, gels or aerosols.

7. The composition for preventing hangover and protecting liver as claimed in claim 5, wherein the specific examples of the pharmaceutically acceptable carrier or some of its components are saccharides such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and methyl cellulose; powdered gum tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyhydric alcohols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting agents, stabilizers; an antioxidant; a preservative; pyrogen-free water; isotonic saline solution; and phosphate buffer, etc., wherein the preferred carrier is physiological saline, or phosphate buffered saline.

8. The composition for preventing hangover and protecting liver as claimed in claim 6, wherein each capsule or tablet contains NAD + supplement 20mg, coenzyme Q10 80mg, superoxide dismutase 250mg, acetylcysteine 90mg, L-theanine 25mg, buffered vitamin C25 mg, silicon dioxide and triacetin (enteric coating) 40 mg; the total weight of each capsule or each tablet is 2.485 g; the balance is pharmaceutically acceptable carrier.