Anti-alcoholism composition, liquid preparation with anti-alcoholism effect and preparation method and application of anti-alcoholism composition and liquid preparation

Abstract

The invention relates to an anti-alcoholism composition, a liquid preparation with an anti-alcoholism effect and a preparation method and application of the liquid preparation, and belongs to the technical field of physiology. The anti-alcohol composition comprises an NAD (nicotinamide adenine dinucleotide) precursor substance and amino acid, and the NAD precursor substance is one or more of nicotinamide, nicotinamide ribose, nicotinamide mononucleotide or nicotinic acid; and the amino acid is one or more of L-glutamine, L-serine or L-lysine. The special combination of the NAD precursor substance and the amino acid can synergistically accelerate ethanol metabolism, and the alcohol effect dispelling liquid preparation prepared on the basis of the alcohol effect dispelling liquid preparation is flexible in use time, controllable in component and outstanding in alcohol effect dispelling effect, and has very wide application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to an alcoholism-relieving composition, a liquid preparation with alcoholism-relieving efficacy and a preparation method and application thereof, and belongs to the technical field of physiology. BACKGROUND

[0002] After drinking, the concentration of ethanol in the blood will increase, and the effect of ethanol on the body is closely related to the concentration of ethanol in the blood. The liver is the main organ for metabolizing ethanol, and its process of metabolizing ethanol mainly relies on the catalysis of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) in hepatocytes, and the process is shown in the following formulas 1 and 2: ADH first oxidizes ethanol to acetaldehyde, and ALDH further oxidizes acetaldehyde to acetic acid, and the latter is finally converted into water and carbon dioxide and discharged out of the body; in the oxidation of ethanol or acetaldehyde, ADH and ALDH need to be coupled to oxidized coenzyme I (NAD + ) to be reduced to reduced coenzyme I (NADH).

[0003]

[0004] The activity of ADH and ALDH in the liver is a key factor affecting the speed of ethanol metabolism in the body. It is generally believed that ethanol is metabolized at a constant speed in the body, because the amount of daily drinking has saturated the total activity of liver ethanol metabolism enzymes, especially ADH. It is a general consensus in the medical community that the speed of ethanol metabolism in the human body mainly depends on the original activity of ethanol metabolism enzymes of the individual, although there are Alda-1 and other ALDH activators, but there is no drug approved for increasing the activity of liver ADH or ALDH at present, so the general sense of alcoholism-relieving drugs cannot accelerate ethanol metabolism[1].

[0005] Among the commonly used drugs for treating alcoholism, naloxone has a hangover effect [2], metadoxine has a protective effect on the liver [3], and also has the effect of accelerating the excretion of ethanol and acetaldehyde from the kidneys [4]. A large part of the alcohol products on the market are mainly composed of nutrients such as vitamins, minerals, amino acids, organic acids and sugars, and the design concept of these products is to use multiple nutrients to superimpose multiple possible beneficial effects to support the therapeutic effect. When taking the above alcohol products, a large amount of water is often needed to be supplemented at the same time, which is similar to the effect of giving alcohol poisoning patients glucose solution, although it can relieve symptoms to a certain extent, but it does not have a clear alcohol treatment target in its mechanism of action. This also includes the concept of symptomatic treatment, when drinking leads to dehydration and hypoglycemia in the body, it should be treated promptly. Another type of alcohol product is represented by L-glycine, which can increase the residence time of alcohol in the stomach when taken at the same time as alcohol. Since a certain amount of ADH and ALDH is also expressed in the gastrointestinal tract, delaying the entry of ethanol into the intestine can reduce the amount of ethanol actually absorbed into the blood circulation through the first-pass effect [5]. Protecting the liver is the mechanism of action of many antioxidants and natural products for alcohol, when alcohol causes oxidative stress in the liver, antioxidants can enhance the metabolic and detoxification capacity of liver cells, especially vitamin C and silymarin with strong antioxidant effect, so they are often used as components of alcohol products [6], but such drugs usually need to be taken before drinking, or even a long time in advance to take effect.

[0006] Looking at the above alcohol products, none of them has the effect of increasing the activity of liver ADH or ALDH, so they cannot accelerate the rate of ethanol metabolism to achieve the true meaning of alcohol. Among the alcohol products that accelerate ethanol oxidation, fructose is the most studied one, and its mechanism of action may be related to the reoxidation of NADH produced during ethanol oxidation [7]. In human experiments, oral fructose and other sugars [8], such as honey rich in fructose [9], have the effect of accelerating ethanol metabolism, however, the results of studies on the acceleration of ethanol metabolism by fructose are not consistent, one of which may be related to the order of administration of fructose and ethanol

[10] . In a controlled clinical study, intravenous injection of fructose also has the effect of accelerating ethanol metabolism

[11] , but fructose has higher risks such as increasing triglyceride levels and increasing cardiovascular metabolic disorders

[12] . In addition, fructose accelerates ethanol metabolism but not acetaldehyde metabolism, which can lead to the accumulation of more toxic acetaldehyde

[13] . Acetaldehyde has a mutagenic effect and is listed as a class 1 carcinogen by the World Health Organization together with ethanol (alcoholic beverages). In a study of patients with a loss-of-function mutation in the ALDH2 gene, the effect of fructose on ethanol metabolism was not observed

[14] . ALDH2*2The effect of fructose therapy varies from person to person, and it is not universally accepted as a routine method for treating acute alcohol poisoning. Amino acids and peptides are another type of hangover remedy. Amino acid injection can accelerate ethanol elimination rate, and the mechanism may be related to the increase of NADH reoxidation in mitochondria

[14] . Peptides such as corn peptides also accelerate ethanol elimination rate [15, 16], but the effect of this type of drug in the treatment regimen of giving it after drinking is not obvious enough, and it is better to take it before drinking

[16] . The mechanism may be the same as amino acids, but it works slower. Literature only reports that amino acids and peptides increase the activity of ADH, and do not mention that they increase the activity of ALDH, so they may increase the accumulation of acetaldehyde and cause more damage to the body.

[0007] In summary, most hangover remedies are composed of multiple nutritional ingredients, common ingredients include vitamins, minerals, amino acids, organic acids, sugars, peptides and plant extracts, which produce effects through the synergistic effect of each component, but do not necessarily have a core mechanism of action. Therefore, it is particularly important to develop a composition with a clear and efficient mechanism of action for hangover relief.

[0008] 1. Pittler MH, et al. Interventions for preventing or treating alcohol hangover: systematic review of randomised controlled trials. BMJ. 2005; 331(7531):1515-1518. 2. Mackenzie AI. Naloxone in alcohol intoxication. Lancet. 1979; 1(8118):733-734. 3. Mao Yimin, et al. Multicenter, randomized, double-blind, placebo-controlled clinical study of metadoxine tablets in the treatment of alcoholic liver disease. Liver. 2006, 11(5):303-306. 4. Gessa GL. Guida al trattamento farmacologico dell'alcolismo [Guidelines for the drug therapy of alcoholism]. Recenti Prog Med. 1990; 81(3):171-175. 5. Iimuro Y, et al. Glycine prevent alcohol-induced liver injury by decreasing alcohol in the rat stomach. Gastroentology 1996; 110(5): 1536-1542. 6. Das SK, et al. Protective effects of silymarin, a milk thistle (Silybium marianum) derivative on ethanol-induced oxidative stress in liver. Indian J Biochem Biophys. 2006; 43(5):306-311. 7. Villalobos-García D, et al. The fructose-dependent acceleration of ethanol metabolism. Biochem Pharmacol. 2021; 188:114498. 8. Pawan GL. Vitamins, sugars and ethanol metabolism in man. Nature. 1968; 220(5165):374-376. 9. Onyesom I. Effect of Nigerian citrus (Citrus sinensis Osbeck) honey on ethanol metabolism. S Afr Med J. 2004; 94(12):984-986. 10. Crownover BP, et al. Activation of ethanol metabolism in humans by fructose: importance of experimental design. J Pharmacol Exp Ther. 1986; 236(3):574-579. 11. Brown SS, et al. A controlled trial of fructose in the treatment of acute alcoholic intoxication. Lancet. 1972; 300(7783):898-900. 12. Uzuegbu UE, et al. Fructose-induced increase in ethanol metabolism and the risk of Syndrome X in man. C R Biol. 2009; 332(6):534-538. 13. Nuutinen HU, et al. Blood acetaldehyde concentration gradient between hepatic and antecubital venous blood in ethanol-intoxicated alcoholics and controls. Eur J Clin Invest. 1984; 14(4):306-311. 14. Lisander B, et al. Enhanced rate of ethanol elimination from blood after intravenous administration of amino acids compared with equicaloric glucose. Alcohol Alcohol. 2006; 41(1):39-43. 15. Yamaguchi M, et al. Effect of corn peptide on alcohol metabolism and plasma free amino acid concentrations in healthy men. Eur J Clin Nutr. 1996; 50(10):682-688. 16. Zhu YQ, et al. Comparative study of anti-alcoholism effects of silk hydrolysate and different groups. Silkworm Bulletin. 2008; 39(3):16-18. SUMMARY The present application provides a kind of alcoholism composition, liquid preparation with alcoholism efficacy and its preparation method and application.The alcoholism composition described in the present application includes one or more of NAD precursor such as nicotinamide (NAM), nicotinamide riboside (NR), nicotinamide mononucleotide (NMN) or nicotinic acid (NA) and one or more of amino acid such as L-glutamine, L-serine or L-lysine, the special combination can synergistically accelerate ethanol metabolism;In addition, other nutritional ingredients that assist the effect in some combinations, including vitamin B1, vitamin B6, citric acid or citrate.

[0009] The main component of the alcoholism composition described in the present application is NAD precursor and amino acid.The mechanism of action of alcoholism includes: increasing the NAD + level and NAD + / NADH ratio of liver, increasing the activity of liver ADH and ALDH, and accelerating the metabolism of ethanol and acetaldehyde.

[0010] An alcoholism composition, the alcoholism composition includes NAD precursor and amino acid, wherein the NAD precursor is one or more of nicotinamide, nicotinamide riboside, nicotinamide mononucleotide or nicotinic acid; the amino acid is one or more of L-glutamine, L-serine or L-lysine.

[0011] In the above technical solution, the mass ratio of the NAD precursor and the amino acid is 1:0.45-24.

[0012] Preferably, the mass ratio of the NAD precursor and the amino acid is 1:0.45-12.

[0013] Further preferably, the mass ratio of the NAD precursor and the amino acid is 1:1.9-5.3.

[0014] In the method described in the present application, when multiple amino acids are selected, they can be compounded in any ratio.

[0015] Preferably, the amino acid is a mixture of L-glutamine and L-serine compounded in a mass ratio of 1:1-2.3.

[0016] Preferably, the amino acid is a mixture of L-glutamine and L-lysine compounded in a mass ratio of 1:0.35-1.43.

[0017] Preferably, the amino acid is a mixture of L-glutamine, L-serine and L-lysine compounded in a mass ratio of 1:1-2.3:0.35-1.43.

[0018] In the method described in the present application, when multiple NAD precursors are selected, they can be compounded in any ratio.

[0019] Preferably, the NAD precursor is a mixture of NA and NAM with a mass ratio of 1:1-5.

[0020] Preferably, the NAD precursor is a mixture of NA and NMN with a mass ratio of 1:1-5.

[0021] Preferably, the NAD precursor is a mixture of NA and NR with a mass ratio of 1:1-5.

[0022] In the above technical solution, the hangover-relieving composition further comprises other nutritional ingredients, and the nutritional ingredients are one or more of vitamin B1, vitamin B6, citric acid or citrate.

[0023] In the method of the present application, when multiple nutritional ingredients are selected, the vitamin B1 and vitamin B6 can be compounded in any ratio.

[0024] In the method of the present application, when one of the multiple selected nutritional ingredients is citric acid or citrate, the mass fraction is 0.44-0.95.

[0025] Preferably, the nutritional ingredients are a mixture of vitamin B1, vitamin B6, and citric acid or citrate.

[0026] Further preferably, in the hangover-relieving composition, the mass ratio of NAD precursor, amino acid, vitamin B1, vitamin B6, and citric acid or citrate is 1:0.45-12:0.046-0.25:0.02-0.12:0.28-1.2.

[0027] More preferably, the hangover-relieving composition contains 0.25 g of nicotinamide, 0.486 g of L-glutamine, 0.346 g of L-serine, 0.5 g of L-lysine, 0.07 g of vitamin B1, 0.03 g of vitamin B6, and 0.294 g of sodium citrate dihydrate, or a combination of ingredients with the same mass ratio.

[0028] The hangover-relieving composition of the present application is prepared into a food or a pharmaceutically acceptable dosage form.

[0029] Further, the dosage form includes but is not limited to capsule preparation, tablet or injection preparation.

[0030] Another object of the present application is to provide a liquid preparation with alcoholism relieving effect, which comprises the above-mentioned alcoholism relieving composition and a liquid medium, and the mass volume ratio of the sum of the mass of the NAD precursor substance and the amino acid to the liquid medium is 1 g:1.6-660 mL.

[0031] Preferably, the mass volume ratio of the sum of the mass of the NAD precursor substance and the amino acid to the liquid medium is 1 g:2.0-632 mL.

[0032] Further preferably, the mass volume ratio of the sum of the mass of the NAD precursor substance and the amino acid to the liquid medium is 1 g:2.0-188 mL.

[0033] Further, the liquid medium is one or more of pure water, physiological saline, Ringer's solution or other physiological balanced solution.

[0034] Still further, the pH of the Ringer's solution is 7.4, and the specific formula composition is as follows: 6.92 g / L NaCl, 0.35 g / L KCl, 0.16 g / L KH2PO4, 0.29 g / L MgSO4·7H2O, 8.1 mM NaHCO3, 11.1 mM D-(+)-glucose, 25 mM HEPES, 2.5 mM CaCl2, 2.5% aqueous solution of fat-free BSA.

[0035] Still another object of the present application is to provide a preparation method of the above-mentioned liquid preparation with alcoholism relieving effect, which comprises adding the NAD precursor substance and the amino acid and / or other nutritional ingredients into the liquid medium, stirring and mixing uniformly at room temperature until completely dissolved.

[0036] Still another object of the present application is to provide the use of the above-mentioned alcoholism relieving composition or the above-mentioned liquid preparation with alcoholism relieving effect in the preparation of products for relieving alcoholism and / or treating acute alcoholism.

[0037] The beneficial effects of the present application are as follows: 1. Flexible use time and wide application range: The use of the alcoholism relieving composition of the present application is not limited by time, and it can effectively accelerate the clearance of ethanol in the blood and significantly promote sobering up before, after or during drinking. This has obvious advantages compared with many alcoholism relieving drugs whose mechanism of action relies on protecting the liver or delaying ethanol absorption. Most of these types of alcoholism relieving drugs need to be taken before drinking. In addition, the alcoholism relieving composition of the present application has a wide range of applications, which can not only relieve post-drinking discomfort, promote sobering up, reduce hangover state and accelerate the recovery of normal functions after drinking, but also relieve the symptoms of acute alcoholism, strengthen the function of liver metabolism of ethanol and reduce the damage of ethanol and its metabolite acetaldehyde to the body.

[0038] 2、Ingredients controllable, in line with laws and regulations: the ingredients of the alcoholism composition described in the application, in addition to NMN and NR, meet the "National Food Safety Standard" or "Standard for Use of Food Nutrient Fortifiers". And, NR has been given the status of "New Food Ingredient (NDI)" by the US FDA, and NMN has been given the status of "Generally Recognized as Safe (GRAS)" dietary supplement raw material by the US FDA. Therefore, all the ingredients used in the application can be safely applied to the human body, with technical specifications to ensure quality, stable source supply, cheap, easy to get, easy to reprocess, which has obvious advantages compared with many alcoholism drugs containing plant or animal extracts.

[0039] 3、Unique mechanism of action, outstanding effect of alcoholism: the alcoholism composition described in the application can accelerate liver metabolism of ethanol by increasing the activity of ADH and ALDH. In vivo experiments show that: in rat models, the composition of the application can significantly improve the blood ethanol clearance rate after intraperitoneal injection of ethanol; in mouse models, it can effectively shorten the duration of drunkenness after gavage of ethanol, proving its excellent wake-up effect. In vitro experiments have confirmed that: in the isolated rat hepatocyte model, the composition can directly accelerate the elimination of ethanol and acetaldehyde by hepatocytes, which provides direct cell level evidence for its alcoholism effect by enhancing liver metabolic function. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 Figure for the effect of NAD precursor on ethanol metabolism of hepatocytes.

[0041] Figure 2 Figure for the effect of NMN and NA on acetaldehyde production / metabolism of hepatocytes when ADH and / or ALDH are inhibited.

[0042] Figure 3 Figure for the effect of NMN and NA on NAD + (A), NADH (B), NADt content (C) and NAD + / NADH ratio (D) of hepatocytes.

[0043] Figure 4 Figure for the effect of NMN and NA on free NAD + / NADH ratio in hepatocyte cytoplasm.

[0044] Figure 5 Figure for the effect of L-serine or fructose on NAD precursor accelerating ethanol metabolism of hepatocytes.

[0045] Figure 6 Figure for the change of blood ethanol concentration of rats with time under the action of different doses of NMN.

[0046] Figure 7Graph of the results of the comparison of the ethanol metabolism of the composition containing nutrients with the control group.

[0047] Figure 8 Graph of the results of the ethanol metabolism of the composition containing a low dose of niacin.

[0048] Figure 9 Graph of the results of the effect of the hangover composition on the duration of intoxication of mice.

[0049] Figure 10 Graph of the results of the effect of the hangover composition on the ethanol (A) and acetaldehyde (B) content in the serum of mice after ethanol exposure. DETAILED DESCRIPTION

[0050] The following non-limiting examples can make the person of ordinary skill in the art more fully understand the present application, but do not limit the present application in any way.

[0051] The test methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0052] Example 1 A hangover composition includes the following ingredient composition: sodium citrate dihydrate 0.444 g (14.9%), L-glutamine 0.7305 g (24.6%), L-serine 0.5205 g (17.5%), L-lysine 0.75 g (25.3%), nicotinamide 0.375 g (12.7%), vitamin B1 (thiamine hydrochloride) 0.105 g (3.5%), vitamin B6 (pyridoxine hydrochloride) 0.045 g (1.5%).

[0053] Preparation of a liquid preparation (compositions 1a, 1e and 1f) with hangover efficacy with pure water or a mixed solution of pure water and physiological saline as the liquid medium, the specific process is as follows: Composition 1a. Weigh the compound powder according to the ingredient composition weight of the above composition, add all at once into 5 mL pure water, stir or mix evenly at room temperature until completely dissolved.

[0054] Composition 1e. Mix the above composition 1a and physiological saline according to the volume ratio of 1:1.

[0055] Composition 1f. Mix the above composition 1e and physiological saline according to the volume ratio of 1:1.

[0056] Comparative Example 1 Preparation of a liquid preparation (compositions 1b-1d, single 1i-1k) with pure water, physiological saline or a mixed solution of pure water and physiological saline as the liquid medium, the specific process is as follows: Composition 1b. Consists of the following compounds in the indicated weights dissolved in 5 mL of pure water: sodium citrate dihydrate 0.444 g, L-glutamine 0.7305 g, L-serine 0.5205 g, L-lysine 0.75 g, nicotinic acid 0.015 g, vitamin B1 (thiamine hydrochloride) 0.105 g, vitamin B6 (pyridoxine hydrochloride) 0.045 g.

[0057] Composition 1c. Consists of the following compounds in the indicated weights dissolved in 5 mL of pure water: sodium citrate dihydrate 0.444 g, L-glutamine 0.7305 g, L-serine 0.5205 g, L-lysine 0.75 g.

[0058] Composition 1d. Consists of the following compounds in the indicated weights dissolved in 5 mL of physiological saline: nicotinamide 0.375 g, vitamin B1 (thiamine hydrochloride) 0.105 g, vitamin B6 (pyridoxine hydrochloride) 0.045 g.

[0059] Single 1i. 10% NMN solution, i.e. 1 g nicotinamide mononucleotide is dissolved in 10 mL of pure water.

[0060] Single 1j. 5% NMN solution, the above single 1i and pure water are mixed in a volume ratio of 1:1.

[0061] Single 1k. 2.5% NMN solution, the above single 1j and pure water are mixed in a volume ratio of 1:1.

[0062] Example 2 A liquid preparation (Composition 2a-2x, Composition 3a-3d) with alcoholism relieving effect using gram-Hen's solution as liquid medium is prepared as follows: (1) Preparation of reaction liquid matrix: an aqueous solution containing 6.92 g / L NaCl, 0.35 g / L KCl, 0.16 g / L KH2PO4, 0.29 g / L MgSO4·7H2O, 8.1 mM NaHCO3, 11.1 mM D-(+)-glucose, 25 mM HEPES, 2.5 mM CaCl2, 2.5% fat-free acid BSA, pH 7.4.

[0063] (2) Preparation method of glutamine working solution: first dissolve L-glutamine powder in the reaction liquid matrix to prepare L-glutamine working solution with concentrations of 16.4, 16.67, 19.05, 19.51 mM.

[0064] (3) Preparation of NAD precursor working solutions: There are four compounds: nicotinamide (NAM), nicotinamide ribochloride (NR·Cl), nicotinamide mononucleotide (NMN), and nicotinic acid (NA). First, a 100 mM NAD precursor working solution was prepared. The specific steps were as follows: individual compound powders were dissolved in the reaction solution matrix, and then diluted with the reaction solution matrix to prepare NAM, NR, and NMN working solutions with concentrations of 50, 20, 10, and 5 mM, and NA working solutions with concentrations of 50, 20, 10, 5, 1, 0.1, 0.01, and 0.001 mM.

[0065] (4) Preparation of composition 2a-2x: Compositions 2a-2e. are composed of the following compounds at the stated final concentrations: L-glutamine (16 mM), nicotinamide (16, 8, 3.2, 1.6, 0.8 mM); preparation method: 2.1 mL of 19.05 mM L-glutamine working solution is mixed with 0.4 mL of 100, 50, 20, 10, and 5 mM NAM working solutions, respectively.

[0066] Composition 2f-2j. is composed of the following compounds at the stated final concentrations: L-glutamine (16 mM), nicotinamide ribochloride (16, 8, 3.2, 1.6, 0.8 mM); preparation method: 2.1 mL of 19.05 mM L-glutamine working solution is mixed with 0.4 mL of 100, 50, 20, 10, and 5 mM NR·Cl working solutions, respectively.

[0067] Composition 2k-2o. is composed of the following compounds at the stated final concentrations: L-glutamine (16 mM), nicotinamide mononucleotide (16, 8, 3.2, 1.6, 0.8 mM); preparation method: 2.1 mL of 19.05 mM L-glutamine working solution is mixed with 0.4 mL of 100, 50, 20, 10, and 5 mM NMN working solutions, respectively.

[0068] Composition 2p-2x is composed of the following compounds at the stated final concentrations: L-glutamine (16 mM), nicotinic acid (16, 8, 3.2, 1.6, 0.8, 0.16, 0.016, 0.0016, 0.00016, 0.000016 mM); preparation method: 2.1 mL of 19.05 mM L-glutamine working solution is mixed with 0.4 mL of 100, 50, 20, 10, 5, 1, 0.1, 0.01, 0.001, and 0.0001 mM NA working solutions, respectively.

[0069] (5) Preparation of 0.4 M serine solution: Weigh 0.126 g L-serine and dissolve it in 6 mL of ultrapure water.

[0070] (6) Preparation of compositions 3a-3d: Compositions 3a-3c. are composed of the following compounds at the stated final concentrations: L-glutamine (16 mM), L-serine (8 mM), and NAD precursor compound (16 mM), wherein there are three NAD precursor compounds, namely NAM, NR·Cl and NMN in that order; preparation method: mix 2.4 mL of 16.67 mM L-glutamine working solution, 0.05 mL of 0.4 M serine solution, and 0.05 mL of 0.8 M NAD precursor working solution.

[0071] Composition 3d. Composed of the following compounds at the stated final concentrations: L-glutamine (16 mM), L-serine (8 mM), nicotinic acid (16 mM); Preparation method: Mix 2.1 mL of 19.51 mM L-glutamine working solution, 0.05 mL of 0.4 M serine solution, and 0.4 mL of 0.1 M NA working solution.

[0072] Comparative Example 2 The preparation process of a liquid formulation (composition 3e-3h) using Kröller-Heinz solution as the liquid medium is as follows: The preparation methods for the glutamine working solution and the NAD precursor working solution are the same as in Example 2.

[0073] The composition, 3e-3g, is composed of the following compounds at the stated final concentration: L-glutamine (16 mM), fructose (16 mM), and NAD precursor compound (16 mM), wherein there are three NAD precursor compounds, namely NAM, NR·Cl, and NMN in that order; preparation method: mix 2.4 mL of 16.67 mM L-glutamine working solution, 0.05 mL of 0.8 M fructose solution, and 0.05 mL of 0.8 M NAD precursor working solution.

[0074] The composition was prepared by mixing 2.1 mL of 19.51 mM L-glutamine working solution, 0.05 mL of 0.8 M fructose solution, and 0.4 mL of 0.1 M NA working solution.

[0075] Comparative Example 3 of Pharmacological Experiments A study on the rate of ethanol elimination from rat hepatocytes by L-glutamine, pyruvate, and the combination of L-glutamine and pyruvate: (1) Experimental methods Instruments: FWS-30 shaking water bath incubator, SpectraMax Paradigm microplate reader; Animals: Male SD rats (200-300 g); Cells: Freshly isolated rat hepatocytes; Reagents: Ethanol, yeast ADH, NAD+ + CCK-8 reagent; Samples: L-glutamine, pyruvate.

[0076] a. Isolation of rat hepatocytes: Rat hepatocytes were isolated using a modified Seglen two-step method, with freshly isolated hepatocytes used in each experiment. Rats were fasted for 48 hours but allowed free access to water. They were then anesthetized by intraperitoneal injection of 320 mg / kg aphthylazine. The abdominal cavity was opened, and a portal vein cannula was inserted. The liver was perfused at 37°C with a solution of calcium-free KH solution (6.92 g / L NaCl, 0.35 g / L KCl, 0.16 g / L KH2PO4, 0.29 g / L MgSO4·7H2O, 8.1 mM NaHCO3, 15 mM glucose, 25 mM HEPES, pH 7.4) at 8 mL / min for 10–15 min. The perfusion solution was then changed to one containing 0.05% type IV collagenase and 1.26 mM CaCl2, and perfused at 5 mL / min for approximately 20 min. The liver cells were then cleaved to release their contents, and the cells were digested by shaking at 37°C and 80 rpm for 10 min. Incubate on ice for 15 min, then resuspend the precipitated cells in 300 mL of 4℃ washing buffer (calcium-containing KH solution: 6.92 g / L NaCl, 0.35 g / L KCl, 0.16 g / L KH₂PO₄, 0.29 g / L MgSO₄·7H₂O, 8.1 mM NaHCO₃, 15 mM glucose, 25 mM HEPES, pH 7.4, 1.25 mM CaCl₂). Incubate on ice for 15 min, then resuspend the precipitated cells in 20 mL of reaction matrix (calcium and BSA-containing KH solution). Check cell viability using trypan blue staining. Centrifuge 1 mL of cell suspension at 12,000 × g for 2 min, discard the supernatant, and weigh the cells wet.

[0077] b. Preparation of the hepatocyte ethanol oxidation reaction: Add 2.45 mL of reaction matrix containing 16.4 mM glutamine (final concentration 10 mM) or without glutamine to each reaction flask, then add 1 mL of freshly isolated rat hepatocytes (wet weight 80 mg / mL ~ 100 mg / mL). Place the reaction flasks in a 37℃ shaking water bath and incubate at 100 rpm for 55 min, providing oxygen to the surface for 1 min every ~30 min. Add 0.05 mL of pure water to each control group and 0.05 mL of 0.8 M sodium pyruvate solution (final concentration 10 mM) to each positive control group. Incubate at 37℃, 100 rpm for 5 min (providing oxygen to each flask for 1 min). Add 0.5 mL of 68 mM ethanol (final concentration 8.5 mM) prepared with the reaction matrix to each reaction flask and continue shaking and incubation. When transferring the liquid, remove the flask briefly, insert the pipette tip below the liquid surface to add the ethanol, and then tighten the rubber stopper.

[0078] c. Ethanol sample extraction (fractional extraction method): After adding ethanol and mixing, add 0.6 mL of 4 M perchloric acid to each 0 min flask. After reacting with ethanol for 60 min, add 0.6 mL of 4 M perchloric acid to each 60 min flask. After vortexing, let stand on ice for 20 min to remove proteins. These samples are used as samples at the 0 min and 60 min time points for ethanol metabolism. Transfer 0.5 mL of the suspension sample from each reaction flask to an EP tube pre-chilled in an ice bath. Centrifuge (12,000×g, 4℃, 2 min). Transfer 200 μL of the supernatant to an EP tube pre-chilled in an ice bath (containing 27 μL of chilled 20% KOH) and mix. Let stand on ice for 10 min to precipitate insoluble matter. Centrifuge again (12,000×g, 4℃, 2 min) to obtain the supernatant. Transfer 50 μL of the supernatant to an EP tube pre-cooled in an ice bath (containing 350 μL of chilled 50 mM potassium phosphate buffer, pH 7.5) to mix as the test sample, and place it on ice for testing.

[0079] d. WST-8 method for ethanol determination: Add 10 μL of standard or sample, 100 μL of reaction mixture (100 mM KCl, 75 mM aminourea, 130 mM glycine, 0.2% Triton X-100, 1 mM NAD) to each well of a 96-well plate sequentially. + Add 10 μL of CCK-8 solution (pH 7.6). Incubate the plate on a shaker at 30°C and 80 rpm for 5 min in the dark. Measure the absorbance at 450 nm at 30°C using a microplate reader and record the reading of the sample or standard well. A n ’ (Corresponding to the NADH content in the system), record the reading of the blank well.B’ Place the plate in a shaker at 30°C and 80 rpm in the dark for 5 min, then allow it to return to 30°C. Add 10 μL of 300 U / mL ADH solution to each well and incubate at 30°C and 80 rpm in the dark for 25 min. Measure the absorbance at 450 nm and record the reading for the sample or standard well. A n ” (Corresponding to the NADH content in the system + the NADH content produced by ethanol), record the reading of the blank well. B” .

[0080] The absorbance value of the sample well is calibrated according to (Formula 1) to obtain the calibrated absorbance value.

[0081] Formula 1 in, n Sample number; i : Hole number; A n ’ : Absorbance of the standard wells before the addition of ADH; A n ” : Absorbance of the standard wells after adding ADH; B’ : Absorbance of the blank well before adding ADH; B” : Absorbance of the blank well after adding ADH.

[0082] calibrate absorbance value A n By substituting the values ​​into the ethanol standard curve, the ethanol content at 0 min and after 60 min of reaction can be obtained. The difference between the two values ​​is used to calculate the ethanol metabolism amount (μmol) during the reaction process. Dividing this by the cell wet weight gives the cell weight-standardized ethanol metabolism amount (μmol / g cell). Dividing this by the reaction time gives the ethanol metabolism rate (μmol / min / g cell).

[0083] (2) Experimental results The experimental results are shown in Table 1. It can be seen that rat hepatocytes did not show ethanol metabolism activity without the addition of substrate; pyruvate significantly increased the ethanol elimination rate of hepatocytes; L-glutamine also increased the ethanol elimination rate of hepatocytes, but the effect was weaker than that of pyruvate; when L-glutamine and pyruvate were used in combination, the ethanol elimination rate of hepatocytes also increased slightly, but the increase was less than that of pyruvate alone.

[0084] Table 1. Ethanol elimination rate and substrate promoting effect in rat hepatocytes (mean ± SD, n = 3)

[0085] * p <0.05 compared with the control group (one-way ANOVA and Dunnett's test) Pharmacological Experiment Example 3 A study on the effects of different NAD precursors on hepatocyte ethanol metabolism in the presence of L-glutamine: (1) Experimental methods Instruments: FWS-30 shaking water bath incubator, SpectraMax Paradigm microplate reader; Animals: Male SD rats (200-300 g); Cells: Freshly isolated rat hepatocytes; Reagents: Ethanol, yeast ADH, NAD+ + CCK-8 reagent; Samples: L-glutamine, NAD precursors (NAM, NR, NMN, NA).

[0086] Rats were fasted for 48 hours but allowed free access to water, and rat hepatocytes were isolated using a modified Seglen two-step method.

[0087] a. Preparation of hepatocyte ethanol oxidation reaction: Add 2.1 mL of 19.05 mM glutamine working solution (final concentration 10 mM) to each reaction flask, then add 0.4 mL of reaction matrix (control) or different concentrations of NAD precursor working solution (final concentrations of 0.5, 1, 2, 5, and 10 mM, with NA concentrations extended downwards to 1 × 10⁻⁶ mM). -8 Finally, add 1 mL of freshly isolated rat hepatocytes (wet weight 80 mg / mL ~ 100 mg / mL) to each reaction flask. Incubate the reaction flasks in a 37°C shaking water bath at 100 rpm in the dark for 1 hour. Add 0.5 mL of 68 mM ethanol (final concentration 8.5 mM) prepared with the reaction matrix to each reaction flask and continue shaking and incubation. When pipetting, remove the flask after a short while, insert the pipette tip below the liquid surface to add ethanol, and then tighten the rubber stopper.

[0088] b. Extraction of ethanol samples (serial sampling method): After mixing, take 0.5 mL of the reaction solution from each bottle after adding ethanol at 0 min and 60 min, respectively, and transfer it to an EP tube pre-chilled in an ice bath (containing 0.6 mL of 5.5% (w / v) perchloric acid solution). Mix and set aside. After vortexing, let stand on ice for 20 min to remove proteins, and use these as samples at 0 min and 60 min time points. Centrifuge (12,000×g, 4℃, 2 min). Transfer 200 μL of the supernatant to an EP tube pre-chilled in an ice bath (containing 30 μL of 4 MKOH solution), mix and set aside. Let stand on ice for 10 min to precipitate insoluble matter. Centrifuge again (12,000×g, 4℃, 2 min) to obtain the supernatant. Transfer 100 μL of the supernatant to an EP tube pre-cooled in an ice bath (containing 300 μL of chilled 50 mM potassium phosphate buffer, pH 7.5), mix them to form the test sample, and place the tube on ice for testing.

[0089] Ethanol was determined using the WST-8 method. The amount of ethanol metabolized was obtained, and the ethanol metabolism rate was calculated.

[0090] (2) Experimental results The experimental results are shown in Figure 1 It can be seen that when used in combination with L-glutamine, all NAD precursors can increase the ethanol metabolism rate of hepatocytes in a dose-dependent manner. Among them, NA has the strongest effect, followed by NR and NMN, while NAM has the weakest effect on increasing the ethanol metabolism rate.

[0091] Figure 1 The height of the lower dashed line is 0.768, corresponding to the average ethanol elimination rate of the control group; the height of the upper dashed line is 2.78, which is the maximum theoretical value of ethanol elimination rate (ethanol elimination rate of rat hepatocytes promoted by 10 mM pyruvate in the reference); the height of the middle dashed line is 1.774, which is the median value between the two. A four-parameter logistic regression model was used for curve fitting, with a minimum value of 0.768 and a maximum value of 2.78, from which the LogEC for each group can be obtained. 50 The values, arranged from smallest to largest, are: NA: -4.036, NMN: -2.643, NR: -2.637, NAM: -2.232.

[0092] Pharmacological Experiment Example 4 A study on the effects of NMN and NA on acetaldehyde production / metabolism in hepatocytes in the presence of L-glutamine when ADH and / or ALDH are inhibited: (1) Experimental methods Instruments: SpectraMax Paradigm microplate reader, Thermo Fisher Scientific Nanodrop One micro-spectrophotometer; Animals: Male SD rats (200-300 g); Cells: Freshly isolated rat hepatocytes; Reagents: 4-methylpyrazole (methylpyrazole, ADH inhibitor), cyanamide (cyanamide, ALDH inhibitor), ethanol, acetaldehyde, yeast ADH, CCK-8 reagent, 3-methyl-2-benzothiazolinone hydrazone (MBTH), ferric ammonium sulfate; Samples: L-glutamine, NMN, NA.

[0093] Rats were fasted for 48 hours but allowed free access to water, and rat hepatocytes were isolated using a modified Seglen two-step method.

[0094] a. Effect of NAD precursors on ADH activity: The experiment was divided into two main groups, A (cyanamide) and B (methopyrazole + cyanamide), each further subdivided into three subgroups: 1 (control group), 2 (NMN group), and 3 (NA group). Each reaction flask was repeated once (replication number: -1, -2). 2.05 ml of preheated 19.51 mM glutamine working solution (final concentration 10 mM) was added to each flask. 0.4 ml of reaction matrix, 0.1 M NMN working solution (final concentration 10 mM), and 0.1 M NA working solution (final concentration 10 mM) were added to groups 1, 2, and 3, respectively. 1 ml of hepatocyte suspension (wet weight ~100 mg / ml) was added to each flask. The flasks were incubated at 37°C and 100 rpm in the dark for 30 min (with rubber stoppers used, and oxygen was introduced into each flask for 1 min every 30 min). Group A was added with 0.05 ml of 0.8 M cyanamide solution (final concentration 10 mM), and Group B was added with 0.05 ml of a mixture of 0.8 M cyanamide and 0.16 M pyrazole (final concentration 10 mM / 2 mM). The mixture was incubated at 37°C and 100 rpm in the dark for 30 min. 0.5 ml of 68 mM ethanol (final concentration 8.5 mM) prepared with the reaction matrix was added to both Groups A and B. When transferring the ethanol, the bottle was removed momentarily, and the pipette tip was submerged below the liquid surface before adding the ethanol. The rubber stopper was then tightened. Acetaldehyde samples were collected at 0, 15, and 30 min from each reaction bottle with a replicate number of -1, and NAD samples were collected at 0 and 30 min from each reaction bottle with a replicate number of -2. + NADH and pyruvate, lactic acid samples.

[0095] b. Effect of NAD precursors on ALDH activity: The experiment was divided into four groups: Group C (methylpyrazole), which was further subdivided into subgroups 1 (control group), 2 (NMN group), 3 (NMN' group), and 4 (NA group). Each reaction flask was repeated twice (replication number: -1, -2). 2.05 ml of preheated 19.51 mM glutamine working solution (final glutamine concentration 10 mM) was added to each flask. Groups 1, 2, and 3 were respectively incubated with 0.4 ml of reaction matrix, 0.1 M NMN working solution, and 0.1 M NA working solution (final concentration 10 mM). 1 ml of cell suspension (wet weight ~10 mg) was added to each flask. The flasks were incubated at 37°C and 100 rpm in the dark for 30 min (with rubber stoppers used, and oxygen was introduced into each flask for 1 min every 30 min). In Group C, subgroups 1, 2, and 4 were each added with 0.05 ml of 0.16 M methylpyrazole solution (final concentration 2 mM). Subgroup C3 was added with 0.05 ml of a mixture of 0.8 M cyanamide and 0.16 M methylpyrazole (final concentration 10 mM / 2 mM). The mixture was incubated at 37°C and 100 rpm in the dark for 30 min. 0.5 ml of 1.6 mM acetaldehyde (final concentration 0.2 mM) prepared with the reaction matrix was added to Group C. During pipetting, the bottle was removed momentarily, and the pipette tip was submerged below the liquid surface before adding the acetaldehyde. The rubber stopper was then tightly closed. Acetaldehyde samples were collected at 0, 10, and 20 min from each reaction bottle with a replicate number of -1, and NAD samples were collected at 0 and 20 min from each reaction bottle with a replicate number of -2. + NADH and pyruvate, lactic acid samples.

[0096] c. Pretreatment of acetaldehyde test samples: After mixing the solutions in the reaction flasks at each time point, immediately take 100 μL of the reaction mixture from each flask and add it to an ice-cold brown EP tube (containing 0.2 mL of 0.1% MBTH solution and 0.7 mL of ice-cold distilled water), and vortex to mix. This serves as the acetaldehyde test sample for each time point.

[0097] d.NAD + Sample pretreatment: After mixing the reaction flasks at each time point, immediately take 300 μL of the reaction mixture from each of the -2 flasks and add it to an ice-cold EP tube. Incubate on ice for 5 min, centrifuge at 2,000×g, 4℃ for 10 min, discard the supernatant, add 0.5 mL of frozen 0.025 M HCl, shake for 45 s, heat in a 60℃ metal bath for 30 min, then incubate on ice for 5 min. Add 8.4 μL of 1 M NaOH to each tube to neutralize the pH, incubate on ice for 5 min, centrifuge at 12,000×g, 4℃ for 5 min, and transfer the supernatant to a new ice-cold brown EP tube as NAD. + The sample to be tested is placed on ice.

[0098] e. Pretreatment of NADH test samples: After mixing the reaction flasks at each time point, immediately take 400 μL of the reaction mixture from each of the -2 flasks and add it to an ice-cold EP tube. Incubate on ice for 5 min, centrifuge at 2,000×g, 4℃ for 10 min, discard the supernatant, add 0.5 mL of frozen 0.05 M NaOH, shake for 45 s, heat in a 60℃ metal bath for 30 min, then incubate on ice for 5 min. Add 11 μL of 2 M HCl to each tube to neutralize the pH, incubate on ice for 5 min, centrifuge at 12,000×g, 4℃ for 5 min, and take 0.2 mL of the supernatant into a new ice-cold brown EP tube as the NADH test sample. Place it on ice for testing.

[0099] f. Pretreatment of pyruvate and lactate samples: After mixing the reaction flasks at each time point, immediately transfer 500 μL of the reaction mixture from each of the two flasks into ice-cold EP tubes. Incubate in an ice-salt bath for 5 min, centrifuge at 1,000 rpm, 4°C for 10 min, discard the supernatant, add 0.5 mL of frozen PBS, gently invert to mix the cells, centrifuge at 1,000 rpm, 4°C for 10 min, discard the supernatant. Add 0.5 mL of PBS to the cell pellet of group A and 0.3 mL of PBS to the cell pellet of group C. Sonicate the cells in an ice-water bath at 300 W for 5 seconds each time, with a 15-second interval, repeating 5 times. Centrifuge at 2,500 rpm, 4°C for 10 min, and collect the supernatant as the pyruvate and lactate samples, placing them on ice for testing.

[0100] g. Acetaldehyde determination: The phenol reagent spectrophotometric method was used. After preparing the acetaldehyde standard and the sample to be tested, they were placed on ice for 30 min. They were then incubated in a metal bath at 34℃ for 10 min. 0.08 mL of 10 g / L ferric ammonium sulfate solution was added at room temperature, vortexed, and incubated again at 34℃ for 12 min. The mixture was then cooled to room temperature by running water. The sample was centrifuged at 12,000×g at 4℃ for 2 min, and 0.2 mL of the supernatant was transferred to a new ice-brown EP tube as the acetaldehyde sample. The absorbance of the standard and sample supernatant at 595 nm was measured using an ultra-micro spectrophotometer.

[0101] h. NAD assay (WST-8 method): Add 20 μL of NADH standard or sample and 100 μL of reaction mixture (containing 100 U / mL ADH in 40 mM Tris-HCl, 120 mM NaCl, 10.9% ethanol, pH 8.0) to each well of a 96-well plate. Incubate the plate at 30°C and 80 rpm in the dark for 5 min. Add 10 μL of CCK-8 solution to each well and incubate at 30°C and 80 rpm in the dark for 25 min. Add 10 μL of stop solution to each well and incubate at 30°C and 80 rpm in the dark for 5 min. Measure the absorbance at 450 nm using a microplate reader and record the readings for each well.

[0102] i. Free NAD+ in the cytoplasm + / NADH ratio determination: Pyruvate and lactate were tested using pyruvate and lactate kits from Nanjing Jiancheng Bioengineering Institute, following the recommended methods. Free NAD+ in the cytoplasm... + The / NADH ratio can be calculated using formula 2, where, K’ The equilibrium constant of the lactate dehydrogenase system ( K’ = 1.11×10 -4 ): Formula 2 (2) Experimental results The experimental results are shown in Figure 2 Table 2 Figure 3 , Figure 4 .

[0103] Figure 2 A represents the change in acetaldehyde content over time in a hepatocyte mixture (100 mg wet weight, 4 ml) after pretreatment with a given NAD precursor (10 mM, 60 min) or a carrier (control) and the ALDH inhibitor cyanamide (10 mM, 60 min followed by 30 min). This was before the reaction was started with the addition of ethanol (8.5 mM) to the mixture. Figure 2 B represents the change in acetaldehyde content over time in a mixture of hepatocytes (100 mg) after pretreatment with a given NAD precursor or carrier, cyanamide, and the ADH inhibitor metoprazole (2 mM) before the addition of ethanol. Figure 2 C represents the change in acetaldehyde content over time in a mixture of hepatocytes (10 mg) after pretreatment with a given NAD precursor or carrier and inhibitor (cyanamide and metoprolol for the NMN' group, and metoprolol only for the other groups) before the reaction was initiated with the addition of acetaldehyde (0.2 mM). The figure shows the results of one of the five independent experiments. The results of the other experiments were similar to those shown in the figure. Figure 2The results showed that when used in combination with L-glutamine, the NAD precursor, representing the effects of NA and NMN, on the production or metabolism of acetaldehyde in hepatocytes. When ALDH was inhibited ( Figure 2 A) Both NA and NMN increase the rate at which hepatocytes convert ethanol to acetaldehyde, with NA having a stronger effect. When ADH and ALDH are inhibited ( Figure 2 B), the rate of ethanol metabolism in hepatocytes is low, and NA and NMN have no promoting effect on this. When ADH is inhibited ( Figure 2 C), both NA and NMN increase the rate at which hepatocytes metabolize acetaldehyde. Among them, NMN has a stronger effect and can be inhibited by ALDH inhibitors (NMN').

[0104] The results in Table 2 show that when used in combination with L-glutamine, both NMN and NA increased the ADH activity of hepatocytes, with NA having a greater effect; both NMN and NA increased the ALDH activity of hepatocytes, with NMN having a greater effect. The effect of NA was mainly to increase the ADH activity of hepatocytes, while the effect of NMN was mainly to increase the ALDH activity of hepatocytes.

[0105] Table 2. Effects of NMN and NA on ADH and ALDH activities in hepatocytes (mean ± SD) n = 5)

[0106] a Figure 2 The acetaldehyde formation rate in A from 0-15 min minus Figure 2 The acetaldehyde production rate in cell B from 0 to 15 minutes was then normalized to cell weight.

[0107] b Figure 2 The acetaldehyde elimination rate in cell C from 0 to 10 min was then normalized to cell weight.

[0108] * p <0.05, *** p <0.001, **** p <0.0001 compared with the control group (one-way ANOVA and Dunnett test).

[0109] Figure 3 Experimental groupings Figure 2 A (Group A: n = 5) Figure 2 Group B: n = 5) and Figure 2 Group C: n = 3), data are mean ± SD. * p <0.05, **p <0.01, *** p <0.001 compared with control 0 min (two-way repeated measures ANOVA and Tukey's test); # p <0.05, ## p <0.01, ### p <0.001, #### p <0.0001 compared with the control at 30 min or 20 min (two-way repeated measures ANOVA and Tukey's test); & p <0.05, && p <0.01, &&& p <0.001 itself 0 min vs 30 min or 20 min (two-way repeated measures ANOVA and Sidak test). Figure 3 The results showed that when used in combination with L-glutamine, NA and NMN had an effect on NAD in hepatocytes. + NADH, NADt (NAD + + NADH) content and NAD + The effect of the NAD / NADH ratio. It was found that NMN increased NAD in hepatocytes. + The addition of acetaldehyde did not change (after the addition of ethanol) or decreased (after the addition of acetaldehyde) the NADt content in hepatocytes; under the experimental conditions, NA did not increase the NADt content in hepatocytes, but only increased it after the addition of ethanol (group A). + The content of NADH was reduced. In the comparison before and after the addition of ethanol (Group A), the control group showed a significant increase in NADH content and a decrease in NADH content. + The NADH / NADH ratio was significantly reduced; NMN could inhibit these changes, while NA could reverse them. The effects of NA could be partially inhibited by ADH inhibitors (Group B). In the comparison before and after the addition of acetaldehyde (Group C), the control group showed a significant increase in NADH content and a decrease in NADDH / NADH content. + The NADH ratio was significantly reduced. NA could inhibit this change, while NMN could reverse it. The effect of NMN could be partially inhibited by ALDH inhibitors (NMN in group C).

[0110] Figure 4 Experimental group A is the same Figure 2 A, a new NA' group containing cyanamide and metoprolol was added. Figure 4 Experiment B group same Figure 2C. After adding ethanol for 0 min and 30 min (A) or acetaldehyde for 0 min and 20 min (B), the pyruvate and lactate content of hepatocytes was measured, and the cytoplasmic free NAD was calculated using Formula 2. + / NADH ratio. Show mean ± SD ( n = 6). # p <0.05, ## p <0.01, #### p <0.0001 compared with the control at 30 min or 20 min (two-way repeated measures ANOVA and Sidak test); & p <0.05, &&&& p <0.0001 Comparison of 0 min with 30 min or 20 min (two-way repeated measures ANOVA and Sidak test). Figure 4 The results showed that when used in combination with L-glutamine, NA and NMN reduced the free NAD+ in hepatocyte cytoplasm. + The effect of the NADH ratio. In the comparison before and after the addition of ethanol ( Figure 4 A), the control group showed free NAD. + / The NADH ratio decreased significantly; NMN could inhibit this change, while NA could reverse it; however, the effect of NA could be inhibited by ADH inhibitors. In the comparison before and after the addition of acetaldehyde (…), Figure 4 B), free NAD in each group + There was no significant difference in the / NADH ratio.

[0111] Human body synthesizes NAD + It relies on three pathways: the salvage pathway using NAM as a substrate, the Preiss-Handler pathway using NA as a substrate, and the de novo synthesis pathway using tryptophan as a substrate. NA and NAM are two forms of vitamin B3. The salvage pathway involves NAD+ in the body. + The shortest of the synthetic pathways involves the primary substrates NR and NAM being converted into the intermediate NMN in the cytoplasm. NMN is then transported into the mitochondria and converted into NAD in the mitochondria. + Mitochondria are the source of NAD+ in cells. + The storage pool of mitochondrial NAD + Increased levels can increase cellular NADt content, which is similar to... Figure 3 As shown in A and C, NMN can increase NAD50 in liver cells. + and NAD+ content. When NAD+ in mitochondria... + Content or NAD +An increase in the ALDH / NADH ratio also enhances ALDH activity in mitochondria, as shown in the NMN group in Table 2. Conversely, the Preiss-Handler pathway substrate NA did not exhibit an increasing effect on NADt levels, because NA synthesizes NAD. + It occurs in the cytoplasm, where excess NAD3 in the cytoplasm... + It will continue to degrade into NAM and be secreted outside the liver cells. Because NA increases NAD in mitochondria... + The effect of content is weak ( Figure 3 A), and its promoting effect on ALDH activity is also weak (Table 2). However, Figure 2 The results showed that norepinephrine (NA) had a greater promoting effect on ethanol metabolism in hepatocytes than NMN. This is because NA had a greater promoting effect on cytoplasmic ADH activity than NMN (Table 2), which is due to NA's ability to increase the free NAD content in the cytoplasm. + The ratio of NADH to NMN is higher, and this effect is greater than that of NMN. Figure 4 A). In summary, NMN (also representing the substrates NR and NAM, which are also salvage pathways) and NA can accelerate the metabolism of ethanol and acetaldehyde by hepatocytes. In particular, NA can increase NAD in the cytoplasm. + A decrease in the NADH / NADH ratio or a reduction in NADH levels can accelerate the oxidation of ethanol to acetaldehyde by ADH. NMN can increase NAD+ levels in mitochondria. + / NADH ratio or increase NAD + The increased content accelerates the oxidation of acetaldehyde to acetic acid by ALDH.

[0112] Pharmacological Experiment Example 5 A study on the effect of L-glutamine in combination with L-serine or fructose on the acceleration of NAD precursor ethanol metabolism in hepatocytes: (1) Experimental methods Instruments: FWS-30 shaking water bath incubator, SpectraMax Paradigm microplate reader; Animals: Male SD rats (200-300 g); Cells: Freshly isolated rat hepatocytes; Reagents: Ethanol, yeast ADH, NAD+ + CCK-8 reagent; Samples: L-glutamine, L-serine, fructose, NAM, NR, NMN, NA.

[0113] Rats were fasted for 48 hours but allowed free access to water, and rat hepatocytes were isolated using a modified Seglen two-step method.

[0114] a. Preparation of hepatocyte ethanol oxidation reaction: First, add 2.4 mL of 16.67 mM glutamine working solution (final concentration 10 mM), 0.05 mL of pure water, 0.4 M serine solution (final concentration 5 mM) or 0.8 M fructose solution (final concentration 10 mM) to the reaction bottle containing NAM, NR or NMN. Then add 0.05 mL of 0.8 M NAM working solution, 0.8 M NR working solution or 0.8 M NMN working solution (final concentration 10 mM each). First, add 2.1 mL of 19.05 mM glutamine working solution (final concentration 10 mM), 0.05 mL of pure water, 0.4 M serine solution (final concentration 5 mM), or 0.8 M fructose solution (final concentration 10 mM) to each reaction flask containing NA. Then add 0.4 mL of 0.1 M NA working solution (final concentration 10 mM). Finally, add 1 mL of freshly isolated rat hepatocytes (wet weight 80-100 mg) to each reaction flask. Incubate the reaction flasks in a 37°C shaking water bath at 100 rpm in the dark for 1 hour. Add 0.5 mL of 68 mM ethanol (final concentration 8.5 mM) prepared with the reaction solution matrix to each reaction flask and continue shaking and incubation. When pipetting, remove the flask after a short while, insert the pipette tip below the liquid surface to add ethanol, and then tighten the rubber stopper.

[0115] b. Residual ethanol in the reaction solution at 0 min and 60 min after the addition of ethanol was extracted using a continuous sampling method. Ethanol was determined by the WST-8 method. The amount of ethanol metabolized was obtained, and the ethanol metabolism rate was calculated.

[0116] (2) Experimental results The experimental results are shown in Figure 5 Rat hepatocytes were first given a given precursor of NAD (10 mM) and / or fructose (10 mM) (A, n =6), L-serine (5 mM) (B, n = 4) Or pretreat the vector for 60 min before performing the ethanol metabolism experiment. Data are expressed as mean ± SD. p <0.01, *** p <0.001, **** p <0.0001 compared with the control group without additives (two-way ANOVA and Dunnett's test); # p <0.05, ## p <0.01, ### p <0.001, #### p <0.0001 compared with controls containing added fructose or L-serine (two-way ANOVA and Dunnett's test);& p <0.05, && p <0.01, &&& p <0.001 Comparison of no fructose or L-serine in each control group or NAD precursor group with fructose or L-serine added (two-way ANOVA and Sidak test). Figure 6 The results showed that, in combination with L-glutamine, the addition of fructose or L-serine could enhance the effect of NAD precursors in promoting hepatocyte ethanol metabolism to some extent. For example, 10 mM fructose significantly promoted the effect of NMN but failed to promote the effect of NAM; 5 mM L-serine significantly promoted the effects of NAM, NMN, and NR but failed to promote the effect of NA. However, it should be noted that 10 mM NA already pushed the ethanol metabolism rate close to its theoretical maximum. The results suggest that L-serine combined with NAM is more effective than fructose combined with NAM.

[0117] Comparative Example 4 of Pharmacological Experiments A study on the changes in blood ethanol concentration in rats over time under different doses of NMN: (1) Experimental methods Instruments: Celian 456-GC gas chromatograph; Column: Restek Stabilwax® polar column (length: 30 m, inner diameter: 0.32 mm, film thickness: 0.25 μm); Animals: Male SD rats (250–300 g); Reagents: Ethanol, EDTA, n-propanol, sulfuric acid, sodium tungstate; Sample: NMN Rats were fasted for 24 hours but allowed free access to water. They were weighed and randomly divided into a control group, a low-dose NMN group, a medium-dose NMN group, and a high-dose NMN group. The control group was administered pure water by gavage at 10 mL / kg. The NMN groups were administered equal volumes of single-component 1k, single-component 1j, and single-component 1i by gavage (dose of 250 mg / kg, 500 mg / kg, and 1000 mg / kg, respectively). 30 minutes later, a saline solution containing 20% ​​ethanol was injected intraperitoneally at a dose of 2 g / kg. Subsequently, tail blood was collected at 0, 10, 20, 30, 60, 120, 180, 240, and 300 minutes after ethanol administration. The blood was mixed with EDTA for anticoagulation, and n-propanol was added as an internal standard. Protein was then precipitated with tungstate. The supernatant was collected by centrifugation, filtered through an aqueous filter membrane, and ethanol was detected by gas chromatography.

[0118] (2) Experimental results The experimental results are shown in Figure 6 . Figure 6 The results showed that the blood ethanol concentrations in the control group and the rats in each drug-treated group were highly synchronized. All experimental groups reached peak blood drug concentrations 10–30 minutes after ethanol exposure.C max After 60 minutes, a typical linear elimination phase was observed, with differences between groups. C max and eliminate phase slope ( K 0) No statistically significant difference. The results suggest that simply supplementing with NAD precursors does not accelerate ethanol metabolism.

[0119] Pharmacological Experiment Example 6 Study on accelerating ethanol metabolism of composition 1a obtained in Example 1 and compositions 1c and 1d obtained in Comparative Example 1: (1) Experimental methods Instruments: Scion 456-GC gas chromatograph; Column: Restek Stabilwax polar gas chromatograph (length: 30 m, inner diameter: 0.32 mm, film thickness: 0.25 μm); Animals: male SD rats (250~300 g); Reagents: ethanol, EDTA, acetonitrile; Samples: [sodium citrate dihydrate, L-glutamine, L-serine, L-lysine, nicotinamide, vitamin B1, vitamin B6] (compositions 1a, 1c, 1d).

[0120] Rats were fasted for 24 hours but allowed free access to water. They were weighed and randomly divided into a control group, a nutritional acid group (dosage: 0.294 g / kg sodium citrate dihydrate, 0.486 g / kg L-glutamine, 0.346 g / kg L-serine, 0.5 g / kg L-lysine), a vitamin group (dosage: 0.25 g / kg nicotinamide, 0.07 g / kg vitamin B1, 0.03 g / kg vitamin B6), and a vitamin + nutritional acid group (dosage: 0.294 g / kg sodium citrate dihydrate, 0.486 g / kg L-glutamine, 0.346 g / kg L-serine, 0.5 g / kg L-lysine, 0.25 g / kg nicotinamide, 0.07 g / kg vitamin B1, 0.03 g / kg vitamin B6). The control group was administered at 16.7... The groups were administered physiological saline at a dose of mL / kg by gavage. The nutrient acid group, vitamin group, and vitamin + nutrient acid group were administered equal volumes of composition 1c, composition 1d, and composition 1a by gavage, respectively. Immediately after gavage, physiological saline solution containing 20% ​​ethanol was injected intraperitoneally at a dose of 2 g / kg. Subsequently, tail blood was collected at 0, 10, 20, 30, 60, 120, 180, 240, 300, and 360 minutes after ethanol administration. The blood was mixed with EDTA for anticoagulation, tert-butanol internal standard was added, and then mixed with acetonitrile at a ratio of 1:1. The supernatant was collected by centrifugation, filtered through an aqueous filter membrane, and gas chromatography was used to detect ethanol.

[0121] (2) Experimental results The experimental results are shown in Figure 7 And Table 3.Figure 7 The results showed that the changes in blood ethanol concentration over time in the nutritional acid group and the vitamin group were not significantly different from those in the control group. However, the ethanol elimination rate in the vitamin + nutritional acid group was faster than that in the control group, and the blood ethanol concentration was significantly lower than that in the control group starting 2 hours after ethanol exposure. Figure 8 The results were analyzed by pharmacokinetic analysis (Table 3), and the time to peak ethanol concentration was found in each group. T max )and C max No significant difference was observed in the vitamin + nutrient acid group compared to the control group. K 0 increased significantly, with an increase of 25%; its ethanol exposure ( AUC () also decreased significantly.

[0122] Table 3. Ethanol metabolism kinetic parameters for each group (Mean ± SEM, n = 6)

[0123] ** p <0.01 compared with the control group (one-way ANOVA and Dunnett test).

[0124] Comparative Example 5 of Pharmacological Experiments Study on ethanol metabolism of composition 1b obtained in Comparative Example 1: (1) Experimental methods Instruments: Scion 456-GC gas chromatograph; Column: Restek Stabilwax polar gas chromatograph (length: 30 m, inner diameter: 0.32 mm, film thickness: 0.25 μm); Animals: male SD rats (250~300 g); Reagents: ethanol, EDTA, acetonitrile; Samples: [sodium citrate dihydrate, L-glutamine, L-serine, L-lysine, nicotinic acid, vitamin B1, vitamin B6] (composition 1b).

[0125] Rats were fasted for 24 hours but allowed free access to water. They were weighed and randomly divided into a control group and an experimental group (administration dosage: 0.294 g / kg sodium citrate dihydrate, 0.486 g / kg L-glutamine, 0.346 g / kg L-serine, 0.5 g / kg L-lysine, 0.01 g / kg niacin, 0.07 g / kg vitamin B1, 0.03 g / kg vitamin B6). The control group was administered physiological saline by gavage at 16.7 mL / kg, while the experimental group was administered an equal volume of composition 1b by gavage. Immediately after gavage, a physiological saline solution containing 20% ​​ethanol was injected intraperitoneally at a dose of 2 g / kg. Subsequently, tail blood was collected at 0, 10, 20, 30, 60, 120, 180, 240, and 300 minutes after ethanol administration. The blood was mixed with EDTA for anticoagulation, tert-butanol was added as an internal standard, and then mixed with acetonitrile at a 1:1 ratio. The supernatant was collected by centrifugation, filtered through an aqueous filter membrane, and gas chromatography was used to detect ethanol.

[0126] (2) Experimental results The experimental results are shown in Figure 8 . Figure 8 The results showed that composition 1b did not accelerate ethanol metabolism in rats. Compared with composition 1a, composition 1b was formulated with 10 mg / kg nicotinic acid instead of 250 mg / kg nicotinamide. These results suggest that nutritional supplementation or lower doses of NAD precursors combined with amino acids do not accelerate ethanol metabolism.

[0127] Numerous studies have shown that oral NMN can be utilized by the body to increase NAD+ levels in the liver. + Therefore, NMN did not accelerate ethanol metabolism in in vivo experiments. Figure 6 The problem isn't with NMN absorption, but rather that NMN alone is ineffective; it simply increases NAD+ in the liver. + Levels of NAD+ do not accelerate ethanol metabolism. In cell experiments, the effect of NAD+ precursors in increasing the rate of ethanol metabolism is achieved when used in combination with L-glutamine and / or L-serine, because when used in combination, in addition to increasing NAD+... + In addition to levels, it can also increase NAD. + / NADH ratio. In subsequent pharmacological experiments, Example 6, neither a combination of nutrient acids containing citric acid, L-glutamine, L-serine, and L-lysine nor a combination of vitamins containing NAM, vitamin B1, and vitamin B6, when used separately, accelerated ethanol metabolism. However, when the vitamin combination and the nutrient acid combination were used together, the ethanol metabolism rate increased significantly. Figure 7 However, if the NAM in this compound is replaced with a low dose of NA, no effect is produced. Figure 8These results indicate that adequate NAD precursors and amino acids are essential for increasing ethanol metabolism. The mechanism was elucidated in pharmacological experiment example 4, as the above combination simultaneously increases hepatic NAD levels. + Levels and NAD + The NADH ratio increases the NAD ratio in hepatocytes. + Levels of NAD+ have a promoting effect on ALDH activity, while increasing NAD+ levels in hepatocyte cytoplasm has a promoting effect on ALDH activity. + The NADH / ADH ratio can increase ADH activity. This will be further confirmed in the following in vivo experiments.

[0128] Pharmacological Experiment Example 7 The composition 1a obtained in Example 1 showed the effects of composition 1a on ADH and ALDH activity, NADt content, and cytoplasmic free NAD in the liver of ethanol-exposed rats. + Study on the / NADH ratio: (1) Experimental methods Instruments: SpectraMax Paradigm microplate reader (MegGU Molecular), Shanghai Instrument & Electronics Analyzer 752N UV-Vis spectrophotometer; Animals: Male SD rats (250-300 g); Reagents: Ethanol, yeast ADH, NAD+ + CCK-8 reagent; Sample: [sodium citrate dihydrate, L-glutamine, L-serine, L-lysine, nicotinamide, vitamin B1, vitamin B6] (composition 1a).

[0129] Rats were fasted for 24 hours but allowed free access to water. They were weighed and randomly divided into a control group and a compound group. The control group was administered physiological saline by gavage at 16.7 mL / kg, while the compound group was administered an equal volume of Composition 1a (dosage: 0.294 g / kg sodium citrate dihydrate, 0.486 g / kg L-glutamine, 0.346 g / kg L-serine, 0.5 g / kg L-lysine, 0.25 g / kg nicotinamide, 0.07 g / kg vitamin B1, and 0.03 g / kg vitamin B6) by gavage. Immediately after gavage, the rats were injected intraperitoneally with physiological saline solution containing 20% ​​ethanol at a dose of 2 g / kg. 210 minutes later, the rats were anesthetized by intraperitoneal injection of afodin. They were then euthanized by exsanguination through the abdominal aorta. The entire liver was collected, the blood was blotted dry with filter paper, and the liver tissue was weighed. Total NAD (NADt), pyruvate, lactate, ADH, and ALDH were extracted from the liver tissue.

[0130] a. NADt assay: Liver tissue was cut into small pieces, frozen in liquid nitrogen, and ground into powder in a mortar. 2–4 mg of the powder was mixed with 1 mL of 10 mM potassium phosphate buffer (pH 7.0) containing 70% ethanol. The mixture was extracted by intermittent vortexing at 25°C for 20 min, centrifuged at 3000 × g for 20 min, and the supernatant was used as the NADt sample. The NADt content was determined using the WST-8 method. After the precipitate was heated to dryness, NaOH was added and vortexed overnight. The dissolved product was neutralized with HCl and used for protein assay. The protein content was determined using the Bradford method. The tissue NADt content was standardized to protein content.

[0131] b. Free NAD+ in liver tissue + / NADH ratio determination: A homogenate was prepared from liver tissue frozen in liquid nitrogen, and the supernatant after centrifugation was used as the test sample. Free NAD in the cytoplasm was measured. + The NADH ratio was determined using a pyruvate kit and a lactate kit.

[0132] c. Determination of hepatic ADH and ALDH activity: For each group, the same liver lobe was taken, weighed approximately 1 g of liver tissue, minced, and added to pre-cooled sucrose buffer (0.25 M sucrose, 0.33 mM DTT, 10 mM Tris-HCl, pH 7.4) at 4°C. The liver tissue was homogenized 10-12 times using a glass homogenizer to prepare a 1:5 liver homogenate, which was then diluted to prepare a 1:10 liver homogenate. 0.5 mL of the 1:5 liver homogenate was centrifuged at 24,900 × g for 60 minutes at 4°C, and the supernatant was used as the ADH sample. Activity was tested at 25°C, containing 2.8 mM NAD. + The reaction was performed using ADH reaction buffer (0.2 M Tris-HCl, 0.106 M phosphate, 0.1 M KCl, pH 7.3) containing 10 mM ethanol. After sample addition, the absorbance change at 340 nm wavelength was measured using a spectrophotometer over 5 minutes. The molar absorptivity of NADH at this wavelength (6,220 L·mol⁻¹) was used as the basis for the determination. -1 ·cm -1The production rate of NADH per minute was calculated, with 1 μmol NADH / min defined as 1 unit of enzyme activity (U). The total liver ADH activity was obtained by combining liver weight and liver homogenate dilution ratio. Mitochondria were extracted from the liver using gradient centrifugation to purify ALDH; the entire process was performed at 4°C. 8–10 mL of a 1:10 liver homogenate was centrifuged at 700 × g for 10 minutes. The supernatant was transferred to a new test tube and centrifuged at 8,000 × g for 10 minutes. The supernatant was discarded. The crude mitochondria were resuspended in 10 mL of sucrose buffer and centrifuged at 8,000 × g for 10 minutes. The supernatant was discarded. The purified mitochondria were weighed, and a small amount of 30 mM sodium phosphate buffer containing 1 mM EDTA and 1 mM DTT was added. The mixture was resuspended at pH 6.0, sonicated, and centrifuged at 24,900 × g for 60 minutes at 4°C. The supernatant was used as the ALDH sample. Activity was tested at 25°C with 0.5 mM NADH. + The reaction was carried out under the conditions of ALDH reaction buffer (0.125 M sodium pyrophosphate, 1.25 mM EDTA, pH 9.0) with 0.1 mM acetaldehyde. After the sample was added, the change in absorbance at 340 nm wavelength was measured with a spectrophotometer within 5 minutes to calculate the amount of NADH produced per minute. ALDH activity was normalized to mitochondrial weight or mitochondrial protein content.

[0133] (2) Experimental results The experimental results are shown in Table 4. Table 4 shows that composition 1a significantly increased ALDH activity in the mitochondria of ethanol-exposed rat livers by 100%, but had no significant effect on increasing total liver ADH activity; it also significantly increased liver NADt content and cytoplasmic free NAD. + The NAD+ / NADH ratio increased by 200% for the former and 300% for the latter. Increased NAD+ content promoted ALDH activity, while cytoplasmic free NAD+... + An increased NADH / ADH ratio suggests that ADH accelerates the oxidation of ethanol to acetaldehyde. The results indicate that the hangover-relieving composition's ability to increase ethanol metabolism is related to its ability to increase ADH and ALDH activity.

[0134] Table 4. Effects of Composition 1a on ADH and ALDH activity, NADt content, and cytoplasmic free NAD in the liver of ethanol-exposed rats. + The effect of the / NADH ratio (Mean ± SEM, n = 5)

[0135] a t test.

[0136] bMann-Whitney U test.

[0137] Pharmacological Experiment Example 8 Study on the duration of intoxication of compositions 1a, 1e and 1f obtained in Example 1: (1) Experimental methods Instruments: SpectraMax Paradigm microplate reader (MegG), Shanghai Instrument & Electronics Analyzer 752N UV-Vis spectrophotometer, Thermo Fisher Scientific Nanodrop One micro-volume spectrophotometer; Animals: Female ICR mice (22-26 g); Reagents: Ethanol, yeast ADH, NAD+ + CCK-8 reagent, 3-methyl-2-benzothiazolidinonehydrazone (MBTH), ferric ammonium sulfate; Samples: metadoxine, [sodium citrate dihydrate, L-glutamine, L-serine, L-lysine, nicotinamide, vitamin B1, vitamin B6] (composition 1a, composition 1e, composition 1f).

[0138] Mice were fasted for 12 hours but allowed free access to water, weighed, and randomly divided into a control group (n=10), a model group (n=12), a positive control group (n=12), a low-dose treatment group (n=12), a medium-dose treatment group (n=12), and a high-dose treatment group (n=12). The control group mice were administered physiological saline by gavage, while the other groups were administered 6.5 g / kg ethanol (50% V / V physiological saline solution) by gavage. Thirty minutes later, the control and model groups were intraperitoneally injected with physiological saline at a dose of 33.3 mL / kg, while the positive control group was intraperitoneally injected with 500 mg / kg metadoxine (dissolved in an equal volume of physiological saline). The low-dose, medium-dose, and high-dose treatment groups were intraperitoneally injected with equal volumes of Composition 1f, Composition 1e, and Composition 1a (i.e., 1 / 2, 1, or 2 times the dose of the hangover remedy), respectively. Subsequently, the disappearance and recovery of the righting reflex were observed. Five and a half hours after ethanol administration, blood was collected by enucleation, and the mice were euthanized by cervical dislocation, and their livers were collected. Blood was used for the determination of ethanol and acetaldehyde; liver tissue was used to extract total NADt, prepare mitochondria, and extract ALDH; and total liver lysate and mitochondrial lysate were prepared. NADt content was determined. + / NADH ratio and ALDH activity.

[0139] a. Righting Reflex Test: The righting reflex test was used to determine the level of alcohol intoxication in mice. Mice other than the control group were tested after being exposed to ethanol and losing their ability to move. The mice were placed in a supine position; if the righting reflex disappeared, it indicated intoxication. The mice were then transferred to a small animal heating pad while still in a supine position for further observation. When the righting reflex disappeared and the mouse rolled over on its own, and then reappeared with the righting reflex 30 seconds later, it was considered sober. The number of intoxicated mice and the number of deaths in each group were recorded, as well as the duration of intoxication and sobering up in sensitive mice, and the duration of intoxication was calculated.

[0140] b. Determination of ethanol and acetaldehyde: Blood was placed at room temperature for 30 minutes, then centrifuged at 4000×g for 10 minutes at 4°C. The serum was transferred to new EP tubes and stored at -80°C. Before testing, the serum was centrifuged again at 4000×g for 5 minutes at 4°C to obtain the sample. The ethanol content in serum was determined using the WST-8 method. The acetaldehyde content in serum was determined using the phenol reagent spectrophotometric method.

[0141] c. Determination of hepatic ALDH activity: Mouse hepatic mitochondrial ALDH activity was determined using the method described in Example 9 of the pharmacological experiments. NADt, NADH, and NAD... + The NADt / NADH ratio was calculated as follows: The NADt content in mouse liver was determined using the method described in Example 9 of the pharmacological experiment. Separately, 0.8 mL of the NADt sample was mixed with 50 μL of 0.25 M NaOH to adjust the pH of the solution to >9.0, and then heated at 80°C for 15 minutes to degrade the NAD+. + After cooling, the pH of the solution was adjusted to 7.0–7.5 with 20 μL of 1 M Tris-HCl (pH 6.8), and the volume was recorded. This volume served as the NADH sample, and the NADH content was determined using the WST-8 method. NAD+ content was calculated by subtracting the NADH content from the NAD+ content. + The content was then calculated, and finally, NAD was determined. + / NADH ratio.

[0142] (2) Experimental results The experimental results are shown in Tables 5 and 6. Figure 9 , Figure 10 The results showed no significant difference in the intoxication rate among the ethanol treatment groups (Table 5). 5.5 hours after ethanol administration, most of the intoxicated mice in the model groups remained unconscious, while all mice in the high-dose treatment group regained consciousness. Figure 9 The results showed that the hangover remedy shortened the duration of intoxication in mice. Figure 10The results showed that the hangover remedy could reduce serum ethanol and acetaldehyde levels in ethanol-exposed mice, and these effects were dose-dependent and stronger than those of metadoxine. Table 6 shows that high-dose hangover remedies could, to some extent, prevent the decrease in hepatic mitochondrial ALDH activity caused by ethanol exposure and also increase hepatic NADt content by approximately 6 times, thus mitigating the NAD3-suppressed by ethanol. + The / NADH ratio has returned to normal.

[0143] Table 5. Intoxication and mortality rates in mice of each group

[0144] Table 6. Effects of the hangover remedy on liver ALDH activity, NADt content, and NAD+ in ethanol-exposed mice. + The effect of the / NADH ratio (Mean ± SEM)

[0145] * p <0.05, *** p <0.001 compared with the control group.

[0146] # p <0.05, ## p <0.01, #### p <0.0001 compared with the model group.

[0147] a Kruskal-Wallis test and Dunn test.

[0148] b One-way ANOVA and Tukey's test.

[0149] To demonstrate the promoting effect of the present invention's hangover-relieving composition on ethanol metabolism in animal experiments, two experimental designs were used to rule out the possibility that the test composition might prolong the residence time of ethanol in the stomach. The first experimental design involved gavage of the composition to animals, followed by intraperitoneal injection of ethanol. The second experimental design involved gavage of the ethanol solution to animals, followed by intraperitoneal injection of the composition 30 minutes later. By bypassing the first-pass effect in the gastrointestinal tract through intraperitoneal injection of ethanol, or by allowing a time interval after gavage to allow ethanol absorption by the intestines before injecting the composition, and by administering the composition via different routes to reduce interactions, the influence of the test composition on ethanol absorption can be minimized.

[0150] In pharmacological experiment Example 6, rats were injected intraperitoneally with 2 g / kg ethanol. The ethanol content in blood collected from the tail was measured hourly from 1 hour to 6 hours to calculate the ethanol elimination rate. The effect of the composition in accelerating the ethanol elimination rate was observed by intraperitoneal injection and gavage administration of the same composition at approximately the same time.

[0151] In Example 8 of the pharmacological experiment, mice were administered 6.5 g / kg of ethanol by gavage. The time of intoxication and sobering up was determined by the righting reflex test, from which the duration of intoxication was calculated. The sobering-up effect of the composition was tested by intraperitoneal injection 30 minutes after gavage administration of ethanol.

[0152] In pharmacological experiments Examples 2, 3, and 5, the ethanol elimination rate of freshly isolated rat hepatocytes was determined by testing the remaining ethanol content in the reaction flasks 0 minutes and 60 minutes after the addition of ethanol. By adding the test component to the test system (reaction solution), pre-incubating the hepatocytes for 1 hour, and then adding ethanol (final concentration 8.5 mM) to conduct the ethanol elimination experiment, the effect of the test component or composition in accelerating the ethanol elimination rate of hepatocytes can be determined.

Claims

1. A hangover remedy composition, characterized in that: The hangover relief composition includes an NAD precursor and an amino acid, wherein the NAD precursor is one or more of nicotinamide, nicotinamide ribose, nicotinamide mononucleotide, or nicotinic acid; and the amino acid is one or more of L-glutamine, L-serine, or L-lysine.

2. The hangover relief composition according to claim 1, characterized in that: The mass ratio of the NAD precursor to the amino acid is 1:0.45~24.

3. The hangover relief composition according to claim 1, characterized in that: The amino acid is a mixture of L-glutamine, L-serine and L-lysine in a mass ratio of 1:1~2.3:0.35~1.

43.

4. The hangover relief composition according to claim 1, characterized in that: The hangover relief composition also includes other nutrients, which are one or more of vitamin B1, vitamin B6, citric acid, or citrates.

5. The hangover relief composition according to claim 4, characterized in that: The nutrients are a mixture of vitamin B1, vitamin B6, and citric acid or citrates.

6. The hangover relief composition according to claim 5, characterized in that: In the hangover relief composition, the mass ratio of NAD precursor, amino acids, vitamin B1, vitamin B6, and citric acid or citrate is 1 : 0.45~12 : 0.046~0.25 : 0.02~0.12 : 0.28~1.

2.

7. A liquid preparation with hangover-relieving effects, characterized in that: The liquid formulation comprises the hangover relief composition according to any one of claims 1 to 6 and a liquid medium, wherein the mass-to-volume ratio of the sum of the NAD precursor and the amino acid to the liquid medium is 1 g : 1.6 to 660 mL.

8. The hangover relief composition according to claim 7, characterized in that: The liquid medium is one or more of pure water, physiological saline, K-Heinz solution, or other physiologically balanced solutions.

9. A method for preparing the liquid preparation with hangover-relieving effects as described in claim 7 or 8, characterized in that: The NAD precursor, along with amino acids and / or other nutrients, are added to a liquid medium and stirred at room temperature until completely dissolved.

10. The use of the hangover relief composition of claim 1 or the liquid preparation with hangover relief effect of claim 7 in the preparation of hangover relief and / or treatment of acute alcohol poisoning products.

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