A composition for protecting liver and relieving alcoholism, preparation and application thereof

Abstract

The present application relates to a kind of composition for protecting liver and resolving alcohol and its preparation and application, it is related to the field of traditional Chinese medicine and health food.The composition for protecting liver and resolving alcohol includes the combination of at least 3 kinds of ingredients in vitamin B1, taurine, threonine, radix puerariae extract, ampelopsis glandulifera leaf extract.The present application finds that vitamin B1, taurine, threonine, ampelopsis glandulifera leaf extract, radix puerariae extract have the function of protecting liver cell and accelerating alcohol metabolism, and these drugs with liver-protecting and alcohol-resolving function are mixed according to certain concentration, and present stronger liver-protecting and alcohol-resolving effect than single component, and the combination of at least 3 kinds can achieve the requirement of liver-protecting and alcohol-resolving.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine and health food, specifically to a composition for protecting the liver and relieving hangovers, its preparation and application. Background Technology

[0002] Alcoholic beverages such as baijiu, red wine, and beer have become indispensable on dining tables in my country and around the world. According to the latest scientific findings, alcohol causes varying degrees of damage to the body. The damage to the liver is mainly manifested in the direct killing of liver cells by alcohol, reducing the liver's ability to metabolize alcohol, leading to a vicious cycle of continuous damage to the liver and other tissues and organs, potentially resulting in alcoholic hepatitis, alcoholic fatty liver, acute liver failure, and cirrhosis. Products that protect the liver and help with hangovers can provide effective protection against daily alcohol consumption and reduce the toxic side effects of alcohol. Therefore, the development of products with liver-protecting and hangover-relieving properties is urgent and has enormous market potential.

[0003] Most hangover remedies and liver-protecting products on the market currently contain traditional Chinese medicine ingredients with unclear mechanisms and unstable effects. Furthermore, these ingredients with unclear mechanisms can increase the burden on the liver and kidneys. For example, a patent titled "A Hangover Relief and Liver-Protecting Composition, Hangover Relief and Liver-Protecting Drink, and its Preparation Method and Application" (application number 201711473069.6) contains ingredients such as Hovenia dulcis, Pueraria lobata, Poria cocos, Atractylodes macrocephala, Phragmites communis, Ligustrum lucidum, Mentha haplocalyx, and Dunaliella salina, which suffer from the drawbacks of potential liver toxicity, difficulty in standardization, and unclear mechanisms associated with traditional Chinese medicine ingredients. Similarly, a patent titled "A Product with Hangover Relief and Liver-Protecting Functions and its Preparation Method" (application number 201410028678.0) contains ingredients such as Ganoderma lucidum, Schisandra chinensis, Hovenia dulcis, Pueraria lobata, Buddleja officinalis, and jujube, which also suffer from the drawbacks of potential liver toxicity, difficulty in standardization, and unclear mechanisms associated with traditional Chinese medicine ingredients. For example, the patent titled "A Hydrogel Tablet for Relieving Hangovers and Protecting the Liver and Its Preparation Method and Application" (application number 201911174637.1) contains 20-40 parts chitosan, 25-55 parts sodium alginate, 3-20 parts gelatin, 1-10 parts calcium carbonate, and 0.05-0.5 parts gallic acid. The composition is relatively controllable. Its main mechanism of action is that the components form a barrier on the gastrointestinal mucosa and slow down the rapid absorption of alcohol. Theoretically, it cannot protect alcohol that has already entered the bloodstream.

[0004] Food and its ingredients possess natural safety. Based on foods and food additives with clearly defined ingredients in the food catalog, this invention develops products that protect the liver and relieve hangovers, avoiding the drawbacks of existing hangover and liver-protecting products. These products directly protect liver cells and accelerate alcohol metabolism. Therefore, this invention provides a composition, formulation, and application for protecting the liver and relieving hangovers. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a composition for protecting the liver and relieving hangovers, as well as its formulation and application. The aim is to provide a composition with liver-protecting and hangover-relieving effects that is entirely composed of food ingredients.

[0006] The present invention addresses the above-mentioned technical problems by providing a liver-protecting and hangover-relieving composition comprising at least three of the following components: vitamin B1, taurine, threonine, kudzu root extract, and Ampelopsis japonica leaf extract.

[0007] This invention, based on food catalogs and basic liver research literature, screened 14 foods with clearly defined components (vitamin B1, vitamin B2, vitamin B6, vitamin B12, niacin, pantothenic acid, taurine, threonine, tryptophan, methionine, leucine, fructose, *Vitis pyrenoidosa* leaf extract (main component is dihydromyricetin), and kudzu root extract (main component is puerarin)). These foods may have liver-protective and hangover-relieving effects, aiming to reduce the harm of alcohol to the human body through food or food supplements, reduce potential toxic side effects, and improve safety. This invention is based on the scientific logic of protecting liver cells to improve hangover-relieving ability, and has undergone repeated testing.

[0008] (1) Toxicity and dose-dependent testing of the liver-protecting and hangover-relieving components: All 14 components showed no significant toxicity to C57BL / 6 primary hepatocytes at various concentration gradients; Vitamin B1 showed an effective concentration range of 0.009-0.576 μg / mL for alcohol-induced hepatocyte damage, with an optimal concentration of 0.036 μg / mL; Taurine showed an effective concentration range of 1.71-109.44 μg / mL for alcohol-induced hepatocyte damage, with an optimal concentration of 6.84 μg / mL; Threonine showed an effective concentration range of... The effective concentration range for kudzu root extract against alcohol-induced hepatocellular damage was 9-576 μg / mL, with an optimal concentration of 36 μg / mL. The effective concentration range for kudzu root extract against alcohol-induced hepatocellular damage was 1024-32768 μg / mL, with an optimal concentration of 8192 μg / mL. The effective concentration range for *Vitis thunbergii* leaf extract against alcohol-induced hepatocellular damage was 0.025-51.2 μg / mL, with an optimal concentration of 1.6 μg / mL. These concentrations were used in subsequent experiments. The other nine components showed no dose-dependent effect in improving hepatocellular damage; their results are not listed here.

[0009] (2) Experiment on the protective effect against alcoholic hepatocyte damage: It was confirmed that five drugs (vitamin B1, taurine, threonine, kudzu root extract, and Aureobasidium candida leaf extract) had the effect of protecting C57BL / 6 primary hepatocytes when a certain concentration of alcohol was added.

[0010] (3) Experiment on the effect of alcohol detoxification: Drugs VB1, taurine, threonine, kudzu root extract, and Ampelopsis japonica leaf extract all have certain alcohol detoxification abilities, among which Ampelopsis japonica leaf extract is the strongest and threonine is the weakest; at the same time, the ability of liver cells to detoxify alcohol can be significantly improved after mixing these 5 components.

[0011] (4) Alcohol metabolism experiment: Combinations of at least three of the following: vitamin B1, taurine, threonine, kudzu root extract, and Ampelopsis japonica leaf extract all have good alcohol-relieving ability, and the combination of three can achieve the desired effect.

[0012] (5) Animal experiments: The combination of at least three of the following: vitamin B1, taurine, threonine, kudzu root extract, and Ampelopsis japonica leaf extract can accelerate the decomposition of alcohol, enabling alcohol to decompose at a faster rate and for a shorter time in C57BL / 6 mice, with significantly lower GPT levels, thus having a significant liver-protective effect.

[0013] In summary, this invention screened five food ingredients (vitamin B1, taurine, threonine, kudzu root extract, and Ampelopsis japonica leaf extract) that are non-cytotoxic and have hepatoprotective and hangover-relieving effects. Furthermore, in further research, combinations of at least three of these hepatoprotective and hangover-relieving food ingredients at certain concentration ratios yielded better hangover-relieving and hepatoprotective effects than single ingredients.

[0014] Vitamin B1, also known as thiamine, was the first water-soluble vitamin to be purified. Its chemical name is 3-[(4-amino-2-methyl-5-pyrimidinyl)-methyl]-5-(2-hydroxyethyl)-4-methylthiazolyl hydrochloride. It plays a role in maintaining normal glucose metabolism and may promote alcohol breakdown by acting as a coenzyme in the tricarboxylic acid cycle. Alcoholics are more prone to vitamin B1 deficiency, increasing their risk of organic amnesia. This condition can lead to brain damage, memory loss, confusion, instability, and intermittent vision loss; therefore, timely supplementation with vitamin B1 can also play a role in protecting the brain after alcohol consumption.

[0015] Taurine is a sulfur-containing amino acid in animals, but it is not a component of proteins. It is an organic osmotic regulator that not only participates in regulating cell volume but also provides a basis for the formation of bile salts and plays an important role in modulating intracellular free calcium concentration. Taurine has a certain protective effect against ethanol-induced hepatocyte damage, alcoholic fatty liver, fatty liver oxidation, alcoholic gastric damage, and alcoholic brain damage in rats.

[0016] Threonine is an important nutritional fortifier that can relieve fatigue, plays a vital role in protecting cell membranes, and promotes phospholipid synthesis and fatty acid oxidation in vivo. Its formulations have medicinal efficacy in promoting human development and combating fatty liver disease, and it is a component of compound amino acid infusions.

[0017] Pueraria lobata extract, also known as puerarin, is an isoflavone derivative isolated from the traditional Chinese medicine kudzu root. It possesses coronary vasodilatory effects and exhibits antipyretic, sedative, and coronary blood flow-increasing properties. Clinically, it is used for angina pectoris and hypertension. Pueraria lobata extract, absorbed through the stomach, can protect against liver damage, induce apoptosis of activated hepatic stellate cells, effectively reverse chemically induced liver fibrosis, and protect against carbon tetrachloride-induced acute liver injury. It also possesses a variety of physiological activities. There are numerous reports on the use of kudzu root and puerarin in the treatment of alcohol poisoning.

[0018] The leaf extract of Amaryllis phalloides mainly contains 98% dihydromyricetin. It is mostly extracted from a woody vine of the Amaryllis genus in the Vitaceae family, and sometimes from Japanese raisin tree. Its main active ingredients are flavonoids, which have a variety of unique effects such as scavenging free radicals, anti-oxidation, anti-thrombosis, anti-tumor, and anti-inflammation. Dihydromyricetin is a special type of flavonoid. In addition to the general characteristics of flavonoids, it also has the effects of relieving alcohol poisoning, preventing alcoholic liver disease and fatty liver, inhibiting the deterioration of liver cells, and reducing the incidence of liver cancer. It is a good product for protecting the liver and relieving hangovers.

[0019] The beneficial effects of this invention are: This invention relates to the discovery of vitamin B1, taurine, threonine, *Vitis pyrenoidosa* leaf extract, and kudzu root extract in the food catalog, which have the functions of protecting liver cells and accelerating alcohol metabolism. Furthermore, when these liver-protecting and hangover-relieving drugs are mixed at a certain concentration, they exhibit a stronger liver-protecting and hangover-relieving effect than a single ingredient. A combination of at least three of them can meet the requirements for liver protection and hangover relief.

[0020] Based on the above technical solution, the present invention can be further improved as follows.

[0021] Furthermore, the liver-protecting and hangover-relieving composition comprises the following components in parts by weight: 0.009-0.576 parts of vitamin B1, 1.71-109.44 parts of taurine, and 1024-32768 parts of kudzu root extract.

[0022] Furthermore, the liver-protecting and hangover-relieving composition comprises the following ingredients in parts by weight: 0.009-0.576 parts of vitamin B1, 1.71-109.44 parts of taurine, and 0.025-51.2 parts of Ampelopsis japonica leaf extract.

[0023] Furthermore, the liver-protecting and hangover-relieving composition comprises the following ingredients in parts by weight: 0.009-0.576 parts of vitamin B1, 1024-32768 parts of kudzu root extract, and 0.025-51.2 parts of Ampelopsis japonica leaf extract.

[0024] Furthermore, the liver-protecting and hangover-relieving composition comprises the following components in parts by weight: 1.71-109.44 parts of taurine, 1024-32768 parts of kudzu root extract, and 0.025-51.2 parts of Ampelopsis japonica leaf extract.

[0025] Furthermore, the liver-protecting and hangover-relieving composition comprises the following ingredients in parts by weight: vitamin B1 0.009-0.576 parts, taurine 1.71-109.44 parts, kudzu root extract 1024-32768 parts, and Amaryllis leaf extract 0.025-51.2 parts.

[0026] Furthermore, the liver-protecting and hangover-relieving composition comprises the following ingredients in parts by weight: vitamin B1 0.009-0.576 parts, taurine 1.71-109.44 parts, threonine 9-576 parts, kudzu root extract 1024-32768 parts, and Ampelopsis japonica leaf extract 0.025-51.2 parts.

[0027] A second aspect of the present invention is to provide a formulation of a liver-protecting and hangover-relieving composition, wherein the formulation of the liver-protecting and hangover-relieving composition is in the form of an oral liquid, capsule, tablet or pill.

[0028] A third aspect of the present invention is to provide an application of a liver-protecting and hangover-relieving composition, wherein the liver-protecting and hangover-relieving composition described in any of the above claims is used in the preparation of a hangover-relieving and liver-protecting product.

[0029] Furthermore, the products include health foods or medicines. Attached Figure Description

[0030] Figure 1 This is a flowchart illustrating the detection of the killing effect of alcohol on primary mouse hepatocytes according to the present invention.

[0031] Figure 2 Black and white image of FDA / PI staining results of primary mouse hepatocytes treated without alcohol according to the present invention;

[0032] Figure 3 This is a black and white image showing the FDA / PI staining results of mouse primary hepatocytes in an alcohol solution with a concentration of 6000 mg / 100 mL.

[0033] Figure 4 This is a flowchart of the toxicity detection process for the liver-protecting and hangover-relieving components to be tested in this invention.

[0034] Figure 5 This is a diagram illustrating the protective effect of the present invention VB1;

[0035] Figure 6 This is a diagram illustrating the protective effect of taurine in this invention.

[0036] Figure 7 This is a diagram illustrating the protective effect of threonine in this invention;

[0037] Figure 8 This is a diagram illustrating the protective effect of the kudzu root extract of the present invention.

[0038] Figure 9 This is a diagram illustrating the protective effect of the *Ampelopsis grossedentata* leaf extract according to the present invention.

[0039] Figure 10 This is a flowchart of the experiment on the hangover relief effect of the present invention;

[0040] Figure 11 The diagram shows the hangover relief effects of the five components of this invention and their combination.

[0041] Figure 12 This is a flowchart of the alcohol metabolism experiment of the present invention. Detailed Implementation

[0042] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0043] Example

[0044] 1. Experimental materials and reagents

[0045] Primary hepatocytes: Primary hepatocytes were isolated and extracted from C57BL / 6 mice and cultured in vitro; C57BL / 6 mice were purchased from Guangdong Provincial Medical Laboratory Animal Center.

[0046] The method for isolating and culturing primary mouse hepatocytes includes the following steps: Following the instructions of the primary hepatocyte isolation kit (LV-PHIK001, two-step collagenase digestion method) used, healthy primary mouse hepatocytes were isolated and extracted according to the steps described in the instructions. The isolated and extracted primary mouse hepatocytes were resuspended in PMH plate-forming medium and cultured at a viable cell count of 1.0*10⁻⁶. 5 / cm 2 The cells were seeded into 96-well collagen-coated plates at a density of 1000 mg / ...

[0047] The liver-protecting and hangover-relieving components to be tested are: Vitamin B1 (VB1) (Aladdin, T104103-100g), Vitamin B2 (VB2) (Aladdin, R104137-25g), Vitamin B6 (VB6) (Aladdin, V108688-25g), Vitamin B12 (VB12) (Aladdin, V104142-1g), Niacin (sigma, N0761-100G), Pantothenic acid (sigma, P5155-100G), Taurine (sigma, T8691-25G), and Threonine (sigma, T8441- 100G), tryptophan (Sigma, T118579-100G), methionine (Sigma, M5308-100G), leucine (Sigma, L8912-100G), fructose (Sigma, F3510-100G), *Ampelopsis grossedentata* leaf extract (Shaanxi Huike Botanical Development Co., Ltd., batch number HK20201106, main component is dihydromyricetin, content is 98%), kudzu root extract (Shaanxi Huike Botanical Development Co., Ltd., batch number HK20201105, main component is isoflavone derivative, content is 98%).

[0048] Other equipment and reagents: PMH plate-laying medium (Livo Biotechnology, LV-WEP005), fetal bovine serum (Biological Industries, 04-001-1ACS), 96-well collagen-coated plate (Livo, LV-Coated96w), anhydrous ethanol (Sinopharm, 10009259); FDA / PI staining (Bestbio, catalog number BB-4217-1000T), alcohol test kit (EnzyChrom) TM Ethanol Assay Kit (Catalog No. ECET-100), Alanine Aminotransferase (ALT) Detection Kit (GPT Detection Kit, Solarbio, Catalog No. BC1555).

[0049] 2. Detection of the killing effect of alcohol on primary hepatocytes

[0050] 2.1 Experimental Methods

[0051] The experimental procedure is as follows Figure 1 In the following experiment, the primary hepatocytes must be mostly dead (more than 2 / 3) within 3 hours. If the number of dead cells is insufficient, false positive results will be easily obtained when testing the protective effect of the drug. If all cells are completely dead, false negative results will be easily obtained.

[0052] First, alcohol solutions with concentrations of 1000 mg / 100 mL, 2000 mg / 100 mL, 3000 mg / 100 mL, 4000 mg / 100 mL, 5000 mg / 100 mL, 6000 mg / 100 mL, 7000 mg / 100 mL, 8000 mg / 100 mL, 9000 mg / 100 mL, 10000 mg / 100 mL, and 20000 mg / 100 mL were designed. All alcohol solutions of different concentrations were obtained by diluting anhydrous ethanol with fetal bovine serum. Then, C57BL / 6 primary hepatocytes cultured in 96-well plates (…) Figure 1 After removing the old culture medium, 100 μL of each of the above-mentioned concentrations of alcohol solution were added and the cells were incubated in a CO2 incubator for 3 hours. Finally, FDA / PI staining and fluorescence photography were performed to confirm the cell viability.

[0053] 2.2 Experimental Results

[0054] Based on the experimental results, in the alcohol-free control group ( Figure 2 No significant cell death occurred; at a concentration of 6000 mg / 100 mL, alcohol had a good killing effect on primary hepatocytes (killing power of more than 2 / 3). Figure 3 While it can kill liver cells, it doesn't completely kill all the cells. Other concentrations of alcohol solutions either fail to kill liver cells or kill all of them.

[0055] 3. Toxicity and dose-dependency testing of the liver-protecting and hangover-relieving components to be tested.

[0056] 3.1 Preparation of gradient concentrations of each component

[0057] First, prepare an alcohol solution with a concentration of 6000 mg / 100 mL using fetal bovine serum. Then, dissolve and prepare 5 mL of each drug using the liver-protecting and hangover-relieving component to be tested and the 6000 mg / 100 mL alcohol solution according to Tables 1 and 2 below.

[0058] Table 1. Working concentration range of the liver-protecting and hangover-relieving components to be tested.

[0059]

[0060] Table 2 Working concentration ranges of Pueraria lobata extract and Ampelopsis japonica leaf extract

[0061]

[0062] 3.2 Group Design

[0063] Design category:

[0064] Serum group: Fetal bovine serum control group;

[0065] Alcohol group: 6000mg / 100mL alcohol solution (prepared with fetal bovine serum);

[0066] Drug group: Fetal bovine serum is formulated with various drug gradients, totaling 14 components;

[0067] Drug + Alcohol Group: 6000mg / 100mL alcohol solution (prepared from fetal bovine serum), containing various drug concentrations, totaling 14 components.

[0068] 3.3 Experimental Methods

[0069] C57BL / 6 primary hepatocytes cultured in 96-well plates ( Figure 4 After removing the old culture medium, 100 μL of each of the above solutions was added, with 3 replicates for each concentration. After incubation in a CO2 incubator for 3 hours, FDA / PI staining and fluorescence photography were performed to confirm the cell viability and determine the toxicity of each component.

[0070] Fourteen components were selected according to their respective concentration gradients, with three replicate wells, to determine whether each component dose-dependently enhanced hepatocyte activity. The ratio of green cells (live cells) to red cells (dead cells) was calculated by analyzing FDA / PI staining results and plotted using Graphpad Prism.

[0071] 3.4 Experimental Results

[0072] Evaluation criteria: The above drug protection experiment was repeated, and the experimental data results of each experiment were statistically analyzed. The FDA / PI staining results of the drug group were compared with those of the serum group and scored. The more viable cells compared with the serum group, the higher the score. 0 points indicated no difference from the alcohol group in all concentration gradients, and 10 points indicated no difference from the serum group in all concentration gradients.

[0073] The results of the toxicity test are shown in Table 3. The results show that none of the 14 components had significant toxicity to C57BL / 6 primary hepatocytes at any concentration gradient.

[0074] Table 3 compares the FDA / PI staining results of the drug group with those of the serum group.

[0075]

[0076] The results of the dose-dependent experiment are as follows Figure 5-9The experimental results show that: VB1 has an effective concentration range of 0.009-0.576 μg / mL for alcohol-induced hepatocellular damage, with an optimal concentration of 0.036 μg / mL; taurine has an effective concentration range of 1.71-109.44 μg / mL, with an optimal concentration of 6.84 μg / mL; threonine has an effective concentration range of 9-576 μg / mL, with an optimal concentration of 36 μg / mL; kudzu root extract has an effective concentration range of 1024-32768 μg / mL, with an optimal concentration of 8192 μg / mL; and *Ampelopsis grossedentata* leaf extract has an effective concentration range of 0.025-51.2 μg / mL, with an optimal concentration of 1.6 μg / mL. Subsequent experiments will use the optimal concentrations. The other nine components showed no dose-dependent effect in improving hepatocellular damage; the corresponding results are not listed here.

[0077] 4. Experiment on the protective effect of drugs against alcoholic hepatocyte damage

[0078] To exclude individual differences in animals, the following drugs were repeatedly tested: VB1 (0.036 μg / mL), VB2 (0.036 μg / mL, null control group 1), taurine (6.84 μg / mL), threonine (36 μg / mL), tryptophan (36 μg / mL, null control group 2), A1 (kudzu root extract, 8192 μg / mL), and A2 (Aureobasidium candida leaf extract, 1.6 μg / mL). The FDA / PI staining results of the drug + alcohol group were compared with those of the alcohol group and scored. A higher score indicated more viable cells compared to the alcohol group, with 0 points indicating no difference from the alcohol group and 10 points indicating no difference from the serum group. The procedure is as follows: Figure 10 As shown.

[0079] Design grouping:

[0080] Serum group: Fetal bovine serum control group;

[0081] Alcohol group: 6000mg / 100mL alcohol solution (prepared with fetal bovine serum);

[0082] Drug + Alcohol Group: 6000mg / 100mL alcohol solution (prepared from fetal bovine serum), containing various drug concentrations, a total of 7 components, with VB2 and tryptophan serving as control groups to determine drug efficacy.

[0083] Table 4. Drug protection scores against alcoholic hepatocellular damage

[0084]

[0085] Table 4 shows the experimental results: VB1 mean score ± standard deviation 7.75 ± 0.433, taurine mean score ± standard deviation 7.75 ± 0.433, threonine mean score ± standard deviation 7.25 ± 0.829, kudzu root extract (A1) mean score ± standard deviation 8.25 ± 0.433, and *Ampelopsis grossedentata* leaf extract (A2) mean score ± standard deviation 7.75 ± 0.433. These five components have relatively high mean scores and low standard deviations, with small differences between the four experimental results, confirming that these five drugs have a protective effect on C57BL / 6 primary hepatocytes. The remaining two components (VB2 and tryptophan) have low mean scores and can be determined to have no protective effect on C57BL / 6 primary hepatocytes.

[0086] 5. Experiment on hangover relief effect

[0087] Based on the results of the protective test, the five food components showed hepatocyte protective effects. Further research can be conducted on the alcohol-detoxifying abilities of VB1, taurine, threonine, A1, and A2. The alcohol-detoxifying abilities of VB1, taurine, threonine, A1, and A2 at a 1000 mg / 100 mL alcohol concentration (volume ratio 1.267%, prepared by diluting anhydrous ethanol with fetal bovine serum) were determined. The experimental procedure is as follows: Figure 10 .

[0088] An alcohol detection kit, EnzyChrom, needs to be added to the alcohol tolerance test experiment. TM EthanolAssay Kit (catalog number ECET-100). Due to the detection limit of the kit, we lowered the alcohol concentration to 1000 mg / 100 mL. An alcohol concentration of 80 mg / 100 mL or higher is considered drunk driving. The 1000 mg / 100 mL alcohol concentration far exceeds this standard, which can indicate the strength of the drug's ability to neutralize alcohol, thereby reducing background interference and improving the sensitivity of alcohol detection.

[0089] 5.1 Group Design

[0090] The designed groups are: serum group (pure serum), alcohol group (1000mg / 100mL alcohol solution), each individual drug + alcohol group (1000mg / 100mL alcohol solution), and D-5 drug mixture + alcohol group (1000mg / 100mL alcohol solution).

[0091] 5.2 Experimental Methods

[0092] C57BL / 6 primary hepatocytes cultured in 96-well plates Figure 1In the culture medium, 100 μL of each concentration was added to three replicates, and the mixture was incubated in a CO2 incubator for 3 hours. The supernatant was then collected, and the alcohol concentration in the collected supernatant was determined using a kit. The above alcohol sobriety test was repeated four times, and the measured alcohol concentrations (vol%) are shown in Table 5 below. Figure 11 .

[0093] 5.3 Experimental Results

[0094] Table 5 shows the alcohol concentration (vol%) measured in the hangover relief experiment.

[0095]

[0096] The experimental results show that the average alcohol concentration in the alcohol group was 0.792 v / v, in the VB1 group it was 0.56875 v / v, in the threonine group it was 0.6455 v / v, in the taurine group it was 0.55275 v / v, in the A1 group it was 0.4395 v / v, in the A2 group it was 0.4315 v / v, and in the mixed group it was 0.1505 v / v. It can be determined that drugs VB1, taurine, threonine, A1, and A2 all have certain alcohol-detoxifying abilities, with A2 being the strongest and threonine the weakest. Furthermore, mixing these five components (mixed 5C) significantly enhances the ability of hepatocytes to detoxify alcohol.

[0097] 6. Alcohol metabolism experiment

[0098] Based on the results of Example 2, all five drugs had varying degrees of alcohol-relieving effects, and the combination of the five drugs resulted in a more significant alcohol-relieving effect. To further confirm the different combinations and exclude threonine, which has the weakest alcohol-relieving ability, we tested the effects of combinations of three, four, and five drugs on alcohol metabolism. The alcohol-relieving ability of different combinations at a 1000 mg / 100 mL alcohol concentration (volume ratio 1.267%, prepared by diluting anhydrous ethanol with fetal bovine serum) was measured. The experimental procedure is as follows: Figure 12 .

[0099] An alcohol detection kit, EnzyChrom, needs to be added to the alcohol tolerance test experiment. TM EthanolAssay Kit (catalog number ECET-100). Due to the detection limit of the kit, we lowered the alcohol concentration to 1000 mg / 100 mL (a concentration greater than or equal to 80 mg / 100 mL is considered drunk driving, and 1000 mg / 100 mL far exceeds this standard, which can indicate the strength of the drug's ability to neutralize alcohol), in order to reduce background interference and improve the detection sensitivity of alcohol.

[0100] 6.1 Group Design

[0101] The specific groups are as follows:

[0102] Three combinations: VB1 + taurine + A1 (3C-1), VB1 + taurine + A2 (3C-2), VB1 + A1 + A2 (3C-3), taurine + A1 + A2 (3C-4);

[0103] Four combinations: VB1 + taurine + A1 + A2 (4C);

[0104] Five combinations: VB1 + taurine + threonine + A1 + A2 (5C).

[0105] The designed groups are: serum group (pure serum), alcohol group (1000mg / 100mL alcohol solution), and drug + alcohol group (1000mg / 100mL alcohol solution).

[0106] 6.2 Experimental Methods

[0107] C57BL / 6 primary hepatocytes cultured in 96-well plates ( Figure 12 After removing the old culture medium, 100 μL of each solution was added, with three replicates for each concentration. The mixture was incubated in a CO2 incubator for 3 hours, and the supernatant was collected. The alcohol concentration in the collected supernatant was determined using a kit. The above alcohol sobriety test was repeated four times, and the measured alcohol concentrations (vol%) are shown in Table 6 below.

[0108] 6.3 Experimental Results

[0109] Table 6. Alcohol concentration (vol%) measured in alcohol metabolism experiment.

[0110]

[0111]

[0112] The experimental results show that the average alcohol concentration was 0.792 v / v for the 3C-1 group, 0.322 v / v for the 3C-2 group, 0.264 v / v for the 3C-3 group, 0.195 v / v for the 3C-4 group, 0.197 v / v for the 3C-4 group, 0.166 v / v for the 4C group, and 0.168 v / v for the 5C group. Therefore, groups 3C, 4C, and 5C all have good hangover-relieving abilities, with the order being: 5C group ≈ 4C group > 3C-3 group ≈ 3C-4 group > 3C-2 group > 3C-1 group. The 3C combination can achieve the desired effect; the specific ratios can be adjusted according to product design requirements.

[0113] 7. Animal experiments

[0114] Based on the results of the liver protection and alcohol detoxification experiments described above, further animal experiments can be conducted. A new reagent kit for animal experiments has been added: the GPT detection kit (Solepro, BC1555). First, the combination of VB1 + taurine + A1, which has relatively weak alcohol detoxification ability in cell experiments, will be used. If this group of animals shows an alcohol detoxification effect, it indicates that other groups also have an alcohol detoxification effect.

[0115] 7.1 Experimental Grouping

[0116] Experimental Groups:

[0117] NS group: physiological saline;

[0118] Alcohol group: 40% alcohol, prepared with physiological saline;

[0119] Group 3C-1: VB1 + taurine + A1 (3C-1) is prepared with physiological saline;

[0120] 3C-1 + 40% alcohol group: VB1 + taurine + A1 (3C-1) was prepared with physiological saline and administered by gavage with 40% alcohol.

[0121] 7.2 Experimental Methods

[0122] An alcoholic liver injury model was established to determine the optimal alcohol concentration for gavage. The gavage dose was increased as much as possible while ensuring the survival of C57BL / 6 mice. Preliminary experiments showed that a 40% alcohol concentration and a gavage volume of 0.015 mL / g were sufficient to maintain the alcoholic liver injury model in C57BL / 6 mice. Exceeding the dose and alcohol concentration easily led to mouse death.

[0123] Forty healthy male C57BL / 6 mice aged 6-8 weeks were randomly divided into four groups of 10 mice each: normal saline (NS) group, 40% alcohol group, 3C-1 group, and 3C-1 + 40% alcohol group.

[0124] ① Prepare a 40% alcohol solution by diluting anhydrous ethanol with NS;

[0125] ② Mix NS with A1 (480mg / kg), taurine (444.6ug / kg), and VB1 (2.34ug / kg) (drug dosage is calculated from the drug concentration at the cellular level, and the total blood volume of mice is calculated as 6.5% of body weight) to prepare mixed 3C-1 group;

[0126] Forty C57BL / 6 mice were fasted but not watered 12 hours prior to the experiment. The 3C-1 group and the 3C-1+40% alcohol group were first administered by gavage, while the alcohol group and the NS group were administered an equal volume of physiological saline. The mice were then returned to their cages and waited for 30 minutes. After gavage, the mice were fasted and not watered until the end of the experiment. 30 minutes later, the alcohol group and the 3C-1+40% alcohol group were administered 40% alcohol, while the NS group and the 3C-1 group were administered an equal volume of physiological saline. Blood was collected from the tail at 1 hour, 2 hours, and 3 hours to measure serum alcohol concentration. Blood was collected from the eyeballs at 16-24 hours to measure GPT, and the mice were then sacrificed. The above animal experiment was repeated 3 times, and the average values ​​of the alcohol concentration and alanine aminotransferase concentration are shown in Tables 7 and 8 below.

[0127] 7.3 Experimental Results

[0128] Table 7 Alcohol Metabolism Rate

[0129] NS Group -0.03083 -0.04818 -0.0247 NA alcohol group 0.50509 0.47957 0.45711 0.01599 3C-1 -0.01552 -0.00837 -0.02981 NA 3C-1 + 40% alcohol group 0.56429 0.41632 0.36017 0.06804

[0130] The alcohol metabolism rate (vol% / h) for each group is calculated as (initial concentration - final concentration) / time (h); NA (NotAvailable) indicates that the alcohol is unavailable.

[0131] As shown in Table 7, the average 3-hour alcohol metabolism rate of the 3C-1 + 40% alcohol group was 0.06804 vol% / h, while that of the alcohol group was 0.01599 vol% / h. It can be seen that the alcohol metabolism rate of the 3C-1 mixed group was 4.2 times higher than that of the alcohol group, indicating that the 3C-1 mixed group can accelerate the breakdown of alcohol, allowing alcohol to be broken down at a faster rate and for a shorter time in C57BL / 6 mice.

[0132] Table 8 GPT Concentration Determination

[0133] Average GPT concentration (U / mL) -0.64678 2.13972 -0.3116 0.9498

[0134] As shown in Table 8, at 21 h, the average GPT concentration in the mixed 3C-1+ alcohol group was 0.9498 U / mL, while the average GPT concentration in the alcohol group was 2.13972 U / mL. The GPT concentration in the 3C-1+ alcohol group was significantly lower than that in the alcohol group, indicating a significant liver-protective effect.

[0135] In summary, this invention relates to the discovery that VB1, taurine, threonine, *Vitis viridissima* leaf extract, and kudzu root extract, as listed in the food catalog, have the function of protecting liver cells and accelerating alcohol metabolism within a certain concentration range. Furthermore, when these liver-protecting and hangover-relieving drugs are mixed at a certain concentration, they exhibit a stronger liver-protecting and hangover-relieving effect than a single ingredient. A combination of at least three of them is sufficient to meet the requirements for liver protection and hangover relief.

[0136] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0137] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A composition for protecting the liver and relieving hangovers, characterized in that, The liver-protecting and hangover-relieving composition comprises the following components in parts by weight: vitamin B1 0.009-0.576 parts, taurine 1.71-109.44 parts, threonine 9-576 parts, kudzu root extract 1024-32768 parts, and Ampelopsis japonica leaf extract 0.025-51.2 parts. The kudzu root extract is puerarin; The extract of *Agrostis dentata* leaves contains 98% dihydromyricetin.

2. A formulation based on the liver-protecting and hangover-relieving composition of claim 1, characterized in that, The dosage form of the preparation is oral liquid, capsule, tablet or pill.

3. The application of a composition for protecting the liver and relieving hangovers, characterized in that, The liver-protecting and hangover-relieving composition of claim 1 is used in the preparation of a hangover-relieving and liver-protecting drug.

Images