CN117224544A - Liver-protecting and alcohol-dispelling composition, preparation and application thereof - Google Patents
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- CN117224544A CN117224544A CN202311024397.3A CN202311024397A CN117224544A CN 117224544 A CN117224544 A CN 117224544A CN 202311024397 A CN202311024397 A CN 202311024397A CN 117224544 A CN117224544 A CN 117224544A
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- taurine
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- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The invention relates to a composition for protecting liver and dispelling effects of alcohol, a preparation and application thereof, and relates to the fields of traditional Chinese medicines and health-care foods. The composition for protecting liver and dispelling effects of alcohol comprises a combination of at least 3 components of vitamin B1, taurine, threonine, kudzuvine root extract and ampelopsis grossedentata leaf extract. The invention discovers that vitamin B1, taurine, threonine, ampelopsis grossedentata leaf extract and radix puerariae extract have the functions of protecting liver cells and accelerating alcohol metabolism, and the medicines with liver protection and alcohol effect dispelling effects show stronger liver protection and alcohol effect dispelling effects than single component after being mixed according to a certain concentration, and at least 3 combinations can meet the requirements of liver protection and alcohol effect dispelling.
Description
Technical Field
The invention relates to the field of traditional Chinese medicines and health-care foods, in particular to a composition for protecting liver and dispelling effects of alcohol, and a preparation and application thereof.
Background
Wine such as white wine, red wine, beer and the like has become indispensable on dining tables in China and even worldwide. According to the latest scientific findings, alcohol can cause different degrees of injury to the body. The damage of alcohol to liver is mainly represented by that the alcohol directly kills liver cells, so that the alcohol dispelling capability of the liver is reduced, the alcohol is continuously damaged to the liver or other tissues and organs, and the vicious circle can cause alcoholic hepatitis, alcoholic fatty liver, acute liver failure, liver cirrhosis and the like. The product for protecting liver and dispelling effects of alcohol can provide effective protection for daily drinking and reduce toxic and side effects of alcohol on human bodies. Therefore, the development of products with liver protection and anti-alcohol effects is urgent, and the products have great market transformation potential.
The existing products for dispelling effects of alcohol and protecting liver in the market mostly contain traditional Chinese medicine components, the mechanism is unknown, the effect is unstable, and meanwhile, the traditional Chinese medicine components with unknown mechanisms increase the burden of livers and kidneys. The application number is 201711473069.6, and the main components of the anti-alcohol liver-protecting composition are medicinal components such as semen hoveniae, flos puerariae lobatae, poria cocos, bighead atractylodes rhizome, reed rhizome, glossy privet fruit, peppermint, brine alga and the like, and the anti-alcohol liver-protecting composition has the defects of potential toxic and side effects of the liver, difficulty in standardization, unclear mechanism and the like of the traditional Chinese medicine components. The main components of the product are ganoderma lucidum, schisandra chinensis, hovenia dulcis thunb, pueraria flower, butterflybush flower and jujube, and the product has the defects of potential toxic and side effects of liver, difficulty in standardization, unclear mechanism and the like of traditional Chinese medicine components. The main components of the anti-alcohol liver-protecting hydrogel sheet are 20-40 parts of chitosan, 25-55 parts of sodium alginate, 3-20 parts of gelatin, 1-10 parts of calcium carbonate and 0.05-0.5 part of gallic acid, the components are relatively controllable, and the main action mechanism is that the components form a barrier effect on gastrointestinal mucosa and slow down the rapid absorption of alcohol, so that the alcohol which has entered into blood circulation cannot be protected theoretically.
The food and the components thereof have natural safety, the products with the functions of protecting liver and dispelling effects of alcohol are developed based on the foods and food additives with definite components in the food catalogue, the defects of the existing anti-alcohol and liver-protecting products are overcome, liver cells are directly protected, and the effect of accelerating alcohol metabolism is achieved. In view of the above, the invention provides a composition for protecting liver and dispelling effects of alcohol, and a preparation and application thereof.
Disclosure of Invention
The invention aims to solve the technical problem of providing a composition for protecting liver and dispelling effects of alcohol, and a preparation and application thereof. Aims to provide a composition which is all food ingredients and has the effects of protecting liver and dispelling alcohol effects.
The present invention solves the above technical problems, and a first aspect provides a composition for protecting liver and alleviating hangover, which comprises a combination of at least 3 components of vitamin B1, taurine, threonine, kudzuvine root extract and ampelopsis grossedentata leaf extract.
Based on food catalogues and liver basic research documents, 14 ingredients (vitamin B1 (VB 1), vitamin B2 (VB 2), vitamin B6 (VB 6), vitamin B12 (VB 12), nicotinic acid, pantothenic acid, taurine, threonine, tryptophan, methionine, leucine, fructose, ampelopsis grossedentata leaf extract (the main ingredient is dihydromyricetin) and pueraria lobata extract (the main ingredient is puerarin)) are screened, and the foods possibly have the effects of protecting liver and dispelling effects of alcohol, so that the harm of alcohol to human bodies is reduced through foods or food supplements, the potential toxic and side effects on human bodies are reduced, and the safety is improved; the invention is based on scientific logic for protecting liver cells so as to improve the anti-alcohol capability, and repeated tests prove that:
(1) Toxicity and dose-dependent detection of liver-protecting and anti-alcohol components to be detected: the 14 components have no obvious toxicity to the C57BL/6 primary liver cells in various concentration gradients; the effective concentration range of the vitamin B1 for the liver cell damage caused by alcohol is 0.009-0.576 micrograms/milliliter, and the optimal concentration is 0.036 micrograms/milliliter; the effective concentration range of the taurine is 1.71-109.44 micrograms/milliliter, and the optimal concentration is 6.84 micrograms/milliliter; the effective concentration range of the threonine to the liver cell damage caused by alcohol is 9-576 micrograms/milliliter, and the optimal concentration is 36 micrograms/milliliter; the effective concentration range of the pueraria extract for the hepatic cell injury caused by alcohol is 1024-32768 micrograms/milliliter, and the optimal concentration is 8192 micrograms/milliliter; the effective concentration range of the ampelopsis grossedentata leaf extract for treating the hepatic cell injury caused by alcohol is 0.025-51.2 micrograms/milliliter, and the optimal concentration is 1.6 micrograms/milliliter; the subsequent experiments were carried out using this concentration; the other 9 components have no dose-dependent effect of improving the liver cell injury, and the corresponding results are not listed here;
(2) Protection effect experiment on alcohol hepatocyte injury: confirm that 5 medicines (vitamin B1, taurine, threonine, kudzuvine root extract and ampelopsis grossedentata leaf extract) have the effect of protecting C57BL/6 primary liver cells on the premise of adding alcohol with a certain concentration;
(3) Alcohol effect test: the medicine VB1, taurine, threonine, radix puerariae extract and ampelopsis grossedentata leaf extract have certain anti-alcohol capability, wherein the ampelopsis grossedentata leaf extract is strongest and the threonine is weakest; meanwhile, the capability of promoting the liver cells to relieve the alcohol after the 5 components are mixed can be obviously improved;
(4) Alcohol metabolism experiment: at least 3 combinations of vitamin B1, taurine, threonine, kudzu root extract and ampelopsis grossedentata leaf extract have good anti-alcohol capability, and 3 combinations can achieve ideal effects;
(5) Animal experiment: the combination of at least 3 of vitamin B1, taurine, threonine, kudzuvine root extract and ampelopsis grossedentata leaf extract can accelerate the decomposition speed of alcohol, so that the alcohol can be decomposed at a higher speed in a C57BL/6 mouse body, the residence time is shorter, the GPT is obviously lower, and the liver protection effect is obvious;
in a word, 5 food components (vitamin B1, taurine, threonine, kudzu root extract and ampelopsis grossedentata leaf extract) which are non-cytotoxic and have liver protecting and anti-inebriation effects are selected, and in further research, at least 3 food components with liver protecting and anti-inebriation effects are mixed according to a certain concentration ratio, so that better anti-inebriation and liver protecting effects are obtained compared with single component.
Among them, vitamin B1, also called thiamine, is a water-soluble vitamin purified by people at the earliest, and has the chemical name of 3- [ (4-amino-2-methyl-5-pyrimidinyl) -methyl ] -5- (2-hydroxyethyl) -4-methylthiazolium chloride, has the function of maintaining normal sugar metabolism, and can promote the decomposition of alcohol by being used as a coenzyme for tricarboxylic acid circulation. Alcoholics also more likely lack vitamin B1, increasing the incidence of organic amnesia syndrome. The disease can cause brain injury, memory loss, confusion, instability and intermittent vision loss, so that the timely VB1 supplementation also has a certain effect on the brain protection after drinking.
Taurine is a sulfur-containing amino acid in animal bodies, but is not a constituent of proteins, is an organic permeation regulating substance, not only participates in regulating cell volume, but also provides a basis for bile salt formation, and plays an important role in the preparation of the concentration of free calcium in cells; taurine has certain protective effects on ethanol-induced rat liver cell injury, alcoholic fatty liver, fatty liver oxidation, alcoholic gastric injury, alcoholic brain injury and the like.
Threonine is an important nutrition enhancer, has the functions of relieving fatigue of human body and protecting cell membranes, and can promote phospholipid synthesis and fatty acid oxidation in vivo. The preparation has the medicinal efficacy of promoting human development and resisting fatty liver, and is a component in compound amino acid transfusion;
the pueraria extract, also called puerarin, is isoflavone derivative separated from radix Puerariae with crown dilating effect, and has effects of relieving fever, tranquilizing, and increasing coronary blood flow. Clinically used for coronary heart disease, angina and hypertension; the radix puerariae extract can protect liver injury through gastric absorption, induce and activate hepatic stellate cell apoptosis, effectively reverse chemically induced hepatic fibrosis, and also has a protective effect on acute liver injury induced by carbon tetrachloride, and simultaneously has multiple physiological activities. The traditional Chinese medicine puerarin has more reports on the aspect of treating alcoholism.
The Ampelopsis grossedentata leaf extract mainly contains 98% dihydromyricetin, is mainly extracted from a woody vine plant of Ampelopsis in Vitaceae, and also is extracted from useful semen Hoveniae, wherein the main active ingredient is flavonoid compounds, and the substances have various peculiar effects of scavenging free radicals, resisting oxidation, resisting thrombus, resisting tumor, diminishing inflammation and the like; the dihydromyricetin is a special flavonoid compound, has the general characteristics of the flavonoid compound, has the functions of relieving alcoholism, preventing alcoholic liver and fatty liver, inhibiting liver cell deterioration, reducing the incidence of liver cancer and the like, and is a good product for protecting liver and dispelling the effects of alcohol.
The beneficial effects of the invention are as follows: the invention relates to the discovery that vitamin B1, taurine, threonine, ampelopsis grossedentata leaf extract and radix puerariae extract in a food catalog have the functions of protecting liver cells and accelerating alcohol metabolism, and the medicines with liver protection and anti-alcohol effects are mixed according to a certain concentration to show stronger liver protection and anti-alcohol effects than single components, and at least 3 combinations of the medicines can meet the requirements of liver protection and anti-alcohol effects.
On the basis of the technical scheme, the invention can be improved as follows.
Further, the composition for protecting liver and dispelling effects of alcohol comprises the following components in parts by weight: 10.009-0.576 parts of vitamin B, 1.71-109.44 parts of taurine and 1024-32768 parts of radix puerariae extract.
Further, the composition for protecting liver and dispelling effects of alcohol comprises the following components in parts by weight: 10.009-0.576 parts of vitamin B, 1.71-109.44 parts of taurine and 0.025-51.2 parts of Ampelopsis grossedentata leaf extract.
Further, the composition for protecting liver and dispelling effects of alcohol comprises the following components in parts by weight: 10.009-0.576 parts of vitamin B, 1024-32768 parts of radix puerariae extract and 0.025-51.2 parts of ampelopsis grossedentata leaf extract.
Further, the composition for protecting liver and dispelling effects of alcohol comprises the following components in parts by weight: 1.71-109.44 parts of taurine, 1024-32768 parts of radix puerariae extract and 0.025-51.2 parts of ampelopsis grossedentata leaf extract.
Further, the composition for protecting liver and dispelling effects of alcohol comprises the following components in parts by weight: 10.009-0.576 parts of vitamin B, 1.71-109.44 parts of taurine, 1024-32768 parts of radix puerariae extract and 0.025-51.2 parts of ampelopsis grossedentata leaf extract.
Further, the composition for protecting liver and dispelling effects of alcohol comprises the following components in parts by weight: 10.009-0.576 parts of vitamin B, 1.71-109.44 parts of taurine, 9-576 parts of threonine, 1024-32768 parts of radix puerariae extract and 0.025-51.2 parts of ampelopsis grossedentata leaf extract.
The second aspect of the invention provides a preparation of the liver-protecting and anti-alcohol composition, wherein the preparation of the liver-protecting and anti-alcohol composition is in the form of oral liquid, capsule, tablet or pill.
In a third aspect, the present invention provides an application of a composition for protecting liver and dispelling effects of alcohol, wherein the composition for protecting liver and dispelling effects of alcohol is used for preparing an anti-alcohol liver-protecting product.
Further, the product includes a health food or a drug.
Drawings
FIG. 1 is a flow chart of the detection of the killing effect of alcohol on primary hepatocytes of mice in the present invention;
FIG. 2 is a black and white chart showing FDA/PI staining results of primary hepatocytes of alcohol-free treated mice of the present invention;
FIG. 3 is a black and white chart showing the FDA/PI staining results of primary hepatocytes of mice in the present invention in an alcohol solution with a concentration of 6000mg/100 mL;
FIG. 4 is a flow chart of toxicity detection of the liver-protecting anti-hangover ingredients to be tested according to the present invention;
FIG. 5 is a diagram showing the protective effect of VB1 of the present invention;
FIG. 6 is a graph showing the protective effect of taurine according to the present invention;
FIG. 7 is a view showing the protective effect of threonine according to the present invention;
FIG. 8 is a graph showing the protective effect of the radix Puerariae extract of the present invention;
FIG. 9 is a graph showing the protective effect of Ampelopsis grossedentata leaf extract according to the present invention;
FIG. 10 is a flow chart of an anti-hangover effect experiment according to the present invention;
FIG. 11 is a graph showing the effects of the five ingredients and their combination in alleviating hangover;
FIG. 12 is a flow chart of an alcohol metabolism test according to the present invention.
Detailed Description
The principles and features of the present invention are described below with examples given for the purpose of illustration only and are not intended to limit the scope of the invention. The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
Examples
1. Experimental materials and reagents
Primary hepatocytes: the primary liver cells of the C57BL/6 mice are isolated and extracted and cultured in vitro to obtain the recombinant strain; wherein the C57BL/6 mouse is purchased from the medical laboratory animal center in Guangdong province;
the method for separating and culturing the primary liver cells of the mice comprises the following steps: according to the instruction of the primary hepatocyte isolation kit (Liwovens, LV-PHIK001, two-step collagenase digestion method), the primary hepatocytes of mice with good conditions were isolated and extracted according to the steps described in the instruction, and the isolated primary hepatocytes of mice were resuspended in PMH plating medium and then expressed in viable cell count of 1.0×10 5 /cm 2 Inoculating into 96-well collagen coated plate at density, and adding 37 deg.C/5% CO 2 Is cultured in an incubator of (2) for 1 hour,the old culture medium is removed gently, a new PMH plating culture medium is added, and after the culture in an incubator is continued for 8 hours, the liver cells can be used for drug detection experiments.
The liver protecting and alcohol relieving component to be tested is as follows: vitamin B1 (VB 1) (Avidine, T104103-100G), vitamin B2 (VB 2) (Avidine, R104137-25G), vitamin B6 (VB 6) (Avidine, V108688-25G), vitamin B12 (VB 12) (Avidine, V104142-1G), niacin (sigma, N0761-100G), pantothenic acid (sigma, P5155-100G), taurine (sigma, T8691-25G), threonine (sigma, T8441-100G), tryptophan (sigma, T118579-100G), methionine (sigma, M5308-100G), leucine (sigma, L8912-100G), fructose (sigma, F3510-100G), ampelopsis grossedentata leaf extract (HK 20201106, major component is dihydromyricetin, content 98%), radix Puerariae extract (HK 20201105, major component is isoflavone content, and the derivative is the derivative of the group.
Other equipment and reagents: PMH plating medium (liwa organism, LV-WEP 005), fetal bovine serum (Biological Industries,04-001-1 ACS), 96-well collagen Coated plates (liwa, LV-Coated96 w), absolute ethanol (national drug, 10009259); FDA/PI staining (Bestbio/Bei Bo, cat# BB-4217-1000T), alcohol detection kit (Enzychrom TM Ethanol Assay Kit, cat# ECET-100), glutamic pyruvic transaminase detection kit (GPT detection kit, soy bao, cat# BC 1555).
2. Detection of killing effect of alcohol on primary liver cells
2.1 Experimental methods
The experimental procedure is shown in FIG. 1. Since the primary hepatocytes should undergo most of the death (2/3 or more) within 3 hours of the following experiment; if the cell death quantity is insufficient, a false positive result is easy to obtain when the subsequent medicine is tested for the protection effect, and if all cells are completely dead, a false negative result is easy to obtain.
First, alcohol solutions having alcohol concentrations of 1000mg/100mL, 2000mg/100mL, 3000mg/100mL, 4000mg/100mL, 5000mg/100mL, 6000mg/100mL, 7000mg/100mL, 8000mg/100mL, 9000mg/100mL, 10000mg/100mL, 20000mg/100mL were designedThe solution is obtained by diluting absolute ethyl alcohol with fetal calf serum together with alcohol solutions with different concentrations; then, the old medium of the C57BL/6 primary hepatocytes (FIG. 1) cultured in the 96-well plate was removed, and 100uL of each of the above-mentioned alcohol solutions, CO, were added 2 Culturing in an incubator for 3 hours; finally, FDA/PI staining was performed for fluorescence photography to confirm the survival status of the cells.
2.2 experimental results
As can be seen from the experimental results, in the alcohol-free control group (fig. 2), no significant cell death occurred; the concentration of the alcohol solution is 6000mg/100mL, and the alcohol has good killing effect (killing power is more than 2/3) on primary liver cells (figure 3), but does not completely kill all cells at the same time. Other concentrations of alcohol either fail to kill hepatocytes or kill hepatocytes altogether.
3. Toxicity and dose-dependent detection of liver-protecting anti-alcohol component to be detected
3.1 preparation of gradient concentration of each component
Alcohol solution with the concentration of 6000mg/100mL is prepared by fetal bovine serum, and then 5mL of each drug is respectively dissolved and prepared by the liver-protecting anti-alcohol component to be tested and the alcohol solution with the concentration of 6000mg/100mL according to the following tables 1 and 2.
TABLE 1 working concentration ranges of liver protecting and anti-hangover ingredients to be tested
TABLE 2 working concentration ranges of Pueraria lobata extract and Ampelopsis grossedentata leaf extract
3.2 group design
The set of designs:
serogroup: a fetal bovine serum control group;
alcohol group: 6000mg/100mL of alcoholic solution (fetal bovine serum preparation);
drug group: preparing each medicine gradient of fetal bovine serum, and adding 14 components;
drug + alcohol group: 6000mg/100mL of alcoholic solution (fetal bovine serum) was prepared, and the concentration of each drug was 14 ingredients in total.
3.3 Experimental methods
Removing old culture medium of C57BL/6 primary hepatocytes (FIG. 4) cultured in 96-well plate, adding 100uL of each group of solutions, each concentration of which has 3 multiple wells, and CO 2 After 3 hours of culture in the incubator, FDA/PI staining and fluorescence photographing were performed to confirm the survival state of the cells, and the toxicity of each component was judged.
The 14 components are subjected to repeated hole 3 according to the respective concentration gradient, and whether the 14 components have dose-dependent liver cell activity improvement is detected. By analyzing the FDA/PI staining results, the ratio of green cells (living cells) to red cells (dead cells) was counted and plotted by Graphpad Prism.
3.4 experimental results
Evaluation criteria: repeating the drug protection experiments, and statistically analyzing the experimental data results of each time: the FDA/PI staining results of the drug group were scored as compared to the serogroup, the more live cells compared to the serogroup, the higher the score, 0 divided into all concentration gradients without differences from the alcohol group, and 10 divided into all concentration gradients without differences from the serogroup.
The results of the toxicity experiments are shown in Table 3; from the experimental results, it is possible to obtain: the 14 components were not significantly toxic to C57BL/6 primary hepatocytes at each concentration gradient.
TABLE 3 FDA/P I staining results for drug groups compared to serogroups
The results of the dose-dependent experiments are shown in figures 5-9; from the experimental results, it is possible to obtain: the effective concentration range of VB1 to the liver cell damage caused by alcohol is 0.009-0.576 micrograms/milliliter, and the optimal concentration is 0.036 micrograms/milliliter; the effective concentration range of the taurine is 1.71-109.44 micrograms/milliliter, and the optimal concentration is 6.84 micrograms/milliliter; the effective concentration range of the threonine to the liver cell damage caused by alcohol is 9-576 micrograms/milliliter, and the optimal concentration is 36 micrograms/milliliter; the effective concentration range of the pueraria extract for the hepatic cell injury caused by alcohol is 1024-32768 micrograms/milliliter, and the optimal concentration is 8192 micrograms/milliliter; the effective concentration range of the ampelopsis grossedentata leaf extract for treating the hepatic cell injury caused by alcohol is 0.025-51.2 micrograms/milliliter, and the optimal concentration is 1.6 micrograms/milliliter; subsequent experiments were performed with optimal concentrations. The other 9 components have no dose-dependent effect on improving hepatic cell injury, and the corresponding results are not listed here.
4. Experiment of the protective Effect of drugs on alcohol liver cell injury
To exclude individual differences in animals, duplicate experiments were performed on 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 (pueraria extract, 8192 μg/ml), A2 (ampelopsis grossedentata leaf extract, 1.6 μg/ml). The FDA/P I staining results of drug+alcohol group were scored as compared to alcohol group, the more living cells compared to alcohol group, the higher the score, 0 as no difference from alcohol group, and 10 as no difference from serum group. The flow is shown in fig. 10.
The design group is as follows:
serogroup: a fetal bovine serum control group;
alcohol group: 6000mg/100mL of alcoholic solution (fetal bovine serum preparation);
drug + alcohol group: 6000mg/100mL alcohol solution (fetal bovine serum) is prepared, and the concentration of each drug is 7 ingredients, wherein VB2 and tryptophan are used as non-effective control of the drugs
Table 4 protection score of drug against alcoholic hepatocyte injury
From the experimental results table 4 it is possible to: VB1 average mean + -standard deviation 7.75 + -0.433, taurine average mean + -standard deviation 7.75 + -0.433, threonine average mean + -standard deviation 7.25 + -0.829, kudzu root extract (A1) average mean + -standard deviation 8.25 + -0.433, ampelopsis grossedentata leaf extract (A2) average mean + -standard deviation 7.75 + -0.433, the 5 average scores are higher, the standard deviation is lower, the difference between 4 experimental results is small, and 5 medicaments can be confirmed to have the effect of protecting C57BL/6 primary hepatocytes. The remaining 2 components (VB 2 and tryptophan) had a low average score, and were judged to have no effect of protecting the C57BL/6 primary hepatocytes.
5. Alcohol effect test
According to the result of the protection test, five food components have the effect of protecting liver cells, and the anti-alcohol capability of VB1, taurine, threonine, A1 and A2 can be further studied on the basis. The alcohol-neutralizing ability of VB1, taurine, threonine, A1 and A2 was measured at an alcohol concentration of 1000mg/100mL (volume ratio: 1.267%, prepared by diluting fetal bovine serum with absolute ethanol). The flow of the experiment is shown in FIG. 10.
The alcohol detection kit is added in the alcohol dispelling capability test experiment: enzychrom TM Ethanol Assay Kit (cat# ECET-100). Because of the problem of detection limit of the kit, the alcohol concentration is reduced to 1000mg/100mL, and the alcohol concentration is 80 mg/100mL or more, which belongs to drunk driving standards, and the alcohol concentration of 1000mg/100mL far exceeds the standards, so that the alcohol dispelling capability of the medicine can be proved to be strong or weak, and the background interference is reduced, and the alcohol detection sensitivity is improved.
5.1 group design
The set of designs: serogroup (pure serum), alcoholic group (1000 mg/100mL alcoholic solution), each drug alone+alcoholic group (1000 mg/100mL alcoholic solution configuration), D-5 drug mixture+alcoholic group (1000 mg/100mL alcoholic solution configuration).
5.2 Experimental methods
Removing old culture medium from C57BL/6 primary hepatocytes (FIG. 1) cultured in 96-well plate, adding 100uL of each concentration of 3 multiple wells, and CO 2 Culturing in an incubator for 3 hours, and collecting a supernatant; the alcohol concentration in the collected supernatant was measured with a kit. The above alcohol-dispelling ability test was repeated 4 times, based on the measured alcohol concentration (vol%) as shown in the following table5 and fig. 11.
5.3 experimental results
TABLE 5 alcohol concentration (vol%) was measured by alcohol effect test
From the experimental results, it can be seen that: the average alcohol concentration was 0.792v/v%, VB1 group was 0.56875v/v%, threonine group was 0.6455v/v%, taurine group was 0.55275v/v%, A1 group was 0.4395v/v%, A2 group was 0.4315v/v% and mixed group was 0.1505v/v%. The medicine VB1, taurine, threonine, A1 and A2 can be judged to have certain anti-alcohol capability, wherein A2 is the strongest and threonine is the weakest; meanwhile, the 5 components are mixed (mixed with 5C) to promote the anti-alcoholic capability of the liver cells to be obviously improved.
6. Alcohol metabolism experiment
According to the results of example 2, 5 drugs all have different degrees of anti-alcohol effect, and 5 drugs can be mixed to have more obvious anti-alcohol effect. To further confirm the different combinations, we tested the effects of 3 mixes, 4 mixes, 5 mixes on alcohol metabolism, respectively, excluding threonine, which is the least potent in alleviating alcohol. The ability of the different combinations to relieve alcohol at 1000mg/100mL alcohol concentration (volume ratio 1.267%, configuration by dilution of absolute ethanol with fetal bovine serum) was measured. The experimental procedure is shown in FIG. 12.
The alcohol detection kit is added in the alcohol dispelling capability test experiment: enzychrom TM Ethanol Assay Kit (cat# ECET-100). Due to the problem of detection limit of the kit, the alcohol concentration is reduced to 1000mg/100mL (more than or equal to 80 mg/100mL belongs to drunk driving standards, and the alcohol concentration of 1000mg/100mL far exceeds the standards, so that the alcohol dispelling capability of the medicine can be proved to be strong or weak) so as to reduce background interference and improve the alcohol detection sensitivity.
6.1 group design
The specific grouping is as follows:
3 combinations: B1+taurine+A1 (3C-1), B1+taurine+A2 (3C-2), B1+A1+A2 (3C-3), taurine+A1+A2 (3C-4);
4 combinations: VB1+ taurine + A1+ A2 (4C);
5 combinations: VB1+ taurine + threonine + A1+ A2 (5C).
The set of designs: serogroup (pure serum), alcoholic group (1000 mg/100mL alcoholic solution), drug+alcoholic group (1000 mg/100mL alcoholic solution).
6.2 Experimental methods
Removing old culture medium from C57BL/6 primary hepatocytes (FIG. 12) cultured in 96-well plate, adding 100uL of each, 3 multiple wells each, and CO 2 Culturing in an incubator for 3 hours, and collecting a supernatant; the alcohol concentration in the collected supernatant was measured using a kit. The above alcohol effect test experiment was repeated 4 times, and the alcohol concentration (vol%) was measured as follows in table 6:
6.3 experimental results
TABLE 6 alcohol metabolism test for alcohol concentration (vol%)
From the experimental results, it can be seen that: the average alcohol concentration was 0.792v/v%, the 3C-1 alcohol concentration was 0.322v/v%, the 3C-2 alcohol concentration was 0.264v/v%, the 3C-3 alcohol concentration was 0.195v/v%, the 3C-4 alcohol concentration was 0.197v/v%, the 4C alcohol concentration was 0.166v/v%, and the 5C alcohol concentration was 0.168v/v%. It can be seen that groups 3C, 4C and 5C all have better anti-alcohol ability, wherein group 5C is approximately equal to group 4C and is approximately equal to group 3C-3, group 3C-4, group 3C-2 and group 3C-1. The 3C combination can achieve ideal effects, and the specific proportion is adjusted according to the design requirement of the product.
7. Animal experiment
According to the liver protection experiment and the anti-alcohol experiment result, a stepping action object experiment can be further performed on the basis. The new kit for animal experiment detection comprises: GPT detection kit (Soy Bao, BC 1555). The combination of VB1, taurine and A1 with relatively weak anti-alcohol capability in the cell experiment is used first, and if the group of animals has anti-alcohol effect in the level, other groups also have anti-alcohol effect.
7.1 Experimental grouping
Experimental grouping:
NS group: physiological saline;
alcohol group: preparing 40% alcohol and physiological saline;
group 3C-1: VB1+ taurine + A1 (3C-1) is configured by normal saline;
3C-1+40% alcohol group: VB1+ taurine + A1 (3C-1) is prepared by normal saline and 40% alcohol is used for lavage.
7.2 Experimental methods
And (3) establishing an alcohol liver injury model, determining the optimal alcohol concentration of the stomach infusion, and improving the stomach infusion dosage as much as possible under the condition of ensuring that the C57BL/6 mice are not dead. The pre-experiment shows that the C57BL/6 mice can meet the requirements of an alcohol liver injury model according to the gastric lavage volume of 0.015mL/g at the alcohol concentration of 40%, and the mice die easily due to the fact that the dosage and the alcohol concentration are exceeded.
40 healthy male C57BL/6 mice of 6-8 weeks of age were randomly divided into 4 groups of 10 animals each, and were classified into physiological saline (NS), 40% alcohol, 3C-1, and 3C-1+40% alcohol groups.
(1) Diluting absolute ethyl alcohol with NS to prepare 40% alcohol solution;
(2) mixing NS according to A1 (480 mg/kg), taurine (444.6 ug/kg), VB1 (2.34 ug/kg) (the drug dosage is converted from the drug concentration at the cell level, and the total blood amount of the mice is calculated according to 6.5% of the weight) to prepare a mixed 3C-1 group;
feeding 40C 57BL/6 mice in advance for 12 hours without water interruption; the 3C-1 group and the 3C-1+40% alcohol group are subjected to gastric lavage treatment, and the alcohol group and the NS group are filled with equal volumes of physiological saline and put back into a cage for waiting for 30min. Feeding and water cutting off after stomach irrigation until the experiment is finished; after 30min, the alcohol group and 3C-1+40% alcohol group were filled with 40% alcohol, and the NS group and 3C-1 group were filled with equal volumes of physiological saline; cutting tails for 1h, 2h and 3h to collect blood and measure serum alcohol concentration; collecting the eyeball and blood to measure GPT in 16-24h, and killing the mice; the above animal experiments were repeated 3 times, and the results of measuring the alcohol concentration and the glutamic pyruvic transaminase concentration were averaged as shown in tables 7 and 8 below.
7.3 experimental results
TABLE 7 alcohol metabolism rate
| 1h | 2h | 3h | Average alcohol metabolism rate (vol%/h) within 3h | |
| 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 |
Wherein, alcohol metabolism rate (vol%/h) = (initial concentration-end concentration)/time h for each group; NA (Not Available) indicates unavailability.
As shown in Table 7, the average metabolism rate of 3h alcohol in the 3C-1+40% alcohol group was 0.06804vol%/h, the average metabolism rate of 3h alcohol in the alcohol group was 0.01599vol%/h, and it was found that the metabolism rate of alcohol in the mixed 3C-1 group was 4.2 times higher than that in the alcohol group, indicating that the mixed 3C-1 can accelerate the decomposition rate of alcohol, so that alcohol can be decomposed at a faster rate in the C57BL/6 mice, and the residence time is shorter.
TABLE 8 GPT concentration determination
| Group of | NS group | Alcohol group | Group 3C-1 | 3C-1+ alcohol group |
| Average GPT concentration (U/mL) | -0.64678 | 2.13972 | -0.3116 | 0.9498 |
As is clear from the GPT concentrations in Table 8, the average GPT concentration of the mixed 3C-1+ alcohol group was 0.9498U/mL at 21 hours, the average GPT concentration of the alcohol group was 2.13972U/mL, and the 3C-1+ alcohol group was significantly lower than the GPT of the alcohol group, and had a significant liver protecting effect.
In summary, the invention relates to the discovery that VB1, taurine, threonine, ampelopsis grossedentata leaf extract and radix puerariae extract in a food catalog have the functions of protecting liver cells and accelerating alcohol metabolism within a certain concentration range, and the medicines with liver protection and alcohol effect dispelling effects are mixed according to a certain concentration to show stronger liver protection and alcohol effect dispelling effects than single components, and at least 3 combinations of the medicines can meet the requirements of liver protection and alcohol effect dispelling.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (10)
1. A composition for protecting liver and dispelling effects of alcohol, which is characterized by comprising a combination of at least 3 components of vitamin B1, taurine, threonine, kudzuvine root extract and ampelopsis grossedentata leaf extract.
2. The liver-protecting and anti-alcohol composition according to claim 1, wherein the liver-protecting and anti-alcohol 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 radix puerariae extract.
3. The liver-protecting and anti-alcohol composition according to claim 1, wherein the liver-protecting and anti-alcohol 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 0.025-51.2 parts of ampelopsis grossedentata leaf extract.
4. The liver-protecting and anti-alcohol composition according to claim 1, wherein the liver-protecting and anti-alcohol composition comprises the following components in parts by weight: 10.009-0.576 parts of vitamin B, 1024-32768 parts of radix puerariae extract and 0.025-51.2 parts of ampelopsis grossedentata leaf extract.
5. The liver-protecting and anti-alcohol composition according to claim 1, wherein the liver-protecting and anti-alcohol composition comprises the following components in parts by weight: 1.71-109.44 parts of taurine, 1024-32768 parts of radix puerariae extract and 0.025-51.2 parts of ampelopsis grossedentata leaf extract.
6. The liver-protecting and anti-alcohol composition according to claim 1, wherein the liver-protecting and anti-alcohol composition comprises the following components in parts by weight: 10.009-0.576 parts of vitamin B, 1.71-109.44 parts of taurine, 1024-32768 parts of radix puerariae extract and 0.025-51.2 parts of ampelopsis grossedentata leaf extract.
7. The liver-protecting and anti-alcohol composition according to claim 1, wherein the liver-protecting and anti-alcohol composition comprises the following components in parts by weight: 10.009-0.576 parts of vitamin B, 1.71-109.44 parts of taurine, 9-576 parts of threonine, 1024-32768 parts of radix puerariae extract and 0.025-51.2 parts of ampelopsis grossedentata leaf extract.
8. Formulation based on a composition for protecting liver and alleviating hangover according to any one of claims 1 to 7, characterized in that it is in the form of an oral liquid, capsule, tablet or pill.
9. Use of a composition for protecting liver against alcohol, characterized in that the composition for protecting liver against alcohol according to any one of claims 1 to 7 is used in the preparation of an anti-alcohol liver-protecting product.
10. The use of a composition for protecting liver and alleviating hangover according to claim 9, wherein said product comprises a health food or a pharmaceutical.
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