CN114748566A

CN114748566A - Composition for dispelling effects of alcohol and protecting liver and application thereof

Info

Publication number: CN114748566A
Application number: CN202210365794.6A
Authority: CN
Inventors: 宋立孝; 谭余庆; 杨米一
Original assignee: Enzhong Shandong Digital Health Development Co ltd
Current assignee: Enzhong Shandong Digital Health Development Co ltd
Priority date: 2022-04-08
Filing date: 2022-04-08
Publication date: 2022-07-15
Anticipated expiration: 2042-04-08
Also published as: CN114748566B

Abstract

The invention provides a composition for relieving alcoholism and protecting liver and application thereof, belonging to the technical field of medicines and foods, wherein the composition for relieving alcoholism and protecting liver comprises 35-45 parts of traditional Chinese medicinal material extract, 0.2-0.4 part of yeast extract I, 0.2-0.4 part of yeast extract II and 0.3-0.5 part of garlic extract, wherein the traditional Chinese medicinal materials comprise the following components in parts by weight: and (3) kudzuvine flower: the mass ratio of the chrysanthemum is 1-3: 1-3: 2-6, and mixing. The composition disclosed by the invention can effectively remove oxygen radicals, reduce the harm of the oxygen radicals to liver cells, and simultaneously has physiological activities of resisting bacteria, resisting inflammation, enhancing immunity and the like, so that a good effect of relieving alcoholism and protecting liver is achieved.

Description

Composition for dispelling effects of alcohol and protecting liver and application thereof

Technical Field

The invention belongs to the technical field of medicines and foods, and particularly relates to a composition for dispelling effects of alcohol and protecting liver and application thereof.

Background

Alcoholic Liver Disease (ALD) is a liver disease caused by long-term heavy drinking, and includes 4 major groups, such as Alcoholic Fatty Liver (AFL), Alcoholic Hepatitis (AH), alcoholic liver fibrosis (AHF), and Alcoholic Cirrhosis (AC). The number of deaths from alcohol abuse worldwide is 250 million, accounting for 4% of all deaths, and 40% of deaths from cirrhosis are due to ALD. Alcohol abuse, particularly ALD resulting from alcohol abuse in young people, has become a major public health problem throughout society.

The Oxidative Stress (OS) generated in the alcohol metabolism plays a crucial role in the pathogenesis of alcoholic liver injury, and the injury of liver cells is closely related to Reactive Oxygen Species (ROS). Alcohol metabolism produces large amounts of Nicotinamide Adenine Dinucleotide (NADH), resulting in an increased ADH/NAD + ratio in the cytoplasm or mitochondria, thereby promoting increased mitochondrial respiratory chain electron transport streams and the production of large amounts of ROS in the liver. The large accumulation of ROS can cause imbalance of oxidation-reduction reaction in liver, further promote the liver cells to generate lipid peroxidation damage, and the ROS can attack DNA chains to cause mutations such as breakage, fragment deletion or insertion and the like of the DNA chains, so that the liver cells can possibly generate variation. ROS can react with unsaturated fatty acid in a biological membrane to reduce the unsaturated fatty acid in the membrane and enhance the permeability of the membrane, so that the structure and the function of liver cells are damaged, and when the liver cells are damaged, AST (in mitochondria) and ALT in the cells can enter blood through cell membranes; lipid peroxidation products such as malondialdehyde, etc., polymerize with proteins or DNA to form adducts, inducing the expression of a number of cellular inflammatory factors, resulting in liver cell damage.

The researches show that the kudzu root, the pueraria flower and the chrysanthemum contain rich flavonoids, and the flavonoids belong to phenolic acid compounds, have the effects of capturing oxygen radicals, eliminating the oxygen radicals, reducing the harm of the oxygen radicals to organisms, and have good physiological activities of inhibiting the free radicals, resisting DNA damage, resisting bacteria, resisting inflammation, protecting the liver, enhancing immunity and the like. The bioactive components such as zinc, B vitamins, reductive glutathione and the like contained in the yeast can be used as synthetic raw materials of body reaction enzyme on one hand, and can effectively remove oxygen ions and free radicals in vivo on the other hand, thereby playing a role in protecting liver cells. The garlic is a kind of plant capable of forming bulb in Allium plant of Liliaceae, is a widely planted vegetable crop, and contains abundant phenolic acid, flavone, flavonol, flavanone and other compounds with physiological activities of protecting liver, regulating immunity, resisting oxidation, resisting blood coagulation, protecting cardiac muscle, resisting virus, reducing blood fat, regulating intestinal flora balance, etc., and in addition, the garlic polysaccharide content in the garlic is up to more than 70%, and the garlic polysaccharide belongs to small molecular heteropolysaccharide and has good biological function. However, no report has been found about whether the synergistic effect can be achieved by mixing the extracts of pueraria root, pueraria flower, chrysanthemum, yeast and garlic to prepare the traditional Chinese medicine composition with the functions of relieving alcoholism and protecting liver.

Disclosure of Invention

In view of the above, the present invention aims to provide a composition for alleviating hangover and protecting liver, which has significant efficacy of alleviating hangover and protecting liver through synergistic effects among the compositions.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a composition for relieving alcoholism and protecting liver, which comprises the following components in parts by weight:

35-45 parts of traditional Chinese medicine extract, 0.2-0.4 part of yeast extract I, 0.2-0.4 part of yeast extract II and 0.3-0.5 part of garlic extract;

the traditional Chinese medicinal materials comprise the following components in parts by weight: and (3) kudzuvine flower: the mass ratio of the chrysanthemum is 1-3: 1-3: 2-6.

Preferably, the preparation method of the traditional Chinese medicine extract comprises the following steps: mixing the traditional Chinese medicinal materials with 60-85% ethanol solution according to the material-liquid ratio of 1: 8-15, soaking for 7-12 h, heating and refluxing for 1.5-3 h to obtain liquid medicine, concentrating and drying.

Preferably, the preparation method of the yeast extract I comprises the following steps: dried yeast and 0.032-0.066 mol/L Na₂HPO₄Mixing the solutions according to the material-liquid ratio of 1: 3-5, extracting for 2.5-4 h, filtering with 200-400 meshes to obtain a supernatant, and freeze-drying.

Preferably, the preparation method of the yeast extract II comprises the following steps: mixing dry yeast and water according to a feed-liquid ratio of 1: 8-12, incubating in a water bath at 35-40 ℃ for 15-30 min to obtain a bacterial suspension, filtering the bacterial suspension with a sieve of 80-200 meshes, and taking a precipitate; and (3) resuspending the precipitate in a buffer solution according to the mass-to-volume ratio of 1: 3-5, homogenizing and crushing the yeast at a high speed, centrifuging the crushed yeast to obtain a supernatant, and freeze-drying the supernatant for 48-72 hours at the temperature of-20 to-40 ℃.

Preferably, the crushing conditions are: stirring for 4-5 min under the condition of 6000-8000 r/min, and then continuously stirring for 6-8 min under the condition of 15000-20000 r/min.

Preferably, the preparation method of the garlic extract comprises the following steps: after primary grinding and crushing of garlic, adding a buffer solution with the volume of 4-6 times that of the garlic, and continuing grinding for 15-30 min; and then heating the mixed solution at 50-60 ℃ for 15-30 min, and filtering with 200-400 meshes to obtain a supernatant, wherein the supernatant is a garlic extract, and the garlic extract is freeze-dried at-20-40 ℃ for 48-72 h.

The invention also provides application of the composition in preparation of food, health-care products or medicines for relieving alcoholism and protecting liver.

The invention also provides application of the composition in preparing a medicament for treating or preventing liver injury, wherein the liver injury is caused by alcohol.

Preferably, the composition is added with pharmaceutically acceptable auxiliary materials to prepare a medicament, and the medicament comprises tablets, pills and powder.

The invention also provides an effervescent tablet for relieving alcoholism and protecting liver, which comprises the composition and pharmaceutically acceptable auxiliary materials.

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

according to the invention, the kudzu root, the flower of kudzuvine and the chrysanthemum extract are mixed with the yeast extract and the garlic extract in proportion, so that the raw materials are synergistic, the obtained composition can promote the elimination of acetaldehyde, reduce the concentration of ethanol in blood, shorten the drunk sleep time and achieve the effect of quickly dispelling the effects of alcohol. Meanwhile, the composition can effectively remove oxygen free radicals, improve the activity of antioxidant enzymes, reduce lipid peroxidation products, further reduce the harm of the oxygen free radicals to organisms, play a role in protecting liver cells, and can be used for preventing or relieving liver injury caused by alcohol.

The composition has the advantages of simple and easily obtained raw materials, low cost, obvious effects of dispelling the effects of alcohol and protecting the liver, less side effects and safe and stable drug effect.

The invention is added with pharmaceutically acceptable auxiliary materials to prepare the effervescent tablet, is convenient to take and carry, and can achieve good effects of dispelling the effects of alcohol and protecting liver.

Drawings

FIG. 1 shows the activity of alanine Aminotransferase (AST) and aspartate Aminotransferase (ALT) in the serum of mice;

FIG. 2 is the Triglyceride (TG) content in liver tissue of mice;

FIG. 3 is the reduced Glutathione (GSH) content in mouse liver tissue;

FIG. 4 is the Malondialdehyde (MDA) content in mouse liver tissue;

FIG. 5 shows pathological tissue changes (HE X100) of mouse liver; A. blank group, B, model group, C, compound liver benefiting tablet group, D, effervescent tablet No. 1 dosage group, E, effervescent tablet No. 2 dosage group, F, effervescent tablet No. 3 dosage group, G, effervescent tablet No. 4 dosage group;

FIG. 6 shows pathological tissue changes of mouse liver (HE X400); A. blank group, B, model group, C, compound liver benefiting tablet group, D, effervescent tablet No. 1 dosage group, E, effervescent tablet No. 2 dosage group, F, effervescent tablet No. 3 dosage group, G, effervescent tablet No. 4 dosage group;

FIG. 7 shows pathological tissue changes of mouse liver (oil red staining X100); A. blank group, B, model group, C, compound liver benefiting tablet group, D, effervescent tablet No. 1 dosage group, E, effervescent tablet No. 2 dosage group, F, effervescent tablet No. 3 dosage group, G, effervescent tablet No. 4 dosage group;

FIG. 8 shows pathological histological changes of mouse liver (oil red staining X400); A. blank group, B, model group, C, compound liver-benefiting tablet group, D, effervescent tablet No. 1 dosage group, E, effervescent tablet No. 2 dosage group, F, effervescent tablet No. 3 dosage group, G, effervescent tablet No. 4 dosage group;

FIG. 9 is a morphogram of mouse liver cells; A. blank group, B, model group, C, compound liver-benefiting tablet group, D, effervescent tablet No. 1 dosage group, E, effervescent tablet No. 2 dosage group, F, effervescent tablet No. 3 dosage group, G, effervescent tablet No. 4 dosage group;

FIG. 10 is a mouse liver subcellular morphology map; A. blank group, B, model group, C, compound liver-benefiting tablet group, D, effervescent tablet No. 1 dosage group, E, effervescent tablet No. 2 dosage group, F, effervescent tablet No. 3 dosage group, G, effervescent tablet No. 4 dosage group;

FIG. 11 is a diagram of mouse liver mitochondrial morphology; A. blank group, B, model group, C, compound liver benefiting tablet group, D, effervescent tablet No. 1 dosage group, E, effervescent tablet No. 2 dosage group, F, effervescent tablet No. 3 dosage group, G, effervescent tablet No. 4 dosage group.

Detailed Description

The invention provides a composition for relieving alcoholism and protecting liver, which comprises the following components: 35-45 parts of traditional Chinese medicine extract, 0.2-0.4 part of yeast extract I, 0.2-0.4 part of yeast extract II and 0.3-0.5 part of garlic extract; the traditional Chinese medicinal materials are obtained by mixing kudzuvine root, kudzuvine flower and chrysanthemum.

The traditional Chinese medicine raw materials used by the invention and the pharmacological activities thereof are as follows:

radix Puerariae is dried root of Pueraria lobata Ohwi of Pueraria of Leguminosae. Mainly contains flavonoids such as daidzin, daidzein, puerarin, etc., daidzein-4, 7-diglucoside, puerarin-7-xyloside, puerarin, isoflavone glycoside and starch. Has the effects of expelling pathogenic factors from muscles, relieving fever, promoting eruption, promoting salivation, quenching thirst, invigorating yang, and relieving diarrhea. Can be used for treating exterior syndrome with fever, strong pain of neck and back, measles without adequate eruption, thirst due to fever, thirst due to yin deficiency, dysentery due to heat-clearing away, and diarrhea due to spleen deficiency.

Flos Puerariae Lobatae is flower of Pueraria lobata (Willd.) Ohwi and Pueraria thomsonii (Willd.) Ohwi of Leguminosae. Mainly contains irisolidone, genistein, biochanin A, pueraria lobata glycosides, triterpenoid saponin and volatile oil. Has effects of relieving alcoholic intoxication, activating spleen, and stopping bleeding, and can be used for treating alcoholic intoxication, dysphoria, thirst, headache, dizziness, abdominal distention, vomiting, acid regurgitation, anorexia, hematemesis, and intestinal wind with hemoptysis.

Flos Chrysanthemi is the dried head-shaped inflorescence of Chrysanthemum morifolium Ramat of Compositae and Chrysanthemum genus. Contains volatile oil, such as Borneolum, Camphora, chrysanthenone, and optionally stevioside, adenine, choline, flavone, stachydrine, vitamin A, vitamin B1, vitamin E, amino acids, and acacetin. Has the effects of dispelling wind and heat, suppressing liver yang, removing liver heat, improving eyesight, and clearing away heat and toxic materials.

The invention comprises the following steps: and (3) kudzuvine flower: the mass ratio of the chrysanthemum is 1-3: 1-3: 2-6 to obtain a traditional Chinese medicine mixture, extracting effective components of the mixture, and finding that a certain synergistic effect exists among active substances in the extract obtained according to the proportion, the scavenging capacity of oxygen free radicals can be remarkably improved, and the better effects of dispelling the effects of alcohol and protecting the liver can be achieved. Kudzu root: and (3) kudzuvine flower: the mass ratio of the chrysanthemum is more preferably 1: 1: 2.

the invention does not limit the specific sources of the kudzu root, the pueraria flower and the chrysanthemum.

In the invention, the preparation method of the traditional Chinese medicine extract comprises the following steps: the traditional Chinese medicinal materials and 60-85% ethanol solution are mixed according to the material-to-liquid ratio of 1: 8-15, soaked for 7-12 hours, and heated and refluxed for 1.5-3 hours to obtain liquid medicine. Wherein the concentration of the ethanol is preferably 80%, the material-liquid ratio is preferably 1: 10-12 g/mL, the soaking time is preferably 8-10 h, and the heating reflux time is preferably 2-2.5 h. The heating reflux operation adopts a conventional heating reflux device, and the heating temperature is 40-60 ℃, and further preferably 50-55 ℃. By the treatment, the flavonoid compounds contained in the traditional Chinese medicine mixture can be effectively extracted, the extraction rate and the flavone content are higher, and meanwhile, the content of impurities in the extract can be reduced, so that the extract is easy to concentrate and dry.

As an optional implementation mode, the traditional Chinese medicine extracting solution is filtered to remove residues, and the liquid medicine obtained by refluxing is directly sieved by a 200-300-mesh sieve to remove residues; as another alternative embodiment, the invention directly adopts vacuum filtration to remove the dregs. The invention does not limit the specific filtering and deslagging mode.

The invention carries out concentration and drying on the obtained traditional Chinese medicine extracting solution. As an optional implementation mode, the obtained liquid medicine is subjected to alcohol recovery treatment, residual liquid is further concentrated, concentrated solution is subjected to vacuum drying to prepare dry paste, and the dry paste is ground into powder and sieved by a 40-60-mesh sieve for later use. The invention does not limit the specific concentration and drying mode of the traditional Chinese medicine extracting solution.

In the present invention, the method for preparing yeast extract I comprises: dry fermentationMother liquor and 0.032-0.066 mol/L of Na₂HPO₄And mixing the solutions according to the feed-liquid ratio of 1: 3-5, extracting for 2.5-4 h, and filtering to obtain a supernatant. Wherein, Na₂HPO₄The concentration of the solution is preferably 0.04-0.06 mol/L, the material-liquid ratio is preferably 1:4g/mL, and the extraction time is preferably 3 h.

The extraction of the invention is as follows: and (3) putting the feed liquid mixture into a water bath at 36-37 ℃ for heat preservation for 2-4 h, and continuously stirring in the heat preservation process, wherein the operation is favorable for the dispersion of yeast in the solution and the transfer of active substances.

The method comprises the steps of screening a mixed extract by a 200-400-mesh sieve, preserving the temperature of a supernatant for 15-20 min at 50-55 ℃, continuously stirring, immediately placing the supernatant in 0 ℃ cold water to cool to room temperature after heat preservation, filtering again at 200-400 meshes (4 ℃), and collecting filtrate. Preferably, the mixed extracts are respectively sieved by a 300-mesh sieve to obtain supernatants. The invention can use a filter screen or a plate filter and other instruments for separating the extracting solution, does not need the specific instruments, and can improve the pharmacological action of the yeast extract I by purifying the active ingredients in the extracting solution in the mode.

The invention freezes the filtered supernatant fluid for 48-72 hours at-20 to-40 ℃, and the dry paste is ground into powder and passes through a 40-60 mesh sieve for later use. The temperature is further preferably-30 ℃ to-35 ℃, the time is further preferably 55-60 h, and the particle size is further preferably 45-55 meshes.

By adopting the preparation method, the yeast extract with higher activity can be obtained, the decomposition of ethanol in a body can be effectively promoted, the concentration of ethanol in blood can be reduced, and the good anti-alcohol effect can be realized after the yeast extract is compatible with other components.

In the present invention, the method for preparing the yeast extract II comprises: mixing dry yeast and water according to a feed-liquid ratio of 1: 8-12, incubating in a water bath at 35-40 ℃ for 15-30 min to obtain a bacterial suspension, centrifuging the bacterial suspension, and taking a precipitate; and (3) suspending the precipitate in a buffer solution according to the mass-to-volume ratio of 1: 3-5, crushing the yeast suspension in an ice-water bath by using a high-speed homogenizer, and filtering to obtain a supernatant after crushing. The material-liquid ratio is preferably 1: 9-10 g/mL, the water temperature is preferably 37-38 ℃, the incubation time is preferably 20-25 min, and the mass-volume ratio is preferably 1:4 g/mL.

The invention carries out centrifugal treatment on the bacterial suspension, and abandons the supernatant and leaves the precipitate. The filtering conditions of the invention are as follows: 80 to 200 mesh, preferably 100 to 150 mesh. As an optional implementation mode, the precipitate obtained by centrifugation can be washed by distilled water for 1-3 times, and then filtered under the condition of 80-200 meshes, and the precipitate is reserved and stored in a refrigerator at 4 ℃ for later use. The invention removes the supernatant by the filtration treatment of the step to obtain a large amount of complete yeast cells.

The present invention resuspends the yeast cell pellet in a buffer. As an alternative embodiment, the buffer of the present invention is Na₂HPO₄-KH₂PO₄，pH 8.0。

The invention utilizes a high-speed homogenizer to crush cell sap, and the crushing conditions are as follows: crushing for 4-5 min at 4 ℃ and 6000-8000 r/min, and then continuously crushing for 6-8 min at 15000-20000 r/min. More preferably, the raw materials are firstly crushed for 4.5min under the condition of 6500-7000 r/min and then are continuously crushed for 7min under the condition of 17000-18000 r/min. The invention breaks the yeast cell through high-speed homogenization treatment, and the yeast cytoplasm matrix component flows out, so as to be convenient for subsequent separation and extraction.

The invention carries out centrifugal treatment on the yeast breaking liquid and collects the supernatant. The filtering condition is 200-400 meshes, and the supernatant is obtained by filtering, and the preferable size is 300-350 meshes. The purpose of filtration here in the present invention is to discard the organelle and nuclear components of the precipitated yeast cells and to collect the cytoplasmic matrix components of the yeast cells.

The invention carries out freeze drying on the supernatant obtained by separation for 48-72 h at the temperature of minus 20 ℃ to minus 40 ℃, and the dry paste is ground into powder and sieved by a sieve of 40-60 meshes for later use. The temperature is further preferably-30 ℃ to-35 ℃, the time is further preferably 55-60 h, and the particle size is further preferably 45-55 meshes.

The yeast extract II can be obtained by adopting the preparation method. The yeast extract II contains a large amount of active ingredients such as intracellular enzymes, so that the yeast extract II has the effects of promoting the decomposition of acetaldehyde in an organism, reducing the damage of acetaldehyde to liver cells, and realizing good effects of relieving alcoholism and protecting liver after being compatible with other components.

In the present invention, the preparation method of the garlic extract comprises: after grinding and crushing the garlic, adding a buffer solution with the volume of 4-6 times that of the garlic, and continuously grinding for 15-30 min; and then heating the mixed solution at 50-60 ℃ for 15-30 min, and filtering with a 200-400-mesh screen to obtain a supernatant. Wherein, the adding volume of the buffer solution is preferably 5 times, the grinding time is preferably 20-25 min, the heating temperature is preferably 55 ℃, the treatment time is preferably 20-25 min, and the filtering pore size is preferably 300 meshes.

The garlic is further ground by adding a buffer solution, and the buffer solution is a phosphate buffer solution (pH7.8) as an alternative embodiment. The invention adds buffer solution to grind, which can dissolve active substance (especially superoxide dismutase) in garlic in buffer solution, and is beneficial to extract subsequent pharmacological active substance.

The invention centrifuges the mixture of garlic and buffer solution after grinding, discards the precipitate and reserves the supernatant fluid containing active ingredients. The filtration conditions are as follows: filtering with 200-400 meshes to obtain supernatant, and further preferably filtering with 300-350 meshes.

The garlic extract obtained by the invention contains abundant active enzymes, micromolecular heteropolysaccharide, flavone, phenolic acid and other substances with pharmacological activity, so that the antioxidant, immunity enhancing and blood fat reducing capabilities of the composition are improved.

The invention carries out freeze drying on the supernatant obtained by separation for 48-72h at the temperature of minus 20 ℃ to minus 40 ℃, and the dry paste is ground into powder and sieved by a sieve of 40-60 meshes for later use. The temperature is further preferably-35 ℃ to-38 ℃, the time is further preferably 50-65 h, and the particle size is further preferably 45-55 meshes.

The preparation method of the hangover-alleviating and liver-protecting composition comprises the following steps: and (3) uniformly mixing the Chinese medicinal material extract, the yeast extract I, the yeast extract II and the garlic extract which are respectively extracted according to the parts by weight. Preferably, the composition comprises the following components in percentage by weight: 38-42 parts of traditional Chinese medicine extract, 0.25-0.35 part of yeast extract I, 0.25-0.35 part of yeast extract II and 0.35-0.45 part of garlic extract; more preferably, 40 parts of traditional Chinese medicine extract, 0.3 part of yeast extract I, 0.3 part of yeast extract II and 0.4 part of garlic extract.

The anti-alcohol and liver-protecting composition can be directly used as a main component in anti-alcohol and liver-protecting food, health-care products or medicines, is used for quickly relieving alcohol effect after drunkenness, and is used for preventing, relieving or treating liver injury caused by alcohol.

The anti-alcoholism and liver-protection composition is used as a main medicinal component, and pharmaceutically acceptable auxiliary materials are added to prepare medicaments, wherein the medicaments comprise but are not limited to tablets, pills and powder. As an optional implementation mode, the effervescent tablet can be prepared, is convenient to carry and take, and has good effects of dispelling the effects of alcohol and protecting liver.

The invention provides an effervescent tablet for relieving alcoholism and protecting liver, which comprises the following components in percentage by mass: 35-45% of traditional Chinese medicine extract, 0.2-0.4% of yeast extract I, 0.2-0.4% of yeast extract II, 0.3-0.5% of garlic extract and the balance of common auxiliary materials for effervescent tablets. The invention does not limit the selection of the specific auxiliary materials.

The invention utilizes the composition for relieving alcoholism and protecting liver to prepare the effervescent tablet for relieving alcoholism and protecting liver. As an optional implementation mode, the traditional Chinese medicine extract, the yeast extract I, the yeast extract II and the garlic extract are weighed and uniformly mixed with auxiliary materials (an acid source, an alkali source, lactose and a sweetening agent), 5-10% of absolute ethyl alcohol PVP solution is used for preparing a soft material, the soft material is granulated through a 10-14-mesh sieve, the soft material is dried in an oven at the temperature of 45-50 ℃ for 50-70 min and taken out, and then the soft material is granulated through a 10-14-mesh sieve and tabletted. The invention does not limit the specific preparation process of the effervescent tablet.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

The animals used in the specific embodiment of the invention are: 70 ICR male mice, SPF grade, 14-16g, purchased from Beijing Wittingle laboratory animal technology, Inc., animal quality certification number: no. 1100112111113775142.

Triglyceride kit (TG, single reagent GPO-PAP method): nanjing was established as a bioengineering institute, lot number: 20210911, respectively; aspartate aminotransferase (AST, microazyme assay): nanjing was established as a bioengineering institute, lot number: 20211117, respectively; alanine aminotransferase (ALT, microazyme assay): nanjing was established as a bioengineering institute, lot number: 20210728, respectively; malondialdehyde test kit (MDA): nanjing was established as a bioengineering institute, lot number: 20210728, respectively; reduced glutathione (GSH, microplate method): nanjing was established as a bioengineering institute, lot number: 20210827, respectively; electron microscope fixative, hundred kilometric biology, lot number: b0012; 4% paraformaldehyde fixing solution, Beijing Soilebao Tech. The specification of the compound liver-benefiting tablet is 0.32g multiplied by 100 tablets, Jiangsu Zhongxing pharmaceutical industry Co., Ltd., Chinese medicine standard: and Z32020664.

In the invention, SPSS 20.0 statistical software is adopted for data processing, the measurement data is represented by mean plus or minus standard deviation, a plurality of groups of samples meet the requirements of normality test and variance homogeneity, variance analysis is adopted, pairwise comparison among the groups is realized by adopting pairing t test, and the difference P less than 0.05 has statistical significance.

In the present invention, denotes P <0.05 as compared to the blank group, denotes P <0.01 as compared to the blank group, and denotes P <0.001 as compared to the blank group; # denotes P <0.05 compared to the model group, # # denotes P <0.01 compared to the model group, # # # denotes P <0.001 compared to the model group.

Example 1

This example orthogonally designed the extraction method of Chinese herbal extracts.

Mixing the radix puerariae, flos puerariae and chrysanthemum mixed powder with an ethanol solution according to the mass ratio of 1:1:2, soaking for 10 hours, and heating and refluxing to obtain a liquid medicine. Wherein the extraction process parameters are subjected to orthogonal experiments according to the table 1, and the optimal extraction method is determined by evaluating the extraction rate of the effective components.

TABLE 1 extraction method orthogonal design sheet

The content of the above effective components in the extract is determined by high performance liquid chromatography with puerarin, tectoridin and rutin as representative components, and the result is shown in Table 2.

TABLE 2 content of effective components (%, w/w) in the extract

As can be seen from table 2, the contents of puerarin, tectoridin and rutin in the traditional Chinese medicine extract extracted by the method described in experiment number 9 are all significantly higher than those in other experiment groups, which indicates that the traditional Chinese medicine extract with higher flavonoid content can be obtained by extracting with 80% ethanol solution for 3 hours in a material-to-liquid ratio of 1:15, and only 1 time of extraction is needed, so the method described in experiment number 9 is an optimal extraction process.

Example 2

The anti-alcohol and liver-protecting composition is prepared in the embodiment.

(1) Preparing a traditional Chinese medicine extract:

kudzu root: and (3) kudzuvine flower: the mass ratio of the chrysanthemum is 1: 1: 2, mixing, adding 80% ethanol solution according to the feed-liquid ratio of 1:15g/mL, soaking for 10h, and heating (55 ℃) and refluxing for 3 h. The obtained liquid medicine is directly screened by a 200-mesh sieve, dregs of a decoction are filtered, and the obtained traditional Chinese medicine extracting solution is concentrated and dried. Grinding the dry paste into powder and sieving with a 40-mesh sieve for later use.

(2) Preparation of yeast extract I:

adding 0.05mol/L Na into dry yeast according to the feed-liquid ratio of 1:4g/mL₂HPO₄Keeping the temperature of the solution in 37 ℃ water bath for 3h, stirring continuously, and extracting at normal temperature for 3h after the temperature is kept. The combined extracts were filtered using a 200 mesh screen. Keeping the temperature of the obtained solution at 55 deg.C for 15min, stirring, immediately cooling in 0 deg.C cold water to room temperature after keeping temperature, filtering again at 400 mesh, freeze drying the obtained solution at-40 deg.C for 48h, grinding the obtained dry extract, and sieving with 40 mesh sieve.

(3) Preparation of yeast extract II:

mixing dry yeast and water according to a feed-liquid ratio of 1:10g/mL, incubating in water bath at 37 ℃ for 20min to obtain a bacterial suspension, filtering the bacterial suspension with a 100-mesh sieve, and discarding the supernatant; washing the precipitate with distilled water for 1 time, filtering again (100 mesh), and keeping the precipitate.

The precipitate was resuspended in 0.05mol/L PBS (Na) at a mass/volume ratio of 1:4g/mL₂HPO₄-KH₂PO₄pH 8.0) buffer solution, stirring the obtained yeast suspension for 4min under 8000r/min in an ice-water bath by a high-speed homogenizer (the crushing is divided into two steps, and then continuously stirring for 8min under 18000 r/min), filtering by using a 400-mesh screen after crushing, and collecting supernatant. Freeze drying the supernatant at-30 deg.C for 72 hr, grinding the dried extract, and sieving with 40 mesh sieve.

(4) Preparing a garlic extract:

grinding and crushing peeled garlic cloves, adding 5 times of 0.05mol/L phosphate buffer (pH7.8) by volume, and continuously grinding for 20 min; then heating the mixed solution at 55 deg.C for 20min, sieving with 400 mesh sieve to obtain supernatant, freeze drying at-30 deg.C for 72h, and pulverizing the obtained dry extract, and sieving with 40 mesh sieve.

(5) Taking 40 parts of the traditional Chinese medicine extract obtained in the step (1), 0.3 part of the yeast extract I obtained in the step (2), 0.3 part of the yeast extract II obtained in the step (3) and 0.4 part of the garlic extract obtained in the step (4) according to parts by weight; mixing uniformly.

Example 3

In the embodiment, the adding proportion of each raw material of the effervescent tablet is optimally designed. Weighing the Chinese medicinal material extract, the yeast extract I, the yeast extract II, the garlic extract and auxiliary materials (acid source, alkali source, lactose and erythritol), uniformly mixing, preparing a soft material by using an absolute ethyl alcohol PVP solution, granulating by using a 14-mesh sieve, drying in an oven at 48 ℃ for 60min, taking out, granulating by using the 14-mesh sieve again, and tabletting.

In the present example, the Chinese herbal medicine extract, the yeast extract I, the yeast extract ii, and the garlic extract were obtained in example 2, respectively. In this example, yeast extract I: and (3) yeast extract II: garlic extract 3:3:4 to give a mixture. The dispersant comprises an acid source and a carbon source according to a proportion, wherein the acid source is citric acid, and the carbon source is sodium bicarbonate.

In this example, the experiment design was optimized according to table 3 for each raw material addition ratio.

TABLE 3 effervescent tablet raw material ratio (%, w/w)

The physicochemical properties of the effervescent tablets obtained in each experimental group were measured according to the following criteria,

(1) and (3) pH value measurement: putting 1 effervescent tablet into a 50mL beaker, adding water with the temperature of 45 ℃ and 20mL, standing, and determining the pH value of the solution after the effervescent tablet is completely disintegrated.

(2) Determination of disintegration time limit: 1 effervescent tablet is put into 200mL 45 deg.C hot water, and the time from the beginning of bubble release to the time when the tablet is completely dispersed and dissolved and the gas in water is released stops.

(3) And (3) gas production rate measurement: the mass of a 100mL beaker (containing 50mL of water at a temperature of 20. + -. 5 ℃) was precisely weighed by a mass loss method. Precisely weighing 1 tablet of effervescent tablet in wine, placing in the beaker until the gas around the tablet stops escaping, completely dissolving or dispersing the tablet in water, and weighing the total mass of the beaker and the solution.

The pH, disintegration time and gas production of the effervescent tablets obtained from each group were tested in parallel for 3 times and the average values were calculated, the results are shown in table 4.

TABLE 4 physicochemical Properties of the effervescent tablets of each group

As can be seen from Table 4, the effervescent tablets obtained in experiment group 7 had an average disintegration time of 76s, a gas production rate of 0.051g, a pH of 5.45, and a better overall result than those obtained in the other experiment groups. Therefore, the related properties of the anti-alcohol and liver-protecting effervescent tablet prepared according to the proportion of 41 percent of the anti-alcohol and liver-protecting composition (Chinese medicinal material extract: mixture: 40:1), 45 percent of dispersant (citric acid: sodium bicarbonate 1:1.5), 5 percent of PVP, 2 percent of erythritol and 7 percent of lactose are the optimal combination.

Example 4

In this embodiment, the Chinese medicinal composition obtained in example 2 is used to prepare an effervescent tablet for alleviating hangover and protecting liver.

The effervescent tablet comprises the following raw materials in percentage by weight: 41% of a composition for relieving alcoholism and protecting liver, 45% of a dispersant, 5% of PVP, 2% of erythritol and 7% of lactose. The dispersing agent is formed by mixing citric acid and sodium bicarbonate according to the mass ratio of 1: 1.5.

Respectively weighing the anti-alcohol and liver-protecting composition, the dispersing agent, the erythritol and the lactose according to the proportion, uniformly mixing, preparing a soft material by using an absolute ethyl alcohol PVP solution, and granulating through a 14-mesh sieve. And (3) drying the soft material particles in an oven at 50 ℃ for 60min, taking out, sieving with a 14-mesh sieve, grading, and directly tabletting in a tabletting machine to obtain the soft material.

Example 5

The experimental animals were randomly divided into 7 groups, which were blank control group, model group, compound YIGANLING tablet group, effervescent tablet No. 1 dosage group, effervescent tablet No. 2 dosage group, effervescent tablet No. 3 dosage group, and effervescent tablet No. 4 dosage group.

The mice of each group were administered with the gastric lavage drug at 8 am on each experimental day, wherein the experimental animals of the blank control group and the model group were administered with gastric lavage purified water according to the administration volume of 15 ml/kg; the positive medicine is perfused according to the administration volume of 15ml/kg, and the administration dosage of the corresponding compound liver benefiting tablet is 0.52g/kg (body weight); the dosage groups of effervescent tablet No. 1, No. 2, No. 3 and No. 4 were separately administered in a volume of 15ml/kg by intragastric administration, and the dosage was 1.0g/kg (body weight), 2.0g/kg (body weight), 4.0g/kg (body weight) and 8.0g/kg (body weight). Each group was gavaged for 28 consecutive days. On the 29 th day, except for the blank group, the animals of other experimental groups were subjected to intragastric alcohol molding by a specific method of 14 th day with 20mL/kg of intragastric 53-degree white spirit, 20 th day with 15mL/kg of intragastric 53-degree white spirit, after 12h intervals, that is, 30 th day of the experiment, 8 th morning, mice were weighed, eyeballs were picked and blood was collected in a 2mL polyethylene centrifuge tube, liver tissues were taken and weighed.

1. Measurement of mouse organ index

All mice were fasted at 8 pm, 12h the day before drawing material and weighed the following day. The whole liver is taken out after the blood is taken out from the eyeball, after the liver is cleaned by normal saline, the fresh weight of the liver is weighed after the liver is sucked dry by using filter paper, and the liver index of the liver is calculated, and the result is shown in table 5.

Liver index ═ liver fresh weight (g)/body weight (g) × 100%

TABLE 5 mouse liver index

It can be seen from table 5 that alcohol molding significantly increased the liver index of the experimental animals, which is consistent with the side effects of alcohol in practice.

2. Determination of mouse serum glutamic-pyruvic transaminase (AST) and serum glutamic-oxaloacetic transaminase (ALT) activities

Placing the collected fresh blood of the mouse in a constant temperature box at 37 ℃ for 2h, centrifuging (3000rpm, l0min), separating serum, and respectively measuring the absorbance (OD value) of each hole at the wavelength of 510nm and the wavelength of 505nm of an enzyme labeling instrument according to the operation of a corresponding kit using instruction so as to obtain corresponding AST and ALT activity data.

AST and ALT activity assays in mice are shown in FIG. 1. As can be seen from fig. 1, the activities of AST and ALT in the serum of the model group were significantly increased compared to the blank group, and it can be seen that the model creation method using alcohol gavage actually caused clear liver injury to the experimental animals. When the compound liver-benefiting tablet and the effervescent tablet are respectively used for treatment at different dosages, the activities of two transaminases are inhibited to a certain degree, and the results prove that the effervescent tablet has a certain protection effect on liver injury caused by alcohol.

3. Determination of protein content in mouse liver tissue

Accurately weighing a certain mass of liver tissue, adding 9 times of volume of physiological saline according to the weight (g) volume (mL) ratio of 1:9, mechanically homogenizing under ice water condition to prepare 10% tissue homogenate, 2500 rpm, centrifuging for 10min, taking supernatant, diluting by 30 times, mixing the diluted tissue homogenate with working solution according to the instruction in a kit, adding the mixture into a pore plate, setting a blank pore and a standard pore, measuring the wavelength at 562nm, carrying out color comparison by an enzyme-labeling instrument, and reading the absorbance. The total protein concentration of the sample was calculated according to the following formula:

protein concentration (μ g/mL) ═ measurement OD value-blank OD value)/(standard OD value-blank OD value) × standard concentration × dilution multiple before sample test

The standard samples were measured according to the instructions and a standard curve was constructed, the function relationship between the UV absorbance (Y) and the protein concentration (C), Y, is 0.7509C +0.0928 (R)²＝0.9954)。

4. (TG) determination of triglyceride content in mouse liver tissue

The liver homogenate was collected and the OD in each well was measured at 510nm wavelength of a microplate reader according to the instructions of the kit, and the results are shown in FIG. 2.

Triglyceride content (mmol/L) × (determination OD-blank OD)/(standard OD-blank OD) × standard concentration/protein concentration of sample to be measured

As shown in fig. 2, the triglyceride content of the animals in the model building group and each treatment group is obviously higher than that of the blank group, and the experimental animal model of alcoholic liver injury can be proved to be obtained in the research. After 28 days of preventive treatment by using different dosages of the compound liver-benefiting tablets and the effervescent tablets, the content of TG in each group is reduced compared with that in an untreated model group, and the effervescent tablets have a certain protection effect on alcoholic liver injury.

5. (GSH) determination of reduced glutathione content in mouse liver tissue

The liver homogenate was collected and the OD in each well was measured at 405nm wavelength of a microplate reader according to the instructions of the kit, and the results are shown in FIG. 3.

GSH content (nmol/gprot) in the tissue (measured OD value-blank OD value)/(standard OD value-blank OD value) × standard concentration × concentration of sample protein to be measured

As shown in fig. 3, the GSH content was decreased in each experimental group compared to the blank group due to liver damage caused by alcohol modeling. Modeling is carried out 30 days after preventive treatment, and the content of GSH in each treatment group is increased compared with that in a model group, and the content of GSH is also increased along with the increase of the administration dosage of the effervescent tablets.

6. (MDA) determination of malondialdehyde content in liver tissue of mouse

The liver homogenate was collected and the OD value of each well was measured at a wavelength of 532nm in a microplate reader according to the instructions of the kit, and the results are shown in FIG. 4.

Tissue MDA content (nmol/gprot) ═(determination OD-blank OD)/(standard OD-blank OD) × standard concentration/protein concentration of sample to be measured

As shown in FIG. 4, the MDA content of each group was increased after the alcohol gavage. After the compound liver-benefiting tablet and the effervescent tablets with different dosages are used for treatment, the MDA content is reduced relative to a model group, the liver injury caused by alcohol can be relieved by adopting a prevention and treatment method, the MDA content reduction effect of the effervescent tablet No. 2 dosage group is most obvious, and the data has significant difference compared with the model group.

7. Pathological histological examination

Respectively taking a part of tissues of the left lobe of the liver, preparing slices according to different requirements, carrying out HE staining and oil red staining, and observing by using an optical microscope, wherein the HE staining result is shown in a figure 5 and a figure 6; the results of oil red staining are shown in FIGS. 7 and 8.

(1) HE staining: a part of liver left lobe tissue was cut into small pieces of 1cm by 1cm, and fixed in 4% paraformaldehyde. Three days later, the liver tissue was removed for sampling and dehydration: flushing with running water, alcohol overnight, alcohol-alcohol putting the dehydrated tissue into xylene, and observing the transparency of the tissue by the xylene while taking care; putting the transparent tissue into melted paraffin and then putting the tissue into the paraffin; pouring the dissolved paraffin into an embedding frame, sequentially placing the tissue blocks into the embedding frame by using heated forceps, and marking; after cooling, pieces were trimmed. Slicing in a slicing machine, spreading the slide with warm water, fishing the clean slide, and carrying out film collection. And (5) putting the slices with the adhered tissues into an oven for baking for about 4 hours.

1) Paraffin section dewaxing to water: placing the slices into xylene I20 min-xylene II 20 min-absolute ethyl alcohol I10 min-absolute ethyl alcohol II 10 min-95% alcohol 5 min-90% alcohol 5 min-80% alcohol 5 min-70% alcohol 5 min-distilled water washing in sequence.

2) Hematoxylin staining nuclei: slicing into Harris hematoxylin, staining for 3-8min, washing with tap water, differentiating with 1% hydrochloric acid alcohol for several seconds, washing with tap water, returning blue with 0.6% ammonia water, and washing with running water.

3) Eosin staining of cytoplasm: the sections were stained in eosin stain for 1-3 min.

4) Dewatering and sealing: placing the slices in 95% alcohol I5 min-95% alcohol II 5 min-absolute ethanol I5 min-absolute ethanol II 5 min-xylene I5 min-xylene II 5min to dehydrate and transparent in sequence, taking out the slices from xylene, air drying, and sealing with neutral gum.

As can be seen from the HE staining of liver tissues shown in FIGS. 5 and 6, the blank group of hepatocytes had a complete morphology and the liver cords were aligned. The model group can see obvious vacuole of liver cells, disorder of liver cords, diffuse water sample degeneration, fuzzy cell nucleus and invisible irregular eosinophilic block-shaped substance deposition (Mallory-Denk corpuscle). The swelling of cells in each group treated with the drug was reduced, the water-like degeneration was reduced and the cells were arranged relatively orderly. The compound liver-benefiting tablet group and the effervescent tablet have obviously lower dosage groups and pathological changes than the model group and the effervescent tablet low dosage group.

(2) Oil red dyeing

1) Freezing the slices and drying at normal temperature;

2) saturated oil red O dye liquor A: pure water 3: 2 preparing oil red working solution, and filtering for later use.

3) And (3) slicing the slices into the oil red O working solution prepared in the step 2), dyeing for 7-10min (in dark place), and preparing the slices for use.

4) Slicing into differentiation liquid B for differentiation for 5-10 s.

5) The solution was washed with distilled water 2 times.

6) Staining the slices with hematoxylin staining solution C for 2-4min, washing with distilled water for 2 times, differentiating with hematoxylin differentiation solution D for 2-3S, washing with distilled water, returning hematoxylin to blue solution E for 2-3S, washing with tap water, and performing microscopic examination.

7) And (5) sealing by using glycerol gelatin.

From fig. 7 and fig. 8, it can be seen that steatosis in the model group is obvious, dense round fat droplets can be seen under the microscope, and fat droplets also exist in liver tissues of other groups of mice, but after 28 days of dryness, the fat droplets in the liver tissues are reduced compared with the model group by the compound liver benefiting tablet group and the effervescent tablet different dosage group, particularly the fat droplets in the dosage group of the effervescent tablet are obviously reduced, while the fat droplets in the effervescent tablet low dosage group are not obviously changed.

8. Morphological observation of liver tissue cytology

The left lobe part tissue of the liver of each group of mice is taken, the size of the grain of rice is about, and is fixed by glutaraldehyde precooled at 4 ℃ and 0.1% mol/L phosphate buffer. 2 days after the block, about 1mm x 1mm, in 0.01M PBS washing 3 times, each time 15 min-1% osmic acid fixed 2h-0.01M PBS washing, each time 15 min-ethanol gradient dehydration-100% acetone 15min, 2 times-acetone: the resin V/V is 1:1, 1 h-acetone: the resin V/V is 1:2, 2 h-pure resin 2h embedding. The embedded resin block is polished and sliced, and then the uranium monoxide and lead citrate are dyed and examined by a microscope.

The intervention effect of the effervescent tablet on acute alcoholic liver injury is observed from two angles of cell nucleus and mitochondria micro-morphology. It can be seen from FIGS. 9 and 10 that the blank control group had smooth cell contour, round and regular nucleus, and clear nucleolus; the cell contour of the model group is irregular, the cell nucleus is shriveled, the nucleolus is fuzzy, and a large amount of vacuoles exist in the plasma; after the medicine is used for treatment, the cell morphology of the compound liver-benefiting tablet group and the effervescent tablet group with dosage No. 2-4 is improved and changed, bubbles in plasma are reduced, nuclei become round, and nucleolus is relatively clear. As can be seen from fig. 11, the blank control group mitochondria had a rounded profile, with a clear abruptness; the model group has the advantages that the mitochondrial cristae is swollen, the outline disappears, and floccules appear in cytoplasm; however, after treatment, the phenomenon is relieved, and particularly, the improvement of the effervescent tablet No. 3 and No. 4 dosage groups is relatively remarkable, although the mitochondria are slightly swollen, the outline is smooth, and the ridge structure is clear.

The result shows that the effervescent tablet has a certain protection effect on the phenomena of liver tissue lesion, fat accumulation and the like caused by alcoholic liver injury, and can effectively reduce liver tissue vacuole and reduce fat deposition in liver cells.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. The composition for relieving alcoholism and protecting liver is characterized by comprising the following components in parts by weight:

the traditional Chinese medicinal materials comprise the following components in parts by weight: and (3) kudzuvine flower: the mass ratio of the chrysanthemum is 1-3: 1-3: 2-6, and mixing.

2. The composition as claimed in claim 1, wherein the preparation method of the extract of Chinese herbs comprises: mixing the traditional Chinese medicinal materials with 60-85% ethanol solution according to the material-liquid ratio of 1: 8-15, soaking for 7-12 h, heating and refluxing for 1.5-3 h to obtain liquid medicine, concentrating and drying.

3. The composition of claim 1, wherein the yeast extract I is prepared by a method comprising: dried yeast and 0.032-0.066 mol/L Na₂HPO₄Mixing the solutions according to the material-liquid ratio of 1: 3-5, extracting for 2.5-4 h, filtering with 200-400 meshes to obtain a supernatant, and freeze-drying.

4. The composition of claim 1, wherein the yeast extract ii is prepared by a method comprising: mixing dry yeast and water according to a material-liquid ratio of 1: 8-12, incubating in a water bath at 35-40 ℃ for 15-30 min to obtain a bacterial suspension, filtering the bacterial suspension with a sieve of 80-200 meshes, and taking a precipitate; and (3) suspending the precipitate in a buffer solution according to the mass volume ratio of 1: 3-5, homogenizing and crushing the yeast at a high speed, centrifuging the crushed yeast to obtain a supernatant, and freeze-drying the supernatant at the temperature of-20 to-40 ℃ for 48-72 hours.

5. The composition according to claim 4, characterized in that the crushing conditions are: stirring for 4-5 min under the condition of 6000-8000 r/min, and then continuously stirring for 6-8 min under the condition of 15000-20000 r/min.

6. The composition according to claim 1, wherein the garlic extract is prepared by a method comprising: after primary grinding and crushing of garlic, adding a buffer solution with the volume of 4-6 times that of the garlic, and continuing grinding for 15-30 min; and then heating the mixed solution at 50-60 ℃ for 15-30 min, filtering with a 200-400 mesh screen to obtain a supernatant, wherein the supernatant is a garlic extract, and freeze-drying the garlic extract at-20-40 ℃ for 48-72 h.

7. The use of the composition according to any one of claims 1 to 6 in the preparation of a food or health product or medicament for alleviating hangover and protecting liver.

8. Use of a composition according to any one of claims 1 to 6 for the preparation of a medicament for the treatment or prevention of liver damage, wherein the liver damage is caused by alcohol.

9. The use of claim 7 or 8, wherein the composition is formulated into a medicament comprising a tablet, a pill, a powder, with the addition of a pharmaceutically acceptable excipient.

10. An effervescent tablet for relieving alcoholism and protecting liver, which is characterized by comprising the composition of any one of claims 1 to 6 and pharmaceutically acceptable auxiliary materials.