Traditional Chinese medicine composition for dispelling effects of alcohol and relieving alcoholic liver injury and application of traditional Chinese medicine composition

Abstract

The invention belongs to the field of medicines, and relates to a traditional Chinese medicine composition for dispelling the effects of alcohol and relieving alcoholic liver injury and application thereof. The pharmaceutical composition is prepared from the following raw materials: curcuma zedoary, fructus amomi, rhizoma alismatis, rhizoma atractylodis macrocephalae and radix angelicae.

Description

Technical Field

[0001] The invention belongs to the field of medicine and relates to a traditional Chinese medicine composition for sobering up and relieving alcoholic liver damage and application thereof. Background Art

[0002] my country is the world's oldest country to practice winemaking. As a unique cultural medium, wine has become an indispensable part of people's lives. With the continuous development of the times, drinking has become more frequent and younger. Moderate drinking can dispel dampness and prevent cold, relax muscles and activate blood circulation, strengthen the spleen and appetite, and promote sleep. However, excessive drinking can cause nausea and vomiting, and in severe cases, even death due to respiratory muscle paralysis. Long-term drinking can lead to diseases such as fatty liver, alcoholic hepatitis, and cirrhosis. Therefore, there is a strong demand for drugs or health products that can relieve alcohol and protect the liver.

[0003] According to Traditional Chinese Medicine, the core pathogenesis of various symptoms after drinking alcohol can be summarized as "accumulation of damp-heat, disharmony of the internal organs, and imbalance of qi, blood, and body fluids." Alcohol is a pungent and hot food with a strong, ascending, drying nature. Upon entering the stomach, it easily generates damp-heat, primarily affecting the spleen and stomach, which obstructs the ascending and descending flow of qi. Stagnation of damp-heat in the spleen and stomach impairs its transport and transformation functions, causing abdominal distension, nausea, vomiting, and loss of appetite. Damp-heat then rises to the head and face, resulting in a heaviness of the head and flushed face and eyes. Furthermore, alcohol's pervasive properties disrupt the liver and gallbladder, causing liver dysfunction and qi stagnation to transform into fire, resulting in flank pain, irritability, headaches, and dizziness. Liver fire, acting recklessly, invades the stomach, exacerbating upward flow of stomach qi and causing belching, heartburn, and increased vomiting. Furthermore, the heat from alcohol depletes body fluids, leading to dry mouth, oliguria, and yin fluid deficiency. It can also disrupt qi and blood, causing excessive blood heat and restlessness. For those who drink alcohol for a long time, the damp heat may cause phlegm to turn into blood stasis, damage the liver and spleen, and even affect the kidney yin, resulting in a mixed syndrome of deficiency and excess.

[0004] Gehua Jiedun Decoction is a classic Chinese medicine formula for hangover relief, originating from Li Dongyuan's Treatise on the Spleen and Stomach during the Yuan Dynasty. It is specifically designed for conditions caused by alcohol accumulation damaging the spleen or damp-heat in the middle. The recipe consists of 13 herbs: kudzu flower, green peel, costus root, tangerine peel, ginseng, umbellate polypore, poria cocos, Shenqu, oriental Chinese medicine, dried ginger, white atractylodes macrocephala, white cardamom, and amomum villosum. Kudzu flower, as the main ingredient, has the function of sobering up and invigorating the spleen; ginseng and white atractylodes macrocephala strengthen the spleen and qi and protect the spleen and stomach; umbellate polypore, poria cocos, and oriental Chinese medicine promote diuresis and eliminate dampness; and white cardamom and amomum villosum eliminate dampness, promote qi circulation, and soothe the stomach and relieve vomiting. Functional analysis of the individual herbs revealed overlapping activities in some, increasing the metabolic burden on the body. Furthermore, some herbs may only play a supporting role. Commonly used alcohol-relief drugs, such as metadoxine and naloxone, also have side effects such as tachycardia and increased blood pressure, and should not be taken long-term or used as preventive medications. Therefore, it is necessary to develop a traditional Chinese medicine composition that has a significant effect in sobering up, relieving the symptoms of "damp-heat accumulation, viscera disharmony, and imbalance of qi, blood, and body fluids" and protecting the liver.

[0005] Curcuma elata (Roxb.), a plant of the genus Curcuma in the Zingiberaceae family, is primarily found in Southeast Asia, including Thailand, Myanmar, Laos, and Yunnan, China. It's used by the general public as a hangover remedy, with remarkable success. Hangover headaches are common symptoms of excessive drinking. Traditional Chinese Medicine believes that excessive drinking can lead to internal accumulation of dampness and heat or disordered qi and blood. Angelica dahurica (Baizhi) is pungent and warm in nature, entering the lung and stomach meridians. It has the effects of dispelling wind, relieving pain, and dispelling cold. Its pungent and warm properties may help dissipate dampness, clear the meridians, and relieve pain.

[0006] The present invention applies modern scientific theories to explain and demonstrate ancient Chinese wisdom. Based on the binding scores of the active ingredients of each herb in Curcuma zedoaria, Angelica dahurica, and the classic formula Gehua Jiezhu Decoction with targets related to acute alcohol poisoning, key components are screened for combination, providing new ideas for the treatment of acute alcohol poisoning with traditional Chinese medicine. Summary of the Invention

[0007] The purpose of the present invention is to provide a traditional Chinese medicine composition for sobering up and alleviating alcoholic liver damage.

[0008] The pharmaceutical composition of the present invention is prepared from the following raw materials in parts by weight:

[0009] 1-4 parts of Curcuma zedoaria, 0.5-2 parts of Amomum villosum, 0.25-1 parts of Alisma orientalis, 0.25-1 parts of Atractylodes macrocephala, and 0.25-1 parts of Angelica dahurica.

[0010] Preferably, the pharmaceutical composition of the present invention is processed from the following raw materials:

[0011] 4 parts of Curcuma zedoaria, 2 parts of Amomum villosum, 1 part of Alisma orientalis, 1 part of Atractylodes macrocephala, and 1 part of Angelica dahurica.

[0012] Another object of the present invention is to provide a method for preparing the Chinese medicine composition.

[0013] The preparation method of the present invention comprises the following steps:

[0014] (1) Medicinal material grinding: Place each medicinal material in a wall-breaking machine and grind it thoroughly, then filter it through a 200-mesh sieve;

[0015] (2) Preparation of suspension: Weigh the corresponding dosage components after sieving, mix them and add appropriate amount of distilled water to prepare the Chinese medicine compound suspension.

[0016] Another object of the present invention is to provide a use of the Chinese medicine composition in the preparation of a medicament for sobering up and alleviating alcoholic liver damage.

[0017] The applications of the present invention include: prolonging alcohol tolerance time, shortening drunkenness time, and significantly accelerating the plasma ethanol clearance rate in acute alcohol poisoning; the Chinese herbal compound significantly increases the activity of liver alcohol-degrading enzymes, such as alcohol dehydrogenase ADH, acetaldehyde dehydrogenase ALDH, cytochrome P450 family member CYP2E1, and catalase CAT, accelerating ethanol metabolism; the Chinese herbal compound significantly reduces the levels of alanine aminotransferase ALT, aspartate aminotransferase AST, and gamma-glutamyl transferase gamma-GT in serum, indicating that the Chinese herbal compound alleviates alcohol-induced liver damage; the Chinese herbal compound has no significant effect on the expression level of serum cystatin Cys-C, indicating that the Chinese herbal compound has no kidney toxicity; the Chinese herbal compound can reduce the production of lipofuscin in the liver of mice with acute alcohol poisoning, and has no significant effect on the tissue structure of the liver and kidneys.

[0018] The pharmaceutical composition of the present invention further contains a pharmaceutically acceptable carrier.

[0019] The pharmaceutical preparation composition of the present invention, its oral administration preparation may contain auxiliary materials, such as binders, fillers, diluents, tableting agents, lubricants, disintegrants, colorants, flavoring agents and wetting agents, and the tablets may be coated if necessary.

[0020] Suitable fillers include cellulose, mannitol, lactose, and other similar fillers. Suitable disintegrants include starch, polyvinylpyrrolidone, and starch derivatives, such as sodium starch glycolate. Suitable lubricants include, for example, magnesium stearate. Suitable pharmaceutically acceptable wetting agents include sodium lauryl sulfate.

[0021] The pharmaceutical preparations of the present invention can be prepared into solid oral compositions by conventional methods such as mixing, filling, tableting, etc. Repeated mixing can distribute the active substance throughout those compositions using a large amount of fillers.

[0022] Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be dry products that can be reconstituted with water or other suitable vehicles before use. Such liquid preparations may contain conventional additives such as suspending agents, for example, sorbitol, syrup, methylcellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel or hydrogenated edible fats, emulsifiers, for example, lecithin, sorbitan monooleate or gum arabic; non-aqueous vehicles (which may include edible oils), for example, almond oil, fractionated coconut oil, oily esters such as glycerol esters, propylene glycol or ethanol; preservatives, for example, methylparaben or propylparaben or sorbic acid, and, if desired, conventional flavorings or coloring agents.

[0023] When the pharmaceutical composition of the present invention is prepared as a solid pharmaceutical preparation in the form of a powder, tablet, dispersible powder, capsule, or cachet, a solid carrier may be used. The solid carrier is preferably one or more substances selected from diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, and bulking agents, or may be an encapsulating material. Suitable solid carriers include magnesium carbonate, magnesium stearate, talc, sucrose, lactose, pectin, dextrin, starch, gelatin, methylcellulose, sodium carboxymethylcellulose, and cocoa butter. Tablets, powders, cachets, and capsules represent the most advantageous oral solid preparations due to their ease of administration.

[0024] The pharmaceutical composition of the present invention may be in any edible dosage form.

[0025] The preparation may be in the form of tablets, capsules, pills, oral solutions, lozenges, granules, pills, powders, pastes, pills, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, drops and patches.

[0026] The present invention applies modern scientific theories to explain and demonstrate ancient Chinese wisdom. Based on the traditional Chinese medicine theory of "clearing away heat and dampness to dispel pathogenic factors, harmonizing the liver and stomach to restore the Qi mechanism, nourishing yin and cooling the blood to strengthen the body", the key components of Curcuma zedoaria, Angelica dahurica and Pueraria lobata hangover-relieving decoction are selected and combined to relieve the symptoms of acute alcohol poisoning such as "accumulation of dampness and heat, disharmony of internal organs, and imbalance of Qi, blood and body fluids".

[0027] The Chinese herbal composition of the present invention has the effects of sobering up and protecting the liver. It can prolong the alcohol tolerance time of mice, shorten the time of intoxication, and significantly accelerate the plasma ethanol clearance rate of mice with acute alcohol poisoning. The Chinese herbal compound significantly increases the activity of liver alcohol-detoxifying enzymes, such as alcohol dehydrogenase ADH, acetaldehyde dehydrogenase ALDH, cytochrome P450 family member CYP2E1, and catalase CAT, accelerating ethanol metabolism. The Chinese herbal compound significantly reduces the levels of alanine aminotransferase ALT, aspartate aminotransferase AST, and γ-glutamyl transferase γ-GT in the serum, indicating that the Chinese herbal compound alleviates liver damage caused by acute alcohol poisoning; the Chinese herbal compound has no significant effect on the expression level of serum cystatin Cys-C, indicating that the Chinese herbal compound has no renal toxicity. The Chinese herbal compound can reduce the production of lipofuscin in the liver of mice with acute alcohol poisoning, without significantly affecting the tissue structure of the liver and kidneys. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1.Screening of key components in Da Ze Zhu, Bai Zhi and Ge Hua Jie Zhu DecoctionA. Intersection of potential therapeutic targets of Da Ze Zhu, Bai Zhi and Ge Hua Jie Zhu Decoction and genes related to acute alcohol poisoningB. Drug component-target networkC. Ranking of active ingredients by degree valueDrug: Da Ze Zhu, Bai Zhi and Ge Hua Jie Zhu DecoctionAAP: Acute alcohol poisoningE: Da Ze ZhuBZ: Bai ZhuZ: Alisma B: Bai ZhiS: Amomum villosum.

[0029] Figure 2 Effect of Chinese herbal compound on hangover reliefA. Alcohol tolerance timeB. Drunkenness timeC. Time in the barD. Plasma ethanol concentration*P<0.05, **P<0.01.

[0030] Figure 3 ELISA assay to detect the activities of liver alcohol-degrading enzymes: A. Alcohol dehydrogenase (ADH) activity B. Aldehyde dehydrogenase (ALDH) activity C. Cytochrome P450 family member CYP2E1 enzyme concentration D. Catalase (CAT) activity *P < 0.05, **P < 0.01, ***P < 0.001.

[0031] Figure 4 .ELISA experiment was used to detect the effects of high-dose Chinese herbal compound on serum liver injury markers. A. Alanine aminotransferase (ALT) activity B. Aspartate aminotransferase (AST) activity C. γ-glutamyl transferase (γ-GT) activity *P < 0.05, **P < 0.01, ***P < 0.001.

[0032] Figure 5 HE staining to evaluate the effects of high-dose Chinese herbal compound on liver and kidney tissues in mice. A. HE staining of liver tissue. B. HE staining of kidney tissue.

[0033] Figure 6 Effects of high-dose Chinese herbal compound on lipofuscin deposition in mouse liver tissue A. HE staining of liver tissue B. Lipofuscin expression level in liver tissue DETAILED DESCRIPTION

[0034] The present invention is further illustrated by the following specific examples, but is not intended to be limiting of the present invention.

[0035] Example 1. Screening of key components in Curcuma zedoaria, Angelica dahurica and Pueraria lobata decoction for treating acute alcoholism

[0036] To identify key components of Dazezhu, Baizhi, and Gehua Jiezhu decoction for the treatment of acute alcoholism, a network pharmacology approach was used for systematic analysis. The main components of each of these herbs were retrieved using the TCMSP database (https: / / old.tcmsp-e.com). Potentially active components were identified based on the bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18. The Swiss Target Prediction database (http: / / swisstargetprediction.ch / ) was used to predict the potential therapeutic targets of these active components. The potential therapeutic targets of these active components were further evaluated based on pharmacokinetics (GI absorption: high) and drug-likeness (Lipinski's rule of five and Ghose's law).

[0037] The key words "acute alcohol poisoning" were used to screen genes related to acute alcohol poisoning in the Genecards database (http: / / www.genecards.org), OMIM database (http: / / omim.org / ), and DisGeNET database (http: / / www.disgenet.org / web / DisGeNET / ). Using the Venny 2.1 tool, the intersection of potential therapeutic targets of Da Ze Zhu, Bai Zhi, and Ge Hua Jie Zhu Tang with genes related to acute alcohol poisoning was constructed, and the key genes in the treatment of acute alcohol poisoning with Da Ze Zhu, Bai Zhi, and Ge Hua Jie Zhu Tang were identified ( Figure 1 A).

[0038] Cytoscape was used to construct a drug component-target network diagram based on the intersection genes corresponding to the active ingredients in Da Zezhu, Baizhi and Gehua Jiezhong Decoction ( Figure 1 B) The top ten active ingredients with great potential for the treatment of acute alcohol poisoning were screened based on the degree value. The results showed that these active ingredients were mainly concentrated in Curcuma zedoaria, Rhizoma alismatis, Fructus amomi, Atractylodes macrocephala and Angelica dahurica ( Figure 1 C), therefore, the present invention combines Rhizoma Curcumae, Rhizoma Alismatis, Fructus Amomi, Rhizoma Atractylodis Macrocephalae and Angelica Dahurica as a traditional Chinese medicine composition to conduct subsequent animal experiments to verify its efficacy.

[0039] Example 2. Construction of acute alcohol intoxication model and drug administration

[0040] Establishment of a mouse model of acute alcohol intoxication

[0041] Eight-week-old male Kunming mice were purchased from the Animal Center of the Second Affiliated Hospital of Harbin Medical University. The mice were gavaged with 56% alcohol liquor at different doses (0.1 mL / 10 g, 0.15 mL / 10 g, 0.17 mL / 10 g, 0.2 mL / 10 g, and 0.25 mL / 10 g). The results showed that mice gavaged with a dose of 0.17 mL / 10 g lost their righting reflex within 1 hour, and the mortality rate of this group of mice was 0, indicating that the model was successfully established. Therefore, 0.17 mL / 10 g was determined to be the standard dose for establishing the model.

[0042] Grouping and dosing

[0043] The experiment was divided into seven groups: a normal control group, an acute alcohol intoxication group, a positive control Gehua Jiezhu decoction group, a Curcuma zedoaria group, a low-dose Chinese herbal compound treatment group, a medium-dose Chinese herbal compound treatment group, and a high-dose Chinese herbal compound treatment group. Each group consisted of 10 mice, housed in two cages, with five mice per cage.

[0044] The recipe for Kudzu Flower Hangover-Relief Decoction consists of 2 parts (7.46g) of Kudzu Flower, 2 parts (7.46g) of Amomum villosum, 2 parts (7.46g) of White Cardamom, 1 part (3.73g) of Aucklandia lappa, 1 part (3.73g) of Polyporus umbellatus, 1 part (3.73g) of Shenqu (Shenqu), 1 part (3.73g) of Alisma Orientalis, 1 part (3.73g) of Dried Ginger, 1 part (3.73g) of Atractylodes macrocephala, 0.75 parts (2.80g) of Citrus aurantium, 0.75 parts (2.80g) of Citrus reticulatae, 0.75 parts (2.80g) of Ginseng, and 0.75 parts (2.80g) of Poria. A water decoction was prepared according to the original recipe (crude drug concentration of 1g / mL), and mice were administered a dose of 0.4g of crude drug per 10g body weight.

[0045] To ensure the safety of medication, the dosage of the Chinese medicine compound administration group is configured with reference to the dosage of Gehua Jiezhu Decoction, wherein, Amomum villosum, Rhizoma Alismatis, and Atractylodes macrocephala are used as adjuvants and are configured with reference to the dosage of Gehua Jiezhu Decoction; Curcuma zedoaria is used as the main drug, and the dosage of reference Pueraria lobata is set to 2 parts; Angelica dahurica is used as an adjuvant, and the dosage is 0.5 parts. The configuration of the Chinese medicine compound of the present invention is 2 parts (7.46g) of Curcuma zedoaria, 1 part (3.73g) of Amomum villosum, 0.5 parts (1.865g) of Rhizoma Alismatis, 0.5 parts (1.865g) of Atractylodes macrocephala, and 0.5 parts (1.865g) of Angelica dahurica. The Chinese medicine compound is converted into a dosage according to the ratio of human and mouse body surface area, and the conversion coefficient of adults and mice is 9.1. According to a 70kg adult's daily medication, the mouse dosage after conversion is 2.18g / kg. The dosage of the low-dose, medium-dose, and high-dose compound groups was set to 1.09 g / kg, 2.18 g / kg, and 4.36 g / kg, respectively. The preparation method was as follows: (1) Medicinal material crushing: Each medicinal material was placed in a wall-breaking machine and fully crushed, and then filtered through a 200-mesh sieve. (2) Suspension preparation: The corresponding dosage components after sieving were weighed separately, mixed, and 4.82 mL of distilled water was added to prepare a Chinese medicine compound suspension. 1 hour before modeling, the corresponding suspension was administered to mice in the low-dose, medium-dose, and high-dose Chinese medicine compound groups by gavage, with a suspension volume of 0.01 mL / g.

[0046] The Curcuma zedoaria group received the same dose of the medium-dose compound group (0.97 g / kg). Preparation of the Curcuma zedoaria suspension: Weigh the sieved Curcuma zedoaria and add distilled water to mix thoroughly. One hour before modeling, administer the suspension to mice via oral gavage at a volume of 0.01 mL / g.

[0047] Blood sample collection

[0048] During the experiment, blood was collected from the mice at 1, 3, 5, 7, and 9 hours after alcohol administration. The blood was placed in a 1.5 mL EP tube and allowed to stand at room temperature for 1 hour until it naturally coagulated. The tube was centrifuged at 4000 rpm for 10 minutes, and the serum was transferred to a new EP tube and stored at -80°C for later use.

[0049] Example 3. Evaluation of the hangover-relieving effect of a traditional Chinese medicine compound

[0050] Effects of Chinese herbal compound on the duration of intoxication in mice

[0051] Mice were gavaged at a dose of 0.17 mL / 10 g to establish an acute alcohol intoxication model. The mice's reactions were timed and observed. If the mouse was unable to right itself three times in a row within 30 seconds, its righting reflex was considered lost, and the mouse was thus intoxicated. The time from administration of the alcohol to the loss of the righting reflex was recorded, which was the alcohol tolerance time. The mice's condition was continuously observed. When the mouse was able to right itself three times in a row within 30 seconds, it indicated that its righting reflex had recovered. The duration of the loss of the righting reflex was recorded, which was the intoxication time. During the observation process, the mice were placed on a rotarod. Every half hour, the mice's stay time on the rotarod and changes in motor coordination were monitored to assess the mice's sobriety. When the mice stayed on the rotarod for 120 seconds, they were considered completely sober. The results showed that mice with acute alcohol intoxication developed motor dysfunction after a few minutes and completely lost their righting reflex within 1 hour. The alcohol tolerance time of the model group mice was about 23 minutes, and the intoxication time was 389 minutes. The alcohol tolerance time of the positive control group Gehua Jiezhu decoction and the Curcuma zedoaria group mice was extended to 34 minutes and 31 minutes, respectively, and the intoxication time was shortened to 279 minutes and 275 minutes. The low-dose Chinese herbal compound had no significant effect on the alcohol tolerance time and intoxication time of the mice. Compared with the Curcuma zedoaria group mice, the medium and high doses of the Chinese herbal compound significantly prolonged the alcohol tolerance time of the acute alcohol poisoning mice to 38 minutes and 47 minutes ( Figure 2 A), the drunkenness time was shortened to 221min and 169min ( Figure 2 B). Compared with the model group, mice in the high-dose Chinese herbal compound group recovered motor function 2 hours earlier ( Figure 2 C), therefore, a high dose of the TCM compound was used in subsequent experiments.

[0052] Effects of Chinese herbal compound on serum ethanol concentration in mice

[0053] A blood ethanol assay kit was used to measure serum ethanol levels in mice 1, 3, 5, 7, and 9 hours after alcohol administration to assess the rate of alcohol metabolism. 1000 μL of alcohol dehydrogenase working solution and 80 μL of anhydrous ethanol standard solution were added to a standard tube. Then, 1000 μL of alcohol dehydrogenase working solution and 80 μL of the sample to be tested were added to a measuring tube. The mixture was immediately mixed and timed. The absorbance value A1 was read at a wavelength of 340 nm after 30 seconds. After 1 minute, the absorbance value A2 was read. ΔA / min was calculated as (A2 - A1) / min. Blood ethanol concentration (mg / dL) = (ΔA sample / min) / (ΔA standard / min) × C standard. Results showed that plasma ethanol concentration in mice reached its peak 1 hour after alcohol administration. Both the positive controls, Gehua Jiezhu Decoction and Curcuma zedoariae, reduced plasma ethanol concentration in mice. Compared with the mice in the Curcuma zedoaria group, the high-dose Chinese herbal compound significantly reduced the serum ethanol concentration at 1, 3, 5, and 7 hours, indicating that the Chinese herbal compound can accelerate the metabolic rate of ethanol ( Figure 2D).

[0054] Example 4. Effect of Chinese herbal compound on the activity of alcohol-detoxifying enzymes in mouse liver

[0055] ELISA kits were used to detect the activity of alcohol detoxification enzymes in the mouse liver, such as alcohol dehydrogenase ADH, acetaldehyde dehydrogenase ALDH, cytochrome P450 family member CYP2E1 enzyme and catalase CAT, to evaluate the alcohol detoxification function of the mouse liver. Alcohol dehydrogenase is involved in the first step of alcohol metabolism, metabolizing alcohol into acetaldehyde in the liver. Acetaldehyde dehydrogenase is involved in the second step of alcohol metabolism and is the only enzyme that metabolizes acetaldehyde, a key molecule that causes alcohol poisoning. For long-term drinkers, cytochrome P450 family member CYP2E1 can convert alcohol into acetaldehyde, while promoting the production of hydroxyethyl free radicals (HER) and hydroxyl free radicals (OH - ) and superoxide (O2 - ) and a series of ROS, while catalase can remove hydrogen peroxide that causes oxidative damage and is also involved in some ethanol metabolism.

[0056] (1) Liver protein extraction: Accurately weigh the liver tissue; add 9 volumes of normal saline at a weight (g): volume (mL) ratio of 1:9; grind into a homogenate in an ice-water bath; centrifuge at 2500 rpm for 10 min at 4°C; collect the supernatant and determine its total protein concentration (C) 总蛋白 .

[0057] (2) Detection of alcohol dehydrogenase ADH: add 10 μL of distilled water to the blank well, add 10 μL of the sample to be tested to the test well, draw 240 μL of working solution into each well, shake the plate gently to mix and time; after 20 seconds, place the plate in a microplate reader and read the absorbance value A1 of each well at 240 nm; place the plate in a 37°C constant temperature box and incubate for 20 minutes; quickly place the plate in a microplate reader and read the absorbance value A2 of each well at 240 nm; calculate the difference between the two absorbances (ΔA = A2-A1) and use it to calculate the ADH activity: ADH enzyme activity (U / mgprot) = (ΔA 测定 -ΔA 空白 ) / (6.22×0.69)×V 总 / (V 样本 ×T)×1000 / C 总蛋白 .

[0058] (3) Detection of aldehyde dehydrogenase (ALDH): The liver weight (g): extract volume (mL) was set at a ratio of 1:10, and the mixture was homogenized in an ice bath, centrifuged at 10,000 rpm for 20 min at 4°C, and the supernatant was collected and its total protein concentration was determined as C. 总蛋白; Add 0.1 mL of the sample to be tested and 0.9 mL of the working solution to the assay tube, mix thoroughly, and read the absorbance value A1 of each well at 340 nm; incubate in a 37°C incubator for 5 minutes, and read the absorbance value A2 of each well; calculate the difference between the two absorbances (ΔA = A2 - A1) and use it to calculate ALDH activity: ALDH enzyme activity (U / mgprot) = (322 × ΔA) / C 总蛋白 .

[0059] (4) Detection of CYP2E1, a member of the cytochrome P450 family: Remove the required plate strips from the aluminum foil bag after equilibration at room temperature for 20 minutes, set up standard wells and sample wells; add 50 μL of standard of different concentrations to each standard well. First add 10 μL of the sample to be tested to the sample well, then add 40 μL of sample diluent; do not add to the blank well; except for the blank well, add 100 μL of horseradish peroxidase-labeled detection antibody to each standard well and sample well, seal the reaction wells with a sealing film, and incubate at 37°C in a constant temperature incubator for 60 minutes; discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let it stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat this washing process 5 times; add 50 μL of substrate A and B to each well, incubate at 37°C in the dark for 15 minutes; add 50 μL of stop solution to each well, measure the OD value of each well at a wavelength of 450 nm within 15 minutes, and draw a standard curve to obtain the curve equation. Substitute the OD value of each sample into the equation to obtain the corresponding value, and then multiply it by 5 to obtain the actual concentration of CYP2E1.

[0060] (5) Detection of catalase (CAT): Take a 1 cm optical path quartz cuvette and adjust it to zero at 240 nm with double-distilled water; first add 0.02 mL of sample to the bottom of the cuvette, then add 3 mL of substrate solution that has been preheated to 25°C and has an OD value between 0.5 and 0.55 to the cuvette; immediately measure the absorbance value A1 at 240 nm; measure the absorbance value A2 again after 1 minute; calculate the CAT activity using the formula: CAT activity (U / mgprot) = log(A1 / A2) × 5.80 / C 总蛋白 .

[0061] The results showed that the enzyme activities of alcohol dehydrogenase ADH, acetaldehyde dehydrogenase ALDH, cytochrome P450 family member CYP2E1 and catalase CAT in the liver of mice in the normal control group were 12.87U / mgprot, 3.92U / mgprot, 7.63ng / mL and 236.66U / mgprot, respectively; the enzyme activities of alcohol dehydrogenase ADH, acetaldehyde dehydrogenase ALDH and cytochrome P450 family member CYP2E1 in the liver of mice with acute alcohol poisoning increased to 23.84U / mgprot, 7.93U / mgprot and 9.13ng / mL, while the activity of catalase CAT did not change significantly (243.42U / mgprot). In the positive control group, the liver enzyme activities of alcohol dehydrogenase (ADH), acetaldehyde dehydrogenase (ALDH), cytochrome P450 family member CYP2E1, and catalase (CAT) were 29.73 U / mgprot, 10.86 U / mgprot, 10.78 ng / mL, and 338.28 U / mgprot, respectively. In the Curcuma zedoaria group, the liver enzyme activities of alcohol dehydrogenase (ADH), acetaldehyde dehydrogenase (ALDH), cytochrome P450 family member CYP2E1, and catalase (CAT) were 29.64 U / mgprot, 10.14 U / mgprot, 9.89 ng / mL, and 308.77 U / mgprot, respectively. Low-dose TCM compound had no significant effect on the activities of these four enzymes. Compared with the Curcuma zedoaria group, the activities of alcohol dehydrogenase ADH, acetaldehyde dehydrogenase ALDH, cytochrome P450 family member CYP2E1 enzyme, and catalase CAT in the liver of mice in the medium-dose Chinese herbal compound group were 33.86U / mgprot, 12.31U / mgprot, 11.42ng / mL, and 366.84U / mgprot, respectively. In the high-dose Chinese herbal compound group, the activities were 38.94U / mgprot, 15.96U / mgprot, 13.49ng / mL, and 432.65U / mgprot, respectively. The above experimental results indicate that the Chinese herbal compound enhances the alcohol-resolving function and antioxidant function of the mouse liver ( Figure 3 AD).

[0062] Example 5. Effect of Chinese herbal compound on liver function in mice

[0063] ELISA kits were used to measure serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and γ-glutamyltransferase (γ-GT) activity in mice to evaluate the hepatotoxic effects of a high-dose traditional Chinese medicine compound. When liver damage occurs, cell swelling and increased membrane permeability lead to the release of ALT from the cytoplasm into the blood. AST is primarily located in the nucleus and is released into the blood upon hepatocyte death and disintegration. γ-Glutamyltransferase reflects hepatocyte integrity and is widely used as a marker for excessive alcohol consumption and alcoholic liver disease.

[0064] (1) Detection of ALT: Preheat the ALT matrix solution to 37°C; add 20 μL matrix solution and 5 μL sample to be tested to the assay wells, and add 20 μL matrix solution to the control wells; gently shake the plate to mix, and react at 37°C for 30 min; add 20 μL 2,4-dinitrophenylhydrazine solution to the assay wells, and add 20 μL 2,4-dinitrophenylhydrazine solution and 5 μL sample to be tested to the control wells; gently shake the plate to mix, and react at 37°C for 20 min; add 200 μL sodium hydroxide solution (0.4 mol / L) to each assay well and control well; gently shake the plate to mix, and let it stand at room temperature for 15 min; set the wavelength to 505 nm, measure the OD value of each well, and subtract the OD value of the control well from the OD value of the assay well to obtain the net OD value; substitute the standard curve to calculate the ALT activity value.

[0065] (2) Detection of aspartate aminotransferase (AST): Preheat the aspartate aminotransferase matrix solution to 37°C; add 20 μL of matrix solution and 5 μL of the sample to be tested to the assay wells, and add 20 μL of matrix solution to the control wells; gently shake the plate to mix, and react at 37°C for 30 min; add 20 μL of 2,4-dinitrophenylhydrazine solution to the assay wells, and add 20 μL of 2,4-dinitrophenylhydrazine solution and 5 μL of the sample to be tested to the control wells; gently shake the plate to mix, and react at 37°C for 20 min; add 200 μL of sodium hydroxide solution (0.4 mol / L) to each of the assay wells and the control wells; gently shake the plate to mix, and let it stand at room temperature for 15 min; set the wavelength to 505 nm, measure the OD value of each well, and subtract the OD value of the control well from the OD value of the assay well to obtain the net OD value; substitute the standard curve to calculate the aspartate aminotransferase (AST) activity value.

[0066] (3) Detection of γ-glutamyltransferase γ-GT: add 25 μL of distilled water, standard solution and serum sample to the blank well, standard well and test well respectively; add 200 μL of γ-glutamyl p-nitroaniline buffer solution to each well; shake the plate gently to mix, and react at 37°C for 5 min; add 50 μL of glycylglycine solution to each well; shake the plate gently to mix, and react at 37°C for 1 min; set the wavelength to 415 nm, and continuously detect the absorbance value of each well at 1 minute and 3 minutes, and calculate ΔA; calculate the activity of γ-glutamyltransferase γ-GT by the formula: γ-GT activity (U / L) = (ΔA 测定 -ΔA 空白 ) / (ΔA 标准 -ΔA 空白 )×Standard activity.

[0067] Results showed that serum ALT, AST, and γ-glutamyltransferase (γ-GT) levels in the normal control group were 6.20, 8.41, and 2.11 U / L, respectively. In mice exposed to acute alcohol intoxication, these levels increased to 29.72, 17.00, and 4.40 U / L, respectively, indicating alcohol-induced liver damage. In the positive control group, Gehua Jiezhu Decoction reduced serum ALT, AST, and γ-glutamyltransferase (γ-GT) levels to 24.31, 15.04, and 3.54 U / L, respectively, alleviating alcohol-induced liver damage. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and γ-glutamyl transferase (γ-GT) in the high-dose TCM compound group were also reduced to 15.07, 14.99, and 2.52 U / L, respectively, indicating that the high-dose TCM compound was non-toxic to the liver and could alleviate liver damage caused by acute alcohol poisoning ( Figure 4 AC).

[0068] Example 6. Effect of the Chinese herbal compound of the present invention on renal function in mice

[0069] An ELISA kit was used to measure serum cystatin C levels in mice to assess the renal toxicity of a high-dose traditional Chinese medicine compound. After equilibration at room temperature for 20 minutes, the desired strips were removed from the aluminum foil pouch and set up with standard and sample wells. 50 μL of a standard of varying concentrations was added to each well. First, add 10 μL of the sample to be tested to the sample well, and then add 40 μL of sample diluent; do not add to the blank well; except for the blank well, add 100 μL of horseradish peroxidase-labeled detection antibody to each well of the standard well and sample well, seal the reaction well with a sealing film, and incubate at 37°C in a constant temperature box for 60 minutes; discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let it stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat this washing process 5 times; add 50 μL of substrate A and B to each well, incubate at 37°C in the dark for 15 minutes; add 50 μL of stop solution to each well, measure the OD value of each well at a wavelength of 450 nm within 15 minutes, and draw a standard curve to obtain the curve equation; substitute the OD value of each sample into the equation to obtain the corresponding value and multiply it by 5 to obtain the actual Cys-C concentration. The results showed that the serum cystatin Cys-C levels of mice in the acute alcohol poisoning group and the high-dose TCM compound treatment group were within the normal range, indicating that the high-dose TCM compound had no abnormal effects on the kidneys (Table 1).

[0070] Table 1. Serum cystatin levels

[0071]

[0072]

[0073] Example 7. Effect of Chinese herbal compound on pathological changes of liver and kidney tissues in mice

[0074] HE staining was used to observe the structural changes in the liver and kidney of mice and to evaluate the effects of a high-dose traditional Chinese medicine compound on the liver and kidney of mice with acute alcohol intoxication. Tissue sections were first dewaxed and immersed in xylene I and II for 10 min each. Sections were then hydrated and immersed in anhydrous ethanol, 95% ethanol, 80% ethanol, and 75% ethanol for 1 min each, followed by a 2-min rinse in distilled water. Sections were then stained with hematoxylin for 1 min, gently rinsed in tap water for 2 min, and then rinsed in running water for 1 min. Sections were then color-separated with 1% hydrochloric acid alcohol for 2 s, rinsed in running water for 5 min, and finally stained with eosin for 10 s and rinsed in running water for 1 min. Sections were then dehydrated and transparentized by immersing in 75% ethanol, 80% ethanol, 95% ethanol, and anhydrous ethanol for 1 min each, followed by immersion in xylene I and II for 2 min each. Finally, sections were mounted with neutral resin and observed and imaged under a microscope. The results showed that the liver plates of mice with acute alcohol poisoning and mice treated with high-dose Chinese herbal compound were arranged regularly, and the liver lobule structure was not obviously disordered ( Figure 5 A), but the liver lipofuscin of mice with acute alcohol poisoning is excessively deposited, and high-dose Chinese herbal compound can significantly alleviate the excessive deposition of lipofuscin, indicating that the metabolic function of liver cells in mice with acute alcohol poisoning is abnormal, and high-dose Chinese herbal compound can improve the metabolic function of liver cells ( Figure 6 A, B). At the same time, there were no obvious abnormalities in the glomeruli and surrounding structures of the mice in the acute alcohol poisoning group and the mice in the high-dose Chinese herbal compound treatment group, and no pathological changes were found in the renal tubules and interstitium, indicating that the high-dose Chinese herbal compound had no significant effect on the renal tissue structure ( Figure 5 B).

[0075] Example 8, formulation screening

[0076] In order to screen out the ratio of traditional Chinese medicine composition with the best hangover-relieving effect, three groups of traditional Chinese medicine compositions with different ratios were set up, and the serum ethanol concentration, alcohol dehydrogenase (ADH), acetaldehyde dehydrogenase (ALDH) and cytochrome P450 family CYP2E1 enzyme activities of mice in each group were measured.

[0077] Proportion 1 is the composition of the present invention:

[0078] Raw material composition (by weight): 4 parts of Curcuma zedoaria, 2 parts of Amomum villosum, 1 part of Alisma orientale, 1 part of Atractylodes macrocephala, and 1 part of Angelica dahurica.

[0079] Ratio 2

[0080] Compared with proportion 1, the proportion of raw materials is different.

[0081] Raw material composition (by weight): 2 parts of Curcuma zedoaria, 2 parts of Amomum villosum, 1 part of Alisma orientale, 1 part of Atractylodes macrocephala, and 1 part of Angelica dahurica.

[0082] Ratio 3

[0083] Compared with proportion 1, the proportion of raw materials is different.

[0084] Raw material composition (by weight): 1 part of Curcuma zedoaria, 2 parts of Amomum villosum, 1 part of Alisma orientale, 1 part of Atractylodes macrocephala, and 0.5 part of Angelica dahurica.

[0085] Comparative Example 1

[0086] The difference between the traditional Chinese medicine composition provided by the present invention is that it does not contain Curcuma zedoaria and Angelica dahurica.

[0087] Comparative Example 2

[0088] The difference between the traditional Chinese medicine composition provided by the present invention is that it does not contain Curcuma zedoaria.

[0089] Comparative Example 3

[0090] The difference between the traditional Chinese medicine composition provided by the present invention is that it does not contain Angelica dahurica.

[0091] The results showed that compared with other Chinese herbal compound formulas, the ratio 1 used in this project significantly reduced the serum ethanol concentration of mice and increased the activity of liver alcohol-detoxifying enzymes, such as alcohol dehydrogenase, acetaldehyde dehydrogenase, and cytochrome P450 family CYP2E1 enzyme, indicating that the Chinese herbal compound with ratio 1 has the best alcohol-detoxifying effect (Table 2).

[0092] Table 2. Effects of different ratios of Chinese herbal compound on the detoxification of alcohol

[0093]

[0094] Note: Compared with the control group, #p<0.05, ###p<0.001. Compared with the model group, *p<0.05, **p<0.01, ***p<0.001.

[0095] Example 9. Evaluation of the hangover-relieving effects of different species of Curcuma zedoaria

[0096] To evaluate the alcohol-detoxifying effects of Curcuma elata, a species of Curcuma zedoaria proposed in this project, three compound formulas containing Curcuma elata, Curcuma zedoaria, and Curcuma phaeocaulis were prepared. Serum ethanol concentrations and liver alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activities were measured in mice. Compound 1 consisted of 2 parts Curcuma elata, 2 parts Amomum villosum, 1 part Alisma orientale, 1 part Atractylodes macrocephala, and 1 part Angelica dahurica. Compound 2 consisted of 2 parts Curcuma zedoaria, 2 parts Amomum villosum, 1 part Alisma orientale, 1 part Atractylodes macrocephala, and 1 part Angelica dahurica. Compound 3 consisted of 2 parts Curcuma phaeocaulis, 2 parts Amomum villosum, 1 part Alisma orientale, 1 part Atractylodes macrocephala, and 1 part Angelica dahurica. The results showed that compared with the mice treated with Compound 2 and Compound 3, the serum ethanol concentration of the mice treated with Compound 1 was significantly reduced, and the activities of liver alcohol dehydrogenase and acetaldehyde dehydrogenase were significantly increased, indicating that the species Curcuma elata proposed in this project has a better alcohol-relieving effect (Table 3).

[0097] Table 3. Effects of different species of Curcuma zedoaria on sobering up

[0098]

[0099] Note: Compared with the control group, #p<0.05, ###p<0.001. Compared with the model group, *p<0.05, **p<0.01, ***p<0.001.

[0100] The above description is only a preferred embodiment of the present invention. It should be noted that the embodiments of the present invention are not limited to the embodiments described. Without departing from the principles of the present invention, a number of changes, modifications, substitutions, combinations, and simplifications may be made, all of which are equivalent replacement methods and should also be considered as the scope of protection of the present invention.

Claims

1. A Chinese medicine composition for sobering up and relieving alcoholic liver damage, which is prepared from the following raw materials in parts by weight: 1-4 parts of Curcuma zedoaria, 0.5-2 parts of Amomum villosum, 0.25-1 parts of Alisma orientalis, 0.25-1 parts of Atractylodes macrocephala, and 0.25-1 parts of Angelica dahurica.

2. The Chinese medicine composition according to claim 1, characterized in that It is made from the following raw materials in parts by weight: 4 parts of Curcuma zedoaria, 2 parts of Amomum villosum, 1 part of Alisma orientalis, 1 part of Atractylodes macrocephala, and 1 part of Angelica dahurica.

3. The Chinese medicine composition according to claim 1, characterized in that It also contains a pharmaceutically acceptable carrier.

4. The Chinese medicine composition according to claim 1, characterized in that Prepared into any edible dosage form.

5. The Chinese medicine composition according to claim 4, characterized in that The dosage forms are tablets, capsules, pills, oral liquids, lozenges, granules, pills, powders, pastes, pills, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, drops and patches.

6. The method for preparing the Chinese medicine composition according to claim 1, comprising the following steps: (1) Medicinal material grinding: Place each medicinal material in a wall-breaking machine and grind it thoroughly, then filter it through a 200-mesh sieve; (2) Preparation of suspension: Weigh the corresponding dosage components after sieving, mix them and add appropriate amount of distilled water to prepare the Chinese medicine compound suspension.

7. Use of the traditional Chinese medicine composition according to claim 1 in the preparation of a medicament for sobering up and alleviating alcoholic liver damage.

8. The use according to claim 7, characterized in that Applications include: prolonging alcohol tolerance time, shortening drunkenness time, and significantly accelerating the plasma ethanol clearance rate in acute alcohol poisoning.

9. The use according to claim 7, characterized in that Applications include: significantly improving the activity of liver alcohol decomposition enzymes such as alcohol dehydrogenase ADH, acetaldehyde dehydrogenase ALDH, cytochrome P450 family member CYP2E1 and catalase CAT, and accelerating ethanol metabolism.

10. The use according to claim 7, characterized in that Applications include: significantly reducing the levels of alanine aminotransferase ALT, aspartate aminotransferase AST and γ-glutamyl transferase γ-GT in serum, and alleviating liver damage caused by acute alcohol poisoning.

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