Traditional Chinese medicine composition and medicinal preparation for clearing liver, resolving dampness and dispelling effects of alcohol as well as preparation method and application of traditional Chinese medicine composition and medicinal preparation

Abstract

The invention relates to the field of traditional Chinese medicines, in particular to a traditional Chinese medicine composition and a medicinal preparation for clearing liver, resolving dampness and dispelling effects of alcohol as well as a preparation method and application of the traditional Chinese medicine composition and the medicinal preparation. The compatibility of ministerial drugs adopts a Sanhuang coke-separating and heat-clearing system. Poria cocos can dredge water dampness and nourish spleen soil in Zhongzhou, and especially can realize dynamic balance of eliminating evil and strengthening body resistance by adjusting water metabolism, and white peony root can nourish blood, astringe yin, soften liver and harmonize blood. The compatibility of the scorched hawthorn fruit can enhance the regulation of lipid metabolism, and the three medicines are combined to moisten the liver, dredge and restore the normal state. Mirabilite is used for relaxing bowel and removing heat; radix astragali is used as a main medicine for tonifying qi and strengthening superficies; pinellia ternate-ginger officinal magnolia bark drug pairs are important for construction of central transmission. The conductant drug honey-fried licorice root is regulated to be neutral, and the drug is introduced into the place. The traditional Chinese medicine has the effects of enlivening the spleen, dispelling the effects of alcohol, clearing heat and promoting diuresis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of traditional Chinese medicine, in particular to a traditional Chinese medicine composition for clearing liver, removing dampness and resolving alcohol and a pharmaceutical preparation, and a preparation method and use thereof. BACKGROUND

[0002] Liver injury induced by chronic alcohol abuse has become an important problem affecting global public health. As the most representative disease among alcohol-related organ damage, the pathological features of alcoholic liver disease (ALD) mainly include persistent inflammatory response in liver parenchyma, imbalance of reactive oxygen metabolism, and intestinal microecosystem disorder. The disease spectrum covers the whole process from simple fatty degeneration to alcoholic steatohepatitis (ASH), and then to liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Most of the long-term alcohol exposure population will experience alcoholic fatty liver stage, and a part of them will progress to alcoholic hepatitis state. The current clinical management strategy still takes the intervention of abstinence, nutritional support, glucocorticoid therapy, and the use of liver-protecting and enzyme-reducing drugs as the cornerstone, but there are still patients with pathological outcome to cirrhosis.

[0003] The pathogenesis of ALD has not been thoroughly elucidated, but the related mechanisms involved behind it, such as oxidative stress, abnormal lipid metabolism, and intestinal microbiota imbalance, have been confirmed to have a key impact. These factors, through multi-level interaction, jointly drive the evolution of the pathological process, and each plays a different role in the occurrence and development of ALD. There is no effective treatment for ALD at present, and the main methods are patient abstinence, nutritional support, glucocorticoid therapy, and liver transplantation.

[0004] Therefore, it is an urgent task to develop effective traditional Chinese medicine for the treatment of alcoholic liver disease by fully utilizing the advantages of traditional Chinese medicine. SUMMARY

[0005] Therefore, the purpose of the present application is to provide a traditional Chinese medicine composition and pharmaceutical preparation for clearing liver, removing dampness and resolving alcohol, and a preparation method and use thereof. The traditional Chinese medicine composition and pharmaceutical preparation can significantly reduce the liver function ALT, AST, GGT and ALP of patients with alcoholic liver disease, and improve the APRI index of patients. It can relieve the symptoms of patients such as stabbing or distending pain in the lateral costal region, abdominal distention, lassitude, bitter taste in the mouth, dry mouth, yellow eyes, loss of appetite, nausea, vomiting, intestinal rumbling, restlessness and irritability, indicating that the compound has the effect of improving ALD.

[0006] The application provides a traditional Chinese medicine composition for clearing liver, removing dampness and resolving alcohol, which comprises, by weight, Gegen 25-35 parts, Jijingsi 13-18 parts, Huangqi 4-8 parts, Huanglian 4-8 parts, Huangbai 4-8 parts, Fuling 18-22 parts, Jianghoupu 7-11 parts, Baishao 25-35 parts, Mangxie 2-4 parts, Jiaoshanzha 13-18 parts, Huangqi 25-60 parts, Fabaixian 7-11 parts and Zhugancao 4-8 parts.

[0007] Further, the traditional Chinese medicine composition comprises, by weight, Gegen 30 parts, Jijingsi 15 parts, Huangqi 6 parts, Huanglian 6 parts, Huangbai 6 parts, Fuling 20 parts, Jianghoupu 9 parts, Baishao 30 parts, Mangxie 3 parts, Jiaoshanzha 15 parts, Huangqi 30 parts, Fabaixian 9 parts and Zhugancao 6 parts; or,

[0008] Gegen 25 parts, Jijingsi 18 parts, Huangqi 4 parts, Huanglian 8 parts, Huangbai 4 parts, Fuling 22 parts, Jianghoupu 7 parts, Baishao 35 parts, Mangxie 2 parts, Jiaoshanzha 18 parts, Huangqi 25 parts, Fabaixian 11 parts and Zhugancao 8 parts; or,

[0009] Gegen 35 parts, Jijingsi 13 parts, Huangqi 8 parts, Huanglian 4 parts, Huangbai 8 parts, Fuling 18 parts, Jianghoupu 11 parts, Baishao 25 parts, Mangxie 4 parts, Jiaoshanzha 13 parts, Huangqi 60 parts, Fabaixian 7 parts and Zhugancao 4 parts.

[0010] The application further provides a preparation method of the traditional Chinese medicine composition, which comprises: weighing Gegen, Jijingsi, Huangqi, Huanglian, Huangbai, Fuling, Jianghoupu, Baishao, Mangxie, Jiaoshanzha, Huangqi, Fabaixian and Zhugancao according to selected weight parts, mixing, and then extracting according to a conventional extraction method or extracting each ingredient according to a conventional extraction method and then mixing.

[0011] Further, the conventional extraction method is selected from one or more of decoction extraction, reflux extraction, immersion extraction, percolation extraction, ultrasonic extraction and water vapor distillation extraction; and / or, the extraction solvent used in the extraction is selected from water or an alcohol solution with a volume percentage of 5-98%; and / or, the extraction is performed at least once; each extraction is performed for at least 10 min; and / or, the ratio of the volume of the extraction solvent to the weight of the raw material is ≥2 L / kg.

[0012] The application further provides a pharmaceutical preparation comprising the traditional Chinese medicine composition according to any of the application or prepared by any of the preparation methods.

[0013] In some preferred embodiments, the pharmaceutical preparation is prepared into a clinically acceptable dosage form by adding or not adding a pharmaceutically acceptable carrier to the traditional Chinese medicine composition or the traditional Chinese medicine composition prepared by the preparation method according to a conventional process.

[0014] In some preferred embodiments, the pharmaceutical preparation is a granule, a powder, a tablet, a capsule, a pill, a decoction, a syrup, a decocted extract or a suspension.

[0015] In some preferred embodiments, the pharmaceutically acceptable carrier is selected from at least one of a pharmaceutically acceptable solvent, a solubilizer, an emulsifier, a binder, a disintegrant, a filler, a lubricant, a glidant, a wetting agent, a flavoring agent, a preservative.

[0016] The present application also provides a use of the traditional Chinese medicine composition, the traditional Chinese medicine composition prepared by the preparation method or any of the above-mentioned pharmaceutical preparations in the preparation of a medicament for preventing and / or treating alcoholic liver disease.

[0017] In some embodiments, the alcoholic liver disease is alcoholic liver disease with liver stagnation and spleen deficiency and internal accumulation of damp-heat.

[0018] In some embodiments, the use of the traditional Chinese medicine composition and / or the pharmaceutical preparation in the preparation of a medicament for improving liver function.

[0019] In some embodiments, the use of the traditional Chinese medicine composition and / or the pharmaceutical preparation in the preparation of a medicament for improving the degree of oxidative stress.

[0020] In some embodiments, the use of the traditional Chinese medicine composition and / or the pharmaceutical preparation in the preparation of a medicament for regulating the Nrf2-Keap1 signal pathway.

[0021] The technical scheme of the present application has the following advantages:

[0022] 1.The traditional Chinese medicine composition provided by the present application is obtained through repeated optimization of the composition and content of the compound traditional Chinese medicine through clinical research and basic research, and uses Folium Pteridi and Fructus Hoveniae as monarch drugs to synergistically play the effects of dispelling alcohol and activating the spleen and dispersing and promoting. The minister drugs are combined to form a Sanhuang Fojiao Qingjie system: Huangqin clears the upper Jiaogong and removes heat from the Qi aspect, Huanglian resolves the damp-heat stagnation in the middle Jiaogong, and Huangbai dredges the turbid evil stagnation in the lower Jiaogong. Fuling can not only dredge water and dampness but also tonify the spleen, and is particularly good at achieving the dynamic balance between eliminating evil and supporting the healthy by regulating water and liquid metabolism. Baishao nourishes blood and astringes Yin and soothes the liver and blood, and forms a synergistic effect of "dredging and astringing" with Fuling. The combination of Fojiaoshanxia can enhance the regulation of lipid metabolism, and the combination of the three drugs can make the liver soft and restore normal function. Mangxiao can clear heat and purge the bowels; Huangqi is a qi-tonifying and surface-anchoring drug; the Banxia-Jiang-Huopu drug pair is essential for the construction of the pivot and transportation, and can harmonize the stomach, remove turbidity, reduce adverse flow, and relieve distension and fullness to dredge the middle Jiaogong. The combination of the above drugs can play the roles of dispelling alcohol and activating the spleen, clearing heat and removing dampness, and the present prescription can tonify deficiency without assisting evil, purge excess without damaging the healthy, clear heat without being bitter and dry, and remove dampness and water without damaging Yin. The magic of the present prescription lies in grasping the "liver disease transmission" rule and integrating the methods of soothing the liver, tonifying the spleen, and harmonizing the stomach into one system to form a unique system for the treatment of liver diseases. The present prescription can treat the symptoms and the root cause of the disease and regulate qi and blood. Clinical use shows that the Qinggan Huashi Jiejiu Granules have remarkable effects in improving the TCM symptoms of patients and reducing liver enzymes.

[0023] According to different stages of disease development, the use strategy of Huangqi can be adjusted. In the early stage of the disease, a small dose of raw Huangqi (for example, 25-35 parts) is used to enhance the body's resistance to evil; in the later stage of the disease, the dosage of Huangqi is increased (for example, 35-60 parts) to achieve the dual effects of eliminating evil and supporting the healthy.

[0024] 2.The traditional Chinese medicine composition provided by the present application has a positive therapeutic effect on alcoholic liver disease, can improve the mental state of an ethanol-induced NIAAA mouse model, reduce serum ALT, AST, DBIL, TBIL, and Fe+ indexes, reduce inflammatory cell infiltration of liver tissue, and reduce the generation of lipid vacuoles. It can improve the degree of oxidative stress indicators of an ethanol-induced NIAAA mouse model, that is, increase the activities of GSH and SOD and reduce the level of MDA. It can also reduce the content of IL-6 and IL-1β inflammatory factors and reduce lipid deposition in liver tissue sections. It can also increase the expression levels of Nrf2, HO-1 protein and mRNA, and GPX4 protein in liver tissue of an ethanol-induced alcoholic liver injury mouse, and reduce the mRNA expression levels of Keap1 and PRDX3 proteins. The Nrf2-Keap1 signaling pathway and ferroptosis may be important signal pathways for the treatment of ALD.

[0025] 3. Through clinical observation, it is found that the liver-damp-resolving and alcohol-resolving traditional Chinese medicine composition can benefit the patients, significantly reduce the liver function ALT, AST, GGT and ALP of the patients with alcoholic liver disease, and improve the APRI index of the patients. The composition can relieve the symptoms of the patients such as rib pain or distending pain, abdominal distension, lassitude, dry mouth, yellow eyes, loss of appetite, nausea, vomiting, intestinal rumbling, irritability and the like. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the description of the embodiments or the prior art will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0027] Figure 1 is the change of the body weight of the mice before and after modeling;

[0028] Figure 2 is the liver index data graph;

[0029] Figure 3 is the determination of the ALT, AST, TBIL, DBIL and Fe+ indicators of the mice;

[0030] Figure 4 is the HE staining graph of the liver tissues of the mice in each group

[0031] Figure 5 is the oxidative stress index data graph;

[0032] Figure 6 is the inflammatory factor data graph;

[0033] Figure 7 is the oil red staining graph of the liver tissues of the mice in each group;

[0034] Figure 8 is the protein expression of Nrf2, Keap1, HO-1, GPX4 and PRDX3 in the liver of the mice in each group; Note: C = CON group, L = QGHSL group, M = QGHSM group, H = QGHSH group. Compared with the CON group, # P<0.05, ## P<0.01; compared with the ALD group, △ P<0.05, △△ P<0.01;

[0035] Figure 9 is the mRNA expression of Nrf2, Keap1, HO-1 and PRDX3 in the liver of the mice in each group; Note: compared with the CON group, # P<0.05,## P<0.01; compared with the ALD group, △ P<0.05, △△ P<0.01. DETAILED DESCRIPTION

[0036] The following examples are provided to better further understand the present application, and are not limited to the best mode, and do not constitute limitations to the content and protection scope of the present application, and any person under the inspiration of the present application or combining the present application with other prior art features to obtain any product same or similar to the present application falls within the protection scope of the present application.

[0037] The specific experimental steps or conditions not indicated in the examples can be performed according to the operation or conditions of the conventional experimental steps described in the literature in the art. The reagents or instruments not indicated by the manufacturer are all conventional reagent products that can be obtained by purchase.

[0038] Example 1

[0039] The present example provides a traditional Chinese medicine composition, comprising, Flos Puerariae 30g, Hoveniae Fructus 15g, Scutellariae Radix 6g, Coptidis Rhizoma 6g, Phellodendri Chinensis Cortex 6g, Poria 20g, Zingiberis Recens Rhizoma and Magnoliae Officinalis Cortex 9g, Radix Paeoniae Alba 30g, Mirabilitum 3g, Focatus Fructus 15g, Radix Astragali 30g, Rhizoma Pinelliae Praeparatum 9g and Radix Glycyrrhizae Praeparata 6g.

[0040] The present example provides a pharmaceutical preparation comprising the above traditional Chinese medicine composition, and the preparation method is as follows: Flos Puerariae, Hoveniae Fructus, Scutellariae Radix, Coptidis Rhizoma, Phellodendri Chinensis Cortex, Poria, Zingiberis Recens Rhizoma and Magnoliae Officinalis Cortex, Radix Paeoniae Alba, Mirabilitum, Focatus Fructus, Radix Astragali, Rhizoma Pinelliae Praeparatum and Radix Glycyrrhizae Praeparata are weighed according to the prescription weight of the traditional Chinese medicine composition. Extraction: add 10 times the mass of water to the medicinal materials of the first decoction, heat to boiling, and keep it slightly boiling for 1.5 hours, and then filter (200 mesh); add 8 times the mass of water to the medicinal materials of the second decoction, heat to boiling, and keep it slightly boiling for 1 hour, and then filter (200 mesh), and combine the filtrates. Concentration: reduce the pressure to concentrate the filtrate into a clear paste of a certain specific gravity. Spray drying: collect the powder in time. Sieving and mixing: sieve the dry paste powder, and mix for 30 minutes to make it uniform. Granulation: the intermediate after extraction is granulated by dry method to form 12-40 mesh granules, and 52.8g of Qinggan Huashi Jiejiu Granules is obtained.

[0041] Example 2

[0042] The present example provides a traditional Chinese medicine composition, comprising, Flos Puerariae 30g, Hoveniae Fructus 15g, Scutellariae Radix 6g, Coptidis Rhizoma 6g, Phellodendri Chinensis Cortex 6g, Poria 20g, Zingiberis Recens Rhizoma and Magnoliae Officinalis Cortex 9g, Radix Paeoniae Alba 30g, Mirabilitum 3g, Focatus Fructus 15g, Radix Astragali 30g, Rhizoma Pinelliae Praeparatum 9g and Radix Glycyrrhizae Praeparata 6g.

[0043] The present embodiment provides a pharmaceutical preparation containing the above-mentioned traditional Chinese medicine composition, and the preparation method is as follows: according to the prescription weight of the traditional Chinese medicine composition, take Gufu, Jijieshi, Huangqi, Huanglian, Huangbai, Fuling, Jianghoupu, Baishao, Jiaoshanzha, Huangqi, Fabaixia and Zhugancao, add water and decoct three times, the first time adds 8 times the amount of water of the quality of medicinal materials, decocts for 2 hours, the second and third times each adds 6 times the amount of water of the quality of medicinal materials, each decocts for 1 hour, combines the decocting liquid, filters, reduces the pressure and concentrates the filtrate to a thick paste with a relative density of 1.20-1.25 (50℃), dries, mixes with mirabilite powder after powdering, and obtains a drug powder. According to the conventional tablet operation method, medicinal accessories are added, the components are uniformly mixed, wet granulation is performed, sieving is performed, the conventional tablet preparation method is used for tablet pressing, and tablets are prepared. The use amount of each component is: 50 parts of drug powder, 30 parts of lactose, 12 parts of microcrystalline cellulose, 12 parts of magnesium stearate, and 8 parts of polyethylene glycol.

[0044] Example 3

[0045] The present embodiment provides a traditional Chinese medicine composition, which includes Gufu 35g, Jijieshi 13g, Huangqi 8g, Huanglian 4g, Huangbai 8g, Fuling 18g, Jianghoupu 11g, Baishao 25g, mirabilite 4g, Jiaoshanzha 13g, Huangqi 60g, Fabaixia 7g and Zhugancao 4g.

[0046] The present embodiment provides a pharmaceutical preparation containing the above-mentioned traditional Chinese medicine composition, and the preparation method is as follows: according to the prescription weight of the traditional Chinese medicine composition, take Gufu, Jijieshi, Huangqi, Huanglian, Huangbai, Fuling, Jianghoupu, Baishao, Jiaoshanzha, Huangqi, Fabaixia and Zhugancao, add water and decoct three times, the first time adds 8 times the amount of water of the quality of medicinal materials, decocts for 2 hours, the second and third times each adds 6 times the amount of water of the quality of medicinal materials, each decocts for 0.5 hours, combines the decocting liquid, filters, reduces the pressure and concentrates the filtrate to a thick paste with a relative density of 1.20-1.25 (50℃), dries, mixes with mirabilite powder after powdering, adds dextrin, granulates, fills into a capsule shell, and prepares a capsule.

[0047] Experimental Example 1 Animal Experiment

[0048] 1. Experimental materials

[0049] 1.1 Experimental animals and grouping

[0050] C57BL / 6J male mice, weighing 20±5g, mouse age 8 weeks. Purchased from Beijing Vantoll Life Experimental Animal Technology Co., Ltd. Animal license number: SCXK (Jing) 2021-0006. Raising in the National Nanometer Science Center, constant temperature and humidity, 12 hours light and 12 hours dark cycle, complete freedom of diet and water. The raising and experimental conditions and the experimental process strictly abide by the animal experiment ethical principles, and are reviewed by the ethics committee of Guang'anmen Hospital, approval number: 2ACUC-GAMY-2024-064.

[0051] Animal grouping: the mice were randomly divided into control group (Control group, CON), ALD model group (ALD group), shugan huashi jiejiu granules treatment group (SF group), qinggan huashi jiejiu granules low, medium and high dose groups (QGHS group, specifically QGHSL, QGHSM and QGHSH) a total of 6 groups. According to the experimental needs, 9 mice in the control group, 9 in the ALD group, 9 in the SF group, 9 in the QGHS low, medium and high dose groups were set up, and they were adaptively fed in the laboratory for one week.

[0052] 1.2 Experimental drugs and reagents

[0053] (1) Preparation of experimental drugs:

[0054] The qinggan huashi jiejiu granules were prepared by the method of Example 1. The safe and suitable intragastric volume of mice was 0.1 ml / 10g body weight, and the above qinggan huashi jiejiu granules were prepared into drug solutions containing 14mg, 28.1mg and 56mg of crude drug per milliliter by adding water. Store in a 4℃ refrigerator.

[0055] (2) Shugan huashi jiejiu granules:

[0056] Shugan huashi jiejiu granules (batch number H20040299) produced by Tianjin Tianrui Pharmaceutical Co., Ltd. were prepared into a shugan huashi jiejiu granules solution with a concentration of 16.38mg / ml by using distilled water as the solvent. The solution concentration is equivalent to 3 times the recommended adult clinical dosage.

[0057] 2. Experimental methods

[0058] 2.1 Preparation method of liquid diet

[0059] 2.1.1 Preparation method of control liquid diet

[0060] Weigh 225g of control liquid diet reagent, mix it thoroughly with 850ml of distilled water, and obtain a final volume of 1000ml of control liquid diet.

[0061] 2.1.2 Preparation method of alcohol liquid diet

[0062] Weigh 133g of alcohol liquid diet reagent (purchased from Beijing Sbeif Biological Technology Co., Ltd., item number: SFD032), 20.3g of maltodextrin, and mix them thoroughly with 850ml of distilled water and 52.6ml of 95% alcohol to obtain a final volume of 1000ml of alcohol liquid diet.

[0063] 2.2 Preparation of intragastric liquid

[0064] 2.2.1 Preparation method of maltose intragastric liquid (9g / kg)

[0065] Before gavage, 9 g of maltodextrin was weighed and dissolved in distilled water to a final volume of 20 ml, mixed well to prepare a 45% (wt / vol) maltose gavage solution.

[0066] 2.2.2 Preparation of high-dose alcohol gavage solution (5 g / kg)

[0067] Before gavage, prepare a 31.5% (vol / vol) alcohol gavage solution: mix 6.6 ml of 95% alcohol with 13.4 ml of distilled water to prepare a 20 ml of 31.5% alcohol gavage solution.

[0068] Table 1 Heat distribution of liquid diet

[0069]

[0070] Table 2 Preparation of 1000 ml of control liquid diet and alcohol liquid diet

[0071]

[0072] 2.3 Specific modeling method

[0073] 54 C57BL / 6J male mice were used in the experiment. After 7 days of basic feed adaptation, they were randomly divided into 6 groups (control group, model group, shugan huashi jiu granules low, medium and high dose groups), 9 mice per cage.

[0074] The modeling process is divided into three stages:

[0075] Initial stage - days 1-5, all experimental mice stop providing regular solid feed and drinking water, and are replaced with control liquid diet, 20 ml per mouse per day. Body weight is monitored daily and the amount of diet provided the next day is adjusted.

[0076] Alcohol induction phase - days 6-15, differential feeding was implemented, the control group continued to receive 20 ml / day of standard liquid diet, the rest of the groups were switched to alcohol liquid feed, 20 ml per day. From day 8, drug intervention was carried out synchronously, silybin group was given corresponding solution (silybin solution 16.38 mg / ml, gavage volume 0.1 ml / 10g body weight), traditional Chinese medicine group was given liver- clearing, dampness-resolving and alcohol-resolving granules at gradient doses (liver-clearing, dampness-resolving and alcohol-resolving granules at medium dose group was given drug solution with crude drug concentration of 28.1 mg / ml (equivalent dose), liver-clearing, dampness-resolving and alcohol-resolving granules at low dose group was given drug solution with crude drug concentration of 14 mg / ml (0.5 times the equivalent dose), liver-clearing, dampness-resolving and alcohol-resolving granules at high dose group was given drug solution with crude drug concentration of 56 mg / ml (2 times the equivalent dose), gavage volume was 0.1 ml / 10g body weight), the control group and the model group were given the same amount of normal saline. Body weight was monitored during this phase and gavage volume and diet amount were adjusted dynamically.

[0077] Terminal processing phase - from 7 am to 9 am on the 16th day, differential gavage was performed, the control group received 45% maltose solution, the rest of the experimental groups were given 31.5% ethanol solution, the drug volume was calculated according to body weight (g) x 20 μl. After 9 hours, the modeling process was completed and all mice were sacrificed.

[0078] This program constructs a liver injury model through stepwise alcohol exposure combined with drug intervention, the key parameter settings refer to the Lieber-DeCarli liquid feeding system to ensure model stability and repeatability.

[0079] 2.4 Sample collection after modeling

[0080] (1) The mice were executed by cervical dislocation, and the eyeballs of the animals were removed with an ophthalmic curved forceps before execution to allow the blood to flow naturally into a centrifuge tube pre-added with an anticoagulant to complete sample collection.

[0081] (2) The distal end of the portal vein was selected as the puncture point, and a sterile indwelling needle was inserted to establish a perfusion channel. Uniform perfusion was performed from the portal vein to the liver through 37°C constant temperature normal saline, and after the liver volume was fully filled, the inferior vena cava was disconnected to establish a reflux channel. The change in liver color was continuously observed, and the perfusion was terminated when the whole organ showed uniform fading. The liver tissue was completely stripped and weighed immediately, and a specific area of the left lobe was selected and placed in 4% paraformaldehyde fixing solution for tissue fixation.

[0082] 3. Mouse model evaluation

[0083] 3.1 Observation of the overall state of mice

[0084] During the experiment, the following indicators need to be collected daily: first, assess the change in hair color; second, monitor the changes in activity and behavior patterns, and pay attention to the morphological characteristics of the excrement, such as texture and color differences. Such data can provide multi-dimensional basis for physiological state analysis.

[0085] 3.2 Record the changes in mouse weight and calculate the liver index

[0086] At 16:30 every day, the weight of the experimental mice is measured and data collection is completed. After measurement, the intragastric volume is dynamically adjusted according to the measured value. After the model construction phase is completed, the body weight data of each group of experimental objects is compared horizontally and statistically analyzed. (Mouse liver index = mouse liver weight / mouse weight x 100%)

[0087] After the animal model is established according to the experimental design, eyeball enucleation is used to collect mouse blood samples, and the obtained blood samples are transferred to an anticoagulant blood collection tube. The sample is allowed to stand at room temperature for 30 minutes, and then centrifuged at 3000 rpm for 15 minutes to separate the plasma component. Alanine aminotransferase (ALT) assay kit (purchased from Shenzhen Leidu Life Science and Technology Co., Ltd., Catalog No.: S03030), aspartate aminotransferase (AST) assay kit (purchased from Shenzhen Leidu Life Science and Technology Co., Ltd., Catalog No.: S03040), total bilirubin (TBIL) assay kit (purchased from Changchun Hui Li Biological Technology Co., Ltd., Catalog No.: C120), direct bilirubin (DBIL) assay kit (purchased from Changchun Hui Li Biological Technology Co., Ltd., Catalog No.: C119), iron determination test kit (purchased from Changchun Hui Li Biological Technology Co., Ltd., Catalog No.: C016) are used to test the concentration levels of ALT, AST, TBIL, DBIL, and iron ions (Fe + ) in the plasma of each group of animals according to the instructions of each kit.

[0088] 3.3 Mouse liver pathological HE staining

[0089] 3.3.1 Sample preparation

[0090] After the experimental mice are executed by cervical dislocation, the liver is first slowly and uniformly infused with preheated 37℃ physiological saline solution. When the liver tissue color is observed to be uniformly faded to light yellow, the perfusion process is terminated, and then immediately samples are taken from the same anatomical region of the left lobe and immersed in a 4% paraformaldehyde solution for fixation. The tissue samples need to be fixed for 24 hours under low temperature conditions (4℃).

[0091] 3.3.2 Embedding

[0092] (1) After 24 hours of fixation, the liver tissue needs to be morphologically adjusted, and then a gradient dehydration process is performed. Specifically, the tissue is sequentially treated with 70%, 80%, 95%, and 100% ethanol. After the dehydration process, the tissue is transparentized in two cylinders of xylene solution for 35 minutes each;

[0093] (2) After the preheated liquid paraffin is injected into the embedding mold, the transparentized tissue is quickly positioned in the mold, and the identification label is attached simultaneously. A -20°C low-temperature solidification system is used to accelerate the wax solidification. After the wax block is completely hardened, morphological adjustment is performed using a sectioning knife to ensure that the edge of the embedding block is flat and a suitable amount of edge paraffin is retained, laying a foundation for the subsequent sectioning process.

[0094] 3.3.3 Sectioning and spreading

[0095] The paraffin-embedded tissue block is placed in a microtome, and the instrument parameters are adjusted to cut a wax section with a thickness of about 4 μm. The wax section is transferred to a thermostatic water bath device using ophthalmic forceps, and the water temperature is controlled within the range of 40-45°C. The liquid surface tension is used to naturally stretch the tissue. After the wax section is completely flattened, a pre-treated glass slide is used to adsorb it from below the liquid surface to ensure that the wax section and the glass slide are tightly attached without air bubbles. Then the glass slide is transferred to a 60°C constant temperature oven, and the drying process is continued until the water is completely evaporated, and a stable bond is formed between the wax and the glass slide. Finally, the dried sample is stored in a dry environment at room temperature for subsequent staining or microscopic observation.

[0096] 3.3.4 HE staining

[0097] The liver tissues of the animals in each group are stained with hematoxylin-eosin (H&E) high-definition constant staining kit (purchased from Servicebio) according to the method described in the kit instructions.

[0098] 3.4 Data statistics and analysis

[0099] In this study, SPSS21 and GraphPad Prism software are used for data processing and visualization. The experimental data are expressed in the form of mean value combined with dispersion index. In terms of statistical method selection, first, the normality test is used to judge the data distribution characteristics. If it conforms to the normal distribution, the parametric test is used, and the independent sample t-test is used for comparison of two independent samples. If the data shows non-normal distribution characteristics, non-parametric methods are selected. For comparison and analysis of multiple data, when the variance homogeneity condition is met, one-way analysis of variance (ANOVA) is performed. If the variance is not homogeneous, non-parametric test is used for difference analysis between groups. Non-normal data are described by median combined with interquartile range P50 (P25-P75). The threshold for significance test is set to P<0.05.

[0100] 4. Results

[0101] 4.1 Survival and general condition of mice

[0102] Before the start of the modeling intervention, all experimental subjects showed stable physiological characteristics. Specifically, the rodent animals had soft and shiny fur, no hair disorder or alopecia; the subcutaneous tissue was well filled, the eyelids opened and closed freely, and the pupils were sensitive to light. At the behavioral level, all groups maintained a high level of autonomous activity, and showed rapid directional response to sound and light stimuli, without abnormal neurological reflexes such as lethargy or excessive excitement.

[0103] On the 10th day of the alcohol induction experiment (i.e. the 5th day of alcohol liquid diet intervention), the model group of rodents showed significant physiological and behavioral changes compared with the normal control group. Through systematic observation, it was found that the model group showed progressive decline in hair luster, significant decrease in behavioral activity, prolonged neurological response time, decreased eye movement sensitivity, and physiological characteristics of decreased excrement viscosity. In addition, the animals in this group began to show aggressive behavior among the same species in group interaction. In contrast to the above-mentioned model group, the SF intervention group and the QGHS group showed relative advantages in overall condition. Specifically, the activity of these experimental group individuals was maintained well, and no obvious group conflict was observed. In terms of surface characteristics, the hair of these individuals was close to that of the control group, the decline in neurological reflex function was smaller, and the digestive system was relatively stable.

[0104] On the 15th day of the alcohol intervention experiment (i.e. after 10 days of continuous intake of alcohol liquid diet), the model group of experimental animals showed significant physiological changes: the hair was fluffy and dull, the response to external stimuli was delayed, the wake-up cycle was shortened and showed a tendency to sleepiness, the eye was weak, and the digestive system function was disordered, showing abnormal fecal water content and intermittent diarrhea. In comparison, the overall condition of the SF group and the QGHS group was significantly better than that of the model group, and the specific characteristics included the following aspects: the hair was clean and elastic, the behavioral activity was high, the circadian rhythm was maintained, and the excrement shape was maintained, the texture was uniform and there was no obvious abnormality, and the incidence of fighting behavior among groups was significantly reduced.

[0105] Survival: During the entire experimental period, no mice in the control group died; 3 mice in the model group died, of which 2 died due to being bitten by another mouse in the same cage; 2 mice in the Shuifeiji Bini group died; 2 mice in the Qinggan Huashi Jiejiu Granules high-dose group died, one of which died due to improper gavage operation, and the other may have died due to the relatively thick drug solution in the high-dose group, which was not tolerated.

[0106] 4.2 Analysis of mouse body weight

[0107] During the experimental period, the initial and final body weights of mice in each group showed no significant difference (P>0.05), indicating that the balance of caloric intake during the modeling stage was effectively controlled, thus reducing the systematic error caused by uneven distribution of dietary calories and ensuring the reliability of experimental data. See Figure 1 .

[0108] 4.3 Analysis of liver index of mice

[0109] Compared with the control group, the liver index of mice in the model group showed a very significant increase (P<0.01), while after intervention with SF aqueous solution or QGHS prescription, the liver index of animals in both groups was effectively reduced (P<0.05), but the statistical difference between the two treatment methods did not reach a significant level. The results confirmed that the liquid diet successfully constructed the ALD animal model. See Figure 2 .

[0110] 4.4 Determination of liver function ALT, AST, TBIL, DBIL and Fe+ indicators of mice

[0111] After 16 days of liquid diet intervention experiment, compared with the normal control group, the liver function detection indexes AST and ALT, TBIL, DBIL, Fe+ of the experimental animals in the model group showed a very significant upward trend (P<0.01). Experimental data showed that SF preparation and QGHS compound could effectively reduce the abnormal increase of AST, ALT, TBIL, DBIL and Fe+ in the peripheral blood of experimental animals, and both groups of intervention measures showed statistically significant improvement effect (P<0.01). See Figure 3 , Table 3.

[0112] Table 3 Summary of index data

[0113]

[0114]

[0115] Note: Compared with the CON group, * P<0.01; compared with the ALD group, △ P<0.05, △△ P<0.01.

[0116] 4.5 Pathological analysis of liver of mice

[0117] The normal control group rats showed complete liver lobule structure, and the hepatocytes were distributed radially around the central vein. No inflammatory cell infiltration or necrosis was observed. The model group showed significant pathological changes, with disordered arrangement of hepatocytes and diffuse fatty degeneration. A large number of atypical lipid vacuoles appeared in the cytoplasm. Compared with the model group, the hepatocyte arrangement of the SF treatment group tended to be regular, and the inflammatory infiltration was significantly reduced. A small amount of lipid vacuole was observed. The liver cord structure of the low-dose traditional Chinese medicine group partially recovered, and a small amount of lipid droplet vacuole residue was occasionally observed. The liver tissue structure of the medium and high-dose traditional Chinese medicine groups was close to normal, with clear liver lobule outline. Only a few areas had mild inflammatory reactions, and a small amount of lipid droplet vacuole was observed (20 times field of view under a microscope). Figure 4 .

[0118] 5. CONCLUSION

[0119] In this study, Lieber-DeCarli liquid diet combined with a single high-dose ethanol gavage method was used to construct an alcoholic liver disease animal model (NIAAA). This experimental design fully simulates the pathogenic mode of long-term alcohol abuse accompanied by acute alcohol intake in humans. During the model construction phase of the experimental period, the body weight data of the experimental animals in each group showed no statistically significant difference (P>0.05), which reflects the good stability of energy metabolism regulation under nutritional intervention conditions. Through histopathological evaluation of liver tissue, it was found that the model group showed typical large-bubble fatty degeneration characteristics, accompanied by obvious inflammatory cell infiltration. At the same time, the activities of ALT and AST in the peripheral blood were significantly increased. The simultaneous increase in the above pathological characteristics and serum transaminase indicators fully verified the successful establishment of the ALD animal model. The experiment verified the repair effect of Qinggan Huashi Jiejiu Granules and Shui Fei Ji Bin on alcoholic liver injury through multiple indicators. In the efficacy evaluation phase, Qinggan Huashi Jiejiu Granules and Shui Fei Ji Bin were used for 8-day intervention treatment, respectively. Experimental data showed that the animal model induced by liquid feed successfully established the pathological state of liver function damage, and the regulation effect of the two intervention schemes on liver enzyme indicators had curative effect. The liver weight index and serum transaminase levels of the treatment group were significantly lower than those of the model group, and the liver histopathology improved significantly, with reduced lipid deposition range and reduced inflammatory cell infiltration.

[0120] In summary, Qinggan Huashi Jiejiu Granules can improve the mental state of ethanol-induced NIAAA mouse models, reduce serum ALT, AST, DBIL, TBIL, Fe + indicators, reduce inflammatory cell infiltration in liver tissue, and reduce the generation of lipid droplet vacuoles.

[0121] Example 2: Intervention effect of Qinggan Huashi Jiejiu Granules on oxidative function and inflammation changes in ALD model mice

[0122] 1. Experimental method

[0123] The liver tissues and blood plasma of each group of mice obtained in Experimental Example 1 were detected by the following method.

[0124] (1) Detection of the contents of IL-6, IL-1β, MDA, SOD and GSH in mouse plasma

[0125] Mouse interleukin 6 (IL-6) enzyme-linked immunoassay (ELISA) kit (purchased from Wuhan Genemei Technology Co., Ltd., JYM0012Mo), mouse interleukin 1β (IL-1β) enzyme-linked immunoassay (ELISA) kit (purchased from Wuhan Genemei Technology Co., Ltd., JYM 0531Mo), mouse malondialdehyde (MDA) enzyme-linked immunoassay (ELISA) kit (purchased from Wuhan Genemei Technology Co., Ltd., JYM0345Mo), mouse glutathione (GSH) enzyme-linked immunoassay (ELISA) kit (purchased from Wuhan Genemei Technology Co., Ltd., JYM0743Mo), and mouse superoxide dismutase (SOD) enzyme-linked immunoassay (ELISA) kit (purchased from Wuhan Genemei Technology Co., Ltd., JYM0346Mo) were used, and the concentration levels of IL-6, IL-1β, MDA, GSH and SOD in the plasma of each group of animals were tested according to the instructions of each kit.

[0126] (2) Oil red O staining of mouse liver tissue

[0127] (1) OCT embedding and sectioning: Fresh liver tissue was taken and OCT embedded, and thin sections were cut using a freezing microtome and temporarily stored in a refrigerator.

[0128] (2) Sample pretreatment: The frozen sections stored in the refrigerator were taken out, dried after rewarming in a normal temperature environment, and then immersed in tissue fixing solution for 15 minutes, followed by rinsing with running water and natural drying.

[0129] (3) Lipid staining procedure: Saturated oil red O mother liquor and deionized water were mixed at a ratio of 6:4, and after double low-temperature standing and qualitative filter paper filtration, a staining working solution was obtained. The treated sections were completely immersed in the staining solution, and staining was performed in the dark for 8-10 minutes.

[0130] (4) Background removal and washing: After staining was completed, the sample was briefly placed for 3 seconds, and then transferred to two groups of 60% isopropanol solution for gradient treatment (3 seconds in the first cylinder and 5 seconds in the second cylinder), followed by immersion and washing in two groups of pure water for 10 seconds each to remove residual reagents.

[0131] (5) Nucleus counterstaining and optimization: The sample was counterstained with hematoxylin staining solution for 3-5 minutes, washed with pure water for three times (5 seconds / 10 seconds / 30 seconds), then treated with acid differentiation solution for a short time (2-8 seconds), washed with distilled water and blued with blue solution for 1 second, and finally developed by two groups of tap water immersion (5 seconds / 10 seconds) to complete the chromatin development.

[0132] (6) Sample storage technology: Glycerol gelatin was selected as the mounting medium, which was evenly covered between the glass slide and the cover glass, and special attention was paid to exclude air bubble interference.

[0133] (7) Microscopic observation and recording: The optical microscope was used to observe the staining results systematically, and high-definition images were collected simultaneously for subsequent quantitative analysis of lipid deposition.

[0134] 2. Results

[0135] (1) Intervention effect of oxidative stress indicators

[0136] Compared with the normal control group, the GSH and SOD levels of the model group mice showed a significant downward trend, while the MDA content increased significantly, and the differences between groups reached the statistical threshold (P<0.05). In the intervention experiment, the experimental groups receiving QGHS and SF treatment showed significant improvement: compared with the untreated model group, the GSH and SOD activities of these two groups were significantly increased, while the MDA level was significantly decreased, all with statistical significance (P<0.05). As shown in Table 4. Figure 5

[0137] Table 4 Data analysis of oxidative stress indicators

[0138]

[0139] Note: compared with the CON group, * P<0.01; compared with the ALD group, △ P<0.05, △△ P<0.01.

[0140] (2) Regulation effect on inflammatory factors

[0141] Experimental data analysis showed that compared with the normal control group, the expression levels of IL-6 and IL-1β in the serum of the model group rats showed a significant upward trend, and the difference between groups was statistically significant (P<0.05). In the intervention experiment, the concentrations of the above two inflammatory factors in the QGHS medium and high dose groups and the SF group were significantly lower than those in the model group, and statistical significance was observed (P<0.01). This result showed that QGHS and SF intervention could effectively regulate the expression level of inflammatory factors. As shown in Table 5. Figure 6 , Table 5.​

[0142] Table 5 Inflammatory factor index data analysis

[0143]

[0144]

[0145] Note: compared with the CON group, * P < 0.01; compared with the ALD group, △ P < 0.05, △△ P < 0.01.

[0146] (3) Oil red staining pathological analysis

[0147] Through histological observation, it was found that a large number of orange-red lipid granules accumulated in the cytoplasm of hepatocytes in the model group, and the degree of lipid deposition was significantly higher than that in the normal control group. The orange-red lipid droplets in the SF group were slightly reduced compared with the model group. After intervention with QGHS, although the low-dose group still had extensive lipid droplet distribution, the deposition amount was reduced compared with the model group. With increasing doses, the number of lipid granules in hepatocytes in the medium and high-dose groups showed a gradient decrease, and the lipid droplets in the cells of the two groups were significantly reduced. For example, Figure 7 .

[0148] 3. Summary

[0149] Qinggan Huashi Jiejiu Granules can improve the degree of oxidative stress indicators in NIAAA mouse models induced by ethanol, that is, increase the activities of GSH and SOD, and reduce the level of MDA. And it can reduce the content of IL-6, IL-1β inflammatory factors, and reduce the lipid deposition of liver tissue sections.

[0150] Example 3: Exploration of the effect of Qinggan Huashi Jiejiu Granules on alcoholic liver injury model mice based on Nrf2-Keap1 signaling pathway

[0151] 1. Experimental materials

[0152] 1.1 Experimental animals and specific modeling methods

[0153] The same as example 1.

[0154] 2. Experimental methods

[0155] 2.1 Mouse liver tissue sampling

[0156] The same as example 1.

[0157] 2.2 Detection of Nrf2, Keap, HO-1, PRDX3 and GPX4 protein expression by WB technology

[0158] 2.2.1 Tissue total protein extraction:

[0159] ①Tissue block with pre-cooled PBS 2-3 times, remove blood, cut into small pieces in the homogenate tube, add 2 4mm homogenate beads, add 10 times the volume of the tissue lysis buffer, set the homogenization program for homogenization;

[0160] ②The homogenate tube is taken out and placed on ice for 30 min, shake every 5 min to ensure complete lysis of the tissue;

[0161] ③12000 rpm, 4℃, centrifuge for 10 min, collect the supernatant, which is the total protein solution.

[0162] 2.2.2 BCA method for protein quantification

[0163] ①According to the number of liver tissue samples, prepare enough BCA working solution according to the ratio of reagent A:B 1:50, and mix thoroughly.

[0164] ②According to the sample quantity, prepare appropriate amount of BCA working solution according to the ratio of 50 volumes of BCA reagent A to 1 volume of BCA reagent B (50:1), and mix thoroughly;

[0165] ③Add 200 μl of BCA working solution to each well, dilute the sample to be tested, so that the total volume of the sample diluent is 10 μl, add 200 μl of BCA working solution, mix thoroughly, and place in a 37℃ constant temperature box for 30 min. Then use the enzyme marker to measure the absorbance of the sample at 562 nm wavelength. According to the standard curve, calculate the content of the extracted liver protein.

[0166] 2.2.3 SDS-PAGE protein electrophoresis

[0167] (1) Gel preparation

[0168] ①Glass plate pretreatment: clean the glass plate for electrophoresis with detergent and distilled water in turn, remove surface impurities, and dry for standby. Then assemble it to the gel preparation support and clamp it tightly to ensure the device is stable and not loose.

[0169] ②Separation and pouring: select the appropriate separation gel formula according to the molecular weight of the target protein, finally introduce APS and TEMED catalyst, and quickly stir to mix the solution thoroughly. Pour the mixed separation gel along the inner wall of the glass plate into the gel chamber,

[0170] ③Seal the gel and polymerize: immediately cover the surface of the separation gel with a layer of deionized water or isopropanol to isolate oxygen, and stand at room temperature for about 30 minutes until a clear interface is formed between the gel and the water layer, indicating that the polymerization is complete.

[0171] ④Treat the surface of the separation gel: pour off the top sealing liquid, absorb the remaining water with filter paper, and ensure that the gel surface is completely dry and free of liquid to avoid interference with subsequent operations.

[0172] ⑤Concentrated gel: Mix according to the concentrated gel recipe, and add APS and TEMED at the last stage. Mix rapidly to form a homogeneous solution. Pour the concentrated gel solution along the inner wall of the glass plate on top of the resolving gel until the liquid surface is level with the top end of the short glass plate. Then insert the sample comb vertically, taking care to adjust the angle to avoid air bubbles between the teeth.

[0173] ⑥Concentrated gel solidification: Let it stand at room temperature for 20-30 minutes until the concentrated gel is fully polymerized. After solidification, gently pull out the comb and rinse the sample well with distilled water to remove the unpolymerized gel fragments. The gel preparation is now complete.

[0174] (2) Electrophoresis

[0175] ①Sample preparation: Add the same volume of 5x loading buffer to each sample with the same total protein content; mix well, and then boil in a water bath for 10 minutes.

[0176] ②Sample loading: Prepare 1x concentration of the electrophoresis buffer for use. After the concentrated gel is fully solidified, transfer the gel tray to the electrophoresis tank and fix it. Pour the buffer into the tank until it completely covers the surface of the gel, then remove the sample comb vertically. When operating, slowly pull it out on one side, and use the calibrated micropipette to slowly inject along the side wall of the sample well.

[0177] ③Electrophoresis: Use the constant voltage mode in stages: connect the electrophoresis tank to the electrophoresis instrument, and use the initial stage of 80V for pre-electrophoresis. After the bromophenol blue tracer migrates to the interface of the resolving gel, adjust it to 120V constant voltage. Continue electrophoresis until the tracer reaches the edge of the bottom of the gel, and then terminate the electrophoresis process.

[0178] 2.2.4 Transfer

[0179] ①Cut the PVDF membrane to the appropriate size, activate it in methanol to make it uniform and transparent, then rinse it with distilled water. Soak the membrane, gel, sponge pad, and filter paper in the pre-cooled transfer solution, and balance for 15 minutes.

[0180] ②Starting from the negative side, stack them in the following order: pre-wetted sponge pad → 3 layers of filter paper soaked in transfer solution → balanced gel → PVDF membrane → 3 layers of filter paper → another sponge pad. When stacking each layer, use a glass rod or roller to remove air bubbles and ensure that each layer is tightly attached. Finally, lock the transfer clamp to form the transfer "sandwich".

[0181] ③Place the assembled transfer clamp vertically in the electrophoresis tank, and add enough pre-cooled transfer solution to cover the device. To control the temperature, place the transfer tank in an ice bath environment or surround it with ice. Connect the power supply and set the constant voltage to 80V for continuous transfer, with a total duration of 3 hours.

[0182] 2.2.5 Blocking and hybridization

[0183] ①Blocking: After the completion of the electrotransferring procedure, the PVDF membrane was transferred into the pre-prepared blocking solution (5% skim milk powder) and placed on a horizontal shaker for room temperature blocking. The shaking condition was maintained for 60 minutes.

[0184] ②Primary antibody incubation: According to the technical parameters of the reference antibody, the primary antibody was diluted with the same concentration of blocking solution. The blocked membrane was transferred into a special incubation bag, and the diluted primary antibody working solution was injected into the bag. After the air bubbles were removed, the bag was sealed and placed on a room temperature shaker for dynamic incubation. After the reaction was completed, the membrane was washed with TBST solution for three times on a shaker for 5 minutes each time to remove the residual antibody.

[0185] ③Secondary antibody incubation: The secondary antibody was diluted at a ratio of 1:5000. The PVDF membrane was completely immersed in the diluted solution and incubated on a room temperature shaker for 120 minutes. After the reaction was terminated, the TBST washing process was repeated three times for 5 minutes each time to ensure that the unbound label was completely removed.

[0186] 2.2.6 Exposure identification

[0187] Take 200 μl of each of the luminescence solutions A and B and place them in a centrifuge tube. Mix well to prepare the working solution. Transfer the rinsed PVDF membrane to the surface of a plastic film and use filter paper to absorb the residual liquid on the membrane surface. Apply the mixed luminescence solution evenly to the surface of the PVDF membrane, avoiding the formation of air bubbles. Then cover and seal the membrane with a plastic film. Place the covered PVDF membrane into a chemiluminescence imager and start the automatic exposure program according to the preset parameters. Capture and save the film image.

[0188] 2.2.7 Scanning and analysis

[0189] After digital imaging of the film, the optical density parameters of the specific bands were measured using Quantity One software.

[0190] 2.3 qRT-PCR method for detecting the expression of liver Nrf2, Keap1, HO-1, PRDX3 mRNA

[0191] 2.3.1 Extraction of total RNA from samples

[0192] TRNzol universal RNA extraction reagent was used according to the manufacturer's instructions. The liver tissue was ground into powder in a pre-cooled mortar and processed according to the following procedure:

[0193] ①Lysis treatment: Add an appropriate amount of Trizol reagent to the tissue powder and let it stand at room temperature for 5 minutes to facilitate cell lysis.

[0194] ② Phase separation: add 0.2 mL chloroform, shake vigorously by hand, mix well and incubate at room temperature for 5-10 minutes. Then centrifuge at 12000 rpm for 15 minutes (4°C), transfer about 70% of the volume of the upper aqueous phase to a new centrifuge tube.

[0195] ③ RNA precipitation: add an equal volume of pre-cooled isopropanol at -20°C to the aqueous phase, mix gently by inverting and then ice-bath for 10 minutes. Centrifuge at 12000 rpm for 15 minutes (4°C), discard the supernatant.

[0196] ④ Wash and purify: add 1 mL of pre-cooled 75% ethanol to the precipitate, shake to suspend and then centrifuge at 12000 rpm for 5 minutes at 4°C, completely remove the wash solution.

[0197] ⑤ Dissolve RNA: dry the precipitate at room temperature for 5 minutes to a translucent gel, and use RNase-Free ddH2O to dissolve the RNA precipitate

[0198] ⑥ Concentration detection: use ND-2000 spectrophotometer, with RNase-Free ddH2O as blank control, take 1 μL sample on the detection base to determine the absorbance value, evaluate the RNA purity and concentration.

[0199] 2.3.2 Primer information

[0200] The primer synthesis was completed by Wuhan Saiver Biotechnology Co., Ltd., the internal reference gene was selected as mouse GAPDH, and the primer sequence and length of the detection gene are shown in the table.

[0201] Table 6 Primer information

[0202]

[0203] 2.3.3 Reverse transcription to synthesize cDNA

[0204] 1) Preparation of reverse transcription reaction system

[0205] Table 7 Reaction system

[0206]

[0207] 2) Mix gently and centrifuge

[0208] 3) Set the reverse transcription program on the ordinary PCR instrument to complete the reverse transcription.

[0209] 2.3.4 Quantification

[0210] The product was taken as GAPDH as an internal reference, 0.1 ml PCR reaction plate was prepared, and the following reaction system was prepared, 3 tubes for each reverse transcription product. After spotting the sample, the PCR sealing film was sealed with a sealing film instrument, and the micro-pore plate centrifuge was centrifuged. The amplification was completed on the fluorescence quantitative PCR instrument. The PCR amplification reaction conditions were 95°C pre-denaturation for 30 s, 95°C denaturation for 15 s, 60°C annealing for 30 s, 40 cycles, and 2 -ΔΔCt The relative quantitative analysis was performed on the target gene.

[0211] Table 8 reaction system

[0212]

[0213] Table 9 PCR amplification conditions and process

[0214]

[0215] 3. Results

[0216] 3.1 Nrf2, Keap1, HO-1, PRDX3 and GPX4 protein expression

[0217] The experimental data analysis showed that, compared with the normal control group, the expression levels of Keap1 and PRDX3 proteins in the model group showed a statistically significant increasing trend (P<0.01), while the expression levels of Nrf2, HO-1 and glutathione peroxidase 4 (GPX4) proteins were statistically significantly decreased (P<0.01).

[0218] In the treatment intervention group, the SF group and the low, medium and high dose groups of traditional Chinese medicine all showed a regulating effect on the abnormal expression of the model group, that is, the Keap1 protein level of the three groups was lower than that of the model group, and the expression levels of Nrf2, HO-1 and GPX4 proteins were up-regulated, and all the differences reached statistical significance (P<0.01). For PRDX3, the SF group and the medium and high dose groups of traditional Chinese medicine all showed a regulating effect on the abnormal expression of the model group, that is, the PRDX3 protein level of the corresponding group was lower than that of the model group and had statistical significance. There was a gradient difference in the expression regulation amplitude between the treatment groups, among which, the QGHS medium and high dose groups showed more obvious regulating effect. See Figure 8 , Table 10.

[0219] Table 10 summary of WB index data of mouse liver tissue in each group

[0220]

[0221]

[0222] Note: compared with the CON group, * P<0.01; compared with the ALD group, △ P<0.05, △△ P<0.01.

[0223] 3.2 mRNA expression of Nrf2, Keap1, HO-1 and PRDX3 genes

[0224] The experimental data analysis showed that, relative to the normal control group, the model group experimental subjects showed significant changes in the mRNA expression levels of Nrf2, Keap1, HO-1 and PRDX3 genes (P<0.05). In addition, after the intervention of different doses of QGHS, the detection data of the low, medium and high dose groups showed that the transcriptional activity of Nrf2 and HO-1 genes was improved compared with the model group. Compared with the model group, the expression fold of the Keap1 gene in the high dose group had a significant decrease, which was statistically significant (P<0.05). Compared with the model group, the expression fold of the PRDX3 gene in each group had a significant decrease, which was statistically significant (P<0.05). See Table 11. Figure 9 , Table 11.

[0225] Table 11 qRT-PCR statistical data table

[0226]

[0227] Note: compared with the CON group, * P<0.01; compared with the ALD group, △ P<0.05, △△ P<0.01.

[0228] 4. Conclusion

[0229] Qinggan Huashi Jiejiu Granules can increase the expression levels of Nrf2, HO-1 protein and mRNA, and GPX4 protein in the liver tissue of alcohol-induced alcoholic liver injury mice, and can reduce the expression levels of Keap1 and PRDX3 protein mRNA. Nrf2-Keap1 signaling pathway and ferroptosis may be important signal pathways for the treatment of ALD by this compound.

[0230] Experimental Example 4 Clinical Experiment

[0231] 1. Clinical data

[0232] 1.1 Basic information

[0233] The research cases come from: 60 patients with liver stagnation and spleen deficiency, internal accumulation of damp-heat in the liver from October 2022 to December 2024 were selected from the Hepatitis Clinic of Guang'anmen Hospital of China Academy of Chinese Medical Sciences. The patients were treated with Qinggan Huashi Jiejiu Granules for 24 weeks. Before and after treatment, the safety indicators such as blood, urine, and stool routine were checked once; liver function, color Doppler ultrasound, and other indicators were checked before treatment, 12 weeks and 24 weeks of treatment, and the patient's TCM syndrome score was recorded once, so as to evaluate the effectiveness and safety of Qinggan Huashi Jiejiu Granules in the treatment of ALD.

[0234] 1.2 Diagnostic criteria

[0235] 1.2.1 Western medicine diagnostic criteria

[0236] Reference Alcohol Liver Disease Prevention and Treatment Guidelines (2018 Update Edition) of Chinese Medical Association Hepatology Branch, Fatty Liver and Alcohol Liver Disease Group. The diagnostic criteria for alcoholic liver disease are as follows: those who meet item (1) and exclude other causes of liver disease, and those who meet items (3) and (4) can be diagnosed with alcoholic liver disease.

[0237] (1) There is a long history of drinking, generally more than 5 years, equivalent to male alcohol consumption of more than or equal to 40g / d, and female alcohol consumption of more than or equal to 20g / d; or a large amount of drinking history within 2 weeks, equivalent to alcohol consumption of more than 80g / d. The alcohol consumption (g) conversion formula = drinking amount (mL) x alcohol content (%) x 0.8;

[0238] (2) Clinical symptoms are non-specific, may be asymptomatic, or have right upper quadrant pain, loss of appetite, fatigue, weight loss, jaundice, etc.; as the disease worsens, there may be neuropsychiatric symptoms, spider veins, liver palms, etc.

[0239] (3) AST, ALT, GGT, TBil, PT, etc. The level of index increases;

[0240] (4) Liver B-ultrasound, CT, magnetic resonance imaging or transient elastography examination has typical manifestations.

[0241] 1.2.2 TCM syndrome differentiation criteria

[0242] Reference Chinese Medical Association Hepatology Branch, Fatty Liver and Alcohol Liver Disease Group. Alcohol Liver Disease Prevention and Treatment Guidelines (2018 Update Edition), Chinese National Standard TCM Clinical Diagnosis and Treatment Terms Part, and Guidelines for Clinical Research on New Drugs of Traditional Chinese Medicine (Trial).

[0243] The diagnostic criteria for liver stagnation and spleen deficiency, internal accumulation of damp-heat in the liver are as follows:

[0244] Main symptoms: ① fatigue and lack of energy; ② right flank discomfort or distension; ③ abdominal distension; ④ sticky and unsmooth stool.

[0245] Secondary symptoms: ① yellowish complexion; ② dry mouth and bitter taste; ③ loss of appetite; ④ nausea and vomiting.

[0246] Tongmai: pale red or red tongue, yellow greasy fur, stringy and slippery pulse.

[0247] The above main symptoms 2 items plus secondary symptoms 1 or 2 items are required, and the tongue and pulse conditions are also referred to.

[0248] 1.3 Inclusion Criteria

[0249] (1) Meet the diagnostic criteria for alcoholic liver disease, and the liver function ALT, AST or γ-GT is greater than 1.5 times the normal value at the time of enrollment;

[0250] (2) Meet the syndrome differentiation criteria of liver stagnation and spleen deficiency, internal accumulation of dampness and heat;

[0251] (3) Age between 18-65 years old, gender unrestricted;

[0252] (4) Willing to participate in this research and sign the informed consent form.

[0253] 1.4 Exclusion Criteria

[0254] (1) Patients with other viral hepatitis such as hepatitis B and C;

[0255] (2) Patients with autoimmune liver disease, drug-induced liver disease, toxic hepatitis, liver malignancy, non-alcoholic fatty liver, occult hepatitis, and patients with unexplained liver disease;

[0256] (3) Pregnant or lactating women, people with allergic constitution;

[0257] (4) Patients with mental abnormalities who cannot cooperate with the study;

[0258] (5) Patients with severe heart, brain, lung, kidney, hematopoietic, immune and other system diseases;

[0259] (6) Other conditions considered unsuitable for enrollment by the researcher (including alcohol-dependent population).

[0260] 1.5 Criteria for withdrawal, exclusion, dropout, and termination of the study

[0261] (1) Withdrawal criteria: ① Patients lost to follow-up; ② Patients request to withdraw from the study; ③ Researchers believe that the study subjects are not suitable for continuing the study.

[0262] (2) Exclusion criteria: study subjects who should not have been enrolled but have been enrolled, including misenrollment, misdiagnosis, one-time drug use, and no detection records. Excluded cases should be explained, CRF table records are kept for reference, and are not included in the efficacy statistical analysis. However, at least one treatment is received, and at least one safety record is included in the adverse reaction analysis.

[0263] (3) Drop-out criteria: cases that have been enrolled but failed to complete clinical observation, including self-withdrawal, loss of follow-up, poor compliance, intercurrent illness, and re-drinking during treatment ordered by the physician. The reasons for drop-out should be explained. If there are baseline efficacy data, the last primary efficacy indicator result of the case will be transferred as the final result for statistical analysis, and the CRF table will be kept for reference.

[0264] (4) Termination criteria: including the following aspects: ① the researcher finds safety problems, poor efficacy, or major errors in the program; ② the sponsor cannot continue the clinical study due to funding or management reasons; ③ the administrative supervisor cancels the trial. When the trial is terminated, all CRF tables should be kept for reference.

[0265] 1.6 Bias control

[0266] The researcher explains the case report form filling and follow-up matters to the subjects. After the subjects give their informed consent, the data collection is carried out. After the data collection is completed, the researcher conducts data verification in a timely manner. If there are errors and omissions, the researcher is required to correct them in a timely manner. When modifying, the original records should be kept clear and visible, and the corrected parts should be signed by the researcher and dated.

[0267] 2. Methods

[0268] 2.1 Study design

[0269] 1. Sample size estimation: Referring to the determination of pre-test sample size in traditional Chinese medicine clinical research, under the condition that the research goal is to explore moderate standard effect size, based on the design principle of minimizing the expected total sample size, the initial sample size is set to 50 cases, to balance the statistical efficiency and resource input efficiency. To ensure the integrity of the research data and pre-estimate the buffer space for sample loss, a 20% drop-out rate is estimated, so the final total sample size is tentatively set to 60 cases.

[0270] 2. A prospective self-before-after paired trial research method is used to select 60 patients with alcoholic liver disease who meet the inclusion and exclusion criteria and are treated at the Hepatitis Clinic of Guang'anmen Hospital of China Academy of Chinese Medical Sciences from October 2022 to December 2024 for 24 weeks. The study has been approved by the ethics committee.

[0271] 2.2 Treatment regimen

[0272] The Qinggan Huashi Jiejiu Granules are prepared by the method of Example 1. Usage and dosage: 26.4g of Qinggan Huashi Jiejiu Granules are taken with 150ml of water each time, twice a day, half an hour after meals. Patients enrolled in the study need to abstain from alcohol during treatment.

[0273] 2.3 Observation indicators

[0274] 2.3.1 General indicators

[0275] (1) Demographic data: gender, age, height, weight, etc.

[0276] (2) General condition: vital signs, physical examination, etc.

[0277] (3) Factors affecting efficacy: disease duration, comorbidities, pre-treatment medication, etc.

[0278] 2.3.2 Safety indicators

[0279] (1) Blood, urine, and stool routine tests.

[0280] (2) Renal function

[0281] 2.3.3 Efficacy indicators

[0282] (1) Primary efficacy indicators: liver function indicators: ALT, AST, GGT, TBIL, ALP.

[0283] (2) Secondary efficacy indicators: liver fibrosis indicators: APRI; TCM syndrome score; B-ultrasound.

[0284] 2.3.4 Detection time points

[0285] ① Symptoms and signs: observed and recorded before treatment, at 12 weeks of treatment, and after 24 weeks of treatment.

[0286] ② Physicochemical indicators: observed and recorded before treatment, at 12 weeks of treatment, and after 24 weeks of treatment.

[0287] ③ Safety indicators: checked before treatment and at 24 weeks of treatment;

[0288] ④ Adverse reactions: observed in real time according to specific adverse reactions.

[0289] 3. Evaluation criteria

[0290] 3.1 Efficacy evaluation criteria

[0291] Symptom quantification score criteria: Refer to the "Guidelines for Clinical Research of New Drugs of Traditional Chinese Medicine" formulated by the State Food and Drug Administration of the People's Republic of China in 2002 for syndrome efficacy evaluation. Use the nimodipine method to calculate the reduction of syndrome score = [(pre-treatment score - post-treatment score) / pre-treatment score] x 100%.

[0292] ① Clinical cure: main symptoms and signs disappear or basically disappear, with a reduction of syndrome score > 95%;

[0293] ② Marked effectiveness: main symptoms and signs significantly improve, with a reduction of syndrome score < 95% but > 70%;

[0294] ③ Effectiveness: main symptoms and signs significantly improve, with a reduction of syndrome score < 70% but > 30%;

[0295] 4. Invalid: The main symptoms and signs are not significantly improved, or even aggravated, and the syndrome score is reduced by <30%.

[0296] 3.2 Safety evaluation criteria

[0297] The time of occurrence, symptoms, degree, whether treated, disappearance time, etc. of adverse reactions were recorded in detail to determine whether they were related to the medication.

[0298] Grade 1: Safe, with no adverse reactions.

[0299] Grade 2: Relatively safe, with adverse reactions that do not require any treatment to continue medication.

[0300] Grade 3: Safety issues with moderate adverse reactions that can continue after treatment.

[0301] Grade 4: Discontinued due to adverse reactions.

[0302] 4. Statistical methods

[0303] All statistical analysis tests used two-sided hypothesis testing with a hypothesis testing level of α = 0.05. A P value less than or equal to 0.05 was considered to be statistically significant. The description of data statistical description and inference, and the principles of hypothesis testing are as follows:

[0304] ① Measurement indicators use mean and standard deviation, minimum, maximum, and median to statistically describe data distribution. Independent sample t-test (normal distribution) or Wilcoxon rank sum test (non-normal distribution) is used for hypothesis testing.

[0305] ② Count indicators use frequency and percentage to statistically describe data distribution. X2 test / precise or approximate Fisher's test is used for hypothesis testing, and Wilcoxon rank sum test is used for ordinal indicators. Statistical analysis is implemented using SPSS 26.0 statistical software

[0306] 5. Results

[0307] 5.1 General information and baseline characteristics

[0308] 5.1.1 Number of inclusions and reasons for dropout

[0309] This study planned to include 60 people, but due to the fact that 3 of them did not strictly abstain from alcohol during medication, leading to an increase in liver function indicators, the research group actively refused to provide them with medication again. Three people dropped out because they refused to recheck their liver function tests, and two people dropped out because they experienced abdominal pain, persistent loose stools, and mild indigestion symptoms after taking the medication. Therefore, a total of 52 patients with alcoholic liver disease were included.

[0310] 5.1.2 Patient gender, age data

[0311] A total of 52 patients with alcoholic liver disease were included in this study, all male. Age 36-65 years, the average age (50.27±8.51) years. Among them, 16 cases of 36-44 years old (30.8%); 21 cases of 45-54 years old (40.4%); 15 cases of 55-65 years old (28.8%). The age of the population included in this study is concentrated between 36 and 54 years old, see Tables 12, 13.

[0312] Table 12 Patient gender, age, disease duration data

[0313]

[0314] Note: After inspection, the patient's age is in normal distribution (P>0.05).

[0315] Table 13 Age distribution

[0316]

[0317] 5.1.3 Drinking history summary

[0318] Table 14 Patient drinking history, average daily alcohol intake

[0319]

[0320] Note: The patient's drinking history is in normal distribution, and the average daily alcohol intake data is not in normal distribution (P<0.05).

[0321] 5.2 Comparison of liver function indicators

[0322] Statistical test revealed that the key indicators of liver function of the study subjects deviated from the normality assumption before and after the intervention (P<0.05), therefore the non-parametric analysis method was selected, and the median value and its interquartile range were used as statistical parameters. The normality test confirmed that the observation data of TBIL, ALB, ALP indicators before and after treatment met the normality requirements (P>0.05), so the mean ± standard deviation As a statistical description parameter, according to the characteristics of the data, the study used paired sample t test to compare the changes of the indicators before and after the intervention. The results showed that: compared with before treatment, the average levels of ALT, GGT, ALP of patients after 12 weeks of treatment were lower than before (P<0.05), and the average levels of ALT, AST, GGT, ALP of patients after 24 weeks of treatment were lower than before (P<0.05); There was no statistically significant difference in TBIL and ALB levels after 12 weeks and 24 weeks of treatment (P>0.05), see the table below.

[0323] Table 15 Comparison of liver function before and after treatment

[0324]

[0325] Note: *P<0.05 compared with before treatment.

[0326] 5.3 Evaluation of therapeutic effect by complete blood count

[0327] Differences in hematological parameters before and after treatment were analyzed. Normality tests showed that white blood cell count (WBC), red blood cell count (RBC), platelet count (PLT), hemoglobin (HGB), and mean corpuscular volume (MCV) all conformed to a normal distribution (P > 0.05). Data are expressed as mean ± standard deviation. Paired t-tests were used to compare pre- and post-treatment levels. Statistical results showed that after 24 weeks of treatment, there were no statistically significant changes in WBC, RBC, HGB, MCV, and PLT levels compared to baseline (P > 0.05). Detailed data are shown in Table 16.

[0328] Table 16 Comparison of blood routine indicators before and after treatment

[0329]

[0330] 5.4 APRI numerical evaluation

[0331] A differential analysis of APRI levels before and after treatment was performed. Normality tests showed that APRI did not conform to a normal distribution (P > 0.05), therefore the median and interquartile range were used for description, and nonparametric statistical methods were selected. Statistical results showed that after 24 weeks of treatment, the patients' APRI index was significantly lower than the baseline level, and the difference was statistically significant (P < 0.05). Detailed data are shown in Table 17.

[0332] Table 17 Comparison of liver elasticity indices before and after treatment

[0333]

[0334] Note: *P<0.05 compared with before treatment.

[0335] 5.5 Evaluation of Therapeutic Effect by Traditional Chinese Medicine Syndrome Scoring

[0336] 5.5.1 Total Score of Traditional Chinese Medicine Syndrome

[0337] The total TCM syndrome scores of the patients did not conform to a normal distribution (P < 0.05), so the median and interquartile range were used for description, and nonparametric statistical methods were selected. The results showed that compared with before treatment, the total TCM syndrome scores of the patients decreased after 12 and 24 weeks of treatment, and the differences were statistically significant (P < 0.001). See Table 18.

[0338] Comparison of TCM syndrome scores in Table 18

[0339]

[0340] Note: Compared with before treatment, *P<0.001.

[0341] 5.5.2 Single item scores of TCM syndromes

[0342] The data of single item scores of TCM syndromes of patients before and after treatment were not in accordance with normal distribution (P<0.05), and were statistically described by median and interquartile range, and were analyzed by non-parametric test. The results showed that compared with before treatment, the single item scores of TCM syndromes of patients after 24 weeks of treatment were decreased, including lateral costal pain or distending pain, abdominal distention, lassitude, dry mouth, dry mouth, yellowish eyes, loss of appetite, nausea, vomiting, borborygmus, and irritability (P<0.05). See Table 19.

[0343] Objective: To evaluate the safety of the treatment. Methods: The safety of the treatment was evaluated by observing the adverse reactions of patients during the treatment.

[0344] Comparison of single item scores of TCM syndromes before and after treatment

[0345]

[0346] Note: Compared with before treatment, *P<0.05.

[0347] 5.5.3 Effective rate of TCM syndrome scores

[0348] According to the method of nimodipine, the effective rate of 52 patients with alcoholic liver disease after 24 weeks of treatment was calculated. According to the calculation, 0 cases were cured, 3 cases (5.8%) were markedly effective, 41 cases (78.9%) were effective, 8 cases (15.3%) were ineffective, and the total effective number was 44, and the total effective rate was 84.7%. See Table 20.

[0349] Evaluation of TCM syndrome scores (treatment for 24 weeks)

[0350]

[0351] 5.6 Safety evaluation

[0352] In the clinical observation of drug use, 5 patients showed intestinal reactions in the early stage of treatment, with specific manifestations of increased defecation frequency, increased flatus volume, and active intestinal sounds, and other changes in intestinal function. Combined with the physical characteristics of patients and the analysis of drug ingredients, such reactions are mainly due to the diarrhea effect caused by the promotion of intestinal peristalsis by mirabilite in patients with spleen-stomach deficiency and cold constitution. In response to this situation, the clinical treatment adopts a two-way adjustment plan: for those with obvious deficiency and cold, it is recommended to add ginger and jujube and other warm medicinal materials during decoction to regulate the spleen and stomach; for those sensitive to mirabilite, a dose reduction strategy is implemented, with maintaining a slight laxative effect as the adjustment benchmark. These intestinal reactions are not accompanied by whole-body symptoms such as physical decline or mental state abnormalities, and are effectively improved through adaptive adjustment. The rest of the subjects did not experience significant drug-related adverse reactions during the treatment period.

[0353] 6. Conclusion

[0354] The results of the analysis showed that the total effective rate was 84.7%, objectively proving that Qinggan Huashi Jiejiu Granules can benefit patients with alcoholic liver disease, significantly reduce liver function and APRI index in patients with alcoholic liver disease, and relieve symptoms such as stabbing or distending pain in the lateral costal region, abdominal distention, lassitude, dry mouth, yellow eyes, loss of appetite, nausea, vomiting, intestinal rumbling, and irritability.

[0355] Obviously, the above embodiments are only examples for clarity, and are not limitations of the embodiments. Based on the above description, other different forms of changes or variations can be made by those of ordinary skill in the art. All embodiments do not need to be exhausted, and obvious changes or variations still fall within the protection scope of the present application.

Claims

1. A traditional Chinese medicine composition for clearing liver heat, resolving dampness, and relieving alcohol intoxication, characterized in that, The raw materials of the traditional Chinese medicine composition, by weight, include 25-35 parts of kudzu flower, 13-18 parts of Japanese raisin tree fruit, 4-8 parts of scutellaria baicalensis, 4-8 parts of coptis chinensis, 4-8 parts of phellodendron chinense, 18-22 parts of poria cocos, 7-11 parts of magnolia officinalis, 25-35 parts of white peony root, 2-4 parts of mirabilite, 13-18 parts of charred hawthorn, 25-60 parts of astragalus membranaceus, 7-11 parts of prepared pinellia ternata, and 4-8 parts of prepared licorice root.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, According to the weight proportions, the raw materials of the traditional Chinese medicine composition include: 30 parts of kudzu flower, 15 parts of Japanese raisin tree fruit, 6 parts of scutellaria baicalensis, 6 parts of coptis chinensis, 6 parts of phellodendron chinense, 20 parts of poria cocos, 9 parts of magnolia officinalis, 30 parts of white peony root, 3 parts of mirabilite, 15 parts of charred hawthorn, 30 parts of astragalus membranaceus, 9 parts of prepared pinellia ternata, and 6 parts of prepared licorice root; or, 25 parts of kudzu flower, 18 parts of Japanese raisin tree fruit, 4 parts of scutellaria baicalensis, 8 parts of coptis chinensis, 4 parts of phellodendron chinense, 22 parts of poria cocos, 7 parts of magnolia officinalis, 35 parts of white peony root, 2 parts of mirabilite, 18 parts of charred hawthorn fruit, 25 parts of astragalus membranaceus, 11 parts of prepared pinellia ternata, and 8 parts of prepared licorice root; or, 35 parts of kudzu flower, 13 parts of Japanese raisin tree fruit, 8 parts of scutellaria baicalensis, 4 parts of coptis chinensis, 8 parts of phellodendron chinense, 18 parts of poria cocos, 11 parts of magnolia officinalis, 25 parts of white peony root, 4 parts of mirabilite, 13 parts of charred hawthorn, 60 parts of astragalus membranaceus, 7 parts of prepared pinellia ternata, and 4 parts of prepared licorice root.

3. A method for preparing the traditional Chinese medicine composition according to claim 1 or 2, characterized in that, Weigh out the following ingredients according to the selected weight proportions: kudzu flower, Japanese raisin tree fruit, scutellaria baicalensis, coptis chinensis, phellodendron chinense, poria cocos, magnolia officinalis, white peony root, mirabilite, charred hawthorn, astragalus membranaceus, prepared pinellia ternata, and prepared licorice root. Mix them together and extract using conventional extraction methods, or extract them separately using conventional extraction methods and then mix them together to obtain the final product.

4. The method for preparing the traditional Chinese medicine composition according to claim 3, characterized in that, The conventional extraction method is selected from one or more of the following: decoction extraction, reflux extraction, maceration extraction, percolation extraction, ultrasonic extraction, and steam distillation extraction; and / or, the extraction solvent used during extraction is selected from water or an alcohol solution with a volume percentage of 5-98%; and / or, the extraction is performed at least once; each extraction lasts at least 10 minutes; and / or, the volume ratio of the extraction solvent used during extraction to the weight of the raw material is ≥2 L / kg.

5. A pharmaceutical preparation, characterized in that, This includes the traditional Chinese medicine composition according to claim 1 or 2, or the traditional Chinese medicine composition prepared by the preparation method according to claim 3 or 4.

6. The pharmaceutical preparation according to claim 5, characterized in that, The pharmaceutical preparation is a traditional Chinese medicine composition prepared by the traditional Chinese medicine composition according to claim 1 or 2 or the preparation method according to claim 3 or 4, with or without a pharmaceutically acceptable carrier, and prepared into a clinically acceptable dosage form according to conventional processes.

7. The pharmaceutical preparation according to claim 6, characterized in that, The pharmaceutical preparation is a granule, powder, tablet, capsule, pill, decoction, syrup, paste, or suspension; And / or, the pharmaceutically acceptable carrier is selected from at least one of pharmaceutically acceptable solvents, solubilizers, emulsifiers, binders, disintegrants, fillers, lubricants, flow aids, wetting agents, flavoring agents, and preservatives.

8. Use of a traditional Chinese medicine composition according to claim 1 or 2, a traditional Chinese medicine composition prepared by the preparation method according to claim 3 or 4, or a pharmaceutical preparation according to any one of claims 5-7 in the preparation of a medicament for the prevention and / or treatment of alcoholic liver disease.

9. The use according to claim 8, characterized in that, The alcoholic liver disease mentioned is an alcoholic liver disease characterized by liver stagnation and spleen deficiency, and internal accumulation of damp-heat.

10. A traditional Chinese medicine composition according to claim 1 or 2 and / or a pharmaceutical preparation according to claim 3 or 4 having at least one of the following uses (1)-(3): (1) Use in the preparation of drugs for improving liver function; (2) Use in the preparation of drugs that improve the degree of oxidative stress; (3) Use in the preparation of drugs that regulate the Nrf2-Keap1 signaling pathway.

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