Chinese herbal medicine combination for alcohol detoxification and liver protection, and its application

Abstract

Chinese herbal medicine combination for alcohol detoxification and liver protection, characterized in that the Chinese herbal medicine combination comprises the following components: Milk thistle extract, kudzu extract, astragalus extract, and salvia extract; the mass ratio of puerarin to silybin contained in the Chinese drug combination is 1:4 to 4:1.

Description

Technical field

[0001] The present invention belongs to the field of functional Chinese drug combinations and relates in particular to a Chinese drug combination for alcohol detoxification and liver protection as well as its application. State of the art

[0002] With improving living standards and increasing social activities, the number of alcohol-consuming people and their consumption are steadily rising. The liver is the most important metabolic organ for alcohol in the human body, processing over 90% of alcohol. Excessive alcohol consumption increases the metabolic burden on the liver and can even impair health. According to current forecasting models, if current drinking patterns continue, over 3.6 million people in China will develop alcoholic cirrhosis and almost 2 million will die from it between 2022 and 2040.

[0003] Alcohol is absorbed through the gastrointestinal tract and enters the bloodstream, with over 95% being metabolized in the liver, primarily via three pathways: the alcohol dehydrogenase (ADH) system, the cytochrome P450 2E1 (CYP2E1) enzyme system, and the catalase system. Acetaldehyde and reactive oxygen species (ROS), produced during ethanol metabolism, are major contributors to liver damage. Acetaldehyde is a highly toxic substance that directly damages the mitochondria and microtubules of liver cells and can also disrupt DNA synthesis. In long-term alcohol consumers, the expression of CYP2E1 is further increased, leading to a massive accumulation of acetaldehyde and exacerbating liver cell damage.Simultaneously, the reactive oxygen species (ROS) produced during ethanol metabolism recruit immune cells and induce the production of pro-inflammatory factors, triggering oxidative stress and inflammation. They can bind to proteins and alter their structure and function, bind to DNA and form highly carcinogenic polymers, exacerbating liver damage. Furthermore, the accumulation of ROS can inhibit the expression of proteins associated with energy metabolism signaling pathways, leading to fat accumulation in the liver. The invention and its advantages

[0004] To overcome the problems and shortcomings of the prior art, the present invention offers a Chinese herbal combination for alcohol detoxification and liver protection, as well as its application. This Chinese herbal combination, through its appropriate composition of Chinese herbs, exhibits a superior effect on alcohol detoxification and liver protection compared to individual herbs and is of great importance for improving liver health.

[0005] According to the first aspect of the present invention, a Chinese medicinal combination for alcohol detoxification and liver protection is provided, comprising the following ingredients: milk thistle extract, kudzu extract, astragalus extract, and salvia extract; wherein the mass ratio of puerarin to silybin contained in the Chinese medicinal combination is 1:4 to 4:1.

[0006] Chinese herbal medicines are derived from extracts of natural plants, many of which can be consumed daily and have virtually no side effects. Scientists have found that some Chinese herbal medicines possess antioxidant, alcohol-detoxifying, liver-protective, and anti-inflammatory effects and perform well in improving alcoholic liver damage. Animal studies have confirmed that they can significantly alleviate the pathological symptoms of alcoholic liver damage, meaning that daily intake of these plants can achieve the goal of preventing and treating alcoholic liver damage. Currently, however, research on the prevention and treatment of alcoholic liver damage with Chinese herbal medicines focuses primarily on the pharmacodynamic evaluation of individual herbal medicines and their active ingredients.Furthermore, existing studies on Chinese formulas often consist of the mechanical combination and accumulation of Chinese medicines with the same alcohol-detoxifying and liver-protective effects, similar to the arbitrary combination of various vitamins, rather than being specifically designed based on the biological mechanisms of alcohol damage. This "layering" method lacks a clear basis for synergistic effects and cannot reflect the hierarchical relationships between the medicines. Therefore, the present invention offers a scientifically formulated Chinese medicine combination with synergistic enhancement that exhibits a significant effect in alcohol detoxification and liver protection.

[0007] Specifically, based on modern medical research, kudzu extract accelerates ethanol metabolism, alleviates intoxication symptoms, and reduces direct alcohol-related damage to liver cells; milk thistle extract (whose main active ingredient is silymarin) is a recognized hepatocyte protectant with antioxidant and anti-inflammatory properties and can inhibit key processes leading to liver fibrosis; salvia extract primarily improves liver microcirculation and blood perfusion, counteracts fibrosis, reduces the accumulation of liver lipids (especially triglycerides), and regulates immune function, thereby slowing the progression of chronic liver diseases; astragalus extract: studies show that it promotes protein synthesis in the liver, contributes to the repair and regeneration of liver cells, while simultaneously stabilizing the liver cell membrane and lowering liver enzyme levels.The Chinese formula, consisting of the simultaneous application of the four aforementioned herbs, targets four key pathophysiological pathways at once: alcohol metabolism (kudzu), cell protection and anti-inflammatory effects (milk thistle), improved circulation and antifibrotic properties (salvia), and promotion of repair and regeneration (astragalus). Therefore, it is not a simple mixture of the above-mentioned components; its design aims to create a coherent chain of protection and repair against alcoholic liver damage through systematic intervention across multiple targets and pathways. Consequently, this Chinese formula, through the combined action of these four components, can generate synergistic effects and exhibit a more pronounced efficacy in alcohol detoxification and liver protection.

[0008] In particular, the Chinese herbal medicine combination provided by the present invention strictly limits the ratio of silybin to puerarin. Silybin and puerarin are the key active ingredients of milk thistle and kudzu, respectively, and both have been described in the literature as having a liver-protective effect. However, no published technology to date has demonstrated that both, in a specific ratio, can produce a synergistic protective effect exceeding expectations, especially in models of alcoholic liver damage. The present invention favors the ratio of puerarin to silybin and offers a Chinese herbal medicine combination for alcohol detoxification and liver protection with a scientifically formulated composition, synergistic enhancement of effect, and robust evidence.Furthermore, validation in vitro on cell models and in the mouse model for acute alcoholic liver damage has confirmed that the Chinese drug combination provided by the present invention can effectively lower indicators such as AST, ALT, LDH, GSH and TG while simultaneously improving the problem of fatty liver.

[0009] Preferably, the Chinese herbal combination for alcohol detoxification and liver protection, calculated by weight, comprises the following ingredients: milk thistle extract 4 to 20 parts, kudzu extract 5 to 30 parts, astragalus extract 4 to 20 parts, and salvia extract 4 to 20 parts.

[0010] Preferably, the mass ratio of puerarin to silybin is 4:1. Rigorous in vitro and in vivo experiments have validated that this specific ratio maximizes the synergistic potentiation of the two core active ingredients, puerarin and silybin, while simultaneously maximizing the synergistic potentiation of the two core active ingredients with astragalus extract and salvia extract, effectively reducing the side effects caused by the aforementioned Chinese medicinal ingredients.

[0011] Preferably, the milk thistle extract is extracted from 3.5 to 5 g of milk thistle; and / or the kudzu extract is extracted from 4 to 8 g of kudzu; and / or the astragalus extract is extracted from 3.5 to 7 g of astragalus; and / or the salvia extract is extracted from 3.8 to 8 g of salvia. Limiting the raw material quantities means that, under the specified process conditions, stable extracts with the expected content of active ingredients (such as puerarin, silybin, astragaloside, salvianic acid, etc.) can be obtained. This directly ensures the achievement of the "core constituent content range" in the previous technical concept and guarantees that the chemical and pharmacological basis of each batch of the final product remains consistent, thereby overcoming quality variations that can occur with Chinese pharmaceutical raw materials due to origin or batch differences.

[0012] Preferably, the milk thistle extract is extracted from 4.32 g of milk thistle; and / or the kudzu extract is extracted from 6 g of kudzu; and / or the astragalus extract is extracted from 5.04 g of astragalus; and / or the salvia extract is extracted from 5.61 g of salvia.

[0013] According to the second aspect of the present invention, a use of the Chinese drug combination according to one of the preceding claims is provided for the manufacture of a product for alcohol detoxification and liver protection.

[0014] When the aforementioned Chinese herbal combination is used in the manufacture of a product for alcohol detoxification and liver protection, the Chinese herbal combination achieves alcohol detoxification and liver protection through at least one of the following aspects: (1) reduction of AST levels; (2) reduction of ALT levels; (3) reduction of LDH levels; (4) increase in body weight; (5) reduction of MDA levels in liver tissue; (6) increase of reduced GSH levels in liver tissue; (7) reduction of TG levels in liver tissue; (8) reduction in the degree of degeneration of liver fat cells. The Chinese herbal combination provided by the present invention has been validated in vitro using cell models and in a mouse model of acute alcoholic liver damage to significantly improve the numerous indicators mentioned above.Therefore, the "alcohol detoxification and liver protection" effect mentioned in the present invention is no longer based on theoretical or traditional descriptions of experience, but has a quantitative empirical basis from modern pharmacological research. This confirms that the Chinese drug combination provided by the present invention does indeed have a significant effect on alcohol detoxification and liver protection.

[0015] When using the above-mentioned Chinese herbal medicine combination in the manufacture of a product for alcohol detoxification and liver protection, the product includes one of the following: food, health food, medicine or pet food.

[0016] When using the above-mentioned Chinese herbal medicine combination in the manufacture of a product for alcohol detoxification and liver protection, the dosage form of the food, health food, medicine or pet food shall include at least one of solid preparations, liquid preparations, instant powders or drops.

[0017] Preferably, the solid preparation comprises at least one of tablets, capsules, granules or pills.

[0018] Preferably, the liquid preparation comprises at least one of oral solutions, drinks or syrups.

[0019] According to the third aspect of the present invention, a pharmaceutical preparation, food preparation or health food preparation is provided which comprises the Chinese drug combination according to one of the preceding claims and a pharmaceutically, food- or health-food compatible carrier.

[0020] In summary, the Chinese herbal formula provided by the present invention, through its four components, can generate a significant synergistic effect and does not simply represent a superimposition of individual components or effects. Through the precise division of labor and synergy of the individual components with regard to their sequence of action and function, it achieves a holistic, multi-target integrated intervention against alcoholic liver damage from its onset and development to its repair, thus embodying a systemic treatment strategy based on an understanding of modern pathology.

[0021] Furthermore, the present invention has creatively discovered and determined through in vitro (HepG2 cell model) and in vivo experiments (mouse model of alcoholic liver disease) that the core active ingredients puerarin and silybin produce a significant synergistic protective effect at a mass ratio in the specific range of (1 to 4): (1 to 4) (particularly 4:1). The combination within this preferred ratio range is significantly more effective in many respects, such as lowering transaminases (ALT / AST), reducing oxidative stress (lowering MDA, increasing GSH), and improving hepatic steatosis, than the use of either ingredient alone or in other ratios, thus solving the key problem of blind combinations in formulations. Example(s) of implementation

[0022] To enable those skilled in this technical field to better understand the concept of the present invention, the technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only a subset of the embodiments of the present invention and not all of them. Example 11.1 Materials and Sources

[0023] Materials: Hep-G2 cell line.

[0024] Equipment: centrifuge, microscope, multi-function microplate reader, CO2 incubator, drying oven, water bath, cell culture consumables.

[0025] Reagents: MEM medium (with NEAA) (Procell, PM150410), fetal calf serum (Procell, 164210), penicillin-streptomycin dual antibiotic (Procell, PB180120), 0.25% trypsin-EDTA digestive solution (Procell, PB180226), PBS solution (Procell, PB180327), DMSO (Sigma-Aldrich, D2650), serum-free cell freezing medium (New Cell & Molecular Biotech, C40100), CCK-8 (Beyotime, C0040), ALT test kit (Nanjing Jiancheng, C009-2-1), AST test kit (Nanjing Jiancheng, C010-2-1), SOD test kit (Nanjing Jiancheng, A001-3-2), LDH test kit (Beyotime, C0019M), BCA test kit (Beyotime, P0010), anhydrous ethanol (Sinopharm, 10009218),

[0026] Samples of the Chinese drug combination, as shown in Table 1. The sources of the Chinese drug raw materials are as shown in Table 2. Table 1 Samples of the Chinese drug combination in example 1 group Milk thistle extract (by weight) Kudzu extract (parts by weight) Astragalus extract (parts by weight) Salvia extract (parts by weight) Puerarin : Silybin (mass ratio) Experimental group 1 5 20 20 20 4:1 Experimental group 2 10 20 20 20 2:1 Experimental group 3 20 5 20 20 1:4 Comparison group 1 5 0 20 20 1:0 Comparison group 2 0 20 20 20 0:5 Table 2 Sources of Chinese pharmaceutical raw materials name source Raw material before boiling boiling operation Kudzu extract Manufactured by Xuancheng CityBaicao PlantIndustry and TradeCo., Ltd. Kudzu, 6 g The kudzu root was set in 50% ethanol at 10 times its weight and extracted twice at 85°C, with each extraction lasting 3 hours. Milk thistle extract Sold by IdinaBiotechnology(Shanghai) Co., Ltd. Milk thistle, 4.32 g The milk thistle was soaked in ethanol at 1.5 times its weight for 2 hours, followed by three reflux extractions at 70 °C, with the first reflux extraction lasting 2 hours, the second reflux extraction lasting 1.5 hours, and the third reflux extraction lasting 1 hour. Salvia extract Manufactured by Xuancheng CityBaicao PlantIndustry and TradeCo., Ltd. Salvia, 5.61 g Salvia was stabilized in 80% ethanol at 10 times the weight of Salvia and extracted twice at 75°C, with the first extraction lasting 1.5 h and the second extraction lasting 1 h. Astragalus extract Manufactured by Xuancheng CityBaicao PlantIndustry and TradeCo., Ltd. Astragalus, 5.04 g Astragalus was set in water containing 12 times its weight and extracted three times at 95 °C, with each extraction lasting 1 hour.

[0027] The extracts mentioned above were each obtained through processes such as grinding and boiling (extraction or reflux extraction), standing, filtering, concentration, and drying. The final form was extract powder / dry extract powder (solid), i.e., a dry powder or granules. 1.2 Test method: HepG2 cytotoxicity experiment and model creation:

[0028] Cells in good condition and in the logarithmic growth phase with a density of approximately 90% were digested and counted to prepare a cell suspension. A cell suspension of 100 µL per well (containing 5000 cells) was placed in a 96-well plate (the outermost ring of the 96-well plate was filled with PBS solution) and cultured for 24 h at 37 °C and 5% CO2 in an incubator;

[0029] Different concentrations of the substances to be tested were prepared (using complete MEM medium as solvent), and different concentrations of the substances to be tested were adjusted by gradient dilution with complete MEM medium, with complete medium serving as a blank control;

[0030] The old medium was discarded, and according to the group, 200 µL of different concentrations of the substances to be tested were added to the wells containing cells, with 3-5 replications per group; the culture plate was incubated in the incubator for a further 24 h;

[0031] The CCK-8 detection solution was prepared using basic MEM medium (100 µL basic medium + 10 µL CCK-8 reagent per well). The solution in the 96-well plate was discarded, and 110 µL of the prepared CCK-8 detection solution was added. Several wells without cells were treated with the prepared CCK-8 detection solution as a CCK-8 blank control. Incubation was performed for 1 hour, and the absorbance was measured at 450 nm using a microplate reader.

[0032] Cell viability was calculated using the formula. Cell survival rate=[(As−Ab) / (Ac−Ab)]×100%; where, As: Experimental in-depth study (containing cell medium, CCK-8, substance to be tested); Ac: Control well (containing cell medium, CCK-8, solvent); From: Empty well (containing medium without cells and substance to be tested, CCK-8).

[0033] Experimental grouping: blank control group, ethanol model group, experimental group (sample). Based on the cell activity results, a sample concentration of 1 mg / mL was chosen, and the ethanol model concentration condition was damage at a concentration of 0.6% for 24 h.

[0034] Healthy cells were seeded into a 12-well plate, and when the cells were 60–70% confluent, they were treated with 0.6% anhydrous ethanol for 24 hours. The medium containing the ethanol-treated cells was then replaced with medium containing the test substance; culture was continued for the required time, depending on the experimental requirements; after completion of the culture, the cells were washed with PBS, digested with trypsin, and the cell pellet was collected.

[0035] The following tests were performed on the cell pellet obtained: (1) ALT test

[0036] The cell pellet was collected, washed with PBS, centrifuged to remove the supernatant, and the pellet was left as is. The cells were lysed with lysis buffer for 40 min on ice. A portion was used to determine protein concentration, and another portion was tested according to the steps of the test kit.

[0037] The alanine aminotransferase substrate solution was preheated to 37 °C. 20 µL of alanine aminotransferase substrate solution and 5 µL of the test sample (the 3 experimental groups and 2 control group samples from Table 1, with a concentration of 1 mg / mL confirmed by previous cytotoxicity tests, with the total concentration of all combinations being a uniform 1 mg / mL) were added to the test wells. 20 µL of alanine aminotransferase substrate solution was added to the control wells. The plate was gently shaken to mix and reacted at 37 °C for 30 min. 20 µL of 2,4-dinitrophenylhydrazine solution were added to the test wells, and 20 µL of 2,4-dinitrophenylhydrazine solution and 5 µL of the sample to be tested were added to the control wells. The plate was gently shaken to mix and reacted at 37 °C for 20 min. 200 µL of sodium hydroxide solution (0.4 mol / L) were added to both the test and control wells.The plate was gently shaken to mix and left to stand at room temperature for 15 minutes. The wavelength was set to 505 nm, and the OD value of each well was measured. The net OD value was obtained by subtracting the OD value of the control well from the OD value of the test well. Using the standard curve, the ALT activity value was calculated.

[0038] In this experiment, an empty control group and a model control group were also established, with the empty control group containing no sample and the model control group being the ethanol model group. ALT activity values ​​were tested using the same method described above. (2) AST test

[0039] The cell pellet was collected, washed with PBS, centrifuged to remove the supernatant, and the pellet was left as is. The cells were lysed with lysis buffer for 40 min on ice. A portion was used to determine protein concentration, and another portion was tested according to the steps of the test kit.

[0040] The aspartate aminotransferase substrate solution was preheated to 37 °C. 20 µL of aspartate aminotransferase substrate solution and 5 µL of the sample to be tested (the 3 experimental groups and 2 control group samples from Table 1, with a concentration of 1 mg / mL confirmed by previous cytotoxicity tests, with the total concentration of all combinations being a uniform 1 mg / mL) were added to the test wells. 20 µL of aspartate aminotransferase substrate solution was added to the control wells. The plate was gently shaken to mix and reacted at 37 °C for 30 min. 20 µL of 2,4-dinitrophenylhydrazine solution were added to the test wells, and 20 µL of 2,4-dinitrophenylhydrazine solution and 5 µL of the sample to be tested were added to the control wells. The plate was gently shaken to mix and reacted at 37 °C for 20 min. 200 µL of sodium hydroxide solution (0.4 mol / L) were added to both the test and control wells.The plate was gently shaken to mix and left to stand at room temperature for 15 minutes. The wavelength was set to 505 nm, and the OD value of each well was measured. The net OD value was obtained by subtracting the OD value of the control well from the OD value of the test well. Using the standard curve, the AST activity value was calculated.

[0041] In this experiment, an empty control group and a model control group were also established, with the empty control group containing no sample and the model control group being the ethanol model group. AST activity values ​​were tested using the same method described above. (3) LDH test

[0042] The cell pellet was collected, washed with PBS, centrifuged to remove the supernatant, and the pellet was left as is. The cells were lysed with lysis buffer for 40 min on ice. A portion was used to determine protein concentration, and another portion was tested according to the steps of the test kit.

[0043] The LDH Release Assay Buffer was thawed, equilibrated to room temperature, and then mixed to prepare for use. Other reagents were kept in an ice bath to prepare for use. 1 to 100 µL of the sample or the diluted sample (the 5 treatment group samples from Table 1, with a concentration of 1 mg / mL confirmed by prior cytotoxicity tests, with the overall concentration of all combinations being uniformly 1 mg / mL) were added to the sample wells of the 96-well plate, and LDH Release Assay Buffer was added to the sample wells to make up to 100 µL. 100 µL each of LDH detection working solution was added to the standard and sample wells, mixed, and incubated at 37 °C in the dark for 10 to 30 minutes. 20 µL of Stop Solution were added to each well, mixed, and then the absorbance was measured at 450 nm. The LDH activity value was calculated using the standard curve.

[0044] In this experiment, an empty control group and a model control group were also established, with the empty control group containing no sample and the model control group being the ethanol model group. LDH activity values ​​were tested using the same method described above. 1.3 Test results

[0045] As shown in Table 3, these are the test results regarding AST, ALT and LDH levels. Table 3 Influence of different ratios of puerarin and silybin on AST, ALT and LDH levels in HepG2 cells (mean ± standard deviation) group AST (U / gprot) ALT (U / gprot) LDH (mU / mL) Empty control group 12,56 ± 2,36 10,74 ± 2,32 76,13 ± 16,28 Model control group 51,21 ± 4,31 46,30 ± 3,51 610,02 ± 17,99 Experimental group 1 21,22 ± 1,3* 9,54 ± 1,61**** 106,19 ± 17,23** Experimental group 2 23,93 ± 3,38 12,51 ± 1,54** 172,11 ± 42,82* Experimental group 3 25,21 ± 5,01 16,93 ± 1,12** 187,48 ± 61,48 Comparison group 1 24,33 ± 0,38* 15,16 ± 2,09** 216,86 ± 27,72 Comparison group 2 32,54 ± 2,59* 8,20 ± 2,03*** 179,28 ±9,95*

[0046] Note: In comparison to the model group, significance is represented by *, p<0.05 is represented as *, p<0.01 as , p<0.001 as and p<0.0001 as***.

[0047] Table 3 shows that, compared to the model control group, the levels in experimental groups 1 to 3 and comparison groups 1 to 2 all decreased, with experimental group 1 exhibiting the most significant reduction and a better effect than the other groups. The above results demonstrate that the combination ratio according to the invention can reduce the leakage rate of the cell membrane damage indicators AST, ALT, and LDH and has a significant protective effect against alcohol-induced liver damage in HepG2 cells. Example 21.1 Materials and Sources

[0048] Animals used: SPF-class KM mice, male, 18 to 22 g, 50 animals, provided by the Experimental Animal Center of Shandong University. Prior to administration of the test substances, all animals were acclimatized for 3 days, with quarantine and acclimatization feeding performed simultaneously.

[0049] Test kits and main instruments used: Malondialdehyde (MDA) determination kit, purchased from the Nanjing Jiancheng Bioengineering Institute. Reduced glutathione (GSH) determination kit, purchased from the Nanjing Jiancheng Bioengineering Institute. Triglyceride (TG) determination kit, provided by Neusoft Weiteman Biotechnology (Nanjing) Co., Ltd. Total protein (TP) determination kit, provided by Neusoft Weiteman Biotechnology (Nanjing) Co., Ltd. Anhydrous ethanol, purchased from Sinopharm Chemical Reagent Co., Ltd. Fully automated biochemical analyzer, Beckman AU680 type. Electronic balance, Mettler-Toledo Instrument (Shanghai) Co., Ltd. PL602-L. High-efficiency tissue cell sample preparation unit, Lawson DHFSTPRP-CL64 type. High-speed benchtop refrigerated centrifuge, H1750R. Multifunctional microplate detector, Synergy H1. Freezing microtome, HM525 type.

[0050] The selected Chinese drug combination and the sources of the Chinese drug raw materials are shown in Table 1 and Table 2 in Example 1. 1.2 Test method

[0051] Fifty male Kunming mice were randomly assigned to five groups of 10 animals each after a three-day acclimatization feeding period. The dosage design included low, medium, and high doses based on 5, 10, and 30 times the daily intake per person (45 mg / kg body weight), respectively: 225 mg / kg body weight, 450 mg / kg body weight, and 1350 mg / kg body weight. Simultaneously, an empty control group and an ethanol model control group (5600 mg / kg body weight) were established. 1.125 g, 2.250 g, and 6.750 g of the sample were weighed out, dissolved in distilled water, and made up to 100 mL. The substance was administered once daily to the animals in each dose group via intragastric gavage at a volume of 0.2 mL / 10 g body weight. The same volume of distilled water was administered to the empty control group and the ethanol model control group.Intratastral administration was continued for 32 days, with body weight measured every 4 days and the dosage adjusted accordingly. On day 32, all three dose groups and the mouse model control group received 50% (v / v) ethanol by gavage at a rate of 0.14 ml / 10 g body weight. After a 16-hour fast, the animals were euthanized, livers were removed, and 10% (w / v) liver homogenates were prepared. MDA, GSH, and TG levels were determined using test kits. Simultaneously, a cross-sectional sample was taken from the midline of the left liver lobe, frozen sections were prepared, stained with Sudan III, the nuclei were counterstained with hematoxylin, the sections were capped, and the distribution and area of ​​the lipid droplets in the liver were observed. The data were analyzed using SPSS 13.0 statistical software with one-way ANOVA and the Dunnet t-test, and the rank sum test was performed with SAS 8.1.The significance level was set at α=0.05. (1) Body weight

[0052] The mice were weighed twice a week, and the body weight of the mice before and after the experiment was measured to calculate the weight gain of the mice in each group. (2) Detection of MDA, TG and reduced GSH in liver homogenates

[0053] 0.5 g of liver tissue was precisely weighed, and nine times the volume of physiological saline was added at a weight-to-volume ratio of 1:9. The mixture was then mechanically homogenized at low temperatures to produce 10% and 5% homogenates. Detection and calculation methods for MDA and reduced GSH were performed according to the instructions for the test kits from the Nanjing Jiancheng Bioengineering Institute. TP and TG were measured using the Beckman AU680 fully automated biochemical analyzer. MDA content (nmol / 100 mg protein) = Standard concentration (10 nmol / mL) × 100 / TP (mg / mL) GSH content (μmol / g liver tissue) = Standard concentration (0.02 μmol / mL) / 0.1 (g / mL) TG (μmol / g liver tissue) = value measured by biochemical analyzer (mmol / L) × 1000 × dilution factor / 1000

[0077] (3) Pathological-histological examination of the liver

[0054] Material for pathological observation: A cross-sectional sample was taken from the center of the left lobe of the liver of each group of animals. Freeze-sectioned sections were prepared and stained with oil red O. The degree of hepatocyte damage was observed under a light microscope. The distribution, extent, and area of ​​lipid droplets in the liver were primarily observed, and the cumulative total lesion score in the observed fields of view was determined.

[0055] The evaluation criteria are as follows: Scattered, sparse lipid droplets in the hepatocytes - 0 points; Hepatocytes with lipid droplets no more than 1 / 4 - 1 point; Hepatocytes with lipid droplets no more than 1 / 2 - 2 points; Hepatocytes with lipid droplets no more than 3 / 4 - 3 points; Liver tissue almost completely replaced by lipid droplets - 4 points.

[0056] How the evaluation criteria are defined: Provided that the model is established, the indicator is rated as positive if the levels of MDA, GSH and TG in the test substance group show a significant difference compared to the model control group. 1.3 Test results(1) Influence on the body weight of the mice

[0057] As shown in Table 4, the influence of the test substance (Chinese drug combination or individual Chinese drugs) on the body weight of the mice in the different dose groups is illustrated. Table 4 Influence of the test substance on the body weight of the mice in the different dose groups (mean ± standard deviation) group Body weight on day 1 Body weight on day 8 Body weight on day 16 Body weight on day 24 Body weight on day 32 Empty control group 19,87±0,44 25,86±0,97 30,94±1,73 33,17±1,78 36,35±1,72 Model control group 19,26±1,25 25,67±0,98 30,17±2,23 33,52±1,63 36,78±1,79 Low-dose group 19,40±1,03 24,8 8±0,6 1 29,38±1,40 33,33±2,12 36,70±2,17 Medium dose group 19,54±0,83 25,38±0,8 6 30,03±1,00 33,71±1,22 37,28±1,67 High-dose group 19,99±1,01 26,17±1,04 29,64±1,24 33,36±1,08 36,21±1,66

[0058] Note: Comparison of each dose group with the model control group and the blank control group yielded P>0.05 in each case.

[0059] Table 4 shows that the test substance had no significant effect on the body weight of the mice in any of the dose groups, and the difference was not significant compared to the model control group and the empty control group (P>0.05). (2) Influence on MDA, reduced GSH and TG in liver homogenates

[0060] As shown in Table 5, the influence of the test substance (Chinese drug combination or individual Chinese drugs) on MDA, reduced GSH and TG in liver homogenates is shown. Table 5 Influence of the test substance on MDA, reduced GSH and TG in liver homogenates (mean ± standard deviation) group MDA (µmol / g liver tissue) Reduced GSH (µmol / g liver tissue) TG (µmol / g liver tissue) Empty control group 0,21±0,04** 4,15±0,53** 26,23±3,09** Model control group 0,32±0,03 2,83±0,08 142,13±13,97 Low-dose group 0,29±0,04 2,81±0,13 95,39±17,09** Medium dose group 0,28±0,05 3,27±0,29** 77,87±11,22** High-dose group 0,24±0,05** 3,70±0,22** 65,56±12,10**

[0061] Note: Compared to the model control group: *P<0.05, **P<0.01.

[0062] Table 5 shows that, compared to the model control group and the empty control group, the levels of MDA, GSH, and TG in the liver tissue were significantly increased, with a statistically significant difference (P<0.01, P<0.05). Compared to the model control group, MDA in the liver homogenates of mice was reduced in the high-dose group, the level of reduced GSH was increased in the medium and high-dose groups, and the TG level in the liver homogenates of mice was reduced in the low-, medium-, and high-dose groups, with a significant difference (P<0.01). (3) Influence on the pathological histology of mouse liver

[0063] As shown in Table 6, the influence of the test substance (Chinese drug combination or individual Chinese drugs) on the pathological histology of the mouse liver is illustrated. Table 6 Influence of the test substance on the pathological histology of the mouse liver (mean ± standard deviation)

[0064] Note: Compared to the model control group: **P<0.01.

[0065] Table 6 shows that, compared to the empty control group, the assessment of liver fat cell degeneration (lipid droplets) was significantly increased in the mice of the model control group, with a significant difference (P<0.01). Combined with the biochemical results of the liver homogenates, this demonstrates that the model for alcoholic liver damage has been established. Compared to the model control group, the degree of hepatic steatosis was significantly reduced in the mice of the medium- and high-dose groups, with a significant difference (P<0.01).

[0066] The exemplary embodiments mentioned above serve only to illustrate the technical solution of the present invention and not to limit its scope of protection. Although the present invention has been described in detail with reference to the above exemplary embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced with an equivalent one, but all such modifications or replacements fall within the scope of protection of the present invention.