A traditional Chinese medicine compound composition for alleviating alcoholic liver damage and a probiotic-fermented traditional Chinese medicine compound composition.

Abstract

This invention discloses a traditional Chinese medicine compound composition and a probiotic-fermented traditional Chinese medicine compound composition for alleviating alcoholic liver damage. The raw materials include Terminalia chebula, Phragmites communis, Morus alba, Polygonatum sibiricum, Hovenia dulcis, Hippophae rhamnoides, Fennel, Mung bean, Glycyrrhiza uralensis, Lycium barbarum, and Jujube. The fermented probiotics are Lactobacillus rhamnosus and Bifidobacterium adolescentis. The traditional Chinese medicine compound and its probiotic fermentation product of this invention can improve hepatocyte vitality, reduce alcohol-induced hepatocyte damage, regulate hepatic lipid metabolism disorders, and achieve the effect of detoxifying and protecting the liver by reducing alcohol-induced oxidative stress in the liver.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a traditional Chinese medicine compound composition for alleviating alcoholic liver damage, a probiotic fermented traditional Chinese medicine compound composition, and its preparation method. Background Technology

[0002] Alcoholic beverages have become an integral part of people's daily lives. Since the liver is the main organ for alcohol metabolism, excessive or long-term drinking can lead to a series of liver diseases, such as alcoholic liver disease (ALD), and may even eventually develop into cirrhosis and liver cancer [Zou J, et al. Journal of pharmaceutical analysis, 2023, 13(9): 999–1012]. In recent years, the incidence of ALD has been rising year by year and has become the second leading cause of liver injury, second only to viral hepatitis. Acute alcoholic liver injury lacks specific clinical symptoms, so there are still no effective treatment methods [Yuan J, et al. Journal of Agricultural and Food Chemistry, 2021, 69(14): 4101–4110].

[0003] Alcohol damages the liver and affects fatty acid metabolism. In the early stages of alcoholic liver injury, triglyceride (TG) levels accumulate in large quantities, leading to fatty liver. As liver damage worsens, pathological examination images show liver edema, congestion, or hyperplasia and hypertrophy, an increased liver coefficient, a large accumulation of fat particles, increased liver cell necrosis, and infiltration of inflammatory cells [Hou RL, et al. BioMed Research International, 2021, 10(4): 5597135].

[0004] Oxidative stress is considered a significant factor in alcohol-induced liver damage. Alcohol metabolism induces the production of large amounts of reactive oxygen species (ROS), preventing the body from effectively scavenging free radicals through its own antioxidant system, thus triggering oxidative stress [Shu GW, et al. Journal of Functional Foods, 2019, 58:105-113]. ROS directly attack biomolecules such as DNA, RNA, and proteins, leading to hepatocyte apoptosis and necrosis. Reactive oxygen species oxidize polyunsaturated fatty acids, disrupting the phospholipid bilayer structure. This, coupled with increased permeability of the mitochondrial and cell membranes of hepatocytes, results in the release of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) into the bloodstream. Furthermore, imbalances in liver redox homeostasis impair normal liver function, affecting lipid metabolism and leading to triglyceride accumulation and fatty degeneration of hepatocytes [Park HS, et al. Journal of Ethnopharmacology, 2019, 239:111912]. The NRF2 pathway, an important antioxidant pathway in the body, is normally located in the cytoplasm and its low-level expression is maintained by ubiquitination degradation. When the body experiences redox homeostasis imbalance, ubiquitination degradation is inhibited, and NRF2 translocates from the cytoplasm to the nucleus, where it binds to antioxidants and initiates the expression of downstream antioxidant genes such as HO-1 and NQO1. The NRF2 / HO-1 pathway plays a crucial role in regulating hepatic oxidative stress levels and alleviating alcoholic liver damage [Sun X, et al. Environmental Toxicology and Pharmacology, 2018, 60:110–117]. Currently, commonly used drugs for treating alcoholic liver disease include levodocin, biphenyl diester, and silymarin, all of which have some therapeutic effect. However, due to their singular mechanisms of action and side effects, they are insufficient to address the diverse clinical manifestations of alcoholic liver disease.

[0005] Traditional Chinese medicine (TCM) possesses the characteristics of multiple components and multiple targets, thus enabling it to treat diseases from different angles. Furthermore, the active substances derived from natural components have fewer toxic side effects, minimizing drug side effects while treating diseases. Further, utilizing microbial fermentation of TCM materials combines the advantages of TCM materials, enzyme preparations, and probiotics, while also offering the advantages of being natural, non-toxic, having no side effects, leaving no residue, being safe, and clean [Liu Yang, et al. Anhui Agricultural Sciences, 2017, 45(34): 123-125]. After fermentation, TCM can achieve the effect of reducing toxicity and increasing efficacy, producing new properties and functions, and improving the utilization rate of TCM resources. Summary of the Invention

[0006] The first objective of this invention is to provide a traditional Chinese medicine compound composition and a probiotic fermented traditional Chinese medicine compound composition for alleviating alcoholic liver damage, based on traditional Chinese medicine theory and modern biological theory.

[0007] The second objective of this invention is to provide an oral preparation containing the above-mentioned traditional Chinese medicine compound composition or probiotic fermented traditional Chinese medicine compound composition and a method thereof for preparation.

[0008] To achieve its objectives, the present invention employs the following technical solution:

[0009] This invention first discloses a traditional Chinese medicine compound composition for alleviating alcoholic liver damage, which includes the following raw materials in parts by weight: 1-50 parts of Terminalia chebula, 1-50 parts of Phragmites communis rhizome, 1-50 parts of Morus alba fruit, 1-50 parts of Polygonatum sibiricum, 1-50 parts of Hovenia dulcis fruit, 1-50 parts of Hippophae rhamnoides fruit, 1-50 parts of Fennel seeds, 1-50 parts of Mung bean, 1-50 parts of Lycium barbarum fruit, 1-50 parts of Jujube, and 1-50 parts of Glycyrrhiza uralensis.

[0010] The present invention further discloses a probiotic fermented traditional Chinese medicine compound composition for alleviating alcoholic liver damage, wherein the probiotic fermented traditional Chinese medicine compound composition is obtained by fermenting the extract of the traditional Chinese medicine compound composition with probiotics.

[0011] In the aforementioned probiotic-fermented traditional Chinese medicine compound composition, the probiotics used are *Lactobacillus rhamnosus* and *Bifidobacterium adolescentis*. The effective viable count of both probiotics in the composition is 1.0 × 10⁻⁶. 7 cfu / mL -1.0×10 9 cfu / mL.

[0012] As a preferred embodiment, the traditional Chinese medicine compound composition includes the following raw materials in parts by weight: 20 parts of Terminalia chebula, 10 parts of Phragmites communis rhizome, 5 parts of Morus alba fruit, 5 parts of Polygonatum sibiricum, 5 parts of Hovenia dulcis fruit, 6 parts of Hippophae rhamnoides fruit, 3 parts of Fennel seeds, 2 parts of Mung bean, 9 parts of Glycyrrhiza uralensis root, 4 parts of Lycium barbarum fruit, and 3 parts of Jujube.

[0013] This invention also discloses an oral preparation, which is prepared from the above-mentioned traditional Chinese medicine compound composition or probiotic fermented traditional Chinese medicine compound composition. The oral preparation can be an oral liquid, powder, capsule, granule, or tablet, and the preparation method of the oral preparation is as follows:

[0014] Step 1: Preparation of the extract of the traditional Chinese medicine compound composition: Mix the raw materials in the traditional Chinese medicine compound composition evenly according to the specified ratio, add 1-20 times the weight of water, soak for 30-240 minutes (preferably 180 minutes), decoct and extract at 80℃-100℃ for 30-120 minutes (preferably 100℃ for 40 minutes) to obtain the extract; repeat the extraction 2-5 times (preferably 2 times), combine the extracts, and then concentrate to a density of 1.00-1.50 g / cm³. 3 The liquid (preferably with a density of 1.00-1.15 g / cm³) 3 ), which is the extract of a compound traditional Chinese medicine composition.

[0015] Step 2, Preparation of Probiotics: The MRS medium and Bifidobacterium O233 medium used are conventional microbial culture media, which can be understood and implemented by those skilled in the art. Lactobacillus rhamnosus and Bifidobacterium adolescentis were streaked onto MRS solid medium and Bifidobacterium O233 solid medium, respectively, and incubated at 37°C in an anaerobic chamber for 24-48 hours. Single colonies were picked and inoculated into the corresponding liquid culture media, and anaerobically incubated at 37°C for 24-48 hours to obtain Lactobacillus rhamnosus and Bifidobacterium adolescentis bacterial suspensions. The effective viable count of both probiotics was adjusted to 1.0 × 10⁻⁶. 9 cfu / mL.

[0016] Step 3: Preparation of the probiotic-fermented traditional Chinese medicine compound composition: The Lactobacillus rhamnosus and Bifidobacterium adolescentis bacterial cultures obtained in the above manner were inoculated into the extract of the traditional Chinese medicine compound composition obtained in Step 1. After all components were stirred evenly, fermentation was carried out under anaerobic conditions at 37℃ for 72 hours to obtain the probiotic-fermented traditional Chinese medicine liquid. The total inoculation volume of the two bacterial cultures was 0.5-20% of the volume of the extract of the traditional Chinese medicine compound composition, and the two bacterial cultures were inoculated in equal volume ratios.

[0017] Step 4: Preparation of oral formulations:

[0018] The extract of the traditional Chinese medicine compound composition obtained in step 1 or the traditional Chinese medicine liquid fermented by probiotics obtained in step 3 is centrifuged, filtered and sterilized to obtain an oral liquid.

[0019] Alternatively: Centrifuge, concentrate, filter, and sterilize the extract of the traditional Chinese medicine compound composition obtained in step 1 or the traditional Chinese medicine liquid fermented by probiotics obtained in step 3, then freeze-dry until the moisture content does not exceed 5%, grind into powder, pass through a No. 6 sieve, and obtain freeze-dried powder.

[0020] Alternatively: Centrifuge, concentrate, filter, sterilize, and spray dry the extract of the traditional Chinese medicine compound composition obtained in step 1 or the traditional Chinese medicine liquid fermented by probiotics obtained in step 3 to obtain a powder.

[0021] Alternatively: the extract of the traditional Chinese medicine compound composition obtained in step 1 or the traditional Chinese medicine liquid fermented by probiotics obtained in step 3 is added to excipients to make soft material, which is then sieved to make wet granules. The wet granules are then dried, sized, compressed into tablets and / or filled into capsules to make oral preparations in granule, tablet or capsule form.

[0022] In solid dosage forms such as powders, granules, tablets, and capsules, each dose is 2-20g (preferably 5g).

[0023] In the traditional Chinese medicine formula of this invention: *Clerodendrum trichotomum* (green fruit) is sweet, sour, and astringent, neutral in nature, and enters the stomach and lung meridians. It has the effects of clearing heat and detoxifying, relieving sore throat and promoting saliva production. *Compendium of Materia Medica* calls it "the fruit of the liver and stomach." Modern pharmacology believes that its total flavonoids and triterpenoids help enhance the activity of antioxidant enzymes such as SOD and GSH-PX in the body, inhibit ROS generation, and alleviate oxidative damage to biological macromolecules, thereby achieving a liver-protective effect. *Phragmites communis* (reed rhizome) enters the lung and stomach meridians, and has the effects of clearing heat and purging fire, promoting saliva production and quenching thirst, relieving irritability, stopping vomiting, and promoting urination. Modern research shows that *Phragmites communis* contains abundant vitamins and a large amount of carbohydrates and proteins. In addition, it also contains fatty acids, amino acids, tocopherols, sterols, polyphenols, and other phenolic acid components. *Phragmites communis* polysaccharides have a liver-protective effect. Mulberries are sweet, sour, and cold in nature, and enter the heart, liver, and kidney meridians. They have the effects of nourishing yin and blood, and promoting body fluid production. Mulberries have been a commonly used traditional Chinese medicine since ancient times. The *Compendium of Materia Medica* records that mulberry juice can be drunk to relieve alcohol poisoning. Mulberries contain various active ingredients, such as resveratrol, flavonoids, polysaccharides, and alkaloids. Among them, flavonoids can combat oxidative stress and liver inflammation. Polygonatum rhizome is sweet and neutral in nature, and enters the spleen, lung, and kidney meridians. It has the effects of tonifying qi and nourishing yin, strengthening the spleen, moistening the lungs, and benefiting the kidneys. It is used for spleen and stomach weakness, fatigue, dry mouth and poor appetite, dry cough due to lung deficiency, insufficient essence and blood, and internal heat and thirst. Hovenia dulcis fruit has the effects of clearing heat and promoting diuresis, quenching thirst and relieving irritability, relieving alcohol poisoning, and promoting urination and defecation. It is suitable for symptoms such as fever, thirst, vomiting, drunkenness, and difficulty in urination and defecation. Its flavonoid components may be related to its alcohol-relieving function. Sea buckthorn has the effects of tonifying qi and strengthening the spleen, resolving phlegm and removing turbidity, and promoting blood circulation and removing blood stasis. It contains a variety of components such as flavonoids, tannins, terpenes, polysaccharides, and vitamins, and mainly has anti-inflammatory and antioxidant effects. Sea buckthorn flavonoids and sea buckthorn polysaccharides can help reduce liver damage. Fennel enters the liver, kidney, spleen, and stomach meridians. It is pungent and warm in nature, and has the effects of dispelling cold and relieving pain, regulating qi and harmonizing the stomach. Fennel water extract can improve liver cell damage. Mung beans are rich in protein, vitamins, flavonoids, alkaloids, phytosterols, and other bioactive substances. They have a variety of physiological effects such as lowering blood sugar, lowering blood lipids, and anti-inflammation. Mung bean flavonoids are one of the active ingredients in mung beans. Pharmacological experiments have shown that mung bean flavonoids can significantly inhibit the formation and development of liver damage. Licorice is often used in traditional Chinese medicine to assist in the treatment of diseases, playing a role in harmonizing various drugs. Moreover, isoglycyrrhizin in licorice has a significant protective effect on liver cells. Goji berries can nourish the kidneys, lungs, liver, and promote sperm production and qi circulation. They are a mild tonic, containing polyphenols, polysaccharides, alkaloids, amino acids, and other substances, possessing antioxidant, liver-protective, and anti-lipid peroxidation effects. Jujubes are a commonly used qi-tonifying and health-promoting herb in Traditional Chinese Medicine. They have anti-allergic effects, central nervous system depressant effects, liver protection, muscle strengthening, and inhibition of cancer cell proliferation. They are particularly effective in treating hepatitis, lowering blood pressure, treating boils, replenishing blood, improving brain function, fighting cancer, and strengthening the spleen and body. The jujube polysaccharides they contain can scavenge oxygen free radicals, enhance immune function, and resist lipid peroxidation.Licorice root is sweet and neutral in nature, entering the spleen, stomach, and lung meridians. It is primarily used to treat spleen and stomach weakness, poor appetite, abdominal pain and loose stools, fatigue and fever, pulmonary tuberculosis and cough, palpitations, and epilepsy. It also treats sore throat, peptic ulcers, carbuncles and sores, and detoxifies drug poisoning and food poisoning. When used together, these herbs have a protective effect on the liver and alleviate liver damage caused by alcohol.

[0024] Meanwhile, the probiotic composition of the present invention, Lacticaseibacillus rhamnosus, can increase the number of beneficial bacteria in the small intestine, restore the concentration of duodenal tight junction protein, and reduce the concentration of inflammatory factors in the liver, thereby playing a preventive and protective role; Bifidobacterium adolescentis can inhibit the number of harmful bacteria that produce toxins, thus playing a good therapeutic role for liver patients.

[0025] The probiotic fermented traditional Chinese medicine composition of this invention is prepared by fermenting traditional Chinese medicine extracts with probiotics. The traditional Chinese medicine used has the effects of protecting the liver and alleviating liver damage caused by alcohol. It works synergistically with probiotics to promote the proliferation and survival rate of probiotics, thus replacing prebiotics. The probiotics degrade the traditional Chinese medicine extracts and reduce the side effects of the drugs. This traditional Chinese medicine probiotic composition can repair damaged liver cells and protect the liver. It can be used to prepare drugs to alleviate alcoholic liver damage, such as for the prevention and / or treatment of alcoholic liver disease. Attached Figure Description

[0026] Figure 1 A diagram illustrating the process of modeling acute alcoholic liver disease in mice.

[0027] Figure 2 Figure 1 shows the effect of a traditional Chinese medicine composition on the levels of biochemical indicators in the serum of mice with acute alcoholic liver disease. Figure 2 shows the results of alanine aminotransferase (ALT), Figure 3 shows the results of aspartate aminotransferase (AST), and Figure 4 shows the results of triglycerides.

[0028] Figure 3 The images show the effects of a traditional Chinese medicine composition on liver pathological sections of mice with acute alcoholic liver disease. In the images, A is an HE staining image and B is an Oil Red staining image.

[0029] Figure 4 The graph shows the effect of traditional Chinese medicine composition on gene expression levels in the liver of mice with acute alcoholic liver disease. A is a quantitative graph of NRF2 gene expression level, and B is a quantitative graph of HO-1 gene expression level.

[0030] Figure 5 Figure 1 shows the effects of probiotic fermented traditional Chinese medicine compound composition and alcohol on HepG2 cell viability. Figure 2 shows the effect of different concentrations of alcohol on HepG2 cell viability, Figure 3 shows the effect of different concentrations of traditional Chinese medicine composition on HepG2 cell viability, and Figure 4 shows the protective effect of probiotic fermented traditional Chinese medicine composition on HepG2 cell viability.

[0031] Figure 6 The graph shows the effect of probiotic fermentation of traditional Chinese medicine compound composition on the content of biochemical indicators in alcohol-induced HepG2 cells. Figure A is the alanine aminotransferase (ALT) graph, and Figure B is the aspartate aminotransferase (AST) graph.

[0032] Figure 7 The graph shows the effect of probiotic fermentation of traditional Chinese medicine compound composition on the gene expression level of alcohol-induced HepG2 cells. A is the quantitative graph of NRF2 gene expression level, and B is the quantitative graph of HO-1 gene expression level. Detailed Implementation

[0033] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to examples. The following content is merely an example and illustration of the concept of the present invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, as long as they do not depart from the inventive concept or exceed the scope defined by the claims, all of which should fall within the protection scope of the present invention.

[0034] The probiotics used in this invention are derived from commercially available probiotics, with no specific brand requirement. The Lactobacillus rhamnosus and Bifidobacterium adolescentis used in the following examples were purchased from Shandong Zhongke Jiayi Biotechnology Co., Ltd.

[0035] I. Preparation of Oral Formulations

[0036] Example 1: Preparation of a compound extract of traditional Chinese medicine and probiotics

[0037] The Chinese medicinal materials used in this embodiment are: 20g of green plum, 10g of reed rhizome, 5g of mulberry, 5g of polygonatum, 5g of jujube fruit, 6g of sea buckthorn, 3g of fennel, 2g of mung bean, 9g of licorice, 4g of wolfberry, and 3g of jujube.

[0038] The extract of the traditional Chinese medicine composition in this embodiment was prepared according to the following steps:

[0039] (1) Mix the above-mentioned Chinese herbal ingredients evenly according to the ratio, add 1L of water and soak for 180 minutes.

[0040] (2) Boil over high heat. Start timing after the liquid boils. Then simmer over low heat for 40 minutes. Filter through a 100-mesh filter and keep the filtrate.

[0041] (3) Add 1L of water to the dregs from step (2), boil over high heat, start timing after the liquid boils, then simmer over low heat for 40 minutes, filter with a 100-mesh filter and keep the filtrate.

[0042] (4) Combine the two filtrates and boil them over high heat to concentrate them to 1.00-1.15 g / mL (where g is the mass of the original Chinese medicinal materials used) to obtain the extract of the Chinese medicine compound composition, which is referred to as Chinese medicine extract.

[0043] The bacterial strains prepared in this embodiment are as follows:

[0044] (1) Lactobacillus rhamnosus and Bifidobacterium adolescentis were streaked onto MRS solid medium and Bifidobacterium 0233 solid medium, respectively, and cultured at 37°C for 24 hours in an anaerobic incubator.

[0045] (2) Pick a single colony and inoculate it into the corresponding liquid culture medium. Incubate anaerobically at 37°C for 24 hours. Expand the culture as needed.

[0046] (3) Measure the OD600 value using a visible spectrophotometer and adjust the viable count to 1.0 × 10⁻⁶. 9 cfu / mL.

[0047] The Lactobacillus rhamnosus bacterial suspension and Bifidobacteriumadolescentis bacterial suspension obtained by the above method were inoculated with the traditional Chinese medicine extract at a volume ratio of 2.5:2.5:100. After the components were stirred evenly, they were fermented under anaerobic conditions at 37℃ for 72 hours. After centrifugation at 5000rpm for 10 minutes, filtration and sterilization were performed to obtain the probiotic fermented traditional Chinese medicine liquid.

[0048] Example 2: Preparation of solid dosage forms

[0049] The Chinese medicinal materials used in this embodiment are: 20g of green plum, 10g of reed rhizome, 5g of mulberry, 5g of polygonatum, 5g of jujube fruit, 6g of sea buckthorn, 3g of fennel, 2g of mung bean, 9g of licorice, 4g of wolfberry, and 3g of jujube.

[0050] The extract of the traditional Chinese medicine composition in this embodiment was prepared according to the following steps:

[0051] (1) Mix the above-mentioned Chinese herbal ingredients evenly according to the ratio, add 1L of water and soak for 180 minutes.

[0052] (2) Boil over high heat. Start timing after the liquid boils. Then simmer over low heat for 40 minutes. Filter through a 100-mesh filter and keep the filtrate.

[0053] (3) Add 1L of water to the dregs from step (2), boil over high heat, start timing after the liquid boils, then simmer over low heat for 40 minutes, filter with a 100-mesh filter and keep the filtrate.

[0054] (4) Combine the secondary filtrates and boil and concentrate them over high heat to 1.00 - 1.15 g / mL (where g is based on the mass of the original Chinese medicinal materials used), obtaining an extract of the Chinese medicine compound composition, simply referred to as the Chinese medicine extract.

[0055] The strain preparation in this example is as follows:

[0056] (1) Inoculate Lactobacillus rhamnosus and Bifidobacterium adolescentis by streaking on MRS solid medium and Bifidobacterium 0233 solid medium respectively, and culture at 37 °C for 24 hours in an anaerobic chamber.

[0057] (2) Pick single colonies and inoculate them into the corresponding liquid media, culture anaerobically at 37 °C for 24 hours, and perform subculture according to requirements.

[0058] (3) Measure the OD600 value with a visible spectrophotometer and adjust the viable cell count to 1.0×10 9 cfu / mL.

[0059] Inoculate the Lactobacillus rhamnosus bacterial liquid, Bifidobacterium adolescentis bacterial liquid obtained by the above method and the Chinese medicine extract according to a volume ratio of 2.5:2.5:100. After stirring each component evenly, ferment at 37 °C under anaerobic conditions for 72 h to obtain the Chinese medicine liquid fermented by probiotics. Centrifuge at 5000 rpm for 10 min, filter, and sterilize to obtain the Chinese medicine liquid fermented by probiotics.

[0060] Freeze-dry or spray-dry the above-prepared Chinese medicine extract or the Chinese medicine liquid fermented by probiotics to obtain a solid powder agent. Or add appropriate binders, wetting agents, other excipients, etc. to make a soft material, pass through a sieve to make wet granules, and the wet granules are respectively made into solid dosage forms such as granules, tablets, capsules, etc. through drying, sizing, tabletting, capsule filling, etc., and each dose in the solid preparation is 5 g.

[0061] II. Pharmacodynamic experiments of the composition

[0062] 1. Experimental materials [[ID=二十八]]

[0063]

[0064] C57BL / 6J male mice (20 - 25 g, 8 weeks old) are purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., with the production license number SCXK (Beijing) 2019 - 0001. The mice are raised in the experimental animal platform of the Hefei Comprehensive National Science Center Institute of Artificial Intelligence, and the animal breeding license is SYXK (Anhui) 2020 - 006. The breeding environment temperature is 22 - 24 °C, and they are allowed to eat and drink freely.

[0065] 1.2 Cell lines and culture media

[0066] Human hepatocellular carcinoma cells (HepG2) were purchased from Sewell Biotech. Cell culture reagents included DMEM / F12 (BasalMedia, China), fetal bovine serum (Wisent, Canada), trypsin (Sigma, USA), and penicillin-streptomycin mixture (Solepro, China).

[0067] 1.3 Primer Sequence

[0068] The primer sequences used in the quantitative PCR experiment are shown in the sequence listing.

[0069] The NRF2 gene in human hepatocytes (HepG2) was quantified by real-time PCR using primers Hs_NRF2F (as shown in SEQ ID NO: 1) and Hs_NRF2R (as shown in SEQ ID NO: 2), and the HO-1 gene in HepG2 was quantified by primers Hs_HO-1F (as shown in SEQ ID NO: 3) and Hs_HO-1R (as shown in SEQ ID NO: 4). Human β-actin was used as an internal reference gene, and the primers used were Hs_β-actinF (as shown in SEQ ID NO: 5) and Hs_β-actinR (as shown in SEQ ID NO: 6). The relative expression levels of NRF2 and HO-1 genes in the control group, model group, drug-treated group, and positive group were detected.

[0070] The NRF2 gene in mouse liver tissue was quantified by real-time PCR using primers Mm_NRF2F (as shown in SEQ ID NO: 7) and Mm_NRF2R (as shown in SEQ ID NO: 8), and the HO-1 gene in mouse liver tissue was quantified by primers Mm_HO-1F (as shown in SEQ ID NO: 9) and Mm_HO-1R (as shown in SEQ ID NO: 10). Mouse β-actin was used as an internal reference gene, and the primers used were Mm_β-actinF (as shown in SEQ ID NO: 11) and Mm_β-actinR (as shown in SEQ ID NO: 12). The relative expression levels of NRF2 and HO-1 genes in the liver tissues of mice in the control group, model group, drug-treated group, and positive group were detected.

[0071] 1.4 Experimental Reagents

[0072] Triglyceride (TG), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total protein (TP) kits (Shenzhen Raydu Life Sciences Co., Ltd.), Formalin, Xylene, Anhydrous ethanol, Hematoxylin reagent, Eosin reagent, Ethanol hydrochloride, Resin mounting medium, Oil Red O staining solution (Shanghai Sangon Biotech Co., Ltd.), CCK-8 (Glpbio, USA), qPCR primers (General Biotech (Anhui) Co., Ltd.), Trizol (Shanghai Sangon Biotech Co., Ltd.), Reverse transcription kit (Beijing TransGen Biotech Co., Ltd.), qPCR kit (Beijing TransGen Biotech Co., Ltd.).

[0073] 1.5 Experimental Apparatus

[0074] Biosafety cabinets, CO2 incubators, fume hoods, induction cookers, benchtop centrifuges, low-temperature centrifuges, liquid nitrogen tanks, -80℃ ultra-low temperature freezers, 4℃ freezers (Thermo Fisher Scientific, USA), fully automated biochemical analyzers (Shenzhen Raydu Life Science Co., Ltd.), electrically heated incubators (Shanghai Yiheng Scientific Instruments Co., Ltd.), fully automated dehydrators, paraffin embedding machines, freezing stages, paraffin microtome, slide spreaders, cryostats (Leica Instruments GmbH, Germany), electronic balances (Sartorius, Germany), grinders (Wuhan Saivee Biotechnology Co., Ltd.).

[0075] 2. Experimental Methods

[0076] 2.1 Protective effect of traditional Chinese medicine composition on mice with acute alcoholic liver disease

[0077] 2.1.1 Grouping and administration of mice with acute alcoholic liver disease

[0078] After one week of acclimatization, male C57BL / 6J mice were randomly divided into 5 groups (n=10 per group): control group, model group, low-dose herbal extract group (15 mg / kg, based on the weight of the raw herbal material used in the extract), high-dose herbal extract group (60 mg / kg, based on the weight of the raw herbal material used in the extract), and positive control silymarin group (100 mg / kg). The entire acute alcoholic liver injury model lasted for 4 days. The control group mice were administered physiological saline by gavage throughout the process. The mice were pretreated with herbal extract and silymarin by gavage (once every 24 hours, for a total of four times). On the third day, the mice were administered 50% ethanol at a concentration of 5 g / kg by gavage (once every 12 hours, for a total of three times).

[0079] 2.1.2 Determination of serum ALT, AST and TG levels

[0080] Twelve hours after the last gavage, the mice were euthanized, and blood was immediately collected. The supernatant was collected by centrifugation at 3500 rpm for 10 minutes, and the serum ALT, AST, and TG levels were measured using a biochemical analyzer.

[0081] 2.1.3 Liver tissue staining

[0082] HE staining: Liver tissue was collected, fixed overnight with 4% paraformaldehyde, and then washed with PBS. Sections were prepared according to standard procedures (dehydration and clearing, paraffin embedding) with a thickness of 5 μm. The sections were then spread, retrieved, and dried. After dewaxing, hematoxylin staining, hydrochloric acid-ethanol differentiation, tap water blueing and eosin staining, the sections were mounted and photographed.

[0083] Oil Red staining: Liver tissue was taken, fixed overnight with 4% paraformaldehyde, and dehydrated continuously with 15% and 30% sucrose. After OCT embedding, frozen sections with a thickness of 10 μm were prepared. The sections were stained with oil red and counterstained with hematoxylin, mounted, and photographed.

[0084] 2.1.4 Quantitative Real-Time PCR

[0085] RNA extraction: Take tissue frozen at -80℃, add 1mL of Total RNA Extractor to every 50mg of tissue, add zirconia beads, and grind the sample using a grinder; after lysis, incubate the sample at room temperature for 5-10min to ensure complete separation of nucleoproteins and nucleic acids; add 0.2mL of chloroform, vortex vigorously for 15sec, incubate at room temperature for 3min, and centrifuge at 12,000rpm at 4℃ for 10min; transfer the upper aqueous phase to a 1.5mL centrifuge tube, add an equal volume of pre-chilled isopropanol, mix well, and incubate on ice for 10min; centrifuge at 12,000rpm at 4℃ for 10min, and discard the supernatant. Wash the precipitate with 75% ethanol, centrifuge at 7500rpm for 5min at 4℃, repeat twice; after drying at room temperature, add 30-50μL of N-ase-free ddH2O to dissolve the RNA sample, which can be stored at -80℃ for long-term storage. Take 1μg of RNA to synthesize cDNA by reverse PCR, and then use a qPCR kit to perform gene quantification analysis using an ABI Piperism 7500fast real-time PCR system.

[0086] 2.2 Protective effect of probiotic-fermented traditional Chinese medicine composition on alcohol-induced HepG2 cell damage

[0087] 2.2.1 Cell Culture

[0088] HepG2 cells were grown in DMEM / F12 medium supplemented with 10% fetal bovine serum and 1% penicillin antibiotics and cultured in a cell culture incubator at 37°C and 5% CO2.

[0089] 2.2.2 Cell viability assay

[0090] HepG2 cells in the logarithmic growth phase were seeded into 96-well plates (1×10⁻⁶ cells per well). 4Cells were cultured overnight with 1, 2, 4, 6, 8, and 10 mg / mL of herbal extract (based on the mass of the raw herbal material used in the extract). After 24 h of culture, the cytotoxicity of the herbal composition was assessed using a CCK-8 assay kit. Under the same conditions, cells were treated with 2%, 4%, 6%, 8%, and 10% ethanol to screen for suitable ethanol-damaged cell concentrations. To determine the safe concentration range and suitable ethanol-damaged cell concentrations of the herbal composition, cells were cultured for 24 h with herbal extract (4 mg / mL) and probiotic-fermented herbal solution (4 mg / mL), followed by 24 h of ethanol exposure to assess the protective effect of the composition.

[0091] 2.2.3 Measurement of ALT and AST in cells

[0092] Cell treatment conditions were the same as in 2.2.2. Cell supernatants from different treatment groups were collected, centrifuged at 500g, 4℃ for 10min, and the supernatants were taken to determine the ALT and AST content in the cells. At the same time, cells were lysed by sonication to determine the total intracellular protein content.

[0093] 2.2.4 Quantitative Real-Time PCR

[0094] Cell processing conditions were the same as in 2.2. RNA was extracted from the cells using Trizol, and 1 μg of RNA was reversed to obtain cDNA for downstream gene qPCR detection.

[0095] 2.3 Data Statistics and Analysis

[0096] Data analysis was performed using GraphPad Prism 9. The independent samples t-test was used for comparisons between groups, and one-way ANOVA was used to analyze the significant differences between multiple groups. A p-value < 0.05 was considered to be statistically significant. # p<0.05, ## p<0.01 and ### p<0.01 indicates a comparison with the control group; *p<0.05, **p<0.01 and ***p<0.001 indicate a comparison with the model group; Δ p<0.05, ΔΔ p<0.01 and ΔΔΔ p<0.001 indicates a comparison with the probiotic-fermented traditional Chinese medicine group.

[0097] 3. Experimental Results

[0098] 3.1 Protective effect of traditional Chinese medicine composition on mice with acute alcoholic liver disease

[0099] 3.1.1 Traditional Chinese medicine composition reduces the levels of ALT, AST, and TG in the serum of mice with acute alcoholic liver disease.

[0100] The mouse modeling process is as follows Figure 1 As shown. Alanine aminotransferase (ALT) is mainly found in the cytoplasm of hepatocytes. Normally, even a small amount released into the bloodstream can significantly increase serum enzyme activity. When the permeability of the hepatocyte membrane changes, ALT leaks from the cell into the circulating blood. Even 1% hepatocyte necrosis can double the serum enzyme activity; therefore, ALT is the most sensitive marker of acute hepatocellular injury. Figure 2 As shown in Figure A, after four drug pretreatments, mice were administered 5 mg / kg of 50% ethanol via gavage three times, every 12 hours. Compared to the control group (administered saline via gavage), the serum ALT levels in the model group mice were significantly increased, indicating that alcohol-induced hepatocyte damage and increased cell permeability. The serum ALT levels in the low-dose group (7.5 mg / kg) and high-dose group (30 mg / kg) of the traditional Chinese medicine composition were significantly lower than those in the model group, indicating that the composition can effectively alleviate alcohol-induced ALT release from hepatocytes.

[0101] Aspartate aminotransferase (AST) is mainly found in the heart, followed by the liver. Alanine aminotransferase (ALT) enters the bloodstream first, followed by AST. Figure 2 As shown in Figure B, the AST content in the model group was slightly higher than that in the control group, and drug pretreatment had a certain regulatory effect on the elevated AST content in serum.

[0102] Alcohol can impair liver mitochondrial function, inhibit the oxidation and utilization of lipids in the liver, and lead to the accumulation of triglyceride levels. For example... Figure 2 As shown in Figure C, compared to the control group, the level of triglycerides in the blood of the model group was significantly increased, while drug pretreatment, including high- and low-dose traditional Chinese medicine groups and silymarin pretreatment, could significantly reduce the increase in serum TG levels induced by alcohol.

[0103] In summary, this traditional Chinese medicine composition can alleviate alcoholic liver damage to some extent by lowering the levels of ALT and TG in alcohol-induced serum.

[0104] 3.1.2 Traditional Chinese medicine composition improves the pathological state of the liver in mice with acute alcoholic liver disease.

[0105] HE staining results analysis: Hematoxylin is an alkaline staining solution, which binds to nucleic acids and turns blue, while eosin is an acidic dye, which binds to cytoplasmic components and turns red. Hematoxylin-eosin staining is mainly used to observe the morphology and structure inside cells. Figure 3As shown in Figure A, in the control group, hepatocytes exhibited intact morphology and structure, with clearly visible hepatic lobules and hepatic cords arranged radially around the central vein. In the model group, hepatocytes were disorganized, with increased gaps between the nucleus and cytoplasm, some showing inflammatory cell infiltration, focal necrosis, indistinct hepatic lobule outlines, and the formation of fat vacuoles. Compared to the model group, the traditional Chinese medicine combination and silymarin groups showed varying degrees of improvement in hepatocyte distribution and reduction in fat vacuole formation.

[0106] Analysis of Oil Red Staining Results: Oil Red O dye specifically stains lipid droplets in cultured adipocytes or neutral lipids such as triglycerides in tissues red or orange-red. For example... Figure 3 As shown in Figure B, the control group exhibited regular hepatocyte morphology and hepatic cord arrangement, with almost no red fat granules within the cells. In contrast, the model group showed a dense distribution of red fat granules in the liver. This is because alcohol metabolism damages hepatocyte mitochondria, affecting the liver's lipid metabolism process and leading to lipid accumulation. Compared to the model group, the high- and low-dose groups of the traditional Chinese medicine composition, as well as silymarin, improved hepatocyte morphology and distribution to varying degrees, significantly reducing the accumulation of red fat granules in the liver.

[0107] 3.1.3 Activation of antioxidant gene expression in liver tissue of mice with acute alcoholic liver disease by traditional Chinese medicine composition

[0108] NRF2, acting as a molecular switch for redox reactions in vivo, translocates from the cytoplasm to the nucleus under oxidative stress, binds to antioxidant response elements, and activates the expression of downstream antioxidant genes. It plays a crucial role in regulating redox homeostasis. Studies have shown that activation of the NRF2 / HO-1 pathway can significantly improve alcohol-induced hepatocyte damage both in vivo and in vitro. qPCR results are shown below. Figure 4 As shown, alcohol stimulation significantly downregulated NRF2 expression levels in liver tissue to 0.69-fold compared to the control group, while the high- and low-dose groups of the traditional Chinese medicine composition and the positive control group restored NRF2 expression levels to 0.90, 0.96, and 1.5-fold, respectively, compared to the control group, with silymarin showing the most significant promoting effect. HO-1, as a downstream target gene of NRF2, has a similar expression level to NRF2. Alcohol stimulation downregulated HO-1 expression levels in liver tissue, while pretreatment with the traditional Chinese medicine composition upregulated HO-1 expression levels. This indicates that the traditional Chinese medicine composition can alleviate alcohol-induced liver injury by activating the NRF2 / HO-1 pathway in vivo and improving the redox homeostasis of liver tissue.

[0109] 3.2 Protective effect of probiotic fermented traditional Chinese medicine composition on alcohol-induced HepG2 cell damage

[0110] 3.2.1 Probiotic fermented traditional Chinese medicine composition upregulates alcohol-inhibited HepG2 cell viability

[0111] The HepG2 liver cancer cell line is viable and easy to culture, and is widely used in studies of hepatotoxicity and metabolism, serving as a good model for investigating alcoholic liver injury. For example... Figure 5 As shown in Figure A, cell viability gradually decreased with increasing alcohol concentration. Generally, a cell viability of approximately 50%-70% of the control group is selected as the modeling condition. This significantly reduces cell survival rate, but avoids excessively low survival rates that could cause irreversible damage and affect subsequent evaluation experiments on the cell-protective effects of the traditional Chinese medicine composition. Therefore, treating cells with 6% alcohol for 24 hours was used as the subsequent cell modeling condition. Figure 5 As shown in Figure B, cells were treated with different concentrations of traditional Chinese medicine extracts for 24 hours. When the concentration of the extract was increased to 6 mg / mL, it exhibited cytotoxicity and significantly reduced cell viability. Therefore, the safe concentration range for this extract should be less than 6 mg / mL. Treatment of cells with 6% alcohol for 24 hours reduced cell viability to 56.92% of the control group. Pretreatment with 4 mg / mL of the extract and with a probiotic-fermented extract restored cell viability to 74.7% and 86.3% of the control group, respectively. Figure 5 C). This indicates that the traditional Chinese medicine composition and its probiotic fermentation composition can protect against alcohol-induced hepatocyte damage and improve cell survival rate. Moreover, the protective effect of the probiotic-fermented traditional Chinese medicine liquid on hepatocytes is significantly higher than that of the unfermented traditional Chinese medicine extract (p<0.05).

[0112] 3.2.2 Probiotic-fermented traditional Chinese medicine combination reduces alcohol-induced ALT and AST leakage in hepatocytes

[0113] When hepatocytes are damaged, cell membrane permeability increases, leading to the leakage of intracellular ALT and AST into the extracellular space. In in vitro cultured cells, the degree of hepatocyte damage can be assessed by measuring the ALT and AST levels in the cell supernatant. Figure 6 As shown, after 24 hours of stimulation with 6% alcohol, the ALT level in the cell culture supernatant increased to 1.8 times that of the control group. Pretreatment with the traditional Chinese medicine composition and its probiotic fermentation products could downregulate the ALT level in the culture supernatant, bringing it close to that of the control group. Furthermore, compared with the control group, the ALT leakage level in the model group was significantly increased. Compared with the model group, pretreatment with the traditional Chinese medicine extract and its probiotic fermentation products could downregulate the AST level in the culture supernatant to 1.45 and 1.14 times that of the control group, respectively, indicating that the traditional Chinese medicine composition and its probiotic fermentation products have a good protective effect against alcohol-induced hepatocellular damage, and the traditional Chinese medicine composition fermented with probiotics has a better protective effect on the liver.

[0114] 3.2.3 Probiotic fermented traditional Chinese medicine composition upregulates the expression of antioxidant genes in HepG2 cells

[0115] like Figure 7 As shown, 6% alcohol stimulation of hepatocytes for 24 hours significantly downregulated NRF2 expression and disrupted intracellular redox homeostasis, while traditional Chinese medicine extracts and their probiotic fermentation products activated NRF2 expression. HO-1, an important downstream antioxidant gene of NRF2, [further details needed]. Figure 7 B found that the expression level of HO-1 in the model group decreased to 0.73 times that of the control group, while the expression of HO-1 in the traditional Chinese medicine group was upregulated to 1.04 times that of the control group. Furthermore, the probiotic-fermented traditional Chinese medicine composition further upregulated HO-1 expression to 1.58 times that of the control group, consistent with in vivo experiments. The NRF2 / HO-1 pathway plays an important regulatory role in protecting hepatocytes against alcohol-induced oxidative damage by the traditional Chinese medicine composition. In addition, the efficacy of the traditional Chinese medicine compound in alleviating alcohol toxicity was significantly enhanced after co-fermentation with the probiotics Lactobacillus rhamnosus and Bifidobacterium adolescentis. This demonstrates that the traditional Chinese medicine compound and probiotics work synergistically and complement each other.

[0116] The above are merely exemplary embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A probiotic fermented traditional Chinese medicine compound composition for alleviating alcoholic liver damage, characterized in that: The probiotic-fermented traditional Chinese medicine compound composition is obtained by fermenting the extract of a traditional Chinese medicine compound composition with probiotics. The traditional Chinese medicine compound composition consists of the following raw materials in parts by weight: 1-50 parts of Terminalia chebula, 1-50 parts of Phragmites communis rhizome, 1-50 parts of Morus alba fruit, 1-50 parts of Polygonatum sibiricum, 1-50 parts of Hovenia dulcis fruit, 1-50 parts of Hippophae rhamnoides fruit, 1-50 parts of Fennel seeds, 1-50 parts of Mung bean, 1-50 parts of Lycium barbarum fruit, 1-50 parts of Jujube, and 1-50 parts of Glycyrrhiza uralensis. The probiotics are Lactobacillus rhamnosus and Bifidobacterium adolescentis .

2. The probiotic fermented traditional Chinese medicine compound composition for alleviating alcoholic liver damage according to claim 1, characterized in that: In the aforementioned probiotic fermented traditional Chinese medicine compound composition, the effective live bacteria count of each probiotic is 1.0 × 10⁻⁶. 7 cfu / mL -1.0×10 9 cfu / mL.

3. The use of the probiotic fermented traditional Chinese medicine compound composition according to any one of claims 1 to 2 in the preparation of a drug for relieving alcoholic liver damage.

4. An oral preparation, characterized in that: The oral preparation is prepared from the probiotic fermented traditional Chinese medicine compound composition according to any one of claims 1 to 2.

5. The oral formulation according to claim 4, characterized in that: The oral preparations are oral liquids, powders, capsules, granules, or tablets.

6. A method for preparing an oral formulation of the probiotic fermented traditional Chinese medicine compound composition according to claim 1 or 2, characterized in that, Includes the following steps: Step 1: Preparation of the extract of the traditional Chinese medicine compound composition: Mix all the raw materials in the traditional Chinese medicine compound composition evenly according to the specified ratio, add 1-20 times the weight of water, soak for 30-240 minutes, decoct at 80℃-100℃ for 30-120 minutes to obtain the extract; repeat the extraction 2-5 times, combine the extracts, and then concentrate to a density of 1.00-1.50 g / cm³. 3 The liquid is the extract of the compound Chinese medicine composition; Step 2, Preparation of probiotics: ... Lactobacillus rhamnosus After purification and screening on MRS solid medium, the cultured and purified samples were inoculated into MRS liquid medium for propagation and anaerobic culture to obtain... Lactobacillus rhamnosus The bacterial suspension was adjusted to have an effective viable count of 1.0 × 10⁻⁶. 9 cfu / mL; Will Bifidobacterium adolescentis After being cultured, screened, and purified on Bifidobacterium 0233 solid medium, it was inoculated into Bifidobacterium 0233 liquid medium for propagation and anaerobic culture to obtain... Bifidobacterium adolescents is The bacterial suspension was adjusted to have an effective viable count of 1.0 × 10⁻⁶. 9 cfu / mL; Step 3, Preparation of probiotic fermented traditional Chinese medicine liquid: ... Lactobacillus rhamnosus bacterial solution and Bifidobacterium adolescentis The bacterial solution was inoculated into the extract of the traditional Chinese medicine compound composition. After the components were stirred evenly, the mixture was fermented under anaerobic conditions at 37°C for 72 hours to obtain the probiotic fermented traditional Chinese medicine solution. Step 4: Preparation of oral formulations: The probiotic-fermented Chinese medicine liquid obtained in step 3 is centrifuged, filtered, and sterilized to obtain an oral liquid. Alternatively, the probiotic-fermented Chinese medicine liquid obtained in step 3 can be centrifuged, concentrated, filtered, and sterilized to produce a powder; the powder is a freeze-dried powder obtained by freeze-drying to a moisture content of no more than 5%, grinding into powder, and sieving, or a powder obtained by spray drying. Alternatively, the probiotic fermented Chinese medicine liquid obtained in step 3 can be added to excipients to form a soft mass, which is then sieved to form wet granules. The wet granules are then dried, sized, compressed, and / or capsule-filled to form granule preparations, tablets, or capsules.

7. The preparation method according to claim 6, characterized in that: In step 3, the total inoculation volume of the two bacterial solutions is 0.5-20% of the volume of the extract of the traditional Chinese medicine compound composition, and the two bacterial solutions are inoculated in equal proportions.

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