Composition and application thereof in preparation of products for improving liver injury

Abstract

The invention relates to the technical field of microecological preparations, in particular to a composition and application thereof in preparation of a product for improving liver injury. The novel composition provided by the invention contains probiotics, a radix puerariae extract and a grape skin extract, and the composition can effectively improve liver injury from an intestine-liver axis mechanism level. Animal tests prove that the effect is very ideal, the main components have a synergistic effect, the effect of remarkably improving liver injury can be achieved from multiple mechanism levels, and the traditional Chinese medicine composition is safe and free of side effects.

Description

Technical Field

[0001] This invention relates to the field of microecological preparations technology, and in particular to compositions and their use in the preparation of products that improve liver damage. Background Technology

[0002] The liver, a vital organ, plays a crucial role in maintaining energy balance, metabolic homeostasis, immune regulation, protein synthesis, and detoxification. It also plays a dominant role in drug metabolism, infection defense, and overall health. However, the liver is highly susceptible to damage from various factors, such as drug overdose, severe infections, chronic alcoholism, and exposure to toxic chemicals like carbon tetrachloride. Inflammatory responses can also contribute to liver damage.

[0003] Treatment of liver injury mainly involves three aspects. First, etiological treatment: For alcoholic liver injury, strict abstinence from alcohol is crucial; for drug-induced liver injury, the causative drug should be discontinued immediately; for liver injury caused by chemical toxins, immediate removal from the toxic environment is essential. Second, hepatoprotective treatment: Commonly used hepatoprotective drugs include hepatocyte membrane repair and protection agents, such as polyene phosphatidylcholine; detoxifying drugs, such as glutathione, N-acetylcysteine, and thioproline; anti-inflammatory drugs, such as magnesium isoglycyrrhizinate and diammonium glycyrrhizate; antioxidant drugs, such as silymarin, bicyclol, and biphenyl diester; and choleretic drugs, such as S-adenosylmethionine and ursodeoxycholic acid. Third, nutritional supplementation: Consuming easily digestible, high-protein, and high-calorie foods helps with liver recovery.

[0004] Despite the existence of various treatment methods, the efficacy of existing treatments for liver injury remains relatively limited. Long-term medication can easily lead to drug resistance in patients, accompanied by drug toxicity and side effects. Given the long treatment cycle and difficulty in curing liver injury, developing effective and side-effect-free agents for the prevention and treatment of liver disease has become an urgent problem to be solved. Although the combined use of multiple active substances may improve the improvement of liver injury, the effects of drug combination include synergistic, additive, and antagonistic effects, and not all combination combinations can produce synergistic effects. If a synergistic effect cannot be achieved, the combination regimen may not be superior to monotherapy. Therefore, exploring drugs that can exert synergistic effects after combination is particularly crucial and urgent for improving the treatment efficacy of liver injury. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a composition and its application in the preparation of products for improving liver injury. The present invention uses a combination of traditional Chinese medicine extracts and Bacillus coagulans to treat liver injury, exerting effects from multiple aspects such as anti-oxidation, reducing inflammatory response, and regulating intestinal flora homeostasis.

[0006] The composition provided by the present invention consists of kudzu root extract, grape skin extract, Bacillus coagulans and yeast powder.

[0007] In the composition provided by this invention, both kudzu root extract and grape skin extract possess antioxidant physiological effects, effectively neutralizing free radicals and reducing oxidative stress-induced liver damage. *Weizmannii coagulans* can inhibit the production of inflammatory cytokines, increase the secretion of anti-inflammatory cytokines, exert immunomodulatory effects, reduce liver inflammation, and alleviate liver damage in patients. *Weizmannii coagulans* can also promote the growth of other beneficial bacteria in the gut, restore gut microbiota homeostasis, restore the barrier function of the intestinal epithelium, and further achieve a protective effect against liver damage through gut-liver axis regulation.

[0008] The scientifically formulated combination of *Weizmannii coagulans*, kudzu root extract, grape skin extract, and yeast powder creates a synergistic effect, resulting in hepatoprotective efficacy superior to that of a single ingredient. Using a mouse CCl4 liver injury model, studies have demonstrated that the composition of this invention exhibits superior hepatoprotective effects compared to certain commercially available drugs, single-function ingredients, and single *Weizmannii coagulans* samples, showing lower liver indices, less liver damage, a stronger reduction in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, and a stronger inhibitory effect on NF-κB-mediated liver inflammation.

[0009] Therefore, the composition provided by this invention, consisting of kudzu root extract, grape skin extract, Bacillus coagulans TBC-169 and yeast powder, can significantly improve liver damage at multiple mechanism levels, and is safe and without side effects. The combination of each component produces a synergistic effect.

[0010] In this invention, the mass ratio of kudzu root extract, grape skin extract, Bacillus coagulans and yeast powder is (36-210):(8-107):(24-160):(6-99).

[0011] Or the mass ratio of the kudzu root extract, grape skin extract, Bacillus coagulans and yeast powder is (45-178):(11-96):(30-145):(8-87);

[0012] Or the mass ratio of the kudzu root extract, grape skin extract, Bacillus coagulans and yeast powder is (56-165):(14-85):(37-132):(9-85);

[0013] Or the mass ratio of the kudzu root extract, grape skin extract, Bacillus coagulans and yeast powder is (68-147):(18-83):(45-124):(11-77);

[0014] Or the mass ratio of the kudzu root extract, grape skin extract, Bacillus coagulans and yeast powder is (75-122):(21-65):(56-103):(13-64);

[0015] Or the mass ratio of the kudzu root extract, grape skin extract, Bacillus coagulans and yeast powder is (79-116):(23-57):(58-92):(16-55);

[0016] Or the mass ratio of the kudzu root extract, grape skin extract, Bacillus coagulans and yeast powder is (85-101):(25-49):(64-85):(17-43);

[0017] In some embodiments, the mass ratio of the kudzu root extract, grape skin extract, Bacillus coagulans and yeast powder is (70-100):(23-37):(50-88):(20-25).

[0018] In a specific embodiment, the mass ratio of kudzu root extract, grape skin extract, Bacillus coagulans and yeast powder is 90.02:29.96:70:21.

[0019] In this invention, the Bacillus coagulans is Bacillus coagulans TBC169, and the yeast powder is LalminB200.

[0020] Furthermore, the present invention also provides the use of the composition as described above in the preparation of products that improve liver damage.

[0021] The improvement of liver injury described in this invention includes reducing liver index, reducing transaminase levels, reducing HE staining score, and / or inhibiting inflammatory pathways.

[0022] In this invention, the transaminase includes ALT and / or AST;

[0023] The inhibition of inflammatory pathways includes: reducing p-IκB protein and / or p-NF-κB protein levels, inhibiting phosphorylation of IκB and NF-κB (p65) and / or inhibiting activation of the NF-κB signaling pathway.

[0024] In this invention, the liver injury includes hepatitis, liver fibrosis, cirrhosis, liver cancer, fatty liver, alcoholic liver disease, or non-alcoholic fatty liver disease.

[0025] The improvements described in this invention also include the prevention or treatment of liver damage, including adjuvant protection against chemically induced liver damage, alcohol detoxification, and prevention and treatment of liver diseases such as hepatitis, liver fibrosis, cirrhosis, liver cancer, fatty liver, alcoholic liver disease, or non-alcoholic fatty liver disease. Treatment of liver diseases mainly includes lowering transaminase levels, reducing liver damage, and inhibiting inflammatory pathways. The transaminases include alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The composition provided by this invention contains probiotics, effectively improving liver damage at the gut-liver axis mechanism level.

[0026] Furthermore, the present invention also provides a product for improving liver damage, comprising the composition and excipients described above.

[0027] In the product described in this invention, the viable count of Bacillus coagulans is greater than 8 × 10⁻⁶. 7 CFU / g.

[0028] In this invention, the excipients include fillers (e.g., starch, pregelatinized starch, dextrin, sucrose, lactose, microcrystalline cellulose, mannitol, sorbitol, dicalcium phosphate, calcium sulfate), lubricants (e.g., magnesium stearate, micronized silica gel, talc, hydrogenated vegetable oil, polyethylene glycol, sodium lauryl sulfate), disintegrants (e.g., sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, croscarmellose sodium, croscarmellose, effervescent disintegrants (sodium bicarbonate and citric acid)), binders (e.g., hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone, gelatin, polyethylene glycol, starch paste, methylcellulose, ethylcellulose), humectants (e.g., glycerin, propylene glycol, sorbitol), and pH adjusters (e.g., citric acid, sodium citrate, phosphoric acid, sodium dihydrogen phosphate, phosphate). The following ingredients are included: disodium hydrogen, sodium hydroxide, hydrochloric acid, sodium bicarbonate; sweeteners (e.g., sucrose, lactose, steviol glycosides, aspartame, sucralose, glycyrrhizin, xylitol); flavoring agents (e.g., vanillin, strawberry flavor, menthol, orange peel tincture, banana flavor, apple flavor); preservatives (e.g., sodium benzoate, potassium sorbate, methylparaben, ethylparaben, propylparaben, benzalkonium bromide); solubilizers (e.g., polysorbate-80, poloxamer, propylene glycol, glycerin, urea); coating materials (e.g., hydroxypropyl methylcellulose, ethyl cellulose, acrylic resins (Eudragit series), cellulose acetate); and thickeners (e.g., sodium carboxymethyl cellulose, gum arabic, agar, sodium alginate, xanthan gum). Preferably, it contains one or more of the following excipients: microcrystalline cellulose, sodium hydroxymethyl starch, magnesium stearate, starch, or silicon dioxide.

[0029] Products made from the compositions of the present invention preferably include pharmaceuticals, foods for special medical purposes, health foods, functional foods, food products, candies, beverages, etc., with the addition of various excipients.

[0030] The dosage forms of the products described in this invention include, but are not limited to: powders, granules, tablets, capsules, soft capsules, oral liquids, etc.

[0031] Acute toxicity tests and 30-day feeding trials have confirmed that the compositions of this invention do not exhibit any toxic side effects.

[0032] The present invention also provides a method for improving liver injury, comprising administering the composition or product as described above.

[0033] The methods of administration include, but are not limited to, swallowing, chewing, holding in the mouth, dissolving in water, or dripping.

[0034] This invention provides a novel composition containing probiotics, kudzu root extract, and grape skin extract. This composition effectively improves liver damage at the gut-liver axis mechanism level. Animal studies have demonstrated its excellent efficacy; the main components work synergistically to significantly improve liver damage at multiple mechanism levels, and it is safe with no side effects. Attached Figure Description

[0035] Figure 1 Image showing the results of HE staining of liver pathological sections;

[0036] Figure 2 The protein band diagram is shown. Since previous experiments found that groups E and F had no significant effect on liver damage, Western blotting was not performed on groups E and F.

[0037] Figure 3 The effect of drug administration on liver index in mice ( (n=10); Note: ****, P<0.0001 compared with group K; #### P<0.0001 compared with group M;

[0038] Figure 4 The effect of drug administration on serum ALT levels in mice ( n=10); Note: ****, P<0.0001 compared with group K; ##, P<0.01 compared with group M; ####, P<0.0001 compared with group M;

[0039] Figure 5 The effect of drug administration on serum AST levels in mice ( (n=10); Note: ****, P<0.0001 compared with group K; #, P<0.05 compared with group M; ##, P<0.01 compared with group M; ###, P<0.001 compared with group M;

[0040] Figure 6 This shows the effect of drug administration on the HE staining score of mouse liver pathological sections. (n=10); Note: ****, P<0.0001 compared with group K; ## P<0.01 compared with group M; ### P<0.001 compared with group M; #### P<0.0001 compared with group M. Detailed Implementation

[0041] This invention provides compositions and their use in the preparation of products that improve liver injury. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0042] Unless otherwise defined in this invention, scientific and technical terms related to this invention shall have the meanings understood by one of ordinary skill in the art.

[0043] The terms “comprising,” “including,” and “having” are used interchangeably to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” herein also provides for schemes “consisting of…”.

[0044] The term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can mean: A exists alone, A and B exist simultaneously, or B exists alone. A and B can be singular or plural.

[0045] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions mean any combination of these items, including any combination of single or multiple items.

[0046] The numerical ranges and parameters involved in this invention have been presented as precisely as possible in the specific embodiments. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise explicitly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have a reasonable deviation within a certain range, such as ±10%, ±5%, ±1%, or ±0.5%.

[0047] The embodiments and comparative examples of this invention describe some cases. These embodiments illustrate certain implementations of the invention. However, this does not mean that the effects of the invention can only be achieved in these cases. In fact, any concentration of the components between the two endpoint values ​​shown in the embodiments can achieve a good effect in improving liver injury.

[0048] Formulas B, C, E, and F only list some cases where the results were not good in the experiments. In addition, many other attempts were made during the research and development process, such as trying to use different probiotics, using different strains of lactic acid bacteria (such as using Bifidobacterium or Lactobacillus), or adjusting the ratio or concentration of each component. However, the effects of these attempts were not as good as those of Formula G, and will not be elaborated here.

[0049] The test materials used in this invention are all common commercially available products, and can be purchased on the market. Among them:

[0050] Kudzu root extract is obtained from kudzu root through conventional water or alcohol extraction methods, with an extraction rate of 4%-20%, and its main component is puerarin.

[0051] Grape skin extract is obtained from grape skins through conventional water or alcohol extraction methods, with an extraction rate of 3%-15%, and its main component is resveratrol.

[0052] *Weizmannii coagulans*, also known as *Bacillus coagulans*, possesses advantages such as heat resistance, stomach acid resistance, easy storage, and rapid growth. *Bacillus coagulans* can produce lactic acid, lowering the pH value of the intestine, increasing the abundance of beneficial bacteria, promoting their growth and reproduction, while reducing the number of harmful bacteria, inhibiting their colonization in the intestine, regulating intestinal flora homeostasis, restoring the barrier function of the intestinal epithelium, reducing intestinal epithelial permeability, reducing the risk of harmful substances entering the liver through the gut-liver axis, and maintaining normal liver function. This invention uses *Bacillus coagulans* TBC169, produced by Qingdao Donghai Pharmaceutical Co., Ltd., which has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 1207.

[0053] The yeast powder mentioned in the examples is yeast extract, purchased from Shanghai Tongyuan Food Technology Co., Ltd. Lalmin B200 is one of the series of yeast powders.

[0054] It should be understood that in the various embodiments of this application, the sequence numbers of the above processes do not imply the order of execution. Some or all steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application. The present invention is further illustrated below with reference to embodiments:

[0055] The composition of the embodiment has an adjuvant protective effect on chemical liver injury

[0056] 1 Test purpose

[0057] In this experiment, a commercially available drug for treating liver injury was used as the positive control. After continuously gavage-feeding the drugs to Kunming (KM) mice for 30 days, a single gavage of 1% carbon tetrachloride solution was used to establish a model. By detecting various liver injury indexes, the protective effect of the invention on chemical liver injury in mice was explored.

[0058] 2 Test materials and test methods

[0059] 2.1 Test animals: 120 male KM mice, 3 - 4 weeks old, weighing 18 - 22 g, provided by Shandong Pengyue Laboratory Animal Breeding Co., Ltd., animal production license number: SCXK(Shandong)20220006. Breeding environment: room temperature is 20℃ - 25℃, relative humidity is 40% - 70%, artificial lighting, day and night light and dark alternation is 12 h, drinking water, feed and bedding are all sterile. All animal experiments were carried out according to the animal experiment guidelines and protocols approved by the Donghai Pharmaceutical Welfare Ethics Committee, and the ethical approval number is: LLSC - 2024 - 001.

[0060] 2.2 Test drugs: The following are the test drugs. The composition provided by the present invention is prepared from the following main raw materials (unit: parts by weight).

[0061] Table 1 Composition formulation and reference substances

[0062]

[0063] 2.3 Test methods

[0064] Grouping: After normal feeding of the mice for one week, the mice with too small or too large body weights were excluded. The remaining 100 mice were randomly divided into 10 groups according to body weight, namely blank control group (K), model control group (M), a commercially available drug group in a certain city (Y), formulation A group (A), formulation B group (B), formulation C group (C), formulation D group (D), formulation E group (E), formulation F group (F), formulation G group (G), with 10 mice in each group.

[0065] Preventive administration: The body weights of the mice in each group were recorded every other day. Each day, they were orally gavaged with purified water (group K and group M) or the corresponding drug (dissolved in purified water and administered in suspension) once a day, and continuously administered for 30 days at a dose of 10 mL / kg BW.

[0066] Modeling: Two hours after the end of administration on day 30, all experimental groups except group K were administered 1% CCl4 (diluted with edible vegetable oil) by gavage at a dose of 5 mL / kg BW. Group K was administered an equal volume of edible vegetable oil by gavage. After the end of the modeling administration, the mice were fasted but allowed free water for 16 hours.

[0067] 2.4 Sample Preparation

[0068] On the day of sampling, mouse weight was recorded. Mice were anesthetized by intraperitoneal injection of 10% chloral hydrate at a dose of 0.1 mL / 10 g. Blood was collected by enucleation. After standing at room temperature for 1 hour, the blood samples were centrifuged (3000 rpm, 15 min) to obtain serum, which was stored at -80°C. Mice were euthanized by cervical dislocation after blood collection. The livers were dissected, weighed, and the liver index was calculated. The left lobe of the liver was soaked in 10% neutral formalin solution for 48 hours, dehydrated using a programmed procedure, embedded in paraffin, sectioned, spread, and stained with hematoxylin and eosin (HE) for liver pathological examination.

[0069] 3. Detection methods

[0070] 3.1 Measurement of liver index

[0071] Before dissection, the mice were weighed and blood was drawn. The livers were then dissected, rinsed with pre-cooled PBS buffer, blotted dry with filter paper, and weighed to calculate the liver coefficient of the mice. The liver coefficient (%) = liver mass (g) / body mass (g) × 100%.

[0072] 3.2 Determination of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST)

[0073] The levels of ALT and AST in serum were detected using alanine aminotransferase (ALT) assay kit and aspartate aminotransferase (AST) assay kit.

[0074] 3.3 Histopathological observation of liver tissue

[0075] The left lobe of the liver was immersed in 10% neutral formalin solution for 48 hours, then dehydrated, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). The area occupied by hepatocyte ballooning degeneration, hepatocyte fatty degeneration, cytoplasmic aggregation, hydropic degeneration, and hepatocyte necrosis in each field of view was recorded. Each lesion type was scored according to Table 1, and the total lesion score was accumulated. The score for hepatocyte necrosis was multiplied by 2 and included in the total lesion score.

[0076] Table 2. HE staining scoring table for liver pathological sections.

[0077] lesion area score Generally normal 0 points A certain type of hepatocyte occupies 1 / 4 of the entire field of view. 1 point A certain type of hepatocyte occupies 1 / 2 of the entire field of view. 2 points A certain type of hepatocyte occupies 3 / 4 of the entire field of view. 3 points A certain type of hepatocyte occupied the entire field of view 4 points

[0078] 3.4 Western Blot Experiment

[0079] The livers of each group of animals were weighed, and total protein was extracted by adding lysis mixture. A standard curve was plotted, and the protein content was measured. Protein samples were then prepared and SDS-PAGE electrophoresis was performed to detect the expression of IκB, NF-κB, p-IκB, and p-NF-κB proteins.

[0080] 4. Statistical processing methods

[0081] Experimental results are expressed as mean ± standard deviation. The data for each group were statistically analyzed using GraphPad Prism 8.0 software. Differences between groups were analyzed using Dunnett's test of one-way ANOVA. A p-value < 0.05 was considered statistically significant.

[0082] 5 Experimental Results

[0083] 5.1 Effects of drug administration on liver index in mice

[0084] After animal exposure to the toxin, the liver index increased, indicating hepatocyte proliferation, liver congestion, and edema. Table 2 shows that the liver index of the model control group (Group M) was 5.15%, while that of the blank control group (Group K) was 3.47%. The liver index of the model control group was 48.41% higher than that of the blank control group, a significant difference (P < 0.05), indicating that oral administration of 1% CCl4 solution can induce liver damage in mice, causing significant liver congestion and swelling. The liver indices of the commercially available drug group, formulation C group, and formulation G group were 4.54%, 4.47%, and 4.31%, respectively, which were 11.84%, 13.25%, and 16.46% lower than those of the model control group, a significant difference (P < 0.05). The liver indices of formulations A, B, D, E, and F groups decreased compared to the model control group, but the difference was not significant. Therefore, it can be seen that the effects of medium and low doses of the active ingredient, Bacillus coagulans (TBC-169) alone, and low doses of the active ingredient in combination with Bifidobacterium and Lactobacillus respectively on the treatment of liver injury are limited. However, high doses of the active ingredient and low doses of the active ingredient in combination with Bacillus coagulans can significantly reduce the liver index in mice and alleviate liver injury in mice.

[0085] Table 3 Effects of drug administration on liver index in mice ( n=10)

[0086] Group Liver index (%) Blank control group (K) 3.47±0.23 Model control group (M) <![CDATA[5.15±0.28 * ]]> A certain group of commercially available drugs (Y) <![CDATA[4.54±0.19 # ]]> Formula A (A) 5.06±0.31 Formula B (B) 4.93±0.26 Formula C (C) <![CDATA[4.47±0.31 # ]]> Formula D (D) 4.89±0.27 Formula E (E) 4.97±0.17 Formula F (F) 4.97±0.41 Formula E (G) <![CDATA[4.31±0.16 # ]]>

[0087] Note: * Compared with group K, P < 0.05, indicating a statistically significant difference; # Compared with group M, P < 0.05, indicating a statistically significant difference.

[0088] 5.2 Effects of drug administration on serum ALT and AST levels in mice

[0089] ALT and AST are the earliest and most sensitive indicators of changes in liver function. Normally, serum ALT and AST levels are very low. However, when hepatocytes are damaged, cell membrane permeability increases, causing ALT and AST to leak out of the hepatocytes, leading to elevated serum ALT and AST levels. Their activity levels reflect the degree of liver damage. In the model control group (M group), the serum ALT and AST levels of mice were 5448.07 and 3121.04, respectively, significantly higher than those in the blank control group (K group), which was statistically significant (P < 0.05). This indicates that oral administration of 1% CCl4 solution disrupted the mouse hepatocyte membrane, causing acute liver injury, and the model was successfully established. The serum ALT levels in the commercially available drug group, formula C group, and formula G group decreased by 16.38%, 17.23%, and 22.80% respectively compared with the model control group, showing a significant difference (P < 0.05); the serum ALT levels in formulas A, B, D, E, and F groups decreased compared with the model control group, but the difference was not significant. The serum AST levels in the commercially available drug group, formula B group, formula C group, and formula G group decreased by 31.32%, 33.91%, 36.30%, and 41.21% respectively compared with the model control group, showing a significant difference (P < 0.05); the serum AST levels in formulas A, D, E, and F groups decreased compared with the model control group, but the difference was not significant. Therefore, it can be seen that the effects of low-dose active ingredients, Bacillus coagulans (TBC-169) alone, and low-dose active ingredients combined with Bifidobacterium and Lactobacillus respectively in treating liver injury are limited. However, high-dose active ingredients and low-dose active ingredients combined with Bacillus coagulans can significantly reduce the levels of ALT and AST in mouse serum and alleviate liver injury in mice.

[0090] Table 4. Effects of drug administration on serum ALT and AST levels in mice ( n=10)

[0091] Group ALT(U / L) AST(U / L) Blank control group (K) 17.60±7.25 5.13±3.43 Model control group (M) <![CDATA[5448.07±464.45 * ]]> <![CDATA[3121.04±938.41 * ]]> A certain group of commercially available drugs (Y) <![CDATA[4555.82±871.55 # ]]> <![CDATA[2143.28±476.22 # ]]> Formula A (A) 4942.8±370.21 2288.40±451.65 Formula B (B) 4856.01±542.15 <![CDATA[2062.62±433.79 # ]]> Formula C (C) <![CDATA[4509.24±590.70 # ]]> <![CDATA[1988.10±854.38 # ]]> Formula D (D) 4998.61±401.69 2311.15±417.80 Formula E (E) 4912.80±381.98 2288.40±543.47 Formula F (F) 4846.01±567.40 2292.62±753.72 Formula E (G) <![CDATA[4205.74±286.14 # ]]> <![CDATA[1834.98±620.20 # ]]>

[0092] Note: * Compared with group K, P < 0.05, indicating a statistically significant difference; # Compared with group M, P < 0.05, indicating a statistically significant difference.

[0093] 5.3 Effect of drug administration on HE staining of mouse liver pathological sections

[0094] from Figure 1As can be seen, in the blank control group, the liver pathological sections stained with HE showed a polyhedral shape under the microscope, with the central cell nucleus appearing as a round vesicle. The hepatocytes were polygonal and arranged orderly in the liver plates around the central vein. In the model control group, the liver lobules of the mice showed obvious damage, manifested as hepatocyte swelling and rupture, cytoplasmic vacuolation, and nuclear fragmentation. As shown in Table 4, compared with the blank control group, the HE staining score of the model control group was significantly higher, with a statistically significant difference (P < 0.05). The HE staining scores of liver pathological sections in the commercially available drug group, formula B group, formula C group, and formula G group decreased by 32.02%, 28.09%, 30.34%, and 41.57% respectively compared with the model control group, showing a significant difference (P < 0.05). The HE staining scores of liver pathological sections in formulas A, D, E, and F groups decreased compared with the model control group, but the difference was not statistically significant. Therefore, it can be seen that the effects of low-dose active ingredients, Bacillus coagulans (TBC-169) alone, and low-dose active ingredients in combination with Bifidobacterium and Lactobacillus respectively on the treatment of liver injury are limited, while the combination of low-dose active ingredients with Bacillus coagulans can significantly improve the liver injury in mice.

[0095] Table 5. Effects of drug administration on HE staining scores of mouse liver histopathological sections ( n=10)

[0096] Group Liver pathological section HE staining score Blank control group (K) 0 Model control group (M) <![CDATA[17.80±1.66 * ]]> A certain group of commercially available drugs (Y) <![CDATA[12.10±3.08 # ]]> Formula A (A) 14.50±2.42 Formula B (B) <![CDATA[12.80±3.09 # ]]> Formula C (C) <![CDATA[12.40±2.06 # ]]> Formula D (D) 14.70±3.66 Formula E (E) 14.35±2.65 Formula F (F) 14.40±1.36 Formula G (G) <![CDATA[10.40±2.24 # ]]>

[0097] Note: * indicates a statistically significant difference compared to group K (P < 0.05); # indicates a statistically significant difference compared to group M (P < 0.05).

[0098] 5.4 Effects of drug administration on the expression of p-IκB and p-NF-κB proteins

[0099] In the NF-κB signaling pathway, phosphorylation of IκB and NF-κB (p65) (p-IκB and p-NF-κB, i.e., p-p65) mediates the activation of the inflammatory pathway. Carbon tetrachloride entering the body stimulates hepatocytes, and a cascade signaling system activates the IκB kinase complex (IKK). Activated IKK phosphorylates IκB (S36), causing IκB to detach from NF-κB. NF-κB phosphorylation induces the release of inflammatory factors such as TNF-α, IL-1β, and IL-6.

[0100] The results showed that, compared with the blank control group, the expression level of p-IκB protein in the model group was increased, and the expression level of p-NF-κB protein in the model group was higher than that in the blank group, indicating that after oral administration of 1% CCl4 solution, IκB and its downstream NF-κB (p65) were phosphorylated, and the NF-κB signaling pathway was activated.

[0101] Compared with the model group, the 6 synergistic effects of each treatment group were observed.

[0102] Based on the Bliss independent model, we analyzed whether the composition produced a synergistic effect, with formulation A as drug A, formulation D as drug B, and formulation G as the combination drug group.

[0103] The efficacy of each drug group is expressed as a percentage change relative to the model group, calculated using the following formula:

[0104] E = (Data for each test item - Data for the model group) / Data for the model group * 100%

[0105] The coefficient Q calculated by the Bliss independent model is: (Percentage decrease due to combination therapy) / (Percentage decrease due to single-drug therapy in group A + Percentage decrease due to single-drug therapy in group D - Percentage decrease due to single-drug therapy in group A × Percentage decrease due to single-drug therapy in group D)

[0106] Table 6

[0107]

[0108] A value greater than 1 calculated by the Bliss independent model indicates synergy. As the previous results showed, the composition had a synergistic effect in improving liver index, reducing ALT levels, and improving HE staining scores.

[0109] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A composition comprising kudzu root extract, grape skin extract, Bacillus coagulans and yeast powder.

2. The composition according to claim 1, characterized in that, The mass ratio of the kudzu root extract, grape skin extract, Bacillus coagulans and yeast powder is (40-200):(13-80):(25-160):(8-70).

3. The composition according to claim 2, characterized in that, The mass ratio of the kudzu root extract, grape skin extract, Bacillus coagulans, and yeast is 90.02:29.96:70:

21.

4. The composition according to any one of claims 1 to 3, characterized in that, The Bacillus coagulans is Bacillus coagulans TBC169, and the yeast powder is LalminB200.

5. Use of the composition according to any one of claims 1 to 4 in the preparation of a product for improving liver damage.

6. The application according to claim 5, characterized in that, The improvement of liver injury includes reducing liver index, reducing transaminase levels, reducing HE staining score and / or inhibiting inflammatory pathways.

7. The application according to claim 6, characterized in that, The transaminases include ALT and / or AST; The inhibition of inflammatory pathways includes: reducing p-IκB protein and / or p-NF-κB protein levels, inhibiting phosphorylation of IκB and NF-κB (p65) and / or inhibiting activation of the NF-κB signaling pathway.

8. The application according to claim 6, characterized in that, The liver injury includes hepatitis, liver fibrosis, cirrhosis, liver cancer, fatty liver, alcoholic liver disease, or non-alcoholic fatty liver disease.

9. A product for improving liver damage, characterized in that, Includes the composition and excipients described in any one of claims 1 to 3.

10. The product according to claim 9, characterized in that, The viable count of Bacillus coagulans was greater than 8 × 10⁻⁶. 7 CFU / g.

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