A composition with liver-protecting efficacy and a preparation method and application thereof

Abstract

The application provides a composition with liver protection efficacy and a preparation method and application thereof. The composition with liver protection efficacy comprises the following ingredients by mass fraction: 10-30 parts of Silybum marianum powder, 15-40 parts of Pueraria lobata powder, 20-35 parts of Salvia miltiorrhiza powder, 5-15 parts of freeze-dried Rubus corchorifolius fruit powder, and 8-20 parts of Hovenia dulcis fruit powder. The preparation method comprises the following steps: mixing the above-mentioned raw materials, subjecting the mixture to enzymolysis by using a composite enzyme, subjecting the enzymolysis product to fermentation by using a composite strain, obtaining a fermentation product, and freeze-drying the fermentation product to obtain the composition with liver protection efficacy. The composite enzyme comprises cellulase and pectinase, and the composite strain comprises Bacillus pumilus and Sporolactobacillus inulinus. The composition with liver protection efficacy obtained by the application can effectively improve the liver function level.

Description

Technical Field

[0001] This invention relates to the field of health products, specifically to a composition with liver-protecting effects, its preparation method, and its application. Background Technology

[0002] In recent years, with the rising incidence of liver diseases, the demand for liver-protecting health products and drugs has continued to grow. Traditional liver-protecting products are mostly based on single active ingredients, which suffer from problems such as single target of action and insufficient synergistic effects. At the same time, the active ingredients in plant raw materials often have low extraction efficiency and limited bioavailability due to their complex molecular structure or cell wall encapsulation. In existing technologies, although there have been attempts to improve the release of plant components by using enzymatic hydrolysis or fermentation processes, problems such as single enzyme types and unreasonable strain compatibility still result in insignificant improvement in product activity. For example, conventional fermentation processes often use single strains (such as lactic acid bacteria or acetic acid bacteria), which are difficult to fully degrade cellulose and pectin in raw materials and lack the ability to target and transform liver-protecting active ingredients. Therefore, developing a composition that enhances liver-protecting efficacy through multi-raw material synergistic formulation, combined enzymatic hydrolysis and targeted fermentation technology has become an urgent technical problem to be solved. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a composition with liver-protective effects, its preparation method and application, which can effectively improve liver function.

[0004] The technical solution of this invention is as follows:

[0005] In a first aspect, the present invention provides a method for preparing a composition with hepatoprotective effects, comprising the following steps:

[0006] a) Enzymatic hydrolysis: The substrate raw materials of the composition are mixed evenly to obtain raw material powder. The raw material powder is prepared into a raw material solution with a concentration of 11-13 wt% using sterile deionized water. A compound enzyme is added to the raw material solution for enzymatic hydrolysis to obtain an enzymatic hydrolysate. The amount of the compound enzyme added is 1-3 wt% of the mass of the raw material solution. The compound enzyme is composed of cellulase and pectinase mixed in a mass ratio of 1:(0.5-0.7). The cellulase activity is 30,000 U / g, the pectinase activity is 30,000 U / g, the enzymatic hydrolysis temperature is 40-55℃, the enzymatic hydrolysis time is 2-4 h, and the enzymatic hydrolysis pH is 4.0-5.5.

[0007] b) In the compound fermentation, the enzymatic hydrolysate is prepared into a mixed solution with a concentration of 10-12 wt% using sterile deionized water. After sterilization of the mixed solution, it is inoculated with a mixed bacterial culture and fermented at 30-37℃ for 48-72 hours to obtain the fermentation broth. The fermentation broth is then sterilized, filtered, concentrated, and spray-dried to obtain the liver-protecting composition. The mass ratio of the mixed bacterial culture to the mixed solution is 1:(11-12). The mixed bacterial culture consists of *Acetobacter pasteurellii* inoculum and *Lactobacillus inulinus* inoculum, with a viable count ratio of 1:(0.5-1.5). The viable count of the *Acetobacter pasteurellii* inoculum is 1×10⁻⁶. 8 -1×10 10 CFU / g, the viable count of the *Lactobacillus inulinus* inoculum culture solution is 1×10⁻⁶. 8 -1×10 10 CFU / g;

[0008] The preservation number of the *Acetobacter pasteurellium* is: CGMCC No. 12930;

[0009] The preservation number of the Lactobacillus inulinus is: CGMCC No. 2185.

[0010] The substrate raw material of the composition includes the following raw materials in parts by weight:

[0011] Milk thistle powder: 10-30 parts;

[0012] Kudzu root powder: 15-40 servings;

[0013] Salvia miltiorrhiza powder: 20-35 parts;

[0014] Wild cherry jelly powder: 5-15 servings;

[0015] Hovenia dulcis powder: 8-20 servings.

[0016] Preferably, the substrate raw material of the composition comprises the following parts by weight:

[0017] Milk thistle powder: 20-30 parts;

[0018] Kudzu root powder: 25-40 servings;

[0019] Salvia miltiorrhiza powder: 30-35 parts;

[0020] Wild cherry jelly powder: 5-10 portions;

[0021] Hovenia dulcis powder: 15-20 servings.

[0022] Furthermore, the substrate raw material of the composition comprises the following raw materials in parts by weight:

[0023] Milk thistle powder: 20 parts;

[0024] Kudzu root powder: 25 portions;

[0025] Salvia miltiorrhiza powder: 30 portions;

[0026] Wild cherry jelly powder: 10 servings;

[0027] Hovenia dulcis powder: 15 portions.

[0028] In a second aspect, the present invention provides the application of a composition with hepatoprotective effects prepared by the preparation method described in the first aspect in the preparation of health products or pharmaceuticals with hepatoprotective effects.

[0029] Thirdly, the present invention provides a liver-protecting preparation comprising a composition having liver-protecting effects prepared by the preparation method described in the first aspect.

[0030] Preferably, the dosage form of the liver-protecting preparation is any one of tablets, capsules, ointments, pills, or oral liquids.

[0031] Fourthly, the present invention provides a liver-protecting tablet, characterized in that it comprises the following components in parts by weight:

[0032] The composition with liver-protective effects prepared by the preparation method described in the first aspect: 30-35 parts;

[0033] Microcrystalline cellulose: 10-30 parts;

[0034] Lactose: 20-40 parts

[0035] Magnesium stearate: 0.01-1 part;

[0036] Skim milk powder: 10-30 servings;

[0037] Sodium carboxymethyl starch: 5-15 parts.

[0038] The preparation method of the liver-protecting tablets includes the following steps:

[0039] Step 1: Mix the microcrystalline cellulose, lactose, skim milk powder, and sodium carboxymethyl starch, then add the mixture to a dry granulator for granulation, and collect 10-120 mesh particles;

[0040] Step 2: Mix the liver-protecting composition, the granules collected in Step 1, and magnesium stearate to obtain a mixture;

[0041] Step 3: Put the mixture into a tablet press and compress it into tablets. Adjust the pressure to 10-40 kN to obtain liver protection tablets.

[0042] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0043] The synergistic effect of the composition of the present invention is achieved through a three-dimensional network of multi-component targeted regulation and process enhancement, as follows:

[0044] 1. In terms of preparation process, cellulase and pectinase at a mass ratio of 1:(0.5-0.7) are used to perform compound enzymatic hydrolysis on the substrate raw material to break the cell wall and release the active ingredients;

[0045] 2. Regarding the compatibility of substrate ingredients, the active components of milk thistle powder, silymarin, flavonoids, and polysaccharides, activate the Nrf2 / ARE antioxidant pathway. Meanwhile, the isoflavones, terpenes, and polysaccharides in kudzu root powder inhibit the NF-κB inflammatory pathway, forming a dual "oxidation-inflammation" blockade that enhances the liver-protective efficacy of the composition. The anthocyanins in wild cheri lyophilized powder chelate metal ions, and the polysaccharides, saponins, and polyphenols in Japanese raisin tree powder synergistically enhance free radical scavenging and toxin metabolism in the body. The polysaccharides and saponins in tanshinone powder activate the PI3K / Akt anti-apoptotic pathway. These ingredients improve microcirculation to form a closed loop of "damage repair-metabolic balance," further increasing the liver-protective efficacy of the composition.

[0046] 3. Simultaneously, co-fermentation with *Acetobacter pasteurellii* (CGMCC No. 12930) and *Bacillus inulinus* (CGMCC No. 2185) is adopted to further release flavonoids, polyphenols, and polysaccharides from the substrate raw materials, and increase the bioavailability by increasing the organic acids and complex enzymes produced by the fermentation. Detailed Implementation

[0047] To enable those skilled in the art to better understand the technical solutions in the embodiments of the present invention, and to make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be further described below.

[0048] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and such ranges or values ​​should be understood to include values ​​close to such ranges or values. With respect to data ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0049] Some of the raw materials used in this experiment came from the following sources:

[0050] Milk thistle powder: Milk thistle was purchased from Qingping Chinese medicinal materials market in Guangzhou. After screening and removing impurities, the milk thistle was ground, passed through an 80-mesh sieve, and then sealed for later use.

[0051] Kudzu root powder: Kudzu root was purchased from Qingping Chinese medicinal materials market in Guangzhou. After screening and removing impurities, the kudzu root was ground, passed through an 80-mesh sieve, and made into powder. It was then sealed and stored for later use.

[0052] Salvia miltiorrhiza powder: Salvia miltiorrhiza was purchased from Qingping Chinese medicinal materials market in Guangzhou. After screening and removing impurities, the purchased Salvia miltiorrhiza was ground, passed through an 80-mesh sieve, and made into powder. It was then sealed and stored for later use.

[0053] Wild cherry berry lyophilized powder: purchased from Ningxia Xiangcao Biotechnology Co., Ltd.

[0054] Hovenia dulcis powder: Hovenia dulcis seeds were purchased from Qingping Chinese Medicinal Herbs Market in Guangzhou. After screening and removing impurities, the seeds were ground, passed through an 80-mesh sieve, and then sealed for later use.

[0055] Cellulase: Purchased from Shaanxi Chenming Biotechnology Co., Ltd., with an enzyme activity of 30,000 U / g.

[0056] Pectinase: Purchased from Shaanxi Chenming Biotechnology Co., Ltd., with an enzyme activity of 30,000 U / g.

[0057] The mass fractions of the substrate raw materials for the liver-protecting composition and their preparation methods are as follows:

[0058] Parts by weight of substrate raw materials for liver-protecting composition:

[0059] Substrate group 1:

[0060] Milk thistle powder: 20 parts;

[0061] Kudzu root powder: 25 portions;

[0062] Salvia miltiorrhiza powder: 30 portions;

[0063] Wild cherry jelly powder: 10 servings;

[0064] Hovenia dulcis powder: 15 portions.

[0065] Substrate group 2:

[0066] Milk thistle powder: 10 parts;

[0067] Kudzu root powder: 15 portions;

[0068] Salvia miltiorrhiza powder: 20 portions;

[0069] Wild cherry berry jelly powder: 15 servings;

[0070] Hovenia dulcis powder: 8 parts.

[0071] Substrate group 3:

[0072] Milk thistle powder: 30 parts;

[0073] Kudzu root powder: 40 servings;

[0074] Salvia miltiorrhiza powder: 35 parts;

[0075] Wild cherry jelly powder: 5 portions;

[0076] Hovenia dulcis powder: 20 portions.

[0077] Substrate ingredient group ①: Unlike composition 1, it does not contain lyophilized cherifolia fruit powder, and missing mass parts are made up with sterile deionized water.

[0078] Substrate ingredient group ②: Unlike composition 1, it does not contain Hovenia dulcis powder, and the missing mass fractions are made up with sterile deionized water.

[0079] Substrate ingredient group ③: Unlike composition 1, it does not contain Hovenia dulcis powder and Cherry berry lyophilized powder, and the missing mass parts are made up with sterile deionized water.

[0080] Substrate ingredient group ④ differs from composition 1 in that it does not contain milk thistle powder, kudzu root powder, or salvia miltiorrhiza powder, and the missing mass fractions are made up with sterile deionized water.

[0081] The method for preparing composition 1 includes the following steps:

[0082] a) Enzymatic hydrolysis: Mix all raw materials in substrate raw material group 1 to obtain raw material powder. Prepare a raw material solution with a concentration of 12wt% using sterile deionized water. Add a compound enzyme to the raw material solution for enzymatic hydrolysis to obtain an enzymatic hydrolysate. The amount of the compound enzyme added is 2wt% of the mass of the raw material solution. The compound enzyme is composed of cellulase and pectinase mixed in a mass ratio of 1:0.6. The cellulase activity is 30,000 U / g, the pectinase activity is 30,000 U / g, the enzymatic hydrolysis temperature is 50℃, the enzymatic hydrolysis time is 3h, and the enzymatic hydrolysis pH is 5.0.

[0083] b) Compound fermentation: The enzymatic hydrolysate was prepared into a 11 wt% mixture using sterile deionized water. After sterilization, the mixture was inoculated with a mixed bacterial culture and fermented at 34°C for 64 hours to obtain a fermentation broth. The fermentation broth was then sterilized, filtered, concentrated, and spray-dried to obtain a liver-protecting composition. The mass ratio of the mixed bacterial culture to the mixed liquid was 1:12. The mixed bacterial culture consisted of *Acetobacter pasteurellii* inoculum and *Bacillus inulinus* inoculum, with a viable cell count ratio of 1:1 between the *Acetobacter pasteurellii* inoculum and *Bacillus inulinus* inoculum. The viable cell count of the *Acetobacter pasteurellii* inoculum was 1 × 10⁻⁶. 9 CFU / g, the viable count of the *Lactobacillus inulinus* inoculum culture solution is 1×10⁻⁶. 9 CFU / g;

[0084] The preservation number of the *Acetobacter pasteurellium* is: CGMCC No. 12930;

[0085] The preservation number of the Lactobacillus inulinus is: CGMCC No. 2185.

[0086] The method for preparing composition 2 includes the following steps:

[0087] a) Enzymatic hydrolysis: Mix all raw materials in substrate raw material group 2 to obtain raw material powder. Use sterile deionized water to prepare a raw material solution with a concentration of 11wt%. Add a compound enzyme to the raw material solution for enzymatic hydrolysis to obtain an enzymatic hydrolysate. The amount of the compound enzyme added is 1wt% of the mass of the raw material solution. The compound enzyme is a mixture of cellulase and pectinase in a mass ratio of 1:0.7. The cellulase activity is 30,000 U / g, the pectinase activity is 30,000 U / g, the enzymatic hydrolysis temperature is 55℃, the enzymatic hydrolysis time is 4h, and the enzymatic hydrolysis pH is 4.0.

[0088] b) Compound fermentation: The enzymatic hydrolysate was prepared into a 10wt% mixture using sterile deionized water. After sterilization, the mixture was inoculated with a mixed bacterial culture and fermented at 30°C for 72 hours to obtain a fermentation broth. The fermentation broth was then sterilized, filtered, concentrated, and spray-dried to obtain a liver-protecting composition. The mass ratio of the mixed bacterial culture to the mixed liquid was 1:11. The mixed bacterial culture consisted of *Acetobacter pasteurellii* inoculum and *Lactobacillus inulinus* inoculum, with a viable count ratio of 1:0.5 between the *Acetobacter pasteurellii* inoculum and *Lactobacillus inulinus* inoculum. The viable count of the *Acetobacter pasteurellii* inoculum was 1 × 10⁻⁶. 8 CFU / g, the viable count of the *Lactobacillus inulinus* inoculum culture solution is 1×10⁻⁶. 8 CFU / g;

[0089] The preservation number of the *Acetobacter pasteurellium* is: CGMCC No. 12930;

[0090] The preservation number of the Lactobacillus inulinus is: CGMCC No. 2185.

[0091] Composition 3:

[0092] a) Enzymatic hydrolysis: Mix all raw materials in substrate raw material group 3 to obtain raw material powder. Use sterile deionized water to prepare a raw material solution with a concentration of 13wt%. Add a compound enzyme to the raw material solution for enzymatic hydrolysis to obtain an enzymatic hydrolysate. The amount of the compound enzyme added is 1wt% of the mass of the raw material solution. The compound enzyme is a mixture of cellulase and pectinase in a mass ratio of 1:0.5. The cellulase activity is 30,000 U / g, the pectinase activity is 30,000 U / g, the enzymatic hydrolysis temperature is 40℃, the enzymatic hydrolysis time is 2h, and the enzymatic hydrolysis pH is 5.5.

[0093] b) Compound fermentation: The enzymatic hydrolysate was prepared into a 12wt% mixture using sterile deionized water. After sterilization, the mixture was inoculated with a mixed bacterial culture and fermented at 37°C for 48 hours to obtain a fermentation broth. The fermentation broth was then sterilized, filtered, concentrated, and spray-dried to obtain a liver-protecting composition. The mass ratio of the mixed bacterial culture to the mixed liquid was 1:12. The mixed bacterial culture consisted of *Acetobacter pasteurellii* inoculum and *Lactobacillus inulinus* inoculum, with a viable count ratio of 1:1.5 between the *Acetobacter pasteurellii* inoculum and *Lactobacillus inulinus* inoculum. The viable count of the *Acetobacter pasteurellii* inoculum was 1 × 10⁻⁶. 10 CFU / g, the viable count of the *Lactobacillus inulinus* inoculum culture solution is 1×10⁻⁶. 10 CFU / g;

[0094] The preservation number of the *Acetobacter pasteurellium* is: CGMCC No. 12930;

[0095] The preservation number of the Lactobacillus inulinus is: CGMCC No. 2185.

[0096] The preparation method of composition ① differs from that of composition 1 in that substrate raw material group ① is used instead of substrate raw material group 1, while the remaining steps and parameters are the same as those of composition 1.

[0097] The preparation method of composition ② differs from that of composition 1 in that substrate raw material group 2 is used instead of substrate raw material group 1, while the remaining steps and parameters are the same as those of composition 1.

[0098] The preparation method of composition ③ differs from that of composition 1 in that substrate raw material group 3 is used instead of substrate raw material group 1, while the remaining steps and parameters are the same as those of composition 1.

[0099] The preparation method of composition ④ differs from that of composition 1 in that substrate raw material group ④ is used instead of substrate raw material group 1, while the remaining steps and parameters are the same as those of composition 1.

[0100] The preparation method of composition ⑤ differs from that of composition 1 in that, in the compound fermentation step, *Acetobacter pasteurellii* inoculum solution is used to replace the compound bacterial solution, the mass ratio of *Acetobacter pasteurellii* inoculum solution to the mixed solution is 1:12, and the viable count of the *Acetobacter pasteurellii* inoculum solution is 1×10⁻⁶. 9 CFU / g.

[0101] The preparation method of composition ⑥ differs from that of composition 1 in that, in the compound fermentation step, *Lactobacillus inulinus* inoculum solution is used to replace the compound bacterial solution, the mass ratio of *Lactobacillus inulinus* inoculum solution to the mixed solution is 1:12, and the viable count of the *Lactobacillus inulinus* inoculum solution is 1×10⁻⁶. 9 CFU / g.

[0102] The preparation method of composition ⑦ differs from that of composition 1 in that it does not involve compound fermentation, but rather filters, concentrates, and spray-dries the enzymatic hydrolysate to obtain the liver-protecting composition.

[0103] The preparation method of composition ⑧ differs from that of composition 1 in that the ratio of viable bacteria count of *Acetobacter pastoris* inoculum to *Lactobacillus inulinus* inoculum in the mixed bacterial solution is 2:1, while the remaining steps and parameters are the same as those of composition 1.

[0104] The preparation method of composition ⑨ differs from that of composition 1 in that commercial Acetobacter pasteurellii CGMCC No. 12930 is used instead of Acetobacter pasteurellii. All other steps and parameters are the same as those of composition 1. The commercial Acetobacter pasteurellii was purchased from Mingzhou Biotechnology, catalog number: B89645.

[0105] The preparation method of composition ⑩ differs from that of composition 1 in that commercial Lactobacillus inulinus CGMCC No. 2185 is used instead of Lactobacillus inulinus CGMCC No. 2185. All other steps and parameters are the same as those of composition 1. The commercial Lactobacillus inulinus was purchased from Mingzhou Biotechnology, catalog number: BMZ146281.

[0106] Efficacy test:

[0107] Animal efficacy verification:

[0108] In an ethanol-induced liver injury model, compositions 1-3 of the present invention showed significant protective effects against ethanol-induced liver injury.

[0109] Test sample preparation: Use deionized water to prepare a 20wt% mixture of compositions 1-3 and compositions ①-⑩.

[0110] Experimental grouping: Female KM mice aged 6-7 weeks and weighing 20±2g were randomly divided into 1 normal group, 1 model group, and 13 experimental groups, with 10 mice in each group. After acclimatization for 1 week, the mice were subjected to subsequent experiments.

[0111] Experimental methods: The normal group and model group were administered 0.2 mL of sterile deionized water by gavage daily, while the experimental group was administered 0.2 mL of the combined solution by gavage daily for two consecutive weeks. After one week of gavage, the mice in the model group and experimental group were administered 5 mL / kg of 56v / v% edible alcohol-water solution by gavage daily for one week. After the last gavage, the mice were fasted for 16 hours, and blood was collected from the ocular veins. Serum was separated, and the activities or contents of ALT (alanine aminotransferase), AST (aspartate aminotransferase), and TG (triglycerides) in the serum were analyzed. The experimental results are expressed as mean ± standard deviation, and the results are shown in Table 1.

[0112] During the experiment, all groups of mice were provided with the same feed and drinking water, allowed free access to food, and followed a 12-hour circadian rhythm.

[0113] Table 1. Efficacy Verification Results

[0114]

[0115]

[0116] Note: "*" indicates p < 0.05 compared to the model group; "#" indicates p < 0.05 compared to the normal group.

[0117] The experimental results show that the activity values ​​of ALT, AST and TG in the model group were significantly higher than those in the normal group, indicating that the modeling was successful.

[0118] The results of comparing the model group with compositions 1-3 and compositions ①-⑩ showed that the compositions had a significant protective effect against acute alcoholic liver injury in mice.

[0119] Comparing the results of composition 1 with those of compositions ①-④, it can be seen that the effects of milk thistle powder, kudzu root powder, salvia miltiorrhiza powder, wild cherry berry lyophilized powder, and Japanese raisin tree powder in the substrates of the present invention are not simply additive. Wild cherry berry lyophilized powder and Japanese raisin tree powder can significantly enhance the liver-protective effects of milk thistle powder, kudzu root powder, and salvia miltiorrhiza powder.

[0120] Comparing the results of composition 1 with those of compositions ⑤-⑥ and ⑧-⑩, it can be seen that the fermentation product produced by the combined use of *Acetobacter pasteurellii* CGMCC No. 12930 and *Lactobacillus inulinus* CGMCC No. 2185 in this invention for fermenting the substrate described in this invention has significant liver-protective effects.

[0121] Comparing the results of composition 1 and composition 7, it can be seen that the fermentation process in this invention can effectively enhance the liver-protective effects of milk thistle powder, kudzu root powder, salvia miltiorrhiza powder, cheri berry freeze-dried powder, and jujube seed powder.

[0122] The mass fractionation and preparation method of liver-protecting tablets are as follows:

[0123] Liver protection tablet 1:

[0124] Composition 1: 33 parts; microcrystalline cellulose: 20 parts; lactose: 30 parts; magnesium stearate: 0.5 parts; skim milk powder: 20 parts; sodium carboxymethyl starch: 10 parts.

[0125] Liver Protection Tablet 2:

[0126] Composition 1: 35 parts; microcrystalline cellulose: 30 parts; lactose: 20 parts; magnesium stearate: 1 part; skim milk powder: 10 parts; sodium carboxymethyl starch: 5 parts.

[0127] Liver protection tablets 3:

[0128] Composition 1: 30 parts; microcrystalline cellulose: 10 parts; lactose: 40 parts; magnesium stearate: 0.01 parts; skim milk powder: 30 parts; sodium carboxymethyl starch: 15 parts.

[0129] Liver protection tablets 4:

[0130] Composition 2: 33 parts; microcrystalline cellulose: 20 parts; lactose: 30 parts; magnesium stearate: 0.5 parts; skim milk powder: 20 parts; sodium carboxymethyl starch: 10 parts.

[0131] Liver protection tablets 5:

[0132] Composition 3: 33 parts; microcrystalline cellulose: 20 parts; lactose: 30 parts; magnesium stearate: 0.5 parts; skim milk powder: 20 parts; sodium carboxymethyl starch: 10 parts.

[0133] Preparation method of liver protection tablets 1-5:

[0134] Step 1: Mix the microcrystalline cellulose, lactose, skim milk powder, and sodium carboxymethyl starch, then add the mixture to a dry granulator for granulation and collect 80-mesh particles;

[0135] Step 2: Mix the liver-protecting composition, the granules collected in Step 1, and magnesium stearate to obtain a mixture;

[0136] Step 3: Put the mixture into a tablet press and compress it into tablets. Adjust the pressure to 40 kN to obtain liver protection tablets.

[0137] Acute oral toxicity test:

[0138] Test samples: Liver-protecting tablets 1-5.

[0139] Using a dose-limiting method, 20 SPF-grade ICR mice weighing 18-22g (half male and half female) were selected. 20.00g of liver-protecting tablets were ground and diluted with water to 60mL to reach the maximum permissible gavage concentration. The mice were administered the tablets orally twice daily at 4-hour intervals, with each gavage volume being 0.3mL / 10g·bw, equivalent to a dose of 20.00g / kg·bw. Mice were fasted for 6 hours before the first gavage. After gavage, mice were observed for two weeks, and signs of toxicity and mortality were recorded.

[0140] Table 2 Results of acute oral toxicity test in mice.

[0141] gender Number of animals (individuals) way Dosage (g / kg·bw) Number of deaths (animals) LD50 (g / kg·bw) male 10 Oral 20 0 >20 female 10 Oral 20 0 >20

[0142] As shown in Table 2, no obvious poisoning symptoms were observed after gavage administration of the liver-protecting tablets at a dose of 20.00 g / kg·bw to both sexes of ICR mice, and no deaths were observed during the 14-day observation period. At the end of the observation period, the test animals were euthanized and dissected. No obvious abnormalities were found in the major organs, including the liver, spleen, kidneys, intestines, heart, and lungs. The median lethal dose (LD50) of the liver-protecting tablets described in this invention for both male and female ICR mice is greater than 20.00 g / kg·bw. According to the acute toxicity dose grading standard in GB15193.3-2014, it belongs to the practically non-toxic category.

[0143] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for preparing a composition with liver-protective effects, characterized in that, Includes the following steps: Enzymatic hydrolysis: The substrate raw materials of the composition are mixed to obtain raw material powder. The raw material powder is prepared into a raw material solution with a concentration of 11-13 wt% using sterile deionized water. A compound enzyme is added to the raw material solution for enzymatic hydrolysis to obtain an enzymatic hydrolysate. The amount of the compound enzyme added is 1-3 wt% of the mass of the raw material solution. The compound enzyme is composed of cellulase and pectinase in a mass ratio of 1:(0.5-0.7). The enzymatic hydrolysis temperature is 40-55℃, the enzymatic hydrolysis time is 2-4 h, and the enzymatic hydrolysis pH is 4.0-5.

5. Compound fermentation: The enzymatic hydrolysate is prepared into a 10-12 wt% mixture using sterile deionized water. After sterilization, the mixture is inoculated with a mixed bacterial culture and fermented at 30-37℃ for 48-72 hours to obtain a fermentation broth. The fermentation broth is then sterilized, filtered, concentrated, and spray-dried to obtain a liver-protecting composition. The mass ratio of the mixed bacterial culture to the mixed liquid is 1:(11-12). The mixed bacterial culture consists of *Acetobacter pasteurellii* inoculum and *Lactobacillus inulinus* inoculum, with a viable count ratio of 1:(0.5-1.5). The viable count of the *Acetobacter pasteurellii* inoculum is 1×10⁻⁶. 8 -1×10 10 CFU / g, the viable count of the *Lactobacillus inulinus* inoculum culture solution is 1×10⁻⁶. 8 -1×10 10 CFU / g; The preservation number of the *Acetobacter pasteurellium* is: CGMCC No. 12930; The preservation number of the *Lactobacillus inulinus* is: CGMCC No. 2185; The substrate raw material of the composition consists of the following parts by weight: Milk thistle powder: 10-30 parts; Kudzu root powder: 15-40 servings; Salvia miltiorrhiza powder: 20-35 parts; Wild cherry jelly powder: 5-15 servings; Hovenia dulcis powder: 8-20 servings.

2. The preparation method according to claim 1, characterized in that, The substrate raw material of the composition consists of the following parts by weight: Milk thistle powder: 20-30 parts; Kudzu root powder: 25-40 servings; Salvia miltiorrhiza powder: 30-35 parts; Wild cherry jelly powder: 5-10 portions; Hovenia dulcis powder: 15-20 servings.

3. The preparation method according to claim 2, characterized in that, The substrate raw material of the composition consists of the following parts by weight: Milk thistle powder: 20 parts; Kudzu root powder: 25 portions; Salvia miltiorrhiza powder: 30 portions; Wild cherry jelly powder: 10 servings; Hovenia dulcis powder: 15 portions.

4. The preparation method according to any one of claims 1-3, characterized in that, The cellulase activity is 10,000-100,000 U / g, and the pectinase activity is 10,000-100,000 U / g.

5. A composition with liver-protective effects, characterized in that, The composition is prepared by the method of preparing the composition with liver-protective effect according to any one of claims 1-4.

6. The use of the composition with hepatoprotective effects according to claim 5 in the preparation of health products or pharmaceuticals with hepatoprotective effects.

7. A liver-protecting preparation, characterized in that, The composition containing the liver-protective effect as described in claim 5.

8. The liver-protecting preparation according to claim 7, characterized in that, The dosage form of the preparation is any one of tablets, capsules, ointments, or oral liquids.

9. A liver-protecting tablet, characterized in that, It contains the following components by weight: The liver-protective composition according to claim 5: 30-35 parts; Microcrystalline cellulose: 10-30 parts; Lactose: 20-40 parts Magnesium stearate: 0.01-1 part; Skim milk powder: 10-30 servings; Sodium carboxymethyl starch: 5-15 parts.

10. A method for preparing a liver-protecting tablet as described in claim 9, characterized in that, Includes the following steps: Step 1: Mix the microcrystalline cellulose, lactose, skim milk powder, and sodium carboxymethyl starch, then add the mixture to a dry granulator for granulation, and collect 10-120 mesh particles; Step 2: Mix the liver-protecting composition, the granules collected in Step 1, and magnesium stearate to obtain a mixture; Step 3: Put the mixture into a tablet press and compress it into tablets. Adjust the pressure to 10-40 kN to obtain liver protection tablets.

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