Use of a composite biological enzyme preparation in the preparation of a product for improving chemical liver injury
By developing compound biological enzyme preparations, the problem of increased liver burden due to long-term use of existing liver protection products has been solved, achieving effective improvement and protection against chemically induced liver damage.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NANNING PANGBO BIOLOGICAL ENG CO LTD
- Filing Date
- 2026-03-16
- Publication Date
- 2026-06-05
Smart Images
Figure CN122140908A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and in particular to the application of a compound bioenzyme preparation in the preparation of products that improve chemically induced liver injury. Background Technology
[0002] Chemical liver injury is a disease caused by long-term or excessive exposure to toxic chemicals, resulting in abnormal liver function. It is commonly seen in drug abuse, alcohol consumption, and exposure to environmental toxins.
[0003] Currently, most liver-protecting products on the market achieve their purpose through the following types of ingredients. One type is plant extracts, such as kudzu root, Japanese raisin tree fruit, turmeric, and vine tea extracts. These natural products are believed to have certain hepatoprotective, choleretic, and alcohol metabolism-promoting effects. However, their active ingredients are mainly flavonoids and polysaccharides. While they show significant effects with short-term use, long-term use requires the liver to metabolize these ingredients, potentially increasing the liver's burden. Their effectiveness in protecting against chemically induced liver damage has a limited duration. Another type consists of proteins and various peptides, including corn oligopeptides, oyster peptides, and soybean peptides. These substances indirectly protect the liver by providing the amino acids needed for liver repair, providing antioxidants, or regulating immunity. A third type utilizes probiotics, such as Bacillus coagulans. For example, the solution described in patent CN117511825A uses probiotics to produce flavonoid aglycones and isoflavone aglycones, thereby producing a liver-protective effect. However, this method has similar problems to natural products, failing to address the issue of increased liver burden with long-term use.
[0004] Patent CN117551626A describes an alcohol-degrading enzyme protein capable of breaking down ethanol and acetaldehyde. It proposes an enzyme protein with a specific amino acid sequence, obtained using molecular biology methods, capable of degrading both ethanol and acetaldehyde. This provides valuable insights for developing novel products to protect against chemically induced liver injury and demonstrates the development potential of bioenzymes in this field.
[0005] With the development of biotechnology, bio-enzyme preparations, due to their high efficiency and specificity, have shown broad application prospects in the health field. In the specific area of assisting in the protection against chemically induced liver injury, some studies have begun to explore the application potential of bio-enzymes.
[0006] While the value of bioenzymes in protecting against chemically induced liver injury has been preliminarily recognized, the inherent advantages of their high efficiency, specificity, and safety have not been directly utilized. More importantly, there are currently no reports on mature products on the market that utilize carefully designed compound bioenzyme preparations as the core active ingredient for protecting against chemically induced liver injury. This indicates that developing a highly efficient, safe compound bioenzyme preparation containing multiple highly complementary enzymes, mimicking the mechanism of action of healthy human bioenzymes, and possessing good oral stability will be an important direction for overcoming the limitations of existing liver-protective products. Such a preparation has the potential to fundamentally reduce the burden on the liver and meet the market's demand for protection against acute and chronic chemically induced liver injury. Summary of the Invention
[0007] The purpose of this invention is to provide an application of a compound bio-enzyme preparation in the preparation of products for improving chemically induced liver injury, thereby addressing the problems existing in the prior art. The compound bio-enzyme preparation developed in this invention can effectively improve chemically induced liver injury. It carefully selects bio-enzymes from plant, animal, and microbial sources, combines essential nutrient components, and simulates the action mechanism of the body's own bio-enzymes to achieve the effect of improving chemically induced liver injury, providing strong technical support for the development of liver-protective products.
[0008] To achieve the above objectives, the present invention provides the following solution: This invention provides the application of a compound biological enzyme preparation in the preparation of products that improve chemically induced liver injury. The raw materials of the compound biological enzyme preparation include 3-14 kinds selected from papain, bromelain, acidic protease, pepsin, trypsin, α-amylase, sucrase, lipase, cellulase, pectinase, glucosylamylase, nuclease, serrepeptidase and β-galactosidase.
[0009] Furthermore, the raw materials of the compound bio-enzyme preparation also include auxiliary components; The auxiliary ingredients are at least one of glucose, maltodextrin, taurine, soybean peptide powder, sodium alginate, lysine, vitamin C, and B vitamins.
[0010] Furthermore, the raw materials of the compound bio-enzyme preparation include bromelain, papain, acidic protease, lipase, cellulase, and glucosylamylase.
[0011] Furthermore, the raw materials of the compound bio-enzyme preparation include bromelain, papain, acidic protease, α-amylase, and β-galactosidase.
[0012] Furthermore, the raw materials of the compound bio-enzyme preparation include neutral protease, α-amylase, β-galactosidase, lipase and cellulase.
[0013] Furthermore, the raw materials of the compound bio-enzyme preparation include bromelain, papain, acidic protease, taurine, lysine, and B vitamins; The B vitamins mentioned are niacin, vitamin B6, and vitamin B1. 12 .
[0014] Furthermore, the compound bio-enzyme preparation comprises the following components in the following amounts: bromelain 25,000-35,000 IU / g, papain 15,000-25,000 IU / g, acidic protease 45,000-55,000 IU / g, taurine 0.2-0.8 mg / g, lysine 0.1-0.3 mg / g, niacin 0.2-0.8 mg / g, vitamin B6 0.1-0.3 mg / g, and vitamin B12 0.1-0.3 mg / 100g.
[0015] Furthermore, the raw materials of the compound bio-enzyme preparation include bromelain, papain, acidic protease, nuclease, taurine, and soybean peptide powder.
[0016] Furthermore, the compound bio-enzyme preparation comprises the following components in the following amounts: bromelain 25,000-35,000 IU / g, papain 15,000-25,000 IU / g, acidic protease 45,000-55,000 IU / g, nuclease 100-300 IU / g, taurine 0.2-0.8 mg / g, and soybean peptide powder 10-50 mg / g.
[0017] Furthermore, the chemically induced liver injury is alcoholic liver injury.
[0018] The present invention discloses the following technical effects: This invention develops a compound bioenzyme preparation that can be used to prepare products that improve chemically induced liver injury. The compound bioenzyme preparation of this invention combines safety and efficacy. Acute toxicity tests have verified that the compound bioenzyme preparation is practically non-toxic, with no significant adverse effects on the body weight or organ indices of experimental animals, and exhibits high oral safety. This compound bioenzyme preparation can significantly improve chemically induced liver injury, effectively reducing serum ALT and AST activities, alleviating hepatocyte degeneration, necrosis, and inflammatory infiltration, and reducing the level of inflammatory factors in liver tissue. For alcoholic liver injury, it can increase serum and liver ADH and ALDH activities, promote alcohol metabolism, regulate four blood lipid indicators, reduce liver indices and liver MDA content, and alleviate oxidative damage to the liver. Compared with existing hepatoprotective ingredients such as silymarin and silymarin phospholipids, the preparation of this invention performs better in improving pathological damage to liver tissue and regulating the activity of key enzymes in alcohol metabolism. It can fundamentally reduce the metabolic burden on the liver and has a good protective effect against both acute and chronic alcoholic liver injury, providing a novel and highly effective core ingredient for the development of hepatoprotective products. Attached Figure Description
[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0020] Figure 1 Figure showing the weight changes of mice in different groups in Example 1 for efficacy verification; Figure 2 Organ and tissue morphology diagrams of mice in different groups in Example 1 for effect verification; Figure 3 Statistical graphs of organ indices of mice in different groups in Example 1 for efficacy verification; where AD represents the heart index, liver index, kidney index, and spleen index, respectively. Figure 4 Liver morphology images of rats in different groups in Example 2 for efficacy verification; where A: normal group; B: model group; C: silymarin group; D: silymarin phospholipid group; E: low-dose biological enzyme group; F: medium-dose biological enzyme group; G: high-dose biological enzyme group; Figure 5 HE staining images of liver pathological tissues from different groups of rats in Example 2 to verify the efficacy; the scale bar in the images is 50 μm. Figure 6 Statistical graph of TNF-α (A), IL-6 (B) and IL-1β (C) levels in liver tissue of rats in different groups in Example 2 for efficacy verification; Figure 7 Statistical graphs of serum ADH (A), serum ALDH (B), liver ADH (C), and liver ALDH (D) levels in different groups of rats in Example 3 for efficacy verification; Figure 8 Statistical chart of serum TG (A), TC (B), LDL-C (C), and HDL-C (D) levels in different groups of rats in Example 3 to verify the efficacy. Detailed Implementation
[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.
[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0026] Example 1 A compound biological enzyme preparation for protecting against chemically induced liver injury, the raw material formula of which is as follows: Bromelain 30,000 IU / g, papain 20,000 IU / g, acidic protease 50,000 IU / g, lipase 5,000 IU / g, cellulase 140 IU / g, and glucoamylase 10,000 IU / g.
[0027] Example 2 A compound biological enzyme preparation for protecting against chemically induced liver injury, the raw material formula of which is as follows: Bromelain 25,000 IU / g, papain 25,000 IU / g, acidic protease 45,000 IU / g, lipase 5,500 IU / g, cellulase 100 IU / g, and glucoamylase 15,000 IU / g.
[0028] Example 3 A compound biological enzyme preparation for protecting against chemically induced liver injury, the raw material formula of which is as follows: Bromelain 35,000 IU / g, papain 15,000 IU / g, acidic protease 55,000 IU / g, lipase 4,500 IU / g, cellulase 200 IU / g, and glucoamylase 5,000 IU / g.
[0029] Example 4 A compound biological enzyme preparation for protecting against chemically induced liver injury, the raw material formula of which is as follows: Bromelain 30,000 IU / g, papain 20,000 IU / g, acidic protease 50,000 IU / g, α-amylase 3,500 IU / g, and β-galactosidase 2,800 IU / g.
[0030] Example 5 A compound biological enzyme preparation for protecting against chemically induced liver injury, the raw material formula of which is as follows: Bromelain 25,000 IU / g, papain 25,000 IU / g, acidic protease 45,000 IU / g, α-amylase 4,000 IU / g and β-galactosidase 2,400 IU / g.
[0031] Example 6 A compound biological enzyme preparation for protecting against chemically induced liver injury, the raw material formula of which is as follows: Bromelain 35,000 IU / g, papain 15,000 IU / g, acidic protease 55,000 IU / g, α-amylase 3,000 IU / g and β-galactosidase 3,200 IU / g.
[0032] Example 7 A compound biological enzyme preparation for protecting against chemically induced liver injury, the raw material formula of which is as follows: Bromelain 30,000 IU / g, Papain 20,000 IU / g, Acidic protease 50,000 IU / g, Taurine 0.5 mg / g, Lysine 0.2 mg / g, Niacin 0.5 mg / g, Vitamin B6 0.2 mg / g, and Vitamin B12 0.2 mg / 100 g.
[0033] Example 8 A compound biological enzyme preparation for protecting against chemically induced liver injury, the raw material formula of which is as follows: Bromelain 25,000 IU / g, Papain 25,000 IU / g, Acidic protease 45,000 IU / g, Taurine 0.8 mg / g, Lysine 0.1 mg / g, Niacin 0.8 mg / g, Vitamin B6 0.1 mg / g, and Vitamin B12 0.3 mg / 100 g.
[0034] Example 9 A compound biological enzyme preparation for protecting against chemically induced liver injury, the raw material formula of which is as follows: Bromelain 35,000 IU / g, Papain 15,000 IU / g, Acidic protease 55,000 IU / g, Taurine 0.2 mg / g, Lysine 0.3 mg / g, Niacin 0.2 mg / g, Vitamin B6 0.3 mg / g, and Vitamin B12 0.1 mg / 100 g.
[0035] Example 10 A compound biological enzyme preparation for protecting against chemically induced liver injury, the raw material formula of which is as follows: Neutral protease 0.4 million IU / g, α-amylase 1.7 million IU / g, β-galactosidase 3000 IU / g, lipase 800 IU / g and cellulase 140 IU / g.
[0036] Example 11 A compound biological enzyme preparation for protecting against chemically induced liver injury, the raw material formula of which is as follows: Neutral protease 0.3 million IU / g, α-amylase 1.8 million IU / g, β-galactosidase 2500 IU / g, lipase 900 IU / g and cellulase 100 IU / g.
[0037] Example 12 A compound biological enzyme preparation for protecting against chemically induced liver injury, the raw material formula of which is as follows: Neutral protease 0.5 million IU / g, α-amylase 1.6 million IU / g, β-galactosidase 3500 IU / g, lipase 700 IU / g and cellulase 180 IU / g.
[0038] Example 13 A compound biological enzyme preparation for protecting against chemically induced liver injury, the raw material formula of which is as follows: Bromelain 30,000 IU / g, papain 20,000 IU / g, acidic protease 50,000 IU / g, nuclease 200 IU / g, taurine 0.5 mg / g, and soybean peptide powder 30 mg / g.
[0039] Example 14 A compound biological enzyme preparation for protecting against chemically induced liver injury, the raw material formula of which is as follows: Bromelain 25,000 IU / g, papain 25,000 IU / g, acidic protease 45,000 IU / g, nuclease 300 IU / g, taurine 0.2 mg / g, and soybean peptide powder 50 mg / g.
[0040] Example 15 A compound biological enzyme preparation for protecting against chemically induced liver injury, the raw material formula of which is as follows: Bromelain 35,000 IU / g, papain 15,000 IU / g, acidic protease 55,000 IU / g, nuclease 100 IU / g, taurine 0.8 mg / g, and soybean peptide powder 10 mg / g.
[0041] Example 1 of effect verification Acute toxicity evaluation (safety evaluation) of different formulations of compound biological enzyme preparations in normal mice: 1. Experimental Methods The maximum tolerated dose (MTD) test was used. Forty Kunming mice were randomly divided into four groups: a male control group, a female control group, a male drug-treated group, and a female drug-treated group. The control group was administered an equal volume of purified water by gavage. For the drug-treated groups, the dosage and administration method were as follows: the designed dose was 15 g / kg b·wt. 15 g of the compound biological enzyme preparation sample (Example 13) was weighed and diluted with purified water to 40 mL to prepare the test substance, which was prepared immediately before use. After fasting for 12 hours with free access to water, the test sample was administered by gavage twice, 4 hours apart, with no food given during the interval. The gavage volume for each dose was 20 mg b·wt. Food was given 4 hours after the last gavage.
[0042] Observe and record the daily condition of the mice: After drug administration, observe the basic condition of the mice, including coat color, food intake, water intake, feces, daily activities, and mental state, for a period of 14 days. Determine whether toxic symptoms appear, and record the weight of the mice every other day from the date of the first administration. Dissect the mice to observe their tissues. 14 days after drug administration, mice in each group were euthanized by cervical dislocation and dissected to observe the pathological changes in their internal organs. If any abnormalities are visible to the naked eye, pathological examination is performed. After dissection, the heart, liver, kidney, and spleen tissues of the mice were weighed, and organ indices were calculated.
[0043] 2. Experimental Results 2.1 Effects of compound biological enzyme preparations on mouse condition Mice were continuously observed after administration. No mice died after gavage administration of the compound bio-enzyme preparation at 15 g / kg. Mice in all groups showed reduced activity after gavage, exhibiting quietness and sluggishness. They responded to auditory stimuli, had normal appetite, smooth fur, and no abnormal secretions from the eyes, nose, or mouth. Their feces were normal. On the second day after administration, one female mouse experienced weight loss without other adverse reactions. Its weight recovered on the third day, likely due to the unpleasant odor of the compound bio-enzyme preparation causing a short-term loss of appetite after gavage. The remaining mice were in normal condition, with no significant differences between the administered and control groups.
[0044] 2.2 Effects of compound biological enzyme preparations on changes in mouse body weight After gavage administration, mice in four groups were observed continuously for 14 days, and their body weight was recorded every other day. Body weight changes are shown in Table 1. Compared with the control group (female mice), there was no significant difference in body weight between the female mice in the treatment group and the control group (P>0.05); compared with the control group (male mice), there was no significant difference in body weight between the male mice in the treatment group and the control group (P>0.05). Throughout the experimental observation period, the body weight of both male and female mice continuously increased, and the increase was good, with a generally consistent trend. Figure 1 This indicates that administering the maximum dose of the compound bio-enzyme preparation to mice via gavage had no significant adverse effect on their body weight.
[0045] Table 1. Effects of gavage administration of compound biological enzyme preparations on body weight in mice. 2.3 Effects of compound biological enzyme preparations on organ indices in mice After dissecting the mice, the morphology of the organs and tissues of each group of mice was observed by the naked eye. It was found that there were no obvious abnormalities in the compound biological enzyme preparation group and the control group. Figure 2 The organ index is the ratio of the weight of a specific organ to the body weight of an experimental animal. Under normal circumstances, this ratio remains relatively constant. However, when an animal is exposed to toxins, the weight of the damaged organ can change, and the organ index will also change accordingly. In this experiment, the major organs of mice were weighed, and the organ index was calculated. The results are shown in […]. Figure 3 There were no statistically significant differences in organ indices between the male administration group and the male control group of the compound bio-enzyme preparation (P>0.05), and no statistically significant differences in organ indices between the female administration group and the female control group of the compound bio-enzyme preparation (P>0.05). This suggests that the compound bio-enzyme preparation has low or essentially no toxicity.
[0046] 2.4 Evaluation results of acute toxicity of compound biological enzyme preparation Acute toxicity tests in mice showed no mortality after oral administration of the compound bio-enzyme preparation at concentrations of 15 g / kg and 20 mL / kg via gavage; therefore, the LD50 could not be calculated. 50 Value, median lethal dose (LD50) 50 LD50 is an important indicator for evaluating acute drug testing and can effectively assess the acute toxicity of drugs. 50 The higher the LD50, the lower the toxicity of the drug. The LD50 of the compound biological enzyme preparation was not detected in the experiment. 50 The values indicate that the compound bio-enzyme preparation has very low toxicity. The results of this experiment show that its maximum dose (MTD) is greater than 15 g / kg. According to the acute toxicity grading standard, the above compound bio-enzyme preparation belongs to the practically non-toxic category.
[0047] Example 2 of effect verification Effects of compound biological enzyme preparations on a rat model of acute liver injury induced by carbon tetrachloride (pharmacological investigation): 1. Experimental Methods 1.1 Establishment of a rat model of acute liver injury caused by carbon tetrachloride Male SD rats (weighing 180-220 g) were acclimatized for one week and then administered 3% carbon tetrachloride (5 mL / kg by gavage) once to establish an acute liver injury model caused by carbon tetrachloride.
[0048] 1.2 Grouping of rats Seventy male SD rats were randomly divided into seven groups after one week of acclimatization: normal group (edible oil), model group (3% carbon tetrachloride), silybin group, silybin phospholipid group, low-dose biological enzyme group (referred to as low-dose enzyme group), medium-dose biological enzyme group (referred to as medium-dose enzyme group), and high-dose biological enzyme group (referred to as high-dose enzyme group), with 10 rats in each group.
[0049] 1.3 Grouping and Sampling of Rats The rats were weighed and their weight recorded daily, and were administered bioenzymes by gavage (Example 13) for 30 days at a volume of 10 mL / kg. On day 30 of the experiment, the animals in each group were fasted overnight for 16 hours. The model group and each drug administration group were given 3% carbon tetrachloride by gavage once (gavage volume of 5 mL / kg). The normal control group was given vegetable oil. The drug administration groups continued to receive the drug until the end of the experiment (with an interval of more than 4 hours between gavage and administration of 3% carbon tetrachloride).
[0050] The dosage is as follows: Silymarin group: silymarin 21 mg / kg; Milk thistle phospholipid group: Milk thistle phospholipid tablets 288mg / kg; Low, medium, and high dose groups of bio-enzyme: 0.0625 g / kg, 0.125 g / kg, and 0.25 g / kg of the compound bio-enzyme from Example 13.
[0051] 1.4 Detection Indicators 1.4.1 Liver biochemical indicators: The activities of ALT, AST, etc. in serum were measured according to the kit instructions.
[0052] 1.4.2 Liver Histopathological Observation: Immediately after the experiment, the rats were dissected and their livers were removed. The livers were rinsed with pre-cooled physiological saline, dried with filter paper, photographed, and weighed. The middle part of the left lobe of the rat liver was immersed in 4% paraformaldehyde for 3 days. Before the second sampling, the tissue was rinsed with running water for more than 4 hours. The tissue was cut into small pieces with a thickness of 3-4 mm, dehydrated by alcohol gradient, embedded in paraffin, pre-cooled, sectioned, stained with HE, mounted with a small amount of neutral resin, and air-dried. The liver tissue morphology, lobular structure, degree of steatosis, hepatocyte degeneration, necrosis, inflammatory cell infiltration, and fibrosis were observed under a 400x optical microscope. According to the scoring criteria for liver histological changes in non-alcoholic liver injury in the "Evaluation Test Items, Test Principles and Result Judgment of Health Food", the area occupied by various lesions in each field of view was evaluated and recorded, and the total score of the lesions in the observed fields of view was accumulated.
[0053] 1.4.3 Serum inflammatory factor assay: The levels of inflammatory factors TNF-α, IL-1β and IL-6 were measured according to the ELISA kit instructions.
[0054] 2. Experimental Results 2.1 Effects of biological enzymes on liver function in rats with carbon tetrachloride-induced liver injury After the experiment, the rats were anesthetized, and blood was collected from the abdominal aorta into blood collection tubes. After the blood was allowed to stand for 2 hours, the serum was separated (4℃, 3500 r / min, centrifuged for 15 min). The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured. The results are shown in Table 2.
[0055] Table 2. Effects of biological enzymes on ALT and AST activities in rats with carbon tetrachloride-induced liver injury ( ±s, n=6) Note: Compared with the normal group △△ P <0.01; compared with the model group, P <0.05, P <0.01.
[0056] Table 2 shows that, compared with the normal group, the ALT and AST levels in the model group were significantly higher ( P The result was <0.01, indicating that the carbon tetrachloride-induced liver injury model was established. Compared with the model group, the silymarin group, silymarin group, and all doses of the bioenzyme significantly reduced ALT activity (P < 0.01). Compared with the model group, the low-dose bioenzyme group significantly reduced AST activity (P < 0.05), while the silymarin group, silymarin group, and high-dose bioenzyme group showed a decrease in AST activity, but the differences were not statistically significant.
[0057] 2.2 Effects of biological enzymes on liver morphology in rats with carbon tetrachloride-induced liver injury like Figure 4 As shown, the livers of rats in the normal group were deep red in color, with a smooth and translucent surface, a rosy appearance, and a soft texture. The livers of rats in the model group were yellowish in color, with a rough, gritty surface, increased in size, and less elastic. Compared to the model group, the livers of rats in the silymarin group, silymarin group, and each dose of the bioenzyme group showed a rosy color, reduced gritty surface texture, and improved damage.
[0058] 2.3 Effects of biological enzymes on pathological changes in liver tissue of rats with carbon tetrachloride-induced liver injury The effects of biological enzymes on the pathological changes in liver tissue of rats with carbon tetrachloride-induced liver injury are shown in Table 3.
[0059] Table 3. Effects of biological enzymes on pathological changes in liver tissue of rats with carbon tetrachloride-induced liver injury (n=4, ±s) Note: Compared with the normal group △△ P <0.01; compared with the model group, P <0.01.
[0060] like Figure 5 HE staining results of liver tissue showed that in the normal group, the liver lobule structure of rats was intact, the hepatocyte nuclei were clear and intact without nuclear pyknosis, the cytoplasm was abundant, and the hepatic cords were neatly arranged. In contrast, the liver tissue structure of the model group rats was significantly disordered, with numerous ballooning degeneration, significant fatty degeneration and hydropic degeneration areas around the central vein, accompanied by a small amount of cytoplasmic aggregation. The results after treatment intervention are as follows: (1) Silymarin group and silymarin group: Compared with the model group, the ballooning degeneration and fatty degeneration of hepatocytes in the above groups were significantly improved.
[0061] (2) Bioenzyme dosage groups: Compared with the model group, the ballooning degeneration, fatty degeneration and hydropic degeneration of hepatocytes in the low, medium and high dose groups were improved. Among them, the low dose group and the medium dose group were better than the high dose group.
[0062] 2.4 Effects of biological enzymes on the levels of TNF-α, IL-6, and IL-1β in liver tissue of rats with carbon tetrachloride-induced liver injury Table 4. Effects of biological enzymes on the levels of TNF-α, IL-6, and IL-1β in liver tissue of rats with carbon tetrachloride-induced liver injury ( ±s, n=6) Note: Compared with the normal group△△ P <0.01; compared with the model group, P <0.05, P <0.01.
[0063] The results are as follows Figure 6 As shown in Table 4, compared with the normal group, the TNF-α content in the liver tissue of rats in the model group was significantly increased, and the difference was statistically significant. P <0.01); compared with the normal group, the IL-1β content in the liver tissue of rats in the model group was significantly increased ( P< 0.01); Compared with the model group, the silymarin group, silymarin group, and low-dose enzyme group all significantly reduced the IL-1β content in rat liver tissue ( P <0.05 or P <0.01).
[0064] Example 3 of effect verification Effects of compound biological enzyme preparations on a rat model of ethanol-induced acute liver injury (pharmacological investigation): 1. Experimental Methods 1.1 Establishment of an alcoholic acute liver injury model in rats A liver injury model was induced in rats using ethanol (analytical grade) at a concentration of 50% (diluted with distilled water). On day 30, the rats were given a single oral gavage dose of 1.2 mL / 100 g of the enzyme sample to induce an alcoholic liver injury model.
[0065] 1.2 Grouping of rats Eighty-four male SD rats were randomly divided into seven groups after one week of acclimatization: blank group, model group, silybin group, silybin phospholipid group, low-dose biological enzyme group (abbreviated as low-dose enzyme group or low enzyme group), medium-dose biological enzyme group (abbreviated as medium-dose enzyme group or medium enzyme group), and high-dose biological enzyme group (abbreviated as high-dose enzyme group or high enzyme group), with 12 rats in each group.
[0066] 1.3 Grouping and Sampling of Rats Rats were weighed and their body weight recorded daily. They were then administered a compound bioenzyme via gavage (Example 13) for 30 days at a volume of 10 mL / kg. On day 30, the silymarin group, silymarin phospholipid group, model group, and all treatment groups were administered the drug via gavage. The control group was given distilled water via gavage. Four hours later, all rats except the control group were given 1.2 mL / 100g BW of 50% ethanol via gavage. The control group was given distilled water via gavage.
[0067] The dosage is as follows: Silymarin group: silymarin 21 mg / kg; Milk thistle phospholipid group: Milk thistle phospholipid tablets 288mg / kg; Low, medium, and high dose groups of the bio-enzyme: 0.0625 g / kg, 0.125 g / kg, and 0.25 g / kg of the compound bio-enzyme from Example 13.
[0068] 1.4 Detection Indicators 1.4.1 Blood biochemical indicators: The levels and activities of four lipid parameters, acetaldehyde dehydrogenase, and alcohol dehydrogenase in serum were measured according to the kit instructions.
[0069] 1.4.2 Liver biochemical indicators: The liver was ground with liquid nitrogen and then added to physiological saline to make a 10% liver tissue homogenate. The homogenate was centrifuged at 4000 r / min for 10 min at 4℃. The supernatant was collected and the content and activity of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in the liver tissue were determined according to the recommended detection methods in the "Methods for Functional Testing and Evaluation of Health Foods (2023 Edition)".
[0070] 2. Experimental Results 2.1 ADH and ALDH activity The results of the detection of the effects of biological enzymes on ADH and ALDH activities in rats with acute alcoholic liver injury are shown in the figure. Figure 7 See Table 5.
[0071] Table 5. Effects of bioenzymes on ADH and ALDH activity in rats with acute alcoholic liver injury ( ±s, n=8) Note: Compared with the blank group, △ P <0.05, △△ P <0.01; compared with the model group, P <0.05, P <0.01.
[0072] 2.2 Four blood lipid tests The results of the four blood lipid tests are shown below. Figure 8 See Table 6.
[0073] Table 6. Effects of biological enzymes (Example 13) on four lipid parameters in rats with acute alcoholic liver injury. ±s, n=8) Example 4 of effect verification 1. Experimental Methods 1.1 Methods for establishing a mouse model of chronic alcoholic liver injury SPF grade C57BL / 6J mice, male, 8 weeks old, were acclimatized with a non-alcoholic control liquid diet for 2 days. The model group was then acclimatized with a liquid alcohol diet for 5 days (during which the alcohol concentration was gradually increased from 1% to 4%). Starting from day 8, the model group was fed a liquid diet containing 5% alcohol for 10 consecutive days. On day 18, the model group was given a high dose of alcohol (5 g / kg) by gavage between 7 and 9 am to induce a chronic alcoholic liver injury model.
[0074] 1.2 Mouse grouping Fifty-six male SPF-grade C57BL / 6J mice were randomly divided into seven groups of eight: blank group, model group, silymarin group, silymarin phospholipid group, low-dose biological enzyme group, medium-dose biological enzyme group, and high-dose biological enzyme group.
[0075] 1.3 Mouse grouping and sampling The blank group was fed the control liquid diet, while the model group, milk thistle group, milk thistle phospholipid group, and biological enzyme group were given liquid alcohol diet daily according to the modeling method, and biological enzyme samples were also fed at the same time.
[0076] The dosage is as follows: Silymarin group: silymarin 30.7 mg / kg; Milk thistle phospholipid group: Milk thistle phospholipid tablets 416mg / kg; Low, medium, and high dose groups of bio-enzyme: 0.09 g / kg, 0.18 g / kg, and 0.36 g / kg of the compound bio-enzyme in Example 13.
[0077] 1.4 Detection Indicators 1.4.1 Liver index Immediately after the experiment, the mice were dissected, their livers were removed, rinsed with pre-cooled saline, dried with filter paper, photographed, and weighed. The liver index (liver weight / body weight) was calculated. 1.4.2 Liver tissue MDA, GSH The liver was dissected and removed, and a portion of the tissue was taken to detect the MDA and GSH activities in the liver tissue using a test kit.
[0078] 2. Experimental Results 2.1 Liver index Table 7 Effects of biological enzymes on liver function in mice with alcoholic liver injury ( ±s, n=8) Note: Compared with the blank group, △△ P <0.01; compared with the model group, P <0.05, P <0.01.
[0079] As shown in Table 7, compared with the blank group, the liver index of mice in the model group was significantly increased ( P <0.01); Compared with the model group, the liver index of mice in the silymarin group and the low, medium and high dose groups of biological enzymes was significantly reduced ( P <0.05, P <0.01).
[0080] 2.2 Liver tissue MDA, GSH Table 8. Effects of biological enzymes on MDA and GSH levels in liver tissue of mice with alcoholic liver injury ( ±s, n=8) Note: Compared with the blank group, △△ P <0.01; compared with the model group, P <0.05, P <0.01 As shown in Table 8, compared with the blank group, the MDA content in the liver tissue of the model group mice was significantly increased ( P <0.01); compared with the model group, the MDA content in the liver tissue of mice in each treatment group was significantly reduced ( P <0.01, P <0.05%. Compared with the control group, the GSH content in the liver tissue of mice in the model group was significantly reduced ( P <0.01), and compared with the model group, there was no statistically significant difference in GSH content among the drug-treated groups.
[0081] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. The application of a compound biological enzyme preparation in the preparation of products for improving chemically induced liver injury, characterized in that, The raw materials of the compound bio-enzyme preparation include 3 to 14 kinds selected from papain, bromelain, acidic protease, pepsin, trypsin, α-amylase, sucrase, lipase, cellulase, pectinase, glucosylamylase, nuclease, serrepeptidase and β-galactosidase.
2. The application according to claim 1, characterized in that, The raw materials for the compound bio-enzyme preparation also include auxiliary components; The auxiliary ingredients are at least one of glucose, maltodextrin, taurine, soybean peptide powder, sodium alginate, lysine, vitamin C, and B vitamins.
3. The application according to claim 1, characterized in that, The raw materials of the compound bio-enzyme preparation include bromelain, papain, acidic protease, lipase, cellulase and glucosylamylase.
4. The application according to claim 1, characterized in that, The raw materials of the compound bio-enzyme preparation include bromelain, papain, acidic protease, α-amylase, and β-galactosidase.
5. The application according to claim 1, characterized in that, The raw materials for the compound bio-enzyme preparation include neutral protease, α-amylase, β-galactosidase, lipase, and cellulase.
6. The application according to claim 2, characterized in that, The raw materials of the compound bio-enzyme preparation include bromelain, papain, acidic protease, taurine, lysine, and B vitamins. The B vitamins mentioned are niacin, vitamin B6, and vitamin B1. 12 .
7. The application according to claim 6, characterized in that, The compound bio-enzyme preparation comprises the following components in the following amounts: bromelain 25,000-35,000 IU / g, papain 15,000-25,000 IU / g, acidic protease 45,000-55,000 IU / g, taurine 0.2-0.8 mg / g, lysine 0.1-0.3 mg / g, niacin 0.2-0.8 mg / g, vitamin B6 0.1-0.3 mg / g, and vitamin B12 0.1-0.3 mg / 100 g.
8. The application according to claim 2, characterized in that, The raw materials of the compound bio-enzyme preparation include bromelain, papain, acidic protease, nuclease, taurine, and soybean peptide powder.
9. The application according to claim 8, characterized in that, The compound bio-enzyme preparation comprises the following components in the following amounts: bromelain 25,000-35,000 IU / g, papain 15,000-25,000 IU / g, acidic protease 45,000-55,000 IU / g, nuclease 100-300 IU / g, taurine 0.2-0.8 mg / g, and soybean peptide powder 10-50 mg / g.
10. The application according to claim 1, characterized in that, The chemical liver injury mentioned is alcoholic liver injury.