Composition for preventing and treating alcoholic liver injury and application

Abstract

The invention relates to a composition for preventing and treating alcoholic liver injury and application thereof. The composition mainly comprises the following active ingredients: wheat peptide, soft-shelled turtle peptide and kudzuvine root powder. According to the invention, zebra fish is used as a model organism, ethanol exposure treatment is utilized to induce and establish an alcoholic liver injury model, and the alcoholic liver injury is identified by means of apparent histomorphology observation, biochemical index detection, gene expression quantity analysis and the like. Two bioactive peptides (wheat peptides and soft-shelled turtle peptides) screened from six bioactive peptides and five plant extracts are compounded with one plant extract (radix puerariae powder), and the effects of dispelling the effects of alcohol and protecting the liver of the compound are explored from the aspects of enteric microbiomics and non-targeted metabonomics. Researches show that the intervention of the compound significantly improves the hepatomegaly induced by alcohol, and has a synergistic effect in the aspects of preventing hepatocyte injury (improving ALDH activity) and oxidative injury (improving SOD activity) caused by alcohol.

Description

Technical Field

[0001] The present invention relates to a composition, and in particular to a composition capable of preventing and treating alcoholic liver damage and its application. Background Art

[0002] Alcoholic liver disease (ALD) is becoming a chronic liver disease problem worldwide. Its pathogenesis involves multiple pathways such as alcohol metabolism, oxidative damage and lipid metabolism. Since there are no specific drugs for the treatment of ALD and the existing treatment approaches may have significant toxic side effects, the development and research of safe and efficient natural functional active substances such as bioactive peptides and plant extracts has gradually received widespread attention, and related compound products have appeared on the market. However, existing research lacks the exploration of the synergistic effect between these food functional factors.

[0003] The study of the hangover-removing effect of corn hydrolysate has opened the prelude to exploring the liver-protective effect of bioactive peptides. To date, there have been many research results on liver-protective peptides from different protein sources. Yu et al. studied the protective effect of corn germ powder albumin peptide fraction (APF4) on acute alcoholic liver injury in mice. The results showed that in the mouse model, APF4 at a dose of 800 mg / kg / bw significantly promoted alcohol metabolism, specifically manifested in a significant decrease in blood alcohol concentration, CYP2E1, alanine aminotransferase (ALT), aspartate aminotransferase (AST), TG and MDA levels; acetaldehyde dehydrogenase (ALDH), SOD and GSH levels increased significantly. In another study using alcoholic liver disease in mice as a model, Wang et al. found that oyster peptides significantly upregulated Nrf-2, heme oxygenase-1 (HO-1), and quinone oxidoreductase 1 (NQO1) at the gene expression level, and significantly reduced the gene expression of NF-κB, TNF-α, and IL-6. Therefore, it is speculated that the possible mechanism of oyster peptides in protecting the liver is through enhancing the antioxidant capacity in the body and inhibiting the occurrence of inflammatory response. In addition to traditional experimental research, emerging technical means such as molecular docking have become an important technology in the field of computer-aided drug research. Molecular docking is a method of drug design based on the characteristics of receptors and the interaction between receptors and drug molecules. It mainly studies the interaction between molecules and predicts their binding mode and affinity. Wang et al. isolated liver-protecting peptides from pig livers and used molecular docking to simulate their binding sites with ADH. The product with the peptide sequence of Asp-Thr-Leu-Pro-His-Pro-Thr-Ala-Pro (NTLPHPTAP) required the lowest energy (-0.88 kcal / mol) to bind to ADH, which means it has the best ADH activation activity, which helps promote ethanol metabolism, scavenge free radicals, and improve alcoholic liver disease. Ren et al. pointed out that the most effective antioxidant active peptides YP-4 and LP-4 purified and identified from river clams significantly increased the viability of LO2 cells damaged by ethanol. These two peptides significantly reduced ROS levels and effectively inhibited the decline of mitochondrial membrane potential. Molecular docking revealed that YP-4 and LP-4 have potential inhibitory activity on CYP2E1, thereby reducing ethanol-induced oxidative stress.

[0004] Flavonoids refer to a series of compounds with two benzene rings with phenolic hydroxyl groups connected to each other by three central carbon atoms, and their basic mother nucleus is 2-phenylchromone. Flavonoids have always been a hot topic in drug research and development due to their diverse physiological activities and low biological toxicity. Puerarin, also known as puerarin, is an isoflavone derivative isolated from the traditional Chinese medicine Pueraria root. Xu et al. studied the differences in metabolites related to alcohol exposure and puerarin based on UPLC-MS / MS. Metabolomics analysis showed that alcohol caused abnormal expression of 82 metabolites, involving a total of 18 metabolic pathways and modules, including apoptosis, necroptosis, nucleotides and fatty acid metabolism. Puerarin reversed 7 metabolites related to necroptosis and sphingolipid metabolism pathways. Hoveniae is a plant of the Rhamnaceae family, Hovenia dulcis. Hoveniadulcis The dried mature seeds of Thunb. are rich in flavonoids. Qiu et al. fed Sprague-Dawley rats with a Lieber-DeCarli diet containing alcohol or isocaloric maltodextrin to establish a chronic alcoholic liver injury model and evaluated the effect of Hovenia dulcis extract (SHE) on improving liver injury and intestinal flora disturbance at the level of the gut-liver axis. Biochemical and histopathological analysis showed that SHE significantly alleviated ethanol-induced liver lipid deposition and inflammatory response. SHE upregulated the expression of tight junction proteins ZO-1 and occludin in the intestine by inhibiting the TLR4 pathway and its downstream inflammatory mediators, and significantly reversed the changes in intestinal microbiota induced by ethanol, reducing the production of intestinal-derived endotoxins. Silymarin is a natural flavonolignan compound. Tvrdý et al. hypothesized that silymarin may prevent alcoholic liver injury by inhibiting ADH, so they tested the zinc chelating activity of pure silymarin flavonolignan. Among the six flavonoid lignins tested, the 2,3-dehydrogenated derivatives (2,3-dehydrosilybin and 2,3-dehydrosilybin) significantly chelated zinc ions and blocked the action of ADH regardless of the presence or absence of zinc ions. Therefore, 2,3-dehydrosilybin may have biologically relevant inhibitory effects on ADH and glutamate dehydrogenase.

[0005] A large number of studies have shown that food-derived bioactive peptides and plant extracts have good effects in preventing alcoholic liver damage. However, most existing studies have only proven the protective effects of food functional factors on alcohol detoxification and alcoholic liver damage, and no comparative studies have been conducted to compare their functional differences. In addition, the market lacks alcohol detoxification compound health foods with clear synergistic effects. Summary of the invention

[0006] In view of the problems existing in the prior art, the purpose of the present invention is to provide a composition and application for preventing and treating alcoholic liver damage. This is specifically achieved through the following technical solutions: A composition for preventing and treating alcoholic liver damage mainly comprises the following active ingredients: wheat peptide, turtle peptide and kudzu root powder.

[0007] Furthermore, the mass percentages of the active ingredients in the composition are: wheat peptide 16.7-66.6%, turtle peptide 16.7-66.6%, and kudzu root powder 16.7-66.6%.

[0008] Furthermore, the mass percentages of the active ingredients in the composition are 16.7% wheat peptide, 66.6% turtle peptide, and 16.7% kudzu root powder.

[0009] Application of the composition in preparing health food or medicine for preventing and treating alcoholic liver damage.

[0010] Furthermore, the health food is a functional food made from the above composition and an edible carrier.

[0011] Furthermore, the medicine is a medicine prepared from the above composition and a pharmaceutically acceptable carrier.

[0012] The present invention compounded the selected active peptides and plant extracts, and explored the alcohol-detoxifying and liver-protecting effects of the compound from the perspectives of intestinal microbiome and non-targeted metabolomics. Studies have shown that the compound intervention significantly improved alcohol-induced liver enlargement, and had a synergistic effect in preventing alcohol-induced liver cell damage (increased ALDH activity) and oxidative damage (increased SOD activity). BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The apparent tissue morphology of acute alcoholic liver disease in zebrafish larvae (magnification 100x, A, control group; B, model group; C, corn peptide; D, wheat peptide; E, turtle peptide; F, primary soybean peptide; G, tertiary soybean peptide; H, sea cucumber peptide; I, kudzu root powder; J, Hovenia dulcis; K, silymarin; L, artichoke; M, curcumin; N, liver area. Different lowercase letters represent significant differences ( p <0.05)); Figure 2 Effects of bioactive peptides and plant extracts on liver cell damage (A, changes in AST activity; B, changes in ALDH activity); Figure 3 Effects of bioactive peptides and plant extracts on acute alcoholic liver oxidative damage (A, changes in SOD activity; B, changes in MDA activity); Figure 4 To investigate the effects of bioactive peptides and plant extracts on lipid metabolism in acute alcoholic liver disease; Figure 5This is the observation of the apparent tissue morphology of acute alcoholic liver disease in zebrafish larvae (magnification 100x, A, control group; B, model group; C, wheat peptide; D, turtle peptide; E, kudzu root powder; F, scheme 1; G, scheme 2; H, scheme 3; I, scheme 4; J, scheme 5; K, scheme 6; L, scheme 7; M, liver area); Figure 6 The effects of the compound on acute alcoholic hepatocyte injury in zebrafish larvae (A, changes in AST activity; B, changes in ALDH activity); Figure 7 The effects of the compound on acute alcoholic liver oxidative damage in zebrafish larvae (A, changes in SOD activity; B, changes in MDA activity); Figure 8 The effect of the compound on lipid metabolism in zebrafish larvae with acute alcoholic liver disease; Fig. 9 The comprehensive score of the compounding solution; Fig.10 The effect of the compound on the expression of alcohol metabolism genes (A, the expression of cyp2y3 gene; B, the expression of cyp3A65 gene); Fig.11 The effect of the compound on the expression of lipid metabolism genes (A, acc1 ; B, fasn ; C, hmgcra ;D, hmgcrb ); Fig.12 The effects of the compound on the expression of oxidative damage genes (A, atf6; B, chop; C, gadd45αa). DETAILED DESCRIPTION

[0014] The present invention will be further described below in conjunction with the accompanying drawings.

[0015] 1. Zebrafish husbandry and breeding The feeding method of zebrafish refers to the Westerfield method. In brief, the environmental conditions are 27.5-28.5 ℃, the light cycle is 14:10 (14 h of light, 10 h of darkness), the culture water is about 400 mg of artificial sea salt and about 10 mg of NaHCO3 per liter of reverse osmosis water, the water conductivity is 450-550 μS / cm, the pH is 6.8-7.5, and the total hardness (calculated as CaCO3) is 50-100 mg / L. Feed once in the morning and evening at a fixed time and quantity every day, and feed freshly hatched brine shrimp and solid fish feed alternately. The mating behavior of zebrafish requires the biological clock control of the light cycle, and at the beginning of each light cycle, it enters the state of mating and spawning. The night before mating, the male and female zebrafish are placed in the mating tank in a ratio of 2:1, and the male and female fish are separated by a partition. After the light cycle starts the next day, the partition is removed to allow the male and female fish to mate and spawn naturally. Collect the fish eggs and place them in a culture dish containing embryo culture medium. Develop them in a constant temperature biochemical incubator at 28 ℃. Observe them daily, remove dead eggs in time, and replace with fresh embryo culture medium.

[0016] 2. Selection of Alcohol Concentration for Modeling Acute Alcoholic Liver Disease in Zebrafish Larvae The liver of zebrafish embryos is fully formed when they develop to 96 hpf. In order to avoid the effect of eating on metabolism, this application uses 5dpf juveniles to establish an acute alcoholic liver model and conduct subsequent experiments. When the embryos develop to 5dpf, 540 juveniles with normal liver development are selected in time and randomly divided into a control group, a 1.0% alcohol treatment group, a 1.5% alcohol treatment group, a 2.0% alcohol treatment group, a 2.5% alcohol treatment group, and a 3.0% alcohol treatment group. They are placed in a six-well cell culture plate, with 30 fish per well and three parallel wells. The control group is cultured with embryo culture medium, and the alcohol treatment group is cultured with embryo culture medium containing corresponding concentrations of alcohol. They are cultured at 28 °C and a light cycle of 14:10 for 48 h. The death of the juveniles is observed and recorded at several time points, including the 6th, 12th, 24th, 36th, and 48th h of culture, and the dead fish are removed in time and the survival rate is calculated.

[0017] 3. Alcohol treatment and active substance intervention in zebrafish larvae 5dpf zebrafish larvae were randomly selected and divided into a control group, a model group, and an active substance intervention group. The intervention group was further divided into 6 bioactive peptide groups: corn peptide, wheat peptide, turtle peptide, primary soybean peptide, tertiary soybean peptide, and sea cucumber peptide; and 5 plant extract groups: kudzu root powder, Hovenia dulcis powder, water-soluble silymarin, artichoke, and curcumin. All zebrafish in all groups were placed in a six-well cell culture plate, 30 per well, and three parallel wells. The control group was cultured with embryo culture medium, the model group was cultured with embryo culture medium containing 350 mM ethanol (2% ethanol solution), and the intervention group was cultured with embryo culture medium containing 350 mM ethanol and 5 μg / mL active substances, and cultured for 48 h at 28 ℃ and a light cycle of 14:10.

[0018] 4. Observation of the Histomorphology of Zebrafish Larvae Liver Tg zebrafish larvae were selected and anesthetized with 0.02% tricaine for 10-20 seconds, then fixed laterally in 4% sodium carboxymethylcellulose and photographed under bright field and fluorescence conditions of a fluorescence microscope. The relative liver area of ​​zebrafish larvae in each group was quantified using ImageJ software.

[0019] 5. Alcohol treatment and compound intervention of zebrafish larvae 5dpf zebrafish larvae were randomly selected and divided into a control group, a model group, and an active substance intervention group. According to the preliminary screening results, wheat peptides, turtle peptides, and kudzu root powder were selected for compound experiments. The intervention group was further subdivided into three single active substance intervention groups: wheat peptides, turtle peptides, kudzu root powder, and seven compound substance intervention groups. The compounding scheme was designed using Minitab Statistical software, and the details are shown in Table 1. All grouped zebrafish were placed in a six-well cell culture plate, with 30 per well and three parallel wells. The control group was cultured with embryo culture medium, the model group was cultured with embryo culture medium containing 350 mM ethanol (2% ethanol solution), and the intervention group was cultured with embryo culture medium containing 350 mM ethanol and 5 μg / mL active substances, and cultured for 48 hours at 28 °C and a light cycle of 14:10. Among them, peptides and kudzu root powder were from Hangzhou Kangyuan Food Technology Co., Ltd.

[0020] Table 1: Compounding scheme design

[0021] 6. Analysis of the synergistic effects of active peptides and plant extracts The drug interaction coefficient (CDI) was used to analyze the synergistic effect of combined drug use. The calculation formula is as follows: ① N ABis the ratio of the parameters of the bioactive peptide and plant extract compound group to the model group; N A or N B is the ratio of the single intervention group to the model group of the corresponding parameter. If CDI < 1, it means that there is a synergistic effect between the drugs; if CDI = 1, it means that there is an additive effect between the drugs; if CDI > 1, it means that there is an antagonistic effect between the drugs.

[0022] 7. Biochemical Analysis of Zebrafish Larvae AB zebrafish larvae were selected and the relevant biochemical indices were determined according to the steps in the kit instructions, including hepatocyte damage (AST and ALDH), oxidative damage (SOD and MDA), and lipid metabolism (TG).

[0023] 8. Data processing The chemical experiment was repeated three times, and the experimental results were statistically analyzed and plotted using GraphPad Prism 9.5 software. Independent sample T test was used to compare two groups of data, and one-way analysis of variance and Tukey's test were used for multiple comparisons of multiple groups of data. Fuzzy comprehensive evaluation matrix operations were calculated using MATLAB2023b. Experimental data are expressed as mean ± standard error (Mean ± SEM). p <0.05 is considered statistically significant difference, * p <0.05,** p <0.01,*** p <0.001, **** p <0.0001.

[0024] Analyze the results 1. Effects of bioactive peptides and plant extracts on the histomorphology of acute alcoholic liver disease in zebrafish larvae By directly observing the liver's surface morphology, the protective effects of bioactive peptides and plant extracts on alcohol-induced liver damage can be compared intuitively and quickly. Figure 1 The liver of the model group was significantly enlarged compared with the control group. It is speculated that the acute alcoholic liver injury model of zebrafish was successfully established after 2% alcohol exposure, and lipid droplets accumulated in the liver. Liver area calculation results ( Figure 1 N in the figure shows that the liver area in the model group increased significantly compared with the control group ( p <0.05). Compared with the model group, the wheat peptide group, turtle peptide group and kudzu root powder group significantly improved the liver area enlargement induced by 2% alcohol exposure ( p <0.05). Compared with the model group, the other bioactive peptides and plant extracts showed an overall trend of inhibiting the increase of liver area, but the trend was not significant, indicating that they also had a certain protective effect on the liver enlargement induced by 2% alcohol.

[0025] 2. Effects of bioactive peptides and plant extracts on acute alcoholic hepatocyte injury in zebrafish larvae The content of AST in normal liver cells is low. When liver cells are damaged, the cell membrane permeability increases, and the release of AST in the cytoplasm leads to an increase in its concentration. Therefore, the change in AST value can be used as an indicator to judge liver cell damage. ALDH is widely present in various animals, plants and microorganisms. Its main function is to oxidize acetaldehyde into acetic acid, which plays an important role in alcohol metabolism. This application measured the above two liver cell damage related indicators, and the results are as follows Figure 2 Compared with the control group, the AST activity in the model group increased significantly ( p <0.001), while ALDH activity decreased significantly ( p <0.0001), which proves that the zebrafish acute alcohol liver injury model was successfully established. Most of the active peptides and plant extracts in the intervention group showed the ability to repair liver cells damaged by alcohol. Among them, the top three groups that significantly reduced AST activity compared with the model group were wheat peptide group, kudzu root powder group and artichoke group ( Figure 2 A in p <0.0001), the wheat peptide group decreased AST activity by 48.8%; and the top three groups with increased ALDH activity were the turtle peptide group, corn peptide group and kudzu root powder group ( Figure 2 B in p <0.05), the ALDH activity of the turtle peptide group was 1.62 times that of the model group. According to the overall trend, the effects of active peptides and plant extracts in reducing AST activity are similar, while the effect of active peptides in increasing ALDH activity is better than that of plant extracts.

[0026] 3. Effects of bioactive peptides and plant extracts on acute alcoholic liver oxidative damage in zebrafish larvae SOD plays a vital role in the body's oxidation and antioxidant balance and can remove superoxide anion free radicals (O 2-• ·), protect cells from damage. The level of SOD activity indirectly reflects the body's ability to eliminate oxygen free radicals. The oxygen free radicals produced by the body will attack the polyunsaturated fatty acids in the biological membrane, induce lipid peroxidation, and thus form lipid peroxides, such as: aldehyde (MDA), ketone, hydroxyl, carbonyl, hydroperoxy or endoperoxy, and new oxygen free radicals. In addition, oxygen free radicals can also cause cell damage through the decomposition products of lipid hydroperoxides. Therefore, the content of MDA can be used as an indicator to reflect the degree of lipid peroxidation in the body, and indirectly reflect the degree of cell damage. This study measured the above two oxidative damage-related indicators, and the results are as follows. Figure 3As shown. Compared with the control group, the SOD activity of the model group decreased significantly and the MDA content increased significantly, which further confirmed the successful establishment of the zebrafish acute alcohol liver injury model. Most of the active peptides and plant extracts in the intervention group showed the ability to prevent and repair oxidative damage to the body under the influence of alcohol. Among them, the top three groups with increased SOD activity compared with the model group were sea cucumber peptide group, kudzu root powder group and silymarin group ( Figure 3 The SOD activity of the sea cucumber peptide group was 1.21 times that of the model group, and the SOD activity of the kudzu root powder group and silymarin group was also 1.15 times higher than that of the model group; the top three groups in reducing MDA content were the wheat peptide group, curcumin group and kudzu root powder group ( Figure 3 B in p <0.0001), the MDA content of wheat peptide group and curcumin group was 65.9% and 64.1% of that of model group respectively. The effect of plant extract on enhancing SOD activity and reducing MDA content was better than that of active peptides.

[0027] 4. Effects of bioactive peptides and plant extracts on lipid metabolism in zebrafish larvae after acute alcoholic liver injury TG is a risk factor for cardiovascular disease. Short-term heavy drinking or long-term drinking can cause symptoms of high blood sugar and high triglycerides in the human body. This application measured the TG content of active peptides and plant extracts in the zebrafish acute alcoholic liver injury model. The results are as follows Figure 4 As shown. The TG content in the model group was significantly higher than that in the control group ( p <0.01), which is consistent with the test results of other biochemical indicators, which strongly proves that the zebrafish acute alcoholic liver model was successfully established. In the intervention group, the top three groups that reduced TG content were Pueraria powder group, turtle peptide group and sea cucumber peptide group, which significantly reduced liver TG content compared with the model group ( p <0.01), the TG content of the kudzu root powder group decreased by 30.2%. The effects of active peptides and plant extracts in reducing TG content were similar.

[0028] 5. Effects of the compound on the histomorphology of acute alcoholic liver disease in zebrafish larvae The results of direct photographing of Tg zebrafish under a fluorescence microscope are as follows: Figure 5 The liver area calculation results ( Figure 5 The M in the data showed that the wheat peptide, turtle peptide and kudzu root powder intervention groups showed significant preventive effects on acute alcoholic liver injury ( p <0.05), the liver area approached that of the control group; compared with the model group, Scheme 1 and Scheme 6 significantly prevented the increase in liver area induced by 2% alcohol exposure ( p <0.05), the liver area showed a decreasing trend after the other combination schemes, but it was not significant, indicating that they also have a certain effect in reversing alcoholic liver damage.

[0029] 6. Effects of the compound on acute alcoholic hepatocyte injury in zebrafish larvae This application measured two liver cell damage related indicators, AST and ALDH, and the results were as follows Figure 6 As shown. Schemes 2, 3, 5 and 6 significantly reduced the AST activity of hepatocytes exposed to 2% alcohol ( Figure 6 A in p <0.05), among which the AST activity of schemes 2 and 3 decreased by nearly 30%; except for scheme 3, the other combined schemes showed a trend of increasing ALDH activity after treatment, among which scheme 1 had a significant difference compared with the model group ( Figure 6 B in p <0.05), ALDH activity was 1.38 times that of the model group.

[0030] 7. Effects of the compound on acute alcoholic liver oxidative damage in zebrafish larvae This application measured two oxidative damage related indicators, SOD and MDA, and the results were as follows Figure 7 The SOD activity of Scheme 4 was 1.17 times that of the model group, and significantly increased the SOD activity of the 2% alcohol exposure treatment ( Figure 7 A in p <0.05), the other compound solutions showed a trend of increasing SOD activity, but not significantly; all solutions significantly reduced MDA content ( Figure 7 B in p <0.05), among which the MDA content of scheme 5 was reduced by 52.5% compared with the model group, indicating that the combination of wheat peptide, turtle peptide and kudzu root powder has a significant effect on reducing the MDA content in the acute alcoholic liver injury model of zebrafish larvae.

[0031] 8. Effects of the compound on lipid metabolism in zebrafish larvae with acute alcoholic liver disease This application measured the TG content of lipid metabolism-related indicators, and the results were as follows Figure 8 All seven compounding schemes significantly reduced the TG content of 2% alcohol exposure treatment ( p <0.05), among which the TG contents of scheme 2 and scheme 4 were reduced by 52.6% and 45.1% respectively compared with the model group, and from the overall trend, the effect of the combined scheme on TG content was better than that of the active substance single intervention group, indicating that the combined use of wheat peptide, turtle peptide and kudzu root powder may have a certain degree of synergistic effect in regulating lipid metabolism.

[0032] 9. Fuzzy comprehensive evaluation (FSE) and synergistic effect (CDI) analysis According to the above-mentioned apparent tissue morphological observations and related biochemical index determination results, it can be concluded that the compound products of wheat peptides, turtle peptides and kudzu root powder have a certain preventive effect on acute alcoholic liver injury. In order to quantify the comprehensive scores of each compounding scheme, the fuzzy comprehensive evaluation (FSE) method was used to analyze and calculate the biochemical index data. First, all the data were standardized, and the results are shown in Table 2. Then, the entropy weight was calculated to obtain the weight vector ω = (0.1690, 0.1870, 0.1824, 0.2051, 0.2565). Finally, the comprehensive score was calculated, and the results are as follows Fig. 9 As shown in the figure, the comprehensive score of scheme 6 is 0.6098, which is higher than that of other schemes, and is significantly higher in the results of apparent tissue morphology observation ( p <0.05) prevented the enlargement of liver area caused by 2% alcohol treatment, so it was determined that the following ratio of wheat peptide 16.7% + turtle peptide 66.6% + kudzu root powder 16.7% would be used for subsequent mechanism exploration and chronic alcoholic liver injury research experiments.

[0033] Table 2: Biochemical index data standardization

[0034] According to formula ①, the CDI values ​​of the five biochemical indicators of each compounding scheme were calculated, and the results are shown in Table 3. The AST activity CDI of the seven compounding schemes was greater than 1, indicating that the compounding products of wheat peptides, turtle peptides and kudzu root powder had no synergistic effect in reducing the increase in AST activity induced by alcohol. The ALDH and SOD activity CDIs of all compounding schemes were less than 1, among which the ALDH activity CDI of scheme six was 0.422657, and the SOD activity CDI was 0.747854, which had a significant synergistic effect. In terms of MDA and TG content, the synergistic trends of the alcohol-detoxifying and liver-protecting effects of each scheme were different. The MDA and TG content CDI of scheme one was less than 1, indicating that the compounding scheme had a synergistic effect in improving the above two indicators. Scheme three also had a synergistic effect on reducing the MDA content, and compound scheme two also showed a certain degree of synergistic effect in lowering the TG content.

[0035] Table 3: CDI value scoring table for different biochemical indicators in each scheme

[0036] 10. Effects of the compound on the expression of alcohol metabolism genes in zebrafish larvae with acute alcoholic liver disease Two genes involved in metabolizing alcohol were evaluated cyp2y3 and cyp3a65 Expression levels in each group of zebrafish. cyp2y3 and cyp3a65It is a member of the cytochrome P450 (CYP) family, mainly found in the liver of zebrafish, and plays a vital role in the alcohol metabolism process. Blocking the expression of CYP homologous genes can reduce the damage caused by alcohol metabolism and oxidative stress. Fig.10 The compound intervention was significant ( p <0.05) down-regulated the expression levels of the above two proteins. It is speculated that the preventive effect of the compound on acute alcoholic liver injury may be to promote alcohol metabolism, reduce the content of ROS induced by CYP, and reduce the degree of liver oxidative damage.

[0037] 11. Effects of the compound on the expression of lipid metabolism genes in zebrafish larvae with acute alcoholic liver disease In order to further evaluate whether the compound has the ability to regulate lipid metabolism, improve lipid homeostasis and resist alcohol-induced liver steatosis, this application selected several key genes related to lipid metabolism, including fatty acid synthesis genes ( acc1 and fasn ) and cholesterol synthesis genes ( hmgcra and hmgcrb ) for detection. RT-qPCR results ( Fig.11 ) showed that compared with the control group, alcohol treatment caused a significant upregulation of the mRNA expression in the above zebrafish larvae ( p <0.05), while the compound intervention was significant ( p <0.05) down-regulated genes related to fatty acid synthesis ( acc1 and fasn ) but for cholesterol synthesis related genes ( hmgcra and hmgcrb ), indicating that the compound may regulate lipid metabolism by regulating the fatty acid synthesis pathway.

[0038] 12. Effects of the compound on the expression of genes in zebrafish larvae with acute alcoholic liver oxidative damage Among the various pathological types of alcoholic liver damage, endoplasmic reticulum stress and DNA damage caused by oxidative damage play a key role. This application compares the mRNA expression levels of genes atf6, chop and gadd45αa related to endoplasmic reticulum stress and DNA damage ( Fig.12 ), confirming that the compound can significantly reduce the elevated chop mRNA level induced by alcohol ( Fig.12 The expression of gadd45αa was significantly increased in the control group (P>0.05), but it did not significantly affect the expression of gadd45αa (P>0.05), and only showed a decreasing trend. Compared with the model group, the expression of atf6 did not change significantly after the compound intervention. Therefore, it is speculated that the compound may protect the liver by inhibiting the DNA damage pathway to alleviate alcohol-induced hepatocyte apoptosis.

Claims

1. A composition for preventing and treating alcoholic liver damage, characterized in that: The composition mainly comprises the following active ingredients: wheat peptide, turtle peptide and kudzu root powder.

2. A composition for preventing and treating alcoholic liver damage according to claim 1, characterized in that: The mass percentages of the active ingredients in the composition are: 16.7-66.6% wheat peptide, 16.7-66.6% turtle peptide, and 16.7-66.6% kudzu root powder.

3. The composition for preventing and treating alcoholic liver damage according to claim 2, characterized in that: The mass percentages of the active ingredients in the composition are 16.7% wheat peptide, 66.6% turtle peptide and 16.7% kudzu root powder.

4. Use of the composition according to any one of claims 1 to 3 in the preparation of health food or medicine for preventing and treating alcoholic liver damage.

5. The use according to claim 4, characterized in that The health food is a functional food prepared from the composition according to any one of claims 1 to 3 and an edible carrier.

6. The use according to claim 4, characterized in that The medicine is a medicine prepared by combining the composition according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

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