A liver peptide with liver-protective and eye-fatigue-relieving properties, its preparation method, and its applications.

Abstract

This application discloses a liver peptide with liver-protective and eye-fatigue-relieving properties, its preparation method, and its applications, belonging to the field of biotechnology. The liver peptide contains at least one of DAIP, SPF, DIAP, SLP, AVGP, or NLP. The prepared liver peptide has multiple physiological functions: it can effectively activate ADH enzyme activity, reduce ALT and AST levels, and protect hepatocytes; it improves lipid metabolism by regulating the expression of SREBP-1c, FAS, and PPAR-α genes; it also has significant antioxidant capacity, reducing ROS levels and increasing SOD and CAT content; and it can upregulate RPE65 and LRAT gene expression, maintaining visual function. The method disclosed in this invention is simple and convenient to operate, and the obtained liver peptide can be widely used in functional products for liver protection and eye fatigue relief, providing an innovative solution for the high-value utilization of liver resources.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a liver peptide that protects the liver and improves eye fatigue, as well as its preparation method and applications. Background Technology

[0002] More than 3 million people die globally each year from alcohol abuse, with alcoholic liver disease being one of the leading causes of death. Current treatments for alcoholic liver disease primarily include alcohol cessation interventions, drug therapy (such as glucocorticoids), nutritional support, and liver transplantation for end-stage liver disease. However, commonly used drugs often have adverse reactions such as diarrhea, nausea, drowsiness, headache, and insomnia, and their long-term efficacy is not ideal. Meanwhile, while liver transplantation is an effective treatment for end-stage liver disease, it faces challenges such as donor shortages, high costs, and lifelong immunosuppression. Therefore, developing novel, highly effective, and low-toxicity hepatoprotective and alcohol-detoxifying drugs has become a critical issue that urgently needs to be addressed in clinical practice.

[0003] The widespread use of electronic devices in modern society has led to a significant increase in the incidence of visual fatigue syndrome, which is mainly manifested as dry eyes, blurred vision, and eye strain. Vitamin A is a key nutrient for maintaining visual function, and animal liver happens to be a natural source of essential nutrients for visual health, such as vitamin A, B12, zinc, and copper. Traditional eye care medications mostly use synthetic vitamin supplements, which have low bioavailability and lack synergistic active ingredients. Therefore, developing multifunctional active ingredients with high bioavailability from natural resources has become a new research direction for solving the above-mentioned health problems.

[0004] Bioactive peptides are protein fragments composed of 2-20 amino acids, possessing unique advantages such as small molecular weight, easy absorption, strong targeting, and low toxicity. Recent studies have found that peptides derived from animal organs (liver, placenta, etc.) exhibit significant health-promoting effects, particularly in the area of ​​hangover relief and liver protection. Their core mechanisms include: promoting alcohol metabolism (increasing the activity of alcohol dehydrogenase ADH), inhibiting oxidative stress (enhancing the activity of antioxidant enzymes such as SOD and GSH-Px), and reducing inflammatory responses.

[0005] Despite significant progress in animal liver peptide research, limitations remain, including a lack of research on mechanisms for improving visual fatigue and a lack of comparative studies using a single liver source. Therefore, this invention uses pig and bovine liver as raw materials to compare the functional effects and differences of different enzyme preparations and their combinations, and while exploring the liver peptides' effects on alcohol detoxification and liver protection, it also examines their effect on a visual fatigue model. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a liver peptide that protects the liver and improves eye fatigue, as well as its preparation method and application.

[0007] According to a first aspect of the present invention, a liver peptide that protects the liver and improves eye fatigue is provided, said liver peptide comprising at least one of DAIP, SPF, DIAP, SLP, AVGP or NLP;

[0008] The specific sequence of DAIP is shown in SEQ ID NO.1, the specific sequence of DIAP is shown in SEQ ID NO.2, and the specific sequence of AVGP is shown in SEQ ID NO.3.

[0009] Furthermore, the hepatic peptide comprises at least one of DAIP or SPF.

[0010] According to a second aspect of the present invention, a method for preparing liver peptides that protect the liver and improve eye fatigue is provided, comprising the following steps:

[0011] S1. Mix fresh liver with water and homogenize to obtain liver slurry;

[0012] S2. Add acidic protease to the liver fluid for hydrolysis to obtain the first hydrolysate;

[0013] S3. Add neutral protease and flavor protease to the first hydrolysate for hydrolysis, and then treat with enzyme inactivation to obtain the second hydrolysate.

[0014] S4. Cool the second hydrolysate, add activated carbon and stir to react, then centrifuge and ultrafilter to obtain liver peptides;

[0015] The prepared liver peptide contains at least one of DAIP, SPF, DIAP, SLP, AVGP, or NLP;

[0016] The specific sequence of DAIP is shown in SEQ ID NO.1, the specific sequence of DIAP is shown in SEQ ID NO.2, and the specific sequence of AVGP is shown in SEQ ID NO.3.

[0017] Furthermore, the liver is bovine liver, and the mass ratio of the liver to water is 1:(3~5).

[0018] Further, the amount of acidic protease added is 0.5% to 2% of the liver mass; the amount of neutral protease added is 0.5% to 1% of the liver mass; the amount of flavor protease added is 0.5% to 1% of the liver mass; and the amount of activated charcoal added is 4% to 6% of the liver mass.

[0019] Furthermore, the acidic protease has an enzyme activity of 150,000 U / g, the neutral protease has an enzyme activity of 100,000 U / g, and the flavor protease has an enzyme activity of 100,000 U / g.

[0020] Furthermore, the hydrolysis conditions for the acidic protease are: adjusting the pH of the system to 2.5–3.0 and hydrolyzing at 40°C–42°C for 2–3 hours; the hydrolysis conditions for the neutral protease and the flavor protease are: adjusting the pH of the system to 7.0–7.5 and hydrolyzing at 55°C–57°C for 1–2 hours.

[0021] Furthermore, the enzyme inactivation treatment conditions are: adjusting the pH to 6 and inactivating the enzyme at 85℃~90℃ for 15~20 min; the ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 5000 Da.

[0022] Furthermore, the preparation method also includes activity detection and cell experiments on the liver peptide.

[0023] Furthermore, the hepatic peptide comprises at least one of DAIP or SPF.

[0024] According to a third aspect of the present invention, the use of the liver peptide as described herein in the preparation of functional products for protecting the liver and improving visual fatigue is proposed.

[0025] According to a fourth aspect of the present invention, the application of a liver peptide prepared by the method described herein in the preparation of functional products for protecting the liver and improving visual fatigue is proposed.

[0026] The beneficial effects of this invention are:

[0027] The liver peptide preparation method provided by this invention significantly improves the yield and bioactivity of liver peptides by optimizing enzymatic hydrolysis process parameters and employing stepwise hydrolysis technology using acidic protease, neutral protease, and flavor protease. The prepared liver peptides possess multiple physiological functions: they can effectively activate ADH enzyme activity, reduce ALT and AST levels, and protect hepatocytes; they improve lipid metabolism by regulating the expression of SREBP-1c, FAS, and PPAR-α genes; they also have significant antioxidant capacity, reducing ROS levels and increasing SOD and CAT content; and they can upregulate RPE65 and LRAT gene expression, maintaining visual function. This method is simple and convenient to operate, requiring only enzymatic hydrolysis, inactivation, decolorization, and ultrafiltration steps for industrial production. The resulting liver peptide products can be widely used in functional product fields such as hangover relief and liver protection, anti-oxidation, and improvement of visual fatigue, providing an innovative solution for the high-value utilization of liver resources. Attached Figure Description

[0028] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention.

[0029] Figure 1 This is a diagram showing the effect of ADH content on HepG2 cells in a specific embodiment of the present invention;

[0030] Figure 2 This is a graph showing the effect of TG content on HepG2 cells in a specific embodiment of the present invention;

[0031] Figure 3 This is a diagram showing the influence of ALT and AST levels on HepG2 cells in a specific embodiment of the present invention.

[0032] Figure 4 This is a diagram showing the influence of SOD content in HepG2 cells according to a specific embodiment of the present invention.

[0033] Figure 5 This is a diagram showing the effect of SREBP-1c gene expression in HepG2 cells according to a specific embodiment of the present invention.

[0034] Figure 6 This is a diagram showing the effect of FAS gene expression in HepG2 cells according to a specific embodiment of the present invention;

[0035] Figure 7 This is a diagram showing the effect of PPAR-α gene expression in HepG2 cells according to a specific embodiment of the present invention;

[0036] Figure 8 This is a diagram showing the influence of ROS content on ARPE-19 cells in a specific embodiment of the present invention.

[0037] Figure 9 This is a diagram showing the effect of CAT content on ARPE-19 cells in a specific embodiment of the present invention.

[0038] Figure 10 This is a diagram showing the effect of Bax gene expression in ARPE-19 cells according to a specific embodiment of the present invention.

[0039] Figure 11 This is a diagram showing the effect of Bcl-2 gene expression in ARPE-19 cells according to a specific embodiment of the present invention.

[0040] Figure 12 This is a diagram showing the effect of Nrf2 / HO-1 gene expression in ARPE-19 cells according to a specific embodiment of the present invention.

[0041] Figure 13 This is a diagram showing the influence of RPE65 and LART gene expression in ARPE-19 cells according to a specific embodiment of the present invention.

[0042] Figure 14 This is a 2D diagram of the docking of DAIP with the ADH1A receptor molecule in a specific embodiment of the present invention;

[0043] Figure 15 This is a 2D diagram of the docking of DAIP and LART receptor molecules in a specific embodiment of the present invention;

[0044] Figure 16 This is a 2D diagram of the docking of DAIP with the Nrf2 receptor molecule in a specific embodiment of the present invention;

[0045] Figure 17 This is a 2D diagram of the docking of DAIP with the ADH1A receptor molecule in a specific embodiment of the present invention;

[0046] Figure 18 This is a 2D diagram of the docking of DAIP and LART receptor molecules in a specific embodiment of the present invention;

[0047] Figure 19 This is a 2D diagram of the docking of DAIP with the Nrf2 receptor molecule in a specific embodiment of the present invention;

[0048] Figure reference numerals: 1-THR A:48; 2-GLY A:204; 3-GLY A:47; 4-ARG A:369.

[0049] Attached image captions: Figure 1-13 Different lowercase letters (a / b / c / d / e / f) represent significant differences between groups (p<0.05), and different uppercase letters (A / B / C / D / E / F) represent significant differences within groups (p<0.05). Detailed Implementation

[0050] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings.

[0051] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0052] Given the current lack of research on the mechanism of visual fatigue improvement of animal liver peptides, and the limitations such as the lack of comparisons due to the single liver source, the first objective of this invention is to propose a liver peptide that can protect the liver and improve visual fatigue.

[0053] The second objective of this invention is to provide a method for preparing liver peptides that protect the liver and improve eye fatigue. The preparation method has low equipment requirements, is simple and easy to operate, and is suitable for large-scale industrial production.

[0054] The third objective of this invention is to propose an application of the liver peptide as described above in the preparation of functional products for protecting the liver and improving visual fatigue.

[0055] The fourth objective of this invention is to propose the application of a liver peptide prepared by the method described above in the preparation of functional products for protecting the liver and improving visual fatigue.

[0056] To achieve the above objectives, the present invention provides a liver peptide that protects the liver and improves eye fatigue. The liver peptide comprises at least one of DAIP, SPF, DIAP, SLP, AVGP, or NLP; the specific sequence of DAIP is shown in SEQ ID NO.1, the specific sequence of DIAP is shown in SEQ ID NO.2, and the specific sequence of AVGP is shown in SEQ ID NO.3. The liver peptide comprises at least one of DAIP or SPF.

[0057] This invention also provides a method for preparing liver peptides that protect the liver and improve eye fatigue, the specific steps of which are as follows:

[0058] Step 1

[0059] Fresh liver is mixed evenly with water at a mass ratio of 1:(3-5) of liver to pure water. The mixed sample is then ground and homogenized to form liver slurry.

[0060] Step Two

[0061] Acidic protease (acidic protease activity 150,000 U / g) was added to liver serous fluid for the first hydrolysis. The pH of the system was adjusted to 2.5-3.0, and the hydrolysis was carried out at 40℃-42℃ for 2-3 hours to obtain the first hydrolysate. The amount of acidic protease added was 0.5-2% of the liver mass.

[0062] Step 3

[0063] Neutral protease and flavor protease (neutral protease activity 100,000 U / g and flavor protease activity 100,000 U / g) were added to the first hydrolysate for a second hydrolysis. The pH of the system was adjusted to 7.0-7.5, and hydrolysis was carried out at 55-57℃ for 1-2 hours. Then, the pH was adjusted to 6 and the enzymes were inactivated at 85-90℃ for 15-20 minutes to obtain the second hydrolysate. The amount of neutral protease added was 0.5-1% of the liver mass, and the amount of flavor protease added was 0.5-1% of the liver mass.

[0064] Step Four

[0065] The hydrolysate was cooled to 57°C, and 4–6% (by weight of the raw material) of activated carbon was added and stirred for 1 hour. The mixture was then centrifuged to separate the solid and liquid phases, yielding a filtrate containing hepatin. The centrifugation speed was 4000–6000 rpm for 5–10 minutes. Subsequently, the filtrate was subjected to ultrafiltration to retain a solution containing hepatin with a molecular weight less than 5000 Da.

[0066] Step 5

[0067] Liver peptide activity was assessed, including in vitro ADH activation rate, TG content in HepG2 cells, alcohol dehydrogenase, transaminase, SOD content, and SREBP-1C, FAS, and PPAR-α gene expression; and ROS and CAT content in retinal pigment epithelial cells, as well as Bax, Bcl-2, Nrf2, HO-1, PRE65, and LRAT gene expression.

[0068] It should be noted that the enzymes with the same name used in the following examples and comparative examples are from the same commercially available company and brand, meaning that the enzyme activities of the enzymes with the same name used in each example and comparative example are consistent. The enzyme activities of the acidic protease (150,000 U / g), neutral protease (100,000 U / g), flavor protease (100,000 U / g), alkaline protease (200,000 U / g), papain (300,000 U / g), lipase (50,000 U / g), trypsin (250,000 U / g), and pepsin (30,000 U / g) used in the following examples and comparative examples refer to the enzyme activity per gram of enzyme (used to characterize the enzyme's ability to catalyze a specific chemical reaction), which has a different meaning from the enzyme addition unit U / g calculated per gram of protein in the liver.

[0069] Example 1

[0070] 1. Take 500g of beef liver and 1500g of pure water and homogenize them to obtain liver slurry;

[0071] 2. Adjust the temperature to 42℃, adjust the pH to 2.5 with hydrochloric acid, and add 5g of acidic protease to hydrolyze for 3 hours;

[0072] 3. Adjust the temperature to 57℃, adjust the pH to 7.0 with sodium hydroxide, add 5g of neutral protease and 2.5g of flavor protease and hydrolyze together for 1 hour; then adjust the pH to 6 and raise the temperature to 88℃ to inactivate the enzyme for 20 minutes.

[0073] 4. Adjust the temperature to 57℃, add 30g of activated carbon and stir for 1 hour;

[0074] 5. Centrifuge and filter (centrifuge speed 5000 rpm, time 10 min) and collect the supernatant. Filter the supernatant through diatomaceous earth to obtain a filtrate containing hepatin.

[0075] Example 2

[0076] 1. Take 500g of beef liver and homogenize it with 2500g of pure water to obtain liver slurry;

[0077] 2. Adjust the temperature to 42℃, adjust the pH to 2.5 with hydrochloric acid, and add 7.5g of acidic protease to hydrolyze for 3 hours;

[0078] 3. Adjust the temperature to 57℃, adjust the pH to 7.0 with sodium hydroxide, add 2.5g of neutral protease and 2.5g of flavor protease and hydrolyze together for 1 hour; then adjust the pH to 6 and heat to 88℃ to inactivate the enzyme for 20 minutes.

[0079] 4. Adjust the temperature to 57℃, add 30g of activated carbon and stir for 1 hour;

[0080] 5. Centrifuge and filter (centrifuge speed 5000 rpm, time 10 min) and collect the supernatant. Filter the supernatant through diatomaceous earth to obtain a filtrate containing hepatin.

[0081] Example 3

[0082] 1. Take 500g of beef liver and 1500g of pure water and homogenize them to obtain liver slurry;

[0083] 2. Adjust the temperature to 42℃, adjust the pH to 2.5 with hydrochloric acid, and add 5g of acidic protease to hydrolyze for 3 hours;

[0084] 3. Adjust the temperature to 57℃, adjust the pH to 7.0 with sodium hydroxide, add 5g of neutral protease and 5g of flavor protease and hydrolyze together for 1 hour; adjust the pH to 6 and raise the temperature to 88℃ to inactivate the enzyme for 20 minutes.

[0085] 4. Adjust the temperature to 57℃, add 30g of activated carbon and stir for 1 hour;

[0086] 5. Centrifuge and filter (centrifuge speed 5000 rpm, time 10 min) and collect the supernatant. Filter the supernatant through diatomaceous earth to obtain a filtrate containing hepatin.

[0087] Comparative Example 1

[0088] The hydrolysis process in this comparative example uses pig liver as raw material. The 500g of beef liver in step 1 is replaced with 500g of pig liver, and the remaining operations and processes are the same as in Example 1.

[0089] In Example 1, the amount of acidic protease added was 5g. Based on the protein content (30%) in bovine liver, the amount of acidic protease added was 5000U / g. The specific calculation process is as follows:

[0090] The enzyme activity of 5g of acidic protease is: 150,000 U / g × 5 = 750,000 U. Converted to the amount of enzyme added per gram of bovine liver protein, it is: 750,000 U / (500g × 30%)g = 5,000 U / g.

[0091] In Example 1, the amount of neutral protease added was 5g, which, based on the protein content of bovine liver, represents 3300 U / g of neutral protease; the amount of flavor protease added was 2.5g, which, based on the protein content of bovine liver, represents 1700 U / g of flavor protease. In this comparative example, the amount of acidic protease added was 5g, which, based on the protein content (30%) of pig liver, represents 5000 U / g of acidic protease; the amount of neutral protease added was 5g, which, based on the protein content of pig liver, represents 3300 U / g of neutral protease; and the amount of flavor protease added was 2.5g, which, based on the protein content of pig liver, represents 1700 U / g of flavor protease. Therefore, the amount of enzymes added in this comparative example is equal to the total amount of acidic protease, neutral protease, and flavor protease used in Example 1.

[0092] Comparative Example 2

[0093] The hydrolysis process in this comparative example uses acidic protease. The amount of acidic protease added in step 2 was changed to 10g. After 4 hours of enzymatic hydrolysis, the pH was adjusted to 6 and the temperature was raised to 88℃ for 20 minutes to inactivate the enzyme. Step 3 was removed, and the remaining operations and processes were the same as in Example 1.

[0094] The amount of acidic protease added in this comparative example is 10g, which is 10000U / g based on the protein content in bovine liver. Therefore, the amount of enzyme added in this comparative example is the same as the total amount of acidic protease, neutral protease and flavor protease used in Example 1.

[0095] Comparative Example 3

[0096] The raw material used in this comparative example was pig liver, and the hydrolysis process employed acidic protease. In step 1, 500g of beef liver was replaced with 500g of pig liver, and the amount of acidic protease added in step 2 was changed to 10g. After 4 hours of enzymatic hydrolysis, the pH was adjusted to 6, and the temperature was raised to 88℃ for 20 minutes to inactivate the enzyme. Step 3 was removed; the remaining operations and processes were the same as in Example 1.

[0097] The amount of acidic protease added in this comparative example is 10g, which is 10000U / g based on the protein content in pig liver. Therefore, the amount of enzyme added in this comparative example is the same as the total amount of acidic protease, neutral protease and flavor protease used in Example 1.

[0098] Comparative Example 4

[0099] The hydrolysis process in this comparative example used alkaline protease, neutral protease, and flavor protease. In step 2, the acidic protease was replaced with 5g of alkaline protease. The temperature was adjusted to 57°C, the pH to 8.5, and the hydrolysis was carried out for 2 hours. In step 3, 2.5g of neutral protease was added. The remaining operations were the same as in Example 1.

[0100] The 5g alkaline protease in this comparative example, based on the protein content of bovine liver, is 6700 U / g; the 2.5g neutral protease and 2.5g flavor protease, based on the protein content of bovine liver, are 3300 U / g, which is the same amount as the total amount of acidic protease, neutral protease and flavor protease used in Example 1.

[0101] Comparative Example 5

[0102] This comparative example uses pig liver as the raw material, and the hydrolysis process employs alkaline protease, neutral protease, and flavor protease. In step 1, 500g of beef liver was replaced with 500g of pig liver. In step 2, the acidic protease was replaced with 5g of alkaline protease. The temperature was adjusted to 57°C, the pH to 8.5, and the hydrolysis time was 2 hours. In step 3, 2.5g of neutral protease was added. The remaining operations were the same as in Example 1. The 5g of alkaline protease in this comparative example, based on the protein content of pig liver, is 6700 U / g; the 2.5g of neutral protease and 2.5g of flavor protease, based on the protein content of pig liver, are 3300 U / g, which is the same as the total amount of acidic protease, neutral protease, and flavor protease used in Example 1.

[0103] Comparative Example 6

[0104] The hydrolysis process in this comparative example used papaya, neutral and flavor protease. The acidic protease in step 2 was replaced with papain, and the amount added was 2.5g. The temperature was adjusted to 57℃, the pH was 7.0, and the enzymatic hydrolysis was carried out for 2 hours. The remaining operations and processes were the same as in Example 1.

[0105] In this comparative example, the amount of papain added (2.5g) based on the protein content of bovine liver was 5000 U / g; the amount of neutral protease (5g) based on the protein content of bovine liver was 3300 U / g; and the amount of flavor protease (2.5g) based on the protein content of bovine liver was 1700 U / g. The amount of enzymes in this comparative example was equal to the total amount of acidic protease, neutral protease, and flavor protease used in Example 1.

[0106] Comparative Example 7

[0107] The raw material used in this comparative example was pig liver. The hydrolysis process employed papaya, neutral, and flavor protease. In step 1, 500g of beef liver was replaced with 500g of pig liver, and in step 2, the acidic protease was replaced with papaya protease at a dosage of 2.5g. The temperature was adjusted to 57℃, the pH to 7.0, and the hydrolysis was carried out for 2 hours. The remaining operations and processes were the same as in Example 1.

[0108] In this comparative example, the amount of papain added (2.5g) based on the protein content of pig liver was 5000 U / g; the amount of neutral protease added (5g) based on the protein content of pig liver was 3300 U / g; and the amount of flavor protease added (2.5g) based on the protein content of pig liver was 1700 U / g. The amount of enzymes in this comparative example was equal to the total amount of acidic protease, neutral protease and flavor protease used in Example 1.

[0109] Comparative Example 8

[0110] The hydrolysis process in this comparative example only used lipase, neutral and flavor protease. The acidic protease in step 2 was replaced with lipase, the amount added was changed to 15g, the temperature was adjusted to 42℃, the pH was 7.0, and the enzymatic hydrolysis was carried out for 2 hours. The remaining operations and processes were the same as in Example 1.

[0111] The comparative example contains 15g of lipase, which is 5000 U / g based on the protein content of bovine liver; 5g of Bacillus subtilis neutral protease, which is 3300 U / g based on the protein content of bovine liver; and 2.5g of Bacillus subtilis flavor protease, which is 1700 U / g based on the protein content of bovine liver. The amount of enzymes in this comparative example is equal to the total amount of acidic protease, neutral protease and flavor protease used in Example 1.

[0112] Comparative Example 9

[0113] The raw material used in this comparative example was pig liver. The hydrolysis process only used lipase, neutral and flavor protease. The 500g of beef liver in step 1 was replaced with 500g of pig liver, the acidic protease in step 2 was replaced with lipase, and the amount added was changed to 15g. The temperature was adjusted to 42℃, the pH was 7.0, and the enzymatic hydrolysis was carried out for 2 hours. The remaining operations and processes were the same as in Example 1.

[0114] The 15g lipase in this comparative example has a protein content of 5000 U / g based on the protein content of pig liver; 5g neutral protease has a protein content of 3300 U / g based on the protein content of pig liver; and 2.5g flavor protease has a protein content of 1700 U / g based on the protein content of pig liver. The amount of enzymes in this comparative example is the same as the total amount of acidic protease, neutral protease and flavor protease used in Example 1.

[0115] Comparative Example 10

[0116] The hydrolysis process in this comparative example uses only alkaline protease. The acidic protease in step 2 is replaced with alkaline protease, the temperature is adjusted to 57°C, the amount added is changed to 7.5g, the pH is adjusted to 6 after 4 hours of enzymatic hydrolysis, and the temperature is raised to 88°C for 20 minutes to inactivate the enzyme. Step 3 is omitted, and the remaining operations and processes are the same as in Example 1.

[0117] The 7.5g alkaline protease in this comparative example is 10,000 U / g based on the protein content in bovine liver; the amount of enzyme in this comparative example is equal to the total amount of acidic protease, neutral protease and flavor protease used in Example 1.

[0118] Comparative Example 11

[0119] The raw material used in this comparative example was pig liver, and the hydrolysis process used only alkaline protease. In step 1, 500g of beef liver was replaced with 500g of pig liver, and in step 2, acidic protease was replaced with alkaline protease. The temperature was adjusted to 57℃, and the amount added was changed to 7.5g. After 4 hours of enzymatic hydrolysis, the pH was adjusted to 6, and the temperature was raised to 88℃ for 20 minutes to inactivate the enzyme. Step 3 was omitted, and the remaining operations and processes were the same as in Example 1.

[0120] The 7.5g alkaline protease in this comparative example is 10,000 U / g based on the protein content in bovine liver; the amount of enzyme in this comparative example is equal to the total amount of acidic protease, neutral protease and flavor protease used in Example 1.

[0121] Comparative Example 12

[0122] In this comparative example, alkaline protease and trypsin were used for hydrolysis. In step 2, the acidic protease was replaced with alkaline protease, the temperature was adjusted to 57°C, the pH to 8.3, and the amount added was changed to 5g. Hydrolysis was carried out for 2 hours. In step 3, the neutral protease and flavor protease were replaced with trypsin, the amount added was 2.5g, the temperature was adjusted to 39°C, the pH to 8.0, and after 2 hours of hydrolysis, the pH was adjusted to 6.0, and the temperature was raised to 88°C for 20 minutes to inactivate the enzyme. The remaining operations and processes were the same as in Example 1.

[0123] The 5g alkaline protease in this comparative example has a protein content of 6700 U / g based on bovine liver protein content; the 2.5g trypsin has a protein content of 3300 U / g based on bovine liver protein content; the amount of enzymes in this comparative example is equal to the total amount of acidic protease, neutral protease and flavor protease used in Example 1.

[0124] Comparative Example 13

[0125] This comparative example uses pork liver as the raw material, and the hydrolysis process employs alkaline protease and trypsin. In step 1, 500g of beef liver was replaced with 500g of pork liver; in step 2, acidic protease was replaced with alkaline protease; the temperature was adjusted to 57℃, the pH to 8.3, and the amount added was changed to 5g; enzymatic hydrolysis was carried out for 2 hours. In step 3, neutral protease and flavor protease were replaced with trypsin, with an addition amount of 2.5g; the temperature was adjusted to 39℃, the pH to 8.0, and after 2 hours of enzymatic hydrolysis, the pH was adjusted to 6.0, and the temperature was raised to 88℃ for 20 minutes to inactivate the enzyme. The remaining operations and processes were the same as in Example 1.

[0126] The 5g alkaline protease in this comparative example has a protein content of 6700 U / g based on the protein content of pig liver; the 2.5g trypsin has a protein content of 3300 U / g based on the protein content of pig liver; the amount of enzymes in this comparative example is the same as the total amount of acidic protease, neutral protease and flavor protease used in Example 1.

[0127] Comparative Example 14

[0128] In this comparative example, only pepsin and trypsin were used in the hydrolysis process. The acidic protease in step 2 was replaced with pepsin, the temperature was adjusted to 39°C, the pH was adjusted to 2.5, the amount added was changed to 8.5g, and the enzymatic hydrolysis was carried out for 2 hours. The neutral protease and flavor protease in step 3 were replaced with trypsin, the amount added was 5g, the temperature was adjusted to 39°C, the pH was adjusted to 8.0, the enzymatic hydrolysis was carried out for 2 hours, the pH was adjusted to 6.0, and the temperature was raised to 88°C for 20 minutes to inactivate the enzyme. The remaining operations and processes were the same as in Example 1.

[0129] The 8.5g pepsin in this comparative example has a protein content of 1700 U / g based on bovine liver protein content; the 5g trypsin has a protein content of 8300 U / g based on bovine liver protein content; the enzyme amount in this comparative example is the same as the total amount of acidic protease, neutral protease and flavor protease used in Example 1.

[0130] Comparative Example 15

[0131] The raw material used in this comparative example was pig liver, and the hydrolysis process used only pepsin and trypsin. In step 1, 500g of beef liver was replaced with 500g of pig liver; in step 2, the acidic protease was replaced with pepsin; the temperature was adjusted to 39℃, the pH to 2.5, and the amount added was changed to 8.5g; the hydrolysis was carried out for 2 hours. In step 3, the neutral protease and flavor protease were replaced with trypsin, and the amount added was 5g; the temperature was adjusted to 39℃, the pH to 8.0, and after 2 hours of hydrolysis, the pH was adjusted to 6.0, and the temperature was raised to 88℃ for 20 minutes to inactivate the enzymes. The remaining operations and processes were the same as in Example 1.

[0132] The 8.5g pepsin in this comparative example, based on the protein content of pig liver, is 1700 U / g; the 5g trypsin, based on the protein content of pig liver, is 8300 U / g. The amount of enzymes in this comparative example is equal to the total amount of acidic protease, neutral protease and flavor protease used in Example 1.

[0133] Example 4

[0134] The performance of the liver peptides prepared in the above examples and comparative examples was tested:

[0135] 1. Antioxidant activity of liver peptides

[0136] The liver peptides from the examples and comparative examples were prepared into 10 mg / mL solutions (using water as the solvent), and their hydroxyl radical scavenging rate and DPPH radical scavenging rate were detected. The test results are shown in Table 1.

[0137] The DPPH free radical scavenging rate of hepatic peptides was determined by the DPPH method for peptide antioxidant determination in GB / T 39100~2020; the hydroxyl free radical scavenging rate was determined by the study on antioxidant activity of Panax notoginseng polysaccharides.

[0138] Table 1. Antioxidant activity of liver peptides processed by different methods

[0139] ;

[0140] As shown in Table 1, the liver peptides from both the examples and comparative examples exhibit certain antioxidant activity, indicating that different enzymes or combinations of enzymatic hydrolysis can produce components with antioxidant activity. However, the hydroxyl radical scavenging rate or DPPH scavenging rate of some of the comparative examples was not ideal (below 30%). Furthermore, liver peptides from different sources showed varying degrees of antioxidant effect under the same enzyme preparation, indicating differences between the raw materials. Therefore, further testing of their in vitro ADH activation rate was necessary.

[0141] 2. In vitro activation rate of hepatic peptides with alcohol dehydrogenase (ADH)

[0142] The ADH kit was operated according to the instructions. The protein concentration of the sample and positive control was adjusted to 1 mg / mL. 50 μL of sample was mixed with 150 μL of working solution and stabilized at 37 ℃ for 5 min. Then, 50 μL of ADH solution (enzyme activity 1 U / mL) was added to initiate the chemical reaction, and the timing began from this point. The absorbance was measured at 340 nm using a microplate reader, with OD at 340 nm recorded every 10 s for 10 min. Distilled water was used as a negative control. The resulting curve was fitted, and the first derivative of the fitted curve at 0 min was calculated to quantify the NADH production rate, i.e., the initial reaction rate. All experiments were repeated three times, and the ADH activation rate was calculated using the formula:

[0143] ADH activation rate % = (Vs - Vo) / Vo × 100%

[0144] Vs sample initial reaction rate;

[0145] Initial reaction rate of the negative control (Vo).

[0146] Table 2. ADH activation rate (%) of liver peptides from different processes

[0147] ;

[0148] ADH is a key initiating enzyme in the metabolism of ethanol in the liver, responsible for oxidizing ethanol to acetaldehyde. Activating ADH can accelerate ethanol clearance and reduce the accumulation and residence time of ethanol and its subsequent toxic products (acetaldehyde, free radicals) in the body, which is one of the core mechanisms of "sobering up." The higher the activation rate, the greater its potential to promote alcohol metabolism.

[0149] As shown in Table 2, the liver peptides in the examples and comparative examples have a certain effect on ADH activation. Among them, the ADH activation rates of comparative examples 9, 10, 12, and 14 are higher, indicating that different enzymatic hydrolysis combinations can promote ADH activation. However, further comprehensive consideration and verification are still needed.

[0150] 3. Cellular experiments on the alcohol-detoxifying and liver-protecting effects of hepatic peptides.

[0151] Based on the above in vitro test results, Example 1, Comparative Example 1, Comparative Example 6, Comparative Example 7, Comparative Example 9, Comparative Example 10, Comparative Example 12 and Comparative Example 14 were selected for further verification.

[0152] 3.1 Hepatic peptide cytotoxicity experiment and modeling in HepG2 cells

[0153] HepG2 cells in the logarithmic growth phase were collected and cultured in DMEM complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) at a concentration of 2.0 × 10⁶ cells / mL. 5 Cell suspensions were prepared at a density of cells / mL and seeded into 96-well plates, with 100 μL added to each well. The plates were then incubated at 37°C for 24 h in a CO2 incubator. Afterward, the 96-well plates were removed, and the culture medium (except for the blank group) was aspirated. Different concentrations of hepatic peptide solution (0, 0.5, 1.0, 1.5, 2.0 mg / mL) or culture medium containing different concentrations of ethanol were added according to the group. The plates were then incubated at 37°C for 24 h in a CO2 incubator. The culture medium was then aspirated, and 10 μL of CCK8 solution (protected from light) was added to each well. The absorbance of each well was measured at 450 nm using a microplate reader, and the average value of three wells was taken for each group. Cell viability was calculated using formula (1):

[0154] Cell viability (%) = (A1 / A0) - (Ac-A0) × 100% (1)

[0155] A0: Absorbance value of the blank group;

[0156] Ac: Absorbance value of the control group;

[0157] A1: Absorbance value of the active peptide sample group or the absorbance value of the model group;

[0158] Experimental groups: blank group, alcohol model group, experimental group (alcohol + sample), positive control group (alcohol + silymarin).

[0159] Based on the cell viability results, the concentration of liver peptide was selected as 1 mg / mL, the alcohol modeling concentration was 400 mM for 24 h of injury, and silymarin at 10 μg / mL was selected as the positive control.

[0160] 3.2 Effects of liver peptides on alcohol-induced ADH levels in HepG2 cells

[0161] Cells with good growth were divided into groups of 5.0 × 10⁻⁶. 5 Cells were seeded at a density of [number] cells / well in 12-well plates and cultured for 24 h in a 37°C, 5% CO2 incubator until cell adhesion was observed. After cell adhesion, the culture medium was discarded, and the cells were stimulated with 400 mM ethanol for 24 h to establish a cell model. After modeling, each experimental group was added to complete culture medium containing 6% of the sample, while the model group was cultured in complete culture medium alone. The cells were cultured at 37°C for 24 h. After intervention, the supernatant was aspirated, and 200 μL of Tris-Triton lysis buffer was added directly to each well. The cells were incubated on ice for 30–40 min. The lysis buffer was transferred to 1.5 EP tubes, vortexed to promote lysis, and centrifuged at 12000 g at 4°C for 10 min. The supernatant was collected. Protein concentration was quantitatively determined using BCA assay according to the ADH kit.

[0162] Depend on Figure 1 It can be seen that: compared with the normal group, the ADH content in the alcohol-induced liver injury model group was significantly reduced, indicating that the alcoholic liver injury model was successfully established; compared with the model group, the ADH content in the silymarin group was significantly increased, verifying its role as a positive control. Compared with the model group, the examples and some comparative examples significantly increased the ADH content, indicating that liver peptide samples can enhance ADH activity and improve ethanol metabolism.

[0163] 3.3 Effect of liver peptides on alcohol-induced triglyceride (TG) levels in HepG2 cells

[0164] Triglycerides (TG) are the main form of fat storage in the liver. Alcohol intake severely disrupts the liver's lipid metabolism balance (promoting fat synthesis and inhibiting fat breakdown), leading to abnormal TG accumulation in hepatocytes and the formation of alcoholic fatty liver (steatohepatitis). Detecting intracellular TG levels is a direct indicator for assessing the degree of lipid accumulation in the liver.

[0165] Cells with good growth were divided into groups of 5.0 × 10⁻⁶. 5Cells were seeded at a density of 1 cell / well in 12-well plates and cultured for 24 h in a cell culture incubator at 37℃ and 5% CO2 until cell adhesion was observed. After cell adhesion, the culture medium was discarded, and the cells were stimulated with 400 mM ethanol for 24 h to establish a model. After modeling, each experimental group was given complete culture medium containing 6% of the sample, while the model group was given complete culture medium and cultured at 37℃ for 24 h. After intervention, the supernatant was aspirated, and 200 μL of Tris-Triton lysis buffer was added directly to the well plate and lysed on ice for 30–40 min. The lysis buffer was transferred to a 1.5 EP tube, vortexed to promote lysis, centrifuged at 12000 g at 4℃ for 10 min, and the supernatant was collected. BCA protein concentration was quantitatively detected and measured according to the TG kit. The TG content was calculated according to formula (2):

[0166] TG content = TG activity (Kamen's units) obtained by substituting into the standard curve × 0.482 / sample protein concentration × 100% (2)

[0167] 0.482: Conversion from Kamen's units to U;

[0168] Depend on Figure 2 It can be seen that: compared with the normal group, the TG content in the alcohol-induced model group was significantly increased, indicating that alcohol successfully induced fatty degeneration; compared with the model group, the silymarin group had a significantly decreased TG content, verifying its role as a positive control. Compared with the model group, the examples and each comparative example significantly reduced the TG content, indicating that the liver peptide samples can improve fat accumulation.

[0169] 3.4 Effects of liver peptides on alcohol-induced alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in HepG2 cells

[0170] ALT and AST are mainly found in the cytoplasm and mitochondria of hepatocytes. When hepatocytes are damaged (e.g., by alcohol toxicity, inflammation, or necrosis), leading to disruption of cell membrane integrity, these enzymes leak into the cell culture supernatant. Therefore, detecting ALT and AST activity is the most commonly used and sensitive biomarker reflecting the degree of hepatocyte damage.

[0171] ALT and AST were used for activity detection according to ALT and AST kits, respectively, and calculated according to formulas (3) and (4):

[0172] ALT activity (U / gprot) = AST activity (Kamen's units) obtained by substituting into the standard curve × 0.482 / sample protein concentration × 100% (3)

[0173] AST activity (U / gprot) = Substitute into the standard curve to get AST activity (Kamen's units) × 0.482 / sample protein concentration × 100% (4)

[0174] 0.482: Conversion from Kamen's units to U;

[0175] Depend on Figure 3 It was found that: compared with the normal group, the ALT and AST levels in the alcohol-induced model group were significantly increased, indicating successful modeling and that alcohol caused significant hepatocellular damage; compared with the model group, the ALT and AST levels in the silymarin group were significantly decreased, proving the effectiveness of the model and the positive control drug. Compared with the model group, the examples and comparative examples all significantly reduced ALT and AST levels, indicating that the liver peptide samples can alleviate alcohol-induced hepatocellular damage and have a cytoprotective effect.

[0176] 3.5 Effect of liver peptides on alcohol-induced superoxide dismutase (SOD) levels in HepG2 cells

[0177] Superoxide dismutase (SOD) is one of the most important antioxidant enzymes in the body, responsible for scavenging excess superoxide anion free radicals produced during alcohol metabolism and protecting cells from oxidative damage. Alcohol metabolism consumes a large amount of antioxidants and generates free radicals, often leading to a decrease in intracellular SOD activity due to consumption or inhibition.

[0178] The SOD content was calculated according to formulas (5) and (6) using the SOD kit:

[0179] SOD inhibition rate (%) = [(OD control - OD control blank) - (OD measurement - OD measurement blank)] / (OD control - OD control blank) × 100% (5)

[0180] SOD activity (U / mgprot) = SOD inhibition rate / 50% × (0.24mL / 0.02mL) / sample protein concentration (mgprot / mL) (6)

[0181] Depend on Figure 4 It was found that: compared with the normal group, the SOD content in the alcohol-induced model group was significantly lower, indicating that alcohol induced a significant oxidative stress state, and the model was successful; compared with the model group, the SOD content in the silymarin group was significantly higher, proving that the positive drug has an antioxidant effect. Compared with the model group, the examples and comparative examples all showed a significant increase in SOD content, indicating that the liver peptide samples can enhance the antioxidant defense capacity of liver cells and resist alcohol-induced oxidative stress. Reducing oxidative damage is one of the important mechanisms of "liver protection".

[0182] 3.6 Effects of liver peptides on gene expression in alcohol-induced HepG2 cells

[0183] Total RNA was extracted from cells using the Simply P Total RNA Extraction Kit (BioFlux). 2 μL of sterile, enzyme-free water was dropped onto a Nanodrop one plate to determine the RNA concentration and purity (purity between 1.8 and 2.1). A 20 μL qRT-PCR reaction system was established. The PCR amplification conditions were: pre-denaturation 95℃ for 30 s; denaturation 95℃ for 15 s, annealing 54℃ for 30 s, extension 72℃ for 45 s, for 40 cycles. The melting curve reaction conditions were: 95℃ for 30 s, 60℃ for 1 min, with fluorescence signals collected every 5℃ increase. Primers were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd., with GAPDH as an internal control. Primer sequence information is listed in Table 3.

[0184] Table 3 Primer Sequence Information-1

[0185] ;

[0186] It should be noted that all of the above primers are existing primers.

[0187] SREBP-1c (sterol regulatory element-binding protein-1c) is a core transcription factor regulating fatty acid and triglyceride synthesis. It activates the expression of a series of lipid synthesis genes, including FAS (fatty acid synthase). FAS is a key enzyme catalyzing the synthesis of long-chain fatty acids and is directly regulated by SREBP-1c. Its activity directly affects the rate of triglyceride (TG) synthesis. PPAR-α (peroxisome proliferator-activated receptor α) is a core transcription factor regulating fatty acid oxidation (β-oxidation). It activates the expression of genes involved in fatty acid uptake, transport, and mitochondrial and peroxisome β-oxidation, promoting fatty acid breakdown for energy.

[0188] Depend on Figure 5 , Figure 6 and Figure 7 It can be seen that, compared with the normal group, the alcohol model group significantly upregulated the expression of SREBP-1c and FAS genes, which are key driving factors for alcoholic fatty liver. Compared with the model group, the examples and some comparative examples (comparative examples 6, 7, 9, 10, 12, and 14) significantly downregulated the expression of SREBP-1c and FAS mRNA, indicating that most liver peptide samples can inhibit the synthesis of fat in hepatocytes.

[0189] Compared with the normal group, the alcohol-induced model group showed significantly downregulated PPAR-α expression and activity, leading to inhibited fatty acid oxidation and exacerbated lipid accumulation. Compared with the model group, the examples and some comparative examples (Comparative Example 1, Comparative Example 6, and Comparative Example 7) significantly upregulated PPAR-α mRNA expression. This indicates that some liver peptide samples can activate the fatty acid oxidation pathway and promote lipolysis, consistent with the results of reducing TG. This is another important mechanism for reversing fatty liver.

[0190] 4. Experiment on the improvement of visual fatigue by liver peptides

[0191] Human retinal pigmented epithelial cells-19 (RPE-19) are located between the outer segments of choroidal capillaries and photoreceptors, forming the blood-retinal barrier and providing essential nutrition and metabolic support to the retinal neuronal layer. RPE-19 cells generate oxygen free radicals during metabolism, making them susceptible to oxidative attack, manifesting as cellular senescence, ROS accumulation, mitochondrial dysfunction, and increased cell necrosis and apoptosis.

[0192] 4.1 Hepatic peptide cytotoxicity experiment and modeling of ARPE-19 cells

[0193] ARPE-19 cells in the logarithmic growth phase were collected and cultured in DMEM complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) at a concentration of 1.0 × 10⁶ cells / mL. 5 Cell suspensions were prepared at a density of cells / mL and seeded into 96-well plates, with 100 μL added to each well. The plates were then incubated at 37°C for 24 h in a CO2 incubator. Subsequently, the 96-well plates were removed, and the culture medium (except for the blank group) was aspirated. Different concentrations of hepateptide solution (0, 0.5, 1.0, 1.5, 2.0 mg / mL) were added. After incubation at 37°C for 24 h in a CO2 incubator, the culture medium was aspirated, and 10 μL of CCK8 solution (protected from light) was added to each well. The absorbance of each well was measured at 450 nm using a microplate reader, and the average value of three wells was taken for each group. Cell viability was calculated using the same formula as formula (1).

[0194] Based on the experimental results, the final selection was a liver peptide sample concentration of 1 mg / mL, an H2O2 modeling concentration of 0.7 mM for 2 h of injury, and a positive control of 10 μM lutein.

[0195] 4.2 Effects of hepatic peptides on H2O2-induced reactive oxygen species (ROS) levels in ARPE-19 cells

[0196] The retina, especially photoreceptor photoreceptor cells (RPE cells), has one of the highest levels of oxidative stress in the body due to its extremely high metabolic rate and exposure to high oxygen and light. Excessive reactive oxygen species (ROS) are a core pathological factor leading to major blinding eye diseases such as age-related macular degeneration, diabetic retinopathy, and retinitis pigmentosa. ROS damage lipids, proteins, and DNA, ultimately causing RPE dysfunction and photoreceptor apoptosis.

[0197] ARPE-19 cells in good growth condition were seeded into 96-well plates at a cell density of 1.0 × 10⁶ cells / well. 5 Cells were cultured per well at 37 ℃ in a 5% CO2 incubator for 24 h. After adhesion, the supernatant was discarded. Except for the NC control group, all other groups were replaced with complete medium containing 0.7 mM H2O2. The NC control group underwent a medium change. After 2.5 h of H2O2 intervention, each group was treated with complete medium containing the corresponding sample and concentration for 24 h. The supernatant was discarded, and 100 μL of diluted DCFH-DA was added, followed by culturing for 30 min. Cells were washed 1-2 times with serum-free medium to remove any DCFH-DA that had not entered the cells. Simultaneously, the fluorescence intensity before and after stimulation was measured using a microplate reader: excitation wavelength 488 nm, emission wavelength 525 nm.

[0198] Experimental groups: blank group, H2O2 model group, positive control group (H2O2 + lutein), experimental group (H2O2 + sample).

[0199] 4.3 Effect of liver peptides on H2O2-induced catalase (CAT) content in ARPE-19 cells

[0200] CAT is a key antioxidant enzyme that specifically catalyzes the breakdown of H2O2 into water and oxygen, serving as the cell's first line of defense in clearing ROS and maintaining redox balance.

[0201] ARPE-19 cells in good growth condition were seeded into 96-well plates at a cell density of 1×10⁻⁶. 5 Each well was incubated with 1 sample per well at 37 ℃ in a 5% CO2 incubator for 24 h. After observation of adhesion, the supernatant was discarded. Except for the NC control group, all other groups were replaced with complete medium containing 0.7 mM H2O2. The NC control group was replaced with fresh medium. After 2.5 h of H2O2 intervention, each group was treated with complete medium containing the corresponding sample and concentration for 24 h. After aspirating the supernatant, 300 μL of Tris-Triton lysis buffer was added directly to the well, and the plate was incubated on ice for 30–40 min. The lysis buffer was transferred to a 1.5 EP tube, vortexed to promote lysis, and centrifuged at 12000 g at 4 ℃ for 20 min. The supernatant was collected. Protein concentration was quantitatively determined using BCA. CAT assay was performed according to the kit.

[0202] Depend on Figure 8 and Figure 9 It can be seen that: compared with the normal group, the ROS content in the H2O2 modeling group was significantly increased and the CAT content was significantly decreased, indicating that the H2O2 modeling was successful; compared with the model group, the ROS content in the lutein group was significantly decreased and the CAT content was significantly increased, proving the free radical scavenging effect of the positive control drug. Compared with the model group, the examples and comparative examples all significantly reduced the ROS content and increased the CAT content, indicating that the hepatic peptide samples have enhanced antioxidant capacity, which helps protect RPE cells from oxidative damage and maintain their normal functions (such as supporting photoreceptors and maintaining the blood-retinal barrier), thereby protecting vision.

[0203] 4.4 Effects of liver peptides on H2O2-induced gene expression in ARPE-19 cells

[0204] ARPE-19 cells in good growth condition were seeded into 12-well plates at a cell density of 10⁵ cells / well and cultured at 37°C in a 5% CO₂ incubator for 24 h. After observation of cell adhesion, the supernatant culture medium was discarded. Except for the NC control group, all other groups were replaced with complete culture medium containing 0.7 mM H₂O₂. The NC control group was simply replaced with fresh culture medium. After 2.5 h of H₂O₂ intervention, each group was treated with complete culture medium containing the corresponding sample and concentration for 24 h.

[0205] After the intervention of samples and positive controls, total RNA was extracted from cells in each group, and the concentration and purity of RNA were measured. Using the extracted RNA as a template, RNA was reverse transcribed into cDNA. The obtained cDNA was stored at -20℃. The original solution was diluted 5 times for subsequent steps in the qPCR reaction. Real-time quantitative PCR was performed. The reaction conditions were: 95℃ pre-denaturation for 30 s; 95℃, 10 s, 60℃, 30 s, for a total of 40 cycles; melting curve 95℃, 15 s, 60℃, 60 s, 95℃, 15 s. The Ct cycle threshold was obtained according to the two-step amplification program, and the relative expression level of RNA in different groups was calculated by formula (7) = 2 - ΔΔCt. Glycerol-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The primer sequences are shown in the table below:

[0206] Table 4 Primer Sequence Information - 2

[0207] ;

[0208] It should be noted that all of the above primers are existing primers.

[0209] Under the premise of ensuring stable cell growth and not affecting proliferation capacity, the effects of liver peptide samples prepared in the examples and comparative examples on H2O2-induced gene expression in ARPE-19 cells were measured as follows: Figure 10 , Figure 11 , Figure 12 and Figure 13 As shown.

[0210] Bax and Bcl-2 are key factors regulating apoptosis (programmed cell death). Bax promotes increased permeability of the mitochondrial outer membrane, releasing pro-apoptotic factors (such as cytochrome C) and initiating the apoptosis cascade. Bcl-2, on the other hand, inhibits Bax activity, stabilizes the mitochondrial membrane, and prevents apoptosis. In various retinal diseases (such as AMD, glaucoma, and retinal detachment), apoptosis of RPE cells and photoreceptors is a direct cause of vision loss.

[0211] Depend on Figure 10 and Figure 11 It was found that, compared with the normal group, H2O2 in the model group significantly upregulated Bax and downregulated Bcl-2, inducing cell apoptosis. Compared with the model group, the lutein control group significantly downregulated Bax and upregulated Bcl-2, indicating that lutein can significantly alleviate the effect of H2O2 on cell apoptosis. Compared with the model group, the examples and some comparative examples (Comparative Example 1, Comparative Example 6, Comparative Example 9, Comparative Example 10, Comparative Example 12, and Comparative Example 14) significantly downregulated Bax and upregulated Bcl-2, indicating that liver peptide samples can protect RPE cell survival and maintain retinal structural integrity and function by inhibiting pro-apoptosis and promoting anti-apoptosis.

[0212] Nrf2 is a core transcriptional regulator of the cellular antioxidant defense system. Under oxidative stress or stimulation by electrophilic substances, Nrf2 translocates from the cytoplasm to the nucleus, binds to antioxidant response elements, and initiates the transcription of a series of downstream protective genes (antioxidant proteins), among which HO-1 is one of its most important target genes. HO-1 catalyzes the degradation of heme, producing products with strong antioxidant, anti-inflammatory, anti-apoptotic, and cytoprotective effects (such as carbon monoxide and iron ions).

[0213] Depend on Figure 12It was found that, compared with the normal group, H2O2 significantly upregulated the expression of Nrf2 and HO-1 in the model group, indicating that H2O2 successfully activated the Nrf2 / ARE signaling pathway in cells, triggering an endogenous antioxidant defense response. Compared with the model group, the lutein control group significantly downregulated the expression of Nrf2 and HO-1, indicating that lutein can significantly alleviate the effects of H2O2-induced oxidative stress on cells. The examples and some comparative examples also significantly downregulated the expression of Nrf2 and HO-1, indicating that the samples may directly neutralize free radicals, reduce oxidative damage, and cells do not need to maintain a high level of endogenous defense, so the activity of the Nrf2 pathway naturally declines. Combined with the above-mentioned ROS scavenging and apoptosis inhibition, this shows that hepatic peptides can effectively protect cells.

[0214] RPE65 (retinal pigment epithelium-specific protein) and LRAT (lecithin retinyl acyltransferase) are core components of the visual cycle, which occurs in RPE cells and is responsible for regenerating 11-cis-retinal, the photochromophore of visual pigments (rhodopsin and cone pigments). The expression levels and functional activities of RPE65 and LRAT directly determine the regeneration rate and supply of 11-cis-retinal, thus affecting the speed of photoregeneration, which is crucial for maintaining night vision adaptation (scotoma vision) and photopic sensitivity.

[0215] Depend on Figure 13 It was found that, compared with the normal group, H2O2 in the model group significantly inhibited the expression of RPE65 and LRAT; compared with the model group, the lutein control group significantly upregulated the expression of RPE65 and LRAT, indicating that lutein can significantly restore the damage to cells caused by H2O2. Compared with the model group, the examples and some comparative examples (Comparative Example 1, Comparative Example 6, Comparative Example 9, Comparative Example 10) significantly upregulated the expression of RPE65 and LRAT, indicating that liver peptide samples can maintain the functional integrity of the visual cycle in RPE cells and are the most direct indicator for assessing the impact of peptides on the molecular basis of core visual function.

[0216] In conclusion, Figure 1 , Figure 3 , Figure 5 , Figure 6 , Figure 8 , Figure 9 , Figure 10 , Figure 11 The results all showed that Examples 1, 9, 10, 12, and 14 had significant effects on multiple aspects, including ADH activation, ALT / AST reduction, reduction of SREBP-1c and FAS gene expression, reduction of ROS accumulation, increase of CAT content, reduction of Bax factor, and increase of Bcl-2 expression. Furthermore, Figure 2The results showed that Example 1 was more effective than Comparative Examples 9, 10, 12 and 14 in reducing TG content; Figure 7 The results showed that Example 1 was more effective in promoting PPAR-α than Comparative Examples 9, 10, 12 and 14; Figure 12 The results showed that Example 1 was more effective than Comparative Examples 9, 10, 12 and 14 in downregulating Nrf2 and HO-1 expression. Figure 13 The results showed that Example 1 was more effective at upregulating PRE65 expression than Comparative Examples 9, 10, 12 and 14.

[0217] Based on the in vitro and cellular experimental results of all the above embodiments and comparative examples, the active peptide prepared by the method in Example 1 has the best overall effect in terms of relieving hangovers, protecting the liver, and improving visual fatigue.

[0218] 5. Peptide screening for the effect of hepatic peptides on the mRNA expression of related genes.

[0219] 5.1 Pretreatment Method

[0220] 5.1.1 Reductive Alkylation

[0221] 1) Accurately transfer 50 μL of sample into an EP tube using a pipette, and add 50 μL of water and mix well;

[0222] 2) Accurately pipette 1 μL of 1M DTT (dithiothreitol) solution into the sample to make the final DTT concentration 10 mmol / L, and reduce it in a 56℃ water bath for 1 h;

[0223] 3) Accurately pipette 2 μL of 1M IAM (2-iodoacetamide) solution into the sample to make the final IAM concentration 20 mmol / L, and react in the dark at room temperature for 40 min;

[0224] 4) Accurately pipette 1 μL of 1M DTT solution into the sample to make the final DTT concentration 10 mmol / L, in order to neutralize unreacted IAM.

[0225] 5.1.2 C18 Desalination (Stage-Tip)

[0226] 1) Desalted using C18 stage-tip and dried under vacuum at 45°C.

[0227] 5.2 Hands-on Practice

[0228] The processed samples were then analyzed using a liquid chromatography-mass spectrometry (LC-MS) system under the following conditions:

[0229] 5.2.1 Liquid Chromatography Conditions

[0230] 1) Pre-column: 150 μm id × 50 mm, packing: Reprosil-Pur 120 C18-AQ 3 μm; Analytical column: 150 μm id × 170 mm, packing: Reprosil-Pur 120 C18-AQ 1.9 μm;

[0231] 2) Mobile phase A: 0.1% FA (formic acid);

[0232] 3) Mobile phase B: 0.1% FA, 80% ACN (acetonitrile);

[0233] 4) Flow rate: 600 nL / min;

[0234] 5) Analysis time for each component: 66 min;

[0235] 6) Specific chromatographic conditions are shown in Table 5:

[0236] Table 5 Chromatographic conditions

[0237] ;

[0238] 5.2.2 Mass Spectrometry Conditions

[0239] The first-order mass spectrometry parameters are as follows: Resolution 70,000; AGC target 3e6; Maximum IT 100 ms; Scan range 100 to 1500 m / z.

[0240] The secondary mass spectrometry parameters are as follows: Resolution 17,500; AGC target 1e5; Maximum IT 50 ms; TopN 20; NCE / steppedNCE 28.

[0241] Raw data was obtained through mass spectrometry.

[0242] 5.3 Search Criteria

[0243] The raw mass spectrometry files were searched using software to retrieve the target protein database. The search parameters were as follows:

[0244] 1) Fixed modifications: Carbamidomethyl (C).

[0245] 2) Variable modifications: Oxidation (M), Acetyl (Peptide N-term).

[0246] 3) Enzyme: Non specific.

[0247] 4) Analyze the database: uniprotkb_Acipenser (genus)_2024_11_13; uniprotkb_Oreochromis (genus)_2024_12_05.

[0248] 5) Peptide Mass Tolerance: 20 ppm;

[0249] 6) Secondary mass spectrometry bias (Fragment Mass Tolerance): 0.02 Da.

[0250] The raw files acquired by mass spectrometry contained over 800 peptide sequences. First, they were screened by abundance to obtain 50 peptide sequences with an abundance greater than 100,000,000. Next, the peptides were screened by activity scoring, and six peptides with a score >0.5 were selected. These six peptides had not been reported in peptide databases. Furthermore, toxicity prediction was performed on these six peptides, and all results showed no toxicity. The peptide list obtained after database searching is shown below. Table 6 below shows the six peptide sequences obtained in Example 1, sorted by abundance after screening based on peptide sequence number, abundance, activity score, and toxicity.

[0251] Table 6 Information on the 6 peptides obtained after screening

[0252] ;

[0253] Molecular docking software was used to perform molecular docking of the above six peptide sequences with ADH1A, LART, and Nrf2 receptors. The three-dimensional structures of ADH1A (PDB code: 1hso), LART (PDB code: 4Q95), and Nrf2 (PDB code: 7k2f) were downloaded from the database. The specific docking binding energies of the six peptide sequences with different receptors are shown in Table 7.

[0254] Table 7. Binding energies of the six peptide sequences to the receptor protein

[0255] ;

[0256] Based on binding energy and peptide abundance, the DAIP and SPF sequences were selected from the peptides in Tables 6 and 7 as characteristic peptide sequences in liver peptides that have the effect of protecting the liver and improving eye fatigue.

[0257] Figure 14 , Figure 15 and Figure 16These are 2D diagrams showing the docking of DAIP with ADH1A, LART, and Nrf2 receptor protein molecules, respectively. Figure 17 , Figure 18 and Figure 19 These are 2D diagrams showing the docking of SPF with ADH1A, LART, and Nrf2 receptor protein molecules, respectively.

[0258] like Figure 14 As shown, hydrogen bonds, salt bridges, van der Waals forces, mutual attraction, and alkyl bonds exist between DAIP and the ADH1A acceptor. Additionally, Sulfur-X interactions are present, primarily found between divalent sulfur and N, O, or S atoms; as... Figure 15 As shown, conventional hydrogen bonds, C-H bonds, alkyl bonds, and van der Waals forces interact between DAIP and the LART receptor; as Figure 16 As shown, hydrogen bonds, van der Waals forces, salt bridges, attractive charges, π-alkyl groups, and π-anionic interactions exist between DAIP and the Nrf2 receptor. Figure 17 As shown, SPF and the ADH1A receptor are connected by hydrogen bonds, salt bridges, van der Waals forces, and alkyl bonds; for example... Figure 18 As shown, SPF and LART acceptors exhibit alkyl bonds, π-π stacking, and van der Waals interactions, as well as π-Sulfur-X interactions, primarily existing between π and N, O, or S atoms; Figure 19 As shown, hydrogen bonds, van der Waals forces, salt bridges, attractive charges, and π-alkyl interactions exist between SPF and the Nrf2 receptor.

[0259] In summary, the liver peptides of this invention possess the following characteristics: (1) they have in vitro ADH activation effects; (2) they reduce ROS accumulation and increase SOD and CAT levels, thereby enhancing antioxidant capacity; (3) they reduce ALT and AST levels to protect the integrity of hepatocyte membrane structure, reduce TG levels and the expression of SREBP-1c and FAS genes, promote the expression of PPAR-α genes, inhibit fat accumulation, promote fat oxidation and decomposition, regulate lipid metabolism, and reduce alcohol toxicity; (4) they upregulate the expression of RPE65 and LRAT genes, maintaining the functional integrity of RPE cell visual circulation. Based on the above functional characteristics, liver peptides can also be used to prepare functional products with obvious effects such as alcohol detoxification, liver protection, and improvement of visual fatigue.

[0260] The specific embodiments of this application have been described above, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for preparing liver peptides that protect the liver and improve eye fatigue, characterized in that, Includes the following steps: S1. Mix fresh liver with water and homogenize to obtain liver slurry; S2. Add acidic protease to the liver fluid for hydrolysis to obtain the first hydrolysate; S3. Add neutral protease and flavor protease to the first hydrolysate for hydrolysis, and then treat with enzyme inactivation to obtain the second hydrolysate. S4. Cool the second hydrolysate, add activated carbon and stir to react, then centrifuge and ultrafilter to obtain liver peptides; The liver is bovine liver, and the mass ratio of the liver to water is 1:(3~5). The amount of acidic protease added is 0.5% to 2% of the liver mass; the amount of neutral protease added is 0.5% to 1% of the liver mass; the amount of flavor protease added is 0.5% to 1% of the liver mass; and the amount of activated charcoal added is 4% to 6% of the liver mass. The acidic protease has an enzyme activity of 150,000 U / g, the neutral protease has an enzyme activity of 100,000 U / g, and the flavor protease has an enzyme activity of 100,000 U / g. The acidic protease is hydrolyzed under the following conditions: the pH of the system is adjusted to 2.5–3.0, and hydrolysis is carried out at 40°C–42°C for 2–3 hours; the neutral protease and the flavor protease are hydrolyzed under the following conditions: the pH of the system is adjusted to 7.0–7.5, and hydrolysis is carried out at 55°C–57°C for 1–2 hours.

2. The preparation method according to claim 1, characterized in that, The enzyme inactivation treatment conditions are: adjusting the pH to 6 and inactivating the enzyme at 85℃~90℃ for 15~20min; the ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 5000 Da.

3. The use of a liver peptide prepared by the method according to any one of claims 1-2 in the preparation of a health product for improving visual fatigue.

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