Anti-inflammatory and antioxidant hepatoprotective short peptide from chicken egg, preparation method and application thereof

Abstract

This invention discloses an egg-derived anti-inflammatory, antioxidant, and liver-protective short peptide, its preparation method, and its applications, relating to the field of bioactive peptide technology. The short peptide is a bioactive short peptide derived from egg white protein, with its amino acid sequence selected from at least one of the sequences shown in SEQ ID NO:1 to SEQ ID NO:9. It has the functions of inhibiting tumor necrosis factor-α and interleukin-1β, increasing superoxide dismutase activity, and reducing malondialdehyde content. The short peptide is prepared using a solid-phase polypeptide synthesis method and purified for confirmation. The bioactive short peptide with a defined sequence is isolated from hydrolyzed egg white protein, filling the technological gap in egg short peptides with both anti-inflammatory and antioxidant effects from natural sources. The short peptide can independently block the downstream inflammatory-oxidative effector cascade of alcohol damage and protect damaged hepatocytes, independent of the ethanol metabolism pathway. It can be used to prepare drugs, health foods, or functional foods for preventing or improving alcoholic liver damage.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide technology, specifically to egg-derived anti-inflammatory, antioxidant, and liver-protective short peptides, their preparation methods, and their applications. Background Technology

[0002] Chronic inflammation and oxidative stress are important contributing factors to the development of various diseases, such as cardiovascular disease, diabetes, neurodegenerative diseases, and liver injury, with alcoholic liver injury being a typical example. Excessive alcohol consumption is widespread globally, and alcoholic liver injury caused by it has become one of the most common chronic liver diseases, placing a heavy burden on patients' health and society. From a pathogenesis perspective, after excessive ethanol intake, ethanol is mainly metabolized in the liver via pathways such as alcohol dehydrogenase and cytochrome P450 2E1, inducing the production of a large number of reactive oxygen species during metabolism, causing oxidative stress. Oxidative stress leads to the accumulation of malondialdehyde, a lipid peroxidation product, and the depletion of the activity of endogenous antioxidant enzyme superoxide dismutase, damaging the integrity and fluidity of hepatocyte membranes. On the other hand, it can activate Kupffer cells in the liver, prompting them to release pro-inflammatory factors such as tumor necrosis factor-α and interleukin-1β, triggering and amplifying the inflammatory response, ultimately mediating inflammatory damage and death of hepatocytes. Oxidative stress and inflammatory response promote and amplify each other, constituting a key downstream link in the development of alcoholic liver injury. Once this injury cascade is initiated, its progression no longer depends linearly on the upstream ethanol concentration itself.

[0003] Currently, widely used synthetic anti-inflammatory drugs (such as ibuprofen and aspirin) and synthetic antioxidants (such as butylated hydroxytoluene and tert-butylhydroquinone) have certain anti-inflammatory or antioxidant effects, but long-term use may cause side effects such as gastrointestinal damage and liver toxicity, making it difficult to meet the needs of long-term dietary intervention and prevention of alcoholic liver injury. Therefore, finding safe, long-term edible natural active ingredients with both anti-inflammatory and antioxidant effects from food sources has become a research hotspot in this field. Eggs, as a high-quality protein source, contain a variety of bioactive peptides in their protein hydrolysates, which are naturally derived and relatively safe, thus attracting widespread attention. However, current research on the development of egg-derived peptides has long focused on applications such as angiotensin-converting enzyme inhibition, blood pressure reduction, and antibacterial properties. Research on specific short peptide components in egg protein that simultaneously possess high anti-inflammatory and antioxidant activities and can directly protect damaged hepatocytes is still insufficient, and the relevant sequences have not been clearly defined, making it difficult to meet the needs of clinical application and product development.

[0004] Furthermore, existing research on developing functional components for combating alcoholic liver injury using food proteins such as eggs largely focuses on whole protein hydrolysates. Whole protein hydrolysates contain a wide variety of bioactive peptides with complex compositions; their effective components are often unclear, their mechanisms of action are ambiguous, and the composition and activity can fluctuate between different batches, making it difficult to achieve stable and controllable quality and definite efficacy. Meanwhile, some schemes using egg white peptides to improve alcoholic liver injury primarily rely on activating alcohol-metabolizing enzymes to accelerate the clearance of ethanol and acetaldehyde. However, there is a lack of short, well-defined bioactive peptides that can directly block the downstream oxidative stress and inflammatory cascades initiated by ethanol damage. Therefore, identifying specific short peptide sequences in egg protein that possess both anti-inflammatory and antioxidant effects and can directly protect damaged hepatocytes is crucial for developing safe, effective, and quality-controlled products for combating alcoholic liver injury.

[0005] In their paper "Preparation, identification, and functional properties of ADH-activating and hepatoprotective potential egg whitepeptides," published in *European Food Research and Technology* (Vol. 252, No. 4, 2026, Article No. 158), Li Shugang et al. proposed an egg white peptide with potential hepatoprotective function. This method involved optimizing a stepwise enzymatic hydrolysis process to prepare the egg white peptide, and using computer-aided analysis to screen for four short peptides: LAPGW, WLF, WPPP, and WFLPR. Its protective effect against ethanol-damaged hepatocytes mainly relies on activating alcohol dehydrogenase to accelerate ethanol metabolism and clearance. However, the short peptides and their pathways of action provided by this method depend on the upstream step of accelerating ethanol metabolism. They fail to provide specific short peptides with well-defined sequences that can directly block the downstream inflammatory-oxidative effector cascade of ethanol damage, thus failing to protect already damaged hepatocytes. Therefore, it is difficult to directly address existing hepatocyte inflammation and oxidative damage without altering the ethanol metabolism rate.

[0006] Therefore, there is an urgent need in this field for an egg-derived short peptide with a well-defined sequence, high safety, significant anti-inflammatory and antioxidant effects, and the ability to independently block the downstream inflammatory-oxidative effector cascade of alcohol damage and protect hepatocytes, independent of the ethanol metabolism pathway. Summary of the Invention

[0007] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide an egg-derived anti-inflammatory, antioxidant, and liver-protective short peptide, its preparation method, and its application, thereby solving the core technical problem of existing egg-derived peptides lacking specific short peptides with well-defined sequences that can directly block the downstream inflammatory-oxidative effector cascade of alcohol damage and protect damaged hepatocytes.

[0008] To achieve the above objectives, a first aspect of the present invention provides an egg-derived anti-inflammatory, antioxidant, and liver-protective short peptide, wherein the short peptide is a bioactive short peptide derived from egg white protein, and its amino acid sequence is selected from at least one of the following groups: VLLPDEVSG (SEQ ID NO:1), YRGGLEP (SEQ ID NO:2), AFKDEDTQAMP (SEQ ID NO:3), ITWEK (SEQ ID NO:4), TVYPI (SEQ ID NO:5), RFDKLPG (SEQ ID NO:6), ITKPNDVYS (SEQ ID NO:7), FGPPGKKDPV (SEQ ID NO:8), and FDKLPGF (SEQ ID NO:9).

[0009] In a preferred embodiment, the amino acid sequence of the short peptide is selected from at least one of the sequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, which are hydrolyzed fragments of ovalbumin or ovotransferrin; more preferably, the short peptide is FGPPGKKDPV shown in SEQ ID NO:8 or VLLPDEVSG shown in SEQ ID NO:1. The short peptide may also include its N-terminal acetylated or C-terminal amidated modified forms, or pharmaceutically or food-acceptable salts thereof.

[0010] The second aspect of the present invention provides a method for preparing the above-mentioned egg-derived anti-inflammatory, antioxidant, and liver-protective short peptide. The method employs a solid-phase polypeptide synthesis method, using fluorene methoxycarbonyl-protected amino acids as raw materials, and sequentially coupling them from the C-terminus to the N-terminus, adding each amino acid residue in the amino acid sequence one by one. Each coupling cycle includes removing the fluorene methoxycarbonyl protection with piperidine solution, washing, activating the amino acids with a condensation reagent and coupling them, and end-capping. After coupling, the short peptide is cleaved from the resin with a cleavage reagent containing trifluoroacetic acid to remove the side chain protecting groups, obtaining a crude peptide. The crude peptide is then purified by reversed-phase high-performance liquid chromatography, freeze-dried, and the sequence and molecular weight are confirmed by liquid chromatography-mass spectrometry to obtain the target short peptide with a purity of not less than 95%. In a preferred embodiment, the deprotection is performed using a 20% (v / v) piperidine solution; the condensation reagent is a combination of O-benzotriazole-tetramethylurea hexafluorophosphate and 1-hydroxybenzotriazole, with a molar ratio of amino acid to condensation reagent of 1:1; the cleavage reagent is a mixture of trifluoroacetic acid, water, and triisopropylsilane in a volume ratio of 95:2.5:2.5. Besides solid-phase synthesis, the short peptide can also be obtained through targeted enzymatic hydrolysis and purification of egg white protein.

[0011] A third aspect of the present invention provides the application of the above-mentioned egg-derived anti-inflammatory, antioxidant, and hepatoprotective short peptides in the preparation of products for preventing or improving alcoholic liver injury, wherein the alcoholic liver injury is manifested by at least one of the following: ethanol-induced decrease in hepatocyte survival rate, increase in serum alanine aminotransferase and aspartate aminotransferase activity, decrease in liver tissue superoxide dismutase activity, increase in malondialdehyde content, and increase in tumor necrosis factor-α and interleukin-1β levels; the product is a drug, health food, or functional food, and its dosage form is oral liquid, tablet, capsule, granule, or powder; the short peptides can be used alone, or two or more of them can be used in combination, or in combination with silymarin.

[0012] The short peptides of this invention differ fundamentally in their mechanism of action from existing egg white peptide protocols that rely on accelerating ethanol metabolism and clearance. The short peptides of this invention do not depend on altering the metabolic rate of ethanol; instead, they directly act on the downstream oxidative stress and inflammatory effector cascade caused by ethanol damage. By increasing superoxide dismutase activity, reducing malondialdehyde content, and inhibiting the release of tumor necrosis factor-α and interleukin-1β, they synergistically protect already damaged hepatocytes. This solves the core technical problem of existing technologies lacking short peptides with well-defined sequences capable of directly blocking the downstream inflammatory-oxidative effector cascade caused by alcohol damage. The short peptides of this invention have a defined sequence, are naturally derived, and possess significant anti-inflammatory and antioxidant effects. Their quality is controllable, and batch-to-batch consistency is good, overcoming the shortcomings of existing whole protein crude hydrolysates, which have complex functions, unclear mechanisms, and large batch-to-batch variations.

[0013] In one specific embodiment of the present invention, the sequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:9 of the short peptide are hydrolyzed fragments of ovalbumin, and the sequence shown in SEQ ID NO:8 is a hydrolyzed fragment of ovotransferrin. These sequences are all derived from the main functional proteins in egg white and have a good safety profile. The present invention, by isolating, screening, and identifying specific short peptide sequences derived from food-derived proteins and their anti-inflammatory, antioxidant, and hepatoprotective functions from egg white hydrolyzed proteins, provides a sequence-defined active molecular basis for developing safe, effective, and quality-controllable anti-alcoholic liver injury products, possessing good clinical application value and market development potential.

[0014] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention isolates and identifies a series of bioactive short peptides with defined sequences from hydrolyzed egg white protein, solving the problem of significant side effects of existing synthetic drugs and filling the technological gap in egg short peptides with both anti-inflammatory and antioxidant effects from natural sources. These short peptides, after systematic screening using in vitro cell models and verification through animal experiments, can significantly alleviate ethanol-induced hepatocyte damage, improve the survival rate of damaged hepatocytes, and effectively reduce pathological changes such as hepatocyte swelling, fatty degeneration, and inflammatory cell infiltration caused by alcohol at the whole animal level, restoring serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities to normal. The short peptides described herein possess both potent antioxidant and significant anti-inflammatory effects: on the one hand, they dose-dependently increase the activity of superoxide dismutase in liver tissue, inhibit lipid peroxidation, and reduce malondialdehyde content, thus mitigating the attack of oxidative stress on hepatocytes from both antioxidant and membrane protection perspectives; on the other hand, they dose-dependently reduce the levels of tumor necrosis factor-α and interleukin-1β, blocking the amplifying effect of the inflammatory cascade driven by oxidative stress, and the high-dose group's reduction effect on tumor necrosis factor-α reaches a level comparable to that of the positive control silymarin. The short peptides of this invention are naturally derived, have high safety, and possess good clinical application value and market development potential. Attached Figure Description

[0015] Figure 1 This is a graph showing the effect of the short peptide of the present invention on the survival rate of L02 hepatocytes induced by ethanol damage.

[0016] Figure 2 This is a graph showing the effect of the short peptide of the present invention on serum alanine aminotransferase activity in mice with alcoholic liver injury.

[0017] Figure 3 This is a graph showing the effect of the short peptide of the present invention on serum aspartate aminotransferase activity in mice with alcoholic liver injury;

[0018] Figure 4 This is a figure showing the effect of the short peptide of the present invention on the activity of superoxide dismutase in the liver tissue of mice with alcoholic liver injury;

[0019] Figure 5 The figure shows the effect of the short peptide of the present invention on the malondialdehyde content in the liver tissue of mice with alcoholic liver injury.

[0020] Figure 6 This is a graph showing the effect of the short peptide of the present invention on the level of tumor necrosis factor-α in mice with alcoholic liver injury;

[0021] Figure 7 This is a graph showing the effect of the short peptide of the present invention on the level of interleukin-1β in mice with alcoholic liver injury;

[0022] Figure 8 This is a diagram showing the effect of the short peptide of the present invention on the pathological (HE staining) liver tissue of mice with alcoholic liver injury;

[0023] Figure 9 The graph shows the effect of different ethanol concentration gradients on the survival rate of L02 hepatocytes. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention. Unless otherwise specified, the reagents and materials used in the following embodiments are commercially available. The amino acid residues involved in the following embodiments are represented by single-letter codes, where A represents alanine, D represents aspartic acid, E represents glutamic acid, F represents phenylalanine, G represents glycine, I represents isoleucine, K represents lysine, L represents leucine, M represents methionine, N represents asparagine, P represents proline, Q represents glutamine, R represents arginine, S represents serine, T represents threonine, V represents valine, W represents tryptophan, and Y represents tyrosine.

[0025] Example 1

[0026] Solid-phase synthesis, purification, and structural confirmation of short peptides. This embodiment uses a solid-phase peptide synthesis method to prepare nine short peptides of the present invention. Using a suitable resin as the solid-phase support and fluorene methoxycarbonyl-protected amino acids as raw materials, the peptides were sequentially coupled according to the amino acid arrangement from the C-terminus to the N-terminus. Each coupling cycle was performed as follows: first, the amino-terminal fluorene methoxycarbonyl protecting group was removed with a 20% (v / v) solution of piperidine in N,N-dimethylformamide for 10 min; then, the peptides were thoroughly washed with N,N-dimethylformamide; subsequently, the next fluorene methoxycarbonyl-protected amino acid was added after activation with a mixture of O-benzotriazole-tetramethylurea hexafluorophosphate and 1-hydroxybenzotriazole at a molar ratio of 1:1, and coupling was carried out at room temperature for 40 min; after coupling, the peptides were capped with acetic anhydride, washed, and then entered the next cycle. After all amino acid residues were coupled, the short peptide was treated with a lysis reagent consisting of trifluoroacetic acid, water and triisopropylsilane in a volume ratio of 95:2.5:2.5 for 2 h to lyse the short peptide on the resin and simultaneously remove the side chain protecting groups. The resulting solution was precipitated with ice-cold ether and collected by centrifugation to obtain the crude peptide.

[0027] The crude peptides were dissolved and purified by reversed-phase high-performance liquid chromatography (RP-HPLC) using a C18 preparative column. Gradient elution was performed using an aqueous solution containing 0.1% trifluoroacetic acid (v / v) as mobile phase A and acetonitrile containing 0.1% trifluoroacetic acid (v / v) as mobile phase B. The target peak was detected and collected at 220 nm. The collected solution was freeze-dried to obtain the pure product. The purity was determined to be no less than 95% by analytical RP-HPLC. The molecular weight and sequence of the purified product were confirmed by liquid chromatography-mass spectrometry (LC-MS / MS). The primary mass spectrometry [M+H] was measured. + The peaks match the theoretical molecular weights of the short peptides, with deviations not exceeding 0.5 Da. The molecular formulas and theoretical values ​​of the monoisotopic molecular weights of the nine short peptides are shown in Table 1.

[0028] Table 1. Molecular formulas and molecular weights of the nine short peptides of this invention

[0029] serial number amino acid sequence Molecular formula Single isotope molecular weight / Da SEQ ID NO:1 VLLPDEVSG <![CDATA[C 41 H 69 N9O 15 ]]> 927.491 SEQ ID NO:2 YRGGLEP <![CDATA[C 35 H 54 N 10 O 11 ]]> 790.397 SEQ ID NO:3 AFKDEDTQAMP <![CDATA[C 53 H 81 N 13 O 20 S]]> 1251.544 SEQ ID NO:4 ITWEK <![CDATA[C 32 H 49 N7O9]]> 675.359 SEQ ID NO:5 TVYPI <![CDATA[C 29 H 45 N5O8]]> 591.327 SEQ ID NO:6 RFDKLPG <![CDATA[C 38 H 61 N 11 O 10 ]]> 831.460 SEQ ID NO:7 ITKPNDVYS <![CDATA[C 46 H 73 N 11 O 16 ]]> 1035.524 SEQ ID NO:8 FGPPGKKDPV <![CDATA[C 49 H 76 N 12 O 13 ]]> 1040.565 SEQ ID NO:9 FDKLPGF <![CDATA[C 41 H 58 N8O 10 ]]> 822.428

[0030] Example 2

[0031] This invention relates to the effect of a short peptide on the survival rate of L02 hepatocytes induced by ethanol. Normal L02 human hepatocytes in the logarithmic growth phase were seeded and cultured in adherent culture plates, and randomly divided into a normal control group, an ethanol model group, and various short peptide intervention groups. The ethanol model group was treated with 5% ethanol (v / v) to construct a hepatocyte injury model. The short peptide intervention groups were administered different concentrations of the short peptide of this invention concurrently with ethanol treatment, with concentration gradients of 75 μmol / L, 150 μmol / L, 300 μmol / L, and 600 μmol / L. The normal control group received neither ethanol nor short peptide treatment. After treatment, cell viability was measured using a cell viability assay. The results are shown below. Figure 1 As shown. Figure 1 As shown, compared with the normal control group, the cell survival rate in the ethanol model group was significantly reduced, confirming that ethanol induced hepatocyte damage and death. After intervention with the short peptide of this invention, the survival rate of damaged hepatocytes showed a significant upward trend, and gradually increased with increasing intervention concentration within the investigated concentration range, with statistically significant differences. Under the condition of 3.5% ethanol treatment, the short peptide of this invention could also significantly reverse the ethanol-induced decrease in cell survival rate. Representative results are listed in Table 2.

[0032] Table 2. Effect of the short peptide of the present invention on the survival rate of L02 cells damaged by ethanol (5% v / v) (with the normal control group as 100%)

[0033] Grouping Cell viability / % Grouping Cell viability / % normal control group 100.0 ITWEK (600 μmol / L) 71.6 Ethanol model group 30.2 TVYPI (600 μmol / L) 57.8 VLLPDEVSG (600 μmol / L) 78.4 RFDKLPG (600 μmol / L) 52.3 YRGGLEP (600 μmol / L) 55.1 ITKPNDVYS (600 μmol / L) 69.7 AFKDEDTQAMP (600 μmol / L) 61.8 FGPPGKKDPV (600 μmol / L) 50.4 FDKLPGF (600 μmol / L) 59.6 FGPPGKKDPV (300 μmol / L) 41.2

[0034] Example 3

[0035] Establishment of a mouse model of alcoholic liver injury and the effect of short peptides on serum transaminases. Healthy C57BL / 6 mice were randomly divided into a normal control group, an ethanol model group, a positive control group, and low- and high-dose short peptide groups after acclimatization. Before model establishment, the cell model was investigated using ethanol concentration gradients of 3%, 3.5%, 4%, 4.5%, and 5% (v / v) to determine suitable modeling conditions. The results are as follows: Figure 9 As shown, cell viability decreased with increasing ethanol concentration, thus determining the ethanol exposure intensity for subsequent whole animal experiments. In the whole animal experiments, alcoholic liver injury was induced in the ethanol model group and all drug-treated groups by gavage with ethanol; the positive control group was given silymarin 100 mg / kg; the low-dose and high-dose short peptide groups were given the short peptide of this invention at 100 mg / kg and 200 mg / kg, respectively; the normal control group was given an equal volume of solvent. After drug administration, blood was collected and serum was separated, and the activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured. The results are shown below. Figure 2 and Figure 3 As shown. Figure 2 and Figure 3As shown, compared with the normal control group, the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in the ethanol model group were significantly increased; after intervention with the short peptide of this invention, the activities of the above-mentioned liver injury markers returned to normal, indicating that hepatocytes were protected. Representative data are listed in Table 3.

[0036] Table 3. Effects of the short peptides of this invention on serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice with alcoholic liver injury.

[0037] Grouping Alanine aminotransferase (ALT) / (mmol / ml) Aspartate aminotransferase (AST) / (μmol / L) normal control group 0.21 5.5 Ethanol model group 0.34 6.0 Silymarin group 0.25 5.4 VLLPDEVSG high-dose group 0.26 5.4 AFKDEDTQAMP high-dose group 0.24 5.3 FGPPGKKDPV high-dose group 0.25 5.3 ITKPNDVYS high-dose group 0.26 5.3

[0038] Example 4

[0039] This invention relates to the antioxidant activity of short peptides and their effects on superoxide dismutase (SOD) and malondialdehyde (MDA) levels in liver tissue. Liver tissue homogenates were prepared from mice in each group of Example 3, and the SOD activity and MDA content in the liver tissue were measured. The results are as follows: Figure 4 and Figure 5 As shown. Figure 4 As shown, compared with the normal control group, the activity of superoxide dismutase in liver tissue of the ethanol model group was significantly decreased, suggesting that ethanol exposure leads to impaired endogenous antioxidant capacity of the liver; while after intervention with the short peptide of this invention, the activity of superoxide dismutase in liver tissue increased significantly in a dose-dependent manner. Figure 5 As shown, the malondialdehyde (MDA) content in the liver tissue of the ethanol model group was significantly increased, while the short peptide intervention of this invention could dose-dependently reduce the MDA content. These results confirm that the short peptide of this invention can effectively activate the liver's endogenous antioxidant enzyme system, clearing harmful byproducts such as superoxide anions generated by oxidative stress; simultaneously, by increasing superoxide dismutase activity, it inhibits the occurrence and progression of lipid peroxidation damage to hepatocyte membranes, thereby maintaining the integrity and fluidity of the hepatocyte membrane structure, exerting an anti-alcoholic liver injury effect from both antioxidant and membrane protection perspectives. Representative data are listed in Table 4.

[0040] Table 4. Effects of the short peptides of the present invention on superoxide dismutase activity and malondialdehyde content in mouse liver tissue.

[0041] Grouping Superoxide dismutase (U / mgprot) Malondialdehyde (nmol / mgprot) normal control group 135.2 0.28 Ethanol model group 65.4 0.85 Silymarin group 130.1 0.48 VLLPDEVSG low-dose group 109.6 0.56 VLLPDEVSG high-dose group 128.7 0.41 FGPPGKKDPV low-dose group 112.3 0.53 FGPPGKKDPV high-dose group 138.5 0.40

[0042] Example 5

[0043] This invention relates to the anti-inflammatory effects of short peptides and their influence on tumor necrosis factor-α (TNF-α) and interleukin-1β. Serum or hepatocyte supernatant from mice in each group of Example 3 was used to determine the levels of pro-inflammatory factors TNF-α and interleukin-1β using enzyme-linked immunosorbent assay (ELISA). The results are as follows: Figure 6 and Figure 7 As shown. Figure 6 and Figure 7As shown, compared with the normal control group, the levels of tumor necrosis factor-α and interleukin-1β in the ethanol model group were significantly increased, suggesting that ethanol exposure had induced a strong inflammatory response and mediated the progression of liver injury. After intervention with the short peptide of this invention, the level of tumor necrosis factor-α decreased significantly in a dose-dependent manner, and the level of tumor necrosis factor-α in the high-dose group could be significantly reduced to near the intervention effect of the positive control silymarin. The level of interleukin-1β also decreased significantly after intervention with the short peptide of this invention. These results confirm that the short peptide of this invention can effectively inhibit the release of ethanol-induced pro-inflammatory factors, block the amplification effect of the inflammatory cascade driven by oxidative stress, and reduce the secondary damage of the inflammatory response to hepatocytes, thereby blocking the progression of alcoholic liver injury from an anti-inflammatory perspective. Representative data are listed in Table 5.

[0044] Table 5. Effects of the short peptides of the present invention on the levels of tumor necrosis factor-α and interleukin-1β in mice.

[0045] Grouping Tumor necrosis factor-α (ng / L) Interleukin-1β / (ng / L) normal control group 530 20 Ethanol model group 805 82 Silymarin group 572 41 FGPPGKKDPV low-dose group 678 53 FGPPGKKDPV high-dose group 610 38 VLLPDEVSG low-dose group 665 44 VLLPDEVSG high-dose group 598 35

[0046] Example 6

[0047] This invention relates to the effects of short peptides on the pathological characteristics of liver tissue in mice with alcoholic liver injury. Liver tissues from each group of mice in Example 3 were fixed, embedded, sectioned, and stained with hematoxylin and eosin. The morphology of the liver tissues was then observed under a light microscope. The results are as follows: Figure 8 As shown. Figure 8 As shown, in the normal control group, the liver lobule structure was clear and the hepatocytes were neatly arranged; in the ethanol model group, obvious hepatocyte swelling, fatty degeneration, and inflammatory cell infiltration were observed, typical pathological changes; after intervention with the short peptide of this invention, the above-mentioned pathological changes caused by alcohol were significantly improved, and the liver structure tended to be normal. This result further confirms the protective effect of the short peptide of this invention on alcoholic liver injury at the histomorphological level, and is corroborated by changes in serum transaminase, antioxidant indicators, and anti-inflammatory factors.

[0048] Comparative Example 1

[0049] Comparison of crude egg white hydrolysate with the purified short peptide of this invention. To illustrate the technical contribution of the short peptide with a well-defined sequence provided by this invention compared to existing crude whole protein hydrolysates, this comparative example uses unpurified crude egg white protein hydrolysate as a control. The effect on the survival rate of ethanol-damaged L02 hepatocytes was investigated using the same cell model and dosage as in Example 2. The results show that, at the same mass concentration as the short peptide of this invention, the crude egg white hydrolysate's effect on improving the survival rate of damaged hepatocytes is significantly weaker than that of the purified short peptide of this invention, and the effect fluctuates significantly between batches. Regarding anti-inflammatory and antioxidant indicators, the crude hydrolysate's inhibitory effect on tumor necrosis factor-α and its restorative effect on superoxide dismutase activity are also weaker than those of the short peptide of this invention. This indicates that the anti-inflammatory, antioxidant, and hepatoprotective activities described in this invention are highly concentrated in specific amino acid sequences, rather than diffused throughout the whole protein hydrolysate. Removing these specific sequences would prevent the achievement of the core technical problem addressed by this invention, thus verifying that these specific sequences are essential technical features for achieving the technical effects of this invention.

[0050] Comprehensive analysis of the test data. Based on the results of Examples 2 to 6 and Comparative Example 1, the protective effect of the short peptide of the present invention against alcoholic liver injury has the following characteristics: First, the effect is dose-dependent: at both the cellular and whole animal levels, the short peptide of the present invention enhances cell survival rate and superoxide dismutase activity, and reduces malondialdehyde, tumor necrosis factor-α, and interleukin-1β, all with increasing dosage. Second, high-dose efficacy reaches the level of benchmark drugs: the high-dose group of the short peptide of the present invention reduces tumor necrosis factor-α levels to a level comparable to the positive control drug silymarin, showing good drug development potential. Third, synergistic convergence through multiple mechanisms: The short peptide of this invention does not rely on accelerating ethanol metabolism and clearance, but rather exerts its effects simultaneously at three levels: anti-inflammatory (inhibiting tumor necrosis factor-α and interleukin-1β), antioxidant (enhancing superoxide dismutase and reducing malondialdehyde), and directly maintaining hepatocyte survival. This allows it to act independently of the ethanol metabolism pathway, directly blocking the downstream inflammatory-oxidative effector cascade of alcohol damage and protecting already damaged hepatocytes. Fourth, sequence specificity: Comparative Example 1 shows that the effects of the technology are highly dependent on the specific amino acid sequence defined in this invention. In summary, the short peptide of this invention is naturally derived, has a well-defined sequence, and is highly safe. It possesses significant anti-inflammatory and antioxidant effects and can be used to prepare drugs, health foods, or functional foods for the prevention or improvement of alcoholic liver injury, demonstrating good clinical application value and market development potential.

[0051] In Example 1, after each amino acid coupling cycle, a small amount of resin was used to monitor the completeness of coupling using the ninhydrin colorimetric method: a negative colorimetric result indicated complete coupling, allowing the next cycle to proceed; a positive colorimetric result was used to repeat the coupling operation until a negative colorimetric result was obtained, ensuring the correct assembly of the target short peptide sequence. After all amino acid residues were coupled and cleaved, the resulting crude peptide solution was repeatedly washed and precipitated with pre-cooled ether to remove soluble impurities. The precipitate was collected by centrifugation and then freeze-dried under vacuum to obtain dry crude peptide powder. Purity was determined using analytical reversed-phase high-performance liquid chromatography (RP-HPLC) with a C18 analytical column. A linear gradient elution was performed using an aqueous solution containing 0.1% trifluoroacetic acid and acetonitrile as the mobile phase. The absorbance at 220 nm was recorded, and the purity was characterized by the percentage of the target peak area to the total peak area. Through the above process, all nine short peptides of this invention were obtained as pure products with a purity of not less than 95%. The first-order mass spectrometry [M+H] obtained by liquid chromatography-mass spectrometry (LC-MS) was measured. + The peaks matched the theoretical molecular weights of the short peptides listed in Table 1, thus confirming the amino acid sequence and molecular weight of the synthesized short peptides.

[0052] In Example 2, L02 normal human hepatocytes were cultured in a medium containing 10% fetal bovine serum at 37°C in a constant temperature incubator containing 5% carbon dioxide. After the cells reached a suitable confluence, they were digested with trypsin, counted, and seeded into culture plates at an appropriate seeding density. After cell adhesion, the cells were treated according to the aforementioned groups: the ethanol model group and each short peptide intervention group were treated with 5% or 3.5% ethanol to construct a damage model. The short peptide intervention groups were treated with 75 μmol / L, 150 μmol / L, 300 μmol / L, or 600 μmol / L of the short peptide of this invention, respectively, while the normal control group received no ethanol or short peptide. All groups were incubated for an appropriate time. After incubation, the culture supernatant was discarded, cell viability assay reagent was added to each well, and incubation continued. The absorbance of each well was then measured at the corresponding wavelength using a microplate reader, and the cell viability of each group was calculated with the absorbance of the normal control group as 100%. Multiple parallel replicates were set up for each group, and the experiment was independently repeated multiple times. The data obtained were expressed as averages. The differences between groups were statistically tested using analysis of variance, and the differences were considered statistically significant.

[0053] like Figure 1As shown in Table 2, under the modeling condition of 5% ethanol (v / v), the cell survival rate of the ethanol model group was significantly reduced to approximately 30% compared to the normal control group, confirming that ethanol induced hepatocyte damage and death. The short peptides of this invention, within a concentration range of 75 μmol / L to 600 μmol / L, all improved the survival rate of damaged cells to varying degrees, exhibiting a clear concentration-dependent relationship; that is, cell survival rate gradually increased with increasing intervention concentration. Among them, VLLPDEVSG (SEQ ID NO:1) and ITWEK (SEQ ID NO:4) showed particularly significant improvements in cell survival rate at 600 μmol / L, while ITKPNDVYS (SEQ ID NO:7) also demonstrated good cell protective activity. Under the relatively mild modeling condition of 3.5% ethanol (v / v), the short peptides such as FGPPGKKDPV (SEQ ID NO:8) also significantly reversed the ethanol-induced decrease in cell survival rate. These results indicate that the series of short peptides of this invention can significantly alleviate ethanol-induced hepatocyte damage, effectively improve the survival rate of damaged hepatocytes, and possess clear anti-alcoholic hepatocyte damage activity.

[0054] In Example 3, healthy C57BL / 6 mice were randomly divided into groups according to body weight after acclimatization, with several mice in each group. During the modeling and drug administration period, each drug administration group was administered the short peptide of this invention or the positive control drug silymarin via oral gavage daily at a set dose. The ethanol model group and the normal control group were administered ethanol or an equal volume of solvent via gavage, respectively. This was continued for an appropriate period to establish a stable alcoholic liver injury model. After the drug administration period, the mice were fasted for a certain period of time, and blood was collected using an appropriate method. After the blood was allowed to stand, the serum was separated by centrifugation, aliquoted, and stored at low temperature for later testing. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured using the corresponding kits according to their instructions. Figure 9 In the ethanol concentration gradient experiment shown, L02 cells were treated with ethanol at volume fractions of 3%, 3.5%, 4%, 4.5%, and 5%, respectively. The results showed that cell survival rate decreased with increasing ethanol concentration. Based on this, the appropriate modeling concentration for in vitro cell models was determined, and a basis was provided for setting the ethanol exposure intensity in whole animal experiments.

[0055] In Example 4, the preparation process of liver tissue homogenate was as follows: An appropriate amount of fresh liver tissue was taken, and pre-cooled physiological saline was added at a certain mass-to-volume ratio. The mixture was thoroughly homogenized under ice bath conditions, centrifuged, and the supernatant was collected for analysis. Simultaneously, the total protein content in the supernatant was measured to normalize the various indicators. The determination of superoxide dismutase activity was based on its ability to scavenge superoxide anion free radicals, while the determination of malondialdehyde (MDA) content was based on its reaction with thiobarbituric acid to generate colored products. From a mechanistic perspective, reactive oxygen species (ROS) generated during ethanol metabolism in the liver attack polyunsaturated fatty acids on cell membranes, triggering a chain reaction of lipid peroxidation, generating lipid peroxidation end products represented by MDA, thus damaging the integrity and fluidity of cell membranes. Simultaneously, excessive accumulation of ROS consumes and inhibits endogenous antioxidant enzymes such as superoxide dismutase, weakening the body's antioxidant defense capabilities. The short peptide of this invention enhances superoxide dismutase activity in a dose-dependent manner, promptly scavenging harmful free radicals such as superoxide anions and blocking the initiation and propagation of lipid peroxidation chain reactions, thereby reducing the damage of oxidative stress to hepatocytes at both the antioxidant and hepatocyte membrane protection levels.

[0056] In Example 5, the levels of tumor necrosis factor-α and interleukin-1β were determined using enzyme-linked immunosorbent assay (ELISA). Following the instructions of the respective kits, the absorbance was measured using an ELISA reader, and the concentrations were converted to the corresponding values ​​based on the standard curve. During the development of alcoholic liver injury, excessive ethanol and its metabolites, along with oxidative stress, can activate Kupffer cells in the liver, prompting the release of pro-inflammatory factors such as tumor necrosis factor-α and interleukin-1β. These pro-inflammatory factors can further amplify the inflammatory response and exacerbate hepatocyte damage, mutually reinforcing each other with oxidative stress, forming a vicious cycle. The short peptide of this invention inhibits the release of these pro-inflammatory factors, blocking the amplification effect of this inflammatory cascade and reducing secondary damage to hepatocytes caused by the inflammatory response. Particularly noteworthy is that the high-dose group of the short peptide of this invention showed an inhibitory effect on tumor necrosis factor-α comparable to that of the positive control silymarin, indicating that the short peptide of this invention has good anti-inflammatory and hepatoprotective effects and considerable development potential.

[0057] In Example 6, the preparation of liver tissue pathological sections included the following steps: sampling, fixation with fixative, gradient dehydration, paraffin embedding, sectioning, and hematoxylin-eosin staining. After preparation, the liver tissue morphology was observed and images were acquired under an optical microscope. Pathologically, the hepatocytes in the ethanol model group showed typical pathological changes such as ballooning enlargement, fatty degeneration reflected by lipid droplets of varying sizes in the cytoplasm, and scattered inflammatory cell infiltration within the liver lobules. After intervention with the short peptide of this invention, the degree of the above pathological changes was significantly reduced, and the morphology of hepatocytes and the structure of liver lobules tended to be normal. These histomorphological results, along with the recovery of serum transaminase activity, the improvement of liver tissue antioxidant indicators, and the decrease in pro-inflammatory factor levels, corroborate each other on multiple levels, jointly confirming the protective effect of the short peptide of this invention on alcoholic liver injury.

[0058] Example 7

[0059] Product formulations containing the short peptide of this invention. The short peptide of this invention can be formulated into various dosage forms suitable for oral administration using conventional methods in the art. When formulating an oral liquid, the short peptide of this invention is dissolved in water for injection or purified water at a suitable concentration, and appropriate amounts of flavoring agents and preservatives are added. After stirring and dissolving, the pH is adjusted to a suitable range, and the solution is filtered, filled, and sterilized. When formulating tablets, the short peptide of this invention is mixed evenly with appropriate amounts of fillers, disintegrants, and lubricants, and then compressed into tablets, with coating if necessary. When formulating capsules, the short peptide of this invention is mixed with suitable excipients and then filled into capsules. When formulating granules or powders, the short peptide of this invention is mixed with suitable excipients and then granulated or packaged. The above dosage forms can be used alone or in combination with silymarin for the prevention or improvement of alcoholic liver damage.

[0060] In a specific example of an oral liquid formulation, the short peptide FGPPGKKDPV shown in SEQ ID NO:8 of this invention is dissolved in an appropriate amount of purified water, and an appropriate amount of sweetener and preservative is added. After stirring and dissolving, the volume is adjusted to approximately 6.8, and the solution is filtered through a 0.22 μm filter membrane for sterilization and then dispensed into clean containers to obtain an oral liquid containing the short peptide of this invention. In a specific example of a tablet formulation, the short peptide VLLPDEVSG shown in SEQ ID NO:1 of this invention is mixed evenly with microcrystalline cellulose, croscarmellose sodium, and magnesium stearate in an appropriate ratio and then compressed into tablets to obtain tablets containing the short peptide of this invention. In a specific example of a capsule formulation, the short peptide of this invention is mixed evenly with lactose, starch, and other excipients and then filled into gelatin capsules to obtain capsules containing the short peptide of this invention. All of the above formulations can be used to prevent or improve alcoholic liver damage.

[0061] Comparative Example 2

[0062] Controls of short peptides with sequences different from those of this invention. To further illustrate the necessity of the specific amino acid sequence defined in this invention for achieving the technical effects, several short peptides with sequences different from those of this invention were selected as controls in this comparative example. Their effects on the survival rate of hepatocytes damaged by ethanol and on the level of tumor necrosis factor-α were examined using the same methods as in Examples 2 and 5. The results showed that, at the same drug concentration, the control short peptides were significantly weaker than the short peptides of this invention in both increasing the survival rate of damaged hepatocytes and inhibiting tumor necrosis factor-α. This indicates that the anti-inflammatory, antioxidant, and hepatoprotective activities of the short peptides of this invention do not derive from the general properties of protein-derived peptides, but are closely related to the specific amino acid sequence defined in this invention; in other words, replacing the specific sequence defined in this invention with other sequences would not achieve the technical effects desired by this invention, thus further verifying that the specific amino acid sequence is a necessary technical feature for achieving the technical effects of this invention.

[0063] Example 8

[0064] Evaluation of the in vitro antioxidant activity of the short peptides of this invention. To characterize the direct antioxidant capacity of the short peptides of this invention at the molecular level, the free radical scavenging ability of each short peptide was determined according to conventional methods in the art. The short peptides of this invention were prepared into a series of solutions of different concentrations, mixed with corresponding free radical reaction systems, and reacted under suitable conditions in the dark. The changes in absorbance were measured, and their antioxidant activity was characterized by the free radical scavenging rate. The results showed that each short peptide of this invention possessed a certain free radical scavenging ability, and the scavenging rate increased with increasing short peptide concentration. Among them, short peptides containing hydrogen-donating amino acid residues such as tyrosine and tryptophan in their sequences exhibited relatively stronger free radical scavenging activity. These in vitro antioxidant results are consistent with the results in Example 4 showing that the short peptides of this invention increased superoxide dismutase activity and reduced malondialdehyde content in vivo, further supporting the antioxidant effect of the short peptides of this invention at the molecular level.

[0065] Furthermore, the nine short peptides described in this invention are derived from different parent proteins and different sequence segments in egg white protein. Their activity levels in anti-inflammatory, antioxidant, and cell-protective indicators vary to some extent, demonstrating a clear correspondence between sequence and function. In practical applications, one of the aforementioned short peptides can be used alone, or two or more can be used in combination to exert a synergistic effect, depending on the specific needs for preventing or improving alcoholic liver injury. Alternatively, the sequences of the short peptides used can be selected and combined according to different dosage forms and routes of administration. By clarifying the aforementioned specific sequences and their anti-inflammatory, antioxidant, and hepatoprotective functions, this invention provides a sequence-defined active molecular basis for developing safe and effective products for treating alcoholic liver injury.

[0066] When using the short peptides of this invention to prevent or improve alcoholic liver injury, the dosage can be adjusted according to the subject's weight, the severity of liver injury, and the dosage form and route of administration. The aforementioned examples investigated the effects at the cellular level using a concentration gradient from 75 μmol / L to 600 μmol / L, and at the whole animal level using both low and high doses. The results showed that the short peptides of this invention have a clear dose-dependent protective effect against alcoholic liver injury within the investigated dose range, and the protective effect increases with increasing dose. Therefore, under the premise of ensuring safety, the protective effect can be enhanced by appropriately increasing the dosage, or the overall efficacy can be optimized by combining different short peptides or by combining them with silymarin. The short peptides of this invention are derived from egg white protein, which is a hydrolyzed fragment of dietary protein. Compared with synthetic anti-inflammatory drugs and synthetic antioxidants, they have the characteristics of being naturally derived and having higher safety, making them suitable for development into health foods or functional foods that can be consumed for a longer period, and also suitable for further development into drugs.

[0067] Based on the combined in vitro and in vivo experimental results of this invention, it can be concluded that the protective effect of the short peptide of this invention against alcoholic liver injury is based on the mechanism of directly intervening in the downstream effector cascade of ethanol injury. Specifically, oxidative stress and inflammatory response induced by ethanol and its metabolic processes are key downstream links mediating hepatocyte damage, and once initiated, this link is relatively independent of changes in upstream ethanol concentration. The short peptide of this invention does not rely on altering the metabolic rate of ethanol, but directly acts on this downstream effector cascade: on the one hand, it interrupts oxidative stress by increasing superoxide dismutase activity and reducing malondialdehyde content; on the other hand, it blocks the amplification of the inflammatory cascade by inhibiting the release of tumor necrosis factor-α and interleukin-1β. The two work synergistically to protect already damaged hepatocytes, improve their survival rate, and improve the pathological damage of liver tissue. It is this mechanism of action that distinguishes the short peptide of this invention from existing egg white peptide schemes that rely on accelerating ethanol metabolism and clearance, and solves the core technical problem of the lack of short peptides with well-defined sequences that can directly block the downstream inflammatory-oxidative effector cascade of alcohol injury. The short peptides of this invention have well-defined sequences and can be stably obtained through solid-phase synthesis or targeted enzymatic hydrolysis of egg white protein, resulting in controllable quality and good batch-to-batch consistency. This overcomes the shortcomings of existing crude hydrolysates of whole protein, such as complex functions, unclear mechanisms, and large batch-to-batch fluctuations, and has good clinical application value and market development potential.

[0068] In this invention, silymarin was used as a positive control in whole animal experiments. Silymarin is a natural ingredient widely used for hepatoprotection, and its hepatoprotective effect is mainly achieved through antioxidant and anti-inflammatory pathways, rather than relying on accelerating ethanol metabolism. Comparing the short peptide of this invention with silymarin allows for an objective evaluation of the protective strength of the short peptide under accepted hepatoprotective standards. Experimental results show that the short peptide of this invention exhibits a similar trend of action to silymarin in terms of antioxidant (increasing superoxide dismutase activity and reducing malondialdehyde content) and anti-inflammatory (reducing tumor necrosis factor-α and interleukin-1β levels). Moreover, the high-dose group achieved a tumor necrosis factor-α reduction effect comparable to that of silymarin, indicating that the short peptide of this invention has good hepatoprotective activity.

[0069] Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are important biomarkers reflecting hepatocellular damage. Under normal physiological conditions, these transaminases are mainly found within hepatocellular cells. When hepatocellular cells are damaged and their membrane integrity is disrupted, intracellular transaminases are released into the bloodstream, leading to elevated serum transaminase activity. Therefore, the degree of elevation in serum transaminase activity can, to some extent, reflect the severity of hepatocellular damage. The results of Example 3 showed that the serum ALT and AST activities in the ethanol model group were significantly elevated, indicating that ethanol caused significant hepatocellular damage. After intervention with the short peptide of this invention, the transaminase activities significantly decreased and tended to normalize, thus confirming the protective effect of the short peptide of this invention on hepatocellular cells at the serum biochemical level. This is consistent with the improvement in cell viability and liver tissue pathology.

[0070] To investigate the protective effect of the short peptide of the present invention under different injury intensities, in Example 2, hepatocyte models with different degrees of injury were constructed using 5% and 3.5% (v / v) ethanol, respectively. Under the stronger injury condition of 5% (v / v) ethanol, the cell survival rate of the ethanol model group decreased more significantly, and the short peptide of the present invention could still significantly improve the survival rate of damaged cells at a higher concentration. Under the relatively mild injury condition of 3.5% (v / v) ethanol, the short peptide of the present invention also showed a significant effect on restoring cell survival rate. The above results indicate that the short peptide of the present invention can exert a protective effect under different injury intensities, suggesting that its activity against alcoholic hepatocyte injury has good robustness.

[0071] The short peptides of this invention can be used alone, in combination of two or more, or in combination with other hepatoprotective components such as silymarin to exert synergistic or complementary protective effects. In one example of combined use, VLLPDEVSG shown in SEQ ID NO:1 and FGPPGKKDPV shown in SEQ ID NO:8 were combined in an appropriate ratio, and their effect on the survival rate of hepatocytes damaged by ethanol was investigated according to the method of Example 2. The results showed that the combined use of the two peptides improved the cell survival rate by no less than that of either short peptide alone, indicating that the short peptides of this invention are suitable for development into anti-alcoholic liver injury products in combination.

[0072] Besides solid-phase peptide synthesis, the short peptides of this invention can also be obtained through targeted enzymatic hydrolysis and purification of egg white protein. Specifically, a suitable protease can be used to hydrolyze egg white protein, and the hydrolysis conditions can be controlled to promote the release of the target sequence. The hydrolysate is then fractionated and purified by ultrafiltration, gel filtration chromatography, reversed-phase high-performance liquid chromatography, etc., and the target short peptide is identified and confirmed by liquid chromatography-mass spectrometry, thereby obtaining the short peptides of this invention with a well-defined sequence. Regardless of whether solid-phase synthesis or targeted enzymatic hydrolysis and purification is used, the obtained short peptides of this invention have a defined amino acid sequence, controllable quality, and can stably reproduce their anti-inflammatory, antioxidant, and hepatoprotective activities.

[0073] The short peptides of this invention have well-defined sequences and structures, facilitating rigorous quality control. During preparation, purity can be determined by reversed-phase high-performance liquid chromatography (RP-HPLC), and molecular weight and sequence can be confirmed by liquid chromatography-mass spectrometry (LC-MS) to ensure consistency between different batches. Compared to existing whole-protein crude hydrolysates, which have complex compositions, unclear active ingredients, and significant batch-to-batch variations, the sequence-defined short peptides provided by this invention offer significant advantages in quality control and batch consistency, making them more suitable for industrial production and standardized application.

[0074] The short peptides described in this invention are derived from the main proteins in egg white. Egg white protein contains a variety of functional proteins, which can release various bioactive peptides after hydrolysis by proteases. This invention isolates, screens, and identifies the above-mentioned nine specific short peptide sequences with anti-inflammatory, antioxidant, and hepatoprotective activities from hydrolyzed egg white protein, each corresponding to different sequence segments in egg white protein. Since these short peptides are derived from dietary egg protein, they have a good safety profile and are suitable for development into functional foods or health foods that can be consumed for extended periods. They can also be further developed into corresponding drugs for the prevention or improvement of alcoholic liver damage.

[0075] The embodiments described above are merely examples illustrating several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. Egg-derived anti-inflammatory, antioxidant, and liver-protective short peptides, characterized in that... The short peptide is a bioactive short peptide derived from egg white protein, and its amino acid sequence is selected from at least one of the following groups: VLLPDEVSG, as shown in SEQ ID NO:1; YRGGLEP, as shown in SEQ ID NO:2; AFKDEDTQAMP, as shown in SEQ ID NO:3; ITWEK, as shown in SEQ ID NO:4; TVYPI, as shown in SEQ ID NO:5; RFDKLPG, as shown in SEQ ID NO:6; ITKPNDVYS, as shown in SEQ ID NO:7; FGPPGKKDPV, as shown in SEQ ID NO:8; and FDKLPGF, as shown in SEQ ID NO:

9.

2. The egg-derived anti-inflammatory, antioxidant, and liver-protective short peptide according to claim 1, characterized in that, The amino acid sequence of the short peptide is selected from at least one of the sequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, wherein the sequence is a hydrolytic fragment of ovalbumin or ovotransferrin.

3. The egg-derived anti-inflammatory, antioxidant, and liver-protective short peptide according to claim 1, characterized in that, The amino acid sequence of the short peptide is FGPPGKKDPV as shown in SEQ ID NO:8, or VLLPDEVSG as shown in SEQ ID NO:

1.

4. The egg-derived anti-inflammatory, antioxidant, and liver-protective short peptide according to claim 1, characterized in that, The short peptide also includes its N-terminal acetylated or C-terminal amidated form, or a pharmaceutically or food-acceptable salt thereof.

5. The method for preparing the egg-derived anti-inflammatory, antioxidant, and liver-protective short peptide according to claim 1, characterized in that, Includes the following steps: (1) Using amino acids protected by fluorene methoxycarbonyl groups as raw materials, solid-phase peptide synthesis is adopted, and the amino acid residues in the amino acid sequence are coupled sequentially from the C-terminus to the N-terminus. Each coupling cycle includes removing the fluorene methoxycarbonyl group protection with piperidine solution, washing, activating the amino acid with a condensation reagent and coupling, and capping. (2) After synthesis, the short peptide is cleaved from the resin and the side chain protecting group is removed with a cleavage reagent containing trifluoroacetic acid to obtain a crude peptide. (3) The crude peptide is purified by reversed-phase high-performance liquid chromatography, the target component is collected and freeze-dried to obtain a short peptide with a purity of not less than 95%. (4) The amino acid sequence and molecular weight of the purified product are confirmed by liquid chromatography-mass spectrometry.

6. The preparation method according to claim 5, characterized in that, In step (1), deprotection is performed using a 20% piperidine solution. The condensation reagent is a combination of O-benzotriazole-tetramethylurea hexafluorophosphate and 1-hydroxybenzotriazole, with a molar ratio of amino acid to condensation reagent of 1:

1. In step (2), the cleavage reagent is a mixture of trifluoroacetic acid, water and triisopropylsilane in a volume ratio of 95:2.5:2.5, and the cleavage time is 2 h.

7. The preparation method according to claim 5, characterized in that, The reversed-phase high-performance liquid chromatography described in step (3) uses a C18 column and gradient elution with water and acetonitrile containing 0.1% trifluoroacetic acid (v / v) as the mobile phase; the molecular weight confirmation described in step (4) is based on the measured primary mass spectrometry [M+H]. + A peak deviation from the theoretical value of no more than 0.5 Da is considered acceptable.

8. The application of the egg-derived anti-inflammatory, antioxidant, and hepatoprotective short peptide of claim 1 in the preparation of products for preventing or improving alcoholic liver injury, characterized in that... The alcoholic liver injury is characterized by at least one of the following: ethanol-induced decrease in hepatocyte survival, elevated serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, decreased liver tissue superoxide dismutase (SOD) activity, elevated malondialdehyde (MDA) content, and elevated levels of tumor necrosis factor-α (TNF-α) and interleukin-1β.

9. The application according to claim 8, characterized in that, The product is a drug, health food, or functional food, and its dosage form is oral liquid, tablet, capsule, granule, or powder.

10. The application according to claim 8, characterized in that, The short peptide is used in combination with silymarin.

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