Antioxidant peptides, methods of making and using the same

Abstract

This invention discloses a class of antioxidant peptides, their preparation methods, and applications. It aims to provide an active peptide with excellent free radical scavenging effects. The amino acid sequence of the antioxidant peptide is one or any combination of WWL, WDRW, WWWV, RSPWR, WPRCQL, and CVKWML. This active peptide is obtained by directly enzymatically hydrolyzing fresh shells to extract bioactive peptides, followed by a two-step purification process to obtain peptides with good activity. This represents a novel strategy for constructing a high-quality bioactive peptide library of shells. Furthermore, computer-aided screening, in vitro activity assays, and cell activity assays are used to rapidly and efficiently screen peptides with good antioxidant activity. The preparation method is simple and belongs to the field of biotechnology.

Description

Technical Field

[0001] This invention discloses a class of antioxidant peptides, and also discloses the preparation method and application of the antioxidant peptides, belonging to the field of biotechnology. Background Technology

[0002] Alcohol-related diseases (ALD) are among the major diseases caused by long-term alcohol consumption. The liver, as the primary site of alcohol metabolism, is the main organ susceptible to alcohol poisoning and is one of the most common causes of liver-related morbidity and mortality. Alcoholic liver disease generally progresses from simple hepatic steatosis to alcoholic hepatitis, liver fibrosis, and cirrhosis, thus posing a serious threat to human health. Increasing evidence suggests that alcoholic liver disease may be associated with increased oxidative stress and free radical-related damage.

[0003] Alcohol is primarily metabolized via the alcohol dehydrogenase system (ADH) in the cytoplasm and the microparticle oxidation pathway (MEOS) in the endoplasmic reticulum, catalyzing the dehydrogenation of alcohol to produce toxic and carcinogenic acetaldehyde, which is then rapidly oxidized to non-toxic acetic acid by acetaldehyde dehydrogenase. Within the ADH metabolic pathway, the metabolism of ethanol leads to the formation of nicotinamide adenine dinucleotide (NAD). + The conversion of NADH to reduced coenzyme I (NADH) in hepatocytes leads to an NADH / NAD ratio. + A high electron ratio leads to excessive electron flow in the mitochondrial respiratory chain, resulting in electron accumulation and leakage in mitochondrial respiratory chain complexes I and III, thus generating reactive oxygen species (ROS). In the MEOS metabolic pathway, ethanol is mainly metabolized to acetaldehyde via CYP2E1, and NADPH is oxidized to oxidized coenzyme II (NADP). + CYP2E1 is a cytochrome reductase that can leak electrons into oxygen to form hydroxyl radicals (·OH) and superoxide anions (·O). 2- Therefore, the metabolism of alcohol produces a large amount of reactive oxygen species (ROS). When the number of free radicals, including ROS, exceeds that of endogenous antioxidants, liver homeostasis is disrupted. Excessive ROS within hepatocytes can damage proteins, lipids, and DNA, leading to abnormal liver structure and function. Therefore, oxidative stress caused by excessive ROS may be a major cause of ALD progression.

[0004] Currently, abstinence from alcohol is the most effective treatment for alcoholic liver disease. However, in patients with alcohol addiction, withdrawal is often accompanied by withdrawal syndrome, leading to a large number of patients failing to complete treatment or relapsing. Although many drugs have been proven effective in treating alcoholic liver disease by reducing oxidative stress levels, such as metoprolol and N-acetylcysteine ​​(NAC), the side effects of long-term use are unavoidable. Given the significant role of oxidative stress in alcoholic liver disease, increasing the liver's antioxidant capacity is considered a promising strategy for prevention or treatment. Bioactive peptides that can resist free radical production are called antioxidants, including endogenous and exogenous antioxidants. The former often plays a crucial role in the body; however, current research mainly focuses on the development and application of exogenous antioxidants, as they can resist free radical-mediated oxidative stress damage and maintain redox homeostasis. Many studies have shown that dietary antioxidants can prevent alcoholic liver disease by scavenging free radicals, restoring antioxidant enzyme activity, and maintaining the homeostasis of the liver's antioxidant defense system. In addition to providing nutrition, food-derived bioactive peptides have been extensively studied and found to be more easily absorbed, more stable, and safer than antioxidants from other sources. Therefore, the development of food-derived peptides has significant practical implications for preventing ethanol-induced liver damage.

[0005] Thin-shelled clams, also known as sea snails, scientifically named *Musculus senhousei*, are a common type of shellfish found in coastal areas. Rich in protein, they contain various essential amino acids and micronutrients, making them a popular and nutritious seafood. However, the processing level of thin-shelled clams is generally low, and there are currently no reports on efficient and rapid identification of antioxidant peptides derived from them.

[0006] Traditional methods for separating and purifying bioactive peptides mainly include gel filtration, ion exchange, and reversed-phase high-performance liquid chromatography (RP-HPLC). These processes are time-consuming and cumbersome, thus necessitating the development of a more convenient and feasible method for screening peptides with antioxidant activity. With the continuous advancement of science and technology, research strategies combining high-performance computer-aided screening of bioactive peptides are gaining widespread application. A method combining size exclusion chromatography and RPI with computer-aided analysis offers a more feasible and effective alternative to traditional methods for screening antioxidant peptides.

[0007] Current research on the preparation of antioxidant peptides mostly focuses on enzymatic hydrolysis using commercial proteases, followed by separation using methods such as gel filtration, ion exchange, and reversed-phase high-performance liquid chromatography, and then identification of the most active components. Existing methods for obtaining new bioactive peptides are mostly cumbersome and time-consuming, involving enzymatic hydrolysis followed by bioactivity-guided separation, purification, and identification. Summary of the Invention

[0008] To address the aforementioned problems, the first objective of this invention is to provide a class of antioxidant peptides that exhibit excellent free radical scavenging effects.

[0009] The second technical solution provided by this invention is a method for preparing the above-mentioned active peptides. This method directly extracts bioactive peptides from fresh thin-shelled shells through enzymatic hydrolysis, and then obtains peptides with good activity through two-step purification, thereby constructing a new strategy for building a high-quality bioactive peptide library of thin-shelled shells. Furthermore, it rapidly and efficiently screens peptides with good antioxidant activity through computer-aided screening, in vitro activity and cell activity assays. The preparation method is simple.

[0010] By calculating its active site using quantum chemical methods, it was found that the antioxidant peptide has a liver-protective function. Therefore, the third technical solution provided by this invention is the application of the above-mentioned antioxidant peptide in the preparation of drugs for the prevention / treatment of ethanol-induced liver injury.

[0011] The method for preparing the antioxidant peptide according to the first technical solution provided by the present invention includes the following steps in sequence:

[0012] 1) Extraction of thin-shell bioactive peptides from thin-shell shells;

[0013] 2) The thin-shell bioactive peptides extracted in step 1) were initially separated by size exclusion chromatography to obtain crude bioactive peptides;

[0014] 3) The antioxidant activity of the crude active peptides separated in step 2) was measured, and two crude active peptides with high antioxidant activity were screened out.

[0015] 4) The crude active peptides screened in step 4) were further separated by reversed-phase high-performance liquid chromatography to obtain active peptides;

[0016] 5) The antioxidant activity of the active peptides obtained in step 4) was measured, and the two active peptides with the highest activity were screened out.

[0017] 6) The amino acid sequence of the active peptides obtained in step 5) was identified by mass spectrometry to establish a thin-shell peptide library;

[0018] 7) Peptides from the thin-shell peptide library were subjected to Peptide Ranker score, CPP Pred score, toxicity prediction, and sensitization prediction to obtain peptides with potential biological activity, namely WWL, WDRW, WWWV, RSPWR, WPRCQL, and CVKWML.

[0019] Furthermore, the preparation method of the above-mentioned antioxidant peptides, step 1) of extracting the bioactive peptides from the shell includes the following steps:

[0020] 1) Thin-shelled shells are steamed to obtain thin-shelled meat. The thin-shelled meat is added to homogenize and extract endogenous peptides under acidic conditions. Then, solvent is added, centrifugation is performed to remove proteins, the supernatant is collected, the solvent is removed, and freeze-drying is performed to obtain freeze-dried peptides.

[0021] 2) Disperse the lyophilized peptides prepared in step 1) in distilled water, adjust the pH to acidic, then add pepsin to simulate gastric digestion and hydrolysis to obtain a mixture, adjust the pH to weakly alkaline again, add pancreatin to continue simulating intestinal digestion, and stop digestion by inactivating enzymes after the reaction is completed; then centrifuge to collect the supernatant, freeze dry, and obtain thin-shell bioactive peptides.

[0022] Furthermore, the above-mentioned method for preparing antioxidant peptides involves the preliminary separation of active peptides using size exclusion chromatography: the thin-shell bioactive peptides are dissolved in ultrapure water, filtered, and then injected into a chromatographic column. A Sephadex G25 column is used, and elution is performed with 0.01N HCl solution at 4°C at a flow rate of 5 mL / 20 min. The eluent is collected using a fraction collector, with 5 mL collected from each tube, up to 195 tubes. After collection, the absorbance values ​​of each component are read at 214 nm and 280 nm. According to the order of collection, the eluents from every 5 tubes are mixed sequentially, starting from the 41st tube, to obtain 31 components, designated as F1, F2, F3, F4...F29, F30, F31. These components are then freeze-dried and stored at -20°C for later use.

[0023] Furthermore, in the above-mentioned method for preparing antioxidant peptides, during step 4) reversed-phase high-performance liquid chromatography separation, an Agilent ZORBAX SB-Aq C18 column is used. The mobile phase consists of component A and component B, where component A is acetonitrile and component B is 0.1% trifluoroacetic acid (v / v). The flow rate is set to 1 mL / min, the column temperature to 30°C, and the detection wavelength to 214 nm.

[0024] The elution gradients for separation are as follows: 0-2 min, 100% B; 2-10 min, 100%-90% B, 0%-10% A; 10-20 min, 90%-75% B, 10%-25% A; 20-25 min, 75%-60% B, 25%-40% A; 25-35 min, 60%-10% B, 40%-90% A; 35-40 min, 10%-100% B, 90%-10% A; 40-45 min, 100% A.

[0025] The eluent was collected using a fraction collector, with each fraction collected every minute, resulting in a total of 30 lyophilized fractions, which were sequentially labeled F8-1, F8-2, F8-3, ..., F8-11, F8-12, F8-13, F8-14, F15-1, F15-2, F15-3, ..., F15-14, F15-15, F15-16. The collected fractions were then lyophilized and stored at -20°C.

[0026] Furthermore, in the above-mentioned method for preparing antioxidant peptides, the antioxidant activity tests described in steps 2) and 4) are all performed by measuring the absorbance values ​​of the crude active peptide and the active peptide at 517 nm in a cuvette and calculating the DPPH free radical scavenging rate; and by measuring the absorbance values ​​of the crude active peptide and the active peptide at 734 nm and calculating the ABTS free radical scavenging rate.

[0027] Furthermore, in the above-mentioned method for preparing antioxidant peptides, step 6) of constructing a thin-shell peptide library includes the following steps:

[0028] 1) Dissolve F8-11 and F15-6 in ultrapure water, filter, and inject into the EASY-nLC1200 nano liquid chromatography system for separation;

[0029] The mobile phase consists of component A and component B, wherein: component A is a 0.1% (v / v) formic acid aqueous solution (FA); component B consists of a formic acid aqueous solution and acetonitrile in a volume ratio of 2:8, wherein the formic acid aqueous solution has a volume concentration of 0.1%; the flow rate is 0.3 μL / min, and the column temperature is 30℃;

[0030] The elution gradient was as follows: 0–2 min, 3–8% B, 97–92% A; 2–48 min, 8–26% B, 92–74% A; 48–53 min, 26–35% B, 74–65% A; 53–57 min, 35–100% B, 65–0% A; 57–60 min, 100% B.

[0031] 2) The amino acid sequences of peptides in the lyophilized samples obtained after separation by Acclaim Pep Map™ RSLC column were identified using a peptidomics strategy. 2216 peptides were obtained and included in the peptide library to establish a thin-shell peptide library.

[0032] Furthermore, in the above-mentioned method for preparing antioxidant peptides, step 7) involves performing PeptideRanker scoring, CPP Pred scoring, toxicity prediction, and sensitization prediction on the peptides from the thin-shell peptide library as follows:

[0033] 1) The potential bioactivity of the 2216 identified peptides was predicted using Peptide Ranker software, and 234 peptides with a score >0.8 were selected.

[0034] 2) The cell permeability of the above 234 peptides was predicted using CPP Pred software, and 20 peptides with a score > 0.5 were selected.

[0035] 3) The potential toxicity of the 20 peptides after screening was predicted using Toxin Pred software, and the potential sensitization of the 20 peptides was predicted using AllerTOP software. Finally, 6 peptides were screened: WWL, WDRW, WWWV, RSPWR, WPRCQL, and CVKWML.

[0036] The third technical solution provided by this invention is the application of the above-mentioned antioxidant peptide as a drug for the prevention / treatment of ethanol-induced liver injury.

[0037] Compared with the prior art, the technical solution provided by the present invention has the following technical advantages:

[0038] 1. The antioxidant peptides ABTS and DPPH provided by this invention have good scavenging effects on free radicals.

[0039] 2. The present invention provides a novel strategy for constructing a high-quality thin-shell bioactive peptide library by directly extracting bioactive peptides from fresh thin-shell shells through enzymatic hydrolysis, followed by two-step purification to obtain peptides with good activity. Then, by using computer-aided screening, in vivo activity and cell activity assays, peptides with good antioxidant activity can be rapidly and efficiently screened. The preparation method is simple.

[0040] 3. By calculating its active site using quantum chemistry, it was proven that the antioxidant peptide provided by this invention has liver-protective function. Attached Figure Description

[0041] Figure 1 These are SEC chromatograms of different components from F1 to F31, and the removal rates of free radicals scavenging ABTS and DPPH.

[0042] Figure 2 The HPLC spectrum of F8 and DPPH free radical scavenging;

[0043] Figure 3 This is the HPLC spectrum of F15 and the ABTS free radical scavenging.

[0044] Figure 4 It refers to the DPPH free radical scavenging ability of six peptides;

[0045] Figure 5 It refers to the ABTS free radical scavenging ability of six peptides;

[0046] Figure 6 It describes the protective effect of six antioxidant peptides against ethanol-induced cell damage.

[0047] Where: A represents the results of constructing the ethanol-damaged HepG2 cell model; B represents the protective effect of six antioxidant peptides on cells; C and D represent the effects of antioxidant peptides on AST and ALT activities; and E represents the effects of antioxidant peptides on ROS levels in ethanol-induced HepG2 cells.

[0048] Figure 7 The results are based on the analysis of the active sites of six peptides using HOMO and Millikan charge distribution and bond length. Detailed Implementation

[0049] The following detailed description of specific embodiments is provided in conjunction with the accompanying drawings, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0050] Unless otherwise specified, all raw materials and reagents used in the examples are commercially available.

[0051] The UV-Vis spectrophotometer used in the examples was a Shanghai Yuanxi Q-6 model; the multi-functional microplate reader was a Guangzhou Lianbo Varioskan LUX model; and the nano-level liquid chromatography-electrospray ionization-ultra-high resolution time-of-flight tandem mass spectrometer was a Thermo Fisher Easy nLC1200 / Q Exactive plus.

[0052] mM refers to mmol / L, 1M HCl refers to 1 mol / L HCl solution, and 1M NaOH refers to 1 mol / L NaOH solution.

[0053] The website link for the Peptide Ranker software is http: / / distilldeep.ucd.ie / PeptideRanker.

[0054] The CPP Pred software website link is: http: / / distilldeep.ucd.ie / CPPpred / ;

[0055] The link to the Toxin Pred software website is: http: / / crdd.osdd.net / raghava / toxinpred / ;

[0056] The link to the AllerTOP software website is: http: / / www.ddg-pharmfac.net / AllerTOP / ;

[0057] The link to the Biopep database website is: https: / / biochemia.uwm.edu.pl / biopep-uwm / .

[0058] Example 1

[0059] The antioxidant peptide provided in this embodiment is obtained by thin-shell antioxidant separation and purification, and its amino acid sequence is one or any combination of WWL, WDRW, WWWV, RSPWR, WPRCQL, and CVKWML.

[0060] Its preparation method includes the following steps in sequence:

[0061] 1) Preparation of thin-shell bioactive peptides

[0062] 1.1) 30g of shellfish meat was obtained by steaming in a 70℃ water bath for 30 minutes, chopped, and then 120ml of 0.01M HCl was added. The mixture was homogenized at 4℃ for 8 minutes using a beater to extract endogenous peptides. Then, 3 times the volume of ethanol was added to obtain an endogenous peptide mixture. The endogenous peptide mixture was allowed to stand at 4℃ for 20 hours, and then centrifuged at 12,000g at 4℃ for 20 minutes to remove proteins. The supernatant was collected, and the added ethanol was removed in a rotary evaporator. The mixture was then frozen at -80℃ to solidify, and then freeze-dried under vacuum in a freeze dryer to obtain 5g of freeze-dried peptide powder. According to GB5009.5-2016, the protein content in the freeze-dried peptide powder was determined to be 0.7885g by the Kjeldahl method. Finally, the extracted freeze-dried peptide powder was stored at -20℃.

[0063] 1.2) Disperse 5g of the lyophilized peptide powder prepared in step 1.1) in 25ml of distilled water and adjust the pH to 2.0 using 1M HCl; add 0.03g of pepsin (4% of the total protein mass in the lyophilized peptide) and perform simulated gastric digestion hydrolysis at 37℃ for 2 hours to obtain a hydrolyzed mixture; then adjust the pH of the mixture to 5.3 using 0.9M NaHCO3, and further adjust it to 7.5 using 1M NaOH; continue to simulate intestinal digestion by adding 0.03g of pancreatin (4% of the protein mass in the lyophilized peptide) and hydrolyzing at 37℃ for 2 hours. After the reaction is completed, boil in a water bath for 10 minutes to inactivate the enzyme and stop digestion; then centrifuge the final hydrolyzed mixture at 4℃ and 8000g for 10min, collect the supernatant, freeze-dry it to obtain thin-shell bioactive peptides and store them at 4℃.

[0064] (ii) Separation of the thin-shell bioactive peptides prepared in step 1), comprising the following separation methods:

[0065] 1) Size exclusion chromatography separation

[0066] First, dissolve 1g of the thin-shell bioactive peptide in 5mL of ultrapure water, filter through a 0.45μm filter membrane, and then inject into a chromatographic column packed with Sephadex G25 packing material (2.5×60cm, 5.0μm). Elute with 0.01M HCl solution at 4℃, with a flow rate of 5mL / 20min. Collect the eluent using a fraction collector, collecting 5mL of eluent from each tube, up to 195 tubes. After collection, read the absorbance values ​​of each component at 214nm and 280nm. Refer to Table 1 for specific data, and then plot the chromatogram. According to the collection order, starting from tube 41, mix the eluent from every 5 tubes in that order, finally obtaining 31 components, labeled F1, F2, F3, F4...F29, F30, F31. Freeze-dry and store at -20℃ for later use.

[0067] Table 1

[0068]

[0069]

[0070]

[0071]

[0072] Antioxidant activity test

[0073] Thirty-one components obtained by size exclusion chromatography, namely F1, F2, F3, F4...F29, F30, and F31 (hereinafter referred to as samples), were subjected to determination of their DPPH and ABTS free radical scavenging abilities. The specific determination methods are as follows:

[0074] DPPH free radical scavenging capacity determination: Mix 100 μL of sample with 500 μL of ethanol, add 125 μL of DPPH ethanol solution (2% w / v), mix well, react in the dark for 60 min, and then add to a cuvette to measure the absorbance value at 517 nm. Use the same volume of water as a control instead of the sample.

[0075] The DPPH radical scavenging rate is calculated using the following equation:

[0076]

[0077] ABTS free radical scavenging ability determination: Add 10 μL of sample to a cuvette and mix with 990 μL of ABTS dilution solution. After reacting in the dark for 6 min, measure the absorbance at 734 nm using a UV spectrophotometer and record it as OD. sampleUsing PBS as a blank control and vitamin E (Trolox) at molar concentrations of 0 μM, 200 μM, 400 μM, 800 μM, and 1200 μM as positive controls, the absorbance values ​​were recorded as OD values. control and OD trolox .

[0078] The ABTS radical scavenging rate was calculated using the following equation:

[0079]

[0080] A standard curve regression equation was obtained by plotting Trolox concentration and ABTS radical scavenging rate using linear fitting. The TEAC value of the samples was calculated based on the equation and the ABTS radical scavenging rate; the results are expressed as μM TE / g sample. The results showed that components F8 and F15 had the highest DPPH radical scavenging capacity compared to other components. Figure 1 As shown.

[0081] 2) Reversed-phase high-performance liquid chromatography separation

[0082] An Agilent ZORBAX SB-Aq C18 column (4.6 mm × 250 mm, 5 μm) was used in a high-performance liquid chromatography (HPLC) system. The mobile phase consisted of component A and component B. Component A was acetonitrile (ACN), and component B was 0.1% trifluoroacetic acid (TFA) by volume. The flow rate was set to 1 mL / min, the column temperature to 30 °C, and the detection wavelength to 214 nm.

[0083] F8 fraction was reconstituted with ultrapure water, filtered through a 0.45 μm filter membrane, and then injected into the chromatographic column. The elution gradients were as follows: 0–2 min, 100% B; 2–10 min, 100%–90% B, 0%–10% A; 10–20 min, 90%–75% B, 10%–25% A; 20–25 min, 75%–60% B, 25%–40% A; 25–35 min, 60%–10% B, 40%–90% A; 35–40 min, 10%–100% B, 90%–10% A; 40–45 min, 100% A.

[0084] Collect the eluent using a fraction collector, collecting each fraction every 1 minute, and label them sequentially as F8-1, F8-2, F8-3, ..., F8-11, F8-12, F8-13, F8-14;

[0085] The F15 fraction was reconstituted with ultrapure water, filtered through a 0.45 μm filter membrane, and then injected into the chromatographic column. The elution gradients were as follows: 0–2 min, 100% B; 2–10 min, 100%–90% B, 0%–10% A; 10–20 min, 90%–75% B, 10%–25% A; 20–25 min, 75%–60% B, 25%–40% A; 25–35 min, 60%–10% B, 4… 0%-90% A; 35-40 minutes, 10%-100% B, 90%-10% A; 40-45 minutes, 100% A; Collect the eluent using a fraction collector, collecting each fraction every 1 minute, and label them F15-1, F15-2, F15-3...F15-14, F15-15, F15-16; Finally, freeze-dry the collected 30 fractions and store them at -20°C.

[0086] Antioxidant activity test

[0087] The DPPH and ABTS free radical scavenging abilities of fractions F8-1, F8-2, F8-3...F8-11, F8-12, F8-13, F8-14, F15-1, F15-2, F15-3...F15-14, F15-15, F15-16 (hereinafter referred to as samples) obtained by reversed-phase high-performance liquid chromatography were determined. The specific determination methods are as follows:

[0088] DPPH radical scavenging capacity determination: Mix 100 μL of sample with 500 μL of ethanol, add 125 μL of DPPH ethanol solution (2% w / v), mix well, react in the dark for 60 min, then add to a cuvette and measure the absorbance at 517 nm. Use the same volume of water as a control instead of the sample. The DPPH radical scavenging rate is calculated using the following equation:

[0089]

[0090] ABTS free radical scavenging ability determination: Add 10 μL of sample to a cuvette and mix with 990 μL of ABTS dilution solution. After reacting in the dark for 6 min, measure the absorbance at 734 nm using a UV spectrophotometer and record it as OD. sample Using PBS as a blank control and vitamin E (Trolox) at molar concentrations of 0 μM, 200 μM, 400 μM, 800 μM, and 1200 μM as positive controls, the absorbance values ​​were recorded as OD values. control and OD trolox .

[0091] The ABTS radical scavenging rate was calculated using the following equation:

[0092]

[0093] A standard curve regression equation was obtained by plotting Trolox concentration and ABTS radical scavenging rate using linear fitting. The TEAC value of the sample was calculated based on the equation and the ABTS radical scavenging rate; the results are expressed as μM TE / g sample. The results are as follows: Figure 2 , Figure 3 As shown, the data are expressed as mean ± standard error; the absence of common letters in the marker characters indicates a significant difference of P < 0.05, which was obtained through one-way ANOVA; it was found that F8-11 had the highest DPPH radical scavenging ability, and F15-6 had the strongest ABTS radical scavenging ability.

[0094] (iv) Construction of thin-shell polypeptide library

[0095] The active peptide fragments were identified using nano-level liquid chromatography-electrospray ionization-ultra-high resolution time-of-flight tandem mass spectrometry.

[0096] The two lyophilized samples F8-11 and F15-6 with the best free radical scavenging rates in step 3) were dissolved in ultrapure water to prepare solutions with concentrations of 1 mg / ml for F8-11 and 1 mg / ml for F15-6, respectively. The solutions were then filtered through a 0.22 μm filter membrane. Each solution was injected at a volume of 2 μL into an EASY-nLC1200 nano liquid chromatography system (Thermo Scientific, MA, USA). The samples were first enriched using an Acclaim PepMap™ 100trap column (75 μm × 2 cm, nano Viper 2Pk C18, 3 μm, 100 μm pore size), and then separated using an Acclaim Pep Map™ RSLC column.

[0097] The mobile phase consists of component A and component B, wherein: component A is a 0.1% (v / v) formic acid aqueous solution (FA); component B consists of a formic acid aqueous solution and acetonitrile in a volume ratio of 2:8, wherein the formic acid aqueous solution has a volume concentration of 0.1%; the flow rate is 0.3 μL / min, and the column temperature is 30℃;

[0098] The elution gradient was as follows: 0–2 min, 3–8% B, 97–92% A; 2–48 min, 8–26% B, 92–74% A; 48–53 min, 26–35% B, 74–65% A; 53–57 min, 35–100% B, 65–0% A; 57–60 min, 100% B.

[0099] The amino acid sequences of peptides in lyophilized samples separated by AcclaimPep Map™ RSLC columns were identified using a peptidomics strategy, yielding 2216 peptides. All identified peptide sequences were included in a peptide library, and a thin-shell peptide library was established. The peptide sequences identified by F8-11 are shown in Table 2; the peptide sequences identified by F15-6 are shown in Table 3.

[0100] Table 2

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114] Table 3

[0115] Serial Number sequence Serial Number sequence Serial Number sequence Serial Number sequence 2193 GAGD 2199 QLT 2205 GCSN 2211 FW 2194 YN 2200 ACNT 2206 ACAN 2212 KE 2195 LS 2201 MGDF 2207 MTFA 2213 ETE 2196 GY 2202 VLLPK 2208 MTF 2214 GL 2197 QGD 2203 CSNN 2209 DTE 2215 LL 2198 SVVPSPK 2204 MNN 2210 FDYF 2216 UE

[0116] (v) Computer-aided screening

[0117] The potential biological activity of the 2216 peptides identified in step four was predicted using the Peptide Ranker software. Specifically, the number and position of certain amino acid residues in the peptide sequence were scored and given a value of 0-1. The larger the value, the greater the probability of potential biological activity. Peptides with a score >0.8 (234 peptides) were selected.

[0118] Cell permeability was predicted using CPP Pred software, with a value of 0-1. The higher the value, the better the cell permeability and the greater the probability of potential bioavailability. Peptides with a score >0.5 (20 peptides) were selected.

[0119] Using Toxin Pred and AllerTOP software, based on machine learning technology, Toxin Pred software was used to predict the potential toxicity of 20 peptides after screening, while AllerTOP software was used to predict the potential sensitization of 20 peptides, ultimately selecting 6 peptides.

[0120] Using the Biopep database, peptide sequences can be compared with previously reported literature to determine whether they have been reported before, and can be preliminarily identified as novel peptide sequences.

[0121] Based on this, peptides WWL, WDRW, WWWV, RSPWR, WPRCQL, and CVKWML, which exhibited potential biological activity and had Peptide Ranker values ​​> 0.8, CPP Pred values ​​> 0.5, were non-toxic, non-sensitizing, and showed potential biological activity, were screened out, as shown in Table 4. This indicates that computer-aided screening identified six novel peptides with potential biological activity, good cell permeability, non-toxicity, and no potential sensitization.

[0122] Table 4 lists 6 peptides with potential biological activity, non-toxicity, and non-allergenicity.

[0123] serial number sequence PeptideRanker rating CPPPred rating Toxicity prediction Allergenicity prediction 1 WWL 0.99 0.65 Non-toxic Non-allergenic 2 WWWV 0.99 0.55 Non-toxic Non-allergenic 3 WDRW 0.97 0.52 Non-toxic Non-allergenic 4 WPRCQL 0.92 0.54 Non-toxic Non-allergenic 5 CVKWML 0.87 0.53 Non-toxic Non-allergenic 6 RSPWR 0.86 0.70 Non-toxic Non-allergenic

[0124] To demonstrate the antioxidant activity of the six peptides screened in this application—WWL, WDRW, WWWV, RSPWR, WPRCQL, and CVKWML—the following antioxidant activity verification experiments are presented:

[0125] 1. Validation of in vitro antioxidant activity

[0126] Six peptide fragments (WWL, WDRW, WWWV, RSPWR, WPRCQL, CVKWML) obtained from screening were subjected to solid-phase synthesis (the synthetic peptides were chemically synthesized by Jier Biochemical (Shanghai) Co., Ltd. using solid-phase method, and the obtained synthetic peptides were polypeptide solid powders with a purity >95%), resulting in different synthetic peptides (polypeptide solid powders with a purity >95%). They were labeled as synthetic peptides WWL, WWWV, WDRW, WPRCQL, CVKWML, and RSPWR. The synthetic peptides were prepared into solutions with concentrations of 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.8mM, 1.0mM, 2mM, 2.5mM, 3.5mM, 4mM, 4.5mM, and 5mM respectively using deionized water, and the free radical scavenging ability of these six synthetic peptides was determined (the method was the same as in (1)).

[0127] Experimental results are as follows Figure 4 and Figure 5 As shown in the figure, the horizontal axis represents the concentration of synthetic peptides WWL, WWWV, WDRW, WPRCQL, CVKWML, and RSPWR. (Data are expressed as mean ± standard error; no common letter indicates a significant difference of P < 0.05, obtained through one-way ANOVA.) The results show that all six peptides have good in vitro DPPH free radical scavenging ability, can scavenge DPPH free radical activity in a dose-effect relationship, and all of them have good in vitro ABTS free radical scavenging ability.

[0128] 2. Verification of intracellular antioxidant activity:

[0129] (a) Construction of an ethanol-damaged HepG2 cell model: 100 μL of cell suspension was seeded in 96-well plates and incubated for 24 hours (2 × 10⁻⁶ cells / well). 4 Cells / well were removed, and the old culture medium was discarded. 100 μL of serum-free culture medium was added and incubated for 24 hours. The old culture medium was then discarded. The experimental groups were incubated with different concentrations of ethanol (0.25M-2M), while the control group was incubated with serum-free culture medium for 24 hours. Cell viability was measured using the CCK-8 assay to determine the ethanol concentration at which cell viability reached 50%.

[0130] CCK-8 assay: HepG2 cells were inoculated at 2 x 10⁻⁸ ppm. 5 Cells were seeded at a density of [number] cells / ml in 96-well plates and cultured at 37°C for 24 hours. After incubation, cells were exposed to a synthetic peptide (0.1 mM) for 24 hours. Subsequently, cells were washed twice with phosphate-buffered saline (PBS) and 100 μL of fresh culture medium and 10 μL of CCK-8 were added. After 1 hour of incubation, absorbance was immediately measured at OD 450 nm using a multi-functional fluorescent microplate reader. Results are expressed as cell viability percentage. Cell viability was calculated using the following formula:

[0131]

[0132] (b) Pre-protection of HepG2 cells damaged by ethanol with thin-shell peptides: Cells were divided into control, model, and experimental groups. 100 μL of cell suspension was seeded into 96-well plates for 24 hours (2 × 10⁶ cells / well). 4 Cells were incubated in 100 μL of serum-free medium (100 cells / well) and the medium was changed. The experimental group received 100 μL of 0.1 mM antioxidant peptide, while the blank and damage control groups received serum-free medium. After 24 hours, the medium was changed again. The control group received 100 μL of serum-free medium, while the model and experimental groups received 100 μL of medium containing ethanol at the concentration selected in (a). After 24 hours of incubation, cell viability was measured using the CCK-8 method described in (a).

[0133] (c) Measurement of intracellular reactive oxygen species (ROS) levels: Relative ROS levels in cells were measured using the DCFH-DA fluorescent probe. Cells were pretreated with thin-shell peptides for 24 hours, followed by an additional 24 hours of ethanol induction. Cells were then collected, washed three times with PBS, and immediately incubated with DCFH-DA (10 μM) for 30 minutes. After washing three times with sterile phosphate-buffered saline, fluorescence values ​​at excitation wavelength of 485 nm and emission wavelength of 525 nm were rapidly measured using a multi-functional fluorescent microplate reader.

[0134] (d) Determination of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities: ALT and AST were measured using the ALT and AST detection kits from Nanjing Construction Biotechnology Institute. Cells were collected and sonicated in an ice-water bath according to the manufacturer's instructions, followed by ALT and AST measurements.

[0135] Experimental results are as follows Figure 6 As shown in the figure (n=6 per group), data are expressed as mean ± standard error. No common letter indicates a significant difference (P<0.05), determined by one-way ANOVA. The results show that all six peptides effectively cleared ROS induced by ethanol in HepG2 cells and effectively alleviated ethanol-induced liver damage.

[0136] 3. Quantum chemical calculations for structure-activity analysis of peptides

[0137] The molecular structure of the antioxidant peptides was determined using ChemDraw (version 20.0) software. The results were input into Chem3D to obtain the spatial coordinates of the antioxidant peptides and saved as a Gaussian 16W input file. DFT calculations were performed using GaussView software (version 6.0) and the Gaussian 16W program (Gaussian Inc., Wallingford, CT, USA). The geometry was obtained using the Gaussian 16W program, and the active sites were analyzed using B3LYP / 6-31(d, p).

[0138] The calculation results are shown in Table 5 and Figure 7 As shown, the results indicate that the active sites of the six antioxidant peptides are all located on the NH group of the indole ring in the tryptophan side chain.

[0139] Table 5. Millikan charge distribution, bond length, and active site of the six peptides.

[0140]

[0141] Ultimately, the six peptides with good in vitro and intracellular antioxidant effects—CVKWML, WWL, WDRW, WWWV, RSPWR, and WPRCQL—are the target peptides of this invention.

Claims

1. A class of antioxidant peptides, characterized in that, The antioxidant peptides are combinations of peptides with the amino acid sequences WWL, WDRW, WWWV, RSPWR, WPRCQL, and CVKWML.

2. The use of the antioxidant peptide according to claim 1 in the preparation of a drug for the prevention / treatment of ethanol-induced liver injury.

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