Antioxidant peptide as well as preparation method and application thereof

Abstract

The invention discloses an antioxidant peptide as well as a preparation method and application thereof, and aims to provide an active peptide with a good free radical scavenging effect, and the amino acid sequence of the antioxidant peptide is one or any combination of WWL, WDRW, WWWV, RSPWR, WPRCQL and CVKWML; according to the thin-shell bioactive peptide, fresh thin shells are directly subjected to enzymolysis treatment to extract thin-shell bioactive peptide, the peptide with good activity is obtained through two-step purification, a new strategy of a high-quality thin-shell bioactive peptide library is constructed, and the thin-shell bioactive peptide library is obtained through a method of computer-aided screening and in-vitro activity and cell activity determination. The peptide with good antioxidant activity is rapidly screened at high throughput, and the preparation method is simple; belongs to the technical field of biology.

Description

Technical Field

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

[0002] Alcohol-related disease (ALD) is one of the main diseases caused by long-term drinking. The liver, as the main site of alcohol metabolism, is the main organ suffering from alcohol poisoning and one of the most common causes of liver-related morbidity and mortality. Alcoholic liver disease generally develops from simple hepatic fatty degeneration to alcoholic hepatitis, liver fibrosis and cirrhosis, causing serious harm to human life and health. Increasing evidence shows that alcoholic liver disease may be related to increased oxidative stress and free radical-related damage.

[0003] Alcohol is mainly dehydrogenated by the alcohol dehydrogenase metabolic pathway (ADH) in the cytoplasm and the cell microsomal oxidation pathway (MEOS) in the endoplasmic reticulum to produce toxic carcinogenic acetaldehyde, which is then rapidly oxidized to non-toxic acetic acid by acetaldehyde dehydrogenase. In the ADH metabolic pathway, the metabolism of ethanol leads to the degradation of nicotinamide adenine dinucleotide (NAD + ) to reduced coenzyme I (NADH), making the NADH / NAD + The ratio is at a high level, resulting in excessive flow of electrons in the mitochondrial respiratory chain, accumulation and leakage of electrons in complex I and complex III of the mitochondrial respiratory chain, and thus the generation of ROS. In the MEOS metabolic pathway, ethanol is mainly metabolized into acetaldehyde by CYP2E1, and NADPH is oxidized into oxidized coenzyme II (NADP + ), CYP2E1 is a cytochrome reductase that can leak electrons to oxygen to form hydroxyl radicals (·OH) and superoxide anions (·O 2- ), so a large amount of ROS is also produced while metabolizing alcohol. When the number of free radicals, including ROS, exceeds the endogenous antioxidant components, liver homeostasis will be destroyed, and excessive ROS in liver cells will damage proteins, lipids and DNA, leading to abnormal liver structure and function. Therefore, oxidative stress caused by excessive ROS may be the main cause of ALD progression.

[0004] Currently, abstinence from alcohol is the most effective means of treating alcoholic liver disease. However, in patients with alcohol addiction, abstinence is often accompanied by withdrawal syndrome, resulting in a large number of patients failing to complete treatment or relapse. Although many drugs have been proven to be effective in treating alcoholic liver disease by reducing oxidative stress levels, such as medivacin and N-acetylcysteine ​​(NAC), side effects caused by long-term use are inevitable. Since oxidative stress has attracted much attention in the process of alcoholic liver disease, increasing the antioxidant protection capacity of the liver is considered to be a promising strategy for preventing or treating alcoholic liver disease. Active peptides that can resist the production of free radicals are called antioxidants, including endogenous antioxidants and exogenous antioxidants. The former often play a vital role in the body, however, current research mainly focuses on the development and application of exogenous antioxidants because they can resist free radical-mediated oxidative stress damage and maintain redox homeostasis. Many studies have shown that food-borne 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 widely studied to be more easily absorbed, more stable, and safer than antioxidants from other sources. Therefore, the development of food-derived peptides has important practical significance in preventing ethanol-induced liver damage.

[0005] Thin shells, also known as clams, are scientifically named Musculus senhousei. They are a common shellfish distributed in coastal areas. They are rich in protein, a variety of essential amino acids and trace nutrients for the human body, and are a popular seafood with high nutritional value. However, the processing level of thin shells is generally low, and there is no report on the efficient and rapid identification of thin shell-derived antioxidant peptides.

[0006] Traditional methods for separating and purifying bioactive peptides mainly include gel filtration, ion exchange, reversed-phase high-performance liquid chromatography separation, etc. The separation and purification process is time-consuming and cumbersome, so there is an urgent need to develop a more convenient and feasible research method for screening peptides with antioxidant activity. With the continuous advancement of science and technology, the research strategy of combining high-performance computer-assisted screening of active peptides has gradually been widely used. The method of combining molecular exclusion chromatography and reversed-phase high-performance liquid chromatography with computer-assisted analysis is a more feasible and effective alternative to the traditional method of screening antioxidant peptides.

[0007] At present, the preparation of antioxidant peptides is mostly concentrated on using commercial proteases for enzymatic hydrolysis, followed by separation by gel filtration, ion exchange, reversed-phase high-performance liquid chromatography, etc., and then identifying the most active components. The existing methods for obtaining new bioactive peptides are mostly based on protease hydrolysis, followed by biological activity-oriented separation and purification, and then identification, which is cumbersome and time-consuming. Summary of the invention

[0008] To solve the above problems, the first object of the present invention is to provide a class of antioxidant peptides, which have good scavenging effects on free radicals.

[0009] The second technical solution provided by the present invention is a method for preparing the above-mentioned active peptides. This method directly extracts the thin shell bioactive peptides by enzymatic hydrolysis of fresh thin shells, and then obtains peptides with good activity through two-step purification, thereby constructing a new strategy for high-quality thin shell bioactive peptide library, and then through computer-assisted screening, in vitro activity and cell activity assay methods, rapid and high-throughput screening of peptides with good antioxidant activity, and the preparation method is simple.

[0010] By calculating its active sites through quantum chemistry, it was found that the antioxidant peptide has liver protection function. Therefore, the third technical solution provided by the present invention is the use of the above antioxidant peptide for preparing a drug for preventing / treating ethanol-induced liver damage.

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

[0012] 1) Extracting bioactive peptides from the thin shells;

[0013] 2) Preliminarily separating the thin shell bioactive peptides extracted in step 1) by size exclusion chromatography to obtain crude active peptides;

[0014] 3) measuring the antioxidant activity of the crude active peptides separated in step 2) to screen out two crude active peptides with high antioxidant activity;

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

[0016] 5) measuring the antioxidant activity of the active peptides obtained in step 4) and screening out the two most active active peptides;

[0017] 6) using mass spectrometry to identify the amino acid sequence of the active peptide obtained in step 5) and establish a thin shell polypeptide library;

[0018] 7) The peptides in the thin-shell peptide library were scored by Peptide Ranker, scored by CPP Pred, and predicted for toxicity and sensitization to obtain the potential bioactive peptides WWL, WDRW, WWWV, RSPWR, WPRCQL, and CVKWML.

[0019] Furthermore, in the above-mentioned method for preparing the antioxidant peptide, step 1) extracting the thin shell bioactive peptide from the thin shell comprises the following steps:

[0020] 1) Steaming the lamina to obtain lamina meat, adding the lamina meat to homogenize under acidic conditions to extract endogenous peptides, then adding a solvent, centrifuging to separate proteins, collecting the supernatant, removing the solvent, and freeze-drying to obtain freeze-dried peptides;

[0021] 2) dispersing the lyophilized peptide prepared in step 1) in distilled water, adjusting the pH to acidic, then adding pepsin to simulate gastric digestion and hydrolyze to obtain a mixture, adjusting the pH to weak alkaline again, adding pancreatic enzyme to continue simulating intestinal digestion, and inactivating the enzyme after the reaction is completed to stop digestion; then centrifuging and collecting the supernatant, lyophilizing, and obtaining the thin-shell bioactive peptide.

[0022] Furthermore, the preparation method of the above-mentioned antioxidant peptides uses molecular exclusion chromatography to initially separate active peptides. The method is as follows: the thin-shell bioactive peptide is dissolved in ultrapure water, filtered, and then injected into a chromatographic column, a chromatographic column filled with Sephadex G25 is used, and 0.01N HCl solution is used for elution at 4°C, and the flow rate during elution is 5 mL / 20 min; the eluate is collected by a component collector, 5 mL of eluate is collected in each test tube, and 195 tubes are collected in sequence, and the absorbance values ​​of each component at 214 nm and 280 nm are read respectively after collection, and the eluates in every 5 test tubes starting from the 41st tube are mixed in the order of collection, and finally 31 components are obtained, recorded as F1, F2, F3, F4...F29, F30, F31, freeze-dried and stored at -20°C for use.

[0023] Furthermore, in the above-mentioned method for preparing antioxidant peptides, in step 4) reverse phase high performance liquid chromatography separation, the chromatographic column adopts an Agilent ZORBAX SB-Aq C18 chromatographic column, the mobile phase consists of component A and component B, the component A is acetonitrile; the component B is trifluoroacetic acid with a volume concentration of 0.1%; the flow rate is set to 1 mL / min, the column temperature is 30°C, and the detection wavelength is 214 nm;

[0024] The elution gradient of the separation is as follows: 0-2 minutes, 100% B; 2-10 minutes, 100%-90% B, 0%-10% A; 10-20 minutes, 90%-75% B, 10%-25% A; 20-25 minutes, 75%-60% B, 25%-40% A; 25-35 minutes, 60%-10% B, 40%-90% A; 35-40 minutes, 10%-100% B, 90%-10% A; 40-45 minutes, 100% A;

[0025] Use a fraction collector to collect the eluate, and collect each fraction every 1 minute to obtain a total of 30 fraction freeze-dried samples, which are named 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 in sequence. The collected fractions are then freeze-dried and stored at -20°C.

[0026] Furthermore, in the above-mentioned method for preparing antioxidant peptides, the antioxidant activity tests described in step 2) and step 4) are both to measure the absorbance values ​​of crude active peptides and active peptides at 517nm in a cuvette, and calculate the DPPH free radical scavenging rate; to measure the absorbance values ​​of crude active peptides and active peptides at 734nm, and calculate the ABTS free radical scavenging rate.

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

[0028] 1) Dissolve F8-11 and F15-6 in ultrapure water, filter, and inject into 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% formic acid aqueous solution (FA) by volume; component B is composed of a formic acid aqueous solution and acetonitrile in a volume ratio of 2:8, and 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°C;

[0030] The elution gradient was: 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 peptide fragments in the freeze-dried samples separated by Acclaim Pep MapTM RSLC column were identified by peptideomics strategy, and 2216 peptides were obtained. The identified 2216 peptide sequences were 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) the method for performing PeptideRanker scoring, CPP Pred scoring, toxicity prediction, and sensitization prediction on the polypeptides in the thin-shell polypeptide library is as follows:

[0033] 1) Peptide Ranker software was used to predict the potential biological activities of the 2216 peptides identified, and 234 peptides with scores > 0.8 were selected;

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

[0035] 3) Toxin Pred software was used to predict the potential toxicity of the 20 peptides after screening, and AllerTOP software was used to predict the potential allergenicity of the 20 peptides. Finally, 6 peptides WWL, WDRW, WWWV, RSPWR, WPRCQL, and CVKWML were screened out.

[0036] The third technical solution provided by the present invention is the use of the above-mentioned antioxidant peptides as a drug for preparing a drug for preventing / treating ethanol-induced liver damage.

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

[0038] 1. The antioxidant peptide free radicals ABTS and DPPH provided by the present invention have good scavenging effects.

[0039] 2. The present invention provides a new strategy for constructing a high-quality thin shell bioactive peptide library by directly extracting thin shell bioactive peptides by enzymatic hydrolysis of fresh thin shells, and then obtaining peptides with good activity through two-step purification. Then, computer-assisted screening, in vivo activity and cell activity assays are used to quickly and high-throughput screen peptides with good antioxidant activity. The preparation method is simple.

[0040] 3. The active sites were calculated by quantum chemistry, proving that the antioxidant peptide provided by the present invention has liver protection function. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 are the SEC chromatograms and free radical scavenging rates of ABTS and DPPH of different components of F1-F31;

[0042] Figure 2 It is the HPLC spectrum of F8 and DPPH free radical scavenging;

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

[0044] Figure 4 is the DPPH radical scavenging ability of the six peptides;

[0045] Figure 5 is the ABTS free radical scavenging ability of the six peptides;

[0046] Figure 6 The protective effects of six antioxidant peptides against ethanol-induced cell damage.

[0047] Among them: A is the result of the construction of the ethanol-damaged HepG2 cell model; B is the protective effect of six antioxidant peptides on cells; C and D are the effects of antioxidant peptides on AST and ALT activities; E is the effect of antioxidant peptides on ROS levels in ethanol-induced HepG2 cells;

[0048] Figure 7 The results of active site analysis of six peptides using HOMO, Mulliken charge distribution and bond length. DETAILED DESCRIPTION

[0049] The specific implementation modes are described in detail below in conjunction with the accompanying drawings, but it should be understood that the protection scope of the present invention is not limited by the specific implementation modes.

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

[0051] The UV-visible spectrophotometer used in the examples is the Shanghai Yuanxi Q-6; the multifunctional microplate reader is the Guangzhou Lianbo Varioskan LUX; and the nanoliter liquid chromatography-electrospray-ultra-high resolution time-of-flight tandem mass spectrometer is the Thermo Fisher Easy nLC1200 / Q Exactive plus.

[0052] mM refers to mmoL / L, 1M HCl refers to 1moL / L HCl solution, and 1MNaOH refers to 1moL / L NaOH solution.

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

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

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

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

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

[0058] Example 1

[0059] The present embodiment provides a class of antioxidant peptides, which are obtained by separation and purification of thin shell antioxidants, and whose amino acid sequence is one of WWL, WDRW, WWWV, RSPWR, WPRCQL, CVKWML or any combination thereof.

[0060] The preparation method comprises the following steps in sequence:

[0061] 1) Preparation of thin shell bioactive peptides

[0062] 1.1) 30 g of thin shell meat was obtained by boiling in a 70°C water bath for 30 minutes, chopped, and then 120 ml of 0.01M HCl was added, and endogenous peptides were extracted by homogenization at 4°C for 8 minutes using a slapping homogenizer. After that, 3 times the volume of ethanol as the endogenous peptide was added to obtain an endogenous peptide mixture, and the endogenous peptide mixture was allowed to stand at 4°C for 20 hours, and then deproteinized by centrifugation at 4°C and 12,000g for 20 minutes; the supernatant was collected, and the added ethanol was removed in a rotary evaporator, placed in a -80°C refrigerator to freeze into a solid state, and then placed in a freeze dryer under vacuum conditions to obtain 5 g of freeze-dried peptide powder; with reference to GB5009.5-2016, the protein content in the freeze-dried peptide powder was determined by the Kjeldahl method to be 0.7885 g; finally, the extracted freeze-dried peptide powder was stored at -20°C.

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

[0064] 2) separating the thin shell bioactive peptide prepared in step 1), including the following separation methods in sequence:

[0065] 1) Size Exclusion Chromatography Separation

[0066] First, 1g of thin shell bioactive peptide was dissolved in 5mL ultrapure water, filtered through a 0.45μm filter membrane, and then injected into a chromatographic column filled with Sephadex G25 filler (2.5×60cm, 5.0μm). 0.01M HCl solution was used for elution at 4°C, and the flow rate was set to 5mL / 20min. The eluent was collected by a component collector, and 5mL of eluent was collected in each test tube, and 195 tubes were collected in sequence. After the collection, the absorbance values ​​of each component at 214nm and 280nm were read respectively. For specific data, see Table 1, and then a chromatogram was drawn. According to the order of collection, the eluents in every 5 test tubes starting from the 41st tube were mixed in accordance with the order of collection, and finally 31 components were obtained, recorded as F1, F2, F3, F4...F29, F30, F31, freeze-dried and stored at -20°C for standby use.

[0067] Table 1

[0068]

[0069]

[0070]

[0071]

[0072] Antioxidant activity test

[0073] The 31 components obtained by size exclusion chromatography: F1, F2, F3, F4...F29, F30, F31 (hereinafter referred to as samples) were tested for DPPH and ABTS free radical scavenging abilities. The specific testing methods are as follows:

[0074] DPPH free radical scavenging ability determination: 100 μL sample was mixed with 500 μL ethanol, 125 μL DPPH ethanol solution (2% w / v) was added, mixed and reacted for 60 min in the dark, then added to a cuvette to determine the absorbance value at 517 nm. The same volume of water was used instead of the sample as a control.

[0075] The DPPH free radical scavenging rate was calculated by the following equation:

[0076]

[0077] Determination of ABTS free radical scavenging ability: Add 10 μL of sample and 990 μL of ABTS diluent to a cuvette, react for 6 minutes in the dark, and measure the absorbance at 734 nm in a UV spectrophotometer, which is recorded as OD samplePBS was used as a blank control, and vitamin E (Trolox) with molar concentrations of 0 μM, 200 μM, 400 μM, 800 μM, and 1200 μM was used as a positive control. The absorbance values ​​were recorded as OD control and OD trolox .

[0078] The ABTS free radical scavenging rate was calculated according to the following equation:

[0079]

[0080] The Trolox concentration and ABTS free radical scavenging rate were plotted and the standard curve regression equation was obtained by linear fitting. The TEAC value of the sample was calculated based on the equation and the ABTS free radical scavenging rate of the sample; the result was expressed in μM TE / g sample. As a result, it was found that the F8 and F15 components had the highest DPPH free radical scavenging ability compared with other components, such as Figure 1 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 included component A and component B; component A was acetonitrile (ACN), and component B was trifluoroacetic acid (TFA) with a volume concentration of 0.1%; the flow rate was set at 1 mL / min, the column temperature was 30°C, and the detection wavelength was 214 nm.

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

[0084] The eluate was collected using a fraction collector, and each fraction was collected every 1 minute, and was sequentially recorded as F8-1, F8-2, F8-3........F8-11, F8-12, F8-13, F8-14;

[0085] The F15 component was reconstituted with ultrapure water, filtered through a 0.45 μm filter membrane, and injected into the chromatographic column. The elution gradient of the separation was as follows: 0-2 minutes, 100% B; 2-10 minutes, 100%-90% B, 0%-10% A; 10-20 minutes, 90%-75% B, 10%-25% A; 20-25 minutes, 75%-60% B, 25%-40% A; 25-35 minutes, 60%-10% B, 4 0%-90% A; 35-40 minutes, 10%-100% B, 90%-10% A; 40-45 minutes, 100% A; use a fraction collector to collect the eluate, collect each fraction every 1 minute, and record them as F15-1, F15-2, F15-3...F15-14, F15-15, F15-16 in sequence; finally, the collected 30 fractions are then freeze-dried and stored at -20°C.

[0086] Antioxidant activity test

[0087] The DPPH and ABTS free radical scavenging abilities of the 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, and F15-16 (hereinafter referred to as samples) obtained by reverse phase high performance liquid chromatography were determined respectively. The specific determination methods are as follows:

[0088] DPPH free radical scavenging ability determination: 100 μL sample was mixed with 500 μL ethanol, 125 μL DPPH ethanol solution (2% w / v) was added, mixed and reacted for 60 minutes in the dark, then added to the cuvette to measure the absorbance value at 517 nm. The same volume of water was used instead of the sample as a control (Control). The DPPH free radical scavenging rate was calculated by the following equation:

[0089]

[0090] Determination of ABTS free radical scavenging ability: Add 10 μL of sample and 990 μL of ABTS diluent to a cuvette, react for 6 minutes in the dark, and measure the absorbance at 734 nm in a UV spectrophotometer, which is recorded as OD sample PBS was used as a blank control, and vitamin E (Trolox) with molar concentrations of 0 μM, 200 μM, 400 μM, 800 μM, and 1200 μM was used as a positive control. The absorbance values ​​were recorded as OD control and OD trolox .

[0091] The ABTS free radical scavenging rate was calculated according to the following equation:

[0092]

[0093] The Trolox concentration and ABTS free radical scavenging rate were plotted and the standard curve regression equation was obtained by linear fitting. The TEAC value of the sample was calculated based on the equation and the ABTS free radical scavenging rate of the sample; the result was expressed in μM TE / g sample. Figure 2 , Figure 3 As shown, data are expressed as mean ± standard error; labeled characters without common letters indicate significant differences at P < 0.05, as determined by one-way analysis of variance; it was found that F8-11 had the highest DPPH free radical scavenging ability, and F15-6 had the strongest ABTS free radical scavenging ability.

[0094] IV) Construction of thin-shell peptide library

[0095] Identification of active peptide fragments by nano-liquid chromatography-electrospray ionization-ultra-high resolution time-of-flight tandem mass spectrometry

[0096] The two freeze-dried samples F8-11 and F15-6 with the best free radical scavenging rate in step 3) were dissolved in ultrapure water to 1 mg / ml F8-11 solution and 1 mg / ml F15-6 solution respectively; filtered with a 0.22 μm filter membrane respectively; injected into an EASY-nLC1200 nano liquid chromatography system (Thermo Scientific, MA, USA) with an injection volume of 2 μL, first enriched with an Acclaim PepMapTM 100trap column (75 μm×2 cm, nano Viper 2Pk C18, 3 μm, pore size 100 μm), and then separated with an Acclaim Pep MapTM RSLC column;

[0097] The mobile phase consists of component A and component B, wherein: component A is a 0.1% formic acid aqueous solution (FA) by volume; component B is composed of a formic acid aqueous solution and acetonitrile in a volume ratio of 2:8, and 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°C;

[0098] The elution gradient was: 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 peptide sequences of the peptides in the freeze-dried samples obtained after separation by AcclaimPep MapTM RSLC column were identified by peptideomics strategy, and 2216 peptides were obtained. All the identified peptide sequences were included in the 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 QDJ 2203 CSNN 2209 DTE 2215 LL 2198 SVVPSPK 2204 MNN 2210 FYF 2216 UE

[0116] 5) Computer-assisted screening

[0117] The potential biological activity of the 2216 peptides identified in step 4) was predicted using Peptide Ranker software, specifically: the number and position of certain specific amino acid residues in the peptide sequence were scored, and a value of 0-1 was given. The larger the value, the greater the probability of having potential biological activity. Peptides with a score of >0.8 (234 peptides) were selected;

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

[0119] Based on machine learning technology, Toxin Pred and AllerTOP software can be used to predict the potential toxicity of the 20 peptides after screening, and AllerTOP software can be used to predict the potential allergenicity of the 20 peptides, and finally 6 peptides can be screened out.

[0120] The Biopep database can be used to compare peptide sequences with reported literature to determine whether they have been reported before and preliminarily determine whether they are new peptide sequences.

[0121] Based on this, peptides WWL, WDRW, WWWV, RSPWR, WPRCQL, and CVKWML with Peptide Ranker values ​​> 0.8, CPP Pred values ​​> 0.5, no toxicity, no sensitization, and potential bioactivity were screened out, as shown in Table 4. This indicates that 6 new peptides with potential bioactivity, good cell permeability, no toxicity, and no potential sensitization were screened out by computer-assisted screening.

[0122] Table 4 Six peptides with potential bioactivity, no toxicity and no allergenicity

[0123] serial number sequence PeptideRanker Rating CPPPred score Toxicity prediction Prediction of sensitization 1 WWL 0.99 0.65 Non-toxic Non-allergenic 2 WWWV 0.99 0.55 Non-toxic Non-allergenic 3 WDJ 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] In order to prove the antioxidant activity of the six peptides WWL, WDRW, WWWV, RSPWR, WPRCQL, and CVKWML screened in this application, the antioxidant activity verification experiment is given below:

[0125] 1. Verification of in vitro antioxidant activity

[0126] The 6 peptide segments (WWL, WDRW, WWWV, RSPWR, WPRCQL, CVKWML) obtained by screening were subjected to solid phase synthesis (synthetic peptides were synthesized by solid phase chemical method by commissioning Gill Biochemical (Shanghai) Co., Ltd., and the obtained synthetic peptides were polypeptide solid powders with purity>95%), and different synthetic peptides (polypeptide solid powders with purity>95%) were obtained. They were labeled as synthetic peptide WWL, synthetic peptide WWWV, synthetic peptide WDRW, synthetic peptide WPRCQL, synthetic peptide CVKWML, and synthetic peptide RSPWR. The synthetic peptides were prepared into synthetic peptide 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 with deionized water, and the free radical scavenging ability of these 6 synthetic peptides was measured (the method is consistent with (1)).

[0127] The experimental results are as follows Figure 4 and Figure 5 As shown in the figure, the abscissa is the concentration of the synthetic peptides WWL, WWWV, WDRW, WPRCQL, CVKWML and RSPWR) (data are expressed as mean ± standard error; marks without common letters indicate significant differences of P < 0.05. The results of one-way analysis of variance showed that the six peptides had good in vitro DPPH free radical scavenging ability, could scavenged DPPH free radical activity in a dose-effect relationship, and all had good in vitro ABTS free radical scavenging ability.

[0128] 2. Verification of intracellular antioxidant activity:

[0129] (a) Construction of ethanol-damaged HepG2 cell model: 100 μL of cell suspension was inoculated in a 96-well plate and incubated for 24 h (2 × 10 4 100 μL serum-free medium was added and incubated for 24 h, the old medium was discarded, different concentrations of ethanol (0.25 M-2 M) were added to the experimental group, and serum-free medium was added to the control group and incubated for 24 h. Cell viability was determined using the CCK-8 assay to explore the ethanol concentration at which cell viability was 50%.

[0130] CCK-8 method: HepG2 cells were cultured at 2 x 10 5 The cells were seeded in 96-well plates at a density of 100 μg / ml and cultured at 37 °C for 24 hours. After the incubation period, the cells were exposed to synthetic peptides (0.1 mM) for 24 hours. Thereafter, the 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 incubation for 1 hour, the absorbance was immediately measured at OD 450nm on a multifunctional fluorescent microplate reader. The results are expressed as a percentage of cell viability. Cell viability is calculated by the following formula:

[0131]

[0132] (b) Pre-protection of HepG2 cells damaged by ethanol by thin-shell peptides: The cells were divided into control group, model group and experimental group. 100 μL cell suspension was inoculated in a 96-well plate for 24 hours (2×10 4 The medium was changed after 24 h. The experimental group was treated with 100 μL of 0.1 mM antioxidant peptide, while the blank group and the injury control group were replaced with serum-free medium. After 24 h, the medium was changed again. The control group continued to add 100 μL of serum-free medium, and the model group and the experimental group added 100 μL of medium containing ethanol at the concentration selected in (a). After incubation for 24 h, the cell viability was determined using the CCK-8 method described in (a).

[0133] (c) Determination of intracellular reactive oxygen species (ROS) levels: The relative level of intracellular ROS was measured using the DCFH-DA fluorescent probe. Cells were pretreated with thin-shell peptide for 24 hours and then induced with ethanol for an additional 24 hours before collection, washed three times with pbs, and immediately added with DCFH-DA (10 μM) and incubated for 30 minutes. After washing three times with sterile phosphate-buffered saline, the fluorescence values ​​at an excitation wavelength of 485 nm and an emission wavelength of 525 nm were quickly measured using a multifunctional fluorescence microplate reader.

[0134] (d) Determination of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities: ALT and AST detection kits from Nanjing Jianshe Bioengineering Institute were used for ALT and AST determination. According to the manufacturer's instructions, cells were collected and ultrasonically disrupted in an ice water bath, and then ALT and AST were measured.

[0135] The experimental results are as follows Figure 6 As shown, (n = 6 per group), data are expressed as mean ± standard error. Marks without common letters indicate significant differences at P < 0.05. The results of one-way analysis of variance showed that the six peptides can effectively remove ROS produced by HepG2 cells induced by ethanol and effectively alleviate ethanol-induced liver damage.

[0136] 3. Quantum chemical calculation of peptide structure-activity analysis

[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 Gaussian input files, i.e., Gaussian 16W input files. DFT calculations were performed using GaussView software (version 6.0) and Gaussian 16W program (Gaussian Inc., Wallingford, CT, USA). The geometry was obtained using Gaussian 16W program, and 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 showed that the active sites of the six antioxidant peptides were all on the NH of the indole ring of the tryptophan side chain.

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

[0140]

[0141] Finally, the six peptide segments CVKWML, WWL, WDRW, WWWV, RSPWR, and WPRCQL with better antioxidant effects in vitro and in cells are the target peptides of the present invention.

Claims

1. A class of antioxidant peptides, characterized in that: The amino acid sequence of the antioxidant peptide is one of WWL, WDRW, WWWV, RSPWR, WPRCQL, CVKWML or any combination thereof.

2. The method for preparing the antioxidant peptide according to claim 1, characterized in that: The steps are as follows: 1) Extracting bioactive peptides from the thin shells; 2) Preliminarily separating the thin shell bioactive peptides extracted in step 1) by size exclusion chromatography to obtain crude active peptides; 3) measuring the antioxidant activity of the crude active peptides separated in step 2) to screen out two crude active peptides with high antioxidant activity; 4) The crude active peptides screened in step 4) are further separated by reverse phase high performance liquid chromatography to obtain active peptides; 5) measuring the antioxidant activity of the active peptides obtained in step 4) and screening out the two most active active peptides; 6) The active peptides obtained in step 5) are separated by a liquid phase system and then sent to a mass spectrometry system for amino acid sequence identification to establish a thin-shell polypeptide library; 7) The peptides in the thin-shell peptide library were scored by Peptide Ranker, scored by CPP Pred, and predicted for toxicity and sensitization to obtain the potential bioactive peptides WWL, WDRW, WWWV, RSPWR, WPRCQL, and CVKWML.

3. The method for preparing the antioxidant peptide according to claim 1, characterized in that: Step 1) extracting the thin shell bioactive peptides from the thin shell comprises the following steps: 1) Steaming the lamina to obtain lamina meat, adding the lamina meat to homogenize under acidic conditions to extract endogenous peptides, then adding a solvent, centrifuging to separate proteins, collecting the supernatant, removing the solvent, and freeze-drying to obtain freeze-dried peptides; 2) dispersing the lyophilized peptide prepared in step 1) in distilled water, adjusting the pH to acidic, then adding pepsin to simulate gastric digestion and hydrolyze to obtain a mixture, adjusting the pH to weak alkaline again, adding pancreatic enzyme to continue simulating intestinal digestion, and after the reaction is completed, inactivating the enzyme to stop digestion; then centrifuging and collecting the supernatant, lyophilizing, and obtaining the thin-shell bioactive peptide.

4. The method for preparing the antioxidant peptide according to claim 1, characterized in that: The method of preliminary separation of active peptides by molecular exclusion chromatography is as follows: the thin-shell bioactive peptides are dissolved in ultrapure water, filtered and then injected into a chromatographic column, a chromatographic column filled with Sephadex G25 is used, 0.01M HCl solution is used for elution at 4°C, and the flow rate during elution is 5mL / 20min; the eluent is collected by a component collector, 5mL of eluent is collected in each test tube, and 195 tubes are collected in sequence, and the absorbance values ​​of each component at 214nm and 280nm are read respectively after collection, and the eluents in every 5 test tubes starting from the 41st tube are mixed in sequence according to the order of collection, and finally 31 components are obtained, which are recorded as F1, F2, F3, F4...F29, F30, F31, freeze-dried and stored at -20°C for future use.

5. The method for preparing the antioxidant peptide according to claim 1, characterized in that: Step 4) During the reverse phase high performance liquid chromatography separation, the chromatographic column is an Agilent ZORBAX SB-Aq C18 chromatographic column; the mobile phase is composed of component A and component B, the component A is acetonitrile; the component B is trifluoroacetic acid with a volume concentration of 0.1%; the flow rate is set to 1 mL / min, the column temperature is 30° C., and the detection wavelength is 214 nm; The elution gradient of the separation is as follows: 0-2 minutes, 100% B; 2-10 minutes, 100%-90% B, 0%-10% A; 10-20 minutes, 90%-75% B, 10%-25% A; 20-25 minutes, 75%-60% B, 25%-40% A; 25-35 minutes, 60%-10% B, 40%-90% A; 35-40 minutes, 10%-100% B, 90%-10% A; 40-45 minutes, 100% A; Use a fraction collector to collect the eluate, and collect each fraction every 1 minute to obtain a total of 30 fraction freeze-dried samples, which are named 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 in sequence. The collected fractions are then freeze-dried and stored at -20°C.

6. The method for preparing the antioxidant peptide according to claim 1, characterized in that: The antioxidant activity tests described in step 2) and step 4) are both to measure the absorbance of crude active peptides and active peptides at 517nm in a cuvette, and calculate the DPPH free radical scavenging; to measure the absorbance of crude active peptides and active peptides at 734nm, and calculate the ABTS free radical scavenging rate.

7. The method for preparing the antioxidant peptide according to claim 5, characterized in that: Step 6) The method for constructing a thin shell polypeptide library comprises the following steps: 1) Dissolving F8-11 and F15-6 selected in claim 5 with ultrapure water, filtering, and injecting into EASY-nLC1200nano liquid chromatography system for separation; The mobile phase consists of component A and component B, wherein: component A is a formic acid aqueous solution with a volume concentration of 0.1%; component B is composed of a formic acid aqueous solution and acetonitrile with a volume ratio of 2:8, and the formic acid aqueous solution in component B has a volume concentration of 0.1%; the flow rate is 0.3 μL / min, and the column temperature is 30°C; The elution gradient was: 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; 2) The peptide fragments in the freeze-dried samples separated by Acclaim Pep MapTM RSLC column were identified by peptideomics strategy, and 2216 peptides were obtained. The identified 2216 peptide sequences were included in the peptide library to establish a thin shell peptide library.

8. The method for preparing the antioxidant peptide according to claim 1, characterized in that: Step 7) The peptides in the thin-shell peptide library are scored by Peptide Ranker, scored by CPP Pred, and toxicity and sensitization prediction methods are as follows: 1) Peptide Ranker software was used to predict the potential biological activities of the 2216 peptides identified, and 234 peptides with scores > 0.8 were selected; 2) The cell permeability of the above 234 peptides was predicted using CPP Pred software, and 20 peptides with scores > 0.5 were selected; 3) Toxin Pred software was used to predict the potential toxicity of the 20 peptides after screening, and AllerTOP software was used to predict the potential allergenicity of the 20 peptides. Finally, 6 peptides WWL, WDRW, WWWV, RSPWR, WPRCQL, and CVKWML were screened out.

9. Use of the antioxidant peptide according to claim 1 as a drug for the prevention / treatment of ethanol-induced liver damage.

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