CN111423495A

CN111423495A - Rapana venosa polypeptide with function of resisting oxidative stress damage as well as preparation method and application of rapana venosa polypeptide

Info

Publication number: CN111423495A
Application number: CN202010313278.XA
Authority: CN
Inventors: 张姗姗; 刘可春; 李晓彬; 张轩铭; 张云; 侯海荣; 夏青; 孙晨
Original assignee: Biology Institute of Shandong Academy of Sciences
Current assignee: Biology Institute of Shandong Academy of Sciences
Priority date: 2020-04-20
Filing date: 2020-04-20
Publication date: 2020-07-17
Anticipated expiration: 2040-04-20
Also published as: CN111423495B

Abstract

The invention relates to a rhodospirillum polypeptide with oxidative stress damage resistance and a preparation method and application thereof.A rhodospirillum polypeptide compound with oxidative stress damage resistance function has an amino acid sequence shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.5 or SEQ ID No. 6. the invention also discloses the application of the polypeptide as a drug effect component in the preparation of a drug for treating diseases caused by oxidative stress damage or as a health-care component in the preparation of an antioxidant health-care product.

Description

Rapana venosa polypeptide with function of resisting oxidative stress damage as well as preparation method and application of rapana venosa polypeptide

Technical Field

The invention relates to a rapana venosa polypeptide with antioxidant stress injury and a preparation method and application thereof, belonging to the technical field of functional polypeptides.

Background

Oxidative stress refers to the condition that a large amount of Reactive Oxygen Species (ROS) and other oxidizing substances are generated in vivo due to endogenous (endogenous reactive oxygen species generated by various metabolic reactions) or exogenous (environmental factors, drugs and body aging) causes imbalance of the redox balance of the body, so that excessive ROS are accumulated in the body, and the excessive ROS in the body can reduce the oxidation resistance of the body per se, generate lipid peroxidation, damage and even apoptosis of cell DNA, promote the generation of inflammatory factors, hinder the metabolism of nutrient substances and damage the functions of tissues, thereby causing diseases. Modern researches show that oxidative stress is crucial to the occurrence and development of chronic non-infectious diseases such as cardiovascular and cerebrovascular diseases, neurological diseases, inflammatory diseases and the like, with the continuous improvement of living standard of people, diseases caused by oxidative stress damage become main diseases affecting the health of people, and with the continuous aggravation of the aging situation of China, the probability of suffering from oxidative stress damage related diseases is higher for the aged population due to the reduction of body functions, and great economic burden is caused to families and lives, so that the early intervention on the oxidative stress state of the organism is performed, the oxidative reduction imbalance state of the organism is improved, the occurrence and development of diseases are reduced, and the research hotspot of the current medicine research and development and the large health industry is formed.

The marine organisms are of various and large numbers, and are derived from active substances of marine organisms, such as peptides and polysaccharidesTerpenoids and the like have good activities of resisting inflammation, oxidation, bacteria and virus^[1]. With the advance of the national policy of "sea-forcing," the development of marine organisms has been more and more emphasized in recent years. The marine organisms have great difference from terrestrial organisms in living environments, and active substances derived from the marine organisms often have novel structures and unique biological activities, so that more possibilities are provided for lead compounds required by the research and development of new drugs.

For example, chinese patent document CN109180781A (application No. 201810915407.5) discloses a polypeptide having a function of repairing oxidative damage, and a preparation method and an application thereof. A polypeptide with function of repairing oxidative damage has an amino acid sequence shown in SEQ ID NO. 1. The invention also discloses the application of the polypeptide as a drug effect component in preparing a drug for treating diseases caused by oxidative damage or as a health-care component in preparing an antioxidant health-care product. The invention discloses a polypeptide compound containing 10 amino acid residues extracted from a Chinese osbeckia for the first time, and the detection shows that the polypeptide compound can be used for inhibiting the generation of angiotensin converting enzyme in blood vessels, inhibiting the increase of blood sugar and repairing oxidative stress injury caused by peroxide by eliminating the generation of ROS in vivo, and can be used for developing a medicament and an antioxidant health-care product for subsequently treating diseases caused by oxidative injury.

The venorula (Raoana venosa) genus Mollusca (molusca), Gastropoda (Gastropoda), gilles (Prosobranchia), Neogastropoda (Neogastropoda), osteanaceae (Muricidae), is a large marine animal of great economic importance. The method is mainly distributed in yellow sea, Bohai sea and east sea in China, Japanese coastal sea, Korean peninsula and other areas. The soft part of the rapana venosa consists of three parts, namely a head part, a foot part and an internal organ ball, so that the rapana venosa has the advantages of fleshy and compact meat quality, delicious taste and high nutritional value. To date, most of the research on the rapana venosa has focused on its biology, such as genome, nutrition, reproductive characteristics, etc., or its nutritional components, such as crude proteins, polysaccharides, crude lipids, etc. Currently, there are few reports relating to the active ingredient of the rapana venosa.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a polypeptide with the function of resisting oxidative stress damage and a preparation method and application thereof.

The technical scheme of the invention is as follows:

the polypeptide with the function of resisting oxidative stress damage has an amino acid sequence shown as SEQ ID NO. 1.

SEQ ID NO.1：Met-Val-Leu-Leu-Gly-Val-Leu-Met-Gly MVLLGLVLMG。

The polypeptide with the function of resisting oxidative stress damage has an amino acid sequence shown as SEQ ID NO. 2.

SEQ ID NO.2：Ala-Arg-Leu-Gly-Leu-Ala-Thr-Leu ARLGLATL

The polypeptide with the function of resisting oxidative stress damage has an amino acid sequence shown as SEQ ID NO. 3.

SEQ ID NO.3：Leu-Leu-Thr-Arg-Ala-Gly-Leu LLTRAGL

The polypeptide with the function of resisting oxidative stress damage has an amino acid sequence shown as SEQ ID No. 5.

SEQ ID NO.5：Lys-Ser-Thr-Glu-Leu-Leu-Ile KSTELLI

The polypeptide with the function of resisting oxidative stress damage has an amino acid sequence shown as SEQ ID NO. 6.

SEQ ID NO.6：Phe-Gly-Ile-Asn-Leu-Ile-Gln FGINLIQ

A polypeptide combination with antioxidant stress injury function comprises polypeptide combination composed of amino acid sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.

A method for extracting the polypeptide with the function of resisting oxidative stress damage comprises the following steps:

(1) removing shells of the rapana venosa, taking all soft tissue parts, grinding, adding an acid solution with the pH value of 1.0-4.0, adding pepsin with the weight of 5-20% of the weight of the soft tissue, performing oscillatory enzymolysis for 1-5 hours at 35-40 ℃, adjusting the pH value to 7.0-9.0, adding trypsin and chymotrypsin with the weight of 5-20% of the weight of the soft tissue, performing oscillatory enzymolysis for 1-5 hours at 35-40 ℃, centrifuging, taking supernatant, concentrating and freeze-drying to obtain the rapana venosa polypeptide extract;

(2) redissolving the rapana venosa polypeptide extract prepared in the step (1) by using a buffer salt solution, carrying out column separation by using Sephadex G25, eluting 5 column volumes by using a buffer salt solution with the pH value of 6.0-8.0 as an eluent, collecting fractions of column volumes of 3 rd-5 th, freeze-drying, dissolving dry powder saline, separating by using Sephadex L H-20, collecting a sample by using the saline as the eluent at the speed of 10m L/45 min, collecting one part every 45min, combining 14 th-20 th parts of active section eluent, and concentrating to prepare a polypeptide active section crude extract;

(3) dissolving the crude extract of the active polypeptide segment prepared in the step (2) by using ammonium acetate buffer solution with the concentration of 10mM and the pH value of 5.8-6.2, filtering the solution by using a 4.5 mu m microporous membrane, and separating the solution by using a Welch HI L IC Amide column, wherein the binary mobile phase comprises Acetonitrile (ACN) and ammonium acetate buffer solution with the concentration of 10mM and the pH value of 5.8-6.2, the volume ratio of the Acetonitrile (ACN) to the ammonium acetate buffer solution is 85:15, and the flow rate is 0.8 ml/min^-1Collecting eluent with an absorption peak with in-vitro DPPH free radical scavenging activity at 210nm, identifying and determining amino acid composition, and freeze-drying to obtain the polypeptide with the function of resisting oxidative stress damage.

Preferably, in the step (1), the enzymolysis pH value of pepsin is 2.0-3.0, and the enzymolysis pH value of trypsin and chymotrypsin is 7.2-8.0; further preferably, in the step (1), the pH regulator is hydrochloric acid and sodium hydroxide.

Preferably, in the step (1), the enzyme activity ratio of trypsin to chymotrypsin is 1: (0.2-5).

According to the present invention, in the step (2), the buffer salt system is a phosphate buffer system, and the pH value is 6.8 to 7.2.

Preferably, in the step (3), L C-MS protein identification technology is adopted for identifying and determining amino acid composition.

The application of one or the combination of more than two of the polypeptides with the function of resisting oxidative stress damage as a drug effect component in preparing the drugs for treating oxidative stress damage diseases.

The application of one or the combination of more than two of the polypeptides with the function of resisting oxidative stress damage as an effective component in preparing antioxidant health-care food.

Advantageous effects

The invention discloses 5 anti-oxidative stress active peptides extracted from rapana venosa for the first time, and the detection shows that the 5 polypeptide compounds can independently remove the generation of ROS in vivo, reduce the macrophage aggregation in zebra fish bodies, inhibit the generation of angiotensin converting enzyme in blood vessels and the generation of inflammatory cytokine interleukin 1(I L-11), repair the body damage caused by oxidative stress, and can be used for developing the medicines and anti-oxidative health-care products for preventing diseases caused by oxidative stress damage in the subsequent process, thereby having wide market prospect.

Drawings

FIG. 1 is a photograph of the raw Rapana venosa used in the example;

in the figure: A. the overall appearance of the venosa; soft tissue of the Bolus omaculatus;

FIG. 2 is a graph of the Molecular Weight (MW) distribution from the active segment of Rapana venosa as determined by gel permeation chromatography;

FIG. 3 is a graph showing the MS/MS mass spectrum results of the amino acid sequence of the active peptide;

wherein: FIG. 3-1 is a graph showing the result of MS/MS mass spectrometry of the amino acid sequence shown in SEQ ID NO. 1;

FIG. 3-2 is a graph showing the result of MS/MS mass spectrometry of the amino acid sequence shown in SEQ ID NO. 2;

FIGS. 3-3 are graphs showing the results of MS/MS mass spectrometry of the amino acid sequence shown in SEQ ID NO. 3;

FIGS. 3 to 4 are graphs showing the results of MS/MS mass spectrometry of the amino acid sequence shown in SEQ ID NO. 4;

FIGS. 3 to 5 are graphs showing the results of MS/MS mass spectrometry of the amino acid sequence shown in SEQ ID NO. 5;

FIGS. 3 to 6 are graphs showing the results of MS/MS mass spectrometry of the amino acid sequence shown in SEQ ID NO. 6;

FIG. 4 is a graph showing the results of activity detection of fractions eluted by the Rapana venosa Sephadex L H-20;

FIG. 5 shows the detection result of HI L IC chromatographic column of crude extract of active segment of Rapana venosa;

FIG. 6 is a graph showing the results of the HI L IC column detection of samples of each fraction section in comparative example 1;

in the figure, A is HI L IC chromatogram of 8 th to 13 th samples, B is HI L IC chromatogram of 21 st to 29 th samples;

FIG. 7 is a graph of the HI L IC column assay results for a sample of the cut fraction from comparative example 2;

FIG. 8 is a graph showing the repairing effect of each sample on the in vivo oxidative damage of zebra fish;

FIG. 9 is a graph of the anti-inflammatory effect of each sample on zebrafish in vivo;

in the figure: blank control; b, model group; c positive control group; group D example 1; e example 2-1; f example 2-2; g examples 2-3; examples 2-4; i examples 2 to 5; j examples 2-6;

FIG. 10 is a graph showing the effect of the docking of each sample with ACE enzyme based on molecular docking;

wherein: FIG. 10-1 is a 3D diagram of the molecular docking of example 2-1 and example 2-4 with ACE enzyme;

FIG. 10-2 is a 2D diagram of the molecular docking of example 2-1 and example 2-4 with ACE enzyme;

FIG. 11 is a graph showing the effect of docking I L-11 on each sample based on molecular docking technology;

FIG. 11-1 is a 3D diagram of the molecular docking of example 2-1 and example 2-4 with I L-11;

FIG. 11-2 is a 2D diagram of the molecular docking of example 2-1 and example 2-4 with I L-11;

Detailed Description

The technical solutions of the present invention are further described below with reference to the following embodiments and the drawings of the specification, but the scope of the present invention is not limited thereto.

Source of biological material

The venosa is purchased from Shandong Jinan seafood, a common commercial product, as shown in figure 1.

Detection method

Method for detecting molecular weight distribution of active component

Using TSK-gel G2000 SW_XLColumn (7.8mm × 250mm) (TOSOH, Yamaguchi, Japan), Molecular Weight (MW) distribution from the active fragment of Rapana venosa (FIG. 2) determined by gel permeation chromatography, mobile phase from 0.1mol L^-1Phosphate bufferLiquid (pH 6.7) and 0.1 mol. L^-1Na₂SO₄Composition, flow rate set at 0.2m L. min^-1。

Using ribonuclease (13700Da), aprotinin hydrochloride (6511Da), angiotensin II (1046Da), HH L (430Da) and L-serine (105Da) as control, and plotting an elution volume-molecular weight curve with In Mw of 17.50-0.089T (R)²0.9785, Mw is molecular weight and T is elution volume).

Method for detecting composition of active component amino acid

Dissolving lyophilized active peptide to be detected in 6mol L^-1Hydrolysis in HCl (1mg peptide/m L HCl), 24 hours in a 110 ℃ drying cabinet filtered hydrolysed samples were evaporated at 45 ℃ by a rotary evaporator the residue was dissolved in distilled water and freeze dried then samples and mixture amino acid standards were derivatised with AQC and passed through RP-HP L C₁₈And (4) measuring. The amino acid composition of the sample fractions was identified and quantified from a standard curve of mixed amino acids (table 1). All samples were assayed in triplicate.

Nano-L C-L TQ-Orbitrap-MS/MS identification of active peptide sequence

Amino acid sequence identification of active peptides using EASY-Nlc1000 chromatography system (Thermo Finnigan, Bremen, Germany), L TQ OrbitrapVelos Pro Mass Spectroscopy (Thermo Finnigan, Bremen, Germany) purified peptides were purified using a concentration of 0.1 mg.m L with 0.1% trifluoroacetic acid^-1Then 2 μ L samples were injected into a capture column (100 μm × mm, RP-C18, thermo Inc.) for preconcentration, after which the preconcentrated samples automatically entered an analytical column (75 μm × mm, RP-C18, thermo Inc.) with 0.1% (v/v) formic acid as eluent in ultrapure water for an Analysis period of 60min in a detection mode of positive ion mode, spray voltage of 1.8kV, ion transport capillary temperature of 250 ℃, corrected with standard calibration solution before use, a parent ion scan range of 350-0.25, activation time: 30ms, dynamic exclusion time: for 30 s. The resolution of MS1 was 60,000 at M/Z400, and MS2 was mass resolved in the ion trap. The primary mass spectrum is acquired in a profile mode, and the secondary mass spectrum is acquired in a centroid mode to reduce the size of a data file. Mascot 2.3 software (Matrix Science, USA) was used for data analysis. The database is a moth spiroid database, the enzyme is trypsin, and the maximum allowable missed cutting site is 2. The fixed modification is as follows: carbammidomethyl (C); the variable modifications are: acetyl (Protein N-term), deamidated (NQ), dioxidation (W), oxidation (M); the MS tolerance is + -30 ppm, and the MSMS tolerance is + -0.15 Da. The NCBInr database was used for peptide identification. Only identified peptides with expected values below 0.05 were considered. The BIOPEP database was used to search for previously identified amino acid sequences with antioxidant properties. The MS/MS mass spectrum result of the amino acid sequence of the active peptide is shown in FIG. 3.

Example 1

The method for extracting the polypeptide with the function of resisting oxidative stress damage comprises the following steps:

(1) removing shells of the rapana venosa, taking all soft tissue parts, grinding, and adopting a multi-digestive tract enzyme semi-bionic preparation technology, namely adding a 5-fold acid aqueous solution (pH value is 2.2) into the rapana venosa tissue, adding pepsin according to the enzyme substrate ratio of 8%, oscillating and extracting for 2 hours at 37.6 ℃, then adjusting the pH value of a reaction system to 7.8, adding trypsin and chymotrypsin according to the enzyme substrate ratio of 8%, wherein the enzyme activity ratio of the trypsin to the chymotrypsin is 1: extracting under oscillation at 37.6 deg.C for 2 hr, centrifuging the reaction solution, collecting supernatant, concentrating, lyophilizing, and storing dry powder at low temperature to obtain Mallotus philippinensis polypeptide extract;

(2) redissolving the polypeptide extract prepared in the step (1) by using a buffer salt solution, carrying out column separation by using Sephadex G25, eluting 5 column volumes by using a phosphate buffer solution with the pH value of 6.8 as an eluent, collecting fractions with the column volumes of 3 rd to 5 th, freeze-drying, dissolving dry powder by using saline, carrying out separation by using Sephadex L H-20, collecting a sample by using the saline as the eluent at the speed of 10m L/45 min, collecting one part every 45min, combining the in-vitro DPPH free radical scavenging activity, combining 14 th to 20 th parts of active segment eluent (figure 4), concentrating to prepare a polypeptide active segment crude extract, and representing that the molecular weight of the active peptide segment of the Neptunella is distributed in a region less than 3000Da by adopting a GPC method;

(3) dissolving the crude extract of the active polypeptide segment prepared in the step (2) by using ammonium acetate buffer solution with the concentration of 10mM and the pH value of 5.8-6.2, filtering the solution by using a 4.5 mu m microporous membrane, and separating the solution by using a Welch HI L IC Amide column, wherein the binary mobile phase comprises Acetonitrile (ACN) and ammonium acetate buffer solution with the concentration of 10mM and the pH value of 5.8-6.2, the volume ratio of the Acetonitrile (ACN) to the ammonium acetate buffer solution is 85:15, and the flow rate is 0.8 ml/min^-1Combining the in vitro DPPH free radical scavenging activity, collecting the eluent with active absorption peak at 210nm (figure 5), lyophilizing to obtain polypeptide with anti-oxidative stress injury function, and determining the amino acid composition by L C-MS protein identification technology.

Through detection, the polypeptide amino acid sequence with the function of resisting oxidative stress damage is shown as SEQ ID No. 1-6:

SEQ ID NO.1：Met-Val-Leu-Leu-Gly-Val-Leu-Met-Gly MVLLGLVLMG。

SEQ ID NO.2：Ala-Arg-Leu-Gly-Leu-Ala-Thr-Leu ARLGLATL

SEQ ID NO.3：Leu-Leu-Thr-Arg-Ala-Gly-Leu LLTRAGL

SEQ ID NO.4：Gly-Thr-Ser-Phe-Thr-Thr-Thr-Ala-Glu-Arg GYSFTTTAER

SEQ ID NO.5：Lys-Ser-Thr-Glu-Leu-Leu-Ile KSTELLI

SEQ ID NO.6：Phe-Gly-Ile-Asn-Leu-Ile-Gln FGINLIQ

example 2-1

The polypeptide with the amino acid sequence shown in SEQ ID NO.1 is artificially synthesized by adopting an Fmoc solid phase synthesis method (Liu Zhen, Huangqiang. Fmoc solid phase synthesis method, Guangxi national academy of sciences, 1999, 5 (2): 110-.

Examples 2 to 2

The polypeptide with the amino acid sequence shown in SEQ ID NO.2 is artificially synthesized by adopting an Fmoc solid phase synthesis method (Liu Zhen, Huangqiang. Fmoc solid phase synthesis method, Guangxi national academy of sciences, 1999, 5 (2): 110-.

Examples 2 to 3

The polypeptide with the amino acid sequence shown in SEQ ID NO.3 is artificially synthesized by adopting an Fmoc solid phase synthesis method (Liu Zhen, Huangqiang. Fmoc solid phase synthesis method, Guangxi national academy of sciences, 1999, 5 (2): 110-.

Examples 2 to 4

The polypeptide with the amino acid sequence shown in SEQ ID NO.4 is artificially synthesized by adopting an Fmoc solid phase synthesis method (Liu Zhen, Huangqiang. Fmoc solid phase synthesis method, Guangxi national academy of sciences, 1999, 5 (2): 110-.

Examples 2 to 5

The polypeptide with the amino acid sequence shown in SEQ ID NO.5 is artificially synthesized by adopting an Fmoc solid phase synthesis method (Liu Zhen, Huangqiang. Fmoc solid phase synthesis method, Guangxi national academy of sciences, 1999, 5 (2): 110-.

Examples 2 to 6

The polypeptide with the amino acid sequence shown in SEQ ID NO.6 is artificially synthesized by adopting an Fmoc solid phase synthesis method (Liu Zhen, Huangqiang. Fmoc solid phase synthesis method, Guangxi national academy of sciences, 1999, 5 (2): 110-.

Comparative examples 1 to 1

The method of example 1, except that the eluate collected in step (2) is concentrated in 8 th to 13 th fractions (A) to obtain a corresponding polypeptide mixture sample. According to the purification method described in step (3), no corresponding chromatographic peak was observed under the same retention time conditions (FIG. 6). The active polypeptide is only present in the 585-900 min fraction. Therefore, the sample prepared in the step (2) is used as a test sample for the next activity evaluation experiment.

Comparative examples 1 to 2

The method of example 1, except that the eluate collected in step (2) is concentrated in 21 st to 29 th fractions (B) to obtain corresponding polypeptide mixture samples. According to the purification method described in step (3), no corresponding chromatographic peak was observed under the same retention time conditions (FIG. 6). The active polypeptide is only present in the 585-900 min fraction. Therefore, the sample prepared in the step (2) is used as a test sample for the next activity evaluation experiment.

Comparative example 2

The process as described in example 1, except that the experimental starting material described in step (1) was the whole soft tissue of the whelk. According to the purification method described in step (3), no corresponding chromatographic peak was observed under the same retention time conditions (FIG. 7). The extract of the whole soft tissue of the cone snail does not contain the active polypeptide. Therefore, the sample prepared in the step (2) is used as a test sample for the next activity evaluation experiment.

Examples of the experiments

DPPH radical scavenging Activity

The DPPH radical scavenging activity of the sample polypeptides was tested according to the method described by L ee et al.

200. mu.l of the fraction was added to a solution containing 200. mu.l of 0.15mmol-L^-1Tubes of DPPH ethanol solution and vortex the mixture for a few seconds. The mixture was then incubated in the dark at 37 ℃ for 12 hours. Ultrapure water was used as a control. The absorbance was measured at 517nm and determined in triplicate. The DPPH radical scavenging activity of the peptide fractions was calculated as follows:

DPPH radical scavenging Activity (%) - (1-As/Ac) × 100

Where As is the absorbance of the sample and Ac is the absorbance of the control. Although DPPH radical scavenging activity of active peptides is expressed as the semi-inhibitory concentration (IC50), IC₅₀Defined as the peptide concentration required to inhibit 50% of free radical formation. DPPH radical scavenging ratio IC of examples and comparative examples₅₀See table 2.

Determination of in vivo antioxidant Activity

The in vivo antioxidant assay of the polypeptide samples was performed by using the transgenic zebrafish line Tg (krt 4: NTR-hKikGR) cy 17.

Transgenic zebrafish embryos developing 24hpf were distributed into 24-well cell culture plates (10 embryos/well) and incubated with 2m L10 mM metronidazole (MTZ, dissolved in zebrafish culture water) and the polypeptide sample at a dose of 100 μ g.m L^-124 hours after drug treatment at 28 ℃. Zebrafish treated with fish water without metronidazole and peptide were used as vehicle control. Zebrafish treated with metronidazole without peptide were used as negative control. The peptide was replaced by Vitmin C as a positive control. Each group was performed in at least three replicates. After incubation, with tricaine (0.16%, w/v)) Zebrafish embryos were anesthetized and then observed for fluorescence and imaged using a FSX100 Bio Imaging Navigator instrument. The number of fluorescent spots was evaluated by using imagepro-plus software. The in vivo antioxidant activity of the polypeptide sample was calculated as follows:

antioxidant activity (%) ═ FS_s-FS_nc)/(FS_vc-FS_nc)×100

Wherein FS_sIs the fluorescent spot of the sample (polypeptide sample), FS_ncFluorescent dot, FS, which is a negative control_vcIs a fluorescent spot (vitamin C). The results of the in vivo antioxidant evaluation of examples 1 and 2 and comparative examples 1 and 2 are shown in FIG. 8.

Determination of in vivo anti-inflammatory Activity

Detection of in vivo anti-inflammatory activity of polypeptide samples was performed by using macrophage fluorescent TG zebrafish (zlyz: EGFP).

The healthy zebra fish which develops for 72 hours are randomly distributed into 24-pore plates, 10 zebra fish in each pore, 2ml in each pore, a blank group, a model group, a positive control group and a dosing group are arranged, the blank group is only treated by adding fish culture water, the model group is treated by adding copper sulfate later, the positive control group is treated by sequentially adding ibuprofen solution with the concentration of 100 mu g/ml and copper sulfate, and the dosing group is treated by sequentially adding sample solution with different concentrations and copper sulfate. The treatment method of the model group, the positive control group and the administration group comprises the steps of firstly treating with a medicament for a certain time, adding a proper amount of copper sulfate solution into each experimental group to enable the final concentration of the copper sulfate solution in the system to reach 20 mu g/ml, then covering and sealing, placing in a 28 ℃ illumination box for incubation for 1h, then washing off copper sulfate and medicament residues on the surfaces of zebra fish juvenile fish with fish culture water, adding a small amount of tricaine, anaesthetizing the juvenile fish, observing the macrophage change condition of each juvenile fish under a body type fluoroscope microscope, taking a picture of the zebra fish juvenile fish under 4 times, processing and analyzing the picture by adopting Image pro-plus software, and counting the number of migration of neutrophils to the spinal cord center line. The results of the in vivo anti-inflammatory evaluations of examples 1 and 2 and comparative examples 1 and 2 are shown in FIG. 9.

ACE inhibitory activity research based on molecular docking technology

The ChemDraw module and Chem3D pro module in chembioffice 2014 were used to map the 3D structures of 6 pure peptides with the polypeptide as the ligand. ACE is used as a blood pressure reduction active target point of molecular docking. The tertiary structures of ACE are derived from Protein Data Bank (PDB) databases, respectively. After the ligand was subjected to charge addition (add value) and electric field application (applied for following), and the receptor was subjected to water removal (remove water), protein treatment (clean protein), charge addition (add value) and electric field application (applied for following), the active site and the docking radius were set, and molecular docking was performed using a CDocker module in Discovery study 2016 software. According to the CDocker energy and CDocker interaction energy values, potential antihypertensive activity of 6 pure polypeptides is discussed.

IF-1 α inhibitory activity research based on molecular docking technology

A ChemDraw module and a Chem3D pro module in ChemBioOffice2014 are adopted to draw a 3D structure of 6 pure peptides, the polypeptides are used as ligands, interleukin cell 1(I L-11) is used as an anti-inflammatory target point of molecular docking, the three-stage structure of I L-11 is respectively derived from a Protein Data (PDB) database, the ligands are subjected to charging (add value), electric field (application for subsequent), water removal (remove water), Protein treatment (clean Protein), charging (add value) and electric field (application for subsequent), the receptors are subjected to molecular docking by a CDocker module in Discovery Studio2016, and the potential anti-inflammatory activity of the 6 pure polypeptides is investigated according to CDocker energy and CDocker interaction energy values.

Determination of in vitro cell oxidative damage repair Activity

By means of H₂O₂The induced macrophage RAW264.7 oxidative damage repair model measures the cellular oxidative damage repair activity of the polypeptide sample.

RAW264.7 cells cultured to logarithmic phase are inoculated into a 96-well plate (the cell density is 2 × 104/m L), each well is inoculated with 190 mu L, a sample group, a negative control group and a damage control group are set, and 10 mu L H is added to the rest groups except the negative control group which is treated by DMSO₂O₂After 4h of treatment, the sample group is added with 1 mu L sample to be tested, the damage control group is added with 1 mu L DMSO, and the mixture is placed in a container 37C, 5% CO₂The samples of the examples were incubated in an incubator for 48h and their effects on cell viability, intracellular oxidation levels and antioxidant enzyme systems of the injury model were determined. The results are shown in Table 3.

Statistical analysis

All tests were repeated three times and the results were expressed as mean ± standard deviation SPSS 16.0(SPSS inc., Chicago, I L) was used for statistical analysis all data was made by Origin 9.0(Origin L ab, north ampton, MA, USA) one-way analysis of variance (ANOVA) was used to analyze the differences P-values less than 0.05 were considered statistically significant Pearson correlation coefficients were used to assess the correlation between content and activity linear regression equations were calculated by linear regression analysis.

Results of the experiment

The amino acid composition of the Neptune-venuoside peptide complex is shown in Table 1, the evaluation results of the activity of DPPH free radical scavenging experiment in vitro in examples and comparative examples are shown in Table 2, the evaluation results of the activity of repairing oxidative damage of cells in vitro in examples and comparative examples are shown in Table 3, the evaluation results of the antioxidant activity in vivo in examples and comparative examples are shown in FIG. 8, the evaluation results of the anti-inflammatory activity in vivo in examples and comparative examples are shown in FIG. 9, the ACE inhibitory effect of examples and comparative examples is shown in FIG. 10, and the inhibitory effect of IF-1 α in examples and comparative examples is shown in FIG.

As can be seen from Table 1, the total amino acid content of the Neptunea venosa active peptide complex is 925.93mg/g, 17 amino acids were detected, wherein serine (Ser), arginine (Arg) and threonine (Thr) are the 3 amino acids with the highest content in Citrogopaludina chinensis, and account for 51.45% of the total amino acids.

TABLE 1 Neptunea venosa active segment amino acid composition identification

As can be seen from Table 2, each sample in the examples has strong in vitro DPPH free radical scavenging experimental activity, and the sample obtained in the comparative example does not have in vitro antioxidant activity.

TABLE 2 evaluation results (IC) of antioxidant Activity in vitro of the examples and comparative examples₅₀The value, n-3,

)

as can be seen from table 3, each of the samples in the examples improves the degree of oxidative damage of cells, and shows significant difference compared to the model group, and each of the samples in the comparative examples does not improve the oxidative damage of cells.

TABLE 3 results of in vitro evaluation of cellular oxidative damage repair (IC) of the samples of examples₅₀The value, n-3,

)

as can be seen from fig. 8, in the examples, each sample can significantly reduce the apoptosis of the fluorescent cells of the skin of the zebra fish caused by metronidazole, and improve the oxidative damage of the zebra fish, and compared with the model group, the comparative example has no significant difference in the number of the fluorescent cells of the skin of the zebra fish, and the experimental results further indicate that the polypeptide of the characteristic peptide fragment sequence prepared by the above method has significant antioxidant activity.

The experimental results show that the polypeptide with the characteristic peptide fragment sequence prepared by the method has obvious in-vivo and in-vitro antioxidant activity and in-vivo anti-inflammatory activity, can inhibit the generation of angiotensin converting enzyme and the generation of cell inflammatory factor IF-1 α, has potential antioxidant stress injury function, and can be used as a medicinal component for preparing oxidative stress injury repair medicines and antioxidant health care products.

Sequence listing

<110> institute of biological research of academy of sciences of Shandong province

<120> rapana venosa polypeptide with antioxidant stress injury, preparation method and application thereof

<160>6

<170>SIPOSequenceListing 1.0

<210>1

<211>10

<212>PRT

<213> Rapana venosa (Rapana venosa)

<400>1

Met Val Leu Leu Gly Leu Val Leu Met Gly

1 5 10

<210>2

<211>8

<212>PRT

<213> Rapana venosa (Rapana venosa)

<400>2

Ala Arg Leu Gly Leu Ala Thr Leu

1 5

<210>3

<211>7

<212>PRT

<213> Rapana venosa (Rapana venosa)

<400>3

Leu Leu Thr Arg Ala Gly Leu

1 5

<210>4

<211>10

<212>PRT

<213> Rapana venosa (Rapana venosa)

<400>4

Gly Tyr Ser Phe Thr Thr Thr Ala Glu Arg

1 5 10

<210>5

<211>7

<212>PRT

<213> Rapana venosa (Rapana venosa)

<400>5

Lys Ser Thr Glu Leu Leu Ile

1 5

<210>6

<211>7

<212>PRT

<213> Rapana venosa (Rapana venosa)

<400>6

Phe Gly Ile Asn Leu Ile Gln

1 5

Claims

1. The polypeptide with the function of resisting oxidative stress damage has an amino acid sequence shown as SEQ ID NO. 1.

2. The polypeptide with the function of resisting oxidative stress damage has an amino acid sequence shown as SEQ ID NO. 2.

3. The polypeptide with the function of resisting oxidative stress damage has an amino acid sequence shown as SEQ ID NO. 3.

4. The polypeptide with the function of resisting oxidative stress damage has an amino acid sequence shown as SEQ ID No. 5.

5. The polypeptide with the function of resisting oxidative stress damage has an amino acid sequence shown as SEQ ID NO. 6.

6. A polypeptide combination with antioxidant stress injury function comprises polypeptide combination composed of amino acid sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.

7. A method for extracting the polypeptide with the function of resisting oxidative stress damage is characterized by comprising the following steps:

8. The method according to claim 7, wherein in the step (1), the pepsin enzymolysis pH value is 2.0-3.0, and the trypsin and chymotrypsin enzymolysis pH value is 7.2-8.0; further preferably, in the step (1), the pH regulator is hydrochloric acid and sodium hydroxide;

preferably, in the step (1), the enzyme activity ratio of trypsin to chymotrypsin is 1: (0.2-5);

preferably, in the step (2), the buffer salt system is a phosphate buffer system, and the pH value is 6.8-7.2;

preferably, in the step (3), the identification and determination of the amino acid composition adopt L C-MS protein identification technology.

9. The use of a combination according to any one or more of claims 1 to 5, and of a combination with a polypeptide having an amino acid sequence as shown in SEQ ID No.4, as a pharmaceutical active ingredient in the manufacture of a medicament for the treatment of oxidative stress injury.

10. The use of any one or combination of two or more of claims 1 to 5, and the combination with a polypeptide having an amino acid sequence as shown in SEQ ID No.4 as an active ingredient in the preparation of antioxidant health food.