CN109180781B - With the polypeptide and the preparation method and application thereof for repairing oxidative damage function - Google Patents
With the polypeptide and the preparation method and application thereof for repairing oxidative damage function Download PDFInfo
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- CN109180781B CN109180781B CN201810915407.5A CN201810915407A CN109180781B CN 109180781 B CN109180781 B CN 109180781B CN 201810915407 A CN201810915407 A CN 201810915407A CN 109180781 B CN109180781 B CN 109180781B
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- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000005040 ion trap Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- FBAFATDZDUQKNH-UHFFFAOYSA-M iron chloride Chemical compound [Cl-].[Fe] FBAFATDZDUQKNH-UHFFFAOYSA-M 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- NNFCIKHAZHQZJG-UHFFFAOYSA-N potassium cyanide Chemical compound [K+].N#[C-] NNFCIKHAZHQZJG-UHFFFAOYSA-N 0.000 description 1
- 230000003334 potential effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 150000003248 quinolines Chemical class 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 230000002000 scavenging effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 235000014102 seafood Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000010339 sodium tetraborate Nutrition 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- AGGKEGLBGGJEBZ-UHFFFAOYSA-N tetramethylenedisulfotetramine Chemical compound C1N(S2(=O)=O)CN3S(=O)(=O)N1CN2C3 AGGKEGLBGGJEBZ-UHFFFAOYSA-N 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 229940072040 tricaine Drugs 0.000 description 1
- FQZJYWMRQDKBQN-UHFFFAOYSA-N tricaine methanesulfonate Chemical compound CS([O-])(=O)=O.CCOC(=O)C1=CC=CC([NH3+])=C1 FQZJYWMRQDKBQN-UHFFFAOYSA-N 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical class OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- BSVBQGMMJUBVOD-UHFFFAOYSA-N trisodium borate Chemical compound [Na+].[Na+].[Na+].[O-]B([O-])[O-] BSVBQGMMJUBVOD-UHFFFAOYSA-N 0.000 description 1
- 208000019553 vascular disease Diseases 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Animal Behavior & Ethology (AREA)
- Toxicology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to a kind of with the polypeptide and the preparation method and application thereof for repairing oxidative damage function.A kind of polypeptide with reparation oxidative damage function, amino acid sequence is as shown in SEQ ID NO.1.The drug of disease caused by treating oxidative damage in preparation as effective component the invention also discloses aforementioned polypeptides is preparing the application in antioxidant health-care product as health-care components.Present invention firstly discloses the polypeptide compounds containing 10 amino acid residues extracted from Neptunea cumingi, it is found by detection, the polypeptide compound can pass through the generation of the internal ROS of removing, inhibit the generation of blood vessel Angiotensin-Converting invertase, inhibit the raising of blood glucose, the oxidativestress damage as caused by peroxide is repaired, the exploitation of the drug and antioxidant health-care product of disease caused by successive treatment oxidative damage can be carried out, had a vast market foreground.
Description
Technical field
The present invention relates to a kind of with the polypeptide and the preparation method and application thereof for repairing oxidative damage function, and it is more to belong to function
Peptide technology field.
Background technique
The Chronic Non-Communicable Diseases of developed country and developing country's death rate and disease incidence ranking front three at present
It (NCDs) is diabetes, cancer, angiocarpy and cranial vascular disease.With the development of aging of population and social economy and life
The disease incidence of the continuous variation of mode, non-communicable diseases will continue growing.The year two thousand thirty is expected, it is related to non-communicable diseases
Death toll will be added to 52,000,000.In order to mitigate the burden of non-communicable diseases, it is necessary to take prevention and treatment non-infectious
Property disease effective measures.One of the main reason for including non-communicable diseases such as diabetes, hypertension, atherosclerosis, it is
The active oxygen (ROS) of internal excess generation.Therefore, ROS is inhibited to generate the potential prevention and treatment measure that can be used as NCDs.So
And many synthetized oxidation preventive agents limit use because it is with the related potential risk of health.Therefore, natural dietary antioxidants because
Its advantages and higher safety and become optimal selection.
Marine animal and plant is various in style, and the bioactive substance being rich in is abundant.Due to compared with the animals and plants in land source,
The significant difference of living environment, therefore marine animal and plant is considered as the treasure-house in novel bioactive compound source.Numerous studies
Show that there is anti-oxidant, anti-hypertension, the oceanic biological active peptides of anti-diabetic, antibacterial and anti-tumor biological have very
High potential value can be used for preventing and treating non-communicable diseases.In addition, with consumer to nutrition and health understanding not
Disconnected to improve, the demand to functional food and nutrient and healthcare products is also increasing.Research is increasingly focused on recently spreads out from diet
The biologically active peptide separated in raw marine organisms.
Mollusk is the second largest animal door on the earth.It is not only played an important role in terms of maintaining the ecological balance,
And there is very big commercial value as a kind of edibility Animal resources.The potential active ingredient of many of mollusk
Object can be used for dietary supplements, functional food, nutriment and drug.However, presently disclosed from the soft of application value
The research that potential activity compound is separated in body animal is less.
Neptunea cumingi (Neptunea arthritica cumingii) belongs to Mollusca (Mollusca), Gastropoda
(Gastropoda), Caenogastropoda (Neogastropoda), Buccinidae (Buccinida) are that a kind of large-scale predatism abdominal foot is dynamic
Object.It mainly lives in China, Japan, the sea area of 10 to 78 meters of depths of Korea and South Korea.In China, Neptunea cumingi is distributed mainly on Huang
Sea and Bohai Sea region, especially Dalian and Penglai, shandong Province.Fleshy hypertrophy compact, the delicious flavour of Neptunea cumingi, nutritive value is high,
But hatching rate is low, therefore commercial value with higher.Up to the present, most of researchs in relation to Neptunea cumingi are concentrated mainly on it
Biologically, such as the genome of Neptunea cumingi, nutrition, reproduction characteristics etc. or its nutritional ingredient, such as crude protein, polysaccharide, thick rouge
Matter, fatty acid and amino acid composition etc..Report relevant to the active constituent of Neptunea cumingi is less at present, and only early stage research discovery is fragrant
Contain Tetramine in spiral shell saliva, histamine and choline derivative have neural activity.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of with the polypeptide for repairing oxidative damage function and its preparation side
Method and application.
Technical solution of the present invention is as follows:
A kind of polypeptide with reparation oxidative damage function, amino acid sequence is as shown in SEQ ID NO.1.
SEQ ID NO.1 is as follows:
Tyr-Ser-Gln-Leu-Glu-Asn-Glu-Phe-Asp-Arg(YSQLENEFDR)。
The above-mentioned derivative with the polypeptide for repairing oxidative damage function, to amino acid sequence as shown in SEQ ID NO.1
Polypeptide in amino acid residue modified, modification include amidation modification, carbonylation modification, phosphorylation modification and/cyclisation
Modification.
It is preferred according to the present invention, the amidation be modified to the 1st amino acids residue of N-terminal carry out acetylation modification or
Person is carrying out amidation modification in the 1st amino acids residue of C-terminal.
Preferred according to the present invention, the phosphorylation modification is to carry out sugar in N-terminal the 2nd and/or the 6th amino acids residue
Base modification and/or phosphorylation modification.
Preferred according to the present invention, the cyclisation is modified to the cyclisation modification being connected with N-terminal amino acid residue in C-terminal, N-terminal
2nd the cyclisation modification being connected between the side chain of the 6th amino acids residue, N-terminal are connected with the 7th amino acids residue side chains of N-terminal
Cyclisation modification, the 9th ammonia of cyclisation modification or N-terminal the 2nd and N-terminal for being connected with the 6th amino acids residue side chains of N-terminal of C-terminal
The N-N of base acid residue connected cyclisation modification.
A method of aforementioned polypeptides being extracted, steps are as follows:
(1) by after Neptunea cumingi decladding, meat part is taken, is ground, the acidic ethanol of 8~12 times of weight is added, extract 2.5~7h,
Filtering takes filtrate, stands 10~60min under condition of ice bath, take supernatant liquor, be concentrated, decolourize, and centrifugation takes supernatant, removes low
Polar impurity is made polypeptide and mixes stoste；
(2) polypeptide made from step (1) is mixed stoste to be separated with sephadex G 25, with 0.018~0.022M
HCl- water is eluant, eluent, collects sample according to the speed of 5mL/20min, every 20min collects portion, by the 34th~36 part of activity
Section eluent merges, concentrated, and polypeptide active section crude extract is made；
(3) by the ammonium acetate buffer of polypeptide active section crude extract concentration 10mM, pH5.8~6.2 made from step (2)
Dissolution, through 4.5 μm of micro-pore-film filtrations, then through Welch HILIC Amide post separation, binary mobile phase is ACN and concentration
The volume ratio of the ammonium acetate buffer of 10mM, pH5.8~6.2, ACN and ammonium acetate buffer is 85:15, flow velocity 1ml
min-1, the eluent at 210nm with absorption peak is collected, active three functional polypeptides are screened, it is concentrated, dry, it is made to have and repair
The polypeptide of multiple oxidative damage function.
Preferred according to the present invention, in the step (1), acidic ethanol is the ethanol solution of volumetric concentration 45~55%,
PH4.8~5.2；It is further preferred that the pH adjusting agent of acidic ethanol is acetic acid in the step (1).
It is preferred according to the present invention, it is described to be extracted as extracting 3 under 28~32 DEG C of stirring condition in the step (1)
~6h.
It is preferred according to the present invention, in the step (1), it is concentrated as under conditions of 38~40 DEG C, rotary evaporation is removed
Ethyl alcohol in solution.
It is preferred according to the present invention, in the step (1), decolourize for mass percent 0.5~1% is added into solution
Diatomite-sodium carboxymethyl starch compound, wherein the mass ratio of diatomite and sodium carboxymethyl starch is 3:2, it is stored at room temperature 1.5~
3h。
Preferred according to the present invention, in the step (1), removing low polar impurity is to be retained using hexane extraction 2~4
Water phase；It is further preferred that the additive amount of hexane is 0.8~1.2 times of liquor capacity.
It is preferred according to the present invention, in the step (2), vacuum-concentrcted condition be vacuum degree be 0.08~
0.1MPa, bath temperature are 40~45 DEG C.
Preferred according to the present invention, in the step (3), Welch HILIC Amide column specification is 4.6mm × 250mm,
5μm。
Preferred according to the present invention, in the step (3), three functional polypeptides of screening are specific to walk using active guiding technique
It is rapid as follows:
It is evaluated using antioxidation activity in vitro evaluation model, ACE inhibitory activity evaluation model and alpha-amylase inhibitory activity
Model measures the activity of each fraction section, filters out the fraction section that the inhibiting rate in 3 kinds of evaluation models is more than 80%.
Preferred according to the present invention, in the step (3), dry is to be freeze-dried under the conditions of -20 DEG C.
It is above-mentioned that there is the polypeptide for repairing oxidative damage function and its derivative to treat oxidation damage in preparation as effective component
Hurt the application in disease medicament.
It is above-mentioned that there is the polypeptide for repairing oxidative damage function to prepare the application in antioxidant health-care product as health-care components.
Beneficial effect
Present invention firstly discloses the polypeptide compounds containing 10 amino acid residues extracted from Neptunea cumingi, pass through detection
It was found that the polypeptide compound can inhibit the generation of blood vessel Angiotensin-Converting invertase by the generation of the internal ROS of removing,
Inhibit the raising of blood glucose, repairs the oxidativestress damage as caused by peroxide, successive treatment oxidative damage can be carried out and caused
Disease drug and antioxidant health-care product exploitation, have a vast market foreground.
Detailed description of the invention
Neptunea cumingi raw material photo used in Fig. 1 embodiment；
In figure: A, Neptunea cumingi overall appearance；B Neptunea cumingi soft tissue；C button of cartilage portion；D Neptunea cumingi internal organ；
Fig. 2 button of cartilage active section crude extract molecular weight distribution testing result figure；
Fig. 3 active section extract amino acid forms result figure；
Fig. 4 activity peptide amino acid sequence MS/MS mass spectral results figure；
Fig. 5 button of cartilage G25 elutes each fraction section Activity determination result figure；
In figure: absorbance value and DPPH free radical inhibiting rate curve graph under each fraction section 280nm wavelength of A；
Absorbance value and ACE inhibiting rate curve graph under each fraction section 280nm wavelength of B；
Absorbance value and alpha-amylase inhibiting rate curve graph under each fraction section 280nm wavelength of C；
Fig. 6 button of cartilage active section crude extract HILIC chromatographic column testing result and different chromatographic peak Activity evaluation figures；
In figure: A is active section extract HILIC chromatogram；B is each chromatographic peak Activity evaluation histogram of crude extract；
The HILIC chromatographic column testing result figure of each fraction section sample in Fig. 7 comparative example 1；
In figure: the HILIC chromatogram of the 31st~33 part of sample of A；The HILIC chromatogram of the 37th~39 part of sample of B；
The HILIC chromatographic column testing result figure of fraction section sample in Fig. 8 comparative example 2；
Fig. 9 each sample is to zebra fish vivo oxidation injury repair effect picture；
In figure: A blank control；B model of oxidative group；C positive controls；D example 1 group；2 groups of E embodiment；F pairs
Ratio 1A group；G comparative example 1B group；2 groups of H comparative example.
Specific embodiment
Technical solution of the present invention is further elaborated below with reference to embodiment and Figure of description, but institute of the invention
Protection scope is without being limited thereto.
Biological material source
Neptunea cumingi described in embodiment be purchased from Jinan, Shandong Province seafood market, ordinary commercial products, as shown in Figure 1.
Detection method
Molecules of active components amount is distributed detection method
Use TSK-gel G2000SWXLColumn (7.8mm × 250mm) (TOSOH, Yamaguchi, Japan), using gel
Molecular weight (MW) distribution (Fig. 2) for the active section that permeation chromatography measurement is obtained from internal organ.Mobile phase is by 0.1molL-1Phosphoric acid
Salt buffer (pH 6.7) and 0.1molL-1Na2SO4Composition, flow rate set 0.2mLmin-1。
With ribalgilase (13700Da), hydrochloric acid Aprotinin (6511Da), Angiotensin II (1046Da), HHL
(430Da) and Serine (105Da) be reference substance, draftings elution volume-Molecular weight plots for In Mw=17.50~
0.089T(R2=0.9785, Mw are molecular weight, and T is elution volume).
Active constituent amino acid forms detection method
Freeze drying activity peptide to be detected is dissolved in 6molL-1In HCl (1mg peptide/mL HCl), in 110 DEG C of drying boxes
Hydrolysis 24 hours.Filtered hydrolyzation sample is evaporated at 45 DEG C by rotary evaporator.Residue is dissolved in distilled water
And it is freeze-dried.Then, sample and mixture amino acid standard with AQC derivatization and are passed through into RP-HPLC18Measurement.From mixed
The amino acid for closing identification and quantification sample fraction in the standard curve of amino acid forms (Fig. 3).All samples measure in triplicate.
The sequence of nano-LC-LTQ-Orbitrap-MS/MS identified activity peptide
Using EASY-Nlc1000 chromatographic system (Thermo Finnigan, Bremen, Germany), LTQ Orbitrap
Velos Pro mass spectrograph (Thermo Finnigan, Bremen, Germany) is to the amino acid sequence identity of active peptide.Purifying
Peptide with the concentration containing 0.1% trifluoroacetic acid be 0.1mgmL-1Ultrapure water dissolution.Then 2 μ L samples are injected into trapping column
Pre-concentration is carried out in (100 μ m 20mm, RP-C18, thermo Inc.).The sample of subsequent pre-concentration is automatically into analytical column
(75 μ m 150mm, RP-C18, thermo Inc.).Using 0.1% (v/v) formic acid in ultrapure water as eluant, eluent, analyze duration:
60min, detection mode: positive ion mode, spray voltage: 1.8kV, ion transfer capillary temperature: 250 DEG C, using preceding through marking
Quasi- correcting fluid correction, precursor scans range: 350-1800m/z, scanning of the mass spectrum mode are the collecting work mode that information relies on
Under (IDA, Information Dependent Analysis), each full scan (full scan) acquires strongest 10 afterwards
Fragment patterns stored (MS2scan), fragmentation pattern: collision induced dissociation (CID, collision-induced dissociation),
Normal state energy 35%, q value 0.25, activation time: 30ms, dynamic exclude the time: 30s.MS1 resolution ratio in M/Z 400 is
60,000, MS2 be unit mass resolution in an ion trap.First mass spectrometric uses profile type collection, and second order ms use
Centroid mode is acquired to reduce data file size.2.3 software of Mascot (Matrix Science, USA) is used for data
Analysis.Database is Buccinidae database, and enzyme is trypsase, and allowing maximum leakage enzyme site is 2.Fixed modification are as follows:
Carbamidomethyl(C)；Variable modification are as follows: Acetyl (Protein N-term), Deamidated (NQ),
Dioxidation(W),Oxidation(M)；MS tolerance is ± 30ppm, and MSMS tolerance is ± 0.15Da.NCBInr database is used
It is identified in peptide.Only consider the peptide of the identification for the desired value for having lower than 0.05.BIOPEP database is used to find previously to have identified
With anti-oxidant, the amino acid sequence of ACE inhibitory activity.Active peptide amino acid sequence MS/MS mass spectral results are shown in Fig. 4.
Embodiment 1
A method of extracting has the polypeptide for repairing oxidative damage function, and steps are as follows:
(1) by after Neptunea cumingi decladding, meat part is taken, is ground, the acidic ethanol of 8 times of weight is added, acidic ethanol is that volume is dense
The ethanol solution of degree 55%, acidic ethanol acetic acid tune pH4.8 extract 2.5h under 32 DEG C of stirring condition, filter, take filter
Liquid stands 30min under condition of ice bath, takes supernatant liquor, and under conditions of 40 DEG C, rotary evaporation removes the ethyl alcohol in solution, so
Diatomite-sodium carboxymethyl starch compound (mass ratio 3:2) is added in 0.5% ratio by mass percentage afterwards, is stored at room temperature 3h,
Centrifugation, takes supernatant, and the hexane extraction of 0.8 times of volume is added, and retains water phase, continuous extraction 4 times, polypeptide is made and mixes stoste；
(2) polypeptide made from step (1) is mixed stoste to be separated with sephadex G 25, with 0.02M HCl- water
For eluant, eluent, sample is collected according to the speed of 5mL/20min, every 20min is collected once, tracks skill using external three functional activity
Art collects the 34th~36 part of active section eluent (Fig. 5), dense under conditions of vacuum degree is 0.08MPa, temperature is 45 DEG C
Polypeptide active section crude extract is made in contracting；
(3) ammonium acetate buffer of polypeptide active section crude extract concentration 10mM, pH6.0 made from step (2) is dissolved,
Through 0.45 μm of micro-pore-film filtration, using Welch HILIC Amide post separation, column specification is 4.6mm × 250mm, 5 μm, flowing
It is mutually the ammonium acetate buffer of ACN and concentration 10mM, pH6.0, the volume ratio of ACN and ammonium acetate buffer is 85:15, and flow velocity is
1ml·min-1, Detection wavelength 210nm, using antioxidation activity in vitro evaluation model, ACE inhibitory activity evaluation model and α-
Amylase inhibiting activity evaluation model measures the activity of each fraction section, filters out the inhibiting rate in 3 kinds of evaluation models and is more than
80% chromatographic peak is the chromatographic peak (Fig. 6) at retention time 16min with strong absworption peak, collects corresponding eluent, dense
It contracts, be freeze-dried under the conditions of -20 DEG C, is made with the polypeptide for repairing oxidative damage function.
It is detected, has the polypeptid acid sequence for repairing oxidative damage function as shown in SEQ ID NO.1.
Embodiment 2
Using Fmoc solid-phase synthesis described in Liu Zhennan etc., (Guangxi Liu Zhennan, Huang Qiang .Fmoc solid-phase synthesis is national
Institute's journal, 1999,5 (2): 110-112), artificial synthesized amino acid sequence polypeptide as shown in SEQ ID NO.1.
Embodiment 3
A method of extracting has the polypeptide for repairing oxidative damage function, and steps are as follows:
(1) by after Neptunea cumingi decladding, meat part is taken, is ground, the acidic ethanol of 12 times of weight is added, acidic ethanol is that volume is dense
The ethanol solution of degree 45%, acidic ethanol acetic acid tune pH5.2 extract 7h under 28 DEG C of stirring condition, filter, take filtrate,
30min is stood under condition of ice bath, takes supernatant liquor, under conditions of 38 DEG C, rotary evaporation removes the ethyl alcohol in solution, then presses
Diatomite-sodium carboxymethyl starch compound (mass ratio 3:2) is added in the ratio of mass percent 1%, is stored at room temperature 1.5h, from
The heart takes supernatant, and the hexane extraction of 1.2 times of volumes is added, and retains water phase, continuous extraction 2 times, polypeptide is made and mixes stoste；
(2) polypeptide made from step (1) is mixed stoste to be separated with sephadex G 25, with 0.01M HCl- water
For eluant, eluent, sample is collected according to the speed of 5mL/20min, every 20min is collected once, tracks skill using external three functional activity
Art is collected the 34th~36 part of active section eluent (Fig. 5), is concentrated under conditions of vacuum degree is 0.1MPa, temperature is 40 DEG C,
Polypeptide active section crude extract is made；
(3) ammonium acetate buffer of polypeptide active section crude extract concentration 10mM, pH6.8 made from step (2) is dissolved,
Through 0.45 μm of micro-pore-film filtration, using Welch HILIC Amide post separation, column specification is 4.6mm × 250mm, 5 μm, flowing
It is mutually the ammonium acetate buffer of ACN and concentration 10mM, pH6.8, the volume ratio of ACN and ammonium acetate buffer is 4:1, and flow velocity is
1ml·min-1, Detection wavelength 214nm, using antioxidation activity in vitro evaluation model, ACE inhibitory activity evaluation model and α-
Amylase inhibiting activity evaluation model measures the activity of each fraction section, filters out the inhibiting rate in 3 kinds of evaluation models and is more than
80% chromatographic peak is the chromatographic peak (Fig. 6) at retention time 16min with strong absworption peak, collects washing under corresponding retention time
De- liquid, it is concentrated, be freeze-dried under the conditions of -20 DEG C, it is made with the polypeptide for repairing oxidative damage function.
It is detected, has the polypeptid acid sequence for repairing oxidative damage function as shown in SEQ ID NO.1.
Embodiment 4
Artificial synthesized amino acid sequence as described in Example 2, the difference is that, the 1st amino acids residue of N-terminal into
Row acetylation modification.
Embodiment 5
Artificial synthesized amino acid sequence as described in Example 2, the difference is that, by the 1st amino acids residue of C-terminal into
Row amidation modification.
Embodiment 6
Artificial synthesized amino acid sequence as described in Example 2, the difference is that, by the 2nd amino acids residue of N-terminal into
Row is glycosylation modified.
Embodiment 7
Artificial synthesized amino acid sequence as described in Example 2, the difference is that, by the 2nd amino acids residue of N-terminal into
Row phosphorylation modification.
Embodiment 8
Artificial synthesized amino acid sequence as described in Example 2, the difference is that, by the 6th amino acids residue of N-terminal into
Row is glycosylation modified.
Embodiment 9
Artificial synthesized amino acid sequence as described in Example 2, the difference is that, by the 6th amino acids residue of N-terminal into
Row phosphorylation modification.
Embodiment 10
Artificial synthesized amino acid sequence as described in Example 2, the difference is that, linear chains of amino acids is subjected to cyclisation and is repaired
Decorations, the cyclisation being connected with N-terminal amino acid residue including C-terminal modify (A), N-terminal the 2nd between the side chain of the 6th amino acids residue
(C), C-terminal and N-terminal the 6th are modified in the cyclisation that connected cyclisation modification (B), N-terminal are connected with the 7th amino acids residue side chains of N-terminal
N-terminal the 2nd in the connected cyclisation modification (D) of amino acid residue side and main chain is connected with the N-N of the 9th amino acids residue
Cyclisation modify (E).
Comparative example 1
Method as described in Example 1, the difference is that, the eluent of collection described in step (2) is respectively
31~33 parts (A) and the 37th~39 part (B) are concentrated respectively, and corresponding mixtures of polypeptides sample is made.By described in step (3)
Purification process has no corresponding chromatographic peak (Fig. 7) under the conditions of identical retention time.Show the active peptides exist only in 660~
In 720min fraction section.Therefore using sample obtained by step (2) as test sample, it to be used for next step active appraisal experiment.
Comparative example 2
Method as described in Example 1, the difference is that, experimental raw described in step (1) is Rapana venosa meat.It presses
Purification process described in step (3) has no corresponding chromatographic peak (Fig. 8) under the conditions of identical retention time.Show Rapana venosa internal organ
In do not contain the active peptides.Therefore using sample obtained by step (2) as test sample, it to be used for next step active appraisal experiment.
Comparative example 3
Artificial synthesized amino acid sequence as described in Example 2, the difference is that, by N-terminal the 5th and the 7th bit amino
Sour residue replaces with Gly amino acid residue.
Experimental example
DPPH free radical scavenging activity
According to the DPPH free radical scavenging activity of the method test sample polypeptide of Lee et al. description.
200 μ l fractions are added to containing 200 μ l0.15mmolL-1In the pipe of DPPH ethanol solution, and by mixture whirlpool
It revolves several seconds.Then, mixture is incubated 12 hours in 37 DEG C in the dark.Ultrapure water is used as control.It measures and inhales at 517nm
Luminosity, and be measured in triplicate.The DPPH free radical scavenging activity of peptide fraction calculates such as following formula:
DPPH free radical scavenging activity (%)=(1-As/Ac) × 100
Wherein As is the absorbance of sample, and Ac is the absorbance of control.Although the DPPH free radical scavenging activity of active peptide
It is indicated with 503nhibiting concentration (IC50), IC50It is defined as that 50% free radical is inhibited to form required peptide concentration.Embodiment and comparative example
DPPH free radical scavenging activity IC50It is shown in Table 1 and table 2.
Reducing power detection
Using the reducing power such as Moayedi et al. the method measurement polypeptide sample.
By the iron of 100 μ l polypeptide samples and 50 μ l kaliumphosphate buffers (0.2M, pH6.6) and 150 μ l concentration 1% (w/v)
Potassium cyanide mixing simultaneously incubates 30 minutes at 50 DEG C.Then, be added into reaction mixture 100 μ l10% trichloroacetic acids (TCA) with
Terminate the reaction.It is centrifuged after ten minutes in 12,000rpm, takes out 150 μ l supernatants.Then, 100 μ l ultrapure waters and 50 μ l are dense
0.1% (w/v) frerrous chloride is spent to be added in supernatant.
Control sample had not both included peptide fraction or had not included iron chloride.Absorbance is measured at 700 nm, it is triplicate to carry out
Measurement.Although measuring the reducing power of active peptide, IC by IC5050The absorbance of sample needed for being defined as 0.5.Embodiment and right
The reducing power IC of ratio50It is shown in Table 1 and table 2.
Hydroxyl radical free radical scavenging capacity
Polypeptide sample Scavenging activity on hydroxyl free radical is detected according to measuring method described in Dong Z.Y..
1mL polypeptide sample is mixed with 0.5ml salicylic acid-ethyl alcohol (10mM) and 0.5ml FeSO 4 (10mM).Then, exist
0.5ml H is added2O2After in (8.8mM), mixture is incubated 30 minutes at 37 DEG C.Ultrapure water is used to replace fraction as blank
Control, and H will be lacked2O2Reaction mixture be used as control.Absorbance is measured at 510nm, and is surveyed in triplicate
It is fixed.The hydroxyl radical free radical scavenging capacity of polypeptide sample calculates such as following formula:
Hydroxyl radical free radical scavenging capacity (%)=[1- (As-Ac)/Abc]×100
Wherein As is the absorbance of sample, and Ac is the absorbance of control, and Abc is the absorbance of blank control.Pass through IC50It comments
Estimate the hydroxyl radical free radical activity of active peptide.The hydroxyl radical free radical clearance rate IC of embodiment and comparative example50It is shown in Table 1 and table 2.
The measurement of internal antioxidant activity
By using transgenic zebrafish system Tg (krt4:NTR-hKikGR) cy17, the internal antioxygen of polypeptide sample is carried out
The detection of change.
The transgenic zebrafish embryo for developing pf for 24 hours is assigned in 24 porocyte culture plates (10 embryo/holes), and with
2mL10mM metronidazole (MTZ is dissolved in zebra fish culture water) and polypeptide sample are incubated with, and dosage is 100 μ gmL-1, 28 DEG C of items
Under part, after drug-treated 24 hours.It is used as intermedium control with the zebra fish that the fish and water of no metronidazole and peptide is handled.With being free of
The zebra fish of the metronidazole processing of peptide is used as negative control.Vitmin C is used to replace peptide as positive control.Every group at least carries out
It is parallel three times to repeat.After incubation, zebrafish embryo is anaesthetized with tricaine (0.16%, w/v), then observes the glimmering of zebrafish embryo
Light is simultaneously imaged using FSX100Bio Imaging Navigator instrument.By using imagepro-plus software evaluation fluorescence
The quantity of spot.The internal antioxidant activity of polypeptide sample calculates such as following formula:
Antioxidant activity (%)=(FSs-FSnc)/(FSvc-FSnc)×100
Wherein FSsIt is the phosphor dot (polypeptide sample) of sample, FSncIt is the phosphor dot of negative control, FSvcIt is phosphor dot (dimension
Raw element C).The internal anti-oxidant evaluation result of Examples 1 and 2 and comparative example 1 and 2 is shown in Fig. 9.
The measurement of ACE inhibitory activity
Polypeptide sample ACE inhibitory activity is measured according to the method for the reports such as Chen.
By 50 μ L peptides and 50 μ LACE solution (25mUmL-1) mix and be incubated for 5 minutes at 37 DEG C.Then, will contain
125 μ L8.3mMHHL in the 50mM sodium borate buffer liquid of 0.5M NaCl (pH8.3) be added in mixture with start reaction and
It is incubated for 60 minutes at 37 DEG C.Then, reaction is terminated by adding the 1M HCl of 125 μ L.It is extracted by using 750 μ L ethyl acetate
Hippuric acid(HA).After centrifugation, 500 upper layers μ L are shifted, are evaporated in vacuo ethyl acetate at room temperature.Residue is dissolved in
In 1.5mL ultrapure water, absorbance is measured at 228nm.ACE inhibitory activity is measured by following formula:
ACE inhibitory activity (%)=[(Ab-As)/(Ab-Ac)] × 100
Wherein Ab and As be not and the absorbance of the mixture with HHL and Ac be the not mixture of ACE extinction
Degree.Pass through IC50Assess the effect of the ACE inhibitory activity of active peptide.The ACE inhibiting rate IC of embodiment and comparative example50It is shown in Table 1 He
Table 2.
The measurement of alpha-amylase inhibitory activity
The alpha-amylase inhibitory activity of polypeptide sample is measured according to the method for the reports such as Uraipong.
1mL alpha-amylase (0.5UmL-1) and 0.5mL freeze drying activity peptide are pre-mixed, dosage 5,4,3,2,1mg
The 20mmolL-1 sodium phosphate buffer (pH value 6.9) of mL-1 is simultaneously incubated for 10 minutes at 25 DEG C.Then, add into mixture
Enter 1% starch solution of 0.5mL.It is incubated at 25 DEG C after five minutes, by the way that 2mL DNS reagent and 2mL 1molL-1N is added
NaOH terminates reaction, then incubates mixture 5 minutes at 100 DEG C.After cooling to room temperature, 200 μ L mixtures are added 96
In the microtest plate of hole, and absorbance is measured at 540nm.Acarbose is used as positive control.Active peptide is replaced with ultrapure water
As control.Use the reaction mixture of shortage alpha-amylase as background.The measurement of all samples carries out in triplicate.Activity
The alpha-amylase inhibitory activity of peptide is indicated with IC50.Alpha-amylase inhibitory activity (%) is calculated using following formula:
Alpha-amylase inhibitory activity (%)=(Ac-Acb)-(As-Asb)/(Ac-Acb) × 100
Wherein Ac is the absorbance of control, and Acb is the absorbance for compareing background, and As is the absorbance of sample, and Asb is sample
The absorbance of background.The alpha-amylase inhibiting rate IC of embodiment and comparative example50See Tables 1 and 2.
The measurement of alpha-glucosaccharase enzyme inhibition activity
According to the alpha-Glucosidase inhibitory activity of Uraipong et al. method measurement polypeptide sample reported.
By 50 μ L freeze drying activity peptides (dosage 5,4,3,2,1mgmL-1) and 50 μ L5mmolL-1PNPG solution exists
100mmol·L-1It is blended in sodium phosphate buffer (pH 6.9) in 96 hole microwell plates, is incubated for 5 minutes at 37 DEG C.Then, will
The 0.1UmL of 100 μ L-1Alpha-Glucosidase is added in each hole in phosphate buffer, and is incubated for 30 minutes at 37 DEG C.?
Absorbance is measured at 405nm.Acarbose is used as positive control.α-is calculated using equation identical with alpha-amylase inhibitory activity
Glucosidase inhibitory active.The alpha-Glucosidase inhibiting rate IC of embodiment and comparative example50See Tables 1 and 2.
The measurement of cell in vitro oxidative damage repairing activity
Using H2O2The cellular oxidation damage of the macrophage RAW264.7 oxidative damage repairing model measurement polypeptide sample of induction
Hurt repairing activity.
By the RAW264.7 cell inoculation of culture to logarithmic phase in 96 orifice plates (cell density is 2 × 104/mL), often
Hole is inoculated with 190 μ L, sets up sample sets, negative control group and damage control group, in addition to negative control group plus DMSO are handled, remaining group
10 μ L H are added2O24h is handled, 1 μ L sample to be tested is added in subsequent sample sets, and damage control group is added 1 μ L DMSO, is put into 37
DEG C, contain 5%CO248h is cultivated in incubator, measurement sample is to damage model cell viability, Cellular Oxidation level and antioxygen
Change the influence of enzyme system.Experimental result is shown in Table 3.
Statistical analysis
All tests in triplicate, are as a result expressed as average value ± standard deviation.SPSS 16.0 (SPSS Inc.,
Chicago, IL, USA) for statisticalling analyze.All data by Origin 9.0 (Origin Lab, Northampton, MA,
USA it) makes.One-way analysis of variance (ANOVA) is for analyzing difference.P value is considered statistically significant less than 0.05.
Pearson correlation coefficient is used to assess the correlation between content and activity.Equation of linear regression passes through linear regression analysis meter
It calculates.
Experimental result
Antioxidation activity in vitro, antihypertensive activity and the hypoglycemic activity of the polypeptide isolated and purified from button of cartilage and Rapana venosa meat
Evaluation results are shown in Table 1, and antioxidation activity in vitro, antihypertensive activity and the hypoglycemic activity evaluation result of the polypeptide of synthesis are shown in Table 2.Various kinds
The cell in vitro oxidative damage repairing activity evaluation result of product is shown in Table 3.According to experimental result it is found that as prepared by the above method
The polypeptide of feature peptide section sequence has significant internal, antioxidation activity in vitro, while being able to suppress angiotensin converting enzyme
Generation, reduce blood glucose, have the function of potentially to repair oxidative damage, can be used as effective component and be used to prepare oxidative damage reparation
Drug and antioxidant health-care product.And it by carrying out structural modification to the particular amino acid residue in above-mentioned sequence, can be improved
The bioactivity of polypeptide.Polypeptide after Glu residue in above-mentioned amino acid sequence is replaced does not have antioxidant activity, table
Bright above-mentioned amino acid sequence is the specificity of antioxidant activity.
1 natural origin sample of table is depressured in vitro and antioxidant activity evaluation result (IC50Value, n=3,)
Representative has significant difference P < 0.01 compared with Example 1,Representative have compared with Example 2 significant difference P <
0.01。
The external decompression of 2 synthesis polypeptide of table and antioxidant activity evaluation result (IC50Value, n=3,)
3 natural origin sample of table is depressured in vitro and antioxidant activity evaluation result (IC50Value, n=3,)
Sequence table
<110>Shandong Province academy sciences Biology Research Institute
<120>there is the polypeptide and the preparation method and application thereof for repairing oxidative damage function
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 10
<212> PRT
<213>Neptunea cumingi (Neptunea arthritica cumingii)
<400> 1
Tyr Ser Gln Leu Glu Asn Glu Phe Asp Arg
1 5 10
Claims (15)
1. a kind of with the polypeptide for repairing oxidative damage function, amino acid sequence is as shown in SEQ ID NO.1.
2. there is the derivative for the polypeptide for repairing oxidative damage function, to amino acid sequence such as SEQ ID described in claim 1
Amino acid residue in polypeptide shown in NO.1 is modified, modification are as follows: is carried out acetylation to the 1st amino acids residue of N-terminal and is repaired
Decorations, the 1st amino acids residue of C-terminal is carried out amidation modification, by the 2nd amino acids residue of N-terminal carry out it is glycosylation modified, by N
Hold the 2nd amino acids residue carry out phosphate modification, by the 6th amino acids residue of N-terminal carry out it is glycosylation modified, by N-terminal the 6th
Amino acids residue carries out phosphorylation modification, the 2nd and the 6th the cyclisation modification that C-terminal is connected with N-terminal amino acid residue, N-terminal ammonia
What is be connected between the side chain of base acid residue is cyclized modification, the cyclisation modification that N-terminal is connected with the 7th amino acids residue side chains of N-terminal, C-terminal
The cyclisation modification that is connected with the 6th amino acids residue side chains of N-terminal and the N-terminal the 2nd on main chain and the 9th amino acids residue
N-N connected cyclisation modification.
3. a kind of method for extracting polypeptide described in claim 1, which is characterized in that steps are as follows:
(1) by after Neptunea cumingi decladding, meat part is taken, is ground, the acidic ethanol of 8~12 times of weight is added, extract 2.5~7h,
Filtering takes filtrate, stands 10~60 min under condition of ice bath, take supernatant liquor, be concentrated, decolourize, and centrifugation takes supernatant,
Low polar impurity is removed, polypeptide is made and mixes stoste；
The acidic ethanol is the ethanol solution of volumetric concentration 45~55%, pH4.8~5.2；
(2) polypeptide made from step (1) is mixed stoste to be separated with sephadex G 25, with 0.018~0.022 M
HCl- water is eluant, eluent, collects sample according to the speed of 5 mL/20 min, every 20 min collects portion, by the 34th~36 part
Active section eluent merges, concentrated, and polypeptide active section crude extract is made；
(3) ammonium acetate buffer of polypeptide active section crude extract concentration 10mM, pH5.8~6.2 made from step (2) is molten
Solution, through 0.45 μm of micro-pore-film filtration, then through Welch HILIC Amide post separation, binary mobile phase is ACN and concentration
The volume ratio of the ammonium acetate buffer of 10mM, pH5.8~6.2, ACN and ammonium acetate buffer is 85:15, and flow velocity is 1ml min-1, the eluent at 210nm with absorption peak is collected, multifunction activity polypeptide is screened, it is concentrated, dry, it is made to have and repairs oxygen
Change the polypeptide of damage function.
4. method as claimed in claim 3, which is characterized in that in the step (1), the pH adjusting agent of acidic ethanol is second
Acid.
5. method as claimed in claim 3, which is characterized in that described to be extracted as stirring at 28~32 DEG C in the step (1)
3~6h is extracted under the conditions of mixing.
6. method as claimed in claim 3, which is characterized in that in the step (1), be concentrated as in 38~40 DEG C of condition
Under, rotary evaporation removes the ethyl alcohol in solution.
7. method as claimed in claim 3, which is characterized in that in the step (1), decolourize for quality hundred is added into solution
Divide diatomite-sodium carboxymethyl starch compound than 0.5~1%, wherein the mass ratio of diatomite and sodium carboxymethyl starch is 3:2,
It is stored at room temperature 1.5~3h.
8. method as claimed in claim 3, which is characterized in that in the step (1), removing low polar impurity is using hexane
Extraction 2~4 times retains water phase.
9. method according to claim 8, which is characterized in that in the step (1), the additive amount of hexane is supernatant volume
0.8~1.2 times.
10. method as claimed in claim 3, which is characterized in that in the step (2), vacuum-concentrcted condition is vacuum
Degree is 0.08~0.1 MPa, and bath temperature is 40~45 DEG C.
11. method as claimed in claim 3, which is characterized in that in the step (3), Welch HILIC Amide column specification
For 4.6mm × 250mm, 5 μm.
12. method as claimed in claim 3, which is characterized in that in the step (3), screened and lived using active guiding technique
Property polypeptide, the specific steps are as follows:
Using antioxidation activity in vitro evaluation model, ACE inhibitory activity evaluation model and alpha-amylase inhibitory activity evaluation model,
The activity for measuring each fraction section filters out the fraction section that the inhibiting rate in 3 kinds of evaluation models is more than 80%.
13. method as claimed in claim 3, which is characterized in that in the step (3), dry is to freeze under the conditions of -20 DEG C
It is dry.
14. having polypeptide derivative described in the polypeptide for repairing oxidative damage function or claim 2 as medicine described in claim 1
Imitate the application in the drug of disease caused by ingredient treats oxidative damage in preparation.
15. having polypeptide derivative described in the polypeptide for repairing oxidative damage function or claim 2 as guarantor described in claim 1
Strong ingredient is preparing the application in antioxidant health-care product.
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