CN105646656B - Griseus shark cartilage protein antioxidant peptide and application thereof - Google Patents
Griseus shark cartilage protein antioxidant peptide and application thereof Download PDFInfo
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- 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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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Abstract
The invention discloses a griseus cartilage protein antioxidant peptide and application thereof, wherein griseus cartilage is used as a raw material, enzymolysis liquid is obtained by total protein extraction, acetone fractional precipitation and trypsin enzymolysis, the enzymolysis liquid is separated and purified by ultrafiltration, ion exchange resin chromatography, gel column chromatography and reversed-phase high performance liquid chromatography to obtain the antioxidant peptide Gly-Glu-Arg-Glu-Ala-Asn-Val-Met, and the molecular weight is 905.00Da when ESI-MS is used for determination; the prepared high-activity antioxidant peptide has stronger free radical scavenging activity and good lipid peroxidation inhibition effect, and can be developed as a medicine, a health food or a food additive and the like.
Description
Technical Field
The invention relates to an animal cartilage active peptide, in particular to a shark griseus cartilage protein antioxidant peptide and application thereof.
Background
Shark of the Ash color (A)Mustelus griseus) The common name is Sha tiao, also called as Gray mink shark, which is a kind of shark belonging to the genus Astrocaryum of the order Fulviformes of the class Chondrichthyes. Distributed in the northwest pacific region including the south China sea, the east China sea, the yellow sea, Taiwan, etc., and the south Korea, the Mupu, and Japan, etc.
The drugs can be used for the treatment of liver, meat, skin, and fin of Astrocarya platyphylla. The Astrocaryum platypomum skin has effects of relieving fish and shrimp toxin, resolving food stagnation, etc. The shark meat has effects of invigorating spleen, invigorating qi, removing blood stasis, relieving swelling, nourishing, and strengthening body constitution, and can be used for treating asthenia, slow wound healing, spleen deficiency, edema, etc. The shark fin has the effects of nourishing and strengthening, tonifying qi, tonifying deficiency and the like. The shark skin has effects of resolving food stagnation and removing fish toxin. The Astrocaryum torvum has effects of clearing heat and detoxicating, detumescence, promoting blood circulation, etc. The shark bone has the effects of dispelling pathogenic wind, removing dampness, invigorating spleen, relieving diarrhea, promoting blood circulation, and relieving pain. The shark hearts have the effects of invigorating spleen and stomach, eliminating phlegm and fluid retention, tonifying qi and opening diaphragm. Can be used for treating retention of phlegm and fluid in the body, indigestion, chest and wrist distention, emesis, diarrhea, etc. caused by spleen and stomach weakness.
Disclosure of Invention
The technical problem to be solved by the invention is to provide the griseus chondroprotein antioxidant peptide capable of eliminating free radicals and inhibiting lipid peroxidation.
The technical scheme adopted by the invention for solving the technical problems is as follows: a griseus shark cartilage protein antioxidant peptide: the griseus shark cartilage protein antioxidant peptide is characterized in that the antioxidant peptide is an octapeptide compound, the amino acid sequence is Gly-Glu-Arg-Glu-Ala-Asn-Val-Met (GERREANVM), and the molecular weight is 905.00Da when ESI-MS is used for determination.
The preparation method of the griseus shark cartilage protein antioxidant peptide comprises the following steps:
1) preparation of crude extract of total protein of griseus shark cartilage: weighing and cutting grifola frondosa cartilage into small fragments, adding a proper amount of distilled water, homogenizing in a high-speed tissue triturator to obtain paste, soaking the paste grifola frondosa cartilage in 1.0 mol/L guanidine hydrochloride solution (containing 0.02 mol/L MES and 0.02 mol/L EDTA and having the pH value of 7-8) with the volume of 4-6 times of that of the paste, and stirring and extracting at the temperature of 4 ℃ for 36-48 h. Centrifuging the extracting solution at the temperature of 4 ℃ at 4000-6000 r/min for 15-25 min, removing residues and collecting supernate; centrifuging the supernatant at 4 ℃ at 10000-12000 r/min for 15-25 min, and filtering the supernatant with a 0.22 mu m microporous filter membrane to remove insoluble impurities. Putting the filtrate into a dialysis bag with molecular cut-off of 8000, dialyzing with Tris-HCl (pH 7.6) buffer solution at 4 ℃ for 24-48 h, and obtaining the solution in the dialysis bag as the crude extract of the total protein of the shark cartilage.
2) Preparation of the protein by acetone fractional precipitation of griseus shark cartilage: slowly adding pre-cooled acetone into the crude extract of the total protein of the griseus frondosus cartilage in an ice bath until the concentration of the acetone is 30%, standing for 4-6 h at the temperature of-20 ℃, centrifuging at the temperature of 4 ℃ and at the speed of 10000-12000 r/min for 20 min, taking supernatant, adding the pre-cooled acetone into the solution until the final concentration of the acetone is 60%, obtaining the 60% acetone precipitated protein of the griseus frondosus cartilage, and freeze-drying to obtain the 60% acetone fractional precipitated protein.
3) Enzymolysis of the protein precipitated by acetone fractionation of griseus shark cartilage:dissolving 60% acetone fractional precipitation protein of griseus platyphylla cartilage into Tris-HCl buffer solution (0.05 mol/L, pH 7.5-8.5) according to a feed-liquid ratio of 1g: 3-5 mL, adding trypsin (1.9 × 10) according to 2.0-3.0% of the weight of the 60% acetone fractional precipitation protein4U/g), carrying out enzymolysis for 3-5 h at 40 ℃, then inactivating enzyme at 90 ℃ for 10 min, centrifuging the enzymolysis liquid at 4 ℃ for 15-25 min at 10000-12000 r/min, discarding residues, and collecting supernatant to obtain the cartilage protein enzymolysis liquid.
4) The preparation of the griseus shark cartilage protein antioxidant peptide: performing ultrafiltration treatment on the enzymolysis liquid by using a 3 kDa ultrafiltration membrane, collecting a part with the molecular weight less than 3 kDa, and freeze-drying to obtain an ultrafiltration zymolyte; purifying the ultrafiltration zymolyte by ion exchange resin chromatography, gel column chromatography and reversed phase high performance liquid chromatography (RP-HPLC) to obtain the antioxidant peptide.
Preferably, the shark of step 1) is a shark ofMustelus griseus）。
Preferably, the specific processes of ion exchange resin chromatography, gel column chromatography and RP-HPLC purification in the step 4) are as follows:
ion exchange resin chromatography: dissolving the ultrafiltration zymolyte in double distilled water to prepare a solution with the concentration of 45-55 mg/mL, passing through a DEAE-52 ion exchange resin chromatographic column, eluting with water, 0.1 mol/L, 0.5 mol/L and 1.0 mol/L NaCl solution at the flow rate of 0.6-0.8 mL/min, collecting one tube of the eluate every 3min, detecting at 280 nm, merging the solutions in the test tube according to peaks, comparing the removal activities of DPPH free radicals and hydroxyl free radicals of each peak, and selecting a component with the strongest activity for freeze-drying to obtain the ion exchange chromatography zymolyte;
gel column chromatography: dissolving the ion exchange chromatography zymolyte in double distilled water to prepare a solution with the concentration of 45-55 mg/mL, separating by Sephadex G-15 column chromatography, eluting by the double distilled water at the flow rate of 0.6-0.8 mL/min, collecting one tube of the eluted solution every 3min, detecting at 280 nm, merging the solutions in the test tube according to peaks, comparing the removal activity of DPPH free radicals and hydroxyl free radicals of each peak, and selecting a component with the strongest activity for freeze-drying to obtain the gel chromatography zymolyte;
RP-HPLC purification: preparing the gel chromatography zymolyte into a solution of 80-100 mu g/mL by using double distilled water, purifying by using RP-HPLC, and obtaining 1 high-activity antioxidant peptide Gly-Glu-Arg-Glu-Ala-Asn-Val-Met (GERARNVM) according to the scavenging activity of DPPH free radicals and hydroxyl free radicals, wherein the molecular weight is 905.00Da by ESI-MS (ESI-MS).
More preferably, the RP-HPLC conditions are: the sample injection amount is 10-15 mu L; chromatographic column Zorbax SB-C18(250 mm. times.4.6 mm, 5 μm); mobile phase: water-acetonitrile gradient elution (acetonitrile concentration is increased from 0 to 50 percent at constant speed in 0-32 min); the elution speed is 0.6-1.0 mL/min; the ultraviolet detection wavelength is 280 nm.
The invention also provides the application of the griseus cartilage protein antioxidant peptide: it has the advantages of safety, no toxic side effect, strong antioxidant activity, easy digestion and absorption, etc., so it can be used as additive for medicine, health food and food.
Compared with the prior art, the griseus shark cartilage protein antioxidant peptide has good scavenging effect on DPPH free radicals, hydroxyl free radicals, ABTS free radicals and superoxide anion free radicals; meanwhile, Gly-Glu-Arg-Glu-Ala-Asn-Val-Met (GERARNVM) also shows good lipid peroxidation inhibition effect; Gly-Glu-Arg-Glu-Ala-Asn-Val-Met (GERARNVM) has the advantages of safety, no toxic or side effect, strong antioxidant activity, easy digestion and absorption and the like, and can be used as an additive of medicines, health-care foods and foods.
Drawings
FIG. 1 is a chromatogram of DEAE-52 ion exchange resin of the present invention.
FIG. 2 is a Sephadex G-15 chromatogram of a Sephadex gel of the invention.
FIG. 3 RP-HPLC analysis of the zymolyte prepared by Sephadex G-15.
FIG. 4 Mass Spectrum of Gly-Glu-Arg-Glu-Ala-Asn-Val-Met (GEREMNVM).
FIG. 5 the lipid oxidation resistance of Gly-Glu-Arg-Glu-Ala-Asn-Val-Met (GERARMINV).
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
Example (b):
a preparation method of the griseus shark cartilage protein antioxidant peptide comprises the following preparation process flows: the antioxidant peptide is prepared by performing ultrafiltration, ion exchange chromatography, gel filtration chromatography and high performance liquid chromatography on 60% acetone precipitated protein trypsin enzymolysis zymolyte of cartilage of hammerhead shark cartilage.
1) Preparation of crude extract of total protein of griseus shark cartilage: weighing sharkMustelus griseus) Cutting cartilage into small pieces, adding appropriate amount of distilled water, homogenizing in high speed tissue triturator to paste, soaking pasty shark cartilage in 4 times volume of 1.0 mol/L guanidine hydrochloride solution (containing 0.02 mol/L MES and 0.02 mol/L EDTA, pH 7.0), and extracting under stirring at 4 deg.C for 48 hr. Centrifuging the extractive solution at 4 deg.C at 5000 r/min for 20 min, removing residue, and collecting supernatant; centrifuging the supernatant at 4 deg.C and 12000 r/min for 20 min, and filtering the supernatant with 0.22 μm microporous membrane to remove insoluble impurities. Putting the filtrate into a dialysis bag with molecular cut-off of 8000, dialyzing with Tris-HCl (pH 7.6) buffer at 4 deg.C for 48 hr to obtain crude extract of total protein of shark cartilage.
2) Preparation of the protein by acetone fractional precipitation of griseus shark cartilage: taking crude extract of the total protein of the griseus cartilage, slowly adding precooled acetone in an ice bath until the acetone concentration is 30%, standing for 5 h at-20 ℃, centrifuging at a high speed of 10000 r/min for 20 min at 4 ℃, taking supernatant, adding precooled acetone until the acetone concentration is 60% to obtain the 60% acetone precipitated protein of the griseus cartilage, and freeze-drying to obtain the 60% acetone fractional precipitated protein.
3) Enzymolysis of the protein precipitated by acetone fractionation of griseus shark cartilage: dissolving shark cartilage 60% acetone fractional precipitation protein in Tris-hydrochloric acid buffer solution (0.05 mol/L, pH 8.0) at a ratio of 1g:4mL, adding trypsin (1.9 × 10) 2.5% of the weight of the protein4U/g), performing enzymolysis at 40 ℃ for 4 h, then inactivating enzyme at 90 ℃ for 10 min, centrifuging the enzymolysis liquid at 4 ℃ for 20 min at 12000 r/min, discarding residues, and collecting supernatant to obtain cartilage protein enzymolysis liquid.
4) The preparation of the griseus shark cartilage protein antioxidant peptide: and (3) carrying out ultrafiltration treatment on the enzymolysis liquid by adopting a 3 kDa ultrafiltration membrane, collecting a part (MP 60-I) with the molecular weight less than 3 kDa to obtain ultrafiltration enzymolysis liquid, and purifying the ultrafiltration enzymolysis liquid by sequentially carrying out ion exchange resin chromatography, gel column chromatography and reversed phase high performance liquid chromatography (RP-HPLC) to obtain the antioxidant peptide.
① ion exchange resin chromatography, dissolving MP60-I in double distilled water to prepare a solution with the concentration of 50 mg/mL, passing through a DEAE-52 ion exchange resin chromatography column, eluting with water, 0.1 mol/L, 0.5 mol/L and 1.0 mol/L NaCl solution at the flow rate of 0.6 mL/min, collecting one tube of the eluate every 3min, detecting at 280 nm, merging the solutions in the tube according to peaks, comparing the DPPH free radical and hydroxyl free radical scavenging capacity of each peak, selecting the component with the strongest antioxidant capacity, and freeze-drying to obtain the ion exchange chromatography zymolyte MP60-3 (figure 1);
② gel column chromatography, dissolving MP60-3 in double distilled water to prepare a solution with the concentration of 50 mg/mL, separating by Sephadex G-15 column chromatography, eluting with double distilled water at the flow rate of 0.6-0.8 mL/min, collecting one tube of the eluate every 3min, detecting at 280 nm, combining the solutions in the tube according to peaks, comparing the DPPH free radical and hydroxyl free radical scavenging capacity of each peak, selecting the component with the strongest antioxidant capacity, and freeze-drying to obtain a gel chromatography zymolyte MP60-3-1 (figure 2);
③ RP-HPLC purification, preparing MP60-3-1 into 80-100 μ g/mL solution with double distilled water, and purifying by RP-HPLC (RP-HPLC conditions are that the sample amount is 15 μ L; and chromatographic column Zorbax SB-C18(250 mm. times.4.6 mm, 5 μm); mobile phase: water-acetonitrile gradient elution (acetonitrile concentration is increased from 0 to 50 percent at constant speed in 0-32 min); the elution speed is 0.8 mL/min; ultraviolet detection wavelength of 280 nm) to obtain 1 high-activity antioxidant peptide DCPE-B (figure 3) based on the scavenging activity to DPPH free radical and hydroxyl free radical.
④ Structure detection comprises collecting antioxidant peptide DCPE-B with highest DPPH free radical and hydroxyl free radical scavenging activity, detecting by RP-HPLC to obtain single peak, determining amino acid sequence by protein/polypeptide sequence analyzer to be Gly-Glu-Arg-Glu-Ala-Asn-Val-Met (GERAMV), and determining molecular weight by ESI-MS to be 905.00Da ([ M + H ]]+906.68 Da) (FIG. 4).
The obtained cartilage of Astrocaryum platypomumThe protein antioxidant peptide Gly-Glu-Arg-Glu-Ala-Asn-Val-Met (GERARNVM) is subjected to a free radical scavenging experiment and a lipid peroxidation inhibition experiment, and the experiment results show that: Gly-Glu-Arg-Glu-Ala-Asn-Val-Met (GERREANVM) vs DPPH free radical (EC)502.76 mg/mL), hydroxyl radical (EC)500.22 mg/mL), ABTS free radical (EC)500.08 mg/mL) and superoxide anion radical (EC)500.13 mg/mL); meanwhile, Gly-Glu-Arg-Glu-Ala-Asn-Val-Met (GERARNVM) also showed good inhibition of lipid peroxidation (FIG. 5).
Compared with the prior art, the griseus shark cartilage protein antioxidant peptide has good scavenging effect on DPPH free radicals, hydroxyl free radicals, ABTS free radicals and superoxide anion free radicals; meanwhile, Gly-Glu-Arg-Glu-Ala-Asn-Val-Met (GERARNVM) also shows good lipid peroxidation inhibition effect; Gly-Glu-Arg-Glu-Ala-Asn-Val-Met (GERARNVM) has the advantages of safety, no toxic or side effect, strong antioxidant activity, easy digestion and absorption and the like, and can be used as an additive of medicines, health-care foods and foods.
Finally, it should also be noted that the above-mentioned list is only one specific embodiment of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
SEQUENCE LISTING
<110> Zhejiang ocean academy
<120> shark cartilage protein antioxidant peptide and application thereof
<130>zjou-wb-201511-401
<160>1
<170>PatentIn version 3.5
<210>1
<211>8
<212>PRT
<213> Artificial Synthesis
<400>1
Gly Glu Arg Glu Ala Asn Val Met
1 5
Claims (2)
1. The griseus shark cartilage protein antioxidant peptide is characterized in that: the antioxidant peptide is an octapeptide compound, the amino acid sequence is Gly-Glu-Arg-Glu-Ala-Asn-Val-Met, and the molecular weight is 905.00Da when measured by ESI-MS;
EC of the antioxidant peptide on DPPH free radical502.76mg/mL, EC for hydroxyl radical50EC for ABTS free radical of 0.22mg/mL50EC for superoxide anion radical of 0.08mg/mL50It was 0.13 mg/mL.
2. The use of the shark cartilage protein antioxidant peptide as described in claim 1, wherein the peptide is selected from the group consisting of: the antioxidant peptide can be used for preparing antioxidant health food or food additive.
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