CN116751248B - Antioxidant peptide and application thereof in preparation of free radical scavenging drugs - Google Patents
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- 101800000068 Antioxidant peptide Proteins 0.000 title claims abstract description 24
- 239000003814 drug Substances 0.000 title abstract description 15
- 229940079593 drug Drugs 0.000 title abstract description 10
- 238000002360 preparation method Methods 0.000 title description 8
- 230000007760 free radical scavenging Effects 0.000 title description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 7
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 29
- 230000003078 antioxidant effect Effects 0.000 abstract description 19
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 17
- 229920001184 polypeptide Polymers 0.000 abstract description 15
- 240000006794 Volvariella volvacea Species 0.000 abstract description 13
- 102000004169 proteins and genes Human genes 0.000 abstract description 12
- 108090000623 proteins and genes Proteins 0.000 abstract description 12
- 150000003254 radicals Chemical class 0.000 abstract description 8
- -1 DPPH free radical Chemical class 0.000 abstract description 4
- 239000000284 extract Substances 0.000 abstract description 3
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- 238000001514 detection method Methods 0.000 abstract description 2
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- 235000006708 antioxidants Nutrition 0.000 description 11
- 239000000047 product Substances 0.000 description 7
- 230000002292 Radical scavenging effect Effects 0.000 description 5
- MGJZITXUQXWAKY-UHFFFAOYSA-N diphenyl-(2,4,6-trinitrophenyl)iminoazanium Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1N=[N+](C=1C=CC=CC=1)C1=CC=CC=C1 MGJZITXUQXWAKY-UHFFFAOYSA-N 0.000 description 5
- 238000010521 absorption reaction Methods 0.000 description 4
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 244000252132 Pleurotus eryngii Species 0.000 description 2
- 235000001681 Pleurotus eryngii Nutrition 0.000 description 2
- 239000012564 Q sepharose fast flow resin Substances 0.000 description 2
- 206010070834 Sensitisation Diseases 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 150000001450 anions Chemical class 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 2
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- RQOCXCFLRBRBCS-UHFFFAOYSA-N (22E)-cholesta-5,7,22-trien-3beta-ol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)C=CCC(C)C)CCC33)C)C3=CC=C21 RQOCXCFLRBRBCS-UHFFFAOYSA-N 0.000 description 1
- OQMZNAMGEHIHNN-UHFFFAOYSA-N 7-Dehydrostigmasterol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)C=CC(CC)C(C)C)CCC33)C)C3=CC=C21 OQMZNAMGEHIHNN-UHFFFAOYSA-N 0.000 description 1
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- DNVPQKQSNYMLRS-NXVQYWJNSA-N Ergosterol Natural products CC(C)[C@@H](C)C=C[C@H](C)[C@H]1CC[C@H]2C3=CC=C4C[C@@H](O)CC[C@]4(C)[C@@H]3CC[C@]12C DNVPQKQSNYMLRS-NXVQYWJNSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
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- 240000001080 Grifola frondosa Species 0.000 description 1
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- 241000282412 Homo Species 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
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- 239000012505 Superdex™ Substances 0.000 description 1
- XBJFCYDKBDVADW-UHFFFAOYSA-N acetonitrile;formic acid Chemical compound CC#N.OC=O XBJFCYDKBDVADW-UHFFFAOYSA-N 0.000 description 1
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- 125000000539 amino acid group Chemical group 0.000 description 1
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- 235000021466 carotenoid Nutrition 0.000 description 1
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- 230000009920 chelation Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
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- DNVPQKQSNYMLRS-SOWFXMKYSA-N ergosterol Chemical compound C1[C@@H](O)CC[C@]2(C)[C@H](CC[C@]3([C@H]([C@H](C)/C=C/[C@@H](C)C(C)C)CC[C@H]33)C)C3=CC=C21 DNVPQKQSNYMLRS-SOWFXMKYSA-N 0.000 description 1
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 description 1
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- 239000007791 liquid phase Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
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- 239000000203 mixture Substances 0.000 description 1
- 238000005319 nano flow HPLC Methods 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000002542 parent ion scan Methods 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
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- 238000010298 pulverizing process Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
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- 210000001995 reticulocyte Anatomy 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
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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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/18—Antioxidants, e.g. antiradicals
-
- 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
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Toxicology (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Dermatology (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses an antioxidant peptide and application thereof in preparing a medicament for removing free radicals. The amino acid sequence of the antioxidant peptide is SGPSFDWPGFK. The invention extracts and purifies polypeptide from straw mushroom fruiting body protein, determines the amino acid sequence of one antioxidant peptide through LC-MS/MS, and has DPPH free radical clearance of 67.15% +/-0.44% and Fe after antioxidant activity detection 2+ The chelating ability is 76.34+/-0.31%, the reducing ability is 0.34+/-0.01, and the polypeptide can be used as a natural antioxidant peptide and has potential application prospect in the fields of preparing medicines, foods, skin care products and the like for removing free radicals.
Description
Technical Field
The invention belongs to the technical field of biological extracts, and relates to an antioxidant peptide and application thereof in preparing a medicament for removing free radicals.
Background
Excessive free radical attack on biomolecules in the human body causes oxidative damage, which to some extent causes potential damage to the body, leading to the development of various diseases such as cancer, aging, atherosclerosis and Alzheimer's disease. Several studies have shown that antioxidants, at relatively low concentrations, can inhibit or delay oxidative damage to cells, and have an important role in human health. The research of efficient and safe natural antioxidants has been the focus of attention of researchers at home and abroad. Antioxidant peptides extracted from natural products are reported to have free radical scavenging activity with less toxicity and side effects to humans than chemically synthesized drugs. The only disadvantage is the low oral bioavailability of polypeptide drugs. Ouyang Junfang et al point out that oral biomacromolecule drugs have very limited stability and absorption degree in the gastrointestinal tract, reviewed the entrapment mode of lipid nanocarriers on protein polypeptide drugs and their corresponding mechanism of overcoming physiological barriers, and introduced important characteristics and recent research progress for improving the oral bioavailability of protein polypeptide drugs (Ouyang Junfang, zhang Yongjie, chen Xijing. Research progress of lipid nanocarriers for oral delivery of protein polypeptide drugs [ J ]. J.China medicine industry J2022,53 (09): 1240-1250.).
Numerous studies have demonstrated that edible fungi are rich in a variety of antioxidants including polysaccharides, phenols, proteins, peptides, carotenoids, ergosterol, vitamins C and E, and the like. In addition, edible mushrooms grow faster than plants, which makes them a relatively abundant source of commercial natural bioactive compounds. Pei Yuncheng the preparation and preliminary analysis of stability of antioxidant peptide of pleurotus eryngii stem have been studied, wherein the DPPH free radical clearance of the antioxidant peptide of pleurotus eryngii is 55.52% + -0.89%, the clearance is lower, and the antioxidant capacity is not strong (Pei Yuncheng, zhu Dan, cui Cailian, et al. Preparation of antioxidant peptide of apricot Bao Gubing and preliminary analysis of stability thereof [ J ]. Food industry technology, 2020,41 (4): 8.). Straw mushrooms contain various active ingredients, especially abundant proteins, but no related report has been made so far on the preparation of antioxidant peptides from straw mushroom fruiting bodies as raw materials.
Disclosure of Invention
The invention aims to provide an antioxidant peptide and application thereof in preparing a medicament for removing free radicals.
The technical solution for realizing the purpose of the invention is as follows:
the amino acid sequence of the antioxidant peptide is SGPSFDWPGFK, namely Ser-Gly-Pro-Ser-Phe-Asp-Trp-Pro-Gly-Phe-Lys, and is shown as SEQ ID NO. 1.
The antioxidant peptide is applied to the preparation of a medicament for removing free radicals.
The free radical is DPPH.
Compared with the prior art, the invention has the following advantages:
the invention firstly extracts and purifies polypeptide from the sporophore protein of straw mushroom, discovers a plurality of antioxidant peptides with good antioxidant capacity, determines the amino acid sequence of one antioxidant peptide as SGPSFDWPGFK through LC-MS/MS, and has DPPH free radical clearance of 67.15 percent +/-0.44 percent and Fe by antioxidant activity detection 2+ The chelating ability is 76.34% + -0.31%; the reducing power is 0.34+/-0.01, can be used as a natural antioxidant peptide, and has potential application prospect in the fields of preparing medicines, foods, skin care products and the like for removing free radicals.
Drawings
FIG. 1 shows the separation of products Q1, Q2 by anion column.
FIG. 2 shows DPPH radical scavenging of isolated products Q1, Q2.
FIG. 3 shows the separation of the products G1, G2, G3, G4, G5 by means of gel columns.
FIG. 4 shows DPPH radical scavenging of isolated products G1, G2, G3, G4, G5.
FIG. 5 is a TIC profile of G3.
FIG. 6 is a secondary mass spectrum of an active polypeptide.
FIG. 7 is a prediction of the structure of an active polypeptide.
Detailed Description
The invention will be described in further detail with reference to specific embodiments and drawings.
1. Measurement of antioxidant Activity:
DPPH Radical Scavenging Activity (DRSA): assays were performed with reference to the kit (purchased from suzhou grissin biotechnology limited).
Fe2+ chelation rate: reference (Ma Mengjiao) preparation of antioxidant peptide of Carnis Trionycis and its anti-aging function research [ D ]. Tin-free: university of Jiangnan 2020:1-68).
3. Measurement of reducing force: the reducing power of the samples was determined by the method of reference (Dong Y R, qi G H, yang Z P, et al preparation, separation and antioxidant properties of hydrolysates derived from Grifola frondosa protein [ J ]. Analytical and Bioanalytical Chemistry,2015,33 (6): 500-506).
Example 1
(1) Repeatedly washing straw mushroom fruiting bodies to remove impurities such as cultivation materials, draining water, freeze-drying, pulverizing into powder, and mixing with the double distilled water according to the mass ratio of 10:1, adding double distilled water to dissolve straw mushroom powder, standing for 4h, centrifuging at 8000r/min at 4 ℃ for 20min, taking supernatant, using 80% saturation according to an ammonium sulfate saturation table, adding ammonium sulfate into the supernatant, fully dissolving, standing for 12h, centrifuging to obtain precipitate, dissolving the precipitate in double distilled water, adding into a dialysis bag, placing into ultrapure water, dialyzing for 48h, and freeze-drying after the completion to obtain straw mushroom protein.
(2) Dissolving straw mushroom protein in double distilled water (substrate mass concentration is 3.11g/100 mL), regulating pH and temperature to the optimal action condition of alkaline protease (purchased from Shanghai Meilin Biochemical technology Co., ltd., enzyme activity unit is 250U/mg), balancing for 30min, adding alkaline protease for enzymolysis for 3.7h, adding enzyme amount is 3.81% (the added amount of alkaline protease accounts for the mass percent of straw mushroom protein), inactivating enzyme after enzymolysis, heating at 90 ℃ for 15min to terminate reaction, centrifuging at 4 ℃ for 15min at 8000r/min, taking supernatant, and freeze-drying to obtain an enzymolysis product. Separating and purifying the enzymolysis product sequentially by using an ultrafiltration centrifuge tube with 10kDa and 3kDa, and lyophilizing to obtain three polypeptide components with different molecular weights, wherein F1 is less than 3kDa, F2 is less than 3kDa and F3 is less than 10kDa>10kDa. The antioxidant activity (DPPH free radical scavenging Activity, fe) of three polypeptide components of different molecular weights were tested separately 2+ Chelating rate and reducing power), and the results are shown in table 1, and it can be seen from table 1 that the polypeptide component having the optimal antioxidant activity is F1.
TABLE 1 antioxidant Activity of three polypeptide Components of different molecular weights
Note that: the different letters represent significant differences (P < 0.05)
Note：Different letters indicate significant differences(P＜0.05)
(3) The polypeptide fraction F1 was isolated and purified using a Q Sepharose FF (1 cm. Times.5 cm) anion exchange column coupled to the AKTA Pure system. Sample F1 was dissolved in Tris-HCl (20 mM pH 7.5) buffer, applied through a 0.22 μm microporous membrane, and the Q Sepharose FF anion column was equilibrated with Tris-HCl (20 mM pH 7.5) buffer. Elution was performed with a linear gradient of 1M NaCl (0-100%) in the same buffer at a flow rate of 1 mL/min. The elution process was monitored at 280nm and the results are shown in FIG. 1, showing two absorption peaks, with absorption peak 375 designated Q1 and absorption peak 50 designated Q2. DPPH radical scavenging activities of Q1 and Q2 were measured, respectively, and the results are shown in FIG. 2, which indicate that the antioxidant activity of component Q1 is highest.
Component Q1 was further purified using a Superdex 30 Increate10/300 GL gel column. Sample Q1 was dissolved in ultrapure water, passed through a microporous membrane of 0.22 μm, and applied to a column, the column was equilibrated with ultrapure water at a flow rate of 0.5mL/min, the column was eluted with the same mobile phase at room temperature at 0.5mL/min, the elution was monitored at 280nm, and 5 components were isolated, as shown in FIG. 3, the DPPH radical scavenging activities of G1, G2, G3, G4 and G5 were measured in the order named G1, G2, G3, G4 and G5 from the order named G3, G4 and G5 from the order named G3, G2, G3, G4 and G5, as shown in FIG. 4, were 32.77.+ -. 0.71%, 58.54.+ -. 1.45%, 68.98.+ -. 0.68%, 49.25.+ -. 0.65%, 42.77.+ -. 0.36% and the results showed that the antioxidant activity of G3 was the highest.
(4) G3 was separated using a Nano-HPLC liquid phase system UltiMate 3000 RSL Cnano (ThermoFisher Scientific). Sample G3 was dissolved in ultrapure water, the sample was loaded by an autosampler and bound to a Trap column (100 μm. Times.20 mm, RP-C18, agilent), mobile phase A was a 0.1% formic acid-water solution, mobile phase B was a 0.1% formic acid-acetonitrile solution, and then separated by Analysis column (75 μm. Times.150 mm, RP-C18, new Objective) under the following conditions: mobile phase B:0-5min,5%;5-30min,5-38%;30-35min,38-95%, and flow rate of 300nL/min. Mass spectrometry was then performed by a Q-actual plus mass spectrometer (ThermoFisher Scientific), parent ion scan range: 350-2000m/z, and performing full scan acquisition spectrum under information dependent acquisition working mode (DDA, date Dependent Acquisition), wherein the TIC spectrum is shown in figure 5. The protein discover 2.1 software is used for analyzing the map, a plurality of antioxidant active peptides are identified and obtained, the amino acid composition and sequence of each antioxidant active peptide are determined, wherein the amino acid sequence of one antioxidant active peptide is SGPSFDWPGFK, the mass spectrum of one antioxidant active peptide is shown as SEQ ID NO.1, the mass spectrum of one antioxidant active peptide is shown as fig. 6, and the molecular weight of one antioxidant active peptide is 1224.3kDa.
(5) The performance of the SGPSFDWPGFK peptide was predicted by bioinformatics.
Bioactive peptide predictor PeptideRanker gave a score for SGPSFDWPGFK peptide with an antioxidant activity score of greater than 0.9. The obtained amino acid sequence SGPSFDWPGFK is subjected to physical and chemical property prediction in a PeptideRanker bioactive peptide prediction server: isoelectric point is between 5.55-5.84; amino acid residues with one positive charge and one negative charge; the stability is good; the predicted half-life of the reticulocyte in the mammal is 1.9h, the half-life in the yeast is more than 20h, and the half-life in the escherichia coli is more than 10h; is a hydrophilic polypeptide.
The safety of the obtained straw mushroom antioxidant peptide is evaluated in a PeptideRanker bioactive peptide prediction server, toxicity and sensitization of the straw mushroom antioxidant peptide are predicted, and the result shows that the straw mushroom antioxidant peptide is nontoxic and has no sensitization and higher safety.
(6) The SGPSFDWPGFK peptide is obtained by solid phase synthesis method in the biological engineering Co., ltd, and the antioxidant activity of SGPSFDWPGFK peptide is measured, the DPPH free radical clearance is 67.15% + -0.44%, fe 2+ The chelating ability is 76.34% + -0.31%; the reducing force is 0.34+/-0.01. The structure of the obtained straw mushroom antioxidant peptide is predicted by PepDraw, and the result is shown in figure 7.
Claims (1)
1. The antioxidant peptide is characterized in that the amino acid sequence is SGPSFDWPGFK and is shown as SEQ ID NO. 1.
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