CN102952177A - Odorrana tiannanensis natural antioxidant peptide reconstructed body and preparation method and application thereof - Google Patents
Odorrana tiannanensis natural antioxidant peptide reconstructed body and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to odorrana tiannanensis natural antioxidant peptide reconstructed bodies and a preparation method and application thereof. The reconstructed bodies comprise two types, i.e. OA-OTVC and OA-OTC, which are linear chain small peptides and respectively contain sixteen amino acid residues and fourteen amino acid residues, the molecular weight of the OA-OTVC and the molecular of the OA-OTC are respectively 1688.06Da and 1489.79Da, and the isoelectric points are respectively 9.85 and 9.62. The polypeptide complete sequence primary structure of the OA-OTVC is valine - valine- lysine - cysteine - serine - cysteine - arginine - proline - glycine - serine - proline - alanine - proline - arginine - cysteine - lysine; the polypeptide complete sequence primary structure of the OA-OTC is lysine - cysteine - serine - cysteine - arginine - proline - glycine - serine - proline - alanine - proline - arginine - cysteine - lysine. The reconstructed bodies have the advantages of good antioxidant activity, small molecular weight and simple structure, and can be used for preparing antioxidant drugs, food additives, cosmetics and the like.
Description
Technical field
The invention provides transformation body OA-OTVC, OA-OTC of two kinds of Natural Antioxidant Peptides that derive from the smelly frog in the southern regions of the Yunnan Province (Odorrana tiannanensis) and preparation method thereof, use, belong to field of biomedicine technology.
Background technology
Free radical refers to any atom or atomic group that comprises a unpaired electron.Organism mainly is active oxygen radical with the free radical that the external world contacts and self produces, and comprises ultra-oxygen anion free radical, hydroxy radical qiao, hydrogen peroxide, lipid peroxy free radical, nitrogen peroxide, nitric oxide free radical, singlet oxygen and ozone.Under higher concentration, active oxygen radical is to cellularstructure, nucleic acid, lipid and the protein injury of biomass cells.Cause dna damage, further cause misreplication, genomic instability, transcribe unexpected initial or termination, thereby cause organism variation or disease to occur; Cause cell membrane fluidity and permeability changes, cause cellularstructure and changing function; Cause protein oxidation, cause protein denaturation and inactivation.
Find at present, active oxygen radical is relevant with multiple human diseases, such as cancer, cardiovascular and cerebrovascular diseases, ischemical reperfusion injury, rheumatoid arthritis, diabetes, alzheimer's disease, Parkinson's disease and aging etc.In order to resist active oxygen radical to the infringement of body, organism forms multiple different free radical scavenging system in very long evolutionary process, the small-molecule substance that comprises the non-genomic coding is such as uric acid, vitamins C, vitamin-E, carotenes, Thioctic Acid and ubiquinone etc.; The macromole antioxidant reductase of genes encoding is such as superoxide-dismutase (SOD), catalase, peroxidase, Trx enzyme system and gsh enzyme system; The small molecules anti-oxidation peptide class of genes encoding is such as the various small molecules antioxidation polypeptides of finding from batrachians ranid skin.Various dissimilar free-radical scavengerss have great application potential at medicine and cosmetic field, such as vitamins C, superoxide-dismutase (SOD) and small molecules antioxidation polypeptide.
China has abundant amphibian animal resource, and it is the important component part of Chinese biological resource, and wherein a lot of species have the value such as edible or medicinal, have very large development and use potentiality.Amphibians is owing to living in for a long time under the environment that humidity, darkness, microorganism grow in a large number, three cover defense mechanisms in long-term natural evolution process, have been formed: (1) physical barriers, mucous gland is secreted a large amount of glycoprotein and proteoglycan, be wrapped in the skin appearance face, hinder the intrusion of pathogenic bacteria; (2) acquired immune system contains IgG antibody in the Amphibians skin; (3) innate immune system mainly is made of a large amount of antibacterial peptides and anti-oxidation peptide.There is long history in China to the application of batrachians medicine, but the research of its activeconstituents and pharmacological properties is mainly concentrated on the organic molecules such as alkaloid, and the research of its skin activity peptide matters is also rarely had report.The smelly frog in the southern regions of the Yunnan Province (Odorrana tiannanensis) belongs to amphibia Anura Ranidae Rana, is the endemic species of China, is distributed in the ground such as Hainan, Yunnan, moves in the dark mountain stream that The turbulent river crashes its way through more.The type locality of these species is at Hekou, Yunnan Province.Have been reported about the smelly frog form in the southern regions of the Yunnan Province, growth and molecular evolution at present, about the natural antibacterial in the smelly frog in the southern regions of the Yunnan Province source-anti-oxidant multifunctional polypeptide, we have been reported in previous work.
Transformation body OA-OTVC, the OA-OTC of the Natural Antioxidant Peptides of the smelly frog in the southern regions of the Yunnan Province of the present invention (Odorrana tiannanensis), molecular weight is little, and is simple in structure, is linear molecule, makes things convenient for chemosynthesis and preparation.Other anti-oxidation peptides and the transformation precursor of comparing and having reported, the oxidation-resistance of OA-OTVC, OA-OTC is stronger, to DPPH, the ABS free radical all has extremely strong reductibility, therefore can become the good template of exploitation anti-oxidation medicine, foodstuff additive, makeup effective constituent.
Summary of the invention
The invention provides two kinds and have very strong resistance of oxidation, derive from transformation body OA-OTVC, OA-OTC and preparation method and the application of the smelly frog in the southern regions of the Yunnan Province (Odorrana tiannanensis) Natural Antioxidant Peptides.
For achieving the above object, the technical solution used in the present invention is:
Transform body OA-OTVC: the little peptide of a kind of straight chain, contain 16 amino-acid residues, molecular weight is 1688.06 Da, iso-electric point is 9.85; Its polypeptide complete sequence primary structure is α-amino-isovaleric acid-α-amino-isovaleric acid-Methionin-halfcystine-Serine-halfcystine-Arg-Pro-glycine-Serine-proline(Pro)-Ala-Pro-arginine-halfcystine-Methionin;
Transform body OA-OTC: the little peptide of a kind of straight chain, contain 14 amino-acid residues, molecular weight is 1489.79Da, iso-electric point is 9.62; Its polypeptide complete sequence primary structure is: Methionin-halfcystine-Serine-halfcystine-Arg-Pro-glycine-Serine-proline(Pro)-Ala-Pro-arginine-halfcystine-Methionin.
According to the aminoacid sequence of the two, with synthetic its complete sequence of automatic Peptide synthesizer; By HPLC reversed phase column chromatography desalting and purifying, and determine that its purity is greater than 95%; Measure its molecular weight with ground substance assistant laser desorption ionization flight time mass spectrum (MALDI-TOF-MS).
Transform the application of body antibacterial peptide, described transformation body antibacterial peptide is for the preparation of the purposes of anti-oxidation medicine, foodstuff additive, makeup effective constituent.
Beneficial effect of the present invention is: the present invention is according to the aminoacid sequence of the southern regions of the Yunnan Province smelly frog Natural Antioxidant Peptides odorranain-A-OT, utilize transformation body OA-OTVC, the OA-OTC of molecular modification method design odorranain-A-OT, this transformation body has extremely strong anti-oxidant activity, has in addition the beneficial features such as molecular weight is little, simple in structure, the preparation method is simple.
Embodiment:
The below further specifies essentiality content of the present invention with embodiment, but content of the present invention is not limited to this.
Embodiment 1
Derive from the transformation body OA-OTVC of the smelly frog in the southern regions of the Yunnan Province (Odorrana tiannanensis) Natural Antioxidant Peptides, the chemical preparation process of OA-OTC:
The preparation method of I, OA-OTVC, OA-OTC: the aminoacid sequence of transforming body OA-OTVC, OA-OTC according to the smelly frog in the southern regions of the Yunnan Province (Odorrana tiannanensis) Natural Antioxidant Peptides, with automatic Peptide synthesizer (433A, Applied Biosystems) synthetic its complete sequence utilizes the desalination of HPLC reversed phase column chromatography.
II, molecular weight determination adopt ground substance assistant laser desorption ionization flight time mass spectrum (MALDI-TOF).
The OA-OTVC of III, purifying, OA-OTC identify its purity with the high-efficient liquid phase chromatogram HPLC method, molecular weight determination adopts ground substance assistant laser desorption ionization flight time mass spectrum (MALDI-TOF), isoelectric focusing electrophoresis is measured iso-electric point, measures the aminoacid sequence structure with automatic Protein Sequencer.
Measurement result is: OA-OTVC, OA-OTC are two kinds of varients of the smelly frog in smelly frog the southern regions of the Yunnan Province, the southern regions of the Yunnan Province (Odorrana tiannanensis) Natural Antioxidant Peptides odorranain-A-OT.It all is straight-chain polypeptide for OA-OTVC, OA-OTC, contains respectively 16,14 amino-acid residues, and molecular weight is respectively 1688.06 Da, 1489.79Da, and iso-electric point is respectively 9.85,9.62.。The OA-OTVC total order is classified as: α-amino-isovaleric acid-α-amino-isovaleric acid-Methionin-halfcystine-Serine-halfcystine-Arg-Pro-glycine-Serine-proline(Pro)-Ala-Pro-arginine-halfcystine-Methionin; The OA-OTC total order is classified as: Methionin-halfcystine-Serine-halfcystine-Arg-Pro-glycine-Serine-proline(Pro)-Ala-Pro-arginine-halfcystine-Methionin.
Embodiment 2
The anti-oxidant activity experiment of OA-OTVC, OA-OTC:
1.DPPH free radical scavenging activity:
Take by weighing a certain amount of DPPH(2,2 '-hexichol is for bitter taste acyl group phenylhydrazine), use dissolve with methanol, be made into the solution of 6 * 10-5 M, now with the current.92 μ l DPPH solution and 8 μ l samples (OA-OTVC, OA-OTC, 2 mg/ml) are mixed, and at room temperature lucifuge leaves standstill 30 min respectively, and measures light absorption value respectively at 517 nm places.The blank group replaces testing sample with the sample dissolution medium.Experiment do three parallel, in ultraviolet spectrophotometer when zeroing, used methyl alcohol.
DPPH clearance rate (%)=(AB-AA)/AB * 100(AB: blank group light absorption value; AA: the sample sets light absorption value).
Table 1. DPPH anti-oxidant activity
The result is as shown in table 1, and OA-OTVC, OA-OTC have extremely strong DPPH free radical scavenging activity, and when sample concentration was 160 μ g/ml, its DPPH clearance rate I% can reach respectively 57.79% and 57.19%.
2. the ABTS+ radical cation is removed active
2,2 '-azino-bis (3-ethylbezothiazoline-6-sulfonic acid) diamonium salt, ABTS, Amresco, the U.S.), Chinese 2,2-azine by name-two (3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) di-ammonium salts.ABTS can oxidized generation quite stable blue-greenish colour radical cation ABTS
+, at 734 nm places maximum absorption band is arranged, ABTS is being arranged in the presence of the antioxidant of hydrogen supply capacity
+With it reaction, generation does not have coloured ABTS, and the variation of measuring 734 nm place absorbancys can reflect antioxidant for clearing ABTS
+Capacity of water.
ABTS storage liquid preparation: ABTS is dissolved in the solution that PBS damping fluid (3.58 g NaHPO411H2O, 0.15 g KCl is dissolved in 1 L deionized water, with 0.1 M NaOH accent pH to 7.4 for 8.18 g NaCl, 0.27 g KH2PO4) is made into 2 mM.
ABTS
+Solution preparation: with ABTS storage liquid and the by volume 250:1 mixing of 70 mM Potassium Persulphate (K2S2O8) aqueous solution, place 15-16 h in the room temperature lucifuge.Before on-test, be 0.80 ± 0.03 with ABTS+ solution with the light absorption value that PBS is diluted to 734 nm wavelength places.
With 2 μ l samples (2 mg/ml) and the above-mentioned corrected ABTS of 48 μ l
+Solution mixes, after room temperature is placed 10 min, in the light absorption value of 734 nm wavelength place detection reaction liquid.The sample dissolution medium is as blank.
ABTS
+Clearance rate I (%)=(AB-AA)/AB * 100 (AB: blank group light absorption value; AA: the sample sets light absorption value).
Table 2. ABTS
+Radical cation is removed active
The result is as shown in table 2, and OA-OTVC, OA-OTC have extremely strong ABTS+ radical cation and remove activity, and when sample concentration was 160 μ g/ml, its ABTS+ radical cation is removed activity can reach respectively 94.38% and 94.46%.We studies show that, anti-oxidation peptide of the same race is different to the removing ability of different sorts free radical, and above-mentioned OA-OTVC, the OA-OTC of experimental results show that is strong to ABTS+ free radical scavenging activity comparison DPPH free radical scavenging activity.
3. reducing power is measured
Reagent: 0.2 M, the sodium phosphate buffer of pH 6.6 (26.7 g NaH2PO4H2O, 53.65 g Na2HPO47H2O add deionized water to 1 L); 1% K3Fe (CN) 6; 10% trichoroacetic acid(TCA); 1% FeCl3.
Experimental technique: 10 μ l samples (2 mg/ml) mix with 50 μ l sodium phosphate buffers and 50 μ l, 1% K3Fe (CN) 6,50 ℃ of water-bath 20 min; Then the trichoroacetic acid(TCA) that adds 50 μ l 10%, centrifugal 10 min of 3000 rpm; Get supernatant 50 μ l, add 50 μ l deionized waters and 10 μ l, 1% FeCl3, absorb in 700 nm photometries.More the strong representation reducing power is stronger in photoabsorption.
Table 3. reducing power is measured
The result is as shown in table 3, and the absorbance of OA-OTVC, OA-OTC is increased significantly than the blank group, and photoabsorption is stronger, and expression sample reducing power is stronger.
Claims (3)
1. the transformation body of the smelly frog Natural Antioxidant Peptides in the southern regions of the Yunnan Province is characterized in that, this transformation body comprises two kinds: OA-OTVC and OA-OTC:
OA-OTVC: the little peptide of a kind of straight chain, contain 16 amino-acid residues, molecular weight is 1688.06 Da, iso-electric point is 9.85; Its polypeptide complete sequence primary structure is α-amino-isovaleric acid-α-amino-isovaleric acid-Methionin-halfcystine-Serine-halfcystine-Arg-Pro-glycine-Serine-proline(Pro)-Ala-Pro-arginine-halfcystine-Methionin;
OA-OTC: the little peptide of a kind of straight chain, contain 14 amino-acid residues, molecular weight is 1489.79Da, iso-electric point is 9.62; Its polypeptide complete sequence primary structure is: Methionin-halfcystine-Serine-halfcystine-Arg-Pro-glycine-Serine-proline(Pro)-Ala-Pro-arginine-halfcystine-Methionin.
2. the chemical synthesis process of transformation body claimed in claim 1 is characterized in that, according to the aminoacid sequence of the two, with synthetic its complete sequence of automatic Peptide synthesizer; By HPLC reversed phase column chromatography desalting and purifying, and determine that its purity is greater than 95%; Measure its molecular weight with ground substance assistant laser desorption ionization flight time mass spectrum (MALDI-TOF-MS).
3. the application of transformation body claimed in claim 1 is characterized in that, transforms body OA-OTVC and OA-OTC and has DPPH free radical scavenging activity and ABTS+ radical cation removing activity, for the preparation of anti-oxidation medicine, foodstuff additive, makeup.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103233012A (en) * | 2013-05-17 | 2013-08-07 | 梁醒财 | Antioxidant peptide of odorrana andersonii skin and gene thereof |
| CN107253977A (en) * | 2017-07-03 | 2017-10-17 | 大连理工大学 | Suppress melanin and generate oxidation resistant small peptide, preparation method and applications |
| CN107266534A (en) * | 2017-07-03 | 2017-10-20 | 大连理工大学 | The anti-oxidant small peptide for suppressing melanin generation, preparation method and applications |
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| US20030087819A1 (en) * | 2001-05-09 | 2003-05-08 | Bielicki John K. | Cysteine-containing peptides having antioxidant properties |
| CN101638432A (en) * | 2009-03-27 | 2010-02-03 | 中国科学院昆明动物研究所 | Rana pleuraden antioxidant peptide and genes and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030087819A1 (en) * | 2001-05-09 | 2003-05-08 | Bielicki John K. | Cysteine-containing peptides having antioxidant properties |
| CN101638432A (en) * | 2009-03-27 | 2010-02-03 | 中国科学院昆明动物研究所 | Rana pleuraden antioxidant peptide and genes and application thereof |
Non-Patent Citations (3)
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| WEIYU HE ET AL.: "Host defense peptides in skin secretions of Odorrana tiannanensis: Proof for other survival strategy of the frog than merely anti-microbial", 《BIOCHIMIE》 * |
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| 周涌等: "林蛙皮活性肽抗氧化活性研究", 《安徽农业科学》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103233012A (en) * | 2013-05-17 | 2013-08-07 | 梁醒财 | Antioxidant peptide of odorrana andersonii skin and gene thereof |
| CN107253977A (en) * | 2017-07-03 | 2017-10-17 | 大连理工大学 | Suppress melanin and generate oxidation resistant small peptide, preparation method and applications |
| CN107266534A (en) * | 2017-07-03 | 2017-10-20 | 大连理工大学 | The anti-oxidant small peptide for suppressing melanin generation, preparation method and applications |
| CN107253977B (en) * | 2017-07-03 | 2020-04-28 | 大连理工大学 | Antioxidant small peptide for inhibiting melanin generation, preparation method and application thereof |
| CN107266534B (en) * | 2017-07-03 | 2020-11-13 | 大连理工大学 | Small peptide for resisting oxidation and inhibiting melanin generation, preparation method and application thereof |
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