CN102952177B - 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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- 229920001184 polypeptide Polymers 0.000 claims abstract description 10
- 125000000539 amino acid group Chemical group 0.000 claims abstract description 7
- 239000003814 drug Substances 0.000 claims abstract description 6
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 claims description 19
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
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 two kinds and derive from transformation body OA-OTVC, OA-OTC of the Natural Antioxidant Peptides of the smelly frog in the southern regions of the Yunnan Province (Odorrana tiannanensis) and preparation method thereof, application, belong to field of biomedicine technology.
Background technology
Free radical refers to any atom that comprises a unpaired electron or atomic group.Organism is mainly 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, cellularstructure, nucleic acid, lipid and the protein of active oxygen radical to biomass cells causes damage.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, 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 the infringement of active oxygen radical to body, in very long evolutionary process, organism forms multiple different free radical scavenging system, comprise the small-molecule substance of non-genomic coding, as uric acid, vitamins C, vitamin-E, carotenes, Thioctic Acid and ubiquinone etc.; The macromole antioxidant reductase of genes encoding, as superoxide-dismutase (SOD), catalase, peroxidase, Trx enzyme system and gsh enzyme system; The small molecules anti-oxidation peptide class of genes encoding, 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, 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 exploitation 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, are formed: (1) physical barriers, mucous gland is secreted a large amount of glycoprotein and proteoglycan, be wrapped in skin appearance face, hinder the intrusion of pathogenic bacteria; (2) acquired immune system, contains IgG antibody in Amphibians skin; (3) innate immune system, is mainly made up 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 to the organic molecules such as alkaloid, and the research of its skin activity peptide matters is also rarely had to report.The smelly frog in the southern regions of the Yunnan Province (Odorrana tiannanensis) belongs to amphibia Anura Ranidae Rana, is Chinese endemic species, 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-anti-oxidant multifunctional polypeptide in smelly frog source, the southern regions of the Yunnan Province, 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, simple in structure, is linear molecule, facilitates chemosynthesis and preparation.Compare other anti-oxidation peptides of having reported and transformation precursor, the oxidation-resistance of OA-OTVC, OA-OTC is stronger, to DPPH, 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 there is 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:
Transformation 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;
Transformation 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).
The application of transformation 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, preparation method is simple.
Embodiment:
Further illustrate essentiality content of the present invention with embodiment below, 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: according to the aminoacid sequence of the smelly frog in the southern regions of the Yunnan Province (Odorrana tiannanensis) Natural Antioxidant Peptides transformation body OA-OTVC, OA-OTC, 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).
OA-OTVC, the OA-OTC of III, purifying identifies its purity by 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, by automatic Protein Sequencer mensuration aminoacid sequence structure.
Measurement result is: OA-OTVC, OA-OTC are two kinds of varients of the smelly frog in Chou Wa the southern regions of the Yunnan Province, the southern regions of the Yunnan Province (Odorrana tiannanensis) Natural Antioxidant Peptides odorranain-A-OT.It is all 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.。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; 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 a certain amount of DPPH(2,2 '-hexichol is for bitter taste acyl group phenylhydrazine), with 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.Blank group replaces testing sample with sample dissolution medium.Experiment do three parallel, in ultraviolet spectrophotometer when zeroing, is used methyl alcohol.
DPPH clearance rate (%)=(AB-AA)/AB × 100(AB: blank group light absorption value; AA: sample sets light absorption value).
Table 1. DPPH anti-oxidant activity
Result is as shown in table 1, and OA-OTVC, OA-OTC have extremely strong DPPH free radical scavenging activity, and in the time that sample concentration is 160 μ g/ml, its DPPH clearance rate I% can reach respectively 57.79% and 57.19%.
2. 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-bis-(3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) di-ammonium salts by name.ABTS can oxidized generation quite stable blue-greenish colour radical cation ABTS
+, there is maximum absorption band at 734 nm places, under the antioxidant that has hydrogen supply capacity exists, ABTS
+reaction with it, generates and 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: the solution that ABTS is dissolved in to PBS damping fluid (3.58 g NaHPO411H2O, 0.15 g KCl is dissolved in 1 L deionized water, with 0.1 M NaOH tune pH to 7.4 for 8.18 g NaCl, 0.27 g KH2PO4) and is made into 2 mM.
ABTS
+solution preparation: by ABTS storage liquid and the 250:1 mixing by volume of 70 mM Potassium Persulphate (K2S2O8) aqueous solution, place 15-16 h in room temperature lucifuge.Before on-test, be 0.80 ± 0.03 by ABTS+ solution with the light absorption value that PBS is diluted to 734 nm wavelength places.
By 2 μ l samples (2 mg/ml) and the above-mentioned corrected ABTS of 48 μ l
+solution mixes, and room temperature is placed after 10 min, in the light absorption value of 734 nm wavelength place detection reaction liquid.Sample dissolution medium is as blank.
ABTS
+clearance rate I (%)=(AB-AA)/AB × 100 (AB: blank group light absorption value; AA: sample sets light absorption value).
Table 2. ABTS
+radical cation is removed active
Result is as shown in table 2, and OA-OTVC, OA-OTC have extremely strong ABTS+ radical cation and remove activity, and in the time that sample concentration is 160 μ g/ml, its ABTS+ radical cation is removed activity can reach respectively 94.38% and 94.46%.Our research shows, the removing ability difference of anti-oxidation peptide of the same race to different sorts free radical above-mentionedly experimental results show that OA-OTVC, OA-OTC are 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 (26.7 g NaH2PO4H2O, 53.65 g Na2HPO47H2O, add deionized water to 1 L) of pH 6.6; 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 DEG C of water-bath 20 min; Then add the trichoroacetic acid(TCA) of 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 strong representation reducing power is stronger in photoabsorption.
Table 3. reducing power is measured
Result is as shown in table 3, and the absorbance of OA-OTVC, OA-OTC is increased significantly compared with blank group, and photoabsorption is stronger, represents that sample reducing power is stronger.
Claims (2)
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:
(1) the first transformation body OA-OTVC: the little peptide of a kind of straight chain, contain 16 amino-acid residues, molecular weight is 1688.06Da, 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;
(2) the second transformation 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.
2. the application of the transformation body of the smelly frog Natural Antioxidant Peptides in the southern regions of the Yunnan Province in described in claim 1, it is characterized in that, transformation body OA-OTVC and OA-OTC have DPPH free radical scavenging activity and ABTS+ radical cation is removed activity, for the preparation of anti-oxidation medicine, foodstuff additive, makeup.
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