CN106854241A - Smelly frog skin antioxidation polypeptide AOP OA1 in Yunnan and preparation method and application - Google Patents
Smelly frog skin antioxidation polypeptide AOP OA1 in Yunnan and preparation method and application Download PDFInfo
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- CN106854241A CN106854241A CN201611043115.4A CN201611043115A CN106854241A CN 106854241 A CN106854241 A CN 106854241A CN 201611043115 A CN201611043115 A CN 201611043115A CN 106854241 A CN106854241 A CN 106854241A
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/463—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from amphibians
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Abstract
The invention discloses smelly frog skin antioxidation polypeptide AOP OA1 in a kind of Yunnan and preparation method and application, the amino acid sequence such as SEQ ID NO of antioxidation polypeptide AOP OA1:Shown in 1.Antioxidant peptide of odorrana andersonii skin AOP OA1 preparation methods of the present invention can be isolated and purified from the smelly frog skin secretion kind in Yunnan and obtained.1 antioxidation polypeptide AOP OA1 of novelty is obtained in the skin of the therefrom domestic smelly frog in Yunnan of the present invention, its antioxidation activity is very strong, about 400 times of vitamin E.The smelly frog skin antioxidation polypeptide AOP OA1 total orders array structure in Yunnan of the invention is entered into line search through Protein Data Bank to compare, any phase homopolypeptide is found no.
Description
Technical field
The invention belongs to biomedical sector, specifically, it is related to a kind of smelly frog skin antioxidation polypeptide AOP-OA1 in Yunnan
And preparation method and application.
Background technology
Antioxidant is to prevent the dysgenic material of oxygen, and it is that a class can help capture and neutralize free radical, so that
Dispel the class material that free radical is damaged to human body.Substantial amounts of antioxidant is had now been found that, these antioxidants can be with
It is divided into following two major class：1) small molecule of non-genomic coding is helpless, such as vitamin E, vitamin C, glutathione；2) gene
The macromolecular substances of coding, such as superoxide dismutase, catalase, thioredoxin.These antioxidants by
It is widely used in various fields, including health, food, cosmetics and various industry, for example as the steady of fuel and lubricant
Determine the anti-oxidation of agent, can also add to play in the oil prevents polymerization so as to the purpose for avoiding engine incrustation from being formed, singly in 2007,
The income for just creating about 3,700,000,000 dollars of the antioxidant of industrial use.Importance based on antioxidant, scientists
Always novel antioxidant is found tireless.
Amphibian be aquatic animal excessively to the important monoid between terrestrial animal, its skin is exposed, and surface does not have hair
Hair covering, directly facing various survival pressures, takes all more important biological functions, such as resist microorganism invasion, body
Tone section, water salt balance regulation etc..There is substantial amounts of research evidence to show that the skin of amphibian can secrete substantial amounts of activity
Material (mainly albumen and polypeptide) enables it to complete these important biological functions.Recently, in the amphibian Yunnan frog
The new antioxidant of a class is found that in skin, its chemical nature is polypeptide, different with two traditional major class antioxidants, its
By gene code, but molecular weight very little.Hereafter, this class material is have also discovered in some amphibian skins, but and is passed
The antioxidant of system is compared, and our 26S Proteasome Structure and Functions to this class novel antioxidant understand also knows little about it.
The smelly frog in Yunnan is a kind of distinctive amphibian of China, is distributed widely in the Yunnan-Guizhou Plateau of strong ultraviolet irradiation, according to
Its habitat, it is presumed that its skin can secrete efficient novel oxidation-resistant polypeptide enables it to resist wound caused by ultraviolet irradiation
Evil.On the one hand the research of excavation, 26S Proteasome Structure and Function to these polypeptides be we have appreciated that amphibian adaptation High aititude is ultraviolet by force
Irradiation habitat provides new molecule clue, still further aspect can for we provide it is new, with the anti-oxidant of independent intellectual property right
Agent.
The content of the invention
In view of this, the present invention is directed to above-mentioned problem, there is provided a kind of smelly frog skin antioxidation polypeptide AOP-OA1 in Yunnan
And preparation method and application, the smelly frog skin antioxidation polypeptide AOP-OA1 in Yunnan for preparing have structure it is novel, simple,
Active strong the characteristics of.
In order to solve the above-mentioned technical problem, the invention discloses a kind of smelly frog skin antioxidation polypeptide AOP-OA1 in Yunnan, resist
The amino acid sequence such as SEQ ID NO of oxidation polypeptide A OP-OA1:Shown in 1.
The invention also discloses a kind of preparation method of the smelly frog skin antioxidation polypeptide AOP-OA1 in above-mentioned Yunnan, including
Following steps：
1) pretreatment of raw material：The smelly frog skin secretion in Yunnan is taken, the physiological saline that mass concentration is 0.9% is dissolved in, very
Vacuum freecing-dry is concentrated into dry powder, and -80 DEG C save backup；
2) Sephadex G50 molecular sieves：By step 1) in the smelly frog skin secretion freeze-dried powder dissolving in Yunnan for preparing
In deionized water, upper Sephadex G50 pillars are eluted with Tris-HCl buffer solutions, and flow velocity is 2.0 milliliters/10 points
Clock, collects one for every 10 minutes and manages, and determines its absorbance under 280 nanometers, collects and combines the eluting peak with antioxidation activity；
3) high performance liquid chromatography reversed phase chromatography：By step 2) in the eluent for preparing be splined on HypersilODS2 posts
Son, laboratory apparatus is the high-pressure liquid phase systems of Waters 1525, under conditions of flow velocity is 1 ml/min, with acetonitrile linear
Eluted under gradient condition, monitoring wavelength is 215 nanometers, collects and combines the eluting peak with antioxidation activity, concentrate drying
After obtain object.
Further, step 2) in Sephadex G50 pillars in advance with 20mM, pH6.8, containing 0.1M NaCl's
Tris-HCl buffer solutions balance 24h.
Further, step 3) in Hypersil ODS2 chromatographic columns need in advance with containing the trifluoro that volumetric concentration is 0.1%
The ultrapure water balance of acetic acid.
Further, step 3) in acetonitrile be the acetonitrile containing 0.1% trifluoroacetic acid.
Further, step 3) in linear gradient elution be specially：Acetonitrile concentration from 0% rise to 60% consumed when
Between be 60 minutes.
Prepared and anti-oxidant phase the invention also discloses a kind of smelly frog skin antioxidation polypeptide AOP-OA1 in above-mentioned Yunnan
Application in medicine, health products, the cosmetics of pass.
Compared with prior art, the present invention can be obtained including following technique effect：
1) 1 new antioxidation polypeptide AOP-OA1, its antioxygen have been obtained in the skin of the therefrom domestic smelly frog in Yunnan of the present invention
Change activity very strong, about the 400 of vitamin E times.By the smelly frog skin antioxidation polypeptide AOP-OA1 complete sequence knots in Yunnan of the invention
Structure enters line search and compares through Protein Data Bank, finds no any phase homopolypeptide.
2) the characteristics of Antioxidant peptide of odorrana andersonii skin AOP-OA1 has simple structure, antioxidation activity is strong.
3) the Antioxidant peptide of odorrana andersonii skin AOP-OA1 that purifying is obtained shows in free radical ABTS+ removes experiment
Stronger antioxidation activity, disclosing Antioxidant peptide of odorrana andersonii skin AOP-OA1 of the present invention has potential application prospect.
Certainly, implement any product of the invention to it is not absolutely required to while reaching all the above technique effect.
Brief description of the drawings
Accompanying drawing described herein is used for providing a further understanding of the present invention, constitutes a part of the invention, this hair
Bright schematic description and description does not constitute inappropriate limitation of the present invention for explaining the present invention.In the accompanying drawings：
Fig. 1 is the G50 sieve chromatography figures of Antioxidant peptide of odorrana andersonii skin AOP-OA1 of the present invention；
Fig. 2 is the anti-phase C18 column chromatographies figures of HPLC of Antioxidant peptide of odorrana andersonii skin AOP-OA1 of the present invention, wherein, 1 is
It is AOP-OA1 peaks；
Fig. 3 is the free radical ABTS+ scavenging capacities that the present invention isolates and purifies Antioxidant peptide of odorrana andersonii skin AOP-OA1
Figure.
Specific embodiment
Describe embodiments of the present invention in detail below in conjunction with embodiment, thereby to the present invention how application technology hand
Section can fully understand and implement according to this to solve technical problem and reach the implementation process of technology effect.
The Antioxidant peptide of odorrana andersonii skin AOP-OA1 of embodiment 1 is isolated and purified and identified
1st, isolate and purify
The smelly frog live body in Yunnan picks up from Baoshan, Yunnan, takes its skin secretion and (is dissolved in the physiology salt that mass concentration is 0.9%
Water, this can maximize and keep its BA), vacuum freeze drying is concentrated into dry powder, and -80 DEG C save backup.
The first step：Sephadex G50 molecular sieves：According to the method described above obtain skin secretion freeze-dried powder, be dissolved in from
In sub- water, take on 1 milliliter (albumen with polypeptide total content be 100 milligrams) in advance with 20mM Tris-HCl buffer solutions (pH=6.8,
NaCl containing 0.1M) 24 hours Sephadex G50 (GE Healthcare, ultra-fine) pillar (40 centimetres of length, internal diameter of balance
1.5 centimetres of width), eluted with same buffer solution, flow velocity is 2.0 milliliters/10 minutes, collects within every 10 minutes one and manages, and is surveyed
Fixed its absorbance under 280 nanometers, gained isolates and purifies collection of illustrative plates as shown in figure 1, wherein AOP-OA1 is present in shown in figure
Peak.
Second step：High performance liquid chromatography reversed phase chromatography：
First step gained sample is splined on and has been balanced with ultra-pure water (containing the trifluoroacetic acid that volumetric concentration is 0.1%) in advance
Hypersil ODS2 pillars (Erie is special, and size is 4.6 millimeters × 300 millimeters), laboratory apparatus is the high pressure liquids of Waters 1525
Phase system, under conditions of flow velocity is 1ml/min, with acetonitrile (containing 0.1% trifluoroacetic acid) in linear gradient (0-60%, 60
Minute) under the conditions of eluted, monitoring wavelength is 215 nanometers, and gained isolates and purifies collection of illustrative plates as shown in Fig. 2 in figure shown in " 1 "
It is the natural Antioxidant peptide of odorrana andersonii skin AOP-OA1 of purifying.
2nd, Molecular Identification
The measure of amino acid sequence：
The Antioxidant peptide of odorrana andersonii skin AOP-OA1 that purifying is obtained is in full-automatic protein sequencing instrument (Shimadzu
PPSQ-31A through Edman edman degradation Edmans on), Antioxidant peptide of odorrana andersonii skin AOP-OA1 overall amino acid sequences are determined, as a result table
Bright Antioxidant peptide of odorrana andersonii skin AOP-OA1 has SEQ ID NO of the present invention：Structure shown in 1.
Embodiment 2：The antioxidation activity of Antioxidant peptide of odorrana andersonii skin AOP-OA1
The present invention draws Antioxidant peptide of odorrana andersonii skin AOP-OA1's using the experiment detection of free radical ABTS+ scavenging capacities
Antioxidation activity, as a result shows that Antioxidant peptide of odorrana andersonii skin AOP-OA1 has stronger antioxidation activity.
Specific steps and result：The ABTS of 7mM mixes in water with the potassium peroxydisulfate of 2.8mM, anti-in room temperature dark surrounds
Answer 6 hours so that ABTS is oxidized into ABTS+ free radicals, afterwards with deionized water dilute 50 times, take 50uL dilutions and
50uL samples mix, and are reacted 30 minutes under room temperature dark surrounds, afterwards, its absorbance are surveyed under 415nm wavelength.Free radical ABTS
+ clearance rate (%) calculated with following formula：(A blank-Asample)×100/Ablank.Various concentrations Yunnan is smelly
The antioxidation activity of frog antioxidant peptide AOP-OA1 is as shown in Figure 3.
Described above has shown and described some preferred embodiments of invention, but as previously described, it should be understood that invention is not
Form disclosed herein is confined to, the exclusion to other embodiment is not to be taken as, and can be used for various other combinations, modification
And environment, and can be carried out by the technology or knowledge of above-mentioned teaching or association area in invention contemplated scope described herein
Change.And the change and change that those skilled in the art are carried out do not depart from the spirit and scope of invention, then all should be in the appended power of invention
In the protection domain that profit is required.
Claims (7)
1. a kind of smelly frog skin antioxidation polypeptide AOP-OA1 in Yunnan, it is characterised in that the ammonia of the antioxidation polypeptide AOP-OA1
Base acid sequence such as SEQ ID NO：Shown in 1.
2. a kind of preparation method of the smelly frog skin antioxidation polypeptide AOP-OA1 in Yunnan as claimed in claim 1, its feature exists
In comprising the following steps：
1) pretreatment of raw material：The smelly frog skin secretion in Yunnan is taken, the physiological saline that mass concentration is 0.9% is dissolved in, vacuum is cold
It is lyophilized it is dry be concentrated into dry powder, -80 DEG C save backup；
2) Sephadex G50 molecular sieves：By step 1) in the smelly frog skin secretion freeze-dried powder in Yunnan for preparing be dissolved in
In ionized water, upper Sephadex G50 pillars are eluted with Tris-HCl buffer solutions, and flow velocity is 2.0 milliliters/10 minutes, often
Collect within 10 minutes one to manage, determine its absorbance under 280nm, collect and combine the eluting peak with antioxidation activity；
3) high performance liquid chromatography reversed phase chromatography：By step 2) in the eluent for preparing be splined on Hypersil ODS2 pillars,
Laboratory apparatus is the high-pressure liquid phase systems of Waters 1525, under conditions of flow velocity is 1 ml/min, with acetonitrile in linear gradient
Under the conditions of eluted, monitoring wavelength is 215 nanometers, the eluting peak with antioxidation activity is collected and combined, after concentrate drying
To object.
3. the smelly frog skin antioxidation polypeptide AOP-OA1 in Yunnan according to claim 2, it is characterised in that step 2) in
Sephadex G50 pillars are balanced 24 hours with 20mM, pH 6.8, the Tris-HCl buffer solutions containing 0.1M NaCl in advance.
4. the smelly frog skin antioxidation polypeptide AOP-OA1 in Yunnan according to claim 2, it is characterised in that step 3) in
Hypersil ODS2 chromatographic columns need in advance with the ultrapure water balance containing the trifluoroacetic acid that volumetric concentration is 0.1%.
5. the smelly frog skin antioxidation polypeptide AOP-OA1 in Yunnan according to claim 2, it is characterised in that step 3) in
Acetonitrile is the acetonitrile containing 0.1% trifluoroacetic acid.
6. the smelly frog skin antioxidation polypeptide AOP-OA1 in Yunnan according to claim 2, it is characterised in that step 3) in
Linear gradient elution is specially：Acetonitrile concentration rose to for 60% time for being consumed for 60 minutes from 0%.
7. a kind of smelly frog skin antioxidation polypeptide AOP-OA1 in Yunnan as claimed in claim 1 is being prepared and anti-oxidant Related Drug
Application in thing, health products, cosmetics.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611043115.4A CN106854241A (en) | 2016-11-24 | 2016-11-24 | Smelly frog skin antioxidation polypeptide AOP OA1 in Yunnan and preparation method and application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611043115.4A CN106854241A (en) | 2016-11-24 | 2016-11-24 | Smelly frog skin antioxidation polypeptide AOP OA1 in Yunnan and preparation method and application |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108530527A (en) * | 2018-04-23 | 2018-09-14 | 昆明医科大学 | Polypeptide OA-G L21 and purification method and application thereof |
| CN108586576A (en) * | 2018-04-23 | 2018-09-28 | 昆明医科大学 | A kind of polypeptide OA-FF10 and its method of purification and application |
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|---|---|---|---|---|
| CN103819541A (en) * | 2014-03-06 | 2014-05-28 | 福州大学 | Microalgae oxidation prevention polypeptide |
| CN104530192A (en) * | 2015-01-19 | 2015-04-22 | 云南民族大学 | Antioxidant peptide AOP-OM1 as well as preparation method and application thereof |
| CN105713072A (en) * | 2016-02-26 | 2016-06-29 | 昆明昂名科技有限公司 | Antioxidant polypeptide (AOP-OL1) and preparation method and application thereof |
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2016
- 2016-11-24 CN CN201611043115.4A patent/CN106854241A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103819541A (en) * | 2014-03-06 | 2014-05-28 | 福州大学 | Microalgae oxidation prevention polypeptide |
| CN104530192A (en) * | 2015-01-19 | 2015-04-22 | 云南民族大学 | Antioxidant peptide AOP-OM1 as well as preparation method and application thereof |
| CN105713072A (en) * | 2016-02-26 | 2016-06-29 | 昆明昂名科技有限公司 | Antioxidant polypeptide (AOP-OL1) and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
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| KOHEN R 等: "Skin low molecular weight antioxidants and their role in aging and in oxidative stress", 《TOXICOLOGY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108530527A (en) * | 2018-04-23 | 2018-09-14 | 昆明医科大学 | Polypeptide OA-G L21 and purification method and application thereof |
| CN108586576A (en) * | 2018-04-23 | 2018-09-28 | 昆明医科大学 | A kind of polypeptide OA-FF10 and its method of purification and application |
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Application publication date: 20170616 |