CN104530192B - A kind of antioxidation polypeptide AOP OM1 and preparation method and application - Google Patents
A kind of antioxidation polypeptide AOP OM1 and preparation method and application Download PDFInfo
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- CN104530192B CN104530192B CN201510025060.3A CN201510025060A CN104530192B CN 104530192 B CN104530192 B CN 104530192B CN 201510025060 A CN201510025060 A CN 201510025060A CN 104530192 B CN104530192 B CN 104530192B
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Abstract
The invention discloses a kind of antioxidation polypeptide AOP OM1 and preparation method and application, described antioxidation polypeptide AOP OM1 amino acid sequence such as SEQ ID：Shown in No.1.Described preparation method is to isolate and purify acquisition using big rana margaretae skin secretion as raw material.The present invention is that a kind of new antioxidant is found that in the skin of the big rana margaretae of amphibian, and its chemical nature is polypeptide, different with two traditional major class antioxidants, and it is by gene code, but molecular weight very little.1 novel antioxidation polypeptide AOP OM1 is obtained in the skin of the therefrom domestic big rana margaretae of the present invention, its antioxidation activity is very strong, about 200 times of vitamin E.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, and in particular to a kind of antioxidation polypeptide AOP-OM1 and preparation method thereof is with answering
With.
Background technology
Antioxidant is to prevent the dysgenic material of oxygen, and it is that a class can help to 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）The small-molecule substance of non-genomic coding, 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 avoid the purpose of engine incrustation formation, 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 resists 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）Enable it to complete these important biological functions.But because of particularity such as crude drug sources,
The research of its Effective Compounds and mechanism of action is also more weak, and the report on its active component is almost nil.Therefore, such as
What makes full use of modern biotechnology means, isolates and purifies active peptides and carries out Structural Identification, carries out research medicinal material effect in a deep going way
Answer material base and study on mechanism, be the field scientific research technical staff make great efforts to seek for many years the research direction that breaks through it
One.
The content of the invention
The first object of the present invention is to provide a kind of antioxidation polypeptide AOP-OM1；Second purpose is described in offer
Antioxidation polypeptide AOP-OM1 preparation method；3rd purpose is the application of the antioxidation polypeptide AOP-OM1 described in offer.
The first object of the present invention is achieved in that described antioxidation polypeptide AOP-OM1 amino acid sequence such as SEQ
ID：Shown in No.1.
The second object of the present invention, which is achieved in that, to be comprised the following steps：
A, take the big rana margaretae skin secretion 100mg of raw material, add 1ml deionized water dissolvings, upper Sephadex G-75
Post, is eluted with Tris-HCl buffer solutions with flow velocity 1.5ml/10min, is collected once per 10min, and eluent is determined respectively and is existed
Absorbance under 280nm, collects the eluent for merging active peak component, medicinal extract a is obtained after concentrate drying；
1ml deionized water dissolvings, upper Hypersil ODS2 chromatographic columns, with acetonitrile with 1ml/min are added in B, 1mg medicinal extract a
Flow velocity is eluted, the absorbance under monitoring 215nm, is collected the eluent for merging active peak component, is obtained after concentrate drying
Object.
The antioxidation polypeptide AOP-OM1 prepared with above-mentioned isolation and purification method structural formula is identified by the following method
Out：
1st, the measure of amino acid sequence：
Obtained big rana margaretae skin anti-oxidation peptide AOP-OM1 is purified in full-automatic protein sequencing instrument（Shimadzu
PPSQ-31A）On through Edman edman degradation Edmans, determine big rana margaretae skin anti-oxidation peptide AOP-OM1 overall amino acid sequences, as a result
Show that big rana margaretae skin anti-oxidation peptide AOP-OM1 has SEQ ID NO of the present invention:Structure shown in 1.
2nd, mass spectroscopy molecular measures fixed：
Substance assistant laser desorpted flight time mass spectrum (MALDI-TOF-MS) have detected the big rana margaretae skin that purifying is obtained
Skin anti-oxidation peptide AOP-OM1 accurate molecular weight, instrument is Bruker companies Autoflex III TOF/TOF mass spectrographs.
The molecular weight of big rana margaretae skin anti-oxidation peptide AOP-OM1 mass spectroscopies is respectively 938.1 dalton, and mass spectral results disclose big green
Posttranslational modification is not present in smelly frog skin anti-oxidation peptide AOP-OM1.
The third object of the present invention is achieved in that described antioxidation polypeptide AOP-OM1 is preparing anti-oxidation medicine
In purposes.
The present invention is the antioxidant for being found that a class is new in the skin of the amphibian Yunnan frog, and its chemical nature is many
Peptide, different with two traditional major class antioxidants, it is by gene code, but molecular weight very little.Hereafter, in some amphibians
This class material is have also discovered in skin, but is compared with traditional antioxidant, our knots to this class novel antioxidant
Structure and function, which understand, also knows little about it.Big rana margaretae is a kind of distinctive smelly frog of China, is distributed widely in the cloud of strong ultraviolet irradiation
Your plateau, according to its habitat, enables it to resist ultraviolet irradiation and is made it is presumed that its skin can secrete efficient antioxidant
Into injury.1 novel antioxidation polypeptide AOP-OM1 is obtained in the skin of the therefrom domestic big rana margaretae of the present invention, it resists
Oxidation activity is very strong, about the 200 of vitamin E times.
Brief description of the drawings
Fig. 1 is present invention rana margaretae skin anti-oxidation peptide AOP-OM1 G-75 sieve chromatography figures greatly；
Fig. 2 is the present invention rana margaretae skin anti-oxidation peptide AOP-OM1 anti-phase C18 column chromatographies figures of HPLC greatly；
Fig. 3 isolates and purifies natural big rana margaretae skin anti-oxidation peptide AOP-OM1 mass spectrogram for the present invention；
Fig. 4 isolates and purifies big rana margaretae skin anti-oxidation peptide AOP-OM1 free radical ABTS+ scavenging capacities for the present invention
Figure.
Embodiment
The present invention is further illustrated below in conjunction with the accompanying drawings, but the present invention is not any limitation as in any way, base
In present invention teach that any conversion or replacement made, belong to protection scope of the present invention.
Antioxidation polypeptide AOP-OM1 of the present invention amino acid sequence such as SEQ ID：Shown in No.1.
Antioxidation polypeptide AOP-OM1 of the present invention preparation method, comprises the following steps：
A, take the big rana margaretae skin secretion 100mg of raw material, add 1ml deionized water dissolving, upper Sephadex G-75
Post, is eluted with Tris-HCl buffer solutions with flow velocity 1.5ml/10min, is collected once per 10min, and eluent is determined respectively and is existed
Absorbance under 280nm, collects the eluent for merging active peak component, medicinal extract a is obtained after concentrate drying；
B, take medicinal extract a 1mg, add 1ml deionized water dissolvings, upper Hypersil ODS2 chromatographic columns, with acetonitrile with 1ml/
Min is eluted, the absorbance under monitoring 215nm, collects the eluent for merging active peak component, mesh is obtained after concentrate drying
Mark thing.
Sephadex G-75 posts described in step A need to use 20mmol/L in advance, and pH7.8 Tris-HCl buffer solutions are put down
Weigh 24h.
Hypersil ODS2 chromatographic columns described in step B need to use the ultrapure water balance containing 0.1% trifluoroacetic acid in advance
20min。
Acetonitrile described in step B is the acetonitrile containing 0.1% trifluoroacetic acid.
Elution described in step B be in linear gradient elution, i.e., acetonitrile concentration from 0% rise to 100% the time spent in be
100min。
The application of the present invention is purposes of the described antioxidation polypeptide AOP-OM1 in anti-oxidation medicine is prepared.
The big rana margaretae skin anti-oxidation peptide AOP-OM1 that the present invention is provided, its preparation method can pass through bioid credit
From purification process, obtained in big rana margaretae skin secretion.Big rana margaretae skin anti-oxidation peptide AOP- prepared by the above method
OM1, can be identified by mass spectrum and antioxidation activity.
The big rana margaretae skin anti-oxidation peptide AOP-OM1 that present invention purifying is obtained is in free radical ABTS+Remove in experiment and show
Stronger antioxidation activity is shown, disclosing the big rana margaretae skin anti-oxidation peptide AOP-OM1 of the present invention has potential application prospect.
The big rana margaretae skin anti-oxidation peptide AOP-OM1 of the present invention has the strong beneficial spy of simple in construction, antioxidation activity
Point.
The big rana margaretae skin anti-oxidation peptide AOP-OM1 of embodiment 1 is isolated and purified and identified
1st, isolate and purify
Big rana margaretae live body picks up from zunyi, guizhou, with 6V galvanic current stimulation 6s, then with its skin of normal saline flushing, obtains
Skin secretion, vacuum freeze drying, -80 ° save backup.
The first step：Sephadex G-75 exclusion chromatographies：Skin secretion freeze-dried powder is obtained according to the method described above, is dissolved in
In deionized water, 1ml is taken（Protein content is 100mg）It is upper to use 20mM Tris-HCl buffer solutions in advance（PH=7.8, containing 0.1 M
NaCl）The balance Sephadex G-75 of 24 hours（GE Healthcare, it is ultra-fine）Pillar（Length 30cm, internal diameter width
1.5cm）, eluted with same buffer solution, flow velocity is 1.5ml/10min, collected once per 10min, determine it in 280nm
Under absorbance, gained isolates and purifies collection of illustrative plates as shown in figure 1, wherein AOP-OM1 is present in peak shown in Fig. 1 arrows.
Second step：High performance liquid chromatography reversed phase chromatography：
1mg is taken to be dissolved in after 1ml deionized waters being splined on and use ultra-pure water in advance after sample vacuum freeze drying obtained by the first step
（Containing 0.1% trifluoroacetic acid）Balance 20min Hypersil ODS2 5mm posts（Dalian Erie is special, size be 4.6mm ×
300mm）, laboratory apparatus is the high-pressure liquid phase systems of Waters 1525, under conditions of flow velocity is 1ml/min, uses acetonitrile（Contain
0.1% trifluoroacetic acid）In linear gradient（0-100% in 100min）Under the conditions of eluted, monitoring wavelength be 215nm, institute
What is obtained isolates and purifies collection of illustrative plates as shown in Fig. 2 arrow show the natural big rana margaretae skin anti-oxidation peptide AOP- of purifying in figure
OM1。
2nd, Molecular Identification
The measure of amino acid sequence：
Obtained big rana margaretae skin anti-oxidation peptide AOP-OM1 is purified in full-automatic protein sequencing instrument（Shimadzu
PPSQ-31A）On through Edman edman degradation Edmans, determine big rana margaretae skin anti-oxidation peptide AOP-OM1 overall amino acid sequences, as a result table
Bright big rana margaretae skin anti-oxidation peptide AOP-OM1 has SEQ ID NO of the present invention:Structure shown in 1.
Mass spectroscopy molecular measures fixed：
Substance assistant laser desorpted flight time mass spectrum (MALDI-TOF-MS) have detected the big rana margaretae skin that purifying is obtained
Skin anti-oxidation peptide AOP-OM1 accurate molecular weight, instrument is Bruker companies Autoflex III TOF/TOF mass spectrographs.
The molecular weight of big rana margaretae skin anti-oxidation peptide AOP-OM1 mass spectroscopies is respectively 938.1 dalton, as shown in Figure 3.Mass spectrum knot
Fruit discloses big rana margaretae skin anti-oxidation peptide AOP-OM1 in the absence of posttranslational modification.
Embodiment 2
The big rana margaretae skin anti-oxidation peptide AOP-OM1 prepared with embodiment 1 antioxidant activity tests：
Method：Tested using free radical ABTS+ scavenging capacities and have detected the anti-of big rana margaretae skin anti-oxidation peptide AOP-OM1
Oxidation activity, as a result shows that big rana margaretae skin anti-oxidation peptide AOP-OM1 has stronger antioxidation activity.
Specific steps and result：Take 7mM ABTS in water and 2.8mM potassium peroxydisulfate is mixed, in room temperature dark surrounds
React 6 hours so that ABTS is oxidized into ABTS+Free radical, dilutes 50 times with deionized water afterwards, takes 50 μ L to dilute
Reacted 30 minutes under liquid and the mixing of 50 μ L samples, room temperature dark surrounds, afterwards, its absorbance is surveyed under 415nm wavelength.Free radical
ABTS+Clearance rate（%）Calculated with following formula：（Ablank-Asample）×100/Ablank.The big rana margaretae of various concentrations resists
The antioxidation activity for aoxidizing peptide AOP-OM1 is as shown in Figure 4.
SEQUENCE LISTING
<110>Ethnic university of Yunnan Province, Beijing Mei Ruikunhua Science and Technology Ltd.s
<120>A kind of anti-oxidation peptide AOP-OM1 and preparation method and application
<130> 2015
<160> 10
<170> PatentIn version 3.3
<210> 1
<211> 1501
<212> PRT
<213>Anti-oxidation peptide AOP-OM1 amino acid sequences
Asn Ser Lys Tyr Leu Cys Asn Pro 8
Claims (5)
1. a kind of antioxidation polypeptide AOP-OM1, it is characterised in that described antioxidation polypeptide AOP-OM1 amino acid sequence is such as
SEQ ID：Shown in No.1, molecular weight is 938.1 dalton.
2. the preparation method of the antioxidation polypeptide AOP-OM1 described in a kind of claim 1, it is characterised in that comprise the following steps：
A, take the big rana margaretae skin secretion 100mg of raw material, add 1ml deionized water dissolvings, upper Sephadex G-75 posts are used
Tris-HCl buffer solutions are eluted with flow velocity 1.5ml/10min, are collected once per 10min, and eluent is determined respectively in 280nm
Under absorbance, collect merge active peak component eluent, medicinal extract a is obtained after concentrate drying；
1ml deionized water dissolvings, upper Hypersil ODS2 chromatographic columns, with containing 0.1% trifluoroacetic acid are added in B, 1mg medicinal extract a
The solution of acetonitrile is eluted with 1ml/min flow velocity, and elution is to use linear gradient elution, i.e. the second containing 0.1% trifluoroacetic acid
The solution concentration of nitrile rose to for 100% time consumed for 100min from 0%, the absorbance under monitoring 215nm, collects and merges activity
Object is obtained after the eluent of peak fraction, concentrate drying.
3. preparation method according to claim 2, it is characterised in that the Sephadex G-75 posts described in step A need pre-
First use 20mmol/L, pH7.8 Tris-HCl buffer solutions balance 24h.
4. preparation method according to claim 2, it is characterised in that the Hypersil ODS2 chromatographic columns described in step B
The ultrapure water balance 20min containing 0.1% trifluoroacetic acid need to be used in advance.
5. purposes of the antioxidation polypeptide AOP-OM1 in anti-oxidation medicine is prepared described in a kind of claim 1.
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Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN106146621A (en) * | 2016-08-24 | 2016-11-23 | 昆明腾森科技有限公司 | Big rana margaretae skin antioxidation polypeptide AOP OA3 and preparation method thereof |
| CN106243190A (en) * | 2016-08-24 | 2016-12-21 | 昆明派尊科技有限公司 | Big rana margaretae skin antioxidation polypeptide AOP OA5 and preparation method thereof |
| CN106117316A (en) * | 2016-08-24 | 2016-11-16 | 昆明珀凡科技有限公司 | Big rana margaretae skin antioxidation polypeptide AOP OA2 and preparation method thereof |
| CN106117317A (en) * | 2016-08-24 | 2016-11-16 | 昆明佰盟科技有限公司 | Big rana margaretae skin antioxidation polypeptide AOP OA4 and preparation method thereof |
| CN106317200A (en) * | 2016-08-24 | 2017-01-11 | 云南昂肽生物科技有限公司 | Odorrana graminea skin antioxidant peptide AOP-OA6 and preparing method thereof |
| CN106854241A (en) * | 2016-11-24 | 2017-06-16 | 云南民族大学 | Smelly frog skin antioxidation polypeptide AOP OA1 in Yunnan and preparation method and application |
| CN106632644A (en) * | 2016-11-24 | 2017-05-10 | 云南民族大学 | Antioxidant polypeptide AOP-OA2 in skin of Yunnan odorous frog as well as prepration method and application of antioxidant polypeptide AOP-OA2 |
| CN110590913A (en) * | 2019-10-12 | 2019-12-20 | 昆明医科大学 | Antioxidant damage skin protection active polypeptide AOP-P1 and preparation method and application thereof |
| CN110590912A (en) * | 2019-10-12 | 2019-12-20 | 昆明医科大学 | Novel antioxidant active polypeptide OA-VI12 as well as preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN101824077A (en) * | 2010-03-12 | 2010-09-08 | 中国科学院昆明动物研究所 | Large green frog antioxidant peptide (antioxidin-RL) as well as gene and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN101824077A (en) * | 2010-03-12 | 2010-09-08 | 中国科学院昆明动物研究所 | Large green frog antioxidant peptide (antioxidin-RL) as well as gene and application thereof |
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