CN102584950A - Natural antioxidant peptide (Rotodorin-D1), and preparation method and application thereof - Google Patents
Natural antioxidant peptide (Rotodorin-D1), and preparation method and application thereof Download PDFInfo
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- CN102584950A CN102584950A CN2012100141195A CN201210014119A CN102584950A CN 102584950 A CN102584950 A CN 102584950A CN 2012100141195 A CN2012100141195 A CN 2012100141195A CN 201210014119 A CN201210014119 A CN 201210014119A CN 102584950 A CN102584950 A CN 102584950A
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Abstract
The invention discloses a natural antioxidant peptide (Rotodorin-D1) and a preparation method and application thereof. The natural antioxidant peptide (Rotodorin-D1) is a gene coded product, a sequence from an N end to a C end is KSSDVKYYLAKCTTDVQ. The natural antioxidant peptide (Rotodorin-D1) is made of skin of Odorrana graminea Boulenger which only exists in China; in Batrachia skin, Odorrana graminea Boulenger has a main role in protecting skin exposed to the outside from being damaged by ultraviolet rays, and the polypeptide (Rotodorin-D1) with antioxidative activity is used in preparation of cosmetics. Origin returning and all natural effects meet consuming requirements of current publics. Compared with the conventional antioxidant (BHT), the Rotodorin-D1 has activity which is improved by 1000 times; besides the antioxidant activity, the Rotodorin-D1 has certain antibacterial activity, immunoregulation activity, and melanin forming restraining activity.
Description
Technical field
The present invention relates to biological technical field, specifically a kind of polypeptide with anti-oxidant activity--natural anti-oxidation peptide Rotodorin-D1 and preparation method thereof and the application in makeup.
Background technology
Radical is the hazardous compound that produces in the body oxidizing reaction, has strong oxidizing property, can damage the tissue and the cell of body, and then causes chronic disease and aging effect.Under the normal physiological state, the generation and the removing of radical are in dynamic balance state in the body, be under the poor environment state but work as body, and with advancing age, intravital radical will produce too much or remove slow.Free-radical scavengers is also referred to as inhibitor, can remove radical unnecessary in the body, alleviates their damages to body.
At present, exist 2 cover antioxidant systems in the nature biotechnology body of always generally acknowledging, first cover is a protein macromolecule, such as SOD; Second cover is for organic molecule, such as Vc, BHT etc.The inhibitor of chemosynthesis such as BHA, BHT etc., itself has toxicity, side effect, national governments one after another to its mandatory provision the ADI value, control its excessive interpolation.Common inhibitor in the makeup, like vitamins C, vitamin E and SOD etc., its oxidation activity is limited again.And the interpolation product of the natural antioxidants of natural astaxanthin and so on, because its attraction is " natural ", price is generally too high, is difficult to accepted by popular.
Summary of the invention
The purpose of this invention is to provide a kind of natural anti-oxidation peptide Rotodorin-D1 and preparation method thereof and the application in makeup; The natural anti-oxidation peptide that exists in the skin with the peculiar smelly frog of China substitutes traditional inhibitor; Development of New Generation is natural, utmost point low toxicity, anti-oxidant type makeup efficiently.
Along with the development of polypeptide group and genomics, the present invention has found the 3rd cover antioxidant system in frog skin, and its polypeptide by genes encoding constitutes, and this peptide species is many to be made up of 10-20 amino-acid residue.The weapon that the skin that this natural anti-oxidation peptide is the highlands Amphibians is effective and powerful to uvioresistant damage be vital to its existence, and its source is natural, and low toxicity, characteristic efficiently more meet the requirement of modern to makeup.
The natural anti-oxidation peptide that the present invention obtained derives from the skin secretion of the distinctive smelly frog of China (Odorrana andersonii).Batrachia is the important transitional type of animal from the aquatic Lu Sheng life of living, and its skin is exposed, is rich in body of gland, can secrete a large amount of active substances, comprises antibacterial peptide, kassinin kinin, analgesia peptide etc., wherein also has the polypeptide of a large amount of unknown function.The smelly frog is batrachian " ancestors ", can secrete the mixture with irritating smell because of its skin when the irriate and gain the name.Show that according to the experience of working in the past choose ancestors and plant the effective constituent of extracting, its activity is stronger, the present invention has selected the smelly frog in ancestors Yunnan of batrachia for use, and the supposition of experimental result accord with expectation.
Natural anti-oxidation peptide Rotodorin-D1 of the present invention prepares by following method:
1. obtaining of skin secretion: the skin 6s that stimulates the smelly frog in Yunnan with the voltage of 6V; Clean the health of the smelly frog afterwards with 25mM Tris-HCl damping fluid (pH=7.8); Collect skin secretion; With the secretory product of collecting in the centrifugal 15min of 4000rpm, with the supernatant vacuum lyophilization, and be stored in-80 ℃ subsequent use;
2. the purifying of polypeptide: freeze dried supernatant samples is dissolved again; On use 25mM Tris-HCl damping fluid (pH=7.8 in advance; Contain 0.1M NaCl) Sephadex G-75 gel-filtration column that balance is good, carry out wash-out with the flow velocity of 0.3ml/min, every 10min collects once.Eluted product is monitored with 280nm, draws the separation and purification collection of illustrative plates, collects oxidation-resistant active ingredient, and uses C
4Vacuum freezedrying behind the salinity inside post is removed.Afterwards; With NaAc-HAc damping fluid (pH=4.0) sample dissolution of 20mM, the Resource S separator column good with the same buffer balance carries out purifying, utilizes the same damping fluid contain 1M NaCl, carries out wash-out with the flow velocity of 1ml/min; Elutriant is monitored with the 215nm wavelength; Collect composition, separate with the good HPLC C18 separator column of ultrapure water balance that contains 0.1% (v/v) TFA afterwards, utilize and contain the flow velocity wash-out of the acetonitrile of 0.1% (v/v) TFA with 0.7ml/min with anti-oxidant activity; Elutriant is monitored under 215nm, finally obtains the pure article of natural anti-oxidation peptide Rotodorin-D1.
The anti-oxidant activity assessment: the assessment of sample anti-oxidant activity is reflected on its clearance rate to radical, and is specific as follows: as at first to dispose the ABTS storage liquid: at aqueous solution dissolving 2.8mM Potassium Persulphate and 7mM ABTS, and lucifuge reaction at least 6 hours; Afterwards, dilute with water is 125 times immediately, and the water-soluble sample of 50 microlitres adds 50 microlitre ABTS diluents; Lucifuge reaction at least 10 minutes; Afterwards, under the 415nm wavelength, read its absorbancy, the sample anti-oxidant activity calculates with following formula: (A
Blank-A
Sample) * 100/A
Blank
Obtain natural anti-oxidation peptide Rotodorin-D1 through method for preparing, it is a gene encoding production.N holds the sequence of C end: KSSDVKYYLAKCTTDVQ.Active: as can to remove 20% radical under the concentration of 1ug/ml, have the activity that certain sterilization, immunomodulatory and water balance are regulated.
The product the key technical indexes: the active evaluation criteria of anti-oxidation peptide is the Cmin (ug/ml or umol/ml) that can play a role.
Natural anti-oxidation peptide Rotodorin-D1 of the present invention; Be applied to the preparation of makeup; The natural anti-oxidation peptide is directly put in the prescription of makeup and gone, have pure natural, characteristics of high efficiency, and have the activity that certain sterilization, immunomodulatory and water balance are regulated.
The present invention has the following advantages compared with prior art:
1. pick up from the skin of the peculiar smelly frog of China, its main effect in batrachia skin is that the exposed skin of protection does not receive ultraviolet injury---return origin, pure natural, meet existing nowadays popular consumption requirement.
2. the antioxidant BHT that specific activity is traditional is strong 1000 times.
3. except having anti-oxidant activity, also have certain anti-microbial activity, immunoregulatory activity, inhibition melanochrome and form activity.
Application of the present invention is an invention of cosmetic industry, will become a new mark post of cosmetic industry.
Description of drawings
Fig. 1 is the process flow sheet of natural anti-oxidation peptide Rotodorin-D1 preparation of the present invention.
Embodiment
1. obtaining of skin secretion: the skin 6s that stimulates the smelly frog in Yunnan with the voltage of 6V; Clean the health of the smelly frog afterwards with 25mM Tris-HCl damping fluid (pH=7.8); Collect skin secretion; With the secretory product of collecting in the centrifugal 15min of 4000rpm, with the supernatant vacuum lyophilization, and be stored in-80 ℃ subsequent use;
2. the purifying of polypeptide: freeze dried supernatant samples is dissolved again; On use 25mM Tris-HCl damping fluid (pH=7.8 in advance; Contain 0.1M NaCl) Sephadex G-75 gel-filtration column that balance is good, carry out wash-out with the flow velocity of 0.3ml/min, every 10min collects once.Eluted product is monitored with 280nm, draws the separation and purification collection of illustrative plates, collects oxidation-resistant active ingredient, and uses C
4Vacuum freezedrying behind the salinity inside post is removed.Afterwards; With NaAc-HAc damping fluid (pH=4.0) sample dissolution of 20mM, the Resource S separator column good with the same buffer balance carries out purifying, utilizes the same damping fluid contain 1M NaCl, carries out wash-out with the flow velocity of 1ml/min; Elutriant is monitored with the 215nm wavelength; Collect composition, separate with the good HPLC C18 separator column of ultrapure water balance that contains 0.1% (v/v) TFA afterwards, utilize and contain the flow velocity wash-out of the acetonitrile of 0.1% (v/v) TFA with 0.7ml/min with anti-oxidant activity; Elutriant is monitored under 215nm, finally obtains the pure article of natural anti-oxidation peptide Rotodorin-D1.Natural anti-oxidation peptide Rotodorin-D1, it is a gene encoding production.N holds the sequence of C end: KSSDVKYYLAKCTTDVQ.Active: as can to remove 20% radical under the concentration of 1ug/ml, have the activity that certain sterilization, immunomodulatory and water balance are regulated.
The anti-oxidant activity assessment: the assessment of sample anti-oxidant activity is reflected on its clearance rate to radical, and is specific as follows: as at first to dispose the ABTS storage liquid: at aqueous solution dissolving 2.8mM Potassium Persulphate and 7mM ABTS, and lucifuge reaction at least 6 hours; Afterwards, dilute with water is 125 times immediately, and the water-soluble sample of 50 microlitres adds 50 microlitre ABTS diluents; Lucifuge reaction at least 10 minutes; Afterwards, under the 415nm wavelength, read its absorbancy, the sample anti-oxidant activity calculates with following formula: (A
Blank-A
Sample) * 100/A
Blank
Natural anti-oxidation peptide Rotodorin-D1 of the present invention is applied to the preparation of makeup, the natural anti-oxidation peptide is directly put in the prescription of makeup and is gone, pure natural, efficient, have an activity that certain sterilization, immunomodulatory and water balance are regulated.
Claims (4)
1. a natural anti-oxidation peptide Rotodorin-D1 is characterized in that natural anti-oxidation peptide Rotodorin-D1, and it is a gene encoding production, and N holds the sequence of C end: KSSDVKYYLAKCTTDVQ.
2. the preparation method of the described natural anti-oxidation peptide of claim 1 Rotodorin-D1 is characterized in that carrying out according to the following steps:
1. obtaining of skin secretion: the skin 6s that stimulates the smelly frog in Yunnan with the voltage of 6V; Clean the health of the smelly frog afterwards with 25mM Tris-HCl pH of buffer=7.8; Collect skin secretion; With the secretory product of collecting in the centrifugal 15min of 4000rpm, with the supernatant vacuum lyophilization, and be stored in-80 ℃ subsequent use;
2. the purifying of polypeptide: freeze dried supernatant samples is dissolved again; On use 25mM Tris-HCl pH of buffer=7.8 in advance; Contain the good Sephadex G-75 gel-filtration column of 0.1M NaCl balance, carry out wash-out with the flow velocity of 0.3ml/min, every 10min collects an elutriant; Elutriant is monitored with 280nm, draws the separation and purification collection of illustrative plates, collects oxidation-resistant active ingredient, and uses C
4Post is removed behind the salinity of the inside after the vacuum freezedrying; With NaAc-HAc pH of buffer=4.0 sample dissolution of 20mM, the Resource S separator column good with the same buffer balance carries out purifying, utilizes the same damping fluid contain 1M NaCl, carries out wash-out with the flow velocity of 1ml/min; Elutriant is monitored with the 215nm wavelength; After collecting composition, separate, utilize and contain the flow velocity wash-out of the acetonitrile of 0.1%v/vTFA with 0.7ml/min with the good HPLC C18 separator column of ultrapure water balance that contains 0.1%v/vTFA with anti-oxidant activity; Elutriant is monitored under 215nm, finally obtains the pure article of natural anti-oxidation peptide Rotodorin-D1.
3. the preparation method of natural anti-oxidation peptide Rotodorin-D1 according to claim 2; It is characterized in that the anti-oxidant activity assessment is specific as follows: at first dispose the ABTS storage liquid: at aqueous solution dissolving 2.8mM Potassium Persulphate and 7mM ABTS, lucifuge reaction at least 6 hours, afterwards; Dilute with water is 125 times immediately; The water-soluble sample of 50 microlitres adds 50 microlitre ABTS diluents, lucifuge reaction at least 10 minutes, afterwards; Under the 415nm wavelength, read its absorbancy, the sample anti-oxidant activity calculates with following formula: (A
Blank-A
Sample) * 100/A
Blank
4. the application of the described natural anti-oxidation peptide of claim 1 Rotodorin-D1 is characterized in that the polypeptide Rotodorin-D1 of anti-oxidant activity is applied to the natural anti-oxidation peptide directly put in the prescription of makeup in the preparation of makeup.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2012100141195A CN102584950A (en) | 2012-01-17 | 2012-01-17 | Natural antioxidant peptide (Rotodorin-D1), and preparation method and application thereof |
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| CN2012100141195A CN102584950A (en) | 2012-01-17 | 2012-01-17 | Natural antioxidant peptide (Rotodorin-D1), and preparation method and application thereof |
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Cited By (1)
| 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 |
-
2012
- 2012-01-17 CN CN2012100141195A patent/CN102584950A/en active Pending
Non-Patent Citations (4)
| Title |
|---|
| XINWANG YANG: "Extremely Abundant Antimicrobial Peptides Existed in the Skins of Nine Kinds of Chinese Odorous Frogs", 《JOURNAL OF PROTEOME RESEARCH》 * |
| 周庆峰等: "蛙皮活性抗菌肽最新研究进展", 《中国农学通报》 * |
| 邬晓勇等: "基于云南臭蛙抗菌肽序列分析的药物设计策略", 《 西 北 师 范 大 学 学 报(自然科学版)》 * |
| 邬晓勇等: "蛙皮抗菌肽活性检测方法的筛选及其分离纯化", 《西南农业学报》 * |
Cited By (1)
| 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 |
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Application publication date: 20120718 |