CN111777662A - Active polypeptide, preparation method and application in anti-aging cosmetics - Google Patents
Active polypeptide, preparation method and application in anti-aging cosmetics Download PDFInfo
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- CN111777662A CN111777662A CN202010727176.2A CN202010727176A CN111777662A CN 111777662 A CN111777662 A CN 111777662A CN 202010727176 A CN202010727176 A CN 202010727176A CN 111777662 A CN111777662 A CN 111777662A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2/00—Peptides of undefined number of amino acids; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
- A61K8/645—Proteins of vegetable origin; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/84—Products or compounds obtained by lyophilisation, freeze-drying
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- Organic Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
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- Dermatology (AREA)
- Birds (AREA)
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- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Cosmetics (AREA)
Abstract
The invention discloses an active polypeptide, a preparation method and application in anti-aging cosmetics. Important manifestations of skin aging include: the skin's antioxidant capacity decreases, leading to aging; the collagen and elastin are gradually lost, and wrinkles and sagging occur. The invention provides an active polypeptide, which can improve the antioxidant capacity of human skin fibroblasts on one hand, and is probably related to the activity of catalase in the human skin fibroblasts improved by the active polypeptide; on the other hand, the content of type I collagen and elastin in human skin fibroblasts can be increased, so that skin wrinkles and sagging can be overcome. Therefore, the active polypeptide provided by the invention has the effect of resisting skin aging, and can be used as an anti-aging active component for preparing anti-aging cosmetics.
Description
Technical Field
The invention belongs to the field of cosmetics, and relates to an active polypeptide, a preparation method and application in anti-aging cosmetics.
Background
Anti-aging is always the focus of long-term attention in the cosmetic industry, and the aim of delaying skin aging is one of the pursuit targets of most people. Many people are keen in the research of anti-aging experiments and develop many anti-aging cosmetics.
Skin aging is a process of making the human body function or develop rapidly or slowly naturally under the influence of internal and external factors, mainly due to endogenous aging and exogenous aging. Endogenous aging is an irresistible complex natural law and mainly shows that cell metabolism is gradually slowed down, collagen is gradually lost, collagen is damaged, hormone secretion is disordered, fat content begins to be reduced, partial cells are naturally killed and the like, and the conditions that skin loses elasticity and luster, skin is dried to generate wrinkles, even more, melanin pigmentation, senile plaque generation and the like are reflected on the skin. Exogenous aging is mainly affected by environmental pollution, excessive ultraviolet irradiation and other factors. Skin aging is mainly gradually shown from an epidermal layer, an epidermis-dermis connecting layer and a dermis layer, for example, wrinkles on the skin are a main mark of skin aging, and the skin aging problems of wrinkles, looseness and the like are caused by loss of collagen and breakage of elastic fibers due to thinning of the skin thickness.
Currently, polypeptides are among the most popular anti-aging ingredients in anti-aging cosmetics. The study of polypeptides having anti-ageing activity is therefore also a endlessly variable.
Disclosure of Invention
The invention aims to provide an active polypeptide, a preparation method and application in anti-aging cosmetics.
The technical scheme of the invention is as follows:
an active polypeptide prepared by the steps of:
step S1, degreasing: pulverizing dried folium Artemisiae Argyi, and defatting with petroleum ether under reflux;
step S2, extracting: leaching degreased and solvent-volatilized folium Artemisiae Argyi with sodium hydroxide water solution with pH of 8.5-9.5 at room temperature, centrifuging the extractive solution at rotation speed of 4000-5000 r/min for 8-12 min;
step S3, decoloring: adding activated carbon into the centrifuged supernatant, stirring and decoloring, centrifuging and collecting the supernatant;
step S4, alcohol precipitation: adding absolute ethyl alcohol into the supernatant after decolorization and centrifugation to enable the final volume percentage concentration of the ethyl alcohol to be 60%, fully stirring, standing at a low temperature for 10-14 h, centrifuging at a rotating speed of 7500-8500 r/min for 15-25 min, collecting the supernatant, removing the ethyl alcohol through rotary evaporation, and freeze-drying to obtain a folium artemisiae argyi polypeptide crude extract;
step S5, gel column chromatography: dissolving folium Artemisiae Argyi polypeptide crude extract with distilled water, loading onto Sephadex G-25 gel chromatographic column, eluting with PBS buffer solution at flow rate of 1mL/min, monitoring with protein nucleic acid detector at wavelength of 280nm, collecting eluate corresponding to the 2 nd elution peak, and freeze drying.
Preferably, in step S1, the folium Artemisiae Argyi is degreased 1-3 times with petroleum ether of 8-12L volume for 1-2 h each time.
Preferably, in step S2, each kilogram of folium Artemisiae Argyi is leached at room temperature for 1-3 times, each time for 6-10 hours, with a volume of 6-10L of sodium hydroxide aqueous solution.
Preferably, in step S3, the amount of activated carbon is 0.2% w/v, the decolorizing temperature is 75 ℃, and the decolorizing time is 30 min.
Preferably, in step S4, the low temperature means 4 ℃.
Preferably, in step S5, the Sephadex G-25 gel chromatography column has an inner diameter and a bed height of 1: 10.
The active polypeptide is used for improving the antioxidant capacity of skin.
The use of the above active polypeptide for increasing the collagen content in skin.
The use of the above active polypeptide for increasing elastin content in skin.
The application of the active polypeptide in preparing anti-aging cosmetics.
The beneficial technical effects are as follows:
the invention provides a polypeptide which is separated from folium artemisiae argyi. On one hand, the active polypeptide can improve the antioxidant capacity of the human skin fibroblasts, which is probably related to that the active polypeptide can improve the activity of catalase in the human skin fibroblasts; on the other hand, the content of type I collagen and elastin in human skin fibroblasts can be increased, so that skin wrinkles and sagging can be overcome. Therefore, the active polypeptide provided by the invention has the effect of resisting skin aging, and can be used as an anti-aging active ingredient for preparing anti-aging cosmetics.
Drawings
FIG. 1 is a chromatogram for monitoring the elution of a gel column;
FIG. 2 is a comparison of human skin fibroblast proliferation activity in each group;
FIG. 3 is a comparison of catalase activity in human skin fibroblasts of various groups;
FIG. 4 is a comparison of the content of type I collagen and elastin in human skin fibroblasts of each group.
Detailed Description
The following examples are intended to illustrate the essence of the present invention, but should not be construed as limiting the scope of the present invention.
Example 1: preparation of active polypeptide
First, test materials
The raw material is dry folium Artemisiae Argyi of Artemisia of Compositae.
The active carbon is conventional active carbon powder for decolorization, and the chemical reagents are conventional reagents.
Second, method and results
The preparation steps are as follows:
step S1, degreasing: pulverizing dried folium Artemisiae Argyi, defatting with petroleum ether under reflux, defatting with 10L petroleum ether for 2 times per kg folium Artemisiae Argyi, each time for 1.5 hr;
step S2, extracting: extracting defatted folium Artemisiae Argyi with pH 9.0 sodium hydroxide aqueous solution at room temperature, centrifuging the extractive solution at 4500r/min for 10 min; wherein, each kilogram of folium Artemisiae Argyi is leached at room temperature for 2 times, each time for 8h, with 8L volume of sodium hydroxide aqueous solution;
step S3, decoloring: adding activated carbon into the centrifuged supernatant, stirring and decoloring, centrifuging and collecting the supernatant; wherein the dosage of the active carbon is 0.2% w/v, the decoloring temperature is 75 ℃, and the decoloring time is 30 min;
step S4, alcohol precipitation: adding anhydrous ethanol into the supernatant after decolorization and centrifugation to make the final volume percentage concentration of ethanol be 60%, fully stirring, standing at 4 ℃ for 12h, centrifuging at the rotating speed of 8000r/min for 20min, collecting the supernatant, performing rotary evaporation at 50 ℃ to remove ethanol, and freeze-drying to obtain folium Artemisiae Argyi polypeptide crude extract;
step S5, gel column chromatography: dissolving folium Artemisiae Argyi polypeptide crude extract with distilled water (10mg/mL), loading onto Sephadex G-25 gel chromatographic column (loading volume is 1/10 of column bed volume) with inner diameter and column bed height ratio of 1:10, eluting with PBS buffer solution at flow rate of 1mL/min, monitoring with protein nucleic acid detector at wavelength of 280nm, and FIG. 1 is gel column chromatography elution monitoring chromatogram, collecting eluate corresponding to the 2 nd elution peak, and freeze drying.
Example 2: anti-aging Activity of active Polypeptides
First, test materials
Active polypeptide was prepared as in example 1 and stored at-20 ℃ until use.
Human skin fibroblasts (HDFs) were purchased from zermer fly, were cryopreserved HDF-a cells, normal human skin fibroblasts from adult skin, and were cryopreserved at the end of primary culture.
Calf serum, DMEM medium, penicillin and streptomycin were purchased from Gibco, USA.
Human Catalase (CAT) enzyme-linked immunoassay kit was purchased from Biyuntian biotechnology Co.
Second, test method
1. Cell culture
HDF cells were cultured in DMEM containing 10% calf serum, 1% penicillin and 1% streptomycin at 37 ℃ with 5% CO2Culturing under the condition of relative saturation humidity, carrying out passage for 2-3 d, and taking cells in logarithmic growth phase for testing.
2. MTT method for measuring cell proliferation activity
Taking HDF cells in logarithmic growth phase, and adjusting the cell density to 5 × 104And (4) inoculating the cells/mL into a 96-well plate, wherein each well is 200 mu L, and randomly dividing the cells into a normal control group, an oxidative damage group and a polypeptide protection group after culturing for 24 hours. The polypeptide protection group is continuously cultured by using complete culture medium containing active polypeptide (5, 10 and 20 mu g/mL) with different final mass concentrations, the normal control group and the oxidative damage group are continuously cultured by using normal complete culture medium, and after 24 hours, the oxidative damage group and the polypeptide protection group are replaced by the medium containing 150 mu M H2O2The complete culture medium is continuously cultured for 12h, the culture solution is discarded, PBS is washed, 100 mu L of MTT solution with the concentration of 0.5mg/L is added into each hole, the incubation is carried out for 4h, the liquid in each hole is carefully absorbed and discarded, 150 mu L of LDMSO is added into each hole, the oscillation is carried out for 10min, the light absorption value A of each hole is measured under the 490nm wavelength of an enzyme labeling instrument, the cell proliferation activity is calculated according to a formula, 5 repeated holes are respectively arranged at each concentration of a normal control group, an oxidation damage group and a polypeptide protection group, and the cell proliferation activity is × 100% of the A oxidation damage group or the polypeptide protection group/A normal control group.
3. Determination of catalase activity by kit
Setting control group and polypeptide group, culturing the polypeptide group with complete culture medium containing active polypeptide (10, 20 μ g/mL) with different final mass concentration, culturing the control group with normal complete culture medium, centrifuging after 72h, discarding supernatant, washing with PBS, and diluting to 1 × 106cell/mL cell solution, repeated freeze thawing to prepare cell pulp, centrifuging to obtain cell supernatant, and determining catalase activity according to the specification of a human Catalase (CAT) enzyme-linked immunoassay kit, and performing parallel experiments for 3 times.
4. Western blot method for determining content of type I Collagen (Collagen I) and elastin (elastin)
Setting control group and polypeptide group, culturing the polypeptide group with complete culture medium containing active polypeptide (10, 20 μ g/mL) with different final mass concentration, culturing the control group with normal complete culture medium, centrifuging after 72h, discarding supernatant, washing with PBS, and diluting to 1 × 106cell/mL cell solution, repeatedly freezing and thawing to prepare cell pulp, centrifuging to obtain cell supernatant, and measuring protein concentration by BCA methodAnd performing SDS-PAGE electrophoresis on equivalent protein, concentrating gel for 80V and 30min, separating the gel for 90min, wet-transferring the gel to a PVDF membrane, sealing 5% skimmed milk at normal temperature for 2h, incubating Collagen I, Elasticin and GAPDH overnight at 4 ℃, washing the membrane for 3 times (about 15min for each time) by PBST, incubating corresponding secondary antibody at normal temperature for 1.5h, washing the membrane for 3 times (15 min for each time) by PBST, developing the PVDF membrane by adopting an ECL luminescence solution on a Bilun day, and taking a picture for analysis.
5. Data processing
All data were processed using SPSS15.0 software, expressed as mean ± s, and comparisons between groups were by one-way anova, with P < 0.05 indicating significant differences.
Third, test results
1. Effect of active polypeptide on human skin fibroblast oxidative damage resistance
The cell proliferation activity of each group is shown in table 1 and fig. 2, and compared with a normal control group, the proliferation activity of the HDF cells of the oxidation injury group is obviously reduced; compared with the oxidative damage group, the proliferation activity of the HDF cells in the polypeptide protection group is obviously improved. This indicates that the proliferation activity of HDF cells in oxidative damage environment is significantly reduced, whereas the resistance of HDF cells to oxidative damage is significantly improved and the ability to resist oxidative damage is significantly enhanced after predrying with the active polypeptide prepared in example 1.
TABLE 1 cell proliferation Activity of each group
2. Effect of active Polypeptides on Catalase Activity in human skin fibroblasts
Catalase activity in each group of cells as shown in table 2 and fig. 3, the catalase activity in the human skin fibroblasts of the polypeptide group was significantly increased compared to the control group, and was positively correlated to the administration concentration of the active polypeptide. As the skilled person knows, the activity of catalase is directly related to the antioxidant capacity of the cell, and the higher the activity of catalase is, the stronger the antioxidant capacity of the cell is.
TABLE 2 Catalase Activity in groups of cells
3. Effect of active Polypeptides on the content of type I collagen and elastin in human skin fibroblasts
The contents of type I collagen and elastin in each group of cells are shown in fig. 4, and compared with the control group, the contents of type I collagen and elastin in the human skin fibroblasts of the polypeptide group are obviously increased.
Important manifestations of skin aging include: the skin's antioxidant capacity decreases, leading to aging; the collagen and elastin are gradually lost, and wrinkles and sagging occur. The tests show that the active polypeptide provided by the invention can improve the antioxidant capacity of human skin fibroblasts on one hand, which is probably related to that the active polypeptide can improve the activity of catalase in the human skin fibroblasts; on the other hand, the content of type I collagen and elastin in human skin fibroblasts can be increased, so that skin wrinkles and sagging can be overcome. Therefore, the active polypeptide provided by the invention has the effect of resisting skin aging, and can be used as an anti-aging active component for preparing anti-aging cosmetics.
Claims (10)
1. An active polypeptide prepared by the steps of:
step S1, degreasing: pulverizing dried folium Artemisiae Argyi, and defatting with petroleum ether under reflux;
step S2, extracting: leaching degreased and solvent-volatilized folium Artemisiae Argyi with sodium hydroxide water solution with pH of 8.5-9.5 at room temperature, centrifuging the extractive solution at rotation speed of 4000-5000 r/min for 8-12 min;
step S3, decoloring: adding activated carbon into the centrifuged supernatant, stirring and decoloring, centrifuging and collecting the supernatant;
step S4, alcohol precipitation: adding absolute ethyl alcohol into the supernatant after decolorization and centrifugation to enable the final volume percentage concentration of the ethyl alcohol to be 60%, fully stirring, standing at a low temperature for 10-14 h, centrifuging at a rotating speed of 7500-8500 r/min for 15-25 min, collecting the supernatant, removing the ethyl alcohol through rotary evaporation, and freeze-drying to obtain a folium artemisiae argyi polypeptide crude extract;
step S5, gel column chromatography: dissolving folium Artemisiae Argyi polypeptide crude extract with distilled water, loading onto Sephadex G-25 gel chromatographic column, eluting with PBS buffer solution at flow rate of 1mL/min, monitoring with protein nucleic acid detector at wavelength of 280nm, collecting eluate corresponding to the 2 nd elution peak, and freeze drying.
2. Active polypeptide according to claim 1, characterized in that: in the step S1, each kilogram of folium Artemisiae Argyi is degreased 1-3 times with petroleum ether with a volume of 8-12L, 1-2 h each time.
3. Active polypeptide according to claim 1, characterized in that: in step S2, each kilogram of folium Artemisiae Argyi is leached at room temperature for 1-3 times, each time for 6-10 hours, with 6-10L of sodium hydroxide aqueous solution.
4. Active polypeptide according to claim 1, characterized in that: in step S3, the usage amount of the activated carbon is 0.2% w/v, the decoloring temperature is 75 ℃, and the decoloring time is 30 min.
5. Active polypeptide according to claim 1, characterized in that: in step S4, the low temperature means 4 ℃.
6. Active polypeptide according to claim 1, characterized in that: in step S5, the inner diameter and bed height of the Sephadex G-25 gel chromatography column were 1: 10.
7. Use of the active polypeptide of claim 1 for increasing the antioxidant capacity of skin.
8. Use of the active polypeptide of claim 1 for increasing collagen content in skin.
9. Use of the active polypeptide of claim 1 for increasing elastin content in skin.
10. Use of the active polypeptide of claim 1 for the preparation of an anti-aging cosmetic.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010727176.2A CN111777662B (en) | 2020-07-27 | 2020-07-27 | Active polypeptide, preparation method and application in anti-aging cosmetics |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010727176.2A CN111777662B (en) | 2020-07-27 | 2020-07-27 | Active polypeptide, preparation method and application in anti-aging cosmetics |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111777662A true CN111777662A (en) | 2020-10-16 |
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| CN113527422A (en) * | 2021-07-10 | 2021-10-22 | 浙江省农业科学院 | Polypeptide FLCQWP with anti-oxidation function and application thereof |
| CN114381488A (en) * | 2022-03-02 | 2022-04-22 | 淮安唯尔美化妆品有限公司 | Active peptide, anti-skin aging application and commercialized application in cosmetics |
| CN114805485A (en) * | 2022-05-26 | 2022-07-29 | 青岛麦迪赛斯医疗技术有限公司 | Chlorella bioactive peptide and application thereof in preparation of anti-aging essence |
| CN115894625A (en) * | 2023-01-13 | 2023-04-04 | 桂林医学院 | Polypeptide with anti-aging effect and application thereof |
| CN118994318A (en) * | 2024-08-29 | 2024-11-22 | 永创恒新生物医学科技(北京)有限公司 | Active peptide, cosmetic containing active peptide and application |
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| CN114805485A (en) * | 2022-05-26 | 2022-07-29 | 青岛麦迪赛斯医疗技术有限公司 | Chlorella bioactive peptide and application thereof in preparation of anti-aging essence |
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| CN115894625B (en) * | 2023-01-13 | 2024-08-27 | 桂林医学院 | A polypeptide with anti-aging effect and its application |
| CN118994318A (en) * | 2024-08-29 | 2024-11-22 | 永创恒新生物医学科技(北京)有限公司 | Active peptide, cosmetic containing active peptide and application |
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