CN114702549A

CN114702549A - Active hexapeptide with antioxidation effect and application thereof

Info

Publication number: CN114702549A
Application number: CN202210356740.3A
Authority: CN
Inventors: 高理钱; 贾艳; 李孟础; 张达; 谢程亮; 肖奇才; 王璇; 孙杰
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2022-04-06
Filing date: 2022-04-06
Publication date: 2022-07-05
Anticipated expiration: 2042-04-06
Also published as: CN114702549B

Abstract

The invention belongs to the technical field of functional raw materials of cosmetics, and particularly relates to an active hexapeptide with an anti-oxidation effect and application thereof, in order to develop bioactive peptide with anti-oxidation activity and capable of promoting proliferation and migration of human skin fibroblasts, the invention discloses a hexapeptide (GHKGHK), which can promote proliferation and migration of human skin fibroblasts, promote type I collagen synthesis, improve skin elasticity and firmness, has anti-oxidation activity and strong capacity of eliminating DPPH free radicals, is low in irritation and high in safety, can be applied to the fields of biological pharmacy, cosmetics and the like, such as preparation of anti-aging cosmetics, medicines for aging-related diseases, wound healing promoting products, anti-oxidation products and the like, and has important application prospects.

Description

Active hexapeptide with antioxidation effect and application thereof

Technical Field

The invention belongs to the technical field of functional raw materials of cosmetics, and particularly relates to an active hexapeptide with an antioxidation effect and application thereof.

Background

The cosmetics are a compound mixture prepared by reasonably blending and processing various raw materials, and the cosmetics have various raw materials and different performances. Wherein, chemical synthesis and plant extraction are always the main sources of functional raw materials of cosmetics. With the continuous updating and development of traditional cosmetic raw materials, biological beauty treatment and gene beauty treatment become new targets of development, and especially peptide raw materials and skin care products containing peptides are more and more favored by people. The bioactive peptide provides a brand new direction and thought for cosmetic raw materials because most biochemical reactions and evolution processes in organisms are regulated and controlled by specific amino acid sequences (polypeptides or proteins) to a certain extent.

Human skin fibroblasts are the most prominent cells in the dermis layer of the skin and have an important role in maintaining the elasticity and tone of the skin. Human skin fibroblasts have strong protein synthesis capacity, can synthesize and secrete a large amount of matrix components such as elastin, collagen, glycosaminoglycan and glycoprotein, and further generate elastic fibers, collagen fibers and reticular fibers, and secrete various cell repair factors, so that the skin has strong renewal and self-repair capacity. Therefore, the cell viability and proliferation properties of human skin fibroblasts are not only closely related to skin aging, but also play a key role in the skin barrier.

Free radicals have a clear relationship with aging, and scientists believe that free radicals are a major cause of aging. The free radicals can promote the generation of lipofuscin in vivo, the accumulation of lipofuscin in skin cells can form senile plaque, and the accumulation in brain cells can cause hypomnesis or dysnoesia, even senile dementia. Free radicals can also cause skin laxity, increased wrinkles, decreased bone regeneration in the elderly, etc. Once the body cannot remove the free radicals in time due to excessive increase of the free radicals caused by internal or external factors, the cells and tissues are damaged, and finally, the loss of body functions and the occurrence of diseases related to aging are caused. Therefore, the elimination of excess free radicals in vivo and in vitro plays an important role in the process of preventing aging and certain diseases.

In conclusion, the bioactive peptide which has antioxidant activity and can promote the proliferation and migration of human skin fibroblasts is excavated, and the bioactive peptide which is used as a cosmetic raw material for preparing the skin care product has important application prospect.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a hexapeptide, which has antioxidant activity, can promote the proliferation and migration of human skin fibroblasts, promotes the synthesis of type I collagen, and can be used for resisting oxidation, resisting aging, promoting wound healing and the like.

In order to realize the purpose, the invention is realized by the following technical scheme:

the invention provides a hexapeptide, wherein the amino acid sequence of the hexapeptide is shown as SEQ ID NO: 1 (GHKGHK). The amino acid sequence of the hexapeptide is Gly-His-Lys-Gly-His-Lys.

The amino acid sequence of the invention is reasonably synthesized by screening appropriate resin by adopting a standard Fmoc scheme. The method specifically comprises the following steps: the C-terminal carboxyl of the target polypeptide is connected with an insoluble polymer resin in a covalent bond form, then the amino group of the amino acid is taken as a starting point to react with the carboxyl of another molecule of amino acid to form a peptide bond, and the process is repeated continuously, so that the target polypeptide product can be obtained. And after the synthesis reaction is finished, removing the protecting group, and separating the peptide chain from the resin to obtain the target product. The polypeptide synthesis is a solid phase synthesis process for repeatedly adding amino acids, and the solid phase synthesis sequence is synthesized from a C end to an N end. The polymer resin is one of crosslinked polystyrene, polyamide, polyethylene-glycol resin, etc.

The invention also provides application of the hexapeptide in preparing an antioxidant and/or anti-aging product.

The invention also provides application of the hexapeptide in preparing a product for promoting wound healing.

Preferably, the anti-aging is the scavenging of aging-causing free radicals.

Preferably, the anti-aging is to improve the activity of human skin fibroblasts.

Preferably, the wound healing is promoting migration of human skin fibroblasts.

The polypeptide is evaluated by human skin fibroblasts for resisting aging, improving skin barrier and skin repair, healing wounds and the like; evaluating the antioxidation of the polypeptide by a DPPH free radical scavenging experiment; the chick embryo chorioallantoic membrane experiment evaluates the safety of the polypeptide. Research shows that the hexapeptide (GHKGHK) can play a role in promoting the growth of human skin fibroblasts at a lower concentration, promote the synthesis of type I collagen, improve the elasticity and the compactness of skin, and effectively promote the migration of cells.

Preferably, the product is a pharmaceutical, cosmetic or nutraceutical.

Preferably, the product contains an effective amount of hexapeptide, the balance being adjuvants or other compatible ingredients.

Further, in order to increase the applicability of the product, the auxiliary materials include conventional diluents, fillers, binders, wetting agents, absorption promoters, surfactants, lubricants and stabilizers.

Further, the other compatible components include natural drugs, chemical drugs or biological drugs.

Preferably, the above medicine for treating aging-related diseases further comprises pharmaceutically acceptable carriers and/or adjuvants, such as sour agent, toner, flavoring agent, sweetening agent, or their combination, in order to improve the applicability of the medicine.

Preferably, the product can be made into different dosage forms, such as tablet, capsule, injection, liposome nanoparticle, controlled release agent, gel ointment, topical liniment, patch, cream, detergent, emulsion, gel or toner.

Compared with the prior art, the invention has the beneficial effects that:

the invention discloses a hexapeptide (GHKGHK), which can promote the proliferation and migration of human skin fibroblasts, promote the synthesis of type I collagen, improve the elasticity and the compactness of skin, has antioxidant activity and strong capacity of eliminating DPPH free radicals, has low irritation and high safety, can be applied to the fields of biological pharmacy, cosmetics and the like, such as preparation of anti-aging cosmetics, medicines for diseases related to aging, products for promoting wound healing, antioxidant products and the like, and has important application prospects.

Drawings

FIG. 1 is a high performance liquid chromatography identification map of a polypeptide;

FIG. 2 is a graph of mass spectrometry of a polypeptide;

FIG. 3 is a graph showing the results of an assay in which hexapeptide affects proliferation of human skin fibroblasts;

FIG. 4 shows the results of measurements of hexapeptides affecting human skin fibroblast mobility (A is a 24h cell mobility growth curve; B is 24h cell mobility);

FIG. 5 shows the measurement results of DPPH radical clearance by hexapeptide;

FIG. 6 shows the measurement results of hexapeptide affecting the content of human type I collagen;

FIG. 7 is a test chart of chick embryo chorioallantoic membrane of hexapeptide.

Detailed Description

The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the respective embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The experimental procedures in the following examples were carried out by conventional methods unless otherwise specified, and the test materials used in the following examples were commercially available by conventional methods unless otherwise specified.

EXAMPLE 1 solid phase Synthesis of hexapeptides

The amino acid sequence of the invention is reasonably synthesized by screening appropriate resin by adopting a standard Fmoc scheme. The method specifically comprises the following steps:

selecting Wang resin (Queen resin) in high molecular resin (cross-linked polystyrene), sequentially adding amino acids from right to left according to the characteristics of an amino acid sequence (GHKGHK) Gly-His-Lys-Gly-His-Lys, firstly connecting a carboxyl of Lys with a resin in a covalent bond form, then performing a shrinking reaction on the amino of Lys and the carboxyl of His, adding Gly after the reaction, reacting the amino of His with the carboxyl of Gly, cutting off the resin after connecting the last Gly amino acid to obtain a target polypeptide, purifying by using High Performance Liquid Chromatography (HPLC) to obtain a final polypeptide product, and performing Mass Spectrum (MS) analysis on the polypeptide.

As shown in fig. 1 and fig. 2, the hexapeptide synthesized in this example has a theoretical molecular weight of 662.75, an actual molecular weight of 662.71, and a sequence of: Gly-His-Lys-Gly-His-Lys. Wherein Gly represents amino acid with English name of Glycine and Chinese name of Glycine and corresponding residue; his represents an amino acid with the English name Histidine and the Chinese name Histidine and the corresponding residue; lys represents an amino acid with the English name Lysine and the Chinese name Lysine and the corresponding residue.

Example 2 Effect of hexapeptides on human skin fibroblast morphology and proliferation

Human skin fibroblasts were cultured in complete medium [ consisting of basal medium high-glucose DMEM, 10% fetal bovine serum (v/v), and 1% diabody (consisting of penicillin and streptomycin, v/v ═ 1:1) ], and placed at 37 ℃ in 5% CO₂Culturing in a constant-temperature incubator until the fusion rate of 80-90% in a cell culture dish, then carrying out passage, and calculating the cell number by using a full-automatic cell counter after the passage reaches two to three generations.

The cells were arranged in a 3X 10 order³The density of each well is inoculated in a 96-well plate (PBS is used as a blank control), when the cells are attached to the wall, the culture medium is removed, 100 mu L of fresh culture medium containing 0.0032 mu M-2 mu M (0.0032 mu M, 0.016 mu M, 0.08 mu M, 0.4 mu M and 2 mu M) polypeptide is added, the same amount of DMEM culture solution is added into a control group, the culture is continued for 24 h-72 h in an incubator, then 10 mu L MTT is added into each well, the incubator is continued to incubate for 4h, then 100 mu L DMSO is added, and finally the culture is continued for 4h in the incubator.

After the incubation is finished, the optical density (namely OD value) of each hole at 570nm is measured on an automatic microplate reader, and each group is provided with 6 multiple holes. The experimental results of the examples are shown in FIG. 3.

As a result, it was found that when the hexapeptide of the present invention was allowed to act on human fibroblasts for 72 hours, the cells of each polypeptide group became long, the fusiform protrusion of the cells increased, the cells became more compact, and the number of adherent cells increased. Meanwhile, the results of fig. 3 show that there is a significant difference in the proliferation of human skin fibroblasts when the concentration of the polypeptide is 0.0032 μ M to 2 μ M (a) ((^**P＜0.01，^***P is less than 0.001), which shows that the hexapeptide can improve the activity of human skin fibroblasts, thereby improving the skin elasticity and having a certain anti-aging effect.

Example 3 cell scratch test

After subculturing human skin fibroblasts as in example 2, approximately 5X 10 cells were added per well in 24-well plates⁴Culturing for 24 hr, vertically cross-scratching each well with 200 μ L suction head, washing the scratched cells with PBS, and sequentially adding 500 μ L solution containing 0.0032 μ M-2 μ M (0.0032 μ M, 0.016 μ M, and 0.08 μ M) to each well0.4 μ M, 2 μ M) polypeptide [ high-sugar DMEM, 1% diabase (consisting of penicillin and streptomycin, v/v ═ 1:1) in basal medium ] were cultured continuously, observed at 0h and 24h, respectively, and the mobility, which is (initial scratch width-scratch width at the corresponding time point)/initial scratch width × 100%, was calculated using ImageJ software.

The experimental result of fig. 4 shows that hexapeptide has a more obvious cell migration promoting effect at a lower concentration (0.0032 μ M), and the cell migration rate reaches 38.04%; at a concentration of 2 μ M, the cell mobility reached 83.38, which was 78.94% higher than that of the blank drug-free group (cell mobility of 4.44% in the blank drug-free group). The hexapeptide of the invention has the functions of promoting the self-repair of the skin, promoting the skin barrier and promoting the wound healing.

EXAMPLE 4 DPPH free radical scavenging Activity assay of hexapeptides

mu.L of DPPH reagent (1mg of DPPH diluted to 20mL with absolute ethanol) and 60. mu.L of polypeptide (concentration 0.08mM, 0.4mM, 2mM, 10mM, 50mM, respectively) were added to a 96-well plate as a test group. Simultaneously setting a blank group and a model group, wherein the blank group comprises 180 mu L of absolute ethyl alcohol and 60 mu L of polypeptide, the model group comprises 180 mu L of DPPH reagent and 60 mu L of ultrapure water, each hole is provided with 3 compound holes, and the reaction is carried out for 15-30 minutes at room temperature in a dark place. After the reaction, the absorbance at 517nm was measured by a microplate reader, and DPPH radical clearance {1- (test set-blank set) ÷ model set) } × 100%.

The experimental result of fig. 5 shows that the hexapeptide of the present invention has a certain radical scavenging effect, and the DPPH clearance rate reaches 78.1% at a concentration of 50mM, which indicates that the hexapeptide of the present invention has certain anti-radical and anti-oxidation effects.

Example 5 Effect of hexapeptides on human type I collagen

After subculturing human skin fibroblasts as in example 2, approximately 5X 10 cells per well were added to 24-well plates⁴Culturing fibroblast for 24 hr, removing culture medium, adding fresh serum-free DMEM medium with final concentration of 0.1 μ M polypeptide, and standing at 37 deg.C and 5% CO₂The incubation was continued for 24 hours. Set negative in the experiment processThe positive control group is an equivalent serum-free DMEM medium, and the negative control group is an equivalent serum-containing DMEM medium. After the culture is finished, collecting cell culture supernatant, centrifuging the cell culture supernatant in a 1.5mL sterile centrifuge tube, and detecting the content of human type I collagen (COL I) by using a human type I collagen enzyme linked immunosorbent assay kit and adopting an ELISA method according to the using instruction.

The experimental result of fig. 6 shows that, compared with the negative control, the hexapeptide has a relatively obvious promoting effect on the synthesis of COL I in cells at a relatively low concentration (0.1 μ M), which indicates that the hexapeptide of the present invention has the effect of promoting the secretion and synthesis of collagen, and has good effects on improving the elasticity and firmness of skin.

EXAMPLE 6 chick embryo chorioallantoic Membrane experiment

Placing 0-day-old hatching eggs for half a day at room temperature, then placing the hatching eggs into an automatic incubator with an upward air chamber, adjusting the temperature to 37.6 +/-0.1 ℃, and adjusting the relative humidity to 46 +/-1%; sun-shine embryo, discarding white protein and dead embryo; hatching till 10-12 days for standby. Removing eggshell from the air chamber of the incubated hatching egg, wetting eggshell membrane with physiological saline, slightly peeling to expose chorioallantoic membrane (CAM), and placing a nonirritating rubber ring with diameter of 1.5cm on the CAM; 1 mu M-50 mM polypeptide aqueous dispersion (50mM) is placed in an incubator for half an hour in advance, then 0.3mL polypeptide aqueous dispersion is directly dripped into the rubber ring, after 30 minutes, the vascular effects of CAM bleeding, vascular lysis, blood coagulation bleeding and the like in the ring are observed, and the evaluation is carried out according to the evaluation standard in the table 1; physiological saline is used as a negative control group, and 1% sodium dodecyl sulfate is used as a positive control group.

TABLE 1 evaluation criteria for vascular Effect

Note: bleeding refers to the flow of blood out of the blood vessels and/or capillaries of the CAM; coagulation refers to the denaturation of intravascular and extravascular proteins, usually found only in large and medium-large blood vessels; vessel thawing refers to the disappearance of blood vessels on the CAM membrane.

The test results in fig. 7 show that the saline negative control group scored 0, the hexapeptide test group scored 0, and the 1% sodium lauryl sulfate positive control group scored 3. The hexapeptide of the invention has extremely low irritation and high safety to human body and skin.

In conclusion, the invention synthesizes the hexapeptide (GHKGHK) for the first time, the hexapeptide can play a role in promoting the growth of human skin fibroblasts at a lower concentration and can effectively promote the migration of cells, in-vitro chemical antioxidant detection also shows that the hexapeptide has antioxidant activity and strong capacity of eliminating DPPH free radicals, can promote the proliferation and migration of the human skin fibroblasts, has low irritation and high safety, and can be applied to the fields of biological pharmacy, cosmetics and the like.

The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.

Claims

1. A hexapeptide having an amino acid sequence as set forth in SEQ ID NO: 1 is shown.

2. Use of the hexapeptide of claim 1 for the preparation of an antioxidant and/or anti-aging product.

3. Use of the hexapeptide of claim 1 for the preparation of a product for promoting wound healing.

4. The use according to claim 2, wherein the anti-aging is the scavenging of aging-causing free radicals.

5. Use according to claim 2, wherein the anti-aging is an increase in the viability of human skin fibroblasts.

6. Use according to claim 2, wherein said wound healing is promoting migration of human skin fibroblasts.

7. Use according to claim 2 or 3, wherein the product is a pharmaceutical, cosmetic or nutraceutical product.

8. The use according to claim 5, wherein the product comprises an effective amount of hexapeptide, the balance being adjuvants or other compatible ingredients.

9. Use according to claim 6, characterized in that the adjuvants comprise conventional diluents, fillers, binders, wetting agents, absorption promoters, surfactants, lubricants and stabilizers.

10. The use of claim 6, wherein said other compatible components comprise natural pharmaceuticals, chemical pharmaceuticals or biological pharmaceuticals.