CN115845026A - Acetyl dipeptide-1 cetyl ester and composition and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biological medicine, and discloses an application of acetyl dipeptide-1 cetyl ester in preparing medicines, health products and cosmetics for promoting healing, resisting oxidation or repairing tissues, and also discloses a composition of acetyl dipeptide-1 cetyl ester, which comprises the following components: 0.1 to 3 weight percent of acetyl dipeptide-1 cetyl ester, 4 to 6 weight percent of emollient, 2 to 5 weight percent of thickener, 2 to 5 weight percent of humectant, 1 to 3 weight percent of emulsifier, 0.3 to 2 weight percent of preservative, 0.05 to 0.2 weight percent of neutralizer and the balance of water. The invention develops a new application of the known compound acetyl dipeptide-1 cetyl ester through a series of researches, has good healing promotion and tissue repair effects, and develops a new application field of the acetyl dipeptide-1 cetyl ester.

Description

Acetyl dipeptide-1 cetyl ester and composition and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to acetyl dipeptide-1 cetyl ester and a composition thereof, and application of the acetyl dipeptide-1 cetyl ester in tissue healing.

Background

Neurotransmitters in the skin include hormones such as cortisol, endorphins, oxytocin, dopamine, etc., which are capable of regulating cellular properties and skin functions (such as immunity, cell differentiation, proliferation, pigmentation, etc.). The release of these substances can be induced by physical, chemical or even emotional stimuli. Peptides are the first active in the skin care industry, and corresponding solutions are provided for this direction. Researchers at Kyoto university, japan, in the 1980's, discovered a "tyrosine arginine dipeptide" with analgesic activity, which is capable of releasing endorphins and stabilizing their degradation, and which may have neurotransmitter properties, intervene in the sensory nerve level released by calcitonin gene-related peptide (CGRP), inducing soothing effects on the skin. However, the hydrophilicity of tyrosine-arginine dipeptide results in its inability to penetrate the dermal layer and is therefore not suitable for use in cosmetics.

In order to solve the problem and improve the stability of the dipeptide, researchers develop a series of derivatives on the basis of the problem. Wherein acetyl dipeptide-1 cetyl ester (CAS: 196604-48-5) is added with lipophilic "acetyl" and "cetyl ester" groups at two ends of the dipeptide respectively to enhance the transdermal absorption capacity of the component. It was reported that, without using any skin care product, capsaicin was applied to the left and right nasolabial folds, and that cosmetics containing acetyl dipeptide-1 cetyl ester showed significantly lower burning/stinging values after one and two minutes after use, and thus proved to have a rapid and significant soothing effect in vivo. In 2006, the company Irelaya proposed that the addition of acetyl dipeptide-1 cetyl ester increased the moisture content of the skin by 39.5% more over four weeks than the product without acetyl dipeptide-1 cetyl ester. In 2012, researchers have shown that acetyl dipeptide-1 cetyl ester has a certain whitening effect on skin after it is added. The importance of acetyl dipeptide-1 cetyl ester in soothing, moisturizing and whitening is well known.

However, the reports and applications of acetyl dipeptide-1 cetyl ester in academia and industry are mainly limited in the aspects of relaxation, and no research and related applications are available on the effects of elasticity, skin tissue healing and the like. Deeply exploring other effects of acetyl dipeptide-1 cetyl ester, expanding the application aspect of the acetyl dipeptide-1 cetyl ester, and having important research significance and practical value.

Disclosure of Invention

In a first aspect of the invention, there is provided a novel use of acetyl dipeptide-1 cetyl ester: application of acetyl dipeptide-1 cetyl ester in preparation of medicines, health products and cosmetics for promoting healing, resisting oxidation or repairing tissues.

In a second aspect of the invention, a composition comprising acetyl dipeptide-1 cetyl ester has healing, antioxidant or tissue repair promoting effects.

The promotion of healing includes the promotion of wound healing, which refers to the natural process of regeneration of human skin and epidermal tissue after the tissue such as skin is separated or damaged due to the damage of human tissue or organ caused by mechanical factors. Tissue repair is the process by which local tissues, cells, are damaged or killed by some pathogenic factor, repaired by the regeneration of adjacent healthy cells, to restore tissue integrity.

Further, the composition also has at least one of skin elasticity enhancing, anti-inflammatory soothing, or moisturizing effects.

In a third aspect of the invention, a composition of acetyl dipeptide-1 cetyl ester is comprised of: 0.1 to 3 weight percent of acetyl dipeptide-1 cetyl ester, 4 to 6 weight percent of emollient, 2 to 5 weight percent of thickener, 2 to 5 weight percent of humectant, 1 to 3 weight percent of emulsifier, 0.3 to 2 weight percent of preservative, 0.05 to 0.2 weight percent of neutralizer and the balance of water.

Further, the emollient is isononyl isononanoate; the thickening agent is carbomer, xanthan gum and cetostearyl alcohol; the humectant is glycerin; the emulsifier is WL912 and EG; the preservative is PHCH and p-hydroxybenzoate; the neutralizing agent is triethanolamine.

Further, the composition is as follows: 0.1 to 3 weight percent of acetyl dipeptide-1 cetyl ester, 4 to 6 weight percent of isononyl isononanoate, 0.1 to 0.3 weight percent of carbomer, 0.05 to 0.2 weight percent of xanthan gum, 1.85 to 4.5 weight percent of cetostearyl alcohol, 2 to 5 weight percent of glycerol, 0.9 to 2.6 weight percent of WL912, 0.1 to 0.4weight percent of EG, 0.25 to 1.5weight percent of PHCH, 0.05 to 0.5 weight percent of p-hydroxybenzoate, 0.05 to 0.2 weight percent of triethanolamine and the balance of deionized water.

Further, the composition is as follows: 0.6wt% acetyl dipeptide-1 cetyl ester, 3wt% isononyl isononanoate, 0.15wt% carbomer, 0.1wt% xanthan gum, 3wt% cetostearyl alcohol, 3wt% glycerol, 1.2wt% WL912, 0.2wt% EG, 0.5wt% PHCH, 0.1wt% paraben, 0.1wt% triethanolamine, the remainder being deionized water.

WL912 is a commercial emulsifier consisting of: cetearyl alcohol, ceteareth-25, glyceryl stearate, sodium lauryl sulfate, mineral oil.

EG is a commercial emulsifier consisting of: sodium acrylate/sodium acryloyldimethyl taurate copolymer, isohexadecane and polysorbate-80.

PHCH is a commercial preservative consisting of: 32wt% of hexanediol, 40wt% of phenoxyethanol, 20 wt% of chlorphenesin and 8wt% of water.

In a fourth aspect of the invention, a process for the preparation of a composition of acetyl dipeptide-1 cetyl ester comprises the steps of:

s1, adding carbomer, glycerol, xanthan gum, p-hydroxybenzoate and deionized water into an emulsifying pot, and heating to 80-85 ℃ for dissolution;

s2, adding isononyl isononanoate, WL912 and cetostearyl alcohol into an oil phase pot, heating to 80-85 ℃ for dissolving, then adding into an emulsifying pot for homogenizing for 3-5 min, preserving heat, stirring for 20-30 min, and cooling;

s3, cooling to 65-70 ℃, adding EG for homogenization for 2-5 min, continuously stirring and cooling to 45-50 ℃, adding triethanolamine and stirring uniformly;

and S4, cooling to 40-45 ℃, adding PHCH, uniformly stirring, cooling to 35-40 ℃, adding acetyl dipeptide-1 cetyl ester, uniformly stirring, testing to be qualified, and discharging.

In a fifth aspect of the invention, the use of a composition of acetyl dipeptide-1 cetyl ester for promoting healing, antioxidation or tissue repair.

Further, the composition of acetyl dipeptide-1 cetyl ester has skin elasticity enhancing, anti-inflammatory soothing or moisturizing effects.

The invention has the following beneficial effects:

1. through a series of researches, the invention develops a new application of a known compound acetyl dipeptide-1 cetyl ester, has good effects of promoting healing, resisting oxidation and repairing tissues, and develops a new application field of the acetyl dipeptide-1 cetyl ester.

2. According to the invention, proper auxiliary materials are selected, and the acetyl dipeptide-1 cetyl ester is prepared into a composition, so that the effects of promoting healing and tissue repair of the composition are further researched, and particularly, the composition shows good healing promoting capability in animal experiments.

3. The invention also finds that the acetyl dipeptide-1 cetyl ester has the effects of enhancing skin elasticity, resisting inflammation, relieving, moisturizing and the like, and can be used in the fields of cosmetics, medicines and the like.

Drawings

FIG. 1 is a bar graph of the elastase inhibition of example 1;

FIG. 2 is a bar graph showing the results of the LPS-induced cell assay of example 2;

FIG. 3 shows the results of the cell scratching method in example 3;

FIG. 4 is a bar graph of the results of the lactic acid challenge test of example 5;

FIG. 5 is a bar graph of the results of the capsaicin challenge test of example 6;

FIG. 6 is a bar graph of the results of SLS stimulation test of example 7;

FIG. 7 is a bar graph of the results of the skin moisture test of example 8;

FIG. 8 is a bar graph of the results of the test for transdermal water loss of example 8;

FIG. 9 is a bar graph of the results of the skin oil test of example 8;

FIG. 10 is a photograph of a wound healing experiment of example 9;

FIG. 11 is a bar graph of the results of the antioxidant test of example 10.

Detailed Description

The present invention will be further described with reference to the following specific examples.

Example 1

Elastase inhibition methods: taking 2mL of 2mg/mL elastase solution, adding samples (acetyl dipeptide-1 cetyl ester) with different concentrations, fully and uniformly mixing in a vortex manner, oscillating for 20min in a shaker at 37 ℃ and 400r/min, immediately adding 5mL of 0.5mol/L phosphate buffer solution with pH of 6.0, uniformly mixing in a vortex manner, taking a proper amount of uniformly mixed solution to a 2mL centrifugal tube, centrifuging for 10min, precisely absorbing 200 mu L of supernatant to a 96-well plate, measuring absorbance by an enzyme-labeling instrument at the wavelength of 495nm, and simultaneously carrying out 400-800 nm spectrum scanning.

And taking a substrate and enzyme solution as a blank control group, taking a substrate and enzyme solution and a sample solution as an enzyme inhibition group, and taking the substrate and sample without the enzyme solution as background. Each group is provided with 3 times of holes. Inhibition (%) = [1- (An-An ')/(A0-A0') ]. Times.100%, wherein A0 is the absorbance of a sample without adding An enzyme, A0 'is the absorbance of a sample without adding only a substrate and An enzyme, an is the absorbance of a solution with only a sample, and An' is the absorbance of a sample without adding An enzyme. When An ' > An exhibits a promoting effect, the promoting rate (%) = [1- (An ' -An)/(A0-A0 ') ] × 100%.

As shown in FIG. 1, the elastase inhibition was concentration-dependent on acetyl dipeptide-1 cetyl ester, indicating that acetyl dipeptide-1 cetyl ester has the potential to enhance skin elasticity.

Example 2

Detection of anti-inflammatory repair efficacy of acetyl dipeptide-1 cetyl ester by LPS induced cell method

Interleukin 6 (IL-6) is the most typical cytokine associated with inflammation. It plays an important role in host defense by modulating immune and inflammatory responses. Inflammation affects the skin barrier, which enhances epidermal water loss, and also affects the growth of keratinocytes, which are difficult to recover after the barrier is damaged. Meanwhile, the skin care product can decompose extracellular matrix to cause skin collapse, inhibit the synthesis of collagen and enable the skin to be loose and wrinkled. Therefore, the preparation method effectively reduces the generation of interleukin IL-6 in keratinocytes and fibroblasts caused by external injury and ultraviolet, reduces inflammatory reaction, and is important for recovering skin barrier and protecting skin elasticity and stability.

Human fibroblasts were cultured at a density of 1X 10 ⁴ Planting in 96-well plate, placing in incubator, adhering to wall overnight, removing supernatant after 24 hr, adding 100 μ L samples (acetyl dipeptide-1 cetyl ester) diluted with DMEM medium with different concentrations, and negative control group is DMEM medium without samples, each group has 3 multiple wells with mass fraction of 5% CO ₂ Incubation at 37 ℃. After 2h of administration, the LPS model group and the experimental group were added with 10. Mu.g/mL LPS and incubated together for 24h. After the reaction, 50. Mu.L of cell supernatant was collected and the expression of IL-6 gene in the cells was detected by using IL-6ELISA kit.

As shown in FIG. 2, acetyl dipeptide-1 cetyl ester has a significant inhibitory effect on the inflammatory factor IL-6, and the IL-6 level is 38.25 times of the basal level under the stimulation of LPS at 10. Mu.g/mL. Under the action of acetyl dipeptide-1 cetyl ester with the concentrations of 0.0125, 0.025, 0.05 and 0.1mmol/L respectively, the level of IL-6 factor is obviously reduced and is respectively 30.12 times, 22.51 times, 15.48 times and 10.08 times of the basic level, which proves that the anti-inflammatory and soothing agent has good anti-inflammatory and soothing effects and has certain potential in other skin diseases caused by inflammatory factors.

Example 3

Detection of wound healing of acetyl dipeptide-1 cetyl ester by cell scratching method

Cell scratch assay is an in vitro assay to study cell migration. When the keratinocytes grow to be fused into a single-layer state, a blank area (scratch) is artificially manufactured on the fused single-layer cells, the cells at the edge of the scratch gradually enter the blank area to heal the scratch, the migration process of the epidermal keratinocytes is simulated to a certain extent, and the migration capability of the cells is judged by observing the cell states of the scratch areas at different periods, so that the method is an important method in an in-vitro experiment for researching the healing and repairing of skin wounds.

The operation method comprises the following steps:

1. the plates were streaked. Firstly, a Marker pen is used on the back of a 6-hole plate, a straight ruler is used for uniformly drawing transverse lines which are about every 0.5-1 cm and cross through holes, each hole at least penetrates through 5 lines, and the lines are not too thick when drawing lines.

2. And (5) cell spreading. About 5X 10 additions to the wells ⁵ And (3) inoculating each cell (the number of different cells is different and is adjusted according to the growth speed of the cells), wherein the inoculation principle is that the fusion rate reaches 100 percent after the overnight inoculation.

3. And (4) cell lineation. The next day, the cell layer was scored with a tip perpendicular to the cell plane along the line drawn on the back of the plate on the first day (preferably the same tip is used between different wells).

4. The cells were washed. After the scoring was completed, the cells were washed 3 times with sterile PBS, the nonadherent cells, i.e., the scored cells during the scoring, were washed away, the gap left after scoring was clearly visible, and then fresh serum-free medium was replaced.

5. And (5) culturing and observing cells. Diluting the sample (acetyl dipeptide-1 cetyl ester) with culture medium, adding to a cell culture dish, placing the cells at 37 deg.C, 5wt% CO ₂ The cells were cultured in an incubator, and then taken out at 48h, observed on a microscope line and measured for the width of the scratch, and photographed, and the healing rate was calculated with Image J software.

The results are shown in fig. 3, the scratch width of the experimental group is narrower than that of the solvent control group, which indicates that acetyl dipeptide-1 cetyl ester is effective in promoting keratinocyte healing; the calculated healing rate after 48 hours is 28.7%, and the acetyl dipeptide-1 cetyl ester has the capacity of enhancing the skin barrier and promoting wound healing.

Example 4

Preparation of acetyl dipeptide-1 cetyl esters composition

The composition of the composition is: 0.6wt% acetyl dipeptide-1 cetyl ester, 3wt% isononyl isononanoate, 0.15wt% carbomer 941, 0.1wt% xanthan gum, 3wt% cetostearyl alcohol, 3wt% glycerol, 1.2wt% WL912, 0.2wt% EG, 0.5wt% PHCH, 0.1wt% paraben, 0.1wt% triethanolamine, and the balance water.

The amount of the above components may be appropriately varied, for example, acetyl dipeptide-1 cetyl ester in an amount of 0.1 to 3wt%, isononyl isononanoate in an amount of 4 to 6wt%, carbomer in an amount of 0.1 to 0.3wt%, xanthan gum in an amount of 0.05 to 0.2wt%, cetostearyl alcohol in an amount of 1.85 to 4.5wt%, glycerin in an amount of 2 to 5wt%, WL912 in an amount of 0.9 to 2.6wt%, EG in an amount of 0.1 to 0.4wt%, PHCH in an amount of 0.25 to 1.5wt%, paraben in an amount of 0.05 to 0.5wt%, triethanolamine in an amount of 0.05 to 0.2wt% are possible.

The composition was prepared according to the following procedure:

s1, adding carbomer, glycerol, xanthan gum, p-hydroxybenzoate and deionized water into an emulsifying pot, and heating to 80-85 ℃ for dissolution;

s2, adding isononyl isononanoate, WL912 and cetostearyl alcohol into an oil phase pot, heating to 80-85 ℃ for dissolving, then adding into an emulsifying pot for homogenizing for 3-5 min, preserving heat, stirring for 30min, and cooling;

s3, cooling to 70 ℃, adding EG, homogenizing for 2min, continuously stirring, cooling to 50 ℃, adding triethanolamine, and uniformly stirring;

and S4, cooling to 45 ℃, adding PHCH, uniformly stirring, cooling to 40 ℃, adding acetyl dipeptide-1 cetyl ester, uniformly stirring, and discharging after the test is qualified.

Example 5

Method for detecting soothing effect of acetyl dipeptide-1 cetyl ester composition by lactic acid stimulation experiment

The operation method comprises the following steps: at room temperature, 50. Mu.L of a 5wt% aqueous lactic acid solution and 0.1g of acetyl dipeptide-1 cetyl ester composition were applied to the nasolabial sulcus and either one side of the cheek of a subject (30 persons in total), and the subjective symptoms of the subject were asked at 0, 2.5, 5 and 8min using a 5wt% aqueous solution of lactic acid-containing PBS (Phosphate Buffered Saline, a Phosphate buffer solution, main components of which are Na HPO, KH PO, naCl and KCl) as a control, and the score was calculated by 4-point method (0: no tingling sensation, 1: mild tingling, 2: moderate tingling, 3: severe tingling) and the average score was calculated. When the skin barrier is damaged, lactic acid enters the skin and stimulates the unmyelinated class C nerves, thereby producing a tingling sensation.

The results are shown in FIG. 4, where the acetyl dipeptide-1 cetyl ester composition had significantly reduced lactic acid stinging compared to the PBS control.

Example 6

Detection of soothing efficacy of acetyl dipeptide-1 cetyl ester compositions using capsaicin sting assay

The operation method comprises the following steps: at room temperature, 50. Mu.L of a capsaicin solution 31.6mg/kg and 0.1g of acetyl dipeptide-1 cetyl ester composition were applied to the nasolabial sulcus and either cheek of the subjects (30 persons in total), and the subjects were asked about subjective symptoms at 15 minutes using a PBS aqueous solution containing capsaicin 31.6mg/kg as a control, and scored by 4 points (0 point no sensation, 1 point mild discomfort, 2 point moderate discomfort, and 3 point severe discomfort), and the average points were calculated.

As shown in FIG. 5, the acetyl dipeptide-1 cetyl ester composition significantly reduced the discomfort of capsaicin and relieved the irritating effects of capsaicin on the skin compared to the blank control.

Example 7

Sodium dodecyl sulfate (SLS) stimulation experiments were used to test the soothing efficacy of acetyl dipeptide-1 cetyl ester compositions

The operation method comprises the following steps: placing 5wt% of SLS and 0.1g of acetyl dipeptide-1 cetyl ester composition in a plaque test device, partially encapsulating it in a test site (inner forearm side) (30 persons each of control group and test group), removing the plaque test device after 24 hours and observing the skin change of the test site at a specified time after removing the plaque test device, and scoring by 4 points (0 points as no tingling sensation, 1 points as mild stinging, 2 points as moderate stinging, and 3 points as severe stinging), and calculating the average point.

The results are shown in FIG. 6, where the acetyl dipeptide-1 cetyl ester composition significantly reduced the discomfort associated with SLS and alleviated the skin irritation associated with SLS as compared to the blank.

Example 8

Detection of skin Water content, TEWL, and skin oil secretion of acetyl dipeptide-1 cetyl esters composition

The operation method comprises the following steps: selecting 3cm part of nasal wing of the subject (30 persons in total) as test part with test area of (5 × 5) cm ² The test site was coated with acetyl dipeptide-1 cetyl esters composition and the other side of the nasal wing was not coated with any product as a control. Applying acetyl dipeptide-1 cetyl ester composition in the same test area in the morning and evening of 20 days, uniformly cleaning the test part of the subject at the same time every day, sitting still for 30min in a constant temperature and humidity environment (temperature 20 +/-2 ℃ and relative humidity 50 +/-10%), and measuring the water content of the skin, the water loss rate TEWL of the test skin and the oil content of the skin. Each test site in the experimental and control groups was measured for 20 seconds and averaged.

Figure 7 is a bar graph of the results of the skin moisture test of example 8. The skin moisture of the control group did not change significantly over the test period. The acetyl dipeptide-1 cetyl esters compositions increased the skin moisture content by 4.8%,8.2%,11.7%,17.9% and 20.1% at days 1, 5, 10, 15 and 20 after application, respectively, compared to application. The acetyl dipeptide-1 cetyl ester composition has obviously enhanced water content of skin stratum corneum and moisturizing effect.

Figure 8 is a bar graph of the results of the test for transdermal water loss of example 8. There was no significant change in the transdermal water loss of the control group over the test period. The skin moisture content of the acetyl dipeptide-1 cetyl ester composition is respectively reduced by 1.3%,3.4%,6.4% and 13.2% on 5 th, 10 th, 15 th and 20 th days after the application. After the acetyl dipeptide-1 cetyl ester composition is used, the water loss in the skin is obviously reduced, and the skin barrier is protected.

FIG. 9 is a bar graph of the results of the skin oil test of example 8. The skin oil content of the control group was 40.0. Mu.g/cm on days 1, 5, 10, 15 and 20 of the test, respectively ² ,41.2μg/cm ² ,44.2μg/cm ² ,40.2μg/cm ² ,43.5μg/cm ² (ii) a The skin oil content of acetyl dipeptide-1 cetyl ester composition on 5 days, 10 days, 15 days and 20 days after application is 40.0 μ g/cm ² ,38.7μg/cm ² ,39.6μg/cm ² ,35.1μg/cm ² ,32.8μg/cm ² . Skin oil secretion is reduced after the acetyl dipeptide-1 cetyl ester composition is used.

Example 9

0.5% acetyl dipeptide-1 cetyl ester is dissolved in 25mL deionized water, added into a 100mL beaker, then added with the cross-linking agent aluminum chloride, the preservative ascorbic acid, ammonia water, urea and glucose in sequence, stirred for 5min, slowly added with 0.5g sodium carboxymethylcellulose, and stirred vigorously to be dissolved completely, thus obtaining the hydrogel.

BALB/C mice, fasted for 4h, were deprived of back hair after anesthesia. Using a skin sampler with the diameter of 6mm to make two full cortex incisions on two sides of the median line of the ridge; a silicone splint having an inner diameter of 1cm and a thickness of 1mm was fixed around the wound.

Mice were randomly divided into a blank control group and an administration group, each group was administered with 100 μ L of gel, changed every 2 days, and wound sizes were recorded on days 0, 4, 8, and 14. The wound area was calculated using Image J, and the rate of healing was calculated from the wound area.

The results are shown in fig. 10, on the 4 th day of wound healing, the wound healing rate of the blank group is 25%, the wound healing rate of the administration group is 36%, on the 8 th day of wound healing, the wound healing rate of the blank group is 43%, the wound healing rate of the administration group is 68%, on the 14 th day of wound healing, the wound healing rate of the blank group is 76%, and the wound healing rate of the administration group is about 95%, which indicates that acetyl dipeptide-1 cetyl ester can promote wound healing and remarkably improve the wound healing efficiency.

Example 10

Zebrafish embryo reactive oxygen scavenging assay

Wrinkle formation is associated with the inactivation of proteins, nucleic acids, lipids and other molecules by oxidation as a result of Reactive Oxygen Species (ROS). In daily life, oxidative stress damage of the skin may increase due to various reasons such as sunlight and compound stimulation, and the skin may abnormally age, thereby causing the skin to lose elasticity and generating wrinkles. The ROS regulation mechanism in the zebra fish body is the same as that of the human body. The specific active oxygen fluorescent staining reagent is used, ROS in zebra fish embryo cells can be efficiently and specifically marked, and the fluorescence intensity of the ROS in the zebra fish embryo cells is positively correlated with the ROS level in the cells. And (3) analyzing and quantitatively reading a green fluorescence signal enhanced along with the increase of ROS in the zebra fish embryos by using software, testing and comparing the ROS level change of the fish embryos of the treatment group and the blank control group, and calculating the ROS clearance so as to evaluate the anti-wrinkle and compact effects of the raw materials or the products.

The method comprises the following steps:

1. test grouping

Healthy zebrafish embryos 2 days after fertilization were selected. The test requires a blank control group (fish embryo culture solution), a positive control group (glutathione working solution) and a test object group (acetyl dipeptide-2 cetyl ester). If organic solvent is used for assisting dissolution, the concentration of the solvent in each group of solution needs to be the same.

2. Blank reference group setting

Fish embryo culture solution: 2940mg of anhydrous calcium chloride, 1233mg of magnesium sulfate heptahydrate, 630mg of sodium bicarbonate were weighed, 55mg of potassium chloride is dissolved in 10L of water to prepare the potassium chloride, and the pH value is 6.5-8.5.

Randomly selected 24 tail fish embryos, transferred to 96-well plates, each well containing one tail fish embryo and 0.2mL fish embryo culture fluid.

3. Positive control set

Glutathione solution: 1g of glutathione powder was weighed and dissolved in 10mL of water to prepare 100g/L stock solution. The working solution is prepared fresh before use by diluting the stock solution to 0.1g/L with fish embryo culture solution.

Randomly selecting 24 tail fish embryos, transferring the embryos to a 96-well plate, wherein each well contains one tail fish embryo and 0.2mL of glutathione working solution.

4. Treatment of test objects

The test solution was prepared by directly dissolving the test substance (acetyl dipeptide-2 cetyl ester) in a fish embryo culture solution.

Randomly selecting 24 tail fish embryos, transferring the embryos to a 96-well plate, wherein each well contains one tail fish embryo and 0.2mL of test substance solution, and the concentrations of the test substance solutions are respectively 5% of formula addition concentration (0.25 g/L), 0.7% of formula addition concentration (0.035 g/L) and 0.1% of formula addition concentration (0.005 g/L).

5. Post-treatment

And placing the blank control group, the positive control group and the test object group in a thermostat with the temperature of 28 +/-1 ℃ for culturing until 72 +/-1 h is obtained after fertilization. Transferred to a 24-well plate containing 12 fish embryos per well and 2mL of Reactive Oxygen Species (ROS) test solution, stained with H2DCFDA, and placed in an incubator at 28 + -1 deg.C for 2 + -1H.

6. Microscopic analysis

Placing the fish embryo on one side. And (4) taking a picture of the fish embryo under a fluorescence stereomicroscope according to the uniform shooting parameters.

7. Data and result calculation

The photograph is opened with analytical software such as Image J, the yolk area of each fish embryo is labeled, then the "measured average intensity" in the "measured" column is selected, and the "average signal intensity" in the test results is taken as the ROS signal intensity.

8. Calculation results

Calculating Reactive Oxygen Species (ROS) clearance: clearance = (C-T)/C × 100%.

T is the average value of ROS (reactive oxygen species) average signal intensity of the test object treated group fish embryos; c is the average value of ROS 'average signal intensity' of fish embryos in the blank control group. And carrying out double-tail T test on the ROS signal intensity of the test object treated fish embryos and the ROS signal intensity of a blank control group to obtain a p value.

As shown in FIG. 11, the ROS clearance of zebrafish embryos at concentrations of 0.25g/L, 0.035g/L and 0.005g/L was respectively 27% (p = 0.0013), 32% (p = 0.00012) and 30% (p = 0.00031) for acetyl dipeptide-2 cetyl palmitate. The acetyl dipeptide-2 cetyl palmitate can obviously eliminate ROS in zebra fish embryos, and has the advantages of antioxidant effect, obvious anti-wrinkle and firming effects.

The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are also within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims (9)

1. Application of acetyl dipeptide-1 cetyl ester in preparation of medicines, health products and cosmetics for promoting healing, resisting oxidation or repairing tissues.

2. A composition comprising acetyl dipeptide-1 cetyl ester, said composition having healing promoting, antioxidant or tissue repair benefits.

3. The composition of claim 2, wherein the composition further has at least one of skin elasticity enhancing, anti-inflammatory soothing, or moisturizing effects.

4. A composition of acetyl dipeptide-1 cetyl ester consisting of: 0.1 to 3 weight percent of acetyl dipeptide-1 cetyl ester, 4 to 6 weight percent of emollient, 2 to 5 weight percent of thickener, 2 to 5 weight percent of humectant, 1 to 3 weight percent of emulsifier, 0.3 to 2 weight percent of preservative, 0.05 to 0.2 weight percent of neutralizer and the balance of water.

5. The composition of claim 4 wherein said emollient is isononyl isononanoate; the thickening agent is carbomer, xanthan gum and cetostearyl alcohol; the humectant is glycerin; the emulsifier is WL912 and EG; the preservative is PHCH and p-hydroxybenzoate, and the neutralizing agent is triethanolamine.

6. The composition according to claim 5, characterized in that it consists of: 0.1 to 3 weight percent of acetyl dipeptide-1 cetyl ester, 4 to 6 weight percent of isononyl isononanoate, 0.1 to 0.3 weight percent of carbomer, 0.05 to 0.2 weight percent of xanthan gum, 1.85 to 4.5 weight percent of cetostearyl alcohol, 2 to 5 weight percent of glycerol, 0.9 to 2.6 weight percent of WL912, 0.1 to 0.4weight percent of EG, 0.25 to 1.5weight percent of PHCH, 0.05 to 0.5 weight percent of p-hydroxybenzoate, 0.05 to 0.2 weight percent of triethanolamine and the balance of deionized water.

7. A method of preparing the composition of claim 6, comprising the steps of:

s1, adding carbomer, glycerol, xanthan gum, p-hydroxybenzoate and deionized water into an emulsifying pot, and heating to 80-85 ℃ for dissolution;

s2, adding isononyl isononanoate, WL912 and cetostearyl alcohol into an oil phase pot, heating to 80-85 ℃ for dissolving, then adding into an emulsifying pot for homogenizing for 3-5 min, preserving heat, stirring for 20-30 min, and cooling;

s3, cooling to 65-70 ℃, adding EG for homogenizing for 2-5 min, continuously stirring, cooling to 45-50 ℃, adding triethanolamine and stirring uniformly;

and S4, cooling to 40-45 ℃, adding PHCH, uniformly stirring, cooling to 35-40 ℃, adding acetyl dipeptide-1 cetyl ester, uniformly stirring, testing to be qualified, and discharging.

8. Use of a composition of acetyl dipeptide-1 cetyl ester according to any of claims 4 to 6 for promoting healing, antioxidation or tissue repair.

9. Use of a composition of acetyl dipeptide-1 cetyl ester according to any of claims 4 to 6 for enhancing skin elasticity, anti-inflammatory soothing or moisturizing.

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