LU502320B1 - Acetylated and amidated hexapeptide, purification method and application thereof - Google Patents

Abstract

The invention discloses an acetylated and amidated hexapeptide, a purification method and an application thereof. An amino acid sequence of the acetylated and amidated hexapeptide is shown as follows: Ac-Gly-Met-Cys-Cys-Ser-Arg-NH2, abbreviated as Ac-GMCCSR-NH2, and having a molecular weight of 696.86 Da. A polypeptide of the present invention is synthesized by a solid-phase synthesis method, and the acetylated and amidated hexapeptide of the present invention has enhanced activity of resisting skin photoaging, and can be applied to the fields of foods, biopharmaceuticals, cosmetics and the like.

Description

ACETYLATED AND AMIDATED HEXAPEPTIDE, PURIFICATION METHOD ANDLU502320

APPLICATION THEREOF Technical Field The invention belongs to the field of biotechnology, and particularly relates to an acetylated and amidated hexapeptide, a purification method and an application thereof. Background With the continuous change of the earth's environment, the atmospheric ozone layer has also been severely damaged, resulting in an increase in the intensity of ultraviolet (UV) radiation.

Meanwhile, opportunities for outdoor sports and sunbathing are gradually increasing along with changes in people's lifestyles; people will thus suffer more ultraviolet radiation, which will cause a gradual increase in photo-damaging skin diseases. Acute damage from sunburn and chronic accumulative damage from photoaging, skin cancer, etc. to the skin caused by ultraviolet radiation pose a potential threat to human health.

Nowadays, the problem of skin aging has attracted more and more attention. Therefore, the development of components or products that can protect the skin from light damage, prevent and delay skin aging has become a hot spot of current medical research.

Small molecular peptides are a class of highly active peptide substances. Small molecular peptides may be absorbed by the body in a complete form; they have the characteristics of active absorption, low energy consumption or no energy consumption; and they can directly enter the blood circulation and be sent to various parts of the human body, and widely used in the field of biomedicine. Due to hydrophilic nature of proteins, it is often difficult for them to be transported across biological membrane; therefore, improving their membrane permeability and improving their cellular internalization rates are important scientific issues. It is also one of the research directions for those skilled in the art to improve the performance of small molecular peptides by modifying them. Wherein, N-terminal acetylation and C-terminal amidation of polypeptides are main forms of their structural modification.

Summary In order to solve the problems existing in the prior art, the present invention provides an acetylated and amidated hexapeptide, which has good antioxidation activity and activity of resisting skin photoaging, and can prevent or reduce skin damage caused by UVB.

In order to achieve its purpose, the technical solution adopted by the present invention is as follows:

An acetylated and amidated hexapeptide is named as Ac-GMCCSR-NH», and has LAJ502320 molecular weight of 696.86 Da, and its chemical structural formula is as follows: x He SH EM PR Wherein, the abbreviation Ac represents that the English name is Acetyl, and the Chinese name is acetyl; NH» represents that the English name is Amide group, and the Chinese name is amide group; Gly represents a corresponding residue of an amino acid whose English name is Glycine and the Chinese name is glycine; Met represents a corresponding residue of an amino acid whose English name is Methionine and Chinese name is methionine; Cys represents a corresponding residue of an amino acid whose English name is Cysteine and Chinese name is cysteine; Ser represents a corresponding residue of an amino acid whose English name is Serine and Chinese name is serine; and Arg represents a corresponding residue of an amino acid whose English name is Arginine and Chinese name is arginine.

The present invention also provides a purification method for the above-mentioned acetylated and amidated hexapeptide, which comprises the following steps: loading the acetylated and amidated hexapeptide to be purified (the crude peptide of the synthesized acetylated and amidated hexapeptide) to a chromatographic column; and performing a gradient elution with a mobile phase A and a mobile phase B as eluents, wherein the mobile phase A is an acetonitrile solution containing 0.08%~0.12% (volume) of trifluoroacetic acid, and the mobile phase B is water containing 0.08%~0.12% (volume) of trifluoroacetic acid; a procedure for the gradient elution is that: a sum of volume proportions of the mobile phase A and the mobile phase B is 100%, an initial volume proportion of the mobile phase A is 8%~12%, the volume proportion of the mobile phase A rises to 30%~40% from 0.01 min to 25 min after loading, the volume proportion of the mobile phase A rises to 100% within 25 min to 25.1 min after loading, the volume proportion of the mobile phase A is kept at 100% till the end of an operation at 30 min, and a detection wavelength is 220 nm; and a polypeptide solution at a target peak is collected.

Further, the chromatographic column is a Cig chromatographic column.

Preferably, the mobile phase A is an acetonitrile solution containing 0.1% (volume) of trifluoroacetic acid, and the mobile phase B is water containing 0.1% (volume) of trifluoroaceti¢J502320 acid; during the gradient elution, the initial volume proportion of the mobile phase A is 10%, and the volume proportion of the mobile phase A rises to 35% from 0.01 min to 25 min after loading, The present invention also provides an application of the acetylated and amidated hexapeptide, which can be used for preparing a skin care product that resists skin photoaging or has skin moisturizing effect.

The present invention also provides an application of the acetylated and amidated hexapeptide in preparing a preparation for preventing or repairing a photodamage of Hacat cells caused by UVB.

The present invention also provides an application of the acetylated and amidated hexapeptide in preparing an antioxidation preparation.

The present invention also provides an application of the acetylated and amidated hexapeptide in preparing a preparation for preventing or reducing skin damage caused by UVB radiation.

The present invention also provides a composition comprising the acetylated and amidated hexapeptide as described above.

Further, the composition is cosmetic, food or medicine.

The present invention adopts methods such as antioxidation chemical model (ABTS free radical), human epidermal immortalized cell (Hacat) and its UVB-aged model, Kunming mice and its back skin UVB-aged model, etc., to test an anti-oxidative activity and activity of resisting skin photoaging of the acetylated and amidated hexapeptide of the present invention. Experiments have confirmed that the acetylated and amidated hexapeptide of the present invention has stronger antioxidation activity, and the protective effect on UVB-aged Hacat cells thereof 1s significantly stronger than that of the hexapeptide before modification. The acetylated and amidated hexapeptide of the present invention has a potential to be applied in cosmetics for resisting skin photoaging, which can not only prevent or reduce skin damage caused by UVB, maintain skin moisture, reduce MDA content in skin, and enhance SOD activity, CAT activity and GSH-Px activity in the skin tissue, but also decrease expression quantities of MMP-1 and MMP-3 in a cytoplasmic matrix.

The synthetic polypeptide acetylated and amidated hexapeptide with antioxidation activity and activity of resisting skin photoaging provided by the invention can be applied to the fields of foods, biopharmaceuticals, cosmetics and the like.

Description of Drawings

Fig. 1 is a HPLC chart of identifying purity for the acetylated and amidated hexapeptid&J502320 (Ac-GMCCSR-NHz).

Fig 2 is an ESI-MS spectrum of the acetylated and amidated hexapeptide (Ac-GMCCSR-NHz). Wherein, the abscissa is m/z (mass-to-charge ratio), and the ordinate is intensity (signal intensity).

In Fig. 3, A-D are macroscopic characterization images of the mouse back skin; A1-D1 are the examination results for histopathological sections of the skin tissue (200 x). Wherein, A and A1 are normal control groups only applied with a solvent; B and B1 are UVB-aged model groups; C and C; are protection groups added with positive controls; and D and Di are protection groups added with the acetylated and amidated hexapeptide.

FIG. 4 is a graph for determining an epidermal thickness of the skin. Note: * represents a significant difference from the normal group; and # represents a significant difference from the model group.

FIG. 5 is a graph for determining moisture content in the skin. Note: * represents a significant difference compared with the normal group; and # represents a significant difference compared with the model group.

Fig. 6 is a graph for determining MDA content in the skin tissue. Note: * represents a significant difference from the normal group; and # represents a significant difference from the model group.

Fig. 7 is a graph for determining SOD activity in the skin tissue. Note: * represents a significant difference from the normal group; and # represents a significant difference from the model group.

Fig. 8 is a graph for determining CAT activity in the skin tissue. Note: * represents a significant difference from the normal group; and # represents a significant difference from the model group.

Fig. 9 is a graph for determining GSH-Px activity in the skin tissue. Note: * represents a significant difference from the normal group; and # represents a significant difference from the model group.

Fig. 10 is a graph for determining MMP-1 content in the skin tissue. Note: * represents a significant difference from the normal group; and # represents a significant difference from the model group.

Fig. 11 is a graph for determining MMP-3 content in the skin tissue. Note: # represents a significant difference from the model group.

Detailed Description of Embodiments LU502320 The technical solutions of the present invention will be further described below in combination with specific examples and drawings, but the implementation and the protection scope of the present invention are not limited thereto.

5 The cells or reagents used in the following examples are commercially available, unless otherwise specified; and the experimental operations that are not specifically described in the examples are all routine experimental operations in this field.

Ac-GMCCSR-NHz is synthesized by a polypeptide solid-phase synthesis method, and a synthesis of the polypeptide can be specifically completed by a commercial biosynthetic company through using conventional polypeptide solid-phase synthesis methods in this field. An amino acid sequence adopts a conventional standard Fmoc scheme in this field, which is introduced below for reference. Polypeptide solid-phase synthesis Rink Amide resin (Shanghai SynPeptide Co., Ltd.) is selected. Since Rink Amide resin has itself amino groups, it can directly react with amino acids to form stable amide bonds. According to the characteristics of the amino acid sequence Ac-Gly-Met-Cys-Cys-Ser-Arg-NH», the carboxyl group of Arg is firstly connected with one resin in the form of a covalent bond, and then the amino group of Arg and the carboxyl group of Ser undergo a dehydration reaction. After a treatment, Cys is added, the amino group of Ser reacts with the carboxyl group of Cys, and amino acids are added from right to left in turn. After adding the last Gly amino acid, the resin is cleaved to obtain the amidated hexapeptide Ac-GMCCSR-NH,. After the synthesis of the amino acid chain is completed, the activated acetic acid reacts with the N-terminal amino group of the peptide chain under the catalysis of acetic anhydride and pyridine, and an acetylation reaction is carried out to obtain an acetylated and amidated hexapeptide. Purification of acetylated and amidated hexapeptide The acetylated and amidated hexapeptide Ac-GMCCSR-NH; synthesized by polypeptide solid-phase synthesis is purified to obtain a product with a purity of 95% or more. The present invention performs purification according to the following method: the acetylated and amidated hexapeptide Ac-GMCCSR-NHz is purified by a high-performance liquid chromatography, and the Ac-GMCCSR-NH; crude peptide is purified by a gradient elution through a kromasil Cig-5 (4.6x250 mm) chromatographic column, with a flow rate of 1.0 mL/min. A solvent A is taken as a mobile phase A, and a solvent B is taken as a mobile phase B, wherein the mobile phase A is an acetonitrile solution containing 0.1% (volume) of trifluoroacetic acid; the solvent B is water containing 0.1% (volume) of trifluoroacetic acid. The gradient elution is as follows: a total volume of A+B is 100%, an initial volume proportion of A is 10%, the proportion of A rises t&J502320 35% from 0.01 min to 25 min after loading, the proportion of A rises to 100% from 25 min to

25.1 min after loading, the proportion of A is kept at 100% till the end of an operation at 30 min, and a detection wavelength is 220 nm. A polypeptide solution at a target peak is collected, quickly cooled in liquid nitrogen, and then lyophilized. A product with a purity of 95% or more is obtained. The HPLC result of identifying the purity is seen in Fig. 1, and a structure thereof is identified by ESI-MS (as shown in Fig. 2). After the identification, its structural formula is: 2 ahi

NG Introduction to evaluation method for antioxidative activity of polypeptide Determination of ABTS free radical scavenging activity: ABTS solution of 5 mmol/L is formulated with PBS (pH 7.4), excess MnO is added to prepare free radicals, and the solution is placed at 30 °C for 12 h, and then centrifuged at a condition of 4500 r/ min for 10 min. A supernatant is collected and filtered through a 0.2 um nylon membrane. A filtrate is stored at -20 °C for use as an ABTS" stock solution. The ABTS" stock solution needs to be diluted to a desired concentration with PBS solution prior to use in the assay. A 96-well plate is used to determine the ABTS free radical scavenging activity: adding 20 uL of water + 180 pL of ABTS solution, and performing a full-wave scanning to determine that the maximum absorption wavelength is 736 nm (microplate reader program: shaking for 30 s, determining wavelength range of 500-800 nm). When testing the ABTS free radical scavenging activity of a sample, an absorbance value of a system is adjusted to 0.70 + 0.02 to obtain a working solution. Determination of sample: adding 20 uL of samples of different concentrations (1-100 pg/mL) and 180 uL of ABTS working solution. The absorbance value of the sample is recorded as Asample, the absorbance value of the model group (20 uL of distilled water + 180 uL of ABTS working solution) is recorded as Amodet, and the blank group is set with the absorbance value being recorded as Aplank. A ABTS free radical scavenging rate 1s calculated according to the following formula: scavenging rate for ABTS free radical = (Amodel - Asample)/(Amodel - Ablank)

Example 1 Test of scavenging activity for ABTS free radical LU502320 The ABTS free radical scavenging activities of the acetylated and amidated hexapeptides and hexapeptides are evaluated with reference to the above-described method. The ABTS free radical scavenging activity is determined by a 96-well plate: a detection wavelength is set at 736 nm, a reaction system is 20 pL of water + 180 uL of ABTS solution, when testing the ABTS free radical scavenging activities of hexapeptide and acetylated and amidated hexapeptide, an Absorbance value of the system 1s adjusted to 0.70 + 0.02 to obtain ABTS working solution. 20 pL of a sample with a concentration of 1-100 pg/mL and 180 pL of ABTS working solution are added, data is collected every 5 min with a total of 40 min for reaction. After the reaction is ended, the ABTS free radical scavenging rate is calculated according to the above formula. In addition, a positive control group is also set up with vitamin C as a test sample. After testing, the half-maximal scavenging concentration (ICso value) of the hexapeptide GMCCSR on ABTS free radical is 16.94 uM, the ICso value of the acetylated and amidated hexapeptide (Ac-GMCCSR-NHz) on ABTS free radical is 17.82 uM, while the ICs value of the positive control vitamin C is 26.18 uM. The results show that the acetylated and amidated hexapeptide (Ac-GMCCSR-NH») has the same strong antioxidation activity as the hexapeptide GMCCSR, and is stronger than the positive control, which has a potential to be used in food, medicine and cosmetic.

Example 2 The following operation is performed: taking a bottle of human epidermal immortalized cells Hacat, digesting it with trypsin containing 0.25% EDTA, centrifuging, suspending the cells in a complete medium, counting with a hemocytometer, formulating a cell suspension with a concentration of 5x10* cells/mL, taking a 96-well plate, and dividing 100 uL to each well, that is, 5000 cells per well. The same is incubated for 48 h at 37 °C in a saturated humidity incubator with a volume fraction of CO; being 5%. At the same time, a positive control pentapeptide (Matrixyl, Palm-KTTKS), the acetylated and amidated hexapolypeptide solution (Ac-GMCCSR-NHz), and the hexapolypeptide solution (GMCCSR) are formulated at a concentration of 100 pg/mL, and 200 pL of sample solutions (for normal control group, replaced with a complete medium) are added to each well, the same is incubated for 48 h, the samples are aspirated and discarded, washed once with PBS, and a survival rate of the cell is detected by MTT method. The experimental results are shown in Table 1. Table 1 Toxicity of acetylated and amidated hexapeptide to Hacat

Sample Cell Survival rate (%) Significance Ranking U502320 © Hexapeptide ~~ 99.I8+697 a Acetylated and amidated hexapeptide 81.52+6.26 b Positive control pentapeptide 5.32+1.83 Cc Note: The lowercase letters a and b indicate the significant difference between survival rates of the cell in different samples at the same concentration, and are arranged in descending order of the alphabet, P<0.05 between adjacent letters, with the difference being significant.

It can be seen from Table 1 that the toxicities of the acetylated and amidated hexapeptide and the hexapeptide to Hacat cells are significantly lower than that of the positive control pentapeptide. Among the synthetic polypeptides, the survival rate of Hacat cells is over 80% after the action of the acetylated and amidated hexapeptide. Therefore, the protective effect of the test sample on Hacat cells can be considered under the condition lower than that concentration.

Example 3 Protective effect of acetylated and amidated hexapeptide on UVB-aged Hacat cells In order to explore the protective effect of polypeptide on UVB-aged Hacat, a UVB-aged Hacat model is established, and a UVB-aged group and a non-UVB normal group are instituted.

Based on the set UVB aging intensity, the optimal UVB dose is determined by determining the survival rate of Hacat via MTT method and the apoptosis rate of cells by means of a flow cytometer. The results show that when the UVB radiation intensity is 35 mJ/cm?, the survival rate of Hacat cells in the model group is 47.56+6.40% of that in the normal group, with a significant difference, approaching half the survival rate. At the same time, the results of apoptosis determined by the flow cytometer show that the apoptosis rate of the model group is significantly higher than that of the Hacat cells in the normal group; and the proportion of the cells in Gi stage in the model group is significantly smaller than that of in the normal group, and the proportion of the cells in S stage in the model group is significantly higher than that of in the normal group. Therefore, UVB can retard the growth of Hacat cells at S stage. The results show that the ultraviolet rays UVB-aged Hacat model is successfully established.

Based on the above-described UVB-aged Hacat model, the protective effect of the acetylated and amidated hexapeptide on Hacat in the concentration range of 1-50 ug/mL is tested and compared with the hexapeptide and the positive control pentapeptide. The specific experimental results are shown in Table 2.

Table 2 Protective effect of acetylated and amidated hexapeptide on Hacat

© Sample Cell Survival rate (%) Significance Ranking LU502320 © Hexapeptide = 11297¢1073 b Acetylated and amidated hexapeptide 137.14+£8 42 a Positive control pentapeptide 121.56+1.42 b “Note: lowercase letters a and b indicate significant differences in cell survival rate between different samples, and are arranged in descending order of the alphabet, P<0.05 between adjacent letters, with the difference 1s significant.

It can be seen from Table 2 that the protective effect of the acetylated and amidated hexapeptide on UVB-aged Hacat is significantly stronger than those of the hexapeptide and the pentapeptide. It can be seen that the acetylated and amidated hexapeptide has a potential to be applied in cosmetics, and can effectively protect UVB-aged human epidermal immortalized cells.

Example 4 56 SPF grade Kunming mice (or KM mice), 7-8 weeks old, all female, weighing 25 g, are selected and divided into 7 groups. These mice are purchased from Laboratory Animal Center of Southern Medical University, Guangzhou, with a production license: SCXK (Guangdong) 2011-0015, of which animal quality certificate is 44002100005930.

Breeding environment is as follows: temperature 23 + 2 °C, humidity 55 + 10%, alternation with 12 h illumination and 12 h darkness, without any UV irradiation, and food and water freely supplied. Mice are acclimated to the environment for 1 week before an experiment. Then, random grouping is performed.

56 KM mice are randomly divided into the following 4 groups: A is a normal control group (Normal control, NC), B is a model control group (Model control, MC), C is a positive control group (pentapeptide Palm-KTTKS, Matrixyl), and D is a acetylated and amidated hexapeptide (Ac-GMCCSR-NHz) prevention group, a total of 4 groups, 8 in each group. Each group is divided into two cages; and the mice are normally fed under the same condition, and they are allowed to be acclimated to the environment for one week before subsequent experiments are carried out.

For the first time, the hair in area of 2.5 x 3 cm? on the back of all mice is cut off with a electric clipper, and the short hair is gently shaved with an automatic lady shaver. Before the formal experiment, the mice are acclimated to the environment for two days. From the first week to the tenth week, the dorsal skin of the mice is administrated and UVB treated for ten weeks.

Before each treatment, depilation is performed. The specific treatment is operated as follows: after shaving, the normal group is administrated 100 pL of the solvent, the model group is administrated 100 uL of the solvent, and the positive control group and the acetylated atd/502320 amidated hexapeptide group are administrated 100 uL (10 mg/mL) of the sample respectively; after the administration, the mice are given enough space to move alone for 2 h, and the samples are allowed to permeate and be absorbed. Subsequently, the model group, the positive control group and the acetylated and amidated hexapeptide group are all irradiated with 60 mJ/cm? of UVB. During the experiment, if erythema, blisters and erosions appear, the irradiation is stopped immediately for 2-3 d; and the experiment is continued after the symptoms disappear. A frequency of the administration in each experimental group is three times one week. At the end of the 10th week, the mice in each group are carefully depilated on the back (with a area of 2.5x3 cm”), photographed, and killed by cervical dislocation. The full-layer skin at the experimental site on their back is quickly taken down, and the connective tissue and subcutaneous fat are stripped off, 1.0 cmx1.0 cm of tissue is quickly cut and fixed in 4% paraformaldehyde for two days or more. Subsequently, the fixed skin tissue is taken out, rinsed with a running water overnight, embedded in paraffin, sectioned, stained, and photographed.

After obtaining HE-stained photos under a 200x field of view, 10 fields of view are randomly selected, and an average value is taken to estimate an average thickness of the mouse skin epidermis represented by this sample. An image analysis software of Image Pro Plus 6.0 is used to analyze the thickness of the skin epidermis of the test mice. The experimental results are shown in Figs. 3-4.

The test for moisture content in skin is determined by a method of drying the skin tissue to a constant weight. The mice are killed by cervical dislocation, the full-layer skin at the experimental site on their back is quickly taken down, and the connective tissue and subcutaneous fat are stripped off. About 0.2 g of the skin is quickly cut off, and its wet weight is accurately weighed. It is quickly placed in an oven and dried to constant weight at 80 °C.

Moisture content in skin is calculated according to formula (1): Moisture content in skin (%) = (wet weight - dry weight) / wet weight x 100 (1) The experimental results are shown in Fig. 5.

At the same time, about 0.5 g of skin is quickly cut and obtained, rinsed twice with pre-cooled normal saline, wiped dry, weighed, and quickly put into a pre-cooled EP tube. A 10% skin tissue homogenate is formulated with 0.9% normal saline. The homogenate is applied on a smear for microscopy, when no complete cells are seen in more than 3 random fields of view, the homogenization is stopped. After being centrifuged, a centrifugate is ready for use after the supernatant being taken away. Protein content is determined by means of BCA kit, followed by malondialdehyde (MDA) kit, superoxide dismutase (SOD) kit, catalase (CAT) kit and glutathione peroxidase (GSH) kit etc. to measure the content of each relevant MDA and th&J502320 activity of antioxidation-related enzyme in the tissue, and the operation is performed in strict accordance with the specification. The experimental results are seen in Figs. 6-9.

0.4 g of skin tissue is taken, added into 9 times of pre-cooled PBS, homogenized at a condition of 4 °C, homogenated at 10000 rpm, for 20 s, to obtain a 10% homogenate, and centrifuged at 3000 x g for 20 min at 4 °C. The obtained supernatant is used to evaluate the secreted MMP-1 and MMP-3, and the absorbance value at 450 nm is determined to obtain the contents of mouse matrix metalloproteinase MMP-1 and MMP-3 in the skin tissue. The specific determination method is performed in strict accordance with the enzyme linked Immunoassay (ELISA) kit specification, and the experimental results are shown in Figs. 10-11.

In Fig. 3, A-D are the macroscopic characterization images of the mouse dorsal skin; A1-D1 are the examination results of histopathological sections in the skin tissue (200%). Wherein, A and A; are normal control groups only applied with a solvent; B and B1 are UVB-aged model groups; C and Cı are protection groups added with positive control; D and D; are protection groups added with the acetylated and amidated hexapeptide.

It can be seen from Fig. 3 that the skin of the back of the mice in the normal group is smooth, ruddy, plump, and full of elasticity, without any slack phenomenon. At the same time, the H&E staining results show that the structure of each layer in the skin of the normal group is complete, the subcutaneous hair follicles and sebaceous glands are plump in shape and full filled, the epidermis is thin, the junction between the epidermis and the dermis is tight, the thickness of the dermis layer is moderate, the wave-like collagen fiber bundles are orderly arranged and evenly distributed, and no pathological changes are found. The surface of the back skin of the mice in the model group is dark, slack in appearance, and deep and thick in wrinkles, and local aging and necrosis phenomenon appear in the superficial cortex. It can be seen preliminarily that all the sample treatment groups have a certain degree of improvement in the skin of the mice compared with those in the model group. The results of H&E staining show that the epidermis in the model group appears phenomena of irregular thickening, fragmentation of cell nucleus, incomplete keratinization of partial epidermis, vacuolar degeneration of a small number of cells from basal layer cells, accompanied by infiltration of inflammatory cells (lymphocytes, monocytes), etc. Relative to the model group, the positive control pentapeptide protection group is improved in the skin color, and it is no longer so gray and dark. The glossiness and the plumpness of the skin are improved to different degrees; however, relatively obvious sunburns could still be seen. The H&E staining results show that in the positive control group, a small number of cells in the basal layer of the epidermis are vacuolated and degenerated, and the dermis layer appears moderate edema accompanied with infiltration of a small number bP502320 inflammatory cells (lymphocytes, monocytes). The local skin damage of the back skin of the mice in the acetylated and amidated hexapeptide protection group is reduced. Compared with the model group, the skin color is brighter, the epidermis is smoother, and no slackness as well as deep and thick wrinkles are found. The results show that the acetylated and amidated hexapeptide has a potential for application in cosmetics for resisting skin photoaging.

FIG. 4 is a graph for determining an epidermal thickness of the skin. Note: * represents a significant difference from the normal group; and # represents a significant difference from the model group.As can be seen from Fig. 4, relative to the normal group, the epidermal thickness of the UVB-treated mice in the model group is significantly increased; relative to the model group, the epidermal thicknesses of the mice in the positive control group and the acetylated and amidated hexapeptide-treated group are all significantly reduced. There is no significant difference between the acetylated and amidated hexapeptide-treated group and the normal group. The results show that the acetylated and amidated hexapeptide has a potential to be applied in cosmetics for resisting skin photoaging.

FIG. 5 is a graph for determining moisture content in the skin. Note: * represents a significant difference compared with the normal group; and # represents a significant difference compared with the model group. After the mice are slaughtered, a part of the whole layer of skin is taken and dried to a constant weight to calculate the moisture content in the skin. The results are seen in Fig. 5. It can be seen from the figured that there is a significant difference between the model group and the normal group. The moisture content in the skin layer of the model group is significantly lower than that of the normal group, and the acetylated and amidated hexapeptide group is significantly higher than the model group. After the acetylated and amidated hexapeptide is applied to the skin, it plays a role of maintaining the moisture content in the entire layer of skin.

Fig. 6 is a graph for determining MDA content in the skin tissue. As can be seen from the figure, the content of MDA in the model group is significantly higher than that in the normal group, and the content of MDA in the acetylated and amidated hexapeptide group 1s significantly lower than that in the model group. Due to the action of ultraviolet UVB, free radicals will be generated, which will damage the lipid layer of the skin and generate excess MDA. Because the acetylated and amidated hexapeptide has stronger antioxidation activity, it can scavenge free radicals in time, thereby reducing MDA content in the skin. The results show that the acetylated and amidated hexapeptide has a potential to be applied in cosmetics for resisting skin photoaging.

Fig. 7 is a graph for determining SOD activity in the skin tissue. It can be seen from th&J502320 figure that the SOD activities of tissues in the model group and all UVB-aged groups decrease significantly, and the SOD activity in the acetylated and amidated hexapeptide treatment group is significantly stronger than that in the model group. The results show that the acetylated and amidated hexapeptide has a potential to be applied in cosmetics for resisting skin photoaging.

Fig. 8 is a graph for determining CAT activity in the skin tissue. It can be seen from the figure that the CAT activities of the tissues of the UVB-aged model group and the positive control group decrease significantly. The CAT activity in the tissues of the acetylated and amidated hexapeptide group is significantly higher than that of the model group. The results show that the acetylated and amidated hexapeptide has a potential to be applied in cosmetics for resisting skin photoaging.

Fig. 9 is a graph for determining GSH-Px activity in the skin tissue. It can be seen from the figure that the GSH-Px activity of the model group is significantly lower than that in the normal group, and the GSH-Px activities in the skin tissues in the positive control pentapeptide and the acetylated and amidated hexapeptide group are significantly higher than that of the model group, indicating that the positive control pentapeptide as well as the acetylated and amidated hexapeptide can both resist skin photoaging by increasing GSH-Px activity in tissues.

Fig. 10 and Fig. 11 are graphs of determining the expression situation of MMP-1 and MMP-3 in the skin tissue, respectively. It can be seen from the figure that relative to the UVB-aged model group, the expression quantities of MMP-1 and MMP-3 in the acetylated and amidated hexapeptide group are significantly decreased. Therefore, the acetylated and amidated hexapeptide achieve its anti-photoaging effect by decreasing the expression quantities of MMP-1 and MMP-3 in the cytoplasmic matrix, and has a potential to be applied in cosmetics for resisting skin photoaging

Claims (10)

Claims LU502320

1. An acetylated and amidated hexapeptide, characterized in that, its chemical structural formula 1s as follows: ? O O O O O O A l OH l OH l OH l OH l OH Il N—CH-C—N—CH-C—N—CH-C—N—CH-C—N—CH-C—N-—CH-C—NH; H CH, CH, CH, CH, CH, CH, SH SH OH CH, ge CH3 NH G—NH NH,

2. A purification method for the acetylated and amidated hexapeptide according to claim 1, characterized in that, it comprises the following steps: loading the acetylated and amidated hexapeptide to be purified to a chromatographic column; and performing a gradient elution with a mobile phase A and a mobile phase B as eluents, wherein the mobile phase A is an acetonitrile solution containing 0.08%~0.12% of trifluoroacetic acid, and the mobile phase B is water containing 0.08%~0.12% of trifluoroacetic acid; a procedure for the gradient elution is that: an initial volume proportion of the mobile phase A is 8%~12%, the volume proportion of the mobile phase A rises to 30%~40% from 0.01 min to 25 min after loading, the volume proportion of the mobile phase A rises to 100% between 25 min and 25.1 min, the volume proportion of the mobile phase A is kept at 100% till the end of an operation at 30 min, and a detection wavelength is 220 nm; and a polypeptide solution at a target peak is collected.

3. The purification method according to claim 2, characterized in that, the chromatographic column is a Cig chromatographic column.

4. The purification method according to claim 2, characterized in that, the mobile phase A is an acetonitrile solution containing 0.1% of trifluoroacetic acid, and the mobile phase B is water containing 0.1% of trifluoroacetic acid, and/or, during the gradient elution, the initial volume proportion of the mobile phase A is 10%, and the volume proportion of the mobile phase A rises to 35% from 0.01 min to 25 min after loading.

5. An application of the acetylated and amidated hexapeptide according to claim 1, characterized in that, the acetylated and amidated hexapeptide is used for preparing a skin care product which resists skin photoaging or has skin moisturizing effect. LU502320

6. An application of the acetylated and amidated hexapeptide according to claim 1, characterized in that, the acetylated and amidated hexapeptide is used for preparing a preparation for preventing or repairing light damage to Hacat cells caused by UVB.

7. An application of the acetylated and amidated hexapeptide according to claim 1, characterized in that, the acetylated and amidated hexapeptide is used for preparing an antioxidation preparation.

8. An application of the acetylated and amidated hexapeptide according to claim 1, characterized in that, the acetylated and amidated hexapeptide is used for preparing a preparation for preventing or improving skin damage caused by UVB radiation.

9. A composition, characterized in that, the composition contains the acetylated and amidated hexapeptide according to claim 1.

10. The composition according to claim 9, characterized in that, the composition is cosmetic, food or medicine.