An active peptide, cosmetics containing the active peptide, and their applications

Abstract

This invention discloses an active peptide, a cosmetic containing the active peptide, and its applications, belonging to the field of cosmetic technology. The amino acid sequence of the active peptide is MQWNCPLGSAVITLHYRGDK. This invention has experimentally demonstrated the use of this active peptide in the preparation of anti-skin aging cosmetics. The polypeptide of this application has antioxidant, cell proliferation enhancement, anti-aging, and skin aging delaying effects, and can be used to develop and prepare anti-skin aging cosmetics.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, specifically to an active peptide, cosmetics containing the active peptide, and their applications. Background Technology

[0002] The skin is the largest organ in the human body, playing many important functions such as protection, temperature regulation, and sensation. However, with age, the skin undergoes a series of structural and functional changes, manifesting as skin laxity, increased wrinkles, decreased elasticity, and pigmentation—all typical characteristics of skin aging. Skin aging is a complex biological process influenced by both internal and external factors. Internal factors include genes and hormone levels; external factors include ultraviolet radiation, environmental pollution, and lifestyle habits.

[0003] Currently, research and applications in anti-skin aging mainly focus on the following aspects:

[0004] Antioxidants: Free radicals are one of the main causes of skin aging. Antioxidants can neutralize free radicals and slow down the skin aging process.

[0005] Promote collagen synthesis: Collagen is an important component of the skin. As we age, collagen synthesis decreases. Ingredients that promote collagen synthesis can improve skin elasticity and firmness.

[0006] Moisturizing: Maintaining skin hydration is an important measure to prevent dry skin and wrinkles;

[0007] Anti-inflammatory: Inflammatory responses accelerate skin aging, while anti-inflammatory ingredients can relieve inflammation and protect the skin;

[0008] Currently, bioactive peptides have become a hot topic in anti-aging cosmetics and drug research due to their excellent bioactivity and safety. Therefore, finding bioactive peptides that combat skin aging has become an important direction. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of existing technologies and provide an active peptide, a cosmetic containing the active peptide, and its application. To achieve the above objective, this invention provides the following technical solution:

[0010] In a first aspect, the present invention provides an active peptide, wherein the amino acid sequence of the active peptide is MQWNCPLGSAVITLHYRGDK.

[0011] Preferably, the present invention provides a method for preparing the above-mentioned active peptide as follows:

[0012] S1. Select clean honeysuckle vines and crush them into 1-2mm using a pulverizer. Mix the honeysuckle vine powder with 4 times the volume of ethanol, let it stand at 4℃ for 12 hours, filter and discard the ethanol, and process the powder until it is completely dry at room temperature.

[0013] S2. First stage of enzymatic hydrolysis: The dried honeysuckle vine powder was mixed with phosphate buffer at pH 8.0 at a ratio of 1:10. Protease AX and trypsin were added, each at 1% of the weight of the honeysuckle vine powder. The mixture was hydrolyzed in a water bath at 55°C for 4 hours, with regular stirring to keep the mixture uniform. Then the mixture was heated to 85°C and held for 10 minutes to inactivate the enzyme.

[0014] S3, Second stage of enzymatic hydrolysis: Adjust the pH of the hydrolysate to 6.5, add glucose protease at a rate of 0.5% of the weight of honeysuckle vine powder, continue enzymatic hydrolysis at 45°C for 2 hours, and then heat to 85°C again for 10 minutes to inactivate the enzyme.

[0015] S4. After the enzymatic hydrolysis is completed, the mixture is immediately centrifuged at 4°C and centrifugation force set at 10000g for 20 minutes to remove undissolved solids and protein precipitates. The supernatant is then treated with an ultrafiltration membrane with a molecular weight cutoff of 3kDa to remove low molecular weight impurities and concentrate the peptides.

[0016] S5. Pre-equilibrate the DEAE-cellulose column with 20mM Tris-HCl buffer, pH 8.0. Load the concentrated peptide sample onto the equilibrated DEAE-cellulose column, wash the column with pH 8.0 and 20mM Tris-HCl buffer, and elute with a NaCl gradient, gradually increasing the gradient from 0M NaCl to 1M NaCl. Collect the eluent.

[0017] S6. Use a C18 column for RP-HPLC purification. Elution program: 0-100% acetonitrile gradient, containing 0.1% trifluoroacetic acid, flow rate 1 ml / min, detection wavelength 220 nm, collect the target peptide peak.

[0018] S7. Final purification was performed using a Sephadex G-25 gel column with 0.1M phosphate buffer, pH 7.0 as the mobile phase.

[0019] S8. Use MALDI-TOF mass spectrometry to confirm the molecular weight and purity of the peptide, and confirm the sequence of the target peptide as MQWNCPLGSAVITLHYRGDK.

[0020] Secondly, the present invention provides the use of an active peptide in the preparation of anti-skin aging cosmetics, wherein the amino acid sequence of the active peptide is MQWNCPLGSAVITLHYRGDK.

[0021] Preferably, the present invention also provides a cosmetic product comprising the above-mentioned active peptide.

[0022] Preferably, the cosmetic is a face cream, serum, lotion, or face mask.

[0023] Thirdly, the present invention also provides the use of an active peptide in the preparation of an anti-skin aging drug, wherein the amino acid sequence of the active peptide is MQWNCPLGSAVITLHYRGDK.

[0024] Preferably, the drug comprises: a therapeutically effective dose of the active peptide and pharmaceutically acceptable excipients.

[0025] The present invention differs from the prior art in that it achieves the following technical effects:

[0026] This invention discovers an active peptide, cosmetics containing the active peptide, and its applications through experiments. The polypeptide of this application has antioxidant, cell proliferation enhancement, anti-aging, and skin aging delay effects, and can be used to develop and prepare anti-skin aging cosmetics. Attached Figure Description

[0027] Figure 1 These are the cell proliferation curves for each group in the experiments of this invention. Detailed Implementation

[0028] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0029] Example 1

[0030] This invention provides an active peptide, the amino acid sequence of which is MQWNCPLGSAVITLHYRGDK, and the three-letter abbreviation corresponding to the sequence is: Met-Gln-Trp-Asn-Cys-Pro-Leu-Gly-Ser-Ala-Val-Ile-Thr-Leu-His-Tyr-Arg-Gly-Asp-Lys.

[0031] Example 2

[0032] This invention provides a method for preparing the active peptide described in Example 1, the method being as follows:

[0033] S1. Select clean honeysuckle vines and crush them into 1-2mm using a pulverizer. Mix the honeysuckle vine powder with 4 times the volume of ethanol, let it stand at 4℃ for 12 hours, filter and discard the ethanol, and process the powder until it is completely dry at room temperature.

[0034] S2. First stage of enzymatic hydrolysis: The dried honeysuckle vine powder was mixed with phosphate buffer at pH 8.0 at a ratio of 1:10. Protease AX and trypsin were added, each at 1% of the weight of the honeysuckle vine powder. The mixture was hydrolyzed in a water bath at 55°C for 4 hours, with regular stirring to keep the mixture uniform. Then the mixture was heated to 85°C and held for 10 minutes to inactivate the enzyme.

[0035] S3, Second stage of enzymatic hydrolysis: Adjust the pH of the hydrolysate to 6.5, add glucose protease at a rate of 0.5% of the weight of honeysuckle vine powder, continue enzymatic hydrolysis at 45°C for 2 hours, and then heat to 85°C again for 10 minutes to inactivate the enzyme.

[0036] S4. After the enzymatic hydrolysis is completed, the mixture is immediately centrifuged at 4°C and centrifugation force set at 10000g for 20 minutes to remove undissolved solids and protein precipitates. The supernatant is then treated with an ultrafiltration membrane with a molecular weight cutoff of 3kDa to remove low molecular weight impurities and concentrate the peptides.

[0037] S5. Pre-equilibrate the DEAE-cellulose column with 20mM Tris-HCl buffer, pH 8.0. Load the concentrated peptide sample onto the equilibrated DEAE-cellulose column, wash the column with pH 8.0 and 20mM Tris-HCl buffer, and elute with a NaCl gradient, gradually increasing the gradient from 0M NaCl to 1M NaCl. Collect the eluent.

[0038] S6. Use a C18 column for RP-HPLC purification. Elution program: 0-100% acetonitrile gradient, containing 0.1% trifluoroacetic acid, flow rate 1 ml / min, detection wavelength 220 nm, collect the target peptide peak.

[0039] S7. Final purification was performed using a Sephadex G-25 gel column with 0.1M phosphate buffer, pH 7.0 as the mobile phase.

[0040] S8. Use MALDI-TOF mass spectrometry to confirm the molecular weight and purity of the peptide, and confirm the sequence of the target peptide as MQWNCPLGSAVITLHYRGDK.

[0041] Example 3

[0042] This invention provides a cosmetic product comprising the active peptides described in Example 1.

[0043] In addition, the cosmetics mentioned are face creams, serums, lotions, or face masks.

[0044] Experimental Example 1

[0045] I. Materials

[0046] The active peptide of this application was prepared by the inventor using the method of Example 2, and the amino acid sequence is MQWNCPLGSAVITLHYRGDK;

[0047] Human dermal fibroblasts (HDFs) were purchased from ATCC;

[0048] Fetal bovine serum (FBS), DMEM high glucose medium (DMEM-HG), and DMEM low glucose medium (DMEM-LG) were purchased from HyClone.

[0049] The CCK-8 kit, cell senescence β-galactosidase staining kit, and DCFH-DA reactive oxygen species (ROS) detection kit were purchased from Beyotime.

[0050] II. Methods

[0051] 1. Cell Culture and Grouping

[0052] HDFs cells were cultured in DMEM-LG containing 10% fetal bovine serum and 1% penicillin antibody at 37°C, 5% CO2, and saturated humidity, with the medium changed every 2-3 days. HDFs in the logarithmic growth phase were divided into five groups: blank group, model group, low-concentration peptide group, medium-concentration peptide group, and high-concentration peptide group.

[0053] Among them, the blank group was cultured in low-glucose medium throughout the process; the model group was cultured in high-glucose medium without intervention from active peptides to simulate senescent cells; the low-concentration peptide group was cultured in high-glucose medium with 50 μg / mL peptides; the medium-concentration peptide group was cultured in high-glucose medium with 100 μg / mL peptides; and the high-concentration peptide group was cultured in high-glucose medium with 200 μg / mL peptides.

[0054] 2. Cell proliferation activity assay

[0055] Cell proliferation activity was detected using a CCK-8 assay kit.

[0056] Logarithmic growth phase HDFs cells were digested with 0.25% trypsin, resuspended in DMEM-LG medium containing 10% FBS and 1% penicillin antibiotics, and counted under a microscope. 5000 cells / 200 μL of culture medium were seeded into 96-well plates and grouped according to the above grouping method, with 5 replicates per group. After culturing at 37℃, 5% CO2, and saturated humidity for 24 hours, the corresponding culture medium was replaced for each group. After culturing for 24, 48, and 72 hours, 10 μL of LCK-8 solution was added to each well, and incubated for 4 hours. The absorbance (OD) value was read at 450 nm using a microplate reader, and a proliferation curve was plotted.

[0057] 3. Detection of cellular senescence levels

[0058] Cell senescence levels were detected using a β-galactosidase staining kit.

[0059] Logarithmic growth phase HDFs cells were digested with 0.25% trypsin, resuspended in DMEM-LG medium containing 10% FBS and 1% penicillin antibiotics, and seeded in culture flasks at a density of 1×10^6 cells / mL. Cells were grouped according to the above method, and after 24 hours of culture, the corresponding medium was replaced for each group, and this was repeated every 48 hours for 12 days. Cells were collected, and the senescence level was detected using a β-galactosidase assay kit. Positive cells were counted under a microscope, and the senescence rate was calculated.

[0060] 4. Detection of reactive oxygen species (ROS) levels

[0061] Intracellular reactive oxygen species (ROS) levels were detected using the DCFH-DA ROS assay kit.

[0062] Log-phase HDF cells were digested with 0.25% trypsin, resuspended in DMEM-LG medium containing 10% FBS and 1% penicillin-dextrose antibody, and seeded in culture flasks at a density of 1×10^6 cells / mL. Cells were grouped according to the above method, and after 24 hours of culture, the corresponding medium was replaced for each group, and this was repeated every 48 hours for 12 days. Cells were collected, washed three times with PBS, and 10 μM DCFH-DA probe was added to each well. Cells were incubated at 37°C for 20 minutes, washed three times, and then observed and photographed using a fluorescence microscope to measure intracellular reactive oxygen species levels.

[0063] III. Data Analysis

[0064] Data are expressed as mean ± SD. The t-test was used for comparisons between groups, and P < 0.05 was considered statistically significant.

[0065] IV. Experimental Results and Analysis

[0066] 1. Cell proliferation activity

[0067] The OD values ​​for each group are shown in Table 1. The cell proliferation curves are referenced in Table 1. Figure 1 .

[0068] Experimental results: Compared with the blank group, the proliferation activity of HDFs cells in the model group was significantly weakened; compared with the model group, the proliferation activity of HDFs cells in the low-concentration peptide group, medium-concentration peptide group, and high-concentration peptide group was significantly enhanced, and the proliferation activity of HDFs cells in the high-concentration peptide group was the strongest.

[0069] Table 1 OD values ​​for each group

[0070]

[0071] 2. Cellular senescence level

[0072] The positive rates of β-galactosidase staining in each group are shown in Table 2:

[0073] Experimental results: Compared with the blank group, the staining positivity rate of HDFs cells in the model group was significantly increased; compared with the model group, the staining positivity rates of HDFs cells in the low-concentration peptide group, medium-concentration peptide group, and high-concentration peptide group were significantly decreased, and the staining positivity rate of HDFs cells in the high-concentration peptide group was the lowest.

[0074] Table 2. Positive rate of β-galactosidase staining in each group

[0075] Group positive cell count Total cell count Positive rate (%) Blank group 23 1000 2.3 Model group 225 1000 22.5 low concentration peptide group 171 1000 17.1 Medium concentration peptide group 137 1000 13.7 High concentration peptide group 82 1000 8.2

[0076] 3. Reactive oxygen species (ROS) level

[0077] The ROS levels for each group are shown in Table 3:

[0078] Experimental results: Compared with the control group, the ROS level of HDFs cells in the model group was significantly increased; compared with the model group, the ROS level of HDFs cells in the low-concentration peptide group, medium-concentration peptide group, and high-concentration peptide group was significantly decreased, and the ROS level of HDFs cells in the high-concentration peptide group was the lowest.

[0079] Table 3 ROS levels of each group

[0080]

[0081]

[0082] V. Conclusion

[0083] Experimental data show that the peptides in this application can significantly improve the proliferation activity of HDFs cells, reduce cell senescence levels and reactive oxygen species levels, and have a good anti-skin aging effect.

[0084] Experiment Example 2

[0085] I. Materials

[0086] The active peptide of this application was prepared by the inventor using the method of Example 2, and the amino acid sequence is MQWNCPLGSAVITLHYRGDK;

[0087] Choose an animal:

[0088] Species: C57BL / 6 mouse;

[0089] Sex: Male and female;

[0090] Age: 2-3 months;

[0091] Weight: 20-25g;

[0092] Adaptation period: Mice were acclimatized to the experimental environment for one week before the experiment to ensure they adapted to the environmental changes and reduce stress response.

[0093] II. Experimental Grouping

[0094] Mice were randomly divided into four groups (10 mice in each group):

[0095] Control group: No peptide treatment was given; the animals were fed and managed according to standard practices.

[0096] Low-dose peptide group: 1 mg / kg of peptide injected subcutaneously daily;

[0097] Medium-dose peptide group: 5 mg / kg of peptide injected subcutaneously daily;

[0098] High-dose peptide group: 10 mg / kg of peptide injected subcutaneously daily.

[0099] The appropriate dose of peptide was administered subcutaneously daily for 28 consecutive days.

[0100] III. Preparation of Skin Aging Models

[0101] Ultraviolet radiation:

[0102] Except for the control group, the skin on the backs of mice in the other groups was irradiated with a low dose of UVB (once a day, 0.5 J / cm²). 2 (This is repeated for 5 consecutive days to induce early skin aging.)

[0103] IV. Key Observation Indicators

[0104] Skin elasticity measurement:

[0105] The elasticity of the skin on the back of mice was measured using a cutometer before and after the experiment, and the skin elasticity coefficient (R0, unit: mm) was recorded.

[0106] Collagen content:

[0107] The content of collagen in the skin was quantified using Sirius Red staining, and the collagen content (OD value) was recorded using optical density measurement.

[0108] Antioxidant enzyme activity:

[0109] The activities of SOD (superoxide dismutase) and GPx (glutathione peroxidase) in skin tissue were measured, and the activities of SOD and GPx (U / mg protein) were recorded respectively.

[0110] V. Experimental Results

[0111] The elastic coefficient R0 measured using a cutometer is as follows:

[0112] Control group: 1.21±0.10 mm;

[0113] Low-dose peptide group: 1.23±0.12 mm;

[0114] Medium-dose peptide group: 1.26±0.14 mm;

[0115] High-dose peptide group: 1.28±0.15mm.

[0116] The OD values ​​after Sirius Red staining are as follows:

[0117] Control group: 0.42±0.03;

[0118] Low-dose peptide group: 0.51±0.04;

[0119] Medium-dose peptide group: 0.68±0.05;

[0120] High-dose peptide group: 0.77±0.06.

[0121] The activity data for SOD and GPx are as follows:

[0122] SOD activity:

[0123] Control group: 82±5 U / mg protein;

[0124] Low-dose peptide group: 85±6 U / mg protein;

[0125] Medium-dose peptide group: 89±7 U / mg protein;

[0126] High-dose peptide group: 94±8 U / mg protein;

[0127] GPx activity:

[0128] Control group: 52±4 U / mg protein;

[0129] Low-dose peptide group: 56±5 U / mg protein;

[0130] Medium-dose peptide group: 58±6 U / mg protein;

[0131] High-dose peptide group: 63±7U / mg protein.

[0132] The skin elasticity of the peptide-treated group was significantly improved compared with the control group. Peptide treatment can significantly increase the collagen content in the skin. The SOD and GPx activities of the high-dose peptide group were significantly higher than those of the control group. Therefore, the peptides of this application have good anti-skin aging effects.

[0133] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An active peptide, characterized in that: The amino acid sequence of the active peptide is MQWNCPLGSA. VITLHYRGDK.

2. The use of an active peptide in the preparation of anti-skin aging cosmetics, wherein the amino acid sequence of the active peptide is MQWNCPLGSAVITLHYRGDK.

3. A cosmetic product, characterized in that, Includes the active peptide as described in claim 1.

4. The cosmetic product as described in claim 3, characterized in that, The cosmetics mentioned are face creams, serums, lotions, or face masks.

5. The use of an active peptide in the preparation of an anti-skin aging drug, wherein the amino acid sequence of the active peptide is MQWNCPLGSAVITLHYRGDK.

6. The use according to claim 5, characterized in that, The drug comprises: a therapeutically effective dose of the active peptide and pharmaceutically acceptable excipients.

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