A 15-peptide based on giant salamander hydrolyzed collagen and its application

Abstract

The present invention belongs to the technical fields of cosmetics and biomedicine, and particularly relates to a fifteen - peptide based on giant salamander hydrolyzed collagen and its application. The amino acid sequence of the fifteen - peptide of the present invention is Gly - Ala - Thr - Gly - Pro - Thr - Gly - Pro - Thr - Gly - Pro - Ala - Gly - Pro - Lys. The fifteen - peptide shows a significant promoting effect on the content of CollagenⅢ in fibroblasts induced by UVA radiation. At the same time, it has a good inhibitory effect on the activity of tyrosinase, and has a strong scavenging effect on DPPH free radicals and ABTS+ free radicals, indicating that the fifteen - peptide has good skin firming, anti - aging and whitening effects and can be used for the preparation of cosmetics.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptide and cosmetic preparation, and particularly relates to a 15-peptide based on giant salamander hydrolyzed collagen and application thereof. Background Art

[0002] The dermis of human skin is rich in connective tissue, mainly collagen and elastin. Wrinkles on the skin are mainly caused by problems with collagen and elastin in the dermis. In the presence of excessive free radicals in the body, excessive cross-linking of collagen can be caused, resulting in a significant decrease in its elasticity, which promotes the formation of wrinkles. On the other hand, as people age, the rate of enzymatic hydrolysis of collagen and elastase in the dermis is greater than their synthesis rate, which accelerates the formation of wrinkles. At the same time, in photoaging and erythema tissues caused by ultraviolet rays, the increase in the activity of collagenase and elastase promotes the enzymatic hydrolysis of collagen and elastin. Therefore, bioactive substances with free radical scavenging activity and promoting the increase of collagen and elastin have a firming effect on the skin.

[0003] CollagenⅢ has a reconstructive property, can strengthen the elasticity and strength of microvessels, can provide sufficient nutrients for cells, directly combine with angioblasts to promote the formation of new blood vessels, and is an important factor in maintaining the plumpness, lubrication, and luster of the skin; it is a bioactive substance that promotes the increase of fibroblast CollagenⅢ and has a skin tightening effect. Tyrosinase is the rate-limiting enzyme in the melanin synthesis pathway. It mainly affects the production of melanin by affecting the conversion of tyrosine into dopa and the oxidation of dopa into dopaquinone. Furthermore, bioactive substances that inhibit tyrosinase are bioactive substances with whitening effects.

[0004] As the "darling" of skin care products, peptides are widely used in the preparation of skin care products. However, peptides composed of different numbers and sequences of amino acids have different effects. For example, dipeptides can relax facial muscles, smooth wrinkles, and have a good anti-wrinkle effect. At the same time, they can protect DNA and have antioxidant properties. Nonapeptides can block the formation of melanin, thereby reducing the chance of melanin production and have a good whitening effect. Finding new bioactive substances with skin tightening and whitening effects is an important research direction in the field of beauty cosmetics. Summary of the invention

[0005] The object of the present invention is to provide a pentapeptide based on giant salamander hydrolyzed collagen and application thereof. The pentapeptide has the effects of tightening and whitening the skin and can be used in the preparation of cosmetics to improve the tightening and whitening effects of the cosmetics.

[0006] The present invention provides a pentapeptide based on hydrolyzed collagen of giant salamander, wherein the amino acid sequence of the pentapeptide is Gly-Ala-Thr-Gly-Pro-Thr-Gly-Pro-Thr-Gly-Pro-Ala-Gly-Pro-Lys.

[0007] The present invention also provides the use of the 15-peptide described in the above technical solution in the preparation of cosmetics.

[0008] Preferably, the cosmetics include special cosmetics.

[0009] Preferably, the special cosmetics include products having any one or more effects of firming, anti-wrinkle and whitening.

[0010] Preferably, the mass concentration of the pentadecapeptide in the cosmetic is 0.0313% to 0.125% m / V.

[0011] Preferably, the mass concentration of the pentadecapeptide in the cosmetic is 0.0313% m / V or 0.0625% m / V or 0.125% m / V.

[0012] The present invention also provides a cosmetic, wherein the active ingredient of the cosmetic includes the pentadecapeptide described in the above technical solution.

[0013] Preferably, the mass concentration of the pentapeptide in the cosmetic is 0.0313% to 0.125% m / V.

[0014] Beneficial effects:

[0015] The present invention provides a pentapeptide based on hydrolyzed collagen of giant salamander, wherein the amino acid sequence of the pentapeptide is Gly-Ala-Thr-Gly-Pro-Thr-Gly-Pro-Thr-Gly-Pro-Ala-Gly-Pro-Lys, and the pentapeptide composed of amino acids significantly increases the content of Collagen III in fibroblasts induced by UVA radiation, and has a good inhibitory effect on the activity of tyrosinase, and has an inhibitory effect on DPPH free radicals and ABTS. + Free radicals have a strong scavenging effect, which means that the 15-peptide has good firming, anti-aging and whitening effects on the skin and can be used in the preparation of cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0017] Figure 1 is the HPLC chromatogram of the 15-peptide in Example 1;

[0018] Figure 2 This is the MS structure identification diagram of the 15-peptide in Example 1;

[0019] Figure 3 This is the trend chart of the change of CollagenⅢ content in fibroblasts induced by UVA radiation in vitro by the 15-peptide in Application Example 1;

[0020] Figure 4 Schematic diagram of the ability of the 15-peptide in Application Example 2 to scavenge DPPH free radicals;

[0021] Figure 5 Schematic diagram of the ability of the 15-peptide to scavenge ABTS free radicals in Application Example 3;

[0022] Figure 6 This is a schematic diagram of the results of the 15-peptide's ability to inhibit tyrosinase in Application Example 4. DETAILED DESCRIPTION

[0023] The present invention provides a pentapeptide based on hydrolyzed collagen of giant salamander, wherein the amino acid sequence of the pentapeptide is Gly-Ala-Thr-Gly-Pro-Thr-Gly-Pro-Thr-Gly-Pro-Ala-Gly-Pro-Lys.

[0024] The 15-peptide of the present invention is preferably derived from hydrolyzed collagen of giant salamander and is a low molecular weight water-soluble polypeptide. In the present invention, the 15-peptide can be abbreviated as GATGPTGPTGPAGPK (SEQ ID NO.1). The molecular weight of the 15-peptide of the present invention is preferably 1265.39 Da; the purity is preferably 93.79%. In the amino acid sequence of the present invention, Gly represents the corresponding residue of the amino acid whose English name is Glycine and whose Chinese name is Glycine; Ala represents the corresponding residue of the amino acid whose English name is Alanine and whose Chinese name is Alanine; Thr represents the corresponding residue of the amino acid whose English name is Threonine and whose Chinese name is Threonine; Pro represents the corresponding residue of the amino acid whose English name is Proline and whose Chinese name is Proline; Lys represents the corresponding residue of the amino acid whose English name is Lysine and whose Chinese name is Lysine.

[0025] The present invention preferably uses the Fmoc synthesis method to synthesize the pentadecapeptide. The present invention does not specifically limit the specific steps of the Fmoc synthesis method, and polypeptides with the same amino acid sequence as the pentadecapeptide of the present invention obtained by the Fmoc synthesis method in the art all fall within the protection scope of the present invention.

[0026] The present invention also provides the use of the pentadecapeptide described in the above technical solution in the preparation of cosmetics. The pentadecapeptide described in the present invention can significantly increase the content of UVA radiation-induced fibroblast collagen III, inhibit the activity of tyrosinase, and have an effect on DPPH free radicals and ABTS.+ Free radicals have a strong scavenging effect and have good firming, anti-aging and whitening effects on the skin.

[0027] In the present invention, the cosmetics preferably include special cosmetics, and further preferably include products with any one or more of the effects of firming, anti-wrinkle and whitening, and more preferably products with firming, anti-wrinkle and whitening effects. The mass concentration of the pentad peptide of the present invention in the cosmetics is preferably 0.0313% to 0.125% m / V, more preferably 0.0313% m / V or 0.0625% m / V or 0.125% m / V. The present invention does not specifically limit the types of the cosmetics, and lotions, emulsions, creams and essential oils containing the pentad peptide all belong to the protection scope of the present invention. In the cosmetics described in the present invention, the amount of the pentad peptide added is preferably 0.1% of the mass of the cosmetics.

[0028] The present invention also provides a cosmetic, the active ingredient of which includes the 15-peptide described in the above technical solution. In the cosmetic of the present invention, the mass concentration of the 15-peptide is preferably 0.0313% to 0.125% m / V, more preferably 0.0313% m / V or 0.0625% m / V or 0.125% m / V. The cosmetic of the present invention preferably includes cosmetics having any one or more of the effects of firming, anti-wrinkle and whitening, more preferably cosmetics having firming, anti-wrinkle and whitening effects. The cosmetic of the present invention preferably includes excipients accepted in cosmetics. The present invention does not specifically limit the type and content of the excipients, which can be added according to the specific preparation of the cosmetics.

[0029] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0030] Example 1

[0031] The preparation method of 15-peptide based on hydrolyzed collagen of giant salamander comprises the following steps:

[0032] A polymer resin (Fmoc-Pro-CTC Resin purchased from Zhongpeptide Biochemical Co., Ltd.) was selected. According to the characteristics of the 15-peptide Gly-Ala-Thr-Gly-Pro-Thr-Gly-Pro-Thr-Gly-Pro-Ala-Gly-Pro-Lys, the carboxyl group of Lys was first connected to the polymer resin in the form of a covalent bond, and then the amino group of Lys and the carboxyl group of Pro were condensed. After treatment, Gly was added, and the amino group of Pro reacted with the carboxyl group of Gly. Amino acids were added from right to left in sequence. After the last Gly was added, the resin was removed to obtain the target polypeptide.

[0033] Purification was performed by high performance liquid chromatography, the chromatographic column model was Kromasil-C18 liquid chromatography column, the specification was 4.6×250 mm, 5 μm; mobile phase A: water containing 0.1% trifluoroacetic acid (TFA) by mass percentage; mobile phase B: acetonitrile containing 0.1% trifluoroacetic acid and 0.1% TFA; within 25 min, phase A decreased from 90% to 65%, and phase B increased from 10% to 35%, the flow rate was 1.0 mL / min, and the detection wavelength was 220 nm. Figure 1 shown.

[0034] The purified liquid was quickly frozen with liquid nitrogen and freeze-dried to obtain the final product with a purity of more than 90%. The structure was identified by MS. Figure 2 shown.

[0035] Depend on Figure 1 and Figure 2 It can be concluded that the present invention synthesized a 15-peptide having an amino acid sequence of Gly-Ala-Thr-Gly-Pro-Thr-Gly-Pro-Thr-Gly-Pro-Ala-Gly-Pro-Lys.

[0036] Application Example 1

[0037] Determination of the Effect of Pentadapeptide on the Content of CollagenⅢ in Fibroblasts Induced by UVA Radiation in Vitro

[0038] Fibroblasts (HFF-1) SCSP-109 (purchased from the Cell Bank of the Chinese Academy of Sciences) were cultured at 5×10 4 Cells were seeded into 24-well plates / well and incubated in an incubator at 37°C and 5% CO 2 Incubate overnight under the conditions. The experiment set up a blank control group (BC), a negative control group (NC), a positive control group (PC) and a sample group. The sample group was prepared by dissolving the 15-peptide prepared in Example 1 in a phosphate buffer with a pH of 7.2, and three concentration gradients were set, namely 0.0313%, 0.0625% and 0.125% (m / V). The drug was administered when the cell plating rate in the 24-well plate reached 40% to 60%.

[0039] 1 mL of DMEM high-glucose culture medium (VivaCell) was added to each well of the blank control group and the negative control group; 1 mL of DMEM high-glucose culture medium containing 100 μg / mL vitamin C and 7 μg / mL vitamin E was added to each well of the positive control group; 1 mL of DMEM high-glucose culture medium containing the corresponding concentration of synthetic peptides was added to each well of the sample group. After the administration, the negative control group, the positive control group and the sample group received a total dose of 9 J / cm 2 At the same time, the blank control group was placed in the same environment (UVA radiation dose of 0 J / cm2 After incubation for 24 h, the cell culture supernatant was collected in an EP tube and stored in a -80°C refrigerator.

[0040] According to the operating instructions of the ELISA test kit (purchased from Shanghai Microspectro Detection Technology Group Co., Ltd.), the content of Collagen III in each group of cells was detected and analyzed. The results are as follows: Figure 3 shown.

[0041] Depend on Figure 3 It can be concluded that the contents of Collagen III in the BC group, NC group, PC group, and the groups with peptide concentrations of 0.0313%, 0.0625%, and 0.125% (m / V) were 10.01, 1.65, 2.38, 2.59, 2.57, and 2.48 ng / mL, respectively.

[0042] Compared with the BC group, the level of CollagenⅢ in the NC group fibroblasts was significantly decreased after receiving UVA radiation with a total dose of 9J / cm2 (p<0.05), indicating that the irradiation stimulation was effective. Compared with the NC group, vitamin C and vitamin E in the PC group can significantly increase the level of CollagenⅢ (p<0.05), indicating that this positive control test is effective. Compared with the NC group, the synthetic peptide group to be tested showed a significant improvement in the content of CollagenⅢ in fibroblasts induced by UVA radiation at concentrations of 0.0313%, 0.0625% and 0.125% (m / V) (p<0.05), indicating that it has anti-wrinkle and firming effects.

[0043] Application Example 2

[0044] The ability of pentapeptide to scavenge DPPH free radicals

[0045] The 15-peptide prepared in Example 1 was prepared into polypeptide solutions of different concentrations with deionized water, 0.2, 0.4, 0.6, 0.8 and 1.0 mg / mL, respectively. 2 mL of polypeptide solutions of different concentrations were taken in test tubes, and 2 mL of the prepared DPPH solution (0.004 g DPPH powder was added to a 50 mL volumetric flask, fixed to volume with 95% ethanol, and stored at 4°C in the dark) was added, mixed evenly, and kept in the dark at room temperature for 30 minutes. Centrifuged at 10000 rpm for 10 minutes, and the supernatant was taken to measure the absorbance A at 517 nm. j , and simultaneously measure the absorbance A of 2 mL of different peptide solutions plus 2 mL of 95% ethanol i , the absorbance of 2mL DPPH plus 2mL distilled water is A 0 .

[0046] DPPH free radical scavenging rate (%) = [A 0 -(A j-A i )] / A 0 ×100%

[0047] Where: A 0 is the absorbance value of the blank control; A j is the absorbance of the sample solution; A i is its own absorbance value.

[0048] The results of the test are as follows Figure 4 shown.

[0049] Depend on Figure 4 It can be seen that the ability of the pentadecapeptide of the present invention to scavenge DPPH free radicals increases with the increase of the concentration of the synthetic polypeptide.

[0050] Application Example 3

[0051] Pentadecepeptide scavenges ABTS + Capabilities

[0052] ABTS was prepared with distilled water to a concentration of 7.4 mmol / L, and then mixed with an equal volume of 2.6 mmol / L K 2 S 2 O 8 Mix evenly, place in the dark at room temperature for 12 hours, and then dilute with 95% ethanol solution so that the absorbance at a wavelength of 734 nm is 0.70 ± 0.02, thus obtaining ABTS. + Free radical stock solution.

[0053] The pentapeptide prepared in Example 1 was prepared into peptide solutions of different concentrations with deionized water, which were 20, 60, 100, 140 and 180 μg / mL, respectively. 0.9 mL of peptide solutions of different concentrations and 2 mL of ABTS were added to the test tubes respectively. + The free radical stock solution was placed at room temperature for 6 minutes, then centrifuged at 10,000 rpm for 10 minutes, and the supernatant was taken to measure the absorbance A at a wavelength of 734 nm. i , and simultaneously measure the absorbance A of 0.9 mL of different peptide solutions plus 2 mL of deionized water j , 2mL ABTS + The absorbance of the free radical stock solution plus 0.9 mL of distilled water is A 0 .

[0054] ABTS free radical scavenging rate (%) = [A 0 -(A i -A j )] / A 0 ×100%

[0055] Where: A 0 The absorbance value of the blank control; Ai The absorbance of the sample solution; A j Its own absorbance

[0056] The results are as follows Figure 5 As shown: The synthetic polypeptide of the present invention removes ABTS + The free radical capacity increases with the concentration of synthetic peptides.

[0057] Application Example 4

[0058] Inhibitory activity test of 15-peptide against tyrosinase

[0059] Skin melanin is mainly caused by the action of tyrosinase contained in the skin on the amino acid tyrosine that makes up the skin, causing it to further react and produce melanin. Inhibition of tyrosinase can inhibit the production of melanin substances from the source.

[0060] A negative control group, a positive control group and three sample groups were set up respectively, wherein the negative control group was deionized water; the positive control group was 0.3% (g / 100mL) kojic acid; the concentrations of the 15-peptide solutions (the solvent was a phosphate buffer solution with a pH of 7.2) in the three sample groups were 0.125%, 0.0625% and 0.0313% (g / 100mL), respectively, and the inhibition rate of the samples in each group on tyrosinase was determined according to the following determination method.

[0061] The determination method is as follows: Accurately pipette A according to Table 1 1 , A 2 , A 3 and A 4 The four reaction solutions were placed in test tubes, kept in a water bath at 30°C for 10 min, 0.5 mL of tyrosinase solution was added, shaken, reacted for 10 min, and quickly transferred to a cuvette to measure the absorbance at 475 nm. The specific operation is shown in Table 1. The results are shown in Table 1. Figure 6 As shown, Figure 6 Middle control represents negative control group.

[0062] Table 1 Tyrosinase inhibition operation table

[0063]

[0064]

[0065] The tyrosinase inhibition rate was calculated as follows:

[0066]

[0067] Depend on Figure 6It can be concluded that the inhibition rates of the negative control group, the three polypeptide groups and the positive control group on tyrosinase are 0, 69%, 61%, 52% and 100% respectively; it can be seen that compared with the negative control group, the 15-peptide aqueous solution of the present invention has a better inhibitory effect on tyrosinase, indicating that it has a whitening effect on the skin.

[0068] It can be concluded from the above examples that the 15-peptide of the present invention can significantly increase the content of Collagen III in UVA-induced fibroblasts, inhibit the activity of tyrosinase and have an inhibitory effect on DPPH free radicals and ABTS. + Free radicals have a strong scavenging effect, which indicates that the 15-peptide has good firming, anti-aging and whitening effects on the skin.

[0069] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A 15-peptide based on hydrolyzed collagen of giant salamander, It is characterized in that The amino acid sequence of the pentadecapeptide is Gly-Ala-Thr-Gly-Pro-Thr-Gly-Pro-Thr-Gly-Pro-Ala-Gly-Pro-Lys.

2. Use of the pentapeptide according to claim 1 in the preparation of cosmetics.

3. The use according to claim 2, It is characterized in that The cosmetics include special cosmetics.

4. The use according to claim 3, It is characterized in that The special cosmetics include products having any one or more effects of firming, anti-wrinkle and whitening.

5. The use according to any one of claims 2 to 4, It is characterized in that The mass concentration of the pentadecapeptide in the cosmetic is 0.0313% to 0.125% m / V.

6. The use according to claim 5, It is characterized in that The mass concentration of the pentadecapeptide in the cosmetic is 0.0313% m / V or 0.0625% m / V or 0.125% m / V.

7. A cosmetic product, It is characterized in that The active ingredient of the cosmetic comprises the pentapeptide according to claim 1.

8. The cosmetic according to claim 7, It is characterized in that The mass concentration of the pentapeptide in the cosmetic is 0.0313% to 0.125% m / V.

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