An antioxidant active polypeptide Cos-16 and its application

Abstract

The present invention discloses an antioxidant active polypeptide Cos-16 and its application, and its amino acid sequence is shown in SEQ ID NO.01. The present invention has high activity, no cytotoxicity, high stability and antioxidant activity, and plays a beneficial role in protecting cells from ultraviolet radiation and oxidative stress caused by hydrogen peroxide stimulation. The present invention can protect the skin from light damage caused by ultraviolet radiation and has great medical application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of functional polypeptides, and specifically relates to an antioxidant active polypeptide Cos-16 and an application thereof. Background Art

[0002] Photoaging is characterized by a complex process of changes in the skin over time caused by UV exposure. It causes premature skin aging and is superimposed on the changes caused by chronological aging. Photoaging clinical signs include wrinkles, moles, mottled hyperpigmentation, loss of translucency and decreased elasticity. These characteristics are further influenced by the individual's ethnicity and genetics.

[0003] Ultraviolet rays are the main culprit of wrinkles caused by photoaging of the skin. After penetrating the skin, fibroblasts will produce a large amount of matrix metalloproteinases (MMPs), which cannot promote the synthesis of collagen, but instead destroy the collagen and elastic fibers in the dermis.

[0004] Anti-aging has always been the focus of the cosmetics industry for a long time, and delaying skin aging is one of the goals pursued by most people. Many people have always been keen on the research of anti-aging experiments and have developed many anti-aging cosmetics.

[0005] Currently, peptides are one of the most popular anti-aging ingredients in anti-aging cosmetics, and research on peptides with anti-aging activity is also emerging in an endless stream. Summary of the invention

[0006] The present invention aims to provide an antioxidant active polypeptide Cos-16.

[0007] Another object of the present invention is to provide the application of the above antioxidant active polypeptide Cos-16.

[0008] The technical solution of the present invention is as follows

[0009] An antioxidant active polypeptide Cos-16, whose amino acid sequence is shown in SEQ ID NO.01.

[0010] The application of the above antioxidant active polypeptide Cos-16 in the preparation of antioxidant health care composition.

[0011] An antioxidant health care composition, the effective ingredients of which include the antioxidant active polypeptide Cos-16.

[0012] In a preferred embodiment of the present invention, the active ingredient is the above-mentioned antioxidant active polypeptide Cos-16.

[0013] Application of the above antioxidant active polypeptide Cos-16 in the preparation of cosmetic compositions.

[0014] A cosmetic composition, the active ingredient of which includes the above-mentioned antioxidant active polypeptide Cos-16.

[0015] In a preferred embodiment of the present invention, the active ingredient is the above-mentioned antioxidant active polypeptide Cos-16.

[0016] Application of the above antioxidant active polypeptide Cos-16 in an anti-ultraviolet composition.

[0017] An anti-ultraviolet composition, the active ingredient of which includes the above-mentioned antioxidant active polypeptide Cos-16.

[0018] In a preferred embodiment of the present invention, the active ingredient is the above-mentioned antioxidant active polypeptide Cos-16.

[0019] The beneficial effects of the present invention are:

[0020] 1. The present invention has high activity, no cytotoxicity, high stability and antioxidant activity, and plays a beneficial role in protecting cells from ultraviolet radiation and oxidative stress caused by hydrogen peroxide stimulation.

[0021] 2. The present invention can protect the skin from light damage caused by ultraviolet radiation and has great medical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the HPLC reverse phase C18 column chromatogram of the antioxidant active polypeptide Cos-16 prepared in Example 1 of the present invention.

[0023] Figure 2 This is the mass spectrum of the antioxidant active polypeptide Cos-16 prepared in Example 1 of the present invention.

[0024] Figure 3 This is one of the in vitro free radical scavenging activity graphs of the antioxidant active polypeptide Cos-16 in Example 2 of the present invention.

[0025] Figure 4 This is the second graph of the in vitro free radical scavenging activity of the antioxidant active polypeptide Cos-16 in Example 2 of the present invention.

[0026] Figure 5 This is a diagram showing that the antioxidant active polypeptide Cos-16 in Example 3 of the present invention protects HaCaT cell viability from UVB-induced damage.

[0027] Figure 6 The antioxidant active polypeptide Cos-16 in Example 4 of the present invention inhibits the accumulation of ROS in UVB-induced HaCaT cells.

[0028] Figure 7This is a diagram showing the regulation of the activity of the antioxidant active polypeptide Cos-16 on the catalase in HaCaT cells in Example 5 of the present invention.

[0029] Figure 8 This is a diagram showing the regulation of glutathione peroxidase activity of HaCaT cells by the antioxidant active polypeptide Cos-16 in Example 5 of the present invention. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further illustrated and described below through specific implementation modes in combination with the accompanying drawings.

[0031] Example 1 Peptide synthesis, purification and identification

[0032] Weigh an appropriate amount of 2-cl-Trt resin and swell it with dichloromethane for 20 minutes. Set the degree of substitution (SD). The target sequence is relatively short and the SD is set to 0.3. Weigh 1.3eq (eq = resin amount × degree of substitution) of the first amino acid Fmoc-L-oh according to the quantitative method. After the resin is swollen, wash it repeatedly with dichloromethane for 2-3 times, and use dichloromethane as the reaction solvent DIEA for catalysis; add the first amino acid to react for 1-2 hours. Dichloromethane is volatile, and dichloromethane is appropriately supplemented during the process. After the reaction is completed, add an appropriate amount of methanol at a volume of 1:1 for 30 minutes. After the end of the head, wash 3 times and remove fmoc with 20% pip for 10 minutes. After the deprotection is completed, wash 5-6 times and test positive, indicating that the first amino acid has been successfully connected to the resin, and the subsequent reaction can be started. The amount of 3eq for the subsequent reaction is added. The reaction method can be HOBT+DIC or HBTU+DIEA, and the reaction time is 1 hour. After the end, if the test is negative, proceed to the next step. If it is positive, repeat the previous step until it is completely completed. The resin was drained and lysed on a shaker for 2 h using TFA:EDTt:TIS:H2O=95:2:2:1 for 1 g of resin and 10 mL of cutting solution. The resin was filtered and centrifuged to obtain a crude polypeptide. The crude polypeptide was purified and identified by HPLC-ESI-MS to obtain polypeptides such as SEQ ID NO.01 and Figure 2 The antioxidant active polypeptide Cos-16 shown has an amino acid sequence of ACFLEGL.

[0033] The above HPLC conditions: inertsil ODS-SPC18 column (4.6mm×250mm, 5um), column temperature is room temperature, injection volume is 30μL. The mobile phase is 0.1% trifluoroacetic acid aqueous solution, isocratic washing, flow rate is 1mL / min. The HPLC purification and identification results are as follows Figure 1As shown in Table 1, the peak at 4.2 min is Cos-16, and the purity of Cos-16 after purification reaches 95.75%. Mass spectrometry conditions: electrospray ionization source positive ion mode ESI, select monitoring mode Scan and SIR, full scan molecular weight range 100-500, characteristic scan molecular weight 297, spray voltage 5.5KV, nebulizer gas flow rate 15Lh, ion source temperature 500℃, cone voltage 15V, collision gas is nitrogen, data acquisition scan time 20s.

[0034] Table 1

[0035]

[0036] Example 2 DPPH detection

[0037] The DPPH free radical scavenging ability detection kit (Biyuntian, item number: BC4750) was used to detect the free radical scavenging activity of the antioxidant active polypeptide Cos-16 prepared in Example 1. Take 500 μL of reagent 2 solution, thaw on ice, add 2625 μL of reagent 1 solution (anhydrous ethanol), mix thoroughly, and place on ice for use. Take 1 mL of the extract provided by the kit and add it to the brown glass tube of reagent 3. After fully mixing, divide and store 50 μL per tube and place it at -20°C; take out the polypeptide mother solution from the -20°C refrigerator, add DMSO to dilute, place on ice for use and prepare the solvent for standby. Preheat the spectrophotometer for more than 30 minutes, adjust the wavelength to 515nm, and zero with anhydrous ethanol. Use the extract to prepare a vitamin C solution greater than 0.3 mg / mL of the 10 mg / mL vitamin C solution (reagent three) for use (add 1.2 mL of the extract to the storage centrifuge tube and mix thoroughly). Add antioxidant active polypeptide Cos-16 solution, DMSO, extract and working solution to a 1.5mL EP tube, respectively, to make the final concentration of the polypeptide 1.25uM, 2.5uM and 5uM. After mixing, stand at room temperature in the dark for 30 minutes, and measure the absorbance at 515nm. The absorbance values ​​of the blank tube, positive control tube, control tube and assay tube are recorded as A blank, A positive control, A control and A assay, respectively. The blank tube only needs to be measured 1-2 times. Turn on the instrument for preheating and debug the parameters. Use the instrument to test, record the test data and analyze the results as follows Figure 3 , Figure 4 As shown, Figure 3 This indicates that the antioxidant peptide Cos-16 has strong free radical scavenging activity and is concentration-dependent. Figure 4 The results showed that when the concentration of the antioxidant active peptide Cos-16 was 5uM, it had significant free radical scavenging activity compared with the control group.

[0038] Example 3 Effect of antioxidant peptide Cos-16 on HaCaT cell viability

[0039] HaCaT cells were plated into 96-well plates (about 1×10 4 After 24 h of complete cell adhesion, the culture medium was removed and 100 μL serum-free culture medium containing different concentrations of antioxidant active peptide Cos-16 was added. After incubation for 24 h, the cells were washed three times with PBS and 50 μL PBS solution was left at the bottom of the well plate. The HaCaT cells were irradiated with 20 mJ / em using a UVB ultraviolet irradiator. 2 After irradiation, fresh culture medium was added immediately, and culture was continued for 24 hours. Then, 10ul of CCK-8 reagent was added to each well. After incubation for 2 hours, the OD value was detected at a wavelength of 450nm. The results are shown in Figure 5 As shown, it can be seen that the antioxidant active peptide Cos-16 has no toxicity to HaCaT cells and when HaCaT cells are damaged by UVB, the viability of HaCaT cells can be improved as the concentration of Cos-16 increases.

[0040] Example 4 Detection of intracellular ROS levels

[0041] Intracellular ROS levels were measured using a fluorescence assay (DCFH-DA assay). HaCaT cells seeded in 96-well plates were pretreated with 5 mM N-acetylcysteine ​​(NAC) or Cos-16 at the indicated concentrations for 1 h. The cells were then incubated in serum-free medium with 10 μM H2DCFDA for 30 min and exposed to 20 mJ / cm 2 UVB, followed by lysis with 0.1% Triton X-100 for 10 min. Fluorescence intensity was measured at 485 nm excitation and 528 nm emission. Intracellular ROS levels were expressed as fold changes in fluorescence intensity. Figure 6 As shown, Cos-16 was able to inhibit UV-induced ROS accumulation in HaCaT cells.

[0042] Example 5 Determination of GPx and CAT enzyme activities in HaCaT cells

[0043] Four different concentration gradient groups were set up in the experiment, and 3 replicate wells were set up in each group. HaCaT in the logarithmic growth phase was inoculated in a 24-well plate, and 1 mL of cell suspension was injected into each well. The inoculation density was 1×10 5 / mL, and then placed it in a 5% CO2 incubator for adherent culture at a temperature of 37°C. After culturing for 24 hours, the culture medium was discarded, and solutions of the antioxidant active polypeptide Cos-16 of different concentrations (2.5, 5, 10 μM) were prepared, and then 600 μL of solutions of the antioxidant active polypeptide Cos-16 of different concentrations were added to the cell culture medium of each well of the sample group. After continuing to culture for 2 hours, the cells were washed twice with PBS. The cells were lysed at 4°C for 10 minutes, the lysed cells were collected, and the supernatant was obtained according to the instructions of the kit. According to the operating procedures of the kit, the GPx and CAT enzyme activities were determined respectively, and the protein concentration was determined by the BCA protein concentration determination method. The results are as follows Figure 7 , Figure 8 As shown, it can be seen that with the increase in the concentration of the antioxidant active peptide Cos-16, the GPx and CAT enzyme activities in HaCaT cells are upregulated.

[0044] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention. Sequence Listing <110> Xiamen University <120> An antioxidant active polypeptide Cos-16 and its application <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence <400> 1 Ala Cys Phe Leu Glu Gly Leu 1 5

Claims

1. An antioxidant active polypeptide Cos-16, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

01.

2. Use of the antioxidant active polypeptide Cos-16 according to claim 1 in the preparation of an antioxidant health care composition.

3. An antioxidant health care composition, characterized in that: The effective ingredient comprises the antioxidant active polypeptide Cos-16 as claimed in claim 1.

4. Use of the antioxidant active polypeptide Cos-16 according to claim 1 in preparing a cosmetic composition.

5. A cosmetic composition, characterized in that: The effective ingredient comprises the antioxidant active polypeptide Cos-16 as claimed in claim 1.

6. Use of the antioxidant active polypeptide Cos-16 according to claim 1 in an anti-ultraviolet composition.

7. An anti-ultraviolet composition, characterized in that: The effective ingredient comprises the antioxidant active polypeptide Cos-16 as claimed in claim 1.

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