A spot-lightening polypeptide, a composition containing the polypeptide and its application

Abstract

The present invention relates to a spot-lightening polypeptide, a composition containing the polypeptide and applications thereof. The present invention provides a spot-lightening fusion polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1) and a cosmetic composition thereof, wherein the polypeptide amino acid sequence is shown in SEQ ID NO:4. The spot-lightening polypeptide has good antioxidant and in vitro tyrosinase inhibitory activities, and the cosmetic composition containing the polypeptide also has the activity of inhibiting melanin synthesis in melanoma cells, thereby being able to achieve a good skin spot-lightening effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetic peptides, and in particular relates to a spot-lightening polypeptide, a composition containing the polypeptide and applications thereof. Background Art

[0002] The color of human skin originates from melanin synthesized in the outermost layer of the skin, namely melanocytes, which determines the color of the skin. When the skin is exposed to sunlight, melanin protects skin cells from UV damage. The formation of melanin is mainly regulated by the expression of various melanin synthesis-related enzymes. Tyrosinase is the rate-limiting enzyme that controls the formation of melanin and is mainly involved in the production of L-dopaquinone. TRP-1 and TRP-2 catalyze specific steps of electron generation and stabilize tyrosinase activity.

[0003] Common cosmetic whitening ingredients are mainly tyrosinase inhibitors, such as arbutin, kojic acid and salicylic acid, but these whitening agents have certain side effects. Consumers prefer safe and effective bioactive peptide products, which are closely related to skin cell chemotaxis and migration, repair and regeneration, tissue cell proliferation, protein synthesis and secretion, metabolism and regulation, etc.

[0004] Nonapeptide-1 (melanostatin-5) is a peptide hormone that competes with α-melanocyte stimulating hormone (α-MSH) for melanocortin receptor-1 (MC1R), weakening the binding efficiency of the two, thereby blocking the activation of tyrosinase and inhibiting the synthesis of melanin.

[0005] As a natural biopolymer material, collagen has good biocompatibility, biodegradability and absorbability, and can promote cell formation and epithelial cell formation. It can be widely used in medicine, beauty, cosmetics, health care products and other fields. Its role in skin damage repair, plastic surgery, and enhancing the body's immunity has also been recognized.

[0006] Current research mainly focuses on the antioxidant and freckle-removing effects of bioactive peptides such as collagen, nonapeptide-1, tetrapeptide-30, and hexapeptide-2. Few studies have constructed fusion proteins of the above-mentioned active peptides to improve their antioxidant and freckle-removing effects. Summary of the invention

[0007] In order to solve the above problems, the present invention first provides a spot-lightening fusion polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1) and a cosmetic composition thereof. The spot-lightening polypeptide has good antioxidant and in vitro tyrosinase inhibitory activities. At the same time, the cosmetic composition containing the polypeptide also has the activity of inhibiting melanin synthesis in melanoma cells.

[0008] The present invention provides a light spot polypeptide, characterized in that the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 4.

[0009] The present invention also provides a spot-lightening composition, which comprises a spot-lightening polypeptide, an emulsifier, a moisturizer, a softener, a thickener and a chelating agent.

[0010] The present invention also provides an application of the spot-lightening composition in the preparation of skin care products.

[0011] The invention also discloses a skin care product, comprising the above-mentioned spot-lightening composition or spot-lightening polypeptide.

[0012] Compared with the prior art, the present invention has at least the following beneficial effects:

[0013] The present invention provides a freckle fusion polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1), the amino acid sequence of which is shown in SEQ ID NO: 4. The freckle polypeptide has good antioxidant and in vitro tyrosinase inhibitory activities;

[0014] The present invention provides a spot-lightening polypeptide composition for the first time, which has a better spot-lightening effect than other spot-lightening polypeptide compositions in the prior art, mainly reflected in the improvement of the inhibition rate of melanin synthesis in melanoma cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 SWISS-MODEL predicted the tertiary structure model of the dark spot fusion polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1);

[0016] Figure 2 SephadexG-25 separation and purification of the freckle fusion polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1) and SDS-PAGE purity detection;

[0017] Figure 3 Determination of in vitro tyrosinase inhibition rate of freckle fusion polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1);

[0018] Figure 4 Determination of the antioxidant capacity of the freckle fusion polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1);

[0019] Figure 5Determination of the inhibition rate of melanin synthesis in melanoma cells by freckle fusion polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1). DETAILED DESCRIPTION

[0020] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0021] Example 1 Construction of the Plasmid of the Light Spot Fusion Polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1)

[0022] A designed light spot fusion polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1) amino acid sequence and the corresponding nucleotide sequence are as follows:

[0023] NPP-1 (nonapeptide-1): YRKSKMVFK (SEQ ID NO: 1);

[0024] Linker: GGGGS (SEQ ID NO: 2);

[0025] Using the NCBI Reference Sequence: NP_001001135.2 recombinant collagen structure data recorded by NCBI, and using biological software analysis and simulation, we can parse the appropriate recombinant collagen fragments:

[0026] rCOL (recombinant collagen): GFPGPRGPPGPQGATGPLGPK (SEQ ID NO: 3);

[0027] Amino acid sequence of spot fusion polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1):

[0028] YRKSKMVFKGGGGSGFPGPRGPPGPQGATGPLGPKGFPGPRGPPGPQGATGPLGPKGFPGPRGPPGPQGATGPLGPKGFPGPRGPPGPQGATGPLGPKGGGGSYRKSKMVFK (SEQ ID NO: 4);

[0029] Nucleotide sequence of light spot fusion polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1):

[0030] tatcgcaaaagcaaaatggtgtttaaaggcggcggcggcagcggctttccgggcccgcgcggcccgccgggcccgcagggcgcga ccggcccgctgggcccgaaaggctttccgggcccgcgcggcccgccgggcccgcagggcgcgaccggcccgctgggcccgaaaggc tttccgggcccgcgcggcccgccgggcccgcagggcgcgaccggcccgctgggcccgaaaggctttccgggcccgcgcggcccgcc gggcccgcagggcgcgaccggcccgctgggcccgaaaggcggcggcggcagctatcgcaaaagcaaaatggtgtttaaataa(SEQ ID NO:5);

[0031] The tertiary structure model of the freckle fusion polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1) was predicted using SWISS-MODEL. Figure 1 .

[0032] Figure 1 The results showed that adding a linker between NPP-1 and (rCOL)4 and (rCOL)4 between NPP-1 and NPP-1 prevented the NPP-1 and rCOL active peptides from losing their functional activity due to mutual folding and entanglement in the tertiary structure of the protein.

[0033] The recombinant gene (NPP-1)-linker-(rCOL)4-linker-(NPP-1) was synthesized and provided by GenScript Biotechnology Co., Ltd., and BamHI and NotI restriction endonuclease sites were designed at both ends of the two DNA fragments, respectively. The recombinant gene fragment (NPP-1)-linker-(rCOL)4-linker-(NPP-1) was cut with BamHI and NotI, and the plasmid pET30a(+) (purchased from Novagen) was also cut with BamHI and NotI. The target fragment was separated by 1.5% agarose gel electrophoresis and excised. The DNA was purified by Gel Extraction Kit (Gel Extraction Kit). After purification using the PCR Kit, (NPP-1)-linker-(rCOL)4-linker-(NPP-1) and linear pET30a(+)DNA were obtained; the linear pET30a(+)DNA was treated with CIAP and connected to (NPP-1)-linker-(rCOL)4-linker-(NPP-1), and then transformed into Escherichia coli DH5α, and cloned and selected using kanamycin. The clones were re-cultured in 1.5 ml LB medium, and the expression plasmid pET30a(+)-(NPP-1)-linker-(rCOL)4-linker-(NPP-1) with the target DNA was extracted and purified by alkaline lysis method.

[0034] Example 2 Expression, purification and identification of freckle fusion polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1)

[0035] The BL21 strain containing the pET30a(+)-(NPP-1)-linker-(rCOL)4-linker-(NPP-1) recombinant plasmid was streaked and activated on LB solid medium containing kanamycin (50 μL / mL), and a single colony was picked and transferred to LB liquid medium containing kanamycin. When the OD600nm was 0.6-0.8, an appropriate amount of IPTG was added to induce the expression of the light spot fusion polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1). The induced bacterial solution was centrifuged, the precipitate was collected, resuspended, and the bacterial cells were disrupted by ultrasound (power 50%, ultrasound 3s, stop 3s, and disruption for 45min). The supernatant was adsorbed on a Ni2+ affinity column and eluted with 400mmol / L imidazole to obtain the eluate containing the light spot fusion polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1). Subsequently, the eluate containing the freckle fusion polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1) was dialyzed twice, each for 5 h. The first dialyzate contained 250 mmol / L NaCl, 10% glycerol, 2 mmol / L Tris-HCl, pH 7.4; the second dialyzate contained 150 mmol / L NaCl, and the other components and contents were the same as those of the first dialyzate. After freeze-drying, the crude freckle fusion polypeptide was finally obtained.

[0036] Take 5.0g of 50-100 mesh Sephadex G-25 particles and disperse them with an appropriate amount of distilled water. Stir while heating to make them swell for 2h, let it stand for a while, pour off the upper clear liquid, and repeat washing 2-3 times; then wash with pH7.0 Tris-acetate buffer 2-3 times, use ultrasonic degassing before perfusion to remove bubbles in the gel, dissolve the crudely extracted freckle fusion polypeptide freeze-dried powder in ultrapure water, prepare 100mg / mL sample solution, first filter through a 0.22μm filter membrane to remove impurities, and slowly add 3mL to the chromatography column with a plastic rubber dropper. The eluent is ultrapure water, the flow rate is set to 0.2-0.5 mL / min, the wavelength is set to 220nm, and the eluent is collected in a continuous tube according to the order of outflow, 2 in each tube. mL, determine the number of tubes to be collected, the collection time for each tube is 10 min, and obtain a spectrum in which the light spot fusion polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1) is concentrated in peak A. Then, the light spot fusion polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1) corresponding to peak A is tested for purity by SDS-PAGE. The results are as follows Figure 2 shown. Figure 2The results showed that the freckle fusion polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1) was concentrated in the peak A area, and the purity was high as detected by SDS-PAGE.

[0037] Example 3 Determination of in vitro tyrosinase inhibition rate of freckle fusion polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1)

[0038] Refer to the method of Geng Guangqing et al. (Geng Guangqing, Study on the Inhibitory Effect of Beauveria bassiana Extract on Tyrosinase Activity, Anhui: Anhui Agricultural University, 2011), and use L-tyrosine as the substrate. Take 40μL of 0.1mg / mL L-tyrosine solution, add 40μL of white spot fusion polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1) sample solution and 80μL of phosphate buffer solution, keep at 37℃ for 10min, then add 40μL of 250U / mL tyrosinase solution to start the reaction, measure the absorbance at 0min and 30min at 475nm, and calculate the inhibition rate of tyrosinase according to formula (1). The results are as follows: Figure 3 As shown. Tyrosinase inhibition rate / %=[1-(A1-A2) / (A3-A4)]×100% (1) Where: A1——absorbance of substrate, sample solution, tyrosinase and PBS system; A2——absorbance of sample solution, tyrosinase and PBS system; A3——absorbance of substrate, tyrosinase and PBS system; A4——absorbance of tyrosinase and PBS system.

[0039] Figure 3 The results showed that the inhibition rate of tyrosine phenolase activity of the spot-lightening fusion polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1) increased with increasing concentration. When the concentration was between 0.2 and 0.6 mg / mL, the inhibition rate was about 60% to 80%, showing a good dose-effect relationship. This indicates that the spot-lightening fusion polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1) has the potential to be developed as a whitening agent at the material level.

[0040] Example 4 Determination of the Antioxidant Capacity of the Spot-Lightening Fusion Polypeptide (NPP-1)-Linker-(rCOL)4-Linker-(NPP-1)

[0041] Prepare 7mmol / L ABTS solution and 140mmol / L potassium persulfate solution with ultrapure water respectively, take 88μL potassium persulfate solution and add it to 5mL ABTS solution, mix well at room temperature and avoid light for 12h, then dilute to an absorbance of 0.7±0.02 at 734nm to obtain ABTS working solution. Take 100μL sample solution and mix with 100μL ABTS working solution, react at room temperature for 10min, and measure the absorbance at 734nm. Use ultrapure water as blank control. Calculate the clearance rate according to formula (2), and the results are as follows: Figure 4 As shown. ABTS free radical scavenging rate / %=[1-(At-Ar) / A0]×100% (2) Where: At——absorbance of sample and ABTS working solution; Ar——absorbance of sample and ultrapure water; Ao——absorbance of ultrapure water and ABTS working solution. Figure 4 The results showed that as the concentration increased, the ABTS free radical scavenging rate of the freckle fusion polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1) increased, showing a good dose-effect relationship. When the concentration was 0.6 mg / mL, the scavenging rate reached 86.32%, indicating that the freckle fusion polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1) also has the potential to develop antioxidants at the material level.

[0042] Example 5 Determination of the inhibition rate of melanin synthesis in melanoma cells by a whitening and spot-lightening skin care product containing spot-lightening fusion polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1)

[0043] Group A contains white spot lightening skin care product components of spot lightening fusion polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1): 40 parts of spot lightening fusion polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1), 30 parts of emulsifier, 10 parts of moisturizer, 10 parts of emollient, 0.5 parts of thickener and 0.5 parts of chelating agent, which are fully stirred and cooled to 30°C by a homogenizer to prepare a whitening and spot lightening skin care product. Group B contains white spot lightening skin care product components of nonapeptide-1: 40 parts of nonapeptide-1, 30 parts of emulsifier, 10 parts of moisturizer, 10 parts of emollient, 0.5 parts of thickener and 0.5 parts of chelating agent, which are fully stirred and cooled to 30°C by a homogenizer to prepare a whitening and spot lightening skin care product. Group C contains the white spot-lightening skin care product components of hexapeptide-2: 40 parts of nonapeptide-1, 30 parts of emulsifier, 10 parts of moisturizer, 10 parts of softener, 0.5 parts of thickener and 0.5 parts of chelating agent, which are fully stirred and cooled to 30°C by a homogenous mixer to prepare a whitening and spot-lightening skin care product. Group D contains the white spot-lightening skin care product components of tetrapeptide-30: 40 parts of nonapeptide-1, 30 parts of emulsifier, 10 parts of moisturizer, 10 parts of softener, 0.5 parts of thickener and 0.5 parts of chelating agent, which are fully stirred and cooled to 30°C by a homogenous mixer to prepare a whitening and spot-lightening skin care product. The whitening and freckle-removing skin care products prepared in the AD group were diluted to a concentration of 0.2 g / mL with RPMI-1640 liquid culture medium, and filtered with a 0.22 μm sterile filter for later use; the second-generation B16 mouse melanoma cells were cultured in a 96-well plate at a density of 1×104 / mL, and the medium was changed after 24 hours. The whitening and freckle-removing skin care products of the AD group were added, with 3 wells for each skin care product and a skin care product concentration of 0.2 g / mL. The skin care products were replaced with RPMI-1640 culture medium in the control group. After incubation at 37°C and 5% CO2 for 72 hours, the supernatant was discarded. 1 mL of 2.5 g / L pancreatin was added to each well and digested for 5 minutes at room temperature. 4 mL of culture medium was added to stop the digestion and blown into a single cell suspension. Take 0.5mL for cell counting, centrifuge the rest of the cell suspension at 1500r / min for 10min, discard the supernatant, add 1mL of 1mol / L NaOH solution, incubate in a 90℃ water bath for 2h, and measure the absorbance (OD) value at 490nm on a microplate reader. Calculate the inhibition rate of melanin synthesis by the whitening and spot-lightening skin care products in the AD group according to the following formula:

[0044] Melanin synthesis inhibition rate / %=[1-(absorbance value of sample well × cell density of control well) / (absorbance value of control well × cell density of sample well)]×100% Figure 5The results showed that the whitening and spot-lightening skin care products prepared by the AD groups can effectively inhibit the synthesis of melanin and have a certain whitening and spot-lightening effect; among them, the whitening and spot-lightening skin care products prepared by the A group have a much higher inhibition rate of melanin synthesis in melanoma cells than the whitening and spot-lightening skin care products prepared by other groups, indicating that the spot-lightening fusion polypeptide (NPP-1)-linker-(rCOL)4-linker-(NPP-1) has a better spot-lightening effect than other spot-lightening polypeptides. The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A spot-lightening polypeptide, characterized in that The amino acid sequence of the polypeptide is shown in SEQ ID NO:

4.

2. A composition containing the spot-lightening polypeptide according to claim 1, characterized in that: The composition also includes emulsifiers, humectants, emollients, thickeners and chelating agents.

3. Use of the spot-lightening polypeptide according to claim 1 in preparing spot-lightening skin care products.

4. Use of the composition according to claim 2 in preparing a spot-lightening skin care product.

5. A spot-lightening skin care product, comprising the spot-lightening polypeptide according to claim 1 or the composition according to claim 2.

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