Polypeptide nigrocin-0a27 with whitening activity and application thereof

Abstract

This invention provides a skin-whitening peptide, Nigrocin-OA27, and its applications. The amino acid sequence of Nigrocin-OA27 is GFLSKPLPVGRKIVPWLSGLC. Its application is in inhibiting tyrosinase activity. This invention provides a novel peptide, Nigrocin-OA27, containing only 21 amino acids. This peptide can inhibit tyrosinase activity, thereby reducing the melanin content in the skin, and has the potential to be developed into skin-whitening products and drugs for the prevention or treatment of melanin-related diseases. Furthermore, the peptide Nigrocin-OA27 provided by this invention is easy to synthesize, low in cost, and easy to promote and apply.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a polypeptide Nigrocin-OA27 and its application in inhibiting tyrosinase activity. Background Technology

[0002] Melanocytes are widely distributed in the basal layer of the epidermis of human skin, and their number and distribution are not significantly different among different ethnic groups. After exposure to stimuli such as ultraviolet radiation, melanocytes can secrete large amounts of melanin to protect the skin from oxidative damage. Melanin is an important preventative factor, especially in preventing skin cancer; therefore, a certain level of melanin is essential for skin protection. However, when melanin secretion is excessive or unevenly distributed, it can cause problems such as age spots, post-inflammatory hyperpigmentation, and even melanoma, resulting in serious aesthetic and health issues. It is estimated that about 15% of the global population invests in skin whitening agents, with Asia dominating the market. Global Industry Analysts (GIA) predict that, driven by new markets in Asia, particularly India, Japan, and China, the global market for skin whitening products reached $23 billion in 2020. In India alone, it is estimated that $432 million was spent on brightening creams and skin care products in 2010. A recent survey shows that 80% of Indian men use whitening face creams, and the number of consumers is growing at a rate of 18% annually. Therefore, safe and effective skin whitening agents have a broad market and clinical demand.

[0003] However, most commonly used skin whitening agents are derived from plant extracts and inorganic substances, which often cause various health problems when applied to the human body. For example, Vitamin C can easily cause allergies and contact dermatitis; hydroquinone and kojic acid are only used in limited concentrations as skin whitening agents in a few countries, and are banned in some countries because they may cause thyroid cancer, nephrotoxicity, genotoxicity, and cytotoxicity to melanocytes; arbutin is hydrolyzed into D-glucose and hydroquinone by the main skin flora (Staphylococcus epidermidis and Staphylococcus aureus), enzymes (such as glycosidases), and temperature. Therefore, the discovery of safe and effective new skin whitening agents is urgently needed.

[0004] In recent years, researchers have been searching for novel skin-whitening agents that are highly active, low in toxicity, and have small molecular weights. Short peptides (SPs) have become a research focus due to their advantages such as short sequences, high activity, low toxicity, and the ability to be artificially designed and synthesized. Naturally derived skin-whitening agents are considered to be less toxic and healthier. Animal-derived natural skin-whitening active peptides offer even more advantages, combining the benefits of both short peptides and naturally derived skin-whitening agents, while also exhibiting better affinity for the human body and providing greater advantages in studying their mechanisms of action. Amphibian skin is known as a "natural peptide library," yet very few peptides with skin-whitening effects have been reported to date.

[0005] The present invention aims to provide a polypeptide with whitening effect derived from the Yunnan stink frog. Summary of the Invention

[0006] The first objective of this invention is to provide a polypeptide Nigrocin-OA27, and another objective of this invention is to provide applications of said polypeptide Nigrocin-OA27.

[0007] The first objective of this invention is achieved by providing a polypeptide, Nigrocin-OA27, with the amino acid sequence GFLSKPLPVGRKIVPWLSGLC.

[0008] Another object of the present invention is achieved by using the polypeptide Nigrocin-OA27 in the inhibition of tyrosinase activity.

[0009] The beneficial effects of this invention are as follows: This invention provides a novel polypeptide, Nigrocin-OA27, containing only 21 amino acids. This polypeptide, Nigrocin-OA27, can inhibit tyrosinase activity, thereby reducing the melanin content in the skin, and has the potential to be developed into skin whitening products and drugs for the prevention or treatment of melanin-related diseases. Furthermore, the polypeptide Nigrocin-OA27 provided by this invention is easy to synthesize, low in cost, and easy to promote and apply. Attached Figure Description

[0010] Figure 1 This is a primary structure diagram of the polypeptide Nigrocin-OA27 of the present invention;

[0011] Figure 2 The diagram shows the effect of the present invention's polypeptide Nigrocin-OA27 on B16 cells, where A is the dopa colorimetric reaction of B16 cells, B is the melanin production content of B16 cells, C is the effect on the tyrosinase activity of B16 cells, and D is the quantification of the change in melanin content of B16 cells.

[0012] Figure 3 This diagram illustrates the preventive effect of the present invention's peptide Nigrocin-OA27 on the melanin model in C57 mice. A represents the apparent image of melanin deposition; B represents an HE staining image of ear tissue; C represents a Masson-Fontana (melanin staining) image of ear tissue; D represents an immunohistochemical staining image of TYR factor; E represents an immunohistochemical staining image of MITF factor; F represents the quantification of HE staining in ear tissue; G represents the quantification of melanin staining in ear tissue; H represents the quantification of immunohistochemical staining of TYR factor; and I represents the quantification of immunohistochemical staining of MITF factor.

[0013] Figure 4This image shows the skin-whitening effect of the present invention's peptide Nigrocin-OA27 on melanin in the ears of C57 mice. A is an apparent image of melanin deposition; B is an image of ear tissue stained with hematoxylin and eosin (HE); C is an image of ear tissue stained with Masson-Fontana (melanin); D is an image of TYR factor immunohistochemical staining; E is an image of MITF factor immunohistochemical staining; F is the quantification of HE staining in ear tissue; G is the quantification of melanin staining in ear tissue; H is the quantification of TYR factor immunohistochemistry; and I is the quantification of MITF factor immunohistochemistry. Detailed Implementation

[0014] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0015] The present invention discloses a polypeptide Nigrocin-OA27, the amino acid sequence of which is GFLSKPLPVGRKIVPWLSGLC, as shown in SEQ ID NO.1.

[0016] The present invention also provides the application of the polypeptide Nigrocin-OA27 in inhibiting tyrosinase activity.

[0017] The present invention further provides the application of the peptide Nigrocin-OA27 in the preparation of a drug for the prevention or treatment of melanin-related diseases.

[0018] The present invention further provides the application of the peptide Nigrocin-OA27 in the preparation of whitening skin care products.

[0019] The whitening skincare products mentioned include skin creams, sunscreens, lotions, serums, toners, facial cleansers, face masks, or shower gels.

[0020] The present invention also provides a whitening skin care product, wherein the whitening skin care product uses the polypeptide Nigrocin-OA27 as the main active ingredient or one of the active ingredients.

[0021] Example 1: Screening of the peptide Nigrocin-OA27

[0022] Step 1: Adult Yunnan stink frogs (from Yunnan Province, China) were kept in a 21 cm × 21 cm × 15 cm tank for 7 days to acclimatize to the environment. The water was changed regularly and the frogs were fed mealworms. After rinsing the skin of the Yunnan stink frogs with deionized water, they were quickly killed by destroying their brains and their skin was collected.

[0023] Step 2: Construction of Yunnan Stink Frog cDNA Library: RNA was extracted from the obtained skin tissue using a total RNA extraction kit (TIANGEN, China). After extraction, the RNA molecules were reversed into cDNA molecules to establish a polypeptide cDNA library derived from Yunnan Stink Frog skin tissue.

[0024] Step 3: To screen for cDNA encoding mature Nigrocin-OA27, polymerase chain reaction (PCR) templates were obtained from cDNA synthesized using SMART technology, along with 5' PCR primers (5'-CCAAA(G / C)ATGTTCACC(T / A)TGAAGAA-3') and 3' PCR primers (5'ATTCAGGCCGAGGCC GACA TG-3') synthesized by BGI (China). The obtained PCR products were cloned into viable E. coli DH5α cells and sequenced using an Applied Biosystems DNA sequencer (ABI 3730XL, Foster City, California, USA). The results are as follows: Figure 1 As shown, the full-length cDNA sequence is 305 bp (containing 305 bases), encoding 67 amino acid residues, and the mature peptide sequence is “GFLSKPLPVGRKIVPWLSGLC”. Figure 1 (Underlined portion of the middle base sequence). A search revealed this peptide to be a novel peptide, which was named Nigrocin-OA27.

[0025] The polypeptide Nigrocin-OA27 was synthesized by Wuhan Baiyixin Biotechnology Co., Ltd.

[0026] Example 2: Activity detection of the peptide Nigrocin-OA27

[0027] 1. Effects of the peptide Nigrocin-OA27 on melanin production in B16 cells

[0028] B16 cells were digested and centrifuged, counted using a hemocytometer, and then diluted to approximately 50,000 cells / mL. 2 mL of cell suspension was added to each well of a six-well plate, divided into five groups: a blank control group, a 20 μM arbutin group (positive control group), and groups containing 5 μM, 10 μM, and 20 μM Nigrocin-OA27 peptides. After 72 h of culture, cells from each group were digested, centrifuged, and collected into centrifuge tubes. 300 μL of 1 mM NaOH solution was added, and after complete dissolution and mixing of melanin, the absorbance was measured at 475 nm using a microplate reader to calculate the effect on melanin production.

[0029] Result: As Figure 2 B and Figure 2As shown in D, the peptide Nigrocin-OA27 reduces the melanin content of B16 cells, and the melanin content of B16 cells is inversely proportional to the peptide concentration.

[0030] 2. Effects of the peptide Nigrocin-OA27 on tyrosinase activity in B16 cells

[0031] Cells in each well of the above groups were lysed with 300 μL of 1% Triton-X100 solution, centrifuged at 12000 rpm for 15 min at 4℃, and the supernatant was collected into a new centrifuge tube. 100 μL of supernatant and 100 μL of 1 mg / ml L-DOPA substrate solution were added to each well of a 96-well plate, and the plates were incubated at 37℃ for 30 min. The absorbance was then measured at 475 nm using a microplate reader to observe the effect on the tyrosinase activity of B16 cells.

[0032] Result: As Figure 2 As shown in Figure C, the peptide Nigrocin-OA27 can significantly reduce the tyrosinase activity of B16 cells, and the tyrosinase activity of B16 cells is inversely proportional to the peptide concentration.

[0033] 3. Dopa colorimetric reaction

[0034] Each well in each group was washed three times with PBS, and then the cells were fixed with 4% formaldehyde solution for 15 min. After rinsing three times with deionized water, 2 mg / ml L-DOPA solution was added and the cells were incubated at 37°C for 3-6 h. The cells were then rinsed once with deionized water and once with PBS, and the dopa colorimetric reaction of B16 cells in each group was observed and photographed under a microscope.

[0035] Result: As Figure 2 As shown in Figure A, the dopa staining of B16 cells treated with the peptide Nigrocin-OA27 was lighter.

[0036] 4. The preventive and whitening effects of the peptide Nigrocin-OA27 on ultraviolet-induced ear pigmentation in C57 mice.

[0037] Prevention group: Female C57 mice (6-8 weeks old, 18-22 g) were divided into 5 groups: blank control group, UVB group, propylene glycol group (negative control), arbutin group (positive control), and Nigrocin-OA27 (1 mM) group. Arbutin, propylene glycol, and propylene glycol-soluble Nigrocin-OA27 were applied to the ears of C57 mice at a dose of 20 μL / ear / day. Five hours after application, except for the blank control group, the other groups were irradiated with a UV lamp at 70 mJ / cm². 2 After 17 consecutive days of irradiation, the pigmentation of the mouse ears was observed and photographed, and the mouse ear tissue was taken for pathological histological analysis.

[0038] Whitening Group: Similar to the prevention group, female C57 mice (6-8 weeks old, 18-22 g) were divided into 5 groups. Except for the blank control group, the remaining mice were irradiated with ultraviolet lamps at 70 mJ / cm². 2 For 23 consecutive days, with a 2-day interval after irradiation, mice were treated with 1 mM Nigrocin-OA27 and arbutin dissolved in propylene glycol, 20 μL / ear / day. The negative control group was treated with the same volume of blank propylene glycol. The UVB group and the blank control group were not treated with any drugs. After 30 days of drug treatment, the pigmentation of the mouse ears was observed and photographed, and mouse ear tissue was collected for histopathological analysis.

[0039] Result: As Figure 3 , 4 As shown, the peptide Nigrocin-OA27 can reduce melanin content by inhibiting the MITF pathway, and can effectively prevent and reduce UVB-induced ear pigmentation in mice.

[0040] Conclusion: The active peptide Nigrocin-OA27 provided by this invention has excellent inhibitory activity against B16 cell tyrosinase, which can inhibit melanin deposition in cells and has the potential to be developed into skin whitening products and drugs for the prevention or treatment of melanin-related diseases.

Claims

1. A polypeptide Nigrocin-OA27, the amino acid sequence of which is GGLSKPLPVGRKIVPWLSGLC.

2. The use of the polypeptide Nigrocin-OA27 of claim 1 in the preparation of whitening skin care products.

3. The application according to claim 2, characterized in that, The whitening skincare products mentioned include skin creams, sunscreens, lotions, serums, toners, facial cleansers, face masks, or shower gels.

4. A whitening skincare product, characterized in that, The whitening skincare product uses the polypeptide Nigrocin-OA27 described in claim 1 as one of its active ingredients.

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