TWI614022B - Peptide, method and composition for decreasing melanin content - Google Patents

Abstract

The present invention provides an isolated peptide for reducing melanin content in a mammalian individual. The isolated peptide is composed of the amino acid sequence of SSASTTED (SEQ ID NO: 1). The invention also provides methods and compositions for inhibiting melanin production in a mammalian individual.

Description

Peptides, methods and compositions for reducing melanin content

The present invention generally relates to a peptide that inhibits melanin production or reduces melanin content in a mammalian individual, as well as methods and compositions thereof.

The most significant cause of human pigmentation is due to the synthesis and distribution of melanin in the skin and hair. Therefore, the color of the skin and hair depends mainly on the type of pigment present and its concentration.

U.S. Patent No. 6,579,848 is directed to the use of the squirrel signal protein and peptide and pharmaceutical compositions thereof and methods for inhibiting melanin production by melanocytes. US Patent No. 8,669,238 discloses a method for treating hyperpigmentation, comprising administering a composition to an individual suffering from hyperpigmentation, the composition comprising at least one inhibitor selected from the group consisting of Inhibitors of kinesin Kif13A inhibitors, inhibitors of the AP-1 linker complex subunit, and interactions between the AP-1 linker complex subunit or the AP-1 linker complex and the kinesin Kif13A . U.S. Patent No. 8,455,023 relates to Cinnamomum subavenium extract and its use in inhibiting melanin production in whitening beauty.

The present invention unexpectedly finds that the peptide having the sequence SSASTTED (SEQ ID NO: 1) is effective in inhibiting or reducing melanin production in a mammalian individual.

Thus, in one aspect, the invention provides an isolated peptide useful for inhibiting melanin production or reducing melanin content of melanocytes in a mammalian individual in a mammalian individual. The isolated peptide is composed of the amino acid sequence of SEQ ID NO: 1.

In another aspect, the present invention provides a method for inhibiting melanin production or reducing melanin content of melanocytes in a mammalian individual, comprising administering to the individual or the melanocyte an effective amount of an individual according to the present invention The peptide. In a preferred embodiment of the invention, the isolated peptide is administered topically to the individual.

In still another aspect, the present invention provides a composition for inhibiting melanin production or reducing melanin content of melanocytes in a mammalian individual in a mammalian individual. The composition comprises an effective amount of an isolated peptide consisting of the amino acid sequence of SEQ ID NO: 1. According to a particular embodiment of the invention, the composition may further comprise an acceptable carrier and may be formulated as a topical formulation.

The above general description and the following detailed description are intended to be illustrative and not restrictive.

The foregoing summary, as well as the following detailed description, will be better understood by For the purpose of illustrating the invention, the present preferred embodiments are shown in the drawings.

In the drawings: Figure 1A shows reduced melanin production by cells of the invention in cells. B16F10 melanoma cells were treated with 10-50 μg/ml of the peptide of SEQ ID NO: 1 for 5 days, and the medium was changed on the third day. The cells and medium were collected in test tubes. The inhibitory effect of citric acid on tyrosinase activity is known, and therefore, it was used as a negative control group in a cell test. It is known that IBMX can increase the content of intracellular cAMP, thereby stimulating melanin production, and therefore, in the cell test, it is used as a positive control group.

Figure 2A shows the melanin content in the cells. Figure 2B shows the secretion of melanin in the medium. Each measurement was three replicates and the data is expressed as mean ± SD. *p<0.05 compared to the control group.

Figure 3 shows the increased tyrosinase activity by the peptide of the present invention. *p<0.05 compared to the control group.

Figure 4 shows the results of DOPA staining.

In one aspect, the invention provides an isolated peptide for use in inhibiting melanin production or reducing melanin content of melanocytes in a mammalian individual, the isolated peptide being SSASTTED (SEQ ID NO: 1) The amino acid sequence consists of.

In another aspect, the present invention provides a method for inhibiting melanin production or reducing melanin content of melanocytes in a mammalian individual in a mammalian individual. The method comprises the steps of: displacing the isolated peptide of SEQ ID NO: 1 in an amount effective to inhibit melanin production in the mammalian individual, or in an amount effective to reduce the melanin content of the melanocyte The mammalian individual or the melanocytes of the mammalian individual. Preferably, the isolated peptide is administered topically.

In a further aspect, the invention provides a composition comprising an effective amount of an isolated peptide consisting of the amino acid sequence of SEQ ID NO: 1. The composition of the present invention can be used to inhibit melanin production or to reduce melanin content in melanocytes of a mammalian individual. The composition can be used for cosmetic purposes, for example, skin whitening.

In a particular embodiment of the invention, the composition further comprises a (physiologically) acceptable carrier.

In a particularly preferred embodiment, the compositions of the invention are formulated as a topical formulation.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning meaning meanings

The term "a" or "an" is used in conjunction with the term "comprising" in the context of the patent application and/or the specification, meaning "one" or "one or more", "at least one" The meaning of "one or more than one" is the same.

The phrase "peptide" is used herein in its ordinary meaning, that is, a polymer whose monomers are amino acids and which are linked to each other by a guanamine bond, or alternatively, Polypeptide. When the amino acid is an α-amino acid, an L-optical isomer or a D-optical isomer can be used. In addition, non-natural amino acids such as β-alanine, phenylglycine and homoarginine may also be included. Use the standard abbreviation for amino acids.

The term "carrier" as used herein refers to a material that is generally used to modulate a pharmaceutical or cosmetic composition that enhances stability, sterility, and delivery. When the peptide delivery system is formulated as a solution or suspension, the delivery system is in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, for example, water, buffer, 0.8% saline, 0.3% glycine, hyaluronic acid, and the like. The composition may contain physiologically acceptable auxiliary substances as needed to approximate physiological conditions, such as pH adjustment and buffering agents, solution tonicity adjusting agents, wetting agents, and the like, for example, sodium acetate, sodium lactate, chlorine Sodium, potassium chloride, calcium chloride, sorbitan monolaurate, oleic acid triethanolamine, and the like.

The term "local" or "locally" is used herein in its ordinary meaning to mean a position that can be in or on any part of the body, including but not limited to the epidermis, any other dermis, or any other. Body tissue. Topical administration or administration means contacting the peptide directly with the tissue, such as a skin or membrane containing melanin producing cells.

The present invention contemplates the use of the isolated peptide of SEQ ID NO: 1 as an active ingredient for various uses. In a preferred embodiment, the isolated peptide of the present invention is combined with an acceptable carrier to form a topical preparation which can be placed on the skin. Topical formulations may include ointments, lotions, pastes, creams, gels, drops, sachets, sprays, solutions, shampoos, hair conditioners, powders, and transdermal patches. A thickener, a diluent, an emulsifier, a dispersing aid or a binder may be used as needed. Preferably, the function of the carrier is to increase the skin permeability of the peptide of the present invention and to deliver the peptide to melanocytes under in vivo conditions. Suitable carriers are well known to those skilled in the art and include, but are not limited to, water, dimethyl hydrazine, ethanol, liposomes, liquid paraffin, paraffin, dimethylformamide, 2-pyrrolidone, Oleic acid, as well as the penetration enhancer of the brand Azone®.

The invention is further illustrated by the following examples, which are provided by way of illustration and not limitation.

Instance

Example 1: The peptide of SEQ ID NO: 1 inhibits melanogenesis in mouse melanoma cells

1. Materials and methods

1.1 Preparation of isolated peptide of SEQ ID NO: 1

The peptide obtained from human lactoferrin and having the amino acid sequence of SSASTTED (SEQ ID NO: 1) (796.75 Da) was synthesized by MD Bio, Inc. (Taipei, Taipei). The purity and composition of the peptide were confirmed by high performance liquid chromatography (HPLC) and mass spectrometry. 10 mg of the peptide powder was dissolved and mixed with 1 ml of double deionized water (ddH ₂ O) to prepare a 10 mg/ml sample of the peptide of SEQ ID NO: 1, which was stored at -20 ° C until use.

1.2 Cell culture

B16F10 mouse melanoma cells were cultured in DMEM without phenol red and containing 10% fetal bovine serum and penicillin/streptomycin (100 IU/50 g/mL) under humidified air containing 5% CO ₂ at 37 °C. .

1.3 Melanin content detection

The method of Lee et al ., J Invest Dermatol 124, 405-411, 2005 was slightly modified to determine the melanin content in cultured B16F10 cells. Briefly, B16F10 cells were seeded in 6-well culture dishes (2 x 10 ⁴ cells/well) and cultured overnight to attach the cells. After 5 days of treatment with various test samples in an incubator, washing, trypsin treatment and counting before pelleting. After boiling for 1 hour in 1 M NaOH, the melanin in each cell was quantified, and the melanin content of each sample was read from a calibration curve for synthetic true melanin at 400 nm and converted into 3 independent experiments. Mean pcDNA/cell mean ± SE.

1.4 Determination of tyrosinase

Tyrase is the rate-limiting enzyme in the melanin production pathway. Its assay provides a high specificity and sensitivity index for the degree of induction of melanin production. The tyrosinase activity of cultured B16F10 cells was determined by a slight modification according to the method of Bellei et al ., J Biol Chem 285, 7288-7299, 2010. Briefly, B16F10 cells were seeded in 6-well culture dishes (2 x 10 ⁴ cells/well) and cultured overnight to attach the cells. After 5 days of treatment with various test samples in an incubator, the cells were washed with PBS, after which the cells were collected using trypsin. At the end of the culture, the cells were lysed with phosphate buffer (pH 6.8) containing 1% Triton X-100. The cells were then broken by freezing and thawing, and the lysate was clarified by centrifugation at 10,000 x g for 10 minutes. After protein quantification and protein concentration adjustment in lysis buffer, 100 μl of each lysate (each containing the same amount of protein) was equally dispensed into a 96-well plate, and then 100 μl of 5 mM L-DOPA was added to each well. . After incubating at 37 ° C for 30 minutes, the absorbance at 475 nm was measured with a spectrophotometer. The assay was repeated three times.

1.5 DOPA staining

DOPA staining was also performed to determine the enzyme activity of tyrosinase. B16F10 cells were seeded in 6-well culture dishes (2 × 10 ⁴ cells/well), and cultured overnight to attach the cells. After 4 days of treatment with various test samples in an incubator, the cells were washed with PBS, fixed with 2% paraformaldehyde, and further washed three times with PBS, and the cells were reacted at 0.1 ° DOPA (dissolved in 0.1 M at 37 ° C). In PBS for 5 hours and observed by light microscopy (Wang et al ., Exp Ther Med 6, 967-972, 2013).

2. Results

2.1 Impact on melanin production

B16F10 cell cultures treated with 50 μg/ml of the peptide of SEQ ID NO: 1 showed less cytochrome (Fig. 1).

2.2 Effect on melanin content

Treatment of B16F10 cells with 10 and 50 μg/ml of the peptide of SEQ ID NO: 1 showed a significantly reduced amount of melanin synthesis ( Fig. 2A and Fig. 2B ).

2.3 Effect on tyrosinase activity

10 and 50μg / ml of SEQ ID NO: 1 peptide treated B16F10 cells exhibit the activity of tyrosinase (FIG. 3) is significantly reduced, and the cells also exhibited weak staining DOPA (FIG. 4).

Those skilled in the art will appreciate that the above-described embodiments may be modified without departing from the broad inventive concept of the invention. Therefore, it is understood that the invention is not limited to the particular embodiments disclosed,

<110> Muscle Life Research Institute Co., Ltd.

<120> peptides, methods and compositions for reducing melanin production

<130> 0766/REN0002TW

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 8

<212> PRT

<213> Homo sapiens

<400> 1

Claims (10)

An isolated peptide for inhibiting melanin production in a mammalian subject, which consists of the amino acid sequence of SEQ ID NO: 1. A use of the isolated peptide as claimed in claim 1 for the preparation of a skin lightening formulation. The use of Patent Range 2, wherein the content of melanin is reduced by inhibiting the production of melanin by the isolated peptide as claimed in claim 1. The use of Patent Rep. 2, wherein the isolated peptide according to claim 1 is administered topically to the mammalian individual. A composition for inhibiting melanin production in a mammalian subject comprising an effective dose of the isolated peptide as claimed in claim 1. The composition of claim 5, which further comprises a physiologically acceptable carrier. The composition of Patent Application No. 5 is prepared as a topical preparation. The composition of claim 7, wherein the topical preparation comprises an ointment, lotion, cream, gel, drop, spray or liquid. The composition of claim 8, wherein the topical preparation is a lotion or cream. The composition of claim 8, wherein the topical preparation is a gel.