KR101250812B1 - Undecenoyl dipeptide derivatives and skin whitening composition containing the same - Google Patents

Abstract

The present invention relates to an undecenoyl dipeptide derivative represented by the following Formula 1 and a cosmetic composition for skin whitening comprising the same. The undecenoyl dipeptide derivatives of the present invention act as an antagonist of MC1R, an alpha melanocyte stimulating hormone (a-MSH) receptor, to inhibit the production of melanin in the cell, and do not cause an inflammatory response that the existing MC1R antagonist has with a whitening effect. In addition, the effect of inhibiting the inflammatory response by UV has been demonstrated. It also inhibited the endothelin-induced stimulation because it cross-reacted during the downstream signaling of MC1R and the endothelin B-type receptor. These undecenoyl dipeptide derivatives are harmless to the human body, and have excellent skin permeability and stability, and thus can be used very effectively as skin whitening cosmetics with anti-inflammatory effects.
[Formula 1]
CH ₂ = CH (CH ₂ ) ₈ -C (= O) -A ₁ -A ₂ -OR
In the formula, -C (= O)-means carbonyl, A ₁ , A ₂ are amino acid residues selected from Leu, Phe, Trp and Gly, respectively, where Leu, Phe and Trp are L- Or a D-form or a mixture thereof, R is H, C ₁ -C ₁₈ alkyl, phenyl, or benzyl, the undecenoyl group binds to an amine group of A ₁ , and OR is a carbon of A ₂ To bind to a carbonyl group.

Description

Undecenoyl dipeptide derivatives and skin whitening composition containing the same}

The present invention relates to an undecenoyl dipeptide derivative and a cosmetic composition for skin whitening comprising the same, and more particularly, acts as an antagonist of MC1R, an alpha melanocyte stimulating hormone (α-MSH) receptor, leading to skin blackening. It binds to its receptor, MC1R, through competitive binding with its alpha melanocyte stimulating hormone, which blocks melanin synthesis pathways and effectively blocks inflammation-related signaling pathways, resulting in undecenoyl depeptides with excellent skin whitening and anti-inflammatory effects. It relates to a derivative and a cosmetic composition for skin whitening comprising the same.

Melanin (melanin) is produced in the melanocytes (Melanosome), which are organelles that exist in cells called melanocytes (Melanocytes) in the basement layer of the skin. The amount is determined. The production of melanin is determined by genetic factors such as congenital factors, female hormones, drugs, pregnancy, ultraviolet rays, stress, etc., which are most influenced by UV rays. Melanin absorbs the light energy of ultraviolet rays and protects cells from damage caused by ultraviolet rays. When abnormally low melanin is produced, skin lesions such as vitiligo are caused, and excessive production not only damages the skin, but also forms blemishes and freckles, and moreover, causes skin cancer. The skin of modern man is damaged by exposure to environmental pollution, external stimuli such as ultraviolet rays and stress. Thus, the damaged skin has a slow recovery rate, wrinkles, decreased elasticity and skin pigmentation. Among these, especially in Asian women, the expectation for the whitening effect is very high, and various cosmetics have been developed to reduce the color of melanin already produced or to suppress the production of new melanin.

Important mechanisms in melanin biosynthesis are largely due to melanocyte stimulating hormone (α-MSH) and endothelin-1.

When the α-MSH binds to the melanocortin-1 receptor (MC1R), which is an α-MSH receptor present in the melanocyte membranes, by stimulating melanin forming cells such as ultraviolet rays, hormones, and drugs, the stimulated MC1R Activates an enzyme called adenylate cyclase, which increases cyclic adenosine monophosphate (cAMP) in the cell and causes a series of processes of melanin biosynthesis to occur continuously by increased cAMP. do. cAMP sequentially activates protein kinase A (PKA) and CREB transcription factors to promote expression of MITF (Microphthalmia associated transcription factor), a precursor of α-MSH. When MITF causes transcriptional activity of melanin synthesis-related genes such as tyrosinase, tyrosinase related protein 1 (TRP1) and dopachrome tautomerase (DCT), tyrosine in melanocytes (tyrosine) in the melanocytes (tyrosinase; Important enzyme) is converted into dopa (DOPA; 3,4-dihydroxyphenylalanine) and dopaquinone.

Endothelin-1 is also increased by stimulation of ultraviolet rays, hormones, drugs, and the like, and binds to endothelin B type receptor (ETbR) present in melanocyte membranes. Phospholipase C (PLC) stimulated by ETbR is used to convert phosphatidylinositol 4,5-bisphosphate (PIP ₂ ) present in the cell membrane into diacyl glycerol (DAG) and inositol. Decompose with 1,4,5-trisphosphate (inositol 1,4,5-trisphosphate: IP ₃ ). IP ₃ binds to the IP ₃ receptors of the endoplasmic reticulum, opening up the calcium channel of the endoplasmic reticulum, and increasing the calcium concentration in the cytoplasm is involved in melanin synthesis through several chain reactions in the cell. (Protein kinase C: PKC) is activated. Endoderin activates PKA, as well as PKC, through cAMP stimulation. It is known that melanogenesis occurs through the cross-talk of these two kinase enzymes. Endothelin-1 / ETbR signaling is also synergistic with SCF / c-KIT, another pathway involved in melanin synthesis, increasing tyrosinase expression, leading to skin blackening.

Through this sequential and complex reaction, pheomelanin (phemelanin (red melanin) is formed), or through enzymatic reactions such as TRP1, DCT, eumelanin (eumelanin (melanin, black or brown melanin)) is formed. The produced melanin is transferred from the melanocytes to the keratinocytes (keratinocytes), which form the epidermis, through the melanosomes, and is deposited on the skin when overproduced according to the amount of melanin produced in this process. It will cause freckles and skin blackening.

Conventionally, as a skin whitening material, a substance having a tyrosinase inhibitory activity such as arbutin, ascorbic acid, kojic acid, glutathione, and the like is added to the cosmetic to give a skin whitening effect, It has improved skin hyperpigmentation such as blemishes and freckles. However, for example, kojic acid effectively inhibits enzymatic activity by adsorbing copper ions present in the active site of tyrosinase, but it is unstable when blended into cosmetics and causes skin troubles. Ascorbic acid and its derivatives have a poor skin penetration. It is difficult, and its efficacy is poor when used as a cosmetic raw material because of the oxidation instability. Hydroquinone causes allergies, high cytotoxicity and irritation and is limited in each country. Arbutin is a derivative in which glucopyranoside is bound to hydroquinone, which has been widely used because it has no side effects of hydroquinone, and has been spotlighted, but it is decomposed by skin enzymes. That is, the existing whitening raw materials are severe skin irritation or toxic, the product stability is not good, the skin penetration is not good, the whitening effect is insignificant, there is a limit in use.

Another cause of melanin biosynthesis is ETbR stimulation. When endodelin-1 is secreted by ultraviolet rays, it promotes the secretion of various inflammation-related cytokines (Cytokine) as well as skin blackening caused by ETbR stimulation. The endothelin-1 / ETbR stimulatory signal stimulates the secretion of cytokines such as IL-1a, IL-1b, COX-1, COX-2, TNF-α, IFN-γ, and PEG2, causing UV-inflammation. , N-cadherin (M-Cherin), MMP-1, MMP-2, MMP-9, Integrin (Integrin) to increase the expression of cancer cells to help metastasis. In other words, the development of ETbR antagonists will have the function of inhibiting the skin inflammatory response caused by ultraviolet light and protect skin cells from skin cancer.

Therefore, in the development of a new whitening agent, the whitening effect is excellent, as well as stabilizing the damaged skin due to ultraviolet rays, excellent skin permeability or stability, bio-friendly development of a non-toxic raw material has emerged as an important key.

Document 1 Imokawa G, Yada Y, Kimura M., Biochem. J. 314 (1996) 305. Sriwiriyanont P, Ohuchi A, Hachiya A, Visscher MO, Boissy RE., Lab Invest. 86 (2006) 1115 3 Hachiya A, Kobayashi A, Ohuchi A, Takema Y, Imokawa G., J Invest Dermatol 116 (2001) 578 [4] Scott MC, Suzuki I, Abodel-Malek ZA., Pigment Cell Res 15 (2002) 433 [Reference 5] Hearing VJ. Tsukamoto K., FASEB J, 5 (1991) 2902. 6 Halaban R, Cheng E, Svedin S, Aron R, Hebert DN., J Biol Chem. 276 (2001) 11933. 7 Imokawa G, Kobayashi T, Miyagishi M, Higashi K, Yada Y., Pigment Cell Res 10 (1997) 218. 8 Hara M, Yaar M, Gilchrest BA., J Invest Dermatol 105 (1995) 744. 9 Imokawa G, Miyagishi M, Yada Y., J Invest Dermatol. 105 (1995) 32. Metz M, Lammel V, Gibbs BF, Maurer M., Am J Pathol 169 (2006) 815. 11 Spinella F, Rosanu L, Di Castro V, Decandia S, Nicotra MR, Natali PG, Bagnato A., Cancer Res 67 (2007) 1725. 12. Lan CC, Wu CS, Cheng CM, Yu CL, Chen GS, Yu HS., Exp Dermatol 14 (2005) 528. 13 Lahav R, Heffner G, Patterson PH., Proc Natl Acad Sci USA 96 (1999) 11496. 14 Kadono S, Manaka I, Kawashima M, Kobayashi T, Imokawa G., J Invest Dermatol 116 (2001) 571. Document 15 Udono-Fujimori R, Totsune K, Murakami O, Arihara Z, Kikuchi K, Tamai M, Shibahara S, Takahashi K., J Cardiovasc Pharmacol. 44 (2004) S471-3. 16 Hachiya A, Kobayashi T, Takema Y, Imokawa G. J Biol Chem 277 (2002) 5395. 17. Channa K. JayawickremeS, J. Mark Quillan§, Gerard F. Graminski, and Michael R. Lerner., J Biol Chem 269 (1994) 29846. [18] Jose 'C. Garcı'a-Borro' n, Berta L, Sa'nchez-Laorden, Celia Jime'nez-Cervantes., Pigment Cell Res 18 (2005) 393. 19. Scotti MC, Suzuki I, Zalafa A.A-M., Pigment Cell Res 15 (2002) 433. 20. Solano F, Briganti S, Picardo M, Ghanem G., Pigment Cell Res. 19 (2006) 550. 21 Houben-Weyl, Methods of Organic Chemistry; Synthesis of Prptides and Peptidomimetics, Workbench edition vol E22a, Georg Thieme Verlag (2004)

The present inventors focused on MC1R, which plays the most important role in skin pigmentation process, and synthesized an antagonist of MC1R, and thus the undecylenoyl dipeptide derivatives according to the present invention was applied to skin blackening caused by MC1R. The inhibitory effect was demonstrated to be very good, the signal transduction process was elucidated, and ETbR blackening stimulation was also demonstrated to be inhibited through MC1R-dependent cross-reaction. In addition, it has been demonstrated by establishing an in vitro experimental system that inhibits the inflammatory response by UV.

Undecenoyl dipeptide derivatives used in the present invention are arbutin, kojic acid, which is widely used as a whitening test material, and DermaPep ™ UL (patent application number PCT / KR2010 / 006387, INCI name: Methyl Undecenoyl) The whitening effect was better than that of Leucinate. Thus, the present inventors have developed a functional whitening material with an anti-inflammatory effect and proved its efficacy.

Accordingly, an object of the present invention is to provide an undecenoyl dipeptide derivative which is harmless to the human body, has an excellent whitening effect and is accompanied by an anti-inflammatory effect, and has excellent skin permeability and stability, and a cosmetic composition for skin whitening containing the same.

In order to achieve the above object, the present invention provides an undecenoyl dipeptide derivative. The undecenoyl dipeptide derivative according to the present invention is represented by the following Chemical Formula 1.

[Formula 1]

CH ₂ = CH (CH ₂ ) ₈ -C (= O) -A ₁ -A ₂ -OR

In the formula, -C (= O)-means carbonyl, A ₁ , A ₂ are amino acid residues selected from Leu, Phe, Trp and Gly, respectively, where Leu, Phe and Trp are L- Or a D-form or a mixture thereof, R is H, C ₁ -C ₁₈ alkyl, phenyl, or benzyl, the undecenoyl group is bonded to an amine group of A ₁ , and OR is a carbon of A ₂ To bind to a carbonyl group.

Particularly, in Formula 1, A ₁ and A ₂ are composed of L- or D-type or a mixed form of leucine, and R is preferably a methyl group, and more preferably, the undecenoyl dipeptide derivative is an undeceno. 1-L- (d) L-OMe, undecenoyl- (d) LL-OMe and undecenoyl- (d) L (d) L-OMe. Where L means l-type leucine and (d) L means d-type leucine.

The present invention also provides a cosmetic composition for skin whitening comprising the above-described undecenoyl dipeptide derivative as an active ingredient.

In such a composition, the undecenoyl dipeptide derivative is preferably 0.0001 to 10% by weight based on the total weight of the cosmetic composition for skin whitening. In addition, the cosmetic composition for skin whitening may be made of any one formulation selected from the group consisting of lotions, creams, foundations, essences, gels, packs, foam cleansing, and soap.

The present invention also provides a cosmetic composition for preventing inflammatory diseases associated with ultraviolet rays, comprising the above-described undecenoyl dipeptide derivative as an active ingredient.

First, according to the present invention, the undecenoyl dipeptide derivative acts as an antagonist of MC1R, effectively inhibits the expression of genes in the lower stage, inhibits the activity of tyrosinase, and inhibits the major enzyme of melanogenesis. It has the effect of inhibiting melanin production by inhibiting expression.

Second, in addition to excellent whitening effect, inhibits inflammation caused by ultraviolet light.

Third, the undecenoyl dipeptide derivative is an MC1R antagonist and shows inhibitory effect on ETbR stimulation.

Fourth, the undecenoyl dipeptide derivatives use biocompatible amino acids, resulting in low cytotoxicity and no skin irritation.

Fifth, the undecenoyl dipeptide derivative has a low molecular weight and high skin penetration due to the organic affinity of fatty acids.

The present invention provides an undecenoyl dipeptide derivative. The undecenoyl dipeptide derivative according to the present invention is represented by the following Chemical Formula 1.

[Formula 1]

CH ₂ = CH (CH ₂ ) ₈ -C (= O) -A ₁ -A ₂ -OR

In the formula, -C (= O)-means carbonyl, A ₁ , A ₂ are amino acid residues selected from Leu, Phe, Trp and Gly, respectively, where Leu, Phe and Trp are L- Or a D-form or a mixture thereof, R is H, C ₁ -C ₁₈ alkyl, phenyl, or benzyl, the undecenoyl group is bonded to an amine group of A ₁ , and OR is a carbon of A ₂ To bind to a carbonyl group.

The undecenoyl dipeptide derivative of the present invention can be used as an active ingredient of a cosmetic composition that inhibits the production of melanin in cells and gives a whitening effect on the skin. Since the undecenoyl dipeptide derivative of the present invention acts as an antagonist of MC1R, a lotion, cream, essence, gel, ointment, pack, stick, powder, and the like, such as skin lotion and milk lotion, are known by a known cosmetic preparation method. Whitening cosmetics can be prepared in the form, and pharmaceutically acceptable carriers, excipients, and diluents can be added to the quasi-drugs and external medicines without any side effects.

Undecenoyl dipeptide derivatives according to the present invention can be synthesized through solid phase synthesis or solution synthesis. In solid phase synthesis, the N-terminus is protected and the reactive side chains are protected. As the protecting group for protecting the N-terminus, 9-fluorenylmethyloxycarbonyl group (Fmoc) is generally used. In the case of solution synthesis, conventionally well-known synthesis methods can be used. For example, the undecenoyl dipeptide methyl ester can be synthesized using a synthetic route as in Scheme 1.

[Reaction Scheme 1]

Undecenoyl chloride (Undecenoyl chloride) and the amino acid (A ₁ -OH) in the KOH aqueous solution as shown in Scheme 1 above (1) can be obtained undecenoyl amino acid (Undecenoyl amino acid). Herein, the carbonyl group of the undecenoyl chloride reacts with the amine group of the amino acid to bind, and OH of A ₁ -OH is OH of the carboxyl group of the amino acid.

On the other hand, the reaction of the second amino acid (A ₂ -OH) with thionyl chloride (Thionyl chloride, SOCl ₂ ) in methanol solution as shown in (2) can be obtained in high yield. Similarly, OH of A ₂ -OH is OH of the carboxyl group of the amino acid.

When the two substances obtained in (1) and (2) are reacted with a coupling agent (Coupling Reagent), the desired undecenoyl dipeptide methyl ester can be obtained as shown in (3). Coupling Reagents that can be used at this time can be used as a general coupling agent widely used in peptide synthesis, for example, dicyclohexyl carbodiimide (DCC), HBTU (N-[(1H- benzotriazol-1-yl) (dimethylamino) methylene] -N-methylmethanaminium hexafluorophosphate N-oxide) or HATU (N-[(dimethylamino) -1-1H-1,2,3-triazolo [4,5-b] pyridin- 1-yl-methylene] -N-methylmethanaminium hexafluorophosphate N-oxide may be used, and HOAt (1-Hydroxy-7-Azabenzotriazole) or HOBt (1-Hydroxybenzotriazole) may be used together as an adjuvant. More detailed synthesis methods are described in Example 1.

The content of the undecenoyl dipeptide derivative contained in the preparation of the composition for skin whitening according to the present invention is preferably 0.0001 to 10% based on the total weight of the composition in consideration of the skin whitening effect and economical efficiency, and more preferably 0.002 1.0 wt%.

Hereinafter, for the specific description of the present invention will be described with examples and experimental examples. However, the following Examples and Experimental Examples may be modified in other forms, and the content of the present invention is not limited to the following Examples and Experimental Examples. Examples and experimental examples of the present invention are provided to more fully explain the present invention.

Experimental Example 1 Preparation of Sample

(1) Synthesis of Undecenoyl Amino Acids

0.28 mol amino acid in a 2 L reactor was added to an aqueous solution of 0.74 mol KOH dissolved in 700 g of water, and then cooled using an ice-salt bath. Subsequently, 0.19 mol of Undecenoyl chloride was added dropwise through a dropping tank over about 2 hours, and then aged for 6 hours in an ice-salt bath, followed by 12% hydrochloric acid solution. Neutralize until pH = 3. After extracting with 300 ml of ethyl acetate, the ethyl acetate layer was washed with 100 ml of brine, and then water was removed using MgSO ₄ , and the ethyl acetate was distilled off to remove undecenoyl amino acid. acid). Yields vary slightly with amino acids, but yields of approximately 70-90% are obtained.

(2) Synthesis of Amino Acid Methyl Ester Hydrochloric Acid

0.38 mol of amino acid and 150 ml of methanol were added to a 500 mL reactor, and then cooled to 0 ° C. using an ice-salt bath. 0.50 mol of thionyl chloride was then added dropwise through the dropping tank over 3 hours. After the dropping was completed, the reaction temperature was raised to 60 ° C. and aged for 2 hours to terminate the reaction. Then, methanol and unreacted thionyl chloride were distilled off using a rotary evaporator. White crude product is dissolved in a minimum amount of methanol, and diethyl ether is added to precipitate. The precipitated solid is filtered, washed with diethyl ether and dried to obtain amino acid Methyl ester hydrochloride. Yields vary slightly depending on the amino acid, but yields of approximately 80-90%.

(3) Synthesis of Undecenoyl Dipeptide Methyl Ester

0.030 mol of amino acid methyl ester hydrochloric acid, 0.027 mol of undecenoyl amino acid, 0.027 mol of HOBt, 0.057 mol of diisopropylethyl amine and 100 ml of tetrahydrofuran (THF) were added to a 500 mL reactor. It is then cooled to 0 ° C. using an Ice-Salt Bath. Subsequently, a solution of 0.03 mol of dicyclohexylcarbodiimide (DCC) dissolved in 10 ml of THF is introduced through a dropping tank over about 40 minutes. The reaction solution was heated to room temperature, aged for 5 hours, filtered, and the filtrate was concentrated using a rotary evaporator. Dissolve by adding 200 mL of ethyl acetate, and then using a separatory funnel, wash twice with 50 mL of 1N hydrochloric acid solution, twice with 50 mL of water, twice with 50 mL of 5% aqueous sodium bicarbonate solution, and finally with 50 mL of water three times. After removing water using MgSO ₄ and then removing the solvent using a rotary evaporator to obtain the Undecenoyl dipeptide Methyl ester. Yields vary depending on the type of peptide but yield approximately 70-80%.

(4) Synthesis of Undecenoyl Dipeptide

Dissolve 0.021 mol of undecenoyl amino acid methyl ester in 300 ml of methanol in a 1 L reactor. Subsequently, 420 ml of 1N NaOH aqueous solution was added thereto, and then aged at 25 ° C. for 1 hour. After concentrating the reaction solution using a rotary evaporator, 200mL of water is added and dissolved. Neutralize using a 10% aqueous citric acid solution until pH = 3. The product starts to precipitate. After adding 200 mL of ethyl acetate, the product is extracted and washed with 50 mL of brine. Undecenoyl dipeptide can be obtained by removing water using MgSO ₄ and removing solvent using a rotary evaporator. Yields vary depending on the peptide but yields approximately 70-90%.

Using the synthesis method as described above, the undecenoyl dipeptide compounds of Examples 1 to 23 were synthesized (Table 1). Examples 1 to 23 are applied in DermaPep ™ UL (Patent Application No. PCT / KR2010 / 006387), which are the inventors of the present invention, of Leucine, Tryptophan, Phenylalanine, or Glycine. Synthesized in combination. Where L is Leucine, F is Phenylalanine, G is Glycine, and W is tryptophan, and these four amino acids are in consideration of the structure showing the whitening effect through prior experiment of the present inventors. It was proved and selected through in vitro experimental system. OH at the peptide C-terminus represents a free acid group, OMe represents a methyl ester group, (d) represents a D-form amino acid, and an amino acid without such a designation is l- Type amino acids.

Example 1 Undecenoyl F-OMe Example 2 Undecenoyl LL-OH Example 3 Undecenoyl LL-OMe Example 4 Undecenoyl LF-OH Example 5 Undecenoyl LF-OMe Example 6 Undecenoyl LW-OH Example 7 Undecenoyl FF-OH Example 8 Undecenoyl FL-OH Example 9 Undecenoyl FL-OMe Example 10 Undecenoyl FG-OH Example 11 Undecenoyl FW-OH Example 12 Undecenoyl GL-OH Example 13 Undecenoyl GL-OMe Example 14 Undecenoyl (d) L-OMe Example 15 Undecenoyl (d) L L -OMe Example 16 Undecenoyl L (d) L -OMe Example 17 Undecenoyl (d) L (d) L -OMe Example 18 Undecenoyl G (d) L-OH Example 19 Undecenoyl G (d) L-OMe Example 20 Undecenoyl F (d) L-OH Example 21 Undecenoyl F (d) L-OMe Example 22 Undecenoyl (d) FL-OMe Example 23 Undecenoyl (d) LF-OMe

Experimental Example 2 Experiment for Confirming Whitening Effect: Determination of Melanin Content and Tyrosinase Enzyme Activity

The whitening efficacy of the undecenoyl dipeptide compounds of Examples 1 to 23 was tested. Skin blackening is due to the production of melanin in the skin's melanocytes, which are released into the epidermis. Therefore, if the melanogenesis and the mechanism of melanocytes blocking the production of melanin, melanin is not produced and skin blackening does not appear. In addition, since Examples 1 to 23 will function as antagonists of MC1R, it was predicted that by binding to MC1R instead of α-MSH, it would inhibit the downstream signaling process of MC1R.

The undecenoyl dipeptide derivatives of Examples 1 to 23 were pretreated with melanin-producing cells, and treated with α-MSH, which artificially synthesizes melanin, to determine how much inhibition of skin blackening ability of α-MSH was detected. It was. In addition, the whitening effect was measured by measuring the inhibitory effect on tyrosinase enzymes involved in the main stage of melanin synthesis. In the demonstration of efficacy of whitening raw materials, measuring the inhibition rate of melanin synthesis and tyrosinase enzyme activity in melanocytes is widely used as a basic experiment.

(1) Cell culture

Mouse melanoma B16F10 cells were used as melanocytes. Cells were cultured at 37 ° C., 5% CO ₂ − using 90% Dulbecco's modified eagle's medium (DMEM, GibcoBRL, USA) medium mixed with 10% fetal bovine serum (FBS; Hyclone, Lagan, UT). Incubated at 95% atmosphere (Incubator; Thermo-scientific, USA). Penicillin G and streptomycin (Phenicilin G sodium, 100 units / L, streptomycin sulfate, 100 mg / L; GibcoBRL, USA) were used as antibiotics.

(2) MTT analysis

In each well of a 96-well plate, cells were placed at 2.5 × 10 ³ cells / well (final volume 100 ml), incubated for 24 hours in a 37 ° C., 5% CO ₂ incubator, and then the medium of each well was removed and fresh DMEM ( Phenol red-free) medium was changed. Each well was treated with a whitening efficacy sample and a conventional whitening material by concentration, and incubated in an incubator for 24 hours. 10 ml of MTT solution (5 mg / ml in PBS) was added to each well 3 hours before the end of incubation. After the incubation, the culture solution of each well was removed, and 100 ml of DMSO solution was added to dissolve the generated Formazin. Absorbance was measured at 570 nm using an ELISA reader (TECAN), and the cell viability (%) was converted by the following Equation 1 based on the negative control group treated with DMSO instead of the sample.

[Equation 1]

% Cell viability = [(OD _sample ) / OD _control ] x 100

Table 2 is a table showing the results of measuring their cytotoxicity by treating various concentrations of Examples 1 to 23.

Cell survival rate (%) of Examples 1 to 23 Concentration (ppm)
Name of sample One 5 10 20 100 250 500 Example 1: Undecenoyl F-OMe 100 100 100 93 82 72 54 Example 2: Undecenoyl LL-OH 100 100 100 95 91 65 61 Example 3: Undecenoyl LL-OMe 100 100 101 100 88 77 65 Example 4: Undecenoyl LF-OH 100 101 99 92 82 76 52 Example 5: Undecenoyl LF-OMe 100 100 98 96 83 69 51 Example 6: Undecenoyl LW-OH 100 99 99 85 79 65 49 Example 7: Undecenoyl FF-OH 100 100 99 92 80 71 47 Example 8: Undecenoyl FL-OH 100 102 100 99 73 70 55 Example 9 Undecenoyl FL-OMe 100 100 99 101 78 65 32 Example 10 Undecenoyl FG-OH 100 100 100 98 79 57 48 Example 11: Undecenoyl FW-OH 100 98 100 95 82 68 56 Example 12 Undecenoyl GL-OH 100 100 101 98 80 64 25 Example 13: Undecenoyl GL-OMe 100 102 100 100 79 64 29 Example 14 Undecenoyl (d) L-OMe 100 102 100 97 90 81 67 Example 15 Undecenoyl (d) L L -OMe 100 100 101 99 89 76 59 Example 16 Undecenoyl L (d) L-OMe 100 100 100 97 88 70 61 Example 17 Undecenoyl (d) L (d) L -OMe 100 99 99 98 85 72 57 Example 18 Undecenoyl G (d) L-OH 100 100 97 101 75 61 48 Example 19 Undecenoyl G (d) L-OMe 100 100 100 99 73 62 49 Example 20 Undecenoyl F (d) L-OH 100 97 99 98 79 59 51 Example 21: Undecenoyl F (d) L-OMe 100 100 102 99 75 55 32 Example 22 Undecenoyl (d) FL-OMe 100 100 105 102 69 56 41 Example 23 Undecenoyl (d) LF-OMe 100 100 97 92 72 69 44 DermaPep ™ UL 100 100 99 93 80 64 51

As a result of measuring the cytotoxicity before testing the whitening efficacy of each of Examples 1 to 23, it was determined that the samples used in the experiment did not affect the cell growth and showed no cytotoxicity at an effective concentration (5 to 30 ppm). After that, the experiment was conducted at an appropriate concentration.

(3) measuring melanin synthesis inhibitory effect

In each well of a 6-well plate, B16F10 melanin producing cells were placed at 1 × 10 ⁵ cells / well (Final volume 3 ml), incubated for 24 hours in a 37 ° C., 5% CO ₂ incubator, and then the medium of each well was removed and a new DMEM (10% FBS). Whitening efficacy samples were prepared for each well by concentration and pretreated for 30 minutes.

Thereafter, 1 ppm of α-MSH was treated and incubated for 48 hours. Thereafter, the culture medium was removed, 1 ml of PBS was added thereto, each cell was scraped with a cell scraper (Corning, USA), and the cells were detached, and each was transferred to a new 1.5 ml tube. Subsequently, the supernatant was removed by centrifugation at 4 ° C. and 13,000 rpm for 3 minutes, and cell pellets were recovered to obtain 200 ml of Lysis buffer (1% Triton X-100, 1 mM PMSF, 50 mM Tris-HCl, pH 7.0). EDTA (pH 8.0), 150 mM NaCl, 4 ° C) was added. The cells were lysed by vortex at 10 minute intervals for 30 minutes, centrifuged at 4 ° C and 13,000 rpm for 30 minutes to remove supernatant, and cell pellets containing melanin were recovered and 100 ml of Ether / Isopropanol 1: 1 solution. Wash once. The supernatant was removed by centrifugation at 13,000 rpm for 10 minutes, dried at 60 ° C. for about 3 hours, and 150 ml of 2N NaOH added with 10% DMSO was added thereto to dissolve intracellular melanin at 60 ° C. for 1 hour. . 100 ml of the supernatant obtained by dissolving each well of a 96-well plate was measured at 405 nm using an ELISA reader. As a negative control, DMSO, a solvent in which the sample was dissolved, was added in the same amount as the sample throughput. The positive control did not process the sample, but treated only α-MSH to maximize melanin production. Melanin synthesis inhibition rate (%) was calculated by the following equation.

&Quot; (2) "

% Melanin synthesis inhibition = [(A-C) / (A-B)] x 100

A. The amount of melanin in the positive control group

B. The amount of melanin in the negative control

C. The amount of melanin in the test substance

Table 3 is a chart measuring the melanin synthesis inhibition rate (%) of the samples.

Melanin synthesis inhibition rate (%) of Examples 1-23 division Treatment concentration (ppm) Melanin Synthesis Inhibition Rate (%) Positive control group (No sample, α-MSH 1ppm) DMSO 0.01% - Arbutin 30 37 Kojic acid 200 29 DermaPep ™ UL 5 8 15 37 30 74 SepiWhite ™ MSH 5 11 15 8 30 63 Example 1 Undecenoyl F-OMe 5 -8 15 23 30 38 Example 2: Undecenoyl LL-OH 5 -2 15 21 30 48 Example 3: Undecenoyl LL-OMe 5 2 15 47 30 67 Example 4: Undecenoyl LF-OH 5 -7 15 -41 30 36 Example 5: Undecenoyl LF-OMe 5 -3 15 29 30 50 Example 6: Undecenoyl LW-OH 5 13 15 11 30 21 Example 7: Undecenoyl FF-OH 5 1.8 15 11 30 23 Example 8: Undecenoyl FL-OH 5 7 15 35 30 57 Example 9 Undecenoyl FL-OMe 5 One 15 36 30 69 Example 10 Undecenoyl FG-OH 5 11 15 23 30 52 Example 11: Undecenoyl FW-OH 5 One 15 28 30 41 Example 12 Undecenoyl GL-OH 5 -2 15 24 30 36 Example 13: Undecenoyl GL-OMe 5 5 15 35 30 51 Example 14 Undecenoyl (d) L-OMe 5 15 15 44 30 69 Example 15 Undecenoyl (d) L L -OMe 5 14 15 42 30 74 Example 16 Undecenoyl L (d) L-OMe 5 13 15 84 30 89 Example 17 Undecenoyl (d) L (d) L -OMe 5 24 15 75 30 93 Example 18 Undecenoyl G (d) L-OH 5 4 15 25 30 54 Example 19 Undecenoyl G (d) L-OMe 5 11 15 33 30 65 Example 20 Undecenoyl F (d) L-OH 5 9 15 15 30 49 Example 21: Undecenoyl F (d) L-OMe 5 14 15 24 30 61 Example 22 Undecenoyl (d) FL-OMe 5 -2 15 5 30 42 Example 23 Undecenoyl (d) LF-OMe 5 One 15 15 30 41

As a result of the experiment, Examples 1 to 23, which are undecenoyl dipeptide derivatives, showed a concentration-dependent melanin synthesis inhibitory effect as the prescription concentration was increased from 5 ppm to 30 ppm. It showed much better melanin inhibitory effect than arbutin or kojic acid, which is known as a conventional whitening cosmetic. As a result, it was confirmed that Examples 1 to 23 are excellent candidate materials as whitening raw materials.

When analyzing the results of Examples 1 to 23, the same dipeptide showed more excellent melanin inhibitory effect in the sample substituted with methyl ester than the -OH group (Examples 2 and 3 [dipeptide sequence LL], Examples 4, 5 [dipeptide sequence LF], Examples 8, 9 [dipeptide sequence FL], Examples 12, 13 [dipeptide sequence GL], Examples 18, 19 [dipeptide sequence G (d) L ], Example 20, 21 [dipeptide sequence F (d) L]).

When comparing the sequences of dipeptides, melanin synthesis inhibitory effect was the best in the repeat sequence of leucine (Leucine), the whitening effect was excellent in the sample containing the D-form amino acid when comparing the leucine repeat sequences (Example 3, 15, 16, 17).

In comparison with the conventional invention DermaPep ™ UL, Examples 14, 15, 16, and 17, which have a terminal substituted with a methyl ester group, have the same leucine repeat sequence, and include a double D-form leucine, show particularly excellent effects. It was. More noteworthy is that the maximum melanin inhibition rate shown at 30 ppm, the highest potency concentration of DermaPep ™ UL, is superior to DermaPep ™ UL 30 ppm prescribing effect even when only half of that is 15 ppm in Examples 16 and 17. That is, Examples 16 and 17 showed a higher whitening effect with a lower effective concentration.

The experiments were all performed three times at a time, and Table 3 shows the results of analyzing five or more sets of experiments.

(4) Experiment of whitening effect through inhibition of tyrosinase enzyme activity

In each well of a 6-well plate, B16F10 melanocytes (or HEMn-LP melanocytes) were added to 1 × 10 ⁵ cells / well (Final volume 3 ml), incubated in 37 ° C., 5% CO ₂ incubator for 24 hours, The medium from each well was removed and replaced with fresh DMEM. Whitening efficacy samples were prepared for each well by concentration and pretreated for 30 minutes.

Then 1 ppm of α-MSH was treated and incubated for 48 hours. Thereafter, the culture medium was removed, 1 ml of PBS was added thereto, and each well was scraped with a cell scraper to separate cells, and then transferred to each 1.5 ml tube. Subsequently, the supernatant was removed by centrifugation at 4 ° C. and 13,000 rpm for 3 minutes, and the cell pellet was recovered and 100 ml sodium phosphate buffer (pH6.8 containing 200 ml of Lysis buffer (1% Triton X-100, 1 mM PMSF) was added. )) The cells were lysed by vortex at intervals of 10 minutes for 30 minutes, and then centrifuged at 4 ° C and 13,000 rpm for 30 minutes to transfer the supernatant to a new 1.5 ml tube. Total protein was quantified using BCA protein assay kit (PIERCE, USA), and 40 ml of each sample was placed in each well of a 96-well plate, followed by phosphate buffer (pH, 6.8). 160 ml of L-3,4-dihydroxyphenylalanine (L-DOPA) dissolved immediately in After reacting for 20 to 60 minutes at 37 ° C, absorbance was measured at 475 nm using an ELISA reader. DMSO, a solvent in which a sample was dissolved instead of a sample, was added as a negative control, and the same amount as the sample throughput was added. The positive control did not process the sample, but only α-MSH was used to maximize the activity of tyrosinase enzyme. The inhibition rate of tyrosinase enzyme activity (%) was calculated by the following equation.

&Quot; (3) "

% Tyrosinase activity inhibition = [(A-C) / (A-B)] x 100

A. Tyrosinase Enzyme Activity Rate of Positive Control

B. Tyrosinase Enzyme Activity Rate of Negative Control

C. Tyrosinase Enzyme Activity Rate of Test Substance

Table 4 is a chart measuring the percent inhibition of tyrosinase activity of the samples.

Inhibition rate of tyrosinase activity of Examples 1 to 23 (%) division Treatment concentration (ppm) Tyrosinase activity inhibition rate (%) Positive control group (No sample, α-MSH 1ppm) DMSO 0.01% - Arbutin 30 47 Kojic acid 200 46 DermaPep ™ UL 5 23 15 53 30 86 SepiWhite ™ MSH 5 3 15 31 30 61 Example 1 Undecenoyl F-OMe 5 0 15 21 30 41 Example 2: Undecenoyl LL-OH 5 0 15 24 30 51 Example 3: Undecenoyl LL-OMe 5 One 15 33 30 71 Example 4: Undecenoyl LF-OH 5 -2 15 11 30 24 Example 5: Undecenoyl LF-OMe 5 -7 15 22 30 33 Example 6: Undecenoyl LW-OH 5 3 15 13 30 15 Example 7: Undecenoyl FF-OH 5 -4 15 -3 30 37 Example 8: Undecenoyl FL-OH 5 4 15 42 30 67 Example 9 Undecenoyl FL-OMe 5 13 15 31 30 72 Example 10 Undecenoyl FG-OH 5 -3 15 0 30 42 Example 11: Undecenoyl FW-OH 5 -5 15 -9 30 21 Example 12 Undecenoyl GL-OH 5 0 15 3 30 52 Example 13: Undecenoyl GL-OMe 5 7 15 49 30 61 Example 14 Undecenoyl (d) L-OMe 5 52 15 67 30 89 Example 15 Undecenoyl (d) L L -OMe 5 9 15 86 10 82 Example 16 Undecenoyl L (d) L-OMe 5 5 15 96 30 107 Example 17 Undecenoyl (d) L (d) L -OMe 5 4 15 97 30 107 Example 18 Undecenoyl G (d) L-OH 5 6 15 42 30 70 Example 19 Undecenoyl G (d) L-OMe 5 16 15 23 30 75 Example 20 Undecenoyl F (d) L-OH 5 -One 15 12 30 39 Example 21: Undecenoyl F (d) L-OMe 5 2 15 10 30 71 Example 22 Undecenoyl (d) FL-OMe 5 0 15 2 30 62 Example 23 Undecenoyl (d) LF-OMe 5 -One 15 41 30 71

As a result of the experiment, Examples 1 to 23, which are undecenoyl dipeptide derivatives, showed an effect of inhibiting concentration-dependent tyrosinase enzyme activity as the prescription concentration was increased from 5 ppm to 30 ppm. It showed much better inhibitory activity of tyrosinase enzyme activity than arbutin or kojic acid, which is known as a conventional whitening cosmetic. As a result, it was confirmed that Examples 1 to 23 were excellent whitening candidates that inhibited melanin production as well as inhibiting tyrosinase enzyme activity, which is an enzyme essential for melanin production.

When analyzing the results of Examples 1 to 23, the same dipeptide showed better tyrosinase enzyme activity inhibiting effect in the sample substituted with methyl ester than the terminal -OH group (Examples 2 and 3 [Dipeptide Sequence LL ], Examples 4, 5 [dipeptide sequence LF], Examples 8, 9 [dipeptide sequence FL], Examples 12, 13 [dipeptide sequence GL], Examples 18, 19 [dipeptide sequence G (d ) L], Example 20, 21 [dipeptide sequence F (d) L]).

When comparing the sequences of dipeptides, tyrosinase enzyme activity was the best inhibitory effect in the leucine repeat sequence, and the whitening effect was excellent in the sample containing D-form amino acids when leucine repeat sequences were compared. Example 3, 15, 16, 17).

In comparison with the conventional invention DermaPep ™ UL, Examples 14, 15, 16, and 17, which have a terminal substituted with a methyl ester group, have the same leucine repeat sequence, and include a double D-form leucine, show particularly excellent effects. It was. More notably, the maximum tyrosinase enzyme activity inhibition rate shown at 30 ppm, the highest potent concentration of DermaPep ™ UL, was superior to the DermaPep ™ UL 30 ppm prescription effect even when only half of that was 15 ppm in Examples 16 and 17. That is, Examples 16 and 17 showed a higher whitening effect with a lower effective concentration.

The experiments were all performed three times at a time, and Table 4 shows the results of analyzing five or more sets of experiments.

Experimental Example 3 Measurement of Inhibition of Melanin Gene Expression

Through Experimental Example 2, it was found that Examples 1 to 23 inhibited melanin synthesis through inhibition of tyrosinase enzyme activity. If so, we examined which intracellular signal transduction pathways inhibited tyrosinase enzyme activity and inhibited melanin synthesis in their whitening efficacy.

The major signaling mechanism that stimulates melanogenesis is initiated by an increase in the amount of cAMP in the cell through adenylate cyclase. Therefore, it was measured how much the amount of intracellular cAMP is inhibited by Examples 1 to 23.

In addition, experiments were carried out to see if Examples 1 to 23 really act as MC1R-specific antagonists to show whitening efficacy. In Examples 1 to 23, Examples 16 and 17, which had the highest whitening effect, increased tyrosinase activity and melanin production when PKA, cAMP, cGMP, PKC, and ETbR stimuli, which are factors causing skin blackening, were increased in addition to MC1R stimulation, respectively. Experiments were conducted to evaluate which signaling pathways showed whitening efficacy by measuring how much inhibition was performed. The present inventors anticipated that the whitening raw materials of the present invention would be specifically targeted to MC-R-stimulating α-MSH, and thus, the whitening raw materials of the present invention were ETbR-stimulated, which is known to cross-react in MC1R and cells. In addition, it was predicted to show some inhibitory effect through intracellular mechanism. In addition, since the whitening raw materials of the present invention will bind to the MC1R receptor and block its lower signaling mechanism, when stimulating intracellular proteins (PKA, cAMP, cGMP, PKC) activated by MC1R stimulation, MC1R present in the cell membrane It could be expected that the whitening material bound to the lysate would not be effective.

Next, when melanocytes are stimulated with skin blackening, the lower signaling system is activated to promote transcription of melanin-related genes such as tyrosinase, TRP1, and TRP2. % Transcription inhibition was measured using RT-PCR.

(1) cAMP inhibitory effect of Examples 1 to 23

The amount of cAMP released into the cells was measured using a cAMP ELISA kit (Assay Design, USA). The amount of cAMP generated during cell signal transduction of skin blackening was measured by ELISA (Enzyme-Linked Immunosorbent Assay) and the results are shown in Table 5. The amount of cAMP in the cells was normalized to the amount of protein for each sample measured using the BCA protein assay.

Intracellular cAMP inhibition rate (%) of Examples 1 to 23 division Treatment concentration (ppm) cAMP inhibition rate (%) Positive control group (No sample, α-MSH 1ppm) DMSO 0.01% - DermaPep ™ UL 30 39 SepiWhite ™ MSH 30 36 Example 1 Undecenoyl F-OMe 30 41 Example 2: Undecenoyl LL-OH 30 44 Example 3: Undecenoyl LL-OMe 30 42 Example 4: Undecenoyl LF-OH 30 38 Example 5: Undecenoyl LF-OMe 30 41 Example 6: Undecenoyl LW-OH 30 27 Example 7: Undecenoyl FF-OH 30 28 Example 8: Undecenoyl FL-OH 30 15 Example 9 Undecenoyl FL-OMe 30 22 Example 10 Undecenoyl FG-OH 30 19 Example 11: Undecenoyl FW-OH 30 14 Example 12 Undecenoyl GL-OH 30 21 Example 13: Undecenoyl GL-OMe 30 31 Example 14 Undecenoyl (d) L-OMe 30 52 Example 15 Undecenoyl (d) L L -OMe 30 55 Example 16 Undecenoyl L (d) L-OMe 30 50 Example 17 Undecenoyl (d) L (d) L -OMe 30 49 Example 18 Undecenoyl G (d) L-OH 30 44 Example 19 Undecenoyl G (d) L-OMe 30 34 Example 20 Undecenoyl F (d) L-OH 30 37 Example 21: Undecenoyl F (d) L-OMe 30 31 Example 22 Undecenoyl (d) FL-OMe 30 33 Example 23 Undecenoyl (d) LF-OMe 30 26

Through the above experimental results, it can be seen that Examples 1 to 23 inhibited the amount of cAMP in the cell, thereby affecting tyrosinase enzyme activity, which is a lower signaling pathway, and inhibited melanin synthesis. Since Examples 1 to 23 are made of MC1R antagonists, it is obvious that adenylate cyclase activity, which is a MC1R subsignal, is inhibited, thereby reducing intracellular cAMP release.

The experiments were all conducted twice at a time, and Table 5 shows the results of analyzing two or more sets of experiments.

(2) Confirmation of function as MC1R-specific antagonist of Example 16, 17

The undecenoyl dipeptide derivative used in the present invention was synthesized by modifying DermaPep ™ UL. It has already been demonstrated that DermaPep ™ UL is an MC1R specific antagonist (Patent Application No. PCT / KR2010 / 006387). Therefore, Examples 16 and 17, which were the most effective among Examples 1 to 23, were selected to determine whether they exhibit whitening efficacy by acting as MC1R-specific antagonists. Different stimulation to stimulate melanin synthesis by stimulating melanocytes, and demonstrated how effectively the blocking of Examples 16, 17 (%) and tyrosinase enzyme activity inhibition (%).

α-MSH (MC1R stimulation), Endothelin (16-21) (ETbR stimulation), Forskolin (PKA stimulation), 8-Bromo-cAMP (cAMP stimulation), 8-Bromo-cGMP (cGMP stimulation), PMA (PKC stimulation) Inhibition rate (%) of melanin synthesis is shown in Table 6, and tyrosinase enzyme activity inhibition rate (%) is shown in Table 6, and the effect of Examples 16 and 17 on the increased melanin synthesis and tyrosinase enzyme activity by treatment.

Reagents used for melanin stimulation were treated at the following concentrations.

α-MSH: 1ppm (Synthesis)

Endothelin (16-21): 10ppm (Synthesis)

Forskolin: 20ppm (Sigma, USA)

8-Bromo-cAMP: 10 ppm (Sigma, USA)

8-Bromo-cGMP: 10ppm (Sigma, USA)

PMA: 20ppm (Sigma, USA)

Inhibition rate of melanin synthesis of Example 16, 17 according to various melanin production stimulation (%) division Treatment concentration (ppm) α-MSH Endothelin (16-21) Forskolin 8-Br-cAMP 8-Br-cGMP PMA Negative control group
(No sample treatment) DMSO 0.01% - - - - - - DermaPep ™ UL 15 38 23 0 2 0 -22 30 65 39 -11 3 -8 -25 Example 16:
Undecenoyl L (d) L -OMe 15 61 44 -13 0 -3 -21 30 73 53 -21 0 -2 -10 Example 17:
Undecenoyl (d) L (d) L -OMe 15 72 39 -9 -5 -5 2 30 81 55 -14 -12 -9 -18

Inhibition rate of tyrosinase enzyme activity of Examples 16 and 17 according to various melanin production stimulation (%) division Treatment concentration (ppm) a-MSH Endothelin (16-21) Forskolin 8-Br-cAMP 8-Br-cGMP PMA Negative control group
(No sample treatment) DMSO 0.01% - - - - - - DermaPep ™ UL 15 39 22 -One -22 -One -2 30 75 42 One -5 0 0 Example 16:
Undecenoyl L (d) L -OMe 15 82 54 0 -2 4 -2 30 92 61 -24 -14 3 -5 Example 17:
Undecenoyl (d) L (d) L -OMe 15 85 42 2 0 -3 -21 30 99 59 -13 0 -6 -12

As can be seen from the results of Tables 6 and 7, Examples 16 and 17 were the most excellent inhibition of melanin production and tyrosinase enzyme activity when the melanin production stimulation by MC1R. Forskolin, 8-Bromo-cAMP, 8-Bromo-cGMP, PKA by PMA, Adenylate cyclase, cGMP, PKC stimulation is a sub-step of the signaling mechanism in which MC1R antagonist acts, Examples 16, 17 Skin blackening irritation could not be prevented. Of particular note, the ETbR stimulatory response by Endothelin (16-21) also showed some inhibitory effect. This assumes that ETbR and MC1R share several molecules in the cell and cross-talk. As a result, the MC1R antagonists of the present invention cross-talk with the ETbR signaling system, and proved to bind to MC1R and show a whitening effect.

(3) mRNA inhibitory effect of skin blackening-related genes of Examples 1 to 23

B16F10 melanogenesis cells were placed in ⁵ × 10 ⁵ cells / well (Final volume 5 ml) in a 60 mm dish, incubated in 37 ° C., 5% CO ₂ incubator for 24 hours, and then the medium of each well was removed and replaced with fresh DMEM. Whitening efficacy samples were prepared for each well by concentration and pretreated for 30 minutes.

Thereafter, 1 ppm of α-MSH was treated and incubated for 48 hours. Thereafter, the culture medium was removed, washed with 1 ml of PBS, and RNA was extracted using an easy-spin ™ (DNA free) Total RNA extraction kit (Intron, Korea). PCR primers were prepared beta-actin, tyrosinase, TRP1, TRP2, MITF, POMC of the mouse, and amplified 30 times in the conditions of 94 ° C 1 minute, 60 ° C 1 minute, 72 ° C 1 minute. PCR products were loaded on 1% agarose gel and electrophoresed for 20 minutes and identified using EC3 UV illuminator (UVP, USA). MRNA quantified by densitometer was quantified by dividing by beta-actin value. Primer sequences are shown in Table 8.

PCR primer sequences of skin blackening-related genes Gene name Forward primer (5 '→ 3') Reverse primer (5 '→ 3') Tyrosinase GGC CAG CTT TCA GGC AGA GGT TGG TGC TTC ATG GGC AAA ATC TRP1 GCT GCA GGA GCC TTC TTT CTC AAG ACG CTG CAC TGC TGG TCT TRP2 GGA TGA CCG TGA GCA ATG GCC CGG TTG TGA CCA ATG GGT GCC Beta-actin TGG AAT CCT GTG GCA TCC ATG AAA C TAA AAC GCA GCT CAG TAA CAG TCC G

The results of measuring the inhibitory effects of the genes mentioned in Table 8 are listed in Table 9 below.

Result of mRNA inhibition rate (%) of skin blackening-related genes of Examples 1 to 23 through RT-PCR assay division Maximum Potential Concentration (ppm) Tyrosinase mRNA
Inhibition Rate (%) TRP1 mRNA
Inhibition Rate (%) TRP2 mRNA
Inhibition Rate (%) Negative control group
(No sample treatment) DMSO 0.01% - - - Arbutin 20 74 49 52 Kojic acid 200 72 71 72 DermaPep ™ UL 30 140 69 125 Example 1 30 121 110 109 Example 2 30 74 71 102 Example 3 30 76 44 65 Example 4 30 83 38 57 Example 5 30 92 46 69 Example 6 30 96 63 88 Example 7 30 97 112 113 Example 8 30 45 33 -21 Example 9 30 78 60 72 Example 10 30 63 64 57 Example 11 30 68 71 -31 Example 12 30 -15 -21 2 Example 13 30 53 76 -2 Example 14 30 98 144 108 Example 15 30 99 156 99 Example 16 30 111 162 69 Example 17 30 115 179 73 Example 18 30 19 49 - Example 19 30 91 104 - Example 20 30 33 -6 - Example 21 30 50 -28 - Example 22 30 56 -15 - Example 23 30 105 17 108

As shown in Table 9, Examples 1 to 23 generally inhibited the mRNAs of Tyrosinase, TRP1, and TRP2. Thus, Examples 1 to 23, which are MC1R antagonists, bind to MC1R to inhibit melanin synthesis and tyrosinase enzyme activity. Inhibiting the mRNA of tyrosinase, TRP1, TRP2 genes, it was proved to inhibit the whitening effect through inhibition at the transcription level. Moreover, the inhibitory effect of Examples 16 and 17 was the most excellent among Examples 1-23.

In this experiment, the results of three or more reproducibility checks were synthesized and expressed as average values.

Thus, through Experimental Examples 2 and 3, Examples 1 to 23, which are MC1R antagonists, act as MC1R-specific antagonists (Tables 6 and 7), reduce the amount of cAMP in cells (Table 5), tyrosinase, Inhibition of transcription of skin blackening-related genes such as TRP1 and TRP2 (Table 9) shows that whitening efficacy is shown by inhibiting tyrosinase enzyme activity and melanin biosynthesis (Tables 3 and 4).

<Experimental Example 4>

When the α-MSH-induced melanogenesis inhibitory reactions described in Examples 1 to 23 were to recover the whitening sample and re-treated only α-MSH, reversibly increased melanin synthesis or irreversibly decreased once for melanin. Experiments proved that the whitening samples were recovered by causing permanent damage to the intracellular melanin synthesis apparatus, and that the melanin synthesis stimulation with α-MSH did not increase melanin synthesis again. In this experiment, Examples 16 to 17 of Examples 1 to 23 were selected and tested.

Already, previous experiments confirmed that the whitening effect of DermaPep ™ UL reversibly restored the melanin synthesis function of normal melanocytes when samples were recovered.

After treating with Example 16, 17 and α-MSH and incubating for 48 hours, replacing the medium with fresh medium, Examples 16, 17 were no longer treated, and only α-MSH was continued to be treated.

Melanin synthesis inhibition rate (%) and tyrosinase enzyme activity inhibition rate (%) are shown in Tables 10 and 11 below.

Examples 16, 17% melanin synthesis inhibition upon recovery, 48 hours after treatment division Treatment concentration (ppm) When the sample was not collected,
Continue processing the sample with α-MSH 48 hours later, sample recovery
Continue processing α-MSH only Negative control group
(No sample treatment) DMSO 0.01% - - DermaPep ™ UL 5 3.7 5 15 31 -27 30 79 44 Example 16:
Undecenoyl L (d) L -OMe 5 23 -One 15 89 24 30 88 31 Example 17:
Undecenoyl (d) L (d) L -OMe 5 6 2 15 77 33 30 92 42

Examples 16, 17% inhibition of tyrosinase activity at recovery, 48 h after treatment division Treatment concentration (ppm) When the sample is not collected,
Continue processing the sample with α-MSH 48 hours later, sample recovery
Continue processing α-MSH only Negative control group
(No sample treatment) DMSO 0.01% - - DermaPep ™ UL 5 4 31 15 32 0 30 70 48 Example 16:
Undecenoyl L (d) L -OMe 5 11 One 15 76 24 30 82 39 Example 17:
Undecenoyl (d) L (d) L -OMe 5 3 -3 15 71 35 30 85 42

As can be seen from the above experimental results, if DermaPep ™ UL, Examples 16, 17 without continuing to recover, concentration-dependent melanin synthesis and tyrosinase enzyme activity was inhibited, but after 48 hours, the time when the whitening effect is sufficient, DermaPep ™ UL, Examples 16 and 17, were recovered and treated only with α-MSH, which again increased melanin content and tyrosinase enzyme activity. These results indicate that the whitening effect of Examples 16 and 17, including DermaPep ™ UL, is a reversible response at the MC1R receptor level, rather than disrupting the mechanisms involved in intracellular melanin synthesis.

<Experimental Example 5>

Although α-MSH causes skin blackening, it is well known to have an inhibitory effect on various inflammatory stimuli. Therefore, it has been pointed out that whitening raw materials having a whitening effect as an MC1R antagonist may cause an inflammatory reaction. In Experimental Example 5, the anti-inflammatory efficacy of Examples 1 to 21 was tested. It was confirmed that the existing invention DermaPep ™ UL inhibits the inflammatory response to ultraviolet rays (Patent Application No .: PCT / KR2010 / 006387), and through Example 3, Examples 16 and 17 are MC1R antagonists and also inhibited against ETbR. Since it was confirmed that the efficacy, the anti-inflammatory effect of the ETbR antagonist could be expected. The anti-inflammatory efficacy of Examples 1 to 23 was confirmed by mRNA gene suppression rate and ELISA (Enzyme-Linked Immunosorbent Assay) method to measure the amount of various inflammation-related cytokines increased by UVB.

(1) Cell culture

Human keratinocyte HaCaT cells were used as keratinocytes. Cells were prepared at 90 ° Dulbecco's modified eagle's medium (DMEM, GibcoBRL, USA) medium containing 10% fetal bovine serum (FBS; Cyclone, Lagan, UT) at 37 ° C, 5% CO ₂ -95% (Incubator; Thermo-scientific, USA). Penicillin G and streptomycin (Phenicilin G sodium, 100 units / L, streptomycin sulfate, 100 mg / L; GibcoBRL, USA) were used as antibiotics.

(2) mRNA Inhibitory Effect of the Inflammation-Related Genes of Examples 1 to 23

Put HaCaT keratinocytes into 6x10 ⁵ cells / well (Final volume 5ml) in a 60 mm dish, incubate for 24 hours in a 37 ° C, 5% CO ₂ incubator, remove the medium from each well and use fresh serum-free DMEM. Replaced. Whitening efficacy samples were prepared for each well by concentration and pretreated for 4 hours.

Thereafter, UV-B (100mJ / cm ² ) was treated in PBS buffer, and then replaced with serum-free DMEM medium. Thereafter, the culture medium was removed, washed with 1 ml of PBS, and RNA was extracted using an easy-spin ™ (DNA free) Total RNA extraction kit (Intron, Korea). PCR Primer was prepared in human gapdh, Interleukin-1a (IL-1a), Interleukin-1b (IL-1b), Interleukin 6 (IL-6), Interleukin 8 (IL-8), 94 ° C 1 minute, After 5 times amplification at 52 ° C. 1 min, 72 ° C. 1 min, amplified 30 times at 56 ° C. 1 min, 72 ° C. 1 min. PCR products were loaded on 1% agarose gel and electrophoresed for 20 minutes and identified using EC3 UV illuminator (UVP, USA). MRNA quantified by densitometer was quantified by dividing by gapdh value. The Primer sequence is shown in Table 12.

PCR primer sequence of skin inflammation related gene Gene name Forward primer (5 '→ 3') Reverse primer (5 '→ 3') IL-1a CTC TCT GAA TCA GAA ATC CTT CTA TC CTA GTC AAA TTT CAC TGC TTC ATC C IL-1b TGG AGA ACA CCA CTT GTT GCT CCA AAA CAG ATG AAG TGC TCC TTC CAG G IL-6 GCC TTC GGT CCA GTT GCC TT GCA GAA TGA GAT GAG TTG TC IL-8 ATG ACT TCC AAG CTG GC GTG GCT TCT CAG CCC TCT TCA AAA ACT TCT C GAPDH CAA AAG GGT CAT CAT CTC TG CCT GCT TCA CCA CCT TCT TG

The results of measuring the inhibitory effects of the genes mentioned in Table 12 are listed in Table 13 below.

Result of mRNA inhibition rate (%) of genes related to skin inflammation of Examples 1 to 21 through RT-PCR assay division Maximum Potential Concentration (ppm) IL-1α mRNA
Inhibition Rate (%) IL-1β mRNA
Inhibition Rate (%) IL-6 mRNA
Inhibition Rate (%) IL-8 mRNA
Inhibition Rate (%) Negative control group
(Sample no treatment) DMSO 0.01% - - - DermaPep ™ UL 30 69 51 38 68 SepiWhite ™ MSH 30 66 41 41 64 α-MSH One 102 26 71 77 Example 1 30 -39 10 31 51 Example 2 30 38 41 20 8 Example 3 30 81 30 33 33 Example 4 30 40 33 25 6 Example 5 30 116 15 42 52 Example 6 30 11 40 65 59 Example 7 30 12 21 -12 -134 Example 8 30 -9 22 -7 15 Example 9 30 62 24 -14 33 Example 10 30 97 16 -8 -12 Example 11 30 96 33 23 2 Example 12 30 12 34 -One 48 Example 13 30 45 14 29 69 Example 14 30 81 29 57 76 Example 15 30 66 43 71 75 Example 16 30 79 42 50 69 Example 17 30 66 48 61 65 Example 18 30 25 6 39 -43 Example 19 30 13 29 29 33 Example 20 30 8 26 -8 73 Example 21 30 39 0 44 -72

As shown in Table 13, Examples 1 to 21, although there is a difference in degree, IL-1a, IL-1b, IL-6, and IL-8, which are cytokines (Cytokine) increased by UV-B as a whole, Was effectively suppressed.

(3) ELISA (Enzyme-linked Immunosorbent Assay) Method

The amount of protein was measured by ELISA (Enzyme-Linked Immunosorbent Assay) method.

Put HaCaT keratinocytes into 6x10 ⁵ cells / well (Final volume 5ml) in a 60 mm dish, incubate for 24 hours in a 37 ° C, 5% CO ₂ incubator, remove the medium from each well and use fresh serum-free DMEM. Replaced. Whitening efficacy samples were prepared for each well by concentration and pretreated for 4 hours.

Thereafter, UV-B (100mJ / cm ² ) was treated in PBS buffer and then replaced with serum-free DMEM medium to process the sample again and incubated for 24 hours. Thereafter, the culture medium was removed, washed with 1 ml of PBS, 1 ml of PBS was added, each well was scraped with a cell scraper to separate cells, and then transferred to each 1.5 ml tube. Subsequently, the supernatant was removed by centrifugation at 4 ° C. and 13,000 rpm for 3 minutes, and the cell pellet was recovered and 200 ml of 0.15 M RIPA buffer (50 mM TRIS, 150 mM NaCl, 0.1% SDS, 0.5% Sodium deoxycholate, 1% Triton X). -100, 1% NP-40, 100 mM PMSF). The cells were lysed by vortex at intervals of 10 minutes for 30 minutes, and then centrifuged at 4 ° C and 13,000 rpm for 30 minutes to transfer the supernatant to a new 1.5 ml tube. Total protein was quantified using a BCA protein assay kit (PIERCE, USA), and samples quantified in the same amount were prepared. Strip for ELISA was inserted into an ELISA plate, coated for 30 minutes (0.1% Glutaraldehyde in PBS), washed three times (0.1% Tween20 in PBS), 100ml each sample was added and reacted at 37 ° C for 3 hours. After the washing was repeated three times, the blocking buffer was added 100ml each reaction for 1 hour at 37 ° C. After washing three times, the primary antibodies IL-1β and IL-6 (antibody titer 1: 1000, Santacruz, USA) diluted in blocking buffer were added to each well 100ml each reaction at 37 ° C for 3 hours, washed three times After that, the secondary antibody (anti-rabbit, titer 1: 2000, Santacruz, USA) was added 100ml each reaction was performed at 37 ° C for 1 hour. After washing the secondary antibody three times or more, 100ml each of the TMB solution (Sigma, USA) was added and reacted at room temperature for 10-30 minutes, 1N H ₂ SO ₄ by 50ml each to terminate the reaction. Absorbance was measured at 450 nm using an ELISA reader (TECAN, USA). Protein expression inhibition rate (%) was calculated by the following equation (4).

&Quot; (4) &quot;

% Protein expression inhibition = [(A-C) / (A-B)] x 100

A. Absorbance of UV-B (100mJ / cm ² ) Treatment Group

B. Absorbance of Negative Control

C. Absorbance of Test Substance

This experiment was carried out by designating Examples 16, 17, and the inhibition rate of IL-1β and IL-6 protein expression increased by UVB is shown in Table 14 below.

Inhibition Rate of IL-1β and IL-6 Protein Expression of Examples 16 and 17 (%) division Treatment concentration (ppm) IL-1β protein expression inhibition rate (%) IL-6 protein expression inhibition rate (%) Negative control group
(Sample no treatment) DMSO 0.01% - - DermaPep ™ UL 15 24 15 30 33 24 Example 16:
Undecenoyl L (d) L -OMe 15 21 18 30 17 19 Example 17:
Undecenoyl (d) L (d) L -OMe 15 32 22 30 21 15

In Table 14, the amount of protein was measured by designating only Examples 16 and 17 as a representative. However, when the total amount of protein is measured by combining Table 13, it can be inferred that the remaining examples also reduce protein amount as well as gene level.

As a result, Examples 1 to 23 can act as an MC1R antagonist to show whitening efficacy, and at the same time inhibit the inflammation-related signaling processes below the ETbR (Table 13, 14) it can be seen that it has an anti-inflammatory effect.

The cosmetic composition for skin whitening of the present invention not only provides an excellent whitening effect even in a small amount, but also shows a superior whitening effect even when compared to DermaPep ^™ UL, which is a conventional whitening raw material. Although the present invention has been described in detail with reference to the experimental examples described above, it will be apparent to those skilled in the art that various modifications and changes are possible within the scope and spirit of the present invention, and such modifications and variations are included in the appended claims. It is natural to belong.

Claims (7)

An undecenoyl dipeptide derivative represented by the following [Formula 1].
[Formula 1]
CH ₂ = CH (CH ₂ ) ₈ -C (= O) -A ₁ -A ₂ -OR
In the above formula, -C (= 0)-means carbonyl, A ₁ , A ₂ are amino acid residues, A ₁ is selected from Leu, Phe and Gly, A ₂ is Leu, Phe, Trp and Selected from Gly, wherein Leu, Phe and Trp are L- or D-forms or a mixture thereof, R is H or CH ₃ , and the undecenoyl group binds to an amine group of A ₁ , OR binds to the carbonyl group of A ₂ . The undecenoyl dipeptide derivative according to claim 1, wherein in Formula 1, A ₁ and A ₂ are composed of L- or D-form or mixed leucine, and R is a methyl group. The method of claim 1, wherein the undecenoyl dipeptide derivatives are undecenoyl-L- (d) L-OMe, undecenoyl- (d) LL-OMe and undecenoyl- (d) L (d) L -OMe, where L means l-type leucine and (d) L means d-type leucine. A cosmetic composition for skin whitening comprising an undecenoyl dipeptide derivative according to any one of claims 1 to 3 as an active ingredient. The cosmetic composition for skin whitening according to claim 4, wherein the undecenoyl dipeptide derivative is 0.0001 to 10% by weight based on the total weight of the cosmetic composition for skin whitening. The cosmetic composition for skin whitening according to claim 4, wherein the cosmetic composition for skin whitening is composed of any one formulation selected from the group consisting of lotion, cream, foundation, essence, gel, pack, foam cleansing, and soap. . Claim 1 to claim 3 comprising an undecenoyl dipeptide derivative according to any one of claims as an active ingredient, cosmetic composition for preventing inflammatory diseases associated with ultraviolet rays.