KR101410897B1

KR101410897B1 - Lipoic acid PEGylated peptide derivative, method for preparing the same, and cosmetic compostion comprising the same for whitening or improving wrinkle

Info

Publication number: KR101410897B1
Application number: KR1020120056060A
Authority: KR
Inventors: 채규윤; 이범철; 김보미; 이덕희; 록치충
Original assignee: 이스켐주식회사
Priority date: 2012-05-25
Filing date: 2012-05-25
Publication date: 2014-07-16
Also published as: KR20130132681A

Abstract

The present invention relates to a lipoic acid-PEG-peptide derivative, a process for producing the same, and a cosmetic composition for whitening or wrinkle improvement containing the same. The lipoic acid-PEG-peptide derivative of the present invention is safe because it has little cytotoxicity, and exhibits excellent tyrosinase inhibitory activity, melanin formation inhibitory activity, MMP-1 inhibitory activity and collagen biosynthesis promoting activity, It can be very useful as a raw material for a cosmetic composition for cosmetics.

Description

[0001] The present invention relates to a lipoic acid-PEG-peptide derivative, a method for producing the same, and a cosmetic composition for improving whitening or wrinkling, which comprises the same.

The present invention relates to a lipoic acid-PEG-peptide derivative, a process for producing the same, and a cosmetic composition for whitening or wrinkle improvement comprising the lipoic acid-PEG-peptide derivative.

The extracellular matrix forms the structural basis of many tissues, especially the skin, which is also a major subject for dermatology and cosmetic applications. Extracellular matrix proteins, including collagen and collagen fibers, play a pivotal role in cell migration, proliferation and gene regulation in wound healing processes. Collagen is the most abundant connective tissue in humans, and collagen fibers provide elasticity and tension so that the skin-like organs can be well organized. Therefore, it is known that a decrease in extracellular matrix components such as loss or collapse of collagen and an excessive accumulation of glycosaminoglycan (GAGs) cause wrinkles on the skin due to aging. With these characteristics in mind, researchers have focused on many products and chemical compounds that enhance aging skin conditions, stimulate the importance of collagen in the process of aging, and stimulate the biosynthesis of extracellular matrix. In particular, some oligopeptides function not only in the human body but also as lifelike messengers such as hormones, neurotransmitters or neurotransmitters in the skin, and some cosmeceutical peptides can be used for therapeutic or cosmetic purposes For medical use, it has been developed to improve skin physiological activity such as anti-aging or moisturizing properties. For example, the hexapeptide of Val-Gly-Val-Ala-Pro-Gly (VGVAPG) stimulates the production of fibroblasts of human skin while simultaneously down-regulating the expression of elastin and the Tyr- The Asp-Asp-Ala (YYRADDA) peptide sequence inhibits collagen degradation by inhibiting procollagen-C-proteinase, an enzyme that separates C-propeptide from type 1 procollagen . In addition, Lys-Thr-Thr-Lys-Ser (KTTKS), Glu-Glu-Met-Glu-Arg- Arg (EEMQRR), and the tripeptide Gly- Lt; RTI ID = 0.0 > type-1 < / RTI > collagen, the predominant form of collagen. The peptides can stimulate collagenogenesis by fibroblasts. On the other hand, since skin functions as a physical and chemical barrier to defend the body from the harmful environment of the outside, typical peptides are degraded by enzymes and the circulating half-life is only a few minutes to a few hours, There is a problem that the bioavailability in vivo is relatively low due to other endogenous factors outside.

On the other hand, lipoic acid (LA, lipoic acid) exists in various animals and plants, and mainly acts as a coenzyme in an enzyme reaction in mitochondria. It is known that octanoic acid becomes a precursor of the C-8 fatty acid chain of lipoic acid, and cyteine becomes a source of sulfur and biosynthesis of lipoic acid occurs. DHLA (dihydrolipoic acid), a form of reduced lipoic acid and lipoic acid, is well-known as an antioxidant. It protects vitamin C, E, and glutathione and enhances the effect of NF-kB. In addition, it acts to promote the generation of degrading enzymes that only remove damaged collagen fibers. It is also known to suppress melanin formation by inhibiting tyrosinase activity by ultraviolet light. However, due to the nature of highly reactive -SH (sulfhydryl) group-containing compounds, they are oxidized to produce disulfides, so that their solubility decreases and there is a problem in stability such as discoloration and decomposition. In addition, although it is soluble in ethanol, it has a low solubility in water, which is problematic in formulation of cosmetics. In order to improve this, it has been suggested that mixing with vitamin C and E is a method capable of stable storage for a long period of time. However, such a method has limitations in improving the stability of lipoic acid and has a disadvantage in that odor, formulation, stability, The application to the external preparation for skin was limited.

Therefore, there is a desperate need to develop a technique for a cosmetic composition that solves the problems of lipoic acid, solubility and cytotoxicity, and improves the chemical stability of lipoic acid and functional peptide, thereby excelling in whitening and wrinkle improvement.

The present inventors have been studying a method for improving solubility, chemical stability, and toxicity while maintaining the functionality of liposan (LA, Lipoic acid) and peptides, and have found that lipoic acid-PEG-peptide derivatives prepared by linking lipoic acid and functional peptides to PEG Was found to be safe with little cytotoxicity, and it was confirmed that the effect of inhibiting tyrosinase activity, inhibition of melanin formation, inhibiting the expression of MMP-1 (matrix metalloproteinase) and promoting collagen biosynthesis were excellent. Respectively.

Accordingly, the present invention is intended to provide a lipoic acid-PEG-peptide derivative and a method for producing the same.

The present invention also provides a cosmetic composition for whitening or wrinkle improvement comprising a lipoic acid-PEG-peptide derivative.

The present invention provides lipoic acid-PEG-peptide derivatives and processes for their preparation.

The lipoic acid-PEG-peptide derivative of the present invention is safe because it has little cytotoxicity, exhibits excellent tyrosinase inhibitory activity, melanin formation inhibitory activity, MMP-1 inhibitory activity and promoting collagen biosynthesis activity, It can be very useful as a raw material for a cosmetic composition for improvement.

Brief Description of the Drawings Fig. 1 is a diagram schematically showing binding of lipoic acid, PEG (polyethylene glycol) and a peptide of the present invention.
2 is a graph showing the ¹³ C-NMR spectrum of the lipoic acid-PEG-peptide derivative (LAP-KTTKS) of the present invention.
3 is a diagram showing a ¹³ C-NMR spectrum of the lipoic acid-peptide derivative (LA-KTTKS) of the present invention.
4 is a graph showing the effect of the lipoic acid-PEG-peptide derivative (LAP-KTTKS) of the present invention on cytotoxicity of human skin fibroblasts.
FIG. 5 is a graph showing the effect of the lipoic acid-PEG-peptide derivative of the present invention on tyrosinase inhibitory activity (a) and melanin pigment production inhibitory activity (b).
6 is a graph showing the degree of inhibition of UVA-induced expression of MMP-1 (matrix metalloproteinase-1) by the lipoic acid-PEG-peptide derivative of the present invention.
7 is a graph showing the effect of the lipoic acid-PEG-peptide derivative of the present invention on collagen biosynthesis.

Hereinafter, the present invention will be described in detail.

The present invention provides a lipoic acid-PEG-peptide derivative (LAP-KTTKS, PEGylated lipoic acid-peptide conjugate) represented by the following formula (1).

[Chemical Formula 1]

In Formula 1, n is an integer of 1 to 1000.

The present invention also provides a method for producing a lipoic acid-PEG-peptide derivative (LAP-KTTKS, PEGylated lipoic acid-peptide conjugate 1) represented by the following Reaction Scheme 1.

[Reaction Scheme 1]

(Ii) is TFA / EDT / TIS / H ₂ O, and n is an integer of 1 to 1000. In the reaction scheme 1, (i) is DIC, HOBt and DCM / DMF.

Lipoic acid derivative represented by the reaction formula 1 -PEG- peptide (LAP-KTTKS, PEGylated lipoic acid -peptide conjugate, 1) production method (a) 2- chloro-trityl chloride resin and Fmoc (9-fluorenylmethyloxycarbonyl), the Boc (N ( tert - butyloxycarbonyl) and serine-lysine-threonine-threonine-lysine, which are protected with a t-Bu ( tert- butyl) protecting group; (b) reacting the compound of formula (5) with LAP (PEGylated lipoic acid) to produce a compound of formula (6); And (c) deprotecting the side chain protecting group of the compound of formula (VI) and isolating the solid phase resin to prepare the compound of formula (1); .

The linear pentapeptide KTTKS bound to the resin of Reaction Scheme 1 can be synthesized by solid-phase peptide synthesis (SPPS, solid) using 2-chlorotrityl chloride resin preloaded with Fmoc-serine -phase peptide synthesis). The Fmoc (9-fluorenylmethyloxycarbonyl) protecting group in the production method of the present invention is used to temporarily protect the N- terminal amino group, the Boc (N - tert - butyloxycarbonyl) protecting group and the t-Bu (tert -butyl) the side chain protecting groups ( -NH ₂ group and -OH group). The binding between the protected amino acids is carried out using a solution of HBTU / HOBt (O-Benzotriazole-N, N ', N'-tetramethyl-uronium-hexafluoro-phosphate / N-Hydroxybenzotriazole) reagent and 20% piperidine / DMF (piperidine / dimethylformamide) is used to remove the Fmoc protecting group. The peptide bound to the resin is washed with a mixed solution of dichloromethane (DMM) and DMF, and bound to the lipoic acid or lipoic acid-PEG ester using DIC ( N, N'- diisopropylcarbodiimide) and HOBt. The prepared liposan-peptide polymer (Formula 6) is placed in a mixed solution of TFA / EDT / TIS / H ₂ O for standard separation to remove the peptide side chain protecting group and separate from the solid phase resin. Further, the separated resin is filtered with a filter, and the filtrate, which has passed through a filter, is precipitated by adding ether, followed by centrifugation, followed by washing with ether and drying. The dried product is subjected to RP-HPLC (reverse phase-high performance liquid chromatography) using acetonitrile containing TFA as an isomer to isolate the lipoic acid-PEG-peptide polymer of formula (1).

The method of preparing LAP (PEGylated Lipoic acid, 4) in Reaction Scheme 1 is shown in the following Reaction Scheme 2.

[Reaction Scheme 2]

And in Scheme 2 (i) is _{t-BuOK / BrCH 2 CO 2} Et, H 2 O / NaOH, (ii) is EDC, DMAP, DCM, n is an integer from 1 to 1,000.

First, the carboxy group is introduced into PEG by a modification of the previously disclosed method (KY Chai et al., 2011) (3). When the reaction of introducing a carboxy group into PEG is carried out, α -hydroxyl- ω -carboxyl PEG, dicarboxylated PEG, and unreacted PEG are produced. Next, the α -hydroxyl- ω -carboxyl PEG (HO-PEG-COOH) was purified by ion exchange chromatography using DEAE-Sephadex and catalytic amount of DMAP (4- (dimethylamino) pyridine) (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) -mediated esterification reaction. The DMAP has a hydroxyl group at the terminal to allow polymerization reaction with lipoic acid, and inhibits the formation of by-products, while providing a carboxy group to be polymerized with the pentapeptide component.

Also, the present invention provides a cosmetic composition for whitening or wrinkle improvement, which comprises the lipoic acid-PEG-peptide derivative of Formula 1 above.

The lipoic acid-PEG-peptide derivative (LAP-KTTKS) according to the present invention has low toxicity to skin cells, high tyrosinase inhibitory activity and high melanin production inhibitory activity, In addition, the effect of improving the wrinkle is excellent because of its excellent inhibitory activity on expression of matrix metalloproteinase-1 (MMP-1) and induction of collagen biosynthesis. In addition, by solubilizing poly ethylene glycol (PEG) with lipoic acid (LA) And chemical stability. Therefore, the lipoic acid-PEG-peptide derivative of the present invention can be usefully used as a cosmetic composition which is effective for whitening or wrinkle reduction.

The tyrosinase of the present invention is a metal enzyme containing copper as an enzyme that forms a dihydroxyphenylalanine (DOPA) by tyrosine hydrolysis and further oxidizes to form dopaquinone. It catalyzes the oxidation of tyrosine and phenols by oxygen, and is said to exist in various plant and animal tissues including mushrooms, potatoes, and apples. Hydroxylation of tyrosine to produce DOPA is the first step in the synthesis of melanin, a dark brown pigment, which is related to the lightness of the skin. If the enzyme is genetically deficient, melanin deficiency in the animal tissue results in white matter.

MMP-1 (matrix metalloproteinase-1) of the present invention is also referred to as an interstitial collagenase, and it is known that when human fibroblasts are irradiated with UV, they are overexpressed in cells within a few hours. MMP-1 is considered to be a key modulator involved in the degradation of the extracellular matrix of skin, i. E., Type 1 and type 3 collagen, and plays an important role in the aging process due to solar light stress. Deficiency of collagen eventually results in the formation of wrinkles . Thus, inhibition of major collagen-degrading enzymes such as MMP-1 can be a useful anti-aging agent.

The cosmetic composition of the present invention may be prepared by mixing with the lipoic acid-PEG-peptide derivative, if necessary, with an excipient for cosmetics such as water, physiological saline, glycerol, a surfactant, a moisturizer, a thickener, a chelating agent, Depending on the method of preparation, it can be prepared in various forms. For example, the cosmetic composition may be a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nutrition lotion, a massage cream, a nutritional cream, It can be manufactured in the form of hand cream, essence, nutrition essence, pack, soap, shampoo, cleansing foam, cleansing cream, body lotion, body cleanser, emulsion, press powder, loose powder, eye shadow.

The cosmetic composition may also contain conventional adjuvants such as stabilizers, solubilizers, vitamins, pigments, and flavorings commonly used in the cosmetic composition arts. In the cosmetic composition, the content of the lipoic acid-PEG-peptide derivative may be an amount effective to achieve a whitening or wrinkle-reducing effect, for example, 0.001 to 10% by weight based on the total weight of the composition, May be contained in an amount of about 0.01 to 1% by weight.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example 1. The lipoic acid- PEG - peptide derivatives ( LAP - KTTKS )

1. Protected KTTKS - Preparation of Resin Polymer (5)

2-Chlorotrityl chloride resin (34 mg, 50 μmol) was added to the reaction vessel, 2 ml of DCM was added, and the mixture was stirred for 2-3 minutes. After removing the solvent, serine (Fmoc-Ser (tBu) -OH: S, 100 μmol) protected with Fmoc was dissolved in 1.5 ml of DMF and 34 μl (200 μl) of DIPEA, and the reaction vessel was charged with 2-chlorotrityl chloride And the mixture was stirred with the resin for 2 to 4 hours. The solvent was removed, washed 5 times with DCM, and a small amount was taken to measure the capacity. In order to cap the reaction in the reaction vessel, DCM / MeOH / DIPEA (17/2/1, (v / v)) was added to the reaction vessel and repeated twice for 15 minutes, And washed. 20% piperidine in DMF solvent was added to the capped reactant, and the mixture was reacted twice for 10 minutes, and then sufficiently washed with DCM so that no piperidine remained. After removing the solvent, lysine (Fmoc-Lys (boc) -OH; K, 100 μmol) protected with Fmoc was dissolved in 1.5 ml of DMF together with DIPEA and HOBt (14 mg, 100 μmol) After stirring for 2 to 4 hours, the solvent was removed. After washing three or more times with DCM, free amines were identified by Kaiser experiment. Lysine (Fmoc-Lys (boc) -OH: Fmoc-Thr (tBu) -OH: T, 100 μmol), threonine K, 100 μmol) was reacted to obtain 0.4 g of a protected yellow lysine-threonine-threonine-lysine-serine (KTTKS) polymer.

2. Lipoic acid and Of polyethyleneglycol concrete( LAP , PEGylated lipoic acid , 4)

Lipoic acid (0.006 mmol) and DCM (12 ml) were added to a round bottom flask and stirred. After the lipoic acid was completely dissolved, EDC (N- (3-Dimethylamino propyl) -N'-ethylcabodiimide hydrochloride, 0.0059 mmol) was added in portions 2 and 3 while stirring, and a nitrogen balloon was prepared. PEG (HO-PEG-COOH: 0.005 mmol), one end of which was oxidized to carboxyl group, and 12 ml of DCM were dissolved in another reaction vessel and dissolved therein. When the oxidized PEG was completely dissolved, a solution in which oxidized PEG was dissolved was added to the reaction vessel in which the lipoic acid was dissolved. The reaction was carried out under stirring in an ice bath condition for 1 hour, then, the ice bath was removed, After the reaction was completed, the reaction mixture was transferred to a separating funnel, washed with distilled water, separated from the DCM layer, and washed again with distilled water five times . The separated DCM layer was dehydrated with MgSO ₄ to remove residual water and filtered. The filtrate was concentrated under reduced pressure to give a pale yellowish solid title compound.

3. Lipoic acid- PEG - peptide derivatives ( LAP - KTTKS )

A mixture of DCM and DMF (4: 1, (v / v)) and LAP (1.6 mmol), DIC (3.2 mmol) and HOBt (3.2 mmol) was added thereto, followed by stirring. After 4 hours, a small amount of reactant was taken in the middle of the stirring, and Kaiser test was performed, and reaction was continued at room temperature with continued stirring until Kaiser test negative reaction occurred. The reaction time is approximately 24 hours. After the reaction was completed, the reaction mixture was filtered through a filter paper, washed with DCM and DMF for about 5 times, and the reactants adhered to the resin were collected and dried, and the dried reaction product was cleavaged again. The cleavage solution was prepared by mixing TFA / EDT / TIS / H ₂ O (trifluoroacetic / 1,2-ethanedithiol / triisopropylsilane / H ₂ O) at a volume ratio of 9.5: 0.2: 0.1: 0.2, 1 ml of the separation solution per g was added and reacted at room temperature for 3 hours to remove the peptide protecting group and solid phase resin. The resin and the protecting group were removed using a filter paper, the filtrate was collected, precipitated in cold ethyl ether, and centrifuged to obtain only a precipitate. Cool ethyl ether was added to the obtained precipitate and centrifuged to collect the precipitate. The precipitate was washed 5 times or more until the odor of the separated solution was no longer observed. Upon completion of the washing procedure, the title compound was obtained as a solid (LAP-KTTKS, PEGylated lipoic acid-peptide conjugate, 1, 880 mg, 49%) under vacuum.

A schematic diagram of the lipoic acid-PEG-peptide derivative (LAP-KTTKS) of the present invention is shown in FIG.

The ¹ H-NMR spectrum of the lipoic acid-PEG-peptide derivative of the present invention is shown below, and the ¹³ C-NMR spectrum is shown in FIG.

^{1 H-NMR (500 MHz,} CD 3 OD): δ (ppm) 4.45-4.43 (m, 4H), 4.35-4.24 (m, 2H), 3.94-3.82 (m, 2H), 3.30 (m, 4H) , 2.96-2.43 (m, 2H), 1.96-1.84 (m, 4H), 1.49-1.30 (m, 8H).

Comparative Example 1. Lipoic acid-peptide derivatives of formula (2) LA - KTTKS )

(I) is DIC, HOBt, DCM / DMF and (ii) is TFA / EDT / TIS / H ₂ O.

(LA-KTTKS, lipoic acid-peptide conjugate, 2, 340 mg, 2 mmol) was obtained in the same manner as in Example 1, except that LAP was used instead of LAP. 58%).

The ¹ H-NMR spectrum of the lipoic acid-peptide derivative (LA-KTTKS) is shown below, and the ¹³ C-NMR spectrum is shown in FIG.

^{1 H-NMR (500 MHz,} CD 3 OD): δ (ppm) 4.46-4.43 (m, 4H), 4.35 (d, 2H, J = 3.5 Hz), 4.29-4.25 (m, 2H), 3.93-3.88 (m, 2H), 3.64 (m, ~ 180H, PEG backbone), 3.30 (m, 4H), 2.97-2.92 (m, 2H), 1.90-1.72 .

Experimental Example 1. The lipoic acid- PEG - peptide derivatives ( LAP - KTTKS ) Cytotoxicity analysis

Human skin fibroblast or mouse melanoma B16F10 cells were purchased from Korean Cell Line Bank and cultured in DMEM supplemented with 10% fetal bovine serum (BioWhittaker, Walkersville, MD, USA), 1% penicillin-streptomycin (Gibco BRL, Were cultured in DMEM (Dulbecco's modified eagle's medium) supplemented with α -melanocyte stimulating hormone ( α- MSH, α- melanocyte stimulating hormone, Sigma) at 37 ° C and humidified 5% CO ₂ atmosphere.

Human dermal fibroblast (HDF) or B16F10 melanoma cells were incubated with various concentrations of the sample (control, LAP, LA-KTTKS, LAP-KTTKS, KTTKS) to determine the cytotoxicity of the lipoic acid peptide polymer. Lt; / RTI >

The results are shown in Fig.

As shown in FIG. 4, the lipoic acid-peptide derivative (LAP-KTTKS) of the present invention showed excellent cell survival rate while the lipoic acid-peptide derivative (LA-KTTKS) decreased the cell survival rate. This is probably because LA-KTTKS did not use a PEG linker. Therefore, it can be seen that the lipoic acid-PEG-peptide derivative (LAP-KTTKS) of the present invention is safe because it has little cytotoxicity.

Experimental Example 2. Whitening effect analysis

1. Tyrosinase inhibitory activity assay ( Tyrosinase inhibition assay )

The degree of inhibition of tyrosinase activity was measured by changing a previously reported method (HYChung et al., 2005) with a spectrophotometer using L-DOPA as a substrate. 0.1 mL of mushroom tyrosinase solution (625 U mL ^{? 1} ), 0.9 mL of 1/15 mM phosphate buffered saline (2.0 mM) and 0.2 mM sample (control, LAP, LA-KTTKS, LAP-KTTKS , KTTKS) were preincubated at 25 ° C for 10-15 minutes. Then, 0.03% of L-DOPA solution was added and reacted. The tyrosinase inhibition rate (%) was then obtained by measuring the absorbance of the reaction using a microplate reader at 475 nm.

The results are shown in FIG. 5A.

As shown in FIG. 5A, LAP, LA-KTTKS, and LAP-KTTKS inhibited tyrosinase activity to 92.7%, 60.2%, and 78.4%, respectively. Therefore, it can be seen that the lipoic acid-PEG-peptide derivative (LAP-KTTKS) of the present invention is superior in tyrosinase inhibitory activity to LA-KTTKS.

2. Melanin pigment inhibitory activity assay

B16F10 cells were treated with 0.2 mM test samples (control, LAP, LA-KTTKS, LAP-KTTKS, KTTKS) and incubated for 72 hours. The cultured cells were counted, washed with PBS, and then collected by centrifugation at 3000 rpm for 5 minutes. The collected cell pellet was washed with PBS and then solubilized with 0.2 mL of 1 N NaOH and 1 mL of homogenization buffer (50 mM sodium phosphate, pH 6.8, 1% Triton X-100, and 2 mM PMSF) ). The absorbance of melanin was monitored at 405 nm and the amount of melanin was measured with a standard curve made using synthetic melanin (Sigma).

The results are shown in FIG. 5B.

As shown in FIG. 5B, the lipoic acid-PEG-peptide derivatives (LAP-KTTKS) and LA-KTTKS of the present invention inhibited the melanin pigment production to 56.3% and 46.1%, respectively. On the other hand, KTTKS reduced the synthesis of melanin pigment by only 7.5%. Therefore, it can be seen that the lipoic acid-PEG-peptide derivative (LAP-KTTKS) of the present invention is superior to LA-KTTKS in inhibiting melanin pigment formation.

Experimental Example 3. MMP -1 inhibitory activity assay

In order to confirm the effect of the lipoic acid-PEG-peptide derivative of the present invention on the expression of UV-induced MMP-1 (matrix metalloproteinase-1) in human dermal fibroblasts, the following experiment was conducted.

First, the fibroblasts were washed twice with PBS and treated with the concentrations (0.01, 0.05, 0.1, 0.25, 0.5 mM) of the samples (control, LAP, LA-KTTKS, LAP-KTTKS, KTTKS) UVA was irradiated to Human Dermal Fibroblast (HDF) at an energy density of 6.3 J / cm ² for 40 minutes. After 72 hours of UV irradiation, the level of MMP-1 protein in the culture medium was analyzed by enzyme-linked immunosorbent assay (ELISA). The anti-MMP-1 monoclonal antibody was added and incubated at 37 ° C for 60 minutes. Secondary antibodies (anti-mouse IgG polymerized with alkaline phosphatase) suspended in PBS were added to each well and incubated for 30 minutes Lt; / RTI > After washing the culture, OD (optical density) value was measured at 405 nm with a spectrophotometer.

The results are shown in Fig.

As shown in FIG. 6, it was confirmed that the lipoic acid-PEG-peptide derivative (LAP-KTTKS) of the present invention showed better MMP-1 inhibitory activity than LA-KTTKS.

Experimental Example 4. Collagen Biosynthesis amount analysis( Collagen synthesis assay )

The following experiment was conducted to confirm the effect of the lipoic acid-PEG-peptide derivative (LAP-KTTKS) of the present invention on collagen synthesis using human dermal fibroblast (HDF).

Human skin fibroblasts were cultured in 96-well plates (5 x 10 ⁴ cells) in DMEM medium containing 10% fetal bovine serum, and then the medium was added to 0.5 mM test samples (control, LAP, LA-KTTKS, LAP-KTTKS , KTTKS). After culturing for 24 hours, the supernatant of each well was collected and the type 1 procollagen was measured with an EIA (enzyme immunoassay) kit (MK101, Takara, Japan).

The results are shown in Fig.

As shown in FIG. 7, the lipoic acid-peptide derivative (LAP-KTTKS) of the present invention was found to increase the amount of collagen biosynthesis more than that of the lipoic acid-peptide derivative (LA-KTTKS).

Examples of formulations for the cosmetic composition of the present invention are illustrated below.

Formulation example One : Cosmetics Manufacture of pharmaceutical preparations

1. Manufacture of flexible lotion

ingredient Content (% by weight) The lipoic acid-PEG-peptide derivative (LAP-KTTKS) 0.5 1,3-butylene glycol 5.2 Oleyl alcohol 1.5 ethanol 3.2 Polysorbate 20 3.2 Benzophenone-9 2.0 Carboxyl vinyl polymer 1.0 glycerin 3.5 incense a very small amount antiseptic a very small amount Purified water Balance system 100

After mixing the above components, they were prepared according to the usual production method of softened lotion.

2. Manufacture of Milk Lotion

ingredient Content (% by weight) The lipoic acid-PEG-peptide derivative (LAP-KTTKS) 0.6 glycerin 5.1 Propylene glycol 4.2 Tocopheryl acetate 3.0 Liquid paraffin 4.6 Triethanolamine 1.0 Squalane 3.1 Macadamia nut oil 2.5 Polysorbate 60 1.6 Sorbitan sesquioleate 1.6 Propylparaben 0.6 Carboxyl vinyl polymer 1.5 incense a very small amount antiseptic a very small amount Purified water Balance system 100

After mixing the above ingredients, they were prepared according to the usual milk lotion production method.

3. Manufacture of nutritional cream

ingredient Content (% by weight) The lipoic acid-PEG-peptide derivative (LAP-KTTKS) 1.0 glycerin 4.0 vaseline 3.5 Triethanolamine 2.1 Liquid paraffin 5.3 Squalane 3.0 Wax 2.6 Cotopheryl acetate 5.4 Polysorbate 60 3.2 Carboxyl vinyl polymer 1.0 Sorbitan sesquioleate 3.1 incense a very small amount antiseptic a very small amount Purified water Balance system 100

After mixing the above ingredients, they were prepared according to the usual nutritional cream manufacturing method.

Claims

(LAP-KTTKS, PEGylated Lipoic acid-peptide conjugate) represented by the following formula (1): < EMI ID =
[Chemical Formula 1]

In Formula 1, n is an integer of 1 to 1000.

(a) 2- chloro-trityl chloride resin and Fmoc (9-fluorenylmethyloxycarbonyl), Boc (N - tert- butyloxycarbonyl) and the t-Bu (tert -butyl) protecting group-protected amino acid (serine-lysine-threonine-threonine -lysine to ) In the presence of a base to produce a compound of formula (5);
(b) reacting the compound of formula (5) with LAP (PEGylated lipoic acid) to produce a compound of formula (6); And
(c) deprotecting the side chain protecting group of the compound of formula (VI) and separating the solid phase resin to prepare the compound of formula (1); (LAP-KTTKS) represented by the following Reaction Scheme 1: < EMI ID =
[Reaction Scheme 1]

In the above Reaction Scheme 1,
(i) is DIC ( N, N'- diisopropylcarbodiimide); HOBt (N-Hydroxybenzotriazole); A mixed solution of dichloromethane (DCM) and dimethylformamide (DMF)
(ii) is a mixed solution of TFA (trifluoroacetic), EDT (1,2-ethanedithiol), TIS (triisopropylsilane) and H ₂ O,
SPPS is a solid-phase peptide synthesis, Fmoc is 9-fluorenylmethyloxycarbonyl,
and n is an integer of 1 to 1000.

A cosmetic composition for whitening comprising a lipoic acid-PEG-peptide derivative represented by the following formula
[Chemical Formula 1]

In Formula 1, n is an integer of 1 to 1000.

4. The whitening cosmetic composition according to claim 3, wherein the content of the lipoic acid-PEG-peptide derivative of Formula 1 is 0.001 to 10% by weight based on the total weight of the composition.

A cosmetic composition for improving wrinkles comprising a lipoic acid-PEG-peptide derivative represented by the following formula (1)
[Chemical Formula 1]

In Formula 1, n is an integer of 1 to 1000.

6. The cosmetic composition for improving wrinkles according to claim 5, wherein the content of the lipoic acid-PEG-peptide derivative of formula (1) is 0.001 to 10% by weight based on the total weight of the composition.