KR20130083103A

KR20130083103A - Anti-aging peptides having depigmentation, anti-wrinkle, anti-oxidation, hair-growth, and angiogenic activities, and uses thereof

Info

Publication number: KR20130083103A
Application number: KR1020120003650A
Authority: KR
Inventors: 김대현; 김국현
Original assignee: (주)와이즈덤레버러토리
Priority date: 2012-01-12
Filing date: 2012-01-12
Publication date: 2013-07-22

Abstract

The present invention relates to a peptide having a skin condition-improving effect and a use thereof, and more particularly to skin wrinkle improvement by collagen biosynthesis promoting action, skin whitening effect by tyrosinase enzyme inhibitory action, and inflammatory cytokines. It can provide an anti-inflammatory effect through the expression inhibiting action and promote the growth of hair papilla cells and promote the growth of blood vessels by promoting growth factor expression and hair follicle growth-promoting growth factor expression.

Description

Anti-aging Peptides having depigmentation, anti-wrinkle, anti-oxidation, hair-growth, and angiogenic activities, peptides for skin whitening, wrinkle improvement, antioxidant, anti-inflammatory, hair growth and anti-aging of angiogenesis and uses pretty}

The present invention relates to a peptide having a skin condition improving effect and a use thereof, and more specifically, to a skin wrinkle improvement effect by promoting collagen biosynthesis, a skin whitening effect by a tyrosinase enzyme inhibitory effect, and an inflammatory cytokine expression inhibitory effect. It can provide an anti-inflammatory effect and hair growth promoting effect by promoting papilla cell proliferation and angiogenesis-promoting growth factor expression and hair follicle growth-promoting growth factor expression.

Skin aging is known to occur due to cell variation, accumulation of free radicals, toxic substances, and modification of skin gap constituent proteins such as collagen. As a result, skin wrinkle formation, decreased skin elasticity and skin spots appear. The most important cause of skin wrinkles is a decrease in the density of the skin support layer due to the alteration and damage of collagen proteins that make up the skin layer, and the improvement of skin wrinkles can be expected by applying an active material that promotes skin collagen protein production to the skin. It is known that it can.

Skin pigment overdeposition diseases such as blemishes, freckles, and senile plaques are a phenomenon caused by pigment production of melanin in pigment cells over-produced due to ultraviolet rays or free radicals, and transferred to the skin keratinocytes. In this melanin biosynthesis process, the enzyme action called tyrosinase plays the most important role, and ingredients that inhibit tyrosinase activity are known to be effective in skin whitening activity.

Atopic dermatitis, contact dermatitis, scalp psoriasis, acne dermatitis Most skin disorders are accompanied by inflammatory reactions such as itching, erythema and edema. do. However, long-term use of topical corticosteroids is known to cause various skin side effects such as skin atrophy and swelling progenitors. Therefore, there is a need for development of active skin-friendly materials with less skin side effects.

Hair growth shows a regular cycle of 2 to 6 years of growth, 2 to 4 weeks of degeneration, and 3 to 4 months of rest periods. If abnormal changes occur, hair loss may occur. Currently, the representative hair loss treatment drug is finasteride, which is a mechanism for inhibiting the production of the male hormone dihydrotestosterone, which is the cause of hair loss, and is being sold. Minoxidil is being sold for its efficacy in treating hair loss due to increased nutrient supply. It is also known that several growth hormone proteins, which function to promote the growth of dermal papilla cells and hair follicle growth in hair follicles, play an important role in hair formation. Growth factors involved in hair regeneration include IGF-1 (Insulin-like Growth Factor), VEGF (Vascular Endothelial Growth Factor), and bFGF (basic Fibroblast Growth Factor), and in the case of VEGF, hair follicles through angiogenesis It is known to strengthen hair follicles by nourishing the cells smoothly, and bFGF is known to play an important role in the differentiation of hair follicles, especially when observed in the inner and outer hair follicle areas of growing hair follicles.

Angiogenesis refers to the mechanism by which new blood vessels are formed from existing blood vessels and is known to play an important role in wound healing and the female reproductive cycle. Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis, and is known to be involved in the development and homeostasis of blood vessels and lymphatic vessels through binding to VEGF receptors.

The problem to be solved by the present invention is to provide a skin external preparation composition comprising a novel peptide and the same as an active ingredient to improve skin wrinkles, skin whitening effect, skin inflammation and hair loss prevention, hair growth promoting effect and angiogenic effect.

The present inventors have shown that each peptide of the present invention improves skin wrinkles and whitening, prevents hair loss and improves hair growth according to aging, and improves skin disease caused by anti-inflammatory acne skin, atopic skin, and promotes angiogenesis protein expression in humans. It was found that the angiogenic effect according to the present invention was completed.

In order to confirm the skin wrinkle improvement effect of the peptide in the present invention, the degree of collagen protein synthesis and the level of collagen protein were identified using skin fibroblasts, and the degree of inhibition of melanin production was analyzed by analyzing the activity of tyrosinase, a melanogenesis enzyme. Analyzed.

In addition, the hair papilla and human mesenchymal stem cell proliferation, vascular endothelial growth factor, epithelial growth factor and tumor necrosis factor-alpha expression level were compared and analyzed to prevent hair loss, promote hair growth, and improve inflammatory skin. In addition, by promoting the expression of vascular endothelial growth factor, epithelial growth factor, it can be seen that the blood vessels are formed through this medium and may also help to improve skin anti-aging of wrinkles and whitening.

In the present specification, the term 'external skin' means that it is used to improve the skin condition or to suppress the deterioration of the skin condition by directly applying to the skin to exhibit various effects. The external skin preparation of the present invention can be used as cosmetics, pharmaceuticals, and in particular external medicines such as ointments. Among them, the use of skin cosmetics or hair cosmetic forms is preferred, and the form of such cosmetics is not particularly limited.

Peptides according to the present invention is effective in improving skin wrinkles by promoting collagen protein biosynthesis, skin whitening effect by inhibiting tyrosinase enzyme activity, anti-inflammatory effect by inhibiting inflammatory cytokine expression and promoting papilla cell proliferation Hair loss prevention and hair growth promoting effect by angiogenesis-promoting growth factor expression and hair follicle growth-promoting growth factor expression.

Fig. Figure shows the promotion of collagen gene expression.
Fig. It is a graph showing the collagen biosynthesis promoting effect.
3. It is a graph showing the degree of inhibition of tyrosinase enzyme activity.
FIG. It is a graph showing the effect of promoting cell proliferation using dermal papilla cells.
Figure 5. It is a graph showing the effect of promoting cell proliferation using human mesenchymal stem cells.
6. It is a graph showing the effect of promoting the production of vascular endothelial growth factor.
7. It is a graph showing the effect of promoting epidermal growth factor production.
Figure 8. It is a graph showing the degree of inhibition of tumor necrosis factor-alpha expression.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not construed as being limited by these embodiments.

Example 1 Collagen Gene Expression Analysis

After culturing skin fibroblasts for 24 hours, each peptide was treated with 10 ^-9 M and after 24 hours, total RNA was extracted and RT-PCR was used to confirm the expression level of collagen mRNA.

Total RNA from cells cultured under conditions was isolated using the TRIzol method (Gibco-BRL). Next, cDNA was synthesized from 3 μg total RNA using Superscript II reverse transcriptase (Promega) and oligo dT as a template using total RNA treated with an RNase-OUT ribonuclease inhibitor (Invitrogen Inc, USA). Each sample was used to amplify the collagen gene using a thermal cycler (ABI Biosystem 9700). Amplification conditions are as follows: (1) 48 degrees Celsius, 50 minutes; (2) 94 degrees C., 1 minute; (3) 94 degrees C., 30 seconds; Annealing 55 degrees Celsius, 45 seconds; 30 cycles repeated at 72 ° C., 1 min amplification; (4) 72 degrees C., 3 minutes; And (5) a final hold at 4 degrees Celsius until the sample is analyzed. The amplified collagen gene was electrophoresed on 1% agarose gel and confirmed under UV light.

As a result, as shown in Figure 1 it can be seen that the collagen gene expression is promoted in the cells treated with each peptide.

Example 2 Measurement of Collagen Biosynthesis Promotion Effect

Human normal fibroblasts were seeded in 6-well microplates (2 × 10 ⁵ cells / well) containing DMEM medium and incubated at 37 ° C. for 24 hours in a CO 2 incubator at 5% concentration. After 24 hours, the medium was removed from each well, and each peptide was treated in DMEM containing 0.05% of FBS, and then cultured again for 24 hours. After 24 hours, the cell culture medium was collected to prepare a sample.

The amount of collagen synthesized was determined using the collagen measurement kit (Procollagen Type I C-peptide EIA kit (MK101), Takara, Kyoto, Japan). The amount of collagen type I C-peptide (Type I C-peptide; PICP) Was measured. After diluting the standard solution included in the collagen kit, absorbance was measured at 450 nm to prepare a standard concentration curve. 100 ul of an antibody solution with POD enzyme was added to each well, and 20 ul of the supernatant of the sample and the standard solution diluted by concentration were added and mixed. The mixture was left at 37 ° C. for 3 hours, and then all contents were removed and each well was washed four times with 400 ul of PBS. 100 ul of substrate solution was added to the wells, and then left at room temperature for 15 minutes. In order to stop the reaction, 100ul of 1N sulfuric acid solution was added and mixed, and the absorbance was measured at 450nm. This experiment was conducted twice at the same time to find the average value.

As a result, each peptide promoted collagen biosynthesis and showed the highest collagen production rate between 10 ^-9 and 10 ^-7 M concentration according to each peptide. The results are shown in Fig.

Example 3 Measurement of Inhibition of Tyrosinase Activity

Tyrosinase was used as Sigma (Sigma) company extracted from the mushrooms. First, tyrosine, a substrate, was dissolved in sodium phosphate buffer (0.1M, pH6.8) to make a solution having a concentration of 0.3 mg / ml, and the solution was added to a test tube with 334 µl, and then sodium phosphate buffered again. 576 μl of solution (0.1M, pH6.8) was added. 70 μl of each sample was taken for each peptide concentration and added to the in vitro solution and allowed to react for 10 minutes in a 37 ° C. incubator.

In the control group, 70 μl of purified water was used instead of the peptide of the present invention. 20 µl of 1700 units / ml of tyrosinase solution was added to the reaction solution of the experimental group and the control group so that the total volume was 1 ml and reacted in a 37 ° C incubator for 30 minutes. The test tube containing the reaction solution was placed in ice water to quench the activity of tyrosinase, and then the absorbance at 470 nm was measured.

The blank of the experimental group was the reactant except the tyrosinase solution, that is, the tyrosine, each peptide, and the reaction solution of sodium phosphate buffer, and the blank of the control group was to add purified water instead of the peptide in the blank of the experimental group.

Inhibition rate of tyrosinase for each peptide concentration and control was calculated using the following Experimental Formula 1. The experimental results are shown in Table 1.

[Experimental Equation 1]

% Tyrosinase inhibition = 100-{[(A-B)-(C-D)] / (A-B)} X 100

A: absorbance of the control group B: blank absorbance of the control group

C: absorbance of the experimental group D: blank absorbance of the experimental group

Peptide
order IC50 (M) DHK <10 ^-11 KHK <10 ^-11 GHK <10 ^-10 KHG <10 ^-10 RHG <10 ^-11 HHK <10 ^-11 HHR <10 ^-11

As shown in Table 1 and Figure 3, each peptide inhibited the activity of tyrosinase.

Example 4 Evaluation of Efficacy of Hair Papillary Cell Proliferation

Evaluation of dermal papilla cell proliferation efficacy was measured by BrdU (bromodeoxyuridine) reagent by the antibody immunoassay (BrdU proliferation assay, CALBIOCHEM) by comparing the amount of cells that divide and label BrdU while culturing cells Was done. The dermal papilla cells were cultured in an incubator at 37 ° C. and 5% CO 2 using DMEM (Cambrex) containing 10% FBS (Gibco BRL) as a medium. Cells were planted at a concentration of 5x10 ³ cells / well and cultured with DMEM (no peptide) and each peptide according to the present invention was added to each concentration to incubate for 3-5 days, and the proliferation was measured.

The result showed that the concentration of each peptide 10- ⁹ M highest dermal papilla cell proliferation accelerating effect in as shown in FIG.

Example 5 Evaluation of Stem Cell Proliferation Promoting Effect

Human mesenchymal stem cells are prepared according to the following method. The mesenchymal stem cell separation is prepared by passing the donor bone marrow to the monocytes from the bone marrow through density gradient centrifugation and subcultured with DMEM culture.

Human mesenchymal stem cell proliferation efficacy evaluation was measured using BrdU (bromodeoxyuridine) reagent immunoassay (BrdU proliferation assay, CALBIOCHEM) as a way to compare the amount of BrdU-labeled and dividing cells while culturing cells . The cells were planted at a concentration of 3x10 ³ / cm ² and cultured with DMEM-serum free and each peptide according to the present invention was added thereto at a concentration of 10 ^-9 M, and cultured for 3 to 5 days, and the proliferation was measured.

As a result, each of the peptide (10- ⁹ M) as shown in Figure 5 is shown a 1.6 ~ 2.2 times as the mesenchymal stem cell growth promoting effect.

Example 6 Vascular Endothelial Growth Factor (VEGF)

Synthesis Promotion Effect Analysis>

Human normal fibroblasts were seeded in 48-well microplates (1 × 10 ⁴ cells / well) containing DMEM medium and incubated at 37 ° C. for 24 hours in a CO 2 incubator at 5% concentration. After 24 hours, the medium was removed from each well, and each peptide (10 ^-9 M) was treated in DMEM-serum free medium, and then cultured again for 72 hours. After 72 hours, cell media were collected to prepare samples.

In order to analyze the amount of VEGF expression was performed using an ELISA kit (VEGF ELISA kit, R & D systems INC., MN, USA). 100 μl of the culture solution was reacted on a plate of an antibody-labeled kit, and washed with phosphate buffer (PBS) to carry out peroxidase-attached goat anti-mouse immunoglobulin G.

(peroxidase-labeled goat anti-mouse IgG, R & D systems INC., MN, USA) and washed again. Color development was carried out using a substrate (TMB; 3,3 ', 5,5'-tetramethylbenzidine) and the reaction was terminated. The absorbance was measured at 450 nm using an ELISA reader.

As a result, as shown in FIG. 6, the VEGF expression was increased in the culture treated with each peptide.

Example 7: Epidermal cell growth factor (bFGF; basic fibroblast growth factor)

Synthesis Promotion Effect Analysis>

To analyze the degree of bFGF expression, the cell culture solution was collected and stored at -70 ° C.

The effect of each peptide on the expression of basic fibroblast growth factor (bFGF) was examined using an enzyme-linked immunosorvent assay (ELISA).

Fibroblasts were cultured in 48-well plates at 1 × 10 ⁴ cell number / well concentration, followed by treatment with each peptide (10 ⁻⁹ M) in each well and incubated for 72 hours. The culture was reacted by dispensing 100 μl into a plate labeled with recombinant human fibroblast growth factor (rhFGF) antibody (bFGF ELISA kit, R & D systems INC., MN, USA). After washing the reacted cells, they were reacted with peroxidase-attached goat anti-mouse immunoglobulin G (R & D systems INC., MN, USA) and washed, followed by TMB (3 After the color development using a 3 ', 5,5'-tetramethylbenzidine substrate, the reaction was terminated, and the absorbance was measured at 450 nm using an ELISA reader (Molecular Devices, USA) to confirm the expression level of bFGF.

As a result, it can be seen that bFGF expression was increased in the culture medium treated with each peptide as compared to the control without any treatment as shown in FIG. 7.

Example 8: Anti-inflammatory efficacy analysis

Inflammatory responses in the human body are mediated mainly by proinflammatory cytokine. In order to evaluate the anti-inflammatory efficacy of each peptide according to the present invention, the expression of TNF-alpha was confirmed by using animal-level Balb / C mice.

Balb / C mice were injected with 300 ug / kg of bacterial lipopolysaccharide (LPS) to induce an inflammatory response. Mice were injected with each peptide (10 ^-9 M) except control (+ LPS & -LPS). After 16 hours of peptide injection, mouse blood was collected and the amount of TNF-alpha was measured using an ELISA kit (Genzyme, Mineapolis, MN). As shown in FIG. 8, it was observed that the production of TNF-alpha sustained by LPS was reduced by each peptide.

Amino acid sequence aspartate, histidine, lysine
Amino acid sequence 2 lysine, histidine, lysine
Amino acid sequence 3 glycine, histidine, leucine
Amino acid sequence 4 Lysine, histidine, glycine
Amino acid sequence 5 arginine, histidine, glycine
Amino acid sequence 6 histidine, histidine, lysine
Amino acid sequence 7 histidine, histidine, arginine

Claims

A skin condition improving peptide comprising 3 amino acids in length and characterized by the following sequence:

X ₁ HK or X ₂ HL or X ₃ HG or X ₄ HR

X ₁ is aspartate, lysine, arginine, or histidine;
X ₂ Is glycine, aspartate, lysine, histidine, or arginine;
X ₃ Is lysine, arginine, aspartate, histidine, or glycine;
X ₄ Is histidine, aspartate, lysine, arginine, or glycine.

Peptide copper complexes that are three in length and improve skin wrinkles, prevent skin aging, improve skin whitening, improve atopic dermatitis symptoms, improve acne skin inflammation, improve scalp psoriasis and prevent hair loss, and promote hair growth.

X ₁ HK or X ₂ HL or X ₃ HG or X ₄ HR

X ₁ is aspartate, lysine, arginine, or histidine;
X ₂ Is glycine, aspartate, lysine, histidine, or arginine;
X ₃ Is lysine, arginine, aspartate, histidine, or glycine;
X ₄ Is histidine, aspartate, lysine, arginine, or glycine.

Peptide showing improvement in skin condition consisting of one amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6 and 7.
The amino acid first sequence is aspartate, histidine, lysine
The second amino acid sequence is lysine, histidine, lysine
The amino acid third sequence is glycine (glycine), histidine (histidine), leucine (leucine)
The fourth amino acid sequence is lysine, histidine, glycine
The fifth amino acid sequence is arginine, histidine, glycine
The sixth amino acid sequence is histidine, histidine, and lysine.
The seventh amino acid sequence is histidine, histidine, and arginine.

The skin external preparation according to claim 1, wherein the improvement of the skin condition is skin wrinkle improvement, skin aging prevention, skin whitening improvement, atopic dermatitis symptom improvement, acne skin inflammation improvement, scalp psoriasis improvement and hair loss prevention, hair growth promotion Composition.

The angiogenic peptide according to claim 1, which is mediated by up-regulation of vascular endothelial growth factor (VEGF) or epithelial cell growth factor (bFGF).

The skin external preparation according to claim 3, wherein the improvement of the skin condition is skin wrinkle improvement, skin aging prevention, skin whitening improvement, atopic dermatitis symptom improvement, acne skin inflammation improvement, scalp psoriasis improvement and hair loss prevention, hair growth promotion Composition.

The angiogenic peptide according to claim 3, which is mediated by up-regulation of vascular endothelial growth factor (VEGF) or epithelial cell growth factor (bFGF).