KR20090046504A - Composition for preventing and improving skin disease comprising capsiate, dihydrocapsidate or salt thereof as an active ingredient - Google Patents

Abstract

The present invention relates to a novel use of capsiate or dihydrocapsiate, and more particularly, to prevent skin diseases comprising capate, dihydrocaptate or salts thereof as an active ingredient. It relates to a composition for improvement. The composition of the present invention exhibits an effect on skin diseases and angiogenesis due to the anti-UV, anti-aging effect through antioxidant activity, inflammatory response inhibition and angiogenesis inhibition. Therefore, the composition of the present invention can be used for the purpose of preventing and improving diseases caused by skin diseases and angiogenesis.

Capsate, Dehydrocapsate, Inflammatory disease, UV protection, Skin aging, Prevention, Treatment

Description

Composition for preventing and improving skin disease comprising capsiate, dihydrocapsidate or salt thereof as an active ingredient

The present invention relates to a novel use of capsiate or dihydrocapsiate, and more particularly, to prevent skin diseases comprising capate, dihydrocaptate or salts thereof as an active ingredient. It relates to a composition for improvement.

Skin disease is any abnormality that occurs in the skin of animals, including humans. Since the skin covers the surface of the body, there are many opportunities for direct contact with various stimuli or external pathogens such as ultraviolet rays and physicochemical stimuli, and are affected by the body. In addition, the skin disease is caused by various causes because it is affected by pathological changes such as heredity, inflammation, benign and malignant tumors, hormones, trauma, degenerative degeneration.

Ultraviolet (UV), one of the causes of skin diseases, is only a fraction of the sunlight that reaches the earth, but acts as a factor causing erythema, pigmentation, wrinkles and most skin cancers. It is the reactive free radicals that are responsible for the damage to skin cells and tissues caused by UV rays, which destroy the antioxidant defenses made up of antioxidant and non-enzymatic antioxidants, eventually leading to decreased elasticity, wrinkle formation, keratin thickening and collagen breakdown. Photoaging due to symptoms such as et al ., Exp Gerontol, 35 (3): 307-316, 2000) or skin cancer (Hurza and Pentland,J Invest Dermatol, 100: 35S-41S, 1993; Brenneisen Pet al ., Ann NY Acad Sci 973: 31-43, 2002; Wlascheck met al ., J Phtochem Photobiol B 63: 41-51, 2001). In order to prevent or treat skin diseases caused by ultraviolet rays, it is necessary to construct a system capable of activating the production of enzymes mainly acting on the skin as well as in vivo.

Inflammatory skin disease is a complex series of physiological factors, including enzyme activation by various inflammation mediators and immune cells, secretion of inflammatory mediators, fluid infiltration, cell migration, and tissue destruction due to various stimulating factors causing a series of inflammatory reactions in the skin epithelium. A disease that causes a series of clinical signs and symptoms, such as itching, swelling, erythema, peeling, fever, and pain. Inflammatory skin diseases include atopic dermatitis, contact dermatitis, seborrhoic dermatitis and acne.

Among them, atopic dermatitis is generally used in the same sense as eczema (eczema), eczema-like skin lesions are also referred to as endogenous eczema or Benny yangjin. The cause of atopic dermatitis has not been identified to date and is known to have a genetic tendency, and now it is persuaded that it is a kind of autoimmune disease. Unlike normal eczema or dermatitis, it shows unusual symptoms and course, and 70 to 80% of pediatric eczema corresponds to this. Contact dermatitis is an inflammation of the skin caused by contact with an external substance. It has the same symptoms as acute eczema, but differs from eczema in that it occurs as a response to certain externally acting substances. Seborrheic dermatitis is a dermatitis that occurs well in areas with high secretion of sebum, such as the head, forehead, and armpits. It is also called seborrheic eczema. A lot of erythema and slender slits (dandruff) occur and appear in many 20s and 40s. Unlike ordinary eczema, the disease is caused by constitution or sebaceous abnormalities, sensitive to sunlight or heat, often worsening in spring and autumn, and easy to recur.

Until now, antihistamines, vitamin ointments, and corticosteroids have been mainly used for the treatment of inflammatory skin diseases. However, these drugs have a temporary effect and most often have severe side effects. Therefore, there has been a demand for the development of new substances effective for inflammatory skin diseases.

On the other hand, capsiate is a kind of capsaicin (capsaicin) is a kind of capsaicinoid (capsaicinoid) mainly found in CH19 Sweet, a spicy pepper variety. Capsate also activates capsaicin receptors and exhibits the same efficacy as capsaicin, such as body fat accumulation inhibition. However, various studies have been conducted on their mechanisms due to their non-spicy characteristics, but have not yet obtained specific results.

Therefore, the inventors of the present invention, while studying the physiological functions of capsate and dihydrocapsate, found that they have new functions such as inhibition of inflammatory reactions and inhibition of angiogenesis, and thus include capsate or dehydrocapsate. The present invention was completed by developing a cosmetic composition for preventing and improving skin diseases.

Accordingly, it is an object of the present invention to provide novel uses of capsates or dihydrocapsates.

In order to achieve the above object, the present invention provides a cosmetic composition for preventing and improving skin diseases comprising a capsate, dihydrocapsate or a salt thereof as an active ingredient.

Hereinafter, the content of the present invention will be described in more detail.

The composition of the present invention is characterized in that it comprises a capate, dihydrocaptate or a salt thereof.

The capsate or dihydrocapsate of the present invention has the structures of Formulas 1 and 2, respectively, and can be separated and purified from nature, used commercially, or prepared by chemical synthesis methods known in the art.

Preferably, the capsate or dihydrocapsate may be separated and purified from nature. More preferably, it can be separated and purified from CH19 Sweet which is not spicy pepper variety. The capsate or dihydrocapsate according to the present invention can be extracted by known methods commonly used in the art, such as organic solvent extraction and chromatography.

In one embodiment of the present invention was examined the inhibitory effect of capsate and dehydrocaptate on the intracellular reactive oxygen group induced by ultraviolet irradiation. As a result, it was found that the active oxygen group generated by ultraviolet irradiation decreased when the capsate or dihydrocapsate was treated, and thus the capsate and the dihydrocapsate had an antioxidant effect (Example 1 Reference).

In another embodiment of the present invention, the expression of COX-2 by UV and TPA, MMP-1 expression by UV, expression of IL-6, IL-8 and TNF-α, which are pro-inflammatory cytokines by UV, The inhibitory effect of capsate or dehydrocaptate was examined on the expression of VEGF, MMP-2 and MMP-9. As a result, the capsate or dehydrocapsate inhibited the expression of COX-2 whose expression was increased by irradiation of ultraviolet rays or TPA, MMP-1, IL-6, a pro-inflammatory cytokine by ultraviolet irradiation. Inhibition of the expression of IL-8 and TNF-α and the expression of angiogenesis products, VEGF, MMP-2 and MMP-9, and the capsate or dehydrocaptate was induced by UV irradiation for inflammatory response It can be seen that the effect of inhibiting the formation of blood vessels (see Examples 2 to 5).

In another embodiment of the present invention, the inflammatory response and skin damage by UV light, COX-2 expression by UV light, IL-6 and TNF-α, which are pro-inflammatory cytokines by UV light, The inhibitory effect of capsate on the expression of angiogenesis products, Ki67, PECAM-1, ICAM-1, VEGF, MMP-2 and MMP-9 and erythema by UV light was examined. As a result, the capsate mitigates the inflammatory reaction, skin damage and erythema caused by ultraviolet light, COX-2 with increased expression, IL-6 and TNF-α as pro-inflammatory cytokines, Ki67 as an angiogenesis product. Inhibition of the expression of PECAM-1, ICAM-1, VEGF, MMP-2 and MMP-9, and thus the capsate has the effect of inhibiting the inflammatory response and neovascularization caused by ultraviolet irradiation even in vivo It was found (see Examples 5 to 9).

Accordingly, the present invention provides a cosmetic composition for the prevention and improvement of skin diseases, including captate, dihydrocaptate or salts thereof.

The cosmetic composition of the present invention may include capate or dihydrocaptate in itself or in the form of a salt. As the salt, an acid addition salt formed by free acid is preferable. The free acid may be an organic acid or an inorganic acid. The organic acid is not limited thereto, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, Glutaric acid and aspartic acid. In addition, the inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid.

The cosmetic composition may be prepared in liquid or solid form using bases, adjuvants and additives commonly used in the cosmetic field. Cosmetics in liquid or solid form may include, but are not limited to, for example, cosmetics, creams, lotions, baths and the like. At this time, bases, adjuvants and additives commonly used in the cosmetic field are not particularly limited, and may include, for example, water, alcohols, propylene glycol, stearic acid, glycerol, cetyl alcohol, liquid paraffin, and the like.

More specifically, the cosmetic composition of the present invention contains the basic cosmetic composition (cosmetics, creams, essences, cleansing foams and cleansing water, containing the capsate or dihydrocapsate of the present invention and with dermatologically acceptable excipients, Pack, body oil), color cosmetic composition (foundation, lipstick, mascara, makeup base), hair product composition (shampoo, rinse, hair conditioner, hair gel), functional cosmetic composition (charity blocker, wrinkle improver) and soap It may be prepared in the form. The excipients include, but are not limited to, emollients, skin penetration enhancers, colorants, fragrances, emulsifiers, thickeners and solvents. In addition, fragrances, pigments, fungicides, antioxidants, preservatives and moisturizing agents may be further included, and may include thickeners, inorganic salts, synthetic polymer materials and the like for the purpose of improving the properties.

For example, when preparing the face wash and soap containing the capate or dihydrocapsate of the present invention, it can be easily prepared by adding capate or dihydrocaptate to the usual face wash and soap base. . When the cream is prepared, it can be prepared by adding a capate or a dehydrocaptate to a general oil-in-water (O / W) cream base. To this, fragrances, chelating agents, pigments, antioxidants, preservatives and the like, or synthetic or cloth burning materials such as proteins, minerals and vitamins for the purpose of improving physical properties may be added.

The content of the captate or dihydrocaptate contained in the cosmetic composition of the present invention may be contained in a range of 0.0001 to 50% by weight, preferably 0.01 to 10% by weight, based on the total weight of the cosmetic composition.

The composition according to the present invention has an excellent effect of preventing skin damage caused by ultraviolet rays, and is effective in skin damage due to ultraviolet rays itself or an inflammatory reaction caused by ultraviolet rays. More specifically, it is very useful for the prevention and improvement of various skin diseases caused by ultraviolet rays or inflammation, and can also be very useful for maintaining the quality by preventing oxidation of the cosmetics themselves due to the antioxidant effect.

The skin disease refers to a skin disease or inflammatory skin disease caused by ultraviolet rays. The skin disease caused by ultraviolet rays is not limited thereto, but may include photoaging, blemishes, freckles, blemishes, dry skin, skin wrinkles, sunburn, photokeratosis, skin cancer, and seborrheic keratosis. Can be. In addition, the 'inflammatory skin disease' is not limited to this, skin irritation, acute and chronic eczema, contact dermatitis, atopic dermatitis, seborrheic dermatitis, chronic simple mammary glands, interrogation, deprivation dermatitis, papular urticaria, psoriasis, Sun dermatitis, acne and the like.

On the other hand, in another embodiment of the present invention, the effect of capsate or dehydrocapsate on the proliferation of endothelial cells induced by VEGF, an angiogenesis inducer, was examined. As a result, it was found that there was an effect of inhibiting the proliferation of captate and dehydrocaptate endothelial cells, and this effect was not due to cytotoxicity (see Example 10).

Thus, in another embodiment of the present invention, the mechanism of inhibition of proliferation of endothelial cells was confirmed by analyzing changes in cell cycle and expression of cyclin protein. As a result, it can be seen that by reducing the expression of cyclin D1 in the transition from the G1 phase to the S phase, the capsate and the dihydrocaptate inhibit the progression of the cell cycle to inhibit proliferation (see Example 11).

In another embodiment of the present invention was confirmed the effect on VEGF-induced endothelial cell metastasis and tube formation. As a result, it was found that the chemotactic mobility and tube formation increased when VEGF was treated, but decreased when treated with captate or dehydrocaptate (see Example 12).

In another embodiment of the present invention was confirmed the effect on the expression of VE-cadherin and endothelial permeability induced by VEGF. As a result, it was found that capsate and dihydrocapsate inhibited VEGF-induced epithelial permeability by inhibiting tyrosine phosphorylation of VE-cadherin (see Example 13).

In another embodiment of the present invention was confirmed the effect on the neovascularization of endothelial cells induced by VEGF. As a result, it was found that capsate and dehydrocaptate inhibited VEGF-induced neovascularization by inhibiting germination of aortic rings, angiogenesis in matrigel plugs, and endothelial cell formation. See Example 14).

Accordingly, the present invention provides a composition for the prevention and improvement of skin diseases comprising capsate, dihydrocapsate or salts thereof as an active ingredient. The composition of the present invention can be used in various forms in the form of cosmetics for the purpose of preventing and improving skin diseases.

Below. The present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

< Example  1>

Induced by ultraviolet irradiation Intracellular To free radicals (intracelluar ROS)  Inhibitory effect

<1-1> Captain's Intracellular  Reactive Oxygen Group Inhibitory Effect

HaCaT cells (Human keratinocyte cell line) (donated by Professor N. Fusenig of Cancer Research, Germany) in 5 x 10 ⁵ cells (1 x 10 for a 100 mm culture dish) ⁶ cells), and then incubated in DMEM (Dulbecco's modified Eagle's medium) medium containing 10% fetal bovine serum for 24 hours.

After incubation, capsiate was treated at 50 μM for 30 minutes, and the cells were washed three times using phosphate buffered saline (PBS). UV lamps (FSX 24 T12 / UVB / HO, pansol ^TM ) to induce reactive oxygen species (ROS) USA) was irradiated with ultraviolet light of 100 J / m ² . Immediately after UV irradiation, the cells were washed again with phosphate buffer and capsid was treated in DMEM medium containing 1% fetal bovine serum at the same concentration as the pretreatment concentration, followed by further incubation for 2 hours. Here, DCFH-DA (2,7-dichlorofliuorescin diacetate) was treated with HBSS (hank's balanceed salt solution, BioWhittaker, CAMBREX, USA) for 30 minutes at a concentration of 25 μM and washed three times with phosphate buffer.

The cells thus treated were measured for UV-induced ROS using a fluorescence microscope (Carl ZEISS, USA), and the experimental group was divided into the following groups: no treatment group-neither UV nor capsiate; Experimental group A-only treated with capsiate, not UV; Control group-only UV treatment; Experimental Group B—Ultraviolet and capsiate treatments respectively (the same as in Examples 1 to 3 below).

As a result, as shown in Fig. 1A, the generation of intracellular ROS is significantly reduced compared to the case of treating the capsate before ultraviolet irradiation (experimental group B) only the ultraviolet rays (control group), and the capsate inhibits ROS induced by ultraviolet rays. It was found that there is an effect. Therefore, it can be seen that capsate has an antioxidant effect in view of the effect of inhibiting ROS induced by ultraviolet rays.

<1-2> Dehydrocapsate Intracellular  Reactive Oxygen Group Inhibitory Effect

Except for the use of dehydrocapsiate (dehydrocapsiate) instead of the capsate in the same manner as in Example <1-1> was measured the inhibitory effect of the intracellular reactive oxygen group induced by ultraviolet light.

As a result, as shown in Fig. 1B, the intracellular ROS generation was significantly reduced compared to the case of treatment with dehydrocaptate prior to ultraviolet irradiation (experimental group B) and the irradiation of only ultraviolet ray (control), so that the capsate was induced by ultraviolet rays. It was found that there is an effect of inhibiting ROS. Therefore, it can be seen that the dehydrocaptate also has an antioxidant effect in view of the effect of inhibiting ROS induced by ultraviolet rays.

< Example  2>

COX The inhibitory effect of -2 expression

<2-1> Captain's  By ultraviolet COX -2 expression inhibition effect

In order to determine the effect of capsiate on COX-2 expression by UV light in human keratinocytes, HaCaT cells were inoculated with 5 x 10 ⁵ cells in a 60 mm culture dish, followed by fetal calf serum. (fetal bovine serum) was incubated for 24 hours in Dulbecco's modified Eagle's medium (DMEM) medium containing 10%.

After culturing, starvation was made by incubating for 24 hours in DMEM medium containing 1% fetal calf serum. After treatment with capsiate at each concentration (10 μM, 25 μM, 50 μM) for 30 minutes, the cells were washed three times with phosphate buffered saline (PBS), and then COX-2 was induced. Ultraviolet rays of 100 J / m ² were irradiated. Immediately after UV irradiation, the cells were washed again with phosphate buffer and incubated for 16 hours by treating capsiate with pretreatment concentration in DMEM medium containing 1% fetal calf serum.

In order to know the expression level of COX-2 in the cultured cells, western blotting was performed by the following method. The cells of the test group treated with each condition were treated with 2 mM EDTA, 137 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM sodium vanadate, 10 mM NaF, and 1 mM PMSF (phenylmethanesulfonyl fluoride) Protein was dissolved in RIPA buffer containing 1% Triton X-100, 10% Glycerol and a protease inhibitor cocktail and separated by SDS-PAGE electrophoresis using a 10% acrylamide gel. It was. It was transferred to a nylon membrane, followed by goat anti-COX-2 antibody (goat anti COX-2 antibody, santacuz, USA) and secondary antibody, anti-goat IgG Horse Radish Peroxidase conjugation antibody, 1; 10000, zymed) was used to isolate the desired protein. This was detected using ECL (Amersham, USA) and then imaged using an image analyzer RAS 3000 Imaging system (FUJI film, Japan).

As a result, as shown in FIG. 2A, in the control group, the expression of COX-2 was increased by UV irradiation, and the increased COX-2 expression was confirmed to decrease in a concentration-dependent manner after treatment with capsiate.

<2-2> COX -2 confirm the mechanism of suppression of expression

MAPK signaling and transcription to control which intracellular pathways inhibit COX-2 expression in capsiate inhibition of COX-2 expression induced by UV irradiation in human keratinocytes. We investigated the transcription factor NF-kB signaling.

MAPKs are mainly involved in the processes of intraflammation, apoptosis and carcinogenesis, as well as cell damage induced by UV irradiation (Peus D, Vasa RA, Beyerle A). , Meves A, Krautmacher C, Pittelkow MR.UVB activates ERK1 / 2 and p38 signaling pathways via reactive oxygen species in cultured keratinocytes. J Invest Dermatol 1999; 112: 7516). The transcription factor NF-kB also plays a role in regulating various intracellular factors, including COX-2, which is expressed in response to cellular inflammatory responses (Paik J, Lee JY, Hwang D. Signaling pathways for TNFa-induced COX-). 2 expression:. mediation through MAP kinases and NFkB, and inhibition by certain nonsteroidal anti-inflammatory drugs Adv Exp Med Biol 2002; 507: 503-508.).

In order to investigate the intracellular factors affecting COX-2 expression regulated by capsiate, capsiate was treated by concentration (10 μM, 25 μM and 50 μM) before UV irradiation for 30 minutes. Secondary with rabbit anti-ERK antibody, rabbit anti-p-ERK antibody, rabbit anti-p38 antibody, rabbit anti-p-p38 antibody, rabbit anti-JNK antibody and rabbit anti-p-JNK antibody (cell signaling), respectively Western blot was performed in the same manner as in Example <2-1> except that an anti-rabbit IgG-HRP binding antibody (zymed) was used as the antibody.

As a result, as shown in Figure 2B, p38 and JNK was confirmed that the phosphorylation of the ERK (capsilate) is suppressed by the capsitite (capsiate) compared with no change.

<2-3> COX -2 confirm the mechanism of suppression of expression

In order to examine the activation of the transcriptional regulator NF-kB, immunostaining was performed using HaCaT cells, which are human keratinocyte lines, in the following manner.

After inoculating the cells on a 14 mm culture slide, the cells were incubated for 24 hours in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum. After incubation, the cells were starved for 24 hours in DMEM medium containing 1% fetal bovine serum (starvation). Thereafter, capsiate was treated at 25 μM for 30 minutes, and the cells were washed three times with phosphate buffer and then irradiated with 100 J / m ² UV. Immediately after UV irradiation, the cells were washed again with phosphate buffer and treated with capsiate at the same concentration as pretreatment in DMEM medium containing 1% fetal bovine serum, followed by incubation for 4 hours, and the cells were fixed with methanol for 5 minutes. Immunostaining was performed.

The fixed cells were blocked with 10% normal goat serum and then washed three times with phosphate buffer. Hoechst (Sigma St. Louis, Mo.), followed by fluorescent staining of NF-kB using a rabbit anti-NF-kB antibody (santacruz, USA) and an anti-rabbit IgG secondary antibody (invitrogen, USA) attached to Alexa 488. Counter staining was performed.

As a result, as shown in Fig. 2C, NF-kB was activated by ultraviolet irradiation and moved into the nucleus, whereas in experimental group B treated with capsate before ultraviolet irradiation, only the untreated group or the experimental group treated with no capsid was treated. As shown by A, the presence of NF-kB in the cytoplasm shows that Capsate inhibits nuclear migration of NF-kB induced by ultraviolet irradiation.

<2-4> Dehydrocapsate  By ultraviolet COX -2 expression inhibition effect

In the same manner as in Example <2-1> except that dihydrocapsate was used instead of capsate to examine the effect of dehydrocapsate on the expression of COX-2 by ultraviolet light in human keratinocytes. To determine the effect on COX-2 expression.

As a result, as shown in FIG. 2D, in the control group, the expression of COX-2 was increased by UV irradiation, and the increased COX-2 expression was confirmed to decrease in a concentration-dependent manner after treatment with dihydrocaptate.

<2-5> Captain's TPA On by COX -2 expression inhibition effect

To determine the effect of capsiate on COX-2 expression by Phorbol 12-myristate 13-acetate (TPA) in human keratinocyte line (HaCaT), HaCaT cells were cultured in a 60 mm culture dish in a 5 x 10 mm dish. After inoculating ⁵ cells, the cells were incubated for 24 hours in DMEM (Dulbecco's modified Eagle's medium) containing 10% fetal bovine serum.

After culturing, starvation was made by incubating for 24 hours in DMEM medium containing 1% fetal calf serum. Capsiate was then treated for 30 minutes at each concentration (10 μM, 25 μM, 50 μM), and then TPA was treated at a concentration of 80 uM to induce COX-2. The cells so treated were then incubated for another 6 hours.

In order to know the expression level of COX-2 in the cultured cells, western blotting was performed by the following method. The cells of the test group treated with each condition were treated with 2 mM EDTA, 137 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM sodium vanadate, 10 mM NaF, and 1 mM PMSF (phenylmethanesulfonyl fluoride). Proteins were dissolved in RIPA buffer containing 1% Triton X-100, 10% Glycerol and protease inhibitor cocktail and separated by SDS-PAGE electrophoresis using 10% acrylamide gel. . It was transferred to a nylon membrane, followed by goat anti-COX-2 antibody (goat anti COX-2 antibody, santacuz, USA) and secondary antibody, anti-goat IgG Horse Radish Peroxidase conjugation antibody, 1; 10000, zymed) was used to isolate the desired protein. This was detected using ECL (Amersham, USA) and then imaged using an image analyzer RAS 3000 Imaging system (FUJI film, Japan).

As a result, as shown in FIG. 3, in the control group, COX-2 expression was increased by TPA treatment, but in the experimental group treated with capsiate, it was confirmed that the COX-2 expression increased by TPA was decreased.

< Example  3>

Induced by ultraviolet light MMP -1 expression inhibitory effect

Ultraviolet irradiation increases MMP-1, a collagen degrading enzyme in skin tissue, which mediates skin aging. To investigate the effect of capsiate on this action, the following experiments were carried out using HaCaT cells and human fibroblasts.

HaCaT cells and human normal fibroblasts were inoculated to 1 x 10 ⁶ cells in a 100 mm dish, followed by DMEM (Dulbecco's modified Eagle's) containing 10% fetal bovine serum. medium) and incubated for 24 hours in medium. After incubation, the cells were starved for 24 hours in DMEM medium containing 1% fetal calf serum. After treatment with capsiate at each concentration (10 μM, 25 μM, 50 μM) for 30 minutes, the cells were washed three times with phosphate buffer, and then HaCaT and human were used to induce collagen degrading enzyme MMP-1. Fibroblasts were irradiated with UV at 100 J / m ² and 200 J / m ² , respectively. Immediately after UV irradiation, the cells were washed again with phosphate buffer and treated with capsiate at the same concentration as pretreatment in DMEM medium containing 1% fetal bovine serum and incubated for 48 hours.

In order to measure the degree of inhibition of enzymes affecting skin aging after culture, RT-PCR was used to determine the mRNA expression of MMP-1 induced in the skin by UV irradiation. RNA was extracted from the cells of each experimental group using trizol (trizol, Invitrogen, USA), and PCR (PTC-225 peltier thermal cycler, MJ Reserch USA) was used as a template to prepare cDNA of each experimental group. The mRNA expression level of MMP-1 was measured using commercially available MMP-1 primers (Quiagen, USA) and real-time PCR (Roter Gene 6000 series, Corbett Reserch, Aus) as templates. The mRNA expression level of MMP-1 in the cell was expressed as compared to the untreated group.

As a result, as shown in Figure 4, as a result of the experiment, it was confirmed that the expression of MMP-1 increased when irradiated with HaCaT cells (control). In contrast, in the experimental group treated with capsiate, the expression of MMP-1 was reduced compared to the control group not treated with the capsiate. Accordingly, it was found that capsiate significantly reduced the expression of MMP-1, a collagen degrading enzyme increased by ultraviolet irradiation.

< Example  4>

Pro-inflammatory cytokines induced by UV irradiation ( pro - inflammatory cytokine Caption for increase capsiate)  Inhibitory effect

Irradiation with ultraviolet light increases cytokines such as IL-6 (interleukin-6), IL-8, and TNF-α (tumor necrosis factor-α), which mediate inflammatory responses. To investigate the effect of capsiate on this action, human keratinocyte lines were tested as follows.

HaCaT cells were inoculated to 1 x 10 ⁶ cells in a 100 mm culture dish, and then cultured in DMEM (Dulbecco's modified Eagle's medium) medium containing 10% fetal bovine serum for 24 hours. After incubation, the cells were starved for 24 hours in DMEM medium containing 1% fetal calf serum. After treatment with capsiate at each concentration (10 μM, 25 μM, 50 μM) for 30 minutes, the cells were washed three times with phosphate buffer and then 100 J / m ² to induce cytokines. UV light was irradiated. Immediately after UV irradiation, the cells were washed again with phosphate buffer and incubated in DMEM medium containing 1% fetal bovine serum with capsiate at the same concentration as the pretreatment concentration for 4 hours.

MRNA level of IL-6, IL-8 and TNF-α induced mainly in the skin by UV irradiation to measure the degree of inhibition of pro-inflammatory cytokine affecting the inflammatory response after culture Was determined by RT-PCR.

RNA was extracted from the cells of each experimental group using trizol (trizol, Invitrogen, USA), and PCR (PTC-225 peltier thermal cycler, MJ Reserch USA) was used as a template to prepare cDNA of each experimental group. Using each of the cDNAs as a template, commercial IL-6, IL-8 and TNF-α primers and real-time PCR (Roter Gene 6000 series, Corbett Reserch, Aus) were used to determine IL-6, IL-8 and TNF-α. mRNA expression level was measured. The mRNA expression level of intracellular pro-inflammatory cytokines was expressed in comparison to the untreated group.

As a result, as shown in Figure 5, the experimental results, it was confirmed that the expression of the pro-inflammatory cytokines IL-6, IL-8 and TNF-α when exposed to the ultraviolet keratinocytes (control). On the contrary, in experimental group B treated with capsiate, the expression of IL-6, IL-8 and TNF-α was decreased compared to the control group not treated with capsiate. Therefore, it was found that capsiate significantly reduced the expression of IL-6, IL-8 and TNF-α increased by ultraviolet irradiation.

< Example  5>

UV-induced angiogenesis substances ( angiogenesis factor For expression Capsiate  Inhibitory effect

<5-1> VEGF  About manifestation Captain's  Inhibitory effect

When an inflammatory response occurs, the blood vessels dilate and the skin becomes red (erythema), and subsequent frequent inflammatory reactions lead to carcinogenesis. One of the most prominent phenomena of these cancers is angiogenesis, which is an essential process for cancer cells to grow (Fukuda R, Kelly B, Semenza GL. Vascular endothelial growth factor gene expression in colon cancer cells exposed to prostaglandin E2). is mediated by hypoxia-inducible factor 1. Cancer Res 2003; 63: 23304). In addition, during the inflammatory process, infiltration of inflammatory cells occurs and secretion of various cytokines causes cell migration or cell proliferation, which leads to the release of neovascular products. do. Therefore, in order to investigate the effect of capsiate on this series of reactions, an angiogenesis product was analyzed.

First, in order to investigate the ability of capsiate to inhibit angiogenesis, an immunostaining method was used to examine capsiate's inhibition of expression of VEGF (vescular edotheilal growth factor), which is a representative intracellular angiogenesis inducer.

To determine the effect on VEGF, HaCaT cells were seeded on 14 mm culture slides, and then cultured in DMEM (Dulbecco's modified Eagle's medium) medium containing 10% fetal bovine serum for 24 hours. After incubation, the cells were starved for 24 hours in DMEM medium containing 1% fetal bovine serum (starvation). Thereafter, capsiate was treated at 25 μM for 30 minutes, and the cells were washed three times with phosphate buffer and then irradiated with 100 J / m ² UV. Immediately after UV irradiation, the cells were washed again with phosphate buffer and treated with capsiate to the pretreatment concentration in DMEM medium containing 1% fetal bovine serum, followed by incubation for 24 hours, and the cells were fixed with methanol for 5 minutes. Immunostaining was performed as in Example <2-3>. At this time, a rabbit anti-VEGF IgG antibody (santacruz. USA) and an anti rabbit-IgG secondary antibody (Invitrogen, USA) attached with Alexa 488 were used for VEGF detection.

As a result, as shown in Fig. 6A, the expression of VEGF was increased in the control group irradiated only with ultraviolet rays, and in the experimental group B further treated with capsid, the expression of VEGF was decreased so that the capsiate was induced by UV irradiation. Inhibition of expression of VEGF in the body was seen.

<5-2> MMP -2 and MMP For -9 Capsiate  Inhibitory effect

MMPs are matrix metalloproteinases that are induced by pro-inflammatory cytokines and are representative of neovascularized substances expressed during the induction of neovascularization in cells by ultraviolet light (Mantena SK, Meeran SM, Elmets CA, Orally administered green tea polyphenils prevent ultraviolet radiation induced skin cancer in mice through cativation of T cells and inhibition of angiogenesis in tumors.J nutr 2005: 135: 2871-7).

In this regard, the inhibition of capsiate on the expression of MMP-2 and MMP-9, which induce angiogenesis, was measured by mRNA expression using real-time PCR and protein expression and activity using zymorgraphy. I found out.

In order to determine the capsiate inhibition of MMP expression, HaCaT cells were inoculated in 5 x 10 ⁵ doses in a 60 mm culture dish, followed by DMEM containing 10% fetal bovine serum. (Dulbecco's modified Eagle's medium) was incubated for 24 hours. After 24 hours of culture, starvation was performed by incubating for 24 hours in DMEM medium containing 1% fetal calf serum. Thereafter, capsiate was treated at each concentration (10 μM, 25 μM, 50 μM) for 30 minutes, and the cells were washed three times with phosphate buffer and then irradiated with 100 J / m ² UV. Immediately after UV irradiation, the cells were washed again with phosphate buffer and treated with capsiate at the pretreatment concentration in DMEM medium containing 1% fetal bovine serum, followed by incubation for 36 hours, followed by intracellular MMP-2 and 9 mRNA. Expression levels were examined using real-time PCR in the same manner as in Example 4. At this time, the mRNA expression level of MMP was expressed as compared to the untreated group and the experimental group is as follows.

As a result, as shown in FIG. 6B, the mRNA expression level of MMP induced by ultraviolet rays was measured. As a result, MMP-2 and MMP were treated in the case of experimental group B irradiated with ultraviolet rays after treatment with capsiate compared to the control group irradiated with ultraviolet rays only. A decrease in the expression level of -9 was seen. Thus, capsiate appears to inhibit mRNA expression of MMP-2 and 9 induced by ultraviolet light.

<5-3> MMP -2 and MMP For -9 Capsiate  Inhibitory effect

In relation to these results, zymorgraphy was performed to investigate the expression level reduction and the activity of MMP-2 and MMP-9 at the protein level.

HaCaT cells were inoculated in 5 x 10 ⁵ volumes in a 60 mm culture dish, and then cultured in DMEM (Dulbecco's modified Eagle's medium) medium containing 10% fetal bovine serum for 24 hours. After incubation, the cells were starved for 24 hours in DMEM medium containing 1% fetal calf serum. Thereafter, capsiate was treated at each concentration (10 μM, 25 μM, 50 μM) for 30 minutes, and the cells were washed three times with phosphate buffer and then irradiated with 100 J / m ² UV. Immediately after UV irradiation, the cells were washed with phosphate buffer again, and treated with capsiate in the DMEM medium containing 1% fetal bovine serum at the same concentration as the pretreatment concentration, followed by further incubation for 36 hours. The culture medium of each cell was harvested and the activity of MMP-2 and MMP-9 protein secreted into the medium was measured. At the time of measurement, the same amount of medium obtained from the same cell number was all concentrated in the same amount and maintained at 4 ° C. in order not to affect the protein activity.

Prepared samples were electrophoresed at 4 ° C using a 7.5% acrylamide gel containing gelatin and no SDS, followed by reconditioning buffer (50 mM TrisHCl, pH 7.4, 2.5). % (v / v) Triton X-100) for 30 minutes followed by zymogram incubation buffer (50 mM TrisHCl, pH 7.6, 50 mM NaCl, 10 mM CaCl ₂ , 0.05% Brij35) in a 37 ° C thermostat. The reaction was carried out for at least 16 hours while being kept in the flask. The reacted gels were then stained with 0.1% corgi to measure the activity of MMP-2 and MMP-9.

As a result, as shown in Figure 6C, it was confirmed that the expression of pro MMP-2, MMP-2 and MMP-9 is significantly increased in the medium of the cell exposed to ultraviolet rays, compared with the capsiate before ultraviolet irradiation In the medium of the experimental group B treated with the expression and activity of pro MMP-2 and MMP-2 and MMP-9 was observed.

These results confirm that capsiate significantly reduces the expression of MMP induced by ultraviolet light. Capsiate has an excellent effect on inhibiting angiogenesis derived from an inflammatory response.

< Example  6>

UV radiation in animal models Capsiate  effect

In order to confirm in vitro animal experiments using human keratinocyte line HaCaT cells in animal models, the following experiments were performed using hairless mouse (SKH-1). Treated only with acetone without irradiation; Experimental group A-1mM capsiate was dissolved in acetone without treatment with ultraviolet rays and treated with 200 µl every 24 hours; Control group-treated only with ultraviolet and acetone; Experimental Group B—Ultraviolet and 1 mM capsiate, respectively, was dissolved in acetone and treated with 200 μl every 24 hours (the same as in Examples 6 and 7 unless otherwise stated).

Capsiate was applied to the back of the mouse 1 hour before ultraviolet irradiation and then irradiated with ultraviolet light of 2.5 KJ / m ² . In order to prevent rash that may occur immediately after ultraviolet irradiation, the same concentration of capsiate was applied to each experimental mouse again after 1 hour. Capsiate was then applied every 24 hours and UV irradiation was carried out four times at a dose of 2.5 KJ / m ² every other day. One hour after the last irradiation to investigate changes in mouse tissue and intracellular signaling. After euthanizing the mice used in the experiment, the skin tissue was biopsied after skin tissue was observed by removing the dorsal skin tissue.

As a result, as shown in Figure 7A, in the control group mice (UV) the dorsal skin was thick blood vessels and a lot of blood, inflammatory reaction due to the wound (wound) also occurred severely. However, the skin tissues of the mice treated with UV after applying capsiate (Experimental Group B, UV + CPT) had decreased blood vessel thickness, blood swelling, and the number of them. And it was confirmed that the depth is less.

In addition, hematocylin-eosin staining was performed to examine the changes in the overall skin tissue. As shown in FIG. 7B, hyperplasia of the untreated group (UV) epidermis was observed. In contrast, the mice exposed to UV rays after treatment with capsiate (Experimental Group B, UV + CPT) showed a significant decrease in epidermal hyperkeratosis and decreased blood vessels and edema. there was.

As a result, it was thought that capsiate could effectively protect against skin damage caused by UV irradiation.

< Example  7>

UV-induced in animal models COX For -2 expression Capsiate  effect

<7-1> COX For -2 expression Capsiate  effect

As described above, it was found that capsiate inhibits inflammatory reactions, angiogenesis and edema due to the overall skin wound caused by ultraviolet irradiation. Thus, in order to determine the effect of capsiate associated with the inflammatory response in the animal model, changes in COX-2 expression and intracellular signaling were confirmed by Western blot using the tissues of the mice tested in Example 6. .

The skin tissue frozen in liquid nitrogen was finely ground using a mortar and pestle, followed by RIPR buffer (2 mM EDTA, 137 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM sodium vanadate, 10 mM NaF, 1 mM PMSF). Proteins were isolated using 1% Triton X-100, 10% glycerol, protease inhibitor cocktail) and then SDS-PAGE electrophoresis using acrylamide gel. It was transferred to a nylon membrane and the COX-2 protein was observed using goat anti-COX-2 IgG antibody (santacuz, USA) and anti-goat IgG HRP attached antibody (zymed) as the secondary antibody. In order to image this, it was detected using ECL (Amersham, Piscataway, NJ) and imaged using an image analyzer (RAS 3000 Imaging system, FUJI film, Japan).

As a result, as shown in Figure 8A, it was confirmed that COX-2 expressed by four times of ultraviolet irradiation is significantly reduced in the skin tissue of the mouse by capsiate treatment.

<7-2> COX For -2 expression Capsiate  effect

In order to confirm this, mouse skin tissues were immunostained for COX-2 expression as follows. Biopsied skin tissues from the mice were fixed in 4% paraformaldehyde for 24 hours and washed again with running water for 24 hours. The tissues were sequentially dehydrated in 70, 80, 90, 95, and 100% ethanol and xylene, and then immersed in the dissolved paraffin to infiltrate the tissue. The paraffin tissue thus cut was thinly sliced to 5 μm, fixed to the slide, and the slide was again paraffin-free and submerged using 100, 95, 90, 80, 70% ethanol and sterile distilled water from xylene. After the epitope retrieval process using 10 mM sodium chloride solution (sodium citric acid buffer, 0.05% Tween 20, pH 6.0), intracellular peroxidase (H ₂ O ₂ ) After removal, blocking was performed for 10 hours with 10% normal goat serum. The blocked tissues were washed three times with phosphate buffer, treated with COX-2 antibody (1: 100, santecruz, USA) diluted in 10% normal goat serum, bound for at least 16 hours at 4 ° C, and then again phosphate buffered. Washed three times. This was treated with a biotinyl secondary (biotinylaton) secondary antibody (DAKO code K0675; Carpinteria, DAKO) for 30 minutes and washed again three times. After treatment with streptavidin-peroxidase (DAKO code K0675) for 30 minutes, it was washed three times with phosphate buffer and finally with DAB-3-3 diaminobenzidine (DAB) (DAKO). I developed a color. The colored tissue was fixed to the slide after dehydration and observed under an optical microscope.

As a result, as shown in FIG. 8B, it was found that COX-2 was significantly increased by ultraviolet rays, and capsiate effectively suppressed the increased COX-2.

<7-3> COX -2 confirm the mechanism of suppression of expression

To investigate intracellular signaling associated with COX-2 expression inhibited by capsiate, proteins isolated from mouse skin tissue (ERK, p-ERK, p38) were used. , p-p38 and JNK, p-JNK) were subjected to western blot as in Example <2-2>.

As a result, as shown in Fig. 8A, P38 and JNK could not see a big difference between the UV irradiation after acetone treatment only (UV) and the UV irradiation after treatment with capsiate (UV + CPT). However, in the case of ERK, the phosphorylation (phospholation) was inhibited in the experimental group irradiated with UV light after the treatment with capsiate.

From these results, as in Example <2-2>, it could be seen that capsiate regulates COX-2 expression and related intracellular changes by inhibiting phosphorylation of ERK.

<7-4> for pro-inflammatory cytokines Captain's  Inhibitory effect

To investigate the effects of capsate on IL-6 and TNF-α produced and induced in skin tissue during inflammatory reactions, the expression level of pro-inflammatory cytokines and the inhibitory ability of capsate were examined as follows. At this time, the experimental group was as follows: untreated group-treated only with acetone without irradiating ultraviolet light; Experimental Group A-200 μl treatment of 1 mM capsiate in acetone without UV treatment; UV alone treatment group-treated only with UV and acetone; Experiment group B-1mM capsiate (capsiate) dissolved in acetone and 200μ treatment and after one hour UV treatment.

To this end, pretreatment with acetone and capsate before UV irradiation was carried out after 5 hours of irradiation at 5 KJ / m ² , followed by euthanasia for 6 hours, followed by RT-PCR using mRNA extracted from each tissue of a mouse without biopsies. It was confirmed as follows. In order to extract RNA from the tissue, each mouse tissue was rapidly frozen in liquid nitrogen after biopsy and finely chopped using a mortar and pestle. RNA was extracted from mouse tissue using Trizol (invitrogen, USA), and then PCR (PTC-225 peltier thermal cycler, MJ Reserch USA) using a commercial kit (reverse transcription system, Quiagen, USA) was used as a template. CDNA of each experimental group was prepared.

Real-time PCR (Roter Gene 6 series, Corbett Reserch, Aus) was performed using IL-6 and TNF-α primers (Quiagen, USA) as a template for each cDNA. The degree was expressed in comparison with the acetone-treated group after normalization with GAPDH (glyceraldehyde-3-phosphate dehydrogenase).

As a result, as shown in Figure 8C it can be seen that the expression of IL-6 and TNF-α induced by ultraviolet light is reduced by the capsiate (capsiate). These results suggest that capsiate can effectively control the inflammatory response by inhibiting the pro-inflammatory cytokines induced in the inflammatory response by ultraviolet rays.

< Example  8>

Animal Models Using Hairless Mice Capsiate In neovascularization  Inhibitory effect

In order to investigate the effect of capsate on various intracellular expression inducers related to neovascularization among a series of intracellular reactions induced by inflammatory reactions, the ultraviolet light of capsiate using mouse tissue in Example 6 was used. Inhibition of angiogenesis induced by the investigation was examined.

<8-1> Ki67 For Captain's  Inhibitory effect

Ki67 is a substance related to cell division and proliferation. It is an intracellular substance whose expression is increased in the epidermis basal layer during inflammatory reactions and cancers (Boland GP, Butt IS, Prasad R, et al. COX- 2 expression is associated with an aggressive phenotype in ductal carcinoma in situ.Br J Cancer 2004; 90: 423429). In order to confirm the effect of the capsiate (capsiate) on the immunostaining was carried out as follows.

Skin tissue obtained by biopsy from mouse skin was immersed in OCT compound (Sakura Finetechnical Co. Ltd, Japan) and then slowly frozen in liquid nitrogen. The tissue thus prepared was sectioned to 7 μm and fixed to acetone on the slide. The tissue was washed with distilled water and phosphate buffer and then blocked with 10% normal goat serum for 1 hour, followed by ki67 antibody (1: 100, neomarker, USA) for 16 hours at 4 ° C. Reacted. The resultant was washed three times with phosphate buffer, and then treated with Alexa 488-conjugated secondary antibody (invitrogen, USA) for 30 minutes and washed three times. It was mounted and observed with a fluorescence microscope (Carl ZEISS, USA).

As a result, as shown in Fig. 9A, it was observed that Ki67 expressing cells significantly increased by ultraviolet rays were significantly inhibited by the treatment of capsiate.

<8-2> PECAM For -1 Captain's  Inhibitory effect

In addition to Ki67, Platelet-Endothelial Cell Adhesion Molecule-1 (CD31), an angiogenic cognitive substance, is an endothial marker, which increases its expression around the vascular wall during neovascularization. My substance is Thus, by examining the expression level of PECAM-1, the effect of capsiate associated with neovascularization can be observed.

To investigate this, the paraffin sections of the prepared tissues as in Example 7 were subjected to the same procedure as in Example 7, and then treated with PECAM-1 antibody (1: 100, BD Pharmingen, USA) on the slide skin tissue. Treatment with attached secondary antibody (DAKO code K0675; DAKO Corp., USA) for 30 minutes followed by three washes again. After treatment with streptavidin-peroxidase (DAKO code K0675, DAKO Corp., USA) for 30 minutes, washed again three times with phosphate buffer and finally DAB-3-3 diaminobenzidine (DAB) (DAKO Corp., Color) to confirm the expression level of PECAM-1.

As a result, as shown in FIG. 9B, the expression of PECAM-1 was increased inside the blood vessel wall around the dermis by dermal irradiation, but the capsiate was treated before UV irradiation. It can be seen that the expression of is significantly reduced.

<8-3> ICAM For -1 Captain's  Inhibitory effect

In Example 7 to investigate the expression level of ICAM-1 (intercellular cell adhesion molecule-1, CD54) related to cell infiltration, cell migration and angiogenesis during the inflammatory process Western blot was performed in the same manner as in Example 7 using the protein isolated from the mouse skin tissue of each experimental group. At this time, a mouse anti-ICAM-1 antibody (mouse anti ICAM-1 antibody, santacruz, USA) and a HRP-attached secondary antibody (1: 10000, zymed) were used to investigate the expression level of ICAM-1.

As a result, as shown in Figure 9C, it was confirmed that the expression of ICAM-1 increased by ultraviolet light is reduced in the mouse skin tissue of experimental group B irradiated with UV light after treatment with capsid.

<8-4> VEGF For Captain's  Inhibitory effect

Western blotting was performed in the same manner as in Example 7 using the protein isolated from the mouse skin tissue of each experimental group for VEGF, which plays an important role in angiogenesis. At this time, VEGF antibody (santacruz. USA) and a secondary antibody (1: 10000, zymed) to which HRP was attached were used to investigate the expression of VEGF.

As a result, as shown in FIG. 9D, VEGF was secreted over the epidermal and dermal layers by UV irradiation, and it was found that the expression of VEGF was reduced in the skin tissues of mice when UV irradiation was performed after capsiate treatment.

<8-5> MMP -2 and MMP For -9 Captain's  Inhibitory effect

In addition, the in vitro cell experiment confirmed the decrease in the expression level by capsiate, and to examine the effects on MMP-2 and MMP-9 induced by angiogenesis and immune response in animal models. MRNA was extracted from mouse tissues of each experimental group using hairless mice.

In order to extract RNA from the tissue, each mouse tissue was rapidly frozen in liquid nitrogen after biopsy, and then chopped using a mortar and pestle, and RNA of mouse tissue was extracted using trizole (invitrogen, USA). As a template, cDNA of each experimental group was prepared by performing PCR (PTC-225 peltier thermal cycler, MJ Reserch, USA) using a commercial kit (reverse transcription system, Quiagen, USA).

Real-time PCR (Roter Gene 6 series, Corbett Reserch, Aus) was performed using MMP-2 and MMP-9 primers (Quiagen, USA) as a template for each cDNA, and mRNA expression level of MMP was leveled with GAPDH. After normalization, the expression level was shown in comparison to the acetone-treated group.

As a result, as shown in Figure 9E, it can be seen that the expression of MMP-2 and MMP-9 is significantly increased during the irradiation of ultraviolet rays, MMP-2 and 9-valent capsiate treatment increased by the ultraviolet irradiation It can be seen that the decrease.

As a result, capsiate can effectively inhibit angiogenesis mediated by an inflammatory response, and furthermore, there is a possibility that the capsiate is an inhibitor of inflammation and anticancer.

< Example  9>

Erythema induced by ultraviolet ( erythema For) Capsiate  effect

<9-1> About erythema Captain's  effect

When ultraviolet rays are irradiated on the skin, erythema occurs, such as reddening of the skin and swelling, followed by various cell changes such as inflammation, apoptosis and cancer (Tadashi Terui, Hachiro Tagami, Mediators of inflammation involved in UVB erythema, Journal of Dermatological Science 23 Suppl. 1 (2000) S1S5). In order to investigate the effect of capsiate on erythema accompanied by inflammatory reactions caused by ultraviolet rays, the following experimental groups were divided using hairless mice: untreated group-only acetone without irradiation Processing; Experimental group A-Capsiate was dissolved in acetone so as to be 1 mM without UV treatment, and 200 µl treatment was performed every 24 hours; UV alone treatment group-treated only with UV and acetone; Experimental Group B-Capsiate was dissolved in acetone to be ultraviolet and 1 mM, respectively, and 200 µl was treated every 24 hours.

Acetone and capsiate diluted therein were applied to the experimental group divided as above. One hour after attached to a cradle designed to only the square section of 1.2 cm diameter each mouse, etc. The UV light can be irradiated or the like of the mice 400, 600, 800, each of the J / m ² Ultraviolet rays were irradiated. After 48 hours, erythema was measured.

As a result, as shown in Fig. 10A, as the amount of UV irradiation increased, the erythema was increased, and the erythema of the mice treated with UV after capsiate treatment was higher than that of the mice irradiated with UV rays. The degree was weak.

<9-2> Changes in Skin Tissue Captain's  effect

In order to observe the change in the skin tissue of the mouse treated in Example <9-1>, hematoclin-eosin staining was performed on the portion irradiated with UV rays of 2.5 KJ / m ² a total of four times.

As a result, as shown in FIG. 10B, the thickness of the epidermis was increased when the ultraviolet rays were irradiated, but the thickness of the epidermis was reduced when the UV treatment was performed after the capsate was applied.

These results indicate that capsiate can effectively inhibit skin erythema induced by UV irradiation.

< Example  10>

VEGF Of endothelial cell proliferation induced by

<10-1> Identification of effects on endothelial cell proliferation

MTT analysis was performed to determine the effect of captate or dehydrocaptate on VEGF-induced endothelial cell (EC) proliferation.

Human umbilical vein endothelial cells (HUVECs) were placed in gelatin-coated 24 well plates at a concentration of 2 x 10 ⁴ cells / well and washed twice with M199 medium (invitrogen, USA) after 24 h incubation. It was then incubated for 6 hours in M199 medium containing 1% fetal bovine serum. Each concentration (1 μM, 5 μM, 10 μM, 25 μM) was added to capate or dehydrocaptate for 30 minutes and then incubated for 10 hours with 10 ng / ml of VEGF (Upstate, USA). MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide salt) solution (0.5 mg / ml) was added thereto and incubated at 37 ° C. for 3 hours. The residual amount of MTT was carefully removed, the resulting crystals were dissolved in DMSO for 30 minutes, mixed well for 5 minutes, and the absorbance was measured at 560 nm.

As a result, as shown in FIG. 11A, it was found that the proliferation of VEGF-induced HUVECs was inhibited in a concentration-dependent manner by capsate or dehydrocapsate. On the other hand, in view of the normal growth of cells when HUVECs were cultured with the capate or dihydrocapsate concentration raised to 50 μM without VEGF-induced stimulation, the inhibitory effect of such capsate or dehydrocapsate was observed. Does not appear to be cytotoxic (results not shown).

<10-2> Identification of effects on endothelial cell proliferation

The inhibitory effect of VEGF-induced endothelial cell (EC) proliferation of capsate or dihydrocapsate is described in Lee OH et al., 1999, Biochem. Biophys. Res. Commun., 264: 743 -750) method ³ was confirmed by performing H- thymidine in corporation analysis ⁽³ H-thymidine incorporation assay) according to according to.

Briefly, HUVEC was added to a gelatin-coated 24-well plate at a concentration of 2 x 10 ⁴ cells / well, washed twice with M199 medium (invitrogen, USA) after 24 hours of incubation, and then contained 1% fetal bovine serum. Incubated for 6 hours in one M199 medium. Pre-incubate for 30 minutes by adding 25μM captate or dehydrocaptate, and incubate for 30 hours with 10ng / ml of VEGF, and then incubate for 6 hours with 0.5μCi / ml of ³ H-thymidine. It was.

In cultured cells, DNA was precipitated with 10% trichloroacetic acid at 4 ° C. for 30 minutes and then washed twice with water. It was dissolved in 0.2N NaOH / 0.1% sodium dodecyl sulfate (SDS) and measured by liquid scintillation counter.

As a result, as shown in Figure 11B, it can be seen that VEGF increases the DNA synthesis of HUVEC, DNA synthesis was inhibited by the capsate or dehydrocapsate.

< Example  11>

Confirmation of Endothelial Cell Proliferation Inhibition Mechanism

<11-1> Cell Cycle Change Analysis

In order to determine the mechanism by which endothelial cell proliferation was inhibited in Example 10, FACS analysis (fluorescence-activated cell sorter analysis) was performed for each cell.

HUVECs were placed in 60 mm plates in an amount of 3 × 10 ⁵ cells, and then washed twice with M199 medium (invitrogen, USA) for 24 hours and then incubated for 6 hours in M199 medium containing 1% fetal calf serum. Each concentration (1 μM, 5 μM, 10 μM, 25 μM) was added to pre-incubate for 30 minutes by caption or dehydrocaptate, followed by incubation for 10 hours with 10 ng / ml of VEGF. It was collected and fixed with ethanol cooled on ice. Immobilized cells were dehydrated in phosphate buffer containing 2% fetal bovine serum, 0.1% Tween20 at 4 ° C. for 30 minutes, centrifuged and re-dissolved in the same buffer. RNase was treated (37 ° C., 1 hour) and then stained with propidium iodide. This was analyzed by FACS analyzer (FACScan DB PharMingen flow cytometer).

As a result, as shown in FIG. 11C, the HUVEC of G1 arrest was decreased compared to the control when VEGF was treated, but the endothelial due to VEGF was treated when capsid or dehydrocaptate was treated. The G1 quiescent state of the cells was decreased and HUVEC of G1 quiescent state was increased again. Therefore, it was found that the capsate or dihydrocapsate affects the transition from the G1 group to the S group.

<11-2> Cyclin D1 Determine impact on

Since the transition from the G1 group to the S group is partially regulated by cyclin D1, it was examined whether the capsate or dihydrocaptate affects it.

HUVECs were placed in 60 mm plates in an amount of 3 × 10 ⁵ cells, and then washed twice with M199 medium (invitrogen, USA) for 24 hours and then incubated for 6 hours in M199 medium containing 1% fetal calf serum. Each concentration (1 μM, 5 μM, 10 μM, 25 μM) was added to capate or dehydrocaptate for 30 minutes before incubation and 10 ng / ml of VEGF was added and incubated for 12 hours. After collecting the protein at each concentration was quantified and the protein level was confirmed by the antibody of cyclin D1, cyclin E, cyclin A using Western blotting.

As a result, as shown in FIG. 11D, when VEGF was treated, the expression of cyclin D1 was increased at the protein level, but it was found to decrease in a concentration-dependent manner by capsate or dihydrocapsate. In addition, in the case of cyclin E and cyclin A, the expression of VEGF was increased in the protein level, but it was found that the concentration-dependent decrease in capsate or dehydrocapsate.

Thus, it was found that the capsate or dihydrocapsate inhibits the progression of the VEGF-induced cell cycle by reducing the expression of cyclin D1 by the transition from the G1 phase to the S group.

< Example  12>

VEGF Inhibition of metastasis and tube formation induced by

<12-1> Chemotaxis  Determine impact on mobility

Transwell assay (Corning Costar, USA) was performed to determine the effect of capsate or dehydrocaptate on the chemotactic motility of HUVEC (Lee OH et al., 1999, Biochem). Biophys Res Commun. 264; 743-750).

Briefly, the bottom surface of the filter was coated with 10 μg gelatin and then M199 medium containing VEGF and 1% fetal calf serum was placed in the lower wells. HUVECs were trypsinized and prepared to a concentration of 1 × 10 ⁶ cells / ml in M199 medium containing 1% fetal calf serum. Each concentration (1 μM, 5 μM, 10 μM, 25 μM) of the captate or dehydrocaptate was pretreated for 30 minutes at room temperature. 100 ml of medium cells were placed in the upper well and incubated at 37 ° C. for 4 hours. The cells were fixed, stained with hematocillin and eosin and the non-migrating cells on the top surface of the filter were removed. Cells migrated to the bottom side of the filter were observed and counted with an optical microscope (x200) to measure mobility.

As a result, as shown in FIG. 12A, the chemotactic mobility increased when VEGF was treated in HUVEC, but concentration-dependently decreased when capsid or dehydrocaptate was treated.

<12-2> Confirmation of influence on tube formation

In order to determine the effect of capsate or dihydrocapsate on the degree of tube formation, according to the method described in known literature (Lee OH et al., 1999, Biochem. Biophys. Res. Commun. 264; 743-750) It was performed as follows.

Briefly, 250 μl growth factor-reduced Matrigel (10 mg protein / ml) was well mixed in a 16 mm diameter tissue culture well and polymerized at 37 ° C. for 30 minutes. HUVECs cultured for 6 hours in M199 medium containing 1% fetal bovine serum were trypsinized, pooled and resuspended in M199 medium containing 1% fetal bovine serum. Each concentration (1 μM, 5 μM, 10 μM, 25 μM) of capsate or dehydrocaptate was treated for 30 minutes at room temperature and then placed in a matrigel at a concentration of 1.8 × 10 ⁵ cells / well and VEGF (10 ng / ml). Put it. After 20 hours of incubation, the culture was photographed (x200), and the area covered by the tube network was measured using optical imaging techniques (scanned with Adobe Photoshop and measured with Image-Pro Plus (Media Cybermetics)). Measured.

As a result, as shown in FIGS. 12B and C, when HUVEC was treated with VEGF, a structure similar to that of a tube was formed, but it was found that their formation was reduced when capsid or dehydrocaptate was treated.

< Example  13>

VEGF Induced by VE - Cadherin  Inhibition of phosphorylation and endothelial cell permeability

<13-1> Confirmation of Inhibitory Effect on Endothelial Cell Permeability

A ^[14 C] sucrose permeability analysis ^([14 C] sucrose permeability assay) to confirm the kaepsi benzoate or dihydro cap when eyiteuyi effect on the endothelial permeability induced by VEGF was carried out.

Briefly, HUVECs were placed in a transwell filter (Corning Costar), and when reached confluence, they were incubated for 3 hours in M199 medium containing 1% fetal calf serum. Treated with caption or dehydrocaptate at each concentration (5 μM, 10 μM, 25 μM) for 30 minutes and then with VEGF (50 ng / ml) for 1 hour. [ ¹⁴ C] Sucrose (0.8 μCi / ml, 50 μl) was placed in the upper compartment, and after 30 minutes, the radiation dose diffused in the lower compartment was measured with a scintillation counter (Wallac, perkinElmer).

As a result, as shown in FIG. 13A, the endothelial cell permeability was increased when VEGF was treated, but the permeability of the endothelial cells was decreased when capseate or dehydrocaptate was treated.

<13-2> VE - Of VE-cadherin  Identify impact on expression

In order to determine whether the change in endothelial cell permeability is related to VE-cadherin, the expression level of VE-cadherin in the cell membrane gap was confirmed.

HUVEC cells were placed in gelatin-coated cover slips at a concentration of 12 × 10 ⁵ cells / well and incubated for 24 hours. After incubation, the cells were washed twice with M199 medium and incubated for 6 hours in M199 medium containing 1% fetal calf serum. Treated with caption or dehydrocaptate at each concentration (5 μM, 10 μM, 25 μM) for 30 minutes and then with VEGF (50 ng / ml) for 30 minutes. Cells were treated with 2% formaldehyde for 10 minutes to allow fixation and permeability, and washed three times with phosphate buffer. It was then treated with 0.1% Triton X-100 and 2% BSA / PBS blocking solution for 30 minutes. It was labeled with VE-cadherin antibody at room temperature for 2 hours and then washed with phosphate buffer and again labeled with FITC-attached secondary antibody for 1 hour and 30 minutes at room temperature. Cover slips were mounted on SloFade (Molecular Probes) and observed with a fluorescence microscope (Carl Zeiss).

As a result, as shown in FIG. 13B, when VEGF is treated, the cell adhesion function of VE-cadherin present in the cell-cell junction is greatly reduced, thereby increasing endothelial permeability and causing cell migration. However, when pretreatment with capsate or dihydrocapsate, it was confirmed that the cell adhesion function returned to its original state.

<13-3> VE - Cadherin  Identify the effect on phosphorylation

Since VEGF induces tyrosine phosphorylation of VE-cadherin, it is known to loosen intercellular contacts and regulate endothelial permeability (Esser S. et al., 1998, J. Cell Sci., 111: 1853-1865). Inhibition of VE-cadherin tyrosine phosphorylation was confirmed.

HUVECs were placed in 60 mm plates in an amount of 3.5 × 10 ⁵ cells, and then washed twice with M199 medium (invitrogen, USA) for 24 hours, and then incubated for 6 hours in M199 medium containing 1% fetal calf serum. Each concentration (5 μM, 10 μM, 25 μM) was added to capate or dehydrocaptate for 30 minutes before incubation, and 50 ng / ml of VEGF was added and incubated for 6 hours. After pooling, the proteins obtained at the respective concentrations were quantified, and then protein levels were confirmed using VE-cadherin antibodies using Western blotting.

As a result, as shown in Figure 13C and D, the treatment of VEGF induced the phosphorylation of VE-cadherin, it can be seen that when phosphorus or dihydrocaptate treatment their phosphorylation decreases in a concentration-dependent manner .

Summarizing these results, it was found that the capsate or the dihydrocaptate inhibited the tyrosine phosphorylation of VE-cadherin, thereby inhibiting the permeability induced by VEGF.

< Example  14>

VEGF Inhibition of Angiogenesis Induced by

<14-1> Identification of effects on neovascularization

When VEGF was treated to determine the effect of capsate or dehydrocaptate on VEGF-induced neovascularization, the degree of spouting from the aortic ring was confirmed as follows.

Arteries were isolated from Sprague Dawley rats (6-8 weeks old) and then prepared for aortic rings. The 48 well plate was coated with 120 μl of Matrigel, and then the aortic ring was placed and 50 μl of Matrigel was added again. To this was added the medium containing only 200 μl of VEGF and VEGF containing capsate or dihydrocapsate and containing VEGF (human endothelial serum-free medium, invitrogen, USA). After 6 days the cells were fixed and stained with Diff-Quick and measured in a double blinded manner at values of 0-5.

As a result, as shown in Figures 14A and B, it can be seen that the treatment of VEGF inhibits germination in a concentration-dependent manner when capsid or dehydrocaptate is treated, whereas germination is better from aortic rings.

<14-2> Identification of effects on neovascularization

Through these results, a mouse Matrigel plug assay was performed to investigate in vivo the effect of CAPGF-induced neovascularization of capsate or dehydrocaptate (Min KJ et. al., 2007, Blood, 15: 1495-1502).

Matrigel containing only 60 μg of capsate or dehydrocaptate, 100 ng of VEGF and 10 units of heparin, and 0.6 ml of Matrigel and 100 ng of VEGF were treated with C57 / BL6 mice (Orient, Korea). Injected via subcutaneous injection. After 6 days the resulting Matrigel plugs were removed.

As a result, as shown in Fig. 14C, when the VEGF only contains a dark red plug, the blood vessels contain a lot of red blood cells, VEGF-induced neovascularization can be seen that a lot of blood vessels generated in the matrigel there was. On the other hand, when the capsate or the dihydrocapsate was light in color, it was similar to that of matrigel alone, and it was found that neovascularization was hardly achieved.

<14-3> Measurement of endothelial cell content

CD31 staining was performed by immunohistochemical analysis to determine the endothelial cell content in the Matrigel plug formed above.

The extracted Matrigel plug is fixed with 2% formaldehyde, and then 15% sucrose and 30% sucrose are used to remove the moisture in the matrigel using the principle of osmotic pressure. . The hardened Matrigel plug was cut to 8 μm to 12 μm in thickness and stained with CD31-antibody (PECAM-1 antibody, BD Biosciences, USA) to check the level of endothelial cells under a microscope.

As a result, as shown in Figures 14D and E, the treatment with VEGF, while a lot of endothelial cells expressing CD31, when treated with capsate or dehydrocapsate is reduced to a similar level as the control group again. there was.

Thus, these results indicate that capsate or dehydrocaptate has the effect of inhibiting angiogenesis in animal models.

As described above, the composition of the present invention exhibits an effect on skin diseases and angiogenesis by having an ultraviolet blocking effect and skin aging prevention effect through antioxidant activity, inflammatory response inhibition and angiogenesis inhibition. Therefore, the composition of the present invention can be used for the purpose of preventing and improving diseases caused by skin diseases and angiogenesis.

Figure 1 shows the inhibitory effect of the intracellular active oxygen group of capsate (A) and dehydrocapsate (B) (Con: no treatment; CAT, deCPT: experimental group A; UV: control; UV + CAT UV + deCPT: experimental group B; CAT: capsate; deCPT: dihydrocapsate).

Figure 2 shows the effect of inhibiting COX-2 expression of capsate (A) and dihydrocapsate (D) and the effect on MAPK (B) and NF-κB (C) signaling (PERK: Phosphorylated ERK, PP38: phosphorylated P38).

Figure 3 shows the effect of inhibiting COX-2 expression induced by TPA of capsate (CPT: capsate).

Figure 4 shows the MMP-1 expression inhibitory effect induced by the ultraviolet irradiation of the capsate (A: UV 100J / m ² irradiation, B: UV 200J / m ² irradiation).

Figure 5 shows the inhibitory effect of capsate on the pro-inflammatory cytokines IL-6 (A), IL-8 (B), and TNF-α (C) (CPT10: Capsate 10 μM, CPT25: Capsate 25 μΜ; CPT50: Captate 50 μΜ).

Figure 6 shows the inhibitory effect of capsate on the expression of VEGF (A), MMP-2 and MMP-9 (B, C) (the numbers in the graph abscissas of Figures 4B and 4C indicate the amount Unit: μM)).

Figure 7 shows the effect of capsate on ultraviolet light in hairless mice (Vehicle: no treatment group)

FIG. 8 shows the effects on capxate COX-2 (A, B) and MAPK signaling (A) and on the expression of pro-inflammatory cytokines (C).

FIG. 9 shows the effects of Ki67 (A), PECAM-1 (B), ICAM-1 (C), VEGF (D) and MMP-2 and MMP-9 (E) expression associated with neovascular induction of capsate It is shown.

Figure 10 shows the effect on the erythema (A) and skin tissue (B) by the ultraviolet irradiation of capsate.

Figure 11 shows the effect on the endothelial cell proliferation of capsate and dehydrocapsate via MTT assay (A) and 3H-thymidine incorporation assay (B) and cell cycle (C) and cyclin of endothelial cells Changes in protein (D) are shown (D-cap: dihydrocaptate).

FIG. 12 shows the effects on chemotactic mobility (A) and tube formation (B, C) of capsate and dihydrocapsate.

FIG. 13 shows the effects on endothelial cell permeability (A) and expression of VE-cadherin (intercellular adhesion) and phosphorylation (C) of capsate and dehydrocaptate.

FIG. 14 shows the effect of capsate and dehydrocaptate on endothelial cell expression (D, E) identified by germination (A, B), Matrigel plug (C) and CD31 from aortic rings.

Claims (5)

A cosmetic composition for preventing and improving skin diseases comprising a capsiate represented by Formula 1 or a salt thereof as an active ingredient. <Formula 1>

A cosmetic composition for preventing and improving skin diseases comprising dihydrocapsiate represented by Formula 2 or a salt thereof as an active ingredient. <Formula 2>

The cosmetic composition according to claim 1 or 2, wherein the skin disease is a skin disease or an inflammatory skin disease caused by ultraviolet rays. The cosmetic composition according to claim 3, wherein the skin disease caused by ultraviolet rays is selected from the group consisting of photoaging, blemishes, freckles, blemishes, dry skin, skin wrinkles, sunburn, photokeratosis, skin cancer and seborrheic keratosis. The method of claim 3, wherein the inflammatory skin disease is skin inflammation, acute and chronic eczema, contact dermatitis, atopic dermatitis, seborrheic dermatitis, chronic simple mammary glands, interrogation, deprived dermatitis, papular urticaria, psoriasis, sun dermatitis and acne Cosmetic composition, characterized in that selected from the group consisting of.

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