KR0155613B1 - Collagenase expression inhibitor containing ursolic acid - Google Patents

Abstract

The present invention provides a novel use of ursolic acid, which exhibits the effect of inhibiting gene expression of collagenase induced by light, and thus preventing aging of the skin by light, and thus, ursolic acid may be used for aging of skin by light It can be usefully used to inhibit the action.

Description

Collagenase Expression Inhibitors Containing Ursolic Acid

1 is a northern hybrid response result showing that the expression of collagenase changes over time in fibroblasts irradiated with ultraviolet rays.

2 is a result of northern hybrid reaction comparing the expression of collagenase in the ursolic acid treated group and the untreated group.

The present invention relates to a collagenase expression inhibitor containing ursolic acid as an active ingredient.

When the human skin is chronically exposed to ultraviolet rays, it causes qualitative or quantitative changes in the dermal extratracelluar matrix, resulting in the drying of the skin, becoming rough and wrinkled, thickening of the stratum corneum, and loss of normal elasticity of the skin. Drooping (keratosis) photoaging occurs. In addition, the blood vessels expand capillaries, senile surpluses, freckles, and the like, irregularly deposited or changed pigment. Kligman reported how to apply retinoic acid to restore photoaged skin (Kligman Kligman. Photodermatol., 3. 5. 1986).

However, retinoic acid has a severe irritation at the time of application, and side effects such as thorax occur, so its use is limited.

When irradiated human fibroblasts with a long wavelength of ultraviolet rays A, an increase in the amount of collagen-degrading enzymes, type I and type III collagenase, was observed, which may cause photoaging of the skin. (Wlaschek et al., J. Invest. Dermatol., 101, 164, 1993).

The present inventors have studied the effect of ursolic acid, which regulates the growth and differentiation of cancer cells and inhibits anti-inflammatory and angiogenesis (Korean Patent Application No. 92-21,117). The phenomenon of suppressing the expression of genes has been found and based on this, the present invention has been completed.

Therefore, it is an object of the present invention to provide a new use of ursolic acid which has the effect of inhibiting gene expression of collagenase induced by light and thus preventing aging of the skin by light.

Other objects, features and applications of the present invention will become apparent to those skilled in the art from the following detailed description.

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

According to the present invention, ursolic acid inhibits the expression of the collagenase gene induced by light irradiation. Collagenase rapidly increases the amount of expression of its genes 12 hours after UV irradiation (FIG. 1). However, administration of ursolic acid before or after light irradiation significantly reduces the expression of the collagenase gene by light irradiation (FIG. 2). Therefore, it can be usefully used for preventing or treating aging of the skin by light irradiation.

Ursolic acid is extracted from loquat (Eriobotrya japonica), and its use as a skin whitening agent is known (Polasa, Japanese Patent Publication No. 90-2848). However, there has been no report or suggestion regarding suppressing the expression of collagenase as in the present invention.

The dosage of ursolic acid used for inhibiting collagenase gene induction according to the present invention is not particularly limited, and may be selected within various ranges according to its body type, administration method, administration purpose, and the like. For example, cosmetics may be applied to the skin in various forms such as skins, lotions, creams, essences, or packs, and pharmaceuticals may be administered by oral, parenteral or topical administration.

When administered topically in formulations such as ointments or creams, the dosage of ursolic acid may be in the range of 0.001 to 1 mg / cm 3 per day. In the case of oral or parenteral administration, it may be administered in a dosage of 0.1 μg to 1 mg / kg per day. When used as a cosmetic formulation, it is contained in an amount of 0.01 to 10 parts by weight based on the total weight of the composition.

In addition, the ursolic acid used in accordance with the present invention may be added to foods, the amount thereof is not particularly limited and may be added to various foods in an amount effective to inhibit collagenase expression.

Collagenase expression inhibitors containing ursolic acid can have a variety of formulations, and the type of such formulations does not limit the scope of the invention. Specific formulations of the various formulations may be made according to various conventional ingredients and compositions well known to those skilled in the art, and may be easily made by those skilled in the art.

Collagenase expression inhibitors may be used for the prevention and treatment of skin aging by light, may be administered before or after light irradiation, and preferably before light irradiation.

In the present invention, it was confirmed that the expression of collagenase gene is increased by light irradiation, and that the expression of such collagenase gene is reduced when ursolic acid is administered before light irradiation. That is, the fibroblasts obtained by removing the human dermis were cultured, the cultured cells were incubated for 48 hours in a dulbecco medium containing ursolic acid, and the ultraviolet ray A was irradiated once in an amount of 20 J / cm 3 for 48 hours. Continue to incubate for time. At this time, the irradiation amount of ultraviolet ray A is the amount that the dermis is damaged when exposed to the air for 20 hours. Stain with 0.4% trypan blue solution and measure the survival and viability of the cells using a hematocytometer.

In addition, when the expression level of collagenase gene was measured by collagenase gene RNA and Northern hybridization reaction from surviving cells using a probe for collagenase gene, expression was suppressed in cells treated with ursolic acid. (Figure 2).

The photoaging protective agent composition of the present invention containing ursolic acid can be utilized as cosmetics, pharmaceuticals, preventive drugs, health foods and the like.

The present invention is described in detail through the following examples. However, the concept of the invention is not limited to the embodiment.

Example 1

[Cultivation of Fibroblasts]

According to the method of culturing dermal fibroblasts (Fleischnajer et al., J. invest.Dermatol., 76, 400, 1981), the epidermis of the newborn is excised, and after removing the fat layer using surgical scissors, Skin sections were made and placed in the dish with the dermal layer in contact with the bottom of the dish. 3 ml of fibroblast growth medium (Gibco: DMEM medium with 10% calf serum) was added and incubated at 37 ° C. for 6 hours, followed by staining with 0.4% trypan blue (Sigma). Observation with a matocytometer confirmed the proliferation of dermal fibroblasts.

Every 5 days, when fibroblasts cover about 80% of the plate and incubated with fresh medium, these cells are washed with phosphate buffer and treated with 0.02% trypsin EDTA (Sigma) for 5 minutes to free individual fibroblasts I was. Trypsin was neutralized with medium supplemented with 10% serum and fibroblasts were transferred to a new dish for passage.

Example 2

[Ursolic Acid Treatment and Light Irradiation]

The fibroblasts obtained in Example 1 were transferred to fibroblast growth medium to which ursolic acid was added at a concentration of 10 ^-3 , 10 ^-4 , 10 ^-5 or 2x10 ^-5 M and incubated for 48 hours, followed by an ultraviolet irradiator (Professor Prof. Mutzhas, Germany). Product name Supuvasun 3000) was irradiated for 2 minutes and 25 seconds to 20J / ㎠ with a light source and incubated for another 48 hours.

On the other hand, in order to determine the effect of treatment with ursolic acid after light irradiation, the fibroblasts were cultured in fibroblast growth medium for 48 hours, and then the fibroblasts irradiated with ultraviolet rays were transferred to the fibroblast growth medium to which ursolic acid was added and cultured again for 48 hours. .

As a result, 10 ^-3 or 10 ^-4 M in a lean ursolic acid-containing medium was observed that the cells are damaged by cytotoxicity by ursolic acid, 10 ^-5 M to about 2 × 10 ^-5 M concentration of ursolic acid Inhibitory effect of collagenase expression was observed at.

Example 3

Based on the result obtained in Example 2, the same experiment as in Example 2 was conducted using ursolic acid at a concentration of 10 ^-5 M.

On the other hand, fibroblasts were cultured using a medium to which ursolic acid was not added in order to compare the expression level of the collagenase gene in the presence of ultraviolet irradiation.

Comparative Example 1

In the same manner as in Example 2, the cells were harvested at 1, 3, 6, 12, 24, or 48 hours after culturing, without irradiating the fibroblasts with ursolic acid. Subsequently, mRNA amount of the collagenase gene was isolated and hybridized from these cells in the same manner as in Example 4 to measure the amount of mRNA. The results are shown in FIG.

In FIG. 1, NC shows the result when light is not irradiated, and when the skin is irradiated from the result of FIG. 1, it is confirmed that mRNA expression of collagenase gene rapidly increases after 12 hours.

Example 4

[Measurement of Expression of Collagenase Gene]

Plasmid pCllase I (ATCC 57685) was digested with restriction enzymes Hind III and Smal and electrophoresed with 1.5% low melting agarose gel to recover 2.0 kb fragments. 50 μl of reaction solution labeled 2.0 kb DNA with 1a-32P1-dCTP (manufactured by Amersham Co. Ltd.) according to the method of Feinberg and Vogelstein (Feinberg Vogelstein.Anal. 150 µl of the reaction stopper (20 mM salt, 2 mM EDTA, 0.2% SDS. 20 mM trisine hydrochloric acid, pH 7,5) was added. Unreacted isotopes were removed by passing through a column packed with P60 (Biorad).

Meanwhile, the fibroblasts cultured above were collected in a flask using a cell scraper, and mRNA was extracted by acid guanidinium-phenol-chloroform extraction method (Chomzynscki Sacchi.Anal. Biochem., 162, 156, 1987). . Quantitation by absorbance at 260 nm of ultraviolet light, 15 μl of RNA was loaded onto a 1.2% agarose 2.2M formaldehyde gel, followed by electrophoresis at 60 volts for 4 hours, transferred to a nitrocellulose membrane, and fixed with ultraviolet light for 30 seconds.

Hybrid reaction was carried out overnight at 42 ° C. with the probe prepared above, and the X-ray film was developed after washing.

As a control, fibroblasts were cultured in a medium to which no ursolic acid was added, and mRNA was separated from the cells in the absence of UV irradiation.

The results are shown in FIG. In Figure 2, NC represents a control. When UVA is irradiated with UV light, UsA is treated with ursolic acid only without UV irradiation, UsA + UVA is pretreated with ursolic acid and irradiated with UV light, and UVA + UsA is post-treated with ursolic acid after UV irradiation. The amount of collagenase gene expression in the case is shown.

As can be seen from the results of FIG. 2, fibroblasts pretreated or post-treated with ursolic acid significantly reduce the amount of collagenase gene expression by ultraviolet irradiation. Therefore, ursolic acid can be usefully used to protect the skin from ultraviolet rays and to prevent photoaging.

Claims (1)

An ultraviolet irradiation-induced collagenase gene expression inhibitor, comprising ursolic acid as an active ingredient.