KR100734039B1 - Pharmaceutical composition for preventing or treating skin diseases - Google Patents

Abstract

A pharmaceutical composition for preventing or treating skin diseases is provided to inhibit wrinkle production, hyperkeratosis and collagen degradation by inhibiting reduction of type I procollagen and expression increase of MMP-1(matrix metaloproteinase-1) and COX-2(cyclooxygenase-2) which are caused by ultraviolet rays. The pharmaceutical composition for preventing or treating skin diseases comprises 1,2,3,4,6-penta-O-galloyl-beta-D-glucose represented by the formula(1) or its pharmaceutically acceptable salt as effective ingredient, wherein the skin disease is a UV-causing skin disease including photoaging, or an inflammatory skin disease including skin inflammation, acute or chronic eczema, contact dermatitis, atopic dermatitis, seborrhoic dermatitis, lichen simplex chronicus, intertrigo, exfoliative dermatitis, papular urticaria, psoriasis, actinic dermatitis and acnes.

Description

Pharmaceutical Composition for preventing or treating skin diseases

1 shows the results of high performance liquid chromatography of ethyl acetate fractions.

Figure 2, 1,2,3,4,6-penta-o-galloyl-beta on the expression patterns of type I procollagen (A) and MMP-1 (B) by ultraviolet A irradiation in human dermal fibroblasts -Graph showing the effect of di-glucose (PGG).

* Indicates significance at 95% confidence.

Figure 3, 1,2,3,4,6-penta-o-galloyl-beta for the expression pattern of type I procollagen (A) and MMP-1 (B) by ultraviolet B irradiation in human dermal fibroblasts -Graph showing the effect of di-glucose (PGG).

* Indicates significance at 95% confidence.

Figure 4 shows the effect of 1,2,3,4,6-penta-o-galloyl-beta-di-glucose (PGG) on the expression pattern of COX-2 protein by ultraviolet B irradiation in human keratinocytes. The result of analysis by blot.

Figure 5 is a result of examining the inhibitory effect of 1,2,3,4,6-penta-o-galloyl-beta-di-glucose on the skin wrinkles induced by ultraviolet irradiation in the mouse model.

A: Skin photograph of the untreated group, which was not treated with both ultraviolet and 1,2,3,4,6-penta-o-galloyl-beta-di-glucose

B: Skin photograph of UV alone treatment group

C: Skin photograph of experimental group treated with both ultraviolet and 1,2,3,4,6-penta-o-galloyl-beta-di-glucose

D: Untreated skin replication template not treated with both ultraviolet and 1,2,3,4,6-penta-o-galloyl-beta-di-glucose

E: skin replication template of UV alone treatment group

F: skin replication template of experimental group treated with both ultraviolet and 1,2,3,4,6-penta-o-galloyl-beta-di-glucose

Figure 6 is a result of examining the inhibitory effect of 1,2,3,4,6-penta-o-galloyl-beta-di-glucose on the skin keratin thickening induced by ultraviolet irradiation in the mouse model.

▲ indicates thin epidermis, ↑ shows panniculus carnosus, * shows keratinizing cysts, and □ area was enlarged 200 times.

A: untreated group not treated with both ultraviolet and 1,2,3,4,6-penta-o-galloyl-beta-di-glucose

B: UV only treatment group

C: Experimental group treated with both ultraviolet and 1,2,3,4,6-penta-o-galloyl-beta-di-glucose

7 shows collagen degradation induced by UV irradiation in a mouse model.

The result of investigating collagen formation promoting effect of 1,2,3,4,6-penta-o-galloyl-beta-di-glucose

▲ indicates epithelial layer, ↑ indicates collagen.

A: untreated group not treated with both ultraviolet and 1,2,3,4,6-penta-o-galloyl-beta-di-glucose

B: UV only treatment group

C: Experimental group treated with both ultraviolet and 1,2,3,4,6-penta-o-galloyl-beta-di-glucose

FIG. 8 shows Type I procollagen expression after treatment with 1,2,3,4,6-penta-o-galloyl-beta-di-glucose in type I procollagen inhibited by UV irradiation in a mouse model. As a result of investigating the effect, the □ region was enlarged 400 times, and ▲ represents type I procollagen.

A: untreated group not treated with both ultraviolet and 1,2,3,4,6-penta-o-galloyl-beta-di-glucose

B: UV only treatment group

C: Experimental group treated with both ultraviolet and 1,2,3,4,6-penta-o-galloyl-beta-di-glucose

Figure 9 shows the expression of MMP-1 increased expression by ultraviolet irradiation in the mouse model

As a result of investigating the inhibitory effect of 1,2,3,4,6-penta-o-galloyl-beta-di-glucose, ▲ represents MMP-1.

A: untreated group not treated with both ultraviolet and 1,2,3,4,6-penta-o-galloyl-beta-di-glucose

B: UV only treatment group

C: Experimental group treated with both ultraviolet and 1,2,3,4,6-penta-o-galloyl-beta-di-glucose

The present invention relates to a pharmaceutical composition for preventing or treating skin diseases. More specifically, the present invention relates to a pharmaceutical composition for preventing or treating skin diseases containing 1,2,3,4,6-penta-o-galloyl-beta-di-glucose as an active ingredient.

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 external stimulation such as ultraviolet rays or direct contact with various pathogens, and the body is strongly affected. In addition, the disease is caused by a variety of causes because it is affected by pathological changes, such as heredity, inflammation, benign and malignant tumors, hormones, trauma, degenerative degeneration.

Ultraviolet (UV) radiation from the sun is a small amount of light that reaches the earth, but it acts as a factor that causes 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 (Scharffetter-Kochanek et al., Exp Gerontol , 35 (3): 307-316, 2000) or skin cancer (Hurza and Pentland, J Invest Dermatol , 100: 35S-41S, 1993; Brenneisen P. et al., Ann NY Acad Sci 973: 31-43, 2002; Wlascheck M et 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 build a system that can activate the production of enzymes mainly acting on the skin as well as in vivo.

Inflammatory skin diseases are diseases in which a series of clinical signs and symptoms, such as itching, swelling, erythema, peeling, etc., are caused by various stimulating factors causing a series of inflammatory reactions in the skin epithelium. Inflammatory skin diseases include atopic dermatitis, contact dermatitis, seborrhoic dermatitis and acne.

Until now, antihistamines, vitamin ointments, and corticosteroids have been mainly used for the treatment of inflammatory skin diseases. However, these drugs are often temporary and often have severe side effects.

Tannin is a compound widely distributed in many plants and is a complex polymer (molecular weight 600 ~ 2000) in which various polyphenols are polymerized instead of one determined compound. Tannins are classified into hydrolysis type which is easily hydrolyzed by acid or enzyme and condensation type which is relatively stable to degradation depending on their characteristics. Hydrolyzed tannins and condensed tannins differ greatly in structure and properties as well as in their degradation form (Hagerman AE, Tannin Chemistry.Department of chemistry and biochemistry, Miami Univesrity, USA, p35, 2002). Hydrolyzed tannins are readily hydrolyzed to produce sugars and phenolic acids and have hydroxyl (-OH) and carboxyl (-COOH) and esters thereof (RCOOR) as reactors. It is also included in gall bladder, acorns, pomegranates, grass roots and the like. Derivatives include 1,2,3,4,6-penta-o-galloyl-beta-di-glucose, 1,2,3,6-tetragalloyl glucose, acetannin Etc. Condensed tannins, on the other hand, are not hydrolyzed without sugar and are formed by the carbon-to-carbon bonds of a number of catechins and epicatechins. It has only hydroxyl (-OH) as a reactor and is included in persimmon fruit, tea leaf, mimosa and the like. In addition, the derivatives include epigallocatechin-3-gallate (EGCG), delphinidin, luteolinidin, apigeninidin and the like.

Recently, it has been found that green tea extract is effective for skin beauty, and its active ingredient has been reported to be a catechin compound (Jin Jin-ho, 6th International Green Tea Symposium, 2001, Seoul). In addition, the results of confirming that condensed tannin derivatives and EGCG, the main component of green tea catechin, have an effect on inhibiting skin aging (Salah N et al., Arch) . Biochem Biophys . 322: 339-346, 1995; Kim SY et al., Arch Pharm Res . 28: 784-90, 2005; Lee J H et al., J Dermatol Sci . 2005). And the Republic of Korea Patent Registration No. 10-0439590 discloses the results of confirming the effect of wrinkle treatment, erythema formation and skin hardening of the cosmetic containing tannin extracted from green tea. However, it has not been reported that hydrolyzed tannins have an effect of preventing or treating skin diseases.

Accordingly, the present inventors have studied for many years to develop a composition that can prevent or treat skin diseases. As a result, 1,2,3,4,6-penta-o-galloyl-beta-di-glucose, which is a derivative of hydrolyzed tannin, has been studied. By inhibiting type I procollagen reduction, MMP-1 expression, COX-2 expression, wrinkle formation, keratin thickening and collagen degradation induced by UV light, to prevent or treat skin diseases. The invention has been completed.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of skin diseases.

In order to achieve the above object, the present invention, 1,2,3,4,6-penta-o-galloyl-beta-di-glucose (1,2,3,4,6-penta-O-galloyl It provides a pharmaceutical composition for the prevention or treatment of skin diseases containing -beta-D-glucose) or a pharmaceutically acceptable salt thereof as an active ingredient.

Hereinafter, the present invention will be described in detail.

1,2,3,4,6-penta-o-galloyl-beta-di-glucose of the present invention is represented by the following formula (1).

The 1,2,3,4,6-penta-o-galloyl-beta-di-glucose according to the invention can be used on its own or in the form of salts, preferably in the form of pharmaceutically acceptable salts. As the salt, an acid addition salt formed by a pharmaceutically acceptable 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.

1,2,3,4,6-penta-o-galloyl-beta-di-glucose of the present invention can be isolated and used according to methods known in the art. Preferably, it can be isolated from tannic acid by conventional metanolysis and normal phase or reverse phase chromatography. In addition, it can be isolated from the peony by conventional extraction processes and chromatography (Korean Patent Publication No. 2003-0075947), and may be chemically synthesized according to chemical methods known in the art.

1,2,3,4,6-penta-o-galloyl-beta-di-glucose according to the present invention can prevent and treat skin diseases by inhibiting various skin disease mediators. The main cause of skin disease caused by ultraviolet rays is the synthesis of matrix metalloproteinase-1 (MMP-1), an enzyme that breaks down the components of the skin's intercellular substances, such as collagen, by signaling to the active oxygen species signaling system. (Angel, P. et al., Cell, 49: 729-739, 1987; Devary, Y., et al., Science , 261: 1442-1445, 1993). MMP-1 breaks down collagen and causes skin disease symptoms such as decreased skin elasticity, skin thickening and wrinkle formation. In addition, MMP-1 is a collagen that is expressed in metabolic processes when it is necessary to break down collagen structure and plays a central role in physiological phenomena such as wound repair, tissue resorption, embryonic development, angiogenesis and cell migration. It regulates metabolism and is involved in the degradation of the basement membrane during rheumatoid and cancer cell metastasis (Bresalier RS et.al., Cancer Res ., 47: 1398-1406, 1987). Therefore, by suppressing the expression of MMP-1 increased by UV irradiation, skin elasticity degradation, keratin thickening of the skin, wrinkle formation and the like can be prevented, and skin diseases caused by UV irradiation can be prevented and treated.

The 1,2,3,4,6-penta-o-galloyl-beta-di-glucose of the present invention reduces the expression of MMP-1 increased by ultraviolet irradiation in vitro and in vivo.

1,2,3,4,6-penta-o-galloyl-beta-di-glucose of the present invention increases the expression of type I procollagen reduced by ultraviolet irradiation in vitro and is increased by ultraviolet irradiation. Reduced expression of MMP-1. At this time, since the skin protection effect of EGCG, a condensed tannin derivative and the main component of green tea catechin, is known, the effect was also confirmed. In the treatment of 1,2,3,4,6-penta-o-galloyl-beta-di-glucose, the expression of type I procollagen increases significantly compared to the treatment of EGCG. In addition, the MMP-1 inhibitory effect of 1,2,3,4,6-penta-o-galloyl-beta-di-glucose was shown to inhibit the MMP-1 effect of EGCG confirmed in the domestic patent (Korean Patent Registration No. 10-0439590). Much better than that (FIGS. 2 and 3).

In vivo experiments also show that 1,2,3,4,6-penta-o-galloyl-beta-di-glucose inhibits the reduction of type I procollagen and the expression of MMP-1 induced by ultraviolet light ( 8 and 9).

COX-2 is a substance involved in the inflammatory response in the body, and is expressed in large quantities by external stimuli such as ultraviolet rays in skin cells, and promotes skin cell damage by converting arachidonic acid in the cell membrane to prostagladin Overexpressed in tissues. Therefore, it is possible to prevent inflammatory skin diseases by inhibiting the expression of COX-2.

In addition, overexpression of COX-2 has been shown in a variety of malignancies and has been reported to inhibit apoptosis and increase the invasiveness of tumor-causing cells (Dempke et al., J. Can. Res. Clin. Oncol. 127: 411 -17, 2001). Therefore, skin cancer can be prevented by suppressing the expression of COX-2.

1,2,3,4,6-Penta-O-galloyl-beta-di-glucose of the present invention exhibits the effect of reducing the expression of COX-2 increased by ultraviolet irradiation in vitro (FIG. 4). .

In vivo experiments have shown that 1,2,3,4,6-penta-o-galloyl-beta-di-glucose of the present invention promotes the formation of collagen when treated with skin tissue with reduced collagen by UV irradiation. Effect is shown (FIG. 7).

In addition, the 1,2,3,4,6-penta-o-galloyl-beta-di-glucose of the present invention exhibits an effect of suppressing keratin thickening caused by ultraviolet rays, and suppresses wrinkle formation caused by ultraviolet rays. Effect is shown (see FIGS. 5 and 6).

Therefore, the present invention is to prevent skin diseases containing 1,2,3,4,6-penta-o-galloyl-beta-di-glucose or a pharmaceutically acceptable salt thereof represented by Chemical Formula 1 as an active ingredient or Provided is a therapeutic pharmaceutical composition.

The skin disease refers to a skin disease or inflammatory skin disease caused by ultraviolet rays.

In the above description, 'skin disease caused by ultraviolet rays' is not limited thereto, and may include photoaging or skin cancer.

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.

The pharmaceutical compositions according to the invention contain 1,2,3,4,6-penta-o-galloyl-beta-di-glucose alone or add one or more pharmaceutically acceptable carriers, excipients or diluents. It may contain. As used herein, "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not normally cause an allergic or similar reaction when administered to a human.

The pharmaceutical composition for preventing or treating skin diseases of the present invention can be administered to a mammal by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, transdermal administration, subcutaneous, intravenous, intramuscular or intraperitoneal administration. Preferably the pharmaceutical composition of the present invention may be administered transdermally. As used herein, 'transdermal administration' refers to the administration of the pharmaceutical composition of the present invention to cells or skin so that the active ingredient contained in the pharmaceutical composition for preventing or treating skin diseases is delivered into the skin. For example, the pharmaceutical composition of the present invention may be prepared in an injectable formulation and administered by lightly pricking the skin with a 30-gauge thin needle or by directly applying it to the skin.

have.

The pharmaceutical composition of the present invention may be formulated into a preparation for oral or parenteral administration according to the route of administration as described above.

In the case of preparations for oral administration, the compositions of the present invention may be formulated using methods known in the art as powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions and the like. Can be. For example, oral formulations can be obtained by tablets or dragees by combining the active ingredients with solid excipients, milling them, adding suitable auxiliaries and then processing them into granule mixtures. Examples of suitable excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol and starch, cellulose, including starch, corn starch, wheat starch, rice starch and potato starch, etc. Fillers such as cellulose, gelatin, polyvinylpyrrolidone, and the like, including methyl cellulose, sodium carboxymethylcellulose, hydroxypropylmethyl-cellulose, and the like. In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid or sodium alginate and the like may optionally be added as a disintegrant. Furthermore, the pharmaceutical composition of the present invention may further include an anticoagulant, a lubricant, a humectant, a perfume, an emulsifier, a preservative, and the like.

For parenteral administration, it may be formulated in the form of injections, creams, lotions, external ointments, oils, moisturizers, gels, aerosols and nasal inhalants in the art. These formulations are described in Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a prescription generally known in all pharmaceutical chemistries.

The total effective amount of 1,2,3,4,6-penta-o-galloyl-beta-di-glucose of the present invention can be administered to a patient in a single dose, and multiple doses ) May be administered by a fractionated treatment protocol which is administered for a long time. The pharmaceutical composition of the present invention may vary the content of the active ingredient according to the degree of skin disease. Preferably the preferred total dose of 1,2,3,4,6-penta-o-galloyl-beta-di-glucose of the present invention is from about 0.01 μg to 1,000 mg, most preferably per kg of patient body weight per day 0.1 μg to 100 mg. However, the concentration of 1,2,3,4,6-penta-o-galloyl-beta-di-glucose is not only the route of administration and the number of treatments of the pharmaceutical composition, but also the age, weight, health condition, sex, and disease of the patient. Since the effective dosage for the patient is determined by considering various factors such as the severity, diet, and excretion rate, those having ordinary knowledge in the art should consider 1,2,3,4,6-penta. Appropriate effective dosages for o-galloyl-beta-di-glucose as a prophylactic or therapeutic agent for skin diseases can be determined. The pharmaceutical composition according to the present invention is not particularly limited to its formulation, route of administration and method of administration as long as the effect of the present invention is shown.

In addition, the present inventors found that 1,2,3,4,6-penta-o-galloyl-beta-di-glucose was produced by the UV-induced wrinkles, keratin thickening, collagen degradation, and type I procollagen. To confirm the effect of inhibiting the decrease, increased MMP-1 expression and increased COX-2 expression and to prepare a cosmetic composition containing the 1,2,3,4,6-penta-o-galloyl-beta-di-glucose It was. The cosmetic composition comprises one or more excipients and additives generally used in the field of preparing cosmetic compositions together with 1,2,3,4,6-penta-o-galloyl-beta-di-glucose of the present invention. It can be manufactured easily according to a method known in the art.

More specifically, the cosmetic composition of the present invention contains 1,2,3,4,6-penta-o-galloyl-beta-di-glucose of the present invention as an active ingredient and a basic cosmetic product with a dermatologically acceptable excipient. Compositions (cosmetics, creams, essences, cleansing foams and cleansing waters such as face washes, packs, body oils), color cosmetic compositions (foundation, lipstick, mascara, makeup bases), hair product compositions (shampoos, rinses, hair conditioners, hair gels) ) And soap. 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 moisturizers may be further included, and for the purpose of improving the properties of thickeners, inorganic salts, synthetic polymer materials

And the like.

For example, when preparing a face wash and soap comprising 1,2,3,4,6-penta-o-galloyl-beta-di-glucose of the present invention, 1,2 may be added to a conventional face wash and soap base. It can be easily prepared by addition of 3,4,6-penta-o-galloyl-beta-di-glucose. When the cream is prepared, it can be prepared by adding 1,2,3,4,6-penta-o-galloyl-beta-di-glucose to a cream base of a general oil-in-water type (O / W). To this, synthetic or natural materials, such as proteins, minerals, vitamins, etc., for the purpose of improving physical properties, such as flavors, chelating agents, pigments, antioxidants, and preservatives, may be added.

The content of 1,2,3,4,6-penta-o-galloyl-beta-di-glucose contained in the cosmetic composition of the present invention is 0.0001 to 50% by weight, preferably 0.01 to the total weight of the cosmetic composition. To 10% by weight.

Hereinafter, 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>

1,2,3,4,6-penta-o- Galloyl -Beta-di- Glucose  Isolation

1,2,3,4,6-penta-o-galloyl-beta-di-glucose was prepared from the tannic acid via a metanolysis reaction. Commercially available tannic acid (tannic acid, Sigma, MO, USA) was used as the reaction material. 10 g of tannic acid was subjected to metanolis reaction for 15 to 18 hours at a temperature of 55 to 65 ° C. in a mixture of acetate buffer and methanol at a ratio of 3: 7 (v / v), respectively. The methanolic reaction solution was adjusted to a weakly acidic state of pH 6.0 and concentrated at 30 ° C. or lower using a vacuum concentrator to remove methanol. The concentrate was calibrated with water, separated with polarity using ethyl ether, and the resultant was separated sequentially with polarity using ethyl acetate. The ethyl acetate fraction was concentrated at a temperature below 30 ° C. using a vacuum concentrator, and the residue layer was lyophilized with a lyophilizer. Then, the degree of metabolism reaction in each solvent fraction was confirmed by high performance liquid chromatography, and the 1,2,3,4,6-penta-o-galloyl-beta-di of the tannin derivatives in the ethyl acetate fraction were examined. -It was confirmed that glucose is contained in a biased manner (see Figure 1).

300 mg of 3.0 g of the ethyl acetate fraction was injected into a reverse phase silica column (1.0 x 300 mm, Waters) of high performance liquid chromatography (GILSON), and then 0.1% trifluoracetic acid (trifluoracetic acid) as a mobile phase solvent. 1.5 mL of eluate was obtained in a test tube while varying the concentration of acetonitrile containing acetonitrile and 0.1% trifluoracetic acid (20:80-> 40:60). The eluate containing 1,2,3,4,6-penta-o-galloyl-beta-di-glucose was concentrated under reduced pressure with a speed vac to obtain 10 mg of isolated pure material. Analysis of the material by mass spectrometry confirmed that the isolated material was 1,2,3,4,6-penta-o-galloyl-beta-di-glucose with a molecular weight of 940 (results not shown).

In addition, 1,2,3,4,6-penta-o- can be used to increase the production yield for commercial use of isolated 1,2,3,4,6-penta-o-galloyl-beta-di-glucose. Drying 3.0 g of the ethyl acetate fraction, which confirmed that galloyl-beta-di-glucose was biased, was mixed with the same amount of water in the course of solvent fractionation of the tannin acid reaction solution after the metanolysis, and then concentrated under reduced pressure to obtain ethyl acetate. The fractions were forcibly dissolved in water and recrystallized at refrigeration temperature to give off-white crystals. The mixture was washed with water and recrystallized with a water-soluble alcohol solution to repeat 200 mg of 1,2,3,4,6-penta-o-gallo. Yl-beta-di-glucose was isolated.

Thus, 1,2,3,4,6-penta-o-galloyl-beta-di-glucose isolated from tannic acid was dissolved in DMSO at a concentration of 100 mg / ml and stored frozen at -20 ° C until use.

< Example  2>

examiner Intracellular  Type I collagen reduction by ultraviolet rays and MMP - 1 increase  For 1,2,3,4,6-penta-o- Galloyl -Beta-di- Glucose  Inhibitory effect

Human normal dermal fibroblasts obtained by separating and culturing in dermal dermal layers excised from infant circumcision have 1 x 10 ⁵ cells in each well of a 6-well plate. After inoculation, the cells were incubated for 24 hours in DMEM (Dulbecco's modified Eagle's medium) containing 10% fetal bovine serum. After incubation, the cells were washed three times with phosphate buffered saline (PBS), and then irradiated with UV A and UV B as follows to induce skin aging. In the case of ultraviolet A, a total of 50 mJ / cm ² was irradiated with an energy amount of 27 mW / cm ² per second using an ultraviolet A lamp (Sankyo Denki FL8BL lamp, Sankyo Denki Co., Japan). A total of 170 mJ / cm ² was irradiated with an energy amount of 1.15 mW / cm ² per second using an ultraviolet B lamp (Sankyo Denki G9T5E lamp, Sankyo Denki Co., Japan). Immediately after UV irradiation, the cells were washed again with phosphate buffer and 1,2,3,4,6-penta-o-galloyl-beta-di-glucose was added to the DMEM medium without fetal calf serum. Incubation was carried out for an additional 24 hours by the addition of a concentration of ml. The experimental group was divided into:

Untreated group-not treated with both ultraviolet and 1,2,3,4,6-penta-o-galloyl-beta-di-glucose

Ultraviolet ray treatment group-only ultraviolet ray treatment

Control-treated with UV and EGCG

Experimental group-treated with both ultraviolet and 1,2,3,4,6-penta-o-galloyl-beta-di-glucose

Inhibition of MMP-1 expression and type I procollagen expression of EGCG, a condensed tannin derivative and the main component of green tea catechin, was reported in the Korean patent and was used as a control. In order to confirm skin aging after culture, the expression level of the type I procollagen (type I procollagen) and its degrading enzyme MMP-1, which are the intercellular skin matrix components, was measured. Peptide EIA kit, TaKaRa, Japan) and MMP-1 ELISA kit (MMP-1 human, ELISA system, Biotrak Assay, Amersham Biosciences, USA) were measured according to the manufacturer's protocol. The concentrations of intracellular type I procollagen and MMP-1 are expressed in ng protein per ml of total cell culture.

As a result, it was confirmed that the expression of type I procollagen was decreased while the expression of MMP-1 was increased when the normal dermal fibroblasts were irradiated with UV light (ultraviolet ray alone). In addition, the increase in the expression of type I procollagen and the decrease in the expression of MMP-1 by 1,2,3,4,6-penta-o-galloyl-beta-di-glucose treatment was found to be concentration-dependent. In addition, it was confirmed that the above effect of 1,2,3,4,6-penta-o-galloyl-beta-di-glucose was greater than the EGCG effect reported in the domestic patent (FIG. 2 and FIG. 3).

From the above results, 1,2,3,4,6-penta-o-galloyl-beta-di-glucose increases the expression of type I procollagen reduced by ultraviolet irradiation and increases MMP- by ultraviolet irradiation. It was confirmed to reduce the expression of 1.

< Example  3>

examiner Intracellular  By ultraviolet COX 1,2,3,4,6-penta-o- for -2 increase Galloyl -Beta-di- Glucose  Inhibitory effect

Human HaCaT cells donated from German N. Fusenig (German Cancer Research, Germany) were inoculated to give 1.3 × 10 ⁶ cells in a 100 mm culture dish, followed by DMEM containing 10% fetal calf serum. Incubated for 24 hours in the medium. After incubation, the cells were washed three times with phosphate buffer and then irradiated with ultraviolet B as follows to induce skin aging. A total of 10 mJ / cm ² was irradiated with an energy amount of 0.6 mW / cm ² per second using an ultraviolet B lamp. Immediately after UV irradiation, cells were washed again with phosphate buffer and 1,2,3,4,6-penta-o-galloyl-beta-di-glucose was added to 1, 10, 30 in DMEM medium without fetal calf serum. , Was added at a concentration of 50 uM and further incubated for 12 hours. The experimental group was divided into:

Untreated group-not treated with both ultraviolet and 1,2,3,4,6-penta-o-galloyl-beta-di-glucose

Ultraviolet ray treatment group-only ultraviolet ray treatment

Experimental group-treated with both ultraviolet and 1,2,3,4,6-penta-o-galloyl-beta-di-glucose

After 12 hours of incubation, the cells were washed with cold phosphate buffer, and then the cell proteins were separated with cell lysis buffer, COX-2 antibody (Santa Cruz Biotch., USA) and horseradish-peroxidase (horseradish). Western blot analysis was performed using secondary antibodies (ZYMED Laboratories, USA) bound to peroxidase. Β-actin was used as a loading control.

As a result, it was confirmed that the expression of COX-2 was increased compared to the untreated group when UV was irradiated to human keratinocytes. In addition, it was confirmed that the expression of COX-2 decreased concentration-dependently by 1,2,3,4,6-penta-o-galloyl-beta-di-glucose treatment (FIG. 4).

From the above results, it was confirmed that 1,2,3,4,6-penta-o-galloyl-beta-di-glucose reduced COX-2 expression by UV irradiation in skin keratinocytes.

< Example  4>

1,2,3,4,6-Penta-O- for Wrinkle Generation by Ultraviolet Rays in a Mouse Model Galloyl -Beta-di- Glucose  Inhibitory effect

We investigated whether 1,2,3,4,6-penta-o-galloyl-beta-di-glucose has an effect of inhibiting wrinkle generation caused by ultraviolet rays. First, seven week-old female hairless mice (albino SKH-1 hairless mice, Hallim Experimental Animal Experimenter, Suwon, South Korea) were divided into three groups: 10 per group:

Untreated group-not treated with both ultraviolet and 1,2,3,4,6-penta-o-galloyl-beta-di-glucose

UV alone treatment group-treated with compositions that do not contain UV and 1,2,3,4,6-penta-o-galloyl-beta-di-glucose

Experimental group-treated with compositions containing ultraviolet and 1,2,3,4,6-penta-o-galloyl-beta-di-glucose

Mice in each group were placed in an ultraviolet box equipped with an ultraviolet B lamp and irradiated with 100 mJ / cm ² of UV, which is the minimum amount of erythema (MED). UV light is once a day, three times a week for four weeks: 100 mJ / cm ² for week 1, 200 mJ / cm ^{2 for} week ² , 300 mJ / cm ^{2 for} week ³ , 400 mJ / cm ^{2 for} week 4 The mice were adapted to ultraviolet light by irradiation with increasing. After 4 weeks, irradiation with 400 mJ / cm ² was performed for 6 weeks to induce skin photoaging by exposing the hairless mice to ultraviolet rays for a total of 10 weeks (Kilgman LH, Clin Dermatol 14: 183-195, 1996). In the experimental group, a composition containing 1,2,3,4,6-penta-o-galloyl-beta-di-glucose after 4 weeks of ultraviolet irradiation was propylene glycol solution containing 30% ethanol (7 : 3 [v / v] propylene glycol: ethanol) and applied to the skin of mice in an amount of 10 mg / 200 μl / 4 cm ² , and the UV-only group was 1,2,3,4,6-penta. Compositions containing no -o-galloyl-beta-di-glucose were treated in mice in the same manner as the experimental group in an amount of 200 μl / 4 cm ² . After 10 weeks, 24 hours after the final UV irradiation, the skin wrinkles of the mice were observed using a skin photograph and skin replica kit (skin replica kit, Courage + Khazaka Electronic, Koln, Germany).

As a result, the incidence of wrinkles was increased by ultraviolet irradiation, and the frequency of wrinkles was reduced when 1,2,3,4,6-penta-o-galloyl-beta-di-glucose was treated (FIG. 5). .

From the above experimental results, it was confirmed that 1,2,3,4,6-penta-o-galloyl-beta-di-glucose inhibited the occurrence of wrinkles.

< Example  5>

Keratin in mouse model After rain  For 1,2,3,4,6-penta-o- Galloyl -Beta-di- Glucose  Inhibitory effect

In the same manner as in Example 4, the hairless mice were treated with ultraviolet and 1,2,3,4,6-penta-o-galloyl-beta-di-glucose, and skin tissues were extracted. After immersing the extracted tissue in 4% neutral buffered formalin solution for at least 24 hours, the tissue is washed, dehydrated and transparent according to a conventional method, and finally embedded in paraffin to form a paraffin block. Tissue sections were prepared to a thickness of 5 μm. In order to determine the thickening of the stratum corneum through the cell arrangement and morphological changes, the tissue sections were subjected to deparaffinization, a sequential hydration process, and commercially available heteroxylin solution according to Mayer and eosin solution ( After the progressive staining process using eosin solution, the tissue sections were stained again with hematoxylin-eosin (H & E), which was subjected to sequential dehydration, clearing, and encapsulation. As a result of observing with an optical microscope, the hematoxylin stain solution stains the nucleus blue, and the eosin stain solution stains the cytoplasm red.

As a result, the thickening of the stratum corneum of the skin was significantly increased in the tissue irradiated with ultraviolet rays (ultraviolet ray alone) compared with the tissue not irradiated with ultraviolet ray (untreated group). However, when 1,2,3,4,6-penta-o-galloyl-beta-di-glucose was treated (experimental group), the thickening of the stratum corneum was significantly reduced (FIG. 6). From the above test results, it was confirmed that 1,2,3,4,6-penta-o-galloyl-beta-di-glucose inhibits the thickening of the stratum corneum caused by ultraviolet light.

< Example  6>

1,2,3,4,6-penta-o- in mouse model Galloyl -Beta-di- Glucose  Collagen formation promoting effect

Skin tissue was extracted after the hairless mice were treated with 1,2,3,4,6-penta-o-galloyl-beta-di-glucose and ultraviolet rays in the same manner as in Example 4. Skin tissue sections were prepared in the same manner as in Example 5 in order to check the collagen change in the skin connective tissue damaged by ultraviolet light. The tissue sections were treated with deparaffin, water, and subjected to Masson trichome special staining, which was stained using three dyes. More specifically, it is immersed in Weigert's Iron Hematoxylin solution to stain the nucleus, and then immersed in Bibrich sacrlet acid fusion solution, and the cytoplasm, keratin and muscle fibers in the tissue. Etc. were dyed. Finally, after staining with 2% light green solution and finally staining collagen, the tissue section is dehydrated, transparent and encapsulated with Masson trichome special dye. After the observation was performed by optical microscope. Cell nuclei are stained in black, cytoplasm, keratin, and muscle fibers in tissue sections are red, and collagen in the dermis is turquoise.

As a result, the thickness of the epidermal layer was increased in the tissue irradiated with ultraviolet rays (ultraviolet ray treatment group) compared with the tissue not irradiated with ultraviolet ray (untreated group), and the fragmentation of collagen fibers was thinned. . On the other hand, when 1,2,3,4,6-penta-o-galloyl-beta-di-glucose was treated (experimental group), collagen was increased in quantity and the amount of denatured collagen was increased as compared with the UV-only group. It was confirmed that the reduction (Fig. 7).

From the above results, it was confirmed that 1,2,3,4,6-penta-o-galloyl-beta-di-glucose inhibited collagen degeneration and showed collagen formation-promoting effects on collagen damage induced by ultraviolet rays. .

< Example  7>

Type I Collagen Reduction by Ultraviolet Rays in Mouse Models and MMP - 1 increase  For 1,2,3,4,6-penta-o- Galloyl -Beta-di- Glucose  Inhibitory effect

In order to confirm the effect of the 1,2,3,4,6-penta-o-galloyl-beta-di-glucose identified in Example 2 in the mouse model, the hairless mouse was prepared in the same manner as in Example 4. Were treated with ultraviolet and 1,2,3,4,6-penta-o-galloyl-beta-di-glucose. After the treatment using the skin tissue obtained from the extraction, the sections were prepared in the same manner as in Example 5, followed by deparaffinization and hydration, followed by the method of Griffiths (Grittiths et al., New Eng J Med . 329: 530-535, 1993). Expression of type I procollagen and MMP-1 using rat anti-human type I procollagen antibody (Chemicon, USA) and rabbit anti-human MMP-1 antibody was confirmed using an optical microscope.

As a result, the expression of type I procollagen was decreased when only ultraviolet rays were irradiated (ultraviolet ray alone), but 1,2,3,4,6-penta-o-galloyl-beta-di-glucose was decreased. In the case of treatment (experimental group), it was found that the expression of type I procollagen was increased as compared to the UV-only treatment group (FIG. 8).

In addition, the expression of MMP-1, a type I procollagen degrading enzyme, was increased by UV rays, but this increase was caused by 1,2,3,4,6-penta-o-galloyl-beta-di-glucose. It was confirmed that the inhibition by (Fig. 9).

From the above experimental results, it was confirmed that 1,2,3,4,6-penta-o-galloyl-beta-di-glucose inhibited type I procollagen reduction and increased expression of MMP-1 by ultraviolet rays.

< Application example  1> skin Photoaging

Skin photoaging occurs due to chronic exposure to ultraviolet rays, which causes harmful oxygen in the human body to form along with the harmful elements of the external environment, resulting in peroxidation of the lipid components of the cell membranes and hardening of the cell membranes, resulting in poor supply of oxygen and nutrients to the cells. do. Due to the slow metabolism of the skin and the turn-over of skin cells, the stratum corneum becomes thicker and collagen (collagen), the collagen fiber of the dermal layer, is destroyed and elastic fibers with the decrease of the substrate, hyaluronic acid. Phosphorus elastin is also denatured, degrading skin elasticity, making skin appear innocuous, dull, dry and rough, skin dryness, loss of elasticity, wrinkle formation, capillary wall weakening, senile pigmentation, etc. Phenomenon occurs. Therefore, 1,2,3,4,6-penta-o-galloyl-beta of the present invention inhibits the expression of collagen degrading enzyme MMP-1 and increases the synthesis of type I procollagen to prevent their skin symptoms. -Di-glucose is effective in treating skin photoaging.

< Application example  2> skin irritation

Inflammation is a response of living tissue with blood vessels to local damage, leading to increased expression of COX-2 by reactive oxygen species produced by ultraviolet light. Inflammatory changes include increased blood flow, increased permeability of blood vessel walls, and infiltration of white blood cells, all of which have been reported to involve cell adhesion (Jackson, JR et al., FASEB J. 11: 457-465, 1997). Inflammation induced histologically increased infiltration of inflammatory cells and biochemically increased collagen degrading enzyme activity. Thus, 1,2,3,4,6-penta-o-galloyl-beta-di-glucose of the present invention which inhibits COX-2 expression responsible for the progression of the inflammatory response is effective in treating inflammation.

< Application example  3> skin cancer

Skin cancer refers to all cancers occurring in the skin, and skin cancers include basal cell carcinoma, squamous cell carcinoma, and melanoma. Basal cell carcinoma is the most common malignant tumor occurring in humans, including skin. The most important trigger is ultraviolet radiation, which can occur where the trauma, scar, or burn was present. The skin symptom is one or several small nodules gradually enlarged, the central part is depressed, the area is ulcer or crust or scab, and the periphery is slightly embankment-shaped. It is primarily related to UV rays, so it is most common on the face, especially on the nose, forehead, ears and cheeks.

Squamous cell carcinoma is the second most common malignancy after basal cell carcinoma. As with basal cell carcinoma, ultraviolet light is an important cause of squamous cell carcinoma. Genetic factors, various chemicals known as carcinogens, radiation, papilloma virus, and burn scars are also caused. Symptoms often occur in places exposed to ultraviolet light, such as the face or hands, and have a hard boil that covers the surface with ulcers.

Therefore, 1,2,3,4,6-penta-o-galloyl of the present invention inhibits the expression of MMP-1, a collagen degrading enzyme increased by ultraviolet rays, and suppresses the expression of COX-2 overexpressed in skin cancer tissues. Beta-di-glucose is effective in treating skin cancer.

< Production Example >

Preparation of a medicament containing a pharmaceutical composition for the treatment or prevention of skin diseases

<1-1> Preparation of Tablet

1,2,3,4,6-penta-o-galloyl-beta-di-glucose 25 mg of the present invention, 26 mg of lactose for excipients and 3.5 mg of avicel (microcrystalline cellulose), sodium starch glyconate as a disintegration aid 1.5 mg and L-HPC (low-

hydrosyprophylcellulose) was added to a U-type mixer with 8 mg and mixed for 20 minutes. After mixing was completed, 1 mg of magnesium stearate was further added as a lubricant and mixed for 3 minutes. Tablets were prepared by tableting and film coating after a quantitative test and a humidity test.

<1-2> Preparation of Syrup

A syrup containing 1,2,3,4,6-penta-o-galloyl-beta-di-glucose or a pharmaceutically acceptable salt thereof of the present invention as an active ingredient of 2% (W / V) is as follows. Prepared in the same manner: 2 g of acid addition salt of 1,2,3,4,6-penta-o-galloyl-beta-di-glucose of the present invention, 0.8 g of saccharin and 25.4 g of sugar were dissolved in 80 g of warm water. . After cooling the solution, 8.0 g of glycerin, 0.04 g of flavor, 4.0 g of ethanol, 0.4 g of sorbic acid, and an appropriate amount of distilled water were mixed thereto. Water was added to the mixture to make 100 mL.

<1-3> Preparation of Capsule

1,2,3,4,6-penta-o-galloyl-beta-di-glucose 50 mg of the present invention, lactose 50 mg, starch 46.5 mg, talc 1 mg and an appropriate amount of magnesium stearate are mixed and hard gelatin Capsules were prepared by filling the capsules.

<1-4> Preparation of Injection Solution

Injection solutions containing 10 mg of the active ingredient were prepared by the following method: 1 g of hydrochloride of 1,2,3,4,6-penta-o-galloyl-beta-di-glucose of the present invention, sodium chloride 0.6 100 ml were prepared by dissolving g and 0.1 g of ascorbic acid in distilled water. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

<1-5> Preparation of Cream

40 g of cream base containing oily ingredients such as 1.5 g of stearic acid, 2.2 g of stearyl alcohol, 0.5 g of butyl stearate, 0.5 g of propylene glycol, 2.0 g of glycerol monostearate, 0.3 g of potassium hydroxide, aqueous components, surfactants, etc. The 1,2,3,4,6-penta-o-galloyl-beta-di-glucose of the invention is mixed at a concentration of 2% (w / w) to emulsify well in a mixer, degassed, filtered and cooled to form a cream composition. Was prepared. Chelating agents, flavoring agents and pigments were added as additives to the composition, and the formulations were prepared in oil / water so that a small amount of oily ingredient was present.

As described above, the pharmaceutical composition containing 1,2,3,4,6-penta-o-galloyl-beta-di-glucose of the present invention as an active ingredient may reduce type I procollagen and express MMP-1. There is an effect that can prevent or treat skin diseases by inhibiting increase, increase in COX-2 expression, wrinkle formation, keratin thickening and collagen degradation.

Claims (4)

1,2,3,4,6-penta-O-galloyl-beta-di-glucose represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof Pharmaceutical composition for the prevention or treatment of skin diseases, containing as an active ingredient. <Formula 1>

The pharmaceutical composition of claim 1, wherein the skin disease is a skin disease or an inflammatory skin disease caused by ultraviolet rays. The pharmaceutical composition according to claim 2, wherein the skin disease caused by ultraviolet light is photoaging. The method of claim 2, wherein the inflammatory skin disease is skin inflammation, acute and chronic eczema, contact dermatitis, atopic dermatitis, seborrheic dermatitis, chronic simple mammary glands, interrogation, deprivation dermatitis, papular urticaria, psoriasis, sun dermatitis and acne Compositions selected from the group consisting of.

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