KR20070107252A - Pharmaceutical composition containing sargaquinoic acid for preventing or treating skin disease and skin cancer - Google Patents

Abstract

A pharmaceutical composition containing Sargaquinoic acid for preventing or treating skin disease and skin cancer is provided to prevent and treat diseases associated with hyperplastic skin cells by inducing cytotoxicity and apoptosis of skin cells, and promotes apoptosis by irradiating ultraviolet rays. A pharmaceutical composition for preventing or treating skin disease and skin cancer contains Sargaquinoic acid represented by the formula(I) or its pharmaceutically acceptable salt as an effective ingredient, wherein the medicinal effects of the pharmaceutical composition are increased by irradiating ultraviolet rays and the skin disease is hyperplastic skin disease including psoriasis, fishskin disease, Bowen's disease, papilloma, eczema, eczema, atopic dermatitis, acne vulgaris, dermatitis, lichen planus, striatus, foot hyperkeratosis, dark spots, actinic keratosis, skin aging and photoaging.

Description

Pharmaceutical composition containing Sargaquinoic acid for preventing or treating skin disease and skin cancer}

Figure 1 is a schematic of the process of extracting the sagaquinoic acid of the present invention from the waddle hatban.

FIG. 2 shows the results of nuclear magnetic resonance (NMR) analysis of Sagaquinoic acid separated from the waddle cap plaque, and results of hydrogen-nuclear magnetic resonance spectrum (a) and carbon-nuclear magnetic resonance spectrum (b).

Figure 3 shows the results of nuclear magnetic resonance (NMR) analysis of Sagaquinoic acid isolated from the waddle moss, COSY (a), HSQC (b) and HMBC (c) spectrum analysis results.

Figure 4 shows the cytotoxic effect of sagaquinoic acid in human keratinocytes, the results of MTT assay (a), FACS assay (b) and TUNEL assay (c).

Figure 5 is the result of Western blot analysis of the effect on the expression of cell death-related factors for each treatment concentration of sagaquinoic acid in human keratinocytes.

Figure 6 is the result of analyzing the apoptosis effect of Sagaquinoic acid by ultraviolet B irradiation in human keratinocytes by MTT analysis method.

Figure 7 is the result of Western blot analysis of the effect on the expression of cell death-related factors of sagaquinoic acid by ultraviolet B irradiation in human keratinocytes. FIG. 7A shows the result after 4 hours after the ultraviolet B treatment, and FIG. 7B shows the result after 8 hours after the ultraviolet B treatment.

8 is a result of analyzing the effect of Saga-quinoic acid apoptosis by the ultraviolet rays in the mouse model by H & E staining method, TUNEL analysis method and immunohistostaining method.

The present invention relates to a pharmaceutical composition for preventing or treating skin diseases and skin cancer. More specifically, the present invention relates to a pharmaceutical composition for the prevention or treatment of skin diseases and skin cancer containing Sargaquinoic acid (Sargaquinoic acid) represented by the formula (I) as an active ingredient.

Skin diseases are located at the outermost layer of the human body and the skin covering the surface of the body is always exposed from the outside, which is caused by various factors and pathogens that irritate the skin, and is caused by genetic, inflammation, benign and malignant tumors and hormones. It is also caused by the effects of pathological changes such as trauma, degenerative degeneration. In particular, since the stratum corneum in the outermost layer of the skin is in direct contact with the external environment, damage to the stratum corneum may be a direct factor of skin disease. The keratinocytes in the stratum corneum of the skin have a certain cycle and when the cells divide and form new cells, the cells fall off the skin after their life is called. This is called the epidermal peeling or turn over. Thus, our skin is not an organ that remains intact once formed, but is an organ that is constantly being recreated and removed. However, if the abnormality occurs at the time of skin formation or dropout of the skin, various problems may occur, and most skin diseases are caused from this.

Psoriasis is a chronic skin disease in which the hyperproliferation of keratinocytes and various inflammatory cells, especially T cells, are infiltrated and activated, rapidly proliferating keratinocytes with angiogenesis. Psoriasis is caused by an unusual constitution or immune response that causes parts of keratinocytes to grow too fast, resulting in abnormally thick stratum corneum, ranging from millet to palm size, and can appear in any part of the body, not just in the body. It is distinctive. In particular, the external stimulation occurs frequently in the knee, scalp, buttocks, elbows, external genitalia. Psoriasis is a skin disease currently suffering from 0.5% to 2% of the world's population, but the exact cause has not been identified.

To date, the treatment of skin diseases caused by psoriasis and cell hyperproliferation consists of methods of rapidly reducing cell proliferation and reducing inflammation, these treatments optionally with irradiation, topical, systemic or both of the active agent. It is done with the use of. However, the active agents and investigations thus far have caused side effects causing ulceration of the skin, severe erythema, edema and blister formation in the treatment area, and tend to recur, so that current treatment is not a target for cure. It is just a dimension. Therefore, in recent years, interest in new therapeutic agents and techniques for inducing apoptosis of skin cells in order to treat skin diseases and skin cancers caused by the hyperproliferation of the skin cells is increasing.

Apoptosis refers to programmed cell death and is a form of cell death that occurs under physiological and pathological conditions. In normal cell tissues, cell proliferation and apoptosis are balanced so that the number of cells remains constant.However, in cancer cell tissues and tumor cell tissues, cell death is unilaterally increased due to inadequate cell death compared to rapid cell proliferation. (Raff. MC, Nature , 356: 397. 1992). Thus, the regulation of apoptosis is closely related to diseases associated with tissue or cell damage, such as cancer, autoimmune diseases, allergies, neurodegenerative diseases (Alzheimer's disease), aging, myocardial infarction and stroke. It plays an important role in the function of the skin, internal organs and immune system. Therefore, therapeutic agents and techniques for treating various diseases and cancers have been developed by using a method of inducing or inhibiting apoptosis, and in particular, skin diseases caused by skin cell proliferation mentioned above may be induced or prevented or treated. Techniques for doing so are being developed. In the related art, nucleotide homologs for topical treatment of proliferative skin diseases for the treatment of psoriasis and skin diseases using antiproliferative nucleoside homologs that inhibit the proliferation of cells (Korean Patent Publication No. 1999-022470). ), 1,25-dihydroxy-16,22,23-trisdehydro-cholecalciferol derivatives (Korean Patent No. 0240541), which are pharmaceutical compositions for inhibiting skin hyperproliferation and tumor cells, skin diseases There is a composition for inducing apoptosis containing phytosphingosine derivatives that induce apoptosis for the treatment of tumors and cancers (Korean Patent Publication No. 2004-0016353).

However, the above-described compounds and compositions are artificially synthesized and have a disadvantage of causing many side effects such as causing headaches, dizziness, abnormal sensations, neurological symptoms, and skin side effects when used for a long period of time. Therefore, there is a need for the development of improved therapeutics without side effects.

The present inventors have found that the natural origin of the low side effect looking for the valuable substances with a therapeutic effect on the skin disease for twisting Sargassum (Sargassum sagaquinoic acid) was isolated and purified, and it was found for the first time that the sagaquinoic acid of the present invention exhibited a marked activity in inducing apoptosis of skin cells.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of skin diseases and skin cancer containing sagaquinoic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

In order to achieve the object of the present invention as described above, the present invention provides a pharmaceutical composition for the prevention or treatment of skin diseases and skin cancer containing sagaquinoic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

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

Sagaquinoic acid of the present invention can be represented by the following formula (I).

<Formula I>

Sagaquinoic acid according to the invention can be used on its own or in the form of salts, preferably in the form of pharmaceutically acceptable salts. The term “pharmaceutically acceptable salts” means salts suitable for use in contact with tissues of humans and lower animals without causing excessive toxicity, irritation, allergic reactions, etc. within the scope of pure medical judgment. The salts may be prepared in situ during the final separation and purification of the sagaquinoic acid of the present invention or separately by reaction with an inorganic base or an organic base. Pharmaceutically acceptable salts include, but are not limited to, for example, alkali metal or alkaline earth based cations such as, but not limited to, lithium, sodium, potassium, calcium, magnesium and aluminum salts and nontoxic quaternary ammonia and amine cations (ammonium, tetra Methylammonium, tetraethylammonium, methylammonium, dimethylammonium, trimethylammonium, triethylammonium, diethylammonium and ethylammonium).

Sagaquinoic acid of the present invention can be isolated and purified from nature. That is, they can be obtained from plants using a method of extracting and separating conventional materials. In order to obtain the extract, it is appropriately dried and macerated or only dried to extract with a suitable organic solvent, and the desired extract can be purified using purification methods known to those skilled in the art. Preferably Saga CUNAULT acid of the invention for twisting Sargassum (Sargassum sagamianum ) can be isolated and purified.

Sargassum of the wobbler sagamianum ) is a seaweed belonging to the Mabanban family and is distributed on the east and south coasts of Korea and the coast of Japan. Sagaquinoic acid extracted from the waddle moss has been reported to have a function of growing nerve cells dependently or non-dependently on nerve growth factor (NGF), a growth factor of nerve cells (Chi Kwan Tsang et al., European J. of Pharmacol ., 2004, Yuto Kamei et al., Int . J. Devl Neuroscience , 2003), there are no reports on the apoptosis control function and mechanism of sagaquinoic acid.

Water or C1-C6 organic solvent may be used as an extraction solvent for the separation of the sagaquinoic acid of the present invention from the waddle cap. The organic solvent is methanol (ethanol), ethanol (ethanol), propanol (propanol), isopropanol (isopropanol), butanol (butanol), acetone (acetone), ether (ether), benzene (benzene), chloroform, Various solvents such as ethyl acetate, methylene chloride, hexane, cyclohexane and petroleum ether may be used alone or in combination. Preferably methanol can be used, most preferably chloroform or ethyl acetate. Separation and purification of the sagaquinoic acid of the present invention from the methanol extract may be performed alone or in combination with column chromatography and TLC filled with various synthetic resins such as silica gel or activated alumina. Can be. Preferably, silica gel column chromatography and octadecyl silica gel column chromatography may be used in combination. However, the extraction and separation and purification method of the active ingredient is not necessarily limited to the above method.

Sagaquinoic acid of the present invention can induce cytotoxicity and apoptosis of skin cells to treat or prevent skin diseases and skin cancer associated with hyperproliferated cells.

Sagaquinoic acid of the present invention has a cytotoxic effect on skin cells in skin diseases caused by overproliferation of skin cells. In one embodiment of the present invention, the human keratinocytes treated with the sagaquinoic acid of the present invention was confirmed to have a cytotoxic effect compared to the human keratinocytes not treated with sagaquinoic acid through the MTT assay method, As a result of confirming cytotoxicity by gradually increasing the concentration of sagaquinoic acid, it was confirmed that there is cytotoxicity in a concentration-dependent manner (see FIG. 4A).

In addition, the sagaquinoic acid of the present invention induces apoptosis of skin cells. In one embodiment of the present invention, when the SAGA was treated to human keratinocytes, it was confirmed by FACS and TUNEL analytical methods that apoptosis was induced in the cells, and to human keratinocytes not treated with sagaquinoic acid of the present invention. It was confirmed that the apoptosis effect is superior to that, and as the concentration and treatment time of sagaquinoic acid were increased, the apoptosis effect was better (see FIGS. 4B, 4C and 4D).

Apoptosis is a genetically programmed cellular phenomenon characterized by well-defined morphological phenomena such as cell contraction, chromatin condensation, nuclear fragmentation, and membrane blebbing. Plays an important role in However, program defects of apoptosis are believed to affect a wide range of human metabolic disorders from neurodegenerative and autoimmune disorders to neoplasms. Factors involved in the apoptosis is Bcl-2, Bax, PARP, it can caspase (caspase), etc. are known (Wyllie, A., Nature, 389 :. 237, 1997; Ferrer I, Planas AM J Neuropathol Exp Neurol. , 62: 329-39, 2003). The caspases include caspase-9, caspase-8, and caspase-3, and when activated by apoptosis mechanisms within cells, caspases appear in truncated form.

 Sagaquinoic acid of the present invention activates apoptosis related factors to induce cell death. After treating the sagamiquinoic acid of the present invention on human keratinocytes, and confirming the activity of apoptosis-related factors, the caspase-9, caspase-8, and caspase-3 were activated and cleaved. In addition, PARP, a substrate of caspase, was also activated. In addition, as the treatment concentration of the sagaquinoic acid of the present invention was confirmed that the activity of apoptosis-related factors increased further (see Fig. 5). On the other hand, Bcl-2 and Bax, which are related to apoptosis, are confirmed to be unaffected by the sagaquinoic acid of the present invention, so that the sagaquinoic acid of the present invention affects the cell death mechanism by caspase in the cell. It was found to exert.

In addition, the present invention can further increase apoptosis of cells by treatment with sagaquinoic acid and ultraviolet light.

Ultraviolet rays may be UVA (200-290nm), UVB (290-320nm), UVC (320-400nm) depending on the wavelength, preferably UVB (ultraviolet B) can be used. In particular, ultraviolet B increases free radicals (ROS) when exposed to cells or cultured cells in vivo, and these free radicals induce oxidative stress, resulting in cell death (Hattori, Y. et al ., J. Invest Dermatol 107:. 733-737, 1997 , Kim, HJ et al, Apoptosis 9:. 449-456, 2004). Therefore, the above-mentioned effects of ultraviolet rays are used in the method of treating skin diseases.

 In the present invention, after treating the sagamiquinoic acid of the present invention on human keratinocytes, the apoptosis was confirmed by irradiation with ultraviolet rays, and it was confirmed that the cell death effect was superior to the cells irradiated with ultraviolet rays than the cells not irradiated with ultraviolet rays ( 6, it was confirmed that the activity of apoptosis related factors was further increased when irradiated with ultraviolet rays (see FIG. 7).

Therefore, the sagaquinoic acid of the present invention activates cytotoxicity and cell death related factors of skin keratinocytes and induces cell death, thereby treating and preventing hyperproliferative skin diseases such as psoriasis and skin cancer.

The present invention provides a pharmaceutical composition for the prevention or treatment of skin diseases and skin cancer containing sagaquinoic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

The skin disease may be a hyperproliferative skin disease, and the term "hyperproliferative skin disease" refers to a disease caused by hyperproliferation of skin epidermal cells. More than 95% of the epidermal cells are composed of keratinocytes, which produce a protein called keratin and continuously differentiate into basal cells, pole cells and granule cells. The skin has a constant cycle of new and disappearing cells, that is, turnover, which can cause hyperproliferative skin disease if new cells overproliferate rapidly or stagnant old cells as compared to the time when the cells fall apart. do. Examples of the hyperproliferative skin disease include, but are not limited to, psoriasis, young, Bowen's disease, papilloma, eczema, addictive eczema, atopic dermatitis, vulgaris, seborrheic dermatitis, lichen planus, glandular line, keratosis May include seborrheic keratosis, actinic keratosis, skin aging, photoaging and the like.

The pharmaceutical compositions according to the invention may contain sagaquinoic acid alone or may further contain one or more pharmaceutically acceptable carriers, excipients or diluents. As used herein, the term “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 and skin cancer 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, the term “transdermal administration” refers to 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 compositions of the present invention may be prepared in an injectable formulation and administered by a method of lightly pricking the skin with a 30 gauge thin injection needle, or by applying directly to the skin.

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.

Formulations for parenteral administration may be formulated by methods known in the art in the form of injections, creams, lotions, external ointments, oils, humectants, gels, aerosols and nasal inhalants. 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 sagaquinoic acid of the present invention may be administered to a patient in a single dose and may be administered by a fractionated treatment protocol that is administered in multiple doses 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 sagaquinoic acid of the invention may be from about 0.001 μg to 100 mg, most preferably 0.1 μg to 1 mg per kg of patient body weight per day. However, the concentration of sagaquinoic acid is determined by taking into consideration various factors such as the age, weight, health condition, sex, severity of the disease, diet and excretion rate, as well as the route and frequency of treatment of the pharmaceutical composition. In this regard, one of ordinary skill in the art will be able to determine an appropriate effective dosage for the specific use of the sagaquinoic acid as a prophylactic or therapeutic agent for skin diseases. 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.

Hereinafter, the present invention will be described in detail by way of examples.

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

< Example  1>

Sagaquinoic acid  Extraction and separation

<1-1> from the wobbler hat Sagaquinoic acid  Separation and Purification

450 g of wobbler Mabban (purchased in Seongsan, Jeju) was pulverized into a powder, and then extracted three times with 4 L of 100% methanol and stirred with a stirrer at room temperature for 24 hours. After extraction, the filtrate was concentrated using a rotary evaporator (Buchi) to obtain about 38 g of methanol extract. All 38 g of the methanol extract were dissolved in 80% methanol and solvent-distributed with n -hexane to remove nonpolar material. The 80% methanol layer of the remaining active fraction after the removal of the nonpolar material was partitioned into 30% methanol and chloroform layers. The 30% methanol layer was again partitioned between water and n -butanol, and the chloroform layer was subjected to octadecyl silica flash chromatography on methanol solvent to obtain 12 g of 80% methanol fraction. About 2 g of the 80% methanol fraction was subjected to silica column chromatography sequentially using 100% chloroform and a mixed solvent having a mixing ratio of chloroform and methanol of 95: 5, 8: 2, and 7: 3 as eluents. Got. 445 mg, which is a part of the third fraction, was subjected to silica flash chromatography using a mixed solvent having a mixing ratio of chloroform and methanol of 99.5: 0.5 as eluent, and a mixed solvent having a mixing ratio of chloroform and methanol of 9: 1. Silica flash chromatography was performed with eluent. Thereafter, silica preparative TLC was performed again using a mixed solvent having a mixing ratio of chloroform and methanol of 98: 2 to obtain 187 mg of sagaquinoic acid as a pure single component of the present invention.

<1-2> Sagaquinoic acid  Structural analysis

In order to confirm whether the compound isolated and purified in Example <1-1> is sagaquinoic acid, a structural analysis experiment was performed. In the structural analysis experiment, MS spectrum, IR spectrum, and NMR spectrum analysis were performed in the following manner. ESIMS spectra were performed using a JEOL JMX-SX 102 mass spectrometer, and high resolution mass spectrometry was performed using a JEOL AX-505H mass spectrometer. IR spectra were performed with Perkin Elmer's 1750 FT-IR spectrophotometer, and ¹ H NMR (400 MHZ) and ¹³ C (100 MHZ) spectra were performed with Bruker ARX-400 NMR spectrometers. The analysis results are as shown below.

Isolated and purified Sagaquinoic acid  Structural Analysis Results

 -Colorless oil

UV λ _max (EtOH) (ε) 250 nm (14,500)

IR (film) ν _maz cm ^-1 1680 (aromatic C = C), 1715 (C = O)

ESIMS m / z 447.2 [M + Na] ⁺

HRFABMS m / z 447.2511 [M + Na] ⁺ calcd for C ₂₇ H ₃₆ O ₄ Na, 447.2502

¹ H-NMR (CDCl ₃ , 400 MHz) δ 6.53 (1H, s), 6.45 (1H, s), 5.99 (1H, t, 7.2), 5.15-5.06 (3H, m), 3.11 (2H, d , 7.2), 2.59 (2H, q, 7.2), 2.25 (2H, t, 7.2), 2.14-2.04 (8H, m), 2.04 (3H, s), 1.66 (3H, s), 1.61 (3H, s ), 1.59 (3H, s), 1.57 (3H, s)

¹³ C-NMR (CDCl ₃ , 100 MHz) δ 188.3 (C-1), 188.2 (C-4), 173.4 (C-18 '), 148.7 (C-2), 146.2 (C-6), 145.7 (C-10 '), 140.1 (C-3'), 134.8 (C-7 '), 133.4 (C-5), 132.5 (C-3), 132.4 (C-15'), 130.8 (C-11 '), 124.8 (C-6'), 123.7 (C-14 '), 118.2 (C-2'), 39.8 (C-5 '), 39.3 (C-8'), 34.8 (C-12 ') , 28.4 (C-9 '), 28.1 (C-13'), 27.8 (C-1 '), 26.6 (C-4'), 25.9 (C-16 '), 17.9 (C-17'), 16.3 (C-7), 16.2 (C-20 '), 16.1 (C-19')

As a result of the analysis, the compound isolated and purified in Example <1-1> is a colorless oil component, high resolution mass spectrometry [HRFABMS (High Resolution Fast Atom Bombardment Mass Spectrometry)] spectrum, [ m / z 447.2511 (M + Na ) ⁺ , calcd for C ₂₇ H ₃₆ O ₄ , 447.2502] and ¹ H, ¹³ C-NMR spectra showed that the molecular formula of the compound was C ₂₇ H ₃₆ O ₄ , and that the unsaturation was 10. It could be predicted.

The results of ¹ H-NMR indicated that the four peaks between 5.99 and 5.06 ppm and the two peaks at 6.53 and 6.45 ppm were olefin hydrogen peaks, and appeared as singlets between 2.04 and 1.57 ppm. Five peaks were confirmed to be hydrogen of the methyl group (see FIG. 2A). In addition, as a result of analysis of ¹³ C-NMR spectrum, it was confirmed that the two peaks appearing at 188.3 and 188.2 ppm is carbonyl carbon, and the peak appearing at 173.4 ppm was the carbonyl group carbon peak of carboxylic acid. Twelve peaks appearing between 148.7 and 118.2 ppm were found to be sp ² methylene carbon of the olefin, and five peaks appearing at 25.9, 16.1, 16.2, 16.3 and 17.9 ppm were methyl carbon. Meanwhile, the E configuration for the double bond of C-2 'and C-3', C-6 'and C-7', and C-14 'and C-15' is the vinyl-methyl group 20 'and 19, respectively. Chemical shift values of ', 17' to δ _{H-20 '} 1.61, δ _C-20' 16.2, δ _{H-19 '} 1.59, δ _C-19' 16.1, δ _{H-17 '} 1.66, δ _C-17' 17.9 Determined by The E, Z configuration [E-δ _H 1.60, δ _C 17.6, Z- determined by chemical shifts of carbon and hydrogen of methyl groups linked to double bonds of geraniol and polyprenol compounds reported. δ _H 1.68, δ _C 25.6] (Cimino, G. et al., Tetrahedron , 1972, 28, 1315-1324; Kusumi, T. et al., Chem . Lett . , 1979, 3, 277-278; Yasuyuki et al., Polymer , 1982, 23, 1087-1090; Koichi, I. et al., Biochem . J .. 1983, 213, 305-311). In addition, the Z configuration [δ _H- 10'5.99] of the C-10 'and C-11' double bonds is reported by C-10 and C-10 of the reported E- and Z-2-methyl-2-pentenoic acids compounds. Determined by the chemical shift values of olefin hydrogen in methylene hydrogen of C-9 [E-δ _H 6.88, Z-δ _H 6.06] (Chan, KC et al., J. Org . Chem . , 1968, 33, 3382) -3385; Kusumi, T. et al., Chem . Lett . , 1979, 3, 277-278) (see FIG. 2B). From the results of the COSY NMR spectrum and the HSQC NMR spectrum, nine quaternary carbons 1, 2, 4, 6, 3 ', 7', 11 ', 15', 18 '[δ 188.3, 148.7, 188.2, 146.2, 140.1 , 134.8, 130.8, 132.4, 173.4] and carbon containing hydrogen could be predicted separately, and olefin hydrogen 3, 5, 2 ', 6', 10 ', 14' [δ 6.45, 6.53, 5.11 × 3, 5.99 ] [Δ 132.5, 133.4, 118.2, 124.8, 145.7, 123.7] linked to. In addition, the area ratio was able to predict carbon [δ 25.9, 17.9, 16.1, 16.2] linked to methyl hydrogen 16 ', 17', 19 ', 20' [δ 1.57, 1.66, 1.59, 1.62]. , olefinic carbon and a methyl group for the remaining carbon other than a carbon sp ³ containing hydrogen through a cross peak in the HSQC spectrum appeared was confirmed that the methine carbon-methylene or sp ^3. In addition, as can be seen from the results of the HMBC NMR spectrum, the partial structure was estimated through the cross-peak of hydrogen appearing for each carbon (see FIG. 3). Therefore, the structural analysis experiments as described above confirmed that the isolated and purified compound of Example <1-1> was a known substance sagaquinoic acid, which is a plastoquinone derivative (Kusumi, T. et al. , Chem . Lett. , 1979, 3, 277-278).

< Example  2>

Sagaquinoic acid  Cytotoxicity Survey

<1-1> MTT Assay  Cytotoxicity Measurement

The isolated and purified sagaquinoic acid of Example 1 of Example 1 was examined for cytotoxicity against HaCaT, a human keratinocyte by MTT cell viability assay. The HaCaT cell line was provided by Professor N. Fuseni of German Cancer Research. The HaCaT cells were incubated in a 37 ° C. and 5% CO ₂ incubator in DMEM medium supplemented with 10% FBS (Hyclone), 100 unit / ml penicillin and 100 μg / ml streptomycin. The viability and cell number during cell culture were measured periodically by trypan blue exclusion using a hemocytometer (Neunauer, Germany), and the initial cell viability was maintained above 95% in all experiments. The cultured HaCaT cells were dispensed into 96-well plates with a cell number of 5 × 10 ⁴ cells / well and incubated for 24 hours in 37 ° C. and 5% CO ₂ incubator. Then, the cultured HaCaT cells were treated with sagaquinoic acid in each well at concentrations of 0, 1, 2, 5, and 10 μg / ml, respectively, and then cultured in a 37 ° C., 5% CO ₂ incubator for 24 hours. . After incubation, 5 mg / ml MTT reagent was added to each well at a concentration of 20 µl / 100 ml in the presence of the medium, and incubated for 2 hours in an incubator. Thereafter, the culture medium was removed, and 150 µl of DMSO was added to each well, and cell viability was analyzed by measuring absorbance at 540 nm using an ELISA reader (Molecular Devices, USA). In this case, the control group was not treated with sagaquinoic acid, and cell viability was expressed as a relative value with the cell viability of the control as 100%.

As a result, the cell viability decreased as the concentration of sagaquinoic acid was increased, and the cell viability was reduced by 20% when 10 ug / ml of sagaquinoic acid was treated (see FIG. 4A). ). Therefore, it was confirmed that the sagaquinoic acid of the present invention is toxic to human keratinocytes.

<1-2> FACS  And TUNEL  ( TdT -mediated dUTP  Cytotoxicity Measurement by Nick-end Labeling Method

Whether the sagaquinoic acid of the present invention has an apoptosis effect on human keratinocytes was investigated by FACS and TUNEL analysis. First, for FACS analysis, HaCaT cells cultured in the same manner as in Example <1-1> were dispensed into 6-well plates with a cell number of 3 × 10 ⁵ cells / well, followed by a 37 ° C., 5% CO ₂ incubator. Incubated for 24 hours at. The cells were treated with sagaquinoic acid at concentrations of 0, 1, and 10 μg / ml, respectively, and then cultured in 37 ° C., 5% CO ₂ incubator for 12 hours and 24 hours. After the incubation was completed, the cells were detached from the wells with trypsin and fixed by treatment with 3.7% paraformaldehyde, and the fixed cells were enhanced with 70% alcohol for 30 minutes with Propidium Iodide (PI) solution. The cells were stained to perform the FACS assay. As a control, only DMSO was used without treating sagaquinoic acid. For the TUNEL analysis method, after culturing the cells for 24 hours as described above, the cells were dispensed on chamber slides according to the method of ApopTag® Flurorescein in situ apoptosis detection kit and adhered to the chamber slides. After washing with PBS solution of pH 7.4. At this time, to completely fix the cells, immersion slide in a PBS solution of 4% paraformaldehyde at room temperature for 30 minutes, washed with PBS to inhibit the action of endogenous peroxidase (blocking solution (1) % H ₂ O ₂ Soaked in methanol) for 30 minutes at room temperature. Thereafter, the cells were washed with PBS to enable cell permeation, and then soaked in 0.1% sodium citrate solution containing 0.1% Triton X-100 at 4 ° C. for 2 minutes. After washing twice more with PBS for labeling, the diluted TUNEL reaction mixture was added to the slides and incubated in a 37 ° C. humidified chamber for 60 minutes, and then the slides were washed with PBS. Then, incubated for 20 minutes in a TBS solution containing 1% BSA, 5% FBS. An anti-fluorescein antibody conjugated with horse-radish peroxidase was added to the culture, reacted at 37 ° C. for 1 hour, washed with PBS, and a DAB-based solution was added thereto. Hematoxylin solution was added for back staining. The slides were then washed with PBS and glass coverslips were placed on the slides and analyzed under a microscope. The nuclei of dead cells positive in the TUNEL reaction were stained dark brown, making it easy to distinguish them from blue staining.

As a result, it was confirmed that FACS analysis showed better apoptosis effect in the cells treated with sagaquinoic acid than in the control group, and the apoptosis effect was increased when 10 ug / ml was treated than 1 ug / ml. It was confirmed that apoptosis was significantly increased when the treatment with sagaquinoic acid was performed for 24 hours longer than 12 hours (see FIG. 4B). As a result of the TUNEL assay, as in the FACS analysis, it was confirmed that the TUNEL-positive cells, that is, the dead cells, increased as the treatment concentration of the sagaquinoic acid of the present invention increased (see FIG. 4C). Therefore, the sagaquinoic acid of the present invention was found to have a cytotoxic effect on human keratinocytes and to kill cells in a concentration-dependent manner.

< Example  3>

Sagaquinoic acid Skin cells  Death effect

HaCaT cells, which are human keratinocyte lines cultured in the same manner as in Example 1, were dispensed into 6-well plates with a cell number of 3 × 10 ⁵ cells / well. After 12 hours of serum starvation, sagaquinoic acid was treated to cells at concentrations of 0, 1, 2 and 5 μg / ml, respectively. After 24 hours, RIPA buffer (12 mM EDTA, 137 mM NaCl, 20 mM Tris-HCl, pH 8.0), 1 mM Na ₃ VO ₄ (Sigma-Aldrich, USA) 10 mM NaF (Sigma), 1 mM PMSF (Sigma), 1 Cells were lysed using% Triton X-100, 10% Glycerol and Protease Inhibition Cocktail (Roche, Germany). The lysed cells were placed in a 1.5 ml tube and pipetted, and left on ice for 20 minutes. The lysed cells were centrifuged at 14000 rpm for 20 minutes to recover the supernatant to separate proteins. The separated protein was subjected to electrophoresis (SDS-PAGE) by determining the concentration of each sample using a BCA protein analysis kit (Pierce, Rockford, Illinois). Western blot analysis was performed using the protein separated by electrophoresis. Western blot analysis transfers proteins from the gel to the polyvinylidene fluoride membrane (Pall Corporation) at the end of the electrophoresis of the sample, and then the membrane is removed for 2 hours at room temperature in a 3% BSA solution (Amesco, USA). ), And the primary antibody was treated and reacted at room temperature for 2 hours. The primary antibody may be an anti-caspase-3 antibody, an anti-caspase-8 antibody, an anti-Bcl-2 antibody, an anti-Bax antibody or an anti-Actin antibody, which are antibodies to cell death-related signaling mechanisms. It was purchased and used from Cell Signaling Technology, Beverly, Mass., And anti-caspase-9 antibody and anti-PARP antibody were purchased from Upstate, NY. After reaction with the primary antibody, the reaction was washed with TBS for about 15 minutes and treated with the secondary antibody for 45 minutes. Rabbit secondary goat antibodies, goat anti-mouse antibodies and goat anti-rabbit antibodies were purchased from Zymed (Zymed, Sanfrancisco, USA) as the secondary antibody. After the reaction, the sample was sufficiently washed with TBS for 30 minutes or more, and the protein was identified using an ECL detection kit (Santa Cruz). In this case, as a control, cells not treated with sagaquinoic acid were treated by the same method as above.

As a result, after 24 hours of treatment with sagaquinoic acid, as the concentration of sagaquinoic acid increased, the apoptosis inducer was activated to confirm the cleaved forms of caspase-8, caspase-9 and caspase-3. , Concentration-dependently increased the amount of cleaved protein. Furthermore, cleavage of PARP, a substrate of caspase, was also activated by sagaquinoic acid. However, the expression of Bax, another apoptosis inducer, was not affected by the sagaquinoic acid of the present invention, and the expression of Bcl-2, an apoptosis suppressor, was not affected. Iksan was found to have no effect on the expression of the Bcl family, including Bax and Bcl-2 (see FIG. 5).

Therefore, from the above experimental results, the sagaquinoic acid of the present invention has an activity of inducing skin cell death by activating caspase-8, caspase-9 and caspase-3, which are specifically related to cell death in human keratinocytes. It was confirmed that there is.

< Example  4>

By ultraviolet Sagaquinoic acid  Apoptosis synergistic effect

We investigated whether ultraviolet rays have an effect of increasing apoptosis by sagaquinoic acid in human keratinocytes. HaCaT cells cultured in the same manner as in Example 1 were dispensed into 6-well plates at a cell number of 3 × 10 ⁵ cells / well. After 12 hours of serum starvation, each well was pretreated with sagaquinoic acid at concentrations of 0, 0.5, 1, 2, 5, and 10 μg / ml, followed by an ultraviolet B irradiator (FS24T12 / UVB / HO, Voltare Co., Fairfield, CT, USA, 290-320nm) using a dose of 150 J / m ² was investigated. After 18 hours of ultraviolet irradiation, 5 mg / ml of MTT reagent was added to each well at a concentration of 20 µl / 100 ml in the presence of medium, and incubated for 2 hours in an incubator. After 2 hours, the culture solution was removed, and 150 μl of DMSO was added to each well, and cell viability was analyzed by measuring absorbance at 540 nm using a spectrophotometer. In this case, the control group was not treated with sagaquinoic acid, and cell viability was expressed as a relative value with the cell viability of the control as 100%.

As a result of the experiment, the cells irradiated with ultraviolet rays together with the cells treated with only the sagaquinoic acid of Example <1-1> (Fig. 4A) without irradiating the ultraviolet rays under the same conditions as in Example 4 (pretreated with sagaquinoic acid) Cells) was found to significantly increase apoptosis (see Figure 6). In particular, cells treated with only sagaquinoic acid without UV irradiation showed more than 60% cell viability at a concentration of 5 μg / ml sagaquinoic acid, while those exposed to ultraviolet light (pretreated cells of sagaquinoic acid) were 1 μg / Cell death increased rapidly in the concentration of sagaquinoic acid in ml, showing only about 25% cell viability. Therefore, it was confirmed that the apoptosis effect of sagaquinone ic acid was greatly increased by ultraviolet rays.

< Example  5>

By ultraviolet Sagaquinoic acid  Cell death On related factors  Impact

In Example 4, it was confirmed that the ultraviolet rays further increase the apoptosis effect by sagaquinoic acid in human keratinocytes. Therefore, it was investigated whether ultraviolet rays also affected factors related to cell death by sagaquinoic acid. First, HaCaT cells, which are human keratinocytes cultured in the same manner as in Example 1, were dispensed into 6-well plates with a cell number of 3 × 10 ⁵ cells / well. After 12 hours of serum starvation (serum starvation), the concentration of sagaquinoic acid was pretreated at 0, 2, 5 μg / ml for 30 minutes before irradiating ultraviolet B. 4 and 8 hours after irradiation with ultraviolet B, RIPA buffer (12 mM EDTA, 137 mM NaCl, 20 mM Tris-HCl, pH 8.0), 1 mM Na ₃ VO ₄ (Sigma-Aldrich, USA) 10 mM NaF (Sigma) , 1mM PMSF (Sigma), 1% Triton X-100, 10% Glycerol, Protease Inhibition Cocktail (Roche, Germany)), and then lysing the cells to determine protein concentration and electrophoresis (SDS-PAGE). ) Western blot analysis was performed using the isolated protein as in Example 3. At this time, as a control, cells not treated with sagaquinoic acid were separated from proteins in the same manner as above, and Western blot analysis was performed.

As a result, after 4 hours of pretreatment with Sagaquinoic acid and irradiation with ultraviolet B, the cells were found to have significantly more cleaved forms of caspase-9 and caspase-3 than cells treated with ultraviolet B only. (FIG. 7A). In addition, after 8 hours of pretreatment with sagaquinoic acid, caspase-8, caspase-9 and caspase-3 were activated by UVB, and thus the cut form was confirmed. However, as a result of FIG. 7A, caspase activity was not observed in cells treated with 2 and 5 μg / ml of sagaquinoic acid alone. However, in the result of FIG. 5, 2 and 5 μg / ml of sagaquinoic acid were observed. After 18 hours of treatment alone, caspase activity was observed. Therefore, in the cell death effect by the sagaquinoic acid it can be seen that by treatment with ultraviolet light shortens the time that the cell death occurs, showing a higher effect. In addition, it was confirmed that caspase activity was observed in cells treated with only ultraviolet light but not treated with sagaquinoic acid, and PARP, a substrate of caspase, was also activated by ultraviolet light (FIG. 7B).

From the above experimental results, cell death-related factors such as caspase-8, caspase-9, caspase-3 and PARP are more activated when treated with UVB alone than when only sagaquinoic acid is treated with human keratinocytes alone. It could be confirmed that this leads to the fact that skin cell death is significantly induced. Therefore, if the sagaquinoic acid and ultraviolet light of the present invention is treated together with the skin disease cells will effectively increase the therapeutic effect by inducing apoptosis of the skin cells.

< Example  6>

In mouse model Sagaquinoic acid  Increased cell death by UV rays

The effects of sagaquinoic acid on skin affected by UV light in hairless mice were investigated. Three 10-week-old male rats (HRS / J-hr + / +, KRIBB, Daejeon, Korea) were categorized into three per group and one group was not treated with both ultraviolet and sagaquinoic acid (untreated group). Was treated with acetone and then irradiated with ultraviolet light (ultraviolet alone treatment group). The other group was treated with sagaquinoic acid followed by ultraviolet light at a dose of 5 KJ / m ² (experimental group). In the experimental group, 100 µl of sagaquinoic acid (1 mg / ml) diluted with acetone was applied to the rat's back skin 30 minutes before UV irradiation. After 30 minutes, ultraviolet rays were irradiated to the back of the rat at a dose of 2.5 KJ / m ² using an ultraviolet B irradiator (FS24T12 / UVB / HO, Voltare Co., Fairfield, CT, USA, 290-320 nm). After 30 minutes of UV irradiation, Sagaquinoic acid was applied to the rat's back skin in the same manner. At this time, in the ultraviolet treatment group, acetone was treated in the same amount in place of sagaquinoic acid. Skin was extracted from the back of each rat 24 hours after ultraviolet irradiation. The extracted tissues were immersed in 4% formalin solution for 16 hours or longer and then embedded in paraffin to prepare tissue sections having a thickness of 5 μm. The tissues were deparaffinized for H & E (Hematoxylin & Eosin) staining and deparaffinized for TUNEL and tissue staining. To facilitate tissue infiltration, the reaction was performed for 20 minutes with 0.1 M citrate, and for 20 minutes at room temperature with 3% hydrogen peroxide, followed by washing, followed by 2 hours with blocking buffer (3% BSA, 0.2% Triton X-100). It was reacted at room temperature for a while. TdT enzyme was used for TUNEL staining and caspase-3 (Cell Signaling Technology, Beverly, MA) was used as a primary antibody for immunohistostaining at 4 ° C. for 16 hours. The biotinylated secondary antibody was then reacted and in turn reacted with streptavidin (DAKO) labeled with peroxidase. The reaction was observed using a DAB substrate chromogen system (DAKO) at 24 hours after UV irradiation. Positive cells killed by TdT enzyme and caspase-3 in this system appear brown.

The experimental results showed that the TdT enzyme and caspase-3 positive cells were increased in the UV-irradiated tissue (UV treatment alone) compared to the UV-irradiated tissue (untreated treatment). However, when irradiated with ultraviolet rays after treatment with sagaquinoic acid (experimental group), it was confirmed that the TdT enzyme and caspase-3 positive cells were significantly increased compared to the ultraviolet treatment alone (see FIG. 8). Therefore, it was confirmed that sagaquinoic acid increased the cell death by further activating the caspase-3 activity by ultraviolet rays.

< Production Example  1>

Preparation of a medicament containing the pharmaceutical composition for the treatment or prevention of skin diseases and skin cancer according to the present invention

<1-1> Preparation of Tablet

25 mg of sagaquinoic acid of the present invention is 26 mg of lactose for excipients and 3.5 mg of avicel (microcrystalline cellulose), 1.5 mg of sodium starch glyconate as a disintegration aid, and 8 mg of low-hydrosyprophylcellulose (L-HPC) for binders. The U-type mixer was put together 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

Syrup containing sagaquinoic acid of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient of 2% (W / V) was prepared by the following method: 2 g of acid addition salt of sagaquinoic acid of the present invention, saccharin 0.8 g 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

A capsule was prepared by mixing 50 mg of sagaquinoic acid, 50 mg of lactose, 46.5 mg of starch, 1 mg of talc and 1 mg of magnesium stearate and filling it into a hard gelatin capsule.

<1-4> Preparation of Injection Solution

Injectable solutions containing 10 mg of the active ingredient were prepared in the following manner: 100 g of 1 g of sagaquinoic acid hydrochloride, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved 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. Sagaquinoic acid of the invention was mixed at a concentration of 2% (w / w) to emulsify well in a mixer, degassed, filtered and cooled to prepare a cream composition. A chelating agent, flavoring agent and pigment were added to the composition as an additive and the formulation was prepared in oil / water so that a small amount of oily ingredient was present.

< Application example  1>

psoriasis

Psoriasis is a skin disease in which the skin surface is covered with silvery white scales, the surrounding skin and the border are clear, and red spot scabs of various sizes are formed and gradually increase in size. Psoriasis causes a combination of genetic and environmental factors. The cause is not yet clear, but the etiology varies due to hormonal changes, stress, trauma, infections such as tonsillitis, climate, dry skin, drugs and heredity. Psoriasis occurs because these factors cause skin cells to proliferate six to seven times faster than normal cells. Thus, psoriasis can be treated by killing skin cells (RajD et al., J Invest Dermatol . 2006, Feb, 126 (2), 243-57; Kruger-Krasagakis S. et al., J Dermatol . 2006, 154 (3), 460-6.), Sagaquinoic acid having an activity of killing the skin cells of the present invention is effective in the treatment of psoriasis.

< Application example  2>

Constellation (acne)

Acne is a kind of skin disease called acne vulgaris and cotton cloth, with narrow rashes on the forehead, spleen, nose, and mouth. As it turns into inflammation, it becomes convex in red. It is a skin disease that becomes yellow when bacteria are infected. Thus, apoptosis of keratinocytes has been reported to be associated with the treatment of acne (Nelson AM, et al., J Invest Dermatol . 2006 30: [Epub ahead of print]). Therefore, sagaquinoic acid, which has an activity of killing the skin cells of the present invention, is effective in the treatment of acne vulgaris.

< Application example  3>

Skin aging and Photoaging

Skin aging and photoaging are caused by chronic exposure to ultraviolet rays, which cause harmful oxygen in the human body to form along with the harmful elements of the external environment. This causes permeation of lipid components in the cell membranes and hardening of the cell membranes, resulting in poor supply of oxygen and nutrients to the cells. Occurs accordingly. Due to this cause, the metabolism of the skin and generation of skin cells are delayed, the skin stratum corneum becomes thicker, and the skin undergoes a change in structure and function. This results in a series of skin aging and photoaging phenomena, with many characteristic features such as aging of the skin, loss of elasticity, wrinkle formation, loss of moisture retention, and sagging (Wulf HC. Et al., Micron . 2004, 35, 185-). 91; Haake AR, et al., J Investigative Dermatol Symp Proc . 1998, 3, 28-35). Therefore, the killing of thick skin keratinocytes is effective in order to prevent these skin symptoms, the sagaquinoic acid of the present invention is effective in the treatment of skin aging and photoaging by smoothing the metabolism and generation crossover of skin cells.

< Application example  4>

cutaneous cancer

Skin cancer refers to all cancers that occur 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 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. Squamous cell carcinoma is the second most common malignancy after basal cell carcinoma. 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. The above-mentioned skin cancers have various causes, but are ultimately caused by an imbalance in cell proliferation and apoptosis mechanisms (Bowen AR, et al., J Dermatopathol . 2004 26: 177-81.). Therefore, it is effective in the treatment of skin cancer because it can inhibit the proliferation of cancer cells due to the apoptosis effect of the sagaquinoic acid of the present invention.

As described above, the pharmaceutical composition containing the sagaquinoic acid of the present invention as an active ingredient induces apoptosis in human skin cells, and when treated with ultraviolet light, has an effect of further enhancing apoptosis, resulting in hyperproliferation of cells and It can be usefully used to prevent or treat related skin diseases and skin cancer.

Claims (4)

A pharmaceutical composition for preventing or treating skin diseases or skin cancer containing sagaquinoic acid represented by the following formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient. <Formula I>

The pharmaceutical composition of claim 1, further comprising ultraviolet irradiation. The pharmaceutical composition according to claim 1 or 2, wherein the skin disease is a hyperproliferative skin disease. The method of claim 3, wherein the hyperproliferative skin disease is psoriasis, young, Bowen's disease, papilloma, eczema, addictive eczema, atopic dermatitis, vulgaris, seborrheic dermatitis, lichen planus, glandular line, foot hyperkeratosis, Pharmaceutical composition, characterized in that selected from the group consisting of seborrheic keratosis, actinic keratosis, skin aging and photoaging.

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