KR101415170B1 - Compositions for preventing or treating skin damage by ultraviolet rays - Google Patents

Abstract

The present invention relates to a composition containing a syakicin compound for preventing, ameliorating, or treating skin damage caused by ultraviolet rays. The present invention relates to a composition containing matrix-metalloproteinases (MMPs) Inhibiting the activity of human neutrophil elastase (HNE) inducing skin inflammation and inducing the production of collagen and causing aging of the skin, so as to prevent, ameliorate, or treat skin damage caused by ultraviolet rays And can be usefully used as a composition.

Description

Technical Field [0001] The present invention relates to a composition for preventing and treating skin damage caused by ultraviolet rays (Compositions for preventing or treating skin damage by ultraviolet rays)

The present invention provides a composition for prevention and treatment of skin damage caused by ultraviolet rays containing a syakicin compound as an active ingredient.

Skin damaged by ultraviolet rays causes various changes in structure and function. Among these changes, it is important that the skin becomes thin due to the low level of collagen, elastin and other components of skin connective tissue. Sun rays and oxygen cause free radicals and reactive oxygen species (ROS) in skin cells, causing DNA damage and cell membrane lipid peroxidation. In addition, it affects the expression of matrix metalloproteinase (MMPs) enzyme involved in destroying extracellular matrix such as collagen, damages connective tissue, induces skin inflammation, destroys tissue, and accelerates aging .

Skin aging is a complex biological process that affects skin function and appearance. The tissue associated with the skin includes several types of collagen, elastin, fibronectin, proteoglycan and other extracellular matrix (ECM) proteins, Type 1 collagen is most abundant (Kligman, 1969; Bernstein et < RTI ID = 0.0 > al ., 1994; Rittie and Fisher, 2002).

Aging of human skin due to ultraviolet irradiation is a cumulative process of exposure to sunlight (Imokawa, 208; Imokawa 2009). Quantitative and qualitative changes in the resulting extracellular matrix proteins are related to photo-damage, Which eventually results in skin aging (Imokawa, 2009). Elastin is a structurally important protein of the extracellular matrix and is a major component of elastic fibers that provide elasticity and elasticity to various tissues including the skin, lungs, ligaments, and arterial wall (Wiedow et al ., 1990; Tsukahara et al ., 2006), a decrease in elastin in the skin causes wrinkling (Tsuji et al., 2001).

Human neutrophil elastase (HNE) is a serine protease that is mainly found in azurophil granules of polymorphonuclear leukocytes. It contains elastin, collagen, paribronectin, laminin, , Proteoglycans (Wiedow et al ., 1990; Tsuji et al ., 2001; Tsukahara et al ., 2006) and several connective tissues such as cartilage tissue (Antonicelli et al., 2007).

Biologically, the activity of elastase is known to increase skin aging and reduce skin elasticity (Labat-Robert et < RTI ID = 0.0 > al ., 2000; Tsukahara et al ., 2001; Tzaphlidou, 2004), inhibition of the activity of elastin degrading enzyme can soon inhibit skin aging (Kim et al ., 2009; Xu et al ., 2010).

Matrix metalloproteinases (MMPs) are enzymes that are expressed by irradiation of ultraviolet-B (290-320 nm) and degrade the components of various extracellular matrix proteins. MMPs are subdivided into subgroups such as collagenase, gelatinases, stromelysins, and membrane-type MMPs, and are most commonly found in unstimulated skin cells and healthy tissues , But expression is induced by a variety of extracellular stimuli such as ultraviolet or infrared irradiation, growth factors, cytokines, and cancer promoting factors (Rittie and Fisher, 2002).

The activity of gelatinase (i.e., MMP-2 and MMP-9) is increased by ultraviolet-B irradiation, and suppression of the activity of the gelatinase can inhibit skin epidermal thickness and wrinkle formation (Inomata et al ., 2003; Suganuma et al ., 2010).

MMP-1 is a major enzyme that degrades collagen 1 (Dong et al ., 2008), keratinocytes of epidermis induced and damaged by sunlight exposure (Fagot et al ., 2004) and fibroblasts (Fagot et al., 2002; Dong et al., 2008).

However, the effect on the skin damaged by ultraviolet irradiation of the compound of the Syracycline is not known.

Accordingly, the present inventors have conducted research to develop a composition for treating skin damage caused by ultraviolet rays, and found that a compound of the present invention inhibits the activity of the HNE enzyme and inhibits the expression of MMP-1 and MMP-2 induced by ultraviolet And pro-collagen expression in human dermal fibroblast cells (HDFCss) is increased. Thus, it can be used as a composition for treating skin damage caused by ultraviolet rays, and the present invention has been completed .

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating skin damage caused by ultraviolet rays.

Another object of the present invention is to provide a cosmetic composition for preventing or improving skin damage caused by ultraviolet rays.

It is still another object of the present invention to provide a health food composition for preventing or ameliorating skin damage caused by ultraviolet rays.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for preventing or treating skin damage caused by ultraviolet rays, which comprises, as an active ingredient, a syakicin compound represented by the following formula 1 or a pharmaceutically acceptable salt thereof do:

[Chemical Formula 1]

(In the formula 1,

R ₁ may form a ring of 5 ring in combination with CH ₃ C = O or a neighboring substituent,

R ₂ is H or CH ₃ ,

R < ₃ > is H or OH.

The present invention also provides a cosmetic composition for preventing or ameliorating skin damage caused by ultraviolet rays, which comprises the compound of formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

Furthermore, the present invention provides a health food composition for preventing or ameliorating skin damage caused by ultraviolet rays, which comprises the compound of formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

The compound of the present invention can effectively inhibit the expression of matrix metalloproteinases (MMPs), which cause skin damage by ultraviolet rays, to induce skin inflammation and collagen production, Inhibits the activity of human neutrophil elastase (HNE), and thus can be usefully used as a composition for preventing, ameliorating or treating skin damage caused by ultraviolet rays.

1 is a fragmentary view according to one embodiment of the present invention.
FIG. 2 is a graph showing the extent of inhibition of MMP-1 by Syrian A in accordance with an embodiment of the present invention. FIG.
FIG. 3 is a graph showing the degree of inhibition of MMP-1 by Syriacsin A according to an embodiment of the present invention.
FIG. 4 is a graph showing the extent of inhibition of MMP-1 by Syialcinsin B according to an embodiment of the present invention. FIG.
FIG. 5 is a graph showing the degree of inhibition of MMP-1 by Syakukshin B according to an embodiment of the present invention.
FIG. 6 is a graph showing the degree of inhibition of MMP-2 and the induction of pro-collagen production according to an embodiment of the present invention.
FIG. 7 is a graph showing the survival rate of cells according to the treatment of a syakicin compound according to an embodiment of the present invention. FIG.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating skin damage caused by ultraviolet rays, which comprises, as an active ingredient, a compound of the following formula (1) or a pharmaceutically acceptable salt thereof.

(In the formula 1,

R ₁ may form a ring of 5 ring in combination with CH ₃ C = O or a neighboring substituent,

R ₂ is H or CH ₃ ,

R < ₃ > is H or OH.

Preferably, the syakicin based compound may be selected from the compounds of the following formulas (Ia-Ic).

[Formula 1a]

[Chemical Formula 1b]

[Chemical Formula 1c]

In this case, the compound of formula (1a)

The compound of formula (Ib) is selected from the group consisting of Syriacins B,

The compound of formula (1c) is referred to as Syriacsin C.

In the composition according to the present invention, the above-mentioned syakicin compound may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Dioleate, aromatic acid, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide Sulfonate, methanesulfonate, propanesulfonate, naphthalene-1-sulphonate, naphthalene-1-sulphonate, , Naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention can be produced by a conventional method, for example, by dissolving a syakicin compound in an excess amount of an acid aqueous solution and then using the salt in a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile Followed by precipitation.

Or by heating an acid aqueous solution or an alcohol and then evaporating the mixture or drying the precipitated salt by suction filtration.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the salt of the undissolved compound, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt.

In the composition according to the present invention, the above-mentioned syakicin compound may be isolated from, but not limited to, Hibiscus japonica extract.

As an example, the above-mentioned syakicin compound may be, as shown in Fig. 1,

A step of extracting Mugunghwa muscle with methanol to obtain an extract of Mugungwha (step 1);

The Hibiscus japonica extract obtained in the above Step 1 was suspended in water and then extracted with n-hexane, chloroform, ethyl acetate and n-butanol in the order of n-hexane, chloroform, ethyl acetate and n- (Step 2);

Separating the active fractions by performing silica gel chromatography from the chloroform fraction obtained in step 2 above (step 3); And

(Step 4) of isolating the syakicin compound from the active fraction obtained in step 3 above.

Hereinafter, the production method of the present invention will be described step by step (see FIG. 1).

Step 1 according to the present invention is a step of obtaining a Hibiscus cunea extract, specifically by performing the following steps.

a) extracting an extract of Solanaceae with an extraction solvent;

b) cooling and then filtering the extract of step a); And

c) concentrating the filtered extract of step b) under reduced pressure and drying.

In step 1, the hibiscus in step a) may be used without limitation, such as cultivated or marketed. The above-mentioned Hibiscus can be used for the entire plant, but it is preferable to use the root, but not limited thereto.

The extraction solvent may be water, alcohol or a mixture thereof. As the alcohol, C ₁ to C ₄ lower alcohol is preferably used, and ethanol or methanol is more preferably used, and methanol is most preferably used, but not limited thereto. Examples of the extraction method include filtration, hot water extraction, immersion extraction, reflux cooling extraction, ultrasonic extraction, and the like, and extraction is preferably performed once to five times by hot water extraction, But is not limited thereto. The extraction solvent may be added in an amount of 1 to 10 times, preferably 1 to 5 times, to the stem weight of the dried Hibiscus. The extraction temperature is preferably 20 to 30, but is not limited thereto. The extraction time is preferably from 24 to 72 hours, but is not limited thereto.

In step 1, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in step c), but the present invention is not limited thereto. The drying is preferably performed under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, but not always limited thereto.

Step 2 according to the present invention is a step of obtaining n-hexane, chloroform, ethyl acetate and butanol fractions of cervus chestnut tree by sequential fractionation of the extracts of Hibiscus japonica obtained in Step 1 with n-hexane, chloroform, ethyl acetate and butanol.

In one embodiment according to the present invention, water and n-hexane are added to the extract of cow's milk alcohols, for example, methanol extract prepared in step 1, and the water layer and the organic layer are separated using a separatory funnel , And the organic layer fraction is concentrated under reduced pressure using a vacuum distillation apparatus to obtain a n-hexane fraction.

Thereafter, chloroform was added to the water layer, the organic layer was separated into a water layer and an organic layer using a separatory funnel, and the obtained organic layer fraction was concentrated under reduced pressure using a vacuum distillation apparatus to obtain a chloroform fraction. Ethyl acetate was added to the water layer The organic layer was separated into a water layer and an organic layer using a separatory funnel, and the obtained organic layer fraction was concentrated under reduced pressure using a vacuum distillation column to obtain an ethyl acetate fraction. Butanol was again added to the water layer, , And the organic layer fraction obtained is concentrated under reduced pressure using a vacuum distiller to obtain a butanol fraction.

Step 3 according to the present invention is a step of isolating the active fractions by performing silica column chromatography on the choline chloroform fraction obtained in step 2 above.

In one embodiment of the present invention, the chloroform fraction of the fractions obtained from step 2 is subjected to silica gel column chromatography using a mixed solvent of n-hexane: EtOAc (10: 1 to 1: 1) as an elution solvent And 10 fractions (A to J) were separated, and fractions A and B were used as active fractions in the next step.

Step 4 according to the present invention is a step of isolating a syakicin compound from the active fraction obtained in step 3 above.

Specifically, the above Syriacin A was subjected to silica gel column chromatography using the mixed solvent of n-hexane: EtOAc (10: 1 to 1: 1) as an elution solvent, and then chloroform: Sepaphase LH-20 column chromatography using a mixed solvent of methanol (1: 1) as an elution solvent and performing prepolymerization on an ODS column (21 x 250 mm) using 70% methanol as an elution solvent. HPLC. ≪ / RTI >

The active fraction B obtained in step 3 was subjected to silica gel column chromatography using a mixed solvent of chloroform: methanol (100: 1 to 1: 1) as an elution solvent, and then 100% methanol was eluted Followed by LH-20 column chromatography as a solvent. At this time, in the course of obtaining the present Syriacusin C, Syriacsin C and the active fraction B2 were separated. The active fraction B2 was used to obtain Syriacsin B.

Further, the active fraction B2 isolated in step 4 of Syriacsin B was dissolved in Prep. Bromoacetate using a mixed solvent of chloroform: methanol (10: 1) as an elution solvent. Silica gel TLC.

The inventors of the present invention found that, in order to examine the effect of improving the skin aging caused by ultraviolet rays of the Syrian quinic acid compound, the present inventors accelerated skin aging by decomposing extracellular matrix proteins (ECM) such as elastin, collagen, fibronectin, laminin, proteoglucan, (Human neutrophil elastase (HNE)) was measured. As a result, it was confirmed that the syakicin compound showed an effect of inhibiting HNE activity (see Table 2).

The present inventors have also found that inhibition of matrix metalloproteinase (MMPs) enzyme activity, which degrades various extracellular components (collagen, gelatin, etc.), in order to examine the effect of treating the skin damage by ultraviolet Respectively. As a result, the Syriacucin-based compound effectively inhibited the expression of MMP-1 and MMP-2 expressed by ultraviolet-B (290-320 nm) irradiation in human dermal fibroblast cells (HDFCs) -Collagen (see Figs. 2, 3, 4, 5 and 6).

Further, the present inventors confirmed the viability of human skin fibroblasts (HDFCs) using tetrazolium discoloration test to examine the cytotoxicity of the syakicin compound. As a result, it was confirmed that the survival rate of human skin fibroblasts (HDFCs) treated with the Syrian nucleic acid compound was more than 90%, and that the survival rate of the cells by the Syakuccine compound was not significantly changed 7).

Accordingly, the compound of the present invention can be used as an effective ingredient of a pharmaceutical composition for preventing or treating skin damage caused by ultraviolet rays.

In the present invention, skin damage caused by ultraviolet rays includes skin inflammation, erythema, keratinocytosis, sunburn or skin aging caused by damage of epidermal tissue or connective tissue of skin.

The present invention provides a pharmaceutical composition for preventing or treating skin damage caused by ultraviolet rays, which comprises the compound of formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

The pharmaceutical composition comprising the above-mentioned syakicin compound of the present invention preferably contains 0.1 to 50% by weight of the composition based on the total weight of the composition, but is not limited thereto.

The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of medicaments.

The pharmaceutical composition according to the present invention can be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like oral preparation, external preparation, suppository and sterilized injection solution according to a conventional method Can be used. Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may be formulated into the compositions of the present invention with at least one excipient such as starch, calcium carbonate, (sucrose), lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol gelatin and the like can be used.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and any parenteral administration method can be used, and systemic administration or topical administration is possible, but systemic administration is more preferable, intravenous administration is most preferable .

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the pharmaceutical composition of the present invention is preferably administered at 0.0001 to 0.03 g / kg per day, preferably 0.001 to 8 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

The present invention also provides a cosmetic composition for preventing or ameliorating skin damage caused by ultraviolet rays, which comprises the compound of formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

Functional cosmetics according to the present invention include, but are not limited to, lotions, skins, gels, creams, patches or sprays. In the production of the functional cosmetic composition containing the above-mentioned syakicin compound of the present invention, 0.1 to 10.0% by weight of the syakicin compound of the present invention may be added to the cosmetic composition usually contained. The cosmetic composition may contain, in addition to the compound of the present invention, a fatty substance, an organic solvent, a solubilizer, a thickening agent and a gelling agent, a softening agent, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, , Ionic or nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, barrier agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or cosmetics And may contain adjuvants conventionally used in the field of dermatology, such as any other ingredient. The components can also be introduced in amounts commonly used in the field of dermatology.

Furthermore, the present invention provides a health food composition for preventing or ameliorating skin damage caused by ultraviolet rays, which comprises the compound of formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

The composition according to the present invention can be added to health supplements such as foods, beverages and the like for the purpose of preventing or improving skin damage caused by ultraviolet rays.

There is no particular limitation on the kind of the food. Examples of the foods to which the composition can be added include dairy products including dairy products such as drinks, meat, sausage, bread, biscuits, rice cakes, candies, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, Beverages, alcoholic beverages and vitamin complexes, dairy products, and dairy products, all of which include health functional foods in a conventional sense.

The composition of the present invention can be added directly to food or used together with other food or food ingredients, and can be suitably used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement). Generally, the amount of the composition in the health food may be 0.1 to 90 parts by weight of the total food. However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

The health functional beverage composition of the present invention has no particular limitation on the other ingredients except that it contains the above-mentioned composition as an essential ingredient in the indicated ratios and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 g of the composition of the present invention.

In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks.

These components may be used independently or in combination. The ratio of such additives is not so important, but is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited thereto.

< Example  1> Preparation of Mugungwha tree extract

As shown in Fig. 1, the syakicin compound represented by formula (1) according to the present invention was prepared by the following steps.

Step 1: Preparation of the extract

1.6 kg of shredded mulberry tree root was cut into pieces and 20 ℓ of methanol was added to the prepared mulberry tree root. The extract was extracted at room temperature for 48 hours and filtered twice. The extract was concentrated using a vacuum distiller 850 g of a methanol extract of Corynebacterium glutinosa was obtained.

Step 2: Fraction  Produce

2 L of water and 2 L of n-hexane were added to the oilseed rape seed oil methanol extract prepared in step 1, and the organic layer was separated into a water layer and an organic layer using a separatory funnel. The organic layer fractions were concentrated under reduced pressure Concentration yielded 210 g of n-hexane fraction.

Further, 2 liters of chloroform (CHCl ₃ ) was added to the water layer, and the water layer and the organic layer were separated using a separatory funnel. The obtained organic layer fraction was concentrated under reduced pressure using a vacuum distillation column to obtain 184 g of a chloroform fraction.

Further, 2 liters of ethyl acetate was added to the water layer, and the organic layer was separated into a water layer and an organic layer using a separatory funnel. The obtained organic layer fraction was concentrated under reduced pressure using a vacuum distillation apparatus to obtain 115 g of an ethyl acetate fraction.

Further, 2 liters of n-butanol was added to the water layer, and the organic layer was separated into a water layer and an organic layer using a separatory funnel. The obtained organic layer fraction was concentrated under reduced pressure using a vacuum distiller to obtain 225 g of n-butanol fraction.

Among the above fractions, human neutrophil elastase (HNE) inhibitory effect assay showed that the chloroform fraction had the best HNE inhibitory effect.

Step 3: Active Fraction  Manufacturing 1

Of the fractions obtained in step 2, 184 g of the chloroform fraction having the most excellent HNE inhibitory effect was loaded on a column of 7 x 57 cm in diameter filled with silica of 70-230 mesh, and n-hexane and ethyl Acetate was carried out by silica gel column chromatography with a concentration gradient of from 10: 1 to 1: 1 (v / v). In addition, the column chromatography was carried out, and the active fractions A and B were separated from each other while observing TLC (thin layer chromatography) analysis and HNE inhibitory activity, and used in the next step.

Step 4-1: Preparation of Syriacsin A

312 mg of the active fraction A obtained in the above step 3 was subjected to the same procedure as in the silica gel column chromatography performed in step 3, and then loaded on a column packed with sephadex LH-20 gel, and the volume fraction of chloroform and methanol Column chromatography using a mixed solvent of 1: 1 as elution solvent. Finally, a column having a diameter of 20 mm × 250 mm packed with Senshu pak ODS (manufactured by YMC, RP-18, Japan) (Waters 515 pump, 2,996 photodiode array detector, USA) under the condition of 8 ml / min using 70% methanol aqueous solution as elution solvent to obtain 35.5 mg of purified Syriacusin A.

Step 4-2: Syriacusin  Manufacture of C

The active fraction B (286 mg) obtained in the above step 3 was diluted with a silica gel of step 3 above, except that a mixed solvent of chloroform and methanol having a concentration gradient of from 100: 1 to 1: 1 (v / v) The column chromatography was carried out in the same manner as in column chromatography, and then loaded on a column packed with sephadex LH-20 gel and subjected to column chromatography using 100% methanol as an elution solvent to obtain 3 mg of purified Syuaxin C. At this time, in this step, Syriacsin C and the active fraction B2 were separated, and the active fraction B2 was used in the next step to obtain Syriacsin B.

Step 4-3: Syriacusin  Manufacture of B

22.5 mg of the active fraction B2 obtained in the above Step 4-2 was subjected to a silica gel prep TLC using a mixed solvent of a chloroform-methanol elution solvent ratio of 10: 1 to obtain 3.5 mg of purified Syriacsin B .

<Analysis> Identification of Syrian Kusin A, B and C

For the structural analysis of the active fractions A, B1 and B2 obtained in Example 1, NMR analysis and mass spectrometry were carried out. Molecular weight and molecular formula were determined by HREI-MS measurement using JEOL's JMS-SX 102A and 1D ( ¹ H, ¹³ C, DEPT) and 2D ( ¹ H- ¹ H COSY, ¹ H- ¹³ C HMQC, ¹ H- ¹³ C HMBC) NMR spectral analysis of the structure revealed that the structure was identified by the chemical formulas 1A, 1B and 1C in Table 1 below. The results of the analysis are shown in Table 1 below.

rescue Spectroscopic analysis data

[Formula 1a]

HREI-MS 233.0808 [M + H] &lt; ⁺ &gt; ¹ H NMR




H-5), 2.29 (6-CH ₃ ), 9.66 (7-OH), and 7.93 3.67 (8-OCH ₃₎ ¹³ C NMR




(C-1), 199.2 (1-CHO), 166.3 (C-2), 116.4 (C-3), 140.0 , 126.3 (C-6), 16.3 (6-CH 3), 151.2 (C-7), 141.7 (C-8), 59.5 (8-OCH 3), 125.9 (C-8a)

&Lt; RTI ID = 0.0 &

HREI-MS 263.0915 [M + H] &lt; ⁺ &gt; ¹ H NMR




H-5), 4.84 (6-CH ₃ ), 4.09 (7-OCH ₃ ), 7,10 (2-OH) , 3.92 (8-OCH ₃₎ ¹³ C NMR




(C-1), 198.6 (1-CHO), 166.5 (C-2), 118.9 (C-6), 61.4 ( 6-CH 2), 148.0 (C-8), 59.7 (8-OCH 3), 127.3 (C-8a)

[Chemical Formula 1C]

HREI-MS 231.0638 [M + H] &lt; ⁺ &gt; ¹ H NMR




7.05 (H-3), 7.86 (H-4), 7.36 (H-5), 2.37 (6-CH 3), 4.31 (7-OCH 3) ¹³ C NMR




119.2 (C-3), 134.8 (C-4), 100.3 (C-4a), 122.4 (C-5), 129.9 (C-6), 17.9 ( 6-CH 3), 140.2 (C-7), 59.9 (7-OCH 3), 131.6 (C-8), 131.7 (C-8a)

As shown in Table 1, the active fraction A was found to be Syriacsin A, the active fraction B1 was Syriacsin C, and the active fraction B2 was Syriacsin B, as shown in Table 1.

< Experimental Example  1 > human neutrality elastase ( HNE )

Human neutrophil elastase (HNE) is an enzyme involved in skin aging that degrades extracellular matrix proteins (ECM) such as elastin, collagen, fibronectin, laminin, proteoglucan and the like. In Experimental Example 1, the following experiments were carried out to examine the effect of the Syakaksin A, B and C prepared in Example 1 on HNE inhibition.

First, 40 μl of substrate solution [1.4 mM N-methoxysuccinyl-ala-ala-pro-val- p- nitroanilide in 10 mM Tris-HCl buffer (pH 7.5)] and 10 μl of enzyme solution EGCG (epigallocatechin-3-gallate) was dissolved in dimethylsulfoxide (DMSO) solution and diluted with Tris-HCl expansion solution to prepare 1, 3, 10 and 30 μM, and 50 μl of the mixture was added to each well. The cells were incubated at 37 ° C. for 1 hour in a shaker cell incubator, and then 100 μl of 0.2 μg / ml soybean trypsin inhibitor was added to terminate the reaction Respectively. The reacted samples were measured for absorbance at a wavelength of 405 nm using an ELISA reader (Model: VersaMax, manufacturer: Molecular devices, USA), and the HNE inhibitory activity was calculated by the following equation.

Ab: absorbance of control group

As: Absorbance of the sample

The HNE inhibitory activities of the Syriacsin A, B and C calculated by the above formula are shown in Table 2 below.

HNE inhibitory activity (%) according to compound concentration IC ₅₀ ([mu] M)
compound 1 [mu] M 3 [mu] M 10 μM 30 μM Syrian Kushin A 19.7 ± 1.6 25.7 ± 1.7 51.9 ± 1.1 76.6 ± 0.7 8.0 Syriacusin B 15.3 ± 1.8 30.1 ± 2.1 86.3 ± 0.6 93.3 ± 1.2 5.2 Syriacusin C 16.5 ± 2.2 22.9 ± 1.0 74.4 ± 1.2 89.6 ± 1.0 6.1 Control group: EGCG 45.3 ± 1.6 63.0 ± 0.5 75.8 ± 0.8 83.2 ± 1.2 1.1

As shown in Table 2, it was confirmed that the Syriacins A, B and C prepared in Example 1 are effective as a skin aging inhibitor which inhibits decomposition of elastin.

< Experimental Example  2> Matrix Of metalloprotease (MMPs)  Expression inhibition measurement

Matrix metalloproteases (MMPs) are enzymes that are expressed by ultraviolet irradiation and decompose various extracellular components (collagen, gelatin, etc.) and are associated with skin aging classified into subgroups such as MMP-1 and MMP-2 It is an enzyme. In Experimental Example 2, the following experiments were carried out to examine the effect of Syriacsin A and B produced in Example 1 on the expression of MMPs in human dermal fibroblast cells (HDFCs).

Human dermal fibroblast cells (HDFCs) (CRL-2076) were purchased from the American Type Culture Collection. The HDFCs is a 6-well culture plates with 100 U / ㎖ penicillin A, 100 U / ㎖ streptomycin to:: (Gibco DMEM, manufacturer) 5% CO _2, 37 ℃ conditions (manufacturer: BD Falcon) to capture cell incubator And cultured for 24 hours. HDFCs cultured for one day were washed twice with PBS (phosphate-buffered saline) solution and irradiated with UV-B (290-320 nm) at 10 mJ / cm ² intensity. A Syrian kusin A and B produced after UV irradiation, washed once again with just PBS solution in Example 1 to 0, 1, 2, 5, 10, in the cell culture medium was added to 20 μM concentration of 5% CO _2, 37 ℃ The samples were cultured for 48 hours.

The sample prepared above was dissolved in a lysis buffer (0.5 v / v% Nonidet P-40 in 20 mM Tris-HCl (pH 8.3); 150 mM NaCl; protease inhibitors (2 ug / ml aprotinin, pepstatin, and chymostatin); 1 [mu] g / ml leupeptin and pepstatin; 1 mM phenylmethylsulfonyl fluoride (PMSF); and 1 mM Na ₄ VO ₃ ], and then cultured at 4 ° C for 30 minutes to obtain total mononuclear cells. Next, the treated sample was centrifuged at 4,000C for 12 minutes at 12,000 gravity acceleration (g) for 5 minutes, and the supernatant was used as a sample of HDFCs induced MMP protein expression.

MMP -1 of Expression level  And pro-collagen synthesis

The HDFCs samples prepared above were placed in sodium dodecyl sulfate (SDS) sample buffer and denatured by heating for 5 minutes. Total protein was separated by size with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS_PAGE) and transferred to a nitrocellulose membrane (manufacturer: Amersham international). Next, the nitrocellulose membrane was coated with a monoclonal anti-MMP-1 antibody (manufactured by Calbiochem) diluted 1: 1000 and polyclonal anti-pro-collagen diluted with 1: 500 Tris-buffered saline and Tween 20 (TBST) containing 5% defatted milk were mixed with Type-1 antibody (manufacturer: Santa Cruz Biotechnology Inc.) 10 mM Tris-HCl, pH 7.6; 150 mM NaCl; 0.5% Tween 20] for 1 hour. At this time, the antibody was used as a secondary antibody, which was cultured together with HRP-conjugated anti-rat IgG antibody diluted at 1: 1000 and anti-goat IgG antibody diluted at 1: 500. The amount of expression of MMP-1 and pro-collagen protein was quantitated by a concentration measurement program (TINA) using an ECL system (manufacturer: Amersham international).

MMP -2 of Expression level  Measure

15 μl of the HDFCs prepared above was placed in a Tris-glycine SDS sample buffer (2 ×) and mixed. Then, 25 μl of the sample was loaded on 10% SDS-PAGE and subjected to electrophoresis. After electrophoresis, the SDS gel was incubated with 1 × zymogram regeneration buffer (manufactured by Invitrogen, USA) at room temperature for 30 minutes. Next, the 1 × eosinophilization buffer (manufacturer: Invitrogen, USA) was added and developed for 30 minutes, and then exchanged for a new 1 × eosinophilization buffer. The SDS gel thus treated was incubated at 37 ° C. for one day and stained with a 0.05% staining reagent (Commassie Brilliant Blue solution). Then, 10% acetic acid and 40% stearic acid were added until the protein bands were clearly visible on the blue background. The stain was removed with a stain removal reagent containing methanol.

The experimental results are shown in Figs. 2, 3, 4, 5 and 6.

FIG. 2 is a graph showing the extent of inhibition of MMP-1 by Syrian A in accordance with an embodiment of the present invention. FIG.

FIG. 3 is a graph showing the degree of inhibition of MMP-1 by Syriacsin A according to an embodiment of the present invention.

FIG. 4 is a graph showing the extent of inhibition of MMP-1 by Syialcinsin B according to an embodiment of the present invention. FIG.

FIG. 5 is a graph showing the degree of inhibition of MMP-1 by Syakukshin B according to an embodiment of the present invention.

FIG. 6 is a graph showing the degree of inhibition of MMP-2 and the induction of pro-collagen production according to an embodiment of the present invention.

As shown in Figs. 2, 3, 4 and 5, Syriacsin A and B were shown to inhibit the expression of MMP-1 in a concentration-dependent manner. Specifically, about 50% of MMP-1 was expressed in HDFCs containing 10 μM of Syakuccine A and only about 20% of MMP-1 was expressed in 20 μM of HDFCs. In addition, it was confirmed that about 60% of MMP-1 was expressed in a sample of HDFCs to which 20 μM of Syiacin B was added. Thus, it can be seen that the syakicin-based compound effectively inhibits the expression of MMP-1 expressed by ultraviolet irradiation.

Further, as shown in Fig. 6, ultraviolet irradiation hinders the synthesis of type-1 pro-collagen and induces the expression of MMP-2. However, Syriacusin A inhibits MMP-2 expression in a concentration-dependent manner, and the synthesis of pro-collagen is markedly increased. In particular, the expression of MMP-2 was significantly reduced in the HDFCs sample to which 20 μM of Syriacsin A was added.

Accordingly, the present invention relates to a compound capable of inhibiting the expression of MMP-1 and MMP-2 expressed by ultraviolet irradiation in human skin fibroblasts (HDFCss) and inducing the synthesis of pro-collagen, Can be usefully used as a composition for inhibiting skin damage caused by the skin.

< Experimental Example  3> Cytotoxicity test

Survival of human skin fibroblasts (HDFCss) was confirmed using tetrazolium discoloration assay (Seaot and Lang, 1986) in order to examine cytotoxicity of the syakicin compound prepared in Example 1.

First, human dermal fibroblast cells (HDFCs) (CRL-2076) were purchased from the American Type Culture Collection. The HDFCs is 100 U / ㎖ A penicillin and 100 U / ㎖ 6- well culture plate with streptomycin (manufacturer: BD Falcon) to capture cell incubator of 5% CO _2, 37 ℃ conditions: in (DMEM, Gibco manufacturer) UV-B at 0, 10, 20 and 30 mJ / cm &lt; ² &gt; After culturing in the intensities, Syriacsin A and B of Example 1 were treated at 0, 3, 10 and 30 μM concentration and cultured for 48 hours. Next, MTT reagent (tetrazolium dye, 2- (4,5-dimethylthiazol-2-yl) -2,5-di-phenyl-tetrazolium bromide) was added and incubated for 3 hours. After incubation, the supernatant was removed and 150 μl of DMSO (dimethyl sulfoxide) was added. When the MTT reagent was added to the cells, the viable cells were developed. The viability (%) of the cells was measured by measuring the absorbance at 570 nm using a microplate reader. The results are shown in Table 3 and FIG.

Survival of cells after treatment with UV-B (10 mJ / cm ² ) extract
Control (μM) Syriacsin A (mu M) Syriacsin B (μM) 0 3 10 30 3 10 30 Cell viability (%) 100 103 101 94 101 106 103

FIG. 7 is a graph showing the survival rate of cells according to the treatment of a syakicin compound according to an embodiment of the present invention. FIG.

As shown in Table 3 and FIG. 7, the survival rate of human skin fibroblasts (HDFCs) treated with the Syrian nucleic acid compound prepared in Example 1 was more than 90%, and the survival rate of the cells by the Syakuccine- And that there was no significant change in

Therefore, the compound of the present invention can be used as a composition for treating skin damage caused by ultraviolet rays.

&Lt; Formulation Example 1 > Preparation of pharmaceutical preparation

1-1. Manufacture of Powder

500 mg of the compound of formula (1)

Lactose 100 mg

10 mg of talc

The above components are mixed and filled in airtight bags to prepare powders.

1-2. Manufacture of tablets

500 mg of the compound of formula (1)

Corn starch 100 mg

Lactose 100 mg

2 mg of magnesium stearate

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

1-3. Manufacture of capsules

500 mg of the compound of formula (1)

Corn starch 100 mg

Lactose 100 mg

2 mg of magnesium stearate

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

1-4. Injection preparation

500 mg of the compound of formula (1)

Sterile sterilized water for injection

pH adjuster

(2 ml) per ampoule in accordance with the usual injection method.

1-5. Manufacture of liquid agent

100 mg of the compound of formula (1)

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, The liquid is prepared by sterilization.

< Formulation example  2> Manufacture of cosmetics

2-1. Production of flexible lotion

Examples of preparations of a soft lotion containing an active ingredient of a syakicin compound are shown in Table 4 below.

Raw material Content (parts by weight) Syuakucine-based compounds of formula (1) 10.00 1,3-butylene glycol 1.00 Disodium iodide 0.05 Allantoin 0.10 Dipotassium glycyrrhizate 0.05 Citric acid 0.01 Sodium citrate 0.02 Glycereth-26 1.00 Arbutin 2.00 Hydrogenated castor oil 1.00 ethanol 30.00 Preservative a very small amount coloring agent a very small amount Flavoring agent a very small amount Purified water Balance

2-2. Manufacture of nutrition cream

Formulation examples of nutritional creams containing a syacycinic compound were prepared according to the composition shown in Table 5 below.

Raw material Content (parts by weight) Syuakucine-based compounds of formula (1) 10.0 1,3-butylene glycol 7.0 glycerin 1.0 D-Panthenol 0.1 Plant extract 3.2 Magnesium aluminum silicate 0.3 PEG-40 stearate 1.2 Stearic acid 2.0 Polysorbate 60 1.5 Chin type glyceryl stearate 2.0 Sorbitan sesquioleate 1.5 Cetearyl alcohol 3.0 Mineral oil 4.0 Squalane 3.8 Carlyric / capric triglyceride 2.8 vegetable oil 1.8 Dimethicone 0.4 Dipotassium glycyrrhizate a very small amount Allantoin a very small amount Sodium hyaruronate a very small amount Tocopheryl acetate Suitable amount Triethanolamine Suitable amount Preservative Suitable amount Flavoring agent Suitable amount Purified water Balance

&Lt; Formulation Example 3 > Preparation of health food

1000 mg of the compound of formula (1)

Vitamin mixture dosage mg

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

0.13 mg of vitamin

Vitamin B₂ 0.15 mg

Vitamin B₆ 0.5 mg

Vitamin B₁₂ 0.2 [mu] g

10 mg vitamin C

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 mg of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

24.8 mg of magnesium chloride

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

&Lt; Formulation Example 4 > Preparation of health drink

1000 mg of the compound of formula (1)

Citric acid 1000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added to a total of 900 ml

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 DEG C for about 1 hour. Then, the resulting solution was filtered to obtain a sterilized 2 L container, sealed sterilized, Were used to prepare beverage compositions.

Although the composition ratio is relatively mixed with the ingredient suitable for the favorite drink, it is also possible to arbitrarily modify the blending ratio according to the regional or national preference such as the demand class, demand country, use purpose, and the like.

&Lt; Formulation Example 5 > Production of other health foods

5-1. Manufacturing of beverages

Honey 522 mg

5 mg &lt; RTI ID = 0.0 &gt;

Nicotinic acid amide 10 mg

3 mg of sodium riboflavin hydrochloride

Pyridoxine hydrochloride 2 mg

Inositol 30 mg

Orthoic acid 50 mg

0.48 to 1.28 mg of the compound represented by formula (1)

200 ml of water

A beverage was prepared using the above-mentioned composition and content by a conventional method.

5-2. Of chewing gum  Produce

Gum base 20%

Sugar 76.36 ~ 76.76%

0.24 to 0.64% of the syndiotactic compound of formula (1)

Fruit flavor 1%

Water 2%

Chewing gum was prepared using the above-mentioned composition and content by a conventional method.

5-3. Manufacture of candy

Sugar 50 to 60%

Syrup 39.26 ~ 49.66%

0.24 to 0.64% of the syndiotactic compound of formula (1)

Orange fragrance 0.1%

The composition and the content of the candy were prepared using a conventional method.

5-4. Manufacture of flour food products

0.5 to 5 parts by weight of the compound of formula (1) was added to 100 parts by weight of wheat flour, and bread, cake, cookies, crackers and noodles were prepared by using this mixture to prepare health improving foods.

5-5. dairy product( dairy products )

5 to 10 parts by weight of the compound of formula (1) is added to 100 parts by weight of milk, and various dairy products such as butter and ice cream are prepared using the milk.

5-6. Solar  Produce

Brown rice, barley, glutinous rice, and yulmu were alphalized by a known method and dried, and the powder was made into a powder having a particle size of 60 mesh by a pulverizer. Black beans, black sesame seeds, and perilla seeds were steamed and dried by a known method, and then powdered with a particle size of 60 meshes by a grinder. The grains and seeds prepared above were mixed with the Syriacic acid compound of formula (1) in the following proportions.

Brown rice 30%

Yulmu 15%

Barley 20%

Perilla 7%

Black soybeans 7%

Black sesame 7%

3% &lt; / RTI &gt; of the &lt; RTI ID =

Manure 0.5%

0.5%

Claims (9)

A pharmaceutical composition for improving skin wrinkles containing an effective amount of a compound of any one of the following formulas (1) to (1c) or a pharmaceutically acceptable salt thereof:
[Formula 1a]

[Chemical Formula 1b]

[Chemical Formula 1c]

.
The pharmaceutical composition for improving skin wrinkles according to claim 1, wherein the skin wrinkles are caused by ultraviolet rays.
delete A cosmetic composition for improving skin wrinkles containing an effective amount of a compound of the following formula (I):
[Formula 1a]

[Chemical Formula 1b]

[Chemical Formula 1c]

.
The cosmetic composition for improving skin wrinkles according to claim 4, wherein the skin wrinkles are caused by ultraviolet rays.
delete A health food composition for improving skin wrinkles containing an effective amount of a compound of any one of the following formulas (1) to (1c) or a pharmaceutically acceptable salt thereof:
[Formula 1a]

[Chemical Formula 1b]

[Chemical Formula 1c]

.
The health food composition according to claim 7, wherein the skin wrinkles are caused by ultraviolet rays.
delete

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