KR102008814B1 - Composition for skin cell regeneration, anti-wrinkle, antioxidant, anti-imflamation, and skin whitening - Google Patents

Abstract

The present invention relates to a composition for skin regeneration, anti-wrinkle, antioxidant, anti-inflammatory and skin whitening, more specifically 4 ', 5,7-trihydroxy-3', 6-dimethoxyflavone according to the present invention Pharmaceutical composition by promoting collagen synthesis of skin fibroblasts, inhibiting skin regeneration, wrinkle improvement, inhibiting free radical production, antioxidant effect, NO production, anti-inflammatory effect and melanin synthesis It can be used as external skin preparation, cosmetic composition or health food.

Description

Composition for skin cell regeneration, anti-wrinkle, antioxidant, anti-imflamation, and skin whitening}

The present invention relates to a composition for skin regeneration, wrinkle improvement, antioxidant, anti-inflammatory and skin whitening.

Collagen is a major substrate protein produced by skin fibroblasts and is present in extracellular epilepsy.Its important functions include mechanical firmness of skin, resistance of connective tissue and tissue binding, support of cell adhesion, cell division and differentiation. Induction of growth or wound healing) is known. This collagen is reduced by age and light aging by ultraviolet irradiation, which is known to be closely related to the wrinkle formation of the skin. In addition, in recent years, extensive research on skin aging has revealed an important function of collagen in skin.

Active ingredients are known that promote collagen synthesis and exhibit wrinkle improvement. For example, retinoic acid, transforming growth factor (TGF) [non-patent document 1], protein derived from animal placenta [patent document 1], betulinic acid (betulinic acid) [patent document 2], chlorella extract [ Patent Documents 3 and 4 are known as collagen synthesis promoting substances. However, the active ingredients are limited in the amount of use due to safety problems such as irritation and redness when applying the skin, or there is a problem in that the effect of improving the skin function is not expected by substantially promoting collagen synthesis of the skin.

On the other hand, free radicals introduced from outside the living body or generated in the living body cause many problems such as promoting aging of the living body or generating cancer. Therefore, many researches and developments on antioxidants that inhibit oxidation by active oxygen have been made. Antioxidants are widely distributed in copper and plant systems, and many phenolic compounds, flavonoids, tocopherols, vitamin C, and selenium are known in fruits and vegetables. However, the antioxidants present in nature are not expected to have a substantial enough effect on skin application. Therefore, many synthetic antioxidants having excellent antioxidant power and low cost are used, but their use is limited due to safety concerns such as human side effects.

In addition, inflammation is an immune response of the human body in response to a wound or disease, and oxidative stress such as ultraviolet rays, free radicals, and free radicals activates inflammatory factors to cause various diseases and aging of the skin. Vascular activating polypeptides such as kinin, plasmin, and complement act on vasodilation, contraction and chemotaxis, as well as lymphokines such as interleukin-6 (IL-6). Phosphorus and arachidonic acid are responsible for the inflammatory response. Arachidonic acid is metabolized to prostaglandin and leukotriene, which are inflammatory mediators, through two pathways, cyclooxygenase or lipooxygenase, to mediate various inflammatory reactions.

On the other hand, the anti-inflammatory agent that acts to remove the inflammatory source, reduce the biological response and symptoms to eliminate inflammation. Materials used for anti-inflammatory purposes to date include nonsteroidal flufenamic acid, ibuprofen, benzydamine, indomethacin, and indonethacin, and prednisolone as a steroid system. , Dexamethasone and the like. In addition, allantoin, azene, hydrocortisone, etc. are known to be effective in anti-inflammatory, but these substances have a limited amount of use as a matter of safety to the skin, or a problem that can not be expected to substantially reduce the effect of the inflammation. There is this.

Also, to have white and fair skin is everyone's constant desire. It is genetically determined by the concentration and distribution of melanin in human skin, but is also influenced by environmental or physiological conditions such as sun ultraviolet rays, fatigue and stress. Melanin is a type of amino acid tyrosine (tyrosine) acts as an enzyme called tyrosinase (tyrosinase) is converted into dopa (DOPA), dopaquinone (dopaquinone) is produced through a non-enzymatic oxidation reaction. The pathway by which melanin is produced is known, but the mechanisms that induce melanin synthesis, the previous stages of tyrosinase action, are still unknown.

On the other hand, as a known whitening component, substances that inhibit tyrosinase enzyme activity such as kojic acid, arbutin, hydroquinone, L-ascorbic acid and derivatives thereof, and There are various plant extracts. By inhibiting the synthesis of melanin pigments, they not only brighten the skin tone to realize skin whitening, but also improve skin hyperpigmentation such as blemishes and freckles due to ultraviolet rays, hormones or heredity. However, when the skin is applied, there are limitations on the amount of use due to safety problems such as irritation and redness, or there is a problem in that a substantial effect cannot be expected due to a slight effect.

Therefore, it is safe for the living body, the active ingredient is stable, and above all, the skin regeneration, anti-wrinkle, antioxidant, anti-inflammatory and whitening activity is superior to the existing skin rejuvenation, wrinkle improvement, antioxidant, anti-inflammatory and whitening substances. There is an urgent need for the development of these components.

Japanese Patent Laid-Open No. 8-231370 Japanese Patent Laid-Open No. 8-208424 Japanese Patent Laid-Open No. 9-40523 Japanese Patent Laid-Open No. 10-36283

 Cardinale G. et al, Adv. Enzymol., 41, p. 425, 1974

Thus, the present inventors have found that 4 ', 5,7-trihydroxy-3', 6-dimethoxyflavone (4 ', 5,7-Trihydroxy-3', 6-dimethoxyflavone) is used to inhibit collagen synthesis of fibroblasts of the skin. Promotes skin regeneration, improves wrinkles by inhibiting collagenase activity, suppresses free radicals, inhibits antioxidant activity, produces NO and anti-inflammatory effects, inhibits melanin synthesis, whitening effect It confirmed that what was shown and completed this invention.

Accordingly, it is an object of the present invention to provide the use of 4 ', 5,7-trihydroxy-3', 6-dimethoxyflavones for skin regeneration, wrinkle improvement, antioxidant, anti-inflammatory or skin whitening.

As a means for solving the above problems, the present invention provides a pharmaceutical composition for skin regeneration, wrinkle improvement, antioxidant, anti-inflammatory and skin whitening comprising a compound represented by the following formula (1) as an active ingredient:

[Formula 1]

As another means for solving the above problems, the present invention provides a skin external preparation for skin regeneration, wrinkle improvement, antioxidant, anti-inflammatory and skin whitening comprising the compound of Formula 1 as an active ingredient.

As another means for solving the above problems, the present invention provides a cosmetic composition for skin regeneration, wrinkle improvement, antioxidant, anti-inflammatory and skin whitening comprising the compound of Formula 1 as an active ingredient.

As another means for solving the above problems, the present invention provides a health food for skin regeneration, wrinkle improvement, antioxidant, anti-inflammatory and skin whitening comprising the compound of Formula 1 as an active ingredient.

4 ', 5,7-trihydroxy-3', 6-dimethoxyflavone according to the present invention promotes collagen synthesis of fibroblasts of skin and inhibits collagenase activity to promote skin regeneration and free radicals It can be used in medicine, cosmetics or health foods by suppressing NO, showing antioxidant effect, inhibiting NO production involved in inflammatory reaction, and having excellent anti-inflammatory effect, and inhibiting melanin synthesis to show whitening effect.

EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.

Skin regeneration, wrinkle improvement, anti-oxidation, anti-inflammatory and whitening ingredients show excellent skin regeneration, wrinkle improvement, anti-oxidation, anti-inflammatory and whitening activity at low concentrations. Excellent absorption ability, low volatility for long time stay sufficient for skin regeneration, wrinkle improvement, antioxidant, anti-inflammatory and whitening effect, stable active ingredient on composition or skin, It is desirable that the formulation be easy and safe for the skin. However, among the known components, components that satisfy all of the above properties are rare. For example, some skin regeneration, anti-wrinkle, antioxidant, anti-inflammatory and whitening ingredients have excellent skin regeneration, anti-wrinkle, antioxidant, anti-inflammatory and whitening activity at low concentrations in vitro, but have the ability to penetrate and absorb skin. It is difficult to apply to real skin apart. Other active ingredients are less hydrophilic and difficult to formulate in medicine or cosmetics. In addition, some skin regeneration, anti-wrinkle, antioxidant, anti-inflammatory and whitening ingredients may decompose or transform into other compounds that are already lost when applied to the skin when exposed to heat, light or oxygen. .

As can be seen in the examples below, 4 ', 5,7-trihydroxy-3', 6-dimethoxyflavones are superior at low concentrations (4 ', 5,7-trihydroxy-3', 6-dimethoxyflavone). Since it shows excellent collagen synthesis promoting effect, antioxidant effect, anti-inflammatory effect and whitening effect, it can be used as an active ingredient in medicines, cosmetics and health foods for skin regeneration, wrinkle improvement, antioxidant, anti-inflammatory and skin whitening.

Accordingly, the present invention provides a pharmaceutical composition for skin regeneration, anti-wrinkle, antioxidant, anti-inflammatory and skin whitening, comprising the compound represented by Formula 1 as an active ingredient:

[Formula 1]

The compound name of the compound of Formula 1 is NSC 271638; Galetin 3,6-dimethyl ether; 6-Methoxy-3-O-methylkaempferol; 6-Methoxykaempferol 3-methylether; 6-Hydroxyapigenin-3,6-dimethylether; 5,7,4'-Trihydroxy-3,6-dimethoxyflavone; 3,6-Dimethoxy-2- (4-hydroxyphenyl) -5,7-dihydroxy-4H-1-benzopyran-4-one, also known as 4 ', 5,7-trihydroxy-3', 6-dimeth It is called oxyflavone (4 ', 5,7-Trihydroxy-3', 6-dimethoxyflavone).

The compound of Formula 1 may be synthesized or used a commercially available compound.

The pharmaceutical composition for skin regeneration, wrinkle improvement, antioxidant, anti-inflammatory and skin whitening of the present invention may include a pharmaceutically acceptable salt of the compound of Formula 1.

The pharmaceutically acceptable salt of the compound of Formula 1 may be an acid addition salt formed using an organic acid or an inorganic acid, and the organic acid may be, for example, formic acid, acetic acid, propionic acid, lactic acid, butyric acid, isobutyric acid or trifluoroacetic acid. , Malic acid, maleic acid, malonic acid, fumaric acid, succinic acid, succinic acid monoamide, glutamic acid, tartaric acid, oxalic acid, citric acid, glycolic acid, glucuronic acid, ascorbic acid, benzoic acid, phthalic acid, salicylic acid, anthranilic acid, dichloroacetic acid, aminooxyacetic acid And benzenesulfonic acid, p-toluenesulfonic acid and methanesulfonic acid salts and inorganic acids include, for example, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid and boric acid salts. It may preferably be in the form of hydrochloride or acetate, more preferably in the form of hydrochloride.

The above-mentioned acid addition salts may be a) directly mixing the compound of formula 1 and an acid, b) dissolving and mixing one of them in a solvent or a hydrous solvent, or c) solvent or submerging the compound of formula 1 It is prepared by a general salt preparation method which is placed in an acid in a solvent and mixed with them.

In addition to the above, salts that can be additionally salted are Gabar salt, Gabapentin salt, Pregabalin salt, Nicotinate, Adipate salt, Hemimalonate, Cysteine salt, Acetylcysteine salt, Methionine salt, Arginine salt, Lysine salt, Ornithine salt, Aspartate.

In addition, when using the compound of Formula 1 of the present invention as a medicine, it may further contain one or more active ingredients exhibiting the same or similar functions. For example, it may include known skin regeneration, wrinkle improvement, antioxidant, anti-inflammatory and skin lightening ingredients. The inclusion of additional skin rejuvenation, wrinkle improvement, antioxidant, anti-inflammatory and skin lightening ingredients will further enhance the skin regeneration, wrinkle improvement, antioxidant, anti-inflammatory and skin lightening effects of the compositions of the present invention. When the ingredient is added, skin safety, ease of formulation, and stability of the active ingredients may be considered. In one embodiment of the invention, the composition is a skin regeneration component known in the art, retinoic acid, TGF, protein from animal placenta, betulinic acid and chlorella extract, as antioxidant ingredients known in the art, tocopherol, selenium , Vitamin C and phenolic compounds, as whitening ingredients known in the art, substances that inhibit tyrosinase enzyme activity such as kojic acid, arbutin, hydroquinone, L-ascorbic acid (L) -Ascorbic acid) and derivatives thereof and various plant extracts may further include one or two or more components selected from the group consisting of. Additional ingredients may be included from 0.0001% to 10% by weight relative to the total composition weight, and the content range may be adjusted according to requirements such as skin safety, ease of formulating the compound of Formula 1, and the like. .

In addition, the pharmaceutical composition for skin regeneration, anti-wrinkle, antioxidant, anti-inflammatory and skin whitening of the present invention may further comprise a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers may contain various components such as buffers, sterile water for injection, general saline or phosphate buffered saline, sucrose, histidine, salts, polysorbates and the like.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and may be administered in the form of a general pharmaceutical preparation, for example, in various dosage forms, orally and parenterally, during clinical administration. , Diluents or excipients such as extenders, binders, wetting agents, disintegrants, surfactants and the like.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient in the pharmaceutical composition of the present invention, for example, starch, calcium carbonate, It may be prepared by mixing sucrose or lactose, gelatin and the like.

In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

In the present invention, the 'skin regeneration effect' refers to the recovery of skin tissue against damage caused by external and internal causes. The damage caused by the external causes may include ultraviolet rays, external pollutants, wounds, traumas, and the like, and the damage caused by the internal causes may include stress.

In the present invention, the 'wrinkle improvement effect' refers to inhibiting or inhibiting the generation of wrinkles on the skin, or alleviating wrinkles already generated.

In the present invention, the 'antioxidative effect' refers to a cell caused by free radicals or reactive oxygen species (ROS) that are highly reactive due to oxidative stress caused by intracellular metabolism or ultraviolet light. Refers to the inhibition of oxidation, including the removal of free radicals or reactive oxygen species, thereby reducing damage to cells.

In the present invention, the "anti-inflammatory effect" refers to inhibiting inflammation, which is one of the defense responses of biological tissues to a certain stimulus, combined with three kinds of tissue degeneration, circulatory disorder and exudation, and tissue proliferation. To say complex lesions. More specifically, inflammation is part of innate immunity and, as in other animals, human innate immunity recognizes patterns of cell surfaces that are specific to pathogens. Phagocytes recognize cells with such surfaces as nonmagnetic devices and attack pathogens. If the pathogen breaks through the body's physical barrier, an inflammatory reaction occurs. Inflammatory reactions are nonspecific defenses that create a hostile environment against invading microbes. In inflammatory reactions, when wounded or an external infectious agent enters the body, white blood cells in the early stages of immune response rush to express cytokines. Therefore, the expression level of cytokines in cells is an indicator of inflammatory response activation. Examples of skin diseases related to inflammation include atopic dermatitis, psoriasis, radiation, chemicals, burns, etc., erythematous disease, acid burns, bullous skin disease, mammary gland-type diseases, allergic itching, seborrheic eczema, rose acne, Vulgaris, polymorphic exudative erythema, nodular erythema, glansitis, vulvitis, inflammatory hair loss such as alopecia areata, cutaneous T-cell lymphoma, and the like.

In the present invention, the term "whitening effect" means not only brightening skin tone by inhibiting the synthesis of melanin pigments, but also improving skin hyperpigmentation such as blemishes and freckles due to ultraviolet rays, hormones or heredity.

The pharmaceutical composition of the present invention may provide a desirable skin regeneration effect, wrinkle improvement effect, antioxidant effect, anti-inflammatory effect and whitening effect when an effective amount of the compound of Formula 1 is included. In the present invention, the term 'effective amount' means an amount of a compound capable of promoting regeneration of damaged skin, improving wrinkles, inhibiting or alleviating oxidation of cells, inhibiting inflammation, or exhibiting a whitening effect. . The effective amount of the compound of formula 1 included in the composition of the present invention will vary depending on the form in which the composition is commercialized, how the compound is applied to the skin, the time it stays on the skin, and the like. For example, when the composition is manufactured as a medicine, it may include the compound of Formula 1 in a higher concentration than when manufactured as a cosmetic that is routinely applied to the skin. Therefore, the daily dosage is 0.1 to 100 mg / kg, preferably 30 to 80 mg / kg, more preferably 50 to 60 mg / kg, 1 to 1 day, based on the amount of the compound of Formula 1 It may be administered six times.

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

The present invention can also be provided in the formulation of the skin external preparation for skin regeneration, wrinkle improvement, antioxidant, anti-inflammatory and whitening comprising the compound of Formula 1 as an active ingredient.

When the compound of Formula 1 is used as an external preparation for skin, it is further added to fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants. Common in water, ionic or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or external preparations for the skin It may contain adjuvants commonly used in the field of dermatology, such as any other ingredients used as. The ingredients may also be introduced in amounts generally used in the field of dermatology.

When the compound of Formula 1 is provided as an external preparation for skin, it is not limited thereto, and may have a formulation such as an ointment, a patch, a gel, a cream, or a spray.

The present invention can also be provided in the formulation of a cosmetic for skin regeneration, wrinkle improvement, antioxidant, anti-inflammatory and whitening comprising the compound of Formula 1 as an active ingredient.

When the compound of Formula 1 is used as a cosmetic, the cosmetic prepared by containing the compound of Formula 1 as an active ingredient may be prepared in the form of a general emulsion formulation and solubilized formulation. For example, lotions such as flexible lotions or nourishing lotions, emulsions such as facial lotions, body lotions, creams such as nourishing creams, moisture creams, eye creams, essences, cosmetic ointments, sprays, gels, packs, sunscreens, makeup bases, liquids Formulations such as foundations, powders, cleansing creams, cleansing lotions, makeup removers such as cleansing oils, cleansing foams, soaps, body washes and the like.

In addition, the cosmetics are in addition to the compound of Formula 1, fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water Commonly used in ionic or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or cosmetics It may contain adjuvants conventionally used in the cosmetic field, such as any other ingredient.

The compound of Formula 1 may be a relatively high concentration of the compound of Formula 1 in the case of a wash-off type cosmetic such as a make-up remover, a detergent, etc., in which the active ingredient stays on the skin within a short time when it is commercialized. It may include. On the other hand, in the case of a leave-on type cosmetic such as a lotion, an emulsion, a cream, an essence, etc., in which the active ingredient stays on the skin for a long time, the compound of Chemical Formula 1 has a lower concentration than the wash-off type cosmetic. It may be included. Although not limited thereto, in one embodiment of the present invention, the composition may include the compound of Formula 1 in an amount of 0.0001% to 10% by weight (preferably 0.0001% to 1% by weight) based on the total weight of the composition. Can be. When the composition of the present invention contains less than 0.0001% by weight of the compound of Formula 1, sufficient skin regeneration, anti-wrinkle, antioxidant, anti-inflammatory and whitening effects cannot be expected, and when it contains more than 10% by weight, allergy This is to prevent unwanted reactions or skin safety problems may occur.

The present invention also relates to a health food for skin regeneration, wrinkle improvement, antioxidant, anti-inflammatory and whitening comprising the compound of Formula 1.

In the present specification, 'health food' is a food prepared by adding the compound of Formula 1 to food materials such as beverages, teas, spices, gums, confectionery, or the like, encapsulated, powdered, suspensions, and the like when ingested This means that there is a specific effect, but unlike the general medicine has the advantage that there is no side effect that can occur when taking long-term use of the drug as a raw material.

Since the health food of the present invention thus obtained can be consumed on a daily basis, high skin regeneration, wrinkle improvement, antioxidant, anti-inflammatory and whitening effects can be expected and are very useful.

When the compound of Formula 1 is used as a food additive, the compound of Formula 1 may be added as it is or used with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient can be suitably determined according to the purpose of use (prevention, health or therapeutic treatment). Generally, in the preparation of food or beverages, the composition of the present invention is added in an amount of up to 15 parts by weight, preferably up to 10 parts by weight based on the raw materials. However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. .

There is no particular limitation on the kind of food. Examples of foods to which the substance may be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, dairy products including gum, ice cream, various soups, beverages, teas, drinks, Alcoholic beverages and vitamin complexes, and the like and include all of the health foods in the conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates, etc. as additional components, as in the general beverage. The above-mentioned natural carbohydrates are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The proportion of said natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the composition of the present invention.

In addition to the above, the health food of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols. And carbonation agents used in carbonated beverages. In addition, the health food of the present invention may contain a flesh for preparing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not critical but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Reference Example  1: 4 ', 5,7- Trihydroxy -3 ', 6-dimethoxyflavone (4', 5,7- Trihydroxy -3 ', 6-dimethoxyflavone) Material Information

[Formula 1]

Compound name: NSC 271638; Galetin 3,6-dimethyl ether; 6-Methoxy-3-O-methylkaempferol; 6-Methoxykaempferol 3-methylether; 6-Hydroxyapigenin-3,6-dimethylether; 5,7,4'-Trihydroxy-3,6-dimethoxyflavone; 3,6-Dimethoxy-2- (4-hydroxyphenyl) -5,7-dihydroxy-4H-1-benzopyran-4-one, also known as 4 ', 5,7-trihydroxy-3', 6-dimeth It is called oxyflavone (4 ', 5,7-Trihydroxy-3', 6-dimethoxyflavone).

CAS No .: 18085-97-7

Where to buy: Tauto Biotech Co., Ltd.

Origin: Arnica chamissonis Less. Or Arnica flowers of longlfolia DCEaton)

Example  1: collagen synthesis effect

The substances shown in Table 1 below were added to the culture solution of human-derived fibroblasts to test collagen synthesis promoting effects at the cellular level. The synthesis of collagen was quantified using a PICP EIA kit (Procollagen Type I C-Peptide Enzyme ImmunoAssay KIT). To evaluate the cytotoxicity at 10 ppm, 1 ppm, 0.1 ppm, 0.01 ppm, and 0.001 ppm of the concentration of the test substance in human-derived fibroblasts before the experiment (method of culturing fibroblasts for MTT test [Reference: Mossman T. (1983) Rapid Colorimetric Assay for Cellular Growth & Survival:.. application to proliferation & cytotoxicity assays Journal of Immunological Methods 65, 55-63], and collagen synthesis was evaluated by selecting a concentration without cytotoxicity. In the experiment, the final concentration of the compound of Formula 1 was set to 1 ppm and 10 ppm, and each sample was added to the culture medium of human fibroblasts, incubated for 1 day, and then the culture medium was taken and collagen synthesized at each concentration with the PICP EIA kit. The extent was measured at 450 nm using a spectrophotometer. For comparison of effects, the collagen synthesis was measured by the same method for the culture medium (control) of the fibroblasts to which nothing was added and the sample to which vitamin C was added to a final concentration of 52.8 μg / mL. Collagen production was measured as UV absorbance, the collagen production increase rate was calculated as the ratio of collagen production relative to the control, the results are summarized in Table 1 below.

Collagen Biosynthesis Effect of Compound of Formula 1 (Repeat Number = 3) sample Absorbance average Collagen Growth Rate (%) Control group (no addition) 782 - Vitamin C (52.8 μg / mL) 946 121 10 ppm of a compound of formula 1 1064 136 1 ppm of a compound of Formula 1 899 115

As can be seen from the results of Table 1, the compound of Formula 1 exhibited better collagen synthesis effect at a lower concentration than when vitamin C, which is generally known to have collagen synthesis ability, was applied.

Example  2: Collagenase  Active inhibitory effect

It was confirmed as follows the collagenase activity inhibitory effect on the material of Table 2. To evaluate the cytotoxicity at 10 ppm, 1 ppm, 0.1 ppm, 0.01 ppm, and 0.001 ppm of the concentration of the test substance in human-derived fibroblasts before the experiment (method of culturing fibroblasts for MTT test [Reference: Mossman T. (1983) Rapid Colorimetric Assay for Cellular Growth & Survival:.. application to proliferation & cytotoxicity assays Journal of Immunological Methods 65, 55-63], and collagenase assay was performed by selecting a concentration without cytotoxicity. Fibroblasts, which are human normal skin cells, were seeded in a 24-well microplate at 2.5 × 10 ⁴ cells per well, incubated for 24 hours at 10% serum DMEM medium and 37 ° C., and then 10% serum DMEM medium was removed. After washing once with phosphate buffer solution, the mixture was further cultured in serum-free DMEM medium containing the compound of Formula 1 and serum-free DMEM medium without the compound of Formula 1 for 30 minutes. Thirty minutes after stimulation at 50 ng / mL of TNF-α (tumor necrosis factor-alpha), a substance known to produce MMP-1, 30 minutes after sample treatment. In this case, the TNF-α untreated group and the treated group were treated as the untreated group not treated with TNF-α and the TNF-α treated group among the controls not containing the compound of Formula 1. The supernatant of each well was collected to measure the amount (ng / mL) of newly synthesized MMP-1 using an MMP-1 assay kit (Amersham, USA), and the collagenase activity inhibition rate was determined according to Equation 1 below. -1 generation inhibition rate (%) was calculated, the results are shown in Table 2 below. The amount of MMP-1 in the control group refers to the amount of MMP-1 in the TNF-α treated group, and the amount of MMP-1 in the experimental group refers to the group to which the substance was added at each concentration.

[Equation 1]

Inhibitory Effect of the Compound of Formula 1 on Collagenase Activity (Repeat Number = 4) sample Amount of MMP-1 (ng / mL) % Inhibition 10 ppm of a compound of formula 1 6.9876 60.9917 1 ppm of a compound of Formula 1 17.2959 6.7248 Negative Control (TNF-α No Treatment) 6.8675 Positive control group (TNF-α treatment) 17.8915

Example 3: whitening effect - confirm melanin production inhibitory effect

The compound of formula 1 was added to the culture medium of mouse B-16 mouse melanoma cells to test the whitening effect at the cellular level (Lotan R., Lotan D. Cancer Res . 40: 3345-3350, 1980 ). Before the experiment, rat melanoma cells were assessed for toxicity, and non-toxic concentrations were selected for whitening evaluation. The compound of formula 1 was tested in the culture solution to the final concentration of 2.5, 5, 10 and 20 ㎍ / mL, Arbutin as a control was added to the medium to 200 ㎍ / mL and treated with B-16 melanoma cells, respectively Incubated for 3 days. Thereafter, cells were trypsinized, removed from the culture vessel, centrifuged, and melanin was extracted. The detached cells were added with 1 mL of sodium hydroxide solution (1 N concentration), boiled for 10 minutes to dissolve the melanin, and the absorbance was measured at 400 nm using a spectrophotometer to measure the amount of melanin produced. The melanin amount was measured by absorbance per unit cell count (1 × 10 ⁶ cells). The relative melanin production relative to the control group was calculated as inhibition rate (%) and the results are summarized in Table 3. In addition, the experiment was repeated three times.

Inhibitory effect of melanin production at the cellular level sample Melanin production % Inhibition Control group (no addition) 0.078 - Control 1: Arbutin (200 μg / mL) 0.044 44 Compound of Formula 1 (20 μg / mL) 0.029 63 Compound of Formula 1 (10 μg / mL) 0.044 44 Compound of Formula 1 (5 μg / mL) 0.061 22 Compound of Formula 1 (2.5 μg / mL) - -

As can be seen from the results of Table 3, the compound of Formula 1 has a superior melanin production inhibitory ability against the cultured mouse melanoma cells compared with the known whitening material Arbutin (Arbutin).

Example  4: antioxidant effect Free radical  Erasure rate

Free radical scavenging activity was measured to confirm the antioxidant activity of the compound of formula (1). Free radical scavenging activity was measured using DPPH. DPPH was purchased from Sigma Co., Ltd, USA. First, a 1.5 mM (0.06 mg / mL) standard DPPH ethanol solution was made. Then, ethanol was added to the ascorbic acid, an antioxidant, as a compound of Formula 1 and a reference material, respectively, to prepare samples at concentrations of 50 µg / ml, 25 µg / ml, 12.5 µg / ml, 6.25 µg / ml, and 3.125 µg / ml. . Then, the sample and the standard DPPH solution were added in the same ratio, stirred well, and then reacted at 37 ° C. for 30 minutes, and the absorbance was measured at 520 nm. At this time, ethanol was added instead of the sample as a control. The free radical scavenging ability was obtained from IC ₅₀ , which is a half maximal inhibitory concentration, and the results are shown in Table 4 below. IC ₅₀ is a general method of expressing free radical scavenging ability as a concentration of ascorbic acid and a compound of formula (1) required to remove 50% of free radicals from an additive-free control group.

Free radical scavenging rate (IC ₅₀ ) sample Free radical scavenging rate (µg / mL; ppm) Ascorbic acid 9 Compound of Formula 1 23

As shown in Table 4, although the activity is lower than that of ascorbic acid (vitamin C), which is a powerful antioxidant, it can be seen that it is suitable for use as an antioxidant effect material because it shows stability with excellent antioxidant effect as a natural substance.

Example  5: anti-inflammatory effect-nitric oxide ( NO ) Inhibitory effect on production

In order to confirm the anti-inflammatory effect and skin trouble alleviating effect of the compound of Formula 1, nitric oxide (NO) formation inhibitory experiment was conducted by GRIESS method using RAW264.7 cell line (ATCC number: CRL-2278). Specifically, RAW264.7 cells, which are macrophages of mice, were passaged several times, placed in a 24-well plate so that 3 × 10 ⁵ cells were put in one well, and then cultured for 24 hours. Subsequently, the compound of formula 1 was diluted with the concentration shown in Table 5 and replaced with the cell medium containing. In this case, in Table 5, 1% means the concentration of 10 mg of the compound of Formula 1 dissolved in 1 mL of medium, and 0.1%, 0.01%, and 0.001%, respectively, are the concentrations of 1 mg, 0.1 mg, and 0.01 mg of the compound. As a positive control, L-NMMA (L-NG-Monomethylarginine), which was treated with NO-producing inhibitors, was incubated together for 30 minutes, and the treatment concentration was set to 0.001% by adding 10 μg per 1 mL of medium. In addition, 1 μg of LPS (Lipopolysaccharide) was treated as a stimulus and incubated for 24 hours. 100 μl of the supernatant was transferred to a 96-well plate, 100 μl of GRIESS solution was added thereto, and reacted at room temperature for 10 minutes. The absorbance at 540 nm was measured to determine the NO inhibitory effect of Compound 1, and the results are shown in the following table. 5 is shown. The inhibition rate of NO production was determined based on the NO production amount of the experimental group treated with LPS only.

NO production inhibitory effect sample NO production inhibition rate (%) L-NMMA (positive control, 0.001%) 31.15 Compound of Formula 1 (0.001%) 13.62 Compound of Formula 1 (0.01%) 22.96 Compound of Formula 1 (0.1%) 30.18 Compound of Formula 1 (1%) 34.73

As shown in Table 5, the activity is low when compared to the representative anti-inflammatory material L-NMMA, but shows excellent anti-inflammatory activity as a natural substance, the anti-inflammatory effect as a secondary role in whitening and wrinkle improvement The synergy effect can be expected.

Formulation example  1: Preparation of Pharmaceutical Formulations

1. Preparation of powder

0.001 g of compound of Formula 1

1g lactose

The above ingredients were mixed and filled in airtight cloth to prepare a powder.

2. Preparation of Tablets

0.2 mg of compound of Formula 1

Corn starch 100 mg

Lactose 100 mg

Stearic acid  Magnesium 2 mg

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

3. Preparation of Capsule

0.2 mg of compound of Formula 1

Corn starch 100 mg

Lactose 100 mg

Stearic acid  Magnesium 2 mg

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

4. Manufacture of rings

0.003 g of compound of Formula 1

Lactose 1.5 g

1 g of glycerin

Xylitol  0.5 g

After mixing the above components, it was prepared to be 4 g per ring according to a conventional method.

5. Preparation of Granules

2 mg of compound of formula 1

Soybean Extract 50 mg

Glucose 200 mg

Starch 600 mg

After mixing the above components, 100 mg of 30% ethanol was added and dried at 60 ° C. to form granules, and then filled in fabric.

Formulation example  2: manufacture of cosmetics

1. Preparation of Soft Cosmetics (Skin Lotion)

Like the following composition, a flexible longevity containing the compound of formula 1 as an active ingredient was prepared according to a conventional method.

0.1 wt% of a compound of Formula 1

Beta-1,3-Glucan 1.0 wt%

Butylene Glycol 2.0 wt%

Propylene Glycol 2.0 wt%

Carboxy vinyl polymer 0.1 wt%

0.2 wt% of PEG-12 nonylphenyl ether

Polysorbate 80 0.4 wt%

Ethanol 10.0 wt%

0.1% by weight of triethanolamine

0.05 wt% preservative

0.05% by weight of pigment

0.05% by weight fragrance

Purified water to  100 wt%

2. Preparation of Nutrients (Milk Lotion)

As shown in the following composition, nutrient longevity containing the compound of Formula 1 as an active ingredient was prepared according to a conventional method.

0.1 wt% of a compound of Formula 1

Beta-1,3-Glucan 1.0 wt%

Beeswax 4.0 wt%

Polysorbate60 1.5 wt%

Sorbanthesquioleate 1.5 wt%

0.5% by weight of liquid paraffin

Caprylic / Capric Triglycerides 5.0 wt%

Glycerin 3.0 wt%

Butylene Glycol 3.0 wt%

Propylene Glycol 3.0 wt%

Carboxy vinyl polymer 0.1 wt%

0.2% by weight of triethanolamine

0.05 wt% preservative

0.05% by weight of pigment

0.05% by weight fragrance

Purified water to  100 wt%

3. Preparation of nutrition cream

As in the following composition, a nourishing cream containing the compound of formula 1 as an active ingredient was prepared according to a conventional method.

0.2 wt% of a compound of Formula 1

Beta-1,3-Glucan 5.0 wt%

Beeswax 10.0 wt%

Polysorbate60 1.5 wt%

PEG-60 Cured Castor Oil 2.0 wt%

0.5 wt% sorbitan sesquioleate

10.0% by weight of liquid paraffin

Squalane 5.0 wt%

Caprylic / Capric Triglycerides 5.0 wt%

Glycerin 5.0 wt%

Butylene Glycol 3.0 wt%

Propylene Glycol 3.0 wt%

0.2% by weight of triethanolamine

0.05 wt% preservative

0.05% by weight of pigment

0.05% by weight fragrance

Purified water to  100 wt%

4. Preparation of Massage Cream

As in the following composition, a massage cream containing a compound of Formula 1 as an active ingredient was prepared according to a conventional method.

0.1 wt% of a compound of Formula 1

Beta-1,3-Glucan 3.0 wt%

Beeswax 10.0 wt%

Polysorbate60 1.5 wt%

PEG-60 Cured Castor Oil 2.0 wt%

Sorbanthesquioleate 0.8 wt%

40.0% by weight of liquid paraffin

Squalane 5.0 wt%

Caprylic / Capric Triglyceride 4.0 wt%

Glycerin 5.0 wt%

Butylene Glycol 3.0 wt%

Propylene Glycol 3.0 wt%

0.2% by weight of triethanolamine

0.05 wt% preservative

0.05% by weight of pigment

0.05% by weight fragrance

Purified water to  100 wt%

5. Manufacture of pack

As in the following composition, a pack containing the compound of formula 1 as an active ingredient was prepared according to a conventional method.

0.2 wt% of a compound of Formula 1

Beta-1,3-Glucan 1.0 wt%

Polyvinyl Alcohol 13.0 wt%

Sodium Carboxymethyl Cellulose 0.2% by weight

Glycerin 5.0 wt%

Allantoin 0.1 wt%

Ethanol 6.0 wt%

0.3 wt% of PEG-12 nonylphenyl ether

Polysorbate 60 0.3 wt%

0.05 wt% preservative

0.05% by weight of pigment

0.05% by weight fragrance

Purified water to  100 wt%

Formulation example  3: External skin preparations  Produce

1. Preparation of Gel

As in the following composition, a gel containing the compound of formula 1 as an active ingredient was prepared according to a conventional method.

0.1 wt% of a compound of Formula 1

Beta-1,3-Glucan 0.1 wt%

0.05% by weight of ethylenediamine sodium acetate

Glycerin 5.0 wt%

Carboxy vinyl polymer 0.3 wt%

Ethanol 5.0 wt%

PEG-60 Cured Castor Oil 0.5% by weight

0.3% by weight of triethanolamine

0.05 wt% preservative

0.05% by weight of pigment

0.05% by weight fragrance

Purified water to  100 wt%

2. Manufacture of ointments

As in the following composition, an ointment containing the compound of formula 1 as an active ingredient was prepared according to a conventional method.

0.5% by weight of compound of formula 1

Beta-1,3-Glucan 10.0 wt%

Beeswax 10.0 wt%

Polysorbate 60 5.0 wt%

PEG-60 Cured Castor Oil 2.0 wt%

Sorbanthesquioleate 0.5 wt%

Vaseline 5.0 wt%

10.0% by weight of liquid paraffin

Squalane 5.0 wt%

Shea Butter 3.0 wt%

Caprylic / Capric Triglycerides 5.0 wt%

Glycerin 10.0 wt%

Propylene Glycol 10.2 wt%

0.2% by weight of triethanolamine

0.05 wt% preservative

0.05% by weight of pigment

0.05% by weight fragrance

Purified water to  100 wt%

3. Preparation of Topical Drug (Gel Ointment)

As the following composition, a gel ointment comprising the compound of formula 1 as an active ingredient was prepared according to a conventional method.

0.5% by weight of compound of formula 1

Beta-1,3-Glucan 10.0 wt%

1.5% by weight of polyacrylic acid (Carbopol 940)

Isopropanol 5.0 wt%

Hexylene glycol 25.0 wt%

Triethanolamine 1.7 wt%

Deionized water to  100 wt%

4. Preparation of medicament (patch) for topical administration

As in the following composition, a patch containing the compound of Formula 1 as an active ingredient was prepared according to a conventional method.

0.5% by weight of compound of formula 1

Beta-1,3-Glucan 3.0 wt%

Hexylene glycol 20.0 wt%

Diethylamine 0.7 wt%

1.0% by weight of polyacrylic acid (Carbopol 934P)

Sodium sulfite 0.1 wt%

1.0 weight% of polyoxyethylene lauryl ether (E.O = 9)

1.0% by weight of polyhydroxyethylene cetyl stearyl ether (Cetomacrogol 1000)

Viscous paraffin oil 2.5% by weight

Caprylic acid ester / capric acid ester (Cetiol LC) 2.5% by weight

Polyethylene glycol 400 3.0 wt%

Deionized water to  100 wt%

Formulation example  4: manufacture of food

Food containing the compound of formula 1 of the present invention was prepared as follows.

1. Preparation of flour food

0.05 to 1.0 parts by weight of the compound of Formula 1 was added to the flour, using the mixture to prepare bread, cake, cookies, crackers and noodles to prepare a health food.

2. Manufacturing of Dairy Products

0.2 parts by weight of the compound of Formula 1 was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

3. Manufacture of wire

Brown rice, barley, glutinous rice, and yulmu were alphanated by a known method to distribute the dried ones, and then prepared into a powder having a particle size of 60 mesh. Black beans, black sesame seeds, and perilla were also steamed and dried by a known method, and then ground to a powder having a particle size of 60 mesh. The compound of Chemical Formula 1 was concentrated under reduced pressure in a vacuum concentrator, and the dried product obtained by drying with a spray and a hot air dryer was pulverized with a particle size of 60 mesh to obtain a dry powder.

The dry powder of the grains, seeds and the compound of Formula 1 prepared in the above was prepared by blending in the following ratio with respect to 100 parts by weight of the mixed powder.

Cereals (30 parts by weight brown rice, 15 parts by weight of barley, 20 parts by weight of barley), seeds (7 parts by weight perilla, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds), compound of formula 1 (0.1 parts by weight),

Ganoderma lucidum (0.5 parts by weight),

Foxglove (0.5 Parts by weight )

Formulation example  5: manufacture of beverages

1. Manufacture of health drinks

0.1 mg of compound of Formula 1

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Purified water  By adding a full 900 mL

After mixing the above components according to the conventional healthy beverage manufacturing method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained by sterilization in a sterilized 2L-container, sealed sterilized and stored in the refrigerator It was used to prepare a healthy beverage composition of the invention.

Although the composition ratio is a composition suitable for a preferred beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and intended use.

2. Preparation of Vegetable Juice

1 g of the compound of Chemical Formula 1 of the present invention was added to 1,000 mL of tomato or carrot juice to prepare a vegetable juice for health promotion.

3. Manufacture of fruit juice

1 g of the compound of Formula 1 was added to 1,000 mL of apple or grape juice to prepare a fruit juice for health promotion.

Claims (24)

A pharmaceutical composition for improving skin regeneration and wrinkles comprising the compound represented by Formula 1 as an active ingredient:
[Formula 1]

Skin external preparation for skin regeneration and wrinkle improvement comprising the compound represented by Formula 1 as an active ingredient:
[Formula 1]

The method of claim 2,
The external preparation is an external preparation for skin in the form of an ointment, patch, gel, cream or spray.
Cosmetic composition for skin regeneration and wrinkle improvement comprising the compound represented by the formula (1) as an active ingredient:
[Formula 1]

The method of claim 4, wherein
Cosmetic composition in the form of a lotion, essence, lotion, cream, pack, gel, powder, foundation or detergent.
Health foods for skin regeneration and wrinkle improvement comprising the compound represented by the formula (1) as an active ingredient:
[Formula 1]

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