KR101304677B1 - A chestnut bur extracts and a cosmetic composition containing the chestnut bur extracts as an active ingredient - Google Patents

Abstract

PURPOSE: A cosmetic composition is provided to improve antioxidation, whitening, anti-inflammation, and anti-wrinkling activities using a chestnut bur extract, thereby preventing and treating oxidative stress and inflammation. CONSTITUTION: A cosmetic composition contains 0.001-10 wt% of a butanol extract of chestnut bur with antioxidation, whitening, anti-inflammation, antibacterial, and anti-wrinkling activities as an active ingredient. The composition is manufactured in a formed selected among a skin lotion, a skin softener, a skin toner, a lotion, a milk lotion, a moisture lotion, a nutrition lotion, a massage cream, a nutrition cream, a moisture cream, a hand cream, an essence, a nutrition essence, a pack, a soap, a shampoo, a cleansing foam, a cleansing lotion, a cleansing cream, a body lotion, a body cleanser, an emulsion, a pressed powder, a powder, and an eye shadow.

Description

A Chestnut bur Extracts and A Cosmetic Composition Containing The Chestnut bur Extracts as An Active Ingredient}

The present invention relates to a functional composition containing chestnut extract as an active ingredient, and more particularly, to a cosmetic composition having antioxidant, whitening, wrinkle improvement and anti-inflammatory activity, containing the solvent extract fraction obtained from chestnut as an active ingredient. It is about.

In modern society, researches are actively conducted to explore various substances that can cure or prevent diseases according to the desire to live healthier. Among them, the research focuses on researching various biological activities using natural products, separating and purifying active ingredients, identifying biochemical functions of the components, and scientifically verifying their efficacy. Although there are various aging factors in humans, active oxygen is found to be one of the causes of promoting aging along with various diseases in the human body, and researches on functional foods and functional cosmetics for removing active oxygen are being actively conducted.

In addition, as the standard of living improves and the life expectancy is extended, interest in skin care is increasing. In particular, the increase in the photoaging of the skin due to the increase of UV exposure of the skin due to environmental pollution further amplifies the interest in skin whitening. have.

The development of functional cosmetics with whitening effect, anti-aging and sun protection, centered on natural products, is being actively conducted. The skin aging phenomenon shows various structural and functional changes.In particular, various internal and external stresses such as age and ultraviolet rays reduce skin elasticity and luster and promote skin aging by depositing skin pigments such as blemishes and freckles. do.

Melanin, a high-molecular natural pigment of phenols that is widely present in nature, is made from the skin's intracellular tyrosinase, which is made during the biosynthesis process of enzymes, and increases the skin's resistance to ultraviolet rays, drying, and extreme temperatures, but produces excessive melanin. Causes pigmentation, such as blemishes, freckles, and spots on the human body, and promotes aging of the skin. Therefore, skin aging can be suppressed by lowering the activity of tyrosinase, which is a major cause of skin aging.

In particular, attention is focused on natural materials, which are highly preferred by consumers. Chemical whitening materials have been side effects such as rejection reactions such as allergic reactions and non-continuous whitening effect through clinical trials, and researches are actively being conducted to find whitening materials that are friendly to skin and stable.

Accordingly, the present inventors have completed the present invention by confirming physiological activities such as antioxidant, anti-inflammatory, whitening, anti-wrinkle, etc. against the chestnut extract solvent-fractionated from chestnut.

The present invention is based on the various functions of the chestnut extract,

The main object of the present invention is to provide a functional cosmetic composition containing the chestnut extract as an active ingredient.

It is another object of the present invention to provide a skin external composition for preventing and improving skin diseases caused by oxidative stress and inflammation, which contains chestnut extract as an active ingredient.

In order to solve the above problems, the present invention provides a functional composition containing the chestnut extract as an active ingredient.

In particular, it provides a cosmetic composition or skin external composition for preventing and improving skin diseases caused by oxidative stress and inflammation, containing chestnut extract as an active ingredient.

At this time, the crude extract of chestnut extract, polar solvent soluble extract or non-polar solvent soluble extract, the crude extract may be extract from a solvent selected from water, including purified water, methanol, ethanol, butanol or a mixed solvent thereof; The polar solvent soluble extract is an extract available from a solvent selected from water, ethanol, butanol or a mixed solvent thereof; And the non-polar solvent soluble extract includes an extract soluble in hexane, chloroform, dichloromethane or ethyl acetate.

Chestnut extract of the present invention is preferably butane extract.

In particular, the composition of the present invention is characterized in that it has antioxidant activity, whitening activity, anti-inflammatory activity, antimicrobial activity and anti-wrinkle activity, the total weight of the composition is 0.001 to 10% by weight, preferably 0.1 to 10 It may be included in% weight.

In addition, the cosmetic composition of the present invention is not limited, but skin lotion, skin softener, skin toner, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, essence, nutrition essence, pack , Soap, shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, emulsion, press powder, loose powder and eye shadow and the like.

The chestnut extract according to the present invention has an antioxidant activity, a whitening activity, an anti-inflammatory activity, an antimicrobial activity, and an anti-wrinkle activity, so it is useful as an external skin composition and a cosmetic composition that can prevent or improve skin diseases caused by oxidative stress and inflammation. There is an effect that can be used.

1 is a photograph of chestnut.
Figure 2 is a graph of electron donating ability according to the extraction solvent of chestnut extract.
3 is a graph of ABTS + cation radical scavenging activity according to the extraction solvent of chestnut extract.
Figure 4 is a graph of Superoxide anion radical scavenging ability according to the extraction solvent of chestnut extract.
5 is a graph of mushroom tyrosinase inhibitory activity of chestnut extract.
6 shows cell viability results of chestnut extract against melanocytes B16F10.
7 is a result of tyrosinase inhibitory activity of chestnut extract against melanocytes B16F10.
8 is a result of the melanin synthesis inhibitory activity of the chestnut extract against melanocytes B16F10.
9 shows the elastase inhibitory activity of chestnut extract.
10 shows the collagenase activity inhibitory activity of chestnut extract.
Figure 11 shows the cell viability of chestnut extract on fibroblast (CCD-986sk).
12 shows Collagen synthesis of chestnut extract on fibroblast (CCD-986sk).
Figure 13 shows the cell viability of chestnut extract on macrophage (Raw264.7).
Figure 14 shows the nitric oxide inhibition of chestnut extract.
Figure 15 shows the antimicrobial activity of chestnut extract against Staphylococcus aures.
Figure 16 shows the antimicrobial activity of chestnut extract against Staphylococcus epidermidis.
Figure 17 shows the antimicrobial activity of the chestnut extract against Escherichia coli.
18 shows the antimicrobial activity of chestnut extract against Propinoibacterium acnes.

The terms used in the present invention are defined as follows.

"Extract" is characterized in that the crude extract of chestnut extract, polar solvent soluble extract or non-polar solvent soluble extract.

"Crude extract" is a solvent selected from water containing purified water, lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, butanol, or a mixed solvent thereof, preferably a mixed solvent of water and methanol, more preferably 50 to 100% Extracts soluble in methanol.

"Polar solvent soluble extract" includes extracts soluble in water, methanol, butanol or a mixed solvent thereof, preferably water or methanol, more preferably methanol.

"Non-polar solvent soluble extract" includes extracts soluble in hexane, chloroform, dichloromethane, or ethyl acetate, preferably hexane, dichloromethane or ethyl acetate, more preferably in hexane or ethyl acetate solvent.

By "active ingredient" is meant a component which alone can exhibit the desired activity or can itself exhibit activity with a carrier which is inactive.

"Pharmaceutical composition" means a mixture of chestnut extract of the present invention with other chemical components such as diluents or carriers.

A "carrier" is defined as a compound that facilitates the addition of a compound into a cell or tissue. For example, dimethyl sulfoxide (DMSO) is a commonly used carrier that facilitates the incorporation of many organic compounds into cells or tissues of an organism. “Acceptable carrier in cosmetic preparation” means a compound or composition already known and used that may be included in a cosmetic preparation or a compound or composition that will be developed in the future and has no toxicity beyond that which the human body is able to adapt upon contact with the skin.

A "diluent" is defined as a compound that not only stabilizes the biologically active form of a compound of interest, but also is diluted in water to dissolve the compound. Salts dissolved in buffer solutions are used as diluents in the art. A commonly used buffer solution is phosphate buffered saline, since it mimics the salt state of the human solution. Since buffer salts can control the pH of the solution at low concentrations, buffer diluents rarely modify the biological activity of the compounds.

"Functional composition" means the product which improved the functionality of a general composition by adding the chestnut extract of this invention. Functionality can be roughly classified into physical properties and physiological functions. When the extract of the present invention is added to a general composition, for example, food or cosmetics, the physical and physiological properties of the general food or cosmetics will be improved, and the present invention can improve such improvement. Products with function are generically defined as 'functional compositions'.

Hereinafter, the present invention will be described in detail.

All technical terms used in the present invention, unless defined otherwise, are used in the meaning as commonly understood by those skilled in the art in the related field of the present invention. Also, preferred methods or samples are described in this specification, but similar or equivalent ones are also included in the scope of the present invention. The contents of all publications referred to herein are incorporated herein by reference.

The present invention relates to the use of chestnut extract, relates to the exercise of the specific physiological activity and function contained in the chestnut extract.

Chestnut (Fig. 1) refers to the thick shell portion surrounding the fruit. Chestnut is a deciduous broad-leaved arboreous tree belonging to the genus Fagusaceae Chestanea, which is distributed around 10 species around the world. There are four species of Chinese chestnut (C. mollisima), American chestnut (C. dentata), and European chestnut (C. savita). To date, only research on chestnut skin, which is the fruit of chestnut, has been actively conducted. Yulpi is known to keep skin clean, beautiful and prevent aging, and it is also known to have a moisturizing effect that moisturizes the skin or prevents excessive evaporation from the skin. Known ingredients are ellagic acid, quercetin, morin, maringenin, flavonol, and Senter reported the highest amount of gallic acid among chestnut ingredients.

Chestnut extract can be prepared and used by a method known in the art, a modified method thereof, or a method according to the present invention. As one embodiment, it can manufacture by the following method.

Chestnut extract or crude extract of the present invention contains about 1 to 30 times the volume of chestnut weight, preferably 5 to 15 times the volume of water (w / v%) including purified water, methanol, ethanol, butanol, etc. A solvent selected from the lower alcohols of 6 to 6 or a mixed solvent thereof, preferably a water and ethanol mixed solvent, more preferably 50 to 100% ethanol, is added at about 0 to 100 ° C., preferably at room temperature for 10 to 60 hours. Preferably, the extraction method such as cold sewage extraction, hot water extraction, ultrasonic extraction, reflux cooling extraction, or heat extraction method for 30 to 50 hours, preferably extracted by hot water extraction method, filtered and concentrated under reduced pressure, chestnut crude extract of the present invention Can be obtained.

In addition, the polar solvent or non-polar solvent soluble extract of the present invention is about 1 to 150 times the weight of the crude extract, preferably 50 to 100% ethanol crude extract, preferably 5 to 100 times the volume (w / v% ), Hexane, ethyl acetate and butanol are sequentially added to a volume of about 1 to 10 times, preferably 1 to 5 times, and fractionated 1 to 5 times, preferably 2 to 4 times. The polar solvent and nonpolar solvent soluble extract of the invention can be obtained. Preferably butane extract can be obtained and used.

The present invention includes a method for preparing the chestnut extract.

According to an embodiment of the present invention the manufacturing process of the chestnut extract fraction is shown in FIG.

The preparation method is merely an exemplary method thereof, and may be suitably modified by various methods based on the art. For example, the non-exemplified extraction method according to the present invention may be used. Successful modifications can be made by those skilled in the art.

If those skilled in the art to which the present invention pertains, specific reaction conditions for the preparation of the chestnut extract according to the present invention can be confirmed through examples to be described later, and a detailed description thereof will be omitted.

Chestnut extract of the present invention has a variety of functions.

Antioxidant activity

Significant amounts of free radicals are produced during oxidation for energy production in vivo. Most of these free radicals are destroyed by in vivo elimination mechanisms, but a large amount of active oxygen is generated instantaneously or chronically free radicals are generated and the balance with the antioxidant defense system causes various diseases. Antioxidants to inhibit the toxicity of free radicals include natural antioxidants such as ascorbic acid, tocopherol, carotenoids, flavonoids, proteins and phospholipids, and synthetic antioxidants such as butylated hydroxy anisol (BHA) and butylated hydroxy toluene (BHT). . Synthetic antioxidants have been reported to cause cancer in the human body due to the mutagenicity and toxicity of biological enzymes and fats, a safer and more potent natural antioxidants are required.

Chestnut extract of the present invention has an excellent antioxidant effect.

In one embodiment, DPPH, ABTS, Superoxide anion radical was measured to see the antioxidant capacity of the chestnut was confirmed that the antioxidant capacity is excellent.

As a result of measuring the electron donating ability of the chestnut extract, the chestnut n-BuOH layer showed an effect of 99.9% at 1,00 / mL, and the ABTS cation radical showed a 99% effect at a concentration of 1,00 / mL. Super anion radical scavenging activity was 87.8% in acetone extract at 1,000 / mL and 99.80% in the n-BuOH layer.

Among the chestnut fractions of the present invention, butanol fraction shows the strongest antioxidant activity and has antioxidant activity in the order of n-BuOH> H ₂ O>EtOAc> n-hexane fraction.

Whitening active

Melanin in the epidermal basal layer plays the most important role in determining human skin color and is synthesized in melanocytes, a special type of brown intracellular organelles in melanocytes. Ultraviolet rays cause skin melanogenesis and are generally made to protect the skin from external stimuli such as ultraviolet rays. However, excessive melanin synthesis and accumulation result in visually detrimental diseases such as blemishes and freckles, and melanocytes contain the enzymes necessary to synthesize normal melanin, which contains enzymes necessary to synthesize melanin. The best known enzymes are tyrosinase, tyrosinase related protein-1 and TRP-2.

The synthesis of melanin is metabolized to 3,4-dihydroxyphenylalanine (DOPA) and DOPA quinone by tyrosinase as a substrate of tyrosine, an amino acid. The reaction after DOPA quinone is divided into two types: pheomelanin, which determines yellow to reddish brown color, and eumelanin production, which determines brown to brownish brown color. In melanin biosynthesis, tyrosinase is an important marker gene for studying the skin pigmentation process because it mediates an important rate-regulating step in which tyrosine is converted to DOPA quinone. Tyrosinase is also known to be involved in the conversion of 5,6-dihydroxyindole (DHI), an intermediate metabolite of eumelanin, to indole-5,6-quinone12). Although the function of TRP-1 is not yet clear, it is reported that it will act as a 5,6-dihydroxyindole-2-carboxylic acid (DHICA) oxidase, and that it will act to stabilize tyrosinase.

In melanin biosynthesis, tyrosinase is an important marker gene for studying the skin pigmentation process because it mediates an important rate-regulating step in which tyrosine is converted to dopaquinone. Tyrosinase is also known to be involved in the conversion of 5,6-dihydroxyindole (DHI), an intermediate metabolite of eumelanin, to indole-5,6-quinone. Although the function of TRP-1 is not yet clear, it is reported that it will act as a 5,6-dihydroxyindole-2-carboxylic acid (DHICA) oxidase, and that it will act to stabilize tyrosinase.

When the skin is irradiated with UV rays, keratinocytes secrete endothelin-1 (ET-1), α-MSH, corticosteroids, and nitric oxide (NO), increasing skin pigment. α-MSH binds to the melanocortin-1 receptors in melanocytes and increases the concentration of cyclic denosine monphosphate (cAMP) to increase melanin production. Nitric oxide increases melanogenesis through the cyclic guanidine monophosphat (cGMP) pathway.

Chestnut extract of the present invention inhibits the expression of enzymes and genes involved in the melanin synthesis.

In one embodiment, the inhibition of melanin biosynthesis and tyrosinase inhibitory activity against n-BuOH layer was confirmed to inhibit melanin biosynthesis.

Specifically, n-BuOH fraction showed 82.1% tyrosinase inhibitory activity at 1,000 μg / mL, and as a result of measuring the whitening effect of chestnut n-BuOH in B16F10 cells, n-BuOH was inhibited at 10 μg / mL in tyrosinase inhibition. Inhibitory activity of 37.6% and melanin biosynthesis showed over 60% biosynthesis inhibition rate at a concentration of 10 μg / mL.

Wrinkle improvement active

As the skin ages, the thickness of the skin decreases, wrinkles increase, and elasticity decreases. This is because the secretion of various hormones that regulate metabolism internally decreases, the activity of the skin cells is reduced and the biosynthesis of skin constituent proteins is reduced. Photoaging skin, also characterized by strong wrinkles and thick skin, is caused by free radicals and harmful free radical species that are increased by ultraviolet light. As aging progresses, the ability to produce structural proteins such as collagen, elastin, and hyaluronic acid, which make up the skin, decreases, and the biosynthesis of type I collagenase (MMP, Matrix metalloproteinase) increases, resulting in more breakdown of skin structural proteins. . Accumulating oxidative stress also promotes this skin aging.

Aging of the skin is manifested by various symptoms such as skin dryness and thickness, skin tone, skin elasticity change, blood vessel dilation and wrinkle formation. Matrix metalloproteinases (MMPs), which break down substrates, play an important role in the development of skin photoaging. Extracellular matrix in the dermis plays an important role in maintaining the structure and elasticity of the skin, of which 80-85% is composed of type I collagen. Type 1 collagen is synthesized in the form of procollagen as a precursor and then discharged out of the cell to cut off the amino and carboxyl ends to form a finished collagen.

There are many biological reactions involved in the reconstitution of matrix proteins, especially MMPs, which play an important role in degrading matrix proteins, including intestitial collagenase (MMP-1), 72 kD gelatinase (MMP-2), 92 kD gelatinase (MMP-9), More than 26 MMPs are known, including neutrophil collagenase (MMP-8) and collagenase (MMP-13). In skin exposed to ultraviolet rays, the expression of MMPs is increased, and the increased MMPs break down collagen constituting the skin, resulting in a deficiency of matrix proteins, leading to skin aging. Skin aging is a natural phenomenon in the human body. The process of aging occurs in all the organs that make up the body, and aging progresses as the skin ages.

Chestnut extract of the present invention inhibits the enzyme activity involved in the skin aging.

In one embodiment, the collagen biosynthesis of chestnut n-BuOH fractionation layer showed more than 50% effect even at 20 μg / ml, and the chestnut n-BuOH extract showed 74% inhibition at 50μg / mL as a result of Elastase inhibition. Collagenase inhibition showed an effect of 59.12% at a concentration of 1,000 μg / mL.

Anti-inflammatory activity

Inflammatory response is a protective action of a host that is rapidly activated through various mechanisms of action to defend against any organic changes in the body or tissues such as physical, chemical or bacterial infections. It is the most important of the immune responses induced by macrophage. Once the stimulation is applied, the vasoactive substance such as inflammatory component is released locally and the vascular permeability is increased to cause inflammation. However, the continuous inflammatory reaction promotes the mucosal injury, and as a result, it causes some diseases .

In particular, various mediators such as cytokines, prostagrandin E2 (PGE2), lysosomal enzymes, and fee radicals are involved in macrophages. In particular, macrophages activate nuclear factor-κB (NF-κB / p65), a transcription factor of the inflammatory response by stimulation such as cytokines, tumor necrosis factor-α (TNF-α), and lipopolysaccharide (LPS). Nitric oxide synthase (iNOS) and cycloxygenase-2 (COX-2) are expressed to produce excess nitric oxide (NO) and PGE2 and cause inflammation.

Chestnut extract of the present invention inhibits the enzyme activity involved in the inflammation.

In one embodiment, it was confirmed that chestnut n-BuOH extract in macrophages reduced NO expression, showing a near 60% inhibition rate at 20μg / mL concentration.

Antimicrobial activity

Furthermore, the chestnut extract of the present invention has antimicrobial activity.

In one embodiment, for the night n-BuOH layer, four bacteria (Staphylococcus aureus, Epidermal cystosis, Escherichia coli, Acne) were selected from among the bacteria that are problematic for the skin, and as a result of all four bacteria It was confirmed that the antimicrobial effect in the n-BuOH layer is excellent.

As such, the chestnut extract of the present invention has various effects of excellent antibacterial, antioxidant, whitening, anti-inflammatory and anti-wrinkle ability.

Therefore, the present invention relates to a functional composition containing the chestnut extract as an active ingredient in another aspect.

As such a functional composition, a health functional food, a cosmetics, or a skin external composition etc. are mentioned, for example. Functionality can be roughly divided into physical properties and physiological functions. When the chestnut extract of the present invention is added, the physical properties and physiological functions of the composition will be improved.

That is, the chestnut extract of the present invention may be used as a main ingredient or additives and auxiliaries in the manufacture of various functional foods, cosmetics, and external skin preparations. In one embodiment, the amount of the extract in the functional composition may generally be added in an amount of 0.01 to 15% by weight of the total composition, and in addition to containing the extract, a person skilled in the art according to the conventional method for preparing a food composition or cosmetic composition may suitably It can be selected and used.

As a most preferred embodiment, the present invention relates to a functional cosmetic composition containing the chestnut extract as an active ingredient.

The composition may comprise 0.001 to 10% by weight, preferably 0.1 to 10% by weight of chestnut extract relative to the total weight of the composition. Cosmetic composition having antioxidant, whitening, anti-wrinkle and / or anti-inflammatory activity, containing chestnut extract according to the present invention can be used in various cosmetics or face washes for skin improvement effects such as anti-aging and anti-wrinkle of the skin. have.

Products to which the composition according to the present invention can be added may include, for example, cosmetics (cosmetics) such as various creams, lotions, skins, and cleansing agents, face washes, soaps, treatments, and essences.

At this time, cosmetics (cosmetics) include lotion, milky lotion, cream, gel, essence (essence), pack cosmetics and the like. More specifically, without limitation, skin lotion, skin softener, skin toner, lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutrition cream, moisturizing cream, hand cream, essence, nutrition essence, pack, soap, shampoo , Cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, latex, press powder, loose powder and eye shadow.

Cosmetics containing an effective amount of the composition of the present invention is promoted the regeneration of the skin tissue of the applied site, in particular, it can maintain a young and healthy skin by the antioxidant, whitening, anti-inflammatory, wrinkle improvement and / or anti-aging effect.

"Cosmetic water" is generally a clear liquid cosmetic applied to the skin surface in order to keep the skin clean and healthy. The basic function of the lotion is to supply moisture or moisturizing ingredients to the stratum corneum of the skin, and the lotion also has the function of softening the skin. Dissolves and stabilizes substances that are difficult to dissolve in water to make the appearance transparent. Transparent or translucent using microemulsion or lipid nanosphere, O / W type (oil in water type), The opaque lotion emulsified by W / O type or W / S type | mold, the thing which mix | blended the water-soluble polymer, etc. are mentioned. As the treatment method of the present invention, a known " lotion " can be suitably used depending on its use and the like.

In addition to the composition of this invention, lotion contains the following components, for example. Namely, purified water for dissolving water-soluble components such as ion-exchanged water and supplying water to the stratum corneum; alcohol for dissolving and sterilizing oil-soluble components such as ethanol and propanol to give a refreshing feeling; glycerin, PEG, hyal Moisturizers for moisturizing the stratum corneum, such as lonic acid; emollients (oil components that prevent moisture from evaporating); moisturizing agents such as ester oils and vegetable oils; and raw material components such as polyoxyethylene oleyl alcohol ethers. Solubilizer for solubilizing; buffering agent for adjusting pH of products such as citric acid, lactic acid, amino acids, etc .; for adding flavors such as vanillin, orange flavor, lemon flavor, milk flavor geraniol, linarol Flavoring agent; Antiseptic for inhibiting microorganisms such as methylparaben and phenoxy ethanol to prevent decay; Colorant for coloring; Metal ion sequestrant, Ultraviolet ray Hand et al., Fading agent to prevent fading or discoloration; a drug, such as astringents and, fungicides, energetic agents, anti-inflammatory agents, or whitening agent, may contain a mixture of two or more.

When the total amount of the lotion is 100% by weight, the composition of the present invention may contain 0.00 to 20% by weight, preferably 0.01 to 10% by weight, more preferably about 0.1 to 5% by weight.

"Emulsion" is an intermediate between a lotion and a cream and is generally a fluid emulsion. Latex is a cosmetic mainly used to supply moisture, moisturizer, oil, etc. to the skin in order to maintain the skin's moisture balance, moisturizing and flexibility. The components contained in the milky milk are similar to the ingredients contained in the creams described later. However, since the milky milk is fluid, the amount of solid oil and lead is less than that of the creams. In the treatment method of the present invention, the known " milky lotion " can be suitably used depending on its use and the like.

The emulsion may contain, for example, 0.001 to 20% by weight, preferably 0.01 to 10% by weight, more preferably about 0.1 to 5% by weight of the composition of the present invention.

"Cream" is a type of emulsion in which one side of a liquid that does not mix with each other, such as water and oil, is dispersed in a stable state in the other dispersion medium. In the treatment method of the present invention, a known " cream " can be appropriately used depending on its use and the like. These creams include emolent creams to moisturize or soften the skin, massage creams to promote blood circulation, cleansing creams for skin cleansing, hair removers for hair removal, deodorant creams for deodorizing, Exfoliated cream, and the like.

The cream contains, for example, the following ingredients: That is, the aqueous phase components included in the cream include purified water such as ion-exchanged water; alcohols for dissolving and sterilizing oil-soluble components such as ethanol and propanol to give a refreshing feeling; Humectants; and mucus such as queen's seed, pectin, cellulose derivatives, and the like. Oil components included in the cream include hydrocarbons such as squalene, liquid paraffin, petrolatum and solid paraffin; oils such as olive oil, almond oil, cacao fat and castor oil; lead such as beeswax, lanolin and jojoba oil; stearic acid, oleic acid and palmitic acid Higher alcohols such as fatty acids such as cetanol and stearyl alcohol, esters such as IPM, glycerin triester, pentaerythritol tetraester, silicone oils such as polysiloxanes, and the like. The amount of each component contained in the cream varies greatly depending on the type and application thereof, but known ones may be suitably used in the treatment method of the present invention.

The cream may contain 0.001 to 20% by weight, preferably 0.01 to 10% by weight, more preferably about 0.1 to 5% by weight of the composition of the present invention.

The gel is a cosmetic which is uniform in the appearance state such as a gel or a sol and is transparent to translucent. As a treatment method of the present invention, a known " gel " can be suitably used depending on its use and the like. And an aqueous gel for replenishing and moisturizing the skin, an oily gel for maintaining the moisture of the skin, and replenishing oil, a water-soluble gel for promoting blood circulation, and a gel for washing. The gel may be prepared by using a gelling agent containing a water-soluble polymer such as carboxyvinyl polymer and methyl cellulose. Moreover, you may use gel massage oil.

Essence (Essence) is basically a component such as the above-mentioned lotion, emulsion and cream, and includes those containing an emulsion, a solubilizing agent, a moisturizing agent, water, and other medicines. The well-known cosmetic liquid in the treatment method of this invention can be used. Examples of the emulsion contained in the serum include natural vegetable oils such as olive oil, camellia oil, sesame oil, sunflower oil, sweet almond oil and jojoba oil; diglycerin esters or triglycerin esters of fatty acids such as capric acid, myristic acid, oleic acid, isostearic acid, And the like. Such emulsions mainly function as emollients. Solubilizers included in the cosmetic liquid include propylene glycol fatty acid esters such as glycerin fatty acid ester, polyglycerol fatty acid ester, sorbitan fatty acid ester, and propylene glycol monostearate, glycerin, diglycerin, ethylene glycol, propylene glycol, 1,3-butyl Polyhydric alcohols, such as len glycol, etc. are mentioned. On the other hand, a solubilizer is preferable because it uses glycerin or diglycerol, and since a thermal effect is heightened in a massage. Distilled water and deionized water are mentioned as water contained in a cosmetic liquid. Other pharmaceutical agents include fungicides, preservatives, vitamins, natural extracts from plants, pigments and flavorings.

The quantity of each component which comprises a cosmetic liquid can employ | adopt a well-known component amount. For example, when the total amount of the cosmetic liquid is 100% by weight, the composition of the present invention may contain 0.001 to 20% by weight, preferably 0.01 to 10% by weight, more preferably about 0.1 to 5% by weight. Moreover, 65 to 90 weight% of an emulsion, 1 to 10 weight% of a solubilizer, 1 to 30 weight% of a moisturizer, about 0 to 10 weight% of other drugs, and the remainder are water. What is necessary is just to manufacture a cosmetic liquid according to a well-known manufacturing method.

"Pack cosmetic" is to apply to the target site for the purpose of moisturizing the skin, promoting blood circulation, and cleansing. Pack cosmetics generally include a jelly, paste, powder, creamy liquid, and the like, and may be applied to the face to form a film and then peeled off, wiped off after application, or washed off after application. . Powdered materials are used by dissolving or suspending uniformly in water or the like during use. Pack cosmetics in which a cosmetic material is impregnated into a nonwoven fabric or collagen sheet having a shape such as an eye mask or pack cosmetics in which the above nonwoven fabric is immersed in a cosmetic composition when used.

Pack cosmetics may contain 0.001 to 20% by weight, preferably 0.01 to 10% by weight, more preferably 0.1 to 5% by weight, most preferably about 0.2 to 2% by weight of the composition of the present invention. The other composition may be a known composition used for a pack cosmetic, and may be prepared according to a known production method.

However, without being limited to the above description, the above cosmetics may be used according to the method in which each is commonly used.

The cosmetic composition according to the present invention may further include selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymer polysaccharides, sphingolipids and seaweed extract in addition to chestnut extract as an active ingredient. The water-soluble vitamin may be any compound that can be incorporated into cosmetics, but preferably vitamin B1, vitamin B2, vitamin B6, pyridoxine, pyridoxine hydrochloride, vitamin B12, pantothenic acid, nicotinic acid, nicotinic acid amide, folic acid, vitamin C, and vitamin H. And salts thereof (thiamine hydrochloride, sodium ascorbate salt, etc.) and derivatives (ascorbic acid-2-sodium phosphate salt, ascorbic acid-2-magnesium phosphate salt, and the like). The water-soluble vitamin can be obtained by a conventional method such as a microorganism conversion method, a purification method from a culture of a microorganism, an enzymatic method, or a chemical synthesis method.

The oil-soluble vitamin may be any compound that can be formulated in cosmetics, but preferably vitamin A, carotene, vitamin D2, vitamin D3, vitamin E (d1-alpha tocopherol, d-alpha tocopherol, d-alpha tocopherol) , Derivatives thereof (ascorbic palmitate, ascorbic stearate, ascorbic acid dipalmitate, dl-alpha tocopherol acetate, dl-alpha tocopherolvitamin E, DL-pantothenyl alcohol, D-pantothenyl alcohol, pantothenyl ethyl) Ethers, etc.). Usability Vitamins can be obtained by a conventional method such as a microorganism conversion method, a purification method from a culture of a microorganism, an enzyme or a chemical synthesis method. The polymer peptide may be any compound as long as it can be formulated in cosmetics, but preferably collagen, hydrolyzed collagen, gelatin, elastin, hydrolyzed elastin, keratin, and the polymer peptide may be purified from a culture medium of a microorganism, an enzyme method. Or it can obtain and refine | purify by conventional methods, such as a chemical synthesis method, or can use it, refine | purifying normally from natural products, such as dermis, such as a pig and a cow, and silk fiber of a silkworm. The polymer polysaccharide may be any compound as long as it can be blended into cosmetics. Preferably, the polymer polysaccharide may be hydroxyethyl cellulose, xanthan gum, sodium hyaluronate, chondroitin sulfate or a salt thereof (sodium salt, etc.). Chondroitin sulfate or a salt thereof may be used after being purified from mammals or fish. The sphingolipid may be any compound that can be formulated into cosmetics, but preferably ceramide, phytosphingosine, sphingolipids, The pingo lipids can usually be purified from mammals, fish, shellfish, yeasts or plants by conventional methods or obtained by chemical synthesis. The seaweed extract may be any compound that can be formulated in cosmetics, but may preferably be brown algae extract, red algae extract, green algae extract, and also calginane, arginic acid, sodium arginate and arginic acid purified from these seaweed extracts. Potassium and the like are also included in the seaweed extract used in the present invention. Seaweed extract can be obtained by purification from seaweed by conventional methods.

In addition, the cosmetic composition of the present invention may include other components that are usually mixed with the cosmetics as needed in addition to the essential components.

Other ingredients that can be added include fats and oils, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, fungicides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, flavorings. , Blood circulation accelerators, cooling agents, restriction agents, purified water and the like. Examples of the fat or oil component include ester fats, hydrocarbon fats, silicone fats, fluorine fats, animal fats, and vegetable fats and oils.

As another embodiment, the present invention relates to an external skin composition for preventing and improving skin diseases caused by oxidative stress and inflammation containing chestnut extract as an active ingredient.

The topical skin composition of the present invention is a kind of pharmaceutical composition, and includes a pharmaceutically acceptable carrier, excipient, etc. in addition to the chestnut extract, which is an active ingredient thereof, and topical formulations such as creams, lotions, ointments (semi-solid external drugs). , Microroemulsions, gels, pastes, transdermal agents (TTS) (such as patches, bandages, etc.) and the like.

The term "pharmaceutically acceptable" as used herein means that the application (prescribing) subject does not have the above-mentioned toxicity that is adaptable without inhibiting the activity of the active ingredient.

Examples of pharmaceutically acceptable carriers include lactose, glucose, sucrose, starch (such as corn starch, potato starch, etc.), cellulose, derivatives thereof (such as sodium carboxymethyl cellulose, ethylcellulose, etc.), malt, gelatin, talc, Solid lubricants (e.g. stearic acid, magnesium stearate, etc.), calcium sulfate, vegetable oils (e.g. peanut oil, cottonseed oil, sesame oil, olive oil, etc.), polyols (e.g. propylene glycol, glycerin, etc.), alginic acid, emulsifiers (e.g. TWEENS), wetting agents (Such as sodium lauryl sulfate), colorants, flavors, stabilizers, antioxidants, preservatives, water, saline, phosphate buffer solutions, and the like. The carrier may be selected from one or more of suitable pharmaceutical formulations according to the formulation of the pharmaceutical composition of the present invention. The excipient may be selected according to the formulation of the pharmaceutical composition of the present invention. For example, suspending agents such as sodium carboxymethyl cellulose, methyl cellulose, hydropropyl methyl cellulose, sodium alginate, polyvinyl pyrrolidone, .

The external dermal composition of the present invention has a daily transdermal dose of 0.001 to 150 mg / kg body weight, and may be administered once or divided into several doses. However, since the dosage of the pharmaceutical composition of the present invention is determined in view of various related factors such as route of administration, age, sex, weight, and patient's severity of the patient, the dose is limited in any aspect to the scope of the present invention Should not be understood to be.

Since the chestnut extract-containing composition of the present invention is a natural vegetable component, there is almost no toxicity and side effects, so that it can be used with confidence even for long-term use.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Reagents, Instruments, and Methods

One. Antioxidant ability  And anti-inflammatory measurement reagents

1,1-diphenyl-2-picryl -hydrazyl (DPPH), xanthine, xanthine oxidase, nitro blue tetrazolium (NBT), tween-20, hyaluronidase, protease inhibitor, griess reagent , 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid), ripa buffer, Hemoglobin and the like were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The primary antibodies used in the experiment were purchased from iNOS BD Bioscience (Sanjose, CA, USA), COX-2 Cayman (Ann Arbor, MI, USA), and β-Actin (Santa Cruz, CA, USA). Anti-rabbit IG-G horseradish peroxidase (HRP) -conjugated antibody, a secondary antibody, was purchased from Santa Cruz, and an ELISA kit for the determination of PGE2, IL-1β, IL-6, IL-8, and TNF-α purchased from saystems Inc., Minneapolis, MN, USA) and from the cDNA polymerase kit, dNTP mixture (Promega, Madison, WI, USA).

2. Reagent for measuring whitening and antiwrinkle effect

Mushroom tyrosinase, tyrosine, L-3,4-dihydroxyphenyl-alanine (L-DOPA), dimethyl sulfoside (DMSO), porcine pancreas elastase (PPE), N-succinyl- (L) -Ala) 3-p-nitroanilide, collagenase and 4-phenylazobenzyl oxycarbonyl-Pro-Leu-Gly-Pro-D-Arg and the like are described in Sigma Chemical Co. (St. Louis, MO, U.S.A.) and used. For cell culture, Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), penicillin / streptomycin, trypsin 250, and 0.4% trypan blue stain were purchased from Gibco (New york, USA). cAMP kit (Cayman, Ann Arbor, MI, USA), MMP-1, MMP-3, MMP-9, MITF, TRP-1, TRP-2, Tyrosinase primary antibody and mouse-anti-goat, rabbit-anti- Secondary antibodies, such as mouse, were purchased from Santacruz (CA, USA). As the other various reagents, an express reagent was used.

3. Cell Lines and Reagents Used to Measure Cytotoxicity

The cell lines used for measuring cytotoxicity were purchased from ATCC, melanoma cells B16F10, fibroblast cells CCD-986k, and macrophage cells Raw 264.7. Cytotoxicity assay reagent 0.4% trypan blue stain was purchased from Gibco BRL Co. (Grand Island, USA) and Haemacytometer (Marienfeld, Germany) and used 3- [4,5-dimethylthiazol-2-yl] -2, 5-diphenyl-tetrazoliumbromide (MTT) is available from Sigma Chemical Co. (St. Louis, MO, U.S.A.) and used.

4. Strains and Media Used to Measure Antimicrobial Activity

Strains used in the antimicrobial activity screening test were Staphylococcus aureus KCTC 1621, Staphylococcus epidermidis KCTC 1917, Escherichia coli. KCTC 1039 was used, and propionibacterium acnes KCTC 3065 was passaged as an acne-producing bacterium. As a liquid medium for pre-culture and main culture, nutrient broth (NB) was purchased from Difco Lab. broth (TSB) was purchased from Difco Lab. (Sparks, US, A.) and used. As a liquid medium for pre-culture and main culture of propionibacterium acnes, it was purchased from Gifu anaerobic medium (GAM, Nissui Co. Japan). The solid medium was tryptic soy agar (TSA), nutrient agar (NA) was purchased from Difco Lab. It was purchased from Japan. In addition, Gifu anaerobi medium (GAM, Nissui Co. Japan) liquid medium was used for the experiment by adding agar powder to the medium.

5. Equipment used in the experiment

The instruments used in the bioactivity experiment were UV / VIS spectrophotometer (Hitachi, Japan), rotary vacuum evaporator (ToKyo, Rikakikai CO. Japan), centrifuge (Hitachi, Japan), freeze drier (Ilsin, Korea), microscope (Olympus, Japan). ), CO2 incubator (Hanbaek Scientific Co. Korea), pH meter (Metrohm, Switzerland), BOD incubator (Hanbaek Co. Korea), autoclave (Hanbaek Scientific CO. Korea), ELISA reader (Tecan Ma nnedorf), PCR Bio-Rad , USA), ABI step one plusTM (Applied Biosystems, CA, USA), and the instrument used in the cosmetic stability experiment was pH meter (Thermo Scientific, Canada, USA), homo mixer (Tokushu Kika Kogyo Co. Japan), It was measured using an agi mixer (Young Hana Tect. Korea) hot plate (Young Hana Tech. Korea).

6. LLCE  ( Lamellar Liquid Crystal Emulsion Reagents Used in Manufacturing

Methylparaben (Rita), Butylene Glycol (Daicel), Hydrogeneted Lecithin (Lipoid), Ceteayrl alcohol (Cognis), Caprylic / Capric triglyceride (Kokyu), Tocopheryl aceteate (Basf), Cyclopentasioxane (shhin Etsu), Phenoxyethanol (Basf), Triethan Purchased from Basf).

7. Cell Culture

DMEM medium containing 10% FBS and 1% penicillin / streptomycin (100 U / mL) was used for the culture of each cell. The cells were passaged at 37 ° C. and 5% CO 2 incubator.

8. MTT assay Cytotoxicity measurement

Cytotoxicity was measured according to the method of Carmichael. Dispense 0.18 mL of each cell line [melanoma (B16F10), macrophage (Raw 264.7) cell, fibroblast (CCD-986sk) cell] into 96 well plates at 5 x 104 cells / well, and add 0.02 mL After incubation for 24 hours at 37 ℃, 5% CO2 incubator. The control group was incubated under the same conditions by adding the same amount of distilled water. After incubation for 4 hours with the addition of 0.02 mL of MTT solution prepared at 5 mg / mL concentration, the culture medium was removed, 0.15 mL of DMSO was added to each well for 30 minutes at room temperature, and the absorbance was measured at 540 nm with an ELISA reader. . The cytotoxicity measurement was expressed as the absorbance decrease rate of the sample solution addition group and no addition group.

Cytotoxicity (%) = (1-absorbance of the sample addition group / absorbance of the no addition group) × 100

Example  1: chestnut extract preparation

Chestnut bur used in this experiment was harvested around October and November 2010 in Cheongdo-gun, Gyeongbuk and used as experimental materials.

Extraction of the sample was carried out as follows.

In the case of acetone extract, 70% acetone was added 10 times the weight of the sample, soaked at room temperature for 24 hours, and the supernatant and the precipitate were separated and extracted three times in the same manner. Each extract was centrifuged, filtered, concentrated, lyophilized, and stored as a sample in the refrigerator. 300 g of chestnuts were extracted with 70% acetone three times a week at room temperature, and then filtered with filter paper (Whatman No. 2). The filtrate was concentrated under reduced pressure to obtain an acetone extract (37.04 g). The acetone extract was suspended in distilled water, and then fractionated by adding different solvents using solvent polarity differences. Chestnut acetone extract and n-hexane were put into a fraction funnel at a 1: 1 ratio and fractionated with n-hexane. The n-hexane layer was concentrated under reduced pressure again to obtain 1.25 g of hexane fraction. Through the same process, an EtOAc layer (6.91 g), a BuOH layer (4.51 g), and H 2 O (18.79 g) were sequentially added to obtain each fraction.

Example  2: Determination of Antioxidant Effect of Chestnut Extract

2-1. Total polyphenol content measurement

Phenolic compounds are one of the secondary metabolites widely distributed in the plant system and have various molecular structures and molecular weights. Because they have phenolic hydroxyl (OH) groups, they easily bind to proteins and other macromolecules, and have various physiological activities such as antioxidants and anticancer. Most of the phenolic substances are plant sources and are found in a wide range of plants, including trees, bark, stems, leaves, fruits, roots, flowers, and seeds.

Polyphenol quantification was based on AOAC. That is, 1 mL of Folin-Ciocalteu phenol reagent reagent was added to 3 mL of a 100-fold diluted sample solution, 0.2 mL of 1N HCl was added, 1 mL of saturated solution Na2CO3 was added, mixed, and the mixture was left to stand at room temperature for 1 hour, followed by absorbance at 640 nm. After the measurement, the polyphenol content was calculated by comparing the absorbance value of the standard curve prepared with tannic acid as a standard substance. Table 1 shows the results of measuring the polyphenol content of chestnut extract after drawing a standard curve using the tannic acid as a reference material.

Sample Content (mg / g) Chestnut bur ext. 60.37 n-Hexane ext. 11.35 EtOAc ext 66.89 n-BuOH ext. 87.05 H ₂ O ext. 70.50

2-2. Electronic Donor  Measure

The electron donating ability was determined to confirm the availability of chestnut extract as a natural antioxidant. Electron donating ability (EDA) was measured by modifying Blois' method.

1,1 diphenyl-2-picrylhycrazyl (DPPH) is a relatively stable free radical with a deep purple color. It is reduced and decolorized by sulfur-containing amino acids such as cystein and glutathion, and L-ascorbic acid and BHA to detect antioxidants from various natural materials. It is used a lot to do it. Free radicals are prone to aging by binding to lipids or proteins in the human body. Phenolic compounds have a strong ability to reduce or offset free radicals and can be used as a measure of inhibiting aging by free radicals in the human body. Endogenous defense mechanisms against such active oxygen include enzymes such as glutathione S-transferase, glutathione peroxidase, superoxide dismutase and catalase. Exogenous defense mechanisms include vitamins A, C, E, flavonoid pigments and polyphenols.

Therefore, 1 mL of 0.2 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) was added to 2 mL of each sample solution, stirred for 30 minutes, and the absorbance was measured at 517 nm. The electron donating ability was expressed as the absorbance reduction rate of the sample solution addition group and no addition group.

Electron donating ability (%) = (1-absorbance of sample addition group / absorbance of no addition group) × 100

The results are shown in FIG. 2. At 100 μg / mL, the acetone extract showed 91.7%, ethyl acetate fraction 87.69%, butanol fraction 96.2%, and water fraction 78.9%. In the control group, BHA showed 96.9% electron donating ability at 100ug / mL. The chestnut butanol fraction showed more than 80% effect at 50 μg / mL, which was similar to that of butylated hydroxyanisole (BHA). Butanol fraction showed the strongest activity in chestnut fraction, and electron donating ability of n-BuOH> H ₂ O>EtOAc> n-hexane fraction.

This result shows that methanol extract showed 50.0% inhibition at 795 μg / mL in the electron donating ability of Yulmu in the conventional Kim et al. (Korea J. Food. Sci. Technol 32, 1409-1413, 2000); Tofu, Mokdanpi, Baekjak, and Bokbunja of Choi et al. (Korean J. Biotechnol.Bioeng 18 (4), 282-288,2003) were fractionated with ethyl acetate solvent, and their electron donating ability was determined to be at least 95.0% at 1,000 mg / mL. It showed better antioxidant capacity than the results indicated. In addition, as a result of measuring the electron donating ability of mugwort water extract and ethanol extract of Park et al. (Korean J.food preservation 9 (2), 248-252, 2002), it was 41% and 39% at 100ug / mL. According to the extraction conditions of turmeric according to the extraction conditions of An BJ et al. (Korean J. Medicinal Crop Sci. 14 (3), 168-172,2006), the effect of 89.0% was obtained at 1,000 μg / mL of supercritical extraction sample.

That is, through these results, the chestnut butanol fraction of the present invention showed more excellent antioxidant effect, it was confirmed that the availability as a natural antioxidant.

2-3. ABTS +. cation radical scavenging activity  Measure

ABTS +. Antioxidant activity using radical is ABTS +. It was measured by cation decolorization assay method.

ABTS + radical cation scavenging ability can measure both hydrogen donating antioxidants and chain-braking antioxidants and is applicable to both aqueous and organic phases. to be. ABTS radical cation scavenging activity is characterized by the reaction of 2,2-azino-bis (3-ethyl- benthiazoline-6-sulfonic acid) diammonium salt (ABTS) with potassium persulfate to produce ABTS + radicals. It is a method to measure the decolorization of light green color with addition, and can measure both hydrogen-donating antioxidant and chain breaking antioxidant.

7 mM 2,2-azino-bis (3-ethyl-benthiazoline-6-sulfonic acid) and 2.4 mM potassium persulfate were mixed and left at room temperature for 24 hours to form ABTS +., Diluted with ethanol and diluted with ABTS +. 100 μL of the sample was added to 100 μL, and left for 1 minute, and then measured at 732 nm absorbance.

% Erasure = (1-absorbance of sample addition group / absorbance of no addition group) × 100

As a result, the ABTS electron donating ability of the acetone extract and the solvent fraction is shown in FIG.

At 100 μg / mL, all the layers except the hexane fraction showed 99%, and at 5 μg / mL the butanol fraction was 93.9% and the water fraction 88.6%, which showed higher electron donating ability than the control butylated hydroxyanisole (BHA) 79.5%. Among the chestnut fractions, butanol fraction showed the strongest activity. This indicates that turmeric and turmeric extracts of the conventional Oh et al. (Kor. J. Herbology. 25 (1), 83-91, 2010) showed an efficacy of at least 75% at a concentration of 1,000 μg / mL in ABTS + radical cation scavenging ability. It shows lower potency than all extracts except chestnut hexane fraction. As a result, the chestnut extract can be seen that has a high scavenging ability of ABTS + cation.

2-4. Superoxide anion radical Scatters  Measure

Superoxide anion radical scavenging ability was measured by nitroblue tetrazolium (NBT) reduction method 46).

Superoxide molecular oxygen produced is reduced _{anion radicla (· O 2 - ·} 2O 2 + 2e- → 2 · 2O 2) key enzyme (2O involved in the first defense mechanism to remove the _{2 - + 2H + → H 2} O 2 + O _2- ), which prevents the generation of the most toxic hydroxy radicals, and is currently used as an additive for cosmetics as an anti-inflammatory material or a cosmetic material for preventing skin aging.

Add 0.1 mL of each sample solution, 0.6 mL of 0.1 M potassium phosphate buffer (pH 7.5), and 1 mL of substrate solution in which NBT (0.24 mM) is dissolved. Add 1 mL of xanthine oxidase (0.049 U / mL), and add 37 mL. After reacting for 20 minutes at 1 N HCl 1 mL to terminate the reaction, the amount of superoxide anion radical generated in the reaction solution was measured at 560nm.

% Erasure = (1-absorbance of sample addition group / absorbance of no addition group) × 100

Superoxide anion radical scavenging activity of the chestnut extract of the present invention is shown in FIG.

Chestnut Acetone extract showed 87.8% scavenging activity at the concentration of 1,000 μg / mL and 42.7% scavenging activity at 1,000 μg / mL for the n-Hexane. EtOAc was 91.89% at 1,000 μg / mL, 99.8% at 1,000 μg / ml n-BuOH, 83.3% at H2O, and 88.4% at 1,000 μg / mL in Vit.C. From the above results, it was confirmed that the superoxide anion radical scavenging ability of EtOAc and n-BuOH was superior to that of the control group Vit.C.

Inhibitory activity of each fraction showed radical scavenging ability in the order of n-BuOH>EtOAc>H2O> n-Hexane.

Example  3: measure the whitening effect

3-1. Tyrosinase  Measurement of inhibitory activity

Human skin color is determined by pigment components such as melanin and carotene, distribution of blood vessels, and thickness of the stratum corneum. Melanin is a pigment that blocks UV rays from the sun's rays, or when it is weakened due to aging, it is deposited on the surface of the skin and appears as spots, freckles, and various pigments. This melanin is biosynthesized in melanocytes in pigment cells called melanocytes in the basal epidermis. Tyrosine is oxidized to L-3,4-dihydroxyl phenylalanine (DOPA) and DOPA quinone by tyrosinase in melanocytes. DOPA quinone is then known as DOPA chrome, 5,6-dihydroxyindole, indole-5,6-quinone, followed by polymerization to indole-5,6-quinone to produce melanin. The aim of this study was to measure tyrosinase inhibitory activity that can effectively inhibit melanin polymer biosynthesis in skin.

Tyrosinase inhibitory activity was measured according to the method of Yagi et al. The reaction sphere was added with 0.2 mL of mushroom tyrosinase (110 U / mL) in a mixture of 0.2 mL of substrate solution dissolved in 10 mM L-DOPA in 0.5 mL of 0.175M sodium phosphate buffer (pH 6.8) and 0.1 mL of sample solution. The reaction was measured at 475 nm DOPA chrome produced in the reaction solution. The tyrosinase inhibitory activity was expressed as the absorbance decrease rate of the sample solution added and non-added.

% Inhibition = (1-absorbance of the sample addition group / absorbance of the no addition group) × 100

The results are shown in Fig.

Chestnut Acetone extract showed 50.4% inhibition at 1,000 μg / mL, and showed the highest n-BuOH with 10.6% n-Hexane fraction, 42.7% Ethyl acetate fraction, 82.1% n-BuOH fraction and 61.6% H2O fraction. Although inhibitory activity was shown, the control group Vit.C showed 99% at 500 ug / mL, which did not reach the effect. As a result of measuring the tyrosinase inhibitory activity of rhododendron extracts such as An, the results showed inhibition of 24.0% and 48.0% in hot water and ethanol extract 1,000ug / mL and tyrosinase inhibitory activity using Jeju plants such as Lee. At 1,000 μg / mL, the chloroform fractions of chicken ragweed, rowancho, acidic peppermint, iron knee, white powder, and ginseng vine showed 4.27%, 5.57%, 6.23%, 4.41% and 6.55%, respectively. Gardenia ethanol extract of Kwak et al. The tyrosinase inhibitory activity of the chloroform fractions was 62.7%, 72.6%, and 59.9% at 10,000 μg / mL, indicating that the activity of chestnut n-BuOH fractions was better.

3-2. Melanoma cell  (B16F10) of Survival rate  Confirm

The MTT screening method for checking cell viability is 96 well plate, and the test results are widely used as a cytotoxicity and cell proliferation screening method to easily read many samples by using an ELISA reader (multiwell microplate reader). One of the methods. In the case of cancer cells, yellow water-soluble MTT tetrazolium is reduced to purple water-insoluble MTT formazan by the action of mitochondrial dehydrogenase during metabolism. The absorbance of MTT formazan peaks at wavelengths near 550 nm, reflecting the concentration of metabolically active cells.

The survival rate of melanoma cells by chestnut extract was confirmed by MTT assay, and the results are shown in FIG. 6.

Cytotoxicity was confirmed as low as 80% in the Acetone extract and ethyl acetate fraction of chestnut extract up to 75 μg / mL, but 50 μg / mL for n-Haxane fraction, 20 μg / mL for n-BuOH fraction, and 10 for H2O fraction. Only up to μg / mL showed no cytotoxicity. The measurement of tyrosinase activity and melanin biosynthesis in melanoma cells (B16F10) was carried out at 10 μg / mL where all extracts were not toxic. The tyrosinase activity, melanin biosynthesis and whitening-related signaling factors of n-BuOH fractions were measured for survival. This was confirmed at concentrations of 5, 10, 15, and 20 μg / mL, which were more than 90%.

3-3. Melanoma cell At (B16F10) tyrosinase  Confirm inhibitory activity

The most important enzyme in the production of melanin is tyrosinase, a tyrosine hydroxylase that oxidizes tyrosine in melanocytes to form 3,4-dihydortyphenylalanine (DOPA) .It acts as a DOPA oxidase that oxidizes DOPA to make dopachrome and finally melanin polymer. Will be synthesized. Therefore, the experiment of inhibiting tyrosinase activity is useful in the development of skin whitening agent.

Cellular tyrosinase  Measurement of inhibitory activity

Melanoma (B16F10) cells were cultured in each culture dish, and the medium was removed and washed with PBS. 100 μL of lysis buffer (67mM sodium phosphate buffer, 1% trito X-100, 0.1mM phenylmethyl sulfonylfluoride) was added to each dish, and the cells were destroyed by standing on ice, and the cells were collected and ultra sonicated. The supernatant obtained by centrifugation at 13,200 rpm for 20 minutes was used as an enzyme solution. Then, 120mL of 8.0mM L-DOPA dissolved in 67mM phosphate buffer (pH6.8) and 40μL of sample solution were added to a 96-well plate, and 40μL of enzyme solution dissolved in 67mM phosphate buffer (pH 6.8) was added. After incubation for 5 minutes, the amount of produced DOPA chrome was measured at 490 nm.

% Inhibition = (1-absorbance of the sample addition group / absorbance of the no addition group) × 100

The results of measuring tyrosinase inhibitory activity of the extracts of the chestnut and the melanoma cells of the fractions are shown in FIG. 7.

Chestnut Acetone extract showed 28% inhibition effect at 10 μg / mL, and n-BuOH fraction showed 37.6% inhibition effect at 10 μg / mL, which was the most effective among the fractions. n-BuOH showed an inhibitory effect in a concentration-dependent manner.

3-4. Melanoma cell  (B16F10) In  Confirmation of melanin biosynthesis inhibition

Melanin is a pigment widely distributed in animals, plants, and microorganisms, and it is responsible for protecting the human body from harmful ultraviolet rays and free radicals, as well as determining skin color. This pigment is produced in the human body by melanocyte cells in the basal layer of the epidermis in the skin, activated by a variety of stimuli including ultraviolet rays, and migrates up together with keratin produced by keratinocyte cells. It exists in the stratum corneum, the outer layer. The biosynthesis of melanin pigment is known to be regulated by several enzymes including tyrosinase enzyme. Melanin, which is a cause of pigmentation and skin blackening, has a positive function of inhibiting and protecting skin photoaging and sun keratopathy by ultraviolet rays, and in terms of cosmetics, not only pigmentation but also toxicity by melanin precursors. It also has a negative function of facilitation. Phenolic derivatives such as hydroquinone and resorcinol, or L-ascrobic acid and its derivatives and Kojic acid, arbutin, lactic acid, glucosamine and tunicamycin, which inhibit melanin production, have been developed to cure pigmentation. There is a problem and only a very limited amount is used.

Melanoma cell Melanin biosynthesis inhibition rate measurement in (B16F10)

Inhibition of melanin biosynthesis from cutaneous melanoma cells was measured according to Hosoi et al. B16F10 cells incubated in DMEM medium were dispensed to 1 × 10 6 cells / dish in a 100mm culture dish, and cultured for 24 hours, 2 mL of the samples were prepared by concentration, and washed 48 hours later with phosphate buffer (pH 7.4). Then, the cells were detached with 0.25M trypsin-EDTA solution, and the harvested cells were treated with 1 mL of 5% TCA per 1 × 10 6 cells, centrifuged twice at 2,500 rpm, and the separated melanin was washed with phosphate buffer. After adding 1 mL of ether: ethanol (1: 3), the mixture was centrifuged twice and washed with ether 1 mL and dried. 1 mL of 1N NaOH was added to the dried melanin and reacted at 80 ° C. for 1 hour, and then the absorbance was measured at 405 nm. Inhibition of melanin biosynthesis was shown by the absorbance decrease rate of the sample solution addition group and no addition group.

Melanin Synthesis Inhibition Rate (%) = (1-Absorbance of Sample Additive / No Absorption) x100

The results of measuring the melanin biosynthesis inhibitory effect in the melanoma cells of the chestnut fraction is shown in FIG.

Chestnut Acetone extract showed a 41% effect at a concentration of 10 μg / mL and n-BuOH showed a 65% effect, which was the most effective among the chestnut fractions.

Example  4: wrinkle improvement effect

4-1. Elastase  Measurement of inhibitory activity

Aging is the functional, structural, and biochemical rice padding that occurs throughout the organs of cells and body tissues. Human skin is exposed to intrinsic aging, externally contaminated air, drugs, and ultraviolet light, which are caused by hormonal secretion decreases over time, and the function and activity of immune cells decreases, thereby reducing the biosynthesis of bio-constitutive proteins. By photo aging, the skin becomes thinner, wrinkles are increased, elasticity is reduced, as well as blemishes, freckles and blotch are increased. Elastase derived from fibroblasts plays an important role in the three-dimensional distortion of skin elastic fibers, and the increase of elastase activity contributes to skin wrinkle formation by reducing skin elastic fibers. Elastase, which is present in human neutrophil granulocytes, is an enzyme that degrades elastin, a substrate protein important for maintaining skin elasticity in the dermis, and a nonspecific hydrolase that can degrade collagen, another important substrate protein. Therefore, elastase inhibitors are effective in improving skin wrinkles, and ursolic acid is used as an elastase inhibitor.

Therefore, the absorbance of elastase activity was measured by measuring the color change caused by the degradation of nitroanilide (NA) using N-succinyl- (Ala) -3-p-nitroanilide, an elastase substrate that is important for maintaining skin elasticity in the dermis. Confirmed.

Elastase inhibitory activity was measured according to the method of Cannell et al. In other words, using N-succinyl- (L-Ala) 3-p-nitroanilide as a substrate, the amount of p-nitroanilide produced from the substrate at 37 ° C. for 20 minutes was measured at 405 nm. That is, each test solution was prepared to a certain concentration, 0.5 mL of each was prepared in a test tube, 0.5 mL of porcine pancreas elastase (2.5 U / mL) solution dissolved in 50 mM tris-HCl buffer (pH 8.6) was added, and then 50mM Tris- as a substrate. N-succinyl- (L-Ala) 3-p-nitroanilide (0.5mg / mL) dissolved in HCl buffer (pH 8.6) was added and reacted for 20 minutes. Elastase inhibitory activity showed a decrease in absorbance of the sample solution added and non-added.

% Inhibition = (1-absorbance of the sample addition group / absorbance of the no addition group) × 100

Wrinkle production and related elastase inhibitory activity measurement results are shown in FIG. 9.

In the case of chestnut Acetone extract, 100 μg / mL and 59.55% showed similar inhibition rate to the positive control of EGCG 1000 μg / mL and 58.08%. In the case of n-BuOH, 50 μg / mL, 74% showed a high inhibitory effect even at low concentrations.

As a result of measuring the elastase inhibitory activity from various medicinal plants of Kwak et al. (Korean J. Medicinal Crop Sci 13 (6), 213-216, 2005), most of them showed less than 30.0% of inhibitory activity at the concentration of 1 mg / mL. Kim, et al. (J. Soc. Cosmet. Scientisis Korea 30 (1), 15-22, 2004) measured the elastase inhibitory activity against safflower seed extract and ursolic acid, genistein, and oleanolic acid known as plant-like hormones. IC50 was 50.8 μg / mL, safflower seed extract was 265 μg / mL, and genistein and ursolic acid showed inhibition rate of 0.5 mg / mL at 31.7% and 4 mg at 4 mg%, respectively. Thus, it was confirmed that the chestnut n-BuOH fraction layer of the present invention showed better elastase inhibitory activity.

4-2. Collagenase  Measurement of inhibitory activity

Collagen, the major constituent of the extracellular matrix, is the major matrix protein produced by skin fibroblasts. It is also an important protein, accounting for about 30% of the total weight of biological proteins, and has a solid triple helix structure. Collagen forms most of the organic matter in the skin, tendons, bones and teeth, especially in the bones and dermis of the skin. Collagen's main functions are known as mechanical firmness of skin, resistance of connective and connective tissues, support of cell adhesion, induction of cell division and differentiation.

As age increases, the number of fibroblasts and cell number decrease, which reduces the amount of fiber (collagen and elastin), decreases the moisture in skin cells, and changes the structure of the stratum corneum. In addition, the action of collagenase is increased to increase the cross-linked form of collagen, thereby reducing the smoothness, moisturizing and tension. Collagen is not affected by proteases such as trypsin, but it has been reported to be degraded by collagenase.

Collagenase inhibitory activity of chestnut extract was measured according to the method of Wunsch et al. In other words, the reaction sphere was added with 4 mM CaCl 2 in 0.1 M tris-HCl buffer (pH 7.5), 0.25 mL of substrate solution in which 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (0.3 mg / mL) was dissolved, and the sample. 0.15 mL of Collagenase (0.2 mg / mL) was added to 0.1 mL of the mixed solution, which was allowed to stand at room temperature for 20 minutes, and then 0.5 mL of 6% citric acid was added to stop the reaction. Absorbance was measured. Collagenase inhibitory activity was indicated by the absorbance decrease rate of the sample solution added and the non-added sample.

% Inhibition = (1-absorbance of the sample addition group / absorbance of the no addition group) × 100

 The results are shown in FIG.

Chestnut Acetone extracts were 54.73% at 1000 μg / mL and solvent fractions were 53.44%, 57.22%, 59.12% and 55.79% at 1000 μg / mL, respectively. At 1000 μg / mL, the inhibitory activity was similar, but at low concentration of 5 μg / mL, n-butyl alcohol was 25%, which was higher than the positive control EGCG. The results of the collagenase inhibitory activity of the extracts of Lee, et al., Showed 27.6% at 100 μg / mL and 40, 37.5%, and 35%, respectively, at 250 μg / mL of Kangsa, Yin and Yang Guk Chestnut n-butyl alcohol was confirmed to show a better inhibition rate.

4-3. Fibroblast cell  ( CCD -986 sk ) Survival rate

The survival rate of fibroblasts by chestnut extract was confirmed by MTT assay and is shown in FIG. 11.

Cytotoxicity was observed from 75 μg / mL in the n-butyl alcohol and water fractions. Collagen biosynthesis and western blot and polymerase chain reaction (PCR) were measured at fractions below 20 μg / mL with low cytotoxicity.

4-4. Fibroblast  ( CCD -986 sk ) In collagen Biosynthesis amount  Confirm

Collagen is a major structural protein that accounts for 75% of the dry weight of skin. It is produced from fibroblasts, which are the main cells of the dermal layer, and then secreted into extracellular matrix to maintain elasticity of the skin through the triple helix structure. The collagen in the body is divided into 20 types, and the skin dermis contains type I, III, V, VI, Ⅶ type collagen, especially collagen type I is 80 ~ 85% of the total collagen, and collagen type III is 10 ~ 15 Occupies% Collagen is synthesized through hydroxylation process with coenzyme of vitamin C, Fe2 + and silicon. Proline and lysine, the major amino acids present in collagen proteins, function to maintain skin elasticity by forming posttranslational modifications, which are hydroxylated, and triple helix structures of collagen, respectively. Stabilize the structure. Hydroxyproline, which is less quantitative than hydroxylproline, plays an important role in stabilizing the fiber, which is the final stage of collagen synthesis, as well as in triple helix stabilization, where the trace element skilicon regulates enzyme activity to stabilize collagen. Let's do it. Collagen (I, II, III, IV and V) are synthesized in the form of precursors called pro-collagen. pro-collagen includes peptide base sequences called propeptides at the amino and carboxy termini. The function of the propeptide is known to assist in the folding of pro-collagen molecules in the endoplasmic reticulum and to be cleaved and separated from collagen molecules when collagen polymerization occurs. Thus, by measuring the amount of isolated propeptide, it is possible to determine the degree of collagen biosynthesis in cells.

Figure 12 shows the results of measuring collagen biosynthesis by procollagen type I C-peptide (PICP) enzyme immunoassay on fibroblasts of chestnut extract.

Chestnut exports showed an effect of 56.71% at 50 μg / mL. As a result of measuring collagen biosynthesis of milk extract of Lee et al., It showed 44.4% biosynthesis at 200 μg / mL. The collagen biosynthesis effect of the n-BuOH layer was found to be high.

Example  5: anti-inflammatory effect measurement result

5-1. Macrophage cell  ( Raw264 .7) survival rate check

Macrophage, which acts as a first-line immune mechanism, is a phagocytic cell produced by bone marrow, which specifically detects and digests foreign substances that enter the body, and various cytotoxic substances such as hydrogen peroxide (H ₂ O ₂ ) and NO. It is an immune cell that secretes and destroys heterologous cells or cancer cells.

The results of confirming the toxicity of macrophage cells by chestnut fraction by MTT assay are shown in FIG. 13.

As a result of determining whether the chestnut fraction reduced the production of LPS-induced nitiric oxide or was caused by the decrease of the cell population due to the cytotoxicity of the compound, n-Hexane, EtOAc, n-BuOH, Water as shown in FIG. Both layers showed a survival rate of 90% even at a concentration of 20 μg / mL. Nitric oxide measurements were performed at concentrations below 20 μg / mL with survival rates of 90% or higher.

5-2. Nitric oxide  Inhibitory Activity Measurement Results

Nitric oxide (NO) was measured by the amount of nitric oxide (NO) in the cell supernatant as nitrite and griess. The griess reagent (Sigma, USA), which is a safe form after reduction to nitrate for nitrite, was used.When 5 × 10 ⁵ cell confluence is 80% in 6 well plates, serum-free medium is washed after washing twice with PBS. After incubation for more than 12 hours using the LPS 1 mμg / mL in all wells except the control group was stimulated. After 2 hours, the samples were treated by concentration. NO production was measured after 24 hours after the supernatant was collected and reacted with griess reagent for 10 minutes at 540 nm. In the group where only LPS was added, the amount of NO produced was 100%, and the absorbance measured when the sample was added was expressed.

In order to measure the degree of NO production inhibition of Raw264.7 cells, the result of measuring the amount of NO produced by treating the cells of the chestnut fraction by concentration is shown in FIG. 14.

LPS treated group showed higher NO expression than LPS untreated group, and it was confirmed that NO expression was reduced in each fraction layer. In the case of EtOAc and n-butanol, it was confirmed that the inhibition rate was nearly 60% at a concentration of 20 μg / mL. Inhibition of inflammatory expression in macrophage lines showed a high effect of the chestnut EtOAc and n-butanol layers. In particular, n-butanol was found to show an effect close to 40% even at low concentrations of 10 μg / mL. Inhibition of NO production in raw 264.7 cells induced by LPS-induced situational hydrothermal extract of Kim et al. And NO production of n-butanol at night was higher than that of Injinho-tang such as Yun 74% at 300 μg / mL. It was confirmed that the inhibitory effect is excellent.

Example  6: Antibacterial effect measurement result

6-1. Bacterial culture

As a liquid medium for preculture and main culture, Staphylococcus aureus KCTC 1621, Staphylococcus epidermis KCTC 1917, Escherichia coli KCTC 1039 were used. Nutrient broth (NB) was used as a liquid medium for Escolichia coli, Staphylococcus aureus, and Staphylococcus epidermidis as a liquid medium for pre-culture and main culture, and agar was added to the liquid medium. Staphylococcus aureus KCTC 1621, Staphylococcus epidermis KCTC 1917, Escherichia coli KCTC 1039 were incubated at 37 ° C in a CO ₂ incubator.

6-2. Growth inhibition ring ( Clear zone ) Confirm

In general, among the skin flora, Staphylococcus aures, Staphylococcus epidermidis, Exdherichia coli, Propinoibacterium acnes, etc. are reported. Staphylococcus aures, Staphylococcus epidermidis, Exdherichia coli, Propinoibacterium acnes It is a common cause of a wide range of diseases in animals. Staphylococcus aures, Staphylococcus epidermidis, Escherichia coli, and Propinoibacterium acnes are the causative agents of purulent disease, causing food poisoning and sepsis, and these bacteria are widely distributed in the nasal cavity and body surface of healthy people and animals as well as purulent. A bacterium called Staphylococcus aureus, which is the most representative strain of the skin flora that normally exists on our skin.It does not harm the human body and causes medical problems.But recently, many strains showing antibiotic resistance have become Sepsis can occur or central nervous system opportunistic infections can occur. Escherichia coli, a pathogenic food poisoning bacterium that has recently emerged in the food industry, can be classified into Enterotoxigenic Escherichia coli (ETEC), Enteropathogenic Escherichia coli (EPEC), Enterohemorrhagic Escherichia coli (EPEC), and Enteroaggregative Escherichia coli, depending on the presence of pathogenic toxins and infection patterns. (EAEC), Enteroinvasive Escherichia coli (EIEC), and Diffusely adherent Escherichia coli (DAEC). Bacteria that reside in the acne of the acne include Propionibacterium acnes, Propionibacterium granulosum, and Staphylococcus epidermidis, and the evidence that P. acnes is involved in inflammation is the action of cytotoxicity by P. acnes, inflammatory mediators secreted from P.acnes, and enzymes. In addition to humoral or cell mediated area transfer has been reported. When the skin pores are blocked by various factors, propinoibacterium acnes, an absolute anaerobic bacterium, proliferates in the pores, secretes lipase, a lipolytic enzyme, and breaks down triglyceride, a major component of sebum, into free fatty acid. The acid stimulates the hair follicle wall epithelial cells to increase keratinocyte formation, and at the same time, stagnant hyperkeratosis to form microsurfaces, an early stage of acne formation, and the secretion of keratin and sebum continues to thin the hair follicle wall. It causes inflammation.

Growth Inhibition ( Clear zone ) Measure

Antibacterial activity was measured using a paper disc method. That is, one platinum is taken from each strain cultured in a plate medium, incubated for 18 to 24 hours in 10 mL of liquid medium, and then inoculated with 0.1 mL of the bacterium solution in 10 mL of liquid medium, followed by 3 to 6 hours of main culture. The cells were inoculated to be about 1 × 10 ⁷ cells per plate and evenly spread with a sterile swab. Place the sterilized filter paper disc (8mm, Tokyo, Japan) on a solid plate medium, absorb the sample by concentration to 0.05 mL / disc, and incubate at 37 ℃ for 18 ~ 24 hours to make clear zone (mm) around the disc. The diameter was measured.

The results of examining the antimicrobial effect of the chestnut fraction are shown in Table 2 and FIGS. 15-18.

All fractions except n-hexane showed antimicrobial effects against Staphylococcus aures, Staphylococcus epidermidis, Escherichia coli and Propinoibacterium acnes. n-BuOH fraction showed growth inhibition at 0.1 mg / disc or higher for all the tested bacteria, and growth inhibition rings at 9.3 ± 4.2mm and 8.2 ± 2.4mm at 1mg / disc for Escherichia coli and Staphylococcus aures, respectively. Indicated.

Example  7: Preparation of Various Formulations Containing Chestnut Extract

7-1. Preparation of Skin Lotion

Skin lotion in the cosmetic preparation containing the chestnut extract of Example 1 was prepared as follows.

7-2. Manufacture of lotions

A lotion in the cosmetic preparation containing the chestnut extract of Example 1 was prepared as follows.

7-3. Serum  Produce

Serum of the cosmetic containing the chestnut extract of Example 1 was prepared as follows.

7-4. Manufacture of cream

The cream of the cosmetics containing the chestnut extract of Example 1 was prepared as follows.

7-5. Check the performance of the formulation

To make sure that dermatitis occurs when using the developed raw materials or products, a patch test is performed on the arm and back of a person as a simple preliminary experiment.

The patch test was performed using Waggoner, Matsumura, and Aberer, Kim's method for 30 hours using a Finn chamber on Scanpor tape on the skin of the inner plate of the formulation containing chestnut n-BuOH stored at room temperature for 30 days. After sealing the patch, the reaction of the skin was visually confirmed. The experiment was conducted on 10 male and female subjects.

The results of the patch test using the formulation prepared above are shown in the table below.

Skin lotion

Lotion

Serum

cream

As such, the human patch test test results confirmed that the composition containing the chestnut extract of Example 1 belongs to a non-irritant range and is a safe substance to the human body.

Claims (15)

Cosmetic composition containing butanol extract of chestnut as an active ingredient having antioxidant activity, whitening activity, anti-inflammatory activity, antibacterial activity and anti-wrinkle activity.
delete delete delete delete delete The method of claim 1,
Cosmetic composition, characterized in that it comprises 0.001 to 10% by weight of the extract relative to the total weight of the composition.
delete The method of claim 1,
The composition includes skin lotion, skin softener, skin toner, lotion, milk lotion, moisturizing lotion, nourishing lotion, massage cream, nourishing cream, moisturizing cream, hand cream, essence, nutrition essence, pack, soap, shampoo, cleansing foam, cleansing A cosmetic composition comprising a formulation selected from the group consisting of lotions, cleansing creams, body lotions, body cleansers, emulsions, press powders, loose powders and eye shadows.
delete delete delete delete delete delete

Images