KR20090098480A - A skin care agent containing azolla imbricata extract - Google Patents

Abstract

A skin external composition containing Azolla imbricate extract is provided to ensure the effects of suppression of tyrosinase activity and melanogenesis and removement of oxygen free radical. A skin external composition comprises 0.001~30.0 weight% of Azolla imbricate extract. The Azolla imbricata extract is isolated using purified water, methanol, ethanol, glycerin, ethyl acetate, butylenes glycol, propylene glycol, or hexane. The composition is used in the form of gel, water soluble liquid, cream, essence, water in oil or oil in water.

Description

A skin care agent containing Azolla imbricata extract}

The present invention relates to an external composition for skin containing water frog leaf extract as a main active ingredient, more specifically, it contains 0.001 to 30.0% by weight of water frog leaf extract, active oxygen scavenging effect, tyrosinase activity inhibitory effect and melanin biosynthesis inhibitory effect, The present invention relates to an external skin composition having excellent skin wrinkle improvement, acne suppression effect and hair growth effect.

As a person ages, his skin is constantly changing due to skin aging and pigmentation. The factors that determine the color of the skin basically include parts such as tanning due to blemishes, freckles and sun exposure, or overall pigmentation, other than acne, scars, and keratin distribution, excluding differences according to race, region, sex and age. And blood circulation, stress, and health. Among the above factors, the most important factor is pigmentation.

The pigments affecting skin color include melanin, carotene, and hemoglobin, the most important of which is melanin. The most important factors affecting melanin biosynthesis are ultraviolet and hormone secretion. Melanin biosynthesis is a series of oxidation processes that begin with the oxidation of tyrosine, a type of amino acid, from melanocytes of melanocytes to tyrosinase and dihydroxy phenylalanine. (eumelanin) polymer. This biosynthesis process is carried out in a special type of brown intracellular organelles called melanosomes. Melanosomes, including melanin granules, move from the periphery of the nucleus to the dendritic end, and then into the cytoplasm by the phagocytosis of keratinocytes. Accumulate around the nucleus of the site. The synthesis of melanin, the number of melanosomes, and the migration to surrounding keratinocytes are primarily genetically affected, in part by hormones and ultraviolet rays. In addition, cytokines (cytokine), copper ions, copper, zinc, iron, and other metal ions involved in the expression of tyrosinase and the synthesis and transfer of melanin are known to be involved in interferon, prostaglandin, histamine, etc. Society of Cosmetic Scientists, 25: 77-127, 1999). Therefore, the skin whitening effect can be expected by inhibiting the synthesis of melanin, which determines the skin color, and the activity of tyrosinase, an enzyme involved therein.

Moreover, the factor which shows the characteristic of aging with skin color is the formation of wrinkles, and the fall of elasticity. Skin aging can be divided into intrinsic, chronological aging and photoaging [Gilchrest BA: J. Am. Acad. Dermatol., 21, 610-613 (1989). Endogenous aging is a naturally occurring aging phenomenon caused by deterioration of physiological functions of living organisms with increasing age [Braverman IM et al .: J. Invest. Dermatol., 78, 434-443 (1982). Photoaging means that the skin is repeatedly exposed to light to change the appearance or function of the skin [Ridder GM et al .: J. Am. Acad. Dermatol., 25, 751-760 (1991).

In addition, skin aging may be caused by the activation of free radicals according to UV rays, stress, disease conditions, environmental factors, wounds, age, and when this condition is intensified, destroys the antioxidant defenses in vivo, cells and Damage to tissues promotes adult disease and aging. More specifically, lipids, proteins, polysaccharides, nucleic acids, and the like, which are major constituents of skin, are oxidized to destroy skin cells and tissues, resulting in skin aging. In particular, the oxidation of proteins is caused by severe cuts in the skin's connective tissue such as collagen, hyaluronic acid, elastin, proteoglycan, and fibronectin, resulting in severe hyperinflammatory reactions and skin elasticity. If this becomes worse, the mutation of DNA causes mutations, cancers, and immune function deterioration. Therefore, the cell membrane is protected by eliminating reactive oxygen species, which are mediated by the body's metabolic process, or by irradiation with ultraviolet rays or inflammatory reactions, and the already damaged cells are regenerated by active metabolism to proliferate the skin. You can recover quickly and maintain healthy skin.

In vivo, the synthesis and degradation of extracellular matrix such as collagen is properly regulated, but its synthesis decreases as aging progresses, and the matrix metalloproteinase (MMP), an enzyme that degrades collagen, Expression is promoted, the elasticity of the skin is reduced, wrinkles are formed. In addition, these enzymes are also activated by ultraviolet radiation and free radicals. Matrix metalloproteinases (MMPs) are secreted from various cells in the body, including keratinocytes and fibroblasts in human skin. They act as calcium and zinc-dependent endopeptidase and operate at neutral pH. Various extracellular substrates are used as substrates. MMPs are classified into MMP-1, MMP-2, and MMP-9 according to their substrate specificity. MMP-1 is a fibroblast type collagenase, an enzyme of epilepsy, and collagen type I, II, III, Ⅶ as substrate. There are, Ⅹ, Ⅹ and gelatin, cut into 3/4 or 1/4 length peptides of original collagen length to facilitate the action of nonspecific enzymes (gelatinase). MMP-2 is a 72 kD gelatinase that degrades gelatin, collagen types IV, V, VIII, VIII, elastin and fibronectin as substrates. Metallolastase also degrades elastin with MMP-13. Collagen types I and III, elastin and fibronectin, which occupy most of the dermal layer of human skin, give strength and tension to the skin. Wrinkles, loss of elasticity, and sagging skin when proteins are decomposed by abnormally activated MMP by ultraviolet rays and free radicals, etc. Aging phenomenon is accelerated. In particular, by inhibiting the activity and biosynthesis of MMP-1, which degrades type I collagen, which accounts for the largest proportion of binding proteins, skin binding proteins can be protected to prevent wrinkle formation and elasticity. Therefore, there is a need for the development of a substance capable of regulating or inhibiting MMP expression in which activation is induced in cells by ultraviolet rays. Until now, most of the raw materials used as external skin preparations simply inhibited MMP enzyme activity.

In addition, male alopecia is male hormone-dependent, and thus has a direct relationship with the amount of male hormone. Accordingly, many studies have been reported for the prevention and treatment of hair loss through the inhibition of male hormone activity. On the other hand, when the function of the sebaceous gland is increased by the increase in the secretion of male hormones, the scalp produced by stagnation in the hair follicles due to the hyperkeratosis of the hair follicle wall is an early stage of acne. When explaining the mechanism of hair loss and acne caused by male hormones, 5 alpha-Reductase is present in testosterone-reactive tissues such as sebaceous glands, hair follicles, prostate and epididymis, and is a testosterone. ) Is an enzyme involved in the metabolism of dihydrotestosterone into dihydrotestosterone, which requires NADPH. In addition, testosterone is involved in skeletal muscle increase, male external genitalia, scrotum growth, spermatogenesis, etc., and dihydrotestosterone is involved in such tissues as acne, sebum increase, hair loss, and enlarged prostate (Diane et al. JID 1995). In particular, excessive secretion of male hormones after puberty leads to acne and hair loss, and to prevent hair loss and anti-acne agents by using 5-alpha-reductase inhibitors to prevent excessive production of dehydrotestosterone by 5 alpha-reductase. There are active researches to develop the system.

Recently, in order to reduce skin irritation caused by various chemicals and the like, much attention has been focused on functional cosmetics having functions such as antioxidant, wrinkle relief, and whitening obtained from natural extracts. Natural materials have less side effects on the skin, and as the consumer's response to the external skin preparations using natural materials has recently increased, the development value of the external skin preparations has increased. For example, US Pat. No. 5,972,341 describes the anti-wrinkle effect of a plant of the genus Commiphora, in particular Commiphora mukul. Japanese Patent Application Laid-open No. Hei 9-672662 describes seaweed extracts having a hyaluroronidase inhibitory effect. Japanese Patent Application Laid-open No. Hei 10-85905 describes the skin texture improvement effect of Fucoidan extracted from Wakame. Japanese Patent Application Laid-Open No. 2-245087 describes the antioxidant effect of Sargassum genus extract. Japanese Patent Application Laid-Open No. 3-294384 describes an antioxidant composition extracted from the genus Hijikia. Japanese Patent Laid-Open No. 7-10769 describes a extract of Phaeophyceae having an astringent effect.

Therefore, an object of the present invention is to select a natural product having a low human hazard, and to confirm its efficacy as an external preparation for skin, to provide an excellent external preparation for skin.

In addition, an object of the present invention is to provide an external preparation for skin that exhibits a predetermined functional effect such as whitening and wrinkle improvement without skin irritation.

That is, an object of the present invention is to provide a natural extract that can be applied to the skin and inhibits the production of melanin and the activity of enzymes related thereto when contained in the external preparation for skin, thereby exhibiting an excellent whitening effect.

In addition, it is an object of the present invention is applicable to the skin external preparations, and when contained in the external preparations of the skin and exhibits an excellent anti-aging effect, such as free radical scavenging effect, MMP-1 biosynthesis and type 1 procollagen biosynthesis effect, skin wrinkle improvement effect To provide an extract.

It is also an object of the present invention to provide a natural extract that exhibits 5 alpha-reductase inhibitory effect with excellent hair loss prevention and acne prevention effect.

Accordingly, the present inventors have studied the possibility of applying as a skin external preparation for various natural substances that have not been known as cosmetics or external preparations for skin, and thus selected natural frog frog rice, which is an outpost of Golanaceae fern family, and prepared extracts therefrom. Satisfying by measuring active oxygen scavenging effect, tyrosinase activity inhibitory effect and whitening effect due to melanin biosynthesis inhibitory effect, skin wrinkle improvement effect, 5 acne-reductase inhibitory effect acne prevention and hair loss prevention and hair growth effect One result was found to be applicable as an external skin preparation.

The external composition for skin of the present invention exhibits excellent whitening effect by inhibiting melanin production and related enzyme activity without skin irritation, free radical scavenging effect, MMP-1 biosynthesis and promoting type 1 procollagen biosynthesis, skin It shows excellent anti-aging effects such as wrinkle improvement, and shows excellent hair loss prevention and acne prevention.

As a raw material used in the composition of the present invention ( Azolla imbricata ) is an evergreen perennial herb of ferns and ferns, and grows on water in paddy fields or ponds. The stem is divided into feathers and forms a triangular whole and 1 ~ 1.5㎝ in length, and has a thin root and hairs. Leaves are petioles, scaly, divided into 2, with fine processes, and the fragments are crusted (translucent like thin paper), triangular, round, and blunt. The leaf surface is green in summer but turns red in winter, and because of its deep color, the water looks red and is also called Manganghong (홍 江 紅). The spore sac comes under the first leaf of the branch extending sideways, and there is one spore in the sac. The vesicle sac has many spores and is wrapped in a round vesicle. It is distributed in Korea, Japan, Taiwan, China, and Indochina.

The present invention provides an external composition for skin containing the frog frog rice extract. In the present invention, "skin external preparation" refers to cosmetics, functional cosmetics and medicines, quasi drugs used for the purpose of applying to the skin to improve wrinkles, whitening, acne prevention and / or treatment, hair growth, hair loss prevention and / or treatment, etc. It used to contain.

The frog frog rice extract of the present invention is characterized in that extracted using one or more solvents selected from purified water, methanol, ethanol, glycerin, ethyl acetate, butylene glycol, propylene glycol, dichloromethane and hexane.

In addition, the present invention is characterized in that the extract is obtained by cold condensation at room temperature, a liquid obtained by heat filtration, further concentrated by a solvent concentrated under reduced pressure or lyophilization.

In addition, the present invention is characterized in that the content of the frog frog rice extract is 0.001 ~ 30.0% by weight based on the dry weight of the whole skin external preparation composition. When the content of the extract is less than 0.001% by weight, there is almost no skin improvement effect, and when the content of the extract is greater than 30.0% by weight, the effect of increasing the content is not economical.

In addition, the present invention, the external composition of the skin, frog frog rice extract, characterized in that selected from the formulation of the lotion, gel, water-soluble liquid, cream, essence, oil-in-water (O / W) type and water-in-oil (W / O) type It provides a topical skin composition containing the main active ingredient.

In addition, the present invention is characterized in that the topical skin composition is a cosmetic composition or a pharmaceutical composition.

Water frog frog extract can be formulated into pharmaceutical compositions such as capsules, liquids, ointments, patches or sustained-release agents using a pharmaceutically acceptable carrier, pharmacologically acceptable bases, carriers, excipients, binders ( Examples: starch, tragacanth gum, gelatin, molasses, polyvinyl alcohol, polyvinylether, polyvinyl pyrrolidone, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose and carboxymethyl cellulose), grinding agents (e.g., agar, Starch, vellatin powder, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, crystalline cellulose, calcium carbonate, sodium bicarbonate and sodium alginate), lubricants (e.g. magnesium stearate, talc, hydrogenated vegetable oils), colorants and the like. The carriers and excipients include lactose, glucose, sucrose, mannitol, potato starch, corn starch, calcium carbonate, calcium phosphate and cellulose. In addition to the above, additives such as adjuvants such as stabilizers, dissolution aids, transdermal absorption accelerators, and preservatives may be further added.

The bottle isocho extract provided by this invention can be made into acid addition salt as needed. As acid addition salts, for example, salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, salts with organic acids such as acetic acid, propionic acid, citric acid, lactic acid, oxalic acid, maleic acid, fumaric acid, succinic acid, tartaric acid and methanesulfonic acid Can be mentioned. Such salts can be easily prepared by conventional methods.

Isocho extract of the present invention has excellent hair growth promoting effect, hair growth effect. Therefore, applying it to the scalp can treat and improve hair loss and prevent hair loss.

Isocho extract of the present invention can be applied to pathological alopecia such as alopecia areata, alopecia areata, seborrheic alopecia in addition to hair loss, or less hair loss, also known as androgenetic alopecia and male hormonal alopecia. As the amount of the medicinal plant extract of the present invention, which is appropriately determined according to sex, age, degree of symptoms such as hair loss and shrinking hair, etc., usually 0.01 to 20 mg / cm 2 is applied to the scalp once a day or several times per adult. do.

In addition, when the frog frog rice extract of the present invention is used in medicines, quasi-drugs, or cosmetics for the purpose of hair growth, hair growth promotion, hair loss prevention, etc., the formulation is not particularly limited as long as the formulation can exert the effects of the present invention. It can be arbitrarily selected, and examples thereof include tonic, lotion, emulsion, cream, ointment, gel, spray, mousse and the like.

In addition, in such a formulation, in addition to the water frog rice extract according to the present invention, it is possible to formulate ingredients that can be conventionally blended into wool in the fields of medicines, quasi-drugs, and cosmetics. For example, drugs, such as vitamin E and its derivatives which have a blood circulation promoting effect, estradiol which has an anti-male-hormone effect, hormone agents, such as estrone, etc. are mentioned.

Vasodilation agents such as alkoxycarbonylpyridine N-oxide and acetylcholine derivatives; Skin hyperfunctional agents, such as ceparantin; Antibacterial agents such as hexachlorophene, undecylenic acid, trichlorocarbanide, and bithionol; Zinc and its derivatives; Lactic acid or alkyl esters thereof; Organic acids such as citric acid; Protease inhibitors, such as a Trane samsamic acid, etc. can be mix | blended.

Moreover, alcohol, such as ethanol and isopropyl alcohol; Polyhydric alcohols such as glycerin, propylene glycol, polyethylene glycol, and the like; Oils such as higher fatty acids, higher alcohols, hydrocarbon oils, natural oils, ester oils and silicone oils; Surfactants; Spices; Chelating agents; Moisturizing agents such as 1,3-butylene glycol, hyaluronic acid and derivatives thereof, maltitol, atherocollagen and sodium lactate; Thickeners such as carboxyvinyl polymers; Antioxidants; Ultraviolet absorbers; Pigments; water; The component normally mix | blended with the wool agent, such as a stabilizer, can be mix | blended in the range which does not impair the effect of this invention.

The pharmaceutical composition prepared as described above may be applied to the skin once or several times a day depending on the symptoms and may adjust the application according to the degree of improvement of the symptoms.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are only illustrative description of the present invention, and the scope of the present invention is not limited to these Examples.

Example  One: Seal frog rice  Extract manufacturer

The dried and dried dry frog frog starch (全 草) was refluxed three times for 5 hours with a 95% ethanol aqueous solution, and then cooled and filtered through Whatman # 5 filter paper. The filtered extract was concentrated under reduced pressure and lyophilized at 50 ° C. or lower, and then dried by using one or more solvents selected from purified water, ethanol, butylene glycol, and propylene glycol to contain 0.001 to 30.0 wt% of the reduced pressure concentrate and lyophilisate. An extract was prepared.

Example  2: NBT Antioxidant Effect Measurement Experiment

In order to confirm the antioxidant effect of the water frog frog extract obtained in Example 1, a comparative sample of BHT (Butylated hydroxytoluene), which is well known as an antioxidant under laboratory conditions, was compared and the antioxidant activity was measured using NBT (Nitro Blue Tetrazolium) method.

In order to measure the antioxidant effect, the active oxygen produced by xanthine and xanthine oxidase is measured by the NBT method, and the effect of the test substance removing the active oxygen, that is, the active oxygen scavenging effect, is evaluated. Free radicals are produced by xanthine and xanthine oxidase. This active oxygen reacts with Nitroblue Tetrazolium (NBT) to measure the active oxygen scavenging rate by measuring the blue color produced by this at a wavelength of 560 nm.

As a measuring method, 0.05M Na ₂ CO ₃ (2.4ml, pH 10.2), 3mM xanthine solution (0.1ml), 3mM EDTA solution (0.1ml), bovine serum albumin (BSA 0.15%) solution (0.1ml), 0.72mM 0.75 mM solution of NBT (nitroblue tetrazolium) (0.1 ml) is added, and 0.1 ml of sample solution is added thereto and left at 25 ° C for 10 minutes. Add 0.1 ml of xanthine oxidase solution (absorbance of the blank test in the range of 0.2 to 0.23, ie, absorbance to be A ₅₆₀ = 0.3 / 20 minutes), stir rapidly, and incubate at 25 ° C for 20 minutes. 0.1 ml of 6 mM CuCl ₂ solution is then added to stop the reaction and the absorbance St at 560 nm is measured. In the blank test, absorbance Bt is measured by using distilled water instead of the sample solution as described above. In the absence of enzyme in the sample solution, 560 nm absorbance So was measured before the reaction, and the blank of the sample solution was operated in the same manner using distilled water instead of the xanthine oxidase solution to measure the absorbance Bo.

The effect was calculated by Equation 1, and the results are shown in Table 1.

% Inhibition = [1- (St-So) / (Bt-Bo)] X 100

St: absorbance at 560 nm after enzyme reaction of sample solution

Bt: absorbance at 560 nm after enzymatic reaction of blank test solution

So: absorbance at 560 nm before reaction without enzyme addition of sample solution

Bo: absorbance at 560 nm before reaction without enzyme in blank test solution

As can be seen in Table 1, the water frog frog extract has been confirmed to have a superior antioxidant power than BHT (Butylated hydroxytoluene).

 Sample Name  Antioxidative effect(%)  Seal Frog Extract 0.1% by weight  92.2%  0.01% by weight of frog frog extract  74.7%  Frog Frog Extract 0.001% by weight  27.6%  0.1 wt% Butylated hydroxytoluene  89.3%

Example  3: Free radical  Scavenging activity measurement experiment

In order to measure the free radical scavenging activity of the water frog frog extract obtained in Example 1, an antioxidant such as BHT (Butylated hydroxytoluene) was compared in a laboratory sample and free radical scavenging activity was measured using DPPH method.

The DPPH method measures free radical scavenging activity by reducing power using a free group called DPPH (2,2-Di (4-tert-octylphenyl) -1-picrylhydrazyl free radical). The free radical scavenging rate is measured at a wavelength of 560 nm by comparing the degree to which the DPPH is reduced by the test substance to reduce the absorbance.

Water frog frog rice extract of 0.1%, 0.01%, 0.001% concentration was prepared for DPPH free radical scavenging activity measurement. Extracts of the above concentrations were placed in 96-well plates, respectively, and DPPH prepared in 100 uM methanol solution was added to obtain a total volume of 200 μl. After leaving it at 37 ° C. for 30 minutes, absorbance was measured at 560 nm. Free radical scavenging activity (%) was calculated by the following equation.

Free radical scavenging activity (%) = 100- (B / A) X100

A: absorbance of the control wells not treated with the frog frog rice extract of the present invention

B: absorbance of the experimental group wells treated with the frog frog rice extract of the present invention

As can be seen in Table 2, the water frog frog extract showed better free radical scavenging activity than BHT (Butylated hydroxytoluene), which is widely used as an antioxidant.

 Sample Name  Free radical scavenging activity (%)  Seal Frog Extract 0.1% by weight  94.4%  Frog Frog Extract 0.05% by weight  93.9%  Frogweed Extract 0.025% by weight  88.9%  0.1 wt% Butylated hydroxytoluene  81%

Example  4: Tyrosinase  Inhibitory effect measurement experiment

This example is to determine the whitening effect by looking at the degree of inhibition of the function of the enzyme tyrosinase (tyrosinase) in order to confirm the whitening effect of the water frog frog extract obtained in Example 1.

Tyrosinase is an enzyme that accelerates the oxidation of tyrosine in vivo and helps melanin production. In this example, the method of inhibiting the function of this enzyme to inhibit the oxidation of tyrosine to form a black polymer called melanin (Pomerantz SH: J. Biochem . , 24: 161-168, 1996) is applied. To determine the whitening effect.

The inhibitory activity against tyrosinase of each sample was determined by placing 15 μl of the sample into a 96 well plate, adding 150 μl of 50 mM phosphate buffer (pH 6.5), 25 μl of 1.5 mM L-tyrosine solution, and then using mushroom tyrosinase (1,500 units / ml, Sigma). G) 10 μl was added and reacted at 37 ° C. for 20 minutes, and then the absorbance was measured at 490 nm using a microplate reader (ELx800, USA) to measure the inhibition rate against tyrosinase. Inhibition rate for tyrosinase (%) was calculated by Equation 3, IC₅₀ The value is the concentration of the substance that inhibits tyrosinase enzyme activity by 50%.

% Inhibition = [(D-C)-(B-A)] / (D-C) × 100

A: Absorbance before reaction of the well containing the sample

B: Absorbance after reaction of the well containing the sample

C: Absorbance before reaction of the well without sample

D: Absorbance after reaction of well without sample

As a result of testing the inhibitory effect of tyrosinase activity, as shown in Table 3, the IC ₅₀ value of the water frog frog extract is 0.11%, which is lower than that of kojic acid and oil-soluble licorice extract, which is known to be known, but it shows superior tyrosinase activity inhibition effect than arbutin. It was.

 Sample Name Tyrosinase Inhibitory Effect (IC ₅₀ )  Frog Frog Extract  0.11%  Kojic acid  0.04%  Arbutin  0.47%  Lettuce extract  10.20%  Soluble Licorice Extract  0.08%

Example  5: B16F1 Melanosite  Experiment to measure melanin production inhibitory effect

This Example is to determine the whitening effect by looking at the degree of melanin production inhibition on B16F1 melanocytes in order to confirm the whitening effect of the water frog frog extract obtained in Example 1.

B16F1 melanocytes used in this example are cell strains derived from mice, and cells that secrete a black pigment called melanin. Samples were treated during the artificial culture of these cells to evaluate the degree of melanin reduction. The B16F1 melanocytes used in this example were used after being sold from the American Type Culture Collection (Accession No .: 6323).

The melanin biosynthesis inhibitory effect of B16F1 melanocytes was measured as follows. B16F1 melanocytes were dispensed in 6 well plates at a concentration of 2 × 10 ⁶ per well, cells were attached, and the samples were incubated for 72 hours by treating the samples at a concentration that does not cause toxicity. After 72 hours of incubation, the cells were detached with trypsin-EDTA, and the cells were counted and centrifuged to recover the cells. Quantification of intracellular melanin was carried out with a slight modification of the method of Rotan: Cancer Res . , 40: 3345-3350, 1980. After washing the cell pellet with PBS once, 1 ml of homogenization buffer solution (50 mM sodium phosphate, pH 6.8, 1% Triton X-100, 2 mM PMSF (phenylmethanesulphonylfluoride)) was added to vortex for 5 minutes to disrupt the cells. Melt the extracted melanin by adding 1N NaOH {including 10% DMSO (dimethyl sulfoxide)} to the cell filtrate obtained by centrifugation (3,000 rpm, 10 minutes), and measure the absorbance of melanin at 405 nm with a microplate reader. Quantitative determination of the melanin production inhibition rate (%) of the sample. Melanin inhibition rate (%) of B16F1 melanocytes was calculated by the equation (4), IC ₅₀ value is the concentration of the substance inhibiting 50% melanin production.

% Inhibition = [(A-B) / A] × 100

A: Melanin amount in wells without sample

B: Melanin amount in wells to which sample was added

As a result of testing the melanin production inhibitory effect of B16F1 melanocytes, as shown in Table 4, the IC ₅₀ value of the water frog frog extract is 0.2%, which is weaker than the existing whitening agent, hydroquinone and oil-soluble licorice extract, but superior to arbutin. Indicated.

 Sample Name Melanin synthesis inhibitory effect (IC ₅₀ )  Frog Frog Extract  0.2%  Hydroquinone  0.03%  Arbutin  0.46%  Lettuce extract  5.32%  Soluble Licorice Extract  0.04%

Example  6: In vitro MMP -1 inhibitory experiment

Fluorescence assay was used to determine the effect of inhibiting MMP-1 activity. The substrate used in the experiment was a fluorescently labeled DQ ^TM -collagen, and an enzyme (collagenase) was used by Molecular Probe (Eugene, OR, USA). The reaction buffer solution (0.5M Tris-HCl, 1.5M NaCl) was used. , 50 mM CaCl ₂ , 2 mM sodium azide, pH 7.6) was used after 10-fold dilution. To 100 µl of reaction buffer, 20 µl of DQ ^TM collagen dissolved in the reaction buffer at 0.25 mg / ml and 40 µl of water frog rice extract (0.01 to 0.1 wt%) were added and 40 µl of collagenase diluted to 0.5 units was added. do. After 20 minutes at room temperature, the fluorescence value was measured using a fluorescence spectrophotometer (Perkin Elmer, UK) at an absorption wavelength of 495 nm and an emission wavelength of 515 nm. The fluorescence value of the sample itself was also measured and corrected when calculating the enzyme activity.

The results are shown in Table 5, and when treated with 0.025% water frog rice extract, it inhibited about 69% of the collagen degradation activity of Clostridium collagenase, which is superior to the inhibitory effect of green tea extract.

 Sample Name  Collagenase activity inhibition rate (%)  Seal Frog Extract 0.1% by weight  96.7%  Frog Frog Extract 0.05% by weight  82.6%  Frogweed Extract 0.025% by weight  68.5%  0.2% by weight of green tea extract  70%

Example  7: after UV irradiation Seal frog rice  By extract MMP -1 expression inhibition evaluation

Enzyme-immunoassay (ELISA) was performed to measure the concentration of MMP-1 after UV irradiation and sample addition of the water frog frog extract obtained in Example 1. Human dermal fibroblasts were irradiated with UVA at an energy of 6.3 J / cm 2 using a UV chamber. UV irradiation amount and incubation time were established through preliminary experiments to maximize the expression of MMP in fibroblasts. The negative control was wrapped in tinfoil and kept in the environment of UVA for the same time. UVA emission was measured using a UV radiometer. The cells during the UVA irradiation were intact with the previously dispensed medium, and were irradiated with UVA, exchanged with the medium containing the sample, and then cultured for 24 hours, and the medium was recovered and coated in 96 wells. The primary antibody (MMP-1 (Ab-5) monoclonal antibody) was treated and reacted at 37 ° C. for 60 minutes. After reacting alkaline antibody phosphatase conjugated anti mouse IgG (whole mouse, alkaline phosphatase conjugated), which is a secondary antibody, for about 60 minutes, alkaline phosphatase substrate solution (1 mg / ml ρ-nitrophenyl phosphate in diethanolamine buffer solution) was added. The reaction was carried out at room temperature for 30 minutes and the absorbance was measured at 405 nm with a microplate reader. As a control, one without addition of a sample was used.

The results are shown in Table 6. Compared with the control group, the water frog frog extract was added more than 73% of the MMP-1 induced expression when irradiated with ultraviolet light, which was similar to the inhibition rate of the retinol used as a control.

 Test group  MMP-1 expression inhibition rate (%)  Control  -  0.01% by weight of frog frog extract  73.6%  Frog Frog Extract 0.005% by weight  39.4%  Retinol 0.005 wt%  38.0%

Example  8: Promoting Type 1 Procollagen Biosynthesis

In this experimental example, a procollagen assay was performed in the following manner to investigate the effect of promoting the biosynthesis of the skin matrix component type 1 procollagen after the addition of the water frog frog extract obtained in Example 1.

Human dermal fibroblasts isolated from neonatal foreskin tissues were obtained from Modern Tissue Technology (MTT, Korea) and were placed in DMEM / F-12 (Dulbecco's MEM: Ham's Nutrient Mixture F-12) 3: 1 medium. % Fetal Bovine Serum was added and cultured at 1 × 10 ⁴ cell / cm 2 concentration. When grown at about 70-80%, the cells were subcultured at a ratio of 1: 3, and the 3-4th passaged cells were used for the experiment.

For experiments to measure the amount of procollagen, fibroblasts were cultured over 48% in 48 well plates, and then the frog frog extract was 0.01% (v / v), 0.05% (v / v) and 0.001% ( v / v) concentration, and after 24 hours, the amount of free collagen in the medium was measured using a procollagen type-1 C-peptide EIA kit (MK101, Takara, Japan). Measured.

The results are shown in Table 7. Promolate collagen biosynthesis 34.5% when 0.005% (v / v) of the frog frog rice extract, which showed a similar effect to TGF (Tissue Growth Factor) -β, a cell signaling material.

 Sample Name  Procollagen biosynthesis promoting effect (%)  Frogweed Extract 0.005% by weight  34.5%  Frogweed Extract 0.0025% by weight  21.8%  Frog Frog Extract 0.001% by weight  12.4%  TGF-β 0.001 wt%  35.2%

Example  9: antimicrobial activity test against acne

This Example was a paper disc test (Paper Disc Test) to confirm the antimicrobial activity of the water frog frog extract obtained in Example 1 against acne bacteria. First, in order to activate propionibacterium acnes, a skin flora of the cause of acne, it was pre-incubated for 48 hours in BHI liquid medium (Brain-Heart Infusion Broth; 3.7%). The culture medium thus prepared was smeared in 0.1 ml each of BHI solid medium (Brain-Heart Infusion Broth; 3.7%; agar 1.5%) and dried. The water frog frog extract obtained in Example 1 was diluted to 12% (w / v) in a 95% ethanol aqueous solution, and 50 µl each was added to a 8 mm diameter paper disk, and the resultant was placed on a solid medium prepared above. The cells were incubated for 3 days. The antimicrobial activity was evaluated by observing the bacterial growth inhibition area around the paper disk and measuring the size of the ring. As a result, the size of the bacterial growth inhibitory ring of the frog frog rice extract was found to be 19 mm.

Example  10: Seal frog rice  5 alpha- of the extract Reductase  Activity inhibitory test

5 alpha-reductase activity inhibitory 5 alpha-reductase used in the experiment was used for the enzyme produced by the fibroblasts derived from the foreskin.

Fibroblasts were inoculated to inoculate 10,000 cells per microplate hole and then cultured. In each hole, 0.1.mμ.Ci of radiolabeled testosterone with ³ H (tritium) was added and cultured to determine whether the fibroblasts used it. As a control, no water frog rice extract was added. After 24 hours of incubation, the supernatant is obtained and steroids are obtained with 1 ml of ethyl acetate-cyclohexane 1: 1 extractant. The obtained steroid is placed on a thin layer chromatography plate and developed with chloroform / methanol mixture 98/2 (v / v). The radioactivity of the points corresponding to testosterone and dihydrotestosterone was measured by using a densitometer to calculate the conversion rate, and the result was compared with the control (conversion rate when no extract was added). Alpha-reductase inhibitory activity was evaluated.

% Inhibition = [A-B] / [A] × 100

A = conversion of testosterone to dihydrotestosterone (without extract)

B = conversion of testosterone to dihydrotestosterone (extract added)

Experimental results indicate that the frog leaf extract has a 5 alpha-reductase inhibitory effect (Table 8).

Frogweed Extract Concentration (%)  One  0.1  0.05  0.025  0.01  0.005  0.0025  % Inhibition  100.0  97.5  92.1  88.7  72.4  63.3  42.3

Example  11: Hair Growth Effect Test

In this example, a 30% hydrous ethanol solution containing 2% of the dry frog extract dried extract obtained in Example 1 was prepared and used for the test. The hair growth effect test was used for 47-53 days after the birth of the mouse (ICR), and the back hair was removed and 10 back skins were used. Applied. The length of hair and the degree of hair growth over time were compared by adding scores from 0 to 2 according to the degree of restoration after removal of hair. In order to compare the degree of hair growth, as a control, 30% alcohol solution was applied to each individual to observe the growth state.

As a result, it can be seen that the frog leaf extract has an excellent hair growth promoting effect (Table 9).

 Elapsed Days / Sample  5  10  15  Frog Frog Extract  0.16 ± 0.02  0.72 ± 0.13  1.48 ± 0.59  Control  0.08 ± 0.03  0.29 ± 0.09  0.97 ± 0.44

Example  12 and Comparative example  One

In this example, the cosmetic preparation containing the water frog frog extract obtained in Example 1 was prepared, and the skin elasticity improving effect and the wrinkle improvement effect were evaluated by carrying out a comparative experiment with Comparative Example 1 in humans.

The cosmetics used in the comparative experiments are in the form of a cream, the composition of which is shown in Table 10. First, the phase b) recorded in Table 10 is heated and stored at 70 ° C. To this, a) phase is added, and after pre-emulsification, it is emulsified uniformly with a homomixer, and then cooled slowly to prepare a cream (Example 12, Comparative Example 1). 20 experimenters (20-35 year old female) were applied to the cream prepared in Example 12 on the right side of the face and the cream prepared in Comparative Example 1 on the left side of the face twice a day for 2 consecutive months.

The skin elasticity improvement effect after the completion of the experiment was measured using a skin elasticity measuring instrument (cutometer SEM 575, C + K Electronic Co., Germany) before and after using the product for 2 months. The experimental results are described in Table 11 below as the ΔR7 value of the cutometer SEM 575, where the R7 value represents the nature of skin viscoelasticity. As shown in Table 11, it can be seen that the skin elasticity improvement effect of the experimenter who applied the cream containing water frog frog extract is excellent.

Example 12 Comparative Example 1 email Stearyl alcohol 8 8 Stearic acid 2 2 Stearic cholesterol 2 2 Squalane 4 4 2-octyldodecyl alcohol 6 6 Polyoxyethylene (25 mol addition) alcohol ester 3 3 Glyceryl monostearic acid ester 2 2 B) award Frog Frog Extract One - Propylene glycol 5 5 Purified water Remaining amount Remaining amount

Note) Unit: weight%

 Experimental product  Skin elasticity effect (△ R7)  Example 12  0.31  Comparative Example 1  0.14

n = 20, p <0.05

20 experimenters (20-35 year old female) were applied to the cream prepared in Example 12 on the right side of the face and the cream prepared in Comparative Example 1 on the left side of the face twice a day for 2 consecutive months.

After completion of the experiment, the skin wrinkle improvement effect is produced by using a silicone replica to examine the wrinkle improvement effect of the skin before and after using the product for two months. SV60, C + K Electronic Co., Germany). The results are shown in Table 12, which shows the average of each parameter value after 2 months minus the parameter value 2 months ago. In other words, the negative the value, the higher the wrinkle improvement effect. Therefore, as shown in Table 12, it could be confirmed that the skin wrinkle improvement effect of Example 12 containing the frog frog rice extract was greatly enhanced.

 Experimental product  R1  R2  R3  Example 12  -0.27  -0.21  -0.10  Comparative Example 1  -0.12  -0.10  -0.03

R1: difference between the highest and lowest of the wrinkle contour

R2: Average of R1 values after randomly dividing the wrinkle contour by 5 spaces

R3: Highest value of R1 divided by 5

Other examples are shown below. That is, the lotion, milky lotion and essence containing the water frog frog extract obtained in Example 1 were prepared in Examples 13 to 15.

Example  13: Seal frog rice  Preparation of lotion containing extract

To 8 g of 95% ethanol, 0.05 g of polypyrrolidone, 0.1 g of oleyl alcohol, 0.2 g of polyoxyethylene monooleate, 0.2 g of fragrance, 0.1 g of paraoxybenzoic acid methyl ester, a small amount of antioxidant, and a small amount of pigment were dissolved. . 0.05 g of water frog frog extract obtained in Example 1 and 5 g of glycerin were dissolved in 85.33 g of purified water, and then the mixed solution was added and stirred to obtain a lotion having a skin improvement effect.

Example  14: Seal frog rice  Preparation of Latex Containing Extract

1.2 g of cetyl alcohol, 10 g of squalane, 2 g of vaseline, 0.2 g of paraoxybenzoic acid ethyl ester, 1 g of glycerin monoesteralide, 1 g of polyoxyethylene (20 mole added) monooleate and 0.1 g of fragrance were dissolved at 70 ° C. 0.5 g of water frog rice extract obtained in Example 1, 5 g of dipropylene glycol, 2 g of polyethylene glycol-1500, 0.2 g of triethanolamine, and 76.2 g of purified water were dissolved by heating to 75 ° C. Both mixtures were emulsified and cooled to obtain an emulsion having an oil-in-water (O / W) type skin improvement effect.

Example  15: Seal frog rice  Containing extract Essence  Produce

To 5 g of 95% ethyl alcohol, 1.2 g of polyoxyethylene sorbitan monooleate, 0.3 g of chitulose, 0.2 g of sodium hyaluronate, 0.2 g of vitamin E-acetate, 0.2 g of sodium licorice, 0.1 g of paraoxybenzoic acid ethyl ester 1 g of the water frog rice extract obtained in Example 1 and a suitable amount of pigment was mixed to obtain a cosmetic liquid having an effect of improving skin.

The skin lotion, milky lotion and essence containing the frog frog rice extract obtained in Examples 13 to 15 are whitening effect due to active oxygen scavenging effect, tyrosinase inhibitory effect and melanin biosynthesis inhibitory effect, skin wrinkle improvement effect, 5 alpha-Lee It showed excellent effect on skin improvement such as acne prevention effect, hair loss prevention and hair growth effect due to ductase inhibitory effect.

Claims (6)

Skin external preparation composition containing the frog frog rice extract. The external preparation composition for skin according to claim 1, wherein the external preparation composition for skin has an anti-wrinkle effect. The external preparation composition for skin according to claim 1, wherein the external preparation composition for skin has an improvement in skin elasticity. The external preparation composition for skin according to claim 1, wherein the external preparation composition for skin has a skin whitening function. According to claim 1, wherein the external application composition for external skin, characterized in that the acne preventive and therapeutic effect. The composition for external application for skin according to claim 1, wherein the composition for external application for skin has a hair loss prevention and hair growth effect.