KR101764807B1 - A cosmetic composition for anti-aging comprising immature fruits extracts of Rosa multiflora - Google Patents

Abstract

The present invention relates to cosmetic compositions containing immature and berry fruit extracts as main active ingredients, and more particularly, to a cosmetic composition containing immature and berry fruit extracts prepared by extracting immature and berry fruit using a solvent, concentrating and freeze- , 0.001 to 1.0% by weight of a cosmetic composition which is excellent in active oxygen scavenging effect, skin wrinkle improving effect, skin elasticity improving effect, photoaging prevention effect, whitening effect, acne prevention effect, hair damage prevention effect, hair loss prevention and hair growth effect .
According to the present invention, not only the active oxygen scavenging effect but also the effect of inhibiting MMP-1 biosynthesis in human fibroblasts and the effect of promoting biosynthesis of type 1 procollagen, skin elasticity and wrinkle improving effect, anti-aging effect, whitening effect , Acne prevention effect, hair damage prevention effect, hair loss prevention and hair growth effect.

Description

The present invention relates to a cosmetic composition comprising an immature and berry fruit extract having an anti-aging effect as an active ingredient,

The present invention relates to a cosmetic composition containing an immature and berry fruit extract as a main active ingredient, and more specifically, it is characterized by containing an immature and berry fruit extract as an active ingredient in an amount of 0.001 to 1.0% by weight, The present invention relates to an anti-aging functional cosmetic composition which is excellent in anti-aging effect, skin wrinkle improving effect, skin elasticity improving effect, photoaging prevention effect, whitening effect, acne prevention effect, hair damage prevention effect, hair loss prevention and hair growth effect.

Skin aging can be divided into two types: intrinsic (chronological aging) and phtoaging (Gilchrest BA: J. Am. Acad. Dermatol ., 21, 610-613 (1989)]. Endogenous aging is a naturally occurring aging phenomenon that is accompanied by decreased physiological function of the body as age increases [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 can be caused by active oxygen species activated by ultraviolet rays, stress, disease states, environmental factors, wounds, and aging. When such conditions are deepened, the antioxidant defense network existing in the living body is destroyed, It damages tissues and promotes adult diseases and aging. More specifically, lipids, proteins, polysaccharides and nucleic acids, which are major components of the skin, are oxidized to destroy skin cells and tissues, resulting in skin aging. In particular, the oxidation of proteins causes severe hyperinflammation of collagen, hyaluronic acid, elastin, proteoglycans, and fibronectin, which are connective tissues of the skin, which interferes with skin elasticity. Mutation, cancer induction, and immune function.

Therefore, it is necessary to protect the cell membranes by abolishing reactive oxygen species that are mediated by the body's metabolic processes, ultraviolet irradiation, and inflammatory reactions, and regenerate already damaged cells by active metabolism to proliferate the cells Can be restored and healthy skin can be maintained. In aging, enzymes such as matrix metalloproteases (MMPs) are involved, as well as active oxygen species. The synthesis and degradation of extracellular matrix such as collagen in vivo is appropriately controlled, but its synthesis decreases as aging progresses And promotes the expression of matrix metalloproteinase (MMP), an enzyme that degrades collagen, resulting in reduced elasticity of the skin and wrinkles. These degrading enzymes are also activated by exposure to ultraviolet light. Therefore, there is a demand for development of a substance capable of controlling the activation of MMP induced in the cell by ultraviolet light or inhibiting its activity.

The following are skin changes caused by pigmentation. The skin normally produces melanin to protect the skin from ultraviolet rays of sunlight. Melanin is produced in melanocytes, a kind of skin cells, and has a natural screening function that is distributed on the surface of the skin to block ultraviolet rays harmful to human body. In the mechanism of melanin production, there is an enzyme called tyrosinase which is synthesized in melanocyte. Tyrosinase produces dopaquinone via dopamine (DOPA) using an amino acid called tyrosine as a substrate, and melanin, which is a black pigment, is produced from dopaquinone by several stages of oxidative condensation reaction. (Japanese Patent Application Laid-open No. Hei 4-9320), hydroquinone (Japanese Patent Laid-Open No. 6-192062), kojic acid (Japanese Patent Laid-open No. 56-7710), arbutin Has been used as a whitening cosmetic composition due to its inhibitory effect on tyrosinase. However, its use has been limited due to poor stability and coloration of the composition, bad odor generation, lack of efficacy and effectiveness at the living body level, to be. In addition, the inhibitory effect of tyrosinase in vitro has been proven, but the effect is lower in experiments similar to actual levels of the living body.

Recently, a lot of attention has been focused on functional cosmetics having functions such as antioxidation, wrinkle reduction, whitening, and itching alleviation obtained from natural extracts in order to reduce skin irritation caused by various chemical substances and the like. In addition to low adverse effects on the skin, natural materials have recently increased their value as a raw material for cosmetics due to increased consumer acceptance of cosmetics using natural materials. For example, U.S. Patent No. 5,972,341 describes the wrinkle-reducing effect of the Commiphora plant, especially the Commiphora mukul extract. Japanese Patent Application Laid-Open No. 9-672662 discloses seaweed extracts having hyaluronidase inhibitory activity. Japanese Patent Application Laid-Open No. 10-85905 discloses an effect of improving skin texture of fucoidan extracted from Wakame. Japanese Patent Laid-Open No. 2-245087 describes the antioxidative effect of Sargassum sp. Extract. Japanese Patent Laid-Open No. 3-294384 discloses an antioxidant composition extracted from a genus of Hizikia. Japanese Patent Application Laid-Open No. 7-10769 discloses Phaeophyceae extract having an astringent effect.

In addition, male-pattern alopecia are male hormone-dependent and have a direct relationship with the amount of male hormone. Accordingly, many studies for prevention and treatment of alopecia through inhibition of male hormone activity have been reported. On the other hand, when the function of the sebaceous gland is activated by the increase of the secretion of the male hormone, the excess sebum produced in the hair follicle is stagnated in the hair follicle due to the overgrowth of the hair follicle wall. 5-alpha-Reductase is one of the male hormones present in male hormone reactive tissues such as sebaceous glands, hair follicles, prostate, and epididymis, as a result of male hormone-induced hair loss and acne. , An enzyme involved in the metabolism of testosterone into dihydrotestosterone, and NADPH is required for its conversion. In addition, testosterone is involved in male sexual dysfunction, skeletal muscle increase, male external genitalia, scrotum growth, spermatogenesis and the like, and dihydrotestosterone is involved in the relevant tissues such as acne, sebum increase, hair loss and enlargement of the prostate. In particular, after puberty, excessive secretion of male hormones causes acne and hair loss. To prevent excessive production of dihydrotestosterone, an active form of male hormone produced by 5-alpha-reductase, 5-alpha-reductase Studies have been actively conducted to develop anti-hair loss and anti-acne agents using an azide inhibitor.

In order to solve such skin problems, many cosmetic products using natural products have been developed to reduce skin irritation caused by various chemical substances and the like. In addition to low adverse effects on skin, natural materials are becoming increasingly valuable as raw materials for cosmetics, as consumers have become more receptive to cosmetics using natural materials.

1. Korean Patent Publication No. 10-2011-0041616 2. Korean Patent Publication No. 10-2015-0025680

An object of the present invention is to provide a cosmetic composition which is applicable to cosmetics and which has excellent anti-aging properties such as active oxygen scavenging effect, reduced MMP-1 biosynthesis and promoting type 1 collagen biosynthesis, skin wrinkle improving effect, skin elasticity improving effect, And to provide a natural extract exhibiting the effect of the present invention.

Another object of the present invention is to provide a natural extract which shows a whitening effect by ultraviolet irradiation or the like when applied to cosmetics.

It is also an object of the present invention to provide a natural extract having excellent hair damage prevention effect, hair loss prevention effect, hair growing effect and acne prevention effect when applied to cosmetics.

Accordingly, the inventors of the present invention, after a long period of research, have selected an immature seed and a briar fruit to prepare an extract from the extract, and found that the active oxygen scavenging effect, the skin wrinkle improving effect, the elasticity improving effect, the photoaging prevention effect, the whitening effect, , Hair loss prevention and hair growth effect were measured. As a result, it was found that the effect was excellent and the efficacy as a cosmetic can be expected.

Briar ( Rosa multiflora ) is 1 ~ 2m in height and has branching and branching and tangling. Leaves are alternate phyllotaxis. Small leaves 5-9, oval or obovate, no hairs on the surface, fine hairs on the back, and small sawtooth on the edge. The stipule is attached to the basal end of petiole at the lower part and has teeth like teeth. Flowers bloom in May, white or somewhat reddish, hanging on cones. In some cases there is a glandular hair on the stem of a small flower, the calyx is rolled up and the hair is denser on the inside. The petal is obovate, the tip is swollen and fragrant. The fruit is round and has a diameter of about 8mm and ripens red in September. Achenes are 3mm long and white and have hairs. ( Rosa multiflora var. Adenochaeta ), leaf blossoms ( Rosa multiflora var. Adenochaeta ), small leaves less than 2 cm in length and small flower briers ( Rosa multiflora var. Quelpaertensis ), stipule edges are almost plain, hairy Jeju Briar (Rosa multiflora luciae) that border briar that is serrated on the edges of the color red flowers and stipules (R osa multiflora jaluana ). It lives in the foothills of Korea, Japan, and China, and in sunny streams and valleys. Efficacy is effective when the elderly can not see the urine and when the whole body is swollen, forgetfulness, insomnia, dreamy, easily fatigued, and decreased sexual function. It can be used for boils and gags, and it is effective for increasing appetite and improving tack. It also dilates coronary arteries, improves fat and protein metabolism in the body, and inhibits the formation of atherosclerosis. It is also used for laxatives, venom, constipation, and nephritis.

The present invention provides a cosmetic composition containing immature and berry fruit extract.

In addition, the immature and briarwood of the present invention is characterized by green fruits unlike maturation.

In addition, the immature and berry fruit extract of the present invention is characterized by containing 0.001 to 1.0% by weight based on the freeze-dried weight of the whole cosmetic composition. When the content of the extract is less than 0.001% by weight, the effect of improving skin is scarcely produced. When the content of the extract is 1.0% by weight or more, the extract is cytotoxic and the effect of increasing the content is insignificant.

The present invention is further characterized in that the immature and berry fruit are extracted with at least one solvent 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 immature and briar fruit extracts are obtained by extracting immature and briarwood at room temperature, cooling and filtering, and then concentrating under reduced pressure and freeze-drying at 50 ° C or less.

The present invention also relates to the cosmetic composition as described above, wherein the cosmetic composition is at least one selected from the group consisting of lotion, gel, water-soluble liquid, cream, ointment, essence, shampoo, hair rinse, hair treatment, hair mousse, lipstick, oil / O) type. ≪ / RTI >

In addition, the immature and berry fruit extract of the present invention can be formulated into a pharmaceutical composition such as a capsule, a liquid preparation, an ointment, a patch or a sustained release formulation using a pharmaceutically acceptable carrier, and a pharmacologically acceptable base, (E.g. starch, tragacanth gum, gelatin, molasses, polyvinyl alcohol, polyvinyl ether, polyvinylpyrrolidone, hydroxypropylcellulose, methylcellulose, ethylcellulose and carboxymethylcellulose) A lubricant such as magnesium stearate, talc, hydrogenated vegetable oil) and a lubricant (for example, agar, starch, bellatin powder, sodium carboxymethylcellulose, carboxymethyl cellulose calcium, crystalline cellulose, calcium carbonate, sodium hydrogencarbonate and sodium alginate) Colorants and the like. Examples of the carriers and excipients include lactose, glucose, sucrose, mannitol, potato starch, cornstarch, calcium carbonate, calcium phosphate and cellulose.

In addition to the above, additives such as stabilizers, solubilizers, perfume fragrances such as transdermal absorption accelerators, and preservatives may be further added.

The pharmaceutical composition thus prepared can be applied to the skin once to several times a day depending on the symptoms, and the application can be controlled according to the degree of symptom improvement.

The immature and berry fruit extract according to the present invention has excellent anti-aging effects such as active oxygen scavenging effect, MMP inhibitory effect, modulating effect of MMP expression by ultraviolet irradiation, and skin wrinkle improving effect and mitigating skin irritation caused by ultraviolet irradiation Respectively.

In addition, the immature and berry fruit extracts showed whitening effects such as melanin formation inhibitory effect, which is a cause of stain, freckle and skin pigmentation.

In addition, the immature and berry fruit extracts exhibited an antimicrobial activity against acne and an inhibitory effect against 5 - alpha - reductase, which have the effect of preventing hair damage and hair loss and preventing acne.

Therefore, cosmetic compositions such as lotion, cream, emulsion, pack, and powder containing such immature and berry fruit extracts have excellent effects of active oxygen scavenging effect and collagenase activity regulating effect, skin wrinkle improving effect, anti-aging effect, whitening, , Prevention of hair damage, prevention of hair loss, and hair growth effect.

Hereinafter, the present invention will be described in more detail by way of examples. It should be noted, however, that these examples are only illustrative examples of the present invention, and the scope of the present invention is not limited to these examples.

Example 1: Preparation of immature and berry fruit extract

The shredded immature and brioche fruit were refluxed for 6 hours in 70% (v / v) ethanol aqueous solution, cooled, and filtered through a Whatman # 5 filter paper. The filtered extract was concentrated under reduced pressure at 50 ° C or less and lyophilized to prepare a powdery extract.

Comparative Example  1: Production of mature and berry fruit extract

The shredded maturity and berry fruit were refluxed for 6 hours in 70% (v / v) ethanol aqueous solution, cooled, and filtered through a Whatman # 5 filter paper. The filtered extract was concentrated under reduced pressure at 50 ° C or less and lyophilized to prepare a powdery extract.

Example  2: Comparison of polyphenol content and flavonoid content

The polyphenol content was measured by Folin-Denis method and the total flavonoid content was measured by Nieva Moreno et al . In order to compare polyphenol content and flavonoid content of the immature and maturation and berry fruit extracts obtained in Example 1 above. (2000) was modified and measured.

First, the polyphenol content was measured by dissolving the sample in distilled water at a concentration of 10 mg / ml and then taking 0.2 ml of the sample in a test tube, adding distilled water to make 2 ml, adding 0.2 ml of Foiln-ciocalteu's phenol reagent, . 0.4 ml of a saturated solution of Na ₂ CO ₃ was added thereto, mixed with 1.4 ml of distilled water, allowed to react at room temperature for 1 hour, and then absorbance was measured at 725 nm using a UV / VIS spectrophotometer (Shimadzu U-1201, Japan). At this time, the total polyphenol compound was dissolved in distilled water at a concentration of 10 mg / ml and tannic acid (Sigma Co, USA) was taken to be a solution of 0, 37.5, 75, 150 and 300 / / The content of polyphenol compounds in the extracts was calculated using this. The flavonoid content was determined by Nieva Moreno et al. (2000) was diluted to a concentration of 10 mg / ml with 80% ethanol as a solvent. 0.1 ml of 10% aluminum nitrate, 0.1 ml of 1M potassium acetate and 4.7 ml of 80% ethanol were added to a sample, After 40 minutes of reaction, the absorbance was measured at 415 nm using a UV / VIS spectrophotometer. The total amount of flavonoids was determined from quercetin (Sigma Chemical Co.) to a final concentration of 0, 10, 25, 50, 100, 250 and 500 / / The content of the flavonoid compound was determined and the content thereof is shown in Table 1.

As shown in Table 1, the total polyphenol content and flavonoid content in the immature and berry extracts were higher than those in the mature ones.

Name of sample Total polyphenol
Content (mg / g) Total flavonoid
Content (mg / g) Example 1
Immature and berry fruit extract
86.26 ± 0.21
64.65 + 0.14 Comparative Example 1
Maturation and berry fruit extract
42.62 + - 0.57
22.01 + - 0.44

Example  3: Assessment of cytotoxicity

This example evaluated the cytotoxicity of the immature and briar fruit extract obtained in Example 1 and the maturation and briar fruit extract of Comparative Example 1 to skin cells.

To the 96-well test plate, 1x10 ⁵ cells / ml of diluted fibroblast was added to the cell culture medium (DMEM supplemented with 10% FBS) and adhered for 24 hours. The extracts were diluted to an appropriate concentration in each well and treated for 24 hours. After 24 hours, the medium was removed, and a cell culture medium 200 containing a solution of MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide) (2.5 mg / And incubated in a 37 ° C CO ₂ incubator for 2 hours. The medium was removed and 100 μl of DMSO (dimethyl sulfoxide) was added. After shaking for 5 minutes to dissolve the cells, absorbance at 565 nm was measured in a microplate reader. Cell viability (%) was determined according to Equation (1) and the concentration of the extract without affecting cell viability was determined.

Bo: 565 nm absorbance value of a well that underwent chromogenic reaction in cell culture medium

Bt: 565 nm absorbance value of the well that underwent chromogenic reaction without treatment of the sample

St: a 565 nm absorbance value of the well treated with the color-developed sample

As shown in Table 2, the 100% survival treatment concentration of the dermal fibroblast was slightly less than 0.01% in the immature of Example 1 and the maturation and berry fruit extract of Comparative Example 1, indicating slight cytotoxicity. However, this generally indicates that it is a safe sample with little potential for skin irritation when considering the concentrations used in cosmetics.

Name of sample
100% cell viability concentration Example 1
Immature and berry fruit extract
0.01% Comparative Example 1
Maturation and berry fruit extract
0.01%

Example  4: NBT Experiment on antioxidant effect test

In order to confirm the antioxidative effect of the immature and maturation and briar fruit extracts obtained in Example 1, the green tea extract, which is well known as an antioxidant under laboratory conditions, was used as a comparative sample and NBT (Nitro Blue Tetrazolium) Antioxidant activity was measured.

In order to measure the antioxidant effect, the active oxygen produced by xanthine and xanthine oxidase was measured by the NBT method and the effect of the test substance on the removal of active oxygen, that is, the effect of scavenging reactive oxygen was evaluated. Active oxygen was produced by xanthine and xanthine oxidase. The active oxygen was reacted with Nitro Blue Tetrazolium (NBT), and the blue color produced by the reaction was measured at a wavelength of 560 nm to measure the active oxygen scavenging ratio.

_{0.05M Na 2 CO 3 (2.4㎖)} , 3mM xanthine solution (0.1㎖), 3mM EDTA solution (0.1㎖), BSA solution (0.1㎖), 0.72mM NBT solution was added to (0.1㎖) sample solution here 0.1㎖ And the mixture was allowed to stand at 25 DEG C for 10 minutes. A solution of xanthine oxidase (0.1 ml) was added and stirred rapidly, and incubation was started at 25 캜 for 20 minutes. Then, 6 mM CuCl ₂ solution (0.1 ml) was added to stop the reaction, and the absorbance St at 560 nm was measured. The blank test was carried out in the same manner as above using distilled water instead of the sample solution, and the absorbance Bt was measured. Also, the blank of the sample solution was measured by the same procedure using distilled water instead of the enzyme, and the absorbance Bo was measured.

The inhibition rate was calculated by the formula (2), and the results are shown in Table 3.

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

 Bt: Absorbance at 560 nm after enzyme reaction in blank test solution

 So: absorbance of 560 nm before reaction in the absence of enzyme in the sample solution

Bo: absorbance at 560 nm before the reaction in the absence of enzyme in the blank test solution

As shown in Table 3, the extracts of immature and berry fruit showed better antioxidative activity than those of matured and showed antioxidative effects similar to those of green tea extract, which is known to have excellent antioxidant activity.

Name of sample content Antioxidative effect (%) Example 1
Immature and berry fruit extract 0.01 wt% 98.44 Comparative Example 1
Maturation and berry fruit extract 0.01 wt% 71.25 Green tea extract 0.01 wt% 87.88

Example  5: Free radical  Measurement of scavenging activity

This example is a comparative sample in which extracts having excellent antioxidative activity, such as green tea extract, under laboratory conditions, are used to measure the free radical scavenging activity of the immature and berry fruit extracts obtained in Example 1 and Comparative Example 1 Free radical scavenging activity was measured by the DPPH method.

Free radical scavenging activity was measured by reducing power using a free radical called DPPH (2,2-Di (4-tert-octylphenyl) -1-picrylhydrazyl free radical). Free radical scavenging was measured at a wavelength of 560 nm by comparing the degree of reduction of the absorbance by DPPH reduction by the test substance with the absorbance of the blank test solution.

The DPPH free radical scavenging activity was determined by concentration. The extracts of the above concentrations were placed in a 96-well plate, and DPPH prepared in a 100 uM methanol solution was added thereto to make the total volume of the solution 200 μl. After incubation at 37 ° C for 30 minutes, absorbance was measured at 560 nm.

The free radical scavenging activity (%) was calculated by the following equation (3).

 A: Absorbance of the control well without treatment of the extract of the present invention

B: Absorbance of the experimental group well treated with the extract of the present invention

As shown in Table 4, the immature and berry fruit extract of Example 1 exhibited superior free radical scavenging activity as compared with the maturation and berry fruit extract of Comparative Example 1, and showed better antioxidative effects than the green tea extract having excellent antioxidant activity there was.

Name of sample content Antioxidative effect (%) Example 1
Immature and berry fruit extract 0.01 wt% 91.32 Comparative Example 1
Maturation and berry fruit extract 0.01 wt% 63.17 Green tea extract 0.01 wt% 85.91

Example  6: After irradiation with ultraviolet light, by immature and berry fruit extract MMP -1 expression inhibition evaluation

Enzyme immunoassay (ELISA) was performed to measure the concentration of MMP-1 after UV irradiation of the immature and berry extracts obtained in Example 1 and Comparative Example 1 and the addition of the sample.

UVA was irradiated to human dermal fibroblasts at an energy of 6.3 J / cm < 2 > using a UV chamber. Ultraviolet irradiation dose and incubation time were established by preliminary experiment to maximize MMP expression level in fibroblasts. Negative controls were wrapped in silver foil to maintain the same time in the UVA environment. UVA emission was measured using a UV radiometer. Cells under UVA irradiation were irradiated with UVA, and then cultured for 24 hours in a medium supplemented with the sample. Then, the medium was recovered and coated on a 96-well plate. The primary antibody (MMP-1 (Ab-5) monoclonal antibody) was treated and reacted at 37 ° C for 60 minutes. The secondary antibody, alkaline phosphatase-conjugated anti-mouse IgG (whole mouse, alkaline phosphatase conjugated) was reacted for about 60 minutes and then incubated with alkaline phosphatase substrate solution (containing 1 mg / ml ρ-nitrophenyl phosphate in diethanolamine buffer solution ) Was reacted at room temperature for 30 minutes and absorbance was measured at 405 nm with a microplate reader. As a control group, a sample to which no sample was added was used.

As shown in Table 5, the inhibition rate of MMP-1 induced by ultraviolet light irradiation was higher than that of the control group not treated with the sample (0.005 wt% to 61%) compared with the control group, and the retinol and maturation Of the inhibition rate of < RTI ID = 0.0 >

Test group MMP-1 expression inhibition rate (%) Negative control group - Example 1
Immature and berry fruit extract 0.005 wt% 61 Comparative Example 1
Maturation and berry fruit extract 0.005 wt% 45 Retinol 49

Example  7: promoting type 1 procollagen biosynthesis

To investigate the effect of the immature and mature and briar fruit extracts obtained in Example 1 and Comparative Example 1 on the biosynthesis of type 1 procollagen as a skin matrix component after the addition of the extracts to skin cell culture media, procollagen assay) was performed in the following manner.

Human dermal fibroblasts isolated from neonatal foreskin tissues were purchased from Modern Tissue Technology (MTT, Korea) and supplemented with 10% fetal bovine serum (FBS) in DMEM / F-12 (3: And cultured at a concentration of 1 x 10 ⁴ cells / ml. When 70 ~ 80% of the cells were grown, the cells were subcultured at a ratio of 1: 3, and the cells cultured in the third to fourth passages were used for the experiment. For the measurement of the amount of procollagen, the fibroblasts were cultured in a 48-well plate at a concentration of 90% or more, the respective samples were added at appropriate concentrations, and the amount of procollagen liberated in the medium after 24 hours (Procollagen type-1 C-peptide EIA kit) (MK101, Takara, Japan).

As shown in Table 6, the immature and maturation and berry fruit extracts of Example 1 showed a 28% promoting effect on the biosynthesis of procollagen at a concentration of 0.005 wt%, which is lower than that of vitamin C used as a positive control, And showed excellent activity.

Name of sample content MMP-1 expression inhibition rate The immature and berry fruit extract of Example 1 0.005 wt% 28.4% The maturation and berry fruit extract of Comparative Example 1 0.005 wt% 18.3% Vitamin C 0.005 wt% 34.2%

Example  8: UV irradiation Skin cell Optical damage  Defense effectiveness verification experiment

In this Example, the effects of the immature and mature and briar fruit extracts obtained in Example 1 and Comparative Example 1 on cytotoxicity by ultraviolet irradiation were evaluated. Fibroblasts were inoculated into 96-well test plates at 1 × 10 ⁵ cells / ml for 24 hours. After 24 hours, the extract to be evaluated was treated and cultured for 24 hours under the same culture conditions. Then the medium was removed and each well was washed once with PBS and 100 쨉 l PBS was added to each well. The cells were irradiated with 10 mJ / cm 2 of ultraviolet light using ultraviolet B (UVB) lamp (Model: F15T8, UVB 15W, Sankyo Dennki, Japan) Was added. The extract was treated and cultured for 24 hours. After 24 hours, the medium was removed, and 200 μl of MTT solution (2.5 mg / ml) containing cell culture medium per well was added and cultured in a 37 ° C CO ₂ incubator for 2 hours. The medium was removed and 100 μl of DMSO (dimethyl sulfoxide) was added. Cells were lysed by shaking for 2 minutes and absorbance at 565 nm was measured in a microplate reader.

The cell viability (%) was measured as shown in Equation (4), and the cytotoxic mitigation rate by ultraviolet was calculated as shown in Equation (5).

Bo: Absorbance at 565 nm of wells that had undergone color development of the cell culture medium

Bt: Absorbance at 565 nm of a well that underwent chromogenic reaction in a well not treated with a sample

St: absorbance at 565 nm of a well subjected to color development reaction in a well treated with a sample

Bo: Cell survival rate of wells not irradiated with ultraviolet light and not treated with sample

Bt: Cell viability of wells irradiated with ultraviolet light and not treated with the sample

St: Cell viability of well treated with ultraviolet light and treated with sample

As shown in Table 7, the immature and berry fruit extract of Example 1 showed a 12.1% reduction in cytotoxicity due to ultraviolet rays at a concentration of 0.001%, thereby preventing cell damage caused by ultraviolet rays.

Name of sample Treatment concentration (%) Cytotoxic Relaxation Rate (%) The immature and berry fruit extract of Example 1 0.005 12.1 The maturation and berry fruit extract of Comparative Example 1 0.005 4.3

Example  9: Tyrosinase  Experiment of inhibition effect measurement

This example confirms the degree of suppression of the function of the enzyme tyrosinase to confirm the whitening effect of the sample obtained in Example 1 and evaluates the whitening effect.

Tyrosinase is an enzyme that helps the production of melanin by stimulating the oxidation process of tyrosine in vivo. This example is a method (Pomerantz SH: J. Biochem., 24: 161-168, 1996) in which the degree of inhibition of the function of the enzyme to inhibit the formation of a black polymer called melanin by oxidation of tyrosine is measured To evaluate the whitening effect.

The inhibitory activity of each sample on tyrosinase was determined by adding 15 μl of a sample to a 96-well plate, adding 150 μl of 50 mM phosphate buffer (pH 6.5) and 25 μl of a 1.5 mM L-tyrosine solution, followed by addition of mushroom tyrosinase (1,500 units / Sigma) was added and reacted at 37 DEG C for 20 minutes. Then, the inhibition rate against tyrosinase was measured by measuring the absorbance at 490 nm using a microplate reader (ELx800, USA). The inhibition rate (%) for tyrosinase was calculated according to Equation (6), and IC ₅₀ The value is the concentration of the substance that inhibits tyrosinase enzyme activity by 50%.

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 well without sample

D: absorbance after reaction of well without sample

As shown in Table 8, the IC ₅₀ of the immature and berry fruit extract of Example 1 Value was 0.01%, indicating the maturation of Comparative Example 1 and the superior inhibitory effect of tyrosinase activity.

Name of sample Tyrosinase inhibitory effect (IC ₅₀ ) The immature and berry fruit extract of Example 1 0.05% The maturation and berry fruit extract of Comparative Example 1 0.1% Arbutin 0.01%

Example  10: B16F1 Melanocyte  Melanin synthesis inhibition experiment

In order to confirm the whitening effect of the extract obtained in Example 1, this example evaluated the degree of inhibition of melanin formation on B16F1 melanocyte and evaluated the whitening effect.

The B16F1 melanocyte used in this example is a cell strain derived from a mouse, and is a cell that secretes a melanin pigment called melanin. During the artificial culture of these cells, samples were treated to compare the degree of reduction of melanin pigment. The B16F1 melanocyte used in this example was purchased from ATCC (American Type Culture Collection, Accession No. 6323). Melanin synthesis inhibitory effect of B16F1 melanocyte was measured as follows. B16F1 melanocytes were dispensed in a 6-well plate at a concentration of 2x10 ⁶ cells / ml per well, and the cells were treated at a concentration that did not cause toxicity after the cells were adhered and cultured for 72 hours. After incubation for 72 hours, the cells were detached with trypsin-EDTA, and the number of cells was measured, followed by centrifugation to recover the cells. Quantification of intracellular melanin was carried out with a slight modification of the method of Lotan: Cancer Res., 40, 3345-3350 (1980). Cell pellet was washed once with PBS (phosphate buffered saline), and 1 ml of homogenization buffer (50 mM sodium phosphate, pH 6.8, 1% Triton X-100, 2 mM PMSF (phenylmethanesulphonylfluoride)) was added and vortexed for 5 minutes to disrupt the cells Respectively. After centrifugation (3,000 rpm, 10 min), 1N NaOH (containing 10% DMSO (Dimethyl sulfoxide)) was added to the cell filtrate to dissolve the extracted melanin, and the absorbance of melanin was measured at 405 nm using a microplate reader. Was measured to determine the inhibition rate (%) of melanin formation in the sample. Melanin formation inhibition rate (%) of B16F1 melanocyte was calculated according to Equation (7).

A: Amount of melanin in wells to which no sample was added

B: Amount of melanin in the well to which the sample was added

As shown in Table 9, the melanin synthesis inhibitory effect of the immature and berry fruit extract was 41%, which was similar to that of arbutin, a conventional whitening agent.

Name of sample Treatment concentration (%) Melanin synthesis inhibitory effect (%) The immature and berry fruit extract of Example 1 0.005 41% The maturation and berry fruit extract of Comparative Example 1 0.005 13% Arbutin 0.1 47%

Example  11: Antimicrobial activity against acne bacteria

A paper disc test was conducted to confirm the antibacterial activity against the acne bacterium of the extract obtained in Example 1. First, the cells were pre-cultured for 48 hours in a BHI liquid medium (Brain-Heart Infusion Broth; 3.7%) to activate the epidermis Propionibacterium acnes, the cause of acne. The bacterial culture thus prepared was plated in 0.1 ml of BHI solid medium (Brain-Heart Infusion Broth; 3.7%; agar 1.5%) and dried. Each of the extracts obtained in Example 1 was diluted to 12% (w / v) in an aqueous 95% ethanol solution, and 50 μl of each of the diluted extracts was dropped on a paper disk having a diameter of 8 mm and placed on the solid medium prepared above. For 3 days. The antimicrobial activity was evaluated by observing the growth inhibition zone around the paper disk and measuring the size of the inhibition zone.

The antimicrobial activity against acne bacterium was measured to be 19 mm, and the inhibition of bacterial growth of maturing and briar fruit extract was 11 mm.

Example  12: Effects of 5 Alpha - Reductase  Activity inhibition evaluation experiment

The 5 alpha-reductase activity inhibitory ability of the extract obtained in Example 1 was evaluated. 5 alpha - reductase activity The 5 - alpha - reductase used in the inhibition assay was produced by the enzyme produced by the forebrain - derived fibroblasts.

After inoculation, 10,000 microbial cells per microplate hole were inoculated and cultured. Radiolabelled testosterone was added to each hole at 0.1 mμCi with 3H (tritium), followed by culturing to determine whether fibroblasts were used. As a control group, those without immature and berry fruit extracts were used. After 24 hours of incubation, the supernatant is obtained and steroids are obtained with 1 ml of 1: 1 ethyl acetate-cyclohexane extraction solvent. The obtained steroid was placed on a thin layer chromatography plate and developed with a chloroform / methanol mixture 98/2 (v / v). The radioactivity of the points corresponding to testosterone and dihydrotestosterone was measured using a densitometer to calculate the conversion rate, and the result was compared with the control group (the conversion rate when the extract was not added) Alpha-reductase inhibitors were evaluated.

 A: Conversion rate of testosterone to dihydrotestosterone (when no extract is added)

B: Conversion rate of testosterone to dihydrotestosterone (upon addition of extract)

As shown in Table 10, it can be seen that the immature and berry fruit extract has 5 alpha-reductase inhibitory effect.

The immature stage of Example 1
Concentration of berry fruit extract (%) 0.05 0.025 0.001 0.0005 0.0025 0.001 0.0005
Inhibition rate (%)
65.4
56.8
41.4
35.3
12.1
10.2
8.2

Example  13: Skin elasticity improvement and wrinkle improvement effect test

A cosmetic composition containing the extract obtained in Example 1 was prepared, and skin elasticity improving effect and wrinkle improving effect were evaluated in comparison with Comparative Example 2 for human skin.

The cosmetics used in the comparative experiment are in the form of a cream, and the composition thereof is shown in Table 11. First, the b) image recorded in Table 11 was heated and stored at 70 ° C. A) was added to this, followed by preliminary emulsification, uniformly emulsified with a homomixer, and then gradually cooled to prepare a cream (Example 13, Comparative Example 2). The cream prepared in Example 13 was applied to the right side of the face and the cream prepared in Comparative Example 2 was applied to the left side of the face for 2 consecutive months for 15 consecutive subjects (female of 20 to 35 years old) . The skin elasticity was measured by using a skin elasticity meter (SEM 575, C + K Electronic Co., Germany) before and after using the product for 2 months. The experimental results are shown in Table 12 as the ΔR7 value of Cutometer SEM 575, where the value of R7 indicates viscoelasticity of the skin.

As shown in Table 12, it can be seen that the cream containing the immature and berry fruit extract of Example 1 has excellent skin elasticity improving effect.

division Example 13 Comparative Example 2 end Stearyl alcohol 8 8 Stearic acid 2 2 Stearic acid cholesterol 2 2 Squalane 4 4 2-octyldodecyl alcohol 6 6 Polyoxyethylene (25 mole addition) alcohol ester 3 3 Glyceryl monostearate Aster 2 2 I Immature and berry fruit extract One - Propylene glycol 5 5 Purified water Balance Balance

Note) Unit: wt%

Experimental product Skin elasticity effect (R7) Example 13
(Containing the immature and berry fruit extract of Example 1) 0.35 Comparative Example 2 0.11

n = 20, p < 0.05

The cream prepared in Example 13 was applied to the right side of the face and the cream prepared in Comparative Example 2 was applied to the left side of the face for 2 consecutive months for 15 consecutive subjects (female of 20 to 35 years old) .

To evaluate the wrinkle improvement effect of the skin before and after the use of the product after the completion of the experiment, a silicone replica was made and the wrinkle state of the designated area was measured with a visiometer SV60, C + K Electronic Co., Germany). The results are shown in Table 13, which shows the average of the parameter values after 2 months minus the parameter values two months ago. That is, as the value becomes negative, it means that the wrinkle improving effect is higher. Therefore, as shown in Table 13, it was confirmed that the effect of improving the wrinkles of the skin of Example 13 containing the immature and berry fruit extract was greatly improved.

Experimental product R1 R2 R3 R4 R5 Example 13 -0.21 -0.17 -0.11 -0.08 -0.06 Comparative Example 1 -0.11 -0.07 -0.06 -0.05 -0.02 R1: Difference between the maximum value and the minimum value of the wrinkle contour line
R2: The wrinkle contour line is arbitrarily divided into 5 squares, and the average of R1 values
R3: Maximum value of R1 divided by 5
R4: Mean value of the baseline of the wrinkle contour minus the value of each apex and valley
R5: Mean value of the value obtained by subtracting the outline of each wrinkle from the base line of the wrinkle contour line

Example  14: Whitening efficacy  Clinical evaluation

The skin whitening effect was evaluated in humans using a cream cosmetic formulation containing the immature and berry fruit extracts obtained in Example 13 by performing a comparative experiment with Comparative Example 2. 20 women (20 to 35 years old) were applied with the cream prepared in Example 13 on the right side of the face and the cream prepared in Comparative Example 2 on the left side of the face for 2 consecutive months, respectively . After completion of the test, the skin of the applied area on both sides of the face was analyzed for change of color brightness (L) by using an image analyzer and a chromometer (Minolta CR300) for change of skin color. Objective visual observation by a plurality of experts Subjective visual observation was performed and the effect was measured according to the following classification. The results are shown in Table 14. At this time, the degree of whitening efficacy was classified into the following seven grades and evaluated.

Whitening efficacy Visual evaluation criteria: -3: very bad, -2: worse, -1: slightly worse, 0: no change, 1: slight improvement, 2:

As shown in Table 14, it can be seen that the whitening effect is excellent in the facial skin of the user who applied the cream containing the immature and berry fruit extracts.

Change in skin color brightness (L) Objective evaluation of expert Subjective evaluation of the subject Example 13 Comparative Example 2 Example 13 Comparative Example 2 Example 13 Comparative Example 2 medium 7.89 3.58 4.3 2.6 4.7 2.7

Other examples are shown below. Namely, lotion, milky lotion and essence solution containing the immature and berry fruit extracts obtained in Example 1 were prepared as in Examples 14 to 16. Lotions and essences containing these immature and berry fruit extracts can be used as active oxygen scavenging effect, skin wrinkle improving effect, skin elasticity improving effect, photoaging prevention effect, whitening effect, acne prevention effect, hair damage prevention effect, And it showed excellent effect on skin improvement.

Example  14: Production of lotion containing immature and berry fruit extract

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 p-hydroxybenzoic acid methyl ester, a small amount of antioxidant and a small amount of pigment are mixed and dissolved in 8 g of 95% ethanol . 0.05 g of the immature and berry fruit extract obtained in Example 1 and 5 g of glycerin were dissolved in 85.33 g of purified water. The mixture was added to the mixture, followed by stirring to obtain a skin lotion having skin-improving effect.

Example  15: Manufacture of emulsion containing immature and berry fruit extract

0.2 g of cetyl alcohol, 10 g of squalane, 2 g of vaseline, 0.2 g of p-hydroxybenzoic acid ethyl ester, 1 g of glycerin monoestearylate, 1 g of polyoxyethylene (20 mols) monooleate and 0.1 g of perfume were mixed and dissolved at 70 캜 0.5 g of the immature and berry fruit extract obtained in Example 1, 5 g of dipropylene glycol, 2 g of polyethylene glycol-1,500, 0.2 g of triethanolamine and 76.2 g of purified water were dissolved by heating at 75 캜. The two were mixed and emulsified and then cooled to obtain an oil-in-water (O / W) type milky lotion.

Example  16: containing immature and berry fruit extract Serum  Produce

To 5 g of 95% ethyl alcohol, 1.2 g of polyoxyethylene sorbitan monooleate, 0.3 g of chitoolose, 0.2 g of sodium hyaluronate, 0.2 g of vitamin E-acetate, 0.2 g of sodium permanganate, 0.1 g of p-hydroxybenzoic acid ethyl ester 1 g of the immature seed, berry fruit extract obtained in Example 1 and an appropriate amount of pigment were mixed to obtain a serum having skin-improving effect.

Claims (14)

A cosmetic composition comprising green immature and berry fruit extract.
delete The method according to claim 1,
The cosmetic composition according to claim 1, wherein the immature and berry fruit extract is contained in an amount of 0.001 to 1.0% by weight based on the total weight of the cosmetic composition.
The method according to claim 1,
Wherein the extract is extracted with at least one solvent selected from purified water, methanol, ethanol, glycerin, ethyl acetate, butylene glycol, propylene glycol, dichloromethane or hexane as an extraction solvent.
The method according to claim 1,
Wherein the immature and berry fruit extracts are obtained by extracting immature and berry fruit at room temperature, cooling and filtering, and then concentrating under reduced pressure and lyophilizing at 50 ° C or lower.
The method according to claim 1,
Wherein the cosmetic composition exhibits an active oxygen scavenging effect.
The method according to claim 1,
Wherein the cosmetic composition has an effect of improving skin wrinkles.
The method according to claim 1,
Wherein the cosmetic composition exhibits an effect of improving skin elasticity.
The method according to claim 1,
Wherein the cosmetic composition exhibits an anti-aging effect.
The method according to claim 1,
Wherein the cosmetic composition has a whitening effect.
The method according to claim 1,
Wherein the cosmetic composition exhibits an anti-acne effect.
The method according to claim 1,
Wherein the cosmetic composition exhibits an effect of preventing hair damage.
The method according to claim 1,
Wherein the cosmetic composition exhibits hair loss prevention and hair growth effect.
The method according to claim 1,
The formulation of the cosmetic composition may be in the form of lotion, gel, water-soluble liquid, cream, ointment, essence, shampoo, hair rinse, hair treatment, hair mousse, lipstick, oil / &Lt; / RTI &gt; by weight of the cosmetic composition.