WO2008078846A1

WO2008078846A1 - Cosmetic composition for anti-aging of skin

Info

Publication number: WO2008078846A1
Application number: PCT/KR2006/005766
Authority: WO
Inventors: Soon Keun Lee; Wie-Jong Kwak; Yong-Baik Cho; Chang-Kyun Han; Jeong Joo Shin; Joo Hyon Kim; Eun Ju Lee; Chil Mann Jung; Se Jun Yun
Original assignee: Sk Chemicals Co., Ltd.
Priority date: 2006-12-27
Filing date: 2006-12-27
Publication date: 2008-07-03

Abstract

The present invention relates to an antiaging cosmetic composition for application to skin and, more particularly, to an antiaging cosmetic composition for application to skin, prepared by extracting, fractionizing, purifying and pulverizing Gentiana scabra Bunge, ginkgo leaves and Agaricus blazei Murrill, respectively, and then mixing the resulting powders in an appropriate ratio to be omogenized, and showing complex effects of improving skin elasticity and regenerating epidermal cells, such as an elastase inhibiting effect, an epidermal cell proliferating effect, a hyaluronic acid biosynthesis effect, a laminin producing effect, a skin immune enhancing effect, a skin turnover promoting effect, an elasticity improving effect, a skin moisturizing effect, and the like.

Description

[DESCRIPTION] [Invention Title]

COSMETIC COMPOSITION FOR ANTI-AGING OF SKIN

[Technical Field] The present invention relates to an antiaging cosmetic composition for application to skin and, more particularly, to an antiaging cosmetic composition for application to skin, prepared by extracting, fractionizing, purifying and pulverizing Gentiana scabra Bunge, ginkgo leaves and Agaricus blazei Murrill, respectively, and then mixing the resulting powders in an appropriate ratio to be homogenized, and showing complex effects of improving skin elasticity and regenerating epidermal cells, such as an elastase inhibiting effect, an epidermal cell proliferating effect, a hyaluronic acid biosynthesis effect, a laminin producing effect, a skin immune enhancing effect, a skin turnover promoting effect, an elasticity improving effect, a skin moisturizing effect, and the like.

[Background Art]

During growth people undergo changes in skin induced by both internal and external factors. Before skin aging process begins, it is noteworthy that a woman's skin is more elastic, soft and moist than that of a man, and also has lower level of sebum secretion and less body hair. Moreover, as a woman's skin cell has relatively high physiological activity, collagen synthesis of and metabolism occur actively in dermal fibroblasts. Further, synthesis of hyaluronic acid, which is essential in maintaining skin moisture, occurs actively in dermal fibroblasts, thereby providing elasticity and moisture to the skin. Particularly, skin elasticity is provided by elastic fibers consisting of elastin present in the dermis. Because the elastic fibers have very low elastic coefficient, they are easily deformed even by small external physical force and also easily restored. However, excessive exposure of skin to UV or skin aging can activate elastase, which specifically responds to elastin associated with skin elasticity, thus decreasing the biosynthesis of elastin fibers decreases, thereby losing skin elasticity. Moreover, when hyaluronidase is activated, hyaluronic acid that maintains moisture level in intercellular spaces, protects cells by forming a gel-phase matrix in tissues and maintains the luster and the elasticity of skin becomes deteriorated. Especially, at the age of 40 or thereabout, as hormonal balance of a human body is destroyed, the abilities of cell regeneration and generation of skin lipids are greatly reduced.

Various types of cosmetic products aimed at preventing skin aging by providing elasticity and luster to skin and preventing wrinkles and sag of skin have been proposed. Typical cosmetics are those where polyhydric alcohols, such as glycerin and sorbitol, are mixed with moisturizing ingredients, such as hyaluronic acid, collagen, elastin, natural moisturizing factors (amino acid, lactate, sodium pyrrolidonecarboxylic add, urea, and intercellular lipid), etc.; and cosmetics where vitamins, such as vitamin A, vitamin C, vitamin E and derivatives thereof, vitamin F (linolenic acid), vitamin H (glutathione), etc., are mixed with cell regenerating ingredients, such as hormones, plant extracts (glycyrrhetic acid and beta-carotene), animal extracts (bovine placenta extracts and royal jelly), etc.

However, the conventional cosmetic materials have several disadvantages in that they cannot meet their effects or may cause side effects to the skin when applied.

Accordingly, to improve the anti-aging effects of the cosmetic materials including improvement in skin elasticity and regeneration of epidermal cells and to solve the conventional drawbacks of unsatisfactory efficacy, unstable preparations and skin side effects of single active materials and single herbal medicines, and to provide a complex composition showing complex effects of improving skin elasticity and regenerating epidermal cells, such as elastase inhibiting effect, epidermal cell proliferating effect, hyaluronic acid biosynthesis effect, laminin producing effect, skin immune enhancing effect, skin turnover promoting effect, elasticity improving effect, skin moisturizing effect, etc. The inventors of the present invention have intensively studied various known herbal extracts and, as a result, completed the present invention by discovering that a cosmetic composition, prepared by extracting, fractionizing, purifying and pulverizing Gentiana scabra Bunge, ginkgo leaves and Agaricus blazei Murrill, respectively, and then mixing the resulting powders in an appropriate ratio to be homogenized, is a safe material not causing any side effects to the skin and maximizes the effects of improving skin elasticity and preventing skin aging.

[Disclosure]

[Technical Problem]

Accordingly, an object of the present invention is to provide a hypoallergenic cosmetic composition capable of preventing skin aging.

[Technical Solution]

The present invention provides a hypoallergenic and skin anti-aging cosmetic composition comprising a complex compound prepared by extracting, fractionizing, purifying and pulverizing Gentiana scabra Bunge, ginkgo leaves and Agaήcus blazei Murrill, respectively, and then mixing the resulting powders in an appropriate ratio followed by homogenization.

[Mode for Invention]

Hereinafter, the present invention as described above will be described in more detail. The present invention relates to a cosmetic composition for skin anti-aging, prepared by extracting, fractionizing, purifying and pulverizing Gentiana scabra Bunge, ginkgo leaves and Agaήcus blazei Murήll, respectively, and then mixing the resulting powders in an appropriate ratio to be homogenized, and showing complex effects of improving skin elasticity and regenerating epidermal cells, such as an elastase inhibiting effect, an epidermal cell proliferating effect, a hyaluronic acid biosynthesis effect, a laminin producing effect, a skin immune enhancing effect, a skin turnover promoting effect, an elasticity improving effect, a skin moisturizing effect, and the like.

Gentiana scabra Bunge, which belongs to the Gentianaceae family, is a perennial herb plant and a medicinal herb distributed in Japan and China as well as all over the Korean Peninsula. Gentiana scabra Bunge contains medicinal ingredients such as gentianine, gentiopicroside, gentisin, gentianose, etc. In the herbal treatment, dried roots and stems of Gentiana scabra Bunge are prescribed as a bitter stomachic for the treatment of indigestion, anorexia, etc. In addition, it has been known that Gentiana scabra Bunge has an anti-inflammatory effect, an antipyretic action and an antibacterial effect.

Ginkgo leaves refer to the leaves of a ginkgo biloba tree and contain various ingredients. Important ingredients of ginkgo leaves are flavonoids, biflavonoids, proanthocyanidins, terpene lactones, and the like. Flavonoid contained in ginkgo leaves contains quercetin, isorhamnetins, 3-O-methylmyristicins, kaempferol, etc. Biflavonoid contains amentoflavone, bilobetin, 5-methoxybilobetin, ginkgetin, isoginkgetin, etc. Further, ginkgo leaves contain proanthocyanidins and terpene lactones containing ginkgolides A, B and C. It has been verified that these substances have physiological effects such as an anti-inflammatory effect, an anti-allergic effect, an anti-rheumatic effect, an anti-tumor effect and an analgesic effect. Also, these substances have been used as an agent for improving blood circulation.

Agaήcus blazei Murrill grows wild in the mountain areas in Brazil called Piedade and belongs to the Agaricaceae family. It has been reported that Agaricus blazei Murrill is distributed in North America, Brazil, Korea and Japan and used as health foods. As medicinal ingredients, Agaricus blazei Murrill contains cerevisterol, antitumor active steroids A, B and C, 12 kinds of fatty acids, trace metal elements K and 10 other kinds, vitamins Bl, B2 and D2, niacin, beta-D-glucan, glucomannan protein complex, heteroglucan protein complex, mannan protein complex, xyloglucan protein complex, lecithin, etc.

In the present invention, the complex composition having an excellent anti- aging effects such as improvement of skin elasticity and regeneration of epidermal cells by using the above three medicinal herbs is prepared by using a conventional method comprising: extracting herbs, concentrating and pulverizing the extracts and mixing the resulting extract powders with a weight ratio of 2:0.5-2:0.5-2 followed by homogenization. Preferably, each of the three herbal extracts is heated with water or an organic solvent and then extracted.

More preferably, the process for preparing a herbal extract complex composition comprises:

(1) cutting roots of dried Gentiana scabra Bunge into small pieces, heating the resulting roots with water or an organic solvent of 5 to 10 times against the weight of the herb, extracting the resulting solution 2 to 4 times, filtering and concentrating the extract under reduced pressure, adding a predetermined amount of distilled water again to remove the organic solvent by an azeotropical concentration, and drying the resultant under vacuum; (2) placing dried ginkgo leaves into an organic solvent, extracting the resulting solution 2 to 4 times, filtering, concentrating the extract under reduced pressure, adding a predetermined amount of distilled water again to remove the organic solvent by an azeotropical concentration, pulverizing the resultant, subsequently, suspending the extract powder with distilled water of 2 to 3 times of volume against the weight of the extract powder, adding ethyl acetate thereto, stirring and settling the resulting solution to obtain fractions, adding a normal hexane of 2 to 5 times of volume to clean and filter the settled fractions, and drying the resultant under vacuum;

(3) cutting dried Agaricus blazei Murrill into small pieces, heating the resulting herb with water or an organic solvent of 5 to 10 times of volume against the weight of the herb, extracting the resulting solution 2 to 4 times, filtering and concentrating the extract under reduced pressure, adding a predetermined amount of distilled water again to remove the organic solvent by an azeotropical concentration, and drying the resultant under vacuum; and (4) mixing the extract powders obtained in the extract processes (1), (2) and (3) in a weight ratio of 2:0.5-2:0.5-2 to yield the concentrated complex composition.

Here, the organic solvent used in the above steps may include at least one selected from the group consisting of: ethanol, propanol, butanol, glycerol, propyleneglycol, 1,3-butyleneglycol, methyl acetate, ethyl acetate, benzene, normal hexane, diethylether and dichloroform.

Especially, in the present invention, a complex composition, prepared by mixing the dried and pulverized three herbal extracts including the Gentiana scabra Bunge ethanol extract, the ginkgo leaf ethylacetate extract and the Agaricus blazei Murrill ethanol extract in a weight ratio of 2:0.5-2:0.5-2, has excellent effects of improving skin elasticity and regenerating epidermal cells due to a synergistic action between the herbal ingredients compared with the respective single extracts. Here, when the respective dried powder extracts are mixed with each other in the above- described composition ratio, the most advantageous medicinal efficacy can be taken. However, if the mixing ratio of the herbal extracts is out of the above range, it is impossible to maximize the complex effects of improving skin elasticity and regenerating epidermal cells, such as an elastase inhibiting effect, an epidermal cell proliferating effect, a hyaluronic acid biosynthesis effect, a laminin producing effect, a skin immune enhancing effect, a skin turnover promoting effect, an elasticity improving effect, a skin moisturizing effect, etc., and the effects are limited to certain effects.

Further, when the complex composition composed of natural extracts in accordance with the present invention is applied to the skin, it rarely causes undesirable side effects to the skin compared with other single materials. Actually, as a result of a skin toxicity test, it has been ascertained that the herbal extract composition has no toxic effect on human body.

As such, the present invention has advantages in that the composition, prepared by extracting, fractionizing, concentrating and pulverizing the three herbal medicines such as the Gentiana scabra Bunge, the ginkgo leaves and the Agaricus blazei

Murrill, respectively, can maximize effects of improving skin elasticity and regenerating epidermal cells.

Accordingly, when the herbal extract complex composition of concentrated powder is applied to cosmetics, it can be prepared in the form of a softening lotion, an astringent, a lotion, an essence, a cream, a gel, a pack, a soap, an essence sheet, a hydrogel patch, a mask, etc. Moreover, it is preferable that the herbal extract complex composition of concentrated powder be mixed in an amount of 0.001 to 10.0 wt % relative to the total cosmetic composition. If the amount of the herbal extract complex composition of concentrated powder is less than 0.001 wt%, the medicinal effects are very tiny, whereas, if it exceeds 10 wt%, it is impossible to maximize the medicinal effects in comparison with the applied concentration and it may cause problems in manufacturing cosmetic preparations.

[Brief Description of the Drawings]

FIG. 1 is a graph depicting a skin immune enhancing effect by T cell activation of herbal extracts in accordance with comparative examples and examples; FIG. 2 is a graph depicting a skin immune enhancing effect by dioxide productivities of herbal extracts in accordance with comparative examples and examples;

FIG. 3 is a graph depicting a skin tissue of a hairless mouse using a Verhoeff stain method in an untreated group; FIG. 4 is a graph depicting an effect of improving elastic fibers on a skin tissue of a hairless mouse using a Verhoeff stain method after applying an extract in accordance with comparative example 2;

FIG. 5 is a graph depicting an effect of improving elastic fibers on a skin tissue of a hairless mouse using a Verhoeff stain method after applying an extract in accordance with comparative example 10; FIG. 6 is a graph depicting an effect of improving elastic fibers on a skin tissue of a hairless mouse using a Verhoeff stain method after applying an extract in accordance with comparative example 12;

FIG. 7 is a graph depicting an effect of improving elastic fibers on a skin tissue of a hairless mouse using a Verhoeff stain method after applying an extract in accordance with example 1; and

FIG. 8 is a graph depicting an effect of improving skin elasticity on a human body by cream compositions in accordance with a prescription example and a comparative prescription example.

Examples

The present invention will be described in more detail based on the following examples, however, they should not be construed as limiting the scope of the present invention.

Comparative Examples 1 to 15

Each 1 1 of the solvents depicted in the following table 1 was added to each 100 g of ground substances obtained by cutting Gentiana scabra Bunge, ginkgo leaves and

Agaricus blazei Murrill into small pieces. The resulting solutions were boiled at 70 to 80 ⁰C for 5 hours in a reflux extractor having a cooling condenser and filtered by a 200- mesh filter cloth. Then, the extracted solutions obtained in the respective steps were filtered by Whatman filter paper at room temperature to remove insoluble ingredients, concentrated under reduced pressure at 55 to 60 °C in a distillation apparatus having a cooling condenser, and dried under vacuum to obtain extract powders. Dry weights of the herbs, solvents, and obtained extract powders used in the respective comparative examples are shown in the following Table 1. [Table 1]

Example 1

Concentrated powder of Gentiana scabra Bunge extracted in comparative example 2, concentrated power of ginkgo leaves extracted in comparative example 10 and concentrated powder of Agaήcus blazei Murrill extracted in comparative example 12 were mixed and homogenized in a ratio of 2:1:1 to obtain a complex composition.

Comparative Example 16

Ground substances obtained by mixing and cutting 100 g of Gentiana scabra Bunge, 50 g of ginkgo leaves and 50 g of Agaήcus blazei Murrill into pieces were extracted with 50% ethanol in the same manner as described in the above comparative examples, thus obtaining three complex herbal extracts.

Prescription Example and Comparative Prescription Example: Preparation of creams After extracting the respective herbs of Example 1 in accordance with the present invention, the resulting extracts were concentrated, pulverized and then mixed in an appropriate ratio to yield a complex composition. Subsequently, a cream containing the complex composition (prescription example) and a cream containing no complex composition (comparative prescription example) were prepared as follows:

(1) Materials of Nos. 1 to 12 were weighed and placed into a oily tank and stirred at 75 ⁰C to be dissolved. (2) Materials of Nos. 13 to 18 were weighted and placed in to an aqueous tank and stirred at 75 °C to be dissolved.

(3) The resulting solution of (2) was transferred to an emulsifying tank under vacuum reduced pressure and then the resulting solution of (1) was transferred thereto and emulsified at 3,500 rpm for 5 minutes.

(4) Materials of Nos. 19 and 20 were weighed and changed into a 1% solution. The 1% solution was injected into an emulsifying tank under vacuum reduced pressure to be mixed and homogenized at 2,500 rpm, stirred using a pedal mixer at 25 rpm while vacuum degassing, and cooled to 55 °C. (5) Complex composition of concentrated powder of example 1 was weighed and heated with a portion of 1,3-butyleneglycol to be dissolved. Subsequently, an aroma is weighed and placed into the respective emulsifying tanks to be homogenized, dispersed and cooled to 30 ⁰C, thus preparing a cream.

In the comparative prescription example, a sample untreated cream was prepared in the same manner as described above.

Experimental Example 1: Skin safety test

Skin safety of the complex herbal extracts prepared in accordance with comparative examples 1 to 16 and the cosmetic composition prepared in accordance with example 1 was measured as follows. Each 15 μg of solutions prepared by dissolving each 0.2 wt% of the extracts of comparative examples 1 to 16 and example 1 with 50% 1,3-butyleneglycol and each 15 μg of the creams of the prescription example and the comparative prescription example were dropped in a Finn Chamber and then fixed to the humeral regions to be tested of thirty subjects (age distribution: 24 to 40 ages, except for skin lesion carriers).

Patches were applied to the test regions for 24 hours and then removed.

Subsequently, the test regions were marked with a marking pen and then observed after 24 hours, 48 hours and 72 hours, respectively, to evaluate the skin irritations.

The skin patch test was performed according to an International Contact Dermatitis Research Group (ICDRG) standard.

[Table 2]

[Table 3]

As a result of the skin safety test on the extracts of comparative examples and example, as the irritation rates were all evaluated 0.2% or less, it was thus confirmed that all of these materials were very safe. Experimental Example 2: Elastase inhibition test (in vitro)

To measure elastase inhibitory effects of the three herbal extracts in accordance with the extract solvents of comparative examples 1 to 15, the complex composition prepared by extracting, concentrating and pulverizing the above three herbal extracts, respectively in accordance with example 1, and the three complex herbal extracts of comparative example 16, an elastase inhibition test was performed to compare the extracts with a negative control group whether they inhibit the elastase activity that acts as a major factor in improving the skin elasticity.

Synthetic substrate methoxysuccinyl-Ala-Ala-Ala-Pro-p-nitroanilide (8.0 mM, 25 μL) was reacted with 25 μL of 0.1 M Hepes buffer solution (pH 7.4, containing 0.5 M

NaCl) containing 5 μg/mL of human neutrophil-derived elastase and the samples of the above comparative examples and example 1 at 37⁰C for 30 minutes and then the absorbances of 4-nitroaniline produced as a decomposition product were measured at

405 nm using a microplate reader. Moreover, the enzyme inhibition rates were calculated according to the following formula 1 and the results are shown in the following table 4:

[Formula 1]

I NH IBI T I ON RATE(%)= -^- X100 wherein A denotes an absorbance when the sample is not added and B denotes an absorbance when the sample is added.

[Table 4]

As shown in Table 4, the inhibitory activity against elastase was shown variously according to the extract solvents and fractional layers. The complex composition of example 1, prepared by extracting, concentrating and pulverizing the three herbal extracts and mixing the concentrated powders in an appropriate ratio to be homogenized, showed a remarkable effect on the elastase inhibitory activity more than the single extracts of comparative examples 1 to 15 and the three complex herbal extract of comparative example 16.

Experimental Example 3: Test for a proliferating effect of human-derived epidermal cells (in vitro)

Proliferating effect of epidermal cells was measured for a treated group in which 0.01% of extracts of comparative examples 2, 10 and 12 and example 1 were diluted on the epidermal cells collected from human skin and a control group in which the sample was not treated.

Epidermal cells of human skin cultured in Dulbecco's modified Eagle's media (DMEM) containing 2.5% fetal bovine serum were dispensed in a 96-well plate culture medium at a density of 5XlO³ cells/well. The resulting cells were cultured by treating each 100 μL of the above samples twice a day for 3 days and then each 50 μL of 0.2% MTT (3-[4,5-Dimethylthiodiazol-2-yl]-2,5-Phenyltertazolium bromide) solution was added to the respective wells and cultured at 37^°C for 4 hours. Subsequently, the resulting formazan, produced by reacting with dehydrogenase of mitochondria and MTT, was dissolved with dimethyl sulfoxide (DMSO). The absorbances of the dissolved formazan were measured at 570 ran using a microplate reader. The proliferating effects of the epidermal cells by the samples were

determined using the absorbance measured. Since the absorbance is increased if the

amount of cells is increased as the cells are proliferated, the absorbance is in

proportion to the amount of cells. The proliferating effects of the epidermal cells

were calculated by the following formula 2, comparing the absorbances of the

respective samples with those of the control group:

[Formula 2]

PROL I FERATI ON RATE(%)= ABSORBANCE OF TREATED GROUP - ABSORBANCE OF CONTROL GROUP

X100

ABSORBANCE OF CONTROL GROUP

[Table 5]

As shown in Table 5, the complex composition of example 1, prepared by

mixing extracting, concentrating and pulverizing the three herbal extracts and mixing

the concentrated powders in an appropriate ratio to be homogenized, showed an

excellent proliferating effect of epidermal cells compared with the single extracts. Experimental Example 4: Test for evaluating an effect of promoting productivity of hyaluronic acid (in vitro)

Hyaluronic acid has numerous functions of protecting cells, maintaining lubrication and softness of tissues, resisting to external forces, preventing bacterial infections, etc. by maintaining moisture in intercellular spaces and forming a gel-phase matrix in tissues. Like this, skin's fibrous proteins have functions of maintaining the shape of tissues, providing elasticity to skin, etc. Accordingly, if the production of fibrous proteins is promoted, it provides elasticity to the skin so as to be elastic skin. A typical aging phenomenon caused by the lack of proteins of skin cells is a decrease in the skin's elasticity. If there is lack of proteins of skin cells, adhesion forces between epidermal cells and between epidermis and dermis are weakened and thereby the skin becomes thin to show the decrease in the skin's elasticity. To improve such a decrease in the skin's elasticity, there is provided a method for promoting the metabolism of adhesion proteins, and one of the test models is to test the promoting effect of the productivity of hyaluronic acid that is one of the skin cell adhesion proteins.

Actions of the extracts of comparative examples 2, 10 and 12 and example 1 for the productivity of hyaluronic acid of epidermal cells were evaluated using human epidermal cells. Epidermal cells (δxlO³ cells/ well) suspended in keratinocyte growth media (KGM) for epidermal cells were inoculated in a 24-well plate culture medium and cultured until confluent in an incubator (37 °C, 5% CO₂). Subsequently, the resulting cells were changed into epidermal cells culture media containing 10 μg/mL, 50 μg/mL and 100 μg/mL of the above extracts and into untreated media and cultured under the same conditions for 48 hours. After 48 hours, the resulting supernatants were collected and the amounts of hyaluronic acid in the culture supernatants were measured using a hyaluronic acid plate that is a kit for the measurement. The results are shown in the following table 6 and the indexes of the evaluation are based on the following standards:

-: No formation (Negative control group) + : Weak formation found

++: Formation found

+++: Obvious formation found

[Table 6]

As seen in Table 6, it could be found that the herbal extract complex composition of example 1 showed an excellent producing effect of hyaluronic acid compared with the single extracts.

Experimental Example 5: Test for evaluating an effect of promoting productivity of laminin (in vitro)

Epidermal horny cells were isolated from human skin and cultured in epidermal cell culture media having a low calcium concentration. Bovine hypophysis extract and EFG were added to the media. Cells were cultured in the culture media and reproduced to the fourth generation. Then, adherent cells were detached by trypsin-EDTA treatment and uniform cell suspensions except for aggregates of cells was obtained by filtration. Cells were collected by centrifugation the collected cells were resuspended in DMEM-F12(2:l)-0.1% BSA to be 8^χlO7mL. The resulting cell suspensions were added to 0.5 mL media containing samples of a concentration of two times. The culture was performed using a 24-well plate at 37⁰C for 24 hours. After the culture, the culture supernatants were transferred to Eppendorf tubes and centrifuged at 15,000 rpm for 5 minutes. The resulting supernatants were transferred to new tubes and preserved at -20 ⁰C until the time of laminin measurement. Since the laminin bound in cells and on culture plastic is solubilized, a tris-hydrochloric acid buffer solution (pH 7.4) containing various surfactants was added to the respective wells and preserved at -20 ⁰C for one night. On the following day, an ultrasound treatment was performed and then lyophilized. On the next day, the resultants were redissolved and centrifuged at 15,000 rpm for 5 minutes. The resulting supernatants were transferred to tubes and preserved at -20 °C until the time of laminin measurement.

Laminins existing in the culture supernatants and in the cell layer were measured by a sandwich ELISA method. A monoclonal antibody for laminin 3-chain of the laminin, BMI 65 was bound to a solid layer of a 96- well ELISA plate. Since the laminins are measured by sandwiching, 6Fl 2, another kind of monoclonal antibody for laminin 3-chain was biotinylated (b-6F12) in advance to use. In the present method, only heterotrimers (332) showing functions were measured but heterodimers

(32) were not detected. Samples were added to the respective wells in which 3% gelatin phosphate buffer solution containing b-6F12 was added in advance. The final dilution rates of the samples in the wells were 1/4 in the culture solution and 1/10 in the cell layer. An antigen-antibody reaction was performed at 37 °C for 2 hours and washed. Then, avidin-HRP (horseradish peroxidase) solution was added to be reacted again for 30 minutes to 1 hour. After washing, an ABTS solution as an HRP substrate was added thereto and the absorbances were measured at 405 nm using a microplate reader. The amounts of laminins produced were expressed by percentages and the results are shown in the following table 7:

[Table 7]

As set forth in Table 7, it could be identified that the herbal extract complex composition in accordance with example 1 showed an excellent producing effect of laminin compared with the single extracts.

Experimental Example 6: Test for a skin immune enhancing effect (in vitro)

Laboratory animals used in this experiment were 4-week-old BALB/ c white mice (body weights: about 30 g). Normal cultures of comparative examples 2, 10 and 12, example 1 in accordance with the present invention and the control group (sample untreated) were dissolved in aqueous 1,3-butylene glycol solution and intraperitoneally injected at a concentration of 5 mg/mL to the mice. After 3 days, abdominal cells of the mice were collected and the cells were counted. Then, the abdominal cells were plated at a density of 2XlO⁴ cells/ well. The cells were cultured at an incubator (37 ⁰C, 5% CO₂) for 48 hours and then cultured for 4 hours by adding MTT. After shaking the resulting cultures at room temperature for 15 minutes, the absorbances (the amount of MTT reduced by cells is in proportion to the number of survival cells existing in the respective wells) were measured at 550 nm using an ELISA reader for 96-well plates.

Using the same mice as described above, abdominal and spleen cells were plated in a 96-well plate at a density of 2x10* cells/ well with the same samples at the same concentration and cultured in an incubator (37°C, 5% CO₂) for 24 hours. Then, the resulting cultures were gently shaken by adding a Griess reagent. Subsequently, the nitrogen dioxide contents were checked by measuring the absorbances at 550 ran. Moreover, the above samples were diluted using a coating buffer solution and dispensed to the wells. After closing the lids, the wells were left for 24 hours. Subsequently, the resulting wells were washed using a buffer solution. An assay diluent was added to the wells and left at room temperature for 1 hour. Each 100 μL of the samples was added to the respective wells and left at room temperature. Then, the resulting wells were washed five times. After adding TMB substrate solution, the wells not lidded were left at darkroom for 30 minutes. Subsequently, a reaction stop solution was put into the respective wells and the productivities of IL-2 (interleukin-2) were determined by measuring the absorbances at 450 nm.

After 3 days from the intraperitoneal injection with each 100 μL of the same samples as described above at a concentration of 5 mg/mL, the spleen cells were stimulated with concanavalin A (a glycoprotein extracted from bean seeds and an immune activating material activating T-cells) to measure IL-2 activities. The results are shown in Fig. 1 (The functions of IL-2 are to induce T-cell activation and B-cell differentiation). <Evaluation of nitrogen dioxide productivity of abdominal cells>

After 3 days from the intraperitoneal injection with each 100 μL of the same samples as described above at a concentration of 5 mg/mL, the abdominal cells were stimulated with lipopolysaccharide (a glycolipid extracted from gram-negative bacteria cell walls and an immune-activating material activating B-cells) to measure nitrogen dioxide productivity in the abdominal cells. The results are shown in Fig. 2. As shown in the test results of Figs. 1 and 2, the complex composition of the three herbal extracts of example 1 showed an excellent skin immune enhancing effect.

Experimental Example 7: Test for an elastin synthesis effect by dyeing skin tissues of hairless mice

UV was irradiated from a UV irradiator onto 1 L of squalene at the maximum UVA dose of J/m²/sec for 4 weeks. Subsequently, 1 L of methanol was added to the UV irradiated squalene and mixed in a shaker at 2,000 rpm for 2 hours. Supernatants were collected and separated in a centrifuge at 2,000 rpm for 3 minutes. The resulting supernatants were collected to prepare Sq-OOH. The oxidized squalene and ethanol were diluted in a ratio of 1:3 to prepare an artificial wrinkle inducing material. Each 100 μL of the artificial wrinkle inducing material was applied to skins of 8-week-old hairless mice twice a day at regular intervals for 6 weeks to induce artificial wrinkles. This experimental example was to evaluate the effect of improving elasticity shown when applying the samples to the artificial wrinkles of hairless mice induced by an elastin dyeing method. Fifty hairless mice having artificial wrinkles induced by the above experiment were divided into five groups. 0.01% samples of comparative examples 2, 10 and 12 and example 1 were dissolved in 50% ethanol solution and applied to four groups and 50% ethanol solution was applied to the other one negative control group for 2 weeks. Then, the skin tissues of hairless mice were extracted and subjected to a Verhoeff stain method to compare the effects of improving elasticity based on the untreated control group of Fig. 3. The results are shown in Figs. 4 to 7.

As depicted in Figs. 4 to 7 showing the elastin synthesis activities of the respective samples based on the control group of Fig. 3, it could be found that the elastin density was shown the highest in example 1 of the present invention compared with the single extracts.

Experimental Example 8: Test for a skin turnover promoting effect in human skin

In order to identify the effects of promoting the skin turnover for comparative examples 2, 10 and 12, example 1, an untreated control group and a positive control group containing lactic acid, twenty healthy male and female subjects (average age: 34-year-old) were participated in this experiment. Colors inside the humeral regions

of the subjects before applying the samples were measured by a colorimeter and then

artificially tanned using a cream containing dihydroxy acetone (DHA). Subsequently,

the color differences between before and after applying the samples were compared.

Next, each 0.2 wt% of the samples of comparative examples, example 1 and the

control groups was dissolved in a mixed solvent (water:ethanol:butyleneglycol =

80:15:5) and applied to the resulting humeral regions twice a day to measure the

decolorization degrees using the colorimeter everyday, thus measuring the times

(color change according to protein discoloration = skin turnover time) taken in

returning to the original colors. The skin turnover promotion rates were calculated

by the following formula 3 and the results are shown in table 8:

[Formula 3]

TURNOVER PROMOTION RATE(%)= TURNOVER TIME WHEN SAMPLE IS UNTREATED - TURNOVER TIME WHEN APPLYING SAMPLE

X100

TURNOVER TIME WHEN APPLYING SAMPLE

wherein the turnover time denotes a time required that the existing horny

layer is changed into a new horny layer, that is, a time required that the skin is

restored to the original state after tanned by the DHA. [Table 8]

As indicated in Table 8, it could be learned that the herbal extract complex composition of concentrated powder of example 1 had an excellent cell regeneration activity as the skin turnover time was taken more shortened compared with the control group containing lactic acid and the single extracts.

Experimental Example 9: Clinical test for a skin elasticity effect in human skin

In order to evaluate the effects of improving the skin elasticity when applying the creams in accordance with the prescription example and the comparative prescription example to faces, twenty healthy male and female subjects (average age: 35-year-old) were participated in this clinical trial. The cream of the prescription example was applied to left regions of the subjects' faces and the cream of the comparative prescription example was applied to right regions of the subjects' faces at room temperature 25°C and at a relative humidity of 50% twice a day in the morning and evening. After 1 week, 2 weeks, 3 weeks and 4 weeks, the skin elasticity was measured using a ballistometer and the results are shown in the following table 9 and in Fig. 8.

[Table 9]

As shown in Table 9, it could be ascertained that the cream containing the complex extract in accordance with the prescription example of the present invention showed an excellent effect of improving the skin elasticity compared with the cream containing no sample in accordance with the comparative prescription example.

Experimental Example 10: Test for evaluating a skin moisturizing effect in human skin

The creams of the prescription example and the comparative prescription example were applied to humeral regions of twenty healthy male and female subjects (average age: 35-year-old) at room temperature 25°C and at a relative humidity of 50% twice a day for 8 weeks. Then, the values of transepidermal water loss (TEWL) were measured using a Tewameter and ΔTEWL values denoting the changes according to time were calculated. Moreover, the skin moisturizing effects in accordance with the changes in electric conductivity according to epidermal water (in a range of 0 to 150) were measured using a Corneometer. [Table 10]

As a result of the above experiment, it could seen that the transepidermal water loss (TEWL) value of the cream in accordance with the prescription example was remarkably improved compared with that of the comparative prescription example and the prescription example showed an excellent skin moisturizing effect compared with the comparative prescription example.

[Industrial Applicability] As described in detail above, it has been ascertained that a complex composition, prepared by extracting and pulverizing Gentiana scabra Bunge, ginkgo leaves and Agaricus blazei Murrill, respectively, and then mixing the resulting powders in an appropriate ratio to be homogenized in accordance with the present invention shows complex effects of improving skin elasticity and regenerating epidermal cells, such as an elastase inhibiting effect, an epidermal cell proliferating effect, a hyaluronic acid biosynthesis effect, a laminin producing effect, a skin immune enhancing effect, a skin turnover promoting effect, an elasticity improving effect, a skin moisturizing effect, and the like. Accordingly, if the complex composition in accordance with the present invention is applied to cosmetics, it is expected that the complex composition is very useful as a cosmetic material for preventing skin aging including an improvement of skin elasticity and regeneration of epidermal cells.

Claims

[CLAIMS]

[Claim 1]

An antiaging cosmetic composition for application to skin prepared by mixing a Gentiana scabra Bunge ethanol extract, a ginkgo leaf ethylacetate extract and an Agaricus blazei Murrill ethanol extract in a weight ratio of 2:0.5-2:0.5-2.

[Claim 2]

In claim 1, the cosmetic composition has an elastase inhibiting effect, an epidermal cell proliferating effect, a hyaluronic acid biosynthesis effect, a laminin producing effect, a skin immune enhancing effect, a skin turnover promoting effect, an elasticity improving effect and a skin moisturizing effect.

[Claim 3]

In claim 1, the complex composition of concentrated powder is contained 0.001 to 10.0 wt % relative to the total cosmetic composition.

[Claim 4]

In claim 1, the cosmetic is prepared in the form of a softening lotion, an astringent, a lotion, an essence, a cream, a gel, a pack, a soap, an essence sheet, a hydrogel patch, or a mask.