KR20140130277A

KR20140130277A - Composition of skin external application comprising glycoprotein

Info

Publication number: KR20140130277A
Application number: KR1020130048192A
Authority: KR
Inventors: 이성현; 이찬우; 성수현; 황태원
Original assignee: 주식회사 두래
Priority date: 2013-04-30
Filing date: 2013-04-30
Publication date: 2014-11-10

Abstract

The present invention relates to a cosmetic composition containing glycoprotein and, more specifically, a composition containing glycoprotein for external application to skin which provides the excellent effects of anti-oxidant, anti-aging, skin whitening, skin regeneration, and anti-inflammation. More specifically, the present invention relates to a composition containing glycoprotein for external application to skin wherein the glycoprotein is a vegetable glycoprotein extracted from at least one of the group consisting of pine leaves, red ginseng, green tea, and silver birch.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a composition for skin external application containing glycoprotein,

The present invention relates to a composition for external application for skin containing a plant glycoprotein, and more particularly, to a composition for external application for skin which can provide excellent antioxidation, skin whitening, anti-aging, skin moisturizing, Skin external composition.

The human skin undergoes aging process as it gets older. Skin aging can be roughly divided into two types depending on the factors. One of them, intrinsic aging, is that the structure and physiological functions of the skin are constantly declining while aging, and the other, extrinsic aging, is caused by accumulated external stresses such as sunlight . Especially, sunlight is one of the well known causes of aging. Skin exposed to ultraviolet rays for a long time thickens the stratum corneum and collagen (collagen) and elastin (skin) become denatured and lose elasticity of skin. This aging of the skin is accompanied by various functional and structural changes.

The epidermis, dermis and subcutaneous tissues constituting the skin become thinner with aging. In addition, the extracellular matrix (ECM) component of the dermal tissue responsible for skin elasticity and tensile changes. The ECM consists of two major components. One is the elastic fiber, which accounts for about 2 to 4% of the total ECM, and the other is the collagen, which accounts for about 70 to 80% of the total ECM. As the aging progresses, the elasticity of the skin decreases greatly, which is due to the decrease of collagen and elastin.

Collagen and elastin are controlled by several factors. Matrix metallo protease (MMP), such as Collagenase and Elastase, degrades collagen and elastin, resulting in reduced collagen content in the skin. When collagen and elastin are reduced in the dermis, the epidermis of the skin becomes coarse and the elasticity decreases, which is the aging phenomenon.

Various studies have been conducted on inhibitors capable of inhibiting the expression or activity of collagenase and elastase in order to effectively suppress collagen and elastin, which are the causes of the decrease in elasticity.

Peroxisome proliferator activated receptors (PPARs) are a class of nuclear hormone receptors. To date, three isotypes of α, β, and γ have been reported. However, in general, it is activated by binding to a specific ligand. By binding to a promoter of a protein and promoting transcription, the expression of the target protein is increased, It induces a physiological change.

PPARs affect the body's anti-inflammatory and regenerating skin tissues through immunity, tissue regeneration and interaction with cells, along with the effects of aging, inflammation and moisturizing. PPARs activate anti-inflammatory actions by inhibiting the production of IL (Interleukin) -6 and the function of NF-κB (Nuclear Factor-kappa B) and AP (Activator protein) -1.

Antihistamines, vitamin ointments and bush corticosteroids are mainly used for the treatment of inflammatory skin diseases such as atopic dermatitis, contact dermatitis, seborrhoic dermatitis and acne. Most cases are temporary and there are many side effects.

Several factors are involved in determining the color of a person's skin. Among them, factors such as activity of melanocyte (melanocyte) which makes melanin pigment, distribution of blood vessels, thickness of skin, presence of pigment inside and outside the body such as carotinoid and bilirubin are important. In particular, the most important factor is melanin, a melanin pigment produced by the action of various enzymes such as tyrosinase in melanocytes in the human body. The formation of melanin pigment affects genetic factors, hormonal secretion, physiological factors such as stress, and environmental factors such as ultraviolet irradiation.

The melanin pigment produced in melanocytes of body skin is a phenolic polymer substance having a complex form of black pigment and protein. It protects the subcutaneous skin organs by blocking ultraviolet rays irradiated from the sun, and at the same time, free radicals And protects proteins and genes in the skin. Melanin, which is produced by stress stimuli inside and outside the skin, is a stable substance that does not disappear until the skin is excreted through skin keratinization even if the stress disappears. However, when melanin is produced more than necessary, it induces hypercholesterolemia such as spots, lobes, and dots, resulting in poor cosmetic results.

Today, oriental women prefer white, clean skin like white whites and use it as an important criterion of beauty, so there is a growing demand for improvement of hyperpigmentation and resolution of cosmetic problems.

The antiallergic effect of glycoprotein isolated from aloe and the effect of anti-aging of glycoprotein extracted from elm have been known, but the pine needle, red ginseng, green tea, tea, red ginseng, gooseberry, Has not been reported on improving the skin condition including skin aging prevention, whitening, skin regeneration, and anti-inflammatory effect on the skin of plant glycoproteins extracted from the plant.

Korean Patent Publication No. 2003-0075412 Korean Patent Publication No. 2001-0062928

Accordingly, the present inventors have found that when phytogenic glycoprotein extracted from at least one selected from the group consisting of pine needle, red ginseng, green tea, tea gourd, Hongikongcheon, gooseberry, Siberian ostrich and white birch is used as an effective ingredient, It has been confirmed that there is antioxidant, whitening, skin regeneration, anti-inflammatory effect and the like, and the present invention can provide an effect of improving the skin condition.

Accordingly, an object of the present invention is to provide a cosmetic and pharmaceutical composition containing plant glycoprotein extracted from at least one organism selected from the group consisting of pine needle, red ginseng, green tea, tea, red ginseng, gooseberry, To provide an external skin composition for skin.

In order to achieve the above object, the present invention provides a skin containing plant glycoprotein extracted from at least one member selected from the group consisting of pine needle, red ginseng, green tea, tea, red ginseng, gooseberry, Thereby providing an external application composition.

The present invention also provides an anti-aging, whitening, skin regenerating or anti-inflammatory skin external composition composition containing the above plant glycoprotein as an active ingredient.

The composition of the present invention contains phytogenic glycoprotein extracted from at least one plant selected from the group consisting of pine needle, red ginseng, green tea, tea gourd, Hongikyungcheon, gooseberry, Siberian ostrich and white birch as effective ingredients, Exhibits an anti-aging effect, and can provide excellent skin regeneration, whitening, and anti-inflammatory skin condition improving effect.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a flow chart showing a process for producing a plant glycoprotein.
Fig. 2 is a graph showing the results of PPAR-a activation of plant glycoprotein.

The composition for external application for skin according to the present invention contains, as an active ingredient, a plant glycoprotein extracted from at least one organism selected from the group consisting of pine needle, red ginseng, green tea, tea, red ginseng, gooseberry, Siberian ostrich and white birch.

The pine needle used in the present invention means the leaf of Pinus densiflora S. et Z.).

The red ginseng used in the present invention means that ginseng, which means non-dried ginseng, is heated and steamed or dried by conventional methods in the related art.

The green tea used in the present invention means Camellia sinensis. In the present invention, the whole or a part of the plant such as the leaves, stems, roots, fruits and flowers of the tea tree can be used, have.

The Inonotus Obliquus used in the present invention is found in birch-parasitic mushrooms, mainly in Siberia, North America, Northern Europe, etc., which is at least 45 degrees north latitude.

Rhodiola rosea used in the present invention is a perennial plant of rhododendron which is native to northern Europe and Asia, and is also called rocky flower. In the present invention, all or a part of plants such as leaves, stems, roots, fruits and flowers of the Rhodiola can be used.

The Ribes grossularia used in the present invention is a deciduous broad-leaved shrub of the primate and is also called a Western currant. In the present invention, the whole or part of plants such as leaves, stems, roots, fruits and flowers of the gooseberry can be used, and the gooseberry fruit can be used.

The Bergenia cordifolia used in the present invention is a perennial plant and a perennial plant originating in Siberia and Mongolia, and is also called a Himalayan rock rice cake. In the present invention, the whole or part of plants such as leaves, stems, roots, fruits, flowers of Siberian ostrich can be used.

Betula papyrifera used in the present invention is a deciduous broad-leaved arboreous tree of birch native to northern Asia and Europe. In the present invention, the whole or part of a plant such as leaf, stem, root, Can be used.

The plant glycoprotein used in the present invention means a glycoprotein which protects a cell wall when a plant is grown or a cell wall is damaged while being present in a primary cell wall in a plant cell. These vegetable glycoproteins act as important natural moisturizing factors and contain more sugar than animal collagen, so they are easy to use and have low molecular weight, so they are absorbed well on the skin. In addition, it is composed of amino acid, which is a biological substance, and it can be safely absorbed to skin and directly provide amino acid to prevent skin aging. These plant glycoproteins are characterized by different skin effects depending on the organism to be extracted.

The glycoprotein extracted from red ginseng has antioxidant, anti-aging effect and collagen synthesis effect, and the glycoprotein extracted from pine needle has an effect of skin regeneration, skin whitening, anti-aging effect.

The plant glycoprotein according to the present invention can be produced by a known method. For example, the organism is dried by any method such as natural drying or forced drying, finely pulverized, and then washed with a polar solvent such as water, methanol, ethanol, butanol or acetone; Non-polar solvents such as ether, hexane, chloroform, and ethyl acetate; A mixed solvent of the non-polar solvent and the polar solvent; A leachate containing an active ingredient can be obtained by leaching by using a solvent such as alkaline water or a vegetable oil such as soybean oil or sesame oil by any method such as cold-water treatment, percolation, warm-up and hot water extraction . The leaching treatment may be carried out at a temperature of about 12 to 96 hours in the case of cold beating and percolation, and at a temperature close to the reflux temperature of the solvent, preferably from 0.5 to 24 It is preferable to perform the operation for about the time. Then, the residue and the filtrate are separated by filtration through a filter cloth and centrifugation, and the separated filtrate is subjected to enzymatic treatment by hydrolysis to obtain a low molecular weight product, which is then concentrated under reduced pressure to obtain glycoprotein. After obtaining the extract by using the solvent, the liquid product can be obtained by freezing, heating and filtering at room temperature by a conventional method known in the art, or the solvent can be further evaporated, spray dried or freeze-dried.

The enzyme used for the hydrolysis may be, for example, an? -Amylase,? -Amylase, a gluco-amylase, a protease, a cellulase, a pectinase or a mixed enzyme thereof.

The plant glycoprotein according to the present invention is easily absorbed to the skin because it is reduced in molecular weight through hydrolysis.

The composition for external application for skin according to the present invention may contain the vegetable glycoprotein in an amount of 0.0001 to 30% by weight, preferably 0.01 to 10% by weight, based on the total weight of the composition. If the content of the glycoprotein is less than 0.0001 wt%, the effect of using the extract is insignificant. If the content of the glycoprotein is more than 30 wt%, the skin stability or the shape of the product may be caused.

The composition for external application for skin according to the present invention may contain phytogenic glycoprotein extracted from at least one organism selected from the group consisting of pine needle, red ginseng, green tea, tea, red ginseng, gooseberry, Siberian oak, and white birch as effective ingredients . When the complex glycoprotein is contained as a complex glycoprotein containing two or more kinds of glycoproteins, the content of each glycoprotein in the complex glycoprotein is preferably contained in the same amount. By containing them as the above-mentioned two or more kinds of complex glycoproteins, skin regeneration, whitening, and anti-inflammatory effect, which are the effects of the present invention, can exhibit a remarkably synergistic effect through interaction of the respective components.

The vegetable glycoprotein used in the present invention provides excellent antioxidative power and promotes biosynthesis of collagen through reduction of biosynthesis of MMP (Matrix metalloproteinase) -1, thereby improving skin elasticity and improving skin wrinkles. Therefore, Can be used as an anti-aging composition.

Further, the present invention can be used as a whitening composition which effectively inhibits biosynthesis of melanin.

In addition, the present invention can be used as a skin regeneration composition that effectively induces differentiation of skin cells.

In addition, the present invention can be used as a composition for anti-inflammation which effectively activates PPAR-a.

The composition for external application for skin of the present invention can be formulated as a cosmetic composition and can be formulated containing a cosmetically or dermatologically acceptable medium or base. These are all formulations suitable for topical application, for example as a solution, a gel, a solid, a paste anhydrous product, an emulsion obtained by dispersing an oil phase in water, a suspension, a microemulsion, a microcapsule, a microgranule or an ionic form (liposome) In the form of an ionic follicular dispersion, or in the form of creams, skins, lotions, powders, ointments, sprays or concealers. It can also be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant. These compositions may be prepared according to conventional methods in the art.

In addition, the composition according to the present invention may further comprise at least one selected from the group consisting of fatty substances, organic solvents, solubilizers, thickening agents, gelling agents, softening agents, antioxidants, suspending agents, stabilizing agents, Such as fillers, emulsifiers, emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blockers, moisturizers, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredient commonly used in cosmetics May contain adjuvants commonly used in the field of cosmetics or dermatology. Such adjuvants are introduced in amounts commonly used in the cosmetics or dermatological fields.

In addition, the composition of the present invention may contain a skin absorption promoting substance to increase the skin improving effect.

Hereinafter, the constitution and effects of the present invention will be described in more detail with reference to examples and test examples. However, these test examples are provided for illustrative purposes only in order to facilitate understanding of the present invention, and the scope and scope of the present invention are not limited by the following examples.

[Preparation Example 1] Preparation of general pine leaf extract

1 kg of dried pine leaf is pulverized and 10 kg of distilled water is added, followed by hot extraction at 80 to 90 ° C for 6 hours. The obtained extract was centrifuged, and the supernatant was separated. The filtrate was concentrated under reduced pressure, and the solvent was distilled off to obtain about 89 g of a solid.

[Preparation Example 2] Preparation of general red ginseng extract

Using 1 kg of red ginseng, red ginseng was extracted in the same manner as in Production Example 1 to obtain about 84 g of solid content.

[Preparation Example 3] Preparation of ordinary green tea extract

Using 1 kg of dried tea leaves, green tea was extracted in the same manner as in Production Example 1 to obtain a solid content of about 96 g.

[Preparation Example 4] Preparation of general cargarifera extract

Using the dried car as 1 kg, the car was extracted in the same manner as in Production Example 1 to obtain a solid content of about 88 g.

[Preparation Example 5] Preparation of general red ginseng extract

Using the dried 1 kg of Hongyeong Streams outgrowth, Hongkryun Stream was extracted in the same manner as in Production Example 1 to obtain a solid content of about 79 g.

[Preparation Example 6] Preparation of general gooseberry fruit extract

Using dried 1 g of gooseberry fruit, the gooseberry was extracted in the same manner as in Production Example 1 to obtain a solid content of about 92 g.

[Preparation Example 7] Preparation of general Siberian oyster extract

Using 1 kg of dried Siberian ostrich outpost, about 91 g of solid content was obtained by extracting Siberian ostrich in the same manner as in Preparation Example 1.

[Preparation Example 8] Preparation of general white birch extract

A total of 1 kg of dried white birch was used to extract white birch in the same manner as in Production Example 1 to obtain a solid content of about 83 g.

[Preparation Example 9] Preparation of pine needle glycoprotein

1 kg of dried pine needle was crushed, 10 kg of distilled water was added, and then subjected to hot water extraction at 80 to 90 ° C for 5 hours. The residue is removed using a 3 μm mesh, and the α-amylase is treated at 20 to 70 ° C. for 20 hours to make the extracted polymer into a low molecular form. The enzyme-treated extract is heated at 80 to 90 ° C for 20 hours to inactivate the enzyme, centrifuged, and the supernatant is concentrated under reduced pressure to obtain a concentrate. 90% ethanol is added to the concentrate 10 times the volume of the concentrate to precipitate the remaining polymer. After the precipitate was removed, the concentrate was concentrated under reduced pressure to obtain lyophilized glycoprotein solid of about 63 g.

[Preparation Example 10] Preparation of red ginseng glycoprotein

1 kg of red ginseng was prepared in the same manner as in Production Example 9 to obtain about 58 g of red ginseng glycoprotein solid.

[Production Example 11] Preparation of green tea glycoprotein

Using 1 kg of dried tea leaves, about 48 g of green tea glycoprotein solid was obtained in the same manner as in Production Example 9.

[Preparation Example 12] Preparation of chaga glycoprotein

The dried tea was prepared in the same manner as in Production Example 9, using 1 kg, to obtain a glycoprotein solid content of about 57 g.

[Preparation Example 13] Preparation of Hong Kyung Chun Glycoprotein

A total of 1 kg of the dried Hong Kyung Stream was used in the same manner as in Production Example 9 to obtain about 43 g of Hong Hong Kyung Glycoprotein solid.

[Preparation Example 14] Preparation of gooseberry fruit glycoprotein

1 kg of dried gooseberry fruit was prepared in the same manner as in Production Example 9 to obtain about 62 g of gooseberry fruit glycoprotein solid.

[Preparation Example 15] Preparation of Siberian ostrich glycoprotein

Using about 1 kg of dried Siberian ostrich, about 55 g of Siberian oyster was extracted in the same manner as in Production Example 9 to obtain a Siberian ostrich glycoprotein solid.

[Production Example 16] Production of white birch glycoprotein

Using 1 kg of whole dried white birch, white birch was extracted in the same manner as in Production Example 9 to obtain about 62 g of white birch glycoprotein solid.

[Test Example 1] PPAR-alpha activation experiment

PPAR-alpha plays a role in promotion of differentiation, proliferation inhibition and lipid biosynthesis of skin cells and anti-inflammation, and the activation tendency of PPAR-alpha of plant glycoprotein used in the present invention was confirmed by the following method.

Monkey kidney epithelial cell line CV-1 cells (ATCC CCL 70) were subcultured in charcoal / dextran-treated DMEM medium containing 10% fetal bovine serum and the effect of estrogen on phenol red No-phenol red medium was used to remove. Plasmids (PPARs, PPARs, QIAGEN) are activated by the PPARs (PPARs Response Element), which is activated by the combination of PPAR- (ligand-linked PPAR-) with a universal promoter that is expressed under normal culture conditions. , A firefly luciferase gene serving as a reporter at the back thereof, and a universal promoter to be used as a reference, and a plasmid to which a Renilla luciferase gene was coupled.

The CV-1 cells were added to a 24-well plate at a concentration of 9 × 10 ⁴ cells / well and cultured for 24 hours. Then, the three kinds of plasmid genes were transiently transfected. Then, the cells were cultured for 24 hours, washed with 1 × PBS (Phosphate Buffered Saline), treated with 1 ppm and 10 ppm of each of the above Preparation Examples 9 to 16, cultured for 24 hours, washed with 1 × PBS, Cells were broken with 1 X PLB (Passive Lysis Buffer) and the activity of luciferase was measured using Dual-Luciferase® Reporter Assay System kit (Promega). For comparison, 1 ppm and 10 ppm of each of the general extracts prepared in Preparation Examples 1 to 8 were treated in place of glycoprotein, and the activity of PPAR-alpha was calculated by measuring the activity of luciferase.

As the negative control group, Wy-14,643, the most potent ligand of PPAR-α, was used at a concentration of 0.5 μM and 1 μM in the positive control group. The results are shown in Table 1.

Treating material PPAR-alpha activation degree 1 ppm 10 ppm Negative control group badge 0.2 Positive control group Wy-14,643 (0.5 [mu] M) 0.57 Wy-14,643 (1 [mu] M) 1.28 Common extract Production Example 1 (pine needle) 0.24 0.29 Production Example 2 (red ginseng) 0.37 0.47 Production Example 3 (Green tea) 0.41 0.52 Production Example 4 (tertiary) 0.31 0.45 Production Example 5 (Hong Kyung Chun) 0.23 0.28 Production Example 6 (Gooseberry) 0.33 0.41 Production Example 7 (Siberian oak) 0.28 0.36 Production Example 8 (white birch) 0.32 0.48 Glycoprotein Production Example 9 (pine needles) 0.86 0.91 Production Example 10 (red ginseng) 0.88 0.93 Production Example 11 (Green tea) 0.84 0.87 Production Example 12 (tert) 0.76 0.82 Production Example 13 (Hong Kyung Chun) 0.57 0.64 Production Example 14 (Gooseberry) 0.74 0.79 Production Example 15 (Siberian oak) 0.63 0.71 Production Example 16 (white birch) 0.65 0.72

In the results shown in the above Table 1, the general extract has a slightly higher degree of activation of PPAR-α than the negative control, but it can be confirmed that glycoprotein exhibits a significantly higher activity of PPAR-α than the general extract.

Thus, it can be seen that the composition of the present invention has a skin anti-inflammatory effect.

[Test Example 2] MMP-1 assay (MMP-1 assay)

In order to examine the effect of reducing the biosynthesis of MMP-1 due to ultraviolet rays related to degradation of the skin substrate of the glycoprotein according to the present invention and promoting the biosynthesis of type 1 procollagen, MMP-1 analysis was performed by the following method Respectively.

First, the epidermis and dermis were separated from collagenase, and epidermis and dermis were placed in 0.25% trypsin solution, respectively, and incubated at 37 ° C in a 5% CO ₂ incubator And treated for 10 minutes. Then vortex was performed to release keratinocytes and fibroblasts, respectively. The cells were collected and washed, and then keratinocyte growth medium (Clonetics, USA) was cultured at a concentration of 1 × 10 ⁴ cells / cm 2. The fibroblasts were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) Lt; / RTI > 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 MMP-1, fibroblasts were cultured in a 48-well plate at a concentration of 90% or more, and then transformed into a DMEM medium containing no FBS to starvate for one day. Then, the plate was washed twice with PBS, and 100 쨉 l of PBS was added. The plate was opened and irradiated with ultraviolet A (Dermite cube 401, UVAtec, USA, equipped with UVA filter) at 15 J / cm 2. Immediately after the irradiation, the cells were washed once with PBS. DMEM containing 10, 100 ppm of fetal bovine serum was added to each of the above Preparation Examples 9 to 16 at 37 ° C and 5% CO ₂ And cultured in an incubator for 48 hours. For comparison, the general extracts prepared in Preparation Examples 1 to 8 were treated at the same concentration as that of glycoprotein and cultured. At this time, the control group was not treated with the extract and UVA (control group 1) and the UVA treated group (control group 2) without the extract. The amount of MMP-1 liberated in the medium was measured using an ELISA spectrophotometer (Multiskan GO, Thermo scientific), corrected for the total amount of protein in the fibroblasts, and calculated as% based on the value of control 1, The expression rates are shown in Table 2.

Treating material MMP-1 expression rate (%) 10 ppm 100 ppm Control 1 No treatment (no UVA treatment) 100 Control group 2 No treatment + UVA 174 Common extract Production Example 1 (pine needle) 170 167 Production Example 2 (red ginseng) 146 124 Production Example 3 (Green tea) 165 158 Production Example 4 (tertiary) 164 149 Production Example 5 (Hong Kyung Chun) 157 134 Production Example 6 (Gooseberry) 161 157 Production Example 7 (Siberian oak) 160 147 Production Example 8 (white birch) 153 131 Glycoprotein Production Example 9 (pine needles) 147 126 Production Example 10 (red ginseng) 119 93 Production Example 11 (Green tea) 140 114 Production Example 12 (tert) 142 121 Production Example 13 (Hong Kyung Chun) 109 87 Production Example 14 (Gooseberry) 132 102 Production Example 15 (Siberian oak) 124 98 Production Example 16 (white birch) 137 109

As can be seen from the results shown in Table 2, when the glycoprotein is treated, the expression of MMP-1, a collagen degrading enzyme, is lower than in the case of treatment with a general extract, thereby effectively preventing collagen degradation.

Therefore, it can be seen from the above results that glycoprotein inhibits collagen degradation and effectively prevents skin aging.

[Test Example 3] Evaluation of melanogenesis inhibitory activity using B16 / F10 melanoma cells

In order to examine the inhibitory effect of glycoprotein on melanogenesis, experiments were conducted using B16 / F10 melanoma cells (Korean Cell Line Bank) as follows.

2 × 10 ⁴ cells / well (final volume: 3 ml), 96 wells (10 μl) were added to 6 well plates (melanin quantitation) with DMEM containing 10% FBS as the test substances in the above-mentioned Production Examples 9 to 16 and Production Examples 1 to 8 2 × 10 ³ cells / well (final volume 200 μl) in a plate (MTTassay) and cultured in a 5% CO ₂ incubator (MCO-20AIC, Sanyo) at 37 ° C. for 24 hours. After the culture, the medium was removed, and DMEM medium containing 10% FBS, 2 μM α-MSH (Melanocyte stimulating hormone) and 2 mM theophylline was used as a new medium, and the test material was added to the medium for 3 days. On day 5, the medium cultured in a 6-well plate was removed and the cell pellet was recovered by treating with 0.25% trypsin-EDTA (Sigma Chemical Co.) solution. The cell pellet was recovered and transferred to a 1.5 ml tube and centrifuged at 10,000 rpm for 10 minutes to remove the supernatant . The resulting pellet was dried at 60 ° C., and 100 μl of 1N NaOH was added to dissolve the melanin in the cells. This solution was diluted with PBS and the absorbance was measured at 475 nm using an ELISA spectrophotometer (Multiskan GO, Thermo scientific) to determine the inhibition rate of melanin formation in the sample. Cells to which the extract had not been added were used as a negative control, and compared with the melanin content in the negative control, the degree of melanin formation inhibition of the test substance was measured to determine the whitening effect. The inhibition rate of melanin production was calculated according to the following formula (1), and the results are shown in Table 3. As a positive control, arbutin was used as a test substance at a concentration of 200 μM.

Treating material Melanin production inhibition rate (%) 10 ppm 100 ppm Negative control group No extract added - Positive control group Arbutin (200 μM) 63 Common extract Production Example 1 (pine needle) 5 7 Production Example 2 (red ginseng) 11 13 Production Example 3 (Green tea) 8 11 Production Example 4 (tertiary) 12 15 Production Example 5 (Hong Kyung Chun) 9 11 Production Example 6 (Gooseberry) 10 12 Production Example 7 (Siberian oak) 4 8 Production Example 8 (white birch) 7 6 Glycoprotein Production Example 9 (pine needles) 21 22 Production Example 10 (red ginseng) 24 33 Production Example 11 (Green tea) 32 42 Production Example 12 (tert) 42 54 Production Example 13 (Hong Kyung Chun) 21 24 Production Example 14 (Gooseberry) 30 38 Production Example 15 (Siberian oak) 39 49 Production Example 16 (white birch) 18 21

In the results of Table 3, it was found that glycoprotein showed better inhibitory effect on melanin formation than general extract, and that the higher the concentration of extract used, the more the inhibition of melanin production was produced.

[Test Example 4] Evaluation of skin regeneration effect using fibroblast cells

The degree of skin regeneration effect by using glycoprotein was evaluated by the following method. (Fibroblast basal medium) containing 10% FBS was added to a 96-well plate at a concentration of 1 × 10 ³ cells / well (final volume: 100 μl) ₂ incubator (MCO-20AIC, Sanyo) for 24 hours. After the culture, the medium was removed, and the medium was replaced with FBS-free FBM medium and cultured in a starvation state. For comparison, control cells were cultured in FBM medium (control 1) containing 10% FBS and cells cultured in medium without FBS (control 2). After culturing, the cells were treated with 10 ppm and 100 ppm of the above-mentioned Production Examples 9, 10, 11, 12, 13, 14, 15 and 16 and Production Examples 1, 2, 3, 4, 5, 6, 7 and 8 And cultured for 48 hours. After 48 hours, the degree of cell increase was confirmed using EZ-Cytox kit (Duzen Biotechnology Research Institute), and the degree of increase of cells was calculated based on the result of Control 2, and the results are shown in Table 4.

Treating material Cell growth rate (%) 10 ppm 100 ppm Control 1 FBM medium containing 10% FBS 152 Control group 2 FBM badge without FBS 100 Common extract Production Example 1 (pine needle) 112 107 Production Example 2 (red ginseng) 102 103 Production Example 3 (Green tea) 108 105 Production Example 4 (tertiary) 103 110 Production Example 5 (Hong Kyung Chun) 105 108 Production Example 6 (Gooseberry) 109 113 Production Example 7 (Siberian oak) 115 111 Production Example 8 (white birch) 107 106 Glycoprotein Production Example 9 (pine needles) 114 121 Production Example 10 (red ginseng) 124 129 Production Example 11 (Green tea) 110 119 Production Example 12 (tert) 109 110 Production Example 13 (Hong Kyung Chun) 114 117 Production Example 14 (Gooseberry) 118 123 Production Example 15 (Siberian oak) 120 127 Production Example 16 (white birch) 112 113

In the results of Table 4 above, it was confirmed that the treatment with glycoprotein increased the cells more than the treatment with the general extract.

Therefore, it can be seen from the above results that glycoprotein can effectively promote skin regeneration.

[Referential Example 1] Preparation of Examples 1 to 11

As a result of the above tests, red ginseng, red ginseng tea, green tea, and chaga glycoprotein having the best skin effect among the preparation examples were selected and mixed at the concentrations shown in Table 5 below. Examples 1 to 6 were obtained by mixing glycoproteins of two kinds of preparation examples in a ratio of 1: 1, and Examples 7 to 10 were obtained by mixing glycoproteins of three production examples at a ratio of 1: 1: 1, , 4 kinds of glycoproteins were mixed at a ratio of 1: 1: 1: 1.

Unit (wt%) Production Example 10 Production Example 11 Production Example 12 Production Example 13 Example 1 50 50 - - Example 2 50 - 50 - Example 3 50 - - 50 Example 4 - 50 50 - Example 5 50 50 Example 6 - - 50 50 Example 7 About 33.3 About 33.3 About 33.3 - Example 8 About 33.3 About 33.3 - About 33.3 Example 9 About 33.3 - About 33.3 About 33.3 Example 10 - About 33.3 About 33.3 About 33.3 Example 11 25 25 25 25

[Test Example 5] PPAR-alpha activation experiment of complex glycoprotein

In order to confirm the anti-inflammatory effect by the combination of two or more kinds of glycoproteins according to the present invention, PPAR-α activation experiment was carried out in the same manner as in Test Example 1, except that the above Examples 1 to 11 were treated at 10 ppm, Are shown in Table 6 and Fig. 2 below. For comparison, 10 ppm of the negative control, the positive control and the 10th, 11th, 12th, and 13th groups of Test Example 1 were also shown.

Treating material PPAR-alpha activation degree Negative control group badge 0.2 Positive control group Wy-14,643 (0.5 [mu] M) 0.57 Wy-14,643 (1 [mu] M) 1.28 Single glycoprotein Production Example 10 (red ginseng) 0.87 Production Example 11 (Green tea) 0.93 Production Example 12 (tert) 0.82 Production Example 13 (Hong Kyung Chun) 0.64 Complex glycoprotein Example 1 (red ginseng, green tea) 0.98 Example 2 (red ginseng, tea) 0.87 Example 3 (Red ginseng, Hongyeongcheon) 0.73 Example 4 (green tea, tea) 0.81 Example 5 (Green Tea, Hong Kyung Chun) 0.62 Example 6 (Cha Cha, Hong Kyung Chun) 0.84 Example 7 (red ginseng, green tea, tea) 0.65 Example 8 (red ginseng, green tea, Hongyeong Stream) 0.86 Example 9 (Red ginseng, Chaaga, Hongikong Stream) 1.01 Example 10 (Green Tea, Cha Cha, Hong Kyung Chun) 0.76 Example 11 (red ginseng, green tea, tea, Hongikong Stream) 0.59

In the results shown in Table 6 and FIG. 2, the activity of PPAR-alpha was highest in the case of treating Example 9 in which the glycoproteins extracted from red ginseng, tea, and Hongikong stream were mixed, and the synergistic effect .

[Experimental Example 6] Expression rate of MMP-1 in the complex glycoprotein

In order to confirm the effect of inhibiting the expression of MMP-1 by the combination of two or more kinds of glycoproteins according to the present invention, MMP-1 was assayed in the same manner as in Test Example 2 except that the above Examples 1 to 11 were treated at 100 ppm And the results are shown in Table 7 below. For comparison, the results of treating 100 ppm of control 1, control 2 and preparations 10 to 13 of Test Example 2 are also shown.

Treating material MMP-1 expression rate (%) Control 1 No treatment (no UVA treatment) 100 Control group 2 No treatment + UVA 174 Single glycoprotein Production Example 10 (red ginseng) 93 Production Example 11 (Green tea) 114 Production Example 12 (tert) 121 Production Example 13 (Hong Kyung Chun) 87 Complex glycoprotein Example 1 (red ginseng, green tea) 112 Example 2 (red ginseng, tea) 102 Example 3 (Red ginseng, Hongyeongcheon) 97 Example 4 (green tea, tea) 117 Example 5 (Green Tea, Hong Kyung Chun) 108 Example 6 (Cha Cha, Hong Kyung Chun) 80 Example 7 (red ginseng, green tea, tea) 119 Example 8 (red ginseng, green tea, Hongyeong Stream) 105 Example 9 (Red ginseng, Chaaga, Hongikong Stream) 73 Example 10 (Green Tea, Cha Cha, Hong Kyung Chun) 107 Example 11 (red ginseng, green tea, tea, Hongikong Stream) 112

From Table 7 above, it can be seen that the expression of MMP-1 is most suppressed when Example 9 is treated. In addition, when Example 6 was treated, it was found that the expression of MMP-1 was significantly suppressed as compared with the case where only the other examples and the production examples were treated.

[Test Example 7] Evaluation of melanin formation inhibitory ability of complex glycoprotein

In order to confirm the whitening effect by the combination of two or more kinds of glycoproteins according to the present invention, melanin using B16 / F10 melanoma cells in the same manner as in Test Example 3, except that the above Examples 1 to 11 were treated at 100 ppm The results are shown in Table 8 below. For comparison, the control group, the positive control group, and the preparation examples 10 to 13 of Test Example 3 were treated at a concentration of 100 ppm.

Treating material Melanin production inhibition rate (%) Negative control group Control group - Positive control group Arbutin (200 μM) 63 Single glycoprotein Production Example 10 (red ginseng) 33 Production Example 11 (Green tea) 42 Production Example 12 (tert) 54 Production Example 13 (Hong Kyung Chun) 24 Complex glycoprotein Example 1 (red ginseng, green tea) 32 Example 2 (red ginseng, tea) 41 Example 3 (Red ginseng, Hongyeongcheon) 38 Example 4 (green tea, tea) 61 Example 5 (Green Tea, Hong Kyung Chun) 22 Example 6 (Cha Cha, Hong Kyung Chun) 32 Example 7 (red ginseng, green tea, tea) 18 Example 8 (red ginseng, green tea, Hongyeong Stream) 20 Example 9 (Red ginseng, Chaaga, Hongikong Stream) 43 Example 10 (Green Tea, Cha Cha, Hong Kyung Chun) 55 Example 11 (red ginseng, green tea, tea, Hongikong Stream) 43

From the results of Table 8, it was shown that the melanin formation inhibiting effect was excellent in Example 4 and Example 10. [ Particularly, in the case of Example 4, the effect is similar to that of arbutin used as a positive control.

[Test Example 8] Evaluation of effect of complex glycoprotein on skin regeneration

Evaluation of skin regeneration effect using fibroblasts was carried out in the same manner as in Test Example 4, except that the skin regeneration effect by the combination of two or more kinds of glycoproteins according to the present invention was examined at 100 ppm in Examples 1 to 11 And the results are shown in Table 9 below. For comparison, the results of treating the control groups 1 and 2 and Preparation Examples 10 to 13 of Test Example 4 with 100 ppm concentration are also shown.

Treating material Cell growth rate (%) Control 1 FBM medium containing 10% FBS 152 Control group 2 FBM badge without FBS 100 Single glycoprotein Production Example 10 (red ginseng) 129 Production Example 11 (Green tea) 119 Production Example 12 (tert) 110 Production Example 13 (Hong Kyung Chun) 117 Complex glycoprotein Example 1 (red ginseng, green tea) 124 Example 2 (red ginseng, tea) 134 Example 3 (Red ginseng, Hongyeongcheon) 143 Example 4 (green tea, tea) 121 Example 5 (Green Tea, Hong Kyung Chun) 124 Example 6 (Cha Cha, Hong Kyung Chun) 113 Example 7 (red ginseng, green tea, tea) 124 Example 8 (red ginseng, green tea, Hongyeong Stream) 116 Example 9 (Red ginseng, Chaaga, Hongikong Stream) 119 Example 10 (Green Tea, Cha Cha, Hong Kyung Chun) 117 Example 11 (red ginseng, green tea, tea, Hongikong Stream) 125

From the results shown in Table 9, it was confirmed that the differentiation of skin cells was induced in Examples 3 and 2 even in the medium without FBS.

Therefore, it can be confirmed that the skin regeneration effect is excellent in the order of Example 3 and Example 2.

Claims

A composition for external application for skin containing glycoprotein,
Wherein the glycoprotein is a plant glycoprotein extracted from at least one selected from the group consisting of pine needle, red ginseng, green tea, tea gourd, Rhodiola griseus, gooseberry, Siberian ostrich, and white birch.

The composition for external application for skin according to claim 1, wherein the glycoprotein is contained in an amount of 0.0001 to 30% by weight based on the total weight of the composition.

The composition for external application for skin according to claim 1 or 2, wherein the composition is for anti-inflammation.

3. The composition for external application for skin according to claim 1 or 2, wherein the composition is for whitening.

The composition for external application for skin according to claim 1 or 2, wherein the composition is for improving skin wrinkles.

The composition for external application for skin according to claim 1 or 2, wherein the composition is for improving skin elasticity.

The composition for external application for skin according to claim 1 or 2, wherein the composition is for PPAR-a activation.

A method for producing a composition for external application for skin containing glycoprotein,
a) hot water extraction of the dried raw material;
b) hydrolyzing the hydrolyzed extract by enzymatic treatment; And
c) concentrating the hydrolyzed extract under reduced pressure,
Wherein the dried raw material is at least one selected from the group consisting of pine needle, red ginseng, green tea, tea, red ginseng, gooseberry, Siberian oak, and white birch.