KR101715200B1 - Composition containing glycoproteins extract from plant - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising a glycoprotein extracted from at least one selected from the group consisting of Verenia cordifolia, Rodiola losia, Comarum parus trita, Avissibirica, Betulacepula dubarb, and Inonotus auricus, ≪ / RTI > is disclosed. The composition has the effect of preventing or improving skin wrinkles, whitening skin, preventing or improving inflammation, moisturizing skin, promoting skin regeneration, preventing or improving scarring, improving atopic skin, improving antioxidation, preventing or improving aging.

Description

[0001] The present invention relates to a composition containing a plant glycoprotein extract,

The present invention relates to a composition containing plant-derived glycoproteins.

As the average life expectancy of men has recently increased, it is not how long they live, but how long they live and stay young. As you get older, aging is an unavoidable phenomenon. In modern society where environmental pollution and stress are getting worse and the frequency of ultraviolet exposure increases, aging is starting earlier than before.

All the organs of the human body are gradually aging at a certain rate. Among them, skin aging is carried out by a complex process in which various natural aging processes are influenced by the external environment. Most of the early skin aging starts with a dryness of the skin, specifically a decrease in skin elasticity due to lack of moisture and wrinkles. As they get older, they lose their elasticity due to the loss of cellular regeneration, and the connective tissues of the skin, which are destroyed by the effects of hormones and ultraviolet rays, are stretched to form wrinkles.

Specific skin aging phenomenon slows skin regeneration, aging skin becomes rough, skin becomes rough, collagen production decreases, wrinkles occur, and defensive power against ultraviolet rays is lowered, skin color is stained, pigmentation such as spots, The skin protection function is lowered and the drug is easily reacted to stimulation.

The present invention provides a composition for preventing or improving skin wrinkles, skin whitening, preventing or improving inflammation, moisturizing skin, promoting skin regeneration, preventing or improving scarring, improving atopic skin, improving antioxidation, preventing or improving aging.

The present invention to achieve the above object bereuge California cor di polyamic (Bergenia cordifolia, Rhodiola rosea , Comarum palustre , Abies Sibirica , Betula pendula Buds , and Inonotus < RTI ID = 0.0 > Obliquus ) as an active ingredient. The present invention also provides a composition comprising the above-mentioned glycoprotein as an active ingredient.

In one embodiment of the present invention, the composition has a use for preventing or improving skin wrinkles.

In one embodiment of the present invention, the composition is characterized by having a skin whitening application.

In one embodiment of the present invention, the composition is characterized by having an anti-inflammatory or improving effect.

In one embodiment of the present invention, the composition is characterized by having a skin moisturizing use.

In one embodiment of the present invention, the composition is characterized by having a skin regeneration promoting purpose.

In one embodiment of the present invention, the composition is characterized by having a use for scar prevention or improvement.

In one embodiment of the present invention, the composition is characterized by having an application for improving atopic skin.

In one embodiment of the present invention, the composition is characterized by having an antioxidant enhancing use.

In one embodiment of the present invention, the composition is characterized by having anti-aging or improving applications.

The composition of the present invention is a composition comprising a glycoprotein extracted from at least one selected from the group consisting of Verenia cordifolia, Rhodiola losia, Comarum parus trita, Avicivirica, Betula pendulabais, and Inonotus auricus It has an effect of preventing or improving wrinkles of skin, whitening skin, preventing or improving inflammation, moisturizing skin, promoting skin regeneration, preventing or improving scarring, improving atopic skin, improving antioxidation, preventing or improving aging.

FIG. 1 is a graph showing the results of Test Example 8 in which DPPH was used to measure antioxidative activities of glycoprotein and vitamin C extracted from six plants.

As used herein, the term "extract" refers to a substance obtained by extracting a component contained therein from a natural product, regardless of the method of extraction or the kind of the component. For example, it is a broad concept including both extracts of components dissolved in a solvent from natural products by using water or an organic solvent, and extracts of specific components of natural products, such as oils, and extracts of specific components.

As used herein, the term "skin" refers to a tissue covering the body surface of an animal, and is a concept of the broadest notion including a scalp and hair as well as a tissue covering a body surface such as a face or a body.

Hereinafter, the present invention will be described in detail.

One embodiment of the present invention relates to a method for the treatment of diabetes mellitus comprising extracting at least one compound selected from the group consisting of < RTI ID = 0.0 > Verenia < / RTI > coridolia, Rodiola losia, Comarum paulastre, Avis, And a protein as an active ingredient.

In the above, Bergenia < RTI ID = 0.0 > cordifolia ), also known as Siberian ostrich, is a perennial plant of rosewood spider mite. Its origin is Siberia and Mongolia, and its height is about 30-40cm. Flowers bloom in late spring or early spring with reddish purple or pale pink, unusual but white. Rhodiola rosea ( Rhodiola rosea ) belongs to the tree limb, stalagmite, and is also known as Hongkongcheon, and grows mainly in the mountains of the Arctic region of Europe and Asia. Comarum palustre ( Comarum palustre ), also known as Shavilinik valotny , belongs to Rosaceae and is a herb that mainly grows in Russia. Abies Sibirica grows mainly in the wet areas of the northern mountains, which are cold with Siberian fir, belonging to the pine tree Pinus densiflora. Betula pendula belongs to the birch of the oak tree. It is also called the pendula birch and the silver birch. It mainly uses the eye part. It grows in mountains across Europe and Asia, and in high altitudes in southern Europe. Inonotus obliquus belongs to the pine scales and is also called mushrooms. Its origin is Siberian, and it is parasitized in birch of more than 45 degrees north latitude such as Siberia, North America, northern Europe.

As used herein, "collagen" refers to an important fibrous protein of animal connective tissue, which has functions of preventing or improving skin wrinkles, regenerating cells, preventing or improving scarring, strengthening skin elasticity and preventing skin aging. "Pro-collagen" is a precursor of collagen found in the skin and other organs of several vertebrates, and consequently has the same function as collagen. Thus, increased levels of collagen and procollagen can prevent or improve skin wrinkles, regenerate cells, prevent or improve scarring, enhance skin elasticity, and prevent or improve skin aging. On the other hand, "collagenase" is an enzyme that decomposes collagen and is known as one of the causes of wrinkles of the skin. Therefore, inhibition of the expression thereof increases the collagen level of the skin, so that the wrinkles of the skin can be prevented or improved, the cells can be regenerated, the skin elasticity can be enhanced, and the skin aging can be prevented or improved. The compositions disclosed herein are useful for promoting procollagen biosynthesis and for inhibiting the production of collagenase (MMP-1), such as Verdenia cordifolia, Rodiola losia, Comarum paulus strain, Tuliphene dulabox, and inonotus auricus as active ingredients to prevent or improve skin wrinkles, to regenerate cells, to prevent or improve scarring, to strengthen skin elasticity, to prevent skin aging .

"Melanin" is a black pigment, which acts to protect the skin from ultraviolet rays because it has a function to block ultraviolet rays of a certain amount or more. Therefore, when the skin is exposed to ultraviolet rays, a large amount of melanin is produced from the body to protect the skin. Since the skin color is determined by the amount of melanin, the more the amount of melanin is, the darker the skin color, and it causes the spots, the spot, the freckles and the black spot. Therefore, such a composition for inhibiting melanin production can prevent the skin from becoming black and have a skin whitening effect. The compositions disclosed herein are useful for inhibiting melanogenesis in a group consisting of Verenia cordifolia, Rodiola rosia, Comarum parus trita, Avis steriqua, Betulace pendulabauz, and Inonotus auricus The present invention can be used for skin whitening purposes.

Interleukin-6 (IL-6) is a cytokine, also called B-cell stimulating factor 2 (BSF2) or interferon β2 (INF-β2), and is known to mediate inflammatory responses in inflammatory diseases. Interleukin-8 (IL-8), also called neutrophil inducing / activating protein-1 (NAP-1), is also known to mediate the inflammatory response and induce histamine release in patients with atopic skin disease. MCP-1 (Momocyte Chemoattractant Protein-1), also called CCL-2, is also known as an inflammatory cytokine. Therefore, when the biosynthesis of the cytokines is inhibited, the anti-inflammatory effect can be obtained, and further, the atopic skin can be prevented or improved. The compositions disclosed herein are useful for inhibiting inflammation induced by LPS and for inhibiting the biosynthesis of IL-6, IL-8, and MCP-1, such as Verenia cordifolia, Rodiola losia, , Avisibirica, Betulacepula dubarubus, and Inonotus auricus as active ingredients to prevent or ameliorate inflammation, improve atopic skin and prevent skin aging Or improving the activity of the compound.

PPARγ is a factor that exists in the skin constituting cells as a whole. It maintains epidermal homeostasis by regulating differentiation of epidermal keratinocytes and lipid metabolism to induce normal epidermal layer formation. Therefore, homeostasis and restoration of skin barrier function, moisturization, inflammation It is known to play an important role in the development of the healing process. Because of this feature, PPARγ can act as a key regulator of various skin diseases such as psoriasis, wound healing, acne and the like due to the superficial epidermal layer as well as inflammation related skin diseases. Especially, when PPARγ is activated, it is expected that the skin moisturizing effect can be enhanced by strengthening the skin barrier function. The compositions disclosed herein may be used in a group consisting of Verenia cordifolia, Rodiola losia, Comarum parus trit, Avicivirica, Betulacepenula vulgaris, and Inonotus auricus effective to activate PPARy By containing the glycoprotein extracted from the selected one or more as an active ingredient, the skin moisturizing effect can be improved, the atopic skin can be improved, and the aging of the skin can be further prevented or improved.

As used herein, "cell regeneration" refers to the phenomenon of regenerating damaged cells, and "skin cell regeneration" refers specifically to regenerating skin cells. If the skin is not regenerated smoothly, the aging of the skin is promoted, and the melanin pigment produced in the pigment cells in the epidermis is not discharged, and the remaining pigment deposits are generated. On the other hand, if the skin is regenerated smoothly, skin scarring can be prevented or improved and the skin can be moisturized. The compositions disclosed herein are useful for treating cell regeneration, particularly skin regeneration, which are effective for promoting skin regeneration, such as, but not limited to, Verenia cordifolia, Rodiola losia, Comarum paulus tere, Avis tsibirica, The present invention relates to a method for preventing or improving wrinkles of skin, having skin whitening and skin moisturizing effect, promoting skin regeneration, preventing or improving scarring, and further improving aging by incorporating a glycoprotein extracted from at least one selected from the group consisting of Prevention or amelioration.

If more oxygen is present in the body than is needed, some of the remaining oxygen becomes a free radical with a high activity. A highly reactive free radical attacks the cell and the cell is damaged by the attack. Oxygen-rich oxygen, which attacks living tissues and damages cells, is called active oxygen, which is normally produced in the body's metabolic processes, and is abnormally increased by excessive oxygen uptake, stress and other causes during respiration. This active oxygen has strong cytotoxicity and needs to be removed. As used herein, "antioxidant" refers to an action to remove active oxygen which is harmful oxygen. The compositions disclosed herein may comprise one or more compounds selected from the group consisting of antioxidant enzymes, such as Verenia cordifolia, Rodiola losia, Comarum parus trita, Avicivirica, Betulacepenula vulgaris, and Inonotus auricus The present invention can prevent cellular damage caused by active oxygen or ultraviolet rays and prevent or improve wrinkles of the skin, thereby preventing or improving aging.

In one embodiment of the present invention, a sugar chain derived from at least one member selected from the group consisting of Verenia cordifolia, Rodiola losia, Comarum parus trita, Avis steriqua, Betulace pendulabauz, and Inonotus auricus The protein may be contained in an amount of 0.1 to 30% by weight based on the total weight of the composition, depending on the form of the composition. If the content is less than 0.1% by weight, the effect is insufficient. If the content is more than 30% by weight, stability of the composition may be problematic.

In an embodiment according to the present invention, the effect to be achieved by the present invention can be achieved by a method of the present invention, which is comprised of Verenia cordifolia, Rodiola losia, Comarum parus trita, Avissibirica, Derived from one or more glycoproteins selected from the group.

The method of extracting glycoproteins from each plant is as follows: Chilled raw plant is chopped with a crusher, and then an organic acid solution having a pH of 2 to 3 is mixed at a ratio of 1: 2 to 1: 5, And the temperature is maintained at 30 to 50 ° C. The pH of the aqueous solution is adjusted to 1.0 to 3.0, the protein hydrolyzing enzyme is added in an amount of 0.1 g to 1 g% by weight based on the aqueous solution of the organic acid, and the mixture is treated at 30 to 50 ° C for 2 to 24 hours. When the concentration of the enzyme-treated aqueous solution reaches the desired concentration, the first filtration process, which is a process of filtering only the glycoprotein aqueous solution, is performed. The temperature of the filtered aqueous solution is rapidly raised to 85 ° C or higher and the reaction is terminated by sterilization at the same time as the enzymes are inactivated by heating for a certain period of time. The sterilized extract is dried by using a centrifuge to remove foreign matters and dried to complete a glycoprotein powder product (see Korean Patent Publication No. 10-2004-27283).

In addition, the glycoprotein may be obtained by a general method of leaching, purifying, and separating each plant material.

The present invention relates to a pharmaceutical composition containing a glycoprotein extracted from at least one selected from the group consisting of Verenia cordifolia, Rodiola losia, Comarum parus trita, Avicivirica, Betulacepula dubarb, and Inonotus auricus To provide a cosmetic composition comprising the composition. The cosmetic composition may be, for example, a cosmetic product, and the cosmetic appearance of the cosmetic product contains a cosmetically or dermatologically acceptable medium or base. It may be in any form suitable for topical application, for example, as a solution, a gel, a solid, an anhydrous product of a paste, an emulsion obtained by dispersing an oil phase in water phase, an emulsion obtained by dispersing water phase in water phase, a suspension, a microemulsion, Or in the form of a cream, a skin, a lotion, a powder, an ointment, a spray, a cleanser or a concealer, in the form of granules or granules or ionic (liposomes) and non-ionic fecal dispersions. These compositions may be prepared according to conventional methods in the art. The composition according to the invention may also be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

The cosmetic compositions may also be in the form of tablets or capsules prepared by dissolving or dispersing the active ingredient in a diluent such as a lipid, an organic solvent, a solubilizer, a thickener, a gelling agent, a softener, an antioxidant, a suspending agent, a stabilizer, a foaming agent, , Cosmetics or dermatology such as metal ion sequestrants, chelating agents, preservatives, vitamins, blocking agents, wetting agents, 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 art. Such adjuvants are introduced in amounts commonly used in the cosmetics or dermatological fields.

The cosmetic composition of the present invention is not particularly limited in its formulation and may be, for example, softening lotion, convergent lotion, nutritional lotion, nutritional cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, Packs, powders, body lotions, body creams, body oils, and body essences.

The present invention also relates to a method for the production of a glycoprotein which is extracted from at least one selected from the group consisting of Verenia cordifolia, Rodiola losia, Comarum parus trita, Avicisibirica, Betula pendulabaeus, and Inonotus auricus Or a pharmaceutically acceptable salt thereof. Such pharmaceutical compositions may additionally contain preservatives, stabilizers, wettable or emulsifying accelerators, pharmaceutical adjuvants such as salts and / or buffers for the control of osmotic pressure, and other therapeutically useful substances, Can be formulated in the form of an administration agent or a parenteral administration agent.

Examples of the oral administration agent include tablets, pills, hard and soft capsules, liquids, suspensions, emulsions, syrups, powders, powders, granules, granules and pellets, , Diluents (such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and glycine), lubricants (such as silica, talc, stearic acid and magnesium or calcium salts thereof and polyethylene glycols) . The tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine, optionally mixed with starch, agar, alginic acid or its sodium salt Such as disintegrants, absorbents, coloring agents, flavoring agents, and sweetening agents. The tablets may be prepared by conventional mixing, granulating or coating methods.

The parenteral administration may be, for example, a formulation such as an injection, an ointment, an ointment, a lotion, a gel, a cream, a spray, a suspension, an emulsion, a suppository, It is not.

The pharmaceutical composition according to the present invention may be administered orally, parenterally, rectally, topically, transdermally, intravenously, intramuscularly, intraperitoneally, subcutaneously and the like.

In addition, the dosage of the active ingredient will depend on the age, sex, body weight of the subject to be treated, the particular disease or condition to be treated, the severity of the disease or condition, the route of administration and the judgment of the prescriber. Dosage determinations based on these factors are within the level of those skilled in the art. Typical dosages are from 0.001 mg / kg / day to 2000 mg / kg / day, more specifically from 0.5 mg / kg / day to 2.5 mg / kg / day.

The present invention relates to a pharmaceutical composition containing a glycoprotein extracted from at least one selected from the group consisting of Verenia cordifolia, Rodiola losia, Comarum parus trita, Avicivirica, Betulacepula dubarb, and Inonotus auricus ≪ RTI ID = 0.0 > a < / RTI > composition. The health food composition may be formulated into tablets, granules, drinks, caramels, diet bars and the like, although the formulations are not particularly limited. The health food composition of each formulation may be formulated by those skilled in the art without difficulty, depending on the purpose of formulation or use, in addition to the active ingredient, and the synergistic effect may occur when the composition is applied simultaneously with other ingredients .

The dosage of the active ingredient is within the level of those skilled in the art, and the daily dose of the drug depends on various factors such as the degree of progress of the subject to be administered, the age of onset, age, health condition, As a standard, the composition may be administered in an amount of 1 to 500 mg / kg, preferably 30 to 200 mg / kg, once or twice a day, and the dose may be administered by any method within the scope of the present invention .

Hereinafter, the constitution and effects of the present invention will be described in more detail with reference to examples and test examples. However, these examples and 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 and test examples.

[Example]

After chopping each of the refrigerated Verneria cordifolia, Rodiola losia, Comarum parus trita, Avis sterigarica, Betulace pendulabauz, and Inonotus auricus with a crusher, a citric acid solution having a pH of 2 to 3 The aqueous solution was mixed with the chopped plants at a ratio of 1: 3 to prepare an aqueous solution. While adjusting the pH of the aqueous solution to 2.0, 0.3 g weight% pepsin was added to the aqueous solution, and the mixture was treated at 40 캜 for 18 hours.

When a glycoprotein having a yield of 5% was obtained as compared with the enzyme solution treated with the enzyme, the temperature was rapidly raised to 85 ° C and treated for 40 minutes. The resulting extract was centrifuged to remove foreign substances, thereby preparing each glycoprotein in a liquid form. The glycoproteins obtained from Verenia cordifolia, Rodiola losia, Comarum paulastre, Abyssibirica, Betula pendulabaeus, and Inonotus auricus were used in Examples 1, 2, and 3 , Example 4, Example 5, and Example 6 were used.

The samples obtained through the above extraction and separation processes had amino acid compositions as shown in Table 1 below.

Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Aspartic acid 45.11 4.20 10.94 10.50 6.24 9.82 Clutamantan 21.59 62.25 33.12 24.38 14.91 14.98 Serine 2.39 1.07 3.49 5.27 2.31 3.67 Glycine 3.00 0.81 2.66 4.24 3.10 2.36 Histidine 1.37 0.43 1.22 1.75 0.68 0.41 Arginine 2.44 3.59 5.33 4.12 0.98 0.39 Threonine 1.09 0.39 1.52 2.75 1.28 2.00 Alanine 5.75 1.14 10.55 7.65 2.54 2.59 Proline 12.72 25.18 26.54 33.93 61.09 58.45 Tyrosine 0.58 0.19 0.55 0.28 1.04 0.38 Balin 1.35 0.13 1.25 1.82 1.33 1.64 Methionine 0.23 0.05 0.20 0.21 0.59 0.28 Cysteine 0.14 0.11 0.23 0.01 0.06 0.05 Isoleucine 0.55 0.15 0.88 0.92 0.74 0.87 Leucine 0.70 0.13 0.76 1.33 1.15 1.36 Phenylalanine 0.43 0.08 0.43 0.51 0.92 0.53 Lysine 0.58 0.11 0.34 0.33 1.04 0.22 synthesis 100 100 100 100 100 100

[Test Example 1] Performance of procollagen biosynthesis

Fibroblasts isolated from human skin were inoculated into 24 wells at 10 ⁶ per well and cultured until they grew to about 90%. After incubation for 24 hours in a serum-free medium, each of the samples was dissolved in serum-free medium and treated at a concentration of 100 ppm for 24 hours in a CO ₂ incubator. These supernatants were released and ascorbic acid (I) Elisa kit (Procollagen type 1) was used to examine the increase or decrease of procollagen. Vitamin C was also tested as a positive control. The test was carried out at a concentration considering cytotoxicity, and the results are shown in Table 2. The sum performance is obtained by setting the untreated group to 100.

ingredient Sum performance (%) Untreated group 100 Vitamin C 128.5 Example 1 122.8 Example 2 123.4 Example 3 117.6 Example 4 119.4 Example 5 126.2 Example 6 107.3

As can be seen from Table 2, the ability to synthesize procollagen of the Examples was superior to that of the untreated group. Accordingly, each of the embodiments can promote collagen production, thereby alleviating the collagen reduction phenomenon caused by skin aging. Therefore, a composition containing a glycoprotein extracted from each plant can prevent or ameliorate skin wrinkles, prevent or improve scarring, and prevent or improve skin aging. The glycoproteins extracted from each plant can be used in combination of two or more.

[Test Example 2] Collagenase (MMP-1)

The test was performed on a 96-well microtiter plate containing 2.5% fetal bovine serum (DMEM) (Dulbecco's Modified Eagle's Medium) and cultured directly on human skin and cultured at 5,000 cells / well well), and the cells were cultured until they grew to about 70 to 80%. Then, the cells were treated with 10 ppm concentration for 24 hours, and then the cell culture solution was sown. The collagenase expression level was measured using a commercially available collagenase measuring apparatus (Amersham Pharmacia Co., Ltd., Catalog #: RPN 2610). First, the cell culture medium, which had been plated on a 96-well plate uniformly coated with primary collagenase antibody, was added and the antigen-antibody reaction was performed in a thermostatic chamber for 3 hours. After 3 hours, the second collagen antibody bound to the chromophore was placed in a 96-well plate and allowed to react for 15 minutes. After 15 minutes, the color development inducing substance was added to induce color development at room temperature for 15 minutes, and 1 M sulfuric acid was added thereto to stop the reaction (color development) so that the color of reaction solution became yellow. At this time, the degree of yellow is different depending on the degree of progress of the reaction. The absorbance of a yellow-colored 96-well plate was measured at 405 nm using a light absorptometer, and the expression of collagenase was measured by the following equation (1) based on the reaction absorbance of the cell culture solution of the untreated group Respectively. A positive control group treated only with retinoic acid and a negative control treated with UV treatment were also tested. The test was carried out at a concentration considering cytotoxicity, and the results are shown in Table 3.

[Equation 1]

(%) = (Absorbance of each test substance / absorbance of the untreated group) X 100

ingredient Inhibition of Collagenase Expression (%) Untreated group 100 UV treatment 151.2 Retinoic acid 73.5 Example 1 38.6 Example 2 21.3 Example 3 40.4 Example 4 86.7 Example 5 75.8 Example 6 67.3

As the suppression of collagenase expression increases, the percentage decreases. As shown in Table 3, each of the examples has an effect of inhibiting collagenase, matrix metalloprotease (MMP-1). That is, the composition containing the glycoprotein extracted from each plant inhibits the biosynthesis of MMP-1, a skin tissue degrading enzyme caused by endogenous aging or external environmental factors, thereby suppressing collagen reduction in the skin and improving wrinkles. The glycoproteins extracted from each plant can be used in combination of two or more.

[Test Example 3] Melan-a cell-mediated inhibition of melanin formation

Melan-a cells (obtained from St. George's Hospital Medical School, UK) were inoculated into 6-well plates (melanin quantitation) in DMEM containing 10% FBS at 2 × 10 ⁴ cells / well (final volume 3 ml) (MTT assay) in 2 X 10 ³ cells / hole (final volume 200μl) to insert and cultured for 24 hours at 37 ℃, 5% CO ₂ incubator (MCO-20AIC, Sanyo). After the culture, the medium was removed and DMEM containing 10% FBS, 2 μM α-MSH (melanocyte stimulating hormone) and 2 mM theophylline was used as a new medium and the medium was added to the medium for 3 days . On day 5, the culture medium was removed from the 6-well plate and the cell pellet was recovered by treatment with 0.25% trypsin-EDTA (Sigma Chemical Co.) solution, transferred to a 1.5 ml test tube, centrifuged at 10,000 rpm for 10 minutes, Respectively. 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 at 475 nm was measured by an ELISA reader to determine the inhibition rate of melanin formation in the examples. The non-treated group in which nothing was treated was used as a negative control group, and the group treated with arbutin was used as a positive control group. The degree of inhibition of melanin formation in the examples was measured in comparison with the melanin content in the non-treated group, and the whitening effect was measured. The inhibition rate of melanin formation was calculated according to the following formula (2), and the results are shown in Table 4.

&Quot; (2) "

Melanin inhibition rate (%) = (absorbance of each test substance / absorbance of untreated group) X 100

Melanin formation inhibitory effect ingredient density Melanin formation inhibition (% control) Untreated group No additives - Arbutin 50 ppm 43.8% Example 1 25 ppm 21.5% 50 ppm 33.8% Example 2 25 ppm 31.5% 50 ppm 36.3% Example 3 25 ppm 20.6% 50 ppm 27.4% Example 4 25 ppm 43.8% 50 ppm 51.3% Example 5 25 ppm 37.5% 50 ppm 43.2% Example 6 25 ppm 17.3% 50 ppm 27.5%

As can be seen from the above Table 4, it was recognized that each of the Examples had an effect of inhibiting melanin formation, and the higher the glycoprotein concentration, the better. Thus, it was confirmed that the glycoproteins extracted from each plant according to the present invention have an excellent whitening effect because they inhibit the production of melanin. The glycoproteins extracted from each plant can be used in combination of two or more.

[Test Example 4] Measurement of anti-inflammatory activity (cell-based analysis)

The untreated 246.7 macrophage cell line (obtained from the Korean Cell Line Bank) was placed in DMEM, and 10% FBS penicillin (100 IU / ml) and streptomycin (100 μg / ml) were added and incubated at 37 ^° C in a 5% CO ₂ incubator Cells were plated on a 48-well plate at a density of 5 × 10 ⁵ cells / ml at a density of 300 μl. After 24 hours, the cells were treated with each concentration. After 1 hour, 1 μg / ml of LPS, which is an inflammation inducing substance, was treated. After 18 hours, the amount of NO released into the culture medium was measured using a Griess reagent. The group treated with 10 μM dexamethasone was used as a positive control for anti-inflammatory activity which inhibits the release of NO, and the group treated with 1 μg / ml of LPS was used as a negative control. The amount of NO was determined by comparison with the measured value of the standard NaNO ₂ , and the results are shown in Table 5 below.

ingredient Extract concentration (ppm) % Inhibition rate 1 mu g / ml of LPS + 10 mu M of dexamethasone - 100 1 [mu] g / ml of LPS - 0 1 μg / ml of LPS + Example 1 50 42.2 1 μg / ml of LPS + Example 2 50 31.6 1 mu g / ml of LPS + Example 3 50 39.1 LPS 1 占 퐂 / ml + Example 4 50 39.6 1 mu g / ml of LPS + Example 5 50 41.1 LPS < RTI ID = 0.0 > llg / ml + 50 29.3

As shown in Table 5, each of the Examples had the effect of inhibiting NO production and inhibiting inflammation induced by LPS as compared with the negative control group. Therefore, it was confirmed that the glycoproteins extracted from each plant had an effect of preventing or improving inflammation. The glycoproteins extracted from each plant can be used in combination of two or more.

[Test Example 5] Confirmation of IL-6, IL-8 and MCP-1 secretion inhibitory effects by stimulation in keratinocyte HaCaT

The test keratinocytes (cell line name: HaCaT available from German Cancer Institute, Heidelberg, Germany) were inoculated into 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin (2 × 10 ⁴ cells / well) in a 48-well plate using DMEM (Dulbecco's Modified Eagle Medium, Lonza, USA) containing penicillin-streptomycin and Gibco Density, and cultured at 37 ° C and 5% CO ₂ for 24 hours. After removing the culture medium, the cells were washed once with 200 μl of phosphate buffered saline (PBS), and 200 μl of DMEM without fetal bovine serum was added thereto and cultured for 24 hours. The samples were then treated with DMEM containing 1% fetal bovine serum for 10 minutes at the concentrations listed in Tables 6, 7 and 8, respectively. Then, retinoic acid (Retinoic acid, available from Sigma-Aldrich, USA) and cultured for 24 hours. The inhibitory effect of these cytokines on the secretion of IL-6, IL-8 and MCP-1 ELISA kit (BD pharmigen, USA) was evaluated. On the other hand, a group in which 1 ppm of hydrocortisone was used as a positive control group, a group in which nothing was treated as a negative control group, and a group in which only retinoic acid was treated alone were also tested. The inhibitory effect of the cytokine secretion was determined by plotting a standard curve based on the absorbance of rh IL-6, rh IL-8 and rh MCP-1 (available from BD Pharmigen, USA) , The absorbance of the examples was substituted to obtain the secretion amount of these cytokines, and the stimulation relaxation rate was obtained by the following equation (3). In addition, the suppression rate of HaCaT cells not treated with retinoic acid and HaCaT cells treated with retinoic acid was calculated by the following equation (4) based on the difference in secretion amount of cytokines, and the cytokine secretion inhibiting effect of each example was evaluated.

&Quot; (3) "

(%) = [1 - (cytokine secretion amount of each test substance / cytokine secretion amount of the group treated with retinoic acid alone)] x 100

&Quot; (4) "

Inhibition rate (%) = {1 - [(amount of cytokine secretion of each test substance - amount of cytokine secretion of negative control) / amount of cytokine secretion of each test substance}} x 100

Table 6, Table 7, and Table 8 below show test results related to IL-6, IL-8, and MCP-1, respectively.

ingredient Extract concentration (ppm) IL-6 secretion amount (pg / ml) Stimulus relaxation rate (%) % Inhibition Negative control group - 20.12 - - Retinoic acid (10 μm) - 78.55 - - Retinoic acid (10 m) +
Hydrocortisone (1 ppm) - 22.58 71.25 89.10 Retinoic acid (10 m) +
Example 1 100 23.62 69.92 85.18 Retinoic acid (10 m) +
Example 2 100 27.45 65.05 73.29 Retinoic acid (10 m) +
Example 3 100 38.27 51.26 52.57 Retinoic acid (10 m) +
Example 4 100 28.13 64.18 71.52 Retinoic acid (10 m) +
Example 5 100 29.55 62.38 68.08 Retinoic acid (10 m) +
Example 6 100 34.62 55.92 58.11

ingredient Extract concentration (ppm) IL-8 secretion amount (pg / ml) Stimulus relaxation rate (%) % Inhibition Negative control group - 128.5 - - Retinoic acid (10 μm) - 521.9 - - Retinoic acid (10 m) +
Hydrocortisone (1 ppm) - 159.7 69.40 80.46 Retinoic acid (10 m) +
Example 1 100 200.3 61.62 64.15 Retinoic acid (10 m) +
Example 2 100 145.6 72.10 88.25 Retinoic acid (10 m) +
Example 3 100 192.6 63.09 66.71 Retinoic acid (10 m) +
Example 4 100 207.5 60.24 61.92 Retinoic acid (10 m) +
Example 5 100 163.3 68.71 78.68 Retinoic acid (10 m) +
Example 6 100 138.3 73.50 92.91

ingredient Extract concentration (ppm) MCP-1 secretion amount (pg / ml) Stimulus relaxation rate (%) % Inhibition Negative control group - 638.83 - - Retinoic acid (10 μm) - 1406.24 - - Retinoic acid (10 m) +
Hydrocortisone (1 ppm) - 710.68 49.46 90.64 Retinoic acid (10 m) +
Example 1 100 835.32 40.59 74.40 Retinoic acid (10 m) +
Example 2 100 763.21 45.73 83.70 Retinoic acid (10 m) +
Example 3 100 782.05 44.39 81.68 Retinoic acid (10 m) +
Example 4 100 738.24 47.50 86.53 Retinoic acid (10 m) +
Example 5 100 724.51 48.48 88.17 Retinoic acid (10 m) +
Example 6 100 789.38 43.87 80.92

As can be seen in Tables 6, 7, and 8, each of the examples shows the effect of suppressing secretion of IL-6, IL-8, and MCP-1. Therefore, it was confirmed that the glycoproteins extracted from each plant have an anti-inflammatory effect by inhibiting the secretion of the cytokines, which are inflammation-inducing factors, and thus have an effect of preventing or improving inflammation. The glycoproteins extracted from each plant can be used in combination of two or more.

[Test Example 6] Evaluation of PPARγ efficacy

1) Cell culture

CV-1 cells (obtained from ATCC), a normal African green monkey kidney epithelial cell, were cultured in DMEM (Dulbecco Modified (R)) supplemented with 10% Fetal Bovine Serum and 100 units / ml penicillin-streptomycin Eagle's Medium) and cultured at 37 ° C and 5% CO ₂ .

2) Plasmid transfection

The plasmid is a PPAR? Plasmid expressing PPAR? Under normal culturing conditions, a firefly luciferase that acts as a reporter and a PPARs response element (PPRE) reaction element (PPARs Response Element (PPRE) The PPRE-Luc plasmid containing the gene and the β-gal plasmid used for the transfection value correction were used.

CV-1 cells were plated on a 24-well plate at a concentration of 5 x 10 ⁴ , cultured for 18 to 24 hours, and a mixture of the plasmids and 1: 3 (μg: μl) of DMEM and PEI (Polyethylenimine) , And then plasmids were introduced into the cells in the same manner as that in which they were sprayed on each of the balls.

3) Measurement of PPAR-γ activity

Plasmids were introduced into the cells and allowed to stabilize by incubation for 24 hours, after which the samples were processed and incubated again for 24 hours. At the end of incubation, the cells were washed twice with cold PBS, and cells were treated with reporter lysis buffer (Promega) for 15 minutes to destroy the cells. The cell eluate thus obtained was transferred to a microtube and centrifuged at 12,000 rpm for 3 minutes at 4 ° C to separate the cell residue to obtain a pure cell eluate.

The PPAR-γ activity of each example was compared by mixing the lysate substrate (Luciferase substrate, Promega) at a ratio of 2: 1 and measuring the amount of light emitted by the luminometer. The cell extract was treated with the same amount of 2X? -Galactosidase enzyme assay buffer (Promega), reacted at 37 占 폚 for 30 minutes, and the absorbance at 420 nm was measured. - galactosidase activity were compared. The PPAR-gamma activity of each example was divided by the activity of each beta-galactosidase to compensate for differences in the degree of plasmid introduction and the significance of the activity was assessed using this sample t-test of Minitab. Meanwhile, TGZ (troglitazone), a PPAR-γ agonist, was also tested as a positive control.

ingredient density Activity (% control) Untreated group No additives 100% TGZ 5 [mu] M 563% Example 1 50 ppm 233% Example 2 50 ppm 319% Example 3 50 ppm 180% Example 4 50 ppm 255% Example 5 50 ppm 384% Example 6 50 ppm 129%

As can be seen in Table 9, the PPARγ activity of each example was superior to the untreated group. Therefore, it was confirmed that glycoproteins extracted from each plant had excellent skin moisturizing effect by activating PPARγ. The glycoproteins extracted from each plant can be used in combination of two or more.

[Test Example 7] Evaluation of cell regeneration efficiency

Keratinocyte, HaCaT (cell line name: HaCaT available from German Cancer Institute, Heidelberg, Germany) was added to each well of a 96-well plate incubator at 10 ⁴ per well. DMEM containing 10% FBS (fetal bovine serum) For 24 hours. Then, the above examples were replaced with DMEM containing 1% FBS at concentrations of 0.5 and 2.8 ppm, respectively, and then cultured for 48 hours. The WST-1 solution (Roche) was added thereto at a ratio of 1/10, followed by reaction at 37 ° C for 2 hours in an incubator. The plate culture incubator after incubation measured the color development degree at 440 nm. When the degree of color development of the untreated group was 100, the degree of color development in each of the examples was evaluated to determine the degree of cell proliferation (%). In addition, the untreated group treated with nothing and the group treated with only 10% FBS were tested together, and the results are shown in Table 10 below.

ingredient density Cell proliferation (% control) Untreated group No additives 100% FBS 10% 188% Example 1 0.5 ppm 157% 2.8 ppm 132% Example 2 0.5 ppm 133% 2.8 ppm 124% Example 3 0.5 ppm 129% 2.8 ppm 131% Example 4 0.5 ppm 144% 2.8 ppm 139% Example 5 0.5 ppm 129% 2.8 ppm 147% Example 6 0.5 ppm 125% 2.8 ppm 109%

As can be seen in Table 10, each of the examples was superior in cell proliferation to the non-treatment group. That is, it was confirmed that the glycoproteins extracted from each plant had an effect of regenerating the cells because the cell proliferation effect was recognized. Therefore, the glycoproteins extracted from each plant may have the effect of preventing or wrinkling the skin, whitening the skin, moisturizing the skin, regenerating the cells, preventing or improving the scarring, preventing or improving the aging. In addition, two or more glycoproteins extracted from each plant can be used in combination.

[Test Example 8] Evaluation of antioxidant capacity (DPPH)

The powdery products were prepared by drying the six kinds of glycoprotein liquid product, Examples 1, 2, 3, 4, 5, and 6, prepared in the above Examples, using a freeze dryer . Thereafter, 100 g of its dried weight was dissolved in DMSO to a concentration of 10%. As a positive control, vitamin C was dissolved in water to make 1%. DPPH (1,1-diphenyl-2-picrylhydrazyl) was prepared in ethanol at a concentration of 100 μM. The prepared glycoprotein solution and vitamin C solution were diluted 1/2 step with ethanol to prepare solutions at five concentrations. Then, 10 μl of each of the prepared solutions and 190 μl of DPPH solution were mixed and incubated at 37 ° C for 30 minutes. The OD value was then measured at 540 nm. On the other hand, the untreated group treated with nothing was tested together as a negative control group. The thus-measured value was evaluated as the antioxidant power of each of the non-treated groups as 100, and is shown in FIG. In FIG. 1, the lower the Y-axis value is, the greater the antioxidant power is.

As can be seen from Fig. 1, each of the examples had an excellent antioxidative effect as compared with the negative control, and the effect was increased as the concentration was higher. Therefore, it was confirmed that the glycoprotein extracted from each plant had an excellent antioxidative effect. The glycoproteins extracted from each plant can be used in combination of two or more.

The glycoprotein extracted from at least one selected from the group consisting of Verenia cordifolia, Rodiola rosia, Comarum parus strain, Avis sterilika, Betula pendulabaeus, and Inonotus auricus according to the present invention Formulation examples of cosmetic compositions, pharmaceutical compositions and health food compositions containing the composition as an active ingredient will be described in more detail. However, cosmetic compositions, pharmaceutical compositions and health food compositions can be applied in various formulations, It is not intended to be limiting but merely to be illustrative.

[Formulation Example 1] Softening lotion (skin lotion)

The softening longevity was prepared by a conventional method according to the composition shown in Table 11 below.

Compounding ingredient Content (% by weight) The glycoprotein extracted from each plant 0.1 glycerin 3.0 Butylene glycol 2.0 Propylene glycol 2.0 Carboxyvinyl polymer 0.1 Phage-12 nonylphenyl ether 0.2 Polysorbate 80 0.4 ethanol 10.0 Triethanolamine 0.1 Preservative, coloring, fragrance Suitable amount Purified water Balance

[Formulation Example 2] Nutritional lotion (Milk lotion)

Nutrition lotion was prepared according to a conventional method according to the composition shown in Table 12 below.

Compounding ingredient Content (% by weight) The glycoprotein extracted from each plant 0.1 glycerin 3.0 Butylene glycol 3.0 Propylene glycol 3.0 Carboxyvinyl polymer 0.1 Wax 4.0 Polysorbate 60 1.5 Caprylic / capric triglyceride 5.0 Squalane 5.0 Sorbitacecequioleate 1.5 Liquid paraffin 0.5 Cetearyl alcohol 1.0 Triethanolamine 0.2 Preservative, coloring, fragrance Suitable amount Purified water Balance

[Formulation Example 3] Nourishing cream

Nutritive creams were prepared according to the compositions shown in Table 13 below in a conventional manner.

Compounding ingredient Content (% by weight) The glycoprotein extracted from each plant 0.1 glycerin 3.0 Butylene glycol 3.0 Liquid paraffin 7.0 Beta Glucan 7.0 Carbomer 0.1 Caprylic / capric triglyceride 3.0 Squalane 5.0 Cetearyl glucoside 1.5 Sorbitan stearate 0.4 Polysorbate 60 1.2 Triethanolamine 0.1 Preservative, coloring, fragrance Suitable amount Purified water Balance

[Formulation Example 4] Massage cream

Massage creams were prepared in a conventional manner according to the composition shown in Table 14 below.

Compounding ingredient Content (% by weight) The glycoprotein extracted from each plant 0.1 glycerin 8.0 Butylene glycol 4.0 Liquid paraffin 45.0 Beta Glucan 7.0 Carbomer 0.1 Caprylic / capric triglyceride 3.0 Wax 4.0 Cetearyl glucoside 1.5 Sesquioleic acid sorbitan 0.9 Vaseline 3.0 paraffin 1.5 Preservative, coloring, fragrance Suitable amount Purified water Balance

[Formulation Example 5] Pack

The pack was prepared in a conventional manner according to the composition shown in Table 15 below.

Compounding ingredient Content (% by weight) The glycoprotein extracted from each plant 0.1 glycerin 4.0 Polyvinyl alcohol 15.0 Hyaluronic acid extract 5.0 Beta Glucan 7.0 Allantoin 0.1 Nonyl phenyl ether 0.4 Polysorbate 60 1.2 ethanol 6.0 Preservative, coloring, fragrance Suitable amount Purified water Balance

[Formulation Example 6] Ointment in external preparation for skin

Ointments were prepared in a conventional manner according to the composition shown in Table 16 below.

Compounding ingredient Content (% by weight) The glycoprotein extracted from each plant 0.1 glycerin 8.0 Butylene glycol 4.0 Liquid paraffin 15.0 Beta Glucan 7.0 Carbomer 0.1 Caprylic / capric triglyceride 3.0 Squalane 1.0 Cetearyl glucoside 1.5 Sorbitan stearate 0.4 Cetearyl alcohol 1.0 Wax 4.0 Preservative, coloring, fragrance Suitable amount Purified water Balance

[Formulation Example 7] Preparation of powders

Glycoprotein extracted from each plant ............ 100 mg

Lactose ................................... 100 mg

Talc ..................................... 10 mg

Maintenance ...................................... 5 mg

The above components are mixed and filled in airtight bags to prepare powders.

[Formulation Example 8] Preparation of tablets

Glycoprotein extracted from each plant ........ 50 mg

Corn starch ...................... 100 mg

Lactose .................................. 100 mg

Magnesium stearate ........ 2 mg

Vitamin C ............................ 50 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

[Formulation Example 9] Preparation of capsule

Glycoprotein extracted from each plant ............... 50 mg

Corn starch ................................ 100 mg

Lactose ............................................. 100 mg

Magnesium stearate ................... 2 mg

Vitamin C ....................................... 50 mg

Serine ................................................ 50 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

[Formulation Example 10] Preparation of liquid agent

Glycoprotein extracted from each plant ............................. 100 mg

Isolation Party ................................................ ... 10 g

Mannitol ................................................. .... 5 g

Vitamin C ................................................ ... 50 mg

Serine ................................................. ....... 50 mg

maintain................................................. .......

Purified water................................................. ......

Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, And sterilized to prepare a liquid preparation.

[Formulation Example 11] Preparation of health food

Glycoprotein extracted from each plant ... 1000 mg

Vitamin mixture

Vitamin A Acetate ............................ 70 ㎍

Vitamin E ...................................... 1.0 mg

Vitamin B1 ....................................... 0.13 mg

Vitamin B2 ....................................... 0.15 mg

Vitamin B6 ........................................ 0.5 mg

Vitamin B12 ........................................ 0.2 ㎍

Vitamin C ..................................... 10 mg

Biotin .......................................... 10 μg

Nicotinic acid amide ....................................... 1.7 mg

Folic acid .............................................. 50 ㎍

Calcium pantothenate ...................................... 0.5 mg

Mineral mixture

Ferrous sulfate .......................................... 1.75 mg

Zinc oxide ............................................ 0.82 mg

Magnesium carbonate ..................................... 25.3 mg

Potassium Phosphate ....................................... 15 mg

Secondary Calcium Phosphate ......................................... 55 mg

Potassium citrate ........................................... 90 mg

Calcium carbonate ..................................... 100 mg

Magnesium chloride ..................................... 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively comparatively mixed with the composition suitable for health food as a preferable formulation, the compounding ratio may be arbitrarily modified, and the above components are mixed according to a conventional method for producing health food , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

[Formulation Example 12] Preparation of health drink

Glycoprotein extracted from each plant ............................. 1000 mg

Citric acid ................................................. ......... 1000 mg

oligosaccharide................................................. ........ 100 g

Plum concentrate ................................................ ......... 2 g

Taurine ................................................. ............. 1 g

Purified water was added to the whole ... 900 ml

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated at 85 DEG C for about 1 hour with stirring, and the solution thus prepared was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, ≪ / RTI >

Although the composition ratio is relatively mixed with the ingredient suitable for the beverage, it is possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, the use purpose, and the like.

Claims (3)

A cosmetic composition for moisturizing the skin, comprising as an active ingredient a glycoprotein extracted from buds of Betula pendula ,
The glycoprotein may be prepared by adding an organic acid solution having a pH of 2 to 3 at a ratio of 1: 2 to 1: 5 to the dry weight of the above-described Betulace Pendula in the eye of Betula Pendula, Wherein the temperature is maintained at 30 to 50 DEG C for 2 to 24 hours, and then the glycoprotein aqueous solution is extracted by filtration. The method according to claim 1,
Wherein skin moisturization is by activation of PPARy. 3. The method according to claim 1 or 2,
And 0.1 to 30% by weight of the glycoprotein extracted from the eye of the above-mentioned Betulacepidula, based on the total weight of the composition.

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