KR101808222B1 - Composition for improving skin elasticity containing sphallerocarpus gracilis extract - Google Patents

Abstract

A pharmaceutical composition, a cosmetic composition, and a health functional food composition containing an Sphallerocarpus gracilis extract as an active ingredient improve skin elasticity of an individual, prevent or reduce skin wrinkles, and prevent or inhibit skin aging.

Description

[0001] The present invention relates to a composition for improving skin elasticity,

A cosmetic composition and a health functional food composition for promoting the elasticity of the skin including an acidic box extract.

Skin is an organ that covers the outside. It protects the organs in the body from external stimuli and plays an important role in keeping the body homeostatic such as body temperature control. Such skin is aged by various internal and external factors and is divided into endogenous aging by genetic cause and exogenous aging by environmental cause. Among them, photoaging refers to aging by ultraviolet (UV). Recently, the destruction of the ozone layer due to environmental pollution has increased the amount of ultraviolet rays, and accordingly, research on photoaging has been attracting attention (Wang SQ et al., Dermatol. Ther. 23 (1): 31-47, 2010). In the photoaged skin, appearance characteristics such as roughness, loss of elasticity, wrinkles and irregular pigmentation are observed. Among them, the main research field of photoaging is the change of the skin wrinkles. There have been many reports on the basic physiological metabolism changes such as synthesis, degradation, and moisture content of collagen, which is a major constituent of skin, in the formation of skin wrinkles by photoaging (Brenneisen et al., Ann. NY Acad. Sci., 973: 31-43, 2002).

The skin is composed of three layers, from the outside to the epidermis, the dermis and the subcutaneous fat layer. The collagen present in the dermal layer of the skin accounts for about 70 to 80% of the dry weight of the skin, and it is known to control the elasticity of the skin together with elastic fibers elastin. Particularly, ultraviolet rays increase the production of reactive oxygen species and collagen degradation and biosynthesis are reduced by the collapse of enzymatic and non-enzymatic antioxidant defenses of the skin, resulting in a marked reduction of collagen in the dermis (Bickers DR, Athar M, J. Invest. Dermatol., 126 (12): 2565-2575, 2006). Important collagen degradation factors are matrix metalloproteinases (MMPs), which are involved in the degradation of extracellular matrix and basement membrane. Studies have been reported that the enzyme increases activity by ultraviolet rays and inhibits it, leading to an increase in skin thickness and wrinkle formation induced by ultraviolet light (Inomata S et al., J. Invest. Dermatol., 120 ): 128-134, 2003). Therefore, it is effective to control MMPs for prevention and treatment of photoaging.

Consumers' expectation and interest in wrinkle improvement has been diversified from retinol to natural products extract, adenosine, and cell growth factor from the 2000s (Lee EJ et al., Journal of the Korean Society of Cosmetology, 17 (1): 127-133, 2011). Conventional methods are to manufacture and use cosmetic products or medicines containing retinoid, ascorbic acid, tocopherol, hyaluronic acid and the like.

Effective ingredients for skin wrinkle improvement or skin aging improvement which are currently being developed can not be used as raw materials for some cosmetics or are very unstable and can not be easily delivered to the skin. Therefore, a special stabilization system and delivery system are required. There is a problem that it is not visible. For example, the retinoid, which is an active ingredient for improving skin wrinkles, has been used to inhibit collagenase and increase collagen synthesis to improve photoaging phenomenon such as wrinkle formation and elasticity reduction. However, retinoids are very sensitive to ultraviolet rays, (Rabe JH, J. Am. Acad. Dermatol., 55: 1-19, 2006), which causes side effects such as skin irritation and long-term use. Therefore, research is continuing on methods to induce skin wrinkles and skin elasticity improvement while minimizing side effects.

One aspect of the present invention provides a pharmaceutical composition for enhancing elasticity of the skin comprising an acid-free box extract as an active ingredient.

Another aspect provides a cosmetic composition for promoting the elasticity of the skin comprising an acid-free box extract as an active ingredient.

Another aspect provides a health functional food composition for promoting the elasticity of the skin comprising an acid-free box extract as an active ingredient.

One aspect of the present invention provides a pharmaceutical composition for enhancing elasticity of the skin comprising an acid-free box extract as an active ingredient.

Musan box (茂山 床子) is a scientific name Sphallerocarpus It means a plant with gracilis . Leaf of flatulence box is flat, bottom part is sheath-shaped and white long hairs are densely. The last branch is linear, the edge is flat and has no hair. The subdivision is about 5mm long and has 8 ridgelines. It is flat on both sides and there are three lipids on the intercostal space and intercostal space. Flowers bloom in August, white, no guns, 5 guns, oval, narrow bottom, pointed like thorns, with fine hairs on the edge. The acid-free box is about 50 to 100 cm. The country of origin is Korea, and it grows in mountainous regions of Korea, China, Russia and Mongolia, and grows mainly in Mt.

The anther box extract may be an extract extracted from the whole or part of the above or below the anoxic box, or a material derived therefrom, by a solvent. The above-ground portion of the anther box may be a stem, a branch, a leaf, a flower, a petal or a combination thereof. The underside of the anaerobic box may be a root. The whole or part of, or a material derived from, the overhead or underground portion of the anoxic container used for the extraction may be ground or chopped or suitably dried.

The solvent may be water, acetone, an alcohol such as a C1-C6 alcohol, or a mixture thereof. The C1-C6 alcohol may be methanol, ethanol, propanol, isopropanol, 1,3-propanediol, butanol, pentanol, hexanol and the like. The solvent may be, for example, a mixture of water and an alcohol, that is, an aqueous solution of an alcohol. The alcohol concentration of the alcohol aqueous solution may range from about 1 to 100 (v / v)%, such as from about 1 to 99.5 (v / v)%, from about 10 to 100 (v / v)%, about 30 to 100 (v / v)%, about 40 to 100 (v / v)%, about 50 to 100 (v / To 100 (v / v)%, or about 75 to 100 (v / v)%. The aqueous alcohol solution may be methanol, ethanol, or an aqueous butanol solution. The acetone may be 100% concentration.

The extraction is carried out in an amount of about 100 ml to about 10 l, about 200 ml to about 5 l, about 400 ml to about 2.5 l of the extraction solvent per kg of the material for the whole or a part of the overhead or underground portion of the anhydrous box, , About 500 mL to about 2 L, about 750 mL to about 1.5 L, or about 750 mL to about 1.25 liter.

The extraction may comprise mixing the above or below the top or bottom portion of the anoxic container with the solvent, and a portion of, or a material derived therefrom, and allowing to stand for a period of time. The setting may include moderate agitation. The extraction may be repeated one or more times, for example, 1 to 5 times, 2 to 4 times, or 3 times.

The extraction may be performed by warmed liquid extraction, pressurized liquid extraction (PLE), microwave assisted extraction (MAE), subcritical extraction (SE), or a combination thereof . The subcritical extraction may be subcritical water extraction (SWE). Sub-critical water extraction is also referred to as superheated water extraction or pressurized hot water extraction (PHWE).

The extraction may be carried out at a temperature of about 4 캜 to 70 캜, such as about 4 캜 to 50 캜, about 4 캜 to 40 캜, about 4 캜 to 30 캜, about 10 캜 to 70 캜, About 10 ° C to about 50 ° C, about 10 ° C to about 50 ° C, about 4 ° C to about 40 ° C, about 4 ° C to 30 ° C, about 10 ° C to 40 ° C, 10 < 0 > C to 30 < 0 > C. The extraction time may vary depending on the selected temperature. The extraction time may be 1 hour to 2 months, 1 hour to 1 month, 1 hour to 15 days, 1 hour to 10 days, 1 hour to 5 days, 5 hours to 1 month, 15 hours, 5 hours to 10 days, 5 hours to 5 days, 10 hours to 1 month, 10 hours to 15 days, 10 hours to 10 days, or 10 hours to 5 days.

The extraction may include separating the plant residue and the extract by a known method such as filtration. The extraction can also include removing the solvent from the resulting extract by known methods such as concentration under reduced pressure. The extraction may also comprise preparing the dried extract by drying, such as lyophilization, of the resulting extract.

The composition may further comprise an acidic box fraction. The non-acidic box fraction means a substance in which the anhydrous box extract is divided into a part of its components, that is, a fractioned substance. The fraction may be obtained by solvent fractionation. The solvent fractionation can be by mixing the anhydrous box extract with a solvent and separating the substance present in the solvent. The fraction may be a hexane fraction, an ethyl acetate fraction, a butanol fraction or a water fraction obtained by suspending the anthracnose box extract and then fractionating it with hexane, ethyl acetate, butanol or water.

The dry skin extract promotes skin elasticity, prevents or improves skin wrinkles, and prevents or prevents aging of the skin.

The skin elasticity refers to the elasticity of elastic fibers and / or collagen fibers, which are composed of elastin and is present in the dermal layer, and refers to the force that the skin keeps tight against external stimuli or the like. Improving skin elasticity means that the amount of collagen in the dermal layer is increased and skin elasticity is improved.

The wrinkles of the skin means wrinkles that are formed when the skin is worn or aged. The wrinkles of the skin can be caused by the cause of the gene, the decrease of collagen and elastin present in the skin dermis, and the external environment. Wrinkle prevention or wrinkle improvement means that the elasticity of the skin is maintained to suppress or inhibit the generation of wrinkles, or to remove or alleviate the wrinkles already generated.

The skin aging involves all the skin changes that occur as the skin ages. For example, the aging of the skin may be, but not limited to, reduction of skin elasticity due to aging, generation of new skin wrinkles, increase in depth and number of skin wrinkles, and drying of skin by aging. Preventing or preventing skin aging means slowing down any changes in skin that occur as you age. It is practically impossible to completely prevent skin aging according to the passage of time, but it is possible to delay the skin aging as much as possible by the above composition.

The composition may be one for promoting expression of a collagen gene in a cell. The composition may increase the synthesis of collagen, increase the activity of the collagen produced by the composition, or increase the amount of collagen secreted by the cells. In addition, the composition may also inhibit the expression, activity, or secretion of collagenase, inhibit the degradation of collagen produced, inhibit the activity of collagen produced, or inhibit collagen secretion Can be prevented from being reduced due to external stimulation or the like. Accordingly, the composition can be used for maintaining or increasing the elasticity of tissues containing collagen by increasing the expression of collagen, thereby promoting regeneration of the skin and preventing aging of the skin. The composition may be to maintain or increase the elasticity of a tissue comprising collagen, e. G., Skin tissue, to reduce wrinkle formation and to remove wrinkles in the skin tissue.

The composition may also increase the activity or expression of an up-stream enzyme or protein in the pathway of synthesis of collagen. The collagen may be selected from the group consisting of type I collagen, type II collagen and type III collagen. The gene encoding the collagen may be selected from the group consisting of COL1A1, COL2A1 and COL3A1.

That is, the composition may increase the expression of the collagen gene itself, or may increase the activity of the collagen produced by the composition. The composition may also increase the activity or expression of enzymes or proteins upstream of the collagen production.

The composition may be present in an amount of from 0.001% to 80%, such as from 0.005% to 79%, from 0.01% to 78%, from 0.05% to 75%, from 0.1% to 73% %, From 0.5 wt% to 70 wt%, from 1 wt% to 70 wt%, from 2 wt% to 65 wt%, from 4 wt% to 63 wt%, from 5 wt% to 60 wt% ≪ / RTI > When the dry skin extract is less than 0.001% by weight based on the total weight of the composition, the skin elasticity improvement effect is insignificant. When the skin lotion content is more than 80% by weight, the content of other components is limited.

The composition may comprise a pharmaceutically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, mannitol, or a combination thereof. The carrier may be an excipient, a disintegrant, a binder, a lubricant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low substituted hydroxy cellulose, or combinations thereof. The disintegrant may be carboxymethylcellulose calcium, starch glycolate sodium, calcium monohydrogen phosphate anhydrate, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or combinations thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

The composition may be formulated into a parenteral dosage form. The parenteral dosage form may be an injection or an external preparation for skin. The external preparation for skin may be a cream, a gel, an ointment, a skin emulsifier, a skin suspension, a transdermal patch, a drug-containing bandage, a lotion, or a combination thereof.

The external preparation for skin is usually used as a component used in external skin preparations such as cosmetics or medicines such as an aqueous component, an oily component, a powder component, an alcohol, a moisturizer, a thickener, an ultraviolet absorber, a whitening agent, an antiseptic, , Coloring agents, various skin nutrients, or a combination thereof, and may be suitably blended as necessary.

The external preparation for skin may be a metal blocker such as sodium edetate, sodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate or gluconic acid, caffeine, tannin, bellapamil, licorice extract, glabridine, Vitamin C, ascorbic acid magnesium phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, fructose, fructose and other herbal medicines, various herbal medicines, tocopherol acetate, glycyrrhizic acid, Saccharides such as trehalose can also be appropriately compounded.

Another aspect provides a method of enhancing the elasticity of the skin of an individual comprising administering the composition to a subject. The composition is the same as described above.

Such administration can be administered by methods known in the art. May be administered directly or indirectly to the subject by any means. The administration can be systemically or locally administered. The administration may be topically or wholly administered to a site where skin elasticity and regeneration are necessary or wrinkled areas.

The subject may be a mammal, such as a person, a cow, a horse, a pig, a dog, a sheep, a goat, or a cat.

The administration can be carried out in an amount of 0.1 mg to 1,000 mg, for example 0.1 mg to 500 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 0.1 mg to 5 mg, 1 from 1 mg to 500 mg, from 1 mg to 100 mg, from 1 mg to 50 mg, from 1 mg to 25 mg, from 5 mg to 1000 mg, from 5 mg to 500 mg, from 5 mg to 100 mg, from 5 mg to 50 mg mg, 1 mg to 5 mg, 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 10 mg to 25 mg.

Another aspect provides a cosmetic composition for promoting the elasticity of the skin comprising an acid-free box extract as an active ingredient. The cosmetic composition enhances skin elasticity, prevents or improves skin wrinkles, and prevents or prevents aging of the skin. The composition may be one for promoting expression of a collagen gene in a cell. The composition may increase the synthesis of collagen, increase the activity of the collagen produced by the composition, or increase the amount of collagen secreted by the cells. In addition, the composition may also inhibit the expression, activity, or secretion of collagenase, inhibit the degradation of collagen produced, inhibit the activity of collagen produced, or inhibit collagen secretion Can be prevented from being reduced due to external stimulation or the like. Accordingly, the composition can be used for increasing or decreasing the elasticity of tissues including collagen by increasing the expression of collagen, thereby improving skin beauty and preventing skin aging. The composition may be to maintain or increase the elasticity of the tissue comprising the collagen, e. G., The skin, to reduce wrinkle formation and wrinkles in the tissue. Therefore, the composition can be used as a cosmetic composition for enhancing skin elasticity, preventing or improving wrinkles of skin, or preventing or preventing skin aging.

The anhydrous box extract is the same as described above.

The cosmetic composition may be provided in any formulation suitable for wholly or topically application. For example, as a solution, an emulsion obtained by dispersing an oil phase in an aqueous phase, an emulsion obtained by dispersing an oil phase in water, a suspension, a solid, a gel, a powder, a paste, a mask pack or a sheet, a foam or an aerosol composition . Compositions of such formulations may be prepared according to conventional methods in the art.

The cosmetic composition may further comprise a moisturizing agent, an emollient agent, an ultraviolet absorber, an antiseptic, a bactericide, an antioxidant, a pH adjuster, an organic and inorganic pigment, a perfume, a cold agent or a limiting agent. The amount of the additional component such as the above-mentioned moisturizing agent can be easily selected by a person skilled in the art within a range not to impair the purpose and effect of the present invention.

The cosmetic composition may be used in an amount of 0.001 to 80% by weight, for example, 0.005 to 79% by weight, 0.01 to 78% by weight, 0.05 to 75% by weight, 0.1 to 73% by weight, From 1 wt% to 70 wt%, from 2 wt% to 65 wt%, from 4 wt% to 63 wt%, from 5 wt% to 60 wt%, or from 10 wt% to 55 wt% % ≪ / RTI > acidic box extract.

Another aspect provides a method for creasing skin comprising applying the composition to the skin of an individual. In the above method, the cosmetic composition is applied to the treatment of the skin, the lips and the hair, particularly the cosmetic treatment, including the scalp. In the method, the step of applying to the skin comprises contacting the composition with the skin to allow the components of the composition to penetrate into the skin. The step of applying to the skin may be combined with means such as electrical or magnetic forces, such as iontophoresis, for other means to help penetrate the composition into the skin.

Another aspect provides a health functional food composition for promoting the elasticity of the skin comprising an acid-free box extract as an active ingredient. The health functional food composition can enhance skin elasticity, prevent or improve skin wrinkles, and prevent or prevent skin aging.

The anhydrous box extract is the same as described above.

The health functional food composition means various types of food additives or functional foods. For example, the health food composition may be processed into fermented milk, cheese, yogurt, juice, probiotics, health supplements, and the like, and various other food additives. The health functional food composition may further include a flavoring agent, a coloring agent, a bactericide, an antioxidant, an antiseptic, a moisturizing agent, a thickening agent, an inorganic salt, an emulsifying agent or a synthetic high molecular substance. The addition amount of the additional component such as the above-mentioned antioxidant and the like can be easily selected by those skilled in the art within a range not to impair the purpose and effect of the present invention.

The health functional food composition may comprise from 0.001% to 80%, such as 0.005% to 79%, 0.01% to 78%, 0.05% to 75%, 0.1% % To 70 wt.%, 1 wt.% To 70 wt.%, 2 wt.% To 65 wt.%, 4 wt.% To 63 wt.%, 5 wt.% To 60 wt.%, Or 10 wt. And 55% by weight of anhydrous box extract.

The composition ratio of the health functional food composition may be arbitrarily varied depending on the regional or national preference such as the demand class, the demanding country, the use purpose, and the like. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

A pharmaceutical composition, a cosmetic composition, and a health functional food composition containing an acidic box extract as an active ingredient can enhance skin elasticity of an individual, prevent or improve skin wrinkles, and prevent or prevent skin aging.

FIG. 1 shows the results of confirming that ethanol extract of non-acidic box inhibits collagenase-1 secretion of human fibroblasts.
Fig. 2 shows the results of confirming that the ethanolic extract of corn oil increased the amount of type-1 procollagen secreted by human fibroblasts.
FIG. 3 shows the results of confirming that the ethanolic extract of corn kernels suppresses the expression of collagenase in human keratinocytes.
FIG. 4 shows the results of confirming that the ethanol extract of non-acidic box increased the amount of collagen expression in human keratinocytes.

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

Example  1: Manufacture of boxwood extract and confirmation of collagen increase

In this Example, anthracnose box extract was prepared from anthracnose box and the effect of anthracnose box extract on collagen production was confirmed.

(1) Manufacture of boxwood extract

Sphallerocarpus gracilis ) were obtained from Mongolia in 2014. The ground portion of the anthracnose box was crushed and placed in an extraction container. Ethanol was added to the extraction container, left at room temperature for 3 days, and filtered through filter paper to obtain an anthracnose box extract. More specifically, 1 L of ethanol was added to 1 kg of dried anhydrous caspase, and the mixture was allowed to stand at room temperature for 3 days. Next, the mixture was passed through a filter paper (Whatman, NO.2, 8 mu m) to obtain a filtrate. The obtained filtrate was concentrated under reduced pressure using a rotary vacuum concentrator (EYELA, N-1100) and freeze-dried (Operon, FDCF-12003) was performed for 48 hours to remove residual solvent. 72 g of box extract was obtained. The thus-obtained extract was dissolved in DMSO (dimethyl sulfoxide) and used in the following experiment.

(2) Confirmation of suppression of collagenase-1 secretion in UV-induced fibroblasts

It was confirmed as follows whether or not the dry cell extract inhibits secretion of collagenase-1 (Matrix Metalloproteinase-1, MMP-1) by human fibroblasts.

Human fibroblasts were inoculated into wells of a 24-well microtiter plate containing 1 ml of DMEM (Dulbecco's Modified Eagle's Media: Hyclone) medium containing 2.5% (v / v) 10 < ⁵ > cells / well. Next, the human fibroblasts were cultured until their confluency reached about 90%.

Next, the dry cell extract of (1) above was dissolved in a serum-free DMEM medium and added to the well containing the human fibroblasts at a culture medium concentration of 5, 10 and 20 μg / ml. As a positive control, epigallocatechin gallate (EGCG, Sigma, USA) was used, and the above-mentioned epigallocatechin gallate was added at a concentration of 10 μM. As a negative control, DMSO was added to the medium in the same volume. Then, human fibroblasts were cultured for 1 hour. After 1 hour of addition, each well was washed with DPBS and irradiated with 15 mJ using a UV irradiator.

Then, the dry cell extract of (1) above was dissolved in a serum-free DMEM medium and further added to the well containing the human fibroblasts at a medium concentration of 5, 10, 20 μg / ml. A positive control group was further added with the above-mentioned epigallocatechin gallate at a medium concentration of 10 [mu] M. As a negative control, DMSO was added to the medium in the same volume. Then, human fibroblasts were cultured for 24 hours. After 24 hours of addition, the cell culture was collected and centrifuged to obtain only the supernatant.

Next, the degree of collagenase-1 production was measured using a collagenase-1 measurement kit (QIA55, Merch & Co., USA). First, 20 μl of the above supernatant was added to a 96-well plate uniformly coated with collagenase primary antibody, and antigen-antibody reaction was performed at room temperature for 2 hours. After 2 hours, 100 쨉 l of the primary collagen antibody bound to the chromophore was added to the 96-well plate incubator and reacted for 1 hour. After 1 hour, 100 [mu] l of color development inducing substance was added and color development was induced at room temperature for 30 minutes. Then, 50 [mu] l of termination buffer was added to stop the color development reaction. The color of the reaction solution was yellow, and the degree of yellowing was different according to the progress of the reaction. The yellow 96-well plate incubator was inserted into the absorber and the absorbance at 450/540 nm was measured. Fig. 1 shows the results of confirming that the anthracnose extract inhibited the secretion of collagenase-1 by human fibroblasts. As shown in Fig. 1, human fibroblasts supplemented with negative control significantly increased the secretion of collagenase-1 by about 60% by UV irradiation, whereas human fibroblasts supplemented with anthracnose Secretion of Edo collagenase-1 was hardly increased.

(3) In fibroblasts induced by UV, type-1 Procollagen (Type-1 procollagen) Confirmation of increased secretion

Whether or not the ascorbic acid extract increased the secretion of type-1 procollagen was confirmed as follows.

Fetal bovine serum is 2.5 (v / v)% of DMEM containing as: human fibroblasts on (Dulbecco's Modified Eagle's Media Hyclone ) 24- well plate culture medium containing the culture medium 1㎖ (24-well microtiter plate) 1x10 5 cells / Well and incubated under the same conditions as described above until confluency reached about 90%.

Next, the dry cell extract of (1) above was dissolved in a serum-free DMEM medium and added to the well containing the human fibroblasts at a culture medium concentration of 5, 10 and 20 μg / ml. As a positive control, epigallocatechin gallate was used, and the epigallocatechin gallate was added at a concentration of 10 μM. As a negative control, DMSO was added to the medium in the same volume. Then, human fibroblasts were cultured for 1 hour. After 1 hour of addition, each well was washed with DPBS and irradiated with 15 mJ using a UV irradiator.

Then, the dry cell extract of (1) above was dissolved in a serum-free DMEM medium and further added to the well containing the human fibroblasts at a medium concentration of 5, 10, 20 μg / ml. A positive control group was further added with the above-mentioned epigallocatechin gallate at a medium concentration of 10 [mu] M. As a negative control, DMSO was added to the medium in the same volume. Then, human fibroblasts were cultured for 24 hours. After 24 hours of addition, the cell culture was collected and centrifuged to obtain only the supernatant.

Next, using an ELISA kit (MK101, Takara Bio Ink., Japan) confirming the peptide (procollagen type-C-peptide, PIP) as an index of collagen synthesis, the effect of anthracnose extract on collagen production was quantitatively Respectively. Since the level of PIP is proportional to the level of collagen, the procollagen type I C-peptide is a peptide that is cleaved in the process of converting collagen from collagen in vivo in the process of collagen synthesis in vivo. Therefore, Collagen levels can be determined from peptides.

First, a mouse anti-PIP monoclonal antibody conjugated with peroxidase was added to a 96-well plate in which a mouse anti-PIP monoclonal antibody was uniformly coated. Thereafter, 100 μl of the above-obtained supernatant was added and reacted for 3 hours in an incubator at 37 ° C. After 3 hours of reaction, 100 μl of a substrate solution containing hydrogen peroxide and tetramethylbenzidine was added, followed by color development at room temperature for 15 minutes. Then, 50 μl of termination buffer was added to stop the color development reaction. The 96-well plate incubator in which the color development reaction was stopped was inserted into the extinction system and absorbance was measured at 450 nm.

FIG. 2 shows the results of confirming that the casein extract promotes the production and secretion of type-1 procollagen in human fibroblasts. As shown in Fig. 2, human fibroblasts supplemented with negative control significantly decreased type-1 procollagen secretion by about 30% by UV irradiation, whereas human fibroblasts supplemented with anthracnose The expression of type-1 procollagen was significantly increased.

(4) Collagenase in keratinocytes induced by UV MMP ) Confirmation of expression level inhibition

Whether or not the anthracnose extract inhibited collagenolytic enzyme expression was confirmed as follows.

Fetal bovine serum is 2.5 (v / v)% a DMEM containing a (Dulbecco's Modified Eagle's Media: Hyclone) 2x10 human keratinocytes on a 6-well plate culture medium (6-well microtiter plate) containing the medium 1㎖ ⁶ cell / Well, and cultured under the same conditions as described above until confluency reached about 90%.

Next, the dry cell extract of (1) was dissolved in a serum-free DMEM medium and added to the well containing the human keratinocytes at a culture medium concentration of 5, 10, and 20 μg / ml. As a positive control, epigallocatechin gallate was used, and the epigallocatechin gallate was added at a concentration of 10 μM. As a negative control, DMSO was added to the medium in the same volume. Human keratinocytes were then incubated for 1 hour. After 1 hour of addition, each well was washed with DPBS and irradiated with 15 mJ using a UV irradiator.

Next, the dry cell extract of (1) above was dissolved in serum-free DMEM medium and further added to the well containing the human keratinocytes at a culture medium concentration of 5, 10, 20 / / ml. A positive control group was further added with the above-mentioned epigallocatechin gallate at a medium concentration of 10 [mu] M. As a negative control, DMSO was added to the medium in the same volume. Human keratinocytes were then incubated for 24 hours. After 24 hours of addition, TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was added to the cells and total RNA was obtained from the cells. The RNA was reverse transcribed with cDNA using reverse transcriptase. RT-PCR was performed using the synthesized cDNA as a template and using the following primer set as a primer. The relative mRNA expression level of each gene was normalized to the beta -actin value.

SEQ ID NO: gene 5`-sequence-3` SEQ ID NO: 1 β-actin Forward 5'-AGCCATGTACGTAGCCATCC-3 ' SEQ ID NO: 2 β-actin Reverse 5'-CTCTCAGCTGTGGTGGTGAA-3 ' SEQ ID NO: 3 MMP-1 Forward 5'-ACAGCTTCCCAGCGACTCTA-3 ' SEQ ID NO: 4 MMP-1 Reverse 5'-CTCTTGGCAAATCTGGCGTG-3 ' SEQ ID NO: 5 MMP-2 Forward 5'-CGCATCTGGGGCTTTAAACAT-3 ' SEQ ID NO: 6 MMP-2 Reverse 5'-CCATTAGCGCCTCCATCGTA-3 ' SEQ ID NO: 7 MMP-9 Forward 5'-CATCCGGCACCTCTATGGTC-3 ' SEQ ID NO: 8 MMP-9 Reverse 5'-CATCGTCCACCGGACTCAAA-3 '

FIG. 3 shows the results of examining the effect of anthracnose extract on the amount of collagenase (MMP) expression in human keratinocytes. As shown in FIG. 3, human keratinocyte cells supplemented with negative control significantly increased collagenolytic enzyme expression by UV irradiation, whereas human keratinocyte cells supplemented with anthracnose box extract showed collagenase Expression was significantly decreased.

(5) Confirmation of increase of collagen expression in keratinocytes induced by UV

Whether or not the ascorbic acid extract increased collagen collagen expression was confirmed as follows.

Fetal bovine serum is 2.5 (v / v)% a DMEM containing a (Dulbecco's Modified Eagle's Media: Hyclone) 2x10 human keratinocytes on a 6-well plate culture medium (6-well microtiter plate) containing the medium 1㎖ ⁶ cell / Well, and cultured under the same conditions as described above until confluency reached about 90%.

Next, the dry cell extract of (1) was dissolved in a serum-free DMEM medium and added to the well containing the human keratinocytes at a culture medium concentration of 5, 10, and 20 μg / ml. As a positive control, epigallocatechin gallate was used, and the epigallocatechin gallate was added at a concentration of 10 μM. As a negative control, DMSO was added to the medium in the same volume. Human keratinocytes were then incubated for 1 hour. After 1 hour of addition, each well was washed with DPBS and irradiated with 15 mJ using a UV irradiator.

Next, the dry cell extract of (1) above was dissolved in serum-free DMEM medium and further added to the well containing the human keratinocytes at a culture medium concentration of 5, 10, 20 / / ml. A positive control group was further added with the above-mentioned epigallocatechin gallate at a medium concentration of 10 [mu] M. As a negative control, DMSO was added to the medium in the same volume. Human keratinocytes were then incubated for 24 hours. After 24 hours of addition, TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was added to the cells and total RNA was obtained from the cells. The RNA was reverse transcribed with cDNA using reverse transcriptase. RT-PCR was performed using the synthesized cDNA as a template and using the following primer set as a primer. The relative mRNA expression level of each gene was normalized to the beta -actin value.

SEQ ID NO: gene 5`-sequence-3` SEQ ID NO: 9 COL1A1 Forward 5'-TGAGCGGACGCTAACCCCCT-3 ' SEQ ID NO: 10 COL1A1 Reverse 5'-CAGACGGGACAGCACTCGCC-3 ' SEQ ID NO: 11 COL3A1 Forward 5'-TGGTGCCCCTGGTCCTTGCT-3 ' SEQ ID NO: 12 COL3A1 Reverse 5'-TACGGGGCAAAACCGCCAGC-3 '

FIG. 4 shows the results of confirming that the extract from the anthracnose box promotes expression of collagen synthesis-related factors COL1A1 and COL3A1 in human keratinocytes. As shown in Fig. 4, human keratinocytes containing negative control group showed decreased expression of COL1A1 and COL3A1 by UV irradiation, whereas human keratinocyte cells supplemented with anthracnose extract showed expression of COL1A1 and COL3A1 even in UV irradiation Respectively.

From the above results, it was confirmed that the casein extract was able to reduce the expression of MMP-1 from human fibroblasts and promote collagen expression under the condition of irradiation with ultraviolet light. That is, in the case of the dry skin extract, the expression of collagenase was promoted while the expression of collagenase was reduced under irradiation of ultraviolet light in human fibroblast. Thus, the anther box extract can promote and enhance the elasticity of the tissue in tissues containing human fibroblasts, for example, by promoting collagen synthesis in the skin. Therefore, the dry skin box extract is effective for improving the texture including human fibroblasts, for example, skin wrinkles or preventing skin aging.

Example  2: pharmaceutical composition comprising an acidic box extract, Cosmetics  Compositions, and health functional food compositions

(One) Soft capsule

The soft capsule was prepared by mixing 150 mg of the extract from the dead skin, 2 mg of palm oil, 8 mg of palm kernel oil, 4 mg of yellow light, and 6 mg of lecithin and filling the capsule with 400 mg per capsule according to a conventional method.

(2) Purification

The granules were formed by mixing 150 mg of extract of Musan casualty, 100 mg of glucose, 50 mg of red ginseng, 96 mg of starch and 4 mg of magnesium stearate and 40 mg of 30% ethanol, followed by drying at 60 ° C., Tablets were tableted.

(3) granules

100 mg of 30% ethanol was added to the mixture to form granules. The granules were then dried at 60 ° C. to form granules. The granules were then filled in the granules. The final weight of the contents was 1 g.

(4) Drinks

150 mg of extract of Musan casualus, 10 g of glucose, 50 mg of red ginseng extract, 2 g of citric acid and 187.8 g of purified water were mixed and filled in a bottle. The final volume of the contents was adjusted to 200 ml.

(5) health functional foods

Vitamin A acetate, vitamin E, vitamin E, vitamin B, vitamin B6, vitamin B12, vitamin C 10 mg, biotin 10 mg, nicotinamide 1.7 mg, vitamin B 0.15 mg, 50 mg of folic acid, 0.5 mg of calcium pantothenate, 1.75 mg of ferrous sulfate, 0.82 mg of zinc oxide, 25.3 mg of magnesium carbonate, 15 mg of potassium phosphate monobasic, 55 mg of calcium phosphate dibasic, 90 mg of potassium citrate, 100 mg of calcium, and 24.8 mg of magnesium chloride were mixed to prepare granules.

(6) Health functional drinks

The final volume of the contents was adjusted to 900 ml by adding 1000 mg of the dead skin cancer extract, 1000 mg of citric acid, 100 g of oligosaccharide, 2 g of the plum concentrate and 1 g of taurine. After stirring and heating at 85 DEG C for about 1 hour, the solution made was filtered and obtained in a sterile 2 liter vessel.

(7) softening longevity

Compounding ingredient Content (% by weight) Musan casualty extract 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

(8) Nourishing lotions

Compounding ingredient Content (% by weight) Musan casualty extract 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

(9) Nourishing creams

Compounding ingredient Content (% by weight) Musan casualty extract 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

(10) Massage creams

Compounding ingredient Content (% by weight) Musan casualty extract 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

(11) Pack

Compounding ingredient Content (% by weight) Musan casualty extract 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 preservative 6.0 Good Preservative, coloring, fragrance Suitable amount Purified water Balance

(12) Ointment

Compounding ingredient Content (% by weight) Musan casualty extract 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

<110> KOREA INSTITUTE OF SCIENCE AND TECHNOLOGY <120> Composition for improving skin elasticity containing          sphallerocarpus gracilis extract <130> PN115343 <160> 12 <170> KoPatentin 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> B-actin Forward <400> 1 agccatgtac gtagccatcc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> B-actin Reverse <400> 2 ctctcagctg tggtggtgaa 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MMP-1 Forward <400> 3 acagcttccc agcgactcta 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MMP-1 Reverse <400> 4 ctcttggcaa atctggcgtg 20 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> MMP-2 Forward <400> 5 cgcatctggg gctttaaaca t 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MMP-2 Reverse <400> 6 ccattagcgc ctccatcgta 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MMP-9 Forward <400> 7 catccggcac ctctatggtc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MMP-9 Reverse <400> 8 catcgtccac cggactcaaa 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COL1A1 Forward <400> 9 tgagcggacg ctaaccccct 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COL1A1 Reverse <400> 10 cagacgggac agcactcgcc 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COL3A1 Forward <400> 11 tggtgcccct ggtccttgct 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COL3A1 Reverse <400> 12 tacggggcaa aaccgccagc 20

Claims (14)

A pharmaceutical composition for preventing or treating skin aging comprising an acid-free box extract as an active ingredient. The composition according to claim 1, wherein the dry box extract is extracted from the ground part of the anoxic box. 2. The composition of claim 1, wherein the anhydrous box extract is extracted from water, acetone, C1-C6 alcohol, or a combination thereof. The composition of claim 1, wherein the dry strength extract is from 0.001% to 80% by weight, based on the total weight of the composition. The composition of claim 1, further comprising a pharmaceutically acceptable diluent or carrier. The composition according to claim 1, which promotes expression of a collagen gene in a cell. A cosmetic composition for promoting the elasticity of the skin containing an acidulant box extract as an active ingredient. 8. The composition of claim 7, wherein the anther box extract is extracted from a top portion of the anoxic container. 8. The composition of claim 7, wherein the anhydrous box extract is extracted from water, acetone, C1-C6 alcohol, or a combination thereof. 8. The composition of claim 7, wherein the dry strength extract is from 0.001% to 80% by weight based on the total weight of the composition. A health functional food composition for promoting the elasticity of skin containing an acid free box extract as an active ingredient. 12. The composition of claim 11, wherein the anther box extract is extracted from a top portion of the anoxic container. 12. The composition of claim 11, wherein the dry strength extract is extracted from water, acetone, C1-C6 alcohol, or a combination thereof. 12. The composition of claim 11, wherein the dry strength extract is from 0.001% to 80% by weight relative to the total weight of the composition.

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