KR20150049078A - Cosmetic Composition including Rosa davurica Pall Callus Cultured in medicum containing mineral water - Google Patents

Abstract

The present invention relates to a cosmetic composition containing Rosa davurica callus, and more specifically, to an anti-aging cosmetic composition containing Rosa davurica callus cultured in a medium containing mineral water. According to the present invention, the cosmetic composition containing Rosa davurica callus has no toxicity in skin cells, excellent synthetic ability for skin collagen, and an effect on skin moisture, pore tightening, inhibition of sebum secretion, improvement in acne, and antioxidative action.

Description

[0001] The present invention relates to a cosmetic composition containing a bovine callus, which is cultured in a mineral water-containing medium, which comprises a cosmetic composition including Rosa davurica Pall Callus Cultured in medicum containing mineral water,

More particularly, the present invention relates to an antioxidant cosmetic composition containing a bioartificial callus cultured in a mineral water-containing medium.

The skin of a person is constantly changing as he ages, and gradually aging. Continuous physical and chemical stimuli received from the outside deteriorate the skin function and accelerate the aging of the skin. Such aging of the skin accompanies the deterioration of the skin constituents as a whole, and as a result, the skin is easily split.

In addition, a decrease in fibroblasts and impaired activity of these cells are also an important part of the skin aging process. In order to prevent such skin aging phenomenon and to maintain healthier and more beautiful skin, cosmetics containing physiologically active substances obtained from various plants, animals and microorganisms have been conventionally used to maintain the inherent functions of the skin, We have been striving to control aging effectively.

However, existing cosmetic raw materials are still insufficient to satisfy satisfactory effects or efficacies, and have a problem of causing skin side effects.

Acne vulgaris (acne vulgaris) is a skin disease caused by the narrowing or clogging of the pores and the loss of sebum. Hair follicles contain sebaceous glands that secrete oil and moisturize the hair. If the sebaceous gland secretion increases or the entrance of the hair follicle becomes clogged, the proliferation of acne bacterium becomes active and causes acne. There are many causes of acne, but the proliferation of the male hormones androgen, acne causing bacteria, Propionibacterium acnes and the increase of sebum secretion are the most important. In patients with acne, androgens stimulate the sebaceous glands to increase sebum secretion, and these increased sebum tangle with dead skin cells and block the fascial entrance. This clogged follicle entrance provides an environment in which the anaerobic bacterium, Propionibacterium acnes, can grow, and the lipases secreted by these bacteria degrade the sebum, which causes the hair follicles to irritate the hair follicle, eventually causing inflammation. Prevention and treatment of acne can be achieved through excessive suppression of sebum secretion and inflammation relief. Therefore, if the cause is removed in a complex manner, the acne can be effectively inhibited. In particular, acne has been increasing in recent years due to external stresses, air pollutants, hormone imbalance, etc., and it is becoming more and more evident in adults. A method for preventing and treating acne by applying salicylic acid, triclosan and trihydroxy palmiic acid to a cosmetic composition as a method for improving skin troubles associated with acne by opening pores and promoting sebum secretion has been disclosed (Korean Patent Laid-Open Publication No. 2004-0089072) discloses a method of suppressing sebum secretion by reducing sebum secretion amount in sebaceous glands by taking oligosaccharide (JP-A-05-294836). In addition, a method for preventing acne by applying a violet extract having antibacterial activity to Propionibacterium acnes has been proposed (Korean Patent Publication No. 2002-0082616)

On the other hand, there is a patent (Korean Patent Registration No. 10-1043813) related to cosmetic composition in the past regarding the hair and ear, but this relates to a cosmetic composition having a protective effect against skin damage caused by ozone, There have been no functional studies on antioxidant effect as a cosmetic material.

Korean Patent Publication No. 2004-0089072 Japanese Patent Application Laid-Open No. 05-294836 Korean Patent Publication No. 2002-0082616 Korean Patent No. 10-1043813

The present inventors have made efforts to produce a cosmetic composition having skin-improving ability, and have found that the extract of Bovine calli extract, especially the bovine callus cultivated in a medium containing mineral water, is excellent in skin moisturizing, whitening, sebum suppression, Efficacy, etc., the present invention has been completed.

Accordingly, an object of the present invention is to provide a cosmetic composition for improving skin comprising an extract of Bacillus subtilis as an active ingredient.

It is another object of the present invention to provide a method for producing the biotite callus.

It is still another object of the present invention to provide a method for producing a cosmetic composition containing the above-mentioned Bio-Radical Callus extract as an active ingredient.

In order to achieve the above object, the present invention provides a cosmetic composition for improving skin comprising an extract of Bacillus subtilis as an active ingredient.

In the present invention, the composition may be used for whitening, wrinkle prevention or improvement, skin moisturizing, pore reduction, sebum secretion suppression, and acne improvement.

In the present invention, the bacteriocidal callus may be characterized by being cultured in a mineral water-containing medium.

In the present invention, the concentration of the mineral water in the culture medium may be 0.5 to 4% (v / v).

The present invention also provides a method for producing Baculovirus callus having a tyrosinase activity inhibitory activity, an antioxidative ability, or a collagen cohesive ability, which comprises culturing Bacillus subtilis callus in a mineral water containing medium.

In the present invention, the concentration of the mineral water in the culture medium may be 0.5 to 4% (v / v).

In the present invention, the mineral water-containing medium comprises 0.5 to 3 mg / l of 6-benzylaminopurine, the pH is 6 to 6.5, the concentration of 6-benzylaminopurine is 0.5 to 3 mg / l, KNO3 is 2.3 to 2.7 g / l, 0.1 to 0.3 g / l of 2H2O, 0.3 to 0.5 g / l of MgSO4.7H2O, 0.2 to 0.4 g / l of NH4H2PO4, 0.04 to 0.015 g / l of KI, 0.04 to 0.06 g / l of H3BO3, 0.005 to 0.015 g of MnSO4- 1, ZnSO 4 揃 7H 2 O 0.0005 to 0.0015 g / l, Na 2 MoO 4 揃 2H 2 O 0.00005 to 0.00015 g / l, CoCl 2 揃 6H 2 O 0.00005 to 0.00015 g / l, FeSO 4 揃 7H 2 O 0.01 to 0.02 g / l, Na 2 EDTA 0.015 to 0.025 g / And inositol (Inositol) in an amount of 0.5 to 1.5 g / l.

The present invention also relates to a method for producing a plant, comprising: (a) inducing a callus from a live seed, and culturing it in a mineral water-containing medium; And (b) preparing a composition containing the extract of the culture obtained in the step (a). The present invention also provides a method for producing a cosmetic composition for skin improvement comprising the extract of Bovine Enteritidis.

The cosmetic composition containing the extract of bacteriocidal extract according to the present invention has excellent skin collagen synthesis ability without toxicity on skin cells and has skin moisturizing effect, reduction of pores, inhibition of sebum secretion, improvement of acne and antioxidative effect.

Fig. 1 is a photograph of the results obtained from germinating seeds obtained on days 7, 8, 9, 10 and 21 after germination.
Fig. 2 is a photograph showing the result obtained by culturing the derived bovine callus in a medium containing no mineral water.
Fig. 3 shows the HPLC results of the Bioartificial callus extract.
FIG. 4 is a graph showing the results of analysis of HPLC component according to concentration of mineral water in mineral water and Bacillus subtilis extract.

The present invention relates to a cosmetic composition for skin improvement comprising a bacteriostatic extract as an active ingredient and a process for producing the same.

In the present invention, Rosa davurica Pall. Is a perennial plant belonging to Rosacea, and is a deciduous broad-leaved shrub distributed in Korea, Japan, Manchuria and Siberia. It has been known for some time as a medicinal effect of roots and fruits, and it has been known that it has a key structure and a blood circulation landscape. It is a useful medicinal plant resource that has been used for treatment of indigestion, anticipation copying, stomach pain and menstrual relief. The content of ascorbic acid was 1030 times higher than that of lemons and the content of beta-carotene was 810 times higher than that of carrots. In the peel of the raw nuts, betulinic acid, alphitolic acid, oleanolic acid, maslinic acid, ursolic acid, Tetracyclic triterpene and quercetin such as pomolic acid, tormentic acid and euscaphic acid, quercetin, hyperin, tilyloside, and flavonoids such as tiliroside have been reported.

In one aspect, the present invention relates to a cosmetic composition for improving skin comprising an extract of Bacillus subtilis as an active ingredient. Specifically, the Bacillus subtilis extract may be contained in an amount of 0.1 to 10% by weight based on the total weight of the composition. These ratios are merely preferred ranges, for example, 10% by weight or more and 20% or more, and 30% or more of the skin is improved, including excellent pore reduction, skin moisturizing, acne improvement, sebum suppression, antiinflammation, Are obvious to those skilled in the art.

In the present invention, it is preferable to use the cultured product of Bio-Radical Callus (culture) itself, to use the culture by filtration, to crush the culture, or to dry the culture to obtain powder, have.

In the present invention, "extract" means an extract obtained by various known extraction methods such as powdering, freeze-drying, or cold water extraction, hot water extraction and ethanol extraction.

The extraction method in the present invention is not particularly limited, and examples thereof include cold extraction, ultrasonic extraction, reflux extraction, hot water extraction and the like. Here, in the case of hot water extraction, the hot water extract is obtained by heating at 80 to 100 ° C for 8 to 48 hours in a hot water distiller.

In the case of cold water extraction, for example, the culture itself or the dried powder thereof is mixed with cold water (15-25 ° C) and extracted for 3 days to obtain a cold water extract.

Alternatively, it can be produced by a method of extracting using water, an organic solvent, or a mixed solvent thereof. The extracted liquid can be used directly or by concentrating and / or drying. When extraction is carried out using an organic solvent, an organic solvent such as methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, (DMSO), 1,3-butylene glycol, propylene glycol, or a mixed solvent thereof. The active ingredient of the herbal medicine may be extracted at room temperature or warmed under the condition that the active ingredient is not destroyed or minimized. Depending on the organic solvent to be extracted, the degree of extraction and the degree of loss of the active ingredient of the medicament may differ. Therefore, an appropriate organic solvent should be selected and used. Particularly, in the present invention, an ethanol extract, preferably a 20-50% ethanol extract, specifically 50% ethanol extract, is preferable, and an extraction method is obtained by mixing with 50% ethanol and stirring for 3 days.

In the present invention, the above extract may be used by concentration or dilution, and a distillate of the extract may be used.

In the present invention, facilitating skin improvement refers to improvement of skin protection and skin condition, skin whitening, prevention or improvement of skin aging and wrinkles, skin moisturizing, suppression of sebum secretion, improvement of acne, reduction of pores, skin protection, Barrier function improvement, skin irritation mitigation, skin cell proliferation and regeneration ability, antioxidant ability, and collagen synthesis promoting ability.

In the present invention, the expression "comprising as an active ingredient" means a cosmetic composition which is capable of exhibiting skin improving effect, for example, antioxidant, pore reduction, skin moisturizing, sebum suppression, acne improvement, Quot; means an amount of effective amount that is capable of exhibiting collagen synthesis or iNOS inhibitory activity, and the like.

Accordingly, the cosmetic composition according to the present invention is particularly useful as a cosmetic composition for aquaporin-3 (hereinafter referred to as " aquaporin-3 ") related to collagen synthesis gene expression, tyrosinase activity inhibition, increase in expression of collagen biosynthetic genes, ) And filaggrin expression can be applied, and it can be applied for skin aging prevention, skin wrinkle improvement / prevention, whitening, antioxidation, pore contraction, sebum suppression, skin moisturization and the like.

In the present invention, callus refers to a specific tissue or cell mass produced when a tissue cut out from a plant is cultured in a medium containing an auxin, a wound is injured to a certain kind of plant, or an auxin is treated with the excrement. Usually callus is an amorphous tissue or cell mass that has lost its ability to cause normal organogenesis or tissue differentiation, and is mostly flowcytosed. In a broad sense, it also includes plant tumor tissues caused by infection such as Agrobacterium.

In the present invention, it is not limited whether a callus derived from a seed of a fresh ear or an adult is a callus. In one embodiment, the callus may be cut out of a bioartificial tissue germinated from a live seed.

Plant tissue culture is a technique in which a living small plant tissue is aseptically cultured in vitro to grow plant cells, organs and plants according to the purpose of culture. Plant tissue culture can be attributed to the basic characteristics of plants. Plant tissue culture technology is based on the use of differentiating totipotency to regenerate plants from plant somatic cells, a unique feature of plants. Namely, plants can be regenerated from these cells and tissues by culturing single cells (including protoplasts), leaves and root sections of plants in a medium supplemented with specific nutrients and growth regulators. The ability of these plants to have differentiating totipotency is a strategy for survival from harsh environments and herbivorous damage for the fixation and long-term survival of plants. Therefore, plants can be said to have the ability to regenerate lost organs again. The plant tissue cells isolated from the parent plant have the potential to be reproduced as a complete plant when cultured in a suitable culture environment, that is, plants having typical performance are of academic importance such as embryology, but they are also utilized for practical use of cosmetic raw materials .

In another aspect, the present invention relates to a method for producing a hair follicle comprising: (a) inducing a callus from a live seed, and culturing it in a mineral water-containing medium; And (b) a step of preparing a composition containing the extract of the culture obtained in the step (a).

In the step (a), at the time of inducing the virgin callus, a suitable medium can be selected. If there is a medium commonly used in plant tissue culture in the technical field, it can be used without limitation. For example, the composition of the MS medium (1 L standard) is 1650 mg NH ₄ NO ₃ , 1900 mg KNO ₃ , 440 mg CaCl ₂ .2H ₂ O, MgSO 4, _{4 .7H 2 O 370 mg, KH} 2 PO 4 170 mg, KI 0.83 mg, H 3 BO 3 6.2 mg, MnSO 4 .4H 2 O 22.3 mg, ZnSO 4 .7H2O 8.6 mg, Na 2 MoO 4 .2H 2 O 0.25 mg, CuSO ₄ , 5H ₂ O 0.025 mg, CoCl ₂ .6H ₂ O 0.025 mg, FeSO ₄ .7H ₂ O 27.8 mg, Na ₂ EDTA.2H ₂ O 37.3 mg, Myoinositol 100 mg, Nicotinic acid 0.5 mg, Pyridoxine -HCl 0.5 mg, Thiamine-HCl 0.5 mg, Glycine 2 mg, and Sucrose 30000 mg.

In the present invention, it is preferable that in the step (b), a composition containing the extract of Bacillus subtilis obtained through the cultivation of step (a) is prepared as a suitable external preparation for skin or the culture is cultured by a known extraction method In the form of an extract to prepare a skin external composition composition in a suitable form.

For example, in the step (b), the bioglass callus culture obtained in step (a) is dried, powdered and mixed with purified water to prepare a composition, or the bioglass callus culture obtained in step (a) , Mixing the mixture into purified water, and then preparing the composition by cold water extraction, hot water extraction or ethanol extraction.

As another example, the culture obtained in the step (a) may be dried by hot air and powdered to evaporate water.

In the present invention, the derived bovine callus may be a callus extract obtained by culturing in a medium containing mineral water.

In another aspect, the present invention relates to a method for producing a biovarial callus having a tyrosinase activity inhibiting ability, an antioxidation ability, or a collagen combining ability, which comprises culturing a bioparticle callus in a mineral water containing medium.

In the present invention, " mineral water " means water taken from 1,011 m underground as the mineral water (Jinjin hot spring water) in the mountain near the beach of Geumjinae in Gangneung, Gangneung, Gangwon Province. The ratio of salinity of such mineral water is 18 ~ The ratio of calcium to magnesium may be 1.6 to 1.7: 1, and preferably the ratio of salinity is 20% of the total weight, and the ratio of Ca (calcium) to Mg (magnesium) is 1.66: 1. It is composed of the best golden ratio.

[Figure 1] Analysis of mineral water composition

The concentration of the mineral water in the medium may be 0.5 to 10% (v / v), preferably 0.5 to 4% (v / v), more preferably 1.0 to 3.0% (v / v) Do not.

In the method according to one embodiment of the present invention, the medium is preferably a medium (Murashige & Skoog), a WPM (McCown Lloyd) or an SH (Shenk & Hildebrant Medium) medium, And more preferably from 2.3 to 2.7 g / l of KNO3, from 0.1 to 0.3 g / l of CaCl2.2H2O, from 0.3 to 0.5 g / l of MgSO4.7H2O, from 0.2 to 0.4 g / l of NH4H2PO4, from 0.0005 to 0.0015 g / , H3BO3 0.04 to 0.06 g / l, MnSO4. H2O 0.005 to 0.015 g / l, ZnSO4.7H2O 0.0005 to 0.0015 g / l, Na2MoO4.2H2O 0.00005 to 0.00015 g / l, CoCl2.6H2O 0.00005 to 0.00015 g / , A medium containing 0.01 to 0.02 g / l of 7H2O, 0.015 to 0.025 g / l of Na2EDTA and 0.5 to 1.5 g / l of inositol, and most preferably 2.5 g / l of KNO3, 0.2 g of CaCl2.2H2O 1, MgSO 4 揃 7H 2 O 0.4 g / l, NH 4 H 2 PO 4 0.3 g / l, KI 0.001 g / l, H3BO 3 0.05 g / / L, 0.0001 g / l CoCl2.6H2O, 0.015 g / l FeSO4.7H2O, 0.02 g / l Na2EDTA and 1 g / l inositol. .

In the method according to an embodiment of the present invention, the medium may comprise a growth regulator, preferably 6-benzylaminopurine, more preferably 0.5-3 mg / l 6-benzylaminopurine , And most preferably 6 mg of 6-benzylaminopurine, but is not limited thereto. In addition, the pH of the medium may be 6 to 6.5, preferably pH 6, but is not limited thereto.

In the method according to an embodiment of the present invention, the cultivation of the virgin callus can be performed at 20 to 32 ° C, preferably at 22 to 28 ° C, but is not limited thereto.

In a method according to an embodiment of the present invention, the preferred concentration of mineral water in the medium is 2 to 5% (v / v), the pH is 6 to 6.5, 1 to 3 mg / l of 6-benzylaminopurine, 2.3 to 2.7 g of KNO3 0.1 to 0.3 g / l of CaCl2 占 H2O, 0.3 to 0.5 g / l of MgSO4 占 H2O, 0.2 to 0.4 g / l of NH4H2PO4, 0.0005 to 0.0015 g / l of KI, 0.04 to 0.06 g / l of H3BO3, MnSO4 占 H2O 0.005-0.015 g / l, ZnSO4.7H2O 0.0005-0.0015 g / l, Na2MoO4.2H2O 0.00005-0.00015g / l, CoCl2.6H2O 0.00005-0.00015g / l, FeSO4.7H2O 0.01-0.02g / l, Na2EDTA 0.015 ~ 0.025 g / l, and inositol (0.5 to 1.5 g / l), and the cultured bovine callus is cultured at 25 to 32 ° C, but is not limited thereto.

The cosmetic composition of the present invention may contain an acceptable carrier in cosmetic preparations. Herein, "an acceptable carrier for a cosmetic preparation" is a known or used compound or composition that can be contained in a cosmetic preparation, or a compound or composition to be developed in the future, which is toxic, instable or irritating It says nothing.

The carrier may be included in the cosmetic composition of the present invention in an amount of about 1% by weight to about 99.99% by weight, preferably about 90% by weight to about 99.99% by weight of the composition, based on the total weight thereof. However, since the ratio varies depending on the formulations described below in which the cosmetic composition of the present invention is prepared, and on the specific application site (face, neck, etc.) of the cosmetic composition and its preferable application amount, And should not be construed as limiting the scope of the invention.

Examples of the carrier include an alcohol, an oil, a surfactant, a fatty acid, a silicone oil, a wetting agent, a moisturizer, a viscous modifier, an emulsifier, a stabilizer, an ultraviolet scattering agent, an ultraviolet absorber, a coloring agent and a perfume. The compounds / compositions which can be used as the above-mentioned alcohol, oil, surfactant, fatty acid, silicone oil, humectant, humectant, viscous modifier, emulsifier, stabilizer, ultraviolet scattering agent, ultraviolet absorber, The person skilled in the art can select and use the appropriate substance / composition.

As an embodiment of the present invention, the cosmetic composition according to the present invention may contain glycerin, butylene glycol, propylene glycol, polyoxyethylene hydrogenated castor oil, ethanol, triethanolamine, etc., Preservatives, dyes, coloring agents, purified water and the like can be contained as needed.

The cosmetic composition according to the present invention can be prepared in various forms such as lotion, essence, gel, emulsion, lotion, cream (water-in-oil type, water-in-water type, multiphase), solution, suspension (Soft capsules, hard capsules) with a coating such as anhydrous products (oil and glycol), gel, mask, pack, powder, or gelatin.

The skin of the present invention includes not only a face but also a scalp and a whole body. The cosmetic composition applicable to such scalp is a shampoo, a rinse, a treatment, a hair removal agent, etc., and a body cleanser And can be manufactured in various forms as an application of the composition.

The method for preparing a cosmetic composition containing the Bacteriocariales extract according to the present invention is not limited to the above-described manufacturing method, and any person skilled in the art may make modifications The cosmetic composition containing the extract of Laotiania cell culture extract of the buttercups according to the present invention can be prepared.

In particular, the above-mentioned cosmetic composition can be produced in the form of a general emulsified formulation and a solubilized formulation, using a conventionally known manufacturing method, in addition to the manufacturing method specifically disclosed in the present invention.

When the cosmetic composition is manufactured from a cosmetic composition, the cosmetic composition of the emulsified formulation includes nutritional lotion, cream, essence and the like. It can also be prepared in the form of adjuvants which can be applied topically or systemically, which are conventionally used in the field of dermatology by containing a dermatologically acceptable medium or base.

In addition, suitable cosmetic formulations include, for example, solutions, gels, solid or kneaded anhydrous products, emulsions obtained by dispersing an oil phase in water, suspensions, microemulsions, microcapsules, microgranules or ionic forms (liposomes) May be provided in the form of a follicular dispersing agent of the type, cream, skin, lotion, powder, ointment, spray or conical stick. It can also be prepared in the form of a foam or an aerosol composition further containing a compressed propellant.

The cosmetic composition of the present invention may further contain at least one selected from the group consisting of fatty substances, organic solvents, solubilizers, thickeners and gelling agents, softening agents, antioxidants, suspending agents, stabilizers, foaming agents, perfumes, Or any other conventionally used in cosmetics such as nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, barrier agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, ≪ RTI ID = 0.0 > cosmetics < / RTI > such as botanical ingredients, or adjuvants conventionally used in the field of dermatology. And, the above ingredients can be introduced in amounts commonly used in the field of dermatology.

The cosmetic composition according to the present invention includes functional cosmetics capable of functioning to prevent skin aging due to whitening, skin moisturization, reduction of pores, suppression of sebum secretion, acne improvement, antioxidation, and collagen synthesis promoting ability.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Example 1-1: Bacteriocidal callus induction according to the present invention

The seeds used in the present invention were harvested at Asean-ri, Jeonseon-eup, Jung-sung, Gangwon-do, and germinated by inoculation in soil. The germinated raw nematodes were immersed in 70% ethanol for 30 seconds, then washed with sterilized water, disinfected with disinfectant (30% Lax + Tween 20) for 20 minutes, rinsed 3 times with sterilized water, The cells were cultured in a 1 / 2MS medium containing 1 mg / L 6-benzylaminopurine, 0.3 mg / L 2,4-D, 3% sucrose and 8 g / Callus was induced (Fig. 1).

Example 1-2: Culture and mass production of Bovine callus tissue grown in a medium containing mineral water according to the present invention

The aseptically induced and cultured bovine calli were cultured in 1 / 2MS (+ 1mg / L 6-benzylaminopurine) containing mineral water (0%, 1%, 3%, 5%, 10% , 0.3 mg / L 2,4-D) at 25 ° C and 70% relative humidity, and subcultured at intervals of 2 to 3 weeks. Thereafter, a balloon-type bioreactor was used to mass-produce liquid culture of the same composition except for agar at a temperature of 25 ° C, a humidity of 70%, and an air supply of 0.1 vvm at intervals of 14 days (FIG. 2) .

Examples 1-3: Preparation of the extract of callus cultured on a medium containing mineral water by concentration according to the present invention

The proliferated cultured callus cultures were harvested and sufficiently dried with a clean tissue, and then dried in a dryer at 60 ° C for 2 days. 50 g of dried bovine callus cultured powder was placed in a vessel, and 1 L of purified water was added thereto, followed by extraction at 121 캜 for 1 hour. After the extraction, the extract was filtered with a mesh to remove the solid content, and the crude extract of callus cultured from the filtrate was lyophilized and dissolved in purified water and used in the present invention.

EXPERIMENTAL EXAMPLE 1 Analysis of Components of Bacteriophage Callus Culture Extracts Grown in a Mineral Water-Containing Medium by HPLC

The components were analyzed by HPLC of the callus culture extract grown on the medium containing the mineral water. The analysis conditions were as follows.

Analysis of the cultured bovine callus culture extracts cultured in medium containing 0%, 1%, 3%, 5% and 10% of mineral waters showed similar useful ingredient patterns in the Bovine callus culture extract, In the cultured bovine callus culture extract cultured in medium containing 1% mineral water, the peak detected at the Rt value of 6.4 min was the highest (Figs. 3 and 4).

Experimental Example 2: Influence of the present invention on the viability of skin cells of cultured bovine callus cultured in medium containing mineral water

Human fibroblast (CCD-986Sk) was cultured in an ATCC (American Type Culture Collection) in order to examine the proliferative activity of skin cells on the extract prepared in Example 1. Each cell was inoculated into a 24-well culture plate at a concentration of 1 × 10 ⁴ cells / ml. The medium was DMEM (Dubelco's Modified Eagle Medium, BRL, USA) containing 10% FBS. When the cells were cultured in DMEM containing 10% FBS for 48 hours and cultured by 25-30% of the surface area of the culture dish, the extract prepared in Example 1 was replaced with FBS-free DMEM containing 0.5-5% Respectively. After culturing, 5 쨉 l of a solution of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT, Sigma M5655, USA) (2.5 mg / ml) (DMSO, Sigma D265O, USA) was added to each well, and the formazan crystals formed were dissolved by stirring for 20 minutes. Then, 100 μl each of the supernatant was dissolved in 96 wells And the absorbance at 570 nm was measured by Enzyme-Linked Immunosorbent Assay (ELISA). The degree of proliferative activity of skin cells was calculated by the following formula based on the absorbance intensity of the control group using pure water and expressed as a percentage. The results are shown in Table 2.

[Equation 1]

Cell proliferation effect (%) = [(absorbance of experiment group - absorbance of control group) / absorbance of control group] 100

As shown in Table 2, the results of the test on the cytotoxicity of human keratinocyte showed that the mineral water was grown in a medium containing 0%, 1%, 3%, 5% and 10% No cytotoxicity was observed by treatment with 0.05 ~ 1 mg / ml of callus cultured extract.

Experimental Example 3: Inhibition of in vitro tyrosinase activity

Tyrosinase is a rate-limiting enzyme that plays an important role in skin melanogenesis. It acts as a dopa oxidase that oxidizes tyrosine in the melanosome of melanocytes, a cell that produces pigment of the skin, to make dopa and then to oxidize dopa again to make dopachrome. tyrosinase catalyzes three distinct reactions in the melanin synthesis stage: 1) hydroxylation of monophenol (L-tyrosine), 2) dehydrogenation of catechol (L-DOPA), and 3) dehydrogenation of DHI. Therefore, inhibition of tyrosinase activity of various whitening functional raw materials can be an important measure of whitening activity. Therefore, the degree of inhibition of mushroom tyrosinase enzyme activity was measured in the culture broth derived from the culture medium containing the mineral water by concentration in vitro .

30 μl of fresh calli culture extract derived from medium containing 1%, 3%, 5% and 10% of mineral water, 210 μl of 100 mM phosphate buffer (pH 6.5), mushroom tyrosinase (1500 U / ml - 2000 U / ml , Fulka Cat. No. 93898) and 40 μl of 1.5 mM tyrosine, and incubated at 37 ° C for 10 to 15 minutes. Absorbance was measured at 490 nm using an ELISA reader.

tyrosinase activity inhibition rate (%) = 100 - [(b-b ') / (a-

a: Absorbance after reaction of blank sample

b: Absorbance after reaction of the sample solution

a ', b': absorbance measured by replacing with tyrosinase buffer

As shown in Table 3, the whitening effect of the inhibitory effect of in vitro Tyrosinase activity was 0.05 to 1 mg / ml of the extract of callus cultured on the medium containing 0%, 1%, 3%, 5% and 10% of mineral water, ml treatment inhibited tyrosinase activity by 15% at 1 mg / ml of the extract of callus cultured from the culture medium containing 1% of mineral water, and overall, it showed better whitening effect than the case without mineral water.

Experimental Example 4: Antioxidant test using DPPH free radical scavenging ability

The ability of 1,1-Diphenyl-2-picryhydrazyl (DPPH, Sigma D9132-1G) to excrete free radicals in ethanol can be tested to determine the direct action of antioxidant activity. The compound 1,1-Diphenyl-2-picryhydrazyl (DPPH, Sigma D9132-1G) generates free radicals in ethanol and is mixed with the callus culture extract derived from the medium containing and without mineral water to determine the amount of free radicals And it was confirmed to some extent.

400 μl of ethanol, 500 μl of 0.1 mM DPPH solution, 100 μl of the extract of callus cultured from the medium containing and without mineral water, vortexed for 10 seconds, and allowed to react in a dark place for 30 minutes. Absorbance was measured at 517 nm using ELISA and the degree of antioxidant activity was expressed as a percentage based on the absorbance of the control group using ethanol.

Free radical activity inhibition rate (%) = 100- (Reaction absorbance of each sample liquid / Reaction absorbance of the blank) X100

As shown in Table 4, the antioxidative effect of DPPH radical scavenging was evaluated by treating 0.05-0.1 mg / ml of the callus culture extract grown from the medium containing 0%, 1%, 3%, 5% and 10% of mineral water The highest antioxidative effect was 30% in 1 mg / ml of callus cultured extract grown in medium containing 3% mineral water. Also, it showed better antioxidative effect when cultured in the absence of mineral water.

EXPERIMENTAL EXAMPLE 5: Expression of collagen-related gene (collagen)

The major component of the skin is collagen. Collagen accounts for 70-80% of the dermal dry weight. Type I and III collagen are the major components of human dermis. Type I is 80% of total collagen and type III is 15% of total collagen. Skin wrinkles are caused by an imbalance in the synthesis and degradation of collagen. In normal skin, the synthesis of type I collagen and its degrading enzyme, MMP-1, are in balance. However, it has been reported that the synthesis of type I and III collagen degrades and the activity of MMP-1 increases in photoaged skin.

This test confirms the expression level of Collagen gene involved in collagen synthesis in human fibroblast (mRNA level). Human fibroblast cells were cultured in a 100 mm / 60.1 cm culture dish at 37 ° C and 5% CO 2 with Dulbecco's Modified Eagle's Medium (DMEM), 10% Fatal bovine serum (FBS) and 1% Antibiotic-Antimycotic. When confluence of human fibroblast cells is over 80%, cells are seeded at 1 × 10 ⁴ cells / well in a 96-well plate, cultured until confluence reaches 80% or higher in cell culture conditions, And callus culture extracts induced by the culture medium containing various concentrations of the extracts for 24 hours. The test method was Real-time PCR, and the test procedure is as follows.

(1) RNA Isolation and cDNA Synthesis

RNA isolation and cDNA synthesis were performed using FastLane Cell one-step buffer set (QIAGEN 216213). The cells were washed with FCW (cell wash buffer), incubated at room temperature for 5 minutes, and reacted at 75 ° C for 5 minutes.

(2) Real-time PCR analysis

In order to analyze the collagen gene involved in collagen synthesis, the cDNA synthesized above was used as a template and subjected to Rotor Gene Q Real-time PCR Machine (Qiagen) according to the manual of 2X QuantiTect SYBR Green RT-PCR Master Mix. The primers used in the experiments were Qiagen QuantiTectprimer assays (18S rRNA QT00199367, -actin QT01680476, Collagen QT01005725). The expression rate was normalized to 18S rRNA. Real-time PCR conditions are as follows.

<Real-time cycler conditions>

As shown in Table 5, the expression rate of the collagen biosynthesis gene by qPCR was 0.05 to 1 mg of the extract of callus cultured from a medium containing 0%, 1%, 3%, 5% and 10% of mineral water / ml treatment, the expression level of collagen synthesis gene was 2.8 times higher than that of the control group at 1 mg / ml of the extract of callus cultured from the culture medium containing 1% of mineral water. In addition, the expression of collagen biosynthesis gene was confirmed by cultured Bacillus subtilis callus cultures containing mineral water by concentration rather than mineral water.

Experimental Example 6: Expression of a gene related to skin moisturizing effect (Aquaporin-3)

If the moisturizing function of the skin does not function properly, it will cause the skin elasticity to decrease. That is, loss of moisture of the skin is an important factor of skin aging. The moisture balance of the skin is controlled by the epidermis. The stratum corneum of the epidermis is an accumulation of dead cells, but plays a very important role in maintaining the moisture of the skin. The human body is composed mostly of water, which plays an important role in the maintenance of life. The moisture content of the normal skin is 70% in the body and 20% in the stratum corneum, which supports the elasticity and flexibility of the skin. When the water content of the stratum corneum is 10% or less, it is known that the skin becomes dry and causes skin troubles. The amount of moisture in the stratum corneum produced by regular keratinization is always 10-20%.

By increasing the amount of aquaporins that regulate epidermal hydration, humectant research has been conducted through a mechanism that increases the absorption of moisture in the skin. In general, 3 billion water molecules per second pass through the skin in humans, through a protein called aquaporin. Aquaporins are divided into several types according to structure and function. Among them, aquaporins-3, which is known to be associated with skin, is present in keratinocytes and is known to exist in the basal layer of epidermis.

This test confirms the expression level of Aquaporin-3 gene, which is involved in skin moisturizing effect, in human keratinocyte (keratinocytes) at mRNA level. Human keratinocyte cell lines were cultured in a 100 mm / 60.1 cm culture dish at 37 ° C and 5% CO 2 with Dulbecco's Modified Eagle's Medium (DMEM), 10% Fatal bovine serum (FBS) and 1% Antibiotic-Antimycotic. When confluence of human keratinocyte was over 80%, cells were plated in 96 well plate at 1 cell / well. Cells were cultured until confluence reached 80% or higher in cell culture conditions, and then exchanged with DMEM free medium. After incubation for 24 h, the culture broth extract of Bacillus subtilis was induced to express the related gene Aquaporin-3. The test method was Real-time PCR, and the test procedure is as follows.

(1) RNA Isolation and cDNA Synthesis

RNA isolation and cDNA synthesis were performed using FastLane Cell one-step buffer set (QIAGEN 216213). The cells were washed with FCW (cell wash buffer), incubated at room temperature for 5 minutes, and reacted at 75 ° C for 5 minutes.

(2) Real-time PCR analysis

In order to analyze Aquaporin-3 gene related to skin moisturization, the synthesized cDNA was used as a template and subjected to Rotor Gene Q Real-time PCR Machine (Qiagen) according to the manual of 2X QuantiTect SYBR Green RT-PCR Master Mix. The primers used in the experiments were Qiagen's QuantiTectprimer assays (18S rRNA QT00199367, -actin QT01680476, Aquaporin-3 QT00212996). The expression rate was normalized to 18S rRNA. Real-time PCR conditions are as follows.

<Real-time cycler conditions>

As shown in Table 6, the expression rate of Aquaporin-3 biosynthetic gene by qPCR was 0.05-0.5%, and the concentration of Aquaporin-3 biosynthetic gene was 0.05 ~ The expression rate of Aquaporin-3 synthetic gene was increased 3.2 times as compared to the control group at 1 mg / ml of the callus culture extract grown from the medium containing 1% of mineral water by 1 mg / ml treatment. In addition, the expression rate of Aquaporin-3 biosynthetic gene was confirmed by cultured bovine callus culture extract containing mineral water by concentration rather than mineral water.

Experimental Example 7: Expression of filaggrin gene

A natural moisturizing factor (NMF) plays an important role in maintaining the water content of the stratum corneum. Amino acids, the major component of NMF, have been reported to be produced by degradation of the filaggrin protein, which is derived from keratohyalin granules. Pilar green is a protein consisting of 317 amino acids. Since the amino acid, the main constituent of NMF, has been found to be derived from filaggrin, studies have been carried out on the relationship between dry skin condition and filula green. It has been found that amino acids in the stratum corneum are decreased and filla green expression is reduced in dry skin such as senile dryness, atopic disease, and the like. It is well known that skin troubles such as skin roughness are caused by a drying environment.

Filaggrin is composed of 317 amino acids and is composed of a linker peptide consisting of 7 amino acids of fillaggrin uint (molecular weight 34 kDa) It is expressed as a propagarin (macromolecule with a molecular weight of about 400 kDa), which is a bundled precursor protein. Fillaggrin is finally decomposed into amino acids by activity of amino peptidase, carboxypeptidase and the like in the course of reaching the stratum corneum.

This test confirms the expression level of the filaggrin gene in human keratinocytes (keratinocytes) at the mRNA level. Human keratinocyte cell lines were cultured in a 100 mm / 60.1 cm culture dish at 37 ° C and 5% CO 2 with Dulbecco's Modified Eagle's Medium (DMEM), 10% Fatal bovine serum (FBS) and 1% Antibiotic-Antimycotic. When confluence of human keratinocyte was over 80%, cells were plated in 96 well plate at 1 cell / well. Cells were cultured until confluence reached 80% or higher in cell culture conditions, and then exchanged with DMEM free medium. After 24 hours of incubation of the calli culture extracts derived from the culture medium containing various factors, the effect of the filaggrin gene was examined. The test method was Real-time PCR, and the test procedure is as follows.

(1) RNA Isolation and cDNA Synthesis

RNA isolation and cDNA synthesis were performed using FastLane Cell one-step buffer set (QIAGEN 216213). The cells were washed with FCW (cell wash buffer), incubated at room temperature for 5 minutes, and reacted at 75 ° C for 5 minutes.

(2) Real-time PCR analysis

In order to analyze the filaggrin gene, the cDNA synthesized above was used as a template and subjected to a Rotor Gene Q Real-time PCR Machine (Qiagen) according to the manual of 2X QuantiTect SYBR Green RT-PCR Master Mix. The primers used in the experiments were Qiagen QuantiTectprimer assays (18S rRNA QT00199367, -actin QT01680476, filaggrin QT02448138). The expression rate was normalized to 18S rRNA. Real-time PCR conditions are as follows.

<Real-time cycler conditions>

As shown in Table 7, the expression rate of filaggrin biosynthetic gene by qPCR was 0.05 to 1 mg / ml, which was obtained from a culture medium containing 0%, 1%, 3%, 5% and 10% of mineral water / ml treatment, the expression level of the filaggrin synthetic gene was increased 2.4 times as compared to the control group at 1 mg / ml of the callus culture extract grown in the medium containing 1% mineral water. In addition, the expression rate of filaggrin biosynthesis gene was confirmed by cultured bovine callus cultured extract containing mineral water by concentration rather than mineral water.

Experimental Example 8: In vitro Test

In order to measure the pore-shrinking effect of the composition of the present invention, the pore-shrinking effect of the extract prepared in Example 1 was tested using hemoglobin as a protein instead of skin. Hemoglobin solution (0.05 g / 50 ml) was prepared with 0.9% Phosphate Buffer Saline (0.1 mM, pH 7.4), 2 ml of the extract obtained in the above Example was added to 2 ml of the hemoglobin solution, The mixture was centrifuged at 3,500 rpm for 10 minutes, and 2 ml of purified water was added to 1 ml of the supernatant, which was measured in a UV-Visible spectrum at 407 nm. As a control, 2 ml of PBS (Phosphate Buffer Saline) was added in place of the extract of the above Example. In the case of the experimental group containing various concentrations of the composition, the shrinkage effect was measured by measuring the amount of hemoglobin settled. The higher the sedimentation rate (%) of hemoglobin was, the better the shrinkage effect of pores was judged.

As shown in the above table, there was significant difference in the culture extract of Bacillus cereus containing 1% and 3% of mineral water and the effect of pore contraction was shown.

Experimental Example 9: Effect of inhibiting sebum

The sebum secretion of the skin was measured using a skin oil analyzer (Sebum meter SM810) 4 weeks after the test. The experiment was performed at 22, 40 humidity. The measurement value for oily skin is 220 / cm ² or more, and for normal skin is 100220 / cm ² . Purified water was used as a control, and the experimental results are shown in the table.

As shown in the results of Table 9, compared with the control group, the group treated with Bacillus cereus culture broth containing 1% and 3% of mineral water showed excellent sebum secretion inhibitory effect.

Experimental Example 10: Effect of improving acne

10 male and female acne sufferers were treated with a lotion containing 1% of callus culture extract grown in a medium containing 1% mineral water throughout the face for 4 weeks in the morning and evening I was able to do well. The results of the evaluation of the acne healing effect were evaluated according to the degree of the person who participated in the experiment.

&Lt; Evaluation of state before test &gt;

1 = a little acne 2 = a little acne 3 = acne severe

&Lt; Determination of acne effect &

-: Almost no effect, +: slight effect, ++: Much mitigated, +++: Completely healed

As can be seen from Table 10 above, the improvement effect of acne was confirmed in the majority of subjects after 4 weeks of using a 1% softened lotion containing a 1% of mineral water extract

Preparation of cosmetic composition

Cosmetics of the emulsified form such as nutrient lotion, cream, essence, etc., and cosmetics of the solubilized form such as the softened longevity were prepared with the cosmetics containing the raw green ginger extract of Example 1 of the present invention as an active ingredient.

Production Example 2-1: Lotion

According to the following formulation, it was prepared according to the conventional lotion preparation method.

Production Example 2-2: Essence

According to the following prescription, it was prepared according to a conventional method for producing essence.

Production Example 2-3: Lotion

According to the following formulation, it was prepared according to a conventional lotion preparation method.

Production Example 2-4: Cream

According to the following formulation, it was prepared according to a conventional cream production method.

Production Example 2-5: Gel

According to the following prescription, it was prepared according to a conventional gel preparation method.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (12)

A cosmetic composition comprising a bovine callus extract. The composition of claim 1, wherein the composition is a whitening agent. The composition of claim 1, wherein the composition is anti-wrinkle or anti-wrinkle. The composition of claim 1, wherein the composition is for skin moisturization. The composition of claim 1, wherein the composition is for shrinking pores. The composition of claim 1, wherein the composition is for inhibiting sebum secretion. The composition of claim 1, wherein the composition is for improving acne. The composition according to claim 1, wherein the biotite callus is induced by culturing in a mineral water-containing medium. The composition according to claim 8, wherein the concentration of mineral water in the culture medium is 0.5 to 4% (v / v). A method for producing a bioluminescent callus having a tyrosinase activity inhibiting ability, an antioxidant ability, or a collagen combining ability, which comprises culturing a bioluminescent callus in a medium containing mineral water. The production method according to claim 10, wherein the concentration of mineral water in the culture medium is 0.5 to 4% (v / v). 12. The method according to claim 11, wherein the mineral water-containing medium comprises 0.5 to 3 mg / l of 6-benzylaminopurine, the pH is 6 to 6.5, and the amount of 6-benzylaminopurine is 0.5 to 3 mg / MgSO4.7H2O 0.3-0.5 g / l, NH4H2PO4 0.2-0.4 g / l, KI 0.0005-0.0015 g / l, H3BO3 0.04-0.60 g / L, KNO3 2.3-2.7 g / l, CaCl2 2H2O 0.1-0.3 g / ZnSO4.7H2O 0.0005 to 0.0015 g / l, Na2MoO4.2H2O 0.00005 to 0.00015 g / l, CoCl2.6H2O 0.00005 to 0.00015 g / l, FeSO4.7H2O 0.01 to 0.02 g / l, MnSO4 占 H2O 0.005 to 0.015 g / 0.0 &gt; g / l, &lt; / RTI &gt; Na2EDTA, 0.015 g / l to 0.025 g / l, and 0.5 to 1.5 g / l of inositol.

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