KR101838354B1 - Anti-aging composition for skin external application comprising Camellia japonica Plant Cell Culture Extract and Method for Preparing the Same - Google Patents

Abstract

The present invention relates to a composition for external application for skin containing a camellia plant cell culture or an extract thereof and a method for producing the same, and more particularly, to an anti-aging external preparation for skin comprising a camellia plant cell culture or an extract thereof as an active ingredient, And a manufacturing method thereof.
The camellia plant cell culture according to the present invention or the composition for external application for skin containing the extract of the present invention is excellent in skin aging resistance and skin collagen synthesis ability without any toxicity to skin cells and is effective for improving wrinkles, Skin protection, skin irritation, skin tightening, skin barrier enhancement, and elasticity improvement.

Description

 [0001] The present invention relates to an anti-aging dermatological composition containing an extract of camellia plant cell culture and a method for preparing the same,

The present invention relates to a composition for external application for skin containing a camellia plant cell culture or an extract thereof and a method for producing the same, and more particularly, to an anti-aging external preparation for skin comprising a camellia plant cell culture or an extract thereof as an active ingredient, And a manufacturing method thereof.

Over time, the secretion of various hormones that regulate metabolism is reduced, and the function of immune cells and the activity of cells are lowered, so that the biosynthesis of immune proteins and biologic proteins necessary for the living body is reduced The skin becomes thinner (in the case of endogenous aging) and the elasticity is decreased by intrinsic aging which is caused by external aging and photoaging due to various pollutants and ultraviolet ray externally, Keratinocytes and melanocytes are damaged by UVB. Skin aging is divided into chronologic aging, which occurs in areas that are not exposed to sunlight, and actinic aging, which is a combination of chronologic aging and degenerative changes in the exposed parts of sunlight. In chronological aging, characteristic clinical signs of skin are fine wrinkles, atrophy of dermis, and decrease of subcutaneous fat layer. In the photoaging process, coatse wrinkling and furrowing appear and abnormal elasticity material accumulates and the skin thickens and loosens like leather. The phenomenon seen in chronic sunlight-damaged skin is an abnormal increase in the elastic elastosis and proteoglycan of the upper collagen of the dermis and a significant decrease in collagen, the main protein of the dermis. Collagen gives strength and tension to the skin, which protects the skin from external stimuli or forces, and it accounts for 90% of the dermis. Collagen reduction is closely related to skin and aging. In cells, cellular activity decreases due to endogenous senescence and photoaging, signal transduction system becomes incomplete, biosynthesis of matrix metalloproteinases (MMPs), which degrade skin tissue, is increased, collagen biosynthesis is reduced, and wrinkles are formed and elasticity , And it is known that melanogenesis leading to skin blackening is increased.

On the other hand, the skin barrier (Stratum corneum, skin barrier) consists of dead keratinocyte (intercellular lipid) and intercellular lipids, protects the skin from external stimuli and protects the skin from evaporating moisture It plays a key role in skin health. In other words, it prevents excessive release of water in the body and prevents harmful substances such as chemicals and microorganisms from entering our bodies. It plays an important role in intercellular lipid stability in keratinocyte envelope, which constitutes the surface of dead keratinocytes. The keratinocyte envelope is composed of protein membranes such as involucrin, loricrin, and filaggrin.

The keratinocytes make the skin barrier through the process of differentiation and currency keratinization. Skin barrier function can be aged or destroyed by external factors, and aged skin is easily destroyed by acetone or tape stripping. Damage to the skin barrier can cause skin moisture loss and wrinkles.

In addition, tight junctions consist of one type of cell junction, claudin, tricellulin, junctional adhesion molecule (JAM) and zona occluden (ZO). The tight junction can be divided into a part contained in the cell membrane and an intracellular part. Occludin and claudin are closely related constitutive proteins contained in cell membranes. Occludin is the first transmembrane protein found in adherent synapses and has four transmembrane domains. Occludin is known to regulate the function of tight junctions as well as constituents of tight junctions. Claudin, a close-coupled conformational protein with Occludin, also has four transmembrane domains. The tight junction is a cell-to-cell linkage that fills the gap between neighboring cells, controls the movement of small molecules, controls the permeation of cells between cells, and maintains the polarity of the cells. Recently, it has been reported that tight junctions play an important role in skin barrier function.

Development of various anti-aging cosmetic materials (Patent Documents 1, 2 and 3) for preventing and strengthening skin barrier which plays a key role in skin health is urgently needed.

Patent Document 1: Korean Patent No. 10-0727743 Patent Document 2: Korean Patent Publication No. 10-2014-0016556 Patent Document 3: Korean Patent Publication No. 10-2013-0001842

The present inventors have found that cell culture of the camellia plant or its extract is useful for improving the firmness of the skin and preventing or reducing the visible symptoms of aging such as wrinkles resulting from external and internal causes such as sun exposure and other environmental stresses To prevent skin aging and improve skin aging including improvement of skin wrinkles, improvement of skin wrinkles, strengthening of skin barrier, skin moisturizing, skin convergence, skin soothing, protection of skin due to ultraviolet rays, , And thus, the present invention has been completed.

Accordingly, an object of the present invention is to provide a method for producing a cell culture of a camellia plant having an excellent anti-aging effect.

It is an object of the present invention to provide a skin external composition for skin improvement comprising a cell culture of a camellia plant or an extract thereof as an active ingredient.

It is still another object of the present invention to provide a composition for external application for skin comprising the cell culture of the camellia plant or an extract thereof as an active ingredient.

The present invention relates to an anti-aging dermatological composition and a method for producing the same, which is safe for human body and has an excellent skin aging-improving effect, and a method for producing the same. The present invention relates to a composition for preventing or aging skin aging and improving skin wrinkles, skin barrier enhancement, skin moisturizing, , Acne improvement, skin protection against ultraviolet rays, prevention of skin aging including improvement of skin damage due to ultraviolet rays, improvement of skin aging, that is, an anti-aging effect, or an extract thereof.

In order to achieve the above object, the present invention provides a method for producing a camellia plant cell culture characterized in that a plant cell is isolated from a tissue of Camellia japonica plant and cultured.

In the present invention, it is possible to separate and culture cells from seeds of Camellia sinensis, placenta, ovary or petal.

In the present invention, cells may be isolated from the seeds, leaves, stems, or roots of the camellia and cultured.

In the present invention, the plant cell may be cultured in a culture medium containing a plant growth regulator. In particular, the culture medium may be cultured in a culture medium containing NAA (alpha -naphthalene acetic acid) or 6-BAP (6-Benzylaminopurine) And then culturing the plant cells in a medium containing all of them.

The present invention also provides a method for producing a composition for external application for skin, which comprises culturing a plant cell culture of the camellia japonica plant or an extract thereof.

In the present invention, it may be characterized in that it comprises the step of drying the plant cell culture of the camellia plant, pulverizing it and mixing it with purified water to prepare a composition.

In the present invention, it may be characterized in that it comprises the step of drying the plant cell culture of the camellia plant, pulverizing it, mixing it with purified water, and then subjecting it to ultrasonic extraction or hot water extraction to prepare a composition.

The present invention also provides a skin external composition for skin improvement comprising a cell culture of the camellia japonica plant or an extract thereof.

In the present invention, the skin improving agent may be at least one selected from the group consisting of antioxidants, antioxidants, antioxidants, antioxidants, antioxidants, antioxidants, antioxidants, antioxidants, antioxidants, For skin protection or skin damage improvement according to the present invention, and for skin soothing.

Hereinafter, the present invention will be described more specifically.

Camellia japonica Linne is an evergreen bark belonging to the tea and camellia. Camellia is distributed in about 100 species in Southeast Asia. One species is distributed in the southern part of Korea, and many kinds of horticultural varieties that make many changes such as flower shape, color and leaf are widely planted as ornamental plants. It grows up to 5 ~ 15 meters in height and grows in mountains of Jeju Island and southern part of Korea. The bark is grayish brown to yellowish brown, smooth, smooth and glossy, eggplant light brown with no hairs. Leaves are alternate, oval to long oval, thick leather, pointed end, wavy sawtooth on edge. The front side of the leaf is glossy and the back side is pale green. The petiole is about 5mm long.

The flowering period of camellia is from 12 to the following April, and the fruit is autumn. One red flower blooms on the tip of branch or leaf axil. There are many calyxes and fallen, and there is no peduncle. Five to seven petals spread diagonally and have many stamens. When petals fall on the petals, they fall together. Anther is yellow and style is divided into 3 pieces. Rounded seeds and berries are 2 to 3 cm long, ripen in red in autumn, and split into three halves, with dark brown seeds in them. The seeds are close to the elliptical shape and have a corner on the back (back) of about 2cm in length and 1.5cm in diameter. In the old days, oil made from seeds was called 'camellia oil' and was used as the head oil of the ladies.

It is called 'Camellia japonica Linne for albipetala H.Chang'. The Camellia sasanqua Thunberg, which originated from Japan, is distinguished from the Camellia japonica var. Hortensis Mak. By the fact that the leaf is smaller than the camellia and the underside of the petal is not attached, and the petal is spread almost horizontally.

Other names of camellia are camphor (camphor), camellia (camphor), camphor (camphor), camellia (camphor), camellia Tree, camellia, interpretation, and camellia oil.

The collection time of the camellia buds is sampled from the spring equinox to the bloom (April 20). Generally, when the bud is convex and the flower is about to bloom, collect it and dry it in the sun or put it in a weak fire, wrap it in paper, seal it, keep it in a dry and well ventilated place.

Camellia japonica contains leucoanthocyanin and anthocyanin. Fruit contains oil, camellin, thachysaponin, hydrolyzed thachysaponin, camelliagenin A, B, C, and leaves contain I-epicatechol and d-catechol.

Telomerase is composed of catalytic subunit telomerase reverse transcriptase (TERT) and telomerase RNA component (TERC) and plays an important role in telomere length regulation, aging and tumorigenesis. In addition, it participates in the regulation of DNA repair, chromatin remodeling, and metabolism through various cellular signaling. Indeed, many recent studies have demonstrated that the function of telomerase is regulated by interactions with various genes.

 Most normal human somatic cells become shorter by telomic DNA every time cell division occurs due to the DNA end replication problem. Increasing shortening of telomere is an important mechanism to regulate cell senescence and cellular replication restriction. Telomerase plays an important role in maintaining telomere length, and nucloeytic degradation and end-to-end fusion occur to prevent cell death or recombination.

Above the role of telomerase in maintaining telomere length, several evidences about the physiological function of telomerase have been suggested. For example, when telomerase is expressed, cell survival increases even if cellular stress is induced, and it is known that TERT plays a role independently of telomerase activity in protecting cells from cell death. Therefore, TERT is involved in aging independently of telomere length control, which is a main function of TERT.

In other words, it is known that telomerase, which is a reverse transcription enzyme that maintains telomere length by inducing cell senescence in normal cells, is closely related to the promotion of differentiation and mitotic organization of cells.

In the present invention, in order to understand the possibility as an anti-aging material, it is confirmed that the cambrian plant cell culture or its extract is effective as an anti-aging material through the activity on the promoting activity of collagen production and the indicators related to the differentiation of keratinocytes In addition, we confirmed the action of promoting filaggrin production, which is an indicator of skin cell differentiation, that is, promoting the proliferation and differentiation of keratinocytes, thereby increasing the production of filamentous green cells.

In one aspect, the present invention relates to a method for producing a camellia plant cell culture characterized in that plant cells are isolated from a tissue of Camellia japonica plant and cultured.

In the present invention, a camellia plant is a concept including a camellia tree and a camellia flower. For example, the structure of a camellia plant may be a camellia or a seed of a tree, a camellia of a camellia, an ovary of a camellia, a camellia, Roots and so on.

In the present invention, the cultivation of the plant cells may be performed by separating and culturing the cells from the seeds, laver, ovary or petal of Camellia sinensis.

The placenta of the plant is an important part that regulates seed breeding, such as an animal-derived placenta. It is a part of the ovary in the pistil (ovary, ovary). It is usually on the edge of the carpel, which forms the ovary. It also occurs in the base of the ovary, or in the shape of a column standing upright in the ovary from the base.

In the present invention, the cultivation of the plant cells may be performed by separating and culturing the cells from seeds, leaves, stems, or roots of the camellia.

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 the present invention, the plant cell may be cultured in a culture medium containing a plant growth regulator. In particular, the culture medium may be cultured in a culture medium containing NAA (alpha -naphthalene acetic acid) or 6-BAP (6-Benzylaminopurine) And then culturing the plant cells in a medium containing all of them.

In the present invention, the NAA (alpha -naphthalene acetic acid) may be contained at 1-10 mg / L, preferably 2-8 mg / L, 4-6 mg / L, and the 6-BAP may be 0.01-1 mg / L, preferably 0.05-0.5 mg / L, and 0.08-0.3 mg / L.

In another aspect, the present invention relates to a composition for external application for skin for improving skin comprising Camellia japonica cell culture or an extract thereof as an active ingredient, and a method for producing the same.

In the present invention, the camellia plant cell culture or its extract may be contained in an amount of 0.1 to 10% by weight based on the total weight of the composition. For example, 1%, 0.5%, 5%, and 10% by weight of the present invention are included in the range of 1%, 0.5%, and 5% Skin aging, improvement of aging, improvement of skin barrier, improvement of skin, convergence of skin, improvement of wrinkles, skin moisturizing, skin soothing, antibacterial, improvement of acne, skin due to ultraviolet rays Protection efficacy and the like are obvious to those skilled in the art.

The present invention relates to a method for producing a skin culture improving skin composition containing a cell culture of a camellia plant or an extract thereof, comprising the steps of:

(a) isolating and cultivating plant cells from tissue of a camellia plant; And (b) preparing a culture or a composition containing the extract obtained in the step (a).

In the present invention, the camellia plant cell culture (culture) itself may be used, the culture may be used by filtration, the culture may be used after being crushed, or the culture may be dried and powdered and then dissolved in purified water have.

In the step (a), specifically, a suitable medium may be selected for culturing the camellia plant cells. 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, in the step (b), it is preferable that a composition containing the cambrian plant cell culture obtained as a result of the cultivation in the step (a) is prepared as a suitable external preparation for skin, or the culture is cultured in a known The extract can be obtained in the form of an extract through an extraction method and can be prepared into an appropriate external preparation for skin.

For example, in step (b), the camellia plant cell culture obtained in step (a) is dried, powdered and mixed with purified water to prepare a composition, or the cultured camellia plant cell 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, "extract" means an extract obtained by powdering the camellia plant cell culture extract, or by various known extraction methods such as cold water extraction method, hot water extraction method and ethanol extraction method.

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, cold water (15-25 ° C), for example, is mixed with the above-mentioned placental tissue culture or its dried powder and extracted for 3 days to obtain 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. In the case of extraction using an organic solvent, an organic solvent such as methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, N-dimethylformamide (DMF) (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. As described in the examples, the extract is mixed with 50% ≪ / RTI >

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, the term "anti-aging" refers to a method of improving skin protection and skin condition, skin whitening, prevention or improvement of skin aging and wrinkles, pore contraction and reduction, skin convergence, skin moisturizing, skin soothing, skin protection, Improve skin function, improve skin barrier function, reduce skin irritation, improve skin cell proliferation and regeneration ability, antioxidant ability, ability to promote collagen synthesis, skin protection by ultraviolet rays, skin damage by ultraviolet rays, antibacterial, acne improvement, etc. .

In the present invention, the term "comprising as an active ingredient" means a composition for external application for skin, which is capable of exhibiting a skin improving effect upon antioxidation including skin convergence, skin soothing, skin barrier enhancement, Composition for external application for skin means an effective amount of such an amount as to exhibit collagen synthesis related to the wrinkle-reducing effect, increase in TERT expression, increase in aquaporin-3 expression, skin convergence, wrinkle improvement, skin barrier enhancement and improvement.

In the present invention, the principle of skin astringent action is a phenomenon in which skin is contracted by binding with polymer flavonoids of skin protein to form crosslinks. Convergence means basically wrinkled or funky. Convergent agent has a function to shrink skin and mucosal blood vessels, and has an effect of inhibiting secretion of mucus by interrupting cell gap and lymphatic gap. In addition, since astringent agent has a property of binding to protein, it can generally judge degree of convergence effect according to degree of binding of hemoglobin protein to extract. Such convergent action may be said to have an effect of forming an insoluble film on the surface of the skin and mucous membranes by external application or contents and consequently protecting the locality or densifying the tissue to reduce the permeability of the cell membrane.

The convergence of the skin is divided into two types: chemical convergence and physical forestation. Chemical convergence refers to temporary constriction of sweat glands and pores by substances that coagulate proteins like tannins. On the other hand, physical convergence refers to the temporary shrinkage of sweat glands and pores by using cold water on the skin or by lowering the temperature of the skin due to the volatilization of ethanol. In addition, such skin convergence is caused by summer stress or stress, skin trouble occurs when more sebum occurs, and the skin trouble becomes more and more reddened. Skin relaxation means relaxation of such skin troubles , And is an action distinct from skin convergence.

In the skin barrier protection function according to the present invention, the skin barrier is composed of a stratum corneum, which is the outermost layer of the epidermis, and a corneocyte, which is mainly non-nucleated. The multilamellar lipid layer, composed of intercellular lipids such as ceramides, cholesterol, and fatty acids synthesized by keratinocytes of the skin barrier maintained through normal division and differentiation of epidermal cells, It plays a role. Meanwhile, omega hydroxyceramide in the intercellular lipids is chemically covalently bound to involucrin, which is a protein of the corneocyte outer layer, to form a corneocyte lipid envelope (CLE) Type interstitial lipids physically stabilizes the barrier function of the role is to play a role.

The composition of the present invention is delivered to the stratum corneum through skin application to promote the differentiation of keratinocytes, thereby improving the thickness of the skin layer to be thick, and also has excellent effect of restoring skin barrier damage, so that it can be utilized as a functional cosmetic composition And can be useful for the treatment and prevention of skin diseases caused by damage to the skin barrier. Skin diseases caused by skin barrier damage include, but are not limited to, atopic dermatitis, xeroderma, psoriasis, ichthyosis, acne and the like.

In addition, the mechanism of skin barrier protection was confirmed by increasing the amount of occludin and claudin-1 protein, which are closely related to each other. Filaggrin is one of several structural proteins that keratinocytes express at the differentiation stage. It is responsible for differentiation from the basal layer to the stratum corneum of the epidermis. It is responsible for the natural moisture factor (NMF) , And is used as a main index of moisture retention and skin membrane function of the skin. Camellia plant cell culture according to the present invention has excellent skin barrier protection, enhancement and improvement function as the gene expression of pillar green is greatly increased .

In addition, the cambrian plant cell culture according to the present invention or a composition containing the extract of the present invention has antimicrobial activity, and thus can be used for antibacterial or acne improvement based on antibacterial activity of acne bacteria.

Furthermore, it has been confirmed that the cambrian plant cell culture according to the present invention has a superior protective effect on skin cells upon irradiation with ultraviolet rays, and thus it can be used for skin protection by ultraviolet irradiation.

In other words, the composition for external application for skin according to the present invention can be applied for prevention or improvement of skin aging due to enhancement or protection of skin barrier function, increase of procollagen, improvement of skin wrinkle based on collagen biosynthesis, In addition, the present invention can be applied to protecting skin from photoaging such as ultraviolet rays, improving skin damage, improving acne, calming skin, converging skin, moisturizing skin, and reducing pores.

In the present invention, the composition for external application for skin may be a cosmetic composition or a pharmaceutical composition.

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 skin topical composition of the present invention in an amount of from about 1% by weight to about 99.99% by weight, preferably from 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 composition for external application for skin of the present invention is prepared, and its specific application site (face, neck, etc.) or its preferable application amount and the like, 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 composition for external application for skin according to the present invention may contain glycerin, butylene glycol, propylene glycol, polyoxyethylene hardened castor oil, ethanol, triethanolamine and the like in addition to the camellia plant cell culture extract , Antiseptic, antioxidant, coloring agent, purified water and the like may be included as needed.

The composition for external application for skin according to the present invention can be prepared in various forms such as lotion, essence, gel, emulsion, lotion, cream (underwater type, water type, multiphase), solution, suspension ), Capsules (soft capsules, hard capsules) having a coating such as anhydrous products (oil and glycol), gel, mask, pack, powder or gelatin.

The skin of the present invention includes not only the face but also the scalp and whole body. The composition for external application for skin which can be applied to such scalp includes shampoo, rinse, treatment, hair removal agent, etc., and a body cleanser And the like can be manufactured in various forms.

The method for manufacturing the composition for external application for skin containing the camellia plant cell culture extract according to the present invention is not limited to the above-described production method, and any person skilled in the art may apply the method , It is also possible to prepare a composition for external application for skin containing the cambial plant cell culture extract according to the present invention.

In particular, the composition for external application for skin may be prepared in the form of a general emulsified formulation and a solubilized formulation, by a 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.

In addition, the composition for external application for skin of the present invention may further comprise 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, As used herein means any emulsion or emulsion which is commonly used in emulsifying or emulsifying agents, fillers, sequestering and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or cosmetics Adjuvants commonly used in the cosmetics or dermatological fields, such as other ingredients. And, the above ingredients can be introduced in amounts commonly used in the field of dermatology.

The composition for external application for skin according to the present invention is useful as an anti-aging functional cosmetic composition which can improve wrinkles due to the ability to promote collagen synthesis, prevent or improve skin aging due to excellent TERT expression ability, protect skin barrier function, .

In the present invention, in the case of a pharmaceutical composition, it can function as a pharmaceutical composition for skin condition improvement as shown in the following examples.

Such a pharmaceutical composition may contain, in addition to an effective component, a camellia plant cell culture or an extract thereof, a "pharmaceutically acceptable carrier ", which carrier is a diluent, a lubricant, a binder, a disintegrant, a sweetener, ≪ / RTI > The pharmaceutical composition may further comprise an additive. The additives may include flavoring agents, vitamins, and antioxidants. Examples of the diluent include lactose, dextrin, tapioca starch, corn starch, soybean oil, microcrystalline cellulose or mannitol as a carrier, magnesium stearate or talc as a lubricant, , And the binding agent may be polyvinylpyrrolidone or hydroxypropylcellulose. Examples of the disintegrant include carboxymethylcellulose calcium, sodium starch glycolate, polacrilin potassium, or crospovidone; examples of sweeteners include white sugar, fructose, sorbitol, or aspartame; stabilizers include sodium carboxymethylcellulose, Or xanthan gum, and the preservative may be methyl paraoxybenzoate, propyl p-hydroxybenzoate, or potassium sorbate.

The pharmaceutical composition may be formulated into conventional pharmaceutical formulations known in the art. The pharmaceutical composition may be formulated and administered in the form of an oral administration preparation, an injection preparation, a suppository, a transdermal preparation, and a dosage form. For example, the formulations may be formulated for oral administration, such as solutions, suspensions, powders, granules, tablets, capsules, pills, or expanses.

The camellia plant cell culture according to the present invention or the composition for external application for skin containing the extract of the present invention has an excellent TERT-expressing ability without toxicity to skin cells, thereby preventing or improving skin aging and having excellent skin collagen synthesis ability, It has skin moisturizing, skin soothing, antibacterial, acne improvement, skin protection by ultraviolet rays, skin damage improvement, skin convergence, and skin barrier enhancement ability.

1 is a photograph showing a plant cell culture of a camellia tree according to the present invention.
2 is a photograph showing germination process from a camellia seed according to the present invention.
FIG. 3 is a photograph showing the cell induction process from leaves, stems and roots from the germinated camellia plant according to the present invention.
4 is a photograph showing the induction process of camellia, flower petal and camellia leaf cells from Camellia sinensis according to the present invention.
FIG. 5 shows the skin cytotoxicity test results of the cambial plant cell culture extract according to the present invention.
FIG. 6 shows the results of the test for the effect of TERT expression on the anti-aging-related gene of the cambial plant cell culture extract according to the present invention.
FIG. 7 shows the results of the test for promoting the synthesis of procollagen from the cultured camellia plant cell culture extract according to the present invention.
8 is a graph showing the effect of the Aquaporin-3 protein expression test on the moisture-related gene of the cambrian plant cell culture extract according to the present invention.
9 is a graph showing the results of component analysis by HPLC of cambial cell culture extract of Camellia sinensis.
FIG. 10 is a graph showing the results of component analysis by HPLC of an ovarian cell culture extract derived from Camellia sinensis.
FIG. 11 is a graph showing the results of analysis of components by HPLC of a petiole cell culture extract derived from camellia.
FIG. 12 is a graph showing the results of component analysis by HPLC of a cambium-derived leaf cell culture extract.
13 is a graph showing the results of component analysis by HPLC of a root cell culture extract of Camellia sinensis.

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.

In particular, the following examples have confirmed the efficacy of the cambrian plant cell culture extracts, but it will be apparent to those skilled in the art that such effects are also obtained for the culture itself, not the extract.

1-1. Preparation of cell extracts of camellia according to the present invention

(1) Germination from camellia seeds

In order to sterilize the surface of the camellia seeds, the camellia seeds (Seogwangri, Andeok-myeon, Seogwipo city) were dipped in 70% ethanol for 30 seconds, sterilized with 2% sodium hypochlorite for 5 minutes and sterilized And washed. The germination was induced by sterilization of the camellia seeds on the soil and cultivation at 25 ° C and 70% humidity for 5 weeks (FIG. 2).

(2) Cell separation

2.1 Isolation of placenta cells from camellia

For the cultivation, Camellia sinensis was sterilized with 70% ethanol and 0.5% sodium hypochlorite for 5 minutes and washed several times with sterilized water. Carefully remove the oocyte wall using a tweezers and separate the embryonic stem cells from MS medium containing 3% sucrose, 5 mg α-naphtalene acetic acid (NAA) and 0.1 mg 6-Benzylaminopurine (6-BAP) Cat # M0256) for 2 to 3 weeks.

2.2 Separation of ovary cells from camellia

For the cultivation, Camellia sinensis was sterilized with 70% ethanol and 0.5% sodium hypochlorite for 5 minutes and washed several times with sterilized water. Cells were separated from ovaries using tweezers carefully and cultured on MS medium (Duchefa, Cat # 5) containing 3% Sucrose, 5 mg α-naphtalene acetic acid (NAA) and 0.1 mg 6-Benzylaminopurine M0256) for 2 to 3 weeks.

2.3 Isolation of Flower Petals

For cultivation, camellia petals were sterilized with 70% ethanol and 0.5% sodium hypochlorite for 1 minute and washed several times with sterilized water. Carefully cut with a tweezers and a sharp knife at once, and cut off the MS medium (Duchefa, Cat) containing 3% Sucrose, 5 mg α-naphtalene acetic acid (NAA) and 0.1 mg 6-Benzylaminopurine # M0256) for 2 to 3 weeks.

2.4 Isolation of Leaf Cell from Camellia

For cultivation, camellia leaves were sterilized with 70% ethanol and 0.5% sodium hypochlorite for 1 minute and washed several times with sterile water. Carefully cut with a tweezers and a sharp knife at one time and cut the leaves in MS medium (Duchefa, Cat) containing 3% Sucrose, 5 mg α-naphtalene acetic acid (NAA) and 0.1 mg 6-Benzylaminopurine # M0256) for 2 to 3 weeks.

2.5 Camellia stem (Stem) cell separation

For cultivation, the camellia stem was sterilized with 70% ethanol and 0.5% sodium hypochlorite for 1 minute and washed several times with sterile water. Carefully cut with a tweezers and a sharp knife at once, and cut the stem in MS medium (Duchefa, Cat) containing 3% Sucrose, 5 mg α-naphtalene acetic acid (NAA) and 0.1 mg 6-Benzylaminopurine # M0256) for 2 to 3 weeks.

2.6 Camellia Root Cell Isolation

For the cultivation, the roots of Camellia sinensis were sterilized with 70% ethanol and 0.5% sodium hypochlorite for 1 minute and washed several times with sterilized water. Carefully cut with a tweezers and a sharp knife at once, and cut the MS medium (Duchefa, Cat) containing 3% Sucrose, 5 mg α-naphtalene acetic acid (NAA) and 0.1 mg 6-Benzylaminopurine (6-BAP) # M0256) for 2 to 3 weeks.

FIG. 3 is a photograph showing the cell induction process from leaves, stems and roots from the germinated camellia plant according to the present invention. 4 is a photograph showing the induction process of camellia cells, petal cells, and camelli leaf cells from Camellia sinensis.

(3) Cultivation and mass production of isolated camellia, ovary, petal, leaf, stem and root cells

The aseptically isolated camellia placenta, ovary, petal, leaf, stem and root cells were subcultured in MS medium containing 3% sucrose at intervals of 2 ~ 3 weeks. Afterwards, balloon bioreactor (Samsung Science) (Fig. 1), the culture was grown at a temperature of 25 ° C and an air supply of 0.1 vvm at 14 days intervals in a liquid medium of the same composition except agar.

(4) Production of camelliaceae, ovary, petal, leaf, stem and root cell culture extract

Camellia thunbergii, ovary, petal, leaf, stem and root cells were harvested using a bioreactor. After removing enough moisture with a clean tissue, they were dried in a dryer at 60 ° C for 2 days. 50 g of cultured camellia thunbergii, ovary, petal, leaf, stem and root cell cultures were placed in a container, and 10 L of purified water was added thereto, followed by heating at 80 to 100 ° C for 8 to 48 hours in a hot water distiller to obtain a hot water extract . After extraction, the extracts were filtered with a mesh to remove solid content, and the extracts of Camelliaceae, Ovary, Petal, Leaf, Stem and Root Cells were prepared.

Experimental Example 1: Cytotoxicity Test on skin cells of the composition for external application for skin according to the present invention

Human skin keratinocyte (HaCaT) was used to examine the toxic effects of camellia thuja, ovary, petal, leaf, stem and root cell culture extracts obtained according to Example 1 on skin cells. Human keratinocytes were inoculated into 24-well culture plates at a concentration of 1 × 10 ⁴ cells / ml. The medium was DMEM (Dubelco's Modified Eagle Medium, BRL, USA) containing 10% FBS. After culturing in DMEM containing 10% FBS for 48 hours and culturing by 25 to 30% of the surface area of the culture dish, the composition prepared in Example 1 was replaced with FBS-free DMEM containing 0.1% to 10% Respectively. 50 μl of a solution (2.5 mg / ml) of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT, Sigma M5655, USA) (DMSO, Sigma D2650, USA) was added to each well, and the formazan crystals formed were dissolved by stirring for 20 minutes. 100 μl of each solution was added to 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 equation (1) based on the absorbance of the control group using pure water and expressed as a percentage.

[Equation 1]

Cytotoxic effect (%) = (absorbance upon treatment with extract / absorbance of control) x 100

As shown in FIG. 5, when treated with a plant cell culture extract at a concentration of 1-20%, which is a culture extract of Camelliaceae, Ovary, Ovary, Petal, Leaf, Stem and Root Cell, Viability was not affected.

Experimental Example 2: Test for the expression of anti-aging-related gene TERT in the composition for external application for skin according to the present invention

2.1 Cell culture

Human keratinocyte line (HaCaT) was cultured in 100 mm / 60.1 cm culture dish with Dulbecco's Modified Eagle's Medium (DMEM), 10% Fatal bovine serum (FBS) and 1% Antibiotic-Antimycotic (GIBCO, Cat. # 15240-062) And cultured under the conditions of 37 ° C and 5% CO ₂ . When human keratinocytes are confluent at a concentration of 80% or more, the cells are seeded at 1 × 10 ⁴ cells / well in a 96-well plate. Cell culture is continued until confluence reaches 80% After replacing with free medium (without FBS), treat the test substance. After 24 hours of material addition, real-time PCR method was used to confirm the effect of each substance on the expression of telomerase reverse transcriptase (TERT), and 1 mM shikimic acid was used as a positive control.

2.2 RNA Isolation and Real-Time PCR Analysis

For RNA isolation and cDNA synthesis, use SuperPrep cell lysis & RT Kit for qPCR (TOYOBO Cat. # SCQ-101). The cells were washed once with DPBS, and incubated at room temperature for 5 minutes with 50 μl cell lysis mixture (gDAN remover added to lysis buffer). Stop the reaction by stopping the reaction and store in ice. 8l of the extracted mRNA is taken in 32 RT of RT reaction mixture and PCR is performed to synthesize cDNA. The PCR is performed at 37 캜 for 15 min, 50 캜 for 5 min, and 98 캜 for 5 min. For comparative analysis of gene expression, proceed to the Rotor Gene Q Real-time PCR Machine (Qiagen) according to the manual of Thunderbird SYBR qPCR Mix (TOYOBO Cat. # QPS-201) using the synthesized cDNA as a template. The primers used in the experiments were Qiagen's QuantiTectprimerassays (Telomerase reverse transcriptase (TERT) Cat. # QT00073409). The relative incidence was normalized by Housekeeping gene (GAPDH, Cat. # QT01192646). Real-time PCR conditions are as follows.

<Real-time cycler conditions>
Step Time Temperature Reverse transcription 30 min 50 PCR initial activation step 15 min 95 3-step cycling Denaturation 15 s 94 Annealing 30 s 60 Extension 30 s 72 Number of cycles 45

As shown in FIG. 6, when the camellia plant cell culture extract obtained according to Example 1 was examined for the evaluation of cell survival efficacy through expression of anti-senescence-related gene TERT (Telomerase Reverse Transcriptase) The plant cell culture extract had excellent TERT expression ability, and especially the expression level of 1 mM Shikimic acid, which was used as a positive control in 5% concentration of Placenta, Ovary, Flower and 1% Leaf, was confirmed.

Experimental Example 3: Procollagen synthesis enhancement test of the composition for external application for skin according to the present invention

Human fibroblasts (Human Skin Fibroblast) for 37 ℃, 5% in a CO ₂ incubator of 10% in the cell culture medium to maintain a constant humidity FBS, Penicillin (50U / ml) , Streptomycin DMEM (Dulbecco's containing (50 / ml) The cells were cultured in a modified Eagle's medium (Gibco, USA) at a concentration of 1 × 10 ⁵ cells / ml in a 48-well plate. The control and the culture medium containing the camellia plant cell culture extract obtained according to Example 1 as the test substance were added and cultured in a 5% CO ₂ incubator at 37 ° C for 48 hours. After 48 hours, the amount of newly synthesized collagen was measured by measuring 20 μl of each supernatant of the culture medium and measuring it using Procollagen Type I C-Peptide EIA Kit (PICP, Takara, Cat No. MK101). The amount of PICP was converted to ng / 1 × 10 ⁵ cells and calculated according to the following equation (2).

&Quot; (2) &quot;

As shown in FIG. 7, the amount of procollagen produced in the synthesis of collagen by the treatment of camellia seedlings, ovary, root, petal and leaf cell culture extract was measured. As a result, the plant cell culture extract of camelliaceae leaf 10, 20ppm, The effect of 10 ppm of extract was significantly higher than that of plant cell culture extracts derived from other sites, indicating a significant effect of promoting collagen synthesis.

Experimental Example 4: Skin barrier function test of the composition for external application for skin according to the present invention

4.1 Cell culture

When the human epidermal keratinocytes were confluent at a concentration of 80% or more, they were divided into 96 well plates at a density of 1 × 10 ⁴ cells / well, and then cultured in the same manner as in Example 1 except that the camellia seedlings, ovary, And the expression of filaggrin gene was confirmed by real-time PCR.

4.2 RNA Isolation and Real-Time PCR Analysis

 For RNA isolation and cDNA synthesis, use SuperPrep cell lysis & RT Kit for qPCR (TOYOBO Cat. # SCQ-101). The cells were washed once with DPBS, and incubated at room temperature for 5 minutes with 50 μl cell lysis mixture (gDAN remover added to lysis buffer). Stop the reaction by stopping the reaction and store in ice. 8l of the extracted mRNA is taken in 32 RT of RT reaction mixture and PCR is performed to synthesize cDNA. The PCR is performed at 37 캜 for 15 min, 50 캜 for 5 min, and 98 캜 for 5 min. For comparative analysis of gene expression, proceed to the Rotor Gene Q Real-time PCR Machine (Qiagen) according to the manual of Thunderbird SYBR qPCR Mix (TOYOBO Cat. # QPS-201) using the synthesized cDNA as a template. The primers used in the experiments were Qiagen QuantiTectprimerassays (Filaggrin Cat. # QT01192646). The relative incidence was normalized by Housekeeping gene (GAPDH, Cat. # QT01192646). Real-time PCR conditions are as follows.

<Real-time cycler conditions>
Step Time Temperature Reverse transcription 30 min 50 PCR initial activation step 15 min 95 3-step cycling Denaturation 15 s 94 Annealing 30 s 60 Extension 30 s 72 Number of cycles 45

Treatment concentration part Filaggrin gene expression rate
(Relative Expression of Filaggrin)  Negative control group - 1.00 One % Taurus 1.52 Ovary 1.38 petal 0.98 leaf 1.09 stem 1.08 Root 1.14 2.5% Taurus 3.38 Ovary 2.89 petal 1.20 leaf 2.01 stem 1.38 Root 1.91 5.0% Taurus 2.21 Ovary 1.50 petal 0.95 leaf 1.51 stem 1.05 Root 1.28

It can be confirmed that the skin barrier function can be improved by promoting normal differentiation of keratinocytes in skin cells and promoting expression of filaggrin in skin converted into natural moisturizing factor in skin. Thus, the expression of the filaggrin gene of the camellia, ovary, petal, leaf, stem and root cell culture extracts derived from the camellia according to Example 1 was measured. As shown in Table 3, And it was confirmed that the amount of gene expression was significantly higher than that of the extracts derived from other parts of the cambium - derived cells and oocyte - derived cells.

Experimental Example 5: Effect of moisturizing related gene Aquaporin-3 protein expression test on the composition for external application for skin according to the present invention

HaCaT (human keratinocyte) was incubated in 100 mm / 60.1 cm culture dish with Dulbecco's Modified Eagle's Medium (DMEM), 10% Fatal bovine serum (FBS) and 1% Antibiotic-Antimycotic (GIBCO, Cat. # 15240-062) , And 5% CO ₂ . When HaCaT was confluent at 90% confluence, 1 × 10 ⁵ cells / 12-well plate was dispensed in a 100 mm / 60.1 cm culture dish and cultured for 24 hours. The test material, a culture extract of the camellia plant cell obtained according to Example 1, . After incubation for 4 hrs, the cells were lysed in 0.1% SDS, 1% NP40, 150 mM NaCl, 0.5% sodium deoxycholate, 50 mM Tris-Cl (pH 7.5) and protease inhibitors (Roche, Mannheim, Germany) And the protein is quantitated by BCA assay. An aliquot of the quantified protein is loaded on SDS-polyacrylamide gel, separated by electrophoresis, and transferred to NC membrane. The membranes were blocked with blocking solution (5% skim milk in TBST (Tris-buffered saline with 0.1% Tween 20) for 30 min, washed with TBST and incubated with primary antibody (AQP3, Santa Cruz, Cat. # Sc- solution at 4 ° C overnight. Thereafter, the cells were washed three times for 10 minutes with TBST and reacted with primary antibody conjugated secondary antibody (AQP3: goat) for 2 hours. The completed blot is analyzed by color development with an ECL solution kit (Amersham, UK).

As shown in FIG. 8, the moisturizing effect of the Aquaporin-3 protein expressed by the moisturizing effect was confirmed. As a result, it was found that the camphor flower petal, leaf, When the cultured cells were applied to skin cells, the best moisturizing effect was confirmed by the expression of Aquaporin 3.

Experimental Example 6: Skin Convergence Effect Test of the composition for external application for skin according to the present invention

Astringency is defined as a binding reaction in Latin derived from as (to) and stringere (bind) in Latin. Convergence in the skin refers to the phenomenon that skin proteins contract with the polymer flavonoids to form a crosslink, causing the skin to contract. Commonly known as astringents are organic acid metal salts, inorganic metal salts and alcohols, and metal salts and inorganic metal salts are not suitable for cosmetic applications because they cause skin toxicity or skin diseases. Ethanol and isoflopanol are used as the astringent agents in alcohol, but when the use of ethanol is more than 20%, it can cause serious skin irritation. Therefore, there is a growing interest in convergence of natural products. Convergence of the skin to the skin is divided into two types: chemical convergence and physical convergence. The chemical convergence is inhibited by excessive substances such as tannin, Refers to the temporary contracting of pores, whereas physical convergence refers to the temporary shrinkage of sweat glands and pores by using cold water on the skin or by lowering the skin temperature by volatilization of ethanol. In cosmetics, astringents function to shrink the enlarged pores in the face area, and are used to temporarily tighten the skin by giving elasticity and pulling to give sensual satisfaction and make-up function. In addition to possessing moisture and moisturizing ingredients in the stratum corneum, it has convergence action and suppression of sebum secretion, has a refreshing feeling of use, and has a function of preventing the erasure of make-up.

In order to measure the skin convergence effect of the cambial plant cell culture extract obtained according to Example 1, blood protein (hemoglobin) similar to skin protein was used. Each sample solution and hemoglobin solution (500ppm) were mixed in a ratio of 1: 1, shaken, and centrifuged at 1,500 rpm for 3 minutes, and the absorbance was measured at 576 nm. The measurement of convergence effect was expressed by the absorbance reduction rate of the sample solution addition group and the no addition group.

&Quot; (3) &quot;

Concentration effect (%) = (1-absorbance of sample-added group / absorbance of no-addition group) x 100

Treatment concentration part Measuring Convergence Effect
(Astringent activity test)
(% of control)  Negative control group - 0 Tannic acid
(500 ppm) 37.9 One % Taurus 32 Ovary 11 petal 28 leaf 12 stem 3 Root 13 2.5% Taurus 28 Ovary 8 petal 21 leaf 6 stem 4 Root 11 5.0% Taurus 25 Ovary 17 petal 19 leaf 8 stem 9 Root 10

As shown in Table 4, the astringent effect of the cambium-derived turmeric, ovary, petal, leaf, stem and root cell culture extracts according to Example 1 was measured. As a result, It was confirmed that the convergence effect of the petal part was superior to other ovary, leaf, stem and root.

Experimental Example 7: Measurement of antimicrobial effect of the external preparation for skin according to the present invention

The antimicrobial effect of acicular, ovary, petal, leaf and root cell extracts obtained from the camellia according to Example 1 and triclosan, a chemical synthetic antibacterial agent, was compared.

The antimicrobial activity test against acne bacteria ( Propionibacterium acnes ATCC 6919) was carried out by liquid dilution method. Each sample (the camellia plant cell culture extract prepared in Example 1) was diluted to a concentration of 0 to 5% at 1% intervals in 2 ml of a liquid BHI medium, 20 μl of 1 × 10 ⁶ / ml of the bacterial solution was inoculated, Lt; / RTI &gt; The minimum inhibitory concentration (MIC) was defined as the lowest growth inhibitory concentration based on turbidity.

part Minimum growth inhibitory concentration (%)
Propionibacterium acnes ATCC 6919 One Taurus One 2 Ovary 3 3 petal 2 4 leaf 2 5 stem 5 6 Root 3

As shown in Table 5, it was found that the extracts of camellia-derived turmeric, ovary, petal, leaf, stem and root cell cultures had antimicrobial activity against acne bacteria.

Experimental Example 8: Skin soothing effect of the composition for external application for skin according to the present invention

After dividing the group into 30 individuals for evaluation group with severe skin troubles, the cell culture extracts of 1% camellia-derived turmeric, ovary, petal, leaf, stem and root plant obtained according to Example 1 were given to each group for 2 weeks After the product was used once a day, the skin trouble-reducing effect and the skin soothing effect after the evaluation and the two weeks after use were evaluated, and the average was calculated to evaluate the skin soothing effect of each test substance.

Criteria for evaluation of skin condition grade 1. Skin
Skin problems such as acne, rashes and atopy are getting worse
Skin problems such as acne, rashes, atopy are slightly worse
Acne, rash, atopy and skin problems are the same as before
Some minor skin problems such as acne, rashes, and atopy
Skin problems such as acne, rashes and atopy are greatly improved
One
2
3
4
5 2. Skin soothing effect
Dryness, itching, and stinging are very sensitive to skin conditions.
Sensation of dryness, itching, stinging, etc.
Dryness, itching, and stinging are the same as before use
Dryness, itching, and stinging are a little calming.
Dryness, itching, burning, and soothing skin conditions
One
2
3
4
5

Camellia-derived site Skin Trouble Relief Skin soothing effect Control group (purified water) 1.9 2.0 Tracheal cell culture extract 3.7 4.2 Ovary cell culture extract 3.9 4.0 Petal cell culture extract 4.3 4.5 Leaf cell culture extract 3.8 4.2 Stem cell culture extract 3.5 2.9 Root cell culture extract 4.8 5.0

As can be seen from the above Table 7, according to the present invention, 1% of the camellia, ovary, petal, leaf, stalk, and root cell culture extracts were more effective than the pure water only for skin trouble relief, skin soothing, It was confirmed that the effects were all improved.

EXPERIMENTAL EXAMPLE 9: Protecting skin cells by ultraviolet rays of the composition for external application for skin according to the present invention

Ultraviolet Cross-Linker CL-1000 (Ultra-Violet Products, CA), which emits ultraviolet rays at a wavelength of 302 nm, was used as a light source for UV irradiation. HaCaT cells were cultured in a 24-well plate at a concentration of 1 × 10 ⁵ cells / ml for 24 hours. Then, the medium was removed and washed with phosphate buffer solution. 500 ml of phosphate buffer solution was added, and ultraviolet rays were irradiated at 10 mJ / cm &lt; ² &gt; and then replaced with a medium not containing FBS. The sample of the cambrian plant cell culture extract obtained according to Example 1 was treated for each concentration, Respectively. After 5 mg / ml of MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, Sigma) was added and incubated for 3 hours in an incubator, the formazan formed was dissolved in DMSO, After transferring to a 96-well plate, absorbance was measured with an ELISA reader at 565 nm.

UV irradiation Camellia part
Plant cell Cell Viability (% of Control) Treatment concentration
0.5% Treatment concentration
One % - Control group 100 100 + Control group 50 52 + Taurus 67 89 Ovary 68 78 petal 65 72 leaf 70 75 Root 62 68 stem 55 57

In order to examine the protective effect of keratinocyte cytotoxic effect of 0.5% and 1% camellia trees according to Example 1 on ultraviolet light of the cell culture extracts of the plant tissues of turf, ovary, leaves, roots and stem cells, As shown, excellent cytoprotective effect against ultraviolet irradiation was confirmed as compared with the control group.

Experimental Example 10: Analysis of Components of Camellia thunbergii, Ovary, Petal, Leaf and Root Cell Extracts by HPLC

Component analysis was carried out by HPLC of the camellia-derived turmeric, ovary, petal, leaf and root cell extracts obtained according to Example 1. The analysis conditions are as follows.

division Analysis condition HPLC device type Waters 1525 Binary HPLC Pump Column type Gemini 5 C18 110 A (Phenomenex) Column Specification 250 * 4.60 mm, 5 micron Solvent conditions - Solvent A: Water (containing 0.1% TFA (Trifluoro acetic acid))
- Solvent B: Acetonitrile (containing 0.1% TFA (Trifluoro acetic acid)) wavelength 245 nm (UV lamp) Flow rate 1 ml / min

As a result, the component analysis results of Figs. 9 to 13 were obtained.

Preparation of cosmetic composition

Cosmetic products of an emulsified formulation such as nutritional lotion, cream, essence and the like and solubilized formulation such as softening water were prepared with the cosmetic product containing the camellia plant cell culture 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.

Raw material name Weight% (w / w) glycerin 5.0 Dipropylene glycol 3.0 Hyaluronic acid 0.5 Polyoxyethylene hardened castor oil 0.1 Polyethylene oleyl ethyl 0.1 ethanol 5.0 antiseptic 0.15 Beauty Suitable amount flash Suitable amount The camellia plant cell culture extract of Example 1 2.0 Purified water to 100

Production Example 2-2: Essence

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

Raw material name Weight% (w / w) Cetostearyl alcohol 1.0 Self emulsifying monostearic acid 1.0 Wax 0.5 Squalane 5.0 Isocetyl octanoate 3.0 Dimethylsiloxane 0.3 Sorbitan monostearate 0.5 Polyethylene glycol monostearate 8.0 glycerin 4.0 Propylene glycol 0.2 Carboxy polymer 0.22 Triethanolamine 0.25 antiseptic Suitable amount Spices Suitable amount flash Suitable amount The camellia plant cell culture extract of Example 1 7.0 Purified water to 100

Production Example 2-3: Lotion

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

Raw material name Weight% (w / w) Cetostearyl alcohol 0.8 Self emulsifying monostearic acid 1.0 Wax 0.5 Stearic acid 0.5 Liquid paraffin 7.0 Squalane 5.0 Macadamia oil 3.0 Isocetyl octanoate 2.0 Dimethylsiloxane 0.3 Sorbitan monostearate 0.5 Polyethylene glycol monostearate 1.2 glycerin 4.0 Propylene glycol 4.0 Betaine 4.0 Carboxy polymer 0.12 Triethanolamine 0.15 antiseptic 0.25 Spices Suitable amount flash Suitable amount The camellia plant cell culture extract of Example 1 5.0 Purified water to 100

Production Example 2-4: Cream

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

Raw material name Weight% (w / w) Cetostearyl alcohol 3.0 Self emulsifying monostearic acid 1.5 Chin type monostearate 1.5 Wax 0.5 Liquid paraffin 8.0 Squalane 7.0 Isocetyl octanoate 4.0 Refined jojoba oil 4.0 Dimethylsiloxane 0.3 Sorbitan monostearate 1.0 Polyethylene glycol monostearate 1.2 glycerin 6.0 Propylene glycol 4.0 Betaine 4.0 Xanthan gum 0.06 Triethanolamine 0.10 antiseptic 0.25 Spices Suitable amount flash Suitable amount The camellia plant cell culture extract of Example 1 7.0 Purified water to 100

Production Example 2-5: Gel

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

Raw material name Weight% (w / w) glycerin 4.0 Propylene glycol 4.0 ethanol 10 Polyoxyethylene hardened castor oil 0.1 Carboxy polymer 0.30 Triethanolamine 0.30 antiseptic Suitable amount Spices Suitable amount flash Suitable amount The camellia plant cell culture extract of Example 1 1.0 Purified water to 100

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 (17)

delete A method for producing a camellia plant cell culture, which comprises culturing a plant cell from a tissue of a Camellia japonica plant and culturing the plant, wherein the cultured camellia plant cell culture is dried and pulverized and mixed with purified water to prepare a composition The method of manufacturing a composition for external application for skin according to claim 1,
The texture of the camellia plant is the placenta of camellias,
Wherein plant cells are cultured in a culture medium containing NAA (alpha -naphthalene acetic acid) and 6-BAP (6-benzylaminopurine) when the plant cells are separated and cultured. A method for producing a camellia plant cell culture, which comprises culturing a plant cell from a tissue of a Camellia japonica plant and culturing the plant cell, Or hydrothermal extraction to prepare a composition for external application for skin,
The texture of the camellia plant is the placenta of camellias,
Wherein plant cells are cultured in a culture medium containing NAA (alpha -naphthalene acetic acid) and 6-BAP (6-benzylaminopurine) when the plant cells are separated and cultured. delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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