KR102108916B1 - Cosmetic Composition for Improving Skin Condition Comprising Plant cell complex cultures to improve skin radiance and vitality - Google Patents

Abstract

The present invention relates to a cosmetic composition for skin improvement containing a plant cell culture extract / filtrate as a complex, and more specifically, Edelweiss callus culture or its extract, seaside erringo callus culture or its extract, and rock saem Fire callus culture, or extract, apple cell culture or its extract, grape cell culture or its extract, desert rose leaf cell extract, lavender leaf cell extract, Chinese chaste tree leaf cell extract, lotus leaf cell culture powder, myrrh By containing any one or more of leaf cell extracts and arabian jasmine leaf cell extracts as an active ingredient, the skin is revitalized, skin vitality is improved, skin wrinkles are prevented or improved, skin elasticity is improved, improvement, skin convergence, skin barrier function protection, Regarding cosmetic composition having functions such as skin protection from UV rays, atopy, anti-inflammatory, antioxidant, and anti-aging A.

Description

Cosmetic Composition for Improving Skin Condition Comprising Plant cell complex cultures to improve skin radiance and vitality}

The present invention relates to a cosmetic composition for skin improvement containing a plant cell complex culture or an extract thereof, and more specifically, an Edelweiss callus culture or an extract thereof, a seaside ergo callus culture or an extract thereof, and rock samfire callus Culture or extract, apple cell culture or extract, grape cell culture or extract, desert rose leaf cell culture or extract, lavender leaf cell culture or extract, Chinese chaste tree leaf cell culture or extract, By containing lotus leaf cell cultures or extracts, myrrh leaf cell cultures or extracts, arabian jasmine leaf cell cultures or extracts as active ingredients, it gives skin vitality, increases skin vitality, prevents or improves skin wrinkles, promotes and improves skin elasticity , Skin convergence, skin barrier function protection, skin protection from UV rays, antioxidant, anti-aging, etc. It relates to a cosmetic composition with the function.

Recent studies have identified a molecular network of biological clocks that governs circadian rhythms. As the biological clock that the expression of genes related to physiological metabolism and body rhythms of organisms are regulated in accordance with the day and night cycle that occurs as the Earth rotates has been gradually revealed in detail, research on disease related to the cycle has been actively conducted. Professor Jeffrey C. Hall of the University of Maine in the United States, Professor of the University of Brandice at Michael Rosbash, and Professor Michael W. Young of the University of Rockefeller have a 24-hour day and night cycle through the model animal Drosophila. In recognition of his achievements in identifying the mechanism of action of the appearing circadian genes, he was honored with the 2017 Nobel Prize. The winners discovered the related genes in the 1980s and discovered the basic mechanism of the biological clock in the 1980s, after the existence of the gene related to the biological clock was first confirmed. In the molecular network of these bioclock genes, there are clock genes that have developed while adapting to changes in day and night. Clock genes called 'Clock (Clock circadian regulator)' and 'PER1 (Period circadian protein)' are present in organs such as the heart, lungs, fats, blood vessels and kidneys, as well as brain tissue such as the cerebellum, midbrain and hypothalamus. .

Thanks to their research, it was found that the biological cycle is a biological phenomenon that occurs when the concentration of the protein (PER) expressed by the gene called period is changed in a 24-hour cycle. As the PER protein produced by the pyriod gene increases and continues to accumulate in the cytoplasm outside the cell nucleus, the PER protein now binds to other enzymes and enters the cell nucleus to suppress the expression of the pyriod gene. Thus, the cycle of the livelihood clock appears to be a molecular vibration that increases and decreases the concentration of PER protein. Later, as other genes (proteins) that regulate the stability and functionality of these basic mechanisms were discovered one after another, the mechanism of the 'bioclock', which is precisely and precisely controlled, became known. At this stage, the dimer of CLOCK and BMAL1 proteins triggers the expression of periodic (PER) and Cryptochrome (CRY) family of feedback genes located in the lower stages as transcription factors, and the gene products inhibit the higher stage transcription factors. It forms a series of molecular cycles that activate. These molecular networks are known to influence various physiological processes by triggering the circadianity of genes that are regulated below (Stratmann and Schibler, 2006, J Biol Rhythms 21: 494). That is, the bioclock is operated by adjusting the rhythm of the living body to be repeated every day through the associated feedback of the CLOCK / PER protein.

Independent bio-clock systems exist for skin cells such as keratinocytes, melanocytes, and fibroblasts. The skin biological clock regulates skin physiological activity. Skin cell proliferation and differentiation, hydration and transepidermal water loss (TEWL), capillary blood flow, sebum production, temperature, surface pH, wrinkle formation are circadian rhythms. Looks like When the rhythm of this process is broken, the homeostasis of the skin is broken, the skin aging is promoted, and skin cancer can be seriously caused while the physiological reaction required at the correct time cannot be caused.

Human skin undergoes changes by various internal and external factors as it ages. Internally, the secretion of various hormones that regulate metabolism decreases, and the function of immune cells and the activity of cells decreases, thereby reducing the biosynthesis of immune proteins and bio-constitutive proteins necessary for living organisms. Externally, due to the destruction of the ozone layer, the content of ultraviolet rays reaching the surface of the sunlight increases and the free radicals and free radicals, etc. increase as the environmental pollution becomes more severe, thereby reducing the thickness of the skin, increasing wrinkles, Not only does the elasticity decrease, but skin troubles often occur, causing various changes.

When the skin aging phenomenon progresses, keratinocytes and fibroblasts have dividing, collagen synthesis decreases, MMP production increases, melanin production signal transmission increases, and thus wrinkles increase and skin elasticity decreases. The spots, freckles and blotch will increase. In particular, the hormonal balance is broken around the forties, and the cell regeneration ability, collagen synthesis ability, and moisture content of the stratum corneum, which is important for skin moisturization, are significantly reduced. It is suggested that the stratum corneum lipid component decreases, the natural moisturizing factor decreases, and the dry skin clinical glycerol amount decreases. Eventually, in aging skin, the amount of water supplied from the lower epidermis to the stratum corneum is insufficient, resulting in severe skin dryness, which causes inflammation and pruritus to occur easily. On the other hand, the wrinkles of the skin are due to the imbalance of collagen synthesis and degradation, and in general, the synthesis of type I collagen in the skin and its degrading enzyme MMP-1 are in balance. However, in photo-aged skin, the synthesis of type I and III collagen decreases and the activity of MMP-1 increases. These matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade extracellular matrix and are known to be involved in wound healing or tissue regeneration in normal conditions.

The present inventors, as a result of diligent efforts to discover materials having various functionalities such as providing skin vitality, enhancing skin vitality, improving skin wrinkles, regenerating skin, enhancing skin barrier function, and improving wrinkles, resulted in Edelweiss callus culture or its extract, seaside Lingo callus culture or extract thereof, Roxemfire callus culture, or extract, apple cell culture or extract, grape cell culture or extract, desert rose leaf cell extract, lavender leaf cell extract, Chinese yeast tree leaf cell Extract, lotus leaf cell culture powder, myrrh leaf cell extract, Arabian Jasmine leaf cell extract revealed that it has a biological clock control effect, confirmed that it can be a variety of functional cosmetic materials, and completed the present invention.

Accordingly, the object of the present invention is Edelweiss callus culture or its extract, seaside ergo callus culture or its extract, Roxamfire callus culture, or extract, apple cell culture or its extract, grape cell culture or its extract , Desert rose leaf cell extract, lavender leaf cell extract, Chinese chaste tree leaf cell extract, lotus leaf cell culture powder, myrrh leaf cell extract, arabian jasmine leaf cell extract It is to provide a composition.

Another object of the present invention, the edelweiss callus culture or its extract, seaside ergo callus culture or its extract, Roxemfire callus culture, or extract, apple cell culture or its extract, grape cell culture or the same Extract, Desert rose leaf cell extract, Lavender leaf cell extract, Chinese chaste tree leaf cell extract, Lotus leaf cell culture powder, Myrrh leaf cell extract, Arabian jasmine leaf cell extract It is to provide a method for producing a cosmetic composition.

In order to solve the above problems, the present invention is edelweiss callus culture or its extract, seaside ergo callus culture or its extract, Roxemfire callus culture, or extract, apple cell culture or its extract, grape cell culture or One or more of the extract, desert rose leaf cell extract, lavender leaf cell extract, Chinese chaste tree leaf cell extract, lotus leaf cell culture powder, myrrh leaf cell extract, arabian jasmine leaf cell extract, and a method of manufacturing the extract, It provides a cosmetic composition containing.

Hereinafter, the present invention will be described in more detail.

In one aspect, the present invention, Edelweiss callus culture or its extract, seaside ergo callus culture or its extract, Roxemfire callus culture, or extract, apple cell culture or its extract, grape cell culture or its extract , Desert rose leaf cell extract, lavender leaf cell extract, Chinese chaste tree leaf cell extract, lotus leaf cell culture powder, myrrh leaf cell extract, arabian jasmine leaf cell extract It relates to a composition. Specifically, the callus culture or its extract is not limited in its content, but may be contained in an amount of 0.1 to 10% by weight based on the total weight of the composition.

In the present invention, the description of each plant of each plant cell as an active ingredient is as follows.

(1) Edelweiss Callus Culture Extract, Leontopodium Alpinum Callus Culture Extract, 高山 火 草 （LEONTOPODIUM ALPINUM）

Edelweiss is a plant belonging to the family Asteraceae and inhabits alpine regions of Europe and Asia, including the Alps. It is 10-20cm in height and is covered with white cotton wool as a whole. The leaves are relatively much from the root, slightly on the stem, and linear. At the end of the stem, guns gather and run, spreading in all directions, with a slight heading in the center.

(2) Seaside erringo callus culture filtrate, Eryngium Maritimum Callus Culture Filtrate, ERYNGIUM MARITIMUM

The Eryngium of the Seaside Eringo genus is derived from the name given to a species of the genus, the old Greek name 'eryggion'. It is a 2 or 2 year plant that grows to about 60cm in height. The upper branch splits a lot. The lower leaf has a heart shape, divided into 3 to 5 pieces, and has a needle bed. The upper leaves have no leaf disease and cover the stem. There are about 6 guns, and a slightly wide oval shape with 2 ~ 3 ferrules on both sides. It becomes a bed at the end. Flowers bloom from summer to autumn, and inflorescences are round and have a metallic light purple color.

(3) Crithmum Maritimum Callus Culture Filtrate

Rock samfire is an edible wild plant belonging to the Apiaceae family, and is a special vegetation plant that inhabits rocky rocks of clean beaches in Europe. It is an extreme plant that has resistance to spray, high salinity, temperature and wind. The flower grows gathered at the end of the stem and is green-yellow in size and has five petals. When the plant is broken, you can smell the fragrant lemon. It has been used for medicinal purposes as well as ingredients for centuries.It is said that drinking seeds, roots, and leaves in wine can help with jaundice and urinary tract pain.It is also effective in scurvy due to lack of vitamin C. I also took it on.

(4) Apple cell culture extract, Malus Domestica Fruit Cell Culture Extract, MALUS DOMESTICA

The fruit of the dicotyledonous rosacea, a deciduous arboreous plant, has a round shape with a diameter of about 5 ~, and the color is usually red or yellow. It is known as the Balkan Peninsula. It is an alkaline food and contains many calories and good for your body. Dietary fiber prevents arteriosclerosis by sending harmful cholesterol accumulated in blood vessels out of the body and increasing beneficial cholesterol.

(5) Vitis Vinifera (Grape) Fruit Cell Extract, 葡萄 (VITIS VINIFERA)

Grapes are fleshy and juicy, with berries inside, ripening in black in July-August. The country of origin is known as the Black Sea coast and Kafka region in western Asia, and there are records in Korea about it in the Goryeo Dynasty.

(6) Desert Rose Leaf Cell Extract, Adenium Obesum Leaf Cell Extract, ADENIUM OBESUM

It is a dicotyledonous evergreen tree and is one of the succulent plants found in desert climates such as tropical Africa and Arabia. The name of the flower originates from Aden in Yemen, one of the native regions, and is said to be 'Desert Rose' because the flower is as pretty as a rose.

(7) Lavandula Angustifolia (Lavender) Leaf Cell Extract, 薰衣草 (LAVANDULA ANGUSTIFOLIA)

The name Lavandula comes from the Latin word for "wash," because the Romans liked to add lavender to the water when bathing or washing. It is a perennial plant of the family Lamiaceae, in which purple flowers of star-shaped flowers bloom on the stalks, producing essential oils with an oily scent emanating from the hairs covering the flowers, leaves, and stems.

(8) Chinese Chest Tree Leaf Cell Extract, Vitex Negundo Leaf Cell Extract, VITEX NEGUNDO

This is a shrub from southern Europe, and the leaves are lush in the shape of hairs, and fragrant pale blue-purple flowers bloom.

(9) Lotus leaf cell culture powder, Nelumbo Nucifera Leaf Cell Culture Powder, NELUMBO NUCIFERA

Kites are native to southern Asia and northern Australia. It is a clean and noble plant that grows in the mud and has been a friendly plant for people from many countries. The rhizome is thick and stretches to the side, has a lot of nodes, and especially in autumn, the tip is thick. Flowers bloom in July-May, red or white, run one at the end of the stalk, 15 ~ in diameter, with thorns on the stalk. The petals are upside down, and there are several stamens. The calyx is large and flat, about 10cm in diameter, and the fruits are nuts. The seed is in a hole in the calyx.

(10) Myrrha Leaf Cell Extract, COMMIPHORA MYRRHA

Myrrh is also called myrrha. It is a pale yellow or dark brown large and small lump substance. Myrrh is a medicinal herb made from the myrrh of the olive tree family or a rubber resin obtained from Hapji.

(11) Arabian Jasmine Leaf Cell Extract, Jasminum Sambac (Jasmine) Leaf Cell Extract, JASMINUM SAMBAC

India is native to China, Taiwan, the Philippines, India and Indonesia, and is mainly distributed in tropical and subtropical regions. The leaves face each other or turn one by one and are oval or broad egg-shaped. The edges are flat, pointed, and shiny.

In the present invention, each of the samples may be included in the composition of the present invention in the form of plant cell culture, extract, filtrate, and the like.

 In the present invention, 'callus (callus)' is an oily tissue in which a cell is damaged when the plant is wounded, the cell recovers its fission ability, and the wound is enlarged, or the tissue cut from the plant is cultured in a medium containing auxin, Refers to an undifferentiated tissue or cell mass formed when a plant is wounded or a wound is treated with auxin, and can be permanently divided and propagated by subculturing in a new medium. The callus is an amorphous tissue or cell mass that has lost the ability to produce normal organ formation or tissue differentiation, and is mostly composed of flow cells. In a broad sense, it also includes plant tumor tissues caused by infections such as agrobacterium. That is, when cells in a plant proliferate, they are immediately orientated and regularly assembled into organs as well as specific tissues. However, callus, which is a cluster of undifferentiated cells formed through tissue culture, is interpreted as releasing control that has been maintained until then when cells stimulate various plant growth hormones from the outside.

When the callus obtained by dedifferentiation is put into a liquid medium having the same composition and shake cultured, the cells are separated apart and continue to grow as a suspended state. This callus or cultured cell is essentially different from that of a differentiated cell in which a complete plant ages and dies because it can permanently divide and proliferate by subculturing in a new medium. When the callus or cultured cells that have been passaged for several generations are applied to a solid medium from which various plant growth hormones have been removed, the eyes and roots come out and the individual plants are restored. This regenerated plant originates from the division of a cultured cell, and the origin of the cultured cell is derived from embryonic tissue or water tissue.

In the present invention, the "callus" can be any one derived from any part of the plant, but in one embodiment, it is part of the petal, calyx, fruit, ovary, throne tissue, or cut off cotyledons from germinated seedlings of seeds. It may be a callus induced by.

In the present invention, "Cellus culture" or "Plant cell culture" or "Plant cell Callus culture" is a culture of callus in a medium. The medium can be used without limitation, as long as there is a medium that is generally used in callus culture in the art.

In the present invention, the skin is used for skin improvement, skin vitality enhancement, skin convergence, wrinkle improvement, skin barrier strengthening or improvement, skin regeneration, moisturizing, maintaining elasticity, skin barrier function protection, improvement, anti-inflammatory, It includes various skin activity improvement functions such as atopy, antioxidant, anti-aging, pore contraction, sebum suppression, and acne improvement.

The present invention, a method for preparing a cosmetic composition for skin improvement containing a plant cell complex culture or an extract thereof includes the following steps:

(a) separating cells from each plant tissue and inducing a callus to cultivate them; And (b) preparing a composition containing the culture obtained in step (a) or an extract thereof.

In the present invention, step (b) may include the steps of drying each plant cell callus culture obtained in step (a), powdering it, and mixing it in purified water to prepare a composition.

In the present invention, step (b), each of the plant cell callus culture obtained in step (a) is dried, powdered and mixed with purified water, followed by ultrasonic extraction or hot water extraction to prepare a composition. It can contain.

In the present invention, the callus culture (culture) itself may be used, the culture may be used by filtration, the culture may be crushed, or the culture may be dried and powdered, and then dissolved in purified water.

In the present invention, "extract" means an extract obtained by various extraction methods known in the art, such as powdering the extract of the callus culture, cold water extraction, hot water extraction, and ethanol extraction.

The extraction method in the present invention is not particularly limited, for example, cold immersion extraction, ultrasonic extraction, reflux extraction, hot water extraction, and the like. Here, in the case of hot water extraction, the hot water extractor is heated at 80 to 100 ° C for 8 to 48 hours to obtain a hot water extract.

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

Alternatively, it may be prepared by extraction using water, an organic solvent, or a mixed solvent thereof. The extracted liquid can be used immediately or concentrated and / or dried. In the case of extraction using organic solvents, methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or a mixed solvent of the organic solvent is used, and the active ingredient of the crude drug is not destroyed or can be extracted by heating at room temperature or in a minimized condition. Depending on the organic solvent to be extracted, the degree of extraction and loss of the active ingredient of the drug may differ, so select and use the appropriate organic solvent. In particular, in the present invention, an ethanol extract, preferably 20-50% ethanol extract, and in one embodiment, 50% ethanol extract is preferred, and the extraction method is as described in the Examples, after mixing with 50% ethanol for 3 days It is obtained by stirring.

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

In the present invention, the meaning of "containing as an active ingredient", as a cosmetic composition, may exhibit skin improvement effects due to anti-aging, including skin vitality, skin vitality enhancement, skin convergence, skin barrier strengthening, improvement, and wrinkle improvement. Existing, for example, as a cosmetic composition, collagen synthesis related to wrinkle improvement efficacy, elastin increase, skin convergence, wrinkle improvement, skin barrier strengthening, improvement, pore contraction, sebum suppression, acne improvement, whitening, anti-inflammatory, etc. It means to contain an effective amount of.

In the present invention, the principle of the skin astringent action refers to a phenomenon in which the skin contracts by forming cross-linking by combining with the polymer protein flavonoids. The meaning of convergence basically means that wrinkles are wrinkled or shrunk, and the astringent agent has the effect of contracting skin and mucosal blood vessels, and has the effect of inhibiting the secretion of mucus by blocking the cell gap and lymph gap. In addition, since the astringent has a property of binding to a protein, in general, it is possible to determine the degree of convergence effect according to the degree to which the protein of hemoglobin binds to the extract. It can be said that such a convergence action has an effect of forming a poorly soluble film on the surface of the skin and mucous membranes by external use or contents, and as a result, protecting a local area or compacting the tissue to decrease the permeability of the cell membrane. The convergence action on the skin is divided into two types: chemical convergence and physical convergence, and chemical convergence refers to the temporary contraction of sweat glands and pores by substances that coagulate proteins, such as tannins. On the other hand, the physical convergence action refers to the temporary contraction of sweat glands and pores by using cold water on the skin or lowering the temperature of the skin by volatilization of ethanol.

In addition, such skin convergence occurs during summer or due to stress, when more sebum occurs, skin trouble occurs and the skin trouble occurs more and more red, and skin soothing means to relieve such skin trouble. .

In the skin barrier protection function according to the present invention, the skin barrier is the outermost layer of the epidermis, and the stratum corneum is mainly composed of non-nuclear flat corneocytes. A multi-lamella lipid layer formed of intercellular lipids such as ceramide, cholesterol, and fatty acids synthesized by keratinocytes of the skin barrier maintained through normal division and differentiation of epidermal cells is a protective film to prevent moisture in the skin from evaporating. Plays a role. On the other hand, among these intercellular lipids, omega hydroxy ceramide is chemically linked to involucrin, a protein of the outer layer of corneocytes, by chemical covalent bonding to form a corneocyte lipid envelope (CLE), thereby forming a multilayer lipid membrane. It plays a role of physically stabilizing the intercellular lipids of the form and strengthens the barrier function.

The composition of the present invention is delivered to the stratum corneum through skin application to promote the differentiation of keratinocytes, and not only has the effect of thickening the thickness of the epidermal layer, but also has an excellent effect of restoring the damage of the skin barrier. It can be useful for the treatment and prevention of skin diseases caused. Skin diseases caused by skin barrier damage include, but are not limited to, atopic dermatitis, xeroderma, psoriasis, ichthyosis, and acne.

In addition, the mechanism of skin barrier protection was confirmed by increasing the protein content of occludin and claudin-1, which are the close junctional constitutive proteins. Filaggrin is one of several structural proteins that keratinocytes express in the differentiation step, and is involved in differentiation from the base layer to the stratum corneum of the epidermis, and is the main body of natural moisture factor (NMF) essential for maintaining moisture in skin tissue. It is also used as a major indicator of skin moisture retention and skin membrane function. The plant cell complex callus culture according to the present invention has excellent skin barrier protection, strengthening, and improvement functions as the gene expression of pilargreen is significantly increased. You may have

In addition, the plant cell complex callus culture or extract thereof according to the present invention can be used for skin protection according to ultraviolet irradiation.

In addition, the plant cell complex callus culture or extract thereof according to the present invention has the ability to increase elastin expression. Elastin is a structural substance present in the extracellular matrix of connective tissue, and is a substance present in the skin to impart elasticity. Elastin is a polypeptide called Tropo elastin, which is synthesized in cells and then produced by cross-linking extracellular tropoelastin. Therefore, elastin has a secondary or three-dimensional elasticity due to the cross-linking of tropoelastin. Therefore, the plant cell complex callus culture or extract thereof according to the present invention can be used for skin elasticity enhancement and improvement.

Therefore, the cosmetic composition according to the present invention has, in particular, an increase in procollagen and an increase in collagen biosynthesis, and thus can be applied for skin aging prevention and skin wrinkle improvement / prevention, as well as skin elasticity enhancement and skin barrier according to elastin expression enhancement It can be applied for reinforcement and improvement.

In another aspect, the present invention, (a) edelweiss, seaside ergo, rock samfire, apples, grapes, desert rose leaves, lavender leaves, Chinese chestnut tree leaves, lotus leaves, myrrh leaves, arabian jasmine each plant tissue Inducing and culturing callus from; And (b) preparing a composition containing each of the plant cell callus cultures or extracts thereof, and a method for preparing a cosmetic composition for skin improvement containing the plant cell complex callus cultures or extracts thereof.

In step (a), specifically, a medium suitable for inducing and culturing callus may be selected. Any medium that is generally used in the technical field can be used without limitation. In plants, MS medium, B5 medium, and the like are mainly used. For example, the composition of MS medium (based on 1 L) is NH ₄ NO ₃ 1650 mg, KNO ₃ 1900 mg, CaCl ₂ .2H ₂ O 440 mg, MgSO 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, Sucrose 30000 mg.

In the present invention, in the step (b), the composition containing each callus culture obtained through the culture of step (a) is prepared as a cosmetic composition in an appropriate form, or the culture is known extraction It can be obtained in the form of an extract through a method to prepare a cosmetic composition in an appropriate form.

For example, in step (b), each callus culture obtained in step (a) is dried, powdered, and mixed in purified water to prepare a composition,

The step (a) may include drying the callus culture obtained in step, powdering it, and mixing it with purified water, followed by cold water extraction, hot water extraction or ethanol extraction to prepare a composition.

As another example, the culture obtained in step (a) may be prepared by drying the powder after hot air drying and evaporating moisture.

In the cosmetic composition, it may include a carrier that is acceptable in cosmetic preparations. Here, the "acceptable carrier in cosmetic preparations" is a compound or composition that is already known and used that may be included in cosmetic preparations, or is a compound or composition to be developed in the future. Say nothing.

The carrier may be included in the cosmetic composition of the present invention in an amount of about 1% to about 99.99% by weight, preferably about 90% to about 99.99% by weight of the weight of the composition relative to its total weight. However, since the ratio varies depending on the formulation as described below, in which the cosmetic composition of the present invention is prepared, and its specific application site (face, neck, etc.) or its preferred application amount, the ratio can be applied to the present invention in any aspect. It should not be understood as limiting the scope of.

Examples of the carrier include alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, moisturizing agents, viscous modifiers, emulsions, stabilizers, ultraviolet scattering agents, ultraviolet absorbers, colorants, fragrances, and the like. The alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, moisturizing agents, viscous modifiers, emulsions, stabilizers, ultraviolet scattering agents, ultraviolet absorbers, colorants, compounds / compositions that can be used as fragrances, etc., are already known in the art. Therefore, those skilled in the art can select and use an appropriate material / composition.

As an embodiment of the present invention, the cosmetic composition according to the present invention may include glycerin, butylene glycol, propylene glycol, rolled polyoxyethylene cured castor oil, ethanol, triethanolamine, etc. in addition to the callus extract as the active ingredient, Preservatives, flavoring agents, coloring agents, purified water, etc. may be included in minor amounts as necessary.

In addition, it may further include a well-known physiologically active ingredient or other plant cell culture or extract. For example, it may further include a plant cell culture or plant cell culture extract of extracellular or loofah or extracellular / loofah hybrids.

The cosmetic composition according to the present invention may be prepared in various forms, for example, lotion, essence, gel, emulsion, lotion, cream (oil-in-water type, water-in-oil type, multi-phase), solution, suspension (anhydrous and water-based) , Anhydrous products (oil and glycol-based), gels, masks, packs, powders, or capsules with soft coatings such as gelatin (soft capsules, hard capsules) may be prepared in the form of a formulation.

The skin in the present invention is a concept that includes not only the face, but also the scalp and the whole body, as a cosmetic composition that can be applied to such scalp, there are shampoo, conditioner, treatment, hair-repellent, etc., body cleanser that can be applied to the whole body, etc. It can be manufactured in various forms for the purpose of.

The manufacturing method of the cosmetic composition according to the present invention is not limited to the above-described manufacturing method, and a person having ordinary skill in the art to which the present invention pertains can also manufacture the method by partially modifying the above manufacturing method.

When prepared as a cosmetic composition, the cosmetics of the emulsifying formulation include nutrient makeup, cream, essence, etc., and the cosmetics of the solubilizing formulation include softening makeup. In addition, by containing a dermatologically acceptable medium or base, it can be prepared in the form of a topical or systemic adjuvant commonly used in the dermatology field.

In addition, suitable cosmetic formulations include, for example, emulsions, suspensions, microemulsions, microcapsules, microgranules or ionic forms (liposomes), nonionics obtained by dispersing an oil phase in a solution, gel, solid or dough anhydrous product, or water phase. It can be provided in the form of a vesicle dispersant in the form, in the form of a cream, skin, lotion, powder, ointment, spray or consylstick. It can also be prepared in the form of a foam or aerosol composition further containing a compressed propellant.

In addition, the cosmetic composition of the present invention may further include fatty substances, organic solvents, solubilizers, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, and ionic forms Or nonionic emulsifiers, fillers, metal ion blockers and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives, lipid vesicles or any other commonly used in cosmetics. It may contain auxiliaries commonly used in cosmetic or dermatological fields such as ingredients. In addition, the above ingredients may be introduced in an amount generally used in the field of dermatology.

The cosmetic composition containing the plant cell complex culture or extract thereof according to the present invention provides skin vitality, improves skin vitality, improves skin wrinkles, regenerates skin, protects skin barrier function, protects skin from UV rays, atopy, anti-inflammatory, antioxidant, As a cosmetic composition having functions such as anti-aging, it can be used as a material for various functional cosmetics.

1 is a result confirming whether or not the toxicity of the plant cell complex composition according to the present invention.
2 is a result of confirming the mRNA expression rate of the skin barrier-related gene of the plant cell complex composition according to the present invention.
3 is a result confirming the wrinkle improvement effect of the plant cell complex composition according to the present invention.
4 is a result confirming the anti-inflammatory effect of the plant cell complex composition according to the present invention.
5 is a result confirming the whitening effect of the plant cell complex composition according to the present invention.
6 is a result confirming the protective effect from ultraviolet rays of the plant cell complex composition according to the present invention.
7 is a result of confirming the results of the gene expression test related to bio-clock regulation of the plant cell complex composition according to the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

Example  1: according to the invention Plant cell Complex  Composition preparation

Example  1-1: Surface disinfection of 11 plant samples

Edelweiss, seaside ergo, rock samfire, apple, grape, desert rose leaf, lavender leaf, Chinese chest tree leaf, lotus leaf, myrrh leaf, arabian jasmine leaf to prepare the cell according to the present invention, the surface To sterilize, immersed in 70% ethanol for 30 seconds, shaken with 2% sodium hypochlorite for 5 minutes, sterilized, and then washed with sterile water.

Example  1-2: Plant cell induction from 11 samples

1) Sterilized edelweiss leaves were cut at a time using a sharp knife, plant growth regulators 1 mg / L 6-Benzylaminopurine, 0.3 mg / L 2,4-D, 3% Sucrose and agar 8 g / L In the basic MS medium containing (Murashige and Skoog 1962, Duchefa, Cat No. M0221), cancer cells were cultured at a growth condition of 25 ° C and humidity of 70% to induce initial plant cells. Each medium was adjusted to pH 5.8 with 1N sodium hydroxide (NaOH). Passage culture was performed every two weeks.

2) Sterilized seaside erringo leaves are cut at once with a sharp knife, 50 mg / L plant growth regulators (indole-3-acetic acid), 5 mg / L zeatin (Zeatin) , 3% Sucrose and agar containing 8 g / L of basic MS medium (Murashige and Skoog 1962, Duchefa, Cat No. M0221) at 25 ° C and humidity of 70% in cancerous conditions. Did. Each medium was adjusted to pH 5.8 with 1N sodium hydroxide (NaOH). Passage culture was performed every two weeks.

3) Sterilized Rocksamfire leaves are cut at once with a sharp knife, 5 mg / L plant growth regulators (indole-3-acetic acid), 10 mg / L zeatin, 3 % Sucrose and agar 8 g / L of basic MS medium (Murashige and Skoog 1962, Duchefa, Cat No. M0221) was cultured at 25 ° C. and humidity of 70%, and cultured to induce initial plant cells. Each medium was adjusted to pH 5.8 with 1N sodium hydroxide (NaOH). Passage culture was performed every two weeks.

4) Sterilized apple leaves are cut at once with a sharp knife, 5 mg / L indole-3-acetic acid (IAA), 10 mg / L zeatin, 3% Sucrose and agar 8 g / L containing the basic MS medium (Murashige and Skoog 1962, Duchefa, Cat No. M0221) in 25 ℃, humidity of 70% in the growth conditions of cancer culture and induced initial plant cells. Each medium was adjusted to pH 5.8 with 1N sodium hydroxide (NaOH). Passage culture was performed every two weeks.

5) Basic MS medium containing plant growth regulators 0.1 mg / L 2,4-D, 3% Sucrose and agar 8 g / L by cutting the sterilized grape leaves at once with a sharp knife ( Murashige and Skoog 1962, Duchefa, Cat No. M0221) induced early plant cells by cancer culture at 25 ° C and 70% humidity. Each medium was adjusted to pH 5.8 with 1N sodium hydroxide (NaOH). Passage culture was performed every two weeks.

6) Sterilized desert rose leaves are cut at once with a sharp knife, 0.5 mg / L plant growth regulators (indole-3-acetic acid), 1 mg / L zeatin, 3 % Sucrose and agar 8 g / L of basic MS medium (Murashige and Skoog 1962, Duchefa, Cat No. M0221) was cultured at 25 ° C. and humidity of 70%, and cultured to induce initial plant cells. Each medium was adjusted to pH 5.8 with 1N sodium hydroxide (NaOH). Passage culture was performed every two weeks.

7) Basic MS medium containing 0.1 mg / L 2,4-D, 3% Sucrose and 8 g / L of plant growth regulators by cutting sterilized lavender leaves at once with a sharp knife ( Murashige and Skoog 1962, Duchefa, Cat No. M0221) induced early plant cells by cancer culture at 25 ° C and 70% humidity. Each medium was adjusted to pH 5.8 with 1N sodium hydroxide (NaOH). Passage culture was performed every two weeks.

8) Sterilized Chinese chaste tree leaves are cut at once with a sharp knife, 0.5 mg / L plant growth regulators (indole-3-acetic acid), 1 mg / L zeatine (Zeatin) , 3% Sucrose and agar containing 8 g / L of basic MS medium (Murashige and Skoog 1962, Duchefa, Cat No. M0221) at 25 ° C and humidity of 70% for cancer culture and inducing early plant cells Did. Each medium was adjusted to pH 5.8 with 1N sodium hydroxide (NaOH). Passage culture was performed every two weeks.

9) Sterilized lotus leaf is cut at once with a sharp knife, Plant Growth Regulators 1 mg / L 6-Benzylaminopurine, 0.3 mg / L 2,4-D, 3% Sucrose and agar 8 g / L-containing 1/2 MS medium (Murashige and Skoog 1962, Duchefa, Cat No. M0221) was incubated at 25 ° C and humidity of 70% in a growing condition to induce early plant cells. Each medium was adjusted to pH 5.8 with 1N sodium hydroxide (NaOH). Passage culture was performed every two weeks.

10) Sterilized myrrh leaves are cut at once with a sharp knife, 0.5 mg / L plant growth regulators (indole-3-acetic acid), 1 mg / L zeatin, 3% Sucrose and agar 8 g / L containing the basic MS medium (Murashige and Skoog 1962, Duchefa, Cat No. M0221) in 25 ℃, humidity of 70% in the growth conditions of cancer culture and induced initial plant cells. Each medium was adjusted to pH 5.8 with 1N sodium hydroxide (NaOH). Passage culture was performed every two weeks.

11) Sterilized arabian jasmine leaves are cut at once with a sharp knife, 0.5 mg / L plant growth regulators (indole-3-acetic acid), 1 mg / L zeatin, 3 % Sucrose and agar 8 g / L of basic MS medium (Murashige and Skoog 1962, Duchefa, Cat No. M0221) was cultured at 25 ° C. and humidity of 70%, and cultured to induce initial plant cells. Each medium was adjusted to pH 5.8 with 1N sodium hydroxide (NaOH). Passage culture was performed every two weeks.

Example  1-3: according to the invention Plant cell  Culture and mass production

Cells of edelweiss, seaside ergo, rock samfire, apple, grape, desert rose leaf, lavender leaf, Chinese chest tree leaf, lotus leaf, myrrh leaf, arabian jasmine leaf cultured through passage culture aseptically, In a liquid medium of the same composition except agar, the temperature was adjusted to 25 ° C, humidity 70%, and the air supply amount of 0.1 vvm, and cultured and proliferated at 14-day intervals to produce mass.

Example  1-4: according to the invention Plant cell  Complex extract preparation

Each of the 11 plant cells obtained by cultivation was harvested, and after removing sufficient moisture with a clean tissue, dried at 60 ° C for 2 days. In order to obtain the composition according to the present invention, edelweiss plant cells 40 g, seaside ergo callus 20 g, rock samfire callus 20 g, apple callus 20 g, grape callus 40 g, desert rose leaf callus 40 g, lavender leaf callus 40 g, Chinese Chest Tree Leaf Callus 20 g, Lotus Leaf Callus 40 g, Myrrh Leaf Callus 20 g and Arabian Jasmine Leaf Callus 20 g were thoroughly washed with purified water and mixed. Using a 20 L purified water in a hot water distiller for 8 to 48 hours, heating to 80 to 100 ° C to obtain a hot water extract of the callus complex. The obtained composition was centrifuged at 12000 rpm for 4 ° C for 30 minutes to remove insoluble solids, and the supernatant was obtained as a callus complex extract.

Experimental Example 1: Effect test on cell viability

In this experiment, the cell proliferation activity of the plant cell complex extract prepared in Example 1 was examined.

To this end, first, HaCaT (human keratinocyte, keratinocyte) and Detroit551 (human fibroblast, fibroblast) were dispensed into a 96 well plate with DMEM medium containing 10% FBS, and in a thermostat at 5% CO ₂ and 37 ° C. Incubated for 24 hours. After 24 hours, cells were treated to a final concentration of 0.5, 1, and 2% of the plant cell complex extract, and further cultured for 24 hours. Thereafter, 4 μL of 5 mg / mL MTT solution was added to each well, and cultured for 4 hours. After removing the culture solution, cells were lysed by adding 100 μL of DMSO (Dimethyl sulfoxide) solution, and absorbance was measured at 540 nm using a spectrophotometer.

The degree of survival of skin cells was calculated and calculated according to the following equation based on the absorbance intensity of the control group using purified water, and the results were shown in FIG. 1.

[Equation 1] Cell viability (%) = [(absorbance in the experimental group-absorbance in the control group) / absorbance in the control group] × 100

As shown in Figure 1, it was confirmed that the plant cell complex extract of the present invention does not show toxicity in three types of skin cell lines within the treatment concentration.

Experimental Example  2: Antioxidant effect ( ABTS  Radical Scavenging Activity)

The measurement of antioxidant capacity using ABTS radical is a method that uses a method that uses a color that discolors blue-green, which is a specific index of radicals, by removing ABTS free radicals generated by reaction with potassium persulfate by antioxidants. It was measured.

1.0 mM AAPH (2,2'-azo-bis 2-amidinopropanedeihydrochloride) was mixed with 2.5 mM ABTS (2,2'-azino-bis 3-ethylbenzenothiazolin-6-sulfonic acid) dissolved in 100 mM PBS buffer, followed by 68 The reaction was carried out in a constant temperature bath at ℃ for 12 minutes. The degree of absorbance decreased at 734 nm was measured by reacting 20 μl of plant cell complex extract with 980 μl ABTS solution in a 37 ° C. water bath for a final concentration of 0.5, 1, 2, 5%. At this time, the positive control was Trolox 0.1. % Was used. The ABTS radical scavenging ability was calculated according to the following equation.

[Mathematics]

Treatment material ABTS  Radical Erasing ability  ( % ) Control 0 Trolox 0.1% 52.67 Plant cell complex extract 0.5% 31.33 Plant cell complex extract 1% 34.67 Plant cell complex extract 2% 47.33

As a result, as shown in the above table, it was confirmed that the plant cell complex extract of the present invention has an antioxidant effect according to the scavenging ability of the ABTS radical in the treatment concentration.

Experimental Example  3: Skin barrier improvement gene expression test

In this experiment, in order to examine the effect of improving the skin barrier of the plant cell complex extract prepared in Example 1, changes in expression levels of AQP3, a water / glycerol transporter, and FLG, known as a natural moisturizing factor, were investigated.

After dispensing HaCaT cells in a 96 well plate, the cells were cultured for 24 hours under cell culture conditions. Thereafter, the plant cell complex extract as a test substance was treated with the cells to a final concentration of 1% together with DMEM medium without serum, and further cultured for 24 hours. Real-time PCR method was performed to confirm at the gene level of AQP3 and FLG, and the test sequence is as follows.

For RNA isolation and cDNA synthesis, SuperPrep ^TM cell lysis & RT Kit for qPCR (TOYOBO, Cat. SCQ-101) was used. The cells from which the medium was removed were washed once with PBS, and 50 μL cell lysis mixture (including gDNA remover) was added and reacted for 5 minutes, and then a stop solution was added. 8 μL of the extracted mRNA was added to 32 μL of the RT reaction mixture, and cDNA was synthesized using PCR at 37 ° C for 15 minutes, 50 ° C for 5 minutes, and 95 ° C for 5 minutes. In order to compare and analyze the expression of genes, cDNA synthesized above was used as a template, and real-time PCR analysis was performed using Thunderbird ^TM SYBR qPCR Mix (TOYOBO, Cat. QPS-201). The primer used in the experiment was Qiagen's QuantiTect®primer assays (GAPDH; Cat. The real-time qPCR conditions were first 95 ° C 15 minutes reaction, and then proceeded to a total of 40 cycles with 94 ° C 15 seconds, 60 ° C 30 seconds, and 72 ° C 30 seconds per cycle.

As a result, as shown in FIG. 2, it was confirmed that in the cells treated with the plant cell complex extract 1% of the present invention, the mRNA expression rates of AQP3 and FLG were increased, thereby improving skin barrier.

Experimental Example  4: Skin regeneration and wrinkle improvement effect test

In this experiment, in order to examine the effect of improving the skin regeneration and wrinkles of the plant cell complex extract prepared in Example 1, the collagen decomposition enzyme MMP-1 (Matrix Metalloproteinase-1) and the precursor of collagen synthesis, procollagen The biosynthetic amount of (Procollagen Type I C-Peptide, PIP) was measured.

<4-1> MMP-1 biosynthesis measurement

After the Detroit551 cells were dispensed into 48 well plates, the cells were cultured for 24 hours under cell culture conditions. Thereafter, the plant cell complex extract as a test substance was treated with the cells to a final concentration of 1% with DMEM medium without serum, and further cultured for 48 hours.

Take 100 μL of the diluted supernatant of the culture medium through the above process and put it into antibody coated microtiter plate with 100 μL of MMP-1 standard in MMP-1 Biotrak Activity Assay Kit (Amersham, Cat. RPN2629) 25 The reaction was carried out for 3 hours at ℃. After removing the medium and washing 4 times with 200 μL of PBS, 100 μL of antiserum was added to each well and reacted at 25 ° C. for 3 hours. Thereafter, the solution was removed, washed 4 times with 200 μL of PBS, and 100 μL of peroxidase-labeled antibody solution was added to each well, and further reacted at 25 ° C. for 1 hour. In addition, after removing the solution, 100 μL of the substrate solution was added to each well and reacted at room temperature for 15 minutes in a shaded state. Then, 100 μL stop solution (1 NH ₂ SO ₄ ) was added and absorbance at 450 nm was measured. After measuring absorbance at 450 nm, the standard concentration curve of MMP-1 was prepared to calculate the amount of MMP-1 in the sample.

<4-2> PIP biosynthesis measurement

After the Detroit551 cells were dispensed into 48 well plates, the cells were cultured for 24 hours under cell culture conditions. Thereafter, the plant cell complex extract as a test substance was treated with the cells to a final concentration of 1% with DMEM medium without serum, and further cultured for 48 hours.

Take 20 μL of the diluted supernatant of the cultured medium through the above process, put in 20 μL of the PIP standard in the PIP EIA Kit (Takara, Cat.MK101), respectively, in an antibody coated microtiter plate, and 100 μL of peroxidase-labeled antibody solution Was also added to each well, and reacted at 37 ° C for 3 hours. After removing the medium and washing 4 times with 200 μL of PBS, 100 μL of the substrate solution was added to each well and reacted at room temperature for 15 minutes in the shaded state. Thereafter, 100 μL stop solution (1 NH ₂ SO ₄ ) was added, and absorbance at 450 nm was measured. After measuring the absorbance at 450 nm, the PIP standard concentration curve was prepared to calculate the amount of PIP in the sample.

As a result, as shown in FIG. 3, it was confirmed that the amount of MMP-1 was decreased and the amount of PIP was increased in cells treated with 1% of the plant cell complex extract of the present invention, thereby increasing anti-wrinkle effect.

Experimental Example  5: anti-inflammatory effect

The amount of NO ₂ present in the cell culture fluid of a nitric oxide (NO) generated from Raw 264.7 ^cell line in the form was measured using the Griess reagent. To measure NO production inhibition in Raw 264.7 cells activated with 1 μg LPS, the experimental group and the control group treated with the medium containing the plant cell complex extract having a final concentration of 1.0% were cultured for 24 hours, and then Griess reagent was subjected to cell culture supernatant. 100 μl and 100 μl of Griess reagent (1% sulfanilamide in 5% phosphoric acid, 1% α-naphthylamide in H ₂ O) were mixed and reacted in 96 well plates for 10 minutes, and absorbance was measured at 540 nm. The concentration of NO ₂ ^- was diluted with sodium nitrate, and absorbance was measured to obtain a standard curve.

As a result, as shown in FIG. 4, it was confirmed that in the cells treated with 1% of the plant cell complex extract of the present invention, the NO content was decreased in the inflammatory reaction induced by LPS, thereby having an anti-inflammatory effect.

Experimental Example  6: Inhibit melanin production Whitening effect  effect

B16F1 cells were added in 2 mL of 1 × 10 ⁵ cells / well in a 6 well plate with DMEM medium containing 10% FBS, and cultured for 24 hours in a constant temperature incubator at 5% CO2 and 37 ° C. After removing all the medium, each well was treated with 2 ml of a plant cell complex extract containing a final concentration of 1%. After treating α-MSH dissolved in DMEM medium to a final 10 nM, the treated cells were reacted for 72 hours in a constant temperature incubator at 5% CO 2 at 37 ° C. Remove the culture solution, wash 3 times with PBS (phosphate buffer saline), treat 100 μL of 1X RIPA buffer containing protease inhibitor, lyse the cells, centrifuge at 15,000 rpm for 20 minutes, and then add the supernatant to a new container. Was recovered. The recovered supernatant is added to each 96 well plate by 50 μL.

To quantify the protein, 150 μL of BCA solution (A: B = 50: 1) was added to each well, reacted for 30 minutes in an incubator at 37 ° C., and absorbance was measured with an ELISA reader at 540 nm. Did. The measured absorbance value was substituted into a standard calibration curve using albumin in a concentration range of 0-1 mg / mL to obtain a protein amount.

Finally, for quantitative analysis of melanin, Pellet was dried at 60 ° C., and 400 μL of 1N NaOH containing 10% DMSO was added to react in a 90 ° C. thermostat. 100 μL of the reacted solution was placed in a 96 well plate, and absorbance was measured at 492 nm using an ELISA reader. The amount of melanin was obtained by substituting the measured absorbance into the melanin standard calibration curve in the concentration range of 0-2.5 mg / mL.

As a result, as shown in FIG. 5, it was confirmed that cells treated with 1% of the plant cell complex extract of the present invention have a whitening effect by inhibiting melanin production.

Experimental Example  7: Cell protection effect by ultraviolet irradiation

As a light source to be used for UV irradiation, an ultraviolet crosslinker CL-1000 (Ultra-Violet Products, CA) emitting ultraviolet rays having a wavelength of 302 nm was used. HaCaT cells, keratinocytes, were placed in 24-well plates at a concentration of 1x10 ⁵ cells / ml, cultured for 24 hours in an incubator, the medium was removed, and washed with a phosphate buffer. After adding 500 μL of phosphate buffer solution, irradiated with ultraviolet light at 10 mJ / cm², replaced with fresh cell culture medium not containing FBS, treated with a plant cell complex extract at a final concentration of 1%, and further cultured for 24 hours. To this, 5 mg / ml of MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, Sigma) was added, followed by incubation for 3 hours in an incubator, and the formed fomazan was dissolved with DMSO, After transferring to a 96-well plate, absorbance was measured with an ELISA reader at 595 nm, and cell viability was compared with a control group that did not process the sample after UV irradiation.

As a result, as shown in FIG. 6, it was confirmed that the cell viability was increased because the reduced cell viability after UVB induction had a cell protective effect by treating the plant cell complex extract 1% of the present invention.

Experimental Example  8: Gene expression test related to biological clock regulation

In this experiment, in order to examine the effect of regulating the bioclock of the plant cell complex extract prepared in Example 1, changes in expression levels of related genes CLOCK and PER1 were investigated.

After dispensing HaCaT cells in a 96 well plate, the cells were cultured for 24 hours under cell culture conditions. After this, after inducing UVB, the cells were treated with the DMEM medium containing no serum to treat the cells as a test substance to a final concentration of 1%, and further cultured for 8, 16, 24, and 32 hours. Real-time PCR method was performed to confirm at the gene level of CLOCK and PER1, and the test sequence is as follows.

For RNA isolation and cDNA synthesis, SuperPrep ^TM cell lysis & RT Kit for qPCR (TOYOBO, Cat. SCQ-101) was used. The cells from which the medium was removed were washed once with PBS, and 50 μL cell lysis mixture (including gDNA remover) was added and reacted for 5 minutes, and then a stop solution was added. 8 μL of the extracted mRNA was added to 32 μL of the RT reaction mixture, and cDNA was synthesized using PCR at 37 ° C for 15 minutes, 50 ° C for 5 minutes, and 95 ° C for 5 minutes. In order to compare and analyze the expression of genes, cDNA synthesized above was used as a template, and real-time PCR analysis was performed using Thunderbird ^TM SYBR qPCR Mix (TOYOBO, Cat. QPS-201). The primers used in the experiment were Qiagen's QuantiTect®primer assays (GAPDH; Cat. The real-time qPCR conditions were first 95 ° C 15 minutes reaction, and then proceeded to a total of 40 cycles with 94 ° C 15 seconds, 60 ° C 30 seconds, and 72 ° C 30 seconds per cycle.

As a result, as shown in FIG. 7, the expression pattern of irregular CLOCK and PER1 by UVB stimulation showed the expression pattern of CLOCK, PER1 of cells that did not give UVB stimulation in cells treated with 1% of the plant cell complex extract of the present invention. It was confirmed to recover closely.

Experimental Example  9: Skin energy activity enhancing effect test

In this experiment, in order to examine the effect of enhancing the skin energy activity of the plant cell complex extract prepared through Example 1, the activity level of the energy (ATP) of the cell was measured.

After the Detroit551 cells were dispensed into 12 well plates, the cells were cultured for 24 hours under cell culture conditions. Thereafter, the plant cell complex extract, a test substance, was treated with the cells to a final concentration of 0.5, 1, 2% with DMEM medium without serum, and further cultured for 24 hours.

Take 50 μL of the diluted lysate of the cells cultured through the above process, put 50 μL of ATP standard in ATP assay kit (Abcam, Cat. Ab83355) into 96 well plates, respectively, and add 50 μL of ATP reaction mix solution In addition, it was added to each well and allowed to react at room temperature for 30 minutes in a shaded state. Then, after measuring the absorbance at 570 nm, ATP standard concentration curve was prepared to calculate the amount of ATP in the sample. The degree of skin energy activity was calculated according to the following equation based on the absorbance intensity of the control group using purified water and expressed as a percentage.

Treatment material Energy active effect (%) Control 0 Plant cell complex extract 1% 44.77

As a result, as shown in the above table, it was confirmed that the energy treatment effect was excellent in cells treated with 1% of the plant cell complex extract of the present invention.

Experimental Example  10: skin vitality enhancing effect test

In this experiment, in order to examine the effect of enhancing the skin vitality of the plant cell complex extract prepared through Example 1, 20 test subjects were administered to 20 female subjects aged 20 to 45 years with a Body Mass Index (BMI) of 21 to 27. After showering once a day for a week, it was used with massage, and it was judged whether the effect was compared before and after 12 weeks of use. As for the evaluation items, the number of subjects who responded that they felt the effect on skin cell activation and skin vitality through questionnaire evaluation is shown in Table 3 below.

Test substance Skin cell activation Skin vitality Control 2 0 Plant cell complex extract 0.5% 6 5 Plant cell complex extract 1% 11 13 Plant cell complex extract 2% 13 17

As a result, as shown in the above table, the plant cell complex extract of the present invention hardly felt the effect of skin cell activation and skin vitality improvement after use for 12 weeks in the case of the control group for the majority of respondents in the evaluation of the use in treatment concentration, It was confirmed that the plant cell complex extract responded that it felt skin cell activation and skin vitality enhancement.

Experimental Example  11: Acne improvement effect

For 10 men and women who had acne, the lotion was applied evenly over the entire face for 4 weeks in the morning and evening with the 1% concentration of plant cell complex extract prepared in Example 1. Determination of the acne improvement effect was evaluated by evaluating the condition before the test with 1-3 points based on the following criteria based on the degree to which the person participating in the experiment felt, and the results are shown in the table.

<Evaluation of the state before the test>

1 = some acne 2 = some acne 3 = severe acne

<Acne effect judgment>

-: Almost no effect, +: little effect, ++: much relief,

+++: completely healed

Subject Pre-test status After 2 weeks After 4 weeks Subject 1 One - - Subject 2 3 + ++ Subject 3 One - + Subject 4 2 + ++ Subject 5 One + + Subject 6 2 + + Subject 7 2 + ++ Subject 8 2 - ++ Subject 9 One - + Subject 10 3 ++ +++

As a result, as shown in the above table, after using the flexible lotion containing 1% of the plant cell complex extract of the present invention for 4 weeks, it was possible to confirm the improvement effect of acne in most subjects.

Experimental Example  12: sebum suppression effect experiment

In order to confirm the sebum inhibiting effect of the plant cell complex extract, sebum secretion of the skin was measured using a skin oil meter (Sebum meter SM810) after 4 weeks of 10 subjects. The experiment was conducted at 22 ± 2 ° C and 40 ± 5% humidity. When the oily and the measured value is more than 220 ㎍ / cm ^2, when the normal skin is 100~220 ㎍ / cm ^2. Purified water was used as a control.

Treatment material 0 days (before use) 28 days (after use) Control 232 225 Plant cell complex extract 0.5% 231 220 Plant cell complex extract 1% 230 191 Plant cell complex extract 2% 227 180

As a result, as shown in the table above, it was confirmed that the sebaceous secretion amount of the plant cell complex extract of the present invention decreased after 4 weeks of use within the treatment concentration.

Experimental Example  13: Pore contraction effect test (In vitro Test)

To measure the pore contraction effect of the plant cell complex extract composition of the present invention, the composition prepared in the above example was tested for the effect of pore contraction using hemoglobin as a protein to replace skin. Prepare a hemoglobin solution (0.05 g / 50 ml) with 0.9% Phosphate Buffer Saline (0.1 mM, pH 7.4), add 2 ml of edelweiss plant cell and arrhythmic cell culture extract to 2 ml of hemoglobin solution, shake and mix for 30 seconds, then After centrifugation at 3,500 rpm for 10 minutes, 2 ml of purified water was added to 1 ml of the supernatant, and the UV-Visible spectrum was measured at 407 nm. As a control, 2 ml of PBS (Phosphate Buffer Saline) was added.

Treatment material Pore contraction effect ( % ) Control 0 Plant cell complex extract 0.5% 28.78 Plant cell complex extract 1% 35.74 Plant cell complex extract 2% 39.15

As a result, as shown in the table above, it was confirmed that the plant cell complex extract of the present invention has a pore contraction effect in the treatment concentration.

Claims (10)

Edelweiss callus culture or extract thereof, seaside ergo callus culture or extract, Roxemfire callus culture or extract, apple cell culture or extract, grape cell culture or extract, desert rose leaf cell culture or Extract, lavender leaf cell culture or extract, Chinese chaste tree leaf cell culture or extract, lotus leaf cell culture or extract, myrrh leaf cell culture or extract, and arabian jasmine leaf cell culture or extract as active ingredients Cosmetic composition for preventing or improving skin wrinkles. delete Edelweiss callus culture or extract thereof, seaside ergo callus culture or extract, rock samfire callus culture or extract, apple cell culture or extract, grape cell culture or extract, desert rose leaf cell culture or Extract, lavender leaf cell culture or extract, Chinese chaste tree leaf cell culture or extract, lotus leaf cell culture or extract, myrrh leaf cell culture or extract, and arabian jasmine leaf cell culture or extract as active ingredients Cosmetic composition for protecting or strengthening the skin barrier function. Edelweiss callus culture or extract thereof, seaside ergo callus culture or extract, Roxemfire callus culture or extract, apple cell culture or extract, grape cell culture or extract, desert rose leaf cell culture or Extract, lavender leaf cell culture or extract, Chinese chaste tree leaf cell culture or extract, lotus leaf cell culture or extract, myrrh leaf cell culture or extract, and arabian jasmine leaf cell culture or extract as active ingredients Cosmetic composition for improving or preventing acne. Edelweiss callus culture or extract thereof, seaside ergo callus culture or extract, Roxemfire callus culture or extract, apple cell culture or extract, grape cell culture or extract, desert rose leaf cell culture or Extract, lavender leaf cell culture or extract, Chinese chaste tree leaf cell culture or extract, lotus leaf cell culture or extract, myrrh leaf cell culture or extract, and arabian jasmine leaf cell culture or extract as active ingredients A cosmetic composition for skin protection according to ultraviolet ray irradiation. Edelweiss callus culture or extract thereof, seaside ergo callus culture or extract, Roxemfire callus culture or extract, apple cell culture or extract, grape cell culture or extract, desert rose leaf cell culture or Extract, lavender leaf cell culture or extract, Chinese chaste tree leaf cell culture or extract, lotus leaf cell culture or extract, myrrh leaf cell culture or extract, and arabian jasmine leaf cell culture or extract as active ingredients Cosmetic composition for skin whitening containing. Edelweiss callus culture or extract thereof, seaside ergo callus culture or extract, Roxemfire callus culture or extract, apple cell culture or extract, grape cell culture or extract, desert rose leaf cell culture or Extract, lavender leaf cell culture or extract, Chinese chaste tree leaf cell culture or extract, lotus leaf cell culture or extract, myrrh leaf cell culture or extract, and arabian jasmine leaf cell culture or extract as active ingredients Contains antioxidant cosmetic composition. Edelweiss callus culture or extract thereof, seaside ergo callus culture or extract, rock samfire callus culture or extract, apple cell culture or extract, grape cell culture or extract, desert rose leaf cell culture or Extract, lavender leaf cell culture or extract, Chinese chaste tree leaf cell culture or extract, lotus leaf cell culture or extract, myrrh leaf cell culture or extract, and arabian jasmine leaf cell culture or extract as active ingredients Containing sebum suppressing cosmetic composition. Edelweiss callus culture or extract thereof, seaside ergo callus culture or extract, Roxemfire callus culture or extract, apple cell culture or extract, grape cell culture or extract, desert rose leaf cell culture or Extract, lavender leaf cell culture or extract, Chinese chaste tree leaf cell culture or extract, lotus leaf cell culture or extract, myrrh leaf cell culture or extract, and arabian jasmine leaf cell culture or extract as active ingredients Containing cosmetic composition for shrinking pores. Edelweiss callus culture or extract thereof, seaside erringo callus culture or extract, rock samfire callus culture or extract, apple cell culture or extract, grape cell culture or extract, desert comprising the following steps: Rose leaf cell culture or extract, lavender leaf cell culture or extract, Chinese chaste tree leaf cell culture or extract, lotus leaf cell culture or extract, myrrh leaf cell culture or extract, and arabian jasmine leaf cell culture Or a method for producing a cosmetic composition according to any one of claims 1 and 3 to 9 containing an extract:
(a) Inducing and culturing callus from each plant tissue of edelweiss, seaside ergo, rock samfire, apple, grape, desert rose leaf, lavender leaf, chinese chaste tree leaf, lotus leaf, myrrh leaf, and arabian jasmine To do; And (b) preparing a composition containing each of the plant cell callus cultures or extracts thereof.

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