KR101781027B1 - Composition for Improving Skin Conditions Comprising Lycium chinense Mill. callus Extract - Google Patents

Abstract

The present invention relates to a composition for improving skin condition comprising an extract of Gujuga callus as an active ingredient. The callus extracts of the present invention used in the composition of the present invention are excellent in preventing aging of skin through molecular mechanisms such as promotion of collagen synthesis, protective action of fibroblasts, proliferation effect of follicular dermal papilla cells, improvement of skin wrinkles, skin regeneration, , Mitigates, improves, prevents or treats skin damage due to ultraviolet rays or active oxygen, improves, prevents or treats atopic dermatitis, and promotes hair growth or hair loss. In addition, the Gugija callus extract has no cytotoxicity and skin side effects and can be safely applied to cosmetic, pharmaceutical or food compositions.

Description

TECHNICAL FIELD The present invention relates to a composition for improving skin condition comprising an extract of Gujuzana callus as an active ingredient. callus Extract}

More particularly, the present invention relates to a composition for improving skin condition comprising an extract of Gugija callus extract as an active ingredient. More specifically, the present invention relates to a composition for preventing skin aging, skin wrinkles, skin regeneration, skin elasticity, , A pharmaceutical composition or a food composition for improving skin condition, which contains Gugija callus extract as an effective ingredient for improving, preventing or treating atopic dermatitis, preventing or treating atopic dermatitis, and preventing hair growth or preventing hair growth.

Skin is a protective membrane with various physiological functions and is an important organ for obtaining beauty appeal from other people. There is a saying that a person has an appearance of 90%. According to a consciousness survey, it is said that the appearance that is concerned with the aging is not the inside but the male and the female. Aging skin is accompanied by visible skin changes and functional dysfunctions of various skin, so active research is underway in the field of cosmetics to delay or prevent it. Skin aging is characterized by various factors such as genetic factors, exposure to ultraviolet rays, and production of reactive oxygen species by physicochemical stress. Active oxygen species attack and damage intracellular proteins, lipids, or nucleic acids, and this damage accumulates and causes aging. As a representative phenomenon of skin aging, sebum secretion is decreased, skin becomes dry, cell regeneration is delayed, and aging skin is piled up and skin becomes rough. In addition, the amount of collagen that supports the epidermis is reduced, and elastin (elastic fiber) is denatured to form wrinkles. In addition, the skin color is stained, spots, black spots and pigmentation symptoms appear, the skin becomes thinner, the skin protection function is weakened. Other phenomena include an increase in skin troubles due to a decrease in the barrier effect associated with a decrease in skin thickness. Researches related to skin aging are classified into materials that protect skin from ultraviolet rays and active oxygen, which are major causes of skin aging, skin wrinkle improving materials, skin elasticity enhancing materials, skin moisturizing materials, have.

As the skin ages, dermal atrophy occurs, and the dermis thickness of the elderly is reduced by about 20%. At this time, the number of the dermis and the number of blood vessels in general decreases and the dermis changes. Changes in the dermis are caused by a change in the number of fibroblasts and a change in the molecular weight of the extracellular matrix due to a decrease in their ability to synthesize. Specific changes include separation of collagen bundles, reduction of point polysaccharide synthesis, reduction in the number and diameter of collagen and elastic fibers, collagen and elastic fiber grinding, and expansion of blood vessels. Generally, among the various complex factors such as the moisture content of the skin, the collagen content and the external environment, the collagenase And the activity and activity of collagenase.

In recent years, development of materials capable of effectively solving phenomena such as wrinkles, spots or freckles, obesity and hair loss caused by exposure to ultraviolet rays due to environmental destruction and pollution as well as endogenous aging has been extensively studied.

Plants were a means of food resources in the past, but they are now being expanded as a source of a wide range of chemicals, including medicines, flavors, colors, pesticides, and dyes. For example, there is a growing interest in plants in the field of cosmetics for the purpose of preventing or preventing skin aging, antioxidation, hair loss, etc. using plant-derived useful substances (e.g., carotenoids, phenol compounds, vitamins and the like).

However, the rate of growth of plants is very slow, the amount of secondary metabolites depends on the kind of plant, and the amount of secondary metabolites varies depending on the kind of plant. In the same species, season, place, geographical climate, The productivity varies depending on the plant parts. In addition, plant resources due to ecosystem pollution and destruction due to rapid industrialization have been exhausted recently.

Thus, many studies have been attempted to produce various useful substances (eg, secondary metabolites) dependent on plants in a biotechnological way. The most effective method of production is plant cell culture. This method can effectively produce secondary metabolites through culture medium, culture environment and various elicitor treatments. In addition, it can be said that it is commercially important because it is possible to continuously mass produce the useful substance produced by plants without receiving natural environment constraints (high / toxic pesticides, heavy metal pollution, seasonal domination, etc.).

On the other hand, Lycium chinense Mill. Is a twin perennial plant and belongs to the branch family. It is a deciduous broad-leaved shrub with a stem length of 4m and has been cultivated in small scale as a medicinal plant since 1925. Betaine, known as the pharmacologically active ingredient of Gujia, is an amine-based substance having the structure of (CH) ₃ NCH ₂ COOH, and it has pharmacological activity in plants and microorganisms as well as various antioxidants, anti- (Korean Patent Publication No. 2006-0025693). Fruit (Gugija) widely used as a Chinese medicinal herb contains not only betaine but also carotene, zeaxanthine, nicotinic acid, vitamin and physalien. And it has nutritional effects such as liver protection, fatigue and fever treatment, prevention of arteriosclerosis, and reduction of cholesterol. In addition, rutin, which is effective for preventing various adult diseases, is contained in the old leaves, so that there is an antipyretic effect on tuberculosis, dissolution, and hematopoiesis (Kang, Kyungil et al., 2006, 103.). In addition, antibiotics, flavonoids, sapogenins, tropane alkaloids, dipeptides lyciumamide, sugiol and (5-α-stigmastane- 3,6-dione) and the like. However, as in the present invention, the effect of preventing the skin aging, improvement of skin wrinkles, skin regeneration, skin elasticity, improvement of atopic dermatitis, prevention or treatment of hair growth, hair growth promoting or hair loss prevention is not disclosed at all in the present invention, There is no example in which a composition containing a callus-derived callus extract having an effect is produced.

Accordingly, the inventors of the present invention have found that the extract of Gujuga callus extract is effective for preventing skin aging, improving skin wrinkles, regenerating skin, improving skin elasticity, alleviating, improving, preventing or treating skin damage due to ultraviolet rays or active oxygen, It was confirmed that the effect of promoting hair growth or hair loss was excellent, thereby completing the present invention.

Accordingly, one object of the present invention is to provide a composition for improving skin conditions comprising Lycium chinense Mill. Callus extract as an active ingredient.

It is another object of the present invention to provide a cosmetic composition, a pharmaceutical composition or a food composition for improving skin condition comprising the composition.

Other objects and advantages of the present invention will be more clearly understood from the following detailed description, claims and drawings.

In one aspect, the present invention provides a composition for improving skin condition comprising Lycium chinense Mill. Callus extract as an active ingredient.

As used herein, the term "callus" is an amorphous tissue or cell mass that has lost the ability to induce normal organogenesis or tissue differentiation and is mostly flowcytosed, and the tissue cut from the plant is cultured in various media, Refers to a specific tissue or cell mass produced when cultured in a tissue culture medium containing cytokinin, injured in a plant, or treated with auxin of a plant body.

As used herein, the term "improvement of skin condition" means improvement and improvement of all forms of skin condition, and preferably includes prevention of skin aging, improvement of skin wrinkles, skin regeneration, skin elasticity enhancement, Amelioration, improvement, prevention or treatment of damage, improvement of atopic dermatitis, prevention or treatment, and prevention of hair growth or hair loss.

In the present invention, the " Lycium chinense Mill. Callus" is composed of any one or more, preferably leaves, stems, branches and fruits selected from the group consisting of leaves, stems, branches, A callus derived from any one or more sites selected from the group consisting of leaves, branches and fruits. In one specific example, the callus callus may be a callus, Cut calligraphy.

In the present invention, the induction of calli can be carried out through conventional methods known in the art. More specifically, the induction of callus is carried out by culturing a plant tissue or a fragment thereof in a tissue culture medium. As the culture medium for tissue culture, medium such as MS (Murashige and Skoog, 1962), N6 (Chu et al. , 1975), B5 (Gamborg et al. , 1968), NN (Nitsch and Nitsch, 1967) A basic medium of various plants can be used. It is preferable that the culture medium for tissue culture is one in which a plant growth regulator is added. The plant growth regulator may be selected from the group consisting of 2,4-D (2,4-dichlorophenoxyacetic acid), 2,4,5-T (2,4,5-trichlorophenoxyacetic acid 2-methoxy-3,6-dichlorobenzoic acid, indole-3-acetic acid, indole-3-butyric acid, MCPA (2-methyl-4-chlorophenoxyacetic acid), NAA Auxin such as 1-naphthylacetic acid, 2-naphthyloxyacetic acid and 4-amino-2,5,6-trichloropicolinic acid; Bip (6-benzylaminopurine), 2iP (N6- (2-isopentyl) adenine), Kinetin (6-furfurylaminopurine), Thidiazuron (1-phenyl- 3- (1,2,3-thiadiazol- (4-hydroxy-3-methyl-trans-2-butenylaminopurine), and derivatives thereof. In addition, the tissue culture medium may further contain alkaloids, terpenoids, carotenoids, phenol compounds, and natural extracts that act on differentiation and proliferation of cells.

As one specific example, the callus of the present invention can be obtained by separating and disinfecting the branches of Gujuga tree and then treating the plant with an organic component (e.g. carbohydrate, amino acid etc.), inorganic nutrients (e.g. nitrogen, phosphoric acid, potassium, etc.) Naphthalene acetic acid, oxine, etc.) for 5 to 8 weeks. When callus, which is a cell mass, is formed by division of branch cells, the callus is separated from the medium and placed on a fresh solid medium. Followed by subculturing.

In the present invention, the callus extract of Gujuga may be obtained by disintegrating the callus itself or the callus callus powder and the callus by a physical method such as a chemical method or a french press method using a general disinfectant, Alternatively, the cells may be cultured by a conventional cell culture method known in the art, and then the pellet may be removed using a physical method such as ultra-high speed centrifugation to extract the remaining culture liquid by a conventional method known in the art, Quot; refers to a substance having a prophylactic or therapeutic activity. The callus extract of Gujuga is not only the extract extracted by a conventional method but also a dry powder or any form which is formulated using the same.

The Gugija callus extract of the present invention can be extracted using water or an organic solvent, and the extracted solution can be used in a liquid form or can be used by concentration and / or drying. The organic solvent may be selected from the group consisting of methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, N-dimethylformamide, dimethylsulfoxide, 1,3-butylene glycol, propylene glycol or a mixed solvent thereof. The extract can be extracted at room temperature or warmed under conditions where the active ingredient of the extract 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 extract may differ. Therefore, an appropriate organic solvent should be selected and used. The extraction method is not particularly limited, and examples thereof include cold extraction, hot water extraction, ultrasonic extraction, and reflux cooling extraction.

In addition, the callus extract of Gujuga may be obtained through a conventional purification process in addition to the method of extracting by the above-mentioned extraction solvent. For example, by separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatographies (made for separation by size, charge, hydrophobicity or affinity), and the like, Fragrant callus extract can also be obtained through fractionation.

According to one specific embodiment, the Gugija callus extract of the present invention has an effect of promoting collagen synthesis, inhibiting collagenase activity, and protecting fibroblasts from oxidative damage due to active oxygen with little cytotoxicity and side effects .

According to another specific embodiment, the guinea callus extract of the present invention increases the proliferation of hair follicular dermis-inducing cells, exhibits an excellent hair growth promoting or hair-removing effect, and is effective in preventing the growth of eotaxin-1 in fibroblasts It was confirmed that there was an effect of reducing the production amount depending on the concentration.

Accordingly, the composition comprising the extract of Gugija callus extract of the present invention as an active ingredient can be used as an effective ingredient for preventing skin aging, improving skin wrinkles, regenerating skin, improving skin elasticity, alleviating, Prevention or treatment of hair growth, and prevention of hair growth or hair loss.

As used herein, the term "hair growth promoting" or "hair loss prevention" is used interchangeably and has the same meaning as another term wool or hair growth promotion used in the art.

The present invention is advantageous in that a useful substance can be obtained without damaging a plant by using a callus callus, not a callus root itself.

In addition, the composition of the present invention is remarkably superior to Gugija extract in preventing skin aging, improving skin wrinkles, regenerating skin, improving skin elasticity, alleviating, improving, preventing or treating skin damage caused by ultraviolet rays or active oxygen, improving atopic dermatitis, Preventing or treating hair growth, and promoting hair growth or preventing hair loss.

The composition of the present invention may contain 0.001 to 15.0% by weight, preferably 0.0001 to 10.0% by weight, more preferably 0.001 to 5% by weight, based on the weight of the whole composition, of an effective ingredient, Gujuza callus extract. When the content of the callus extract is less than 0.00005% by weight, the skin aging prevention, skin wrinkle improvement, skin regeneration, skin elasticity enhancement, mitigation, improvement, prevention or treatment of skin damage due to ultraviolet rays or active oxygen, improvement or prevention of atopic dermatitis The hair growth promoting or hair loss prevention effect is insufficient, and when it is more than 15% by weight, the increase of the effect is not proportional to the increase of the content, which may be inefficient and the stability of the shape is not ensured .

As one specific application of the present invention, the composition of the present invention is useful for preventing skin aging, improving skin wrinkles, regenerating skin, improving skin elasticity, alleviating, improving, preventing or treating skin damage due to ultraviolet rays or active oxygen, improving atopic dermatitis , Prophylactic or therapeutic treatments, and hair cosmetic compositions for promoting hair growth or preventing hair loss.

When the composition of the present invention is prepared with a cosmetic composition, it may further contain ingredients commonly used in cosmetic compositions in addition to the extract of Gugija callus as an active ingredient. For example, conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavors, and carriers.

The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used as a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be prepared as a nutritional cream, a convergent lotion, a soft lotion, a lotion, an essence, a nutritional gel or a massage cream.

When the formulation of the cosmetic composition is a paste, a cream or a gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide .

When the formulation of the cosmetic composition is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In particular, in the case of spray, a mixture of chlorofluorohydrocarbons, Propane / butane or dimethyl ether.

When the formulation of the cosmetic composition is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid esters of sorbitan.

When the formulation of the cosmetic composition is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspension such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Crystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the cosmetic composition is an interface-active agent-containing cleansing, the carrier component is selected from aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid Amide ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester.

As another specific application of the present invention, the composition of the present invention is useful for preventing skin aging, improving skin wrinkles, regenerating skin, improving skin elasticity, alleviating, improving, preventing or treating skin damage due to ultraviolet rays or active oxygen, Improvement, prophylaxis or treatment, and prevention of hair growth or hair loss.

When the composition of the present invention is manufactured from a pharmaceutical composition, it may contain a pharmaceutically acceptable carrier in addition to the extract of Calli extract as an active ingredient. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include carbohydrate-based compounds such as lactose, amylose, dextrose, sucrose, sorbitol, mannitol, starch, cellulose, etc. ), Acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, salt solution, alcohol, gum arabic, vegetable oil But are not limited to, vegetable oils, soybean oil, soybean oil, olive oil, coconut oil), polyethylene glycol, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical compositions of the present invention may be administered by various routes, such as oral or parenteral, and any manner of administration may be expected, for example, by oral, rectal or intravenous, muscular, subcutaneous injection have. At this time, transdermal administration is preferable for the parenteral route, and topical application by application is most preferable.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . Oral doses of the pharmaceutical compositions of the invention are in the range of 0.001-100 mg / kg (body weight) on an adult basis. When the composition is an external preparation, it is preferable to apply the composition in an amount of 1.0 to 3.0 ml on an adult basis once to five times a day for one month or longer. However, the dose is not intended to limit the scope of the present invention.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

As another specific application of the present invention, the composition of the present invention is useful for preventing skin aging, improving skin wrinkles, regenerating skin, improving skin elasticity, alleviating or improving skin damage due to ultraviolet rays or active oxygen, improving atopic dermatitis, And can be manufactured and used as a food composition for promoting hair growth or preventing hair loss.

When the composition of the present invention is prepared as a food composition, it may contain ingredients commonly added in the manufacture of food, in addition to the Gugija callus extract as an active ingredient. For example, proteins, carbohydrates, fats, nutrients, flavoring agents, and flavoring agents. Examples of the above-mentioned carbohydrates are monosaccharides such as glucose, fructose, and the like; Daisaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors such as tau martin and stevia extract (e.g., rebaudioside A and glycyrrhizin) and synthetic flavors (saccharine, aspartame, etc.) can be used as flavorings.

There is no particular limitation on the kind of the food. Examples of the food to which the Gugija callus extract can be added include dairy products such as meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, Drinks, alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

When the food composition of the present invention is prepared as a drink, it may further contain citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, mulberry extract, jujube extract, licorice extract etc. in addition to the Gugija callus extract of the present invention .

In addition, the food composition may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin , Alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the food composition of the present invention may contain flesh for the production of natural fruit juices, beverages and vegetable drinks. These components may be used independently or in combination.

The cosmetic, pharmaceutical or food composition according to the present invention can be administered to an individual to improve the skin condition of the individual.

The term "individual" as used herein is intended to ameliorate, prevent or treat poor skin conditions such as wrinkles, hair loss, skin damage caused by ultraviolet or active oxygen, inflammatory diseases including atopic dermatitis, Refers to mammals, including, for example, monkeys, cows, horses, pigs, sheep, dogs, cats, rats, mice, chimpanzees, etc., which can achieve these objectives by the administration of other compositions.

As used herein, the term "prophylactic " means inhibiting the occurrence of a disease or disorder in an individual who has never been diagnosed as having a disease or condition, but is likely to be susceptible to such disease or disease.

As used herein, the term " improvement "or" treatment "means (a) inhibiting the development of a disease or disease, (b) relieving the disease or disease, or (c)

The term "administering" as used herein means introducing a predetermined substance into an individual by any suitable method, and the administration route of the composition of the present invention may be administered orally or parenterally ≪ / RTI > The composition of the present invention may also be administered by any device capable of transferring the active substance to a target subject, for example, a cell.

The above-described skin condition can achieve the object of improvement, improvement, prevention or treatment by administration of the composition of the present invention to the individual. Through the above-mentioned contents and the examples described below, As will be understood by those skilled in the art.

The callus extract of Guojia callus, which is used as an active ingredient in the composition of the present invention, can be used for preventing skin aging, improving skin wrinkles, regenerating skin, improving skin elasticity, promoting collagen synthesis and protecting fibroblasts, Improvement, prevention or treatment of skin damage due to skin irritation, improvement of atopic dermatitis, prevention or treatment, and prevention of hair growth or hair loss. In addition, the Gugija callus extract has no cytotoxicity and skin side effects and can be safely applied to cosmetic, pharmaceutical or food compositions.

1 is a graph showing the effect of promoting collagen production by the callus extract of Gujuga.
Fig. 2 is a graph showing the effect of suppressing the activity of collagenase (collagenase) by the callus extract of Gugija.
Fig. 3 is a graph showing the effect of inhibiting eotaxin-1 activity by the callus extract of callus.
FIG. 4 is a graph showing the inhibitory effect of oxidative stress induced cell senescence by Gujuga callus extract. FIG.
FIG. 5 is a graph showing the effect of follicular callus cultured extract on the follicular dermal papilla cell proliferation.

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 explaining the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention.

Example 1: Preparation of Gugija callus extract

The Gugija callus extract of the present invention can be prepared as follows.

The perennial branches of P. gugija were soaked in 70% ethanol for 30 seconds and then shaken in 2% sodium hypochlorite solution for 30 minutes. Then, the branches of the sterile gugija were washed with sterilized water more than 5 times and made into small pieces (0.5 ~ 1 cm in the transverse direction). Then, the branches of the killed gugija were treated with MS (Murashinge and Skoog, USA) containing 3 mg / L growth regulator of NAA (1-Naphthaleneacetic acid), 30 g / L of sucrose and 2.5 g / L of Gelite, 1962), and then callus was induced by culturing in a culture room at 25 ° C for 8 weeks. The induced calli were proliferated and purified by subculture at intervals of 5 weeks. The induced callus was inoculated into an air floating biological reactor and cultured and harvested. At this time, the culture medium was inoculated into the same medium except for 2.5 g / L of Gelite in the basic medium, and then suspended in a dark state for 6 weeks in a culture room at 25 ° C. The cultured callus was propagated by subculture at 6-week intervals.

The proliferated callus was then harvested to remove moisture, dried for 2 days in a dryer at 55 ° C and then milled. 100 g of dried Gugija callus powder was dipped in 10 L of 80% methanol and ultrasonically extracted at room temperature for 2 hours. Thereafter, the obtained extract was filtered using a filter paper (Advantes, No. 2), and concentrated under reduced pressure to prepare a callus extract of Gujuga.

Example 2: Measuring effect of enhancing collagen synthesis of Gugija callus extract

Collagen biosynthesis performance can be measured by cell experiments on human fibroblasts. Collagen biosynthesis was performed by inoculating human normal fibroblast (a dermatologist of Ajou University) into a 24-well plate containing DMEM medium (2 × 10 ⁵ cells / well), adding 5% Of CO ₂ incubator at 37 ° C for 24 hours. After 24 hours, the medium was removed from each well and treated with 10 ppm, 50 ppm, and 100 ppm of Gugija callus extract, respectively, and then re-cultured for 24 hours. After 24 hours, the cell culture medium was collected to prepare a sample. The amount of collagen synthesis was determined by measuring the amount of procollagen type I C-peptide (Type I C-peptide: PICP) using a collagen measurement kit (Procollagen Type I C-peptide EIA kit MK101, Takara, Japan). The detailed test method was performed according to the instructions of the Takara Co., Ltd. kit. Fibroblasts treated with 10 ng / ml TGF-β (transforming growth factor) alone were not used as a positive control but fibroblasts treated with 10 ng / ml of callus extract Respectively. The results are shown in Fig.

As shown in Fig. 1, the fibroblasts treated with the callus extracts of 10 ppm, 50 ppm and 100 ppm increased collagen production by 10%, 20% and 40%, respectively, as compared with the control group.

Example 3: Measurement of collagenase activity inhibitory effect of Gugija callus extract

Antibodies against collagenase were used to measure the activity of collagenase, an enzyme that degrades collagen. (Tumor necrosis factor, TNF-alpha, Sigma, USA), which is known to induce collagenase activity. Concentration ranges of Gugija callus extract were determined as 0 ppm, 10 ppm and 100 ppm. The overall test was conducted according to the protocol of the sales company. Type I collagenase assay kit (Amersham Biosciences, RPN2629) was used and the absorbance was measured with an ELISA reader (Bio-Tek ELx808 ^™ Series Ultra Microplate Reader, UK). The measured values were expressed as mean ± SD and the significance was tested by t-test using SPSS / PC + program. As control (-), fibroblasts without tumor necrosis factor alpha and callus extracts were used. The results are shown in Fig.

As shown in Fig. 2, the Gujuga callus extract inhibited the collagenase activity induced by tumor necrosis factor alpha in human-derived fibroblasts in a concentration-dependent manner.

Example 4 Measurement of Skin Elasticity Enhancement Effect of Cosmetics Containing Gugija Callus Extract

4-1. Manufacture of nutritional cream containing Gugija callus extract

The composition of the nutritional cream containing the Gugija callus extract is shown in Table 1 below.

First, water, purified water, triethanolamine and propylene glycol were heated and dissolved at 70 DEG C, and a fatty acid, an oily component, an emulsifier and an antiseptic agent, which had been heated by heating at 70 DEG C, were added thereto and emulsified. After the emulsification is complete, the solution is cooled to 45 ° C and 0.01%, 0.05% or 1.00% gugija callus extract and flavor are added and dispersed and then cooled to 30 ° C.

ingredient Content (% by weight) Control group Test group 1 Test group 2 Test group 3 Gugija callus extract - 0.01 0.05 1.00 Jojoba oil 5.0 5.0 5.0 5.0 Liquid paraffin 7.0 7.0 7.0 7.0 Cetylaryl alcohol 2.0 2.0 2.0 2.0 Polyglyceryl-3 methylglucose distearate 2.0 2.0 2.0 2.0 Glyceryl stearate 0.5 0.5 0.5 0.5 Squalane 3.0 3.0 3.0 3.0 Propylene glycol 4.0 4.0 4.0 4.0 glycerin 5.0 5.0 5.0 5.0 Triethanolamine 0.3 0.3 0.3 0.3 Carboxyvinyl polymer 0.3 0.3 0.3 0.3 Tocopheryl acetate 0.2 0.2 0.2 0.2 Preservative, incense a very small amount a very small amount a very small amount a very small amount Purified water 70.7 70.69 70.65 69.7 Sum 100 100 100 100

4-2. Measurement of skin elasticity enhancement effect of cosmetics containing Gugija callus extract

The wrinkle-reducing effect was evaluated by measuring skin elasticity change in 30 healthy women, which were prepared by the method of Example 4-1.

Specifically, 30 healthy women (25 to 35 years old) under the condition that the temperature was kept constant at 25 DEG C and the humidity of 40% were subjected to three types of cream of Preparation Example 1 and the nutritional cream of Comparative Preparation Example to 1 The skin elasticity was measured using a cutometer SEM 474 (Courage + Khazaka, Cologne, Germany). The criteria for skin elasticity were 0 for no elasticity, 5 for many The values were compared. The results are shown in Table 2 below.

Test Items
(Gugija callus extract) Test group 1
(0.01%) Test group 2
(0.05%) Test group 3
(One%) Control group
(0%) Skin elasticity 2.57 2.77 2.84 2.53

As shown in Table 2, test group 1, test group 2, and test group 3 of the present invention showed improved skin elasticity as compared with the control group. As a result, it was confirmed that the skin elasticity of the callus extract was enhanced by the increase of the content of callus extract.

Example 5: Anti-aging effect by oral administration

Aging is a phenomenon that occurs in all organisms and is present in all tissues and organs. Skin is the largest organ of the body that forms the outermost part of an individual in the case of animals. Wrinkles appearing on the skin are the easiest indicators to determine the degree of aging. In particular, since ultraviolet irradiation can artificially accelerate the aging process, it is possible to confirm whether or not the substance has an actual anti-aging effect by measuring the effect of the test substance administered orally or transdermal .

To investigate the effect of the composition of the present invention on photoaging symptoms, hairless mice were selected as animal models. Eight female mice (hairless mouse-Skh: HR-1) at 6-7 weeks of age were divided into the normal group, the UV control group and the UV / callus extract group. In the normal and UV control groups, 0.5 ml of physiological saline was orally administered. In the UV / Gujugus callus extract administration group, 100 mg of Gugija callus extract per kg of body weight was mixed with 0.5 ml of saline and administered orally Respectively.

The administration period was 5 weeks in total and 5 days in the same time. From 2 to 5 weeks after the oral administration, the UV control group and the UV / callus extract group were irradiated with UV light similar to sunlight three times a week. The total UV exposure was 600 mJ / cm ² during the experiment. As a main phenomenon of anti-aging effect, skin replicas were collected from the dorsal side of a hairless mouse before autopsy to determine the effect of improving wrinkles. Likewise, after 5 weeks, The molds were collected to compare the anti-aging effect, that is, the wrinkle-improving effect. The results are shown in Table 3.

R-parameter UV / Control UV / Gugija callus extract R1 value 0.67 ± 0.010 0.52 0.003 R2 value 0.59 + 0.011 0.45 ± 0.008 R3 value 0.31 ± 0.007 0.20 ± 0.005 R4 value 0.22 0.004 0.13 + 0.013 R5 value 0.41 ± 0.009 0.36 ± 0.009

R1 value: Skin roughness, R2 value: Maximum roughness of skin, R3 value: Average roughness of skin, R4 value: Smoothness depth, R5 Value: Arithmetic average roughness (International Journal of Cosmetic Science, 27 (3): 155-60 (2005)).

As shown in Table 3, it was observed that R1, R2, R3, R4, and R5, which indicate degree of wrinkle improvement, were significantly decreased compared to the UV / control group not administered with hairless mouse administered with callus extract Respectively. These results indicate that the composition of the present invention has an effect of improving skin wrinkles. In other words, it was confirmed that the wrinkle improving effect of Gugija callus extract was exhibited when it was orally administered.

Example 6: Safety test for human skin of Gugija callus extract

6-1. Preparation of external skin preparation containing Gugija callus extract

In order to confirm that the extract of Gugija callus was safe for human skin, a skin external preparation containing Gugija callus extract was prepared by the ingredients and contents of Table 4 below, and then a skin safety test was conducted through cumulative skin irritation test. First, squalene was mixed with purified water, glycerin, and butylene glycol, and dissolved at a temperature of about 70 DEG C (water part). Then, the three components and the other components except for trimethanolamine were dissolved at a temperature of about 70 DEG C Part). Thereafter, the oil phase part was added to the water phase part, and the mixture was firstly emulsified with a homomixer (Tokushu Kika, Japan) and trimethanolamine was finally added.

ingredient content (%) Control group Test group 1 Test group 2 Test group 3 Purified water 72 71.9 71.5 71 glycerin 8.0 8.0 8.0 8.0 Butylene glycol 4.0 4.0 4.0 4.0 Gugija callus extract 0 0.1 0.5 1.0 Caprylic Capri Triglyceride 8.0 8.0 8.0 8.0 Squalane 5.0 5.0 5.0 5.0 Cetearyl glucide 1.5 1.5 1.5 1.5 Sorbitan stearate 0.4 0.4 0.4 0.4 Cetearyl alcohol 1.0 1.0 1.0 1.0 Trimethanolamine 0.1 0.1 0.1 0.1 Gross weight 100 100 100 100

6-2. Experiment of cumulative stimulation of skin

The skin external preparation prepared in Example 6-1 was applied to 30 healthy adults for 9 times for 24 hours cumulative epidermal patches on the upper forearm every other day to determine whether or not the callus extracts stimulate the skin. A pin chamber (Finn chamber, Epitest Ltd, Finland) was used as the deposition method. 15 [mu] l of each of the above external preparations for skin was dropped into the chamber, followed by applying a patch. The degree of the skin reaction on each occasion was scored using the following Equation 1, and the results are shown in Table 5 below.

[Equation 1]

(Number of total subjects × maximum score (4 points))}] × 100] ÷ number of tests (9 times)

A score of ±, a +, and a ++ is given as 1, 2, and 4, respectively, and a safe composition is determined when the average degree of reaction is less than 3.

Test substance Number of subjects who responded Average
Reactivity 1 week 2 weeks 3 weeks Primary Secondary Third Fourth 5th 6th Seventh 8th car 9th ± + ++ ± + ++ ± + ++ ± + ++ ± + ++ ± + ++ ± + ++ ± + ++ ± + ++ Control group
(Squalene) 0 - - 0 - - - - - - - - - - - - - - - - - - - - - - - 0.00 Gujuza callus extract (0.1%)
[Test group 1] 0 - - 0 - - - - - - - - - - - - - - - - - - - - - - - 0.00 Callus extract (0.5%) [Test group 2] 0 - - 0 - - - - - - - - - - - - - - - - - - - - - - - 0.00 Gugija callus extract (1%)
[Test group 3] 0 - - 0 - - - - - - - - - - - - - - - - - - - - - - - 0.00

As shown in Table 5, the numbers of persons corresponding to ±, + and ++ in test groups 1, 2 and 3 were 0, 0 and 0, respectively, and the average reactivity was 0.00, 0.00 and 0.00, respectively. Since the average degree of reactivity is less than 3, the callus extract of Gugija was judged to be a substance safe for human skin which does not exhibit a distinct cumulative stimulus pattern.

Example  7: Evaluation of anti-inflammatory efficacy

To investigate the anti-inflammatory effect, NIH / 3T3 cells (fibroblasts) were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37 ° C and 5% CO _2. ⁵ / well, and cultured for 24 hours. After cultivation, the cells were replaced with fresh DMEM containing no FBS, and 10 ppm or 100 ppm of Gugija callus extract dissolved in 100% DMSO was pretreated and preincubated for 1 hour. After incubation, interleukin-4 (IL-4) was treated at a concentration of 50 ng / mL and cultured for 24 hours. Culture supernatant was recovered and subjected to enzyme-linked innumosorbent assay (ELISA) The level of secretion of eotaxin-1 was measured comparatively. As a control (-), NIH / 3T3 cells (fibroblasts) without IL-4 and callus extracts were used. IL-4 was treated as a positive control, whereas NIH / 3T3 cells (Fibroblast) was used. The results are shown in FIG. 3 and Table 6.

sample Eotaxin-1 activity (%) A positive control [IL-4 (50 ng / ml)] 100 IL-4 (50 ng / ml) + Gujuza callus extract (10 ppm) 91.5 IL-4 (50 ng / ml) + Gugija callus extract (100 ppm) 77

As shown in FIG. 3 and Table 6, the fibroblasts treated with the callus extracts of 10 ppm and 100 ppm had activity of 8.5% and 23% of eotaxin-1, respectively, as compared with the positive control , And thus it can be seen that the extract of Gugija callus has the effect of improving, preventing or treating atopic dermatitis.

Example 8 Effect of Fibroblast Protection from Active Oxygen and Anti-Aging Effect

Human normal fibroblasts were inoculated into a 24-well plate containing DMEM medium (2 × 10 ⁵ cells / well) and cultured in a 5% CO 2 incubator for 37 days Lt; 0 > C for 24 hours. After 24 hours, the cells were exchanged with serum-free DMEM medium. The callus extract of callus was treated with concentrations (0, 10 and 100 ppm) and treated with 1 μM of rotonone, which is known to induce mitochondrial reactive oxygen species. The cell viability was measured by 3-day treatment with rothenone, and the 6-day treatment group was subjected to the SA-beta-Gal staining test, which is an indicator of cell senescence. Each microscopic image was analyzed by Image J program and the degree of staining was measured. As control (-), normal fibroblasts not treated with rottennon and callus extracts were used. The results are shown in Fig.

As shown in Fig. 4, it was observed that the SA-beta-Gal staining intensity increased by rotenone was reduced in the group treated with callus extract.

Three days after the treatment of the test material, cell viability was observed using MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) method. MTT was cleaved by respiratory chain enzymes in living mitochondria as a pale yellow substrate and produced dark blue formazan. Since the reaction does not occur in dead cells, the amount of formazan produced is used to measure viable cell count. The measurement results are shown in Table 7. The cell regeneration rate was calculated by the following equation (2).

&Quot; (2) "

Measurement of cell regeneration rate = {(sample treatment group - treated with rotenone) / treated with rotenone} x 100

Test Items Gugija callus extract
(100 ppm) Gugija callus extract
(500 ppm) Cell Regeneration (%) 15 37

As can be seen in Table 7, the callus extracts of rosemary extract relaxed the fibroblast toxicity effect induced by rotenone. This shows that the callus extract of gujuga protects fibroblasts by a mechanism that prevents mitochondria from being damaged by reactive oxygen species.

Example 9: Effect of proliferation of hair follicle dermal papilla cells

In order to investigate the prevention of hair loss and hair dressing effect of the extract of Calli gingeria according to the present invention under in vitro experimental conditions, the following experiment was conducted

The effect of gugija callus extract on cell viability was examined by culturing the dermal papilla cells in serum - free medium containing callus extract. The hair follicle dermal papilla cells were purchased from Cell Application Company and cultured in the medium of hair follicle dermal papilla cell growth medium (Cell Apllications, Inc. USA).

Specifically, 3 × 10 ⁵ follicular dermal papilla cells cultured using a hair follicle dermal papilla cell growth medium were inoculated into a 6-well plate, After one wash with the medium, the cells were incubated for 72 hours in a serum-free medium containing 1, 10 or 50 μg / mL of Gugija callus extract. After incubation, the cells were washed once with serum-free medium, treated with 10% MTT, and incubated with the cells for 3 hours. The O · D value was measured and converted into a percentage. As a positive control, FGF2, a fibroblast growth factor, and follicular dermal papilla cells treated with complete medium (C, M: complete medium) containing 10% FBS were used instead of Gugija callus extract. The results are shown in Fig.

As shown in Fig. 5, the hair follicle dermal papilla cells treated with Gujuga callus extract showed an increase in cell growth in a concentration-dependent manner.

Example 10: Hair loss prevention and hair growth effect

The following experiments were carried out in order to examine the hair loss prevention and hair dressing effect of the Gugija callus extract according to the present invention.

10-1. Preparation of hair growth inhibitor composition

The hair growth inhibitor composition containing Gujuza callus extract was prepared in the following manner. Test group 1 and test group 2 are respectively hair growth inhibitor compositions containing callus extract.

ingredient content(%) Test group 1 Test group 2 Gugija callus extract 0.1 1.0 Preservative (Gramben) 0.8 0.1 Xanthan-Gum < / RTI > 0.3 0.1 Gross weight 100 100

10-2. Prevention of hair loss and hair growth effect measurement

Forty patients in the late 40s and early 60s were shrunken, 40 patients were divided into four groups, including patients with mild scalp baldness alopecia, typical male alopecia, and acute alopecia areata, and 10 patients were allocated to each group. The hair growth inhibitor prepared in Example 10-1 was applied to the hair loss region of alopecia patients for 3 months each time for 6 months, twice a day. Hair loss and hair growth were observed on a monthly basis. As a comparative group, Moxidil (Hanmi Pharm), which is commercially available, was used and only 50% ethanol was used as a control group. The results are shown in Table 9.

The criteria are as follows:

4: High Effective = Newborn Eye

3: moderate effect = newborn eye (fluffy)

2: slightly effective = reduced number of hair loss

1: No effect = No change at all

- Control group Comparative group Test group 1 Test group 2 Efficacy / Effect 2.2 3.5 2.7 3.0

As shown in Table 8, new hair of the bristles including hairs began to appear in the alopecia treated with the hair growth agent containing the extract of Gujia callus extract of the present invention at one or two months after treatment, and at the 6th month of treatment, 80% or more There was a hair growth effect in the patient. In addition, we observed that newborn babies grew continuously and no hair loss phenomenon was found.

Examples of formulations for the composition of the present invention are illustrated below.

Formulation Example 1: Cosmetic preparation

1-1. Softening longevity

ingredient weight% Gugija callus extract 0.01 glycerin 3.0 Butylene glycol 2.0 Propylene glycol 2.0 Carboxyvinyl polymer 0.1 ethanol 10.0 Triethanolamine 0.1 Preservatives, micronutrients, trace fragrances and trace water 82.79 sum 100.0

1-2. Nutritional lotion

ingredient weight% Gugija callus extract 0.01 Wax 4.0 Polysorbate 60 1.5 Sorbitan sesquioleate 0.5 Liquid paraffin 5.0 Squalane 5.0 Caprylic / capric triglyceride 5.0 glycerin 3.0 Butylene glycol 3.0 Propylene glycol 3.0 Carboxyvinyl polymer 0.1 Triethanolamine 0.2 Preservatives, micronutrients, trace fragrances and trace water 69.69 sum 100.0

1-3. Nourishing cream

ingredient weight% Gugija callus extract 0.01 Wax 10.0 Polysorbate 60 1.5 Sorbitan sesquioleate 0.5 Liquid paraffin 10.0 Squalane 5.0 Caprylic / capric triglyceride 5.0 glycerin 5.0 Butylene glycol 3.0 Propylene glycol 3.0 Triethanolamine 0.2 Preservatives, micronutrients, trace fragrances and trace water 56.79 sum 100.0

1-4. Massage cream

ingredient weight% Gugija callus extract 0.01 Wax 10.0 Polysorbate 60 1.5 Sorbitan sesquioleate 0.8 Liquid paraffin 40.0 Squalane 5.0 Caprylic / capric triglyceride 4.0 glycerin 5.0 Butylene glycol 3.0 Propylene glycol 3.0 Triethanolamine 0.2 Preservatives, micronutrients, trace fragrances and trace water 27.49 sum 100.0

1-5. pack

ingredient weight% Gugija callus extract 0.01 Polyvinyl alcohol 13.0 Sodium carboxymethylcellulose 0.2 Allantoin 0.1 ethanol 5.0 Nonyl phenyl ether 0.3 Preservatives, micronutrients, trace fragrances and trace water 81.39 sum 100.0

Formulation example  2: Pharmaceutical preparation

2-1. Manufacture of Powder

ingredient g Gugija callus extract 2 Lactose One

The above components were mixed and packed in airtight bags to prepare powders.

2-2. Manufacture of tablets

ingredient Mg Gugija callus extract 100 Corn starch 100 Lactose 100 Magnesium stearate 2

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

2-3. Preparation of capsules

ingredient Mg Gugija callus extract 100 Corn starch 100 Lactose 100 Magnesium stearate 2

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (5)

A cosmetic composition for promoting hair growth or preventing alopecia including a callus extract ( Lycium chinense Mill. ) As an active ingredient.
delete Lymechinense Mill. A food composition for promoting hair growth or preventing hair loss comprising an extract of callus as an active ingredient.
The cosmetic composition according to claim 1, wherein the callus is originated from at least one site selected from the group consisting of leaves, stems, branches, fruits and roots of Gujuga tree.
The cosmetic composition according to claim 1, wherein the callus extract is contained in an amount of 0.00005 to 15.0% by weight based on the total weight of the composition.

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