KR20170109705A

KR20170109705A - Method for culturing Lilium Candidum Cell and Functional skin external application comprising Lilium Candidum Cell Culture Extract

Info

Publication number: KR20170109705A
Application number: KR1020160033147A
Authority: KR
Inventors: 모상현; 이정훈; 서효현; 김수윤; 모지홍; 박정곤; 김혜인
Original assignee: 주식회사 바이오에프디엔씨
Priority date: 2016-03-21
Filing date: 2016-03-21
Publication date: 2017-10-10
Also published as: KR101852252B1

Abstract

More particularly, the present invention relates to a method for culturing a plant cell of Madonna lily (Lilium Candidum), a plant derived from a lily plant, and a method for producing the same. The present invention relates to a composition for external application of a skin containing a plant cell culture extract of Lilium Candidum, Skin wrinkles, wrinkle healing, skin regeneration, anti-inflammation, skin whitening, skin moisturization, skin pore contraction, acne suppression, skin damage protection from ultraviolet rays, skin barrier function enhancement including cell culture or extract thereof The present invention relates to a composition for external application for skin and a method for producing the same.
It is possible to mass-produce plant cells of madonna lily containing excellent cosmetic functions including antioxidative activity through the cultivation method of Madonna lily plant cell according to the present invention, The composition for external application has various cosmetic effects such as improvement of wrinkles of skin, wound healing, anti-inflammation, shrinkage of pores, skin moisturization and improvement of acne without toxicity to skin cells.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for culturing Madonna lily plant cells and a method for culturing Madonna lily plant cell,

The present invention relates to a method for culturing a plant cell of Lilium Candidum and a composition for external application of a functional skin containing a plant cell culture extract of Madonna lily. More particularly, the present invention relates to a plant cell cultivation method of Madonna lily and a plant derived from a lily plant The present invention relates to a skin having a use for cell culture or an extract containing the extract, such as anti-aging, wound healing, anti-inflammation, skin whitening, skin moisturizing, skin pore contraction, acne suppression, skin damage protection from ultraviolet rays, An external application composition and a method for producing the same.

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

Although studies on wound healing, anti-aging and anti-inflammatory substances have been actively conducted, existing chemical substances are limited in their use due to various problems such as toxicity, low activity, limitations of application and side effects due to overdose In order to develop a material with a low possibility of side effects, research on extracting active ingredients from natural plants is active. However, even in the case of natural products, there are many problems in using cosmetics or medicines at an effective concentration or more in terms of safety, stability, and discoloration possibility.

Recently, a lot of studies have been made on the development of functional foods using plant resources and utilization of them as cosmetic materials. However, regarding the lilies, there is a prior patent technology related to a cosmetic composition containing an extract of Liliaceae as an active ingredient (patent document 1) However, there is no research on a method for culturing a plant cell that significantly increases the functionality of Madonna lily as a cosmetic material, a cultured plant cell culture or a cosmetic composition containing the extract itself.

Patent Document 1: Korean Patent No. 10-1207557

The present inventors have found that a plant cell culturing method of Madonna lilies and a plant cell culture or an extract thereof are useful for antioxidation, wrinkle improvement, skin moisturizing, skin whitening, skin protection against ultraviolet rays, pore contraction, sebum suppression, Aging, wound healing, etc., and completed the present invention.

Accordingly, an object of the present invention is to provide a method for culturing plant cells of Lilium Candidum.

Another object of the present invention is to provide a skin external composition for improving skin comprising a plant cell culture of Lilium Candidum or an extract thereof as an active ingredient.

In the present invention, the skin improving agent may be used for improving skin wrinkles, wound healing, skin regeneration, skin irritation, suppression, improvement, skin moisturizing, suppressing sebum secretion, inhibiting or improving acne, Prevention, relief, relief, recovery, skin barrier function protection or enhancement.

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

In order to achieve the above object, the present invention provides a method for culturing a plant cell of Lilium Candidum.

The present invention also provides a skin external composition for skin improvement comprising a plant cell culture of Lilium Candidum or an extract thereof as an active ingredient.

The present invention also provides a composition for external application for suppressing or improving skin wrinkles containing a plant cell culture of Lilium Candidum or an extract thereof as an active ingredient.

The present invention also provides a composition for skin external application for skin moisturizing comprising a plant cell culture of Lilium Candidum or an extract thereof as an active ingredient.

The present invention also provides a composition for external application for wound healing or skin regeneration comprising a plant cell culture of Lilium Candidum or an extract thereof as an active ingredient.

The present invention also provides a dermatological composition for alleviating or alleviating inflammation of the skin containing a plant cell culture of Lilium Candidum or an extract thereof as an active ingredient.

The present invention also provides a skin external composition for skin whitening comprising a plant cell culture of Lilium Candidum or an extract thereof as an active ingredient.

The present invention also provides a composition for external application for skin for preventing, alleviating, alleviating, and restoring skin damage caused by ultraviolet rays, which comprises a plant cell culture of Lilium Candidum or an extract thereof as an active ingredient.

The present invention also provides a composition for external application for skin for shrinking pores comprising a plant cell culture of Lilium Candidum or an extract thereof as an active ingredient.

The present invention also provides a skin external composition for sebum suppression comprising a plant cell culture of Lilium Candidum or an extract thereof as an active ingredient.

The present invention also provides a composition for external application for skin inhibiting or improving an acne comprising a plant cell culture of Lilium Candidum or an extract thereof as an active ingredient.

The present invention also provides a composition for external application for skin protecting or enhancing a skin barrier by ultraviolet rays containing a plant cell culture of Lilium Candidum or an extract thereof as an active ingredient.

The present invention also provides a method for preparing a skin external composition for skin improvement comprising a Lilium Candidum plant cell culture or extract thereof comprising the steps of:

(a) cultivating plant cells by separating the apical meristem, leaf or petal of Madonna lily plant; And (b) preparing a skin external composition containing the cell culture of the lily plant or an extract thereof.

In the present invention, the step (b) may be performed by drying the cell cultures of the lily plants obtained in step (a), pulverizing them and mixing them with purified water to prepare a composition, Extraction, ultrasonic extraction or hot water extraction to prepare a composition.

The present invention also relates to a method for producing Lilium Candidum (Lilium Candidum L.) which is obtained by isolating leaves, petals, or mesophyll of a lily plant and cultivating the medium in a medium containing -Benzylaminopurine (6-BAP) ) Plant cell.

In the present invention, the culture medium may be characterized by containing Myo-inositol. In the present invention, the myoinositol content may be in the range of 3 to 7 g / L.

In the present invention, the Madonna lily plant cell culture may be treated with high-frequency waves.

In the present invention, the high frequency may be a wavelength range of 220 to 260 kHz or 360 to 400 kHz, and the high frequency wave having a wavelength range of all of them may be processed.

In the present invention, the high frequency may be characterized by treating the high frequency waves after 3 to 6 weeks, preferably 4 weeks, from the date of inoculation of Madonna lily plant cells.

In the present invention, the high-frequency waves may be treated for 5 to 20 days, preferably for 1 to 10 days, and 2 to 4 times per day, and the high frequency treatment interval is preferably 10 minutes to 1 hour, It can be processed for about 40 minutes.

The method for culturing Madonna lily plant cells according to the present invention enables the mass production of plant cells of madonna lilies containing antioxidative and cosmetic excellent functions including the antioxidative ability, The composition for external application has various cosmetic effects such as improvement of wrinkles of skin, wound healing, anti-inflammation, shrinkage of pores, skin moisturization and improvement of acne without toxicity to skin cells.

Fig. 1 is a photograph showing the induction results of Madonna lily plant cells.
Fig. 2 shows the results of cytotoxicity test of the composition according to the present invention.
FIG. 3 is a graph showing the results of an increase in the biosynthesis of Procollagen Type I C-Peptide (PIP) of the composition according to the present invention.
FIG. 4 shows the skin moisturizing effect of the composition according to the present invention.
5 is a graph showing the result of a wound healing assay analysis of the composition according to the present invention.
FIG. 6 shows the anti-inflammatory effect of COX-2 gene expression inhibition by the composition of the present invention.
FIG. 7 shows the skin whitening effect of the composition according to the present invention.

The present invention relates to a plant cell culturing method of Lilium Candidum, and a composition for external application for skin containing a madonna lily plant cell culture or its extract as an active ingredient, and a method for producing the same.

In one aspect, the present invention relates to a plant cell culture method of Lilium Candidum.

In the present invention, leaves or stem tissues of the lily plants can be used separately.

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

Although there is little difference in the basic MS medium composition when inducing plant cell culture from plant leaf-derived plant stem or the like, the kind of plant growth hormone most important in the induction of plant cell is different.

In the case of plant cell culture according to the present invention, in the case of plant cell culture derived from a leaf or a stem, 6-benzylaminopurine (6-BAP) and thiamine.HCl are contained and cultured to obtain a plant cell culture .

Each combination ratio can be characterized by 6-benzylaminopurine (6-BAP) and thiamine.HCl being 1: 0.3-0.7, or 1: 0.3-0.5. That is, thiamine · HCl may be 30 to 70 parts by weight or 30 to 50 parts by weight based on 100 parts by weight of 6-benzylaminopurine (6-BAP).

For example, 6-benzylaminopurine (6-BAP) and thiamin.HCl may be contained at 0.8-1.2 mg / ml, and 0.3-0.6 mg / ml, respectively.

Such additional additives, such as myoinositol treatment and high frequency treatment, exhibit excellent functionality as a cosmetic material and have an increased antioxidant activity and an increased physiologically active substance content.

In the present invention, Madonna lily plant cell cultures are obtained by culturing a part of Madonna lily plant, for example, whole or part of apical meristem, stems, leaves and petals in a cut induction medium.

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

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

Therefore, it is possible to be derived from any part of the apical meristem, stem, or leaf of the Madonna lily according to the present invention. However, in one embodiment, it is possible to use the apical meristem, stem, petal, It may be a plant cell in which cotyledons are cut off from germinated seedlings of a tissue part or a lily seed, and a plant cell may be formed if some of the lily pieces are cut out and placed in a medium in which the content of auxin and cytokinin is regulated have.

In another aspect, the present invention relates to a composition for external application for skin for skin improvement comprising a plant cell culture of Lilium Candidum or an extract thereof as an active ingredient.

In the present invention, the term "for skin improvement" refers to an agent for improving skin condition, for example, in the case of anti-inflammation, alleviating inflammation, preventing or reducing skin aging, Skin regeneration, skin moisturization, whitening of skin, reduction of pores, enhancement of skin elasticity, suppression of skin damage by ultraviolet rays, suppression of sebum, suppression of acne, and strengthening of skin barrier function.

In the present invention, the term "containing as an active ingredient" means that the composition for external application for skin can exhibit various skin improving effects as described above, for example, wrinkle improvement, wound healing through antioxidant ability, &Lt; / RTI > by weight of the composition.

In the present invention, the plant cell or culture or the extract thereof of the lily may be contained in an amount of 0.1 to 10% by weight based on the total weight of the composition.

Such a ratio is merely a preferable range. For example, in the following examples, 1% to 5% by weight of the test results are included. However, 10% of the test results showed excellent efficacy, and the cell survival rate was also 10 %. It is obvious to a person skilled in the art that, in addition to the above, 20% and 30% or more of skin have excellent skin improving effect and antioxidative activity.

In the present invention, the skin for improving skin can be used for anti-inflammation, anti-aging, skin wrinkle improvement, wound healing, skin elasticity enhancement, skin damage suppression by ultraviolet rays, sebum suppression, acne suppression, skin regeneration, , Skin pore reduction, and the like.

In the present invention, the plant cell or culture (culture) of the Madonna lily itself is used, the culture is used by filtration, the culture is crushed or used, or the culture is dried and powdered, It can be melted and used.

In the present invention, "extract" means an extract obtained by powdering the plant cell or the culture extract of the lily, or by various known extraction methods such as cold water extraction, hot water extraction, ethanol, organic solvent and jojoba oil extraction .

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

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

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

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

In another aspect, the present invention provides a method for preparing a skin external composition for skin improvement comprising a plant cell culture of a plant of Lilium Candidum or an extract thereof:

(a) separating leaves, flowers (leaves), tissues or apical meristems of Madonna lily plants and culturing them into plant cells; And

(b) preparing a skin external composition containing the cell culture of the lily plant or an extract thereof.

The step (a) is a step of obtaining a plant tissue culture, that is, a plant cell culture, or a culture, with some tissue isolated from a Madonna lily plant. This process includes all the processes and technical characteristics described in the method for culturing Madonna lily plant cells.

In the present invention, in the step (b), a plant cell obtained through culturing in step (a) or a composition containing the culture as it is may be prepared from a suitable external preparation for skin composition, The extract can be obtained in the form of an extract through an extraction method and can be prepared into an appropriate external preparation for skin.

For example, in step (b), the plant cell culture of the lily obtained in step (a) is dried, powdered and mixed with purified water to prepare a composition,

the plant cell or culture of the lily obtained in step (a) is dried, powdered and mixed with purified water, and then extracted with cold water, hot water, jojoba oil, vegetable oil, inorganic solvent or ethanol And extracting with an organic solvent such as alcohol to prepare a composition.

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

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

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

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

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

As an embodiment of the present invention, the composition for external application for skin according to the present invention may contain glycerin, butylene glycol, propylene glycol, polyoxyethylene hardened castor oil, ethanol, triethanolamine and the like in addition to the above- Preservatives, flavors, coloring agents, purified water and the like can be included as needed.

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

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

The method for manufacturing the external composition for skin containing Madonna lily plant cell extract or extract thereof according to the present invention is not limited to the above-described manufacturing method. In some modified methods, the external composition for skin containing madonna lily plant cell culture or its extract according to the present invention can be prepared.

In particular, the composition for external application for skin may be prepared in the form of a general emulsified formulation and a solubilized formulation, by a known manufacturing method, in addition to the manufacturing method specifically disclosed in the present invention.

When the composition is prepared with a composition for external application for skin, examples of the cosmetic composition of the emulsified form include nutritional lotion, cream, essence and the like. It can also be prepared in the form of adjuvants which can be applied topically or systemically, which are conventionally used in the field of dermatology by containing a dermatologically acceptable medium or base.

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

In addition, the composition for external application for skin of the present invention may further comprise at least one selected from the group consisting of fatty substances, organic solvents, solubilizers, thickeners and gelling agents, softening agents, antioxidants, suspending agents, stabilizers, foaming agents, As used herein means any emulsion or emulsion which is commonly used in emulsifying or emulsifying agents, fillers, sequestering and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or cosmetics Adjuvants commonly used in the cosmetics or dermatological fields, such as other ingredients. And, the above ingredients can be introduced in amounts commonly used in the field of dermatology.

The composition for external application for skin according to the present invention is useful as an anti-aging agent for preventing or treating skin aging, skin moisturization, skin pore reduction, skin convergence, anti-inflammation, wound healing, skin regeneration, skin whitening, Skin barrier function enhancement, and the like.

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

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

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

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

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

Example 1-1: According to the present invention, From an apical meristem Plant cell Judo

The Madonna lily plant used in the present invention was obtained from Sangbong-myeon, Jinju-si, Gyeongsangnam-do and dipped in 70% ethanol for 30 seconds, washed several times with sterilized water, disinfected with disinfectant solution (30% lactose + 0.05% Tween 20) And washed three times with water. The tissues were taken out of the apical meristems of the sterilized Madonna lily plants, and the tissues were taken out from the apical meristems of the sterilized Madonna lily plants to obtain 1 mg / L 6-benzylaminopurine, 0.4 mg / L Thiamine.HCl, 3% Sucrose and Gelite 2.3 (Murashige and Skoog 1962, Duchefa, Cat. No. M0221) containing 10% g / L of L-glutamic acid at a temperature of 25 ° C and a humidity of 70%. Each medium was adjusted to pH 5.8 with 1 N sodium hydroxide (NaOH). Subculture was performed at intervals of two weeks.

Example 1-2: Induction of plant cells from leaves of Madonna lily plants according to the present invention

The leaves of sterilized Madonna lily plants were wounded with a sharp knife at a time to obtain 1 mg / L 6-benzylaminopurine, 0.4 mg / L Thiamine · HCl, 3% Sucrose and Gelite 2.3 g / L (Murashige and Skoog 1962, Duchefa, Cat. No. M0221), which was cultured under a growth condition at 25 ° C and a humidity of 70% to induce early plant cells. Each medium was adjusted to pH 5.8 with 1 N sodium hydroxide (NaOH). Subculture was performed at intervals of two weeks.

Example 1-3: Derivation of plant cells from flower of Madonna lily plant according to the present invention

The petals of the sterilized Madonna lily plants were scratched at once with a sharp knife to obtain a standard MS medium (Murashige and L.) containing 1 mg / L 6-benzylaminopurine, 0.4 mg / L Thiamine HCl, 3% sucrose and 2.3 g / Skoog 1962, Duchefa Co., Cat No. M0221) under an incubation condition of 25 ° C and 70% relative humidity. Each medium was adjusted to pH 5.8 with 1 N sodium hydroxide (NaOH). Subculture was performed at intervals of two weeks.

Examples 1-4: Mass production of plant cells according to the Madonna lily region according to the present invention

In order to mass-produce aseptically induced cells from apical meristems, leaves and petals of Madonna lily plants, MS medium was cultured on medium containing 1 mg / L 6-benzylaminopurine and 0.4 mg / L Thiamine HCl The culture conditions were incubated for 5 weeks at an air supply of 0.1 vvm in a 20 L balloon type bioreactor (Samsung Sci.) Under conditions of a temperature of 25 ± 2 ° C. and a humidity of 70%.

Example 1-5: Increasing activity according to further treatment of Madonna lily plant cells according to the present invention

An object of the present invention is to add an additive to MS base medium or to use high-frequency treatment as a method for increasing activity of apical meristem, leaf and petal plant cells derived from Madonna lily plant.

1) Addition of additives to MS base medium

In order to increase the activity of apical meristem, leaf and flower plant cells derived from Madonna lily plants, 5 g / L Myo-Inositol (Duchefa, Cat no.) Was added to MS base medium containing 30 g / L Sucrose I0609) was cultured in a culture room of 25 占占 폚 and 70% of humidity (culture conditions were the same as in Example 1-4).

The content of carotenoids, flavonoids and phenolic compounds, which are secondary metabolites, was checked as follows. The measurement method is as follows.

The total carotenoid content was measured according to the AOAC method. (1: 9, v / v) mixture of acetone: nucleic acid (3: 7, v / v) was added to the lyophilized Madonna lily plant cell sample, and the extract was filtered, washed with distilled water, And the absorbance was measured at 436 nm with acetone: nucleic acid (1: 9, v / v) mixture while the extract was injected and aspirated.

Total flavonoid content was determined by the method of Sakanaka et al. (2005) based on the colorimetric method. 1.25 ml of distilled water was added to 0.25 ml of Madonna lily plant cell culture extract and standard solution, and 0.075 ml of 5% (w / v) NaNO ₂ solution was added. After 6 minutes, 0.15 ml of a 10% (w / v) AlCl ₃ solution was added to the mixture, left for 5 minutes, and then 0.5 ml of 1 N NaOH solution was added. The distilled water was added so that the final volume of the mixed solution became 2.5 ml, and the absorbance was measured at a wavelength of 510 nm. (±) catechin standard solution (Sigma Chemical Co., St. Louis, Mo., USA) was used as the reference material. The total flavonoid content was expressed as mg · g ^-1 · DW.

Total phenol content was determined by the method of Ali et al. (2006a) based on the Foline-Ciocalteu method (Folin and Ciocalteu, 1927) in which the Foline-Ciocalteu reagent was reduced to molybdenum blue by reduction of the total phenolic material of Madonna lily plant cell culture extract And the total phenol content was measured. After adding 2.55 ml of distilled water to 0.05 ml of extract and standard solution, 0.1 ml of 2N Folin-Ciocalteu reagent solution (10 time dilution; Sigma chemical CO., St. Louis, Mo., USA) was added. After 6 minutes, 0.5 ml of a 20% (w / v) Na2CO3 solution was added to the mixed solution, and the mixture was allowed to stand for 30 minutes in a dark state and absorbance was measured at a wavelength of 760 nm. Garlic acid was used as a reference material and total phenol content was expressed as mg · g ^-1 · DW.

(Unit: mg · g ^-1 ) Myo-Inositol
(/ L) Induction site Carotenoid content Flavonoid content Phenolic compound content - 104 106 100 5g Apex divisional organization 142 140 138 leaf 139 133 135 petal 145 143 142

② Antioxidant test with DPPH free radical scavenging ability

Compound 1, 1-diphenyl-2-picryl hydrazyl (DPPH, Sigma D9132-1G, USA) produces free radicals in ethanol, which has been shown to be free radicals in the apical meristems derived from the Madonna lilies produced in the examples according to the present invention, (Free radical scavenging activity test) of the free radical scavenging activity of leaf and flower plant cell culture extracts. The free radical scavenging activity was modified by the method of Kim et al. (Kor. J. Pharmacogn., 24 (4), 299-303 (1993)) and the stable free radical DPPH (1,1-diphenyl- picryl hydrazyl, Sigma D9132-1G, USA) reagent was used.

150 μl of the test sample was diluted with 150 μl of a 0.2 mM DPPH solution (ethanol in the case of Blank), and 150 μl of the diluted sample was added. After incubation at room temperature for 30 minutes, the absorbance at 517 nm was measured. Was used. After the absorbance of the test group and the control group was measured, the degree of antioxidant activity was expressed as a percentage based on the absorbance of the control group using pure water.

Culture conditions
Myo-Inositol
(/ L) process
density Induction site Free radical activity inhibition (%) - 0 - One % Madonna lily
extract 12 5g Apex divisional organization 52 leaf 69 Flower 55

As a result, the Madonna lily plant cell culture extract cultured in the medium supplemented with 5 g / L myoinositol showed an inhibition rate of free radical activity, that is, an antioxidant effect, which is far superior to the Madonna lily extract at 1% concentration.

2) Culture on MS base medium and high frequency treatment

The present inventors treated high-frequency waves with apical meristems, leaves and petal plant cells derived from the above-mentioned Madonna lily plant. Specifically, the plant cells derived from the Madonna lily plant were cultivated in a bioreactor (Samsung Science Co., Ltd.) for 4 weeks and then connected to a high frequency wave generator to produce 240 (± 10) kHz or 380 For 3 or 30 minutes.

The contents of carotenoids, flavonoids and phenolic compounds as secondary metabolites, which are physiologically active substances, were checked as follows.

(Unit: mg · g ^-1 ) RF 처리
(Intensity: 20 J / cm 2) Induction site Carotenoid content Flavonoid content Phenolic compound content Not treated (Control) Apex divisional organization 2.40 3.30 80.42 Flower 2.44 3.98 91.85 leaf 2.52 4.01 75.78 240 (± 10) kHz

30mins / times x 3 times / day x 3day Apex divisional organization 25.14 39.42 250.68 Flower 12.00 59.00 320.22 leaf 26.32 20.27 190.96 380 (± 10) kHz

30mins / times x 3 times / day x 3day Apex divisional organization 32.88 50.88 92.99 210.48 38.04 Flower 49.69 368.62 82.97 leaf 30.52

② Antioxidant test with DPPH free radical scavenging ability

process
density RF 처리
(Intensity: 20 J / cm 2) Induction site Free radical activity inhibition (%) One % - Madonna lily
extract 12 Not treated (Control) Apex divisional organization 12 leaf 10 Flower 15 240 (± 10) kHz

30mins / times x 3 times / day x 3day Apex divisional organization 42 leaf 69 Flower 85

As a result, it was confirmed that the extract of Madonna lily plant cell cultured at high frequency had an inhibition rate of free radical activity, that is, an antioxidant effect, which is far superior to Madonna lily extract at 1% concentration.

Example 1-6: Preparation of Extract of Madonna Lily Plant Cell Culture According to the Present Invention

The cultivated cells, leaf and flower plant cell cultures derived from Madonna lily plants cultivated in the examples were harvested, washed several times, and dried in a vacuum freeze dryer (Ilshin, Cat no. LP-50) for 3 days. 1 Kg of dried Madonna lily plant cell culture was put into a container, and 10 L of purified water was added thereto, followed by hot water extraction at 90 to 120 ° C for 48 hours. After extraction, the mixture was filtered with a mesh to remove solid content, and a culture extract was used in the present invention. For comparative experiments, 70% ethanol was extracted under the same conditions as the hot water extraction with ethanol for the ethanol extraction.

Experimental Example 1: Cell Viability for skin cells

Human fibroblast (CCD-986Sk) was cultured in an ATCC (American Type Culture Collection) in order to examine the skin cell survival rate of the composition prepared in the examples. Each cell was inoculated into a 24-well culture plate at a concentration of 1 × 10 ⁴ cells / ml. The medium was DMEM (Dubelco's Modified Eagle Medium, BRL, USA) containing 10% FBS. After culturing in DMEM containing 10% FBS for 48 hours and culturing by 25 to 30% of the surface area of the culture dish, the composition prepared in Example was replaced with FBS-free DMEM containing 0.1 to 5% and further cultured for 24 hours . 50 μl of a solution of 2.5 mg / ml of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT, Sigma M5655, USA) (DMSO, Sigma D2650, USA) was added to each well, and the formazan crystals formed were dissolved by stirring for 20 minutes. Then, 100 μl of each of the formazan crystals was dissolved in 96 wells And the absorbance at 570 nm was measured by Enzyme-Linked Immunosorbent Assay (ELISA). The survival rate of skin cells was calculated by the following formula based on the absorbance of the control group using pure water and expressed as a percentage. The results are as follows.

[Equation 1]

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

As shown in FIG. 2, the cytotoxicity of human skin fibroblast (CCD-986Sk) was examined by 0.1, 1.0, 3.0 and 5.0% And did not show cytotoxicity. That is, it did not affect cell viability.

Experimental Example 2: Improvement of wrinkles by promotion of procollagen synthesis

Human fibroblasts (Human Skin Fibroblast) to 37 degrees, 5% CO ₂ incubator of 10% in the cell culture medium to maintain a constant humidity FBS, Penicillin (50U / ml) , Streptomycin DMEM (Dulbecco's containing (50 / ml) Modified Eagle's Medium, Gibco, USA). Cells were inoculated into 48-well plates at 1 × 10 ⁵ cells / ml in ^500- μl aliquots and cultured for 24 hours. The medium containing the control (DMEM medium) and the plant cell culture extract of Madonna lily flower at a concentration of 0.1-5% was added and cultured in a 5% CO ₂ incubator at 37 ° C for 48 hours. After 48 hours, the amount of newly synthesized collagen was measured by measuring 20 ul of each supernatant of the culture medium using Procollagen Type I C-Peptide EIA Kit (PICP, Takara, Cat No. MK101).

As shown in FIG. 3, it was confirmed that the collagen biosynthetic amount was increased at the 1% content of the hot water extract of Madonna lily flower plant cell culture extract, and the collagen biosynthetic amount increased at 3% and 5% . Thus, it can be seen that the composition of the present invention exhibits excellent collagen synthesis enhancing effect.

Experimental Example 3: Moisturizing effect by increasing AQP3 expression

Human keratinocyte line (HaCaT) was cultured in 100 mm / 60.1 cm² culture dish with Dulbecco's Modified Eagle's Medium (DMEM), 10% Fatal bovine serum (FBS) and 1% Antibiotic-Antimycotic (GIBCO, Cat. # 15240-062) And cultured at 37 ° C and 5% CO 2. When human keratinocytes are confluent at a concentration of 80% or more, the cells are seeded at 1 × 10 4 cells / well in a 96-well plate. Cell culture is continued until confluence reaches 80% or more. When confluence reaches 80% Treat the test material after exchange with medium (without FBS). After 24 hours of material treatment, further culture is performed to see how each substance affects the expression of Aquaporin3.

The test method is Real-time PCR, and the test procedure is as follows. RNA isolation is performed using FastLane Cell one-step buffer set (QIAGEN Cat. # 216213). The cells were washed with FCW (cell wash buffer), and incubated at room temperature for 5 minutes with 50 μl cell processing mixture (Buffer FCPL-supplemented Buffer FCPW, gDAN Wipeout buffer) After transferring, react at 75 ℃ for 5 minutes. In order to analyze Aquaporin3 gene related to moisturization, the RNA extracted from the above was used as a template and subjected to a Rotor Gene Q Real-time PCR Machine (Qiagen) according to the manual of 2X QuantiTect SYBR Green RT-PCR Master Mix. The primers used in the experiments were normalized with Qiagen's QuantiTect® primerassays (Aquaporin3, Cat. # QT00212996) and GAPDH (Cat. # QT01192646). Real-time PCR conditions are as follows.

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

Expression (fold) = (Expression rate of sample treated group) / (Expression rate of control group)

As shown in FIG. 4, as the content of the treatment concentration of the plant extract of Madonna lily flower of the present invention increased by hot water extraction and ethanol extraction, the AQP-3 (Aquaporin-3) gene biosynthesis And the increase in the amount of water.

Experimental Example 4: Wound healing effect

The wound healing process is an important process involved in inflammation, new tissue formation, and remodeling. Skin cell migration is essential for wound healing. To investigate the effect of Madonna lily flower plant cell culture extract on wound healing in human skin cells, HaCaT cells were treated with plant extracts of Madonna lily flower cell cultures and the degree of wound healing was measured. EGF, known to be effective for wound healing as a positive control, was used.

Specifically, human epidermal keratinocyte (HaCaT) was inoculated into a 6-well dish and cultured at 37 ° C, 5% CO 2 until the cells became 100% on the dish surface. After incubation, the cell surface was scratched with a 100 μl pipette tip and wounded. The cells were rinsed once with the new culture medium, and the cells were stained with EGF (Sigma-Aldrich, St. Louis, Mo., USA) The culture broth (2 ml) containing the plant cell culture extract at a concentration of 1% was added and cultured. During the treatment, the concentration of EGF was 100 ng / ml in 2 ml of the culture. After a certain incubation time, cell images were measured at 10 magnification using an AxioObserver FL microscope (Advanced Microscopy Group, Bothell, WA, USA). At the wound site, the cell image was measured in more than three sections, and the percentage of wound closure was calculated using ImageJ software. All experiments were repeated three times.

In Fig. 5, the left side shows the T-20 control group (without treatment of the flower plant cell culture extract) after 24 hours after the wound. As a result, it was confirmed that the plant cell culture extract of Madonna lily flower stimulates wound healing mechanism in human skin cells to promote wound healing.

Experimental Example 5: Inhibition of inflammation by inhibition of COX-2 expression

Human keratinocytes were seeded at 2 × 10 ⁵ cells / well in a 24-well plate, cultured for 24 hours, and treated with UVB irradiation (50 mJ / cm 2). As a positive control, 5 μM hydrocortisone (Sigma, MO, US) was treated. (COX-2) expression after 4 hours of additional culturing.

The test method is Real-time PCR, and the test procedure is as follows. RNA isolation is performed using FastLane Cell one-step buffer set (QIAGEN Cat. # 216213). The cells were washed with FCW (cell wash buffer), and incubated at room temperature for 5 minutes with 50 μl cell processing mixture (Buffer FCPL-supplemented Buffer FCPW, gDAN Wipeout buffer) After transferring, react at 75 ℃ for 5 minutes. In order to analyze COX-2 gene related to inflammation, the RNA extracted from the above was used as a template and subjected to a Rotor Gene Q Real-time PCR Machine (Qiagen) according to the manual of 2X QuantiTect SYBR Green RT-PCR Master Mix. The primers used in the experiments were normalized to Qiagen's QuantiTect® primerassays (COX-2, Cat. # QT00040586) and GAPDH (Cat. # QT01192646). Real-time PCR conditions are as follows.

As shown in Fig. 6, inhibition of COX-2 gene expression was confirmed at a content of 3% of hot-water extract of Madonna lily flower cell culture extract. Therefore, it was confirmed that the composition according to the present invention has anti-inflammatory activity such as relieving, improving and inhibiting inflammation.

Experimental Example 6: Whitening effect by inhibition of MITF and TYR expression

Human keratinocytes are seeded at a density of 1 × 10 ⁵ cells / well in a 96-well plate, cultured for 24 hours, treated with 100 μM α-MSH and the appropriate concentration, and cultured for 3 days. Related transcription factor (MITF) and tyrosinase (TYR) expression associated with the whitening effect.

The test method is Real-time PCR, and the test procedure is as follows. RNA isolation is performed using FastLane Cell one-step buffer set (QIAGEN Cat. # 216213). The cells were washed with FCW (cell wash buffer), and incubated at room temperature for 5 minutes with 50 μl cell processing mixture (Buffer FCPL-supplemented Buffer FCPW, gDAN Wipeout buffer) After transferring, react at 75 ℃ for 5 minutes. The extracted RNA was used as a template and subjected to a Rotor Gene Q Real-time PCR Machine (Qiagen) according to the manual of 2X QuantiTect SYBR Green RT-PCR Master Mix. The primers used in the experiments were normalized with Qiagen's QuantiTect® primerassays (MITF, Cat. # QT00037737), TYR, Cat. # QT00080815 and GAPDH (Cat. # QT01192646). Real-time PCR conditions are as follows.

As shown in FIG. 7, both the hot water extraction and the ethanol extraction of Madonna lily flower cell culture extract inhibited the gene expression of MITF and TYR at a treatment concentration of 0.1% or more, thereby exhibiting a whitening effect. As a positive control, kojic acid was used.

Experimental Example 7: Effect of ultraviolet radiation on cell protection

Ultraviolet crosslinker CL-1000 (Ultra-Violet Products, CA), which emits ultraviolet rays at a wavelength of 302 nm, was used as a light source for UV irradiation. HaCaT cells were cultured in a 24-well plate at a concentration of 1 × 10 ⁵ cells / ml for 24 hours, and then the medium was removed and washed with a phosphate buffer solution. After adding 500 μL of phosphate buffer, the cells were irradiated with ultraviolet light at 10 mJ / cm 2, replaced with fresh cell culture medium containing no FBS, treated with 1% concentration of the sample, and further cultured for 24 hours. After 5 mg / ml of MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, Sigma) was added and incubated in an incubator for 3 hours, the fomazan formed was dissolved in DMSO, After transferring to a 96-well plate, the absorbance was measured with an ELISA reader at 595 nm, and cell viability was compared with that of the untreated control group after UV irradiation.

division Treatment concentration (%) Presence of ultraviolet radiation Cell protection effect by ultraviolet irradiation
(% of Control) Control group - - 100 Control group - + 52 Madonna lily
extract 0.1 51 1.0 50 5.0 51 Madonna lily
Apex divisional organization
Plant cell
Culture extract 0.1 68 1.0 75 5.0 89 Madonna lily
Leaf plant cell
Culture extract 0.1 53 1.0 67 5.0 78 Madonna lily
Flower plant cell
Culture extract 0.1 67 1.0 75 5.0 82

To evaluate the keratinocyte protective effect of Madonna lily plant cell extracts, human keratinocytes (HaCaT) were exposed to ultraviolet light at 20 mJ / cm 2 and then cultured for additional 24 hours. MTT As a result of the assay, the cell viability of the Madonna lily plant cell extract increased compared with Madonna lily extract. Therefore, the cell protection effect of Madonna lily plant cell culture extract was confirmed by ultraviolet irradiation.

Experimental Example 8: In vitro Test

In order to measure the pore-shrinking effect of the Madonna lily plant cell extract composition of the present invention, the composition prepared in the above example was tested for the pore-shrinking effect using hemoglobin as a protein to replace skin.

Hemoglobin solution (0.05g / 50ml) was prepared with 0.9% Phosphate Buffer Saline (0.1mM, pH 7.4). 2ml of hemoglobin solution was added with 2ml of Madonna lily plant cell culture extract, mixed with shaking for 30 seconds, For 10 minutes, 2 ml of purified water was added to 1 ml of the supernatant, and the resultant was measured in a UV-Visible spectrum at 407 nm. As a control, 2 ml of PBS (Phosphate Buffer Saline) was added.

Concentration (%) Precipitation of hemoglobin (%) PBS
(Negative control) 0 0.1 Tannic acid
(Positive control group) One 36 Madonna lily
extract 0.1 One 1.0 3 5.0 3 Madonna lily
Apex divisional organization
Plant cell culture extract 0.1 11 1.0 18 5.0 31 Madonna lily
leaf
Plant cell culture extract 0.1 6 1.0 20 5.0 25 Madonna lily
Flower
Plant cell culture extract 0.1 21 1.0 38 5.0 42

The shrinkage effect was evaluated by measuring the amount of hemoglobin deposited in the composition containing the extract composition of Madonna lily plant cell culture at a concentration of 0.1 to 5.0%, and it was determined that the higher the sedimentation rate (%) of hemoglobin, the better the shrinkage effect of pores. In other words, compared to the Madonna lily extract, it was confirmed that the apical meristem, leaf and flower culture extracts derived from the Madonna lily plant had excellent pore shrinking effect.

Experimental Example 9: Effect of inhibiting sebum

In order to confirm the sebum inhibitory effect of the Madonna lily plant cell culture extract, sebum secretion amount of skin was measured using a skin oil analyzer (Sebum meter SM810) 4 weeks after the test. Experiments were performed 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, and the experimental results are shown in the table.

sample Sebum suppression effect 0 day (before use) 28 days (after use) Control group 230 223 One 1% Madonna Lily Extract 231 193
2 1% madonna lily apex divisional tissue
Plant cell culture extract 230 173
3 1% Madonna lily leaves
Plant cell culture extract 232 161
4 1% madonna lily flower
Plant cell culture extract 230 155

As shown in the above results, the 1% Madonna lily plant cell culture extract showed a superior inhibitory effect on sebum secretion compared with the Madonna lily extract, as compared with the control group.

Experimental Example 10: Effect of improving acne

Acne is a skin lotion prepared in Example 1 as a target man and woman 10 people occurred in the morning, a full four weeks to face the evening of 1% Lt; RTI ID = 0.0 > of Madonna < / RTI > lily flower cell culture extract. The results of the evaluation of the acne healing effect were evaluated according to the degree of the person who participated in the experiment.

&Lt; Evaluation of state before test >

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

&Lt; Determination of acne effect &

-: Little effect, +: Little effect, ++: Much relaxation,

+++: fully healed

Subject Pre-test condition Effect after 2 weeks Effect after 4 weeks Subject 1 2 + + Subject 2 One + ++ Subject 3 One - + Subject 4 2 - - Subject 5 2 + ++ Subject 6 2 + + Subject 7 3 ++ +++ Subject 8 2 - ++ Subject 9 3 + + Subject 10 2 + ++

As shown in Table 11 above, the improvement effect of acne was confirmed in the majority of subjects after using the soft lotion containing 1% Madonna lily flower cell culture extract for 4 weeks.

Experimental Example 11: Skin barrier function test

Human epidermal keratinocytes were incubated with Dulbecco's Modified Eagle's Medium (DMEM), 10% Fatal bovine serum (FBS) and 1% Antibiotic-Antimycotic (GIBCO, Cat. # 15240-062) culture dishes at 37 ° C and 5% CO 2.

When the human epidermal keratinocytes were confluent at a concentration of 80% or more, the cells were cultured for 24 hours at a density of 3 × 10 6 cells / well in a 12-well plate. Lt; / RTI > for 24 hours. When human epidermal keratinocytes are confluent at a concentration of 80% or more, they are divided into 96 well plates at a density of 1 × 10 4 cells / well and treated with the cell culture extracts of Madonna lily plants. After 24 hours of incubation, the expression of filaggrin gene was confirmed by real-time PCR.

RNA isolation and cDNA synthesis are performed using FastLane Cell one-step buffer set (QIAGEN, Cat. # 216213). The cells were washed with FCW (cell wash buffer), and incubated at room temperature for 5 min with 50 μl cell processing mixture (Buffer FCPL supplemented with Buffer FCPL and gDAN Wipeout buffer) And reacted at 75 ° C for 5 minutes. In order to analyze the expression of the filaggrin gene, the cDNA synthesized above is used as a template and subjected to a Rotor Gene Q Real-time PCR Machine (Qiagen) according to the manual of 2X QuantiTect SYBR Green RT-PCR Master Mix. The filaggrin primer used in the experiment was Qiagen Cat. # QT01192646) and the expression rate was Normalization with Housekeeping genes (GAPDH, Cat. # QT01192646). Real-time PCR conditions are as follows.

division Treatment concentration (%) Filaggrin gene expression rate
(Relative Expression of Filaggrin) Control group - 1.00 Madonna lily
extract 0.1 0.78 1.0 0.83 5.0 0.92 Madonna lily
Apex divisional organization
Plant cell
Culture extract 0.1 1.54 1.0 2.19 5.0 2.55 Madonna lily
leaf
Plant cell
Culture extract 0.1 1.09 1.0 1.29 5.0 1.57 Madonna lily
Flower
Plant cell
Culture extract 0.1 1.77 1.0 1.99 5.0 2.3

Increased expression of filaggrin promotes the normal differentiation of keratinocytes by the apical meristem, leaf and flower plant cell culture extracts derived from the Madonna lily plant and promotes the expression of filaggrin in the skin, which is converted into a natural moisturizing factor in the skin Skin barrier function can be improved. That is, the expression of filaggrin gene in Madonna lily apical meristem, leaf and flower cell culture extracts showed that the gene expression rate of madonna lily plant cell culture extract was higher than that of Madonna lily extract Respectively.

Preparation of composition for external application for skin

Cosmetics of emulsified form such as nutritional lotion, cream, essence, etc., and cosmetics of solubilized form such as softened longevity were prepared with the cosmetic containing the culture extract of lily plant of the present invention as an active ingredient.

Production Example 2-1: Lotion

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

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

Production Example 2-2: Essence

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

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

Production Example 2-3: Lotion

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

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

Production Example 2-4: Cream

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

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

Production Example 2-5: Gel

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

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

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

Claims

Lilium Candidum plant cells which are cultured in a medium containing 6-benzylaminopurine (6-BAP) and thiamine HCl by separating leaf, petal, or apical meristem of Madonna lily plant Lt; / RTI >

The method according to claim 1,
Wherein the culture medium contains myoinositol.

The method according to claim 1,
Wherein the medium contains myoinositol in a range of 3 to 7 g / L.

The method according to claim 1,
A method for culturing a plant of Madonna lily plant cell characterized by high frequency treatment of the plant cell culture.

5. The method of claim 4,
Wherein the high frequency has a wavelength range of 220 to 260 kHz or 360 to 400 kHz.

A plant skin cell culture of Lilium Candidum cultured according to the method of claim 1, or an extract thereof.

The method according to claim 6,
The skin improving agent is used for skin wrinkle suppression or improvement, for alleviating or improving inflammation of the skin, for reducing pores, for whitening skin, for suppressing sebum secretion, for suppressing or improving acne, for suppressing skin damage by ultraviolet rays, For skin regeneration or for skin barrier function protection or enhancement.

8. The method of claim 7,
Wherein the extract is selected from the group consisting of organic solvent extracts, ultrasonic extracts or hot water extracts selected from the group consisting of methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform and ethyl acetate of plant cell cultures of Lilium Candidum Wherein the external preparation is a composition for external application for skin.

A method for producing a skin external composition for skin improvement according to claim 6, comprising a culture of plant cells of Lilium Candidum or an extract thereof containing the following steps:
(a) isolating leaf, petal, or apical meristem of a Madonna lily plant and culturing the plant cell; And
(b) preparing a skin external composition containing the Madonna lily plant cell culture or an extract thereof.

10. The method of claim 9,
In step (b), the cell culture of Madonna lily plant cell obtained in step (a) is dried, powdered and mixed with purified water to prepare a composition, or powdered and mixed with purified water, followed by organic solvent extraction, ultrasonic extraction Or hot water extraction to produce a composition.