KR101693923B1 - Anti-inflammation and Anti-aging composition for skin external application comprising Zostera marina Cell Culture Extract and Methods for preparing the Same - Google Patents

Abstract

The present invention relates to a composition for external application for skin having anti-inflammatory and anti-aging effects containing a plant cell culture extract of Zostera marina and to a method for preparing the same, and more particularly to a plant cell culture derived from a rare plant, Anti-aging dermatological composition containing the adventitious roots culture or extract thereof, and a method for producing the same.
INDUSTRIAL APPLICABILITY The herb plant cell or adventitious root culture or the composition for external application of skin containing the extract according to the present invention has the effects of improving skin wrinkles, whitening, shrinking of pores, suppressing secretion of sebum and improving acne without toxicity to skin cells.

Description

[0001] The present invention relates to a composition for external application for skin having anti-inflammatory and anti-aging effects and a method for preparing the same,

The present invention relates to an anti-inflammatory, anti-aging dermatological composition containing a Zostera marina plant cell culture extract and a method of preparing the same, and more particularly, to a plant cell culture derived from a zucchini plant, To a composition for external application for skin having anti-aging, whitening, anti-inflammation, suppression of sebum, inhibition of acne and protection of skin damage from ultraviolet rays, and a preparation method thereof

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

On the other hand, it refers to the plants that live well adapted to the seawater as the well - bloomed flowering plants, and there are a total of 8 species of bark in our country. It plays a very important role in improving the productivity of the coastal fisheries by providing food, habitat and spawning ground for many marine animals with economical value in the coastal and estuarine ecosystem based on well-bred high productivity. In addition, The underground tissue plays a very important role in improving the quality of water and purification of the coastal environment by stabilizing the subsurface structure. The research on the alfalfa is only started in the world only a few decades.

In recent years, many studies have been made on the development of functional foods using plant resources and the utilization of them as cosmetic materials. However, there are prior patented technologies (Patent Document 1) in which the bark extract is used as an active ingredient of cosmetic composition, There is no research as a cosmetic material for the plant cell, the adventitious root culture or the extract thereof cultured therefrom.

Patent Document 1: Korean Patent Publication No. 10-2003-0086933

The inventors of the present invention have confirmed that plant cells, adventitious roots, or extracts thereof of the alfalfa plant are effective for anti-inflammation, anti-aging, whitening and the like, and completed the present invention.

Accordingly, an object of the present invention is to provide a skin external composition for improving skin comprising plant cells of Zostera marina , cultured adventitious roots or extract thereof as an active ingredient.

In the present invention, the skin improving agent may be used for skin wrinkle improvement, whitening, skin inflammation relief, inhibition, improvement, suppression of sebum secretion, inhibition or improvement of acne, prevention of skin damage by ultraviolet rays, .

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

It is still another object of the present invention to provide a method of propagating and transplanting the bark of the bark.

In order to achieve the above object, the present invention provides a composition for external application for skin for improving skin comprising plant cell of Zostera marina , cultured adventitious roots or extract thereof as an active ingredient.

The present invention also provides a composition for preventing or improving skin wrinkles comprising Zostera marina plant cells, adventitious roots cultures or extracts thereof as an active ingredient.

The present invention also provides a whitening dermatological external preparation composition comprising, as an active ingredient, a plant cell of Zostera marina , a culture of adventitious roots or an extract thereof.

The present invention also provides a composition for alleviating or alleviating inflammation of the skin containing plant cells, adventitious roots or extract thereof of Zostera marina as an active ingredient.

The present invention also provides a composition for skin external application for shrinking pores containing plant cells of Zostera marina , cultured adventitious roots or an extract thereof as an active ingredient.

The present invention also provides a composition for external skin application for sebum suppression containing plant cell of Zostera marina , culture of adventitious roots 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 Zostera marina plant cells, adventitious roots cultures or extracts 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 containing plant cells, adventitious roots or extracts thereof of Zostera marina as an active ingredient.

The present invention also provides a method for preparing a skin-improving external skin application composition containing Zostera marina plant cells or adventitious roots cultures or extracts thereof, comprising the steps of:

(a) isolating leaves or stem tissues of a bark plant and culturing the plant cells or adventitious roots; And (b) preparing a composition for external application for skin containing the bark plant cell culture or the adventitious root culture or the extract thereof.

In the present invention, the step (a)

(i) The leaves or stem tissues of the perennial plants are separated to obtain 6-benzylaminopurine (6-BAP), 2,4-dichlorophenoxyacetic acid, 2,4-D ) To obtain a plant cell culture, or alternatively,

(ii) isolating the leaf or stem tissue of the perennial plant and culturing it in a medium containing indole butyric acid (IBA) to obtain an adventitious culture,

.

In the present invention, the method may further comprise the step of culturing in the step (a) in which UVA is treated for 3 to 6 hours or adding 150 mM to 400 uM medium to the medium.

In the present invention, the step (b) may further comprise the steps of: drying the bark plant cell culture or the adventitious root culture obtained in step (a), pulverizing and mixing the resultant with purified water to prepare a composition; Next, the method may include a step of preparing a composition by ultrasonic extraction or hot water extraction.

The present invention provides a method of propagating a zucchini plant characterized by using plant cells of Zostera marina comprising the following steps:

(a) extracting a leaf or stem section of a herbaceous plant;

(b) The extracted leaves or stem sections were cultured in MS medium (Murashige and Skoog) containing 0.5-1 mg / L 6-benzylaminopurine and 0.3-0.6 mg / L 2,4-D Culturing the bacteriophage cell culture according to claim 1;

(c) inducing shoots by culturing the bovine plant cell culture of step (b) in MS medium containing 0.2-0.5 mg / L zeatin; And

(d) inducing shoots induced by step (c) in MS medium containing 0.2-0.5 mg / L indole-3-butyric acid (IBA).

The present invention also provides a method for transplanting an alfalfa plant, characterized in that the alfalfa plant obtained according to the method is transplanted into a river or sea.

The present invention relates to a composition for preventing or treating dermatophyte or adventitious roots or an external skin composition containing the extract of the present invention, which is free of toxicity to skin cells and can reduce skin wrinkles, reduce pores, reduce skin whitening, Efficacy and so on. Also, mass proliferation is possible through the method of propagation of the herbaceous plant according to the present invention.

1 is a photograph showing the appearance of a bare-bodied adult plant.
Fig. 2 is a photograph showing the process of proliferating the herbaceous plant.
Fig. 3 is a photograph of a plant cell derived from an alfalfa plant according to the present invention.
FIG. 4 is a photograph showing a large-scale culture process of a plant cell derived from a bivalinized plant according to the present invention in a 5 L bioreactor.

The present invention relates to a composition for external application for skin containing plant cells or adventitious roots of Zostera marina or an extract thereof as an active ingredient, and a process for producing the same.

In one aspect, the present invention relates to a composition for external application for skin for improving skin comprising plant cell or adventitious roots of Zostera marina or an extract thereof as an active ingredient.

In the present invention, the term "for skin improvement" is intended to improve the condition of the skin, for example, in the case of anti-inflammation, alleviation of inflammation, prevention, alleviation, anti-aging, improvement of skin wrinkles, whitening, reduction of pores, Inhibition of sebum, inhibition of acne, and the like.

In the present invention, the term "comprising as an active ingredient" means a composition capable of exhibiting various skin improving effects as described above, such as wrinkle improvement, whitening through antioxidant ability, anti- ≪ / RTI >

In the present invention, a bark plant cell culture is obtained by culturing a part or all of a stem of an alfalfa plant, or all or part of a leaf, in a cut-off inducing 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 induce any part derived from any part such as the stem of the bark of the present invention or the leaf, but in one embodiment, the cotyledon is cut from the stem of the bark of the alfalfa and part of the leaf, Derived plant cells, and plant cells may be formed by cutting off some of the tissues of alfalfa and placing the contents of auxin and cytokinin in a regulated medium. In the case of adventitious root cultivation, the same process as that of plant cell cultivation is carried out, but the combination of plant hormones is different so that the adventitious roots can be induced.

In the present invention, the plant cell or adventitious roots or extract thereof 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, it is confirmed that 0.1% by weight to 5% by weight of the test results are included. However, it is confirmed that 5% Survival rate was also not affected by 10%. It is obvious to a person skilled in the art that even in the case of 20% and 30% or more, excellent skin improving effect, wrinkle improvement, whitening, anti-inflammatory function and the like are apparent.

In the present invention, the skin improving agent includes anti-inflammatory, anti-aging, skin wrinkle improvement, whitening, skin elasticity enhancement, skin damage suppression by ultraviolet rays, sebum suppression and acne suppression.

In the present invention, it is preferable to use the plant cell or the adventitious root culture (culture) itself, to use the culture by filtration, or to crush the culture, or to dry the culture to obtain a powder, It can be melted and used.

In the present invention, "extract" means an extract obtained by powdering the plant cell or adventitious root culture extract of the herpes zoster or by various known extraction methods such as cold water extraction method, hot water extraction method, ethanol and jojoba oil extraction method.

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

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

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

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

In another aspect, the present invention provides a method for preparing a skin external composition for skin improvement comprising plant cells or adventitious roots of Zostera marina. Plant or extract thereof:

(a) isolating leaves or stem tissues of a bark plant and culturing the plant cells or adventitious roots; And

(b) preparing a composition for external application for skin containing the bark plant cell culture or adventitious roots culture or extract thereof.

The step (a) is a step of obtaining a plant tissue culture, that is, a plant cell culture, or an adventitious root culture, with some tissue isolated from a perennial plant.

In the present invention, leaves or stem tissues of the perennial plant may be used separately.

In the present invention, a suitable medium may be selected for deriving the plant cell culture or the adventitious root culture from the separated tissue. In the step (a), it is possible to select a suitable medium for cultivating the alfalfa or adventitious roots. 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 almost no difference in the basic MS medium composition when inducing plant cell culture from plant leaf-derived or plant stem-derived plants, the kinds of plant growth hormone that are most important in inducing plant cells and adventitious roots are different.

In the case of the plant cell culture according to the present invention, in the case of plant cell culture derived from a leaf or stem, 6-benzylaminopurine (6-BAP), 2,4-dichlorophenoxyacetic acid (2, 4-D) and then cultured to obtain a plant cell culture.

The combination ratio of the respective hormones may be 1: 0.4-0.8 or 1: 0.5-0.6 for 6-benzylaminopurine (6-BAP) and 2,4-dichlorophenoxyacetic acid (2,4-D). That is, it may be characterized by being 40 to 80 parts by weight, or 50 to 60 parts by weight, of 2,4-dichlorophenoxyacetic acid based on 100 parts by weight of 6-benzylaminopurine (6-BAP).

For example, 6-benzylaminopurine (6-BAP) and 2,4-dichlorophenoxyacetic acid (2,4-D) 0.6 mg / ml.

On the other hand, in the case of adventitious root culture, the adventitious root culture can be obtained by culturing indole-3-butyric acid (IBA). For example, an adventitious culture can be obtained by containing 0.2-1 mg / ml IBA.

In the present invention, the step (a) may further include a step of treating UVA for 3 to 6 hours or adding 150 mM to 400 uM medium to a culture medium.

In the present invention, it may include a process of inducing treatment to increase the efficacy of whitening, anti-aging, anti-inflammation, suppression of sebum, improvement of acne and prevention of skin damage by ultraviolet rays.

This attractant treatment includes UVA treatment, shikimic acid treatment, and it is also possible to increase phenolic compound, carotenoid, flavonoid content. The detailed conditions are as follows.

1. UVA: A UV-A fluorescent lamp (20 W, Sankyo Denki, Japan), which is a physical attractant, is cultured for 0.5 to 8 hours each day.

2. Shikimic acid: 200 ~ 600 M Shikimic acid is cultured in MS medium.

In the present invention, it is preferable that in the step (b), the composition containing the plant cell or the adventitious root culture obtained as a result of the cultivation in the step (a) is prepared from a suitable external preparation for skin composition, And can be prepared in the form of an external preparation for skin in a suitable form.

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

The herbaceous plant cells or adventitious root cultures obtained in step (a) are dried, powdered and mixed with purified water, and then extracted with cold water, hot water, jojoba oil, vegetable oil, inorganic solvent or ethanol And then extracting the solution 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 addition, the herbaceous plant cells, adventitious roots or extracts thereof according to the present invention have excellent melanogenesis inhibiting ability and can be used for whitening.

In addition, the bark plant cell, the adventitious root culture or the extract thereof according to the present invention can be used for shrinking pores.

Also, the herbaceous plant cells, adventitious roots or extracts thereof according to the present invention have an effect of inhibiting sebum secretion, inhibiting or improving acne, and preventing skin damage due to ultraviolet rays.

In addition, the bark plant cell, the adventitious root culture, or the extract thereof according to the present invention has antiinflammatory effects through inhibition of iNOS activity, that is, mitigation, alleviation, and inhibition of inflammation.

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.

In 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 hydrogenated castor oil, ethanol, triethanolamine, etc. in addition to the herbaceous plant cells, And preservatives, antioxidants, coloring agents, purified water, and the like may be included as needed.

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

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

The method for manufacturing the composition for external application of dermatophyte containing skin cells, adventitious roots or extract thereof according to the present invention is not limited to the above-described manufacturing method, and any person skilled in the art will recognize that, May also be used to prepare a composition for external application of dermatophyte containing the dicotyledonous plant cell, adventitious roots or extract thereof according to the present invention.

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

When the 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 includes forms of functional cosmetics such as skin astringency, anti-inflammation, wound healing, and anti-aging.

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 bark plant cell, an adventitious root culture or an extract thereof, a "pharmaceutically acceptable carrier ", which may be a diluent, a lubricant, a binder, a disintegrant, And a preservative. 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.

In another aspect, the present invention relates to a method for propagating a zyban plant characterized by using the plant cell of the Zostera marina , comprising the steps of:

(a) extracting a leaf or stem section of a herbaceous plant;

(b) The extracted leaves or stem sections were cultured in MS medium (Murashige and Skoog) containing 0.5-1 mg / L 6-benzylaminopurine and 0.3-0.6 mg / L 2,4-D To obtain a plant cell culture;

(c) inducing shoots by culturing the bovine plant cell culture of step (b) in MS medium containing 0.2-0.5 mg / L zeatin; And

(d) inducing shoots induced by step (c) in MS medium containing 0.2-0.5 mg / L indole-3-butyric acid (IBA).

In the present invention, the plant cell cultivation in step (b) above is followed by culturing the plant cells contained in the composition for external application for skin, wherein the culture conditions are carried out by light cultivation.

In detail, in the present invention, the herbaceous plant can be cultured and propagated in a large amount through the following procedure (FIG. 2).

1) extracting leaf slices

The alfalfa leaf slices used in the present invention include leaf slices of alfalfa grown in an aseptic condition or alfalfa slices grown in the external environment and preferably alfalfa slices grown in an external environment for 1 year or more are used . There is no particular limitation on the method of extracting the leaf slices from the slices, and it is preferable that the extracted leaf slices are used after removing the foreign substances on the leaf slices by using distilled water.

The stem section is subjected to the same process.

2) Sterilization step

In case of using the leaf slices grown in sterile condition, it is not necessary to pass the sterilization step, but in case of using leaf slices grown in the external environment for 1 year or more, it is preferable to pass the sterilization step. In the sterilization step, the leaf slices harvested under light or dark conditions were immersed in 70% ethanol for 30 seconds, washed with sterilized water, disinfected again with disinfectant (30% Lax + Tween 20) for 20 minutes, washed 3 times with sterilized water And sterilization is performed. The sterilized leaf slices are washed with distilled water and then cut to an appropriate size to induce plant cells.

3) Steps to induce plant cells

The step of inducing plant cells from the sterilized leaf slices was carried out in a basal MS medium (Murashige < (R) >) containing 1 mg / L 6-Benzylaminopurine, 0.3 mg / L 2,4-D, 3% sucrose and 8 g / and Skoog 1962, Duchefa Co., Cat No. M0221) under the conditions of growth temperature of 25 ° C and humidity of 70% to induce early plant cells. Each medium was adjusted to pH 5.8 with 1 N sodium hydroxide (NaOH). Subculture is performed every two weeks.

4) Steps to induce shoots

The step of inducing shoots comprises culturing the induced plant cells or the proliferated plant cells in a solid medium and culturing under a normal condition. The incubation temperature is not particularly limited, but is preferably 25 ± 2 ° C.

The culture medium for inducing shoots was prepared by adding Sucrose 30 g / L and Gelite 2.0 g / L to MS base medium, adding 0.2-0.5 mg / L Zeatin for growth regulator, adjusted to pH 5.7 ~ 5.8 Using MS (Murashige & Skoog) medium, incubate at 25 ± 2 ℃ for 4 weeks under growth condition of 25 ℃ and 70% humidity. Preferably, the pH is adjusted to 5.8 by using 1N sodium hydroxide (NaOH).

5) Steps to induce roots

The step of inducing roots consists of culturing the induced shoots in a solid medium and then culturing them. More specifically, the shoots that are not needed such as plant cells around the shoots are discarded and the shoots generated in a large number of plant cells are divided into several shoots And cultivation is subcultured in each medium. The incubation temperature is not particularly limited, but it is preferably 25 ± 2 ° C.

Sucrose 30 g / L and Gelite 2.0 g / L were added to the basal medium of MS and the growth regulators were supplemented with 0.2-1 mg / L Indole-3-butyric acid (IBA) And cultured in a culture room at 25 ± 2 ° C for 4 weeks under a growth condition at a temperature of 25 ° C and a humidity of 70%. Preferably, the pH is adjusted to 5.8 by using 1N sodium hydroxide (NaOH).

6) Propagation stage of the herbaceous plant

Sucrose 10 ~ 30 g / L and Gelite 2.0 g / L are added to MS base medium and cultured in a culture room at 25 ± 2 ℃ to proliferate shoot regeneration and root regeneration-induced plants from dicotyledonous plant cells. Cultivate in the incubation room at 25 ± 2 ℃ for 4 weeks under the condition of growth temperature of 25 ℃ and humidity of 70%.

7) Steps to purify the plant differentiated in the cabin

In the step of purifying differentiated plants in the cabin, the plants differentiated in the cabin are transferred to a culture soil mixed with 1: 1 sand and vermiculite, planted at 22 ~ 28 ℃ and maintained at a relative humidity of 60 ~ 90% And further comprising the step of secondary refining while maintaining a temperature of 15 to 25 DEG C and a relative humidity of 60 to 90% after the further primary refining step.

In the case of transporting a plant propagated through a plant tissue culture to an external environment with a constant temperature and humidity and nutrient optimum, most individuals can not adapt to the poor environment and fail, so purification treatment is also an important part of the tissue culture process . 0.5 to 2 g / L of salt water can be added to purify in the cabin.

Further, from another aspect of the present invention, it relates to a transplantation method of a transplanting of an alfalfa plant, which is characterized in that the above-obtained alfalfa plant is transplanted into a river or sea. Such plant transplantation methods include all plant transplantation procedures well known in the art.

In particular, it additionally comprises the step of propagation:

8) Steps to transplant the plant into the river or sea

The proliferated sheath plant can be transplanted into the river or the sand or soil in the sea.

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

In particular, the following examples have confirmed the efficacy of the herbaceous plant cells, adventitious roots, or extracts thereof, but it will be apparent to those skilled in the art that such effects are obtained not only in the extract but also in the culture itself.

Example 1-1: Derivation of Plant Cells from Zucchini according to the Present Invention

The bivalves used in the present invention were obtained from the RIS project of Incheon Metropolitan City. The bivalves were dipped in 70% ethanol for 30 seconds, washed with sterilized water, and disinfected with disinfectant (30% Lax + Tween 20) for 20 minutes And rinsed three times with sterilized water. (MS) medium (Murashige and Skoog 1962, Duchefa, CA, USA) containing 1 mg / L 6-benzylaminopurine, 0.3 mg / L 2,4-D, 3% sucrose and 8 g / L agar No. M0221) under an incubation condition of 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. The result of the plant cell culture thus obtained was as shown in Fig.

Example 1-2: Induction of adventitious roots of the herbaceous plant according to the present invention

Sucrose 30 g / L and Gelite 2.0 g / L were added to the MS base medium to induce adventitious roots from the perennial plant cells and 0.5 mg / L of indole-3-butyric acid (IBA) Lt; 0 > C for 4 weeks. The distal end of the induced adventitia was cut about 1 cm and inoculated into a liquid medium supplemented with 0.5 mg / L IBA (indole-3-butyric acid) and Sucrose 50 g / L in 1/2 MS medium. And cultured in a culture room at 25 ± 2 ° C for 4 weeks. The harvested adventitious roots were filtered with stainless steel to remove the medium, and the water was removed with a clean tissue and dried at 60 ° C for 2 days. Each medium was adjusted to pH 5.8 with 1 N sodium hydroxide (NaOH).

Examples 1-3: Mass production of bark plant cells and adventitious roots according to the present invention

As described above, in the case of herbaceous plant cells, 1 mg / L 6-Benzylaminopurine, 0.3 mg / L 2,4-D-glucopyranoside was added to the MS medium for mass production of cultured bark plant cells and alfalfa adventitious roots, D in the medium containing 25 mg / L IBA (indole-3-butyric acid) 0.5 mg / L and Sucrose 50 g / L in the 1/2 MS medium. At a humidity of 70% for 5 weeks in a 20 L balloon type bioreactor (Samsung Sci., Ltd.) with an air supply of about 0.1 vvm. Such mass production is shown in FIG.

Examples 1-4: Treatment of dahlia plant cells and adrenaline inducers according to the present invention

It is an object of the present invention to provide a method for increasing the content of carotenoids, flavonoids, and phenolic compounds, which are secondary metabolites of dicotyledonous plant cells and adventitious roots

1. A UV-A fluorescent lamp (20 W, Sankyo Denki, Japan), a physical attractant, is cultured for 0.5 h to 8 h each day.

2. Incubate 200 ~ 600 M Shikimic acid, a chemical inducer, in MS medium.

Methods for measuring the contents of carotenoids, flavonoids, and phenolic compounds, which are secondary metabolites, are as follows.

The total carotenoid content was measured according to the AOAC method. (1: 9, v / v) was added to the lyophilized dicotyledonous plant cells and the adventitious root samples. The extracted liquid was extracted with acetone: nucleic acid (3: 7, v / v) ), And the absorbance was measured at 436 nm with a mixture of acetone: nucleic acid (1: 9, v / v) 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 the alfalfa plant cell and adventitious culture extract and the reference material solution, and then 0.075 ml of a 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 mgg ^-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 the plant cell and adventitious root culture extract And the total phenolic 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 the total phenol content was expressed as mgg ^-1 DW.

(1) Influence of physical attractant, UV-A

1 mg / L of Zeatin and 0.2 mg / L of growth regulator were added to the MS basic medium and cultured at 25 ° C for 5 weeks. From day 6 of culture, the cells were irradiated with 0 h (hour, hour), 0.5 h, 1 h, 4 h, and 8 h daily using a UV-A fluorescent lamp (20 W, Sankyo Denki, Japan).

(Unit: mgg ^-1 ) UV-A Carotenoid
content Flavonoid
content Phenol compound
content 0h 34 32 45 0.5h 37 37 63 2h 43 36 72 4h 46 37 72 8h 54 38 79

(2) Influence of Sikimin, a chemical attractant,

Based on these facts, growth regulators of 0.5 mg / L of IAA and 0.2 mg / L of Zeatin were added to the MS base medium and cultured at 25 ° C. for 5 weeks. Sikimic acid was added to the culture medium at the concentration of 200, 400 and 600 μM at the 6th week of culture.

(Unit: mgg ^-1 ) Concentration of sikimic acid
(μM) Carotenoid
content Flavonoid
content Phenol compound
content 0 43 37 57 100 42 39 67 200 45 36 80 400 46 35 89 600 42 35 62

Example 1-5: Preparation of an extract of bark plant cell culture according to the present invention

The bark plant cells and adventitious root cultures obtained in Example 1-3 were harvested, and the water was sufficiently removed with a clean tissue and dried in a dryer at 60 ° C for 2 days. 50 g of dried bark plant cells and adventitious root culture powder were placed in a container and 1 L of purified water was added thereto, followed by hot extraction at 90 to 120 ° C for 48 hours. After the extraction, the filter was removed by filtration through a mesh to remove solid components, and the filtration of the herbaceous plant cells and the adventitious root culture extract were used in the present invention. For the comparative experiments, the extracts were extracted by hot water extraction in the same manner.

Method of propagation of a bivalve plant using plant tissue culture technology according to the present invention

According to the plant cell induction process described in Example 1 above, plant cells were obtained by culturing in place of the dark culture under the growth conditions, and the herbaceous plants were propagated through the following procedure.

2-1. Shoot Regeneration Induction Process from Plant Cells

Sucrose 30 g / L and Gelite 2.0 g / L were added to the MS basal medium to induce shoot regeneration from the herbaceous plant cells obtained above, and 0.22 mg / L of Zeatin, a growth regulator, Lt; / RTI > Shoot renalization was induced in the proliferating plant cells and cultured in a 252 culture room for 4 weeks at a temperature of 25 and a humidity of 70%. The pH was adjusted to 5.8 using 1N sodium hydroxide (NaOH).

2-2. Root Regeneration Induction Process

Sucrose 30 g / L and Gelite 2.0 g / L were added to MS base medium to induce Root Regeneration in the shoot-regeneration induced region, and 0.2 mg / L Indole-3-butyric acid (IBA) And cultured in a culture room at 25 ± 2 ° C. Root regeneration, which can absorb nutrients to grow into a single plant in the shoot-regeneration-induced area, was induced by culturing in a culture room at 25 ± 2 ° C for 4 weeks under the growth conditions of temperature 25 ° C and humidity 70% . The pH was adjusted to 5.8 using 1N sodium hydroxide (NaOH).

2-3. Propagation process of alfalfa plant

In order to proliferate shoot regeneration and root regeneration-induced plants from herbaceous plant cells, Sucrose 30 g / L and Gelite 2.0 g / L are added to MS base medium and cultured in a culture room at 25 ± 2 ° C. Cultivate in the incubation room at 25 ± 2 ℃ for 4 weeks under the condition of growth temperature of 25 ℃ and humidity of 70%. After 4 weeks, they are transplanted into the river or sea and raised (Fig. 2).

Experimental Example 1: Cytotoxicity of plant extracts of Abalone plant and adventitious roots

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 Example 1-5. 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 for 25-30% of the surface area of the culture dish, the composition prepared in Example 1-5 was replaced with FBS-free DMEM containing 0.1-5% Lt; / RTI > 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) solution was added to each well and the mixture was stirred for 20 minutes to dissolve the formazan crystals. 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 the 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 shown in Table 3.

[Equation 1]

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

Treatment concentration (%) Cytotoxicity%
(Cytotoxicity) Control group 98 Alfalfa
extract 0.5% 82 1.0% 81 5.0% 75 Alfalfa
Plant cell
Culture extract 0.5% 100 1.0% 102 5.0% 105 Alfalfa
Adrenaline
Culture extract 0.5% 100 1.0% 103 5.0% 108

As shown in Table 3, no cytotoxicity was observed with the 0.5%, 1.0% and 5.0% bark plant cells and adventitious root culture extracts, as shown in Table 3, as a result of the test on the cytotoxicity using the human keratinocytes. That is, it did not affect cell viability. On the other hand, the extract of Bombyx mori was 20 ~ 30% cytotoxic according to the treatment concentration.

Experimental Example 2: Mushroom Tyrosinase Activity Measurement of Mushroom Plant Cell and Root Extract (Mushroom Tyrosinase Inhibition Assay)

The L-DOPA oxidation activity of tyrosinase was measured by ELISA reader. Mushroom tyrosinase was purchased from Sigma-Aldrich. 10 μl of 5 mM L-dopa, 90 μl of 0.1 M sodium phosphate buffer (pH 6.8), and various concentrations of calli were placed in a 96-well plate. Finally, 40 μl of tyrosinase enzyme was added and mixed, followed by reaction at 37 ° C for 15 minutes. The initial percentage of Dopachrome formation from the reaction is measured by an increase in absorbance at 490 nm using an ELISA reader.

Tyrosinase converts tyrosine, an important tyrosine in melanin synthesis, to L-DOPA and oxidizes L-DOPA to form Dopaquinone. Dopa oxidase activity was measured by Mushroom tyrosinase and was inhibited by adding various concentrations of test materials.

&Quot; (2) "

Inhibition of tyrosinase (%) = [1 - (Ab sample / Ab control)] × 100

Treatment concentration (%) Tyrosinase Activity
(% of control) Purified water
(Negative control) 100 Alfalfa
extract 0.5% 97 1.0% 94 5% 90 Alfalfa
Plant cell
Culture extract 0.5% 95 1.0% 90 5% 80 Alfalfa
Adrenaline
Culture extract 0.5% 95 1.0% 92 5% 83

As shown in Table 4, the inhibitory activity of tyrosinase inhibitory activity of up to 5% of bark extract, bark plant cell culture extract, and barky bark root culture extract was inhibited by treatment with extracts of Bark plant cell culture and adventitious roots culture, Activity was inhibited.

Experimental Example 3 Procollagen Synthesis Enhancement Test of Bark Plant Cell and Root Extract

Human fibroblasts (Human Skin Fibroblast) for 37 ℃, 5% in a CO ₂ incubator of 10% in the cell culture medium to maintain a constant humidity FBS, Penicillin (50U / ml) , Streptomycin DMEM (Dulbecco's containing (50 / ml) (Eagle's Medium, Gibco, USA). Cells were seeded at 1 × 10 ⁵ cells / ml in a 48-well plate and cultured for 24 hours. The medium containing the control (DMEM medium) and 0.1 ~ 5% bark plant cells and adventitious root culture extract was added and incubated for 48 hours at 37 ° C in a 5% CO ₂ incubator. After 48 hours, the amount of newly synthesized collagen was measured by measuring 20 μl of each supernatant of the culture medium and measuring using Procollagen Type I C-Peptide EIA Kit (PICP, Takara, Cat No. MK101). Calculated according to the following formula and the results are shown in the table.

&Quot; (3) "

Treatment concentration (%) Procollagen biosynthetic amount
(% of control) Control group 100 Alfalfa
extract 0.5% 99 1.0% 105 5% 110 Alfalfa
Plant cell
Culture extract 0.5% 108 1.0% 125 5% 130 Alfalfa
Adrenaline
Culture extract 0.5% 105 1.0% 115 5% 135

As can be seen from the above table, it can be seen that the amount of biosynthesis of collagen is increased with an increase of the content of compositional alfalfa cells and adventitious root culture extract of the present invention. Thus, it can be seen that the composition of the present invention exhibits excellent collagen synthesis promoting effect have.

Experimental Example 4: Effect of inhibiting MMP-1 expression on the wrinkle-improving effect of the plant cell and adventitious roots extract

Skin wrinkles are caused by an imbalance in the synthesis and degradation of collagen. In normal skin, the synthesis of type I collagen and its degrading enzyme, MMP-1, are in balance. However, in photoaged skin, the synthesis of type I and III collagen is reduced and the activity of MMP-1 is increased. These MMPs (matrix metalloproteinases) are proteolytic enzymes that degrade the extracellular matrix and are known to be involved in wound healing and tissue regeneration under normal conditions.

In order to confirm whether the composition of the present invention has an inhibitory effect on MMP-1 production, the following experiment was conducted.

Human fibroblasts (Human Skin Fibroblast) for 37 ℃, 5% in a CO ₂ incubator of 10% in the cell culture medium to maintain a constant humidity FBS, Penicillin (50U / ml) , Streptomycin DMEM (Dulbecco's containing (50 / ml) The cells were cultured in a modified Eagle's medium (Gibco, USA) at a density of 1 × 10 ⁶ cells / ml in a 48-well plate and cultured for 24 hours. The cells were cultured in a 5% CO ₂ incubator at 37 ° C for 48 hours in a control medium (DMEM medium) and a medium containing 0.1% to 5.0% of bifidobacterial cells and adventitious cell culture extracts. After 48 hours, the amount of newly synthesized MMP-1 was measured by measuring 20 μl of each supernatant of the medium using Matrix Metalloproteinase-1, Biotrack Activity (Amersham, Cat No. RPN2629) And the results are shown in the table. ≪ tb >< TABLE >

&Quot; (4) "

UVB
Whether or not to process Treatment concentration (%) Inhibition rate of MMP-1 biosynthesis (%) - Purified water
(Negative control) 0 + 1% Ascorbic Acid
(Positive control group) 30 Alfalfa
extract 0.5% 3 1.0% 5 5% 19 Alfalfa
Plant cell
Culture extract 0.5% 12 1.0% 20 5% 32 Alfalfa
Adrenaline
Culture extract 0.5% 8 1.0% 15 5% 30

As shown in Table 6, the inhibitory effect of MMP-1 on the biosynthesis of MMP-1 was suppressed by treatment with 0.1 ~ 5.0% of bovine plant cells and adventitious root culture extract. In particular, it exhibits more excellent effect by suppressing the expression of MMP-1 biosynthesis gene by plant cell and adventitious root extract,

Experimental Example 5: Anti-inflammatory effect test on inhibition of iNOS activity

The amount of nitric oxide (NO) produced from the Raw 264.7 cell line was determined using the Griess reagent as the NO ₂ ^- form present in the cell culture medium. In order to measure inhibition of NO production in Raw 264.7 cells activated with 1 g LPS, the test group treated with the medium containing the bacteriophage cells and the adventitious cell culture extracts obtained at the concentration of 0.1-5.0% obtained in Example 1 and the control group were cultured for 24 hours After incubation, Griess reagent was mixed with 100 μl of cell culture supernatant and 100 μl of Griess reagent (1% sulfanilamide in 5% phosphoric acid, 1% -naphthylamide in H ₂ O) for 10 min in 96-well plates. The absorbance was measured. The concentration of NO ₂ ^- was obtained by measuring the absorbance by diluting sodium nitrate.

LPS
Whether or not to process sample
density
(%) Cell Viability
(%) NO Production
(M) - Control group (purified water) 100 1.3


+ Control group (purified water) 760 3.6
Alfalfa
extract 0.5 99 3.6 1.0 100 3.5 5.0 101 3.4 Alfalfa
Plant cell
Culture extract 0.5 101 3.4 1.0 100 3.3 5.0 101 3.2 Alfalfa
Adrenaline
Culture extract 0.5 100 3.3 1.0 102 3.2 5.0 105 3.1 Indomethacin 100M 95.8 3.1

As shown in Table 7, the activity of iNOS inhibited the production of NO compared to the control group by treatment with the extracts of Bifidobacterium acanthomaceae and adventitious roots of 0.1 ~ 5.0% concentration, and showed an excellent antiinflammatory effect. In particular, plant cell and adventitious root culture extracts were superior to ibuprofen extracts in inhibiting inflammation by iNOS activity.

Experimental Example 6: In vitro Test

In order to measure the pore-shrinking effect of the herbal plant cells and adventitious roots extract composition of the present invention, the composition prepared in the above examples was tested for hemoglobin shrinkage using hemoglobin as a protein to replace skin.

Hemoglobin solution (0.05 g / 50 ml) was prepared with 0.9% Phosphate Buffer Saline (0.1 mM, pH 7.4), and 2 ml of the hemicellulose solution obtained in Example 1 and 2 ml of the dorsal root extract cultured in Example 1 were added thereto. And then centrifuged at 3,500 rpm for 10 minutes. 2 ml of purified water was added to 1 ml of the supernatant, which was measured in a UV-Visible spectrum at 407 nm.

As a control, 2 ml of PBS (Phosphate Buffer Saline) was added.

 Concentration (%)  Precipitation of hemoglobin (%) PBS
(Negative control) 0 0.1 Tannic acid
(Positive control group) One 35 Alfalfa
extract 0.5 One 1.0 3 5.0 5 Alfalfa
Plant cell
Culture extract 0.5 10 1.0 17 5.0 30 Alfalfa
Adrenaline
Culture extract 0.5 5 1.0 20 5.0 33

The shrinkage effect was evaluated by measuring the sedimentation amount of hemoglobin in the composition containing 0.1% to 0.5% of the herbaceous plant cells and the adriamycin culture extract composition. The higher the sedimentation rate (%) of hemoglobin was, the better the shrinkage effect of pores was judged. In other words, it was confirmed that the extracts of dicotyledonous plant cells and adventitious roots culture were excellent in reducing the pores compared with the extracts.

EXPERIMENTAL EXAMPLE 7: Experiment for inhibiting sebum

In order to confirm the sebum inhibitory effect of the herbaceous plant cells and the adventitious root culture extract, sebum secretion amount of the 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. The measurement value for oily skin is 220 ㎍ / cm ² or more and for normal skin is 100 to 220 ㎍ / cm ² . As a control, purified water was used. The results of the experiment are shown in Table 9.

sample Sebum suppression effect 0 day (before use) 28 days (after use) Control group 230 225 One 1% bark extract 231 195 2 1%
Culture extract 230 171 3 1%
Culture extract 232 165

As shown in the results of Table 9, 1% bark plant cells and adventitious roots extract showed better sebum secretion inhibitory effect than the bark extract extracts, as compared with the control group.

Experimental Example 8: Effect of improving acne

The 10% male and female acne sufferers were allowed to apply the 1% concentration of herbal plant cell culture extract prepared in Example 1 throughout the face for 4 weeks in the morning and evening. The results of the evaluation of the acne healing effect were evaluated according to the degree of the person who participated in the experiment by evaluating the pre-test condition by 1-3 points with reference to the following criteria, and the results are shown in the table.

≪ Evaluation of state before test >

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

≪ 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 10 above, the improvement effect of acne was confirmed in the majority of subjects after using the 1% extract of plant cell culture extract for 4 weeks.

Experimental Example 9: Cell protection effect by ultraviolet irradiation

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 phosphate buffer. After adding 500 L of phosphate buffer, the cells were irradiated with ultraviolet rays at 10 mJ / cm 2, and then replaced with fresh cell culture medium without FBS. The extracts of the bark plant cell culture extract and adventitious root culture extract obtained in Example 1 were treated at a concentration of 1% Followed by further incubation 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.

UV irradiation
The presence or absence Treatment concentration Cell protection effect by ultraviolet irradiation
(% of Control) UV
(-) Control group - 1.0 UV
(+) Control group - 0.5 One % Alfalfa
extract 0.52 Alfalfa
Plant cell
Culture extract 0.72 Alfalfa
Adrenaline
Culture extract 0.81

In order to evaluate the keratinocyte protective effect of the herbaceous plant and adventitious root extracts, human keratinocytes (HaCaT) were exposed to UV irradiation at 20 mJ / cm 2 and then cultured for additional 24 hours, As a result of MTT assay, cell viability was increased compared to control group treated with 1% concentration of UV - irradiated bark plant and adventitious root culture extracts. Therefore, it was possible to confirm the cytoprotective effect of the herbaceous plant cell and the adventitious root culture extract by ultraviolet irradiation.

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 extract of the present invention as an active ingredient.

Production Example 3-1: 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 Herbaceous plant, adventitious root culture extract 2.0 Purified water to 100

Production Example 3-2: 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 Herbaceous plant, adventitious root culture extract 7.0 Purified water to 100

Production Example 3-3: 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 Herbaceous plant, adventitious root culture extract 5.0 Purified water to 100

Production Example 3-4: 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 Herbaceous plant, adventitious root culture extract 7.0 Purified water to 100

Production Example 3-5: 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 Herbaceous plant, adventitious root 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 (14)

A cosmetic composition for inhibiting skin damage by shrinking pores or ultraviolet rays containing a plant cell or adventitious muscle culture of Zostera marina or an extract thereof.
The cosmetic composition according to claim 1, wherein the composition further comprises a skin wrinkle suppressing or improving agent.
The cosmetic composition of claim 1, wherein the composition further comprises a whitening application.
The cosmetic composition according to claim 1, wherein the composition further comprises an anti-inflammatory or anti-inflammatory effect.
The cosmetic composition of claim 1, wherein the composition further comprises an anti-sebum application.
The cosmetic composition of claim 1, wherein the composition further comprises an anti-acne treatment or an improvement.
A method for preparing a cosmetic composition according to claim 1, comprising a Zostera marina plant cell or adventitious root culture or extract thereof comprising the steps of:
(a) isolating leaves or stem tissues of a bark plant and culturing the plant cells or adventitious roots; And
(b) preparing a cosmetic composition containing the bark plant cell culture or the adventitious root culture or the extract thereof.
8. The method of claim 7, wherein step (a)
(i) The leaves or stem tissues of the perennial plants are separated to obtain 6-benzylaminopurine (6-BAP), 2,4-dichlorophenoxyacetic acid, 2,4-D ) To obtain a plant cell culture, or alternatively,
(ii) isolating the leaf or stem tissue of the perennial plant and culturing it in a medium containing indole butyric acid (IBA) to obtain an adventitious culture,
≪ / RTI >
The method according to claim 7, further comprising the step of culturing in the step (a) by adding UVA for 3 to 6 hours or adding 150 mM to 400 uM of a cinnamic acid to the medium.
[8] The method of claim 7, wherein the step (b) comprises: culturing the bark plant cell culture or adventitious root culture obtained in step (a), drying, pulverizing and mixing with purified water to prepare a composition, , Followed by sonication or hot water extraction to produce a composition.

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