KR102016062B1 - Callus extracts from Citrus junos Siebold ex Tanaka and Pyrus pyrifolia Nakai with Whitening and Anti-wrinkle effect - Google Patents

Abstract

The present invention relates to an external composition for skin containing pear or citron callus culture or extract thereof, and more particularly, to an anti-aging external composition for skin containing pear or citron callus culture or extract thereof and carbomer as an active ingredient. It is about.
A pear or citron cell culture according to the present invention or an extract containing the external preparation composition for skin has skin collagen synthesis ability, elasticity improvement and antioxidant ability without toxicity to skin cells, and has high utility value as a functional cosmetic material.

Description

Callus extracts from Citrus junos Siebold ex Tanaka and Pyrus pyrifolia Nakai with Whitening and Anti-wrinkle effect

The present invention relates to a pear callus and citron callus material and carbomer having a whitening and anti-wrinkle effect, more specifically, a functional skin external preparation containing a pear or citron callus culture or its extract and a carbomer as an active ingredient. It is about material.

The cosmetics industry is a technology-intensive and high-value-added industry in which the basic science and applied technologies such as chemistry, biology, physiology, and pharmacy are applied in a technical aspect. Today, cosmetics are products that are used directly on human skin. They must have safety, efficacy, and ease of use. They are not only for the beauty of beauty, but also for protecting, cleaning, moisturizing, supple and physiological functions. It must also have a biological function. In particular, with the rapid industrialization of the cosmetics industry and the aging of society, the importance of the environment and health has been highlighted, and well-being culture has become a mega trend of modern society. According to the development of high-level industries, various skin diseases caused by environmental changes are increasing, and the whitening and anti-aging researches using the high functionality of natural materials are actively conducted in the bioventure, cosmetics, and biotechnology industries.

With the development of cutting-edge biotechnology and the growth of the national economy, cutting-edge technology has been introduced in the cosmetics industry, and the development of cosmetics as a semi-therapeutic concept (synthetic term for cosmetics and pharmaceutics as a therapeutic drug) It is emerging as a mega trend of the industry.

In particular, attempts to develop products with high biological effectiveness by incorporating highly functional biomaterials have been actively conducted mainly in the cosmetics industry and bio companies. Existing cosmetic materials have been used in cosmetics in the traditional way based on information from plants or herbal extracts or folk remedies. On the other hand, there is a trend that the need for functional cosmetic materials with excellent functionality and excellent effect and safety on the skin is applied by applying extracts or extraction methods that have been validated on a biological basis.

On the other hand, plant callus (plant stem cells) refers to the undifferentiated atypical cell mass of the plant, which is representative of the tumor tissue formed by the meristem generated when the plant is injured. Plants are composed of meristems that divide into cells and permanent tissues that do not, and callus is formed when the cells of the first meristem are put in a nutrient medium and grown. After that, the embryo is formed and differentiated into plants. Callus is defined as "plant stem cells" because it divides into each tissue of the plant. Plant tissue culture technology, which has a close relationship with eco-friendly green growth, has been widely used for the purpose of disease resistance and accumulation of certain secondary metabolites by the Rural Development Administration and university districts in Korea since the 1990s. Recently, plant stem cell (calus) material is being actively made centering on bioventure companies.

Under these circumstances, attempts have been made to develop a cosmetic composition for pears or citrons. (Patent Documents 1 and 2) The callus extracts and carbomers of pears or citrons have been developed as anti-aging materials with skin physiological activity. There is no case.

Patent Document 1: Republic of Korea Patent Publication No. 2015-0139688 Patent Document 2: Republic of Korea Patent Publication No. 2015-0081201

In an effort to develop functional cosmetic natural materials, the present inventors induced plant callus through plant tissue culture technology of pear or citron plants, and their supernatant extracts or hot water extracts and carbomers activate the growth and proliferation of skin cells. The present invention was completed by confirming that there are anti-aging, anti-aging, and antioxidant effects including whitening, anti-wrinkle effect, and the like.

Accordingly, it is an object of the present invention to provide a whitening or anti-aging skin external composition comprising pear or citron callus culture or its extract and / or carbomer as an active ingredient.

Still another object of the present invention is to provide a method for preparing an external preparation for skin containing the pear or citron callus culture or its extract as an active ingredient.

In order to achieve the above object, the present invention provides a whitening and anti-aging external composition for skin containing pear or citron callus culture or extract thereof as an active ingredient.

In the present invention, the external composition for skin may be characterized in that it further comprises a carbomer as an active ingredient.

In the present invention, the composition may be characterized in that for preventing or improving wrinkles, for whitening, or for improving elasticity, antioxidant.

The present invention also comprises the steps of (a) inducing callus of the pear or citron plant; And (b) preparing a callus culture or extract thereof derived in step (a); It provides a method for preparing the external preparation for skin composition comprising a.

In the present invention, it provides a method for producing an external composition for skin composition containing a pear or citron callus culture or its extract and carbomer as an active ingredient comprising the step of preparing a composition by mixing the extract and carbomer.

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

The present invention relates to an external preparation composition for skin containing a pear or citron callus culture or its extract as an active ingredient and a method for producing the same.

In one aspect, the present invention relates to an anti-aging or whitening skin external preparation material containing a pear or citron callus culture or its extract as an active ingredient, and an anti-aging or whitening skin external preparation composition.

In this invention, it is related with the external composition for skin containing carbomer further as an active ingredient.

In the present invention, Pyrus pyrifolia Nakai ) Pyrus is a genus of Rosaceae, pear fruit is called "pear". The pear tree is believed to have originated in western or southwestern China.

In the present invention, citrus junos Siebold ex Tanaka ) is a citrus fruit and plant that is bright yellow in color and crusty. The fragrance is good and unlike the hard quince, the pulp is tender.

In the present invention, the carbomer is a thickener, a phase stabilizer that is most commonly used in cosmetics, and the structure is an acidic polymer compound, mainly acrylic acid polymerized. Carbomer's advantage is that it can grow very transparently over a wide pH range, with little irritation to the skin and less contamination by microorganisms. In addition, gelation is very easy and the viscosity can be freely adjusted over a wide range of viscosities. These carbomers are the most widely used thickeners in cosmetics and household goods, from essences to cream formulations, mask packs, hair and hand washes. However, benzene, which is known as a carcinogen, may be used as a solvent in the solubility of water, the blowing of powders, and the synthesis process.

In addition, the pear or citron callus culture or extract thereof in the present invention may contain 0.01 to 1 or 0.01 to 10% by weight or volume% relative to the total weight of the composition, such a ratio is only a preferred range, for example, The following examples include the results of experiments at 0.01 mg / ml, 0.05 mg / mg, 0.1 mg / ml, 0.5 mg / ml, 1 mg / ml, etc. It is apparent to those skilled in the art having regeneration, wrinkle improvement, elasticity improvement, antioxidant function, and the like.

In addition, the carbomer in the present invention may contain 0.01 to 2% by weight or volume% relative to the total weight of the composition.

In the present invention, the embryo or citron callus culture (culture) itself is used, the culture is used by filtration, the culture is used by crushing, the culture is dried and powdered, and then dissolved in purified water to use Can be.

In the present invention, "extract" refers to an extract obtained by pulverizing the pear or citron callus culture extract, or by various extraction methods known in the art, such as cold water extraction, hot water extraction, ethanol extraction.

The extraction method in the present invention is not particularly limited, and for example, cold extraction, ultrasonic extraction, reflux extraction, hot water extraction and the like. Here, in the case of hot water extraction, by heating to 80 ~ 100 ℃ for 8 to 48 hours in a boiling water distillation to obtain a hot water extract.

In the case of cold water extraction, for example, the callus culture itself or dried powder thereof is mixed with cold water (15-25 ° C.) and extracted for 1-10 days to obtain a cold water extract.

Or by extraction using water, an organic solvent, or a mixed solvent thereof. The extracted solution can be used directly or can be concentrated and / or dried. When extracted with an organic solvent, methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or a mixed solvent thereof may be used and extracted by room temperature or warming under conditions where the active ingredient of the herbal medicine is not destroyed or minimized. Depending on the organic solvent to be extracted, the degree of extraction and loss of the active ingredient of the drug may vary, so select an appropriate organic solvent.

In particular, in the present invention, the callus culture was agitated in a refrigerated state (about 4 ° C.) through crushing, followed by centrifugation to obtain a supernatant, "Calus (Cultural) Supernatant Extract".

After crushing the callus culture, a hot water extract was obtained through hot water heating of a bath. The specific form of the extract according to the present invention may be various forms including these.

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

In the present invention, anti-aging means skin protection and skin condition improvement, skin aging and wrinkle prevention or improvement, pore contraction, reduction, skin protection and alleviation of skin inflammatory response, immune disease improvement ability, or skin barrier function improvement. It is a concept that includes alleviation of skin irritation, skin cell proliferation and regeneration ability, antioxidant activity, collagen synthesis enhancement ability, and skin regeneration.

In the present invention, the meaning of "contained as an active ingredient" is a skin external preparation composition, which can exhibit skin improvement effect due to whitening and anti-aging, for example, as a external preparation composition for skin, collagen synthesis and melanogenesis inhibition associated with the anti-wrinkle effect. Means containing an effective amount of whitening effect, skin regeneration, elasticity improvement, antioxidant and the like can be represented.

In this regard, the external preparation composition for skin according to the present invention includes a whitening, wrinkle improvement, prevention, elasticity improvement, skin regeneration, antioxidant composition.

On the other hand, plant tissue culture is a technique for culturing live small plant tissues in vitro ( in vitro ) to proliferate plant cells, organs and plants according to the purpose of culture. Plant tissue culture can be said to use the basic characteristics of the plant. Plant tissue culture technology is based on the use of the totipotency that can regenerate the plant from the somatic cells of the plant, a unique feature of the plant. That is, by culturing single cells (including protoplasts), leaf and root sections of plants in a medium to which specific nutrients and growth regulators are added, the plants can be regenerated from these cells and tissues. The ability of these plants to totipotency is a strategy for surviving from the damage of the environment and herbivores for the fixed life and long-term survival of the plants. Thus, plants can be said to have the ability to regenerate lost organs. Plant tissue cells isolated from the parent plants can be regenerated into complete plants when cultured in an appropriate culture environment, that is, plants with typical properties are not only academically important in embryology but also for practical use of cosmetic raw materials. Is large.

The present invention in another aspect, the method comprising the steps of (a) inducing callus from pear or citron plants; And (b) relates to a method for producing a topical skin composition comprising the step of preparing the induced embryo or citron callus culture or its extract.

In addition, in the present invention, after the step (b) further, (c) containing a pear or citron callus culture or its extract and carbomer comprising the step of preparing a composition by mixing the extract and carbomer It relates to a method for preparing a topical skin composition.

In step (a), specifically, an appropriate medium may be selected for culturing embryo or citron callus. If there is a medium generally used for plant tissue culture in the art, it can be used without limitation. In plants, MS medium and B5 medium are generally used. For example, the composition of MS medium (1 L standard) is NH ₄ NO ₃ 1650 mg, KNO ₃ 1900 mg, CaCl ₂ H ₂ O 440 mg, MgSO _{4 .7H 2 O 370 mg, KH} 2 PO 4 170 mg, KI 0.83 mg, H 3 BO 3 6.2 mg, MnSO 4 .4H 2 O 22.3 mg, ZnSO 4 .7H2O 8.6 mg, Na 2 MoO 4 .2H 2 O _{0.25 mg, CuSO 4, 5H 2} O 0.025 mg, CoCl 2 .6H 2 O 0.025 mg, FeSO 4 .7H 2 O 27.8 mg, Na 2 EDTA.2H 2 O 37.3 mg, Myoinositol 100 mg, Nicotinic acid 0.5 mg, Pyridoxine -HCl 0.5 mg, Thiamine-HCl 0.5 mg, Glycine 2 mg, Sucrose 30000 mg.

In the present invention, in culturing the callus, first, the medium for inducing callus may contain about 0.5 to 5 mg / L of 2,4-dichlorophenoxy-acetic acid and 6-benzylaminopurine, In the dark cycle and 12 to 18 hours photoperiod can be induced.

The induced callus may be cultured in a solid medium medium containing picloram at about 1 to 5 mg / L. In addition, it can be cultured in a liquid medium culture step after the culture on the solid medium.

In the present invention, in the step (b), the pear or citron callus culture obtained through the culture of step (a) as it is, or the culture may be prepared in the form of extract through a known extraction method.

For example, in step (b), the pear or citron callus culture obtained in step (a) is dried, powdered and mixed in purified water,

The pear or citron callus culture obtained in step (a) is dried, powdered and mixed in purified water, followed by cold water extraction, hot water extraction or ethanol extraction,

Or crushing the pear or citron callus culture obtained in step (a), stirring in purified water or PBS, and obtaining a supernatant extract obtained by centrifugation.

Or pulverizing the pear or citron callus culture obtained in step (a), and then stirring in purified water or PBS, and obtaining a hydrothermal extract obtained by heating with a water bath.

Through the steps up to (b) it can be prepared a topical skin composition.

In the present invention, when mixed with carbomer,

As the step (c), it may comprise the step of mixing the extract and carbomer obtained in the step (b) to prepare an external preparation composition for the skin.

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

In the cosmetic composition, an acceptable carrier may be included in the cosmetic formulation. Herein, "acceptable carrier in cosmetic preparation" means a compound or composition already known and used that may be included in a cosmetic preparation or a compound or composition which will be developed in the future, and which has no toxicity, instability or irritation that can be adapted to the human body upon contact with the skin. Say nothing.

The carrier may be included in the topical skin composition of the present invention in an amount from about 1% to about 99.99% by weight, preferably from about 90% to about 99.99% by weight of the composition. However, since the ratio depends on the formulation as described below in which the external preparation composition for skin of the present invention is prepared and its specific application site (face, neck, etc.) or its preferred application amount, the ratio may be viewed in any aspect. It should not be understood as limiting the scope of the invention.

Examples of the carrier include alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, moisturizers, viscosity modifiers, emulsions, stabilizers, ultraviolet scatterers, ultraviolet absorbers, colorants, fragrances, and the like. Alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, humectants, viscosity modifiers, emulsions, stabilizers, ultraviolet scatterers, ultraviolet absorbers, colorants, compounds / compositions that can be used as fragrances are already known in the art. As such, those skilled in the art can select and use appropriate materials / compositions.

In one embodiment of the present invention, the composition for external application for skin according to the present invention may include glycerin, butylene glycol, propylene glycol, rolled polyoxyethylene hydrogenated castor oil, ethanol, triethanolamine, etc. in addition to the pear or citron callus culture extract and carbomer. It may include, and may contain a small amount of preservatives, pharmaceuticals, coloring, purified water and the like as necessary.

The external preparation composition for skin according to the present invention can be prepared in various forms, for example, lotion, essence, gel, emulsion, lotion, cream (oil-in-water, water-in-oil, multiphase), solutions, suspensions (anhydrous and aqueous) ), Anhydrous products (oil and glycol-based), gels, masks, packs, powders, or gelled capsules (soft capsules, hard capsules) formulations and the like.

The skin in the present invention is a concept that includes not only a face but also a scalp and a whole body. As a skin external composition that can be applied to such a scalp, there are shampoos, rinses, treatments, hair regrowths, etc., and a body cleanser that can be applied to the whole body. It may be produced in various forms for the purpose of.

Method for preparing the external preparation for skin containing pear or citron callus culture extract and carbomer according to the present invention as an active ingredient is not limited to the above-described manufacturing method, if one of ordinary skill in the art The method for partially modifying the preparation method may also prepare an external composition for skin containing pear or citron callus culture extract and carbomer according to the present invention.

In particular, the external preparation composition for the skin may be prepared in the form of general emulsion formulations and solubilized formulations using a conventionally known production method, in addition to the production method specifically disclosed in the present invention.

When the cosmetic composition is prepared, cosmetics of the emulsion formulations include nutrient cosmetics, creams, essences, etc., and cosmetics of the solubilized formulations include soft cosmetics. In addition, by containing a dermatologically acceptable medium or base, it can be prepared in the form of topical or systemic adjuvants commonly used in the field of dermatology.

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

In addition, the topical skin composition of the present invention may further comprise fatty substances, organic solvents, solubilizers, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ions Any commonly used in type or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or cosmetics It may contain adjuvants conventionally used in the cosmetic or dermatology field as other ingredients. In addition, the above components may be introduced in an amount generally used in the dermatology field.

The composition containing a pear or citron callus according to the present invention and a composition containing carbomer have excellent skin collagen synthesis ability without skin toxicity, and have skin wrinkle improvement, skin elasticity improvement, whitening, antioxidant effect, Applicable as a material for external skin preparations.

1 is a photograph showing the induction process of callus using the citron and pear plant tissue according to the present invention.
Figure 2 is a diagram showing the preparation of the citron or pear callus supernatant and extract according to the present invention.
Figure 3 is a result confirming the effect of citron callus supernatant extract and citron callus hydrothermal extract on human fibroblast growth. (A: citron callus supernatant extract, B: citron callus hydrothermal extract)
4 is a result confirming the effect of pear callus supernatant extract and pear callus hydrothermal extract on human fibroblast growth (A: pear callus supernatant extract, B: pear callus hydrothermal extract)
5 is a result confirming the effect of citron callus supernatant extract and citron callus hot water extract on the growth of human keratinocytes. (A: citron callus supernatant extract, B: citron callus hydrothermal extract)
6 is a result confirming the effect of pear callus supernatant extract and pear callus hot water extract on the growth of human keratinocytes (A: pear callus supernatant extract, B: pear callus hydrothermal extract)
7 is a result of confirming the tyrosinase enzyme inhibitory activity of citron callus hydrothermal extract and pear callus hydrothermal extract (A: citron callus hydrothermal extract, B: pear callus hydrothermal extract)
8 is a result showing the L-DOPA oxidation inhibitory activity of citron callus hydrothermal extract and pear callus hydrothermal extract. (A: citron callus hydrothermal extract, B: pear callus hydrothermal extract)
9 is a result showing melanin biosynthesis inhibitory activity of citron callus supernatant extract and citron callus hot water extract. (A: citron callus supernatant extract, B: citron callus hydrothermal extract)
10 is a result showing melanin biosynthesis inhibitory activity of pear callus supernatant extract and pear callus hot water extract. (A: Pear callus supernatant extract, B: Pear callus hydrothermal extract)
11 is a result confirming the antioxidant effect of the citron callus supernatant extract and citron callus hot water extract. (Ascorbic acid: Ascorbic acid, Citrus Callus Solution: Citron callus supernatant extract, Citrus Callus Extract: Citron callus hydrothermal extract)
12 is a result confirming the antioxidant effect of the pear callus supernatant extract and pear callus hydrothermal extract. (Ascorbic acid: Ascorbic acid, Pear Callus Solution: Pear callus supernatant extract, Pear Callus Extract: Pear callus hydrothermal extract)
13 is a result confirming the elastase inhibitory activity of the citron callus supernatant extract and citron callus hot water extract. (Ursolic acid: Ursolic acid, Citrus Callus Solution: Citron callus supernatant extract, Citrus Callus Extract: Citron callus hydrothermal extract)
14 is a result confirming the elastase inhibitory activity of pear callus supernatant extract and pear callus hot water extract. (Ursolic acid: Ursolic acid, Pear Callus Solution: Pear callus supernatant extract, Pear Callus Extract: Pear callus hydrothermal extract)
15 is a result confirming the collagen production promoting activity of the citron callus supernatant extract and citron callus hot water extract. (Citrus Callus Solution: Citron Callus Supernatant Extract, Citrus Callus Extract: Citron Callus Hot Water Extract)
16 is a result confirming the collagen production promoting activity of pear callus supernatant extract and pear callus hydrothermal extract. (Pear Callus Solution: Pear Callus Supernatant Extract, Pear Callus Extract: Pear Callus Hydrothermal Extract)
17 is a result confirming the skin cell proliferation activity of the citron callus supernatant extract and citron callus hot water extract. (Citrus Callus Solution: Citron Callus Supernatant Extract, Citrus Callus Extract: Citron Callus Hot Water Extract)
18 is a result confirming the skin cell proliferation activity of the pear callus supernatant extract and pear callus hot water extract. (Pear Callus Solution: Pear Callus Supernatant Extract, Pear Callus Extract: Pear Callus Hydrothermal Extract)
19 is a result confirming the effect of the mixing treatment of pear callus extract, citron callus extract and carbomer on the expression of prolyl hydroxylase (prolyl hydroxylase).
20 is a result confirming the effect of the mixing treatment of pear callus extract, citron callus extract and carbomer on the expression of lysyl hydroxylase (lysyl hydroxylase).
21 is a result confirming the effect of the mixing treatment of pear callus extract, citron callus extract and carbomer on collagen synthesis.

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

In particular, the following examples confirm the efficacy of the pear or citron callus culture extract, but it will be apparent to those skilled in the art that such an effect is also in the culture itself, not the extract.

1. Pears and Citrons According to the Present Invention Plant callus  Induction and culture

(1) Plant material preparation

As a plant for inducing citron callus, the leaves, fruit juice and seeds of citron seedlings purchased from Isaac Farm were used. Plants for induction of pear callus were used to germinate seeds received from good farming associations. After sterilization for 1 minute 30 seconds using 70% EtOH (Merck, cat.603-002-00-5) solution, the solution was stirred for 30 minutes in 2% sodium hypochloride (Duksan, cat. 7681-52-9) solution. It was then washed four times with distilled water sterilized in a clean bench. After sterilization, plant tissue samples were used to induce citron and callus as follows.

(2) citron and pear callus induction

  The leaves, fruit juice, and seeds of citron and pear were cut to a certain size, and then wound on callus induction medium. For the induction of citron and pear callus, 2,4-dichlorophenoxy-acetic acid (2,4-D, Duchefa Cat.D0911.0100) and 6- in basic MS medium (Murashige and Skoog, Duchefa Cat. M0222.0050), respectively. Media containing 1 mg / L of Benzylaminopurine (BA, Duchefa Cat. B0904.0001) was used and induced with a cancer cycle and a 16 hour photoperiod (FIG. 1).

(3) Exploration of Citron and Pear Callus Growth Conditions

  To grow induced citron and pear callus in a solid phase medium, a medium loaded with 2 mg / L of Picloram (Duchefa, Cat. P0914.0005), which is mainly used for plant callus growth, was used in the basic MS medium.

(4) citron and pear callus liquid culture

  The step of culturing on solid medium was converted to liquid medium culturing step for culture scale-up of citron and pear callus grown in solid medium. To this end, 100 ml of MS basal medium to which Picloram 2 mg / L was added was added to a 500 ml Erlenmeyer flask using the optimized culture conditions identified on solid media, and then inoculated with three well-cultured solid culture plates. Incubated at 25 ° C., 100 rpm (FIG. 2).

2. Preparation of Citron and Pear Callus Extract Sample

With citron callus and pear callus culture obtained in the above 1,

Firstly, the extract is extracted by cooling the callus in the refrigerated state ("Calus supernatant extract"), and secondly, the extract is extracted by hydrothermal extraction by heating the water of the callus ("Calus hydrothermal extract") Respectively obtained.

More specifically, the yuja callus was poured into a mortar and poured into liquid nitrogen to finely grind. 1 ml of PBS was added per 0.2 g of citron or pear callus and stirred at 4 ° C. for at least 1 hour to mix well.

First, the preparation of the citron or pear callus supernatant extract was prepared by centrifuging the stirred sample at 8000 rpm for 10 minutes and then taking the supernatant and filtering using a 0.8 um syringe filter ("Calus supernatant extract").

Second, the preparation of citron or pear callus hot water extract is heated for 2 hours in a water bath (90 ~ 95 ℃ ), centrifuged for 10 minutes at 8000rpm, and the supernatant is taken and filtered using a 0.8um syringe filter Prepared ("Calus hydrothermal extract").

Experimental Example  1: pear or citron according to the present invention Callus  Skin safety test of culture extract

Human skin fibroblasts (fibroblasts) were evaluated using the MTT assay to test for the presence of safety or cytotoxicity against the cell line CCD-986sk. Specifically, the fibroblast CCD-986sk 5 × 10 ⁴ cells / well were counted in a 24-well plate using a heamacytometer and then aliquoted and cultured. After 48 hours of incubation in DMEM containing 10% FBS and cultivation of ˜50% of the surface of the culture vessel, lyophilized citron or pear callus supernatant extract and hot water extracts prepared by the above method, and the powdered samples 0.01 mg / 48 hours were further incubated with the treatment of ml ~ 1 mg / ml concentration. After further incubation, 50 μL of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT, Sigma M5655, USA) solution (2.5 mg / mL) was added and incubated for an additional 3 hours. . Subsequently, discard all the cell cultures, treat 200 μL of dimethyl sulfoxide (DMSO, Sigma D2650, USA) with 200 μL of each well and stir, take 100 μL of 96 wells with Enzyme-Linked Immunosorbent Assay (ELISA). Absorbance was measured at 570 nm. For relative evaluation, the degree of promoting toxicity or proliferation of cells was expressed as a percentage based on the absorbance intensity of the control group using pure water.

As a result, the supernatant (cold-hot extract) and extract (hot water extract) of citron callus were not toxic to human fibroblast growth and had a growth promoting effect, as shown in the table below. (FIG. 3, and Tables 1 and 2).

Citron callus supernatant extract (mg / ml) control One 0.5 0.1 0.05 0.01 Result value 100 111.1812 124.4364 117.2227 115.78 105.2299

Citron callus hot water extract (mg / ml) control One 0.5 0.1 0.05 0.01 Result value 100 104.7162 113.0131 103.0568 104.8035 99.21397

As a result, pear callus supernatant and pear callus extract prepared by the above method were very safe for human fibroblast cells CCD-986sk and no cytotoxicity was observed. In particular, the cell growth promoting effect was observed around -6.2 ~ 35.0% in all treated groups compared to the untreated control (Fig. 4, Table 3 and Table 4).

Bacalus supernatant extract (mg / ml) control One 0.5 0.1 0.05 0.01 Result value 100 133.156 135.0177 115.2482 106.117 93.88298

Bacalus hot water extract (mg / ml) control One 0.5 0.1 0.05 0.01 Result value 100 128.2342 128.2342 122.474 112.3702 104.7214

Experimental Example  2: pear or citron according to the present invention Callus  Safety Evaluation of Cultured Extracts on in vitro Keratinocytes

The human dermal constituent keratinocyte cell line HaCaT was evaluated using the MTT assay to test for the presence of safety or cytotoxicity. Specifically, human keratinocyte line HaCaT cells 5 × 10 ³ cells / well were counted in a 24 well plate using a heamacytometer and then aliquoted and cultured. After 48 hours of incubation in DMEM containing 10% FBS and cultivation of ˜50% of the surface of the culture vessel, freeze-dried powders of citron or pear callus supernatant extract and hot water extract prepared in the above method were added to the culture medium at 0.01 mg / ml. Treatment at a concentration of 1 mg / ml and incubation for an additional 48 hours. After further incubation, 50 μL of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT, Sigma M5655, USA) solution (2.5 mg / mL) was added and incubated for an additional 3 hours. . Subsequently, discard the cell culture medium, and 200 μL of dimethyl sulfoxide (DMSO, Sigma D2650, USA) was treated with 200 μL of each well, followed by stirring. Then, 100 μL of 96 wells was taken and 570 nm with Enzyme-Linked Immunosorbent Assay (ELISA). Absorbance was measured at. For relative evaluation, the degree of promoting toxicity or proliferation of cells was expressed as a percentage based on the absorbance intensity of the control group using pure water.

[Equation 1]

Cell safety (%) = (absorbance of test group / absorbance of control group) x 100

As a result, citron callus supernatant and citron callus extract prepared by the above method were very safe for HaCaT, a human keratinocyte cell line, and no cytotoxicity was observed. In particular, the cell growth promoting effect was observed around -4.5 ~ 38.6% in all treated groups compared to the untreated control (Fig. 5, Table 5 and Table 6).

Citron Callus Supernatant Extract
(mg / ml) control One 0.5 0.1 0.05 0.01 Result value 100 134.2618 128.7337 113.6062 130.8764 95.99314

Citron Callus Hot Water Extract
(mg / ml) control One 0.5 0.1 0.05 0.01 Result value 100 138.6101 124.7113 110.4136 105.8577 95.50703

As a result, the pear callus supernatant and pear callus extract prepared by the above method were very safe for HaCaT, a human keratinocyte cell line, and no cytotoxicity was observed. In particular, the effect of promoting cell proliferation around 0 to 35% was observed in all of the treated groups compared to the untreated control (FIG. 6, Table 7 and Table 8).

Bacalus supernatant extract
(mg / ml) control One 0.5 0.1 0.05 0.01 Result value 100 172.78 197.7365 158.5026 145.5891 139.321

Bacalus hot water extract
(mg / ml) control One 0.5 0.1 0.05 0.01 Result value 100 161.1568 157.3007 133.012 113.9901 103.9737

Experimental Example  3: pear or citron Callus  Evaluation of Whitening Activity of Culture Extracts 1 ( Tyrosinase  Activity inhibition)

  Tyrosinase is a rate-limiting enzyme that plays a very important role in skin melanin production. It acts as a dopa oxidase, which oxidizes tyrosine to form dopa, and then oxidizes dopa to form dopachrome in the melanocyte pigment-forming cells (melanocyte). More precisely, tyrosinase (EC 1.14.18.1) plays the most important role in the synthesis of melanin, a human skin pigment, and is expressed by TYR or c-locus, located on human chromosome 11q14-21. The tyrosinase catalyzes three distinct reactions in the melanin synthesis stage: 1) hydroxylation of monophenol (L-tyrosine), 2) dehydrogenation of catechol (L-DOPA), and 3) dehydrogenation of DHI. Therefore, inhibition of tyrosinase activity of various whitening functional raw materials may be an important measure of whitening activity.

In order to confirm the whitening activity of citron or pear callus supernatant and extract prepared by the above method, the degree of inhibition of melanin synthase and melanogenesis process using mushroom-derived tyrosinase and 3,4-dihyrroxyphenylalanine (L-DOPA) as enzyme substrate We examined whether it inhibits the oxidation of L-DOPA, which is an important step.

① in vitro tyrosinase  Activity inhibition test method

More specifically, the freeze-dried citron or pear callus extract was diluted with sodium phosphate buffer (0.1 M, pH 6.8) to prepare a final concentration of 0.5 mg / ml to 10 mg / ml. 290 μL of sodium phosphate buffer, 100 μL of prepared sample, and 100 μL of 10 mM substrate were added to a 2 mL tube, followed by pre-incubation at 37 ° C. for 5 minutes. Thereafter, 10 μL of 2,500 unit / mL tyrosinase solution was added thereto, followed by reaction at 37 ° C. for 10 minutes. After the reaction, absorbance was measured at 475 nm, and arbutin and vitamin C were used at a concentration of 0.5 mg / ml to 10 mg / ml as a positive control. Add 0.1M phosphate buffer solution (pH 7.0) instead of sample solution.

% inhibition of tyrosinase activity = 100-[(b-b ') / (a-a') X 100]

a: absorbance after reaction of blank sample liquid

b: absorbance after reaction of the sample liquid

a ', b': absorbance measured by replacing buffer with tyrosinase

As a result, it was confirmed that significant inhibition of tyrosinase enzyme activity such as 23.1% in the 5 mg / ml treatment group and 36.2% in the 10 mg / ml treatment group was observed in the citron callus hydrothermal extract compared to the untreated control group (Table 9, FIG. 7). .

Citron Callus Hydrothermal Extract
(mg / ml) control 10 5 One Result value 0 36.190 23.095 -2.381

In case of pear callus hydrothermal extract, significant tyrosinase enzyme activity was confirmed in the 10 mg / ml treatment group (Table 10, FIG. 7).

Bacalus hydrothermal extract
(mg / ml) control 10 5 One Result value 0 10.952 -2.381 -8.095

② in vitro L- DOPA  Oxidation Inhibition Test Method

  This test method evaluates the effect of whitening components by measuring the inhibition of the activity of DOPA oxidation of tyrosinase catalyzing the rate-determination step of melanin synthesis. More specifically, the freeze-dried citron or pear callus hydrothermal extract was diluted with sodium phosphate buffer (0.1 M, pH 6.8) to prepare a final concentration of the total reaction solution from 0.5 mg / ml to 10 mg / ml. As a test method, 850ul of sodium phosphate buffer (0.1 M, pH 7.0), 50ul of diluted callus sample and 50ul of mushroom tyrosinase solution were added to the test tube and reacted at 37 ℃ for 6 minutes. 50ul of 0.06mM L-DOPA (L-3,4-dihydroxyphenylalanine, Sigma D9628-25G) solution was added to the solution and reacted at 37 ° C for 1 minute. After the reaction is completed, the absorbance is measured at 475 nm using an ELISA reader. Add 0.1M phosphate buffer solution (pH 7.0) instead of sample solution.

[Equation]

DOPA oxidative activity inhibition rate (%) = 100-{(reaction absorbance of each sample solution / reaction absorbance of blank sample solution) × 100}

As a result, very strong L-DOPA oxidation inhibitory activity was observed in the treatment of citron callus hydrothermal extract compared to the untreated control, 9.2% at 0.5 mg / ml, 18.3% at 2.5 mg / ml, 5 mg / ml The strong inhibitory activity was confirmed around 25.2% upon treatment (Table 11, Figure 8).

Citron Callus Hydrothermal Extract
(mg / ml) control 5 2.5 0.5 Result value 0 25.157 18.339 9.169

In addition, the treatment of pear callus hydrothermal extract was weak but significant L-DOPA oxidation inhibitory activity compared to the untreated control group, 13.5% at 5mg / ml treatment, 3.8% at 2.5mg / ml treatment. (Table 12, FIG. 8).

Bacalus hot water extract
(mg / ml) control 5 2.5 0.5 Result value 0 13.558 3.840 2.665

Experimental Example  4: pear or citron Callus  Evaluation of Whitening Activity of Culture Extracts 2 ( B16F10  Inhibition of melanin production in cells)

In order to confirm the whitening activity of the citron and pear callus supernatants and extracts developed by the above method, the cells were treated with B16F10 (rat melanoma) cells, which are melanin-forming skin constituent cells, and compared with the amount of melanin produced by the cells in culture. Evaluated. More specifically, B16F10 cells were dispensed at 2 × 10 ⁴ cells / well in 96 well plates and treated with 1 mg / ml at 0.05 mg / ml in culture using freeze-dried callus supernatant and extract. Samples were treated at appropriate concentrations and further incubated for 72 hours to induce inhibition of melanin biosynthesis. After further incubation, the supernatant of each well was aspirated, washed with PBS, and 150 μL of 1N NaOH was added to each well. The melanin formed in the cells was completely dissolved by standing at 65 ° C. for 45 minutes, and then 100 μL of the supernatant of each well was taken and measured for absorbance at 405 nm. The amount of melanin produced was evaluated as a percentage of the relative decrease in the non-treated group as a negative control group to evaluate the whitening activity.

[Equation]

Melanin synthesis inhibition rate (%) = 100-{(reaction absorbance of each sample solution / reaction absorbance of negative control group) × 100}

As a result, the treatment of citron callus supernatant extract compared to the non-treated control group showed more than 50% melanin biosynthesis inhibitory effect in the tested concentration, confirming the whitening effect. In the case of citron callus, a strong whitening effect was observed in both the citron callus supernatant extract and the citron callus hydrothermal extract (Table 13 and Table 14, FIG. 9).

Citron callus
Supernatant Extract (mg / ml) control One 0.5 0.1 0.05 Result value 0 51.25581 52.46512 60 53.11628

Citron callus hydrothermal extract (mg / ml) control One 0.5 0.1 0.05 Result value 0 55.87967 50.31905 59.34366 55.33273

As a result, the treatment of pear callus supernatant extract compared to the untreated control group was observed to inhibit melanin biosynthesis from 37.7% to 51.4% within the tested concentration. In addition, the treatment of pear callus hydrothermal extract was increased in a concentration-dependent manner from 7.1% to confirm about 44.6% melanin inhibitory effect at the treatment of 1 mg / ml concentration (Table 15 and Table 16, Figure 10).

Pear callus supernatant extract (mg / ml) control One 0.5 0.1 0.05 Result value 0 41.68297 46.57534 51.36986 37.67123

Pear callus hydrothermal extract (mg / ml) control One 0.5 0.1 0.05 Result value 0 44.60363 38.01337 13.56256 7.067813

Experimental Example  5: pear or citron Callus  Evaluation of Antioxidant Activity of Cultured Extracts ( DPPH  Radical scavenging effect)

Antioxidative effect experiments of citron and pear plant callus supernatants and extracts developed by the above method were measured by the free radical scavenging effect of the sample using DPPH (2,2-diphehyl-pictyl-hydrazyl). As a positive control, ascorbic acid was dissolved in tertiary distilled water to prepare a solution with a concentration of 50 to 1,000 μM. It was. 100 μL of 0.2 mM DPPH methanol solution was added to 100 μL of each sample solution, followed by reaction at room temperature for 30 minutes. Thereafter, 100 μL of each sample solution was added with 100 μL of methanol, and the absorbance was measured at 520 nm using a DPPH methanol solution as a negative control, and DPPH inhibition rate (antioxidation rate) was calculated by the following equation.

[Equation]

DPPH inhibition rate (%) = 100-[(absorbance of sample-absorbance of each sample blank) / absorbance of negative control × 100]

Experimental results showed the maximum antioxidant activity of the citron plant callus supernatant extract of 22.4-4.99%, while the citron plant callus hydrothermal extract showed the maximum antioxidant rate (68.5-9.49%) corresponding to the positive control 125 μM ascorbic acid (Table 17). , FIG. 11).

Ascorbic acid Citron Plant Callus Supernatant Extract Citron plant callus hydrothermal extract Treatment concentration (μM) Free radical scavenging activity (%) Treatment concentration (mg / mL) Free radical scavenging activity (%) Treatment concentration (mg / mL) Free radical scavenging activity (%) 500 98.5 ± 2.64 10 22.4 ± 4.99 10 68.5 ± 9.49 250 80.1 ± 1.93 5 0.00 ± 0.00 5 21.2 ± 1.41 125 62.3 ± 1.77 2 0.00 ± 0.00 2 10.1 ± 4.16 50 17.7 ± 4.37 One 0.00 ± 0.00 One 5.13 ± 3.67 25 0.00 ± 0.00 0.1 0.00 ± 0.00 0.1 0.00 ± 0.00

The pear plant callus supernatant extract showed an antioxidant rate of up to 46.8-6.68% and the pear plant callus hydrothermal extract showed a maximum antioxidant rate (78.7-4.76%) corresponding to the positive control 500 μM ascorbic acid (Table 18, FIG. 18). 12).

Ascorbic acid Pear Plant Callus Supernatant Extract Pear Plant Callus Hydrothermal Extract Treatment concentration (μM) Free radical scavenging activity (%) Treatment concentration (mg / mL) Free radical scavenging activity (%) Treatment concentration (mg / mL) Free radical scavenging activity (%) 1000 95.1 ± 1.40 10 46.8 ± 6.68 10 78.7 ± 4.76 500 82.1 ± 1.13 5 2.14 ± 3.04 5 78.6 ± 1.86 250 67.5 ± 6.05 2 0.00 ± 0.00 2 34.8 ± 14.2 100 52.2 ± 6.05 One 0.92 ± 1.83 One 46.3 ± 4.66 50 24.9 ± 9.25 0.1 2.82 ± 3.28 0.1 35.9 ± 19.3

Experimental Example  6: pear or citron Callus  Elastase Inhibitory Activity of Cultured Extracts ( elastase  inhibition assay)

The elastase inhibitory activity of the citron and pear callus supernatant and extract developed by the above method was evaluated by the EnzChek elastase assay kit. DQ ^TM elastin (soluble bovine neck ligament elastin labeled with fluorescence dye) was used as a substrate and N -Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone was used as an inhibitor. Add 50 μL of ursolic acid (0.0001 ~ 0.1 mM) or 50 μL of citron and pear callus supernatant or extract to each well of the 96 well opti-plate, and 50 μL of DQ elastin working solution (100 μg / mL) to each well. And 100 μL of porcine pancreatic elastase (0.2 U / mL) was added. After reacting at room temperature for 1 hour, the fluorescence intensity of each well was measured in a fluorescence multiplate reader. (excitation: 485 nm, emission: 530 nm) Elastase inhibitory activity of each sample was calculated by the following equation.

[Equation]

Elastase Inhibition Rate (%) = (Ac-As) × 100 / (Ac-An)

Here, Ac is the fluorescence intensity of the well without the measurement sample, As is the fluorescence intensity of the well containing the measurement sample, An is the fluorescence intensity of the blank well without the measurement sample and enzyme.

Experimental results showed that the extract of citron plant callus supernatant showed up to 15.1 to 0.28% of elastase inhibitory activity. (Table 19, FIG. 13).

Ursolic acid Citron Plant Callus Supernatant Extract Citron plant callus hydrothermal extract Treatment concentration (mM) Elastase inhibition (%) Treatment concentration (mg / mL) Elastase inhibition (%) Treatment concentration (mg / mL) Elastase inhibition (%) 0.1 67.0 ± 1.16 10 9.81 ± 0.36 10 15.1 ± 1.01 0.01 44.8 ± 1.59 5 9.51 ± 2.49 5 14.7 ± 0.53 0.0025 30.1 ± 4.43 2 12.2 ± 0.70 2 10.1 ± 1.33 0.001 19.0 ± 1.29 One 11.1 ± 0.87 One 20.3 ± 1.17 0.0001 10.9 ± 0.54 0.1 15.1 ± 0.28 0.1 11.4 ± 0.28

In the case of pear plant callus, the elastase inhibitory activity of the supernatant extract and the hydrothermal extract was not dependent on the treatment concentration. Maximum inhibitory activity (45.9-1.18%) corresponding to 0.001 mM ursolic acid, a positive control, was shown (Table 20, FIG. 14).

Ursolic acid Pear Plant Callus Supernatant Extract Pear Plant Callus Hydrothermal Extract Treatment concentration (mM) Elastase inhibition (%) Treatment concentration (mg / mL) Elastase inhibition (%) Treatment concentration (mg / mL) Elastase inhibition (%) 0.1 96.3 ± 0.55 10 39.6 ± 3.06 10 37.9 ± 1.60 0.01 81.7 ± 2.40 5 42.8 ± 0.97 5 39.9 ± 2.28 0.0025 60.8 ± 1.23 2 43.1 ± 1.67 2 42.0 ± 1.10 0.001 49.9 ± 1.10 One 42.0 ± 0.61 One 44.3 ± 1.70 0.0001 39.3 ± 2.46 0.1 47.6 ± 1.35 0.1 45.9 ± 1.18

Experimental Example  7: pear or citron Callus  Collagen Production Promoting Activity of Cultured Extracts

The collagen production promoting activity of the citron and pear plant callus supernatant extract and hot water extract obtained by the above method was evaluated as follows. Human fibroblast cells (CCD-986sk) were dispensed in 96 well plates at 1 × 10 ⁴ cells / well and incubated in DMEM medium containing 10% FBS for 24 hours, and then diluted with serum free medium. And pear plant callus supernatant and extract or recombinant human transforming growth factor-beta (rhTGF-β) were added to each well by 100 μL and incubated in a CO ₂ incubator for 24 hours. Cell cultures were then taken and measured for collagen biosynthesis using Procollagen type I C-peptide EIA kit (Takara, Japan). First, 100 μL of the anti-POD conjugate solution was added to each well, 20 μL of the sample or standard solution was added thereto, stirred, and then reacted at 37 ° C. for 3 hours. The contents were then removed and washed four times with 400 μL of phosphate buffer (pH 7.4). After washing, 100 μL of the substrate solution was added to each well, followed by reaction at 20˜30 ° C. for 15 minutes, and 100 μL of ELISA stop solution was added thereto to stop color development and stirred. After measuring the absorbance at 450 nm, the amount of collagen biosynthesis was calculated using the standard concentration curve.

The results showed that collagen biosynthesis was significantly increased compared to the untreated negative control group when treated with citron and pear plant callus supernatant or extract. % Increased (Table 21, Figure 15).

rhTGF-β Citron Plant Callus Supernatant Extract Citron plant callus hydrothermal extract Treatment concentration (ng / mL) Procollagen type I C-peptide (ng / mL) Treatment concentration (mg / mL) Procollagen type I C-peptide (ng / mL) Treatment concentration (mg / mL) Procollagen type I C-peptide (ng / mL) 0 379.7 ± 66.83 5 489.6 ± 40.44 5 507.2 ± 38.40 10 571.0 ± 71.52 2 744.6 ± 88.49 2 620.9 ± 72.01 100 617.5 ± 97.41 One 658.3 ± 73.08 One 668.1 ± 22.40 500 561.6 ± 98.9 1,000 504.2 ± 71.7

In contrast, citron plant callus extract (1 mg / mL) increased collagen biosynthesis by 176% compared to untreated negative control and pear plant callus extract (5 mg / mL) increased collagen biosynthesis by 204% compared to untreated negative control (Table 22 , FIG. 16).

rhTGF-β Pear Plant Callus Supernatant Extract Pear Plant Callus Hydrothermal Extract Treatment concentration (ng / mL) Procollagen type I C-peptide (ng / mL) Treatment concentration (mg / mL) Procollagen type I C-peptide (ng / mL) Treatment concentration (mg / mL) Procollagen type I C-peptide (ng / mL) 0 379.7 ± 66.83 5 785.0 ± 27.40 5 776.5 ± 54.00 10 571.0 ± 71.52 2 763.4 ± 33.69 2 681.7 ± 99.70 100 617.5 ± 97.41 One 807.1 ± 46.61 One 560.4 ± 72.78 500 561.6 ± 98.9 1,000 504.2 ± 71.7

Experimental Example 8: Skin Cell Proliferation Activity of Cultured Pear or Citron Callus Extract

The skin cell proliferation activity of the citron and pear plant callus supernatants and extracts developed by the above method was evaluated as follows. Human fibroblast cells (CCD-986sk) were dispensed in 96 well plates at 5 × 10 ³ cells / well and incubated with 10% FBS / DMEM medium for 24 hours, then the culture medium was removed and DMEM containing 0.05% FBS. The culture was again incubated for 24 hours. Then, 100 μL of citron and pear plant callus supernatants and extracts diluted with DMEM culture medium containing 0.5% FBS, and recombinant human transforming growth factor-beta (rhTGF-β) were added to each well. After the addition was incubated for 24 hours in a CO ₂ incubator. After 5 mg / mL WST-1 (Roche Diagnostics) solution was treated with 10 μL of each well, and then reacted for 4 hours at 37 ° C. Absorbance was measured at 450 nm. Was evaluated.

Experimental results showed that the extract of citron and pear plant callus supernatant significantly increased the proliferation of human fibroblasts compared to the untreated negative control at the concentration of 5 mg / mL or less, and when treated with the positive control rhTGF-β at the concentration of 1,000 ng / mL Skin cell proliferation was increased by 135% compared to the untreated negative control. On the other hand, skin cell proliferation was increased by 154% compared to untreated negative control when treated with citron plant callus hot water extract (5 mg / mL) and 126 compared with no treatment negative control when treated with pear plant callus hydrothermal extract (5 mg / mL). % Increased (Tables 23 and 24, FIGS. 17 and 18).

rhTGF-β Citron Plant Callus Supernatant Extract Citron plant callus hot water extract Treatment concentration (ng / mL) Relative cell proliferation (%) Treatment concentration (mg / mL) Relative cell proliferation (%) Treatment concentration (mg / mL) Relative cell proliferation (%) 0 100.0 ± 5.426 10 26.16 ± 1.664 10 27.44 ± 0.952 10 115.2 ± 2.146 5 50.35 ± 3.907 5 153.9 ± 8.878 50 132.5 ± 12.81 2 95.71 ± 4.217 2 130.2 ± 12.93 100 131.0 ± 12.59 One 99.00 ± 4.944 One 133.3 ± 16.58 500 127.9 ± 13.58 0.1 102.8 ± 8.434 0.1 114.2 ± 8.894 1,000 134.8 ± 13.53 0.01 103.1 ± 9.888 0.01 115.7 ± 12.56

rhTGF-β Pear Plant Callus Supernatant Extract Pear Plant Callus Hot Water Extract Treatment concentration (ng / mL) Relative cell proliferation (%) Treatment concentration (mg / mL) Relative cell proliferation (%) Treatment concentration (mg / mL) Relative cell proliferation (%) 0 100.0 ± 5.426 5 104.1 ± 5.122 5 126.4 ± 9.000 10 115.2 ± 2.146 2 110.5 ± 4.119 2 114.5 ± 0.796 50 132.5 ± 12.81 One 118.5 ± 5.040 One 115.2 ± 3.535 100 131.0 ± 12.59 0.1 112.2 ± 8.019 0.1 96.02 ± 5.596 500 127.9 ± 13.58 0.01 134.1 ± 13.69 0.01 106.0 ± 2.882 1,000 134.8 ± 13.53

Experimental Example  9: pear or citron Callus  Skin Regeneration Activity of Cultured Extracts

The skin regeneration activity of the citron and pear callus supernatants and extracts developed by the above method was evaluated as follows. Human fibroblast cells (CCD-986sk) were dispensed in 96 well plates at 3.5 × 10 ⁴ cells / well and incubated for 72 hours in 10% FBS / DMEM medium to form human fibroblast monolayers. Using a wound maker, cell-free zones (scratch wounds) were formed in each well. Each well was then washed four times with 100 μL of phosphate buffer (pH 7.4) and diluted in serum-free DMEM medium and in citron and pear callus supernatant extracts and hydrothermal extracts or recombinant human transforming growth factor. 100 μL of beta and rhTGF-β were added to each well, and the wound wound recovery (regeneration) rate was measured for 72 hours at 4 hour intervals with an Incucyte Zoom microscope (Essen Bioscience) in a CO ₂ incubator.

Experimental results showed that the regeneration activity of skin cells was significantly increased compared to the untreated negative control group in the treatment of citron and pear plant callus supernatant extracts, and rhTGF-β, a positive control group, was treated at 1,000 ng / mL. Activity increased by 155%. On the other hand, treatment with citron plant callus hydrothermal extract (0.01 mg / mL) showed a 155% increase in skin regeneration activity compared to the untreated negative control and treatment with pear plant callus hydrothermal extract (0.01 mg / mL) was 126%. % Improvement was made.

rhTGF-β (ng / mL) Relative wound recovery (%) Plant Callus (mg / mL) Relative wound recovery (%) 0 ng / mL 36.0 ± 0.99 Citron Plant Callus Supernatant Extract
(0.01 mg / mL) 43.6 ± 3.32 10 ng / mL 44.8 ± 1.06 Citron plant callus hydrothermal extract
(0.01 mg / mL) 55.7 ± 1.41 50 ng / mL 43.6 ± 3.04 Pear Plant Callus Supernatant Extract
(0.01 mg / mL) 45.3 ± 0.50 100 ng / mL 49.3 ± 1.11 Pear Plant Callus Hydrothermal Extract
(0.01 mg / mL) 45.3 ± 0.91 500 ng / mL 54.6 ± 3.68
1,000 ng / mL 55.7 ± 6.93

Experimental Example  10: Wrinkle improvement efficacy test of the external composition for skin according to the present invention

Collagen, a key component that maintains skin's elasticity, among the intercellular materials produced by the fibroblasts that make up the human skin, is required for hydorxyproline, a derivative of proline amino acid, and hydroxyllysin, a derivative of lysin amino acid. Required as an element. These two amino acid derivatives are enzymatically converted by proline hydroxlase and lysyl hydroxylase enzymes. How do pear callus extracts, citron callus extracts, and carbomer mixtures affect the expression of two enzymes in fibroblast cells? It was investigated by RT-PCR.

1.1 CCD-986sk Dermal Fibroblast Culture

Human dermal fibroblast cells (CC-986sk skin fibroblasts) were attached to a polystyrene cell culture plate and cultured using DMEM (Gibco, USA) added with 10% FBS (Gibco, USA). Humidity was maintained at 95%, the temperature was maintained at 37 ℃ 5% CO2 was continuously supplied.

1.2 Reverse transcriptase PCR  Of enzymes used mRNA  Expression measurement

RNA was isolated using Trizol reagent in cells treated with pear callus extract, citron callus extract, carbomer (Happy Call (Korea), Polygel CS) alone or mixed for 2 or 3 days at each concentration, and wavelength 260 Quantification at nm. 10 μL of total RNA (0.5 μg / μL) was heated at 65 ° C for 10 minutes and left at 4 ° C for 10 minutes, followed by 1 μL oligo (dT) 15 primer (0.5 μg / μL), 1 μL M-MLV RT (10 unit / μL; promega, USA), 1 μL RNase inhibitor (20-40 unit / μL; promega, USA), 10μL 5X RT buffer (250 mM Tris-HCl, pH8.3, 375 mM KCl, 15 mM MgCl2), 5 μL 2.5 mmol / L dNTP mixture, 0.1% diethylpyrocarbonate (DEPC) was added thereto, and cDNA was synthesized by heating at 37 ° C. for 1 hour. RT-PCR was performed on the synthesized cDNA to measure mRNA expression levels of prolyl hydroxylase and lysyl hydroxylase. Table 1 shows the primer sequences of GAPDH (Glyceraldehyde-3-phosphate dehydrogenase), Prolyl hydroxylase and Lysyl hydroxylase. 1 μL (0.5 μg / μL) of primer hydroxylase was added, and 0.1% DEPC-Water was added to the synthesized cDNA with PCR-premix tube (Bionia), followed by denaturation at 95 ° C for 30 seconds. 30 seconds at 72 ℃, the extension proceeded to 30 cycles for 2 minutes at 72 ℃. Lysyl hydroxylase proceeded with 40 cycles of denaturation at 94 ℃ for 30 seconds, annealing at 52 ℃ for 45 seconds, and extention at 72 ℃ for 30 seconds. PCR product was confirmed that there is no non-specific PCR product or primer dimer in 2% agarose gel, and the amount of the product was measured by imaging densitometer [Labworks ver.4.6. (image acquisition and analysis software), UVP, CA].

Primer sequence for RT-PCR Gene Primer sequence GAPDH Sense 5'-TTG TCA AGC TCA TTT CCT GGT ATG-3 ' Anti-sense 5'-GCC ATG TAG GCC ATG AGG TC-3 Prolyl hydroxylase Sense 5'-CCA CAG CAG AGG AAT TAC AG-3 ' Anti-sense 3’-ACA CTA GCT CCA ACT TCA GG-5 ’ Lysyl hydroxylase Sense 5'-GGA ACC TGG CCT ATG ACA CCC T-3 ' Anti-sense 5'-TGC CAT GCT GTG CCA GGA ACT-3 '

Experimental results showed that pear callus and citron callus extracts were treated with 0.1%, 0.5%, 1.0%, 1.25%, and 1.5% of carbomer alone based on cell culture for prolyl hydroxlase expression. The increase and decrease were repeated compared to the treatment group, and did not show a significant increase effect. In the case of lysyl hydroxylase expression, carbomer alone treated with 0.1%, 0.5%, 1.0%, 1.25%, 1.5% increased and decreased compared to the untreated group, resulting in a large change. It could not be triggered. (FIGS. 19 and 20) However, when the 0.5% concentration of carbomer was mixed with pear callus extract and / or citron callus extract, proline hydroxlase and lysyl hydroxylase expressions were significantly higher than those of the control group or the carbomer alone group. It could be confirmed that the increase.

These results indicate that pear callus extract and citron callus extract did not significantly affect the intracellular gene expression of two enzymes important for synthesizing collagen when treated alone in the tested concentration range, but were mixed with 0.5% carbomer. In cases, a significant increase in the amount of expression was observed. Experimental results showed that carbomer alone was not significantly different from 0.1% to 1.5% in the test group, but proline hydroxylase expression was mixed with 1.0% pear callus extract and 0.5% carbomer. Compared to the untreated group, 159%, citron callus extract 1.0% and carbomer 0.5% were mixed and increased by about 179%. The effects of carbomer, carbomer, pear callus extract and citron callus were increased. Even when 0.5% of the extracts were mixed and treated, the amount increased to 168%.

1. Control group 2.Carbomer 0.1% 3.Carbomer 0.5% 4.Carbomer 1.0% 5.Carbomer 1.25% 6.Carbomer 1.25% 7.Pear callus extract 1.0% + carbomer 0.5% 8. Citron callus extract 1.0% + carbomer 0.5% 9.Pear callus extract 0.5% + citron callus extract 0.5% + carbomer 0.5% Average: 100 106 94 97 105 89 159 179 168 Standard Deviation: 35 16 45 31 9 21 43 33 35

The effect of lysyl hyroxylase on the expression level was similar to that on proline hydroylase. Experimental results showed that carbomer alone was not significantly different from 0.1% to 0.5% in the test group, but the amount of lysyl hydroxylase expression was mixed with 1.0% pear callus extract and 0.5% carbomer. Compared to the untreated group, 189% and 1.0% citron callus extract and 0.5% carbomer were mixed and increased by about 178%. The effects of carbomer, carbomer, pear callus extract and citron callus were increased. Even when 0.5% of the extracts were mixed and treated, the amount increased to 180%.

1. Control group 2.Carbomer 0.1% 3.Carbomer 0.5% 4.Carbomer 1.0% 5.Carbomer 1.25% 6.Carbomer 1.25% 7.Pear callus extract 1.0% + carbomer 0.5% 8. Citron callus extract 1.0% + carbomer 0.5% 9.Pear callus extract 0.5% + citron callus extract 0.5% + carbomer 0.5% Average: 100 88 106 104 121 108 189 178 180 Standard Deviation: 21 56 37 46 53 14 41 58 43

1.3 Collagen production promoting activity confirmation test

The collagen production promoting activity of the citron and pear plant callus supernatants and extracts developed by the above method was evaluated as follows. Human fibroblast cells (CCD-986sk) were dispensed in 96 well plates at 1 × 10 ⁴ cells / well and incubated in DMEM medium containing 10% FBS for 24 hours, and then diluted with serum free medium. And pear plant callus supernatant and extract or recombinant human transforming growth factor-beta (rhTGF-β) were added to each well by 100 μL and incubated in a CO ₂ incubator for 24 hours. Cell cultures were then taken and measured for collagen biosynthesis using Procollagen type I C-peptide EIA kit (Takara, Japan). First, 100 μL of the anti-POD conjugate solution was added to each well, and 20 μL of the sample or standard solution was added thereto, stirred, and reacted at 37 ^o C for 3 hours. The contents were then removed and washed four times with 400 μL of phosphate buffer (pH 7.4). After washing, 100 μL of the substrate solution was added to each well, followed by reaction at 20˜30 ^o C for 15 minutes, and 100 μL of ELISA stop solution was added thereto to stop color development and stirred. After measuring the absorbance at 450 nm, the amount of collagen biosynthesis was calculated using the standard concentration curve.

As a result, in order to confirm whether increased expression of prolyl hydroxylase and lysyl hydroxylase in the cell leads to an increase in the collagen content of the fibroblast, the amount of total collagen synthsis was added to the untreated group (negative control), carbomer 0.1. %, 0.5%, 1.0%, 1.25%, 1.5% treatment group, pear callus extract 1.0% + carbomer 0.5% mixed treatment group, citron callus extract 1.0% + carbomer 0.5% mixed treatment group, and pear callus extract 0.5% + Citron callus extract 0.5% + carbomer 0.5% mixed treatment group. As a result, the carbomer alone treatment group did not show a significant increase effect, such as 106, 100, 104, 121, 108%, respectively. Excellent efficacy in increasing collagen production could be confirmed (FIG. 21).

1. Control group 2.Carbomer 0.1% 3.Carbomer 0.5% 4.Carbomer 1.0% 5.Carbomer 1.25% 6.Carbomer 1.25% 7.Pear callus extract 1.0% + carbomer 0.5% 8. Citron callus extract 1.0% + carbomer 0.5% 9.Pear callus extract 0.5% + citron callus extract 0.5% + carbomer 0.5% Average: 100 106 100 104 121 108 189 178 180 Standard Deviation: 21 56 37 15 19 14 41 58 43

The specific parts of the present invention have been described in detail above, and for those skilled in the art, these specific descriptions are merely preferred embodiments, and the scope of the present invention is not limited thereto. Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

An external composition for skin containing pear callus culture or citron callus culture or extract thereof as an active ingredient, wherein the composition further contains carbomer as an active ingredient.
delete The composition of claim 1, wherein the composition is for preventing or improving wrinkles.
According to claim 1, wherein the composition is a composition, characterized in that for enhancing elasticity.
The composition of claim 1, wherein the composition is for skin regeneration.
The composition of claim 1, wherein the composition has antioxidant capacity.
(a) inducing callus from pear or eugenic adult plants;
(b) drying the pear or citron callus culture obtained in step (a), powdered and mixed in purified water,
Centrifugation to obtain a supernatant, cold water extraction, hot water extraction, or ethanol extraction to prepare a skin external preparation composition; And extracting the extract to obtain an extract, followed by mixing with carbomer to prepare an external composition for skin.
A method for preparing the external preparation for skin according to claim 1.
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