KR20110088197A - Composition for skin external application comprising extract of pharbitis nil callus - Google Patents

Abstract

PURPOSE: An external use skin composition containing Pharbitis nil callus extract is provided to activate skin cells by cell proliferation and to ensure antioxidation and anti-inflammation. CONSTITUTION: An external use skin composition for improving skin condition contains 0.1-10 weight% of Pharbitis nil callus extract as an active ingredient. The Pharbitis nil callus extract is the callus itself, Pharbitis nil callus powder, or extract thereof. The composition is a cosmetic composition or pharmaceutical composition. The composition contains cinnamaldehyde, capsaicin, EGCG, vanillin, or gallic acid.

Description

Composition for Skin External Application Comprising Extract of Pharbitis nil Callus}

The present invention relates to an external composition for skin composition containing morning glory callus extract and a method for preparing the same, and more particularly, to an external composition for improving skin containing a morning glory callus extract as an active ingredient and a method for preparing the same.

Modern man is always threatened from the diseases associated with it by various internal and external stresses, and the skin is not free from such stresses. Various studies have been attempted to keep the skin healthy from such skin stress, and in recent years, interest in cosmetics using natural extracts obtained from natural materials having little skin irritation is increasing.

Plants have long been a source of various natural substances that help human well-being. Most of these substances are secondary metabolites and are used for pharmaceuticals, food additives, cosmetic raw materials, fragrances, and pigments. Despite the synthesis of numerous new compounds by the development of the field of organic synthesis, plants are still recognized as a source of new substances. Considering that there are thousands of new substances with new chemical structures that are found in plants every year, many of them are biologically active, making plants an important ingredient.

 Many chemicals produced by plants are classified as primary metabolites because they are essential for cellular function. Primary plant metabolites are universally present in plant populations and constitute the majority of chemicals produced by plants, but other compounds are classified as secondary metabolites and are the focal point of both scientific research and commercial use because of their many uses. Has become.

In some cases secondary metabolites are produced by plants, for example in response to stress induced by microbial infection, UV irradiation, mechanical damage and also by treatment with organic and inorganic materials. The diversity of secondary metabolites produced under such stress conditions is broad and includes alkaloids, terpenoids, prevonoids, carotenoids, phenolic compounds and phenols. Glycosides. However, not all secondary metabolites are produced in large quantities by all plants, and the kinds of metabolites depend largely on the type of plant.

Plants have excellent totipotency, in which the entire individual is formed even when only a part of the leaves, stems, and roots constituting the plant are transplanted. Plant cells are callus derived from the tissues of the plant. Callus is an oily tissue that cuts and enlarges the cells of the wound when the plant is injured. Or a special mass of tissue produced by a method of culturing in a medium containing Cytokinins.

Callus can be derived from any part of the plant such as leaves, stems, roots, rhizomes, and fruits through plant cell culture, especially tissue culture, and is characterized by being continuously produced by subculture. The basic principle of plant cell culture utilizes the biological fact that every plant cell has the ability to build the whole plant from which the cell is derived. This pluripotency is comparable to the pluripotency of embryonic stem cells in animals. It is therefore acceptable to accept that plant cells have a positive effect on the protection and activation of skin stem cells.

Therefore, there is a recent interest in the use of such callus, and is being studied in various aspects. In addition, as a patent related to callus, there is Korea Patent Publication No. 10-2008-0096411.

 On the other hand, the composition containing the morning glory seeds as a raw material for cosmetics, Korean Patent Registration No. 0475594 discloses a method for producing a cosmetic composition for cosmetic packs containing herbal and natural extracts, but inducing callus from the morning glory seeds callus extract There was no example of using a cosmetic composition to use or induce a useful substance in the morning glory callus.

The present inventors have conducted extensive studies to prepare the external preparation composition for skin improvement, and confirm that morning glory callus extract has an excellent effect on skin moisturizing and soothing, including antioxidant activity and anti-inflammatory activity, and completes the present invention. It became.

Accordingly, it is an object of the present invention to provide a skin external composition for skin improvement comprising morning glory callus extract as an active ingredient.

Another object of the present invention is to provide a method for preparing a skin external composition for skin improvement comprising the morning glory callus extract as an active ingredient.

The present invention, in one aspect, relates to a skin external composition for skin improvement containing morning glory callus extract as an active ingredient. Specifically, the morning glory callus extract may be contained in an amount of preferably 0.01 to 30% by weight, more preferably 0.1 to 10% by weight based on the total weight of the composition. When the extract is contained in less than 0.01% by weight, it is difficult to exert the desired skin improvement effect, when containing more than 30% by weight has a disadvantage in showing the formulation problem.

In the present invention, "morning glory callus extract" means an extract of the morning glory callus itself, morning glory callus powder, or morning glory callus itself or morning glory callus powder extracted with water or alcohol, most preferably ethanol. At this time, the extraction temperature is preferably from room temperature to 100 ℃, most preferably room temperature.

In the present invention, the meaning of "contained as an active ingredient" is meant to be contained to the extent that it can exhibit a skin improvement effect, such as a moisturizing effect, a skin soothing effect, etc. as a skin external preparation composition.

In the present invention, "morning glory" ( Pharbitis nil ) is a perennial vine plant with the dicotyledon moth plant, and is planted for ornamental purposes, but is also wild on the roadside or on the glade. The stem is dense, with long hairs facing downwards, winding up 3m to the left of other plants or objects. The leaves are alternate, have long petioles, round heart-shaped, and the ends of the leaf body are usually divided into three. The edges of the shards are flat, jagged and hairy on the surface. Flowers bloom in a variety of colors, including blue purple, red purple, white and red, in July-August, and hang one to three on the flower stalks from the axilla. Calyx is deeply divided into 5 pieces, the fragment is thin and long, pointed at the end, and has long hairs on the back side. Corolla is 10 ~ 13cm in diameter and looks like a funnel. The bud is curled to the right in the shape of a brush tip. There are five surgeries and one pistil. The fruit is in the calyx and is a round capsule divided into three spaces. Each of the three compartments contains two seeds. Morning glory is used as a medicinal herb. In oriental medicine, dried morning glory seeds are called 牽 牛 子. Blue or red morning glory seeds are called black axis, and white morning glory seeds are called white axis. It is effective in urine and swelling and swelling (積聚: sickness caused by long body mass in the stomach) and low back pain. The effect of black axis is faster than white axis. In civilian, when there are many leaves on morning glory, cut about 20cm from the root and dry it.

 In the present invention, "callus" refers to a special tissue or a mass of cells generated when a tissue cut out of a plant is cultured in an auxin-containing medium, a certain kind of plant is wounded, or an upper mouth is treated with auxin. Normally, callus is an amorphous tissue or cell mass that has lost its ability to cause normal organogenesis or tissue differentiation, and is mostly composed of flow cells. In a broad sense, it may also include plant tumor tissue caused by infections such as agrobacterium.

As the cells in the plant proliferate, they are oriented immediately and regularly assembled into specific tissues and organs. However, if various plant growth hormones are given from the outside, the control that has been maintained until then is released, and the cells are interpreted to proliferate freely. When the callus, an undifferentiated cell mass obtained by this dedifferentiation, is placed in a liquid medium having the same composition and shaken in culture, the cells fall apart and continue to proliferate in a suspended state. The callus or cultured cells are subdivided and proliferated permanently by passage in fresh medium, which is essentially different from the differentiated cells in which complete plants age and die. Callus or cultured cells, which have been passaged for several generations, are applied to a solid medium from which various plant growth hormones have been removed. In other words, the plant is restored in one somatic cell constituting the plant. Plant tissue cells isolated from the parent plants have the potential to be regenerated into complete plants when cultured in an appropriate culture environment. This morphogenic ability is of great value for the practical use of cosmetic raw materials, as well as for academic importance such as plant development.

In addition, cultivation of callus, which is an undifferentiated cell mass through tissue culture using the properties of these plants, and the use of foreign useful substances in these callus, skin moisturizing, soothing and converging, sun protection, whitening, exfoliation, skin aging prevention, antioxidant, Inducing secondary metabolites that have the effects of skin wrinkle improvement, antibacterial, anti-inflammatory and anti-cancer effects can be regarded as valuable as cosmetic raw materials with excellent physiological activity.

In the present invention, the plant tissue for inducing callus may be any part of the seed or the plant. For example, germinated or sterilized seeds can be germinated to cut away parts of the plant, or to cut and sterilize parts of the plant. In this way callus is induced through sterile tissue culture methods. Preferably, the plant fragments are cultured in a tissue culture medium. The medium includes MS (Murashige & Skoog, 1962) medium, N6 (Chu et al., 1975) medium, B5 (Gamborg et al., 1968), NN (Nitsch and Nitsch, 1967) medium, etc. Medium can be used. Plant growth regulators should be added to these basal media. The main plant growth regulators used are Auxin, 2,4-D (2,4-dichlorophenoxyacetic acid), 2,4,5-T (2). , 4,5-trichlorophenoxyacetic acid), Dicamba (2-methoxy-3,6-dichlorobenzoic acid), IAA (indole-3-acetic acid), IBA (indole-3-butyric acid), MCPA (2-methyl-4 -chlorophenoxyacetic acid), NAA (1-naphthylacetic acid), NOA (2-naphthyloxyacetic acid), Picloram (4-amino-2,5,6-trichloropicolinic acid) and Cytokinin family BAP (6-benzylaminopurine) , 2iP (N ^6- (2-isopentyl) adenine), Kinetin (6-furfurylaminopurine), Thidiazuron (1-phenyl-3- (1,2,3-thiadiazol-5-yl) urea), Zeatin (4-hydroxy -3-methyl-trans-2-butenylaminopurine is used at a concentration suitable for the plant, taking into account the species, age, site, and ripening of the plant. In addition, secondary metabolites such as alkaloids, terpenoids, prebonoids, carotenoids, phenolic compounds, and glycosides can be added to plant types to enhance the various physiological activities of callus induced by growth regulators. Can be added accordingly.

A summary of the preferred callus induction and culture method is as follows. A part of the tissue of the plant is separated and sterilized, and then cultured in a medium containing growth regulators of auxin or cytokinin in the plant basic medium. After 2-3 weeks of culturing, when the cells of the tissue divide and the callus, which is a mass of cells, is separated, only the callus is separated and placed on a new medium, and then passaged while observing a constant time interval or callus state.

In another aspect, the present invention

(a) inducing and incubating callus from the morning glory seeds; And

(b) a method for preparing a skin external composition for skin improvement comprising a morning glory callus extract comprising the step of preparing a composition containing a morning glory callus.

In the step (a), specifically, the appropriate medium may be selected to induce callus by germinating seeds of morning glory. If there is a medium generally used in the callus culture in the art, it can be used without limitation.

In the present invention, as a medium for inducing callus from morning glory seeds, MS (Murashige & Skoog, 1962) medium can be used. In addition to this, a variety of media conventionally known as plant callus induction media such as N6, B5, NN described above will also be applicable in the present invention.

When the callus is cultured in a medium containing useful substances, the callus absorbs the useful substances added to the medium and thus contains the useful substances in the callus. It can exhibit physiological activity. The useful substance in the callus may be introduced into the cultured callus by additionally performing the step of densifying the cultured callus in a medium containing the useful substance. Examples of the useful substance include cinnamic aldehyde (cinnamaldehyde), capsaicin (capsaicin), EGCG, vanillin, gallic acid and the like, in particular, secondary metabolites of plants including polyphenols.

In step (b), the callus obtained in step (a) is powdered and prepared in the composition, or the extract extracted by hydrothermal extraction, ethanol extraction, or the like is appropriate concentration, preferably 0.01 to 30, based on the total weight of the composition. It may be prepared by containing in a weight%, more preferably 0.1 to 10% by weight.

In an external composition for skin containing morning glory callus extract according to the present invention, in addition to the aqueous phase component and the oil phase component, additives may be included. The additives include alcohols, colorants, fragrances and the like.

In addition, the topical skin composition according to the invention can be prepared in various forms, for example emulsions, lotions, creams (oil-in-water, water-in-oil, multiphase), solutions, suspensions (anhydrous and aqueous), anhydrous products (oil And glycol-based), gels, masks, packs, powders, and the like.

The method for preparing the external preparation for skin containing the morning glory callus extract according to the present invention is not limited to the above-described manufacturing method, and those skilled in the art to which the present invention pertains may include some modification of the manufacturing method. In addition, the composition for external application for skin containing the morning glory callus extract according to the present invention may be prepared.

Meanwhile, the external preparation composition for skin according to the present invention may be a cosmetic composition or a pharmaceutical composition.

When the external preparation composition for skin is a pharmaceutical composition, it may further include a pharmaceutically acceptable carrier that is already known and used.

When the external preparation composition for skin is a cosmetic composition, it may include an acceptable carrier in a cosmetic preparation. 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 to be developed in the future, 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 from about 1% to about 99.99% by weight, preferably from about 90% to about 99.99% by weight relative to its total weight. However, since the ratio is dependent on the formulation as described above in which the external preparation composition for skin of the present invention is prepared, and its specific application site (face, neck, etc.), its preferred application amount, etc., the ratio is 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, sunscreens, colorants, fragrances, and the like. The alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, moisturizers, viscosity modifiers, emulsions, stabilizers, sunscreens, colorants, compounds / compositions that can be used as fragrances and the like are already known in the art, Appropriate materials / compositions may be selected and used.

As an embodiment of the present invention, the external preparation composition for skin according to the present invention may include glycerin, butylene glycol, propylene glycol, polyoxyethylene hydrogenated castor oil, ethanol, triethanolamine, etc., in addition to the morning glory callus extract, A trace amount may be included if necessary such as a medicine, a pigment, purified water, and the like.

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 systemically applicable 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 cosmetic or dermatology field.

The external composition for skin containing the morning glory callus extract according to the present invention activates the skin cells due to the growth and proliferation of the skin cells, which is safe for the skin and has an antioxidant and anti-inflammatory effect as well as moisturizing and soothing effects on the skin, It helps, and has an excellent effect on skin moisturizing, regenerating and soothing.

1 is a photograph of morning glory callus cultured in Example 1 of the present invention.
Figure 2 is a graph showing the cell proliferation effect by the morning glory callus extract according to the present invention.
Figure 3 is a graph showing the iNOS activity inhibitory effect by the morning glory callus extract according to the present invention.
Figure 4 is a photograph after the LPS and morning glory callus extract according to the present invention to the macrophage of Raw264.7 mice in the iNOS activity inhibition test of Experimental Example 3.
5 is a graph analyzing cytokine expression by the morning glory callus extract according to the present invention.
6 is a graph showing the free radical scavenging effect of the morning glory callus extract and the comparison group grape callus extract and rich callus extract according to the present invention.

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

Example 1 Preparation of Morning Glory Callus Extract According to the Present Invention

The morning glory seeds were sterilized by placing them in 70% ethanol for 1 minute and in 0.25% sodium hypochlorite solution for 45 minutes. After sterilization, the seeds were washed 5 times or more with sterile water, seeded in MS (Murashige & Skoog, 1962) medium and germinated in a culture room of 25 ± 2 ° C., 3000 lux, 16 / 8h photoperiod.

Part of the germinated morning glory was 4.4 g / L in MS medium (with vitamins, Duchefa Cat No. M0406 and M0409), 60 g / L sucrose, 3 mg / L NAA (1-naphthylacetic acid) 3 mg / L, pitagel (phytagel) 2 g / L was added to the medium adjusted to pH 5.8 and subcultured at intervals of about 3 weeks at light conditions of 25 ℃ to induce callus (Fig. 1). The induced callus was finely ground on liquid nitrogen and dissolved in water.

Experimental Example 1: Safety Test

Fifty subjects (mean age 35 years, age 20 to 50 years) were subjected to a skin patch test on the upper arm using a fin chamber. However, Psoriasis, Eczema, and other skin lesion holders, or those who are pregnant, nursing or contraceptives, and antihistamines, were excluded from this experiment.

Wipe the upper arm area of the test site with primary distilled water and dry it for 50 subjects, and then, 0.5% by weight, 1% by weight, and 5% by weight of the morning glory callus extract according to Example 1 in purified water (negative control) and purified water. 25 μl each of the composition (positive control) containing 10% by weight of SLS (Sodium Lauryl Sulfate) was added to the pin chamber and fixed on the upper arm. In general, when 10% SLS touches the skin, it swells red and is frequently used as a positive control in skin irritation experiments.

The patch was applied to the skin for 48 hours, and after removing the patch, the test area was marked with a marking pen (marking pen), and after 4 hours, the skin condition was visually read and the results are shown in Table 1 below. Stimulus degree was calculated according to the following equation (1).

[Equation 1]

 sample Number of test subjects  Judgment result  Irritation degree ++ + + - - 10% SLS 50 5 10 25 10 1.20 Morning Glory Callus Extract
0.5 weight% 50 0 3 One 46 0.14 Morning Glory Callus Extract
Contains 1 wt% 50 One 0 2 47 0.1 Morning Glory Callus Extract
5 weight% 50 0 3 2 45 0.16 * Criteria *
-: No erythema or unusual phenomenon
+-: Slightly reddish
+: Significantly redder than the surroundings
++: reddening and swelling worse than the surroundings

As can be seen in Table 1, the morning glory callus extract according to the present invention was shown to have no safety problems. The result of experimenting with purified water itself was shown as 0 stimulus, which is not separately indicated in Table 1 above.

Experimental Example 2: Cytotoxicity Test

In order to examine the cell proliferation and activity of the morning glory callus extract according to the present invention, human keratinocytes HaCaT (Cell Lines Service, Germany) was inoculated in a 24-well culture plate at a concentration of 1 × 10 ⁴ cells / ml. . Medium was used DMEM (Dubelcco's Modified Eagle Medium, BRL, USA) containing 10% fetal bovine serum (FBS). When cultured in DMEM containing 10% FBS for 48 hours and incubated by 25-30% of the surface of the culture vessel, the morning glory callus extract prepared in Example 1 was 0.1 wt%, 0.5 wt%, 1 wt%, 5 wt%. It was incubated for another 24 hours by replacing with FBS-free DMEM containing%. After incubation, 50 μl of MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide, Sigma M5655, USA) solution (2.5 mg / ml) was added and further incubated for 3 hours. The supernatant was then removed. 200 μl of DMSO (Sigma D2650, USA) solution was added to each well, followed by stirring for 20 minutes to form formazan (formazan) crystals. Then, 100 μl of each was taken in a 96-well plate and the absorbance was measured at 570 nm by ELISA. The cell proliferation effect (cytotoxicity) was calculated according to Equation 2 below based on the absorbance intensity of the control group using pure water and expressed as a percentage, and the results are shown in FIG. 2.

[Equation 2]

Cell proliferation effect (%) = (absorbance at extract treatment) / (absorbance of control) × 100

As shown in Figure 2, the morning glory callus extract according to the present invention showed a cell growth of 112% and 108%, respectively, when treated with 5% by weight and 1% by weight compared to the control group without showing cytotoxicity.

Experimental Example 3: iNOS activity inhibition test

To investigate the inhibitory effects of artificially induced inflammation using macrophage raw264.7 cells, 10% FBS-DMEM (Fetal Bovine Serum) -Dulbecco's Modified Eagle Medium, which does not contain iNOS GIBCO) 1 × 10 ⁵ cells were suspended in medium and inoculated in 24 well plates.

After one day, the extract prepared in Example 1 was treated by concentration (0.5% by weight, 1% by weight, 5% by weight) and incubated for 18 hours, and then 1 μg / ml of LPS (Lipopolysaccharide, Sigma) Treated and incubated for 24 hours. Thereafter, the supernatant was collected, 100 µl each was added to a 96 well plate, and the same amount of grease reagent (Griess reagent, Sigma) was added thereto, and gently shaken at room temperature for 10 minutes, and the absorbance was measured at 570 nm. A calibration curve was prepared using sodium nitrite as a standard product, and the amount of nitric oxide produced in the culture solution was determined for each sample. The yield of each sample was obtained by setting the amount of nitrogen oxide produced in the LPS-treated group to 100%, and the results are shown in Table 2 and FIG. 3.

 sample  % Yield  Control (DMEM Medium) 30.5  LPS 100  Contains 0.5% by weight of LPS + morning glory callus extract 56.79  Contains 1% by weight of LPS + Morning Glory Callus Extract 60.47  Contains 5% by weight of LPS + Morning Glory Callus Extract 62.46

As shown in Table 2 and Figure 3, it was found that the morning glory callus extract according to the present invention inhibits the activity of iNOS.

Experimental Example 4: Cytokine expression analysis test

In order to determine the expression rate of cytokine for the morning glory callus extract according to the present invention, fibroblast (Cell Lines Service, Germany), a human fibroblast, was inoculated in a 24-well culture plate at a concentration of 1 × 10 ⁵ cells / ml. When cultured in DMEM (Dubelcco's Modified Eagle Medium, BRL, USA) containing 10% FBS and cultured by 25-30% of the surface of the culture vessel, FBS-free containing 1% by weight of the extract prepared in Example 1 It was further incubated for 48 hours by replacing with DMEM and FBS-free DMEM used as a control. The expression rates of cytokines of VEGF, EGF, FGF, TGF-β1, IL-1α, IL-6, IL-8 and TNF-α were investigated using HUMAN CYTOKINE ASSAY KIT (Millipore). Experimental method was carried out according to the instructions of HUMAN CYTOKINE ASSAY KIT.

As a result, as shown in Figure 5, when treated with morning glory callus extract according to the present invention 1% by weight, the expression rate of the cytokine was significant compared to the control group, EGF, TGF-β1, the expression of other cytokines Was insignificant.

Experimental Example 5: Moisturizing effect test

Moisturizer (Corneometer CM 825, Courage + Khazaka) was used to measure the moisturizing effect of the morning glory callus extract-containing skin external preparation composition according to the present invention in the following manner.

Twenty subjects in their 20s and 30s were washed several times with water in the upper arm 2 × 2 cm ² , 12 sections, and water was removed without irritation. After 5 minutes, 10 μl of purified water and morning glory callus extract prepared according to the method described in Example 1 were applied to each of the two compartments and applied evenly. After 10 minutes, five measurements were taken per compartment using a moisture meter. Six measurements were made at intervals of 5 minutes for one compartment, which was calculated by the following Equation 3, and the data values were expressed as average values for 20 people. The room temperature was 22 ℃ and the relative humidity was 30%.

&Quot; (3) "

Moisture content (%) = Moisture meter after sample application-Moisture meter before sample application

 Hours (minutes) Purified water 0.5 wt%
Morning Glory Callus
extract 1 wt%
Morning Glory Callus
extract 5 wt%
Morning Glory Callus
extract 10 25 29 30 29 15 7 28 26 27 20 3 20 23 24 25 One 17 19 22 30 0 15 16 19 35 0 13 15 16 40 0 13 15 16

As shown in Table 3, compared with the purified water, it was confirmed that the composition containing 5% by weight of the morning glory callus extract according to the present invention has the most excellent moisturizing effect. Therefore, as the content of morning glory callus extract increased, the moisturizing effect was excellent.

Experimental Example 6: skin soothing effect test

In order to examine the skin irritation relief effect (skin soothing effect) when the morning glory callus extract containing composition according to the present invention was applied, skin irritation relaxation test was performed on 20 men and women in their 20s and 40s.

As a stimulant, 5% Lactic acid (L-lactic acid), which is generally used as a positive control in skin sedation experiments, such as SLS, was used for 10 days by adding to the morning glory callus extract-containing composition prepared in Example 1 To compare the mitigating effect on the stimulation caused by lactic acid. Each morning and evening were used, and then questionnaires were marked for irritation and intensity. Test results were evaluated once a day, and divided into subjective and objective stimuli, and the results are shown in Table 4 below.

Test substance Subjective stimulation Objective stimulus Sum 5 wt% morning glory callus extract 7 5 12 1 wt% morning glory callus extract 5 5 10 0.5 wt% morning glory callus extract 6 6 12 5% Lactic Acid 30 19 49

The morning glory callus extract in Table 4 can be seen that the composition containing 0.5% by weight, 1% by weight and 5% by weight is very excellent in alleviating the skin irritation caused by 5% lactic acid.

Comparative Example 1: Preparation of Grape Callus Extract

The leaves of Viitis vinifera were sterilized, cut into 1 cm × 1 cm, and then dentified on MS medium (Sigma) to which plant hormones (IAA, IPA or coconut milk) were added. After about two weeks, when amorphous callus tissues were induced, they were separated separately and placed on fresh solid medium and passaged at intervals of about two weeks. The induced callus was finely ground in liquid nitrogen and dissolved in purified water.

Comparative Example 2: Preparation of Rich Callus Extract

Aseptically treated lobular seeds were seeded in hormone-free MS medium (Duchefa Cat No. M0221) and germinated under conditions of long time (long day) at 25 to 28 ° C for 18 hours. Cut the cotyledon with 3 ~ 5 cm from the germinated seedlings with a sharp knife to cut sucrose into MS medium. Sucrose 40.0 g / L, Hyponex 3.0 g / L, Peptone 4.0 g / L, Banana powder 35.0 It was wounded in MS medium of pH 5.7-5.8 containing g / L, 2.0 g / L char, 5-10.0 g / L agar, 0.1-1 mg / L NAA and 0.01-0.1 mg / L BA. After about 20-25 weeks, callus was induced in the cut cotyledons dentified in the medium. The induced callus was finely ground in liquid nitrogen and dissolved in purified water.

Experimental Example 7: Antioxidant Test Using DPPH

Compound DPPH (1,1-diphenyl-2-picryl hydrazyl) generates free radicals in ethanol, and the amount of free radicals produced by mixing with a morning glory callus extract according to the present invention in a proportion is reduced (free Radical scavenging activity test) to determine whether it has an antioxidant effect. Free radical scavenging activity test is described by Kim et al. J. Pharmacogn., 24 (4), 299-303 (1993)], a stable free radical DPPH (Sigma D9132-1G, USA) reagent was used.

150 μl of a 0.2 mM DPPH solution (ethanol in the case of Blanc) was used to extract the morning glory callus extract prepared in Example 1 and the grape callus extract prepared in Comparative Example 1 as the comparative group and the rich callus extract prepared in Comparative Example 2, respectively. It was prepared to contain at various concentrations, and 150 μl of each of them was added and mixed, and the absorbance was measured at 517 nm after being allowed to stand at room temperature for 30 minutes. The control group was set to the case of using purified water.

After absorbance measurement for the experimental group and the control group, the degree of antioxidant capacity was expressed as a percentage based on the absorbance intensity of the control group using pure water.

The free radical scavenging activity was obtained using Equation 4 below and the results are shown in FIG. 6.

&Quot; (4) "

Free radical scavenging effect (%) = 1- [absorbance of experimental group / absorbance of control group] X 100

As shown in Figure 6, compared to the control using purified water, when containing 5% by weight of morning glory callus extract, 32% and 0.5% by weight of DPPH free radicals of the reaction system when 40%, 1% by weight It was reduced by 21% and showed a significantly superior antioxidant effect compared to grape and rich callus extract.

Claims (11)

An external composition for skin improvement comprising morning glory callus extract as an active ingredient. The composition of claim 1, wherein the morning glory callus extract is contained in an amount of 0.1 to 10% by weight based on the total weight of the composition. The composition of claim 1, wherein the morning glory callus extract is an extract of morning glory callus itself, morning glory callus powder, or morning glory callus itself or morning glory callus powder extracted with water or alcohol. The skin external composition for skin improvement according to claim 3, wherein the alcohol is ethanol. The skin external composition for skin improvement according to claim 3, wherein the extraction temperature is room temperature. The skin external composition for skin improvement according to claim 1, which is a cosmetic composition or a pharmaceutical composition. (a) inducing and incubating callus from the morning glory seeds; And
(B) a method for producing a skin external composition for skin improvement comprising a morning glory callus extract comprising the step of preparing a composition containing the callus. 8. The method of claim 7, wherein in step (a), the callus is cultured in a medium containing a useful substance. The method of claim 7, further comprising introducing a useful substance into the cultured callus. 10. The method according to claim 8 or 9, wherein the useful substance is cinnanamaldehyde, capsaicin, EGCG, vanillin or gallic acid. The method according to claim 7, wherein in step (b), the callus is powdered to be included in the composition, or the callus itself or the callus powder is extracted by hot water extraction or ethanol extraction.

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