KR20160149183A - Composition for improving skin containing callus of Broussonetia plant - Google Patents

Abstract

Disclosed in the present invention is a composition containing one or more selected from the group consisting of crushed callus of a Broussonetia plant, a callus-cultured medium, a callus extract, crushed cells differentiated from the callus, a cultured medium of cells differentiated from the callus, and an extract of cells differentiated from the callus as an active ingredient. The composition promotes skin regeneration, prevents or mitigates skin wrinkles, improves skin whitening, skin moisturization, and skin tone, and prevents or mitigates skin aging.

Description

TECHNICAL FIELD The present invention relates to a skin improving composition containing a callus of a mulberry plant,

The present invention relates to a composition containing callus of a Mammalian plant.

Plants were a means of food resources in the past, but they are now being expanded as a source of a wide range of chemicals, including medicines, flavors, colors, pesticides, and dyes. In particular, most of the plant-derived useful substances have been attracted attention as an ideal resource capable of developing as a novel active substance due to their physiological activities such as antiviral, antibacterial, anticancer, and antioxidant ability. Since they are derived from plants, It also has the advantage of being friendly.

However, there are many practical difficulties in commercially developing new effective substances from plant resources. The reason for this is as follows. Plants are very slow to grow, have very limited efficacy in plants, have only a small amount of efficacy in plants, and have a problem of natural degradation when plants are used as a resource. Finally, the plant-derived efficants have a very complicated chemical structure and require a multistage polymerization process, resulting in an economical problem of high production costs. For this reason, it has been difficult to stably supply plant-derived efficacious substances.

Korean Patent Publication No. 10-2005-0041259 (April 5, 2005) Chinese Patent Laid-Open Publication No. 1122688 (May 22, 1996) Korean Patent Laid-Open Publication No. 10-1992-21129 (Dec. 18, 1992) Japanese Patent Application Laid-Open No. 04-053495 (Feb. 21, 1992)

The present invention relates to a method for promoting skin regeneration, wherein at least one selected from the group consisting of callus lumps of Mammalia plants, callus culture, callus extract, cell lysate differentiated from callus, culture of cells differentiated from callus and cells differentiated from callus, The present invention provides a method for preventing or improving wrinkles of skin, skin whitening, skin moisturizing, skin tone improvement, anti-aging or improving effect, A culture of cells differentiated from callus, and an extract of cells differentiated from callus, as an active ingredient.

The present invention relates to a method for the production of callus, which comprises using as an active ingredient at least one selected from the group consisting of callus lumps of Mulberry plants, callus cultures, callus extracts, cell lysates differentiated from callus, culture of cells differentiated from callus and cells differentiated from callus ≪ / RTI >

In one embodiment of the present invention, the composition is characterized by having a skin regeneration promoting purpose.

In one embodiment of the present invention, the composition has a use for preventing or improving skin wrinkles.

In one embodiment of the present invention, the composition is characterized by having a skin whitening application.

In one embodiment of the present invention, the composition is characterized by having a skin moisturizing use.

In one embodiment of the present invention, the composition is characterized by having a skin tone improving application.

In one embodiment of the present invention, the composition is characterized by having anti-aging or improving applications.

The present invention relates to a method for the production of callus, which comprises using as an active ingredient at least one selected from the group consisting of callus lumps of Mulberry plants, callus cultures, callus extracts, cell lysates differentiated from callus, culture of cells differentiated from callus and cells differentiated from callus Thereby promoting skin regeneration, preventing or improving wrinkles of the skin, whitening the skin, moisturizing the skin, improving the skin tone, preventing aging, or improving the effect.

1 is a graph showing the results of Experimental Example 5 for the inhibition rate (%) of melanin production of the composition according to the present invention.

As used herein, the term "skin" refers to a tissue covering the body surface of an animal, and is a concept of the broadest notion including a scalp and hair as well as a tissue covering a body surface such as a face or a body.

Hereinafter, the present invention will be described in detail.

As biotechnology techniques have been developed, the field of plant cells that have been induced and differentiated from undifferentiated cells is newly emerging, and many experiments related to the induction differentiation of undifferentiated cells such as the study of signals involved in the development are proceeding. The plant cell culturing method using the plant has become an ideal technology to stably supply plant-derived useful materials without causing environmental problems. The production of useful materials by plant cell cultures has many advantages over direct extraction from plants. In particular, unlike the existing extraction method, it is possible to produce continuously without being influenced by the external environment, and it seems to be the optimum method to solve the pending problems such as the destruction of the ecosystem.

When leaves or stems are harvested to obtain an efficacy substance from a plant, the harvesting can be continued without damaging the plant, but if the root is to be harvested, the whole plant is damaged and the plant can no longer survive . However, when differentiated cells are used, it is possible to obtain a large amount of cells differentiated into root tissue easily with only a very small amount of plant cells, so that the raw material of the effective substance can be obtained without damaging the plant, Can be established as a raw material.

Mulberry tree is a plant of mulberry and Broussonetia kaempferi , Broussonetia kazinoki (mulberry), Broussonetia kurzii , Broussonetia Includes papyrifera . In the present invention, the Mammalia plants include the above four species, more specifically, Mushroom ( Broussonetia kazinoki ) and Cucumber ( Broussonetia papyrifera ). These are deciduous broad-leaved shrubs in the mulberry tree, distributed in most parts of Korea (mainly distributed in the southern part), China, Taiwan and Japan, and are geographically distributed on the mountain side, Mackerel has a long history of using the bast fiber as a raw material for paper, and it has been known to have various medicinal effects from the past. It has been reported that it has effects on tonic, nominal action, darkness, species, waxing, diuretic, etc., eliminating wind, clearing blood and reducing visual acuity.

The present invention relates to a method for the production of callus, which comprises using as an active ingredient at least one selected from the group consisting of callus lumps of Mulberry plants, callus cultures, callus extracts, cell lysates differentiated from callus, culture of cells differentiated from callus and cells differentiated from callus ≪ / RTI > The composition has the effect of promoting skin regeneration, preventing or improving skin wrinkles, skin whitening, skin moisturizing, skin tone improvement, anti-aging or improving effect. There are a lot of kazinol in the cells differentiated from the callus or callus of the Mammalia plant. It has the advantage of obtaining the raw material of the effector without damaging the plant by using the cell differentiated from the callus or callus of the plant of the mulberry tree, not the mulberry plant itself. It can also have better efficacy than the mulberry plant itself.

As used herein, the term " callus " refers to an initial stage cell mass in which a callus is induced and cultured in a solid medium. The term " cells differentiated from callus " refers to the stage from callus-induced and cultured in solid medium to the step of treating plant hormones and differentiating them into specific organs. In one embodiment of the present invention, the specific organs may be leaves, stems or roots. In one embodiment of the present invention, the cells differentiated from callus include cells differentiated from callus to leaf, cells differentiated from callus stem, cells differentiated from callus root. Typically, the time required for differentiation into specific organs of woody plants is about 1 to 60 days, and may have a differentiation period of between 1 and 45 days.

The term " disruption " as used herein means a cell lysate obtained by breaking a cell by a chemical method using a general detergent or the like or a physical method using a French Press or the like. Can be produced according to a known ordinary method, and the method is not particularly limited.

The term "culture medium" used herein means a culture medium obtained by culturing cells for 6 hours to 72 hours by a general method in the art, and then removing the pellet using a physical method such as ultracentrifugation , And the method can be produced according to a conventional method known in the art, and the method is not particularly limited.

As used herein, the term " extract " means a substance which is isolated by dissolving cells in a solvent and is concentrated using distillation or evaporation, and the method can be produced according to a conventional method known in the art, And is not particularly limited. Generally, after drying and pulverizing, an extraction solvent is added and the mixture is heated to extract an active ingredient and concentrated under reduced pressure to obtain an extract. As the extraction solvent, a solvent which can be generally used can be used, and it includes water, an anhydrous or a lower alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, butyl acetate and 1,3-butylene glycol.

The cell culture method used in the present invention is a known solid culture method. Solid culture is a culture of callus cells on a solid medium. The callus can be derived as follows: Sterilized leaves and stems of sodium hypochlorite and ethyl alcohol are cut to an appropriate size and cultured aseptically on a solid culture medium do. When the callus cells start to grow on the cut surface of roots and leaves after the completion of the culturing, the callus is subcultured continuously to establish a stable cell line.

The medium used for callus cell induction may be MS (Murashige & Skoogs) medium. The agar medium is added to the MS medium at 0.5-1.0% and used as a solid medium. Plant hormone auxin is added as a growth regulator. As auxin, 1-naphthaleneacetic acid (NAA) can be used instead of 2,4-D, which has serious human health hazards. Also, when the concentration of sucrose in the medium is maintained at 30 g / l or less, the growth of the cells is good.

The culture temperature is suitably 20-25 ° C, and the growth of cells is excellent under light conditions, under dark conditions. Under these conditions, callus cells growing in solid medium are subcultured to new medium every 4 to 5 weeks, and the amount of cells increases by about 2 to 3 times during this period.

Cells differentiated from the callus of the present invention can be prepared by a conventional method known in the art from a callus to stem, stem or leaf differentiated cells, wherein the medium is supplemented with auxin, gibberellic acid, Etc. may be added at different concentrations to suit the organ to be differentiated at a concentration of between 0.1 and 3 mg / L.

The skin is largely divided into three layers of epidermis, dermis and subcutaneous adipose tissue in order from the outside. It protects the internal organs of the body from changes in temperature and humidity, ultraviolet rays, pollutants, It plays an important role in maintaining the homeostasis of the host. However, ultraviolet rays, excessive physical and chemical stimuli from the outside, stress and nutritional deficiency deteriorate the normal functions of the skin and promote skin aging phenomenon such as loss of elasticity, keratinization and wrinkle formation. In particular, if the keratinization process in which the keratinocytes are successively made in sequence and the outermost keratinocytes from the outermost layer are occasionally deviated, the epidermis keeps a constant thickness. If the keratinization process is not smooth, abnormal keratinocytes are formed, The skin becomes rough and dull, fine wrinkles are formed, and the skin ages in the end. Therefore, when the growth of keratinocytes is promoted, not only skin regeneration can be promoted, but also wrinkles can be prevented and improved, skin whitening effect can be obtained, skin tone can be improved, and aging can be prevented or improved.

Collagen is an important fibrous protein of animal connective tissue, which has the function of preventing or improving skin wrinkles, regenerating skin, preventing or improving scarring, strengthening skin elasticity, and preventing skin aging. Procollagen is a precursor of collagen found in the skin and other organs of many vertebrates, and consequently has the same function as collagen. Thus, increased levels of collagen and procollagen can prevent or improve skin wrinkles, regenerate skin, prevent or improve scarring, enhance skin elasticity, and prevent or improve skin aging. On the other hand, "collagenase" is an enzyme that decomposes collagen and is known as one of the causes of wrinkles of the skin. Matrix metalloproteinase-1 (MMP-1) can be biosynthesized by ultraviolet light associated with degradation of the skin matrix, which results in a decrease in the biosynthesis of type 1 procollagen. Therefore, inhibition of MMP-1 expression increases the collagen level of the skin, thereby preventing or improving the wrinkles of the skin, regenerating the skin, strengthening the skin elasticity, and preventing or improving the aging of the skin. In addition, the degradation ability of fibroblasts present in the dermal layer of the skin is also related to skin aging.

Melanin is a pigment that determines the color of skin and hair. Melanin from melanocytes migrates to the outside of the skin and is distributed in the stratum corneum. When exposed to ultraviolet light, the number of melanocytes increases and melanin production also increases, causing pigmentation, staining, and freckles. Becker's nevus, lentigines, and Nevus spilus are also caused by excessive deposition of epidermal melanin.

Melanin is a complex in which tyrosine is produced by complex enzymatic and nonenzymatic oxidation, and tyrosinase is known to play a pivotal role in this process. Tyrosinase is a metalloprotein enzyme bound to copper and widely distributed in animals, plants, microorganisms and humans, tyrosine is converted into dihydroxyl-L-phenylalanine (L-DOPA), dihydroxyphenylalanine To dopaquinone (Dopaquinone). Dopaquinone is converted into dopachrome, Dopachrome is converted into dihydroxyindole, and finally melanin is synthesized through oxidative polymerization of dihydroxyindole. Thus, suppressing tyrosinase activity or inhibiting oxidation of L-DOPA may inhibit melanin synthesis. Suppression of melanin production can have a skin whitening effect, improve skin tone, and prevent or prevent aging.

Currently most commonly used whitening agents are those containing a tyrosinase inhibitor or a tyrosinase inhibitor, such as arbutin, hydroquinone, hydroxyanisole, ascorbic acid derivatives, kojic acid ), Corticosteroids, and retinoids. However, some studies have reported that some inhibitors cause mutagenesis, leading to the possibility of carcinogenesis. In addition, there are problems such as safety and economical efficiency, There is a difficulty. Particularly, hydroxyanisole and hydroquinone have strong melanin formation inhibitory activity, but exhibit adverse effects such as denaturation of pigment cells or death of the pigment cells and impairment of original function of the cells. In addition, kojic acid has problems such as low inhibitory activity, discoloration during use, and instability of the substance itself.

The present invention relates to a method for the production of callus, which comprises using as an active ingredient at least one selected from the group consisting of callus lumps of Mulberry plants, callus cultures, callus extracts, cell lysates differentiated from callus, culture of cells differentiated from callus and cells differentiated from callus Promoting keratinocyte growth, inhibiting MMP-1, increasing procollagen, promoting tyrosinase activity, inhibiting oxidation of L-DOPA, inhibiting melanin production, promoting skin regeneration, preventing skin wrinkles Skin whitening, skin moisturization, skin tone improvement, anti-aging or improving effect.

At least one selected from the group consisting of callus lumps of Mammalia plants of the present invention, callus cultures, callus extracts, cell lysates differentiated from callus, cultures of callus-differentiated cells, and callus-differentiated cells, By weight based on the total weight of the composition. If the content is less than 0.0001% by weight, the effect is insignificant. If the content is more than 25.0% by weight, there is a problem in feeling of use of the product and stability of the formulation.

The present invention relates to a method for the production of callus, which comprises using as an active ingredient at least one selected from the group consisting of callus lumps of Mulberry plants, callus cultures, callus extracts, cell lysates differentiated from callus, culture of cells differentiated from callus and cells differentiated from callus A cosmetic composition containing the composition of the present invention can be provided. The cosmetic composition contains a cosmetically or dermatologically acceptable medium or base. It may be any formulation suitable for topical application, for example, a solution, a gel, a solid, an anhydrous product of a paste, an emulsion obtained by dispersing an oil phase in an aqueous phase, an emulsion obtained by dispersing a water phase in an oil phase, a multi-emulsion, Capsules, microgranules or in the form of ionic (liposome) and non-ionic follicular dispersions, or in the form of creams, skins, lotions, powders, ointments, sprays, cleansers or conceal sticks. These compositions may be prepared according to conventional methods in the art. The composition according to the invention may also be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

In addition to the above-mentioned materials, the above-mentioned cosmetic composition may contain other ingredients which can give a synergistic effect to the main effect, within the range not impairing the intended main effect of the present invention. The other ingredients can be mixed and selected without difficulty by those skilled in the art depending on the formulation or purpose of the other cosmetic composition.

For example, the cosmetic composition may contain a skin absorption promoting substance to increase its effect. In addition, the cosmetic composition of the present invention may comprise a material selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymeric polysaccharides, sphingolipids and seaweed extracts. In addition, the cosmetic composition of the present invention may contain other essential ingredients, if necessary, in combination with the cosmetic composition. Examples thereof include a moisturizing agent, an emollient, a surfactant, an organic and inorganic A pigment, an organic powder, an ultraviolet absorber, an antiseptic, a bactericide, an antioxidant, a plant extract, a pH adjuster, an alcohol, a pigment, a flavor, a blood circulation accelerator, a cold agent, an antiperspirant agent and purified water. The other ingredients to be contained in the cosmetic composition of the present invention are not limited to these, and the amount of the above ingredients may be adjusted within a range that does not impair the objects and effects of the present invention.

The formulation of the cosmetic composition is not particularly limited, and the formulation can be appropriately selected according to the purpose. For example, it is possible to use a lotion such as a soft lotion (skin lotion and milk lotion), a nutrition lotion, an essence, a nutritional cream, a massage cream, a powder, a lipstick, a makeup base, a foundation, a patch, But is not limited to, one or more formulations selected from the group consisting of cleansing cream, cleansing foam, cleansing water, cleanser, body lotion, body cream, body oil and body essence.

The present invention relates to a method for the production of callus, which comprises using as an active ingredient at least one selected from the group consisting of callus lumps of Mulberry plants, callus cultures, callus extracts, cell lysates differentiated from callus, culture of cells differentiated from callus and cells differentiated from callus Or a pharmaceutically acceptable salt thereof.

When the composition according to the present invention is applied to medicines, it may be formulated into an oral or parenteral dosage form in the form of solid, semi-solid or liquid by adding an inorganic or organic carrier to the composition as an active ingredient. The active ingredient of the present invention can be easily formulated according to the conventional method, and can be easily formulated by adding a surfactant, an excipient, a colorant, a spice, a stabilizer, an antiseptic, a preservative, a wetting agent, an emulsification accelerator, Buffers, and other adjuvants that are commonly used.

Examples of the agent for oral administration include tablets, pills, granules, soft capsules, powders, granules, powders, liquids, emulsions, syrups, pellets, etc. . These formulations may contain diluents (such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and glycine), lubricants (such as silica, talc, stearic acid and magnesium or calcium salts thereof and polyethylene glycols) ≪ / RTI > The tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine, optionally mixed with starch, agar, alginic acid or its sodium salt Such as disintegrants, absorbents, coloring agents, flavoring agents, and sweetening agents. The tablets may be prepared by conventional mixing, granulating or coating methods.

The parenteral administration may be, for example, an external preparation for skin, and may be an injection, a drip, a lotion, an ointment, a gel, a cream, a suspending agent, an emulsion, a suppository, a patch or a spray formulation, But is not limited thereto.

The pharmaceutical composition according to the present invention may be administered orally, parenterally, rectally, topically, transdermally, intravenously, intramuscularly, intraperitoneally, subcutaneously and the like.

In addition, the dosage of the active ingredient will vary depending on the age, sex, and weight of the subject to be treated, the specific disease or condition to be treated, the severity of the disease or condition, the route of administration, and the judgment of the prescriber. Dosage determinations based on these factors are within the level of those skilled in the art. Typical dosages are from 0.001 mg / kg / day to 2000 mg / kg / day, more specifically from 0.5 mg / kg / day to 2.5 mg / kg / day.

Hereinafter, the constitution and effects of the present invention will be described in more detail with reference to Production Examples, Experimental Examples, Examples and Comparative Examples. However, these examples, experimental examples, examples and comparative examples are provided only for illustrative purposes in order to facilitate understanding of the present invention, and the scope and scope of the present invention are not limited to the following production examples, experimental examples, But is not limited to.

[Production Example] Production of callus culture and callus lysate, culture solution and extract

The collected roots and leaves were used for the callus induction. Biological samples were cut to a length of about 10 cm, washed, and then placed in 0.5% sodium hypochlorite solution containing 0.1% Tween-20 and sterilized for 20 minutes with stirring. During this period, the microbubbles on the surface of the biological sample were removed by sonication for 3 seconds. After washing once with sterilized water, it was sterilized again in 70% ethanol solution for 1 minute, and the sterilized and washed biological sample was cut to a size of about 0.5 cm, and 0.5-1.0% of agar was added to the MS medium The culture was started by transferring onto a mixed solid culture medium. 1-naphthaleneacetic acid (NAA), a plant hormone auxin, was used as a growth regulator. The concentration of sucrose in the culture medium was maintained at 30 g / l or less.

All manipulations were performed aseptically in aseptic controls, and callus was induced after 2-3 weeks of incubation at 25 ℃. Callus was well induced from various biological samples, and the best one was transferred to new medium every month and subcultured to establish a cell line.

The callus extract was prepared by centrifuging the cell line, washing it with sterile purified water, aseptically drying the cells (weight 100 g) obtained by pulverizing the cells, adding 0.5 L of 95% ethanol to the pulverized product, By heating at 45 ° C for 3 days. The extract was filtered with a filter cloth of 350 mesh, and then filtered with filter paper of No. 2 filter paper.

On the basis of the established cell line, callus pulp and callus cultures of mulberry and cucumber trees were prepared by a general method.

 [Experimental Example 1] Evaluation of accelerated keratinocyte growth (MTT assay)

Effects of callus pulp, callus extract, callus extract, callus extract, callus extract, callus extract, callus extract and culture of stem differentiated cells from callus on the growth of keratinocytes Were measured by MTT method.

First, keratinocytes were cultured in a 96-well plate at a concentration of 5 × 10 ³ per 200 μl per well, and cultured for 24 hours. Then, callus pulp, callus culture, callus extract and callus of mulberry and cucumber Cells differentiated from roots were treated with 0.001, 0.01 and 0.1% of cell lysate, culture medium, extract, and cell lysate, culture and extract of callus differentiated stem cells for 48 hours, After washing once with PBS, 100 쨉 l of a solution of MTT (Methylthiazolyldiphenyl-tetrazolium bromide, Sigma, USA) (0.5 ㎎ / ml) was added to the cells and cultured at 37 ° C and 5% CO ₂ for 4 hours. Thereafter, the culture solution was inhaled and removed, and then 200 μl of a dimethyl sulfoxide (DMSO) solution was added. After shaking for 10 minutes in a shaker, the absorbance at 540 nm was read in an ELISA reader (DIbiotech, Korea) . At this time, as a control group, a non-treated group in which nothing was treated was used, and the results are shown in Table 1 below. The higher the absorbance, the more effective it is to promote the growth of keratinocytes.

density(%) Absorbance Mulberry Cedar wood Callus scrap 0.001% 135 138 0.01% 136 141 0.1% 143 154 Callus culture 0.001% 148 135 0.01% 152 137 0.1% 162 140 Callus extract 0.001% 113 115 0.01% 115 119 0.1% 118 124 Cells of callus differentiated into roots 0.001% 125 128 0.01% 126 131 0.1% 138 137 Cultures of cells differentiated from roots in callus 0.001% 122 121 0.01% 125 125 0.1% 129 126 Extract of root differentiated cells from callus 0.001% 112 114 0.01% 115 118 0.1% 121 123 Disruption of stem-differentiated cells in callus 0.001% 115 121 0.01% 121 127 0.1% 127 129 Cultures of stem-differentiated cells from callus 0.001% 128 128 0.01% 130 132 0.1% 138 139 Extract of stem-differentiated cells from callus 0.001% 103 106 0.01% 103 108 0.1% 106 110 Untreated control group 0% 100 101

As shown in Table 1, in the present invention, callus pulp, callus pulp, callus extract, callus pulp differentiated from callus, culture broth, extract, The cell lysates, culture medium and extracts were found to be excellent in promoting keratinocyte cell growth. In addition, as the concentration of each component increased, the effect of promoting cleavage of keratinocytes also increased.

As described above, callus pulp, callus extract, callus extract, cellulase differentiated from callus, culture solution, extract, callus of cell differentiated from callus in the callus of mulberry and cucumber which have excellent promoting effect of keratinocyte growth, , The culture solution and the extract promote the regeneration of the skin, prevent and improve the wrinkles of the skin, improve the skin tone, and further prevent and improve the aging. These can be used in combination of two or more.

 [Experimental Example 2] MMP-1 inhibition and procollagen production promotion assay (procollagen assay)

In order to investigate the effect of MMP-1 biosynthesis and promoting biosynthesis of type 1 procollagen in the callus of crushed mulberry and cedar trees, the callus culture, the differentiation of root-differentiated cells into callus and the differentiation of root-differentiated cells into callus, MMP-1 and procollagen assay were performed as follows.

After removing epidermis and dermis by collagenase, the epidermis and dermis were placed in 0.25% trypsin solution and incubated at 37 ° C in a 5% CO ₂ incubator for 10 Respectively. Then vortex was performed to release keratinocytes and fibroblasts, respectively. Cells were collected and washed, and then keratinocyte growth medium (Clonetics, USA) was cultured at a concentration of 1 × 10 ⁴ cells / cm ^2. Fibroblasts were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) And cultured in DMEM medium. When 70-80% of the cells were grown, the cells were subcultured at a ratio of 1: 3, and the cells were subcultured 3-4 times.

For the measurement of the amount of MMP-1, fibroblasts were cultured in a 48-well plate at a concentration of 90% or more and starvated for one day. Then, the plate was washed twice with PBS, and then 100 쨉 l of PBS was added. The plate was opened and irradiated with ultraviolet A (Dermite cube 401 equipped with UVA filters, UVAtec, USA) at 15 J / cm ² . Immediately after the irradiation, the cells were washed once with PBS. The cells were washed with 0.001%, 0.01%, 0.1% of callus of mulberry and cedar trees, callus culture broth, disruption of differentiated cells in callus, Fetal bovine serum was inoculated into DMEM containing the same and incubated in a 5% CO ₂ incubator at 37 ° C for 48 hours. The amount of released MMP-1 in the medium was measured using an MMP-1 human ELISA system system) (RPN2610, Amersham Pharmacia Biotech, UK) and corrected for the total protein content of fibroblasts. At this time, as a control group, no treatment group was used, and the results are shown in Table 2.

On the other hand, in order to measure the amount of procollagen, 0.001%, 0.01%, and 0.1% of callus lumps of mulberry and cucumber trees, respectively, were irradiated under starvation conditions under the same conditions as those for measuring the amount of MMP- , A callus culture, a cell lysate of root differentiated cells in callus, and a culture of cells differentiated from callus in roots were added. After 24 hours, the amount of procollagen liberated in the medium was measured with a procollagen type-1 C-peptide EIA kit type-1 C-peptide EIA kit (MK101, Takara, Japan). At this time, as the control group, a non-treated group in which nothing was treated was used, and the results are shown in Table 3.

density(%) Amount of MMP-1 Mulberry Cedar wood Callus scrap 0.001% 70 75 0.01% 66 68 0.1% 62 60 Callus culture 0.001% 72 75 0.01% 71 71 0.1% 66 64 Cells of callus differentiated into roots 0.001% 65 74 0.01% 61 61 0.1% 54 53 Cultures of cells differentiated from roots in callus 0.001% 67 70 0.01% 63 64 0.1% 61 60 Untreated control group 0% 100 100

density(%) Amount of procollagen Mulberry Cedar wood Callus scrap 0.001% 117 115 0.01% 125 125 0.1% 129 134 Callus culture 0.001% 107 117 0.01% 115 124 0.1% 127 129 Cells of callus differentiated into roots 0.001% 126 127 0.01% 130 132 0.1% 137 137 Cultures of cells differentiated from roots in callus 0.001% 125 123 0.01% 129 130 0.1% 135 136 Untreated control group 0% 100 100

As shown in the results of Table 2, the callus pulp of callus and cedar wood used as an active ingredient in the present invention, the callus culture solution, the cell lysate differentiated into callus from callus, MMP-1. ≪ / RTI > Also, as the concentration of each component increased, the amount of MMP-1 decreased proportionally.

As can be seen from the results in Table 3, the callus pulp of callus and cedar wood used as an active ingredient in the present invention, the callus culture solution, the cell lysate of callus differentiated into roots, and the culture solution of callus- It was found that the biosynthesis of type 1 procollagen was increased. Also, the amount of procollagen produced increased proportionally as the concentration of each component increased.

As described above, the callus pulp, callus culture solution, crushed pulp differentiated from callus, and cell differentiated into callus root from mulberry and cucumber trees, which have excellent MMP-1 inhibitory effect and promoting procolagen biosynthesis, To prevent or improve wrinkles, and further to prevent or improve aging. These can be used in combination of two or more.

[Experimental Example 3] Evaluation of inhibitory activity of tyrosinase activity

The enzyme tyrosinase was extracted from Mushroom and was obtained from Sigma (Sigma). First, trypsin is dissolved in distilled water to make 0.3 mg / ml solution. 1.0 ml of the solution is put into a test tube, and 1.0 ml of potassium phosphate buffer solution (0.1 mol concentration, pH 6.8) and 0.7 ml of distilled water are added Respectively. 0.2 ml of Callus pulp, culture, extract, extracts of mulberry and cedarwood, cell lysate of callus differentiated from callus of mulberry and cucumber, lysate of callus differentiated from callus of callus, Each of the cell lysate, mulberry extract, and cedar wood extract was added to the reaction solution and reacted for 10 minutes at 37 ° C in a thermostat. In the control group, only 0.2 ml of the solvent was added instead of the test substance. To the reaction solution was added 0.1 ml of 2500 Unit / ml of tyrosinase solution, and the mixture was reacted in a 37 ° C thermostat for 10 minutes. The reaction tube was quenched by placing it in ice water, and the absorbance at 475 nm was measured with a spectrophotometer. Arbutin and ascorbic acid, which are conventional whitening agents, were used in the positive control group.

The tyrosinase inhibitory effect of each test substance was evaluated by evaluating the inhibition rate of tyrosinase by the following Equation 1, and the results are shown in Table 4.

[Equation 1]

Tyrosinase inhibition rate (%) = 100 - {(reaction absorbance of each test substance / reaction absorbance of control group) x 100}

Treatment concentration (%) Inhibition rate of tyrosinase (%) Mulberry callus 0.001% 23.1 0.01% 29.0 0.1% 34.6 Calligraphy calligraphy 0.001% 23.2 0.01% 27.4 0.1% 36.1 Mulberry callus culture 0.001% 12.8 0.01% 16.4 0.1% 21.7 Cedarwood callus culture 0.001% 14.3 0.01% 17.9 0.1% 21.1 Mulberry callus extract 0.001% 12.1 0.01% 14.7 0.1% 19.2 Cedarwood callus extract 0.001% 14.2 0.01% 16.3 0.1% 17.8 Disruption of cell differentiated into roots in callus of mulberry tree 0.001% 75.3 0.01% 88.7 0.1% 96.3 Cultures of cells differentiated from roots in callus of mulberry 0.001% 15.6 0.01% 19.2 0.1% 31.9 Extract of root-differentiated cells from callus of mulberry tree 0.001% 14.2 0.01% 21.2 0.1% 26.9 Disruption of cell differentiated into roots in calli of cedar wood 0.001% 95.2 0.01% 96.3 0.1% 99.1 Cells of leaf-differentiated cells in callus of callus 0.001% 4.1 0.01% 8.7 0.1% 10.0 Stem cell differentiation in calli of cedar wood 0.001% 3.2 0.01% 6.5 0.1% 23.5 Mulberry extract 0.01% 33.1 Cedarwood extract 0.01% 40.4 Ascorbic acid 0.01% 39.2 Arbutin 0.01% 50.4

As can be seen from the above Table 4, callus pulp, culture broth, extract, mulberry leaf and cedar wood pulp, differentiated into callus, culture broth, extract, Of the callus of callus, and of the cell differentiated from the callus of calli of the cedar tree. Among them, the effect of inhibiting the tyrosinase activity of the cell lysate differentiated from the callus of the mulberry tree and the cell differentiated from the root differentiated from the callus of the mulberry and cucumber trees rather than the lysate of the stem differentiated from the callus of the cucumber tree was superior They showed a remarkable inhibition of tyrosinase activity compared to the extracts of mulberry and cucumber trees. They also had a tyrosinase inhibiting effect superior to that of ascorbic acid and arbutin at the same concentration. The activity of tyrosinase was also inhibited as the amount of active ingredient increased.

As described above, the cell differentiated into callus of callus of mulberry and cucumber trees having the effect of inhibiting tyrosinase activity, the culture solution and extract of cell differentiated into callus of calli of callus, Have the skin whitening effect, improve the skin tone, and have the effect of preventing or improving skin aging. These can be used in combination of two or more.

[Experimental Example 4] Evaluation of oxidation inhibiting ability of L-DOPA

The effect of inhibiting the activity of lysate, mulberry extract, and coconut tree extract of root-differentiated cells from callus of mulberry and cucumber trees against L-DOPA (el-dopa) oxidation of tyrosinase, which catalyzes the rate-determining step of melanin synthesis. Respectively.

Mushroom extracts from the callus of mulberry and cedar trees were extracted with DMSO and then diluted with 0.1 M sodium phosphate buffer solution (pH 6.5) to give final concentrations of 16.67, 33.34, 66.67, 166.67, 333.33, and 666.67 占 퐂 / ml, respectively. 50 μl of 0.1 M phosphate buffer solution (pH 7.0), 50 μl of the prepared solution and 50 μl of a solution of mushroom tyrosinase (SIGMA) (1500 to 2,000) U / ml were sequentially added to the test tube, and L-DOPA , 4-dihydroxyphenylalanine) was added, and the mixture was reacted at 37 ° C for 15 minutes. After completion of the experiment, the absorbance was measured at 475 nm using a spectrophotometer. The inhibition rate (%) of L-DOPA oxidation activity was calculated by the following formula (2), and the results are shown in Table 5.

&Quot; (2) "

L-DOPA oxidation activity inhibition rate (%) = 100 - {(reaction absorbance of each test substance / reaction absorbance of control group) 100}

Treatment concentration (%) L-DOPA inhibitory activity (%) Disruption of cells differentiated into roots in Mulberry callus 0.001% 77.1 0.01% 80.4 0.1% 90.9 Disruption of cell differentiated into root in calli 0.001% 87.8 0.01% 93.3 0.1% 97.9 Mulberry root extract 0.01% 63.7 Root Extract 0.01% 75.8 Ascorbic acid 0.01% 51.1 Arbutin 0.01% 89.1

As can be seen from Table 5, it was recognized that the cell lysate differentiated into root from callus of mulberry and cedar wood used as an active ingredient in the present invention had excellent L-DOPA oxidation inhibitory effect. They had an L-DOPA oxidation inhibitory effect superior to the root extract of mulberry and cedarwood. In particular, the oxidation of L-DOPA was inhibited by increasing the amount of cell differentiated into callus from callus of mulberry and cedar trees. The addition of 0.1% inhibited the oxidation of el-dopa by almost 100%. The L-DOPA oxidation inhibitory effect of the cell lysate differentiated from callus of mulberry and cucumber trees was superior to that of albutin, which is widely used as a conventional whitening agent.

As described above, disruption of cells differentiated into roots in callus of mulberry and cucumber trees having an el-dopa oxidation inhibitory effect has a skin whitening effect, has an effect of improving skin tone and preventing or improving aging. These can be used in combination of two or more.

[Experimental Example 5] Melanin production inhibitory activity

Melanin production inhibitory effects of cell differentiation, root extract, mulberry extract and cucumber seed extract from callus of mulberry and cucumber trees were evaluated. A control group treated with only the untreated control group and the solvent DMSO was also tested.

After adding 5-6 ml of DMEM medium to a 25 cm 2 T-flask, the mouse-derived B-16 melanoma (KCLB 80008) cell line in freeze storage was inoculated and cultured at 37 ° C under 5% CO ₂ for 24 hours. The cells were then treated with 0.05% trypsin containing 0.02% EDTA, and the cells were inoculated at a rate of 1 × 10 ⁵ per well on a 6-well plate and cultured until the cells were attached to the bottom of the wells at about 80% or more. After the culture, the medium was removed, and the cell lysate, mulberry extract, and cedar wood extract differentiated from roots in callus of mulberry and cucumber melted in DMSO were replaced with media diluted to 0.5, 5 and 50 μg / 3 days were cultured in the% CO _2, 37 ℃. After 3 days, the cells with the medium removed were washed with PBS (phosphatized buffer saline), treated with trypsin to recover the cells, centrifuged at 5,000 rpm for 10 minutes, and the supernatant was removed to obtain a pellet. The cell pellet was dried at 60 ° C for 2 hours and then dissolved in 100 μl of 1 M sodium hydroxide solution containing 10% DMSO. The absorbance at 490 nm was measured with a spectrophotometer to determine the amount of melanin per cell protein. Synthetic melanin (Sigma, USA) was dissolved in 100 μl of 1 M sodium hydroxide solution containing 10% DMSO at various concentrations, from which standard concentration curves were obtained to determine the concentration of melanin. Based on this value, the inhibition rate (%) of melanin formation in the cell differentiated from the callus of mulberry and cucumber trees, mulberry extract, and cucumber extract was determined based on the untreated group. Fig. 1 shows the results obtained by adding a crushed product of callus of mulberry and cedar trees to the mouse-derived B-16 melanoma culture to a final concentration of 50 mu g / ml of a cell lysate, a mulberry extract, .

As can be seen from Fig. 1, the amount of melanin produced when mouse broth B-16 melanoma cultures were added to a final concentration of 50 mu g / ml of cell lysate from callus of mulberry and cedar trees was added Which was significantly lower than the no-added control group. It was confirmed that melanin inhibition rate of melanin formation was superior to that of arbutin, mulberry tree extract and cucumber tree extract which are commonly used as whitening agents because of low melanin content of cell lysates differentiated into roots in callus of mulberry and cucumber trees. Therefore, it was found that the cell lysate of callus of mulberry and cucumber trees differentiated into roots behaves as an excellent whitening agent.

As described above, the cells of the callus of mulberry and cucumber having different melanin production inhibitory effects and differentiated into roots have a skin whitening effect, and have an effect of improving skin tone and preventing or improving aging. These can be used in combination of two or more.

 [Examples 1 to 8 and Comparative Example 1]

The cosmetic compositions of Examples 1 to 8 and Comparative Example 1 were prepared with the compositions shown in Table 6 below.

Component (content:% by weight) Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Example 8 Comparative Example 1 1. Beads wax 2 2 2 2 2 2 2 2 2 2. Stearyl alcohol 3 3 3 3 3 3 3 3 3 3. Stearic acid 8 8 8 8 8 8 8 8 8 4. Squalene 10 10 10 10 10 10 10 10 10 5. Propylene glycol monostearate 3 3 3 3 3 3 3 3 3 6. Polyoxyethylene ether One One One One One One One One One 7. Preservatives Suitable amount Suitable amount Suitable amount Suitable amount Suitable amount Suitable amount Suitable amount Suitable amount Suitable amount 8. Glycerine 5 5 5 5 5 5 5 5 5 9. Propylene glycol 4 4 4 4 4 4 4 4 4 10. Triethylamine One One One One One One One One One 11. Small Dip Polyacrylate 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 12. Purified water To 100 To 100 To 100 To 100 To 100 To 100 To 100 To 100 To 100 13. Mulberry callus lump (Example 1) 5 - - - - - - - - 14. Cedarwood callus pulp (Example 2) - 5 - - - - - - - 15. Mulberry callus culture (Example 3) - - 5 - - - - - - 16. Cedarwood callus culture (Example 4) - - - 5 - - - - - 17. Loss of cell differentiated into roots in callus of mulberry (Example 5) - - - - 5 - - - - 18. Loss of cell differentiated into roots in calli of cedar wood (Example 6) - - - - - 5 - - - 19. Culture medium of cells differentiated from roots in callus of mulberry (Example 7) - - - - - - 5 - - 20. Culture medium of cells differentiated from roots in callus of a cedar tree (Example 8) 5 - 21. Carbomer Suitable amount Suitable amount Suitable amount Suitable amount Suitable amount Suitable amount Suitable amount Suitable amount Suitable amount

The components 1 to 7 were heated and dissolved at 65 to 75 캜 to prepare an oil phase part. Components 8 to 12 were dissolved by heating at 65 to 75 占 폚 to prepare a water-based part. The oil phase and component 21, which were prepared while stirring the prepared water phase, were slowly added and emulsified at 7000-8000 rpm for 5 minutes to prepare a cream formulation. After cooling to 45 캜, one of the components 13 to 20 was added and dispersed at 1500 rpm for 1 minute to prepare Examples 1 to 8. In the case of Comparative Example 1, no additional components were added.

[Experimental Example 6] Pigment deposition inhibitory effect

The area of 2 × 2 cm ² was set in both lower arms of a healthy male and female subjects. Experimental target site only to be irradiated with ultraviolet light putting an aluminum foil on the arm, at a distance of 10cm Toshiba Co. claim FL20S BLB lamp and FL20S E-30 lamp 2 at the same time ^{0.8 × 10 - 7 erg / cm} 3 / circuit 1 Day, and three times a day. Before the irradiation, the test site was washed well with 70% isopropyl alcohol aqueous solution. After the irradiation, the prescriptions of Examples 1 to 8 containing the Mucinaceae plants at the ultraviolet ray irradiation site and Comparative Example 1 not containing them were applied three times a day. After three weeks of application, the degree of pigmentation was determined visually, and the degree of inhibition of pigment deposition by the Examples compared to the Comparative Example was evaluated in three stages of remarkable effect, validity and invalidity. The results are shown in Table 7.

Application group Number of Respondents Remarkable effect available invalidity Example 1 2 10 8 Example 2 One 11 8 Example 3 3 6 11 Example 4 2 9 9 Example 5 4 15 One Example 6 3 11 6 Example 7 4 13 3 Example 8 3 10 7 Comparative Example 1 0 7 13

As can be seen from the results of Table 7, many of the Examples 1 to 8 were evaluated as remarkable effects or effective stages as compared with Comparative Example 1. Therefore, callus pulp of mulberry and cedar wood, callus culture of mulberry and cucumber, callus of cell differentiated into root in callus of mulberry and cedarwood, culture of cells differentiated into callus of callus of mulberry and cedar wood have excellent pigment It was confirmed that it had a deposition inhibiting effect.

Callus pulp of mulberry and cedar wood, callus culture of mulberry and cedar wood having the excellent pigment inhibiting effect as described above, pulp of cell differentiated into roots in callus of mulberry and cedar wood, root of callus of mulberry and cedar wood The culture medium of the differentiated cells has a skin whitening effect, improves skin tone, and has an effect of preventing or improving aging. These can be used in combination of two or more.

[Experimental Example 7] Evaluation of restoration effect on skin barrier function

In order to measure the skin barrier function recovery effect of Examples 1 to 6, acetone was repeatedly applied to the skin of a hairless mouse to deteriorate the barrier function, and TEWL (transdermal water loss) After reaching 4.0 g / m 2 / h as measured by an evaporimeter EP 1 from servomed (Sweden), the above Examples 1 to 6 and Comparative Example 1 were applied to an area of 5 cm 2, and after 1, 2, 4 and 8 hours, To assess the extent to which the barrier function is restored by evaluating the degree of reduction. At this time, a normal lipid mixture (ceramide: 2: 1: 1 mixture of cholesterol: fatty acid) was used as a positive control. The skin moisture loss in the initial damaged barrier was 100%, and the changes over time were compared. The results are shown in Table 8 below.

sample Skin moisture loss After 1 hour After 2 hours After 4 hours After 8 hours Example 1 57 47 35 29 Example 2 68 60 42 34 Example 3 65 51 40 32 Example 4 62 57 41 31 Example 5 69 52 47 33 Example 6 69 54 50 33 Comparative Example 1 76 67 61 58 Lipid mixture 65 46 35 26

As can be seen from the results in Table 8, Examples 1 to 6, which contain callus pulp of mulberry and cedar wood, callus cultures of mulberry and cedar wood, and lysates of roots differentiated from callus of mulberry and cucumber trees, The skin moisture loss was smaller than Comparative Example 1 which did not contain the active ingredient and lipid mixture which was a positive control. Therefore, it was confirmed that the callus pulp of mulberry and cedarwood, the callus culture of mulberry and cedarwood, and the cell lysate of root differentiated from the callus of mulberry and cucumber have excellent skin barrier function recovery effect.

Callus pulp of mulberry and cedar woods, callus culture of mulberry and cucumber trees, and cell lysate of root differentiated from callus of mulberry and cucumber trees have skin moisturizing effect and can prevent and improve aging. . These can be used in combination of two or more.

[Experimental Example 8] Evaluation of skin tone improvement effect

108 adults who were thought to require skin whitening were divided into 9 groups, and each group was provided with Examples 1 to 8 and Comparative Example 1 and applied to the face for 2 weeks each day for 4 weeks.

After 4 weeks of use, a skin tone improvement effect was evaluated through a questionnaire about usability. The results are shown in Table 9 below.

Application group Number of Respondents Very good Good usually Inadequate Example 1 5 6 One 0 Example 2 4 7 3 0 Example 3 3 6 3 0 Example 4 4 4 4 0 Example 5 2 4 5 0 Example 6 3 4 5 0 Example 7 2 4 6 0 Example 8 3 6 3 0 Comparative Example 1 One 3 7 One

As can be seen from the results shown in Table 9, those who applied Examples 1 to 8 felt that the whitening effect was more excellent than those of Comparative Example 1.

Therefore, callus pulp of mulberry and cedarwood, callus culture of mulberry and cedarwood, cell differentiation of root differentiated from callus of mulberry and cedarwood, culture of cells differentiated into roots from callus of mulberry and cedar wood, To improve skin tone and prevent or improve aging. These can be used in combination of two or more.

The present invention relates to a method for the production of callus of a callus of a plant of the present invention, a callus culture solution, a callus extract, a disruption of cells differentiated into callus in callus, a culture of cells differentiated from callus, and an extract of cells differentiated from callus The cosmetic composition and the pharmaceutical composition according to the present invention can be applied to various formulations. However, the present invention is not limited to the specific formulation .

[Formulation Example 1] Suspension emulsion lotion

Suspended emulsion lotion was prepared with the composition shown in Table 10 below.

Constituent Content (% by weight) Methylparaben
Spices
Cetyl octanoate
Polyoxyethylene hardened castor oil
Polysiloxane
Tocopheryl acetate
ethanol
glycerin
Propylene glycol
Carboxyvinyl polymer
Mulberry callus
Purified water Suitable amount
Suitable amount
1.0
0.6
0.4
0.1
12.0
6.0
2.0
0.12
2.0
TO 100

[Formulation Example 2] Essence

Essences were prepared with the compositions shown in Table 11 below.

Constituent Content (% by weight) Methylparaben
Spices
Cetyl octanoate
Octyldodec-16
Coles-24 / Ceteth-24
Polysiloxane
Tocopheryl acetate
ethanol
glycerin
Xanthan gum
Propylene glycol
Carboxyvinyl polymer
Mulberry callus culture
Purified water Suitable amount
Suitable amount
1.0
0.5
0.2
0.4
0.1
6.0
15.0
0.07
4.0
0.12
6.0
TO 100

[Formulation Example 3] Nourishing cream

Nutritive creams were prepared with the compositions shown in Table 12 below.

Constituent Content (% by weight) Liquid paraffin
Cetostearyl alcohol
Wax
Polysiloxane
Chin type monostearate stearate
Stearic acid
Cetearyl glucoside
Polyoxyethylene sorbitan monostearate (20 EO)
Hydrogenated lecithin
Cetyl octanoate
Methylparaben
Propylparaben
Spices
Ethylenediamine sodium tetraacetate
glycerin
Carboxyvinyl polymer
Propylene glycol
Calligraphy calligraphy
Purified water 15.0
2.0
3.0
0.5
2.0
2.0
1.5
1.3

2.0
1.0
0.2
0.1
Suitable amount
0.02
5.0
0.10
2.0
2.0
TO 100

[Formulation Example 4]

The gel was prepared with the composition shown in Table 13 below.

Constituent Content (% by weight) antiseptic
Spices
Cetearyl glucoside
Cetyl octanoate
glycerin
Jojoba wax
Di-l-phenanthinol
ethanol
Carboxyvinyl polymer
Propylene glycol
Cultures of cells differentiated from roots in callus of mulberry
Purified water Suitable amount
Suitable amount
1.5
1.0
5.0
3.0
1.0
7.0
0.6
3.0
6.0

TO 100

[Formulation Example 5] Pack

Packs were prepared with the compositions shown in Table 14 below.

Constituent Content (% by weight) Methylparaben
Spices
Cetyl octanoate
Polyoxyethylene hardened castor oil
Polysiloxane
Cellulose sword
ethanol
glycerin
Propylene glycol
Polyvinyl alcohol
Disruption of cell differentiated into roots in calli of cedar wood
Purified water Suitable amount
Suitable amount
1.0
0.6
0.2
0.2
6.0
5.0
3.0
15.0
3.0

TO 100

[Formulation Example 6] Ointment in external preparation for skin

Ointment was prepared by a conventional method according to the composition shown in Table 15 below.

ingredient Content (% by weight) Purified water Balance glycerin 8.0 Butylene glycol 4.0 Liquid paraffin 15.0 Beta Glucan 7.0 Carbomer 0.1 Cultures of cells differentiated from roots in callus of mulberry 1.0 Caprylic / capric triglyceride 3.0 Squalane 1.0 Cetearyl glucoside 1.5 Sorbitan stearate 0.4 Cetearyl alcohol 1.0 antiseptic Suitable amount incense Suitable amount Pigment Suitable amount Wax 4.0

Claims (3)

An antioxidant cosmetic composition comprising at least one selected from the group consisting of a cell lysate, a culture and an extract thereof of a cell differentiated from callus of Broussonetia papyrifera as an active ingredient. The method according to claim 1,
Wherein the cell differentiated from the callus comprises cells differentiated into leaves, stems, or roots in the callus. The method according to claim 1,
Wherein the composition contains the active ingredient in an amount of 0.0001 to 25.0% by weight based on the total weight of the composition.

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