KR102105001B1 - Method for mass production of whitening functional ingredients using Damnacanthus major cell - Google Patents

Abstract

The present invention relates to a mass production method after increasing the content of a whitening functional useful ingredient by treating an elicitor with Damnacanthus major cells, to a cell culturing method of a Damnacanthus major, and to a method for stably extracting a large amount of useful components from the cells. Furthermore, a value of wild plants is further improved by being applied to the cell culture method of various wild plants containing useful ingredients.

Description

Method for mass production of whitening functional ingredients using Damnacanthus major cell}

The present invention relates to a method of mass production after increasing the content of the whitening functional useful ingredient by treating the elixirs in the capillary cells.

Skin darkening (melanogenesis) is a mechanism of defense against stimulation of ultraviolet rays from melanin-derived cells (melanocytes), which increases melanin production activity, and a large amount of melanin produced by this is transferred to keratinocytes and accumulates in the skin epidermal layer. Is the result. Although melanin protects the skin, hyperpigmentation of the skin causes blemishes, freckles, darkening of the skin after skin irritation, senile pigment spots, etc., which may cause not only cosmetic discomfort to the parties, but also social discomfort. It has a negative effect. In the melanin production process, an enzyme called tyrosinase acts on tyrosine, which is a kind of amino acid, and is converted into dopa (DOPA) and dopaquinone, followed by a non-enzymatic oxidation reaction. It is known that calm, spots, and spots are formed. To alleviate, prevent, and treat pigmentation, freckles, and spots, substances that inhibit melanin production, such as hydroquinone, arbutin, vitamin C, and derivatives thereof, have been developed, and whitening cosmetics containing them have been released. In Korean Patent Publication No. 2005-0509848, whitening cosmetics containing kelactone derivatives separated from the previous issue are disclosed. In addition, Korean Patent Publication No. 2005-0479741 discloses a whitening cosmetic containing a glucose acylated derivative. Recently, efforts have been made to use natural plant extract-derived substances for whitening to prevent side effects of chemically synthesized substances. However, natural plant extract-derived substances have very low extraction concentrations and differ in efficacy depending on the plant's production area. There was a problem that it was difficult to maintain homogeneity.

Meanwhile, the Damnacanthus major is an evergreen broad-leaved tree belonging to the family Rubiaceae, Damnacanthus, and the anthraquinone, Damnacanthal, Damnacanthol, and Dams It has been reported that it contains Nadin. Accordingly, the present inventors developed a method of stably extracting a large amount of anthraquinone from a crystal tree, and completed the present invention to use it as a useful functional ingredient for whitening.

The method of the present invention has the effect of improving the usefulness of the crystal tree because it can produce a lens tree with an enhanced content of the rubidin component by eliminator treatment. In addition, it is expected that the method of the present invention may be applied to a cell culture method of various wild plants containing useful components to further improve the value of wild plants.

The present invention was derived by the above-described needs, and the present inventors extracted various methods of elixirs during the culturing of the capsular cells in a bioreactor to extract rubiadine, a whitening functional useful component, and rubavidin compared to the elixirs-free treatment group. The present invention was completed in view of the fact that it is stably extracted in large quantities.

Accordingly, one object of the present invention is to provide a method for stably extracting a large amount of rubidine, which is a useful component of whitening, by treating an elite cell with a lens.

Another object of the present invention is to provide a composition for skin whitening comprising rubiadine extracted by the above method.

Another object of the present invention is to provide a cosmetic composition for skin whitening comprising the composition for skin whitening.

Another object of the present invention is to provide a pharmaceutical composition for the treatment or prevention of pigmentation diseases comprising the composition for skin whitening.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

Hereinafter, various embodiments described herein are described with reference to the drawings. In the following description, for the full understanding of the present invention, various specific details, such as specific forms, compositions and processes, have been described. However, certain embodiments may be practiced without one or more of these specific details, or in combination with other known methods and forms. In other instances, well-known processes and manufacturing techniques are not described in specific details in order not to unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, form, composition or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Thus, the context of “in one embodiment” or “an embodiment” expressed in various places throughout this specification does not necessarily represent the same embodiment of the invention. Additionally, special features, shapes, compositions, or properties can be combined in any suitable way in one or more embodiments.

Unless otherwise specified in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains.

In one embodiment of the present invention, "Damnacanthus major" refers to an evergreen broad-leaved shrub belonging to the family Rubiaceae and Damnacanthus, and has poor cold resistance, making it impossible to overwinter in the inland region, and to Jeju Island and Jeollanam-do, Korea. In some areas, it grows naturally in forests of less than 700m above sea level and is also distributed in China and Japan. The tree is 30 ~ 70cm in height, and the roots are divided into several and thickened in several places. Stem grayish white, with branched branches, short thorns shorter than leaves. The leaves are opposite, wide egg-shaped, shiny, and flat at the edges. The bottom of the leaf is rounded, the tip is sharp, and the hair disappears, so the thorn comes out from the axilla and the length is about lcm. The flowers are from April to June, with 1 to 2 white flowers running on the axilla and short peduncles. The flower beak looks like a barrel, less than 1.5cm long, splits into 4 ends, and petals are triangular. The upper part of the inside of the tube of flowers has long hairs, with 4 stamens, 1 pistil, and 4 pistils. The fruit is a nuclear fruit, rounded about 5mm in diameter, and ripened in autumn with a red color. At the end of the fruit, a calyx branch remains.

It has been reported that the root of the crystal tree contains anthraquinone, damnacanthal, damnacanthol, and damnidin. The anthraquinone component is reported to have anti-aging effects. According to Xiliang Yang (Journal of Ethnopharmacology, 2012), the crystal root extract has excellent nerve cell protection.

In one embodiment of the present invention, "Elicitor (Elicitor)" is a plant-inducing disease resistance and growth-promoting substances for inducing plants to produce plant fungicides as a defense system against microbial invasion. In other words, like the animal's immune system, plants also have a defense system, and elisitators are substances that stimulate the plant's immune system to produce secondary metabolites in response. The eliminator includes an endogenous eliminator derived from a plant and an exogenous eliminator derived from the outside, and the exogenous eliminator is classified into a biological eliminator derived from microorganisms and an inanimate eliminator. Inanimate eliminators include UV, cold, heat, ethylene, fungicides, pesticides, and heavy metals. By using an appropriate eliminator in the bioreactor, it is possible to increase the amount of the desired component, and the quality and amount of secondary metabolites differ significantly depending on the species and eliminator, so the appropriate organisms according to the desired secondary metabolites It is important to select the species and the appropriate eliminator.

In one embodiment of the present invention, “Rubiadin” is a type of biologically active anthraquinone, C ₁₅ H ₁₀ O ₄ , or “1,3-dihydroxy-2-methyl-9,10-dihydroanthracene-9, 10-dione ”. In the present invention, it is represented by the following formula (1).

[Formula 1]

In one embodiment of the present invention, "whitening" means that the skin is continuously exposed to ultraviolet rays to increase melanocytes, thereby preventing excessive melanin pigmentation on the skin, or thinning the existing melanin pigmentation. Refers to Accordingly, generation of freckles and freckles caused by excessive melanin pigmentation can be suppressed.

In one embodiment of the present invention, "treatment" means to improve or advantageously improve skin pigmentation and other symptoms caused by using the whitening functional useful component extracted in large quantity by using an eliminator from the capillary cells according to the present invention. It means all activities If you have ordinary knowledge in the technical field to which this application belongs, you can grasp the exact standard of skin pigmentation by referring to the data presented by the Korean Medical Association, and judge the degree of improvement, improvement and treatment. There will be.

In one embodiment of the present invention, "prevention" refers to any act of suppressing or delaying skin pigmentation and other symptoms resulting therefrom by using a whitening functional useful ingredient extracted in large quantities by using an eliminator from a capillary cell according to the present invention. Means It will be apparent to those skilled in the art that the composition of the present application having a therapeutic effect on skin pigmentation disease can prevent such diseases using the whitening functional useful ingredient according to the present invention before the initial symptoms or symptoms of skin pigmentation appear.

As used herein, "pharmaceutical composition" means a composition administered for a specific purpose. For the purpose of the present invention, the pharmaceutical composition of the present invention includes a whitening functional useful component extracted in large quantities by using an eliminator from a capillary cell as an active ingredient, and proteins and pharmaceutically acceptable carriers, excipients or proteins involved therein Diluents may be included. The above "pharmaceutically acceptable" carriers or excipients are those approved by the governmental regulatory department, or listed in the government or other generally approved pharmacopeia for use in vertebrates, and more particularly in humans. do.

For parenteral administration, the pharmaceutical compositions of the present invention may be in the form of suspensions, solutions or emulsions in oily or aqueous carriers, and may be prepared in solid or semi-solid form. In addition, the pharmaceutical composition of the present invention may include a formulation agent such as a suspending agent, stabilizer, solubilizer and / or dispersant, and may be sterilized. The pharmaceutical composition can be stable under the conditions of manufacture and storage, and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. Alternatively, the pharmaceutical composition of the present invention may be in sterile powder form for reconstitution with a suitable carrier prior to use. The pharmaceutical composition may be in unit-dose form, in microneedle patches, in ampoules, or in other unit-dose containers, or in multi-dose containers. Alternatively, the pharmaceutical composition may be stored in a freeze-dried (lyophilized) state requiring only the addition of a sterile liquid carrier, for example water for injection just prior to use. Immediate injection solutions and suspensions can be prepared as sterile powders, granules or tablets.

In some non-limiting embodiments, the pharmaceutical compositions of the present invention can be formulated or included in the form of microspheres in a liquid. In certain non-limiting embodiments, the pharmaceutical compositions of the present invention may contain pharmaceutically acceptable compounds and / or mixtures thereof at concentrations between 0.001 and 100,000 U / kg. Also in certain non-limiting embodiments, the pharmaceutical compositions of the invention include suitable excipients including preservatives, suspending agents, additional stabilizers, dyes, buffers, antibacterial agents, antifungal agents, and isotonic agents, such as sugar or sodium chloride. You can. As used herein, the term “stabilizer” refers to a compound selectively used in the pharmaceutical composition of the present invention to increase shelf life. In a non-limiting implementation, the stabilizing agent can be a sugar, amino acid, or polymer. In addition, the pharmaceutical composition of the present invention may include one or more pharmaceutically acceptable carriers, and the carrier may be a solvent or a dispersion medium. Non-limiting examples of pharmaceutically acceptable carriers include water, saline, ethanol, polyols (eg, glycerol, propylene glycol and liquid polyethylene glycol), oils, and suitable mixtures thereof. Non-limiting examples of sterilization techniques applied to the pharmaceutical compositions of the present invention include filtration through bacterial-inhibiting filters, terminal sterilization, coalescence of sterile preparations, radiation, sterilization gas irradiation, heating, vacuum drying and freeze drying. do.

As used herein, “administration” means introducing the composition of the present invention to an individual in any suitable way, and the route of administration of the composition of the present invention can be administered through any general route as long as it can reach the target tissue. have. Oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, intrapulmonary administration, rectal administration, intraperitoneal administration, intraperitoneal administration, intrathecal administration may be made, but the pharmaceutical of the present invention Most preferably, the composition is applied on the skin or provided in the form of subcutaneous or intradermal injection, but is not limited thereto.

The treatment method of the present invention may include administering the pharmaceutical composition in a pharmaceutically effective amount. The effective amount in the present invention is the type of disease, the severity of the disease, the type and content of the active ingredients and other ingredients contained in the composition, the type of formulation and the patient's age, weight, general health status, sex and diet, administration time, route of administration And various factors including the secretion rate of the composition, the duration of treatment, and drugs used simultaneously.

In the present specification, the “cosmetic composition” is a composition for improving or improving skin pigmentation and other symptoms. The cosmetic composition containing the composition of the present invention as an active ingredient is lotion, nutrient lotion, nutrient essence, massage cream, beauty bath additive, body lotion, body milk, bath oil, baby oil, baby powder, shower gel, shower cream, sun Screen lotion, sunscreen cream, suntan cream, skin lotion, skin cream, sunscreen cosmetics, cleansing milk, hair loss agent (for cosmetics), face and body lotion, face and body cream, skin whitening cream, hand lotion, hair lotion, Cosmetic Cream, Jasmine Oil, Bath Soap, Water Soap, Beauty Soap, Shampoo, Hand Wash (Hand Cleaner), Medicinal Soap (Non-Medical), Cream Soap, Facial Wash, Hair Rinse, Cosmetic Soap, Gel for Tooth Whitening, Toothpaste, etc. It can be manufactured in the form of. To this end, the composition of the present invention may further include a suitable carrier, excipient or diluent commonly used in the manufacture of cosmetic compositions. As a carrier, excipient, or diluent that can be further added to the cosmetic composition of the present invention, purified water, oil, wax, fatty acid, fatty acid alcohol, fatty acid ester, surfactant, humectant, thickener, antioxidant, viscosity stabilizer, kill Rating agents, buffers, lower alcohols, etc., but are not limited thereto. In addition, whitening agents, moisturizers, vitamins, sunscreens, perfumes, dyes, antibiotics, antibacterial agents, and antifungal agents may be included as necessary. Hydrogenated vegetable oil, castor oil, cottonseed oil, olive oil, palm oil, jojoba oil, avocado oil may be used as the oil, and waxes include beeswax, mellow, carnauba, candelilla, montan, ceresin, liquid paraffin, and lanolin. Can be used. As fatty acids, stearic acid, linoleic acid, linolenic acid, and oleic acid may be used, and cetyl alcohol, octyldodecanol, oleyl alcohol, panthenol, lanolin alcohol, stearyl alcohol, and hexadecanol may be used as fatty acid alcohol. In addition, isopropyl myristate, isopropyl palmitate, and butyl stearate may be used as fatty acid esters. As the surfactant, cationic surfactants, anionic surfactants, and nonionic surfactants known in the art can be used, and surfactants derived from natural products are preferred if possible. In addition, it may include a hygroscopic agent, a thickener, an antioxidant, etc., which are widely known in the cosmetic field, and their types and amounts are as known in the art.

In order to solve the above problems, the present invention is a step of sterilizing a crystal plant; Inducing and proliferating cells from the root slices of the sterilized quartz plant; And culturing the cells by treating the eliminator in a bioreactor. A method for mass production of capsular cells with increased content of a whitening functional useful ingredient, rubiadine, is provided.

In addition, the present invention provides a cell of a crystal plant plant with an increased content of a whitening functional useful component cultured by the above method.

In addition, the present invention is a step of sterilizing a crystal plant; Inducing and proliferating cells from the root slices of the sterilized quartz plant; And culturing the cells by treating the eliminator in a bioreactor. It provides a method for stably extracting a whitening functional useful component, including rubiadine, a step of extracting the functional whitening useful component from the cell.

In addition, the present invention provides a cosmetic composition comprising rubiadine extracted by the above method as an active ingredient.

More specifically, in one embodiment of the present invention, (a) proliferating the cells from a section of a tree plant (Damnacanthus major); And (b) treating the cells with an eliminator; providing a method for culturing a capillary cell with an increased rubiadin content, wherein the section is the root, stem, or leaf portion of the capsular plant. It provides a method for culturing a capillary cell with an increased content of rubiadine, which is taken from, and the eliminator is selected from the group consisting of methyl-jasmonate, salicylic acid, and yeast extract. Provided is a method for culturing a lens of a lens with increased rubidin content, which is at least one, and the eliminator is provided with a method for culturing a lens of a corrected lens with increased rubidin content, which is treated at a concentration of 10 to 990 μM. And, the rubiadine provides a method for culturing a lens of a capillary cell with an increased amount of rubiadine represented by the following Chemical Formula 1.

[Formula 1]

In another embodiment of the present invention, there is provided a lens with an increased content of rubiadine, which is cultured by a method of culturing a lens of corrected trees with increased rubiadine content.

In another embodiment of the present invention, provides a cosmetic composition comprising a modified globular cell having an increased content of rubiadine as a useful component, and the cosmetic composition provides a cosmetic composition having a whitening effect.

In another embodiment of the present invention, there is provided a pharmaceutical composition for the prevention or treatment of darkening of the skin, comprising the modified globular cells having an increased content of rubidin as an active ingredient.

In another embodiment of the present invention, (a) proliferating the cells from a section of a tree plant (Damnacanthus major); (b) treating the cells with an eliminator and further culturing them; And (c) drying the cells and treating the spirits; providing a method for extracting rubiadin from a capsular cell comprising the root, stem, or leaf portion of a capsular plant. It provides a method for extracting rubiadine from a capillary cell that is collected from, the eliminator is any one selected from the group consisting of methyl jasmonate (methyl-jasmonate), salicylic acid (salicylic acid), and yeast extract Provides a method for extracting rubidin from a capsular cell that is abnormal, and the elisitator provides a method for extracting rubidin from a capsular cell that is treated at a concentration of 10 to 990 μM, wherein the rubidin is It provides a method for extracting rubiadine from a capsular tree cell represented by the following formula (1), the treatment of alcohol is carried out at 25 ℃ to 80 ℃ Provides a method for extracting a Dean rubiah from chome cells, treatment of the alcohol provides a method for extracting a rubiah Dean from the neck fix the cells to perform for 1 hour to 4 hours.

[Formula 1]

In another embodiment of the present invention, a method for extracting the rubiadine provides rubiadine extracted from a plant of a crystal tree cultured by treating an elisitator.

In another embodiment of the present invention, to provide a cosmetic composition comprising rubiadine extracted from a crystal tree plant cultured by treating the eliminator as a useful component, the cosmetic composition is a cosmetic composition having a whitening effect to provide.

In another embodiment of the present invention, there is provided a pharmaceutical composition for the prevention or treatment of skin darkening, comprising as an active ingredient rubiadine extracted from a tree plant grown by treating the eliminator.

The method of the present invention has the effect of improving the usefulness of the crystal tree because it can produce a lens tree with an enhanced content of the rubidin component by eliminator treatment. In addition, it is expected that the method of the present invention may be applied to a cell culture method of various wild plants containing useful components to further improve the value of wild plants.

1 is a process diagram of a cell cultivation method and useful component analysis method of a crystal tree plant having an increased content of useful components by an eliminator treatment according to an embodiment of the present invention.
FIG. 2 is a photograph of induction and selection of capsular cells in a solid medium according to an embodiment of the present invention.
3 is a photograph showing a state in which the cultured wood cells are cultured in a bioreactor and treated with an eliminator according to an embodiment of the present invention.
Figure 4 is a photograph showing the analysis spectrum and content results for useful components of the capillary cell, according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only intended to illustrate the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

In order to achieve the object of the present invention, the present invention,

Sterilizing a crystal tree plant; Inducing and proliferating cells from root, stem, and leaf sections of the sterilized crystal tree plant; And culturing the cells by treating the eliminator in a bioreactor. A method for mass production of fertilized wood cells with increased content of a whitening functional useful ingredient is provided.

In the cultivation method of the present invention, it is preferable that the plant is a root segment, but is not limited thereto.

In addition, in the culture method of the present invention, the whitening functional useful component of the capillary cell may be anthraquinone, damnacanthal, damnacanthol, or damnidin, preferably. Is anthraquinone, and more preferably, it is a rubidin, but is not limited thereto.

More specifically, the cultured cell culture of the present invention, (1) 1x MS (Murashige & Skoog) medium containing 30 g / L of sucrose and 3 mg / L of NAA (1-Naphthylacetic Acid) is used, (2 ) Adjust the medium pH to 5.8, (3) Inoculate the cells to 20.0g / L, (4) Set the air supply to 0.1vvm, (5) Incubate the culture temperature to 20 ± 1 ℃, (6) ) 15 days after cultivation is harvested, but is not limited thereto, and it is possible to allow some modifications if the culture conditions are changeable within the scope of maintaining the same effect by ordinary scientists in the field of the present invention.

In addition, in the culture method of the present invention, the eliminator is preferably methyl jasmonate, salicylic acid, or yeast extract, and more preferably methyl jasmonate, but is not limited thereto.

In the method of culturing cells according to an embodiment of the present invention, when methylzasmonate is used as an eliminator to increase the content of the whitening functional useful component, rubiadine, from 2 weeks after the start of cell culture It may be supplied at a concentration within 400 μM, and preferably may be supplied at a concentration of 100 μM to 400 μM, but is not limited thereto.

In the method for culturing a cell according to an embodiment of the present invention, when salicylic acid is used as an eliminator to increase the content of a whitening functional useful ingredient, rubiadine, salicylic acid is supplied at a concentration within 300 μM from 2 weeks after the start of cell culture. It may be, and may be preferably supplied at a concentration within 100 μM, but is not limited thereto.

In the method for culturing cells according to an embodiment of the present invention, when the yeast extract is used as an eliminator to increase the content of the whitening functional useful ingredient, rubiadine, the yeast extract is within 300 mg / L from 2 weeks after the cell culture starts. It may be supplied at a concentration, and preferably may be supplied at a concentration of 100mg / L to 200mg / L, but is not limited thereto.

The present invention also provides a cell of a crystal plant plant with an increased content of a whitening functional useful ingredient, cultured by the above method. The cells of the capsular tree plant with increased content of the useful component according to the present invention may be, but are not limited to, the capsular (Damnacanthus major) cells with an increased total content of rubiadine.

The present invention also, the step of removing and drying the moisture of the cells; Treating the liquor with the cell dry matter; And extracting rubiadine; provides a method for stably mass-producing rubiadine from a capillary cell comprising a.

In the extraction method of the present invention, the drying is preferably performed at 60 ° C. for 2 days, and the alcohol is preferably used at 60% alcohol, and the extraction is performed at 25 ° C. to 80 ° C. for 1 hour to 4 hours. It is preferred, but is not limited thereto.

The present invention also provides a composition for preventing, treating, or improving whitening comprising rubiadine extracted from a capillary cell as a useful component by the above extraction method.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only to illustrate the present invention, the content of the present invention is not limited to the following examples.

Example 1. Cell induction and proliferation

Crystal trees used in the present invention were used as seedlings germinating seeds purchased from Jeju Island, a native place. For surface sterilization before inoculation, the root, stem, or leaf parts of the plant are cut to an appropriate size, immersed in 70% ethanol for 30 seconds, sterilized by shaking with 2% sodium hypochlorite for 5 minutes, and then sterilized. Washed with a small section (0.5cm ² ) was used as a culture material.

Growth-inducing substance naphthaleneacetic acid (NAA) 3mg / L, sucrose 30g / L, and gelrite for inducing cells from the root, stem, or leaf sections of a crystal plant ) After inoculation in MS (Murashinge and Skoog) solid medium to which 2.5 g / L was added, the cells were cultured for 2 to 4 weeks in a culture room with cancer conditions of 20 ± 1 ° C. When cells are induced from the explant in the solid medium, cells are transferred to the same new solid medium every two weeks and cultured to continuously select cells in which growth is actively performed (FIG. 2). Cells selected from the solid medium were inoculated into an air-lifting bioreactor having a total volume of 3 L, and 2 L of a liquid medium containing no gelite was added, and the cells were cultured for 10 days in a culture room with cancer conditions of 20 ± 1 ° C. Proliferation (FIG. 3). As the liquid medium, a medium consisting of 3 mg / L of NAA and 30 g / L of sucrose was used as the MS medium.

Example 2. Investigation of the effect of elixirs on the increase of useful components of cells during the culture of a bioreactor

Each 3L bioreactor was inoculated with 20.0g of cells after adding medium consisting of 3mg / L of NAA and 30g / L of sucrose to 1.0 MS basic medium for 2L each. The air supply in the bioreactor was 0.1 vvm, the temperature was 20 ± 1 ° C., and culture was performed under dark conditions. In order to maximize cell growth and accumulation of useful components at the same time, a two-stage culture was performed in a bioreactor. Cells were cultured in a bioreactor for 10 days to achieve a certain level of growth, and after 10 days, elite (methyl jasmonate, salicylic acid, yeast extract) was treated at various concentrations to further culture for 5 days. Finally, the effect of elixirs on the growth and whitening functional useful components of the cell, the content of rubidin in the culture of the bioreactor was investigated.

The method for measuring cell growth and rubidin content in each step is as follows. The live weight of the cells was filtered by harvesting the cells with a stainless steel plate, the medium was separated, the moisture was sufficiently removed with a clean tissue, and the weight was measured. The weight in the building was dried at 60 ° C for 2 days and then weighed. In addition, the growth rate of arrhythmia was calculated by Equation 1 below.

[Equation 1]

Growth rate = [(In the building of the last grown cell)-(In the building of the initial inoculated cell)] / (In the building of the initial inoculated cell)

Rubiadine content was analyzed by HPLC for extracts of cell dry matter extracted for 1 hour at 25 ° C and 40 ° C in 60% alcohol (6: 4 ratio of alcohol and water). In the case of the methyl jasmonate treatment, the rubiadine content of the extracts for 1, 2, and 4 hours was analyzed together with extraction at 60 ° C and 80 ° C. Agilent Eclipse XDB-C18 (5 μm, 4.5 mm × 150 mm) was used for the stationary phase, and acetonitrile (AC N) and water were used for the mobile phase, and phosphoric acid was added to adjust the pH. To optimize the detection wavelength, chromatograms with four UV wavelengths were compared and the optimal detection wavelength was set to 280 nm so that the most peaks could be detected with the highest sensitivity.

2-1. Effect of Salicylic Acid on the Increase of Useful Components of Crystal Tree Cells

In order to investigate the effect of salicylic acid on the growth of cells and the increase of useful components, treatment was performed at concentrations of 50, 100, 200, and 300 μM, respectively, after 10 days of culture, and the bioreactor culture conditions were carried out in the same manner as in the above example. Did. Table 1 shows the effect of salicylic acid on cell growth and rubidin content.

Salicylic acid concentration (μM) Live weight (g / L) Building (g / L) Growth rate
Rubiadine content
(25 ℃ extraction group)
(mg / g DW) Rubiadine content
(40 ℃ extraction group)
(mg / g DW) Control (0) 117.90 7.45 4.91 0.053 0.110 50 105.96 7.39 4.87 0.101 0.213 100 104.45 7.21 4.72 0.068 0.172 200 102.23 6.94 4.51 0.062 0.167 300 100.08 6.63 4.26 0.057 0.151

As shown in Table 1, in all treatments of salicylic acid, the weight and weight of the capillary cells were decreased compared to the control. However, the useful ingredient content was found to be higher than that of the control in all salicylic acid treatments. In particular, it was found that the rubiadine content was 0.101 mg / g DW in the salicylic acid 50 μM treatment, which was 1.9 times higher than the control. When extracted at 40 ° C, the average showed 2.4 times higher rubidin content than when extracted at 25 ° C. In this case, the rubidin content was also confirmed to be 0.213 mg / g DW in the salicylic acid 50 μM treatment group, which was 1.9 times higher than the control. Turned out to be.

2-2. Effect of Yeast Extracts on Increased Useful Components of Fertilized Tree Cells

In order to investigate the effect of yeast extract on the growth of cells and the increase of useful components, 10 days after culture, treatment was performed at concentrations of 50, 100, 200, and 300 μM, respectively, and the culture conditions of the bioreactor were carried out in the same manner as in the above example. Table 2 shows the effect of the yeast extract on cell growth and rubidin content.

Yeast extract (mg / L) Live weight (g / L) Building (g / L) Growth rate Rubiadine content
(25 ℃ extraction group)
(mg / g DW) Rubiadine content
(40 ℃ extraction group)
(mg / g DW) Control (0) 119.42 7.24 4.75 0.052 0.108 50 130.41 8.72 5.92 0.058 0.112 100 134.99 8.32 5.60 0.072 0.133 200 106.03 7.58 5.02 0.060 0.129 300 98.38 6.91 4.48 0.043 0.106

As shown in Table 2, the high concentration of yeast extract (300mg / L) inhibited the growth of capillary cells, but the low concentration of yeast extract showed higher growth compared to the untreated control. In addition, the useful component content of the cell was the highest in the yeast extract 100mg / L treatment, and the rubiadine content was 0.072mg / g DW, respectively, which was confirmed to be 1.4 times higher than the control. When extracting at 40 ℃, the average showed 2.1 times higher rubidin content than extraction at 25 ℃, and in this case, the rubidin content was also found to be 0.133mg / g DW in the yeast extract 100μM treatment, 1.2 times compared to the control. Increased.

2-3. The effect of methyl jasmonate on the increase of useful components in capsular cells

In order to examine the effect of methyl jasmonate (methyl-jasmonate) on the growth of cells and the increase of useful components, treatment was performed at concentrations of 50, 100, 200, and 300 μM, respectively, after 10 days of culture, and the bioreactor culture conditions were as described above. It was carried out in the same manner as. Table 3 shows the effect of the methyl jasmonate on cell growth and rubidin content.

Methyl jasmonate
Concentration (μM) Live weight (g / L) Building (g / L) Growth rate
Rubiadine content
(25 ℃ extraction group)
(mg / g DW) Rubiadine content
(40 ℃ extraction group)
(mg / g DW) Control (0) 115.21 7.24 4.75 0.053 0.109 50 91.28 5.63 3.47 0.101 0.278 100 82.86 5.06 3.02 0.113 0.356 200 79.32 4.61 2.66 0.114 0.386 300 71.23 4.09 2.25 0.123 0.389

As shown in Table 3, in all the treatment groups of methyl jasmonate, the results showed that the biomass and the building weight of the lens cells were decreased compared to the control. However, it was found that the useful ingredient content was increased in all methyl jasmonate treatments compared to the control, and in particular, the rubiadine content in the methyl jasmonate 300 μM treatment was confirmed to be 0.123 mg / g DW, which was 2.3 times higher than the control. . When extracted at 40 ° C, the average showed a 2.9-fold higher rubiadine content than when extracted at 25 ° C, and in this case, the rubiadine content was also confirmed to be 0.389 mg / g DW in the methyl jasmonate 300 μM treatment, compared to the control. The increase was 3.6 times.

2-4. Effect of extraction temperature and extraction time on the increase of useful components of cells

From Examples 2-1 to 2-3, it was confirmed that the contents of useful components were generated differently according to the type of the eliminator even under the same plant group cells and the same culture conditions. The use of methyl jasmonate as an elisitator for the capillary cells increased the content of rubiadine the most. Hereinafter, the effect of extraction temperature and extraction time on the increase of useful components of cells was investigated.

After 10 days of culture, the concentration of methyl jasmonate was treated at concentrations of 50, 100, 200, 300, and 400 μM, respectively, and the culture conditions of the bioreactor were carried out in the same manner as in the above example. Rubiadine extraction temperature was set to 25 ℃, 40 ℃, 60 ℃, 80 ℃, the extraction time was set to 1, 2, 4 hours. Table 4 shows the effects of the extraction temperature and the extraction time on the rubidin content.

Extraction time
(hour) Methyl jasmonate
Concentration (μM) Rubiadine content (mg / g DW) 25 ℃ extraction group 40 ℃ extraction group 60 ℃ extraction group 80 ℃ extraction group One Control (0) 0.053 0.109 0.156 0.157 50 0.101 0.278 0.267 0.191 100 0.113 0.356 0.345 0.275 200 0.114 0.386 0.362 0.345 300 0.123 0.389 0.393 0.189 400 0.108 0.359 0.369 0.165 2 Control (0) 0.048 0.142 0.165 0.157 50 0.094 0.269 0.352 0.198 100 0.107 0.319 0.359 0.278 200 0.116 0.350 0.372 0.349 300 0.124 0.512 0.383 0.188 400 0.110 0.486 0.372 0.163 4 Control (0) 0.049 0.155 0.188 0.161 50 0.095 0.286 0.380 0.211 100 0.108 0.335 0.433 0.310 200 0.117 0.353 0.525 0.356 300 0.126 0.515 0.569 0.200 400 0.115 0.487 0.528 0.160

As shown in Table 4, when extracting the methyl jasmonate 300 μM treatment group at 60 ° C. for 4 hours, the rubiadine content was 0.569 mg / g, showing the highest useful ingredient content. When treating 400 μM of methyl jasmonate, it showed a lower rubidine content than when treating 300 μM, and at 80 ° C, the highest extraction temperature, it had a lower rubidine content than when extracting at 60 ° C.

Example 3. Investigation of the effect of useful components of the present invention on cells

MTT tetrazolium is a yellow water-soluble solution that utilizes the principle that when treated with cells, the ring structure of tetrazolium is broken by dehydrogenase in the mitochondria of cells whose metabolic processes are intact, resulting in a blue-violet, insoluble MTT formazan crystal. It is an experimental method that is mainly used to evaluate the survival rate of cells in a sample. Cell viability measurement according to the present invention was evaluated by measuring MTT formazan as absorbance.

Human keratinocytes (HaCaT) in 10% fetal calf serum (FBS, Gibco, USA) and 1% penicillin in order to evaluate the effect of useful components of the modified wood cells obtained in Examples 1 and 2 on animal cells -Cultured in Dulbecco Modified Eagle Medium (DMEM, Welgene, Korea) to which streptomycin (penicillin-streptomycin, Gibco, USA) was added. Human keratinocytes (HaCaT) were respectively dispensed into 1Ⅹ10 ⁴ cells / well in a 96-well plate and cultured at 37 ° C and 5% CO ₂ for 24 hours. Thereafter, useful components obtained in Examples 1 and 2 were diluted in DMEM medium, added to human keratinocytes (HaCaT), and cultured at 37 ° C. and 5% CO ₂ conditions for an additional 24 hours. After incubation, the MTT solution was added to each well to have a concentration of 0.5 mg / mL. After 4 hours, the formed MTT formazan was dissolved with 100 μl of DMSO (Dimethyl sulfoxide), and absorbance (SpectraMax i3, Molecular devices, CA, USA) was measured at 420 nm. Cell viability was measured by Equation 2 below, and the results are shown in Table 5 below.

[Equation 2]

Cell activity (%) = (absorbance of the sample-treated group / absorbance of the sample-free group) X 100

Cell viability (%) Useful ingredient concentration
(mg / ml) Useful ingredients from untreated cells Useful ingredients from salicylic acid treated cells Useful ingredients from yeast extract-treated cells Useful ingredients from methyl jasmonate treated cells 10 81.84 ± 0.24 82.94 ± 3.32 83.62 ± 1.33 87.19 ± 0.12 5 84.92 ± 0.33 87.53 ± 7.44 88.76 ± 4.56 89.43 ± 1.33 2.5 87.19 ± 1.22 88.19 ± 3.12 90.76 ± 0.22 90.55 ± 0.42 1.25 88.61 ± 2.11 90.51 ± 0.32 91.51 ± 2.23 92.51 ± 1.23 0.625 91.37 ± 5.44 92.89 ± 5.44 92.62 ± 1.76 95.51 ± 1.76

As a result, as shown in Table 5, all of the useful components extracted from fertilized cells treated with salicylic acid, yeast extract, or methyl jasmonate with or without elisitator did not affect the viability of animal cells. Confirmed.

Example 4. Investigation of the effect of useful components of the present invention on whitening

Tyrosinase (Tyrosinase) acts as a dopa oxidase that produces dopachrome by oxidizing tyrosine in the process of melanin synthesis that occurs in melanosomes and oxidizing dopa again. Therefore, the whitening function can be evaluated by measuring the activity inhibitory ability of tyrosinase. The whitening efficacy of the extract according to the present invention was evaluated by measuring the activity inhibitory ability of Tyrosinase.

First, 0.1M sodium phosphate buffer (pH 6.5) was prepared, and then 1.5mM L-tyrosine and 1000U / ml tyrosinase were prepared with the solution to prepare reagents necessary for the experiment. To each well of the 96 well plate, 170 μL of useful components obtained in Examples 1 and 2 were added, and then 10 μL of Tyrosinase (1000 U / ml) was added, followed by reaction at 37 ° C. for 10 minutes. Thereafter, 20 μL of 1.5 mM L-tyrosine was added, reacted at 37 ° C. for 10 minutes, and absorbance (SpectraMax i3, Molecular devices, CA, USA) was measured at 490 nm. Tyrosinase activity inhibitory ability was measured by the following equation (3), and the results are shown in Table 6 below.

[Equation 3]

Tyrosinase activity inhibition (%) = (1-absorbance of the sample-treated group / absorbance of the sample-untreated group) X 100

Tyrosinase inhibition activity (%) Useful ingredient concentration
(mg / ml) Useful ingredients from untreated cells Useful ingredients from salicylic acid treated cells Useful ingredients from yeast extract-treated cells Useful ingredients from methyl jasmonate treated cells 10 52.43 ± 0.09 63.84 ± 2.13 62.84 ± 0.09 72.89 ± 1.59 5 29.91 ± 2.02 50.81 ± 0.01 48.57 ± 0.68 54.19 ± 0.29 2.5 21.39 ± 0.68 38.25 ± 5.77 33.11 ± 1.24 40.57 ± 2.12 1.25 9.87 ± 0.29 17.32 ± 2.13 12.19 ± 0.91 20.32 ± 1.56 0.625 4.51 ± 2.13 8.23 ± 0.29 7.11 ± 0.13 10.11 ± 0.19

As a result, as shown in Table 6, all of the useful components extracted from fertilized cells treated with salicylic acid, yeast extract, or methylzasmonate with or without elicitor were excellent in inhibiting tyrosinase activity, but methylzasmo The tyrosinase activity inhibitory ability of the experimental group treated with the useful component extracted from the nate-treated cells was measured to be remarkably high, confirming that the whitening effect was the best.

As a result, even in the case of the normal crystal tree extract without treatment with the eliminator, it was confirmed that the extract of the crystal tree cells cultured by treating the eliminator has better whitening effect, and methyl Jasmonate as an eliminator. When using it was confirmed that the whitening efficacy of the most crystal tree extract is remarkably excellent.

Since the specific parts of the present invention have been described in detail above, it is clear that for those skilled in the art, these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (20)

(a) proliferating the cells from sections of a tree plant (Damnacanthus major); And
(B) a step of treating the cells with the eliminator; containing rubiadin (rubiadin) culture method of culturing the lens of the modified wood containing,
The eliminator is any one or more selected from the group consisting of methyl-jasmonate, salicylic acid, and yeast extract,
When methyl jasmonate is selected as the eliminator, the concentration of the eliminator treatment is 300 μM or more.
According to claim 1,
The section is taken from the root, stem, or leaf portion of a crystal tree plant.
delete delete According to claim 1,
The method, wherein the rubiadine is represented by the following formula (1).
[Formula 1]

Crystalline cell with increased rubiadin content cultured by the method of claim 1.
delete delete delete (a) proliferating the cells from sections of a tree plant (Damnacanthus major);
(b) treating the cells with an eliminator and further culturing them; And
(C) drying the cells, and processing the alcohol; comprising, rubiadin (rubiadin) method of extracting from the cells.
The method of claim 10,
The section is taken from the root, stem, or leaf portion of a crystal tree plant.
The method of claim 10,
The eliminator is any one or more selected from the group consisting of methyl-jasmonate, salicylic acid, and yeast extract.
The method of claim 10,
The eliminator is treated with a concentration of 10 to 990 μM, method.
The method of claim 10,
The method, wherein the rubiadine is represented by the following formula (1).
[Formula 1]

The method of claim 10,
The treatment of the alcohol is carried out at 25 ℃ to 80 ℃, method.
The method of claim 10,
The treatment of alcohol is to be carried out for 1 to 4 hours.
delete delete delete delete

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