KR102234296B1 - COMPOSITION FOR SKIN WHITENING CONTAINING CALLUS EXTRACT FROM Pinellia ternata AND ITS CULTURE MEDIUM - Google Patents

Abstract

The present invention relates to a composition for skin whitening comprising a Banha-derived callus extract and a culture solution thereof as an active ingredient.
Accordingly, as callus derived from Banha is used, it is not toxic when used in the human body, so high stability can be ensured. In addition, it has excellent effects on skin whitening activity, such as inhibiting melanin formation, and can be applied to cosmetics such as lotions, water-soluble liquids, creams, gels, essences, and emulsions. In addition, since raw materials are obtained through plant cell culture rather than collecting plants, the productivity and efficacy of raw materials can be maintained.

Description

Skin whitening composition containing callus extract derived from Banha and its culture as active ingredients {COMPOSITION FOR SKIN WHITENING CONTAINING CALLUS EXTRACT FROM Pinellia ternata AND ITS CULTURE MEDIUM}

The present invention relates to a composition for skin whitening comprising a Banha-derived callus extract and a culture thereof as an active ingredient, and more particularly, to a skin using an extract of a callus derived from Pinellia ternata, a perennial herbaceous plant, or a culture solution thereof. It relates to a composition for skin whitening that can improve the whitening condition of.

The skin has the function of primarily protecting the body from external physical and chemical stimuli. Melanin in the skin is a biopolymer of phenols widely distributed in nature, and is a complex of black pigment and protein. Melanin is known as a substance that protects the body from external environments such as ultraviolet rays, and increases the viability and competitiveness of cells against the environment. On the other hand, it is reported that overproduction of melanin produces spots, freckles, and age spots, promotes skin aging, and is also related to skin cancer induction.

Pigmentation, which is closely related to skin whitening, occurs in melanocytes in the base layer of the skin's epidermis. Pigmentation is known to be greatly affected by the activity of tyrosinase, and hyperpigmentation is induced mainly by ultraviolet rays, inflammation, DNA damage, and free radicals.

As the interest and demand for beauty increased, various methods for managing the skin, which are at the center of beauty, have emerged. Especially, since Korea has a high preference for white and clean skin, whitening, anti-aging, anti-wrinkle, UV protection, etc. The demand for functional cosmetics with various functions has increased. In line with this demand, various types of cosmetics were released in a rainy order, and sales volume was recorded at a high level.

However, as the cosmetics market grows, competition is excessive and in the process of increasing price competitiveness, there are cases where substances included in cosmetics are genetically modified substances or contain environmental hormones. Accordingly, expectations for functional cosmetics mainly containing extracts of natural substances that are not harmful to the human body and can be safely used in the long term are increasing (see Patent Document 1).

However, plant-derived useful ingredients directly extracted from natural products have a high advantage in that their stability is guaranteed when used in the human body, but the yield of extract is low, and various environmental conditions such as season, place, climate, etc. There is a disadvantage in that it is difficult to supply stable raw materials because there may be differences in productivity and efficacy depending on factors.

KR 10-1997-0005272 A1 (1997. 02. 19.)

The present invention has been conceived to solve the above problems, and the problem to be solved in the present invention is to provide a composition capable of improving skin whitening by using a material extracted from a plant having high stability to the human body, the composition It is to provide a composition that can maintain the uniformity of the supply and quality of the product.

In order to solve the above problems, the composition for skin whitening comprising the callus extract derived from Banha and its culture medium as an active ingredient according to the present invention uses the callus extract cultured by the cell tissue of the plant called Banha and the callus culture solution, respectively or together. It is a technical feature to provide a composition capable of improving skin whitening.

The composition for skin whitening comprising the Banha-derived callus extract and the culture medium thereof according to the present invention as an active ingredient is not toxic when used in the human body as the Banha-derived callus is used, and thus high stability can be ensured.

In addition, the composition for skin whitening comprising the Banha-derived callus extract and its culture medium according to the present invention as an active ingredient has excellent effects on skin whitening activity such as inhibiting melanin formation, and it is a lotion, water-soluble liquid, cream, gel, essence. , It can be applied to cosmetics such as emulsions.

In addition, the composition for skin whitening comprising the Banha-derived callus extract and its culture medium according to the present invention as an active ingredient does not collect plants, but acquires raw materials through plant cell cultivation, so that the productivity and efficacy of the raw materials can be maintained. have.

1 is a graph showing the relationship between the melanin synthesis rate of Banha-derived callus extract and B16F10 cells
Figure 2 is a graph showing the effect of Banha-derived callus extract on the survival rate of B16F10 cells
3 is a graph showing the relationship between the melanin synthesis rate of Banha-derived callus extract and SK-MEL-5 cells
Figure 4 is a graph showing the effect of Banha-derived callus extract on the survival rate of SK-MEL-5 cells
Figure 5 is a graph showing the relationship between the melanin synthesis rate of B16F10 cells and Banha-derived callus culture
Figure 6 is a graph showing the effect of the callus culture medium derived from Banha on the survival rate of B16F10 cells
7 is a photograph taken through a Leica inverted microscope after processing a zebrafish embryo by concentration of callus extract derived from Banha
Figure 8 is a graph showing the relationship between the melanin synthesis rate of the callus extract derived from Banha and zebrafish embryos

Terms and words used in the present specification and claims conform to the technical idea of the present invention based on the principle that "the inventor can appropriately define the concept of terms in order to describe his or her invention in the best way." It should be construed as a meaning and concept, but not limited to a conventional or dictionary meaning.

Therefore, the embodiments described in the present specification and the configurations shown in the drawings are only the most preferred embodiments of the present invention, and do not represent all the technical spirit of the present invention, and thus various equivalents that can replace them at the time of application It should be understood that there may be water and variations.

In the present invention, "extract" refers to a callus itself derived from Banha (Pinellia ternata ), or refers to an extract obtained by various extraction methods known conventionally, such as hot water extraction method and alcohol extract by powdering callus.

Suitable as polar solvents are (i) water, (ii) alcohols having 1 to 6 carbon atoms (preferably methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal-butanol, 1-pentanol, 2-butoxyethanol or ethylene glycol), (iii) acetic acid, (iv) dimethyl-formamide (DMFO) and (v) dimethyl sulfoxide (DMSO).

Suitable non-polar solvents include acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1- Pentene, 1-chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene, diethyl ether, diethyl sulfide, chloroform, dichloro Methane, 1,2-dichloroethane, aniline, diethylamine, ether, carbon tetrachloride, methylene chloride, petroleum ether, and THF.

In the present invention, the extraction method is not particularly limited to one method, and various methods of extraction may be used. For example, cold needle extraction, ultrasonic extraction, reflux extraction, hot water extraction, and the like can be used. Specifically, in the case of hot water extraction, the hot water extract is obtained by heating at a temperature of 80 to 100°C for 8 to 48 hours in a hot water distiller. In the case of cold water extraction, a cold water extract is obtained by mixing the callus culture itself or its dried powder with cold water (15~25℃) and then extracting for 3 days. In addition, it may be prepared by extraction using water, an organic solvent, or a mixed solvent thereof. The extracted liquid can be used immediately or concentrated and/or dried after filtering.

The cosmetic composition of the present invention is in the form of an aqueous, aqueous-alcoholic or oily solution, oil-in-water or distilled water or multiple emulsion, aqueous or oily gel, liquid, pasty or solid anhydrous product, an oil dispersion in an aqueous phase using globules. May be, and may be in the form of an ionic and/or nonionic form of lipid endoplasmic reticulum.

The cosmetic composition of the present invention may be a fluid, and specifically, may be in the form of a white or colored cream, ointment, milk lotion, serum, essence, paste, or mousse. It may optionally be applied to the skin in the form of an aerosol, or it may be a solid such as a stick. It can also be used as a skin care product and/or a makeup product.

The composition according to the invention may also contain adjuvants customary in the cosmetic field, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic activators, preservatives, antioxidants, solvents, fragrances, fillers, blockers, pigments, odorants and dyes. I can. The amounts of these various adjuvants are those commonly used in the art. Depending on their nature, these adjuvants can be introduced into the fatty phase, aqueous phase, lipid vesicles and/or nanoparticles.

In addition, the composition according to the present invention may be composed of external preparations such as ointments and creams as therapeutic agents.

The present invention can provide a composition having skin whitening effect using callus derived from Banja, and in particular, callus obtained through plant cell culture rather than using wild or cultivated plants is used.

Plant cell culture can be supplied constantly under stable conditions without being affected by local or seasonal environmental influences, and production efficiency can be increased by promoting the generation of specific useful ingredients through control of the culture conditions. In addition, it has the advantage of securing the types of useful ingredients and plant cells with high productivity through continuous cell selection.

The plant tissue of the origin of the callus used in the present invention is derived from the leaves of the half. The extract of the present invention refers to a culture medium thereof after culturing and extracting callus from half a leaf.

Example 1. Acquisition of callus through tissue culture of half-bottom plant section

Section samples for each half of the tissue were cultured. About 2 weeks after the initiation of culture, yellow callus began to be formed as swelling was made on the cut surface, and about 4 weeks after the initiation of culture, the size of the structure reached about 1 mm as the size of the structure was elongated along with the growth of yellow callus.

On the other hand, the sample was cultured for about 4 weeks to find out the effect of the plant growth regulator on the establishment of the tissue culture system of the section samples for each half of the tissue. As a result of investigating the efficiency of callus formation from the cultured half-bottom section samples, the efficiency of forming callus derived from half-bottom root fragments was the highest in the treatment group in which 1 mg/L 2,4-D was added to the MS base medium. About 6 weeks after cultivation, passages were preserved with new media of the same composition, and an artificial cell line of Banha-derived callus was secured by repeating the passages every 4 weeks.

An extract was prepared using the obtained Banha-derived callus. In this example, n-hexane extract, methanol extract, ethanol extract, and hot water extract were prepared. Specifically, a hot water extract was prepared by reflux extraction of the cultured Banha-derived callus powder and water at a temperature of 80° C. in a ratio of 1:1 to 1:10 for 3 hours. Further, the cultured Banha-derived callus dry powder and n-hexane, methanol, and ethanol solvent were immersed in a ratio of 1:1 to 1:10, followed by extraction and concentration at room temperature for 24 hours. And by removing the solvent, n-hexane extract, methanol extract, and ethanol extract were prepared.

Example 2. Banha-derived callus suspension culture

1 mg/L 2,4-D{(2, 4-dichlorophenoxy) acetic acid} was added to 20 mL of MS liquid medium added to a 250 mL erlenmeyer flask, and about 1 grown in Example 1 g of the callus derived from half a half was transferred to the medium.

Suspension culture was performed in dark culture at 25 °C in a gyratory shaker (100 rpm). After incubation for about 2 weeks, 20 mL of a new liquid medium was added. After culturing for about 4 weeks thereafter, the suspension cultured cells of Banha-derived callus and 5 mL of the culture medium were transferred to another Erlenmeyer flask with 20 mL of fresh liquid medium to perform subculture. The new liquid medium is MS liquid medium with 1 mg/L 2,4-D added. And subculture was carried out at about 4 weeks intervals.

Suspension culture like this is an initial step for in-flight mass production and is a work to establish a suspension culture system.

Hereinafter, it is proved whether the composition according to the present invention has a whitening effect through an experimental example. Test target cells to be used in Experimental Examples 1 to 3 are B16F10 mouse melanoma cells derived from mice (hereinafter referred to as “B16F10 cells”) and SK-MEL-5 human melanoma cells derived from humans (hereinafter “SK-MEL- 5 cells”). Each experimental example was carried out focusing on the cell viability and melanin synthesis inhibitory activity.

Experimental Example 1. Test for whitening effect in B16F10 cells of Banha-derived callus extract 1

1-1. Preparation for experiment (cultivation and quantification of B16F10 cells)

B16F10 cells used in the present invention were cultured in RPMI-1640 medium containing 10% fetal bovine serum (hereinafter referred to as “FBS”). Specifically, the melanoma cell line was placed in a 175 cm2 plastic flask (SPL life science Co., Ltd. Korea) at 37°C, 5% in RPMI-1640 medium containing 10% FBS and 1% antibiotic/antimycotics. It was cultured under the condition of CO₂. Cell lines were maintained by secondary culture once every 2-3 days.

Then, the medium was removed from the 175 cm2 plastic flask in which melanoma cells were grown, washed with CMF-PBS (calcium magnesium free-phosphate buffered saline, pH 7.2), and then treated with 0.25% trypsin/EDTA to remove the cells from the bottom of the flask. After removal, the cells were neutralized with a cell culture solution and then centrifuged (1000 rpm, 3 min). A culture solution was added to the remaining cell pellet, and then a single cell suspension was prepared by repeatedly aspirating with a sterile pipette. The prepared cell suspension and trypan blue were mixed at a ratio of 1:1, and cell quantification was performed using a hemocytometer on an optical microscope.

1-2. Experimental process

The B16F10 cells prepared in 1-1 ^{were suspended in RPMI-1640 medium containing 5% FBS at a concentration of 1×10 3} cells/well, placed in a 96-well cell culture plate, and placed for 1 day to plate the cells. Attach to. Samples (Example 1) at concentrations of 100, 50, 25, 12.5, and 6.25 ug/mL were added to the adhered cells, respectively, and after 24 hours, α-MSH (melanin stimulating hormone) was treated, and then cultured again for 4 days. After cultured B16F10 cells were washed with phosphate buffered saline (hereinafter referred to as “PBS”), dissolved in 1N NaOH solution, and then measured the absorbance at 490 nm with a microplate reader. The rate of inhibition of melanogenesis was determined. Cells treated with only pure DMSO were treated with α-MSH to induce melanin synthesis as a control group, calculated according to Equation 1, and expressed as a percentage.

1-3. Experiment result

As shown in Figure 1, Banha-derived callus extract inhibited melanin synthesis in B16F10 cells. When the Banha-derived callus extract was at a concentration of 29.8 ug/mL, it _{was confirmed that the melanin synthesis rate was reduced to 50% (IC 50} ), and as shown in FIG. 2, the Banha-derived callus extract was at a concentration of 50 ug/mL or less. It was confirmed that there was no toxicity.

Experimental Example 2. Experiment to confirm the whitening effect in SK-MEL-5 cells of Banha-derived callus extract

2-1. Preparation for experiment

Prepared in the same manner as in Experimental Example 1-1, but SK-MEL-5 cells were prepared instead of B16F10 cells.

2-2. Experimental process

The SK-MEL-5 cells prepared in 2-1 were suspended in RPMI-1640 medium containing 10% FBS at a concentration of 1×10 ^{3 cells/well and placed in a 96-well cell culture plate for 1 day.} To attach the cells to the plate. Samples (Example 1) at concentrations of 100, 50, 25, 12.5, and 6.25 ug/mL were added to the attached cells, respectively, and after 24 hours, α-MSH was treated and then cultured for 4 days again. After the cultured SK-MEL-5 cells were washed with PBS, 1N NaOH solution was added to dissolve the cells, and the absorbance was measured at 490 nm with a microplate reader to determine the melanin synthesis inhibition rate. Cells treated with only pure DMSO were treated with α-MSH to induce melanin synthesis as a control group, calculated according to Equation 1, and expressed as a percentage.

2-3. Experiment result

As shown in Figure 3, Banha-derived callus extract inhibited melanin synthesis in SK-MLE-5 cells. When the Banha-derived callus extract was at a concentration of 30.3 ug/mL, it _{was confirmed that the melanin synthesis rate was reduced to 50% (IC 50} ), and as shown in FIG. 4, the Banha-derived callus extract was at a concentration of 50 ug/mL or less. It was confirmed that there was no toxicity.

Experimental Example 3. Checking the whitening effect in B16F10 cells of Banha-derived callus culture solution Experiment 2

3-1. Preparation for experiment

B16F10 cells were prepared in the same manner as in Experimental Example 1-1.

3-2. Experimental process

The B16F10 cells prepared in 3-1 ^{were suspended in RPMI-1640 medium containing 5% FBS at a concentration of 1×10 3} cells/well, placed in a 96-well cell culture plate, and placed for 1 day to plate the cells. Attach to. A culture solution (Example 2) at concentrations of 0.5, 0.2, 0.1, 0.05, 0.01, 0.001, 0.0001, 0.00001, 0.000001, and 0.0000001 uL/uL was added to the attached cells, respectively, and the melanin synthesis inhibition rate was calculated after 24 hours. Cells treated with only pure DMSO were treated with α-MSH to induce melanin synthesis as a control group, and calculated according to the following equation and expressed as a percentage.

3-3. Experiment result

As shown in Figure 5, the callus culture medium derived from Banha inhibited melanin synthesis in B16F10 cells. _{It was confirmed that the melanin synthesis rate was reduced to 50% (IC 50} ) when the Banha-derived callus culture medium was at a dilution of 6/1,000,000, and there was no cytotoxicity at a concentration diluted to 1/10 or less as shown in FIG. Confirmed.

Experimental Example 4. Confirmation of whitening effect in zebrafish embryo of Banha-derived callus extract

4-1. Preparation for experiment

After breeding zebrafish and inducing spawning by turning on the light in the morning, the embryos discharged during spawning are collected and prepared.

4-2. Experimental process

Add 12-15 embryos to each well of a 6-well plate. Banha-derived callus extract was administered at a concentration of 100, 200, 400 μg/mL from 9 hpf (postfertilization time, postfertilization) to 72 hpf (63 hours), and then photographed through a Leica inverted microscope to obtain an image. The images were analyzed through mage J to quantify the melanin formed, and each whitening effect was compared with the control group (DMSO).

4-3. Experiment result

The results are shown in FIG. 7. That is, it was confirmed that melanin formation decreased by 28% and 12%, respectively, in the group treated with the callus extract derived from Banha at 400 and 200 μg/mL, as shown in FIG. 8.

As described, the composition according to the present invention has a skin whitening function by inhibiting the activity of melanin, and can be used to prevent or treat skin pigmentation caused by spots, age spots, acne, freckles, and birthmarks.

Claims (8)

Banha ( Pinellia ternata )-derived callus extract is contained as an active ingredient, but the extract is contained in 30 ug/mL ~ 50 ug/mL based on ^{B16F10 cells 1X10 3 cells/well, for improving skin whitening} Cosmetic composition.
Banha ( Pinellia ternata ) derived callus (callus) culture medium containing as an active ingredient, but B16F10 cells 1X10 ³ cells / well based on the culture medium containing 6/1,000,000 ~ 1/10 dilution concentration for skin whitening improvement Cosmetic composition.
The method according to claim 1 or 2,
The Banha-derived callus is a cosmetic composition for improving skin whitening, characterized in that it is derived from Banha leaf.
The method according to claim 1 or 2,
The skin whitening improvement cosmetic composition for improving skin whitening, characterized in that the improvement by inhibiting the synthesis of melanin.
Banha ( Pinellia ternata )-derived callus extract is contained as an active ingredient, but the extract is contained at 30 ug/mL to 50 ug/mL based on ^{B16F10 cells 1X10 3 cells/well.} Or a therapeutic composition.
Banha ( Pinellia ternata )-derived callus culture solution is contained as an active ingredient, but the culture solution is contained in a dilution concentration of 6/1,000,000 ~ 1/10 based on ^{B16F10 cells 1X10 3 cells/well.} Or a therapeutic composition.
The method according to claim 5 or 6,
The Banha-derived callus is a composition for preventing or treating skin pigmentation, characterized in that it is derived from Banha leaves.
The method according to claim 5 or 6,
The skin pigmentation is a composition for preventing or treating skin pigmentation, characterized in that due to spots, age spots, acne, freckles, and birthmarks.

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