KR102050650B1 - Composition for preventing or treating migratory skin cell comprising Spatholobi Caulis extract - Google Patents

Abstract

The present invention relates to a composition for preventing or treating mobile skin cell diseases, including extracts such as cinnamon as an active ingredient. Extracts such as cinnamon according to the present invention has the activity of proliferating melanoma cells and inhibiting the survival rate of melanoma cells. In addition, by promoting the expression of tyrosinase of melanocytes, there is an activity to promote the production of melanin of the skin can be usefully used as a composition that can prevent or treat mobile skin disease.
In addition, there is no cytotoxicity, there is no toxicity and side effects for the drug can be used safely even when taking long-term, there is a stable effect in the body.

Description

Composition for preventing or treating migratory skin cell comprising Spatholobi Caulis extract

The present invention relates to a composition for preventing or treating mobile skin cell disease, including the extract such as cinnamon as an active ingredient.

Vitiligo, albinoosis, and hair whitening are caused by a variety of causes, including the death of melanocytes by macrophages and stress, the lack of innate melanocytes or melanin synthesis-inducing proteins, and aging. In Korea, according to the craze of whitening cosmetics, various natural materials have been discovered that have whitening effects, but the discovery of materials that promote melanin synthesis is slow. As aging society enters aging society and life expectancy increases, interest in anti-aging is increasing. Of these, dyeing is mainly used to blacken bleaching of hair, but it requires toxicity, time consuming and periodic dyeing of dyes. Inconvenience of use is great.

Therefore, the discovery of materials that can prevent and treat whitening of hair is a socially important point. Melanoma, which is a cancer of melanocytes, has a low incidence in Korea, but the incidence of melanoma increases as ultraviolet rays become stronger due to global warming and climate change. Melanoma can occur not only on exposed skin, but also on all areas of melanocytes such as the inside of the eye, mouth, rectum, and brain, and is characterized by easy metastasis and accounts for less than 5% of all skin cancers diagnosed in the United States. However, mortality is the most dangerous disease (Merk, MSD manual).

Therefore, in order to promote melanin synthesis and combat melanoma outbreaks, it is necessary to discover natural materials to treat them, but the research is still insufficient.

1. Korean Patent Publication No. 10-2009-0052652

An object of the present invention to provide a pharmaceutical composition for the treatment or prevention of mobile skin cell disease comprising an extract such as cinnamon as an active ingredient.

Still another object of the present invention is to provide a cosmetic composition for preventing or improving mobile skin disease comprising a cinnamon extract as an active ingredient.

Still another object of the present invention is to provide a skin external preparation for preventing or improving mobile skin cell disease, including an extract such as cinnamon as an active ingredient.

Furthermore, it is an object of the present invention to provide a health functional food for preventing or improving mobile skin disease comprising the extract such as cinnamon as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for treating or preventing mobile skin cell disease, including the extract such as cinnamon as an active ingredient.

In one embodiment of the present invention, the cinnamon and the like extract may be extracted with any one or more solvents selected from the group consisting of ethanol, methanol, distilled water and mixtures thereof.

In one embodiment of the present invention, the mobile skin cell disease may be one or more diseases selected from the group consisting of melanoma, skin pigmentation, skin pigmentation, vitiligo and leukemia.

In one embodiment of the present invention, the composition may promote the melanin production of the skin by promoting the expression of tyrosinase of melanocytes.

In one embodiment of the invention, the composition can inhibit the proliferation and survival of melanoma cells.

In addition, the present invention provides a cosmetic composition for preventing or improving mobile skin disease comprising a cinnamon extract as an active ingredient.

In one embodiment of the present invention, the mobile skin cell disease may be one or more diseases selected from the group consisting of melanoma, skin pigmentation, skin pigmentation, vitiligo and leukemia.

In addition, the present invention provides a skin external preparation for preventing or improving mobile skin disease comprising a cinnamon extract as an active ingredient.

In one embodiment of the present invention, the mobile skin cell disease may be one or more diseases selected from the group consisting of melanoma, skin pigmentation, skin pigmentation, vitiligo and leukemia.

Furthermore, the present invention provides a health functional food for preventing or improving mobile skin disease comprising the extract of cinnamon as an active ingredient.

In one embodiment of the present invention, the mobile skin cell disease may be one or more diseases selected from the group consisting of melanoma, skin pigmentation, skin pigmentation, vitiligo and leukemia.

Extracts such as cinnamon according to the present invention is active to inhibit the proliferation and survival of melanoma cells. In addition, by promoting the expression of tyrosinase of melanocytes, there is an activity to promote the production of melanin of the skin can be usefully used as a composition that can prevent or treat mobile skin disease.

In addition, there is no cytotoxicity, there is no toxicity and side effects for the drug can be used safely even when taking long-term, there is a stable effect in the body.

Figure 1 confirms the antioxidant efficacy of extracts such as cinnamon. (A: Flowcytometric profiles of B16F10 melanoma cells and NIH3T3 fibroblasts. B: Antioxidant efficacy of extracts such as cinnamon)
Figure 2 confirms the anticancer efficacy of B16F10 melanoma cells of the extract such as cinnamon. (A: Cell growth curve of C16 blood extracts treated with B16F10 melanoma cells. B: Cell survival rate of B16F10 melanoma cells treated with extracts such as cinnamon)
Figure 3 confirms the tyrosinase activity and melanin synthesis enhancement effect of the extract such as cinnamon. (A, B: Tyrosinase activity assay in gel, cinnamon, etc. B16F10 melanoma cells in the treatment of the extract, the effect of increasing the tyrosinase activity.
Figure 4 confirms the mRNA expression control efficacy of extracts such as cinnamon. (A: confirming the changes in the expression of tyrosinase, TRP2, Bax mRNA of B16F10 melanoma cells when treated with extracts such as cinnamon through qPCR)

The skin is largely composed of keratinocytes cells, which actually make up the skin, and melanocytes (melanocytes) that produce melanocytes in relation to skin color. In contrast to the identity of keratinocytes, melanocytes are representative mobile skin cells, and abnormal increase or decrease in their mobility causes various forms of disease, collectively referred to as mobile skin disease.

The research on mobile skin cell disease is much more active in Korea than in Korea because of much more serious social needs than in Korea. Indeed, in 2002, in Europe, about 60,000 new melanoma patients were diagnosed and 17,000 died. In 2004, nearly 60,000 new cases were diagnosed in the United States, and nearly 7,000 were diagnosed. Is expected to die.

The mobility of all cells depends on their migration potential and extracellular matrix remodeling ability, which is signaled by adhesion of the cell adhesion receptor and extracellular matrix on the cell surface. It is tightly controlled. This failure to regulate cell mobility in vivo is expressed in a variety of diseases, for example, the decrease in cell mobility of melanocytes by changes in extracellular matrix with aging causes cell aging and pigmentation, while melanin Abnormal decrease in cell mobility causes vitiligo and abnormal increase in melanocytes causes malignant melanoma.

Vitiligo is an acquired depigmentation disease in which white spots of various sizes and shapes appear on the skin due to a lack of melanocytes (Le Poole IC, Boissy RE, Sarangarajan R, Chen J, Forristal JJ, Sheth P, Westerhof W, Babcock G, Das PK, Saelinger CB.In Vitro Cell Dev Biol Anim. 2000; 36 (5): 309-19; Yu HS.J Biomed Sci. 2002; 9 (6): 564-73.), The most common of skin diseases. Representative disease. Vitiligo is present in about 1% of the population (Whitton ME, Ashcroft DM, Barrett CW, Gonzalez U. Cochrane Database Syst Rev. 2006; 1: CD003263), and 400,000 people in Korea suffer from vitiligo. Nevertheless, vitiligo has not yet been found to be a satisfactory therapeutic effect and has been recognized as an extremely refractory disease.

Melanocytes, the main cause of vitiligo, are cells in the lower part of the epidermis. One is every 10 keratinocytes. Under normal conditions, melanocytes play a role in making and delivering pigments to keratinocytes. Deficiency causes depigmentation in various tissues including skin.

The cause of vitiligo is still unknown, but the cause of the disease has been explained to date. It is known that the immune system is caused by autoimmunity and the excess of the chemical mediators that are released near melanocytes. It is a phenol complex, which is the humoral theory that leads to the production of melanin, and melanocyte self-destruction, which accumulates in melanocytes and destroys melanocytes (Sanlı H, Akay BN, Arat M, KoP, Akan). H, Beksac M, Ilhan O. Dermatology. 2008; 216 (4): 349-354; Machado Filho CD, Almeida FA, Proto RS, Landman G. Vitiligo: Sao Paulo Med J. 2005; 123 (4): 187- 91; Schallreuter KU, Bahadoran P, Picardo M, Slominski A, Elassiuty YE, Kemp EH, Giachino C, Liu JB, Luiten RM, Lambe T, Le Poole IC, Dammak I, Onay H, Zmijewski MA, Dell'Anna ML, Zeegers MP, Cornall RJ, Paus R, Ortonne JP, Westerhof W. Exp Dermatol. 2008; 1 7 (2): 139-40). There is also a genetic predisposition that melanocytes are more easily damaged in the presence of birth defects.

However, this theory is thought to cause vitiligo by inducing melanocyte deficiency by acting in combination rather than independently.

In order to effectively treat mobile skin cell disease, there is a need for a method capable of promoting the proliferation of acquired melanocytes and inducing proliferation of melanocytes proliferated to the lesion site of the disease.

In addition, the development of new materials with enhanced melanin synthesis and anti-cancer effect against melanoma is very necessary, but the research is still insufficient.

The present invention provides a composition for treating or preventing mobile skin cell disease, including the extract such as cinnamon as an active ingredient.

The present inventors paid attention to cinnamon, etc., while trying to develop a natural material with enhanced melanin synthesis and anti-cancer effect against melanoma. Cinnamon lamps are stems of soybeans and bushes. Say.

It is a commonly used herbal medicine in China. It is named after blood red-brown sap in the cross section. Mainly known for its effects on diseases such as menstrual pain, anemia, ischemia, hypertension, arteriosclerosis, etc., and is known to be effective in improving wrinkles, antioxidants, and inhibiting arthritis.

However, there is no mention in the prior art that the use of cinnamon or the like can be used for the prevention or treatment of mobile skin cell disease.

Therefore, the present invention first identified the fact that the extract such as cinnamon can be used for the prevention or treatment of mobile skin cell disease, in particular it was confirmed that it can inhibit the proliferation and survival rate of melanoma cells, tyrosine melanosite It was confirmed that the melanin production of the skin was promoted by promoting the expression and activity of cinases.

Therefore, the present inventors were able to know that the extract such as cinnamon of the present invention can prevent or treat mobile skin cell disease.

Spatholobi Caulis according to the present invention can be obtained by extraction and separation from nature using extraction and separation methods known in the art, 'extract' is defined in the present invention using an appropriate solvent By extracting from cinnamon, etc., for example, hot water extracts such as cinnamon, polar solvent soluble extract or non-polar solvent soluble extract may be included.

As a suitable solvent for extracting the extract from the cinnamon and the like, any solvent that is acceptable in the art may be used, and water or an organic solvent may be used. For example, alcohol having 1 to 4 carbon atoms, acetone, ether including purified water, methanol, ethanol, propanol, isopropanol, butanol, etc. , Solvents such as benzene, chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane may be used alone or in combination. It is not limited.

As the extraction method, any one of hot water extraction method, cold leaching extraction method, reflux cooling extraction method, solvent extraction method, steam distillation method, ultrasonic extraction method, elution method and compression method can be used. In addition, the desired extract may further be subjected to a conventional fractionation process, it may be purified using conventional purification methods. There is no limitation in the preparation method of the extract such as cinnamon of the present invention, any known method may be used.

For example, the cinnamon lamp extract included in the composition of the present invention may be prepared in a powder state by the additional process such as distillation under reduced pressure, freeze drying or spray drying, etc. have. The primary extract may be further purified using various chromatography such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography, and the like. You can also get

Therefore, in the present invention, the extract such as cinnamon is a concept including all the extracts, fractions and purified products obtained at each step of extraction, fractionation or purification, their dilutions, concentrates or dried products.

The composition of the present invention containing such an extract such as cinnamon may be a pharmaceutical composition.

The pharmaceutical composition of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant may include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, Lubricants, flavors and the like can be used.

The pharmaceutical composition may be preferably formulated into a pharmaceutical composition including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredient for administration.

Formulation forms of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, nontoxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders include, but are not limited to, natural and synthetic gums such as starch, gelatin, glucose or beta-lactose, corn sweeteners, acacia, trackercance or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like. Acceptable pharmaceutical carriers in compositions formulated in liquid solutions are sterile and physiologically compatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA can be formulated according to each disease or component, as appropriate in the art.

In one embodiment of the present invention, the cinnamon and the like extract of the present invention may be included in a concentration of 0.1ug / ml to 500ug / ml with respect to the total weight of the composition.

The composition of the present invention may also be a food composition, and in addition to containing an extract such as cinnamon, which is an active ingredient, such a food composition may contain various flavors or natural carbohydrates, and the like as an additional ingredient, as in general food compositions.

The composition of the present invention may also be a cosmetic composition, which may be prepared in the form of general emulsion formulations and solubilizing formulations. Cosmetics of the emulsified formulations include nutrient cosmetics, creams, essences, etc., and cosmetics of the solubilized formulations are flexible cosmetics. In addition to the cosmetic containing the extract such as cinnamon of the present invention by containing a dermatologically acceptable medium or base may be prepared in the form of adjuvants that can be applied topically or systemically applied commonly used in the field of dermatology.

Suitable cosmetic formulations include, for example, emulsions, suspensions, microemulsions, microcapsules, microgranules or ionics (liposomes), nonionics obtained by dispersing an oil phase in a solution, gel, solid or pasty anhydrous product, aqueous phase. It may be provided in the form of a vesicle dispersant, in the form of a cream, skin, lotion, powder, ointment, spray or conceal stick.

It may also be prepared in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

In addition to the peptides, genes or compounds, the cosmetic composition of the present invention is in addition to fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, interfaces Common to active agents, water, ionic or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or cosmetics It may contain adjuvants conventionally used in the cosmetic or dermatological field, such as any other ingredients used as. And the above ingredients may be introduced in amounts generally used in the field of dermatology.

Specific formulations of the cosmetic composition of the present invention include skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, essence, nutrition essence, pack, Formulations such as soaps, shampoos, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, emulsions, press powders, loose powders, eye shadows and the like.

Hereinafter, examples will be described in detail to help understand the present invention. However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

 <Example 1>

Experimental Materials and Methods

1. Manufacturing method of extract such as cinnamon

Put 30 g (Daemyung pharm, Seoul, Korea) and 300 mL of distilled water (DW) into a 1 L Erlenmeyer flask, and then autoclave (Wiseclave WAC-60 for 15 minutes at 110 ℃ and 0.4kg / cm ³ ). , Daihan Scientific, Wonju-si, Gangwon-do, Korea). Thereafter, the extract was filtered by using 3 ~ 5 μfilter paper (Quantitative Ashless, Hyundai Micro, Seoul, Korea) and 0.2 μsyringe filter (Minisart, Sartorius, Goettingen, Germany).

The extract was dried with Speed-Vac concentrator (Modul 408C, Hanil Science, Gimpo-si, Gyeonggi-do, Korea) at 65 ° C and 1,800 rpm, and then dissolved in 7 mL of cinnamon, etc. It was.

2. Cell Culture

Mouse B16F10 melanoma cells and mouse NIH3T3 fibroblasts were 10% (v / v) thermal-inactivated fetal bovine serum (Gemcell, GEMINI Bio-products, Woodland, Calif.) And 1% (v / v). CO ² incubator at 37 ° C. (Thermo Fisher Scientific) using Dulbecco's modified eagle's medium high glucose (DMEM, 4.5 g / L glucose; Corning, Corning, NY) with penicillin-streptomycin solution (Thermo Fisher Scientific, Waltham, Mass.) 95% air and 5% CO ² ).

3. Measurement of the amount of reactive oxygen species produced in cells

B16F10 melanoma cells and NIH3T3 fibroblasts (2 × 10 ⁵ cellseach) were incubated for 12 hours in a 60 mm cell culture dish (SPL Life Sciences, Pocheon-si, Gyeonggi-do, Korea). , 0.07 ng / mL) and vehicle (DW) were incubated for 12 hours.

Subsequently, 10 μfluorogenic dichlorofluorescein diacetate (H ₂ DCFDA, Invitrogen, Carlsbad, CA) was added, incubated for 30 minutes, treated with trypsin-EDTA, transferred to 1.5 mL tubes, and then 1 mL phosphate buffered saline (PBS, pH 7.4) Washing, resuspension in 400 μPBS and reactive oxygen species changes were measured with Guava EasyCyte (Millipore, Temecula, Calif.).

Median fluorescence intensity (MFI) was calculated by FlowJo Version 10.0.7.2 (TreeStar, Ashland, OR).

4. Cell Growth Curve

B16F10 melanoma cells (5 × 10 ⁴ cellseach) were incubated in a 60 mm cell culture dish for 12 hours, and then treated with cinnamon extract (700, 7, 0.07 ng / mL) and vehicle (DW) and incubated for 72 hours. After the first cinnabar lamp extract treatment, the medium was replaced at 24 hour intervals, and the cinnabar lamp extract and vehicle were retreated, and the cells were transferred to a 1.5 mL tube by trypsin-EDTA treatment, followed by 0.4% (w / v) trypan blue dye ( Thermo Fisher Scientific) was stained for 1 minute and the cell growth curve was measured by counting the number of cells with a hemocytometer.

5. Cell viability measurement

After incubating B16F10 melanoma cells (2 × 10 ³ cells / well 100 μm in a 96 well cell culture plate (SPL Life Sciences) for 12 hours, the cinnamon lamp extract (700, 7, 0.07 ng / mL) and vehicle (DW) were treated. After incubation for 24 hours, 10 μEz-cytox cell viability assay reagent (WST-1 assay kit, DoGenBio, Seoul, Korea) was added to each well and incubated for 2 hours, followed by microplate reader (Sunrise ™, Tecan, MaSwitzerland). The cell viability was calculated by measuring the absorbance at 450 nm.

6. Measurement of Tyrosinase Activity

B16F10 melanoma cells (1 × 10 ⁶ cellseach) were incubated for 12 hours in a 100 mm cell culture dish (SPL Life Sciences), and then treated with cinnamon lamp extract (700, 7, 0.07 ng / mL) and vehicle (DW). Incubated for 24 hours.

Subsequently, the cells were transferred to a 1.5 mL tube by treatment with Trypsin-EDTA, washed with 1 mL PBS, all supernatant was removed, and then 100 μLysis buffer (0.1 M sodium phosphate buffer, pH 6.8, 5 mM EDTA, 1% (V / V) triton X-100, 0.1M PMSF) was added and then resuspended, reacted at 4 ° C for 30 minutes, and centrifuged (12,000 rpm, 4 ° C, 15 minutes). Then, the supernatant was transferred to a new 1.5 mL tube, and 75 μl of protein was quantified by Bradford assay.

Thereafter, electrophoresis was carried out at 10% SDSPAGE gel, 100 V for 1 hour, and the gel was transferred to a tray. After adding 10 mL Rinse buffer (0.1 M sodium phosphate buffer, pH 6.8) and reacting for 30 minutes, the buffer was removed and 10 mL L-3,4-dihydroxyphenylalanine (L-DOPA) staining buffer (0.1 M sodium phosphate buffer, pH 6.8, 5 mM L-DOPA) was added and reacted in a 37 ° C incubator for 1 hour. Thereafter, tyrosinase activity was measured by densitometry analysis through ImageJ Version 1.50b (National Institutes of Health, Bethesda, MD).

7. Intracellular Melanin Measurement

B16F10 melanoma cells (1 × 10 ⁵ cellseach) were incubated in a 60 mm cell culture dish for 12 hours, and then treated with cinnamon extract (700, 7, 0.07 ng / mL) and vehicle (DW) and incubated for 24 hours. . After treatment with Trypsin-EDTA, the cells were transferred to a 1.5 mL tube, stained with 0.4% (w / v) trypan blue dye (Thermo Fisher Scientific) for 1 minute, and the number of cells was counted with a hemocytometer. .

Wash the remaining cells with 1 mL PBS, remove all supernatant, and add 1 mL Homogenization buffer (50 mM sodium phosphate, pH 6.5, 1% (v / v) triton X-100, 0.1 M PMSF). Resuspend, resuspend, react for 20 minutes, centrifuge (12,000 rpm, 4 ° C, 15 minutes), remove supernatant and remove 200 μlysis buffer (1N NaOH, 10% (v / v) DMSO ) Was resuspended. After reacting for 30 minutes and transferring 100 μm each to a 96well plate, the absorbance of 405 nm was measured with a microplate reader.

8. qPCR Analysis

B16F10 melanoma cells (2 × 10 ⁵ cellseach) were incubated in a 60 mm cell culture dish for 12 hours, and then treated with cinnamon extract (7 ng / mL) and vehicle (DW) and incubated for 24 hours. Total RNA was extracted with Isol-RNA Lysis Reagent (2302700, SPRIME, Gaithersburg, MD, USA) and cDNA was synthesized with M-MLV reverse transcriptase (RT001, Enzynomics, Yuseong-gu, Daejeon, Korea).

And 5 μ2 × Real-Time PCR Master Mix including SYBR green (DQ385-40H, Biofact, Yuseong-gu, Daejeon, Korea), 3 μDW, cDNA (on a 96-well plate (Applied Biosystems, Foster City, CA, USA)). 10 μ10 pM primers 1 μ (tyrosinase: forward-5'-TTC TGT CCA GTG CAC CAT CT-3 ', Reverse-5'-ATG AAG TTG CCT GAG CAC TG-3'; TRP2: forward-5'-CTT GGC TCA CTC CTT CCT GA-3 ', reverse-5'-AGT GGA GGA CCA CAA ACA CA-3'; Bax: forward-5'-GTT TCA TCC AGG ATC GAG CAG-3 ', reverse-5'-CAT CTT CTT CCA GAT GGT GA-3 '; GAPDH: forward-5'-TGC MTC CTG CAC CAC CAA CT-3', M = A or C, reverse-5'-YGC CTG CTT CAC CAC CTT C-3 ', Y = T or C), pre-denaturing (95 ° C, 10 min) with a StepOnePlus Real-Time PCR System (Applied Biosystems), amplification 40 cycles (95 ° C for 15 secs and 60 ° C for 1 min) ^QPCR was performed at, and mRNA expression level was measured by 2 ^-ΔΔCt method of Kenneth et al.

 <Example 2>

Antioxidant Effect of Extracts from Cinnamon, etc.

As a result of measuring the antioxidant effect on the cinnamon lamp extract of the present invention, when treated with cinnabar lamp extract (7 ng / mL) for 12 hours, the intracellular ROS concentration was reduced by 75.47 ± 22.36% compared to vehicle in B16F10 melanoma cells (P <0.05), 71.66 ± 24.93% decrease in NIH3T3 fibroblasts (P <0.01, see FIG. 1).

ROS is produced by various stresses such as inflammation, high temperature, and apoptosis, and is known to affect intracellular nuclei and mitochondria, thereby causing various negative effects such as mutations, cancer induction, and cell death. It is essential for the survival of cells by regulating action, wound healing, etc. In particular, in the case of cancer cells, the expression of antioxidant proteins such as superoxide dismutase (SOD) and glutathione is suppressed, resulting in higher ROS concentration than normal cells, which is essential for survival and rapid proliferation of cancer cells.

Therefore, the cinnamon and the like extract of the present invention has an antioxidant effect on B16F10 melanoma cells can be effectively used to inhibit the survival and proliferation of cancer cells.

 <Example 3>

Inhibitory Effect of Extracts from Cinnamon and Melanoma Cell Proliferation and Survival Rate

The cell growth curves showed that B16F10 melanoma cell proliferation inhibitory effect of cinnamon lamp extract was reduced by 25.00 ± 10.83% and 51.04 ± 18.84% when treated with cinnabar lamp extract at concentrations of 700 and 7 ng / mL for 48 hours. After 72 hours of treatment, it was found that 23.63 ± 12.59%, 53.85 ± 8.24% decreased (see Fig. 2, P <0.01).

In addition, as a result of confirming the effect of inhibiting cell viability against B16F10 cells of the extracts such as cinnamon, it was found that the cell viability was reduced by 17.58 ± 0.93%, 22.48 ± 4.96% compared to the vehicle at concentrations of 700 and 7 ng / mL.

Cancer cells continue to grow at a faster rate of proliferation than normal cells, and the survival rate and growth inhibition effect of cancer cells are essential for anticancer treatment.

Therefore, the cinnamon lamp extract of the present invention has the effect of reducing the survival and proliferation of B16F10 melanoma cells up to 53.85 ± 8.24%, 53.85 ± 8.24%, respectively, it can be seen that it is very effective in preventing or treating cancer.

<Example 4>

Enhancement of Tyrosinase Activity and Melanin Synthesis of Extracts from Cinnamon, etc.

Tyrosinase is one of the most important enzymes in the regulation of melanin synthesis and is used as a labeling protein in the regulation of melanin synthesis.

The present inventors treated the cinnamon lamp extract (7 ng / mL) for 24 hours in B16F10 melanoma cells to determine whether there is an effect to enhance the tyrosinase activity and melanin synthesis in the extract such as cinnamon, tyrosinase activity 93.93 ± 16.73% increase compared to the vehicle (see Fig. 3, P <0.001).

In addition, as a result of treatment for 48 hours with the extract such as cinnamon (7 ng / mL), the amount of melanin synthesis increased 62.04 ± 7.34% compared to the vehicle.

Therefore, it was found that the extract such as cinnamon of the present invention enhances the activity of tyrosinase and thus increases the melanin synthesis.

Example 5

Regulation of mRNA Expression of Extracts from Cinnamon, etc.

The present inventors compared the mRNA expression level through qPCR after 24 hours treatment of B16F10 melanoma cells with cinnamon lamp extract to determine whether there is an effect of controlling the expression of mRNA in cinnamon lamp extract.

As a result, the expression levels of tyrosinase (36.59 ± 10.94%) and TRP2 (53.33 ± 8.61%), which induce melanin synthesis, were increased compared to vehicle, and Bax (27.82 ± 10.38%), which induces cell death, was also increased. See FIG. 4, P <0.01).

Tyrosinase is one of the most important enzymes in the melanin synthesis process. It was confirmed that tyrosinase expression was increased through qPCR and DOPA oxidase activity was increased through tyrosinase activity assay. In addition, in the present invention, the expression level of TRP2 mRNA was increased, TRP2 has a dopachrome tautomerase activity and produces DHICA by reacting with DOPA-chrome, and DHICA reacts with tyrosine related protein 1 (TRP1) to produce melanin.

As a result, it was confirmed that the amount of melanin synthesis increased, such as cinnamon extract of the present invention induces the expression of tyrosinase and TRP2 mRNA and enhances the activity of tyrosinase to effectively promote melanin synthesis.

Bax is known as an apoptosis-inducing protein that acts on mitochondria and releases cytochrome C inside the cell membrane to induce caspase activity.

Extracts of cinnamon, etc. of the present invention induced the expression of Bax mRNA, and as a result, it was confirmed that the extract of cinnamon, etc. is effective in treating melanoma by reducing cell proliferation and survival rate.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Claims (12)

Pharmaceutical composition for the prevention or treatment of melanoma, including hot water extracts such as cinnamon as an active ingredient. delete delete The method of claim 1,
The composition is characterized in that to reduce the concentration of ROS in melanoma cells, increase the expression of Bax, apoptosis inducing factor. The method of claim 1,
The composition is characterized in that to inhibit the proliferation and survival of melanoma cells. Cosmetic composition for the prevention or improvement of vitiligo comprising hot water extracts such as cinnamon as an active ingredient. The method of claim 6,
The composition is characterized in that to promote the expression and activity of tyrosinase to increase melanin synthesis. Skin external preparation for melanoma prevention or improvement comprising hot water extracts such as cinnamon as an active ingredient. delete Health functional food for the prevention or improvement of melanoma containing hot water extracts such as cinnamon as an active ingredient. delete A method of inhibiting the proliferation of melanoma cells comprising the step of treating melanoma cells with hot water extracts such as cinnamon in vitro .

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