KR20130096055A - Composition comprising extract of hirudo nipponica whitman for inhibiting synthesis of melanin - Google Patents

Abstract

PURPOSE: A composition which includes Hirudo Nipponica Whitman extract is provided to be used for inhibiting an activation of tyrosinase; suppressing a melanogenesis; preventing, reducing and treating a disease from an excessive formation of the melanin; and whitening effect. CONSTITUTION: A cosmetic composition includes Hirudo Nipponica Whitman extracts as an active ingredient. The cosmetic composition is for whitening effect. The cosmetic composition has an antioxidant activity. The Hirudo Nipponica Whitman extract is selected from a group which comprises a methanol extract of Hirudo Nipponica Whitman and an ethanol extract of Hirudo Nipponica Whitman. The melanogenesis hindering medicinal composition includes the Hirudo Nipponica Whitman extract as the active ingredient.

Description

Composition comprising extract of Hirudo nipponica Whitman for inhibiting synthesis of melanin}

The present invention ( Hirudo) nipponica Whitman) relates to a cosmetic composition, a pharmaceutical composition and a food composition utilizing the tyrosinase inhibitory activity, melanin production inhibitory activity, antioxidant activity of the extract.

Among the cells that make up the skin epidermis, melanocytes increase melanin synthesis by exposure to sunlight, and the increased melanin protects the skin from external stimuli, but erythema, pigmentation, skin blackening, skin aging May cause skin cancer, toxicity by melanin precursors and promote cell death.

Melanin is produced through the process of converting the amino acid tyrosine into 3,4-dihydroxyphenylalanine (DOPA) and DOPAquinone by using tyrosinase as the main action enzyme in the immature melanosome. Inhibiting the activity of tyrosinase, a major enzyme to inhibit melanin synthesis, can induce melanin synthesis inhibition and whitening effect [Kim et al., Korean J. Medicinal Crop . Sci . 16 , 255-260, 2008].

Although inhibitors of melanogenesis have been developed, there are some side effects such as weak activity, degeneration or lethality of pigment cells, or impaired cell intrinsic function. Need development

The skin is subjected to oxidative damage from reactive oxygen species (ROS) produced by UV exposure. All cells that use oxygen produce ROS, and ROS cause lipid peroxidation reactions, and the by-products damage proteins and DNA, which are components of cells, resulting in changes in cell membrane structure, decreased enzyme activity, and cell mutations. . In addition, compared to fine lines, dry skin, reduced skin elasticity, endogenous aging, irregular and thick pigmentation occurs on the skin epidermis exposed to sunlight, and pigment diseases such as solar lentigo increase.

The present invention is to solve the problem to find and provide a new cytotoxic and less tyrosinase inhibitory activity, melanin production inhibitory activity.

The present invention to solve the above problems provides a composition comprising a water extract as an active ingredient.

In one embodiment of the present invention provides a composition having a tyrosinase inhibitory activity comprising a water extract as an active ingredient.

As another embodiment of the present invention provides a composition having a melanin production inhibitory activity comprising a water extract as an active ingredient.

In one embodiment of the present invention provides a composition having a whitening effect comprising the water extract as an active ingredient.

In one embodiment of the present invention provides a composition for the prevention, improvement, treatment of diseases caused by the excessive production of melanin containing water extract as an active ingredient.

In addition, another embodiment of the present invention provides a composition having antioxidant activity.

The composition provided by the present invention includes a cosmetic composition, a pharmaceutical composition, a food composition, and a food additive composition as one embodiment.

The composition comprising the water extract provided through the present patent application has tyrosinase inhibitory activity, melanin production inhibitory activity, antioxidant activity, and there is no cytotoxicity against B16-F10 melanoma cell line.

The composition containing the water extract may be used as a safe tyrosinase inhibition, melanin production inhibition, cosmetic composition having a whitening effect, a pharmaceutical composition, a food or food additive composition.

1 is a graph showing the tyrosinase inhibitory activity of water extract. The assay solution contains water methanol extract or ethanol extract and 1500 units / mL tyrosinase, 1.5 mM tyrosine at each concentration. (A: methanol extract administration group B: ethanol extract administration group Arb: arbutin Arbutin). The assay mixture was reacted at 37 ° C. for 15 minutes and the absorbance was measured at 490 nm. Each experimental value is the mean ± SD of three experimental results.
FIG. 2 shows the effect on melanin content of water methanol extract in a-MSH-induced B16-F10 melanoma cell line. Cells were incubated for 24 hours, and treated with 1 M of a-MSH followed by 96 hours of treatment with aqueous methanol extract. (A: extracellular melanin content B: intracellular melanin content). ***: p <0.001, *: p <0.005 (compared to a-MSH alone control)
Figure 3 shows the viability (cell viability) of B16-F10 melanoma cell line by water extract treatment. (A: MTT reduction assay, B: LDH release assay). Cells were incubated for 24 hours with water concentration methanol extract of each concentration. (A) After the MTT reductioin assay, the MTT reduction rate (mean ± SD of three experimental values) was calculated based on the survival rate of each control group. (B) The results of the LDH release assay (mean ± SD of three experiments) were normalized to 100% LDH release in the vehicle-administered group and expressed as% values for the control group.
4 shows monitored morphological changes over 24 hours of B16-F10 melanoma cell line by water methanol extract. Cells were treated with water methanol extract for 24 hours (A: control group, B: 5 μg / mL treatment group, C: 10 μg / mL treatment group, D: 50 μg / mL treatment group). The picture was taken 100 times using a phase-contrast microscope.
Figure 5 shows the effect of apoptosis on the B16-F10 melanoma cell line of the water methanol extract. Cells were treated with water methanol extract for 24 hours (A: control group, B: 5 μg / mL treatment group, C: 10 μg / mL treatment group, D: 50 μg / mL treatment group). The immobilized cells were stained with hoechst 33342 (10 μM) and observed 400 times using a fluorescence microscope.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention screened natural products to find a substance having tyrosinase inhibitory activity, melanin production inhibitory activity and low cytotoxicity, and found that the water extract has such activity.

Water quality belongs to the large group of hiraniidae annulus, and is typically Japanese Hirudo nipponica Whitman), ephedra / quality gold wire tube (Whitmania pigra Whitman) and Ephedra / Multicolor acranulata Whitman) dry body.

The inventors of the present invention water (Hirudo nipponica Methanol extracts and ethanol extracts of Whitman) were prepared and their physiological activities were verified. The water ethanol extracts (HNE) had 18.7% inhibitory activity against tyrosinase at 500 μg / mL and methanol at the same concentration. The extract (HNM) was confirmed to have an inhibitory activity of 28.6%. As a result of investigating the effect of the water methanol extract on the production of melanin in the B16-F10 melanoma cell line, it was confirmed that the melanin content was inhibited by about 50% compared to the a-MSH treated control at the final concentration of 50 μg / mL.

In addition, the total phenolic substance content and DPPH radical scavenging ability were measured to investigate the antioxidant activity of water extracts. The total phenolic content was 165.5 μg / 100 mg GAE and water ethanol extract for water methanol extract (HNM). (HNE) was analyzed to be 147.4 μg / 100 mg GAE. DPPH radical scavenging activity was 15.1% in water ethanol extract (HNE) at 5 mg / mL, 3.3% in water methanol extract (HNM), and ethanol extract showed more than 5 times higher activity than methanol extract.

In order to measure cytotoxicity of B16-F10 melanoma cell line of water extract, MTT reduction assay, LDH release assay, morphological changes of cells, and hoechst staining were performed using water methanol extract. It was confirmed that there is no cytotoxicity by having a survival rate corresponding to the untreated control even at the concentration of.

These results indicate that water extracts have excellent tyrosinase inhibitory activity, melanin production inhibitory activity, antioxidant activity and safety against B16-F10 melanoma cell line, and have side effects such as genetic mutation, instability and skin cancer caused by toxicity. This suggests that alternatives to existing tyrosinase inhibitors are possible.

Accordingly, the present invention provides a composition having a tyrosinase inhibitory activity, including water extract as an active embodiment.

As another embodiment of the present invention provides a composition having a melanin production inhibitory activity comprising a water extract as an active ingredient.

In one embodiment of the present invention provides a composition having a whitening effect.

Another embodiment of the present invention provides a composition for the prevention, improvement, treatment of diseases caused by the excessive production of melanin containing water extract as an active ingredient.

In addition, another embodiment of the present invention provides a composition having antioxidant activity.

The composition provided by the present invention includes a cosmetic composition, a pharmaceutical composition, a food composition, and a food additive composition as one embodiment.

Water extracts are added to the water quality by about 1 to 30 times the weight of water, and if necessary, more solvents are used, soaking, boiling water extraction, reflux circulation, and pressure extraction for about 1 to 5 days at about 10 ^o C to 100 ^o C After extraction by such a method, it can be prepared by filtration (or centrifugation and filtration), it may be further used a method such as concentrated under reduced pressure, lyophilization. The solvent may be one or more mixed solvents selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms, propylene glycol, ethyl acetate, butylene glycol, ether, and chloroform.

The water extract may be in the form of liquid, powder, or granules, but is not limited thereto.

The composition of the present invention can be used for medicine, food, cosmetics and the like and can be administered by any method to suit the purpose. For example, parenteral or oral administration is possible and is prepared in a suitable dosage form depending on the method of administration. The composition according to the invention can be prepared by conventional methods depending on the formulation.

Water extracts have melanin production inhibitory activity, have antioxidant activity, and have low cytotoxicity, so they can be used in cosmetic compositions. In particular it can be used as a cosmetic composition for whitening.

Cosmetic composition of the present invention may comprise 0.001 to 90% by weight, preferably 0.01 to 70% by weight of the water extract relative to the total weight. In addition, the cosmetic of the present invention may further include vitamins, peptides, polysaccharides, lipids, etc., in addition to the water extract, and other fats and oils, moisturizers, surfactants, pigments, ultraviolet absorbers, preservatives, fungicides, antioxidants, etc. It may include, but is not limited to, plant extracts, pH adjusters, alcohols, flavorings, purified water, and the like.

The cosmetic composition of the present invention may be in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, oil, wax, powder, spray, soap, cleansing, , But is not limited to.

In addition, the water extract may be included in the pharmaceutical composition for preventing and treating diseases caused by excessive activity of tyrosinase, excessive production of melanin, a pharmaceutical composition for inhibiting melanin production, or a pharmaceutical composition for antioxidant.

Diseases caused by the excessive production of melanin include erythema, pigmentation, skin blackening, skin aging, skin cancer, and cell death due to melanin precursors, but are not limited thereto. Water extracts can be used for this purpose, respectively or simultaneously, systemic (eg oral) and topical (eg skin).

In the present invention, the water extract may be included in the total amount of the composition, 0.0005 to 30% by weight, preferably 0.01 to 10% by weight, but can be adjusted according to the purpose. The optimal dosage of the composition of the present invention is appropriately determined according to age, individual difference, symptoms, etc., but the dosage when administering to a human is usually 0.01-100 mg / kg, preferably 0.1-10 mg / kg, and this amount It can be administered once or several times a day.

The compositions of the present invention may be used in the form of solids, solutions, emulsions, dispersants, micelles, liposomes, ointments, and the like, and may include organic or inorganic carriers or excipients suitable for oral or parenteral application. The compositions of the present invention are generally for tablets, powders, pellets, capsules, pills, suppositories, solutions, emulsions, suspensions, solutions, jelly, injections and any other form suitable for use. It can be mixed with a nontoxic pharmaceutically acceptable carrier. Usable carriers include solid, semisolid or liquid glucose, lactose, gum arabic, gelatin, mannitol, starch paste, magnesium trisilicate salt, talc, corn starch, keratin, colloidal silica, potato starch, urea, chain Medium length triglycerides, dextran and other carriers suitable for use in the preparation of the formulations are included. In addition, auxiliaries, stabilizers, thickeners, colorants and flavoring agents can be used.

The present invention also provides a food composition or food additive composition for inhibiting tyrosinase activity, inhibiting melanin production, and antioxidant containing water extract as an active ingredient. The present invention also provides a food composition or food additive composition for the prevention and improvement of diseases caused by melanin overproduction. The food composition or food additive composition comprising the water extract of the present invention may be suitably used alone or in combination with other food or food ingredients according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use thereof. In general, the water extract may be added in an amount of 0.0001 to 30% by weight, preferably 0.1 to 10% by weight based on the raw materials in the manufacture of food or beverage, but the amount of the water extract can be adjusted as desired depending on the purpose Do. There is no particular limitation on the kind of the food. Examples of the food include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, Drinks, tea, drinks, alcoholic beverages, and vitamin complexes.

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

Reagents and Experimental Materials

Water quality used in this experiment ( Hirudo nipponica Whitman) was provided by Jirisan Daedang Agricultural Co., Ltd. located in Sancheong-gun, Gyeongnam, South Korea, and used for the experiment. DMEM medium, fetal bovine serum (FBS), penicillin (penicillin), streptomycin, and the like were purchased from Invitrogen (Grand Island, NY, USA). Tyrosinase, tyrosine, arbutin, folin-Ciocalteau's phenol, 2,2-diphenyl-1-picrylhydrazyl (DPPH), Na ₂ CO ₃ , 3- [4,5-dimethylthiazol-2-yl ] -2,5-diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), a-melanocyte stimulating hormone (a-MSH) (st. Louis, Mo, USA) was purchased and used. Lactate dehydrogenase (LDH) release assay kit is available from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The solvents and reagents used in the other experiments all used first grade or higher grades.

Cell culture

Pigment forming cell line used in this experiment was used as a B16-F10 murine melanoma obtained from the Korea Cell Line Bank (KCLB, Seoul). For cell culture, 10% fetal bovine serum (FBS) and 100 units / mL penicillin and 100 mg / mL streptomycin were added to DMEM medium, and 37 ° C. and 5% CO ₂ were maintained at 95% humidity. The cells were cultured in an incubator (MCO-18AIC, SANYO, Osaka, Japan).

Statistical processing

All data processing was done using the SPSS-PC + statistical package. Percentage and mean ± standard deviation (SD) were calculated according to each item, and statistical significance of the mean value among the experimental groups was verified by the Student t-test for the difference in total phenol content according to the extraction solvent. Differences in DPPH radical scavenging activity, tyrosinase inhibitory activity, cell viability, and melanin content by extraction solvent or concentration were analyzed by variance analysis using one-way ANOVA method.

Experimental Example 1 Preparation of Extract of Sample

50 mL of methanol or ethanol was added to 5 g of water, and the mixture was left to stand at room temperature for 3 days, followed by extraction with a filter paper (Advantec, Tokyo, Japan). The filtered extract was concentrated under reduced pressure at 40 ℃ using a rotary pressure reducer (EYELA N-1000, Tokyo, Japan) to obtain an extract. The methanol extract was named HNM and the ethanol extract was named HNE. Each extract was dissolved in methanol and ethanol and diluted to appropriate concentrations for tyrosinase inhibitory activity, melanin inhibitory activity, antioxidant activity, and cytotoxicity experiments.

<Experimental Example 2> Tyrosinase  Measurement of inhibitory activity

In order to investigate the inhibitory effect of water extract on tyrosinase activity, an enzyme catalyzing an important step in the production of melanin pigment, we compared the inhibitory activity of tyrosinase according to the concentration of arbutin and water extract. Water Methanol Extract (HNM) and Water Ethanol Extract (HNE) in To determine the ability to inhibit in vitro mushroom tyrosinase activity, 100 μL of sample and 140 μL of 0.05 M sodium phosphate buffer (pH 6.8) were added to each well of a 96-well plate, 20 mL of enzyme (1500 units / mL mushroom tyrosinase), substrate (1.5). 40 μL of mM tyrosine was added sequentially, followed by incubation at 37 ° C. for 15 minutes, and then absorbance at 490 nm was measured using an ELISA reader. As a control, 100 μL of methanol and ethanol, respectively, and 1 mM of arbutin were used as a positive control.

As a result of measuring the activity of water extracts at concentrations of 10, 50, 100 and 500 μg / mL, tyrosinase activity was inhibited by 20.9, 23.6, 24.4 and 28.6% in the water methanol extract (HNM), respectively. Ethanol extract (HNE) treatment group was inhibited by 10.4, 11.0, 13.0, 18.7%, respectively, showing a concentration-dependent and statistically significant inhibitory effect. Water methanol extract (HNM) was higher tyrosinase inhibitory activity than water ethanol extract (HNE) (Fig. 1A, B). Water quality tyrosinase inhibitory activity is lower than that of arbutin 1 mM, but considering the state of extract seems to have a sufficiently high inhibitory activity.

Experimental Example 3 Measurement of Melanin Production

In order to evaluate the melanin production inhibitory effect of the water methanol extract (HNM) on the B16-F10 melanoma cell line, the cell line was added to a 6-well plate 2 mL to 5x10 ⁴ cells / mL and incubated for 24 hours. Next, the aqueous methanol extract (HNM) was treated at a concentration of 10 and 50 μg / mL, respectively, and the control group was treated with 1% methanol, and then treated with 1-M of a-MSH, a melanin production inducer, and incubated for 96 hours. Production was induced to measure the cell culture and the production of melanin in the cells. After culturing the cells treated with 0.25% trypsin-EDTA solution, the cells were harvested and centrifuged. 1 N NaOH was added to the cell pellet, and the cells were dissolved at 80 ° C. for 10 minutes and the absorbance was measured at 415 nm.

As a result of measuring the melanin content in the culture medium, when the untreated group was 100%, the a-MSH treated group was found to be 151%, indicating that melanin production was induced by the a-MSH treatment, and the water methanol extract (HNM) was obtained. It was confirmed that melanin production was significantly lower than the a-MSH-treated group at 101.5% when treated at the concentration of 50 μg / mL, and showed higher inhibition rate when treated with 100 μM of arbutin, the positive control group. (FIG. 2A).

As a result of measuring the amount of melanin produced in the cells, the a-MSH treated group was 147.4% when the untreated group was 100%. Water Methanol Extract (HNM) significantly decreased to 113.2% in the 10 μg / mL treatment group and 69.0% in the 50 μg / mL treatment group, about 60% more than the 100% treatment of arbutin, the positive control group (129.8%). It was confirmed that further inhibit the melanin production (Fig. 2B). When considering the following cytotoxicity results (Experimental Examples 6 to 9), the cells did not grow due to cytotoxicity did not reduce melanin production, melanin synthesis is proportional to tyrosinase activity in melanocytes and melanin content measurement results In view of the same tendency to inhibit the activity of tyrosinase, water extract can be said to have a protective effect against melanogenesis.

Experimental Example 4 Measurement of Total Phenolic Material Content

Since most of the substances having antioxidant activity contain phenol, physiologically active functions such as antioxidant activity can be known by measuring the amount of phenol in the extract state. In the present invention, gallic acid was selected as a standard to measure the content of polyphenolic substances in water extracts. The content of each extracted phenolic substance was measured by Singleton and Rossi [Park, S, N. Journal of Korean Industrial and Engineering Chemistry. 14 (5) , 657-665, 2003] were adapted to the experiment. Allowed to stand in water methanol extract (HNM) and water ethanol extract (HNE) by the addition of 1N-Folin-Ciocalteau reagent 200 μL to 200 μL at room temperature for 3 minutes, and 10% Na ₂ CO ₃ 200 μL was added and left in the dark for 1 hour, followed by centrifugation at 13,400 × g for 5 minutes, and the reaction solution was measured for absorbance at 690 nm. The standard calibration curve was prepared using gallic acid, and the total phenol content was calculated from the standard calibration curve.

The total phenolic content was 165.5 μg / 100 mg GAE based on 100 mg of water methanol extract (HNM) and water ethanol extract (HNE) was 147.4 μg / 100 mg GAE (Table 1). GAE stands for gallic acid equivalents.

Water quality Hirudo nipponica Whitman ) Total phenolic content of the extract

Extraction solvent
HNM
HNE μg / 100 mg GAE ^a
165.5 ± 23.17
147.4 ± 1.55

^a GAE: gallic acid equivalents

The numerical values of all results are the mean ± SD of the three experimental values.

<Experimental Example 5> DPPH Radical Scatters  Measure

DPPH radical scavenging activity (RSA) is highly related to antioxidant activity, and it is an experiment to check the degree of hydrogen donating ability of antioxidants by checking the degree of discoloration of the dark purple reagent through the extract. Thitilerdecha et al. [Thitilertdecha, N. et al., Food , Inc.] for the DPPH radical scavenging activity of water methanol extract (HNM) and water ethanol extract (HNE) science and Technol . 41 , 2029-2035, 2008] was used to modify the experiment. 80 μL of 0.2 mM DPPH solution was added to 20 μL of the extracted water sample, mixed for 10 seconds, and the absorbance was measured at 492 nm after standing at room temperature for 10 minutes.

The water extracts were measured at concentrations of 0.1, 0.5, 1, and 5 mg / mL, respectively. The DPPH radical scavenging ability was 0.9, 1, 2.8, and 3.3%, respectively, and the water ethanol extract (HNE), respectively. The concentration-dependent and statistically significant results were found to be 5.6, 7.5, 8.4, and 15.1%. The water ethanol extract (HNE) was relatively higher in DPPH radical scavenging ability than the water methanol extract (HNM) (Table 2). ).

Water extract DPPH Radical Scatters
Extraction solvent mg / mL HNM HNE
DPPH RSA (%) 0.1 0.92 ± 0.005 5.58 ± 0.117 0.5 1.03 ± 0.013 7.52 ± 0.267 One 2.76 ± 0.055 8.43 ± 0.394 5 3.28 ± 0.058 15.15 ± 1.485

The numerical values of all results are the mean ± SD of the three experimental values.

<Experimental Example 6> MTT reduction assay

The cytotoxic effect of water methanol extract (HNM) on cell proliferation of B16-F10 melanoma cell line was investigated.

Cell viability was measured by MTT reduction assay of water methanol extract (HNM) on B16-F10 melanoma cell line. 100 μL of B16-F10 melanoma cell line was injected into 96-well plate at a concentration of 5 × 10 ⁴ cells / mL and incubated in 37 ° C., 5% CO ₂ incubator for 24 hours, followed by water methanol extract (HNM). Prepared at concentrations of 1, 5, 10 and 50 μg / mL, respectively, the cells were treated and incubated for 24 hours. Then, 10 mL of MTT (5 mg / mL) solution dissolved in PBS buffer was added to each well and reacted for 30 minutes to confirm formazan formation. Then, 100 μL of the medium was completely removed to dissolve the formazan formed at the bottom of the well. The absorbance was measured at 540 nm with the addition of DMSO using an ELISA reader (Model 680, Bio Rad, USA). Relative cell viability was obtained when 100% of control cells cultured without water extract were treated.

Methanol extract showed high cell viability of 101.4% in 1 μg / mL treatment group, 99.8% in 5 μg / mL treatment group, 102.0% in 10 μg / mL treatment group and 108.8% in 50 μg / mL treatment group. It was confirmed that the water extract did not exhibit cytotoxicity (FIG. 3A).

Experimental Example 7 LDH release assay

LDH release assay was performed to determine the cytotoxicity of the water methanol extract (HNM) against B16-F10 melanoma cell line. 100 μL of B16-F10 melanoma cell lines were injected into 96-well plates at a concentration of 5x10 ⁴ cells / mL, and incubated in 37 ° C., 5% CO ₂ incubator for 24 hours. , B16-F10 melanoma cell line at a concentration of 5, 10, 50 μg / mL and incubated for 24 hours. 50 μL of the upper portion of the culture solution was dispensed into a new 96-well plate, 50 μL of LDH reagent was added thereto, and allowed to stand at room temperature, followed by reaction for 20 minutes. After confirming that the reaction was completed by the change of color, the stop solution was added by 100 μL each to stop the reaction, and the absorbance at 540 nm was measured by ELISA reader to measure the amount of LDH emission.

To measure the LDH in the cells, remove the culture medium, add 50 μL of 0.5% Triton X-100 solution, shake for 10 minutes, remove the cell wall, and react with 50 μL of LDH reagent. After the stop solution was added, the absorbance at 540 nm was measured. The percentage of cytotoxicity against LDH was calculated as the percentage of LDH liberated from the culture against the total LDH liberated in culture and cells as a percentage of the value of the control that was not treated with aqueous methanol extract (HNM).

As shown in FIG. 3B, the aqueous methanol extract (HNM) was treated at concentrations of 1, 5, 10, and 50 μg / mL, respectively. As a result, LDH emissions were 19.4%, 18.8%, 19.1%, and 23.3%, respectively. %), And showed no cytotoxicity.

Experimental Example 8 B16-F10 Melanoma  Observing Morphological Changes in Cell Lines

Melanocytes connect with adjacent keratinocytes through dendritic processes to form epidermal melanin units, which play an important role in the transport of melanosomes. Morphological changes of B16-F10 melanoma cell lines were observed using an inverted microscope to investigate the effects of water methanolic extract (HNM) on dendritic processes and apoptosis.

Add 2 mL of B16-F10 melanoma cell line to 5x10 ⁴ cells / mL in a 6-well plate, incubate for 24 hours, and incubate the aqueous methanol extract (HNM) at concentrations of 5, 10 and 50 μg / mL, respectively. Treatment, and in the case of the control was treated to 1% methanol and observed by culturing for 24 hours. Morphological features were photographed 100 times using a phase contrast microscope (Nikon, Tokyo, Japan).

As a result, when comparing the untreated control and the group treated with water methanol extract (HNM) at concentrations of 5, 10, and 50 μg / mL, respectively, it was confirmed that there was no difference in the density and morphological difference of the cells. (FIG. 4).

Experimental Example 9 Hoechst  33342 dye

To study the mechanism of cytotoxic effect, we confirmed whether apoptosis activity of B16-F10 melanoma cell line was active. In order to observe the change of the nucleus, one of the morphological characteristics of apoptosis, the nucleus was stained using a fluorescent dye, hoechst 33342, which specifically binds to DNA in the nucleus, and observed under a fluorescence microscope.

Cells were incubated for 24 hours at 5x10 ⁴ cells / mL in a 6-well plate and treated with methanol extracts (HNM) at concentrations of 5, 10, and 50 μg / mL, respectively. After incubation for 24 hours, washed twice with PBS buffer, fixed with 10% formalin, stained with hoechst 33342 (10 μM) and observed the nucleus 400 times under a fluorescence microscope.

As shown in FIG. 5, the cell nucleus of the untreated control group showed an oval intact nucleus shape, and the cell nucleus of the experimental group treated with 5, 10, and 50 μg / mL of water methanol extract (HNM) also maintained an oval intact nucleus shape. It was.

The results of Experimental Examples 6 to 9 show that the water extract is not toxic to cells.

Hereinafter, one embodiment of the formulation of the present invention is illustrated, but is not limited thereto.

<Formulation Example 1> Preparation of nutrition cream containing water extract

0.5% by weight of water methanol extract, 6% by weight of proylene glycol, 4% by weight of glycerin, 5% by weight of liquid paraffin, 3% by weight of squalene, 1.5% by weight of polysorbate 60 and the remaining amount of purified water, Cream was prepared.

<Formulation Example 2> Ointment

Water methanol extract 1 g, dexpanthenol 1.5 g, stearic acid 1.0 g, liquid paraffin 5.0 g, braze 4.0 g, cetanol 3.0 g, propylene glycol 13.0 g, triethanolamine 1.5 g, dibutylhydroxytoluene 0.025 g, propyl benzoate 0.015 g, polysorbate 800.1 g, purified water 63.0 g of the composition of the composition according to the conventional ointment preparation method to prepare an ointment.

<Formulation Example 3> Drink

9% by weight of the water methanol extract, 10% by weight fructose, 1% by weight per white sugar, 0.2% by weight of sodium citrate was added to a total of 100% by weight of purified water and stirred to prepare a conventional beverage production method.

Claims (12)

Cosmetic composition comprising water extract as an active ingredient. The cosmetic composition according to claim 1, for whitening. The cosmetic composition according to claim 1, having an antioxidant activity. A solution, suspension, emulsion, paste, gel, cream, lotion, oil, wax, powder, spray, soap, cleansing, pack, foundation, makeup base, and hair according to claim 1. Cosmetic composition prepared in a formulation selected from the group consisting of cosmetics. The cosmetic composition according to any one of claims 1 to 3, wherein the water extract is selected from the group consisting of methanol extract and ethanol extract of water. Pharmaceutical composition for inhibiting melanin production comprising water extract as an active ingredient. Pharmaceutical composition for the prevention or treatment of diseases caused by the excessive production of melanin containing water extract as an active ingredient. The pharmaceutical composition according to claim 6 or 7, which is prepared in a dosage form selected from the group consisting of tablets, powders, pellets, capsules, pills, suppositories, solutions, emulsions, suspensions, solutions, jelly, injections. . The pharmaceutical composition according to claim 6 or 7, wherein the water extract is selected from the group consisting of methanol extract of water and ethanol extract. Food composition or food additive composition for inhibiting melanin production comprising water extract. Food composition or food additive composition for the prevention or improvement of diseases caused by the excessive production of melanin containing water extract as an active ingredient. The food composition or the food additive composition according to claim 10 or 11, wherein the water extract is selected from the group consisting of methanol extract of water and ethanol extract.

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