KR20240002018A - Composition for Skin Whitening Comprising Extract or Fraction of Cissus Quadrangularis as an Active Ingredient - Google Patents

Abstract

The present invention relates to a composition for skin whitening containing Cissus quadrangularis extract or a fraction thereof as an active ingredient, and more specifically, to a cosmetic composition for skin whitening containing Cissus hydrothermal extract or an ethyl acetate fraction thereof as an active ingredient. It relates to composition.
In the present invention, it was confirmed that Cissus thermal water extract or its ethyl acetate fraction significantly inhibits melanin production and tyrosinase activity in melanin-producing cells without cytotoxicity. Therefore, the composition of the present invention is a cosmetic for skin whitening. , it can be usefully used as health functional food for skin whitening, medicine for preventing or treating skin pigmentation disease, and quasi-drugs for preventing or improving skin pigmentation disease.

Description

A composition for skin whitening comprising Cissus extract or a fraction thereof as an active ingredient {Composition for Skin Whitening Comprising Extract or Fraction of Cissus Quadrangularis as an Active Ingredient}

The present invention relates to a composition for skin whitening containing Cissus quadrangularis extract or a fraction thereof as an active ingredient, and more specifically, to a cosmetic composition for skin whitening containing Cissus hydrothermal extract or an ethyl acetate fraction thereof as an active ingredient. It relates to composition.

A person's skin color is determined by the amount of melanin, carotene, and hemoglobin, of which melanin is the most decisive factor. Melanin is synthesized in melanocytes present in the basal layer of the skin and is transferred to surrounding keratinocytes, giving human skin color. Melanin has an important function of protecting the body from ultraviolet rays, but when overproduced, it is known to not only form spots and freckles, but also accelerate skin aging and play an important role in causing skin cancer.

Melanin is produced through enzymatic and non-enzymatic oxidation reactions of tyrosine in melanocyte cells. The enzymes involved in melanin synthesis are tyrosinase and tyrosinase related protein 1 (TRP). -1) and tyrosinase related protein 2 (TRP-2) are known. In addition, the main intracellular signaling pathway is the cAMP/PKA (cyclic monophosphate/protein kinase A) pathway, and cAMP promotes the expression of MITF (Microphthalmia-associated transcription factor) via PKA and CREB1 (cAMP resposive element binding protein 1). do. MITF is an important transcriptional regulator in the melanin synthesis process and is known to promote the transcription of tyrosinase, tyrosinase-related protein 1, and tyrosinase-related protein 2. In addition, α-MSH (alpha-melanocyte stimulating hormone) increases cAMP (cyclic AMP) and PKA (protein kinase A) through MC1R (melanocortin-1-receptor), a cell membrane receptor, and increases tyrosinase production by activated MITF. By regulating expression, it is involved in the proliferation and pigmentation of melanocytes (Curto, EV et. al. , Biochem. Pharmacol. , 57:663-672, 1999; Bentley, NJ et. al. , Mol. Cell. Biol. , 14:7996-8006, 1994).

Accordingly, much research is being conducted on cosmetic compositions for skin whitening using melanin synthesis inhibition mechanisms. In particular, plant-derived materials or herbal medicine ingredients are excellent in terms of stability, and the development of functional materials using them is actively being conducted.

Meanwhile, Cissus ( Cissus quadrangularis ) is an evergreen perennial plant belonging to the genus Cissus of the Buckthorn order, Grape family, and refers to a type of herb also called Indian ivy. Cissus is known to act as a natural appetite suppressant by regulating the secretion of leptin hormone in the body, and is known to have a psychological stabilizing effect by promoting the secretion of the hormone serotonin (Korean Patent Publication No. 10-2021-0020438). In addition, the effect of Cissus methanol extract on promoting melinin production has been reported (Rao GV et. al. , Research Journal of Chemical Sciences , 1(2): 25-29, 2011).

Accordingly, in the present invention, we made diligent efforts to select plant-derived extracts that are more effective and can stably reduce melanin production in the skin. As a result, Cissus stem or leaf extracts inhibit melanin production and tyrosinase in melanocytes. ) It was confirmed that the activity was suppressed to a significant level, and the present invention was completed.

Therefore, the purpose of the present invention is to provide a cosmetic composition for skin whitening containing Cissus thermal water extract or its ethyl acetate fraction as an active ingredient.

Another object of the present invention is to provide a health functional food composition for skin whitening containing Cissus thermal water extract or an ethyl acetate fraction thereof as an active ingredient, a pharmaceutical composition for preventing or treating skin pigmentation diseases, and a quasi-drug for preventing or improving skin pigmentation diseases. To provide a composition.

To achieve the above purpose,

The present invention provides a cosmetic composition for skin whitening containing Cissus quadrangularis thermal water extract or its ethyl acetate fraction as an active ingredient.

In addition, the present invention provides a health functional food composition for skin whitening containing Cissus thermal water extract or its ethyl acetate fraction as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating skin pigmentation disease, comprising Cissus thermal water extract or an ethyl acetate fraction thereof as an active ingredient.

In addition, the present invention provides a quasi-drug composition for preventing or improving skin pigmentation disease, comprising Cissus thermal water extract or an ethyl acetate fraction thereof as an active ingredient.

In a specific embodiment of the present invention, the Cissus may be any one or more selected from the group consisting of Cissus stem and Cissus leaf.

In another specific embodiment of the present invention, the Cissus hydrothermal extract or its ethyl acetate fraction may be included at a concentration of 10 to 500 μg/ml.

In another specific embodiment of the present invention, the Cissus hot water extract can be extracted by adding 5 to 15 liters of water per 100 parts by weight of Cissus and then applying heat for 1 to 3 hours.

In another specific embodiment of the present invention, the ethyl acetate fraction is

(a) adding hexane solvent to the Cissus hydrothermal extract and then obtaining an aqueous phase;

(b) adding chloroform solvent to the aqueous phase and then obtaining the aqueous phase; and

(c) adding ethyl acetate solvent to the aqueous phase and then obtaining an ethyl acetate fraction.

In another specific embodiment of the present invention, the Cissus hydrothermal extract or its ethyl acetate fraction can inhibit melanin production or tyrosinase activity.

In another specific embodiment of the present invention, the Cissus hot water extract or its ethyl acetate fraction inhibits melanin production or tyrosinase activity through inhibition of MITF (Microphthalmia-associated transcription factor) production. can do.

In the present invention, it was confirmed that Cissus thermal water extract or its ethyl acetate fraction significantly inhibits melanin production and tyrosinase activity in melanin-producing cells without cytotoxicity. Therefore, the composition of the present invention is a cosmetic for skin whitening. , it can be usefully used as health functional food for skin whitening, medicine for preventing or treating skin pigmentation disease, and quasi-drugs for preventing or improving skin pigmentation disease.

Figure 1 is a view showing Cissus stems and leaves before drying (left) and Cissus stems and leaves after drying (right).
Figure 2 shows (A) extracellular melanin content, (B) intracellular melanin content, when melanoma cells (B16F1 cells) stimulated with α-melanocyte stimulating hormone (α-MSH) were treated with various Cissus extracts. and (C) data confirming cell viability. Kojic acid is a positive control, WE means hot water extract, 50E means 50% ethanol extract, 70E means 70% ethanol extract, and 100E means 100% ethanol extract. In all figures, data are presented as mean ± standard deviation values (n = 3).
Figure 3 shows data confirming (A) extracellular melanin content and (B) intracellular melanin content when B16F1 cells stimulated with α-MSH were treated with various concentrations of Cissus hot water extract.
Figure 4 shows data confirming tyrosinase activity when B16F1 cells stimulated with α-MSH were treated with various concentrations of Cissus hot water extract.
Figure 5 shows data confirming the effect of suppressing the expression of proteins related to melanin production when B16F1 cells stimulated with α-MSH were treated with various concentrations of Cissus hot water extract. (A) is Western blot data, and (B) is data confirming the relative expression level.
Figure 6 is a schematic diagram of a method for producing fractions of Cissus hot water extract.
Figure 7 shows (A) extracellular melanin content, (B) intracellular melanin content, (C) tyrosinase activity, and (C) when α-MSH-stimulated B16F1 cells were treated with various fractions of Cissus hot water extract. D) Data confirming cell survival rate. Kojic acid is a positive control, WE means hot water extract, HF means hexane fraction, CF means chloroform fraction, EF means ethyl acetate fraction, BF means n-butanol fraction, and WF means water fraction.
Figure 8 shows (A) extracellular melanin content, (B) intracellular melanin content, and (C) tyrosinase when α-MSH-stimulated B16F1 cells were treated with various concentrations of ethyl acetate fraction of Cissus hot water extract. Data confirming activity, and (D) cell viability.
Figure 9 shows data confirming the effect of suppressing the expression of proteins related to melanin production when α-MSH-stimulated B16F1 cells were treated with various concentrations of ethyl acetate fraction of Cissus hot water extract. (A) is Western blot data, and (B) is data confirming the relative expression level.

Hereinafter, the present invention will be described in detail.

The present invention generally relates to a cosmetic composition for skin whitening containing Cissus quadrangularis thermal water extract or its ethyl acetate fraction as an active ingredient.

In the present invention, the Cissus may be any one or more selected from the group consisting of Cissus stem and Cissus leaf.

In the present invention, the Cissus extract may be included at a concentration of 10 to 500 μg/ml. If the concentration of the extract is less than 10 ㎍/㎖, the whitening effect may be weak, and if it is more than 500 ㎍/㎖, the degree of increase in effect as the content increases is negligible, and problems such as cytotoxicity may appear.

In the present invention, the Cissus hot water extract can be extracted by adding 5 to 15 liters of water per 100 parts by weight of Cissus and then applying heat for 1 to 3 hours.

In the present invention, the Cissus extract can inhibit melanin production or tyrosinase activity.

In the present invention, the Cissus extract can inhibit melanin production or tyrosinase activity by inhibiting MITF (Microphthalmia-associated transcription factor) production.

In a specific embodiment of the present invention, hot water extract, 50% ethanol extract, 70% ethanol extract, and 100% ethanol extract were prepared using dried Cissus including stems and leaves. As a result of confirming the effectiveness of the prepared Cissus extract in suppressing melanan production, it was confirmed that Cissus thermal water extract, 50% ethanol extract, 70% ethanol extract, and 100% ethanol extract all reduced the amount of extracellular melanin released and the amount of intracellular melanin produced. . However, Cissus 70% ethanol extract and 100% ethanol extract were found to be cytotoxic, and the melanin production effect of these extracts is believed to be due to cytotoxicity.

On the other hand, since cytotoxicity was not observed in the Cissus hot water extract and the 50% ethanol extract even at a concentration of 200 μg/ml, it was confirmed that these extracts, especially the Cissus hot water extract, were excellent in their efficacy in inhibiting melinin production (Figure 2).

In another specific embodiment of the present invention, as a result of confirming the melanin production inhibition effect of the Cissus thermal water extract, the melanin production inhibition and tyrosinase activity inhibition effects were confirmed, and as a result, the melanoma cells stimulated with α-MSH were confirmed in a concentration-dependent manner. It was confirmed that both extracellularly released melanin and intracellular melanin production as well as tyrosinase activity were suppressed (Figures 3 and 4).

In addition, as a result of confirming the inhibitory effect on the expression of whitening-related proteins of the Cissus hot water extract, it was confirmed that the expression of MITF, a key enzyme in the melanin production process, was suppressed by the Cissus hot water extract (Figure 5). Microphthalmia-associated transcription factor (MITF) is an important transcriptional regulator in the melanin synthesis process and is known to promote the transcription of tyrosinase, TRP-1, and TRP-2.

In the present invention, the ethyl acetate fraction of the Cissus hot water extract is

(a) adding hexane solvent to the Cissus hydrothermal extract and then obtaining an aqueous phase;

(b) adding chloroform solvent to the aqueous phase and then obtaining the aqueous phase; and

(c) adding ethyl acetate solvent to the aqueous phase and then obtaining an ethyl acetate fraction.

In another specific embodiment of the present invention, hexane fraction, chloroform fraction, ethyl acetate fraction, butanol fraction, and water fraction were prepared using Cissus hydrothermal extract, which has excellent whitening activity (FIG. 6). As a result of confirming the cytotoxicity and melanin production inhibition efficacy of each fraction, no cytotoxicity was observed in any of the fractions, and the ethyl acetate fraction was confirmed to have the best efficacy in inhibiting melanin production and tyrosinase activity (Figure 7).

In addition, as a result of confirming the effect of the ethyl acetate fraction of Cissus thermal water extract on concentration-dependent inhibition of melanin production and tyrosinase activity, extracellular melanin and intracellular melanin production in melanoma cells stimulated with α-MSH were found to be concentration-dependent. In addition, it was confirmed that all tyrosinase activities were inhibited (Figures 8A, 8B, and 8C). Furthermore, cytotoxicity of the ethyl acetate fraction of Cissus hot water extract did not appear depending on the concentration, and rather, cell viability was confirmed to increase (Figure 8D).

In another specific embodiment of the present invention, as a result of confirming the inhibitory effect on the expression of whitening-related proteins of the ethyl acetate fraction of Cissus thermal water extract, it was found that the expression of MITF, a key enzyme in the melanin production process, was suppressed by the ethyl acetate fraction. Confirmed (Figure 9).

In other words, the Cissus hot water extract or its ethyl acetate fraction of the present invention not only exhibits the efficacy of inhibiting melanin production and tyrosinase activity at a significant level in melanin-producing cells, but also inhibits the expression of proteins related to melanin production and tyrosinase activity. Suppression was confirmed. Therefore, the Cissus thermal water extract or its ethyl acetate fraction of the present invention can be used in cosmetic compositions for skin whitening, compositions for health functional foods for skin whitening, pharmaceutical compositions for preventing or treating skin pigmentation diseases, and quasi-drugs for preventing or improving skin pigmentation diseases. It can be used as a composition.

The cosmetic composition for skin whitening of the present invention may be in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation or spray. It can be done, but is not limited to this.

In another aspect, the present invention relates to a health functional food composition for skin whitening containing Cissus thermal water extract or its ethyl acetate fraction as an active ingredient.

The health functional food composition for skin whitening may additionally contain various flavoring agents or natural carbohydrates, like a typical food composition.

The formulation form of the health functional food may be tablets, capsules, powders, granules, liquids, pills, etc. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, etc. Additionally, 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, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracacance or sodium oleate, sodium stearate, magnesium stearate, sodium Includes benzoate, sodium acetate, sodium chloride, etc. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, etc. Acceptable pharmaceutical carriers for compositions formulated as liquid solutions include those that are sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these ingredients can be mixed and used, and other common additives such as antioxidants, buffers, and bacteriostatic agents can be added as needed.

In another consistent aspect, the present invention relates to a pharmaceutical composition for preventing or treating skin pigmentation disease, comprising Cissus thermal water extract or an ethyl acetate fraction thereof as an active ingredient.

In another consistent aspect, the present invention relates to a quasi-drug composition for preventing or improving skin pigmentation disease, comprising Cissus thermal water extract or an ethyl acetate fraction thereof as an active ingredient.

In the present invention, the skin pigmentation disease may include at least one selected from the group consisting of melasma, freckles, and melanoma, but is not limited thereto.

The pharmaceutical composition of the present invention can be formulated and used in various forms according to conventional methods. For example, it can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, etc., and can be formulated and used in the form of external preparations, suppositories, and sterile injection solutions. Depending on each dosage form, it may further include pharmaceutically acceptable carriers, excipients, and diluents. In addition, it can be formulated and used in the form of external preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., and sterile injection solutions according to conventional methods.

The carriers, excipients and diluents include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, These include microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oil. When formulating or formulating the pharmaceutical composition, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

It is obvious to those skilled in the art that the therapeutically effective dosage and frequency of administration of the pharmaceutical composition of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by a person skilled in the art, depending on the type of disease, the severity of the disease, the content of the active ingredient and other ingredients contained in the composition, the type of dosage form, the patient's age, weight, and general health condition. , gender and diet, administration time, administration route and secretion rate of the composition, treatment period, and various factors including concurrently used drugs. The pharmaceutical composition of the present invention can be administered to an individual through various routes. For example, it may be administered intravenously, intraperitoneally, intramuscularly, intraarterially, bucally, intracardiacally, intramedullary, intrathecally, transdermally, enterally, subcutaneously, sublingually, or topically, but is not limited thereto. The pharmaceutical composition of the present invention can be administered in an amount of 1 to 10,000 mg/kg/day, and may be administered once a day or divided into several times.

The term "quasi-drug" used in the present invention refers to products that have a milder effect than pharmaceuticals among products used for the purpose of diagnosing, treating, improving, alleviating, treating, or preventing diseases in humans or animals. For example, according to the Pharmaceutical Affairs Act, quasi-drugs exclude products used for medicinal purposes and include products used to treat or prevent diseases in humans and animals, and products that have a mild or no direct effect on the human body.

The quasi-drug composition of the present invention is used for the purpose of improving skin pigmentation, and there is no particular limitation in its formulation, for example, softening lotion, nourishing lotion, massage cream, nutritional cream, pack, mask pack, mask sheet. , it may be a cosmetic composition having a gel or skin-adhesive cosmetic formulation, and may also be a transdermal formulation such as lotion, ointment, gel, cream, patch, or spray.

Additionally, in each dosage form, the quasi-drug composition may contain other ingredients selected arbitrarily depending on the formulation or purpose of use of the other quasi-drug composition. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (suppression or alleviation). For example, it may include conventional auxiliaries such as thickeners, stabilizers, solubilizers, vitamins, pigments and flavors, and carriers.

Hereinafter, the present invention will be described in more detail through examples.

These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Cissus Extract Manufacturing

Cissus used in the present invention was domestically produced. Fresh Cissus was purchased from Nokwoo Compound, dried, extracted, and used as a sample (Figure 1).

Using dried Cissus, four types of extracts were prepared by extraction with water (hot water extraction), 50% (v/v) ethanol, 70% (v/v) ethanol, and 100% (v/v) ethanol.

For the hot water extract, 1 liter of distilled water was added per 10 g of sample, boiled for 2 hours using an electric water boiler (Daewoong Medicine Water Boiler, Daewoong Bio Appliances, Korea), filtered using filter paper (Advantec, No. 41), and concentrated under reduced pressure. It was freeze-dried. For the 50% (v/v) ethanol extract, 70% (v/v) ethanol extract, and 100% (v/v) ethanol extract, add 1 liter of distilled water per 10 g of sample, sonicate for 1 hour, and then cool to room temperature. Primary extraction was performed by reacting overnight, and the next day, after removing the primary extraction solvent, 1 liter of solvent was added again and secondary extraction was performed in the same manner. The primary and secondary extracts were collected, filtered, concentrated under reduced pressure, and then freeze-dried to prepare each extract.

Cissus extract yield according to extraction solvent Extraction method Sample amount (g) Extraction amount (g) transference number(%) thermal water extraction 10 4.453 4.45 50% ethanol extraction 10 3.075 3.07 70% ethanol extraction 10 2.216 2.21 100% ethanol extraction 10 0.301 0.30

The yield of the hot water extract using dried Cissus was 4.45%, the yield of the 70% ethanol extract was 3.07%, the yield of the 70% ethanol extract was 2.21%, and the yield of the 100% ethanol extract was 0.3%. It was confirmed to be the highest.

Confirmation of whitening efficacy of Cissus extract according to solvent

2-1: Melanin production inhibition effect

The experiment was conducted using melanoma (B16F1; Mouse melanoma cell line) cells. B16F1 cells were cultured in an incubator using DMEM medium containing FBS and 1% penicillin streptomycin, maintaining 5% CO ₂ and 37°C. did.

To measure melanin content, B16F1 cells were inoculated into a 60 mm culture dish at a density of 8Х10 ⁴ and then treated with Cissus hydrothermal extract (200 ㎍/㎖), 50% ethanol extract (200 ㎍/㎖), and 70% ethanol extract ( 200 ㎍/㎖), 100% ethanol extract (200 ㎍/㎖), and kojic acid (1 mM) as a positive control, respectively, followed by treatment with α-melanocyte stimulating hormone (α-MSH, 200 nM). And melanin production was induced at 37°C for 72 hours. After 72 hours, 150 ㎕ of cell culture medium was transferred to a 96-well plate and the extracellular melanin content was measured. After the cells were recovered, 1M sodium hydroxide (NaOH) containing 10% dimethyl sulfoxide (DMSO) was added and stored at 80°C for 1 hour to dissolve melanin, and then the content of melanin in the cells was measured at an absorbance of 405 nm. .

As a result, when melanin synthesis was induced by treatment with α-MSH, the amount of extracellular melanin released increased to 179%. On the other hand, when treated with Cissus hot water extract, 50% ethanol extract, 70% ethanol extract, and 100% ethanol extract, the amount of extracellular melanin released decreased by 135%, 136%, 149%, and 193%, respectively, and kojic acid, a positive control, When treated, the amount of extracellular melanin released was 128% (Figure 2A).

Additionally, as a result of measuring the amount of melanin produced within the cells, the amount of melanin produced increased by 182% when treated with α-MSH. On the other hand, when treated with Cissus hot water extract, 50% ethanol extract, 70% ethanol extract, and 100% ethanol extract, the amount of melanin production in cells was suppressed by 115%, 122%, 138%, and 111%, respectively, and the positive control group When treated with kojic acid, the amount of melanin produced by cells was confirmed to be 129% (Figure 2B).

2-2: Confirmation of cytotoxicity

Cytotoxicity evaluation of Cissus extract was confirmed by MTT assay method.

The next day after inoculating B16F1 cells in a 24-well plate at a density of 1Х10 ⁴ , Cissus hydrothermal extract (200 ㎍/㎖), 50% ethanol extract (200 ㎍/㎖), 70% ethanol extract (200 ㎍/㎖), 100 % ethanol extract (200 ㎍/㎖) and kojic acid (1 mM) as a positive control, respectively, followed by treatment with α-melanocyte stimulating hormone (α-MSH, 200 nM), and incubated at 37°C for 72 hours. It was cultured in .

After 72 hours, the medium was replaced with MTT reagent and cultured at 37°C for another hour. The medium containing the MTT reagent was removed, and 400 ㎕ of dimethyl sulfoxide (DMSO) was added to dissolve the purple-colored formazan. After transferring 100 μl of the dissolved material to a 96 well plate, the absorbance was measured at 540 nm. Cell survival rate was calculated by taking the survival rate of the control group that was not treated with α-MSH as 100%.

As a result, it was confirmed that Cissus hot water extract and 50% ethanol extract were not cytotoxic at a concentration of 200 μg/ml. On the other hand, when treated with 70% ethanol extract and 100% ethanol extract at the same concentration, cell survival rates were 73% and 55%, respectively, showing cytotoxicity (Figure 2C). That is, in Example 2-1, the melanin production-suppressing effect of the 70% ethanol extract and the 100% ethanol extract was attributed to cytotoxicity, which means that in reality, the melanin production-suppressing effect was minimal.

In the present invention, it was confirmed that Cissus hot water extract and 50% ethanol extract were excellent in melanin production inhibition efficacy. In particular, Cissus hot water extract was found to have the best extraction yield and melanin production inhibition efficacy.

Confirmation of the efficacy of Cissus thermal water extract in inhibiting melanin production

In the present invention, the melanin production inhibitory effect according to the concentration of Cissus thermal water extract, which has the best melanin production inhibitory effect without cytotoxicity, was confirmed.

Extracellular melanin release and intracellular melanin production were measured in the same manner as in Example 2-1, and Cissus thermal water extract was treated at concentrations of 50 μg/ml, 100 μg/ml, and 200 μg/ml, respectively.

In the case of extracellular melanin release, when melanin synthesis was induced with α-MSH, the amount of melanin increased to 173% compared to the untreated group, and when treated with Cissus hot water extract at different concentrations, it increased by 166%, 156%, and 133%, respectively. The amount of extracellular melanin released decreased in a concentration-dependent manner, and when treated with kojic acid, a positive control, the amount of extracellular melanin released decreased to 103% (Figure 3A).

In the case of intracellular melanin production, the amount of melanin production increased to 193% when treated with α-MSH. When Cissus hot water extract was treated at different concentrations, the amount of melanin production decreased in a concentration-dependent manner by 179%, 170%, and 151%, respectively, and when treated with kojic acid, a positive control, the amount of intracellular melanin production decreased to 138% (Figure 3B).

In other words, it was confirmed that the Cissus hot water extract of the present invention effectively inhibits melanin production, and it was found to be significantly superior to kojic acid, a positive control.

Confirmation of inhibitory effect on tyrosyanase activity of Cissus thermal water extract

In the present invention, we sought to determine whether the melanin production inhibition effect of Cissus thermal water extract was due to inhibition of tyrosinase activity, a key enzyme in the melanin production process.

First, B16F1 cells were inoculated into a 60 mm culture vessel at a density of 8 Х 10 ⁴ and then various concentrations of Cissus hydrothermal extract (50 ㎍/㎖, 100 ㎍/㎖ and 200 ㎍/㎖) and kojic acid (kojic acid) as a positive control were added. acid, 1 mM), and then treated with α-melanocyte stimulating hormone (α-MSH, 200 nM) and cultured at 37°C for 72 hours.

After 72 hours of incubation, cells were obtained by washing with PBS (phosphate buffered saline), and then proteins were extracted using lysis buffer (PRO-PREP, iNtRON, Korea). After quantifying the extracted protein, put the same amount of protein in a 96-well plate, add L-dopa (2 mg/ml), react at 37°C for 1 hour, and measure the absorbance at 450 nm. did. Tyrosinase activity was calculated as a percentage of the absorbance of the control group that was not treated with α-MSH.

As a result, it was confirmed that inhibition of tyrosinase activity was suppressed by Cissus hot water extract in a concentration-dependent manner (Figure 4). When treated with α-MSH, tyrosinase activity increased by 359% compared to the untreated group, while when treated with Cissus thermal water extract at 50 ㎍/㎖, 100 ㎍/㎖, and 200 ㎍/㎖, tyrosinase activity increased by 359% compared to the untreated group. It decreased by 337%, 315%, and 203%, respectively, depending on the concentration of Cissus hot water extract. In particular, it was confirmed that when Cissus hot water extract was treated at a concentration of 200 ㎍/㎖, it showed a superior tyrosinase activity inhibition effect than the positive control, kojic acid.

Confirmation of the efficacy of Cissus thermal water extract in regulating whitening-related protein expression

In the present invention, in order to determine whether Cissus thermal water extract regulates the expression of whitening-related proteins, the expression of tyrosinase and MITF (Microphthalmia-associated transcription factor), which are key enzymes in the melanin production process, was observed. MITF is an important transcriptional regulator in the melanin synthesis process and is known to promote the transcription of tyrosinase, TRP-1, and TRP-2.

First, to confirm changes in tyrosinase protein expression, B16F1 cells were inoculated into a 60 mm culture dish at a density of 8Х10 ⁴ and then treated with Cissus hot water extract (50 ㎍/㎖, 100 ㎍/㎖, 200 ㎍/㎖) and Each was treated with kojic acid (1 mM), a positive control, and then treated with α-melanocyte stimulating hormone (α-MSH, 200 nM), and cultured at 37°C for 72 hours. For changes in MITF protein expression, each sample was treated in the same manner as above and then incubated at 37°C for 4 hours.

After sample processing was completed, cells were washed with PBS and proteins were extracted using lysis buffer. The extracted proteins were quantified using the Bradford assay method, and the quantified proteins were separated using 10% SDS-PAGE (SDS polyacrylamide gel electrophoresis) and then transferred to a PVDF (polyvinylidene difluoride) membrane. The membrane was blocked with 5% skim milk dissolved in TBS-T (Tris-buffered saline-Tween 20 solutions) for 1 hour, washed with TBS-T, and then incubated with tyrosinase, MITF, and β-actin primary antibodies at 4°C. The reaction was allowed to occur overnight. The next day, the membrane was washed with TBS-T and reacted with secondary antibodies (anti-mouse and anti-rabbit IgG) at room temperature for 1 hour. Protein bands were confirmed using HRP substrate and ImageQuant LAS 500 (GE Healthcare Life Science, USA).

As a result, when only α-MSH was treated, tyrosinase and MITF proteins increased by about 500% and 350%, respectively, compared to the untreated group, while Cissus hot water extract was treated at 50 ㎍/㎖, 100 ㎍/㎖, and 200 ㎍/㎖. When treated with ㎖, it was confirmed that tyrosinase and MITF proteins were reduced in a concentration-dependent manner of Cissus hot water extract (FIG. 5). Therefore, it was confirmed that Cissus hot water extract inhibits melanin production through inhibition of tyrosinase and MITF protein expression.

Preparation of Cissus hydrothermal extract fractions

Cissus hydrothermal extract fractions were obtained as hexane fraction, chloroform fraction, ethyl acetate fraction, butanol fraction, and water fraction in the same manner as the schematic diagram shown in FIG. 6.

Specifically, 1.3 g of Cissus hot water extract was mixed with 130 ml of each fractionation solvent, then the mixture was vigorously mixed and allowed to stand to separate layers three times. Fractions for each solvent were concentrated under reduced pressure, freeze-dried, and used as samples.

The yield of each fraction was hexane fraction (44.43%), chloroform fraction layer (14.86%), ethyl acetate fraction (0.22%), butanol fraction (1.39%), and water fraction (26.5%): hexane fraction > water fraction > chloroform fraction. > Butanol fraction > ethyl acetate fraction showed high yield.

Confirmation of whitening efficacy of fractions of Cissus thermal water extract

7-1: Confirmation of melanin production inhibition effect

Each fraction prepared in Example 6 was treated with 200 μg/ml of kojic acid (1 mM), then treated with α-melanocyte stimulating hormone (α-MSH, 200 nM), and incubated for 37 hours for 72 hours. Cultured at ℃. Next, the amount of extracellular melanin released and the amount of intracellular melanin produced were measured in the same manner as in Example 2-1.

As a result, as shown in Figures 7A and 7B, the melanin content of the group treated only with a-MSH was significantly increased compared to the control group, while the hexane fraction (HF), chloroform fraction (CF), and butanol fraction (BF) were excluded. The ethyl acetate fraction (EF) and water fraction (WF) were shown to inhibit the amount of extracellular melanin and intracellular melanin production.

The amount of extracellular melanin released when stimulated with a-MSH was 176.8±5.6% when the untreated control was set as 100%, the hexane fraction was 161.5±2.2%, the chloroform fraction was 162.1±3.6%, and the ethyl acetate fraction was 137.6±5.6%. 5.0%, butanol fraction was 174.3 ± 0.8%, and water fraction was 167.7 ± 2.6%.

In addition, the amount of intracellular melanin production in the a-MSH treated group was 207.1 ± 1.6% when the control group was set at 100%, the hexane fraction was 186.3 ± 0.8%, the chloroform fraction was 182.8 ± 2.5%, and the ethyl acetate fraction was 113.01 ± 2.5%. , the butanol fraction was 176.3 ± 0.0%, the water fraction was 209.4 ± 5.0%, and when treated with kojic acid, it was 159.1 ± 2.5%.

In particular, when the ethyl acetate fraction was treated, the amount of melanin production was significantly suppressed, and it was confirmed that the effect of suppressing intracellular melanin production was superior to that of kojic acid, which was a positive control.

7-2: Measurement of tyrosinase activity

Each fraction prepared in Example 6 was treated in the same manner as Example 4 to determine tyrosinase activity.

As a result, as shown in Figure 7C, the tyrosinase activity increased by a-MSH treatment was decreased by each fraction of Cissus hot water extract, and in particular, the ethyl acetate fraction inhibited tyrosinase activity the best. It was confirmed that

7-3: Confirmation of cytotoxicity

In order to confirm the cytotoxicity of each fraction of Cissus hot water extract, cell viability was measured in the same manner as in Example 2-2.

As shown in Figure 7D, the cell survival rate was as follows: butanol fraction (94.1 ± 0.46%) < chloroform fraction (94.1 ± 0.9%) < hexane fraction (95.2 ± 2.8%) < water fraction (98.5 ± 1.8%) < ethyl acetate fraction. (108.2±0.6%), and the cell viability of all fractions was over 94% and no cytotoxicity was observed.

Confirmation of whitening effect of ethyl acetate fraction of Cissus thermal water extract

In the present invention, we sought to confirm the whitening effect according to the concentration of the ethyl acetate fraction (EF), which has the best melanin production inhibition effect among the fractions of Cissus thermal water extract.

8-1: Melanin production inhibition effect

The amount of extracellular melanin released and the amount of intracellular melanin produced were measured in the same manner as in Example 2-1, and the ethyl acetate fraction of Cissus thermal water extract was treated at concentrations of 50 μg/ml, 100 μg/ml, and 200 μg/ml, respectively. did.

In the case of extracellular melanin release, when melanin synthesis was induced with α-MSH, the amount of melanin increased to 231.8% compared to the untreated group, whereas when the ethyl acetate fraction of Cissus thermal water extract was treated at different concentrations, it increased to 216.7% and 173.0%, respectively. %, the amount of extracellular melanin released decreased in a concentration-dependent manner by 156.5% (Figure 8A).

In the case of intracellular melanin production, when melanin synthesis was induced with α-MSH, the amount of melanin increased to 178.2% compared to the untreated group, whereas when treated with the ethyl acetate fraction of Cissus thermal water extract at different concentrations, it increased to 152.0% and 141.1%, respectively. %, the amount of melanin produced in cells decreased in a concentration-dependent manner by 133.1% (Figure 8B).

8-2: Tyrosinase activity inhibition effect

Tyrosinase activity was measured in the same manner as in Example 4, and the ethyl acetate fraction of Cissus hot water extract was treated at concentrations of 50 μg/ml, 100 μg/ml, and 200 μg/ml, respectively.

As a result, as shown in Figure 8C, when treated with α-MSH, the tyrosinase activity increased to 441.5%, while when the ethyl acetate fraction of Cissus hot water extract was treated at different concentrations, the tyrosinase activity increased to 378.8%. , was suppressed by 303.3% and 162.7%.

In particular, similar to the results in FIG. 7, it was confirmed that the efficacy of inhibiting intracellular melanin production and inhibiting tyrosinase activity was superior when the ethyl acetate fraction was treated at a concentration of 200 μg/ml compared to kojic acid, which was a positive control.

8-3: Confirmation of cytotoxicity

In order to confirm cytotoxicity according to the concentration of the ethyl acetate fraction of Cissus hot water extract, cell viability was measured in the same manner as in Example 2-2.

As a result, as shown in Figure 8D, when the fractions of ethyl acetate were treated at different concentrations, the cell survival rate was all over 100%, and no cytotoxicity was observed due to the fractions. Rather, it was confirmed that the cell survival rate increased. .

Confirmation of the efficacy of ethyl acetate fraction of Cissus thermal water extract in regulating whitening-related protein expression

In the present invention, it was confirmed whether the ethyl acetate fraction of Cissus thermal water extract regulates the expression of tyrosinase and MITF, which are key enzymes in the melanin production process.

The experiment was performed in the same manner as in Example 5, and the ethyl acetate fraction of Cissus hot water extract was treated at concentrations of 50 μg/ml, 100 μg/ml, and 200 μg/ml, respectively.

As a result, as shown in Figure 9, it was confirmed that tyrosinase and MITF expression was suppressed in a concentration-dependent manner in the ethyl acetate fraction of the Cissus hot water extract. In the case of the ethyl acetate fraction at a concentration of 200 μg/ml, kojic acid, a positive control, It was confirmed that the efficacy of inhibiting tyrosinase expression was superior compared to the treatment group.

Claims (12)

A cosmetic composition for skin whitening comprising Cissus quadrangularis thermal water extract or its ethyl acetate fraction as an active ingredient.
The cosmetic composition for skin whitening according to claim 1, wherein the Cissus is a Cissus stem or Cissus leaf.
The cosmetic composition for skin whitening according to claim 1, wherein the Cissus hydrothermal extract or its ethyl acetate fraction is contained in a concentration of 10 to 500 μg/ml.
The cosmetic composition for skin whitening according to claim 1, wherein the Cissus hot water extract is extracted by adding 5 to 15 liters of water per 100 parts by weight of Cissus and then applying heat for 1 to 3 hours.
The method of claim 1, wherein the ethyl acetate fraction is
(a) adding hexane solvent to the Cissus hydrothermal extract and then obtaining an aqueous phase;
(b) adding chloroform solvent to the aqueous phase and then obtaining the aqueous phase; and
(c) adding an ethyl acetate solvent to the aqueous phase and then obtaining an ethyl acetate fraction. A cosmetic composition for skin whitening, characterized in that it is prepared by a method comprising:
The cosmetic composition for skin whitening according to claim 1, wherein the Cissus hydrothermal extract or its ethyl acetate fraction inhibits melanin production or tyrosinase activity.
The method of claim 1, wherein the Cissus hot water extract or its ethyl acetate fraction inhibits melanin production or tyrosinase activity by inhibiting MITF (Microphthalmia-associated transcription factor) production. Cosmetic composition for skin whitening.
A health functional food composition for skin whitening comprising Cissus thermal water extract or its ethyl acetate fraction as an active ingredient.
A pharmaceutical composition for preventing or treating skin pigmentation disease, comprising Cissus thermal water extract or an ethyl acetate fraction thereof as an active ingredient.
The pharmaceutical composition for preventing or treating skin pigmentation disease according to claim 9, wherein the skin pigmentation disease is melasma, freckles, or melanoma.
A quasi-drug composition for preventing or improving skin pigmentation disease, comprising Cissus thermal water extract or an ethyl acetate fraction thereof as an active ingredient.
The quasi-drug composition for preventing or improving skin pigmentation disease according to claim 11, wherein the skin pigmentation disease is melasma, freckles, or melanoma.