KR20210023155A - Cosmetic composition comprising luffa cylindrica sap as active ingredient - Google Patents

Abstract

An embodiment of the present invention provides a cosmetic composition for prevention and alleviation of wrinkles, anti-UV damage, and skin whitening, containing a Luffa cylindrica sap as an active component. Another embodiment of the present invention provides a cosmetic composition for prevention and alleviation of wrinkles, anti-UV damage, and skin whitening, containing 4-bromo-3-methylisoxazol-5-amine as an active component. The composition of the present invention can exhibit various effects such as wrinkle prevention, wrinkle alleviation, anti-UV damage, and skin whitening while containing a small amount of the Luffa cylindrica sap or 4-bromo-3-methylisoxazol-5-amine, which is the active component thereof.

Description

Cosmetic composition using scrubber sap as an active ingredient {COSMETIC COMPOSITION COMPRISING LUFFA CYLINDRICA SAP AS ACTIVE INGREDIENT}

The present invention relates to a cosmetic composition using a scrubber sap as an active ingredient, and more particularly, to a cosmetic composition for anti-wrinkle, wrinkle improvement, anti-UV damage and whitening, using the scrubber sap as an active ingredient.

Skin, as an organ surrounding the outside of the body, plays an important biological role in primarily protecting the body from the environment, as well as a social role as an element constituting most of the outer skin visible to others. In all tissues of living organisms, aging progresses with age, and skin is no exception. In general, skin aging is divided into intrinsic aging and exogenous aging. The former refers to aging that proceeds naturally according to genetically determined according to age, and the latter refers to sunlight, pollution (nicotine, etc.), and repetitive muscle use. , It refers to the aging phenomenon induced by diet, general health condition, etc.

Representative phenomena that appear as skin ages are pigmentation such as wrinkles and spots, age spots, and blemishes. In the case of skin wrinkles, the number of fibroblasts present in the dermal layer of the skin due to various causes and the decrease in the ability to synthesize these cells change and decompose the extracellular matrix, represented by collagen and elastin, and appear as elasticity decreases. Pigmentation is a hormone. It appears as melanin pigment is excessively produced by abnormalities or ultraviolet rays. Humans have been pursuing beauty and youth for a long time, and in particular, there has always been a need for raw materials that have the effect of slowing and improving wrinkles and pigmentation of the skin, which are representative symptoms of external aging as described above. Cosmetics with such effects are forming a large market, and while a number of functional cosmetics are being released, the demand for cosmetics with better effects and raw materials for them is also increasing.

Loofah ( Luffa cylindrica (L.) Roem ) is a perennial plant in the gourd family, and grows with the development of vine-like stems and reticulated fibers, forming a fruit with black seeds. It has been cultivated for a long time in the tropical and subtropical regions of Asia, and depending on the parts such as fruits, shells, seeds, sap, etc., not only materials for food and beverages and sponges used as kitchen and bath products, but also for the treatment of various symptoms in private and traditional medicine. It has been used as a medicine for medicine. For example, in China, it has been used as anthelmintic, antipyretic, and health medicine. In oriental medicine, it has an effect on menstrual impurities and is known as a medicinal for expectorant, hemostasis, and detoxification.

The loofah has been used for skin care in the private sector, and in recent studies, it has been reported that the pulp and peel extracts of the loofah have anti-inflammatory, antioxidant, and anti-moisture loss effects. However, the effect of the scrubber's sap on the skin has not been specifically elucidated. In this regard, Korean Laid-Open Patent Publication No. 10-2010-0061902 claims that a cosmetic composition containing a loofah solution can prevent itching of atopy and can aid in skin regeneration to exhibit atopic treatment effect. It does not provide evidence. In addition, Korean Laid-Open Patent Publication No. 10-2014-0078070 discloses a composition for improving skin in which a mixture of oriental herbal solutions and scrubber sap concentrating herbal ingredients such as yugeunpi, wild ginseng and ginseng, etc. Not only is it difficult to know, it is not known from which material the effect is coming from.

The inventor of the present invention completed this invention by discovering that the scrubber sap has an improvement effect related to wrinkles, UV damage and pigmentation while studying the effect of the scrubber sap on the skin.

The present invention has been devised to solve the above-described needs, and an embodiment of the present invention provides a cosmetic composition for anti-wrinkle, anti-wrinkle, anti-UV damage and whitening, using a scrubber sap as an active ingredient.

In addition, another embodiment of the present invention provides a cosmetic composition for anti-wrinkle, anti-wrinkle, anti-UV damage and whitening using 4-bromo-3-methylisoxazol-5-amine as an active ingredient.

The technical problem to be achieved by the present invention is not limited to the technical problems mentioned above, and other technical problems that are not mentioned can be clearly understood by those of ordinary skill in the technical field to which the present invention belongs from the following description. There will be.

In order to achieve the above technical problem, an embodiment of the present invention provides a cosmetic composition for anti-wrinkle, anti-wrinkle, anti-UV damage and whitening, using a scrubber sap as an active ingredient.

Here, the scrubber sap may be contained in 3 (v/v)% or more in the composition.

The cosmetic composition may increase one or more of the expression of EGFR in fibroblasts, phosphorylation of EGFR, phosphorylation of ERK1/2, and phosphorylation of AKT.

The cosmetic composition may increase one or more of collagen in fibroblasts, elastin in fibroblasts, fat in adipocytes, and C/EBP in adipocytes.

The cosmetic composition is to recover the skin damage induced by UV, and the skin damage is at least one selected from the reduction of collagen in fibroblasts, reduction of elastin in fibroblasts, modification of fibroblasts, and melanin production of melanocytes. It may be accompanied by a phenomenon that includes.

The cosmetic composition may have one or more effects of promoting a decrease in melanin production by HGF and inhibiting an increase in melanin production by FGF.

The scrubber sap may contain 4-bromo-3-methylisoxazol-5-amine. The content of 4-bromo-3-methylisoxazol-5-amine in the scrubber sap may be 3% or more.

Anti-wrinkle, anti-wrinkle, anti-UV damage and whitening cosmetic composition according to another aspect of the present invention uses 4-bromo-3-methylisoxazol-5-amine as an active ingredient.

Here, the composition may contain 3 to 20 μM of 4-bromo-3-methylisoxazol-5-amine.

According to an embodiment of the present invention, there is provided a cosmetic composition that contains a scrubber sap as an active ingredient and has anti-wrinkle, anti-wrinkle, anti-UV damage and whitening effects of the skin without skin irritation. These compositions can produce the same effect as described above while containing a small amount of the scrubber sap.

The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

1 is data on the effect of the scrubber sap on the proliferation (A, B), mobility (C), and antioxidant activity (D, E) of fibroblasts.
2 is data on the effect of the scrubber sap on signal transduction in fibroblasts.
3 is data on the effect of the scrubber sap on the amount of matrix metalloprotase in fibroblasts.
Figure 4 is data on the effect of the scrubber sap on the amount of collagen (A, B) and elastin (C, D) in fibroblasts.
5 is data on the effect of the scrubber sap on fat accumulation in adipocytes.
6 is data on the effect of the scrubber sap on the reduction of collagen (A) and elastin (B) and cell transformation (C) of fibroblasts by UV.
7 is data on the effect of each growth factor on the amount of melanin in melanocytes.
8 is data on the effects of FGF and HGF on the expression of antioxidant proteins (A) and melanogenesis-related proteins (B) in melanocytes, respectively.
9 is data on the effect of the scrubber sap on the survival of melanocytes (A), the amount of melanin (B), and intracellular signaling (C, D).
FIG. 10 is data on the correlation between FGF and HGF mRNA and age (A, B) and the effect of scrubber sap on the change in the amount of melanin (C) according to FGF and HGF treatment of melanocytes.
11 is a data (B) of the effect of the scrubber sap on the survival of melanocytes (A) and the increase in the amount of melanin by UV.
Figure 12 is the HPLC and MS/MS result data of the scrubber sap.
13 is data on the effect of treatment of 4-bromo-3-methylisoxazol-5-amine, a component of the scrubber sap, on the amount of collagen and elastin in fibroblasts.
14 is data on the effect of treatment of 4-bromo-3-methylisoxazol-5-amine, a component of the scrubber sap, on fat accumulation in differentiation-induced adipocytes.
Fig. 15 is a data showing the effect of treatment of 4-bromo-3-methylisoxazol-5-amine, a component of the scrubber sap, on the amount of melanin in melanoma cells.
FIG. 16 is data on the effect of treatment of 4-bromo-3-methylisoxazol-5-amine, a component of the scrubber sap, on UV-treated fibroblasts.
Fig. 17 is data on the effect of treatment of 4-bromo-3-methylisoxazol-5-amine, a component of the scrubber sap, on the proliferation of fibroblasts, adipocytes, and melanoma cells.

Hereinafter, the present invention will be described with reference to the accompanying drawings. However, the present invention may be implemented in various different forms, and therefore is not limited to the embodiments described herein. In the drawings, parts irrelevant to the description are omitted in order to clearly describe the present invention, and similar reference numerals are attached to similar parts throughout the specification.

Throughout the specification, when a part is said to be "connected (connected, contacted, bonded)" with another part, it is not only "directly connected", but also "indirectly connected" with another member in the middle. "Including the case. In addition, when a part "includes" a certain component, this means that other components may be further provided, not excluding other components, unless specifically stated to the contrary.

The terms used in the present specification are only used to describe specific embodiments, and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In the present specification, terms such as "comprise" or "have" are intended to designate the presence of features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, but one or more other features. It is to be understood that the presence or addition of elements or numbers, steps, actions, components, parts, or combinations thereof does not preclude in advance.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings.

An embodiment of the present invention provides a cosmetic composition for anti-wrinkle, anti-wrinkle, anti-UV damage and whitening, using a scrubber sap as an active ingredient.

Here, the scrubber sap may be 3 (v/v)% or more contained in the composition, preferably 5 (v/v)% or more, and most preferably 10 (v/v)% or more. .

The scrubber sap may preferably be collected from a Korean native scrubber. The scrubber sap may be obtained by cutting a portion having a length of about 1 meter exposed from the ground in a stem of a scrubber grown preferably 3 meters or more, and collected from the cut. At this time, it is preferable to clean and disinfect the container receiving the sap and the stem part inserted into the container, and collect the sap in a sealed state to prevent impurities such as rain or dust from entering the sap. It is known that the period during which the sap of the same quality can be collected from the cut part as described above is within 6 days. The collection process may be performed using a sap collector.

The cosmetic composition of the present invention may include ingredients commonly used in cosmetic compositions in addition to the scrubber sap as an active ingredient, and the commonly used ingredients include, for example, antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances. Such conventional adjuvants, and carriers.

The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, It may be formulated as an oil, a powder foundation, an emulsion foundation, a wax foundation, and a spray, but is not limited thereto.

More specifically, it may be prepared in the form of a flexible lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, or a powder.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component. I can.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additionally chlorofluorohydrocarbon, propane / May contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Sex cellulose, aluminum metahydroxide, bentonite, agar or tracanth, and the like can be used.

When the formulation of the present invention is a surfactant-containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide ether Sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters may be used.

When the cosmetic composition of the present invention is a soap, a cleansing formulation containing a surfactant, or a cleansing formulation containing no surfactant, it may be applied to the skin and then wiped off, removed, or washed off with water. As a specific example, the soap is liquid soap, powder soap, solid soap and oil soap, the surfactant-containing cleansing formulation is a cleansing foam, cleansing water, cleansing towel and cleansing pack, and the surfactant-free cleansing formulation is a cleansing cream , Cleansing lotion, cleansing water, and cleansing gel, but is not limited thereto.

The cosmetic composition is to increase one or more of the expression of epidermal growth factor receptor (EGFR) in fibroblasts, phosphorylation of EGFR, phosphorylation of extracellular signal regulated kinase (ERK1/2), and phosphorylation of AKT (AKT serine/threonine kinase). I can.

The skin is located on the outermost shell and includes the epithelium and connective tissue that have waterproof and antibacterial functions, and is composed of the dermis that provides elasticity, volume, and structural rigidity, and the subcutaneous that mainly consists of adipocytes. In particular, the dermis plays an important role in skin aging. As skin aging progresses, not only the dermis thins but also collagen and elastin decrease, and this change results in wrinkles and sagging of the skin. Dermal fibroblasts are known as major cells that structurally fill the dermis and produce collagen, elastin, and the like that act as a barrier.

In particular, intracellular phenomena that affect the amount of collagen and elastin, which are highly related to wrinkles, can appear in various directions, such as inhibiting the synthesis of collagen and elastin, promoting decomposition, and reducing expression, and a signaling system that induces these results. In addition, a plurality of systems such as NF-κB, TGF-β, and MAPK signaling are known, and methods that affect the signaling system also exist in various directions such as changes in expression and post-translational regulation such as phosphorylation. It has been reported to do. The inventors of the present invention believe that the loofah sap, an active ingredient in the cosmetic composition of the present invention, acts in the direction of increasing the activity of the MAPK signaling system in increasing the amount of collagen and elastin in fibroblasts, and in particular, EGFR among MAPK signaling. As a signal initiating molecule, it was confirmed that it not only activates this EGFR, but also increases its expression. When the MAPK signaling is activated, it is known to increase collagen transcription, and it has been demonstrated through experiments to be described later by the inventors of the present invention that it reduces MMP, which promotes the degradation of collagen and elastin through PPARγ.

The cosmetic composition may increase one or more of collagen in fibroblasts, elastin in fibroblasts, fat in adipocytes, and C/EBP (CCAAT enhancer binding protein) in adipocytes. It has been shown that the amount of collagen in fibroblasts, elastin in fibroblasts, and the amount of fat in adipocytes induced differentiation into adipocytes can be increased in a concentration-dependent manner. In addition, according to an embodiment of the present invention, it was confirmed that the change in the adipocytes was accompanied by an increase in the amount of C/EBPα and a decrease in phosphorylation of ERK1/2.

The cosmetic composition is to restore skin damage induced by UV, and the skin damage is a reduction of collagen in fibroblasts, reduction of elastin in fibroblasts, transformation of fibroblasts, and melanin production of melanocytes. It may be accompanied by a phenomenon including one or more selected from among the. The UV may be UV-A.

The cosmetic composition may have one or more effects of promoting a decrease in melanin production by HGF (Hepatocyte growth factor) and inhibiting an increase in melanin production by FGF (Fibroblast growth factor). The inventors of the present invention have confirmed the effect of several growth factors on melanogenesis in melanocytes, and among them, HGF has found that melanin production decreases and FGF increases. It has been shown that the loofah sap, an active ingredient of the composition of the present invention, amplifies the melanin reduction effect of HGF and inhibits the facilitation of melanin production of FGF. This indicates that the whitening effect can be further enhanced when HGF is additionally included in the cosmetic composition.

The scrubber sap may contain 4-bromo-3-methylisoxazol-5-amine. In particular, the scrubber sap may contain 4-bromo-3-methylisoxazol-5-amine as an active ingredient. 4-bromo-3-methylisoxazol-5-amine may be contained in the cosmetic composition at least 3 μM, preferably at least 5 μM, and more preferably at least 10 μM. The content of 4-bromo-3-methylisoxazol-5-amine in the scrubber sap may be 3% or more, preferably 5% or more, and more preferably 7% or more.

Anti-wrinkle, anti-wrinkle, anti-UV damage and whitening cosmetic composition according to another aspect of the present invention uses 4-bromo-3-methylisoxazol-5-amine as an active ingredient.

Here, the composition may contain 3 to 20 μM, preferably 3 to 10 μM, and more preferably 5 to 7 μM of 4-bromo-3-methylisoxazol-5-amine.

Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily implement the present invention. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

Example 1. Scrubber sap collection

After sowing loofah seeds of Korean native varieties (Wonjin Herb Farm, Ulsan, Korea), they were watered every two days and cultured in the open field so that the plants could grow about 3 meters. Scrubber sap was collected from the root of the scrubber by cutting the upper stem of about 1m, and receiving it from the cut for about 12 hours.

Human dermal fibroblasts (Human dermal fibroblasts, Detroit-551 and CCD986-SK), adipocytes (preadipocytes, 3T3-L1) and melanoma cells (B16F10) used in the following cell experiments were the Korea Cell Line Bank (KCLB). , Seoul, Korea). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) in 5% CO ₂ , 37 °C, and humidity controlled conditions. At this time, 10% (v/v) fetal bovine serum for human dermal fibroblast lines and 10% (v/v) fetal calf serum (Gibco-BRL, Gaithersburg, MD, USA) were added to DMEM. , Commonly 1×10 ⁵ unit/L penicillin and 100 mg/L streptomycin (Invitrogen, Carlsbad, CA, USA) were added.

In the following western blot experiments, GAPDH was used as a control marker, and the resulting relative pixel intensity was analyzed by ImageJ.

Experimental Example 1. Analysis of the effect of scrubber sap on fibroblast proliferation, migration, and ROS

Fibroblasts (Detroit-551 and CCD986-SK) were cultured for 72 hours, including 0, 1, 3, 5 and 10% (v/v) of scrubber sap, respectively.

To confirm the effect of the scrubber sap on the proliferation of fibroblasts, 10 μl of a WST-1 reagent (Nalgene, Rochester, NY) reagent was added to the fibroblasts per well, and after 2 hours, the absorbance of the culture was 450 Measured in nm (Bio-Rad Laboratories, Richmond, CA). As a result, it was not statistically significant, but it was found that the cell proliferation was more active as the concentration of the scrubber sap increased in general (Fig. 1A). Regarding the proliferation of cells, the results of additionally confirming the amount of PCNA (proliferating cell nuclear antigen) in the fibroblast lines by western blot (Fig. 1B), the effect of loofah sap promoting the proliferation of fibroblasts It could be confirmed that there is.

To confirm the effect of the scrubber sap on the migration of fibroblasts, the two cell lines were tested according to the manufacturer's protocol using a 24-Transwell device (Corning, NY, USA), and ELISA reader (Bio-Rad ) Was measured at 560 nm. As a result, it was confirmed that both Detroit-551 and CCD986-SK had statistically significant increases in mobility in a concentration-dependent manner of the scrubber sap (Fig. 1C).

Next, in order to confirm the effect of the scrubber sap on ROS (reactive oxygen species), which is known to be highly related to skin aging such as wrinkle formation, ROS detection reagent (Invitrogen, Carlsbad, CA, USA) on the two fibroblast cell lines. After obtaining the stained cells using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA), the data were analyzed using FlowJo software (Tree Star, Ashland, OR). I did. As a result, it was found that the ROS level of the cells treated with the scrubber sap was significantly lower than that of the control group (Fig. 1D). In addition, as a result of performing western blot on proteins known to lower the intracellular oxidative stress, Catalase, SOD1 (Superoxide dismutase 1), SMA (Smooth muscle actin), and TRX (Thioredoxin), a scrubber sap treatment group It was confirmed that the expression of the four proteins was increased in (Fig. 1E), which means that the scrubber sap was able to increase the ability of the cells to remove ROS.

Experimental Example 2. Analysis of the effect of scrubber sap on the intracellular signaling system of fibroblasts

Fibroblasts (Detroit-551 and CCD986-SK) were cultured for 0, 24, 48, and 72 hours, including 0, 10% (v/v) loofah sap, respectively.

As a result of analysis using a phospho-RTK array kit (R&D Systems, Minneapolis, MN) to investigate the effect of scrubber sap on the intracellular signaling system of fibroblasts, scrubber sap was found to induce phosphorylation of EGFR (Fig. 2A). In order to investigate the mechanism of the effect of scrubber sap on fibroblasts in more detail, the molecules in the EGFR signaling system were analyzed by western blot. At this time, the analysis targets were EGFR, phosphor-ERK1/2, ERK1, phospho-AKT, and AKT, and as a result of the analysis, it was confirmed that the amount of EGFR, phosphorylation of ERK1/2 and phosphorylation of AKT increased in a concentration-dependent manner. There was (Fig. 2B).

As there have been reports on the relationship between EGFR signaling and PPARγ, the effect of scrubber sap on the expression and activity of PPARγ was examined. In the scrubber sap treatment group, the Western blot result showed that the expression of PPARγ was decreased (FIG. 2C), and the PPARγ luciferase reporter assay showed that the activity of PPARγ was also decreased (FIG. 2D). To reconfirm the relationship between EGFR and PPARγ, siRNA was treated for each, and western blot for EGFR and PPARγ was performed.As a result, when si-EGFR was treated, the expression of PPARγ increased, and when si-PPARγ was treated It was confirmed that the expression of EGFR was increased.

Taken together, it has been shown that scrubber sap increases the expression and phosphorylation of EGFR in fibroblasts, increases the level of phosphorylation of lower signaling molecules, and decreases the expression and activity of PPARγ through EGFR signaling.

Experimental Example 3. Analysis of the effect of scrubber sap on intracellular MMP of fibroblasts

Fibroblasts (Detroit-551 and CCD986-SK) were cultured for 48 and 72 hours, including 0 and 10% (v/v) loofah sap, respectively.

PPARγ and MMP (Matrix metalloproteases) and the relationship between MMP and skin have been reported, respectively. To examine the effect of scrubber sap on the expression of proteases including MMP in fibroblasts, analysis with a human protease array kit (R&D Systems, Minneapolis, MN) showed that ADAM8(a), ADAM9(b), MMP1(c) , MMP2 (d), MMP7 (e), MMP9 (f) and elastase (g) expression was found to be lower than that of the control group not treated with the scrubber (Fig. 3A). Among these proteases, MMP1, MMP2, and MMP9, which are known to be particularly related to wrinkle formation, were analyzed through additional western blot, and as a result, it was confirmed that the expression of the three MMPs was inhibited in both fibroblast cell lines treated with scrubber sap ( Figure 3B). To reconfirm the relationship between PPARγ and MMP, siRNA treatment for PPARγ and western blot for PPARγ, MMP1, MMP2 and MMP9 were performed.As a result, it was confirmed that the expression of the three MMPs was reduced when si-PPARγ was treated. Could.

Taken together, it was found that the scrubber sap can reduce the expression and activity of PPARγ in fibroblasts, thereby inhibiting the expression of MMP, which is related to wrinkle formation.

Experimental Example 4. Analysis of the effect of scrubber sap on the ECM production of fibroblasts

Fibroblasts (Detroit-551 and CCD986-SK) were cultured for 0, 24, 48, 72, and 96 hours, including 0, 1, 3, 5 and 10% (v/v) of scrubber sap, respectively.

To investigate the effect of the scrubber sap on extracellular matrix (ECM) molecules produced by fibroblasts, representative ECM molecules, collagen type I and elastin, were analyzed. First, as a result of comparing the amount of type 1 procollagen, a precursor of type 1 collagen, by sandwich ELISA by concentration of scrubber sap (72 hours treatment), it was increased in a concentration-dependent manner (Fig. 4A), and type 1 procollagen by 10% scrubber sap treatment time and As a result of analyzing the amount of collagen by western blot, it was confirmed that the amount of collagen increased depending on the treatment time (Fig. 4B). Next, as a result of comparing the amount of elastin according to the concentration of the scrubber sap (72 hours treatment) by sandwich ELISA, the amount of elastin increased in a concentration-dependent manner (Fig. 4C), and the amount of elastin for each 10% scrubber sap treatment time was analyzed by western blot. It was shown as a time-dependent increase (FIG. 4D), and it was confirmed that the scrubber sap structurally fills the dermis to increase the elasticity of the skin and increase the amount of extracellular matrix capable of removing wrinkles.

Experimental Example 5. Analysis of the effect of scrubber sap on fat accumulation in adipocytes

First, it was confirmed through analysis using the WST-1 reagent that the scrubber sap did not affect the survival of adipocytes (Fig. 5A).

To investigate the effect of loofah sap on adipocyte differentiation and adipogenesis, the adipocyte progenitor cell line 3T3L1 was cultured in a medium containing MDI (isobutyl-methylxanthine, dexamethasone and insulin) for 6 days to induce differentiation. At this time, the differentiation-inducing culture and the culture without MDI were each cultured by treating the loofah sap at various concentrations, and after the culture for 6 days, the cells were subjected to oil red O staining for analysis. As a result, a large amount of fat was produced in the differentiation-induced adipocyte progenitor cells, and it was found that fat accumulation was increased in a concentration-dependent manner, especially when the scrubber sap was treated (FIG. 5B). As a result of analyzing the lipid droplet area of the differentiation-inducing culture, when 10% of the scrubber sap was added, the area was significantly increased compared to the control without the scrubber sap (Fig. 5C). It was confirmed that it has the effect of promoting differentiation and adipogenesis of progenitor cells.

In order to find out the mechanism of this effect, the intracellular signaling molecules of adipocytes cultured with scrubber sap in differentiation induction medium and MDI-free medium were analyzed by western blot analysis. In the cells to which the scrubber sap was added, the expression of C/EBPα increased and the phosphorylation of ERK1/2 was decreased (Figure 5D), and the scrubber sap inhibited the ERK1/2 signal in adipocytes, and as a result, C/EBPα It was found that by increasing the expression of, differentiation and adipogenesis were induced.

Experimental Example 6. Analysis of the effect of scrubber sap on the function of fibroblasts by UV

Fibroblasts were seeded on a 60mm plate (Nunc, Roskilde, Denmark) and cultured for 2 days. Before UV irradiation, cells with 80% confluency were cultured in 0.2% FBS medium for 18 hours and starvation. During UV irradiation using a UV-A lamp (365 nm, BoTeck, Seoul, Korea), the light source was placed at a distance of 15 cm from the cells, and 6.3 J/cm with a UV-meter (International Light Inc., Newburyport, MA, USA) ^{UV of 2} was applied. After UV irradiation, the contents of collagen and elastin in cells were analyzed by ELISA and western blot, and as a result, the levels were reduced to about 60% and 65%, respectively, compared to the non-irradiated cell group. It was confirmed that the reduction in collagen and elastin concentration-dependently was recovered by the scrubber solution (FIG. 6).

Experimental Example 7. Analysis of the effects of various growth factors on melanogenesis and ROS elimination ability of melanocytes

For melanoma cells (B16F10), FGF (Fibroblast growth factor), HGF (Hepatocyte growth factor), EGF (Epidermal growth factor), TGF (Transforming growth factor), PDGF (Platelet-derived growth factor), VEGF (Vascular endothelial growth factor) ) Was treated with 20 ng/mL, and after 72 hours, the cell pellet was dissolved in a 0.1 M NaOH solution containing 10% DMSO for 1 hour, and the melanin content was measured at 490 nm. As a result, it was found that, among the growth factors, except for FGF and HGF, it was found that it did not have a significant effect on melanin production, and it was confirmed that FGF promotes melanin production and inhibits HGF (FIG. 7).

To confirm the effect of FGF and HGF on the ROS elimination ability of melanoma cells, 20 ng/mL of melanoma cells were treated, and after 0, 12, 24, 48 hours, the Catalase, SMA and SOD1 proteins related to the defense against ROS. Expression was analyzed by western blot. As a result, it was found that FGF decreased the expression of the three proteins as the treatment time increased, while HGF increased (FIG. 8A).

To find out how FGF and HGF affect melanogenesis, melanoma cells were treated with 20 ng/mL of FGF and HGF, and after 0, 12, 24, 48 hours, Tyrosinase, MITF, TP1, proteins involved in melanogenesis. , Western blot analysis of the expression of TRP2. As a result, FGF increased the expression of all four proteins, and HGF was found to decrease the expression of Tyrosinase, TRP1 and TRP2 (FIG. 8B).

Experimental Example 8. Analysis of the effect of scrubber sap on melanogenesis in melanocytes

First, it was confirmed through analysis using the WST-1 reagent that the scrubber sap did not affect the survival of melanoma cells (Figure 9A), but it was found that there was no toxic effect even when the scrubber sap was treated with 10% or more (data not shown).

Melanoma cells were treated with 0, 0.1, 0.3, 0.5, 1, 3, 5, 10% (v/v) of a scrubber solution, and after 72 hours, the cell pellet was placed in a 0.1 M NaOH solution containing 10% DMSO for 1 hour. After melting, the melanin content was measured at 490 nm. As a result, it was confirmed that the production of melanin decreased in a concentration-dependent manner in the scrubber sap (Fig. 9B).

To reveal the mechanism by which the scrubber sap inhibits melanin production, melanoma cells were treated with 10% (v/v) scrubber sap, and after 72 hours, a Phospho-MAPK array (R&D Systems, Minneapolis, MN) was performed according to the manufacturer's manual. . As a result, it was confirmed that phosphorylation of proteins involved in various MAPK signals was inhibited by the scrubber sap (Fig.9C), and to confirm this again, MAPK signaling proteins JNK, CREB, and MAPK signaling proteins in cells treated with the scrubber sap at different concentrations. As a result of confirming the phosphorylation of AKT, it was found that phosphorylation of the three proteins was inhibited in a concentration-dependent manner (Fig. 9D).

Experimental Example 9. Analysis of the effect of scrubber sap on the promotion/inhibition of melanin production by growth factors

In order to find out how the expression of FGF and HGF affecting melanogenesis changes, the mRNA expression was analyzed by age using the GEO database. As a result, it was found that both FGF and HGF mRNA expression levels statistically significantly decreased as age increased (FIGS. 10A-B).

Next, in order to find out how the scrubber sap affects melanin reduction by HGF and melanin increase by FGF, melanoma cells were cultured in a medium containing 50 ng/mL of HGF and FGF and 10% of scrubber sap for XX hours. After the melanin production was confirmed. As a result, the scrubber sap further strengthened the reduction of melanin by HGF and showed an effect of inhibiting melanin production by FGF (FIG. 10C).

Experimental Example 10. Analysis of the effect of scrubber sap on the production of melanin by UV

Melanoma cells were seeded on a 60mm plate (Nunc, Roskilde, Denmark) and cultured for 2 days. Before UV irradiation, cells with 80% confluency were cultured in 0.2% FBS medium for 18 hours and starvation. During UV irradiation using a UV-A lamp (365 nm, BoTeck, Seoul, Korea), the light source was placed at a distance of 15 cm from the cells, and 6.3 J/cm with a UV-meter (International Light Inc., Newburyport, MA, USA) ^{UV of 2} was applied.

First, it was confirmed through analysis using a WST-1 reagent that the scrubber sap and UV treatment did not affect the survival of melanoma cells (FIG. 11A).

Next, the melanoma cells to which 0, 1, 3, 5, 10% (v/v) of scrubber sap was added were treated with UV, and melanin production of each experimental group was analyzed. As a result, it was found that UV-induced melanin production was inhibited in a concentration-dependent manner in the scrubber sap (FIG. 11B), and in summary with the results of Experimental Example 6, the scrubber sap was collagen in fibroblasts induced by UV and It can be seen that it can inhibit the synthesis and degradation of elastin, cell transformation, and melanogenesis of melanocytes.

Experimental Example 11. Analysis of active ingredients in scrubber sap

For HPLC analysis, an HPLC system (Shimadzu Scientific Instruments, Columbia, MD) equipped with a Photo diode array (PDA) detector and Luna C18(2) column (5 μm particle size, 4.6 mm X 250 nm; Phenomenex, Torrance, CA, USA) ) Was used. The analysis was 0 minutes 20% acetonitrile, 10 minutes 40% acetonitrile, 70% 20 minutes acetonitrile, 25 minutes 70% acetonitrile, 27 minutes 20% acetonitrile, 30 minutes 20% acetonitrile in the sequence. Proceed to the step gradient mode of (column flow rate 1 mL/min), and the peak was analyzed at 270 nm.

For MS analysis, the prepared sample was resuspended in 0.1% FA aqueous solution, and then a Q-Exactive Orbitrap hybrid mass spectrometer (Thermo Fisher Scientific) and Ultimate 3000 UPLC system (Thermo Fisher Scientific) were used. For metabolite analysis, an analysis column with an inner diameter of 50 cm x 75 µm filled with 2 µm C18 resin was used.

The capillary was heated to 250° C. and the spray voltage was set to 2.0 kV. Full MS scans were made with a high mass resolution of 70,000 in the range of 100.0 to 1,000.0 m/z. Automatic gain control (AGC) target was 1E6 and maximum injection time (MIT) was 60 ms. For the peaks scanned at 17,500 resolution for m/z 400 MS ² , precursor ions were fragmented with 30 normalized collisional energy (NCE), at this time, the isolation window was 2.0 m/z. The dynamic exclusion setting was set to 90.0 s.

At equilibrium, the flow rate of the column mobile phase solution was 0.15 mL/min. In gradient elution, 0.1% FA aqueous solution was used as mobile phase solution A, and 80% ACN was used as mobile phase solution B, respectively. The gradient step was a total of 60 minutes, consisting of a linear gradient of 12.5% B 20 minutes, 12.5-25.0% B 5 minutes, 25.0-37.5% B 5 minutes, 37.5-80.0% B 6 minutes, and 2.5% B until the end.

As a result, 4-bromo-3-methylisoxazol-5-amine was identified as a major component, and it was calculated that about 8.86% was contained in the scrubber sap from the peak area (FIG. 12).

To determine whether the 4-bromo-3-methylisoxazol-5-amine is an active ingredient inducing the effect of the above-described scrubber sap, the effects of extracellular matrix molecule formation, fat accumulation, whitening, and anti-UV-damage by the substance were examined. saw. In detail, first, as a result of treatment with 4-bromo-3-methylisoxazol-5-amine on fibroblasts, it was found to increase the amount of collagen and elastin in a concentration-dependent manner, especially from 1-3 μM, showing a statistically significant effect ( Figure 13). Next, as a result of treating the adipocytes with 4-bromo-3-methylisoxazol-5-amine, it was found that when the differentiation-induced adipocytes were treated with a scrubber sap, the fat accumulation was increased in a concentration-dependent manner (Fig. 14). . The increase in the accumulation of extracellular matrix molecules and fat by the 4-bromo-3-methylisoxazol-5-amine indicates that the substance is an active ingredient related to wrinkle improvement and prevention. In addition, as a result of treatment with 4-bromo-3-methylisoxazol-5-amine on melanoma cells, concentration-dependent inhibition of melanin production was observed, and the effect of reducing the amount of tyrosinase, MITF, and TRP1 known to increase melanin production. It could be confirmed that there is (Fig. 15), which means that the substance is an active ingredient that induces a whitening effect. Finally, as a result of treatment with 4-bromo-3-methylisoxazol-5-amine on UV-irradiated cells, it was found that concentration-dependently, it recovered the decrease in the amount of collagen caused by UV and also prevented cell transformation (Figure 16). It was confirmed that this is an active ingredient inducing anti-UV-damaging effect.

Finally, as a result of confirming the effect of the substance on cell growth, there was no effect on the growth of fibroblasts, and there was a slight growth inhibition in the case of adipocytes and melanoma cells, but it was found to be insignificant (FIG. 17).

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that other specific forms can be easily modified without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative and non-limiting in all respects. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as being distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the claims to be described later, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be construed as being included in the scope of the present invention.

Claims (10)

Cosmetic composition for anti-wrinkle, anti-wrinkle, anti-UV damage and whitening using a scrubbing brush as an active ingredient.
The method of claim 1,
The cosmetic composition that the scrubber sap is contained in 3 (v/v)% or more in the composition.
The method of claim 1,
The cosmetic composition is a cosmetic composition that increases one or more of the expression of EGFR in fibroblasts, phosphorylation of EGFR, phosphorylation of ERK1/2 and phosphorylation of AKT.
The method of claim 1,
The cosmetic composition is to increase one or more of one or more of collagen in fibroblasts, elastin in fibroblasts, fat in adipocytes and C/EBP in adipocytes.
The method of claim 1,
The cosmetic composition is to recover skin damage induced by UV,
The skin damage is accompanied by a phenomenon comprising at least one selected from the reduction of collagen in fibroblasts, reduction of elastin in fibroblasts, modification of fibroblasts, and melanogenesis of melanocytes.
The method of claim 1,
The cosmetic composition is a cosmetic composition that has one or more effects of promoting reduction of melanin production by HGF and inhibition of increasing melanin production by FGF.
The method of claim 1,
The cosmetic composition that the scrubber sap contains 4-bromo-3-methylisoxazol-5-amine.
The method of claim 1,
The cosmetic composition that the content of 4-bromo-3-methylisoxazol-5-amine in the scrubber sap is 3% or more.
Cosmetic composition for anti-wrinkle, anti-wrinkle, anti-UV damage and whitening using 4-bromo-3-methylisoxazol-5-amine as an active ingredient.
The method of claim 9,
The composition is a cosmetic composition containing 3 to 20 μM of 4-bromo-3-methylisoxazol-5-amine.

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