KR102294967B1 - Cosmetic composition comprising luffa cylindrica sap as active ingredient - Google Patents

Abstract

One embodiment of the present invention provides a cosmetic composition for anti-wrinkle, anti-wrinkle, anti-UV damage and whitening, comprising a loofah sap as an active ingredient. Another embodiment of the present invention provides a cosmetic composition for anti-wrinkle, anti-wrinkle, anti-UV damage and whitening containing 4-bromo-3-methylisoxazol-5-amine as an active ingredient. The composition as described above can produce various effects such as anti-wrinkle, anti-wrinkle, anti-UV damage and whitening while containing only a small amount of loofah sap or its active ingredient, 4-bromo-3-methylisoxazol-5-amine.

Description

A cosmetic composition comprising a loofah sap as an active ingredient {COSMETIC COMPOSITION COMPRISING LUFFA CYLINDRICA SAP AS ACTIVE INGREDIENT}

The present invention relates to a cosmetic composition comprising a loofah sap as an active ingredient, and more particularly, to a cosmetic composition for anti-wrinkle, anti-wrinkle, anti-UV damage and whitening containing loofah sap as an active ingredient.

As an organ that surrounds the outside of the body, the skin plays an important biological role, primarily protecting the body from the environment, but also plays a social role as an element constituting most of the integument visible to others. In all tissues of living things, aging progresses with age, and the skin is no exception. In general, skin aging is divided into intrinsic aging and extrinsic aging. The former refers to aging that occurs naturally according to genetically determined age according to age, and the latter refers to sunlight, pollution (nicotine, etc.), and repetitive muscle use. , refers to the aging phenomenon induced by diet, general health, etc.

A typical phenomenon that occurs with aging of the skin is pigmentation such as wrinkles, blemishes, age spots, and blemishes. In the case of skin wrinkles, the number of fibroblasts present in the dermal layer of the skin and the synthesis ability of these cells decrease due to various causes, resulting in the change and decomposition of the extracellular matrix represented by collagen and elastin and loss of elasticity. It appears when the melanin pigment is excessively produced by abnormalities or ultraviolet rays. Humans have long been pursuing beauty and youth, and in particular, there has always been a need for raw materials that have the effect of slowing down and improving skin wrinkles and pigmentation, which are typical symptoms of external aging as described above. Cosmetics having such an effect are forming a large market, and while many functional cosmetics are being released, the demand for cosmetics having better effects and raw materials for the same is also increasing.

Loofah ( Luffa cylindrica (L.) Roem ) is an annual plant of the Cucurbitaceae family. It grows by developing vine stems and reticulated fibers and forming fruits with black seeds. It has been cultivated for a long time in tropical and subtropical regions of Asia, and has been used as a material for food and beverages and sponges used for kitchen and bath products, depending on the parts such as fruits, skins, seeds, and sap, as well as in folk and traditional medicine for the treatment of various symptoms. It has been used as a medicine for For example, in China, it has been used as an antiparasitic, antipyretic, and gastric agent, and in oriental medicine, it has an effect on menstrual irregularities and is known as a medicine for expectoration, hemostasis, and detoxification.

Loofah was used for skin care in the private sector, and in recent studies, it was reported that the pulp and peel extracts of loofah had anti-inflammatory, antioxidant and skin moisture loss effects. However, the effect of the loofah's sap on the skin has not been specifically identified. In this regard, Korean Patent Application Laid-Open No. 10-2010-0061902 claims that a cosmetic composition containing a loofah sap can prevent itching of atopic dermatitis and help skin regenerate, thereby exhibiting atopic therapeutic effect. No evidence is presented. In addition, Korean Patent Application Laid-Open No. 10-2014-0078070 discloses a composition for improving skin in which oriental medicinal ingredients such as Yugeunpi, wild ginseng, and ginseng are mixed with a loofah sap, but what are the specific effects of skin improvement? Not only is it difficult to know, it is not known from which material the effect is coming.

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

The present invention has been devised to solve the above needs, and an embodiment of the present invention provides a cosmetic composition for anti-wrinkle, anti-wrinkle, anti-UV damage and whitening, comprising a loofah 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 containing 4-bromo-3-methylisoxazol-5-amine as an active ingredient.

The technical problems to be achieved by the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned can be clearly understood by those of ordinary skill in the art to which the present invention belongs from the description below. 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, comprising a loofah sap as an active ingredient.

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

The cosmetic composition may increase at least one of EGFR expression, EGFR phosphorylation, ERK1/2 phosphorylation, and AKT phosphorylation in fibroblasts.

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

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

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

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

The cosmetic composition for anti-wrinkle, anti-wrinkle, anti-UV damage and whitening according to another aspect of the present invention contains 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 having anti-wrinkle, anti-wrinkle, anti-UV damage and whitening effects without skin irritation by containing loofah sap as an active ingredient. Such a composition can produce the same effect as described above while containing a small amount of loofah sap.

It should be understood that the effects of the present invention are not limited to the above-described effects, and 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 loofah sap on the proliferation (A, B), mobility (C), and antioxidant capacity (D, E) of fibroblasts.
2 is data on the effect of loofah sap on signaling in fibroblasts.
3 is data on the effect of loofah sap on the amount of matrix metalloprotase in fibroblasts.
4 is data on the effect of loofah sap on the amount of collagen (A, B) and elastin (C, D) in fibroblasts.
5 is data on the effect of loofah sap on fat accumulation in pre-adipocytes.
6 is data on the effect of loofah sap on the reduction of collagen (A) and elastin (B) in fibroblasts and cell transformation (C) by UV.
7 is data on the effect of each growth factor on the amount of melanin in melanocytes.
8 is data on the effect of FGF and HGF on the expression of antioxidant protein (A) and melanogenesis-related protein (B) in melanocytes, respectively.
9 is data on the effect of loofah sap on the survival of melanocytes (A), the amount of melanin (B), and intracellular signaling (C, D).
Figure 10 is data on the correlation between FGF and HGF mRNA and age (A, B) and the effect of loofah sap on the change in the amount of melanin according to FGF and HGF treatment of melanocytes (C).
11 is data on the effect of the loofah sap on the survival of melanocytes (A) and the increase in the amount of melanin by UV (B).
12 is HPLC and MS/MS result data of loofah sap.
13 is data on the effect of treatment with 4-bromo-3-methylisoxazol-5-amine, a component of the loofah sap, on the amount of collagen and elastin in fibroblasts.
14 is data on the effect of treatment with 4-bromo-3-methylisoxazol-5-amine, a component of loofah sap, on fat accumulation in differentiation-induced adipocytes.
15 is data on the effect of treatment with 4-bromo-3-methylisoxazol-5-amine, a component of the loofah sap, on the amount of melanin in melanoma cells.
16 is data on the effect of treatment with 4-bromo-3-methylisoxazol-5-amine, a component of the loofah sap, on UV-treated fibroblasts.
17 is data on the effect of treatment with 4-bromo-3-methylisoxazol-5-amine, a component of loofah sap, on the proliferation of fibroblasts, preadipocytes and melanoma cells.

Hereinafter, the present invention will be described with reference to the accompanying drawings. However, the present invention may be embodied in several different forms, and thus is not limited to the embodiments described herein. And in order to clearly explain the present invention in the drawings, parts irrelevant to the description are omitted, 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, coupled)” with another part, it is not only “directly connected” but also “indirectly connected” with another member interposed therebetween. "Including cases where In addition, when a part "includes" a certain component, this means that other components may be further provided without excluding other components unless otherwise stated.

The terms used herein are used only to describe specific embodiments, and are not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise. In this specification, terms such as "comprises" or "have" are intended to designate that the features, numbers, steps, operations, components, parts, or combinations thereof described in the specification exist, but one or more other features It should be understood that this does not preclude the existence or addition of numbers, steps, operations, components, parts, or combinations thereof.

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

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

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

The loofah sap may preferably be collected from a Korean native loofah. The sap of the loofah may be obtained by cutting a part having a length of about 1 m from the surface of a loofah grown from the stem of a loofah that has grown to 3 meters or more, and collecting it from this cut. At this time, it is preferable to clean and disinfect the container for receiving the infusion and the stem part inserted into the container, and to collect it in a sealed state to prevent impurities such as rainwater or dust from entering the infusion. It is known that within 6 days, the same quality of sap can be collected from the above-mentioned cuts. 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 loofah 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, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, It may be formulated as an oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto.

More specifically, it may be prepared in the form of a flexible lotion, a nourishing lotion, a nourishing 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, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component. can

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional 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, solubilizer or emulsifier is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylglycol oil, glycerol fatty esters, fatty acid esters of polyethylene glycol or 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, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Adult cellulose, aluminum metahydroxide, bentonite, agar or tracanth may be used.

When the formulation of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether as carrier components Sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester may be used.

When the cosmetic composition of the present invention is a soap, surfactant-containing cleansing or surfactant-free cleansing formulation, 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 not limited thereto.

The cosmetic composition is to increase at least one of EGFR (Epidermal growth factor receptor) expression, EGFR phosphorylation, ERK1/2 (Extracellular signal regulated kinase) phosphorylation, and AKT (AKT serine/threonine kinase) phosphorylation in fibroblasts. can

The skin exists in the outermost layer and is divided into the epithelium, which has waterproof and antibacterial functions, the dermis, which contains connective tissue, which provides elasticity, volume, and structural rigidity, and the subcutaneous, which is mainly composed of fat cells and has an insulation function. In particular, the dermis plays an important role in skin aging. As skin aging progresses, the dermis becomes thinner and collagen and elastin decrease, and these changes result in wrinkling 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 serve as barriers.

In particular, intracellular phenomena that affect the amount of collagen and elastin, which are highly related to wrinkles, may appear in various directions, such as inhibition of synthesis of collagen and elastin, promotion of degradation, reduction of expression, etc. Signal transduction system leading to 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 have various directions such as changes in expression and post-translational modification of proteins such as phosphorylation. has been reported to do. The inventors of the present invention found 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 particularly, among MAPK signaling, EGFR is used as a signal initiator molecule, and it was confirmed that it not only activates EGFR but also increases its expression. When the MAPK signaling is activated, it is known to increase collagen transcription, and the inventors of the present invention have proven through experiments to be described later that MMP, which promotes the degradation of collagen and elastin through PPARγ, is decreased.

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 loofah sap, an active ingredient of the cosmetic composition, can increase the amount of collagen in fibroblasts, the amount of elastin in fibroblasts, and the amount of fat in preadipocytes induced to differentiate into adipocytes in a concentration-dependent manner. In addition, according to an embodiment of the present invention, it was confirmed that the change in the preadipocytes was accompanied by an increase in the amount of C/EBPa and a decrease in phosphorylation of ERK1/2.

The cosmetic composition is to restore the skin damage induced by UV, and the skin damage is a decrease in collagen in fibroblasts, a decrease in elastin in fibroblasts, transformation of fibroblasts and melanin production in melanocytes. It may be accompanied by a phenomenon including one or more selected from among. 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 of melanocytes, and among them, HGF decreases melanogenesis, and FGF has found that it increases. It was found that the loofah sap, an active ingredient of the composition of the present invention, amplifies the melanin-reducing effect of HGF and inhibits the promotion of melanin production of FGF. This indicates that when HGF is additionally included in the cosmetic composition, the whitening effect can be further enhanced.

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

The cosmetic composition for anti-wrinkle, anti-wrinkle, anti-UV damage and whitening according to another aspect of the present invention contains 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, preferably 3 to 10 μM, more preferably 5 to 7 μM.

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

Example 1. Loofah sap collection

After sowing the loofah seeds (Wonjin Herb Farm, Ulsan, Korea) of a Korean native variety, watered once every 2 days and cultivated in the open field to make the plants grow to about 3 meters. The sap of the loofah was collected by cutting the stem 1 m above the root of the loofah and receiving it from the cut part for about 12 hours.

Human dermal fibroblasts (Detroit-551 and CCD986-SK), preadipocytes (3T3-L1) and melanoma cells (B16F10) used in the following cell experiments were obtained from the Korea Cell Line Bank (KCLB). , Seoul, Korea). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) at 5% CO ₂ , 37 °C, and humidity-controlled conditions. At this time, 10% (v/v) fetal bovine serum for human dermal fibroblasts and 10% (v/v) fetal calf serum (Gibco-BRL, Gaithersburg, MD, USA) for preadipocytes were added to DMEM, respectively. , 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 relative pixel intensity was analyzed using ImageJ.

Experimental Example 1. Analysis of the effects of loofah sap on fibroblast proliferation, migration, and ROS

Fibroblasts (Detroit-551 and CCD986-SK) were cultured for 72 hours by including 0, 1, 3, 5 and 10% (v/v) loofah sap in the culture medium, respectively.

To confirm the effect of the loofah sap on the proliferation of fibroblasts, 10 μl of WST-1 reagent (Nalgene, Rochester, NY) was added to the fibroblasts per well, and the absorbance of the culture was adjusted to 450 after 2 hours. nm (Bio-Rad Laboratories, Richmond, CA). As a result, although not statistically significant, cell proliferation was generally more active as the concentration of loofah sap increased (Fig. 1A). Regarding the proliferation of cells, the results of confirming the amount of proliferating cell nuclear antigen (PCNA) in the fibroblast lines by western blot were also the same (Fig. 1B), and the effect of the sap of loofah to promote the proliferation of fibroblasts was able to confirm that there is

In order to check the effect of the loofah sap on the migration of fibroblasts, an experiment was performed according to the manufacturer's protocol using a 24-Transwell device (Corning, NY, USA) for the above two cell lines, and an ELISA reader (Bio-Rad ) to measure the absorbance at 560 nm. As a result, it was confirmed that both Detroit-551 and CCD986-SK showed a statistically significant increase in mobility in a concentration-dependent manner of the loofah sap (Figure 1C).

Next, in order to confirm the effect of the loofah sap on ROS (reactive oxygen species), which is known to be highly related to skin aging such as wrinkle generation, ROS detection reagent (Invitrogen, Carlsbad, CA, USA) in the two fibroblast cell lines The experiment was carried out according to the manufacturer's protocol using did. As a result, it was found that the ROS level of the cells treated with the loofah sap was significantly lower than that of the control group (Fig. 1D). In addition, as a result of western blot for catalase, SOD1 (Superoxide dismutase 1), SMA (Smooth muscle actin) and TRX (Thioredoxin), which are proteins known to lower the oxidative stress in the cells, the result of the loofah sap treatment group It could be confirmed that the expression of the four proteins was increased in the (Fig. 1E), which means that the loofah sap was able to increase the ROS removal ability of the cells.

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

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

As a result of analysis using the phospho-RTK array kit (R&D Systems, Minneapolis, MN) to investigate the effect of the loofah sap on the intracellular signaling system of fibroblasts, it was found that the loofah sap induced phosphorylation of EGFR (Fig. 2A). To investigate in more detail the mechanism of the effect of loofah sap on fibroblasts, 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. As a result of the analysis, it was confirmed that the amount of EGFR, phosphorylation of ERK1/2, and phosphorylation of AKT increased in the loofah sap in a concentration-dependent manner. There were (Figure 2B).

Previously, there was a report on the relationship between EGFR signaling and PPARγ, and the effect of loofah sap on the expression and activity of PPARγ was investigated. Western blot showed that the expression of PPARγ was decreased in the loofah sap-treated group (Fig. 2C), and the PPARγ luciferase reporter assay showed that the activity of PPARγ was also decreased (Fig. 2D). In order to confirm the relevance of EGFR and PPARγ, siRNA for each was treated 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 was found that loofah sap increased the expression and phosphorylation of EGFR in fibroblasts, increased the phosphorylation level of lower signaling molecules, and decreased the expression and activity of PPARγ through EGFR signaling.

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

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

PPARγ and matrix metalloproteases (MMP) and the association between MMP and skin have been reported, respectively. As a result of analysis with human protease array kit (R&D Systems, Minneapolis, MN) to examine the effect of loofah sap on the expression of proteases including MMP in fibroblasts, ADAM8(a), ADAM9(b), MMP1(c) , the expression of MMP2(d), MMP7(e), MMP9(f) and elastase(g) was found to be lower than that of the control group not treated with loofah sap (Figure 3A). As a result of additional western blot analysis of MMP1, MMP2 and MMP9, which are known to be involved in wrinkle formation, among these proteases, it was confirmed that the expression of the three MMPs was inhibited in both fibroblast lines treated with loofah sap ( Figure 3B). In order to confirm the relationship between PPARγ and MMP again, it was confirmed that the expression of the three MMPs was reduced when si-PPARγ was treated as a result of processing siRNA for PPARγ and western blot for PPARγ, MMP1, MMP2 and MMP9. could

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

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

Fibroblasts (Detroit-551 and CCD986-SK) were cultured for 0, 24, 48, 72, and 96 hours, respectively, with 0, 1, 3, 5 and 10% (v/v) loofah sap in the culture medium.

In order to investigate the effect of loofah 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 at each concentration of loofah sap (72 hours treatment), it increased in a concentration-dependent manner (Fig. 4A), and type 1 procollagen and As a result of analyzing the amount of collagen by western blot, it was confirmed that the amount of collagen increased in a time-dependent manner (Fig. 4B). Next, as a result of comparing the amount of elastin for each concentration of loofah sap by sandwich ELISA (processing for 72 hours), it increased in a concentration-dependent manner (Figure 4C). As it was shown to increase in a time-dependent manner (Figure 4D), it was confirmed that the loofah sap could structurally fill the dermis to increase the elasticity of the skin and increase the amount of extracellular matrix that can eliminate wrinkles.

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

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

To investigate the effect of loofah sap on the differentiation and adipogenesis of preadipocytes, the 3T3L1 progenitor cell line 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 treated with various concentrations of loofah sap, respectively, and cultured, and after 6 days of culture, the cells were analyzed by oil red O staining. As a result, it was found that a large amount of fat was produced in the differentiation-induced preadipocytes, and in particular, when the loofah sap was treated, fat accumulation was increased in a concentration-dependent manner (Fig. 5B). As a result of analyzing the lipid droplet area of the differentiation-inducing culture, it was found that when 10% of the loofah sap was added, the area significantly increased compared to the control group without the loofah sap (Fig. 5C). It was confirmed that there was an effect of promoting differentiation of progenitor cells and adipogenesis.

To investigate the mechanism of this effect, western blot analysis of intracellular signaling molecules of preadipocytes cultured with loofah sap in a differentiation induction medium and a medium without MDI was analyzed. In the cells to which the loofah sap was added, the expression of C/EBPa increased and phosphorylation of ERK1/2 was decreased (Figure 5D). It was found that differentiation and adipogenesis were induced by increasing the expression of

Experimental Example 6. Analysis of the effect of loofah sap on UV-induced fibroblast dysfunction

Fibroblasts were seeded on a 60 mm plate (Nunc, Roskilde, Denmark) and cultured for 2 days. Before UV irradiation, cells at 80% confluency were cultured in 0.2% FBS medium for 18 hours for 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 cell, and 6.3 J/cm with a UV-meter (International Light Inc., Newburyport, MA, USA) ^{UV of 2} was applied. As a result of analyzing the intracellular collagen and elastin contents after UV irradiation by ELISA and western blot, they were reduced to about 60% and 65% levels, respectively, compared to the non-irradiated cell group. It was confirmed that these collagen and elastin reductions were recovered by the loofah sap in a concentration-dependent manner (Fig. 6).

Experimental Example 7. Analysis of the effects of various growth factors on melanin production and ROS removal ability of melanocytes

Melanoma cells (B16F10) in 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 0.1 M NaOH solution containing 10% DMSO for 1 hour, and then the melanin content was measured at 490 nm. As a result, it was found that the growth factors except for FGF and HGF did not have a significant effect on melanogenesis, and it was confirmed that FGF promotes melanogenesis and inhibits HGF (FIG. 7).

In order to determine the effect of FGF and HGF on the ROS clearance ability of melanoma cells, after 0, 12, 24, 48 hours after 20 ng/mL treatment on melanoma cells, Catalase, SMA, and SOD1 proteins related to ROS protection were reduced. Expression was analyzed by western blot. As a result, it was shown that FGF decreased the expression of the three proteins as the treatment time increased, whereas HGF increased (Fig. 8A).

To find out how FGF and HGF affect melanogenesis, melanoma cells were treated with FGF and HGF 20ng/mL each and after 0, 12, 24, and 48 hours, Tyrosinase, MITF, and TP1, proteins involved in melanogenesis, , TRP2 expression was analyzed by western blot. As a result, it was found that FGF increased the expression of all four proteins, and HGF decreased the expression of Tyrosinase, TRP1 and TRP2 (Fig. 8B).

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

First, it was confirmed through analysis using WST-1 reagent that the loofah sap did not affect the survival of melanoma cells (Fig. 9A). not shown).

Melanoma cells were treated with 0, 0.1, 0.3, 0.5, 1, 3, 5, 10% (v/v) of loofah sap, and after 72 hours, the cell pellet was incubated with 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 melanin production was decreased in a concentration-dependent manner in the loofah sap (Fig. 9B).

To elucidate the mechanism by which loofah sap inhibits melanin production, melanoma cells were treated with 10% (v/v) of loofah sap, and 72 hours later, Phospho-MAPK array (R&D Systems, Minneapolis, MN) was performed according to the manufacturer's manual. . As a result, it was confirmed that the phosphorylation of proteins involved in various MAPK signals was inhibited by the loofah sap (Fig. 9C). To confirm this again, the MAPK signaling proteins JNK, CREB, As a result of confirming 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 loofah sap on the promotion/inhibition of melanin production by growth factors

To find out how the expression of FGF and HGF that affects melanogenesis changes, the mRNA expression was analyzed by age using the GEO database. As a result, it was found that the mRNA expression levels of both FGF and HGF decreased statistically significantly with increasing age (Fig. 10A-B).

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

Experimental Example 10. Analysis of the effect of loofah sap on UV-induced melanin production

Melanoma cells were seeded on a 60 mm plate (Nunc, Roskilde, Denmark) and cultured for 2 days. Before UV irradiation, cells at 80% confluency were cultured in 0.2% FBS medium for 18 hours for 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 cell, 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 the WST-1 reagent that the loofah sap and UV treatment did not affect the survival of melanoma cells (Fig. 11A).

Next, UV-treated melanoma cells to which 0, 1, 3, 5, and 10% (v/v) of loofah sap were added, melanin production in each experimental group was analyzed. As a result, it was shown that the UV-induced melanin production was inhibited in a concentration-dependent manner in the loofah sap (Fig. 11B). Combining with the results of Experimental Example 6, the loofah sap contained UV-induced fibroblast collagen and It can be seen that both inhibition and degradation of elastin synthesis, cell transformation, and melanin production of melanocytes can be inhibited.

Experimental Example 11. Analysis of active ingredients of loofah sap

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

For MS analysis, the prepared sample was resuspended in 0.1% FA aqueous solution and then 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 high mass resolution of 70,000 in the 100.0 to 100.0 m/z range. The automatic gain control (AGC) target was 1E6 and the maximum injection time (MIT) was 60 ms. For peaks scanned at 17,500 resolution at m/z 400 MS ² , precursor ions were fragmented with 30 normalized collisional energy (NCE), with an isolation window of 2.0 m/z. The dynamic exclusion setting was set to 90.0 s.

The flow rate of the column mobile phase solution at equilibration was 0.15 mL/min. During 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. Gradient step was a total of 60 minutes, 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 a linear gradient of 2.5 % B was performed 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 loofah sap from the peak area (Fig. 12).

In order to find out whether the 4-bromo-3-methylisoxazol-5-amine is an active ingredient that induces the effect of the aforementioned loofah sap, the extracellular matrix molecule formation, fat accumulation, whitening, and anti-UV-damage effects of the substance are examined. saw. In detail, as a result of treatment with fibroblasts with 4-bromo-3-methylisoxazol-5-amine, it was found to increase the amount of collagen and elastin in a concentration-dependent manner. In particular, it showed a statistically significant effect from 1-3 μM ( 13). Next, as a result of treatment with 4-bromo-3-methylisoxazol-5-amine on preadipocytes, it was found that the concentration-dependent increase in fat accumulation in the differentiation-induced preadipocytes was treated with loofah sap (Fig. 14). . The increase in extracellular matrix molecules and fat accumulation 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 in melanoma cells, melanogenesis was inhibited in a concentration-dependent manner, and the effect of decreasing the amount of tyrosinase, MITF and TRP1, which is known to increase melanogenesis, was observed. It could be confirmed that there is (FIG. 15), which means that the material is an active ingredient that induces a whitening effect. Finally, as a result of treatment with UV-irradiated cells with 4-bromo-3-methylisoxazol-5-amine, it was shown that the concentration-dependent reduction in the amount of collagen caused by UV was restored and also prevented cell transformation (FIG. 16), the substance It was confirmed that this is an active ingredient that induces the anti-UV-damage 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 in the case of preadipocytes and melanoma cells, there was a slight growth inhibition, but it was found to be insignificant (FIG. 17).

The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms 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 in all respects and not restrictive. For example, each component described as a single type may be implemented in a dispersed form, and likewise components described as distributed may be implemented in a combined form.

The scope of the present invention is indicated by the following claims, and all changes or modifications 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)

delete delete delete delete delete delete delete delete A cosmetic composition for anti-wrinkle, anti-wrinkle, anti-UV damage and whitening, comprising 4-bromo-3-methylisoxazol-5-amine as an active ingredient.
10. The method of claim 9,
The composition is a cosmetic composition containing 3 to 20 μM of 4-bromo-3-methylisoxazol-5-amine.

Images