KR102257089B1 - Cosmetic composition for moisturizing comprising diphylleia grayi extract - Google Patents

Abstract

The present invention relates to a cosmetic composition for moisturizing, comprising a Diphylleia grayi extract as an active ingredient.

Description

Moisturizing cosmetic composition containing mountain hazel leaf extract {COSMETIC COMPOSITION FOR MOISTURIZING COMPRISING DIPHYLLEIA GRAYI EXTRACT}

The present invention relates to a cosmetic composition for moisturizing, comprising an extract of mountain haze as an active ingredient.

The skin is divided into three layers: the epidermis, the dermis and the subcutaneous fat, and it protects and supports all stimuli from the external environment. In particular, the amount of moisture contained in human skin is about 15% to 20%. It is present in the skin along with oil, carries various physiological activities necessary for the human body, maintains moisture in the skin, and controls the evaporation of moisture out of the body. It plays an important role. Moisture is adequately present in the stratum corneum to give the skin elasticity and flexibility. The skin is composed of four layers of keratin, epidermis, dermis and subcutaneous tissue. Cells and matrices related to skin aging are mainly present in the epidermis and the dermis.

Collagen and elastin, the constituents of the dermis, give the skin strength and tension, and affect skin elasticity. Collagen is synthesized by the action of fibroblasts and is degraded by collagenase, and elastin is degraded by elastase.

As the skin ages, an internal factor is an imbalance of various hormones related to metabolism, and an external factor is an increase in free radicals and free radicals caused by ultraviolet rays, resulting in a decrease in skin thickness, wrinkles, elasticity and moisturizing effect. Falls.

In addition, as the skin ages, the content of collagen, elastin, hyaluronic acid, and glycoproteins, which are skin components, decreases, and oxidative stress occurs. The biosynthesis of matrix metalloproteinase (MMP), an enzyme that degrades skin tissue by inflammatory factors, increases. In other words, the biosynthesis of substrate materials is reduced due to the decrease in cell activity and inflammation due to natural aging, and the pathway of lipid peroxidation due to oxidative stress caused by the external environment causes functional disorders due to accumulation of high lipid peroxides, destruction of the skin substrate and skin aging A phenomenon occurs. Antioxidants such as compounds capable of scavenging free radicals or substances that inhibit peroxide generation are developed as inhibitors or therapeutic agents for aging and various diseases.

In recent years, according to the trend of naturalism, the demand for natural cosmetics is increasing. In order to solve this, as in Korean Patent Laid-Open No. 10-1800880, various extract manufacturing methods have been developed, and accordingly, development of functional foods and cosmetics using natural extracts has been continued. However, development of a natural cosmetic composition having an excellent anti-aging effect on the skin is still insufficient.

Republic of Korea Patent Publication No. 10-1800880

It is an object of the present invention to provide a cosmetic composition comprising one or more uses selected from skin moisturizing, skin whitening, wrinkle improvement, and antioxidant by including a natural material, Sanha leaf extract as an active ingredient.

However, the problem to be solved by the present application is not limited to the problems mentioned above, and other problems that are not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

In order to achieve the above object, the present invention provides a moisturizing cosmetic composition comprising an extract of mountain hazel leaves as an active ingredient.

The moisturizing cosmetic composition according to the present invention provides skin aging prevention effects, including skin whitening, wrinkle improvement, and antioxidants, as well as skin moisturization by including an extract of the mountain hazel leaf.

Specifically, the cosmetic composition according to the present invention may provide a moisturizing effect by increasing the amount of hyaluronic acid produced in cells that regulates skin barrier function and maintains skin elasticity.

In addition, the cosmetic composition according to the present invention may provide a whitening effect by inhibiting the activity of tyrosinase, an enzyme that promotes melanin production.

In addition, the cosmetic composition according to the present invention may provide an antioxidant effect by increasing free radical scavenging activity, which is an oxygen compound that causes aging, arteriosclerosis, and cancer.

In addition, the cosmetic composition according to the present invention can provide an effect of improving wrinkles by inhibiting the intracellular activity of collagenase (MMP-1), a collagen-degrading enzyme that imparts strength and tension to the skin.

The acid lower leaf extract according to the present invention has excellent antioxidant, anti-wrinkle, whitening and moisturizing effects, and can provide a cosmetic composition having an excellent effect when applied to a cosmetic base as an active ingredient.

1 is a graph showing cell viability in human skin fibroblasts according to concentrations of a mountain lower lobe extract according to an embodiment of the present application.
Figure 2 is a graph showing the hyaluronic acid content by concentration of the mountain lower leaf extract according to an embodiment of the present application.
Figure 3 is a graph showing the hyaluronic acid content by concentration of the mountain lower leaf extract according to the Examples and Comparative Examples of the present application.
Figure 4 is a graph showing the tyrosinase activity by concentration of the mountain lower leaf extract according to an embodiment of the present application.
Figure 5 is a graph showing the tyrosinase activity by concentration of the acid lower leaf extract according to the Examples and Comparative Examples of the present application.
Figure 6 is a graph showing the DPPH scavenging activity by concentration of the mountain lower leaf extract according to an embodiment of the present application.
7 is an image showing the DPPH reaction of the solvent control group, the acid lower leaf extract according to the example, and the positive control group in the examples of the present application.
Figure 8 is a graph showing the DPPH scavenging activity by concentration of the mountain lower leaf extract according to the Examples and Comparative Examples of the present application.
9 is a graph showing the cell viability in human skin fibroblasts according to concentrations of the mountain lower lobe extract according to an embodiment of the present application.
10 is a graph showing intracellular collagenase activity according to concentrations of a mountain lower lobe extract according to an embodiment of the present application.
11 is a graph showing intracellular collagenase activity according to concentrations of a mountain lower leaf extract according to Examples and Comparative Examples of the present application.

The present invention relates to a cosmetic composition having a skin moisturizing, skin whitening, anti-wrinkle, antioxidant, and anti-aging effect, comprising a mountain lower leaf extract as an active ingredient, and in particular, a moisturizing cosmetic composition, as well as a moisturizing effect, skin whitening It has anti-wrinkle, anti-oxidation, and anti-aging effects.

Hereinafter, the present invention will be described in detail.

The cosmetic composition for moisturizing of the present invention, as containing the acid lower leaf extract as an active ingredient, the acid lower leaf extract according to the present invention may be extracted through various extraction methods, anti-aging, skin whitening, skin moisturizing, and wrinkles It is most preferable to extract by shaking extraction in terms of the improvement effect.

The cosmetic composition of the present invention may include one or more uses selected from anti-aging, skin whitening, skin moisturizing, and wrinkle improvement, and may have all of anti-aging, skin whitening, skin moisturizing, and wrinkle-improving effects.

The cosmetic composition according to the present invention may act as an accelerator for hyaluronic acid production by including the extract of the mountain lower leaf as an active ingredient, and specifically, by increasing the amount of hyaluronic acid produced in cells that regulates the skin barrier function and maintains skin elasticity. It can have a moisturizing effect.

Furthermore, by filling moisture in the epidermis through the moisturizing effect, the skin contracts, so that the elasticity improvement of the sagging skin can be dramatically improved. In addition, it is possible to improve skin wrinkles and/or improve skin aging by filling the skin wrinkles and constricting the skin.

In addition, the cosmetic composition according to the present invention may act as a melanin production inhibitor by including the acid lower leaf extract as an active ingredient, and specifically, whitening by inhibiting the activity of tyrosinase, an enzyme that promotes melanin production. It can have an effect.

In addition, the cosmetic composition according to the present invention may act as an active oxygen inhibitor by including the acid lower leaf extract as an active ingredient, and specifically, free radicals, which are oxygen compounds that cause aging, arteriosclerosis, and cancer. ) By increasing the DPPH scavenging activity can exhibit antioxidant effects.

In addition, the cosmetic composition according to the present invention may act as a collagen decomposition inhibitor by including an extract of the mountain lower leaf as an active ingredient, and specifically, collagenase (MMP-1), an enzyme that decomposes collagen that imparts strength and tension to the skin. ) Can exhibit the effect of improving wrinkles by inhibiting intracellular activity.

The cosmetic composition of the present invention is a skin ointment, cream, soft lotion, nutrient lotion, pack, essence, hair tonic, shampoo, conditioner, hair conditioner, hair treatment, gel, skin lotion, skin softener, skin toner, astringent, lotion , Milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, eye cream, moisture cream, hand cream, foundation, nutrition essence, sunscreen, sunscreen cream, mist, aerosol, soap, cleansing foam, cleansing lotion, cleansing It may be provided in a formulation selected from the group consisting of cream, body lotion, and body cleanser, but is not limited thereto.

In addition, in the cosmetic composition of each dosage form, other ingredients other than the acid lower leaf extract may be arbitrarily selected and blended according to the dosage form or purpose of use of other cosmetics, and the type and amount of the ingredients may be appropriately selected by those skilled in the art. For example, a colorant (pigment), a flavoring agent (perfume), a suspending agent, an emulsifier, a dissolution aid, a stabilizer, an isotonic agent, a pH adjuster, a viscosity adjuster, a solvent, and the like may be included.

When the formulation of the cosmetic composition according to the present invention is a paste, cream, or gel, animal fibers, plant fibers, wax, paraffin, starch, tracant, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, or zinc acid are used as carrier components. Etc. can be used.

When the formulation of the cosmetic composition according to 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, additional chlorofluoro Propellants such as hydrocarbon, propane/butane or dimethyl ether.

When the formulation of the cosmetic composition according to the present invention is a solution or emulsion, a solvent, a solvating 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 cosmetic composition according to 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 , Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, and the like may be used.

When the formulation of the cosmetic composition according to 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, sarcosy Acid, fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty diethanolamide, vegetable oil, linoline derivative or ethoxylated glycerol fatty acid ester, and the like may be used.

The acid lower leaf extract may be included in an amount of 0.001 to 20% by weight based on the total weight of the composition, and is preferably contained in an amount of 0.01 to 10% by weight. When the content of the mountain lower leaf extract is less than 0.001% by weight, antioxidant, wrinkle improvement, whitening and moisturizing effects cannot be sufficiently obtained, so that the function as a cosmetic composition decreases, and when the content of the mountain lower leaf extract is more than 20% by weight, the It is uneconomical because it can exhibit sufficient efficacy even if it does not contain abnormalities.

In addition, the cosmetic composition of the present invention may further include a pharmaceutically or cosmetically acceptable additive.

In addition, the mountain lower leaf extract according to the present invention can be obtained through various extraction methods, but it is most preferably extracted by shaking extraction, and specifically, the mountain lower leaf extract is 10 to 40° C. after mixing the mountain lower leaf and a solvent. Extracting by aging for 24 hours to 2 weeks can most effectively improve the yield of the extract.

For example, the shaking extraction is more preferably obtained by shaking extraction for 24 to 72 hours at a temperature of 25 to 40 °C to obtain the mountain lower leaf extract, at this time, the shaking extraction is stirred at a speed of 100 to 200 rpm It includes, and is preferably stirred at a speed of 130 to 150 rpm.

There is no particular limitation on the site of use of the lower leaves used for the extraction of the lower leaves, and each or all of the parts such as leaves, stems, flowers, fruits, and roots may be used, and a plant tissue culture may be used. In addition, the lower leaves may be used as they are without any other treatment, but may be extracted after drying or pulverizing treatment in terms of extraction efficiency.

In addition, the solvent may be one selected from the group consisting of water, a lower alcohol having 1 to 4 carbon atoms, propylene glycol, glycerin, and a mixed solvent thereof, but it is most preferable to use water alone.

Specifically, the content of the extraction solvent may be 1 to 50 times, preferably 1 to 20 times, more preferably 5 to 15 times, most preferably 10 times the weight of the solid content of the lower leaves.

In addition, it can be extracted by heating or at room temperature under conditions in which the active ingredient of the extract of the mountain lower leaf is not destroyed or the destruction is minimized. The extraction method may be, for example, shaking extraction, cold needle extraction, ultrasonic extraction, reflux cooling extraction, room temperature extraction, hot water extraction, ultra-high pressure extraction, incubator extraction, etc., depending on the extraction method, but preferably, a shaking extraction method is used. It is most preferred to extract. Filtration is a process of removing suspended solid particles from the extract. The particles may be filtered using cotton, nylon, paper, etc., or ultrafiltration, cryofiltration, centrifugal separation, etc. may be used, but is not limited thereto.

The step of concentrating and then drying the filtrate includes, but is not limited to, freeze drying, vacuum drying, hot air drying, spray drying, vacuum drying, foam drying, high frequency drying, infrared drying, and the like. In some cases, a process of pulverizing the final dried extract may be added.

According to one embodiment of the present invention, the mountain lower leaf extract may be processed and used in various forms, such as an extract, a powder obtained by drying the extract, and a concentrate obtained by concentrating and filtering the extract.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are for explaining the present invention in more detail, and the scope of the present invention is not limited by the following examples.

Example 1: Mountain lower leaf extract preparation 1 (shaking extraction)

100 mL of water was added to 2 g of dried lower leaves, and then sufficiently aged for 24 hours at 140 rpm at 25°C using a shaking incubator (VS-8480SRN, VISION SCIENTIFIC, Korea), and then filtered paper (5C, 185㎜, Toyo Toshi Kaisha, Ltd, Japan), and the filtrate obtained by filtration was concentrated under reduced pressure to obtain an extract of mountain lower leaves.

Comparative Example 1: Preparation 2 of mountain lower leaf extract (ethanol extraction)

2.0 kg of 50 mass% ethanol aqueous solution was added to 100 g of the dried pulverized product of the leaves and/or stems of the lower leaves and extracted for 2 hours at room temperature. The extract was collected by filtration, concentrated under reduced pressure, and then lyophilized to obtain an extract of hawthorn.

Experimental Example 1: Moisturizing power evaluation (in vitro)

The moisture of the skin is maintained by the sebaceous membrane, which is a mixture of lipids produced in the epidermis, and the water-soluble components present in the stratum corneum. It is reported that the stratum corneum of the epidermis contains 15-20% of moisture. Fillagrin secreted from the granular layer and stratum corneum of the epidermis is a differentiation promoting factor, and by agglomerating keratin filament in the stratum corneum, it forms a hard and flat structure of the cornified cell envelope, thereby physically and permeating the skin. Contributes to the barrier function. As filaggrin moves from the lower layer of the stratum corneum to the surface layer, the natural moisturizing factor (NMF), Pyrrolidone carboxylic acid (PCA), is digested by proteolytic enzymes (e.g., amino peptidase, carboxypeptidase, etc.). , Trans-urocanic acid, amino acid, etc. play an important role in skin moisturizing. In addition, moisturizing factors such as Hyaluronic acid (hyaluronic acid, HA), Urea, Citrate, Lactate, and Glycerol also function as a skin moisturizing barrier.

Hyaluronic acid present in the skin is synthesized by Hyaluronic acid synthase (HAS) in fibroblasts and keratinocytes and accumulated in the extracellular matrix. Three types of HAS genes known so far have been reported, including HAS-1, HAS-2, and HAS-3, and the defects in the moisturizing barrier due to their decreased expression are epidermal atrophy, wrinkle formation, skin moisture reduction, and elasticity reduction. It has been reported to cause aging.

Hyaluronic acid is glycosaminoglycans, in which disaccharides of two types of monosaccharides (D-glucuronic acid, N-acetyl-D-glucosamine) are repeated as a unit, and has a polysaccharide structure that forms a negative charge, so it can contain a significant amount of water. There is a characteristic that there is. The production cycle of hyaluronic acid is 2 to 4.5 days, and it combines with moisture that is 1,000 times the weight of itself, regulates the skin barrier function and hydrates the extracellular matrix to maintain the homeostasis of moisture in the tissues to maintain the elasticity of the skin as well as nutrition. It has been reported to be involved in the storage and diffusion of components, and cell migration.

Accordingly, in this experimental example, the amount of hyaluronic acid produced in the cells for the extract of the mountain lower leaf was measured to evaluate the moisturizing power.

1-1. Experiment method

1-1-1. Experimental material

In this experiment, Dulbecco'Modified Eagle', Antibiotic-Antimycotic (Gibco, USA), Trypsin-EDTA (Gibco, USA), Phosphate Buffered Saline (Gibco, USA), Fetal Bovine Serum (FBS; Gibco, USA), 3- (4,5-Dimethylthiazol-2-yl)-2,5 Diphenyl-2H-tetrazolium Bromide (MTT) (Sigma-Aldrich, USA), Retinoic acid (Sigma-Aldrich, USA), Albumin from Bovine Serum (BSA; Sigma -Aldrich, USA), Bradford Reagent (BIORAD, USA), 1 M Tris-HCl pH 8.0 (Elpis-biotech, Korea), Triton X-100 Yakuri Pure Chemicals, Japan), Dimethyl Sulfoxide (DMSO; Amresco, USA), And Human Hyaluronic Acid (HA) ELISA kit (R&D System, USA) was used.

1-1-2. Experimental material treatment concentration and method

A fresh medium to which FBS was not added was used as a solvent using the extract of the mountain lower leaf at a concentration of 100%, and serial dilution was used. At this time, the experiment was performed after confirming that the test material was dissolved in the solvent. For cell viability analysis, the cells were treated so that the final concentrations were 0.0013%, 0.0025%, 0.0049%, 0.0098%, 0.0196%, and 0.0392%, and among them, a concentration that was not toxic to the cells was selected to measure the amount of hyaluronic acid produced in the cells. . As a solvent control, a solvent containing no test material and FBS was used, and retinoic acid was used as a positive control. Retinoic acid was dissolved in DMSO at 0.01% and diluted in a medium containing no FBS.

1-1-3. Cell line selection and cell culture

Human dermal fibroblasts (HDFs; ATCC, USA) are primary cells isolated from adult skin and are used in which Mycoplasma, Hepatitis B, Hepatitis C, HIV-1, Bacteria, Yeast, and other fungi are not detected. I did. HDFs are inoculated on the bottom of a culture dish and then added to a DMEM/F12 3:1 mixed medium containing 1% Antibiotic-Antimycotic and 10% FBS, and then in a 37°C, 5% CO ₂ incubator (HERAcell 150i, Thermo, USA). Cultured in.

1-1-4. Cell viability evaluation method

Cell viability was analyzed using the principle of MTT. HDFs were inoculated into a 24-well plate at a concentration of 5×104 cells/well, cultured for 24 hours, exchanged for FBS-free medium containing the experimental substance diluted for each concentration, and cultured for 24 hours. After culturing for 24 hours, the presence or absence of changes in the shape of cells due to treatment of each concentration of the test substance was observed under a microscope, and then the MTT solution was added to each well so that the final treatment concentration was 0.05%, and cultured for 4 hours. After removing the culture medium, 1,000 μL of DMSO was added and shaken for 10 minutes, and the absorbance was measured at 540 nm (BioTek, USA).

1-1-5. Method for measuring the amount of hyaluronic acid produced in cells

HDFs were inoculated into a 24-well plate at 5×104 cells/well, cultured for 24 hours, exchanged for FBS-free medium containing the experimental substance diluted by concentration, and cultured for 24 hours. After 24 hours, each culture supernatant was centrifuged at 10,000 rpm for 10 minutes, and the amount of hyaluronic acid produced in the medium as a supernatant from which Debris was removed was measured at 450 nm using a Human Hyaluronic Acid (HA) ELISA Kit. The absorbance was measured at. The degree of hyaluronic acid production was evaluated by correcting it with the total amount of protein, and compared with retinoic acid (0.0015%) as a positive control.

1-1-6. Protein quantification

The amount of protein was quantified according to the Bradford protein quantification method using BSA as a standard solution. BSA is gradually diluted with distilled water to 0, 2, 5, 10, 20 μg/mL, and 20 μL of each concentration of BSA solution is added to 0.98 mL of Bradford reagent and reacted for 5 minutes at 595 nm. The absorbance was measured to obtain a BSA standard curve. In the same way, the absorbance of a mixture of HDFs cultured in a 24-well plate and a mixture of Bradford reagent was measured, and the amount of protein was calculated using a BSA standard curve.

1-2. Experiment result

All experimental results were expressed as mean value ± standard deviation (n=3), and statistical analysis was performed using the SPSS® Package Program (IBM, USA). All results were the results of three or more independent experiments, and statistical significance was verified at the p<0.05 significance level by t-test of independent samples.

1-2-1. Cell viability analysis

As can be seen in Table 1 and FIG. 1 below, it was confirmed that there was no change in cell shape when compared with the solvent control at a concentration of 0.0025% or less of the acid lower leaf extract, and it was also found that there was no cytotoxicity.

matter Concentration (%) Cell viability (%) Standard Deviation p-value Compared to the solvent control group (%) (Solvent control group) 0 100.00 0.00 - - Mountain lower leaf extract
(Example 1) 0.0013 99.54 0.76 0.358 0.46▽ 0.0025 99.62 0.34 0.125 0.38▽ 0.0049 96.86 1.23 0.011 3.14▽ 0.0098 91.16 1.16 0.000 8.84▽ 0.0196 83.14 2.37 0.000 16.86▽ 0.0392 58.09 1.26 0.000 41.91▽ Retinoic acid
(Positive control group) 0.0015 99.86 0.93 0.558 0.14▽

1-2-2. Analysis of intracellular hyaluronic acid production

As can be seen in Table 2 and FIG. 2 below, the amount of hyaluronic acid produced increased as the concentration of the lower leaf extract increased. The production of hyaluronic acid (HA) was significantly increased (p<0.05).

matter Concentration (%) HA concentration
(ng/μg) Standard Deviation p-value Compared to the solvent control group (%) (Solvent control group) 0 54.46 1.07 - - Mountain lower leaf extract
(Example 1) 0.0001 56.88 3.17 0.279 4.45▲ 0.0002 58.33 4.31 0.205 7.10▲ 0.0004 58.43 1.33 0.016 7.28▲ 0.0007 64.10 1.66 0.001 17.69▲ 0.0013 65.61 2.10 0.001 20.47▲ 0.0025 67.75 2.43 0.001 24.40▲ Retinoic acid
(Positive control group) 0.0015 69.20 2.26 0.001 27.07▲

1-2-3. Comparison of intracellular hyaluronic acid production according to the extraction method

As can be seen in Table 3 and FIG. 3, the amount of hyaluronic acid produced in the cells was further increased by the extract of the lower leaf extract extracted by the shaking extraction method according to Example 1 compared to the extract of the lower leaf extract extracted by the ethanol extraction method of Comparative Example 1. .

matter Concentration (%) Comparative Example 1 (ethanol extraction) Example 1 (shaking extraction) HA concentration
(ng/μg) Standard Deviation HA concentration
(ng/μg) Standard Deviation Mountain lower leaf extract 0 53.91 0.09 54.46 1.07 0.0001 54.90 0.72 56.88 3.17 0.0002 55.82 0.33 58.33 4.31 0.0004 57.08 0.82 58.43 1.33 0.0007 59.64 0.73 64.1 1.66 0.0013 61.86 0.66 65.61 2.1 0.0025 62.53 0.88 67.75 2.43

Experimental Example 2: Evaluation of whitening effect (in vitro)

Skin color is determined by the amount and type of melanin synthesized by melanin-producing cells located at the boundary between the epidermis and the dermis. When the physiological function of the skin decreases due to overproduction of melanin, melanin pigment is deposited on the skin surface, resulting in hyperpigmentation.

Causes of hyperpigmentation are caused by ultraviolet rays and heat. Among these, when the skin is exposed to UV rays for a long time, factors promoting melanogenesis such as α-Melanocyte stimulating hormone (α-MSH), Endothelin-1 (ET-1), and Nitric oxide (NO) are secreted from keratinocytes. Α-MSH, one of the factors controlling melanogenesis and skin pigmentation, is one of the locally produced Melanocortin peptides. When α-MSH is attached to the Melanocortin-1 receptor (MC1R), Adenylate cyclase is activated in the cell, increasing the concentration of intracellular cyclic adenosine monophosphate (cAMP), and melanin through the protein kinase A (PKA) cell signaling pathway. It activates tyrosinase, an enzyme that regulates the rate of production pathways.

Melanin production occurs in melanosomes, and melanosomes are organs wrapped with specific endoplasmic reticulum originating from the endoplasmic reticulum. During the development of melanosomes, tyrosinase, Tyrosinase-related protein-1 (TRP-1) and Tyrosinase-related protein-2 (TRP-2) are produced, among which tyrosinase is from L-tyrosine to Dopaquinone. It is a key enzyme involved in melanogenesis, which accelerates the transformation process.

Tyrosinase has several enzymatic activities such as tyrosine hydroxylase, dihydroxyphenylalanine (DOPA) oxidase, and 5,6-dihydroxyindole (DHI) oxidase. Skin whitening effect appears by inhibiting all or part of the enzyme activities such as tyrosinase, TRP-1, TRP-2, and peroxidase, among which tyrosinase and TRP-1 are related to the biosynthesis of eumelanin, which has a dark brown color. It is a key enzyme protein. Most skin whitening materials specifically act on tyrosinase through various mechanisms, preventing their function.

As such, melanin is produced through auto-oxidation and enzymatic oxidation using Tyrosine as a substrate, so tyrosinase inhibitors are used as a whitening substance and treatment for abnormal pigmentation. In addition, tyrosinase inhibition assay and DOPA (3-4-dioxyphenylalanine) oxidation inhibition assay are widely used to verify the whitening effect of the material. These two experimental methods are tyrosinase-induced Tyrosine to DOPA and DOPA to DOPA-chrome. Based on the oxidation reaction, the degree of inhibition is evaluated by the amount of the final reaction product, DOPA-chrome.

Therefore, it is believed that the effect of the skin whitening material can be supported by evaluating the inhibition of the activity of tyrosinase, and thus, in this experimental example, the activity of mushroom tyrosinase against the extract of the lower lobe was measured to evaluate the whitening effect.

2-1. Experiment method

2-1-1. Experimental material

Phosphate Buffered Saline (PBS; Gibco, USA), Tyrosinase from mushroom (Sigma-Aldrich, USA), L-tyrosine (Sigma-Aldrich, USA), and Arbutin (Sigma-Aldrich, USA) were used in this experiment.

2-1-2. Experimental material treatment concentration and method

The acid lower leaf extract was used at a concentration of 100%, followed by serial dilution using PBS as a solvent. At this time, the experiment was performed after confirming that the test material was dissolved in the solvent. The test material was diluted to 0.1%, 0.21%, 0.42%, 0.83%, 1.67%, 3.33%, and 6.66% of final concentration using PBS, and the ability to inhibit mushroom tyrosinase activity was measured. As a solvent control group, PBS containing no test material was used, and as a positive control group, arbutin was used. Arbutin used as a positive control was dissolved in D.W. at 5 mg/mL and diluted with PBS to be used.

2-1-3. Measurement of tyrosinase activity inhibition

In each well of a 96-well plate, 220 μL of 0.1 M PBS (pH 6.8), 20 μL of sample solution, and 20 μL of mushroom tyrosinase solution (2,000 U/mL) were sequentially added. 40 μL of 1.5 mM tyrosine solution was added to this solution, reacted at room temperature for 15 minutes, and absorbance was measured at 490 nm (BioTek, USA). As a solvent control group, PBS was added instead of the test material, and compared with Arbutin (0.01%) as a positive control group.

2-2. Experiment result

All data were expressed as mean value ± standard deviation, and statistical analysis was performed using the SPSS® Package Program (IBM, USA). All results were the results of three or more independent experiments, and statistical significance was verified at the p<0.05 significance level by t-test of independent samples.

2-2-1. Analysis of tyrosinase activity inhibition

As can be seen in Table 4 and FIG. 4, the activity of tyrosinase decreased as the concentration of the lower leaf extract increased, and specifically, the lower leaf extract was from 7.44% to 68.27% at a concentration of 0.21% to 6.66%. Until then, the activity of tyrosinase was significantly decreased in a concentration-dependent manner (p<0.05).

matter Concentration (%) Tyrosinase activity (%) Standard Deviation p-value Compared to the solvent control group (%) (Solvent control group) 0 100 0 - - Mountain lower leaf extract
(Example 1) 0.1 97.75 0.95 0.07 2.25▼ 0.21 92.56 0.1 0.000 7.44 ▼ 0.42 91.53 1.47 0.003 8.47 ▼ 0.83 85.31 0.73 0.033 14.69▼ 1.67 63.52 1.31 0.001 36.48▼ 3.33 42.77 4.24 0.000 57.23▼ 6.66 31.73 2.23 0.001 68.27 ▼ Arbutin
(Positive control group) 0.01 64.55 0.94 0.000 35.45▼

2-2-2. Comparison of tyrosinase activity inhibition by extraction method

As can be seen in Table 5 and Figure 5 below, compared to the extract extracted by the ethanol extraction method of Comparative Example 1, the extract extracted by the shaking extraction method according to Example 1 of the present application further reduced the activity of tyrosinase. .

matter Concentration (%) Comparative Example 1 (ethanol extraction) Example 1 (shaking extraction) Tyrosinase activity (%) Standard Deviation Tyrosinase activity (%) Standard Deviation Mountain bottom
extract 0 100 0 100 0 0.1 97.81 0.63 97.75 0.95 0.21 95.51 0.38 92.56 0.1 0.42 93.27 0.70 91.53 1.47 0.83 89.57 0.63 85.31 0.73 1.67 76.31 0.70 63.52 1.31 3.33 67.27 1.04 42.77 4.24 6.66 53.89 2.16 31.73 2.23

Experimental Example 3: Evaluation of antioxidant effect (in vitro)

Antioxidation means protecting the body from oxidative stress. Oxidants, which induce oxidative stress, are naturally generated during normal metabolic processes such as the process of generating energy using nutrients and immune reactions that destroy bacteria or viruses that invade from the outside. Oxidation-causing substances are very unstable, and if they are generated or accumulated more than necessary, they damage DNA and cells, and as a result, cause harmful effects such as aging and chronic diseases. It exists, so it doesn't matter.

, Hydroxyl radical (OH

, Hydrogen peroxide (H₂O₂), Singlet oxygen (O2)과 같이 매우 높은 것에 이르기까지 다양하다. 이들은 생체에 치명적인 산소 독성을 일으키며, 세포막 분해, 단백질 분해, 지질산화, DNA 변성 등을 초래하여 세포의 기능장애를 유발하고 암을 비롯한 뇌졸중, 파킨슨병 등의 뇌질환과 심장질환, 동맥경화, 염증, 노화, 자가면역질환 등의 각종 질병을 일으키는 것으로 알려져 있다. Free radicals are oxygen compounds produced during the metabolism of cells in the body of animals and plants, and are known to be related to the causes of aging, arteriosclerosis, and cancer. The reactivity of free radicals ranges from very low like oxygen molecules to Superoxide radicals (O₂

, Hydroxyl radical (OH

, Hydrogen peroxide (H ₂ O ₂ ), and singlet oxygen (O2). They cause fatal oxygen toxicity to the living body, cause cell membrane decomposition, protein degradation, lipid oxidation, and DNA degeneration, leading to cell dysfunction, brain diseases such as cancer, stroke, Parkinson's disease, heart disease, arteriosclerosis, and inflammation. , It is known to cause various diseases such as aging and autoimmune diseases.

In particular, it is known that accumulation of lipid peroxide generated by attacking unsaturated fatty acids, which is a constituent of a biological membrane, causes a decrease in biological function, aging, and adult diseases. DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2'-azino-bis (3-ethylbe nzthiazoline-6-sulfonic acid)) can be used to search for such free radical scavenging activity.

Among them, DPPH is a relatively stable free radical with a dark purple color. It is reduced and decolored by sulfur amino acids such as Cysteine and Glutathione, ascorbic acid, and BHA, so its antioxidant activity can be easily observed with the naked eye. It is widely used to search.

Accordingly, in this experimental example, the antioxidative effect was evaluated by measuring the DPPH free radical scavenging ability for the extract of the mountain lower leaves.

3-1. Experiment method

3-1-1. Experimental material

In this experiment, 2,2-diphenyl-1-picrylhydrazyl (DPPH, Sigma-Aldrich, USA), L-ascorbic acid (Sigma-Aldrich, USA) and Methanol (Merck, Germany) were used.

3-1-2. DPPH free radical scavenging ability measurement method

To evaluate the antioxidant activity, the free radical scavenging rate was measured using DPPH. DPPH was prepared and used at a concentration of 300 μM, and the test material was diluted in distilled water so that the final concentrations were 0.04%, 0.08%, 0.16%, 0.31%, 0.63%, 1.25%, 2.5%, and 5%. After adding 100 μL of each test substance and 100 μL of DPPH solution to a 96-well plate in order, react in an oven at 37°C (JSOF-150, JSR, Korea) for 30 minutes and use a Microplate reader (BioTek, USA). Absorbance was measured at 517 nm. Distilled water was used instead of the test material as the solvent control group, and ascorbic acid (0.005%) was used as the positive control group.

3-2. Experiment result

All data were expressed as mean value ± standard deviation, and statistical analysis was performed using the SPSS® Package Program (IBM, USA). All results were the results of three or more independent experiments, and statistical significance was verified at the p<0.05 significance level by t-test of independent samples.

3-2-1. DPPH free radical scavenging ability analysis result

As can be seen in Table 6 and FIG. 6 below, DPPH scavenging ability increased as the concentration of the mountain lower leaf extract increased. It showed significant DPPH free radical scavenging activity (p<0.05, see FIGS. 6 and 7).

matter Concentration (%) DPPH scavenging activity (%) p-value 1st 2nd 3rd Mean±standard deviation (Solvent control group) 0 0 0 0 0.00±0.00 - Mountain lower leaf extract
(Example 1) 0.04 3.3 2.78 4.42 3.50±0.84 0.002* 0.08 10.85 10.15 10.94 10.65±0.43 0.000* 0.16 27.13 26.18 27.55 26.95±0.70 0.000* 0.31 41.49 42.31 42.59 42.13±0.57 0.000* 0.63 63.4 62.93 63.3 63.21±0.25 0.000* 1.25 84.26 84.19 84.44 84.29±0.13 0.000* 2.5 91.38 91.45 91.38 91.40±0.04 0.000* 5 92.23 91.35 92.32 91.97±0.54 0.000* Ascorbic acid
(Positive control group) 0.005 89.15 89.1 89.59 89.28±0.27 0.000*

* p-value: Significant difference at p<0.05 compared to the solvent control group

3-2-2. Comparison of DPPH free radical scavenging ability according to extraction method

As can be seen in Table 7 and Figure 8 below, compared to the extract extracted by the ethanol extraction method of Comparative Example 1, the extract extracted by the shaking extraction method according to Example 1 of the present application further improved DPPH scavenging ability.

matter Concentration (%) Comparative Example 1 (ethanol extraction) Example 1 (shaking extraction) DPPH clearing
activation (%) Standard Deviation DPPH clearing
activation (%) Standard Deviation Mountain bottom
extract 0 0 0 0 0 0.04 1.39 0.13 3.5 0.84 0.08 4.97 0.12 10.65 0.43 0.16 12.96 0.40 26.95 0.7 0.31 22.80 1.10 42.13 0.57 0.63 40.96 0.34 63.21 0.25 1.23 61.76 0.44 84.29 0.13 2.5 75.90 0.25 91.4 0.01 5 86.84 0.38 91.97 0.54

Experimental Example 4: Evaluation of wrinkle improvement effect (in vitro)

Skin wrinkles are caused by an imbalance in the synthesis and decomposition of collagen. Collagen gives strength and tension to the skin, thereby protecting the skin from external stimuli or forces.

Collagen occupies 70-80% of the dermis layer, and Type I, III collagen is the main component of human dermis, and Type I accounts for 80% of total collagen, and Type III accounts for 15%. Collagen is synthesized in the form of a precursor called pro collagen, and contains a peptide sequence called propeptide at the amino and carboxyl ends, which are known to be separated from the collagen molecule when collagen polymerization occurs. Therefore, by measuring the amount of propeptide, the degree of intracellular collagen biosynthesis can be predicted. Effective substances and necessary factors for collagen synthesis include vitamin A (Retinol), which is effective for inhibiting collagen synthesis and decomposition, and vitamin C (Ascorbic acid), which is essential for collagen synthesis and is effective for pigmentation.

Collagen is decomposed by the action of collagen-degrading enzymes (Matrix metalloproteinases, MMPs), so in order to prevent skin wrinkles, it is necessary to increase collagen synthesis and inhibit the action of collagen-degrading enzymes (MMPs). Collagen-degrading enzymes (MMPs) are secreted from many cells, including keratinocytes and fibroblasts in the skin, and support the extracellular matrix (ECM) and basement membrane (BM). It is involved in the breakdown of the major protein components that make up. In addition, according to its structure and functional characteristics, it is divided into four subfamily: Collagenase (MMP-1), Stromelysin (MMP-3), Gelatinase (MMP-2, -9), and Membrane type MMPs (MT-MMPs).

The decrease in connective tissue in the dermis is caused by Collagenase (MMP-1), Gelatinase (MMP-2, -9), etc., and MMP-1 is a protease that acts specifically on collagen, and the activity of MMP-1 It is known that suppressing collagen degradation can reduce the breakdown of collagen to maintain the elasticity of skin tissue and prevent the formation of wrinkles. In addition, studies on the regulatory mechanisms and inhibitors of MMPs are increasing. Retinoic acid, vitamin A (Retinol) derivatives, green tea extract and its main components (-)-Epigallocatechin-3-gallate (EGCG), TGF-β, etc. It is known as a very effective defense material.

Accordingly, in this experimental example, the ability to inhibit collagenase (MMP-1) activity with respect to the extract of the mountain lower leaf was measured to evaluate the effect of improving wrinkles.

4-1. Experimental method

4-1-1. Experimental material

In this experiment, Dulbecco'Modified Eagle'Saline (Gibco, USA), Fetal Bovine Serum (FBS; Gibco, USA), 3-(4,5-Dimethylthiazol-2-yl)-2,5 Diphenyl-2H-tetrazolium Bromide (MTT) (Sigma-Aldrich, USA), TGF-β (Sigma-Aldrich, USA), Albumin from Bovine Serum (BSA; Sigma-Aldrich, USA), Bradford Reagent (BIORAD, USA), 1 M Tris-HCl pH 8.0 (Elpis-biotech, Korea), Triton X-100 Yakuri Pure Chemicals, Japan), and Dimethyl Sulfoxide (DMSO; Amresco, USA) were used.

4-1-2. Experimental material treatment concentration and method

A new medium to which FBS was not added was used as a solvent with 100% concentration of the mountain lower leaf extract, and serial dilution was used. At this time, the experiment was performed after confirming that the test material was dissolved in the solvent. Cells were treated to be 0.0013%, 0.0025%, 0.0049%, 0.0098%, 0.0196%, and 0.0392% for cell viability analysis, and intracellular collagenase activity was measured by selecting a concentration that was not toxic to cells. I did. As the solvent control group, a solvent containing no test material and FBS was used, and as a positive control group, TGF-β was used. TGF-β was dissolved in D.W. at 10 μg/mL and diluted in FBS-free medium to be used.

4-1-3. Cell line selection and cell culture

Human dermal fibroblasts (HDFs, ATCC, USA) were used as primary cells isolated from adult skin, in which Mycoplasma, Hepatitis B, Hepatitis C, HIV-1, Bacteria, Yeast, and other fungi were not detected. HDFs are inoculated on the bottom of a culture dish and then added to a DMEM/F12 3:1 mixed medium containing 1% Antibiotic-Antimycotic and 10% FBS, and then in a 37°C, 5% CO ₂ incubator (HERAcell 150i, Thermo, USA). Cultured in.

4-1-4. Cell viability evaluation method

Cell viability was analyzed using the principle of MTT. HDFs were inoculated into a 24-well plate at a concentration of 5×104 cells/well, cultured for 24 hours, exchanged for FBS-free medium containing the experimental substance diluted for each concentration, and cultured for 48 hours. After 48 hours of cultivation, the presence or absence of changes in the shape of cells due to the treatment of each concentration was observed under a microscope, and then the MTT solution was added to each well so that the final treatment concentration was 0.05%, and incubated for 4 hours. After removing the culture medium, 1,000 μL of DMSO was added and shaken for 10 minutes, and the absorbance was measured at 540 nm (BioTek, USA).

4-1-5. Method for measuring intracellular collagenase (MMP-1) activity inhibition

HDFs were inoculated into a 24-well plate at 5×104 cells/well and cultured for 24 hours. After incubation, it was exchanged for a medium containing no FBS containing the experimental substance diluted for each concentration, and cultured for 48 hours. After incubation for 48 hours, the supernatant was centrifuged at 10,000 rpm for 10 minutes, and the absorbance was measured at 450 nm using the MMP-1 Human ELISA Kit as the supernatant from which debris was removed. MMP-1 activity was evaluated by correcting for the total amount of protein, and compared using TGF-β (final treatment concentration 0.000001%) as a positive control.

4-1-6. Protein quantification

The amount of protein was quantified according to the Bradford protein quantification method using BSA as a standard solution. BSA is gradually diluted in distilled water to 0, 2, 5, 10, 20 μg/mL, and 20 μL of each concentration of BSA solution is added to 0.98 mL of Bradford reagent and reacted for 5 minutes at 595 nm. The absorbance was measured to obtain a BSA standard curve. In the same way, the absorbance of a mixture of HDFs cultured in a 24-well plate and a mixture of Bradford reagent was measured, and the amount of protein was calculated using a BSA standard curve.

4-2. Experiment result

All data were expressed as mean value ± standard deviation, and statistical analysis was performed using the SPSS® Package Program (IBM, USA). All results were the results of three or more independent experiments, and statistical significance was verified at the p<0.05 significance level by t-test of independent samples.

4-2-1. Cell viability analysis result

As can be seen in Table 8 and FIG. 9 below, it was confirmed that there was no change in cell shape when compared with the solvent control group at a concentration of 0.0025% or less of the acid lower leaf extract, and it was also found that there was no cytotoxicity.

matter Concentration (%) Cell viability (%) Standard Deviation p-value Compared to the solvent control group (%) (Solvent control group) 0 100 0 - - Mountain lower leaf extract
(Example 1) 0.0013 100.57 0.28 0.024* 0.57▲ 0.0025 98.94 1.17 0.19 1.06▽ 0.0049 96.28 1.19 0.006* 3.72▽ 0.0098 89.99 1.46 0.000* 10.01▽ 0.0196 80.47 1.92 0.000* 19.53▽ 0.0392 52.99 1.28 0.000* 47.01▽ TGF-β1
(Positive control group) 0.000001 102 1.81 0.128 2.00▲

* p-value: Significant difference at p<0.05 compared to the solvent control group

4-2-2. Intracellular collagenase (MMP-1) activity inhibition assay results

As can be seen in Table 9 and FIG. 10 below, as the concentration of the acid lower leaf extract was increased, intracellular collagenase activity was inhibited, and specifically, the acid lower leaf extract was 8.41% compared to the solvent control group at a concentration of 0.0013% to 0.0025%. Intracellular collagenase activity was significantly reduced by 14.77% (p<0.05).

matter Concentration (%) MMP-1 concentration (pg/μg) Standard Deviation p-value Compared to the solvent control group (%) (Solvent control group) 0 9967.7 218.21 - - Mountain lower leaf extract
(Example 1) 0.0001 10156.3 110.8 8 0.253 1.89△ 0.0002 10242 42.02 0.099 2.75△ 0.0004 10040.4 72.48 0.613 0.73△ 0.0007 9632.6 295.64 0.189 3.36▼ 0.0013 9129.2 134.66 0.005* 8.41 ▼ 0.0025 8495.8 450.54 0.007* 14.77▼ TGF-β1
(Positive control group) 0.000001 4330.3 215.68 0.000* 56.56▼

* p-value: Significant difference at p<0.05 compared to the solvent control group

4-2-3. Comparison of inhibitory ability of intracellular collagenase (MMP-1) activity according to extraction method

As can be seen in Table 10 and Figure 11 below, compared to the extract of the lower lobe extracted by the ethanol extraction method of Comparative Example 1, the extract of the lower lobe extracted by the shaking extraction method according to Example 1 of the present application further reduced intracellular collagenase activity Made it.

matter Concentration (%) Comparative Example 1 (ethanol extraction) Example 1 (shaking extraction) MMP-1 concentration (pg/μg) Standard Deviation MMP-1 concentration (pg/μg) Standard Deviation Mountain bottom
extract 0 10088.8 54.77 9967.7 218.21 0.0001 10202.4 90.25 10156.3 110.88 0.0002 10198.4 84.00 10242 42.02 0.0004 10032.6 116.99 10040.4 72.48 0.0007 9950.1 46.41 9632.6 295.64 0.0013 9878.9 65.04 9129.2 134.66 0.0025 9691.1 58.99 8495.8 450.54

Example 2: Preparation of a cosmetic composition of a cream formulation containing a mountain hazel leaf extract

In the cosmetic formulation, the formulation of the cream formulation as a formulation example of the cosmetic composition containing the acid lower leaf extract of Example 1 is shown in Table 11 below as Example 2.

Comparative Examples 2 and 3: Preparation of a cosmetic composition of a cream formulation containing no mountain lower leaf extract

In order to confirm the effect of the mountain lower leaf extract, as shown in Table 11 below, a cosmetic composition in a cream formulation not containing the mountain lower leaf extract was used as Comparative Example 2, and containing an aloe extract known to have a conventional moisturizing effect. The cosmetic composition of the cream formulation was used as Comparative Example 3.

ingredient
(Unit: wt%) Example 2
(Sanha leaf extract
Containing cream) Comparative Example 2
(Sanha leaf extract
Non-contained cream) Comparative Example 3
(Aloe extract
Containing cream) Mountain lower leaf extract 2.00 - - Aloe extract - - 2.00 Butylene Glycol 2.00 2.00 2.00 glycerin 4.00 4.00 4.00 Pota?㎸舅絹藥矩瑛絹? 0.10 0.10 0.10 Tocopheryl acetate 1.0 1.0 1.0 Floating paraffin 5.0 5.0 5.0 Squalane 5.0 5.0 5.0 Macadamia Nut Oil 10.0 10.0 10.0 Polysorbate 60 1.0 1.0 1.0 Sorbitan Sesquioleide 1.0 1.0 1.0 Carbomer 0.3 0.3 0.3 antiseptic 0.5 0.5 0.5 incense 0.5 0.5 0.5 Purified water Balance Balance Balance Sum 100.00 100.00 100.00

Experimental Example 5: Evaluation of the moisturizing effect of a cream-formed cosmetic composition

In order to investigate the moisturizing effect of the cosmetic composition comprising the extract of the mountain lower leaf according to the present invention, the moisture content of the skin was measured using a skin moisture meter (Corneometer CM 825, Germany).

Skin moisture before and after application to 20 women aged 20 to 50 after preparing the formulation containing the acid lower leaf extract (Example 2) and the formulation not containing (Comparative Example 2) as shown in Table 12 The content was measured as follows.

From 1 hour before the start of the test, uniformly apply ^{each sample at a concentration of 2.0 mg/cm 2} to the upper arm of the applicant who has been waiting indoors under constant temperature and humidity conditions (20 to 22°C, relative humidity 40 to 60%). The other upper arm that was not applied was used as a control. The skin moisture content and the skin moisture loss amount immediately before application were measured, and the skin moisture content at 1 hour, 3 hours, and 5 hours after application was measured, respectively, and are shown in Table 12 below.

Kinds Elapsed time (hr) Application site
Moisture content Non-applied area
Moisture content Non-application
Part contrast Example 2
(Sanha leaf extract
Containing cream) 0 91 81 10▲ One 108 78 30▲ 3 116 80 36▲ 5 90 79 11▲ Comparative Example 2
(Sanha leaf extract
Non-contained cream) 0 82 76 6▲ One 92 79 13▲ 3 93 81 12▲ 5 88 80 8▲ Comparative Example 3
(Aloe extract
Containing cream) 0 85 77 8▲ One 95 79 16▲ 3 96 80 16▲ 5 92 79 13▲

As shown in Table 12, it can be confirmed that the cream containing the mountain lower leaf extract according to the present invention (Example 2) has an excellent moisturizing effect compared to the cream containing no extract or aloe extract (Comparative Examples 2 and 3). there was. In particular, in the case of applying the cream according to Example 2, the skin moisture content significantly increased when 1 to 3 hours elapsed after application.

Experimental Example 6: Evaluation of the effect of improving the elasticity of a cream-formed cosmetic composition

In order to evaluate the effect of improving skin elasticity of the cosmetic composition comprising the extract of the mountain lower leaf according to the present invention, an experiment was conducted as follows.

Thirty women aged 20 to 50 were divided into three groups of 10 women each, and the subjects were thoroughly absorbed by evenly applying cream-type cosmetic formulations to the face after washing their faces every morning and evening for 4 weeks (1 month).

The elasticity of the facial area was measured using a cutometer (SEM474, Courage+Khazaka electronic GmbH. Germany) that can measure skin elasticity, and before and after the use of cosmetics was compared after 1 month of use.

The results of the clinical trials 1 month after the use of cosmetics are shown in Table 13 below, and the skin elasticity improvement values in Table 13 are the average values of each group.

Classification Skin elasticity improvement (%) Example 2
(Cream containing mountain lower leaf extract) 81 Comparative Example 2
(Extract-free cream) 43 Comparative Example 3
(Cream containing aloe extract) 52

As can be seen from Table 13 above, it was confirmed that the skin elasticity was improved the most when the cosmetic composition containing the mountain lower leaf extract was used.

Through this experiment, it can be confirmed that the extract of the mountain lower leaves has an excellent effect in improving skin elasticity as well as a moisturizing effect, so that it is effective in improving wrinkles and aging.

Formulation Example: Preparation of soft cosmetic water (skin) containing the extract of the mountain lower leaves

Among the formulation examples of the cosmetic composition containing the extract of the mountain lower leaf according to Example 1, the formulation of the softening lotion is shown in Table 14 below.

ingredient Content (% by weight) Mountain lower leaf extract 2.00 Butylene Glycol 2.00 glycerin 3.00 Oleyl alcohol 2.00 Polysorbate 20 1.00 ethanol 6.00 antiseptic 0.5 incense 0.5 Purified water Balance system 100.00

The foregoing description of the present application is for illustrative purposes only, and those of ordinary skill in the art to which the present application pertains will be able to understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present application. Therefore, it should be understood that the embodiments described above are illustrative and non-limiting in all respects.

Claims (11)

After mixing the lower leaf and the solvent, stirring at a speed of 100 to 200 rpm for 24 hours to 2 weeks at a temperature of 10 to 40 ℃ containing the extract extracted by shaking as an active ingredient, moisturizing, anti-aging, skin whitening and wrinkles As a cosmetic composition having an improvement effect,
Containing the acid lower leaf extract extracted by the shaking extraction at a concentration of 0.0007 to 0.0025%,
The cosmetic composition has a cell viability of phosphate skin fibroblasts of 98.94% or more, acts as a collagen decomposition inhibitor, improves the ability to inhibit the intracellular activity of collagenase MMP-1, and acts as an accelerator of hyaluronic acid production, for moisturizing Cosmetic composition.
The method according to claim 1,
A cosmetic composition for moisturizing, comprising 0.001 to 20% by weight of the acid lower leaf extract based on the total weight of the cosmetic composition.
delete delete The method according to claim 1,
The acid lower leaf extract is characterized in that it acts as a melanin production inhibitor, moisturizing cosmetic composition.
delete The method according to claim 1,
The sanha leaf extract is characterized in that it acts as an active oxygen inhibitor, moisturizing cosmetic composition.
The method according to claim 1,
The cosmetic composition includes skin ointment, cream, softening lotion, nutrient lotion, pack, essence, hair tonic, shampoo, conditioner, hair conditioner, hair treatment, gel, skin lotion, skin softener, skin toner, astringent, lotion, milk Lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, eye cream, moisture cream, hand cream, foundation, nutrition essence, sunscreen, sunscreen cream, mist, aerosol, soap, cleansing foam, cleansing lotion, cleansing cream, A cosmetic composition for moisturizing, characterized in that it is a formulation selected from the group consisting of a body lotion and a body cleanser.
delete delete The method according to claim 1,
The solvent is water, a cosmetic composition for moisturizing, characterized in that one selected from the group consisting of water, a lower alcohol having 1 to 4 carbon atoms, propylene glycol, glycerin, and a mixed solvent thereof.

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