KR102689846B1 - Cosmetic Composition Comprising Potentilla Chinensis, Taraxacum Officinale, Cornus officinalis, Narcissus Tazetta for Improving Skin - Google Patents

Abstract

The present invention relates to a cosmetic composition for improving skin containing scab flower extract, dandelion leaf extract, Cornus officinalis fruit extract, and daffodil scale stem extract as active ingredients. More specifically, it relates to a cosmetic composition for skin improvement containing scab flower extract, dandelion leaf extract, cornelian cherry fruit extract, and daffodil scale stem extract. By containing cornelian cherry fruit extract and daffodil scale stem extract as active ingredients, it moisturizes the skin, protects against skin stress, improves skin barrier function, protects the skin from ultraviolet rays, protects the skin from blue light, anti-inflammatory, antioxidant, and anti-aging. , relates to a cosmetic composition with functions such as pore reduction and sebum suppression.

Description

Cosmetic composition for improving skin containing scab flower, dandelion leaf, Cornus officinalis fruit, and daffodil scale stem as active ingredients {Cosmetic Composition Comprising Potentilla Chinensis, Taraxacum Officinale, Cornus officinalis, Narcissus Tazetta for Improving Skin}

The present invention provides a cosmetic composition for improving skin containing scab flowers, dandelion leaves, Cornus officinalis fruit, and daffodil scale stems as active ingredients; And it relates to a method of manufacturing the cosmetic composition for improving skin.

The skin is the largest organ in the body by weight and is made up of three layers: the keratinized outer epidermis, the dermis, which is an inner connective tissue rich in blood vessels, and the innermost subcutaneous tissue, and various appendages (hair) associated with the skin. , nails, sensory receptors, and glands) form the skin system (integumentary system). The skin forms the boundary between the inside and outside of the body to carry out the main functions of various organs within the body.

The skin is essential for maintaining homeostasis and is an important organ that performs a variety of functions, including regulating body temperature, suppressing moisture loss, containing sensory receptors, synthesizing various biochemical substances, and excreting small amounts of waste in addition to protective functions.

However, compared to other organs, the skin has many opportunities to come into direct contact with external stimulation or various pathogens, and in particular, when ultraviolet rays are absorbed into the skin, a photochemical reaction occurs, causing skin lesions. In addition, the number of patients with skin diseases caused by factors commonly occurring in modern society, such as mental stress, obesity, alcohol, and smoking, is rapidly increasing.

Accordingly, the development of various skin protectants to suppress skin damage and protect skin cells is actively being developed. However, the developed skin protectors mainly contain synthetic compounds as active ingredients, which may cause skin damage or cause itchiness and spots. It has the disadvantage of causing side effects.

Under this background, the present inventors have made extensive research efforts to develop natural cosmetic materials useful for improving various skin conditions, and as a result, a composition containing scab flower, dandelion leaf, Cornus officinalis fruit, and daffodil scale stem has been developed to improve various skin conditions. After confirming that it was effective, the present invention was completed.

Republic of Korea Patent No. 10-1811411

The present invention aims to solve the above-described problems and other problems associated therewith.

The present inventors, skin moisturizing, skin aging prevention, anti-inflammatory, skin barrier function strengthening, skin soothing, skin protection from ultraviolet rays or blue light, pore reduction, antioxidant, anti-skin stress, wound healing, skin regeneration, acne or sebum improvement, pores As a result of diligent efforts to discover materials with various functionalities, such as shrinkage, it was found that a composition containing scab flowers, dandelion leaves, Cornus officinalis fruit, and daffodil scale stems has various skin-improving effects and can be used as a material for various functional cosmetics. After confirmation, the present invention was completed.

Therefore, the purpose of the present invention is to provide a cosmetic composition for improving skin containing scab flower extract, dandelion leaf extract, Cornus officinalis fruit extract, and daffodil scale stem extract as active ingredients.

Another object of the present invention is to provide a method for producing a cosmetic composition for skin improvement containing the extract of the flower of the present invention, the extract of the dandelion leaf of the present invention, the extract of the cornelian cherry fruit of the present invention, and the extract of the narcissus bulb of the present invention as effective ingredients.

The technical problem to be achieved according to the technical idea of the invention disclosed in this specification is not limited to the problem to solve the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Additionally, the scope of the present application cannot be considered limited by the specific description described below.

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

In one aspect for achieving the above object, the present invention relates to a cosmetic composition for improving skin containing scab flower extract, dandelion leaf extract, Cornelian cornelian fruit extract, and daffodil scale stem extract as active ingredients. Specifically, the extract is not limited in content, but may be contained in an amount of 0.1 to 10% by weight based on the total weight of the composition.

In the present invention, the scab flower extract, dandelion leaf extract, Cornus officinalis fruit extract, and daffodil scale stem extract are used in amounts of 30 to 50 parts by weight, 10 to 20 parts by weight, 10 to 20 parts by weight, and 1 to 5 parts by weight, respectively. It can be.

In the present invention, a description of the plant of each extract serving as an effective ingredient is as follows.

(1) scab flower

The scientific name of scab flower is Potentilla Chinensis , and it is a type of Rosaceae. It is a species that characterizes the low-lying herbaceous vegetation that lives in the mooring section of the upper reaches of very dry rivers under direct sunlight or on the gravel-sand bottom of the beach. This plant is rarely observed on artificial embankments or roads near its main habitat. The back of the leaf is covered with white fluff, which helps it overcome extreme dryness caused by radiant heat.

(2) Dandelion leaves

The scientific name of dandelion is Taraxacum officinale , and it belongs to the order Campanula. It is a naturalized plant native to Europe and can often be seen on the roadside.

(3) Cornus officinalis fruit

The scientific name of Cornus officinalis is Cornus officinalis , and it belongs to the Umbrella family. It refers to the fruit of Cornus officinalis, a deciduous tree of the dogwood family. It is an oval-shaped drupe that is green at first and then ripens red from August to October. The seeds are long oval shaped.

(4) Daffodils

Daffodil is a perennial herb in the family Daffodil family. The scientific name is Narcissus Tazetta , and the roots of daffodils have overlapping scaly stems and are egg-shaped and spherical. The English name for Narcissus Tazetta Bulb Extract is Narcissus Tazetta Bulb Extract.

In the present invention, “skin improvement” refers to all actions that improve or benefit the skin by using the scab flower extract, dandelion leaf extract, Cornus officinalis fruit extract, and daffodil scale stem extract of the present invention.

The skin improvement of the present invention is an example, skin moisturizing, skin barrier improvement, skin aging prevention, skin regeneration or wound healing improvement, skin immunity improvement and defense enhancement, skin inflammation relief or improvement, skin soothing, protection from ultraviolet rays or blue light. This may include, but is not limited to, skin protection, antioxidant, skin protection from skin stress, skin cell energy enhancement, skin pore reduction, sebum suppression, or acne improvement.

The term “skin moisturizing” in the present invention means increasing moisture in the skin and maintaining a moist state. Increasing the skin moisturizing effect can have a beneficial effect on improving skin wrinkles and increasing elasticity.

The term "skin barrier improvement and strengthening" of the present invention refers to improving the thickness of the epidermal layer by promoting differentiation of keratinocytes in the skin barrier composed of keratinocytes.

The term “skin aging” of the present invention is a concept that includes both intrinsic and extrinsic aging. Such skin aging also includes the improvement, prevention, and prevention of various skin aging phenomena such as wrinkle improvement, wrinkle prevention, and elasticity improvement.

The term "skin protection from ultraviolet rays or blue light" in the present invention refers to the prevention or improvement of damage or symptoms caused by exposure of the skin to ultraviolet rays, such as wrinkles caused by ultraviolet rays, loss of skin elasticity, photoaging, or skin keratinization. Blue light is commonly referred to as blue light. It is a type of visible light that occurs frequently in electronic devices in everyday life and can be perceived by the eyes. It has a short wavelength in the range of 380 to 550 nm, increases eye fatigue and makes the skin dull. It is known that it can induce skin aging, and this means protecting the skin from blue light stimulation.

The term “pore reduction” of the present invention refers to reducing the size of pores on the skin surface where hair grows. When pores become large, waste products and bacteria can easily penetrate into the body, which can easily lead to bacterial infection and acne. Therefore, by reducing pores, you can maintain clean skin and protect against bacterial infection.

The term “sebum suppression” in the present invention refers to reducing the amount of oil exuded from the skin. When sebum is secreted excessively, inflammation and acne occur on the skin surface, so suppressing sebum secretion can improve the health of the skin and maintain a fresh skin condition.

The term "acne suppression or improvement" in the present invention means reducing the occurrence of acne caused by excessive sebum secretion and pore closure, or subsiding the acne that occurs.

The term "skin cell damage protection" of the present invention means protecting skin cells from cell damage by blocking causes that may cause damage, for example, increasing resistance to stress, which is one of the causes of skin cell damage. Examples include, but are not limited to, increased gene expression. For example, it means suppressing skin aging or protecting the skin from various environmental stresses, and such environmental stresses may include ultraviolet rays, pollution, wind, or temperature. In the present invention, it can be confirmed by increasing the amount of LEA expression through changes in the expression of LEA (Late embryogenesis abundant), which is known to protect cells from damage against stress.

The term "including as an active ingredient" in the present invention means that the skin cosmetic composition contains an effective amount that can exhibit various skin improvement effects as described above.

Specifically, in an experimental example of the present invention, various skin improvement effects of scab flower extract, dandelion leaf extract, Cornus officinalis fruit extract, and daffodil scale stem extract were confirmed. Specifically, AQP3 (Aquaporin 3), a moisture/glycerol transporter, and An increase in the expression level of FLG (Filaggrin), CLDN6 (Claudin 6), and IVL (Involucrin), known as natural moisturizing factors, was confirmed, and an increase in the expression level of TERT (Telomerase Reverse Transcriptase) gene, an anti-aging related gene, was confirmed. In addition, to confirm the improvement of skin immunity and defense, increased expression of LL37 and hBD-2 was confirmed, and activation of autophagy was confirmed through increased expression of LC-3. It can be seen that it is effective in suppressing inflammation through a decrease in COX-2 gene expression and iNOS gene expression, and the skin soothing effect confirmed on subjects also showed that it has excellent skin soothing ability. It was confirmed that this composition had an excellent skin protection effect on skin cells even when irradiated with ultraviolet rays and blue light. In addition, the cell damage protection effect was confirmed by increasing the expression level of LEA protein, which has an excellent antioxidant effect and increases resistance to stress as the expression level increases. It also has a wound healing effect, pore reduction effect due to hemoglobin precipitation, and sebum suppression. The effect of increasing cellular energy and increasing ATP concentration was confirmed. In addition, it was confirmed that an acne improvement effect was observed in test subjects who applied lotion containing scab flower extract, dandelion leaf extract, Cornus officinalis fruit extract, and daffodil scale stem extract. Based on these test examples, the cosmetic composition of the present invention moisturizes the skin, strengthens the skin barrier, protects cells from damage caused by anti-stress, improves skin immunity, enhances defense, anti-aging by wound healing, wound healing, scar improvement, pore reduction, It was confirmed that it can be usefully used for skin improvement as it has excellent effects such as suppressing sebum, improving cell energy, and suppressing acne.

In another aspect, the present invention includes the steps of (a) preparing scab flower extract, dandelion leaf extract, Cornus officinalis fruit extract, and daffodil scale stem extract; and (b) preparing a composition containing the extract obtained in step (a).

In another aspect, the present invention includes the steps of: (a) preparing scab flowers, dandelion leaves, Cornus officinalis fruit, and daffodil scale stems to obtain an extract; and (b) preparing a composition containing the extract obtained in step (a).

In the present invention, “extract” refers to an extract obtained by various conventionally known extraction methods such as cold water extraction, hot water extraction, and ethanol extraction.

In the present invention, the extraction method is not particularly limited and includes, for example, cold needle extraction, ultrasonic extraction, reflux extraction, hot water extraction, etc. Here, in the case of hot water extraction, the hot water extract is obtained by heating at 80 to 100°C for 8 to 48 hours in a hot water distiller.

Alternatively, it may be prepared by extraction using water, an organic solvent, or a mixed solvent thereof. The extracted liquid can be used directly or after being concentrated and/or dried. When extracting using an organic solvent, methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, N-dimethylformamide (DMF), dimethyl sulfoxide An organic solvent such as (DMSO), 1,3-butylene glycol, propylene glycol, or a mixed solvent thereof can be used, and the active ingredients of the herbal medicine can be extracted at room temperature or by heating under conditions where the active ingredients are not destroyed or minimized. The degree of extraction and loss of the active ingredient of the drug may vary depending on the organic solvent being extracted, so select and use an appropriate organic solvent.

In the present invention, the extract can be used concentrated or diluted, and a distillate of the extract can also be used.

In the present invention, the cosmetic composition may include a carrier acceptable in cosmetic formulations. Here, “acceptable carrier in cosmetic preparations” refers to compounds or compositions already known and used that can be included in cosmetic preparations, or compounds or compositions to be developed in the future, which have no toxicity, instability or irritation beyond what the human body can adapt to upon contact with the skin. It says something that doesn't exist. The carrier may be included in the cosmetic composition of the present invention in an amount of about 1% by weight to about 99.99% by weight based on the total weight, preferably about 90% by weight to about 99.99% by weight of the weight of the composition. However, since the ratio varies depending on the formulation in which the cosmetic composition of the present invention is manufactured, its specific application area (face, neck, etc.), and its preferred application amount, the ratio limits the scope of the present invention in any respect. It should not be understood as doing so.

Examples of the carrier include alcohol, oil, surfactant, fatty acid, silicone oil, wetting agent, humectant, viscosity modifier, emulsion, stabilizer, ultraviolet scattering agent, ultraviolet absorber, coloring agent, fragrance, etc. Compounds or compositions that can be used as alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, moisturizers, viscosity modifiers, emulsions, stabilizers, UV scattering agents, UV absorbers, coloring agents, and fragrances are already known in the art. Therefore, a person skilled in the art can select and use an appropriate material or composition.

The cosmetic composition according to the present invention can be manufactured in various forms, such as lotion, essence, gel, emulsion, lotion, cream (oil-in-water type, water-in-oil type, multi-phase), solution, suspension (anhydrous and aqueous), It can be manufactured in the form of anhydrous products (oil and glycol-based), gels, masks, packs, powders, or capsules (soft capsules, hard capsules) with a coating such as gelatin.

In addition, suitable cosmetic formulations include, for example, solutions, gels, solid or pasty anhydrous products, emulsions obtained by dispersing the oil phase in the water phase, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes), non-ionic. It may be provided in the form of a vesicular dispersion, cream, skin, lotion, powder, ointment, spray or conceal stick. Additionally, it can be prepared in the form of foam or in the form of an aerosol composition further containing compressed propellant.

In addition, the cosmetic composition of the present invention further contains fatty substances, organic solvents, solubilizers, thickening and gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic type. or non-ionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any of those commonly used in cosmetics. It may contain auxiliaries commonly used in the fields of cosmetology or dermatology, such as other ingredients. Additionally, the above ingredients can be introduced in amounts commonly used in the field of dermatology.

In the present invention, skin includes not only the face, but also the scalp and the entire body. Cosmetic compositions that can be applied to the scalp include shampoos, rinses, treatments, hair growth agents, etc., and body cleansers that can be applied to the entire body. It can be manufactured in various forms for various purposes.

Therefore, the cosmetic composition according to the present invention includes the form of functional cosmetics for improving various skin conditions, for example, skin moisturizing, skin immunity enhancement, skin barrier function strengthening/improvement, cell protection by blue light or ultraviolet rays, anti-inflammatory It includes a form of functional cosmetics with effects such as protection of cell damage caused by aging and anti-stress, skin soothing, skin regeneration, wound healing, anti-inflammatory, pore reduction, sebum suppression, cell energy enhancement, and acne suppression.

The cosmetic composition according to the present invention contains scab flower extract, dandelion leaf extract, Cornus officinalis fruit extract, and daffodil scale stem extract as active ingredients, thereby moisturizing the skin, protecting against skin stress, strengthening and improving the skin barrier function, and protecting against ultraviolet rays. It can be used as a material for various functional cosmetics with functions such as skin protection, skin protection from blue light, anti-inflammatory, antioxidant, anti-aging, pore reduction, and sebum suppression.

Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

Example 1: Preparation of compositions according to the invention

Wash 500 g of dried scab flowers, 200 g of dandelion leaves, 200 g of Cornus officinalis fruit, and 50 g of daffodil scale stems grown in Jinju, Gyeongsangnam-do several times with purified water, then wash with 20 L of purified water for 6 to 8 hours, 65 to 100 g. The extract was obtained by extraction under reduced pressure at 90°C.

Experimental Example 1: Test to confirm non-irritating effect through skin cell culture

To determine whether the composition of the present invention is toxic to skin cells and causes skin irritation, an MTT assay was performed. For this purpose, HaCaT cells (keratinocytes, keratinocytes) and Detroit cells (fibroblasts, fibroblasts) were cultured at 96°C with DMEM (Dubelcco's Modified Eagle's Medium) containing 10% FBS (Fetal Bovine Serum) and 1% Antibiotic-Antimycotic, respectively. It was dispensed into a well plate and cultured in a thermostat at 5% CO ₂ and 37°C for 24 hours. Afterwards, the cells were treated with a control group to which purified water was added and a test substance composition, and further cultured for 24 hours. Then, 4 μL of 5 mg/mL MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) solution was added to each well, and incubated for 4 hours to react. . After removing the culture medium, 100 μL of DMSO (dimethyl sulfoxide) solution was added to lyse the cells, and the absorbance was measured at 540 nm.

<Cell survival rate measurement results according to concentration of the composition of the present invention in HaCaT cells>

<Cell survival rate measurement results according to concentration of the composition of the present invention in Detroit cells>

As a result, the composition of the present invention not only showed no toxicity within keratinocyte (HaCaT) and fibroblast (Detroit) cells at a treatment concentration of 0.1 to 5%, but also increased cell viability, making it a safe substance with a low possibility of causing skin irritation due to non-irritating properties. It was confirmed that it was.

Experimental Example 2: Test to confirm moisturizing and skin barrier improvement

In order to determine the effectiveness of the composition according to the present invention in moisturizing and improving the skin barrier, the expression levels of AQP3 (Aquaporin 3), a moisture/glycerol transporter, FLG (Filaggrin), known as natural moisturizing factors, CLDN6 (Claudin 6), and IVL (Involucrin) were measured. I noticed the change. For this purpose, HaCaT cells were dispensed into a 96-well plate and cultured for 24 hours under cell culture conditions. Afterwards, the cells were treated with a control group to which purified water was added and a test substance composition, and further cultured for 24 hours. Real-time PCR was performed to confirm the expression of AQP3 and FLG at the gene level, and the test sequence was as follows. SuperPrep™ cell lysis & RT Kit for qPCR (TOYOBO, Cat. SCQ-101) was used for RNA isolation and cDNA synthesis. Cells from which the medium was removed were washed once with PBS, 50 μL cell lysis mixture was added, reacted for 5 minutes, and 10 μL stop solution was added. 8 μL of previously extracted lysate was added to 32 μL of RT reaction mixture, and cDNA was synthesized using PCR under the conditions of 37°C for 15 minutes, 50°C for 5 minutes, and 95°C for 5 minutes. To comparatively analyze gene expression, the cDNA synthesized above was used as a template, and real-time PCR analysis was performed using Thunderbird™ SYBR qPCR Mix (TOYOBO, Cat. QPS-201). The primers used in the experiment were Qiagen's QuantiTect primer assays (AQP3; Cat. QT00212996, FLG; Cat. QT02448138, CLDN6; Cat. QT01679195, IVL; Cat. QT00082586), and the expression levels of AQP3, FLG, CLDN6, and IVL mRNA in the sample were Quantification was performed with GADPH (Cat. QT01192646). Real-time qPCR conditions were first at 95°C for 1 minute, then at 94°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds per cycle, for a total of 40 cycles.

As a result, by treatment with 1% of the composition of the present invention, the expression of AQP3, known as a skin moisturizing factor, increased by 30% compared to the control group, FLG, a factor related to skin barrier strengthening, increased by 20%, CLDN6 increased by 30%, and IVL In the case of , a 30% increase was confirmed, confirming that it is a composition that contributes to moisturizing and strengthening the skin barrier.

Experimental Example 3: Test to confirm anti-aging effect due to increased TERT expression

In order to examine the anti-aging efficacy of the composition according to the present invention, changes in the expression level of TERT (Telomerase reverse transcriptase), known as an enzyme that extends the length of telomeres at the ends of DNA strands, were examined. We examined changes in the expression level of TERT (Telomerase reverse transcriptase), known as an enzyme that extends the length of telomeres at the ends of DNA strands.

HaCaT cells were dispensed into a 96-well plate and cultured for 24 hours under cell culture conditions. Afterwards, the cells were treated with the control and test substance compositions and further cultured for 24 hours. Real-time PCR method was performed to confirm TERT at the gene level, and the test sequence is as follows.

SuperPrep™ celllysis&RTKitforqPCR (TOYOBO, Cat.SCQ-101) was used for RNA isolation and cDNA synthesis. Cells from which the medium was removed were washed once with PBS, 50 μL cell lysis mixture was added, reacted for 5 minutes, and stop solution was added. 8 μL of previously extracted lysate was added to 32 μL of RT reaction mixture, and cDNA was synthesized using PCR under the conditions of 37°C for 15 minutes, 50°C for 5 minutes, and 95°C for 5 minutes. To comparatively analyze gene expression, the cDNA synthesized above was used as a template, and real-time PCR analysis was performed using Thunderbird™ SYBRqPCRMix (TOYOBO, Cat.QPS-201). The primers used in the experiment were Qiagen's QuantiTect primer assays (GAPDH; Cat. QT01192646, TERT; Cat. QT00073409), and the TERT mRNA expression level in the sample was quantified using GADPH. Real-time qPCR conditions were 95°C for 1 minute, followed by 94°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds per cycle, for a total of 40 cycles.

As a result, it was confirmed that the composition of the present invention is effective in preventing aging by increasing the mRNA expression level of TERT compared to the control group.

Experimental Example 4: Test to confirm skin immunity improvement and defense enhancement

It was confirmed whether the composition of the present invention improves skin immunity and defense through the activity of LL37 (CAMP, Cathelicidin-related antimicrobial peptides) and hBD-2 (human β-Defensin-2), which have antibacterial effects. For this purpose, HaCaT cells were dispensed into a 96-well plate and cultured for 24 hours under cell culture conditions. Afterwards, the cells were treated with a control group to which purified water was added and a composition as a test substance, and further cultured for 24 hours. Real-time PCR method was performed to confirm LL37 and hBD-2 at the gene level, and the test sequence is as follows. SuperPrep™ cell lysis & RT Kit for qPCR (TOYOBO, Cat. SCQ-101) was used for RNA isolation and cDNA synthesis. Cells from which the medium was removed were washed once with PBS, 50 μL cell lysis mixture was added, reacted for 5 minutes, and 10 μL stop solution was added. 8 μL of previously extracted lysate was added to 32 μL of RT reaction mixture, and cDNA was synthesized using PCR under the conditions of 37°C for 15 minutes, 50°C for 5 minutes, and 95°C for 5 minutes. To comparatively analyze gene expression, the cDNA synthesized above was used as a template, and real-time PCR analysis was performed using Thunderbird™ SYBR qPCR Mix (TOYOBO, Cat. QPS-201). The primers used in the experiment were Qiagen's QuantiTect primer assays (LL37; Cat. QT00010458, hBD-2; Cat. QT00204617), and the LL37 and hBD-2 mRNA expression levels in the samples were quantified using GADPH (Cat. QT01192646). Real-time qPCR conditions were first at 95°C for 1 minute, then at 94°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds per cycle, for a total of 40 cycles.

As a result, it was confirmed that the composition of the present invention contributes to improving skin immunity and defense by increasing the expression of LL37 and hBD-2 compared to the control group.

Experimental Example 5: Test to confirm expression of autophagy activity

In order to determine the efficacy of the composition of the present invention in its autophagic activity, which naturally decomposes unnecessary or non-functional organelles or cell components during the intracellular regulation process, LC-3 (Microtubule-associated protein 1A), which is involved in the formation of autophagy, was used. Changes in the expression level of /1B-light chain 3) were investigated. For this purpose, HaCaT cells were dispensed into a 96-well plate and cultured for 24 hours under cell culture conditions. Afterwards, the cells were treated with a control group to which purified water was added and a composition as a test substance, and further cultured for 24 hours. Real-time PCR method was performed to confirm LC-3 at the gene level, and the test sequence is as follows. SuperPrep™ cell lysis & RT Kit for qPCR (TOYOBO, Cat. SCQ-101) was used for RNA isolation and cDNA synthesis. Cells from which the medium was removed were washed once with PBS, 50 μL cell lysis mixture was added, reacted for 5 minutes, and 10 μL stop solution was added. 8 μL of previously extracted lysate was added to 32 μL of RT reaction mixture, and cDNA was synthesized using PCR under the conditions of 37°C for 15 minutes, 50°C for 5 minutes, and 95°C for 5 minutes. To comparatively analyze gene expression, the cDNA synthesized above was used as a template, and real-time PCR analysis was performed using Thunderbird™ SYBR qPCR Mix (TOYOBO, Cat. QPS-201). The primer used in the experiment was Qiagen's QuantiTect primer assays (LC-3; Cat. QT00055069), and the level of LC-3 mRNA expression in the sample was quantified using GADPH (Cat. QT01192646). Real-time qPCR conditions were first at 95°C for 1 minute, then at 94°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds per cycle, for a total of 40 cycles.

As a result, it was confirmed that the composition of the present invention contributes to autophagy activation by increasing LC-3 expression compared to the control group.

Experimental Example 6: Test to confirm inflammation inhibition effect

To determine whether the composition of the present invention has the effect of suppressing skin inflammation, changes in the expression levels of COX-2 (Cyclooxygenase-2) and iNOS (Inducible nitric oxide synthase), which are involved in inflammatory reactions, were investigated. For this purpose, HaCaT cells were dispensed into a 96-well plate and cultured for 24 hours under cell culture conditions. Afterwards, an inflammatory response was induced through UVB irradiation, and the cells were treated with a control group to which purified water was added and a composition as a test substance, and cultured for an additional 4 hours. Real-time PCR method was performed to confirm COX-2 and iNOS at the gene level, and the test sequence is as follows. SuperPrep™ cell lysis & RT Kit for qPCR (TOYOBO, Cat. SCQ-101) was used for RNA isolation and cDNA synthesis. Cells from which the medium was removed were washed once with PBS, 50 μL cell lysis mixture was added, reacted for 5 minutes, and 10 μL stop solution was added. 8 μL of previously extracted lysate was added to 32 μL of RT reaction mixture, and cDNA was synthesized using PCR under the conditions of 37°C for 15 minutes, 50°C for 5 minutes, and 95°C for 5 minutes. To comparatively analyze gene expression, the cDNA synthesized above was used as a template, and real-time PCR analysis was performed using Thunderbird™ SYBR qPCR Mix (TOYOBO, Cat. QPS-201). The primers used in the experiment were Qiagen's QuantiTect primer assays (COX-2; Cat. QT00040586, iNOS; Cat. QT01323189), and the COX-2 and iNOS mRNA expression levels in the samples were quantified using GADPH (Cat. QT01192646). Real-time qPCR conditions were first at 95°C for 1 minute, then at 94°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds per cycle, for a total of 40 cycles.

As a result, the composition of the present invention was confirmed to have an effect on alleviating inflammatory response by reducing the expression of COX-2 and iNOS compared to the control group.

Experimental Example 7: Test to confirm skin soothing effect

In order to determine the skin soothing effect of the essence formulation using the composition according to the present invention according to the skin temperature reduction rate, squares (2cm It was exposed to direct sunlight for 20 minutes. Body heat is measured at intervals of 0 minutes (before sample application), 5 minutes (after sample application), and 10 minutes (after sample application) using a computer infrared thermographic imaging (DITI), and the temperature reduction rate is measured. Calculated.

As a result, it was confirmed that the composition of the present invention maintains a continuous heat reduction rate and has a skin soothing effect.

Experimental Example 8: Test to confirm the effect of protecting skin cells from ultraviolet ray stimulation

In order to examine the effect of the composition of the present invention on protecting skin cells from ultraviolet rays, changes in cell viability were examined. For this purpose, HaCaT cells were dispensed into a 96-well plate and cultured for 24 hours under cell culture conditions. Afterwards, UVB was irradiated, and the cells were treated with a control group to which purified water was added and a composition as a test substance, and further cultured for 24 hours. The light source used for UV irradiation was an ultraviolet crosslinker (Ultra-Violet Products, Cat No. CL-1000) that emits ultraviolet rays with a wavelength of 302 nm. Next, cell survival rate was measured using MTT assay to check whether damage to skin cells caused by ultraviolet rays was protected.

As a result, the composition of the present invention increased cell survival rate compared to the control group irradiated with ultraviolet rays, confirming the cell protection effect of ultraviolet irradiation.

Experimental Example 9: Test of skin cell protection effect by Blue Light

To determine whether the composition of the present invention protects skin cells from irradiation of blue light, which has the shortest wavelength and strongest energy among visible light that can be seen by humans, Topview 5450 SMD LED (ITSWELL, Cat. No. IWS-L5056-VRI-K3) was used to measure cell viability. For this purpose, HaCaT cells were dispensed into a 96 well plate and cultured for 24 hours under cell culture conditions. Afterwards, the cells were irradiated with a wavelength of 460 nm at 10 mJ/cm ² , and the cells were treated with a control group to which purified water was added and a composition as a test substance, and further cultured for 24 hours. Next, MTT assay was performed to measure the survival rate of skin cells after blue light induction.

As a result, it was confirmed that, as in the present invention, the composition according to the present invention can protect the skin by specifically reflecting and blocking light in the blue light range, which is the visible light range.

Experimental Example 10: H ₂ O ₂ Test to confirm antioxidant effect from oxidative toxicity

In order to examine the antioxidant effect of the composition according to the present invention from H ₂ O ₂ oxidation toxicity, changes in cell viability due to H ₂ O ₂ were examined.

For this purpose, HaCaT cells were dispensed into a 48-well plate and cultured for 24 hours under cell culture conditions. Afterwards, 1 mM H ₂ O ₂ was irradiated, and the cells were treated with the control and test substance compositions and further cultured for 12 to 16 hours. Afterwards, cell survival rate was measured using MTT assay to check whether damage to skin cells caused by H ₂ O ₂ oxidative toxicity was protected.

As a result, it was confirmed that cell death caused by H ₂ O ₂ treatment was inhibited in cells treated with the composition of the present invention compared to the positive control group (H ₂ O ₂ treatment), resulting in an antioxidant effect from H ₂ O ₂ oxidation toxicity.

Experimental Example 11: Test to confirm anti-stress protein activation effect

To determine whether the composition of the present invention has a protective effect against intracellular stress, the activity level of LEA (Late embryogenesis abundant) protein was examined. It is known that LEA protects against cell damage by increasing resistance to stress as the expression level increases. For this purpose, HaCaT cells were dispensed into a 6-well plate and cultured for 24 hours under cell culture conditions. Afterwards, the cells were treated with the control group and the test substance composition to which purified water was added, the cells were further cultured for 24 hours, and the In-Cell ELISA Colorimetric Detection kit (Invitrogen, Cat No. 62200) was used to measure the level of LEA protein expression. ) was used. After the culture was completed, the medium of the cells was removed, and the cells were fixed by incubating at room temperature for 15 minutes with 100 μL fixing solution. After washing with PBS, 100 μL of Permeabilization solution was added and reacted at room temperature for 15 minutes. After washing with PBS, 100 μL Quenching solution was added and reacted at room temperature for 20 minutes. After washing with PBS, 100 μL Blocking solution was added and reacted at room temperature for 30 minutes. The solution was removed and 50 μL of LEA primary antibody was added and reacted at 4°C for 24 hours. After removing the antibody, it was washed with PBS, and 50 μL of HRP conjugated secondary antibody was added and reacted for 1 hour. After removing the antibody and washing with PBS, 100 μL of TMB substrate solution was added and reacted at room temperature for 15 minutes while blocking light. Afterwards, 100 μL stop solution (1 NH ₂ SO ₄ ) was added, and absorbance was measured at 450 nm.

As a result, it was confirmed that treatment with the composition of the present invention at different concentrations protects cells from damage by increasing the expression level of LEA protein.

Experimental Example 12: Test to confirm wound healing effect

Wound healing assay was performed to determine whether the composition of the present invention has wound healing ability by promoting cell proliferation and mobility. For this purpose, a culture insert for wound healing assay was added to each well of a 24-well plate, and 70 μL of HaCaT cells were dispensed into the insert at 90% < confluence and cultured for 24 hours. Afterwards, the insert was removed and the cell pattern was obtained through a microscope at time 0, and the cells were treated with a control group to which purified water was added and a composition as a test substance. After further culturing for 16 hours, the medium was removed, 4% PFA (Paraformaldehyde) was added, and reaction was performed at room temperature for 15 minutes. After the fixation process, the cells were washed three times with PBS, and after further culturing for 16 hours, the cell pattern was measured through micrographs. The rate of increase in healing area was calculated by measuring the area between cell intervals at 0 hours and at 16 hours using a program called Image J.

As a result, it was confirmed that the composition of the present invention indirectly contributed to wound healing by increasing the healing area ratio.

Experiment 13: Test to confirm the energy-enhancing effect of cells

In order to determine whether the composition of the present invention has an effect in improving cellular energy, the activity level of ATP released by cells during metabolic processes was measured. For this purpose, Detroit cells were dispensed into a 12-well plate and cultured for 24 hours under cell culture conditions. Afterwards, the cells were treated with a control group to which purified water was added and a test substance composition, and the cells were further cultured for 24 hours. Take 50 μL of the diluted lysate of cells cultured through the above process and place it in each 96-well plate along with 50 μL of ATP standard in the ATP assay kit (Abcam, Cat. ab83355) and add 50 μL of ATP reaction mix. The solution was also added to each well and reacted at room temperature for 30 minutes while blocking light. After the reaction was completed, the absorbance was measured at 570 nm, and an ATP standard concentration curve was created to calculate the amount of ATP in the sample.

As a result, it was confirmed that treatment with the composition of the present invention increased the amount of ATP, resulting in an energy activity enhancement effect.

Experimental Example 14: Test to confirm pore reduction effect (in vitro)

To determine whether the composition of the present invention has a pore-reducing effect, hemoglobin was tested using hemoglobin as a protein to replace skin. For this purpose, purified water was added to 2 mL of 1 mg/mL hemoglobin solution, and 2 mL each of the test substance composition and positive control group (Tannic acid) were added, mixed by shaking for 30 seconds, and then centrifuged at 3,500 rpm for 10 minutes. did. 2 mL of purified water was added to 1 mL of the supernatant, and the absorbance was measured at 407 nm.

As a result, the composition of the present invention increased the precipitation rate of hemoglobin, thereby indirectly confirming the effect of shrinking pores.

Experimental Example 15: Test to confirm sebum inhibition effect

In order to confirm the sebum suppression effect of the essence formulation using the composition according to the present invention, the amount of sebum secretion in the skin of 10 test subjects was measured after 4 weeks using a skin oil meter (Sebum meter SM810). The test was conducted at 22±2°C and 40±5% humidity. For oily skin, the measured value is 220 μg/cm ² or more, and for normal skin, it is 100 to 220 μg/cm ² .

As a result, in the case of the control group, there was little change in the amount of sebum oil after 4 weeks of use, but the composition of the present invention showed a reduced amount of skin oil, confirming the sebum suppression effect.

Experimental Example 16: Test to confirm acne improvement effect

In order to confirm the acne-improving effect of a lotion formulation using the composition of the present invention, 10 men and women with acne were asked to apply the lotion evenly over their entire face in the morning and evening for 4 weeks. To judge the effect of improving acne, the condition before the test was evaluated with 1 to 3 points, referring to the judgment criteria below, depending on the degree felt by the person participating in the experiment, and then the acne healing effect was evaluated, and the results are shown in the table.

<Evaluation of the pre-examination status>

1 = Some acne 2 = Some acne 3 = Severe acne

<Acne effect judgment>

-: Little effect, +: Slight effect, ++: Much relief, +++: Completely healed

As a result, it was confirmed that the composition of the present invention improved acne in most subjects after 2 and 4 weeks of use.

Example 2: Preparation of cosmetic composition

In order to prepare various types of cosmetic compositions containing the composition of the present invention as an active ingredient, lotions, essences, lotions, creams, and gels were prepared according to the composition ratios in the table below.

lotion manufacturing

Essence manufacturing

lotion manufacturers

cream making

gel manufacturing

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the patent claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present invention.

Claims (13)

A cosmetic composition for improving skin containing scab flower extract, dandelion leaf extract, Cornus officinalis fruit extract, and daffodil scale stem extract as active ingredients. The cosmetic composition according to claim 1, wherein the composition is used to protect or strengthen the skin barrier function. The cosmetic composition of claim 1, wherein the composition is for moisturizing skin. The cosmetic composition according to claim 1, wherein the composition is used to protect the skin from ultraviolet rays or blue light. delete The cosmetic composition according to claim 1, wherein the composition is for antioxidant purposes. The cosmetic composition according to claim 1, wherein the composition is used to prevent or relieve skin stress. The cosmetic composition of claim 1, wherein the composition is for soothing skin. The cosmetic composition of claim 1, wherein the composition is used to relieve or prevent skin inflammation. delete The cosmetic composition according to claim 1, wherein the composition is used to reduce pores. The cosmetic composition according to claim 1, wherein the composition is for suppressing sebum. A method for producing the cosmetic composition according to any one of claims 1 to 4, 6 to 9, 11 and 12, comprising the following steps:
(a) preparing scab flower extract, dandelion leaf extract, Cornus officinalis fruit extract, and daffodil scale stem extract; Or, (a) preparing scab flowers, dandelion leaves, Cornus officinalis fruit, and daffodil scale stems to obtain an extract; and
(b) preparing a cosmetic composition containing the extract.