KR102676799B1 - External Composition Comprising Natural Complex Extract for Improving Skin - Google Patents

Abstract

The present invention relates to a composition for external application for skin containing complex extracts of fig, mango, grape, and eggplant as active ingredients.
The complex extract of figs, mango, grapes, and eggplant according to the present invention has skin improvement effects such as skin wrinkle improvement, antioxidant, skin barrier strengthening, anti-aging, anti-inflammatory, skin regeneration, and wound healing. Complex extracts of grapes and eggplant can be useful as skin improvement materials in cosmetics and medicine.

Description

External Composition Comprising Natural Complex Extract for Improving Skin}

The present invention relates to a composition for external use for skin improvement, including skin wrinkle improvement, anti-inflammatory, skin moisturizing, skin barrier improvement, anti-aging, antioxidant, sebum suppression, and itching relief, containing a natural complex extract as an active ingredient.

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. Therefore, there is a need to develop new natural materials that have no side effects on the skin and have excellent skin damage prevention and skin strengthening functions.

Republic of Korea Patent Publication No. 10-2022-0141467

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

An exemplary object of the present invention is to provide a composition for external application for improving skin containing a natural complex extract as an active ingredient, for example, a composition for external application for improving skin containing complex extracts of fig, mango, grape, and eggplant as an active ingredient. It is done.

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 this 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.

In one aspect for achieving the above object, the present invention provides a composition for external application for skin improvement containing complex extracts of fig, mango, grape, and eggplant as active ingredients.

In the present invention, the complex extract of fig, mango, grape and eggplant is obtained by extracting fig, mango, grape and eggplant together in a solvent to obtain a complex extract, or mixing the fig extract, mango extract, grape extract and eggplant extract together. Thus, a complex extract can be obtained.

In the present invention, “extract” refers to extracts obtained by extraction treatment of figs, mangoes, grapes and eggplants, diluted or concentrated liquids of the extracts, dried products obtained by drying the extracts, crude or purified products of the extracts, or these. This includes extracts of all formulations that can be formed using the extract itself and the extract.

In the present invention, the extraction is not particularly limited and can be extracted according to methods commonly used in the art. Non-limiting examples of the extraction method include hot water extraction, cold immersion extraction, solvent extraction, steam distillation, elution, compression, ultrasonic extraction, filtration, and reflux extraction, which are performed alone or using a combination of two or more methods. It can be performed by doing this.

In one embodiment, the extraction step of the present invention may be by hot water extraction. Preferably, hot water extraction may be performed at 70°C to 140°C for 36 to 52 hours, and more preferably, hot water extraction may be performed at 90°C to 120°C for 48 hours.

The extract of the present invention is extracted using an appropriate solvent, and may include, for example, a crude extract, a polar solvent-soluble extract, or a non-polar solvent-soluble extract. The type of extraction solvent used to prepare the extract is not particularly limited, and any solvent known in the art can be used. Non-limiting examples of the extraction solvent include water; lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, propanol, isopropanol, butanol, propyl alcohol, and butyl alcohol; Polyhydric alcohols such as glycerin, butylene glycol, and propylene glycol; Hydrocarbon solvents such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane;　Or a mixture thereof can be used. Additionally, a solvent extract can be prepared by extracting the extract one or more times using the solvent.

The extract of the present invention can be used by filtering, for example, mesh filtration to remove floating solid particles, or by drying it using freeze-drying, hot air drying, spray drying, etc. Additionally, the extract may further undergo a conventional fractionation process or may be purified using a conventional purification method.

In the present invention, “skin improvement” means any action that improves or benefits the skin using the composition of the present invention.

The skin improvement of the present invention is an example, skin protection against ultraviolet irradiation, skin moisturizing, anti-aging, skin barrier strengthening, itching relief, skin (cell damage) protection by anti-stress, anti-wrinkle, skin regeneration, wound healing. This may include, but is not limited to, anti-aging, 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 protection against (from) ultraviolet irradiation and blue light" in the present invention refers to damage or symptoms that occur as the skin is exposed to ultraviolet rays or blue light, such as wrinkles caused by ultraviolet rays, decreased skin elasticity, photoaging, or Prevents or improves skin keratinization.

The term "skin barrier strengthening" in 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 "itching improvement" in the present invention refers to the alleviation of skin symptoms that cause the urge to scratch or rub the skin, and is a concept that includes alleviating itching by reducing the expression of genes that cause itching.

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 “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 “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 protection (protection from skin cell damage)" of the present invention means protecting skin cells from cell damage by blocking causes that may damage skin cells, for example, against stress, which is one of the causes of skin cell damage. Examples include, but are not limited to, increased expression of genes that increase resistance. 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.

The term "anti-inflammatory" in the present invention is used to mean improving, alleviating, suppressing, or delaying the inflammatory response. This inflammatory response involves activation of various inflammatory mediators and enzymes related to immune cells (e.g., iNOS, COX-2, etc.), secretion of inflammatory mediators (e.g., secretion of NO, TNF-α, IL-6, etc.), body fluid infiltration, It involves a series of complex physiological reactions such as cell movement and tissue destruction, and is externally manifested by symptoms such as erythema, pain, swelling, and fever.

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

Specifically, in an experimental example of the present invention, a composition containing complex extracts of fig, mango, grape and eggplant was confirmed to have an improvement and prevention effect on skin wrinkles due to an increase in procollagen biosynthesis, and an increase in AQP3 and filaggrin expression to moisturize the skin, The skin barrier improvement effect was confirmed, the anti-inflammatory effect was confirmed by reducing the expression of COX-2 and iNOS, the antioxidant effect was confirmed by reducing the expression of SOD1, SOD2, and CAT, the antioxidant effect was confirmed from H2O2 oxidation toxicity, and UV stimulation was confirmed. , Skin protection from blue light (skin cell protection) was confirmed, and wound healing and anti-aging effects were confirmed. In addition, the whitening effect was confirmed by reducing the melanin content, and it was also confirmed to have excellent pore shrinkage, sebum suppression, acne improvement, itching alleviation effect, and stress suppression effect through increased HMOX1, NQO1, FTL, and TXNRD1 gene expression. Through these excellent effects, it was confirmed that the composition of the present invention can be usefully used for skin improvement purposes.

In the present invention, the complex extract of fig, mango, grape and eggplant 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 external skin composition may be a cosmetic composition or a pharmaceutical composition.

The cosmetic composition may contain a carrier acceptable in cosmetic preparations. 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 that exhibit toxicity, instability or irritation beyond what the human body can adapt to upon contact with the skin. It says there is no such thing. The carrier may be included in the external skin preparation composition of the present invention in an amount of about 1% to about 99.99% by weight based on the total weight, preferably about 90% by weight to about 99.99% by weight of the composition. However, since the above ratio varies depending on the formulation in which the external skin composition of the present invention is manufactured, its specific application area (face, neck, etc.), and its preferred application amount, the above ratio does not in any way define the scope of the present invention. It should not be understood as limiting.

Examples of the carrier include alcohol, oil, surfactant, fatty acid, silicone oil, wetting agent, moisturizing agent, 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 external skin composition according to the present invention can be prepared in various forms, such as lotion, essence, gel, emulsion, lotion, cream (oil-in-water type, water-in-oil type, multi-phase), solution, and suspension (anhydrous and aqueous). , 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 dispersant, cream, skin, lotion, powder, ointment, spray or stick. It can also be prepared in the form of foam or an aerosol composition further containing compressed propellant.

In addition, the external skin 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, and ions. Formal 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 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 is a concept that includes not only the face, but also the scalp and the whole body. External skin 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 whole body. It can be manufactured in various forms for purposes such as:

Therefore, the composition for external application for skin according to the present invention includes the form of functional cosmetics such as skin regeneration, wound healing, wrinkle improvement, anti-aging, skin barrier strengthening, and antioxidant.

In the present invention, the composition for topical skin application can function as a pharmaceutical composition for improving skin condition.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient, or diluent according to a conventional method. Pharmaceutically acceptable carriers are well known in the art depending on the administration route or dosage form, and for specifics, each country's pharmacopoeia, including the 'Korean Pharmacopoeia', can be referred to. Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, Examples include, but are not limited to, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Additionally, carriers, excipients, and diluents that may be included in the composition of the present invention may be unnatural carriers, but are not limited thereto.

The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, or sterile injectable solutions according to conventional methods. . In detail, it can be prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are made by adding at least one excipient to the compound, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. It can be prepared by mixing. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, they contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. You can. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, wethepsol, macrogol, Tween 61, cacao, laurin, glycerogelatin, etc. can be used. Regarding the specific formulation of pharmaceutical compositions, it is known in the art, and references can be made to, for example, Remington's Pharmaceutical Sciences (19th ed., 1995). The above documents are considered a part of this specification.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The pharmaceutically effective amount refers to an amount that is sufficient to treat the disease at a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects. The effective dose level refers to the patient's health status, type and severity of the disease, It can be determined based on factors including the activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, drugs combined or used simultaneously, and other factors well known in the medical field. The dosage and frequency of administration do not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, dogs, cats, cows, horses, pigs, and humans through various routes, and humans may be preferable. All modes of administration are contemplated and may include, but are not limited to, oral, intravenous, intramuscular or subcutaneous injection.

The composition for topical skin application of the present invention may be a quasi-drug composition.

In addition to the above ingredients, the quasi-drug composition of the present invention may further include pharmaceutically acceptable carriers, excipients, or diluents as needed. The pharmaceutically acceptable carrier, excipient, or diluent is not particularly limited as long as it does not impair the effect of the present invention, and includes, for example, fillers, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, sweeteners, fragrances, preservatives, etc. may include.

Quasi-drug compositions may include, but are not limited to, disinfectant cleaners, shower foams, ointments, wet tissues, coating agents, etc., and the formulation method, dosage, usage method, and components of quasi-drugs can be determined from conventional techniques known in the technical field. It can be selected appropriately.

The natural complex extract according to the present invention has skin improvement effects such as wrinkle improvement, anti-aging, skin barrier strengthening, antioxidant, skin regeneration, and wound healing, and can be usefully used as a material for cosmetics and medicines for skin improvement.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1: Preparation of compositions according to the invention

To obtain the composition according to the present invention, hot water extraction was performed by heating 80 g of cleanly washed figs, 200 g of mango, 200 g of grapes, and 200 g of eggplant in 20 L of purified water at 104°C for 4 hours. After extraction, it was filtered through mesh to remove solids, and a composite extract was obtained.

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

In order to determine whether the composition of the present invention is toxic to skin cells and causes skin irritation, an MTT assay was performed to measure cell viability.

HaCaT cells (keratinocytes, keratinocytes) and Detroit cells (fibroblasts) were cultured in 96-well plates with DMEM (Dubelcco's Modified Eagle's Medium) containing 10% FBS (Fetal Bovine Serum) and 1% Antibiotic-Antimycotic, respectively. and cultured in a thermostat at 5% CO ₂ and 37°C for 24 hours. Afterwards, the cells were treated with the control group to which purified water was added and the test substance composition to a final concentration of 1, 5, and 10%, 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.

sample Treatment concentration (%) HaCaT cell survival rate (%) Detroit cell survival rate (%) control group - 100.00 100.00 test group 0.5 101.02 105.6 One 103.80 110.2 2 108.26 109.8

As a result, it was confirmed that the composition of the present invention did not show any toxicity in skin cells at a treatment concentration of 1 to 10% and thus was not irritating. Therefore, this means that the composition has a low possibility of causing skin irritation and is a safe material.

Experimental Example 2: Test to confirm wrinkle improvement effect due to increased PIP production

In order to examine the wrinkle-improving effect of the composition according to the present invention, the amount of biosynthesis of procollagen (Procollagen Type I C-Peptide, PIP), a precursor for collagen synthesis, was measured.

For this purpose, Detroit551 cells were dispensed into a 48-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 48 hours. At this time, TGF-beta 5 ng/mL was used as a positive control. Then, take 20 μL of the diluted supernatant of the culture medium and place it in each antibody coated microtiter plate along with 20 μL of the PIP standard in the PIP EIA Kit (Takara, Cat. MK101), and add 100 μL of peroxidase-labeled antibody solution to each. It was added to the well and reacted at 37°C for 3 hours. After removing the medium and washing four times with 200 μL of PBS, 100 μL of substrate solution was added to each well and reacted at room temperature for 15 minutes while blocking light. Afterwards, 100 μL stop solution (1 NH ₂ SO ₄ ) was added, and the absorbance at 450 nm was measured.

sample Treatment concentration (%) PIP synthesis ability (%) control group - 100.00 positive control - 130.20 test group 0.5 110.09 One 129.20 2 118.81

As a result, the amount of PIP increased in cells treated with the composition of the present invention at different concentrations compared to the control group, confirming that the composition is effective in improving wrinkles.

Experimental Example 3: Test to confirm the effect of improving moisturizing and skin barrier due to increased expression of AQP3 and FLG

In order to examine the moisturizing and skin barrier improvement efficacy of the composition according to the present invention, changes in the expression levels of AQP3 (Aquaporin), a water/glycerol transporter, and FLG (Filaggrin), known as a natural moisturizing factor, were investigated.

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. At this time, 1% Glyceryl glucoside was used as a positive control. Real-time PCR method was performed to confirm AQP3 and FLG at the gene level, and the test sequence is as follows.

SuperPrep ^TM 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 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 ^TM SYBR qPCR Mix (TOYOBO, Cat.QPS-201). The primers used in the experiment were Qiagen's QuantiTect primer assays (GAPDH; Cat. QT01192646, AQP3; Cat. QT00212996, FLG; Cat. QT02448138), and the AQP3 and FLG mRNA expression levels in the samples were 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.

sample Treatment concentration (%) AQP3 FLG control group - 1.00 1.00 Positive control group - 1.59 3.12 test group 0.5 1.12 1.93 One 1.50 2.31 2 1.72 2.80

As a result, the mRNA expression levels of AQP3 and FLG increased in cells treated with the composition of the present invention at different concentrations compared to the control group, confirming that the composition is effective in moisturizing and improving the skin barrier.

Experimental Example 4: Test to confirm anti-inflammatory effect by reducing COX-2 and iNOS expression

In order to examine the anti-inflammatory effect of the composition according to the present invention, changes in the expression levels of COX-2 (Cyclooxygenase-2) and iNOS (inducible Nitric Oxygen Synthase), which are involved in the inflammatory response, were examined.

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, the cells were treated with the control and test substance compositions, and the cells were further cultured for 4 hours. At this time, Dexamethasone 5 μM was used as a positive control. Real-time PCR method was performed to confirm COX-2 at the gene level, and the test sequence is as follows.

SuperPrep ^TM 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 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 ^TM SYBR qPCR Mix (TOYOBO, Cat.QPS-201). The primers used in the experiment were Qiagen's QuantiTect primer assays (GAPDH; Cat. QT01192646, COX-2; Cat. QT00040586, iNOS; Cat. QT01323189), and the expression levels of COX-2 and iNOS mRNA in the samples were 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.

sample Treatment concentration (%) UVB COX-2 iNOS Control group (-) - - 0.23 0.75 control group - + 1.00 1.00 Positive control group - + 0.72 0.85 test group 0.5 + 0.72 0.83 One + 0.92 0.87 2 + 0.72 0.71

As a result, the mRNA expression levels of COX-2 and iNOS decreased in cells treated with the composition of the present invention at different concentrations compared to the control group (UVB treatment), confirming that the composition has an anti-inflammatory effect.

Experimental Example 5: Experiment to confirm antioxidant effect

Experimental Example 5-1: Test to confirm antioxidant effect by reducing SOD1, SOD2, and CAT expression

In order to examine the moisturizing and skin barrier improvement efficacy of the composition according to the present invention, SOD1 (Superoxide Dismutase), which is known to be involved in the decomposition of H ₂ O ₂ and reactive oxygen species (ROS), which are toxic substances produced in aerobic metabolism, was used. 1), we investigated changes in the expression levels of SOD2 (Superoxide Dismutase 2) and CAT (Catalase).

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. At this time, 2 mM N-acetyl-L-cysteine (NAC) was used as a positive control. Real-time PCR method was performed to confirm at the gene level of SOD1, SOD2, and CAT, and the test sequence is as follows.

SuperPrep ^TM 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 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 ^TM SYBR qPCR Mix (TOYOBO, Cat.QPS-201). The primers used in the experiment were Qiagen's QuantiTect primer assays (GAPDH; Cat. QT01192646, SOD1; Cat. QT01008651, SOD2; Cat. QT01008693, CAT; Cat. QT00079674), and the expression levels of SOD1, SOD2, and CAT mRNA in the samples were quantified using GADPH. did. 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.

sample Treatment concentration (%) SOD1 SOD2 CAT Control group (-) - 1.00 1.00 1.00 Positive control group - 1.49 1.52 1.62 test group 0.5 1.92 1.26 1.23 One 2.10 2.30 1.89 2 1.98 1.46 2.10

As a result, the mRNA expression levels of SOD1, SOD2, and CAT increased in cells treated with the composition of the present invention at different concentrations compared to the control group, confirming that the composition is effective in antioxidant efficacy.

Experimental Example 5-2: 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. At this time, 2 mM N-acetyl-L-cysteine (NAC) was used as a positive control. 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.

sample Treatment concentration (%) HaCaT cell survival rate (%) Control group (-) - 100.00 Control group (+) - 60.33 Positive control group - 88.8 test group 0.5 66.56 One 76.73 2 79.46

As a result, cell death caused by H ₂ O ₂ treatment was inhibited in cells treated with the composition of the present invention at different concentrations compared to the control group (+H ₂ O ₂ ), suggesting that the composition would have an antioxidant effect from H ₂ O ₂ oxidation toxicity. Confirmed.

Experimental Example 6: Test to confirm the effect of protecting skin cells from ultraviolet rays stimulation

In order to examine the effect of the composition according to 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 the control and test substance compositions and further cultured for 24 hours. Next, cell survival rate was measured using MTT assay to check whether damage to skin cells caused by ultraviolet rays was protected.

sample Treatment concentration (%) UVB HaCaT cell survival rate (%) control group - - 100.00 control group - + 45.63 test group 0.5 + 45.69 One + 50.23 2 + 49.85

As a result, cell death caused by UVB treatment was inhibited in cells treated with the composition of the present invention at different concentrations compared to the control group (+UVB), confirming that the composition has a cell protection effect from ultraviolet rays.

Experimental Example 7: Test to confirm skin cell protection effect from blue light induction

In order to examine the effect of the composition according to the present invention on protecting skin cells from blue light stimulation, 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, the cells were treated with the control group and the test material composition, and after 2 hours, blue light was irradiated for 6 hours using a Topview 5450 VRI SMD LED (ITSWELL, Cat. IWS-L5056-VRI-K3), and the cells were incubated for 24 hours. Additional culture was performed. Next, cell survival rate was measured using MTT assay to check whether skin cell damage caused by blue light was protected.

sample Treatment concentration (%) blue light HaCaT cell survival rate (%) control group - - 100.00 control group - + 75.23 test group 0.5 + 75.90 One + 82.10 2 + 83.42

As a result, cell death caused by blue light treatment was inhibited in cells treated with the composition of the present invention at different concentrations compared to the control group (+ blue light), confirming that the composition has a cell protection effect from blue light.

Experimental Example 8: Wound healing and anti-aging efficacy test using wound healing

In order to examine the skin cell regeneration effect of the composition according to the present invention, the anti-aging effect was evaluated by observing whether the test substance promotes cell proliferation and migration using a wound healing assay.

For this purpose, HaCaT cells were dispensed into a 24-well plate and cultured with the insert 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. At this time, 200 ng/ml EGF was used as a positive control. Cell images were taken under a microscope at 0 hours after material treatment and cultured under conditions of 37°C and 5% CO2. After further incubation for 18-20 hours after material treatment, the medium was removed, added to fixing solution (4% paraformaldehyde), incubated at room temperature for 15 minutes, and washed three times with PBS. Fixed cells could be stored at 4°C for one month. The extent of wound healing was calculated by taking pictures under a microscope and using a program called Image J.

sample Treatment concentration (%) Healing area (%) control group - 23.50 Positive control group - 45.23 test group 0.5 28.61 One 33.69 2 35.20

As a result, it was confirmed that the composition of the present invention increased the healing area in cells treated at different concentrations, and it was confirmed that the composition would have a skin cell regeneration effect.

Experimental Example 9: Test to confirm whitening effect by measuring melanin content

In order to confirm the whitening effect of the composition according to the present invention, the effect on melanin content, which plays an important role in melanin formation, was tested.

For this purpose, B16F1 cells were dispensed into 6-well plates at 70% confluence and cultured for 24 hours under cell culture conditions. The sample is then treated with cells and further cultured for 72 hours. At this time, 100 ppm of Kojic acid as a positive control group is also treated. Additionally, 100 nM α-MSH is added with the sample to promote melanin production. After the culture is over, the supernatant of the medium is removed, and the cells are harvested by adding 1X Trypsin-EDTA. (After 72 hours of treatment, if some cells are floating, harvest by dropping all cells on the bottom with a scraper.) Add 200-300 μL of 1 N NaOH solution to the cell pellet, boil at 100°C for 30 minutes, and melanin dissolves. A portion of the dissolved melanin solution is placed in a 96-well plate and the absorbance is measured at 405 nm. The absorbance value of each sample was quantified as protein amount, and melanin synthesis ability was expressed as a percentage.

sample Treatment concentration (%) melanin inhibition ability (%) control group - 0 Positive control group - 36.24 test group 0.5 5.80 One 7.96 2 13.26

As a result, the amount of melanin decreased in cells treated with the composition of the present invention at different concentrations compared to the control group, confirming that the composition is effective in whitening.

Experimental Example 10: Pore contraction effect test (In vitro Test)

In order to measure the pore shrinking effect of the composition according to the present invention, the composition prepared in the above example was tested for pore shrinking effect using hemoglobin as a protein to replace skin.

Prepare a hemoglobin solution (0.05g/50ml) with 0.9% Phosphate Buffer Saline (0.1mM, pH 7.4), add 2ml of edelweiss plant cell and adventitious root cell culture extract to 2ml of hemoglobin solution, and mix by shaking for 30 seconds. It was centrifuged at 3,500 rpm for 10 minutes, and 2 ml of purified water was added to 1 ml of the supernatant, which was measured by UV-Visible spectrum at 407 nm.

As a control, 2 ml of PBS (Phosphate Buffer Saline) was added.

sample Treatment concentration (%) Precipitation of hemoglabin (%) control group
(PBS) - 0.5 Positive control group (tannic acid) One 30 test group 0.5 One One 5 2 9

As a result, the shrinking effect was evaluated by measuring the amount of hemoglobin precipitation of the composition of the present invention. It was determined that the higher the hemoglobin precipitation (%), the better the pore shrinking effect, and it was confirmed that the composition would have a pore shrinking effect.

Experimental Example 11: Sebum inhibition effect experiment

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

sample Treatment concentration (%) Sebum suppression effect 0 days (before use) 28 days (before use) control group - 230 221 test group 0.5 231 190 One 229 175 2 232 171

As a result, sebum was reduced in skin treated with the composition of the present invention at different concentrations compared to the control group, confirming that the composition is effective in suppressing sebum.

Experimental Example 12: Acne improvement effect

Ten men and women with acne were asked to evenly apply the composition prepared in Example 1 to their entire face in the morning and evening for four 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 pre-test condition>

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

<Acne effect judgment>

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

+++: Fully healed

subject Status before test Effect after 2 weeks Effect after 4 weeks Subject 1 2 + Subject 2 One + ++ Subject 3 One - + Subject 4 2 + - Subject 5 2 + ++ Subject 6 2 + + Subject 7 3 ++ +++ Subject 8 2 - ++ Subject 9 3 ++ +++ Subject 10 2 + ++

As a result, the acne improvement effect was confirmed in most subjects after using the softening lotion containing the composition of the present invention for 4 weeks.

Experimental Example 13: Effect of alleviating itching by cytokine gene expression analysis

In order to confirm the itching inhibition and alleviation effect of the composition according to the present invention, the expression levels of IL-4, IL-33, and TSLP mRNA were analyzed.

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 IL-4, IL-33, and TSLP at the gene level, and the test sequence is as follows.

SuperPrep ^TM 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 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 ^TM SYBR qPCR Mix (TOYOBO, Cat.QPS-201). The primers used in the experiment were Qiagen's QuantiTect primer assays (GAPDH; Cat. QT01192646, Interleukin 4 (IL-4); Cat. #QT00012565, Thymic Stromal Lymphopoietin (TSLP); Cat. #QT00051464, Interleukin 33 (IL-33); Cat. #QT00041559), and the expression levels of IL-4, IL-33, and TSLP mRNA in the samples were 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.

sample Treatment concentration (%) IL-4 TSLP IL-33 Control group (-) - 1.00 1.00 1.00 test group 0.5 1.10 0.98 1.05 One 0.76 0.92 0.91 2 0.67 0.88 0.82

As a result, the mRNA expression levels of IL-1, TSLP, and IL-33 were reduced in cells treated with the composition of the present invention at different concentrations compared to the control group, confirming that the composition would be effective in alleviating itching.

Experimental Example 14: Stress suppression effect by HMOX1, NQO1, FTL, TXNRD1 gene expression

In order to confirm the stress suppressing effect of the composition according to the present invention, the expression levels of HMOX1, NQO1, FTL, and TXNRD1 mRNA were analyzed.

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 HMOX1, NQO1, FTL, and TXNRD1 at the gene level, and the test sequence is as follows.

SuperPrep ^TM 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 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 ^TM SYBR qPCR Mix (TOYOBO, Cat.QPS-201). The primers used in the experiment were Qiagen's QuantiTect primer assays (GAPDH; Cat. QT01192646, HMOX1; Cat. #QT00092645, NQO1; Cat. #QT00050281, FTL; Cat. #QT00055860, TXNRD1; Cat. #QT00055902) and HMOX1, NQO1, FTL, and TXNRD1 mRNA expression levels were 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.

sample Treatment concentration (%) HMOX1 NQO1 FTL TXNRD1 Control group (-) - 1.00 1.00 1.00 1.00 test group 0.5 1.45 1.40 1.48 1.39 One 1.68 1.57 1.52 1.49 2 2.43 1.83 1.61 1.68

As a result, the mRNA expression levels of HMOX1, NQO1, FTL, and TXNRD1 increased in cells treated with the composition of the present invention at different concentrations compared to the control group, confirming that the composition is effective in suppressing stress.

Example 2: Preparation of external skin composition

In order to prepare an external skin composition containing complex extracts of fig, mango, grape, and eggplant according to the present invention as active ingredients, lotion, essence, lotion, cream, and gel were prepared respectively.

Preparation Example 2-1: Toner

The lotion was prepared according to the usual method for producing lotion according to the following prescription.

Raw material name Weight % (w/w) glycerin 5.0 Dipropylene glycol 3.0 Hyaluronic acid 0.5 Polyoxyethylene hydrogenated castor oil 0.1 polyethylene oleyl ethyl 0.1 ethanol 5.0 antiseptic 0.15 perfume Appropriate amount flash Appropriate amount Complex extracts of fig, mango, grape, and eggplant 1.0 Purified water to 100

Preparation Example 2-2: Essence

It was prepared according to a conventional essence manufacturing method according to the following prescription.

Raw material name Weight % (w/w) Cetostearyl alcohol 1.0 Self-emulsifying monostearic acid 1.0 beeswax 0.5 squalane 5.0 Isocetyl Octanoate 3.0 dimethylsiloxane 0.3 Sorbitan monostearate 0.5 Polyethylene glycol monostearate 8.0 glycerin 4.0 propylene glycol 0.2 carboxypolymer 0.22 Triethanolamine 0.25 antiseptic Appropriate amount Spices Appropriate amount flash Appropriate amount Complex extracts of fig, mango, grape, and eggplant 1.0 Purified water to 100

Preparation Example 2-3: Lotion

It was prepared according to a conventional lotion manufacturing method according to the following prescription.

Raw material name Weight % (w/w) Cetostearyl alcohol 0.8 Self-emulsifying monostearic acid 1.0 beeswax 0.5 stearic acid 0.5 liquid paraffin 7.0 squalane 5.0 macadamia oil 3.0 Isocetyl Octanoate 2.0 dimethylsiloxane 0.3 Sorbitan monostearate 0.5 Polyethylene glycol monostearate 1.2 glycerin 4.0 propylene glycol 4.0 Betaine 4.0 carboxypolymer 0.12 Triethanolamine 0.15 antiseptic 0.25 Spices Appropriate amount flash Appropriate amount Complex extracts of fig, mango, grape, and eggplant 1.0 Purified water to 100

Preparation Example 2-4: Cream

It was prepared according to a conventional cream manufacturing method according to the following prescription.

Raw material name Weight % (w/w) Cetostearyl alcohol 3.0 Self-emulsifying monostearic acid 1.5 Lipophilic monostearic acid 1.5 beeswax 0.5 liquid paraffin 8.0 squalane 7.0 Isocetyl Octanoate 4.0 Refined jojoba oil 4.0 dimethylsiloxane 0.3 Sorbitan monostearate 1.0 Polyethylene glycol monostearate 1.2 glycerin 6.0 propylene glycol 4.0 Betaine 4.0 xanthan gum 0.06 Triethanolamine 0.10 antiseptic 0.25 Spices Appropriate amount flash Appropriate amount Complex extracts of fig, mango, grape, and eggplant 1.0 Purified water to 100

Preparation Example 2-5: Gel

It was prepared according to a conventional gel production method according to the following prescription.

Raw material name Weight % (w/w) glycerin 4.0 propylene glycol 4.0 ethanol 10 Polyoxyethylene hydrogenated castor oil 0.1 carboxypolymer 0.30 Triethanolamine 0.30 antiseptic Appropriate amount Spices Appropriate amount flash Appropriate amount Complex extracts of fig, mango, grape, and eggplant 1.0 Purified water to 100

From the above description, those skilled in the art to which the present invention pertains will be able to 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 interpreted 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 (14)

A cosmetic composition for skin improvement comprising complex extracts of fig, mango, grape, and eggplant as active ingredients, wherein the skin improvement is sebum suppression.
According to paragraph 1,
A cosmetic composition wherein the skin improvement is wrinkle improvement or prevention.
According to paragraph 1,
A cosmetic composition wherein the skin improvement is skin moisturizing or skin barrier improvement.
According to paragraph 1,
A cosmetic composition wherein the skin improvement is anti-inflammatory.
According to paragraph 1,
A cosmetic composition wherein the skin improvement is for skin antioxidant purposes.
According to paragraph 1,
A cosmetic composition wherein the skin improvement is skin protection from ultraviolet rays or blue light.
delete According to paragraph 1,
A cosmetic composition wherein the skin improvement is skin regeneration.
According to paragraph 1,
A cosmetic composition wherein the skin improvement is skin whitening.
According to paragraph 1,
A cosmetic composition in which the skin improvement is pore contraction.
delete According to paragraph 1,
A cosmetic composition wherein the skin improvement is improvement or prevention of acne.
According to paragraph 1,
A cosmetic composition wherein the skin improvement is alleviation of skin itching.
According to paragraph 1,
A cosmetic composition wherein the skin improvement is suppressing skin stress.