KR102646545B1 - Cosmetic composition for skin whitening or skin barrier improvement comprising Hosta longipes extract - Google Patents

Abstract

The present invention relates to a cosmetic composition for improving the skin barrier, comprising Bibitracus extract, Bibitracus extract, Peach Blossom extract, Rhubarb extract, Macmundong extract, lotus extract, Chaga extract, and Manuka honey. The cosmetic composition of the present invention, which includes Bibitracus extract, Bibichu extract, Arboschwort extract, Rhubarb extract, Macmoon-dong extract, lotus extract, chaga extract, and Manuka honey, has excellent skin barrier improvement effects, moisturizing, glowing, sebum reduction, and dead skin cells. It can be used as a cosmetic composition for improvement.
This study is the result of research conducted with support from the “Local Enterprise Innovative Growth Support (R&D, detailed project number P0010100)” project of the Ministry of SMEs and Startups and the Korea Institute for Advancement of Technology.

Description

Cosmetic composition for skin whitening or skin barrier improvement comprising Hosta longipes extract}

The present invention relates to a cosmetic composition for improving the skin barrier containing Bibitracus extract, Bibichocus extract, Prickly pear extract, Rhubarb extract, Macmundong extract, lotus extract, chaga extract, and Manuka honey.

Skin aging manifests itself as changes in skin color or elasticity, and is divided into intrinsic aging, which occurs naturally due to intrinsic factors, and extrinsic aging, which occurs due to external factors. Intrinsic aging is mainly genetically determined, which determines structural changes in skin cells and tissues. Meanwhile, the main causes of extrinsic aging are environmental or physiological factors such as lack of nutrition, fatigue, stress, and ultraviolet rays, which, unlike intrinsic aging, are preventable and manageable factors. In relation to this, as interest in skin care and related products has recently increased, consumer demand for functional cosmetic compositions for skin care purposes such as whitening, improving elasticity, and blocking ultraviolet rays is also increasing.

The color or brightness of the skin is determined by the concentration and distribution of melanin in the skin. Melanin is catalyzed by tyrosinase, which sequentially converts tyrosine into DOPA and dopaquinone, which then undergoes a non-enzymatic oxidation reaction. It is known to be created through . In addition, collagen is a major matrix protein produced by skin fibroblasts, and is known to play a role in maintaining the mechanical robustness of the skin barrier, maintaining connective tissue resistance and tissue adhesion, and supporting cell adhesion. Collagen decreases due to factors such as aging and ultraviolet irradiation, which is closely related to the formation of wrinkles on the skin.

Among the various functions of the skin, the barrier function of the skin is mainly expressed by the stratum corneum located on the outermost layer of the skin. This stratum corneum not only has a simple barrier function, but also affects the function, role, and structure of the inner layer, such as the epidermal layer or dermal layer. It has been reported that its importance is continuously increasing. The stratum corneum is composed of dead keratinocytes and intercellular lipids, and functions as a skin protective film that protects the skin from external stimuli and prevents moisture from evaporating from the inside.

Substances recently used for skin whitening and wrinkle improvement include arbutin, hydroquinone, and ascorbic acid; Alternatively, retinoic acid, TGF (transforming growth factor), etc. are known, but the amount of use is limited due to skin safety issues, or the effect is minimal, making it difficult to achieve the desired effect.

Meanwhile, " Hosta longipes " is a perennial plant of the monocot family Liliaceae, and grows well in mountain streams or humid places. The tender shoots of Bibichu are mainly eaten, and are known to have diuretic and neuroprotective effects, but there is no known activity for skin whitening or skin barrier improvement.

Accordingly, the inventors of the present invention made diligent efforts to find a material that can practically achieve the anti-aging purpose while ensuring skin safety, and as a result, they found B. birch extract, B. rhubarb extract, Zenbok flower extract, rhubarb extract, Macundong extract, lotus extract, and chaga mushroom extract. The present invention was completed by discovering that a complex extract containing and manuka honey has an excellent effect on improving the skin barrier.

Republic of Korea Patent Publication No. 10-2014-0140396

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

An exemplary object of the present invention is to include skin whitening, skin barrier improvement, antioxidant, anti-inflammatory or To provide a cosmetic composition for improving wrinkles.

Other exemplary objects of the present invention include skin whitening, skin barrier improvement, antioxidant, anti-inflammatory or To provide a method for manufacturing a cosmetic composition for improving wrinkles.

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.

One aspect of the present invention for achieving the above object includes skin whitening, skin barrier improvement, Provided is a cosmetic composition for antioxidant, anti-inflammatory or wrinkle improvement purposes.

Specifically, the composition contains 60 to 80 parts by weight of BB extract based on 100 parts by weight of the total cosmetic composition; 1 to 10 parts by weight of Bibitracus extract; 0.5 to 3 parts by weight each of Seonbokhwa extract, Jong rhubarb extract, Macmundong extract, lotus extract, and chaga extract; and 0.05 to 3 parts by weight of Manuka honey as an active ingredient.

In the present invention, the term " Hosta longipes " is a perennial plant of the monocot plant Liliaceae family, which grows well in mountain streams or humid places. The tender shoots of Bibichu are mainly eaten, and its diuretic and neuroprotective effects are known, but its activities in skin whitening and skin barrier improvement are not yet known. The above bibichus can be purchased commercially, or those collected or cultivated from nature can be used without limitation. Additionally, the bibichu can be used without limitation on parts such as flowers, leaves, fruits, seeds, roots, or stems.

In addition, in the present invention, the term "babichusu (water)" refers to adding 1000 mL of distilled water to 100 g of dried bibichuu, reacting at 121°C for 1 hour, and then reacting the reactant with 2G3 as in one embodiment of the present invention. Thermal water extract, i.e. Bibisu extract, can be produced and obtained by filtration using a glass-filter (PYREX 32940 FNL, JAPAN).

In the present invention, the term " Inula britannica var. japonica )" is a perennial plant of the dicotyledonous plant Campanula order Asteraceae, also called 'Geumbulcho' and mainly grows in wetlands. Herbal medicine is known to have anti-tussive and expectorant effects, but its activities in skin whitening and skin barrier improvement are not yet known. The above-mentioned Seonbokhwa can be purchased commercially, or those collected or cultivated in nature can be used without limitation. In the present invention, extracts of the flowers, leaves, and stems of the flowers, leaves, and stems of the flowers and stems of the flowers and leaves were used to prepare the extracts and cosmetic compositions.

In the present invention, the term " Rheum undulatum L. " is a rhizome that has a unique odor and an astringent and bitter taste. The activities of rhubarb for skin whitening and skin barrier improvement are not yet known, and it can be purchased commercially or collected or cultivated from nature and used without restrictions. In the present invention, rhubarb root, leaf, and stem extracts were used to prepare rhubarb extract and cosmetic compositions.

In the present invention, the term "maekmundong" belongs to the perennial herb of the monocot plant family Liliaceae, and in the present invention, it may mean 'lobular maekmundong ( Ophiopogon japonicas )'. It grows in the shade of mountainous areas and its tuber roots are used medicinally like Macmundong, but its activities in skin whitening and skin barrier improvement are not yet known. This can be purchased commercially, or collected or cultivated from nature and used without restrictions. In the present invention, the leaf extract of the root of the root of McMoondong was used to prepare the extract and cosmetic composition.

In the present invention, the term "lotus ( Indian lotus )" refers to a perennial aquatic plant of the order Ranunculaceae and is also called lotus ( Nelumbo nucifera ). It is known to be used as an astringent and hemostatic agent and to treat enuresis in the private sector, but its activities in skin whitening and skin barrier improvement are not yet known. The lotus flower can be purchased commercially or collected or cultivated in nature and used without limitation.

In the present invention, the term "Chaga ( Inonotus Obliquus )" is a medicinal mushroom that grows on birch trees and is known to contain a large amount of beta-glucan. Chaga mushroom's skin whitening and skin barrier improvement activities are not yet known. Chaga mushrooms can be purchased commercially or collected or cultivated from nature and used without restrictions.

In the present invention, the term "Manuka Honey" refers to honey collected from the flowers of a wild shrub called Manuka ( Leptospermum scoparium ), which grows naturally in New Zealand, and contains a unique natural substance called UMF (Unique Manuka Factor). It is known to have high antibiotic and antibacterial efficacy. However, the activities of Manuka honey in terms of skin whitening and skin barrier improvement are not yet known. It can be purchased commercially or collected from nature and used without restrictions.

In the present invention, the term "extract" refers to a liquid component obtained by immersing the desired material in various solvents and then extracting it at room temperature, low temperature, or heated state for a certain period of time, and a solid content obtained by removing the solvent from the liquid component. it means. In addition, it can be comprehensively interpreted to include all diluted solutions of the above results, concentrated solutions thereof, dried products obtained by drying them, or crude products, purified products, and fractions thereof.

The extract may be extracted from natural, hybrid, or variant plants without limitation. In the present invention, 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; Alcohols having 1 to 4 carbon atoms, such as methanol, ethanol, propyl alcohol, and butyl alcohol; Polyhydric alcohols such as glycerin, butylene glycol, and propylene glycol; and hydrocarbon solvents such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane; Or a mixture thereof can be used, preferably water, alcohol with 1 to 4 carbon atoms, 1,3-butylene glycol, and ethyl acetate can be used alone or in a mixture of two or more, but there are no special restrictions on this. no.

In addition, the method for obtaining the extract is not particularly limited as long as the B. birch extract can be obtained, but specifically, the cold immersion extraction method of immersing the B. c. Methods such as extraction, ultrasonic extraction using ultrasonic waves, and reflux extraction using a reflux cooler can be used. In one example of the present invention, all parts of the dried B. birch were extracted with 95% ethanol (EtOH) and evaporated under reduced pressure to obtain B. c. EtOH extract (HLE).

In the present invention, the term "skin whitening" refers to not only brightening skin tone by inhibiting the synthesis of melanin pigment, but also improving skin hyperpigmentation such as spots and freckles caused by ultraviolet rays, hormones, or genetics.

In the present invention, the term "skin barrier improvement" refers to strengthening the barrier of the stratum corneum located on the outer layer of the skin, and is a concept that includes improving skin moisturization, improving gloss, reducing sebum, and improving dead skin cells.

In the present invention, the term “skin moisturizing” means increasing moisture in the skin and maintaining a moist state.

In the present invention, the term "skin gloss" means maintaining the skin in a shiny, smooth, elastic and glossy state.

In the present invention, the term “sebum reduction” refers to a reduction in secretions from sebaceous glands in the skin. Excessive secretion of sebum can cause inflammation and dermatitis.

In the present invention, the term “keratin improvement” refers to a decrease in the amount of keratin, which is a white cellular foreign body mass in the epidermal layer of the skin.

Specifically, in an embodiment of the present invention, when a cosmetic composition containing B. birch extract, B. c. extract, P. rhododendron sinensis extract, Jong rhubarb extract, Macmundong extract, lotus flower extract, chaga extract, and Manuka honey is applied to human skin, moisturizing is improved, It was confirmed that the effects of improving gloss, reducing sebum, and improving dead skin cells were observed.

The cosmetic composition includes astringent, softening, and nutritious face lotions, nutritional cream, massage cream, essence, pack, skin adhesive patch, skin adhesive gel, powder, ointment, paste, gel, suspension, emulsion, spray, and serum. Alternatively, it may be formulated as a capsule, but is not particularly limited thereto.

In addition, the cosmetic composition of the present invention may additionally include one or more cosmetically acceptable carriers that are blended with general skin cosmetics, and common ingredients include, for example, oil, water, surfactant, moisturizer, lower alcohol, Thickeners, chelating agents, pigments, preservatives, fragrances, etc. may be appropriately mixed, but are not limited thereto. Cosmetically acceptable carriers included in the cosmetic composition of the present invention vary depending on the formulation.

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

When the formulation of the present invention is a suspension, the carrier ingredients include water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, and microcrystals. Cellulose, aluminum metahydroxide, bentonite, or tracant may be used.

When the formulation of the present invention is an ointment, paste, cream or gel, the carrier ingredients include animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide. It can be used.

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

When the formulation of the present invention is a surfactant-containing cleansing agent, the carrier ingredients include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, and fatty acid amide. Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester may be used. Additionally, the ingredients included in the cosmetic composition may be included in amounts generally used in the field of dermatology.

In addition, the cosmetic composition of the present invention contains commonly used auxiliaries, such as hydrophilic or lipophilic gelling agents, hydrophilic and hydrophilic gelling agents, in addition to Bibitracus extract, Bibichu extract, Peach Blossom extract, Rhubarb extract, Macmundong extract, lotus extract, chaga extract and Manuka honey. Alternatively, it may include lipophilic activators, preservatives, antioxidants, solvents, fragrances, fillers, blocking agents, pigments, odorants and dyes.

In another aspect of the present invention, the present invention relates to skin whitening, skin containing B. birch extract, B. rhubarb extract, P. rhododendron extract, Rhubarb extract, Macmundong extract, lotus extract, chaga extract, and Manuka honey, including the following steps: A method for manufacturing a cosmetic composition for improving barrier properties, anti-oxidation, anti-inflammatory or wrinkle improvement is provided.

(a) dissolving and mixing BB fruit, betaine, manuka honey, and disodium EDTA;

(b) dispersing Bibiharva and carbomer and then adding them to the mixture of step (a);

(c) adding and dissolving niacinamide to the bibi harvest and then adding it to the mixture of step (b) and stirring;

(d) adding and dissolving arginine in the bibi harvest and then adding it to the mixture of step (c) and stirring; and

(e) 1,2-Hexanediol, Ethylhexylglycerin, McMoondong extract, lotus flower extract, Chaga mushroom extract, Chanbok extract, Jong rhubarb extract, Bibichu extract and Glutathione Adding and stirring to the mixture of step (d) to prepare a cosmetic composition.

In the present invention, in step (c), niacinamide is added and dissolved in the birch water, and then added to the mixture in step (b) at 50°C to 70°C and maintained at 65°C to 70°C. It can be stirred for 20 to 40 minutes.

In the present invention, in step (d), arginine may be added and dissolved in the birch harvest, and then added to the mixture in step (c) and stirred for 20 to 40 minutes.

In the present invention, in step (e), 1,2-hexanediol, ethylhexylglycerin, McMoondong extract, lotus extract, chaga mushroom extract, Chanbok flower extract, and Jong rhubarb A cosmetic composition can be prepared by adding the extract, Bibitracus extract, and glutathione to the mixture in step (d) at 30°C to 50°C and stirring for 10 to 30 minutes.

In the present invention, the BB harvesting ratio of step (a), the BB harvesting ratio of step (b), the BB harvesting ratio of step (c), and the BB harvesting ratio of step (d) are 8 to 10:3 to 5: It may be characterized as being included in a weight ratio of 1 to 2:1 to 2.

The cosmetic composition of the present invention, which includes Bibitracus extract, Bibichu extract, Arboretum extract, Rhubarb extract, Macmoondong extract, lotus extract, chaga extract, and Manuka honey, has an excellent skin barrier improvement effect, improving moisturizing, improving shine, and reducing sebum. And it can be used as a cosmetic composition for improving keratin.

In order to more fully understand the drawings cited in this specification, a brief description of each drawing is provided.
Figure 1 shows the melanin content in B16F10 cells according to treatment with different concentrations of Bibitra extract.
Figure 2 shows the melanin content in B16F10 cells according to treatment of Bibitra extract and fractions.
Figure 3 shows the melanin content in B16F10 cells according to each treatment of the n-hexane fraction of Bibichu.
Figure 4 shows the change in mushroom tyrosinase activity according to treatment of Bibitra extract and n-hexane fraction at different concentrations.
Figure 5 shows the results of measuring intracellular tyrosinase activity after incubation with B16F10 cells at different concentrations of Bibitra extract and n-hexane fraction.
Figure 6 shows the results of Western blotting of melanin biosynthesis-related proteins (MITF, tyrosinase, TRP1 and TRP2, etc.) according to treatment with B. birch extract and n-hexane fraction.
Figure 7 shows the effects of Bibitra extract and n-hexane fraction on MC1R, MITF, tyrosinase, TRP1, and TRP2 protein expression, respectively.
Figure 8 shows the results of Q-PCR confirming the mRNA expression levels of MITF, tyrosinase, TRP-1, and TRP-2 according to treatment of B. birch extract and n-hexane fraction.
Figure 9 shows the shape and distribution of black spots in zebrafish embryos as a result of treatment with Bibitra extract and n-hexane fraction.
Figure 10 shows the results of measuring the size of black spots in zebrafish embryos as a result of treatment with Bibitra extract and n-hexane fraction.
Figure 11 shows specific ingredients and their contents contained in the cosmetic composition of the present invention.
Figure 12 shows a specific process for producing the cosmetic composition of the present invention.
Figure 13 shows the results of confirming the moisturizing effect of the cosmetic composition of the present invention.
Figure 14 shows the results of confirming the gloss effect by the cosmetic composition of the present invention.
Figure 15 shows the results of confirming the sebum reduction effect of the cosmetic composition of the present invention.
Figure 16 shows the results of confirming the effect of improving keratin by the cosmetic composition of the present invention.

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1: Whitening activity of Bibitracus extract, fractions and main compounds

1. Experimental materials and methods

1-1. Acquisition of ethanol extract from Bibichu

Hosta longipes was purchased from the Hantaek Botanical Garden Foundation in Yongin, Korea. The entire dried portion (730 g) of Bibichuran was extracted with 95% ethanol (EtOH) (10 L

1-2. Obtaining bivitreous fractions and compounds 1 to 10

HLE was suspended in 1 L of distilled water and partitioned sequentially between n-hexane (1 L), EtOAc (1 L), and n-BuOH (1 L). The activated n-hexane soluble fraction (4.4 g) was chromatographed on a silica gel column using CH ₂ Cl ₂ -MeOH (20:1 → MeOH, gradient system) as eluent to give 10 fractions (WDE1 to WDE12).

Among them, fraction WDE3 (492.0 mg) was fractionated into five subfractions (WDE3-1 to WDE3-5) by Sephadex LH-20 column chromatography using MeOH, and subfraction WDE3-3 (74.9 mg) was CH ₂ Cl ₂ -MeOH (40:1) was used as an eluent and applied to silica gel isocratic CC to obtain four fractions (WDE3-3-1 to WDE3-3-4). A small fraction of WDE3-3-4 (63.9 mg) was fractionated into two fractions (WDE3-3-4-1 and WDE3-3-4-2) by Sephadex LH-20 column chromatography using MeOH. Afterwards, Compound 1 (padmatin, 12.3 mg) was obtained as a pure powder by filtering WDE3-3-4-1.

Fraction WDE4 (492.0 mg) was fractionated into five subfractions (WDE4-1 to WDE4-5) by silica gel CC using EtOAc-Hex (5:1 → EtOAc, gradient system). A small fraction of WDE4-3 (84 mg) was suspended in MeOH and filtered to obtain compound 2 (aromadendrin, 35.1 mg). Subsequently, the filtrate of WDE4-3 was fractionated into five small fractions (WDE4-3-1 to WDE4-3-5) by Sephadex LH-20 using MeOH, and the small fraction WDE4-3-4 was filtered. Thus, compound 3 (apigenin, 5.6 mg) was obtained as a pure powder. Small fraction WDE4-4 (62.9 mg) was fractionated into 5 small fractions (WDE4-4-1 to WDE4-4-5) by Sephadex LH-20 using MeOH. Afterwards, RP HPLC (Watchers 120 ODS-BP, S-10 m, 150 Υ 10 mm; detection, UV at 254 nm; flow rate, 2 mL/min) was applied to the small fraction WDE4-4-4 (16.3 mg). and eluted with an AcCN-H ₂ O isocratic system (5:5, 50 minutes) to obtain compound 4 (wikstaiwanone C, 3.5 mg).

Fraction WDE6 (4.7 g) was chromatographed on silica gel CC using CH ₂ Cl ₂ -MeOH (10:1 → MeOH, gradient system) as eluent to obtain five subfractions (WDE6-1 to WDE6-5). . The filtrate of fraction WDE6-3 was recrystallized from MeOH to obtain compound 5 (taxifolin, 35.1 mg) as a pure powder. Small fraction WDE6-2 (467.4 mg) was fractionated into 6 small fractions (WDE6-2-1 to WDE6-2-6) by Sephadex LH-20 using MeOH, and fractions WDE6-2-3, WDE6- Compound 6 (neochamaejasmine B, 3.3 mg), compound 7 (chamaejasmine, 93 mg), and compound 8 (naringenin, 88.5 mg) were obtained as pure powders by filtering 2-5 and WDE6-2-6, respectively.

Fraction WDE7 (5.0 g) was applied to silica gel CC using CH ₂ Cl ₂ -MeOH (10:1 → MeOH, gradient system) as eluent to obtain three subfractions (WDE7-1 to WDE7-3). Five fractions (WDE7-3-1 to WDE7-3-5) were prepared by using CH ₂ Cl ₂ -MeOH (8:1 → MeOH, gradient system) for the small fraction WDE7-3 (2.0 g) and applying silica gel CC. was obtained, and by filtering the fraction WDE7-3-1, compound 9 (afzelechin, 102.3 mg) was obtained as a pure powder.

Finally, fraction WDE8 (2.6 g) was applied to silica gel CC using CH ₂ Cl ₂ -MeOH (20:1 → MeOH, gradient system) as eluent to obtain 5 fractions (WDE8-1 to WDE8-5). did. The small fraction WDE8-3 (149.6 mg) was fractionated into 5 small fractions (WDE8-3-1 to WDE8-3-5) by Sephadex LH-20 (MeOH), and the small fraction WDE8-3-3 (71.1 mg) ) was applied to silica gel CC using CH ₂ Cl ₂ -MeOH (10:1 → MeOH, gradient system) as eluent to obtain two small fractions (WDE8-3-3-1 and WDE8-3-3- 2). Among them, fraction WDE8-3-3-2 was filtered to obtain compound 10 (catechin, 38.2 mg) as a pure powder.

1-3. HPLC analysis

High performance liquid chromatography (HPLC) analysis was performed by an Agilent 1200 series HPLC/MS system (Agilent Technologies, Palo Alto, CA, USA). Baboon extract (HLE) was dissolved in 50% methanol and then filtered through a 0.45 μm PTFE membrane filter. The column used for the separation was a YMC triart C18 column (5 μm, 4.6 Х 150 mm), and the mobile phase was 0.05% trifluoroacetic acid (TFA) in methanol and 0.05% TFA in water at a flow rate of 1.0 mL/min under gradient conditions. flowed to

1-4. Mushroom tyrosinase activity assay

Mushroom tyrosinase activity assay was performed according to Bilodeau et al (2001). That is, an aqueous mushroom tyrosinase solution (110 units/ml) was added to a 96-well microplate to obtain a mixture with a total volume of 200 μL containing L-DOPA (10 mM), which was incubated in sodium phosphate-buffer (100 mM, pH 6.8) was dissolved. Afterwards, B. extract and arbutin (10, 30, and 100 μg/ml) were added to the mixture, and the mixture was incubated at 37°C for 30 minutes.

1-5. cell culture

Mus musculus skin melanoma (B16-F10) cells were obtained from the American Type Culture Collection (ATCC; Rockville, MD). Cells were cultured in DMEM medium (GIBCO, Grand Island, NY) supplemented with 2 mM L-glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin, and 10% heat-inactivated fetal bovine serum (GIBCO, Grand Island, NY). It was cultured in . Thereafter, cells were grown in air at 37°C, completely humidified with 5% CO ₂ and subcultured twice weekly.

1-6. Cell viability measurements

Cell viability was tested by MTT assay. Cells were seeded in a ²⁴ -well plate (5 After 72 hours of incubation, MTT reagent was added to each well and the plate was incubated at 37°C for 2 hours. Afterwards, the medium was removed and the plate was washed with PBS. Intracellular formazan was dissolved in DMSO (Sigma Aldrich Co.) and absorbance was measured at 595 nm using a microplate reader (BIO-TEK Power Wave XS, Winooski, VT, USA).

1-7. Melanin content measurement

B16F10 cells were seeded in a 6-well plate ( ¹ After 72 hours, cells were washed with PBS and scraped. After centrifugation of the collected cells, the supernatant was discarded and lysed with 1N NaOH. Lysed cells were transferred to a 96-well plate, and melanin content was measured by measuring absorbance at 405 nm using a microplate reader (BIO-TEK Power Wave XS, Winooski, VT, USA).

1-8. Measurement of intracellular tyrosinase activity

Cells were seeded in 6-well plates ( ¹ After 72 h of culture, cells were washed with PBS and lysed in 1% Triton X-100. The lysed cells were then cooled on ice for 10 minutes and centrifuged, and the supernatant was collected to determine the enzyme source for the tyrosinase assay. The reaction mixture contained 100 mL of 0.1 M phosphate buffer (pH 6.5), 100 mL of 20mM L-DOPA, and 40 μg of cell lysate in each well of a 96-well microplate, and the reaction mixture was incubated at room temperature. After 1 hour, final absorbance was measured at the same wavelength, and intracellular tyrosinase activity was estimated as a ratio to the control.

1-9. Western blotting analysis

For protein expression analysis, B16F10 cells were harvested and homogenized in lysis buffer at 4°C. After centrifugation, cell debris was discarded and protein concentration was measured using the BCA (bicinchoninic acid) assay. 20 mg of protein was separated on a 10% SDS-PAGE gel and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). PVDF membranes were blocked with 5% skim milk at room temperature for 2 hours and then incubated with primary antibodies. Anti-tyrosinase, anti-TRP-1, anti-TRP-2 (Santa Cruz, CA, USA), anti-MC1R (Melanocortin 1 receptor, abcam) and anti-MITF (Microphthalmia-associated transcription factor, Cell signaling Technology, Beverly) , MA, USA) was used as the primary antibody. Anti-goat IgG-horseradish peroxidase (HRP) and anti-rabbit IgG-HRP (Santa Cruz) were used as secondary antibodies. The reaction was then carried out using Super Signal® West Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL, USA). Immunoreactive bands were visualized using enhanced LAS 4000 film (Fuji film, Tokyo, Japan). Protein loading was monitored in each lane using an anti-GAPDH antibody. Densitometric analysis was performed using Image J software.

1-10. Q-PCR analysis of mRNA expression

Total RNA was extracted using the RNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol and stored at -80°C until use. cDNA was amplified using the ImProm-II reverse transcription system (Promega, Madison, WI, USA) according to the manufacturer's instructions. Afterwards, SYBR green-based quantitative PCR was performed using the Applied Biosystems 7500 Fast Real-Time PCR System and Fast SYBR® Green Master Mix (Life Technologies, UK). All reactions were performed in triplicate and data were analyzed using the 2 ^-ΔΔ CT value method.

The sequences of the primers used in this study are MITF forward: 5'-ATG GAC GAC ACC CTT TCT C-3' (SEQ ID NO: 1); MITF reverse: 5'-GGA GGA TTC GCT AAC AAG TG-3' (SEQ ID NO: 2); Tyrosinase forward: 5'-GGC CAG CTT TCA GGC AGA GGT-3' (SEQ ID NO: 3); Tyrosinase reverse: 5'-TGG TGC TTC ATG GGC AAA ATC-3' (SEQ ID NO: 4); TRP1 forward: 5'-AAG CAG ACA TCC AAC AAC ACT AG-3' (SEQ ID NO: 5); TRP1 reverse: 5'-GCA AGA GTT CAG AAC ACA GGT C-3' (SEQ ID NO: 6); TRP2 forward: 5'-GCA AGA GAT ACA CGG AGG AAG-3' (SEQ ID NO: 7); TRP2 reverse: 5'-CTA AGG CAT CAT CAT CAT CAC TAC-3' (SEQ ID NO: 8); β-actin forward: 5'-GAC AGG ATG CAG AAG GAG ATT ACT-3' (SEQ ID NO: 9); β-actin reverse: 5'-TGA TCC ACA TCT GCT GGA AGG T-3' (SEQ ID NO: 10). The amount of each transcript was calculated as described in the instrument manual and normalized to the amount of β-actin.

1-11. Confirmation of depigmentation effect of Bibichu extract on zebrafish

The experiment was performed according to the standard zebrafish protocol approved by the Kyung Hee University Animal Care Committee [KHUASP(SE)-15-10]. Adult zebrafish under the following conditions were obtained from the Zebrafish Resource Bank (Kyungpook University, Korea); 28.5℃, 14/10 h light/dark cycle at Zebrafish System S type [1500 (W) Х 400 (D) Х 2050 (H) mm] (Genomic Design, Korea). Zebrafish were fed live brine shrimp ( Artemia salina ) twice a day. Embryos were obtained from natural oviposition induced in the morning by exposure to white light. The collected embryos were washed three times using 0.03% sea salt solution. Embryos at 9 hpf were then placed individually into wells of a 96-well plate filled with 100 μl 0.03% sea salt solution, baboon extract, fractions, or isolated compounds. Embryos were treated with 1 μg/ml Bibitra extract and fractions, or 1 μM isolated compounds dissolved in sea salt solution containing 1% DMSO. 1-Phenyl-2-thiourea (PTU) was used as a positive control for depigmentation.

2. Experiment result

2-1. Effect of Bibichurian extract on melanin synthesis

To identify the depigmenting components in the baboon extract, all fractions and isolated compounds were subjected to an anti-melanogenesis assay against B16F10 melanoma cells. α-Melanocyte stimulating hormone (α-MSH) is a peptide hormone and is responsible for melanin production by melanocytes through activation of the melanocortin 1 receptor. In this experiment, B16F10 cells were stimulated with α-MSH (100 nM) and simultaneously treated with each extract, fraction, and compound.

After culturing the cells with the B. birch extract for 72 hours, melanin production was measured. As shown in Figure 1, the melanin level was significantly reduced by 300 μg/ml B. c. extract. Additionally, as a result of measuring melanin production in each fraction, the n-hexane fraction was most effective, and the BuOH fraction showed cytotoxicity (Figure 2). As a result of measuring melanin production by the most effective peak of the n-hexane fraction, peak 6 showed the best melanin production inhibition effect, and peak 3 showed cytotoxicity at 30 uM (Figure 3).

2-2. Effect of Bibitracus extract on tyrosinase activity

Tyrosinase activity was analyzed to understand the possible underlying mechanisms involved in the reduction of melanin synthesis induced by B. birch extract in B16F10 cells. Tyrosinase is one of the most important enzymes involved in regulating melanin production. Specifically, the direct effects of B. chinensis extract and n-hexane fraction on tyrosinase activity were investigated using mushroom tyrosinase in a cell-free system. The extract and n-hexane fraction showed a tendency to inhibit mushroom tyrosinase activity in a concentration-dependent manner (Figure 4).

Intracellular tyrosinase activity was measured after incubating B16F10 cells with the B. birch extract and n-hexane fractions at different concentrations for 72 hours. The results showed that B. c. extract and n-hexane fractions 1, 3, and 10 μg/ml had tyrosinase activity in the cells. was inhibited by 1, 3, 9%, 2, 4, and 10%, respectively (Figure 5). These results indicate that the down-regulation of intracellular tyrosinase activity and direct inhibition of tyrosinase activity in B16F10 cells is due to the anti-melanin role of the B. birch extract/fraction.

2-3. Effects of Bibimbap extract on melanin biosynthesis-related protein and gene expression

Western blotting using cell lysates treated with B. birch extract was used to investigate the effect of B. c. extract on the expression of melanin biosynthesis-related proteins, MITF, tyrosinase, TRP1, and TRP2. As a result, the B. c. extract and n-hexane fraction had tyrosinase and It reduced the expression of TRP2 protein, inhibited MITF, and not only the Bibitra extract but also the n-hexane fraction also reduced the expression of MC1R protein (Figures 6 and 7). In other words, this means that the downregulation of melanin biosynthesis-related proteins by Bibitra extract is related to MC1R signaling.

To investigate whether inhibition of these protein expressions was associated with decreased mRNA levels of melanin biosynthesis-related genes, the mRNA expression levels of MITF, tyrosinase, TRP-1, and TRP-2 were confirmed using Q-PCR. As a result, the Bibitra extract reduced the gene expression of MITF, and the n-hexane fraction also reduced the gene expression of tyrosinase, TRP-1, and TRP-2 (Figure 8).

2-4. In vivo zebrafish pigmentation analysis

To confirm the depigmentation effect of B. c. extract, n-hexane fraction, and major compounds (rp1, rp2), embryos were treated with 1 μg/ml of B. c. extract or n-hexane fraction. As shown in Figure 9, it was confirmed that black spots in zebrafish embryos were significantly reduced as a result of treatment with the Bibitra extract and n-hexane fraction. The size of black spots was significantly reduced in embryos treated with 1 μg/ml of Bibitra extract and n-hexane fraction compared to the control group (Figure 10).

Example 2: Preparation of bibi harvest

For the cosmetic composition of the present invention, purified water was replaced with bibichus extract to prepare bibichus. First, 1000 mL of distilled water was added to 100 g of dried Bibichu (based on total dry weight) and reacted at 121°C for 1 hour. Afterwards, the reactant was filtered using a 2G3 Glass-filter (PYREX 32940 FNL, JAPAN) to obtain a hot water extract, that is, Bibichu. The number was prepared.

Example 3: Preparation of Extract of Peach Blossom Extract

The extract raw material was prepared by mixing 5 parts by weight of Seonbokhwa (flowers, leaves, stems) with 100 parts by weight of purified water. The raw material was subjected to hot water extraction at 90 to 100°C for 4 to 8 hours to obtain an extract, and then filtered through a filter membrane with an average pore size of 25 ㎛ to obtain a primary filtrate. Afterwards, 5 ml of 1,2-hexanediol and 0.1 ml of ethylhexylglycerin were added to 94.9 ml of the filtrate, then sufficiently stirred, and the stirred solution was secondarily filtered using a filter membrane with an average pore size of 10 ㎛ to obtain the P. lily vulgaris extract. was manufactured.

Example 4: Preparation of rhubarb extract

The extract raw material was prepared by mixing 5 parts by weight of rhubarb (root, leaf, stem) with 100 parts by weight of purified water. The raw material was subjected to hot water extraction at 90 to 100°C for 4 to 8 hours to obtain an extract, and then filtered through a filter membrane with an average pore size of 25㎛ to obtain a primary filtrate. Afterwards, 5 ml of 1,2-hexanediol and 0.1 ml of ethylhexylglycerin were added to 94.9 ml of the filtrate, stirred sufficiently, and the stirred solution was filtered a second time using a filter membrane with an average pore size of 10 ㎛ to produce the rhubarb extract. was manufactured.

Example 5: Preparation of Macmundong extract

An aqueous ethanol solution with a concentration of 70% by volume was added to the roots of the lobular valvular sinus, followed by cold soaking at 26 to 27°C for 48 hours, followed by filtration and concentration under reduced pressure to obtain a crude extract. The crude extract was dissolved in an aqueous methanol solution with a concentration of 80% by volume and placed in a separatory funnel. An equal amount of hexane was added thereto, shaken vigorously, and left to stand. Within a few minutes, it was separated into a hexane layer on top and a methanol layer with a concentration of 80% by volume below, and was left for an additional hour or more to completely separate. The separated hexane layer was set aside, and the distribution was repeated by adding hexane again to the methanol layer at a concentration of 80% by volume. For the second time, the separated hexane layer is also collected, and hexane is added again to the methanol layer at a concentration of 80% by volume for distribution. The hexane layers separated three times in this way were combined and concentrated under reduced pressure to perform hexane fractionation. Next, the methanol layer with a concentration of 80% by volume was concentrated to make a methanol solution with a concentration of about 30% by volume and this time distributed with chloroform. Chloroform has a heavy specific gravity, forming the lower layer, and the methanol layer with a concentration of 30% by volume became the upper layer. The chloroform layer was separated and the same method was repeated twice more, and the separated chloroform layers were combined and concentrated under reduced pressure to form a chloroform fraction. The methanol layer with a concentration of 0% by volume was concentrated to completely remove methanol, and then distributed three times with equal amounts of butanol and water. The separated butanol layers were combined and concentrated under reduced pressure to obtain a butanol fraction, thereby obtaining a Macmundong extract.

Example 6: Preparation of lotus extract

An extract raw material was prepared by mixing 13 parts by weight of dried lotus powder with 100 parts by weight of purified water. The raw material was subjected to hot water extraction at 85°C for 4 hours to obtain an extract, and then filtered through a filter membrane with an average pore size of 25㎛ to obtain a primary filtrate. Afterwards, 5 ml of 1,2-hexanediol and 0.1 ml of ethylhexylglycerin were added to 94.9 ml of the filtrate, and after sufficient stirring, the stirred solution was secondarily filtered using a filter membrane with an average pore size of 10 ㎛ to obtain lotus extract. Manufactured.

Example 7: Preparation of chaga mushroom extract

Raw materials were prepared by mixing 2.2 parts by weight of dried chaga powder with 100 parts by weight of purified water. The raw material was subjected to reflux-cooled extraction at 35°C for 6 hours to obtain an extract, and then filtered through a filter membrane with an average pore size of 25㎛ to obtain a primary filtrate. Afterwards, 5 ml of 1,2-hexanediol and 0.1 ml of ethylhexylglycerin were added to 94.9 ml of the filtrate, and after sufficient stirring, the stirred solution was secondarily filtered using a filter membrane with an average pore size of 10㎛ to obtain chaga extract. was manufactured.

Example 8: Preparation of the cosmetic composition of the present invention containing Bibitracus extract, etc.

The cosmetic composition for improving the skin barrier of the present invention includes the Bibitracus extract, Bibitracus extract, Zenbok flower extract, Jong rhubarb extract, Macmundong extract, lotus extract, chaga mushroom extract, and Manuka honey as described in Examples 1 to 7 (Figure 11), based on 100 parts by weight of the total cosmetic composition, 60 to 80 parts by weight of Bibitracus extract, 1 to 5 parts by weight of Bibitracus extract, 0.5 to 3 parts by weight each of Seonbok flower extract, Jong rhubarb extract, Macmundong extract, lotus extract and chaga extract. , Manuka honey is prepared by mixing and stirring each material to produce 0.05 to 3 parts by weight (Figure 12).

Specifically, a mixture was prepared by dissolving and mixing 46.5 g of BBW, 2 g of betaine, 3 g of Manuka honey, and 0.03 g of disodium EDTA. Afterwards, 20 g of Bibisu and 1 g of carbomer were dispersed and added to the mixture. Afterwards, 5 g of birch extract and 2 g of niacinamide were mixed, completely dissolved, added to the mixture at 60°C, and stirred for 30 minutes while maintaining the temperature at 65°C to 70°C. Afterwards, 5 g of birch extract and 5 g of arginine were mixed, completely dissolved, added to the mixture, and stirred for 30 minutes. Afterwards, 0.99g of 1,2-hexanediol, 0.5g of ethylhexylglycerin, 3g of Macmundong extract, 3g of lotus extract, 3g of Chaga mushroom extract, 3g of Zenbok extract, 3g of Rhubarb extract, 5g of Bibichurian extract and 1g of glutathione were added at 40℃. It was added to the mixture and stirred for 20 minutes to prepare the cosmetic composition of the present invention.

Example 9: Confirmation of the skin barrier improvement effect of the cosmetic composition of the present invention

9-1. Selection of test subjects

In order to confirm the skin barrier improvement effect of the cosmetic composition of the present invention prepared in Example 8, a total of 21 test subjects (average age 47.714 ± 7.976 years) who met the test subject selection criteria were selected. The criteria for selecting test subjects are: i) healthy people aged 20 to 60, ii) people with sensitive skin, iii) people who voluntarily signed consent after receiving a thorough explanation of the purpose and content of the test, and iv) people with follow-up observations during the test period. It consists of those who are possible, and v) those who are not included in the test subject exclusion criteria. The exclusion criteria for test subjects are: i) those who do not wish to participate or who do not fill out the consent form, ii) those with psychiatric disorders, iii) those with infectious skin diseases, and iv) those who have received immunosuppressant treatment within 3 months of participating in the study. v) If you have received systemic steroids or light therapy within 1 month of participating in the test; vi) If you have lesions in the test area that make measurement difficult; vii) If you are pregnant or lactating or have the potential to become pregnant; viii) If you have used cosmetics, pharmaceuticals, or This includes cases where there is a severe reaction or allergy to routine light exposure, ix) and other cases where, in addition to the above, it is deemed difficult to conduct human testing at the discretion of the lead researcher or person in charge of testing.

In order to confirm whether the test subject had sensitive skin, the test was conducted after selecting test subjects with sensitive skin through a lactic acid prick test. For the lactic acid prick test, 50㎕ of lactic acid diluted to 10% was soaked in a cotton swab using a micropipette and then applied to the test subject's nasolabial fold. One minute after application of the sample, the test subject's subjective degree of stinging (itching, stinging, burning) was evaluated with a score (0: none, 1: mild, 2: moderate, 3: severe), and if the sample showed a positive reaction, it was considered sensitive. The subject was determined to be a skin subject. Specific test subject information is shown in Table 1.

Test start test subject 21 people dropout 0 people Reason for dropping out - Subject who completed the test 21 people gender 21 women average age 47.714±7.976 years

9-2. Test Methods

(1) Test subject visit and progress schedule

1) Visit 1: Confirmation of test subject consent and test subject selection/exclusion criteria, confirmation of sensitive skin through lactic acid prick test, skin measurement before use of the cosmetic composition containing the Bibitracus extract of the present invention, and distribution of compliance logs

2) Visit 2: Skin measurement after 2 weeks of use, check for adverse reactions and complete questionnaire evaluation

3) Visit 3: After 4 weeks of use, skin is measured to check for adverse reactions, questionnaires are completed, and compliance logs are collected.

(2) Skin measurement methods and procedures

All human application tests are conducted based on the Good Clinical Practice (GCP) and the Declaration of Helsinki, and the ethical validity is reviewed by the KC Skin Clinical Research Center Human Application (Clinical) Institutional Review Board (IRB). did.

To check the condition of the skin, the test subject washed his face with a prepared face wash and allowed his skin to settle for about 30 minutes in a room with constant temperature and humidity conditions (temperature 20-24°C, humidity 40-60%) to adapt to the measurement environment. The application area was photographed or measured, and each time was conducted by the same researcher under the same conditions.

(3) Statistical result analysis method

In order to determine the significance of the change in skin measurements before and after using the cosmetic composition of the present invention prepared in Example 8, it was verified using SPSS 23.0, a statistical analysis program. Statistical significance was confirmed when the probability of significance was p<0.05 in the 95% confidence interval.

1) Comparison before and after use (single measurement)

- Satisfies normality test: Paired samples T-test (parametric method)

- Unsatisfactory normality test: Wilcoxon signed ranks test (non-parametric method)

9-3. Check moisturizing effect

In order to confirm the moisturizing effect of the cosmetic composition of the present invention prepared in Example 8, the skin moisturizing power was measured.

Specifically, the skin moisturizing power was measured using a corneometer to measure the skin moisture content on the cheek area of the face before, 2 weeks after, and 4 weeks after use of the cosmetic composition containing the Bibichu extract of the present invention. The unit of measured skin moisture content is A.U. (Arbitrary Unit), and the average of three measurements was used as skin moisturizing power evaluation data. Since the measurement value and the level of skin moisture content are proportional, it means that skin moisturization improves as the measurement value increases.

As a result of the measurement, as shown in Table 2 and Figure 13, the skin moisture content measurement value was statistically significantly increased after 2 weeks and 4 weeks after use compared to before use of the cosmetic composition of the present invention prepared in Example 8. confirmed. (p<0.05)

Changes in skin moisture content part Mean±standard deviation (A.U.) Rate of change ^a (%) Significance probability ^b (p value) Before use 57.565±8.922 - - After 2 weeks of use 66.157±8.701 15.450 0.000* After 4 weeks of use 62.073±8.882 8.127 0.000*

Rate of change ^a (%) = [(measured value after use - measured value before use)/measured value before use]x100

Significance probability ^b (p value) *: p<0.05 by Paired samples T-test

9-4. Check the shine effect

In order to confirm the gloss effect of the cosmetic composition of the present invention prepared in Example 8, skin gloss was measured.

Specifically, skin gloss was measured using a Skin Glossmeter on the cheek area of the face before using the test product, 2 weeks after using the product, and 4 weeks after using the product. The unit of skin gloss measured is S.G.U. (Skin Gloss Unit), and the average value of three measurements was used as skin gloss evaluation data. Since the measurement value and the degree of skin gloss are proportional, it means that skin gloss improves as the measurement value increases.

As a result of the measurement, as shown in Table 3 and Figure 14, the skin gloss measurement value increased statistically significantly after 2 weeks and 4 weeks after use compared to before using the cosmetic composition of the present invention prepared in Example 8 ( p<0.05).

changes in skin radiance part Mean±Standard Deviation (Skin Gloss Uint) Rate of change ^a (%) Significance probability ^b (p value) Before use 63.921±7.877 - - After 2 weeks of use 68.587±8.993 7.350 0.000* After 4 weeks of use 66.810±8.594 4.497 0.000*

Rate of change ^a (%) = [(measured value after use - measured value before use)/measured value before use]x100

Significance probability ^b (p value) *: p<0.05 by Paired samples T-test

9-5. Confirm sebum reduction effect

In order to confirm the sebum reduction effect of the cosmetic composition of the present invention prepared in Example 8, skin sebum was measured.

Specifically, skin sebum was measured using a Sebumeter to measure the skin sebum content in the area between the eyebrows before using the test product, 2 weeks after using the product, and 4 weeks after using the product. The unit of measured skin sebum content is ㎍/cm ² , and the value measured once was used as skin sebum evaluation data. Since the measured value and the degree of skin sebum are proportional, it means that the skin sebum improves as the measured value decreases.

As a result of the measurement, as shown in Table 4 and Figure 15, the skin sebum content measurement value was statistically significantly reduced after 2 weeks and 4 weeks after use compared to before use of the cosmetic composition of the present invention prepared in Example 8. (p<0.05).

Changes in skin sebum content part Mean±standard deviation (μg/cm ² ) Rate of change ^a (%) Significance probability ^b (p value) Before use 52.476±28.177 - - After 2 weeks of use 43.095±22.980 -15.641 0.000* After 4 weeks of use 43.619±22.333 -15.350 0.000*

Rate of change ^a (%) = [(measured value after use - measured value before use)/measured value before use]x100

Significance probability ^b (p value) *: p<0.05 by Paired samples T-test

9-6. Confirm the effect of improving dead skin cells

In order to confirm the keratin reduction effect of the cosmetic composition of the present invention prepared in Example 8, skin keratin was measured.

Specifically, skin keratin was measured using Corneofix, a special adhesive tape, to collect keratin from the cheek area of the face before using the test product, 2 weeks after using the product, and 4 weeks after using the product, and photographed with a Visioscan VC98. The average keratin index (DI: Desquamation Index) of the taken photos was used as evaluation data, and the unit of the measured keratin analysis value is %. Since the analysis value and the degree of skin keratin are proportional, it means that as the analysis value decreases, the skin keratin improves.

As a result of the measurement, as shown in Table 5 and Figure 16, the skin keratin analysis value decreased statistically significantly after 2 weeks and 4 weeks after use compared to before using the cosmetic composition of the present invention prepared in Example 8 ( p<0.05).

changes in skin keratin part Mean±standard deviation (%) Rate of change ^a (%) Significance probability ^b (p value) Before use 4.630±1.662 - - After 2 weeks of use 3.836±1.393 -15.523 0.001* After 4 weeks of use 4.291±1.531 -5.304 0.015*

Rate of change ^a (%) = [(measured value after use - measured value before use)/measured value before use]x100

Significance probability ^b (p value) *: p<0.05 by Paired samples T-test

9-7. Evaluation of adverse skin reactions

In order to determine whether adverse skin reactions occurred when using the cosmetic composition of the present invention prepared in Example 8, a survey and visual evaluation of adverse reactions were conducted at each visit to determine whether or not they occurred and the degree of symptoms. As a result, as shown in Table 6, it was confirmed that no adverse reactions occurred 2 weeks and 4 weeks after using the cosmetic composition of the present invention prepared in Example 8.

Adverse skin reactions (persons, level average) hour erythema edema scaly itch sting burning sensation stiffness tingling After 2 weeks of use - - - - - - - - After 4 weeks of use - - - - - - - -

9-8. Test subject survey evaluation

A questionnaire evaluation was conducted to confirm the test subjects' skin improvement effectiveness, suitability for use on sensitive skin, and preference for the cosmetic composition of the present invention prepared in Example 8. Test subjects filled out a questionnaire on a 5-point scale (5: very satisfied, 4: satisfied, 3: average, 2: dissatisfied, 1: very dissatisfied), and the number of test subjects who answered 3 or more was calculated as a percentage ( expressed as positive response rate).

As a result, as shown in Tables 7 and 8, it was confirmed that the positive response rate exceeded 90% after 2 weeks and 4 weeks after use of the cosmetic composition of the present invention prepared in Example 8.

Survey of test subjects after 2 weeks of use item Mean±standard deviation Positive response rate (%) Improved skin moisturizing ability 4.095±0.539 100 Improved skin glow 4.143±0.573 100 Improving skin sebum 3.952±0.590 100 Improved skin exfoliation 4.238±0.700 100 Suitable for use on sensitive skin 4.143±0.854 95.238 feeling of use 4.095±0.831 95.238 degree of scent 3.810±0.814 95.238 recommended doctor 4.048±0.740 95.238 purchase intent 3.905±0.889 95.238 overall satisfaction 4.143±0.655 95.238

5-point scale - 5: very satisfied, 4: satisfied, 3: average, 2: dissatisfied, 1: very dissatisfied

Positive response rate (%) - Percentage of subjects who responded with a score of 3 or higher (neutral, satisfied, very satisfied)

Survey of test subjects after 4 weeks of use item Mean±standard deviation Positive response rate (%) Improved skin moisturizing ability 4.286±0.644 100 Improved skin glow 4.429±0.746 95.238 Improving skin sebum 4.286±0.561 100 Improved skin exfoliation 4.286±0.561 100 Suitable for use on sensitive skin 4.286±0.717 95.238 feeling of use 4.095±0.889 95.238 degree of scent 3.857±1.195 90.476 recommended doctor 4.286±0.784 95.238 purchase intent 4.190±0.928 95.238 overall satisfaction 4.190±0.680 95.238

5-point scale - 5: very satisfied, 4: satisfied, 3: average, 2: dissatisfied, 1: very dissatisfied

Positive response rate (%) - Percentage of subjects who responded with a score of 3 or higher (neutral, satisfied, very satisfied)

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 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.

<110> NEWLAND ALL NATURE CO.,LTD. <120> Cosmetic composition for skin whitening or skin barrier improvement comprising Hosta longipes extract <130> KPA01577 <150> KR 1020200063930 <151> 2020-05-27 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 1 atggacgaca ccctttctc 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 2 ggaggattcg ctaacaagtg 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 3 ggccagcttt caggcagagg t 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 4 tggtgcttca tgggcaaaat c 21 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 5 aagcagacat ccaacaacac tag 23 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 6 gcaagagttc agaacacagg tc 22 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 7 gcaagagata cacggaggaa g 21 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 8 ctaaggcatc atcatcatca ctac 24 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 9 gacaggatgc agaaggat tact 24 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 10 tgatccacat ctgctggaag gt 22

Claims (9)

A cosmetic composition for skin whitening, improving the skin barrier, and improving sebum, comprising Bibitracus extract, Bibichu extract, Chanbok flower extract, rhubarb extract, McMoondong extract, lotus extract, chaga extract, and Manuka honey.
According to paragraph 1,
A cosmetic composition wherein the skin barrier improvement includes improvement of skin moisturization.
According to paragraph 1,
A cosmetic composition wherein the skin barrier improvement includes improving skin gloss.
delete According to paragraph 1,
A cosmetic composition wherein the skin barrier improvement includes improvement of skin keratin.
According to paragraph 1,
60 to 80 parts by weight of BB extract based on 100 parts by weight of the total cosmetic composition; 1 to 10 parts by weight of Bibitracus extract; 0.5 to 3 parts by weight each of Seonbokhwa extract, Jong rhubarb extract, Macmundong extract, lotus extract, and chaga extract; and 0.05 to 3 parts by weight of Manuka honey as an active ingredient.
According to paragraph 1,
A cosmetic composition, wherein the extract is prepared by extraction with a solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, and mixed solvents thereof.
According to paragraph 1,
The composition is formulated as astringent, softening, and nutritious face lotions, nourishing cream, massage cream, essence, pack, skin adhesive patch, skin adhesive gel, powder, ointment, suspension, emulsion, spray, serum, or capsule. A cosmetic composition.
Method for producing the cosmetic composition according to claim 1, comprising the following steps:
(a) dissolving and mixing BB fruit, betaine, manuka honey, and disodium EDTA;
(b) dispersing BB extract and carbomer and then adding them to the mixture of step (a);
(c) adding and dissolving niacinamide to the bibi harvest and then adding it to the mixture of step (b) and stirring;
(d) adding and dissolving arginine in the bibi harvest and then adding it to the mixture of step (c) and stirring; and
(e) 1,2-hexanediol, ethylhexylglycerin, McMoondong extract, lotus flower extract, chaga extract, Peach blossom extract, Jong rhubarb extract, Bibitracus extract, and glutathione were added to the mixture in step (d) above and stirred. Steps for preparing a cosmetic composition.