WO2024128370A1 - Cosmetic composition for whitening or wrinkle reduction, comprising fermentation product of pueraria lobata root extract - Google Patents
Cosmetic composition for whitening or wrinkle reduction, comprising fermentation product of pueraria lobata root extract Download PDFInfo
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- WO2024128370A1 WO2024128370A1 PCT/KR2022/020659 KR2022020659W WO2024128370A1 WO 2024128370 A1 WO2024128370 A1 WO 2024128370A1 KR 2022020659 W KR2022020659 W KR 2022020659W WO 2024128370 A1 WO2024128370 A1 WO 2024128370A1
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- cosmetic composition
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- whitening
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- skin
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Images
Definitions
- the present invention relates to a cosmetic composition for whitening or wrinkle improvement containing a fermented product of arrowroot extract.
- This invention was made with support from the Korean government under the Ministry of Health and Welfare's project number 1465032918, "Development of microbiome materials with skin-improving efficacy.”
- Causes of skin aging include internal aging and external aging.
- Internal aging is caused by a decrease in the secretion of various hormones that control metabolism as one ages and a decrease in immune cell function and cell activity
- external aging is caused by free radicals and active harmful substances caused by external factors such as ultraviolet rays and environmental pollution. This is caused by an increase in oxygen.
- various changes occur, including decreased elasticity and increased wrinkles, poor skin complexion, frequent skin troubles, and increased age spots, freckles, and spots.
- MMP matrix metalloproteinase
- melanin is one of the natural pigments contained in the skin and hair of mammals, and has various beneficial functions, such as protecting the skin from harmful ultraviolet rays (UV) and preventing the development of cancer.
- melanin production activity increases in melanocytes as a defense mechanism against stimulation such as ultraviolet rays, and the large amount of melanin produced can be transferred to keratinocytes and accumulate in the epidermal layer of the skin.
- Excessive production and accumulation of melanin causes skin diseases such as blemishes, freckles, skin darkening after skin inflammation, and age-related pigment spots, and not only causes cosmetic inconvenience to the person involved, but also has a negative psychological impact, causing inconvenience in social activities. Additionally, these skin diseases are problematic because they may be accompanied by inflammation such as allergic contact dermatitis or irritant contact dermatitis.
- Substances known to be effective in improving skin wrinkles include adenosine and retinoic acid, but adenosine has minimal clinical efficacy, retinoic acid cannot be used in women of childbearing age, and has a high risk of irritation such as erythema.
- hydroquinone, arbutin, ascorbic acid, kojic acid, and glutathione are known to be substances that inhibit melanin production, but hydroquinone is highly irritating to the skin. In general, use is restricted. Because ascorbic acid is easily oxidized, discoloration and off-odor are problems in cosmetics containing it.
- Thiol-based compounds such as glutathione and cysteine not only have a unique unpleasant odor but also have problems with transdermal absorption, and their glycosides and derivatives Because they are highly polar, there is a problem in that they are difficult to use as ingredients in cosmetics.
- the present inventor conducted research to discover a material effective for skin whitening and skin wrinkle improvement and completed the present invention.
- One object of the present invention is to provide a cosmetic composition for whitening or wrinkle improvement containing a fermented product of arrowroot extract.
- One aspect of the present invention provides a cosmetic composition for whitening or wrinkle improvement comprising a fermented product of arrowroot extract.
- the extraction solvent of the extract may be a solvent selected from the group consisting of alcohols having 1 to 4 carbon atoms and mixtures thereof.
- the extraction solvent for the extract may be an aqueous ethanol solution.
- the volume ratio of ethanol and water in the ethanol aqueous solution may be 1:9 to 9:1.
- the fermented product may be fermented with the strain Pediococcus pentosaseus SEQ0315 (Accession Number: KACC92051P).
- the composition may be included in an amount of 0.01 to 30.0 parts by weight based on 100 parts by weight of cosmetics.
- the cosmetic composition may be a solution, mist, suspension, emulsion, paste, gel, cream, serum, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion. It may be any one formulation selected from the group consisting of foundation, wax foundation, and spray.
- a cosmetic composition for whitening or wrinkle improvement containing a fermented product of arrowroot extract, it not only effectively inhibits melanin biosynthesis but also has an excellent effect of producing type I procollagen, making it a cosmetic composition for skin whitening and wrinkle improvement. It can be effectively used as a material.
- Figure 1 is a manufacturing process diagram of Pediococcus/kudzu root fermentation filtrate according to an embodiment of the present invention.
- Figure 2 is a graph showing the results of HPLC chromatography analysis of Pediococcus/kudzu root fermentation filtrate according to an embodiment of the present invention.
- Figure 3 is a graph showing the results of analyzing and comparing the daidzein content before (A) and after fermentation (B) of the filtrate fermented without adding arrowroot powder by HPLC chromatography.
- Figure 4 is a graph showing the results of analyzing and comparing the daidzein content before (A) and after (B) fermentation of the Pediococcus/kudzu root fermentation filtrate according to an embodiment of the present invention by HPLC chromatography method. am.
- Figure 5 is a graph showing the survival rate of B16-F10 melanoma cells treated with Pediococcus/kudzu root fermentation filtrate according to an embodiment of the present invention.
- Figure 6 shows (A) extracellular melanin contents and (B) extracellular melanin inhibition rate of B16-F10 melanoma cells treated with Pediococcus/kudzu root fermentation filtrate according to an embodiment of the present invention ( This is a graph showing the inhibition of extracellular melanin contents.
- Figure 7 shows (A) intracellular melanin contents and (B) intracellular melanin inhibition rate of B16-F10 melanoma cells treated with Pediococcus/kudzu root fermentation filtrate according to an embodiment of the present invention ( This is a graph showing inhibition of intracellular melanin contents.
- Figure 8 shows (A) total melanin contents and (B) total melanin inhibition rate of B16-F10 melanoma cells treated with Pediococcus/kudzu root fermentation filtrate according to an embodiment of the present invention. This is a graph showing total melanin contents.
- Figure 9 is a graph showing the survival rate of CCD-986sk cells treated with Pediococcus/kudzu root fermentation filtrate according to an embodiment of the present invention.
- Figure 10 is a graph showing type I procollagen production in CCD-986sk cells treated with Pediococcus/kudzu root fermentation filtrate according to an embodiment of the present invention.
- Figure 11 is a graph showing (A) the degree of melanin index improvement and (B) the melanin index improvement rate according to the human application test of the Pediococcus/kudzu root fermentation filtrate according to an embodiment of the present invention.
- Figure 12 is a graph showing (A) the degree of improvement in wrinkle index R1 (roughness) and (B) the improvement rate of wrinkle index R1 according to the human application test of the Pediococcus/kudzu root fermentation filtrate according to an embodiment of the present invention. .
- Figure 13 shows (A) the degree of improvement in the wrinkle index R2 (Maximum Roughness) and (B) the improvement rate of the wrinkle index R2 according to the human application test of the Pediococcus / arrowroot ferment filtrate according to an embodiment of the present invention. It's a graph.
- Figure 14 shows (A) the degree of improvement in the wrinkle index R3 (average roughness) and (B) the improvement rate of the wrinkle index R3 according to the human application test of the Pediococcus / arrowroot ferment filtrate according to an embodiment of the present invention. It's a graph.
- Figure 15 shows (A) the degree of improvement in the wrinkle index R4 (smoothness depth) and (B) the improvement rate of the wrinkle index R4 according to the human application test of the Pediococcus / arrowroot ferment filtrate according to an embodiment of the present invention. It's a graph.
- Figure 16 shows (A) the degree of improvement in the wrinkle index R5 (Arithmetic Average Roughness) and (B) the improvement rate of the wrinkle index R5 according to the human application test of the Pediococcus/kudzu root fermentation filtrate according to an embodiment of the present invention. This is a graph showing .
- One aspect of the present invention provides a cosmetic composition for whitening or wrinkle improvement comprising a fermented product of arrowroot extract.
- Pueraria lobata used in the production of fermented product included as an active ingredient in the cosmetic composition of the present invention, is a vine plant of the dicotyledonous Rosaceae legume family, and has been used as an edible crop for a long time and as a health food such as a nutritional tonic.
- the root is used as a medicinal herb called kudzu root, and it is known to have effects such as sweating and fever reduction, but there is nothing known about the skin whitening and wrinkle-improving effects of fermented arrowroot extract.
- the cosmetic composition of the present invention further contains fatty substances, organic solvents, solubilizers, thickening and gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or non-ionic.
- the cosmetic composition of the present invention may contain various bases and/or additives necessary and appropriate for the formulation of the formulation, and may include non-ionic surfactants, silicone polymers, extenders, fragrances, preservatives, Disinfectants, oxidation stabilizers, organic solvents, ionic or non-ionic thickeners, softeners, antioxidants, free radical destroyers, opacifiers, stabilizers, emollients, silicones, ⁇ -hydroxy acids, anti-foaming agents, humectants, It can be prepared by further containing known compounds such as vitamins, insect repellent, fragrance, preservative, surfactant, anti-inflammatory agent, substance P antagonist, filler, polymer, propellant, alkalinizing or acidifying agent, or colorant.
- the extraction solvent for the extract may be selected from the group consisting of alcohols having 1 to 4 carbon atoms and mixtures thereof.
- the extraction solvent for the extract may be an aqueous ethanol solution.
- the volume ratio of ethanol and water in the ethanol aqueous solution may be 1:9 to 9:1.
- the arrowroot extract used for producing the fermented product according to the present invention can be obtained as follows.
- the kudzu root is washed with water to remove foreign substances, dried in the shade, and an appropriate amount of solvent is added to ensure complete immersion.
- the kudzu root can be used in its dried state or pulverized and used in powder form.
- Kudzu root can be extracted using a common extraction solvent, preferably (a) anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (e.g.
- volume ratio of ethanol and water in the ethanol aqueous solution is not within the range of 1:9 to 9:1, daidzein may not be sufficiently extracted during the fermentation process of the extract.
- the fermented product may be fermented with the strain Pediococcus pentosaseus SEQ0315 (Accession Number: KACC92051P).
- Pediococcus pentosaceus SEQ0315 strain was isolated from the feces of 20 people 1 month after taking kudzu, and the strain was deposited with the National Institute of Agricultural Sciences on April 9, 2015 (microorganism accession number: KACC92051P).
- the fermented product of the arrowroot extract fermented by Pediococcus pentosaceus SEQ0315 strain contains about twice the amount of Daidzein, an indicator component, compared to the unfermented arrowroot root extract, so Pediococcus Fermented kudzu root extract fermented with Pentosaceus SEQ0315 strain can be used more effectively for skin whitening and skin wrinkle improvement compared to unfermented kudzu root extract.
- the composition may be included in an amount of 0.01 to 30.0 parts by weight based on 100 parts by weight of cosmetics.
- the cosmetic composition may be a solution, mist, suspension, emulsion, paste, gel, cream, serum, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion. It may be any one formulation selected from the group consisting of foundation, wax foundation, and spray.
- the cosmetic compositions include solutions, suspensions, emulsions, pastes, gels, creams, serums, lotions, powders, soaps, surfactant-containing cleansing products, oils, powder foundations, emulsion foundations, wax foundations and sprays. It may be formulated, but is not limited thereto.
- softening lotion nourishing lotion, lotion, body lotion, nourishing cream, serum, massage cream, moisture cream, hand cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, gel, patch
- Basic cosmetics such as oil-in-water (O/W) type, water-in-oil (W/O) type, color cosmetics such as lipstick, makeup base or foundation, hair tonic, gel or It can be formulated as a hair cosmetic composition such as hair fixatives such as mousse, hair straightener, or hair dye.
- the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier ingredient. You can.
- 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.
- chlorofluorohydrocarbon, propane/ It may contain a propellant such as butane or dimethyl ether.
- a solvent, solubilizing agent, or emulsifying agent may be used as a carrier component.
- a solvent, solubilizing agent, or emulsifying agent may be used as a carrier component.
- 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 can be used. .
- the carrier component includes 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 miso.
- 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 miso.
- Crystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used.
- 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.
- the cosmetic composition of the present invention is a soap, a surfactant-containing cleansing formulation, or a surfactant-free cleansing formulation
- the soap may be, for example, liquid soap, powdered soap, solid soap, and oil soap.
- Cleansing formulations containing surfactants may be cleansing foam, cleansing water, cleansing towels, and cleansing packs. Cleansing formulations without surfactants may be cleansing formulations. It may be cream, cleansing lotion, cleansing water, and cleansing gel, but is not limited thereto.
- the crushed arrowroot root was mixed with 20% ethanol aqueous solution (v/v) at a weight ratio of 1:20, extracted at 25°C for 24 hours, filtered under reduced pressure, and the solvent was removed using a rotary vacuum concentrator to obtain arrowroot powder.
- v/v 20% ethanol aqueous solution
- the solvent was removed using a rotary vacuum concentrator to obtain arrowroot powder.
- the Pediococcus pentosaseus SEQ0315 Pediococcus pentosaseus SEQ0315; Accession number: KACC92051P
- strain subcultured in 1L of culture media was grown at 1 ⁇ 10 5 to 1 ⁇ 10 6 CFU/ml.
- the arrowroot powder before fermentation and the Pediococcus/Kudzu root fermentation filtrate after fermentation were subjected to HPLC chromatography.
- the Daidzein content of the Pediococcus/kudzu root fermentation filtrate after fermentation increased by about 2 times compared to the arrowroot powder before fermentation (Table 1 and Figure 2). It was set as an indicator component.
- Pediococcus pentosaceus SEQ0315 strain subcultured in 10L of culture media was inoculated to 1 ⁇ 10 5 to 1 ⁇ 10 6 CFU/ml and incubated at 37°C without the addition of arrowroot powder. After fermentation for 48 hours, sterilization (121°C, 15 minutes) and filtration (1 ⁇ m, 0.8 ⁇ m, 0.45 ⁇ m) were performed to prepare a fermented filtrate without adding arrowroot powder. Afterwards, the daidzein content of the filtrate fermented without adding the prepared arrowroot powder and the Pediococcus/kudzu root fermentation filtrate prepared in Example 1-1 were analyzed and compared using HPLC chromatography, respectively.
- B16-F10 melanoma cells were grown in DMEM medium containing 10% Fetal bovine serum (FBS) and 1% Penicillin/streptomycin (P/S) in a 150 mm cell culture dish at 37°C and 5% CO. Cultured in a CO 2 incubator. Cells that reached confluence were subcultured using Trypsin-EDTA.
- FBS Fetal bovine serum
- P/S Penicillin/streptomycin
- Dehydrogenase present in the mitochondria of living cells produces a coloring substance called formazan from Tetrazolium Salt (WST), so by measuring this and measuring the number of living cells, the effect of pediococcus/kudzu root fermentation filtrate on cell survival rate can be determined. was analyzed.
- B16-F10 cells cultured in Example 2-1 at a concentration of 2 ⁇ 10 3 cells/well in a 96 well cell culture plate were added to 10% FBS and 1% penicillin/streptomycin (P/ S) was cultured for 24 hours in DMEM culture medium added. Afterwards, the culture was replaced with DMEM medium containing the Pediococcus/kudzu root fermentation filtrate prepared in Example 1 at a concentration of 15.625, 31.25, 62.5, 125, or 250 ⁇ g/ml, and cultured for 72 hours. DMEM medium containing 10% EZ-CYTOX was added to the cultured cells and reacted for 2 hours. Then, the absorbance was measured at 450 nm using a plate multi-reader and the cell viability was calculated using the formula below. evaluated.
- Cell survival rate (%) (absorbance of sample added group/absorbance of untreated group) ⁇ 100
- the Pediococcus/kudzu root fermentation filtrate showed a cell viability of more than 90% at a concentration of 250 ⁇ g/ml or less (FIG. 5), confirming that it did not affect the cell viability.
- the B16-F10 cells cultured in Example 2-1 were cultured in DMEM medium supplemented with 10% FBS and 1% P/S at a concentration of 1 ⁇ 10 5 cells/well in a 6-well cell culture plate for 24 hours.
- 100 ⁇ M ⁇ -MSH ⁇ -Melanocyte stimulating hormone
- 100 ⁇ M ⁇ -MSH was diluted in DMEM/Modified medium to prepare DMEM/Modified medium containing 100 nM ⁇ -MSH. After removing the culture medium from cells cultured for 24 hours, the cells were washed with 2 ml of DPBS.
- samples were prepared by diluting the Pediococcus/kudzu root fermentation filtrate prepared in Example 1 in DMEM/Modified medium (100 nM ⁇ -MSH) at a concentration of 15.625, 31.25, 62.5, 125 or 250 ⁇ g/ml. After treatment, 3 ml each was cultured in an incubator at 37°C and 5% CO 2 for 48 to 72 hours, and ⁇ -Arbutin was used as a positive control. After the culture was completed, the cells and culture medium were recovered and centrifuged, and then 100 ⁇ l of the supernatant was dispensed into 96 well plates, and the amount of extracellular melanin released was measured by measuring the absorbance at 490 nm.
- DMEM/Modified medium 100 nM ⁇ -MSH
- the remaining supernatant was removed, the cells were washed with 1 ml of DPBS, and dried at 40°C. Then, 120 ⁇ l of 1N NaOH (10% DMSO) was added to the dried cells, and incubated in a 60°C water bath for 1 hour. Cells were lysed by boiling in a bath and then centrifuged at 15000 rpm for 1 hour. 100 ⁇ l of the supernatant was dispensed into 96 well plates, and then the absorbance was measured at 490 nm to measure the amount of melanin produced in the cells.
- the measured amount of melanin was corrected with the total protein amount obtained by BCA method (Bicinchronic acid assay) and a synthetic melanin standard curve.
- the BCA method was performed as follows. After measuring the amount of melanin produced in the cells, the remaining supernatant was diluted to 1/10 for each concentration using distilled water, and then 20 ⁇ l of the diluted sample was distributed to each concentration in a 96-well plate and Pierce BCA Protein Assay Reagent mixed reagent 200 was added. ⁇ l was added and mixed, then left at 37°C for 30 minutes, absorbance was measured at 570 nm, and protein content was confirmed using BSA (Bovine Serum Albumin) as a standard.
- BSA Bovine Serum Albumin
- Melanin production inhibition rate (%) 100 - [(melanin production amount in the sample addition group/melanin production amount in the ⁇ -MSH treated control group) ⁇ 100]
- the Pediococcus/kudzu root fermentation filtrate contained 12.44% (p ⁇ 0.05) and 5.10% (p ⁇ 0.05) of extracellular (Figure 6) and intracellular (Figure 7) melanin, respectively, at a concentration of 250 ⁇ g/ml. It was confirmed that it showed a production inhibition rate. Finally, the Pediococcus/kudzu root fermentation filtrate was confirmed to exhibit a total melanin production inhibitory activity of 9.67% (p ⁇ 0.05) at a concentration of 250 ⁇ g/ml (FIG. 8).
- CCD-986sk cells a human-derived fibroblast cell line
- IMDM medium containing 10% FBS (fetal bovine serum) and 1% P/S
- FBS fetal bovine serum
- P/S fetal bovine serum
- Dehydrogenase present in the mitochondria of living cells produces a coloring substance called formazan from Tetrazolium Salt (WST). Therefore, the effect of Pediococcus/kudzu root fermentation filtrate on cell survival rate was analyzed by measuring the number of living cells.
- WST Tetrazolium Salt
- CCD-986sk cells cultured in Example 3-1 in a 96-well cell culture plate were cultured at a concentration of 3 ⁇ 10 3 cells/well in IMDM medium supplemented with 10% FBS and 1% P/S for 24 hours. . After 24 hours, starvation was performed for more than 6 hours with FBS free IMDM supplemented with 1% P/S. Afterwards, the Pediococcus/kudzu root fermentation filtrate sample prepared in Example 1 was dissolved in distilled water to prepare a stock diluted by concentration, and the final solution was added to IMDM (FBS free, P/S 1%) medium.
- IMDM FBS free, P/S 1%) culture solution prepared by diluting it to a concentration of 15.625, 31.25, 62.5, 125 or 250 ⁇ g/ml and cultured for 48 hours. Replacement of the culture medium was performed by suctioning all of the previous culture medium and adding the same prepared medium. IMDM (FBS free, P/S 1%) medium containing 10% of EZ-CYTOX was added to the cultured cells, reacted for 2 hours, and the absorbance was measured at 450 nm using a plate multi-reader. Cell viability was evaluated by measuring and calculating using the formula below.
- Cell survival rate (%) (absorbance of sample added group/absorbance of untreated group) ⁇ 100
- the Pediococcus/kudzu root fermentation filtrate showed a cell viability of more than 90% at a concentration of 250 ⁇ g/ml or less (FIG. 9), confirming that it did not affect the cell viability.
- the CCD-986sk cells cultured in Example 3-1 were cultured in IMDM medium supplemented with 10% FBS and 1% P/S at a concentration of 1.5 ⁇ 10 4 cells/well in a 24-well cell culture plate for 24 hours. .
- the Pediococcus/kudzu root fermentation filtrate sample was treated in the same manner as in Example 4-2.
- 10ng/ml of TGF- ⁇ was used as a positive control.
- ELISA analysis was performed using the reacted cell culture.
- ELISA analysis was performed using the Procollagen Type I C-peptide (PIP) EIA Kit (TAKARA MK101) according to the protocol provided by the manufacturer.
- PIP Procollagen Type I C-peptide
- TMBZ Substrate Solution
- the total amount of protein contained in each sample was quantified by the same method as the BCA method in Example 2-3, and the expression level of type I procollagen in the untreated group and the sample treatment group corrected for the total protein amount was compared. , the expression promotion rate of type I procollagen was calculated.
- Type I procollagen expression promotion rate (%) (corrected expression level of sample treatment group/corrected expression level of untreated group) ⁇ 100
- the Pediococcus/kudzu root fermentation filtrate was confirmed to exhibit activity in promoting type I procollagen expression at a statistically significant level (p ⁇ 0.05) in the concentration range of 15.625 to 250 ⁇ g/ml, In particular, it was confirmed that the concentration of 62.5 ⁇ g/ml promoted type I procollagen expression by about 26.33% (p ⁇ 0.05) compared to the untreated control group (FIG. 10).
- Example 4 Confirmation of skin whitening and skin wrinkle improvement effect of pediococcus/kudzu root fermentation filtrate according to human application test
- test sample and control sample were prepared with a serum composed of the ingredients in Table 2, and the test sample was prepared by mixing 6% by weight of the Pediococcus/kudzu root fermentation filtrate prepared in Example 1 with the control sample.
- Sample name Ingredient name Pediococcus/Kudzu root fermentation filtrate serum (test sample) Purified water, pediococcus/kudzu root fermentation filtrate, glycerin, Butylene glycol, phenyltrimethicone, 1,2-hexanediol, Polymethylsilsesquioxane, cyclopentasiloxane, dimethiconol, Panthenol, polyglyceryl-10 laurate, dimethicone, Acrylate/C10-30 alkyl acrylate crosspolymer, propanediol, Tromethamine, fragrance, disodium EDTA control serum (control sample) Purified water, glycerin, butylene glycol, phenyltrimethicone, 1,2-hexanediol, Polymethylsilsesquioxane, cyclopentasiloxane, dimethiconol, Panthenol, polyglyceryl-10 laurate, dime
- the human application test was conducted on 24 women with an average age of 51 years, and the study subjects were trained to self-use the samples twice a day in the same manner as actual use under the supervision of the tester.
- one lesion on the left and right side of the pigmented area of the face was designated, one side was designated as a test area, and the other side was designated as a control area. Meanwhile, in order to confirm the effect of improving skin wrinkles, one side of the left and right eye wrinkle areas of the face was designated as a test area, and the other side was designated as a control area.
- a double-blind person who did not participate in the trial used the statistical program IBM SPSS Statistics ver.28.0 (IBM Corp., Armonk, NY, USA) to identify the left and right test sites of the recruited study subjects, using the test sample and control sample. Randomization was performed by site and recorded on the double-blind confirmation.
- IBM SPSS Statistics ver.28.0 IBM Corp., Armonk, NY, USA
- a double-blind person in charge placed the test and control samples in the same container and marked the container with a designated sample code so that neither the research subject nor the tester could know the contents of the sample.
- the research subjects and investigators were prevented from knowing information about the parts used for the test and control samples.
- a dermatologist confirmed the presence of adverse reactions (irritation symptoms such as pruritus and erythema) and checked compliance with sample use during the test period.
- replicas were created using the Skin-Visiometer SV700 in the wrinkles around the eyes on both sides.
- the horizontal axis is set as a virtual extension of the outer corner of the eye
- the vertical axis is set as a virtual vertical line drawn from the outer corner of the eye.
- the point where the horizontal and vertical axes meet is set as the 0 point, and the flat and smoothest surface of the temple area of the square created is as flat as possible. was selected and marked at the center point. The distance from the zero point to the horizontal and vertical axes was measured and recorded so that it could be used for the next measurement.
- the replica production area could be moved without moving to the vertical axis. It was divided. However, since the compartment area must correspond to the center of a range sufficient to manufacture a replica, if it is not suitable for manufacturing a replica due to convex points, scars, or curves, the vertical distance is measured and recorded after moving up and down.
- the melanin index of the bilateral pigmentation lesions marked on the first visit day was measured using a Mexameter, replicas were made using the Skin-Visiometer SV700 in the wrinkles around the eyes on both sides, and the presence or absence of adverse skin reactions was visually evaluated by a dermatologist. evaluated.
- the wrinkle index analysis of the replicas was conducted on the last visit date when production of all replicas was completed to reduce measurement errors. On the last visit day, the test was terminated and the samples were collected, the double-blindness of the collected samples was canceled, and the data from the test site and control site were classified separately.
- the improvement rate of the melanin index of the test area and the control area, where the skin melanin index was measured instrumentally using a Mexameter was calculated using the following equation.
- the wrinkles of the replica in the test area and the control area were quantitatively analyzed using the Skin-Visiometer SV700, and the wrinkle index improvement rate was calculated using the following equation. did.
- the change in wrinkle index ⁇ R3 was from 2 weeks after the test, and ⁇ R1, ⁇ R2, ⁇ R4, and ⁇ R5 were from 4 weeks. Corrected significance between the test site and the control site. It was confirmed that there was a statistically significant difference at the level (p ⁇ 0.017) (Table 6).
- the Pediococcus/kudzu root fermentation filtrate of the present invention is safe for the skin and has excellent skin whitening and skin wrinkle improvement effects, confirming that it can be effectively used as a cosmetic material.
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Abstract
The present invention relates to a cosmetic composition for whitening or wrinkle reduction, comprising a fermentation product of a Pueraria lobata root extract, and effectively inhibits melanin biosynthesis and has an excellent effect of producing type I procollagen, and thus can be effectively used as a material for cosmetics for skin whitening or skin wrinkle reduction.
Description
본 발명은 칡뿌리 추출물의 발효물을 포함하는 미백 또는 주름 개선용 화장료 조성물에 관한 것이다.The present invention relates to a cosmetic composition for whitening or wrinkle improvement containing a fermented product of arrowroot extract.
본 발명은 보건복지부의 과제번호 1465032918, "피부개선 효능을 가진 마이크로바이옴 소재 개발"에 따른 한국 정부의 지원을 받아 이루어졌다.This invention was made with support from the Korean government under the Ministry of Health and Welfare's project number 1465032918, "Development of microbiome materials with skin-improving efficacy."
피부 노화의 원인으로는 내적 노화와 외적 노화가 있다. 내적 노화는 나이가 들어감에 따라 신진대사를 조절하는 각종 호르몬 분비의 감소, 면역 세포의 기능 및 세포 활성 저하 등이 원인이며, 외적 노화는 자외선, 환경오염 등 외부적인 요인에 의해 자유 라디칼 및 활성 유해 산소의 증가 등이 원인이다. 피부 노화가 일어나면, 탄력이 감소하고 주름의 증가뿐 아니라 피부 혈색이 나빠지고, 피부 트러블이 자주 발생하며, 기미와 주근깨 및 검버섯 또한 증가하는 등 여러 가지 변화를 일으키게 된다.Causes of skin aging include internal aging and external aging. Internal aging is caused by a decrease in the secretion of various hormones that control metabolism as one ages and a decrease in immune cell function and cell activity, while external aging is caused by free radicals and active harmful substances caused by external factors such as ultraviolet rays and environmental pollution. This is caused by an increase in oxygen. When skin aging occurs, various changes occur, including decreased elasticity and increased wrinkles, poor skin complexion, frequent skin troubles, and increased age spots, freckles, and spots.
생체 내에서 콜라겐, 엘라스틴과 같은 세포외기질의 합성과 분해는 적절하게 조절되나, 노화가 진행되면서 그 합성이 감소하며 콜라겐을 분해하는 효소인 기질 금속단백질분해효소(matrix metalloproteinase, MMP), 엘라스틴을 분해하는 효소인 엘라스틴분해효소(elastase)의 발현이 촉진되어 피부의 탄력이 저하되고 주름이 형성된다. 또한, 자외선에 의한 활성산소의 생성, 및 피부기질의 분해에 관련된 MMP-1의 생합성에 의한 타입 I 프로콜라겐의 생합성 감소도 피부 노화와 관련이 있다. 또한, 노화의 진행이나 자외선에 등에 의해 피부 세포에서 염증 반응이 증가하며, 이러한 염증 반응들로 인해 기질 금속단백질분해효소(MMP)의 생합성이 증가하여 콜라겐 분해가 나타나게 된다.In vivo, the synthesis and decomposition of extracellular matrix such as collagen and elastin are properly regulated, but as aging progresses, the synthesis decreases and matrix metalloproteinase (MMP), an enzyme that decomposes collagen, decomposes elastin. The expression of elastase, an enzyme that decomposes skin, is promoted, which reduces skin elasticity and causes wrinkles to form. In addition, the production of active oxygen caused by ultraviolet rays and a decrease in the biosynthesis of type I procollagen due to the biosynthesis of MMP-1, which is related to the decomposition of the skin matrix, are also related to skin aging. In addition, inflammatory reactions increase in skin cells due to aging or ultraviolet rays, etc., and these inflammatory reactions increase the biosynthesis of matrix metalloproteinases (MMPs), leading to collagen degradation.
한편, 멜라닌은 포유 동물의 피부와 모발에 포함된 천연 색소 중 하나로써, 유해 자외선(UV)으로부터 피부를 보호하고 암 발병을 방지하는 등 여러 가지 유익한 기능을 나타낸다. 그러나, 멜라닌 색소생성세포(melanocyte)에서 자외선 등의 자극에 대한 방어기작으로 멜라닌 생성활동이 증가되고, 다량 생산된 멜라닌은 각질형성세포(keratinocyte)로 전이되어 피부 표피층에 축적될 수 있다. 멜라닌의 과잉 생산 및 축적은 기미, 주근깨, 피부염증 후의 피부흑화 및 노인성 색소반점 등의 피부 질환을 초래하며, 당사자에게 미용상의 불편뿐만 아니라 정신적으로 부정적인 영향을 미쳐 사회활동에 불편을 초래하기도 한다. 또한, 이러한 피부 질환은 알러지성 접촉성 피부염 또는 자극성 접촉성 피부염과 같은 염증을 동반할 수 있어 문제된다. Meanwhile, melanin is one of the natural pigments contained in the skin and hair of mammals, and has various beneficial functions, such as protecting the skin from harmful ultraviolet rays (UV) and preventing the development of cancer. However, melanin production activity increases in melanocytes as a defense mechanism against stimulation such as ultraviolet rays, and the large amount of melanin produced can be transferred to keratinocytes and accumulate in the epidermal layer of the skin. Excessive production and accumulation of melanin causes skin diseases such as blemishes, freckles, skin darkening after skin inflammation, and age-related pigment spots, and not only causes cosmetic inconvenience to the person involved, but also has a negative psychological impact, causing inconvenience in social activities. Additionally, these skin diseases are problematic because they may be accompanied by inflammation such as allergic contact dermatitis or irritant contact dermatitis.
종래 피부 주름 개선에 효과적이라고 알려진 물질로는 아데노신, 레티노인산(retinoic acid) 등이 있으나, 아데노신은 임상에서의 효능이 미미하고, 레티노인산은 가임여성에게 사용할 수 없고, 홍반 등 자극의 우려가 높다. 한편, 하이드로퀴논(hydroquinone), 알부틴(arbutin), 아스콜빈산(ascorbic acid), 코지산(kojic acid) 및 글루타티온(glutathione) 등이 종래 멜라닌 생성 억제 물질로 알려져 있으나, 하이드로퀴논은 피부 자극성이 심하여 일반적으로 사용이 제한되고 있다. 아스콜빈산은 쉽게 산화되기 때문에, 이를 배합한 화장품에서 변색 및 변취가 문제되고 있으며, 글루타티온 및 시스테인 등의 티올계 화합물은 특유의 불쾌한 냄새를 가질 뿐만 아니라 경피흡수에도 문제점이 있고, 이들의 배당체 및 유도체들도 극성이 높으므로 화장료의 배합 성분으로 사용하기 어렵다는 문제가 있다.Substances known to be effective in improving skin wrinkles include adenosine and retinoic acid, but adenosine has minimal clinical efficacy, retinoic acid cannot be used in women of childbearing age, and has a high risk of irritation such as erythema. Meanwhile, hydroquinone, arbutin, ascorbic acid, kojic acid, and glutathione are known to be substances that inhibit melanin production, but hydroquinone is highly irritating to the skin. In general, use is restricted. Because ascorbic acid is easily oxidized, discoloration and off-odor are problems in cosmetics containing it. Thiol-based compounds such as glutathione and cysteine not only have a unique unpleasant odor but also have problems with transdermal absorption, and their glycosides and derivatives Because they are highly polar, there is a problem in that they are difficult to use as ingredients in cosmetics.
이에, 본 발명자는 피부 미백 및 피부 주름 개선에 효과적인 소재를 발굴하기 위한 연구를 수행하여, 본 발명을 완성하였다.Accordingly, the present inventor conducted research to discover a material effective for skin whitening and skin wrinkle improvement and completed the present invention.
본 발명의 하나의 목적은 칡뿌리 추출물의 발효물을 포함하는 미백 또는 주름 개선용 화장료 조성물을 제공하는 것이다.One object of the present invention is to provide a cosmetic composition for whitening or wrinkle improvement containing a fermented product of arrowroot extract.
본 발명의 일 양상은 칡뿌리 추출물의 발효물을 포함하는 미백 또는 주름 개선용 화장료 조성물을 제공한다.One aspect of the present invention provides a cosmetic composition for whitening or wrinkle improvement comprising a fermented product of arrowroot extract.
본 발명의 일 구체예에 따르면, 상기 추출물의 추출용매는 탄소수 1 내지 4의 알코올 및 이들의 혼합물로 이루어진 군에서 선택되는 용매일 수 있다.According to one embodiment of the present invention, the extraction solvent of the extract may be a solvent selected from the group consisting of alcohols having 1 to 4 carbon atoms and mixtures thereof.
본 발명의 일 구체예에 따르면, 상기 추출물의 추출용매는 에탄올수용액일 수 있다.According to one embodiment of the present invention, the extraction solvent for the extract may be an aqueous ethanol solution.
본 발명의 일 구체예에 따르면, 상기 에탄올수용액의 에탄올 및 물의 부피비는 1:9 내지 9:1일 수 있다.According to one embodiment of the present invention, the volume ratio of ethanol and water in the ethanol aqueous solution may be 1:9 to 9:1.
본 발명의 일 구체예에 따르면, 상기 발효물은 페디오코쿠스 펜토사세우스(Pediococcus pentosaseus) SEQ0315(수탁번호: KACC92051P) 균주로 발효된 것일 수 있다.According to one embodiment of the present invention, the fermented product may be fermented with the strain Pediococcus pentosaseus SEQ0315 (Accession Number: KACC92051P).
본 발명의 일 구체예에 따르면, 상기 조성물은 화장품 100 중량부에 대하여 0.01 내지 30.0 중량부로 포함될 수 있다.According to one embodiment of the present invention, the composition may be included in an amount of 0.01 to 30.0 parts by weight based on 100 parts by weight of cosmetics.
본 발명의 일 구체예에 따르면, 상기 화장료 조성물은 용액, 미스트액, 현탁액, 유탁액, 페이스트, 겔, 크림, 세럼, 로션, 파우더, 비누, 계면활성제-함유 클렌징, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이로 이루어진 군에서 선택되는 어느 하나의 제형일 수 있다.According to one embodiment of the present invention, the cosmetic composition may be a solution, mist, suspension, emulsion, paste, gel, cream, serum, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion. It may be any one formulation selected from the group consisting of foundation, wax foundation, and spray.
칡뿌리 추출물의 발효물을 포함하는 미백 또는 주름 개선용 화장료 조성물에 따르면, 멜라닌 생합성을 효과적으로 억제할뿐만 아니라, 타입 I 프로콜라겐을 생성하는 효과가 우수하므로, 피부 미백 및 피부 주름 개선을 위한 화장품용 소재로 효과적으로 활용될 수 있다.According to a cosmetic composition for whitening or wrinkle improvement containing a fermented product of arrowroot extract, it not only effectively inhibits melanin biosynthesis but also has an excellent effect of producing type I procollagen, making it a cosmetic composition for skin whitening and wrinkle improvement. It can be effectively used as a material.
도 1은 본 발명의 일 구체예에 따른 페디오코쿠스/칡뿌리발효여과물의 제조공정도이다.Figure 1 is a manufacturing process diagram of Pediococcus/kudzu root fermentation filtrate according to an embodiment of the present invention.
도 2는 본 발명의 일 구체예에 따른 페디오코쿠스/칡뿌리발효여과물의 HPLC 크로마토그래피 분석 결과를 나타낸 그래프이다.Figure 2 is a graph showing the results of HPLC chromatography analysis of Pediococcus/kudzu root fermentation filtrate according to an embodiment of the present invention.
도 3은 칡뿌리 분말을 첨가하지 않고 발효한 여과물의 발효 전(A) 및 발효 후(B)의 다이드제인 함량을 HPLC 크로마토그래피 방법으로 분석하여 비교한 결과를 나타낸 그래프이다.Figure 3 is a graph showing the results of analyzing and comparing the daidzein content before (A) and after fermentation (B) of the filtrate fermented without adding arrowroot powder by HPLC chromatography.
도 4는 본 발명의 일 구체예에 따른 페디오코쿠스/칡뿌리발효여과물의 발효 전(A) 및 발효 후(B) 후의 다이드제인 함량을 HPLC 크로마토그래피 방법으로 분석하여 비교한 결과를 나타낸 그래프이다.Figure 4 is a graph showing the results of analyzing and comparing the daidzein content before (A) and after (B) fermentation of the Pediococcus/kudzu root fermentation filtrate according to an embodiment of the present invention by HPLC chromatography method. am.
도 5는 본 발명의 일 구체예에 따른 페디오코쿠스/칡뿌리발효여과물이 처리된 B16-F10 melanoma 세포의 생존율을 나타낸 그래프이다.Figure 5 is a graph showing the survival rate of B16-F10 melanoma cells treated with Pediococcus/kudzu root fermentation filtrate according to an embodiment of the present invention.
도 6은 본 발명의 일 구체예에 따른 페디오코쿠스/칡뿌리발효여과물이 처리된 B16-F10 melanoma 세포의 (A) 세포 외 멜라닌 함량(extracellular melanin contents) 및 (B) 세포 외 멜라닌 억제율(inhibition of extracellular melanin contents)을 나타낸 그래프이다.Figure 6 shows (A) extracellular melanin contents and (B) extracellular melanin inhibition rate of B16-F10 melanoma cells treated with Pediococcus/kudzu root fermentation filtrate according to an embodiment of the present invention ( This is a graph showing the inhibition of extracellular melanin contents.
도 7은 본 발명의 일 구체예에 따른 페디오코쿠스/칡뿌리발효여과물이 처리된 B16-F10 melanoma 세포의 (A) 세포 내 멜라닌 함량(intracellular melanin contents) 및 (B) 세포 내 멜라닌 억제율(inhibition of intracellular melanin contents)을 나타낸 그래프이다.Figure 7 shows (A) intracellular melanin contents and (B) intracellular melanin inhibition rate of B16-F10 melanoma cells treated with Pediococcus/kudzu root fermentation filtrate according to an embodiment of the present invention ( This is a graph showing inhibition of intracellular melanin contents.
도 8은 본 발명의 일 구체예에 따른 페디오코쿠스/칡뿌리발효여과물이 처리된 B16-F10 melanoma 세포의 (A) 총 멜라닌 함량(total melanin contents) 및 (B) 총 멜라닌 억제율(inhibition of total melanin contents)을 나타낸 그래프이다.Figure 8 shows (A) total melanin contents and (B) total melanin inhibition rate of B16-F10 melanoma cells treated with Pediococcus/kudzu root fermentation filtrate according to an embodiment of the present invention. This is a graph showing total melanin contents.
도 9는 본 발명의 일 구체예에 따른 페디오코쿠스/칡뿌리발효여과물이 처리된 CCD-986sk 세포의 생존율을 나타낸 그래프이다.Figure 9 is a graph showing the survival rate of CCD-986sk cells treated with Pediococcus/kudzu root fermentation filtrate according to an embodiment of the present invention.
도 10은 본 발명의 일 구체예에 따른 페디오코쿠스/칡뿌리발효여과물이 처리된 CCD-986sk 세포의 타입 I 프로콜라겐 발현(type I procollagen production)을 나타낸 그래프이다.Figure 10 is a graph showing type I procollagen production in CCD-986sk cells treated with Pediococcus/kudzu root fermentation filtrate according to an embodiment of the present invention.
도 11은 본 발명의 일 구체예에 따른 페디오코쿠스/칡뿌리발효여과물의 인체적용시험에 따른 (A) 멜라닌 지수 개선 정도 및 (B) 멜라닌 지수 개선율을 나타낸 그래프이다.Figure 11 is a graph showing (A) the degree of melanin index improvement and (B) the melanin index improvement rate according to the human application test of the Pediococcus/kudzu root fermentation filtrate according to an embodiment of the present invention.
도 12는 본 발명의 일 구체예에 따른 페디오코쿠스/칡뿌리발효여과물의 인체적용시험에 따른 (A) 주름 지수 R1(거칠기, Roughness) 개선 정도 및 (B) 주름 지수 R1 개선율을 나타낸 그래프이다.Figure 12 is a graph showing (A) the degree of improvement in wrinkle index R1 (roughness) and (B) the improvement rate of wrinkle index R1 according to the human application test of the Pediococcus/kudzu root fermentation filtrate according to an embodiment of the present invention. .
도 13은 본 발명의 일 구체예에 따른 페디오코쿠스/칡뿌리발효여과물의 인체적용시험에 따른 (A) 주름 지수 R2(최대 거칠기, Maximum Roughness) 개선 정도 및 (B) 주름 지수 R2 개선율을 나타낸 그래프이다.Figure 13 shows (A) the degree of improvement in the wrinkle index R2 (Maximum Roughness) and (B) the improvement rate of the wrinkle index R2 according to the human application test of the Pediococcus / arrowroot ferment filtrate according to an embodiment of the present invention. It's a graph.
도 14는 본 발명의 일 구체예에 따른 페디오코쿠스/칡뿌리발효여과물의 인체적용시험에 따른 (A) 주름 지수 R3(평균 거칠기, Average Roughness) 개선 정도 및 (B) 주름 지수 R3 개선율을 나타낸 그래프이다.Figure 14 shows (A) the degree of improvement in the wrinkle index R3 (average roughness) and (B) the improvement rate of the wrinkle index R3 according to the human application test of the Pediococcus / arrowroot ferment filtrate according to an embodiment of the present invention. It's a graph.
도 15는 본 발명의 일 구체예에 따른 페디오코쿠스/칡뿌리발효여과물의 인체적용시험에 따른 (A) 주름 지수 R4(매끄러운 깊이, Smoothness Depth) 개선 정도 및 (B) 주름 지수 R4 개선율을 나타낸 그래프이다.Figure 15 shows (A) the degree of improvement in the wrinkle index R4 (smoothness depth) and (B) the improvement rate of the wrinkle index R4 according to the human application test of the Pediococcus / arrowroot ferment filtrate according to an embodiment of the present invention. It's a graph.
도 16은 본 발명의 일 구체예에 따른 페디오코쿠스/칡뿌리발효여과물의 인체적용시험에 따른 (A) 주름 지수 R5(산술평균 거칠기, Arithmetic Average Roughness) 개선 정도 및 (B) 주름 지수 R5 개선율을 나타낸 그래프이다.Figure 16 shows (A) the degree of improvement in the wrinkle index R5 (Arithmetic Average Roughness) and (B) the improvement rate of the wrinkle index R5 according to the human application test of the Pediococcus/kudzu root fermentation filtrate according to an embodiment of the present invention. This is a graph showing .
본 발명의 일 양상은 칡뿌리 추출물의 발효물을 포함하는 미백 또는 주름 개선용 화장료 조성물을 제공한다.One aspect of the present invention provides a cosmetic composition for whitening or wrinkle improvement comprising a fermented product of arrowroot extract.
본 발명의 화장료 조성물의 유효성분으로 포함되는 발효물의 제조에 사용되는 칡(Pueraria lobata)은 쌍떡잎식물 장미목 콩과의 덩굴식물로써, 오래전부터 구황작물로 식용되었고 자양강장제 등 건강식품으로 이용되기도 하였다. 한방에서는 뿌리를 갈근(葛根)이라는 약재로 쓰는데, 발한·해열 등의 효과가 있는 것으로 알려져 있으나, 칡뿌리 추출물 발효물의 피부 미백 및 주름 개선 효과에 대해서는 알려진 바가 없다. Pueraria lobata , used in the production of fermented product included as an active ingredient in the cosmetic composition of the present invention, is a vine plant of the dicotyledonous Rosaceae legume family, and has been used as an edible crop for a long time and as a health food such as a nutritional tonic. In oriental medicine, the root is used as a medicinal herb called kudzu root, and it is known to have effects such as sweating and fever reduction, but there is nothing known about the skin whitening and wrinkle-improving effects of fermented arrowroot extract.
본 발명의 화장료 조성물은 추가로 지방 물질, 유기 용매, 용해제, 농축제 및 겔화제, 연화제, 항산화제, 현탁화제, 안정화제, 발포제(foaming agent), 방향제, 계면활성제, 물, 이온형 또는 비이온형 유화제, 충전제, 금속 이온 봉쇄제 및 킬레이트화제, 보존제, 비타민, 차단제, 습윤화제, 필수 오일, 염료, 안료, 친수성 또는 친유성 활성제, 지질 소낭 또는 화장품에 통상적으로 사용되는 임의의 다른 성분과 같은 화장품 분야에서 통상적으로 사용되는 보조제를 더 포함할 수 있다.The cosmetic composition of the present invention further contains fatty substances, organic solvents, solubilizers, thickening and gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or non-ionic. 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 other ingredients customarily used in cosmetics. It may further include auxiliaries commonly used in the same cosmetic field.
본 발명의 화장료 조성물은 그 제형의 제제화에 필요하고 적절한 각종 기제 및/또는 첨가물을 포함할 수 있으며, 그 효과를 떨어트리지 않는 범위 내에서 비이온 계면활성제, 실리콘 폴리머, 체질안료, 향료, 방부제, 살균제, 산화 안정화제, 유기 용매, 이온성 또는 비이온성 증점제, 유연화제, 산화방지제, 자유 라디칼 파괴제, 불투명화제, 안정화제, 에몰리언트(emollient), 실리콘, α-히드록시산, 소포제, 보습제, 비타민, 곤충 기피제, 향료, 보존제, 계면활성제, 소염제, 물질 P 길항제, 충전제, 중합체, 추진제, 염기성화 또는 산성화제, 또는 착색제 등 공지의 화합물을 더 포함하여 제조될 수 있다.The cosmetic composition of the present invention may contain various bases and/or additives necessary and appropriate for the formulation of the formulation, and may include non-ionic surfactants, silicone polymers, extenders, fragrances, preservatives, Disinfectants, oxidation stabilizers, organic solvents, ionic or non-ionic thickeners, softeners, antioxidants, free radical destroyers, opacifiers, stabilizers, emollients, silicones, α-hydroxy acids, anti-foaming agents, humectants, It can be prepared by further containing known compounds such as vitamins, insect repellent, fragrance, preservative, surfactant, anti-inflammatory agent, substance P antagonist, filler, polymer, propellant, alkalinizing or acidifying agent, or colorant.
본 발명의 일 구체예에 따르면, 상기 추출물의 추출용매는 탄소수 1 내지 4의 알코올 및 이들의 혼합물로 이루어진 군에서 선택될 수 있다.According to one embodiment of the present invention, the extraction solvent for the extract may be selected from the group consisting of alcohols having 1 to 4 carbon atoms and mixtures thereof.
본 발명의 일 구체예에 따르면, 상기 추출물의 추출용매는 에탄올수용액일 수 있다.According to one embodiment of the present invention, the extraction solvent for the extract may be an aqueous ethanol solution.
본 발명의 일 구체예에 따르면, 상기 에탄올수용액의 에탄올 및 물의 부피비는 1:9 내지 9:1일 수 있다.According to one embodiment of the present invention, the volume ratio of ethanol and water in the ethanol aqueous solution may be 1:9 to 9:1.
본 발명에 따른 발효물의 제조에 사용되는 칡뿌리 추출물은 하기와 같이 수득될 수 있다. 칡뿌리를 물로 세척하여 이물질을 제거한 후 그늘에서 건조하고, 적당한 양의 용매를 첨가하여 완전히 침지되도록 하며, 이때, 칡뿌리는 건조된 상태 그대로 사용되거나 분쇄하여 분말 형태로 사용될 수 있다. 칡뿌리는 통상의 추출용매를 이용하여 추출될 수 있으며, 바람직하게는, (a) 탄소수 1~4의 무수 또는 함수 저급 알코올(예: 메탄올, 에탄올, 프로판올, 부탄올, 노말-프로판올, 이소-프로판올 및 노말-부탄올 등), (b) 상기 저급 알코올과 물과의 혼합용매, (c) 아세톤, (d) 에틸 아세테이트, (e) 클로로포름, (f) 1,3-부틸렌글리콜, (g) 헥산, (h) 디에틸에테르, (i) 부틸아세테이트 (j) 클로로포름-메탄올 또는 (k) 증류수를 이용하여 추출될 수 있고, 바람직하게는 에탄올 및 물이 1:9 내지 9:1, 가장 바람직하게는 2:8의 부피비로 혼합된 에탄올수용액(20% 에탄올)으로 추출될 수 있다. 추출시 실온에서 함침하거나 가온, 멸균될 수 있다.The arrowroot extract used for producing the fermented product according to the present invention can be obtained as follows. The kudzu root is washed with water to remove foreign substances, dried in the shade, and an appropriate amount of solvent is added to ensure complete immersion. At this time, the kudzu root can be used in its dried state or pulverized and used in powder form. Kudzu root can be extracted using a common extraction solvent, preferably (a) anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (e.g. methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol) and normal-butanol, etc.), (b) mixed solvent of the lower alcohol and water, (c) acetone, (d) ethyl acetate, (e) chloroform, (f) 1,3-butylene glycol, (g) It can be extracted using hexane, (h) diethyl ether, (i) butylacetate, (j) chloroform-methanol, or (k) distilled water, preferably ethanol and water in a ratio of 1:9 to 9:1, most preferably. Alternatively, it can be extracted with an aqueous ethanol solution (20% ethanol) mixed at a volume ratio of 2:8. During extraction, it can be impregnated at room temperature or heated and sterilized.
상기 에탄올수용액의 에탄올 및 물의 부피비가 1:9 내지 9:1의 범위에 포함되지 않는 것일 경우, 추출물의 발효 과정에서 다이드제인이 충분히 추출되지 않을 수 있다.If the volume ratio of ethanol and water in the ethanol aqueous solution is not within the range of 1:9 to 9:1, daidzein may not be sufficiently extracted during the fermentation process of the extract.
본 발명의 일 구체예에 따르면, 상기 발효물은 페디오코쿠스 펜토사세우스(Pediococcus pentosaseus) SEQ0315(수탁번호: KACC92051P) 균주로 발효된 것일 수 있다.According to one embodiment of the present invention, the fermented product may be fermented with the strain Pediococcus pentosaseus SEQ0315 (Accession Number: KACC92051P).
페디오코쿠스 펜토사세우스 SEQ0315 균주는 칡 복용 1개월 후 20명의 분변으로부터 분리된 것으로, 상기 균주는 국립농업과학원에 2015년 4월 9일자로 기탁되었다(미생물 수탁번호: KACC92051P).Pediococcus pentosaceus SEQ0315 strain was isolated from the feces of 20 people 1 month after taking kudzu, and the strain was deposited with the National Institute of Agricultural Sciences on April 9, 2015 (microorganism accession number: KACC92051P).
페디오코쿠스 펜토사세우스 SEQ0315 균주에 의해 발효된 칡뿌리 추출물의 발효물에는 발효되지 않은 칡뿌리 추출물 대비 지표성분인 다이드제인(Daidzein)이 약 2배의 함량으로 포함되어 있으므로, 페디오코쿠스 펜토사세우스 SEQ0315 균주로 발효된 칡뿌리 추출물의 발효물은 발효되지 않은 칡뿌리 추출물 대비 피부 미백 및 피부 주름 개선 용도에 더욱 효과적으로 활용될 수 있다.The fermented product of the arrowroot extract fermented by Pediococcus pentosaceus SEQ0315 strain contains about twice the amount of Daidzein, an indicator component, compared to the unfermented arrowroot root extract, so Pediococcus Fermented kudzu root extract fermented with Pentosaceus SEQ0315 strain can be used more effectively for skin whitening and skin wrinkle improvement compared to unfermented kudzu root extract.
본 발명의 일 구체예에 따르면, 상기 조성물은 화장품 100 중량부에 대하여 0.01 내지 30.0 중량부로 포함될 수 있다.According to one embodiment of the present invention, the composition may be included in an amount of 0.01 to 30.0 parts by weight based on 100 parts by weight of cosmetics.
본 발명의 일 구체예에 따르면, 상기 화장료 조성물은 용액, 미스트액, 현탁액, 유탁액, 페이스트, 겔, 크림, 세럼, 로션, 파우더, 비누, 계면활성제-함유 클렌징, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이로 이루어진 군에서 선택되는 어느 하나의 제형일 수 있다.According to one embodiment of the present invention, the cosmetic composition may be a solution, mist, suspension, emulsion, paste, gel, cream, serum, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion. It may be any one formulation selected from the group consisting of foundation, wax foundation, and spray.
예를 들어, 상기 화장료 조성물은 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 세럼, 로션, 파우더, 비누, 계면활성제-함유 클렌징, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이 등으로 제형화될 수 있으나, 이에 한정되는 것은 아니다. 보다 상세하게는, 유연화장수, 영양화장수, 로션, 바디 로션, 영양 크림, 세럼, 마사지 크림, 모이스처 크림, 핸드크림, 에센스, 아이 크림, 클렌징 크림, 클렌징 폼, 클렌징 워터, 팩, 젤, 패치, 수중유(oil-in-water, O/W)형, 유중수(water-in-oil, W/O)형 등의 기초 화장료, 립스틱, 메이크업베이스 또는 파운데이션 등의 색조 화장료, 헤어토닉, 젤 또는 무스 등의 정발제(hair fixatives), 양모제 또는 염모제 등의 모발용 화장료 조성물로 제형화될 수 있다.For example, the cosmetic compositions include solutions, suspensions, emulsions, pastes, gels, creams, serums, lotions, powders, soaps, surfactant-containing cleansing products, oils, powder foundations, emulsion foundations, wax foundations and sprays. It may be formulated, but is not limited thereto. More specifically, softening lotion, nourishing lotion, lotion, body lotion, nourishing cream, serum, massage cream, moisture cream, hand cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, gel, patch, Basic cosmetics such as oil-in-water (O/W) type, water-in-oil (W/O) type, color cosmetics such as lipstick, makeup base or foundation, hair tonic, gel or It can be formulated as a hair cosmetic composition such as hair fixatives such as mousse, hair straightener, or hair dye.
본 발명의 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로써, 동물성유, 식물성유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 사용될 수 있다.When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier ingredient. You can.
본 발명의 제형이 파우더 또는 스프레이인 경우에는 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄 히드록시드, 칼슘 실리케이트 또는 폴리아미드 파우더가 사용될 수 있고, 특히 스프레이인 경우에는 추가적으로 클로로플루오로히드로카본, 프로판/부탄 또는 디메틸에테르와 같은 추진체를 포함할 수 있다.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, propane/ It may contain a propellant such as butane or dimethyl ether.
본 발명의 제형이 용액 또는 유탁액인 경우에는 담체 성분으로서 용매, 용해화제 또는 유탁화제가 사용될 수 있다. 예를 들어, 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌글리콜, 1,3-부틸글리콜 오일, 글리세롤 지방족 에스테르, 폴리에틸렌글리콜 또는 소르비탄의 지방산 에스테르가 사용될 수 있다.When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent, or emulsifying agent may be used as a carrier component. For example, 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 can be used. .
본 발명의 제형이 현탁액인 경우에는 담체 성분으로써, 물, 에탄올 또는 프로필렌 글리콜과 같은 액상의 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소 결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가 또는 트라칸트 등이 사용될 수 있다.When the formulation of the present invention is a suspension, the carrier component includes 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 miso. Crystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used.
본 발명의 제형이 계면활성제 함유 클렌징인 경우에는 담체 성분으로써, 지방족 알코올 설페이트, 지방족 알코올 에테르 설페이트, 설포숙신산 모노에스테르, 이세티오네이트, 이미다졸리늄 유도체, 메틸타우레이트, 사르코시네이트, 지방산 아미드 에테르 설페이트, 알킬아미도베타인, 지방족 알코올, 지방산 글리세리드, 지방산 디에탄올아미드, 식물성유, 라놀린 유도체 또는 에톡실화 글리세롤 지방산 에스테르 등이 사용될 수 있다.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.
본 발명의 화장료 조성물이 비누, 계면활성제 함유 클렌징 또는 계면활성제 비함유 클렌징 제형일 경우, 피부에 도포한 후 닦아내거나 떼거나 물로 씻어낼 수 있다. 상기 비누는 예를 들어, 액상비누, 가루비누, 고형비누 및 오일비누일 수 있고, 계면활성제 함유 클린징 제형은 클렌징폼, 클렌징 워터, 클렌징 수건 및 클렌징 팩일 수 있으며, 계면활성제 비함유 클렌징 제형은 클렌징크림, 클렌징 로션, 클렌징 워터 및 클렌징 겔일 수 있으나, 이에 한정되는 것은 아니다.When the cosmetic composition of the present invention is a soap, a surfactant-containing cleansing formulation, or a surfactant-free cleansing formulation, it can be applied to the skin and then wiped off, removed, or washed with water. The soap may be, for example, liquid soap, powdered soap, solid soap, and oil soap. Cleansing formulations containing surfactants may be cleansing foam, cleansing water, cleansing towels, and cleansing packs. Cleansing formulations without surfactants may be cleansing formulations. It may be cream, cleansing lotion, cleansing water, and cleansing gel, but is not limited thereto.
이하 본 발명을 하나 이상의 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through one or more examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.
실시예 1. 페디오코쿠스/칡뿌리발효여과물의 제조 및 지표성분 확인Example 1. Preparation of Pediococcus/kudzu root fermentation filtrate and confirmation of indicator components
1-1. 페디오코쿠스/칡뿌리발효여과물의 제조방법1-1. Method for producing Pediococcus/kudzu root fermentation filtrate
분쇄된 칡뿌리를 20% 에탄올수용액(v/v)과 1:20의 중량비로 혼합하고, 25℃에서 24시간 동안 추출한 다음, 감압 여과하고 회전진공농축기를 사용하여 용매를 제거하여 칡뿌리 분말을 제조하였다. 그 후, 배양배지(culture media : LB 또는 GAM) 1L에 계대 배양된 페디오코쿠스 펜토사세우스 SEQ0315(Pediococcus pentosaseus SEQ0315; 수탁번호: KACC92051P) 균주를 1×105 내지 1×106CFU/㎖ 가 되도록 접종하고 멸균(121℃, 15분) 처리된 칡뿌리 분말 100g을 투입한 다음, 37℃에서 48시간 동안 발효시켰다. 이후 멸균(121℃, 15분) 및 여과(1㎛, 0.8㎛, 0.45㎛)하여 페디오코쿠스/칡뿌리발효여과물 0.8L를 수득하였다(도 1). The crushed arrowroot root was mixed with 20% ethanol aqueous solution (v/v) at a weight ratio of 1:20, extracted at 25°C for 24 hours, filtered under reduced pressure, and the solvent was removed using a rotary vacuum concentrator to obtain arrowroot powder. Manufactured. Afterwards, the Pediococcus pentosaseus SEQ0315 ( Pediococcus pentosaseus SEQ0315; Accession number: KACC92051P) strain subcultured in 1L of culture media (LB or GAM) was grown at 1×10 5 to 1×10 6 CFU/ml. 100 g of arrowroot root powder that had been sterilized (121°C, 15 minutes) was added and fermented at 37°C for 48 hours. Afterwards, it was sterilized (121°C, 15 minutes) and filtered (1㎛, 0.8㎛, 0.45㎛) to obtain 0.8L of Pediococcus/kudzu root fermentation filtrate (Figure 1).
1-2. 페디오코쿠스/칡뿌리발효여과물의 지표성분 함량 분석1-2. Analysis of indicator component content of Pediococcus/kudzu root fermentation filtrate
실시예 1-1에서 제조된 페디오코쿠스/칡뿌리발효여과물의 지표성분을 확인하기 위하여, 발효되기 전 칡뿌리 분말과 발효 후 페디오코쿠스/칡뿌리발효여과물을 HPLC 크로마토그래피(Chromatography) 방법으로 분석하여 비교한 결과, 발효 후 페디오코쿠스/칡뿌리발효여과물의 다이드제인(Daidzein) 함량이 발효 전 칡뿌리 분말 대비 약 2배 정도 증가된 것으로 확인되어(표 1 및 도 2), 이를 지표성분으로 설정하였다.In order to confirm the indicator components of the Pediococcus/Kudzu root fermentation filtrate prepared in Example 1-1, the arrowroot powder before fermentation and the Pediococcus/Kudzu root fermentation filtrate after fermentation were subjected to HPLC chromatography. As a result of analysis and comparison, it was confirmed that the Daidzein content of the Pediococcus/kudzu root fermentation filtrate after fermentation increased by about 2 times compared to the arrowroot powder before fermentation (Table 1 and Figure 2). It was set as an indicator component.
| 지표성분Indicator component | 발효 전 Area 값Area value before fermentation | 발효 후 Area 값Area value after fermentation |
| 다이드제인(Daidzein)Daidzein | 814,634814,634 | 1,617,0631,617,063 |
1-3. 발효 과정에서 칡뿌리 분말의 첨가 유무에 따른 다이드제인 함량 분석1-3. Analysis of daidzein content according to the presence or absence of arrowroot powder during the fermentation process
배양배지(culture media : LB 또는 GAM) 10L에 계대 배양된 페디오코쿠스 펜토사세우스 SEQ0315 균주를 1×105 내지 1×106CFU/㎖ 가 되도록 접종하고, 칡뿌리 분말의 첨가 없이 37℃에서 48시간 동안 발효시킨 다음, 멸균(121℃, 15분) 및 여과(1㎛, 0.8㎛, 0.45㎛)하여 칡뿌리 분말을 첨가하지 않고 발효한 여과물을 제조하였다. 그 후, 준비한 칡뿌리 분말을 첨가하지 않고 발효한 여과물과 실시예 1-1에서 제조된 페디오코쿠스/칡뿌리발효여과물의 다이드제인 함량을 각각 HPLC 크로마토그래피 방법으로 분석하여 비교하였다.Pediococcus pentosaceus SEQ0315 strain subcultured in 10L of culture media (LB or GAM) was inoculated to 1×10 5 to 1×10 6 CFU/ml and incubated at 37°C without the addition of arrowroot powder. After fermentation for 48 hours, sterilization (121°C, 15 minutes) and filtration (1㎛, 0.8㎛, 0.45㎛) were performed to prepare a fermented filtrate without adding arrowroot powder. Afterwards, the daidzein content of the filtrate fermented without adding the prepared arrowroot powder and the Pediococcus/kudzu root fermentation filtrate prepared in Example 1-1 were analyzed and compared using HPLC chromatography, respectively.
그 결과, 칡뿌리 분말을 첨가하지 않고 발효한 여과물(도 3)에서는 다이드제인이 생성되지 않는 것으로 나타난 반면, 페디오코쿠스/칡뿌리발효여과물(도 4)에서는 발효 후 48시간까지 다이드제인 함량이 증가하는 것으로 확인되었다.As a result, it was shown that daidzein was not produced in the filtrate fermented without adding arrowroot powder (Figure 3), whereas in the Pediococcus/kudzu root fermentation filtrate (Figure 4), it was produced up to 48 hours after fermentation. It was confirmed that idzein content increased.
이와 같은 결과를 통하여, 균주 발효 시 칡뿌리 추출물을 적용할 경우 특이적으로 다이드제인이 형성됨을 확인하였다.Through these results, it was confirmed that daidzein was specifically formed when kudzu root extract was applied during strain fermentation.
실시예 2. B16-F10 melanoma 세포에서 페디오코쿠스/칡뿌리발효여과물의 멜라닌 생합성 저해 활성 확인Example 2. Confirmation of melanin biosynthesis inhibitory activity of Pediococcus/kudzu root fermentation filtrate in B16-F10 melanoma cells
2-1. B16-F10 melanoma 세포 배양2-1. B16-F10 melanoma cell culture
B16-F10 melanoma 세포를 10% Fetal bovine serum(FBS) 및 1% Penicillin/streptomycin(P/S)이 함유된 DMEM 배지를 이용하여 150mm 셀컬쳐 디쉬(Cell culture dish)를 사용하여 37℃, 5% CO2 배양기에 배양하였다. 컨플루언스(Confluence)에 도달한 세포는 Trypsin-EDTA를 사용하여 계대배양을 유지하였다.B16-F10 melanoma cells were grown in DMEM medium containing 10% Fetal bovine serum (FBS) and 1% Penicillin/streptomycin (P/S) in a 150 mm cell culture dish at 37°C and 5% CO. Cultured in a CO 2 incubator. Cells that reached confluence were subcultured using Trypsin-EDTA.
2-2. B16-F10 melanoma 세포의 생존률 분석2-2. Survival rate analysis of B16-F10 melanoma cells
살아있는 세포내의 미토콘드리아에 존재하는 탈수소효소는 Tetrazolium Salt(WST)에서 formazan이라는 발색 물질을 생성하므로, 이를 측정하여 살아있는 세포의 수를 측정함으로써, 페디오코쿠스/칡뿌리발효여과물이 세포 생존율에 미치는 영향을 분석하였다.Dehydrogenase present in the mitochondria of living cells produces a coloring substance called formazan from Tetrazolium Salt (WST), so by measuring this and measuring the number of living cells, the effect of pediococcus/kudzu root fermentation filtrate on cell survival rate can be determined. was analyzed.
구체적으로, 96 웰 세포 배양 플레이트(well cell culture plate)에 2×103cells/well 농도의 실시예 2-1에서 배양한 B16-F10 세포를 10% FBS 및 1% 페니실린/스트렙토마이신(P/S)이 첨가된 DMEM 배양액으로 24시간 배양하였다. 그 후, 실시예 1에서 제조한 페디오코쿠스/칡뿌리발효여과물이 15.625, 31.25, 62.5, 125 또는 250㎍/㎖ 농도로 포함된 DMEM 배지로 교체하여 72시간 배양하였다. 배양이 끝난 세포에 EZ-CYTOX를 10% 함유한 DMEM 배지를 넣고 2시간 반응한 뒤 플레이트 멀티-리더(Plate multi-reader)를 이용하여 450nm에서 흡광도를 측정하고 아래의 식으로 계산하여 세포 생존율을 평가하였다.Specifically, B16-F10 cells cultured in Example 2-1 at a concentration of 2×10 3 cells/well in a 96 well cell culture plate were added to 10% FBS and 1% penicillin/streptomycin (P/ S) was cultured for 24 hours in DMEM culture medium added. Afterwards, the culture was replaced with DMEM medium containing the Pediococcus/kudzu root fermentation filtrate prepared in Example 1 at a concentration of 15.625, 31.25, 62.5, 125, or 250 μg/ml, and cultured for 72 hours. DMEM medium containing 10% EZ-CYTOX was added to the cultured cells and reacted for 2 hours. Then, the absorbance was measured at 450 nm using a plate multi-reader and the cell viability was calculated using the formula below. evaluated.
세포 생존률(%) = (시료 첨가군의 흡광도/무처리군의 흡광도) × 100 Cell survival rate (%) = (absorbance of sample added group/absorbance of untreated group) × 100
그 결과, 페디오코쿠스/칡뿌리발효여과물은 250㎍/㎖ 이하의 농도에서 90% 이상의 세포 생존율을 나타내는 것으로 나타나(도 5), 세포의 생존율에 영향을 미치지 않는 것으로 확인되었다.As a result, the Pediococcus/kudzu root fermentation filtrate showed a cell viability of more than 90% at a concentration of 250 ㎍/ml or less (FIG. 5), confirming that it did not affect the cell viability.
2-3. 세포 내/외 멜라닌 생성 저해활성 분석2-3. Analysis of intra/extracellular melanin production inhibition activity
페디오코쿠스/칡뿌리발효여과물이 B16-F10 melanoma 세포에서 멜라닌 생성을 저해하는 활성을 나타내는지 분석하였다.We analyzed whether Pediococcus/kudzu root fermentation filtrate exhibits activity to inhibit melanin production in B16-F10 melanoma cells.
구체적으로, 실시예 2-1에서 배양한 B16-F10 세포를 6 웰 세포 배양 플레이트에 1×105cells/well 농도로 10% FBS 및 1% P/S가 첨가된 DMEM 배양액에서 24시간 배양하였다. 100μM α-MSH(α-Melanocyte stimulating hormone)을 DMEM/Modified 배지에 희석하여 100nM α-MSH를 함유하는 DMEM/Modified 배지를 제조하였다. 24시간 배양한 세포의 배양액을 제거한 후 DPBS 2㎖로 세포를 세척하였다. 그 후, 실시예 1에서 제조한 페디오코쿠스/칡뿌리발효여과물을 15.625, 31.25, 62.5, 125 또는 250㎍/㎖ 농도로 DMEM/Modified 배지(100 nM α-MSH)에 희석하여 시료를 준비하고, 3㎖씩 처리한 다음 37℃, 5% CO2 배양기에서 48 내지 72시간 배양하였으며, 양성대조군으로는 β-알부틴(β-Arbutin)을 사용하였다. 배양이 완료된 후 세포와 배양액을 회수하여 원심분리한 다음, 상층액을 96 웰 플레이트에 100㎕씩 분주하고, 490nm에서 흡광도를 측정하여 세포 외 멜라닌 유리량을 측정하였다.Specifically, the B16-F10 cells cultured in Example 2-1 were cultured in DMEM medium supplemented with 10% FBS and 1% P/S at a concentration of 1 × 10 5 cells/well in a 6-well cell culture plate for 24 hours. . 100 μM α-MSH (α-Melanocyte stimulating hormone) was diluted in DMEM/Modified medium to prepare DMEM/Modified medium containing 100 nM α-MSH. After removing the culture medium from cells cultured for 24 hours, the cells were washed with 2 ml of DPBS. Afterwards, samples were prepared by diluting the Pediococcus/kudzu root fermentation filtrate prepared in Example 1 in DMEM/Modified medium (100 nM α-MSH) at a concentration of 15.625, 31.25, 62.5, 125 or 250㎍/㎖. After treatment, 3 ml each was cultured in an incubator at 37°C and 5% CO 2 for 48 to 72 hours, and β-Arbutin was used as a positive control. After the culture was completed, the cells and culture medium were recovered and centrifuged, and then 100 μl of the supernatant was dispensed into 96 well plates, and the amount of extracellular melanin released was measured by measuring the absorbance at 490 nm.
그 후, 남은 상층액을 제거하고, DPBS 1㎖로 세포를 세척한 다음 40℃에서 건조시킨 다음, 건조시킨 세포에 1N NaOH(10% DMSO)를 120㎕ 첨가하고, 60℃ Water bath에서 1시간 중탕하여 세포를 용해시킨 다음, 15000rpm에서 1시간 동안 원심분리하였다. 상층액을 96 웰 플레이트에 100㎕씩 분주한 다음 490nm에서 흡광도를 측정하여 세포 내 멜라닌 생성량을 측정하였다. Afterwards, the remaining supernatant was removed, the cells were washed with 1 ml of DPBS, and dried at 40°C. Then, 120 ㎕ of 1N NaOH (10% DMSO) was added to the dried cells, and incubated in a 60°C water bath for 1 hour. Cells were lysed by boiling in a bath and then centrifuged at 15000 rpm for 1 hour. 100 μl of the supernatant was dispensed into 96 well plates, and then the absorbance was measured at 490 nm to measure the amount of melanin produced in the cells.
측정된 멜라닌 양은 BCA법(Bicinchronic acid assay)으로 구한 총 단백질 양과 합성 멜라닌 표준 곡선(Melanin standard curve)로 보정하였다. BCA법은 다음의 방법으로 수행하였다. 세포 내 멜라닌 생성량을 측정하고 남은 상층액을 증류수를 이용하여 농도별로 각 1/10로 희석하여 준비한 다음, 96 웰 플레이트에 희석한 시료를 농도별로 20㎕씩 분주하고 Pierce BCA Protein Assay Reagent 혼합시액 200㎕을 가해 혼합한 다음, 37℃에서 30분 정치하고, 570nm에서 흡광도를 측정하였으며, BSA(Bovine Serum Albumin)를 표준물질로 단백질 함량을 확인하였다.The measured amount of melanin was corrected with the total protein amount obtained by BCA method (Bicinchronic acid assay) and a synthetic melanin standard curve. The BCA method was performed as follows. After measuring the amount of melanin produced in the cells, the remaining supernatant was diluted to 1/10 for each concentration using distilled water, and then 20㎕ of the diluted sample was distributed to each concentration in a 96-well plate and Pierce BCA Protein Assay Reagent mixed reagent 200 was added. ㎕ was added and mixed, then left at 37°C for 30 minutes, absorbance was measured at 570 nm, and protein content was confirmed using BSA (Bovine Serum Albumin) as a standard.
최종적으로, 아래의 식으로 계산하여 멜라닌 생성 억제율을 평가하였다.Finally, the melanin production inhibition rate was evaluated by calculating with the formula below.
멜라닌 생성 억제율(%) = 100 - [(시료 첨가군의 멜라닌 생성량/α-MSH 처리 대조군의 멜라닌 생성량) × 100] Melanin production inhibition rate (%) = 100 - [(melanin production amount in the sample addition group/melanin production amount in the α-MSH treated control group) × 100]
그 결과, 페디오코쿠스/칡뿌리발효여과물은 250㎍/㎖ 농도에서 각각 12.44%(p<0.05) 및 5.10%(p<0.05)의 세포 외(도 6) 및 세포 내(도 7) 멜라닌 생성 억제율을 나타내는 것으로 확인되었다. 최종적으로, 페디오코쿠스/칡뿌리발효여과물은 250㎍/㎖ 농도에서 9.67%(p<0.05)의 총 멜라닌 생성 억제 활성을 나타내는 것으로 확인되었다(도 8).As a result, the Pediococcus/kudzu root fermentation filtrate contained 12.44% (p<0.05) and 5.10% (p<0.05) of extracellular (Figure 6) and intracellular (Figure 7) melanin, respectively, at a concentration of 250㎍/㎖. It was confirmed that it showed a production inhibition rate. Finally, the Pediococcus/kudzu root fermentation filtrate was confirmed to exhibit a total melanin production inhibitory activity of 9.67% (p<0.05) at a concentration of 250㎍/㎖ (FIG. 8).
실시예 3. 인간 유래 섬유아세포주 CCD-986sk 세포에서 페디오코쿠스/칡뿌리발효여과물의 콜라겐 생합성 촉진 효과 확인Example 3. Confirmation of collagen biosynthesis promoting effect of Pediococcus/kudzu root fermentation filtrate in human-derived fibroblast cell line CCD-986sk cells
3-1. 인간 유래 섬유아세포주 CCD-986sk 세포 배양3-1. Human-derived fibroblast cell line CCD-986sk cell culture
인간 유래 섬유아세포주인 CCD-986sk 세포는 10% FBS(fetal bovine serum) 및 1% P/S가 함유된 IMDM 배지에서 150mm 셀컬쳐 디쉬(cell culture dish)를 사용하여 5% CO2 배양기에서 배양하였다. 컨플루언스(Confluence)에 도달한 세포는 마일드 트립신(Mild trypsin)을 사용하여 계대배양을 유지하였다.CCD-986sk cells, a human-derived fibroblast cell line, were cultured in IMDM medium containing 10% FBS (fetal bovine serum) and 1% P/S using a 150 mm cell culture dish in a 5% CO 2 incubator. . Cells that reached confluence were subcultured using mild trypsin.
3-2. 인간 유래 섬유아세포주 CCD-986sk 세포의 생존률 분석3-2. Survival rate analysis of human-derived fibroblast cell line CCD-986sk cells
살아있는 세포내의 미토콘드리아에 존재하는 탈수소효소는 Tetrazolium Salt(WST)에서 formazan이라는 발색물질을 생성한다. 따라서, 이를 측정하여 살아있는 세포의 수를 측정함으로써, 페디오코쿠스/칡뿌리발효여과물이 세포 생존율에 미치는 영향을 분석하였다.Dehydrogenase present in the mitochondria of living cells produces a coloring substance called formazan from Tetrazolium Salt (WST). Therefore, the effect of Pediococcus/kudzu root fermentation filtrate on cell survival rate was analyzed by measuring the number of living cells.
구체적으로, 96 웰 세포 배양 플레이트에 실시예 3-1에서 배양한 CCD-986sk 세포를 3×103cells/well 농도로 10% FBS 및 1% P/S이 첨가된 IMDM 배양액에서 24시간 배양하였다. 24시간 후, 1% P/S이 첨가된 FBS free IMDM으로 6시간 이상 starvation시켰다. 그 후, 실시예 1에서 제조한 페디오코쿠스/칡뿌리발효여과물 시료를 증류수에 용해시켜 농도별로 희석한 스탁(stock)을 준비하고, IMDM(FBS free, P/S 1%) 배지에 최종농도가 15.625, 31.25, 62.5, 125 또는 250㎍/㎖ 농도가 되도록 희석하여 준비한 IMDM(FBS free, P/S 1%) 배양액으로 교체하여 48시간 배양하였다. 배양액의 교체는 이전 배양액을 모두 흡입(suction)하고, 제조한 배지를 동일하게 넣어주어 수행하였다. 배양이 끝난 세포에 EZ-CYTOX를 10% 함유한 IMDM(FBS free, P/S 1%) 배지를 넣고 2시간 반응시킨 다음, 플레이트 멀티-리더(Plate multi-reader)를 이용하여 450nm에서 흡광도를 측정하고 아래의 식으로 계산하여 세포 생존율을 평가하였다.Specifically, CCD-986sk cells cultured in Example 3-1 in a 96-well cell culture plate were cultured at a concentration of 3 × 10 3 cells/well in IMDM medium supplemented with 10% FBS and 1% P/S for 24 hours. . After 24 hours, starvation was performed for more than 6 hours with FBS free IMDM supplemented with 1% P/S. Afterwards, the Pediococcus/kudzu root fermentation filtrate sample prepared in Example 1 was dissolved in distilled water to prepare a stock diluted by concentration, and the final solution was added to IMDM (FBS free, P/S 1%) medium. It was replaced with IMDM (FBS free, P/S 1%) culture solution prepared by diluting it to a concentration of 15.625, 31.25, 62.5, 125 or 250㎍/㎖ and cultured for 48 hours. Replacement of the culture medium was performed by suctioning all of the previous culture medium and adding the same prepared medium. IMDM (FBS free, P/S 1%) medium containing 10% of EZ-CYTOX was added to the cultured cells, reacted for 2 hours, and the absorbance was measured at 450 nm using a plate multi-reader. Cell viability was evaluated by measuring and calculating using the formula below.
세포 생존률(%) = (시료 첨가군의 흡광도/무처리군의 흡광도) × 100 Cell survival rate (%) = (absorbance of sample added group/absorbance of untreated group) × 100
그 결과, 페디오코쿠스/칡뿌리발효여과물은 250㎍/㎖ 이하의 농도에서 90% 이상의 세포 생존율을 나타내는 것으로 나타나(도 9), 세포의 생존율에 영향을 미치지 않는 것으로 확인되었다.As a result, the Pediococcus/kudzu root fermentation filtrate showed a cell viability of more than 90% at a concentration of 250 ㎍/ml or less (FIG. 9), confirming that it did not affect the cell viability.
3-3. 타입 I 프로콜라겐 생성 촉진 활성 분석3-3. Type I procollagen production promotion activity analysis
페디오코쿠스/칡뿌리발효여과물이 CCD-986sk 세포에서 타입 I 프로콜라겐(Type I procollagen)의 발현을 촉진하는 활성을 나타내는지 분석하였다.We analyzed whether Pediococcus/kudzu root fermentation filtrate exhibits the activity of promoting the expression of Type I procollagen in CCD-986sk cells.
구체적으로, 실시예 3-1에서 배양한 CCD-986sk 세포를 24 웰 세포 배양 플레이트에 1.5×104cells/well 농도로 10% FBS 및 1% P/S이 첨가된 IMDM 배양액에서 24시간 배양하였다. 1% P/S이 첨가된 FBS free IMDM으로 6시간 이상 starvation 시킨후, 실시예 4-2와 동일한 방법으로 페디오코쿠스/칡뿌리발효여과물 시료를 처리하였다. 양성 대조군으로는 10ng/㎖의 TGF-β를 사용하였다. 48시간 처리 후, 반응한 세포 배양액을 이용하여 ELISA 분석을 수행하였다.Specifically, the CCD-986sk cells cultured in Example 3-1 were cultured in IMDM medium supplemented with 10% FBS and 1% P/S at a concentration of 1.5 × 10 4 cells/well in a 24-well cell culture plate for 24 hours. . After starvation for more than 6 hours with FBS free IMDM supplemented with 1% P/S, the Pediococcus/kudzu root fermentation filtrate sample was treated in the same manner as in Example 4-2. As a positive control, 10ng/ml of TGF-β was used. After 48 hours of treatment, ELISA analysis was performed using the reacted cell culture.
ELISA 분석은 Procollagen Type I C-peptide(PIP) EIA Kit(TAKARA MK101)를 이용하여 제조사에서 제공한 프로토콜에 따라 수행하였다. 키트에서 제공된 웰-스트립(well-strip)에 Antibody-POD Conjugate 용액 100㎕/well을 각각 분주하고, 스탠다드 용액과 시료를 각각 20㎕/well씩 혼합한 후, 37℃에서 3시간 반응시켰다. 상등액을 제거한 후, 세척 버퍼(wash buffer)를 이용해 웰을 4회 세척하였다. 각각의 웰에 Substrate Solution(TMBZ)을 100㎕/well씩 가하고 실온(20 내지 30℃)에서 15분간 정치한 다음, 정지시약(stop solution)을 100㎕/well씩 첨가해 반응을 정지시키고, 플레이트 멀티-리더를 이용하여 450nm에서의 흡광도를 측정하였다.ELISA analysis was performed using the Procollagen Type I C-peptide (PIP) EIA Kit (TAKARA MK101) according to the protocol provided by the manufacturer. 100㎕/well of Antibody-POD Conjugate solution was dispensed into each well-strip provided in the kit, and the standard solution and sample were mixed at 20㎕/well each, followed by reaction at 37°C for 3 hours. After removing the supernatant, the wells were washed four times using wash buffer. Add 100㎕/well of Substrate Solution (TMBZ) to each well and let stand for 15 minutes at room temperature (20 to 30℃). Then, add 100㎕/well of stop solution to stop the reaction, and plate. Absorbance was measured at 450 nm using a multi-reader.
별도로, 각 시료에 포함되어 있는 총 단백질량을 실시예 2-3의 BCA법과 동일한 방법으로 정량하고, 총 단백질량으로 보정한 무처리군과 시료 처리군의 타입 I 프로콜라겐의 발현량을 비교하여, 타입 I 프로콜라겐의 발현 촉진율을 환산하였다.Separately, the total amount of protein contained in each sample was quantified by the same method as the BCA method in Example 2-3, and the expression level of type I procollagen in the untreated group and the sample treatment group corrected for the total protein amount was compared. , the expression promotion rate of type I procollagen was calculated.
타입 I 프로콜라겐 발현 촉진율(%) = (보정된 시료처리군의 발현량/보정된 무처리군의 발현량) × 100Type I procollagen expression promotion rate (%) = (corrected expression level of sample treatment group/corrected expression level of untreated group) × 100
그 결과, 페디오코쿠스/칡뿌리발효여과물은 15.625 내지 250㎍/㎖ 농도범위에서, 통계적으로 유의한 수준(p<0.05)으로 타입 I 프로콜라겐 발현을 촉진하는 활성을 나타내는 것으로 확인되으며, 특히, 62.5㎍/㎖ 농도에서 무처리 대조군 대비 타입 I 프로콜라겐 발현을 약 26.33%(p<0.05) 촉진하는 것으로 확인되었다(도 10).As a result, the Pediococcus/kudzu root fermentation filtrate was confirmed to exhibit activity in promoting type I procollagen expression at a statistically significant level (p<0.05) in the concentration range of 15.625 to 250㎍/㎖, In particular, it was confirmed that the concentration of 62.5㎍/㎖ promoted type I procollagen expression by about 26.33% (p<0.05) compared to the untreated control group (FIG. 10).
실시예 4. 인체적용시험에 따른 페디오코쿠스/칡뿌리발효여과물의 피부 미백 및 피부주름 개선 효과 확인Example 4. Confirmation of skin whitening and skin wrinkle improvement effect of pediococcus/kudzu root fermentation filtrate according to human application test
4-1. 인체적용시험 준비4-1. Preparation for human application testing
시험 시료 및 대조 시료는 표 2의 성분으로 구성된 세럼으로 준비하였으며, 시험 시료는 대조 시료에 실시예 1에서 제조한 페디오코쿠스/칡뿌리발효여과물을 6중량% 혼합하여 준비하였다.The test sample and control sample were prepared with a serum composed of the ingredients in Table 2, and the test sample was prepared by mixing 6% by weight of the Pediococcus/kudzu root fermentation filtrate prepared in Example 1 with the control sample.
| 시료 명칭Sample name | 성분명Ingredient name |
|
페디오코쿠스/칡뿌리발효여과물 세럼 (시험 시료)Pediococcus/Kudzu root fermentation filtrate serum (test sample) |
정제수, 페디오코쿠스/칡뿌리발효여과물, 글리세린, 부틸렌글라이콜, 페닐트리메치콘, 1,2-헥산디올, 폴리메틸실세스퀴옥세인, 사이클로펜타실록산, 디메치콘올, 판테놀, 폴리글리세릴-10 라우레이트, 디메치콘, 아크릴레이트/C10-30알킬아크릴레이트크로스폴리머, 프로판디올, 트로메타민, 향료, 다이소듐이디티에이Purified water, pediococcus/kudzu root fermentation filtrate, glycerin, Butylene glycol, phenyltrimethicone, 1,2-hexanediol, Polymethylsilsesquioxane, cyclopentasiloxane, dimethiconol, Panthenol, polyglyceryl-10 laurate, dimethicone, Acrylate/C10-30 alkyl acrylate crosspolymer, propanediol, Tromethamine, fragrance, disodium EDTA |
|
대조군 세럼 (대조 시료)control serum (control sample) |
정제수, 글리세린, 부틸렌글라이콜, 페닐트리메치콘, 1,2-헥산디올, 폴리메틸실세스퀴옥세인, 사이클로펜타실록산, 디메치콘올, 판테놀, 폴리글리세릴-10 라우레이트, 디메치콘, 아크릴레이트/C10-30알킬아크릴레이트크로스폴리머, 프로판디올, 트로메타민, 향료, 다이소듐이디티에이Purified water, glycerin, butylene glycol, phenyltrimethicone, 1,2-hexanediol, Polymethylsilsesquioxane, cyclopentasiloxane, dimethiconol, Panthenol, polyglyceryl-10 laurate, dimethicone, Acrylate/C10-30 alkyl acrylate crosspolymer, propanediol, Tromethamine, fragrance, disodium EDTA |
인체적용시험은 평균 연령 51세의 여성 24명을 대상으로 수행하였으며, 연구 대상자는 시험자의 관리 감독 하에 시료를 실제 사용법과 동일하게 1일 2회 자가 사용하도록 교육받았다.The human application test was conducted on 24 women with an average age of 51 years, and the study subjects were trained to self-use the samples twice a day in the same manner as actual use under the supervision of the tester.
피부 미백 효과 확인을 위하여, 안면부의 색소침착 부위 중 좌측과 우측 각각 한 병변을 지정하여 일측은 시험 부위, 다른 일측은 대조 부위로 지정하였다. 한편, 피부 주름 개선 효과 확인을 위하여, 안면부의 좌측과 우측 눈가 주름 부위 중 일측은 시험 부위, 다른 일측은 대조 부위로 지정하였다.To confirm the skin whitening effect, one lesion on the left and right side of the pigmented area of the face was designated, one side was designated as a test area, and the other side was designated as a control area. Meanwhile, in order to confirm the effect of improving skin wrinkles, one side of the left and right eye wrinkle areas of the face was designated as a test area, and the other side was designated as a control area.
시험에 참여하지 않은 이중맹검 담당자가 통계 프로그램 IBM SPSS Statistics ver.28.0(IBM Corp., Armonk, NY, USA)을 이용하여 모집된 연구 대상자의 좌측 및 우측 시험 부위를 시험 시료 사용 부위 및 대조 시료 사용 부위로 무작위 배정하고, 이중맹검 확인서에 기록하였다. 시료의 배포는 이중 맹검 담당자가 연구 대상자와 시험자 모두 시료의 내용을 알 수 없도록 시험 시료와 대조 시료를 동일한 용기에 담아 지정된 시료 코드를 용기에 표기하여 배포하였다. 시험이 종료될 때까지 연구 대상자와 시험자는 시험 시료 및 대조 시료 사용 부위에 대한 정보를 알 수 없도록 하였다.A double-blind person who did not participate in the trial used the statistical program IBM SPSS Statistics ver.28.0 (IBM Corp., Armonk, NY, USA) to identify the left and right test sites of the recruited study subjects, using the test sample and control sample. Randomization was performed by site and recorded on the double-blind confirmation. When distributing samples, a double-blind person in charge placed the test and control samples in the same container and marked the container with a designated sample code so that neither the research subject nor the tester could know the contents of the sample. Until the end of the test, the research subjects and investigators were prevented from knowing information about the parts used for the test and control samples.
연구 대상자는 시험 시작 1주 전부터 평가 결과에 영향을 미칠 수 있는 피부 개선을 목적으로 하는 치료제, 화장품 및 의약외품 등의 사용, 의학적 처치, 마사지 등을 금하도록 하였으며, 방문 12시간 전부터 기초 제품 사용 및 화장을 금지하였다.Research subjects were prohibited from using treatments, cosmetics and quasi-drugs aimed at improving the skin, medical treatment, massage, etc. that may affect the evaluation results from 1 week before the start of the test, and were prohibited from using basic products and applying makeup 12 hours before the visit. was banned.
시험 기간 중 및 시험 종료 직후 피부과 전문의가 이상반응(소양증, 홍반 등의 자극 증상) 유무를 확인하였으며, 시험기간 동안 시료 사용의 순응도를 체크하였다.During the test period and immediately after the end of the test, a dermatologist confirmed the presence of adverse reactions (irritation symptoms such as pruritus and erythema) and checked compliance with sample use during the test period.
4-2. 첫 번째 방문(시험 전)4-2. First visit (before exam)
연구 대상자는 시험 방법과 일정 및 위험성과 가능한 이상반응 등에 대해 설명을 듣고 기초정보를 작성하고 동의서에 서명하였다. 시험자가 제공하는 기준 세안제를 이용하여 세안 후 페이퍼 타올로 가볍게 두드려 물기를 제거한 후 30분간 항온항습 조건(20 내지 24℃, 40 내지 60% 상대습도)에서 안정을 취하도록 한 후, 안면 및 양쪽 눈가 주름 부위의 사진 촬영을 실시하였다.Study subjects received explanations about the test method, schedule, risks, and possible adverse reactions, filled out basic information, and signed a consent form. After washing the face using a standard face wash provided by the tester, lightly pat with a paper towel to remove moisture, allow to rest in constant temperature and humidity conditions (20 to 24°C, 40 to 60% relative humidity) for 30 minutes, and then wash the face and both eye areas. Photographs were taken of the wrinkle area.
안면의 경우, 안면부 좌측과 우측 각각의 색소침착증 병변 한 곳을 사진상에 표기한 후 Mexameter를 이용하여 양측 색소침착증 병변의 멜라닌 지수를 측정하였다. In the case of the face, one pigmentation lesion on the left and right sides of the face was marked on the photograph, and then the melanin index of both pigmentation lesions was measured using a Mexameter.
눈가 주름의 경우, 양측 눈가 주름 부위에서 Skin-Visiometer SV700을 이용하여 레플리카(Replica)를 제작하였다. 레플리카 제작을 위하여, 가로축은 눈꼬리선의 가상 연장선으로, 세로축은 외측 눈꼬리에서 가상으로 그은 수직선으로 설정한 후, 가로축과 세로축이 만나는 지점을 0점으로 삼고, 만들어진 사각형의 관자놀이 부위 중에서 최대한 편평하고 매끄러운 면을 선정하여 가운데 지점에 표기하였다. 0점으로부터 가로축과 세로축까지의 거리를 측정 후 기록하여 다음 측정 시 이용할 수 있도록 하였으며, 눈꼬리선의 가상 연장선(가로축)이 관자놀이 부위 중 편평하고 매끄러운 면에 해당할 경우, 세로축으로 이동 없이 레플리카 제작 부위를 구획하였다. 단, 구획 부위가 레플리카를 제작하기에 충분한 범위의 정중앙에 해당하여야 하므로, 볼록한 점이나 흉터 또는 굴곡이 있어 레플리카를 제작하기에 적절하지 않을 경우, 상하로 이동후에 세로거리를 측정하여 기록하였다.In the case of wrinkles around the eyes, replicas were created using the Skin-Visiometer SV700 in the wrinkles around the eyes on both sides. To create a replica, the horizontal axis is set as a virtual extension of the outer corner of the eye, and the vertical axis is set as a virtual vertical line drawn from the outer corner of the eye. The point where the horizontal and vertical axes meet is set as the 0 point, and the flat and smoothest surface of the temple area of the square created is as flat as possible. was selected and marked at the center point. The distance from the zero point to the horizontal and vertical axes was measured and recorded so that it could be used for the next measurement. If the virtual extension line of the outer corner of the eye (horizontal axis) corresponds to the flat and smooth side of the temple area, the replica production area could be moved without moving to the vertical axis. It was divided. However, since the compartment area must correspond to the center of a range sufficient to manufacture a replica, if it is not suitable for manufacturing a replica due to convex points, scars, or curves, the vertical distance is measured and recorded after moving up and down.
사진 활영 후, 전문가 2인에 의한 육안 평가를 실시하였고(0-9 scale, 표 3), 두 평가자의 점수 판정이 다른 경우 더 높은 값을 채택하였으며, 연구 대상자에게 주의사항과 시료 사용법을 교육한 후 시료를 배포하였다.After taking the photos, a visual evaluation was conducted by two experts (0-9 scale, Table 3). If the two evaluators' scores were different, the higher value was adopted, and the study subjects were educated on precautions and sample use. Afterwards, the samples were distributed.
| 안면face | Bright & Clear(맑고 투명함) ← → Dark& Dull(어둡고 칙칙함)Bright & Clear ← → Dark& Dull | |||||||||
| 00 | 1One | 22 | 33 | 44 | 55 | 66 | 77 | 88 | 99 | |
|
눈가 주름around the eyes wrinkle |
주름 없음 ← → 주름 많음No wrinkles ← → |
|||||||||
| 00 | 1One | 22 | 33 | 44 | 55 | 66 | 77 | 88 | 99 | |
4-3. 두 번째 방문(시험 2주 후) 및 세 번째 방문(시험 4주 후)4-3. Second visit (2 weeks after exam) and third visit (4 weeks after exam)
시험자가 제공하는 기준 세안제를 이용하여 세안 후 페이퍼 타올로 가볍게 두드려 물기를 제거한 후 30분간 항온항습 조건(20 내지 24℃, 40 내지 60% 상대습도)에서 안정을 취하도록 한 후, 안면 및 양쪽 눈가 주름 사진 촬영을 실시하였다. Mexameter를 이용하여 첫 번째 방문일에 표기한 양측 색소침착증 병변의 멜라닌 지수를 측정하였고, 양측 눈가 주름 부위에서 Skin-Visiometer SV700을 이용하여 레플리카를 제작하였으며, 피부과 전문의에 의한 육안 평가 및 피부 이상반응 유무를 평가하였다.After washing the face using a standard face wash provided by the tester, lightly pat with a paper towel to remove moisture, allow to rest in constant temperature and humidity conditions (20 to 24°C, 40 to 60% relative humidity) for 30 minutes, and then wash the face and both eye areas. Wrinkle photography was performed. The melanin index of the bilateral pigmentation lesions marked on the first visit day was measured using a Mexameter, replicas were made using the Skin-Visiometer SV700 in the wrinkles around the eyes on both sides, and the presence or absence of adverse skin reactions was visually evaluated by a dermatologist. evaluated.
4-4. 네 번째 방문(마지막 방문, 시험 8주차)4-4. Fourth visit (last visit, week 8 of exam)
시험자가 제공하는 기준 세안제를 이용하여 세안 후 페이퍼 타올로 가볍게 두드려 물기를 제거한 후 30분간 항온항습 조건(20 내지 24℃, 40 내지 60% 상대습도)에서 안정을 취하도록 한 후, 안면 사진 촬영을 실시하였다. Mexameter를 이용하여 첫 번째 방문일에 표기한 양측 색소침착증 병변의 멜라닌 지수를 측정하였고, 양측 눈가 주름 부위에서 Skin-Visiometer SV700을 이용하여 레플리카를 제작하였으며, 피부과 전문의에 의한 육안 평가 및 피부 이상반응 유무를 평가하였다. 레플리카의 주름 지수 분석은 측정 오차를 줄이기 위해 모든 레플리카의 제작이 완료된 마지막 방문일에 실시하였다. 마지막 방문일에 시험을 종결하고 시료를 회수하였으며, 수거한 시료의 이중맹검을 해제하고 시험 부위 및 대조 부위의 데이터를 각각 분류하였다.After washing the face using a standard facial cleanser provided by the tester, lightly pat with a paper towel to remove moisture, allow to stabilize under constant temperature and humidity conditions (20 to 24°C, 40 to 60% relative humidity) for 30 minutes, and then take facial photographs. It was carried out. The melanin index of the bilateral pigmentation lesions marked on the first visit day was measured using a Mexameter, replicas were made using the Skin-Visiometer SV700 in the wrinkles around the eyes on both sides, and the presence or absence of adverse skin reactions was visually evaluated by a dermatologist. evaluated. The wrinkle index analysis of the replicas was conducted on the last visit date when production of all replicas was completed to reduce measurement errors. On the last visit day, the test was terminated and the samples were collected, the double-blindness of the collected samples was canceled, and the data from the test site and control site were classified separately.
4-5. Mexameter를 이용한 멜라닌 지수 평가4-5. Melanin index evaluation using Mexameter
시험 전, 시험 2주 후, 시험 4주 후 및 시험 8주 후에 Mexameter를 이용하여 피부 멜라닌 지수를 기기적으로 측정한 시험 부위와 대조 부위의 멜라닌 지수 개선율을 다음의 식으로 산출하였다.Before the test, 2 weeks after the test, 4 weeks after the test, and 8 weeks after the test, the improvement rate of the melanin index of the test area and the control area, where the skin melanin index was measured instrumentally using a Mexameter, was calculated using the following equation.
시험 전 연구 대상자의 시험 부위와 대조 부위 멜라닌 지수의 동질성을 확인한 결과, 시험 전 두 그룹 간 통계적으로 유의한 수준(p<0.05)의 차이를 확인할 수 없어 시험 전 동질성을 확인할 수 있었다. 8주간의 시험 기간 동안 시험 시료를 사용한 시험 부위의 멜라닌 지수(M-value)는 시험 2주 후부터 시험 전에 비해 통계적으로 유의한 수준(p<0.05)으로 감소하였다. 이에 비해 대조 시료를 사용한 대조 부위의 경우 사후 검정에서 차이를 확인할 수 없었다. 또한 시험 시료를 사용한 시험 부위와 대조 시료를 사용한 대조 부위를 그룹 간 비교한 결과에서도 통계적으로 유의한 수준의 차이(p<0.05)를 확인하였다(표 4). As a result of confirming the homogeneity of the melanin index of the test and control areas of the study subjects before the test, no statistically significant difference (p<0.05) was confirmed between the two groups before the test, so the homogeneity could be confirmed before the test. During the 8-week test period, the melanin index (M-value) of the test area where the test sample was used decreased to a statistically significant level (p<0.05) from 2 weeks after the test compared to before the test. In comparison, in the case of control sites using control samples, no differences could be confirmed in the post-hoc test. In addition, a statistically significant difference (p<0.05) was confirmed in the results of comparing the test area using the test sample and the control area using the control sample between groups (Table 4).
| 구분division | 평균 ± 표준편차Mean ± standard deviation | 개선율improvement rate |
p-value (그룹 내)p-value (within group) |
p-value (그룹 간)p-value (between groups) |
|
|
시험 부위test part |
시험 전before exam | 173.65 ± 36.15a 173.65 ± 36.15 a | -- | <0.0011) <0.001 1) | <0.0012) <0.001 2) |
| 시험 2주 후2 weeks after exam | 168.15 ± 34.72b 168.15 ± 34.72 b | 3.13%3.13% | |||
| 시험 4주 후4 weeks after exam | 166.13 ± 34.72c 166.13 ± 34.72 c | 4.34%4.34% | |||
| 시험 8주 후8 weeks after exam | 164.94 ± 35.01c 164.94 ± 35.01 c | 5.00%5.00% | |||
|
대조 부위contrast part |
시험 전before exam | 173.53 ± 33.82a 173.53 ± 33.82 a | -- | 0.0431) 0.043 1) | |
| 시험 2주 후2 weeks after exam | 173.09 ± 34.01a 173.09 ± 34.01 a | 0.29%0.29% | |||
| 시험 4주 후4 weeks after exam | 173.15 ± 33.79a 173.15 ± 33.79 a | 0.22%0.22% | |||
| 시험 8주 후8 weeks after exam | 173.88 ± 33.61a 173.88 ± 33.61 a | -0.22%-0.22% | |||
|
abc : 그룹 내에서 문자를 공유하지 않는 평균들은 통계적으로 유의한(p<0.05) 차이를 가짐. By LSD test 1) By Repeated measures ANOVA. 측정 시점을 요인으로 분석 2) By Repeated measures ANOVA. 시료 사용 기간에 따른 변화 그룹간 비교abc: Means that do not share a letter within the group have statistically significant (p<0.05) differences. By LSD test 1) By Repeated measures ANOVA. Analysis of measurement time as a factor 2) By repeated measures ANOVA. Comparison between groups of changes according to sample use period |
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또한, 시점 별 멜라닌 지수 변화량(△)의 시험 부위와 대조 부위 간 통계적 유의성을 검정하기 위해 군간 추가 통계 분석을 실시한 결과, 시험 2주 후, 4주 후 및 8주 후 변화량이 모두 보정된 유의 수준(p<0.017)에서 통계적으로 유의한 차이가 있음을 확인하였다(표 5 및 도 11).In addition, as a result of conducting additional statistical analysis between groups to test the statistical significance between the test and control areas in the melanin index change (△) at each time point, the changes after 2 weeks, 4 weeks, and 8 weeks after the test were all corrected for the significance level. It was confirmed that there was a statistically significant difference (p<0.017) (Table 5 and Figure 11).
| 구분division | 평균 ± 표준편차Mean ± standard deviation |
p-value (그룹 간)p-value (between groups) |
|
| 시험 부위test site |
대조 부위 | ||
| 2주차 △M-valueWeek 2 △M-value | 5.50 ± 2.185.50 ± 2.18 | 0.44 ± 1.100.44 ± 1.10 | <0.0011) <0.001 1) |
|
4주차 △M-value |
7.52 ± 3.607.52 ± 3.60 | 0.38 ± 1.740.38 ± 1.74 | <0.0012) <0.001 2) |
|
8주차 △M-value |
8.71 ± 5.438.71 ± 5.43 | -0.34 ± 1.47-0.34 ± 1.47 | <0.0012) <0.001 2) |
|
1) By Wilcoxon signed rank test. 2) By Paired t-test.1) By Wilcoxon signed rank test. 2) By Paired t-test. |
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4-6. Skin-Visiometer SV700을 이용한 주름 개선 효과 평가4-6. Evaluation of wrinkle improvement effect using Skin-Visiometer SV700
시험 전, 시험 2주 후, 시험 4주 후 및 시험 8주 후에 Skin-Visiometer SV700을 이용하여 시험 부위와 대조 부위의 레플리카(replica)의 주름을 정량 분석하고, 주름 지수 개선율을 다음의 식으로 산출하였다.Before the test, 2 weeks after the test, 4 weeks after the test, and 8 weeks after the test, the wrinkles of the replica in the test area and the control area were quantitatively analyzed using the Skin-Visiometer SV700, and the wrinkle index improvement rate was calculated using the following equation. did.
시험 전 연구 대상자의 시험 부위와 대조 부위 주름 지수(R1 내지 R5)의 동질성을 확인한 결과, 시험 전 R1, R2, R3, R4 및 R5 모두 두 그룹 간 통계적으로 유의한 수준(p<0.05)의 차이를 확인할 수 없어 시험 전 동질성을 확인할 수 있었다. 8주간의 시험 기간 동안 시험 시료를 사용한 시험 부위의 주름 지수 R1, R2, R3 및 R4는 시험 2주 후부터, 주름 지수 R5는 시험 4주 후에 시험 전과 비교하여 통계적으로 유의한 수준(p<0.05)으로 감소한 것으로 확인된 반면, 대조 시료를 사용한 대조 부위의 경우 주름 지수 R1, R2, R3, R4 및 R5는 모두 통계적으로 유의한 수준(p<0.05)의 변화를 나타내지 않은 것으로 확인되었다. 또한 시험 시료를 사용한 시험 부위와 대조 시료를 사용한 대조 부위를 비교한 결과, 모든 주름 지수 R1, R2, R3, R4 및 R5에서 시험 부위와 대조 부위 간 통계적으로 유의한 차이(p<0.05)가 확인되었다(도 12 내지 도 16).As a result of confirming the homogeneity of the wrinkle index (R1 to R5) in the test and control areas of the study subjects before the test, there was a statistically significant difference (p<0.05) between the two groups in all R1, R2, R3, R4 and R5 before the test. could not be confirmed, so homogeneity could be confirmed before testing. During the 8-week test period, the wrinkle index R1, R2, R3, and R4 of the test area using the test sample were statistically significant (p<0.05) after 2 weeks of the test, and the wrinkle index R5 after 4 weeks of the test compared to before the test. On the other hand, in the case of the control area using the control sample, the wrinkle indices R1, R2, R3, R4, and R5 were all confirmed to show no statistically significant change (p<0.05). Additionally, as a result of comparing the test area using the test sample and the control area using the control sample, a statistically significant difference (p<0.05) was confirmed between the test area and the control area in all wrinkle indices R1, R2, R3, R4, and R5. (Figures 12 to 16).
시료 사용 후 시점 별 주름 지수 변화량을 그룹 간 비교한 결과, 주름 지수 변화량 △R3는 시험 2주 후부터, △R1, △R2, △R4 및 △R5는 4주 후부터 시험 부위와 대조 부위 간 보정된 유의수준(p<0.017)에서 통계적으로 유의한 차이가 나타난 것으로 확인되었다(표 6).As a result of comparing the change in wrinkle index at each time point after using the sample between groups, the change in wrinkle index △R3 was from 2 weeks after the test, and △R1, △R2, △R4, and △R5 were from 4 weeks. Corrected significance between the test site and the control site. It was confirmed that there was a statistically significant difference at the level (p<0.017) (Table 6).
| 구분division | 평균 ± 표준편차Mean ± standard deviation |
p-value (그룹 간)p-value (between groups) |
||
| 시험 부위test site | 대조 부위control area | |||
|
△R1△ |
시험 2주 후2 weeks after exam | 0.04 ± 0.030.04 ± 0.03 | 0.01 ± 0.050.01 ± 0.05 | 0.0181) 0.018 1) |
| 시험 4주 후4 weeks after exam | 0.06 ± 0.050.06 ± 0.05 | 0.00 ± 0.040.00 ± 0.04 | <0.0012) <0.001 2) | |
| 시험 8주 후8 weeks after exam | 0.08 ± 0.050.08 ± 0.05 | 0.01 ± 0.040.01 ± 0.04 | <0.0012) <0.001 2) | |
|
△R2△ |
시험 2주 후2 weeks after exam | 0.03 ± 0.040.03 ± 0.04 | 0.02 ± 0.050.02 ± 0.05 | 0.0441) 0.044 1) |
| 시험 4주 후4 weeks after exam | 0.04 ± 0.040.04 ± 0.04 | 0.01 ± 0.030.01 ± 0.03 | 0.0071) 0.007 1) | |
| 시험 8주 후8 weeks after exam | 0.06 ± 0.040.06 ± 0.04 | 0.01 ± 0.040.01 ± 0.04 | <0.0012) <0.001 2) | |
|
△R3△ |
시험 2주 후2 weeks after exam | 0.02 ± 0.020.02 ± 0.02 | 0.01 ± 0.030.01 ± 0.03 | 0.0101) 0.010 1) |
| 시험 4주 후4 weeks after exam | 0.02 ± 0.020.02 ± 0.02 | 0.01 ± 0.020.01 ± 0.02 | 0.0051) 0.005 1) | |
| 시험 8주 후8 weeks after exam | 0.03 ± 0.020.03 ± 0.02 | 0.01 ± 0.020.01 ± 0.02 | <0.0012) <0.001 2) | |
|
△R4△ |
시험 2주 후2 weeks after exam | 0.02 ± 0.030.02 ± 0.03 | 0.00 ± 0.040.00 ± 0.04 | 0.0382) 0.038 2) |
| 시험 4주 후4 weeks after exam | 0.03 ± 0.050.03 ± 0.05 | -0.02 ± 0.04-0.02 ± 0.04 | <0.0012) <0.001 2) | |
| 시험 8주 후8 weeks after exam | 0.04 ± 0.040.04 ± 0.04 | -0.01 ± 0.04-0.01 ± 0.04 | <0.0012) <0.001 2) | |
|
△R5△ |
시험 2주 후2 weeks after exam | 0.00 ± 0.020.00 ± 0.02 | 0.00 ± 0.010.00 ± 0.01 | 0.1531) 0.153 1) |
| 시험 4주 후4 weeks after exam | 0.01 ± 0.020.01 ± 0.02 | 0.00 ± 0.010.00 ± 0.01 | 0.0071) 0.007 1) | |
| 시험 8주 후8 weeks after exam | 0.01 ± 0.020.01 ± 0.02 | 0.00 ± 0.010.00 ± 0.01 | 0.0061) 0.006 1) | |
|
1) By Wilcoxon singed rank test. 2) By Paired t-test.1) By Wilcoxon singed rank test. 2) By Paired t-test. |
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4-7. 연구 대상자에 의한 주관적 설문 평가, 순응도 평가 및 안전성 평가4-7. Subjective questionnaire assessment, compliance assessment, and safety assessment by study subjects
마지막 방문일에 피부 이상반응 유무에 대한 연구 대상자 자가 설문조사를 실시하여, 시험 시료 및 대조 시료 사용 부위의 과색소 호전 정도를 다음과 같은 척도로 주관적 평가하였다: 악화됨(worsened), 변화 없음(no change), 조금 호전되었음(improved), 매우 호전되었음(much improved).On the last visit day, a self-survey was conducted on the study subjects regarding the presence or absence of adverse skin reactions, and the degree of improvement in hyperpigmentation in the test and control sample areas was subjectively evaluated using the following scale: worsened (worsened), no change (no change). ), slightly improved, much improved.
그 결과, 시험 부위에서 과색소의 호전 정도(표 7) 및 눈가 주름 호전 정도(표 8)가 더욱 우수한 것으로 나타났다.As a result, the degree of improvement in hyperpigmentation (Table 7) and the degree of improvement in wrinkles around the eyes (Table 8) was found to be superior in the test area.
| 구분division | 악화됨worsened | 변화 없음no change | 조금 호전a little better |
매우 호전very |
| 시험 부위test site | 00 | 55 | 1010 | 99 |
|
대조 부위 |
00 | 77 | 1111 | 66 |
| 구분division | 악화됨worsened | 변화 없음no change | 조금 호전a little better |
매우 호전very |
| 시험 부위test site | 00 | 33 | 1414 | 77 |
|
대조 부위 |
00 | 44 | 1515 | 55 |
시험 종료 후, 아래의 식으로 시료 사용의 순응도를 확인한 결과, 순응도는 99.74%로 확인되었다.After completing the test, compliance with sample use was checked using the formula below, and compliance was confirmed to be 99.74%.
순응도(%)= 실제 시료 사용 횟수 / 시료를 사용하여야 할 횟수Compliance (%) = Actual number of sample uses / Number of times the sample should be used
또한, 피부과 전문의에 의하여 시험기간 중 시료 사용에 의한 부작용(홍반, 부종, 인설, 가려움, 자통, 작열감, 뻣뻣함, 따끔거림 및 기타 이상증) 발생 여부를 평가한 결과, 시험기간 동안 특별한 이상 증상은 발생하지 않은 것으로 확인되었다.In addition, as a result of evaluating the occurrence of side effects (erythema, swelling, scaling, itching, stinging, burning, stiffness, tingling, and other abnormalities) due to the use of the sample during the test period by a dermatologist, no special abnormal symptoms occurred during the test period. It was confirmed that it was not done.
이와 같은 결과를 통하여, 본 발명의 페디오코쿠스/칡뿌리발효여과물은 피부에 안전하면서도 피부 미백 및 피부 주름 개선 효과가 우수한 것으로 확인되어, 화장료 소재로 효과적으로 활용될 수 있음이 확인되었다.Through these results, it was confirmed that the Pediococcus/kudzu root fermentation filtrate of the present invention is safe for the skin and has excellent skin whitening and skin wrinkle improvement effects, confirming that it can be effectively used as a cosmetic material.
이제까지 본 발명에 대하여 그 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far, the present invention has been examined focusing on its embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.
Claims (7)
- 칡뿌리 추출물의 발효물을 포함하는 미백 또는 주름 개선용 화장료 조성물.A cosmetic composition for whitening or wrinkle improvement comprising a fermented product of arrowroot extract.
- 제 1 항에 있어서, 상기 추출물의 추출용매는 탄소수 1 내지 4의 알코올 및 이들의 혼합물로 이루어진 군에서 선택되는 용매인 것인 미백 또는 주름 개선용 화장료 조성물.The cosmetic composition for whitening or wrinkle improvement according to claim 1, wherein the extraction solvent of the extract is a solvent selected from the group consisting of alcohols having 1 to 4 carbon atoms and mixtures thereof.
- 제 1 항에 있어서, 상기 추출물의 추출용매는 에탄올수용액인 것인 미백 또는 주름 개선용 화장료 조성물.The cosmetic composition for whitening or wrinkle improvement according to claim 1, wherein the extraction solvent for the extract is an ethanol aqueous solution.
- 제 3 항에 있어서, 상기 에탄올수용액의 에탄올 및 물의 부피비는 1:9 내지 9:1인 것인 미백 또는 주름 개선용 화장료 조성물.The cosmetic composition for whitening or wrinkle improvement according to claim 3, wherein the volume ratio of ethanol and water in the ethanol aqueous solution is 1:9 to 9:1.
- 제 1 항에 있어서, 상기 발효물은 페디오코쿠스 펜토사세우스(Pediococcus pentosaseus) SEQ0315(수탁번호: KACC92051P) 균주로 발효된 것인 미백 또는 주름 개선용 화장료 조성물.The cosmetic composition for whitening or wrinkle improvement according to claim 1, wherein the fermented product is fermented with the strain Pediococcus pentosaseus SEQ0315 (Accession Number: KACC92051P).
- 제 1 항에 있어서, 상기 조성물은 화장품 100 중량부에 대하여 0.01 내지 30.0 중량부로 포함되는 것인 미백 또는 주름 개선용 화장료 조성물.The cosmetic composition for whitening or wrinkle improvement according to claim 1, wherein the composition is contained in an amount of 0.01 to 30.0 parts by weight based on 100 parts by weight of the cosmetic.
- 제 1 항에 있어서, 상기 화장료 조성물은 용액, 미스트액, 현탁액, 유탁액, 페이스트, 겔, 크림, 세럼, 로션, 파우더, 비누, 계면활성제-함유 클렌징, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이로 이루어진 군에서 선택되는 어느 하나의 제형인 것인 화장료 조성물.The cosmetic composition of claim 1, wherein the cosmetic composition is a solution, mist, suspension, emulsion, paste, gel, cream, serum, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax. A cosmetic composition that is any one formulation selected from the group consisting of foundation and spray.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2022-0174644 | 2022-12-14 | ||
| KR1020220174644A KR20240091578A (en) | 2022-12-14 | Cosmetic composition for whitening or wrinkle improvement comprising fermented kudzu root extract |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2024128370A1 true WO2024128370A1 (en) | 2024-06-20 |
Family
ID=91486049
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/KR2022/020659 WO2024128370A1 (en) | 2022-12-14 | 2022-12-17 | Cosmetic composition for whitening or wrinkle reduction, comprising fermentation product of pueraria lobata root extract |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2024128370A1 (en) |
-
2022
- 2022-12-17 WO PCT/KR2022/020659 patent/WO2024128370A1/en unknown
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