Cosmetic composition comprising fermented carrot leaf extract

Abstract

The present invention relates to a cosmetic composition comprising a carrot leaf fermentation extract fermented with lactic acid bacteria. Since the content of luteolin in the carrot leaf fermentation extract is high, the cosmetic composition with physiological and nutritional functions can be obtained. The cosmetic composition containing the carrot leaf fermentation extract of the present invention can exhibit excellent anti-inflammatory and lipid inhibition effects.

Description

Technical Field

[0001] The present invention relates to a cosmetic composition containing a carrot leaf fermentation extract fermented with lactic acid bacteria.

[0002] This patent application claims the priority of Korean Patent Application No. 10-2022-0186241, filed on December 27, 2022, and all the contents described in the specification and drawings of this application are incorporated into this application by reference. Background Art

[0003] Carrot leaves contain abundant β-carotene, which enhances immunity, and stress-resistant vitamins A, C, and E. Therefore, they have the effect of eliminating reactive oxygen species that damage human tissues and alleviating stress symptoms that stimulate various diseases. As green and yellow vegetables that are rich in vitamins and minerals and are used as nutritional supplements, they are rich in various minerals such as potassium, calcium, magnesium, iron, and phosphorus, have ingredients comparable to general nutritional supplements, and enhance immunity. In addition, the abundant dietary fiber improves constipation and prevents the intestine from absorbing cholesterol, etc., thus also showing good effects on preventing cardiovascular diseases such as hyperlipidemia or arteriosclerosis. Carrot leaves are rich in zinc and chromium, which are mineral components required to activate insulin that regulates blood sugar. Therefore, they are effective against diabetes such as postprandial hyperglycemia, and the zinc and chlorophyll components inhibit gastrointestinal inflammation and are also effective against indigestion.

[0004] Regarding such carrots, a cosmetic composition containing a carrot leaf extract is disclosed in Korean Patent Publication No. 2017-0001201, and a composition for improving blood circulation, anti-inflammation, anti-obesity, and enhancing immune function containing carrot leaf powder is disclosed in Korean Patent Publication No. 2022-0125943, and a composition for a color hair conditioner containing a carrot leaf extract as one component is disclosed in Korean Patent No. 2303467.

[0005] However, it is known that when directly applying a carrot leaf extract, the effect is significantly weakened compared to general chemicals. Therefore, a fermentation method, etc. for improving the physiological components and their activities of the carrot leaf extract is required.

[0006] Prior Art Documents

[0007] Patent Documents

[0008] Patent Document 0001: Korean Patent Publication No. 10-2017-0001201 (January 4, 2017)

[0009] Patent Document 0002: Korean Patent Publication No. 10-2022-0125943 (September 15, 2022)

[0010] Patent Document 0003: Korean Patent Gazette No. 10-2303467 (September 13, 2021) Summary of the Invention

[0011] Technical Problem

[0012] Therefore, the inventors of the present invention have studied and made efforts to obtain a fermented product of carrot leaves that ensures physiological and nutritional functions, and finally found that a carrot leaf fermented extract fermented with a specific lactic acid bacterium exhibits excellent anti-inflammatory and lipid inhibitory effects, thus completing the present invention.

[0013] Therefore, an object of the present invention is to provide a cosmetic composition containing a carrot leaf fermented extract fermented with a lactic acid bacterium.

[0014] Technical Solution

[0015] The present invention provides a cosmetic composition containing a carrot leaf fermented extract fermented with a lactic acid bacterium.

[0016] The lactic acid bacterium may be one or more strains selected from the group consisting of the genus Bacillus, the genus Streptococcus, the genus Lactococcus, the genus Enterococcus, the genus Lactobacillus, the genus Pediococcus, the genus Leuconostoc, the genus Weissella, and the genus Bifidobacterium.

[0017] The lactic acid bacterium may be Bacillus coagulans KK7 (KCTC 19023P).

[0018] The carrot leaf fermented extract contains luteolin.

[0019] The cosmetic composition may be used for anti-inflammation and lipid inhibition.

[0020] On the other hand, the present invention provides a method for preparing a carrot leaf fermented extract and a carrot leaf fermented extract prepared by the method. The method includes the step of inoculating Bacillus coagulans KK7 (KCTC19023P) onto the carrot leaves and fermenting to obtain a carrot leaf fermented extract.

[0021] The fermentation may be carried out at a temperature of 20°C to 60°C for 1 day to 5 days.

[0022] Advantages of the Invention

[0023] Because the luteolin content of the fermented extract of carrot leaves of the present invention is high, a cosmetic composition having physiological and nutritional functions can be obtained.

[0024] The cosmetic composition comprising the fermented extract of carrot leaves of the present invention can exhibit excellent anti-inflammatory and lipid inhibitory effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A graph showing the results of measuring the amount of nitric oxide (NO) generated by treating the examples and comparative examples of the present invention.

[0026] Figure 2 A graph showing the results of measuring the lipid content by treating the examples and comparative examples of the present invention.

[0027] Figure 3 An image showing intracellular lipid droplets (Oil Red O) observed according to the treatment concentration of the fermented extract of carrot leaves of the examples of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0028] Hereinafter, the present invention will be described in detail. Prior to this, the terms or words used in this specification and the claims should not be construed as being limited to the ordinary or dictionary meanings, and based on the principle that the inventor can appropriately define the concept of the terms to best explain his invention, should be construed as meanings and concepts conforming to the technical idea of the present invention. Therefore, the structures described in the examples recorded in this specification are only one most preferred example of the present invention and do not represent all the technical ideas of the present invention. Therefore, it should be understood that various equivalents and modifications capable of replacing these may exist at the time of filing this application.

[0029] The cosmetic composition of the present invention comprises a fermented extract of carrot leaves fermented with lactic acid bacteria.

[0030] The fermented extract of carrot leaves can be obtained by fermenting carrot leaf extract with lactic acid bacteria or by fermenting carrot leaf raw materials with lactic acid bacteria.

[0031] The carrot leaf extract can be extracted using water or C1-C4 alcohols as a solvent, but preferably, it can be extracted with hot water.

[0032] The amount of the solvent used can be 5 to 20 times that of carrot leaves, and preferably, a weight of 7 to 15 times can be used. When the amount of the solvent used is less than the defined range, there is a problem that the extraction of active ingredients is insufficient and the extraction rate is reduced, and when the amount used is greater than the defined range, there is a problem of low efficiency of the extraction process.

[0033] The extraction can be carried out by using solvent extraction methods well-known in the art such as maceration extraction, ultrasonic extraction, vacuum extraction, etc., and the method is not limited.

[0034] In this case, when hot water is used for the extraction, it can be extracted at a temperature of 40°C to 100°C for 0.5 hours to 24 hours. Preferably, it can be extracted at a temperature of 60°C to 90°C for 1 hour to 12 hours. When the extraction is carried out at a temperature lower than the defined temperature range, the extraction of the active ingredient is insufficient.

[0035] In this case, when hot water is used for the extraction, it can be extracted at a temperature of 40°C to 100°C for 0.5 to 24 hours. Preferably, it can be extracted at a temperature of 60°C to 90°C for 1 to 12 hours. When the extraction is carried out within the defined temperature range, the extraction of the active ingredient is insufficient.

[0036] As the lactic acid bacteria for fermenting the carrot leaf extract, one or more strains selected from the group consisting of the genera Bacillus, Streptococcus, Lactococcus, Enterococcus, Lactobacillus, Pediococcus, Leuconostoc, Weissella, and Bifidobacterium can be used. Preferably, strains of the genera Bacillus, Lactobacillus, Pediococcus, or Lactococcus can be used. More preferably, Bacillus coagulans KK7 (KCTC 19023P) can be used.

[0037] The content of luteolin in the carrot leaf fermented extract fermented by lactic acid bacteria increases, and the content of luteolin in the fermented extract can be 1000 - 5000 mg / kg.

[0038] The luteolin is contained in various plant groups such as mosses, ferns, conifers, angiosperms, etc., as well as dandelions, carrots, peppers, celery, olive oil, etc. It is known that the luteolin affects various biological reactions such as antioxidant, anti-inflammatory, anti-cancer, neuroprotection, and cardioprotection as an antioxidant and a very excellent free radical scavenger, and it is also known to inhibit enzymatic lipid peroxidation reaction, non-enzymatic lipid peroxidation reaction, and CCl4-induced lipid peroxidation reaction.

[0039] The fermented extract of carrot leaves of the present invention exhibits a higher luteolin content than ordinary carrot leaf extracts, and can contain about 1000 to 3000 mg / kg of luteolin, and thus can exhibit excellent anti-inflammatory and lipid-inhibiting effects.

[0040] The cosmetic composition of the present invention may contain components commonly used in cosmetic compositions, for example, may contain conventional adjuvants and carriers such as antioxidants, stabilizers, co-solvents, vitamins, pigments, and fragrances.

[0041] The cosmetic composition of the present invention can be prepared into any dosage form commonly prepared in the art. For example, it can be formulated into solutions, suspensions, emulsions, ointments, gels, creams, lotions, powders, soaps, surfactant-containing detergents, oils, powder foundations, emulsion foundations, wax foundations, and sprays, etc., but not limited thereto.

[0042] More specifically, it can be prepared into soft lotions, nourishing lotions, nourishing creams, massage creams, essences, eye creams, cleansing balms, facial cleansing foams, makeup removers, masks, sprays, or powder dosage forms.

[0043] When the dosage form of the present invention is an ointment, cream, or gel, animal oils, vegetable oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silica, talc, or zinc oxide, etc. can be used as carrier components.

[0044] When the dosage form of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as carrier components. In particular, when it is a spray, propellants such as chlorofluorocarbons, propane / butane, or dimethyl ether can be additionally included.

[0045] When the dosage form of the present invention is a solution or an emulsion, solvents, co-solvents, or emulsifiers are used, for example, including water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol oil, glycerol esters, polyethylene glycol, or fatty acid esters of sorbitan.

[0046] When the dosage form of the present invention is a suspension, liquid diluents such as water, ethanol, or propylene glycol; suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitan esters, and polyoxyethylene sorbitan anhydride esters; microcrystalline cellulose, partial aluminum hydroxide, bentonite, agar, or tragacanth, etc. can be used as carrier components.

[0047] When the dosage form of the present invention is a surfactant-containing cleaner, fatty alcohol sulfates, fatty alcohol ether sulfates, sulfosuccinic acid monoesters, hydroxyethyl sulfonates, imidazoline derivatives, methyl taurine, sarcosinates, fatty acid amide ether sulfates, alkyl amide betaines, fatty alcohols, fatty acid glycerol esters, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acids can be used as carrier components.

[0048] When the cosmetic composition of the present invention is a soap, a surfactant-containing cleaner, or a surfactant-free cleaner, it can be wiped off, removed, or washed with water after being applied to the skin. As specific examples, the soap is a liquid soap, a powder soap, a solid soap, and an oil soap, the surfactant-containing cleaner is a facial cleansing foam, a makeup remover, a makeup remover wipe, and a cleansing mask, and the surfactant-free cleaner is a makeup remover balm, a makeup remover milk, a makeup remover water, and a makeup remover gel, but not limited thereto.

[0049] Moreover, the houttuynia fermentation extract can be included in a topical skin pharmaceutical composition, and the pharmaceutical composition can be a cream, a gel, a patch, a spray, an ointment, a plaster, a lotion, a liniment, a paste, or a poultice, but not limited thereto.

[0050] On the other hand, the present invention provides a method for preparing a carrot leaf fermentation extract, which includes the step of inoculating Bacillus coagulans KK7 (KCTC 19023P) onto carrot leaves and fermenting to obtain the carrot leaf fermentation extract.

[0051] In this case, the inoculation can be carried out with a carrot leaf extract, and the carrot leaf extract is preferably a hot water extract, but not limited thereto, and the inoculation can be carried out with carrot leaf raw materials.

[0052] Relative to the weight of the carrot leaf extract or carrot leaf raw materials, the Bacillus coagulans KK7 (KCTC 19023P) strain can be inoculated at a concentration of 1% to 10%, and preferably, it can be 3% to 7%. When the inoculation concentration of the lactic acid bacteria is less than 1%, it may cause insufficient fermentation, and when it is greater than 10%, it may cause over-fermentation.

[0053] The carrot leaf extract contains a large amount of carotene and luteoline-7-glucoside. Carotene is a hydrocarbon in the carotenoid class, and luteoline-7-glucoside is a glycoside of glucose, which is the main sugar component of blood and the main intermediate product of substance metabolism. Through the fermentation process, bioconversion of luteoline-7-glucoside, which is a glycoside of glucose, can be carried out, so that a carrot leaf fermentation extract with a high luteolin content can be obtained.

[0054] The fermentation can be carried out for 1 to 5 days under the temperature condition of 20°C to 60°C. Preferably, it can be carried out for 3 to 4 days at a temperature of 30°C to 40°C. When the fermentation is carried out below the said range, it may lead to insufficient fermentation. When the fermentation is carried out above the said range, it may lead to over-fermentation.

[0055] Hereinafter, the present invention will be described in detail with reference to examples and experimental examples to specifically illustrate the present invention. However, the embodiments of the present invention can be changed into various different forms, and the scope of the present invention should not be construed as being limited to the following embodiments. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

[0056] Example: Preparation of carrot leaf fermentation extract by lactic acid bacteria

[0057] Suspend 1 g of kimchi sample in sterile physiological saline and then dilute it to 10 -6To prepare a suspension solution. Then, 100 μL of the suspension solution was inoculated onto BDTM Tryptic Soy Agar (15 g·L-1 Pancreatic Digest of Casein, 5 g·L-1 Papaic Digest of Soybean Meal, 5 g·L-1 Sodium Chloride, 5 g·L-1 Agar; Product No 236950) medium and spread. Single colonies were subcultured in the medium cultured under dark conditions at 30 ± 1 °C and pH 7.3 ± 0.2 for 48 hours to screen and purify the isolated microbial strains, and were finally identified as Bacillus coagulans KK7 by 16S-rRNA base sequence analysis. The Bacillus coagulans KK7 strain was deposited at the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology on July 27, 2022, with the deposit number KCTC 19023P.

[0058] The screened and isolated strain was subcultured in Tryptic Soy Agar medium at intervals of 24 hours under dark conditions at 30 ± 1 °C and pH 7.3 ± 0.2.

[0059] Next, 10 times the volume of pure water was added to the dried carrot leaves, and then hot water extraction was carried out at a temperature of 80 °C for 60 minutes to obtain an extract solution after removing the carrot leaves. The cultured Bacillus coagulans KK7 strain was inoculated into the extract solution at 5.0% of the extract weight, and fermentation was carried out at a temperature of 35 °C for 3 days, and then sterilized to obtain a fermented extract of carrot leaves. The filtered extract solution was concentrated under reduced pressure and the water was completely removed using a freeze dryer, and then an extract powder was obtained and used for experiments.

[0060] Comparative Example 1: Preparation of carrot leaf extract

[0061] 10 times the volume of pure water was added to the dried carrot leaves, and then hot water extraction was carried out at a temperature of 80 °C for 60 minutes to obtain an extract solution after removing the carrot leaves. Then, the extract solution was concentrated under reduced pressure and the water was completely removed using a freeze dryer, and then an extract powder was obtained and used for experiments.

[0062] Experimental Example 1: Analysis of luteolin content

[0063] To compare the physiological components of the extracts of the said examples and comparative examples, the index components were analyzed. The luteolin content in the extracts of the said examples and comparative examples was confirmed by HPLC analysis using a Shim-pack GIS C18 column. 10 mg of the extracts of the said examples and comparative examples were accurately weighed respectively, 1 mL of methanol and dimethyl sulfoxide (6:4, v:v) solution were added, and they were dissolved by an ultrasonic extractor for 30 minutes, then filtered with a syringe filter and the luteolin content was measured. The standard was prepared by dissolving it in methanol and dimethyl sulfoxide (6:4, v:v) solution to make the concentration range from 1 μg / mL to 200 μg / mL, and then filtered with a syringe filter before use. After measurement, the peak area was calculated, and a standard calibration curve was constructed using the regression equation. The mobile phase used distilled water containing 0.1% formic acid and acetonitrile, the flow rate was 1.0 mL / minute, the detector used a PDA detector (347 nm), the elution condition was the gradient mode, and the proportion of acetonitrile was changed within 40 minutes for measurement.

[0064] Table 1

[0065] Sample Name Luteolin Content (mg / kg) S.D Example 1933.79 4.42 Comparative Example 441.67 1.21

[0066] As shown in Table 1 above, it was shown that the luteolin content in the fermented extract of carrot leaves of the examples was very high, so it was confirmed that the content of luteolin was significantly increased by lactic acid bacteria fermentation.

[0067] Experimental Example 2: Cell culture

[0068] Experimental Example 2-1: Macrophage culture

[0069] RAW264.7 cells as macrophages were purchased from the American Type Cell Culture (ATCC), and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) medium containing 100 units / mL of penicillin-streptomycin and 10% (v / v) fetal bovine serum (FBS) in an incubator at 37°C and 5% CO2, and subcultured at intervals of 2 to 3 days.

[0070] Experimental Example 2-2: Sebaceous gland cell culture

[0071] Sebocytes as sebaceous gland cells were purchased from Celprogen, and cultured in a serum-containing complete human sebocyte medium (Human Sebocyte Complete Media with Serum), which is a dedicated medium for sebocytes, in an incubator at 37°C with 5% CO2, and subcultured at 7-day intervals.

[0072] Experimental Example 3: Cytotoxicity Evaluation

[0073] EZ-cytox assay

[0074] The EZ-cytox assay is a representative method for measuring cell viability based on the principle that a water-soluble tetrazolium salt (WST) reacts with the dehydrogenase of living cells to produce orange water-soluble formazan.

[0075] Experimental Example 3-1: Cytotoxicity Evaluation against Macrophages

[0076] To confirm cytotoxicity, RAW264.7 cells were seeded at 1.0 - 1.5×10 4 cells / well in a 96-well plate and cultured in DMEM medium supplemented with 10% FBS at 37°C and 5% CO2 for 18 hours. The medium of the cultured cells was replaced with DMEM medium supplemented with 10% FBS and treated with the sample to be evaluated. Then, EZ-cytox was added to each well and reacted at 37°C and 5% CO2 for 30 minutes, and the absorbance at 450 nm was measured using a microplate reader. The average absorbance value of each sample group was calculated, and this value was compared with the absorbance value of the control group to evaluate cell viability.

[0077] Table 2

[0078]

[0079] As shown in Table 2 above, no cytotoxicity was observed in macrophages in the examples and comparative examples.

[0080] Experimental Example 3-2: Cytotoxicity Evaluation against Sebocytes

[0081] To confirm cytotoxicity, sebocytes were seeded at 3.0×10 4Cells / well were seeded in a 24-well plate and cultured in Human Sebocyte Complete Media with Serum at 37 °C and 5% CO2 for 48 hours. The medium of the cultured sebocytes was replaced with Human Sebocyte Serum-free Media and treated with the samples to be evaluated. Then, EZ-cytox was added to each well and reacted at 37 °C and 5% CO2 for 30 minutes, and then the absorbance at 450 nm was measured using a microplate reader. The average absorbance value of each sample group was calculated and compared with the absorbance value of the control group to evaluate cell viability.

[0082] Table 3

[0083]

[0084] As shown in Table 3, no cytotoxicity was observed in the sebocytes of the examples and comparative examples.

[0085] Experimental Example 4: Inhibitory Activity of Nitric Oxide (NO) Generation

[0086] RAW264.7 cells were seeded at 1.5×10 5 cells / well in a 24-well plate and cultured at 37 °C and 5% CO2 for 18 hours. The cultured cells were treated with different concentrations of the samples to be evaluated and cultured for 24 hours. 100 μL of the supernatant of each experimental group was taken into a 96-well plate, 100 μL of griess reagent was added and reacted at room temperature for 10 minutes. Then, the absorbance at 540 nm was measured using a microplate reader. The control group was treated with 2-amino-4-methylpyridine at a concentration of 2 μM. Figure 1 The relative results of the NO generation amounts treated with the examples and comparative examples based on the NO generation amount of the stimulant (LPS) are shown.

[0087] As Figure 1 shown, it was confirmed that the examples more inhibited the generation of NO compared to the comparative examples.

[0088] Experimental Example 5: Confirmation of Sebum Secretion Regulation Effect

[0089] Experimental Example 5-1: Lipid Content Determination

[0090] Sebocytes were seeded at 3.0×10 4 cells / well in a 24-well plate and cultured at 37 °C under 5% CO2 for 48 hours. The medium was replaced with serum-free Human Sebocyte Serum-free Media, and the cells were treated with the extract to be evaluated and cultured for 5 days. Then, the culture medium was removed from each group and the cells were washed with PBS, and then analyzed using a Lipid Extraction kit (abcam) and a Lipid Assay kit (abcam) according to the method provided by the manufacturer. Figure 2 The results of lipid content determination by treatment with the examples and comparative examples are shown.

[0091] As Figure 2 shown, it was confirmed that the examples more strongly inhibited lipid content compared to the comparative examples.

[0092] Experimental Example 5-2: Observation of Lipid Droplets

[0093] Sebocytes were seeded at 3.0×10 4 cells / well in a 24-well plate and cultured at 37 °C under 5% CO2 for 48 hours. The medium was replaced with serum-free Human Sebocyte Serum-free Media, and the cells were treated with the sample to be evaluated and cultured for 5 days. Then, the culture medium was removed from each group and the cells were washed with PBS, and then analyzed using Oil Red O (Lipid stain kit) (abcam) according to the method provided by the manufacturer. Figure 3 Images showing a reduction in lipid droplets by treatment with the examples are shown.

[0094] As Figure 3 shown, it was confirmed that lipid droplets were reduced by the examples.

Claims

1. A cosmetic composition, characterized in that, It contains a fermented carrot leaf extract fermented with lactic acid bacteria.

2. The cosmetic composition according to claim 1, characterized in that, The lactic acid bacteria are one or more strains selected from the group consisting of the genus Bacillus, the genus Streptococcus, the genus Lactococcus, the genus Enterococcus, the genus Lactobacillus, the genus Pediococcus, the genus Leuconostoc, the genus Weissella, and the genus Bifidobacterium.

3. The cosmetic composition according to claim 2, characterized in that, The lactic acid bacteria are Bacillus coagulans KK7, and its deposit number is KCTC 19023P.

4. The cosmetic composition according to claim 1, characterized in that, The fermented carrot leaf extract contains luteolin.

5. The cosmetic composition according to claim 1, characterized in that, The composition has excellent anti-inflammatory and lipid inhibitory effects.

6. A preparation method of a fermented extract of carrot leaves, characterized in that, It includes the step of inoculating Bacillus coagulans KK7 with a deposit number of KCTC 19023P on carrot leaves and fermenting to obtain a fermented carrot leaf extract.

7. The preparation method of the fermented extract of carrot leaves according to claim 6, characterized in that, The fermentation is carried out at a temperature of 20 to 60 °C for 1 to 5 days.

8. A fermented extract of carrot leaves, characterized in that, It is prepared by the method for preparing a fermented carrot leaf extract according to claim 6.

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