A cosmetic compositon using lactic acid bacteria derived from giant centella asiatica

Abstract

The present invention relates to a cosmetic composition using lactic acid bacteria, comprising a giant Centella asiatica fermented extract as an active ingredient, wherein the fermented extract may be obtained by inoculating with lactic acid bacteria isolated from Centella asiatica.

Description

Technology Field

[0001] The present invention relates to a cosmetic composition comprising a fermented giant Centella asiatica material as an active ingredient, which can exhibit excellent skin improvement effects. Background Technology

[0002] In modern society, interest in grooming one's appearance is steadily increasing and has been industrialized in various fields such as cosmetics, makeup techniques, plastic surgery, hair, clothing, and accessories.

[0003] Amidst growing interest in appearance, clear skin is one of the key factors in making one look beautiful; consequently, functional cosmetics that prevent or treat skin aging, as well as cosmetics with the effect of shrinking pores, are widely consumed for the aesthetic improvement of the skin.

[0004] Recently, there has been an active trend in the development of eco-friendly functional cosmetics by introducing physiologically active substances derived from natural sources into cosmetic products.

[0005] Furthermore, for the aesthetic improvement of the skin, functional cosmetics that prevent or treat skin aging, as well as cosmetic or food ingredients that have the effect of shrinking skin pores, are being widely consumed by people of all ages and genders.

[0006] The cosmetics industry is developing numerous products using natural ingredients to reduce skin irritation caused by various chemicals.

[0007] Natural materials not only have few side effects on the skin, but their value as cosmetic ingredients is also gradually increasing as consumer demand for cosmetics using natural materials grows recently.

[0008] Centella asiatica Centella asiaticaIt is a perennial plant belonging to the family Apiaceae, and is also known as tiger grass, gotu kola, asiatic pennywort, or Indian pennywort.

[0009] Centella asiatica grows in the tropical regions of Asia, Oceania, Africa, and the Americas, and is used in folk medicine as a wound healing remedy due to its excellent antioxidant, antibacterial, and anti-inflammatory properties (Gohil, KJ, et al., 2010).

[0010] The major active ingredients of Centella asiatica are known to be asiatic acid, asiaticoside, madecassic acid, and madecassoside, among which asiatic acid has been found to have anticancer effects in various types of cancer.

[0011] Meanwhile, Giant Centella asiatica is a new variety registered with the Korea Forest Service under registration number 288, developed by improving domestic Centella asiatica. It is known to have a higher content of major active ingredients, such as madecassoside and asiaticoside, than existing Centella asiatica, and its leaves and stems are 2 to 3 times larger than existing Centella asiatica. It is registered under the name 'BT-Care'.

[0012] As such, Giant Centella asiatica contains a large amount of major active ingredients compared to ordinary Centella asiatica, and since significant changes in activity can occur through biological processing such as fermentation, the inventors have conducted various studies on natural ingredients for skin improvement. The problem to be solved

[0013] The present invention is intended to solve the aforementioned problems, and the objective of the present invention is to identify skin improvement activity using raw materials with verified skin safety, and thereby provide a cosmetic composition with excellent biosafety along with functionality. means of solving the problem

[0014] According to one aspect of the present invention, a cosmetic composition is provided comprising a giant Centella asiatica fermented extract as an active ingredient, wherein the fermented extract is obtained by inoculating lactic acid bacteria isolated from giant Centella asiatica.

[0015] In one embodiment, the lactic acid bacteria is Lactobacillus plantarum ( Lactobacillus plantarum ) and Lactobacillus pentosus ( Lactobacillus pentosus It may consist of one or more of ).

[0016] In one embodiment, the product may further include a camellia flower petal fermentation extract obtained by inoculating lactic acid bacteria isolated from the giant Centella asiatica.

[0017] In one embodiment, the cosmetic composition may include 100 parts by weight of the giant Centella asiatica fermented extract and 0.5 to 5.0 parts by weight of the camellia flower petal fermented extract.

[0018] In one embodiment, the cosmetic composition may be used for skin whitening and wrinkle improvement.

[0019] In one embodiment, the cosmetic composition may be used for skin moisturizing or antioxidant purposes. Effects of the invention

[0020] According to the present invention, the cosmetic composition has excellent skin improvement effects, such as skin whitening and anti-aging effects, and has excellent human safety.

[0021] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description of the invention or the claims. Brief explanation of the drawing

[0022] Figure 1 is the result of measuring the change in total flavonoid and polyphenol content of a fermented extract according to one embodiment of the present invention. Specific details for implementing the invention

[0023] The terms used in this specification have been selected based on currently widely used general terms whenever possible, taking into account their functions in the present invention; however, these terms may vary depending on the intent of those skilled in the art, case law, the emergence of new technologies, etc. Additionally, in specific cases, terms have been arbitrarily selected by the applicant, and in such cases, their meanings will be described in detail in the relevant description of the invention. Therefore, the terms used in this invention should be defined not merely by their names, but based on their meanings and the overall content of the invention.

[0024] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the present invention pertains. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this application.

[0025] Numerical ranges include the values ​​defined in the above ranges. All maximum numerical limits given throughout this specification include all lower numerical limits as clearly written. All minimum numerical limits given throughout this specification include all higher numerical limits as clearly written. All numerical limits given throughout this specification will include all better numerical ranges within a wider numerical range, as clearly written.

[0026] The embodiments of the present invention are described in detail below, but it is obvious that the present invention is not limited by the following embodiments.

[0027] One aspect of the present invention provides a cosmetic composition comprising a fermented giant Centella asiatica extract as an active ingredient, wherein the fermented extract is obtained by inoculating lactic acid bacteria isolated from giant Centella asiatica.

[0028] The inventors have discovered that by fermenting a giant Centella asiatica material using lactic acid bacteria isolated from giant Centella asiatica, superior skin improvement activity can be exhibited compared to conventional Centella asiatica extracts or fermented extracts, and the cosmetic composition can exhibit various skin functionalities, including a skin whitening effect, through the fermented extract of giant Centella asiatica.

[0029] The above “containing as an active ingredient” may mean containing an effective amount capable of exhibiting a skin improvement effect, for example, skin whitening activity, wrinkle improvement activity, antioxidant activity, etc.

[0030] The above Centella asiatica ( Centella asiatica It is also called Centella asiatica or tiger herb, and its titrated extract (TECA) is used for various purposes in the pharmaceutical field. The above-mentioned giant Centella asiatica was developed by improving domestic Centella asiatica and is registered with the Korea Forest Service as a new variety, registered under registration number 288, under the name 'BT-Care'.

[0031] The above lactic acid bacteria refers to bacteria that break down sugars to produce lactic acid, and typically produce more than 50% lactic acid from glucose consumed and do not cause disease.

[0032] The above lactic acid bacteria is Lactobacillus ( Lactobacillus ) genus, Streptococcus( Streptococcus ) genus, Lactococcus( Lactococcus ) genus, Enterococcus( Enterococcus ) genus, Pediococcus( Pediococcus ) genus, Leuconostoc ( Leuconostoc ) genus, Weissella( Weissella) genus and Bifidobacterium ( Bifidobacterium It may be one or more strains selected from a group consisting of strains of the genus ) but is not limited thereto.

[0033] The above-mentioned giant Centella asiatica fermented extract contains not only the active ingredients of giant Centella asiatica but also bioactive substances added through the fermentation process, which can significantly enhance the skin improvement effect, and the skin improvement effect can be maximized through the fermentation process using the above-mentioned lactic acid bacteria.

[0034] At this time, various types of microorganisms inhabit the giant Centella asiatica, and a fermented extract produced using lactic acid bacteria isolated from the giant Centella asiatica can exhibit excellent skin improvement activity.

[0035] The above-mentioned fermented Centella asiatica extract may be obtained by fermenting giant Centella asiatica.

[0036] The above-mentioned giant Centella asiatica contains a large amount of major active ingredients compared to ordinary Centella asiatica, thereby enabling it to exhibit more beneficial effects on the skin.

[0037] The skin improvement effect of the above-mentioned giant Centella asiatica fermented extract can be maximized when obtained by inoculating it with lactic acid bacteria isolated from the above-mentioned giant Centella asiatica.

[0038] Specifically, the above lactic acid bacteria is Lactobacillus plantarum ( Lactobacillus plantarum ) and Lactobacillus pentosus ( Lactobacillus pentosus It may consist of one or more of the above, and by using the above lactic acid bacteria, the production of physiologically active substances can be significantly enhanced.

[0039] At this time, the above lactic acid bacteria is Lactobacillus plantarum isolated from giant Centella asiatica ( Lactobacillus plantarum ) and Lactobacillus pentosus ( Lactobacillus pentosus It may be ), and Lactobacillus plantarum isolated from the above giant Centella asiatica ( Lactobacillus plantarum ) and Lactobacillus pentosus ( Lactobacillus pentosus ) deposited at the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology Lactobacillus pentosus AL-G1 and Lactobacillus plantarum It could be the AL-G2 strain.

[0040] In one embodiment, the product may further include a camellia flower petal fermentation extract obtained by inoculating lactic acid bacteria isolated from the giant Centella asiatica.

[0041] Camellia tree Camellia japonica L. Camellia is a broad-leaved evergreen tree belonging to the genus Camelliae of the family Theaceae, and is mainly used as a horticultural resource such as an ornamental tree. Its seeds contain high-quality fat and have traditionally been used as raw materials for edible oil and cosmetic oil, while its stems have been used as raw materials for high-quality charcoal. Camellia flowers were also used in temples to make flower pancakes, and dried petals were prescribed for symptoms such as hemoptysis or used for diuretic effects.

[0042] The above-mentioned giant Centella asiatica fermented extract can exhibit superior skin improvement activity through synergistic action when acting together with the above-mentioned camellia flower petal fermented extract.

[0043] The above cosmetic composition may include 100 parts by weight of the giant Centella asiatica fermented extract and 0.5 to 5.0 parts by weight of the camellia flower petal fermented extract.

[0044] The synergistic activity of the above complex fermented extract can be maximized when the proportion of giant Centella asiatica fermented extract is high. However, if the mixing ratio deviates significantly from the above range, the skin improvement effect derived from the active ingredients of each extract may not be properly realized; therefore, the mixing ratio can be appropriately controlled considering the working environment and the quality of the final product.

[0045] The above “extract” is obtained by isolating active ingredients contained in the extraction raw material by contacting a solvent and the extraction raw material under specific conditions, and the extract may contain various active ingredients contained in the extraction raw material.

[0046] The method of obtaining the above extract is not particularly limited and can be obtained through conventional methods in the art, such as cold maceration, hot maceration, or heating using a solvent, for example, by adding natural raw materials to a solvent and heating.

[0047] Specifically, the extract can be prepared by washing the raw materials with water, drying them, and grinding them finely, then extracting them for about 2 to 5 hours using conventional methods such as reflux circulation extraction, pressurized extraction, and ultrasonic extraction with a volume of solvent equal to 5 to 20 times the weight of each raw material, and filtering.

[0048] In addition, the extract can be obtained in a powder state by additional processes such as vacuum distillation or freeze-drying.

[0049] The above extract may also include an extract that has undergone a conventional purification process. For example, the above extract may include fractions obtained through various additional purification methods, such as separation using an ultrafiltration membrane having a certain molecular weight cut-off value, or separation by various chromatographs (designed for separation based on size, charge, hydrophobicity, or affinity).

[0050] The above cosmetic composition may be used for skin whitening and wrinkle improvement.

[0051] The aforementioned "skin whitening" refers to the effect of improving skin brightness and uniformity by suppressing the production of various biomolecules that affect skin color, such as melanin, redox hemoglobin, carotene, and melanoids, thereby alleviating skin pigmentation phenomena like blemishes, freckles, and age spots, and reducing skin yellowness and redness.

[0052] The aforementioned "wrinkle improvement" refers to the phenomenon of inhibiting or hindering the formation of wrinkles on the skin, or alleviating existing wrinkles.

[0053] The above cosmetic composition may be used for skin moisturizing or antioxidant purposes.

[0054] The aforementioned "skin moisturization" refers to all actions that maintain the homeostasis of skin tissue by appropriately regulating moisture loss (moisture evaporation) from the skin. The aforementioned skin moisturizing effect may entail additional skin improvement effects in various aspects, such as exfoliation improvement and skin irritation reduction.

[0055] The aforementioned "antioxidant" refers to the action of inhibiting oxidation. Although the human body maintains a balance between oxidation-promoting substances and oxidation-inhibiting substances, if this balance is lost due to various factors and the system shifts toward promoting oxidation, oxidative stress is induced within the body, which can lead to cell damage and pathological diseases.

[0056] Reactive oxygen species (ROS), which are the direct cause of the above-mentioned oxidative stress, are chemically unstable and highly reactive, and can easily react with various biomaterials such as DNA, proteins, lipids, and carbohydrates. They attack macromolecules in the body, causing irreversible damage to cells and tissues, or leading to mutations, cytotoxicity, and cancer, and are a direct cause of aging. Therefore, by removing or reducing these reactive oxygen species, aging can be prevented and health maintained.

[0057] The above cosmetic composition may be formulated into one or more selected from the group consisting of softening lotion, nourishing lotion, moisturizing cream, nourishing cream, massage cream, essence, ampoule, gel, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray, and powder.

[0058] The above cosmetic composition can be formulated by conventional methods. In formulating the above cosmetic composition, reference may be made to the contents disclosed in the International cosmetic ingredient dictionary, 6th ed (The cosmetic, Toiletry and Fragrance Association, Inc., Washington, 1995).

[0059] Specifically, the cosmetic composition may be prepared in general emulsion formulations and solubilization formulations. For example, it may be formulated as a lotion such as a softening lotion or a nourishing lotion; a fluid such as a facial lotion or a body lotion; a cream such as a nourishing cream, a moisturizing cream, or an eye cream; an essence; a cosmetic ointment; a spray; a gel; a pack; a sunscreen; a makeup base; a foundation such as a liquid type, a solid type, or a spray type; a powder; a makeup remover such as a cleansing cream, a cleansing lotion, or a cleansing oil; or a cleanser such as a cleansing foam, a soap, or a body wash, but is not limited thereto.

[0060] In each formulation of the above cosmetic composition, other ingredients may be appropriately incorporated in addition to the essential ingredients, depending on the type of formulation or purpose of use, within a range that does not impede the purpose according to the present invention.

[0061] The above cosmetic composition may include a conventionally acceptable carrier, and may appropriately incorporate, for example, oils, water, surfactants, humectants, lower alcohols, thickeners, chelating agents, colorants, preservatives, fragrances, etc., but is not limited thereto.

[0062] The above acceptable carriers may vary depending on the formulation. For example, when formulated as an ointment, paste, cream, or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, or extracts thereof may be used as carrier components.

[0063] When the above cosmetic composition is formulated as a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, or extracts thereof may be used as carrier components, and in the case of a spray, it may further include a propellant such as chlorofluorohydrocarbon, propane, butane, or dimethyl ether.

[0064] When the above cosmetic composition is formulated as a solution or emulsion, a solvent, a solubilizing agent, or an emulsifying agent may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan may be used.

[0065] When the above cosmetic composition is formulated as a suspension, liquid diluents such as water, ethanol, or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracanth may be used as carrier components.

[0066] When the above cosmetic composition is formulated into soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolyzates, isethionates, lanolin derivatives, aliphatic alcohols, vegetable oils, glycerol, sugars, etc. may be used as carrier components.

[0067] The above cosmetic composition may additionally contain auxiliary agents commonly used in the fields of cosmetic science or dermatology, such as fatty substances, organic solvents, solvents, thickeners, gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, metal ion blocking agents, chelating agents, preservatives, blockers, humectants, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, and any other ingredients commonly used in cosmetics, depending on the quality or function of the final product.

[0068] However, the above auxiliary agent and its mixing ratio can be appropriately selected so as not to affect the desirable properties of the cosmetic composition according to the present invention.

[0069] The present invention will be further described in detail through the following examples, but it is obvious that the present invention is not limited by the following examples.

[0070] Example 1

[0071] Giant Centella asiatica leaves were washed, completely dried at room temperature, and ground to obtain the ground material.

[0072] The optimal composition of the medium for lactic acid bacteria is 0.5 to 2 w / v% of one or more of glucose or lactose, 1 to 5 w / v% of one or more of yeast extract or soytone, 0.001 to 0.4 w / v% of malt extract, and 0.001 to 2 w / v% of each inorganic salt of dipotassium hydrogen phosphate, sodium acetate hydrate, triammonium citrate, magnesium sulfate hydrate, and manganese sulfate hydrate. The medium is added at 30 times the volume of the crushed giant Centella asiatica and sterilized at 121°C for 15 minutes to prepare a hot water extract medium.

[0073] Lactic acid bacteria were inoculated into the prepared medium and fermented at 25°C to 35°C for 24 to 96 hours using a static culture method.

[0074] After the fermentation was finished, the fermentation supernatant was recovered by centrifuging at 4,000 to 12,000 rpm for 15 to 30 minutes, and the fermentation liquid was obtained by filtering through a 0.2 μm membrane filter.

[0075] The pellet produced after centrifugation was extracted with 30 to 100 times 30 to 100% ethanol at 40 to 80°C for 30 minutes to 3 hours, and the extract and the supernatant were mixed.

[0076] The mixture used activated carbon, ion exchange resin, white clay, diatomite, and magnesium silicate for deodorization and decolorization.

[0077] A remover used for deodorization and decolorization was added in an amount of 0.1% to 10% and stirred at 25°C to 35°C for 20 to 90 minutes, then removed using a 0.2㎛ membrane filter, concentrated under reduced pressure at 30°C, and freeze-dried to prepare a fermented extract.

[0078] At this time, the above-mentioned Giant Centella asiatica used was 'BT Care', a new variety of Giant Centella asiatica improved by Ask Company using domestic Centella asiatica, and the fermentation strain was Bacillus plantarum isolated from Giant Centella asiatica ( Lactobacillus plantarum ) strain was used.

[0079] Example 2

[0080] A fermented extract was prepared by mixing crushed Centella asiatica and camellia flower leaves in a weight ratio of 99:1, using the same method as in Example 1.

[0081] Example 3

[0082] Prepared in the same manner as in Example 1, but using Lactobacillus pentosus isolated from giant Centella asiatica as the fermentation strain ( Lactobacillus pentosus ) strain was used.

[0083] Example 4

[0084] A fermented extract was prepared by mixing crushed Centella asiatica and camellia flower leaves in a weight ratio of 99:1, using the same method as in Example 3.

[0085] Comparative Example 1

[0086] Giant Centella asiatica leaves were washed, completely dried at room temperature, and ground to obtain the ground material.

[0087] A hot water extract of Centella asiatica was obtained by extracting the crushed giant Centella asiatica with 30 times the volume of distilled water as a solvent at a temperature of 121°C for 15 minutes, and then centrifuging at 4,000 to 12,000 rpm for 15 to 30 minutes to recover the supernatant.

[0088] The above extract was filtered through a 250 mesh and 0.2 μm filter, concentrated under reduced pressure at 30°C, and freeze-dried to obtain the solid content of the extract.

[0089] Comparative Example 2

[0090] Prepared in the same manner as Comparative Example 1, but using ordinary Centella asiatica obtained from the market as the extraction material ( Centella asiatica ) was used.

[0091] Comparative Example 3

[0092] Prepared in the same manner as in Example 1, but as a fermentation strain, Bacillus subtilis ( Bacillus subtilis ) was used.

[0093] Comparative Example 4

[0094] It was prepared in the same manner as in Example 1, but yeast was used as the fermentation strain.

[0095] Comparative Example 5

[0096] Prepared in the same manner as in Example 1, but using commercially available Leuconostoc mesenteroides ( Leuconostoc mesenteroides ) was used.

[0097] Comparative Example 6

[0098] Prepared in the same manner as in Example 1, but using Lactobacillus plantarum obtained from the market as the fermentation strain ( Lactobacillus plantarum ) was used.

[0099] Comparative Example 7

[0100] Prepared in the same manner as in Example 1, but using ordinary Centella asiatica instead of giant Centella asiatica as the fermentation material ( Centella asiatica ) was used.

[0101] Comparative Example 8

[0102] Prepared in the same manner as Comparative Example 7, but using Lactobacillus plantarum obtained from the market as the fermentation strain ( Lactobacillus plantarum ) was used.

[0103] Experimental Example 1: Skin Safety Test

[0104] To verify the skin safety of the samples in the examples and comparative examples, an MTT assay was performed on fibroblasts (HDF).

[0105] The samples of the examples and comparative examples were suspended in purified water at concentrations of 0.1, 0.5, 1, and 5 mg / mL, respectively, and then the cell viability was measured.

[0106] Cytotoxicity was performed by modifying Mosmann's method of measuring cell viability using the MTT{3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide} reagent.

[0107] Fibroblasts (HDFs) were placed in each 96-well plate at a ratio of 1 × 10⁶ 4 Cells were dispensed at a concentration of cells / well and cultured at 37°C, 5% CO2 for 48 hours.

[0108] After removing the culture medium, the sample was cultured in a medium treated with different concentrations for 48 hours, then the medium was removed and the sample was washed repeatedly with PBS (Phosphate buffered saline).

[0109] 50 μL of MTT dissolved in PBS at 5 mg / mL was added and incubated for 48 hours at 37°C and 5% CO2. 100 L of DMSO (Dimethyl sulfoxide) was added to each well, stirred for 10 minutes, and the absorbance was measured at 540 nm.

[0110] As a result of the measurements, no cytotoxicity was confirmed at any concentration for the samples of the examples and comparative examples.

[0111] Experimental Example 2: Measurement of Total Flavonoid and Polyphenol Content

[0112] Total polyphenols were quantified for the samples of the examples and comparative examples using the Folin-Ciocalteu method, and total flavonoids were quantified by referring to 3-48 flavonoids in the Korean Health Functional Food Codex (Notification No. 2022-69 of the Ministry of Food and Drug Safety).

[0113] 1) Method for Quantifying Polyphenols

[0114] Gallic acid was used as a standard material. The concentrations of the gallic acid were set to 10 µg / mL, 25 µg / mL, 50 µg / mL, 100 µg / mL, 250 µg / mL, 500 µg / mL, and 1,000 µg / mL, and to each of these, 0.2 mL of 2N Folin-Ciocalteu's reagent and 2 mL of distilled water were added and reacted for about 3 minutes.

[0115] Afterwards, about 2 mL of a 20 wt% aqueous sodium carbonate solution was added to each reacted solution and reacted for about 1 hour.

[0116] Subsequently, a standard curve (R2 = approximately 0.999) was constructed using optical density measured by a spectrometer at approximately 765 nm.

[0117] The above standard curve was obtained using linear regression analysis of data where the x-axis represents the concentration of gallic acid and the y-axis represents optical density.

[0118] 0.2 mL of 2N Folin-Ciocalteu's regant and 2 mL of distilled water were added to 0.1 μL of the sample from the above examples and comparative examples and reacted for about 3 minutes.

[0119] Afterwards, about 2 mL of a 20 wt% sodium carbonate solution (solvent: water) was added to each reacted solution and reacted for about 1 hour. Afterwards, the optical density was measured at about 765 nm using a spectrometer.

[0120] On the above standard curve, the concentration corresponding to the measured optical density value was determined.

[0121] The total polyphenol content contained in the sample was calculated using the corresponding concentrations above, and the polyphenol concentration (µg / mL) of the samples of the example and comparative example was calculated using the calculated polyphenol content.

[0122] 2) Method for Quantifying Flavonoids

[0123] Quercetin was used as a standard material. The concentrations of the quercetin were set to 10 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, and 1,000 μg / mL. To each of these, 0.1 mL of a 10 wt% aqueous aluminum nitrate solution, 0.1 mL of a 1 M aqueous potassium acetate solution, and 4.3 mL of an 80 wt% aqueous ethanol solution were added, and the mixture was reacted for about 40 minutes.

[0124] Subsequently, a standard curve (R2 = approximately 0.999) was constructed using optical density measured by a spectrometer at approximately 415 nm.

[0125] The above standard curve was obtained using linear regression analysis of data where the x-axis represents the concentration of the quercetin and the y-axis represents optical density.

[0126] 0.5 mL of the sample from the above examples and comparative examples was mixed with 0.1 mL of a 10 wt% aluminum nitrate aqueous solution, 0.1 mL of a 1 M potassium acetate aqueous solution, and 4.3 mL of an 80 wt% ethanol aqueous solution, and reacted for about 40 minutes, and the optical density was measured at about 415 nm using a spectrometer.

[0127] On the above standard curve, the concentration corresponding to the measured optical density value was determined.

[0128] The total flavonoid content contained in the sample was calculated using the corresponding concentrations above, and the flavonoid concentration (µg / mL) of the samples of the example and comparative example was calculated using the calculated flavonoid content.

[0129] division Total polyphenol content (µg / mL) Total flavonoid content (µg / mL) Example 1 98.6 196.7 Example 2 103.7 208.7 Example 3 97.3 195.2 Example 4 102.9 204.4 Comparative Example 1 84.1 158.5 Comparative Example 2 80.2 153.3 Comparative Example 3 88.6 166.2 Comparative Example 4 87.2 165.4 Comparative Example 5 91.4 176.2 Comparative Example 6 92.7 179.5 Comparative Example 7 86.8 174.2 Comparative Example 8 85.3 168.4.

[0130] Referring to Table 1, the total polyphenol and flavonoid content of the sample in the example was found to be high, which is attributed to increased extraction of active ingredients due to interaction with microorganisms.

[0131] In particular, in the case of Examples 1 to 4, which underwent a fermentation process using lactic acid bacteria isolated from giant Centella asiatica, the total polyphenol and total flavonoid content were evaluated to be at a higher level, and the flavonoid content increased further when giant Centella asiatica extract was used compared to general Centella asiatica extract.

[0132] The above results suggest that the production of physiologically active substances can be significantly enhanced by varying the fermentation materials and fermentation strains.

[0133] Experimental Example 3: Evaluation of Melanin Production Inhibitory Activity

[0134] Melanocyte B16F10 was cultured to evaluate the melanin production inhibitory effect of the samples in the examples and comparative examples.

[0135] B16F10 is 2 x 10 4 After dispensing cells into each well of a 48-well plate, they were cultured for 18 hours in a cell culture incubator under conditions of 5% CO2 and 37°C.

[0136] After 18 hours, the medium of the B16F10 cells was removed, and the samples of the example and comparative example were treated at the same concentration and cultured for 3 days in a cell culture incubator under conditions of 5% CO2 and 37℃.

[0137] After 3 days, the medium was removed and the cells were washed with PBS, then 1N NaOH was added to the cells and reacted at 50°C for 60 minutes to dissolve the melanin.

[0138] After 60 minutes, the amount of melanin produced was measured at a wavelength of 475 nm using a microplate reader, and the amount of protein was measured at a wavelength of 595 nm using a BCA assay.

[0139] Each experimental group was quantified as “absolute amount of melanin / absolute amount of protein,” and the experimental and control groups were relatively quantified.

[0140] α-MSH was used as a melanogenesis inducer, and arbutin was used as a positive control.

[0141] division Relative melanin value (%) Example 1 42.7 Example 2 43.4 Example 3 41.5 Example 4 42.4 Comparative Example 1 59.2 Comparative Example 2 61.6 Comparative Example 3 53.7 Comparative Example 4 54.9 Comparative Example 5 51.3 Comparative Example 6 50.5 Comparative Example 7 55.4 Comparative Example 8 57.3 positive control group 68.3 control group 100.0

[0142] Referring to Table 2, the samples of Examples 1 to 4 showed superior melanin production inhibitory activity compared to Comparative Examples 1 to 8.

[0143] In particular, in the case of Examples 1 to 4, which underwent a fermentation process using lactic acid bacteria isolated from giant Centella asiatica, the whitening activity was evaluated to be at a higher level, and in the case of Comparative Examples 1 and 2, the giant Centella asiatica extract showed relatively superior whitening activity compared to the general Centella asiatica extract.

[0144] The above results suggest that whitening activity can be significantly enhanced by varying the fermentation material and fermentation strain.

[0145] Experimental Example 4: Evaluation of tyrosinase inhibitory activity

[0146] The tyrosinase inhibitory activity of the samples in the above examples and comparative examples was evaluated. Inhibitory activity was measured by treatment at the same concentration, and L-DOPA was used as the substrate.

[0147] Mushroom tyrosinase was dissolved in 68 mM sodium phosphate buffer (pH 6.8) to a concentration of 125 U / ml, stored at -20℃, and used in the experiment.

[0148] 120 μL of 40 μM and 8 mM L-DOPA diluted samples were added to a 96-well plate.

[0149] After adding 40 μL of mushroom tyrosinase and reacting for 20 minutes, the absorbance was measured at 490 nm using an ELISA reader.

[0150] Kojic acid was used as a control. Tyrosinase inhibitory activity was measured by the ratio of absorbance of the sample-added group to the sample-free group.

[0151] division Tyrosinase inhibition rate (%) Example 1 53.3 Example 2 58.5 Example 3 52.9 Example 4 56.1 Comparative Example 1 40.9 Comparative Example 2 35.4 Comparative Example 3 44.5 Comparative Example 4 44.8 Comparative Example 5 45.1 Comparative Example 6 46.3 Comparative Example 7 42.6 Comparative Example 8 39.1 positive control group 49.2 control group 100.0

[0152] Referring to Table 3, the samples of Examples 1 to 4 showed significantly superior Tyrosinase inhibitory activity compared to Comparative Examples 1 to 8.

[0153] In particular, in the case of Examples 1 to 4, which underwent a fermentation process using lactic acid bacteria isolated from giant Centella asiatica, the tyrosinase inhibitory activity was evaluated to be at a higher level, and in the case of Comparative Examples 3 to 8, although the tyrosinase inhibitory activity was enhanced by fermentation, the rate of increase compared to the examples was evaluated to be at a lower level.

[0154] The above results suggest that whitening activity can be significantly enhanced by varying the fermentation material and fermentation strain.

[0155] Experimental Example 5: Evaluation of Free Radical Scavenging Activity

[0156] The extracts of the examples and comparative examples were suspended in purified water, and their free radical scavenging activity was evaluated.

[0157] The DPPH assay is a method of measuring the degree of decolorization caused by an inhibitor scavenging the stable radical DPPH (2,2-Diphenyl-1-picrylhydrazyl radical) by measuring the absorbance at 540 nm.

[0158] Vitamin C was used as a positive control, and the same experiment was repeated three times to ensure the accuracy of the experiment. The results (sample concentration 10 mg / mL) are shown in Table 4 below.

[0159] division Free radical scavenging ability (% of control) Example 1 74.8 Example 2 82.1 Example 3 75.7 Example 4 81.4 Comparative Example 1 53.5 Comparative Example 2 50.7 Comparative Example 3 58.4 Comparative Example 4 59.5 Comparative Example 5 62.6 Comparative Example 6 64.3 Comparative Example 7 56.2 Comparative Example 8 54.9 Control group (Vitamin C) 84.2

[0160] Referring to Table 4, the samples of Examples 1 to 4 showed superior antioxidant activity compared to Comparative Examples 1 to 8.

[0161] In particular, in the case of Examples 1 to 4, which underwent a fermentation process using lactic acid bacteria isolated from giant Centella asiatica, the antioxidant activity was evaluated to be at a higher level.

[0162] The above results suggest that not only is the antioxidant activity of Giant Centella asiatica superior to that of conventional Centella asiatica materials, but excellent antioxidant activity can also be achieved by fermenting using lactic acid bacteria isolated from Giant Centella asiatica.

[0163] Experimental Example 6: Evaluation of Elastase Inhibitory Activity

[0164] The wrinkle improvement effect of the samples in the examples and comparative examples was evaluated through the inhibitory activity of elastase.

[0165] The samples of the examples and comparative examples were suspended in purified water, and their elastase inhibitory activity was measured as follows.

[0166] 1.6 mM elastase substrate (N-succinyl-(Ala)3-p-nitroanilide; SANA) and 0.2 mM Tris-HCl buffer (pH 8.0) were prepared. 0.165 mL of the above Tris-HCl buffer, 0.005 mL of SANA, and 0.01 mL of elastase were dispensed into a 96-well plate.

[0167] Samples (1 mg / mL) of the examples and comparative examples were added along with a positive control and reacted at 25°C for 15 minutes, after which the absorbance was measured at 405 nm.

[0168] The ratio (%) of elastase inhibitory activity was measured as the ratio of the difference between the absorbance with the sample added and the absorbance without the sample added, and is shown in Table 5.

[0169] division Elastase inhibition rate (% of control) Example 1 36.2 Example 2 41.2 Example 3 35.5 Example 4 39.5 Comparative Example 1 21.1 Comparative Example 2 19.6 Comparative Example 3 24.6 Comparative Example 4 25.3 Comparative Example 5 27.1 Comparative Example 6 28.5 Comparative Example 7 24.3 Comparative Example 8 22.1

[0170] Elastase is an enzyme that breaks down elastin, a crucial matrix for maintaining skin elasticity within the dermis. Additionally, elastase is a non-specific hydrolytic enzyme capable of breaking down collagen, another important matrix protein.

[0171] Therefore, a high elastase activity inhibition rate implies that elastin and collagen, which are important for maintaining skin elasticity, are retained for a long time, suggesting excellent skin elasticity improvement effects.

[0172] Referring to Table 5, the samples of Examples 1 to 4 showed superior elastase inhibitory activity compared to Comparative Examples 1 to 8.

[0173] In particular, Examples 1 to 4, which underwent a fermentation process using lactic acid bacteria isolated from giant Centella asiatica, were evaluated to have excellent elastase inhibitory activity, and the giant Centella asiatica extract was evaluated to have a higher level of elastase inhibitory activity compared to the general Centella asiatica extract.

[0174] The above results suggest that not only is the elastase inhibitory activity of Giant Centella asiatica superior compared to conventional Centella asiatica materials, but excellent wrinkle improvement effects can also be achieved by fermenting using lactic acid bacteria isolated from Giant Centella asiatica.

[0175] Experimental Example 7: Evaluation of Anti-aging Activity

[0176] The samples of the examples were suspended in purified water, diluted to different concentrations, and the wrinkle improvement effect was evaluated.

[0177] MMP1 inhibitory activity was measured to confirm the skin wrinkle effect.

[0178] MMP is a matrix metalloproteinase, a zinc-dependent endoprotease, and MMP-1 is an interstitial collagenase that increases upon UV exposure to the skin and is an enzyme that breaks down collagen.

[0179] To evaluate the degree of inhibition of MMP-1 (Matrix metalloproteinase-1) expression in fibroblasts, 2 x 10⁶ HaCaT cells were used. 5 Cells were seeded into 60mm dishes at a concentration of cells / well, and NHF cells were 3 x 10⁶ 4 After dispensing into a 24-well plate at a cells / well concentration, the cells were cultured for 24 hours under cell culture conditions.

[0180] Afterward, the culture medium was removed, and the cells were starved for 24 hours. HaCaT cells were washed with DPBS, and then DPBS was added to provide UV 15 mJ / cm² 2 investigated.

[0181] The sample was diluted to an appropriate concentration in the supernatant of HaCaT cells obtained after UV irradiation, treated with NHF cells, and then cultured under cell culture conditions.

[0182] After 24 hours, the culture medium of the cultured cells was collected, and the amount of MMP-1 was measured using the Human total MMP-1 kit (DY901; R&D systems).

[0183] Retinoic acid was used as the positive control. MMP-1 inhibitory activity was expressed as the rate of reduction in MMP-1 expression in the control sample treatment group irradiated with UV (Table 6). The MMP1 inhibitory activity of the samples was measured at a concentration of 10 mg / mL.

[0184] division MMP1 inhibition rate (%) Example 1 84.6 Example 2 87.9 Example 3 80.9 Example 4 86.1 Comparative Example 1 54.9 Comparative Example 2 49.6 Comparative Example 3 58.8 Comparative Example 4 57.2 Comparative Example 5 60.1 Comparative Example 6 61.7 Comparative Example 7 55.2 Comparative Example 8 53.8 Positive control (Retinoic acid, 10μM) 73.9

[0185] Referring to Table 6, the samples of Examples 1 to 4 showed superior MMP1 inhibitory activity compared to Comparative Examples 1 to 8.

[0186] In particular, in the case of Examples 1 to 4, which underwent a fermentation process using lactic acid bacteria isolated from giant Centella asiatica, the MMP1 inhibitory activity was evaluated to be at a higher level.

[0187] Experimental Example 8: Evaluation of Skin Moisturizing Activity

[0188] The samples of the examples and comparative examples were suspended in purified water to evaluate the skin moisturizing effect. The expression levels of the HAS2 and AQP3 genes, which are involved in skin moisturization, were measured at the mRNA level in human keratinocytes.

[0189] Human keratinocytes (HaCaT) were cultured with Dulbecco's Modified Eagle's Medium (DMEM), 10% Fatal bovine serum (FBS), and 1% Antibiotic-Antimycotic in a 100 mm / 60.1 cm culture dish at 37°C and 5% CO2.

[0190] 1.0 x 10 keratinocytes 6 Cells were dispensed into a 6-well plate at a concentration of cells / mL and cultured for 24 hours.

[0191] After exchanging the medium for DMEM-free medium, the diluted samples of the example and comparative example were treated and cultured for 24 hours.

[0192] Cells were recovered, washed with cooled phosphate-buffered saline (PBS), 1 mL of TRIzol™ reagent [TRIzol™ reagent, Life Technologies, Inc.] was added, and total RNA was extracted.

[0193] Changes in gene expression were measured using qRT-PCR (quantitative real-time PCR), which binds a fluorescent substance to the DNA product amplified in the polymerase chain reaction (PCR) and continuously detects the fluorescent substance.

[0194] To quantify changes in HAS2 and AQP3 gene expression, the fluorescence values ​​emitted by the PCR products were measured using SYBR green I (Invitrogen).

[0195] A reaction solution was prepared by mixing 0.2 μM primer, 50 mM KCl, 20 mM Tris / HCl pH 8.4, 0.8 mM dNTP, 0.5 U Extaq DNA polymerase, 3 mM MgCl2, and 1×SYBR green in a PCR tube.

[0196] Primary denaturation was performed using Linegene K (BioER, China) at 94°C for 3 minutes, followed by denaturation, annealing, and polymerization for a total of 40 cycles at 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds, and the fluorescence intensity was measured after each cycle.

[0197] PCR results were verified using melting curves for each result. After standardizing the threshold cycle (Ct) value of each gene to the Ct value of β, the change in Ct values ​​was analyzed by comparing them.

[0198] The Ct value is the number of cycles at which the amount of fluorescence generated by the PCR product (the number of amplified PCR products) reaches a certain threshold value above the baseline, and the gene expression level can be verified through the Ct value.

[0199] The results of measuring HAS2 and AQP3 mRNA expression levels are as shown in Table 6 below, and mRNA expression levels were measured after treatment with the samples of the examples and comparative examples (concentration of 10 mg / mL).

[0200] The higher the ability to promote HAS2 and AQP3 expression, the better the skin moisturizing effect can be evaluated.

[0201] [Fold of control] division HAS2 expression level AQP3 expression level Example 1 1.56 1.64 Example 2 1.62 1.76 Example 3 1.57 1.72 Example 4 1.64 1.70 Comparative Example 1 1.30 1.36 Comparative Example 2 1.24 1.32 Comparative Example 3 1.32 1.38 Comparative Example 4 1.35 1.41 Comparative Example 5 1.38 1.45 Comparative Example 6 1.41 1.48 Comparative Example 7 1.33 1.40 Comparative Example 8 1.29 1.37 Negative control group 1.00 1.00

[0202] Referring to Table 7, the expression levels of the HAS2 gene and AQP3 gene increased according to the sample treatment of the example.

[0203] The above results suggest that not only is the skin moisturizing activity of Giant Centella asiatica superior to that of conventional Centella asiatica materials, but the skin moisturizing activity can also be further improved by fermenting using lactic acid bacteria isolated from Giant Centella asiatica.

[0204] In addition, fermented extracts of giant Centella asiatica and camellia flower leaves can effectively enhance skin hydration by promoting the expression of aquaporin proteins and the synthesis of hyaluronic acid through synergistic action.

[0205] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single unit may be implemented in a distributed manner, and components described as distributed may likewise be implemented in a combined form.

[0206] The scope of the present invention is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the present invention.

Claims

Claim 1 It contains giant Centella asiatica fermented extract and camellia flower petal fermented extract as active ingredients, wherein the fermented extract is obtained by inoculating with lactic acid bacteria isolated from giant Centella asiatica, and the lactic acid bacteria are Lactobacillus plantarum and Lactobacillus pentosus ( Lactobacillus pentosus A cosmetic composition comprising one or more of ). Claim 2 delete Claim 3 delete Claim 4 A cosmetic composition according to claim 1, comprising 100 parts by weight of the giant Centella asiatica fermented extract and 0.5 to 5.0 parts by weight of the camellia flower petal fermented extract. Claim 5 A cosmetic composition according to claim 1 or 4, which is intended for skin whitening and wrinkle improvement. Claim 6 A cosmetic composition according to claim 1 or 4, which is for skin moisturizing or antioxidant use.

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