KR102319686B1 - Composition for skin whitening or anti-wrinkle comprising fermented extract of Ambrosia trifida and Annual fleabane - Google Patents

Abstract

The present invention relates to a fermented extract of Maple Leaf ragweed and Albaniasis, and more particularly, to a skin whitening or anti-wrinkle composition comprising the fermented extract. The fermented maple leaf ragweed extract according to the present invention inhibits the activity of tyrosinase and has the effect of inhibiting the production of melanin in melanin-producing cells, and has excellent inhibitory activity of elastase, skin whitening or It can be used in various fields such as cosmetics for wrinkle improvement, external preparations for skin, and food.

Description

Composition for skin whitening or anti-wrinkle comprising fermented extract of Ambrosia trifida and Annual fleabane

The present invention relates to a fermented extract of Maple Leaf ragweed and Albaniasis, and more particularly, to a skin whitening or anti-wrinkle composition comprising the fermented extract.

Environmental pollution caused by industrialization causes obstacles in general life. In particular, interest in skin-related beauty and disease alleviation treatment materials is increasing due to changes in the perception of improving quality of life as well as increasing skin diseases as a protective film for the human body. In general, the preference for white skin is increasing due to skin damage caused by ultraviolet rays along with the deepening of environmental pollution, and as people pursue clearer and cleaner skin with transparent makeup, interest in methods to obtain skin whitening effect is increasing. . In addition, as the diversification and individualization of modern values stand out, the increase in interest as above has led to the development of functional cosmetics containing ingredients such as whitening, moisturizing, cell regeneration, and wrinkle improvement, and the cosmetic industry aiming for product safety is actively conducting R&D on natural materials.

The epidermis, located at the outermost part of the body, is arranged in the order of the basal layer, the stratum corneum, and the stratum corneum. Among the various cell types that make up it, melanocytes are phenolic biopolymers that determine hair color and skin color. They synthesize and secrete melanin, a complex of black proteins, and only prevent skin damage caused by UV rays. It also performs several important functions, including absorption of toxic drugs. However, excessive production of melanin causes skin diseases such as melasma and freckles, and in severe cases, it is reported to be related to the induction of skin cancer.

In general, skin color is determined according to the content and distribution of melanin, and is related to the number and distribution of melanosomes produced in melanocytes and released to the outside of the cells. Melanin is produced in the organelles of skin cells called melanosomes of melanocytes and moves to surrounding epidermal cells called keratinocytes. Melanin is produced through an oxidation process by tyrosinase, which oxidizes tyrosine using tyrosine as a substrate. Hyperpigmentation of the skin can be caused by several factors, such as hormonal abnormalities, genetic diseases, and ultraviolet irradiation after the inflammatory reaction of the skin. The main factors are due to abnormalities in the synthesis and distribution of melanin pigments.

Research on various materials for the purpose of whitening by regulating the activity and expression of the tyrosinase enzyme from the viewpoint of emphasizing the aesthetic function is being conducted. Hydroquinone and ascorbic acid are known so far , kojic acid, retinol, and arbutin. However, most materials exhibiting strong melanin biosynthesis inhibitory activity have a problem in that they cause side effects such as degeneration or death of pigment cells and impairing the original function of cells.

On the other hand, an ecosystem-disrupting species is a wildlife that disturbs or is likely to cause disturbance in the ecosystem. Exotic species with strong fertility can cause serious problems such as encroaching on native habitats as they enter the natural ecosystem, disrupting the balance of the ecosystem and reducing species diversity. Accordingly, the government regulates and regulates wildlife disturbing the ecosystem by law for species that are likely to adversely affect the ecosystem, establishes a management organization, or implements an ecosystem impact assessment system for exotic species to determine whether to import them.

Some invasive species that cause ecological, economic, industrial, and public health damage are defined as ecosystem disturbance wildlife according to Article 2 (4) of the Act on the Conservation and Use of Biodiversity. Also, among organisms that do not fall under the category of alien organisms, organisms that disturb or are likely to disturb the balance of the ecosystem in a specific region, or organisms that are likely to disturb or disturb the balance of the ecosystem among genetically modified organisms produced through genetic modification belong Ecosystem disturbance wildlife designated by the Biodiversity Conservation and Utilization Act includes one mammal (nutria), two amphibians and reptiles (bullfrog, all species of the genus Red-eared tortoise), and two species of fish (blue sea bass, bigmouth bass). ), 1 species of insects (flower locust), 15 species of plants (pigweed, maple leaf ragweed, sagebrush, fern sparrow, water sparrow, goblin branch, baby swimming, thorn gourd, sagebrush, American mugwort, There are a total of 21 species of yangmi yeokchwi, spiny lettuce, chrysanthemum, British chrysanthemum, algae).

Maple leaf ragweed ( Ambrosia trifida ) is a naturalized plant native to North America, and pollen of flowers blooming in summer causes allergic rhinitis or dermatitis. It spreads quickly, causing damage to crops.

On the other hand, annual fleabane is the most common naturalized plant in Korea. It spreads rapidly, and if weeding is not done in time, it will seriously damage the field farming.

Accordingly, the present inventors completed the present invention by confirming the remarkable skin whitening and wrinkle-improving effects of maple leaf ragweed and ragweed fermented extract as a result of research to utilize them as functional raw materials, which are classified as ecosystem disturbing species. became

Accordingly, it is an object of the present invention to provide a cosmetic, external preparation for skin or food composition for skin whitening or wrinkle improvement, comprising a fermented extract of maple leaf ragweed and ragweed as an active ingredient.

In order to achieve the above object, the present invention provides a cosmetic composition for skin whitening or wrinkle improvement comprising a fermented extract of maple leaf ragweed and ragweed as an active ingredient.

In addition, the present invention provides a composition for external application for skin whitening or wrinkle improvement comprising a fermented extract of maple leaf ragweed and ragweed as an active ingredient.

In addition, there is provided a food composition for skin whitening or wrinkle improvement comprising a maple leaf ragweed and fermented extract of fermented ragweed as an active ingredient.

The fermented maple leaf ragweed extract according to the present invention inhibits the activity of tyrosinase and has the effect of inhibiting the production of melanin in melanin-producing cells, and has excellent inhibitory activity of elastase, skin whitening or It can be used in various fields such as cosmetics for wrinkle improvement, external preparations for skin, and food.

1 is a view showing the results of measuring the electron donating ability of maple leaf ragweed and fermented fermented extracts of Amanita fermented in accordance with the present invention.
Figure 2 is a diagram showing the results of measuring the tyrosinase inhibitory activity of the fermented extracts of maple leaf ragweed and A. ragweed according to the present invention.
Figure 3 is a diagram showing the result of measuring the elastase inhibitory activity of the fermented extract of maple leaf ragweed and A. ragweed according to the present invention.
Figure 4 is a diagram showing the results of measuring the cell toxicity of the fermented extract of maple leaf ragweed according to the present invention through MTT analysis.
5 is a view showing the results of confirming the effect of the fermented extract of Maple Leaf ragweed and Alfalfa root according to the present invention on the inhibitory activity of pigmentation-related proteins through Western blotting.
6 is a view showing the results of confirming the effect of the fermented extracts of maple leaf ragweed and ragweed according to the present invention on the mRNA expression of pigmentation-related factors through RT-PCR.
7 is a diagram showing the results of comparing the electron donating ability of a maple leaf ragweed extract, a ragweed extract, and a maple leaf ragweed extract and a fermented ragweed extract.
Figure 8 is a diagram showing the results of comparing the elastase inhibitory activity of the maple leaf ragweed extract, the ragweed extract and the fermented maple leaf ragweed extract.

Hereinafter, the present invention will be described in detail.

According to one aspect of the present invention, the present invention provides a cosmetic composition for skin whitening or wrinkle improvement comprising a fermented extract of maple leaf ragweed ( Ambrosia trifida ) and annual fleabane as active ingredients.

In the present invention, the "extract" is extracted from the fermented product of Maple Leaf ragweed and Alfalfa root using an appropriate solvent, for example, a crude extract, a polar solvent-soluble extract or a non-polar solvent-soluble extract include all In addition, the extract is a concept including all extracts, fractions, dilutions, concentrates or dried products obtained in each step of extraction, fractionation or purification.

The active ingredients of the composition of the present invention, the fermented extract of Maple Leaf ragweed and Alfalfa root, may be obtained by a conventional extraction method, or may be purchased and used commercially.

The fermented maple leaf ragweed and fermented extract may be extracted or fractionated from various parts of the raw material plant, and is preferably extracted from the leaves, stems and roots of maple leaf ragweed and ragweed.

The fermented maple leaf ragweed extract and fermented ragweed extract may be obtained by extraction, separation and fractionation from nature using methods known in the art for extraction, separation and fractionation.

In one embodiment of the present invention, the fermented extract of maple leaf ragweed and ragweed was prepared by mixing maple leaf ragweed and ragweed, and then fermenting it by adding Onyang hot spring water, followed by extraction.

The maple leaf ragweed and ragweed may be mixed in the same amount, that is, in a weight ratio of 1:1.

In one embodiment of the present invention, the hot spring water preferably has a pH of 7 to 9, preferably a pH of 8 to 9, but is not limited thereto.

In an embodiment of the present invention, the hot spring water is potassium 1 to 2 mg/L, sodium 40 to 50 mg/L, calcium 5 to 10 mg/L, silicon 20 to 30 mg/L, lithium 0.05 to 0.20 mg/L, strontium 0.1 to 0.5 mg/L and magnesium 0.1 to 0.4 mg/L, more preferably potassium 1.59 mg/L, sodium 45.5 mg/L, calcium 6.75 mg/L, silicon 25.85 mg/L, lithium 0.12 mg/L, strontium 0.27 mg/L and magnesium 0.21 mg/L.

In the present invention, fermentation means that organic matter is decomposed and changed by microbial action to produce useful substances. The type of microorganism that can be used for fermentation in the present invention is not limited, as long as it is a microorganism capable of enhancing the activity of maple leaf ragweed and aloe vera through fermentation, but is preferably a Lactobacillus sp. The Lactobacillus sp include, for example, Lactobacillus ramno suspension (Lactobacillus rhamnosus), Lactobacillus para Kasei (Lactobacillus paracasei), Lactobacillus casei (Lactobacillus casei), Lactobacillus Planta room (Lactobacillus plantarum), Lactobacillus Bulgaria Cus ( Lactobacillus bulgaricus ), Lactobacillus lactis ( Lactobacillus lactis ), Lactobacillus fermentum ( Lactobacillus fermentum ), and the like are included, but are not limited thereto. In exemplary embodiments of the present invention, but used as a microorganism for fermentation mixture of Lactobacillus Lactobacillus ramno suspension (Lactobacillus rhamnosus), and Lactobacillus para Kasei (Lactobacillus paracasei), but is not limited thereto.

Fermentation in the present invention may be carried out at 25 to 40 ℃ for 1 to 5 days, preferably it may be carried out at 32 to 40 ℃ for 2 to 4 days, but is not limited thereto. In one embodiment of the present invention, after inoculating the Lactobacillus sp. strain, it was fermented at 37° C. for 3 days to prepare a fermented maple leaf ragweed according to the present invention.

Water or an organic solvent may be used as a suitable solvent for obtaining the fermented extract of maple leaf ragweed and fermented ragweed, and any pharmaceutically acceptable organic solvent may be used. For example, as the solvent, an alcohol having 1 to 4 carbon atoms, such as water, methanol, ethanol, propanol, isopropanol, butanol, etc. alone or two or more It can be used by mixing, and it is most preferable to use water.

The extraction temperature is preferably 50 to 100 ℃. In addition, as the extraction method, various methods such as hot water extraction method, cold extraction method, reflux cooling extraction method, solvent extraction method, steam distillation method, ultrasonic extraction method, elution method, compression method, etc. may be used, but it is not limited thereto, and preferably hot water extraction method or Reflux cooling extraction method is used.

The fermented maple leaf ragweed extract and fermented ragweed extract can be obtained by cooling, heating and filtering at room temperature by a conventional method known in the art, and additionally evaporating the solvent, spray-drying or freeze-drying. In addition, the fermented extracts of maple leaf ragweed and ragweed may be prepared in a powder state by an additional process such as distillation under reduced pressure and freeze-drying or spray-drying. In addition, the extract is further purified by using various chromatography methods such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography, etc. can be obtained

Therefore, the fermented maple leaf ragweed extract used in the present invention is a concept including all extracts, fractions and purified products obtained in each step of extraction, fractionation or purification, and their dilutions, concentrates or dried products.

According to an embodiment of the present invention, the fermented extract of Maple Leaf ragweed and fermented ragweed inhibits tyrosinase activity in a tyrosinase enzyme experiment, and has an excellent effect of inhibiting melanin production in a cell model in which melanin accumulation is induced. Therefore, the fermented extracts of Maple Leaf ragweed and Alfalfa root of the present invention can be usefully used as a cosmetic composition for skin whitening. In addition, according to another embodiment of the present invention, the fermented extract of maple leaf ragweed and aloe vera has an excellent effect of inhibiting the activity of elastase. Therefore, the fermented extract of Maple leaf ragweed and Aerica larvae of the present invention can be usefully used as a cosmetic composition for wrinkle improvement.

The fermented extract of maple leaf ragweed and fermented ragweed may be included in an amount of 0.01 to 95% by weight, more preferably 1 to 80% by weight, based on the total weight of the cosmetic composition. If the content is less than 0.01% by weight, the effect of skin whitening or wrinkle improvement may be insignificant, and if it exceeds 95% by weight, the effect increase rate is low compared to the amount used, which may be uneconomical.

The cosmetic composition of the present invention may further include conventional adjuvants and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments, fragrances, and the like, which are commonly used in cosmetic compositions in addition to the active ingredients. For example, the cosmetic composition may further include auxiliary components such as glycerin, butylene glycol, polyoxyethylene hydrogenated castor oil, tocopheryl acetate, citric acid, panthenol, squalane, sodium citrate, and allantoin. .

Since the cosmetic composition of the present invention is basically applied to the skin, it may be prepared in any formulation conventionally prepared with reference to a cosmetic composition in the art. For example, it may be formulated as a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation and spray, etc. The present invention is not limited thereto. More specifically, it may be prepared in the form of a flexible lotion, a nourishing lotion, a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a mask pack, a spray or a powder.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. may be included as carrier components. have.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. may be included as carrier components, and in particular, in the case of a spray, additional chlorofluorohydrocarbon, propane / May contain propellants such as butane and dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer, emulsifier, etc. may be included as carrier components, and specifically, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene Glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol, fatty acid ester of sorbitan, and the like may be included.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol, propylene glycol; suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester; Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tracanth, and the like may be included.

When the formulation of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide as carrier components ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, ethoxylated glycerol fatty acid esters, and the like may be included.

According to another aspect of the present invention, the present invention provides a composition for external application for skin for skin whitening or wrinkle improvement comprising a fermented extract of maple leaf ragweed and fermented ragweed as active ingredients.

When the composition of the present invention is used as a composition for external application to the skin, additionally, a fatty substance, an organic solvent, a solubilizer, a thickening agent and a gelling agent, an emollient, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, a surfactant , water, ionic or non-ionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or common for external applications for the skin It may contain adjuvants commonly used in the field of dermatology, such as any other ingredients used as a dermatome. In addition, the above ingredients may be introduced in an amount generally used in the field of dermatology.

When the composition of the present invention is provided as a formulation for external application to the skin, the present invention is not limited thereto, but may have formulations such as ointments, patches, gels, creams or sprays.

According to another aspect of the present invention, the present invention provides a food composition for skin whitening or wrinkle improvement, comprising the fermented extract of maple leaf ragweed and fermented ragweed as an active ingredient.

When the composition of the present invention is used as a food composition, the food composition of the present invention means a food having a skin whitening effect, and should be harmless to the human body when taken for a long time.

There is no particular limitation on the type of the food. Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages, vitamin complexes, and the like, and includes all health foods in the ordinary sense.

In an embodiment of the present invention, the food composition of the present invention may be a food additive. The food additive may be added as it is, or used with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment).

In an embodiment of the present invention, the food composition of the present invention may be a health drink composition. The health beverage composition may include various flavoring agents or natural carbohydrates as additional components, such as a conventional beverage, in addition to the fermented extract of Maple Leaf ragweed and Alfalfa root. As the above-mentioned natural carbohydrates, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharin and aspartame may be used. The proportion of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, Carbonating agents used in carbonated beverages, etc. may be included. In addition, the composition of the present invention may contain the pulp for the production of natural fruit juice, fruit juice beverage, and vegetable beverage. These components may be used independently or in combination. The proportion of these additives is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example One. Maple Leaf Pig Grass and Manufacture of fermented extract

In order to prepare a fermented extract of maple leaf ragweed and ragweed, the maple leaf ragweed and algae were cut into 1 to 3 cm, respectively, and mixed in equal amounts, and then pulverized to 1,300 mesh. A mixture of crushed maple leaf ragweed and ragweed was sterilized at 127° C. for 30 minutes. In order to ferment the mixture, hot spring water was mixed at a ratio of 1:1 (w/v) and then homogenized to prepare a hot spring water mixture. The components of the hot spring water are shown in Table 1.

ingredient Onyang hot spring water pH 8.75 potassium 1.59mg/L salt 45.5mg/L calcium 6.72mg/L silicon 25.85mg/L lithium 0.12mg/L strontium 0.27mg/L manganese - zinc - magnesium 0.21mg/L steel -

3% by weight of sucrose, 2% by weight of yeast extract and 0.2% by weight of dipotassium phosphate were further added based on the total weight of the hot spring water mixture. Next, the Lactobacillus sp. strain, which is a highly activated lactic acid bacterium, was inoculated at a concentration of 10% in the hot spring water mixture prepared through the above process, and this was fermented for 72 hours at 37°C and 120rpm conditions. Lactobacillus sp. strains were used commercially, specifically, Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) and Lactobacillus paracasei ( Lactobacillus paracasei ) is a mixture.

After the fermentation is complete, the fermented maple leaf ragweed and fermented ragweed are transferred to an extraction flask with a vertical reflux cooler, distilled water 10 times the weight of the sample is added as a solvent, and extraction is repeated 3 times at 100° C. for 60 minutes to extract hot water. Thus, a fermented extract of maple leaf ragweed and ragweed was prepared.

Example 2. Measurement of electron donating ability of maple leaf ragweed and fermented extract

To measure electron donating abilities (EDA), 60 μl of DPPH (1,1-diphenyl-2-picrylhydrazyl) and 120 μl of fermented extracts of maple leaf ragweed and fermented ragweed per concentration were mixed, left for 15 minutes, and then microplate Absorbance was measured at 517 nm using a reader. The electron donating ability was calculated as in Equation 1 below, and the results are shown in FIG. 1 .

[Equation 1]

Electron donating ability (%) = (1 - absorbance of sample added group / absorbance of no addition group) × 100

As shown in FIG. 1 , the effect of increasing the electron donating ability was confirmed as the concentration of the fermented extracts of Maple Leaf ragweed and fermented ragweed increased.

Example 3. Maple Leaf Pig Grass and tyrosinase inhibitory activity measurement of fermented extract

Tyrosinase inhibitory activity to confirm the whitening effect was measured as follows. After preparing a mixed solution of 40 μl of substrate solution and 40 μl of sample solution (fermented extract of Maple leaf ragweed and Astragalus fermented extract) in which 10 mM L-DOPA (Sigma, USA) was dissolved in 80 μl of 67 mM phosphate buffer (pH 6.8), 200 U/ml mushroom tyrosinase (Sigma, USA) 40 μl was added and reacted at 37° C. for 10 minutes. Thereafter, absorbance was measured at 492 nm to confirm the DOPA chromium generated in the reaction solution. Tyrosinase inhibitory activity was calculated as in Equation 2 below, and the results are shown in FIG. 2 .

[Equation 2]

Inhibition rate (%) = (1 - absorbance of sample added group / absorbance of no addition group) × 100

As shown in FIG. 2 , it was confirmed that the whitening effect was excellent, such as the increase in the tyrosinase inhibitory activity as the concentration of the fermented extract of Maple Leaf ragweed and Algae fermented extract increased.

Example 4. Elastase inhibitory activity measurement of maple leaf ragweed and fermented extract

In order to confirm the wrinkle improvement effect, the inhibitory activity of elastase, a representative enzyme that promotes skin aging, was confirmed. Specifically, a sample solution (a fermented extract of maple leaf ragweed and fermented ragweed) was prepared to a certain concentration, and 40 μl of each was dispensed into a 96-well plate, and 2.5 U/ml of porcine pancreas dissolved in 50 mM tris-HCl buffer (pH8.6). After adding 40 μl of elastase (Sigma, USA) solution, 80 μl of N-succinyl-(L-Ala)3-p-nitroanilide (0.5 mg/ml) dissolved in 50 mM tris-HCl buffer (pH 8.6) as a substrate was added. did. This was reacted at 37° C. for 30 minutes, and absorbance was measured at 445 nm to confirm the amount of p-nitroanilide produced from the substrate. Elastase inhibitory activity was calculated as in Equation 3 below, and the results are shown in FIG. 3 .

[Equation 3]

Inhibition rate (%) = (1 - absorbance of sample added group / absorbance of no addition group) × 100

As shown in FIG. 3 , as the concentration of the fermented extracts of Maple Leaf ragweed and A. fermented extract increased, the elastase inhibitory activity increased, and it was confirmed that the wrinkle improvement effect of the fermented extract was excellent.

Example 5. Maple Leaf Pig Grass and cytotoxicity measurement of fermented extract

B16F10, a melanoma cell used in this experiment, was purchased from ATCC (USA). For B16F10 cell culture, DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin (100 U/ml) was used, and _{subcultured at 37° C. and 5% CO 2} adapted to an incubator.

For cell viability measurement, 180 μl of B16F10 cells were aliquoted in a 96-well plate to 1×10 ⁵ cells/well, and samples (maple leaf ragweed and fermented fermented fermented extracts) were prepared by concentration and 20 μl was added at 37°C. , 5% CO ₂ Incubated for 24 hours in an incubator. Here, 40 μl of MTT solution prepared at a concentration of 2.5 mg/ml was added and incubated for 4 hours. Thereafter, the culture medium was removed, and 100 μl of DMSO was added to each well, followed by reaction at room temperature for 10 minutes, and absorbance was measured at 540 nm with an ELISA reader. Cell viability was calculated as in Equation 4 below, and the results are shown in FIG. 4 .

[Equation 4]

Cell viability (%) = (1 - Absorbance of the sample added group / Absorbance of the non-added group) × 100

As shown in FIG. 4 , it was confirmed that the fermented extracts of Maple leaf ragweed and A. fermented ragweed exhibited no cytotoxicity, such as showing a cell viability of about 97% even at a high concentration of 500 μg/ml.

Example 6. Confirmation of pigmentation-related factor inhibitory activity of maple leaf ragweed and fermented extract

6-1. Confirmation of inhibitory activity of pigmentation-related factor protein

To investigate the activity of factors related to pigmentation and whitening, such as MITF, TRP-1, TRP-2, and tyrosinase, at the protein level, 25, 50, and 100 μg/ml Treated by concentration. The control group was treated with α-MSH and set to the section in which melanin was overexpressed, and the normal group was set to the section not treated with α-MSH. At this time, the expression level of β-actin, a housekeeping gene with little difference in expression level, was confirmed and quantified under various cell conditions.

Specifically, B16F10 cells were aliquoted in a 100 mm tissue culture dish at 1×10 ⁶ cells/well, and then cultured for 24 hours to stabilize the cells. After removing the medium, after treatment with α-MSH (100 nm) for 2 hours, each ferment extract was replaced with a medium treated for each concentration, and after culturing for 24 hours, the medium was removed again and washed twice with 1× PBS. Each cell was dissolved in 10 ml of RIPA buffer to which 1 complete mini tab was added, and centrifuged at 4° C. and 13,200 rpm for 20 minutes. The supernatant obtained by centrifugation was quantified with a BCA protein analysis kit, and 20 μl of the protein was separated by electrophoresis on a 10% acrylamide gel. The separated protein was transferred to a polyvinylidene fluoride (PVDF) membrane using a transfer device, and then incubated in blocking buffer (5% skim milk in TBST) at room temperature for 1 hour. The primary antibody was diluted and incubated at 4° C. overnight, and then washed three times with TBST (tris-buffered saline and tween 20) at 10-minute intervals again. The secondary antibody was diluted 1:1,000 and incubated for 2 hours at room temperature, washed 3 times with TBST, and then the band was confirmed using the Davinch-Chemi™ Imager CAS-400SM system. The results of the inhibitory activity of the pigmentation-related factor protein are shown in FIG. 5 .

As shown in FIG. 5 , it was confirmed that the protein expression levels of MITF, TRP-1, TRP-2, and tyrosinase increased by α-MSH were reduced by the treatment of the fermented extracts of Maple leaf ragweed and fermented ragweed.

6-2. Confirmation of mRNA expression of pigmentation-related factors

After the B16F10 cells were seeded in a 100 mm culture dish, the fermented extracts of maple leaf ragweed and fermented ragweed were treated by concentration and cultured for 24 hours. After removing the medium supernatant, 1 mL of trizol lysis buffer was dispensed into each well to dissolve the cells, and 200 μL of chloroform was dispensed and shaken up and down for 20 seconds. After centrifugation at 13,200 rpm for 20 minutes, the supernatant was transferred to a tube containing 0.5 mL of isopropanol and mixed. Centrifuged again at 13,200 rpm for 20 minutes, and after removing the supernatant, 1 mL of 75% EtOH-DEPC water was dispensed into each tube, centrifuged at 13,200 rpm for 5 minutes, and the supernatant was removed and dried at room temperature. . After dissolving 50 μL of DEPC, 5 μL of RNA and 195 μL of sterile water were added to a 96-well plate, and absorbance was measured at 260 and 280 nm, respectively, to measure the total RNA amount. Oligo (dT) 15 primer (500 µg/mL) 1 µL, extracted RNA (2 µg) and nuclease-free water in 10 µL, react at 75 ° C for 5 minutes, 5X reaction buffer, MgCl ₂ , PCR nucleotide mix, RNasin ⓡ Inhibitor, reverse transcriptase, and nuclease-free water were added and reacted at 25° C. for 5 minutes, 42° C. for 60 minutes, and 70° C. for 15 minutes to synthesize cDNA.

In order to confirm the mRNA expression for MITF and tyrosinase, real time-polymerase chain reaction (RT-PCR) was performed. 5× green Go Taq Flexi buffer, MgCl ₂ , PCR nucleotide mix (10 mM), primer, Go Taq DNA polymerase, nuclease-free water, and the synthesized cDNA were added to a PCR tube and mixed, followed by PCR. At this time, PCR was carried out for 35 cycles of 94 ℃ 30 sec, 56 ℃ 60 sec, 72 ℃ 1 min each. After PCR, the PCR product was put into a 1.5% agarose gel containing 0.002% EtBr and electrophoresed at 100V for 40 min. Thereafter, the band was confirmed and quantitatively analyzed using a UV transmission contrast machine. Primers used for RT-PCR are shown in Table 2, and the results of confirming the mRNA expression of pigmentation-related factors are shown in FIG. 6 .

gene primer Sequence (5' → 3') GAPDH sense TGA AGG TCG GTG TGA ACG GAT TTG GC antisense CAT GTA GGC CAT GAG GTC CAC CAC MITF forward AGC GTG TAT TTT CCC CAC AG reverse TAG CTC CTT AAT GCG GTC GT tyrosinase forward GAC GGT CAC TGC ACA CTT TG reverse GCC ATG ACC AGG ATG AC

As shown in FIG. 6 , it was confirmed that the mRNA expression of MITF and tyrosinase was significantly inhibited as the concentration of the fermented extract of Maple Leaf ragweed and fermented ragweed was increased.

Example 7. Comparison of physiological activity of maple leaf ragweed and fermented ragweed extract and maple leaf ragweed or fermented ragweed extract

7-1. Manufacture of extract of maple leaf ragweed or ragweed

Maple leaf ragweed was cut into 1 to 3 cm and mixed and then pulverized to 1,300 mesh. The crushed maple leaf ragweed was sterilized at 127° C. for 30 minutes. The sterilized maple leaf ragweed was transferred to an extraction flask equipped with a vertical reflux cooler, distilled water 10 times the weight of the sample was added as a solvent, and hot water extraction was performed at 100° C. for 60 minutes three times to prepare a maple leaf ragweed extract.

The extract of aloe vera was also prepared in the same way as the extract of Maple Leaf ragweed.

7-2. electron donating ability measurement

In the same manner as in Example 2 for measuring electron donating ability, the electron donating ability of the maple leaf ragweed extract, the algae extract, and the maple leaf ragweed and fermented fermented ragweed extracts prepared in Example 1 were compared, and the results are shown in FIG. 7 .

As shown in FIG. 7 , it was confirmed that the fermented maple leaf ragweed extract prepared in Example 1 had superior electron donating ability compared to the maple leaf ragweed extract or the maple leaf ragweed extract alone. That is, it was confirmed that the fermented maple leaf ragweed and fermented ragweed extract obtained by mixing and fermenting maple leaf ragweed and ragweed alone, compared to maple leaf ragweed or ragweed alone, had significantly superior physiological activity effects due to the synergistic effect of the two substances. .

7-3. Elastase inhibitory activity

In the same manner as in Example 4, the elastase inhibitory activity of the maple leaf ragweed extract, the A. ragweed extract, and the maple leaf ragweed extract and the fermented Amanita fermented extract prepared in Example 1 were compared, and the results are shown in FIG. 8 .

As shown in FIG. 8 , it was confirmed that the fermented maple leaf ragweed extract prepared in Example 1 was superior to the elastase inhibitory activity, that is, the wrinkle improvement effect, compared to the maple leaf ragweed extract or the ragweed extract alone.

Overall, through the above experimental results, the present inventors found that the fermented extracts of maple leaf ragweed and ragweed inhibit the activity of tyrosinase, have an excellent effect of suppressing the expression of pigmentation related factors, inhibit the activity of elastase, etc. It was confirmed that the whitening and wrinkle improvement effects were excellent. In particular, the fermented maple leaf ragweed and ragweed extract according to the present invention is significantly more effective than the maple leaf ragweed or ragweed extract alone, so it can be used variously in cosmetics, external preparations for skin and food fields for skin whitening or wrinkle improvement. have.

Hereinafter, the present invention will be described in more detail through formulation examples. The formulation examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by the formulation examples.

Formulation Example 1. Preparation of a cosmetic formulation

1-1. Manufacture of soft lotion

0.1% by weight of fermented extract of maple leaf ragweed and algae fermented extract, 5.2% by weight of 1,3-butylene glycol, 1.5% by weight of oleyl alcohol, 3.2% by weight of ethanol, 3.2% by weight of polysorbate 20, 2.0% by weight of benzophenone-9, A softening lotion was prepared by a conventional method by mixing 1.0% by weight of carboxyl vinyl polymer, 3.5% by weight of glycerin, a small amount of fragrance, a small amount of preservative, and the remaining amount of purified water.

1-2. Milk lotion manufacturing

Maple leaf ragweed and fermented fermented extract 0.1% by weight, glycerin 5.1% by weight, propylene glycol 4.2% by weight, tocopheryl acetate 3.0% by weight, liquid paraffin 4.6% by weight, triethanolamine 1.0% by weight, squalane 3.1% by weight, macadamia nut oil 2.5 Weight %, polysorbate 60 1.6% by weight, sorbitan sesquirolate 1.6% by weight, propylparaben 0.6% by weight, carboxyvinyl polymer 1.5% by weight, trace amount of fragrance, trace amount of preservative, and the remaining amount of purified water are mixed with conventional Milk lotion was prepared by this method.

1-3. Nutrient cream manufacturing

0.5% by weight of fermented extract of maple leaf ragweed and algae fermented extract, 4.0% by weight of glycerin, 3.5% by weight of petrolatum, 2.1% by weight of triethanolamine, 5.3% by weight of liquid paraffin, 3.0% by weight of squalane, 2.6% by weight of beeswax, 5.4% by weight of tocopheryl acetate, 3.2 wt% of polysorbate 60, 1.0 wt% of carboxyl vinyl polymer, 3.1 wt% of sorbitan sesquioleate, a trace amount of fragrance, a trace amount of preservative and the remaining amount of purified water were mixed to prepare a nourishing cream in a conventional manner.

1-4. massage cream manufacturing

0.5% by weight of fermented extract of maple leaf ragweed and sagebrush, 4.0% by weight of glycerin, 3.5% by weight of petrolatum, 0.5% by weight of triethanolamine, 24.0% by weight of liquid paraffin, 3.0% by weight of squalane, 2.1% by weight of beeswax, 0.1% by weight of tocopheryl acetate, A massage cream was prepared in a conventional manner by mixing 2.4 wt% of polysorbate 60, 1.0 wt% of carboxyl vinyl polymer, 2.3 wt% of sorbitan sesquioleate, a trace amount of fragrance, a trace amount of preservative and the remaining amount of purified water.

1-5. Manufacture of body cleanser for cleaning

A body cleanser for cleansing by mixing 1 g of fermented maple leaf ragweed extract, 18 g of anionic surfactant, 5 g of nonionic surfactant, 7 g of glycerin, 3 g of sodium chloride, 1.5 g of natural olive liquid soap, 1 g of fragrance and 100 g of water. was prepared.

Above, specific parts of the present invention have been described in detail, for those of ordinary skill in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

A cosmetic composition for skin whitening or wrinkle improvement comprising a fermented extract of hot spring water of a maple leaf ragweed ( Ambrosia trifida ) and annual fleabane mixture as an active ingredient.
The cosmetic composition for skin whitening or wrinkle improvement according to claim 1, wherein the fermentation is made by a Lactobacillus sp.
delete According to claim 1,
The hot spring water has a pH of 7 to 9, a cosmetic composition for skin whitening or wrinkle improvement.
5. The method of claim 4,
The hot spring water is potassium 1 to 2 mg / L, sodium 40 to 50 mg / L, calcium 5 to 10 mg / L, silicon 20 to 30 mg / L, lithium 0.05 to 0.2 mg / L, strontium 0.1 to 0.5 mg / L, and magnesium 0.1 to A cosmetic composition for skin whitening or wrinkle improvement, comprising 0.4 mg/L.
According to claim 1,
The extract is a cosmetic composition for skin whitening or wrinkle improvement, which is extracted with one or more solvents selected from water, alcohols having 1 to 4 carbon atoms, and mixed solvents thereof.
A composition for external application for skin whitening or wrinkle improvement, comprising, as an active ingredient, a fermented extract of hot spring water of a mixture of maple leaf ragweed and ragweed.
A food composition for skin whitening or wrinkle improvement, comprising a fermented extract of hot spring water of a mixture of maple leaf ragweed and ragweed as an active ingredient.

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