KR20200015423A - 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 Ambrosia trifida and Erigeron annuus. More particularly, the present invention relates to a composition for improving skin whitening or reducing wrinkles, comprising the fermented extract. The fermented extract of Ambrosia trifida and Erigeron annuus of the present invention not only has an effect of inhibiting the activity of tyrosinase and inhibiting melanin production in melanocytes, but also has an excellent effect of inhibiting elastase, and thus can be variously used in cosmetics, external dermal agents, food fields, etc. for improving skin whitening or reducing wrinkles.

Description

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

The present invention relates to a maple leaf ragweed and Gaecho fermented extract, and more particularly, to a composition for improving skin whitening or wrinkles comprising the fermented extract.

Environmental pollution caused by industrialization causes obstacles to general life. Especially, as skin protection is increasing as a human protective film, the interest in skin-related beauty and disease mitigation treatments is increasing due to a change in perception that quality of life is improved. In general, the preference for white skin is increasing due to the deepening of environmental pollution and skin damage caused by ultraviolet rays, and as the pursuit of clearer and clearer skin with transparent makeup, there is increasing interest in how to obtain skin whitening effect. . In addition, as the diversification and individualization of modern values stand out, the increase in interest 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 the safety of products. Is actively researching and developing natural materials.

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

In general, the skin color is determined according to the content and distribution of melanin, and is related to the number and distribution of melanosomes generated in melanocytes and released to the outside of cells. Melanin is produced in the organelles of skin cells called melanosomes of melanocytes and migrates to the surrounding epidermal cells called keratinocytes. Melanin is produced by the oxidation process by tyrosinase, which oxidizes tyrosine with tyrosine as a substrate. Hyperpigmentation of the skin may be caused by various factors such as hormonal abnormalities, genetic disorders, ultraviolet irradiation, etc. after the inflammatory response of the skin, mainly due to melanin synthesis abnormalities and distribution abnormalities.

Research on various materials aimed at whitening has been conducted by regulating the activity and expression of tyrosinase enzymes from the point of view of aesthetic function, and to date, hydroquinone and ascorbic acid have been known. , Kojic acid, retinole, and arbutin. However, most of the materials exhibiting strong melanin biosynthesis inhibitory activity cause side effects such as denaturation or lethality of pigment cells, impairing the intrinsic function of cells.

Ecological disturbances, on the other hand, are wildlife that can cause disturbances or cause disturbances in ecosystems. Invasive alien species can cause serious problems, such as encroaching native habitats, disrupting the balance of ecosystems and reducing species diversity as they enter native ecosystems. As a result, governments have regulated and regulated wildlife disturbances by law, established a management organization, or implemented an alien species ecosystem impact assessment system to determine whether imports are likely to adversely affect ecosystems.

Some invasive species that cause ecological, economic, industrial and public health damage are defined as ecosystem disturbance wildlife under Article 2, paragraph 4 of the Act on Biodiversity Conservation and Use. In addition, organisms that do not belong to foreign organisms may disturb or disturb the balance of ecosystems in certain regions, and those that may disturb or disturb the balance of the ecosystem among genetically modified organisms produced through genetic modification. Belong. Ecosystem disturbances specified by the Act on the Conservation and Utilization of Biodiversity include one species of mammals (Nutria), two species of amphibians and reptiles (yellow-billed copper and all species of red eared turtles), and two species of fish (blue-balled rockfish, largemouth bass). ), 1 species of insects (flower cicada), 15 species of plants (pork grass, maple leaf ragweed, western spinach sprouts, hairy sparrows, water sparrows, goblins, baby swimming, prickly pear, oleander, wormwood, Yangmi odor, spiny lettuce, barley, English coccyx, Gaechocho) are 21 species.

Maple leaf scab ( Ambrosia trifida ) is a naturalized plant native to North America. Pollen of flowers blooming in summer causes allergic rhinitis or dermatitis. The rate of spread is also fast, damaging crops.

On the other hand, Gaemancho ( Annual fleabane ) is the most common naturalized plant in Korea. The rate of spreading is so high that if we do not weed in time, it will cause severe damage to farming.

Therefore, the present inventors have studied to utilize the maple leaf ragweed and forget-me-not, which are classified as ecosystem disturbance species, as a functional raw material, thereby completing the present invention by confirming the remarkable skin whitening and wrinkle improvement effect of the fermented extract of the maple leaf ragweed and forget-me-not. It became.

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

In order to achieve the above object, the present invention provides a cosmetic composition for improving skin whitening or wrinkles, including maple leaf ragweed and Gaechocho fermented extract as an active ingredient.

The present invention also provides a skin external preparation composition for improving skin whitening or wrinkles, including a maple leaf ragweed and Gaecho fermented extract as an active ingredient.

In addition, the present invention provides a food composition for improving skin whitening or wrinkles, including maple leaf ragweed and Gazelweed fermented extract as active ingredients.

Maple leaf ragweed and Ganoderma lucidum extract according to the present invention not only has the effect of inhibiting the activity of tyrosinase and inhibiting melanogenesis in melanocytes, but also has excellent inhibitory activity of elastase, skin whitening or It may be variously used in cosmetics, skin external preparations and food fields for wrinkle improvement.

1 is a view showing the results of measuring the electron donating ability of the fermented extract of Maple Leaf Porcine and Gaechocho according to the present invention.
Figure 2 is a view showing the results of measuring the tyrosinase inhibitory activity of the maple leaf ragweed and Gaechocho fermented extract according to the present invention.
Figure 3 is a view showing the results of measuring the elastase inhibitory activity of the maple leaf ragweed and Gaechocho fermented extract according to the present invention.
Figure 4 is a diagram showing the results of measuring the cytotoxicity of the maple leaf botanical herb and Gaechocho fermentation extract according to the present invention through the MTT analysis.
Figure 5 is a view showing the results confirming the effect on the inhibitory activity of the pigments related to the pigmentation of maple leaf botanical herb and Gaechocho fermentation according to the present invention through Western blotting.
Figure 6 is a view showing the results confirming the effect on the mRNA expression of pigmentation related factors of maple leaf ragweed and Gaechocho fermentation according to the present invention through RT-PCR.
Figure 7 is a view showing the results of comparing the electron donating ability of the maple leaf ragweed extract, Gaechocho extract and maple leaf ragweed and Gaecho fermentation extract.
Figure 8 is a view showing the results of comparing the elastase inhibitory activity of the maple leaf ragweed extract, Gaechocho extract and maple leaf ragweed and Gaecho fermented extract.

Hereinafter, the present invention will be described in detail.

According to an aspect of the present invention, the present invention provides a cosmetic composition for improving skin whitening or wrinkles, including as an active ingredient Maple leaf ragweed ( Ambrosia trifida ) and Gazelweed ( Annual fleabane ) fermentation.

In the present invention, "extract" is extracted from the fermentation of Maple leaf ragweed and Gazelweed using an appropriate solvent, for example, crude extract, polar solvent soluble extract or nonpolar solvent soluble extract of Maple leaf ragweed and Gazelweed It includes everything. In addition, the extract is a concept that includes all the extracts, fractions, their dilutions, concentrates or dried products obtained in each step of extraction, fractionation or purification.

Maple leaf ragweed and Ganoderma lucidum fermented extract which are the active ingredients of the composition of the present invention may be obtained by a conventional extraction method or may be purchased and used commercially.

The maple leaf ragweed and forget-me-not fermented extract may be extracted or fractionated from various parts of the raw material plant, preferably from the leaves, stems and roots of the maple leaf ragweed and forget-me-not.

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

In one embodiment of the present invention, the maple leaf ragweed and Gaecho fermentation extract was prepared by mixing the maple leaf ragweed and Gaechocho, followed by fermentation by adding hot spring water.

The maple leaf ragweed and forget-me-not can be preferably mixed in the same amount, that is, a weight ratio of 1: 1.

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

In one 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 substances are decomposed and changed by microbial action to produce useful substances. What can be used for the fermentation in the present invention is not limited if the microorganisms that can enhance the activity of the maple leaf and the forget-me-notweed through the fermentation, preferably the strain of the genus Lactobacillus. 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 Lactobacillus bulgaricus , Lactobacillus lactis , Lactobacillus fermentum , and the like, but are not limited thereto. In an embodiment of the present invention, the microorganisms for fermentation, but mixed with Lactobacillus Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) and Lactobacillus paracasei ( Lactobacillus paracasei ) , but is not limited thereto.

In the present invention, the fermentation may be performed for 1 day to 5 days at 25 to 40 ℃, preferably at 2 to 4 days at 32 to 40 ℃, but is not limited thereto. In one embodiment of the present invention, after inoculating the strain of the genus Lactobacillus, by fermentation at 37 ℃ for 3 days to prepare a maple leaf ragweed and Gaechocho fermentation according to the present invention.

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

It is preferable that the said extraction temperature is 50-100 degreeC. In addition, as the extraction method, various methods such as hot water extraction, cold extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, elution, and compression may be used, but are not limited thereto, and preferably, hot water extraction or The reflux cooling extraction method is used.

The maple leaf ragweed and Gaecho fermented extract may be obtained by cooling, heating and filtration at room temperature in a conventional manner known in the art, and may further evaporate, spray-dried, or lyophilized solvent. In addition, the maple leaf ragweed and Gaecho fermented extract 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 may be further purified by various chromatography such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography, and the like. You can get it.

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

According to one embodiment of the present invention, the maple leaf ragweed and Ganoderma lucidum fermentation extracts inhibit tyrosinase activity in tyrosinase enzyme experiments, and have an excellent effect of inhibiting melanin production in a cell model induced melanin accumulation. Therefore, the maple leaf ragweed and Ganoderma lucidum fermentation extract 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, maple leaf ragweed and Gaechocho fermented extract have an excellent effect of inhibiting the activity of elastase. Therefore, the maple leaf ragweed and Gaechocho fermented extract of the present invention can be usefully used as a cosmetic composition for improving wrinkles.

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

The cosmetic composition of the present invention may further include conventional auxiliaries and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments, fragrances and the like that are commonly used in cosmetic compositions in addition to the active ingredient. For example, the cosmetic composition may further include auxiliary ingredients such as glycerin, butylene glycol, polyoxyethylene hardened 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 cosmetic compositions 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, spray, and the like. It is not limited to this. More specifically, it may be prepared in the form of a flexible lotion, nutrition lotion, nutrition cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, mask pack, spray or powder.

When the formulation of the present invention is a paste, cream or gel, the carrier component may include animal oil, vegetable oil, wax, paraffin, starch, trakant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide and the like. have.

When the formulation of the present invention is a powder or a spray, carrier components may include lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, and the like, and in particular, in the case of spray, additionally chlorofluorohydrocarbon, propane Propellants, such as butane, dimethyl ether, and the like.

When the formulation of the present invention is a solution or emulsion, a carrier component may include a solvent, a solubilizer, an emulsion, and the like, and specifically, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, and propylene. Glycols, 1,3-butylglycol oils, glycerol aliphatic esters, polyethylene glycols, fatty acid esters of sorbitan, and the like.

When the formulation of the present invention is a suspension, liquid carrier diluents such as water, ethanol and propylene glycol as carrier components; Suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester; Microcrystalline cellulose, aluminum metahydroxy, bentonite, agar, tracant and the like.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component is an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, isethionate, an imidazolinium derivative, a methyltaurate, a sarcosinate, a fatty acid amide. Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, ethoxylated glycerol fatty acid esters, and the like.

According to another aspect of the present invention, the present invention provides a skin external preparation composition for improving skin whitening or wrinkles, including maple leaf ragweed and Gaecho fermented extract as an active ingredient.

When the composition of the present invention is used as a topical skin composition, it is further added to fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants. Common to water, ionic or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or external preparations for the skin It may contain adjuvants commonly used in the field of dermatology, such as any other ingredients used as. The ingredients may also be introduced in amounts generally used in the field of dermatology.

When the composition of the present invention is provided in a topical skin formulation, it may have a formulation such as, but not limited to, an ointment, patch, gel, cream or spray.

According to another aspect of the present invention, the present invention provides a food composition for skin whitening or wrinkle improvement comprising the maple leaf ragweed and Gaecho fermented extract 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 lightening effect, and should be harmless to the human body when taken for a long time.

There is no particular limitation on the kind of the food. Examples of the food to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, dairy products including gum, ice cream, various soups, beverages, tea, drink, Alcoholic beverages and vitamin complexes, and the like and include all of the health foods in the conventional 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 used as it is, or may be used together with other food or food ingredients, or may be used as appropriate according to conventional methods. The mixed amount of the active ingredient may be appropriately determined depending on 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 beverage composition. The health beverage composition may include various flavoring agents or natural carbohydrates as additional components, such as ordinary drinks, in addition to maple leaf ragweed and Gaecho fermented extract. The natural carbohydrates described above may be used as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and natural sweeteners such as dextrin and cyclodextrin, and synthetic sweeteners such as saccharin and aspartame. The proportion of said natural carbohydrates 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 salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, And carbonizing agents used in the carbonated beverage. In addition, the composition of the present invention may include a pulp for the production of natural fruit juice, fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not critical but is usually 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 with reference to Examples. These examples are only for illustrating the present invention, 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  And Ganoderma lucidum fermentation extract preparation

In order to prepare the maple leaf ragweed and forget-me-not fermented extract, cut the maple leaf ragweed and forget-me-not in 1 to 3cm, respectively, were mixed in equal amounts and then ground to 1,300 mesh. The ground maple leaf and forage mixture were sterilized at 127 ° C. for 30 minutes. To ferment the mixture, hot spring water was mixed at 1: 1 (w / v) and homogenized to prepare a hot water mixture. The components of the warm 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 0.27 mg / L manganese - zinc - magnesium 0.21 mg / L iron -

3% by weight of sucrose, 2% by weight of yeast extract, and 0.2% by weight of dipotassium phosphate were further added to the total weight of the hot water mixture. Next, inoculated with a high concentration of lactic acid bacteria Lactobacillus strain in 10% concentration prepared by the above process at a concentration of 10%, it was fermented for 72 hours at 37 ℃, 120rpm conditions. Lactobacillus genus strain was used commercially available, specifically, Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) and Lactobacillus paracasei ( Lactobacillus paracasei ) is a mixture.

After the end of fermentation, the maple leaf and the forget-me-not fermented product were transferred to an extraction flask equipped with a vertical reflux condenser, and 10 times distilled water of the sample weight was added as a solvent, followed by extraction repeated three times at 100 ° C. for 60 minutes. To prepare a maple leaf ragweed and Gaecho fork fermented extract.

Example 2 Determination of Electron Donating Ability of Fermented Extracts from Maple Leaf Porcine Grass

To measure electron donating abilities (EDA), add 60 μl of DPPH (1,1-diphenyl-2-picrylhydrazyl) and 120 μl of Maple Leaf Porcine and Ganoderma Lucidum per each concentration, mix, and leave for 15 minutes before 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 addition group / absorbance of no addition group) × 100

As shown in Figure 1, the effect of increasing the electron donating ability as the concentration of the maple leaf ragweed and Gaecho fermented extract increased.

Example  3. Maple Leaf  Determination of Tyrosinase Inhibitory Activity of Fermented Extracts

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

[Equation 2]

% Inhibition = (1-absorbance of sample addition group / absorbance of no addition group) x 100

As shown in FIG. 2, it was confirmed that the tyrosinase inhibitory activity was increased as the concentrations of the maple leaf herb and Ganoderma lucidum fermentation extract were increased, and thus the whitening effect was excellent.

Example 4 Determination of Elastase Inhibitory Activity of Fermented Extracts from Maple Leaf Porcine and Ganoderma Lucidum

In order to confirm the anti-wrinkle effect, it was confirmed that the elitase inhibitor activity, a representative enzyme promoting skin aging. Specifically, a sample solution (foliar leaf and Ganoderma lucidum fermentation extract) was prepared to a certain concentration, and 40 μl of each was dispensed in a 96-well plate, and 2.5 U / ml pork pancreas dissolved in 50 mM tris-HCl buffer (pH8.6). After adding 40 μl of an 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) was added as a substrate. It was. The reaction was carried out at 37 ° C. for 30 minutes, and the 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, the results are shown in FIG.

[Equation 3]

% Inhibition = (1-absorbance of sample addition group / absorbance of no addition group) x 100

As shown in Figure 3, as the concentration of the maple leaf ragweed and Gaecho fermentation extract increased, the lyasease inhibitory activity was increased, and through this it was confirmed that the wrinkle improvement effect of the fermentation extract is excellent.

Example  5. Maple Leaf  Cytotoxicity of Korean Fermented Extracts

B16F10, a melanoma cell used in this experiment, was purchased from ATCC (USA). B16F10 cell culture was performed using DMEM medium added with 10% FBS and 1% penicillin / streptomycin (100 U / ml), and was passaged to 37 ℃, 5% CO ₂ incubator.

In order to measure cell viability, B16F10 cells were divided into 180 μl to 1 × 10 ⁵ cells / well in a 96-well plate, and the samples (foliar leaf and Forget-me-not fermented extract) were prepared by concentration and added 20 μl, respectively, at 37 ° C. Incubated for 24 hours in a 5% CO ₂ incubator. 40 μl of the MTT solution prepared at a concentration of 2.5 mg / ml was added thereto 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 the absorbance was measured at 540 nm using an ELISA reader. Cell viability was measured as in Equation 4 below, and the results are shown in FIG. 4.

[Equation 4]

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

As shown in Figure 4, the maple leaf botanical herb and Forget-me-not fermented extract showed no cytotoxicity such as a cell viability of about 97% up to a high concentration of 500μg / ml.

Example 6 Confirmation of Inhibitory Activity of Pigmentation Related Factors of Maple Leaf Porcine and Fermented Forage Extract

6-1. Confirmation of Inhibitory Activity of Pigmentation Related Factor Proteins

In order to investigate the activity of pigmentation and whitening factors such as MITF, TRP-1, TRP-2, tyrosinase, etc. at the protein level, Maple leaf and Forsythia fermented extracts of B16F10 cells at 25, 50, 100 μg / ml Treatment was done by concentration. The control group was treated with α-MSH and the overexpression of melanin, and the normal group was set with no treatment with α-MSH. At this time, the expression level of β-actin, which is a housekeeping gene having almost no difference in expression level even under various conditions of cells, was identified and quantified.

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

As shown in Figure 5, it was confirmed that the amount of protein expression of MITF, TRP-1, TRP-2 and tyrosinase increased by α-MSH was reduced by the treatment of maple leaf and forage fermented extract.

6-2. MRNA expression of pigmentation related factors

B16F10 cells were seeded in a 100 mm culture dish, and then treated with concentrations of maple leaf and forage fermented extract, and incubated 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 13,200rpm for 20 minutes, the supernatant was transferred to a tube containing 0.5 mL of isopropanol. After centrifugation at 13,200rpm for 20 minutes, the supernatant was removed, and 1 mL of 75% EtOH-DEPC water was dispensed into each tube and centrifuged at 13,200rpm for 5 minutes, and then the supernatant was removed and dried at room temperature. . 50 μL of DEPC was dispensed and dissolved, and then 5 μL of RNA and 195 μL of sterile water were added to a 96-well plate, and the absorbance was measured at 260 and 280 nm, respectively. 1 μL of Oligo (dT) 15 primer (500 μg / mL), 10 μL of extracted RNA (2 μg) and nuclease free water, and reacted at 75 ° C. for 5 minutes, followed by 5X reaction buffer, MgCl ₂ , PCR nucleotide mix, RNasin The cDNA was synthesized by adding the inhibitor, reverse transcriptase and nucleated free water at 5 ° C. for 5 minutes, at 42 ° C. for 60 minutes, and at 70 ° C. for 15 minutes.

In order to confirm mRNA expression for MITF and tyrosinase, a real time polymerase chain reaction (RT-PCR) was performed. PCR was performed by adding 5 × green Go Taq Flexi buffer, MgCl ₂ , PCR nucleotide mix (10 mM), primers, Go Taq DNA polymerase, nucleated free water, and cDNA synthesized above to the PCR tube. At this time, PCR was performed for 35 cycles of 94 ° C. 30 seconds, 56 ° C. 60 seconds, and 72 ° C. for 1 minute. After completion of PCR, PCR products were added to 1.5% agarose gel to which 0.002% EtBr was added and electrophoresed at 100V for 40 minutes. The band was then quantitatively analyzed using a UV penetrometer. Primers used to perform RT-PCR are shown in Table 2, and the results of confirming mRNA expression of pigmentation related factors are shown in FIG.

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 direction AGC GTG TAT TTT CCC CAC AG Reverse TAG CTC CTT AAT GCG GTC GT Tyrosinase Forward direction GAC GGT CAC TGC ACA CTT TG Reverse GCC ATG ACC AGG ATG AC

As shown in FIG. 6, it was confirmed that mRNA expression of MITF and tyrosinase was significantly inhibited as the concentrations of the maple leaf and Ga. Fermented extracts increased.

Example 7 Comparison of Physiological Activities of Fermented Extracts from Maple Leaf and Forget-weed

7-1. Manufacture of Maple Leaf Pigweed or Ganoderma Lucidum Extract

Cut the maple leaf pork to 1-3cm, mixed and pulverized to 1,300 mesh. The pulverized maple leaf was sterilized for 30 minutes at 127 ℃. The sterilized maple leaf ragweed was transferred to an extraction flask equipped with a vertical reflux condenser, and 10 times distilled water of the sample weight was added as a solvent, followed by performing hot water extraction three times at 100 ° C. for 60 minutes to prepare a maple leaf extract.

Cauliflower extract was prepared in the same manner as the maple leaf ragweed extract.

7-2. Electron donating ability  Measure

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

As shown in FIG. 7, the maple leaf ragweed and Ganoderma lucidum fermented extract prepared in Example 1 were confirmed to have excellent electron donating ability as compared to the maple leaf ragweed extract or Gazelweed extract alone. In other words, compared to maple leaf ragweed or gazelweed alone, maple leaf ragweed and fermented green leaf extract obtained by mixing and fermenting maple leaf ragweed and Gazelweed were confirmed that the physiological activity effect was remarkably excellent by synergistic effect of the two substances. .

7-3. Elastase inhibitory activity

In the same manner as in Example 4 was compared with the elastase inhibitory activity of the maple leaf ragweed extract, Gaechocho extract and the maple leaf ragweed and Gaecho fermented extract prepared in Example 1, the results are shown in FIG.

As shown in FIG. 8, the maple leaf ragweed and Ganoderma lucidum fermentation extract prepared in Example 1 were found to have an excellent elastase inhibitory activity, that is, wrinkle improvement, as compared to the maple leaf ragweed extract or Gazelweed extract alone.

Based on the above experimental results, the present inventors have shown that the fermented maple leaf and Forget-me-not fermented extracts inhibit the activity of tyrosinase, inhibit the expression of pigmentation-related factors, inhibit the activity of elastase, and the like. It was confirmed that the whitening and wrinkle improvement effect is excellent. In particular, the maple leaf ragweed and Gaechofer fermented extract according to the present invention is significantly superior to the maple leaf ragweed or Gazelweed extract alone, it can be used in a variety of cosmetics, skin external preparations and food fields for skin whitening or wrinkle improvement have.

Hereinafter, the present invention will be described in more detail with reference to the 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 Cosmetic Formulation

1-1. Flexible Cosmetics Manufacturing

0.1% by weight of Maple Leaf and Fermented Forage 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, 2.0% by weight of benzophenone-9, 1.0% by weight of carboxyvinyl polymer, 3.5% by weight of glycerin, trace amount of fragrance, trace amount of preservative, and residual amount of purified water were mixed to prepare flexible cosmetic water in a conventional manner.

1-2. Wheat Crop Maker

0.1% by weight of Maple Leaf and Fermented Fermented Leaf, Glycerin 5.1%, Propylene Glycol 4.2%, Tocopheryl Acetate 3.0%, Liquid Paraffin 4.6%, Triethanolamine 1.0%, Squalane 3.1%, Macadamia Nut Oil 2.5 Weight%, polysorbate 60 1.6% by weight, sorbitan sesquirrolate 1.6% by weight, propyl paraben 0.6% by weight, carboxyvinyl polymer 1.5% by weight, trace fragrance, trace amount of preservative, the balance of purified water The milk lotion was prepared by the method.

1-3. Nutrition Cream

0.5% by weight of Maple Leaf and Fermented Fermented Leaf, 4.0% by weight of Glycerin, 3.5% by weight of Vaseline, 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, A nutrition cream was prepared in a conventional manner by mixing 3.2% by weight of polysorbate 60, 1.0% by weight of carboxyvinyl polymer, 3.1% by weight of sorbitassesquioleate, a small amount of fragrance, a small amount of preservative, and a residual amount of purified water.

1-4. Massage cream manufacturer

0.5% by weight of Maple Leaf and Fermented Forage Extract, 4.0% by weight of glycerin, 3.5% by weight of petroleum jelly, 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, Massage cream was prepared in a conventional manner by mixing 2.4% by weight of polysorbate 60, 1.0% by weight of carboxyvinyl polymer, 2.3% by weight of sorbitassesquioleate, a small amount of fragrance, a small amount of preservative, and a residual amount of purified water.

1-5. Manufacture of Body Cleanser for Cleaning

Fermented extract of Maple Leaf and Ganoderma lucidum 1g, Anionic surfactant 18g, Nonionic surfactant 5g, Glycerin 7g, Sodium chloride 3g, Natural olive liquid soap 1.5g, Flavor 1g and Water 100g Was prepared.

As mentioned above, specific parts of the present invention have been described in detail, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

A cosmetic composition for improving skin whitening or wrinkles comprising maple leaf ( Ambrosia trifida ) and Gaechocho ( Annual fleabane ) fermented extract as active ingredients.
According to claim 1, The fermentation is made of a strain of the genus Lactobacillus, cosmetic composition for skin whitening or wrinkle improvement.
The method of claim 2,
The fermentation will be made after adding hot spring water to the maple leaf ragweed and Gaechocho mixture, skin cosmetic or wrinkle improvement cosmetic composition.
The method of claim 3,
The hot spring water is pH 7 to 9, cosmetic composition for improving skin whitening or wrinkles.
The method of claim 4, wherein
The hot spring water is potassium 1-2 mg / L, sodium 40-50 mg / L, calcium 5-10 mg / L, silicon 20-30 mg / L, lithium 0.05-0.2 mg / L, strontium 0.1-0.5 mg / L and magnesium 0.1 Containing to 0.4 mg / L, cosmetic composition for improving skin whitening or wrinkles.
The method of claim 1,
The extract is extracted with water, one or more solvents selected from alcohols having 1 to 4 carbon atoms and mixed solvents thereof, cosmetic composition for skin whitening or wrinkle improvement.
Skin whitening or anti-wrinkle composition for improving skin whitening or wrinkles including fermented maple leaf and fermented forage.
Food composition for skin whitening or wrinkle improvement comprising the maple leaf botanical herb and Forget-me-not fermented extract as an active ingredient.

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