KR20210068197A - Mugwort fermentation product and a manufacturing method for improving the active ingredient content of the mugwort fermentation produced therefrom, and a cosmetic composition comprising a composition and fermented product effective in improving skin wrinkles and anti-aging containing the same as an active ingredient - Google Patents

Abstract

Disclosed is a cosmetic composition capable of inhibiting the activity of a collagen degrading enzyme (Matrix Metalloproteinase-1), promoting the generation of intracellular collagen, and proliferating epidermal cells. The present invention provides a cosmetic composition comprising a mugwort fermented product or a purified mugwort fermented product as an active ingredient.

Description

A manufacturing method for improving the active ingredient content of a mugwort fermented product and a fermented mugwort produced therefrom, and a composition containing the same as an active ingredient to improve skin wrinkles and anti-aging and a cosmetic composition comprising a fermented product manufacturing method for improving the active ingredient content of the mugwort fermentation produced therefrom, and a cosmetic composition comprising a composition and fermented product effective in improving skin wrinkles and anti-aging containing the same as an active ingredient}

The present invention relates to a cosmetic composition, and more particularly, to a cosmetic composition comprising a fermented mugwort fermented product or purified mugwort fermented product, and a method for producing a fermented mugwort fermented product and purified mugwort fermented product.

One of the biggest areas of interest in cosmetics is aging-related wrinkles, pigmentation, loss of elasticity, dry skin, hair loss, and lack of luster of hair. The skin undergoes various changes through aging. First, the thickness of the epidermis, dermis, and subcutaneous tissue, which are components of the skin, becomes thinner and the extracellular matrix (ECM) component that gives elasticity to the skin changes. With age, its production decreases rapidly, and it has a close relationship with wrinkle formation, and collagen, elastin, proteoglycans, and glucosaminoglycans that make up the skin connective tissue , laminin, fibronectin, etc. are oxidized and lose their functions, so that the skin loses elasticity and wrinkles are excessively formed, changing into senile skin.

The connective tissue fibers of the extracellular matrix include glue fibers (collagen fibers), reticular fibers, and elastic fibers (elastic fibers), of which collagen accounts for about 70% of the skin connective tissue. Most are formed in fibroblasts of the skin. Collagen content in the skin connective tissue decreases with age, which is due to a decrease in collagen synthesis and promotion of decomposition. Therefore, the decrease in collagen biosynthesis and the promotion of collagen decomposition are the biggest causes of skin wrinkles.

The process of collagen biosynthesis is regulated by many factors involved in transcription level and post-translation level, and collagen degradation is caused by collagen degradation by ultraviolet rays. Collagen decomposition is promoted by the promotion of expression of matrix metalloproteases (MMP) such as collagenase, thereby reducing the collagen content. In addition, as the transformation of collagen is accelerated by the external environment, the skin becomes wrinkled and deep. As a result, skin aging is caused by non-homogeneity of cells, loss of elastin, destruction of collagen, reduction and delay of collagen synthesis, and the like. Therefore, although skin aging occurs in the epidermis, it can be seen as a phenomenon that occurs in the dermis rather than this.

Various cosmetic compositions are being studied to solve the problems of skin aging, and the effect of improving skin wrinkles is showing visible results in some cases. For example, clinical results of skin wrinkle removal for retinoids, which are widely used to improve skin wrinkles, blemishes, and pigmentation, among others, retinol have been reported variously, and cosmetics containing retinol are used for skin wrinkles formed by sunlight. It effectively improved skin sagging and loss of elasticity. Retinol is a raw material whose effectiveness has been verified enough to be registered as a raw material for wrinkle function, but it has been constantly raised about specific skin irritation and stability issues. It is used in expensive products at a high price and is difficult to use as a universal material. part exists.

Therefore, in order to effectively prevent or improve skin aging and wrinkles, it is necessary to develop a new antioxidant and anti-aging cosmetic composition using raw materials whose antioxidant efficacy has been confirmed and raw materials whose effects have been accurately verified for preventing skin aging.

Registered Patent No. 10-1784201 discloses a composition for improving skin comprising a fermented mugwort extract as an active ingredient, but does not mention improving the efficacy of a fermented mugwort fermented product.

Accordingly, the present invention has been devised to solve the above problem, and provides a cosmetic composition capable of inhibiting the activity of a collagen degrading enzyme (Matrix Metalloproteinase-1), promoting the generation of intracellular collagen, and proliferating epidermal cells want to

In order to solve the above problems, the present invention provides a cosmetic composition comprising a mugwort fermented product or a purified mugwort fermented product as an active ingredient.

In addition, the mugwort fermented product provides a cosmetic composition characterized in that it is fermented with a Bacillus sp . strain, a Lactobacillus sp . strain, or a mixed strain thereof.

In addition, the Bacillus genus ( Bacillus sp . ) The strain is Bacillus methylotrophicus ( Bacillus methylotrophicus ) or Bacillus subtilis ( Bacillus subtilis ), and the Lactobacillus genus ( Lactobacillus sp . ) The strain is Lactobacillus paracasei ( Lactobacillus paracasei ) ) or Lactobacillus acidophilus (Lactobacillus acidophilus) provides a cosmetic composition, characterized in that.

In addition, the purified mugwort fermented product provides a cosmetic composition, characterized in that it is a fraction separated by sequential solvent fractionation of the mugwort fermented product with hexane, dichloromethane, ethyl acetate, butanol and water.

In addition, the cosmetic composition provides a cosmetic composition comprising 1 to 90% by weight of the mugwort fermented product or the purified mugwort fermented product.

In addition, the cosmetic composition provides a cosmetic composition characterized in that it inhibits the activity of collagen degrading enzyme (Matrix Metalloproteinase-1), promotes intracellular collagen production, and promotes the proliferation of epidermal cells.

In addition, in order to solve another problem, the present invention comprises the steps of preparing a fermented product by inoculating and fermenting the mugwort extract with microorganisms; and removing the microorganisms from the fermented product; provides a method for producing a mugwort fermented product comprising.

In addition, in order to solve another problem, the present invention comprises the steps of preparing a fermented product by inoculating and fermenting the mugwort extract with microorganisms; removing the microorganisms from the fermented product to prepare a mugwort fermented product; and solvent-fractionating the mugwort fermented product.

The present invention provides a cosmetic composition capable of inhibiting the activity of a collagen-degrading enzyme (Matrix Metalloproteinase-1), promoting the generation of intracellular collagen, and proliferating epidermal cells by including a fermented mugwort fermented product and a purified mugwort fermented product as an active ingredient can provide

1 is a schematic diagram of a method for producing a purified mugwort fermented product according to the present invention;
2 is a graph showing the quantitative value of polyphenol of Experimental Example 2 of the present invention;
3 is a photograph taken of the TLC result of Experimental Example 3 of the present invention;
4 is a graph showing the MMP-1 inhibitory ability measurement result of Experimental Example 4 of the present invention;
5 is a graph showing the results of the intracellular collagen production promoting effect of Experimental Example 5 of the present invention;
6 and 7 are graphs showing the light stability and thermal stability measurement results of Experimental Example 6 of the present invention;
8 and 9 are graphs showing the results of measuring the cell proliferation capacity of Experimental Example 7 of the present invention in HDF cells and HACAT cells.

Hereinafter, the present invention will be described in detail through preferred embodiments. Prior to this, the terms or words used in the present specification and claims should not be construed as being limited to conventional or dictionary meanings, and the inventor should properly understand the concept of the term in order to best describe his invention. Based on the principle that can be defined, it should be interpreted as meaning and concept consistent with the technical idea of the present invention. Therefore, since the configuration of the embodiments described in the present specification is only the most preferred embodiment of the present invention and does not represent all the technical spirit of the present invention, various equivalents and modifications that can be substituted for them at the time of the present application It should be understood that there may be

In order to effectively prevent or improve skin aging and wrinkles, the present inventors have repeatedly studied to develop raw materials with confirmed antioxidant efficacy and cosmetic compositions effective for skin aging, as a result of which the mugwort extract is fermented or purified after fermentation In this case, it was found that the efficacy of inhibiting the activity of Mugwort's collagen degrading enzyme (Matrix Metalloproteinase-1), promoting intracellular collagen production, and promoting epidermal cell proliferation could be improved, leading to the present invention.

Accordingly, the present invention discloses a cosmetic composition comprising a mugwort fermented product or a purified mugwort fermented product as an active ingredient.

In the present invention, the mugwort ( Artemisia Vulgaris ) is a perennial herb belonging to the Asteraceae (Compositae), and about 300 species of plants of the genus Artemisia reported so far are native to Korea. In the private sector, mugwort has been used for the treatment of bruises caused by poisonous insect bites, eczema, and wounds. Also, mugwort has been widely cultivated for a long time because it is suitable for domestic soil and climatic conditions to the extent that it appears in the myth of Dangun in Korea. In particular, antioxidant activity, anti-wrinkle improvement, antibacterial, etc. have been reported to exert various effects in cosmetic compositions.

In addition, it is confirmed that the physiologically active effect of mugwort is due to the various polyphenols and flavonoids contained therein. Esculin, which is present in mugwort, is a type of flavonoid and has the activity of vitamin P and absorbs ultraviolet rays. and shows fluorescence when irradiated with 365 nm UV light. In addition, esculin is known as an inhibitor of 5-α-reductase and 12-lipoxygenase with antioxidant action, and leukotriene through the pathway of lipoxygenase. and 5-hydroxyeicosa tetraenoic acid, reducing inflammation by interfering with the production of vasoactive substances.

The present invention provides a method for producing a fermented mugwort fermented product in another aspect, and a method for producing a purified mugwort fermented product in another embodiment.

The method for producing a mugwort fermented product includes the steps of inoculating a mugwort extract with microorganisms and fermenting it to prepare a fermented product; and removing the microorganisms from the fermented product; the method for producing a purified mugwort fermented product includes: inoculating and fermenting the mugwort extract with microorganisms to prepare a fermented product; removing the microorganisms from the fermented product to prepare a mugwort fermented product; and solvent fractionating the mugwort fermented product.

Hereinafter, the present invention will be described in detail through the method for producing the purified mugwort fermented product.

In the present invention, the step of inoculating the mugwort extract with microorganisms and fermenting it to prepare a fermented product is a step for increasing the content of functional substances in mugwort.

The mugwort extract may be prepared using a conventional extraction method known in the art, for example, a solvent extraction method. The extraction solvent used in the preparation of the mixed extract using the solvent extraction method is water, a lower alcohol having 1 to 4 carbon atoms (eg, methanol, ethanol, propanol and butanol) or a mixture thereof, which is a hydrous lower alcohol, propylene glycol, 1, 3-butylene glycol, glycerin, acetone, diethyl ether, ethyl acetate, butyl acetate, dichloromethane, dichloromethane, hexane, and mixtures thereof may be selected from the group consisting of, and selected from water, alcohol, or mixtures thereof Preferably, it may be hot water extraction using hot water more preferably. In addition, when alcohol is used as the extraction solvent, the alcohol is preferably a lower alcohol having 1 to 4 carbon atoms, and the lower alcohol is more preferably selected from methanol or ethanol.

In addition, when extracting the mugwort extract by hot water extraction, for example, purified water can be added in an amount 5 to 20 times the weight of mugwort leaves, and extracted at 100 to 140° C. for 4 to 8 hours, The sugar content may be 4 to 10 brix, preferably 6 to 8 birx.

In the present invention, the microorganism is a Bacillus genus ( Bacillus sp . ) strain, Lactobacillus sp . It may be fermented as a strain or a mixture thereof, and the Bacillus sp . strain is Bacillus methylotropicus ( Bacillus methylotrophicus ) Or Bacillus subtilis ( Bacillus subtilis ) It may be, the Lactobacillus genus ( Lactobacillus sp . ) The strain is Lactobacillus paracasei ( Lactobacillus paracasei ) or Lactobacillus acidophilus ( Lactobacillus acidophilus ) It may be , preferably a mixed strain of Bacillus subtilis and Bacillus methylotropicus.

The fermentation refers to a process in which enzymes possessed by microorganisms change raw materials to create beneficial ingredients, and during the fermentation process, active ingredients are extracted and the content of functional substances increases. In addition, in the present invention, the fermentation can be performed ^{by adjusting the culture medium of the microorganism strain to 10 7} to 10 ¹⁰ cfu/ml, and inoculating 0.1 to 10% by weight, preferably 0.5 to 5% by weight of the mugwort extract, , 1 to 5 days, preferably 2 to 4 days, 25 to 45 ℃, preferably 30 to 40 ℃ can be carried out.

In addition, the medium composition during the microbial culture and fermentation may be glucose 0.2 to 1% by weight, enzyme extract 0.1 to 0.5% by weight, soytone 0.05 to 0.3% by weight, and distilled water (residual amount).

In the present invention, the step of preparing a mugwort fermented product by removing the microorganism from the fermented product is a step of removing microorganisms to prevent further fermentation of the mugwort fermented product, and the microorganism removal may be removed through centrifugation and filtration, , For example, after separating the microorganisms through a centrifuge in the fermented product, it is possible to remove the microorganisms by filtering with a 0.1 ~ 0.3 ㎛ filter.

1 is a schematic diagram of a method for producing a purified mugwort fermented product according to the present invention.

Referring to FIG. 1 , the step of solvent fractionating the mugwort fermented product is a step for purifying the mugwort fermented product to prepare a fraction with a high polyphenol content. The solvent fraction is hexane, dichloromethane, ethyl acetate, butanol and water. It may be performed sequentially.

In addition, in the present invention, the purified mugwort fermented product may be a fraction separated by solvent fractionation of the mugwort fermented product, preferably an ethyl acetate fraction, wherein the ethyl acetate fraction is preferably eluted from a silica gel column.

In the present invention, the cosmetic composition may contain 1 to 90% by weight of the mugwort fermented product or the fermented product, preferably 1 to 20% by weight. In addition, the cosmetic composition may include the mugwort fermented product and the fermented purified product, and may each contain 1 to 20% by weight.

It was confirmed that the cosmetic composition according to the present invention has a very excellent effect of inhibiting the activity of collagen-degrading enzymes such as collagenase and MMP-1 (Matrix Metalloproteinase-1), and also has an effect of promoting intracellular collagen production and skin It was confirmed that the effect of improving the cell regeneration ability was also very excellent.

The cosmetic composition according to the present invention may be applied in a gel type, a skin type, a cream type, an ointment type, etc., but is not limited thereto. The above composition can be suitably prepared by a known method by adding an appropriate conventional softening agent, emulsifying agent, thickening agent or other substances known in the art according to its type.

The gel-type composition may be prepared by adding an emollient such as trimethylolpropane, polyethylene glycol, or glycerin, a solvent such as propylene glycol, ethanol, or isostatic alcohol, and purified alcohol.

The skin type composition includes fatty alcohols such as stearyl alcohol, myristyl alcohol, behenyl alcohol, arachidyl alcohol, isostearyl alcohol, isocetyl alcohol, butylene glycol, glycerin, allantoin, methyl paraben, EDTA- It can be prepared by adding 2-sodium, xanthan gum, dimethicone, polyethylene glycol-60 hydrogenated castol oil, polysorbate 60 and purified water.

The cream-type composition comprises fatty alcohols such as stearyl alcohol, myristyl alcohol, behenyl alcohol, arachidyl alcohol, isostearyl alcohol, isocetyl alcohol, lecithin, phosphatidylcholine, phosphatidylethanolamine, sofatizylserine, sofphatidyl. Lipids such as inositol, derivatives thereof, emulsifiers such as glyceryl stearate, sorbitan palmitate, and sorbitan stearate, natural fats or oils such as avocado oil, apricot oil, babassu oil, lucidium oil, camellia oil, It can be prepared by adding a solvent such as propylene glycol and purified water.

The ointment-type composition may be prepared by adding a softening agent, an emulsifying agent, and waxes such as microcrystalline lead, paraffin, ceresin, beeswax, beeswax, and vaseline.

The cosmetic composition of the present invention may further contain one or more skin wrinkle-improving ingredients exhibiting the same or similar function in addition to the active ingredient. The skin wrinkle improvement component may be any one or more selected from the group consisting of vitamin C, retinoic acid, TGF, animal placental-derived protein, betulinic acid, and chlorella extract, but is not limited thereto. In addition, the cosmetic composition of the present invention may further contain excipients including fluorescent substances, fungicides, hydrotropes, humectants, fragrances, fragrance carriers, proteins, solubilizers, sugar derivatives, sunscreens, vitamins, plant extracts, etc. can

In the present invention, the cosmetic composition is a softening lotion, astringent lotion, nourishing lotion, eye cream, serum, nourishing cream, massage cream, cleansing cream, cleansing lotion, cleansing foam, cleansing water, powder, essence, pack, hair tonic, hair treatment It can be formulated as a conditioner, shampoo or rinse.

The cosmetic composition may be formulated by a conventional method. In the formulation of external skin preparations, reference may be made to the contents disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA, and in the formulation of cosmetic compositions, the International cosmetic ingredient dictionary, 6th ed (The cosmetic, Toiletry and Fragrance Association) , Inc, Washington, 1995) may be referred to.

Specifically, the cosmetic composition can be prepared in general emulsified formulations and solubilized formulations. For example, lotion, such as a softening lotion or a nourishing lotion; emulsions such as facial lotions and body lotions; creams such as nourishing creams, moisturizing creams, and eye creams; essence; makeup ointment; spray; gel; pack; sunscreen; makeup base; foundations such as liquid type, solid type or spray type; powder; makeup removers such as cleansing creams, cleansing lotions, and cleansing oils; Alternatively, it may be formulated as a cleaning agent such as a cleansing foam, soap, body wash, but is not limited thereto. In addition, the external preparation for skin may be formulated as an ointment, patch, gel, cream or spray, but is not limited thereto.

In the cosmetic composition, in addition to the essential components in each formulation, other components may be appropriately blended within the range that does not impair the purpose according to the present invention, depending on the type of formulation or purpose of use.

The cosmetic composition may include a conventionally acceptable carrier, for example, oil, water, surfactant, humectant, lower alcohol, thickener, chelating agent, colorant, preservative, fragrance, etc. may be appropriately mixed, but is limited thereto no.

The acceptable carrier may vary depending on the formulation. For example, when formulated into 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 their Mixtures may be used.

When the cosmetic composition is formulated as a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, or mixtures thereof may be used as a carrier component, and in the case of a spray, chlorofluoro It may further comprise a propellant such as hydrocarbon, propane, butane or dimethyl ether.

When the cosmetic composition is formulated as a solution or emulsion, a solvent, solubilizer, or emulsifier may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl benzoate, propylene glycol, 1,3-Butylglycol oil may be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol fatty esters, fatty acid esters of polyethylene glycol or sorbitan may be used.

When the cosmetic composition is formulated as a suspension, a liquid diluent such as water, ethanol or propylene glycol as a carrier component, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester; Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth and the like can be used.

The cosmetic composition may contain fatty substances, organic solvents, solubilizers, thickeners, gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, and fragrances commonly used in the industry depending on the quality or function of the final product. , surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, commonly used in cosmetics It may additionally contain adjuvants commonly used in cosmetic or dermatological fields, such as any other ingredients. However, the adjuvant and its mixing ratio may be appropriately selected so as not to affect the desirable properties of the cosmetic composition according to the present invention.

The cosmetic composition comprising the mugwort fermented product or purified mugwort fermented product prepared according to the present invention as an active ingredient inhibits the activity of collagen degrading enzyme (Matrix Metalloproteinase-1) by including the mugwort fermented product or purified mugwort fermented product, and It promotes the production of inner collagen and can proliferate epidermal cells.

Hereinafter, specific examples according to the present invention will be described.

Preparation Example 1

After adding 2 kg of purified water to 200 g of dried mugwort leaves, hot water extraction was performed at 120° C. for 6 hours. After extraction, mugwort extract having a sugar content of 6-8 birx was used. Extracted after Lactobacillus para Kasei to (Lactobacillus paracasei, purchased from Korea Cell Line Bank) culturing the strain and has a density measured with a spectrophotometer in the mugwort extract inoculated with 1.0 × 10 ⁹ cfu / ml of culture solution 1% by weight, from 37 ℃ 3 It was fermented for one day, and the medium used for the culture was a medium composed of glucose 0.6% by weight, enzyme extract 0.3% by weight, soyton 0.1% by weight, and distilled water. After fermentation, the fermented broth was centrifuged at 4000 rpm for 10 minutes to separate lactic acid bacteria and the culture broth, and then the culture broth was filtered through a 0.2 μm filter to prepare a mugwort fermented product.

Preparation 2

A fermented mugwort was prepared in the same manner as in Preparation Example 1, except that Lactobacillus acidophilus (purchased from Korea Cell Line Bank) was used as a microorganism.

Preparation 3

As a microorganism in Preparation Example 1, Lactobacillus paracasei (Lactobacillus paracasei, purchased from Korea Cell Line Bank) and Lactobacillus acidophilus (Lactobacillus acidophilus, purchased from Korea Cell Line Bank) were mixed in 1:1 parts by weight using a mixed strain. Except for that, fermented mugwort was prepared in the same manner.

manufacturing example 4

As a microorganism in Preparation Example 1, Bacillus methylotrophicus, Apep microrganism S001, Preparation Example 4

As a microorganism in Preparation Example 1, Bacillus methylotrophicus (Bacillus methylotrophicus, Apep Microrganism S001 ( purchased from Korea Cell Line Bank) was used to prepare fermented mugwort in the same manner.

Preparation 5

A fermented mugwort fermented product was prepared in the same manner as in Preparation Example 1, except that Bacillus subtilis ( Apep microrganism K007, purchased from Korea Cell Line Bank) was used as a microorganism.

Preparation 6

As a microorganism in Preparation Example 1, Bacillus methylotrophicus (Bacillus methylotrophicus, Apep microrganism S001, purchased from Korea Cell Line Bank) and Bacillus subtilis ( Apep ) A fermented mugwort fermented product was prepared in the same manner except that a mixed strain mixed with microrganism K007 (purchased from the Korea Cell Line Bank) in 1:1 parts by weight was used.

Preparation 7

2 kg of 70% (v/v) ethanol was added to 200 g of dried mugwort leaves and extracted at 25° C. for 24 hours. After extraction, mugwort extract having a sugar content of 6-8 birx was used. Extracted after Lactobacillus para Kasei to (Lactobacillus paracasei, purchased from Korea Cell Line Bank) culturing the strain and has a density measured with a spectrophotometer in the mugwort extract inoculated with 1.0 × 10 ⁹ cfu / ml of culture solution 1% by weight, from 37 ℃ 3 It was fermented for one day, and the medium used for the culture was a medium composed of glucose 0.6% by weight, enzyme extract 0.3% by weight, soyton 0.1% by weight, and distilled water. After fermentation, the fermented broth was centrifuged at 4000 rpm for 10 minutes to separate lactic acid bacteria and the culture broth, and then the culture broth was filtered through a 0.2 μm filter to prepare a mugwort fermented product.

Preparation 8

A fermented mugwort fermented product was prepared in the same manner as in Preparation Example 7, except that Lactobacillus acidophilus (purchased from Korea Cell Line Bank) was used as a microorganism.

Preparation 9

In Preparation Example 7, Lactobacillus paracasei (Lactobacillus paracasei, purchased from Korea Cell Line Bank) and Lactobacillus acidophilus (Lactobacillus acidophilus, purchased from Korea Cell Line Bank) as microorganisms in Preparation Example 7 were mixed in 1:1 parts by weight using a mixed strain. Except for that, fermented mugwort was prepared in the same manner.

Preparation 10

As a microorganism in Preparation Example 7, Bacillus methylotrophicus (Bacillus methylotrophicus, Apep Microrganism S001 ( purchased from Korea Cell Line Bank) was used to prepare fermented mugwort in the same manner.

Preparation 11

A fermented mugwort fermented product was prepared in the same manner as in Preparation Example 7, except that Bacillus subtilis ( Apep microrganism K007, purchased from Korea Cell Line Bank) was used as a microorganism.

Preparation 12

As a microorganism in Preparation Example 7, Bacillus methylotrophicus (Bacillus methylotrophicus, Apep microrganism S001, purchased from Korea Cell Line Bank) and Bacillus subtilis ( Apep ) A fermented mugwort fermented product was prepared in the same manner except that a mixed strain mixed with microrganism K007 (purchased from the Korea Cell Line Bank) in 1:1 parts by weight was used.

Preparation 13

2 kg of purified water was added to 200 g of dried mugwort leaves, and hot water extraction was performed at 120° C. for 6 hours to prepare a mugwort extract with a sugar content of 6-8 birx.

Preparation 14

2 kg of 70% (v/v) ethanol was added to 200 g of dried mugwort leaves and extracted at 25° C. for 24 hours to prepare a mugwort extract having a sugar content of 6-8 birx.

Preparation 15

n-hexane, dichloromethane, ethyl acetate and butanol were sequentially added to the mugwort fermented product prepared in Preparation Example 3, and separated into an n-hexane fraction, a dichloromethane fraction, an ethyl acetate fraction, a butanol fraction and a water fraction. Water was prepared.

Experimental Example 1

In order to confirm the skin wrinkle improvement effect of the mugwort extract prepared in Preparation Examples 1 to 14, an in vitro collagenase activity inhibition experiment was performed according to the following method, and the results are shown in Table 1 below. indicated.

<Test method>

(1) Prepare by dissolving collagen in phosphate buffer (100ppm). At this time, adenosine is prepared at the same concentration as a positive control.

(2) 100mM Tris-cl buffer, 100mM CaCl ₂ and 0.25 mg/ml of collagenase, prepare a mixture, and proceed the reaction by adding each of the test substances and the positive control to the mixture at a concentration of 5% (v/v).

(2) The reaction is terminated by adding 25 mM citric acid to 400 μl/tube.

(3) Thereafter, 1.5 ml/tube of ethyl acetate is added, and after vortexing, the supernatant is taken and transferred to sodium sulfate 150 mg/ep-tube, and centrifuged after vortexing (10,000 rpm, 3 minutes).

(4) Take 300 μl/ep-tube of the supernatant, transfer it to a quartz 96-well plate, and measure the absorbance at 320 nm.

Referring to Table 1, it can be seen that all fermented products (Preparation Examples 1 to 12) have a collagenase inhibitory ability, and it can be confirmed that the collagenase inhibitory ability is superior to that of the case without fermentation (Preparation Examples 13 and 14).

In addition, when using the Lactobacillus sp. strain (Preparation Examples 1 to 3 and Preparation Examples 7 to 9) than when using the Bacillus sp. strain (Preparation Examples 4 to 6 and Preparation Examples 10 to 12), the collagenase inhibitory ability is excellent, , when using a mixed strain (Preparation Examples 3, 6, 9 and 12) than when using a single strain (Preparation Examples 1, 2, 4, 5, 7, 8, 10 and 11) that the collagenase inhibitory ability is excellent Able to know.

In addition, it can be seen that the case of hot water extraction (Preparation Examples 1 to 6) is superior to the case of ethanol extraction (Preparation Examples 7 to 12) than the collagenase inhibitory ability.

Experimental example 2

In order to measure the polyphenol content of the mugwort fermented product of Preparation Example 3, the mugwort extract of Preparation Example 13, and the purified mugwort fermented product of Preparation Example 15, an experiment was conducted according to the following method, and the results are shown in Table 2 and FIG. 2 below. and the total polyphenol content was measured using the Folin-Denis method (Folin et al., 1912). The total phenol content was obtained from a standard curve using tannic acid as a standard material, and the result was measured three times and presented as an average value.

<Test method>

(1) Mix 5 ml of distilled water with 1 ml of each diluted sample, add 0.5 ml of Folin-Ciocalteu's phenol reagent, and then react at room temperature for 8 minutes.

(2) After the reaction, 10 ml of 7 wt% sodium carbonate was added, distilled water was added to prepare a final volume of 25 ml, and then left for 2 hours in a dark place at room temperature, followed by a spectrophotometer (BIO-TEK, PowerWave) HT)) to measure the absorbance at 760 nm.

Referring to Table 2 and FIG. 2, it can be seen that the quantitative value of polyphenol in the mugwort fermented product (Preparation Example 3) is increased by about 135% compared to the mugwort extract (Preparation Example 13), and the purified mugwort fermented product (Preparation Example 13) Example 15) can confirm that the polyphenol value of the ethyl acetate fraction is the highest.

In addition, it can be seen that the polyphenol content of the purified mugwort fermented product (Preparation Example 15, ethyl acetate) is more than twice that of the mugwort fermented product (Preparation Example 3).

Experimental Example 3

In order to analyze the material of the mugwort fermented purified product (ethyl acetate) according to Preparation Example 15, additional separation and purification were performed as follows, and the polyphenol content was measured according to the method of Experimental Example 2, and the result is shown. 3 and Table 3 below.

<Experiment method>

(1) The ethyl acetate fraction separated in Preparation Example 15 was subjected to silica column chromatography, in which case a silica column was used, and the mobile phase solvent was dichloromethane from 100% dichloromethane: ethyl acetate 90: 10 , 80: 20 was changed by 1L in the order, and a total of 5 fractions were separated, respectively, fr1(1~2), fr2(3~5), fr3(6~7), fr4(8~14) and isolated with fr5(15).

(2) Silica gel thin film chromatography (Thin Layer Chromatography, TLC) was performed on the separated fr3.

Referring to FIG. 3 , it can be seen that the compound of fr3 is a polyphenol, and referring to Table 3, it can be confirmed that the most polyphenol is present in fr3 among the ethyl acetate fractions.

Experimental example 4

Substances that inhibit the production of matrix methaloprotease-1 (MMP-1, collagenase) protect collagen and maintain the mechanical properties of the skin tissue to prevent elasticity and sagging of the skin. It can be evaluated that raw materials having activity by measuring can be usefully used in the development of cosmetics for wrinkle improvement and elastic skin. Therefore, the MMP-1 production inhibition ability test of the mugwort fermented product of Preparation Example 3, the mugwort extract of Preparation Example 13, and the mugwort fermented purified product of Preparation Example 15 was conducted in the following manner, and the results are shown in FIG. . Retinol was used as a control.

<Experiment method>

Human dermal fibroblasts (HDF) cells were cultured in DMEM (Dulbecco's modified Eagle's media, Sigma) in a medium supplemented with 10% fetal bovine serum (Sigma) at 37°C and 5% CO ₂ conditions. . After culturing the cells at a concentration of 2 X 10 ⁵ cells/well in a 24-well plate and confirming cell adhesion, the control group was treated with nothing and only a solvent was added, and retinol was added to the dish at 10 ug/ml for the experimental group and control group. It was processed so that it became a density|concentration. After adding the test substance to each dish, it was cultured for 2 days. After 2 days, the medium was collected and the inhibition of MMP-1 production was measured using Matrix Metalloproteinase-1 (MMP-1), Human, biotrak, and ELISA kit (GE healthcare RPN2610).

Referring to FIG. 4 , it was confirmed that the expression amount of MMP-1 generated in the cells decreased in most of the experimental groups, and it was confirmed that there was a difference depending on the composition in the experimental group as well. In particular, it seems that the substance active in inhibiting MMP-1 expression is most distributed in the ethyl acetate fraction of the mugwort fermented purified product (Preparation Example 15), and the overall efficacy is judged to be proportional to the content of polyphenols in the fraction. From the results, when UV that induces MMP-1 production is applied to the skin as a factor, it is more useful to use a mixture of mugwort extract and mugwort fermented purified product to inhibit MMP-1 production and inhibit skin aging of the skin. It is presumed that it can

Experimental example 5

The fermented mugwort fermented product of Preparation Example 3, the purified mugwort fermented product (ethyl acetate) of Preparation Example 15, and the intracellular collagen production ability of fr1-5 isolated in Experimental Example 3 were performed in the following manner, and the results were 5 is shown. Ascorbic acid was used as a control.

<Experiment method>

Human dermal fibroblst (HDF) cells to be used for the test are medium 106 (cascade biologics, M-106-500) added with 1X LSGS (Low Serum Growth Supplement, cascade biologics S-003-10). 37 ℃, 5% CO ₂ Incubated under conditions. Cells were cultured in a 24-well plate at ^{a concentration of 1X 10 5} cells/well, cell adhesion was confirmed, and ascorbic acid was treated to a concentration of 10 μg/ml as an experimental group and a control group. After adding the test substance to each dish, it was cultured for 2 days. After 2 days, the medium was collected and the amount of collagen produced was measured using the Procollagen Type I C-peptide (hereinafter PIP) EIA kit (takara MK101).

Referring to FIG. 5 , it was confirmed that the amount of collagen production increased in all the experimental groups and the control group, and the purified case (Preparation Example 15, ethyl acetate, fr1-5) had a superior collagen production effect than the unrefined case (fermented mugwort). It can be confirmed that there is, and it can be seen that the most purified fr3 has the highest collagen production effect.

From these results, it is estimated that a large amount of ingredients that promote collagen production in the skin are contained in the polyphenol of mugwort, and it is believed that the cosmetic composition according to the present invention can inhibit skin aging by increasing collagen production.

Experimental example 6

In order to confirm the thermal and photostability of the mugwort fermented purified product (ethyl acetate fraction) prepared in Preparation Example 15, the experiment was conducted in the following manner, and the results are shown in FIGS. 6 and 7 .

<Experiment method>

Dissolve the purified mugwort fermented substance in 50 mM Tris-HCl (pH 8.0) buffer so that the purified mugwort fermented substance is 10 mg/mL, aliquot it into a glass vial, and store at 50°C for 7, 14, 21 and 28 days. Loss of polyphenols in the composition due to heat was measured and stability under sunlight conditions was measured in the same manner.

6 and 7, as a result of measuring polyphenol loss due to heat and measuring whether stability in sunlight conditions, it shows only less than 10% loss of fermentation purified product up to 28 days, high stability in heat and sunlight storage period It was confirmed that it has

Experimental example 7

In order to confirm the cell proliferation ability of the mugwort extract of Preparation Example 13, the mugwort fermented product of Preparation Example 3, the purified mugwort fermented product (ethyl acetate) of Preparation Example 15, and the cell proliferation ability of fr3 stopped in Experimental Example 3, the experiment was conducted as follows. and the results are shown in FIGS. 8 and 9 . Cell proliferation was measured using the MTT assay.

<Experiment method>

^{Fibroblasts (HDF) and HACAT cells were seeded to a cell number of 4 X 10 4} cells/24 well per well in a 24 well plate, and then incubated at 37° C. and 5% CO ₂ conditions for 24 hours. It was washed twice with PBS (phosphate buffer saline), and each sample was treated with each concentration (1mg~1ug/mL) and incubated for 24 hours. After incubation, 0.5% of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) in DPBS (Dulbecco's Phosphate-Buffered Saline) was mixed with the culture medium at 1:9 (v/v). After addition, incubate in a _{CO 2 incubator for 2 hours.} Then, the generated formazan (frmazan) was dissolved in DMSO (dimethyl sulfoxide) and absorbance was measured at 570 nm using ELISA.

8 and 9, for HACAT cells and fibroblasts (HDF), which are a kind of keratinocyte line, a decrease in the number of cells by the composition treated from a low concentration to a high concentration was not observed, and the cell proliferation was proportional to the concentration. Changes that promote In particular, it was confirmed that the cell proliferation effect was large in fibroblasts. The proliferation rate differed according to the purification of mugwort, and the highest cell proliferation effect was shown in fr.3, when the purified mugwort fermented product was once again purified, and no significant change was observed in the microscopic observation of the cells.

Through this, it can be seen that the cosmetic composition of the present invention has no toxicity to skin-related cells, and it appears to have a skin cell proliferation effect. Therefore, it can be predicted that even if the cosmetic composition of the present invention is treated on the skin, it will not have a significant effect.

< Example >

Each of the components listed in Table 4 was added according to the ratio described below based on 100% by weight of the cosmetic composition, and 1 to 20% by weight of fermented mugwort and purified mugwort was added to the cream type composition.

Preferred embodiments of the present invention described above are disclosed to solve the technical problem, and various modifications, changes, additions, etc. will be possible within the spirit and scope of the present invention by those skilled in the art to which the present invention pertains. , such modifications and changes should be regarded as belonging to the following claims.

Therefore, the scope of the present invention is indicated by the claims to be described later rather than the above detailed description, and all changes or modifications derived from the meaning, scope, and equivalent concept of the claims are included in the scope of the present invention. should be interpreted

Claims (8)

A cosmetic composition comprising a mugwort fermented product or purified mugwort fermented product as an active ingredient. According to claim 1,
The mugwort fermented product is a Bacillus genus ( Bacillus sp . ) A cosmetic composition, characterized in that it is fermented with a strain, a Lactobacillus sp ., a strain or a mixture thereof. 3. The method of claim 2,
The Bacillus genus ( Bacillus sp . ) The strain is Bacillus methylotrophicus ( Bacillus methylotrophicus ) or Bacillus subtilis ( Bacillus subtilis ), and the Lactobacillus genus ( Lactobacillus sp . ) The strain is Lactobacillus paracasei ( Lactobacillus paracasei ) or Lactobacillus acidophilus ( Lactobacillus acidophilus) Cosmetic composition, characterized in that. According to claim 1,
The cosmetic composition, characterized in that the purified mugwort fermented product is a fraction separated by sequential solvent fractionation of the mugwort fermented product with hexane, dichloromethane, ethyl acetate, butanol and water. According to claim 1,
The cosmetic composition comprises 1 to 90% by weight of the mugwort fermented product or the purified mugwort fermented product. According to claim 1,
The cosmetic composition inhibits the activity of a collagen degrading enzyme (Matrix Metalloproteinase-1), promotes intracellular collagen production, and promotes the proliferation of epidermal cells. preparing a fermented product by inoculating and fermenting the mugwort extract with microorganisms; and
removing the microorganisms from the fermented product;
A method for producing a mugwort fermented product comprising a. preparing a fermented product by inoculating and fermenting the mugwort extract with microorganisms;
removing the microorganisms from the fermented product to prepare a mugwort fermented product; and
solvent fractionating the mugwort fermented product;
A method for producing a purified mugwort fermented product comprising a.

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