KR20160074116A - Cosmetic composition containing Crataegis fructus fermented extract - Google Patents

Abstract

The present invention relates to a method for producing a fermented extract of Crataegus pinnatifida BUNGE, and to a functional cosmetic composition containing the fermented extract. The cosmetic composition of the present invention shows improved antioxidant ability by increasing elution of effective bioactive components such as polyphenol and flavonoid contained in Crataegus pinnatifida BUNGE, by extracting and fermenting Crataegus pinnatifida BUNGE by means of a skin-derived fermenting lactobacillus strain and intestinal lactobacillus. Thus, the composition exhibits effects such as skin antiinflammation, promotion of skin cell proliferation, wrinkle prevention, skin moisturization, and skin-whitening.

Description

The present invention relates to a cosmetic composition containing Crataegis fructus fermented extract as an active ingredient,

The present invention relates to a process for producing an acid fermentation extract and a functional cosmetic composition containing the fermentation extract.

Sansa is round and red in appearance, with white spots. Outer surface is yellowish brown or grayish reddish brown with many thin wrinkles. On one side there is a concave 5mm diameter, with occasional calyx around it, and on the other side there are scars or marks. On the side cut, there are 5 ovens with one seed in each room. The seeds are divided into several balls with a length of 6-8 mm, the outer surface is light brown, hard, and the quality is hard. Other names include Sansa, Sanri Hong, Red Pepper, Northern Sansa, Red Pepper, Sanzong Red, Red Pepper, Yanggu There are three kinds of characters: 羊 拳 子, billiard 棠 子, mosa, 羊 里 果, narrative, narrative, sheep, red 实, (), A child, and a plum (梅).

Sansa promotes digestion by warming the noodles. It is effective when it eats meat. It is used for abdominal pain, diarrhea, gastric hyperplasia, chronic enteritis. It also acts on the blood stream to help the flow of blood, eliminates the eosinophilia, and has been reported to have pharmacological actions such as arteriosclerosis, blood circulation improvement, and blood pressure reduction. Recently, polyphenols in Sansa have been reported to prevent skin cancer, and anti-inflammatory and antioxidant effects are known.

On the other hand, due to the development of fermentation engineering, there has been a growing interest in the industrial utilization of GRAS strains capable of fermenting natural materials, due to an increase in the conversion and content of active ingredients by fermentation, As a result, research and development on the fermentation technology of herbs and plant materials using the fraction and identification of lactic acid bacteria in the intestines have been actively carried out. However, as a result of investigating the domestic research trend of fermenting natural products by applying the fermentation process, there are limitations that the strains used are limited to lactobacillus or bifidus lactic acid bacteria, such as Bacillus subtilis and intestinal microorganisms used for fermenting chungkukjang.

Korea Patent Publication No. 2011-0057954

Accordingly, the present inventors have found that a mutant strain selected from the group consisting of Lactobacillus ramnosus HK-9 KCCM11254P, Lactobacillus acidophilus and Lactobacillus bulgaricus isolated from the human body The present invention has been accomplished by confirming that it is suitable for use as a cosmetic composition because it promotes cell proliferation, antioxidant efficacy, wrinkle prevention, and skin moisturizing effect when fermented with the above-mentioned lactic acid bacteria.

Therefore, an object of the present invention is to (a) anaerobically culturing lactic acid bacteria at a concentration of 3-7% (v / v) at a temperature of 34-40 ° C for 12-36 hours in a medium sterilized by a high pressure sterilizer; (b) anaerobically culturing the fermented product in an amount of 1: 8 to 1:12 based on the total weight of the culture medium and culturing the fermented product in the step (a) ; (c) The fermented broodstock fermented from the step (b) is separately collected, and the remaining fermented broodstock is prepared at a ratio of 1: 8 to 1:12 with respect to the total culture medium weight. Extracting the extract with shaking at a temperature of 70 ° C for 20 to 28 hours, filtering the extract with a filter paper to prepare an extract of Acanthopanax senticosus; And (d) mixing the fermented product of the horse mackerel obtained in the step (b) with the extract of the horse mackerel obtained in the step (c).

It is another object of the present invention to provide a cosmetic composition comprising the fermented sea urchin fermentation extract prepared by the method of the present invention as an active ingredient.

It is another object of the present invention to provide a cosmetic composition for preventing skin aging which comprises an acid-fast bacilli extract as an active ingredient.

Another object of the present invention is to provide a cosmetic composition for skin whitening comprising an acid-fast fermented extract as an active ingredient.

Another object of the present invention is to provide a cosmetic composition for improving skin wrinkles comprising an acid-licensed fermented extract as an active ingredient.

It is still another object of the present invention to provide a cosmetic composition having a skin anti-inflammatory effect or a skin moisturizing effect, which comprises an acid-fast bacilli extract as an active ingredient.

In order to accomplish the above object, the present invention provides a method for producing a microorganism, which comprises (a) inoculating a microbial strain sterilized with a high pressure sterilizer at a concentration of 3-7% (v / v) Culturing; (b) anaerobically culturing the fermented product in an amount of 1: 8 to 1:12 based on the total weight of the culture medium and culturing the fermented product in the step (a) ; (c) The fermented broodstock fermented from the step (b) is separately collected, and the remaining fermented broodstock is prepared at a ratio of 1: 8 to 1:12 with respect to the total culture medium weight. Extracting the extract with shaking at a temperature of 70 ° C for 20 to 28 hours, filtering the extract with a filter paper to prepare an extract of Acanthopanax senticosus; And (d) mixing the fermented product of the horse mackerel obtained in the step (b) with the extract of the horse mackerel obtained in the step (c).

In one embodiment of the present invention, the lactic acid bacterium of step (a) is selected from the group consisting of Lactobacillus ramnosus HK-9, KCCM11254P, Lactobacillus acidophilus , and Lactobacillus bulgaricus ) may be used.

The present invention provides a cosmetic composition comprising the granular extract obtained by the above method as an active ingredient.

In addition, the present invention provides a cosmetic composition for preventing skin aging comprising an acid-fast fermented extract as an active ingredient.

In one embodiment of the present invention, the fermented sea urchin fermentation extract is selected from the group consisting of Lactobacillus ramnosus HK-9 KCCM11254P, Lactobacillus acidophilus and Lactobacillus bulgaricus And may be at least one selected from the group consisting of

In addition, the present invention provides a cosmetic composition for skin whitening comprising an acid-fast fermented extract as an active ingredient.

In one embodiment of the present invention, the fermented sea urchin fermentation extract is selected from the group consisting of Lactobacillus ramnosus HK-9 KCCM11254P, Lactobacillus acidophilus and Lactobacillus bulgaricus And may be at least one selected from the group consisting of

The present invention also provides a cosmetic composition for improving skin wrinkles comprising an acid-fast berry extract as an active ingredient.

In one embodiment of the present invention, the fermented sea urchin fermentation extract is selected from the group consisting of Lactobacillus ramnosus HK-9 KCCM11254P, Lactobacillus acidophilus and Lactobacillus bulgaricus And may be at least one selected from the group consisting of

Further, the present invention provides a cosmetic composition having a skin anti-inflammatory effect or a skin moisturizing effect comprising an acid-fast bacilli extract as an active ingredient.

In one embodiment of the present invention, the fermented sea urchin fermentation extract is selected from the group consisting of Lactobacillus ramnosus HK-9 KCCM11254P, Lactobacillus acidophilus and Lactobacillus bulgaricus And may be at least one selected from the group consisting of

The present invention relates to a method for producing an acid fermentation extract and a cosmetic composition comprising the acid fermentation extract as an active ingredient, wherein the cosmetic composition of the present invention ferments and extracts the sea lye using the skin-derived fermented lactic acid bacteria and the intestinal lactic acid bacteria, And enhances the antioxidant ability by promoting dissolution of effective physiologically active ingredients such as polyphenols and flavonoids contained in the skin, and has effects of skin anti-inflammation, promotion of proliferation of skin cells, prevention of wrinkles, skin moisturization and skin whitening.

FIG. 1 is a graph showing DPPH scavenging activity of a fermented sea urchin fermented extract fermented with various lactic acid bacteria.
FIG. 2 is a graph showing the inhibition rate of MMP-1 expression of the acid fermentation extract fermented with various lactic acid bacteria in CCD-986sk cells irradiated with UVA.
FIG. 3 is a graph showing melanin synthesis inhibition rates of the fermented Sansafura fermentation extract fermented with various lactic acid bacteria.
FIG. 4 shows the results of measurement of the amount of nitrogen oxides (NO) produced by fermented extracts of various fermented lactic acid bacteria.

The present invention is characterized by providing a process for producing an acid fermentation extract and a cosmetic composition comprising the acid fermentation extract as an active ingredient.

In recent years, not only many women but also men have become increasingly interested in cosmetics, and various functional cosmetics for skin whitening, acne, atopic dermatitis and wrinkle are being released. However, most functional cosmetics sold on the market are products using synthetic compounds, and cosmetic products containing synthetic compounds can cause sensitive skin reactions depending on the skin, which causes various skin side effects.

Accordingly, the present inventors paid attention to lactic acid bacteria and sasanghae which were studied for the production of functional cosmetics having no side effects and low cost and excellent stability.

Lactic acid bacteria are important microorganisms widely used and applied worldwide, and they have been used for a long time in fermented dairy products such as cheese, fermented milk and lactic acid bacteria drink, and food industry such as kimchi and chungkukjang. Lactobacillus has a long-lasting effect on the treatment and prevention of intestinal diseases, thus preventing the growth of harmful bacteria in the intestines and making good conditions for the growth of beneficial bacteria, thereby preventing food poisoning, preventing intestinal decline, preventing constipation, And is used in various dairy products, foods, and cosmetics industries.

In addition, 'Sansa' is a small species belonging to the Rosaceae apple tree, which is called horsetail or mountain hawthorn. It is hard and much smaller than thorny apple tree and has thorns. It is originated in Asia and North America. The taste is sour, but it has a clear color and moderate sweetness, and it is made with jelly, canned food, alcohol and tea. In herbal medicine, herbal medicine is used as a gastrointestinal medicine.

Sansa is effective in eating meat, and is known to have a particularly good efficacy in the treatment of hypertension, heart disease, indigestion and hyperlipidemia.

The inventors of the Crataegus skin-derived lactic acid bacteria of the study were for the functional cosmetic composition having excellent production stability and low cost without the side effects Lactobacillus ramno suspension HK-9 (Lactobacillus Ramnosus HK-9) KCCM11254P and Lactobacillus acidophilus Lactobacillus acidophilus and Lactobacillus bulgaricus to improve the antioxidant activity by promoting the elution of effective physiologically active ingredients such as polyphenols and flavonoids contained in spermatozoa during fermentation extraction, Promoting, preventing wrinkles, skin moisturizing, and skin whitening, and is therefore suitable for use as a cosmetic composition.

According to one embodiment of the present invention, the skin cell proliferation effect of the granular extract of the present invention fermented with various lactic acid bacteria was examined. As a result, compared with the control group not treated with the granular extract, The highest proliferation effect was obtained by the fermented extract of horseshoe crab using HK-9, which is a skin-derived lactic acid bacterium, 80.9% (see Table 7).

According to another embodiment, DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity was measured for the antioxidative activity of the fermented extract of Sansafura prepared by the method of the present invention. As a result, , The DPPH radical scavenging activity tended to increase with increasing concentration, and showed a high scavenging activity of over 70% at the highest concentration of 1000 μg / ml. Among them, the scavenging ability of the fermented sea tangle extract using HK-9, which is a skin-derived lactic acid bacterium, was 93.19%, indicating that the antioxidant activity was excellent (see FIG. 1).

According to another embodiment, in order to investigate the moisturizing effect of the fermented sea tangle extract prepared by the method of the present invention, the amount of collagen type I produced was measured, (Ascorbic acid) as compared with the positive control (1.0 mg / mL) (see Table 8).

According to another embodiment, in order to investigate the effect of suppressing the wrinkle on the fermented extract of Sansafura prepared by the method of the present invention, the human fibroblast CCD-986sk was UV-irradiated and then the inhibition of MMP-1 expression As a result of the ELISA, the inhibition rate of MMP-1 expression was increased in a concentration-dependent manner in the acid fermentation extract, and the highest concentration of 1 mg / mL was 60.15%, HK-9 As a result, the inhibition rate of MCP-1 expression by the present invention was 69.55%, that of L. bulgaricus was 64.02%, that of L. bulgaricus was 64.02%, and that of B. longum was 35.81% And effectively suppressed the appearance of wrinkles (see FIG. 2).

According to another embodiment, melanin production was measured using Clone-M3, a melanocyte-producing melanocyte which produces melanin, in order to investigate whether skin-whitening effect produced by the method of the present invention is skin-whitening effect , The inhibition rate of melanin synthesis was increased as the concentration of fermented soybean curd extract increased. In the highest concentration of 100 ㎍ / ㎖, 34.11% of general crustacean, HK - 9, 41.21% of L. bulgaricus (35.87%) and B. longum (36.3%), respectively (see FIG. 3).

According to another embodiment of the present invention, the amount of nitric oxide (NO) produced by macrophage was measured in order to confirm the anti-inflammatory activity of the fermented extract produced by the method of the present invention. As a result, . However, the treatment with LPS showed significantly higher NO production than the treatment with LPS alone (4.2 μM). In particular, in the case of the acid fermentation extract of the present invention using the skin-derived lactic acid bacterium HK-9 10 < 5 > .mu.M, indicating an excellent anti-inflammatory activity (see FIG. 4).

Therefore, based on these results, the Sansa fermented extract prepared by the method of the present invention improves the antioxidant ability by promoting the elution of effective physiologically active ingredients such as polyphenols and flavonoids contained in sasan, and promotes skin inflammation, promotion of skin cell proliferation , Anti-wrinkle, skin moisturizing and whitening effect, so that it can be used as a functional cosmetic composition.

Lactobacillus Lymphosus HK-9 ( Lactobacillus < RTI ID = 0.0 > Ramnosus HK-9) KCCM11254P, Lactobacillus ( Lactobacillus acidophilus or Lactobacillus bulgaricus may be added in an amount of 0.001 to 20% by weight based on the total weight of the cosmetic composition.

When the above-mentioned strain is added in an amount of less than 0.001% by weight based on the total weight of the cosmetic, the cosmetic composition of the present invention does not exhibit various physiological activity effects. On the other hand, when added in an amount exceeding 20% by weight, The desired effect can not be obtained in proportion to the added amount. Therefore, it is preferable to add the cosmetic composition within the above-described range in consideration of economical efficiency, and the cost of the cosmetic composition according to the amount of the cosmetic composition increases.

When the fermented extract of the present invention is prepared by a cosmetic composition, the composition of the present invention may include not only fermentation and extraction of the above-mentioned persimmon but also components commonly used in cosmetic compositions. Examples thereof include antioxidants, , Customary adjuvants such as solubilizers, vitamins, pigments and flavoring agents, and carriers.

The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used as a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

In the case where the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. Especially, in the case of a spray, a mixture of chlorofluorohydrocarbons, propane / Propane or dimethyl ether.

When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

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

The present invention also provides a cosmetic method characterized by applying the cosmetic composition of the present invention to human skin.

The cosmetic process of the present invention refers to all the cosmetic processes for applying the cosmetic composition of the present invention to human skin. That is, all methods known in the art for applying the cosmetic composition to the skin belong to the cosmetic method of the present invention.

The cosmetic composition of the present invention may be used alone or in combination, or may be used by overlapping with other cosmetic compositions other than the present invention. Further, the cosmetic composition having excellent skin wrinkle-improving effect according to the present invention can be used according to a conventional method of use, and the use frequency of the cosmetic composition can be changed according to a user's skin condition or taste.

When the cosmetic composition of the present invention is a soap, a surfactant-containing cleansing agent, or a surfactant-free cleansing agent, it may be applied to the skin and then wiped off or removed or washed with water. The surfactant-containing cleansing formulation is a cleansing foam, a cleansing water, a cleansing towel, and a cleansing pack. The surfactant-free cleansing formulation may be a cleansing cream, , Cleansing lotion, cleansing water and cleansing gel, but is not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

< Example  1>

Derived from skin  The use of lactic acid bacteria and common intestinal lactic acid bacteria Fermentation product  Produce

Lactic acid bacteria were cultured in MRS medium to ferment the by-products of the present invention. The culture medium was prepared by adding 55% of MRS medium and 0.5% of L-cysteine hydrochloride to the culture medium. The incubation temperature was 37 ° C, and the fermentation broth In order to conduct experiments under anaerobic conditions due to their characteristics, they were cultivated at a minimum stirring speed (50 to 60 rpm) and then subjected to a 1 L volume in a 2 L flask for 72 hours.

In the present invention, Lactobacillus lambosus HK-9 KCCM11254P ( Lactobacillus rhamnosus HK-9) and the representative general intestinal lactobacillus Lactobacillus ( Lactobacillus bulgaricus ), Bifidobacterium Longum ) was inoculated at a concentration of 5% in a completely sterilized medium by a high-pressure sterilizer and cultured at 37 ° C for 48 hours to perform fermentation. In addition, the dried horseshoe used in the present invention was dried horseshoe containing less than 7% water.

The amount of the culture medium and the culture medium treated with the microorganism were cultured at a ratio of 1: 1 by weight to the total weight. After the fermentation, the fermented broth was collected, Ethanol was used for shaking extraction at 60 DEG C for 24 hours. The obtained extract obtained by extracting the fermented sea tangle was simultaneously subjected to concentration under reduced pressure and lyophilization to prepare a powder.

After fermentation, the obtained fermentation broth was collected, and the remaining fermented product was added to the dried fermented product at a ratio of 1:10 (fermented product: ethanol) using 70% ethanol. The extract was shaken at 60 ° C for 24 hours, After removing the sludge sludge, the filtrate was concentrated under reduced pressure and frozen to prepare powder form and mixed with the fermented powder. In the case of general hunting, 10% of 70% ethanol was added to perform the above extraction, filtration, concentration under reduced pressure, and freezing.

< Example  2>

The Sansa extract  And fermented extract Cosmetics  Method of manufacturing composition

The inventors of the present invention prepared the cosmetic composition containing the fermented extract of Sansafura prepared by the method of Example 1 with the composition shown in Table 1 below and used Lactobacillus rhamnosus HK-9 of the present invention Lactobacillus lambosus HK-9 KCCM11254P In order to compare the efficacy of the cosmetic composition containing the fermented Sansafura fermented extract, the cosmetic composition was prepared using only the components other than the fermented extract of Sansha as the control group in Table 1.

Hereinafter, various formulation examples of a cosmetic composition containing a fermented sea tangle extract using HK-9 as a skin-derived lactic acid bacterium according to the present invention are presented. However, the present invention is not limited thereto.

<2-1> Softening longevity skin )

The softened longevity water containing the fermented sea tangle extract of HK-9, which is the skin-derived lactic acid bacterium of the present invention prepared by a conventional method for producing softened longevity, was prepared as shown in Table 2 below.

<2-2> Nutrition lotion ( lotion )

Nutritional lotion containing the fermented sea tangle extract using the skin-derived lactic acid bacterium HK-9 of the present invention produced by a conventional method of producing a nutritive lotion with the composition shown in Table 3 below was prepared.

<2-3> Nourishing cream

Nutritive creams containing the fermented sea tangle extract using the skin-derived lactic acid bacterium HK-9 of the present invention produced by the conventional nutrition cream preparation method as shown in Table 4 below were prepared.

<2-4> Massage cream

A massaging cream containing an acid-fast-fermented extract of HK-9, which is a skin-derived lactic acid bacterium of the present invention prepared by a conventional method for producing a massage cream, was prepared as shown in Table 5 below.

<2-5> pack

A pack containing the fermented sea tangle extract of HK-9, which is the skin-derived lactic acid bacterium of the present invention prepared by a conventional pack preparation method, was prepared as shown in Table 6 below.

< Experimental Example  1>

Confirmation of skin cell proliferation effect of Sansa fermented extract

To investigate the effect of the present invention on the skin cell proliferation effect, human normal fibroblast (Korean Cell Line Bank) was added to each well of a 96-well microplate at 1 x 10 &lt; ⁴ cells, and cultured in RPMI1640 medium (Sigma) for 24 hours at 37 占 폚. Then, the control group was replaced with the serum-free RPMI1640 medium containing 0.5% and 1% of the acid fermentation extract and the serum-free RPMI1640 medium without the fermentation extract of Sansafura, and further cultured for 24 hours .

Then, 10 μl each of the MTT solution (3- (4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide: 5 mg / ml) was added and cultured for 4 hours, The medium was removed, 100 μl of DMSO solution was added to each well, shaken for 20 minutes, and the absorbance of the supernatant was measured at 517 nm using a microplate reader (Molecular Devices, USA). The cell proliferation effect was calculated by substituting the measurement value into the following equation (1), and the results are shown in Table 7.

[Equation 1]

Cell proliferation effect (%) = [(absorbance of test group - absorbance of control group) / absorbance of control group] × 100

As can be seen from Table 7 above, compared with the control group not treated with the fermented sea urchin fermented extract, all the experimental groups of the fermented sea urchin fermented extract showed a high proliferation effect of 60% or more. Among them, the fermented soybean fermented with HK-9 The extract showed the highest proliferation effect of 80.9%.

< Experimental Example  2>

Identification of Antioxidative Effect of Sansa Fermented Extract

The present inventors measured DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity to examine the antioxidative activity of the fermented extract of Sasa sp.

The DPPH radical scavenging assay is one of the experiments for measuring antioxidant activity and is a method for measuring antioxidant activity by using the degree of depigmentation of purple color as an index as reduced by aromatic compounds and aromatic amines due to the electron donating ability of the antioxidants.

To this end, 2 μL of the granular extract of the present invention was dissolved in ethanol to prepare 2 mL of each concentration, and 1 mL of 200 μM DPPH solution was added. The reaction solution was vortexed for 10 seconds to mix thoroughly, and then allowed to react at room temperature for 30 minutes. The absorbance was measured at 517 nm using a UV-vis spectrophotometer (852A Diode Array Spectrophotometer, Hewlett Packard) to measure the amount of DPPH remaining. The results are shown in FIG.

As a result, the DPPH radical scavenging ability tended to increase with increasing concentration in all the fermented sea urchin extracts, and showed a high sorption capacity over 70% at the highest concentration of 1000 μg / ml. The scavenging ability of the present invention using the skin-derived lactic acid bacterium HK-9 was 93.19%, indicating that the antioxidative activity was excellent (see FIG. 1).

< Experimental Example  3>

Identification of moisturizing effect of Sansa fermented extract

The present inventors measured the amount of collagen type I produced in order to investigate the moisturizing effect of the fermented sea urchin extract.

For this purpose, 0.2% DMSO was used as a negative control, 0.2 mM ascorbic acid was used as a positive control, and test substance concentrations were adjusted to 0.2, 0.4, 0.6, 0.8 and 1.0 mg / mL, respectively. The cultured human fibroblast CCD-986sk was dispensed in 96 well pellet at 2 × 10 ⁴ cells / well. Cells cultured for 24 hours were washed three times with serum-free DMEM, 10% FBS was added to the DMEM solution, which was dissolved in each concentration, and the cells were further cultured for 24 hours. Procollagen type I C-peptide was quantitated using an enzyme-linked immunosorbent assay kit (Takara Bio Inc., Otsu, Japan).

Table 8 shows the results of the collagen synthesis performance of the acid-fast fermented extract according to CCD-986sk cell proliferation.

As a result of comparing the collagen synthesis performance of the acid fermentation extract and ascorbic acid as a positive control, the collagen production amount in all conditions tended to increase in a concentration dependent manner by adding the fermented extract of Sasa japonica, and compared with the positive control group of 1.0 mg / mL And high collagen amount (see Table 8).

On the other hand, since the collagen layer does not undergo an enzyme action other than collagenase and plays an important role in skin health such as waterproofing, moisturizing and elasticity maintenance, the result of stimulating the synthesis of collagen of the present invention is effective for skin immunity and moisturizing Respectively.

< Experimental Example  4>

Measurement of wrinkle inhibition effect of Sansa fermented extract

Experiments were carried out as follows to investigate whether wrinkle suppression effect of the present invention was inhibited.

First, a human fibroblast, CCD-986sk, was cultured in a 35 mm dish at a concentration of 1.5 x 10 ⁵ cells / mL until a confluency of about 80% was reached. After the medium was removed from the UV irradiation, the serum was removed from the medium by washing with PBS (phosphate buffered saline), and the medium was adjusted to 6.3 J / cm ² using LUX metter (DM-28, Takemura, Japan) Respectively. After UVA irradiation, the fraction collected by molecular weight was added to DMEM medium without FBS at a concentration of 1 mg / mL for 24 hours.

The amount of MMP-1 expression induced by UVA irradiation was measured by Dunsmore et al. After irradiation with UVA, the fermented extract was treated for 24 hours. After washing three times with TBS (phosphate buffered saline + 0.05% Tween 20) and blocking with 3% PBS at 37 ° C for 1 hour, the monoclonal anti-MMP-1 was blocked with Blocking buffer blocking buffer) at a ratio of 1: 3,000, followed by reaction at 37 ° C for 90 minutes. After washing with PBS, the secondary antibody (alkaline phosphatase conjugated goat anti-mouse IgG) was diluted 1: 3,000 in Blocking buffer, incubated at 37 ° C for 90 minutes, washed with PBS and incubated with alkaline phosphatase ) Substrate solution (1 mg / mL, p-nitrophenyl phosphate in diethanolamine buffer) was added and reacted at room temperature for 30 minutes. The reaction was stopped completely with 3 N NaOH and absorbance was measured at 405 nm using a microplate reader.

The expression of MMP-1, which plays an important role in skin wrinkle formation due to photoaging of skin, is induced by UVA to increase JNK / p38 activity and increase the activity of AP-1, a transcription factor, -1 expression, leading to a deficiency of collagen in the skin. In order to investigate the effect of the fermented extract on the growth of MMP-1 induced by UVA, UVA was irradiated on HDFs, and the culture inhibitor of MMP-1 was cultured for 24 hours after the addition of the fermentation extract Respectively.

As a result, the inhibition rate of MMP-1 expression was increased in a concentration-dependent manner in the fermented sea tangle fermentation extract, and compared with the control fermented sea tangle extract at the highest concentration of 1 mg / mL, 60.15% 69.55%, and L. bulgaricus showed 64.02% inhibition rate and 35.81% inhibition rate, respectively, of B. longum. Thus, it was found that the expression of MMP-1 effectively inhibited wrinkle expression (See Fig. 2).

< Experimental Example  5>

Measurement of skin whitening effect of Sansa fermented extract

Melanin production was measured by using mouse-derived melanocyte Clone-M3, which produces melanin, in order to examine whether the extract of the present invention has skin whitening effect.

First, Clone-M3 was inoculated in 96 wells, and after 24 hours, each sample was treated for 3 days for 3 days. After 3 days, the medium of each well was removed, followed by washing with PBS. 1 ml of 1N NaOH was added to each well, followed by stirring to allow the melanin component to elute. Absorbance was measured at 400 nm using a light absorber to measure the content of eluted melanin.

As a positive control, melasolv was used, and the treatment was carried out at a maximum concentration of 100 μg / ml.

As a result, it was confirmed that the inhibition rate of melanin synthesis was increased as the concentration was increased. In the highest concentration of 100 ㎍ / ㎖, 34.11% of general crustacean extract, 41.21% of Crassulaceae fermentation extract using HK-9, and L. bulgaricus 35.87% of the fermented mushroom extract and 36.99% of the fermented mushroom extract using B. longum (see FIG. 3).

The above fermented extracts showed similar values when compared with the melanin synthesis inhibitory rate of the positive control group melasolve, indicating that there is an excellent whitening activity.

< Experimental Example  6>

Measurement of skin anti-inflammatory effect of Sansa fermented extract

The amount of nitric oxide (NO) production was measured using Macrophage in order to confirm the anti-inflammatory activity of the present invention.

The cell lines used for measuring the amount of NO production were mouse-derived J774.1 macrophages. The cells were cultured in 10% FBS and RPMI 1640 medium and placed in a 24-well plate at a concentration of 4.5 x 10 4 cells / well Then, the cells were cultured in a 5% CO2 incubator at 37 ° C for 48 hours. LPS (lipipilysaccharide) was treated at a concentration of 200 ng / well, and the cells were cultured for 48 hours Respectively. 50 μg of the supernatant was added and the same volume of Griess reagent (1% sulfanilamide / 0.1% N- (1-naphthyl) -ethylenediamine dihydrochloride / 2.5% h3po4) was added and reacted at room temperature for 10 minutes. Was measured. Sodium nitrite was used as a standard for nitrite, and the concentration was calculated by comparing the standard curve obtained by doubling the concentration from 32 μM to 0.25 μM in RPMI 1640 medium.

FIG. 4 is a graph showing the results of the measurement of the anti-inflammatory activity of the acid fermentation extract. As a result, the NOS (nitric oxide synthase) of macrophages was expressed in various bacteria such as TNF-γ and TNF- α and lipopolysaccharide Since the NOS gene is induced by the influence of the toxin, the NO production ability was confirmed by administering the sample and LPS alone or together in order to confirm this.

As a result, no significant difference was observed in the case of administration of the sample alone compared with the control group. However, the treatment with LPS showed significantly higher NO production than 4.2 μM treated with LPS alone, (10.5 [mu] M) of the present invention showed an excellent anti-inflammatory activity (see FIG. 4).

< Experimental Example  7>

Test for skin stability of Sansa fermented extract

In order to confirm the skin stability of the granular extract of the present invention, the following experiment was carried out.

First, the skin was washed with clean water for 10 persons in each of 20 to 30 male and female subjects, and the diluted solution of 1% of the crude extract of Example 1 and the dexamethasone ointment of Example 1 were applied to the skin, And irradiated with ultraviolet rays (100 mJ / cm 2). The above materials were applied to the skin once every day until day 7 and the average value was measured before and after the irradiation by measuring the erythema index with Mexameter.

As shown in Table 9, it was found that the extract of Sansafura fermented by the method of the present invention shows a protective effect against skin irritation and has a similar effect to that of dexamethasone tested as a positive control. (See Table 9).

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (11)

(a) inoculating lactic acid bacteria into a medium sterilized by a high-pressure sterilizer at a concentration of 3 to 7% (v / v) and anaerobically culturing at a temperature of 34 to 40 ° C for 12 to 36 hours;
(b) anaerobically culturing the cultured suspension of the lactic acid bacteria obtained in step (a) at a ratio of 1: 8 to 1:12 based on the total culture medium weight to produce a fermented product step;
(c) The fermented broodstock fermented from the step (b) is separately collected, and the remaining fermented broodstock is prepared at a ratio of 1: 8 to 1:12 with respect to the total culture medium weight. Extracting the extract with shaking at a temperature of 70 ° C for 20 to 28 hours, filtering the extract with a filter paper to prepare an extract of Acanthopanax senticosus; And
(d) mixing the fermented product of the horse mackerel obtained in the step (b) with the extract of the horse mackerel obtained in the step (c). The method according to claim 1,
The (a) step of the lactic acid bacteria is Lactobacillus ramno suspension HK-9 (Lactobacillus Ramnosus HK-9) KCCM11254P, Lactobacillus ( Lactobacillus acidophilus , and Lactobacillus bulgaricus. &lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt; A cosmetic composition comprising an acid value fermented extract prepared by the method according to any one of claims 1 to 2 as an active ingredient. A cosmetic composition for preventing skin aging comprising an extract of Sansa japonica as an active ingredient. 5. The method of claim 4,
The above fermented sea tangle extract contains Lactobacillus Lambosus HK-9 ( Lactobacillus Ramnosus HK-9) KCCM11254P, Lactobacillus ( Lactobacillus acidophilus , and Lactobacillus bulgaricus . The composition for preventing skin aging according to claim 1, wherein the composition is fermented with at least one lactic acid bacterium selected from the group consisting of Lactobacillus acidophilus and Lactobacillus bulgaricus . A cosmetic composition for skin whitening comprising an extract of Fermented sea tangle as an active ingredient. The method according to claim 6,
The above fermented sea tangle extract contains Lactobacillus Lambosus HK-9 ( Lactobacillus Ramnosus HK-9) KCCM11254P, Lactobacillus ( Lactobacillus acidophilus , and Lactobacillus bulgaricus . The cosmetic composition for skin whitening according to claim 1, wherein the composition is fermented with at least one lactic acid bacterium selected from the group consisting of Lactobacillus acidophilus and Lactobacillus bulgaricus . A cosmetic composition for improving skin wrinkles, comprising an acid fermentation extract as an active ingredient. 9. The method of claim 8,
The above fermented sea tangle extract contains Lactobacillus Lambosus HK-9 ( Lactobacillus Ramnosus HK-9) KCCM11254P, Lactobacillus ( Lactobacillus acidophilus , and Lactobacillus bulgaricus . The cosmetic composition for skin whitening according to claim 1, wherein the composition is fermented with at least one lactic acid bacterium selected from the group consisting of Lactobacillus acidophilus and Lactobacillus bulgaricus . A cosmetic composition having a skin anti-inflammatory effect or a skin moisturizing effect comprising an acid-fast-fermented extract as an active ingredient. 11. The method of claim 10,
The above-mentioned fermented sea tangle fermented extract was prepared by mixing Lactobacillus Ramnosus HK-9 KCCM11254P, Lactobacillus acidophilus , and Lactobacillus bulgaricus . The cosmetic composition for skin whitening according to claim 1, wherein the composition is fermented with at least one lactic acid bacterium selected from the group consisting of Lactobacillus acidophilus and Lactobacillus bulgaricus .

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