KR101426593B1 - Functional cosmetic composition comprising fermented complex marine extracts and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a functional cosmetic composition comprising a complex fermented extract of marine material and a preparation method thereof, and more particularly, to a functional cosmetic composition comprising a complex fermented extract of marine material, To a cosmetic composition for improving dermatitis and whitening, and a method for producing the same.

Description

TECHNICAL FIELD The present invention relates to a functional cosmetic composition comprising a complex fermented extract of marine material and a functional cosmetic composition comprising the fermented extract,

The present invention relates to a functional cosmetic composition comprising a complex fermented extract of marine material and a preparation method thereof, and more particularly, to a functional cosmetic composition comprising a complex fermented extract of marine material, To a cosmetic composition for improving dermatitis and whitening, and a method for producing the same.

Due to the advancement of the Korean economy, western type skin diseases such as atopic dermatitis are increasingly becoming big social issues. Atopic dermatitis is a chronic skin disease that is common in children and continues to be symptomatic even after becoming an adult. The number of patients with atopic dermatitis is increasing worldwide. In recent years, it has become more common in children and more than 20% . Although atopic dermatitis has been reported to be a cause of atopic dermatitis, it has not been completely elucidated yet. It is presumed that atopic dermatitis occurs as a result of various factors and various immunological reactions as well as various clinical features. Immunological abnormalities, environmental factors, immunologic abnormalities of patients, and abnormalities of skin barrier have been considered as major causes of the disease. In the case of atopic dermatitis, it is difficult to provide precise treatment guidelines for the patient as well as for the patient who is a medical consumer because of the difference of treatment between the oriental medicine and the both sides.

Patients with atopic dermatitis are generally known to develop bacterial, viral and fungal skin infections as compared to normal people. Especially skin infections caused by staphylococcus are frequent. Staphylococci are cultured in the skin of atopic dermatitis patients by about 95%, but the difference of cultures of normal persons is about 5%, indicating that the occurrence of atopic dermatitis is closely related. In patients with atopic dermatitis, it is known that the congenital antimicrobial peptides present in the skin are less or less functional than normal people and that the bacteria propagate well on the skin.

Topical steroids have been used as the most basic and effective treatment for atopic dermatitis for more than 50 years, and there are currently no treatments that are as effective as topical steroids. However, when used for a long period of time, side effects such as dilatation of the right part, skin atrophy, capillary dilatation, steroid acne, hirsutism, and vagina may occur. Recently, as these side effects have become known in newspapers, magazines and the Internet, many patients with atopic dermatitis or their caregivers are reluctant to use topical steroids (steroid phobia). Therefore, it is necessary to identify the cause of atopic dermatitis and to develop a more fundamental treatment based on it.

In addition, atopic dermatitis is caused by pigmentation of the skin caused by skin darkness, and the suffering of atopic dermatitis patients is doubling. Therefore, it would be more desirable to develop a novel substance having a whitening effect on post-inflammatory hyperpigmentation as well as the therapeutic effect of atopic dermatitis.

The inventors of the present invention have found that a fermentation broth produced by fermenting a complex extract of a marine material with a lactic acid bacterium can be used for the improvement of atopic dermatitis and the whitening of the deposited skin due to the atopic dermatitis The present invention has been completed.

Korean Patent Publication No. 2010-0111066 (Published on October 14, 2010) Korean Published Patent Application No. 2009-0043115 (Published May 5, 2009)

It is an object of the present invention to provide a cosmetic composition for improving and whitening atopic dermatitis, which comprises a complex fermentation extract of moxa, mung bean, mackerel, squid skin, rhubarb and rhizome.

It is still another object of the present invention to provide a method for improving atopic dermatitis and a method for producing a cosmetic composition for whitening.

The present invention as an embodiment for achieving the above-mentioned problem is gompi (Ecklonia stolonifera , Sargassum fulvellum ), acne ( Ecklonia cava ), squid bark, rhubarb ( Eisenia bicyclis ) and stone chess ( Gigartina The present invention relates to a cosmetic composition for improving and whitening atopic dermatitis,

As used herein, the term "multiple extract" means an extract obtained from a moth, moth, mung bean, squid skin, rhubarb and stone scabbard. As the extraction method, a conventional extraction method can be used, and methods such as extraction with an extraction solvent, supercritical fluid extraction, ultrasonic extraction, and hot water extraction can be exemplified, but the present invention is not limited thereto.

The complex fermentation extract may further comprise 0.1 to 20% by weight of the mugwort extract, 0.1 to 20% by weight of the mugwort extract, 0.1 to 20% by weight of the mugwort extract, 0.1 to 20% by weight of the squid bark extract, 0.1 to 20% And 0.1 to 20% by weight of the extract.

For example, in the case of extracting using an organic solvent, a polar solvent such as ethanol or methanol or a mixed solution of them and purified water is added 10 times as much as the pulverized product to a dry matter of a mugwort, mackerel, mackerel, squid skin, rhubarb, The extract is heated at a temperature of about 60 to 90 ° C for 1 to 2 days in an extractor equipped with a cooling condenser, filtered and concentrated under reduced pressure to obtain a concentrated extract. Then, purified water corresponding to 2 to 3 times the weight of the concentrated extract is added, and a nonpolar solvent such as ethyl acetate or methylene chloride is added in a weight of 3 to 5 times, and the mixture is vigorously shaken. Then, the nonpolar solvent layer as an upper layer is separated, After that, the final dry powder can be obtained. The organic solvent to be extracted and extracted by a conventional extraction method such as the above method may be selected from methanol, ethyl acetate, ethanol, chloroform, butanol, acetone, or a mixed solvent thereof and purified water. In addition, tannin can be removed beforehand by using sodium chloride for effective extraction. It is also possible to immerse the tannin for 2-7 days at room temperature instead of heating.

As another example, the combined extract of the present invention can be extracted using supercritical extraction technology. Supercritical extraction refers to the extraction of a complex extract using carbon dioxide as a carrier gas and ultrahigh pressure over a critical point. Unlike conventional heat extraction or solvent extraction, supercritical extraction is a method of extracting only necessary components of a plant at a high concentration at a low temperature and at a high concentration. The extract can be extracted without destroying the active components of the extract. Instead of an organic solvent, It is not toxic to the skin. As an example of the supercritical extraction method, the supernatant extraction apparatus is filled with the dry matter of moxa, mung bean, mackerel, squid skin, rhubarb and rhizome, firstly extracted with carbon dioxide at 70 ~ 90 ° C and 4,000 ~ 6,000 PSI pressure, 1 to 20 parts by weight of at least one alkaline cosolvent selected from methanol, ethanol, water or a mixed solvent thereof in which 2 to 20 (v / v)% of diethylamine or triethylamine is dissolved in 100 parts by weight of the skin, The mixture can be extracted and then column chromatography can be used to obtain an optional complex extract fraction.

Preferably, the cosmetic composition comprising the complex fermentation extract of the present invention is a cosmetic composition which is produced by fermenting an extract of Myxia mellifera, Namhae, Matsutake, squid skin, rhubarb and stone livers with lactic acid bacteria.

As used herein, the term "produced by fermentation with lactic acid bacteria" refers to the denaturation or modification of raw materials by physical or chemical methods using lactic acid bacteria. In natural terms, the term "fermented by lactic acid bacteria" It means the decomposition process that can get the result. In particular, fermentation using lactic acid bacteria in the present invention means that lactic acid bacteria selectively extract and release only the weakness and components contained in the raw material without changing the external shape.

The term "fermented extract" used herein refers to a fermentation broth obtained by fermenting a complex extract with a lactic acid bacterium, or a filtrate from which lactic acid bacterium cells have been removed from the medium, and includes components contained in a medium in which the complex extract is fermented and cultured using lactic acid bacteria . Preferably, the cosmetic composition according to the present invention comprises a fermentation broth produced by mixing an extract of Mycophyllum auriculariae, Mycobacterium tuberosum, Quercus husk, squid skin, rhubarb and rhizome with a culture medium for lactic acid bacteria and inoculating and fermenting lactic acid bacteria.

In the present invention, the fermentation broth obtained through fermentation is used as it is, or after the fermentation broth is dried, 1 to 20 times (by volume) of the extraction solvent is added to the extract, and the mixture is immersed at 4 to 30 ° C for 3 to 20 days The active ingredient may be extracted and used.

The lactic acid bacteria to be used in the present invention are preferably Enterococcus faecium), Enterococcus faecalis (Enterococcus faecalis , Lactobacillus plantarum , Lactobacillus < RTI ID = 0.0 > fermentum , Lactobacillus lactis , Lactobacillus < RTI ID = 0.0 > rhamnosus , Lactobacillus < RTI ID = 0.0 > sakei), Lactobacillus brevis (Lactobacillus brevis , Lactobacillus < RTI ID = 0.0 > graminis , Leuconostoc citreum , Weissella hellenica), Lactococcus taught rain (Lactococcus garvieae), Peddie Oh Caucus pen Tosa three mouse (Pediococcus pentosaceus ), Pediococcus ( Pediococcus) acidilactici), Bifidobacterium No Marlies (Bifidobacterium animalis ) and Bifidobacterium ( Bifidobacterium < RTI ID = 0.0 > infantis ). < / RTI > The microorganisms used in the present invention are known bacteria and are available from the BRCs of the Korea Research Institute of Bioscience and Biotechnology, the Belgian Co-ordinated Collections of Microorganisms (BCCM) or the American Type Culture Collection (ATCC).

More specifically, the bacteria used in the present invention include Enterococcus faecium (Enterococcus faecium) Include the Enterococcus faecium (Enterococcus faecium) ATCC 19634 or Enterococcus faecium (Enterococcus faecium) ATCC 6057 can be selected and used, and Enterococcus faecalis (Enterococcus faecalis) Was an Enterococcus faecalisEnterococcus faecalis) ATCC 6057 can be used, and Lactobacillus plantarum (Lactobacillus plantarum) Were Lactobacillus plantarum (Lactobacillus plantarum) ATCC 10241, or Lactobacillus plantarum (Lactobacillus plantarum) ≪ / RTI > NCTC 7220, and Lactobacillus fermentum (Lactobacillus fermentum) Include Lactobacillus fermentum (Lactobacillus fermentum) ATCC 14931, or Lactobacillus fermentum (Lactobacillus fermentum) ≪ / RTI > NCDO 1750, and one or more of Lactobacillus lactis (Lactobacillus lactis) Include Lactobacillus lactis (Lactobacillus lactis) ATCC 11454, or Lactobacillus lactis (Lactobacillus lactis) NCIB 8586, and Lactobacillus lambosus (Lactobacillus rhamnosus), Lactobacillus lambatus (Lactobacillus rhamnosus) ATCC 7469, or Lactobacillus lambosus (Lactobacillus rhamnosus) KCTC 3326, and Lactobacillus casei (Lactobacillus sakei), Lactobacillus casei (Lactobacillus sakei) ATCC 31063, or Lactobacillus saccharum (Lactobacillus sakei) ATCC 15521, and one or more of Lactobacillus brevis (Lactobacillus brevis), Lactobacillus brevis (Lactobacillus brevis) ATCC 15521 can be used, and the Lactobacillus graminis (Lactobacillus graminis), Lactobacillus graminis (Lactobacillus graminis) ≪ / RTI > ATCC 51150 can be used,Leuconostoc citreum) Include leucono-stearate (Leuconostoc citreum) ≪ / RTI > ATCC 49370,Weissella hellenica) Include Biscea Helenica (Weissella hellenica) ATCC 51523 can be used, and Lactococcus garvie (Lactococcus garvieaeLactococcus garvie (Lactococcus garvieae) CCUG 11672, Lactococcus garbiera (Lactococcus garvieae) ATCC 11454, or Lactococcus garvie (Lactococcus garvieae) LMG 8162 can be selected and used, and Pediococcus pentosaceus (Pediococcus pentosaceus) Include Pediococcus pentosaceus (Pediococcus pentosaceus) ≪ / RTI > ATCC 33316 can be used,Pediococcus acidilactici) Include Pediococcus with Cydilitici (Pediococcus acidilactici) ATCC 33314 can be used, and the Bifidobacterium anilis (Bifidobacterium animalis) Include Bifidobacterium ani malis (Bifidobacterium animalis) ATCC 27672 can be used, and Bifidobacterium infantis (Bifidobacterium infantis) Include Bifidobacterium infantis (Bifidobacterium infantis) ATCC15697 can be used.

Also preferably, the composition of the present invention may comprise bacteriocin produced by said lactic acid bacteria.

The method for preparing a cosmetic composition comprising the complex fermentation extract according to the present invention will now be described.

(1) preparing a complex extract by extracting a moxa, a moxa, a moxa, a squid skin, a rhubarb and a rhizome;

(2) mixing the complex extract with a culture medium for lactic acid bacteria; And

(3) To the lactic acid bacteria culture medium, Enterococcus faecium , Enterococcus faecalis , Lactobacillus < RTI ID = 0.0 > plantarum), Lactobacillus spread lactofermentum (Lactobacillus fermentum), Lactobacillus lactis (Lactobacillus lactis , Lactobacillus rhamnosus , Lactobacillus < RTI ID = 0.0 > sakei , Lactobacillus brevis , Lactobacillus < RTI ID = 0.0 > graminis , Leuconostoc citreum , Weissella < RTI ID = 0.0 > hellenica), Lactococcus taught rain (Lactococcus garvieae), Peddie Oh Caucus pen Tosa three mouse (Pediococcus pentosaceus ), Pediococcus ( Pediococcus) acidilactici), Bifidobacterium No Marlies (Bifidobacterium animalis ) and Bifidobacterium ( Bifidobacterium < RTI ID = 0.0 > infantis ) and fermenting the same.

Step (1) is a step of preparing a complex extract, preferably a step of preparing an extract of a mushroom, a mushroom, a cuttlefish, a squid skin, a rhubarb and a rockfish.

More preferably, in the present invention, the complex extract is preferably prepared by pulverizing a ginseng, mackerel, cuttlefish, squid skin, rhubarb and rhizome with a nanoparticle followed by ultrasonic extraction. The size of the nanoparticles is in the range of 100 to 500 nm Homogenized particles are preferable. In order to produce nano-extracts, it is preferable to prepare nanoparticles after preparing powders of moxa, manganese, persimmon, squid skin, rhubarb and rhizome, and the powder is physically pulverized using a nano mill. The supercritical fluid extraction process can be widely used in pharmaceuticals, foods, cosmetics, etc., because it has no toxic solvent or surfactant in the particles compared to other extraction processes. RESS (Rapid Expansion of Supercritical Solutions), GAS (Gas Anti-Solvent) (SAS), Aerosol Solvent Extraction System (ASES), Solution Enhanced Dispersion by Supercritical Fluids (SEDS), Particles from Gas Saturated Solutions (PGSS), and Reactive Precipitation in Supercritical Solution (RPSS).

In the step (2), the complex extract is mixed with the lactic acid bacteria culture medium for fermentation of the lactic acid bacteria. The culture medium for lactic acid bacteria may be a conventional medium known in the art. The medium may comprise various carbon sources, nitrogen sources and trace element components. Examples of carbon sources that can be used include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose, fats such as soybean oil, sunflower oil, castor oil, coconut oil, fatty acids such as palmitic acid, stearic acid, linoleic acid, Alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These carbon sources may be used alone or in combination. Examples of nitrogen sources that may be used include organic nitrogen sources such as peptone, yeast extract, gravy, malt extract, corn steep liquor, and soybean wheat, and inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, . These nitrogen sources may be used alone or in combination. The medium may include potassium dihydrogenphosphate, dipotassium hydrogenphosphate and the corresponding sodium-containing salts as a source. It may also include metal salts such as magnesium sulfate, manganese sulfate or iron sulfate. In addition, sodium acetate, ammonium stearate, calcium chloride, amino acids, vitamins and suitable precursors may be included. During the culture, the pH can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid in a suitable manner. Preferably, Lactobacillus culture media include MRS (de Man, Rogosa and Sharpe) liquid medium, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F- 12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium) and Isocove's Modified Dulbecco's Medium.

In step (3), fermentation refers to a general process in which a microorganism changes its organic substance while decomposing. Through such a fermentation process, the microorganism selectively extracts the effective ingredient contained in the raw material of the herbal medicine, These metabolites are called fermentation broth.

Lactic acid bacteria for fermentation of lactic acid bacteria are Enterococcus faecium , Enterococcus faecalis , Lactobacillus < RTI ID = 0.0 > plantarum), Lactobacillus spread lactofermentum (Lactobacillus fermentum), Lactobacillus lactis (Lactobacillus lactis , Lactobacillus rhamnosus , Lactobacillus < RTI ID = 0.0 > sakei , Lactobacillus brevis , Lactobacillus < RTI ID = 0.0 > graminis , Leuconostoc citreum , Weissella < RTI ID = 0.0 > hellenica), Lactococcus taught rain (Lactococcus garvieae), Peddie Oh Caucus pen Tosa three mouse (Pediococcus pentosaceus ), Pediococcus ( Pediococcus) acidilactici), Bifidobacterium No Marlies (Bifidobacterium animalis) and Bifidobacterium Infante Tees (Bifidobacterium infantis ) can be used. The above-mentioned lactic acid bacteria can be used preferably by concentration at 10 ¹⁰ to 10 ¹¹ CFU / ml.

In the present invention, the cultivation and fermentation of the lactic acid bacteria can be carried out according to a suitable culture medium and culture conditions known in the art. Such a process can be easily adjusted by those skilled in the art according to the selected strain.

During the culture, bubbles can be inhibited by using an expanded body such as fatty acid polyglycol ester. In addition, oxygen or an oxygen-containing gas (e.g., air) may be injected to maintain the exhalation state. The temperature at the time of culturing the lactic acid bacteria is usually from 25 to 40 DEG C, preferably from 35 to 40 DEG C, and preferably from 7 to 40 hours. The lactic acid bacteria preferably have a concentration of from 10 ¹⁰ to 10 ¹¹ CFU / ml Can be used.

By fermentation using these lactic acid bacteria, the polymer which can not be absorbed into the skin due to its large size is decomposed into small molecules by the action of lactic acid bacteria. During the decomposition process, the activity of active ingredients is increased by various enzymes of lactic acid bacteria, There is also an effect of height. In addition, toxic substances such as pesticides and heavy metals can be decomposed or inactivated to remove elements that are harmful to the skin. After the fermentation process, the lactic acid bacteria decompose and digest the molecules that make up the extract, and excrete them. Since the fermentation process of the present invention is carried out at a low temperature, lactic acid bacteria can not effectively lose the appearance of the raw material at room temperature without effectively losing or modifying components caused by conventional hot water extraction or vacuum extraction, have.

The cultivation and fermentation of the lactic acid bacterium can be carried out according to a suitable culture medium and culture conditions known in the art. Such a process can be easily adjusted by those skilled in the art according to the selected strain. These various methods are disclosed in various documents. Suspension cultures and adherent cultures can be divided into batches, fed-batch and continuous-batch cultures according to the culture method. During the culture, bubbles can be inhibited by using an expanded body such as fatty acid polyglycol ester. Also, oxygen or an oxygen-containing gas (e.g., air) may be injected to maintain the exhalation state. The temperature at the time of culturing is usually 25 ° C to 40 ° C, preferably 35 ° C to 40 ° C, and the culturing time may be 7 hours to 40 hours. The complex extract may be contained in an amount of 1 to 2% by weight based on the culture medium for lactic acid bacteria.

After completion of the incubation, the culture can be subjected to a process of contrifugation or filtration in order to separate the cells from the culture, and these steps can be carried out as required by those skilled in the art. At this time, the removal of the cells indicates that the lactic acid bacterium cells have been substantially removed through the conventional method for removing the cells.

The thus-produced complex fermentation extract of the present invention can be used for whitening purposes.

In addition, the cosmetic composition of the present invention can provide an improvement effect of atopic dermatitis and a skin whitening effect on a skin pigmented with atopic dermatitis.

In a preferred embodiment of the present invention, the content of the complex fermentation extract is 0.001 to 10.0% by weight, preferably 0.1 to 1% by weight, with respect to the total cosmetic composition. If the content of the complex fermentation extract is less than 0.001% by weight, a whitening effect and atopy improvement effect can not be expected. If the content of the complex fermentation extract is more than 10.0% by weight, skin irritation is caused and the formulation is not stable. May be undesirable.

The cosmetics according to the present invention may be manufactured and commercialized in any form of these formulations in order to achieve the object of the present invention, such as creams, packs, lotions, essences, cosmetics, foundation oils and makeup bases, . Preferably, the cosmetic composition may comprise one formulation selected from the group consisting of softening agents, nutritional lotions, nutritional creams, massage creams, essences, eye creams, cleansing creams, cleansing foams, cleansing waters, packs, have.

In addition, the cosmetic composition according to the present invention can optionally be added with purified water, a moisturizing agent, a higher alcohol, a higher fatty acid, an emollient, a chelating agent, an increasing agent, a permanent component, a pH adjusting agent, have.

The present invention can be utilized as an auxiliary or alternative means for treatment or whitening of atopic dermatitis using a composite fermented extract of marine material, and the active ingredient can penetrate deeply into the skin through nanoporous method.

1 shows the content of fluorotannin in the organic solvent fraction of the complex extract.
FIG. 2A shows the result of confirming the toxicity of a mixed alcohol (ethanol) extract to B16 melanoma cells.
FIG. 2b shows the result of confirming the toxicity of the complex ethyl acetate fraction to B16 melanoma cells. Figure 3 shows the effect of the combined alcoholic extract and ethyl acetate fraction on the production of melanin in B16 cells.
Fig. 4A shows the results of confirming toxicity of diacetate to B16 melanoma cells. Fig.
4B shows the result of confirming the toxicity of dioxinodihydrocoke to B16 melanoma cells.
4C shows the results of confirming the toxicity of flurofucofuroform A to B16 melanoma cells.
FIG. 5 shows the effect of phlorotannines on the production of melanin in B16 cells. (A) is a control group, (B) is a group treated with α-MSH, (E), α-MSH and dioxinodihydrocoke (20 μM) treated group, (F) treated group with α-MSH and di- MSH and fluro fucopurea ECol A (20 uM).
6A shows the effect of diacetal on the production of melanin in B16 cells in B16 melanoma cells treated with? -MSH.
6B shows the effect of dioxinodihydrocule on the production of melanin in B16 cells in B16 melanoma cells treated with? -MSH.
Fig. 6C shows the effect of flurofucofuroecol A on the production of melanin in B16 cells in B16 melanoma cells treated with? -MSH.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example 1. Preparation of complex fermented extract

1-1. Manufacture of complex extracts

20kg of each of Momopsis mackerel, mung bean, mung bean, squid, rhubarb and stone lobster, which are native to the sunshine of Busan, Gyeonggi-gun, Busan, were harvested, dried, pulverized and stored at -20 ℃. A certain amount (2.5 kg) of crushed mackerel, mung bean, mackerel, squid skin, rhubarb and rhododendron were placed in a bowl with a reflux condenser, and then 10 L of alcohol was added thereto and then extracted for 3 hours in a water bath. The extract was filtered and concentrated using a rotary vacuum evaporator (40 ° C). The above procedure was repeated one more time to obtain a total of 670 g of the alcohol extract. The extract was dissolved in a mixed solvent of H ₂ O: ethanol (9: 1, v / v), poured into a separatory funnel and equilibrated with equal volume of n-hexane by shaking the separatory funnel. After that, the upper portion of the soluble portion of n-hexane is collected, treated with sodium sulfate (anhydrous), filtered and concentrated. This procedure was repeated four more times to obtain 77 g of n-hexane fraction. In the same manner, CH ₂ Cl ₂ was added to the H ₂ O layer to collect the CH ₂ Cl ₂ soluble portion in the lower layer to obtain 8.5 g of CH ₂ Cl ₂ fraction. EtOAc was added to the H ₂ O layer, and the EtOAc-soluble fraction of the upper layer was collected to obtain 49 g of EtOAc fraction. Also, n-BuOH was subjected to the same procedure to obtain 72 g of the upper n-BuOH fraction and 353 g of the lower H ₂ O fraction

1-2. Preparation of complex fermented extract

The combined extracts obtained in 1-1 above were added to a medium based on MRS medium to adjust the pH to 5.5 and sterilized with a high pressure sterilizer at 100 ° C or higher for 10 minutes. After the sterilized sample was cooled to 42 ° C, the culture of the mixed culture of the lactic acid bacteria was inoculated at 2% by volume and fermented in a thermostat at 37 ° C for 15 hours. Mixed strains of Lactobacillus plantarum ATCC 10241 and Lactococcus garvieae ATCC 11454 (Production Example 1), Lactobacillus plantarum ATCC 10241 and Lactococcus garvieae LMG 8162 (Preparation Example 2), Lactobacillus rhamnosus ATCC 7469 and Lactococcus garvieae ATCC 11454 (Preparation Example 3), Lactobacillus rhamnosus ATCC 7469 and Lactococcus garvieae LMG 8162 (Production Example 4), Lactococcus garvieae ATCC 11454 and Pediococcus pentosaceus ATCC 33316 (Production Example 5) were respectively used. Lactococcus garvieae LMG 8162 was purchased from the Belgian Co-ordinated Collections of Micro-organisms (BCCM) and the rest were all sold by the American Type Culture Collection (ATCC).

35 kg of the present culture medium was prepared in the same manner as the above culture broth in the fermenter, 3.5 kg of the preculture was inoculated, and cultured at 30 to 37 ° C for 12 to 20 hours to prepare a culture. After the cultivation was completed, the temperature of the fermenter was raised to about 70 캜 or higher and maintained for 30 minutes to inactivate the bacteria. Thereafter, the temperature of the fermenter was cooled to about 50 캜, and the culture broth was centrifuged to separate the cells and the culture filtrate. Activated carbon corresponding to about 1.0 to 2.0% of the total volume of the culture filtrate was added to the separated culture filtrate and decolorized and deodorized for about 3 hours. The filter cake was composed of filter press, diatomite + filter cloth, filter press temperature The filter press pressure is about 1 to 2 atmospheres, the pore size of the filter cloth is about 5 μm, and the amount of diatomaceous earth used is about 5% of the total volume of the filtrate to be treated) to remove activated carbon and impurities And a purified culture filtrate was obtained. The purified culture filtrate was concentrated to obtain about 3.5 kg of concentrated culture filtrate having a solid concentration of about 20 Brix. The concentrated culture filtrate was lyophilized at a trap temperature of about -80 占 폚 or lower for about 40 hours or longer to obtain a dry powder of the complex fermentation extract.

1-3. Analysis of phlorotannin content in extracts

The concentration of phlorotannin in the fractions of the complex extracts was considered to be high, and the content of phlorotannin in each fraction was measured and compared. The results showed that the EtOAc fraction had the highest amount of phlorotannin and was 4 times higher than the other fractions (FIG. 1).

Example 2. Evaluation of whitening efficacy of complex fermented extract

2-1. Antioxidant activity evaluation (DPPH radical scavenging ability)

Highly antioxidant compounds such as α-tocopherol and vitamin C derivatives have also been shown to play an important role in inhibiting melanin production, which can be used as an indicator of whitening effects. Therefore, DPPH radical scavenging activity was measured in order to confirm antioxidant activity of the extracts and fractions. As a result, the extracts and fractions of the complex were found to be excellent in DPPH radical scavenging activity and the highest activity in the ethyl acetate fraction (Table 1).

Fraction EC ₅₀ ( ug / ml ) EtOH 23.6 ± 0.5 n-Hexane 12.9 ± 0.5 CH ₂ Cl ₂ 12.0 ± 2.3 EtOAc 4.4 ± 0.2 n-BuOH 12.7 ± 0.6 H ₂ O > 200 Ascorbic acid 1.8 ± 0.0

2-2. Mushroom Tyrosinase  Activity inhibition assessment

The effect of inhibiting tyrosinase activity was examined to confirm the whitening activity of the extracts and fractions of the complex. Complex alcohol extract and a 10 ul sample and the mushroom tyrosinase diluted fractions (1,000 U / ml, 50 mM phosphate buffer, pH 6.5) 20 ul, and _L -DOPA solution _{(1 mM L -DOPA: 50 mM} phosphate buufer: DW = 10: 10: 9), and reacted at room temperature for 30 minutes. Then, the absorbance was measured at 490 nm to measure tyrosinase activity. As shown in Table 2, the tyrosinase inhibitory activity was observed in the extracts of the combined alcoholic beverages, the dichloromethane fraction and the ethyl acetate fraction, and the ethyl acetate fraction showed the highest activity (Table 2).

Fraction IC ₅₀ ( ug / ml ) EtOH 352.2 + - 4.4 n-Hexane > 500.0 CH ₂ Cl ₂ 176.5 ± 8.2 EtOAc 58.7 ± 1.2 n-BuOH > 500.0 H ₂ O > 500.0 Arbutin 112.2 ± 2.4

2-3. Intracellular Tyrosinase  Activity inhibition assessment

The tyrosinase inhibitory activity of the mushroom of the combined extracts was confirmed. To confirm the effect of these compounds on the tyrosinase activity of the mammals, B16 melanoma cells were used to confirm the effect on intracellular tyrosinase activity. B16 melanoma cells cultured according to the method of Tsuboi et al. (1998) were washed twice with PBS and then disrupted using lysis buffer (0.1 M sodium phosphate, pH 6.8, 0.5% Nonidet P-40) Respectively. The cell lysate was centrifuged at 4,000 rpm for 10 minutes at 13,000 rpm to recover the supernatant containing the intracellular enzyme. The effect of tyrosinase in 10 μl of cell lysate and _L- DOPA solution (2 mg / ml, 0.1 M sodium phosphate, pH 6.8) was observed in 200 μl of a sample diluted in 0.1 M sodium phosphate (pH 6.8). As shown in Table 3, inhibition of tyrosinase activity was observed in the combined alcohol extract, dichloromethane fraction and ethyl acetate fraction (Table 3).

Fraction IC ₅₀ ( ug / ml ) EtOH 301.2 ± 10.1 CH ₂ Cl ₂ 155.4 ± 14.4 EtOAc 55.9 ± 6.7

2-4. Evaluation of melanin production efficacy

Derived B16 melanoma cells showing a specific response to melanin production were used in order to examine the effect of the combined extracts of the extracts exhibiting tyrosinase inhibitory activity and the ethyl acetate fraction having the highest effect on melanogenesis in mammalian skin cells. Experiments were carried out to examine the production of melanin in the cells after culturing the cells by treating α-MSH and diluted samples inducing melanin production.

B16 cells were cultured for a predetermined period of time on a sample-treated medium to confirm the toxicity of B16 melanoma cells to the extracts of the combined alcoholic extracts and the ethyl acetate, followed by the addition of MTS, 3- (4,5-dimethylthiazol-2 -yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2-H-tetrazolium reagent and reacted for 1 hour. The resulting purple insoluble formazan was analyzed by spectrophotometer ELISA The enzyme-linked immunosorbent assay was used to determine the cell viability by measuring absorbance at 490 nm. As a result, the combined alcohol extract and ethyl acetate fraction showed no toxicity to B16 cells at a concentration of 100 ug / ml (FIG. 2).

The effect of the combined alcohol extract and ethyl acetate fraction on melanin production in B16 cells was determined by the method developed by Hosoi et al. B16 cells cultured for 72 hours in a medium containing diluted samples and α-MSH were collected, dissolved in 1 N NaOH (10% DMSO) and heated at 60 ° C for 30 minutes. The lysates were centrifuged at 5,000 rpm for 5 minutes, and the absorbance of the supernatant was measured at 490 nm. As a result, it was shown that the combined alcohol extract and ethyl acetate fraction inhibited melanin production by α-MSH in a concentration-dependent manner (FIG. 3).

2-5. Isolation of active ingredients and evaluation of whitening efficacy

As described above, the content of the polyphenolic compound in the ethyl acetate fraction was three times higher than that of the other fractions, and the antioxidant activity, mushroom tyrosinase enzyme inhibitory activity, B16 cell tyrosinase enzyme inhibitory activity, and B16 cell melanin production inhibitory activity appear. Therefore, ethyl acetate (EtOAc) fractions were tried to isolate compounds with excellent activity. The EtOAc fractions were analyzed using an HPLC system (pump, Shimadzu LC-6AD; Shimadzu SPD-M20A; degasser, Shimadzu DUG-20A ₃ ; Shimadzu LC-10A; system controller, CBM-20A; Shimadzu LC solution, ver.1.22sp). The EtOAc fractions were separated by molecular weight using an Asahipak GS-310 column. Five fractions (GS1-GS5) were obtained. Reversed phase chromatography of the active GS3 and GS4 fractions with Shim-pack PREP-ODS (5 μm, 100 Å, 250 mm × 20 mm, Shimadzu Co., Tokyo, Japan) resulted in eight phlorotannin compounds, The structure was analyzed by ¹³ C- and ³ H-NMR. As a result, it was found that eckol, 2-phloroeckol, phlorofucofuroeckol A and B, 6 ', 6'- (6 ', 6'-bieckol), dieckol, dioxinodehydroeckol, and phloroglucinol have been identified.

In order to confirm the whitening activity of dioctol, dioxinodihydrocoke and flurofucofuroxecol A among the isolated fluorotannines, mushroom tyrosinase inhibitory activity and B16 cell tyrosinase inhibitory activity were measured. As shown in Table 4, the tyrosinase inhibitory activity of diacetone was the highest among the isolated fluorotannines, and the inhibitory activity of the other two compounds was found to be low.

ingredient EC ₅₀ ( uM ) Mushroom
Tyrosinase B16 cells
Tyrosinase Dai Ecol 2.9 ± 0.2 3.8 ± 0.7 Dioxinodihydrocoke > 200 > 200 Florofuco furo ecol A > 200 > 200

Tyrosinase enzymes, which play an important role in intracellular melanin production, are regulated by MITF transcription factors. This means that even if the enzyme does not directly affect the activity of the tyrosinase enzyme, it can participate in intracellular melanin production if it can regulate the transcription of the enzyme through a variety of signal transduction systems from outside the cell. Therefore, three kinds of phlorotannin compounds isolated from complex extracts were treated with α-MSH to confirm the production of melanin in the cells after culturing B16 cells.

In order to confirm the toxicity of phlorotannins to B16 melanoma cells, B16 cells were cultured for a certain period of time on a sample-treated medium, treated with MTS reagent, and reacted for 1 hour to obtain purple insoluble formazan The cell viability was determined by measuring the absorbance under a wavelength of 490 nm with a spectrophotometer ELISA reader. As a result, no significant toxicity was observed for B16 cells in the concentration range of 10, 20, and 40 uM, respectively, for diacetal, dioxinodihydrococcal, and flurofucofurococcole A (FIG. 4).

B16 cells were cultured in the same manner as described above to confirm the effect of the phlorotannines isolated from the complex extract on the production of melanin in B16 cells. When the cells were observed under an optical microscope, it was confirmed that the three kinds of the fluorotannines inhibited intracellular melanin production within a range that does not cause toxicity. In addition, as a result of measuring the absorbance of the cell eluate at 490 nm, all three kinds of phlorotannins isolated from the complex extract inhibited the melanin production by α-MSH in a concentration-dependent manner (FIGS. 5 and 6 ).

Among the fluorotannines isolated from the complex extracts, dioxinodihydrocoke and fluorofucofuroxole A showed low inhibitory activity of tyrosinase enzyme but effectively inhibited intracellular melanin production. Therefore, the whitening effect of these three compounds was analyzed by the concentration - dependent method. As a result, the whitening activity was about 1-10 higher than that of the arbutin. Therefore, these three compounds are thought to modulate the activity of MITF transcription factors, which regulate melanogenesis, rather than direct tyrosinase inhibitory activity.

Example  3. Assessment of improvement effect of complex fermented extract on atopic dermatitis

3-1. Atopic dermatitis reduction effect

Twenty-five NC / Nga mice (SPF, 5 weeks, female, Japan) weighing 13-22g were separated from each other in a general slaughterhouse where they could freely eat water and food, and kept growing for 12 hours. Dermatophagoides pteronyssinus crude extract (DPE) was applied to 15 ears of NC / Nga mice for 1 week (total 3 times) to induce the parotitis. In addition, (1) DPE treated group (5), DPE treated group (5), and DPE treated group (3) were treated with other atopic dermatitis remedy (5), (5), (5), and (5) groups that were not treated at all, were administered once a week for a total of 4 weeks, Before topical application, the degree of ear dermatitis was evaluated according to the following criteria, and the results are shown in Table 5. Table 5 shows that the complex extract of the present invention is superior to the conventional atopic dermatitis in the treatment of inflammation.

Evaluation criteria: No inflammation, 0; Slight inflammation or scarring, 1; Moderate inflammation, scarring or slight bleeding, 2; There is inflammation or hemorrhage or ulcer or loss of ear tissue, 3.

n A week later Two weeks later After 3 weeks After 4 weeks After DPE treatment
Compound extract application group 5 2 One 0 0 After DPE treatment
Group applied only base 5 3 3 3 3 After DPE treatment
Other atopic dermatitis treatments 5 3 2 2 One Only the complex extracts 5 0 0 0 0 Untreated group 5 0 0 0 0

3-2. Ear edema reduction effect

In the experimental procedure of Example 3-1, ear thickness was measured weekly using dial caliper (KORI SEIKI MFG.Co, Japan) for each group. As a result, as shown in Table 6, ear swelling increased to about 70% over time in the group treated with DPE only, but after the DPE treatment, The ear edema increased to about 30% and gradually decreased to 0, indicating a superior ear edema suppression effect than the conventional atopic dermatitis remedy.

n A week later Two weeks later After 3 weeks After 4 weeks After DPE treatment
Compound extract application group 5 32 21 13 2 After DPE treatment
Group applied only base 5 55 62 65 70 After DPE treatment
Other atopic dermatitis treatments 5 39 35 28 19 Only the complex extracts 5 0 0 0 0 Untreated group 5 0 0 0 0

3-3. Skin Humidity  Measure

In the experiment of Example 3-1, the humidity of the back skin was measured weekly using a corneometer CM820 (Courage & Khazaka, Cologne, Germany) and a tewameter TM210 (Courage & Khazaka, Cologne, Germany) for each group.

As a result, as shown in Table 7, in the group to which only the base was applied after the DPE treatment, the humidity of the skin decreased with time and reached about 20%. However, after the DPE treatment, At first, as the time passed, the moisture content of the skin gradually recovered, gradually increasing the skin moisturizing effect compared with the conventional treatment for atopic dermatitis.

(unit : %) n A week later Two weeks later After 3 weeks After 4 weeks After DPE treatment
Compound extract application group 5 55 56 59 63 After DPE treatment
Group applied only base 5 52 39 31 25 After DPE treatment
Other atopic dermatitis treatments 5 50 47 52 55 Only the complex extracts 5 78 74 75 75 Untreated group 5 70 68 65 62

Claims (9)

Ecklonia stolonifera , Sargassum fulvellum ), Ecklonia cava , squid shell, rhubarb ( Eisenia bicyclis , and Gigartina tenella . The present invention relates to a cosmetic composition for improving and whitening atopic dermatitis.
[3] The combination fermented extract according to claim 1, wherein the complex fermentation extract comprises 0.1 to 20% by weight of a mugwort extract, 0.1 to 20% by weight of mugwort extract, 0.1 to 20% And 0.1 to 20% by weight of a rock extract.
[Claim 7] The cosmetic composition according to claim 1, wherein the complex fermentation extract is produced by fermenting Mycobacterium tuberculosis, mung bean, mackerel, squid skin, rhubarb and rhizome extract with lactic acid bacteria.
4. The method of claim 3 wherein the lactic acid bacterium is Enterococcus par when Titanium (Enterococcus faecium), Enterococcus faecalis (Enterococcus faecalis), Lactobacillus Planta Room (Lactobacillus plantarum), Lactobacillus spread lactofermentum (Lactobacillus fermentum), Lactobacillus lactis (Lactobacillus lactis), Lactobacillus ramno Saskatchewan (Lactobacillus rhamnosus), Lactobacillus four K (Lactobacillus sakei), lactose Lactobacillus brevis , Lactobacillus graminis , Leuconostoc citreum , Weissella hellenica , Lactococcus garvieae , Pediococcus penne, Lactobacillus spp ., Lactobacillus spp . Tosa three-house two or more kinds selected from the group consisting of (Pediococcus pentosaceus), Phedi O Lactococcus CD rakti when (Pediococcus acidilactici), Bifidobacterium no Marlies (Bifidobacterium animalis) and Bifidobacterium Infante tooth (Bifidobacterium infantis) in ≪ / RTI >
The method of claim 4, wherein the Enterococcus par when Titanium (Enterococcus faecium) is at least one selected from the group consisting of Enterococcus par when Titanium (Enterococcus faecium) ATCC 19634 or Enterococcus par when Titanium (Enterococcus faecium) ATCC 6057,
The Enterococcus faecalis ) is an Enterococcus faecalis ) ATCC 6057,
The Lactobacillus and Bacillus Planta room (Lactobacillus plantarum) is Lactobacillus Planta room (Lactobacillus plantarum) ATCC 10241, or Lactobacillus Planta room (Lactobacillus plantarum) one or more selected from the NCTC 7220,
The Lactobacillus < RTI ID = 0.0 > fermentum a) is Lactobacillus buffer lactofermentum (Lactobacillus fermentum) ATCC 14931, or Lactobacillus buffer lactofermentum (Lactobacillus fermentum) one or more selected from NCDO 1750,
The Lactobacillus and lock teeth (Lactobacillus lactis) is Lactobacillus lactis (Lactobacillus lactis) ATCC 11454, or Lactobacillus lactis (Lactobacillus lactis) one or more selected from the group consisting of NCIB 8586,
The Lactobacillus ramno a suspension (Lactobacillus rhamnosus) is at least one selected from the group consisting of Lactobacillus ramno suspension (Lactobacillus rhamnosus) ATCC 7469, or Lactobacillus ramno suspension (Lactobacillus rhamnosus) KCTC 3326,
The Lactobacillus and Bacillus four K (Lactobacillus sakei) is at least one selected from the group consisting of Lactobacillus four K (Lactobacillus sakei) ATCC 31063, or Lactobacillus four K (Lactobacillus sakei) ATCC 15521,
The Lactobacillus brevis is Lactobacillus brevis ATCC 15521,
The Lactobacillus graminis is Lactobacillus graminis ATCC 51150 and the Lactobacillus graminis is Lactobacillus graminis ATCC 51150,
The Leuconostoc citreum is Leuconostoc citreum ATCC 49370, the Leuconostoc citreum is Leuconostoc citreum ATCC 49370,
And the by-Cellar helre NIKA (Weissella hellenica) is by Cellar helre NIKA (Weissella hellenica) ATCC 51523,
The Lactococcus garvieae is selected from the group consisting of Lactococcus garvieae CCUG 11672, Lactococcus garvieae ATCC 11454, or Lactococcus garvieae LMG 8162 One or more,
The Pediococcus pentosaceus is Pediococcus pentosaceus ATCC 33316, the Pediococcus pentosaceus is Pediococcus pentosaceus ATCC 33316,
Wherein the Pediococcus acidilactici is Pediococcus acidilactici ATCC 33314, the Pediococcus acidilactici is Pediococcus acidilactici ATCC 33314,
The Bifidobacterium animalis is Bifidobacterium animalis ATCC 27672,
The Bifidobacterium Infante Tees (Bifidobacterium infantis) are Bifidobacterium Infante Tees (Bifidobacterium infantis) ATCC15697 of the cosmetic composition.
The cosmetic composition according to claim 1, wherein the composition comprises bacteriocin produced by lactic acid bacteria.
The cosmetic composition according to claim 1, wherein the cosmetic composition comprises one formulation selected from the group consisting of softening agents, nutrition lotions, nutritional creams, massage creams, essences, eye creams, cleansing creams, cleansing foams, cleansing waters, packs, ≪ / RTI >
A step of preparing a complex extract by extracting a mushroom, a mushroom, a mushroom, a squid skin, a rhubarb and a rhizome;
Mixing the complex extract with a culture medium for lactic acid bacteria; And
Enterococcus faecium , Enterococcus faecalis , Lactobacillus plantarum , Lactobacillus fermentum , Lactobacillus lactis , and Lactobacillus lactis are added to the lactic acid bacteria culture medium, ), Lactobacillus rhamnosus , Lactobacillus sakei , Lactobacillus brevis , Lactobacillus graminis , Leuconostoc citreum , Bacillus subtilis, Weissella hellenica , Lactococcus garvieae , Pediococcus pentosaceus ), Pediococcus ( Pediococcus) acidilactici), Bifidobacterium No Marlies (Bifidobacterium animalis) and Bifidobacterium Infante Tees (Bifidobacterium infantis , and fermenting at least two kinds of lactic acid bacteria selected from the group consisting of < RTI ID = 0.0 >
A method for producing a cosmetic composition according to claim 1.
The method according to claim 8, wherein the lactic acid bacteria are cultured at a temperature of 25 ° C to 40 ° C, a culturing time is 7 hours to 40 hours, and the lactic acid bacteria are used at a concentration of 10 ¹⁰ to 10 ¹¹ CFU / Way.

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