KR20120138308A - A cosmetic composition for anti-atopic skin desease comprising mitol and fucoidan mixture as an efficient component isolated from ecklonia cava - Google Patents

Abstract

PURPOSE: A composition containing Ecklonia cava-derived mitol and fucoidan and an external use skin formulation are provided to ensure an anti-inflammatory effect and to treat atopic dermatitis. CONSTITUTION: A composition with an anti-inflammatory effect contains 0.3 wt% of Ecklonia cava-derived mitol and 0.5-2 wt% of fucoidan. The mitol contains polyphenol derived from Ecklonia cava as an active ingredient. A method for preparing fucoidan comprises: a step of drying Ecklonia cava and extracting Ecklonia cava with 80% ethyl alcohol at room temperature for 12 hours; a step of centrifuging the extract and collect pellet; a step of mixing the pellet with 80% ethyl alcohol and stirring at room temperature for 12 hours; a step of centrifuging and collecting pellet; a step of removing pigment, lipid, and low molecular proteins using acetone and drying to prepare Ecklonia cava powder; a step of adding distilled water to the Ecklonia cava powder and extracting at 60 Deg.C. for 2 hours, centrifuging, and collecting supernatant; a step of adding CaCl_2 to supernatant and incubating at 4 Deg.C. for 18 hours; a step of centrifuging and collecting supernatant; and a step of concentrating the supernatant and adding ethyl alcohol.

Description

A cosmetic composition for anti-atopic skin desease comprising mitol and fucoidan mixture as an efficient component isolated from Ecklonia cava} Eclonia cava}

The present invention relates to the development of a mixed material of mithol and fucoidan derived from Ecklonia cava having anti-inflammatory activity, and more specifically, atopy containing the mixture as an active ingredient at a constant ratio of mititol and fucoidan derived from Ecklonia cava. It relates to a cosmetic composition for improving dermatitis.

Algae usually refers to the giant algae that grow or grow in the sea, and are classified into three types of brown algae, green algae, and red algae depending on the depth of inhabitants and the color of the algae. Most of the seaweeds sold commercially in Korea are mainly produced by aquaculture, and seaweeds such as seaweed, kelp, and seaweed are typically distributed in Korea. Among algae, brown algae are generally multicellular algae, with about 250 genera and 1,500 species known throughout the world. Algae has the most developed system, and there are few single cells or colonies, and most of them are divided into filamentous or stick-like tree branches. A typical feature of brown algae is the differentiated organelles within the cell, as well as chlorophyll a and chlorophyll c as anabolic pigments. In addition, assimilation storage materials are mannitol, laminarin, etc., which are a kind of carbohydrate, and the host cells have two long and short flagella on the side. Among brown algae, Ecklonia cava grows at a depth of about 10m and grows up to 1 ~ 2m long. The stem is cylindrical, the root is root-shaped, and at the end of the stem is a flat leaflet with side strips. The leaf is about 1 m long and brown, but when it is dried, it becomes black, thick, leathery, with pinnate small leaves on both sides. Ecklonia vulgaris is an important algae plant constituting marine forests, and is mainly used to feed abalone and conch. Like brown seaweed and seaweed, it is the main raw material for making alginic acid, iodine, and potassium, and can be collected and used for food, but it is rarely consumed. In Korea, it is mainly distributed in the south coast and Jeju island. In recent years, "mytol", a component of polyphenols contained in large amounts in Ecklonia cava, has been used as a raw material for medicines and health functional foods.

Anti-inflammatory (anti-inflammatory) substances are used to suppress inflammatory symptoms such as edema, erythema, pain, and fever, and to reduce tissue damage caused by inflammation. Inflammation is a normal immune response for biological defense. Cell damage leads to the release of histamine, serotonin, kinin, prostagladins, and leukotrienes, causing fever and pain. Then, redness and swelling due to fibrillation membrane expansion and capillary permeability hyperactivity occur. Atopic dermatitis is a representative disease of sensitive and dry skin and is the result of abnormal secretion of inflammation-causing substances and the abnormal operation of the immune system, a bioprotective mechanism. Steroid-containing ointments should not be used for atopic dermatitis, and treatments using natural-derived substances having an inhibitory effect on inflammatory secretion have recently been received. Mititol, a polyphenol derived from Ecklonia cava, has been reported to be a natural compound having not only excellent antioxidant activity, but also excellent anti-inflammatory activity. Looking at the patents derived from Ecklonia cava so far, the whitening cosmetics containing Ecklonia cava extract of Republic of Korea Patent No. 2002-0015816 is a whitening cosmetic composition excellent in tyrosinase inhibitory effect using the extract obtained after solvent extraction In Korean Patent No. 2001-0098018, 'New material separated from Ecklonia cava, a method for extracting and purifying it and using it as an antioxidant,' is used for extracting new material from Ecklonia cava and using it as an antioxidant. A method for extracting and purifying novel substances; Solvent fractionating the extracted material; And purifying the solvent fractions by chromatography. In addition, the material extracted from Ecklonia cava according to the present invention has a high antioxidant activity and excellent thermal stability, it is disclosed that it can be usefully applied as a natural antioxidant. In addition, recently, only the lowest polar compound among the composite materials included in the mithol was fractionated from ecchimatitol hexane using a nonpolar solvent hexane. The substance was named "Mitol" and mititol was also found to have excellent antioxidant and anti-inflammatory activity. To date, studies on the anti-inflammatory properties of mixed materials of E. coli mititol-derived mititol and fucoidan have been conducted. none.

Accordingly, the present inventors carried out the present invention to industrially use the Ecklonia native to Korea, but hardly edible. In particular, as a result of intensive research to extract nonpolar substances from polyphenols of Ecklonia cava, and to industrially use the remaining by-products of polyphenols, Ecklonia cava mitol and fucoidan are mixed when mititol and fucoidan are derived from E. coli mititol. When used alone it was confirmed to exhibit higher anti-inflammatory activity. Therefore, by using the mixed material of Ecklonia mitol and fucoidan in an appropriate ratio to find out the availability of anti-inflammatory and atopic improvement cream to complete the present invention.

Accordingly, an object of the present invention is to provide a novel use of a material in which mititol-derived mititol and fucoidan are mixed.

Another object of the present invention is to provide a topical cosmetic composition and external preparation for improving atopic dermatitis having an anti-inflammatory activity containing a mixed material prepared by the above method.

The above object of the present invention is Ecklonia cavit ), after extracting the mititol-derived mithol, to extract the fucoidan from the remaining residue to prepare a mixed material of these, in vitro and in vivo when using the prepared mixed material and the mithol and fucoidan alone It was achieved by confirming anti-inflammatory activity and atopic improvement effect in.

The method according to the present invention comprises the steps of mixing the mithol and fucoidan derived from Ecklonia; And applying and evaluating the prepared mixed material as an external skin preparation for anti-inflammatory activity and atopic improvement.

The mixed material of mititol and fucoidan derived from Ecklonia cava according to the present invention has an excellent effect of improving anti-inflammatory activity and atopy than when Ecklonia mititol or fucoidan is used alone.

1 shows Effect of cavanol, mitol and mitol + fucoidan treatment on the viability of Raw 264.7 cells.
Figure 2 shows the Effect of cavanol, mitol and mitol + fucoidan treatment on NO production from LPS induced Raw 264.7 cells.
Figure 3 shows the Suppression effect of cavanol, mitol and mitol + fucoidan treatment on inflammation-associated gene expression in Raw 264.7 cells.
4 is a macroscopic skin appearance observation result.
5 shows the results of observation of the microscopic skin appearance.
6 shows the result of skin thickness measurement.
7 shows the Spleen index measurement results.
8 is a result of measuring serum IgE concentration.
9 is a measurement result of Interleukin-4 concentration.
10 is a result of measuring Interferon-γ concentration.

The present invention provides a cosmetic composition for improving atopic dermatitis comprising a mixed material of Ecklonia mitol and fucoidan as an active ingredient. In the present invention, the fucoidan is a polysaccharide means a sulfur-containing polysaccharide obtained by using Ecklonia cava with water and ethyl alcohol, but is not limited thereto, and may be another known polysaccharide.

In the present invention, fucoidan, a sulfur-containing polysaccharide having antioxidant and anticancer activity, was extracted from Ecklonia cava. First, the Ecklonia cava is dried, and the dried sample is subjected to stirring extraction at room temperature for 12 hours with 80% ethyl alcohol, and centrifuged to obtain pellets. The obtained pellet was stirred and extracted at room temperature for 12 hours using 80% ethyl alcohol, followed by centrifugation to obtain pellets. The obtained pellet is acetone to remove the pigment, lipids and low molecular weight protein, and then dried at room temperature to obtain dried Ecklonia cava powder.

Distilled water was added to the above-mentioned Ecklonia cava powder, stirred extraction at 60 ° C. for 2 hours, and a supernatant was obtained by centrifugation. The pellet was again extracted by stirring with distilled water at 60 ° C. for 2 hours, and then obtained a supernatant by centrifugation. 1% concentration of CaCl ₂ was added to the supernatant obtained in the above two times, and then stored at 4 ^° C. for 18 hours, and only the supernatant was obtained by centrifugation. The obtained supernatant was concentrated using a vacuum concentrator, and then fucoidan was precipitated by adding ethyl alcohol, and fucoidan was obtained by filtration.

In addition, in the extraction and separation process of mitol, Ecklonia cava is first washed with distilled water to remove foreign substances, dried in the shade, and then crushed.

500 g of the powder was refluxed for 2 hours using 20 times of 10% ethyl alcohol by weight. This extraction process was repeated twice. Then, the residue was filtered off and the ethyl alcohol extract was concentrated under reduced pressure using a rotary evaporator. The concentrated solution was suspended in 20 times distilled water, extracted three times using the same amount of ethyl acetate solvent, and the ethyl acetate fractions were collected and concentrated under reduced pressure. After loading the ethyl acetate fraction concentrate on 15 times the amount of silica gel, using a solvent mixed with ethyl acetate and acetone in a volume ratio of 9: 1, the fractions of the active ingredient is collected and concentrated to obtain an algae extract. The total polyphenol content was measured using the Pauline reagent and found to be 95.6%.

The obtained fucoidan was mixed with mititol and used to measure anti-inflammatory activity in vitro and in vivo. As a result of observing the anti-inflammatory activity, the mixed material of Ecklonia mititol and fucoidan was found to have better anti-inflammatory activity than when Ecklon mititol and fucoidan were used alone.

Hereinafter, preferred embodiments of the present invention will be described in more detail with reference to Examples. However, the scope of the present invention is not limited to these examples.

< Experimental Example  1> Ecklonia  Mytol and Fucoidan  On the effects of mixed materials on anti-inflammatory in vitro  Active investigation

Mytol and Fucoidan  Of mixed materials Macrophage proliferation Impact on

In this experiment, we investigated the anti-inflammatory effects of mithol, fucoidan, mithol and fucoidan-containing products (cavanol) and commercial atopy creams (Atofam). The expression of iNOS mRNA, a gene involved in the production of and nitrate, was investigated. Preparation of the mixed sample was 0.5% by weight of fucoidan and 2.0% by weight based on 0.3% by weight of itol.

Figure 1 shows the effect of cavanol, atopam, mititol, mithol + fucoidan mixed material on the proliferation of raw cells. As can be seen in Figure 1, compared with the no addition group, cavanol, atofam, mititol, mititol + fucoidan mixed material did not appear to have a significant effect on the proliferation of raw cells. Thus, cavanol, atopham, mititol, and mititol + fucoidan mixtures are not considered to be toxic to raw cells.

Mytol and Fucoidan  Mixed material Raw cell Secreted from NO  Inhibitory effect

In this experiment, we investigated the effect of nitro oxide, an inflammation-inducing substance secreted from raw cells, to be inhibited by cavanol, atofam, mititol, and mititol + fucoidan mixtures. As shown in FIG. 2, the NO inhibitory effect generated from the LPS-treated raw cell was suppressed depending on the concentration in all samples. Among the samples, the inhibitory effect of the commercial atopy cream, Atopame, was shown to be the lowest, and the inhibitory effects of Cavanol and Mytol were similar. On the other hand, the mixed samples containing fucoidan showed a very good effect at low concentrations (25-50 ug / mL), but showed a similar inhibitory effect to cavanol & mytol at 100 ug / mL.

Mytol and Fucoidan  Mixed material Raw cell Secreted from NO  Inhibitory effect

In this experiment, we investigated whether the release of nitro oxide and other proinflammatory cytokines, which are inflammation-producing substances secreted from raw cells, is related to mRNA gene expression. In FIG. 3, mRNA genes expressed from LPS-treated raw cells are amplified by PCR, and the genes are processed by gel-electrophoresis. As shown in FIG. 3, cavanol, atofam, mititol, and mitol + fucoidan mixtures were significantly suppressed in the production of mRNAs that cause other cytokine secretion as well as iNOS. Therefore, it could be inferred that the inhibitory effect of cavanol, atopamem, mititol, and mithol + fucoidan mixtures was a result of the inhibition of expression of inflammatory genes.

< Experimental Example  2> Anti-inflammatory activity and atopic dermatitis improvement effect in vivo  Research

Fucoidan  Anti-Atopy Containing Cosmetics  Composition Preparation and Sample Preparation

It was prepared by dissolving 1-Chloro-2,4-dinitrobenzene (DNCB), a pseudo-atopic inducing reagent used in the present invention, in acetone to 1% and 0.5%. The method for producing a lotion (lotion) of the present cosmetic composition containing Fucoidan and mititol is as follows. First, water-soluble moisturizer glycerin, water and xanthan gum (2% solution) as a thickener was mixed and dissolved at 75 ~ 80 ℃. Next oil was mixed with cetyl alcohol fatty acid and oil squalane, medowform seed oil was dissolved at 75 ~ 80 ℃. The oil phase was slowly added to the aqueous phase while stirring at 5,000 rpm with a homogenizer and emulsified for 7 minutes. After cooling, the base lotion was prepared without the active ingredient. Into the bass lotion, the natural compound of 0.3%, mititol 0.3% and 0.5% Fucoidan, 0.3% and 2% Fucoidan were added to the bass lotion, and then homogenized at 3,000 rpm for 30 minutes using a homomixer. Was prepared. As a control, Atopalm of Neopharm Co., Ltd. and Cavanol anti atopy lotion of Botamedi Co., Ltd., which are currently commercially available, were used. Finally, phosphate buffer saline (PBS) was used at pH 7.4.

In vivo Laboratory Animal Preparation

In this experiment, 6 week old male hairless mice (26 ± 2 g) supplied from Orient Bio Co., Ltd. (Sungnam, Korea) were used as experimental animals. The experimental animals were allowed to acclimate for one week at the experimental animal kennel and then experimented. During the experiment period, the feed and water were supplied shortly and freely ingested. The temperature of the experimental animal kennel was 23 ± 2 ℃, the relative humidity was 55 ± 5%, and the contrast was maintained for 12 hours. Since the experiments were carried out in nine groups, four dogs were kept in each of nine cages, and the tail of the hairless mice was marked one day before the experiment to identify the individuals.

Induced atopic dermatitis and sample processing

The experiment was divided into nine groups of four animals each. 4 normal groups without treatment (Group.1), 4 control groups coated with DNCB only (Group.2), 4 experimental groups coated with DNCB and treated with phosphate buffer solution (pH 7.4) (Group.3), Four experimental groups coated with DNCB and treated with bass lotion (Group.4), four experimental groups coated with DNCB and treated with lotion containing 0.3% by weight of mitol (Group.5), and applied with DNCB 0.3 Four experimental groups treated with lotions containing 0.5% by weight and 0.5% by weight of Fucoidan (Group.6), four experimental groups treated with lotion containing 0.3% by weight of itol and 2% by weight of Fucoidan applied with DNCB. 7), 4 experimental groups coated with DNCB and treated with Cavanol (containing 0.3 wt% of thytol) (Group 8), 4 experimental groups coated with DNCB and treated with lotion containing Atopalm (Group 9), A total of 36 hairless mice were tested. First, the hairless mice were acclimated to the kennel environment for one week, and then 100 μL of 1% DNCB was applied to each group except Group.1 evenly on the back of the experimental animals at the same time every day. After inducing atopic dermatitis for 7 days in this way, 100 μL of 0.5% DNCB was applied to the back of the experimental animals every two days from the second week of the experiment, and the samples in each group were treated with 100 μL evenly at the same time every day. gave. On the day when the day of processing the DNCB and the day of the sample overlap, 0.5% of the DNCB was treated first, and then each sample was treated after 7 hours. The experiment was for a total of six weeks.

Observation of atopic dermatitis symptoms and skin thickness measurement

(1) Observation of changes in Dorsal skin

From the start of the experiment, the changes in the dorsal skin of hairless mice with atopic dermatitis were observed. When 1% DNCB was applied daily to the dorsal skin of the experimental animals after the adaptation period, for one week, except for Group.1, the DNCB-coated area became very dry, causing erythema similar to atopic symptoms. Changes in appearance such as sulphation, skin, and thymus were observed, and experimental animals were observed to scratch the area where DNCB was applied. And even with the naked eye, the skin thickness gradually became thicker than normal skin. In order to measure these skin changes more precisely during the experiment, the day before the start of a new week during the experiment, each group of subjects had a digital camera (SINY DSLR-A203) and a skin diagnosis system (Aramo TS, Aram). HUVIS Co., Ltd. Sungam. Korea) was used to observe the change in appearance and was recorded. The digimatic indicator (No.99MAH007B, Series No.543) was used to observe the change in skin thickness.

(A) Observation of Macroscopic skin appearance

The appearance of atopic dermatitis-induced experimental animals during the experiment was observed with a digital camera (SINY DSLR-A203) (FIG. 4). The normal group, Group 1, did not differ significantly from week 1 to week 6. On the other hand, all subjects of Group 2 to Group 9 showed atopy-like symptoms such as skin redness, whitening, thickening and rough skin due to DNCB applied during the first week. Afterwards, each sample was treated with 100 μL daily until week 6, the end of the experiment, and atopic dermatitis was improved in the other groups except Group.2, where only DNCB was applied to maintain atopic symptoms, but the difference was slightly different. In Group.3 treated with phosphate buffer solution (PBS, pH7.4), the skin appeared to disappear as the 6th week, but a little bit of sulphate and erythema remained. In the experimental group treated with Cavanol and Atopalm, which were commercialized with the sample containing Mytol and Fucoidan, all symptoms of atopic dermatitis were effective. Especially after 3 weeks, it started to show a clear effect. At the end of the sixth week of the experiment, there was some tearing and dryness, but the degree of atopic dermatitis was remarkably improved compared to the initial state of atopic dermatitis. . Among them, the normalization of skin was noticeable in Group.8 and Group.9 treated with Cavanol and Atopalm. Group.4 treated with bass lotion was also effective in improving atopic symptoms because the treatment of moisturizing lotion with dry skin replenished insufficient water and stabilized the skin. However, the improvement effect was slower and slower than the experimental group treated with lotion with anti-atopic efficacy from Group.5 to Group.9. In the groups treated with the samples, it was confirmed that the symptoms of atopic dermatitis generally improved from week 1 to week 6, but the difference could not be accurately measured.

(B) Microscopic skin appearance observation

Atopic dermatitis induced skin was magnified 60 times using a diagnosis system (FIG. 5). The skin of the normal group was not significantly different for each measurement as shown in FIG. By comparing the skin of the normal group with that of the other experimental groups, we could compare how many keratin, sessile, and lichen scabs appeared in the sample processing area of each group. The control group, Group 2, maintained the symptoms of atopic dermatitis from week 1 to week 6 without the effect of atopic dermatitis improvement. In the rest of the group, by 2 weeks, the skin covering the skin was removed and over time the atopic symptoms were alleviated. In the case of Group.3 treated with phosphate buffer solution, in FIG. 4, keratin was removed from the naked eye at the end of the experiment, and dermatitis caused by atopy appeared to be relatively clean, but the diagnosis system confirmed that the skin still remained. The tissues were lost due to the loss of moisture, and whitening was observed. The experimental groups treated with each sample from Groups 4 to 9 showed improved atopic dermatitis even in the diagnosis system. By the end of the sixth week, the skin was improved to a near, but not nearly as clean, condition as the normal group, and Group.4 found less skin-improving effect than other products containing anti-atopic ingredients. Overall, the difference in skin improvement was slight, but Cavanol and Atopalm improved relatively quickly and moisturized. Compared to Group 5, Group 6 and Group 7, the lotion with Fucoidan was more effective than the lotion containing only 0.3% of mititol, and 0.5% by weight of Fucoidan was added. Addition of 2% by weight was more effective in terms of skin improvement rate and degree of skin improvement.

(2) Skin thickness measurement

The skin of the same area was measured for 6 weeks in mm through each digimatic indicator (No. 99MAH007B, Series No. 543) (FIG. 6). By measuring the skin thickness, the correlation between atopy and skin thickness could be identified. In the normal group, there was no significant difference in skin thickness from week 1 to week 6. In the experimental group treated with 1% of DNCB for a week, the skin thickness increased from 2 to 3 times higher than that of the normal group. After 6 weeks, the skin thickness tended to decrease gradually. In the dermatitis induced by atopic dermatitis, the dermal stratum corneum such as cuticle, sessile, and thymus gland increased due to the increase of percutaneous moisture loss, but the skin thickness increased in the experimental group treated with each sample. At the end of the day, the value did not decrease to the thickness of normal skin, but it was steadily decreasing. Here, Group.2, which had only applied DNCB, also noticed a decrease in skin thickness, which was improved after week 1 because the amount of DNCB was reduced to 0.5% and treated every two days. will be. However, after 2 weeks, atopy symptoms remain similar. The final results showed that Atopalm had the highest skin improvement, but the difference was small between the groups.

Immunological Observations of Atopic Dermatitis

At the end of the experiment and sample collection for the immunological evaluation, the animals were withdrawn from the orbit and heart using heparinized capillary tubes and syringes. Blood was collected and centrifuged at 10,000 rpm for 30 minutes at 4 ° C to separate serum and stored at -70 ° C deep freezer until analysis. After that, the spleen was extracted by sacrificing the experimental animal through the cervical vertebra and its weight was measured through analytical balances (Sartorius / Cp324s, USA).

(1) Spleen index measurement

When allergens enter the skin from the outside, certain T lymphocytes release inflammatory cytokines. This results in an increase in immunoglobulin E (IgE), which leads to an increase in eosinophils, resulting in various symptoms of atopic disease. In this way, eosinophils increase when atopy is induced. Since eosinophils are stored in the spleen, changes in eosinophils were determined by measuring the level of the spleen (FIG. 7). To examine the immune response of mice to DNCB, the mice were sacrificed at the end of the experiment and spleens were removed and weighed. Spleen index was obtained by dividing by body weight (g) of atopic dermatitis mice. As a result, the average values of 3.7663, 7.6144, 6.2357, 5.452, 4.492, 4.5872, 5.127, 4.7641 and 5.2636 were measured in order from Group.1 to Group.9. As shown in FIG. 7, the normal group showed a value of about 3.77, but in Group.2, which induced atopy with DNCB, the value increased by about 2 times to about 7.61. This indicates that eosinophil count was abnormally increased due to atopic dermatitis. In addition, Group.3 treated with phosphate buffer solution was significantly higher than the normal group. This also indicates a significant increase in T lymphocytes in the spleen by DNCB, and it can be seen that the phosphate buffer solution did not properly suppress the increase of T lymphocytes. In group.4, the atopy symptoms were slightly alleviated by the water supply of bass lotion.In experimental animals treated with anti-atopic samples from Group.5 to Group.9, the inhibitory effect of T lymphocytes was excellent. Or lower spleen levels compared to bass lotion. Although the difference in the spleen value is small, the lotion value of the lotion containing 0.3 wt% of mititol was the lowest, followed by the lotion containing 0.3 wt% of mititol and 0.5 wt% of fucoidan, Atopalm, Cavanol, and 0.3 wt% of mititol. The spleen values were measured in the order of lotion and Fucoidan lotion containing 2% by weight to compare the anti-atopic efficacy of each sample.

(2) Immunoglobulin E concentration measurement

IgE induces allergic reactions by binding to receptors on the surface of mast cells or white blood cells (basophil). This is because mast cells and white blood cells have mediators that cause allergic reactions such as histamine. When cells are activated by IgE binding, histamine is secreted and an inflammatory reaction occurs. People with atopic dermatitis usually have high serum IgE levels and are sensitive to various antigens around them. In addition, the more severe the skin symptoms, the higher the IgE levels, so alleviation of skin symptoms is associated with a decrease in IgE levels. Therefore, in the present invention, the anti-atopic efficacy was evaluated by measuring the level of IgE in the serum of experimental animals (FIG. 8). For this immunological evaluation, blood samples collected from the heart and orbit of experimental animals were centrifuged and serum IgE levels were measured by ELISA. 5μL of serum taken from hairless mice and 45μL of dilution buffer were mixed on each 96 well plates. 50μL was dispensed into each well, placed on a shaker at room temperature for 2 hours, and washed three times with washing buffer solution. The biotin-conjugated anti-IgE anitbody was added again, incubated at room temperature for 2 hours, and washed again three times. 50 μL of HRP-conjugated avidin was added to the shaker at room temperature, incubated for 1 hour, and washed again. 50 μL of chromogenic substrate (TMB) reagent was added thereto, followed by incubation for 5 minutes, and then 50 μL dml stop solution was added thereto and the absorbance was measured in an ELISA reader (450 nm). 8 is a result of measuring the final concentration of IgE in atopic dermatitis mice. From Group.1 to Group.9, the mean was 62.735, 615.764, 530.634, 512.931, 369.55, 314.459, 256.138, 141.907, 191.625 ng / mL. When comparing Group.1, which is a normal group, and Group.2, where atopy is induced, it can be seen that the serum IgE concentration is increased by 10 times compared to the normal group. In the case of Group.3 treated with phosphate buffer solution, the inhibition rate of IgE was found to be significantly lower than that of other anti-atopic products. The same was true for the experimental group treated with bass lotion. When the sample containing anti-atopic substance was processed, the expression of total IgE was remarkably reduced compared to Group.2. Among them, Cavanol-treated Group.8 showed the lowest IgE concentration, which showed the highest anti-atopic efficacy. It is reported that the expression level of IgE is significantly lowered as Th1 cell is activated and Th2 cell activity is decreased. Next, Atopalm, lotion containing 0.3% by weight of mititol and 2% by weight of fucoidan, lotion containing 0.3% by weight of mititol and 0.5% by weight of fucoidan, and lotion containing 0.3% by weight of mititol. appear. Comparing Group.5, Group.6, and Group.7, the more Fucoidan content, the less IgE expression, that is, the more anti-atopic effect was found.

(3) Cytokine concentration measurement

Serum IL-4 and INF-γ levels were measured using an enzyme immunometric assay kit (Assay Designs, Inc. USA). 100 μL of the capture antibody diluted 180-fold with PBS in a 96 well plate was aliquoted overnight at room temperature, and then 300 μL of block buffer was dispensed and reacted at room temperature for at least 1 hour. Next, 100 μL of each sample and standard solution diluted with reagent diluent were dispensed, and then reacted at room temperature for 2 hours. After diluting Streptavidin-HRP by working dilution, each 100μL was dispensed into each well and reacted at room temperature for 20 minutes. The substrate solution was dispensed by 100μL and reacted at room temperature for 20 minutes. Each step was washed with 0.05% PBS-Twin, and finally, the reaction was stopped by adding 50 μL of 2N H ₂ SO ₄ after color development. Through the same process, purified cytokine was measured as an ELISA reader (450 nm) as a standard.

(A) Interleukin-4 concentration measurement

In an immunologically normal state, the cytokine of Th1 cells, IFN-γ and IL-4 of Th2 cells, are balanced by regulating each other. However, in the atopic state, IL-4 is overexpressed, resulting in imbalance by inhibiting the expression of IFN-γ. High production of IL-4 stimulates B-cells to induce overexpression of IgE, resulting in excessive release of histamine. IL-4 and IgE are important immunological factors because they not only directly affect or induce atopic symptoms. The results of the expression level of IL-4 showed a general trend similar to that of FIG. 9 (FIG. 9). The expression level of IL-4 was determined to be 24.839, 106.022, 85.679, 79.055, 40.433, 47.349, 35.346, 29.249, 26.633 pg / mL, respectively, from Group.1 to Group.9. As shown in FIG. 9, the expression of IL-4 increased about 4 times higher than normal when atopy was induced. It can be seen that due to atopic dermatitis, the balance between Th1 and Th2 cells is broken. From week 2, the groups treated with each anti-atopic drug significantly reduced the expression level of IL-4, which suppressed the overexpression of cytokine in Th2 cells, thus maintaining a balance between T cells. It has been proven to work. In particular, the IL-4 level of Group.9 treated with Atopalm in the experimental group in which atopic dermatitis was induced was similar to the normal value, indicating the best anti-atopic effect. The anti-atopic effect of Cavanol, lotion containing 0.3% by weight of mititol and 2% by weight of fucoidan, lotion containing 0.3% by weight of mititol, lotion containing 0.3% by weight of mititol and 0.5% by weight of fucoidan. It was observed.

(B) Interferon-γ concentration measurement

As described above, when atopic dermatitis is induced, IL-4 is overexpressed, and the expression of IFN-γ is suppressed by the overexpressed IL-4. Therefore, in order to determine how each sample affects the expression level of IFN-γ, serum of the experimental animals was collected at the end of the experiment, and the expression level of IFN-γ in each group was measured (FIG. 10). The average of 35.178, 18.752, 16.877, 22.698, 46.717, 49.851, 54.215, 62.821, 58.867 pg / mL was measured. In case of atopic dermatitis, IFN-γ expression level was suppressed and therefore, IFN-γ concentration of Group.2 was lower than that of normal group. Antiatopic products can increase the expression level of IFN-γ and balance IL-4, thereby immunologically relieving symptoms of atopic dermatitis. Therefore, the higher the value of the expression level of IFN-γ in Figure 10 can be seen that the anti-atopic effect is excellent. From this point of view, the highest anti-atopic efficacy is Group.8 treated with Cavanol, followed by the present invention lotion containing 0.3% by weight of Atopalm, mititol and 2% by weight of fucoidan, This invention lotion containing 0.5 wt% of Fucoidan, lotion using 0.3 wt% of mititol alone.

The present invention is very useful in the biopharmaceutical industry because it has an excellent effect of providing a cosmetic composition for improving anti-inflammatory activity and atopic dermatitis, which contains a mixture of mithol and fucoidan derived from Ecklonia cava as an active ingredient.

Claims (3)

Anti-inflammatory active composition comprising a mixture of mititol and fucoidan isolated from Ecklonia cava.
The composition of claim 1, wherein the mititol contains a polyphenol derived from Ecklonia cava as an active ingredient.
An external preparation composition for improving atopic dermatitis, comprising 0.3 wt% of derived mitol and 0.5-2.0 wt% of fucoidan isolated from Ecklonia cava as an active ingredient.

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