KR100964018B1 - A method for producing extract of fermented fucoxanthin and Cosmetic composition containing thereof - Google Patents

Abstract

The present invention relates to a method for extracting fucoxanthin (Fucoxanthin) from brown algae using fermented microorganisms and a cosmetic composition containing the same, the brown algae such as kelp, seaweed, moth and scent, bacillus subtilis (Bacillus subtilis) It relates to a cosmetic composition, characterized in that it is obtained in high yield through the fermentation using and containing 0.001 to 30.0% by weight as the main active ingredient. This can provide a multifunctional cosmetic composition having a skin moisturizing effect, irritation relief effect, whitening effect, wrinkle improvement effect, atopy improvement effect.

According to the present invention, fermented fucoxanthin extract is not only excellent in moisturizing effect, but also has a skin wrinkle improvement effect, a whitening effect, and atopy improvement effect.

fucoxanthin, brown seaweed, fermented microorganism, moisturizing, wrinkle improvement, atopic dermatitis

Description

A method for producing extract of fermented fucoxanthin and cosmetic composition containing thereof}

The present invention relates to a method for extracting fucoxanthin (Fucoxanthin) obtained from brown algae using fermented microorganisms and a cosmetic composition containing the same, more specifically, Bacillus subtilis seaweeds such as kelp, seaweed, moth, 반 ( bacillus subtilis , Bacillus subtilis) obtained through the fermentation of fucoxanthin in a high yield and it contains 0.001 to 30.0% by weight as the main active ingredient, characterized by moisturizing effect of skin, whitening effect, wrinkle improvement effect, atopy improvement effect There is to provide a multifunctional cosmetic composition.

Sea algae, which are plants inhabiting the ocean, are classified into green algae (Chlorophyta), red algae (Red Algae, Rodophyta), brown algae (Brown Algae, Phaeophyta), and blue algae (Blue Algae). . Brown algae include brown seaweed, sea bream, mazaban, and bamboo shoots, and red algae include seaweed, loach, stone fern, and haeincho. Green algae include green, auditory, cheontae, cholera, haecam, and half moon horses. There is this.

In addition to chlorophyll, the seaweeds contain various pigments. For example, red algae contain phycobilin, a red-blue pigment, and brown algae, fucoxanthin and carotene, which are brown pigments. It is contained.

In particular, the fucoxanthin contained in brown algae is a natural carotenoid has been published in the journal that antioxidant, anticancer and antimutagenic properties are superior to beta carotenoids. Due to the properties of fucoxanthin, carotenoids containing fucoxanthin extracted from brown algae are expected to be widely used in clinical, nutritional and industrial fields.

delete

In the human body, the skin acts as a protective film to protect the living body from the external environment. It prevents the loss of biological components such as water or electrolytes and prevents harmful substances from entering the body. .

Such skin is largely divided into epidermal layer, dermal layer and subcutaneous fat layer. The epidermal layer is composed of keratinocytes and melanocytes. Since the outermost keratinocyte layers of skin are in direct contact with the external environment, they require strong resistance to physical, chemical, or permeation of substances, and prevent the loss of moisture from the body to the outside. At the same time, it should maintain flexibility by retaining adequate moisture on its own.

In general, the moisture content of the skin is about 70% in the dermal layer, but decreases toward the epidermal layer, which is about 10 to 30% in the keratinocyte layer. The water supplied from the dermal layer is mainly moved up the stratum corneum by passive diffusion and finally discharged to the outside. This is called Trans Epedermal Water Loss (TEWL), and it is known that it is the sebaceous component and the epidermal lipid which are the lipid membrane components of the keratinocyte layer to maintain the appropriate level of transdermal moisture loss. In addition, the keratinocyte layer is known to play an important role in moisturizing the skin due to the presence of a substance having a hydrophilic water retention ability called a natural moisturizing factor (NMF). When the moisture of the normal keratinocyte layer is maintained about 10 to 30%, the skin is smooth and soft, and the body protects normally.

However, when the moisture content of the keratinocyte layer is less than 10%, the skin becomes rough and the body loses its protective function, causing aging. For example, in the case of dry skin, the aggregation force of keratinocytes is weakened, and scaling phenomenon in which keratinocytes are peeled off from the surface of the skin, such as scale fragments, occurs. Thus, the drying of the skin is the moisture content of the keratinocyte layer. This is because it is less than normal skin. In addition, even healthy skin may suffer from a lack of moisture due to harsh external environment, ie, wind, cold weather, sunlight, face wash, shaving, and the like, and thus worsens the skin condition.

Therefore, it is very important to properly maintain the moisture content of the keratinocyte layer of the skin. To this end, cosmetics such as sebum-like components, NMF components, or moisturizers such as polyols were added and used. For example, glycerin, solitol, and the like having three or more hydroxyl groups (OH groups) as water-soluble polyols exhibit excellent moisturizing properties, but are highly sticky, making them unpleasant when used, and having two hydroxyl groups (OH groups), 1, 3-butylene glycol may cause side effects of the skin. In addition, other natural moisturizing factors, such as sodium pyrrolidone carboxylate (PCA-Na), sodium lactate, urea, etc., are highly electrolytic and thus impair the emulsion stability of cosmetics.Amino acids, collagen, elastin, etc. Moisturizing ability, but the moisturizing ability was limited. The ability to retain moisture in the stratum corneum is controlled by a natural moisturizing factor (NMF) consisting of sebum, amino acids, lactic acid, urea and inorganic salts. The development of high-efficiency substances is one of the major research challenges in cosmetics.

Recently, in order to reduce skin irritation caused by various chemicals, much attention has been focused on functional cosmetics having functions such as antioxidant, wrinkle relief, whitening, and moisturizing. Natural ingredients have less side effects on the skin, and as the consumer's response to cosmetics using natural ingredients has increased recently, the development value of cosmetics as a raw material for cosmetics is increasing. For example, US Pat. No. 5,972,341 describes the antiwrinkle effect of a plant of the genus Commiphora, in particular Commiphora mukul extract. Japanese Patent Application Laid-open No. Hei 9-672662 describes seaweed extracts having a hyaluroronidase inhibitory effect. Japanese Patent Application Laid-open No. Hei 10-85905 describes the skin texture improvement effect of Fucoidan extracted from Wakame.

delete

Therefore, the present inventors have studied the possibility of applying to cosmetics in various natural products, but selected fucoxanthin which is brown seaweed, but in the general method of extracting fucoxanthin from brown seaweed, the process of drying and extracting fucoxanthin from brown seaweed The fucoxanthin effective at is not only decomposed and lost, but also in the process of removing organic solvents and the like, resulting in poor yield. Therefore, the present inventors ferment brown algae using fermenting microorganisms bacillus subtilis and Bacillus subtilis and lactobacillus acidophilus , lactic acid bacteria to obtain effective fucoxanthin in high yield and extracts from them. As a result of measuring the skin moisturizing effect, skin irritation relieving effect, whitening effect, wrinkle improvement effect, atopy improvement effect, anti-inflammatory effect, it was found that the efficacy as a cosmetic can be expected.

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for extracting fucoxanthin using brown algae, which enables to obtain fucoxanthin from brown algae such as seaweed, maban, 톳, kelp, etc. in high yield, and also applies it to skin cosmetics containing the main active ingredient. It is possible to provide a natural extract exhibiting excellent effects such as skin moisturizing effect, irritation relaxing effect, whitening effect, wrinkle improvement effect, atopy improvement effect, anti-inflammatory effect when contained in cosmetics.

In order to achieve the above object, the present invention is effective by pulverizing brown algae of a living body collected from the sea and thoroughly washed, and fermenting microorganism Bacillus subtilis ( bacillius subtilis ) for 7 days at room temperature. It provides a method for extracting fucoxanthin using brown algae characterized in that the extracting of the components.

Hereinafter, the present invention will be described in more detail.

In order to obtain a high yield of fucoxanthin from brown algae, the present invention is first subjected to a crushing step of pulverizing brown algae of fresh vegetables that have been collected from the sea and sufficiently washed.

The brown algae may be selected from commonly known seaweed, kelp, mabanban, 톳, etc., these are preferably washed immediately after harvesting and then crushed raw vegetables. This is because when the brown alga is sun-dried, fucoxanthin which is an active ingredient contained in brown algae is decomposed and lost. Therefore, in order to obtain fucoxanthin in high yield, it is preferable to immediately wash and grind the brown algae collected. Bacillus subtilis was used to make fermented fucoxanthin . Bacillus subtilis ( bacillus subtilis ) is a Gram-positive microorganism, capable of forming endospores, and has a rod shape. In the present invention, it was possible to grow under aerobic or anaerobic anaerobic conditions. As the growing condition, the brown algae pulverized was finely added and 10 g / 1 L was added to the conventional microbial culture. Fermentation conditions were grown at about 37 degrees, pH was grown at 5-7.

Fermented fucoxanthin used in the present invention was obtained as follows. After incubation under the same conditions as above for 7 days incubation in a fermentor. After the culture was obtained, the solution was filtered using 0.25 uM of filter paper. The filtrate was further aged for 10 days at low temperature, filtered and used in the present invention, and the final solid concentration was maintained at 10 g / L before use.

The fermented fucoxanthin is added to conventional basic cosmetics, that is, flexible lotion (skin), nourishing lotion (milk lotion), nourishing cream, massage cream, essence, pack, etc., where the amount is 0.0001 based on the dry weight. 20 weight%, Preferably 0.001-10 weight% is added to cosmetics. The effect is less than 0.0001% when the effect is less than 20% is more likely to cause irritation to the skin and may have a significant effect on the stabilization of the formulation.

Examples and Experimental Examples below describe the contents of the present invention, but the contents of the present invention are not limited thereto.

First, the specific method for producing fermented fucoxanthin is as follows.

Brown algae were used in the seaweed, kelp, maban, 톳, etc. generally known, these were washed thoroughly immediately after harvesting and then pulverized raw vegetables and finely made using 300 mesh. This was added to 10 g / L Bacillus subtilis culture. To this, glucose was added as a carbon source, and additionally, peptone and NaCl were incubated. Cultivation was carried out using a 5L fermenter for 7 days at 37 degrees, pH 5-7. After incubation, the culture solution was centrifuged to remove the culture cells first, and then filtered through 0.25uM filter paper. The filtrate thus obtained was aged for about 10 days in a low temperature warehouse at 4 degrees, and then the filtrate was finally filtered using a filter of 0.25 uM. When the filtration is completed, the present invention is characterized in that the extract obtained in the extraction step is transferred to a concentration tank to obtain a concentrated or freeze-dried under reduced pressure at about 60 ℃. The temperature condition in the concentration step is very important, but if the concentration of the extract exceeds 70 ℃, there is a problem that fucoxanthin, an active ingredient contained in the extract is decomposed and lost, so that the extract is concentrated at a temperature not exceeding 70 ℃ Let's do it.

Thus fermented fucoxanthin according to the present invention obtained by freeze drying alone or mixed with other ingredients, such as conventional basic cosmetics, that is, flexible lotion (skin), nutritious longevity (milk lotion), nutrition cream, massage cream, essence, It can be used in various fields including packs and the like. However, when the concentrate obtained in the above-mentioned concentration step is dried as it contains a small amount of impurities in addition to fucoxanthin, in order to solve this problem, it is also possible to produce and use more refined fucoxanthin before or after drying to undergo a purification step. have.

The identification of fucoxanthin can be confirmed by reacting the yellow fraction with DPPH (1,1-Diphenyl-2- Picrylhydrazyl), the fraction obtained at low temperature of 40 degrees Celsius in the dark without light to prevent the decomposition of fucoxanthin Concentration under reduced pressure is preferred to obtain high purity fucoxanthin.

As described above, the present invention has the advantage that the extract can be obtained in a high yield by a relatively simple procedure while minimizing the loss of fucoxanthin, an active ingredient, from brown algae such as seaweed, maban, 톳, and kelp.

Hereinafter, the present invention will be described in more detail with reference to the following examples, which are only presented to aid the understanding of the present invention, but the present invention is not limited thereto.

≪ Example 1 >

2 kg (dry weight 200 g) of fresh kelp washed with water was rinsed several times in a blender (Blender), finely prepared using 300 mesh, and then cultured using Bacillus subtilis (Bacillus subtilis) as described above. After incubation, the culture solution was centrifuged to remove the culture cells first, and then filtered through 0.25uM filter paper. The filtrate thus obtained was aged for about 10 days in a low temperature warehouse at 4 degrees, and then the filtrate was finally filtered using a filter of 0.25 uM. In the present invention, when the filtration is completed, the extract obtained in the extraction step is transferred to a concentration tank and concentrated or freeze-dried at about 60 ℃ to extract the fermentation fucoxanthin.

Comparative Example 1

Add 220g of dried powdered kelp (200g except water 10%) to a 2L Erlenmeyer flask, add 75% ethanol to 1000ml, shake it every 10 minutes while placing it in a cow, extract it for 12 hours, and concentrate 25g of concentrate. It was obtained by drying with a rotary vacuum concentrator and the yield and yield of the dried product are shown in Table 1 below.

division Dry weight Yield yield(%) Example 1 200 g 97 g 48.5% Comparative Example 1 200 g 13.4g 6.7%

delete

As shown in Table 1, in Example 1 extracted using the biological kelp according to the present invention, the yield is very excellent compared to Comparative Example 1 extracted the active ingredient by a general extraction method using dry kelp (about 7 times )can confirm.

delete

Example 2 Experiment of Measuring Antioxidant Effect Using NBT Method

In this example, the antioxidant activity of the fucoxanthin extract obtained in Example 1 was compared with other antioxidants, namely, retinol and BHT, under laboratory conditions, and the antioxidant activity was measured using the NBT method.

In order to measure the antioxidant effect, the active oxygen produced by xanthine and xanthine oxidase is measured by the NBT method, and the effect of the test substance removing the active oxygen, that is, the active oxygen scavenging effect, is evaluated. Free radicals are produced by xanthine and xanthine oxidase, and the reactive oxygen is measured by reacting with nitro blue tetrazolium (NBT) to measure the blue color produced thereby at wavelength 560 nm. do.

As a measuring method,

0.05M Na2CO3 ------------------------------------ 2.4ml

2.3mM xanthine solution ---------------------------------- 0.1ml

3.3mM EDTA Solution ------------------------------------ 0.1ml

4.BSA solution ---------------------------------------- 0.1ml

5.0.72 mM NBT solution ---------------------------------- 0.1ml

6.Xanthine Oxidase Solution ----------------------------- 0.1ml

7.6 mM CuCl2 Solution ----------------------------------- 0.1ml

① Add 1,2,3,4,5 to Bayer's bottle and add 0.1ml of sample solution to it and leave at 25 ℃ for 10 minutes.

② Add 6 and stir quickly, and start incubation for 20 minutes at 25 ℃.

③ After that, add 7 to stop the reaction and measure the absorbance St at 560nm.

④ For the blank test, measure the absorbance Bt by using distilled water instead of the sample solution as above.

⑤ Also, the blank of the sample solution is operated in the same manner using distilled water instead of 6 to measure the absorbance Bo.

St: absorbance at 560 nm after enzyme reaction of sample solution

Bt: absorbance at 560 nm after enzymatic reaction of blank test solution

So: absorbance at 560 nm before reaction without enzyme addition of sample solution

Bo: absorbance at 560 nm before reaction without enzyme in blank test solution

Antioxidant effect was calculated by Equation 1, the results are shown in Table 2.

Sample Name Treatment concentration (%) Antioxidative effect(%) Example 1 0.1 95 Retinol 0.1 93 BHT 0.1 90

delete

All samples tested at 0.1% concentration showed excellent antioxidant effect, and fucoxanthin extract was found to have similar or superior antioxidant effect to retinol and BHT.

Example 3: Antioxidant Effect Measurement Experiment Using DPPH Method

In order to measure the antioxidant effect of the fucoxanthin extract obtained in Example 1, the antioxidant activity of the green tea extract and tocopherol as a comparative sample was measured using a DPPH method.

DPPH method measures the antioxidant activity by reducing power using a free group called 2,2-Di (4-tert-octylphenyl) -1-picrylhydrazyl free radical (DPPH). The free radical scavenging rate is measured at a wavelength of 560 nm by comparing the degree to which the DPPH is reduced by the test substance to reduce the absorbance.

As a reagent used, a 0.1 mM solution of 2,2-Di (4-tert-octylphenyl) -1-picrylhydrazyl free radical (Aldrich Chem. Co., MW = 618.76) was dissolved in methanol to make 100 ml.

As a measuring method,

① Add 0.15ml of sample solution to 0.15ml of 0.1mM DPPH solution in 96-well plate, stir rapidly, and incubate for 10 minutes at 25 ℃.

(2) Then measure the absorbance St at 560nm.

③ In the blank test, measure the absorbance Bt by using distilled water instead of the sample solution as above.

④ Again, the blank of the sample solution was operated in the same manner using methanol instead of the 0.1 mM DPPH solution to measure the absorbance Bo.

The result of the effect was calculated by Equation 2, the results are shown in Table 3.

St: absorbance at 560 nm after free radical scavenging of sample solution

Bt: absorbance at 560 nm after free radical scavenging of blank test solution

So: Absorbance at 560 nm before reaction without free radicals in sample solution

Bo: absorbance at 560 nm before reaction without free radicals in blank test solution

Sample Name Treatment concentration (%) Antioxidative effect(%) Example 1 0.01 90 Green Tea Extract 0.01 51 Tocopherol 0.01 49

delete

Experimental results As shown in Table 3, fucoxanthin extract had better antioxidant effects than green tea extract and tocopherol at 0.01% concentration.

Example 4: in vitro MMP-1 inhibition assay

Substrate metalloproteinase (MMP-1) inhibitory activity was determined in a biochemical model based on the use of collagen and gelatin conjugated to purified collagenase and its substrate, fluorescein (EnzChek ™ gela) Tinase / collagenase kit, by Mocula Probe). Collagenase purified from Clostridium histiocom was supplied in the EnzChek ™ gelatinase / collagenase kit.

A reaction buffer consisting of DQ-collagen purified from porcine skin and conjugated to fluorescein with 0.05M Tris-HCl, 0.15M NaCl, 5mM CaCl2 and 0.2mM sodium azide (pH 7.6) was found in EnzChek® gelatinase / Collagenase kit (Molcula Probes) was used. Fucoxanthin extract was dissolved in the reaction buffer. It is 4; 2; 0.4; 0.2; Test at 0.1% (w / v). Dilutions of the test extracts were incubated with 25 ug / ml of DQ-collagen and 0.1 U / ml of collagenase for 15 minutes, 45 minutes, and 120 minutes at room temperature. In each experimental condition, the control corresponding to the collagenase and DQ-collagen mixture was also incubated in the same manner. In each experimental condition, a blank, hereinafter referred to as 'enzyme-free blank', was incubated in the presence of DQ-collagen and in the absence of collagenase. Each experiment was performed three times.

After 15, 45 and 120 minutes, the signal corresponding to the degradation of DQ-collagen was measured with a fluorometer (excitation: 485 nm, emission 505 nm). The fluorescence value of each sample was measured based on the 'enzyme free blank' fluorescence value. Results are expressed as percent variance for fluorescence units per sample and control.

In conclusion, the fucoxanthin extract tested at 0.04 to 4% (v / v) under selected experimental conditions retained anti-collagenase / gelatinase activity in a dose dependent manner.

Experimental Sample Inhibition rate 0.02% (treatment concentration) 0.04% (treatment concentration) Example 1 43 76 Retinol 0 6 Green Tea Extract 51 59

delete

Experimental results showed that fucoxanthin extract inhibited 76% of the collagen degrading activity of Clostridium collagenase when treated with 0.04%, which was better than the inhibitory effect of green tea extract.

Example 5 Evaluation of Inhibition of MMP-1 Expression by Fucoxanthin Extract After UV Irradiation

In this example, ELISA was performed to measure the concentration of MMP-1 after UV irradiation and sample addition of the fucoxanthin extract obtained in Example 1.

Human dermal fibroblasts are irradiated with UVA at an energy of 5 J / cm 2 using a UV chamber. UV irradiation dose and culture time were established through preliminary experiments to maximize the expression of MMP in fibroblasts. The negative control was wrapped in tinfoil and kept in the environment of UVA for the same time. UVA emission was measured using a UV radiometer. The cells during the UVA irradiation are intact in the previously dispensed medium and irradiated with UVA and then exchanged with the medium containing the sample for 24 hours of incubation, and then the medium is recovered and coated in 96-well.

delete

The primary antibody (MMP-1 (Ab-5) monoclonal antibody and MMP-2 (Ab-3) monoclonal antibody) is treated and reacted at 37 ° C. for 60 minutes. After reacting the secondary antibody anti-mouse IgG (whole mouse, alkaline phosphatase conjugated) for about 60 minutes, the alkaline phosphatase substrate solution (1mg / mlρ-nitrophenyl phosphate in diethanolamine buffer solution) was reacted at room temperature for 30 minutes and then the microplate reader was reacted. Measure the absorbance at 405 nm. As a control, the one without addition of the sample is used.

The fucoxanthin extract showed more than 20% inhibition rate compared to the control group that did not process the MMP-1 expression induced by UV irradiation, which is superior to the inhibition rate of the retinol used as a control (Table 5).

Test group Treatment concentration (%) MMP-1 expression inhibition rate (%) Control - - Example 1 0.1 23 Retinol 0.1 17

delete

Example 6: Experiment to measure melanin production inhibitory effect using B16F1 melanocytes

In this example, to confirm the whitening effect of the fucoxanthin extract obtained in Example 1, the degree of inhibition of melanin production against B16F1 melanocytes was tested.

B16F1 melanocytes used in this example are cell strains derived from mice, and cells that secrete a black pigment called melanin. Samples were treated during the artificial culture of these cells to evaluate the degree of melanin black pigment reduction. B16F1 melanocytes were distributed from ATCC (American Type Culture Collection, Accession No .: 6323). The melanin biosynthesis inhibitory effect of B16F1 melanocytes was measured as follows.

B16F1 melanocytes were dispensed in 6 well plates at a concentration of 2 × 10 ⁶ per well, cells were attached, and the samples were incubated for 72 hours by treating the samples at a concentration that does not cause toxicity. After 72 hours of incubation, the cells were detached with trypsin-EDTA, the cell number was measured, and the cells were recovered by centrifugation. Quantification of intracellular melanin was carried out with a slight modification of the method of Rotan: Cancer Res., 40: 3345-3350, 1980.

The cell pellet was washed once with PBS, and then 1 ml of homogenization buffer solution (50 mM sodium phosphate, pH 6.8, 1% Triton X-100, 2 mM PMSF) was added and vortexed for 5 minutes to disrupt cells. Melt the extracted melanin by adding 1N NaOH (10% DMSO) to the cell filtrate obtained by centrifugation (3,000 rpm, 10 minutes), and measure the absorbance of melanin at 405 nm with a microplate reader. Percent inhibition of production was measured. Melanin inhibition rate (%) of B16F1 melanocytes was calculated by the equation (3), IC50 value is the concentration of a substance that inhibits melanin production by 50%.

A: Melanin amount in wells without sample

B: Melanin amount in the wells to which the sample was added

Sample Name Treatment concentration (%) Melanin synthesis inhibitory effect (IC50) Example 1 0.1 0.09% Arbutin 0.1 0.3% Hydroquinone 0.1 0.07% Soluble Licorice Extract 0.1 0.1%

delete

As a result, the IC50 value of fucoxanthin extract was 0.09%, which showed similar or superior effect to the existing whitening agents hydroquinone, arbutin, oil-soluble licorice extract, and the like.

Example 7 Cytotoxic Alleviation Effect by Ultraviolet Irradiation

This example was carried out to evaluate the alleviation effect of cytotoxicity by ultraviolet irradiation of the fucoxanthin extract obtained in Example 1. Fibroblasts (fibroblasts) were placed in 24-well test plates 1 × 10 ⁵ and attached for 24 hours. Each well was washed once with PBS and 500 ul of PBS was added to each well. The fibroblasts were irradiated with UV 10mJ / cm 2 using an ultraviolet B (UVB) lamp (Model: F15T8, UV B 15W, Sankyo Dennki, Japan), and then PBS was removed and 10% FBS was added to the cell culture medium (DMEM). 1 ml) was added. It was incubated for 24 hours after the treatment of fucoxanthin extract to be evaluated here. After 24 hours, the medium was removed, and 500 μl of cell culture medium and 60 μl of MTT solution (2.5 mg / ml) were added to each well, followed by 37 ° C. CO ₂ for 2 hours. Cultured in the incubator. The medium was removed and 500 μl of isopropanol-HCl (0.04 N) was added. After shaking for 5 minutes, the cells were lysed and 100 μl of the supernatant was transferred to a 96-well test plate, followed by measurement of 565 nm absorbance in a microplate reader.

Cell survival rate (%) was measured by the equation (4) and cytotoxicity relaxation by ultraviolet light was calculated by the equation (5).

Bo: 565 nm absorbance of wells that only developed cell culture media

Bt: 565 nm absorbance of the wells that developed the colorless reaction wells

St: 565 nm absorbance of the wells that reacted the sample treated wells

Bo: cell viability of wells without UV irradiation and no sample treatment

Bt: cell viability of wells that were not irradiated with UV light

St: Cell survival rate of wells treated with UV

Sample Name Treatment concentration (%) Cytotoxic alleviation rate (%) Example 1 0.013 35 0.025 63

delete

Experimental results show that fucoxanthin extract effectively reduces cytotoxicity by UV rays at 0.013% concentration by 35%, effectively protecting against UV toxicity.

Example 8 Inhibitory Effect of Inflammatory Cytokine Expression by Ultraviolet Irradiation

In this Example, Fibroblasts isolated from human epidermal tissue were evaluated in 24-well test plates to evaluate the inhibitory effect of inflammatory cytokine expression expressed by UV irradiation of the fucoxanthin extract obtained in Example 1. × 104 pieces were added and attached for 24 hours. Each well was washed once with PBS and 500 μl of PBS was added to each well. The fibroblasts were irradiated with 10 mJ / cm 2 of ultraviolet rays using an ultraviolet B (UVB) lamp (Model: F15T8, UV B 15W, Sankyo Dennki, Japan), after which PBS was removed and cell culture medium (FMEM was not added to DMEM). Medium) 350 μl was added. After treatment with the fucoxanthin extract to be evaluated here was incubated for 5 hours. 150 μl of the culture supernatant was taken to quantify IL-1α, and thus the inhibitory effect of inflammatory cytokine expression was determined. The amount of IL-1α was quantified using Enzyme-linked Immunosorbent Assay, and the production rate of IL-1α was calculated by Equation 6.

Bo: amount of IL-1α produced in wells not irradiated with UV

Bt: amount of IL-1α produced in wells that were not irradiated with UV light

St: Production of IL-1α in Wells Treated by Ultraviolet Irradiation

Sample Name Treatment concentration (%) Inhibitory rate of inflammatory cytokine expression (%) Example 1 0.013 37 0.025 69

delete

Experimental results show that fucoxanthin extract inhibits the generation of inflammatory cytokines, IL-1α, by 37% at a concentration of 0.013%, effectively preventing the occurrence of inflammation caused by UV at low concentrations (Table 8).

Example 9: Paper Disc Test

This Example was a paper disc test (Paper Disc Test) to check the antibacterial activity of the fucoxanthin extract obtained in Example 1 against acne bacteria. First, in order to activate propionibacterium acnes, a skin flora of the cause of acne, it was preincubated for 48 hours in BHI liquid medium (Brain-Heart Infusion Broth; 3.7%). The prepared culture medium was smeared in 0.1 ml each of BHI solid medium (Brain-Heart Infusion Broth; 3.7%; agar 1.5%) and dried.

The fucoxanthin extract obtained in Example 1 was diluted to 12% (W / v) in a 95% ethanol aqueous solution, and 50 µl each was added to an 8 mm diameter paper disc and placed on the solid medium prepared above. The cells were incubated for 3 days. The antimicrobial activity was evaluated by observing the growth inhibition zones around the paper disc and measuring the size of the ring. As a result, the size of the bacterial growth inhibitory ring of the fucoxanthin extract was found to be 18 mm.

Example 10 and Comparative Example 2

This Example prepared a cosmetic containing the fucoxanthin extract obtained in Example 1 and evaluated the skin whitening effect in a comparative experiment with Comparative Example 2 in humans.

The cosmetics used in the comparative experiments are in the form of a cream, the composition of which is shown in Table 9. First, the phase b) recorded in Table 9 is heated and stored at 70 ° C. To this, a) phase is added, and after pre-emulsification, it is emulsified uniformly with a homomixer, and then cooled slowly to prepare a cream (Example 10, Comparative Example 2). 20 experimenters (women aged 20-35 years) were applied to the right side of the face with the cream prepared in Example 10 and the left side of the face with the cream prepared in Comparative Example 2 twice a day for 2 consecutive months. . After completion of the test, the color of the applied areas on both sides of the face was measured using an image analyzer and the color of the skin using chromameter (Minolta CR300) to measure the change in brightness (ΔL) of the color. Subjective visual observation was performed and the effect was measured according to the following classification. The results are shown in Table 10 below. At this time, the degree of whitening efficacy was classified into the following seven ratings.

* Evaluation criteria for whitening efficacy

-3: very worse -2: worse -1: slightly worse 0: no change

1: slightly improved 2: improved 3: highly improved

Raw material Example 10 Comparative Example 2 end Stearyl alcohol 8 8 Stearic acid 2 2 Stearic cholesterol 2 2 Squalane 4 4 2-octyldodecyl alcohol 6 6 Polyoxyethylene (25 mol addition)
Alcohol ester 3 3 Glyceryl Monostearic Acid
Aster 2 2 I Fucoxanthin Extract One - Propylene glycol 5 5 Purified water Quantity Quantity

delete

Note) Unit: Weight%

Change in brightness of skin color (ΔL) Objective evaluation of the skilled person Subjective Evaluation of the Subject Example 10 Comparative Example 2 Example 10 Comparative Example 2 Example 10 Comparative Example 2 medium 6.52 3.23 2.7 1.4 3.3 2.6

delete

As shown in Table 10, it can be seen that the whitening effect is excellent in the facial skin of the experimenter who applied the cream containing fucoxanthin extract.

Example 11

In this embodiment, the cosmetics containing the fucoxanthin extract obtained in Example 1 were prepared and evaluated by performing comparative experiments on skin wrinkle improvement and skin beauty effects. The creams prepared in Example 10 and Comparative Example 2 were prepared in Comparative Example 2 on the right side of the face of the experimenter (20-35 year old female), and the cream prepared in Example 10 on the left side of the face. The cream was applied twice a day for two consecutive months.

After completion of the experiment, the skin wrinkle relief effect is collected before and after using the product for 2 months by using a silicone resin copy (replica) around the face of the eye, and comparing the state of the eye wrinkles with a skin fine wrinkle device and a skin image analyzer.

Table 11 compares the average eye wrinkle depth and wrinkle reduction rate of the experimenter using the cream prepared in Example 10 with the experimenter using the cream of Comparative Example 2. As shown in Table 11, it can be seen that the skin wrinkle improvement effect is excellent in the skin around the face of the experimenter applying the cream containing the fucoxanthin extract.

Example 10 Comparative Example 2 Before use 2 months after use Before use 2 months after use Average fine wrinkle depth 0.187 0.154 0.186 0.181 Wrinkle Reduction (%) 17.6% 2.7%

delete

n = 20, p <0.01

Example 12 and Comparative Examples 3-6

This Example prepared the cosmetics containing the fucoxanthin extract obtained in Example 1 and evaluated the skin moisturizing effect in a comparative experiment with Comparative Examples 3 to 6 in humans.

The cosmetics used in the comparative experiments are in the form of a cream, the composition of which is shown in Table 12. First, the phase b) recorded in Table 12 is heated and stored at 70 ° C. To this, a) phase is added, and after pre-emulsification, it is emulsified uniformly with a homomixer, and then it is cooled slowly to prepare a cream (Example 12, Comparative Examples 3-6). Example 20 and Comparative Examples 3-6 were apply | coated to the lower part for 20 experimenters, and after about 90 minutes, (DELTA) Hydration was evaluated by the Corneometer (CM 820) for the moisture retention ability before and after application | coating.

Raw material Example 12 Comparative Example 3 Comparative Example 4 Comparative Example 5 Comparative Example 6 end Stearyl alcohol 8 8 8 8 8 Stearic acid 2 2 2 2 2 Stearic cholesterol 2 2 2 2 2 Squalane 4 4 4 4 4 2-octyldodecyl alcohol 6 6 6 6 6 Polyoxyethylene (25 mol addition)
Alcohol ester 3 3 3 3 3 Glyceryl Monostearic Acid
Aster 2 2 2 2 2 I Fucoxanthin Extract 5 - - - - glycerin - - 5 - - Na-hyaluronic acid salt - - - 5 - 1,3-butylene glycol - - - - 5 Purified water Quantity Quantity Quantity Quantity Quantity

delete

Note) Unit: Weight%

division Example 12 Comparative Example 3 Comparative Example 4 Comparative Example 5 Comparative Example 6 Moisturizer Types Fucoxanthin Extract - glycerin Na-hyaluronic acid salt 1,3-butylene glycol Coneometer
(△ Hydration) 19 6 12 8 11

delete

As shown in Table 13 it can be seen that the moisturizing effect is excellent in the facial skin of the experimenter applying the cream containing the fucoxanthin extract.

The present invention relates to a method for extracting fucoxanthin (Fucoxanthin) obtained from brown algae using fermented microorganisms and to a cosmetic composition containing the same, wherein the brown algae such as seaweed, seaweed, moths and sorghum bacillus subtilis ( bacillus subtilis , Bacillus subtilis) ) And lactobacillus acidophilus ( lactobacillus acidophilus , lactic acid bacteria) through the fermentation to obtain a high yield of fucoxanthin, which contains 0.001 to 30.0% by weight as the main active ingredient, skin moisturizing effect, irritation A multifunctional cosmetic composition having an alleviating effect, a whitening effect, an anti-wrinkle effect, an atopy improving effect, and an anti-inflammatory effect can be provided.

According to the present invention, the fermented fucoxanthin extract is excellent in moisturizing effect, improves skin wrinkles, prevents acne, and alleviates skin irritation. Thus, various functional cosmetics can be prepared using this substance.

Claims (9)

A method of extracting fermented fucoxanthin from brown algae using a bacillus subtilis bacterium. A cosmetic composition comprising the fermented fucoxanthin extracted by the method according to claim 1. The method of claim 2, Cosmetic composition characterized in that it contains the fermented fucoxanthin in an amount of 0.001 to 30.0% by weight based on the total weight of the composition. The method of claim 3, The cosmetic composition is a cosmetic composition, characterized in that used for moisturizing the skin. The method of claim 3, The cosmetic composition is a cosmetic composition, characterized in that used for stimulating relaxation. The method of claim 3, The cosmetic composition is a cosmetic composition, characterized in that it is used for skin whitening. The method of claim 3, The cosmetic composition is a cosmetic composition, characterized in that used for improving wrinkles. The method of claim 3, The cosmetic composition is a cosmetic composition, characterized in that used for improving atopy. The method of claim 3, The cosmetic composition is a cosmetic composition, characterized in that selected from the lotion, gel, water-soluble liquid, cream, essence, oil-in-water (O / W) or water-in-oil (W / O) type emulsion, ointment formulation.