KR20130097870A - Ulva spp seaweed hydrolysates that have high glucuronic acid cotent, preparation method thereof and antiaging cosmetic composition containing the same - Google Patents

Abstract

PURPOSE: A functional cosmetic composition for anti-aging is provided to suppress MMP-1 biosynthesis and to promote biosynthesis of type 1 procollagen, thereby improving skin elasticity and anti-wrinkling effect. CONSTITUTION: A method for preparing an Ulva spp. seaweed hydrolysate comprises the steps of: extracting dried and pulverized Ulva spp. seaweed using hot water and preparing a soluble extract; adding ethyl alcohol to the soluble extract and precipitating polysaccharides and proteins; filtering and centrifuging the soluble extract to collect the precipitate; selectively collecting Ulva spp. Seaweed-containing polysaccharides; dispersing the polysaccharides in a volatile acid solution and filtering to obtain an Ulva spp. seaweed hydrolysate; and removing acid and solvents from the Ulva spp. seaweed hydrolysate. A cosmetic composition contains 0.001-30.0 wt% of the Ulva spp. seaweed hydrolysate as an active ingredient, containing 1.0 % of glucoronic acid.

Description

Ulva spp seaweed hydrolysates that have high glucuronic acid cotent, preparation method according to anti-aging cosmetic composition containing the same

The present invention relates to a method for producing an anti-aging functional cosmetic composition from the alveolar (Ulvaceae) algae, and to an anti-aging functional cosmetic composition containing the same as an active ingredient, more specifically to separating the polysaccharide from the algae (Ulvaceae) algae, Acid-decomposing these to prepare a glucuronic acid-containing algae decomposition products, and active oxygen scavenging effect containing the glucuronic acid-containing green algae and algae decomposition products as an active ingredient, skin fine wrinkles improvement effect, skin elasticity improvement effect The present invention relates to an anti-aging functional cosmetic composition having an excellent anti-aging effect.

Photo aging means that the skin is repeatedly exposed to light such as ultraviolet rays to change the appearance or function of the skin [Ridder GM et al .: J. Am. Acad. Dermatol., 25, 751-760 (1991)]. More specifically, photoaging is the damage of lipids, proteins, polysaccharides and nucleic acids which are the main constituents of the skin due to ultraviolet rays, resulting in the destruction of skin cells and tissues, resulting in skin aging. In particular, collagen, hyaluronic acid, elastin, proteoglycan, and fibronectin, which are the connective tissues of the skin, are cut to interfere with skin elasticity and form deep wrinkles, a major phenomenon of photoaging. It leads to a situation of induction and a decrease in immune function.

In vivo, the synthesis and degradation of extracellular matrix such as collagen is properly regulated, but as aging progresses, especially in areas exposed to ultraviolet light, the synthesis decreases and the degradation is accelerated, and enzymes that degrade collagen by ultraviolet light Phosphorus matrix metalloproteinase (hereinafter referred to as 'MMP') expression is promoted to reduce skin elasticity and wrinkles are formed.

MMPs are broadly classified into MMP-1, MMP-2, and MMP-9 according to their substrate specificity. MMP-1 is a fibroblast type collagenase, an enzyme of epilepsy, and its substrates are collagen types I, II, III, Ⅶ, Ⅷ, Ⅹ and gelatin, and are originally 3/4 or 1/4 length peptides of collagen length. The cleavage facilitates the action of nonspecific enzymes (gelatinase, etc.). Collagen types I and III, elastin and fibronectin, which occupy most of the dermal layer of human skin, give strength and tension to the skin.When proteins are broken down by MMPs that are abnormally activated by UV rays and free radicals, wrinkles and elasticity decrease, Aging phenomenon such as sag is accelerated. Inhibiting the activity and biosynthesis of MMP-1, which degrades type I collagen, which accounts for the highest proportion of binding proteins, can protect skin binding proteins and prevent wrinkle formation and elasticity. Therefore, there is a need for the development of a substance capable of regulating or inhibiting MMP expression in which activation is induced in cells by ultraviolet rays.

Recently, in order to reduce skin irritation caused by various chemicals, a lot of attention has been focused on functional cosmetics having functions such as wrinkle relaxation and whitening obtained from natural extracts. Natural materials have less side effects on the skin, and as the consumer's response to the external skin preparations using the natural materials has increased recently, the development value of the photo-aging anti-aging cosmetic composition is further increased.

The Ulvaceae is also called the Cheongtaedae and is a seaweed of algae that grows where freshwater flows from the upper tidal flats of the seashore. Cylindrical fronds are divided into several branches in the lower part and the upper part, and some are hollow. The color is blueish green. Ulva pertusa, Ulva lactuca, Ulva conglobata Kjellman, Ulbraulva japonica, Enteromorpha prolifera, Enteromorpha compressa, Enteromorpha intestinalis lattice This includes Enteromorpha clathrata and Enteromorpha linza, which are common on all coasts of Korea. Usually eaten and used as feed and compost. It is distributed all over the world including Korea.

Among them, Ulva pertusa is classified as a representative harmful seaweed that damages the scenery of summer beaches and damages the aquaculture industry.

Brown seaweed and algae in the surface of the ocean and in the intertidal zones that connect the ocean and land are in harsh environments with direct contact with UV and salt water. Therefore, brown seaweed and algae produce various antioxidants in order to cope with damage to tissues and cells caused by ultraviolet rays and dry environment, and also establish various defense mechanisms such as moisturizing ingredients such as polysaccharides to carry out life activities. .

In other words, by utilizing the self-protective ingredients of the seaweed, it is expected to develop a functional cosmetic material having a high added value.

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. Japanese Patent Application Laid-Open No. 2-245087 describes the antioxidant effect of Sargassum genus extract. Japanese Patent Application Laid-Open No. 3-294384 describes an antioxidant composition extracted from the genus Hijikia. Japanese Patent Laid-Open No. 7-10769 describes a extract of Phaeophyceae having an astringent effect. Patent Publication No. 2009-0020216 discloses a whitening cosmetic composition comprising an extract of Diticota brown algae. Patent No. 0858358 is an extract of Dictyota dichotoma, which is one of NO production inhibitory activity, early inflammatory molecule inhibitory activity, iNOS expression inhibitory activity, COX-2 expression inhibitory activity, and inflammatory PGE2 production inhibitory activity It describes a chamomile extract based on anti-inflammatory activity characterized by exhibiting the above activity. Patent Publication No. 2005-0095298 suppresses the release of mediators such as histamine and β-hexosaminidase in mast cells, and cell-activated substances such as IL-8 and TNF-α (Tumor Necrosis Factor). By suppressing the release of the skin protection and skin disease improvement composition comprising a leather nettle extract extract showing the improvement and treatment effect of inflammatory skin diseases including psoriasis, eczema, acne as well as allergic skin diseases such as atopic dermatitis. It starts. Patent No. 0762181 discloses that the extract of Ecklonia cava has a specific antimicrobial activity against Propionibacterium acnes, the main cause of acne, water-soluble skins for preventing and treating acne, viscosity and hardness modifiers, It describes a cosmetic composition characterized by producing a UV absorber, a pigment, a moisturizer, a detergent, a preservative or a mixture of electrical substances. The cosmetic composition for improving skin transparency containing seaweed extract contains seaweed extract extracted from Dunaliella Salina, supplying energy to skin cells and turning-over of the epidermal cells. It describes a cosmetic composition for acne skin using the chamomile of the extract of chamomile purple in the cosmetic composition to promote clear, clear skin and clear. Patent No. 0745023 discloses that the extract of the red algae symphyocladia latiuscula has specific antibacterial activity against propionibacterium acnes, the main cause of acne, and is a water-soluble skin agent for preventing and treating acne. And a hardness modifier, an ultraviolet absorber, a pigment, a moisturizer, a detergent, a preservative, or a mixture of electrical substances. Patent No. 0728443 relates to a cosmetic additive composition containing a seaweed (Laminaria Saccharina) extract and a cosmetic comprising the same, and contains a seaweed extract, phytospingosine, and extract of Machi, to enhance skin elasticity, pore reduction effect and skin. The cosmetic composition characterized by the antibacterial inhibitory effect is described. Patent No. 0542446 discloses a cosmetic composition containing extract of Maeseng as a main active ingredient, and has excellent cosmetic composition with an inhibitory effect on melanin biosynthesis and tyrosinase activity, an antioxidant effect, and an improvement on skin wrinkles. Korean Patent No. 0429590 provides a cosmetic composition for improving wrinkles containing ECKLONIA CAVA extract, and a method for preparing the same, a method for alleviating wrinkles on the skin, a composition for promoting collagen synthesis, and a method for promoting collagen synthesis using the same. The present invention describes a composition that promotes cellular activity and collagen synthesis of fibroblasts, relieves skin wrinkles and imparts elasticity to skin. However, these seaweed extracts are low in content even though the identification of the main ingredient and the active ingredient or the main ingredient that can be used as an indicator ingredient, or information on the main ingredient is low, the content is low and economical and functional as a raw material of anti-aging functional cosmetic composition This is quite low.

An object of the present invention is to select anti-aging functional cosmetics by selecting the algae of brown seaweed that is classified as a low-utility or harmful seaweed among various natural products.

In addition, it is an object of the present invention to provide a functional cosmetic composition exhibiting an anti-aging effect by using brown seaweed and algae degradation products containing 1.0% or more of glucuronic acid as a raw material.

Accordingly, the present inventors have prepared a degradation product having a high content of glucuronic acid, which can be used as a main component and an index component of anti-aging functional cosmetics from brown seaweed and algae after a long study, thereby promoting the inhibition of MMP-1 biosynthesis and the biosynthesis of type 1 procollagen. As a result of experiments on the effect of improving skin elasticity and fine wrinkles, and the effect of inhibiting photoaging promoted by ultraviolet rays, it has been found that a desirable result can be obtained and the efficacy as an anti-aging functional cosmetic composition can be expected.

The present invention

Step 1: Add a brown seaweed and seaweed as an extraction solvent, add a basic aqueous solution, an acidic aqueous solution and neutral salts such as sodium chloride to adjust pH and salt concentration, and then heat or cool to 20 to 100 ° C. Manufacturing process,

Step 2: After adding filtrate ethyl alcohol in the range of 50% by weight to 500% by weight to the prepared extract of brown seaweed and seaweed aqueous solution to precipitate the polymer polysaccharide and protein, the precipitate is recovered by filtration and centrifugation to recover the brown seaweed and seaweed. Selectively recovering the containing polysaccharide;

Step 3: Hydrochloric acid, acetic acid, formic acid, butyric acid, propionic acid, propionic acid, trifluoroacetic acid, TFA ), Pentafluoropropionic acid (PFPA), heptafluorofluorobutyric acid (HFBA), nonnafluoro pentanoic acid (NFPA), tridecafluoroheptanoic acid (TDFHA), Dispersed in one or more volatile acids 1 to 6N solution selected from pentadecafluorooctaonoic acid (PDFOA), maintaining the reaction temperature of 80 to 125 ℃ in a sealed container, and the acid decomposition after filtration to filter the water-soluble polysaccharide decomposition products Manufacturing process,

Step 4: Concentrated decomposed brown seaweed and algae polysaccharide decomposition product to remove volatile acid and solvent, and then dissolve it in purified water again, repeating the process of dissolving the dried product in purified water at a concentration of 5% may exhibit a pH of 3.0 or more It relates to a method for producing brown seaweed and algae decomposition products containing 1.0% or more of glucuronic acid, so as to remove volatile acids and impurities.

Next, the prepared degradation product can be used as it is, and in some cases, it can be fractionated into brown algae and algae decomposition products having increased glucuronic acid content by using an ultrafiltration device and a gel chromatogram, and can be used for various purposes.

Next, the decomposed product can be concentrated and used by vacuum concentration method, and powdered brown seaweed and algae decomposition products can be obtained using a spray dryer, or dried by lyophilization and pulverized to a suitable size. Can be obtained.

In addition, in the method for producing a low molecular weight pectin decomposition product of the present invention, the volatile acids used for acid decomposition are hydrochloric acid, acetic acid, formic acid, butyric acid, propionic acid, propionic aci1d. , Trifluoroacetic acid (TFA), pentafluoropropionic acid (PFPA), heptafluorofluorobutyric acid (HFBA), nonafluoro pentanoic acid (NFPA), tridecafluoro It is characterized by one or more volatile acids selected from tridecafluoroheptanoic acid (TDFHA), pentadecafluorooctaonoic acid (PDFOA), which is difficult to remove during the manufacturing process, such as concentrated under reduced pressure. Remains in the form of impurities in the finished raw material, if neutralized by the neutralization process, the stability of the emulsion and gel formulation in the cosmetic formulation On a profound effect has the disadvantage that the salt concentration is increased.

In addition, the method for preparing brown seaweed and seaweed polysaccharide decomposition products of the present invention, characterized in that the concentration of the volatile acid solution to disperse and dissolve the brown seaweed and seaweed-containing polymer polysaccharide is 1 to 6N, when the reaction is carried out at 1N or less, Low decomposition reaction rate and degradation rate of polysaccharides may cause increased process cost due to lower glucuronic acid content of browned seaweed and algae polysaccharide decomposition products and increased time required for decomposition process, and the reaction rate is too fast for 6N or more to obtain desired product. It is difficult to control the reaction rate, and there is a possibility of generating impurities such as dehydration condensation reaction, it is difficult to produce the brown seaweed and algae polysaccharide decomposition products containing more than 1.0% glucuronic acid required by the present invention, and has the disadvantage of low economic efficiency.

In addition, the method for producing brown seaweed and algae polysaccharide decomposition products of the present invention, characterized in that the temperature conditions for dispersing and acid-decomposing the brown seaweed and seaweed-containing polymer polysaccharide is 80 to 125 ℃, below 80 ℃ decomposition rate and decomposition rate of polysaccharides This lower process may lead to an increase in process costs due to a decrease in the glucuronic acid content of the prepared brown seaweed and algae polysaccharide decomposition products, and an increase in the time required for the decomposition process. It is difficult to produce brown seaweed and algae polysaccharide decomposition products containing 1.0% or more of glucuronic acid required by the present invention, such as loss of raw materials due to volatilization of volatile acids and costs for removing environmental pollution factors. .

In addition, in the method for preparing brown seaweed and seaweed polysaccharide decomposition products of the present invention, in the process of dispersing the brown seaweed and seaweed-containing polymer polysaccharide by acid decomposition, in the process of removing volatile acids and impurities, the dried product prepared by decompression concentration to 5% concentration It is characterized by removing volatile acids so that pH 3.0 or more can be seen when dissolved in purified water, and when the dried product prepared by decompression concentration of acidic acid is dissolved in purified water at a concentration of 5%, it is volatile in the raw material. The presence of a large number of acids, when applied to cosmetics not only destroys the appearance of the cosmetics, but also causes a specific odor and becomes unsuitable as a raw material for cosmetics. Increased concentrations of salts that have a profound effect on the stability of heavy fluids and gel formulations. There point.

In addition, the ethyl alcohol used in the step of selectively recovering the two-component brown seaweed and seaweed-containing polymer polysaccharide in the method for preparing the brown seaweed and seaweed polysaccharide decomposition product in the range of 50% by weight to 500% by weight relative to the weight of the brown seaweed and seaweed aqueous extract It is characterized by recovering the brown polysaccharides and algae-containing polymer polysaccharide, when the addition amount of ethyl alcohol is less than 50% by weight of the reaction liquid, it is difficult to efficiently precipitate the brown polyps and seaweed-containing polymer polysaccharides, selectively There is a disadvantage in that it is difficult to separate, and when adding more than 500% by weight of ethyl alcohol relative to the weight of the reaction solution, the reaction vessel is too large and the amount of the reaction solution is too large, not only excessive process cost, but also excess ethyl alcohol There is a disadvantage that is not economical to use.

In addition, the method for preparing brown seaweed and algae polysaccharide decomposition products of the present invention, characterized in that the content of glucuronic acid contained in the prepared brown seaweed and seaweed-containing polymer polysaccharide decomposition products, 1.0% by weight glucuronic acid content If less than, there is a disadvantage that the content is low in order to utilize as an active ingredient for improving wrinkles functional cosmetics for the present invention.

In addition, the present invention provides a cosmetic composition, characterized in that the content of the brown seaweed and seaweed degradation products prepared by the method for preparing brown seaweed and seaweed polysaccharide decomposition products is 0.001-30.0% by weight based on the freeze-dried weight of the whole cosmetic composition. When the content of the extract is less than 0.001% by weight, there is almost no skin improvement effect, and when the content of the extract is more than 30.0% by weight, the degree of increase in the effect of increasing the content is insignificant and thus it is not economical.

The present invention relates to brown seaweed and seaweed degradation products prepared by the method for preparing brown seaweed and seaweed degradation products having a glucuronic acid content of 1.0% by weight or more. Brown seaweed and seaweed degradation products according to the present invention is usually in the range of 1.0 to 40% by weight of glucuronic acid, depending on the extraction time, the maximum value is expected to not exceed 50% by weight.

The present invention relates to a cosmetic composition exhibiting anti-aging effect, which contains brown seaweed and seaweed degradation products prepared by the method for preparing brown seaweed and seaweed degradation products having a high glucuronic acid content.

The present invention relates to a cosmetic composition having an effect of improving skin wrinkles, which comprises brown seaweed and seaweed degradation products prepared by a method for producing brown seaweed and seaweed decomposition products having high glucuronic acid content.

The present invention relates to a cosmetic composition having a skin elasticity-improving effect containing the brown seaweed and algae decomposition products prepared by the method for producing brown seaweed and seaweed decomposition products having high glucuronic acid content as a main active ingredient.

In addition, the present invention is a cosmetic composition is characterized in that the glucuronic acid is selected from the formulation of the lotion, gel, water-soluble liquid, cream, essence, oil-in-water (O / W) type and water-in-oil (W / O) type Provided is an anti-aging functional cosmetic composition containing the green seaweed and seaweed degradation products containing 1.0% or more as a main active ingredient.

The composition of the present invention exhibits the effect of inhibiting MMP-1 biosynthesis and improving skin elasticity and fine lines by promoting biosynthesis of type 1 procollagen.

In addition, the composition of the present invention exhibits the effect of suppressing photoaging damage of skin cells promoted by ultraviolet rays or the like.

MMP-1 biosynthesis inhibitory effect, type 1 procollagen effect of synthesizing the skin elasticity and fine wrinkles effect, and the effect of inhibiting photoaging promoted by ultraviolet light, the results were obtained and confirmed the results as an anti-aging functional cosmetic composition It was.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be noted, however, that these examples are only illustrative examples of the present invention, and the scope of the present invention is not limited to these examples.

Example  One: Perforated Heat number  Extract preparation

After dividing and drying the dried perilla in 5% by weight in purified water, it was transferred to a sealed container, extracted at 80 ° C. for 3 hours with stirring, and filtered with Whatman # 5 filter paper. The filtered extract was concentrated under reduced pressure and freeze-dried at 50 ° C. or lower to prepare a powdered hot water extract.

Example  2: Perforated Heat number  extract resolvent  Produce

After dispersing 10 g of hot water extract obtained in Example 1 in 1 liter of trifluoroacetic acid (TFA) 2N solution, acid decomposition was performed for 2 hours while maintaining the reaction temperature at 100 ° C. in a closed container. . The filtered digest was concentrated under reduced pressure at 50 ° C. or lower, and then dissolved in purified water and concentrated under reduced pressure three times to remove volatile acids and solvents. When the dried product of the produced acid degradation product was dissolved in purified water at a concentration of 5%, pH 3.2 was shown, and the prepared cracked hot water extract decomposition product was 2.4 g.

Example  3: Perforated  Polymer Polysaccharide Preparation

After dividing and drying the dried perilla by 5% by weight in purified water, it was transferred to a sealed container, extracted at 80 ° C. for 3 hours with stirring, and filtered with Whatman # 5 filter paper. After adding and stirring the same amount of ethyl alcohol as the weight of the filtered extract, the polymer polysaccharide and protein were precipitated by standing in a 4 ° C. low temperature storage tank, centrifugal filtration, and the like, and the precipitate was recovered by filtration and centrifugation. The hydrothermal extract-containing polymer polysaccharide was selectively recovered. The recovered polysaccharide was redissolved in purified water, concentrated under reduced pressure and lyophilized at 50 ° C. or lower to prepare a powdery polysaccharide.

Example  4: Perforated  Polymer polysaccharide resolvent  Produce

10 g of the green polysaccharide polysaccharide obtained in Example 3 was dispersed in 1 liter of trifluoroacetic acid (TFA) 2N solution, and then acid decomposition was performed for 2 hours while maintaining the reaction temperature at 100 ° C. in a sealed container. . The filtered digest was concentrated under reduced pressure at 50 ° C. or lower, and then dissolved in purified water and concentrated under reduced pressure three times to remove volatile acids and solvents. When the dried product of the produced acid degradation product was dissolved in purified water at a concentration of 5%, pH 3.2 was shown, and the prepared cracked hot water extract decomposition product was 3.6 g.

Experimental Example  1: Cytotoxicity of Samples

Cytotoxicity to skin cells of the soy blue lysate or extract sample obtained in Examples 1 to 4 was evaluated.

Fibroblasts (fibroblasts) were placed in a 24-well test plate 1 x 10 ⁵ and attached for 24 hours. Each well was washed once with PBS (Phosphate buffered saline), and 1 mL of cell culture medium (10% FBS added to DMEM) was diluted to an appropriate concentration in each well. Each was treated and incubated for 24 hours. After 24 hours, the medium was removed, and a cell culture medium (500 μl) and a solution of MTT {3- (4,5-dimethylthiazol-2-yl) -2,5- diphenyl tetrazolium bromide} (2.5 mg / And then cultured in a 37 ° C CO ₂ incubator for 2 hours. 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 쨉 l of the supernatant was transferred to a 96-well test plate, and the absorbance at 565 nm was measured in a microplate reader. The cell viability (%) was determined by Equation (1) and the concentration of the sample that did not affect cell survival was determined.

Bo: 565 nm absorbance value of a well that underwent chromogenic reaction in cell culture medium

Bt: 565 nm absorbance value of the well that underwent chromogenic reaction without treatment of the sample

St: Absorbance value of 565 nm of the well treated and reacted with the sample

As can be seen from Table 1, the 100% viable treatment concentration of skin fibroblasts of each sample was 1.0% in the red hot water extract of Example 1 and 0.1% of the red hot water extract of Example 2 through the acid decomposition process. A slight cytotoxic effect was confirmed. However, this is a safe sample with a low probability of causing skin irritation given the concentrations generally used in cosmetics. In addition, it was confirmed that Example 3 in which only the green polysaccharide polysaccharide was isolated and Sample Example 4 in which the polysaccharide was acid-decomposed had less cytotoxicity than Example 1, which is a hydrothermal extract, and Example 2, which is an acid decomposed product of the hydrothermal extract, respectively. . Therefore, when preparing the hot water extract and polysaccharide separated from them also has a relatively low cytotoxicity, it was confirmed that the possibility of skin irritation is small and safe when considering the concentration used in cosmetics.

Name of sample 100% cell survival concentration Whole green sea green water extract of Example 1 1.0% Cracked Green Water Extract Degradant of Example 2 0.1% The brownish green polymer polysaccharide of Example 3 2.5% The brownish green polymer polysaccharide decomposition product of Example 4 0.5%

Experimental Example  2: NBT Experiment on antioxidant effect test

In order to confirm the antioxidant effect of the sample of green onion decomposition and extracts obtained in Examples 1 to 4 as a comparative example, butylated hydroxytoluene (BHT), which is well known as an antioxidant under laboratory conditions, was used for antioxidant activity using Nitro Blue Tetrazolium (NBT) method. Was measured.

In order to measure the antioxidant effect, the active oxygen produced by xanthine and xanthine oxidase is measured by NBT method, and the effect of removing the active oxygen by the test substance, that is, the active oxygen scavenging effect, is evaluated. Xanthine and xanthine oxidase produce free radicals. This active oxygen reacts with Nitro Blue Tetrazolium (NBT) to measure the active oxygen scavenging rate by measuring the blue color produced by this at a wavelength of 560 nm.

As a measuring method, 0.05M Na2CO3 (2.4ml), 3mM xanthine solution (0.1ml), 3mM EDTA solution (0.1ml), BSA solution (0.1ml) and 0.72mM NBT solution (0.1ml) were added thereto and 0.1ml of sample solution. Was added and left at 25 ° C. for 10 minutes. Xanthine oxidase solution (0.1 ml) is added and stirred quickly and incubation at 25 ° C. for 20 minutes is started. Then 6 mM CuCl ₂ solution (0.1 ml) was added to stop the reaction and the absorbance St at 560 nm was measured. In the blank test, absorbance Bt was measured by using distilled water instead of the sample solution as described above. Again, the blank of the sample solution was operated in the same manner using distilled water instead of 6 to measure the absorbance Bo.

Inhibition rate was calculated by Equation 2, the results are shown in Table 2.

St: 560nm absorbance after enzyme reaction of sample solution

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

So: 560nm absorbance before reaction without enzyme in sample solution

Bo: 560nm absorbance before reaction in the absence of enzyme in blank test solution

As can be seen in Table 2, all the samples showed a lower antioxidant activity than BHT, but the antioxidant activity was relatively increased through the acid decomposition process of pericarp hot water extract and polysaccharide. In particular, the sample of Example 4 acid-decomposed of the reddish green polymer polysaccharide of Example 3 exhibited about 76.1% antioxidant capacity when treated with 0.2% by weight, thereby significantly improving the antioxidant capacity of the reddish green onion and algae polysaccharide degradation products through the manufacturing process of the present invention. It can be seen that.

Name of sample content Antioxidative effect(%) Whole green sea green water extract of Example 1 0.5 wt% 41.3% Cracked Green Water Extract Degradant of Example 2 0.5 wt% 66.4% The brownish green polymer polysaccharide of Example 3 0.5 wt% 32.1% Example  4 of Perforated  Polymer polysaccharide resolvent 0.2 wt% 76.1% BHT 0.1 wt% 89.3%

Experimental Example  3: Free radical  Measurement of scavenging activity

In order to determine the free radical scavenging activity of the black green lysate and extract sample obtained in Examples 1 to 4, an antioxidant such as BHT was compared with a sample and the free radical scavenging activity was measured by DPPH method.

The DPPH method is a method of measuring free radical scavenging activity by reducing force using a free group called DPPH (2,2-Di (4-tert-octylphenyl) -1-picrylhydrazyl free radical). The degree of reduction of the absorbance by reduction of DPPH by the test substance is compared with the absorbance of the blank test solution and the free radical scavenging ratio is measured at a wavelength of 560 nm.

Samples were placed in 96-well plates, respectively, and DPPH prepared in 100 uM methanol solution was added to obtain a total volume of 200 μl. After incubation at 37 ° C for 30 minutes, absorbance was measured at 560 nm. Free radical scavenging activity (%) was calculated by the following equation.

A: Absorbance of control wells without sample

B: Absorbance of Experimental Wells Treated with Samples

As can be seen in Table 3, all four samples showed a relatively lower free radical scavenging activity than BHT, but it was confirmed that the free radical scavenging activity was relatively increased through the acid decomposition process of the hot water extract and polysaccharide. In particular, the sample of Example 4 acid-decomposed of the green polysaccharide polysaccharide of Example 3 exhibits about 93.4% free radical scavenging activity when treated with 0.2% by weight, the free of the green rind extract through the manufacturing process of the brown and seaweed polysaccharide decomposition products of the present invention It can be seen that the radical scavenging activity is significantly improved.

Name of sample content Antioxidative effect(%) Whole green sea green water extract of Example 1 0.5 wt% 9.9% Cracked Green Water Extract Degradant of Example 2 0.5 wt% 59.1% The brownish green polymer polysaccharide of Example 3 0.2 wt% 10.7% Example  4 of Perforated  Polymer polysaccharide resolvent 0.2 wt% 93.4% BHT 0.1 wt% 81.0%

Experimental Example  4: after UV irradiation Perforated On degradation products  by MMP -1 expression inhibition evaluation

Enzyme-immunoassay (ELISA) was performed to measure the concentration of MMP-1 expressed in the skin cells after UV irradiation and sample addition of the greenish green lysate and extract samples obtained in Examples 1 to 4.

Human dermal fibroblasts were irradiated with UVA at an energy of 6.3 J / cm 2 using a UV chamber. UV irradiation amount and incubation time were established through preliminary experiments to maximize the expression of MMP in fibroblasts. Negative controls were wrapped in tinfoil to maintain the same time in the UVA environment. UVA emission was measured using a UV radiometer. The cells during the UVA irradiation were intact in the previously dispensed medium and irradiated with UVA, exchanged with the medium containing the sample, and then cultured for 24 hours, and the medium was recovered and coated in 96 wells. The primary antibody (MMP-1 (Ab-5) monoclonal antibody) was 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 (1 mg / ml ρ-nitrophenyl phosphate in diethanolamine buffer solution) was reacted at room temperature for 30 minutes. The reaction was carried out for a minute and the absorbance was measured at 405 nm with a microplate reader. As a control group, a sample to which no sample was added was used.

The results are shown in Table 4, and the weight of the shredded green water extract of Example 2 and the shredded green polymer polysaccharide of Example 4 were 0.01 wt. It has been shown to inhibit MMP-1 expression of at least 39% at concentrations of at least%. In particular, the poached green polymer polysaccharide degradation product of Example 4 exhibited a inhibition rate of 47% or more at a concentration of 0.005% by weight, which was similar to the inhibition rate of retinol used as a control. However, the hot-water greenish water extracts of Example 1 and Example 3 and the polymer polysaccharide isolated therefrom exhibited relatively low MMP-1 expression inhibitory activity.

In other words, it can be seen that the inhibitory activity against the skin cell MMP-1 expression was significantly improved by the ultraviolet irradiation of the green lard and seaweed polysaccharide degradation products of the present invention.

Name of sample content MMP-1 expression inhibition rate Whole green sea green water extract of Example 1 0.01 wt% 12.3% Cracked Green Water Extract Degradant of Example 2 0.01 wt% 39.1% The brownish green polymer polysaccharide of Example 3 0.01 wt% 15.7% Example  4 of Perforated  Polymer polysaccharide resolvent 0.005 wt% 47.4% Retinol 0.005 wt% 38.0%

Experimental Example  5: Type 1 Procollagen Biosynthesis Promoting Effect

In order to investigate the effect of adding the greenish green lysate and extract samples obtained in Examples 1 to 4 to the skin cell culture solution and to promote the biosynthesis of the type 1 procollagen as a skin substrate component, a procollagen assay was performed as follows. It carried out in the same way.

Human dermal fibroblasts isolated from neonatal foreskin tissue were obtained from Modern Tissue Technology (MTT, Korea) and added 10% fetal bovine serum (FBS) to DMEM / F-12 (3: 1) medium. 1 × 10 ⁴ cell / cm ² Incubated at concentration. When 70 ~ 80% of the cells were grown, the cells were subcultured at a ratio of 1: 3, and the cells cultured in the third to fourth passages were used for the experiment. For the measurement of the amount of procollagen, the fibroblasts were cultured in a 48-well plate at a concentration of 90% or more, the respective samples were added at an appropriate concentration, and after 24 hours, the amount of procollagen liberated in the medium was measured with procollagen Type 1 C-peptide EIA kit (MK101, Takara, Japan).

As shown in Table 5, the shredded green water hot water extract of Example 2 and the shredded green polymer polysaccharide of Example 4 showed a procollagen biosynthesis promoting effect at a concentration of 0.001% by weight. In particular, the poached green polymer polysaccharide degraded product of Example 4 exhibited a biosynthetic promoting effect of about 30% procollagen at a concentration of 0.001% by weight, which is lower than that of TGF-β, a cell signaling material used as a positive control group, but has very good activity. Indicated.

Name of sample content MMP-1 expression inhibition rate Whole green sea green water extract of Example 1 0.001 wt% - Cracked Green Water Extract Degradant of Example 2 0.001 wt% 19.3% The brownish green polymer polysaccharide of Example 3 0.001 wt% - Example  4 of Perforated  Polymer polysaccharide resolvent 0.001 wt% 29.6% TGF-beta 0.001 wt% 33.4%

Experimental Example  6: Experiment for confirming skin keratinocyte activation effect

The effect of skin cell activation by the treatment of the greenish green lysate and extract sample obtained in Examples 1 to 4 was evaluated.

HaCaT cells, human keratinocyte cell lines, were cultured for 24 hours after dispensing 100 μl into 96 well plates at a concentration of 1 × 10 ⁴ / well, and then, each sample was diluted with various concentrations in serum-free medium and treated for 48 hours. Remove the medium, add 500 μl of cell culture medium and 60 μl of MTT {3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide} solution (2.5 mg / ml) per each well. The cells were incubated in a 37 ° C. CO ₂ incubator for hours. 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, and then 565 nm absorbance was measured in a microplate reader, and cell viability (%) was determined by Equation 1.

Name of sample 0.05% treatment
Survival rate 0.1% treatment
Survival rate 0.5% treatment
Survival rate Control group 100 100 100 Whole green sea green water extract of Example 1 99.8 106.8 110.7 Cracked Green Water Extract Degradant of Example 2 102.4 103.8 110.2 The brownish green polymer polysaccharide of Example 3 103.3 120.6 125.8 Example  4 of Perforated  Polymer polysaccharide resolvent 111.1 126.7 145.9

As can be seen in Table 6, when the cell survival rate of the control group to 100% all four samples showed a skin cell activation effect of a concentration-dependent increase in the concentration range of 0.05 to 0.5%. In particular, in the case of Example 3 and Example 4 in which the polymer polysaccharide was isolated, it was found that the 0.5% treatment had more than 25% of skin cell growth promoting effect, and in Example 4, which is a degradation product of the polymer polysaccharide, When treated with 0.5%, the skin cell survival rate was 45% or more, indicating a very good skin cell activation effect.

From the above results, it was confirmed that the brown lard polymer polysaccharide degradation product according to the present invention is an excellent material capable of activating skin cells and skin while exhibiting high anti-aging effect while having high safety.

Experimental Example  7: by ultraviolet irradiation Skin cell Optical damage  Defense effectiveness verification experiment

The effect of the greenish green lysate and extract obtained in Examples 1 to 4 on the skin cell phototoxicity and the cell viability resulting from UV irradiation was evaluated.

Keratinocytes (keratinocytes) were placed in a 24-well test plate and each 1 × 10 ⁵ cells were attached for 24 hours. Each well was washed once with PBS (Phosphate buffered saline) and 500 μl of PBS was added to each well. The keratinocytes were irradiated with UV 10 mJ / 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. After the sample was treated, it was cultured for 24 hours. After 24 hours, the medium was removed, and 500 μl of cell culture medium and MTT {3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide} solution (2.5 mg / ml) per each well 60 After the addition of μl and incubated for 2 hours at 37 ℃ CO ₂ 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, and then 565 nm absorbance was measured in a microplate reader, and cell viability (%) was measured by Equation 2.

Name of sample 0.1% treatment
Contrast to control
Survival rate 0.5% treatment
Contrast to control
Survival rate 1.0% treatment
Contrast to control
Survival rate UV alone treatment group 49.2% Whole green sea green water extract of Example 1 50.1 49.5 54.3 Cracked Green Water Extract Degradant of Example 2 50.6 44.6 32.5 The brownish green polymer polysaccharide of Example 3 49.8 54.7 70.9 Example  4 of Perforated  Polymer polysaccharide resolvent 51.3 66.6 82.7

First, the survival rate of the cells irradiated with ultraviolet rays decreased to 49.2% compared with the cells not irradiated with ultraviolet rays. As can be seen in Table 7, in the case of poached green hot water extract of Example 1, there was no effect of increasing or decreasing the concentration-dependent or significant level of cell viability in the concentration range of 0.1 to 1.0%. In addition, the decayed hot water extract of Example 2 did not show the skin cell photodamage protection effect by ultraviolet rays in the same concentration range, and when treated at 1.0% concentration, the cell survival rate was 32.5%, compared to the control group. Confirmed.

On the other hand, both the green polymer polymer polysaccharide of Example 3 and the green polymer polymer polysaccharide degradation material of Example 4 in the range of 0.1 to 1.0% treatment concentration increased the cell viability compared to the control group and the concentration-dependent increase in the cell survival rate It showed a cell damage defense effect by. In particular, the polymer polysaccharide degradation product of Example 4 was found to have an excellent effect of protecting the skin cells against UV damage by protecting 1.0% of cell viability to 82.7%, thereby preventing the death of skin cells by UV rays. The reddish green algae and the algae polysaccharide degraded product of the present invention is prepared through the process of manufacturing the green algae polysaccharide degrading product is an excellent material showing the anti-aging function that can delay the photoaging of the skin by defending the optical damage caused by ultraviolet rays of the skin cells while high safety Confirmed.

Experimental Example  8: Glucuronic acid  Content Confirmation Experiment

This Experimental Example is for confirming the content of glucuronic acid contained in the poach green digests and extracts obtained in Examples 1 to 4. 1.0 g of each sample was precisely weighed and purified water was added to make 100 ml, and the resultant was treated with an ultrasonic grinder at 30 ° C. for 20 minutes and filtered through a 0.45 μm filter. 10 ml of the filtrate was taken and purified water was diluted to 100 ml to obtain a sample solution. Separately, 100 mg of glucuronic acid standard reagent (C ₆ H ₁₀ O ₇ ; 194.1) was treated in the same manner as the sample solution to prepare a standard solution. 25 μl of the sample solution and the standard solution were each accurately taken and injected into a column packed with a 5 μm pellicle-type anion exchange resin in a Peek material having a diameter of about 4 mm and a length of about 250 mm, and a 30 mM sodium hydroxide solution was used as a mobile phase. Was added to the mobile phase gas and developed at a rate of 1.0 ml / min to detect an electrochemical detector (conductivity detector) to confirm glucuronic acid. The glucuronic acid was tested according to the liquid chromatography method. Peak areas A _T and A _S were measured.

A _T : Glucuronic acid peak area of specimen

A _S : Glucuronic acid peak area of standard product

As a result, as shown in Table 8, the poached green polymer polysaccharide degraded product of Example 4 prepared through the method for preparing browned seaweed and seaweed digested products of the present invention showed a glucuronic acid content of 15.1%, and all other samples of the detector were It showed a content below the detection limit, or a glucuronic acid content of less than 1.0%.

Name of sample Content of Glucuronic Acid (%) Whole green sea green water extract of Example 1 Not detected Cracked Green Water Extract Degradant of Example 2 0.12% The brownish green polymer polysaccharide of Example 3 Not detected Example  4 of Perforated  Polymer polysaccharide resolvent 15.1%

Experimental Example  9: Examination for skin elasticity improvement and wrinkle improvement

The cosmetics containing the brownish green polymer polysaccharide decomposition product obtained in Example 4 were prepared, and the skin elasticity improving effect and the wrinkle improving effect were evaluated by performing a comparative experiment with Comparative Example 1 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 was 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 5, Comparative Example 1). 15 experimenters (women aged 20-35 years) were applied to the right side of the face with the cream prepared in Example 13 and the left side of the face with the cream prepared in Comparative Example 1 twice a day for 2 consecutive months. . The skin elasticity was measured by using a skin elasticity meter (SEM 575, C + K Electronic Co., Germany) before and after using the product for 2 months. The experimental results are shown in Table 9 below as the ΔR7 value of Cutometer SEM 575, where the R7 value indicates viscoelasticity of the skin.

As shown in Table 10, it can be seen that the cream containing the brownish green polymer polysaccharide decomposition product of Example 4 has an excellent skin elasticity improving effect.

division Example 5 Comparative Example 1 end Stearyl alcohol 7.5 7.5 Stearic acid 2 2 Stearic acid cholesterol 2.5 2.5 Squalane 5 5 2-octyldodecyl alcohol 5 5 Polyoxyethylene (25 mole addition) alcohol ester 3 3 Glyceryl monostearate Aster 2 2 I The brownish green polymer polysaccharide decomposition product of Example 4 0.5 - Propylene glycol 5 5 antiseptic Suitable amount Suitable amount Purified water Balance Balance

Note) Unit: wt%

Experimental product Skin elasticity effect (△ R7) Example  5 ( Example  4 Perforated  Polymer polysaccharide resolvent  contain) 0.36 Comparative Example 1 0.14

n = 20, p < 0.05

The cream prepared in Example 5 was applied to the right side of the face and the cream prepared in Comparative Example 1 was applied twice a day for 2 consecutive months to 15 test subjects (women aged 20 to 35 years). To evaluate the wrinkle improvement effect of the skin before and after the use of the product after the completion of the experiment, a silicone replica was made and the wrinkle state of the designated area was measured with a visiometer SV60, C + K Electronic Co., Germany). The results are shown in Table 8, which shows the average of two parameter values after two months minus the parameter values two months ago. In other words, the negative the value, the higher the wrinkle improvement effect. As shown in Table 10, it can be seen that the cream of Example 5 containing the greening polymer polysaccharide decomposition product of Example 4 significantly improved skin wrinkles.

Experimental product R1 R2 R3 Example  5 -0.30 -0.24 -0.12 Comparative Example 1 -0.11 -0.12 -0.04

R1: Difference between the maximum value and the minimum value of the wrinkle contour line

R2: The wrinkle contour line is arbitrarily divided into 5 squares, and the average of R1 values

R3: Maximum value of R1 divided by 5

Other examples are shown below. That is, the lotion, emulsion and cosmetic liquid containing the greenish green polymer polysaccharide decomposition product obtained in Example 4 were prepared in Examples 6 to 8. The lotion, milky lotion and essence containing these perforated macromolecular polylysates showed excellent effects on skin improvement including active oxygen scavenging effect, skin elasticity improvement effect and wrinkle improvement effect.

Example  6: Perforated The degradation product  Containing lotion

8 g of 95% ethanol, 0.05 g of polypyrrolidone, 0.1 g of oleyl alcohol, 0.2 g of polyoxyethylene monooleate, 0.2 g of fragrance, 0.1 g of paraoxybenzoic acid methyl ester, a small amount of antioxidant, and a small amount of pigment It was. The mixed solution was added to a solution of 0.05 g of the brownish green polymer polysaccharide decomposition product obtained in Example 4 and 5 g of glycerin in purified water of 85.33 g, followed by stirring to obtain a skin lotion having a skin improvement effect.

Example  7: Perforated The degradation product  Manufacture of emulsion containing

1.2 g of cetyl alcohol, 10 g of squalane, 2 g of vaseline, 0.2 g of paraoxybenzoic acid ethyl ester, 1 g of glycerin monoesteralide, 1 g of polyoxyethylene (20 mole addition) monooleate and 0.1 g of fragrance were heated, mixed, and It melt | dissolved, and 0.5g of the brownish green polymer polysaccharide degradation products obtained in Example 4, 5g of dipropylene glycol, 2g of polyethyleneglycol-1500, 0.2g of triethanolamines, and 76.2g of purified water were melt | dissolved by heating at 75 degreeC. The two were mixed and emulsified and then cooled to obtain an oil-in-water (O / W) type milky lotion.

Example  8: Perforated The degradation product  Containing Serum  Produce

To 5 g of 95% ethyl alcohol, 1.2 g of polyoxyethylene sorbitan monooleate, 0.3 g of chitulose, 0.2 g of sodium hyaluronate, 0.2 g of vitamin E-acetate, 0.2 g of sodium licorice, 0.1 g of paraoxybenzoic acid ethyl ester 0.5g of the brownish green polymer polysaccharide decomposition product obtained in Example 1 and an appropriate amount of pigment were mixed to obtain a cosmetic solution having an effect of improving skin.

Claims (13)

Step 1: extracting the dried crushed brown seaweed and algae into hot water to prepare a water-soluble extract,
Step 2: adding ethyl alcohol to the prepared brown seaweed and algae aqueous extract to settle the polymer polysaccharide and protein, and then recovering the precipitate by filtration and centrifugation to selectively recover the brown seaweed and algae-containing polymer polysaccharide,
Step 3: dispersing the prepared polysaccharide and seaweed-containing polymer polysaccharide in a volatile acid solution to decompose the acid, and then filtering to prepare a water-soluble polysaccharide decomposition product, and
Step 4: A method for preparing brown seaweed and seaweed degradation products containing 1.0% or more of glucuronic acid, including removing acid and solvent from the prepared brown seaweed and seaweed water-soluble polysaccharide degradation products.
The method according to claim 1,
In the second step, the ethyl alcohol is a method for producing brown seaweed and algae decomposition products containing 1.0% or more of glucuronic acid, characterized in that the range of 50% to 500% by weight relative to the weight of the water-soluble extract.
The method according to claim 1,
In step 3, the volatile acid is hydrocholoric acid, acetic acid, formic acid, butyric acid, propionic acid, propionic acid, trifluoroacetic acid (TFA), pentafluoro Pentafluoropropionic acid (PFPA), heptafluorofluorobutyric acid (HFBA), nonafluoro pentanoic acid (NFPA), tridecafluoroheptanoic acid (TDFHA) and pentadecafluoroocta Method for producing brown seaweed and algae decomposition products containing 1.0% or more of glucuronic acid, characterized in that at least one selected from the group consisting of non-acid (pentadecafluorooctaonoic acid, PDFOA).
The method according to claim 1,
The volatile acid solution of step 3 is a method for producing brown seaweed and seaweed degradation products containing 1.0% or more of glucuronic acid, characterized in that the concentration is 1 to 6N.
The method according to claim 1,
In the step 3, the acid decomposition temperature is 80 to 125 ℃ glucuronic acid, characterized in that the method for producing brown seaweed and algae decomposition products containing 1.0% or more.
The method according to claim 1,
In the step 4, the acid and solvent removal step is carried out to remove the volatile acid and impurities so that the resulting dried solids dissolved in purified water at a concentration of 5% so as to remove the volatile acid and impurities, so that the green onions containing more than 1.0% Method for preparing seaweed digests.
Brown seaweed and algae degradation products containing 1.0% or more of glucuronic acid manufactured by the method of any one of Claims 1-6.
A cosmetic composition comprising the green seaweed and seaweed degradation products as an active ingredient, comprising 0.001 to 30.0% by weight of brown seaweed and seaweed degradation products containing 1.0% or more of glucuronic acid, based on the total composition.
The method according to claim 8,
The cosmetic composition is a cosmetic composition containing the blue seaweed and seaweed decomposition products as an active ingredient, characterized in that it exhibits photoaging prevention effect.
The method according to claim 8,
The cosmetic composition is a cosmetic composition containing the blue seaweed and seaweed decomposition products as an active ingredient, which has an effect of improving the skin wrinkles.
The method according to claim 8,
The cosmetic composition is a cosmetic composition containing the blue seaweed and seaweed decomposition products as an active ingredient, characterized in that it has a function to improve skin elasticity.
The method according to claim 8,
The cosmetic composition is a cosmetic composition containing the blue seaweed and seaweed decomposition products as an active ingredient, characterized in that it has an anti-aging function of the skin.
The method according to claim 8,
The cosmetic composition is a cosmetic composition containing the blue seaweed and algae decomposition products as an active ingredient, characterized in that the protective effect against the active oxygen.