KR20160054727A - Ecklonia stolonifera hydrolysates that have high glucuronic acid cotent, preparation method thereof and antiaging cosmetic composition containing the same - Google Patents

Abstract

The present invention relates to a preparation method for Ecklonia stolonifera hydrolysates containing 1% or more of glucuronic acid, which can be used as a main ingredient or a marker substance for anti-aging cosmetic products, and a composition for external application to skin containing the same as an active ingredient. More specifically, the present invention relates to a preparation method for Ecklonia stolonifera hydrolysates containing 1% or more of glucuronic acid, comprising: a process for isolating polysaccharides from Ecklonia stolonifera and a process for decomposing the isolated polysaccharides using fermentation of Laetiporus sulphureus var. miniatus (Jungh.) Imaz., and a functional composition for external application to skin, which comprises 0.001-90.0 wt% of Ecklonia stolonifera hydrolysates containing 1% or more of glucuronic acid as an active ingredient and thereby serves to suppress photoaging due to UV-rays, has antioxidant activity, promotes the generation of collagen, suppresses biosynthesis of MMP-1, , alleviates fine lines, improves moisturization, and alleviates irritation of the skin.

Description

[0001] The present invention relates to a method for producing a decomposition product of an insecticide containing a large amount of glucuronic acid and an anti-aging functional cosmetic composition containing the same,

The present invention relates to a method for separating a polysaccharide from a worm, decomposing the polysaccharide through reddish brown fermentation to produce a degraded product containing a low molecular weight glucuronic acid, and a method for producing a degraded product containing a large amount of glucuronic acid as an active ingredient To a composition for external application for skin.

Skin aging can be divided into two types: intrinsic (chronological aging) and phtoaging (Gilchrest BA: J. Am. Acad. Dermatol ., 21, 610-613 (1989)]. Endogenous aging is a naturally occurring aging phenomenon that is accompanied by decreased physiological function of the body as age increases [Braverman IM et al . : J. Invest. Dermatol ., 78, 434-443 (1982)]. Photoaging means that the skin is repeatedly exposed to light to change the appearance or function of the skin [Ridder GM et al . : J. Am. Acad. Dermatol ., 25, 751-760 (1991)]. In addition, skin aging can be caused by active oxygen species activated by ultraviolet rays, stress, disease states, environmental factors, wounds, and aging. When such conditions are deepened, the antioxidant defense network existing in the living body is destroyed, It damages tissues and promotes adult diseases and aging. More specifically, lipids, proteins, polysaccharides and nucleic acids, which are major constituents of the skin, are oxidized to destroy skin cells and tissues, resulting in skin aging. In particular, the oxidation of proteins causes severe hyperinflammation of collagen, hyaluronic acid, elastin, proteoglycans, and fibronectin, which are connective tissues of the skin, which interferes with skin elasticity. Mutation, cancer induction, and immune function.

Therefore, it is necessary to protect the cell membranes by abolishing reactive oxygen species that are mediated by the body's metabolic processes, ultraviolet irradiation, and inflammatory reactions, and regenerate already damaged cells by active metabolism to proliferate the cells Can be restored and healthy skin can be maintained. In aging, not only active oxygen species but also MMP (Matrix metallopretease) is involved. In vivo, the synthesis and degradation of extracellular matrix such as collagen are properly regulated, but the synthesis is reduced as aging progresses and the collagen- Expression of matrix metalloproteinases (MMPs) is promoted, and skin elasticity is lowered and wrinkles are formed. These degrading enzymes are also activated by exposure to ultraviolet light. Therefore, there is a demand for development of a substance capable of controlling the activation of MMP induced in the cell by ultraviolet light or inhibiting its activity. Until now, the raw materials used as cosmetic materials mostly inhibited only the activity of degrading enzyme (MMP).

Recently, much attention has been focused on functional cosmetics having functions such as antioxidation, wrinkle reduction, whitening, and itching relief obtained from natural extracts in order to reduce skin irritation caused by various chemical substances and the like. For example, U.S. Patent No. 5,972,341 discloses a wrinkle-reducing effect of an extract of Commiphora mukul. Japanese Patent Application Laid-Open No. 9-672662 discloses an extract of seaweed having a hyaluronidase inhibitory effect, Japanese Patent Application Laid-Open No. Hei 2-245087 discloses an antioxidative effect of Sargassum sp. Extract on the skin texture improvement effect of Fucoidan extracted from Wakame, Patent No. 0431076 discloses a cosmetic composition for improving the healing of atopic skin including a white, black, echinacea, and fermented soybean extract, Korean Patent No. 0511494 discloses an extract and composition for treating atopic dermatitis including at least one of Hashuo, .

Sargassum thunbergii (Sargassum thunbergii) belongs to the family Phaeophyta (Sargassaceae). It is a common seaweed in the coastal area of Korea. It lives in the lower part of the intertidal zone. Young plants are used as feeds but have not received much attention. 2004), and the study of physiological activities such as antioxidant (Choi et al., 2006), anti-inflammatory (Kang et al., 2008) and antibacterial (Lee et al., 2009) et al., 2007). Physiologically active and norisoprenoid compounds such as (+) - epiloliolide, (-) - loliolide and apo-9'-fucoxanthinone have been reported.

To cope with damage to tissues and cells due to ultraviolet rays and harsh environment, it produces a variety of antioxidants. In addition, it creates various protective mechanisms such as polysaccharide to create a moisturizing component. That is, it is expected that functional alcohols having high added value can be developed by utilizing the self-defense component of seaweeds.

Korean Patent No. 1170166 discloses a composition comprising an extract of seaweed selected from the group consisting of a kidney bean paste, a visibly skeleton net, a red squid, a wrinkled red leaf, a true gambling, a worm, a wrinkled hair, An external preparation for skin for promoting collagen production is described.

It is an object of the present invention to provide a composition for external application for skin having natural skin effect inhibiting photoaging damage, antioxidation, promoting collagen synthesis, inhibiting MMP-1 biosynthesis, improving skin wrinkles and moisturizing effect.

Another object of the present invention is to provide a functional cosmetic composition which exhibits an anti-aging effect by using a fermented polysaccharide decomposition product of witches containing 1.0% or more of glucuronic acid as a raw material.

In order to accomplish the above object, the present invention provides a composition for external application for skin, which comprises preparing and dissolving a degradation product having a high content of glucuronic acid from an insect.

In order to provide a composition for external application for skin excellent in inhibition of photoaging damage, antioxidation, promotion of collagen synthesis, inhibition of MMP-1 biosynthesis inhibition, improvement of skin wrinkles and moisturizing effect, the composition for external use of the present invention contains decomposition products of glucuronic acid 1% As an active ingredient.

Hereinafter, the present invention will be described in detail for each step.

Step (a): Step of extracting ultra-high pressure to obtain extract

In this step, the pH and salt concentration are adjusted by adding a basic aqueous solution, an acidic aqueous solution, a neutral salt such as sodium chloride as an extraction solvent, and the resultant is subjected to ultrahigh pressure extraction and filtration for 12 to 24 hours at 500 to 1500 MPa, .

On the other hand, in the present invention, the insect as a material for extraction may be used as it is, but it may be dried naturally or by hot air drying and then pulverized. This is because the advantage of being able to facilitate the extraction is that it is used by pulverizing it.

On the other hand, the ultra high pressure extract is a non-heat treatment method, which can overcome the maintenance of the activity of the physiologically active substance of the extract by the conventional heat treatment, and the texture and flavor of the food. This technique is based on Pascal's principle of uniformly delivering a pressure of 100 to 1000 MPa instantaneously using water or oil as a pressure medium. As one of the latest extraction techniques, heat treatment and pressure treatment are both methods of extracting desired physiologically active substances in the development of cosmetic materials. However, the heat treatment is advantageous in that the chemical treatment does not cause a significant chemical change, whereas the chemical treatment causes a severe chemical change.

Step (b): selectively collecting the liposome-containing polymer polysaccharide

The prepared water soluble insect was added to the extract solution with ethyl alcohol in an amount ranging from 50% by weight to 500% by weight based on the weight of the extract to precipitate the polymer polysaccharide and protein, and the precipitate was recovered by filtration and centrifugation to obtain a lipopolysaccharide- This is a step of collecting selectively.

Step (c): the step of producing the fermented polysaccharide degradation product of the worm through redderived fermentation

The resulting polysaccharide-containing polymer is fermented, concentrated and sterilized with the mycelial (KFCC 11494P) of Laetiporus sulphureus CS0218 to produce a fermented polysaccharide degradation product. According to the following experimental example, it was confirmed that the glucuronic acid content of the fermented polysaccharide degraded by the fermented fermented by mycelium (KFCC 11494P) of Laetiporus sulphureus CS0218 was higher than that of the polysaccharide containing the insecticide of step (b).

The hydrolyzate prepared in the next step can be used as it is. In some cases, the hydrolyzate having high glucuronic acid content can be fractionated into hydrolyzate by Utrafiltration apparatus and gel chromatogram, and it can be used for various purposes.

The hydrolyzate prepared in the next step can be concentrated by vacuum concentration, dried using the freeze drying method, and pulverized to an appropriate size, so that the decomposed granules and fine granules can be obtained.

On the other hand, the fermentation polysaccharide degradation product of the present invention is obtained by extracting the insect worm, and the extraction method can be a commonly used method in the technical field to which the present invention belongs. For example, it may be a hot-water extract of the powder obtained by freeze-drying and pulverizing the insect, or an organic solvent extract obtained by using methanol, ethanol, butanol or a mixed solvent thereof.

On the other hand, the fermented polysaccharide decomposition product of the worm can be used as it is, or dried (or lyophilized) and used in the form of powder. Preferably, the culture solution may be used by filtration. Specifically, the mycelial body is removed by centrifugation after liquid cultivation of a reddish brown mycelium, and the filtrate obtained by filtering the culture filtrate with a paper filter can be used. At this time, the culture means 'fermentation'.

The fermented polysaccharide degraded product of the present invention thus obtained exhibits antioxidant activity, inhibition of matrix metalloproteinase (MMP-1) activity, inhibition of substrate metalloproteinase (MMP-1) expression, promotion of procollagen biosynthesis, An effect of accelerating the production of elastin, an effect of alleviating cytotoxicity due to ultraviolet irradiation, and a water absorbing effect, and thus can be used in a multifunctional skin topical composition.

On the other hand, the composition for external application for skin of the present invention preferably contains 0.001 to 90.0% by weight of the fermented polysaccharide degradation product in the composition as a whole. When the content is less than 0.001% by weight, the skin improving effect is insignificant, If it exceeds, it is not economical because efficiency is less than material input amount.

In the present invention, the term "included as an active ingredient" means that a fermented polysaccharide degradation product is added to the composition for external application for skin to such an extent that the effect of preventing or suppressing skin diseases can be exhibited from the composition for external application for skin of the present invention , And includes various components in a variety of formulations for the purpose of drug delivery and stabilization, and the like.

Examples of the cosmetic composition include cosmetics, gels, water-soluble liquids, creams, essences, oil-in-water (O / W) O) type; A color cosmetic formulation consisting of an underwater type or a water-in-oil type makeup base, a foundation, a skin cover, a lipstick, a lip gloss, a face powder, a two way cake, an eye shadow, a cheek color and an eyebrow pencil; .

On the other hand, the cosmetic composition is most preferably used for anti-aging, skin irritation, skin moisturizing, and wrinkle improvement.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to obtain a fermented polysaccharide degradation product in a high yield in a combination of antioxidation, promotion of collagen synthesis, prevention of aging, improvement of skin wrinkles and moisturizing effect, have.

Brief Description of the Drawings Fig. 1 is a diagram showing a method for producing a fermented polysaccharide degradation product of a worm.
Fig. 2 is a graph showing the results of analysis of glucuronic acid changes in the polymer polysaccharide degradation products by using an HPLC / GPC analyzer.
Fig. 3 is a graph showing the results of analysis of glucuronic acid changes in the fermented polysaccharide degraded product of worms using an HPLC / GPC analyzer.

Hereinafter, the present invention will be described in more detail in the following Examples. However, the scope of the present invention is not limited to the following embodiments, and includes modifications of equivalent technical ideas.

Hereinafter, the present invention will be described in more detail in the following Examples. However, the scope of the present invention is not limited to the following embodiments, and includes modifications of equivalent technical ideas.

Example 1: Preparation of Hot Water Extract from Lemurs

The wandering worms obtained from the Institute of Marine Science and Technology were cut and shredded and dispersed in purified water at 5% by weight, and then subjected to ultrahigh pressure extraction at 1,000 MPa for 12 hours and filtered through Whatman # 5 filter paper. The filtered extract was concentrated under reduced pressure and freeze-dried at 50 or lower to prepare a hot-water extract powder.

Example 2: Preparation of a lipopolysaccharide

10 g of the hot water extract obtained in Example 1 was dispersed in 1 liter of a 2N solution of trifluoroacetic acid (TFA), followed by acid decomposition for 2 hours while maintaining the reaction temperature at 100 in a closed container. The filtrate was concentrated under reduced pressure at 50 or lower, dissolved again in purified water, and concentrated under reduced pressure. The volatile acid and solvent were removed three times. The prepared acid degradation product was dissolved in purified water at a concentration of 5%, and the same amount of ethyl alcohol as that of the solution was added and stirred. Then, the polymer polysaccharide and protein were precipitated in a low-temperature storage tank at 4, The precipitate was recovered by filtration and centrifugation, and the polymeric polysaccharide containing the ultrahigh pressure extract was selectively recovered. The recovered polysaccharide was redissolved in purified water and concentrated under reduced pressure at a concentration of 50 or less and lyophilized to prepare a powdery polymer polysaccharide.

Example 3: Preparation of a fermented polysaccharide degradation product

Seed culture Laetiporus sulphureus CS0218 mycelium (KFCC 11494P) was added to 250 ml flask culture conditions in working vol. (PDA, potato dextrose agar) containing 3% of glucose, 0.6% of yeast extract, 0.2% of polypeptone, 0.05% of MgSO47H2O and 0.05% of K2HPO4 on a 50 ml basis After incubation at 25 to 150 rpm for 7 days, the seed culture was obtained by subculturing at 2-month intervals with 4 refrigerated storage.

Thereafter, 10 g of the polysaccharide of the insecticide obtained in Example 1 was dispersed in 1 liter of purified water, and 0.3% of yeast extract and 0.1% of polypeptone were added. The seed culture was inoculated 10% to the sterilized medium and then cultured at 25 to 150 rpm for 8 days. The cultured product was concentrated and sterilized to prepare a fermented polysaccharide decomposition product.

Experimental Example 1: Measurement of antioxidative effect using NBT

In this Experimental Example, the antioxidative effect of the hydrolyzate obtained in Example 1 above, the polysaccharide obtained from Example 2 above, and the hydrolyzate of the fermented polysaccharide degraded product obtained in Example 3 were measured using the NBT method .

The NBT method is a method in which active oxygen is produced by xanthine and xanthine oxidase, and blue produced by reacting the produced active oxygen with Nitro Blue Tetrazolium (NBT) is measured at a wavelength of 560 nm. In this Experimental Example, the active oxygen scavenging rate was measured in the following manner. BHT, well known as an antioxidant, was used as a positive control.

0.05 M sodium carbonate (Na ₂ CO ₃ ) 2.4, 3 mM xanthine solution 0.1, 3 mM EDTA solution 0.1, BSA solution 0.1 and 0.72 mM NBT solution 0.1 were added to Bayer disease solution, 0.2% by weight was added, and the mixture was allowed to stand at 25 for 10 minutes. After the incubation, 0.1 ml of xanthine oxidase solution was added and the mixture was stirred rapidly. Then, the mixture was incubated at 25 ° C for 20 minutes. After the addition of 0.1 ml of 6 mM copper chloride (CuCl ₂ ) solution, the absorbance at 560 nm (St) Were measured. The blank test was performed using distilled water instead of the sample solution, and the absorbance (Bt) was measured in the same manner as described above. The blank of the sample solution was measured for absorbance (Bo) in the same manner as above using distilled water instead of xanthine oxidase solution. In addition, the active oxygen scavenging ratio (%) was calculated by the following equation (1).

[Equation 1]

(%) = [1- (St-So) / (Bt-Bo)] 100

St: Absorbance at 560 after enzyme reaction of sample solution

Bt: Absorbance at 560 after enzyme reaction of blank test solution

So: absorbance at 560 before enzymatic reaction of enzyme solution-free sample solution

Bo: Absorbance at 560 before enzymatic reaction of enzyme-free blank test solution

Name of sample Active oxygen scavenging rate (antioxidant effect;%) When the larvae contained 0.2 wt% 68 0.1% < RTI ID = 0.0 > 53 When the larvae contained 0.05 wt% 39 BHT 0.1 wt% 89

Name of sample Active oxygen scavenging rate (antioxidant effect;%) The insecticide was 0.2% by weight of the polymer polysaccharide, 77 0.1% by weight of the polymeric polysaccharide 62 When the worm is 0.05% by weight of the polymer polysaccharide, 51 BHT 0.1 wt% 89

Name of sample Active oxygen scavenging rate (antioxidant effect;%) 0.2% by weight of the fermented polysaccharide degradation product 91  ≪ RTI ID = 0.0 > 0.1% < / RTI & 86 0.0 >% < / RTI > by weight fermented polysaccharide degradation product 76 BHT 0.1 wt% 89

The antioxidative effect was measured using NBT method (Table 1, Table 2, Table 3). The hydrothermal extracts of the insects, the polysaccharides of the insecticides, and the degraded polysaccharides of the insecticides showed excellent antioxidative ability.

In particular, the fermented polysaccharide degraded product of Example 3 showed 86% of active oxygen content at a concentration of 0.1%, showing antioxidative activity similar to that of the positive control, 0.1% BHT.

From the above results, it was confirmed that the worms had excellent free radical scavenging ability of the fermentation polysaccharide degradation product, and that the composition for external application for skin of the present invention exhibited excellent antioxidative ability, thereby effectively preventing skin aging.

Experimental Example 2: Measurement of antioxidative effect by DPPH method

In this Experimental Example, the antioxidative effect of the hydrolyzate obtained in Example 1 above, the polysaccharide obtained from Example 2, and the hydrolyzate of the fermented polysaccharide degraded product obtained in Example 3 were measured using the DPPH method .

The DPPH method is a method of measuring free radical scavenging activity by reducing power using a free radical called DPPH (2,2-Di (4-tert-octylphenyl) -1-picrylhydrazyl free radical). In this Experimental Example, the degree of reduction of the absorbance by DPPH reduction by the test substance was compared with the absorbance of the blank test solution, and the free radical scavenging rate was measured at a wavelength of 560 nm. BHT, well known as an antioxidant, was used as a positive control.

Polymeric polysaccharide at a concentration of 0.05 to 0.2% by weight was introduced into the 96-well plate, and the fermented polysaccharide degraded product was introduced into the 96-well plate. To this was added DPPH prepared in 100 uM methanol solution so that the total volume of the solution became 200. After standing at 37 for 30 minutes, the absorbance (St) was measured at 560 nm and the free radical scavenging activity (%) was calculated using the following equation (2).

&Quot; (2) "

Free radical scavenging activity (%) = 100- (B / A) 100

A: Absorbance of the control wells in which the worms were not treated with the polymer polysaccharide or the wormy fermentation polysaccharide degradation product

B: Absorbance of the experimental group well treated with the macromolecule polysaccharide or insecticidal fermented polysaccharide digest

Name of sample Free radical scavenging ratio (antioxidant effect;%) When the larvae contained 0.2 wt% 65 0.1% < RTI ID = 0.0 > 52 When the larvae contained 0.05 wt% 43 BHT 0.1 wt% 85

Name of sample Free radical scavenging ratio (antioxidant effect;%) The insecticide was 0.2% by weight of the polymer polysaccharide, 79 0.1% by weight of the polymeric polysaccharide 52 When the worm is 0.05% by weight of the polymer polysaccharide, 47 BHT 0.1 wt% 85

Name of sample Free radical scavenging ratio (antioxidant effect;%) 0.2% by weight of the fermented polysaccharide degradation product 92 ≪ RTI ID = 0.0 > 0.1% < / RTI & 83 0.0 >% < / RTI > by weight fermented polysaccharide degradation product 64 BHT 0.1 wt% 85

The antioxidant effect was measured by the DPPH method (Table 4, Table 5, Table 6), and the hydrothermal extract, insecticidal polysaccharide, and fermented polysaccharide digest of the insecticide showed excellent antioxidant ability.

In particular, the fermented polysaccharide degradation product of Example 3 showed 83% free radical scavenging ability at a concentration of 0.1%, showing antioxidative activity similar to that of the positive control, 0.1% BHT.

From the above results, it was confirmed that the worms had excellent free radical scavenging ability of the fermentation polysaccharide degradation product, and that the composition for external application for skin of the present invention exhibited excellent antioxidative ability, thereby effectively preventing skin aging.

Experimental Example 3 Measurement of Inhibitory Activity of Metal Protease (MMP-1) in vitro)

In this Experimental Example, the fluorescence analysis was carried out to determine whether the insect wort obtained in Example 1 was a hot-water extract, the insect wort obtained in Example 2 was a high-molecular polysaccharide, the insect wort obtained in Example 3 was in vitro (MMP-1) activity in vitro was investigated.

DQ-Collagen labeled with a fluorescent substance was used as a substrate for the experiment, and commercially available collagenase was used as a enzyme in a molecular probe (Molecular probe, Eugene, OR, USA) Reaction buffer 0.5 M Tris-HCl, 1.5 M NaCl, 50 mM CaCl ₂ , 2 mM sodium azide, pH 7.6 was used after 10-fold dilution. DQ collagen 20 dissolved in a reaction buffer at a concentration of 0.25 / L in the reaction buffer solution 100, a polyglyceride of liposomes and 40 of fermented polysaccharide degradation products (0.02, 0.01, 0.005 wt%) were added to each other, The collagenase 40 was added. After 20 minutes at room temperature, the cow was measured for fluorescence with an absorption wavelength of 495 nm and an emission wavelength of 515 nm using a fluorescence spectrophotometer (Perkin Elmer, UK). Green tea extract was used as a positive control. In the control group, fluorescence value was measured after addition of enzyme buffer in the same amount as the enzyme buffer, and the fluorescence value of the sample itself was also measured to calculate the enzyme activity.

Name of sample MMP-1 activity inhibition rate (%) Control group 0 0.02% < / RTI > by weight of hot- 37  When the insect is 0.01 wt% 24  0.005% by weight of hot water extract, 8 Green tea extract 0.2 wt% 68

Name of sample MMP-1 activity inhibition rate (%) Control group 0 0.02% by weight of a polymeric polysaccharide 53  When the worm is a polymeric polysaccharide containing 0.01% 34  When the insect is a polymer polysaccharide of 0.005 wt% 19 Green tea extract 0.2 wt% 68

Name of sample MMP-1 activity inhibition rate (%) Control group 0 0.02% < tb > < tb > 74  0.01% < RTI ID = 0.0 > wt% < 56 0.005 wt% < tb > < tb > 41 Green tea extract 0.2 wt% 68

Inhibition rate of metalloproteinase (MMP-1) activity in vitro was measured (Table 7, Table 8, Table 9), and the result of hydrothermal extraction of worms, macromolecule polysaccharide, Protein-degrading enzyme (MMP-1) in a concentration-dependent manner. In particular, the fermented polysaccharide degradation product of Example 3 exhibited an inhibition rate of 74% at a concentration of 0.02%, which effectively inhibited the activity of metalloproteinase (MMP-1) at a lower concentration than that of the green tea extract as a positive control there was.

From the above results, it was confirmed that the insecticidal effect of the fermented polysaccharide degradation product was excellent in the inhibition of metalloproteinase (MMP-1) activity, and based on this, the skin external composition composition of the present invention effectively inhibited the metalloproteinase, It is possible to prevent degradation and wrinkle formation.

Experimental Example 4: Measuring effect of promoting type 1 procollagen biosynthesis

In this Experimental Example, it was confirmed by the procollagen assay that the insect wort obtained in Example 1 was a hot-water extract, the insect wort obtained in Example 2 was a polymer polysaccharide, and the insect wort obtained in Example 3 was a fermented polysaccharide decomposition product And to promote the biosynthesis of type 1 procollagen, a skin substrate component.

Human dermal fibroblasts isolated from the epidermal tissue of newborns were purchased from Modern Tissue Technology (MTT, Korea) and supplemented with 10% fetal bovine serum (FBS) in DMEM / F-12 (3: And cultured at a concentration of 1 ⁴ cells / cm ² . When 80% of the cells were grown, the cells were subcultured at a ratio of 1: 3, and the fourth subcultured cells 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, and then the insecticidal polysaccharide and the insecticide were added in concentrations of 0.005% to 0.02% , The amount of procollagen liberated in the medium was measured using a procollagen type-1 C-peptide EIA kit (MK101, Takara, Japan). In addition, TGF- was used as a positive control.

Name of sample Promoting effect of procollagen biosynthesis (%) 0.02% < / RTI > by weight of hot- 114 When the insect is 0.01 wt% 108 0.005% by weight of hot water extract, 102 TGF-0.001 wt% 125

Name of sample Promoting effect of procollagen biosynthesis (%) 0.02% by weight of a polymeric polysaccharide 124 When the worm is a polymeric polysaccharide containing 0.01% 112 When the insect is a polymer polysaccharide of 0.005 wt% 105 TGF-0.001 wt% 125

Name of sample Promoting effect of procollagen biosynthesis (%) 0.02% < tb > < tb > 148 0.01% < RTI ID = 0.0 > wt% < 137 0.005 wt% < tb > < tb > 129 TGF-0.001 wt% 125

As a result of measuring the promoting effect of procollagen biosynthesis (Table 10, Table 11, Table 12), the hydrothermal extract of the insecticide, the polysaccharide of the insecticide, and the fermented polysaccharide decomposition of the insecticide showed the promoting effect of procollagen biosynthesis. In particular, the fermented polysaccharide degradation product of worms showed a similar effect to the cell signaling substance TGF- at a concentration of 0.005% by promoting the production of 129% of the prolactin biosynthesis.

From the above results, it was confirmed that the insecticidal polysaccharide degradation product promoted the excellent procoagulant biosynthesis promoting effect, and it was confirmed that the skin external application composition of the present invention has an excellent skin aging improving effect.

Experimental Example 5: Measurement of mitigation effect of cytotoxicity by ultraviolet irradiation

In this Experimental Example, the effects of the hot water extract obtained in Example 1, the macromolecular polysaccharide obtained in Example 2, and the mitochondrial cytotoxicity by UV irradiation of the fermented polysaccharide degraded product obtained in Example 3 Respectively.

Fibroblasts were placed in 24-well test plates at 1 × 10 ⁵ cells and adhered for 24 hours. Each well was washed once with PBS and 500 wells of PBS were added to each well. After irradiating the fibroblasts with ultraviolet light (10 mJ / uv) using a UVB lamp (Model: F15T8, UVB 15W, Sankyo Dennki, Japan), the PBS was removed and the cell culture medium (DMEM supplemented with 10% FBS 1) was added. Herein, the polymeric polysaccharide was treated with 0.005 to 0.02% by weight of the polysaccharide, and the fermented polysaccharide degraded by the insect was cultured for 24 hours. After 24 hours, the medium was removed, cell culture medium 500 and MTT solution (2.5 /) 60 were added per well, and the cells were cultured in a 37 CO ₂ incubator for 2 hours. After incubation, the medium was removed and 500 isopropanol-HCl (0.04 N) was added. The cells were lysed by shaking for 5 minutes and 100 supernatants were transferred to a 96-well test plate and absorbance was measured at 565 nm using a microplate reader. The cell viability (%) was calculated by the following equation (3), and the cytotoxic relaxation rate by ultraviolet was calculated by the following equation (4).

&Quot; (3) "

Cell survival rate (%) = [(St-Bo) / (Bt-Bo)] 100

Bo: Absorbance at 565 nm of wells reacting with cell culture medium

Bt: Absorbance at 565 nm of a well that underwent chromogenic reaction in a sample not treated with the sample

St: absorbance at 565 nm of wells treated with the sample

&Quot; (4) "

(%) = [1- (St-Bo) / (Bt-Bo)] 100

Bo: Cell survival rate of wells not irradiated with ultraviolet light and not treated with sample

Bt: Cell viability of wells irradiated with ultraviolet light and not treated with the sample

St: Cell viability of well treated with ultraviolet light and treated with sample

Name of sample Treatment concentration (% by weight) Cytotoxic Relaxation Rate (%) Hot water extract 0.02 29 0.01 21 0.005 13

Name of sample Treatment concentration (% by weight) Cytotoxic Relaxation Rate (%) Polysaccharide 0.02 44 0.01 29 0.005 18

Name of sample Treatment concentration (% by weight) Cytotoxic Relaxation Rate (%) Lycopene is a fermentation polysaccharide degradation product 0.02 52 0.01 34 0.005 16

(Table 13, Table 14, Table 15). The hydrolyzate of the insecticide, the polysaccharide of the insecticide, and the polysaccharide of the insecticidal fermented polysaccharide excellently mitigated the cytotoxicity caused by ultraviolet rays, Which is an effective inhibitor of cytotoxicity. In particular, the fermented polysaccharide degraded product of Example 3 showed a cytotoxic relaxation rate of 52% by ultraviolet light at a concentration of 0.02%.

From the above results, it was confirmed that the insecticidal polysaccharide degradation product was excellent in mitigating cytotoxicity by ultraviolet irradiation. Based on this, it was confirmed that the skin external stimulant composition of the present invention has excellent skin irritation mitigation effect by ultraviolet rays.

Experimental Example 6: Measurement of water hygroscopicity

In this Experimental Example, the water hygroscopicity of the fermented polysaccharide degraded product obtained in Example 1, the hydrolyzate obtained in Example 2, and the fermented polysaccharide degraded product obtained in Example 3 were measured.

In the dry epidermal cells of the humans, the polysaccharides and lipids were saturated and absorbed in the distilled water, respectively, and the amount of absorbed water was measured. After 48 hours, the mass change was measured. The amount of water absorbed per skin cell unit mass was compared from each measured mass change. This experiment is a method for comparing the amount of water absorbed to the epidermal cells, and is used as one method of predicting the moisturizing effect even in the human skin.

Specifically, human epidermal cells were dried to make the amount of water contained per unit mass constant, and then the weight of each epidermal cell sample and the amount of water contained were measured using the Kaiser method. The epidermal cells in which the water content is measured are carried on the polymer polysaccharide, the liposome, the fermentation polysaccharide decomposition product and the purified water for 24 hours each, and then the skin cells in which the water is saturated and absorbed are taken out and weighed. To measure the amount of water per unit mass contained in the Kaiser Moisture Analyzer. Then, after drying for 48 hours under reduced pressure, the weight was added, and a part of the weight was measured. The water content was measured by the Kaiser method and the amount of water per unit mass was measured (mg / epidermal cell mass g).

Name of sample Moisture content Initial dry state Saturation After re-drying Hot water extract 400 790 450 Purified water 400 780 400

Name of sample Moisture content Initial dry state Saturation After re-drying Polysaccharide 400 810 590 Purified water 400 780 400

Name of sample Moisture content Initial dry state Saturation After re-drying Lycopene is a fermentation polysaccharide degradation product 400 840 630 Purified water 400 780 400

The results of measurement of water hygroscopicity (Table 16, Table 17, Table 18) show that the epidermal cells saturated with hot water extract, hyperpolarised polysaccharide, and hydrolyzed polysaccharide degraded by worms were re-dried It was confirmed that the water contained much water in the process. In particular, the fermented polysaccharide degradation product of Example 3 showed 840 mg / g, showing excellent skin hygroscopicity of epidermal cells.

From the above results, it was possible to confirm excellent skin hygroscopicity of the decomposition product of fermented polysaccharide, and it was confirmed that the excellent moisturizing effect of the present invention.

Experimental Example 7: Confirmation of glucuronic acid content

 In this Example, the hot water extract obtained from Example 1, the polysaccharide obtained from Example 2, and the glucuronic acid from the fermented polysaccharide degraded product obtained in Example 3 were examined.

1.0 g of each sample was precisely weighed, and purified water was added thereto to give 100. The resulting solution was treated with an ultrasonic pulverizer for 30 minutes at 30 ° C and filtered through a 0.45 filter. The filtrate 10 was taken and purified water was added to dilute to 100 to obtain a sample solution. Separately, 100 g of glucuronic acid standard reagent (C6H10O7; 194.1) was treated in the same manner as the sample solution to prepare a standard solution. Precisely take each of the sample solution and the standard solution 25 and inject them into a column packed with a pellicle type anion exchange resin having a pellet size of about 4 mm and a length of about 250 mm to prepare a mobile phase of 30 mM sodium hydroxide solution, (Conductivity detector) was detected to confirm the glucuronic acid. The test was carried out according to the liquid chromatography method and the peak area of the glucuronic acid was measured using the equation (4) AT and AS were measured.

&Quot; (5) "

AT: peak area of glucuronic acid in the sample

AS: Peak area of glucuronic acid in standard product

Name of sample Content of glucuronic acid (%) Hot water extract Non-detection Polysaccharide 32% Lycopene is a fermentation polysaccharide degradation product 55%

The content of glucuronic acid was measured (Table 19). The content of glucuronic acid in the fermented polysaccharide was 14.3%, and the content of polysaccharide in the fermented polysaccharide was below the detection limit of the detection device.

Claims (5)

Step 1: A process for producing a water-soluble extract by ultra-high pressure extraction of dry crushed worms,
Step 2: After acidic decomposition of the water-soluble extract of the produced insecticide, ethyl alcohol is added to the solution to precipitate the polymer polysaccharide and protein, the precipitate is recovered by filtration and centrifugation, and the polymer polysaccharide containing the insecticide is selectively recovered ,
Step 3: a process for producing a water-soluble polysaccharide decomposition product by fermenting and filtering the prepared polysaccharide-containing polymeric polysaccharide by inoculating mycelium of Laetiporus sulphureus CS0218 (KFCC 11494P) and
Step 4: A method for producing a decomposition product of an insect comprising at least 1.0% of glucuronic acid by sterilizing and concentrating the fermented water-soluble polysaccharide degraded product.
The method according to claim 1,
Wherein said mycelium (KFCC 11494P) of Laetiporus sulphureus CS0218 is characterized by fermentation in step 3 above 1.0% or more of glucuronic acid.
A decomposition product of a worm which contains 1.0% or more of glucuronic acid, which is produced by the method of any one of claims 1 to 2.
The cosmetic composition according to claim 4, wherein the insect comprising 1.0% or more of glucuronic acid contains the degradation product in an amount of 0.001 to 90.0% by weight based on the whole composition.
[Claim 5] The cosmetic composition according to claim 4, wherein the cosmetic composition exhibits an anti-aging effect or a skin anti-aging effect, a protective effect against active oxygen, or a wrinkle and elasticity-improving effect.

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