KR102491017B1 - Composition for Anti-glycation, Anti-oxidation, Anti-Wrinkle, and Moisturizing Property Comprising Seaweed and Peptide as Active Ingredient - Google Patents

Abstract

The present invention relates to a composition for anti-saccharide, anti-oxidation, improvement of skin wrinkles, and skin moisturizing including three complex ingredients of irish moss, corallina officinalis, and acetylhexapeptide-8 as active ingredients. The three complex ingredients of the present invention are not cytotoxic, show anti-saccharide activity equivalent to aminoguanidine known as representative anti-saccharide, show excellent active oxygen species elimination activity at a level similar to that of an ultraviolet untreated negative control group, significantly inhibit expression of MMP-1 protein, effectively increase synthesis of hyaluronan in skin cells; and show excellent activity in regeneration of skin wounds. Therefore, the three complex ingredients are usefully used for the anti-saccharide, the anti-oxidation, the improvement of the skin wrinkles, the skin moisturizing, and the regeneration of the skin wounds.

Description

Composition for Anti-glycation, Anti-oxidation, Anti-Wrinkle, and Moisturizing Property Comprising Seaweed and Peptide as Active Ingredient}

The present invention relates to a composition for anti-glycation, anti-oxidation, skin wrinkle improvement and skin moisturizing, containing seaweed and peptides as active ingredients, and more specifically, three complex components of Irish moss, true coral and acetyl hexapeptide-8 It relates to a composition for anti-glycation, anti-oxidation, skin wrinkle improvement and skin moisturizing comprising as an active ingredient.

Glycation generally refers to a reaction in which a simple sugar such as glucose or fructose forms a covalent bond with a protein or fat that occurs without the involvement of enzymes. Glycation occurs through a series of complex chemical reactions known as Amadori-rearrangements and Maillard-reactions, resulting in advanced glycation endproducts (AGEs).

On the other hand, the glycosylation process of protein occurs slowly because enzyme action is not involved, and it has a greater effect on structural proteins with a long half-life, such as collagen. As a result of the analysis of advanced glycation end products, it was shown that the older the age, the more glycated collagen was accumulated, and the accumulated glycated end products changed into a hard and more brittle state. Glycosylated collagen fails to form an appropriate structure in the extracellular matrix of the dermal layer, and is reported to be a cause of losing skin elasticity and promoting wrinkle formation (Dyer, D. G. et al., The Journal of Clinical Investigation, 9 , p. 2463, 1993).

In addition, it is known that the final glycation products produced by glycation are brownish substances and accumulate in the upper dermal layer as skin aging progresses. It is thought to be a causative substance that gradually turns yellow as skin aging progresses, and it is known that as specific final saccharification products accumulate, reflected light from the skin decreases. While the amount of melanin pigment in the skin has a weak correlation with skin aging, the amount of advanced glycation products accumulated in the dermal layer of the skin gradually increases as skin aging progresses and can darken the complexion (Hiroshi, O. et al., Skin Research and Technology, 15, p. 96, 2009).

Aminoguanidine, Pyridoxamine, and Aspirin are known as substances that inhibit glycation, but these substances have limitations in usage due to safety issues on the skin, or are ineffective due to insignificant effects. There are problems that cannot be expected.

On the other hand, cosmetic materials must balance safety, efficacy, and physical properties. In addition, securing economic feasibility is also an important factor, and cosmetic materials are developed by optimizing skin permeation, aging inhibition, physiological activity, safety, and physical properties using various development methods, natural product extraction and fermentation by microorganisms. As the boundary between cosmetics and pharmaceuticals is collapsing, various cosmeceutical products continue to grow rapidly through complex functional raw materials and bio-materials that combine the stability of cosmetics and the effectiveness of pharmaceuticals.

Under this background, the present inventors have made diligent research efforts to discover natural materials that are effective in antiglycation, and as a result of various studies to enhance their antiglycation efficacy by combining natural seaweed components, Irish Moss and Cham The present invention was completed by finding that the antiglycation ability was significantly improved when two components of coral powder were combined with acetyl hexapeptide-8, while antioxidation, skin wrinkle improvement, moisturizing, and skin regeneration activity were also excellent. I did.

(0001) Korean Patent Publication No. 10-2017-0080094 (2017.07.10.), Composition for improving skin condition containing 4-(4-hydroxyphenyl)-2-butanone (0002) Korean Patent Registration No. 10-1339915 (2013.12.04), Manufacturing method of carob bean extract with excellent anti-glycation and anti-aging activity and anti-aging cosmetic composition containing the extract as an active ingredient

Therefore, the main object of the present invention is to use the three complex components of Irish moss extract, true coral extract and acetyl hexapeptide-8, which have excellent effects on antiglycation, antioxidant, skin wrinkle improvement, skin moisturizing and skin regeneration, as active ingredients. It is to provide a composition containing as.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention includes three complex components of Irish Moss extract, true coral extract, and acetyl hexapeptide-8 as active ingredients, wherein the three complex components are 10:5 to 15:2.5 It provides a cosmetic composition for anti-glycation, antioxidant, skin wrinkle improvement and skin moisturizing, characterized in that it is composed of a weight ratio of ~ 7.5.

The present inventors conducted research to find natural extracts with excellent effects on skin condition improvement, such as anti-glycation, anti-oxidation, skin wrinkle improvement, skin moisturization, and skin regeneration promotion. As a result of diligent research efforts, when three complex ingredients consisting of Irish Moss extract, true coral extract and acetyl hexapeptide-8 are combined in a weight ratio of 10:5~15:2.5~7.5, excellent antiglycation, antioxidant, and skin It was confirmed that it has wrinkle improvement, skin moisturizing, and skin regeneration promoting activities, and it was also confirmed that there is no safety problem when applied to the skin.

The Irish Moss ( Chondrus Crispus ) of the present invention is a seaweed belonging to the genus Dolphinaceae Carragin, also called carraigin, and is a red algae that grows in rocky areas of the Atlantic coast of Europe and North America. Irish Moss is characterized by containing a large amount of MAA (Mycosporine like Amino Acids) ingredients such as Porphyra-334 and Palythene.

In addition, the true coral ( Corallina Officinalis ) is a red algae belonging to the order Jinuari, distributed in the east coast of Korea and Ulleungdo, etc., and contains a large amount of MAA (Mycosporine like Amino Acids) components such as Asterina-330 and Porphyra-334. It is characterized by doing.

In addition, Acetyl Hexapeptide-8 (CAS NO. 616204-22-9) is a peptide component having the chemical structure of Formula 1 below, and is a substance listed as a cosmetic ingredient.

[Formula 1]

The acetyl hexapeptide-8 of the present invention has effects of further improving antiglycation, antioxidant, skin wrinkle improvement, skin moisturizing, and skin regeneration promoting activity in combination with Irish moss and true coral. Copper tripeptide-1, tripeptide-29, tripeptide-1, hexapeptide-12, nicotinoyl tripeptide-35, palmitoyl tripeptide-1, palmitoyl tripeptide-1, palmitoyl tripeptide-1 There are peptides such as Toyl Pentapeptide-4, Nonapeptide-7, Palmitoyl Tripeptide-29, Palmitoyl Tetrapeptide-7, and Hexapeptide-9, but most preferably Acetyl Hexapeptide-8 is used. And it can show the best synergistic effect in antiglycation, antioxidant, skin wrinkle improvement, skin moisturizing, and skin regeneration promoting activity in combination with true coral.

In order to further improve the antiglycation effect of natural seaweed components, the present inventors conducted in-depth research on their combinations and combination ratios, and found that the Irish Moss ( Chondrus Crispus ) and true corals ( Corallina Officinalis ) and acetyl hexapeptide When -8 (Acetyl Hexapeptide-8) is used in combination, it was confirmed that there is a synergistic effect of a certain level or more, and when the above three complex components are combined at a weight ratio of 10:5 to 15:2.5 to 7.5, antiglycation . It has been confirmed that the activities of improving skin wrinkles, moisturizing skin, and promoting skin regeneration are maximized.

As used herein, the term "extract" refers to an extract obtained by extraction of Irish moss ( Chondrus Crispus ) and / or true coral ( Corallina Officinalis ), a diluted solution or concentrate of the extract, a dried product obtained by drying the extract, and the above It includes extracts of all formulations that can be formed using the extract itself and the extract, such as a refined product or a purified product of the extract, or a mixture thereof. The extract of the present invention may be preferably prepared and used in the form of a dry powder after extraction. In the extraction of the Irish Moss ( Chondrus Crispus ) and / or true coral ( Corallina Officinalis ), a method for extracting the extract is not particularly limited, and may be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method include a hot water extraction method, an ultrasonic extraction method, a filtration method, a reflux extraction method, and the like, which may be performed alone or in combination with two or more extraction methods.

In the present invention, the type of extraction solvent used to extract the Irish Moss ( Chondrus Crispus ) and true coral ( Corallina Officinalis ) is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the extraction solvent include water; C1 to C4 lower alcohols such as methanol, ethanol, propyl alcohol and butyl alcohol; polyhydric alcohols such as glycerin, butylene glycol, and propylene glycol; and hydrocarbon solvents such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane; Or a mixture thereof may be used, and preferably, water, lower alcohol, 1,3-butylene glycol, and ethyl acetate may be used alone or in combination of two or more.

In the present invention, the extract obtained by hot water extraction or cold brewing is filtered to remove floating solid particles by filtering out the particles using, for example, nylon or by using cryofiltration, and then used as it is or freeze-dried, hot-air-dried, It can be used after being dried using spray drying or the like.

As used herein, the term "fraction" refers to a product obtained by performing fractionation to separate a specific component or a specific component group from a mixture containing various components.

Non-limiting examples of the fractionation method include a method of obtaining fractions from the extract by treating an extract obtained by extracting Irish Moss ( Chondrus Crispus ) and/or true coral ( Corallina Officinalis ) with a predetermined solvent. In the present invention, the type of solvent used to obtain the fraction is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the fractionation solvent include polar solvents such as water and alcohol; and non-polar solvents such as hexane, ethyl acetate, chloroform, and dichloromethane. These may be used alone or in combination of two or more. When alcohol is used among the fractionation solvents, C1 to C4 alcohols may be specifically used.

The cosmetic composition of the present invention can be used by combining Acetyl Hexapeptide-8 with the Irish Moss ( Chondrus Crispus ) and true coral ( Corallina Officinalis ) extracts. It is not particularly limited thereto, but preferably may be used in combination at a weight ratio of 10:5 to 15:2.5 to 7.5, and most preferably may be used in combination at a weight ratio of 10:10:5.

In the cosmetic composition of the present invention, the three complex components of the Irish moss extract, the true coral extract, and acetyl hexapeptide-8 scavenge reactive oxygen species (ROS) and inhibit the expression of MMP-1 protein. It is characterized in that it suppresses the formation of skin wrinkles and promotes the synthesis of hyaluronic acid to exhibit skin moisturizing activity. In a preferred embodiment of the present invention, when these three components are mixed in a weight ratio of 10:5 to 15:2.5 to 7.5, they exhibit antiglycation activity equal to or higher than that of aminoguanidine, known as a representative anti-glycation substance (see FIG. 2), It exhibits excellent reactive oxygen species scavenging activity similar to that of the negative control group without UV treatment (see FIG. 3), significantly inhibits MMP-1 protein expression (see FIG. 4), and inhibits the synthesis of hyaluronan in skin cells. It has been confirmed that it increases effectively (see FIG. 5).

In addition, in the cosmetic composition of the present invention, the three complex components of the Irish moss extract, true coral extract, and acetyl hexapeptide-8 are characterized by exhibiting excellent activity in promoting skin wound regeneration. In a preferred embodiment of the present invention, it was confirmed that when damaged skin cells were treated with three complex components of Irish Moss extract, true coral malt, and acetylhexapeptide-8, regeneration of damaged skin cells was directly promoted, and these effects has been confirmed to be more excellent at a weight ratio of 10:5 to 15:2.5 to 7.5 (see FIG. 6).

The cosmetic composition of the present invention is harmless to the human body by combining three complex components: Irish moss extract and true coral extract derived from natural seaweed materials, and acetyl hexapeptide-8, known as a safe material without skin side effects. Since it does not impair the quality of the material of cosmetics due to addition, it is suitable for being used in a cosmetic composition. In particular, since it has functions related to antiglycation, antioxidant, skin wrinkle improvement, skin moisturizing, and skin wound regeneration promotion, it is preferable to be included in a functional cosmetic composition and used.

The cosmetic composition of the present invention is skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrient lotion, massage cream, nutrient cream, moisture cream, hand cream, foundation, essence, nutrient essence, mask pack, It may be prepared in any one formulation selected from soap, cleansing foam, cleansing lotion, cleansing cream, body lotion and body cleanser, but is not limited thereto.

The cosmetic composition of the present invention may further include one or more cosmetically acceptable carriers formulated in general skin cosmetics, and as typical ingredients, for example, oil, water, surfactants, moisturizers, lower alcohols, thickeners , A chelating agent, a colorant, a preservative, a flavoring agent, etc. may be appropriately mixed, but is not limited thereto.

Cosmetically acceptable carriers included in the cosmetic composition of the present invention vary depending on the formulation of the cosmetic composition.

When the formulation of the present invention is ointment, paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. This may be used, but is not limited thereto. These may be used alone or in combination of two or more.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohard propellants such as, but not limited to, low carbon, propane/butane or dimethyl ether. These may be used alone or in combination of two or more.

When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, Propylene glycol, 1,3-butyl glycol oil and the like may be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic esters, polyethylene glycol or fatty acid esters of sorbitan Can be used, but is not limited thereto. These may be used alone or in combination of two or more.

When the formulation of the present invention is a suspension, water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Crystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth may be used, but is not limited thereto. These may be used alone or in combination of two or more.

When the formulation of the present invention is a soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolyzates, isethionates, lanolin derivatives, aliphatic alcohols, vegetable oils, glycerol, sugar, etc. are used as carrier components. It may be, but is not limited thereto. These may be used alone or in combination of two or more.

In the cosmetic composition of the present invention, the three complex components of the Irish Moss extract, true coral extract, and acetyl hexapeptide-8 are specifically dry weight, 0.001% to 50% by weight of the total weight of the cosmetic composition. And, more specifically, it may be included in 0.005% by weight to 10% by weight. Within the above range, there is an advantage of exhibiting excellent efficacy for antiglycation, antioxidant, skin wrinkle improvement, skin moisturizing, and promotion of skin wound regeneration, and there is an advantage that the formulation of the composition is stabilized.

According to another aspect of the present invention, the present invention includes three complex components of Irish moss extract, true coral extract, and acetylhexapeptide-8 as active ingredients, wherein the three complex components are 10:5 to 15:2.5 It provides a food composition for anti-glycation, antioxidant, skin wrinkle improvement and skin moisturizing, characterized in that it consists of a weight ratio of ~ 7.5.

The three complex components of the Irish Moss extract, true coral extract, and acetylhexapeptide-8 of the present invention have antiglycation equivalent to or better than aminoguanidine, known as a representative antiglycation substance, at a weight ratio of 10:5 to 15:2.5 to 7.5. activity, exhibits excellent reactive oxygen species scavenging activity similar to that of the UV-untreated negative control group, significantly inhibits MMP-1 protein expression, effectively increases the synthesis of hyaluronan in skin cells, and improves skin wounds Since it exhibits excellent activity in regeneration, it can be usefully used as a food composition for improving skin conditions related to antiglycation, antioxidant, skin wrinkle improvement, skin moisturizing and skin wound regeneration.

The three complex components of the present invention, Irish moss extract, true coral extract, and acetyl hexapeptide-8, and their effects on antiglycation, antioxidant, skin wrinkle improvement, skin moisturization, and promotion of skin wound regeneration are as described above. The food composition may be used in the form of health functional food, but is not limited thereto.

The food composition of the present invention may be included in the form of a fraction or a processed product thereof for the three complex components of Irish Moss extract, true coral extract and acetylhexapeptide-8. In addition, the composition may include a food additive that is acceptable in food science in addition to the active ingredient.

In the present invention, "food supplement additive" refers to a component that can be added to food supplementally, and can be appropriately selected and used by those skilled in the art as being added to prepare a health functional food of each formulation. Examples of food additives include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners , pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, etc. are included, but the types of food additives of the present invention are not limited by the above examples.

The food composition of the present invention may include health functional food. In the present invention, "health functional food" refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients having useful functionality for the human body. Here, "functionality" means adjusting nutrients for the structure and function of the human body or obtaining useful effects for health purposes such as physiological actions, etc., specifically antiglycation, antioxidant, skin wrinkle improvement, skin moisturizing and skin wounds. means a regenerative effect. The health functional food of the present invention can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and components commonly added in the art during the preparation.

In addition, the formulation of the health functional food may also be manufactured without limitation as long as the formulation is recognized as a health functional food. The food composition of the present invention can be prepared in various types of formulations, and unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long time by using edible plant extracts as raw materials, and has excellent portability, It exhibits antiglycation activity equal to or higher than that of aminoguanidine, exhibits excellent reactive oxygen species scavenging activity similar to that of the UV untreated negative control group, significantly suppresses MMP-1 protein expression, and synthesizes hyaluronan in skin cells. It effectively increases and exhibits excellent activity in skin wound regeneration, so it can be taken as an adjuvant to obtain these effects.

There is no limit to the form that the health functional food of the present invention can take, and it may include all foods in a conventional sense, and may be used interchangeably with terms known in the art, such as functional foods. In addition, the health functional food of the present invention may be prepared by mixing suitable other auxiliary ingredients and known additives that may be included in food according to the selection of those skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and There is a vitamin complex, etc., and it can be prepared by adding the three components of Irish moss extract, true coral extract and acetyl hexapeptide-8 according to the present invention to juice, tea, jelly, juice, etc. prepared as main components. Also included are foods used as feed for animals.

The features and advantages of the present invention are summarized as follows:

(1) The present invention provides a cosmetic composition for anti-glycation, anti-oxidation, skin wrinkle improvement and skin moisturizing, comprising three complex components of Irish moss extract, true coral extract, and acetyl hexapeptide-8 as active ingredients.

(2) The three complex components composed of Irish moss extract, true coral extract, and acetyl hexapeptide-8 of the present invention have no cytotoxicity, show antiglycation activity equal to or higher than aminoguanidine known as a representative antiglycation substance, and It exhibits excellent reactive oxygen species scavenging activity similar to that of the untreated negative control group, significantly inhibits MMP-1 protein expression, effectively increases the synthesis of hyaluronan in skin cells, and has excellent activity in skin wound regeneration. Therefore, it can be usefully used for the purpose of antiglycation, antioxidant, skin wrinkle improvement, skin moisturizing and skin wound regeneration in cosmetics, food, and pharmaceutical fields.

(3) In addition, the composition of the present invention is not only very safe to the human body, but also has excellent stability.

1 is a graph showing normal human dermal fibroblasts (NHDF) treated with single or combined components of Irish moss extract, true coral extract, and acetylhexapeptide-8, and measuring cell viability.
Figure 2 is a graph showing the measurement of the anti-glycation effect according to the treatment of single or multiple components of Irish Moss extract, true coral extract, and acetylhexapeptide-8.
Figure 3 is a graph showing the treatment of single or complex components of Irish Moss extract, true coral extract, and acetylhexapeptide-8 to human skin fibroblasts (NHDF, normal human dermal fibroblasts) and measuring the active oxygen species scavenging activity. .
Figure 4 is a graph showing the treatment of Irish moss extract, true coral extract, and single or combined components of acetylhexapeptide-8 to human skin fibroblasts (NHDF, normal human dermal fibroblasts) and measuring the expression of MMP-1 protein to be.
5 is a graph showing changes in hyaluronic acid production after treatment of single or multiple components of Irish moss extract, true coral extract, and acetylhexapeptide-8 to human keratinocytes (HaCaT, human immortalized keratinocyte).
6 is a graph showing normal human dermal fibroblasts (NHDF) treated with single or combined components of Irish moss extract, true coral extract, and acetylhexapeptide-8, and cell regeneration activity measured.

Hereinafter, the present invention will be described in more detail through examples. Since these examples are intended to illustrate the present invention only, the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Preparation of Complex of Irish Moss, True Coral and Acetyl Hexapeptide-8

1-1. Preparation of Irish Moss Extract

After pulverizing 100 g of dried Irish moss, 3L of purified water was used as a solvent for 12 hours of heating and reflux extraction, followed by cooling and filtering with a filter paper having a 1.2 μm permeation size to obtain a filtrate. The obtained filtrate was concentrated under reduced pressure and dried under reduced pressure to prepare Irish Moss dried powder.

1-2. Preparation of true coral algae extract

After pulverizing 100 g of the dried true coral malt, it was heated and refluxed for 12 hours using 3 L of purified water as a solvent, then cooled and filtered with a filter paper having a 1.2 μm permeation size to obtain a filtrate. The obtained filtrate was concentrated under reduced pressure and dried under reduced pressure to prepare a dried powder of true coral powder.

1-3. Preparation of a complex of Irish moss, true coral and acetyl hexapeptide-8

Acetylhexapeptide-8 was mixed with the Irish Moss extract prepared in Examples 1-1 and 1-2 and true coral in the weight ratio shown in [Table 1] below to obtain Preparation Examples 1 to 7 consisting of three components. A complex extract was prepared. For comparative experiments, each single component of Comparative Examples 1 to 3 was prepared.

[Table 1]

Example 2. Cell survival test according to the addition of three components

The effects of single or combined components of Irish moss extract, true coral and acetylhexapeptide-8 on the growth of normal human dermal fibroblasts (NHDF) were confirmed by the following method.

First, human dermal fibroblasts were inoculated at a concentration of 1.2×10 ⁵ in a 40 mm cell culture dish using Dulbeccos modified Eagles medium (DMEM) medium containing 10% FBS (fetal bovine serum, Cambrex). For 24 hours after inoculation, 37 ℃, 5% CO ₂ It was cultured under humid conditions. Thereafter, the medium was removed, 300 μl of PBS was added, and UVB (UVB peak range: 312 nm, UV dose: 144 mJ/cm ² ) was irradiated.

UV-treated human dermal fibroblasts were treated with serum-free DMEM medium by diluting the multi-component samples of the preparation example prepared in Example 1 by 10-fold, and the single-component samples of Comparative Example were diluted 3.3-fold, respectively, and then treated with 72 incubated for hours. After 72 hours, 1 mg/mL of MTT was treated, and after 2 hours, formazan produced by MTT treatment was dissolved in DMSO, and absorbance was measured at 570 nm.

As a result of the experiment, it was confirmed that when human dermal fibroblasts were treated with the three complex components of the present invention, the survival rate of human dermal fibroblasts was increased. It was confirmed that the three complex components composed of 8 can be safely used on human skin (see FIG. 1).

Example 3. Antiglycation activity

In order to confirm the anti-glycation effect of the three complex components of Irish moss extract, true coral and acetyl hexapeptide-8, the glycation inhibitory activity was investigated using L-arginine and glucose. measured.

First, 1M L-arginine and 1M glucose were dissolved in 1M phosphate buffer solution (pH 7.4), and the composite component samples of Preparation Examples 1 to 7 were diluted 10-fold using 1M phosphate buffer solution. , Samples of Comparative Examples 1 to 3 were prepared by diluting single component samples by 3.3 times (see [Table 1]).

1M L-arginine and 1M phosphate buffer solution were mixed at a ratio of 1:4, and then 80 μl was dispensed into a 96-well plate. Samples diluted to 50 ppm and 100 μl of 0.01 M aminoguanidine to be used as a positive control were added thereto and mixed well. Next, after adding glucose diluted with 1M phosphate buffer so that the final concentration of glucose is 0.1M, the mixture was reacted at 70° C. for 4 hours. The degree of glycosylation was measured by measuring the absorbance at 420 nm of the 96-well plate using a spectrophotometer.

The glycation experimental group was an experimental group in which 1M L-arginine and 1M glucose were added to induce glycation.

The glycation inhibition rate (%) was calculated using the following [Equation 1], and after performing the experiment three times, the average value was calculated and summarized in [Table 2] below.

[Equation 1]

[Table 2]

Referring to the experimental results in [Table 2], the antiglycation activity of Preparation Examples 1 to 7 in which the three components of Irish Moss extract, true coral malt and acetylhexapeptide-8 were combined were comparative examples consisting of each single component The anti-glycation activity of 1 to 3 showed a significantly superior tendency.

In addition, the mixing ratio of the three components of Irish Moss extract, true coral malt, and acetyl hexapeptide-8 was also found to be important. It was confirmed that the antiglycation activity was equivalent to that of aminoguanidine, which is known as an antiglycation substance, and when these three components were mixed at a weight ratio of 10:10:5, it was confirmed that they exhibited superior antiglycation activity compared to aminoguanidine ( see Table 3 and Figure 2).

[Table 3]

Through the above results, the three complex components composed of Irish moss extract, true coral and acetylhexapeptide-8 of the present invention showed antiglycation compared to each single component at a weight ratio of 10:5 to 15:2.5 to 7.5. It was confirmed that it can be effectively used for anti-glycation-related purposes because it has a synergistic effect and exhibits excellent anti-glycation activity equal to or higher than that of aminoguanidine, a positive control group.

Example 4. Antioxidant activity

An experiment was performed to confirm the reactive oxygen species (ROS) scavenging ability of three complex components composed of Irish moss extract, true coral malt, and acetyl hexapeptide-8 in human skin fibroblasts.

In the experiment, human skin fibroblasts were irradiated with UVB (UVB Peak range: 312 nm, UV dose: 144 mJ/cm ² ), and then the Irish Moss extract prepared in Example 1, true coral and acetyl hexapeptide-8 complex components (preparation Examples 1 to 7, 10-fold dilution) and their single components (Comparative Examples 1 to 3, 3.3-fold dilution) were diluted and treated with serum-free DMEM medium, respectively, and incubated for 30 minutes, excitation 480 nm, emission ( emission) was performed by measuring fluorescence under 530 nm conditions and comparing DCF fluorescence intensity. Statistical processing was performed using the GraphPad Prism program, and the significance of the average value was examined with a limit of less than 5% using ANOVA (one-way analysis of variance).

Referring to [Figure 3], which summarizes the experimental results, the sample treatment groups of Comparative Examples 1 to 3 treated with single components of Irish Moss extract, true coral and acetyl hexapeptide-8 were 15% (Comparative Example 1) , 11% (Comparative Example 2), and 9% (Comparative Example 3) of active oxygen species scavenging activity was measured, and the antioxidant effect of single components was determined to be not very high. On the other hand, the sample treatment groups of Production Examples 1 to 7 in which these three components were combined were 2% (Production Example 1), 39% (Production Example 2), 47% (Production Example 3), and 53% (Production Example 4), respectively. ), 43% (Preparation Example 5), 34% (Preparation Example 6), and 6% (Preparation Example 7) of reactive oxygen species scavenging activity were measured, and it was observed that there was a synergistic effect of the combination, 10:5 to 15:2.5 At a weight ratio of ~7.5, a level similar to that of the negative control group without UV treatment was observed, and further improved reactive oxygen species scavenging activity was observed.

Through the above experimental results, the complex components composed of Irish Moss extract, true coral malt and acetyl hexapeptide-8 of the present invention maximized the synergistic effect of these combinations at a weight ratio of 10:5 to 15:2.5 to 7.5, resulting in antioxidant It was analyzed that the activity was further improved.

Example 5. Skin wrinkle improvement activity

The effects of single components or complex components of Irish moss extract, true coral malt and acetylhexapeptide-8 on MMP-1 gene expression were analyzed.

In the experiment, 2mL of DMEM culture medium was put in a 40mm cell culture dish, human skin fibroblasts were inoculated at a concentration of about 1.2x10 ⁵ , and cultured for 24 hours in a 37°C, 5% CO ₂ environment. Thereafter, the multi-component samples of Preparation Examples 1 to 7 were diluted 10 times and then cultured for 3 days, and the single component samples of Comparative Examples 1 to 3 were diluted 3.3 times and then cultured for 3 days. Thereafter, the culture medium was harvested, centrifuged for 5 minutes at 4°C and 7,500 rpm, and MMP-1 (Human Total MMP-1 kit, R&D Systems, Inc., Minneapolis, MN, USA) was used by ELISA method. ) Changes in protein expression were confirmed. All experiments were measured in triplicate.

As a result of the experiment, the test groups treated with single components of Irish Moss extract, true coral malt and acetylhexapeptide-8 did not clearly show an inhibitory effect on MMP-1 protein expression. On the other hand, in the test group treated with the complex components of the Irish moss extract, true coral malt, and acetylhexapeptide-8 of the present invention, it was confirmed that the amount of MMP-1 protein production was significantly reduced.

Referring to [Figure 4], which summarizes the experimental results, based on the MMP-1 protein content (Con.) increased by UVB irradiation (144 mJ/cm ² ), Irish Moss extract, true coral and acetyl hexapeptide-8 In the test group treated with a single component sample, MMP-1 protein expression decreased by 1%, 5% and 16%, indicating that the effect of inhibiting MMP-1 protein expression was insignificant.

In contrast, the composite of Irish moss extract, true coral malt, and acetylhexapeptide-8 of the present invention was found to have an excellent effect on inhibiting MMP-1 protein expression according to the combination ratio. Measured at 40% (Production Example 2), 31% (Production Example 3), 60% (Production Example 4), 38% (Production Example 5) and 47% (Production Example 6), respectively, 10:5 to 15: It was observed that MMP-1 protein expression was effectively suppressed at a weight ratio of 2.5 to 7.5.

Through the above experimental results, the complex components composed of Irish moss extract, true coral and acetyl hexapeptide-8 of the present invention maximized the synergistic effect of these combinations at a weight ratio of 10:5 to 15:2.5 to 7.5, It was observed to effectively inhibit the expression of MMP-1 protein in cells, and it was confirmed that it could be effectively used for skin wrinkle improvement.

Example 6. Skin moisturizing activity

In order to verify the skin moisturizing effect of the complex components composed of the Irish Moss extract, true coral and acetyl hexapeptide-8 of the present invention, their effect on the promotion of hyaluronan synthesis was tested.

Hyaluronic acid (HA, Hyaluronan, Hyaluronic acid) is synthesized mainly by epidermal keratinocytes and dermal fibroblasts, and is a substance that plays an important role in creating an environment in which cells can function normally by combining with moisture. A decrease in hyaluronic acid in the skin is one of the direct causes of a decrease in skin elasticity and a decrease in moisture content. Therefore, in this experiment, the amount of hyaluronic acid produced in human keratinocytes was confirmed.

Human keratinocytes (HaCaT, human immortalized keratinocyte) were put in 1 ml of the culture medium in a 24 well cell culture dish, and the cells were inoculated at a concentration of about 1×10 ⁵ , and then maintained at 37° C. in a 5% CO ₂ environment for 24 hours. cultured. Then, single components (Comparative Examples 1 to 3, 3.3-fold dilution) of Irish Moss extract, true coral and acetyl hexapeptide-8 prepared according to Example 1 and their complex components (Preparation Examples 1 to 7, 10 fold dilution) and cultured for 24 hours. Afterwards, the culture solution was harvested, centrifuged for 5 minutes at 4°C, 7,500 rpm, and the expression level of Hyaluronan (Hyaluronan kit, R&D Systems, Inc., Minneapolis, MN, USA) was used by ELISA method. Changes were confirmed, and compared to the untreated negative control group, summarized in [Figure 5].

Referring to [Figure 5], which summarizes the experimental results, in the test group treated with a single component of Irish Moss extract, true coral and acetylhexapeptide-8 as a sample, compared to the untreated negative control group, hyaluronan It was found that there was no significant difference in yield. In contrast, in the test group treated with the complex components of Irish Moss extract, true coral and acetyl hexapeptide-8, it was confirmed that the amount of hyaluronan produced increased in a concentration-dependent manner according to the combination ratio, 10: 141% (Production Example 3), 167% (Production Example 4), 144% (Production Example 5) and 148% (Production Example 6), respectively, compared to the untreated negative control at a weight ratio of 5 to 15:2.5 to 7.5. The amount of hyaluronic acid produced was confirmed.

Through the above results, the combination of Irish moss extract, true coral and acetyl hexapeptide-8 of the present invention maximizes the synergistic effect of these combinations at a weight ratio of 10:5 to 15:2.5 to 7.5, so that skin cells was observed to effectively increase the synthesis of hyaluronan, and through this, it was confirmed that it can be effectively used for skin moisturizing purposes.

Example 7. Skin regeneration activity

The skin regeneration activity of the complex component consisting of Irish moss extract, true coral and acetyl hexapeptide-8 of the present invention was verified.

In the experiment, when human skin fibroblasts, which are primary cultured skin cells, proliferated by 80% in a 100 mm cell culture dish, the skin cells were wounded using the tip of a sterilized tip (200ul), and the Irish prepared in Example 1 A sample containing a single component of Moss extract, true coral and acetylhexapeptide-8 (3.3-fold dilution) or a sample containing these complex components (10-fold dilution) was injected respectively. After 48 hours of injection, the cell regeneration effect was confirmed.

At this time, the cell proliferation was measured using the MTT (methylthiazole-2-yl-2.5-diphenyl tetrazolium bromide) analysis method and the Diff quic stain method, followed by cell staining and microscopic confirmation, and cell regeneration rate compared to the untreated group was measured.

Referring to [Figure 6], which summarizes the experimental results, when damaged skin cells are treated with the complex components of Irish Moss extract, true coral malt, and acetyl hexapeptide-8, it can be confirmed that regeneration of damaged skin cells is directly promoted. This effect was found to be more excellent at a weight ratio of 10:5 to 15:2.5 to 7.5, specifically 131% (Production Example 3), 152% (Production Example 4), and 146%, respectively, compared to the untreated negative control group. (Preparation Example 5) and 132% (Preparation Example 6) increased cell regeneration activity was confirmed.

Based on the above results, it was confirmed that the complex components of Irish Moss extract, true coral malt, and acetyl hexapeptide-8 had a direct effect of promoting skin cell regeneration.

Having described specific parts of the present invention in detail above, it is clear that these specific techniques are merely preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (5)

It contains three complex components of Irish Moss extract, true coral extract and acetyl hexapeptide-8 as active ingredients, wherein the three complex components are composed in a weight ratio of 10:5 to 15:2.5 to 7.5,
A cosmetic composition for anti-glycation. According to claim 1,
Characterized in that the three composite components are composed of a weight ratio of 10: 10: 5,
A cosmetic composition for anti-glycation. According to claim 1,
Characterized in that the three complex components scavenge reactive oxygen species (ROS), suppress skin wrinkles by suppressing MMP-1 protein expression, and promote hyaluronan synthesis,
A cosmetic composition for anti-glycation. According to claim 1,
Characterized in that the three complex components exhibit skin wound regeneration promoting activity,
A cosmetic composition for anti-glycation. It contains three complex components of Irish Moss extract, true coral extract and acetyl hexapeptide-8 as active ingredients, wherein the three complex components are composed in a weight ratio of 10:5 to 15:2.5 to 7.5,
A food composition for anti-glycation.

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