KR102583304B1 - Composition for Anti-glycation, Whitening and Skin Condition Improvement Comprising Plant Complex Extracts as Active Ingredient - Google Patents

Abstract

The present invention contains three complex extracts of white willow bark, hyssop, and Iceland moss as active ingredients, and exhibits anti-glycation, skin wrinkle improvement, skin exfoliation improvement, skin pore reduction, skin whitening anti-inflammatory, and skin regeneration promotion activities. It relates to composition.
The composition of the present invention has no cytotoxicity, exhibits anti-glycation activity equivalent to that of aminoguanidine, a representative anti-glycation substance, significantly inhibits MMP-1 protein expression, and has excellent effects on improving skin keratin and reducing skin pores. , effectively suppresses the production of melanin pigment, effectively suppresses the production of inflammatory cytokine IL-6 caused by fine dust, and has excellent activity in skin wound regeneration, so it is used for the purpose of improving skin condition in the cosmetics and food fields. It can be usefully used.

Description

Composition for anti-glycation, whitening and skin condition improvement comprising plant complex extracts as active ingredients {Composition for Anti-glycation, Whitening and Skin Condition Improvement Comprising Plant Complex Extracts as Active Ingredient}

The present invention relates to a composition for anti-glycation, whitening and improving skin condition containing a complex plant extract as an active ingredient, and more specifically, a composition containing three complex extracts of white willow bark, hyssop and Iceland moss as an active ingredient, It relates to a composition having anti-glycation, skin wrinkle improvement, skin exfoliation improvement, skin pore reduction, skin whitening anti-inflammatory and skin regeneration promotion activities.

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 without the involvement of enzymes. Glycation occurs through a series of complex chemical reactions known as Amadori rearrangement and Maillard reaction, resulting in the production of advanced glycation endproducts (AGEs).

Meanwhile, the glycation process of proteins occurs slowly because enzyme action is not involved, and it has a greater impact on structural proteins with a long half-life, such as collagen. Analysis of advanced glycation end products showed that glycated collagen accumulates with age, and advanced glycation end products change the accumulated proteins into a harder and more brittle state. This glycated collagen is unable to form an appropriate structure in the extracellular matrix of the dermal layer, and is reported to cause loss of skin elasticity and promote wrinkle formation (Dyer, D. G. et al., The Journal of Clinical Investigation, 9 , p. 2463, 1993).

In addition, advanced glycation products generated through glycation are brown-colored substances and are known to accumulate in the upper dermis layer as skin aging progresses. It is thought to be the cause of the complexion gradually turning yellow as skin aging progresses, and it is known that as certain advanced glycation end products accumulate, the reflected light reflected from the skin decreases. Although the amount of melanin pigment in the skin has a weak correlation with skin aging, it has been reported that advanced glycation end products accumulating in the dermal layer of the skin gradually increase in amount 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 limits on the amount of use due to safety issues on the skin, or their effects are so minimal that they are practically effective. There is a problem that cannot be expected.

Meanwhile, cosmetic materials must balance safety, efficacy, and physical properties. In addition, securing economic efficiency is an important factor, and cosmetic materials are being developed by optimizing skin penetration, anti-aging, physiological activity, safety, and physical properties using various development methods, such as extraction of natural products and fermentation by microorganisms. As the boundary between cosmetics and pharmaceuticals is collapsing, various cosmeceutical products are continuing to grow rapidly through complex functional raw materials and biomaterials that combine the stability of cosmetics and the effectiveness of pharmaceuticals.

Under this background, the present inventors made extensive research efforts to discover natural materials with excellent anti-glycation effects, and as a result of various research attempts to strengthen the anti-glycation efficacy of natural plant materials by combining them, white willow bark , when combining the three ingredients of hyssop and Iceland moss, the anti-glycation ability was significantly improved, while it was also found that there was a synergistic effect in improving skin wrinkles, improving skin exfoliation, reducing skin pores, skin whitening, anti-inflammatory, and promoting skin regeneration. Thus, the present invention was completed.

(0001) Korean Patent Publication No. 10-2017-0013149 (2017.02.06), Acne skin improvement cosmetic composition and mask pack using the same (0002) Korean Patent Registration No. 10-1618162 (2016.04.28), Natural fragrance composition containing health functional natural plant extracts (0003) Korean Patent Registration No. 10-1822043 (2018.01.19), Cosmetic composition containing complex extracts of blue lotus and Icelandic moss as active ingredients

Therefore, the main purpose of the present invention is white willow ( Salix alba ) bark and hyssop ( Hyssopus officinalis ), which have excellent effects in anti-glycation, improving skin wrinkles, improving skin exfoliation, reducing skin pores, whitening skin, anti-inflammatory, and promoting skin regeneration. ) and Iceland moss ( Cetraria islandica ) to provide a composition containing three complex extracts as active ingredients.

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

According to one aspect of the present invention, the present invention provides a cosmetic composition containing three complex extracts of white willow ( Salix alba ) bark, hyssop ( Hyssopus officinalis ), and Iceland moss ( Cetraria islandica ) as active ingredients.

The present inventors conducted research to find natural extracts that have excellent effects in improving skin conditions such as anti-glycation, improvement of skin wrinkles, improvement of skin exfoliation, reduction of skin pores, skin whitening, anti-inflammatory, and promotion of skin regeneration, especially natural plant extracts. As a result of extensive research efforts to further improve the anti-glycation ability using , it was confirmed to have synergistic activity in improving skin wrinkles, improving skin exfoliation, reducing skin pores, whitening skin, anti-inflammation, and promoting skin regeneration, and also confirmed that there were no safety issues when applied to the skin.

The white willow ( Salix alba ) of the present invention is a plant of the willow genus of the willow family of the order Malpighi order, is also called white willow, grows in Europe, western and central Asia, and is well known to contain a large amount of salicin.

In addition, Hyssop ( Hyssopus officinalis ) is a perennial plant of the Lamiaceae family, native to Central Asia and Southern Europe, and is used as a spice or herbal tea due to the characteristic minty smell of the entire plant. In addition, it is effective for indigestion, colds, and bronchitis, so it is used as a medicinal herb.

In addition, Iceland moss ( Cetraria islandica ) is a lichen of the order Ascus lichen and grows in the form of a shrub attached to the surface of trees or rock surfaces in the boreal and alpine regions of the Northern Hemisphere. In Northern Europe, Iceland moss is produced in large quantities, so it is known to be collected, ground into powder, and eaten. It is also used as a bitter stomach medicine and as a raw material for glycerin, soap, and cold cream.

In order to further improve the anti-glycation effect of plant extracts, the present inventors conducted in-depth research on the combination of white willow ( Salix alba ) bark, hyssop ( Hyssopus officinalis ), and Iceland moss ( Cetraria islandica ). When using the three types of plant extracts in combination, it was confirmed that there is an excellent synergy effect in anti-glycation, and additionally, it has a synergy effect in improving skin wrinkles, improving skin exfoliation, shrinking skin pores, skin whitening, anti-inflammation, and promoting skin regeneration. It was confirmed that it can be usefully used as a cosmetic composition for improving skin condition.

As used in the present invention, the term "extract" refers to an extract obtained by extraction treatment of white willow ( Salix alba ) bark, hyssop ( Hyssopus officinalis ), and/or Iceland moss ( Cetraria islandica ), a diluted or concentrated liquid of the extract, or the extract. This includes extracts of the extract itself and all formulations that can be formed using the extract, such as dried products obtained by drying, crude or purified products of the extract, or mixtures thereof. The extract of the present invention can preferably be prepared in the form of a dry powder after extraction. In the extraction of the white willow ( Salix alba ) bark, hyssop ( Hyssopus officinalis ), and/or Iceland moss ( Cetraria islandica ), the method of extracting the extract is not particularly limited, and is a method commonly used in the art. It can be extracted according to . Non-limiting examples of the extraction method include hot water extraction, ultrasonic extraction, filtration, and reflux extraction, which may be performed alone or by combining two or more extraction methods.

In the present invention, the type of extraction solvent used to extract the white willow ( Salix alba ) bark, hyssop ( Hyssopus officinalis ), and/or Iceland moss ( Cetraria islandica ) is not particularly limited, and any known in the art Solvents can be used. Non-limiting examples of the extraction solvent include water; C ₁ to C ₄ 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 can be used, and preferably, water, lower alcohol, 1,3-butylene glycol, and ethyl acetate can be used alone or in a mixture of two or more.

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

In the present invention, fractions produced by fractionation treatment of extracts of white willow ( Salix alba ) bark, hyssop ( Hyssopus officinalis ), and/or Iceland moss ( Cetraria islandica ) may be used.

As used in the present invention, the term “fraction” refers to the result obtained by performing fractionation to separate a specific component or specific group of components from a mixture containing various components.

As a non-limiting example of the fractionation method, an extract obtained by extracting white willow ( Salix alba ) bark, hyssop ( Hyssopus officinalis ), and/or Iceland moss ( Cetraria islandica ) is treated with a predetermined solvent to produce a fraction from the extract. Here's how to get it. In the present invention, the type of solvent used to obtain the fraction is not particularly limited, and any solvent known in the art can be used. Non-limiting examples of the fractionating solvent include polar solvents such as water and alcohol; Nonpolar solvents such as hexane, ethyl acetate, chloroform, and dichloromethane may be included. These may be used alone or in combination of two or more types. When alcohol is used among the fractionating solvents, alcohols of C ₁ to C ₄ can be specifically used.

In the cosmetic composition of the present invention, the complex extract is characterized by exhibiting anti-glycation activity. In a preferred embodiment of the present invention, it was confirmed that these three complex extracts exhibit anti-glycation activity equal to or greater than that of aminoguanidine, known as a representative anti-glycation substance (see Figure 2).

In the cosmetic composition of the present invention, the complex extract is characterized by exhibiting skin wrinkle improvement activity. In a preferred embodiment of the present invention, the composite ingredient consisting of white willow bark, hyssop, and Iceland moss was observed to maximize the synergistic effect of the combination to effectively suppress the expression of MMP-1 protein in skin cells, thereby reducing wrinkles in the skin. It was confirmed that it can be effectively used for improvement purposes (see Figure 3).

In the cosmetic composition of the present invention, the complex extract is characterized by improving skin exfoliation and reducing skin pores. In a preferred embodiment of the present invention, the hemoglobin precipitation rate of the test group treated with the three complex extracts of white willow bark, hyssop, and Iceland moss was found to be more increased compared to the group treated with the single extracts of white willow bark, hyssop, and Iceland moss, indicating a synergistic effect due to the combination of these three types. It was confirmed that the test group treated with the complex extract at concentrations of 0.1, 0.5, and 1.0v/v% had hemoglobin precipitation of 22.1%, 28.5%, and 32.9%, respectively, confirming the excellent pore shrinkage effect. (see Table 1). In addition, when using a patch product containing complex extracts of white willow bark, hyssop, and Iceland moss, it was clinically confirmed that the amount of dead skin cells was significantly reduced, and it removed dead skin cells better than commercially available products. It was confirmed that it was active (see Table 3).

In the cosmetic composition of the present invention, the complex extract is characterized by skin soothing and skin moisturizing activities. In a preferred embodiment of the present invention, the complex extract composed of white willow bark, hyssop, and Iceland moss has a skin soothing effect by increasing moisture content in the skin, reducing moisture loss, and reducing the occurrence of skin erythema. was confirmed, and it was confirmed that there was a further improved skin soothing and moisturizing effect compared to conventionally developed and commercially available products (see Table 4 and Figure 4).

In the cosmetic composition of the present invention, the complex extract is characterized by exhibiting skin whitening activity. In a preferred embodiment of the present invention, it was confirmed that the three complex extracts of white willow bark, hyssop, and Iceland moss of the present invention can be effectively used for purposes related to skin whitening by effectively inhibiting melanin production in skin cells (Figure 5).

In the cosmetic composition of the present invention, the complex extract is characterized by having anti-inflammatory activity through inhibiting the production of inflammatory cytokine IL-6 caused by fine dust. In a preferred embodiment of the present invention, the three complex extracts of the present invention exhibit a significantly improved inhibitory effect on IL-6 protein production compared to a single extract, and the combined treatment of the three components reduces inflammation mediators caused by fine dust. It was confirmed that there was an effect of improving the inhibition of production of the cytokine IL-6 (see Figure 6).

In the cosmetic composition of the present invention, the complex extract is characterized by exhibiting skin wound regeneration activity. In a preferred embodiment of the present invention, it was confirmed that when damaged skin cells were treated with a complex extract of white willow bark, hyssop, and Iceland moss, the regeneration of damaged skin cells was directly promoted, and this effect was observed in each single extract. It was confirmed to be more excellent than that (see Figure 7).

The cosmetic composition of the present invention is a combination of three ingredients derived from natural plant materials, white willow bark, hyssop, and Iceland moss, and is not harmful to the human body, and its addition does not damage the quality of cosmetic materials, so it is suitable for use in cosmetic compositions. It is suitable for inclusion and use. In particular, it has functions related to anti-glycation, skin wrinkle improvement, skin exfoliation improvement, skin pore reduction, skin whitening anti-inflammatory and skin regeneration promotion, so it is preferable to be included and used in a functional cosmetic composition.

The cosmetic composition of the present invention includes skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutritional lotion, massage cream, nutritional cream, moisture cream, hand cream, foundation, essence, nutritional essence, mask pack, It can be manufactured 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 that are blended with general skin cosmetics, and common ingredients include, for example, oil, water, surfactant, moisturizer, lower alcohol, and thickener. , chelating agents, pigments, preservatives, fragrances, etc. may be appropriately mixed, but are 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 an ointment, paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. are used as carrier ingredients. may be used, but is not limited thereto. These may be used alone or in combination of two or more types.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silcate, polyamide powder, etc. may be used as carrier ingredients, and especially in the case of a spray, chlorofluorohydride may be additionally used. It may include, but is not limited to, propellants such as low carbon, propane/butane or dimethyl ether. These may be used alone or in combination of two or more types.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent, or 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, etc. can be used, especially 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. may be used, but is not limited thereto. These may be used alone or in combination of two or more types.

When the formulation of the present invention is a suspension, the carrier components include water, liquid diluents such as ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, and miso. Crystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used, but are not limited thereto. These may be used alone or in combination of two or more types.

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

In the cosmetic composition of the present invention, the three complex extracts of white willow bark, hyssop, and Iceland moss may be included in an amount of 0.001% to 50% by weight of the total weight of the cosmetic composition in dry weight, and more specifically, May be included from 0.005% by weight to 10% by weight. Within the above range, it has the advantage of showing excellent efficacy for anti-glycation, skin wrinkle improvement, skin exfoliation improvement, skin pore reduction, skin whitening anti-inflammatory and skin regeneration promotion, and has the advantage of stabilizing the formulation of the composition.

According to another aspect of the present invention, the present invention provides a food composition containing three complex extracts of white willow ( Salix alba ) bark, hyssop ( Hyssopus officinalis ), and Iceland moss ( Cetraria islandica ) as active ingredients.

As mentioned above, the three complex extracts of white willow ( Salix alba ) bark, hyssop ( Hyssopus officinalis ), and Iceland moss ( Cetraria islandica ) have excellent anti-glycation properties, improvement of skin wrinkles, improvement of skin exfoliation, reduction of skin pores, and skin whitening. Because it exhibits anti-inflammatory and skin regeneration promoting activities, it can be useful as a food composition for improving skin conditions related to anti-glycation, skin wrinkle improvement, skin exfoliation, skin pore reduction, skin whitening anti-inflammatory and skin wound regeneration. .

The three complex ingredients of the present invention, white willow ( Salix alba ) bark extract, hyssop ( Hyssopus officinalis ) extract, and Iceland moss ( Cetraria islandica ) extract, anti-glycation, skin wrinkle improvement, skin exfoliation improvement, skin pore reduction, and skin whitening. The anti-inflammatory and skin wound regeneration effects are as described above. The food composition may be used in the form of a 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 processed product of the three complex components of white willow ( Salix alba ) bark extract, hyssop ( Hyssopus officinalis ) extract, and Iceland moss ( Cetraria islandica ) extract. Additionally, the composition may include food additives that are foodologically acceptable in addition to the active ingredients.

In the present invention, “food supplement” refers to a component that can be added to food as an auxiliary ingredient, and can be appropriately selected and used by a person skilled in the art as it is added to manufacture each type of health functional food. Examples of food supplements include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, and protective colloidal thickeners. , pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc., but the types of food supplements of the present invention are not limited to the above examples.

The food composition of the present invention may include health functional foods. 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 with functional properties useful to the human body. Here, “functionality” refers to obtaining useful effects for health purposes such as regulating nutrients or physiological effects on the structure and function of the human body, specifically anti-glycation, improvement of skin wrinkles, improvement of skin exfoliation, and reduction of skin pores. , refers to the effects of skin whitening, anti-inflammatory and skin regeneration promotion. The health functional food of the present invention can be manufactured by a method commonly used in the art, and can be manufactured by adding raw materials and components commonly added in the art.

Additionally, the formulation of the health functional food can also be manufactured without limitation as long as it is a formulation recognized as a health functional food. The food composition of the present invention can be manufactured in various forms, and unlike general drugs, it is made from edible plant extracts, so it has the advantage of not having side effects that may occur when taking the drug for a long time, and is highly portable. It exhibits anti-glycation activity equivalent to or higher than that of aminoguanidine, significantly inhibits MMP-1 protein expression, has excellent effects on improving skin keratin and reducing skin pores, effectively inhibits the production of melanin pigment, and reduces the risk of inflammation caused by fine dust. It effectively suppresses the production of the inflammatory cytokine IL-6 and has excellent activity in skin wound regeneration, so it can be taken as an adjuvant to achieve these effects.

There is no limit to the form that the health functional food of the present invention can take, and it can include all foods in the conventional sense, and can be used interchangeably with terms known in the art, such as functional food. In addition, the health functional food of the present invention can be manufactured by mixing known additives with other appropriate auxiliary ingredients that can be included in the food according to the selection of a person skilled in the art. Examples of foods that can be added include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and There are vitamin complexes, etc., and juices, teas, jellies, juices, etc. prepared using the three complex extracts of white willow ( Salix alba ) bark, hyssop ( Hyssopus officinalis ), and Iceland moss ( Cetraria islandica ) according to the present invention as main ingredients. It can be manufactured by adding. It also includes 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 and a food composition containing three complex extracts of white willow ( Salix alba ) bark, hyssop ( Hyssopus officinalis ), and Iceland moss ( Cetraria islandica ) as active ingredients.

(2) The composition of the present invention has no cytotoxicity, exhibits anti-glycation activity equivalent to or higher than that of aminoguanidine, a representative anti-glycation substance, significantly inhibits MMP-1 protein expression, and is excellent for improving skin exfoliation and reducing skin pores. It effectively suppresses the production of melanin pigment, effectively suppresses the production of inflammatory cytokine IL-6 caused by fine dust, and has excellent activity in skin wound regeneration, improving skin condition in the cosmetics and food fields. It can be usefully used for the following purposes.

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

Figure 1 is a graph showing treatment of single or combined extracts of white willow bark, hyssop, and Iceland moss to human fibroblasts (NHDF, normal human dermal fibroblasts) at different concentrations and measuring cell survival rate.
Figure 2 is a graph showing the anti-glycation effect measured by treatment with single or combined extracts of white willow bark, hyssop, and Iceland moss.
Figure 3 is a graph showing treatment of single or combined extracts of white willow bark, hyssop, and Iceland moss on human dermal fibroblasts (NHDF, normal human dermal fibroblasts) and measuring the expression of MMP-1 protein.
Figure 4 shows skin moisture content (Skin hydration) and moisture loss (TEWL, Trans Epidermal Water Loss) using skin measurement equipment after treating the skin with single or complex extracts of white willow bark, hyssop, and Iceland moss. This is a graph showing the measurement of erythema.
Figure 5 is a graph showing melanin content measured after treating melan-A cells with single or combined extracts of white willow bark, hyssop, and Iceland moss.
Figure 6 is a graph showing treatment of single or combined extracts of white willow bark, hyssop, and Iceland moss to HaCaT cells at different concentrations and measuring changes in IL-6 production.
Figure 7 is a graph showing treatment of single or combined extracts of white willow bark, hyssop, and Iceland moss on normal human dermal fibroblasts (NHDF) and measuring cell regeneration activity.

Hereinafter, the present invention will be described in more detail through examples. Since these examples are merely for illustrating the present invention, the scope of the present invention is not to be construed as limited by these examples.

Example 1. Preparation of plant extract

1-1. Preparation of white willow bark extract

100 g of bark of white willow ( Salix alba ) was pulverized, extracted under reflux for 12 hours using 3 L of purified water as a solvent, cooled, and filtered through filter paper with a penetration size of 1.2 μm to obtain a filtrate. The obtained filtrate was concentrated under reduced pressure and dried under reduced pressure to prepare white willow bark dry powder.

1-2. Preparation of hyssop extract

After pulverizing 100 g of hyssop ( Hyssopus officinalis ) whole plant, reflux extraction was performed for 12 hours using 3 L of purified water as a solvent, followed by cold soaking and filtering through filter paper with a penetration size of 1.2 μm to obtain a filtrate. The obtained filtrate was concentrated under reduced pressure and dried under reduced pressure to prepare hyssop dry powder.

1-3. Preparation of Iceland Moss Extract

100 g of Iceland moss ( Cetraria islandica ) whole plant was pulverized, extracted under reflux for 12 hours using 3 L of purified water as a solvent, cooled, and filtered through filter paper with a penetration size of 1.2 μm to obtain a filtrate. The obtained filtrate was concentrated under reduced pressure and dried under reduced pressure to prepare dried Iceland moss powder.

1-4. Preparation of mixed extract of three types of plants

100 g of the mixture obtained by mixing equal amounts of white willow ( Salix alba ) bark, hyssop ( Hyssopus officinalis ) whole plant, and Iceland moss ( Cetraria islandica ) whole plant was pulverized and extracted under reflux for 12 hours using 3 L of purified water as a solvent. Afterwards, it was cooled and filtered through filter paper with a 1.2㎛ transmission size to obtain a filtrate. The obtained filtrate was concentrated under reduced pressure and dried under reduced pressure to prepare a mixed dried powder of the three plants.

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

The effects of single or combined components of white willow bark, hyssop, and Iceland moss on the growth of human dermal fibroblasts (NHDF, normal human dermal fibroblasts) were confirmed using the following method.

First, human skin fibroblasts were inoculated at a concentration of 1.2×10 ⁵ in a 40 mm-sized cell culture dish using DMEM (Dulbeccos modified Eagles medium), a dedicated medium containing 10% FBS (fetal bovine serum, Cambrex). For 24 hours after inoculation, the cells were cultured at 37°C and 5% CO ₂ humidified conditions. Afterwards, the medium was removed, 300㎕ of PBS was added, and UVB (UVB Peak range: 312nm, UV dose: 144mJ/cm ² ) was irradiated.

UV-treated human skin fibroblasts were treated with the single or combined extracts of white willow bark, hyssop, and Iceland moss prepared in Example 1 in serum-free DMEM medium at various concentrations, and then cultured for 72 hours. After 72 hours, 1 mg/mL of MTT was treated, and 2 hours later, formazan produced by MTT treatment on the cells was dissolved in DMSO and the absorbance was measured at 570 nm.

As a result of the experiment, it was confirmed that when human skin fibroblasts were treated with the three composite ingredients of the present invention, the survival rate of human skin fibroblasts was significantly increased, and through this, white willow bark, hyssop, and Iceland moss of the present invention were used. It was confirmed that the three mixed extracts composed could be safely used on human skin (see Figure 1).

Example 3. Anti-glycation activity

To confirm the anti-glycation effect of three mixed extracts consisting of white willow bark, hyssop, and Iceland moss, the anti-glycation activity was measured using L-arginine and glucose.

First, prepare by dissolving 1M L-arginine and 1M glucose using 1M phosphate buffer solution (pH 7.4), and then prepare the single or complex extracts of Examples 1-1 to 1-4 using 1M phosphate buffer solution. , were treated at concentrations of 10 and 100 μg/ml.

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

The Glycation experimental group is an experimental group in which 1M L-arginine and 1M glucose were added to induce glycation. To measure the absorbance of the sample itself, only 1M L-arginine and the sample were added and the absorbance was measured at 420 nm without adding glucose.

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

[Equation 1]

Referring to the experimental results in [Figure 2], the anti-glycation activity of the three mixed extracts consisting of white willow bark, hyssop, and Iceland moss tended to be significantly superior to the anti-glycation activity of each single extract.

Specifically, the three mixed extracts consisting of white willow bark, hyssop, and Iceland moss showed 44%, 55%, and 62% glycation inhibition rates at concentrations of 1, 10, and 100 ㎍/㎖, respectively, showing an anti-glycation effect compared to each single ingredient. There was a synergistic effect, and at a concentration of 10㎍/㎖ or higher, it showed an equally excellent anti-glycation activity compared to aminoguanidine, a positive control, so the mixed extract of these three components can be effectively used for purposes related to anti-glycation. was able to confirm.

Example 4. Wrinkle improvement activity

To verify the wrinkle-improving activity of three mixed extracts consisting of white willow bark, hyssop, and Iceland moss, the effect of these complex ingredients on MMP-1 gene expression was analyzed.

In the experiment, 2 mL of DMEM culture medium was placed in a 40 mm cell culture dish, human skin fibroblasts were inoculated at a concentration of approximately 1.2x10 ⁵ and cultured for 24 hours at 37°C in a 5% CO ₂ environment. Thereafter, the single or combined extract samples of white willow bark, hyssop, and Iceland moss prepared in Example 1 were diluted to concentrations of 1, 10, and 100 μg/ml, respectively, and cultured for 3 days. Afterwards, the culture medium was harvested and centrifuged for 5 minutes at 4°C at 7,500 rpm to detect MMP-1 (Human Total MMP-1 kit, R&D Systems, Inc., Minneapolis, MN, USA) using the ELISA method. ) Changes in protein expression were confirmed. All experiments were measured in triplicate.

As a result of the experiment, the effect of suppressing MMP-1 protein expression in the test groups treated with single extracts of white willow bark, hyssop, and Iceland moss was not evident. In contrast, in the test group treated with the three complex extracts of white willow bark, hyssop, and Iceland moss of the present invention, it was confirmed that the amount of MMP-1 protein production was significantly reduced.

Referring to [Figure 3], which summarizes the experimental results, the test group treated with a single component sample of white willow bark based on the MMP-1 protein content (Con.) increased by UVB irradiation (144 mJ/cm ² ) MMP-1 protein expression decreased by 9% (10㎍/㎖) and 16% (100㎍/㎖), and in the test group treated with hyssop single-component sample, MMP-1 protein expression decreased by 3% (1㎍/㎖). ), decreased by 11% (10㎍/㎖) and 16% (100㎍/㎖), and in the test group treated with Iceland moss single-component sample, MMP-1 protein expression decreased by 7% (1㎍/㎖) and 13%. (10㎍/㎖) and 20% (100㎍/㎖) decreased.

On the other hand, the three complex extracts of white willow bark, hyssop, and Iceland moss of the present invention were found to have an excellent inhibitory effect on MMP-1 protein expression depending on the combination, with concentrations of 23% (1 μg/ml), 47%, respectively. It was observed that MMP-1 protein expression was effectively suppressed by decreasing % (10㎍/㎖) and 60% (100㎍/㎖).

Through the above experimental results, it was observed that the composite ingredient composed of white willow bark, hyssop, and Iceland moss of the present invention effectively inhibits the expression of MMP-1 protein in skin cells by maximizing the synergistic effect of their combination. Through this, it was confirmed that it can be effectively used to improve skin wrinkles.

Example 5. Pore contraction activity

To confirm the pore-shrinking effect of three complex extracts consisting of white willow bark, hyssop, and Iceland moss, the pore-shrinking effect was tested using hemoglobin as an experimental protein to replace skin.

Skin is mostly composed of protein, and protein ingredients combine with polyphenols to pull and shrink pores. In this way, the pore contraction effect was measured using the chemical convergence effect in which sweat glands and pores are temporarily contracted by a protein coagulating substance. Hemoglobin was used as a protein to replace skin.

A hemoglobin solution (0.05g/50ml) was prepared by adding 0.9% PBS (Phosphate Buffer Saline, 0.1mM, pH 7.4) solution to hemoglobin, and the hemoglobin solution in Examples 1-1 to 1-4 was added to 2ml of the hemoglobin solution. 2 ml of the obtained extract was added at a concentration of 1 mg/ml, mixed by shaking for 30 seconds, and centrifuged at 3,500 rpm for 10 minutes. Afterwards, 2ml of purified water was added to 1ml of the supernatant, and the absorbance was measured using UV-Visible spectrum equipment at 407nm. As a control, 2 ml of PBS (Phosphate Buffer Saline) was added instead of the composition of the above example. The shrinkage effect was evaluated by measuring the amount of hemoglobin precipitation at various concentrations, and it was determined that the higher the hemoglobin precipitation (%), the better the pore shrinkage effect.

The precipitation amount of hemoglobin was measured according to [Equation 2] below and summarized in [Table 1].

[Equation 2]

Pore shrinkage effect (%) = (1 - absorbance of test group/absorbance of control group) × 10

[Table 1]

Referring to [Table 1] above, which summarizes the experimental results, it was observed that the overall pore shrinkage effect was excellent in the test groups treated with three types of complex extracts of white willow bark, hyssop, and Iceland moss.

Specifically, hemoglobin precipitation was observed at 0.7%, 2.9%, and 7.5% in the test group treated with white willow bark single extract at concentrations of 0.1, 0.5, and 1.0v/v%, respectively, and hyssop single extract at 0.1, 0.5, and 1.0%. In the test group treated with v/v% concentration, hemoglobin precipitation was observed at 0.7%, 5.1%, and 11.2%, respectively, and in the test group treated with Iceland moss single extract at 0.1, 0.5, and 1.0v/v% concentration, hemoglobin precipitation was observed. This was observed at 5%, 8%, and 14%, respectively, showing that Iceland moss extract had the best pore tightening effect among single extracts.

Compared to the group treated with these single extracts, the hemoglobin precipitation rate of the test group treated with the three complex extracts of white willow bark, hyssop, and Iceland moss was found to increase further, confirming the synergistic effect caused by the combination of these three types. In the test group treated with the complex extract at concentrations of 0.1, 0.5, and 1.0v/v%, hemoglobin precipitation was observed at 22.1%, 28.5%, and 32.9%, respectively, confirming an excellent pore shrinkage effect.

Example 6. Skin exfoliation activity

The samples of Examples 1-4, which were confirmed to exhibit excellent skin wrinkle improvement and pore shrinkage activity in the above experimental examples, were collected in hydrogel at a concentration of 100 μg/ml, and then applied to a plate-shaped nonwoven fabric with a thickness of 2 mm. A patch for skin exfoliation was manufactured. The exfoliating patch was used to remove dead skin cells from the facial area and its ability to remove dead skin cells was tested.

The subjects were selected from men and women in their 20s and 30s who voluntarily signed a consent form to participate in the test after receiving test information. At this time, healthy subjects without acute or chronic physical diseases, including skin diseases, were selected, and a large number of valid data were secured to enable statistical comparison of the number of subjects. Even among test subjects selected according to the above criteria, cases where adverse reactions such as itching or erythema occurred in the test area were excluded at the discretion of the principal investigator and researcher during the experiment, and the test was conducted on a total of 20 subjects.

In order to objectively evaluate the subjects during the survey process, information about the products used and skin measurement data were not disclosed to the subjects. Additionally, in this test, a survey was conducted after the subjects used the product and measured their skin.

The skin exfoliation experiment was evaluated using the Simple Monadic Test method to evaluate the quality on a 5-point scale in [Table 2] below, and is shown in [Table 3] below. To compare the performance of the three complex extracts of white willow bark, hyssop, and Iceland moss of the present invention, commercially available products A and B were used as comparative examples.

[Table 2]

[Table 3]

As a result of the test, as shown in [Table 3], it was clinically confirmed that the amount of dead skin cells was significantly reduced when the patch product containing the three mixed extracts of the present invention was used for 7 days, and it was confirmed clinically that the amount of dead skin cells was significantly reduced. It was confirmed that it has excellent skin exfoliation ability compared to existing products.

Example 7. Measurement of skin soothing and moisturizing effect using skin measurement equipment

The samples of Examples 1-4 were collected in hydrogel at a concentration of 100 μg/ml, and then applied to a plate-shaped nonwoven fabric with a thickness of 2 mm to prepare a patch for exfoliating skin.

In order to determine whether problems occur when applying the skin exfoliation patch to the skin, erythema, skin hydration, and moisture loss (Trans Epidermal) are measured non-invasively using skin measurement equipment. Water Loss) items were measured to confirm skin soothing and moisturizing effects.

The subjects were 20 men and women in their 20s and 30s. After receiving the test information, those who voluntarily signed the consent form to participate in the test were selected. At this time, healthy subjects without acute or chronic physical diseases, including skin diseases, were selected, and the number of subjects was determined to secure a large number of valid data to enable statistical comparison. Even among test subjects selected according to the above criteria, if adverse reactions such as itching or erythema occurred in the test area, they were excluded at the discretion of the principal investigator and researcher during the experiment, and the data was excluded from calculating the experiment results.

In order to accurately measure skin condition, the specific test was performed by washing the subjects in order and stabilizing them for about 30 minutes under constant temperature and humidity conditions (room temperature 19°C, room humidity 55%).

The first measurement was conducted 30 minutes after washing the face, and after the measurement, the subjects were asked to use a patch for removing dead skin cells. During this process, information about the test product was not provided to the subjects. After the subject attached the patch, 25 minutes later, the patch was removed, rubbed for 5 minutes, and allowed to penetrate the skin to conduct a second measurement. The third measurement was performed 6 hours after the second measurement.

The measurement area was divided into the most protruding parts of the left and right cheekbones (3cm from the side of the nose and 3cm below the eyes), and recorded using a video scope of the same device.

Changes in skin indices for erythema, skin hydration, and trans epidermal water loss were measured using skin measurement equipment (DermaLab Series; Skin Lab Combo, Cortex Technology, Denmark). After measuring each 8 times, the average value of 6 measurements was calculated, excluding the maximum and minimum values. In the case of ultrasonic imaging, one image each of the left and right measurement points was taken.

The SE value (standard error value), which is an estimate of the standard deviation of statistics calculated from data (samples) such as arithmetic mean, was calculated, and the calculation results are shown in [Table 4] below. When trying to calculate the population mean from data, the arithmetic mean is only an estimate of the population mean and there is an error. In this case, the average error of the arithmetic mean as an estimate of the population mean is the sample standard deviation divided by the square root of the sample number. . If this estimate of the arithmetic mean is normally distributed, the 95% confidence interval for the population mean was calculated to be [(point estimate)±1.96×(standard deviation)].

[Table 4]

Referring to [Table 4] and [Figure 4], which summarize the test results, the three complex extracts of the present invention consisting of white willow bark, hyssop, and Iceland moss increase moisture content in the skin and reduce moisture loss. , it was confirmed that it was effective in reducing the occurrence of skin erythema, and it was confirmed that it had a more improved skin soothing and moisturizing effect compared to previously developed and commercially available products.

Through the above results, it was confirmed that the three complex extracts of the present invention consisting of white willow bark, hyssop, and Iceland moss are very effective in reducing skin pores and removing dead skin cells, and are also effective in soothing and moisturizing the skin.

Example 8. Skin whitening activity

In order to verify the whitening effect of the three complex extracts of white willow bark, hyssop, and Iceland moss of the present invention, the method reported in prior literature (Hosoi et al., 1985) was modified to measure melanin content. First, Melan-A cells were treated with single extracts of white willow bark, hyssop, and Iceland moss prepared in Example 1 and their complex extracts at concentrations of 1, 10, and 100 μg/ml for 3 days, and then cultured in medium. was removed and washed twice with PBS (phosphate buffer saline). Then, to dissolve melanin, 400 μl of 1N NaOH was added, and the melanin content was measured at an absorption wavelength of 405 nm using a microplate reader. All experiments were measured in triplicate.

Referring to [Figure 5], which summarizes the experimental results, unlike the test groups treated with a single extract, the amount of melanin produced in the test group treated with three complex extracts of white willow bark, hyssop, and Iceland moss increased significantly in a concentration-dependent manner. A decreasing pattern was confirmed. Specifically, compared to the hyssop single extract treatment group, which is the most effective of the single extracts, the melanin pigment production inhibition effect was improved by 4%, 15%, and 27% at doses of 1, 10, and 100㎍/㎖, respectively. indicated.

Through the above results, it was confirmed that the three complex extracts of white willow bark, hyssop, and Iceland moss of the present invention can be effectively used for purposes related to skin whitening by effectively suppressing melanin production in skin cells.

Example 9. Anti-inflammatory activity

In order to confirm the inhibitory effect of the three complex extracts of white willow bark, hyssop, and Iceland moss of the present invention on the secretion of inflammatory cytokines induced by fine dust, an experiment was conducted on human keratinocytes (HaCaT, human immortalized keratinocytes). carried out.

Human keratinocyte cells were inoculated into a 96 well plate at a concentration of 1.0×10 ⁵ and then cultured for 24 hours at 37°C and 5% CO ₂ humidified conditions. After washing with a PBS solution, 25㎍/㎖ of ultrafine dust (PM 2.5, Particulate Matter 2.5) with a diameter smaller than 2.5㎛ was added to each well as a single extract of white willow bark, hyssop, and Iceland moss, or a combination thereof. Treated with extracts (10 and 100 μg/ml) and incubated for 24 hours. The normal control group was treated with medium only. At the end of the culture, the supernatant was taken and the amount of IL-6 protein produced was analyzed using an enzyme-linked immunosorbent assay (ELISA) kit.

Referring to [Figure 6], which summarizes the experimental results, it was confirmed that the complex extract of white willow bark, hyssop, and Iceland moss showed a better effect in inhibiting the production of IL-6 protein compared to the single extracts.

Specifically, the hyssop extract, which had the best inhibitory effect among the single extracts, inhibited the production of IL-6 protein by 7% and 11% compared to the normal control at concentrations of 10 and 100 ㎍/㎖, respectively, but the three types of complex of the present invention The extract inhibited the production of IL-6 protein by 45% and 62% compared to the normal control at concentrations of 10 and 100㎍/㎖, respectively, and IL-6 depending on the combination of three components of white willow bark, hyssop, and Iceland moss. -6 It was confirmed that the ability to inhibit protein production was significantly improved.

Based on the above results, the three complex extracts of the present invention show a significantly improved effect of suppressing IL-6 protein production compared to the single extract, and the combined treatment of the three ingredients reduces the inflammation-mediated cytotoxicity caused by fine dust. It was confirmed that it was effective in improving the inhibition of production of IL-6, which is a cain.

Example 10. Skin wound regeneration activity

The skin regeneration promoting activity of the three complex extracts of the present invention consisting of white willow bark, hyssop, and Iceland moss was verified.

In the experiment, when human skin fibroblasts, which are primary cultured skin cells, had proliferated 80% in a 100 mm cell culture dish, the skin cells were injured using the end of a sterilized tip (200ul), and the white Single extracts of willow bark, hyssop, and Iceland moss or their combined extracts were injected at concentrations of 1, 10, and 100 μg/ml, respectively. After 48 hours of injection, the cell regeneration effect was confirmed.

At this time, cell proliferation was measured by staining cells using the MTT (methylthiazole-2-yl-2.5-diphenyl tetrazolium bromide) analysis method and the Diff quic stain method and then confirming them under a microscope, and then measuring the cell regeneration rate compared to the untreated group. was measured.

Referring to [Figure 7], which summarizes the experimental results, it was confirmed that when damaged skin cells are treated with a complex extract of white willow bark, hyssop, and Iceland moss, the regeneration of damaged skin cells is directly promoted, and this effect was confirmed to be superior to each single extract. Specifically, the test group treated with Iceland moss extract, which was shown to be most effective in cell regeneration, had 101% (1㎍/㎖), 103% (10㎍/㎖), and 108% (100㎍/㎖) compared to the untreated negative control group. ㎖) showed increased cell regeneration activity, while the test group treated with the three complex extracts had 115% (1㎖/㎖), 146% (10㎍/㎖), and 152% (100㎍/㎖) compared to the untreated negative control group. ㎖) Increased cell regeneration activity was observed, confirming the synergistic effect of the combination of the three plants.

Based on the above results, it was confirmed that the complex extract of white willow bark, hyssop, and Iceland moss had a direct effect in promoting the regeneration of skin cells.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (10)

Contains three complex extracts of white willow ( Salix alba ) bark, hyssop ( Hyssopus officinalis ), and Iceland moss ( Cetraria islandica ) as active ingredients,
The complex extract is characterized in that it simultaneously exhibits anti-glycation activity, skin pore shrinkage activity, and skin keratin improvement activity.
Cosmetic composition for anti-glycation, pore shrinkage, and skin exfoliation improvement. delete According to paragraph 1,
The complex extract is characterized in that it exhibits skin wrinkle improvement activity,
Cosmetic composition for anti-glycation, pore shrinkage, and skin exfoliation improvement. delete According to paragraph 1,
The complex extract is characterized in that it exhibits skin soothing and skin moisturizing activities.
Cosmetic composition for anti-glycation, pore shrinkage, and skin exfoliation improvement. According to paragraph 1,
The complex extract is characterized in that it exhibits skin whitening activity,
Cosmetic composition for anti-glycation, pore shrinkage, and skin exfoliation improvement. According to paragraph 1,
The complex extract is characterized in that it has anti-inflammatory activity through inhibiting the production of IL-6, an inflammatory cytokine caused by fine dust.
Cosmetic composition for anti-glycation, pore shrinkage, and skin exfoliation improvement. According to paragraph 1,
The complex extract is characterized in that it exhibits skin wound regeneration promoting activity,
Cosmetic composition for anti-glycation, pore shrinkage, and skin exfoliation improvement. Contains three complex extracts of white willow ( Salix alba ) bark, hyssop ( Hyssopus officinalis ), and Iceland moss ( Cetraria islandica ) as active ingredients,
The complex extract is characterized in that it simultaneously exhibits skin wrinkle improvement, skin exfoliation improvement, skin whitening, anti-inflammatory, and skin regeneration promotion activities.
Food composition for anti-glycation, pore shrinkage, and skin exfoliation improvement. delete