KR102623383B1 - Composition for moisturizing skin and strengthening skin barrier comprising robinin of robinia pseudoacacia flower extract - Google Patents

Abstract

According to an embodiment of the present invention, a cosmetic composition for moisturizing skin and strengthening the skin barrier containing robinin from acacia flower extract is provided. The acacia flower extract may contain 3% by weight or more of robinin based on the total weight of the acacia flower extract.

Description

Cosmetic composition for moisturizing skin and strengthening skin barrier containing robinin of acacia flower extract {COMPOSITION FOR MOISTURIZING SKIN AND STRENGTHENING SKIN BARRIER COMPRISING ROBININ OF ROBINIA PSEUDOACACIA FLOWER EXTRACT}

The present invention relates to a cosmetic composition for moisturizing skin and strengthening the skin barrier containing robinin from acacia flower extract.

The skin plays an important role in protecting the human body from physical and chemical stimuli from the external environment while maintaining moisture and body temperature, and can perform its function when the stratum corneum of the skin epidermis is normally formed.

This stratum corneum is composed of differentiated keratinocytes and a lipid layer surrounding them. In addition, as the stratum corneum is formed by producing natural moisturizing factor (NMF) and intercellular lipids during the keratinization process of keratinocytes, it becomes firm and flexible and functions as a skin barrier. do.

However, the stratum corneum can easily lose its function due to lifestyle factors such as excessive washing or bathing, environmental factors such as dry air and pollutants, or endogenous factors such as atopic skin or senile skin. Additionally, when the moisture content of keratinocytes decreases, the skin becomes rough and keratinocytes peel off.

Therefore, in order to maintain appropriate skin moisture, various methods are being studied to increase moisture supply, prevent moisture loss, or strengthen the skin barrier.

The present invention is intended to solve the above problems, and its purpose is to provide a cosmetic composition for moisturizing the skin and strengthening the skin barrier containing robinin from acacia flower extract.

The technical problems of the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned will be clearly understood by those skilled in the art from the description below.

According to an embodiment of the present invention, a cosmetic composition for moisturizing skin and strengthening the skin barrier containing robinin from acacia flower extract is provided.

Additionally, the acacia flower extract may contain 3% by weight or more of robinin based on the total weight of the acacia flower extract.

Additionally, the acacia flower extract may be obtained by extracting acacia flowers with at least one of ethanol, methanol, and water.

Additionally, the concentration of ethanol may be 60 to 90% (v/v).

Additionally, the extraction may be performed for 2 to 4 days.

Additionally, the extraction may be repeated 2 to 4 times.

Additionally, the extraction can be performed at room temperature.

Additionally, the robinin may be in the form of liquid or powder form after the liquid has been concentrated and dried.

According to an embodiment of the present invention, a skin external composition for moisturizing the skin and strengthening the skin barrier containing robinin from an acacia flower extract is provided.

According to an embodiment of the present invention, a pharmaceutical composition for treating or preventing dry skin disease comprising robinin from an acacia flower extract is provided.

According to the present invention, robinin from acacia flower extract can be included as an active ingredient to provide skin moisturizing and skin barrier strengthening effects.

Additionally, according to the present invention, the extraction conditions of acacia flowers can be appropriately adjusted to increase the content of robinin in the acacia flower extract.

Additionally, according to the present invention, acacia flower extract can be obtained in high yield.

In order to more fully understand the drawings cited in the detailed description of the present invention, a brief description of each drawing is provided.
Figures 1 and 2 are graphs showing the HPLC analysis results of Experimental Example 1.
Figures 3a and 3b are graphs showing the results of the cytotoxicity test in Experimental Example 2.
Figures 4a and 4b are graphs showing the mRNA expression level (relative value) of HAS2 in Experimental Example 2.
Figures 5a and 5b are graphs showing the mRNA expression level (relative value) of LOR in Experimental Example 2.
Figures 6a and 6b are graphs showing the mRNA expression level (relative value) of TGM-1 in Experimental Example 2.
Figure 7 is a graph showing the results of the cytotoxicity test in Experimental Example 3.
Figures 8a and 8b are graphs showing the mRNA expression levels (relative values) of HAS2 and AQP3 in Experimental Example 3.
Figures 9a and 9b are graphs showing the mRNA expression levels (relative values) of FLG and LOR in Experimental Example 3.

Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art. Additionally, when describing embodiments of the present invention, if detailed descriptions of related known configurations or functions are judged to impede understanding of the embodiments of the present invention, the detailed descriptions will be omitted. In addition, embodiments of the present invention will be described below, but the technical idea of the present invention is not limited or limited thereto and may be modified and implemented in various ways by those skilled in the art.

In this specification, when a part includes a certain element, this does not mean excluding other elements, but may further include other elements, unless specifically stated to the contrary. In this specification, the term and/or includes a combination of a plurality of related items or any one item among a plurality of related items.

According to an embodiment of the present invention, a cosmetic composition for moisturizing skin and strengthening the skin barrier is provided.

The cosmetic composition according to an embodiment of the present invention may include robinin from acacia ( Robinia pseudoacacia ) flower extract as an active ingredient.

Robinin is a flavonol glycoside composed of one galactose and two rhamnose bound to the aglycone kaempferol and is known to have antioxidant and anti-inflammatory effects. Robinin has been mainly used as a raw material for compositions with antioxidant and anti-inflammatory activities, but the present inventor confirmed the activity of robinin in moisturizing the skin and strengthening the skin barrier, and completed the present invention by applying it to cosmetic compositions.

In an embodiment, robinin may be derived from acacia flower extract. Acacia is a tree belonging to the legume family and is known to have anti-inflammatory, diuretic, and choleretic activities. Here, a flower may be referred to as a term that includes petals, stamens, pistils, and sepals. For example, an acacia flower may include one or more selected from the group consisting of acacia petals, stamens, pistils, and sepals.

In an embodiment, acacia flower extract may mean extracting acacia flowers using a solvent. For example, acacia flowers may be raw materials that have not gone through a crushing or grinding process, or they may be cut products that have gone through a crushing or grinding process. Additionally, for example, acacia flowers can be used fresh or dried. In this case, drying can be done, for example, by receiving an appropriate temperature and heat energy, or by drying in the sun at room temperature.

In an example, the acacia flower extract may be obtained by extracting acacia flowers using at least one of ethanol, methanol, and water as a solvent, and may specifically be extracted using ethanol as a solvent. When ethanol, methanol and/or water are used as the extraction solvent, the extraction efficiency of the acacia flower extract, the content of robinin in the extract, and the effectiveness of skin moisturizing and skin barrier strengthening can be improved. However, it is not limited to this, and various solvents applicable in the relevant technical field can be used alone or in combination. For example, water (or distilled water) as an extraction solvent; lower alcohols having 1 to 4 carbon atoms such as methanol, propyl alcohol, and butyl alcohol; polyhydric alcohols such as glycerin, butylene glycol, propylene glycol, methylpropanediol, and 1,2-hexanediol; and hydrocarbon solvents such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane; Or a mixture thereof can be used.

In an example, the concentration of ethanol as an extraction solvent may be 60 to 90% (v/v), specifically 65 to 80% (v/v), and more specifically 65 to 75% (v/v). ) can be. As ethanol as an extraction solvent has a concentration within the above range, the extraction efficiency of the acacia flower extract and the content of robinin in the extract can be improved. However, it is not limited to this.

Additionally, acacia flowers and extraction solvent may be mixed in a ratio of 1:15 to 45, specifically 1:20 to 40, more specifically 1:25 to 35.

In an embodiment, extraction of acacia flowers may be performed for 2 to 4 days, and specifically may be performed for 2 to 3 days. As the extraction of acacia flowers is performed for a period of time within the above range, the extraction efficiency of the acacia flower extract and the content of robinin in the extract can be maximized. However, it is not limited to this.

In an embodiment, the extraction of acacia flowers may be repeated 2 to 4 times, specifically 2 to 3 times. As the extraction of acacia flowers is repeatedly performed within the above range, the extraction efficiency of the acacia flower extract and the content of robinin in the extract can be improved. However, it is not limited to this.

In embodiments, extraction of acacia flowers may be performed at room temperature. For example, the room temperature may be 10 to 30°C, specifically 15 to 25°C. However, it is not limited to this, and extraction of acacia flowers can be performed using extraction methods commonly used in the art, such as cold needle extraction, reflux cooling extraction, or ultrasonic extraction.

Additionally/alternatively, the yield of the acacia flower extract may be at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to the mass of the acacia flowers used, specifically 35% or more. It may be more than %. Also, for example, the upper limit of the yield of the acacia flower extract is not particularly limited, but may be 90% or less, 85% or less, 80% or less, 75% or less, or 70% or less. However, it is not limited to this.

In an embodiment, the acacia flower extract contains robinin in an amount of at least 1.5%, at least 2.0%, at least 2.5%, at least 3.0%, at least 3.5%, at least 4.0%, and at least 4.3% by weight, based on the total weight of the acacia flower extract. It may be included in % by weight or more, 4.5 % by weight or more, or 5.0 % by weight or more, specifically 3.0 % by weight or more, and more specifically 4.3 % by weight or more. In addition, for example, the upper limit of the content of robinin is not particularly limited, but the acacia flower extract contains robinin at 90% by weight or less, 80% by weight or less, 70% by weight or less, and 60% by weight based on the total weight of the acacia flower extract. It may be included in less than or equal to 50% by weight. When the acacia flower extract contains robinin in the above content range, the effectiveness of the cosmetic composition in moisturizing the skin and/or strengthening the skin barrier can be improved. However, it is not limited to this.

In embodiments, the robinin and/or acacia flower extract may be in the form of a liquid or a concentrated and dried liquid. For example, the concentration method may be various methods commonly used in the art, such as a vacuum rotary concentrator or a vacuum rotary evaporator. Additionally, various drying methods commonly used in the art, such as reduced pressure drying, vacuum drying, boiling drying, spray drying, or freeze drying, may be applied. Specifically, freeze drying may be used. For example, drying may be performed for 2 to 5 days, and specifically may be performed for 2 to 4 days. However, it is not limited to this.

In embodiments, the cosmetic composition has a concentration of 0.1 μg/mL or more, 0.5 μg/mL or more, 1 μg/mL or more, 2 μg/mL or more, 3 μg/mL or more, 4 μg/mL or more, or 5 μg/mL or more. Alternatively, it may include 45 μg/mL or less, 40 μg/mL or less, 35 μg/mL or less, 30 μg/mL or less, 25 μg/mL or less, or 20 μg/mL or less of robinin. Here, the concentration may be measured based on 25°C unless specifically limited.

Additionally/alternatively, the cosmetic composition may have a concentration of 0.01 μg/mL or higher, 0.05 μg/mL or higher, 0.1 μg/mL or higher, 0.5 μg/mL or higher, 1 μg/mL or higher, or 450 μg/mL or lower, 400 μg/mL or higher. It may include an acacia flower extract of ㎍/mL or less, 350 ㎍/mL or less, 300 ㎍/mL or less, 250 ㎍/mL or less, or 200 ㎍/mL or less.

The cosmetic composition can provide better skin moisturizing and/or skin barrier strengthening effects without cytotoxicity by containing robinin and/or acacia flower extract having the above-mentioned concentration range. Here, the unit of concentration of robinin and/or acacia flower extract is mass/volume, where mass refers to the mass of robinin and/or acacia flower extract and the volume refers to the mass of the cosmetic composition (e.g. at 25° C. ) can refer to volume.

In an embodiment, the cosmetic composition may be manufactured into functional cosmetics such as nutritional cream, essence, or ampoule, or basic cosmetics such as lotion or lotion. In addition, it can be manufactured into soap, detergent, cleansing cream, cleansing water, etc., or color cosmetics such as lipstick, lip balm, mascara, blusher, shading, highlighter, makeup base, foundation, pact, concealer, skin cover, etc. . It can also be manufactured into hair cleaning products such as shampoo, rinse, hair conditioner, hair gel, etc. Additionally, for example, it may be manufactured as a product that is attached to the skin, such as a pack, hydrogel slimming patch, etc., or may be manufactured as a product that is sprayed on the skin, such as a mist or aerosol.

In an embodiment, the cosmetic composition may contain other ingredients commonly used in the art, such as thickeners, pH adjusters, isotonic agents, surfactants, stabilizers, preservatives, etc., within the range that does not impair the effect of the present invention. It may further include preservatives, ultraviolet absorbers, disinfectants, moisturizers, blockers, antioxidants, organic pigments, inorganic pigments, fragrances, vitamins, etc. The mixing amount of the above components can be easily selected by a person skilled in the art within a range that does not impair the purpose and effect of the present invention.

According to an embodiment of the present invention, a composition for topical skin application for moisturizing skin and strengthening the skin barrier is provided. The composition for topical skin application may include the above-described robinin of the acacia flower extract and/or the acacia flower extract. As described above, robinin of acacia flower extract and/or acacia flower extract may exhibit skin moisturizing and/or skin barrier strengthening activities.

The external skin preparation composition refers to a preparation that acts on the skin externally among external preparations, and may include products such as drugs and quasi-drugs, but is not limited thereto. For example, the external skin composition may have a formulation such as a cream, gel, skin emulsifier, patch, or spray.

In an embodiment, the composition for external skin application includes ingredients commonly used in external skin preparations such as cosmetics or medicines, such as aqueous ingredients, oil-based ingredients, powder ingredients, alcohols, moisturizers, thickeners, sunscreens, whitening agents, preservatives, antioxidants, Surfactants, fragrances, colorants, fatty substances, solubilizers, thickeners, gelling agents, softeners, foaming agents, fragrances, metal ion sequestrants, chelating agents, vitamins, various skin nutrients, or combinations thereof can be appropriately mixed as needed. You can. The mixing amount of the above components can be easily selected by a person skilled in the art within a range that does not impair the purpose and effect of the present invention.

According to an embodiment of the present invention, a pharmaceutical composition for preventing or treating dry skin disease is provided. The pharmaceutical composition may include robinin and/or acacia flower extract as described above. As described above, robinin of acacia flower extract and/or acacia flower extract can exhibit skin moisturizing and/or skin barrier strengthening activities, and therefore can be applied to dry skin diseases.

Dry skin disease refers to a disease that is caused by dry skin or whose symptoms are worsened by dry skin, and may include, for example, dry skin, pruritus, dry eczema, atopic dermatitis, or psoriasis. .

In an embodiment, the pharmaceutical composition may further include a pharmaceutically acceptable salt within a range that does not impair the effect of the present invention. Here, a 'pharmaceutically acceptable salt' may be a salt that does not cause serious irritation to the organism to which the compound is administered and does not impair the biological activity and physical properties of the compound.

For example, pharmaceutically acceptable salts include those of inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid or acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid. , benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcysteine, etc.

Additionally, for example, pharmaceutically acceptable salts can be prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases may include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium salts, and the like. Salts derived from organic bases include, but are not limited to, primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins such as isopropyl amines. Includes salts of amine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyimine resin, etc.

In an embodiment, the pharmaceutical composition may further include a pharmaceutically acceptable carrier or additive within the range that does not impair the effect of the present invention. Here, a 'pharmaceutically acceptable carrier' is one that is commonly used in formulations, does not inhibit the activity of the active ingredient, does not have toxicity beyond what is acceptable for the subject of application or prescription, and does not allow the addition of the compound into cells or tissues. It may be a compound that facilitates.

In embodiments, the pharmaceutical composition may be formulated as a solid formulation or a liquid formulation. For example, when formulated as a solid formulation, it may further include a carrier, flavoring agent, binder, preservative, disintegrant, lubricant, filler, etc., and when formulated as a liquid formulation, water, propylene glycol, etc. It may further include solutions such as solutions, suspensions, emulsions, etc., but is not limited thereto, and may further include colorants, flavoring agents, stabilizers, viscosifiers, etc.

In embodiments, solid preparations may include powders, granules, tablets, capsules, suppositories, etc. For example, a powder can be prepared by simply mixing a mixture of oregano extract and centella asiatica-derived exosomes, which are active ingredients of the present invention, with a carrier such as lactose, starch, or microcrystalline cellulose. The granules are made by mixing the mixture of oregano extract and centella asiatica-derived exosomes of the present invention with a carrier, a binder such as polyvinylpyrrolidone, hydroxypropyl cellulose, and then using a wet granulation method using a solvent such as water, ethanol, or isopropanol, or It can be manufactured using a dry granulation method using compression force. Additionally, tablets can be manufactured by mixing the granules with a lubricant such as magnesium stearate and then compressing them into tablets using a tablet press.

In an embodiment, the pharmaceutical composition may be an oral agent, an injectable agent (e.g., intramuscular injection, intraperitoneal injection, intravenous injection, infusion, subcutaneous injection, implant), an inhalant agent, or a nasal agent, depending on the disease or condition of the individual to be treated. It may be administered as an oral agent, vaginal agent, rectal agent, sublingual agent, transdermal agent, topical agent, etc., but is not limited thereto. Additionally, the pharmaceutical composition of the present invention may be formulated into a suitable dosage unit formulation containing pharmaceutically acceptable carriers, excipients, and vehicles that are commonly used and non-toxic depending on the route of administration.

In an embodiment, the dosage of the pharmaceutical composition varies depending on the patient's condition and weight, degree of disease, drug form, administration route and period, but may be appropriately selected by a person skilled in the art, and may be appropriately selected by the person skilled in the art. and the administration method may be independent, and the method is not particularly limited, and any administration route and method may be used as long as the pharmaceutical composition can reach the target area. For example, the dosage form of the pharmaceutical composition according to the present invention may be used in the form of its pharmaceutically acceptable salt, and may be used alone or in combination with other pharmaceutically active compounds, as well as in an appropriate combination. In addition, for example, the method of administration of the pharmaceutical composition according to the present invention includes not only intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration, or subcutaneous administration, but also methods of applying, spraying, or inhaling the composition to the diseased area. It can be used, but is not limited to this.

According to an embodiment of the present invention, a method for producing an acacia flower extract containing robinin is provided. This method can obtain the final product, robinin, in high yield.

In embodiments, the method may include extracting acacia flowers with a solvent. For example, an acacia flower may include one or more selected from the group consisting of acacia petals, stamens, pistils, and sepals. Additionally, for example, acacia flowers can be used fresh or dried. Additionally, acacia flowers and extraction solvent may be mixed in a ratio of 1:15 to 45, specifically 1:20 to 40, more specifically 1:25 to 35.

In an embodiment, the extraction solvent may be at least one of ethanol, methanol, and water, and may specifically be ethanol. For example, the concentration of ethanol may be 60 to 90% (v/v), specifically 65 to 80% (v/v), and more specifically 65 to 75% (v/v). there is. When ethanol, methanol and/or water are used as the extraction solvent, the extraction efficiency of the acacia flower extract can be increased and a high content of robinin, an active ingredient that achieves the desired effect in the present invention, can be obtained. However, it is not limited to this.

In an embodiment, the extracting step may be performed for 2 to 4 days, and specifically may be performed for 2 to 3 days. As the extraction step is performed for a period of time within the above range, the extraction efficiency of the acacia flower extract and the content of robinin in the extract can be maximized. However, it is not limited to this.

In an embodiment, the extraction step may be repeated 2 to 4 times, and specifically, may be performed 2 to 3 times. As the extraction step is repeatedly performed within the above range, the extraction efficiency of the acacia flower extract can be increased and a high content of robinin, an active ingredient that achieves the desired effect in the present invention, can be obtained. However, it is not limited to this.

In embodiments, the extracting step may be performed at room temperature. For example, the room temperature may be 10 to 30°C, specifically 15 to 25°C. However, it is not limited to this, and the extraction step may be performed using extraction methods commonly used in the art, such as cold needle extraction, reflux cooling extraction, or ultrasonic extraction.

Depending on the embodiment, the step of removing acacia flowers may be further included. For example, the removing step can be performed using a filter plate, filter cloth, or filter paper. For example, filtration can be performed at a size of 0.40 to 0.50 μm. In this case, residues other than the acacia flower extract can be filtered out to increase the purity of the acacia flower extract and minimize contamination of the acacia flower extract.

Depending on the embodiment, the step of concentrating and drying the liquid obtained in the extraction step may be further included. For example, various methods commonly used in the art, such as a vacuum rotary concentrator or a vacuum rotary evaporator, can be applied to concentrate the liquid. Additionally, concentration of the liquid may be performed at 30 to 60 °C, specifically 30 to 55 °C, and more specifically 35 to 50 °C. However, it is not limited to this.

In embodiments, various methods commonly used in the art, such as reduced pressure drying, vacuum drying, boiling drying, spray drying, or freeze drying, may be applied to drying the liquid material, and specifically, freeze drying may be used. For example, the concentrating and drying steps may be performed for 2 to 5 days, and specifically, may be performed for 2 to 4 days. However, it is not limited to this.

In embodiments, the yield of the acacia flower extract may be at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45%, and specifically at least 35%, based on the mass of the acacia flowers used. there is. Also, for example, the upper limit of the yield of the acacia flower extract is not particularly limited, but may be 90% or less, 85% or less, 80% or less, 75% or less, or 70% or less. However, it is not limited to this.

In embodiments, robinin is present in the prepared acacia flower extract in an amount of at least 1.5 wt.%, at least 2.0 wt.%, at least 2.5 wt.%, at least 3.0 wt.%, at least 3.5 wt.%, at least 4.0 wt.%, at least 4.3 wt.%, and at least 4.5 wt.%. It may be included in % or more or 5.0% by weight or more, specifically 3.0% by weight or more, and more specifically, 4.3% by weight or more. In addition, for example, the upper limit of the content of robinin is not particularly limited, but robinin is contained in the acacia flower extract at 90% by weight or less, 80% by weight or less, 70% by weight or less, 60% by weight or less, or 50% by weight or less. It can be included.

For example, if the extraction step is repeated twice over 2 days using 70% ethanol as the extraction solvent, the robinin content can be improved. In this case, robinin may be at least 5.0% by weight, at least 5.1% by weight, at least 5.2% by weight, at least 5.3% by weight, or at least 5.4% by weight in the acacia flower extract.

[Experimental example]

Below, examples are shown to further explain the present invention in more detail, but the present invention is not limited thereto.

Experimental Example 1: Analysis of extraction efficiency and robinin content according to extraction method

(1) Preparation of Preparation Examples 1 to 5

A production example of an acacia flower extract according to the present invention was prepared using the extraction conditions shown in Table 1 below.

Extraction conditions Manufacturing Example 1 Production example 2 Production example 3 Production example 4 Production example 5 extraction solvent 70% Ethanol 70% ethanol methanol acetone Acetonitrile extraction time 2 days 3 days 2 days 2 days 2 days number of extractions Episode 2 3rd time Episode 2 Episode 2 Episode 2

The specific manufacturing method is as follows.

10 g of dried acacia flowers are extracted with 300 g of extraction solvent for each extraction time (2 or 3 days), and the extraction process is repeated for each number of extractions (2 or 3 times). When extraction was completed, the raw material was removed, filtered to 0.45 ㎛, concentrated at about 45°C, and freeze-dried for 3 days to prepare the acacia flower extracts of Preparation Examples 1 to 5.

(2) Preparation of Preparation Example 6

10 g of dried acacia flowers are extracted with 300 g of purified water at 80°C for 2 hours. When extraction was completed, the raw material was removed, filtered to 0.45 ㎛, concentrated at about 45°C, and freeze-dried for 3 days to prepare the acacia flower extract of Preparation Example 6.

(3) Preparation of Comparative Example 1

After mixing 1% of the acacia flower extract and 1% of Viscozyme of Preparation Example 1 with purified water, the reaction proceeds for about 18 hours at 35°C and 150 rpm. Afterwards, Viscozyme was inactivated by heating at 90°C for 10 minutes, centrifugation was performed to remove precipitated Viscozyme, and the obtained supernatant was freeze-dried to prepare an enzyme-treated acacia flower product of Comparative Example 1.

(4) Experimental example: Analysis of extraction efficiency of acacia flower extract

For Preparation Examples 1 to 6, the yield of acacia flower extract was calculated based on the mass of the acacia flower used, and the results are shown in Table 2.

division extraction solvent extraction time number of extractions transference number(%) Manufacturing Example 1 70% Ethanol 2 days Episode 2 35 Production example 2 70% ethanol 3 days 3rd time 45 Production example 3 methanol 2 days Episode 2 34.2 Production example 4 acetone 2 days Episode 2 2.486 Production example 5 Acetonitrile 2 days Episode 2 0.752 Production example 6 Purified water 2 hours 1 time 28.8

As a result of the experiment, it was found that the yield of the acacia flower extract of Preparation Examples 1 and 2 using 70% ethanol as the extraction solvent was measured to be the highest, at 35% or more. In particular, it was confirmed that the yield of the acacia flower extract in Preparation Example 2, in which the extraction was repeated three times over three days, was measured at 45% or more.

In other words, it can be seen that the yield of acacia flower extract can be increased depending on the extraction conditions including the extraction solvent, extraction time, and number of extractions used when extracting acacia flowers.

(5) Experimental example: analysis of robinin content

Robinin was quantified for Preparation Examples 1 to 6 and Comparative Example 1 through High Performance Liquid Chromatography (HPLC), and the results are shown in Table 3 and Figures 1 and 2.

Specific HPLC analysis conditions are as follows.

- Concentration gradient of mobile phase solvent: 0.1% phosphoric acid in Water (solvent A), Acetonitrile (solvent B), 15% solvent B (50min; Isocratic)

- Flow rate: 1 mL/min

- Column: Luna 5u C18(2) 100A New Column (250 x 4.6 mm)

- Detection: Analysis wavelength 350 nm

The HPLC analysis results are shown in Figures 1 and 2. Referring to Figure 1, the HPLC analysis results for Preparation Examples 1 to 6 and the analysis results (Ref.) corresponding to 10 ppm of robinin are shown. In addition, referring to Figure 2, the analysis results for Preparation Example 1 and Comparative Example 1 and the analysis results corresponding to 10 ppm robinin (Ref.) are shown. A calibration curve was prepared using the robinin standard product to quantify the content of robinin in each sample.

division extraction solvent extraction time number of extractions Robinin content (%) Manufacturing Example 1 70% Ethanol 2 days Episode 2 5.50 Production example 2 70% ethanol 3 days 3rd time 4.35 Production example 3 methanol 2 days Episode 2 4.00 Production example 4 acetone 2 days Episode 2 1.41 Production example 5 Acetonitrile 2 days Episode 2 0.89 Production example 6 Purified water 2 hours 1 time 3.84 Comparative Example 1 enzyme-treated product 0

As a result of the experiment, it was confirmed that the acacia flower extracts of Preparation Examples 1 to 6 all contained more than 0.8% robinin, while the acacia flower enzyme-treated product of Comparative Example 1 did not detect robinin.

In particular, it was found that the robinin content of the acacia flower extracts of Preparation Examples 1 and 2 using 70% ethanol as the extraction solvent was measured to be the highest at 4.3% or more. In addition, it was confirmed that the robinin content of the acacia flower extract of Preparation Example 1, in which the extraction was repeated twice over 2 days, was measured to be 5.5% or more.

In other words, it can be seen that the content of robinin contained in the acacia flower extract can be increased depending on the extraction conditions including the extraction solvent, extraction time, and number of extractions used when extracting acacia flowers.

Experimental Example 2: Analysis of the effectiveness of robinin in acacia flower extract for skin moisturizing and skin barrier strengthening

(1) Experimental example: cytotoxicity test

For Preparation Example 1 and Comparative Example 1, a cytotoxicity test was performed using the HaCaT MTT assay, and the results are shown in Figures 3a and 3b. Specifically, Figures 3A and 3B are graphs showing cell proliferation (%) for Preparation Example 1 and Comparative Example 1 prepared at each concentration (unit: ㎍/mL).

The specific experimental method is as follows.

200 ㎕ of the HaCaT cell line was dispensed at an appropriate concentration per well in a 96-well plate and cultured for 24 hours under 37°C and 5% CO ₂ incubator conditions. The cultured cells were treated with the samples at different concentrations and then cultured for 24 hours under 37°C and 5% CO ₂ incubator conditions. 3 hours before the completion of culture, 20 ㎕ of 5mg/mL MTT was treated and cultured under the same conditions for 3 hours. After completion of incubation, the culture medium was discarded, 150 ㎕ of DMSO was dispensed, and the mixture was stirred at room temperature for 10 minutes. Absorbance was measured at 570 nm using a microplate reader (Thermo, UV/Vis). Cell viability was derived using the equation (absorbance of experimental group/absorbance of control group) x 100. When the cell viability was 80% or more, it was judged that there was no cytotoxicity.

As a result of the experiment, referring to FIGS. 3A and 3B, Preparation Example 1 can be evaluated as having no cytotoxicity at 200 ㎍/mL or less, and Comparative Example 1 can be evaluated as having no cytotoxicity at 500 ㎍/mL or less.

(2) Experimental example: moisturizing efficacy test

A moisturizing efficacy test was performed on Preparation Example 1 and Comparative Example 1 by measuring the mRNA expression level of HAS2, and the results are shown in Table 4 and Figures 4a and 4b. Specifically, Figures 4a and 4b are graphs showing the mRNA expression levels (relative values) of HAS2 for Preparation Example 1 and Comparative Example 1, respectively.

The specific experimental method is as follows.

4 mL of HaCaT cell line was dispensed at an appropriate concentration per well in a 60 mm dish and cultured in a 5% CO ₂ incubator at 37°C for 24 hours. After serum starvation was performed on the cultured cells for 24 hours, samples were treated at different concentrations and cultured for 24 hours under 37°C and 5% CO ₂ incubator conditions. After completion of incubation, RNA was extracted by treatment with 1 mL of Trizol (Invitrogen), and RNA condensation and washing were performed using chloroform, 2-propanol, 75% EtOH, and a centrifuge. After drying the RNA, an appropriate amount of nuclease free water was added and quantification was performed using Nano drop (ALLSHENG). After preparing each RNA to the same concentration, reverse transcription was performed using a cDNA Synthesis Kit (PhileKorea). After mixing the synthesized cDNA, Primer (HAS2 Oligonucleotide), SYBR green, and Nuclease free in an appropriate ratio, RT-qPCR was performed using Real time PCR equipment (Applied Biosystem, Quantstudio 5). In the analysis of PCR results, ΔΔCt values were calculated to compare gene expression.

The blank was cultured under the same conditions as the sample by adding the same amount of distilled water. The control group was cultured under the same conditions as the sample by adding the same amount of panthenol. Based on the fact that the mRNA expression level of HAS2 measured in the blank was 1, the mRNA expression level of HAS2 measured in the sample and control group was measured as a relative value, and the increase/decrease ratio (effect, %) for the relative value was measured. The increase/decrease rate of the relative value of the mRNA expression level of HAS2 is calculated by subtracting the mRNA expression level of HAS2 measured in the blank from the mRNA expression level (relative value) of HAS2 measured in each sample, and then measuring the subtracted value on the blank. The percentage value was calculated by dividing by the mRNA expression level of HAS2 and multiplying by 100.

HAS2 is a gene expressed by keratinocytes and is responsible for moving moisture outside the cell into the cell or preserving moisture within the skin epidermis. Therefore, the higher the mRNA expression level of HAS2 is measured, the better moisturizing effect can be evaluated. there is.

division Concentration (㎍/mL) mRNA expression level of HAS2 (relative value, %) Manufacturing Example 1 100 12 200 62 Comparative Example 1 200 2 500 -5

As a result of the experiment, in Preparation Example 1, the mRNA expression level of HAS2 increased significantly compared to the blank, and in particular, at a concentration of 200 ㎍/mL, it was confirmed that it increased by 62% compared to the blank. That is, Preparation Example 1 was found to have excellent moisturizing efficacy.

Meanwhile, in Comparative Example 1, the mRNA expression level of HAS2 was almost similar to that of the blank, and was confirmed to be reduced compared to the blank, especially at a concentration of 500 μg/mL. That is, unlike Preparation Example 1, Comparative Example 1 did not contain robinin, so it was found that the skin moisturizing effect was also minimal.

(3) Experimental example: barrier efficacy test

A barrier efficacy test was performed on Preparation Example 1 and Comparative Example 1 by measuring the mRNA expression levels of LOR and TGM-1, and the results are shown in Table 5 and Figures 5a, 5b, 6a, and 6b. Specifically, Figures 5A and 5B are graphs showing the mRNA expression levels (relative values) of LOR for Preparation Example 1 and Comparative Example 1, respectively, and Figures 6A and 6B are graphs showing the mRNA expression levels (relative values) of TGM-1 for Preparation Example 1 and Comparative Example 1. This is a graph showing the mRNA expression level (relative value).

1) Method for measuring LOR mRNA expression level

HaCaT cell line was dispensed at an appropriate concentration of 4 mL per well into a 60 mm dish and cultured in a 37°C, 5% CO ₂ incubator for 24 hours. After serum starvation was performed on the cultured cells for 24 hours, samples were treated at different concentrations and cultured for 24 hours under 37°C and 5% CO ₂ incubator conditions. After completion of incubation, RNA was extracted by treating with 1 mL of Trizol (Invitrogen), and RNA was condensed and washed using chloroform, 2-propanol, 75% EtOH, and centrifuge. After drying the RNA, an appropriate amount of nuclease free water was added and quantification was performed using Nano drop (ALLSHENG). After preparing each RNA to the same concentration, reverse transcription was performed using a cDNA Synthesis Kit (PhileKorea). After mixing the synthesized cDNA, Primer (LOR Oligonucleotide), SYBR green, and Nuclease free in an appropriate ratio, RT-qPCR was performed using Real time PCR equipment (Applied Biosystem, Quantstudio 5). In the analysis of PCR results, ΔΔCt values were calculated to compare gene expression.

The blank was cultured under the same conditions as the sample by adding the same amount of distilled water. The control group was cultured under the same conditions as the sample by adding the same amount of panthenol. Based on the fact that the mRNA expression level of LOR measured in the blank was 1, the mRNA expression level of LOR measured in the sample and control group was measured as a relative value, and the increase/decrease ratio (effect, %) with respect to the relative value was measured. The increase/decrease rate of the relative value of the mRNA expression level of LOR is calculated by subtracting the mRNA expression level of LOR measured in the blank from the mRNA expression level (relative value) of LOR measured in each sample, and then measuring the subtracted value on the blank. The percentage value was calculated by dividing by the mRNA expression level of the LOR and multiplying by 100.

LOR (loricrin) is a protein that constitutes a protein-lipid complex in differentiated keratinocytes of the epidermis. It plays a role in maintaining skin homeostasis and maintaining the skin barrier. Therefore, the higher the mRNA expression level of LOR is measured, the better the skin barrier is strengthened. It can be evaluated as having efficacy.

2) Method for measuring the mRNA expression level of TGM-1

The HaCaT cell line was dispensed at an appropriate concentration of 4 mL per well into a 60 mm dish and cultured in a 37°C, 5% CO ₂ incubator for 24 hours. After serum starvation was performed on the cultured cells for 24 hours, samples were treated at different concentrations and cultured for 24 hours under 37°C and 5% CO ₂ incubator conditions. After completion of incubation, RNA was extracted by treating with 1 mL of Trizol (Invitrogen), and RNA was condensed and washed using chloroform, 2-propanol, 75% EtOH, and centrifuge. After drying RNA, an appropriate amount of nuclease free water was added and quantification was performed using Nano drop (ALLSHENG). After preparing each RNA to the same concentration, reverse transcription was performed using a cDNA Synthesis Kit (PhileKorea). After mixing the synthesized cDNA, Primer (TGM-1 Oligonucleotide), SYBR green, and Nuclease free in an appropriate ratio, RT-qPCR was performed using Real time PCR equipment (Applied Biosystem, Quantstudio 5). In the analysis of PCR results, ΔΔCt values were calculated to compare gene expression.

The blank was cultured under the same conditions as the sample by adding the same amount of distilled water. The control group was cultured under the same conditions as the sample by adding the same amount of panthenol. Based on the fact that the mRNA expression level of TGM-1 measured in the blank was 1, the mRNA expression level of TGM-1 measured in the sample and control group was measured as a relative value, and the increase/decrease ratio (effect, %) for the relative value was measured. . The increase/decrease ratio for the relative value of the mRNA expression level of TGM-1 is calculated by subtracting the mRNA expression level of TGM-1 measured in the blank from the mRNA expression level of TGM-1 (relative value) measured in each sample. The value was divided by the mRNA expression level of TGM-1 measured in the blank and calculated as a percentage value multiplied by 100.

Transglutaminase-1 (TGM-1) is mainly expressed in the spinous layer, granular layer, and basal layer of the epidermis and is a key gene in the composition of the skin barrier involved in epidermal differentiation. Therefore, the higher the mRNA expression level of TGM-1 is measured, the better. It can be evaluated as having the effect of strengthening the skin barrier.

division Concentration (㎍/mL) LOR mRNA expression level (relative value, %) mRNA expression level of TGM-1 (relative value, %) Manufacturing Example 1 200 56 180 Comparative Example 1 200 2 -12 500 -50 -80

As a result of the experiment, it was confirmed that in Preparation Example 1, the mRNA expression level of LOR increased by 56% compared to the blank, and the mRNA expression level of TGM-1 increased by 180% compared to the blank. That is, Preparation Example 1 was found to have excellent skin barrier strengthening efficacy.

Meanwhile, in Comparative Example 1, at a concentration of 200 μg/mL, the mRNA expression level of LOR was almost similar to that of the blank, and in particular, the mRNA expression level of TGM-1 was confirmed to be decreased compared to the blank. In addition, at a concentration of 500 μg/mL, the mRNA expression levels of both LOR and TGM-1 were confirmed to be decreased compared to the blank. That is, unlike Preparation Example 1, Comparative Example 1 did not contain robinin, so it was found that the skin barrier strengthening effect was also minimal.

Experimental Example 3: Analysis of the efficacy of robinin as a single ingredient in moisturizing the skin and strengthening the skin barrier

(1) Experimental example: cytotoxicity test

A cytotoxicity test was conducted on the single component of robinin in the same manner as in Experimental Example 2, and the results are shown in Figure 7. Specifically, Figure 7 is a graph showing the cell proliferation (%) for robinin at each concentration (unit: ppm).

As a result of the experiment, referring to FIG. 7, robinin can be evaluated as having no cytotoxicity at 20 ppm or less.

(2) Experimental example: moisturizing efficacy test

A moisturizing efficacy test was performed by measuring the mRNA expression levels of HAS2 and AQP3 for the single component robinin, and the results are shown in Table 6 and Figures 8a and 8b. Specifically, Figure 8a is a graph showing the mRNA expression level (relative value) of HAS2 with respect to robinin, and Figure 8b is a graph showing the mRNA expression level (relative value) of AQP3 with respect to robinin.

The method for measuring the mRNA expression level of HAS2 is the same as Experimental Example 2, and the method for measuring the mRNA expression level of AQP3 is as follows.

The HaCaT cell line was dispensed at an appropriate concentration of 4 mL per well into a 60 mm dish and cultured in a 37°C, 5% CO ₂ incubator for 24 hours. After serum starvation was performed on the cultured cells for 24 hours, samples were treated at different concentrations and cultured for 24 hours under 37°C and 5% CO ₂ incubator conditions. After completion of incubation, RNA was extracted by treatment with 1 mL of Trizol (Invitrogen), and RNA was condensed and washed using chloroform, 2-propanol, 75% EtOH, and a centrifuge. After drying the RNA, an appropriate amount of nuclease free water was added and quantification was performed using Nano drop (ALLSHENG). After preparing each RNA to the same concentration, reverse transcription was performed using a cDNA Synthesis Kit (PhileKorea). After mixing the synthesized cDNA, Primer (AQP3 Oligonucleotide), SYBR green, and Nuclease free in an appropriate ratio, RT-qPCR was performed using Real time PCR equipment (Applied Biosystem, Quantstudio 5). In the analysis of PCR results, ΔΔCt values were calculated to compare gene expression.

The blank was cultured under the same conditions as the sample by adding the same amount of distilled water. The control group was cultured under the same conditions as the sample by adding the same amount of panthenol. Based on the fact that the mRNA expression level of AQP3 measured in the blank was 1, the mRNA expression level of AQP3 measured in the sample and control group was measured as a relative value, and the increase/decrease ratio (effect, %) for the relative value was measured. The increase/decrease rate of the relative value of the mRNA expression level of AQP3 is calculated by subtracting the mRNA expression level of AQP3 measured in the blank from the mRNA expression level of AQP3 (relative value) measured in each sample, and then measuring the subtracted value on the blank. The percentage value was calculated by dividing by the mRNA expression level of AQP3 and multiplying by 100.

AQP3 (aquaporin3) is a transport protein that actively moves water molecules from the cell membrane into the cell. Since it plays an important role in moisturizing the skin and healing wounds, the higher the mRNA expression level of AQP3 is measured, the better moisturizing efficacy can be evaluated. .

division Concentration (ppm) mRNA expression level of HAS2 (relative value, %) AQP3 mRNA expression level (relative value, %) Robinin 5 - 27 10 46 -

As a result of the experiment, it was confirmed that robinin as a single component increased the mRNA expression levels of HAS2 and AQP3 compared to the blank. In other words, it was found that robinin has excellent moisturizing efficacy.

(3) Experimental example: barrier efficacy test

A barrier efficacy test was performed by measuring the mRNA expression levels of FLG and LOR for the single component robinin, and the results are shown in Table 7 and Figures 9a and 9b. Specifically, Figure 9a is a graph showing the mRNA expression level (relative value) of FLG with respect to robinin, and Figure 9b is a graph showing the mRNA expression level (relative value) of LOR with respect to robinin.

The method for measuring the mRNA expression level of LOR is the same as Experimental Example 2, and the method for measuring the mRNA expression level of FLG is as follows.

The HaCaT cell line was dispensed at an appropriate concentration of 4 mL per well into a 60 mm dish and cultured in a 37°C, 5% CO ₂ incubator for 24 hours. After serum starvation was performed on the cultured cells for 24 hours, samples were treated at different concentrations and cultured for 24 hours under 37°C and 5% CO ₂ incubator conditions. After completion of incubation, RNA was extracted by treatment with 1 mL of Trizol (Invitrogen), and RNA was condensed and washed using chloroform, 2-propanol, 75% EtOH, and a centrifuge. After drying the RNA, an appropriate amount of nuclease free water was added and quantification was performed using Nano drop (ALLSHENG). After preparing each RNA to the same concentration, reverse transcription was performed using a cDNA Synthesis Kit (PhileKorea). After mixing the synthesized cDNA, Primer (FLG Oligonucleotide), SYBR green, and Nuclease free in an appropriate ratio, RT-qPCR was performed using Real time PCR equipment (Applied Biosystem, Quantstudio 5). In the analysis of PCR results, ΔΔCt values were calculated to compare gene expression.

The blank was cultured under the same conditions as the sample by adding the same amount of distilled water. The control group was cultured under the same conditions as the sample by adding the same amount of panthenol. Based on the fact that the mRNA expression level of FLG measured in the blank was 1, the mRNA expression level of FLG measured in the sample and control group was measured as a relative value, and the increase/decrease ratio (effect, %) with respect to the relative value was measured. The increase/decrease rate of the relative value of the mRNA expression level of FLG is calculated by subtracting the mRNA expression level of FLG measured in the blank from the mRNA expression level (relative value) of FLG measured in each sample, and then measuring the subtracted value on the blank. The percentage value was calculated by dividing by the mRNA expression level of FLG and multiplying by 100.

FLG (filagrin) is a protein contained in keratinocytes that strengthens the weakened skin barrier and generates natural moisturizing factor (NMF). Therefore, the higher the mRNA expression level of FLG is measured, the more effective it is in strengthening the skin barrier. It can be evaluated as having.

division Concentration (ppm) FLG mRNA expression level (relative value, %) LOR mRNA expression level (relative value, %) Robinin 5 20 - 10 51 56 20 28 One

As a result of the experiment, it was confirmed that robinin as a single ingredient increased the mRNA expression levels of FLG and LOR compared to the blank, and in particular, at a concentration of 10 ppm, the mRNA expression levels of FLG and LOR both increased by more than 50%. In other words, it was found that robinin has an excellent skin barrier strengthening effect.

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

Claims (11)

Contains robinin as an active ingredient,
The robinin is derived from acacia ( Robinia pseudoacacia ) flower extract,
The acacia flower extract is obtained by extracting acacia flowers with at least one of ethanol, methanol, and water,
A cosmetic composition for moisturizing skin and strengthening the skin barrier, wherein the concentration of ethanol is 60 to 90% (v/v). delete According to claim 1,
The acacia flower extract is a cosmetic composition for moisturizing skin and strengthening the skin barrier, including 3% by weight or more of robinin based on the total weight of the acacia flower extract. delete delete According to claim 1,
A cosmetic composition for moisturizing the skin and strengthening the skin barrier, wherein the extraction is performed for 2 to 4 days. According to claim 1,
A cosmetic composition for moisturizing skin and strengthening the skin barrier, wherein the extraction is repeated 2 to 4 times. According to claim 1,
A cosmetic composition for moisturizing skin and strengthening the skin barrier, wherein the extraction is performed at room temperature. According to claim 1,
A cosmetic composition for moisturizing the skin and strengthening the skin barrier, wherein the robinin is in the form of a liquid or a concentrated and dried powder. Contains robinin derived from acacia flower extract as an active ingredient,
The extract is obtained by extracting acacia flowers with at least one of ethanol, methanol, and water,
A composition for external application for skin for moisturizing the skin and strengthening the skin barrier, wherein the concentration of ethanol is 60 to 90% (v/v). Contains robinin derived from acacia flower extract as an active ingredient,
The extract is obtained by extracting acacia flowers with at least one of ethanol, methanol, and water,
A pharmaceutical composition for the treatment or prevention of dry skin disease, wherein the ethanol concentration is 60 to 90% (v/v).