KR102606380B1 - Composition for enhancing skin barrier containing Bolboschoenus planiculmis extract - Google Patents

Abstract

The present invention relates to a composition containing an extract of Saeseommae porcelain, wherein the extract induces differentiation of keratinocytes, skin barrier enhancers such as Occludin and Claudin-1, and keratin 10 ( It can be easily used as a cosmetic composition for strengthening the skin barrier by activating differentiation-inducing factors of the skin barrier composition, such as Keratin 10), Filaggrin, Involucrin, and Loricrin.

Description

Cosmetic composition for enhancing skin barrier containing extract of Saeseommae porcelain {Composition for enhancing skin barrier containing Bolboschoenus planiculmis extract}

The present invention relates to a cosmetic composition for strengthening the skin barrier containing extract of Saeseommae porcelain.

Keratinocytes are newly created according to a certain cycle and continuously divide and regenerate on their own. As time goes by, they die and fall off, and are replaced by newly created cells. The normal skin regeneration cycle is 28 days, but this lasts until the mid-20s and increases to 4 to 90 days after the age of 40. Keratinocytes make up most of the epidermis, and as new cells are generated in the basal layer, some are pushed upward and move toward the stratum corneum and begin cell differentiation. As the differentiated cells form a uniform keratinized layer, the skin barrier becomes stronger. As it falls off through peeling, it shows a skin protection effect. This skin regeneration cycle is called turnover. The normal turnover cycle forms a solid barrier structure of the skin, and the differentiation process of keratinocytes is closely related to skin barrier factors. Therefore, a search process is being conducted for various natural products that activate skin barrier factors within keratinocytes.

Accordingly, while screening various natural products for the activity of skin barrier factors, the present inventors confirmed that the Saeseommae china extract has the effect of activating skin barrier factors by acting on the differentiation process of skin keratinocytes, and developed a cosmetic composition for skin improvement. By confirming that it can be used, the present invention was completed.

Republic of Korea Registered Patent No. 10-1914656 (Title of invention: Composition for preventing or treating allergic diseases containing extract of Saeseommae china, Applicant: Konkuk University Glocal Industry-Academic Cooperation Foundation, Date of registration: 2017.07.07.)

Medical Newspaper, ‘Halophyte ‘Saeseommaegi’ whitening and wrinkle improvement efficacy discovered’, 2023.02.10

The purpose of the present invention is to provide a cosmetic composition for strengthening the skin barrier containing Saeseommae porcelain extract.

The present invention relates to a cosmetic composition for strengthening the skin barrier containing extract of Saeseommae porcelain.

The extract can be extracted from Saeseommae porcelain using water, C1 to C4 alcohol, or a mixed solution thereof as a solvent.

The extract is characterized by enhancing the expression of one or more types selected from occludin and Claudin-1, which are adhesive proteins constituting the skin barrier in keratinocytes.

The extract is characterized by inducing skin differentiation of keratinocytes. Preferably, the extract is selected from the group consisting of Keratin 10, Filaggrin, Involucrin, and Loricrin, which are differentiation-inducing factors that constitute the skin barrier in keratinocytes. It is characterized by enhancing the expression of one or more types.

More preferably, the Saeseommaegi extract is characterized by enhancing the expression of one or more genes selected from Occludin and Claudin-1 by 1.5 to 2 times at 100 to 200㎍/㎖. . In addition, the Saeseommae porcelain extract is characterized in that at least one selected from the group consisting of Keratin 10, Filaggrin, Involucrin, and Loricrin is enhanced by about 1.5 to 4.0 times. Do it as

The extract can be extracted from Saeseommaegi using water, C1~C4 alcohol, or a mixed solution thereof as a solvent, and the C1~C4 alcohol may be selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, and isobutanol. You can. The extract may also be water or 30-90% (v/v) alcohol aqueous solution extract, preferably water or 50-80% (v/v) alcohol aqueous solution extract.

Water, C1 to C4 alcohol, or a mixed solution thereof used in the production of the Saeseommae porcelain extract can be used in an amount 1 to 40 times the volume (1 to 40 liters per 1 kg) or 1 to 40 times the weight based on the weight of the Saeseommae porcelain, and is preferred. In other words, 5 to 20 times the volume or 5 to 20 times the weight can be used. The extraction conditions for the Saeseommae china extract may be 4 to 8 hours at 40 to 70°C. The above process can be repeated 2 to 3 times.

As a device for extracting the extract, a conventional extraction device, an ultrasonic grinding extractor, or a fractionator can be used. The extract prepared in this way can be dried with hot air, dried under reduced pressure, or freeze-dried to remove the solvent. Additionally, the extract can be purified and used using column chromatography.

The extract may be prepared alone or appropriately using known methods used for separation and extraction of plant components, such as extraction with organic solvents (alcohol, ether, acetone, etc.), distribution of hexane and water, and column chromatography, according to commercial methods. It can be used after fractionation or purification using a combined method.

The formulation of the cosmetic composition may be any formulation commonly manufactured in the art, including essence, lotion, emulsion, pack, hand cream, foot cream, lip balm, lipstick, eye shadow, eyeliner, and eyebrow pencil. , blusher, highlighter, general lotion, skin, cream, serum, beauty soap, softening lotion, medicated lotion, body cleanser, cleansing foam, cleansing lotion, gel, cleansing oil, cleansing cream, shampoo, conditioner, hair treatment, hair You can provide a selection from lotions, cleansing tissues and cleansing water.

More specifically, when the formulation of the cosmetic composition of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, and silica are used as carrier ingredients. , talc or zinc oxide can be used. When the formulation of the cosmetic composition of the present invention is powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as the carrier ingredient. In particular, when the cosmetic composition is a spray, chlorofluorohydride may be used as a carrier ingredient. It may contain propellants such as carbon, propane-butane or dimethyl ether. When the formulation of the cosmetic composition of the present invention is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene. These include fatty acid esters of glycol, 1,3-butylglycol oil, glycerol aliphatic esters, polyethylene glycol or sorbitan. When the formulation of the cosmetic composition of the present invention is a suspension, the carrier component includes water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used. When the formulation of the cosmetic composition of the present invention is a surfactant-containing cleansing agent, the carrier ingredients include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, acethionate, imidazolinium derivative, methyl taurate, and sarcosinate. , fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linoline derivative, or ethoxylated glycerol fatty acid ester can be used. The cosmetic composition of the present invention may further contain excipients including fluorescent substances, fungicides, hydrotropes-inducing substances, moisturizers, fragrances, fragrance carriers, proteins, solubilizers, sugar derivatives, sunscreens, vitamins, plant extracts, etc. . The amount of the above ingredients can be selected depending on the formulation or purpose of use within a range that does not impair the inherent effect of the cosmetic composition. The amount of the ingredients added may be, for example, 0.1 to 10% by weight, preferably 0.1 to 6% by weight, based on the total weight of the composition, but is not limited thereto.

The present invention relates to a composition containing an extract of Saeseommae porcelain, wherein the extract induces differentiation of keratinocytes, skin barrier enhancers such as Occludin and Claudin-1, and keratin 10 ( It can be easily used as a cosmetic composition for strengthening the skin barrier by activating differentiation-inducing factors of the skin barrier composition, such as Keratin 10), Filaggrin, Involucrin, and Loricrin.

Figure 1 is a graph of the cytotoxicity results of the Saeseommaegi extract of the present invention.
Figure 2 is a photograph of the Western block results confirming the expression levels of Keratin 10, Filaggrin, Involucrin, and Loricrin in keratinocytes of the Saeseommae china extract of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, it is provided to ensure that the content introduced here is thorough and complete, and to sufficiently convey the spirit of the present invention to those skilled in the art.

<Example 1. Preparation of Saeseommae porcelain extract>

The extract sample was prepared according to the extract preparation method of the Natural Products Bank of the National Institute of Biological Resources' Biological Materials Research Group. Fresh whole shoots of Bolboschoenus planiculmis are dried in the shade and cut. The sample was extracted twice for 5 hours at 50°C while shaking with 10 times the weight of the sample in a 70 (v/v)% ethanol aqueous solution. Afterwards, the extract was concentrated under reduced pressure to obtain an extract sample.

<Example 2. Cell culture>

Human epidermal keratinocytes (KC) supplied by Life Technologies (Carlsbad, CA) were cultured under non-differentiating conditions using serum medium containing 0.07mM calcium chloride and growth supplements (Invitrogen, Carlsbad, CA). .

Afterwards, to induce differentiation of KCs cultured under non-differentiating conditions, the medium was replaced with 2mM calcium chloride and further cultured for up to 3 days to construct a KC differentiation model. The results of repeating the same experiment a total of three times were statistically processed.

<Example 3. Cytotoxicity evaluation>

An evaluation of the skin cell toxicity of Saeseommae porcelain extract was conducted. After treating Saeseommaegi extracts at different concentrations and culturing them for 24 hours, cytotoxicity was measured using a WST-1 formazan dye-based cell viability assay kit (CCK-8) (Dojindo, Rockville, MD).

The WST-1 test method is one of the test methods used to measure survival rate. When succinate-tetrazolium reductase, a dehydrogenase, decomposes tetrazolium salts (WST-1) corresponding to the substrate, This is a test method that applies the principle of producing a coloring substance called formazan. Briefly, 1 × 10 ⁴ keratinocytes were dispensed into a 96-well plate and reacted in a cell incubator for 24 hours. Afterwards, the Saeseommaegi extract was treated with 100 or 200 μg/ml, respectively, and further cultured for 24 hours. 10 mL of WST-1 solution per well was added to the culture medium and reacted in a cell incubator for 2 hours. The degree of formazan production was measured at absorbance at 450 nm. The results of repeating the same experiment a total of 6 times were statistically processed.

The results are shown in Figure 1 and Table 1, and no cytotoxicity was observed at each extract treatment concentration.

division density Average cytotoxicity results (%) S.D. Untreated group (Control) 100 4.51 Saeseommae porcelain extract 100 μg/ml 99.66 3.81 200 μg/ml 98.47 5.84

<Example 4. Expression analysis of skin barrier function-related adhesion proteins and skin keratinocyte differentiation markers>

The expression of junctional proteins (Occludin, Claudin-1) and keratinocyte differentiation markers (Keratin 10, Filaggrin, Involucrin, Loricrin), known as indicators of skin barrier function, were confirmed in keratinocytes.

Among these, adapter proteins are known to be mainly expressed before differentiation of keratinocytes, and differentiation markers are known to be expressed at the end of cell differentiation, so the state of keratinocytes was prepared in detail through treatment with CaCl ₂ .

To confirm the expression of adapter proteins, 100 μg/ml of Saeseommae china extract was treated for 24 hours to prepare conditions for non-differentiation of keratinocytes, and 100 μg/ml of Saeseommae china extract was treated with 2mM CaCl ₂ for 24 hours to prepare differentiation conditions. Time was processed.

To confirm differentiation markers, CaCl ₂ 2mM and Saeseommaegi extract 200μg/ml were treated for 3 days.

Afterwards, the expression of each marker was analyzed using Western blotting. To explain this in more detail, keratinocytes were lysed in RIPA buffer containing protease inhibitor and phosphatase inhibitor (Sigma). Total cell lysates were quantified using the BCA protein assay kit (Thermo Scientific). Next, cell lysates (50 μg) were loaded on a 4–15% gradient SDS-PAGE gel (Biorad) under denaturing conditions and transferred to a PVDF membrane (iBlot2 Dry Blotting System, Thermo Scientific). Membranes were blocked with 5% skim milk for 1 h at room temperature and reacted with primary antibodies for BAX, BAD, BCL2, Actin (Santa Cruz), and Cleaved caspase3 (cell signaling) overnight at 4°C. After reacting the HRP-conjugated secondary antibody at room temperature for 2 hours, the membrane was subjected to Western ECL treatment, and the luminescence image was analyzed using LAS500 (GE Healthcare). The results of repeating the same experiment a total of three times were statistically processed. .

Each result is shown in Tables 2 to 6 below, and the differentiation marker results are representatively shown in Figure 2.

Conditions before differentiation of keratinocytes Conjugated protein - Occludin expression (%) average S.D. untreated 100 5.68 Saeseommaegi extract 100μg/ml 185.11 5.47

Differentiation conditions for keratinocytes Conjugated protein - Occludin expression (%) average S.D. CaCl ₂ 2mM (Control) 100 4.96 CaCl ₂ 2mM + Saeseommaegi extract 100μg/ml 121.4 5.83

Conditions before differentiation of keratinocytes Conjugated protein - Claudin-1 expression (%) average S.D. untreated 100 5.68 Saeseommaegi extract 100μg/ml 179.6 3.97

Differentiation conditions for keratinocytes Conjugated protein - Claudin-1 expression (%) average S.D. CaCl ₂ 2mM (Control) 100 3.67 CaCl ₂ 2mM + Saeseommaegi extract 100μg/ml 125.8 5.36

division Expression (%) K10 (Keratin 10) Filaggrin Involucrin Loricrin average S.D. average S.D. average S.D. average S.D. Veh
(CaCl ₂ 2mM) 100 3.82 100 6.17 100 9.62 100 7.77 CaCl ₂ 2mM +
Saeseommaegi
extract
200μg/ml 174.15 10.71 212.85 11.55 363.91 21.64 308.88 6.10

Looking at Tables 2 and 3, it appears that Occludin is more greatly influenced by the extract of keratinocytes before differentiation. This was also proven in Tables 4 and 5 that the expression of Claudin-1 was further enhanced through treatment of Saeseommaegi extract in the pre-differentiation state of keratinocytes.

The results in Table 6 and Figure 2 show that the expression levels of Keratin 10, Filaggrin, Involucrin, and Loricrin were approximately 1.5 to 4.0 due to the treatment of Saeseommaegi extract. It was shown to be significantly enhanced by about a factor of 2, showing that the Saeseommae porcelain extract of the present invention plays a very important role in strengthening the skin barrier.

Claims (5)

A cosmetic composition for strengthening the skin barrier, characterized in that it contains Saeseommae porcelain extract. According to paragraph 1,
The extract is a cosmetic composition for strengthening the skin barrier, characterized in that the extract is extracted from Saeseommaegi using water, C1 to C4 alcohol, or a mixed solution thereof as a solvent. According to paragraph 1,
The extract is a cosmetic composition for strengthening the skin barrier, characterized in that it enhances the expression of one or more types selected from Occludin and Claudin-1, which are adhesive proteins constituting the skin barrier in keratinocytes. According to paragraph 1,
The extract is a cosmetic composition for strengthening the skin barrier, characterized in that it induces skin differentiation of keratinocytes. According to paragraph 1,
The extract contains at least one selected from the group consisting of Keratin 10, Filaggrin, Involucrin, and Loricrin, which are differentiation-inducing factors that constitute the skin barrier in keratinocytes. A cosmetic composition for strengthening the skin barrier, characterized in that it enhances expression.