KR102287728B1 - Composition for protecting skin from ultraviolet ray comprising Syringa dilatata callus extract as effective component - Google Patents

Abstract

The present invention relates to a composition for protecting the skin against ultraviolet rays containing a millet flower callus extract as an active ingredient, wherein the millet flower callus extract inhibits cell death induced by ultraviolet irradiation, It increases the expression of collagen types I and III (pro-collagen type I, III) and has the effect of significantly reducing the synthesis of MMP-1, 2 and 3 increased by UV irradiation, so the extract of sorghum flower as an active ingredient The composition of the present invention containing as can be usefully used as a material for functional cosmetics or health functional food for preventing and improving skin damage caused by ultraviolet rays.

Description

Composition for protecting skin from ultraviolet ray comprising Syringa dilatata callus extract as effective component

The present invention relates to a composition for protecting the skin against ultraviolet rays containing an extract of sorghum callus as an active ingredient.

The phenomenon of pursuing 'well-being', which greatly affects modern society, has not only the primary meaning of 'eat well and live well', but also the meaning of 'the health of the body is expressed through the beauty of the skin'. This phenomenon is also frequently seen in the field of skin care, and the approach for skin beauty now requires a complex function and a scientific approach rather than pursuing a fragmentary effect.

Skin aging can be broadly divided into two types. One of them is intrinsic aging, which refers to the aging phenomenon that cannot be avoided with the passage of time. The second is photoaging, which refers to the aging phenomenon observed in the skin of the face, back of hands, and back of the neck exposed to sunlight for a long time.

Pores are distributed throughout the skin of the face, but in particular, the nose and cheeks can be identified with the naked eye. The reason for the enlarged pores is that the skin loses elasticity and sags as the collagen fibers and elastic fibers that support the pore walls degenerate and decrease as sebum is secreted excessively or skin aging begins.

It is known that the cause of such skin aging is that matrix proteins such as collagen and elastic fibers are damaged, the amount of collagen in the skin is insufficient, and elastic fibers are denatured. In addition, during the process of skin aging, collagen synthesis is reduced through various signal transduction pathways, and the expression of matrix metalloproteinases (MMPs), an enzyme that decomposes extracellular matrix proteins including collagen, is increased. known As such, collagen is recognized as a major factor related to skin aging, and collagen synthesis enhancement and decomposition inhibition are important indicators in relation to skin aging inhibition and skin beauty.

As such, various studies are being conducted to enhance collagen synthesis and improve skin beauty by inhibiting its decomposition, and in particular, natural products having relatively superior skin stability compared to compounds are attracting attention.

On the other hand, Syringa dilatata is a plant that mainly grows in South Pyongan Province, South Hamgyong Province, and Hwanghae Province. The leaves are opposite and heart-shaped, and the upper side is dark green and the back side is yellowish green. The flowers bloom in spring and are usually lotus purple, and the corolla has a long tube and has a good fragrance. There are two stamens, attached to the flower tube, and there is only one pistil. The fruit is a capsule (a fruit divided into several compartments and each compartment has many seeds) and ripens in early autumn.

The sorghum flower is a Korean lilac that is often planted for ornamental purposes. There are 7 species, such as cloves and flowering trees, that are similar in shape to sorghum peduncle, and about 30 species are growing in Europe.

As a prior art related to the skin protection effect of ingredients derived from natural products, Korean Patent No. 0734040 discloses a cosmetic composition for skin protection against UV rays containing gingerol as an active ingredient, and Korean Patent No. 1718190 Although a composition for protecting or treating skin cells against ultraviolet rays containing an extract of genus oleifera as an active ingredient has been disclosed, there is no disclosure regarding a composition for protecting the skin against ultraviolet rays containing the syringa flower callus extract of the present invention as an active ingredient.

The present invention has been derived from the above needs, and has completed the present invention by providing a millet flower callus extract and confirming the cellular protection effect and wrinkle improvement effect of the millet flower callus extract due to UV damage.

In order to solve the above problems, the present invention provides a cosmetic composition for protecting the skin against ultraviolet rays containing an extract of sorghum callus as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving skin damage caused by ultraviolet rays containing an extract of sorghum callus as an active ingredient.

The present invention relates to a composition for protecting the skin against ultraviolet rays containing a millet flower callus extract as an active ingredient, wherein the millet flower callus extract inhibits cell death induced by ultraviolet irradiation, It increases the expression of collagen types I and III (pro-collagen types I and III), and has the effect of significantly reducing the synthesis of MMP-1, 2 and 3 increased by UV irradiation.

1 is a result of confirming the cytotoxicity of a sorghum callus extract. Blank is an untreated control, and NAC (acetylcysteine, N-Acetyl-L-Cysteine) is a positive control.
Figure 2 is the result of confirming the cell death inhibitory ability of the sorghum callus extract by ultraviolet irradiation. Blank is an untreated control group, CON is a control group that is not treated with anything after UV irradiation, and NAC (acetylcysteine, N-Acetyl-L-Cysteine) is a positive control group treated with NAC after UV irradiation. *** means that the cell viability of CON was statistically significantly decreased compared to Blank, and p<0.001. ## and ### mean that the cell viability of the NAC or sorghum callus extract treatment group increased statistically significantly compared to CON, ## is p<0.01, ### is p<0.001.
Figure 3 is a result confirming the expression change of pro-collagen type I (A) and MMP-1 (B) induced by ultraviolet irradiation when treated with a millet flower callus extract. Blank is an untreated control, CON is a control that is not treated with anything after UV irradiation, and NAC (acetylcysteine, N-Acetyl-L-Cysteine) is a positive control treated with NAC after UV irradiation. *** means that the expression of pro-collagen type I in CON was statistically significantly decreased or the expression of MMP-1 was statistically significantly increased compared to Blank, and p<0.001. #, ### means that the expression of pro-collagen type I in the NAC or sorghum callus extract treated group was significantly increased or the expression of MMP-1 was statistically significantly decreased compared to CON, # is p<0.05, ### is p<0.001.
4 is a pro-collagen type I (COL-1), Ⅲ (COL-3) (A) and MMP-1, 2, 3 (B) proteins induced by ultraviolet irradiation when treated with a millet flower callus extract. It is the result of confirming the expression level. Blank is an untreated control, CON is a control that is not treated with anything after UV irradiation, and NAC (acetylcysteine, N-Acetyl-L-Cysteine) is a positive control treated with NAC after UV irradiation.

The present invention relates to a cosmetic composition for protecting the skin against ultraviolet rays containing an extract of sorghum callus as an active ingredient.

The millet flower callus can be formed from millet leaf, stem, petiole, root, petal, etc., and there is no particular limitation on the site of callus formation, but preferably, a millet flower petal section is used to form a callus. , more preferably 1 to 5% (w / v) sucrose, 80 to 120 mg / L myo-inositol, 0.2 to 0.6 mg / L thiamine, 0.2-0.6% (w/v) gelrite and 0.5-1.5 mg/L 2,4-D (2,4-Dichlorophenoxyacetic acid) as a plant growth regulator can be used to form MS medium. , more preferably, 3% (w / v) sucrose, 100 mg / L myo-inositol, 0.4 mg / L thiamine, 0.4% (w / v) ) Gelrite and 1mg/L 2,4-D (2,4-Dichlorophenoxyacetic acid) as a plant growth regulator is formed using MS medium added, but is not limited thereto.

The extraction solvent of the millet flower callus extract may be water, a C ₁ ~ C ₄ lower alcohol or a mixture thereof, preferably ethanol, but is not limited thereto.

The composition inhibits apoptosis of fibroblasts by ultraviolet light, increases the expression of pro-collagen types I and III reduced by ultraviolet light, and reduces the expression of MMP-1, 2 and 3 increased by ultraviolet light.

The skin protection against UV rays has a skin protection effect by at least one action selected from moisturizing, wrinkle improvement and elasticity enhancement, and protects the skin from UV damage, thereby preventing or improving photoaging, which is skin aging caused by UV rays. can

As used herein, the term 'photoaging' refers to a phenomenon in which the skin is wrinkled by exposure to a large amount of ultraviolet rays frequently and for a long period of time. In addition, pigmentation such as freckles, blemishes, and blemishes may occur, and the capillaries of the skin may increase and the skin may become red.

In one embodiment of the present invention, the cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, a solution, suspension, emulsion, paste, gel, cream, lotion, powder , oil, powder foundation, emulsion foundation, wax foundation and spray, and more specifically, skin, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage Cream, nourishing cream, eye cream, moisture cream, hand cream, essence, nourishing essence, pack, cleansing foam, cleansing water, cleansing lotion, cleansing cream, body lotion, body cleanser, soap and powder may be manufactured in cosmetic formulations. The present invention is not limited thereto.

When the formulation of the cosmetic composition of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide, etc. This can be used.

When the formulation of the cosmetic composition of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydro It may contain a propellant 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, solubilizer or emulsifier is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene fatty acid esters of glycol, 1,3-butylglycol oil, glycerol fatty esters, polyethylene glycol or sorbitan.

When the formulation of the cosmetic composition of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester , microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracanth may be used.

The cosmetic composition of the present invention includes ingredients commonly used in cosmetic compositions in addition to the active ingredients, for example, fatty substances, organic solvents, solubilizers, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents ( foaming agents), fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents , conventional adjuvants such as lipid vesicles, and carriers.

In addition, the present invention relates to a health functional food composition for preventing or improving skin damage caused by ultraviolet rays, which contains an extract of sorghum flower as an active ingredient.

The health functional food composition may be prepared in any one formulation selected from powder, granule, pill, tablet, capsule, candy, syrup and beverage, but is not limited thereto.

When the health functional food composition of the present invention is used as a food additive, the health functional food composition may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The amount of the active ingredient may be appropriately used depending on the intended use thereof. In general, in the production of food or beverage, the health functional food composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the raw material. However, in the case of long-term ingestion for health purposes, the amount may be less than or equal to the above range, and may be used in an amount greater than or equal to the above range within a range where there is no problem in terms of safety.

There is no particular limitation on the type of the health functional food. Examples of foods to which the health functional food composition can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea There are drinks, alcoholic beverages, vitamin complexes, and the like, and includes all health foods in the ordinary sense.

In addition, the health functional food composition of the present invention may be prepared as a food, particularly a functional food. The functional food of the present invention includes ingredients commonly added during food production, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when manufactured as a drink, natural carbohydrates or flavoring agents may be included as additional ingredients in addition to the active ingredient. The natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, etc.), oligosaccharides, polysaccharides (eg, dextrin, cyclodextrin, etc.) or sugar alcohols (eg, , xylitol, sorbitol, erythritol, etc.). As the flavoring agent, natural flavoring agents (eg, taumatine, stevia extract, etc.) and synthetic flavoring agents (eg, saccharin, aspartame, etc.) may be used.

In addition to the health functional food composition, various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonic acid It may further contain a carbonation agent and the like used in beverages.

Hereinafter, the present invention will be described in more detail using Preparation Examples and Examples. These preparations and examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited thereto.

Preparation Example 1. Preparation of sorghum callus extract

For the development of plant cell lines through establishment of an in-flight culture system of sorghum flower plants, samples of sorghum flower plants collected in Korea were prepared. To secure sterile samples for in-flight culture, remove contaminants from the collected sorghum flower plants, wash them under running water for 30 minutes to 1 hour, drain the water, cut the petals, put them in a glass culture bottle, 70 Disinfect with shaking in % (v/v) ethanol solution for 1 minute, wash 1 or 2 times with sterile water, and dilute commercial sodium hypochlorite solution with 0.4-0.8% (v/v). A sterile sample was prepared by putting it in the solution and sterilizing the surface for 20 minutes while shaking it well.

The surface sterilized sorghum flower sample removes the remaining moisture with sterilized filter paper, then cuts it into an appropriate size of about 1 cm in width and length within a sterile work surface, prepares sections, and then punctures 3 repetitions of 10 each on a previously prepared culture medium. It was cultured at 25°C in dark conditions.

As a result of the previous experiment, the culture medium was 3% (w/v) sucrose, 100 mg/L myo-inositol, 0.4 mg/L in MS medium (Murashige and Skoog, 1962) with excellent callus induction efficiency. After sterilizing a medium containing thiamine, 0.4% (w/v) gelrite, and 1 mg/L 2,4-D (2,4-Dichlorophenoxyacetic acid) as a plant growth regulator at 120° C. for 15 minutes used for culture.

The sorghum flower plant section samples began to form white callus as they expanded at the tissue cut surface from 2 weeks after the initiation of culture. After culturing for 4 weeks, the size of the structure was extended with proliferation of the white callus, reaching about 1 mm in size. After 6 weeks, it was observed that the formed multiple callus became more active. The callus was subcultured in a new medium of the same composition at a cycle of 4 weeks to obtain a millet flower stem-derived plant cell line (BP1347304, hereinafter referred to as millet flower callus) (KCTC No. PC4392).

After that, the millet flower callus was dried and powdered, and then 3.5 mL of 100% ethanol was added to 50 mg of the millet flower callus dry powder, followed by sonication at room temperature (25±5° C.) for 1 hour to extract. . The sorghum callus ethanol extract was centrifuged at 5,000 rpm at 10° C. for 5 minutes, the supernatant was taken and concentrated using a nitrogen concentrator, and then used as a sample.

Example 1. Confirmation of cytotoxicity and cytoprotective effect of sorghum callus extract by UV irradiation

Human fibroblast Hs68 (CRL-1635) cells were purchased from ATCC (ATCC, Manassas, VA, USA), and 10% (v/v) fetal calf in DMEM (Dulbecco's modified Eagle medium, Gibco, Grand Island, NY). After the addition of serum (fetal bovine serum, Gibco, Grand Isaland, NY) and 1% (v/v) penicillin, it was cultured at _{37° C., 5% CO 2 in an incubator.}

MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide to confirm the cytotoxicity and cytotoxic effect of the ethanol extract of millet flower callus in the cultured Hs68 cells and UV irradiation ) was analyzed.

After the cells ^{were dispensed in a 96-well cell culture vessel at 1×10 4} cells/mL and cultured for 24 hours for stabilization, the cells were replaced with serum-deficient DMEM medium and cultured for 24 hours to equalize the cell cycle. After that, the sorghum callus ethanol extract was treated at different concentrations (100, 250 and 500 μg/mL) for 24 hours, washed twice with PBS, and then a small amount of PBS was added and a UV crosslinker (Analytik Jena) AG, Jena, Germany) was irradiated with UVB 100 mJ/cm 2 . Immediately after UV irradiation, serum-deficient DMEM medium containing ethanol extract of sorghum callus by concentration was added and cultured for 24 hours. Control cells were treated with the same medium lacking serum without UVB irradiation. Thereafter, MTT reagent at a concentration of 5 mg/mL was put into each well, incubated for 4 hours, the supernatant was removed, and 100 μl DMSO (dimethyl sulfoxide) was added to dissolve formazan crystals, and then microplate reader (microplate). The absorbance was measured at 590 nm using a reader, Multiskan Go, Thermo Scientific, Waltham, MA, USA).

As a result, cytotoxicity was not observed even when 500 μg/mL of sorghum callus extract was treated as shown in FIG. 1 .

As a result of confirming the cytoprotective effect of UV irradiation within the range in which cytotoxicity does not appear, it was confirmed that the cell viability decreased by UV irradiation as shown in FIG. 2 was recovered upon treatment with the sorghum callus extract. Through this, it was confirmed that the sorghum callus extract can protect skin cells from cell damage caused by UV irradiation.

Example 2. Wrinkle improvement effect of sorghum callus extract (pro-collagen type I and MMP-1 expression change)

Pro-collagen, which is related to skin adhesion and elasticity, is a component of collagen and is synthesized in fibroblasts. In the case of skin photoaging due to UV exposure, the reduction of type I and III collagen is accelerated, the destruction of collagen is caused by MMPs, and matrix metalloproteinases-1 (MMP-1) and MMP-3, which are directly related to skin aging, are UV-rays. reported to increase by

Therefore, in the present invention, how the expression of pro-collagen type I and MMP-1 induced from UV-induced skin damage is changed in the fibroblast supernatant with human MMP-1 and human pro-collagen type I ELISA kit (Abcam, Cambridge, UK) ) was used for measurement. The fibroblast supernatant was centrifuged at 12,000 rpm for 10 minutes, and the experiment was performed according to the manufacturer's instructions.

As a result, as shown in FIG. 3 , the content of pro-collagen type I inhibited by UVB irradiation was increased in a concentration-dependent manner upon treatment with the syringa flower callus extract. On the other hand, the expression of MMP-1 increased by ultraviolet irradiation was effectively inhibited by the treatment of the ethanol extract of millet flower callus.

Example 3. Wrinkle improvement effect of millet flower callus extract (change in intracellular pro-collagen and MMPs protein expression)

Western blot analysis was performed on pro-collagen proteins and MMP enzymes involved in the intracellular signal transduction process induced by UV light.

Cells in cold RIPA buffer (10 mM Tris-HCl, pH 7.5, 0.1% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 1 mM sodium orthovandate, 120 mM sodium chloride, 1 mM phenylmethylsulfonyl floride, 10 μg/mL leupeptin, 1 μg/mL aprotonin) was added and dissolved, and the supernatant was separated by centrifugation at 10,000Xg for 10 minutes.

After protein quantification, the same amount of protein (20㎍/mL) was electrophoresed using SDS-polyacrylamide gel and then transferred to a PVDF membrane (Bio-Rad Lab., Hercules, CA, USA). ) was done. After blocking the non-specific binding site for 1 hour in 5% (w/v) skim milk (PBST) solution containing 0.1% (v/v) Tween 20, 1 After reacting for 16 hours or more (overnight) with a primary antibody [MMPs, COLs (Cell Signaling, Beverly, MA)], and reacting for 2 hours at room temperature with a secondary antibody corresponding to the primary antibody, protein expression using an ECL solution The amount was measured (amersham imager 600; GE Healthcare, Buckinghamshire, UK).

As a result, as shown in FIG. 4 , the protein content of pro-collagen types I and III inhibited by UVB irradiation was increased when the millet flower callus extract was treated at a concentration of 100 to 500 μg/mL. On the other hand, the expression of MMP-1, 2 and 3 increased by UV irradiation was effectively inhibited by the treatment of the ethanol extract of millet flower callus.

Therefore, from the above results, it was confirmed that MMP protein expression was reduced by the ethanol extract of millet flower callus, and that there was an effect of protecting collagen fibers.

Claims (6)

It contains sorghum callus extract as an active ingredient, suppresses apoptosis of fibroblasts by ultraviolet rays, enhances the expression of pro-collagen types I and III, and reduces the expression of MMP-1, 2 and 3 A cosmetic composition for skin protection against UV rays. According to claim 1, wherein the sorghum callus is 1-5% (w/v) sucrose, 80-120 mg/L myo-inositol, 0.2-0.6 mg of sorghum flower petal fragments MS medium supplemented with /L thiamine, 0.2-0.6% (w/v) gelrite, and 0.5-1.5 mg/L 2,4-D (2,4-Dichlorophenoxyacetic acid) as a plant growth regulator A cosmetic composition for skin protection against ultraviolet rays, characterized in that it was formed using The cosmetic composition for skin protection against ultraviolet rays according to claim 1, wherein the extraction solvent of the sorghum callus extract is water, C ₁ ~ C _{4 lower alcohol, or a mixture thereof.} delete The cosmetic composition for skin protection against UV rays according to claim 1, wherein the skin protection is performed by at least one effect selected from moisturizing, wrinkle improvement, and elasticity enhancement. It contains sorghum callus extract as an active ingredient, suppresses apoptosis of fibroblasts by ultraviolet rays, enhances the expression of pro-collagen types I and III, and reduces the expression of MMP-1, 2 and 3 A health functional food composition for preventing or improving skin damage to UV rays.

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