KR102313083B1 - An anti-oxidative composition comprising an extract of Hydrangea petiolaris or a fraction thereof as an active ingredient - Google Patents

Abstract

The present invention relates to a cosmetic composition and a food composition for antioxidants containing an extract or a fraction thereof as an active ingredient, wherein the extract of Hydrangea petiolaris or a fraction thereof has an antioxidant effect and a skin cell protective effect, It can be used as an active ingredient in cosmetic compositions and food compositions.

Description

An anti-oxidative composition comprising an extract of Hydrangea petiolaris or a fraction thereof as an active ingredient

The present invention relates to an antioxidant cosmetic composition, and food composition containing the rank station (Hydrangea petiolaris) extract or fractions thereof as an active ingredient.

Recently, modern people suffer from various diseases due to environmental pollution and exposure to harmful substances as well as excessive stress and irregular lifestyle. As one of the causes of these diseases, the importance of free radicals is emerging. A significant amount of active oxygen is produced during the oxidation process for energy production in the living body, and these active oxygen are mostly destroyed by the elimination mechanism in the living body. However, when exposed to UV rays, smoking, drinking, or stress, free radicals are excessively or chronically generated. Active oxygen is unstable and highly reactive, so it has the property of easily reacting with various biological materials. Due to this, an appropriate amount of active oxygen has a biological defense function that removes bacteria and viruses that have invaded the body, but an excess of active oxygen attacks cell molecules in the body, and various diseases occur due to damaged cells.

This in order to remove the free radicals are antioxidants developed many synthetic, such as BHA (butylated hydroxyanisole) and BHT (Butylated Hydroxytolune) been used such as functional foods and cosmetics, due to safety problems and has been used in a limited, the food and cosmetics (Korean J Food Preserv , 2012, 19(3), 393-399). Due to this, there is a lot of interest in finding a safer and more effective natural antioxidant.

Korean Patent Publication No. 10-2014-0082555

Under this background, the present inventors have made intensive research efforts to find natural products that exhibit antioxidant effects and have fewer side effects. As a result, extracts and fractions of hydrangea leaves are excellent in extracts and fractions of DPPH (1,1-diphenyl-2-picryl-hydrazyl) free radicals. ) scavenging effect, ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) free radical scavenging effect, skin cell protection effect from oxidative stress, and antioxidant effect, thereby completing the present invention did it

Accordingly, it is an object of the present invention to provide a cosmetic composition for antioxidants containing a leaf extract or a fraction thereof as an active ingredient, Hydrangea petiolaris.

In addition, another object of the present invention is to provide a food composition for antioxidants containing a leaf extract or a fraction thereof as an active ingredient, Hydrangea petiolaris.

According to an aspect of the present invention as described above, the present invention provides an antioxidant cosmetic composition containing extracts or fractions thereof rank station (Hydrangea petiolaris) as an active ingredient, in one embodiment.

The term "Hydrangea petiolaris " of the present invention is a creeper of the family Pantheraceae, and grows about 20 m on rocks and tree trunks by descending the roots from the branches above the ground. It mainly grows in Ulleungdo, Jejudo, and the coasts and mountains of Japan. Flowers bloom in June-July, and form coaxial chrysanthemums with a diameter of 14-25 cm running at the end of branches, with 3-4 sepals like petals on the neutral flower at the edge, sawtooth on the edge, and about 3cm in diameter. . The inner bisexual flower has 5 sepals and petals each, 15-20 stamens, 2-3 styles, and 2-3 ovaries. The fruits are capsules, spherical, with the back part cut flat, truncated, and ripen in brown in September-October. The leaves are opposite, wide egg-shaped or round, with a sharp tip, and the bottom is round or heart-bottomed, with serrated edges on the edge. The length of the petiole is 3~9cm. Propagation is achieved through seed and cuttings propagation, and a large amount of seedlings can be obtained by sowing and seeding seeds on moss.

In the present invention, the cosmetic composition may be applied to extraction of dried and crushed water hydrangea powder, and may include leaves, stems, roots, flowers, or both of water hydrangea, specifically, leaves may be used. can

As used herein, the term "extract" refers to an extract obtained by extraction treatment, a diluted or concentrated liquid of the extract, a dried product obtained by drying the extract, a prepared or purified product of the extract, or a mixture thereof, such as the extract itself and the extract Contains extracts of all formulations that can be formed using

Drying of the extract in the present invention may be carried out by a known method in the range in which useful components from the collected plant are not destroyed, for example, it may be carried out by a method of natural drying in the shade. In addition, crushing or pulverization can be pulverized by crushing or pulverizing to an extent that useful components of plants can be sufficiently extracted in the subsequent extraction process. The drying, crushing, or pulverizing processes may be performed in reverse order or repeated if necessary.

In the extraction of the present invention, the extraction method is not particularly limited, and extraction may be performed according to a method commonly used in the art.

Hydrangea hydrangea ( Hydrangea petiolaris ) in the present invention can be obtained by extracting the extract with water, C ₁ to C ₄ lower alcohol or a mixed solvent thereof. In addition, the lower alcohol may be ethanol or methanol.

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

The fractionation method for obtaining the fraction in the present invention is not particularly limited, and may be performed according to a method commonly used in the art. Non-limiting examples of the fractionation method include a method of obtaining a fraction from the extract by treating an extract of Hydrangea petiolaris with a predetermined solvent.

The kind of solvent used to obtain the fraction in the present invention is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the fractionation solvent include water, alcohol having 1 to 4 carbon atoms, hexane, ethyl acetate, chloroform, dichloromethane, or a mixed solvent thereof.

In an embodiment of the present invention, after drying and crushing the leaves of water hydrangea, extraction was repeated three times with 70% (w/v) ethanol to obtain an ethanol extract of the leaves of water hydrangea, which was n -hexane ( n -hexane, Hex ), ethyl acetate (ethyl acetate, EtOAc), n - butanol (n -butanol, BuOH), water (water, H ₂ O) in sequence fractions in order to a total of four layers of the solvent fraction in normal hexane, ethyl acetate, n A fraction of the butanol, residual water layer was prepared.

In the present invention, the fraction is an n- hexane fraction, an ethyl acetate fraction, an n- butanol fraction or a water fraction.

In addition, the ethanol extract of the hydrangea leaf and the extract of n -hexane ( n -hexane, Hex), ethyl acetate (ethyl acetate, EtOAc), n -butanol ( n -butanol, BuOH), water (water, H ₂ O) It was confirmed that the fraction had an antioxidant effect by scavenging DPPH free radicals and ABTS ^{+ free radicals.} In particular, ethyl acetate (ethyl acetate, EtOAc) and n of rank station ethanol extract-butanol (n -butanol, BuOH) fraction of the DPPH free-radical (free radical) and the ABTS ⁺ scavenging free radicals (free radical) scavenging activity superior confirmed that. In addition, it was confirmed that the human HaCaT cells had a skin cytoprotective effect by inhibiting skin cell damage to oxidative stress induced by hydrogen peroxide.

In the present invention, the ethyl acetate fraction of the hydrangea extract has a skin cell or skin protective effect.

The present invention is provided based on the experimental results, and the cosmetic composition for antioxidant of the present invention is characterized in that it contains an extract of hydrangea or a fraction thereof as an active ingredient. The hydrangea leaf extract and its fractions can be applied as functional cosmetic raw materials, and by using natural plants, side effects and stability caused by synthetic raw materials can be solved.

In addition, in the present invention, the term "active ingredient" means a component that can exhibit the desired activity alone or can exhibit activity together with a carrier that is not itself active.

In addition, in one embodiment of the present invention, as a result of separation and structural identification through MPLC and open column from the ethyl acetate fraction, which had the best antioxidant effect, 6 types of compounds were isolated from the ethyl acetate fraction, and compound 1 was epicatechin to (epi- catechin) with, compound 2 is Hi Perrin (hyperin), compound 3 is a quartz citrine (quercitrin), compound 4 is ill with jerin (afzelin), compound 5 is 1-O- para-one in Kumar -Beta-D-glucopyranoside (1-O-ρ-coumaroyl-β-D-glucopyranoside), compound 6 was identified as asiatic acid, and compoun 7 was identified as corosolic acid. .

Therefore, the rank station extracts or fractions thereof are epicatechin (epi- catechin), Hi Perrin (hyperin), citrine quartz (quercitrin), sick jerin (afzelin), 1-O- para-beta -D- gluconic -yl into Kumano It may include one or more compounds selected from the group consisting of copyranoside (1-O-ρ-coumaroyl-β-D-glucopyranoside), asiatic acid, and corosolic acid.

The compounds were confirmed to have antioxidant effects by scavenging DPPH free radicals and ABTS ⁺ free radicals, and inhibiting skin cell damage due to oxidative stress induced by hydrogen peroxide in human HaCaT cells. Thus, it was confirmed that there is a skin cell protective effect. Accordingly, the compounds have an antioxidant effect or a skin cell protective effect.

As described above, the hydrangea leaf extract, fractions thereof, and compounds isolated therefrom of the present invention can be very usefully used in antioxidant cosmetic compositions.

In the present invention, the term "antioxidant" refers to the action of inhibiting oxidation, in the human body, a prooxidant and an antioxidant are in balance, but this balance is caused by various factors. When an imbalance is achieved and the oxidative stress is induced, potential cellular damage and pathological diseases are caused. Reactive oxygen species (ROS), which is a direct cause of oxidative stress, is unstable and highly reactive, so it easily reacts with various biomaterials and attacks the body's polymers to cause irreversible damage to cells and tissues. NO, HNO ₂ , ONOO ^- such as reactive nitrogen species (RNS), and reactive oxygen species such as ROS oxidize and destroy cells in the body, thereby exposing them to various diseases.

The antioxidant refers to the function of inhibiting the oxidation of cells by free radicals or reactive oxygen species (ROS), which are highly reactive according to oxidative stress caused by intracellular metabolism or ultraviolet rays, It involves the removal of free radicals or reactive oxygen species, thereby reducing damage to cells.

The antioxidant cosmetic composition of the present invention may include a compound or natural extract known to have an antioxidant effect so as to enhance or reinforce the antioxidant effect, in addition to the active ingredient, hydrangea extract or a fraction thereof.

On the other hand, in the cosmetic composition for antioxidant of the present invention, the active ingredient may be included in any amount (effective amount) according to the use, formulation, purpose of mixing, etc. as long as it can exhibit antioxidant activity, and a typical effective amount is the total weight of the composition. It may be included within the range of 0.001 weight % to 99.99 weight % based on the . Here, "effective amount" refers to the amount of an active ingredient capable of inducing an antioxidant effect. Such effective amounts can be determined empirically within the ordinary ability of one of ordinary skill in the art.

The antioxidant cosmetic composition of the present invention may be prepared in various forms, for example, 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, soap, cleansing, oil, powder foundation, emulsion foundation, wax foundation, spray, etc., but is not limited thereto. In addition, specifically, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, essence, nourishing essence, pack, soap, shampoo, It may have a formulation selected from the group consisting of cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, emulsion, lipstick, makeup base, foundation, press powder, and loose powder, but is not limited thereto.

The cosmetic composition for antioxidants of the present invention may include an acceptable carrier in the cosmetic preparation in addition to the active ingredient thereof. Herein, "acceptable carrier for cosmetic preparations" refers to compounds or compositions that are already known and used that can be included in cosmetic preparations, or compounds or compositions to be developed in the future that do not have toxicity, instability, or irritation beyond what the human body can adapt to when in contact with the skin. say that The carrier may be included in the antioxidant cosmetic composition of the present invention in an amount of from about 1% to about 99.99% by weight, preferably from about 5% to about 99% by weight of the total weight of the composition. However, since the above ratio varies depending on the formulation of the cosmetic of the present invention and its specific application site (face or hand) or its desired application amount, the above ratio is intended to limit the scope of the present invention in any aspect. should not be understood

On the other hand, as the carrier, alcohol, oil, surfactant, fatty acid, silicone oil, humectant, humectant, viscosity modifier, emulsion, stabilizer, sunscreen, color developer, fragrance, and the like may be exemplified. Alcohols, oils, surfactants, fatty acids, silicone oils, humectants, humectants, viscosity modifiers, emulsifiers, stabilizers, sunscreens, coloring agents, compounds/compositions, etc. that can be used as the carrier that can be used as the carrier are already known in the art. Therefore, those skilled in the art can select and use an appropriate material/composition.

In another one embodiment the invention provides a food antioxidant composition containing extracts or fractions thereof rank station (Hydrangea petiolaris) as an active ingredient.

In the present invention, Hydrangea petiolaris extract or a fraction thereof, antioxidant, skin cell protective effect, and the description of the compound isolated from the fraction are the same as described above.

Food composition of the present invention is a pill, a powder, a granule, the infiltrant, a tablet, capsule, or may comprise the form of a liquid or the like, of the invention rank station (Hydrangea petiolaris) extract or the type of food that fraction to be added thereof include There are no restrictions. Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, Alcoholic beverages and vitamin complexes are available.

The food composition may be added to the other ingredients in addition to rank station (Hydrangea petiolaris) extracts or fractions thereof, the kind is not particularly limited. For example, it may contain, as an additional component, various herbal extracts, food-logically acceptable food supplements or natural carbohydrates, such as conventional food, but is not limited thereto.

As used herein, the term "food supplement additive" refers to a component that can be supplementally added to food, and is added to prepare food of each formulation, and those skilled in the art can appropriately select and use it. Examples of food supplement additives include various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners , pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, etc., but the above examples are not limited to the types of food supplement additives of the present invention.

Examples of the natural carbohydrate include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Hygin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used.

The food composition of the present invention may include a health functional food. The term "health functional food" as used in the present invention refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients useful in the human body. Here, the term "functionality" refers to obtaining useful effects for health purposes, such as regulating nutrients or physiological effects with respect to the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art. In addition, unlike general drugs, food is used as a raw material, so there are no side effects that may occur when taking the drug for a long time, and it can be excellent in portability.

The mixed amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, in the production of food, the active ingredient of the present invention may be added in an amount of 1 to 50% by weight, preferably 5 to 10% by weight of the raw material composition, but is not limited thereto. However, in the case of long-term ingestion for health and hygiene purposes or for health control, the above amount may be used below the above range.

The health functional food has the advantage of having better antioxidant and skin cell protection effects by ingesting it in the form of inner beauty food. The inner beauty is an inner beauty food called 'eating cosmetics or beauty food'. You can choose and consume the inner beauty food that is right for you, considering your skin condition and lifestyle. More specifically, when mixing the cosmetics containing the cosmetic composition and the inner beauty food containing the Hydrangea petiolaris extract or a fraction thereof, the antioxidant and skin protection effects are significantly increased compared to using only cosmetics, which is more effective am.

According to the present invention, it is possible to prepare a cosmetic composition having an antioxidant effect and a skin cell protective effect, containing a hydrangea leaf extract or a fraction thereof.

1 is a diagram schematically showing a method for preparing an ethanol extract of hydrangea leaves of the present invention and a fraction for each solvent thereof.
Figure 2 is a graph showing the concentration-dependent DPPH free radical scavenging effect of the ethanol extract of hydrangea leaves of the present invention and its solvent-specific fractions.
^{3 is a graph showing the concentration-dependent ABTS +} free radical scavenging effect of the ethanol extract of hydrangea leaves of the present invention and its solvent-specific fractions.
Figure 4 is a graph showing the cell viability of the human HaCaT cells of the ethyl acetate fraction of hydrangea leaves of the present invention.
5 is a graph showing the skin cell protective effect against oxidative stress induced by hydrogen peroxide in human HaCaT cells of the ethyl acetate fraction of hydrangea leaves of the present invention.
6 is a separation diagram schematically showing a method for isolating compounds from the ethyl acetate fraction of hydrangea leaves of the present invention.
7 is a graph showing the DPPH free radical scavenging effect of the compound isolated from the ethyl acetate fraction of hydrangea leaves of the present invention.
^{8 is a graph showing the ABTS +} free radical scavenging effect of the compound isolated from the ethyl acetate fraction of hydrangea leaves of the present invention.
9 is a graph showing the cell viability of the compound isolated from the ethyl acetate fraction of hydrangea leaves of the present invention for human HaCaT cells.
10 is a graph showing the cytoprotective effect of the compound isolated from the ethyl acetate fraction of hydrangea leaves of the present invention against oxidative stress induced by hydrogen peroxide in human HaCaT cells.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

<Experimental Example 1> Preparation of extracts and fractions from hydrangea leaves

The sample used in the present invention, Hydrangea petiolaris leaves, was purchased from Jeju Biological Resources Co., Ltd. in January 2018 and used. After pulverizing 120 g of dried and pulverized hydrangea leaves, it was added to 20 L of 70% (v/v) ethanol (EtOH) and stirred at room temperature for 24 hours. Only the filtrate of the leached sample was collected using a reduced pressure filtration device, and the residue separated in this way was repeated twice more under the same conditions. Then, the filtrate obtained by filtration was concentrated with a rotary evaporator on a water bath at 40° C. or less to obtain 38.5 g of a 70% ethanol extract.

30.0 g of the 70% ethanol extract obtained above was suspended in 1 L of distilled water, and sequentially fractionated according to the polarity order using a separatory funnel, n -hexane ( n -hexane, n- Hex.), ethyl acetate (ethyl acetate, EtOAc) ), n - butanol (n -butanol, n -BuOH) and water (water, H ₂ O) in order to obtain a total of 4 fractions solvent layer (Fig. 1). The resulting fractions were each concentrated under reduced pressure to prepare a powdery hydrangea leaf fraction.

<Experimental Example 2> DPPH radical scavenging activity experiment of hydrangea leaf extract and fractions

2-1. Experimental method

In order to measure the free radical scavenging activity of the 70% ethanol (EtOH) extract and the four solvent fractions obtained in Experimental Example 1, the Blois method (Blois, MS, Nature, 1958, 181; 1199) -1200) was applied to the DPPH radical scavenging activity experiment. At this time, DPPH was used by dissolving in ethanol to a concentration of 0.2 mM.

Specifically, the ethanol extract of hydrangea leaf and its n- hexane ( n- Hex.) fraction, ethyl acetate (EtOAc) fraction, n- butanol ( n- BuOH) fraction, and water (H ₂ O) fraction samples were each 3.125 μg For each concentration of /mL, 6.25 μg/mL, 12.5 μg/mL, 25 μg/mL, 50 μg/mL and 100 μg/mL, it was prepared by dilution using DMSO:EtOH (1:1) solvent.

Then, 180 μl of 0.2 mM DPPH solution and 20 μl of each of the diluted sample solutions were mixed in a 96-well plate and reacted at room temperature for 20 minutes, and then 515 nm using a spectrophotometer. absorbance was measured.

The scavenging rate (%) was calculated by Equation 1 below, and the concentration (SC ₅₀ ; Scavenging concentration of 50%) when each sample inhibited DPPH by 50% was obtained. Each sample was repeated three times to obtain an average value. At this time, vitamin C was used as an antioxidant to compare the activity.

<Equation 1>

Free radical scavenging ability (%) =

A: Absorbance at 515 nm of the reaction solution to which the sample was added

B: Absorbance at 515 nm of sample only

C: Absorbance at 515 nm of the reaction solution to which no sample was added

2-2. Experiment result

The experimental results are shown in Table 1 and FIG. 2 below.

sample SC ₅₀ (μg/mL) EtOH 25.0 n -Hex. 66.2 EtOAc 16.8 n- BuOH 18.0 H ₂ O 87.8 Vitamin C 6.9

As shown in Table 1 and Figure 2, DPPH free radical scavenging activity was shown in both extracts and fractions, and in particular, in the ethyl acetate (EtOAc) fraction and the butanol (n-BuOH) fraction, the DPPH SC ₅₀ value was 16.8 μg / It was confirmed that the free radical scavenging activity was excellent at mL and 18.0 μg/mL (Table 1 and FIG. 2).

<Experimental Example 3> ABTS of hydrangea leaf extract ⁺ Radical scavenging activity experiment

3-1. Experimental method

In order to measure the free radical scavenging activity of the 70% ethanol (EtOH) extract and the four solvent fractions obtained in Experimental Example 1, ABTS ⁺ radical cation decolourisation assay method (Re R, Pellegrain N, Proteggente A Pannala A, Yang M, Rice-Evans C., Free Radical Biol. Med., 1999, 26;1231-1237) was applied to ABTS ⁺ radical scavenging activity test.

At this time, ABTS was dissolved in distilled water to a concentration of 7.0 mM, potassium persulfate was dissolved in distilled water to a concentration of 2.45 mM, mixed to a final concentration, and left for 16 hours in a dark place at room temperature ^{to form ABTS +} radical before use.

Specifically, the ethanol extract of hydrangea leaf and its n- hexane ( n- Hex.) fraction, ethyl acetate (EtOAc) fraction, n- butanol ( n- BuOH) fraction, and water (H ₂ O) fraction samples were each 3.125 μg It was prepared by dilution with DMSO:EtOH (1:1) solvent at concentrations of /mL, 6.25 μg/mL, 12.5 μg/mL, 25 μg/mL, 50 μg/mL and 100 μg/mL.

Then, it was prepared by diluting with EtOH so that the absorbance value of the ^{ABTS +} solution at 700 nm was 0.78 ± 0.03.

In a 96-well plate, ^{180 μl of the diluted ABTS +} solution and 20 μl of each of the diluted sample solutions were mixed and reacted at room temperature for 20 minutes, and then 700 μl using a spectrophotometer. Absorbance was measured in nm.

The scavenging rate (%) was calculated by Equation 2 below, and _{the concentration (SC 50} ; Scavenging concentration of 50%) ^{when each sample inhibited ABTS +} radical by 50% was obtained. Each sample was repeated three times to obtain an average value.

In this case, BHT and vitamin C were used as antioxidants to compare the activity.

<Equation 2>

Free radical scavenging ability (%) =

A: Absorbance at 700 nm of the reaction solution to which the sample was added

B: absorbance at 700 nm of the sample alone

C: Absorbance at 700 nm of the reaction solution to which no sample was added

D: Absorbance of the sample and the reaction solution without addition of ABTS

3-2. Experiment result

The experimental results are shown in Table 2 and FIG. 3 below.

sample SC ₅₀ (μg/mL) EtOH 11.4 n -Hex. 33.6 EtOAc 7.1 n- BuOH 8.8 H ₂ O 57.7 BHT 7.2 Vitamin C 7.2

As shown in Table 2 and FIG. 3, ABTS ⁺ free radical scavenging activity was shown in both the extracts and fractions. In particular, _{the SC 50} value ^{of ABTS +} radical in the ethyl acetate (EtOAc) fraction and the butanol (n- BuOH) fraction was At 7.10 μg/mL and 8.8 μg/mL, respectively, the positive control group, vitamin C, and BHT (Butylated hydroxytoluene) showed similar free radical scavenging activity (Table 2 and FIG. 3).

<Experimental Example 4> Evaluation of skin cytotoxicity of ethyl acetate fraction of hydrangea leaves

4-1. Experimental method

Cell viability was measured using human HaCaT keratinocytes in order to measure the cytotoxicity of the ethyl acetate fraction of the hydrangea leaf prepared in Experimental Example 1. Viability was measured using the MTT assay. Human HaCaT cells were cultured at _{37° C. and 5% CO 2} incubator using DMEM medium containing 1% Penicillin-streptomycin and 10% fetal bovine serum (FBS).

Specifically, the ethyl acetate (EtOAc) fraction of hydrangea leaves was prepared by diluting it with a serum-free medium at concentrations of 2.5 μg/mL, 5 μg/mL, 10 μg/mL, and 20 μg/mL. The cultured human HaCaT cells were seeded in a 96-well plate at 3×10 ⁴ cells/well and cultured with serum medium for 24 hours. Thereafter, the prepared sample was treated and cultured for 24 hours after exchange with a serum-free medium, and the cell viability was evaluated using MTT reagent (FIG. 4). At this time, a serum-free medium was used as a control, and is indicated by (-), and cell viability was measured for each concentration of the sample compared with (-).

4-2. Experiment result

The experimental results are shown in Table 3 and FIG. 4 below.

Concentration (μg/mL) Cell viability (%) Example (-) 100.0 2.5 90.9 5 88.2 10 84.3

As shown in Table 3 and Figure 4, it was confirmed that the ethyl acetate fraction of hydrangea leaves did not show cytotoxicity up to 10 μg/mL.

<Experimental Example 5> Skin cell protective effect of ethyl acetate fraction of hydrangea leaves

5-1. Experimental method

The cultured human HaCaT cells were seeded in a 96-well plate at 3×10 ⁴ cells/well and cultured for 24 hours. The cultured human HaCaT cells were treated with 4 mM _{hydrogen peroxide (H 2} O _{2 ) diluted with DPBS and incubated for 30 minutes.} Thereafter, hydrogen peroxide (H ₂ O ₂ ) was removed, washed with DPBS, and the ethyl acetate fraction of the hydrangea leaf was diluted using a serum-free medium to a concentration of 2.5 μg/mL, 5 μg/mL, and 10 μg/mL. . After culturing this for 24 hours, the MTT reagent was treated and the absorbance was measured to calculate the cell viability. In the cell viability calculation, the case where hydrogen peroxide was not treated was (-), and the case where hydrogen peroxide was treated and the sample was not treated was (+). The sample treated (Example) was used as an experimental group, and cell viability was measured by concentration of the sample by comparison with (-).

5-2. Experiment result

The experimental results are shown in Table 4 and FIG. 5 below.

Treatment concentration (㎍/mL) Cell viability (%) Example (-) 100.0 (+) 65.0 2.5 67.8 5 70.8 10 84.9

As shown in Table 4, as a result of measuring the protective effect of human HaCaT cells against oxidative stress induced by hydrogen peroxide in the ethyl acetate fraction of hydrangea leaves, the higher the concentration of the sample, the higher the concentration-dependent cell viability. Confirmed. This means that even in normal human skin keratinocytes, it has a skin cell protective effect by inhibiting skin cell damage caused by oxidative stress. Therefore, since the ethyl acetate fraction of hydrangea leaves inhibits or improves skin damage, it can be used in the development of cosmetics for skin cell protection. In addition, it is interpreted that there is an antioxidant effect on oxidative stress, and it is considered to be useful for the development of functional cosmetic materials using this.

<Experimental Example 6> Compound separation and structure identification from ethyl acetate (EtOAc) fraction of hydrangea leaves

In order to isolate the compound from the solvent fraction obtained in Experimental Example 1 and identify the structure, an ethyl acetate (EtOAc) fraction of the leaves of water hydrangea was selected, and a physiologically active compound was isolated therefrom and the structure was identified. did.

In order to sequentially subdivide 5.0 g of the ethyl acetate (EtOAc) fraction obtained in Experimental Example 1 according to polarity, medium pressure liquid chromatography (MPLC) was performed. 5.0 g of the ethyl acetate fraction was dissolved in 7 mL of methanol (MeOH) and 3 mL of DMSO, filtered using a 0.45 μm PVDF filter, and injected. As a column, reverse phase silica gel (C ₁₈ ) was used. Methanol (MeOH):water (H ₂ O) (10→50%, 100 min), methanol (MeOH):water (H ₂ O) (50→100%, 20 min) using gradient elution, methanol (MeOH) ) (100%, 10 min) by sequentially lowering the polarity ratio, each eluted by 40 mL to obtain a total of 48 fractions (fraction, Fr.). Among the MPLC fractions, compound 1 (64.5 mg) was obtained from Fr.19, compound 2 (117.5 mg) from Fr.27, compound 3 (104.1 mg) from Fr.36, and compound 4 (100.7 mg) from Fr.41 , compound 7 was obtained from Fr.48, respectively.

In addition, Fr.12-Fr.15 (329.6 mg) was subjected to Sephadex LH-20 column chromatography under a solvent condition of _{chloroform (CHCl 3 ):methanol (MeOH) = 2.5:1.} to obtain compound 5 (5.1 mg), and Fr.46-Fr.47 (141.8 mg) was purified by Sephadex LH-20 column chromatography (Sephadex) under a solvent condition of _{chloroform (CHCl 3 ):methanol (MeOH) = 15:1.} LH-20 column chromatography) was performed to obtain compound 6 (5 mg).

A total of 7 active compounds were separated from the ethyl acetate (EtOAc) fraction as described above, and ¹ H-nuclear magnetic resonance spectrum ( ¹ H NMR spectrum) and ¹³ C-nuclear magnetic resonance spectrum ( ¹³ C-NMR spectrum) were measured to obtain a structure result, compound 1 was identified as epicatechin is the (epi- catechin) with, compound 2 is Hi Perrin (hyperin), compound 3 is a quartz citrine (quercitrin), compound 4 is a sick jerin (afzelin), compound 5: 1 -O-para-coumaroyl-beta-D-glucopyranoside (1-O-ρ-coumaroyl-β-D-glucopyranoside), compound 6 is asiatic acid, compoun 7 is corosol It was identified as corosolic acid (Table 5).

Compound 1 Compound 2 Compound 3 epi-catechin hyperin quercitrin

Compound 4 Compound 5 Compound 6 afzelin 1-O-ρ-coumaroyl-β-D-glucopyranoside asiatic acid

Compound 7 corosolic acid

<Experimental Example 7> DPPH radical scavenging activity experiment of the compound isolated from the ethyl acetate (EtOAc) fraction of hydrangea leaves

7-1. Experimental method

To measure the free radical scavenging activity of the compounds obtained in Experimental Example 6, a DPPH radical scavenging activity experiment was performed using the Blois method (Blois, MS, Nature, 1958, 181; 1199-1200). did. At this time, DPPH was used by dissolving in ethanol to a concentration of 0.2 mM.

Specifically, Compound 1 ( epi -catehin), Compound 2 (hyperin), Compound 3 (quercitrin), Compound 4 (afzelin), Compound 5 (1-O-ρ-coumaroyl-β- D-glucopyranoside), Compound 6 (asiatic acid), and Compound 7 (corosolic acid) in a concentration of 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM using DMSO:EtOH (1:1) solvent. It was prepared by dilution.

Then, 180 μl of 0.2 mM DPPH solution and 20 μl of each of the diluted sample solutions were mixed in a 96-well plate and reacted at room temperature for 20 minutes, and then 515 nm using a spectrophotometer. absorbance was measured. The scavenging rate (%) was calculated by Equation 1 above, and the concentration (SC ₅₀ ; Scavenging concentration of 50%) when each sample inhibited DPPH by 50% was obtained. Each sample was repeated three times to obtain an average value. In this case, BHT and vitamin C were used as antioxidants to compare the activity.

7-2. Experiment result

The experimental results are shown in Table 6 and FIG. 7 below.

sample SC ₅₀ (μM) Compound 1 45.9 Compound 2 35.0 Compound 3 51.6 Compound 4 54.9 Compound 5 >100 Compound 6 >100 Compound 7 >100 BHT 62.1 Vitamin C 31.2

As shown in Table 6 and FIG. 7, DPPH free radical scavenging activity was shown in Compounds 1 to 4 , and both showed superior activity than the control, BHT. In particular, _{the SC 50} value of DPPH radical in Compound 2 was 35.0 μM, confirming that the activity was similar to that of the control group, Vitamin C (Table 6 and FIG. 7).

<Experimental Example 8> ABTS of the compound isolated from the ethyl acetate (EtOAc) fraction of hydrangea leaves ⁺ Radical scavenging activity experiment

8-1. Experimental method

In order to measure the free radical scavenging activity of the compounds obtained in Experimental Example 6, ABTS ⁺ radical cation decolourisation assay method (Re R, Pellegrain N, Proteggente A Pannala A, Yang M, Rice-Evans C) ., Free Radical Biol. Med., 1999, 26;1231-1237) applied to ABTS ⁺ radical scavenging activity test was performed. At this time, ABTS was dissolved in distilled water to a concentration of 7.0 mM, potassium persulfate was dissolved in distilled water to a concentration of 2.45 mM, mixed to a final concentration, and left for 16 hours in a dark place at room temperature ^{to form ABTS +} radical before use.

Specifically, Compound 1 (epi-catehin), Compound 2 (hyperin), Compound 3 (quercitrin), Compound 4 (afzelin), Compound 5 (1-O-ρ-coumaroyl-β- D-glucopyranoside), Compound 6 (asiatic acid), and Compound 7 (corosolic acid) in a concentration of 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM using DMSO:EtOH (1:1) solvent. It was prepared by dilution.

Then, it was prepared by diluting with EtOH so that the absorbance value of the ^{ABTS +} solution at 700 nm was 0.78 ± 0.03. In a 96-well plate, ^{180 μl of the diluted ABTS +} solution and 20 μl of each of the diluted sample solutions were mixed and reacted at room temperature for 20 minutes, and then 700 μl using a spectrophotometer. Absorbance was measured in nm. The scavenging rate (%) was calculated by Equation 2 above, and _{the concentration (SC 50} ; Scavenging concentration of 50%) ^{when each sample inhibited ABTS +} radical by 50% was obtained. Each sample was repeated three times to obtain an average value. In this case, BHT and vitamin C were used as antioxidants to compare the activity.

8-2. Experiment result

The experimental results are shown in Table 7 and FIG. 8 below.

sample SC ₅₀ (μM) Compound 1 28.5 Compound 2 15.1 Compound 3 83.5 Compound 4 38.8 Compound 5 >100 Compound 6 >100 Compound 7 >100 BHT 43.1 Vitamin C 48.7

As shown in Table 7 and Figure 8, ABTS ⁺ free radical scavenging activity was shown in Compounds 1 - 4 , and in particular, the SC ₅₀ ^{values of ABTS +} radical in Compounds 1, 2, and 4 were 28.5 μM, 15.1 μM, and 38.8 μM, respectively. ^{As a result, ABTS +} free radical scavenging ability was superior to that of BHT and Vitamin C as controls (Table 7 and FIG. 8).

<Experimental Example 9> Cytotoxicity evaluation of compounds isolated from hydrangea leaves

9-1. Experimental method

In order to measure the cytotoxicity of the compound obtained in Example 6, cell viability was measured using human HaCaT keratinocytes. Viability was measured using the MTT assay. Human HaCaT cells were cultured at _{37° C. and 5% CO 2} incubator using DMEM medium containing 1% Penicillin-streptomycin and 10% fetal bovine serum (FBS).

Specifically, Compounds 1 - 6 of hydrangea leaves were prepared by diluting them at concentrations of 50 μM and 100 μM using a serum-free medium. The cultured human HaCaT cells were aliquoted in a 96-well plate at 5×10 ³ cells/well and incubated with a serum medium for 24 hours. Thereafter, the samples diluted with serum-free medium were treated and cultured for 24 hours, and then cell viability was evaluated using MTT reagent. At this time, a serum-free medium was used as a control, and is indicated by (-), and cell viability was measured for each concentration of the sample compared with (-).

9-2. Experiment result

The experimental results are shown in Table 8 and FIG. 9 below.

Concentration (μM) Cell viability (%) Example (-) 100.0 Compound 1 50 94.7 100 95.9 Compound 2 50 61.4 100 50.0 Compound 3 50 107.3 100 83.6 Compound 4 50 64.5 100 63.1 Compound 5 50 96.4 100 97.7 Compound 6 50 59.2 100 19.9

As shown in Table 8 and FIG. 9, it was confirmed that hydrangea leaf Compounds 1 , 3 , and 5 did not show cytotoxicity up to 100 μM.

<Experimental Example 10> Cell protective effect of a compound isolated from hydrangea leaves

10-1. Experimental method

The cultured human HaCaT cells were seeded in a 96-well plate at 5×10 ³ cells/well and cultured for 24 hours. The cultured human HaCaT cells were treated with 6 mM _{hydrogen peroxide (H 2} O _{2 ) diluted with DPBS and incubated for 30 minutes.} Thereafter, hydrogen peroxide (H ₂ O ₂ ) was removed, washed with DPBS, and then Compounds 1 , 3 , and 5 were treated at concentrations of 50 μM and 100 μM diluted using a serum-free medium. After culturing this for 24 hours, the MTT reagent was treated and the absorbance was measured to calculate the cell viability. In the calculation of cell viability, the case of treatment with only DPBS was defined as (-), and the case of treatment with hydrogen peroxide and DPBS was defined as (+). The sample treated (Example) was used as an experimental group, and cell viability was measured by concentration of the sample by comparison with (-).

10-2. Experiment result

The experimental results are shown in Table 9 and FIG. 10 below.

Concentration (μg/mL) Cell production rate (%) Example (-) 100.0 (+) 59.9 Compound 1 50 104.3 100 136.2 Compound 3 50 78.6 100 84.7 Compound 5 50 52.7 100 50.7

As shown in Table 9, the cytoprotective effect was measured using Compounds 1 , 3, and 5 isolated from the leaves of hydrangea on human HaCaT cells against oxidative stress induced by hydrogen peroxide. As a result, compound 1 was At the concentrations of 50 μM and 100 μM, 44.4% and 76.3% of cytoprotective effects were observed, and compound 3 was At the concentrations of 50 μM and 100 μM, it was confirmed that there was a cytoprotective effect of 18.7% and 24.8%, respectively. This is interpreted as having an antioxidant effect on normal human skin keratinocytes, and it is thought to be useful for the development of functional cosmetic materials using this.

Claims (10)

As an antioxidant cosmetic composition comprising an extract or a fraction thereof as an active ingredient, Hydrangea petiolaris,
The compounds isolated from the extracts or fractions are epicatechin rank station (epi- catechin), Hi Perrin (hyperin), citrine quartz (quercitrin), sick jerin (afzelin), 1-O- para-coumarone to one-beta -D- Glucopyranoside (1-O-ρ-coumaroyl-β-D-glucopyranoside), asiatic acid (asiatic acid) and corosolic acid (corosolic acid) will be at least one selected from, the cosmetic composition. The cosmetic composition according to claim 1, wherein the hydrangea extract is _{extracted with water, C 1} -C ₄ alcohol, or a mixed solvent thereof. The cosmetic composition according to claim 2, wherein the alcohol is ethanol. The cosmetic composition according to claim 1, wherein the fraction is an n- hexane fraction, an ethyl acetate fraction, an n- butanol fraction, or a water fraction. The cosmetic composition according to claim 1, wherein the ethyl acetate fraction of the hydrangea extract has a skin cell protective effect. delete According to any one of claims 1 to 5, wherein the cosmetic composition is skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand A formulation selected from the group consisting of cream, essence, nourishing essence, pack, soap, shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, emulsion, lipstick, makeup base, foundation, press powder and loose powder Having, a cosmetic composition. As an antioxidant food composition containing an extract or a fraction thereof as an active ingredient, Hydrangea petiolaris,
The compounds isolated from the extracts or fractions are epicatechin rank station (epi- catechin), Hi Perrin (hyperin), citrine quartz (quercitrin), sick jerin (afzelin), 1-O- para-coumarone to one-beta -D- Glucopyranoside (1-O-ρ-coumaroyl-β-D-glucopyranoside), asiatic acid (asiatic acid) and corosolic acid (corosolic acid) will be at least one selected from, the food composition. The food composition of claim 8, wherein the food composition is a health functional food. 10. The method of claim 9,
The health functional food is an inner beauty food (inner beauty food), the food composition.

Images