KR20240013514A - Compositions for Improving Skin Wrinkles and Anti-oxidation Using an Extract of Schizophragma hydrangeoides - Google Patents

Abstract

The present invention is a skin treatment using rock hydrangea extract, which has the activity of inhibiting the production of MMP-1 induced by ultraviolet irradiation in human fibroblasts without cytotoxicity, and has been confirmed to have antioxidant activity by the DPPH method and the ABTS method. Disclosed is a composition for improving wrinkles and anti-oxidation.

Description

Compositions for Improving Skin Wrinkles and Anti-oxidation Using an Extract of Schizophragma hydrangeoides}

The present invention relates to a composition for improving skin wrinkles and anti-oxidation using Schizophragma hydrangeoides extract.

The skin makes up about 16% of the human body, and is in direct contact with the external environment, playing an important role in protecting the human body from external harmful factors such as temperature, humidity, and ultraviolet rays. The skin is composed of the epidermis, dermis, and subcutaneous tissue under the keratin. The epidermis is a thin protective layer composed mainly of keratinocytes, which are made of keratin, melanocytes that produce and secrete melanin, immune cells such as Langerhans cells, and sensory-related cells (Merkel's copuscles). It prevents the invasion of external stimuli and pathogens, regulates body temperature, and maintains moisture and lipid components. The dermis is a connective tissue beneath the epidermis and is mostly composed of a network structure of macromolecules called extracellular matrix. This extracellular stroma is made from fibroblasts and consists of fibrous proteins such as collagen and elastin and polysaccharides such as hyaluronic acid.

As we age, skin cells are damaged due to various pollutants, strong ultraviolet rays, stress, and nutritional deficiencies, and cell proliferation is impaired, causing wrinkles, loss of elasticity, pigmentation, and keratinization in the skin. Gilchrest B.A., J. Am. Acad. Dermatol, 21, 610. 1989).

Collagen is the most important protein in maintaining skin moisture and skin flexibility and elasticity, making up 90% of the dermal layer (J Am Acad Dermatol, 17(4):610-613. 2001), and giving the skin strength and tension. This serves to protect the skin from external stimulation. In addition, elastin in the dermal layer accounts for about 2%, which is less than collagen, but plays a role in supporting collagen fibers. Many recent studies have reported that the deficiency, aggregation, and loss of elastin fibers play an important role in the skin wrinkle formation mechanism. (Weihermann AC et al., Int J Cosmet Sci. 2017 Jun;39(3):241-247; Mora Huertas AC et al., Biochimie. 2016 Sep-Oct;128-129:163-73).

Collagen and elastin are synthesized in fibroblasts and decomposed by collagenase or elastase such as MMP-1 (Matrix metalloproteinase-1). As we age or are exposed to ultraviolet rays, the function of fibroblasts and the number of cells decreases, resulting in a decrease in the amount of collagen or elastin synthesis and an increase in the biosynthesis of collagenase or elastase, which decomposes them. Once the elasticity of the skin caused by collagen and elastin is reduced, it is very difficult to recover. Accordingly, research is being actively conducted to improve skin wrinkles by suppressing the biosynthesis of collagenase, which decreases with age.

Superoxide radical (O ₂ ㆍ), hydroxyl radical (OH ㆍ), and hydrogen peroxide (H ₂ O) generated during normal cellular metabolism in the body or due to exposure to various environmental pollution and chemicals. ₂ ), singlet oxygen ( ¹ O ₂ ), etc. are collectively called reactive oxygen species (ROS).

These free radicals are very reactive, so they react quickly with biomolecules, causing lipid oxidation of biological membranes, protein denaturation, and DNA damage, and can cause various diseases such as cancer, arteriosclerosis, diabetes, autoimmune disease, aging, stroke, and Parkinson's disease. It is known to increase the risk of disease ( Chem. Res. 14: 393 (1981); Arch. Biophys. 247: 1-11 (1986); Science 221: 1256-1264 (1983)).

Living organisms are equipped with an antioxidant system to respond to this continuously occurring oxidative stress. This antioxidant system is largely enzymatic by superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione S-transferase. It is divided into a non-enzymatic system based on antioxidants such as vitamin A, vitamin C, vitamin E, and antioxidant minerals consumed through the system and diet ( Annu. Rev. Nutr. , 16, pp33-50 (1996)). The enzymatic and non-enzymatic systems work complementary to each other to detoxify oxidative substances produced in living organisms.

Antioxidants inhibit or minimize biological damage caused by free radicals by reacting with free radicals, and include butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and propyl gallate ( Synthetic antioxidants such as PG, Propyl galate) and Teritiarybutyl hydroquinone (TBHQ), as well as chlorophyll, green tea extract, Prololis extract, chlorella, vitamin E, vitamin C, squale, flavonoids, bamboo leaf extract, etc. They can be classified as natural antioxidants. As synthetic antioxidants are known to cause carcinogenesis and cardiovascular side effects, research is being actively conducted on plant-derived antioxidants that are safer and more effective ( Korean J. Food Sci. Technol. 34:889-892 (2002)).

Recently, as antioxidant activity measurement techniques to search for substances showing antioxidant activity, TRAP method ( Anal Biochem. , 28, pp300-306. 1989), DPPH method ( Nature 181:1198-1200 (1958)), and ABTS method ( Methods Enzymol 234 : 279-293 (1994); Clin. Sci. 84: 407-412 (1993); Free Rad. Biol. Med. 20: 933-956 (1996)), ORAC Act ( Free Rad. Biol. Med. 18:29-36 (1995)), TOSC Law ( Free Rad. Biol. Med. 24(3):480-493 (1998); Toxicol. Appl. Pharmacol. 156(2):96-105 (1999)) , Potassium ferricyanide method (Jpn, J. of Nutr. 44:307-315, 1986), and SOD-like activity measurement method ( Eur J Biochem 47:469-474, 1974) have been developed.

The present invention discloses the skin wrinkle-improving activity and antioxidant activity of rock hydrangea extract, etc.

The purpose of the present invention is to provide a composition for improving skin wrinkles and anti-oxidation using rock hydrangea extract.

Other or specific purposes of the present invention will be presented below.

In the present invention, as confirmed in the examples and experimental examples below, the rock hydrangea extract and the four compounds of formulas 1 to 4 isolated therefrom are effective against ultraviolet irradiation in human fibroblast HDFn without showing any particular cytotoxicity. It was completed by confirming that it has the activity of suppressing the production of MMP-1 induced by the drug and also has antioxidant activity by the DPPH method and the ABTS method.

<Formula 1>

<Formula 2>

<Formula 3>

<Formula 4>

Considering the above, the present invention can be viewed in one aspect as a composition for improving skin wrinkles containing rock hydrangea extract as an active ingredient, and in another aspect as an antioxidant composition containing rock hydrangea extract as an active ingredient. You can.

In this specification, “rock hydrangea extract” refers to rock hydrangea outgrowth, leaves, stems, above-ground parts, roots, rhizomes, underground parts, seeds, or mixtures thereof, which are the extraction target, in water and a lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.) ), methylene chloride, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butylacetate, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or It refers to extracts obtained by leaching using mixed solvents, extracts obtained using supercritical extraction solvents such as carbon dioxide and pentane, or fractions obtained by fractionating the extracts. The extraction method is determined by determining the polarity of the active substance, degree of extraction, and degree of preservation. Considering this, arbitrary methods such as cold immersion, reflux, heating, ultrasonic radiation, and supercritical extraction can be applied. In the case of a fractionated extract, the extract is suspended in a specific solvent and then mixed with a solvent of different polarity to form a fraction obtained by adsorbing the crude extract onto a column filled with silica gel and then mixed with a hydrophobic solvent, a hydrophilic solvent, or a mixture of these. It is meant to include fractions obtained using the mobile phase. In addition, the meaning of the extract includes concentrated liquid extract or solid extract from which the extraction solvent has been removed by methods such as freeze-drying, vacuum drying, hot air drying, and spray drying. Preferably, it refers to an extract obtained using water, a lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.), or a mixed solvent thereof as an extraction solvent. More preferably, the extract obtained using a mixed solvent of water and ethanol is used as an extraction solvent. Fractions of any solvent layer (including fractions of the water layer) obtained by fractionation with ethyl acetate or water and butanol, and ethyl obtained by suspending the mixed solvent extract of water and ethanol in solid form in water and sequentially fractionating it with hexane, ethyl acetate or butanol. refers to the acetate fraction, butanol fraction or fraction of the residual water layer.

In addition, in this specification, “active ingredient” refers to an ingredient that exhibits the desired activity alone or can exhibit activity in combination with a carrier that is not active on its own.

In addition to the active ingredients, the composition of the present invention is suitable for taking, ingesting, or using for the purpose of improving skin wrinkles or increasing/reinforcing antioxidant effects, or by adding similar activities such as skin whitening activity, UV protection activity, and skin hypersensitivity reaction inhibition activity. To improve convenience, any compound or natural extract that has already been proven safe in the art and is known to have the corresponding activity may be additionally included.

These compounds or extracts include the Pharmacopoeia of each country (in Korea, “Korea Pharmacopoeia”), each country’s Code of Health Functional Foods (in Korea, it is “Standards and Specifications for Health Functional Foods” notified by the Ministry of Food and Drug Safety), and the Code of Functional Cosmetics of each country (in Korea, it is the “Standards and Specifications for Health Functional Foods” notified by the Ministry of Food and Drug Safety). Compounds or extracts listed in compendial documents such as “Functional Cosmetics Standards and Test Methods”), compounds or extracts approved as items in accordance with the laws of each country governing the manufacture and sale of pharmaceuticals (in Korea, this is the Pharmaceutical Affairs Act), health Compounds or extracts recognized as functional in accordance with the laws of each country that govern the manufacture and sale of functional foods (in Korea, this is the “Health Functional Foods Act”), and the laws of each country that govern the manufacture and sale of functional cosmetics (in Korea, it is the “Cosmetics Act”) 」) includes compounds or extracts whose functionality has been recognized.

These ingredients include, for example, arbutin, niacinamide, ascorbyl glucoside, alpha-bisabolol, and Oil Soluble Licorice (Glycyrrhiza) Extract, etc., and retinol, retinyl palmitate, adenosine, polyethoxylated amide, etc., which are recognized as 'skin wrinkle improvement' ingredients by the Korea Cosmetics Convention, and other Korean cosmetics. A complex of drometrizole, drometrizole trisiloxane, digalloyl trioleate, dimethycodiethylbenzalmalonate, diethylhexylbutamidotriazone, and pine bark extract, which are internationally recognized as 'UV protection' ingredients. , phosphatidylserine, finger root extract powder, and complex extracts of red ginseng and Cornus officinalis, etc., and L. sakei Probio 65, which is recognized as a functional ingredient for 'improving sensitive skin conditions' according to the Korean 「Health Functional Foods Act」 , oil containing gamma-linolenic acid, L.plantarum CJLP133, a lactic acid bacteria derived from fruit and vegetables, probiotic ATP, etc., complexes such as pine bark extract recognized as an ingredient for 'improving skin damage from ultraviolet rays', melon extract, phosphatidylserine, probiotic HY7714, and finger root extract. Examples include powders, green tea extract, bamboo leaf extract, melon extract, bokbunja extract (powder), beeswax alcohol, ubiquinol, coenzyme Q10, tomato extract, and grape seed extract, which are recognized as antioxidant ingredients. One or more of these compounds or natural extracts may be included in the composition of the present invention together with the active ingredient.

The composition of the present invention may contain the active ingredient in any amount (effective amount) as long as it can exhibit anti-wrinkle activity and antioxidant activity intended for treatment depending on the use, formulation, purpose of formulation, etc., and the typical effective amount is the total amount of the composition. Based on weight, it will be determined within the range of 0.001% by weight to 15% by weight. Here, “effective amount” means that when the composition of the present invention is administered to mammals, preferably humans, to which it is applied during the administration period recommended by medical or skin experts, the intended dermatological and medical effects, such as wrinkle improvement effect and antioxidant effect, etc. It refers to the amount of the active ingredient contained in the composition of the present invention that can exhibit an effect. Such effective amounts can be determined experimentally within the scope of the ordinary ability of those skilled in the art.

In a specific aspect, the composition of the present invention can be viewed as a food composition.

The food composition of the present invention can be manufactured in any form, for example, beverages such as tea, juice, carbonated drinks, and isotonic drinks, processed oils such as milk and yogurt, gums, rice cakes, Korean snacks, bread, snacks, noodles, etc. It can be manufactured into health functional food preparations such as foods, tablets, capsules, pills, granules, liquid, powder, flakes, paste, syrup, gel, jelly, and bars.

In addition, the food composition of the present invention can have any product classification in terms of legal and functional classification as long as it complies with the enforcement laws and regulations at the time of manufacture and distribution. For example, it is a health functional food in accordance with the laws of an excluded country regulating the manufacture and sale of health functional foods (in Korea, this is the “Act on Health Functional Foods”), or it is a health functional food in accordance with the laws of an excluded country regulating the manufacture and sale of food (in Korea, it is the “Act on Health Functional Foods”). According to the Food Sanitation Act (the Food Sanitation Act) (in Korea, it is the Korean Ministry of Food and Drug Safety Notification “Food Standards and Specifications”), it can be confectionery, beans, soy milk, fermented beverages, special purpose foods, etc. according to each food type.

The food composition of the present invention may contain food additives in addition to the active ingredients. Food additives can generally be understood as substances that are added to, mixed with, or infiltrated into food when manufacturing, processing, or preserving food. Since they are consumed daily and for a long period of time with food, their safety must be guaranteed. In the Food Additives Code of Laws of each country that regulates the manufacturing and distribution of food (in Korea, it is the Food Sanitation Act), food additives with guaranteed safety are limited in terms of ingredients or functions. In the Korea Food Additive Code (Ministry of Food and Drug Safety Notification “Food Additive Standards and Specifications”), food additives are classified into chemical synthetics, natural additives, and mixed preparations in terms of composition. These food additives are classified into sweeteners and flavors in terms of function. It is classified into preservatives, emulsifiers, acidulants, thickeners, etc.

Sweeteners are used to impart an appropriate sweetness to foods, and either natural or synthetic ones can be used in the composition of the present invention. Preferably, a natural sweetener is used, and natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Flavoring agents can be used to improve taste or aroma, and both natural and synthetic ones can be used. Preferably, natural products are used. When using natural products, they can serve the purpose of enhancing nutrition in addition to flavor. Natural flavoring agents may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, etc., or may be obtained from green tea leaves, coriander leaves, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, etc. You can also use things obtained from ginseng (red ginseng), bamboo shoots, aloe vera, and ginkgo nuts. Natural flavoring agents may be liquid concentrates or solid extracts. In some cases, synthetic flavoring agents may be used, and the synthetic flavoring agents may include esters, alcohols, aldehydes, terpenes, etc.

Preservatives include calcium sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc., and emulsifiers include acacia gum, carboxymethyl cellulose, xanthan gum, Examples include pectin, and acidulants include acidic acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, and phosphoric acid. In addition to improving taste, acidulants may be added to ensure that the food composition has an appropriate acidity for the purpose of suppressing the growth of microorganisms.

As a thickening agent, a suspending agent, settling agent, gel forming agent, bulking agent, etc. may be used.

In addition to the food additives described above, the food composition of the present invention may contain bioactive substances or minerals known in the art and whose safety is guaranteed as food additives for the purpose of supplementing and reinforcing functionality and nutrition.

Such physiologically active substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, dibenzoylthiamine, etc., and minerals include calcium preparations such as calcium citrate and magnesium stearate. Magnesium preparations such as iron citrate, iron preparations such as chromium chloride, potassium iodine, selenium, germanium, vanadium, zinc, etc. are included.

The food composition of the present invention may contain the above-described food additives in an appropriate amount to achieve the purpose of addition depending on the product type.

Regarding other food additives that can be included in the food composition of the present invention, each country's food code or food additive code can be referred to.

The composition of the present invention may be considered a pharmaceutical composition in other specific embodiments.

In particular, when the composition for skin whitening of the present invention is considered a pharmaceutical composition, its use can be understood as preventing or treating skin hyperpigmentation, and specific examples of such hyperpigmentation include freckles, senile spots, liver spots, freckles, brown or Sunspots, solar pigment spots, cyanic melasma, hyperpigmentation after use of drugs such as calcium antagonists, sequelae from sclerotherapy, gravidic chloasma, in women taking oral contraceptives. This may include, but is not limited to, melasma, post-inflammatory hyperpigmentation due to wounds or dermatitis, including abrasions and burns, phototoxic reactions, or other similar small fixed pigmented lesions.

The pharmaceutical composition of the present invention contains a pharmaceutically acceptable carrier in addition to the active ingredient and can be prepared into an oral formulation or a parenteral formulation depending on the route of administration by a conventional method known in the art. Here, the route of administration may be any suitable route, including topical route, oral route, intravenous route, intramuscular route, and direct absorption through mucosal tissue, and two or more routes may be used in combination. An example of a combination of two or more routes is a case where two or more dosage forms of drugs according to the administration route are combined, for example, when one drug is administered firstly through an intravenous route and the other drug is administered secondarily through a local route.

Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or dosage form, and for specifics, reference can be made to each country's pharmacopoeia, including the "Korean Pharmacopoeia".

When the pharmaceutical composition of the present invention is prepared as an oral dosage form, it can be prepared as powder, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, suspensions, and wafers using a suitable carrier according to methods known in the art. It can be manufactured in a dosage form such as: Examples of suitable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, and wheat starch, cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, Cellulose such as hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol, grease. Serol, etc. can be mentioned. When formulating, appropriate binders, lubricants, disintegrants, colorants, diluents, etc. can be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch ferrite, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, and wax, and as a lubricant, oleic acid. Examples include sodium, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, its magnesium and calcium salts, and polydethylene glycol. Disintegrants include starch and methyl cellulose. , agar, bentonite, xanthan gum, starch, alginic acid or its sodium salt, etc. Additionally, diluents include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, etc.

When the pharmaceutical composition of the present invention is prepared as a parenteral formulation, it can be formulated in the form of injections, transdermal administration, nasal inhalation, and suppositories along with a suitable carrier according to methods known in the art. When formulated as an injection, an aqueous isotonic solution or suspension can be used as a suitable carrier. Specifically, an isotonic solution such as PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, or 5% dextrose can be used. . When formulated for transdermal administration, it can be formulated in the form of ointments, creams, lotions, gels, external solutions, paste preparations, linear preparations, and aerol preparations. In the case of nasal inhalation, it can be formulated in the form of an aerosol spray using suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and carbon dioxide. When formulated as a suppository, the carrier is Wethepsol ( witepsol), Tween 61, polyethylene glycols, cocoa fat, laurel paper, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, etc. can be used.

Regarding the specific formulation of pharmaceutical compositions, it is known in the art, and references can be made to, for example, Remington's Pharmaceutical Sciences (19th ed., 1995). The above documents are considered a part of this specification.

The preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001 mg/kg to 10 g/kg per day, preferably 0.001 mg/kg to 1 g, depending on the patient's condition, weight, gender, age, patient's severity, and administration route. It may be in the /kg range. Administration can be done once a day or divided into several times. These dosages should not be construed as limiting the scope of the invention in any respect.

In another specific aspect, the composition of the present invention can be considered a cosmetic composition.

Even if the composition of the present invention is identified as a cosmetic composition, the cosmetic composition may be classified as an arbitrary product in terms of its use and by law, and is specifically a functional product with uses such as improving skin troubles, improving skin whitening, and improving atopic dermatitis. It may be cosmetics, non-functional general cosmetics, etc. The product form can take any product form, specifically solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax. It can take the form of products such as foundation or spray. Specific product forms may include softening lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray, or powder.

In addition to the active ingredient, the cosmetic composition of the present invention may contain ingredients commonly used in cosmetic compositions, such as stabilizers, solubilizers, surfactants, vitamins, conventional auxiliaries such as pigments and flavorings, and carriers.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier ingredient. You can.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as the carrier ingredient. In particular, when the formulation is a spray, chlorofluorohydrocarbon and propane may be used as carrier ingredients. /May contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent, or emulsifying agent is used as a carrier component, specifically water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, fatty acid ester of sorbitan, etc. can be used.

When the formulation of the present invention is a suspension, the carrier ingredients include water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, polyoxyethylene sorbitan ester, and microcrystals. Cellulose, aluminum metahydroxide, bentonite, agar, etc. can be used.

When the formulation of the present invention is a surfactant-containing cleansing agent, the carrier ingredients include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, and fatty acid amide. Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester can be used.

The cosmetic composition of the present invention can be manufactured according to a cosmetic composition manufacturing method commonly used in the art, except that it contains active ingredients that exhibit skin wrinkle-improving activity, etc.

As described above, according to the present invention, it is possible to provide a composition for improving skin wrinkles and an antioxidant composition using rock hydrangea extract.

This composition of the present invention can be commercialized into foods, cosmetics, drugs, etc.

Figure 1 is a schematic diagram of separation of active ingredients from rock hydrangea extract.
Figure 2 is the structural formula of four active ingredients isolated from rock hydrangea extract.
Figures 3 and 4 show the cytotoxicity evaluation results and the MMP-1 production inhibition activity evaluation results of the Rock Hydrangea extract and the four active ingredients isolated therefrom, respectively.
Figures 5 and 6 show the results of antioxidant activity evaluation by the DPPH method and the ABTS method, respectively.

Hereinafter, the present invention will be described with reference to examples and experimental examples. However, the scope of the present invention is not limited to these examples and experimental examples.

<Example> Preparation of rock hydrangea extract and separation and identification of active ingredients

<Example 1> Preparation of rock hydrangea extract

3.67 kg of rock hydrangea used in the experiment was collected from around Muljanggyo Bridge in Jocheon-eup, Jeju in May 2021, and the sample was dried and crushed. 624 g of dried and crushed rock hydrangea (extraction part: leaves) was immersed in 24 L of 70% ethanol and leached for 24 h at room temperature. Only the filtrate of the leached sample was taken using a reduced pressure suction filter, and the process was repeated two more times under the same conditions for the residue separated in this way. The filtrate obtained by filtration was concentrated under reduced pressure in an aqueous solution at 40°C to obtain 143 g of extract. 100 g of the obtained extract was suspended in distilled water and sequentially fractionated in order of solvent polarity to produce n-hexane (Hex), ethyl acetate (EtOAc). , n-butanol (BuOH), and H ₂ O fractions were obtained.

<Example 2> Isolation and identification of active ingredients

The BuOH fraction was subdivided according to polarity using MPLC. 7.0 g of the BuOH fraction was dissolved in 10 mL of methanol, filtered using a 0.45 μm PTFE syringe filter, and then injected into a C18 column. Water (solvent A) and methanol (solvent B) were used as mobile phases, and the flow rate was 15 mL/min. The elution conditions were gradient mode, where solvent B was changed at a rate of 10 - 70% for 100 min, and then 100% solvent B was eluted for 10 min to obtain a total of 75 fractions (Fr. MP 1 - 75). Among the MPLC fractions, Fr. MP 51 (120 mg) was identified as a single compound, compound 2 , and was identified by Fr. Compound 1 (38 mg) was separated from MP 16 - 19 using a Sephadex LH-20 column (MeOH = 100). Fr. MP 53 performed a normal silicagel column (CHCl ₃ :MeOH = 2:1) to obtain compounds 3 (14 mg) and 4 (10 mg). Figure 1 shows a schematic diagram of the separation process of the active ingredient.

To identify the structures of the separated compounds, nuclear magnetic resonance (NMR) was performed using JNM-ECX 400 (FT-NMR system, JEOL, Japan), and CD ₃ OD, a solvent exclusively for NMR from CIL, was used as the NMR measurement solvent.

The 1H and 13C-NMR results of each isolated compound are as follows.

Compound 1: colorless, oily; 1H NMR (400MHz, CD ₃ OD): 7.56 (1H, d, J=16.0Hz, H-7), 7.03(1H, d, J=1.8Hz, H-2), 6.93(1H, dd, J= 1.8, 7.8Hz), 6.77(1H, d, J=8.2Hz, H-5), 6.29(1H, d, J=16.0Hz, H-8), 5.37(1H, m, H-3'), 4.13(1H, brs, H-4'), 3.69(1H, dd, J=10.0, 3.2Hz, H-5'), 2.17-1.96(4H, m, H-2', H6'); 13C NMR (100MHz, CD ₃ OD): 180.8 (COOH quin.), 169.1 (COO caff.), 149.6 (C-4), 146.9 (C-7), 127.8 (C-1), 123.1 (C-6) ), 116.8 (C-5), 115.8 (C-2), 115.3 (C-8), 77.8 (C-1'), 75.1 (C-3'), 73.1 (C-4'), 72.6 (C -5'), 40.7 (C-6'), 39.2 (C-2')

Compound 2: brown, powdery; 7.35 (1H, d, J=2.3Hz, H-2'), 7.32(1H, dd, J=2.1, 8.2Hz H-6'), 6.92(1H, d, J=8.2, H-5') , 6.37(1H, d, J=2.3Hz, H-8), 6.20(1H, d, J=2.1Hz, H-6), 5.64(1H, brs, H-1"), 4.38(1H, d , J=7.8Hz, H-1"'), 3.15-3.88(9H, m, sugar protons), 0.98(3H, d, J=6.4Hz, H-6"); 13C NMR (100MHz, CD ₃ OD ): 179.6 (C-4), 166.3 (C-7), 163.2 (C-5), 159.3 (C-2), 158.6 (C-9), 150.0 (C-4'), 146.6 (C-3) '), 136.6 (C-3), 122.9 (C-1', 6'), 117.0 (C-2'), 116.6 (C-5'), 107.3 (C-1"'), 106.0 (C- 10), 102.7 (C-1"), 100.0 (C-6), 94.9 (C-8), 83.0 (C-2"), 77.9 (C-3"'), 77.9 (C-5"') , 75.4 (C-2"'), 73.6 (C-4"), 72.1 (C-3"), 71.9 (C-4"'), 70.8 (C-5"), 62.3 (C-6"') ), 17.8 (C-6")

Compound 3: yellow, powdery; 7.35 (1H, d, J=2.3Hz, H-2'), 7.30(1H, dd, J=2.1, 8.2Hz H-6'), 6.92(1H, d, J=8.2, H-5') , 6.36(1H, d, J=2.3Hz, H-8), 6.19(1H, d, J=2.1Hz, H-6), 5.34(1H, brs, H-1"), 4.24(1H, d , J=7.3Hz, H-1"'), 3.15-3.88(9H, m, sugar protons), 1.02(3H, d, J=5.9Hz, H-6"); 13C NMR (100MHz, CD ₃ OD ): 179.9 (C-4), 166.0 (C-7), 163.3 (C-5), 159.2 (C-2), 158.6 (C-9), 150.0 (C-4'), 146.6 (C-3) '), 136.9 (C-3), 122.9 (C-1'), 122.8 (C-6'), 116.9 (C-2'), 116.6 (C-5'), 107.9 (C-1"') , 105.9 (C-10), 103.4 (C-1"), 99.9 (C-6), 94.9 (C-8), 82.9 (C-2"), 77.9 (C-3"'), 75.4 (C -2"'), 73.8 (C-2"'), 72.0 (C-4"), 71.9 (C-3"), 71.1 (C-5"), 67.2 (C-5"'), 17.8 ( C-6")

Compound 4 yellow, powdery; 7.34 (1H, d, J=2.3Hz, H-2'), 7.32(1H, dd, J=2.1, 8.2Hz H-6'), 6.91(1H, d, J=8.2, H-5') , 6.37(1H, d, J=2.3Hz, H-8), 6.20(1H, d, J=2.1Hz, H-6), 5.34(1H, brs, H-1"), 4.21(1H, dd , J=1.3, 3.1Hz, H-2"), 3.31-3.74(3H, m, sugar protons), 0.93(3H, d, J=5.9Hz, H-6"); 13C NMR (100MHz, CD ₃ OD): 179.8 (C-4), 166.1 (C-7), 163.4 (C-5), 159.5 (C-2), 158.7 (C-9), 150.0 (C-4'), 146.6 (C- 3'), 136.4 (C-3), 123.1 (C-1'), 123.0 (C-6'), 117.1 (C-2'), 116.5 (C-5'), 106.0 (C-10), 103.7 (C-1"), 100.0 (C-6), 94.9 (C-8), 73.4 (C-4"), 72.3 (C-2"), 72.2 (C-3"), 72.1 (C- 5"), 17.8 (C-6")

The NMR results were compared to existing literature (Pharmaceutical Biology, 53(10): 1505-1510, (2015); Phytochemistry, 31(4):1391-1394, (1992); Pharmaceutica Acta Helvetiae 71:199-204, (1996) ; Natural Product Sciences, 23(4):274-280, (2017)), Compound 1 is chlorogenic acid, and Compound 2 is quercetin-3-glucosyl-(1-2)-rhamnoside ( Quercetin-3-glucosyl-(1-2)-rhamnoside), Compound 3 is Quercetin-3-xylosyl-(1-2)-rhamnoside), Compound 4 was confirmed to be quercitrin, and the structure of each compound is shown in Figure 2.

<Experimental example> Wrinkle improvement activity test and antioxidant activity test

<Experimental Example 1> Wrinkle improvement activity experiment

The cell viability of the Hydrangea hydrangea extract, fractions, and four isolated compounds was measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. HDFn cells were cultured in DMEM (Thermo Fisher Scientific, MA, USA) containing 10% FBS (Fetal Bovine Serum, Thermo Fisher Scientific, MA, USA) and 20% Ham's F-12 Nutrient Mixture (Thermo Fisher Scientific, MA, USA). The medium was dispensed into 24 well-plates at a concentration of 7 × 10 ⁴ cells/well and then stabilized for 24 hours in an incubator at 37°C and 5% CO ₂ . Immediately after 3 J/cm ² UV-A irradiation, samples were treated using DMEM supplemented with 2% FBS. Then, all supernatants were removed, and 500 μL of 0.4 mg/mL MTT reagent (Amresco, OH, USA) was added to each well and reacted for 4 hours. The generated formazan crystals were dissolved in DMSO (Dimethyl Sulfoxide, Amresco, OH, USA). Absorbance was measured at 570 nm using a microplate reader (Tecan, Salzburg, Austria). Cell viability was expressed as a percentage (%) based on the untreated group.

MMP-1 (Matrix metalloproteinase-1) breaks down collagen, a structural protein that fills connective tissues such as skin. To confirm the inhibition of MMP-1 production by the Rock Hydrangea extract, fractions, and four isolated compounds, it was measured using the Human MMP1 ELISA Kit (ab215083, Abcam, MA, USA). HDFn cells were distributed in a 24 well-plate at a concentration of 7 × 10 ⁴ cells/well and stabilized in an incubator at 37°C and 5% CO ₂ for 24 hours. After washing once using 1XPBS (phosphate buffered saline) buffer (pH 7.4, Thermo Fisher Scientific, MA, USA), it was irradiated with 3 J/cm ² UV-A on 1XPBS. Immediately after UV-A irradiation, samples were treated using DMEM supplemented with 2% FBS and cultured for 24 hours. The culture supernatant was taken, and the produced MMP-1 was quantified using ELISA method. The MMP-1 production rate was expressed as a percentage (%) based on the UV-A treatment group alone. Adenosine (AD) was used as a positive control.

The results are shown in Figure 3 for the Rock Hydrangea extract and fractions, and in Figure 4 for the four isolated compounds. Referring to Figures 3 and 4, except for the hexane fraction, both the extract and the remaining fractions showed activity in inhibiting the production of MMP1. In the case of the four compounds, Compound 2 and Compound 4 showed activity in suppressing the production of MMP1.

In addition, with regard to cytotoxicity, except for the hexane fraction, the Hydrangea hydrangea extract, the remaining fractions, and the four isolated compounds did not show any particular cytotoxicity.

<Experimental Example 2> Antioxidant activity

<Experimental Example 2-1> DPPH radical scavenging activity (electron donating ability) analysis

DPPH (2,2-diphenyl-1-picrylhydrazyl, Sigma-Aldrich chemical Co., St. Louis, MO, USA), a nitrogen-centered radical that appears in a stable state and displays a dark purple color when the radical is delocalized, reacts with antioxidants. The DPPH radical scavenging activity of 70% ethanol extract and fractions of Rock Hydrangea was measured using the DPPH method, which changes color to yellow. 0.1 mL of extracts and fractions of each concentration and 0.1 mL of 0.2 mM DPPH solution were mixed in the dark, left for 10 minutes, and absorbance was measured at a wavelength of 517 nm using a microplate reader (Tecan, Salzburg, Austria). The control group was measured in the same manner as above using 70% ethanol as a sample extraction solution instead of the extract, and the DPPH radical scavenging activity was calculated using the formula below. Ascorbic acid was used as the positive control group.

※ Inhibition rate (%) = [1-(A-C)/(B-D)] × 100

A: Absorbance of sample treatment area

B: Absorbance of control

C: Absorbance of sample treated without DPPH

D: Absorbance of control without DPPH and sample treatment

The results are shown in Figure 5. Both rock hydrangea extracts and fractions showed DPPH radical scavenging activity.

<Experimental Example 2-2> ABTS radical scavenging activity (electron donating ability) analysis

ABTS ⁺ (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid, Sigma-Aldrich chemical Co., St. Louis, MO, USA), which shows blue-green color when radical delocalized, reacts with antioxidants to decolorize. The ABTS radical scavenging activity of rock hydrangea extract and its fractions was measured using the ABTS method. First, the reagent was prepared by mixing an equal amount of 7.0mM ABTS with 2.45mM Potassium persulfate and reacting in the dark in refrigeration for 16 hours. The reagent was used by diluting it 25 to 30 times in methanol. 0.1 mL of Hydrangea hydrangea extract and fractions of each concentration and 0.1 mL of ABTS solution were mixed in the dark, left for 10 minutes, and then printed on a microplate reader (Tecan, Salzburg, Austria). The absorbance was measured at a wavelength of 517 nm using . The control group was measured in the same manner as above using 70% ethanol as the sample extraction solution instead of the extract, and the ABTS radical scavenging activity was calculated using the formula below.

※ Inhibition rate (%) = [1-(A-C)/(B-D)] × 100

A: Absorbance of sample treatment area

B: Absorbance of control

C: Absorbance of sample treated without ABTS

D: Absorbance of ABTS and control without sample treatment

The results are shown in Figure 6. Both rock hydrangea extracts and fractions showed ABTS radical scavenging activity.

Claims (8)

A composition for improving skin wrinkles comprising an extract of rock hydrangea or any one of the compounds of formulas 1 to 4 below as an active ingredient.
<Formula 1>

<Formula 2>

<Formula 3>

<Formula 4>

According to paragraph 1,
The extract is (i) a water, ethanol, or a mixed solvent extract thereof, (ii) a fraction of any solvent layer obtained by fractionating a water and ethanol mixed solvent extract with water and ethyl acetate or water and butanol, or (iii) water. A composition characterized in that it is an ethyl acetate fraction, a butanol fraction, or a fraction of the remaining water layer obtained by suspending the mixed solvent extract of and ethanol in solid form in water and sequentially fractionating it with hexane, ethyl acetate, or butanol.
According to claim 1 or 2,
The composition is characterized in that it is a food composition.
According to claim 1 or 2,
The composition is characterized in that it is a cosmetic composition.
An antioxidant food composition containing rock hydrangea extract as an active ingredient.
According to clause 5,
The extract is (i) a water, ethanol, or a mixed solvent extract thereof, (ii) a fraction of any solvent layer obtained by fractionating a water and ethanol mixed solvent extract with water and ethyl acetate or water and butanol, or (iii) water. A composition characterized in that it is an ethyl acetate fraction, a butanol fraction, or a fraction of the remaining water layer obtained by suspending the mixed solvent extract of and ethanol in solid form in water and sequentially fractionating it with hexane, ethyl acetate, or butanol.
An antioxidant food composition containing rock hydrangea extract as an active ingredient.
According to clause 5,
The extract is (i) a water, ethanol, or a mixed solvent extract thereof, (ii) a fraction of any solvent layer obtained by fractionating a water and ethanol mixed solvent extract with water and ethyl acetate or water and butanol, or (iii) water. A composition characterized in that it is an ethyl acetate fraction, a butanol fraction, or a fraction of the remaining water layer obtained by suspending the mixed solvent extract of and ethanol in solid form in water and sequentially fractionating it with hexane, ethyl acetate, or butanol.