External preparation for skin

Abstract

To find out an agent containing an active ingredient derived from a natural product for improving blood flow and / or improving dark circles of eyes.SOLUTION: A compound represented by the following formula (I): wherein R1 represents hydrogen or a rhamnosyl group, and R2 represents a glucosyl group or a rhamnosyl group. The blood flow improving agent and / or the dark circle improving agent of the eyes contains a compound expressed by general formula (1), its organic acid ester or their salts.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a blood flow improving agent and / or an agent for improving dark circles under the eyes, which contains kaempferol glycoside, its derivatives or salts thereof as an active ingredient, and to an external skin preparation containing said agent. [Background technology]

[0002] Human skin loses its natural functions, such as contractility, flexibility, and moisture retention, due to internal factors such as aging and external factors such as ultraviolet rays and active oxygen, resulting in various skin problems. For example, wrinkles under the eyes and at the corners of the eyes are thought to be primarily caused by a loss of elasticity due to an aging-related decrease in collagen and elastin (loss of firmness), a decrease in the number of cells producing extracellular matrix in the dermis, a decrease and degeneration of collagen fibers, and a decrease in subcutaneous adipose tissue. Conventional methods for improving such wrinkles include, for example, cosmetics and quasi-drugs containing neutrophil elastase inhibitors (trifluoroisopropyloxopropylaminocarbonylpyrrolidinecarbonylmethylpropylaminocarbonylbenzoylaminoacetate sodium (NilOne)), moisturizing ingredients (hyaluronic acid, collagen, elastin), highly moisturizing ingredients (petrolatum, ceramide, hyaluronic acid), firming ingredients (vitamin C derivatives, retinol, collagen), and collagen-promoting ingredients (retinol).

[0003] Kaempferol is a type of natural flavonoid found in many edible plants, such as tea, broccoli, grapefruit, cabbage, kale, beans, arkroot, leek, tomato, strawberry, grapes, Brussels sprouts, apple, quinoa, horseradish, etc. Research has been conducted on natural flavonoids, including kaempferol, focusing on their diverse physiological effects. For example, it has been reported that kaempferol analogs or their glycosides have effects such as improving exercise efficiency, reducing fatigue, and improving dynamic visual acuity (see Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] WO2019 / 044964 Summary of the Invention [Problem to be solved by the invention]

[0005] The problem to be solved by the present invention is to find a material (agent) containing an active ingredient that improves blood flow and / or reduces dark circles under the eyes. [Means for solving the problem]

[0006] In order to solve the above problems, the present inventors have conducted extensive research and discovered that kaempferol glycoside, which is contained in desired natural products (such as carnation flowers), has the effect of improving blood flow and / or alleviating dark circles under the eyes, and have completed the present invention. That is, the present invention includes the following embodiments.

[0007] [1] Formula (I): [ka] (In the formula, R 1 represents a hydrogen atom or a rhamnosyl group, and R 2 represents a glucosyl group or a rhamnosyl group.) A blood flow improver and / or an agent for improving dark circles under the eyes, comprising a compound represented by the formula (I), its organic acid ester, or a salt thereof. [2] The compound represented by formula (I) is a compound represented by the following formula (II):

[0008] [ka] The blood flow improver and / or eye dark circle improver according to [1], wherein the compound is represented by the formula: [3] The compound represented by formula (I) is a compound represented by the following formula (III):

[0009] [ka] or (IV):

[0010] [ka] The blood flow improver and / or eye dark circle improver according to [1], wherein the compound is represented by the formula: [4] The agent according to any one of [1] to [3], wherein the organic acid ester is a malate ester of a compound of any one of formulas (I) to (IV). [5] An external skin preparation for improving blood flow and / or dark circles under the eyes, comprising the agent according to any one of [1] to [4]. [Effects of the Invention]

[0011] The agent of the present invention has the effect of improving blood flow and / or alleviating dark circles under the eyes when used, for example, as an ingredient in cosmetics or quasi-drugs. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows an outline of the method for fractionating the active ingredient of the present invention from carnation flower extract. [Figure 2] FIG. 2 shows the results of measuring the collagen production promoting effect in normal adult human skin fibroblasts. [Figure 3] FIG. 3 shows the results of measuring the proliferation ability of human fibroblasts. [Figure 4] Figure 4 shows the measurement sites in a human skin monitoring test. [Figure 5] FIG. 5 shows the results of confirming the wrinkle improving effect of Test Example 3. [Figure 6] FIG. 6 shows the results of confirming the dermal collagen fiber density improving effect of Test Example 4. [Figure 7] FIG. 7 shows the method for measuring the mechanical parameters of the skin used in Test Example 5. [Figure 8] FIG. 8 shows the results of Test Example 5. [Figure 9] FIG. 9 shows the results of confirming the preventive effect of staining in Test Example 6. DETAILED DESCRIPTION OF THE INVENTION

[0013] Next, various embodiments of the present invention will be described with reference to the drawings. Note that the various embodiments described below do not limit the scope of the invention as claimed, and not all of the elements and combinations thereof described in the various embodiments are necessarily essential to the solution of the present invention.

[0014] (blood flow improver and / or eye dark circle reducer) In one embodiment, the present invention provides a blood flow improving agent and / or an agent for improving dark circles under the eyes, which contains the following active ingredient. The agent of this embodiment is a compound represented by the following formula (I):

[0015] [ka] The compound represented by the formula (I), its organic acid ester, or a salt thereof, is contained as an active ingredient. 1 represents a hydrogen atom or a rhamnosyl group, and R 2 represents a glucosyl group or a rhamnosyl group. In the above formula (I), R 1 is a rhamnosyl group, and R 2 When is a glucosyl group, the compound can be represented by the above formula (II) and is also called kaempferol-3-(6'''-rhamnosyl-2'''-glucosyl-glucoside) or kaempferol-3-O-[2-O-β-D-glucosyl-6-O-α-rhamnosyl]-β-D-glucoside (CAS No. 55696-58-7). It is a kaempferol glycoside in which one rhamnose and two glucoses are linked in a branched chain. In the Examples below, it is referred to as KMP-2. The hydroxy group of the compound of formula (I) may be bound to an organic acid such as malic acid, succinic acid, and / or malonic acid. The addition of these sugars and organic acids stabilizes the aglycone kaempferol and makes it more soluble in water.

[0016] In addition, in formula (I), R 1 is a hydrogen atom and R2 The compound in which R is a glucosyl group can be represented by the above formula (III), and is referred to as KMP-3 in the examples described below. 1 is a hydrogen atom and R 2 When is a rhamnosyl group, the compound can be represented by the above formula (IV), and is referred to as KMP-5 in the examples below.

[0017] The compounds represented by the formulas (I) to (IV) may be in the free form, or may be in the form of a salt if the hydroxy group has a suitable acidity. Examples of the salt of the hydroxy group include alkali metal salts such as lithium salt, sodium salt, and potassium salt; alkaline earth metal salts such as magnesium salt and calcium salt; aliphatic amine salts such as trimethylamine salt, triethylamine salt, dicyclohexylamine salt, ethanolamine salt, diethanolamine salt, triethanolamine salt, and brocaine salt; aralkylamine salts such as N,N-dibenzylethylenediamine; heterocyclic aromatic amine salts such as pyridine salt, picoline salt, quinoline salt, and isoquinoline salt; basic amino acid salts such as arginine salt and lysine salt; quaternary ammonium salts such as tetramethylammonium salt, tetraethylammonium salt, benzyltrimethylammonium salt, benzyltriethylammonium salt, benzyltributylammonium salt, methyltrioctylammonium salt, and tetrabutylammonium salt; and ammonium salts. The free form or a salt of the hydroxy group of Compound (I) of this embodiment may also exist as a hydrate.

[0018] When extracting the active ingredient from carnation, examples of parts that can be used as the extraction material include the petals. Extraction methods include direct extraction using a solvent, as well as extraction by adding a solvent to the squeezed liquid and / or residue obtained after squeezing. A 50% aqueous ethanol solution is preferably used as the extraction solvent. In this embodiment, an ethanol extract using carnation petals is preferably used. The content of the active ingredient in the extract can be measured as a kaempferol glycoside equivalent. In this specification, "kaempferol glycoside equivalent" refers to the amount of the active ingredient of the present invention converted into the mass of a specific standard. Examples of standard include readily available kaempferol glycosides, such as kaempferol-3-glucoside. Specifically, the extract is analyzed, for example, by reverse-phase HPLC (column: Imtakt UK-C18), and the peak area detected by absorbance at 340 nm when separated with an eluent of methanol:phosphoric acid = 37:63 can be converted by applying it to a calibration curve prepared using various concentrations of kaempferol-3-glucoside.

[0019] The blood flow improver and / or dark circle ameliorator of this embodiment may be the active ingredient itself, or may be a composition containing the active ingredient at a predetermined concentration or higher. Preferably, it may be in the form of a plant extract, such as a carnation flower. The amount of the active ingredient contained in the agent of this embodiment is not particularly limited, as long as it is an amount that can improve blood flow and / or alleviate dark circles when added to a topical skin preparation or the like. For example, when adding 0.1% by mass of the blood flow improver and / or dark circle ameliorator to a topical skin preparation, the active ingredient should be contained at a concentration that can still achieve the effects of improving blood flow and / or alleviating dark circles even when diluted 1000 times. Therefore, the lower and upper limits of the amount of the above-mentioned active ingredient contained in the blood flow improver and / or dark circle improver of this embodiment are, in terms of kaempferol glycoside, the lower limit of the converted value is preferably 1 μg / mL, more preferably 5 μg / mL, and even more preferably 10 μg / mL, and the upper limit of the converted value is preferably 1000 μg / mL, more preferably 750 μg / mL, more preferably 500 μg / mL, and even more preferably 250 μg / mL.

[0020] The amount of each of the above-mentioned active ingredients contained in the blood flow improver and / or dark circle improver of this embodiment is expressed as a kaempferol glycoside equivalent value, with the lower limit of this equivalent value being preferably 0.05 μg / mL, more preferably 0.5 μg / mL, and even more preferably 5 μg / mL, and the upper limit of this equivalent value being preferably 500 μg / mL, more preferably 250 μg / mL, and even more preferably 100 μg / mL.

[0021] The blood flow improver and / or dark circle improving agent of this embodiment can be contained in, for example, a skin external preparation, a food or beverage, or a pharmaceutical composition. The food or beverage or pharmaceutical composition can be used as, for example, a food or beverage or oral composition such as a functional food, a specified health food, a health food, a nutritional supplement (supplement), or a medical food. In this case, in addition to the active ingredient, a pharmaceutically acceptable base or carrier, an additive usable in food, or the like can be added to formulate an oral preparation. The skin external preparation containing the blood flow improver and / or dark circle improving agent of this embodiment is described in detail below.

[0022] (blood flow improver and / or eye dark circle reducer) Localized skin conditions (aging, rough skin, transient hot flashes, etc.) and poor physical conditions (stress, lack of sleep, etc.) can impair blood flow (circulation, blood flow). The skin has a network of capillaries known as microcirculation, which supplies nutrients and oxygen to the epidermis and dermis and plays a role in regulating body temperature. This microcirculatory blood flow is reflected on the skin's surface. The skin's vascular structure is complex, and blood flow can vary depending on the depth of the blood vessels within the dermis. Blood flow can be measured with a laser blood flowmeter, but by modifying the probe, it is possible to measure it at different depths in the skin. For example, poor circulation due to a decrease in capillaries in the shallow layers near the skin's surface is thought to be one of the causes of dull skin due to aging and menopause. It has been reported that rough skin is also caused by poor circulation in deeper layers. Dark circles around the eyes are also caused by poor circulation, and by repeatedly cooling and warming the area, the dark circles will fade and disappear if poor circulation is improved. Furthermore, while the capillary network in the surface and middle layers of the skin is not innervated, the arteriovenous anastomoses (bypasses that shunt arterial blood to veins) in the deeper layers of the skin are innervated, which can lead to poor circulation due to stress. On the other hand, the flushing sensation felt when entering a warm environment from a hot or cold place is due to increased blood circulation, which is thought to be due to an expansion of the capillary network in the surface layer and vasodilation in the deeper layers. Decreased blood flow increases the amount of reduced hemoglobin, which appears blue to purple-red. In such conditions, areas where the blood vessels are easily visible, such as those with thin skin or low collagen and elastin content, appear darker than normal. In particular, the color of blood vessels is easily visible around the eyes, so if the blood vessel structure weakens, the skin's appearance becomes dull, making it more likely that dark circles (dullness) will form under the eyes, damaging the overall appearance (quoted from the Japan Society of Cosmetic Chemists website, glossary, poor circulation: https: / / www.sccj-ifscc.com / library / glossary_detail / 512). The blood flow improving agent and / or the agent for improving dark circles under the eyes is an agent for improving the decrease in blood flow, etc., for improving dark circles under the eyes (for example, reducing the size of dark circles (dullness)), etc. In the present invention, improvement includes not only improvement but also, for example, prevention.

[0023] (External skin preparation form) The topical skin preparation according to one embodiment of the present invention is characterized by containing the blood flow improver and / or the dark circle improving agent. The topical skin preparation according to this embodiment may contain only one of the active ingredients, or a combination of two or more of them. By containing the blood flow improver and / or the dark circle improving agent, the topical skin preparation according to this embodiment exhibits excellent wrinkle prevention or improvement effects. Furthermore, the topical skin preparation according to this embodiment may exhibit effects other than the wrinkle prevention or improvement effects. Such effects include moisturizing effect, improvement of actinic or non-actinic keratosis, skin peeling, stimulation of epidermal renewal, and improvement of aging.

[0024] The topical skin preparation of this embodiment may contain optional ingredients that are typically used in topical skin preparations, in addition to the essential ingredients of the blood flow improving agent and / or the agent for improving dark circles under the eyes. Such optional ingredients may include hydrocarbons such as squalane, petrolatum, and microcrystalline wax; esters such as jojoba oil, carnauba wax, and octyldodecyl oleate; triglycerides such as olive oil, beef tallow, and coconut oil; fatty acids such as stearic acid, oleic acid, and retinoic acid; higher alcohols such as oleyl alcohol, stearyl alcohol, and octyldodecanol; anionic surfactants such as sulfosuccinate esters and polyoxyethylene alkyl sulfate sodium; amphoteric surfactants such as alkyl betaine salts; cationic surfactants such as dialkylammonium salts; nonionic surfactants such as sorbitan fatty acid esters, fatty acid monoglycerides, polyoxyethylene adducts thereof, polyoxyethylene alkyl ethers, and polyoxyethylene fatty acid esters; polyhydric alcohols such as polyethylene glycol, glycerin, and 1,3-butanediol; thickeners and gelling agents; antioxidants; ultraviolet absorbers; colorants; preservatives; and powders.

[0025] The blood flow improver and / or dark circle improver of this embodiment and any optional ingredients are processed according to a conventional method to produce 1) pharmaceuticals, 2) quasi-drugs, 3) topical or whole-body skin preparations (for example, basic cosmetics such as lotion, emulsion, cream, ointment, lotion, oil, and pack, face wash and skin cleanser such as solid soap, liquid soap, and hand wash, massage preparation, cleansing agent, hair removal agent, depilatory agent, shaving treatment, aftershave lotion, preshave lotion, shaving cream, foundation, lipstick, blush, eye shadow, eyeliner, etc.) in an appropriate form for use. makeup cosmetics such as nail polish, mascara, perfumes, nail polish, nail polish remover, poultices, plasters, tapes, sheets, patches, aerosols, etc.); 4) medicinal and / or cosmetic preparations to be applied to the scalp and hair (for example, shampoos, rinses, hair treatments, pre-hair treatments, permanent solutions, hair dyes, hair styling products, hair tonics, hair growth and tonics, poultices, plasters, tapes, sheets, aerosols, etc.); 5) bath additives to be added to bathwater; 6) other products such as underarm odor prevention agents, deodorants, antiperspirants, hygiene products, sanitary cotton, wet tissues, etc. The topical skin preparation of this embodiment can be in any dosage form that can be adapted to the skin, but since the active ingredient penetrates the skin to exert its effect, dosage forms that are easily absorbed by the skin, such as lotion, emulsion, cream, and essence, are preferred.

[0026] The lower and upper limits of the amount of the blood flow improver and / or dark circle improving agent contained in the topical skin preparation of this embodiment are, in total, relative to the total amount of the topical skin preparation. The lower limit is preferably 0.001% by mass, more preferably 0.01% by mass, and even more preferably 0.1% by mass, and the upper limit is preferably 10% by mass, more preferably 5% by mass, and even more preferably 2% by mass.

[0027] The lower and upper limits of the content of the above-mentioned active ingredient contained in the topical skin preparation of this embodiment are, in terms of kaempferol glycoside, relative to the total volume of the topical skin preparation. The lower limit of the converted value is preferably 10 ng / mL, more preferably 50 ng / mL, and even more preferably 100 ng / mL, and the upper limit of the converted value is preferably 10 μg / mL, more preferably 7.5 μg / mL, more preferably 5 μg / mL, and even more preferably 2.5 μg / mL.

[0028] The amount of the above-mentioned active ingredient contained in the topical skin preparation of this embodiment is expressed as a kaempferol glycoside equivalent, with the lower limit of this equivalent being preferably 0.05 μg / mL, more preferably 0.5 μg / mL, and even more preferably 5 μg / mL, and the upper limit of this equivalent being preferably 5 μg / mL, more preferably 2.5 μg / mL, and even more preferably 1 μg / mL.

[0029] The present invention will now be described in more detail with reference to the following examples, but the present invention is not limited to these examples. In the following examples, the unit % used to indicate the amount of each component added means % by mass. [Example]

[0030] [Example 1] Preparation of carnation flower extract 100 g of dried fresh carnation flowers (above the calyx) were soaked in 1 kg of 50% aqueous ethanol at room temperature for one week, then filtered to obtain approximately 0.95 kg of crude extract. The crude extract was then concentrated to remove the ethanol, and 50% aqueous butylene glycol was added to obtain 0.95 kg of liquid carnation flower extract. The concentration of the active ingredient in this extract was calculated by converting the peak area obtained by HPLC analysis under the following analytical conditions using a calibration curve with kaempferol-3-glucoside (MERCK) as the standard. The concentration of the active ingredient in the resulting extract was 102.1 μg / mL (90 μg / mL-110 μg / mL) in terms of kaempferol glycoside.

[0031] LC1 Equipment: SHIMADZU LC-20A Column: Mightysil RP-18 GP 250 x 4.6 mm (5 μm) Mobile phase: CH3CN:0.1%H3PO4=(10:90)→(100:0) / 30 min Flow rate: 1mL / min Temperature: 40℃ Detector: SPD M10Avp 340nm

[0032] [Example 2] Purification of kaempferol glycoside The 50% ethanol aqueous solution of carnation flower extract prepared in Example 1 was purified using a hydrophobic adsorption resin. Figure 1 shows an outline of the method for fractionating the active ingredients of carnation flower extract. The 50% ethanol aqueous solution of carnation flower extract was first crudely fractionated using a column packed with Diaion HP20 (Mitsubishi Chemical Corporation). Fraction 03, eluted with 40% ethanol, was diluted with water and further adsorbed on the same column. The collagen production-promoting activity described below was recovered in fraction 13, eluted with 30% ethanol aqueous solution. This fraction 13 was subjected to preparative liquid chromatography (LC1) under the following conditions, and the main peak was recovered.

[0033] Preparative LC1 Equipment: Agilent Technologies (Agilent 1260 InfinityII) Column: Develosil ODS, 7 μm, 20 × 250 mm Flow rate: 10mL / min Detector: Diode Array Detector WR 340nm Mobile phase: MeOH: 0.1% formic acid = 37:63

[0034] This main peak contained multiple components, so this fraction was further subjected to preparative liquid chromatography LC2 under the following conditions, resulting in two separate peaks (fractions 21 and 22).

[0035] Preparative LC2 Equipment: SHIMADZU 10A Column: Mightysil RP-18 GP 4.6 x 250 mm Flow rate: 1mL / min Detector: SPD M10Avp 340nm Mobile phase: MeOH: 0.1 formic acid = 35:65

[0036] When the concentrations of the compounds contained in fractions 21 and 22 were measured under the following conditions (LC2), the values ​​calculated as kaempferol glycoside were 43.4 μg / mL and 58.7 μg / mL, respectively.

[0037] LC2 Equipment: SHIMADZU 10A Mobile phase: methanol: phosphoric acid = 37:63 Flow rate: 0.7mL / min Column: Imtakt Unison UK-C18 Temperature: 40℃ Detector: SPD M10Avp 340nm

[0038] The compounds recovered in fractions 21 and 22 were analyzed by NMR and identified as kaempferol-3-(6'''-rhamnosyl-2'''-(6-malyl-glucosyl)-glucoside) (KMP-1) and kaempferol-3-(6'''-rhamnosyl-2'''-glucosyl-glucoside) (KMP-2), respectively. KMP-1 is a compound in which malic acid is added to the 6-position of the terminal glucosyl group of KMP-2. The measurement data for KMP-2, measured using a JEOL (JEOL Ltd.) JNM-ECA500 nuclear magnetic resonance spectrometer, are shown in Table 1 below. The "a" in Table 1 indicates overlapping signals.

[0039] [Table 1]

[0040] [Test Example 1] Measurement of collagen production We investigated whether administering the active ingredients contained in carnation flower extract would affect collagen production (synthetic ability). Normal human adult skin fibroblasts were used. Pre-culture was performed using DMEM containing 5% FBS, while in this experiment, DMEM containing 1% FBS was used. Culture was performed under conditions of 5% CO2 and 37°C.

[0041] 5×10 4 Normal human adult dermal fibroblasts (Cell Applications) were seeded onto a 24-well plate and cultured until they reached 75% confluence. The medium was then replaced with DMEM containing 1% FBS and cultured for 24 hours. After that, the medium was replaced with fresh DMEM containing 1% FBS, and samples (the carnation flower extract prepared in Example 1 and the KMP-1 and KMP-2 purified in Example 2) were added and cultured for 72 hours. The concentration of type III collagen in the medium was then evaluated using a Human PIIINP (N-Terminal Procollagen III Propeptide) ELISA kit (Elabscience). The results are shown in Figure 2. The concentrations of the active ingredients in the samples were measured by HPLC and adjusted with 50% butylene glycol aqueous solution to the same concentration as the respective compounds contained in the carnation flower extract.

[0042] Figure 2 shows the type III collagen synthesis ability of each sample, with the type III collagen synthesis ability of the untreated group (labeled cnt) set at 1. In Figure 2, the group to which the extract of Example 1 was added (labeled crude) had a value of 1.30, the KMP-1 added group (labeled KMP-1) had a value of 1.21, and the KMP-2 added group (labeled KMP-2) had a value of 1.52. As is clear from Figure 2, the addition of carnation flower extract, KMP-1, and KMP-2 promoted type III collagen production.

[0043] [Test Example 2] Measurement of human fibroblast proliferation ability The carnation flower extract obtained in Example 1 and the two fractions (KMP-1 and KMP-2) purified in Example 2 were evaluated for their ability to proliferate human fibroblasts. Normal human dermal fibroblasts (Cell applications) were cultured in a 96-well plate at 2.5 × 10 cells per well using DMEM medium containing 5% FBS. 3 The normal human dermal fibroblasts were seeded at a cell count of 100 cells / well. After seeding, the normal human dermal fibroblasts were cultured for 24 hours at 37°C and 5% CO2. The DMEM medium containing 5% FBS was then replaced with DMEM medium containing 1% FBS, and the following samples were administered to each well. The normal human dermal fibroblasts were cultured for 5 days at 37°C and 5% CO2. After 5 days of culture, the number of normal human dermal fibroblasts in each group (Sample 1 to Sample 7) was measured using Cell Counting Kit-8 (DOJINDO). Six samples were measured for each group (Sample 1 to Sample 7).

[0044] Sample 1 group: As a control, a predetermined amount of 50% butylene glycol aqueous solution was administered. Sample 2 group: The carnation flower extract obtained in Example 1 was administered at 0.5%. Sample 3 group: The carnation flower extract obtained in Example 1 was administered at 1%. Sample 4 group: KMP-1 was administered at 0.5%. Sample 5 group: KMP-1 was administered at 1%. Sample 6 group: KMP-2 was administered at 0.5%. Sample 7 group: KMP-2 was administered at 1%. The concentrations of the active ingredients contained in samples 4 to 7 correspond to the group containing carnation flower extract (sample 2 or 3), and samples 4 and 6 were prepared in 50% butylene glycol aqueous solution to have the same concentration as the compounds contained in sample 2, and samples 5 and 7 were prepared in 50% butylene glycol aqueous solution to have the same concentration as the compounds contained in sample 3.

[0045] The measurement results are shown in Figure 3. When the fibroblast proliferation potential of sample 1 (labeled cnt) was set to 1, sample 2 and 3 groups (labeled crude) showed fibroblast proliferation potentials of 1.33 and 1.75, respectively, sample 4 and 5 groups (labeled KMP-1) showed 1.03 and 1.34, respectively, and sample 6 and 7 groups (labeled KMP-2) showed 1.16 and 1.10, respectively. As is clear from Figure 3, it was found that carnation flower extract promoted fibroblast proliferation in a concentration-dependent manner, and KMP-1 promoted fibroblast proliferation at 1%. On the other hand, KMP-2 had no effect on cell proliferation.

[0046] [Example 3] Preparation of lotion The lotions used in the monitoring test below were prepared as follows: A lotion containing 1% of the carnation flower extract prepared in Example 1 was prepared with the composition shown in Table 2 (Table 2 lotion). A placebo (lotion not containing the carnation flower extract) was prepared with the composition shown in Table 3.

[0047] [Table 2]

[0048] [Table 3]

[0049] [Test Example 3] Human skin monitoring test (confirmation of wrinkle improvement effect) The study was conducted between December and January 2020 by applying Table 2 Lotion (a lotion described in Table 2 containing carnation flower extract) and placebo (a lotion described in Table 3 without carnation flower extract) to the corners of the mouth (nasolabial folds) of 12 healthy women in their 30s and 40s (mean age 41.2 years) between December and January 2020. The subjects reported subjective symptoms of sagging skin, fine wrinkles, and / or fragile skin. Table 2 Lotion was applied to the left half of the subjects' faces at specified intervals, and placebo was applied to the right half of the subjects' faces at specified intervals. The specified intervals were twice daily (morning and evening) for one month. Then, on day 0 (baseline) before application of these lotions and four weeks after application of the lotions, the surface of the designated area was irradiated with light of a specific wavelength in 3D, photographed, and the images were processed to measure the overall size of wrinkles, as well as the average width and depth of wrinkles (measurement device: skin analyzer "ANTERA3D"). TM ", Gadelius Medical Inc.). The results are shown in Figure 5.

[0050] Figure 5 shows the relative values ​​of the overall wrinkle size, average wrinkle width, and average wrinkle depth when applying placebo (labeled "Placebo" in Figure 5) and Table 2 Lotion (labeled "Table 2 Lotion" in Figure 5), with the value on day 0 set to 100. As shown in Figure 5(a), the overall wrinkle size was 101.4 in the placebo group and 94.7 in the Table 2 Lotion group. As shown in Figure 5(b), the average wrinkle width was 100.0 in the placebo group and 97.5 in the Table 2 Lotion group. Furthermore, as shown in Figure 5(c), the average wrinkle depth was 101.6 in the placebo group and 93.6 in the Table 2 Lotion group. These results indicate that application of Table 2 Lotion reduced the overall wrinkle size, average wrinkle width, and average wrinkle depth. Note that each value in the bar graph is the mean and standard error of measurements from 12 subjects. In the figure, * and ** indicate significant differences of p<0.05 and p<0.01, respectively, by Wilcoxon test when compared with the measurement value on day 0, and † and †† indicate significant differences of p<0.05 and p<0.01, respectively, by Student's t-test.

[0051] [Test Example 4] Human skin monitoring test (dermal collagen fiber density improvement test) Each lotion was applied under the same conditions as in Test Example 3, except that the measurement site was changed from the side of the corner of the mouth (nasolabial folds) to the cheek (side of the nostrils, see Figure 4), and the dermal collagen fiber density at that site was measured using a DermaLab (registered trademark) Combo. The results are shown in Figure 6. This is a device that can be used to visualize and quantify collagen fiber density in the depth direction of the skin based on differences in the reflection intensity (echo) of ultrasound incident from the skin surface.

[0052] The condition of the lateral nasal alar of a subject who applied Table 2 Lotion on Day 0 and Week 4 was observed using DermaLab, and the resulting intradermal images are shown in Figure 6(A). The Intensity Score (echo intensity), a parameter of this measuring device, was visualized as an index of collagen fiber density; the higher this value, the more collagen there was and the better the condition of dermal collagen. Bright areas in the image (the areas indicated by the arrows in Figure 6) indicate high density, while dark areas indicate low density. The epidermis has high ultrasound reflectivity, resulting in highly reflective echoes, which are imaged as white. As is clear from this image, the number of bright areas increased compared to Day 0, indicating an increase in collagen fiber density within the dermis. This Intensity Score was quantified to represent collagen density. Figure 6(B) shows the collagen density relative to the value on Day 0, set at 100, when applying placebo (labeled "Placebo" in Figure 6(B)) and Table 2 Lotion (labeled "Table 2 Lotion" in Figure 6(B)). As shown in Figure 6(B), the placebo group had a density of 96.8, while the group treated with the lotion in Table 2 had a density of 105.2. These results show that application of the lotion in Table 2 significantly increased dermal collagen fiber density. Note that each value in the bar graph represents the mean and standard error of the measurements from 12 people. In the figure, †† indicates a significant difference of p<0.01 in the Student's t-test.

[0053] [Test Example 5] Human skin monitoring test Each lotion was applied under the same conditions as in Test Example 3, except that the measurement site was changed from the side of the corner of the mouth (nasolabial folds) to the lower eyelid (see Figure 4). The softness and resilience of the skin at that site were measured using a Cutometer MPA580. Elasticity was measured using a 2mm probe with a suction port, applying 500mb suction pressure for 5 seconds, and then analyzing the displacement of the deformed skin when the suction was released. Figure 7 shows the displacement on the vertical axis against the measurement time on the horizontal axis. In the figure, (A) represents softness, (B) represents resilience, and B / A represents elasticity (the tendency of a deformed object to return to its original shape). Elasticity decreases with age, so the higher the better.

[0054] The results are shown in Figure 8. Figure 8(A) shows the results for softness, Figure 8(B) shows the results for restoring force, and Figure 8B / A shows the results for elasticity. Each shows the relative values ​​when the placebo (labeled "Placebo" in Figure 8) and the Table 2 Lotion (labeled "Table 2 Lotion" in Figure 8) were applied, with the value on day 0 being 100. As shown in Figure 8(A), the softness of the placebo-applied group was 99.5, while the softness of the Table 2 Lotion-applied group was 104.6. As shown in Figure 8(B), the restoring force of the placebo-applied group was 99.6, while the restoring force of the Table 2 Lotion-applied group was 109.4. Furthermore, as shown in Figure 8(B / A), the elasticity of the placebo-applied group was 99.1, while the elasticity of the Table 2 Lotion-applied group was 105.8. These results show that application of the lotion in Table 2 tended to increase softness, and significantly increased resilience and elasticity. Each value in the bar graph represents the average and standard error of the measurements taken by 12 people. In the figure, * indicates a significant difference of p<0.05 in the Wilcoxon test when compared to the measurement taken on day 0, and † indicates a significant difference of p<0.05 in the Student's t-test.

[0055] [Test Example 6] Human skin monitoring test (preventive effect test with rash) The study was conducted between December and January 2020 by applying the lotion in Table 2 and a placebo to the inner side of either the left or right forearm (see Figure 4) of 12 healthy women in their 30s and 40s (mean age 41.2 years). The subjects were those who reported subjective symptoms of sagging skin, fine wrinkles, and / or fragile skin. The lotion in Table 2 was applied to a designated area on the subjects' left arm at designated intervals, and the placebo was applied to a designated area on the subjects' right arm at designated intervals. This designated interval was twice daily (morning and evening) for one month. Then, on day 0 before application of these lotions and four weeks after application, rubber bands were wrapped around both arms for a designated period, and the volume of the depressions left by the rubber bands was measured after the rubber bands were removed (measurement device: skin analyzer "ANTERA3D"). TM ", Gadelius Medical Inc.).

[0056] The results are shown in Figure 9. The value on day 0 of the depression volume is set to 100, and the relative values ​​when the placebo (labeled "Placebo" in Figure 9) and the lotion in Table 2 (labeled "Lotion in Table 2" in Figure 8) were applied are shown. As shown in Figure 9, the elasticity of the placebo-application group was 96.8, while the elasticity of the lotion in Table 2 application group was 73.2. These results show that application of the lotion in Table 2 significantly reduced the depression volume compared to day 0. Note that each value in the bar graph is the mean and standard error of the measurements from 12 people. In the figure, ** indicates a significant difference of p<0.01 in the Wilcoxon test when compared to the measurement on day 0.

[0057] [Test Example 7-1] Human skin monitoring test (evaluation of dark circles under the eyes) The study involved applying the lotion shown in Table 2 (a lotion containing carnation flower extract as described in Table 2) and a placebo (a lotion containing carnation flower extract as described in Table 3) to the under-eye area of ​​12 healthy women in their 30s and 40s (mean age 41.2 years). The subjects reported subjective symptoms of sagging skin, fine wrinkles, and / or fragile skin. The lotion shown in Table 2 was applied under the left eye of each subject at a predetermined interval, and the placebo was applied under the right eye of each subject at a predetermined interval. The predetermined interval was twice daily (morning and evening) for one month. The surface of the designated area was then photographed with a facial image analyzer (VISIA, Canfield) and image processed to measure the size of dark circles under the eyes on day 0 (baseline) before application of the lotions and four weeks after application. The following measurement results are the average values ​​of the 12 subjects, with relative values ​​calculated below.

[0058] The size of dark circles on day 0 for each group (lotion application group in Table 2, lotion application group in Table 3) is set at 100, and the measurement results (relative values) are shown below. The value for the placebo application group (lotion application group in Table 3) was 102.3, while the value for the lotion application group in Table 2 was 95.8 (*). * indicates a significant difference (p<0.05) compared to the value on day 0 (100) using the Wilcoxon test. It was confirmed that application of the lotion in Table 2 reduced the size of dark circles (improvement of dark circles).

[0059] [Example 4] Preparation of carnation flower extract 100g of dried fresh carnation (cucumber) flowers (above the calyx) was soaked in 1kg of 50% ethanol solution at room temperature for one week, and then filtered to obtain approximately 0.95kg of crude extract. The crude extract was then concentrated to remove the ethanol, and 50% butylene glycol solution was added to obtain 0.95kg of liquid carnation (cucumber) flower extract.

[0060] [Example 5] Purification of kaempferol glycoside (KMP-3) The 50% ethanol aqueous solution of carnation (cucumber) flower extract obtained in Example 4 was purified using a hydrophobic adsorption resin in the same manner as in Example 2. That is, as shown in Figure 1, the 50% ethanol aqueous solution of carnation flower extract was first crudely fractionated using a column packed with Diaion HP20 (Mitsubishi Chemical Corporation). Fraction 03 (Fr03) eluted with 40% ethanol was diluted with water and further adsorbed on the same column. Fraction 14 (Fr14) eluted with 40% ethanol aqueous solution was then subjected to preparative liquid chromatography (LC3) under the following conditions to recover the main peak (fraction 31).

[0061] Preparative LC3 Equipment: Agilent Technologies (Agilent 1260 InfinityII) Column: Develosil ODS, 7 μm, 20 × 250 mm Flow rate: 10mL / min Detector: Diode Array Detector WR 340nm Mobile phase: MeOH: 0.1% formic acid = 37:63

[0062] The concentration of the compound contained in fraction 31 was measured under the following conditions (LC3), and was found to be 48.66 μg / mL in terms of kaempferol glycoside.

[0063] LC3 Equipment: SHIMADZU 10A Mobile phase: methanol: phosphoric acid = 35:65 Flow rate: 0.8mL / min Column: Imtakt Unison UK-C18 Temperature: 40℃ Detector: SPD M10Avp 340nm

[0064] The compound recovered in fraction 31 was analyzed by NMR and found to be kaempferol-3-(2''-glucosyl-glucoside) (KMP-3). The measurement data for KMP-3 measured using a JEOL (JEOL Ltd.) JNM-ECA500 nuclear magnetic resonance spectrometer are shown in Table 6 below.

[0065] [Table 4]

[0066] [Example 6] Purification of kaempferol glycoside (KMP-5) The 50% aqueous ethanol solution of the carnation (quein) flower extract prepared in Example 4 was subjected to preparative liquid chromatography (preparative LC4) under the following conditions to recover the main peak.

[0067] Preparative LC4 Equipment: Agilent Technologies (Agilent 1260 InfinityII) Column: Develosil ODS, 7 μm, 20 × 250 mm Flow rate: 10mL / min Detector: Diode Array Detector WR 340nm Mobile phase: MeOH: 0.1% formic acid = 35:65

[0068] When the concentration of the compound contained in the aqueous solution was measured under the following conditions (LC4), it was found to be 53.53 μg / mL in terms of kaempferol glycoside.

[0069] LC4 Equipment: SHIMADZU 10A Mobile phase: methanol: phosphoric acid = 35:65 Flow rate: 0.8mL / min Column: Imtakt Unison UK-C18 Temperature: 40℃ Detector: SPD M10Avp 340nm

[0070] The compound recovered in fraction 41 was analyzed by NMR and found to be kaempferol-3-neohesperidoside (KMP-5). The measurement data for KMP-5 measured using a JEOL (JEOL Ltd.) JNM-ECA500 nuclear magnetic resonance spectrometer are shown in Table 7 below.

[0071] [Table 5]

[0072] [Test Example 7-2] Human skin monitoring test (evaluation of dark circles under the eyes) The test was conducted by applying the Table 6 pack (a lotion sheet soaked in 1.5 mL of a liquid containing carnation flower extract with the composition shown in Table 6 per sheet) and a control (a lotion sheet soaked in 1.5 mL of a liquid with the composition shown in Table 7 (liquid not containing carnation flower extract) per sheet) under the eyes of a healthy woman in her twenties. The lotion sheets used were lotion sheets (for spot use) manufactured by MUJI. The subject was a person who was subjectively troubled by dark circles under her eyes.

[0073] The Table 6 pack was applied under the left eye of each subject at a predetermined interval, and the control was applied under the right eye of each subject at a predetermined interval. The predetermined interval was set to apply (stick) the Table 6 pack or the control for 10 minutes once a day (after washing the face at night) for 2 weeks (14 days). The surface of the predetermined area was then measured using a skin analyzer (ANTERA3D) on day 0 (baseline) before application of these lotions and on day 2 (14 days) after application of the lotions. TM , Gadelius Medical Co., Ltd.) and the brightness of dark circles under the eyes (L * values) were measured.

[0074] The brightness (L) of each group (Table 6 pack group, control group) on day 0 * The brightness of the control group was 99.59, while the brightness of the Table 6 pack group was 101.18(*). The more dark circles there are, the greater the brightness (L* The L value) is said to be low (Article: J.Soc.Cosmet.Chem.Japan, Vol.38, No.3, 2004, p202-210, Dermatological study of dark circles around the eyes and how to deal with them, Fig.9, etc.). Table 6: The application (sticking) of the pack increased the brightness (L value) compared to the control group. * The high value confirmed improvement in dark circles under the eyes.

[0075] [Table 6]

[0076] [Table 7]

[0077] [Test Example 8] Measurement of collagen production We investigated whether the addition of the two fractions (KMP-3 and KMP-5) purified in Examples 4 and 5 to cells, and the administration of the active ingredients contained in carnation flower extract, would affect collagen production (synthetic ability). Normal human adult skin fibroblasts were used. Preculture was performed using DMEM containing 5% FBS, whereas in this experiment, DMEM containing 1% FBS was used. Culture was performed under conditions of 5% CO2 and 37°C.

[0078] 5×10 4 Normal human adult dermal fibroblasts (Cell Applications) were seeded onto a 24-well plate and cultured until they reached 75% confluence. After that, the medium was replaced with DMEM containing 1% FBS and cultured for 24 hours. After that, the medium was replaced with fresh DMEM containing 1% FBS, and the following samples were added and cultured for 72 hours.

[0079] Sample 1 group: As a control, a predetermined amount of 50% butylene glycol aqueous solution was administered. Sample 2 group: KMP-3 was administered at 0.5%. Sample 3 group: KMP-5 was administered at 0.5%. The concentrations of the active ingredients contained in Samples 2 and 3 were adjusted with a 50% butylene glycol aqueous solution so that they were the same as the concentrations of the respective compounds contained in the carnation (cucumber) flower extract.

[0080] The concentration of type III collagen in the medium was then measured using a Human PIIINP (N-Terminal Procollagen III Propeptide) ELISA kit (manufactured by Elabscience). This measurement was performed on five samples for each group (groups of sample 1 to sample 3).

[0081] The measurement results are described below. The measurement results show the average values ​​measured in each group. If the average value of the Sample 1 group is set to 1, the average value of the Sample 2 group was 1.283, and the average value of the Sample 3 group was 1.379. The average values ​​of the Sample 2 and Sample 3 groups were found to be significantly different (p<0.01) from the average value of the Sample 1 group and the control group using Dunnett's test. It was found that the addition of Sample 2 and Sample 3 groups promoted type III collagen production.

[0082] The above has described the embodiments (including examples) of the present invention with reference to the drawings, but the specific configuration of the present invention is not limited to this, and even if there are design changes, etc., within the scope that does not deviate from the gist of the present invention, they are included in the present invention. [Industrial Applicability]

[0083] An external skin preparation containing the agent of the present invention may be useful as a cosmetic product for improving blood flow and / or dark circles under the eyes.

Claims

1. The following formula (I): 【Chemistry 1】 (In the formula, R １ represents a hydrogen atom or a rhamnosyl group, and R ２ represents a glucosyl group or a rhamnosyl group, or an organic acid ester thereof, or a salt thereof.

2. The compound represented by formula (I) is represented by the following formula (II): 【Chemistry 2】 The agent according to claim 1, which is a compound represented by the formula:

3. The compound represented by formula (I) is a compound represented by formula (III): 【Transformation 3】 or (IV): 【Chemistry 4】 The agent according to claim 1, which is a compound represented by the formula:

4. The agent according to any one of claims 1 to 3, wherein the organic acid ester is a malate ester of a compound of any one of formulas (I) to (IV).

5. An external skin preparation for improving blood flow and / or dark circles under the eyes, comprising the agent according to any one of claims 1 to 4.

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