JP7166577B1 - Collagen production promoter and external preparation for skin containing the same - Google Patents

Abstract

An object of the present invention is to find an agent containing an active ingredient derived from a natural substance that maintains softness of the skin and imparts resilience (firmness) to the skin. A compound represented by the following formula (I): JPEG0007166577000018.jpg69170 (wherein R1 represents a hydrogen atom or a rhamnosyl group and R2 represents a glucosyl group or a rhamnosyl group), an organic acid ester thereof, or them An agent for promoting collagen production or promoting female hormone action containing a salt of [Selection drawing] Fig. 2

Description

cross reference

This application claims priority based on Japanese Patent Application No. 2021-073458 filed on April 23, 2021 and Japanese Patent Application No. 2021-155274 filed on September 24, 2021 in Japan. , the entire contents of which are incorporated herein by reference in their entirety. Also, the entire contents of all patents, patent applications and publications cited in this application are hereby incorporated by reference in their entirety.

TECHNICAL FIELD The present invention relates to an agent for promoting collagen production containing kaempferol glycoside, a derivative thereof or a salt thereof as an active ingredient, and an external preparation for skin containing this agent.

Due to internal factors such as aging and external factors such as ultraviolet rays and active oxygen, the skin's originally maintained functions such as contractility, flexibility, and moisture retention deteriorate, causing various troubles. For example, wrinkles under the eyes and wrinkles at the corners of the eyes are mainly caused by a decrease in collagen and elastin due to aging (a decrease in firmness), a decrease in the number of cells that produce extracellular matrix in the dermis, a decrease in collagen fibers, and a decrease in collagen fibers. It is believed that loss of elasticity occurs due to degeneration, reduction of subcutaneous adipose tissue, and the like. Conventionally, as a method for improving such wrinkles, for example, neutrophil elastase inhibitory ingredients (trifluoride isopropyl oxopropyl aminocarbonyl pyrrolidine carbonyl methyl propyl amino carbonyl benzoyl amino acetate Na (Niel One) as cosmetics / quasi-drugs ), moisturizing ingredients (hyaluronic acid, collagen, elastin), highly moisturizing ingredients (vaseline, ceramide, hyaluronic acid), firmness-improving ingredients (vitamin C derivatives, retinol, collagen), collagen-promoting ingredients (retinol), etc. there is

Kaempferol is a type of natural flavonoid contained in many edible plants such as tea, broccoli, grapefruit, cabbage, kale, legumes, horse chestnut, leek, tomato, strawberry, grape, Brussels sprouts, apple, quinoa, and horseradish. Natural flavonoids, including kaempferol, have been studied with a focus on their various physiological actions. Non-Patent Document 1), and that kaempferol analogues or glycosides thereof have effects such as improving exercise efficiency, alleviating fatigue, and improving dynamic visual acuity (see Patent Document 1).

Y. Fukui, Y.; Tanaka, T.; Kusumi, T.; Iwashita, K.; Nomoto, Phytochemistry. 2003 May;63(1):15-23.

WO2019/044964

The problem to be solved by the present invention is to find a material (agent) containing an active ingredient that maintains the softness of the skin and gives the skin a resilience (firmness).

As a result of intensive studies by the present inventors in order to solve the above problems, it was found that kaempferol glycosides contained in desired natural products (such as carnation flowers) promote collagen production, maintain skin softness, The present invention was completed by discovering that it has an effect of giving elasticity to the skin. That is, the present invention includes the following embodiments.

（式中、Ｒ^１は水素原子又はラムノシル基を示し、Ｒ^２はグルコシル基又はラムノシル基を示す。）で表される化合物、その有機酸エステル又はそれらの塩を含有するコラーゲン産生促進用の剤。
［２］式（Ｉ）で表される化合物、その有機酸エステル又はそれらの塩を含有する、女性ホルモン作用の促進用の剤。
［３］式（Ｉ）で表される化合物が、下記式（ＩＩ）：[1] Formula (I) below:

(In the formula, R ¹ represents a hydrogen atom or a rhamnosyl group, and R ² represents a glucosyl group or a rhamnosyl group.) An agent for promoting collagen production containing a compound represented by the formula, an organic acid ester thereof, or a salt thereof .
[2] An agent for promoting female hormone action, containing a compound represented by formula (I), an organic acid ester thereof, or a salt thereof.
[3] The compound represented by formula (I) is represented by the following formula (II):

で表される化合物である［１］に記載のコラーゲン産生促進用の剤又は［２］に記載の女性ホルモン作用の促進用の剤。
［４］式（Ｉ）で表される化合物が、下記式（ＩＩＩ）：

The agent for promoting collagen production according to [1] or the agent for promoting female hormone action according to [2], which is a compound represented by:
[4] The compound represented by formula (I) is represented by the following formula (III):

又は（ＩＶ）：

or (IV):

で表される化合物である［１］に記載のコラーゲン産生促進用の剤又は［２］に記載の女性ホルモン作用の促進用の剤。
［５］有機酸エステルが、式（Ｉ）～（ＩＶ）の何れかの化合物のリンゴ酸エステルである［１］～［４］のいずれか1つに記載の剤。
［６］［１］～［５］のいずれか１つに記載の剤を含有する皮膚外用剤。
［７］シワ改善用である［６］に記載の皮膚外用剤。
［８］皮膚のコラーゲン密度を増加させるための［６］又は［７］に記載の皮膚外用剤。

The agent for promoting collagen production according to [1] or the agent for promoting female hormone action according to [2], which is a compound represented by:
[5] The agent according to any one of [1] to [4], wherein the organic acid ester is a malic acid ester of any one of formulas (I) to (IV).
[6] An external skin preparation containing the agent according to any one of [1] to [5].
[7] The external preparation for skin according to [6], which is for improving wrinkles.
[8] The external preparation for skin according to [6] or [7] for increasing the collagen density of the skin.

The agent of the present invention, for example, when used as a component of cosmetics or quasi-drugs, has effects such as keeping the skin soft and imparting resilience (firmness) to the skin.

FIG. 1 shows an outline of the method for fractionating the active ingredient of the present invention from carnation flower extract. FIG. 2 shows the results of measuring collagen production-promoting activity in normal human adult skin fibrospores. FIG. 3 shows the results of measuring human fibroblast proliferation ability. FIG. 4 shows the measurement sites of the skin monitor test in humans. 5 shows the results of confirming the wrinkle-improving effect of Test Example 3. FIG. 6 shows the results of confirming the dermal collagen fiber density improving effect of Test Example 4. FIG. The white in the figure is FIG. 7 shows a method for measuring mechanical parameters of the skin used in Test Example 5. 8 shows the results of Test Example 5. FIG. FIG. 9 shows the results of confirming the effect of preventing blemishes in Test Example 6. FIG.

Next, each embodiment of the present invention will be described with reference to the drawings. It should be noted that each embodiment described below does not limit the invention according to the scope of claims, and all of the elements described in each embodiment and combinations thereof are means for solving the present invention. is not necessarily required for

(agent for promoting collagen production)
In one embodiment, the present invention provides an agent for promoting collagen production containing the following active ingredients. The agent of this embodiment has the following formula (I):

で表される化合物、その有機酸エステル又はそれらの塩、を有効成分として含む。ここで、上記式中、Ｒ^１は水素原子又はラムノシル基を示し、Ｒ^２はグルコシル基又はラムノシル基を示す。上記式（Ｉ）において、Ｒ^１がラムノシル基であり、かつＲ^２がグルコシル基の場合の化合物は上述した式（ＩＩ）で表すことができ、ケンフェロール－３－（６’’’－ラムノシル－２’’’－グルコシル－グルコシド）、又はケンフェロール－３－Ｏ－［２－Ｏ－β－Ｄ－グルコシル－６－Ｏ－α－ラムノシル］－β－Ｄ－グルコシド（ＣＡＳ Ｎｏ．５５６９６－５８－７）とも称する。１つのラムノースと２つのグルコースが分岐鎖状に結合したケンフェロール配糖体である。後述する実施例では、ＫＭＰ－２と称する。式（Ｉ）の化合物のヒドロキシ基には、リンゴ酸、コハク酸及び／又はマロン酸等の有機酸が結合していてもよく、これらの糖や有機酸が付くことでアグリコンであるケンフェロールを水に溶けやすく安定化している。

A compound represented by, an organic acid ester thereof, or a salt thereof is included as an active ingredient. Here, in the above formula, ^R1 represents a hydrogen atom or a rhamnosyl group, and ^R2 represents a glucosyl group or a rhamnosyl group. In the above formula (I), when R ¹ is a rhamnosyl group and R ² is a glucosyl group, the compound can be represented by the above formula (II), 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. It will be referred to as KMP-2 in the examples to be described later. An organic acid such as malic acid, succinic acid and/or malonic acid may be bound to the hydroxy group of the compound of formula (I). Soluble and stable in water.

In formula (I), a compound in which R ¹ is a hydrogen atom and R ² is a glucosyl group can be represented by the above-described formula (III), and will be referred to as KMP-3 in the examples described later. . Furthermore, in formula (I), a compound in which R ¹ is a hydrogen atom and R ² is a rhamnosyl group can be represented by the above-described formula (IV), and will be referred to as KMP-5 in the examples described later. .

The compounds represented by the above formulas (I) to (IV) may be in the free form, or may be in the form of a salt when the hydroxy group has moderate acidity. Examples of hydroxy group salts include alkali metal salts such as lithium salts, sodium salts and potassium salts; alkaline earth metal salts such as magnesium salts and calcium salts; trimethylamine salts, triethylamine salts, dicyclohexylamine salts, ethanolamine salts, Aliphatic amine salts such as diethanolamine salts, triethanolamine salts and brocaine salts; aralkylamine salts such as N,N-dibenzylethylenediamine; heterocyclic aromatic amine salts such as pyridine salts, picoline salts, quinoline salts and isoquinoline salts; basic amino acid salts such as arginine salts and lysine salts; Quaternary ammonium salts; ammonium salts and the like. In addition, the free form of compound (I) of the present embodiment or the salt of the hydroxy group may exist as a hydrate.

In the case of extracting the active ingredient from carnations, the part that can be used as an extraction raw material is, for example, petals. As an extraction method, in addition to direct extraction using a solvent, extraction can be performed by adding a solvent to the press liquid and/or residue obtained after the pressing treatment. Preferably, a 50% aqueous ethanol solution is used as the extraction solvent. In this embodiment, it is preferable to use an ethanol extract using carnation petals. The content of active ingredients in the extract can be measured in terms of kaempferol glycosides. As used herein, "kaempferol glycoside equivalent value" means a value obtained by converting the amount of the active ingredient of the present invention into the mass of a specific standard product. Standards include, for example, 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 the absorbance at 340 nm when separated with an eluent of methanol: phosphoric acid = 37:63. can be converted by applying a calibration curve prepared using various concentrations of kaempferol-3-glucoside.

The agent for promoting collagen production of the present embodiment may be the active ingredient itself or 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 carnation flower. The amount of the active ingredient contained in the agent of the present embodiment is not particularly limited as long as the agent exhibits a collagen production promoting action when added to an external skin preparation or the like. For example, when adding 0.1% of a collagen production-promoting agent in terms of mass to an external skin preparation, if the active ingredient is contained at a concentration that can exhibit a collagen production-promoting effect even when diluted 1000 times. good. Therefore, the lower limit and upper limit of the amount of the active ingredient contained in the agent for promoting collagen production of the present embodiment are the kaempferol glycoside conversion value, and the lower limit of the conversion value is preferably 1 μg/mL, more preferably is 5 μg/mL, more preferably 10 μg/mL, and the upper limit of the conversion value is preferably 1000 μg/mL, more preferably 750 μg/mL, more preferably 500 μg/mL, and still more preferably 250 μg/mL. be.

The amount of each of the above active ingredients contained in the agent for promoting collagen production of the present embodiment is preferably 0.05 μg/mL, more preferably 0.05 μg/mL, more preferably 0.05 μg/mL as the lower limit of the equivalent value of kaempferol glycoside. It is 5 μg/mL, more preferably 5 μg/mL, and the upper limit of this converted value is preferably 500 μg/mL, more preferably 250 μg/mL, and still more preferably 100 μg/mL.

The agent for promoting collagen production of the present embodiment can be contained in, for example, external skin preparations, food and drink, or pharmaceutical compositions. The food, drink or pharmaceutical composition can be used as food or drink such as food with function claims, food for specified health use, health food, nutritional supplement (supplement), medical food, or oral composition. In that case, in addition to the above active ingredients, pharmaceutically acceptable bases and carriers, additives that can be used in foods, and the like can be added to formulate orally administered preparations. The external preparation for skin containing the agent for promoting collagen production of the present embodiment will be described in detail below.

(female hormone)
Female hormones are steroid hormones that govern reproductive functions, and are a general term for estrogen (follicle hormone) and progesterone (luteinizing hormone). Conjugates, or female hormone-like substances similar thereto. Among them, estrogen plays a role in the development and maintenance of functions of female reproductive organs, and is used as a general term for hormones exhibiting estrous action and substances having similar actions. Estrone, estradiol, estriol, and estetrol exist in nature, and estradiol is the main one. Synthetic estrogens having biological activities equivalent to those of them are also known, and estrogens have a stilbene structure, except for some synthetic forms such as stilbene sterols.

Estrogen is mainly secreted from the ovarian granulosa cells, but is also secreted from the fetal placental system, adrenal glands, ovaries, etc. during pregnancy. Estrogen secretion from the ovary is governed by gonadotropin secreted from the anterior pituitary gland, but conversely, estrogen has a feedback effect on the diencephalic pituitary type, and the interaction between the two establishes the estrous cycle. .

Estrogen acts through its receptors, and its main physiological actions are the proliferation of the endometrium, the development of the uterine muscle, the expression of secondary sexual characteristics, the mediation of the establishment of the menstrual cycle, the induction of maternal changes during pregnancy, and the mammary glands. It promotes proliferation and secretion of ducts. Furthermore, since estrogen has receptors not only in the target tissues of the diencephalon-anterior pituitary gland-reproductive organs and mammary glands, but also in various tissues, it has effects not only on reproductive function but also on brain, cardiovascular, Acting on the skeleton, skin, and other tissues throughout the body, it is deeply involved in the health of adult women and is regarded as the hormone that creates women.
Therefore, in the present specification, an agent that promotes one or more of the effects of female hormones as exemplified above is referred to as "an agent for promoting female hormone action" or "an agent for promoting female hormone-like action". are used interchangeably.

(Form of external preparation for skin)
An external preparation for skin in one embodiment of the present invention is characterized by containing the aforementioned agent for promoting collagen production. The external preparation for skin of the present embodiment may contain only one of the above active ingredients, or may contain two or more in combination. The external preparation for skin of the present embodiment exhibits an excellent preventive or ameliorating effect on wrinkle formation by containing the collagen production-promoting agent. In addition, the external preparation for skin of the present embodiment may exert an action other than the action of preventing or improving wrinkles. Such actions include moisturizing action, action for improving actinic keratosis or non-actinic keratosis, exfoliation of skin, stimulating epidermal renewal, and improving aging action.

The external preparation for skin of this embodiment can contain optional ingredients that are usually used in external preparations for skin, in addition to the agent for promoting collagen production, which is an essential component. Examples of such optional ingredients 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, and stearic acid. , fatty acids such as oleic acid and retinoic acid, higher alcohols such as oleyl alcohol, stearyl alcohol and octyldodecanol, anionic surfactants such as sulfosuccinic acid esters and sodium polyoxyethylene alkylsulfate, and amphoteric surfactants such as alkyl betaine salts. agents, cationic surfactants such as dialkylammonium salts, sorbitan fatty acid esters, fatty acid monoglycerides, polyoxyethylene adducts thereof, nonionic surfactants such as polyoxyethylene alkyl ethers and polyoxyethylene fatty acid esters, polyethylene Glycol, glycerin, polyhydric alcohols such as 1,3-butanediol, thickening/gelling agents, antioxidants, ultraviolet absorbers, colorants, preservatives, powders, and the like can be contained.

The agent for promoting collagen production of the present embodiment and optional ingredients are processed according to a conventional method, and are used in suitable forms such as ampoules, capsules, powders, granules, liquids, gels, bubbles, emulsions, sheets, mists, and sprays. 1) pharmaceuticals, 2) quasi-drugs, 3) topical or systemic external skin preparations (e.g. lotions, milky lotions, creams, ointments, lotions, oils, packs and other basic cosmetics, solid soaps, Liquid soap, facial cleanser such as hand wash, skin cleanser, massage agent, cleansing agent, hair remover, depilator, shaving agent, aftershave lotion, preshave lotion, shaving cream, foundation, lipstick, blusher, eye Make-up cosmetics such as shadows, eyeliners, mascara, perfumes, nail agents, nail enamels, nail enamel removers, poultices, plasters, tapes, sheets, patches, aerosols, etc.), 4) Medicinal and/or cosmetic formulations applied to the scalp and hair (e.g., shampoos, rinses, hair treatment agents, pre-hair treatment agents, permanent liquids, hair dyes, hair styling agents, hair tonics, hair restorers, hair tonics, poultices, plasters, tapes, sheets, aerosols, etc.), 5) Bathing agents to be used in bath water, 6) Other anti-armpit odor agents, deodorants, antiperspirants, sanitary products , sanitary cottons, wet tissues, and the like. The external preparation for skin of the present embodiment can be in any dosage form as long as it can be applied to the skin. Usable formulations such as lotions, milky lotions, creams, and essences are preferred.

The lower and upper limits of the amount of the agent for promoting collagen production contained in the external skin preparation of the present embodiment are the total amount with respect to the total amount of the external skin preparation, and the lower limit is preferably 0.001% by mass, more preferably. is 0.01% by mass, 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.

The lower limit and upper limit of the content of the active ingredient contained in the external skin preparation of the present embodiment are preferably 10 ng/ mL, more preferably 50 ng/mL, more preferably 100 ng/mL, and the upper limit of the conversion value is preferably 10 μg/mL, more preferably 7.5 μg/mL, more preferably 5 μg/mL, and even more preferably is 2.5 μg/mL.

The amount of the active ingredient contained in the external skin preparation of the present embodiment is calculated as a kaempferol glycoside conversion value, and the lower limit of this conversion value is preferably 0.05 μg/mL, more preferably 0.5 μg/mL. , more preferably 5 μg/mL, and the upper limit of this conversion value is preferably 5 μg/mL, more preferably 2.5 μg/mL, and even more preferably 1 μg/mL.

EXAMPLES Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples. Incidentally, in the following examples, the unit % of numerical values indicating the addition amount of various components means % by mass.

[Example 1] Preparation of carnation flower extract 100 g of dried fresh carnation flower parts (above the calyx) were immersed in 1 kg of a 50% ethanol aqueous solution at room temperature for 1 week, filtered, and filtered to give a crude extract of about 1 kg. 0.95 kg was obtained. Next, after concentrating the crude extract and evaporating the ethanol, a 50% butylene glycol aqueous solution was added to obtain 0.95 kg of liquid carnation flower extract. The concentration of the active ingredient in this extract was converted from the peak area of the HPLC analysis under the following analytical conditions using a calibration curve using kaempferol-3-glucoside (MERCK) as a standard. The concentration of the active ingredient in the resulting extract is 102.1 μg/mL (90 μg/mL to 110 μg/mL) in terms of kaempferol glycosides.

LC1
Device: Shimadzu LC-20A
Column: Mightysil RP-18 GP 250×4.6 mm (5 μm)
Mobile phase: CH3CN: 0.1% _H3PO4 = ₍ 10:90) → (100:0)/30 min Flow rate: 1 mL/min Temperature: 40°C
Detector: SPD M10Avp 340nm

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

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

Since this main peak contained multiple components, this fraction was further subjected to liquid chromatography preparative LC2 under the following conditions to obtain two single peaks (fractions 21 and 22). was taken.

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

When the concentrations of the compounds contained in fractions 21 and 22 were measured under the following conditions (LC2), they were 43.4 μg/mL and 58.7 μg/mL, respectively, in terms of kaempferol glycosides.

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

NMR analysis of the compounds recovered in fractions 21 and 22 revealed kaempferol-3-(6'''-rhamnosyl-2'''-(6-malyl-glucosyl)-glucoside) (KMP-1), respectively. and kaempferol-3-(6''-rhamnosyl-2'''-glucosyl-glucoside) (KMP-2). KMP-1 is a compound in which malic acid is added to the 6-position of the terminal glucosyl group of KMP-2. Table 1 below shows measurement data of KMP2 measured with a nuclear magnetic resonance apparatus JEOL (JEOL Ltd.) JNM-ECA500. Note that "a" shown in Table 1 indicates overlapping signals.

[Test Example 1] Measurement of Collagen Production Amount It was confirmed whether or not the amount of collagen production (synthesis ability) changed when the above-mentioned active ingredients contained in the carnation flower extract were administered. Normal human adult skin fibroblasts were used as cells. DMEM containing 5% FBS was used for preculture, and DMEM containing 1% FBS was used for this experiment. Culturing was carried out under conditions of 5% CO ₂ and 37°C.

5×10 ⁴ normal human adult skin fibroblasts (Cell applications) were seeded in 24-well plates and cultured to 75% confluence. After that, replace with DMEM containing 1% FBS, culture for 24 hours, replace with new DMEM containing 1% FBS, sample (carnation flower extract prepared in Example 1 and KMP purified in Example 2 -1 and KMP-2) were added and cultured for 72 hours. Thereafter, the concentration of type 3 collagen in the medium was evaluated using Human PIIINP (N-Terminal Procollagen III Propeptide) ELISA kit (manufactured by Elabscience). The results are shown in FIG. The concentrations of the active ingredients in the samples were measured by HPLC, and prepared with a 50% butylene glycol aqueous solution so as to have the same concentration of each compound contained in the carnation flower extract.

FIG. 2 shows the type 3 collagen synthesizing ability of each sample when the type 3 collagen synthesizing ability of the non-addition group (indicated by cnt) is set to 1. FIG. In FIG. 2, the extract addition group of Example 1 indicated as crude was 1.30, the KMP1 addition group indicated as KMP1 was 1.21, and the KMP-2 addition group indicated as KMP-2 was 1.52. there were. As is clear from FIG. 2, it was found that the addition of carnation flower extract, KMP-1 and KMP-2 promoted type 3 collagen production.

[Test Example 2] Measurement of human fibroblast proliferation ability Human fibroblasts of the carnation flower extract obtained in Example 1 and the two fractions (KMP-1 and KMP-2) purified in Example 2 The presence or absence of proliferation ability was evaluated. Using DMEM medium containing 5% FBS, normal human skin fibroblasts (Cell applications) were seeded in a 96-well plate at 2.5×10 ³ cells/well. After seeding, the normal human skin fibroblasts were cultured for 24 hours in an environment of 37° C. and 5% CO ₂ . After that, after replacing the DMEM medium containing 5% FBS with the DMEM medium containing 1% FBS, the following samples were administered to each well, and the normal human dermal fibers were kept for 5 days in an environment of 37 ° C. and 5% CO ₂ . Blast cells were cultured. After culturing for 5 days, the number of normal human skin fibroblasts in each group (sample 1 to sample 7 groups) was measured using Cell Counting Kit-8 (DOJINDO). The measurement was performed on 6 samples in each group (groups of samples 1 to 7).

- Sample 1 group: As a control, a predetermined amount of 50% butylene glycol aqueous solution was administered.
- Group of sample 2: The carnation flower extract obtained in Example 1 was administered so as to be 0.5%.
- Group of sample 3: The carnation flower extract obtained in Example 1 was administered to 1%.
• Sample 4 group: KMP-1 was administered to 0.5%.
• Sample 5 group: KMP-1 was administered at 1%.
• Sample 6 group: KMP-2 was administered to 0.5%.
• Sample 7 group: KMP-2 was administered at 1%.
The concentration of the active ingredient contained in samples 4 to 7 corresponds to the group containing the carnation flower extract (sample 2 or 3), samples 4 and 6 are contained in sample 2, and samples 5 and 7 are contained in sample 3. A sample prepared with a 50% butylene glycol aqueous solution so as to have the same concentration as each compound used was used as a sample.

The measurement results are shown in FIG. When the fibroblast proliferation ability of the sample 1 group (indicated as cnt) is set to 1, the sample 2 and 3 groups (indicated as crude) are 1.33 and 1.75, respectively, and the sample 4 and 5 groups (indicated as KMP-1) showed a fibroblast proliferation potency of 1.03 and 1.34, respectively, and the groups of samples 6 and 7 (labeled KMP2) showed a fibroblast proliferation potency of 1.16 and 1.10, respectively. As is clear from FIG. 3, it was found that the carnation flower extract dose-dependently promoted the proliferation of fibroblasts, and KMP-1 at 1% promoted the proliferation of fibroblasts. On the other hand, KMP-2 had no effect on cell proliferation.

[Example 3] Preparation of lotion A lotion used in the following monitor test was prepared as follows. A lotion having the composition shown in Table 2 and containing 1% of the carnation flower extract prepared in Example 1 (lotion shown in Table 2) was prepared. A placebo (lotion containing no carnation flower extract) was prepared with the composition shown in Table 3.

[Test Example 3] Human skin monitor test (confirmation of wrinkle improvement effect)
12 healthy women in their 30s to 40s (average age 41.2 years old) were applied to the corners of the mouth (nasolabial folds, see Fig. 4) on the face, using the lotion shown in Table 2. Carnation flower extraction The study was conducted between December and January 2020 by applying a lotion containing carnation flower extract) and a placebo (a lotion listed in Table 3, but without carnation flower extract). The subjects are those who feel sagging skin, fine wrinkles and/or fragile skin as subjective symptoms. Table 2 lotion was applied to the left half of the subject's face at predetermined intervals, and the placebo was applied to the right half of the subject's face at predetermined intervals. This predetermined interval was applied twice a day (morning and evening) for one month. Then, on the 0th day (baseline) before the application of these lotions and 4 weeks after the application of the lotions, the surface of the predetermined site was photographed in 3D with light of a specific wavelength, and image processing was performed. , the size of the entire wrinkle, the average width and average depth of wrinkles were measured (measurement device: skin analyzer "ANTERA3D ^™ ", Gadelius Medical Co.). The results are shown in FIG.

Figure 5 shows the overall wrinkle size, average width and average depth of wrinkles, with the value on day 0 set to 100, placebo (labeled "placebo" in Figure 5) and Table 2 lotion ("Table 2" in Figure 5) It shows the relative value when applying "lotion"). As shown in FIG. 5( a ), the overall size of wrinkles was 101.4 for the placebo-applied group and 94.7 for the lotion-applied group in Table 2. As shown in FIG. 5(b), the average wrinkle width was 100.0 for the placebo-applied group and 97.5 for the lotion-applied group in Table 2. In addition, as shown in FIG. 5(c), the average wrinkle depth was 101.6 for the placebo application group and 93.6 for the lotion application group in Table 2. From these results, application of the lotion shown in Table 2 reduced the overall size of wrinkles, the average width and average depth of wrinkles. Each value in the bar graph is shown as the average value and standard error of the measured values of 12 people. In the figure, * and ** indicate a significant difference of p<0.05 and p<0.01, respectively, by Wilcoxon's test when compared to day 0 measurements, † and †† indicates a significant difference with p<0.05 and p<0.01 by Student's t-test.

[Test Example 4] Human skin monitoring test (dermis 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 fold) to the cheek (side of the nasal alar, see FIG. 4), and the dermal collagen of the site was measured. Fiber density was measured using the DermaLab® Combo. The results are shown in FIG. This device can be used as a device for visualizing and quantifying the density of collagen fibers in the depth direction of the skin based on the difference in reflected intensity (echo) of ultrasonic waves incident from the skin surface.

The condition of the side of the nasal alar of the subject to whom the lotion was applied in Table 2 was observed on day 0 and week 4 with DermaLab, and the obtained intradermal image is shown in FIG. 6(A). Intensity Score (echo intensity), which is a parameter of this measuring device, is imaged as an index of collagen fiber density, and the larger this value, the richer the collagen and the better the state of the dermal collagen. Bright areas in the image (areas indicated by arrows in FIG. 6) indicate high density, and dark areas indicate low density. The epidermis is highly reflective to ultrasound, becomes a highly reflected echo, and is imaged white. As is clear from this image, the bright areas increased compared to day 0, and the density of collagen fibers in the dermis increased. This Intensity Score was quantified and used as the collagen density. FIG. 6(B) shows the collagen density, with the value on day 0 set to 100, placebo (indicated as "placebo" in FIG. 6(B)) and Table 2 lotion ("Table 2 lotion" in FIG. 6(B) ”) is applied. As shown in FIG. 6(B), the placebo application group was 96.8, while the Table 2 lotion application group was 105.2. From these results, application of the lotion shown in Table 2 significantly increased the dermal collagen fiber density. Each value in the bar graph is the average value and standard error of the measured values of 12 people. In the figure, †† indicates a significant difference of p<0.01 by Student's t-test.

[Test Example 5] Human skin monitor test Each lotion 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 fold) to the lower eyelid (see Fig. 4) was applied, and the softness and resilience of the skin at the site were measured using a cutometer MPA580. Elasticity was measured using a cutometer by applying suction pressure of 500 mb for 5 seconds using a probe with a suction port of 2 mm, and then analyzing the displacement of the deformed skin when released. FIG. 7 shows displacement on the vertical axis against measurement time on the horizontal axis. In the figure, (A) indicates softness, (B) indicates restoring force, and B/A indicates elasticity (property of a deformed object returning to its original shape). The higher the elasticity, the better, since it declines with age.

The results are shown in FIG. FIG. 8(A) shows the results for softness, FIG. 8(B) for restoring force, and FIG. 8B/A for elasticity. With the value on day 0 set to 100, relative values are shown when placebo (indicated as "placebo" in FIG. 8) and lotion in Table 2 (indicated as "lotion in Table 2" in FIG. 8) were applied. As shown in FIG. 8(A), the softness of the placebo-applied group was 99.5, while the softness of the lotion-applied group in Table 2 was 104.6. As shown in FIG. 8(B), the restoring force of the placebo-applied group was 99.6, while the restoring force of the lotion-applied group in Table 2 was 109.4. Further, as shown in FIG. 8(B/A), the elasticity of the placebo-applied group was 99.1, while the elasticity of the lotion-applied group in Table 2 was 105.8. From these results, the application of the lotion shown in Table 2 tended to increase the softness and significantly increased the restoring force and elasticity. Each value in the bar graph is shown as the average value and standard error of the measured values of 12 people. In the figure, * indicates a significant difference of p < 0.05 by Wilcoxon test when compared to day 0 measurements, † indicates a significant difference of p < 0.05 by Student's t-test. indicates that there is

[Test Example 6] Skin monitor test in humans (preventive effect test with atto)
12 healthy women in their 30s to 40s (average age 41.2 years old) on either the left or right inner forearm (see Figure 4) by applying Table 2 lotion and placebo, 12 in 2020 The test was conducted between May and January. The subjects are those who feel sagging skin, fine wrinkles and/or fragile skin as subjective symptoms. The lotion in Table 2 was applied to a predetermined area on the left arm of the subject at predetermined intervals, and the placebo was applied to a predetermined area on the right arm of the subject at predetermined intervals. This predetermined interval is application twice a day (morning and evening) for one month. Then, on the 0th day before application of these lotions and on the 4th week after application of the lotions, a rubber band was wrapped around both arms for a predetermined time, and the volume of the indentation left by the rubber band after removing the rubber band was measured. (Measuring device; skin analyzer "ANTERA3D ^™ ", Gadelius Medical, Inc.).

The results are shown in FIG. Regarding the pit volume, the value on day 0 is set to 100, and the placebo (indicated as "placebo" in FIG. 9) and Table 2 lotion (indicated as "Table 2 lotion" in FIG. 8) are shown. . As shown in Figure 9, the elasticity of the placebo-applied group was 96.8, while the elasticity of the Table 2 lotion-applied group was 73.2. From these results, the application of the lotion in Table 2 significantly decreased the volume of the pits compared to the 0th day. Each value in the bar graph is the average value and standard error of the measured values of 12 people. In the figure, ** indicates significant difference with p<0.01 by Wilcoxon test when compared to day 0 measurements.

[Test Example 7] Confirmation test of female hormone-like action (5α-reductase enzyme activity)
In dermal papilla cells, depending on the presence or absence of the addition of carnation flower extract (CFE) and the addition of KMP-1 and KMP-2 contained in the extract, androgenic hormone Testosterone (T) is transformed into Dihydrotestosterone (DHT ) and confirmed changes in the amount secreted.

First, the following were prepared.
- Carnation flower extract of Example 1 above: In the description of Test Example 7, hereinafter referred to as "extract".
- The composition of the main peak (containing KMP-1 and KMP-2) obtained in the purification process of kaempferol glycoside in Example 2 above: described as "KMPs" hereinafter in the description of Test Example 7.
- Adult human dermal papilla cells: Cell Applications, San Diego, CA, USA (HFDPC, human hair dermal papilla cells (adult), CA60205a)
Dermal papilla cell culture medium: PromoCell, Heidelberg, Germany (dermal papilla cell growth medium kit: Follicle Dermal Papilla Cell Growth Medium Kit (product code: C-26502)). FCS (Footal Calf Serum) listed as a supplement included in this product code was also added to this medium.
- FBS (Fetal Bovine Serum): Merck, Fetal Bovine Serum (Cat#173012).
· Dulbecco's Modified Eagle Medium (DMEM) Medium: Fuji Film Wako Pure Chemical Industries, Ltd., D-MEM (low glucose) (containing L-glutamine and phenol red), 041-29775.
• Testosterone (T): Fujifilm Wako Pure Chemical (Cat#201-20551).
- β-estradiol (E2): (FUJIFILM Wako Pure Chemical Industries, Ltd., 052-04041).
• Dihydrotestosterone ELISA Kit: Abnova, KA1886.
- Human 3-oxo-5-alpha-steroid 4-dehydrogenase 2 (SRD5A2) ELISA kit: CUSABIO, CSB-EL022654HU.

(1) Step of Raising Adult Human Dermal Papilla Cells Adult human dermal papilla cells were seeded in a 24-well plate at a cell number of 2×10 ⁴ cells/well. After the seeding, the cells were cultured under conditions of 5% CO ₂ and 37° C. for 24 hours. After the culture, the medium was replaced with DMEM medium containing 1% FBS. The cells were cultured in the replaced medium under conditions of 5% CO ₂ and 37° C. for 24 hours.

(2) Step of adding T etc. after step (1) After step (1), the medium was replaced with a 1% FBS/DMEM medium. At that time, in the 1% FBS/DMEM medium, each group was added (or not added) with T etc. as follows. Six samples (n=6) were prepared for each group for the measurement of the amount of DHT described below, and four samples (n=4) were prepared for each group for the measurement of the amount of 5α-reductase type II. .
- Unadded group: A group to which T was not added.
• Control group: group to which T (final concentration 10 nM) was added.
- E2 group: Group to which T (final concentration 10 nM) and E2 (final concentration 10 nM) were added. In this Test Example 7, an experimental group positioned as a positive control (Reference: Eur. J. Dermatol. 2001, 11, 195-198, Influence of estrogens on the androgen metabolism in different subunits of human hair follicle.)
• Extract 0.5 group: group to which T (final concentration 10 nM) and extract 0.5% were added.
• Extract 1.0 group: Group to which T (final concentration 10 nM) and extract 1.0% were added.
- KMPs 0.025 group - Group to which T (final concentration 10 nM) and KMPs 0.025% were added. This addition (addition of 0.025% KMPs) was performed assuming the same amount of KMPs contained in the 0.5 extract group.
- KMPs 0.050 group: Group to which T (final concentration 10 nM) and KMPs 0.050% were added. This addition (addition of 0.050% KMPs) was performed assuming the same amount of KMPs contained in the extract 1.0 group.

After creating these groups, they were cultured under conditions of 5% CO ₂ and 37° C. for 72 hours. After that, the supernatant was collected, and the amount of DHT in the supernatant was measured using Dihydrotestosterone ELISA Kit. The measurement results are shown in Table 4 below.

Numerical values (relative values) shown in Table 4 were calculated as follows. First, the average value of the measurement results for each group (n=6) was calculated. Next, regarding the average value, the numerical value of each group was calculated relative to the control group, with the numerical value of the control group of each group being set to 1. ** indicates data showing a significant difference (p<0.01) from the control group by Student's T-test. †† indicates data showing a significant difference (p<0.01) from the control group in Dunnett's test.

Furthermore, using a cell suspension homogenized with a Passive Lysis Buffer (manufactured by Promega) to which protease and phosphatase inhibitors were added after the culture, the amount of 5α-reductase type II was adjusted to Human 3-oxo-5- Measured using an alpha-steroid 4-dehydrogenase 2 (SRD5A2) ELISA kit. The obtained values were corrected by determining the amount of protein in the cell suspension by the Bradford method. The results of this measurement are shown in Table 5 below. This measurement was performed with reference to a paper (J. Cosmet. Dermatol. 2019, 18, 414-421.).

Numerical values (relative values) shown in Table 5 were calculated as follows. First, the average value of the measurement results for each group (n=4) was calculated. Next, regarding the average value, the numerical value of each group was calculated relative to the control group, with the numerical value of the control group of each group being set to 1. ** indicates data showing a significant difference (p<0.01) from the control group by Student's T-test. †† indicates data showing a significant difference (p<0.01) from the control group in Dunnett's test.

From the results shown in Tables 4 and 5, it was confirmed that the addition of the extract and KMPs had the same effect as the E2 addition group, that is, the female hormone-like action.
Estrogen (estradiol) increases sex hormone binding globulin (SHBG) and suppresses activation of active testosterone (free testosterone) and androgen receptor (Reference: J Am Acad Dermatol. 2016, 74, 945-973, Reference: Endocr Rev. 2000, 21, 363-392). Estrogen suppresses 5α-reductase activity and reduces the conversion of testosterone to DHT (Reference: J Am Acad Dermatol, 2019, 80, 1509-1521, Reference: Eur J Obstet Gynecol Reprod Biol. 2004, vol 112, 136-141). DHT is related to female pattern baldness (FPHL), and the amount of 5α-reductase in the hair follicle shows a higher value than healthy subjects (Reference: Arch Dermatol Res. 2018, 310, 77- 83).
Therefore, also from the descriptions in these references, the results shown in Tables 4 and 5 are considered to be estrogenic effects by antagonizing androgenic effects.

[Example 4] Preparation of carnation flower extract 100 g of the dried carnation flower part (above the sepal) was immersed in 1 kg of a 50% ethanol aqueous solution at room temperature for 1 week, filtered, and then filtered. About 0.95 kg of extract was obtained. Next, after concentrating the crude extract and evaporating ethanol, 50% aqueous butylene glycol solution was added to obtain 0.95 kg of liquid carnation flower extract.

[Example 5] Purification of kaempferol glycoside (KMP-3) From the 50% ethanol aqueous solution of the carnation flower extract obtained in Example 4, using a hydrophobic adsorption resin, Example 2 and Purification was performed in the same manner. That is, as shown in FIG. 1, a 50% ethanol aqueous solution of carnation flower extract was first crudely fractionated with a column filled with Diaion HP20 (Mitsubishi Chemical Co., Ltd.). Fraction 03 (Fr03) eluted with 40% ethanol was diluted with water and adsorbed on the same column, and then fraction 14 (Fr14) eluted with 40% ethanol aqueous solution was collected under the following conditions: A chromatographic preparative LC3 was performed to collect the main peak (fraction 31).

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

When the concentration of the compound contained in fraction 31 was measured under the following conditions (LC3), it was 48.66 μg/mL in terms of kaempferol glycoside.

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

NMR analysis of the compound recovered in fraction 31 revealed it to be kaempferol-3-(2″-glucosyl-glucoside) (KMP-3). Table 6 below shows measurement data of KMP3 measured with a nuclear magnetic resonance apparatus JEOL (Japan Electronics Co., Ltd.) JNM-ECA500.

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

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

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

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

The compound recovered in fraction 41 was analyzed by NMR and found to be kaempferol-3-neohesperidoside (KMP-5). Table 7 below shows measurement data of KMP-5 measured with a nuclear magnetic resonance apparatus JEOL (Japan Electronics Co., Ltd.) JNM-ECA500.

[Test Example 8] Measurement of collagen production amount When the two fractions (KMP-3 and KMP-5) purified in Examples 4 and 5 were added to the cells, and the above active ingredients contained in the carnation flower extract It was confirmed whether the amount of collagen production (synthesis ability) changed when Normal human adult skin fibroblasts were used as cells. DMEM containing 5% FBS was used for preculture, and DMEM containing 1% FBS was used for this experiment. Culturing was carried out under conditions of 5% CO ₂ and 37°C.

5×10 ⁴ normal human adult skin fibroblasts (Cell applications) were seeded in 24-well plates and cultured to 75% confluence. Thereafter, the cells were replaced with DMEM containing 1% FBS, cultured for 24 hours, then replaced with new DMEM containing 1% FBS, and the following samples were added and cultured for 72 hours.

- 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 to 0.5%.
The concentrations of the active ingredients contained in Samples 2 and 3 were prepared with a 50% butylene glycol aqueous solution so that the concentrations of the respective compounds contained in the carnation flower extract were the same.

Thereafter, the concentration of type 3 collagen in the medium was measured using Human PIIINP (N-Terminal Procollagen III Propeptide) ELISA kit (manufactured by Elabscience). The measurement was performed on 5 samples in each group (groups of samples 1 to 3).

The measurement results are described below. The measurement result indicates the average value of the values measured in each group. When the average value of the sample 1 group was 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. A significant difference (p<0.01) was observed between the mean values of the sample 2 and sample 3 groups and the control group in Dunnett's test with respect to the mean value of the sample 1 group. was the data. It was found that the addition of the sample 2 and sample 3 groups promoted the production of type 3 collagen.

Although the embodiments (including examples) of the present invention have been described above with reference to the drawings, the specific configuration of the present invention is not limited thereto, and is within the scope of the present invention. , even if there is a design change, etc., it is included in the present invention.

It was confirmed that the external preparation for skin containing the agent of the present invention is effective in improving wrinkles by promoting collagen production and improving the softness and resilience of the skin. Therefore, the external preparation for skin of the present invention may be used as cosmetics for improving wrinkles.

Claims (2)

Formula (I) below:

(Wherein, R ¹ represents a hydrogen atom or a rhamnosyl group, and R ² represents a glucosyl group or a rhamnosyl group.), an organic acid ester thereof, or a salt thereof, promoting estrogen action agent for The compound represented by the formula (I) is
Formula (II) below:

Formula (III) below:

and / or the following formula (IV)

The agent according to claim 1, which is a compound represented by

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