Topical anti-aging agent, and topical composition containing the active ingredient thereof.

Abstract

To provide topical anti-aging agents comprising a vanilla-derived compound, and topical compositions.SOLUTION: A vanilla-derived compound represented by the general formula (1) is used as an active ingredient of topical anti-aging agents and topical compositions. (In the formula, R1 is a hydrogen atom or a hydroxyl group.)SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to novel uses for compounds isolated from the above-ground parts (leaves and stems) of vanilla (hereinafter referred to herein as "vanilla-derived compounds" or "the compounds of the present invention"). More specifically, it relates to topical anti-aging agents and topical compositions containing vanilla-derived compounds useful for anti-aging of the skin. [Background technology]

[0002] The skin is composed of layers, from the outside in, in the order of epidermis, dermis, and subcutaneous tissue. The dermis, located beneath the epidermis, has an average thickness of 2 mm and is mainly composed of collagen and elastin. Collagen and elastin are produced by fibroblasts and play an important role in maintaining skin flexibility and elasticity. Elastase, an enzyme that breaks down elastin, is known to be induced by aging, UV radiation, reactive oxygen species, and stress. When elastin is broken down by elastase, the cross-linked structure between collagen and elastin becomes brittle, leading to a loss of skin elasticity and causing wrinkles. Inhibiting elastase activity can be expected to restore or maintain skin elasticity.

[0003] Furthermore, fibroblasts in the dermis of the skin produce collagen and hyaluronic acid, which are important components that make up the skin. Collagen is a structural protein that forms connective tissue, such as animal skin. Collagen is important for maintaining skin strength and keeping skin wrinkle-free and youthful. Hyaluronic acid is a major component of the extracellular matrix and is involved in various intercellular interactions. Hyaluronic acid is an essential element for maintaining skin moisture, and it is thought that the skin prevents the loss of water from the body and protects against physical stimuli by producing and accumulating hyaluronic acid. Therefore, it is thought that if collagen production and hyaluronic acid production by fibroblasts can be promoted, it will be possible to maintain fresh and firm skin.

[0004] Conventionally, plant extracts from orchidaceae plants, including the genus Vanilla, have been known to have reactive oxygen species scavenging and lipid peroxide production inhibitory effects, making them useful as antioxidants, and also to have moisturizing effects, making them useful for improving rough and dry skin (Patent Document 1). Furthermore, vanilla extract is known to have synthesis stimulating activity for PDGF growth factor and to be useful in preventing or treating cutaneous aging (Patent Document 2). However, Patent Document 1 does not describe or suggest which part of the Vanilla plant the plant extract used to evaluate antioxidant and moisturizing effects comes from, and this remains unclear. The same is true for Patent Document 2. Furthermore, Patent Document 2 only mentions "vanilla extract," and the extraction solvent used is also unknown. Moreover, Patent Documents 1 and 2 do not explain which components in the plant extract are the active ingredients that exert antioxidant and moisturizing effects, or preventive and therapeutic effects against aging.

[0005] Furthermore, the extracts from the fruits of the Vanilla genus (also called pods, green pods, seed pods, vanilla beans, etc.) contain many aromatic compounds (vanillin, vanillic acid, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, etc.), and the complex interplay of these scents creates a sweet and distinctive aroma (vanilla fragrance). The vanilla fruit extract is known to be useful not only as a fragrance but also as a moisturizing and active ingredient in cosmetics (Patent Document 3, Non-Patent Document 1). Specifically, Patent Document 3 describes that vanilla planifolia PFA (vanilla fruit oil prepared by subjecting vanilla planifolia fruit to Chanel's unique separation and extraction process [polyfractionating: PFA]) and vanilla planifolia intense water (vanilla fruit water) can be used as active ingredients in cosmetics. Non-Patent Document 1 also describes that Chanel's serum (oil cream) contains the aforementioned vanilla planifolia PFA (vanilla fruit oil) and vanilla planifolia intense water (vanilla fruit water) as moisturizing ingredients. Furthermore, Patent Document 4 describes that the lipid-soluble fraction of the fruit of Vanilla planifolia (a fraction that dissolves in the oil phase but not in the aqueous phase) is effective against aging, physiological mechanisms related to aging, or symptoms caused by abnormalities at the epidermal and / or dermal levels related to said physiological mechanisms, and specifically exhibits preventive and ameliorative effects against skin deterioration caused by aging. Thus, the fruit extracts of vanilla plants are currently widely used. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2002-205933 [Patent Document 2] Special Publication No. 2009-539925, paragraph

[0011] [Patent Document 3] Special Publication No. 2014-517029, Claim 14, Paragraph

[0047] [Patent Document 4] Japanese Patent Publication No. 2009-508914, Claim 1, paragraphs

[0006] ,

[0008] , and

[0015] [Non-patent literature]

[0007] [Non-Patent Document 1] This is a website introducing Chanel's "Sublimage La Crème Emollient Cream (Serum / Oil / Cream) Skincare Emollient Cream" (P144290 1): https: / / webcache.googleusercontent.com / search?q=cache:Loj-Lp6w7T8J:https: / / www.duniabangla.com / scrutinyproofeec1907816.htm+&cd=18&hl=ja&ct=clnk&gl=jp [Non-Patent Document 2] Han, S.W.; Wang, X.J.; Cui, B.S.; Sun, H.; Chen, H.; Ferreira, D.; Li, S.; Hamann, M.T. Hepatoprotective Glucosyloxybenzyl 2-Hydroxy-2-Isobutylsuccinates from Pleione Yunnanensis. Journal of Natural Products 2021, 84, doi:10.1021 / acs.jnatprod.0c01117. [Non-Patent Document 3] Li, Y.M.; Zhou, Z.L.; Hong, Y.F. New Phenolic Derivatives from Galeola Faberi. Planta Medica 1993, 59, doi:10.1055 / s-2006-959702. [Non-Patent Document 4] Leyva, V.E.; Lopez, J.M.; Zevallos-Ventura, A.; Cabrera, R.; Canari-Chumpitaz, C.; Toubiana, D.; Maruenda, H. NMR-Based Leaf Metabolic Profiling of V. Planifolia and Three Endemic Vanilla Species from the Peruvian Amazon. Food Chemistry 2021, 358, doi:10.1016 / j.foodchem.2021.129365. [Non-Patent Document 5] Palama, T.L.; Fock, I.; Choi, Y.H.; Verpoorte, R.; Kodja, H. Biological Variation of Vanilla Planifolia Leaf Metabolome. Phytochemistry 2010, 71, doi:10.1016 / j.phytochem.2009.12.011. [Summary of the Invention] [Problems to be Solved by the Invention]

[0008] An object of the present invention is to provide a vanilla-derived compound useful for anti-aging of the skin, and to provide an external anti-aging agent and an external composition containing the vanilla-derived compound. The external anti-aging agents targeted by the present invention include an elastase inhibitor, a promoter for promoting collagen production in skin fibroblasts, and a promoter for promoting hyaluronic acid production in skin fibroblasts. [Means for Solving the Problems]

[0009] The present inventors conducted intensive studies to solve the above problems. As a result, they found that a compound isolated from an aqueous alcohol extract of the above-ground parts (leaves, stems) other than the fruit part of the orchid genus Vanilla plant (Vanilla pompona) produced in Kurume, Japan has an elastase inhibitory action, a promoting action for promoting collagen production in skin fibroblasts, and a promoting action for promoting hyaluronic acid production in skin fibroblasts, which are useful for anti-aging of the skin. In addition, since these compounds do not affect the viability of human skin fibroblasts even at high concentrations, it was suggested that they have high safety for the skin.

[0010] The present invention has been completed based on such findings and includes the following embodiments.

[0011] (I) Topical anti-aging agents (I-1) An external anti-aging agent containing a compound represented by the general formula (1): [Chemical Formula] (In the formula, R1 is a hydrogen atom or a hydroxyl group.). (I-2) The external anti-aging agent according to (I-1), wherein the external anti-aging agent is at least one selected from the group consisting of an elastase inhibitor, a promoter for promoting collagen production in skin fibroblasts, and a promoter for promoting hyaluronic acid production in skin fibroblasts. (I-3) The topical anti-aging agent according to (I-1) or (I-2), wherein the topical anti-aging agent contains an extract of the leaves and / or stems of a plant of the genus Vanilla (Vanilla pompona) that contains a compound represented by general formula (1).

[0012] (II) Topical compositions (II-1) External compositions containing a compound represented by general formula (1): [ka] (In the formula, R1 is a hydrogen atom or a hydroxyl group.) (II-2) A topical composition as described in (II-1), which is a cosmetic, a quasi-drug for topical use, or a topical drug. (II-3) The topical composition according to (II-1) or (II-2), wherein the topical composition contains an extract of the leaves and / or stems of a plant of the genus Vanilla (Vanilla pompona) that contains a compound represented by general formula (1). (II-4) The topical composition described in (II-3), wherein the Vanilla pompona plant is native to Kurume City, Fukuoka Prefecture, Japan. [Effects of the Invention]

[0013] According to the present invention, it is possible to provide a vanilla-derived compound that has elastase inhibitory activity, collagen production promoting activity in skin fibroblasts, and hyaluronic acid production promoting activity, which are useful for anti-aging of the skin, and an extract of the above-ground parts of vanilla (excluding the fruit) containing the same (hereinafter, this may be abbreviated as "vanilla extract").

[0014] The vanilla-derived compound provided by the present invention is useful as an active ingredient in esterase inhibitors, and a topical composition containing the vanilla-derived compound or a vanilla extract containing it can be expected to maintain skin elasticity, suppress the decline in skin elasticity, or restore skin elasticity due to its esterase inhibitory effect.

[0015] Furthermore, the vanilla-derived compound provided by the present invention is useful as an active ingredient in collagen production promoters in skin fibroblasts. By using the vanilla-derived compound or an external composition containing it or vanilla extract, it is possible to prevent wrinkles and improve skin firmness through its collagen production-promoting effect.

[0016] Furthermore, the vanilla-derived compound provided by the present invention is useful as an active ingredient in hyaluronic acid production promoters in skin fibroblasts. A topical composition containing the vanilla-derived compound or a vanilla extract containing it can be expected to improve skin moisture retention (moisturizing) and create fresh, supple skin due to its hyaluronic acid production-promoting effect.

[0017] In addition, the vanilla-derived compound of the present invention does not affect the viability of human dermal fibroblasts even at high concentrations, making it suitable for use as an ingredient in topical compositions. [Brief explanation of the drawing]

[0018] [Figure 1] The MPLC chromatogram of fractions C10-15, performed in Experimental Example 1, "Isolation of the Compound of the Invention," is shown. The numbers shown above the frame in Figure 1 indicate the fractions that were separated. In the figure, the solid line labeled "AB" shows the gradient curve (methanol concentration) of the eluent. Of the large peaks detected in the chromatogram, fractions 5-9, which are included in the first peak, contain compound 2, the target of the present invention, and fractions 20-23, which are included in the third peak, contain compound 1, the target of the present invention, as single components. [Figure 2] The results of the evaluation of the elastase inhibitory activity of the vanilla-derived compounds (Compound 1 and Compound 2) conducted in Experimental Example 2 are shown below. [Figure 3] (A) shows the results of the evaluation of the collagen production promoting effect of the vanilla-derived compounds (compound 1 and compound 2) conducted in Experiment Example 3, and (B) shows the results of the evaluation of the hyaluronic acid production promoting effect of the vanilla-derived compounds (compound 1 and compound 2) conducted in the same Experiment Example. [Figure 4]The results of the safety evaluation of the vanilla-derived compounds (Compound 1 and Compound 2) conducted in Experimental Example 3 are shown. Specifically, the survival rate (%) of human fibroblasts with the addition of the vanilla-derived compounds is shown in comparison to the control. [Modes for carrying out the invention]

[0019] (I) Vanilla-derived compounds The vanilla-derived compounds targeted by the present invention have a structure in which two glucose residues are bonded to 2-isopropylmalic acid (when R1 is a hydrogen atom) or isopropyltartaric acid (when R1 is a hydroxyl group) via benzyl groups, as shown in the general formula (1) below. [ka]

[0020] In this specification, a compound in which R1 is a hydrogen atom in formula (1) is referred to as compound 1. Compound 1 includes bis [4-(β-DO-glucopyranosyloxy)-benzyl]-2-isopropyl malate. Furthermore, in formula (1) above, the compound in which R1 is a hydroxyl group is referred to herein as compound 2. Compound 2 includes bis [4-(β-DO-glucopyranosyloxy)-benzyl]-2-isopropyl tartrate. This compound is also known as vanilla glucoside A.

[0021] These compounds are found in the leaves and stems of the vanilla plant (Vanilla pompona) from Kurume, Fukuoka Prefecture, Japan, and can be isolated from the aqueous alcohol extract. Details of the isolation method are described in Experimental Example 1 below.

[0022] Compounds 1 and 2 (hereinafter referred to as "the vanilla-derived compounds" without distinction) have the effect of inhibiting elastase activity derived from human dermal fibroblasts, as shown in Experimental Example 2 described later. Therefore, they are expected to contribute to maintaining skin elasticity, suppressing the decrease in skin elasticity, and / or restoring skin elasticity. Furthermore, as shown in Experimental Example 2 described later, the vanilla-derived compounds have the effect of promoting collagen production and hyaluronic acid production in human dermal fibroblasts. Therefore, they are expected to give the skin firmness and moisture and contribute to wrinkle prevention.

[0023] (II) Topical anti-aging agents The topical anti-aging agent of the present invention is characterized by containing at least one of the aforementioned compounds 1 and 2 (the vanilla-derived compound) as an active ingredient. Based on the action of the vanilla-derived compound, which is the active ingredient, the topical anti-aging agent of the present invention has at least one action selected from elastase inhibitory action, collagen production promoting action in skin fibroblasts, and hyaluronic acid production promoting action in skin fibroblasts, thereby acting to suppress the decrease in skin elasticity and / or moisture that occurs with aging.

[0024] In this invention, the term "topical anti-aging agent" includes elastase inhibitors, collagen production promoters in dermal fibroblasts, and hyaluronic acid production promoters in dermal fibroblasts. In other words, the topical anti-aging agent of this invention can be used as an elastase inhibitor based on the elastase inhibitory effect of the vanilla-derived compound. Furthermore, the topical anti-aging agent of this invention can be used as a collagen production promoter and a hyaluronic acid production promoter, respectively, based on the collagen production promoting effect and hyaluronic acid production promoting effect of the vanilla-derived compound in dermal fibroblasts.

[0025] The topical anti-aging agent of the present invention can be used as a raw material (active ingredient-containing raw material) for preparing topical compositions described later, preferably topical compositions for anti-aging that have the effect of suppressing or improving skin aging associated with aging by maintaining skin elasticity and water retention and preventing wrinkles.

[0026] The topical anti-aging agent of the present invention may contain the vanilla-derived compound in a purified state. However, as mentioned above, the vanilla-derived compound is a component found in the leaves and stems, which are the above-ground parts of Kurume vanilla (Vanilla pompona), so the agent may also contain an extract or crude product of its leaves and / or stems, to the extent that the effects described above are not impaired. However, the plant extract or crude product may contain the vanilla-derived compound, have the effects described above, and be suitable for topical use in terms of safety, and the raw material and the plant used do not necessarily have to be vanilla (Vanilla pompona). The topical anti-aging agent of the present invention preferably contains purified vanilla-derived compound, or contains a vanilla-derived compound-containing fraction obtained by fractionating and concentrating the vanilla-derived compound from a plant extract.

[0027] The amount of this vanilla-derived compound contained in the topical anti-aging agent should be set appropriately within the range of 1 to 100% by mass, depending on its intended use as an elastase inhibitor, collagen production promoter, and hyaluronic acid production promoter, as long as it exerts the respective effects when added to the topical composition.

[0028] (III) External composition The topical composition of the present invention is characterized by containing at least one of the aforementioned compounds 1 and 2 (the vanilla-derived compound) as an active ingredient. As a raw material containing the vanilla-derived compound, the aforementioned topical anti-aging agents (elastase inhibitors, collagen production promoters, or hyaluronic acid production promoters) can also be used. Based on the anti-aging effects (elastase inhibitory effect, collagen production promoting effect, or hyaluronic acid production promoting effect) of the vanilla-derived compound, the topical composition of the present invention can be prepared as an anti-aging topical composition that suppresses or improves skin aging by maintaining skin elasticity and moisture retention and preventing wrinkles.

[0029] The proportion of the vanilla-derived compound contained in the topical composition of the present invention is not particularly limited, as long as it is such that, when the topical composition is applied to human dermal fibroblasts, it exerts an effect of suppressing elastase activity in said cells, or promotes collagen production and / or hyaluronic acid production in said cells, thereby increasing the amount of collagen and / or hyaluronic acid. It can usually be appropriately selected from the range of 0.00001 to 80% by weight. Preferably, it is 0.0001 to 30% by weight, more preferably 0.001 to 20% by weight. The methods for measuring and evaluating elastase inhibitory activity, collagen production, and hyaluronic acid production in dermal fibroblasts are as described in Experimental Examples 2 and 3 below, and can be carried out in accordance with this description. Preferably, it is an amount that can suppress and / or improve age-related skin deterioration symptoms (skin aging symptoms) based on the effects of the vanilla-derived compound.

[0030] The external compositions of the present invention include external pharmaceuticals, quasi-drugs, or cosmetics. Preferably, they are cosmetic compositions.

[0031] The external composition of the present invention can be prepared in various dosage forms, such as solid preparations, semi-solid preparations (including cream preparations), liquid preparations, and emulsion preparations, depending on the type and application.

[0032] Examples of external pharmaceutical or quasi-drug forms include ointments, creams, patches, liniments, lotions, emulsions, liquids, and aerosols.

[0033] The form of the cosmetic is not particularly limited, but for example, skincare cosmetics may include lotions, serums, masks, massage creams, emulsions, moisturizing creams, lip balms, etc.; and makeup cosmetics may include foundations, face powders, lipsticks, blushes, eyeshadows, etc. Various cosmetic ingredients commonly used in cosmetics may be appropriately incorporated into the above cosmetics. Preferably, although not limited, the composition is in the form of an emulsion such as an emulsion or cream for the reasons mentioned above.

[0034] The topical composition of the present invention may also contain ingredients commonly used in topical compositions, such as oils and fats, waxes, hydrocarbons, fatty acids, alcohols, esters, amino acids, vitamins, surfactants, pH adjusters, preservatives, fragrances, humectants, powders, UV absorbers, thickeners, pigments, antioxidants, whitening agents, anti-inflammatory agents, skin roughness improving agents, acne medications, alkalis, and chelating agents, to the extent that the desired effect is not impaired. Furthermore, a skin absorption enhancer (skin penetration enhancer) may also be included. As the skin absorption enhancer (skin penetration enhancer), one that enhances the transdermal penetration of the compound of the present invention and promotes absorption into skin fibroblasts can be used. Examples of such substances include, without limitation, various surfactants (anionic, cationic, and nonionic surfactants), fatty acids and fatty acid esters (oleic acid, isopropyl myristate, medium-chain triglycerides, etc.), biodegradation accelerators (alkyl esters, aminocaproic acid), and other substances such as amino acids, terpenes, pyrrolidones, urea, phospholipids, and various solvents (polyols, lower alcohols, higher alcohols, dimethyl sulfoxides, etc.).

[0035] In this specification, the terms “contains” and “includes” include the meanings of “consisting of” and “substantially consisting of.” [Examples]

[0036] The present invention will be described below using experimental examples to aid in understanding its structure and effects. However, the present invention is not limited in any way by these experimental examples. Unless otherwise specified, the following experiments were conducted at room temperature (25±5℃) and under atmospheric pressure conditions. Unless otherwise specified, "%" below means "mass percent" and "parts" means "parts by mass".

[0037] Experimental Example 1: Isolation and Identification of the Compound of the Invention (1) Isolation of the compound of the present invention Vanilla pompona leaves from Kurume City, Fukuoka Prefecture, Japan were freeze-dried (-50°C, 40 Pa) and then pulverized in a mill mixer. A 90% methanol aqueous solution was added to the pulverized sample, and Soxhlet extraction was performed. The solvent after extraction was removed using a rotary evaporator to obtain a methanol extract.

[0038] For fractionation of methanol extracts, an ultrafast flash automated purification system (Biotage® selekt: manufactured by Biotage Japan Co., Ltd.) equipped with normal-phase silica gel or reverse-phase C18 flash column chromatography was used. Compound purification was performed using a preparative chromatography system (MPLC) Pure C-850 Flash prep® (manufactured by BUCHI) with UV and ELSD detectors. Analytical TLC was performed on pre-coated silica gel 60 GF254 (20 × 20 cm, 0.2 mm film thickness) or pre-coated RP-C18 F254 plates on aluminum sheets (5 × 7.5 cm, 0.2 mm film thickness). The analytical TLC plates were developed with a suitable solvent, and spots detected under UV absorption at 254 nm and 366 nm were observed. Furthermore, after visualization by spraying with vanillin / sulfuric acid reagent, the plates were heated in a preheated oven at 110°C for 5 minutes, and spots detected by sulfuric acid color development were observed.

[0039] The solvent was removed from the obtained fractions using a rotary evaporator, and the yield of each fraction was measured. The fraction with the highest yield was then subjected to further rough fractionation using MPLC under different solvent conditions, yielding 85 fractions (A1-15, B1-15, C1-9, C10-15, D1-15, E1-15, F1-10). Fractions C10-15, in which UV (254 nm) spots were observed in normal-phase TLC, were again fractionated by reverse-phase MPLC. Water and methanol were used as the eluent, and the contained components were eluted under gradient conditions. A UV detector (detection wavelengths: 220, 254, 280, 365 nm) and an ELSD detector were used as MPLC detectors.

[0040] The MPLC chromatograms for fractions C10-15 are shown in Figure 1. Fifty-nine fractions were separated at the intervals shown on the frame in Figure 1, and each fraction was subjected to reverse-phase TLC to confirm its profile (UV wavelength: 254 nm). As a result, among the large peaks detected in the MPLC chromatogram (Figure 1), the UV spots detected in fractions 5-9, which are included in the first peak, and fractions 20-23, which are included in the third peak, were all single, confirming that each of these fractions contains a single component.

[0041] (2) Identification of isolated components The optical rotation of the components isolated as described above (fractions 5-9 and 20-23) was measured using a Jasco P-2200 polarimeter (manufactured by JASCO Corporation). One-dimensional NMR spectra were also acquired using a Bruker DRX 600 NMR spectrometer (manufactured by Bruker) (internal standard: tetramethylsilane). Furthermore, high-resolution MS data were acquired using LC-MS-IT-TOF. Next, identification was performed by comparing the one-dimensional NMR data, mass spectrometry results, and NMR spectra reported in previous literature. As a result, as described below, the components of fractions 20 - 23 were identified as bis [4-(β-D-O-glucopyranosyloxy)-benzyl]-2-isopropylmalate (Compound 1) having the structure shown in the following formula (a). Also, the components of fractions 5 - 9 were identified as bis [4-(β-D-O-glucopyranosyloxy)-benzyl]-2-isopropyl tartrate (Compound 2) having the structure shown in the following formula (b).

[0042]

Chemical formula

[0043] 1H and13C NMR data (DMSO-d6, δ in ppm) of Compounds 1 and 2 are shown in Table 1. 13

[0044]

Table 1

[0045] (a) Identification of Compound 1 Compound 1 was obtained as an amorphous white powder, and its specific rotation was [α]20D -42.3 (c 0.48, methanol solvent). In high-resolution MS analysis, a deprotonated molecular ion peak [M-H] at m / z: 711.2509 corresponding to the molecular formula C 33 H 44 O 17 was observed. - 1 1H and 13 13C-NMR data showed that two methylene doublets at δ H 2.61 (J = 15.6) and 2.85 (J = 15.6), two ester carbonyls at δ c 173.9 and 170.0, and two carbons at δ c 41.8 and 77.3 were observed. Furthermore, two methyl doublets at 0.73 (6.9) and 0.82 (6.9), δ H ​Methine was observed at 1.83. Previous data suggest the presence of a 2-isopropylmalate moiety (Non-Patent Literature 2). 1 In 1H-NMR, two sets of AA'BB' signals are δ H 7.27, 2H (J=8.8) 7.28, 2H (J=8.8), 7.01, 4H (J=8.7) overlap, and the four methylene protons are δ H This was shown as 4.94 (J=12.4). The values ​​were 94 (J=12.4), 4.95 (J=12.4), 4.9 (J=12.0), 5.05, H, d, (J=12.0), suggesting the presence of two 1,4-disubstituted benzyl units. Also, δ H Two anomeric atoms were observed at 4.85 ppm (J=7.2) and 4.86 ppm (J=7.6). δ c The signals at 100.28, 100.30, 73.2, 76.6, 69.7, 77.0, and 60.7 suggested glucose as the sugar moiety (Non-Patent Literature 2). Based on these data and prior art (Non-Patent Document 3), compound 1 was identified as bis [4-(β-DO-glucopyranosyloxy)-benzyl]-2-isopropylmalate, as described above.

[0046] (b) Identification of compound 2 Compound 2 was obtained as an amorphous white powder with an optical rotation of [α]20D -33.8 (c 0.48, methanol solvent). High-resolution MS analysis revealed that the molecular formula was C 33 H 44 O 18 A deprotonated molecular ion peak [MH]- was observed at m / z: 727.2458, corresponding to the above. 1 H and 13 C-NMR data, δ H Two methylene doublets, 2.61 (J=15.6) and 2.85 (J=15.6), δ HThe NMR spectrum of compound 2 was similar to that of compound 1, except that it was substituted with an oxygen-containing methine at 4.47, H, d (J=7.7). From this, compound 2 was identified as bis 4-[β-DO-glucopyranosyloxy)-benzyl]-2-isopropyl tartrate, as described above. Compound 2 is also known by the common name vanilla glucoside A (Non-patent documents 4 and 5).

[0047] Experimental Example 2: Evaluation of the elastase inhibitory activity of this vanilla-derived compound. In this experiment, the skin elasticity-retaining effect (including the effect of suppressing the decrease in skin elasticity) of the vanilla-derived compounds (compounds 1 and 2) was evaluated by measuring their esterase inhibitory activity. The esterase inhibitory activity of the vanilla-derived compounds was measured using elastase derived from human dermal fibroblasts (NHDF-Ad) and the amount of nitroaniline produced with N-Succinyl-Ala-Ala-Ala-p-nitroanilide as a substrate in the presence of the vanilla-derived compounds. The esterase activity (measured value) was then evaluated by comparing it with the esterase activity (control value, positive control value) measured separately using dimethyl sulfoxide (DMSO) as a control and phosphoramidone (a known elastase inhibitor) as a positive control, instead of the vanilla-derived compounds.

[0048] (1) Experimental method (1-1) Preparation of cell lysate Human dermal fibroblasts (NHDF-Ad) were cultured in Dulbecco's Modified Eagle Medium (DMEM) (high glucose) (containing 1% penicillin-streptomycin and 10% fetal bovine serum (FBS)) in a φ10 cm dish in a CO2 incubator (37°C, 5% CO2) until confluence. Pre-cultured cells were washed with phosphate-buffered saline (PBS) and lysed in cell lysate (0.2 M Tris-HCl (pH 8.0) containing 1 mM PMSF and 0.5% Triton X-100) at a rate of 1.0 × 10⁻¹⁴.6 The cells were resuspended in cells / ml. After sonication, the cells were centrifuged (13,000 rpm, 4°C, 15 minutes), and the supernatant (cell lysate) was collected.

[0049] (1-2) Measurement of elastase activity To 12.5 μl of the supernatant collected as described above, 35.5 μl of 0.2 M Tris-HCl buffer (pH 8.0) and 2 μl each of the test substances (compound 1, compound 2, phosphoramidone, DMSO) were mixed to the concentrations shown below, and incubated at 37°C for 15 minutes (test samples: test sample 1, test sample 2, positive control sample, control sample).

[0050] [Final concentration of each test substance] Compound 1: 4, 40, 200, 400, 800 μM (corresponding to 3, 28, 142, 285, and 570 μg / mL respectively) Compound 2: 4, 40, 200, 400, 800 μM (corresponding to 3, 29, 145, 291, and 582 μg / mL respectively) Phosphoramidone (a known esterase inhibitor): 10, 25, 50, 100 μM

[0051] Subsequently, 50 μl of 5 mM substrate (N-Succinyl-Ala-Ala-Ala-p-nitroanilide) was added to each test sample, and the samples were incubated at 37°C for 24 hours. Next, the absorbance of each test sample at 405 nm was measured using a microplate reader, and the elastase activity of each test sample was calculated.

[0052] (2) Experimental results Figure 2 shows the elastase activity of the positive control sample (phosphoramidon), test sample 1 (compound 1), and test sample 2 (compound 2) (mean ± SD, n=3, *p<0.05, **p<0.01). The elastase activity (%) shown in Figure 2 is the relative ratio with the elastase activity of the control sample (control value) set to 100%. As shown in Figure 2, the addition of compounds 1 and 2 resulted in a significant decrease in elastase activity (elastase inhibitory activity) compared to the control. Both compounds 1 and 2 exhibited concentration-dependent elastase inhibitory activity. IC of elastase inhibition 50 The concentrations were 0.04 ng / mL (64.4 nM) for phosphoramidone, 296.09 μg / mL (415.7 μM) for compound 1, and 426.10 μg / mL (585.1 μM) for compound 2.

[0053] Based on these results, it is suggested that compounds 1 and 2, isolated from vanilla (Vanilla pompona), possess elastase inhibitory activity, contribute to the restoration or maintenance of skin elasticity, and have anti-wrinkle effects.

[0054] Experimental Example 3: Evaluation of the collagen and hyaluronic acid production-promoting effect of this vanilla-derived compound, and safety evaluation. In this experiment, human fibroblasts were treated with the vanilla-derived compounds (compounds 1 and 2), and changes in collagen and hyaluronic acid production in the human fibroblasts were measured to evaluate the collagen and hyaluronic acid production-promoting effect, the resulting anti-wrinkle effect, and the skin moisture retention effect of the vanilla-derived compounds. Furthermore, the safety of this vanilla-derived compound was evaluated by measuring the viability of human fibroblasts cultured in the presence of this compound.

[0055] (1) Experimental method (1-1) Cell preparation Adult human dermal fibroblasts (NHDF-Ad) were used as a model cell. NHDF-Ad cells were pre-cultured in Dulbecco's Modified Eagle Medium (DMEM) (high glucose) (containing 1% penicillin-streptomycin and 10% fetal bovine serum (FBS)) in φ10 cm dishes until confluent. Afterward, they were washed with phosphate-buffered saline (PBS), resuspended in culture medium, and then loaded into 96-well plates in 0.5 × 10⁶ wells. 4 Seeds were seeded at a concentration of cells / well and cultured overnight in a CO2 incubator (37°C, 5% CO2).

[0056] (1-2) Addition of test substance After the overnight culture described above, each test substance was replaced with a serum-free medium (containing 1% penicillin-streptomycin) at the following concentrations and cultured in a CO2 incubator for 72 hours.

[0057] [Final concentration of each test substance] Compound 1: 50 μM (equivalent to 35.6 μg / mL) Compound 2: 50 μM (equivalent to 36.4 μg / mL) Control: DMSO

[0058] (1-3) Hyaluronic acid production promotion evaluation test and collagen production promotion evaluation test The culture supernatant was collected 72 hours after the addition of each test substance, and the amounts of collagen and hyaluronic acid in the culture supernatant were measured using the commercially available ELISA kits described below. Collagen content: Human Collagen Type I ELISA, ACEL Hyaluronic acid quantity: QnE Hyaluronic acid Quantitative ELISA assay, Biotech Trading Partner

[0059] (1-4) Safety evaluation (measurement of cell viability) The cell viability in the culture medium 72 hours after the addition of each test substance was measured using Cell Counting Kit-8 (Dojin Chemical). 100 μL of DMEM (high glucose, containing 1% penicillin-streptomycin) was added to a 96-well plate from which the culture supernatant had been removed. Then, 3 μL of CCK-8 solution was added to each well, and the cells were incubated in a CO2 incubator for 1 hour. 90 μL of the cell supernatant was transferred to the 96-well plate, and the absorbance at 450 nm was measured using a microplate reader to calculate cell viability.

[0060] (2) Experimental results (2-1) Evaluation of hyaluronic acid production promotion and collagen production promotion Figures 3(A) and (B) show the collagen (collagen production) and hyaluronic acid (hyaluronic acid production) amounts in the culture supernatant to which the vanilla-derived compounds (Compound 1 and Compound 2) were added as test substances (mean ± SD, n=3, *p<0.1, **p<0.05). The collagen and hyaluronic acid production amounts shown in Figures 3(A) and (B) are relative ratios to the collagen and hyaluronic acid amounts in the culture supernatant to which DMSO was added instead of the vanilla-derived compounds (control values) are set to 100%. As shown in Figures 3(A) and (B), both of these vanilla-derived compounds showed collagen and hyaluronic acid production-promoting effects at 50 μM.

[0061] (2-2) Safety evaluation (cell viability) Figure 4 shows the survival rate (%) of adult human dermal fibroblasts (NHDF-Ad cells) after the addition of these vanilla-derived compounds (compound 1, compound 2) (n=3, mean ± SD). As shown in Figure 4, when this vanilla-derived compound was added to NHDF-Ad cells, it did not affect cell viability even at high concentrations, confirming the safety of this vanilla-derived compound.

[0062] Based on these results, compounds 1 and 2, isolated from vanilla (Vanilla pompona), are highly safe substances with no cytotoxicity, and are suggested to have collagen and hyaluronic acid production-promoting effects, as well as anti-wrinkle and moisture-retaining (moisturizing) effects on the skin.

Claims

1. A topical composition containing a compound represented by general formula (1): 【Chemistry 1】 (In the formula, R １ (This is a hydrogen atom or a hydroxyl group.) The topical composition is at least one topical anti-aging agent selected from the group consisting of elastase inhibitors, collagen production promoters in dermal fibroblasts, and hyaluronic acid production promoters in dermal fibroblasts. External composition.

2. The topical composition according to claim 1, wherein the topical composition contains an extract of the leaves and / or stems of a plant of the genus Vanilla (Vanilla pompona) containing a compound represented by general formula (1).

3. The topical composition according to claim 1 or 2, wherein the topical composition is a cosmetic, a quasi-drug for topical use, or a pharmaceutical product for topical use.

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