JP7451005B1 - Preadipocyte proliferation and/or differentiation promoter containing a helenalin derivative - Google Patents

Abstract

[Problem] We have discovered a compound contained in arnica extract that promotes the proliferation and/or differentiation of preadipocytes, and provide cosmetics, foods, medicines, etc., and/or materials to be incorporated therein, that use this compound to increase the volume of the fat layer and create a beautiful appearance. [Solution] An agent for promoting the proliferation and/or differentiation of preadipocytes, which contains as an active ingredient a compound represented by the following formula (I): JPEG0007451005000011.jpg54170 (wherein R is a hydrogen atom or an acyl group having 1 to 5 carbon atoms). [Selected Figure] Figure 1

Description

cross reference

This application claims priority based on Japanese Patent Application No. 2022-082055 filed in Japan on May 19, 2022, and the contents described in this application are incorporated herein by reference.

The present invention relates to an agent for promoting proliferation and/or differentiation of preadipocytes containing a helenalin derivative.

Adipocytes are found in the lowest layer of the skin structure, which consists of three layers: epidermis, dermis, and subcutaneous tissue, and constitute an energy storage tissue that stores fat as triglycerides. Adipose tissue stimulates other tissues by secreting various adipokines, such as adiponectin, which is known as a longevity hormone. In addition to its physiological functions, adipose tissue also has the function of creating a beautiful appearance in women by creating plumpness and improving wrinkles through local fat accumulation.

Despite the fact that fat cells have physiologically and aesthetically important functions as described above, there is a tendency for fat cells to be viewed as bad due to the recent trend toward dieting. Therefore, cosmetics, foods, and pharmaceuticals containing ingredients that exclusively suppress the proliferation of preadipocytes and suppress their differentiation into adipocytes are being developed. On the other hand, a composition for external use on the skin containing arnica extract and red algae extract is used to promote the proliferation of myoblasts, which form the muscles on the inner side of the skin, in order to suppress the aging phenomenon of the skin. (For example, see Patent Document 1).

Extracts from arnica flowers from the Asteraceae family have been used since ancient times as a medical herb with anti-inflammatory and blood circulation effects. It has also been reported that this arnica extract has the function of increasing fat cells, increasing the volume of the fat layer, and improving the firmness and elasticity of the skin (see, for example, Patent Document 2). However, the active ingredients contained in arnica flower extract that are involved in promoting proliferation and differentiation of preadipocytes are still unclear.

JP2011-32233A JP2020-023476A

The problem to be solved by the present invention is to find a compound contained in arnica extract that promotes the proliferation and/or differentiation of preadipocytes, and to use this compound to increase the volume of the fat layer and create a beautiful appearance. Our goal is to provide cosmetics, foods, pharmaceuticals, etc., and/or materials used in these products.

The present invention has been made to solve the above problems. The present invention was created based on the discovery that a specific compound fractionated and purified from an arnica flower extract using column chromatography or the like promotes the proliferation and/or differentiation of preadipocytes.
That is, the present invention includes the following embodiments.

(1) The following formula (I):
(In the formula, R is a hydrogen atom or an acyl group having 1 to 5 carbon atoms.) An agent for promoting proliferation and/or differentiation of preadipocytes, which contains a compound represented by the following as an active ingredient.
(2) The preadipocyte proliferation and/or differentiation promoting agent according to (1), wherein the compound is 6-O-methacryloylhelenaline or 6-O-isobutyrylhelenaline.
(3) The preadipocyte proliferation and/or differentiation promoter according to (1), wherein the compound is helenalin or 6-O-acetylhelenaline.
(4) The preadipocyte according to (1), wherein the compound does not contain 6-O-tigloylhelenaline or contains 6-O-tigloylhelenaline in a mass ratio equal to or less than the total amount of the active ingredients. A proliferation and/or differentiation promoting agent.
(5) An external skin preparation for promoting the proliferation and/or differentiation of preadipocytes, which contains the compound represented by the above formula (I) as an active ingredient.
(6) The skin external preparation according to (5), wherein the content of each active ingredient is at least 0.1×10 ⁻⁹ % by mass.
(7) The skin external preparation according to (5) or (4), which does not contain 6-O-tigloylhelenaline or contains it at a concentration of about 1 x 10 ^-7 % by mass or less.

The preadipocyte proliferation and/or differentiation promoting agent of the present invention can be used in cosmetics, foods, pharmaceuticals, etc. for promoting the proliferation and differentiation of preadipocytes, increasing the volume of the fat layer and creating a beautiful appearance, and/or We can provide materials to be mixed with these.

FIG. 1 shows a scheme for fractionating and purifying active ingredients from arnica extract. FIG. 2 shows the results of a proliferation test and a differentiation test of human subcutaneous preadipocytes for each fraction obtained by extracting the arnica extract with diethyl ether and fractionating it by silica gel open column chromatography. FIG. 3 shows DEFr. The results of a proliferation test and a differentiation test on human subcutaneous preadipocytes for Compound 1 and Compounds 3 to 10 isolated from the 4-2 fraction using silica gel open column chromatography, preparative HPLC, etc. are shown. FIG. 4 shows the results of a proliferation test and a differentiation test of human subcutaneous preadipocytes using Compounds 7, 8, and 9 alone or in combination. FIG. 5 shows DEFr. 3 and DEFr. 5~DEFr. A fractionation scheme for further purification of 8 and isolation of new compounds 12-19 is shown. FIG. 6 shows the results of human subcutaneous preadipocyte proliferation and differentiation tests for compounds 11 (Cmpd11) to 19 (Cmpd19).

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 claimed invention, and all of the various elements and combinations thereof described in each embodiment are the solution of the present invention. is not necessarily required.

(definition)
As used herein, "preadipocytes" refer to cells derived from adipose tissue that have the ability to differentiate into adipocytes. Unlike mature adipocytes, preadipocytes do not yet contain fat within their cells and are proliferative fibroblast-like cells. Mature adipocytes refer to cells that have differentiated from these precursor adipocytes so that they can accumulate fat within the cells, and are in a state in which fat is accumulated within the cells. One of the causes of skin aging is the regression and reduction of subcutaneous fat due to aging and ultraviolet rays. When the volume of the subcutaneous fat layer decreases, the skin loses its firmness and the skin surface becomes uneven, leading to wrinkles and depressions. Therefore, by increasing preadipocytes in living organisms such as humans and differentiating them into adipocytes, we can prevent and/or treat local skin volume loss (bust, lips, tear troughs, back of hands, etc.). It has an improving effect. The increase in subcutaneous fat cells is thought to improve the clarity and luster of the skin as well as the nasolabial folds.

In the present embodiment, the "preadipocyte proliferation and/or differentiation promoting agent" (hereinafter sometimes simply referred to as "the agent of the present embodiment") refers to an agent containing active ingredients used for these purposes. It refers to compositions, etc., and is a material that can be added to cosmetics and medicines. This agent may be not only a liquid but also a solid, for example, and specific applications include not only a skin external preparation but also an oral composition (for example, it can be prepared as either a solid or a liquid).

(active ingredient)
The preadipocyte proliferation and/or differentiation promoting agent of this embodiment has the following formula (I):

(wherein, R is a hydrogen atom or an acyl group having 1 to 5 carbon atoms) as an active ingredient. Here, the acyl group in the "acyl group having 1 to 5 carbon atoms" means a substituted carbonyl group, and is a linear, branched, cyclic, saturated, or unsaturated aliphatic hydrocarbon group, Specific examples include acetyl group, propionyl group, isopropionyl group, isobutyryl group, tigloyl group, isovaleryl group, pivaloyl group, methacryloyl group, acroyl group, crotonoyl group, and 2-methylcrotonoyl group. In a preferred embodiment, R in the above formula (I) is a hydrogen atom or an acyl group having 1 to 4 carbon atoms. In a more preferred embodiment, R is a hydrogen atom, an acetyl group, a methacryloyl group, or an isobutyryl group.

In one embodiment, the active ingredient of the invention is 6-O-methacryloylhelenaline and/or 6-O-isobutyrylhelenaline. Here, 6-O-methacryloylhelenaline is Compound 8 in which R represents a methacryloyl group in the compound represented by the above general formula (I). Further, 6-O-isobutyrylhelenaline is a compound 9 in which R represents an isobutyryl group in the compound represented by the above general formula (I).

In another embodiment, the active ingredient of the invention is helenaline or 6-O-acetylhelenaline. Here, helenalin is the compound 12 in which R represents a hydrogen atom in the compound represented by the above general formula (I). Further, 6-O-acetylhelenaline is Compound 13 in which R represents an acetyl group in the compound represented by the above general formula (I).

These compounds may be extracted directly from Arnica flowers, or may be synthesized by chemical modification using helenalin extracted from medicinal plants such as Arnica as a parent compound. For example, a method has been reported in which the hydroxyl group at the 6-position of helenalin is acetylated using acetic anhydride (Maria F Beer et al., Molecules. 2019; 24(6): 1113, see Scheme 2). Based on these, those skilled in the art can easily synthesize Compound 8 and Compound 9 using helenalin and acid anhydride.

When the active ingredient of the present embodiment is extracted from Arnica flowers, "Arnica" is a plant belonging to the Asteraceae family and the genus Arnica (scientific name: Arnica montana). When producing an arnica extract, for example, roots, rhizomes, leaves, stems, flowers, fruits, pericarp, seeds, whole plants, or mixtures thereof can be used. An extract of arnica is produced, for example, by pulverizing the raw or dried material and then extracting it with a solvent. For example, 50 g of dried arnica flowers is immersed in 1 kg of 30% 1,3-butylene glycol solution and extracted in an environment of about 10° C. to about 30° C. for 5 to 10 days. Alternatively, 50 g of dried arnica flowers may be immersed in 1 kg of warm water at about 30° C. to about 50° C. for extraction for 2 to 10 hours. The solution obtained through this immersion is filtered using a predetermined filter material (Glass Fiber File retention time (Gf-75 manufactured by ADVANTEC), Mixed Cellulose ester (A045A047A manufactured by ADVANTEC), etc.). The filtered solution is left standing in an environment of about 0°C to about 10°C for 5 to 10 days. The solution is filtered again using a predetermined filter material and the resulting solution is used as an arnica extract.

(Isolation and purification operation of active ingredient)
The active ingredient of this embodiment is extracted from an arnica extract using a solvent, and chromatography using various separation modes (ion exchange, hydrophilic adsorption, hydrophobic adsorption, size exclusion, ligand exchange, affinity, etc.). fractionation, molecular weight fractionation filtration using filter paper, membrane filters, ultrafiltration membranes, etc., pressurization or reduced pressure, heating or cooling, drying, pH adjustment, deodorization, decolorization, long-term static storage, etc. It is also possible to arbitrarily select and combine these methods. In one embodiment, it is preferable to carry out the reaction in combination with column chromatography, preparative HPLC, etc. used in the examples described later. The structure of the purified compound can be determined by NMR or mass spectrometry.

(Preadipocyte proliferation and/or differentiation promoter)
The content ratio of each active ingredient in the agent of this embodiment is not particularly limited. For example, the agent of the present embodiment may contain only one of 6-O-methacryloylhelenaline and 6-O-isobutyrylhelenaline, or may contain both in any ratio. . Preferred embodiments include both 6-O-methacryloylhelenaline and 6-O-isobutyrylhelenaline, more preferably in approximately a 1:1 ratio. The agent of this embodiment may contain any compound in addition to these active ingredients. For example, plants of the Asteraceae family, including arnica, contain many types of helenalin, its derivatives, or its analogs. These are collectively called sesquiterpene lactones, which are lipophilic 15-carbon terpenoid compounds having a lactone ring.

In folk medicine, sesquiterpene lactones are used as herbal medicines for various conditions. Their diversity is matched by the diversity of their biological activities, and they have been shown to exert anti-inflammatory, cytotoxic, anticancer, antibacterial, antifungal, insecticidal, and antiprotozoal effects. . Therefore, when the agent of the present embodiment contains sesquiterpene lactones as other components, it is necessary to pay attention to their various physiological activities, especially cytotoxicity. For example, as shown in the examples below, arnica extract contains many types of sesquiterpene lactones, but it is preferable to reduce the content ratio of highly cytotoxic components among them. Examples of highly cytotoxic helenalin derivatives include 6-O-tigloylhelenaline. On the other hand, by including a smaller amount of 6-O-tigloylhelenaline than the active ingredient of the present embodiment, differentiation of the adipocytes may be promoted. Therefore, it is preferable that the agent of the present embodiment does not contain 6-O-tigloylhelenaline or contains 6-O-tigloylhelenaline in a mass ratio equal to or less than the total amount of the active ingredients. This is because, in addition to suppressing the cytotoxic effects of other ingredients, a synergistic effect with the active ingredients of this embodiment can also be expected.

(Content of active ingredients contained in external skin preparations)
One use of the agent of this embodiment is as a skin external preparation. The content of each active ingredient in the skin external preparation is not particularly limited, as long as it can achieve the effect of promoting the proliferation and/or differentiation of preadipocytes, taking into account its dosage form and administration method. isn't it. For example, the content of each active ingredient is preferably 0.1 x 10 ^-9 mass % or more, more preferably 0.1 x 10 ^-7 mass % (used as an aqueous solution), based on the total weight of the skin external preparation. 0.1 ng/mL) or more, more preferably 0.3×10 ⁻⁷ % by mass or more, still more preferably 1×10 ⁻⁷ % by mass or more. Further, in consideration of cytotoxicity as a helenalin derivative, the amount is preferably 1×10 ⁻² mass % or less, more preferably 1×10 ⁻³ mass % or less, and still more preferably 1×10 ⁻⁴ mass % or less. In one embodiment, when 6-O-tigloylhelenaline (compound 7) is contained, it is preferably contained at a concentration of about 1×10 ⁻⁷ mass % or less, or not contained.

(Form of external skin preparation)
The skin external preparation according to the present invention includes 1) pharmaceuticals, 2) quasi-drugs, in an appropriate form for use such as ampoules, capsules, powders, granules, liquids, gels, bubbles, emulsions, sheets, mist, and sprays. 3) Topical or systemic skin preparations (for example, basic cosmetics such as lotion, milky lotion, cream, ointment, lotion, oil, pack, etc., facial cleansers and skin cleansing such as bar soap, liquid soap, hand wash, etc.) Make-up cosmetics such as cosmetics, massage agents, cleansing agents, hair removal agents, depilatory agents, shaving agents, aftershave lotions, pre-shave lotions, shaving creams, foundations, lipsticks, blushers, eye shadows, eyeliners, mascara, etc. , perfumes, nail polishes, nail enamels, nail enamel removers, poultices, plasters, tapes, sheets, patches, aerosols, etc.), 4) Medicinal and/or applied to the scalp and hair. Cosmetic preparations (e.g. shampoos, conditioners, hair treatments, pre-hair treatments, permanent solutions, hair dyes, hair styling products, hair tonics, hair growth/nourishing products, poultices, plasters, tapes, etc.) (sheet preparations, aerosol preparations, etc.), 5) bath preparations that are added to bathwater, and 6) other products such as underarm odor preventers, deodorants, antiperspirants, sanitary products, sanitary cotton, and wet tissues.

(Components of external skin preparations)
In addition, such agents can be manufactured by arbitrarily selecting and combining the ingredients and additives listed below as long as they do not impair the effects of the present invention. Although there is no particular restriction on the amount to be added, it is generally considered preferable to be about 0.0001 to 50%.

(1) Various oils and fats: avocado oil, almond oil, fennel oil, perilla oil, olive oil, orange oil, orange laffa oil, sesame oil, cacao butter, chamomile oil, carrot oil, cucumber oil, beef tallow fatty acid, kukui nut oil, safflower oil , shea butter, liquid shea butter, soybean oil, camellia oil, corn oil, rapeseed oil, persic oil, castor oil, cottonseed oil, peanut oil, turtle oil, mink oil, egg yolk oil, palm oil, palm kernel oil, Japanese oak, palm oil Oil, beef tallow, lard, squalene, squalane, pristane, hydrogenated products of these fats and oils (hardened oil, etc.), etc.

(2) Waxes Beeswax, carnauba wax, spermaceti wax, lanolin, liquid lanolin, reduced lanolin, hard lanolin, candelilla wax, montan wax, shellac wax, rice wax, etc.

(3) Mineral oil Liquid paraffin, petrolatum, paraffin, ozokeride, ceresin, microcrystal wax, etc.

(4) Fatty acids Lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, linoleic acid, linolenic acid, docosahexaenoic acid, eicosapentaenoic acid, 12-hydroxystearic acid, undecylenic acid, tall oil, lanolin fatty acid Natural fatty acids such as isononanoic acid, caproic acid, 2-ethylbutanoic acid, isopentanoic acid, 2-methylpentanoic acid, 2-ethylhexanoic acid, and synthetic fatty acids such as isopentanoic acid.

(5) Alcohols Natural alcohols such as ethanol, isopropanol, lauryl alcohol, cetanol, stearyl alcohol, oleyl alcohol, lanolin alcohol, cholesterol, phytosterol, phenoxyethanol, synthetic alcohols such as 2-hexyldecanol, isostearyl alcohol, 2-octyldodecanol, etc. .

(6) Polyhydric alcohols Ethylene oxide, ethylene glycol, diethylene glycol, triethylene glycol, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, polyethylene glycol, propylene oxide, propylene glycol, polypropylene glycol , 1,3-butylene glycol, pentyl glycol, glycerin, pentaerythritol, threitol, arabitol, xylitol, ribitol, galactitol, sorbitol, mannitol, lactitol, maltitol, etc.

(7) Esters Isopropyl myristate, Isopropyl palmitate, Butyl stearate, Hexyl laurate, Myristyl myristate, Oleyl oleate, Decyl oleate, Octyldodecyl myristate, Hexyldecyl dimethyloctoate, Cetyl lactate, Myristyl lactate, Diethyl phthalate, dibutyl phthalate, lanolin acetate, ethylene glycol monostearate, propylene glycol monostearate, propylene glycol dioleate, etc.

(8) Metal soaps Aluminum stearate, magnesium stearate, zinc stearate, calcium stearate, zinc palmitate, magnesium myristate, zinc laurate, zinc undecylenate, etc.

(9) Gummies, sugars or water-soluble polymer compounds Gum arabic, benzoin gum, gum dammar, guaiac butter, Irish moss, gum karaya, gum tragacanth, gum carob, quinseed, agar, casein, lactose, fructose, sucrose or its ester, Trehalose or its derivatives, dextrin, gelatin, pectin, starch, carrageenan, carboxymethyl chitin or chitosan, hydroxyalkyl (C2-C4) chitin or chitosan to which alkylene (C2-C4) oxide such as ethylene oxide is added, low-molecular-weight chitin or chitosan, chitosan salt, sulfated chitin or chitosan, phosphorylated chitin or chitosan, alginic acid or its salt, hyaluronic acid or its salt, chondroitin sulfate or its salt, heparin, ethylcellulose, methylcellulose, carboxymethylcellulose, carboxyethylcellulose, carboxyethylcellulose sodium , hydroxyethyl cellulose, hydroxypropyl cellulose, nitrocellulose, crystalline cellulose, polyvinyl alcohol, polyvinyl methyl ether, polyvinyl pyrrolidone, polyvinyl methacrylate, polyacrylates, polyalkylene oxides such as polyethylene oxide and polypropylene oxide or crosslinked polymers thereof, carboxy Vinyl polymer, polyethyleneimine, etc.

(10) Surfactant Anionic surfactant (alkyl carboxylate, alkyl sulfonate, alkyl sulfate, alkyl phosphate), cationic surfactant (alkyl amine salt, alkyl quaternary ammonium salt), amphoteric Surfactants: Carboxylic acid type amphoteric surfactants (amino type, betaine type), sulfate ester type amphoteric surfactants, sulfonic acid type amphoteric surfactants, phosphate ester type amphoteric surfactants, nonionic surfactants ( Ether type nonionic surfactants, ether ester type nonionic surfactants, ester type nonionic surfactants, block polymer type nonionic surfactants, nitrogen-containing type nonionic surfactants), other surfactants ( natural surfactants, protein hydrolyzate derivatives, polymer surfactants, surfactants containing titanium and silicon, fluorocarbon surfactants), etc.

(11) Various vitamins Vitamin A group: retinol, retinal (vitamin A1), dehydroretinal (vitamin A2), carotene, lycopene (provitamin A), vitamin B group: thiamine hydrochloride, thiamine sulfate (vitamin B1), Riboflavin (vitamin B2), pyridoxine (vitamin B6), cyanocobalamin (vitamin B12), folic acids, nicotinic acids, pantothenic acids, biotin, choline, inositols, vitamin C group: vitamin C acid or its derivatives, vitamin D group: Ergocalciferol (vitamin D2), cholecalciferol (vitamin D3), dihydrotachysterol, vitamin E group: vitamin E or its derivatives, ubiquinones, vitamin K group: phytonadione (vitamin K1), menaquinone (vitamin K2), menadione (vitamin K3), menadiol (vitamin K4), other essential fatty acids (vitamin F), carnitine, ferulic acid, γ-oryzanol, orotic acid, vitamin P (rutin, eriocitrin, hesperidin), vitamin U, etc.

(12) Various amino acids valine, leucine, isoleucine, threonine, methionine, phenylalanine, tryptophan, lysine, glycine, alanine, asparagine, glutamine, serine, cysteine, cystine, tyrosine, proline, hydroxyproline, aspartic acid, glutamic acid, hydroxylysine , arginine, ornithine, histidine, etc., their sulfates, phosphates, nitrates, citrates, or amino acid derivatives such as pyrrolidone carboxylic acid.

(13) Various additives derived from plant- or animal-based raw materials These can be processed by conventional methods (e.g., crushing, milling, washing, hydrolysis, fermentation, purification) depending on the type and form of the product to which they are added. , compression, extraction, fractionation, filtration, drying, pulverization, granulation, dissolution, sterilization, pH adjustment, deodorization, decolorization, etc.). All you have to do is offer it.

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 in any way. In addition, in the following examples, the unit % of the numerical value showing the addition amount of various components means mass %.

[Example 1]
(Component fractionation of arnica flower hot water extract)
Dried arnica flowers were soaked in warm water at about 50°C for 5 hours. The solution obtained through this immersion was filtered using a predetermined filtering material (glass filter paper, Gf-75, manufactured by ADVANTEC) and a cellulose mixed ester membrane filter, A045A047A, manufactured by ADVANTEC. The filtered solution was concentrated using an evaporator, then turned into powder using a freeze dryer, and used as an arnica extract. This arnica extract was fractionated according to the fractionation scheme shown in FIG.

Arnica flower hot water extract (211.8 g) was suspended in distilled water (500 mL) and mixed with diethyl ether (5 x 250 mL, S01), ethyl acetate (5 x 250 mL, S02), butanol (5 x 250 mL, S03). Liquid-liquid partitioning was performed using a diethyl ether fraction (DEFr. 3.9 g), an ethyl acetate fraction (EFr. 2.8 g), a butanol fraction (BFr. 10.8 g), and a water fraction (WFr. (yield not measurable) was obtained.

The most active DEFr. (3.9 g) was subjected to silica gel open column chromatography (57 mmφ x 355 mm L (diameter 57 mm, length 355 mm), S04) and eluted using hexane:acetone = 3:1, 3:2, 1:1. The eluate was analyzed by TLC, and 13 fractions (DEFr.1 to DEFr.13) were obtained. DEFr. 9 (281.9 mg) was subjected to preparative HPLC (conditions 1, S06) to obtain Compound 1 (6.4 mg, retention time 19.7 min). DEFr. which showed high activity among the sub-fractions. 4 (659.2 mg) was subjected to LH-20 gel open column chromatography (20 mmφ x 515 mmL, S05) and eluted using chloroform:methanol = 1:1. The eluate was analyzed by TLC, and 7 fractions (DEFr.4-1 to DEFr.4-7) were obtained.

DEFr. 4-2 (462.6 mg) was subjected to silica gel open column chromatography (35 mmφ x 363 mmL, S07), and Hexane:Chloroform:Ethyl acetate=1:1:0.1, 1:1:0.3, 1:1 :1, and eluted using Chloroform:MeOH=1:1. The eluate was analyzed by TLC to obtain 13 fractions (DEFr.4-2-1 to DEFr.4-2-13).

DEFr. 4-2-6 (66.5 mg) was subjected to preparative HPLC (conditions 2, S09), compound 2 (5.1 mg, retention time 31.2 min), compound 3 (13.5 mg, retention time 31.8 min) , Compound 4 (19.3 mg, retention time 32.0 min) was obtained.

DEFr. 4-2-2 (348.4 mg) was subjected to ODS gel open column chromatography (30 mmφ×112 mmL, S08) and eluted using water:methanol=1:1, 1:2, 0:1. The eluate was analyzed by TLC, and 13 fractions (DEFr.4-2-2-1 to DEFr.4-2-2-13) were obtained.

DEFr. 4-2-2-4 (50.9 mg) was subjected to preparative HPLC (conditions 3, S10), and compound 5 (5.1 mg, retention time 30.8 min) and compound 6 (10.8 mg, retention time 31. 6 min) was obtained.

DEFr. 4-2-2-8 (77.2 mg) was subjected to preparative HPLC (conditions 4, S12) to obtain Compound 7 (43.2 mg, retention time 33.3 min).

DEFr. 4-2-2-6 and 7 (80.9 mg) were subjected to preparative HPLC (conditions 5, S11), and compound 8 (9.5 mg, retention time 31.6 min), compound 9 (10.9 mg, retention time 32.2 min) and Compound 10 (15.9 mg, retention time 32.5 min) were obtained.

(Structural analysis of isolated compounds)
Structural analysis of compound 1 DEFr. 9-1 (6.4 mg) was dissolved in heavy methanol and subjected to FT-NMR analysis. Further, it was dissolved in methanol and subjected to MALDI-TOF-MS analysis.

Structural analysis of compound 2 DEFr. 4-2-6-1 (5.1 mg) was dissolved in deuterated acetone and deuterated chloroform and subjected to FT-NMR analysis. Further, it was dissolved in methanol and subjected to MALDI-TOF-MS analysis.

Structural analysis of compound 3 DEFr. 4-2-6-2 (13.5 mg) was dissolved in deuterated acetone and deuterated chloroform and subjected to FT-NMR analysis. Further, it was dissolved in methanol and subjected to MALDI-TOF-MS analysis.

Structural analysis of compound 4 DEFr. 4-2-6-3 (19.1 mg) was dissolved in deuterated acetone and deuterated chloroform and subjected to FT-NMR analysis. Further, it was dissolved in methanol and subjected to MALDI-TOF-MS analysis.

Structural analysis of compound 5 DEFr. 4-2-2-4-1 (5.1 mg) was dissolved in deuterated chloroform and subjected to FT-NMR analysis. Further, it was dissolved in methanol and subjected to MALDI-TOF-MS analysis.

Structural analysis of compound 6 DEFr. 4-2-2-4-2 (10.8 mg) was dissolved in deuterated chloroform and subjected to FT-NMR analysis. Further, it was dissolved in methanol and subjected to MALDI-TOF-MS analysis.

Structural analysis of compound 7 DEFr. 4-2-2-8-1 (43.2 mg) was dissolved in deuterated chloroform and subjected to FT-NMR analysis. Further, it was dissolved in methanol and subjected to MALDI-TOF-MS analysis.

Structural analysis of compound 8 DEFr. 4-2-2-6-1 (9.5 mg) was dissolved in deuterated chloroform and subjected to FT-NMR analysis. Further, it was dissolved in methanol and subjected to MALDI-TOF-MS analysis.

Structural analysis of compound 9 DEFr. 4-2-2-6-2 (10.9 mg) was dissolved in deuterated chloroform and subjected to FT-NMR analysis. Further, it was dissolved in methanol and subjected to MALDI-TOF-MS analysis.

Structural analysis of compound 10 DEFr. 4-2-2-6-3 (3.2 mg) was dissolved in deuterated chloroform and subjected to FT-NMR analysis. Further, it was dissolved in methanol and subjected to MALDI-TOF-MS analysis.

The purity of the target compound in the sample used in the above structural analysis was analyzed by high performance liquid chromatography (HPLC), which will be described later, and the results are shown in Table 1 below.

<High performance liquid chromatography (HPLC)>
Equipment: SHIMADZU
Pump: LC-20AD
Diode array detector: SPD-M20A
Column oven: CTO-20AD
System controller: CBM-20A
Degasser: DGU-20A
Auto sampler: SIL-20A
Column: Inertsil (registered trademark) ODS-3, 5 (4.6 mmφ x 250 mmL)
Injection volume: 10μl
Analysis time: 60min
Detector wavelength: 210-600nm
Flow rate: 1.0ml/min
Gradient program: MeOH: 0.05% TFAaq. =5%:95%→100%:0% (40min)

<Preparative high performance liquid chromatography (Preparative HPLC)>
Equipment: JASCO
Pump: 880-PU
Detector: 870-UV
Degassa: 880-51
System controller: 880-30
Column: Inertsil (registered trademark) ODS-3, 5 (20 mmφ x 250 mmL)
Injection volume: 350μl
Detector wavelength: 280.0nm
Flow rate: 9.0ml/min

(Condition 1)
Gradient program: MeOH: 0.05% TFA aq. =25:75→100:0 (30min)

(Condition 2)
Gradient program: MeOH: 0.05% TFA aq. =60:40→85:15 (30min)

(Condition 3)
Gradient program: MeOH:Water=60:40 (30min)

(Condition 4)
Gradient program: Acetonitrile: Water = 60:40 → 85:15 (30min)

(Condition 5)
Gradient program: Acetonitrile: 0.05% TFA aq. =50:50 (30min)

(Condition 6)
Gradient program: MeOH:Water=65:35 (30min)

<Transformation nuclear magnetic resonance apparatus (FT-NMR)>
Equipment: JEOL EC 600MHz (JEOL, TOKYO, JAPAN)
Solvent: Chloroform-d (Cambridge Isotope Laboratories, Inc. CAS865-49-6)
Methanol-d4 (Kanto Kagaku Co., Ltd., CAS25221-96)
Acetone-d6 (SIGMA-ALDRICH, CAS666-52-4)

<Matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS)>
Equipment: SHIMADZU BIOTECH AXIMA RESONANCE
Ion mode: positive ion mode
Mass range: Low100+, Low300+
Power: 120-150
Matrix: 2,5-dibenzoic acid (DHB)

<Polarimeter>
Equipment: JASCO-P2300 system
Solvent: Methanol, Chloroform

Structures of Compound 1 and Compounds 3 to 10 estimated from the ¹³ C-NMR spectrum, ¹ H-NMR spectrum, COSY spectrum, HMBC spectrum, HMQC spectrum, and MALDI-TOF-MS spectrum obtained from the above measurements for each sample. is shown below.

(Proliferation test of human subcutaneous preadipocytes)
It was confirmed whether cell proliferation was promoted by adding the purified fraction to human subcutaneous preadipocytes. Each purified fraction used in this evaluation was prepared based on the mass recovered from the arnica flower hot water extract and in the proportions present in the original extract, as described in Tables 2 and 3. did. First, each purified fraction was dissolved in DMSO to prepare a storage solution. This was dissolved in 30% BG to a concentration of 0.75%, and then added to the cells at a 1000-fold or 3000-fold dilution. The parent arnica flower hot water extract powder was prepared by dissolving 50 mg in 500 μL of 30% BG, diluting it in 30% BG to 0.75%, and then adding 0.75% DMSO. It was added to the cells at a 1000-fold or 3000-fold dilution. For comparison, 30% BG with 0.75% DMSO was prepared and added to cells at a 1000-fold or 3000-fold dilution.

Human subcutaneous preadipocytes (manufactured by Lonza, PT-5020) were grown at 1×10 ³ cells/well in a 96-well plate using Preadipocyte Growth Medium (PT-8002, manufactured by Lonza) containing 10% FBS. The cells were spread and cultured at 37° C. for 16 hours in a 5% CO ₂ incubator. Thereafter, each purified fraction was added to the above concentration and cultured at 37° C. for 72 hours in a 5% CO ₂ incubator. After culturing, the number of cells in the wells was measured. As a control for this experiment, wells to which 30% 1,3-butylene glycol, an extraction solvent containing no purified fractions, had been added were set up, and the number of cells in these wells was also measured. The ratio (relative value) of the number of cells in the well to which each purified fraction was added was calculated, assuming that the number of cells in the set well was 100.

(Human subcutaneous preadipocyte differentiation test)
It is known that when preadipocytes differentiate into adipocytes, lipid droplets are stored within the cells (Obesity Research Vol. 13, No. 1, 2007, pages 84-86). Whether addition of each of the purified fractions described above to human subcutaneous preadipocytes promotes differentiation into adipocytes was confirmed by the formation of lipid droplets. Each purified fraction used in this evaluation was prepared to the concentration present in the original extract based on the mass recovered from the arnica flower hot water extract, and diluted 1000-fold or 3000-fold with respect to cells. Added with.

Human subcutaneous preadipocytes (manufactured by Lonza, PT-5020) were grown at 1×10 ⁴ (cells)/well in a 96-well plate using Preadipocyte Growth Medium (manufactured by Lonza, PT-8002) containing 10% FBS. and cultured for 16 hours at 37°C in a 5% CO ₂ incubator. After visually confirming confluence using a microscope, a differentiation induction medium (PT-8002, manufactured by Lonza) was added at 1/2 the concentration recommended by Lonza. At that time, each purified fraction was added to the above concentration and cultured at 37° C. for 10 days in a 5% CO ₂ incubator. After visually confirming the accumulation of lipid droplets using a microscope, the culture solution was removed and the wells were washed twice with PBS. After fixing cells with 10% neutral buffered formalin (Wako Pure Chemical Industries, Ltd., 062-01661), lipid droplets were fixed using Oil Red O (154-02072, Wako Pure Chemical Industries, Ltd.) saturated with 60% isopropyl alcohol. stained. After washing the wells with purified water, the stained lipid droplets were extracted using 100% isopropyl alcohol, and the absorbance was measured at 510 nm. The absorbance value of the well to which 30% 1,3-butylene glycol, which is an extraction solvent, was added to the above concentration instead of each purified fraction was set as 1, and the absorbance value of the well to which arnica extract was added (relative). value) was calculated and used as the differentiation rate (relative value).

The results are shown in Figures 2-4. In FIGS. 2 to 4, "Parent" represents the arnica flower hot water extract used as a raw material for purification, and "Cont" represents 30% 1,3-butylene glycol. From the results shown in FIG. 2, the fraction DEFr. And this fraction was subjected to silica gel open column chromatography fractionation (S04) to obtain DEFr. Activities to promote proliferation and differentiation of human subcutaneous preadipocytes were found in 4 fractions. Therefore, DEFr. The four fractions were further fractionated by silica gel open column chromatography and preparative HPLC. The results of measurement of the obtained fractions (DEFr.2, Cmpd1, Cmpd3, Cmpd4, Cmpd5, Cmpd6, Cmpd7, Cmpd8, Cmpd9, and Cmpd10 listed in Table 3) by the above method are shown in FIG. 3. From the results shown in FIG. 3, it was found that Cmpd7 (containing compound 7), Cmpd8 (containing compound 8), and Cmpd9 (containing compound 9) have proliferation and differentiation promoting activity of human subcutaneous preadipocytes. The reason why the proliferation activity of human subcutaneous preadipocytes decreases with a 1000-fold dilution of Cmpd7 is thought to be due to the cytotoxicity of this compound.

Therefore, Figure 4 shows the results of the proliferation test and differentiation test of human subcutaneous preadipocytes when Cmpd7 (containing compound 7), Cmpd8 (containing compound 8), and Cmpd9 (containing compound 9) were used alone or in combination. Shown below. In addition, in FIG. 4, "Cmpd7+8" is a combination of Cmpd7 and Cmpd8, "Cmpd7+9" is a combination of Cmpd7 and Cmpd9, "Cmpd8+9" is a combination of Cmpd8 and Cmpd9, and "Cmp7+8+9" is a combination of Cmpd7, Comp8, and Cmpd9. Addition is indicated. From the results shown in FIG. 4, it was found that the combination of Cmpd8 and Cmpd9 exhibited the strongest activity in both proliferation and differentiation promotion of human subcutaneous preadipocytes. In Figures 2 to 4, * has a significant difference of p<0.05 with Dunnett's test (one side only) for Cont, and ** has a significant difference of p<0.05 with Dunnett's test (one side only) for Cont. Indicates a significant difference of <0.01.

[Example 2]
Of the 13 fractions (DEFr.1 to DEFr.13) obtained by fractionating the diethyl ether soluble portion obtained in Example 1 by silica gel open column chromatography, DEFr. 3 and DEFr. Each fraction 5-8 was further purified and new compounds 12-19 were isolated. The fractionation scheme for isolating this new compound is shown in Figure 5.

DEFr. 3 (340.0 mg) was subjected to ODS gel open column chromatography (30 mmφ×112 mmL, S14) and eluted using water:methanol=3:2. Among the sub-fractions, DEFr. 3-8 (156.0 mg) was subjected to preparative HPLC (acetonitrile:water = 39:61, S15) to obtain compound 17 (8.3 mg). On the other hand, the obtained fraction DEFr. 3-8-5 was further subjected to preparative HPLC (hexane:2-propanol=90:10, S16) to obtain Compound 16 (15.8 mg).

DEFr. 5 (504.6 mg) was subjected to LH-20 gel open column chromatography (20 mmφ x 515 mmL, S17) and eluted using methanol:chloroform=3:2. The eluate was analyzed by TLC and two fractions (DEFr.5-2 and DEFr.5-3) were obtained. This fraction DEFr. 5-2 (167.0 mg) was subjected to preparative HPLC (methanol:water = 45:55, S18) to obtain Compound 13 (11.2 mg). On the other hand, the obtained fraction DEFr. 5-2-1 was further subjected to preparative HPLC (acetonitrile:water=30:70, S19) to obtain Compound 14 (2.1 mg). Fraction DEFr. 5-3 (130.1 mg) was subjected to ODS gel open column chromatography (S20). Elution was performed using water:methanol=2:3, 1:1, and 1:0, respectively. The obtained fraction DEFr. 5-3-23 (5.0 mg) was further subjected to preparative HPLC (methanol:water = 40:60, S21) to obtain compound 15 (2.9 mg) in fraction DEFr. 5-3-4 (40.0 mg) was further subjected to preparative HPLC (methanol:water = 25:75, S22) to obtain Compound 15 (13.4 mg).

DEFr. 6 (299.0 mg) was subjected to ODS gel open column chromatography (30 mmφ×112 mmL, S23) and eluted using water:methanol=1:1, 1:2, 0:1. The obtained fraction DEFr. 6-3 (59.1 mg) was subjected to silica gel column chromatography and eluted with chloroform:methanol=20:1 (S24), and one eluted fraction DEFr. 6-3-1 (40.0 mg) was further subjected to preparative HPLC (methanol:water = 40:60, S25) to obtain Compound 12 (9.5 mg).

DEFr. 7 (515.0 mg) was subjected to LH-20 gel open column chromatography (20 mmφ x 515 mm L, S05) and eluted using chloroform:methanol = 1:1. The eluate was analyzed by TLC to obtain 6 fractions (DEFr.7-1 to DEFr.7-6). DEFr. 7-4 (32.0 mg) was subjected to preparative HPLC and eluted with methanol:water (0.05% TFA) = 30:70, S27) to obtain Compound 18 (18.2 mg).

DEFr. 8 (548.2 mg) was subjected to LH-20 gel open column chromatography (20 mmφ×515 mmL, S05) and eluted using chloroform:methanol=1:1. One fraction obtained DEFr. 8-7 (25.3 mg) was subjected to preparative HPLC and eluted with methanol:water (0.05% TFA) = 25:75, S29) to obtain Compound 19 (11.3 mg).

(Structural analysis of isolated compounds)
The structures of compounds 11 to 19 estimated from ^{the 13} C-NMR spectrum, ¹ H-NMR spectrum, COSY spectrum, HMBC spectrum, HMQC spectrum, and MALDI-TOF-MS spectrum by the same method as in Example 1 are shown below. .

By the same method as in Example 1, a proliferation test and a differentiation test of human subcutaneous preadipocytes were conducted. The results are shown in Table 4 and FIG. 6 below. In Table 4 and FIG. 6, "KS0019" and "KS0020" described as sample names represent different arnica extract lots. "BG/DMSO" represents a group containing 30% 1,3-butylene glycol and DMSO, which is an extraction solvent that does not contain purified fractions. Cmpd11 to Cmpd19 contain compounds 11 to 19, respectively. The concentration of each sample was adjusted so that the concentration of the compound was the same as that in the arnica extract, and it was added to the cells at a 1000-fold dilution.

From the results in Table 4 and FIG. 6, Cmpd12 (containing compound 12) and Cmpd13 (containing compound 13) significantly promoted the differentiation of human subcutaneous preadipocytes.
In addition, in FIG. 6, * has a significant difference of p<0.05 with Dunnett's test (one side only) for BG/DMSO, and ** has a significant difference of p<0.05 with Dunnett's test (one side only) for BG/DMSO. indicates a significant difference of p<0.01, and *** indicates a significant difference of p<0.001 by Dunnett's test (one-sided only) for BG/DMSO.

The preadipocyte proliferation and/or differentiation promoting agent of the present invention can be used industrially as cosmetics, foods, medicines, etc., and/or materials to be mixed therein.

Claims (5)

The following formula (I):
(In the formula, R is a hydrogen atom, an acetyl group, a methacryloyl group, or an isobutyryl group.) An agent containing a compound represented by the following as an active ingredient, wherein the mass ratio exceeds the total amount of the active ingredients. - A preadipocyte proliferation and/or differentiation promoting agent that does not contain O-tigloylhelenaline . The preadipocyte proliferation and/or differentiation promoting agent according to claim 1, wherein the compound is 6-O-methacryloylhelenaline or 6-O-isobutyrylhelenaline. The preadipocyte proliferation and/or differentiation promoting agent according to claim 1, which contains both 6-O-methacryloylhelenaline and 6-O-isobutyrylhelenaline. A skin external preparation for promoting proliferation and/or differentiation of preadipocytes, which contains an effective amount of the preadipocyte proliferation and/or differentiation promoting agent according to any one of claims 1 to 3 , The external preparation does not contain 6-O-tigloylhelenaline in an amount exceeding the total amount of the active ingredients . The skin external preparation according to claim 4 , wherein the content of 6-O-methacryloylhelenaline and/or 6-O-isobutyrylhelenaline is at least 0.1 x 10 ^-7 % by mass.