JP6152735B2 - White adipocyte brown-like adipocyte differentiation inducer composition - Google Patents

Description

  The present invention induces brown-like adipocyte differentiation of white adipocytes, which can be applied to humans for the first time by using an extract containing a resveratrol cyclization reaction product obtained by a very simple method. It is related with an agent composition.

  There are two types of adipocytes that constitute mammalian adipose tissue: white adipocytes and brown adipocytes. White adipocytes have the role of accumulating energy ingested mainly in the form of triglycerides, while brown adipocytes originate from myoblasts and turn brown when they contain many mitochondria. It is an adipocyte having a role of generating heat by the action of an uncoupling protein 1 (UCP1) expressed therein and converting the accumulated energy into heat (Non-patent Document 1). A large number of brown adipose tissues composed of brown adipocytes are observed in early childhood, and the amount of the tissue decreases as it grows. Conventionally, it has been considered that brown adipocytes do not change from white adipocytes to brown adipocytes because stem cells derived from white adipocytes are different. However, in recent years, the existence of “brown-like adipocytes” that show the same response as brown adipocytes in white adipose tissue has been revealed (Non-patent Document 2). Brown-like adipocytes are adipocytes that are derived from white adipocytes but have much more mitochondria than white adipocytes and can efficiently convert the accumulated lipid energy into heat energy. This report reveals that human adults may also be able to increase brownish adipose tissue, and the induction of differentiation of white adipocytes into brownish adipocytes is new to obesity and diabetes, and Attention has been rapidly increasing as a fundamental treatment method.

  However, it is difficult to induce differentiation of white adipocytes into brown-like adipocytes, and methods that have been confirmed include long-term cold stimulation, stimulation with adrenaline (Non-patent Document 1), peroxime proliferator-responsive reception. Only the addition of a body (PPARγ) agonist (Non-patent Document 3) or the like, and in addition, the differentiation-inducing effect is limited only to subcutaneous fat cells. Further, Patent Document 1 reports a compound having an effect of amplifying UCP1 gene expression on human visceral fat cells, but browning of white adipocytes is not observed, and the concentration of action is very high. The browning of white adipocytes has not been realized at present, such as it is difficult to achieve an effective concentration in the body.

  In addition, it has been clarified that 22 types of fibroblast growth factors (FGFs) currently exist in humans based on amino acid sequence homology (Non-patent Document 4). FGF-21 has been reported to be involved in the promotion of fat degradation and suppression of accumulation, hypercholesterolemia, diabetes (hyperglycemia, insulin resistance) and obesity (Non-Patent Document 6). . For example, Patent Document 2 describes a compound that promotes the production of FGF-21 and has an effect of reducing visceral fat. Patent Document 3 describes FGF-21 and a glucagon-like peptide-1 receptor agonist, antidiabetics. It is described that a therapeutic agent for type 2 diabetes comprising a drug, a dipeptidyl peptidase-4 inhibitor, is useful. Alternatively, Patent Document 4 describes that it is useful for treating metabolic diseases to extend the half-life of FGF-21 using an FGF-21 derivative. In recent years, there has been a report that cold stimulation promotes an increase in blood FGF-21 concentration and promotes browning of white fat, and FGF-21 has attracted attention as one of the browning inducers of white fat. (Non-Patent Document 7). However, so far, reports targeting FGF-21 are only for the purpose of improving metabolic disorders, and no specific proposal has been made as a means for browning white adipocytes.

  In addition, the useful physiological effect of red wine, so-called “French paradox”, is attributed to various physiologically active functions including resveratrol's antioxidant ability. Resveratrol is a kind of hydroxystilbene that is abundant in grape skin and peanut red skin, and is known as a plant-derived compound with various activities such as anti-fungal, anti-bacterial, anti-inflammatory, etc. (Non-patent Document 8). Although resveratrol has been reported on the gene expression enhancing effect of uncoupling protein UCP2 specifically expressed in white adipocytes (Patent Document 5), until now, resveratrol has been reported from white adipocytes to brown-like fat. There is no mention of differentiation effects on cells.

  Thus, to date, metabolic syndrome is prevented and treated by changing the properties of fat cells themselves from white adipocytes to brown-like adipocytes, rather than suppressing appetite or suppressing fat absorption. Development of a fundamental therapeutic / prophylactic agent has been desired, but so far no substance has been found to have a sufficient effect on all white adipocytes, and early development is desired. ing.

JP 2011-241195 A International Publication No. 2011/037223 Japanese Patent Publication No. 2013-518035 Japanese Patent Publication No. 2012-515747 JP 2010-24208 A

Cell Biosci. 2011 Oct 28; 1:35 Cell. 2012 Jul 20; 150 (2): 366-769 Cell Metab. 2012 Mar 7; 5 (3): 395-404. Genome Biol. 2001; 2 (3): REVIEWWS3005 Am J Clin Nutr. 2010 January; 91 (1): 254S-257S Genes Dev. 2012 Feb 1; 26 (3): 271-81 Resveratrol basics and applications, CMC Publishing Nature. 2004 June 17; 429 (6993): 771 Nature. 2012 Sep 13; 489 (7415): 318

In view of the above situation, the present inventors have conducted a search for a compound that has an action of inducing differentiation of white adipocytes into brown-like adipocytes, and have intensively studied to establish a production method thereof. Surprisingly, it succeeded in producing a composition showing an action of inducing differentiation of white adipocytes into brown-like adipocytes by a simple and safe method of heat treatment under the conditions, leading to the completion of the present invention. It was.
Therefore, an object of the present invention is to produce a new effect that has not been seen in resveratrol so far, which has an excellent differentiation-inducing action of white adipocytes into brown-like adipocytes, and can be produced safely and inexpensively. It is an object to provide a composition for inducing differentiation of brown-like adipocytes capable of white adipocytes, and pharmaceuticals and quasi-drugs containing the compositions.

That is, the gist of the present invention is as follows.
[1 ] Formula (1):

、式（２）：

Formula (2):

And formula (3):

In three resveratrol polymer compound differentiation inducer composition to brownish adipocytes in white color adipocytes you containing indicated,
[2] it relates to the [1] Symbol mounting of white adipocytes pharmaceutical or quasi drug comprising the differentiation inducer composition to brownish adipocytes.

The composition for inducing differentiation of white adipocytes into brown-like adipocytes according to the present invention (hereinafter also referred to as the composition of the present invention) can be produced safely and inexpensively by heating resveratrol under alkaline conditions. In addition, because it has a remarkably excellent white fat cell-to-brown adipocyte differentiation-inducing action that is a new action that the resveratrol precursor does not have, it can be used for novel treatment and prevention of metabolic syndrome Useful.
In addition, the composition of the present invention can be obtained from an excellent white adipocyte to a brown-like adipocyte without performing a compound isolation and purification step as described in Japanese Patent Application No. 2013-071836 filed earlier by the present applicant. Therefore, it is possible to provide an agent for inducing differentiation from white adipocytes to brown-like adipocytes at a lower cost than in the prior invention.
In addition, a novel pharmaceutical or quasi-drug for preventing or improving metabolic syndrome can be provided by blending the composition of the present invention with a pharmaceutical or quasi-drug.

1 shows a chromatogram of a reaction solution of resveratrol carried out in Example 1. FIG. FIG. 2 shows FGF-21 in adipocytes immediately after differentiation induction performed in Example 2, peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a mitochondrial marker gene, which is a transcriptional coactivator. Cytochrome c oxidase polypeptide 7A1 (Cox7a1), a transcription coactivator whose enhanced expression is observed in brown-like adipocytes, and peroxisome proliferator-activated receptor γ coactivator 1β (PGC-1β) It is a graph which shows the relative expression level of a gene. FIG. 3 shows FGF-21, PGC-1α, PGC-1β, cell death-inducing DFFA-like effector a (Cidea), which is a brown-like adipocyte marker gene in matured adipocytes carried out in Example 3, and transcription core. It is a graph which shows the relative expression level of each gene of the peroxisome proliferator activated receptor (gamma) co-activator 1β (PGC-1β) which is a activator. FIG. 4 shows PGC-1β, UCP1, COX7a1, and brown-like adipocyte marker gene CBP / p300-interacting transactivator (CITED1) in adipocytes after induction of normal subcutaneous fat differentiation performed in Example 4 It is a graph which shows the relative expression level of each gene.

  Hereinafter, the present invention will be described in detail.

  Resveratrol used as a raw material compound in the composition of the present invention may be naturally derived or may be a chemically synthesized chemical product with high purity. The naturally occurring raw material compound does not need to be completely purified, and a mixture containing other compounds can also be used.

  However, from the viewpoint of increasing the reaction efficiency by heating, the resveratrol is preferably contained as a raw material containing a total of 5% by weight or more in terms of resveratrol. As such a raw material, for example, an extract from a raw material such as grape skin, wine, wine concentrated powder, meringo, lingonberry, peanut peel, itadori, a freeze-dried product of the extract, or the like may be used.

  In the present invention, resveratrol is dissolved in a suitable solvent. At this time, if the solvent is only water, the solubility of resveratrol in water is low, and therefore, it is preferable to dissolve in only a mixed solution of water and an organic solvent or only an organic solvent. There is no restriction | limiting in particular about the compounding ratio of water and an organic solvent, and the kind of organic solvent, Resveratrol should just fully melt | dissolve. Among them, it is preferable from the viewpoint of safety and cost to use a solvent containing only methanol or ethanol, or a mixed solution of water and methanol, water and ethanol, or the like. When the final purification is not sufficiently applied to the composition obtained after the heating reaction, and the composition is used for a pharmaceutical product, quasi-drug, etc., ethanol or water as a solvent from the viewpoint of safety or regulations. It is desirable to use hydrous ethanol.

  The concentration of resveratrol in the mixed solution (hereinafter also referred to as resveratrol-containing solution) obtained by dissolving resveratrol in a solvent as described above is not particularly limited, but the concentration of resveratrol The higher the value, the smaller the amount of solvent used, and the more the resveratrol concentration is preferably adjusted to be close to the concentration at which resveratrol is saturated with respect to each solvent.

  Next, the pH of the resveratrol-containing solution is adjusted to be alkaline. As an adjustment method, for example, after preparing a resveratrol-containing solution, the pH may be adjusted by adding an alkalizing agent, or the pH of the solvent is adjusted in advance when preparing the aforementioned resveratrol-containing solution. Also good. If the pH at the start of the heating reaction of the resveratrol-containing solution is 8.0 or more, it is preferable because the reaction for efficiently producing a plurality of useful resveratrol polymer compounds proceeds as described later. If exceeded, other reactions and decomposition of the target compound occur at the same time as the reaction, and the final recovered amount of resveratrol polymer compound tends to decrease. Accordingly, the pH at the start of the reaction is desirably 8.0 to 13.0.

  The alkalinizing agent is not particularly limited, but sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodium carbonate and the like are desirable from the viewpoint of safety, efficiency and cost. When the case where the pH change during the reaction is suppressed as much as possible occurs, a buffer solution may be used, but this is not always necessary.

  Next, the resveratrol-containing solution adjusted to be alkaline is heated. By this heating, resveratrol is cyclized to produce a useful compound. Such useful compounds include those represented by the formula (1)

Formula (2):

And formula (3):

3 types of resveratrol polymerization compounds represented by

  In the resveratrol polymer compound, the carbon-carbon double bond may be trans or cis, and includes a mixture of a cis isomer and a trans isomer.

  The resveratrol polymerization compound may be a pharmaceutically acceptable salt thereof.

  Examples of the pharmaceutically acceptable salt of the resveratrol polymer compound include alkali metal salts such as lithium salt, sodium salt and potassium salt; alkaline earth metal salts such as magnesium salt, calcium salt and barium salt; aluminum Salts; metal hydroxide salts such as aluminum hydroxide salts; alkylamine salts, dialkylamine salts, trialkylamine salts, alkylenediamine salts, cycloalkylamine salts, arylamine salts, aralkylamine salts, heterocyclic amine salts, etc. Amine salts; amino acid salts such as α-amino acid salts and ω-amino acid salts; peptide salts or primary, secondary, tertiary, or quaternary amine salts derived from them. These pharmacologically acceptable salts can be used alone or in admixture of two or more.

In the present invention, the cyclization reaction for forming the resveratrol polymer compound refers to a reaction in which resveratrol forms a 6-membered ring by polymerization reaction.
In order to efficiently advance the cyclization reaction, the heating temperature of the resveratrol-containing solution is preferably adjusted to 110 ° C. or higher. Also, considering the boiling point of the solvent used, pressure heating is desirable. For example, a resveratrol-containing solution is put in an open container, the container is heated at a high temperature exceeding the boiling point of the solvent, a resveratrol-containing solution is put in a sealed container, and the container is heated, using a retort apparatus or an autoclave. It is preferable to heat at least partially such that the temperature of the resveratrol-containing solution reaches 110 ° C. or higher, such as by heating under pressure, using a device such as a supercritical device or a pressure cooker. From the viewpoint of recovery efficiency of the resveratrol polymer compound and the like, it is more preferable that the temperature of the resveratrol-containing solution is uniformly 120 ° C. to 180 ° C. The heating time is not limited as in the case of the heating temperature, and may be a time condition in which the target reaction efficiently proceeds. In particular, the heating time depends on the heating temperature, and it is desirable to set the heating time according to the heating temperature. For example, when heating near 130 ° C., a heating time of 10 minutes to 120 minutes is desirable. Further, the heating may be performed once or may be repeated repeatedly in a plurality of times. When heating in multiple steps, it is preferable to add a new solvent.

The completion of the production reaction of the resveratrol polymer represented by the formulas (1) to (3) by the heating may be judged by confirming the production amount by component analysis by HPLC, for example.
In addition, about the production amount of the resveratrol polymerization compound represented by said Formula (1)-(3), the differentiation induction effect | action to a brown-like adipocyte of a white fat cell is so strong that there are many in the composition of this invention. However, specifically, the total amount of each may be 1% by weight or more in the composition of the present invention.
Moreover, there is no limitation in particular about each content rate of the resveratrol polymerization compound represented by Formula (1), Formula (2), and Formula (3).

In order to improve the flavor and further enhance the functionality, the reactant is concentrated to increase the concentration of the resveratrol polymerization compound represented by formulas (1) to (3), or the reactant is removed. The salt can be refined to obtain a compound having an increased concentration of the resveratrol polymer represented by the formulas (1) to (3). The concentration and purification can be performed by a known method. For example, the resveratrol polymerization compound represented by the formulas (1) to (3) can be concentrated by extraction using a solvent extraction method such as chloroform, ethyl acetate, ethanol, methanol, or the like, or a supercritical extraction method using carbon dioxide gas. It is also possible to perform concentration and purification using column chromatography. For the concentration and purification, a membrane treatment method such as a recrystallization method or an ultrafiltration membrane can be used.
Among them, by using a synthetic adsorbent, the resveratrol polymer compound represented by the above formulas (1) to (3) is adsorbed and then eluted and then can be easily concentrated and purified. Examples of the synthetic adsorbent include aromatic synthetic adsorbents such as Diaion (registered trademark) HP series and Sepabeads (registered trademark) SP series manufactured by Mitsubishi Chemical Corporation, and Amberlite (registered trademark) manufactured by Organo Corporation. ) Styrenic synthetic adsorbents such as XAD series.

  Moreover, a powdery solid can be obtained by removing the solvent by subjecting the concentrate or purified product to reduced-pressure drying or freeze-drying as necessary.

  The composition of the present invention containing the resveratrol polymerization compound represented by the formulas (1) to (3) obtained as described above is a powerful white adipocyte not found in resveratrol as a raw material Has the effect of inducing differentiation into brown-like adipocytes, and by this brown-like adipocyte differentiation-inducing action, metabolic syndrome can be prevented and treated, the composition of the present invention is novel. It is useful for preventing and / or treating metabolic syndrome.

  The dosage of the composition of the present invention may vary widely depending on the patient's sex, age, physiological state, pathological condition (such as progress of obesity), formulation, administration route, number of administrations, active ingredient concentration in the drug, etc. For example, the content of the composition of the present invention may be about 0.01 to 500 mg / kg, preferably about 0.1 to 100 mg / kg per day for an adult. Administration may be divided, for example, once or several times per day.

  The composition of the present invention may be formulated as a pharmaceutical product. There are no particular limitations on the form of the preparation, and examples include parenteral preparations such as injections, suppositories, eye drops, ointments, aerosols, tablets, coated tablets, powders, fine granules, granules, capsules, and liquids. , Pills, suspensions, emulsions, lozenges, chewable tablets, syrups and other oral preparations. For formulation, pharmaceutically acceptable carriers, excipients, lubricants, binders, disintegrants, diluents, stabilizers, tonicity agents, pH adjusters, buffers, etc. are used. It is done.

  Carriers and excipients include, for example, lactose, sucrose, sodium chloride, glucose, maltose, mannitol, erythritol, xylitol, maltitol, inositol, dextran, sorbitol, albumin, urea, starch, calcium carbonate, kaolin, crystalline cellulose , Silicic acid, methylcellulose, glycerin, sodium alginate, gum arabic and mixtures thereof.

Examples of the lubricant include purified talc, stearate, borax, polyethylene glycol, and a mixture thereof.
Examples of the binder include simple syrup, glucose solution, starch solution, gelatin solution, polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, carboxymethyl cellulose, shellac, methyl cellulose, ethyl cellulose, water, ethanol, potassium phosphate, and a mixture thereof. Can be mentioned.

  Examples of the disintegrant include dry starch, sodium alginate, agar powder, laminaran powder, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose and mixtures thereof. Is mentioned.

Examples of the diluent include water, ethyl alcohol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, and mixtures thereof.
Examples of the stabilizer include sodium pyrosulfite, ethylenediaminetetraacetic acid, thioglycolic acid, thiolactic acid, and a mixture thereof.

Examples of the isotonic agent include sodium chloride, boric acid, glucose, glycerin and a mixture thereof.
Examples of the pH adjuster and buffer include sodium citrate, citric acid, sodium acetate, sodium phosphate, and mixtures thereof.

  Furthermore, the composition of the present invention may contain a bulking agent, a solubilizer, a dispersant, a suspending agent, an emulsifier, an antioxidant, a bacteria inhibitor, a colorant, a corrigent, a flavoring agent and the like.

  In addition, the composition of the present invention may be formulated into a quasi-drug form. Although it does not specifically limit as a quasi-drug, For example, nutritional supplement quasi-drugs, such as a drink, are preferable. In this case, the content of the extract as an active ingredient in the quasi drug may be about 0.001 to 30% by weight as the solid content.

  The composition of the present invention is not only for humans but also for non-human animals such as rats, mice, guinea pigs, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees, and other mammals, You may mix | blend with therapeutic agents or feed, such as birds, amphibians, and reptiles. As feed, for example, livestock feed used for sheep, pigs, cattle, horses, chickens, etc., feed for small animals used for rabbits, rats, mice, etc. The pet food used for a cat, a small bird, a squirrel, etc. is mentioned.

  EXAMPLES Next, although this invention is demonstrated in detail based on an Example, this invention is not limited only to this Example.

(Example 1: Production and purification of a composition containing a resveratrol polymerization compound)
Preparation and purification of a composition containing a resveratrol polymerization compound were performed according to the following procedure. 1 g of trans-resveratrol (manufactured by TECNO SCIENCE Co., Ltd.) is dissolved in 10 mL of ethanol, and 10 mL of a 10% aqueous solution of sodium hydrogen carbonate (manufactured by Wako Pure Chemical Industries, Ltd.) is added to prepare a resveratrol-containing solution ( pH 9.9) was obtained. This resveratrol-containing solution was heated at 130 ° C. for 90 minutes in an autoclave (manufactured by Sanyo Electric, “SANYO LABO AUTOCLAVE”, the same shall apply hereinafter) to prepare a resveratrol-polymerized compound-containing solution.
Next, the resveratrol polymerization compound-containing solution was diluted and dissolved with 1 L of distilled water, and supplied to 400 g of synthetic adsorbent Diaion (registered trademark) HP-20 (manufactured by Mitsubishi Chemical Corporation). After washing with 1 L of distilled water, elution was performed with 1 L of 100% ethanol. The solvent was removed by drying under reduced pressure to obtain 200 mg of a resveratrol polymerization compound-containing composition (hereinafter referred to as a derivative-containing composition). The obtained polymerization compound composition was dissolved in methanol at a concentration of 2 mg / mL, 10 μL of which was analyzed by HPLC.

HPLC analysis was performed under the following conditions.
Column: CAPCELL PAK UG80 column (4.6 mm ID × 250 mm, manufactured by Shiseido Co., Ltd.)
Mobile phase A: 0.1% trifluoroacetic acid (TFA, manufactured by Wako Pure Chemical Industries, Ltd.) / H2O
Mobile phase B: 0.1% TFA / acetonitrile (manufactured by Wako Pure Chemical Industries, Ltd.)
Gradient (volume%): 100% A / 0% B to 0% A / 100% B for 33 minutes, 0% A / 100% B for 7 minutes (all straight lines)

The obtained chromatogram is shown in FIG. When the obtained reaction solution was analyzed by HPLC, a plurality of peaks different from the peak of resveratrol could be detected, and thus a composition containing a plurality of produced compounds could be obtained.

  Next, among the obtained reactants, the compounds contained in the peaks shown in (1), (2) and (3) of FIG. 1 were isolated by preparative HPLC and dried by a conventional method. ), The compound contained in the peak shown in (2) was a light brown powdery substance, and the compound contained in the peak shown in formula (3) was a brown powdery substance.

  Subsequently, the molecular weights of the three kinds of compounds were measured with a high resolution negative-FAB-MS (Fast Atom Bombardment-Mass Spectrometry) according to the method described in Example 2 of JP 2011-251914 A, and nuclear magnetic field was measured. As a result of the resonance (NMR) measurement, the compound contained in the peak of (1) is the compound represented by the above formula (1) (hereinafter referred to as UHA4002), and the compound contained in the peak of (2) is the above. It was found to be a compound represented by the formula (2) (hereinafter referred to as UHA4003).

On the other hand, the compound contained in the peak of (3) was a novel compound that has not been reported so far.
That is, the measured value by high resolution Negative-FAB-MS was 647.2144, and the following molecular formula was obtained from the comparison with the theoretical value.
Theoretical value C ₄₂ H ₃₁ O ₇ (M−H ⁻ ): 647.2148
Molecular formula C ₄₂ H ₃₂ O ₇

And it uses for a nuclear magnetic resonance (NMR) measurement, From the analysis of < ¹ > H-NMR, < ¹³ > C-NMR, and various two-dimensional NMR data, the compound contained in the peak of said (3) is represented by said Formula (3). It was confirmed that the compound was a novel compound having the structure (hereinafter referred to as UHA4022).

  For the NMR measurement value, UHA4022 is used.

Table 1 shows the ¹ H nuclear magnetic resonance spectrum and ¹³ C nuclear magnetic resonance spectrum.
The values were δ and ppm, and the solvent was measured with DMSO-d ₆ .

The physicochemical properties of UHA4022 were as follows.
(Properties)
Brown powder (soluble)
Water: Slightly soluble methanol: Dissolved ethanol: Dissolved DMSO: Dissolved chloroform: Slightly soluble ethyl acetate: Slightly soluble

  In addition, about UHA4002 contained in a derivative containing composition, the present inventors have so far anticancer, antibacterial action (Unexamined-Japanese-Patent No. 2011-251914), sirtuin expression promotion action (Unexamined-Japanese-Patent No. 2012-246242) , APMK activity inhibitory activity (JP 2013-112656 A), sugar absorption inhibitory action (JP 2013-136547 A), ApoB secretion inhibitory action (Japanese Patent Application No. 2012-256369), LDL cholesterol metabolism promoting action (special Application No. 2012-256378), UHA4003, anti-cancer action (JP 2011-251914 A), leptin resistance improving action (JP 2013-28560), mature fat cell hypertrophy suppression action (special No. 2013-95693), anti-inflammatory action (Japanese Patent Application No. 2012-04175) ), And has been confirmed to have an action of promoting the expression of UCP-2 in mature adipocytes (Japanese Patent Application No. 2012-218619). Is not yet known.

(Example 2: Verification of brown-like adipocyte differentiation-inducing action index gene expression of white adipocytes of a derivative-containing composition)
In order to evaluate the brown adipocyte differentiation-inducing action of white adipocytes, evaluation was performed using 3T3-L1 cells (mouse-derived preadipocytes). 3T3-L1 preadipocytes usually differentiate and mature into white adipocytes through a differentiation induction process. However, the expression of the Cidea gene, which is hardly observed in white adipocytes by being induced to differentiate into brownish adipocytes, and the transcriptional coactivator PGC-1β and mitochondria whose expression is enhanced in brown adipocytes and brownish adipocytes An increase in the expression level of the Cox7a1 gene and an increase in the expression of the UCP1 gene are observed. In addition, as mentioned in Non-Patent Document 6, FGF-21 is known as one of the factors that promote differentiation into brown-like adipocytes, and there are several effects brought about by the enhanced expression of FGF-21. As one, it is thought that white adipocytes are induced into brown adipocytes by promoting the expression increase of PGC-1α. Thus, differentiation induction of white adipocytes into brown-like adipocytes was confirmed using the expression levels of each gene of FGF-21, PGC-1α and Cox7a1 as an index.

  Two types of samples, rosiglitazone, which is a synthetic agonist of PPARγ, and the composition prepared in Example 1 were used. Each sample was dissolved in dimethyl sulfoxide (DMSO, manufactured by Wako Pure Chemical Industries, Ltd.) at a concentration of 0.2 mM and 20 mg / mL and used for the test.

  The culture is 10% fetal bovine serum (manufactured by FBS Biologic industries), 1% antibiotic-antimycotic (“Antibiotic-Antimycotic”, Gibco (GIBCO)) “Dulbecco's edifico” "Eagle medium" (DMEM, trade name, manufactured by Sigma) was used. Adipocytes used in the test were prepared according to a conventional method.

The test was conducted as follows. 1 mL of 3T3-L1 cells were seeded at a concentration of 5 × 10 ⁴ cells / mL in a 12-well dish (manufactured by Corning) for cell culture and cultured at 37 ° C. under 5% CO ₂ for 24 hours. After 24 hours, the DMEM medium was replaced with a DMEM medium adjusted to a final composition concentration of 20 μg / mL or rosiglitazone to a final concentration of 1 μM, and the culture was continued to 100% confluence, followed by further incubation for 48 hours. Next, the medium was added to 1 mL of differentiation DMEM supplemented with 1%, 0.5%, and 0.1% of insulin, dexamethasone, and isobutylmethylxanthine attached to “AdipoInducer Reagent” (trade name, manufactured by Takara Bio Inc.), respectively. Each sample was added and exchanged to a final concentration of 20 μg / mL and 1 μM, and differentiation was induced for 48 hours under conditions of 37 ° C. and 5% CO ₂ . In addition, what added 0.5% of only DMSO which is a solvent was set as control.

  After completion of the culture, the total amount of RNA was extracted and purified from the cells using an RNA extraction kit (trade name: TRIzol (registered trademark), manufactured by life technologies). The obtained RNA was subjected to a reverse transcription reaction in accordance with the instruction manual of a reverse transcription reagent for 2-step real-time RT-PCR (trade name: PrimeScript (registered trademark) RTM Master Mix, manufactured by Takara Bio Inc.).

That is, 2 μL of 5 × (Primescript RT Master Mix) and 500 ng of total RNA were mixed, and the total amount was adjusted to 10 μL with RNase Free dH ₂ O. Using a thermal cycler for PCR (trade name: GeneAmp (registered trademark) PCR System 9700, manufactured by Applied Biosystem), reverse transcription reaction with a program in which one cycle is “37 ° C. × 15 minutes → 85 ° C. × 5 seconds” Went. A diluted solution obtained by diluting the reverse transcription reaction solution 5-fold with a dilution reagent for real-time RT-PCR (trade name: EASY Dilution, manufactured by Takara Bio Inc.) was used for real-time RT-PCR analysis.

  Real-time RT-PCR analysis was performed according to a conventional method. For the analysis, “ECO Realtime RT-PCR system” (trade name, manufactured by Illumina) was used. As the primer, FGF-21 forward primer (primer ID: MA-126886-F), FGF-21 reverse primer (primer ID: MA-126866-R), PGC-1α forward primer (primer ID: MA-114509-F) ), PGC-1α reverse primer (primer ID: MA-114509-R), Cox7a1 forward primer (primer ID: MA106801-F) and Cox7a1 reverse primer (primer ID: MA106801-R). The internal standard of the intracellular gene is β-actin, and as its primers, an ACTB forward primer (primer ID: MA050368-F) and an ACTB reverse primer (primer ID: MA050368-R) (all of the above 8 types of primers are Takara Bio). Used).

A real-time RT-PCR reagent (trade name: SYBR (registered trademark) Select Master Mix, manufactured by life technologies) was used for the reaction. In a 48-well PCR plate (manufactured by Illumina), the reaction solution was 2 × (SYBR Select Master Mix) 5 μL, forward primer (50 μM) 0.08 μL, reverse primer (50 μM) 0.08 μL, reverse transcription reaction solution 2 μL. And dH ₂ O 2.84 μL (total amount 10 μL) were mixed, and “50 ° C. × 2 minutes → 95 ° C. × 2 minutes →“ 95 ° C. × 15 seconds → 60 ° C. × 1 minute ”× 40 cycles → 95 ° C. × 15 seconds PCR reaction was carried out with the program “→ 55 ° C. × 15 seconds → 95 ° C. × 15 seconds”.

  From the obtained Ct values of β-actin and FGF-21, PGC-1α and Cox7a1 in each cell (Threshold Cycle: the number of cycles to reach a certain amplification amount (threshold)), FGF-21, PGC-1α and Cox7a1 The relative value of each gene expression level was calculated. The results are shown in FIG.

  As a result, as with the addition of rosiglitazone, the expression level of the FGF-21 gene, which is one of the inducing factors of brown-like adipocytes, and the expression level of the PGC-1α gene with the expression of the FGF-21 gene are added to the derivative-containing composition. It was found that it sometimes increased significantly, and further, it was revealed that the expression level of Cox7a1, which is a mitochondrial marker gene, was significantly increased. Rosiglitazone is a synthetic agonist and its effect is stronger compared to the derivative-containing composition. However, thiazolidine compounds such as rosiglitazone have strong side effects such as edema and heart failure, and are problematic in safety. On the other hand, the derivative-containing composition is a composition having a merit obtained by a much simpler method, and its precursor compound resveratrol is widely distributed in nature and is an extremely safe compound. Furthermore, since hydroxystilbenes such as resveratrol are widely distributed in nature including polymers, they are rich in food experience. Therefore, it was shown that the product of the present invention is a useful composition that is highly safe and has a high possibility of inducing differentiation of white adipocytes into brown-like adipocytes, which is extremely strong like rosiglitazone.

(Example 3: Verification of expression index gene for induction of brown-like adipocyte differentiation of white adipocytes in a derivative-containing composition)
In Example 2, it was evaluated that the derivative-containing composition induced differentiation of white adipocytes into brown-like adipocytes with cells immediately after differentiation induction. In Example 3, the induced cells were brown-like adipocytes even after maturation. The maintenance of the traits was evaluated using mature adipocytes. The evaluation was based on the expression levels of FGF-21, PGC-1α, and the cell death-inducing DFFA-like effector a (Cidea), which is a brown-like adipocyte marker gene. In addition, brown-like adipocytes respond to stimulation with adrenaline. Since the expression level of the UCP1 gene was enhanced, the induction of differentiation of white adipocytes into brown-like adipocytes was confirmed using the expression level of UCP1 gene when adrenaline was added to the matured adipocytes as an index.

  Evaluation compositions and compounds were added in the same manner as in Example 2 to induce differentiation of 3T3-L1 preadipocytes. After induction of differentiation for 48 hours, the culture medium was replaced with 2 mL of maintenance culture DMEM supplemented with 1% of insulin, and further cultured for 1 week to mature adipocytes. For stimulation with adrenaline, matured cells were replaced with 2 mL of maintenance culture DMEM supplemented with 1 μM of L-adrenaline (manufactured by Wako Pure Chemical Industries, Ltd.), cultured for 2 hours, and the response was verified. .

  After maturation for 1 week or after stimulation with adrenaline, RNA was extracted and purified in the same manner as in Example 2, followed by reverse transcription reaction and real-time RT-PCR analysis. The same primers as those used in Example 2 were used for the FGF-21, PGC-1α, and ACTB genes, and the PGC-1β forward primer (primer ID: MA125505-F) was used for the PGC-1β gene. ), PGC-1β reverse primer (primer ID: MA125505-R), Cidea gene for Cidea forward primer (primer ID: MA104629-F), Cidea reverse primer (primer ID: MA104629-R) UCP1 gene for UCP1 A forward primer (primer ID: MA027561-F) and a UCP1 reverse primer (primer ID: MA027561-R) (both manufactured by Takara Bio Inc.) were used.

  FGF-21 from β-actin and FGF-21, PGC-1α, PGC-1β, Cidea and UCP1 Ct values (Threshold Cycle: number of cycles reaching a certain amplification amount (threshold)) in each cell obtained The relative value of each gene expression level of PGC-1β, PGC-1α, Cidea and UCP1 was calculated. The results are shown in FIG.

  As a result, as with the addition of rosiglitazone, the expression level of the FGF-21 gene, which is one of the inducers of brownish adipocytes, and the expression of the PGC-1α gene significantly increased with the expression of the FGF-21 gene. Furthermore, it was found that the expression level of Cidea, a brown-like adipocyte marker gene, and the expression level of PGC-1β, whose expression is enhanced in brown adipocytes, are significantly increased. It was. In addition, the effect of enhancing the expression of the UCP1 gene by the addition of adrenaline is 3.1 times that of the control by the addition of adrenaline, compared with the presence or absence of adrenaline, whereas rosiglitazone is 6.2 times the present invention product. When the derivative-containing composition was added, the expression level was greatly increased by 6.7 times, and the characteristics observed only in brownish adipocytes were observed. That is, by adding the composition of the present invention, it becomes clear that 3T3-L1 preadipocytes are induced to differentiate into brown-like adipocytes and maintain the traits of brown-like adipocytes after maturation, It was shown that the composition of the present invention is highly likely to have an action of inducing differentiation of white adipocytes into brown-like adipocytes.

(Example 4: Verification of brown-like adipocyte differentiation-inducing action indicator gene expression for normal human subcutaneous fat of a derivative-containing composition)
In order to evaluate the brown-like adipocyte differentiation-inducing action of white adipocytes observed using 3T3-L1 cells, human subcutaneous fat-derived normal preadipocytes (Lonza Japan) were used for evaluation. Normal preadipocytes usually differentiate into white adipocytes, but are induced to differentiate into brown-like adipocytes, so that expression of UCP-1 gene that is hardly observed in white adipocytes and expression of Cox7a1 gene expressed in mitochondria Increased amount, PGC-1β, which is a marker gene of brown adipocytes, and enhanced expression of CBP / p300-interacting transactivator (CITED1) gene expressed specifically in brown-like adipocytes are observed. Therefore, differentiation induction of white adipocytes into brown-like adipocytes was confirmed using the expression levels of the CITED1, Cox7a1, and UCP1 genes as indices.

  Two types of samples, rosiglitazone, which is a PPARγ agonist, and a derivative-containing composition were used. Each sample was dissolved in dimethyl sulfoxide (DMSO, manufactured by Wako Pure Chemical Industries, Ltd.) at an appropriate concentration and used for the test.

Subcutaneous fat-derived normal human preadipocytes (manufactured by Lonza Japan), 10% FBS, 2 mM glutamine, and GA-1000, preadipocyte basal medium-2 (PMB-2 medium, Lonza Japan) Cells were collected in a pre-adipocyte culture medium (PGM-2) added to (manufactured) for 4 days, and the cells were collected with EDTA-trypsin solution, suspended in PGM-2 medium at a rate of 8 × 10 ⁴ cells / ml, and cell culture. 0.5 ml each was planted in a 12-well plate. After 24 cultures at 37 ° C. in the presence of 5% CO ₂ , rosiglitazone was added to PGM-2 medium to a final concentration of 2 μM or UHA4003 to a final concentration of 40 μM, added in 0.5 ml increments, and further cultured for 48 hours. . After culture, PGM-2 differentiation medium (PGM-2 medium supplemented with various additional factors (human insulin, IBMX, dexamethasone, indomethacin) attached to the kit) and rosiglitazone at a final concentration of 1 μM or derivatives 1 ml of a differentiation medium supplemented with the composition to a final concentration of 20 μg / mL was added to differentiate and mature the adipocytes. What was cultured for 10 days after the additional addition was used for the test. As a control, a solvent to which only 0.5% of DMSO as a solvent was added was used.

  RNA was extracted and purified in the same manner as in Example 2, and reverse transcription reaction and real-time RT-PCR were performed. Primers include CITED1 forward primer (primer ID: HA204404-F), CITED1 reverse primer (primer ID: HA204404-R), PGC-1β forward primer (primer ID: HA172103-F), PGC-1β reverse primer (primer ID) : HA172103-R), COX7A1 forward primer (primer ID: HA133646-F) and COX7A1 reverse primer (primer ID: HA133646-R), UCP1 forward primer (primer ID: HA158451-F) and UCP1 reverse primer (primer ID: HA158451) -R) was used. The internal standard of the intracellular gene is β-actin, and as its primers, an ACTB forward primer (primer ID: HA067803-F) and an ACTB reverse primer (primer ID: HA067803-R) (all of the above 10 primers are Takara Bio). Used).

  In each of the obtained cells, CIT1 and PGC-1β, COX7A1, and UCP1 were determined from the Ct values (Threshold Cycle: the number of cycles to reach a certain amplification amount (threshold)) of CTED1, PGC-1β, COX7A1, and UCP1. The relative value of each gene expression level was calculated. The results are shown in FIG.

  As a result, as with the addition of rosiglitazone, it was found that the expression level of the CITED1 gene, which is a marker gene of brownish adipocytes, was significantly increased when the derivative-containing composition was added, and further, the mitochondrial marker gene COX7A1 It was revealed that the expression level of was significantly increased. It was also revealed that the expression levels of the UCP1 gene and PGC-1β gene were significantly increased. That is, it shows that human subcutaneous fat-derived normal preadipocytes that originally differentiate into white adipocytes differentiated into brownish adipocytes in the presence of the derivative-containing composition. That is, it was shown that the derivative-containing composition of the present invention has a very strong white adipocyte differentiation-inducing action into brown-like adipocytes.

From the results of Examples 2, 3, and 4 above, the addition of the derivative-containing composition significantly increased the amount of mitochondria in the cell and markedly induced differentiation of white adipocytes into brown-like adipocytes. Thus, it was shown that the derivative-containing composition has an excellent effect of inducing differentiation into brownish adipocytes.
It can also be seen that the derivative-containing composition is superior in that it can be prepared at low cost from food ingredients in one step, compared to rosiglitazone, which is a synthetic agonist that is expensive to manufacture.

(Example 5: Pharmaceutical containing the composition of the present invention)
1 g of the derivative-containing composition obtained in the same manner as in Example 1 was dissolved in ethanol, and the resulting solution was adsorbed on microcrystalline cellulose and dried under reduced pressure. By mixing 10 parts of the adsorbent of the present invention, 23 parts of corn starch, 12 parts of lactose, 8 parts of carboxymethyl cellulose, 32 parts of microcrystalline cellulose, 4 parts of polyvinylpyrrolidone, 3 parts of magnesium stearate, 8 parts of talc, and tableting, A compressed tablet containing the product of the present invention was obtained.

(Example 6: Quasi-drug containing the composition of the present invention)
10 mL of an ethanol solution obtained by dissolving 1.2 g of the derivative-containing composition obtained in the same manner as in Example 1 in ethanol, 20 g of taurine, 0.12 g of vitamin B1 nitrate, 0.6 g of sodium benzoate, 4 g of citric acid 60 g of sugar and 10 g of polyvinyl pyrrolidone were dissolved in purified water, and the whole amount was made up to 1000 mL. The pH was adjusted to 3.2 using dilute hydrochloric acid. 50 ml of 1000 ml of the obtained solution was filled in a glass bottle and sterilized at 80 ° C. for 30 minutes to obtain a drink as a quasi drug.

Claims (2)

Formula (1):

Formula (2):

And formula (3):

A composition for inducing differentiation of white adipocytes into brown-like adipocytes, which comprises the three types of resveratrol polymerization compounds represented by Pharmaceutical or quasi drug comprising the differentiation inducer composition to brownish adipocytes white adipocytes according to claim 1 Symbol placement.

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