JP5821366B2 - Leptin resistance improver - Google Patents

Description

  The present invention relates to a leptin resistance improving agent containing a resveratrol derivative. The present invention also relates to a leptin gene expression inhibitor containing a resveratrol derivative.

  Leptin is a peptide hormone secreted by fat cells and secreted when the energy storage state of the living body is sufficient. Secreted leptin acts on a leptin receptor located in the hypothalamus of the brain to strongly suppress the feeding center. This shows an appetite suppression effect and an energy metabolism enhancement effect, brings about weight loss, and suppresses obesity and weight gain. Leptin having such physiological functions has attracted attention as an anti-obesity agent.

  However, the leptin concentration in blood of obese animals and obese patients is higher than that of healthy individuals, and it has been clarified that there is a positive correlation with body weight (Non-patent Document 1). As a result of administering leptin to this obese patient, no anti-obesity effect was found (Non-patent Document 2). Thus, it has been clarified that there is “leptin resistance” in which the physiological function of leptin is not exhibited despite the high leptin concentration in blood.

Obesity has become a major social problem today, many of which are said to be due to leptin resistance. It has been found that leptin dysfunction affects obesity as well as taste disorders that make it difficult to feel sweet taste (Non-patent Document 3) and reproductive function (Non-Patent Document 4). In addition, as an physiological function of leptin, there are angiogenic action (Non-Patent Document 5), growth hormone secretion promoting action (Non-Patent Document 6), influence on immune function (Non-Patent Document 7), and antidepressant action (Non-Patent Document 8). It is known that leptin resistance is expected to adversely affect these physiological functions, and non-patent document 8 also reports that leptin resistance does not actually show the antidepressant action of leptin.
It is said that the cause of the leptin resistance is a fundamental cause due to an excessive amount of leptin in the blood. Therefore, it is considered that one way to improve leptin resistance is to reduce blood leptin concentration.

As a method for lowering the blood leptin concentration, there is a reduction of adipose tissue by exercise or diet, that is, a diet. However, there are limits to exercise and dietary restrictions in today's lifestyle. Therefore, there is a need for a technique for improving leptin resistance, and several prior arts have been reported so far. For example, a leptin resistance improving agent containing a neurotrophic factor or trk receptor ligand as an active ingredient (Patent Document 1), a leptin resistance improving agent containing diacylglycerol as an active ingredient (Patent Document 2), and a glyceride as an active ingredient A leptin resistance improving agent (Patent Document 3) and a leptin resistance improving agent containing a GABA _B receptor agonist as an active ingredient (Patent Document 4) are known.

  As described above, a plurality of compounds and materials having leptin resistance have been proposed because of the usefulness of the leptin resistance improving agent, and further development of new materials is desired.

On the other hand, resveratrol is a compound having an antibacterial action that exists as a phytoalexin, which protects plants from pathogenic bacteria, and is known to be contained in grape skins. Recent studies are revealing that resveratrol has a useful effect on mammals as well. The useful physiological effect of red wine called so-called “French paradox” is attributed to various physiologically active functions including the antioxidant ability of resveratrol. Furthermore, resveratrol is being clarified to be effective for many diseases (Non-Patent Document 9), and one of them is reported that resveratrol has a strong anticancer activity (non-patent document 9). Patent Document 10).
In addition, many resveratrol derivatives have been isolated and structurally determined so far. Naturally, resveratrol polymers such as ε-biniferin (dimer), α-viniferin (trimer), Vaticanol C (tetramer) and the like have been reported. There is also a report about a non-natural type resveratrol derivative (patent document 5). Resveratrol has been reported to have a leptin secretion promoting action (Patent Document 6).
However, as a result of investigations by the present inventors, resveratrol does not show a leptin resistance improving action, and there is no report that a resveratrol derivative has a leptin resistance improving action.

No. 01/066133 JP 2002-003376 A JP 2002-322052 A No. 2008/018275 Table 2008/136173 JP 2008-231080 A

N. Engl. J. et al. Med. , 1996, 334: 292-295. JAMA. 1999, 282: 1568-1575. Proc. Natl. Acad. Sci. , 2000, 97: 1104-111049. Nat. Genet. , 1996, 12: 318-320. Science, 1998, 281: 1683-1686. Endorinology, 1997, 138: 2203-2206 J. et al. Clin. Endocrinol. Metab. 1999, 84: 3686-3695. Obesity Study, 2010, 16 (3): 188-191 Drug Discovery, 2006, 5: 493-506 Science, 1997, 275 (10): 218-220.

  In view of the above situation, the present inventors searched for a novel leptin resistance improving agent. As a result, the newly prepared resveratrol derivatives were found to be a compound having an excellent leptin expression inhibitory action not found in resveratrol, and the present invention was completed.

  Therefore, an object of the present invention is to provide an excellent leptin resistance improving agent and leptin gene expression inhibitor.

The gist of the present invention is as follows:
[1] The following formula (1):

Leptin resistance improving agent characterized by containing one or more compounds selected from resveratrol derivatives and Ranaru group thereof or a pharmaceutically acceptable salt thereof represented in,
[2] the formula leptin gene expression inhibitor characterized by containing resveratrol derivatives and pharmaceutically acceptable salts or Ranaru least one compound selected from the group represented by (1),
About.

  The leptin resistance-improving agent of the present invention is useful as a novel leptin resistance-improving agent because it has an excellent leptin gene expression-inhibiting action not found in resveratrol. In addition, the leptin gene expression inhibitor of the present invention is useful as a novel leptin gene expression inhibitor because it can reduce leptin gene expression in adipocytes even at low concentrations.

FIG. 1 shows the chromatogram obtained in Example 1. The main compound peaks are indicated by R1 to R5. UHA4003 which is the product of the present invention corresponds to R4. FIG. 2 shows the results of real-time RT-PCR analysis performed in Example 2. The result of having quantified the leptin gene expression level in the fat cell processed with resveratrol and UHA4003 is shown.

Hereinafter, the present invention will be described in detail.
The leptin resistance improving agent and leptin gene expression inhibitor (hereinafter abbreviated as the leptin resistance improving agent of the present invention) of the present invention is represented by the following formula (1):

Characterized in that it contains an in resveratrol derivatives and pharmaceutically acceptable salts or Ranaru least one compound selected from the group indicated.

In the present invention, the “leptin resistance improving agent” or “leptin gene expression inhibitor” refers to an agent capable of reducing the expression level of a gene encoding leptin in adipocytes of human or non-human animals. Thus, by reducing the amount of leptin gene expression in adipocytes, for example, by reducing the leptin concentration that is excessively present in the blood of patients suffering from hyperleptinemia, hyperleptinemia It becomes possible to improve the leptin resistance and various diseases associated therewith.
Specifically, the leptin gene expression inhibitory action can be confirmed by measuring by the method described in Examples described later.

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

  Examples of the pharmaceutically acceptable salt of the resveratrol derivative 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 salt Metal hydroxide salts such as aluminum hydroxide salts; amines such as alkylamine salts, dialkylamine salts, trialkylamine salts, alkylenediamine salts, cycloalkylamine salts, arylamine salts, aralkylamine salts, heterocyclic amine salts 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 therefrom. These pharmacologically acceptable salts can be used alone or in admixture of two or more.

In the production of the resveratrol derivative, resveratrol is used as a precursor. Resveratrol has structural isomers of trans form and cis form, but some conversion of trans form and cis form occurs by heating or ultraviolet rays. Therefore, resveratrol may be a trans isomer, a cis isomer, or a mixture of a trans isomer and a cis isomer. Resveratrol may be a naturally derived product extracted and purified from grape skin, or a chemically synthesized chemical product synthesized with high purity. When using naturally occurring resveratrol, it is not necessary to be completely purified, because the desired production reaction proceeds as described later, and finally the resveratrol derivative used in the present invention is obtained. Mixtures containing ingredients other than resveratrol can also be used. Resveratrol also includes derivatives such as salts, ethers and esters, but these derivatives can also be used as raw materials in the production method of the present invention.
However, from the viewpoint of the recovery rate of the resveratrol derivative, a mixture containing 1% by weight or more in terms of resveratrol is desirable as a raw material.
As said resveratrol, you may use the extract from raw materials, such as grape peel and a peanut, a freeze-dried product, etc.

In the present invention, resveratrol is dissolved in a suitable solvent. At this time, if the solvent is only water, the solubility of resveratrol is remarkably low, and therefore, the solvent may be dissolved only in a mixed solution of water and an organic solvent or in 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 refining composition containing a new resveratrol derivative is not fully applied to the final product and used in food, ethanol or hydrous ethanol is used as a solvent for safety and legal purposes. It is desirable to use it.
There is no particular limitation on the concentration of resveratrol in the solution containing the obtained resveratrol, but the higher the respective concentration, the more advantageous there is that the amount of the solvent used is smaller. A concentration close to the concentration at which resveratrol is saturated with respect to the solvent is preferable.
Resveratrol may not be completely dissolved in the solution before the formation reaction.

  Next, it is preferable to adjust the pH of the solution containing resveratrol (hereinafter referred to as resveratrol-containing solution) to less than 8. As an adjustment method, for example, after preparing a resveratrol-containing solution, a pH adjuster may be added to adjust the pH, or the pH of the solvent may be adjusted in advance when the solution is prepared. If the pH of the resveratrol-containing solution at the start of the reaction is 8.0 or more, other reactions and decomposition of the target compound also occur on the other hand, so that the recovered amount of the resveratrol derivative produced decreases. Therefore, the pH at the start of the reaction is desirably 3 or more and less than 8.

In the present invention, a metal salt is added to the resveratrol-containing solution. The metal salt may be any of an acid salt, a basic salt, and a normal salt, and may be any of a single salt, a double salt, and a complex salt. Furthermore, the metal salt may be one kind or a mixture of plural kinds. As an example of the metal salt, those approved as food additives are preferable in terms of safety. For example, magnesium salt, calcium salt, sodium salt, potassium salt, zinc salt, copper salt and the like that are permitted to be added to foods can be mentioned.
Moreover, as a mixture of the metal salts, for example, a mineral premix (Tanabe Seiyaku Co., Ltd., a mineral mixture mainly composed of zinc gluconate, ammonium iron citrate, calcium lactate, copper gluconate, and magnesium phosphate) In addition, substances containing several kinds of metal salts are listed. Moreover, mineral water can also be mentioned as a mixture containing a some metal salt.
In addition, as content of the said metal salt, what is necessary is just the quantity which can produce | generate the said resveratrol derivative, and there is no limitation in particular.

  Next, the resveratrol-containing solution is heated in the presence of a metal salt. By this heat treatment, a formation reaction of a novel resveratrol derivative is performed. In order to advance the production reaction efficiently, the heating temperature of the resveratrol-containing solution is preferably adjusted to 110 ° C. or higher. Further, considering the boiling point of the solvent to be used, pressure heating is desirable. For example, a resveratrol-containing solution is placed in an open container and the container is heated at a high temperature exceeding the boiling point of the solvent. A resveratrol-containing solution is placed in a sealed container and the container is heated. It is preferable to heat the solution so that the solution temperature reaches 110 ° C. or higher at least partially, such as by using and heating. From the viewpoint of recovery efficiency, it is more preferable that the solution temperature be 110 ° C. to 150 ° C. uniformly. 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 5 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 a plurality of times, it is preferable to add a solvent or, if necessary, the metal salt to supplement the evaporated solvent.

  The completion of the resveratrol derivative production reaction by the heat treatment may be judged by, for example, confirming the production amount of the resveratrol derivative by component analysis by HPLC.

The obtained reaction solution contains the resveratrol derivative used in the present invention.
In addition, when a resveratrol derivative is produced by a process using only safe raw materials, the resveratrol derivative can be used in foods, pharmaceuticals, or quasi drugs in the state of a mixture containing the resveratrol derivative. For example, when natural resveratrol is dissolved in a water-containing ethanol solvent and heated with mineral water or mineral premix added, the resulting reaction solution can be used as one of the food ingredients. is there.

  Also, if you want to improve the flavor and enhance the functionality, concentrate the reaction solution to increase the concentration of resveratrol derivative, or purify the reaction solution to obtain a pure product of resveratrol derivative. Can do. Concentration and purification can be performed by a known method. For example, the resveratrol derivative can be concentrated by extraction by a solvent extraction method using chloroform, ethyl acetate, ethanol, methanol or the like, a supercritical extraction method using carbon dioxide gas, or the like. It is also possible to perform concentration and purification using column chromatography. A membrane treatment method such as a recrystallization method or an ultrafiltration membrane can also be applied.

  Further, when the resveratrol derivative represented by the formula (1) is separated and recovered from the reaction solution, column chromatography, HPLC or the like may be used.

  A powdered resveratrol derivative can be obtained by removing the solvent from the concentrate or purified product by drying under reduced pressure or lyophilization, if necessary.

  In addition, the obtained resveratrol derivative may be converted to a salt of a resveratrol derivative, or the hydroxy group of the resveratrol derivative may be etherified or esterified by a method known in the art, if necessary.

The resveratrol derivative has an excellent leptin gene expression inhibitory action not seen in resveratrol.
Therefore, the present invention can provide an excellent leptin resistance improving agent and leptin gene expression inhibitor by containing the resveratrol derivative as an active ingredient.

  The leptin resistance improving agent or the like of the present invention may be composed only of the resveratrol derivative as an active ingredient, but for example, powders, tablets, pills, capsules, fine granules, granules, etc. When blended in a liquid preparation such as a solid preparation, a liquid preparation, a suspension, an emulsion, or a gel preparation, it may further contain optional components according to each dosage form. In addition, tablets, pills, granules, and capsules containing granules may be sugar-coated with sugars such as sucrose and sugar alcohols such as maltitol, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose as necessary. It can also be applied with a coating. Further, it may be covered with a film of a gastric or enteric substance. Moreover, in order to improve the solubility of a formulation, a well-known solubilization process can also be performed. Based on a conventional method, the resveratrol derivative may be blended with an injection or infusion.

  When using the leptin resistance improving agent or the like of the present invention, for example, the intake of the resveratrol derivative is not particularly limited as long as the desired effect can be obtained, and usually its mode, patient age , Sex, constitution and other conditions, the type and degree of disease, and the like. In terms of the resveratrol derivative, the amount is preferably about 0.1 mg to 1,000 mg per day, which can be taken in 1 to 4 times a day.

  In addition, since the resveratrol derivative is excellent in safety, not only for humans, for example, non-human animals such as rats, mice, guinea pigs, rabbits, sheep, pigs, cows, horses , Cats, dogs, monkeys, chimpanzees, and other mammals, birds, amphibians, reptiles, etc. As feed, for example, livestock feed used for sheep, pigs, cattle, horses, chickens, etc., feed for small animals used for rabbits, rats, mice, etc., feed for seafood used for eel, Thailand, yellowtail, shrimp, etc., dogs, Pet foods used for cats, small birds, squirrels, etc. are listed.

  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, isolation and purification of UHA4003)
700 mg of trans-resveratrol (Tokyo Kasei) was dissolved in 14 mL of ethanol, and 14 mL of 2.5% NaHCO ₃ aqueous solution was added to obtain a resveratrol-containing solution (pH 9.9). This resveratrol-containing solution was heated in an autoclave (SANYO LABO AUTOCLAVE) at 130 ° C. for 20 minutes. Subsequently, 14 mL of ethanol and 14 mL of 5.0% NaHCO ₃ aqueous solution were added to the reaction solution obtained by the first autoclave treatment, and the mixture was again heated in an autoclave (SANYO LABO AUTOCLAVE) at 130 ° C. for 20 minutes. 1 mL of the obtained reaction solution was diluted to 50 mL with methanol, and 10 μL of this was analyzed by HPLC.

HPLC analysis was performed under the following conditions.
Column: Column for reverse phase “Develosil (registered trademark) C-30-UG-5” (4.6 mm.d. × 250 mm)
Mobile phase: A: H ₂ O (0.1% trifluoroacetic acid (TFA)), B: Acetonitrile (0.1% TFA)
Flow rate: 1 mL / min
Injection: 10 μL
Detection: 254 nm
Gradient (% by volume): 30 minutes from 80% A / 20% B to 20% A / 80% B, 5 minutes from 20% A / 80% B to 100% B, 10 minutes at 100% B (all linear)

  The obtained chromatogram is shown in FIG. The main compound peaks are indicated by R1 to R5.

Of the peaks obtained, the compound R4 was purified by preparative HPLC. The isolated and purified compound R4 was dried by a conventional method to obtain a brown powdery substance. This was named UHA4003.
Further, the physicochemical properties of the UHA4003 were as follows.
(Properties)
Brown powder (soluble)
Water: Slightly soluble methanol: Dissolved ethanol: Dissolved DMSO: Dissolved chloroform: Slightly soluble ethyl acetate: Slightly soluble

Next, when the molecular weight of UHA4003 was measured by high resolution FAB-MS (Fast Atom Bombardment-Mass Spectrometry), it was 439.4765, and the following molecular formula was obtained from comparison with the theoretical value.
Theoretical value C28H23O5 (M + H) ⁺ : 439.44792
Molecular formula C ₂₈ H ₂₂ O ₅

  Next, the UHA4003 is subjected to nuclear magnetic resonance (NMR) measurement, and from the analysis of 1H-NMR, 13C-NMR and various two-dimensional NMR data, the UHA4003 has a structure represented by the formula (1). confirmed.

(Example 2: Verification of leptin gene expression inhibitory effect)
In order to evaluate the leptin gene expression inhibitory effect, evaluation was performed using 3T3-L1 cells (mouse-derived adipose precursor cells).

  Two kinds of samples, resveratrol and UHA4003 which is the product of the present invention, were used. Each sample was dissolved in dimethyl sulfoxide (DMSO, manufactured by Wako Pure Chemical Industries, Ltd.) at a concentration of 4 mM, 2 mM, 1 mM, and 0.2 mM and used for the test.

The culture is 10% fetal bovine serum (manufactured by FBS Biologic industries), 1% antibiotic-antimycotics (manufactured by Gibco (GIBCO)) DMEM, manufactured by Sigma) was used. Adipocytes used in the test were prepared according to a standard method. That is, a 6-well dish for cell culture (manufactured by BD Japan) was seeded with 2 mL of 3T3L1 cells at 5 × 10 ⁴ cells / mL, cultured at 37 ° C. under 5% CO ₂ for 48 hours, and 100% confluent. The cells were further cultured for 48 hours while changing the medium every day. Thereafter, the medium was replaced with 2 mL of differentiation DMEM supplemented with 1%, 0.5%, and 0.1% of insulin, dexamethasone, and isobutylmethylxanthine attached to AdipoInducer Reagent (manufactured by Takara Bio Inc.), 37 ° C., What was differentiated and cultured for 48 hours under 5% CO ₂ was used.

  The test was conducted as follows. The differentiated adipocyte medium was replaced with DMEM (maintenance medium) containing 1% insulin, and 10 μL of each sample (final concentrations 20 μM, 10 μM, 5 μM, 1 μM) was added thereto, and the medium was changed every two days ( The compound-containing maintenance medium was cultured for 7 days. In addition, what added 0.5% of only DMSO which is a solvent was set as control.

After adding the sample, the cells were cultured for 24 hours under conditions of 37 ° C. and 5% CO ₂ , and RNA was extracted from the cells using an RNA extraction kit “NucleoSpin (registered trademark) RNA II” (manufactured by Takara Bio Inc.). Purified. The obtained RNA was subjected to a reverse transcription reaction according to the instruction manual of the reverse transcription reagent “PrimeScript (registered trademark) RT Master Mix” (manufactured by Takara Bio Inc.) for two-step real-time RT-PCR. In other words, 4 μL of 5 × Primescript RT Master Mix and 1 μg of total RNA were mixed and adjusted to 20 μL with RNase Free dH ₂ O. Using a thermal cycler for PCR “GeneAmp (registered trademark) PCR System 9700” (Applied Biosystem Co., Ltd.), reverse transcription reaction was performed with a program of {37 ° C. 15 minutes → 85 ° C. 5 seconds} × 1 cycle. It was. A diluted solution obtained by diluting the reverse transcription reaction solution 10-fold with a dilution reagent “EASY Dilution” (manufactured by Takara Bio Inc.) for real-time RT-PCR 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” (manufactured by Illumina) was used. Leptin forward primer (primer ID: MA073409-F) and leptin reverse primer (primer ID: MA073409-R) were used as primers. The internal standard of the intracellular gene is β-actin, and as its primer, ACTB forward primer; (primer ID: MA050368-F), ACTB reverse primer; (primer ID: MA050368-R) (both are Takara Bio Inc.) Made). For the reaction, a real-time RT-PCR reagent “SYBR (registered trademark) Premix EX taq II” (Tli RNase H Plus) (manufactured by Takara Bio Inc.) was used. The reaction solution was 5 × L of 2 × SYBR Premix EX taq II (Tli RNase H Plus), 0.08 μL of forward primer (50 μM), 0.08 μL of reverse primer (50 μM), reverse transcription in 48 well PCR plate (manufactured by Illumina) 2 μL of reaction solution and 2.84 μL of dH ₂ O (total amount: 10 μL) were mixed [95 ° C. 30 seconds → {95 ° C. 15 seconds → 60 ° C. 1 minute} × 40 cycles → 95 ° C. 15 seconds → 55 ° C. 15 seconds → 95 PCR reaction was performed with a program of [15 ° C. for 15 seconds].

  The relative value of the leptin expression level was calculated from the Ct values of β-actin and leptin (Threshold Cycle: the number of cycles reaching a certain amplification level (threshold)) in each cell obtained. The results are shown in FIG.

From the results shown in FIG. 2, it was confirmed that UHA4003 has a strong leptin gene expression-inhibiting action not found in resveratrol.
Therefore, a drug containing UHA4003 as an active ingredient is used as a leptin resistance improving agent that significantly reduces the leptin concentration in the blood of a human or non-human animal or a leptin gene expression inhibitor in a fat cell of a human or non-human animal I understand that I can do it.

  The leptin resistance improving agent of the present invention has an excellent inhibitory effect on leptin gene expression. Therefore, the leptin resistance improving agent of the present invention can also be used as a hyperleptinemia therapeutic agent, an obesity improving / treating agent, or a therapeutic agent for depression caused by leptin resistance.

Claims (2)

Following formula (1):

Leptin resistance improving agent characterized by containing resveratrol derivatives and pharmaceutically acceptable one or more compounds selected from the salts or Ranaru group shown in.
Following formula (1):

Leptin gene expression inhibitor characterized by containing resveratrol derivatives and pharmaceutically acceptable one or more compounds selected from the salts or Ranaru group shown in.

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