JP5940261B2 - RXR and / or PPAR activator - Google Patents

Description

  The present invention relates to a retinoid X receptor (RXR) activator and / or a peroxisome proliferator-activator-receptor (PPAR) activator.

  Retinoid X receptor (Retinoid X Receptor, RXR) is one of the nuclear receptors, forming a homodimer between RXRs and a heterodimer with retinoic acid receptor (RAR), It is known to act as a receptor for agonists such as retinoic acid. And the dimer couple | bonded with the agonist acts as a transcriptional activator of various genes. RAR and RXR have α, β, and γ subtypes, respectively.

  Moreover, RXR forms a heterodimer with each subtype of peroxisome proliferator-activated receptor (PPAR), which is the same nuclear receptor, and binds to various agonists in a ligand-dependent manner. It is also known to induce the expression of a gene having a PPAR response element (PPRE) in the promoter region.

  PPAR has three known subtypes of α, δ, and γ in mammals, and there is tissue specificity in the expression between these subtypes. For example, PPARα is mainly expressed in liver, kidney, heart, skeletal muscle, etc., PPARγ is mainly expressed in adipose tissue and liver, and PPARδ is universally expressed in each tissue.

  When these nuclear receptors are activated, the expression of various genes is activated, which can be expected to prevent or treat skin diseases, allergic diseases, lifestyle-related diseases (diabetes, obesity, etc.), cancer, etc. For this reason, development of nuclear receptor activating components and methods that are highly safe and highly effective, as typified by food-derived ingredients and natural products, has been performed (patents). Documents 1-7, Non-Patent Document 1).

  On the other hand, sugar beet is a useful plant that is used not only as a sugar extraction raw material but also as an extraction raw material for various functional components (raffinose, betaine, etc.). And although some uses are also known about the residue (beet fiber) after sugar extraction from sugar beet roots (patent documents 8-9), development of these further uses is desired.

JP 2010-100551 A JP 2007-119429 A JP 2007-119430 A JP 2007-119431 A JP 2007-119432 A JP 2009-242333 A JP 2010-1111588 A JP-A-10-276663 JP-A-10-295288

Bulletin of the Institute for Biological Function Development, 9: 55-61, 2009

  An object of the present invention is to provide a component having agonist activity for a retinoid X receptor and / or a peroxisome proliferator-responsive receptor and having high safety.

  In order to achieve the above object, as a result of intensive studies, the present inventors have made an ethanol extract from sugar beet root residue (beet fiber) of sugar beet root or a processed / purified product as an active ingredient, so that the retinoid X The present inventors have found that a receptor (RXRα) and a peroxisome proliferator-responsive receptor (PPARα, PPARδ, PPARγ) can be activated, thereby completing the present invention.

That is, the embodiment of the present invention is as follows.
(1) A retinoid X receptor (RXRα) activator comprising a beet fiber ethanol extract or a processed and purified product thereof as an active ingredient.
(2) The agent according to (1), which prevents and / or ameliorates at least one (1 or 2 or more) of skin inflammation, breast cancer, lung cancer, alopecia (dysregulation), and biological rhythm ataxia .
(3) A peroxisome proliferator-responsive receptor α (PPARα) activator characterized in that a beet fiber ethanol extract or a processed and purified product thereof is used as an active ingredient.
(4) Hyperlipidemia, inflammatory diseases (including skin inflammation), insulin resistance diseases (diabetes, arteriosclerosis, etc.), stroke, biological rhythm ataxia (biological rhythm disorders), epidermal cell differentiation and melanocyte The agent according to (3), which prevents and / or improves at least one of differentiation (induction of epidermal cell differentiation, whitening effect by inhibiting differentiation of melanocytes) (1 or 2 or more).
(5) A peroxisome proliferator-responsive receptor δ (PPARδ) activator, characterized in that a beet fiber ethanol extract or a processed and purified product thereof is used as an active ingredient.
(6) Prevent and / or improve at least one (1 or more) of inflammatory diseases, insulin resistance diseases (diabetes, arteriosclerosis, etc.), skin function decline (skin barrier ability, water retention ability, etc.) The agent according to (5), characterized in that
(7) A peroxisome proliferator-responsive receptor γ (PPARγ) activator characterized in that a beet fiber ethanol extract or a processed and purified product thereof is used as an active ingredient.
(8) Hyperlipidemia, hypertension, inflammatory diseases (including allergic diseases), insulin resistance diseases (diabetes, arteriosclerosis, etc.), cerebral infarction, Alzheimer's disease, neurological diseases (one or more) ) Is prevented and / or improved. The agent according to (7).
(9) Beet fiber ethanol extract or processed / purified beet fiber is added 0.1 times or more (preferably 5 times or more) ethanol to beet fiber dried at 80 to 90 ° C or higher. Extracted at a temperature of 0 to 78 ° C. (preferably 10 to 30 ° C.) and obtained by drying the liquid layer after solid-liquid separation, (1) to (8) The agent as described in any one.
(10) Beet fiber ethanol extract or processed / purified beet fiber is 0.1 to 5 times the amount of ethanol added to the dried beet fiber at 80-90 ° C. The agent according to any one of (1) to (8), wherein the agent is obtained by drying the liquid layer after the treatment.
(11) Ethanol extract of beet fiber or processed and purified product thereof is ceramide, cerebroside, glycosylceramide, sphingoid base, sphingolipid, glycerolipid, steryl glycoside, sterol glycoside, sterol, fatty acid, saponin Any one of (1) to (10), comprising at least one selected from steroid glycosides, steroids, retinoids, retinoic acid, retinol, retinal, carotenoids and terpenoids Agent described in 1.

  According to the present invention, it is possible to provide an RXRα activator, a PPARα activator, a PPARδ activator, a PPARγ activator, and a composition (pharmaceutical, food and drink, feed, etc.) containing these. And these activators are insulin resistance syndrome, inflammatory disease, neurological disease, biological rhythm disorder disease, hyperlipidemia, glucose metabolism abnormality, allergic disease, obesity, cancer prevention and improvement, or hair growth / hair growth Useful for skin care, whitening, etc. In particular, the activator of the present invention is very effective, and is therefore characterized in that a sufficient effect can be obtained even with a small dose. Furthermore, since the active ingredient of the present invention is a food-derived natural material, it is highly safe, avoids serious side effects as seen in synthetic drugs, and can be taken for a long time.

It is a graph which shows the influence on the expression of various nuclear receptors by the exposure of a beet fiber ethanol extract in a nuclear receptor activity test. The vertical axis represents Renilla luciferase activity value (fluorescence intensity), and the horizontal axis represents the concentration of beet fiber ethanol extract (sample concentration). It is a graph which shows the activity value of RXR (alpha), PPAR (alpha), PPAR (delta), and PPAR (gamma) by beet fiber ethanol extract exposure in a nuclear receptor activity test. The vertical axis shows the activity value compared with the negative control, and the horizontal axis shows the concentration of the beet fiber ethanol extract.

  In the present invention, an ethanol extract of sugar beet root residue from beetroot (beet fiber) or a processed and purified product thereof is used as an active ingredient of a nuclear receptor activator. Ethanol is used as the extraction solvent, but other alcohols may be used as long as there is no difference in properties such as polarity. However, it is not preferable to use a solvent (such as water) other than alcohols. The form of the extract can be any liquid, dry powder, granule, paste or the like, and is not particularly limited.

  The beet fiber in the present invention means a fiber-containing material obtained from the root of beet (sugar beet), and the koji after collecting sugar content such as sucrose from sugar beet root known as a sugar-making raw material. Or dried and / or pulverized thereof. Here, it is preferable that the beet fiber used for the raw material for producing the ethanol extract of the present invention is sufficiently extracted (removed) with sugar. For example, the beet fiber using a beet sugar production apparatus used in a sugar factory or the like is used. What removed this is preferable. When the removal of sugar is not sufficient, there may be a case where a sufficient effect cannot be exhibited even if an ethanol extract produced using such a raw material is used as an active ingredient.

  As a method for preparing the beet fiber, the sugar beet root is cut into pieces or ground, squeezed, then immersed in warm water, and the residue obtained by sufficiently extracting and removing soluble components such as sucrose is decolored, deodorized, Examples thereof include methods obtained by performing necessary treatments such as drying, pulverization, and sieving. In addition, what was fully dried and / or crushed has destroyed most of the fiber structure of beet fiber, and is more suitable as an active ingredient extraction raw material by ethanol.

  As a method for producing a beet fiber ethanol extract, ethanol (concentration of 90% or more) is added to beet fiber dried under a condition of 80 to 90 ° C. or higher (preferably 100 ° C. or higher). Ethanol after mixing at a temperature of 0 to 78 ° C., preferably 10 to 60 ° C., more preferably 10 to 30 ° C. (room temperature). Examples thereof include a method of removing the extraction residue (such as filtration with a filter). When the amount of ethanol as the solvent is less than 5 times, reflux extraction can be performed by a Soxhlet extractor or the like in order to extract a sufficient amount of the active ingredient. Further, it is preferable to perform reflux extraction even when 5 times or more of ethanol is used.

  In the present invention, “processing” of beet fiber ethanol extract means concentration, grinding, milling, washing, hydrolysis, fermentation, purification, pressing, extraction, fractionation, filtration, drying, powdering, granulation, dissolution, This refers to a treatment that is arbitrarily selected and combined with sterilization, pH adjustment, deodorization, decolorization, etc. "Purification" refers to chromatography / adsorption / desorption using ion exchange resin or silica gel, ion exchange resin or electrodialysis membrane. Desalting used, separation using electrodialysis membrane and / or RO membrane and / or MF membrane and / or UF membrane, crystallization / precipitation using concentration and / or cooling, solid-liquid separation using centrifugation, etc. The process which arbitrarily selected etc. and combined is shown.

  The beet fiber used in the present invention, beet fiber ethanol extract and processed / purified product thereof include ceramide, cerebroside, glycosylceramide, sphingoid base, sphingolipid, glycerolipid, steryl glycoside, sterol glycoside More preferably, it contains at least one or more selected from sterol, fatty acid, saponin, steroid glycoside, steroid, retinoid, retinoic acid, retinol, retinal, carotenoid and terpenoid. In addition, these acquisitions are not limited to the above-described preparation methods, production methods, and the like, and various modifications can be made so that the same quality can be acquired.

  In the present invention, an RXRα activator, a PPARα activator, a PPARδ activator, comprising, as an active ingredient, a beet fiber ethanol extract obtained as described above or a processed and purified product thereof, and A PPARγ activator is provided. Examples of the dosage form include tablets, capsules, granules, powders, syrups and the like. These various preparations may be formulated only with beet fiber ethanol extract, and according to conventional methods, excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspension agents, It can also be formulated using known adjuvants such as coating agents.

  In the present invention, the above preparation is not only used as a pharmaceutical product, but may be used in combination with other drugs as desired, or may be provided as a so-called functional food by mixing with food or drink. It is also possible to do.

  In the present invention, the above preparation and the like are orally or parenterally administered to humans or animals other than humans. The dose (dose) is, for example, 0.1 to 50 g / 60 kg (body weight), preferably 0.5 to 10 g / 60 kg (body weight) per day as a beet fiber ethanol extract or processed / purified product thereof in humans. However, safety is not a problem at all, and the effectiveness is maintained even if it is administered beyond this range. In animals other than humans, the dose may be set by performing a predetermined conversion from the dose to humans.

  Thus, by using beet fiber ethanol extract or processed / purified one as an active ingredient, it is possible to provide an RXRα activator, a PPARα activator, a PPARδ activator, and a PPARγ activator, As a result, insulin resistance syndrome, inflammatory disease, neurological disease, biological rhythm disorder disease, hyperlipidemia, glucose metabolism abnormality, allergic disease, obesity, cancer, etc. (2 or more of diabetes, hypertension, hyperlipidemia (Including combined lifestyle-related diseases) prevention, improvement, hair growth / hair growth, skin care, whitening, etc. can be expected.

  Here, since RXRα is mainly involved in the control of cell proliferation / differentiation, the RXRα activator is particularly effective for skin care (skin inflammation), various cancers, hair growth / hair growth (hair loss), and biological rhythm ataxia. . In addition, since PPARα is involved in a wide range of control centering on triglycerides, PPARα activators are hyperlipidemias, inflammatory diseases, insulin resistance diseases, stroke, biological rhythm disorders, induction of epidermal cell differentiation / It is particularly effective for inhibiting differentiation of melanocytes (that is, whitening effect). Furthermore, PPARδ activators are for inflammatory diseases (including skin care) and insulin resistance diseases, and PPARγ activators are hyperlipidemia, hypertension, inflammatory diseases, insulin resistance diseases, cerebral infarction, Alzheimer's Particularly effective for diseases and neurological diseases.

  The present invention is characterized by the use of beet fiber derived from sugar beet roots, and the effects expected of the present invention can be obtained by using sugar beet leaves or the like as raw materials (using extracts from sugar beet leaves). Absent.

  Examples of the present invention will be described below, but the present invention is not limited to these examples, and various modifications can be made within the technical idea of the present invention.

(Beat fiber nuclear receptor activity test)
In order to investigate the effect of beet fiber on the transcriptional activity of nuclear receptor protein, the following test was performed.

  The beet fiber used as a raw material is ground and squeezed sugar beet root, then dipped and stirred in warm water to obtain a residue from which the soluble components made of sucrose are sufficiently extracted and removed, and this is decolored, deodorized, and 100 ° C. It was obtained by drying, grinding and sieving treatment under the above temperature conditions.

  A hot water extract and an ethanol extract were obtained from the beet fiber thus obtained and used as a sample. Specifically, the hot water extract used was a supernatant obtained by adding 10 times the amount of hot water to beet fiber and stirring for 10 minutes, followed by filtration through a 0.2 μm membrane filter. To extract the ethanol extract, add 10-fold volume of 100% ethanol solution to beet fiber, stir at room temperature for 3 hours, and then dry the supernatant filtered through a 0.2 μm membrane filter under reduced pressure to obtain 200 mg / ml. A solution in which DMSO (dimethyl sulfoxide) was added and dissolved was used.

Each sample was subjected to a nuclear receptor activation test using a reporter assay. As cells to be used, CV-1 and Hep G2 (both sold by National Institute of Biomedical Innovation) were used. First, these cell lines are seeded on a ^6- well plate so as to be 2.0 × 10 ⁵ / well and cultured in Dulbecco's modified Eagle medium (10% FBS, 2 mM L-glutamine added, manufactured by Nissui Pharmaceutical Co., Ltd.) for 1 day. did. Chiral protein expression plasmid (pGal4DBD / NR (LBD)) of Gal4 gene DNA binding domain (Gal4-DBD) and ligand binding domain (NR-LBD) of various human nuclear receptors, Gal4 gene DNA response element and firefly luciferase gene A weight ratio of a reporter plasmid (pGal4-Luc) containing an internal standard plasmid (pRL-CMV, pRL-SV40) in which a gene constitutive expression promoter (CMV, SV40) is linked upstream of the Renilla luciferase gene as an internal standard The mixture was mixed at a ratio of 1: 0.9: 0.1, and dissolved in Opti-MEM medium (GIBCO) at a concentration of 20 mg / ml (total DNA amount). A gene transfer reagent FuGENE (manufactured by Roche) was added in an amount of 1/20, and allowed to stand for 15 minutes, and then 100 μl of this mixture was added to each well and cultured for 6 hours to introduce the gene. Table 1 shows a list of relationships between cell lines and introduced plasmids (nuclear receptor chimeric protein, internal standard, reporter).

Next, transgenic cells were dispersed by trypsin, the CV-1 in 96well plate 1.6x10 ⁴ / well, were seeded again so as to be Hep G2 in 2.0x10 ⁴ / well. At this time, the culture solution was replaced with Dulbecco's modified Eagle's medium (containing no phenol red, 10% activated carbon treated FBS, manufactured by SIGMA) containing each concentration of the test substance. The final concentration of the sample in the culture medium was 0.5, 2, 10% hot water extract, 0.1, 0.25, 0.5% ethanol extract. Water (final concentration 10%) and DMSO (final concentration 0.5%) were used as the solvent for the ethanol extract.

  As negative control (solvent control) in this test, 10% ultrapure water (for hot water extract) and 0.5% DMSO (for ethanol extract) were used. Moreover, as positive control used for the quality control of this test, 1 μM β-estradiol (manufactured by Wako Pure Chemical Industries, Ltd., ERα, ERβ), 0.1 μM GW4064 (manufactured by Tocris Bioscience, FXR), 10 nM dexamethasone (Japanese sum) Kojun Pharmaceutical Co., Ltd., GR), 1 μM TO901317 (manufactured by Cayman Chemical, LXRα), 100 μM WY14643 (manufactured by Tocris Bioscience, PPARα), 0.1 μM GW501516 (manufactured by ENZO, PPARδ), 10 μM Pioglitz Manufactured by PGZ, PPARγ), 25 μM Rifampicin (manufactured by Wako Pure Chemical Industries, Ltd., PXR), 100 μM Am80 (Wako) Made medicine Industry Co., Ltd., RARα), 0.1μM 9cRA (manufactured by Wako Pure Chemical Industries, Ltd., RXRα), 0.1μM Vitamin D3 (ALEXIS Biochemicals Co., Ltd., was used according to the classification of the VDR). For the above substance addition, DMSO (final concentration 0.5%) was used as a solvent.

  After culturing for 48 hours, the cells were washed with phosphate buffered saline (PBS), and the cells were lysed using a dual luciferase assay system (Promega). Further, a substrate solution containing luciferin was added, and firefly and Renilla luciferase activities were measured with a plate reader (ARVO MX, manufactured by Perkin Elmer). The above operation was performed using 3 wells per sample (including positive and negative controls), and the average value of 3 wells was adopted as data.

  The nuclear receptor-dependent gene transcriptional activity (luciferase activity) was defined as the following equation (1). The evaluation of the activity is expressed as a ratio to the negative control (activity value in the sample / activity value in the negative control), and no significant damage is observed in the cells (no significant decrease in Renilla luciferase activity value). A case where this value was 2 or more was defined as significant activity. Further, the ratio of positive control (activity value of the sample with respect to the value where the maximum activity value of the positive control was 100%) was also calculated.

  The results are shown in FIGS. Of the two samples with different extraction methods tested, ethanol extracts were 0.5% exposed in all nuclear receptor assay systems, 0.25% exposed in FXR, LXRα, PXR, and 0. A marked decrease in the activity value of the internal standard (Renilla luciferase) was confirmed in the 1% exposure group, suggesting cytotoxicity (FIG. 1). For this reason, in this study, the numerical value of the exposure group was excluded from the analysis (Table 3). For the hot water extract, no cytotoxicity was observed at all concentrations (Table 2).

  The activity value of the hot water extract for each nuclear receptor was 2 or less at all concentrations, indicating that it did not contain a ligand substance for the receptor (Table 2). On the other hand, the ethanol extract showed significant activity values of 3.34 for PPARα, 4.62 for PPARδ, 7.58 for PPARγ, and 16.16 for RXRα in the 0.25% exposed group (Table 3 and Table 3). FIG. 2) and these showed significant activity values even in relative activity compared to each positive control (Table 3). In addition, in PPARγ and RXRα, significant activity values (PPARγ: 3.52, RXRα: 2.15) were recognized even in the 0.1% exposure group, and these two types of receptors have particularly remarkable activity. (Table 3 and FIG. 2).

  From the above results, it was shown that the beet fiber ethanol extract has agonist activity against PPARα, PPARδ, PPARγ and RXRα.

(Manufacture of beet fiber ethanol extract-containing preparations)
20 parts by weight of beet fiber ethanol extract (dried under reduced pressure) obtained in Example 1, 70 parts by weight of corn starch and 10 parts by weight of glucose were mixed, and the resulting mixture was fluidized-bed granulated with a 5% aqueous solution of hydroxypropylmethylcellulose. To obtain granules.

  The present invention is summarized as follows.

  An object of the present invention is to provide a highly safe component having agonist activity for a retinoid X receptor (RXR) and / or a peroxisome proliferator-responsive receptor (PPAR).

  And, the agent containing beet fiber ethanol extract or processed / purified product as an active ingredient activates the expression of retinoid X receptor (RXRα) and peroxisome proliferator-responsive receptors (PPARα, PPARδ, PPARγ) It is possible to prevent and treat various diseases safely and effectively.

Claims (5)

Sugar beet root is ground and squeezed, then immersed and stirred in warm water to obtain a residue from which soluble components have been extracted and removed, and this is decolorized, deodorized, dried at a temperature of 80 to 90 ° C., ground, and sieved A beet fiber ethanol extract obtained by adding 10 to 50 times the amount of ethanol to beet fiber obtained by separate treatment and extracting at a temperature of 0 to 78 ° C. and drying the liquid layer after solid-liquid separation is obtained. characterized in that is contained as an active ingredient, breast, lung, alopecia, at least one preventing and / or ameliorating a retinoid X receptor biorhythm ataxia (RXR [alpha) production method of the active agent. Sugar beet root is ground and squeezed, then immersed and stirred in warm water to obtain a residue from which soluble components have been extracted and removed, and this is decolorized, deodorized, dried at a temperature of 80 to 90 ° C., ground, and sieved A beet fiber ethanol extract obtained by adding 10 to 50 times the amount of ethanol to beet fiber obtained by separate treatment and extracting at a temperature of 0 to 78 ° C. and drying the liquid layer after solid-liquid separation is obtained. characterized in that is contained as an active ingredient, a manufacturing method of insulin resistance diseases, stroke, peroxisome proliferator-activated receptor to prevent and / or ameliorate at least one of the biological rhythm ataxia alpha (PPARa) activator . Sugar beet root is ground and squeezed, then immersed and stirred in warm water to obtain a residue from which soluble components have been extracted and removed, and this is decolorized, deodorized, dried at a temperature of 80 to 90 ° C., ground, and sieved A beet fiber ethanol extract obtained by adding 10 to 50 times the amount of ethanol to beet fiber obtained by separate treatment and extracting at a temperature of 0 to 78 ° C. and drying the liquid layer after solid-liquid separation is obtained. characterized in that is contained as an active ingredient, a manufacturing method of preventing insulin resistance disease and / or ameliorating peroxisome proliferator activated receptor [delta] (PPAR [delta) activator. Sugar beet root is ground and squeezed, then immersed and stirred in warm water to obtain a residue from which soluble components have been extracted and removed, and this is decolorized, deodorized, dried at a temperature of 80 to 90 ° C., ground, and sieved A beet fiber ethanol extract obtained by adding 10 to 50 times the amount of ethanol to beet fiber obtained by separate treatment and extracting at a temperature of 0 to 78 ° C. and drying the liquid layer after solid-liquid separation is obtained. characterized in that is contained as an active ingredient, hypertension, insulin resistance diseases, cerebral infarction, Alzheimer's disease, peroxisome proliferator-activated receptor to prevent and / or ameliorate at least one of the neurological disorders gamma (PPARy) activity A method for producing an agent. Beet fiber ethanol extract is ceramide, cerebroside, glycosylceramide, sphingoid base, sphingolipid, glycerolipid, steryl glycoside, sterol glycoside, sterol, fatty acid, saponin, steroid glycoside, steroid, retinoid, retinoic acid The method according to any one of claims 1 to 4, comprising at least one or two or more selected from the group consisting of retinal, retinol, retinal, carotenoid, and terpenoid.

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