JP4266386B2 - PPARα activator - Google Patents

Description

  The present invention relates to a health food and a PPAR activator having a PPAR activating action, which can be taken daily since safe natural materials are used.

  Peroxisome proliferator-activated receptor (hereinafter referred to as “PPAR”) is a type of hormone receptor protein present in the nucleus of a cell. When a ligand binds to this receptor, retinoid X receptor is received. Forms a complex with the body (retinoid X receptor, RXR). This complex induces the expression of the gene by binding to the upstream sequence of the specific gene. That is, PPAR plays an important role in vivo as one of the factors that regulate the expression of specific genes.

  A typical metabolic system in which PPAR is involved as a regulator is a lipid metabolic system. That is, PPAR controls the expression of genes involved in lipid oxidation (β oxidation) in the liver and the like.

In addition, it has become clear that PPAR is also involved in the maintenance of homeostasis. For example, PGJ ₂ (15-deoxy-Δ12,14-PGJ ₂ etc.) recently attracting attention among prostaglandins which are otachoids (local signal transmitters) is an intrinsic factor of PPARγ which is an isoform of PPAR. It is a sex ligand and is known to be an important transcription factor for adipocyte differentiation. It has also been revealed that PPAR is involved in metabolism related to various diseases such as regulation of expression of hepatocyte growth factor. Therefore, a drug (ligand) capable of activating PPAR can be used as a therapeutic or preventive drug for inflammation and certain types of cancer as well as hyperlipidemia and diabetes.

  As synthetic drugs that are PPAR activators, so-called thiazolidinedione derivatives such as troglitazone, rosiglitazone, and pioglitazone are known and have been used as therapeutic agents for diabetes. However, these synthetic drugs always have the problem of side effects. For example, troglitazone has been killed by its administration and has already been released on a global scale. In addition to thiazolidinedione derivatives, it is known that lactofen, which is a herbicide, and trichlorethylene, which is used for cleaning semiconductors and the like, have a PPAR activating action. It is impossible to take any other medicine.

  By the way, the present inventors have been studying for a long time about new medicinal effects and their applications regarding natural products used in traditional medicine around the world. These natural products are safe and can be taken daily, unlike synthetic drugs where the problem of side effects is inevitable. Therefore, it is particularly effective to use it as a prophylactic agent for constantly preventing the development of so-called lifestyle-related diseases.

  Some of the results of such research have already been filed as patents for inventions. For example, Patent Document 1 discloses an antidiabetic agent and a dieting agent containing, as active ingredients, an extract composed of three species of a cedar plant, a red spider plant, and a Salacia plant. Patent Document 2 describes an anti-diabetic agent containing an extract of pomegranate flowers, a plant belonging to the genus Salacia as an active ingredient.

However, the main causes of diabetes are insulin secretion deficiency and insulin resistance, but the more fundamental causes of type I diabetes are not only genetic factors but also malignant tumors, infections, and β Cell destruction and type II diabetes include overeating, lack of exercise, and obesity, which are not always clear. That is, since the cause is various, the therapeutic agent for diabetes is not always a PPAR activator.
JP 2002-316938 A (Claim 1) JP 2002-20308 A (Claim 1)

  Under the circumstances as described above, the problem to be solved by the present invention is to be safe and can be taken every day in order to prevent lifestyle-related diseases or reduce intractable symptoms such as intestinal hypersensitivity, Another object of the present invention is to provide a PPAR activator that can be expected to have a wide range of action effects and a health food having a PPAR activation action.

  In order to solve the above problems, the present inventor has used not only herbal medicines and natural products that have been used for many years in traditional medicine around the world, especially Chinese medicine and Chinese medicine, or described in Ayurveda and Yunani, but also fruits and vegetables. Were also examined, and those having PPAR activation ability were searched. As a result, the present invention was completed by finding that a specific substance shows an appropriate ability to activate PPAR.

  The PPARα activator according to the present invention is characterized by containing an aqueous solvent extract of Zingiber officinale Rosc as an active ingredient.

  The aqueous solvent is preferably water, a lower alcohol, or a mixed solvent of water and a lower alcohol, and examples of the lower alcohol include methanol, ethanol, isopropanol, and t-butyl alcohol.

  The PPARα activator is further one selected from the group consisting of Salacia roots, Mangifera indica fruits, Garcinia mangostana peels, and Alpina galangal. Alternatively, it may contain two or more aqueous solvent extracts and / or asenyaku.

  The health food according to the present invention contains the PPARα activator.

  As demonstrated in the examples described later, the extracts of mango, mangosteen, Salacia plant, pomegranate, Kintoki ginger and Odaka Ryokan and Asenyaku according to the present invention are PPARα, PPARα, β, γ. At least one of them can be specifically activated. Since these PPAR isoforms have different expression tissues and related enzyme systems in vivo, these extracts can be selected or combined according to symptoms and body trends. In addition, the effect is milder than that of the synthesized PPAR activator, and since these materials have only been used for herbal medicine or food since ancient times, they can be taken daily.

  Therefore, the health food and the PPAR activator according to the present invention are extremely useful not only as a therapeutic agent for various diseases involving PPAR but also as a preventive agent that can be taken every day.

  Hereinafter, embodiments of the present invention and effects thereof will be described.

  The mango (Mangifera indica) used as a material in the present invention is an evergreen shrub belonging to the family Ursiaceae that is widely cultivated in various places in the tropics and subtropics. Used as material.

  Mangosteen (Garcinia mangostana) is a Hypericaceae plant mainly cultivated in the tropics including Southeast Asia. In the present invention, pericarp is used as a main material.

  The plants of the genus Salacia (genus Salacia, Euphoridae) are plants that naturally grow in India, Sri Lanka, etc., and typical examples thereof include Salacia oblonga and Salacia prinoides. In the present invention, the root or trunk skin can be used as a main material.

  Pomegranate (Punica granatum) is a plant belonging to the family Pomegranate, which has traditionally been used as a nosebleed medicine in Arabian, Indian, and Chinese medicine, and even in Japan, it is used as an eye cleanser. The fact is that little research has been done. In the present invention, a flower is mainly used as a material, but the type of flower is not particularly limited, and it is preferable to use a flower taken from the corolla before falling, regardless of whether it is single or double.

  Kintoki ginger (Zingiber officinale Rosc) is an ancient Japanese ginger, but it is now rarely cultivated in Japan due to the small amount of ginger that is difficult to cook and the inflow of cheap, large foreign ginger. However, it is a variety that has been cultivated in Vietnam and the like through technical guidance by the present inventors.

  Alpina galangal is widely used in southern China, Taiwan, and tropical Asia, and is a rhizome of Antarctic spruce that is cultivated as a spice and gastrointestinal medicine in Vietnam. Such nankyosou fruit is called Kuzuku and is also used for gastrointestinal drugs and spices.

  Asenyak is a solid-dried compressed liquid of Gambir (Uncaria gambir, a vine shrub that is widely distributed in tropical Asia, a vine-like shrub that wraps around other trees and is a companion of the key lizard that grows naturally in Japan). Is. In other words, Asenyak puts young branch leaves of gun beer into a kettle, boils them for several hours, takes out and presses them, cools the compressed liquid that exudes, cuts the solidified pieces into 4-5 cm dice and temperate them. Sun-dried. Recently, some factories produce vacuum-dried compressed liquid to produce asenyaku, and the method for producing asenyak used in the present invention is not particularly limited. In addition to being used as a herbal medicine, such as an astringent in the mouth, this asenyak is used for industrial dyes and tanning, but its use as a health food is not known. In the present invention, asenyaku is used as a powder or dissolved in hot water.

  In producing the extract of the present invention, it is preferable to first dry or roughly dry the material. The method of “drying” is not particularly limited, and examples thereof include sun drying, half sun drying, shade drying, heat drying, room temperature drying, freeze drying, vacuum drying, and the like. , Shade drying is preferred. However, the material may be roughly cut without drying.

  Next, the material itself, a dried material, or a material obtained by roughly cutting or powdering the material is extracted with a solvent. As the solvent for extraction, an aqueous solvent is mainly used. This is in consideration of the convenience of extraction work, safety if it is extracted from an aqueous solvent because it is water-soluble, and safety when the solvent remains. Examples of such aqueous solvents include water; lower alcohols such as methanol, ethanol, isopropanol, and t-butyl alcohol; mixed solvents of water and lower alcohols. Or a mixed solvent of water and ethanol is preferred.

  The amount of the solvent to be used is generally 2 to 50 times the mass of the material if the extracted material is dried, and 0.5 to 30 times if the extracted material is not dried. Further, the extraction temperature is not particularly limited, but it is preferable to heat in consideration of efficiency.

  The extraction time is not particularly limited, but is preferably 1 hour to 12 hours. In addition, the extraction may be left standing or stirred.

  The processing after the completion of extraction follows a conventional method. For example, after filtration, the residue is washed with the solvent used, and after combining with the filtrate, the solvent may be distilled off under reduced pressure or heating. However, excessive heating is not preferred, and it is preferably concentrated under reduced pressure at room temperature to about 50 ° C.

  The extract thus obtained may be taken as it is, but it may also be made into a composition by adding an appropriate amount of additive components. That is, examples of the dosage form of the health food and the PPAR activator according to the present invention include powders, tablets, granules, capsules, and teas, but are not particularly limited. Moreover, you may use as a structural component of processed foods, such as adding to confectionery.

  The dose varies depending on the purpose, the symptom of the user, the number of doses, etc. For example, when taking powder several times a day, the dose can be taken from 100 mg to 5 g at a time.

  The PPAR activated by the agent of the present invention has α, β and γ isoforms, which are different in expression tissues, related metabolic systems, expression levels, and the like. For example, it is considered that PPARα is related to lipid metabolism in the liver, and PPARγ is related to fat synthesis and immune response. Therefore, if any PPAR is activated, it treats chronic inflammation such as diabetes (especially type II diabetes), diabetic complications, hypertension, cerebral hemorrhage, arteriosclerosis, intestinal hypersensitivity, allergic diseases or cancer. Can be prevented. Moreover, as the result of the Example mentioned later, the PPAR activation ability of this invention agent may have selectivity with respect to PPAR isoform. Therefore, the material type of the agent of the present invention can be determined according to the symptoms of the user.

  In addition, as described above, the material used in the present invention is a safe natural product such as fruit, and as a result of Examples described later, the PPAR activation ability of the agent of the present invention always involves a risk of side effects. The effect is milder than the synthetic activator of PPAR. Therefore, by constantly taking the health food and the PPAR activator according to the present invention, it is possible to maintain a healthy body and prevent diseases related to PPAR.

  According to the results of Examples described later, some health foods and PPAR activators according to the present invention contain xanthone derivatives. However, since the PPAR activation ability of the agent of the present invention is equivalent to or superior to that of the standard xanthone derivative, the agent of the present invention contains an excellent component other than the xanthone derivative, or the component and xanthone It is conceivable that the derivative exhibits a synergistic effect.

  Hereinafter, the present invention will be described in more detail by showing production examples and test examples, but the scope of the present invention is not limited thereto.

Production Example 1 Production of PPAR Activator Mango fruits (commercially produced in the Philippines) were purchased and the core (tissue containing the seeds) was removed with a stainless steel knife and immediately freeze-dried. As a result of drying, the weight became 1/10 to 1/12 before drying. In addition, the skin of mangosteen (from Vietnam), Salacia oblonga root, pomegranate flower, Kintoki ginger (from Shizuoka or Vietnam), and Odaka Ryo (from Vietnam) were used as raw materials. Asenyak was used in powder form.

  Roughly cut the above material 500 g of each was added to 5000 mL of 50% aqueous ethanol solution, followed by heating and extraction for 3 hours, followed by filtration. The obtained filtrate was concentrated under reduced pressure at 45 ° C. or lower, and the solvent was completely distilled off to obtain extracts of each specimen. The yield of each extract was as a percentage of each dried product: mango fruit: 13%, mangosteen peel: 8%, Salacia oblonga root: 5%, pomegranate flower: 20%, golden ginger: 9.8% It was.

About each obtained extract, the xanthone derivative estimated to be a main active ingredient was quantified. Mangiferin (manufactured by Sigma, C ₁₉ H ₁₈ O ₁₁ ) and mangosteen (compound name instead of fruit name, C ₂₄ H ₂₆ O ₆ , melting point: 182 ° C.) were used as preparations of xanthone derivatives. LC-10ATVP, SPD-10ADVP, and CBM-10A manufactured by Shimadzu Corporation were used as the HPLC apparatus, detector, and automatic dispenser. As the column, MAX-RP80A (250 × 4.6 mm) also manufactured by Shimadzu Corporation was used, and the HPLC measurement conditions were temperature: 35 ° C., flow rate: 1.0 mL / min, detector wavelengths: 270 nm and 360 nm. .

  Each extract was dissolved in water at a concentration of 1.0 mg / mL, and 50 μL thereof was injected onto the column. As a mobile phase, a mixed solution of acetonitrile and 0.1% (v / v) phosphoric acid water was used. Initial 10:90, 5 minutes later 15:85, 15 minutes later 20:80, 30 to 35 minutes. Later, a 40:60 gradient was applied.

  According to the results, 1.4% of mangiferin was contained in the Salacia root extract, and 0.9% of mangosteen (compound name) was contained in the mangosteen peel extract.

Test Example 1 PPARα Activation Test First, DMEM / F-12 (manufactured by Introgen), a basic medium for cells, bovine serum (final concentration: 10%), penicillin (100 units / mL), streptomycin (100 μg). / ML), 1-glutamine (1%) and HEPES (15 mM) were added to prepare a cell culture medium. Human embryo-derived kidney cells (HEK-293) were added to the cell culture medium and cultured.

  The cultured human embryonic kidney cells are transfected using three types of plasmids (tK-RDRE × 3-Luc, pBI-G-hPPAR-α and pSV-β-galactosidase) and used for the PPARα activation test. It was. That is, when a PPARα gene and a reporter gene are introduced into cells using these three types of plasmids, the expression of luciferase, which is a reporter enzyme, is promoted when PPARα is activated. On the other hand, the expression of β-galactosidase is not affected by PPARα. Therefore, if the luciferase activity of the cells treated with each sample is corrected by the β-galactosidase activity, more accurate PPARα activation ability can be measured.

Specifically, 1 × 10 ⁵ human embryonic kidney cells were inoculated into a 5 mL T25 flask containing a nutrient solution 48 hours before transfection. To this, a transfection solution containing the above plasmid (Roche, FvGENE6) was added so that the plasmid was 3 times equivalent to the cells (DNA). After incubation for 24 hours, the cells were dispensed into a 96-well plate at 5 × 10 ⁴ cells per well, and further incubated for 2 hours.

Each extract and asenyaku obtained in Production Example 1 were dissolved in dimethyl sulfoxide (hereinafter referred to as “DMSO”) to prepare a solution having a concentration of 25 to 100 μg / mL. For comparison, a 0.1 μM solution of GW7647 (manufactured by Sigma-Aldrich), which is a PPARα synthesis activator, and mangiferin, mangosteen (compound), epicatechin, epitaxy known to have a PPARα activation action. A solution of gallocatechin was also prepared. 1 μL of each sample solution was added to the wells and incubated for 48 hours. Thereafter, the cells were thawed using Cell Lysis Buffer (manufactured by Cell Signaling Technology), and the luciferase activity of each sample was measured using a Promega luciferase quantification system (Bright-Glo ^™ Luciferase Assay System). Further, β-galactosidase activity was measured using a β-galactosidase assay system (Beta-Glo ^™ Assay System) manufactured by Promega, and the transformation rate was corrected. Using the above measurement results, the PPARα activation ability of each sample was evaluated by the ratio of luciferase activity to β-galactosidase. The results are shown in FIG.

  Although the pomegranate flower extract showed only weak PPARα activation ability, Salacia root extract, mango fruit extract, mangosteen peel extract, Kintoki ginger extract, Odaka Ryokan extract and Asenyaku Has been demonstrated to have high PPARα activation ability.

Test Example 2 Specificity test of PPARα activation ability To the 96-well plate similar to that prepared in Test Example 1 above, 0 of PPARα synthetic antagonist (synthetic antagonist) MK-866 (manufactured by BIOMOL Laboratories) After adding 1 μM aqueous solution and incubating for 1 hour, each sample was added and processed in the same manner as in Test Example 1 above. The result is shown in FIG.

  From the results, it was revealed that the PPARα activation ability of each extract and asenyaku according to the present invention was highly specific because the activation ability was almost lost by the PPARα antagonist.

Test Example 3 PPARγ Quantitative Test RPMI1640 (manufactured by Nissui Pharmaceutical Co., Ltd.) which is a basic medium for cells, bovine serum (final concentration: 10%), penicillin (100 units / mL), streptomycin (100 μg / mL) Glutamine (1%) was added to prepare a cell culture medium. Separately, THP-1 human monocyte cells purchased from ATCC (American Type Culture Collection) were humidified at 95% O ₂ , 5% CO ₂ , 37 ° C., and cultured using an incubator manufactured by GIBCO. In the following experiment, monocytes with a passage of 5 to 20 μm were used. In addition, for the sake of comparison, PPARγ was synthesized for each of the extracts of Salacia root, mango fruit, mangosteen peel, pomegranate flower, Kintoki ginger, Odaka Ryo and 50% or 100 μg / mL DMSO solution of asenyaku obtained in Production Example 1 above. A 3 μg / mL aqueous solution of an activator GW1929 (manufactured by Sigma-Aldrich) was prepared. Further, in the same manner as in Test Example 1, a solution such as mangiferin was also prepared.

The monocytes prepared above were inoculated into a 10 mL T25 flask at a concentration of 5 × 10 ⁵ cells / mL, 1 mL of DMSO solution of each sample was added, and incubated for 48 hours.

  The ability of each sample to activate PPARγ was determined by G. Davies et al., Journal of Pharmacology and Experimental Therapeutics (J. Pharmacol. Exp. Ther.), Vol. 72, p. 300- (2000 Year). Specifically, first, a melt was added to monocytes on which each sample was allowed to act, followed by centrifugation for 30 minutes using a centrifuge (SS-340-g, manufactured by Sorvall). The obtained supernatant was melted in SDA-PAGE (10% polyacrylamide), Dunn-Carbonate Transfer Buffer (10 mM sodium bicarbonate + 3 mM sodium carbonate + 15% methanol, 4 ° C.) was added, and this was added to polyvinylidene difluoride. The contained protein was immobilized on the membrane by passing through the membrane, transferring the contained protein to the membrane, adding 5% skim milk, and allowing to stand overnight. Anti-human PPARγ antibody-anti-rabbit antibody (1: 500 dilution, manufactured by Santa Cruz Biotechnology) was added to this membrane. After washing the membrane, an anti-rabbit secondary antibody conjugated with horseradish-derived peroxidase (1: 8000 dilution, Promega) was added.

  The bound antibody was detected with a highly sensitive chemiluminescence kit (Roche, Lum-Light Western Blotting Substrate). This film is photographed with an X-ray film (manufactured by Kodak) and developed with an X-ray developing machine (SRX-101A), and then molecular analysis software NIH image version 1.62 (version 2.1.2, Biorad) is used. Thus, PPARγ was quantified. In addition, β-actin and anti-actin antibody (1: 1000 dilution, A5060, manufactured by Sigma) were added to the above membrane and treated with 0.2M aqueous sodium hydroxide solution for 5 minutes to serve as a control for quantitative determination of PPARγ. Was used. The results are shown in FIG.

  According to the results, the extract of Salacia root, mango fruit, mangosteen peel, Kintoki ginger and Odaka Ryo has no clear PPARγ enhancement, but pomegranate flower extract and Asenyaku have clear PPARγ enhancement. Show. Therefore, since the response ability to the PPARγ ligand is improved when the expression level of PPARγ is increased, it has been demonstrated that the pomegranate flower extract and asenyaku of the present invention are excellent in PPARγ activation ability.

It is a figure which shows the PPAR (alpha) activation ability of the extract which concerns on this invention, and asenya. It is a figure which shows the specificity with respect to PPAR (alpha) of the extract which concerns on this invention, and asenya. It is a figure which shows the PPAR (gamma) activation ability of the extract which concerns on this invention, and Asenya.

Claims (4)

  A PPARα activator comprising an aqueous solvent extract of Zingiber officinale Rosc as an active ingredient.   The PPARα activator according to claim 1, wherein the aqueous solvent is water, a lower alcohol, or a mixed solvent of water and a lower alcohol.   The PPARα activator according to claim 1 or 2, wherein the lower alcohol is methanol, ethanol, isopropanol, or t-butyl alcohol.   In addition, one or more aqueous solvents selected from the group consisting of Salacia roots, Mangifera indica fruits, Garcinia mangostana peels, and Alpina galangal. The PPARα activator according to any one of claims 1 to 3, comprising an extract and / or asen yak.

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