JP2005298379A - IkappaB KINASE INHIBITOR - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an IκB kinase inhibitor inhibiting IκB kinase (a) causing NF-κB activation causing transcriptional enhancement of genes relating to immunity, inflammatory reaction, cell growth control and apoptosis, and (b) having inhibition of intracellular signaling of insulin and such properties as causing IRS-1 hypofunction (phosphorylation) causing inhibition of sugar intake into cells. <P>SOLUTION: The IκB kinase inhibitor containing at least one selected from extracts, extracted from fruits or pericarps of a mangosteen (Garcinia mangostana L.), α-mangosteen and γ-mangosteen is provided, and a composition containing the inhibitor is provided. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an IκB kinase inhibitor and a composition comprising the inhibitor, wherein the inhibitor or the composition inhibits IκB kinase and inhibits the action of NF-κB.

  IκB kinase is a protein recently identified as an important kinase on the NF-κB pathway and is an essential component of the basic immune response in mammals. NF-κB is known to cause various gene transcription controls as intracellular transcription factors. Normally, NF-κB binds to IκB and stays in the cytoplasm, but the cells are lipopolysaccharide (LPS), inflammatory cytokines such as TNF-α and IL-1, T cell activation signals, growth factors, When stimulated by a stress inducer or the like, IκB kinase is activated and phosphorylates IκB. The phosphorylated IκB is released from NF-κB, and NF-κB is activated and moves into the nucleus. Activated NF-κB transferred into the nucleus binds to a specific gene sequence and promotes transcription of genes related to immunity, inflammatory response, cell proliferation control, and apoptosis. Therefore, activation of NF-κB is rheumatic, asthma, thyroid dysfunction syndrome and cachexia, arteriosclerosis, HIV, EB virus, apoptosis, cell death in ischemia / reperfusion stroke model, Alzheimer's disease, stroke, microbial infection It is associated with the occurrence of various diseases such as viral carcinogenesis, renal sclerosis, and diabetes. Although the IκB kinase inhibitor inhibits the activity of the IκB kinase and suppresses the occurrence of these diseases, the search has been conducted, but a safe and effective inhibitor has not yet been developed. It has also been reported that IκB kinase inhibits glucose uptake into cells by insulin and is involved in insulin resistance and type II diabetes.

  On the other hand, mangosteen (Garcinia mangostana L.) is a food preservative (for example, see Patent Documents 1 and 2), a 5α-reductase inhibitor (for example, see Patent Document 3), Antibacterial agents (for example, see Patent Document 4), anti-Helicobacter pylori drugs (for example, see Patent Document 5), ultraviolet absorbers (for example, see Patent Document 6), serine protease inhibitors (for example, Patent Documents) 7) is known. Furthermore, with respect to the water-soluble extract of mangosteen (Garcinia mangostana L.) peel, whitening and anti-inflammatory action by inhibiting histamine release from mast cells (see, for example, Patent Document 8), mangosteen (Garcinia mangostana L.) peel As for the polar solvent extract and α-mangosteen and γ-mangosteen, which are components thereof, anti-allergic action by antagonism against histamine and serotonin (see, for example, Patent Document 9) is known. Furthermore, it is also known that the mangosteen extract suppresses the production of prostaglandins (see, for example, Patent Document 10). However, as in the present invention, nothing is known about inhibiting IκB kinase or suppressing gene expression via NF-κB activation.

JP-A-6-98738 Japanese Unexamined Patent Publication No. 7-147951 JP-A-5-17365 JP-A-7-250658 JP-A-8-208501 JP-A-9-87155 JP-A-10-120586 JP-A-4-244004 Japanese Patent Laid-Open No. 10-72357 JP 2002-47180 A

  The present invention has been made to solve the above-mentioned problems, and (a) NF-κB, which is a cause of promoting the transcription of genes related to immunity, inflammatory reaction, cell proliferation control, and apoptosis, has been developed. An IκB kinase having properties such as (b) inhibiting intracellular signal transduction of insulin and causing a decrease in function (phosphorylation) of IRS-1 which is a cause of inhibition of glucose uptake into cells. Inhibiting IκB kinase inhibitors and compositions containing the inhibitors are provided. In the present invention, a plant extract obtained by extraction from mangosteen (Garcinia mangostana L.) fruit or pericarp with a water-containing organic solvent or an organic solvent is used as a medicinal component, thereby inhibiting safe and powerful IκB kinase. Also provided are agents and compositions containing the inhibitors.

  In order to solve the above-mentioned problems, the present inventors have conducted intensive research and obtained extracts from various kinds of solvents from mangosteen (Garcinia mangostana L.) fruits and pericarps, which are fruits of the family Hypericaceae and are used for food. Mangosteen extract obtained by extracting with water-containing organic solvent or organic solvent as a result of extraction and further isolating several components contained in the extract, and as a result of intensive studies on the extract and several components, α -It was found that mangosteen and γ-mangostin have an IκB kinase inhibitory effect and a gene expression inhibitory effect through NF-κB activation.

  That is, as a result of confirming the effect as an IκB kinase inhibitor, the present inventors have obtained an extract obtained by extraction from a mangosteen fruit or pericarp with a water-containing organic solvent or an organic solvent,

Α-mangosteen represented by the following formula:

It has been found that γ-mangostin represented by the formula has excellent IκB kinase inhibitory activity and gene expression suppression activity through NF-κB activation, and the present invention has been completed based on these findings.

  Accordingly, the present invention contains, as an active ingredient, at least one selected from an extract obtained by extraction from a fruit or skin of mangosteen (Garcinia mangostana L.) with a water-containing organic solvent or an organic solvent, α-mangostin, and γ-mangostin. An IκB kinase inhibitor and a composition comprising the same.

  An extract obtained by extracting from the fruit and pericarp of the mangosteen of the present invention with a water-containing organic solvent or an organic solvent, an IκB kinase inhibitor comprising α-mangosteen and / or γ-mangostin as an active ingredient has an excellent IκB kinase inhibitory action And NF-κB has a gene transcription suppressing effect. In addition, the extract obtained by extracting from the fruit and pericarp of the mangosteen of the present invention with a water-containing organic solvent or an organic solvent can be prepared from a natural product and is therefore safe with no side effects. Furthermore, since the IκB kinase inhibitor of the present invention is excellent in taste, a composition for inhibiting IκB kinase having excellent taste can be provided by containing these in pharmaceuticals and foods and drinks.

  The present invention will be described in detail below.

  The extract obtained by extracting from the fruit or skin of mangosteen, which is an active ingredient of the IκB kinase inhibitor of the present invention, with a water-containing organic solvent or organic solvent is obtained from the fruit or skin of a Hypericaceae plant, Garcinia mangostana L. Extracted. Although the mangosteen fruit or skin may be used as it is, it is preferable to use dried and crushed powder because the extraction efficiency is improved. Moreover, before extracting the fruit and pericarp of mangosteen with a water-containing organic solvent or an organic solvent, you may degrease with nonpolar solvents, such as n-hexane and petroleum ether.

  The extraction solvent is an organic solvent such as methanol, ethanol, n-propanol, isopropanol, n-butanol, acetone, ethyl acetate, diethyl ether, chloroform, glycerin, ethyl glycol, propyl glycol, preferably a polar solvent, particularly ethanol. It is recommended to use a simple alcohol or an organic solvent mixed with water. In consideration of the fact that the inhibitor and composition of the present invention may be used as an orally administered drug or food and drink, it is preferable to use only ethanol or a combination of water and ethanol as an extraction solvent from the viewpoint of safety. preferable. The mixing ratio with water is not particularly limited, but the ratio of water is preferably 70% (v / v) or less, more preferably 60% (v / v) or less. The extraction temperature is preferably from room temperature to the reflux temperature of the solvent in consideration of extraction efficiency. The extraction time varies depending on the type of the extraction solvent, the crushed state of the fruit or skin, and the extraction temperature, but 0.5 to 24 hours is preferable.

  If necessary, the extract obtained by extraction with the extraction solvent may be concentrated or removed by an evaporator or the like. In addition, the extract can be used after being purified by solvent fractionation or chromatography, if necessary.

  In addition, α-mangostin and γ-mangostin, which are active ingredients of the IκB kinase inhibitor of the present invention, can be produced by extraction and purification, for example, from the fruit of mangosteen (Garcinia mangostana L.) by a known method. However, the synthesized one can also be used.

  In addition, Yin MJ, Yamamoto Y, and Gaynor RB (1998) The anti-inflammatory agents aspirin and salicylate inhibit the activity of drugs that are currently suggested to treat or prevent diseases by inhibiting IκB kinase. “Aspirin and sodium salicylate” disclosed in IkB-b. Nature 396: 77-80 are known. On the other hand, the IκB kinase inhibitor of the present invention is a naturally-occurring inhibitor of IκB kinase. Therefore, it can be safely used for the treatment / prevention of diseases for which the activity inhibiting action of IκB kinase and the effect of suppressing gene expression by NF-κB are effective. Diseases effective in inhibiting the activity of IκB kinase and diseases effective in suppressing transcriptional expression by NF-κB include, for example, asthma, allergic rhinitis, atopic dermatitis, urticaria, conjunctivitis, spring catarrh, psoriasis Ulcerative colitis, systemic inflammatory response syndrome, sepsis, polymyositis, dermatomyositis, nodular polyarthritis, mixed connective tissue disease, Sjogren's syndrome, gout, Alzheimer's dementia, Parkinson's disease, amyotrophic lateral sclerosis Disease, rheumatoid arthritis, type I diabetes, multiple sclerosis, Crohn's disease, Hashimoto's disease, celiac disease, myasthenia gravis, pemphigus vulgaris, systemic lupus erythematosus, viral disease (HIV, herpes, Sendai rotavirus, influenza) Virus, rabies virus, vesicular stomatitis virus, rhinovirus, hepatitis A virus, hepatitis B virus , Rubella virus, etc.), bacterial diseases, radiation damage, arteriosclerosis, reperfusion injury, cardiac hypertrophy, cancer, etc.

  In addition, IκB kinase is known to directly phosphorylate IRS-1, and this action reduces the function of IRS-1, thereby inhibiting intracellular signal transduction of insulin and sugar uptake into cells. Therefore, the present invention can be used for the treatment and prevention of insulin resistance and type II diabetes.

  The IκB kinase inhibitor of the present invention can be used as a medicine alone or mixed with other medicines or any formulation carriers, diluents and the like in any dosage form. Examples of dosage forms include tablets, granules, powders, fine granules, aerosols, hard capsules, soft capsules, oral liquid preparations, injections, and the like.

  Examples of the composition for inhibiting IκB kinase of the present invention include foods and drinks such as beverages, confectionery, frozen desserts, dairy products, alcoholic beverages, and meats. These IκB kinase-inhibiting compositions contain one or more of fruits of mangosteen (Garcinia mangostana L.), water-containing organic solvent or organic solvent extract of skin, α-mangosteen and γ-mangostin in various foods and drinks. Is obtained. The effective content of mangosteen extract, α-mangosteen and γ-mangostin in this composition for inhibiting IκB kinase varies depending on the administration method and necessary treatment, and it is difficult to define it in general. When calculated as a dose, the dry weight is 0.5 mg to 5 g, preferably 5 mg to 5 g, more preferably 10 mg to 1 g, and 1 mg / L to 100 mg / L for cultured cells. -For mangosteen and γ-mangostin, the dose of 0.1 mg to 1 g per kg of human and animal body weight is preferably 1 mg to 1 g, more preferably 2 mg to 200 mg, and 1 μmol / L to 30 μmol / L for cultured cells. is there.

  Hereinafter, the present invention will be described in more detail with reference to test examples.

Test example

  In this test, an extract prepared from mangosteen (Garcinia mangostana L.), α-mangostin and γ-mangostin inhibit IκB kinase, suppress phosphorylation of IκB, and suppress gene expression via NF-κB activation. I went to find out.

(1) Sample preparation The following samples were used for this test.

1) Sample 1
1 kg of undried pericarp of mangosteen (Garcinia mangostana L.) was immersed in 10 L of methanol and extracted at room temperature for 24 hours. After filtration, the filtrate was dried under reduced pressure with an evaporator to obtain 80 g of an extract.

2) Sample 2
500 g of dried fruit skin powder of mangosteen (Garcinia mangostana L.) was immersed in 5 L of 40% ethanol and extracted at room temperature for 24 hours. After filtration, the filtrate was dried under reduced pressure with an evaporator to obtain 104 g of an extract.

3) Sample 3
480 g of dried fruit skin powder of Mangosteen (Garcinia mangostana L.) was stirred and extracted with 5 L of 70% ethanol for 4 hours (60 ° C.). After filtration, the filtrate was dried under reduced pressure with an evaporator to obtain 128 g of an extract.

4) Sample 4
1.1 kg of dried fruit skin powder of Garcinia mangostana L. was stirred and extracted with 11 L of ethanol for 4 hours (60 ° C.). After filtration, the filtrate was dried under reduced pressure with an evaporator to obtain 128 g of an extract.

5) Sample 5
Mangosteen (Garcinia mangostana L.) fruit (1.1 kg) was ground and extracted with 5 L of ethanol for 0.5 hour (70 ° C.). After filtration, the filtrate was dried under reduced pressure with an evaporator to obtain 88 g of an extract.

6) Samples 6 and 7
80 g of the extract of Sample 1 was dissolved in 350 ml of ethyl acetate and then washed twice with 200 ml of water. The solvent was distilled off from the ethyl acetate fraction with an evaporator to obtain 20 g of a dried product. This dried product was purified by silica gel column chromatography. Elution was performed in a hexane-ethyl acetate system, and gradient elution was carried out gradually to obtain three fractions. The first fraction (5 g) obtained was purified again by silica gel column chromatography (hexane-ethyl acetate, 10: 90 → 30: 70 → 50: 50), and sample 6 (α-mangostin) in the form of yellow crystals 2 g was obtained. The second fraction (2 g) was purified again by silica gel column chromatography (hexane-ethyl acetate, 30: 70 → 50: 50, followed by ethyl acetate only, finally ethyl acetate-methanol, 50:50), yellow 500 mg of amorphous sample 7 (γ-mangosteen) was obtained.

(2) Test method 1) LPS-induced IκB phosphorylation test C6 cells were cultured with a test sample for 1 hour, 10 μg / ml LPS was added, and the mixture was incubated for 30 minutes for stimulation. Cells were lysed with a sample buffer for SDS-PAGE, subjected to SDS-PAGE, and transferred to a polyvinylidene difluoride membrane. Rabbit anti-phosphorylated IκB antibody (0.2 μg / ml) was added and incubated at 25 ° C. for 2 hours. After washing, it was incubated with horseradish peroxidase-conjugated anti-rabbit IgG antibody. Table 1 shows the relative values when phosphorylated IκB was measured by chemiluminescence and the amount of phosphorylated IκB in C6 cells not added with LPS was taken as 1.

2) In vitro IκB kinase test C6 cells were stimulated with LPS (10 μg / ml) for 10 minutes, washed with PBS, and then lysis buffer (2 mM EGTA, 150 mM NaCl, 2 mM DTT, 1 mM APMSF, 10 μg / ml leupeptin, 10 μg / ml) ml aprotinin, 1 mM Na3VO4, 10 mM Tris-HCl, pH 7.5) was added to prepare a cell extract. 6 μg of anti-IΚKα / β antibody was added, and the immune complex was recovered with protein A-Sepharose 4B beads. After washing the IK protein-bound beads three times with a lysis buffer, 10 mM MgCl 2 .6H 2 O, 0.1 mM Na 3 VO 4, 2 mM DTT, 5 mM β-glycerophosphate, [γ-32P] -ATP, 25 mM Tris-HCl, pH 7.5 In 25 μL of the kinase reaction solution. After adding a test sample and incubating at 30 ° C. for 10 minutes, 1 μg of IκBα was added and incubated for 30 minutes. SDS-PAGE sample buffer was added to stop the reaction, proteins were separated by SDS-PAGE electrophoresis, phosphorylated IκBα was quantified by Molecular Imager (GS363, Bio-Rad), and substrate IκB was not added Table 2 shows the relative values with respect to the amount of phosphorylated IκBα.

3) NFκB signaling activation test by dual luciferase measurement C6 cells were seeded in a 24-well plate, and two days later, 0.475 μg / well of pNFκB-Luc (firefly luciferase reporter plasmid containing a repeated NFκB response element) or dN-Luc (Reporter plasmid lacking the NFκB sequence in pNFκB-Luc, Hirai et al., 1994) was transfected. Furthermore, C25 cells were cotransfected with 0.025 μg of control vector Renilla luciferase. After transfection, the cells were cultured for 13 hours and incubated with the test sample for 3 hours. The cells were further added with 1 μg / ml LPS and cultured for 8 hours, and the cells were collected. Firefly luciferase and Renilla luciferase activities were measured with MiniLumat LB9506. Table 3 shows the effect of the test sample as a relative value of the firefly luciferase activity relative to the Renilla luciferase activity.

(3) Test results From the results of this test example, extracts obtained by extraction from the fruit or skin of mangosteen (Garcinia mangostana L.) with a water-containing organic solvent or organic solvent, α-mangosteen and γ-mangostin inhibit IκB kinase. It became clear that it has an action and suppresses gene expression through activation of NF-κB. Therefore, the product of the present invention is useful as an IκB kinase inhibitor, and may be useful for the treatment and prevention of diseases involving mammalian IκB kinase and diseases involving NF-κB.

  Next, the best mode for carrying out the present invention will be described in detail by way of examples. However, the present invention is not limited to the following examples.

  1 kg of undried pericarp of mangosteen (Garcinia mangostana L.) was immersed in 10 L of methanol and extracted at room temperature for 24 hours. After filtration, the filtrate was dried under reduced pressure with an evaporator to obtain 80 g of an IκB kinase inhibitor.

  500 g of dried fruit skin powder of mangosteen (Garcinia mangostana L.) was immersed in 5 L of 40% ethanol and extracted at room temperature for 24 hours. After filtration, the filtrate was dried under reduced pressure with an evaporator to obtain 104 g of an IκB kinase inhibitor.

  480 g of dried fruit skin powder of Mangosteen (Garcinia mangostana L.) was stirred and extracted with 5 L of 70% ethanol for 4 hours (60 ° C.). After filtration, the filtrate was dried under reduced pressure with an evaporator to obtain 128 g of an IκB kinase inhibitor.

  1.1 kg of dried fruit skin powder of Garcinia mangostana L. was stirred and extracted with 11 L of ethanol for 4 hours (60 ° C.). After filtration, the filtrate was dried under reduced pressure with an evaporator to obtain 128 g of an IκB kinase inhibitor.

  Mangosteen (Garcinia mangostana L.) fruit (1.1 kg) was pulverized and extracted with 5 L of ethanol for 0.5 hours (70 ° C.). After filtration, the filtrate was dried under reduced pressure with an evaporator to obtain 88 g of an IκB kinase inhibitor.

  80 g of the extract described in Example 1 was dissolved in 350 ml of ethyl acetate and then washed twice with 200 ml of water. The solvent was distilled off from the ethyl acetate fraction with an evaporator to obtain 20 g of a dried product. This dried product was purified by silica gel column chromatography. Elution was performed in a hexane-ethyl acetate system, and gradient elution was carried out gradually to obtain three fractions. The initially obtained fraction (5 g) was again purified by silica gel column chromatography (hexane-ethyl acetate, 10: 90 → 30: 70 → 50: 50) to obtain 2 g of yellow crystalline α-mangostin. . The second fraction (2 g) was purified again by silica gel column chromatography (hexane-ethyl acetate, 30: 70 → 50: 50, followed by ethyl acetate alone, finally ethyl acetate-methanol, 50:50), yellow 500 mg of amorphous γ-mangosteen was obtained.

The IκB kinase inhibitor obtained in Example 1 was formulated according to the following formulation to prepare an IκB kinase inhibitor-containing powder.
Lactose 25 parts potato starch 10 parts IκB kinase inhibitor 5 parts obtained in Example 1

The IκB kinase inhibitor obtained in Example 2 was formulated according to the following formulation to prepare an IκB kinase inhibitor-containing powder.
Lactose 25 parts potato starch 10 parts IκB kinase inhibitor 5 parts obtained in Example 2

The IκB kinase inhibitor obtained in Example 3 was blended according to the following formulation to prepare an IκB kinase inhibitor-containing powder.
Lactose 25 parts potato starch 10 parts IκB kinase inhibitor 5 parts obtained in Example 3

The IκB kinase inhibitor obtained in Example 4 was formulated according to the following formulation to prepare an IκB kinase inhibitor-containing powder.
Lactose 25 parts potato starch 10 parts IκB kinase inhibitor 5 parts obtained in Example 4

The IκB kinase inhibitor obtained in Example 5 was blended according to the following formulation to prepare an IκB kinase inhibitor-containing powder.
Lactose 25 parts potato starch 10 parts IκB kinase inhibitor 5 parts obtained in Example 5

The α-mangostin obtained in Example 6 was formulated according to the following formulation to prepare an IκB kinase inhibitor-containing tablet.
D-mannitol 10 parts Lactose 10 parts Crystalline cellulose 2 parts Hydroxypropyl cellulose 1 part α-Mangosteen 4 parts

The γ-mangosteen obtained in Example 6 was blended according to the following formulation to prepare an IκB kinase inhibitor-containing syrup.
Simple syrup 10 parts Carboxymethylcellulose 1 part γ-Mangosteen 0.3 part

The IκB kinase inhibitor obtained in Example 2 was formulated according to the following formulation to prepare an injection containing an IκB kinase inhibitor.
Chlorobutanol 0.5 parts Sodium chloride 0.9 parts Water for injection 80 parts IκB kinase inhibitor obtained in Example 2 20 parts

The IκB kinase inhibitor obtained in Example 5 was formulated according to the following formulation to prepare an IκB kinase inhibitor-containing ointment.
Beeswax 3 parts hydrogenated lanolin 8 parts squalane 34 parts solid paraffin 2 parts microcrystalline wax 9 parts white petrolatum 5 parts hexyldecyl adipate 13 parts sorbitan sesquioleate 1 part polyoxyethylene (50) hydrogenated castor oil 1 part glycerin 10 parts Ethanol 1 part Water 10 parts IκB kinase inhibitor 30 parts obtained in Example 5

The γ-mangosteen obtained in Example 6 was blended according to the following formulation to prepare an IκB kinase inhibitor-containing candy.
Granulated sugar 45 parts Minamata (DE 42) 50 parts Water 20 parts γ-Mangosteen 0.5 parts Lemon flavor 1 part

The IκB kinase inhibitor obtained in Example 2 was formulated according to the following formulation to prepare an IκB kinase inhibitor-containing chocolate.
Cocoa bitter 20 parts Cocoa butter 17 parts Sugar 43 parts Whole milk powder 20 parts IκB kinase inhibitor obtained in Example 1 1 part Vanilla flavor 0.1 parts

The IκB kinase inhibitor obtained in Example 3 was blended in the following formulation to prepare an IκB kinase inhibitor-containing chewing gum.
Gum base 20 parts Sugar 56 parts Minamata 13 parts Glucose 10 parts Softener 1 part IκB kinase inhibitor obtained in Example 3 0.5 parts Mint flavor 0.5 parts

The IκB kinase inhibitor obtained in Example 4 was formulated according to the following formulation to prepare an IκB kinase inhibitor-containing tablet confection.
Sugar 75 parts Glucose 19 parts Sucrose fatty acid ester 0.2 parts IκB kinase inhibitor obtained in Example 4 0.5 parts Water 4 parts

Claims (2)

  An IκB kinase inhibitor containing, as an active ingredient, at least one selected from an extract obtained by extraction from a fruit or skin of mangosteen (Garcinia mangostana L.) with a water-containing organic solvent or an organic solvent, α-mangostin, γ-mangostin .   A composition for inhibiting IκB kinase, comprising the IκB kinase inhibitor according to claim 1.