JP2006248939A - Drug for preventing and treating fatty liver - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a drug effective for preventing and treating fatty liver. <P>SOLUTION: The drug for preventing and treating fatty liver contains a processed product of Allium sativum L. , for example, a powder of Allium sativum L. in a dried form as an active ingredient. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a medicament for the prevention and / or treatment of fatty liver.

  The dry powder extracted from garlic has been confirmed to have medicinal effects such as tonicity, healthy stomach, bowel management, and fatigue recovery. In addition, components such as allicin, ajoene, and diallyl disulfide (DADS) contained in garlic inhibit Bcl-2 expression and induce Bax expression, and enhance apoptosis by increasing caspase-3 activity (Exp. Mol. Med. 32 (3), pp. 127-34, 2000; Leukemia, 16 (1), pp. 74-83, 2002), contribution to iron homeostasis by inducing ferritin and transferrin receptor expression (J. Nutr. , 132 (12), pp. 3638-41, 2002), and lipid lowering action by inducing the expression of the microsomal triglyceride transfer protein gene (J. Nutr., 132 (6), pp. 1165-8, 2001) It has been reported. However, there is no previous report regarding processed garlic products such as garlic extract showing effectiveness in the prevention and treatment of fatty liver.

On the other hand, Hsp (heat shock protein) is a group of proteins that normally folds functional proteins existing in the body, and is involved in the repair and protection of biomolecules caused by various stresses. (Physiol. Rev., 72 (4), pp.1063-81, 1992; J. Cell Sci., 104, pp.11-17, 1993; J. Neurosci., 19 (23) , pp. 10338-47, 1999). Among them, Hsp40 folds proteins in cooperation with Hsp70, but plays an important role as a molecule that regulates the activity at that time. In addition, Hsp40 has been reported to reduce the damage caused by carbon tetrachloride under high expression in rats treated with carbon tetrachloride (oxidative stress load), a fatty liver model (Cell Stress Chaperones., 9 (1 ), pp.58-68, 2004), it is considered that prevention and treatment of fatty liver can be achieved by activating the expression of this gene. There has been no report on the effect of processed garlic on Hsp40.
Exp. Mol. Med. 32 (3), pp.127-34, 2000 Leukemia, 16 (1), pp.74-83, 2002 J. Nutr., 132 (12), pp.3638-41, 2002 J. Nutr., 132 (6), pp.1165-8, 2001 Physiol. Rev., 72 (4), pp.1063-81, 1992 J. Cell Sci., 104, pp.11-17, 1993 J. Neurosci., 19 (23), pp. 10338-47, 1999 Cell Stress Chaperones., 9 (1), pp.58-68, 2004

  An object of the present invention is to provide a medicament for the prevention and / or treatment of fatty liver.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that processed garlic products such as garlic extract have the action of promoting the expression of the gene product of Hsp40. Or it was found useful for treatment. The present invention has been completed based on the above findings.

That is, according to the present invention, there is provided a medicament for preventing and / or treating fatty liver, comprising a processed garlic product as an active ingredient.
According to a preferred embodiment of the present invention, there is provided the above medicament, wherein the processed garlic product is a garlic extract; and the above medicament, wherein the processed garlic product is garlic powder in a dry form.

From another aspect, use of a processed garlic product for the manufacture of the above-mentioned medicament; and a method for preventing and / or treating fatty liver, wherein an effective amount of the processed garlic product is administered to a mammal including humans A method comprising the steps is provided.
Furthermore, an Hsp40 expression promoter containing processed garlic as an active ingredient, a method of promoting Hsp40 expression in a mammal including human, wherein an effective amount of the processed garlic is administered to a mammal including human A method comprising the steps is provided by the present invention.

  The medicament of the present invention has an action of promoting the expression of Hsp40 gene and is useful for the prevention and / or treatment of fatty liver.

  The kind of processed garlic used as the active ingredient of the pharmaceutical of the present invention is not particularly limited, and any garlic processed product may be used as long as it is obtained by processing bulbs of Allium sativuml. . In this specification, the term “processing” means that raw garlic is subjected to some processing such as heating, drying, grinding, or extraction, but the type of processing is not particularly limited, and in any sense This term should not be interpreted in a limited way.

  Processing includes, for example, a process of drying raw garlic or powdering after drying, a process of steam distillation of raw garlic, a process of extracting raw garlic with oil, water, hot water, a water-soluble organic solvent, or the like, or A process of heating raw garlic is included, and a process of extracting with a water-soluble organic solvent is particularly preferable. Examples of oils used for extraction include edible vegetable oils such as rapeseed oil, olive oil, and soybean oil. Examples of water-soluble organic solvents include lower alcohols such as ethanol or isopropanol; or glycols such as propylene glycol or diethylene glycol. Is mentioned.

  As the garlic processed product, for example, processed potato, garlic extract, garlic extract, or dried garlic are preferable, and processed potato is particularly preferable. Processed daikon means garlic powder or extract prepared by subjecting a heat-treated garlic extract to a lower alcohol extraction or the like. For example, oxoamidin (registered trademark) (manufactured by Riken Chemical Industry Co., Ltd.), oxoamidin (registered) Trademark) powder (manufactured by Riken Chemical Industry Co., Ltd.), oxoresin (registered trademark) (manufactured by Riken Chemical Industry Co., Ltd.), or oxoresin (registered trademark) powder (manufactured by Riken Chemical Industry Co., Ltd.) are commercially available. As the garlic extract, for example, garlic extract (manufactured by Alps Pharmaceutical Co., Ltd.) or garlic extract (manufactured by Nippon Powder Chemical Co., Ltd.) is commercially available. As dried garlic, for example, garlic powder or roasted garlic powder EX (manufactured by Riken Chemical Industry Co., Ltd.) is commercially available. Among these garlic processed products, oxoamidin (registered trademark) (manufactured by Riken Chemical Industry Co., Ltd.), oxoamidin (registered trademark) powder (manufactured by Riken Chemical Industry Co., Ltd.), oxoresin (registered trademark) (manufactured by Riken Chemical Industry Co., Ltd.) Or, Oxo Resin (registered trademark) powder (manufactured by Riken Chemical Industry Co., Ltd.) is preferred.

  The processed product of garlic, which is an active ingredient of the medicament of the present invention, has the effect of significantly promoting the expression of Hsp40 among heat shock proteins (Hsp, heat shock protein), as specifically shown in the following examples. ing. With regard to Hsp40, it has been reported that carbon tetrachloride-treated rats (oxidative stress load), which is a model of fatty liver, alleviates impaired changes due to carbon tetrachloride under high expression (Cell Stress Chaperones., 9 (1 ), pp. 58-68, 2004), the pharmaceutical of the present invention can exert a preventive and / or therapeutic effect on fatty liver by promoting the expression of this gene.

  As the medicament of the present invention, the above processed garlic product, which is an active ingredient, may be administered as it is, but a pharmaceutical composition containing one or more pharmaceutical additives together with the processed garlic product is prepared and administered. May be. The medicament of the present invention is usually preferably administered orally, but when a garlic extract is used as a processed garlic product, it can be administered parenterally such as intravenous administration or rectal administration. It is also possible to prepare a pharmaceutical composition in the form and administer it parenterally.

  The pharmaceutical composition suitable for oral administration may be either a solid or liquid pharmaceutical composition. The solid pharmaceutical composition for oral use is, for example, an excipient added to the processed product of garlic, which is an active ingredient, and if necessary, for preparations such as binders, disintegrants, lubricants, coloring agents, or flavoring agents. After adding an additive, it can prepare as a tablet, a granule, a powder, a capsule by a conventional method. As a pharmaceutical additive, those commonly used in the art can be used. For example, excipients such as lactose, sodium chloride, glucose, starch, microcrystalline cellulose, silicic acid; binders such as water, ethanol, propanol, simple syrup, gelatin solution, hydroxypropylcellulose, methylcellulose, ethylcellulose, shellac, polyvinylpyrrolidone Disintegrants such as agar powder, sodium lauryl sulfate, stearic acid monoglyceride; lubricants such as purified talc, stearate, borax, polyethylene glycol; colorants such as β-carotene, yellow ferric oxide, caramel; and Examples include flavoring agents such as sucrose and orange peel.

  Oral liquid pharmaceutical compositions are prepared by adding one or more additives for pharmaceutical preparation such as a corrigent, stabilizer, or preservative to processed garlic, which is an active ingredient. It can be prepared as an elixir. As a pharmaceutical additive, those commonly used in the art can be used. Examples include flavoring agents such as sucrose; stabilizers such as tragacanth; and preservatives such as paraoxybenzoic acid esters.

  Examples of the pharmaceutical composition suitable for parenteral administration include dosage forms such as injections, drops, suppositories, transdermal absorption agents, transmucosal absorption agents, and the like. For injections, for example, one or more of pharmaceutical additives such as stabilizers or tonicity agents are added to an aqueous garlic extract, and are used for subcutaneous, intramuscular, or intravenous administration by conventional methods. It can be manufactured as an injection. As a pharmaceutical additive, those commonly used in the art can be used. For example, stabilizers such as sodium pyrosulfite; and isotonic agents such as sodium chloride can be exemplified. Suppositories can be mentioned as pharmaceutical compositions for rectal administration. The suppository can be produced by a conventional method by adding additives for pharmaceutical preparation such as a carrier and a surfactant to the processed garlic. As a pharmaceutical additive, those commonly used in the art can be used. Examples thereof include carriers such as polyethylene glycol and hard fat; and surfactants such as polysorbate 80.

  The dosage of the medicament of the present invention is not particularly limited, and can be appropriately selected depending on the age, weight, and symptoms of the patient, the dosage form, the administration route, the number of administrations, and the like. Usually, about 0.1 mg to 10 g per day can be administered as the mass of the processed garlic. The medicament of the present invention can be administered orally or parenterally once a day or several times a day, but the number of administrations can also be appropriately selected.

Example 1
(1) Method
(a) Test substance:
Oxoamidin powder (registered trademark, manufactured by Riken Chemical Industry Co., Ltd.)
Medium: Saline, concentration: 5 mg / mL
(b) Animal experiment Rats fasted overnight were given a single oral dose of oxoamidin powder to 100 mg / kg (for control animals, a single oral dose of vehicle). After 24 hours, each animal was decapitated without anesthesia, and then the liver was removed and frozen in liquid nitrogen. The frozen liver was stored at −80 ° C. until the next operation.

(c) Total RNA extraction and fluorescent labeling
For extraction of total RNA, SV total RNA isolation system (registered trademark, manufactured by Promega) was used. That is, weigh about 30 mg of frozen sample, add 175 μL of SV RNA Lysis Buffer, homogenize the liver with ultrasonic treatment for 30 minutes and injection needle (22G), add 350 μL of SV RNA Dilution Buffer, and mix by inverting. It was. After incubating at 70 ° C. for 2 minutes, centrifugation was performed twice at 10000 × g and 4 ° C. for 10 minutes, and the supernatant was collected. After adding 200 μL of ice-cooled 95% ethanol and mixing by pipetting, the solution was transferred to a Spin Column Assembly consisting of Spin Basket and Collection tube, and centrifuged at 10000 × g and 4 ° C. for 1 minute. After adding 600 μL of SV RNA Wash Solution to Spin Column Assembly, washing was performed by centrifugation at 10,000 × g and 4 ° C. for 1 minute. Add the DNase I reaction solution (including Yellow Core Buffer 40 μL, MnCl ₂ 5 μL, DNase I 5 μL) prepared in advance to the Spin Column Assembly, let stand at room temperature for 5 minutes, and then add 200 μL of SV DNase Stop Solution to the Spin Column Assembly. And centrifuged at 10000 × g and 4 ° C. for 1 minute. After washing by adding 250 μL of SV RNA Wash Solution, 100 μL of Nuclease-Free Water was added, and total RNA was eluted by centrifuging at 10000 × g and 4 ° C. for 2 minutes.

  Using 400 ng of the total RNA obtained, labeling was performed using the Low RNA Input linear amplification & labeling kit. That is, 400 ng of total RNA, 1.2 μL of T7 promoter primer, and Nuclease-free water were mixed to make a total of 11.5 μL, then incubated at 65 ° C. for 10 minutes, ice-cooled for 5 minutes, and preheated at 65 ° C. 4 μL of 5 × First strand buffer, 2 μL of 0.1 M DTT, 1 μL of 10 mM dNTP mix, 1 μL of MMLV-RT and 0.5 μL of RNaseOUT were added and incubated at 40 ° C. for 2 hours. The reaction was stopped by incubating at 65 ° C. for 10 minutes, followed by ice cooling for 5 minutes. After ice cooling, 15.3 μL of Nuclease-free water, 20 μL of 4 × Transcription buffer, 6 μL of 0.1 M DTT, 8 μL of NTP mix, 6.4 μL of 50% PEG preheated at 40 ° C., 0.5 μL of RNaseOUT, Inorganic 0.6 μL pyrophosphatase and 0.8 μL T7 RNA polymerase were added and mixed. After mixing, 2.4 μL of 10 mM Cyanine 5-CTP (hereinafter Cy5) for RNA derived from the liver of animals administered with oxoamidin powder, and 10 mM Cyanine 3-CTP (hereinafter referred to as Cy3) for RNA derived from the liver of animals administered with vehicle 2.4 μL was added and incubated at 40 ° C. for 2 hours in the dark.

  After completion of the reaction, the reaction product (labeled cRNA) was purified using RNeasy mini (manufactured by QIAGEN). That is, 350 μL of RLT buffer and 250 μL of ethanol were added, mixed, applied to a column, and centrifuged at 10,000 × g for 30 seconds. The column was washed by adding 500 μL of RPE buffer to the column and centrifuging at 10,000 × g for 30 seconds. This washing operation was performed twice. 30 μL of RNase-free water was added, allowed to stand for 1 minute, and then centrifuged at 10000 × g for 30 seconds to elute the labeled cRNA. This elution operation was performed twice. Using the obtained labeled cRNA, hybridization with rat microarray oligo (Agilent, hereinafter referred to as rat oligo) using In situ hybridization kit plus (Agilent) was performed. That is, 0.75 μg of Cy3 labeled cRNA, 0.75 μg of Cy5 labeled cRNA, 50 μL of 10 × control target and nuclease-free water were mixed to make a total of 215 μL, 2 × target solution 215 μL, 25 × fragmentation buffer 9 μL were added. Incubated at 60 ° C. for 30 minutes in the dark. After stopping the reaction by adding 225 μL of 2 × hybridization buffer, it was applied to the rat oligo in the microarray chamber, and hybridization was performed at 60 ° C. for 17 hours. After completion of the reaction, the rat oligo was washed with 6 × SSC containing 0.005% TritonX-102 and 0.1 × SSC containing 0.005% TritonX-102 and dried with nitrogen gas. The dried rat oligo was subjected to a microarray scanner (manufactured by Agilent) to capture an image, and then imaging of a spot and quantification of signal intensity were performed using imaging software Feature Extraction. The obtained numerical values were analyzed with Microsoft EXCEL.

(2) Results and Discussion Among the probes mounted on the rat oligo, control probes and probes with low reliability of measurement values due to noise were removed, and the remaining 20,000 genes were analyzed. As a result, 1464 genes showed an increase in expression level more than 2-fold compared to vehicle administration by administration of oxoamidin powder, and 1714 gene decreased expression to 1/2 or less by administration of oxoamidin powder. Met. Among these, 1113 genes increased in expression and 1222 genes decreased in the known function genes. Genes with increased expression include rat DnaJ (Hsp40) homologue, and the amount of increased expression of this gene was 3.7 times that of vehicle administration (Table 1). Hsp40 has been reported to reduce the impaired changes caused by carbon tetrachloride under high expression in rats treated with carbon tetrachloride (oxidative stress load), a fatty liver model. It is thought to have a protective effect on the liver. From the above results, it was concluded that administration of oxoamidin powder resulted in high expression of Hsp40 in vivo, and the effect of preventing and / or treating fatty liver was exhibited.

Claims (3)

A medicament for the prevention and / or treatment of fatty liver, comprising a processed garlic product as an active ingredient. The medicament according to claim 1, wherein the processed garlic is a garlic extract. The medicament according to claim 1, wherein the processed garlic is garlic powder in a dry form.