JP2013241392A - Absorption inhibitor for cholesterol - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a medicine for inhibiting the absorption of cholesterol by binding with NPC1L1 and an improver of hypercholesterolemia.SOLUTION: There is provided an absorption inhibitor for cholesterol, obtained by formulating an extract of a carpophore or a cultured mycelium of a mushroom selected from Lycoperdon perlatum, Fomitopsis nigra, Hericium ramosum and Pholiota lubrica.

Description

  The present invention relates to a cholesterol absorption inhibitor.

Increased blood cholesterol is a major risk factor for coronary heart disease. In order to lower blood cholesterol, it is basically limited to intake of low cholesterol foods, bile acidic sequestrants (cholestyramine and colestipol), nicotinic acid (niacin), fibrates and probucol. It was. However, these drugs and therapies have limited treatment.
Recently, lovastatin, simvastatin and pravastatin (HMG-CoA reductase inhibitors) have been shown to delay the progression of atheromatous lesions in the coronary and carotid arteries. Furthermore, simvastatin and pravastatin were found to reduce the risk of coronary heart disease.

In arteriosclerotic diseases, cholesteryl esters are a major component of atherosclerotic lesions and a major storage form of cholesterol in arterial wall cells. The formation of this cholesteryl ester is also an important step in the intestinal absorption of dietary cholesterol.
It is known that cholesterol taken into small intestinal epithelial cells is converted into cholesteryl ester in the endoplasmic reticulum in small intestinal epithelial cells, and then formed into chylomicron and absorbed into the body. Therefore, it is speculated that the inhibition of cholesteryl ester formation prevents intestinal absorption of dietary cholesterol, and the concomitant reduction in blood cholesterol suppresses the progression of atheromatous lesion formation, preventing or improving arteriosclerosis. It is thought to be connected.

Also, certain hydroxy substituted azetidinones such as Ezetimibe (US Pat. No. 5,767,115) may be useful as cholesterol lowering agents in the treatment and prevention of atherosclerosis. Are known. Ezetimibe's cholesterol-lowering action is inhibited by the binding of ezetimibe with Niemann Pick Type C 1 like 1 (hereinafter referred to as NPC1L1), a membrane protein responsible for cholesterol absorption in the small intestine, from studies on knockout mice and transgenic mice Became clear (Patent Document 2: JP 2010-252798 A).
Therefore, a substance that inhibits the binding of ezetimibe and NPC1L1 is expected to inhibit cholesterol absorption in the gastrointestinal tract and improve atherosclerosis.

  Mushroom is a general term for fruiting bodies of basidiomycetes and ascomycetes. In recent years, substances having a cholesterol-lowering effect and those having an effect of preventing arteriosclerosis have been obtained from fruit bodies of mushrooms. Patent Document 3 discloses that mushrooms of the genus Pleurotus such as Pleurotus eryngi var. Ferulae and Pleurotus osutreatus produce lovastatin, which is a therapeutic agent for hypercholesterolemia (patent). Document 3: Japanese Translation of PCT International Publication No. 2003-520576). Further, Patent Document 4 discloses that basidiomycetous fungi, shiitake, jellyfish, maitake, and litchi promote secretion of adiponectin and show an effect on lowering cholesterol (Patent Document 4: Japanese Patent Application Laid-Open No. 2008-297256). Thus, it has been known for a long time that the fruiting bodies of specific species of mushrooms have the effect of lowering cholesterol and reducing arteriosclerosis.

  However, it is not known that a component contained in a specific mushroom binds to NPC1L1 protein and suppresses absorption of cholesterol.

US Pat. No. 5,767,115 JP 2010-252798 A Japanese translation of PCT publication No. 2003-520576 JP 2008-297256 A

  An object of the present invention is to provide a drug that binds to NPC1L1 and inhibits cholesterol absorption and an agent for improving hypercholesterolemia.

  As a result of investigations to solve the above problems, the present inventors have found that an extract of a specific mushroom fruit body is strongly bound to NPC1L1 involved in cholesterol absorption among a large number of mushrooms, thereby completing the present invention. It came.

The configuration of the present invention is as follows.
(1) Cholesterol characterized by containing a mushroom fruit body or cultured mycelium extract selected from Lycoperdon perlatum, Fomitopsis nigra, Hericium ramosum, Pholiota lubrica Absorption inhibitor.
(2) NPC1L1 characterized in that it contains a mushroom fruit body or cultured mycelium extract selected from Lycoperdon perlatum, Fomitopsis nigra, Hericium ramosum, Pholiota lubrica That inhibit the function of
(3) Hypercholesterolemia containing mushroom fruit body or cultured mycelium extract selected from Lycoperdon perlatum, Fomitopsis nigra, Hericium ramosum, and Phelliota lubrica as active ingredients Improver.

  According to the present invention, a cholesterol absorption inhibitor and a hypercholesterolemia improving agent are provided.

It is a figure which shows the food intake of the test animal of Test Example 2. It is a figure which shows the blood cholesterol level of the test animal of Test Example 2. It is a figure which shows the amount of excretion cholesterol in the feces of the test animal of Test Example 2.

  The mushrooms used in the present invention are Lycoperdon perlatum, Fomitopsis nigra, Hericium ramosum, Pholiota lubrica, and mushrooms are fruit bodies or mycelia or dried products thereof. It may be a whole or a part of a mushroom. Extracts can be easily produced by water, lower aliphatic alcohol (methanol, ethanol, isopropyl alcohol, etc.), lower aliphatic ketone (acetone, etc.), or a solvent containing these, by an extraction process usually performed by those skilled in the art. Can do. The produced extract can be made into a concentrated extract or a dried extract by a treatment such as heat treatment, freeze-drying or reduced-pressure drying.

  Each mushroom extract can be used as it is, but if necessary, any carrier (water-soluble vitamin, fat-soluble vitamin, vitamin derivative, mineral, herbal medicine) within the qualitative and quantitative range that does not impair the effects of the invention. Herbal extracts, organic acids, coenzymes, etc.).

  The inhibitor or preventive or ameliorating agent of the present invention can be administered orally in a dosage form such as a tablet, granule, powder, capsule, or liquid containing any commonly used optional ingredient. Examples of optional components include excipients, binders, coating agents, lubricants, sugar coatings, disintegrants, extenders, flavoring agents, emulsifying / solubilizing / dispersing agents, stabilizers, pH adjusting agents, and the like. Examples include tonicity agents. The inhibitor of this invention etc. can be manufactured by processing these according to a conventional method.

The dosage of the extract when used as a cholesterol absorption inhibitor or hypercholesterolemia-improving agent varies depending on age, sex, body weight, etc., but is usually about 50 mg to 100 g per day for an adult in terms of the original extract, preferably Is 500 mg to 100 g.
The agent includes not only pharmaceutical products but also quasi drugs and health foods.

Hereinafter, the present invention will be described more specifically with reference to production examples and test examples.
(Production example)
About 4 times the undried fruit bodies of Lycoperdon perlatum, Fomitopsis nigra, Hericium ramosum, and Phelliota lubrica collected from nature After immersing and extracting sequentially with ethanol (Wako) and 1 volume of acetone (Wako) at room temperature, the ethanol and acetone extracts were mixed, and the solvent was concentrated under reduced pressure using a rotary evaporator so that insolubles did not precipitate. Some were concentrated to dryness and used as the ethanol extractor. Next, 1 L of hexane was added to 500 ml of the concentrated liquid, and the liquid was partitioned using a separatory funnel. The recovered hexane phase was concentrated to dryness using a rotary evaporator to form a hexane extractor. In the same manner, the residue was further separated sequentially with 1 L of chloroform (Nacalai tesque), 1 L of ethyl acetate (Nacalai tesque), and 1 L of n-butanol (Nacalai tesque) to obtain each extraction unit. The last residue was lyophilized to form a water-soluble part. The above operation was repeated several times according to the amount of ethanol / acetone extract, and the whole amount of ethanol / acetone extract was processed.
Each extract was evaluated for the binding inhibitory activity of NPC1L1 and [ ³ H] Ezetimibe-glucuronide according to the test method shown below. As a result, 4.8 g (extract 1) of hexane extract from dust mushroom, 40.3 g (extract 2) of hexane from kurosarokoshitake, and 9.1 g of ethanol extract from coral agaric are extracted fractions having the desired inhibitory activity. 3) Chloroform extract part 43g (extract 4) was obtained from chanametsumutake.

(Test Example 1)
The various solvent extracts obtained in the production examples were tested to evaluate the binding inhibitory activity between NPC1L1 and ezetimibe by the following test method.
(1) Cloning of rat NPC1L1 gene Hind III, 3 'end at the 5' end by PCR using iProof High-Fidelity DNA polymerase (Biorad) using rat jejunum-derived single-stranded cDNA (Geno Staff Co., Ltd.) as a template. The full length of rNPC1L1 with Xba I added thereto was amplified and cloned into the pCR4 Blunt TOPO vector using the Zero Blunt TOPO PCR cloning kit (Invitrogen). The cloned DNA sequence is shown in SEQ ID NO: 1.
The primers and PCR conditions used for rNPC1L1 full-length amplification are as follows.
Sense: 5'- aagcttaccatggcagctgcctggctgggatggc-3 '(SEQ ID NO: 2)
Antisense: 5'- tctagattagaacttttggtcacttttgggc-3 '(SEQ ID NO: 3)

PCR conditions 1.98 ° C. for 30 seconds, 2.98 ° C. for 10 seconds, 3.68 ° C. for 30 seconds, 4.72 ° C. for 1.5 minutes, 5.72 ° C. for 10 minutes, 2 to 4 cycles are performed.

(2) Production of rat NPC1L1 stably expressing cells
pCR4 Blunt TOPO rNPC1L1 vector treated with Hind III and Xba I, agarose electrophoresis, rNPC1L1 extracted using QIAquick Gel Extraction Kit (QIAGEN), and Hind III and Xba I sites of expression vector pcDNA3.1 (Invitrogen) Subcloned into The sequence of the prepared pcDNA3.1 rNPC1L1 was confirmed using a premix sequence analysis service (Takara Bio).
Sequencer primer
Sense: 5'-cactgcttactggcttatcg-3 '(SEQ ID NO: 4)
Sense: 5'-gccttttatcagcgcagcttt-3 '(SEQ ID NO: 5)
Sense: 5'-aactctcaccccataccatc-3 '(SEQ ID NO: 6)
Sense: 5'-cagtacttccagaacaaccg-3 '(SEQ ID NO: 7)
Sense: 5'-ttctactcctacctgggtgt-3 '(SEQ ID NO: 8)
Sense: 5'-cttcttccgcaagatatacgc-3 '(SEQ ID NO: 9)
Sense: 5'-ccacagcggaacagtttcat-3 '(SEQ ID NO: 10)
Sense: 5'-tccctcatcaaccttgtcac-3 '(SEQ ID NO: 11)
Sense: 5'-gtccatgcttcttgctgatg-3 '(SEQ ID NO: 12)

  Subsequently, pcDNA3.1 rNPC1L1 was transfected into human kidney-derived HEK293 cells (ATCC) using Fugene HD (Roche), and selection was performed at a final concentration of 1 mg / ml G418-sulfate (Wako). Further, selection was performed by limiting dilution, and a single clone of HEK / rNPC1L1 stably expressing cells was obtained.

(3) Preparation of membrane fraction HEK293 / rNPC1L1 stably expressing cells are seeded on φ150 mmdish (Nunc), cultured in DMEM (Nacalai tesque) + 10% FBS (Gibco) until confluent, and final concentration of 4 mM Sodium Butyrate ( (Sigma Aldrich) Cells treated for 24 hours were collected using Cell Dissociation Buffer (Invitrogen). After centrifugation at 800 rpm for 3 minutes, the remaining cells were suspended in 20 mM HEPES / Tris Buffer (pH 7.4) + proteinase inhibitor (Roche) containing 8% Sucrose and disrupted using ultrasound. After centrifugation at 4000 rpm, 4 ° C. for 10 minutes, the supernatant was ultracentrifuged at 48000 rpm (125000 G), 4 ° C. for 3 hours, and the residue was collected as a membrane fraction. The membrane fraction was resuspended in 20 mM HEPES / Tris Buffer containing 160 mM NaCl and 5% Glycerol, and the protein concentration was determined by BCA assay (Thermo Scientific Pierce) and stored at −80 ° C. until use.

(4) Binding inhibition experiment
In a 96-well plate (Nunc), a final concentration of 25 nM [ ³ H] Ezetimibe-glucuronide (American Radiolabeled Chemicals, Inc.), 1.25 mg / ml HEK / rNPC1L1-derived membrane protein, test substances of each concentration were added to buffer A (26 mM NaHCO ₃ , 0.96mM NaH ₂ PO ₄ , 5mM HEPES, 5.5mM Glucose, 117mM NaCl, 5.4mM KCl, 0.03% Sodium taurocholate, 0.05% Digitonin, pH 7.4) in addition to a total volume of 30μl, react at room temperature for 1 hour I let you. The reaction solution was trapped in UniFilter-96 GF / C (PerkinElmer) using Harvester (PerkinElmer), and free [ ³ H] Ezetimibe-glucuronide was washed using MilliQ water (Millipore). After drying, 15 μl / well of Microscint-20 (PerkinElmer) was added, and the radioactivity of [ ³ H] Ezetimibe-glucuronide bound to rNPC1L1 was measured by Topcount (PerkinElmer).

(5) Results Dust hexane extract (extract 1) and chanametsumutake chloroform extract (extract 4) are dissolved in dimethyl sulfoxide (DMSO) to produce a 2-fold dilution series of 8 steps from a final concentration of 0.5 mg / ml. IC ₅₀ was calculated. Also, black Polyporaceae hexane extraction unit (Extract 2), Sango Hari bamboo ethanol extractor (extract 3) after similarly dissolved in DMSO, prepared 2-fold dilution series of eight steps from a final concentration 2 mg / ml IC ₅₀ Was calculated. The results are shown in Table 1 below.

  Each extract strongly suppressed the reaction of ezetimibe binding to NPC1L1. Therefore, similar to ezetimibe, it was suggested that it inhibits the function of NPC1L1 and prevents intestinal absorption of cholesterol.

(Test Example 2)
The therapeutic effect on hypercholesterolemia that occurs when a high-cholesterol-containing diet is ingested was confirmed using the hexane extract (extract 2).
1. Test animals After acclimatization for 1 week, body weights were measured, and 8-week-old Wistar male rats (Japan SLC) grouped by stratified continuous randomization were used for the experiment.
Hypercholesterolemia was induced by giving a high cholesterol diet (added with 1% cholesterol and 0.5% cholic acid based on CRF-1; Oriental Yeast Co., Ltd.). In order to confirm hypercholesterolemia, a normal group fed with a normal diet (CRF-1; Oriental Yeast Co., Ltd.) was set up.
The group setting is a control group in which corn oil is administered in a state in which a high cholesterol diet is induced in addition to a normal group fed with a normal diet, 0.3 mg / kg ezetimibe (Cosmo Bio Inc.) Control ezetimibe group administered with 50 mg / kg extract group 2 administered with 50 mg / kg of kurosarnokoshika hexane extract (extract 2) produced in the above production example, 150 mg / kg extract 2 administered 150 mg / kg A total of 6 groups, 2 groups of 500 mg / kg extract administered with 500 mg / kg, were set to 6 animals in each group.

2. Test method Regular diet, high-cholesterol diet, and water are allowed to freely take for 3 days, the test substance is dissolved in corn oil, once a day for 2 days (no administration on the first day) from the second day of the test, stomach tube Forcibly orally (2 ml / kg). The normal group and the control group were administered corn oil. The breeding was individual breeding (1 animal / cage), and food intake for 3 days was recorded. Further, on the second day from the start of administration of the test substance, the flooring (Palsoft; Oriental Yeast Co., Ltd.) was replaced, and the entire amount of feces was collected after the test was completed. The collected feces were extracted for lipid according to the Folch method and subjected to analysis. The blood was fasted for 2 hours after the final administration of the test substance (water was freely ingested) and then collected using a vacuum blood collection tube (Terumo Corporation) heparinized from the abdominal aorta under diethyl ether anesthesia. The collected blood was immediately centrifuged at 3,000 rpm for 15 minutes at 4 ° C., and the obtained plasma was stored at −80 ° C. until analysis. The amount of cholesterol in feces and plasma was measured using Cholesterol E Test Wako (Wako Pure Chemical Industries, Ltd.).

3. Test results All results are expressed as mean ± SE. Statistical analysis was performed on 5 groups with high cholesterol diet intake, excluding the normal group, using Excel Statistics 2010. Each measurement data was subjected to one-dimensional analysis of variance and Dunnett's multiple test. The significance level was less than 5% on both sides in all cases.
As a result of the test, there was no difference in the amount of food intake among the five groups fed with a high cholesterol diet (FIG. 1), and there was no change in body weight. From this, it was evaluated that induction of hypercholesterolemia was performed without any difference between groups.
With regard to the blood cholesterol lowering effect, a significant difference was observed in the positive control ezetimibe administration group and the closarno shiitake extract 500 mg / kg administration group compared with the plasma cholesterol in the control group, and the cholesterol lowering effect became clear ( Figure 2).
In addition, as a result of measuring fecal cholesterol, a significant increase was observed in the positive control ezetimibe administration group and the 500 mg / kg extract 2 administration group (FIG. 3).
From the above results, it has been clarified that the decrease in plasma cholesterol in the extract of kurosarnokoshika hexane (extract 2) is due to the cholesterol absorption inhibitory action as in the case of the positive control ezetimibe. Therefore, it became clear that the mushroom extract having the inhibitory action of NPC1L1 is useful as a therapeutic agent for improving hypercholesterolemia.

Claims (3)

  Cholesterol absorption inhibitor comprising a mushroom fruit body or cultured mycelium extract selected from Lycoperdon perlatum, Fomitopsis nigra, Hericium ramosum, and Pholiota lubrica .   The function of NPC1L1, characterized by the combination of mushroom fruit bodies or cultured mycelium extracts selected from Lycoperdon perlatum, Fomitopsis nigra, Hericium ramosum, Pholiota lubrica An inhibitor. A hypercholesterolemia-improving agent comprising a mushroom fruit body or a cultured mycelium extract selected from Lycoperdon perlatum, Fomitopsis nigra, Hericium ramosum, and Phoriota lubrica as an active ingredient.