JPH0859461A - ACAT inhibitor - Google Patents

Abstract

PURPOSE: To obtain the ACAT inhibitor comprising osthol contained in Cnidium monnieri of an umbelliferous plant as an active ingredient. CONSTITUTION: This ACAT contains 1-95wt.% of osthol of the formula as an osthol contained in active ingredient. Osthol is extracted from Cnidium monnieri, belonging to the genus Cnidium, of an umbelliferous plant. Osthol specifically inhibits ACAT (acyl coenzyme A: cholesterol acyltransferase) of the small intestine, the liver and macrophage. The ACAT inhibitor is useful as an absorptior) inhibitor for cholesterol, arteriosclerosis or arteriosclerotic lesion caused by arteriosclerosis. The use of thc ACAT inhibiting substance can suppress absorption of cholesterol in a food from the small intestine, subdue reesterification cholesterol in the liver and lower blood cholesterol by catabolism and discharge promoting action by the inhibitions.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to an acyl coenzyme A: cholesterol acyl transferase (ACA).
T) The inhibitor, more specifically, A containing osthole contained in the fruit of Okazeli (snake bed) (snake bed) as an active ingredient.
It relates to CAT inhibitors.

[0002]

2. Description of the Related Art With the westernization of eating habits, the frequency of ischemic heart disease in Japan has significantly increased. The relationship between blood cholesterol levels and arteriosclerosis, which are elevated by Western diet,
It has been revealed in many epidemiological studies. That is, there is a significant correlation between the serum cholesterol level and the occurrence frequency of ischemic heart disease, and it is said that treating hypercholesterolemia prevents arteriosclerosis. Arteriosclerosis is a lesion characterized by intimal thickening due to lipid accumulation in blood vessels. Recently, macrophage-derived cells store cholesterol ester as lipid droplets in the lesion of atherosclerosis. End-cells are observed and are presumed to be deeply involved in the development of lesions.

On the other hand, acyl coenzyme A: cholesterol acyltransferase (ACAT) is a fatty acid esterifying enzyme of cholesterol, and an inhibitor of this enzyme is expected as a therapeutic drug for arteriosclerosis (Dra).
go R. et al. , Tips 194, (1
2), 1991). That is, it has been reported that ACAT activity is high in the blood vessel wall at the site of arteriosclerotic lesion, and cholesterol ester is accumulated in the blood vessel wall. Therefore, if a substance having an activity of suppressing ACAT activity is used, the esterification of cholesterol can be inhibited, and free cholesterol present in cells is removed from the deposition site by high-density lipoprotein (HDL) and is transported to the liver. Since it is exposed and metabolized, the accumulation of cholesterol ester at the lesion site is suppressed, and a direct anti-atherogenic effect can be obtained.

Cholesterol contained in food is esterified by ACAT in small intestinal mucosal cells and then released into the bloodstream as a component of chylomicrons. Therefore, when the ACAT inhibitor is used, blood cholesterol can be lowered by inhibiting the absorption of cholesterol in food in the intestinal epithelial cells, and as a result, arteriosclerosis can be suppressed.

Cholesterol ester carried to the liver by chylomicrons is decomposed into free cholesterol by cholesterol esterase, and then converted into cholesterol ester by ACAT together with free cholesterol synthesized in the liver, and ultra-low-density lipoprotein ( It is incorporated into VLDL) and released into the blood.
Therefore, if the esterification of cholesterol is blocked by an ACAT inhibitor, the amount of free cholesterol in the liver increases, the cholesterol synthesis system is suppressed, and 7α-, which is a rate-limiting enzyme in the process of converting cholesterol into bile acid. The hydroxylase activity is enhanced, and the catabolic excretion of cholesterol to bile acids is enhanced.

[0006] Thus, by using an ACAT inhibitor, inhibition of absorption of cholesterol in food from the small intestine,
By suppressing the re-esterification of cholesterol in the liver and further promoting the catabolic excretion of cholesterol, blood cholesterol can be lowered.

[0007] Conventionally, some naturally occurring ACAT inhibitors are known to be produced by microorganisms.
A plant-derived ACAT inhibitor has not yet been found.

[0008]

In such circumstances, the inventors of the present invention continued to search for an active ingredient having an ACAT activity-regulating action in the plant ingredients, and as a result, it was a plant of the Seriaceae family, Ocaseri ( Serpent's floor: Cnidium mo
It has been found that an alcohol extract of the fruit (nabieri) of Nnieri) has an ACAT inhibitory effect. Further, as a result of further isolating and purifying a substance having an ACAT inhibitory activity from the extract, the active substance is represented by the following formula (1)

[0009]

Embedded image

The present invention was completed by discovering that it is osthole represented by

That is, the present invention relates to an ACAT inhibitor containing osthole as an active ingredient.

[0012] The present invention also relates to an ACAT inhibitor containing an extract of Tortilis edulis as an active ingredient.

The osthole used in the present invention is
The origin is not particularly limited, and any of naturally derived, fully synthetic and semi-synthetic products can be used.

When a plant is used as the source of osthole, examples of the plant containing osthole include plants of the genus Spermaceae, and particularly fruits of Chinese okazeri (Cnidium monnieri) are preferable. Used for.

Okazeri is a perennial herb native to northeastern China and distributed in Mongolia, Siberia, Ussuri, and the Korean Peninsula. The dried fruit is called a serpentine. Serpentine is a traditional Chinese medicine used for the purpose of anti-inflammation, astringency, seizure, etc., and its extract has been reported to have antitrichomonal action, sex hormone-like action and the like. However, its ACAT inhibitory action has not been reported at all.

Extraction of the extract or osthole from the serpentine is performed as follows, for example. First, the extracted extract can be obtained by extracting the mussels with a polar organic solvent such as methanol, ethanol or acetone, and concentrating the obtained extract under reduced pressure. Here, this extract can be used as it is as an ACAT inhibitor, but if necessary, it is purified as follows and the purified product of each step is ACA.
It can also be used as a T inhibitor. For purification of the extracted extract, for example, the extracted extract is first distributed between ether and water, the ether layer is concentrated and overlaid on a silica gel column, and n-hexane, n-hexane: ethyl acetate (1:
Fractionation of the active ingredient is carried out by eluting sequentially with 1), 4 solvents of ethyl acetate and methanol. Then, the active fraction of the eluate is dried under reduced pressure to obtain a crude active substance. This was subjected to reversed-phase C ₁₈ column using high performance liquid chromatography and acetonitrile: water (80:50).
Ostol can be obtained by separating and purifying from the solvent system. If necessary, purification by preparative thin layer chromatography can be performed.

The thus-obtained extract of osthole or snake bed extract is A of the small intestine, liver and macrophage.
It specifically inhibits the activity of CAT. Therefore, the ACAT inhibitor of the present invention containing these as active ingredients is useful as a cholesterol absorption inhibitor, or a prophylactic / therapeutic agent for arteriosclerosis or arteriosclerotic lesions resulting therefrom.

[0018] Moreover, since the serpentine is conventionally used as a herbal medicine, and osthole is its component,
The ACAT inhibitor of the present invention has no problem in safety.

It is preferable that the osthole or the extract of Torex radix is orally administered alone or together with a pharmacologically acceptable carrier or the like. As oral preparations, for example, tablets,
Capsules, granules, powders, troches, syrups and the like can be mentioned. Tablets, capsules, granules, powders,
In solid preparations such as lozenges, in addition to osthole or extract of Tortilla radix, excipients such as starch, lactose, carboxymethyl cellulose, precipitated calcium carbonate;
Gum arabic, tragacanth gum, gelatin, methyl cellulose, carboxymethyl cellulose and other binders, alginic acid, corn starch and other disintegrants; magnesium stearate, hydrous silicon dioxide, light anhydrous silicic acid and other lubricants, saccharose and other sweeteners, menthol, etc. Other flavoring agents can be added. In liquid preparations such as syrups, sorbitol, gelatin, methylcellulose, vegetable oil, in addition to osthole or snake bed extract
In addition to emulsifiers and the like, sweeteners, flavoring agents, coloring agents and the like can be added. 1 to 95% by weight in these formulations
It is desirable to add the osthole.

[0020]

[Action]

(1) the effect on ACAT activity inhibitory action osthol rat-derived ACAT, using a crude enzyme prepared from the microsome protein fraction rat liver, ¹⁴
It was examined by in vitro reaction of C-oleoyl CoA as a substrate to produce ¹⁴ C-cholesteryl oleate from endogenous cholesterol.

The reaction solution composition was 20 μl of microsome protein.
White (1.83 mg / ml), 20 μl ¹⁴C-oleoyl C
oA (5 μCi / ml, BSA: 96 nmol / ml), 106
μl 100 mM phosphate buffer (5 mM dithiothreitol)
PH 7.4), and 4 μl of methanol or
Is a total of 1 of osthole dissolved in dimethyl sulfoxide.
The volume was 50 μl.

The reaction is carried out at 37 ° C., and after a predetermined reaction time (usually 18 minutes), 5.65 ml of chloroform: methanol (2: 1) is added to stop the reaction, and then 0.0
0.98 ml of a 4N hydrochloric acid aqueous solution and cholesteryl oleate as a carrier were added, and the mixture was extracted with a shaker for 20 minutes and then left overnight in a cool dark place. The next day, the total amount of the separated lower chloroform layer was extracted and dried under a nitrogen stream,
To the remaining upper layer, 5.65 ml of the lower layer of Holchi's partition solution was added and the mixture was extracted again. This was left overnight in a dark place, then the whole amount of the lower layer was extracted and combined with the previously dried chloroform lower layer fraction to dryness.

Next, this was dissolved in 200 μl of chloroform, and the entire amount was applied to a silica gel thin layer chromatography G60 plate (aluminum sheet). 1 with diethyl ether: petroleum ether (10: 190)
After developing 0 cm, autoradiography was performed to confirm the produced ¹⁴ C-cholesteryl oleate. Next, this portion was cut off, and the radioactivity was measured for 30 minutes in toluene by liquid scintillation counting for 5 minutes or by betascope (manufactured by Beta Gen). As a result of measuring the concentration at which this enzyme was inhibited by 50%, the IC ₅₀ value of osthole was 20 μM.

(2) Inhibitory Effect of Cholesterol Ester Accumulation on Macrophages The inhibitory effect of osthole on cholesterol macrophage accumulation on macrophages was examined using the J771.4 strain, which is a mouse macrophage-established cell.

The metabolism of LDL in endothelial cells and smooth muscle cells is caused by the excess intracellular free cholesterol.
Although down-regulation of the L receptor is involved, uptake of denatured LDL in macrophages is via the scavenger receptor. This pathway is non-legal and allows an unlimited accumulation of cholesterol esters.

Therefore, liposomes composed of ¹⁴ C-labeled cholesterol and phosphatidylcholine are incorporated into macrophage cells, and ACA present in the rough endoplasmic reticulum.
It was investigated whether or not osthole suppresses intracellular cholesterol ester accumulation by acting on T.

Macrophage cells were prepared with 10% fetal bovine serum,
Penicillin 50U / ml and streptomycin 50μg
RPMI 1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.)
Incubation was carried out for 2 days under the condition of 5% CO ₂ . Then, trypsin treatment is performed to remove the cells attached to the bottom surface of the well to obtain a cell suspension, and the cells are washed with the above fresh medium, and then the 24-well multiple well plate (Corning 25820-24). Each well was inoculated with 750 μl of a cell solution of 2 × 10 ⁶ cells / ml. After culturing at 37 ° C. under 5% CO ₂ for 37 hours, the medium was replaced with the fresh medium, and 80 μg of cholesterol (containing 0.4 μCi of ³ H-cholesterol) and 1 were added.
40 μl of a 0.3 M glucose solution containing 60 μg of phosphatidylcholine and 10 μl of a methanol solution in which osthole was dissolved were added, and the mixture was cultured for 12 hours. These operations were performed aseptically.

After completion of the culture, RPMI 1 containing no serum
The cells attached to the bottom surface were washed 5 times with a 640 medium using a Komagome pipette, and 1 ml of 2-propanol was added to extract intracellular cholesterol and produced cholesterol ester. The total amount of this extract was applied to silica gel thin-layer chromatography (Art. 5553, manufactured by Merck & Co., Inc.), and hexane: ether: formic acid (80:20:
After development in 2), autoradiography was performed. Next, the cholesterol ester and the cholesterol moiety labeled with ³ H were cut out, and the radioactivity was measured with a liquid scintillation counter to obtain the cholesterol ester accumulation inhibition rate as compared with the control. as a result,
The IC ₅₀ value for osthole was 20 μM.

[0029]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Production Example 1 500 g of a serpentine bark was cold-soaked and extracted with 10 liters of methanol for 1 week at room temperature. The extract was filtered, concentrated under reduced pressure and dried to obtain 38.4 g of crude extract. The crude extract was then partitioned between ether and water. The ether layer is concentrated and the silica gel column (internal diameter 5 cm, length 9
(0 cm), and the active ingredient was fractionated by sequentially eluting with 1 liter of each of four kinds of solvents of n-hexane, n-hexane: ethyl acetate (1: 1), ethyl acetate and methanol.

The eluted solution was collected in 150 ml portions, and the active fraction was dried under reduced pressure to obtain 4.85 g of crude active substance. Next, silica gel column chromatography (5 cmφ x 6
(0 cm) with the whole crude extract and elution with 600 ml of chloroform, and then 95% chloroform: methanol, 9
Active fractions were collected by sequentially eluting with 600 ml each of 0% chloroform: methanol and 80% chloroform: methanol. The amount of crude active substance obtained was 3.5 g.

Next, the total amount of this crude active substance was subjected to chromatography fractionation again by silica gel column chromatography (5 cmφ × 100 cm) using chloroform: methanol (90:10) as an eluent. Eluent is 150
The active fraction was dried under reduced pressure to obtain 2.6 g of crude active fraction.

1 g of this was subjected to high performance liquid chromatography (System 600E manufactured by Waters Co.) using a reversed phase C ₁₈ column and acetonitrile: water (80: 2).
As a result of separation and purification by the solvent system of 0), 0.53 g of a crude active fraction was obtained.

The component exhibiting this ACAT inhibitory activity is further purified by preparative thin layer chromatography (manufactured by Merck: silica gel 60F ₂₅₄ , Art. 5717) using n-hexane: ethyl acetate (3: 1) as a developing solvent. I went. Finally, the active ingredient was recrystallized in hexane-ethyl acetate to obtain 267 mg of colorless columnar crystals. Its purity is 95 as determined by high performance liquid chromatography.
% Or more.

The physicochemical properties of the active substance obtained as described above were as follows. (1) Molecular formula: C ₁₅ H ₁₆ O ₃ (2) Molecular weight: 244 (m / z 2 by mass spectrum)
44 (M ⁺ ) was observed. (3) Ultraviolet absorption spectrum (in ethanol): As shown in FIG. (4) Infrared absorption spectrum (in KBr): As shown in FIG. (5) Solubility in solvent: methanol, ethanol,
Soluble in acetonitrile, ethyl acetate, chloroform, benzene, insoluble in water. (6) Basic, acidic, and neutral: neutral. (7) Color and shape of substance: white, powder. (8) Proton nuclear magnetic resonance spectrum: As shown in FIG. ¹ H-NMR (DMSO-d ₆ , 400MHz, 37c) δ: 1.67 (3H, s), 1.84 (3H,
s), 3.54 (2H, d, J = 8Hz), 3.92 (3H, s), 5.22 (1H, t, J = 8Hz),
6.22 (1H, d, J = 8Hz), 6.83 (1H, d, J = 8Hz), 7.28 (1H, d, J = 12H
z), 7.60 (1H, d, J = 12Hz). (9) Chemical structure:

[0036]

[Chemical 3]

From the above, this active substance was identified as osthole.

Example 1 Tablets Ostol 40 g Lactose 360 g Talc stearate 40 g Starch 60 g The above ingredients were mixed in a mixer and compressed into 0.5 g tablets 100.
I got 0.

[0039]

EFFECT OF THE INVENTION Osthole specifically inhibits the activity of ACAT in the small intestine, liver and macrophages. Therefore,
The ACAT inhibitor of the present invention containing this as an active ingredient is useful as a cholesterol absorption inhibitor or a prophylactic / therapeutic agent for arteriosclerosis or arteriosclerotic lesions resulting therefrom.

[Brief description of drawings]

FIG. 1 is a view showing an ultraviolet absorption spectrum of a substance (osthole) showing an ACAT inhibitory activity extracted from Tortilis edulis.

FIG. 2 is a diagram showing an infrared absorption spectrum of a substance (osthole) having an ACAT inhibitory activity, which is extracted from Torpedo bark.

FIG. 3 is a diagram showing a proton nuclear magnetic resonance spectrum of a substance (osthole) having an ACAT inhibitory activity, which was extracted from Torpedo bark.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Tsunekazu Watanabe 1-1-19 Higashishinbashi, Minato-ku, Tokyo Yakult Honsha Co., Ltd. (72) Inventor Teruo Yokokura 1-1-1 Higashishinbashi, Minato-ku, Tokyo No. 19 Stock Company Yakult Head Office

Claims (4)

[Claims] 1. The following formula (1) An ACAT inhibitor containing osthole as an active ingredient represented by: 2. A containing the extract of Jingdoko as an active ingredient.
CAT inhibitor. 3. A cholesterol absorption inhibitor containing the ACAT inhibitor according to claim 1 or 2. 4. A preventive / therapeutic agent for arteriosclerosis containing the ACAT inhibitor according to claim 1 or 2.