JPS62132878A - Novel physiologically active substance monacolin m and production thereof - Google Patents

Abstract

NEW MATERIAL:A compound expressed by the formula. USE:A blood cholesterol lowering agent, hypolipemic agent and antiarteriosclerotic agent. PREPARATION:A microorganism, belonging to the genus Monascus and capable of producing monacolin M, e.g. Monascus ruber 1005 strain., is cultivated in a culture medium containing glucose, starch, peptone, meat extract, soybean flour, etc., as nutrient sources, by an aerobic culture method at 20-35 deg.C to afford the aimed monacolin M.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a new physiologically active substance having a cholesterol-lowering effect and a method for producing the same.

[Conventional technology]

As a substance that has a blood cholesterol lowering effect, for example, dihydromonacolin L produced by the microorganism Monascus loupel is known (Japanese Patent Application Laid-Open No. 1094-1094).
80).

[Problems to be solved by the present invention]

Regarding blood cholesterol lowering agents, the emergence of active ingredients with strong medicinal efficacy and weak side effects is expected.

[Means for solving problems]

As a result of various studies, the present inventors found that microorganisms belonging to the genus Monascus have a blood cholesterol-lowering effect? It was discovered that monacolin M, a new physiologically active substance represented by the following formula (1), was produced.

The present invention was completed based on the above findings.

Next, the physical and chemical properties of Monacolin M will be described.

Monacolin M is a white crystalline powder that contains lower alcohols such as methanol, ethanol, propatool, acetone,
Soluble in chloroform, ethyl acetate, benzene, etc., but insoluble in hexane, petroleum ether, etc. This substance is a neutral substance and does not dissolve in neutral or acidic water, but by normal alkali treatment, the lactone structure is converted into a ring-opened acidic substance and is transferred to water. This acidic substance is extracted with ethyl acetate, chloroform, etc. in an acidic pH region, and reconverted to monacolin M by drying.

Elemental analysis values for Monacolin M are 67.96% carbon and 8% hydrogen.
．． 43%, oxygen 23.61%.

The molecular weight is 406.5 and the molecular formula is C23H3<Os.

The wavelength showing the absorption peak in the infrared absorption spectrum (c
m-') is as follows.

3386.2962.2927.2871, 2856°1734, 1716. 1703. 1458. 1
375°1261.866.800° Also, the NMR spectrum diagram is shown in FIG.

Thin layer chromatography using silica gel (Merck & Co., Ltd., A 5715, Kieselgel 60 F254, using hexane-acetone (4:1) as the developing solvent gives a single sporatra with an Rf value of 0.4. In addition, this spot is detected by sulfuric acid spray (color develops reddish brown with mild heating) and iodine.

Monacolin M of the present invention can be obtained by culturing a monacolin M producing bacterium belonging to the genus Monascus, and then extracting monacolin M from the culture.
It can be obtained by collecting. A typical example of Monacolin M-producing bacteria is Monascus rubel 1oo.
S strain (Feikoken Bacillus fI4822) is mentioned.

This strain is a known strain described in JP-A-55-111790.

Monacolin M is produced aerobically in a culture using a bacterial strain that produces monacolin M by a method known as a mold culture method. For example, monacolin M producing bacteria are subcultured on a medium consisting of 2% soluble starch, 1% glucose, 2% peptone, and 2% agar, and for monacolin M production, the grown bacterial cells on this agar medium are directly inoculated into the production medium. can be cultured. Furthermore, the bacterial cells grown on the production medium can be cultured on a new production medium, and Monacolin M can be produced there.

Monacolin M-producing bacteria grow at 7 to 40°C, but 20 to 35°C is usually preferred for monacolin M production. To cultivate Monascus bacteria that produces Monacolin M,
All known nutrient sources for culturing mold and other microorganisms can be used to kill them. For example, glucose, malto-, kistrin, starch, lactose, sucrose, glycerin, etc. can be used as carbon sources. Among these carbon sources, glucose and starch are the preferred carbon sources for monacolin M production.

All known sources of nitrogen for the growth of Monascus and other microorganisms to produce Monacolin M can be used. For example, peptone, meat extract, yeast, yeast extract, soy flour,
Peanut flour, corn steep liquor, rice bran, inorganic nitrogen sources, etc. can be used. When producing monacolin M by culturing monacolin M-producing bacteria, inorganic salts and metal salts are added if necessary. In addition, trace amounts of heavy metals can be added when necessary. Monacolin M can be obtained by aerobically culturing its producing bacteria, but commonly used aerobic culture methods such as solid state culture and shaking culture are used. , an aerated agitation culture method is used. When antifoaming is required during culture or medium sterilization, use silicone oil,
Antifoaming agents such as surfactants can be used. Culture temperature is 20~
35°C is preferred.

The culture was continued until monacolin M was substantially accumulated, and the extraction of this substance from the culture was performed as shown in the Examples below (
Depending on the properties of the present substance revealed by the present inventors, various methods can be suitably combined. That is, for example, extraction with ether, ethyl acetate, chloroform, benzene, etc., dissolution in a highly polar solvent such as acetone or alcohol, removal of impurities with a less polar solvent such as petroleum ether or hexane, gel filtration with a Sephadex column, activated carbon, This includes adsorption chromatography using silica gel and the like. By using a suitable combination of these means, the substance can be isolated from the culture as a homogeneous substance.

[For production]

The physiological activity of Monacolin M can be assayed by the following method, which examines its blood cholesterol lowering effect on rats. That is, 5 rats per group received Triton WR-1339.
(Brand name) (This substance has the effect of increasing blood cholesterol levels) 400mg/kg was injected, and at the same time Monacolin M100/kg was orally administered, and 14 hours later the blood was killed by exsanguination. Measure (
administration group). - Rats intravenously injected with 10,000 Tridon WR-1339 alone were treated in the same manner to measure blood cholesterol (control group). By comparing the cholesterol values of both groups, the effect of Monacolin M can be quantitatively determined.

As a result, the blood cholesterol level decreased by 73 (34.2%) when Monacolin M was administered compared to when only Triton WR-1339 was intravenously injected.

In addition, the acute toxicity (LD) of monacolin M when administered orally to mice
so) has low toxicity of more than Ig/kg.

〔effect〕

As described above, monacolin M has the effect of lowering blood cholesterol levels and can be used in medicine, for example, as an antilipidemic agent or an antiarteriosclerotic agent.

These compounds can be administered orally or parenterally, for example, in the form of capsules, tablets, injections, and the like. Oral preparations are usually preferred. The dosage varies depending on age, symptoms, body weight, etc., but it is usually about 50 to 500 doses per day for adults.
(2) is administered in 1 to 3 divided doses. However, larger amounts can be used if necessary.

〔Example〕

Glucose 3%, Beftone 0.8%, Meat extract 3%, Glycerin 7%, NaNO 30.2%, Mg S 04.
Monascus ruberum 1005 strain was inoculated into a liquid medium containing 1% 7H200 and cultured aerobically at a temperature of 25° C. for 10 days. 10A of the filtrate from which bacterial cells were separated from the culture solution was
5 after adjusting pH to 6 with N-HCI! of dichloromethane, and the aqueous layer was adjusted to pH with 6N HCl and a trace amount of trifluoroacetic acid.
6 as 2! The mixture was extracted with ethyl acetate.

The ethyl acetate layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, washed with an equal amount of 5% NaHCOa, and further concentrated to dryness.

The dried product was subjected to silica gel chromatography (gradient elution of hexane-acetone=95:5→70:30), and an effective fraction was collected. The dried product concentrated from the effective one column is subjected to preparative high performance liquid chromatography (column:
ODS, eluent niacetonitrile-water = 45:55
) to obtain an effective fraction, which was extracted with ethyl acetate under acidic conditions, dried with anhydrous sulfuric acid, and dried over IJum, followed by drying under reduced pressure to obtain crude monacolin M8. This was further purified by analytical high performance liquid/body chromatography (column: silica gel, solvent dichloromethane-isopropanol = 97:3) to obtain 2 wg of a purified product of Monacolin M.

[Brief explanation of drawings]

Figure 1 shows the 'H-NMR spectrum of the compound of the present invention (2
00MHz, CDC1g).

Claims (2)

[Claims] (1) Formula ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ Monacolin M is a new physiologically active substance expressed by (2) A method for producing monacolin M, which comprises culturing monacolin M-producing bacteria belonging to the genus Monascus and collecting monacolin M from the culture.