DK149095B - PROCEDURE FOR PREPARING A CHOLESTEROL SYNTHESIS INHIBITIVE COMPOUND called MONACOLIN K - Google Patents

Description

in 149055

The present invention relates to a process for preparing a novel cholesterol synthesis inhibitory compound called monacoline K of the formula: HO'y '-' Y0 6 which is characterized in that a strain of Monascus rubs is grown in a culture substrate containing assimilable carbon and nitrogen sources, whereafter monacoline K is isolated from the culture mixture.

High blood cholesterol levels are well known to be one of the major causes of cardiopathy, such as heart failure or arteriosclerosis. As a result, numerous scientific studies have been conducted to find physiologically acceptable compounds capable of inhibiting cholesterol biosynthesis and thereby reducing blood cholesterol levels. One of these compounds is ML-236, which is disclosed in English Patent No. 1,453,425 and in Danish Patent No. 136,485. ML-236 is produced by growing a microorganism of the genus Penicillium.

It has now been found that strains of the species Monascus rubber, especially Monascus rubber strain 1005 (FERM 4822), produce a closely related compound with ML-236 that has significantly better efficacy than ML-236.

Monascus ruber strain 1005 (FERM 4822) is a recently isolated microorganism exhibiting the following microbiological properties. It was isolated from feed produced in Thailand and deposited on February 16, 1979 under the accession number FERM 4822 in the Fermentation Research Institute, Agency of Industrial Science 2 149095 and Technology, Ministry of International Trade and Industry, Japan, and under the accession number NRRL 12073 in the Agricultural Research Service , Northern Regional Research Laboratory, USA.

1. Growth The growth of a potato-glucose-agar substrate at 25 ° C is rapid and the diameter of the colonies reaches 6 - 6.5 centimeters 10 days after seeding. The colony is flat and a relatively thin basal layer of hyphae develops. Airline development is poor; the air hyphae are white and most of them are woolly. Kleistotesia form on the basal layer of the hyphae and they turn reddish brown upon maturation. Both the surface and the back of the colony are brown to reddish brown in color.

The growth of Sabouraud's agar substrate at 25 ° C is very rapid and the diameter of the colonies reaches 6 - 6.5 centimeters 10 days after sowing. The surface of the colony is very flat and basal hyphae and aerial hyphae develop better than on the potato-glucose-agar substrate. Kleistotesia is very sparse. The surface of the colonies is reddish-yellow to reddish-brown in color, and the reverse is reddish-brown to dark brown.

The growth of oatmeal agar at 25 ° C is slow and the diameter of the colonies reaches 1.5 - 2 centimeters 10 days after sowing.

The colony is flat. The development of air hyphae and the formation of cleistothecia are very poor for both. Both the surface and the opposite side of the colony are dark red to reddish-brown in color.

The growth of Czapek's agar substrate at 25 ° C is very slow and the diameter of the colonies reaches 1.6 - 1.8 centimeters 10 days after seeding.

The growth rates of each of the above substrates at 37 ° C are substantially equal to the growth rates at 25 ° C.

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2. MORPHOLOGICAL PROPERTIES

Cleistotesia are spherical and 30 - 60 microns in diameter; their walls are thin and membranous; their stems have partitions, and each consists of a hypha - with a diameter of 3> 5 - 4.5 microns and a length of 15 - 80 microns. The spore bag consists of 8 spores and it is almost spherical and disappears quickly. The ascospores are colorless and ovoid or ellipsoid; they have a size of 4-5x4-7 microns; and their surface is smooth. Conidia are colorless and spherical or bulbous; their size is 6 - 9 x 6 - 11 microns; their base is just cut off and their walls are relatively-thick and smooth. Conidia is linked mid-point floating to each other as a kind of formation tissue arthrospore. The conidiophore is like a vegetative hyphae and is branched or unbranched; Conidia forms at the top. The mycelia are colorless and branched and have partitions; most of them have a diameter of 3 - 5 microns.

Based on these observations of the properties, the microorganism was identified as a strain of Monascus ruber van Tieghem.

The microbiological properties of Monascus rubs are reported in the following literature: Takada, Transactions of the Micological Society of Japan, 9, 125-130 (1969) [Materials for the Fungus Flora of Japan (7)], and van Tieghem, Bull. Soc.Botan.France, 31. 227 (1884). Ascospore formation of the species is discussed by Cole et al in the Canadian Journal of Botany, 46, 987 (1968), "Conidium ontogeny in hyphomyceres. The imperfect state οι: Monascus ruber and its meristem arthrospores".

Other strains of the species Monascus rubs capable of producing monacoline K can be used in the process of the invention.

In cultivation, methods well known to those skilled in the art of cultivating fungi and other microorganisms are used. Eg. the monacoline K-producing microorganism may first be grown on a suitable substrate, and then the microorganisms produced may be collected and seeded and grown in another growth substrate to produce the desired monacoline K; the culture substrate 4 149095 used for propagating the microorganisms and for the production of monacoline K may be the same or different.

Any culture substrate known to those skilled in the art of growing mushrooms may be used, provided that, as is known, it contains the necessary nutrients, in particular a source of assimilable carbon and a source of assimilable nitrogen. Examples of suitable sources of assimilable carbon are glucose, maltose, dextrin, starch, lactose, sucrose and glycerol. Examples of suitable sources of assimilable nitrogen are peptone, meat extract, yeast, yeast extract, soybean meal, peanut flour, corn mold liquid, rice husks and inorganic nitrogen. Of these nitrogen sources, peptone is particularly preferred. When producing monacoline K, an inorganic salt and / or a metal salt may optionally be added to the culture substrate. Further, a smaller amount of heavy metal can also be added.

Preferably, the microorganism is grown under aerobic conditions, using cultivation methods well known to those of skill in the art, e.g. solid cultures, shaking cultures or cultures under aeration and stirring. The microorganisms grow within a wide temperature range, e.g. from 7 to 40 ° C, but especially for the formation of monacoline K, the preferred culture temperature is in the range 20 to 35 ° C.

During the cultivation of the microorganisms, the production of monacoline K can be controlled by sampling the culture substrate and measuring the physiological activity of monacolin K in the culture substrate by experiments described below. Cultivation is continued until a substantial accumulation of monacoline K is obtained in the culture substrate, at which time monacolin K can be isolated and recovered from the culture broth by any combination of isolation methods selected for its physical properties. Eg. any or all of the following isolation techniques may be used: Extraction of liquid from the culture broth with a hydrophilic solvent (e.g., diethyl ether, ethyl acetate, chloroform or benzene); extracting the organism itself with a hydrophilic solvent (e.g., acetone or an alcohol); concentration; dissolving in a more polar solvent (e.g., acetone or an alcohol); removing impurities with a less polar solvent (e.g., petroleum ether or hexane); gel filtration through a column of a material consisting of "Sephadex1 * (trade name for a material sold by Pharmacia Co., Ltd., USA); For example, the subject monacoline K can be isolated from the culture broth as a pure compound.

Monacolin K has been found to have the following properties: 1. Color and shape: - colorless crystals 2. Melting point: - 157-159 ° C (under decomposition) 3. Elemental analysis: C 71.56 $; H, $ 8.85; O $ 19.59.

4. Molecular weight: - 404 (by mass spectrometry): Molecular formula - C24®3gO * 6. Ultraviolet adsorption spectrum (methanol):

As shown in FIG. 1 there is a neximum at 232, 238 and 246 μιη.

7. Infrared adsorption spectrum (KBr):

The spectrum is as shown in FIG. 2nd

8. Nuclear Magnetic Resonance Spectrum (60 MHz proton):

The spectrum is shown in FIG. 3 in deuterated chloroform, using tetramethylsilane as the internal standard.

9. Nuclear Magnetic Resonance Spectrum (3C):

The spectrum is shown in FIG. 4 in deuterated methanol.

6. Solubility:

Soluble in alcohols (eg methanol, ethanol and propanol), acetone, chloroform, ethyl acetate and benzene.

Insoluble in petroleum ether and hexane.

11. Specific rotation: [α] D = 307.6 (c = 1, methanol).

12. Thin-layer chromatography: Rf = 0.47 [No. 5715 "Silica gel silica gel (Merck & Co., Ltd) which is elicited with a 4: 1 volume mixture of methylene chloride and acetone, visible as an ultraviolet radiation absorbent stain, 50 v / v sulfuric acid (a slight red to reddish brown color produced by heating) or with iodine].

The compound is neutral and insoluble in neutral or acidic aqueous substrates. It is converted to an acidic compound by treatment with an alkali, and then it can be dissolved in water. The acidic compound can be extracted with ethyl acetate or chloroform at an acidic pH and it will be converted to monacoline K by evaporation of the solvent.

The physiological effect of monacoline K can be tested and quantitatively determined by the following in vivo experiments.

In vivo testing with rabbits.

In this experiment, monacolin K's ability to reduce cholesterol levels in rabbit blood was measured. The animals used weighed from 2.5 to 3.0 kg. Just before the start of the experiment, blood was collected from a vein in the rabbit's ear and blood cholesterol levels were measured by the commonly used method.

A predetermined amount of monacoline K was then administered orally continuously for 1 to 5 days, and the cholesterol level in the blood serum after administration was measured. The potency of monacoline K or the monacolin K-containing culture substrate can be determined quantitatively from the cholesterol values obtained before and after the administration of monacolin K.

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The ability of monacoline K to lower blood and liver cholesterol levels is demonstrated in various in vivo experiments.

Reduction of blood cholesterol levels in rats

The animals used were rats of the Wistar Imamichi strain, each having a body weight of approx. The tests were performed on groups of rats, each. group consisted of five animals. Each animal was injected intravenously with 400 'mg / kg of Triton® WR-1339. (a trade name for a compound known to raise blood cholesterol levels) while simultaneously administering 10 mg / kg of monacolin K intra-, peritoneally. 24 hours after it. Intraperitoneal administration, the rats were sacrificed by bleeding and blood was collected and the cholesterol level is determined by conventional methods. This showed that blood cholesterol levels had been reduced by 23.9% over the control group of animals to which only "Triton®" WR-1339 had been administered.

Reduction of blood cholesterol levels in rabbits

The test animals used were rabbits having a body weight of 2.7 to 2.9 kg. Each rabbit was orally administered 1 mg / kg of monacoline K twice daily (morning and evening) for 5 days. Prior to the administration and at 3 and 5 days after the administration, blood was collected from a vein in the ear and cholesterol levels in the blood serum were determined.

From this, it was found that at the 3rd and 5th day after the administration of monacolin K, cholesterol levels were 15 and 29% lower than the pre-administration levels, respectively. monacolin K.

In addition to the valuable inhibitory effect on the biosynthesis of cholesterol, monacoline K has a very low toxicity.

The acute oral toxicity (LD 2) of monacolin K in mice is 1 g / kg body weight or more.

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Monacolin K may be administered orally or parenterally in the form of capsules, tablets, injectable preparations or in another well-known formulation, although it is usually preferred to administer it orally.

The dose varies depending on the patient's age and body weight and the severity of the disease, but usually a daily dose of 0.5 to 50 mg for adults is administered either as a single dose or in two or three divided doses. However, due to the low toxicity of the compound, higher doses can be used if desired.

The invention is further illustrated in the following example.

EXAMPLE

The Monascus rubber 1005 strain was seeded on a liquid liquid substrate containing 6% w / v. glucose, 2.5% w / v. peptone, 0.5% w / v. corn molding liquid and 0.5 wt / vol. ammonium chloride. Culture was continued under aerobic conditions at a temperature of 28 ° C for 10 days. The resulting filtrate (5 L) from the culture broth was adjusted to a pH of 3 by the addition of 6N hydrochloric acid, then extracted with an equal volume of ethyl acetate. The solvent was evaporated under reduced pressure from the extract and the resulting residue was dissolved in 100 ml of benzene. Insoluble residues were filtered off.

The filtrate was washed twice, each time with 100 ml of a 5 w / v. aqueous solution of sodium bicarbonate. 100 ml of a 0.2N aqueous solution of sodium hydroxide was then added to the washed filtrate and the mixture was stirred at room temperature. After investigating that monacoline K had disappeared from the benzene layer by thin layer chromatography, the aqueous layer was separated. The pH of the aqueous layer was adjusted to 5 with 6N hydrochloric acid and the resulting solution was extracted twice, each time with 100 ml of ethyl acetate. The extract was evaporated to dryness under reduced pressure to give 260 mg of oil. This oil was dissolved in benzene and allowed to crystallize