CH645890A5 - MONACOLIN K, ITS PRODUCTION AND ANTI-HYPERCHOLESTERINEEMIC MIXTURE CONTAINING IT. - Google Patents

Description

The present invention relates to a new compound with antihypercholesterolemic activity called Monacolin K. Monacolin K can be produced by cultivating various microorganisms of the genus Monascus.

Thus, the present invention relates to the compound monacolin K of the following formula:

HO

The invention further relates to a process for the production of Monacolin K, which consists in cultivating a Monacolin K-producing microorganism of the genus Monascus in a suitable culture medium.

The invention further relates to an antihypercholesterolemic pharmaceutical preparation which contains monacolin K and a pharmaceutically acceptable carrier or diluent.

High blood cholesterol levels are known to be a major cause of ICardiopathy, such as heart attack or arteriosclerosis. Therefore, a considerable amount of research has been done to identify physiologically acceptable substances that can inhibit cholesterol biosynthesis and, consequently, lower blood cholesterol levels. One such connection is ML-236, which is the subject of British Patent No. 1463 425. ML-236 is obtained by growing microorganisms of the genus Pénicillium.

In experiments with fungi of the genus Monascus, it was found that these fungi and in particular Monascus ruber strain 1005 (FERM 4822) produce an antihypercholesterolemic agent which has significantly better efficacies than ML-236. This remedy is called Monacolin K.

All microorganisms of the genus Monascus, which are able to produce Monacolin K, can be used for the method according to the invention. The strains of Monascus ruber, particularly Monascus ruber strain 1005 (FERM 4822), are particularly valuable.

Monascus ruber strain 1005 (FERM 4822) is a new isolated microorganism with the following microbiological properties. It was isolated from food produced in Thailand and on February 16, 1979 under No. FERM 4822 at the Fermentation Research Institute, Agency of Industriai Science and Technology, Ministry of International Trade and Industry, Japan, and under No. NRRL 12073 at the Agricultural Research Service , Northern Regional Research Laboratory, USA.

1. Growth

The growth on potato-glucose agar medium at 25 ° C is rapid and the diameter of the colonies reaches 6 to 6.5 cm 10 days after vaccination. The colony is flat and a relatively thin base layer of hyphae develops. The development of aerial hyphae is poor. The aerial hyphae are white and most aerial hyphae are wool-like. Some closed fruiting bodies form on the base layer of the hyphae and change to reddish brown when ripened. Both the surface and the back of the colony are brown to reddish brown.

The growth on a Sabouraud agar medium at 25 ° C is very rapid and the diameter of the colony reaches 6 to 6.5 cm 10 days after the vaccination. The surface of the colony is extremely flat, and the hyphae of the base layer and the air hyphae develop better than on an ICar-potato-glucose agar medium. Very little closed fruiting bodies are formed. The surface of the colony is reddish yellow to reddish brown and the back is reddish brown to dark brown.

The wheat flour agar grows slowly at 25 ° C and the diameter of the colony reaches 1.5 to 2 cm after 10 days after vaccination. The colony is flat. The development of aerial hyphae and the formation of closed fruiting bodies is extremely weak. Both the surface and the back of the colony are dark red to reddish brown.

The growth on Czapek agar medium at 25 ° C takes place very slowly and the diameter of the colony reaches 1.6 to 1.8 cm after 10 days after the vaccination.

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The course of growth on each of the media mentioned above at 37 ° C is practically the same as at 25 ° C.

2. Morphological properties

The closed fruiting bodies are spherical and have a diameter of 30 to 60 microns. Their walls are thin and membrane-like. The stems of which have partitions and consist of hyphae with a diameter of 3.5 to 4.5 microns and a length of 15 to 80 microns. The askus consists of 8 spores and is almost spherical and delicate or transparent. The askus spores are colorless and ovoid or ellipsoid. They are 4 to 5x4 to 7 microns in size. Their surfaces are smooth. The conidia are colorless and spherical or pear-shaped. Their size is 6 to 9x6 to 11 microns. The lower parts are blunted, while the walls are relatively thick and smooth. The conidia are connected basipetal to divisible arthrospores. The conidiophore resembles a vegetative hyphe and is branched or unbranched, with conidia formed at the upper end. The mycelia are colorless and branched and have septum, most of which are 3 to 5 microns in diameter.

Because of the properties observed above, this microorganism was identified as a strain of Monascus ruber van Tieghem.

The microbiological properties of Monascus ruber have been disclosed in the following literature: vergi.

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. Offered. France, 31, 277 (1884). The generation of ascospores by the strain was described by Cole et aüm Canadian Journal of Botany 46,987 (1968), "Conidium Ontogeny in hyphomycetes, The imperfect state of Monascus ruber and its meristem arthrospores".

The use of Monascus ruber strain 1005 is described in particularly great detail below. However, it should be specifically noted that any strains of the genus Monascus, as well as the varieties and mutants thereof,

Which monacolin K are capable of producing can also be used for the process according to the invention.

Monacolin K can be prepared by culturing the selected microorganism in a culture broth under aerobic conditions using the same procedures as are well known for the cultivation of fungi and other microorganisms from the prior art. For example, the monacolin K-producing microorganism can first be grown on a suitable medium and then the generated microorganisms can be collected and vaccinated in another culture medium or grown in another culture medium in order to obtain the desired monacolin K in this way. The growth media used for the propagation of the microorganism and for the production of Monacolin K can be the same or different media.

Any culture media known for the cultivation of fungi can be used, provided that, as is also well known, they contain the necessary nutrient materials, in particular an assimilable carbon source and an assimilable nitrogen source. Examples of suitable assimilable carbon sources are glucose, maltose, dextrin, starch, lactose, sucrose and glycerin. For the production of Monacolin K, preference will be given to the carbon sources just mentioned, in particular glucose, glycerol and starch. Examples of suitable assimilable nitrogen sources are

Peptone, meat extract, yeast, yeast extract, soybean meal, peanut meal, corn steep liquor, bran and inorganic nitrogen sources. Peptone will preferably be used as the nitrogen source. In the production of Monacolin K, an inorganic salt and / or a metal salt can be added to the culture medium if necessary. Furthermore, if necessary, a small amount of a heavy metal can be added.

The microorganism is preferably grown under aerobic conditions using known breeding methods, for example with a solid culture, a shaking culture or a culture kept under aeration and shaking. The microorganism grows within a wide temperature range of, for example, 7-40 ° C. However, the preferred breeding temperature for the production of Monacolin K is in the range from 20 to 35 ° C.

During the growth of the microorganism, the extent of the production of Monacolin K can be recorded by taking samples from the culture medium and by measuring the physiological activity of the Monacolin K present in the culture medium with the aid of the test described below. The cultivation can then be continued until a substantial enrichment of Monacolin K in the culture medium has taken place. At this time, the monacolin K can be obtained from the culture broth by any combination of isolation methods, which are selected depending on the physical and chemical properties. So one or more of the following isolation techniques can be used, namely the extraction of the liquid from the culture broth with a hydrophilic solvent, e.g. Diethyl ether, ethyl acetate, chloroform and benzene, extraction of the organism with a hydrophilic solvent such as e.g. Acetone or an alcohol, concentration, dissolving in a more polar solvent such as e.g. Acetone or an alcohol, removing the contaminants using a less polar solvent such as e.g. Petroleum ether or hexane, gel filtration through a column which is coated with a material such as e.g. Sephadex (trademark of a material available from Pharmacia Co., Ltd., USA, USA), absorptive chromatography with activated carbon or silica gel etc. When using a suitable combination of these working methods, monacolin K can be isolated as a pure substance from the culture broth.

Monacolin K has been found to have the following properties:

1. Color and shape: colorless crystals.

2nd melting point: 157-159 ° C (with decomposition).

3. Elemental analysis: C = 71.56%; H = 8.85%; 0 = 19.59%.

4. Molecular weight: 404 (using mass spectrometry).

5. Molecular formula: C24H36O5.

6. Ultraviolet absorption spectrum (methanol): As can be seen from Fig. 1 of the accompanying drawings, the maximum values are 232.238 and 246 mil.

7. Infrared absorption spectrum (KBr): According to the information in FIG. 2 of the accompanying drawings.

8. Proton resonance spectrum (60 MHz proton): According to FIG. 3 of the accompanying drawings, in deuterized chloroform using tetramethylsilane as the internal standard.

9. Magnetic carbon 13 resonance spectrum (13C): According to FIG. 4 of the accompanying drawings, in deuterated methanol.

10. Solubility: In lower alcohols, e.g. Methanol, ethanol and propanol, acetone, chloroform, ethyl acetate and benzene soluble; insoluble in petroleum ether and hexane.

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11. Specific rotation: [<x] d = +307.6 (c = 1, methanol).

12. Thin layer chromatography: Rf = 0.47 [No. 5715 silica gel 6OF254 silica gel (Merck & Co., Ltd), developed in a mixture of 4 parts by volume of methylene chloride and 1 part by volume of acetone, perceptible as an ultraviolet ray-absorbing stain, 50% by volume of sulfuric acid (it develops when heated a pale red to reddish brown color) or with iodine].

The compound is neutral and insoluble in neutral and acidic aqueous media. When treated with an alkali, it turns into an acidic substance and can be dissolved in water. This acidic substance can be extracted with ethyl acetate or chloroform at an acidic pH. When the solvent evaporates, it again passes into the monacolin K.

The physiological activity of Monacolin K can be quantified using the following in vivo test.

In vivo test on rabbits

In this test, the ability to lower cholesterol levels by monacolin K in rabbit blood was measured. The animals used weighed 2.5 to 3.0 kg. Immediately after the start of the test, the blood was collected from the vein in the ear of a rabbit and the cholesterol level in the blood serum was measured in a known manner. A predetermined amount of Monacolin K was then administered orally continuously for 1 to 5 days, and after the administration, the cholesterol level in the blood serum was measured. The potency of the monacolin K or a culture medium containing monacolin K can be determined quantitatively from the cholesterol values which were obtained before and after the administration of monacolin K.

Various in vivo tests have confirmed that monacolin K is able to lower blood and liver cholesterol levels.

Lowering blood cholesterol levels in rats

The rates used were from the Wistar-Ima-michi strain, each with a body weight of approximately 300 g. The tests were carried out on different groups of rats, each group consisting of 5 animals. Each animal was intravenously injected with 400 mg / kg of Triton WR-1339 (a brand name for a known agent for increasing blood cholesterol), while 10 mg / kg of Monacolin K was administered intraperitoneally at the same time. 14 hours after intraperitoneal administration, the rats were killed by bleeding and the blood was collected and its cholesterol level was determined in a manner known per se. It was found that blood cholesterol levels were reduced by 23.9% compared to a comparison group of animals that had only been administered Triton WR-1339.

Lowering blood cholesterol levels in rabbits

The test animals used were rabbits with a body weight of 2.7 to 2.9 kg. Each rabbit was orally administered twice daily, morning and evening, 1 mg / kg of Monacolin K for 5 days.

Before the administration and 3 or 5 days after the administration, blood was drawn from a vein of the ear and the cholesterol levels in the blood serum were determined. It was found that the cholesterol levels when Monacolin K was administered for 3 and 5 days were 15% and 29% lower than the blood level value measured before Monacolin K was administered.

In addition to its inhibitory effect on the biosynthesis of cholesterol, monacolin K is also extremely low toxic. The acute oral toxicity in a mouse of 1 g / kg body weight or more is (LD50) of Monacolin K.

Monacolin K can be administered orally or parenterally in the form of capsules, tablets, injectable preparations or in any other known form, but it will normally be added orally. The dose level varies depending on the age and body weight of the patient and the severity of the disease. However, the daily dose for an adult is generally 0.5 to 50 mg, the agent being administered either in a single dose divided into two or three doses. Because of the low toxicity of the compound, higher doses can be used if necessary.

The invention is illustrated by the example below.

example

Monascus ruber strain 1005 was in a liquid culture medium containing 6 parts by weight / 100 parts by volume glucose, 2.5 parts by weight / 100 parts by volume peptone, 0.5 parts by weight / 100 vol. Parts of corn steep liquor and 0.5 part by weight / 100 parts by volume of ammonium chloride were inoculated. The cultivation was carried out under aerobic conditions for 10 days at a temperature of 28 ° C. The filtrate (5 liters) of the culture broth thus obtained was adjusted to a pH of 3 by adding a 6N hydrochloric acid solution and then extracted with an equal volume of ethyl acetate. The solvent was evaporated from the extract under reduced pressure and the residue formed was dissolved in 100 ml of benzene. The insoluble matter was filtered off.

The filtrate was washed twice with 100 ml of a 5 parts by weight / 100 parts by volume aqueous sodium bicarbonate solution. Then 100 ml of an aqueous 0.2N sodium hydroxide solution was added to the washed filtrate and the mixture was stirred at room temperature. After the disappearance of monacolin K from the benzene layer was determined by thin layer chromatography, the aqueous layer was separated. The pH of the aqueous layer was then adjusted to 3 by adding 6N hydrochloric acid and the solution thus obtained was extracted twice with 100 ml of ethyl acetate each time. The extract was evaporated to dryness under reduced pressure, leaving 260 mg of an oil. This oil was dissolved in benzene and allowed to crystallize. The crystals were then recrystallized from an aqueous acetone solution, giving 87 mg of colorless needles of monacolin K, which had the abovementioned properties.

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Claims (7)

645 890 PATENT CLAIMS 1. Monacolin K of the formula with antihypercholesterolemic activity: HO 2. A process for the preparation of monacolin K, characterized in that a microorganism of the genus Monascus producing monacolin K is grown in a suitable culture medium. 3. The method according to claim 2, characterized in that a strain of Monascus ruber is used as the microorganism. 4. The method according to claim 3, characterized in that strain 1005 is used as strain Monascus ruber. 5. The method according to any one of claims 2 to 4, characterized in that one carries out the cultivation at a temperature in the range of 7 to 40 ° C. 6. The method according to claim 5, characterized in that the cultivation takes place at a temperature of 20 to 35 ° C. 7. Antihypercholesterolemic preparation containing Monacolin K of formula I and a pharmaceutically acceptable carrier or diluent.