SU1158048A3 - Method of obtaining k monacolin possessing antihypercholesterol effect - Google Patents

Abstract

METHOD OF OBTAINING MONACOLINE TO THE OXIDATION OF ANTI-SCHOLHOLESTERYMICHESKY EFFECT OF THE GENERAL FORMULA BUT. About the fact that Monascus ruber 1005 (FERM - P4822) is grown in a nutrient medium containing a source of nitrogen, carbon and mineral salts, under aerobic conditions at 20 ° C, p. the subsequent distribution of the target product from the cupral liquid. with CZ ate 00

Description

measures that produce monacolin K the microorganism can be first cultured on a suitable medium, and then the microorganisms obtained can be collected and used for inoculation and cultured on another culture medium to accumulate the desired monacolin K; the culture media used to propagate the microorganism and to produce minicolin K may be identical or equal.

Any kind of lime can be used on a culture medium suitable for the cultivation of fungi, provided that it contains the necessary nutritional materials, especially a source of assimilable carbon and a source of assimilable nitrogen. Examples of suitable sources of digestible carbon are glucose, maltose dextrin, starch, lactose, sugar and glycerol, of which glucose, glycerin and starch are suitable for monac / lin K production. Examples of suitable sources of assimilable nitrogen are peptone, meat extract, yeast, yeast extract, soybean flour, peanut flour, liquid after soaking corn, rice bran and inorganic nitrogen sources, of which peptone is preferred. In the production of monacolin K, an inorganic salt and / or metal salt may, if necessary, be added to the culture medium. In addition, if required, a small amount of heavy metal may be added.

The microorganism is preferably cultured under aerobic conditions using known cultivation methods, for example, solid culture, agitated culture, or culture under aeration and mixing conditions. The microorganism grows in a wide temperature range, for example, 7–40 ° C, but from the point of view of monacolin K production, the preferred cultivation temperature is 20–30 C. During the cultivation of the microorganism, the production of monacolin K can be controlled by selecting the culture medium 55 and measuring the physiological activity of the monacolin K into the culture medium using the described testov. Cultivation can then be continued to accumulate a significant amount of monacolin K in the culture medium, after which monacolin K can be extracted and removed from the culture broth using any suitable combination of isolation techniques, taking into account its physical and chemical properties. For example, any or all of the following isolation methods may be used: extraction of the solution from the culture broth with a hydrophilic solvent (for example, diethyl ether, ethyl acetate, chloroform or benzene); extraction of the body with a hydrophilic solvent (such as acetone or alcohol); concentration; dissolving in a more polar solvent (e.g. acetone or alcohol); removing impurities with a less polar solvent (such as petroleum ether or hexane); gel filtration through a column with such material as Sephadex; absorption chromatography using activated carbon or silica gel, and the like. By applying the appropriate combination of these methods, the desired monacolin K can be isolated from the culture broth as a pure substance.

Monacolin K, as it is isolated from the Monascus ruber α -culture broth strain 1005, is characterized by the following properties (series A).

Color and shape: colorless. Crystals.

Melting point: 157-159 ° C (with decomposition).

Elementary analysis,%: C 71,56; H 8.85; About 19.59.

Molecular weight: 404 (according to mass spectrometry).

Molecular formula: C is y

UV absorption spectrum (methanol): maxima at 232, 238 and 246 nm.

IR Absorption Spectrum (KB g).

NMR spectrum (proton 60 MHz; in deuterated chloroform using tetramethylsiline as an internal standard). NMR spectrum (C; in deuterated methanol). Solubility: soluble in lower alcohols (for example, methanol, ethanol and propanol), acetone, chloroform. ethyl acetate and benzene. Insoluble in petroleum ether and hexane. Specific rotation: + 307.6 (, methanol). Thin layer chromatography; Rf 0.47 Г№ 5715 Kieselgel (silica gel (Merck & Co.), a mixture of 4: 1 methylene chloride and acetone, detection in the form of UV absorbed mass, 50% (v / v) sulfuric acid (color from pale red to reddish brown appears when heated) or iodine}. Example 1. Monascus ruber strain 1005 is inoculated onto a liquid culture medium containing 6% by volume glucose, 2.5% by volume peptone, 0.5% by volume. % liquid after soaking maize and 0.5% w / v% ammonium chloride. Cultivation is continued under aerobic conditions at 28 ° C for 10 days. The resulting filtrate (5 culture The broth is adjusted to pH 3 with 6N hydrochloric acid and then extracted with an equal volume of ethyl acetate. The solvent is distilled off from the extract under reduced pressure and the resulting residue is dissolved in 100 ml of benzene. The insoluble matter is filtered. The filtrate is washed twice with 100 ml of 5 weight each The aqueous solution of sodium bicarbonate. Then 100 m of a 0.2N sodium hydroxide solution of sodium hydroxide is added to the washed filtrate and the mixture is stirred at room temperature. After confirming the disappearance of monacolin K from the benzene layer by TLC, the aqueous layer is separated. The pH of the aqueous layer was then adjusted to pH-3 with 6N hydrochloric acid and the resulting solution was extracted twice with 100 ml of ethyl acetate each time. The extract was evaporated to dryness under reduced pressure to obtain 260 mg of oil. Monacolin K can reduce cholesterol in the blood and liver. Example 2: Reducing the level of cholesterol in the blood and liver of rats. WistarIma rats were used, each weighing about 300 g. Tests were carried out on a group of rats, each group consisting of 5 animals. Each animal is injected with 300 mg / kg Triton (trade name for a material that increases blood cholesterol levels) with the simultaneous administration of 10 mg / kg monacolin K, either orally or intraperitoneally. Twenty hours after oral administration or 14 hours after intraperitoneal administration, the rats are killed by exsanguination, blood is collected from the liver and the cholesterol level in them is determined by conventional methods. As a result, it was found that the level of cholesterol in the blood is lower compared with the control group of animals to which only Triton WR-1339 is administered, by 22.4% in the case of oral administration and by 23.9% in the case of intraperitoneal administration. The level of cholestat rin in the liver is reduced by 16.7% in animals in which monacolin K is administered orally. Example 3. Reduction of blood cholesterol levels in rabbits. Experimental rabbits with a body weight of 2.7 2, 9 kg are used. 1 mg / kg of monacolin K was orally administered to each rabbit twice a day (in the morning and in the evening) continuously for 5 days. Before administration and on the third and fifth day after administration, blood is collected from the ear vein and the serum cholesterol content is determined. As a result, the cholesterol content on the third and fifth day after administration of monacolin K was obtained by 15 and 29%, respectively, lower than before administration of minicolin K. Monacolin K provides 50% inhibition of cholesterol biosynthesis at a concentration of 0.002 µg / ml using an experiment wherein acetic acid is reacted with a rat liver enzyme. Monacolin K was found to inhibit MI C-6oA reductase, which is a rate-regulating enzyme for cholesterol biosynthesis, and that it competitively inhibits substrate NM G-CoA and has K, a value equal to 5, M. K - value is a constant inhibiting and, more specifically, the dissociation constant of the enzyme-inhibitor complex. This value is obtained by multiplying the concentrations 711580 of the enzyme and inhibitor dividing by the concentration of the enzyme inhibitor complex. In addition to the highly valuable 5 inhibitory effect on cholesterol biosynthesis, monacolin K is characterized by extremely low toxicity. Thus, acute oral toxicity (LDjo) monacolin K for H 488 menspeps is 1 g / kg body weight or even greater, Monacolin K can be administered orally or parenterally in the form of capsules, tablets, injectable drugs in the form of any other known composition, So get the drug monacolin K, which has antihyper-. cholesteric effect.

Claims (1)

METHOD FOR PRODUCING MONACOLINE K, ι POSSESSING ANTI-HYPERCHOLESTERIMIC ACTION of the general formula> 1158048 2