KR830002801B1 - Process for preparing antihypercholesteramic agent monacolink and its preparation - Google Patents

Abstract

Monacolin K was obtained from Monascus ruber. Thus, M.ruber 1005 was cultured in a medium contg. glucose 6, peptone 2.5, corn ext. 0.5 and NH4Cl 0.5 (W/V)%. After 10 days at 28°C, the culture supernatant was adjusted to pH 3 and extd. with EtAc. This soln. was evaporated and dissolved in C6H6The resulting soln. was washed with 5(W/V)% of NaHCO3 soln, treated with 0.2N-NaOH, and stirred at room temp. to give Monacolin K.

Description

Method for preparing high cholesterol, monacoline K

1 is an ultraviolet spectrum of Monacholine K according to the present invention.

2 is an infrared spectrum of Monacholine K according to the present invention.

3 is a nuclear magnetic resonance spectrum of monacoline K according to the present invention using 60 MHz protons in deuterium chloroform using tetramethylsilane as an internal standard.

4 is a nuclear magnetic resonance spectrum of Monacrine K according to the present invention using Bc in small and medium methanol.

The present invention relates to a method for producing a novel compound produced by a microorganism belonging to the genus Monascus, and having a high cholesterol treatment.

Since hyperlipidemia, particularly hypercholesterolemia, is known as one of the main causes of heart disease, such as myocardial infarction or arteriosclerosis, considerable efforts have been made to develop compounds that have the ability to reduce blood fat, especially cholesterol content. . One such compound is described in British Patent No. 1,453,425, which is named ML-236 as isolated from microorganisms belonging to the genus Penicillium.

As a result of systematically studying the microorganisms, the inventors have discovered a new compound named Monacholine K that can be obtained by culturing the genus Monascus and fungi, and more specifically, the Monascus rubber 1005 fungus.

We find the structural formula of monacoline K, which is similar in structure to that of compound ML-236 B, which is the subject of British Patent No. 1,453,425, but monacoline K is two to five times greater than ML-236B. Because of the surprising effect, it was found that even a small amount of administration compared to the ML-236B compound can reduce the cholesterol content by the same amount.

Accordingly, an object of the present invention is to provide a novel compound represented by the following general formula, and designated as Monacholine K, and another object of the present invention is a microorganism belonging to the genus Monascus, preferably Monascus rubber monarch Preferably it provides a method for producing Monacoline K by culturing Monascus rubber 1005.

Still another object of the present invention is to provide a pharmaceutical composition comprising mixing and mixing an active ingredient of monacoline K with a pharmaceutically acceptable carrier or diluent.

Monacholine K-generating monarch according to the present invention is a substance isolated from food produced in Thailand, and investigated its characteristics and named as Monascus rubber and deposited in the Fermentation Research Institute of the Ministry of Commerce, Industry and Energy Deposited as US No. 12073 in the United States Agricultural Research Service, Northern Regional Research Lab-oratory. The bacteriological properties of this novel microorganism are as follows.

1. Growth

The microorganisms grew rapidly on potato-glucose-agar medium at 25 ° C. After 10 days of inoculation, the colonies ranged from 6 to 6.5 cm in diameter, and their surface was flat and fairly thin. This occurred, and the development of aerial hyphae was poor.

The mycelia were white in color, and most of them were hairy. In addition, a lot of closed spore follicles were formed on the basal mycelia of the mycelia, and it turned reddish brown at aging.

Both the surface and the backside of the cells were brown to reddish brown.

The microorganism also grew very fast on saboraud agar medium at 25 ° C. After 10 days of inoculation, the microorganism had a diameter of 6 to 6.5 cm, and the surface of the microorganism was very flat. The growth of basal and aerial mycelium was better than that on -glucose-agar medium. Very few were produced from the occlusive spores, and the surface of the cells was reddish yellow to reddish brown, and the back surface of the cells was reddish brown to dark brown. The growth on oatmeal agar medium at 25 ° C was slow, and after 10 days of inoculation, the cell size ranged from 1.5 to 2cm, and the surface of the cell was flat, and the development of airborne hyphae and the formation of closed spores Were all extremely poor. The color of the surface and the backside of the cells was dark brown to reddish brown. The growth rate on the czapek's medium at 25 ° C was very slow, and after 10 days of inoculation, the cells reached a diameter of 1.6 to 1.8 cm.

The growth rate at 37 ° C. on each medium described above was substantially the same as the growth rate at 25 ° C.

2. Morphological features

The collection of obstructed spores is spherical, 30 to 60 microns in diameter, the stems having a septum, and each stem is composed of mycelium from 3.5 to 4.5 microns in diameter and 15 to 80 microns in length. In addition, the sperma of eight spores is spherical in shape, which gradually disappears.

The scapula is colorless, its shape is oval or elliptical, they are 4-5 × 4-7 microns in size and the surface is smooth. Conidia are colorless, spherical or western-shaped, their size is 6-9 × 6-11 microns, their undersides are cut and their walls are fairly thick and smooth. The conidia are formed in a downwardly connected form, such as a mitotic splitting hole, and the conidia bag is not branched or branched like nutrient hyphae, and forms conidia on the top. Mycelia are colorless, branched, and have red walls, most of which are 3 to 5 microns in diameter.

From the above observations, the microorganism was identified as Monascus rubber van Tiegheam strain.

For ecological features of Monascus rubber, see van Tieghem, Bull. Soc. Botan. France, 31, 227 (1884); Cole et at Canadian Journal of Botany, 46, 987 (1968) and Takada Trans. Mycol. Soc. Japan, 9, 128 (1969).

Monacholine K can be obtained by culturing monacoline K-producing microorganisms in culture broth under oxygenic conditions using the same conditions as those commonly used in fungal culture techniques. In other words, the desired monacoline K can be prepared by culturing the microorganisms in a suitable incubator and then inoculating and culturing the microbial population on another incubator. In this case, the incubator composition used for the preparation of monacoline K may be the same as or different from the composition used for the initial and microbial culture.

Monacoline K-producing microorganisms, in particular Monascus rubber 1005, can be grown over a wide range of temperatures, such as between 7 and 40 ° C. In general, the most preferred temperature for the production of monacoline K is in the range of 20 to 30 ° C.

The incubator used for culturing the microorganism contains known nutrients used for culturing fungi and other microorganisms, and in general, the incubator contains at least an assimilable carbon source and an assimilable nitrogen source. Anabolic carbon sources and preferred examples are glucose, maltose, dextrin, starch, lactose and sucrose. Glycerin and the like, and among these, glucose and starch monacoline K are particularly preferable.

Preferred examples of the assimilable nitrogen source include peptone, gravy, yeast, yeast juice, soy flour, peanut jelly, corn steep liquor, rice arsenic nitrogen source, and among these, peptone is preferred.

In the case of culturing the microorganisms for the purpose of producing monacoline K, inorganic salts and metal salts can be mixed in the incubator if necessary, and a small amount of heavy metals can also be mixed. Preferred compositions of the incubator are, for example, 2% (soluble / volume)% of starch, 1% (weight / volume) of glucose, 2% (weight / volume) of peptone 2, and 2% (weight / volume) of agar.

Monacholine K can be prepared by culturing monacoline K-producing microorganisms in an oxygenous atmosphere using standard techniques of oxygenous culturing (eg, solid culture, vibrating culture, or agitation with aeration).

Antifoaming agents (silicone oils or surfactants) can be incorporated into the medium during the incubation or when antifoams are required during sterilization of the incubator.

The physiological activity of monacoline K can be evaluated by the following experiment which measures the ability of cholesterol to decrease in content in rabbit blood. In this experiment, a rabbit weighing 2.5 to 3.Okg should be used. Before the experiment, blood is drawn from the artery of the rabbit's ear, and the cholesterol content of serum is measured by a conventional method.

Next, a prescribed amount of monacoline K is intermittently administered orally over 1 to 5 days, and after administration, the cholesterol content in the herb is measured. The titer of monacol ben K can be measured by comparing cholesterol content before and after monacoline K administration.

Cultivation of monaclinic K-producing microorganisms can be carried out until the actual amount of monacoline K accumulates in the incubator as measured by the experiments described above. At this stage, monacoline K is used in combination with the known well-known techniques using physical properties. Can be isolated and recovered as culture broth. for example. Isolation is carried out by the following techniques: extraction with diethyl ether, ethyl acetate, chloroform or benzene, dissolution in a less polar solvent (such as acetone or alcohol), less polar solvent (such as petroleum ether). Or a method of removing impurities in hexane), gel filtration through a column such as Sephadex (trade name of Pharmacia Co., LTD.), Adsorption chromatography using activated carbon or silica gel, or the like. have.

By appropriately using the above-described technical methods, the desired monacholine K can be isolated from the culture broth as a pure substance.

Physiological-chemical characteristics of monacoline K are as follows.

Monaklin K has a melting point of 157 to 159 ° C (decomposition), a colorless crystal, soluble in lower alcohols (methane, ethanol and propanol), acetone chloroform, ethyl acetate and benzene, and insoluble in hexane and petroleum ether . In addition, foreign matters are neutral and can be dissolved in water after being converted to an acidic substance by performing an insoluble or alkaline treatment in a neutral or acidic aqueous medium. This acidic route can be extracted with ethyl acetate or chloroform at an acidic pH value and then reduced to monacoline K by neutralizing the solvent.

Elemental Analysis Molecular Formula.

C, 71.56%; H, 8.85%; 0, 19.59% C ₂₄ H ₃₆ O ₅

Molecular Weight. Non-luminescence

404 (by mass spectrometry) [α] ²⁵ _D = + 307.6 (c = 1, meranol)

The mixture of methylene chloride and acetone (4: 1 volume ratio) was developed on silica gel (merck & Co. LTD, NO. 7515 KieseIgeI 60 F254) thin layer chromatography. 50% (volume / volume) Sulfuric acid (light reddish to reddish brown when heated) Sats were observed with an ultraviolet radiation-absorbing lamp after spraying with iodine and showed a single constant at Rf of 0.7. Acute orthotoxic toxicity (LD50) of monacoline K in juvenile rats is less than 1 g / kg body weight.

The inventors have demonstrated through various in vivo experiments that monacoline K has the ability to lower blood cholesterol content.

Blood cholesterol reduction in rats:

Wistar imamichi rats were used as experimental animals for each group consisting of five rats.

Each mouse was injected intravenously with 400 mg / kg of Triton WR1339 (a trade name known to increase blood cholesterol content). Intraperitoneally administered 10 mg / kg, the rats bleeding died 14 hours after administration.

Cholesterol content was measured by conventional methods, demonstrating a 23.9% reduction in blood cholesterol content as compared to another group of mice treated with Triton WR 1339 alone.

Blood Cholesterol Reduction in Rabbits:

Using rabbits of 2.7 kg to 2.9 kg body weight as test animals, each rabbit was orally administered 1 mg / kg of monacoline K twice daily for 5 days in the tidal phase.

Cholesterol was collected from rabbit arteries before and after 3 and 5 days of administration, and the serum cholesterol was measured.These cholesterol levels after 3 and 5 days after monacoline K were administered. The content was 15% and 29% lower than the content prior to the administration of Monaklin K.

Thus, Monacholine K is useful as a treatment for high cholesterol blood or for treating atherosclerosis.

In order to compare Monacholine K and ML-236B, the following formula merinoline (Mevinolin) of the same formula as Monacholine K is as follows.

Lowering Cholesterol by ML-236B (12-day Administration)

The effect of monacoline K (Mevinolin) on the mall is shown in the table below.

Acute inhibition of cholesterol synthesis from acetate [ ¹⁴ C] in rats after oral administration of sodium mebinolinate (O) or ML236B sodium salt (·) alone. Each point is the average of inhibition rates in 10 rats compared to the control analyzed on the same day.

The above table shows that the effect of monacoline K is 6 times that of ML-236B. Plasma cholesterol decreased by 30.4% when ML-236B was administered at 20 mg / kg / day for 12 days, but 8 mg / kg / Since the rate of plasma cholesterol reduction when administered from days to 12 days was 24.2%, the dosage of mebinoline may be reduced. In addition, the usage and dosage of monacoline K in humans is as follows. In humans, 5,1550mg twice a day for 7-11 days, the decrease rate of serum cholesterol was 14,25,24% after 7 days, respectively, showing safety and its effects.

(4% reduction in placebo)

* Average percentage change from petrolatum in total serum cholesterol after mevinolin treatment

○ ‥‥‥‥‥ ○ placebo; ￣ ● 5mg b.i.d; ▲ ＿ ▲ 15 mg b.i.d; ■ ＿ ■ 50mg b.i.d. Bars represent standard letters.

Adverse reactions to the drug are acceptable in the literature (The American Society for Chemical Inve-stigation Inc, volume 69 April 1982, 918-919).

The compounds according to the invention can be administered orally or parenterally in capsules, tablets, injections or other known formulations, but are preferably administered orally. Dosage depends on the patient's age, weight and health condition. In general, the two-day dose is 0.5 to 50 mg in adults, divided into two or three divided doses.

However, because the compound is low toxicity, it is irrelevant to increase the dosage as needed.

Although an Example is given to the following and this invention is demonstrated in detail, the scope of the present invention is not limited only to this Example.

EXAMPLE

Monascus rubber 1005 strain on a liquid incubator containing 6% glucose by weight, 2.5% by weight peptone, 0.5% by weight corn soak and 0.5% by weight ammonium chloride. After inoculation, the culture was continued for 10 days at a temperature of 28 ° C. under oxygenic conditions. 6N hydrochloric acid was added to the resulting culture broth filtrate (5 L) to adjust the pH to 3, and then extracted with an equal volume of ethyl acetate. The residue obtained by extracting the solvent from the extract under reduced pressure was dissolved in 10 ml of benzene, and the insolubles were filtered off.

The filtrate was washed two times with 100 ml of 5 (weight / volume)% aqueous sodium bicarbonate solution, and 100 ml of 0.2 N aqueous sodium hydroxide solution was added to the washed filtrate, and the resulting mixture was stirred at room temperature. After confirming that monacoline K disappeared from the benzeneworms by larva chromatography, the aqueous layer was filtered.

Next, 6N hydrochloric acid was added to adjust the pH of the aqueous layer to 3, and then the resulting solution was extracted twice with 100 ml of ethyl acetate once. The extract was evaporated to dryness under reduced pressure to give 260 mg of an oily substance. This oily substance was dissolved in benzene, crystallized and then recrystallized in acetone aqueous solution to give a colorless needle-shaped monacoline K87 mg having the characteristics described above.

Claims (1)

A method for producing monacoline K, characterized by culturing a monacoline K-producing microorganism belonging to the genus Monascus in a culture medium and then isolating monacoline K from the culture medium.

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