JPS5925599B2 - New physiologically active substance monacolin K and its production method - Google Patents

Description

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

Hyperlipidemia, particularly hypercholesterolemia, is known to be one of the major causes of heart diseases such as myocardial infarction and arteriosclerosis.

Therefore, the present inventor conducted a search for an excellent new physiologically active substance having a cholesterol-lowering effect among microbial products. As a result, the active substance Monacolin was collected from the culture of one strain of the mold. This substance was found to be effective as a blood cholesterol lowering agent through animal experiments using rabbits and rats. Furthermore, we investigated the physical and chemical properties of this substance and found that it is a new substance. This substance is hereinafter referred to as monacolin. The present invention relates to a method for culturing mold and collecting monacolin from the culture, and particularly to a method for culturing Monascus and collecting monacolin from the culture.

The microorganism that can be used in the present invention is a Monacolin-producing bacterium belonging to the genus Monascus, and a strain that the present inventor recognizes as particularly effective is, for example, Monascus Ruberya 100.
5 strains, and this strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry for entrustment of storage on February 16, 1978, and its accession number is FAIKEN Bacteria Ichiyori No. 4822.

It goes without saying that any species of the genus Monascus other than those mentioned above may be used, regardless of their variants or mutant strains, as long as they have the ability to produce monacolin K.

The mycological properties of the monacolin K-producing bacteria isolated from food products produced in Thailand by the present inventors are as follows. 1> Growth Growth on potato glucose agar medium (25° C.) is fast, the diameter of the colony is 5-6 CTIL0 colonies are flat in 10 days, and a relatively thin basal hyphal layer develops.

Aerial hyphae are poorly developed, white and mostly woolly. Numerous ascocarps (CleistOthecia) are present on the basal hyphal layer.
), which turns reddish-brown as it matures.

Both the front and back sides of the colony are brown to reddish brown. Growth on Sabouraud agar medium (25℃) is extremely fast, and the diameter of the colony is 6 to 6.5 Cf in 10 days! Reach L.

The colony surface is extremely flat, and both basal and aerial hyphae develop better than on potato-glucose agar medium. The number of asci formed is extremely small. The surface of the colony is reddish-orange to reddish brown, and the underside is reddish brown to dark brown. Growth on oatmeal agar medium (25°C) is slow, the colony diameter is 1.5 to 2 cfn0 flat after 10 days, and the development of aerial mycelia and ascocarp formation are extremely poor.

Both the front and back sides of the colony are dark red to reddish brown. Growth on Czapetsk agar medium (25°C) is extremely slow, and the diameter of droplets is 1.6 to 1.8 cm in 10 days.The growth rate at 37°C on the various agar mediums listed above is comparable to that at 25°C. do.

2) Form The asci are spherical and 30-60μ in diameter.

The ascocarp wall is thin and membranous. The ascus stalk has septa, diameter 3
．． It consists of hyphae of 5-4.5μ and 15-80μ in length. Ascus has 8 spores, almost spherical and effacement. Ascospore (Asc
OspOres) are colorless, oval to oval, 4 to 5 x 4 to
7μ, smooth surface. Conidia are colorless,
Spherical to pear-shaped, 6-9 x 6-11μ, base cut-like, wall relatively thick and smooth, segmented with basophilic chains. Conidiophores resemble vegetative hyphae, are unbranched to branched, and form conidia at the tips. Mycelium is colorless, branched, septate, and most are 3 to 5 in diameter.
μ. As a result of the above observations, this bacterium is Monascus rubel [
MOnascus rubber (Van Tieghem)
] was identified.

The mycological description of Monascus rubel is detailed in the following paper.

That is, the Journal of the French Botanical Society (Bull
．． SOc. BOtan. France) Volume 31, 227
Page, 1884, Cole et al.: Canadian Journal op.
fBOtany) vol. 46, p. 987, 1968, Takada: Bulletin of the Japanese Mycological Society, vol. 9, p. 128, 1969. As mentioned above, this strain has been submitted to the Institute of Microbial Technology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry as Monascus rubel black 1005 strain.

Monacolin K is produced by aerobically culturing a monacolin K-producing bacterial strain using a method known as a mold culture method. For example, monacolin K-producing bacteria are subcultured on a medium consisting of 2% soluble starch, 1% glucose, 2% peptone, and 2% agar, and for production of hemonacolin K, the cells grown on this agar medium are directly inoculated into the production medium. can be cultured. Furthermore, the bacterial cells grown in the production medium can be cultured in a new production medium, and monacolin K can be produced there. Monacolin K-producing bacteria grow at a temperature of 7 to 40°C, but a temperature of 20 to 35°C is usually preferred for monacolin K production.

For culturing Monascus bacteria that produce monacolin K, any nutrient source known for culturing molds and other microorganisms can be used. For example, glucose, maltose, dextrin, starch, lactose, sutucarose, glycerin, etc. can be used as carbon sources. Among these carbon sources, glucose and starch are the preferred carbon sources for monacolin K production. Any nitrogen source known for the growth of Monascus or other microorganisms to produce monacolin K can be used.

For example, peptone, meat blemishes, yeast, yeast blemishes, soybean flour, peanut flour, corn steep liquor, rice bran, inorganic nitrogen sources, etc. can be used. When culturing monacolin K-producing bacteria to produce monacolin K, inorganic salts and metal salts are added if necessary. Also, trace amounts of heavy metals can be added if necessary. Monacolin K can be obtained by aerobically culturing its producing bacteria, and commonly used aerobic culture methods such as solid state culture, shaking culture, and aerated agitation culture may be used.

When antifoaming is required during culture or medium sterilization, antifoaming agents such as silicone oil and surfactants can be used. The culture temperature is preferably 20 to 35°C. The physiological activity of monacolin K can be assayed by the following method, which examines the rabbit blood cholesterol lowering effect.

That is, blood is collected from the ear vein of a rabbit weighing 2.5 to 3.2K9 immediately before the start of the experiment, and the serum cholesterol level is measured by a conventional method. Next, a fixed amount of monacolin K is continuously administered for 1 to 5 days by oral administration, and the serum cholesterol level after administration is determined. The effect of monacolin K can be quantitatively determined from serum cholesterol levels before and after monacolin K administration. Cultivation is continued until monacolin K is substantially accumulated, and this substance can be extracted from the culture in various ways based on the properties of this substance revealed by the inventor, as shown in the Examples below. This can be done by appropriately combining the following methods. For example, extraction with ether, ethyl acetate, chloroform, benzene, etc., acetone,
Dissolution in alcohols with high polarity, removal of impurities with low polarity solvents such as petroleum ether and hexane,
Gel filtration using a Sephadex column, adsorption chromatography using activated carbon, silica gel, etc. are the first. By using a suitable combination of these means, the substance can be isolated from the culture as a homogeneous substance. Next, the physical and chemical properties of Monacolin K will be described.

Monacolin K is a colorless crystal with a melting point of 157 to 159°C, and is soluble in lower alcohols such as methanol, ethanol, and propanol, acetone, chloroform, ethyl acetate, benzene, etc., and insoluble in hexane, petroleum ether, etc.

This substance is a neutral substance and does not dissolve in neutral or acidic water, but
It is converted into an acidic substance by normal alkali treatment and dissolved in water. This acidic substance is extracted into ethyl acetate, chloroform, etc. in an acidic PH region, and reconverted to monacolin K by drying. The elemental analysis value of Monacolin K is carbon 71.56
%,. Hydrogen is 8.85% and oxygen is 19.59%.

The specific light intensity is [α = +307.6 (C = 1, methanol). From mass spectrometry, the molecular weight is 404 and the molecular formula is C24.
It is H36O5. FIGS. 1 and 2 show the ultraviolet absorption spectrum and infrared absorption spectrum of this substance. Moreover, FIG. 3 shows a 60 MHz proton nuclear magnetic resonance spectrum in deuterated chloroform with tetramethylsilane added as an internal standard. Furthermore, FIG. 4 shows a 1d nuclear magnetic resonance spectrum in deuterated methanol. Thin layer chromatography using silica gel (manufactured by Talc, black 571)
5. Rf value 0 when using dichloromethane-acetone (8:2) as the developing solvent with Kieselgel 60F254)
．． Give 47 a single spot.

Please note that this spot cannot be used with ultraviolet absorption lamps or sulfuric acid spray (
Detected by light pink to reddish brown color upon gentle heating) and iodine. The acute toxicity (LD5O) of monacolin K in mice is 19/Kf or higher, which is low toxicity.

As a result of examining the effect of monacolin K on lowering blood cholesterol using animals using various methods, its usefulness was confirmed. For example, Triton WR-1339 (trade name) was applied to a group of 5 Wistar and Imamichi rats.
(This substance has the effect of increasing blood cholesterol levels) Inject 400 m7/Kf intravenously, and at the same time monacolin Kl.
O~/K7 was administered intraperitoneally, and 14 hours later, the mice were sacrificed by exsanguination and blood cholesterol was measured by a conventional method. the result,
Blood cholesterol was reduced by 23.9% when Monacolin K was administered compared to when only Triton WR-1339 was intravenously injected. Also, weight 2.7-2.9Kf
Monacolin Kll9/K9 was orally administered to rabbits twice a day (morning and evening) for 5 consecutive days.
After 1 day and 5 days, blood was collected from the ear vein and serum cholesterol was measured. As a result, serum cholesterol levels decreased by 15% after 3 days and by 29% after 5 days when monacolin K was administered compared to before the start of administration. As described above, monacolin K 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 5 to 50 mg per day for adults.
! ! f is administered in 1 to 3 divided doses. However, larger amounts can be used if necessary. Next, examples of the present invention will be shown. Now that the above-mentioned properties have been clarified by the present invention, based on these findings, it is necessary to collect monacolin K from cultures or related substances. Various means of modification are possible.

The present invention is not limited to the examples, but includes all methods that can be easily deduced from the knowledge already described. Example 1 Monascus rubel AlOO5 strain was inoculated into a liquid medium containing 6% glucose, 2.5% peptone, 0.5% corn stabilica, and 0.5% ammonium chloride and aerobically maintained at a temperature of 28°C for 10 days. 6M hydrochloric acid was added to the obtained culture suspension 5e to adjust the pH to 3, and the mixture was extracted with an equal amount of ethyl acetate.

The extract was concentrated to dryness, dissolved in 100 Tn1 of benzene, and the insoluble material was removed. The F solution was washed twice with 100 d of 5% sodium bicarbonate solution, and then diluted with 0.2N benzene solution.
100 ml of caustic soda solution was added and stirred at room temperature. After confirming by thin layer chromatography that monacolin K had disappeared from the benzene layer, the aqueous layer was collected. This aqueous layer was converted to Pll3 with 6N hydrochloric acid and extracted twice with 100171t of ethyl acetate. The extract was concentrated to dryness to form an oily substance 26
0711fi was obtained. This oil was dissolved in benzene and crystallized, and then recrystallized from aqueous acetone to obtain 87 colorless needle-like crystals.

[Brief explanation of the drawing]

FIG. 1 shows the ultraviolet absorption spectrum of a methanol solution of monacolin K.

Claims (1)

[Claims] 1. Monacolin K, a new physiologically active substance having the following physicochemical properties. 1) Elemental analysis value: C, 71.56%: H, 8.85%:
O, 19.59% 2) Molecular weight: 404 (by mass spectrometry) 3) Molecular formula: C_2_4H_3_6O_54) Melting point: 1
57-159°C (decomposed) 5) Specific light absorption: [α]D=+307.6 (C=1, methanol) 6) Ultraviolet absorption spectrum: As shown in FIG. 7) Infrared absorption spectrum: As shown in Figure 2 (KBr
). 8) 60 MHz proton nuclear magnetic resonance spectrum: As shown in Figure 3 (deuterochloroform and tetramethylsilane as internal standards). 9) C nuclear magnetic resonance spectrum: As shown in Figure 4 (heavy methanol). 10) Solubility: Soluble in methanol, ethanol, propanol, acetone, ethyl acetate, chloroform, benzene, insoluble in hexane, petroleum ether. 11) Properties and appearance of substance: Neutral, colorless crystal. 12) Chromatography: Silica gel thin layer chromatography using dichloromethane:acetone (4:1) as the developing solvent (manufactured by Merck & Co., No. 5715 Kieselgel 6)
0F_2_5_4), a single spot at Rf 0.47 can be detected with iodine, UV absorption lamp and 50% sulfuric acid spray (warmed). 2. Monacolin K obtained by culturing a monacolin K-producing bacterium belonging to the genus Monascus and isolating monacolin K.
manufacturing method. 3. The production method according to claim 2, wherein the monacolin K-producing bacterium belonging to the genus Monascus is Monascus rubel. 4 Monacolin K-producing bacteria belonging to the genus Monascus is Monascus rubel No. 1005 strain according to claim 2.