JPH0525190A - Novel pharmacologically active substance chrysosporin and production thereof - Google Patents

Abstract

PURPOSE:To provide the subject novel substance hopeful as an agent for preventing or treating arterial sclerosis because of having a cholesterol synthesis- inhibiting activity, a lipid peroxide production-inhibiting activity and a superoxide anion radical-scavenging activity. CONSTITUTION:A novel pharmacologically active substance chrysosuporin. The appearance; dark brown powder; molecular weight: 502;FAB-MS(m/z)501 08323 (M-H); rational formula: C27H18O10; solubility: soluble in lower alcohols, chloroform, etc. The substance is produced by culturing a chrysosporine- producing bacteria [e.g. novel Chrysosporium panolam M10539 strain (FERN P-12279)] belonging to the genus Chrysosporium preferably at a temperature near to 25 deg.C and at a pH of 6-8, removing bacterial cells from the cultured solution and subsequently isolating and purifying the substance from the remained solution by a known method.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to chrysosporin, a method for producing the substance, and a strain producing the substance. Chrysosporin is a rate-limiting enzyme in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-Co.
A (HMG-CoA) reductase (EC1,1,1,
34), lowers blood cholesterol and removes active oxygen species in the body, has the effect of preventing or reducing lipid peroxides, and is thought to be due to arteriosclerosis preventing or therapeutic agents or active oxygen species Expected as a therapeutic drug for various diseases.

[0002]

2. Description of the Related Art Conventionally, mevallotin, progcol and the like have been used as therapeutic agents for arteriosclerosis.

[0003]

Although the above-mentioned therapeutic agents are excellent in potency and toxicity, they are novel compounds suitable for a more useful therapeutic drug for arteriosclerosis that have both cholesterol synthesis inhibitory ability and antioxidant activity. The long-awaited discovery.

[0004]

As a result of various searches for microbial metabolites, the present inventors have found that one strain belonging to an incomplete bacterium inhibits cholesterol synthesis, inhibits lipid peroxide production, and superoxide anion radicals. It was found that it produces a physiologically active substance, chrysosporin, which has a scavenging action. The present invention has been completed based on the above findings.

That is, the present invention relates to a novel pharmacologically active substance, chrysosporin, which has the following physicochemical properties. (A) Exterior: Dark brown powder (b) Molecular weight: 502 FAB-MS (m / z)
^{501,08323 (M-H) - (} c) rational _{formula; C 27 H 18 O 10 (} d) solubility; lower alcohol, chloroform,
Soluble in acetic ester (e) Ultraviolet absorption spectrum; shown in FIG. (F) Red external absorption spectrum: The spectrum measured with a potassium bromide tablet is shown in FIG. (G) Hydrogen nuclear magnetic resonance spectrum: The spectrum measured in deuterated dimethyl sulfoxide is shown in FIG. (H) Carbon nuclear magnetic resonance spectrum: The spectrum measured in deuterated dimethyl sulfoxide is shown in FIG.

The above novel physiologically active substance chrysosporin is
It is obtained by culturing a chrysosporin-producing bacterium belonging to an incomplete bacterium in a nutrient medium to produce and accumulate chrysosporin, and collecting chrysosporin from this culture. M10539 isolated from the soil in the suburbs of Tokyo (Akigawa City, Itsukaichi Town) is a typical chrysosporin-producing bacterium.
Strain (Microtech Lab, No. 12279 "FERMP-1227"
9) ”.

The bacteriological characteristics of the M10539 strain are shown below. Giant colonies by potato-glucose agar plate culture, macroscopic observation of slide culture, and morphological observation by microscope were performed. The colonies reach a diameter of 1.5 to 2 cm after culturing at 25 ° C. for 14 days, swell with dense mycelium without spreading, and have various powdery and fluffy surfaces. The color was brown on both the front and back sides, and the agar was colored brown. Microscopic observation shows that hyphae are thin with 1 to 1.5 μm and have septa, and conidia stalks and hyphae cannot be distinguished, and conidia stalks branch in a steep angle in a ring-like manner. It was shown to have a shape of 2-4 × 2-3 μm. As a result of observation of growth at 4, 20, 25, and 30 ° C, growth was slightly suppressed at 4, 20, 25 ° C, and no growth was observed at 30 ° C. These results and Carmichael's description (Carmichel, JW Can. J. Bot 4
0 , 1137 (1962) to identify the M10539 strain as a chrysosporium panoram (link) fuse.

The chrysosporium panoram strain used in the present invention includes, for example, irradiation treatment with ultraviolet rays and ⁶⁰ Co, nitrogen mustard, nitrous acid, N-methyl-N'-nitro-nitrosoguanidine, 2 aminopurine and the like. The production capacity of chrysosporin can be enhanced by commonly used breeding means such as mutation treatment with a mutagen, transduction, transformation, and cell fusion.

In order to produce the chrysosporin of the present invention, a microorganism having the ability to produce chrysosporin is cultured in a nutrient medium so that chrysosporin is produced and accumulated in the culture and then collected. As the culture method, a deep culture method by liquid culture is advantageous. The medium used for the culture may be any medium containing a nutrient source used by the Chrysosporin panoram hughes strain. As the nutrient source, known ones conventionally used for culturing mold can be used. For example, the carbon source can be glucose or D-.
Xylose, D-fructose, sucrose, lactose, glycerol, dextrin, starch, starch syrup, soybean oil and the like can be used alone or in combination. As inorganic and organic nitrogen sources, ammonium chloride, ammonium sulfate, ammonium nitrate, urea, sodium nitrate, peptone, meat extract,
Yeast extract, dry yeast, corn steep liquor,
Soybean flour, pharma media, oatmeal, casamino acid, bacto soyton, sorible vegetable protein, etc. can be used alone or in combination.

In addition, inorganic salts such as sodium chloride, magnesium sulfate, copper sulfate, zinc sulfate, manganese chloride, calcium carbonate and phosphate can be added, if necessary.
Organic substances such as nucleic acids, amino acids, vitamins and inorganic substances that promote the growth of the strain and the production of chrysosporin can be appropriately added. When foaming is remarkable during culture, vegetable oils such as soybean oil and flaxseed oil and pronal 1
(Toho Chemical Co., Ltd.), silicon KM-70 (Shin-Etsu Chemical Co., Ltd.) and other petroleum antifoaming agents may be added appropriately. The culture temperature is preferably in the range of 18 to 28 ° C, and preferably 25.
It is desirable to carry out the culture at a temperature of around 0 ° C. and a pH of the medium of 5 to 10, preferably 6 to 8. The culturing period varies depending on the composition and temperature of the medium, but generally, when culturing is performed for 3 to 12 days, chrysosporin is produced and accumulated in the culture.

When the production amount in the culture reaches the maximum, the culture is stopped, and the cells are removed by centrifugation or filtration.
The active substance is extracted from the filtrate. For the purification / isolation of the substance, a separation / purification method generally used for isolating a microbial metabolism product from the culture is used. That is, extraction with various solvents, gel filtration, high performance liquid chromatography (HPLC) and the like can be appropriately combined and used. For example, the culture is filtered and the filtrate is extracted with ethyl acetate three times under acidity. The ethyl acetate solution is dehydrated with inorganic sodium sulfate and then concentrated under reduced pressure. It is extracted 3 times with a 5% sodium hydrogen carbonate solution (pH 8.5). The pH of the 5% sodium hydrogen carbonate layer is adjusted to 2.5 with hydrochloric acid, the generated precipitates are collected by centrifugation, washed with water, and then freeze-dried.

The obtained dried precipitate is dissolved in a weak alkaline solution,
Solubility Extract 3 times with butyl acetate under hydrochloric acid acidity (pH 3). The butyl acetate layer is dehydrated with anhydrous sodium sulfate and then concentrated under reduced pressure to dryness. The obtained crude powder is dissolved in a 20 mM potassium phosphate solution (pH 7.4) and purified by Bio-gel P6 column chromatography equilibrated with the same buffer solution. The developing solution is eluted with the same buffer to obtain the active compartment. This is adjusted to pH 3 with hydrochloric acid and then extracted 3 times with ethyl acetate. The separated ethyl acetate layer is dried over anhydrous sodium sulfate and then concentrated under reduced pressure to dryness. Dissolve this powder acetonitr / 0.1% phosphoric acid solution (40:60),
Apply to HPLC using a silica ODS column, elute with the same developing solvent, and collect the active fraction. This is extracted with ethyl acetate, dehydrated with anhydrous sodium sulfate, and then concentrated under reduced pressure to dryness to obtain a purified sample.

The activity of chrysosporin in the culture and the crude fraction was measured by the inhibition rate of HMG-CoA reductase activity. The compound of the present invention, chrysosporin, is expected as a drug such as an arteriosclerosis therapeutic agent, an anti-inflammatory agent, an autoimmune disease therapeutic agent, an anti-myocardial infarction agent, an antiarrhythmic agent, and a cerebral edema improving agent.

When the compound of the present invention is used as a medicine, it is administered as an injection, an oral preparation, a suppository, etc. alone or as an excipient. Any excipient may be used as long as it is pharmacologically acceptable, and its type and composition depend on the administration route and administration method. For example, as the liquid excipient, water, alcohol, or animal or vegetable oil such as soybean oil, peanut oil, mineral oil, or synthetic oil can be used. As the solid excipient, sugars such as maltose and sucrose, various amino acids, cellulose derivatives such as hydroxypropyl cellulose, organic acid salts such as magnesium stearate and the like can be used.

In the case of injections, physiological saline, various buffer solutions, sugar solutions such as glucose, inositol and mannitol, and glycol solutions such as ethylene glycol and polyethylene glycol are desirable as excipients. Also, a lyophilizer together with sugars such as inositol, mannitol, glucose, mannose, maltose, sucrose and excipients of amino acids such as phenylalanine, and a suitable solvent for injection at the time of administration, for example, sterile water, glucose solution Alternatively, it can be dissolved in an electrolyte solution, an amino acid solution or the like and administered intravenously or intramuscularly.

In the case of oral preparations, it is preferable to form tablets, capsules, powders, granules, liquids, dry syrups, etc. together with the liquid or solid excipients.
In addition, as a pellet, a transdermal agent, a topical agent such as a mucosal agent may be used.

The content of the present compound in the preparation is usually 0.001-1% by weight, preferably 0.01-.
It is 0.1% by weight. For example, in the case of an injection, it is usually 0.01 to 0.05% by weight. In the case of an oral preparation, the amount is 0.005 to 10% by weight, preferably 0.05 to 5% by weight, and the balance is the excipient.

The dose is determined according to the patient's age, body weight, symptom, purpose of treatment and the like. Generally, for parenteral administration, for example, as a suppository, 0.1 mg to 10 mg / kg / day, The injection is 0.1 mg to 10 mg / kg / day, and the oral administration is 0.5 mg to 100 mg / kg / day.

[0019]

[Work]

1. Measurement of inhibitory activity of HMG-CoA reductase 1) Preparation of crude enzyme solution This enzyme system was prepared by the method of Knauss et al. (J. Biol. Chem. 2) .
34 , 2835 (1959)) by modifying Endo et al.'S method (Biochim. Biophys Acta 489 , 119 (197).
According to 9)), it was prepared from rat liver as follows. About 200 g of Wistar male rats were used, and cholestyramine (Dowex 1-X) was used to obtain an enzyme with high specific activity.
After free ingestion of commercially available powdered feed (Oriental yeast) containing 2, C1 type, Dow Chemical) for 5 days, decapitated at midnight, the liver was taken out, and immediately after ice cooling, 2 mM Mg was added.
The plate was washed with 0.1 M phosphate buffer (pH 7.4) containing Cl ₂ and 15 mM nicotinamide for 5 seconds. The following operations were all performed at 4 ° C. or under ice cooling. After washing, remove water with filter paper, and chop the liver with a scalpel.
The same buffer solution (10 ml) was added to, and the mixture was lightly ground (4 strokes) with a potter-type homogenizer of "loose fitting". At this time, since HMG-CoA degrading enzyme exists in mitochondria, be careful not to overly grind the liver and destroy the mitochondria. Then, the supernatant obtained by centrifugation at 700 xg for 5 minutes was further centrifuged at 12,000 xg for 30 minutes to remove the nucleus and mitochondria. The fat was removed by cotton filtration, and the supernatant was subjected to ultracentrifugation at 105,000 xg for 60 minutes to obtain a microsome fraction. The microsome fraction was suspended in 0.1 M potassium phosphate buffer (pH 7.4) and then ultracentrifuged at 105,000 × g for 60 minutes, and suspended in the same buffer at 3 g / ml. The resulting microsomal fraction was aliquoted, stored at -80 ° C and thawed immediately before use. The protein was quantified by the Lowry method.

2) Method for measuring HMG-CoA reductase inhibitory activity 10 mM dithiothreitol (DTT), 10 mM E
DTA, 100 mM potassium phosphate buffer (pH 7.
4), microsome fraction 0.6 mg / ml, 10 as substrate
^{0mM DL- [3- 14 C] HMG} -CoA (6.66
A total of 25 μl of the reaction solution containing MBg / mmol) is added to a microtube (snap seal tube 1.5 ml volume) and the following procedure is performed. 37 ° C under aerobic conditions, 30
After incubation for 5 minutes, the reaction was stopped by adding 5 μl of 2N HCl, and further adding 10 μl of DL- [5- ³ H] meva.
lonolactone (100,000 dpm) was added as an internal standard and incubated for 15 minutes. 30 μl of the reaction solution was added to a silica gel TLC plate (Kieselgel 60
(F ₂₅₄ , Merck) spotted several times. For this, 20 mM mevalonola as a carrier was prepared beforehand.
5 μl of ctone was spotted and placed. After development with benzene / acetone (1: 1), the solvent was evolved and the plate was developed with iodine. Rf value of mevalonolacton 0.
Scrap the 4 to 0.6 part into a vial with a micro spatula, add 4 ml of a toluene scintillator, and add ³ H and ¹⁴
Measure the radioactivity of C (Packard Tri-Carb Liquid S
cintillation Spectrometer 3255 type), and the amount of [ ¹⁴ C] mevalonolactone produced was quantified to determine the enzyme activity. The results of the inhibitory activity are shown in Table 1.

[0021]

As is apparent from this table, the HMG-CoA reductase inhibitory activity value (I of chrysosporin of the present invention
C ₅₀ ) is about 50 μg / ml.

2. Antioxidant activity The antioxidant activity of chrysosporin of the present invention is shown together with the measuring method. A total of 600 μl of a reaction solution containing 30 mM Tris-hydrochloric acid buffer, 0.2 mM bleomycin sulfate, 0.2 mM ferric sulfate and 1.3 mM sodium arachidonate is reacted at 37 ° C. for 5 minutes to produce lipid peroxide. The Yagi method (vitamin 37 , 105-112,
1968) and quantified with thiobarbituric acid (TBA). The results of antioxidant activity are shown in Table 2.

[0024]

As is apparent from this table, the chrysosporin of the present invention has an antioxidant activity which is about 3.5 times as high as that of vitamin E in specific activity.

[0026]

As is clear from the above results, the chrysosporin of the present invention can be expected as a novel therapeutic agent for arteriosclerosis by inhibiting HMG-CoA reductase and having an antioxidant action. Examples of the present invention will be shown below, but this is merely an example and does not limit the present invention in any way, and various modifications are possible.

[0027]

【Example】

Example 1. (1) 80 ml of potato-dextrose medium containing 2% of glucose was dispensed into a fermentation 500 ml Sakaguchi flask, and the temperature was kept at 120 ° C for 2
It was autoclaved for 0 minutes. A platinum loop of Chrysosporium panorum Hughes M10539 strain (Microtechnological Research Institute No. 12279) was ingested into this, and cultured at 120 strokes / min at 25 ° C for 4 days on a reciprocal shaker. To do. Add 1 medium of the same composition as above.
0 liter was prepared, and this was dispensed into a 500 ml Sakaguchi flask for each 80 ml and sterilized at 120 ° C. for 20 minutes, and seed culture was inoculated at a rate of 2%. Shaking was carried out at 25 ° C for 12 days. The obtained culture solution is centrifuged (10,000 ×
(g, 20 min) to obtain a supernatant.

(2) Purification By adding hydrochloric acid to 5 liters of the above-mentioned supernatant to adjust the pH to 2.5,
It was extracted 3 times with 2 liters of ethyl acetate. After dehydration with anhydrous sodium sulfate, the mixture was concentrated under reduced pressure to 1 liter with a rotary evaporator. Add 1 liter of 5% NaHC
It was extracted 3 times with an O ₃ solution (pH 8.5). 5% NaH
The CO ₃ layer was adjusted to pH 2.5 with hydrochloric acid, and the resulting precipitate was collected by centrifugation, washed with distilled water, and dried by a freeze dryer (5.53 g). The dried precipitate obtained was dissolved by adding 100 ml of distilled water while adding 3N NaON solution little by little, adjusting the pH to 3 with 3N HCL, and extracting 3 times with an equal amount of butyl acetate. The butyl acetate layer was dehydrated with anhydrous sodium sulfate, and then concentrated under reduced pressure with a rotary evaporator.
It was dried (1.88 g).

The butyl acetate extract was added to 20 mM potassium phosphate.
Dissolve in 100 mg / ml with a glucose solution (pH 7.4)
And add 1 ml of it to 300 mM NaCl and 0.02% N
aN ₃20 mM potassium phosphate buffer (pH 7.
Biogel P-6 equilibrated in 4) (fractionation range)
1,000-6,000 Dalton) column (26 x 40
0 mm) and eluted with the above equilibration buffer (25 ml)
/ H). Divided into 150 fractions of 4.2 ml each
I took it. Fraction No. with strong inhibitory activity on the low-molecular side
Collect 100 to 125 and pH 3 with 6N HCl.
It was adjusted to 0 and extracted 3 times with an equal volume of ethyl acetate. separated
After dehydrating the ethyl acetate layer with anhydrous sodium sulfate, rotate the rotor.
It was concentrated under reduced pressure with an evaporator and dried (310.4m
g). Bio-Gel P-6 active fraction dried product was acetonited.
Lil / dissolved in 0.1% phosphoric acid water (40:60) (20 mg
/ Ml). 200 μl each of which is silica ODS color
Mu (10 x 250^mm) Was used to
Elute with nitrile / 0.1% phosphoric acid water (40:60),
The active fraction was collected. The active fraction collected was taken up with ethyl acetate.
Extracted, dehydrated with inorganic sodium sulfate, and then rotary evaporated.
Concentrate to dryness under reduced pressure with a porator and purify chrysosporin.
35 mg of the product was obtained.

[Brief description of drawings]

FIG. 1 shows an ultraviolet absorption spectrum of chrysosporin.

FIG. 2 shows an infrared absorption spectrum of chrysosporin (measured with a potassium bromide tablet).

FIG. 3 shows a hydrogen nuclear magnetic resonance spectrum of chrysosporin (measured in deuterated dimethyl sulfoxide).

FIG. 4 shows a carbon nuclear magnetic resonance spectrum of chrysosporin (measured in deuterated dimethyl sulfoxide).

Continuation of front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display area C12R 1: 645)

Claims (4)

[Claims] 1. A novel pharmacologically active substance, chrysosporin, having the following physicochemical properties: (a) appearance; dark brown powder (b) molecular weight; 502FAB-MS (m / z) 5
^{01,08323 (M-H) - (} c) rational _{formula; C 27 H 18 O 10 (} d) solubility; lower alcohol, chloroform,
Soluble in acetic ester (e) Ultraviolet absorption spectrum; shown in FIG. (F) Red external absorption spectrum: The spectrum measured with a potassium bromide tablet is shown in FIG. (G) Hydrogen nuclear magnetic resonance spectrum: The spectrum measured in deuterated dimethyl sulfoxide is shown in FIG. (H) Carbon nuclear magnetic resonance spectrum: The spectrum measured in deuterated dimethyl sulfoxide is shown in FIG. 2. A method for producing chrysosporin, which comprises culturing a chrysosporin-producing bacterium belonging to the genus Chrysosporium, accumulating chrysosporin in the medium, and then collecting chrysosporin from the medium. 3. A strain belonging to the genus Chrysosporium is Chrysosporium panoram M10539 strain
279). 4. Chrysosporium panoram M10539 strain (Microtechnological Research Institute No. 12279) and its mutant strain producing chrysosporium