JPH05255304A - New substance cl190y1, its production and antioxidant containing the same substance as the active component - Google Patents

Abstract

PURPOSE:To provide the subject new substance CL190Y1 capable of stabilizing a biomembrane, edible salt, edible oil, a fat, wax, vitamin, a perfume, etc., and useful as an antioxidant. CONSTITUTION:A new substance CY190Y1 represented by the formula. The new substance CY190Y1 represented by the formula can be obtained by culturing a CL190Y1-producing microorganism (Streptomyces aeriouvifer-CL-190 is especially suitable. Bikoken stipulation No. 2545, FERM-BP-2545) belonging to Streptomyces and separating and collecting the objective CL190Y1 from the cultured material.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to a novel substance having an antioxidant property, a method for producing the same, and an antioxidant having the same as an active ingredient. BACKGROUND OF THE INVENTION In the case of storing foods, in addition to spoilage by microorganisms, deterioration due to oxygen in the air poses a serious problem. This change appears as an off taste and an offensive odor due to rancidity of oil and fat, discoloration and oxidative discoloration of pigments. In order to prevent this oxidative deterioration phenomenon, there are a method of putting it in a packaging container such as a bottle and a canned product to cut off contact with air, and a method of adding an antioxidant. Antioxidants include water-soluble antioxidants such as ascorbic acid and erythorbic acid, and oil-soluble antioxidants such as gallic acid esters. The former is mainly used to prevent the oxidation of pigments, and the latter is used to prevent the oxidation of fats and oils.
Oils and fats are easily oxidized in the presence of enzymes, water, metal salts, heat, light, etc., and gradually progress to a gradual initial stage, and after this period, they proceed rapidly. Oxidation is due to the action of oxygen on the double-bonded carbon of unsaturated fatty acids in fats and oils, producing free radicals or peroxides,
It is believed that this causes a chain reaction to be decomposed into aldehydes, ketones, acids and the like. At this time, enzymes, metal salts, etc. act as catalysts, and heat and light supply energy. The higher the degree of unsaturation, the faster this reaction rate. Antioxidants act on this free radical or peroxide, interrupting the chain reaction and oxidizing itself. On the other hand, antioxidants have hitherto been used, for example, various diseases caused by platelet aggregation, inflammation, liver damage, arteriosclerosis, hemolysis, aging or senile dementia, retinopathy, lung damage, heart and lung damage caused by certain drugs. , Ischemic vascular disease, stroke, myocardial infarction, cerebral hemorrhage, cerebral infarction, cerebral thrombosis, cataract, etc., have been proposed and are widely used (JP-A-57-14).
5871, JP-A-57-188586, JP-A-60
-142919, JP-A-63-30415, JP-A-63-117090, JP-A-63-218649,
JP-A-63-130548, JP-A-63-13059
No. 0). [Problems to be Solved by the Invention] An object of the present invention is to provide a novel substance having an antioxidant property, a method for producing the same, and an antioxidant having the same as an active ingredient. [Means for Solving the Problems] The present invention provides a novel substance CL190Y1 represented by the following general formula (I), a method for producing the same, and an antioxidant containing the same as an active ingredient.

[Chemical 2] The present invention will be described in detail below. Novel substance CL1 of the present invention
90Y1 is CL190Y belonging to the genus Streptomyces
Cultivate 1 production bacterium and separate CL190Y1 from the culture.
It can be advantageously produced by collecting. CL
As the 190Y1 producing bacterium, any one can be used as long as it belongs to the genus Streptomyces and has CL190Y1 producing ability. Specifically, the present inventors separated Streptomyces erio- Ubiffer ( Strepto
myces aeriouvifer ) CL-190
(Hereinafter referred to as “CL-190 strain”) can be advantageously used. In addition, it is also possible to isolate a suitable substance from the natural world by a conventional method for isolating an antibiotic-producing strain. In addition, CL190Y1 including Streptomyces CL-190 strain
The production bacteria of C
It goes without saying that a product with an increased productivity of 190Y1 can also be used. The CL-190 strain The CL-190 strain found by the present inventors as a strain of the genus Streptomyces having CL190Y1 production ability is
It has the following contents. 1) Origin and Deposit No. CL-190 strain is an actinomycete isolated from soil collected in Okinawa Prefecture, and was deposited on August 5, 1991 at the Institute of Microbial Science and Technology of the Agency of Industrial Science, Article No. 2545 "
(FERM BP-2545) number. 2) Mycological properties The mycological properties of the CL-190 strain are as follows. "Characteristics" of the actinomycete CL-190 strain are "Experimental method for identifying actinomycetes" (edited by the Japan Actinomyces research group) and International Streptomyces Project (Inter-national).
Streptomyces Project, ISP)
Was performed according to the method of. Morphological characteristics : The vegetative (basic) hyphae of the strain grow well on various agar media while growing without fragmentation of hyphae. The hyphae later give rise to unequal length segments, which may partially result in empty cells devoid of cytoplasm. Even in various liquid media,
Occasionally, similar empty cells are partially produced, and they may be divided into rod-shaped cells by long-term culture. The aerial mycelium extends while uniaxially branching, and when straight, in the middle of the main axis or branch, a large number of curved or looped short branches branch into dense tufts. The aerial hyphae do not form any special sporulated hyphae, but the whole hyphae are segmented into spore chains, and are segmented so as to be broken at the segmental parts to produce segmented spores (spore-like bodies). The segmental spore chain consists of 10 to 50 or more spores, and usually exhibits a straight shape (RF form), and sometimes a curved shape or a loop shape (RA form). Segmental spores are chopped cylindrical, size 0.5-1.0x0.7-
1.6 μm, smooth surface, non-motile. Sometimes spherical to oval, size 0.4-0.6 x 0.6-1.4μ
m, smooth surface, long sac-like sheaths sparsely containing nonmotile spore-like bodies are observed, but no true sporangia.
No other special morphology is observed. Chemical characteristics : Diaminopimelic acid in whole cell hydrolyzate is LL-type and contains no meso-type isomer (cell wall type is type I). Culture characteristics : Table 1 shows the culture properties. The colony color on the surface of the village is pale blue (18ec) to light grayish blue (19dc to 19)
fe) to light grayish blue-green (22fe), and is a blue series. The backside color of the settlement is unclear or olive or light brown, and both colors change depending on pH. The diffusible pigments in the medium slightly produce melanin pigment and light yellow or light brown pigment in a certain medium. Physiological characteristics : Table 2 shows the physiological properties. This strain is mesophilic, positive for melanin, sucrose, raffinose,
Do not use D-mannite.

[Table 1]

[Table 2]

[Table 3] Taxonomic position : Based on the above characteristics, the genus and species to which this strain belongs were searched for taxonomy of known actinomycetes.
The known taxon is “Bacterial Name Approval List, 1980” (Ap
probed Lists of Bacterial
Names; Int. J. Syst. Bacteri
ol. , 30, 225-420, 1980) and its supplements (ibid. 35, 382-407, 1985 and ibid. 35).
Listed in each issue after the volume).
The features of the genus rank of this strain are summarized as follows;
The cell wall type is I, aerial hyphae are formed, and the whole hyphae are segmented to form nonmotile segmented spores (nocardioform). An actinomycete with this feature is Nocardioides.
Oides) Although the genus is only genus differs from the present strain in that the two mycelia in a nutrient-air exhibit easily disintegrate Corynebacterium form bacteria like on the agar medium. The spore chain of this strain is 10-5
Focusing on the feature of 0 or more spores, streptomycetes
It corresponds to the genus Strepto-myces . Although the same genus is defined as not dividing and dividing both nutritional and aerial hyphae other than spore chains, there are a few species showing nocardioform among the belonging species. The fungal species that had been classified as a separate genus due to its special morphology, such as and sclerotium, were also integrated into the same genus. Taking these things into consideration, it was decided that this strain also belong to the genus Streptomyces . The characteristics of the species rank of this strain are summarized as follows; spore chains are usually RA form when RF form, spore surface is smooth, flora color is blue series, colony backside color is unclear and olive or bright. It is brown and changes color depending on pH, melanin pigment formation is positive, diffusible pigment slightly produces light yellow or light brown color, and sucrose, raffinose and D-mannite are not used. No known species matching these characteristics were searched. As the most approximate species, S. Cerestis (S.
(caelestis) was found, but there is no segmentation of the whole hyphae, the spore chain does not show the RF form in the spiral form from the RA form, and differs from this strain in that it utilizes sucrose and raffinose. From the above results, it is judged that this strain should be a new strain. Therefore, this strain is strept
Mrs. Elio Ubiffer (Strepto-myc
es aeriouvifer) , CL-19
0 stock is designated as standard stock. Cultivation / CL190Y1 production compound CL190Y1 is obtained by aerobically culturing CL190Y1 producing bacteria belonging to the genus Streptomyces in an appropriate medium,
It can be produced by separating and collecting the target substance from the culture. The medium can contain any nutrient source that can be utilized by CL190Y1 producing bacteria. Specifically, for example, glucose as a carbon source, maltose,
Starch and oils and fats can be used, and as nitrogen sources, organic substances such as soybean flour, cottonseed meal, dried yeast, yeast extract and corn steep liquor and ammonium salts or nitrates such as ammonium sulfate, sodium nitrate and ammonium chloride are used. it can. Further, if necessary, inorganic salts such as sodium chloride, potassium chloride, phosphate, and heavy metal salt can be added.
In order to suppress foaming during fermentation, a suitable antifoaming agent, such as silicone oil, can be added according to a conventional method. As the culture method, the aerobic liquid culture method is most suitable, as in the generally used antibiotic production method. A suitable culture temperature is 20-37 ° C, preferably 25-30 ° C. With this method, the production amount of CL190Y1 reaches the maximum after 3 days of culture by shaking culture and 2 days of culture by aeration and agitation culture. In this way, a culture in which CL190Y1 is accumulated is obtained. In the culture, CL190Y1 is mostly present in the bacterial cells. CL19 from such cultures
Any purposeful method can be used to collect 0Y1. One of the methods is based on the principle of extraction. Specifically, for CL190Y1 in the bacterial cells, the bacterial cell aggregates obtained by filtration, centrifugation, etc. are treated with methanol, ethanol, acetone, etc. and recovered. There are ways. The culture itself may be subjected to the above extraction operation without separating the cells. Countercurrent partitioning with an appropriate solvent can also be included in the extraction category. The cell extract is C
L190Y1 is dissolved and extracted with a water immiscible solvent such as ethyl acetate. CL thus obtained
If the 190Y1 solution is concentrated to dryness under reduced pressure, CL190Y1
A crude sample is obtained. CL190 thus obtained
In order to further purify the crude preparation of Y1, the extraction method and the adsorption method described above may be combined with a gel filtration method, etc., if necessary, and carried out a required number of times. For example, column chromatography using an adsorbent such as silica gel and a gel filtration agent such as “Sephadex LH-20” (manufactured by Pharmacia) can be appropriately combined and carried out. Specifically, for example, a crude product of CL190Y1 is purified by applying a crude product of CL190Y1 to a “Sephadex LH-20” column, eluting the active fraction with a mixed solution of chloroform-methanol (1: 1), and concentrating to dryness. Is obtained. The CL190Y1 thus obtained had the following physicochemical properties, and as a result of X-ray crystallography, it was found to have the chemical structure represented by the above formula (I). Compound 7 of the present invention
-Demethylnaphtherpine (7-demethyl na
The physicochemical properties of pterpin) are as follows. Appearance Yellow powder Melting point 217-218 ° C (decomposition) [Α] ²¹ _D (C0.15%, CHCl ₃ ) -58
6 ° molecular formula C ₂₀ H ₂₀ O ₅ high resolution FAB mass spectrum measurement value 341.1396 (M + H) ⁺ calculated value 341.1389 UV absorption spectrum λ max nm (ε) (shown in FIG. 1) in MeOH (ε) 266 nm ( 18800), 306
(12400) 410 (4200) 0.01N-NaOH-MeOH (ε) 229 (25200), 292 (20700) 328 (8600), 505 (4200) Infrared absorption spectrum (KBr disk method) (Fig. 2) Shown) (KBr) ν, cm ⁻¹ 3400, 1620, 16
10, 1580, 1280, 1220 Nuclear magnetic resonance spectrum (in deuterated chloroform) Proton (500 MHz) (shown in FIG. 3) Carbon 13 (125 MHz) (shown in FIG. 4) 1.183.1 2.153. 0 3.123.8 4.183.6 4a. 135.0 5.18.3 (107.2) 7.09 (1H) 6.164.3 7.107.2 (108.3) 6.53 (1H) 8.162.9 8a. 108.9 9.31.1 3.48 (1H) 10.120.0 6.04 (1H) 11.136.1 12.29.7 1.95 (2H) 13.20.4 1.28, 1.95 (2H) 14.39.7 1.76 (1H) 15.80.6 16.23.5 1.66 (3H) 17.25.7 1.55 (3H) 18.25.01 .33 (3H) 6-OH 6.37 (1H) 8-OH 11.92 (1H) The compound CL190Y1 of the present invention has antioxidant activity as shown in the following biological activity test. Therefore, CL190Y1 of the present invention can be used as an antioxidant. Biological activity CL190Y1 showed lipid peroxidation inhibitory activity in rat liver microsomes. The produced lipid peroxide was detected by the thiobarbituric acid method. For example, 0.174MKC
0.75 ml of the same buffer was added to 10% rat liver microsomes (0.5 ml) suspended in 25 mM Tris-HCl buffer (pH 7.4) containing 15 μg / m 2.
CL190Y1 0.0 at a concentration of 1 to 200 μg / ml
5 ml and 0.015 M ascorbic acid 0.5 ml
Is added. (Radicals are generated by trivalent iron and ascorbic acid in the reaction solution to oxidize the lipids in liver microsomes.) After incubating this reaction solution at 37 ° C for 1 hour, 0.5 ml of 20% trichloroacetic acid was added to stop the reaction. Then, the mixture was centrifuged at 3000 rpm for 10 minutes, 1 ml of the reaction supernatant was dispensed, and 0.67% thiobarbituric acid was added.
After incubating with 5 ml at 100 ° C for 20 minutes 5
The extinction coefficient was measured at a wavelength of 30 nm. CL190Y1
Had an antioxidant activity at a final concentration of 3.5 μg / ml or more, and had an IC ₅₀ value of 5.27 μg / ml. on the other hand,
Other than using Vitamin E instead of CL190Y1,
The antioxidant activity of vitamin E was measured by the same method as above. As a result, the IC ₅₀ value of vitamin E was 9.44 μg.
/ Ml. CL190Y1 showed lipid peroxidation inhibitory activity against a rat brain homogenization suspension. The produced lipid peroxide was detected by the thiobarbituric acid method. For example, rat brain cells containing 0.174M KCl 25m
Homogenize in M Tris-HCl buffer pH 7.4 and dilute to 10% (W / V). 0.5 ml of this brain homogenizing solution and 0.75 ml of 25 mM Tris-hydrochloric acid buffer containing 0.174 M KCl and 125 μg / ml-
CL190Y1 solution with a concentration of 250 μg / ml 0.05
ml and 0.5 ml 0.015M ascorbic acid are added. (Radicals are generated by trivalent iron and ascorbic acid in the reaction solution to oxidize lipid in liver microsomes.) After incubating the reaction solution at 37 ° C. for 1 hour,
The reaction was stopped by adding 0.5 ml of 20% trichloroacetic acid, centrifuged at 3000 rpm for 10 minutes, and 1 ml of the reaction supernatant was dispensed to give 0.67% thiobarbituric acid 0.5.
After incubation with ml at 100 ° C for 20 minutes 53
The extinction coefficient was measured at a wavelength of 0 nm. As a result 3.5μ
An inhibitory effect on lipid peroxide was observed at a concentration of g / ml or more. The antioxidant of the present invention can be used to stabilize biological membranes, foods, edible oils, fats, waxes, vitamins, flavors and the like. In that case, about 1.
A concentration of 0 to 1000 ppm, preferably about 2.5 to 20
Disperse at a concentration of 0 ppm (based on the weight of the substance to be stabilized). In the following, "%" is "W / V%"
Is. Example 1) Preparation of Seed Mother The medium used was prepared by dissolving the components having the following composition in 1 liter of water and adjusting the pH to 6.2. Glucose 25.0 g Soybean flour 15.0 g Yeast extract 2.0 g Calcium carbonate 4.0 g Dispense 100 ml of the above medium into a 500 ml Erlenmeyer flask equipped with warts, and after sterilization, inoculate 1 platinum loop of Streptomyces CL-190 strain from a slant. The seeds were cultured with shaking at 27 ° C for 2 days. 2) Culture The medium used was prepared by dissolving the components having the following composition in 1 liter of water and adjusting the pH to 6.2. Glucose 25.0 g Soybean flour 15.0 g Yeast extract 2.0 g Calcium carbonate 4.0 g Dispense and sterilize the above medium into 30 liter 50 liter jar fermenters, and 600 ml of the above seeds
Was added at 27 ° C for 2 days, 200 rpm, 0.4V
Aeration-agitation culture of VM was performed. 3) Collection of CL190Y1 After culturing under the above conditions, the culture solution (60 liters) was filtered, the filtrate was extracted with an equal amount of acetone, and the extract was concentrated to dryness. This was dissolved in a small amount of hexane-ethyl acetate (4: 1) to remove insolubles, and then a column of silica gel (“Wakogel C200” manufactured by Wako Pure Chemical Industries) (5 cmφ × 34c).
m) and eluted with hexane-ethyl acetate (4: 1), and then the developing solvent was chloroform / methanol (1
It changed to 00/1) and eluted. Chloroform / methanol eluate fractions were collected, concentrated, and subjected to silica gel thin layer chromatography under the condition of chloroform / methanol (50/1).
Elution with methanol (50/1). The eluate was filtered to remove silica gel and concentrated to obtain 10 mg of CL19.
Yellow crystals of 0Y1 were obtained. The thin layer used for thin layer chromatography is 5 mm thick manufactured by Merck.

[Brief description of drawings]

FIG. 1 is an ultraviolet absorption spectrum of CL190Y1 in CL190Y1 in methanol (solid line) and 0.01 N sodium hydroxide-methanol (dotted line).

FIG. 2 is an infrared absorption spectrum of CL190Y1 by a KBr disk method.

FIG. 3: 5 of CL190Y1 in deuterated chloroform
It is a 00 megahertz proton nuclear magnetic resonance spectrum.

FIG. 4 1 of CL190Y1 in deuterated chloroform
It is a 25 MHz carbon 13 nuclear magnetic resonance spectrum.

[Procedure amendment]

[Submission date] July 10, 1991

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Claim 1

[Correction method] Change

[Correction content]

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display area // (C12P 17/06 C12R 1: 465)

Claims (3)

[Claims] 1. A novel substance CL190 represented by the following formula (I):
Y1. [Chemical 1] 2. A compound of formula (I), CL190Y1.
Is an antioxidant. 3. CL190Y belonging to the genus Streptomyces
Cultivate 1 production bacterium and separate CL190Y1 from the culture.
A method for producing CL190Y1 which is characterized by collecting.