JPS62164679A - Antibiotic oxaspirol c - Google Patents

Abstract

NEW MATERIAL:Novel antibiotic oxaspirol C shown by the formula. USE:An antibiotic having antimicrobial activity against a mold of certain kind, showing antitumor action. PREPARATION:Variant No.54-1 (FERM P-8530) of Rhodotorula glutinis T-110 is cultivated and oxaspirol is collected from a culture mixture. The antibiotic has the following physical and chemical properties. Properties of substance: oily state at normal temperature. Specific rotatory power [alpha]D<20>-430 deg. (C=0.8, CH3OH). Solubility: readily soluble in chloroform, ethyl acetate, acetone and methanol, slightly soluble in benzene, insoluble in water and n-hexane. Color reaction: positive in iodine gas and KMnO4 reagent, negative in FeCl3, ninhydrin, and indophenol reagent, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antibiotic oxaspirole C and a method for producing the same.

The present inventors succeeded in isolating a new antibiotic from the culture solution of Rhodotorula glutinis T-110 mutant strain No. 54-1 by Co irradiation, and named this substance oxaspirole C. did.

Oxaspirole C has the following physical and chemical properties.

l) Properties of substance: Oily at room temperature 2) Specific optical rotation [α) -430° (C=0.8, CH3
0H) 3) Molecular formula Cn05 4) Molecular weight 306 Measured by high-resolution mass spectrometry 5) Ultraviolet absorption spectrum The ultraviolet absorption spectrum measured in methanol was 237 nm (El(, r, 10) shows maximum absorption.

6) Infrared absorption spectrum The infrared absorption spectrum measured by the liquid film method is as shown in FIG.

7) Nuclear magnetic resonance spectrum δppm CD CJ! Measurements were made using tetramethylsilane as a reference substance in 3 solutions.

As a result, the IH-NMR spectra are shown in Table 3.
-NMR spectra are shown in Table 4.

(Margin below) Table 3 IH-NMR spectrum 60.89ppm 3Ht l, 39 2H5extet 2.03 2Hq 2.19 3.33 1Hbr, dd 3.41 28 br 3.92 1Hbr, d 4.37 2+1 4.76 18 t 4.93 Ill t 5.28 11 (br, q 5.33 ill dd 5.67 Ill 5extet5.76
1Hdd 5.83 1tl br, dd5.93
2) 1 rn Table 4 13C-NMR spectrum 6 13.567 22.169 34.548 40.258 56.089 65.447 68.614 90.010 124.003 126.586 129, 121 132, 191 133. 556 135.627 155.317 173.349 8) Soluble in chloroform, ethyl acetate, acetone, methanol Easily soluble in benzene Slightly soluble in water, insoluble in n-hexane 9) Color reaction iosogas, positive for KMnO4 reagent FeC1, 3+ Negative for ninhydrin and indophenol reagents 10) Thin layer chromatography adsorbent Silica gel developing solvent system Rf Benzene: Acetone: Methanol = 8:1:1 0.76 Butyl acetate: Benzene = 95:5 0.47 Chloroform: Ethyl acetate = 9:1 0.18 diethyl ether: acetone = 97.5: 2.5 0.67 By analyzing the above data, the planar structure of oxaspirole C was determined as shown in the following formula.

According to the present invention, Oxaspirole C is
It is produced by culturing mutant strain No. 54-1 of T. glutinis T-110 (Feikoken Kyoiku No. 8530) and collecting oxaspirole C from the culture.

Rhodotorula glutinis T-110 No. 54-1 strain which produces the antibiotic oxaspirole C of the present invention has the following mycological properties.

1) Morphological characteristics Culture is usually carried out at 30°C. Rhodotorula glutinis T.
The morphological characteristics of the -110No and 54-1 strains exhibit a hypha-like shape and propagate by budding.

2) Growth conditions on various culture media Growth conditions on malt extract agar media are good, producing candida-shaped felt-like, yellowish white, and pseudohyphae.

3) Physiological properties The physiological properties of Rhodotorula glutinis T-110 No. 54-1 strain are shown in Table 1, and the carbon source utilization is shown in Table 2.

Table 1 Physiological properties T-11 Acid production Negative Negative vitamin requirement Negative Liquefaction of biotin, B1 zetylan
Positive Negative Arbutin resolution Positive
Positive Carotene production Positive Positive Table 2 Assimilation of carbon sources
+maltose +
+Lactose +Raffinose + +Inositol
+Culture in the present invention is carried out according to the culture method for -PII yeast, and is preferably carried out by shaking culture or aerated agitation culture in a liquid medium. Examples of medium components include glucose, galactose, sucrose, maltose, lactose, raffinose, inositol, mannitol molasses, glycerin, dextrin, starch, and soybean oil as carbon sources.

Cottonseed oil and the like are particularly preferred, with glucose as the nitrogen source, soybean flour, fallen raw flour, cottonseed flour, Farmamine, fishmeal, cornstew liquor, peptone, meat extract, yeast,
Yeast extract, asparagine, sodium nitrate, ammonium nitrate, ammonium sulfate, etc., particularly preferably asparagine, and as inorganic salts, salt, phosphate, calcium carbonate.

Calcium chloride, trace metal salts, etc. are added as appropriate. Silicone oil for liquid culture.

Vegetable oils, surfactants, etc. are appropriately used as antifoaming agents.

The pH of the medium is near neutral, the culture temperature is 25°C to 33°C,
In particular, the temperature is preferably around 30°C. Changes over time in the titer of Oxaspirole C produced over the course of culture were measured using a paper disc (manufactured by Toyo Kagaku Sangyo *@, diameter ** Th1ck) using Staphylococcus aureus as the test bacterium.
Measured by assay method.

Oxaspirole C production usually reaches its maximum value after 72 to 216 hours of culture.

Oxaspirole C was added to the bacterial cells after cultivation.

It can be obtained by removing the solid portion by a filtration operation using diatomaceous earth or the like as a filtration aid or by centrifugation, and then extracting and purifying the 0 liquid or supernatant.

Oxaspirole C can be collected by utilizing its physicochemical properties, for example, using an adsorbent.

As the adsorbent, for example, activated carbon or adsorption resin Amberlite XAD-2°XAD-4, XAD-7
etc. (manufactured by Rohm and Haas) or Diaion IP
IO, 1IP20.11P20AG, HP50, etc. (Mitsubishi Chemical Industries type) are used.

Oxaspirole C can be obtained by passing a liquid containing oxaspirole C through a layer of adsorbent as described above.
After adsorbing and removing impurities contained in the liquid containing oxaspirole C, methanol water,
It can be obtained by elution using acetone water, n-butanol water, etc.

Neutral fat-soluble substances can also be extracted from a culture medium or an aqueous solution by using organic solvents that are immiscible with water, such as chloroform, ethyl acetate, n-butanol, alone or in combination. It is possible. Furthermore, it is also possible to remove mixed acidic or basic substances by washing the organic solvent layer containing oxaspirole C with dilute alkaline water, dilute acidic water, or the like.

In order to purify the oxaspirole C obtained in this way, partition column chromatography using cellulose such as Avicel (Asahi Kasei Kogyo (4151)) or Sephadex Ll+-20 (manufactured by Pharmacia), silica gel, alumina, etc. , adsorption ram chromatography using a carrier such as Florisil, reversed phase column chromatography using a reversed phase carrier, or extraction using the difference in the distribution ratio of oxaspirole C and mixed impurities to the solvent. Oxaspirole C can be purified by repeatedly using the above purification methods alone or in appropriate combinations.Alternatively, Oxaspirole C can be purified by As with lipophilic antibiotics, depending on the culture conditions, bacterial cells may exist in the culture solution.In this case, extraction is performed using a hydrophilic organic solvent such as alcohol or acetone, the solvent is removed from the extract, and then an aqueous solution is extracted. After culturing, it can be extracted and purified in the same manner as from Nitten.

Although the antibacterial activity of Oxaspirol C is generally weak, it has antibacterial activity against certain types of bacteria, and has been shown to have a survival effect on Ehrlich ascites cancer in mice, and has shown antitumor activity.

1) Antibacterial Spectrum The minimum inhibitory concentration of oxaspirole C against general Durham-positive and Durham-negative bacteria was measured by the agardilution method. The results are shown in Table 5.

Table 5 Test bacteria MICμg/adult Escherichia coli K-12> 20
0B 200 G > 200 Pseudomonas aeruginosa
> 200e Proteus vulgaris
＞200Serratia marcesce
ns > 200 Aerobacter aer
Bacillus 5 > 200 Bacillus 5
ubtilis 50brevis
50 Arthrobacter simplex
50 Corynebacterium xerosis
50M1crococcus roseus
501ysodeikticus 50
Staphylococcus aureus
50Candida albicans >
200 tropicalis > 200 Sac
Charomycopsis Jipolytica
> 200 Saccharomyces ce
revisiae > 200 Asper
gillus oryzea ＞
200 niger > 2
00Penicillium chrysogenum
＞200urticae
> 200 Fusarium oxysp
orum > 2002) Antitumor Effect The therapeutic effect of oxaspirol C on Ehrlich carcinoma in mice was tested by the following method.

The results are shown in Table 6.

In addition, the antitumor activity in the table is the average survival days of the untreated group (C)
The ratio of mean survival days (T) of the treatment group to that of the treatment group was expressed as a percentage.

Experimental method 2XIO6 pieces (7) t! ! ICR mice with tumor III cysts (♀.

After 24 hours, Oxaspirol C was intraperitoneally administered once a day for a total of 7 times.

Table 6 3) Toxicity LD5.60■/kg Next, the present invention will be explained in more detail with reference to Examples.

Example Oxaspirole C producing bacterium Rhodotorula glutinis T
-110No, 54-1 strain medium composition -1 Medium 51 Medium composition -1 Glucose 30g Asparagine 2g KtlzPO+ 1 gMgSO
4. 7H201g Naα 0.5gCaα2 ・2
H200, 5g Yeast extract 0.5g FeCJlz ・6820 2mgZnS
203 mg of O4.2H in distilled water was inoculated into 1,000 cells, and cultured with shaking at 30°C for 6 days. After the completion of the culture, the culture solution is centrifuged, and 3β ethyl acetate is added to the obtained oral extract 51 for extraction three times.

Meanwhile, methyl alcohol 31 was added to the bacterial cells, stirred, and mixed. After concentrating the methyl alcohol of this extract under reduced pressure,
Water 31 was added to the residue, and this was extracted three times with 31 ethyl acetate. This was combined with the extract from the above-mentioned oral presentation, the solvent was concentrated under reduced pressure, and benzene 10- was added to the obtained oily residue to remove insoluble materials, followed by silica gel column chromatography. The benzene solution was passed through a column of silica gel (Wako Pure Chemical Industries, Ltd.) 1cn+X75c+m filled with benzene in advance, and benzene:ethyl acetate=
The mixture was eluted with a ratio of 85:15, and then subjected to thin layer chromatography. Both fractions containing oxaspirole C were collected and concentrated under reduced pressure to obtain oxaspirole C50.

[Brief explanation of drawings]

FIG. 1 shows the ultraviolet absorption spectrum of the antibiotic Oxaspirole C (in methyl alcohol solution). FIG. 2 shows the infrared absorption spectrum (liquid film method) of the antibiotic Oxaspirole C. Figure 1 Roll 1 Second love “Outside R monument 4 collection 7R7 To 1shi) O350400nm

Claims (2)

[Claims] (1) Novel antibiotic oxaspirole C. (2) Mutant strain N of Rhodotorula glutinis T-110
o. 54-1, and oxaspirole C was extracted from the culture.
A method for producing a novel antibiotic oxaspirole C, which comprises collecting oxaspirole C.