JPS6210517B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to new antibiotic substances 248-Sq-2A and 248-Sq-2B having anti-lactic acid bacterium activity and anti-tumor activity, and a method for producing the same. Below, simply
248-Sq-2A when describing 248-Sq-2 substance
substance or 248-Sq-2B substance or a mixture thereof. The present inventor discovered a 248-Sq 2 strain of Streptomyces isolated from a soil sample collected in Nagano Prefecture.
It was discovered that two substances with anti-lactic acid bacterium activity existed in the culture solution of the microorganism given the number 248-Sq-2A substance. , named the substance 248-Sq-2B, and studied its physicochemical and biological properties. The 248-Sq-2A and 248-Sq-2B materials of the present invention each have the following properties in common. They are white amorphous powders, readily soluble in water, sparingly soluble in alcohols such as methanol, virtually insoluble in ketones such as acetone, and ethyl acetate, chlorinated hydrocarbon solvents such as chloroform, their melting point is 220 °C (with decomposition). Ultraviolet absorption spectrum of 248-Sq-2A substance (in water)
(measured at a concentration of 25 mg/3 c.c.) showed no characteristic absorption up to 220 nm, and its infrared absorption spectrum (measured with a potassium bromide tablet) is shown in Figure 1 of the accompanying drawings.
The main infrared absorption bands [wave numbers (cm ^-1 )] are as follows. 3260 (s) 2880 (w) 1660 (s) 1530 (m) 1410 (m) 1230 (m) 1165 (w) 1155 (w) 1050 (m) 960 (w) However, s = strong, m = medium , w=weak. When the 248-Sq-2A substance is developed with the mixed solvent system shown below, it gives a single spot in ascending chromatography on a thin layer of silica gel as shown below. (1) Rf0.75 with a developing solvent of chloroform-methanol-ammonia water (2:3:1 volume ratio) (2) Rf0.25 with a developing solvent of butanol-acetic acid-water (4:1:2) 248-Sq -2A substances include carbon, hydrogen, nitrogen and oxygen. Its elemental composition is approximately as follows. C51.10%, H7.14%, N15.83%, O26.37% From its elemental analysis, proton NMR spectrum (Figure 3 of the attached drawing), and carbon ¹³ C NMR spectrum, the 248-Sq-2A substance is The following empirical formula C ₃₁ H ₅₂ N ₈ O ₁₂
It seems to have the following. Furthermore, the proton NMR spectrum and carbon ¹³ C NMR spectrum are 248−Sq−
Shows the presence of six methyl groups in the molecule of 2A. Ultraviolet absorption spectrum of 248-Sq-2A substance (in water)
(measured at a concentration of 25 mg/3 c.c.) shows no characteristic absorption up to 220 nm, and its infrared absorption spectrum (measured with potassium bromide tablets) is shown in Figure 2 of the accompanying drawings. The main infrared absorption bands [wave numbers (cm ^-1 )] are as follows. 3260 (s) 2880 (w) 1660 (s) 1530 (m) 1410 (m) 1230 (m) 1165 (w) 1155 (w) 1050 (m) 960 (w) However, s = strong, m = medium , w=weak. The 248-Sq-2B material yields a single spot on silica gel thin layer ascending chromatography using the mixed solvent system described below. (1) Rf0.70 with a developing solvent of chloroform-methanol-ammonia water (2:3:1 volume ratio) (2) Rf0.20 with a developing solvent of butanol-acetic acid-water (4:1:2) 248-Sq -2B substances include carbon, hydrogen, nitrogen and oxygen. Its elemental composition is approximately as follows. C48.98%, H6.71%, N16.33%, O27.99% From its elemental analysis, proton NMR spectrum (Figure 4 of the attached drawing), and carbon ¹³ C NMR spectrum, 248-Sq-2B is as follows. It appears to have the empirical formula of C ₂₈ H ₄₆ N ₈ O ₁₂ . Furthermore, the proton NMR spectrum and carbon ^13C NMR spectrum are 248−Sq−2B
shows that there are five methyl groups in the molecule of The 248-Sq-2 substance of the present invention has the following antibacterial properties. 248-Sq-2A and 248-Sq-2A substances exhibit bacteriostatic activity against certain Gram-positive bacteria. 248−Sq−2A and 248−Sq− against some microorganisms
The bacteriostatic activity of 2B is shown in the following table as the minimum inhibitory concentration (MIC) (mcg/ml) determined by the conventional multiple dilution method.

【table】

[Table] Furthermore, substances 248-Sq-2A and 248-Sq-2B exhibit strong anti-lactic acid bacteria activity when lactic acid bacteria are grown on a synthetic medium with the composition shown in the following table.

[Table] Lactic acid bacteria (Lactobacillus) on this synthetic medium
The minimum inhibitory concentration for (casei) is 248−Sq−
0.3mcg/ml for 2A substance and 248−Sq−2B substance
It is 0.5mcg/ml. Furthermore, in order to examine the antitumor activity of the 248-Sq-2 substance of the present invention, monkey tumor virus
When tested according to the standard method of evaluating anti-tumor activity by observing the difference in the changes in the morphology of the cells after applying the supplied substance to cells that had undergone tumor transformation due to SV-40 infection and normal cells, 248- Sq−2A and
Substance 248-Sq-2B was found to have antitumor activity at a concentration of 50mcg/ml, and the novel substance of the present invention is expected to be used as an antitumor agent. The second gist of the present invention is to culture a 248-Sq-2 substance-producing bacterium belonging to the genus Streptomyces, and collect a 248-Sq-2A substance, a 248-Sq-2B substance, or a mixture thereof from the culture. A method for producing an antibiotic 248-Sq-2 substance is provided. 248
An example of a -Sq-2 substance-producing bacterium is the aforementioned 248-Sq-2 strain of the genus Streptomyces, which was isolated by the present inventor from a soil sample collected in Nagano Prefecture. This strain was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology on February 18, 1982, with deposit number 5882. This strain was obtained by suspending a small amount of soil sample in sterile distilled water and diluting this suspension to various concentrations.
A small amount of each dilution was then spread on the surface of a Petri dish containing nutrient agar and incubated at 27°C for 2 hours to allow microbial growth.
After ~3 days of incubation, colonies were picked and transplanted onto slanted nutrient agar to create a more abundant culture and isolated according to conventional bacterial isolation methods. The mycological properties of this 248-Sq-2 strain are described below, and this strain was recognized to be a new strain of Streptomyces syiratus. (a) Morphological characteristics The vegetative hyphae are 0.5 to 0.9 μm wide, uniaxially branched, and do not divide on agar medium, but when cultured with shaking in liquid medium, they divide into corynebacteria. Aerial hyphae have short sporophytes branching uniaxially in clusters on a relatively short main axis, and long spore chains are attached to them. Spore chains are usually straight, rarely curved or looped, and consist of 10 to 50 or more spores. Spores are oblong (0.6-0.8 x 1.3-2.0μ
m) or phalange form (phalangiform; 0.3
~0.5×1.3~2.0 μm), exhibiting a smooth surface. As a special form, concentrated hyphae and aerial sclerotium-like objects are formed, and sporangia and motile spores are not observed. (b) Cell wall type The sugar components of diaminopimelic acid (DAP) were investigated using whole cell hydrolysis method. Contains LL-DAP, does not contain meso-DAP, and does not contain sugars that serve as classification indicators. That is, it indicates the cell wall type. (c) Culture properties Culture properties were observed according to the ISP method and are shown in Table 3 below. (d) Physiological properties Physiological properties are shown in Table 4 below.

【table】

【table】

[Table] (e) Assimilation of carbon source Observation was made using Pridham-Godelive agar medium, and the results are shown in Table 5.

【table】

[Table] Actinomycetes strain 248-Sq2 is judged to be included in the genus Streptomyces based on its morphological characteristics and cell wall type I. This strain has a flora color that spans both the red and gray series, and the morphology of the spore chain varies from straight (RF) to curved to looped (RA), forming aerial sclerotium-like objects. Smooth spores are attached. Based on these characteristics and assimilability of carbon sources, a search was conducted using Mr. Burgee's bacterial identification handbook and ISP descriptions. As a result, Streptomyces siratus (S.
S. cirratus) and Streptomyces polychromogenes (S. polychromogenes) are closely related to this strain, and the former, whose bacterial flora colors span both red and gray series, is judged to be the most closely related. This strain differs from the standard strain of Streptomyces siratus in that its spore chains usually exhibit RF, it is not a typical RA, and it does not form melanin-like pigments. in general
It is known that in strains that exhibit both RF and RA forms, the degree of expression of both forms changes depending on culture conditions such as medium composition, pH, and temperature, and the degree of positivity for melanin-like pigment production in the standard strain of Streptomyces siratus is not very high. For reasons such as lack of clarity, these differences cannot be judged as criteria for clearly separating species. Therefore, this bacterial strain was identified as a new strain of Streptomyces cirratus, and is related to Streptomyces cirratus KOSHIYAMA et.al.
It is called 248−Sq2 strain. In the method of the present invention, a 248-Sq-2 substance-producing bacterium, such as the 248-Sq-2 strain (FERM-P5882), is cultured in a medium containing nutrients that can be used by ordinary microorganisms. As the nutrient source, any known nutrient source that has been conventionally used for culturing Streptomyces bacteria can be used. For example, glucose, starch, glycerin, sucrose, starch syrup, molasses, etc. can be used as the carbon source. Also, soybean flour, wheat germ, meat extract, peptone, dry yeast, cornstarch liquor, ammonium sulfate, sodium nitrate, etc. can be used as the nitrogen source. Calcium carbonate, salt, etc. as necessary.
In addition to adding inorganic salts such as potassium chloride and phosphates,
Organic and inorganic substances that aid the growth of bacteria and promote the production of 248-Sq-2 substances can be added as appropriate. As for the culture method, the liquid culture method, especially the deep culture method, is most suitable, as is the case with general antibiotic production methods. Cultivation is carried out under aerobic conditions, and the appropriate temperature for cultivation is 25 to 35°C, but in most cases it is cultured at around 27°C. Thus, the production of 248-Sq-2 substance reaches its maximum in 3 to 5 days in both shaking culture and tank culture. The 248-Sq-2 substance mainly exists in the liquid part of the culture, but as shown in the physical and chemical properties below, it is a water-soluble substance, so when extracting and purifying it, a microorganism such as activated carbon or Diaion HP is used. Porous non-ionic adsorption resin or Amberlite IR-
120, Dowex 50W or the like can be used for adsorption, and this can be eluted using an organic solvent or a suitable acid solution. For example, passing the culture solution through a Dowex 50W (H type) resin column, adsorbing the active ingredient to the resin, and eluting it with a 0.5M pyridine-acetic acid solution is an effective method for purifying this substance. be. The crude powder of 248-Sq-2 material obtained in this manner can be further purified by chromatography using cellulose, silica gel, alumina or Sephadex. The 248-Sq-2 material thus obtained is a white amorphous powder. The 248-Sq-2A substance and the -2B substance are separated by adsorption onto a column of silica gel or Sephadex G-25, followed by development with butanol-acetic acid-water (4:1:2 volume ratio). The method of the invention will now be explained by way of examples. Example 1 (a) One platinum loop was taken from a slant of Streptomyces siratus strain 248-Sq2 (FERM-P5882), inoculated into a 15 ml test tube containing sterilized YH medium, and shaken at 27°C for 48 hours. A pre-previous culture was obtained by culturing. 25g dextrin, 20g soybean flour
Put 100 ml of a sterilized medium (PH 6.4) containing 5 g of NaCl, 4 g of CaCO ₃ to 1 volume with water into a 500 ml Erlenmeyer flask, inoculate with 2 ml of the above pre-prepared culture, and shake at 27°C for 72 hours. A preculture solution was obtained by culturing. (c) Introduce a production medium consisting of the following ingredients into 30 fermenters. Dextrin 375g Soy flour 300g NaCl 75g CaCO ₃ 60g Tap water (enough to make 13) Adjust PH to 0.4 by adding 4N hydrochloric acid solution. The medium is sterilized by passing water vapor at 122°C through it for 20 minutes. After cooling, the volume of the culture medium will change due to condensation of water vapor during the sterilization process.
15 and the pH is 7.0. Next, inoculate this with the above preculture solution (300 ml). Culture at 27℃
I went for 72 hours. During this time, the air is stirred and sterilized by a turbine rotating at 400 rpm (30 rpm).
minutes). At the end of this operation the pH of the culture medium is
7.8 and the volume was 14. (c) Add a supernatant (500 g) to the culture solution 14 obtained as described above and filter it to obtain a culture solution.
The solution is passed through a Diaion HP-20 column (3) for adsorption, and then eluted with methanol (10). The eluate obtained as described above is concentrated under reduced pressure to remove methanol, and the concentrated solution is passed through a Dowex 50 column. After washing the column with water, 5%
Elute the adsorbed substance with an aqueous pyridine solution (10). After concentration under reduced pressure, adjust the pH to 7.0, then pass through a Diaion HP-20 column and perform gradient elution from water to 100% methanol. Here 2
Two active sections A and B are obtained. Each active fraction was collected, concentrated under reduced pressure, and then individually chromatographed on Dowex 50 resin. Elute with 0.5M pyridine-acetic acid solution, collect active fractions, and concentrate under reduced pressure. Next, partition chromatography was performed using Sephadex G-25. Elution was performed with butanol-acetic acid-water (4:1:2), and the active fractions were collected and concentrated under reduced pressure. Next, the active fractions were passed through a Sephadex G-50 column, concentrated, and freeze-dried to 248-Sq-
Obtain 50 mg of 2A substance and 30 mg of 248-Sq-2B substance. Example 2 A supporting agent (500 g) is added to the culture solution 14 obtained in the same manner as in Example 1 (b), and the medium is filtered to obtain a culture solution. Add 500 g of activated carbon to liquid (13), stir for 20 minutes, and filter. Next, mix activated carbon into 70% acetone water (10) (PH2.0), stir and filter to obtain a liquid. Add hydrochloric acid aqueous solution to this to make the pH 7.0,
Concentrate under reduced pressure to remove acetone to obtain a crude extract (3). Dowex the crude extract obtained as above.
Pass through 50 columns. After washing the column with water, the adsorbed substance is eluted with a 5% aqueous pyridine solution (10). After concentration under reduced pressure, adjust the pH to 7.0, then use Diaion HP20.
column and perform gradient elution of water → 100% methanol. Here, two types of activity categories A and B are obtained. Each active fraction was collected and concentrated under reduced pressure, followed by chromatography using Dowex 50 resin. 0.5M pyridine
Elute with acetic acid solution, collect active fractions, and concentrate under reduced pressure. Next, partition chromatography was performed using Sephadex G-25. For elution, a mixture of butanol-acetic acid-water (4:1:2) is used, and the active fractions are collected and concentrated under reduced pressure. Subsequently, it was passed through a column of Sephadex G-50, and each active fraction was concentrated and lyophilized to 248
-50 mg of Sq-2A substance, 30 mg of 248-Sq-2B substance
I got it.

[Brief explanation of the drawing]

Figures 1 and 2 respectively show 248-Sq- of the present invention.
It is an infrared absorption spectrum diagram of substances A and -B,
3 and 4 are proton NMR spectra.

Claims (1)

[Claims] 1. White amorphous powder, easily soluble in water, slightly soluble in alcohols such as methanol, virtually insoluble in ketones such as acetone and ethyl acetate, chlorinated hydrocarbon solvents such as chloroform, melting point 220
°C (accompanied by decomposition), does not show any characteristic absorption in the ultraviolet absorption spectrum, and has wave numbers of 3260, 2880, 1660, and
1530, 1410, 1230, 1165, 1155, 1050 and 960
It shows an absorption band at 0.75 cm ^-1 and was determined by thin layer ascending chromatography on silica gel using chloroform-methanol-aqueous ammonia (2:3:1 volume ratio).
Thin layer elevation chromatography on silica gel using butanol-acetic acid-water (4:1:2 volume ratio) has an Rf of 0.25, and its elemental composition is approximately C51.10%, H7.14 %, N15.38%, O26.37%, 248-Sq-2A substance with anti-lactic acid bacterium activity;
White amorphous powder, readily soluble in water, sparingly soluble in alcohols such as methanol, virtually insoluble in ketones such as acetone and ethyl acetate, chlorinated hydrocarbon solvents such as chloroform, melting point 220°C
(accompanied by decomposition), and does not show any characteristic absorption in the ultraviolet absorption spectrum, and the infrared absorption spectrum (measured with potassium bromide tablets) shows wave numbers of 3260, 2880, 1660,
1530, 1410, 1230, 1165, 1155, 1050 and 960
It shows an absorption band at 0.70 cm ^-1 and a
Rf, butanol-acetic acid-water (4:1:2
It has an Rf of 0.20 in thin layer ascending chromatography of silica gel using (volume ratio), and its elemental composition is
An antibiotic 248-Sq-2 substance selected from the group consisting of 248-Sq-2B substance having anti-lactic acid bacterium activity of C48.98%, H6.71%, N16.33%, O27.99%. 2. Cultivate 248-Sq-2 substance-producing bacteria belonging to the genus Streptomyces, and use the culture to produce 248-Sq-2A.
Antibiotics 248-Sq-2 characterized by collecting the substance or 248-Sq-2B substance or a mixture thereof
Substance manufacturing method.